Bispecific antibody capable of simultaneously targeting G and F glycoproteins of Hunipah virus and application of bispecific antibody

Abstract

The invention discloses a group of bispecific antibodies capable of simultaneously targeting adhesion glycoprotein G and fusion glycoprotein F of Hunipah virus. The bispecific antibody comprises a complete first monoclonal antibody, and a heavy chain variable region and a light chain variable region of a second monoclonal antibody which are respectively connected to the N end of the heavy chain variable region and the N end of the light chain variable region of the first monoclonal antibody; comprising a complete first monoclonal antibody and a single-chain antibody which is connected to an N end or a C end of a heavy chain variable region of the first monoclonal antibody and is formed by connecting a heavy chain variable region and a light chain variable region of a second monoclonal antibody. The bispecific antibody provided by the invention has good expression efficiency, and retains good binding activity with G protein and F protein. The bispecific antibody can effectively cross-neutralize Hendea pseudovirus and Nipah pseudovirus, and shows higher neutralizing activity than a parent antibody. The invention also discloses an application of the anti-Henipah virus bispecific antibody in preparation of Henipah virus treatment drugs.

Description

Technical Field

[0001] This invention discloses a bispecific antibody, belonging to the field of polypeptide technology. Background Technology

[0002] Nipah virus (NiV) and Hendra virus (HeV) belong to the genus Hennipahvirus in the family Paramyxoviridae. They are single-stranded, negative-sense RNA viruses that can cause severe encephalitis and respiratory illnesses in humans, with a mortality rate as high as 50%–100%. These viruses have a wide host range and are continuously expanding geographically, potentially exposing about a quarter of the global population. Therefore, they are listed as priority pathogens for research by the World Health Organization. In recent years, Hennipah virus outbreaks in South Asia have shown a periodic outbreak trend, posing a serious threat to my country's biosecurity, and there is an urgent need to develop effective prevention and control measures.

[0003] The Hennipa virus genome is approximately 18 kb in length, containing six transcription units and untranslated regions at both ends, encoding six structural proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), attachment glycoprotein (G), and large protein (L). Viral invasion of host cells is mediated by the G and F proteins located on the viral envelope. The G protein exists as a homotetramer and is a type II transmembrane protein; the F protein is a trimeric type I transmembrane protein. When the globular head domain of the G protein recognizes and binds to the host cell surface receptors—liver ligands B2 / B3—it activates a conformational change in the F protein, transitioning it from a pre-fusion state to a post-fusion state, thereby driving viral fusion with the cell membrane. Syncytial formation is a typical pathological feature of Hennipa virus-infected cells.

[0004] Given the high pathogenicity of Hennipa virus, live virus experiments must be conducted in a biosafety level 4 (BSL-4) laboratory, which significantly limits the development of vaccines and antibodies. Currently, there are no approved Hennipa virus vaccines or specific treatments for humans worldwide. Existing intervention strategies primarily focus on the G and F proteins, with only one G protein-based VSV vector vaccine and one mRNA-1215 vaccine having entered Phase I clinical trials. Some broad-spectrum antiviral small molecule drugs have limited efficacy in animal models, often requiring high doses and long treatment courses to show protective effects. In contrast, antibody drugs, due to their high specificity and good safety profile, have demonstrated excellent therapeutic effects in animal experiments and are considered ideal treatment candidates. For example, the monoclonal antibody m102.4, targeting the G protein receptor-binding region, has shown protective effects in ferrets and non-human primates and has been used as an emergency measure in 15 high-risk exposed individuals; it has now completed Phase I clinical trials. Another mouse-derived monoclonal antibody, 5B3, targeting the F protein, has also demonstrated protection against lethal doses of the virus in animal models. However, the limited number of existing antibodies, the uneven neutralizing activity of antibodies targeting the G or F proteins in different Hennipa virus strains, and the existence of related antibody escape mutant strains highlight the necessity of developing more efficient and broad-spectrum anti-Hennipa virus antibodies.

