An anti-chikungunya bispecific antibody 8D1-7H9 and its application

Abstract

The present invention provides a camel-derived nanoantibody 7H9 that binds to the E2 protein of the chikungunya virus, and a bispecific antibody 8D1-7H9 based on the camel-derived nanoantibody 7H9 and the anti-chikungunya virus human monoclonal antibody 8D1, and the use of the camel-derived nanoantibody 7H9 and the bispecific antibody 8D1-7H9 in the preparation of a drug for preventing or treating chikungunya virus disease. Compared with the parent monoclonal antibody 8D1, the neutralizing activity of the bispecific antibody provided by the present invention is significantly improved, making it possible to develop it into a specific drug for chikungunya fever.

Description

Technical Field

[0001] 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

[0002] 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.

[0003] At present, some studies have identified a variety of polyclonal or neutralizing monoclonal antibodies (mAbs) that have a protective effect against CHIKV infection. Most of the identified neutralizing monoclonal antibodies against CHIKV target the surface-exposed domain 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 also 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 highly neutralizing fully human monoclonal antibody 8D1 against Chikungunya virus from the plasma cells of the peripheral blood of a person infected with Chikungunya fever 15 days after recovery by flow sorting-single-cell PCR technology. IC 50 The value is 0.12 μg / mL (see CN 110903386 A), but its binding activity and affinity to CHIKV E2 protein are relatively weak. There is currently no approved antibody against CHIKV, so it is urgent to develop a new generation of neutralizing antibody drugs that can provide protective activity.

[0004] 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".

[0005] 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

[0006] 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, 51-58 and 97-118 of SEQ ID NO: 1, respectively.

[0007] 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 "7H9".

[0008] 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 present invention incorporates its disclosure 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 7H9 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: 4.

[0009] 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.

[0010] 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.

[0011] 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 7H9 that binds to the CHIKV E2 protein, that is, the "VHH(H)-IgG" form. In the present invention, the bispecific antibody is named 7H9-8D1-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,

[0012] 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 7H9-8D1-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,

[0013] 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-7H9; 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,

[0014] 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-7H9; 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.

[0015] Third, the present invention provides a polynucleotide. In a preferred technical scheme, the polynucleotide is a polynucleotide encoding the above-mentioned camel-derived nanoantibody 7H9 that binds to CHIKV E2 protein, and the sequence of the polynucleotide encoding the camel-derived nanoantibody 7H9 that binds to CHIKV E2 protein is shown in SEQ ID NO: 2.

[0016] 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 "7H9-8D1-H"; or,

[0017] 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 "7H9-8D1-κ"; or,

[0018] 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-7H9"; or,

[0019] The polynucleotide sequences encoding the heavy chain and light chain of the bispecific antibody against Chikungunya virus 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-7H9".

[0020] 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.

[0021] 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 7H9 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 7H9 that binds to the CHIKV E2 protein described in the present invention.

[0022] 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.

[0023] 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 7H9 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 7H9 that binds to the CHIKV E2 protein described in the present invention.

[0024] 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.

[0025] Sixth, the present invention provides the use of the camel-derived nanoantibody 7H9 that binds to CHIKV E2 protein in the preparation of a drug for preventing or treating Chikungunya virus disease.

[0026] 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.

[0027] The present invention provides a camel-derived nanoantibody 7H9 that binds to CHIKV E2 protein. Due to the different binding activities of the human monoclonal antibody 8D1 and nanoantibody 7H9 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 nano antibody 7H9 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 7H9-8D1-H, 7H9-8D1-κ, 8D1-H-7H9, and 8D1-κ-7H9. The purpose is to utilize the advantages of bispecific antibodies, not only to better retain the specific functions of parent monoclonal antibody 8D1 and nano antibody 7H9, 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, thereby providing candidate drugs for the prevention and treatment of CHIKF. The data of the examples of the present invention show that the bispecific antibodies have good affinity with the CHIKV E2 protein, among which 7H9-8D1-κ has the strongest affinity with the CHIKV E2 protein, reaching less than 1.0E-12, and the protective effect and IC 50 In the evaluation, compared with the antibodies in the prior art, the neutralizing activities of the bispecific antibodies 8D1-H-7H9, 7H9-8D1-κ, 8D1-κ-7H9 and monoclonal antibody 8D1 were significantly improved; compared with the parent monoclonal antibody 8D1, the neutralizing activities of the bispecific antibodies 7H9-8D1-H, 8D1-H-7H9 and 7H9-8D1-κ were improved, which were approximately 2, 3.4 and 4.3 times that of the monoclonal antibody 8D1, respectively, making it possible to develop them into specific drugs for Chikungunya fever. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : SDS-PAGE detection results of CHIΚV E2 protein (separation gel concentration 12%);