[0005] Bispecific antibodies can simultaneously target two antigens or recognize different epitopes, and are expected to enhance neutralizing activity and reduce viral escape probability through complementary or synergistic mechanisms, thus becoming an important development direction for next-generation antibody drugs. Currently, no highly effective bispecific antibodies against Hennipa virus have been reported. The purpose of this invention is to provide a bispecific antibody against Hennipa virus. Summary of the Invention

[0006] Based on the above objectives, in a first aspect of the present invention, a bispecific antibody that simultaneously targets the G and F glycoproteins of Hennipa virus is provided, characterized in that the bispecific antibody comprises an intact first monoclonal antibody and the heavy chain variable region and light chain variable region of a second monoclonal antibody respectively linked to the N-terminus of its heavy chain variable region and the N-terminus of its light chain variable region; or, the bispecific antibody comprises an intact first monoclonal antibody and a single-chain antibody linked to the N-terminus or C-terminus of its heavy chain variable region, wherein the single-chain antibody is formed by linking the heavy chain variable region and light chain variable region of the second monoclonal antibody. The first monoclonal antibody and the second monoclonal antibody are combinations targeting different proteins. The sequence combination of the heavy chain variable region and the light chain variable region of the first monoclonal antibody and the sequence combination of the heavy chain variable region and the light chain variable region of the second monoclonal antibody are selected from the combination of SEQ ID NO.1 and SEQ ID NO.5 and the combination of SEQ ID NO.3 and SEQ ID NO.6.

[0007] In this invention, SEQ ID NO.1 and SEQ ID NO.5 are combined to form a monoclonal antibody 5C8 targeting the Hennipa virus fusion glycoprotein F, and SEQ ID NO.3 and SEQ ID NO.6 are combined to form a monoclonal antibody 1E5 targeting the Hennipa virus adhesion glycoprotein G.

[0008] In a preferred embodiment, the sequence combination of the heavy chain variable region and the light chain variable region of the first monoclonal antibody is the combination of SEQ ID NO.1 and SEQ ID NO.5 (heavy chain variable region and light chain variable region of monoclonal antibody 5C8), and the sequence combination of the heavy chain variable region and the light chain variable region of the second monoclonal antibody is the combination of SEQ ID NO.3 and SEQ ID NO.6 (heavy chain variable region and light chain variable region of monoclonal antibody 1E5).

[0009] In a more preferred embodiment, in the bispecific antibody simultaneously targeting Hennipa virus G and F glycoproteins, the C-terminus of the heavy chain constant region of the first monoclonal antibody contains tandem sequences of peptides as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.2, and SEQ ID NO.6. In this invention, the bispecific antibody having the aforementioned structure is named "5C8-1E5scFv". This bispecific antibody is essentially formed by fusing the C-terminus of the heavy chain constant region of the monoclonal antibody 5C8 targeting Hennipa virus fusion glycoprotein F with a linker of sequence as shown in SEQ ID NO.2, to a single-chain antibody formed by tandemly connecting the heavy chain variable region, linker, and light chain variable region of monoclonal antibody 1E5 targeting Hennipa virus adhesion glycoprotein G, as shown in SEQ ID NO.3, SEQ ID NO.2, and SEQ ID NO.6.

[0010] In another, more preferred embodiment, in the bispecific antibody simultaneously targeting Hennipa virus G and F glycoproteins, the sequence from the N-terminus to the C-terminus of the heavy chain variable region of the first monoclonal antibody is formed by tandemly linked peptides shown in SEQ ID NO.3, SEQ ID NO.2, SEQ ID NO.6, and SEQ ID NO.2. In this invention, the bispecific antibody having the aforementioned structure is named "1E5scFv-5C8". This bispecific antibody is essentially a single-chain antibody formed by tandemly linking the heavy chain variable region, linker, and light chain variable region of monoclonal antibody 1E5 (which targets Hennipa virus adhesion glycoprotein G, as shown in SEQ ID NO.3, SEQ ID NO.2, and SEQ ID NO.6) to the N-terminus of the heavy chain variable region of monoclonal antibody 5C8 (which targets Hennipa virus fusion glycoprotein F) using a linker with the sequence shown in SEQ ID NO.2.

[0011] In a preferred embodiment, the sequence combination of the heavy chain variable region and the light chain variable region of the first monoclonal antibody is the combination of SEQ ID NO.3 and SEQ ID NO.6 (heavy chain variable region and light chain variable region of monoclonal antibody 1E5), and the sequence combination of the heavy chain variable region and the light chain variable region of the second monoclonal antibody is the combination of SEQ ID NO.1 and SEQ ID NO.5 (heavy chain variable region and light chain variable region of monoclonal antibody 5C8).