[0029] Figure 2 : SDS-PAGE detection results of nanobody 7H9-Fc (separation gel concentration 12%);

[0030] Figure 3: A schematic diagram of the structure of a bispecific antibody; wherein, Figure 3 A is 7H9-8D1-H, Figure 3 B is 8D1-H-7H9, Figure 3 C is 7H9-8D1-κ, Figure 3 D is 8D1-κ-7H9;

[0031] Figure 4 : SDS-PAGE test results of bispecific antibody and monoclonal antibody 8D1 (separation gel concentration 12%);

[0032] Figure 5 : ELISA binding curves of bispecific antibody, monoclonal antibody 8D1, nanoantibody 7H9 and CHIKV E2 protein;

[0033] Figure 6 : Binding kinetic curves of monoclonal antibody 8D1, bispecific antibodies 8D1-H-7H9 and 8D1-κ-7H9 to CHIKV E2 protein;

[0034] Figure 7 : Binding kinetic curves of nanobody 7H9, bispecific antibodies 7H9-8D1-H and 7H9-8D1-κ to CHIKV E2 protein;

[0035] Figure 8 : Neutralization activity of bispecific antibodies, parental antibodies and control antibodies against true CHIKV virus. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1: Screening and preparation of nanobodies against CHIKV E2 protein

[0038] 1. Expression and purification of CHIKV E2 protein

[0039] The present invention uses an insect cell-baculovirus system to express CHIΚV E2 protein. First, the CHIΚV E2 protein gene sequence (GenBank: AAU43881.1) is optimized according to the insect cell's preference for codons, and 6 histidine tags (N-terminal) are added. Then, the gene fragment is synthesized by an artificial method and cloned into the pFastBac1 vector (insect cell baculovirus expression vector) by a seamless cloning method. The recombinant plasmid is transformed into DH10Bac competent cells, and after blue-white screening and PCR identification, a recombinant baculovirus bacmid (Bacmind) is obtained. The recombinant baculovirus bacmid is extracted by isopropanol precipitation, and then Bacmind is transfected with a transfection reagent (ExpiFectamine TM Sf Transfection Reagent, A38915) was transfected into Expi Sf21 insect cells and cultured at 27°C and 120 rpm for 96 hours. The culture supernatant was collected, centrifuged, and stored at -80°C to obtain CHIΚV E2 protein P0 virus. The P0 virus was subsequently transferred to Expi Sf9 cells to amplify the virus. After 4 days of continuous culture, the supernatant was collected and purified by nickel ion affinity chromatography (HisTrap TM After purification by excel, CHIΚVE2 protein was obtained. Its expression and purity were identified by SDS-PAGE. Figure 1 As shown, the results show that the molecular weight of the purified CHIΚV E2 protein is 50 kDa, which is in line with expectations and the purity also meets the requirements.

[0040] 2. Camel immunization and establishment of VHH nanoantibody library

[0041] 1 mg of CHIKV E2 protein with 6 histidine tags prepared earlier 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, serum titers were tested using ELISA. Serum titers >10 5, then collect 100 mL of blood, separate peripheral blood mononuclear cells (PBMC), and extract total RNA 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 gel. The recovered DNA was used as a template for secondary amplification, and VHH-FOR and VHH-REV primers (primer sequences are shown in Table 1) were used to run gel to recover the corresponding band of VHH. 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 instructions, transformed into competent cells TG1, and spread on a selective culture medium containing ampicillin. After overnight culture at 37°C, all colonies were collected in LB culture medium, centrifuged and the supernatant was discarded, and the cells were resuspended in LB. This is the antibody library.

[0042] Table 1. PCR reaction primers

[0043]

[0044] 3. Phage display technology to screen specific nanoantibodies

[0045] Take 100 μL of phage display vector library and inoculate it into 100 mL 2×YT / AMP GLU medium. Cultivate at 37°C and 200 r / min until the logarithmic phase (OD 600 value was 0.6~0.8), and 90 μL M13Κ07 helper phage (1.7×10 13 PFU / mL), mix gently, culture at 37 °C overnight, divide the culture medium into 50 mL centrifuge tubes, centrifuge at 3800 g, 4 °C 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 upside down, and let stand on ice for 2 h; centrifuge at 3800 g, 4 °C 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 measure the phage titer.

[0046] The CHIΚV E2 protein was diluted to 4 μg / mL with PBS buffer. Three duplicate wells were selected for a 96-well ELISA plate. 100 μL (400 ng / well) was added to each well and coated overnight at 4 °C. 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 wells were blocked for 1 h at room temperature. The wells were washed with PBST for 4 times. The phage solution collected above was diluted to 5×10 with 2% skim milk powder. 11 PFU / mL, add to the ELISA plate, 100 µL / well, incubate at room temperature for 2 h; discard the phage sample, wash 10 times with PBST, then wash 5 times with PBS, add 100 µL of freshly prepared 0.1M triethylamine to each well, let stand at room temperature for 10 min, aspirate the eluate and quickly neutralize with an equal volume of 1 M Tris-HCl (pH7.4); then take 400 µL of the eluate, infect 4 mL of logarithmic phase TG1 cells, and amplify the phage. Prepare a 96-well plate coated with CHIΚV E2 protein again for the second and third rounds of panning. Randomly pick 96 monoclonal clones from the bacterial plate after the third round of screening. After inducing VHH antibody expression with IPTG, prepare soluble recombinant VHH antibody crude extracts for ELISA detection to preliminarily identify the reactivity of VHH antibody clones with CHIΚV-E2 protein. When OD 450 The value was more than 3 times greater than the PBS control and was judged as positive. A portion of the bacterial solution of the positive clone was sequenced to obtain the nucleic acid coding sequence of the 7H9 clone as shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1, and the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 1, amino acids 26-33, 51-58, and 97-118, respectively.

[0047] 4. Expression and purification of nanobody 7H9

[0048] The Fc sequence was added to the C-terminus of the nucleic acid coding sequence of 7H9, and it was constructed into the pcDNA3.4 vector through the restriction enzyme sites Eco RI and Eco RV, and transfected into Expi293F cells. After 5 days of culture, the supernatant was collected, centrifuged at 3000g 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 as follows Figure 2 As shown, the purified nanobody 7H9-Fc was obtained, and the molecular weight was 40 kDa, which was in line with expectations.

[0049] Example 2. Construction and preparation of bispecific antibodies

[0050] 1. Construction of bispecific antibodies

[0051] 8D1 is a monoclonal antibody against Chikungunya virus, the heavy chain variable region sequence is shown in SEQ ID NO:3, the nucleic acid coding sequence is shown in SEQ ID NO:5, the light chain variable region sequence is shown in SEQ ID NO:4, 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 SEQ ID NO:10 (see CN110903386 A). The VHH of 7H9 and 8D1 were connected by (G4S)2linker (the coding sequence is shown in SEQ ID NO:23), and the sequence was synthesized and cloned into the eukaryotic expression vector pcDNA3.4 by enzyme digestion and homologous recombination. The construction forms include VHH(H)-IgG, IgG(H)-VHH, VHH(L)-IgG, IgG(L)-VHH (175kDa), as shown in Figure 3 As shown (dark gray is the 8D1 heavy chain constant region, dark blue is the 8D1 heavy chain variable region, light gray is the 8D1 light chain constant region, light blue is the 8D1 light chain variable region, and red is the 7H9 heavy chain variable region), the key maps are as follows:

[0052] (1) VHH connected to the N-terminus of the IgG heavy chain (VHH(H)-IgG):

[0053] Eco RI restriction site-GCCGCCACC-start codon-signal peptide-7H9-VHH-(G4S)2-8D1-VH-CH-stop codon-Eco RV restriction site ( Figure 3 A); 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 7H9-8D1-H (the heavy chain sequence is shown in SEQ ID NO: 11, the nucleic acid encoding sequence is shown in SEQ ID NO: 12, the light chain sequence is shown in SEQ ID NO: 13, and the nucleic acid encoding sequence is shown in SEQ ID NO: 14).

[0054] (2) The C-terminus of the IgG heavy chain is connected to VHH (IgG(H)-VHH):

[0055] Eco RI restriction site-GCCGCCACC-start codon-signal peptide-8D1-VH-CH-(G4S)2-7H9-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-7H9 (the heavy chain sequence is shown in SEQ ID NO: 19, the nucleic acid encoding sequence is shown in SEQ ID NO: 20, the light chain sequence is shown in SEQ ID NO: 13, and the nucleic acid encoding sequence is shown in SEQ ID NO: 14).

[0056] (3) Connection between VHH and the N-terminus of IgG light chain (VHH(L)-IgG):

[0057] Eco RI restriction site-GCCGCCACC-start codon-signal peptide-7H9-VHH-(G4S)2-8D1-VL-CL-stop codon-Eco RV restriction site ( Figure 3 C); 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: 7H9-8D1-κ (the heavy chain sequence is shown in SEQ ID NO: 15, the nucleic acid encoding sequence is shown in SEQ ID NO: 16, the light chain sequence is shown in SEQ ID NO: 17, and the nucleic acid encoding sequence is shown in SEQ ID NO: 18).

[0058] (4) VHH connected to the C-terminus of IgG light chain (IgG(L)-VHH):

[0059] Eco RI restriction site-GCCGCCACC-start codon-signal peptide-8D1-VL-CL-(G4S)2-7H9-VHH-stop codon-Eco RV restriction site ( Figure 3 D); 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-7H9 (the heavy chain sequence is shown in SEQ ID NO: 15, the nucleic acid encoding sequence is shown in SEQ ID NO: 16, the light chain sequence is shown in SEQ ID NO: 21, and the nucleic acid encoding sequence is shown in SEQ ID NO: 22).

[0060] 2. Transient Expression and Affinity Chromatography Purification of Bispecific Antibodies

[0061] 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. It was balanced with 20 mM PBS before loading. The sample was injected after the conductivity reached the baseline. After loading, 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. When UV280 was close to the baseline, the collection was stopped. Subsequently, the column was ultrafiltered through an ultrafiltration concentration tube, and the buffer was replaced with PBS to obtain the pure recombinant protein of the bispecific antibody. Its expression and purity were identified by SDS-PAGE. Figure 4 As shown: the heavy chain of antibodies 7H9-8D1-H and 8D1-H-7H9 is about 65 kDa, the light chain is about 25 kDa, the heavy chain of antibodies 7H9-8D1-κ and 8D1-κ-7H9 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.

[0062] Example 3. Identification of bispecific antibody binding activity

[0063] 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:

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 6. Detect the OD value at 450-630 nm on an ELISA instrument, draw the curve using the Logistic four-parameter fitting method, and calculate the EC value of the antibody. 50 value.

[0070] ELISA test results Figure 5 The results showed that the bispecific antibody bound to CHIKV E2 protein in a dose-dependent manner. Compared with monoclonal antibody 8D1, EC 50 (half maximal effect concentration) was significantly improved compared with the nanobody 7H9, EC 50 There was no significant difference, as shown in Table 2.

[0071] Table 2. ELISA binding EC of bispecific antibodies and monoclonal antibodies to CHIKV E2 protein 50

[0072]

[0073] From Table 2, the EC 50 The results show that 7H9-8D1-κ has the best binding activity. “-” indicates EC 50 >1000ng / mL).

[0074] Example 4. Affinity identification of bispecific antibodies

[0075] 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:

[0076] 1. Prepare buffer: Running Buffer (PBS + 0.02% Tween 20 + 0.2% BSA), Regeneration buffer (10 mM Gly, pH 1.75).

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] BLI test results Figure 6-Figure 7 The results showed that the affinity constants KD values ​​of 8D1, 7H9, 8D1-H-7H9, 7H9-8D1-H, 8D1-κ-7H9, and 7H9-8D1-κ were 13.1, 0.93, 4.91, 3.38, 1.21, and <0.001 nM, respectively. The results showed that the constructed bispecific antibodies had good affinity with the CHIKV E2 antigen, among which 7H9-8D1-κ had the strongest affinity with the CHIKV E2 antigen, reaching below 1.0E-12, making it possible to develop into a specific drug for Chikungunya fever. The detailed kinetic parameters are shown in Table 3 below.

[0082] Table 3. Binding kinetic data of bispecific antibodies and parental monoclonal antibodies to CHIKV E2 protein

[0083]

[0084] Example 5. Analysis of the neutralization activity of bispecific antibodies against real viruses

[0085] 1. One day before the experiment, dilute Vero cells to 1.5×10 5 cells / mL concentration, inoculated into 96-well cell culture plates, inoculated with a volume of 200 μL / well, and cultured in a 37°C, 5% CO2 cell culture incubator;

[0086] 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;

[0087] 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;

[0088] 4. After 72 h, discard the cell culture supernatant, 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;

[0089] 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, and measure the OD 630 As a reference, measure OD with a microplate reader 570 Values; cell viability was calculated using (OD sample well-OD death control) / (OD survival control-OD death control). Cell viability and antibody concentration were fitted using GraphPad Prism 8 to calculate antibody IC 50 value;

[0090] 6. Protective effect and IC of dual antibodies in cell models 50 The results, see Figure 8 and Table 4.

[0091] result: Figure 8 is a graph showing the change in concentration of neutralizing activity of 7H9-8D1-H, 8D1-H-7H9, 7H9-8D1-κ, 8D1-κ-7H9, 8D1, AB2 (control antibody, see CN 110903386 A) and CHIΚV true virus, according to the IC in Table 4 50 IC values ​​of bispecific antibodies 7H9-8D1-H, 8D1-H-7H9, 7H9-8D1-κ, and 8D1-κ-7H9 in neutralizing CHIΚV 50 The IC values ​​of monoclonal antibody 8D1 for neutralizing CHIΚV were 0.84, 0.05, 0.04, and 1.86 μg / mL, respectively.50 The IC value of control antibody AB2 in neutralizing CHIΚV is 0.17μg / mL. 50 The results showed that the neutralization activity of bispecific antibodies 8D1-H-7H9, 7H9-8D1-κ, 8D1-κ-7H9 and monoclonal antibody 8D1 was significantly improved compared with the control antibody AB2; compared with the parent monoclonal antibody 8D1, the neutralization activity of bispecific antibodies 7H9-8D1-H, 8D1-H-7H9 and 7H9-8D1-κ was improved, among which 7H9-8D1-κ was more significantly improved, about 4.3 times that of monoclonal antibody 8D1, 8D1-H-7H9 was 3.4 times that of monoclonal antibody 8D1, and 7H9-8D1-H was 2 times that of monoclonal antibody 8D1. The neutralization activity of bispecific antibodies, parent antibodies and control antibodies against CHIKV true virus is shown in Figure 2. Figure 8 shown.

[0092] Table 4. IC values ​​of bispecific antibodies, parental monoclonal antibodies, control antibody AB2 and CHIΚV true virus 50

[0093] .

Claims

1. A camel-derived nanobody that binds to the E2 protein of Chikungunya virus, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the camel-derived nanobody are shown in positions 26-33, 51-58 and 97-118 of SEQ ID NO: 1, respectively.

2. The camel-derived nanobody that binds to the Chikungunya virus E2 protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the camel-derived nanobody is shown in SEQ ID NO:

1.

3. A bispecific antibody against Chikungunya virus, characterized in that: The bispecific antibody uses an anti-Chikungunya virus monoclonal antibody as a parent antibody, and is fused with the camel-derived nanoantibody that binds to the Chikungunya virus E2 protein as described in claim 2 at one end of its heavy chain or light chain, wherein the amino acid sequence of the heavy chain variable region of the anti-Chikungunya virus monoclonal antibody is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region of the anti-Chikungunya virus monoclonal antibody is as shown in SEQ ID NO:

4.

4. The anti-Chikungunya virus bispecific antibody 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 anti-Chikungunya virus bispecific antibody according to claim 4, characterized in that: The fusion is fused in the form of (GGGGS)n connecting peptide connection, wherein n is a natural number of 1-4.

6. The anti-Chikungunya virus bispecific antibody according to claim 5, characterized in that: The bispecific antibody uses an anti-Chikungunya virus monoclonal antibody as a parent antibody, and the C-terminus of the camel-derived nanobody that binds to the Chikungunya virus E2 protein is fused at the N-terminus of the heavy chain of the bispecific antibody, and 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 its light chain is fused to the C-terminus of the camel-derived nanobody that binds to the E2 protein of the Chikungunya virus, and 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 is fused to the N-terminus of the camel-derived nanobody that binds to the E2 protein of the Chikungunya virus, and 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 its light chain is fused to the N-terminus of the camel-derived nanoantibody that binds to the E2 protein of the Chikungunya virus, and 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.

7. A polynucleotide encoding the camel-derived nanobody that binds to the Chikungunya virus E2 protein according to claim 2, characterized in that: The sequence of the polynucleotide is shown in SEQ ID NO:

2.

8. A polynucleotide encoding the anti-Chikungunya virus bispecific antibody according to claim 6, characterized in that: 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, 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, 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, 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.

9. A vector comprising the polynucleotide according to claim 7 or 8.

10. A host cell containing the vector according to claim 9.

11. Use of the camel-derived nanobody that binds to the E2 protein of the Chikungunya virus according to claim 1 or 2 in the preparation of a drug for preventing or treating Chikungunya virus disease.

12. Use of the anti-Chikungunya virus bispecific antibody according to any one of claims 3 to 6 in the preparation of a drug for preventing or treating Chikungunya virus disease.

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