[0012] In a more preferred embodiment, in the bispecific antibody that simultaneously targets Hennipa virus G and F glycoproteins, the N-terminal to C-terminal sequence of the heavy chain variable region of the first monoclonal antibody is composed of tandem peptides shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and the N-terminal to C-terminal sequence of the light chain variable region of the first monoclonal antibody is composed of tandem peptides shown in SEQ ID NO.5, SEQ ID NO.2, and SEQ ID NO.6. In this invention, the bispecific antibody having the aforementioned structure is named "5C8V-1E5". This bispecific antibody is essentially fused at the C-terminus of the heavy chain variable region of the monoclonal antibody 5C8 targeting the Hennipa virus fusion glycoprotein F with a linker of the sequence as shown in SEQ ID NO.2 to the N-terminus of the heavy chain variable region of the monoclonal antibody 1E5 targeting the Hennipa virus adhesion glycoprotein G, and fused at the C-terminus of the light chain variable region of the monoclonal antibody 5C8 targeting the Hennipa virus fusion glycoprotein F with a linker of the sequence as shown in SEQ ID NO.2 to the N-terminus of the light chain variable region of the monoclonal antibody 1E5 targeting the Hennipa virus adhesion glycoprotein G.

[0013] In a preferred embodiment of the present invention, the heavy chain constant region of the complete first monoclonal antibody is of the IgG1 heavy chain constant region as shown in SEQ ID NO.4, and the light chain constant region is of the IgG1 light chain constant region as shown in SEQ ID NO.7.

[0014] In a second aspect of the invention, a polynucleotide is provided that encodes a bispecific antibody that simultaneously targets the G and F glycoproteins of Hennipa virus.

[0015] In one specific embodiment of the present invention, the heavy chain variable polynucleotide sequence encoding monoclonal antibody 5C8 is shown in SEQ ID NO.8, and the light chain variable polynucleotide sequence encoding monoclonal antibody 5C8 is shown in SEQ ID NO.12.

[0016] The heavy chain variable polynucleotide sequence encoding monoclonal antibody 1E5 is shown in SEQ ID NO.10, and the light chain variable polynucleotide sequence encoding monoclonal antibody 1E5 is shown in SEQ ID NO.13.

[0017] The polynucleotide sequence encoding the constant region of the IgG1 heavy chain is shown in SEQ ID NO.11, the polynucleotide sequence encoding the constant region of the IgG1 light chain is shown in SEQ ID NO.14, and the polynucleotide sequence encoding the linker is shown in SEQ ID NO.9.

[0018] In a third aspect of the invention, an expression vector containing the aforementioned polynucleotide is provided. The vector is used to clone and / or express the encoding gene of the bispecific antibody simultaneously targeting the Hennepa virus G and F glycoproteins. In one specific embodiment of the invention, the vector is pcDNA3.4. Other vectors known to those skilled in the art, particularly eukaryotic cell expression vectors, can also be used for the cloning and expression of the encoding gene described in this invention.

[0019] In a fourth aspect of the invention, a host cell containing the above-described expression vector is provided. The host cell is used to express the above-described bispecific antibody simultaneously targeting the G and F glycoproteins of Hennipa virus. In one specific embodiment of the invention, the host cell is an Expi293F cell. Other host cells known to those skilled in the art, especially eukaryotic host cells, can also be used for the expression of the bispecific antibody described in this invention.

[0020] Finally, this invention provides the application of the above-mentioned bispecific antibody that simultaneously targets the G and F glycoproteins of Hennipa virus in the preparation of a therapeutic drug for Hennipa virus disease.

[0021] This invention, based on previously obtained parental monoclonal antibodies with strong cross-neutralizing activity—including 1E5 targeting the G protein and 5C8 and 1D6 targeting the F protein—employs a bispecific antibody construction strategy. Through structure-guided molecular design, the variable regions of the antibodies are combined and sequenced, successfully constructing nine bispecific antibodies capable of simultaneously recognizing and binding to both the G and F proteins. Systematic functional characterization, including: assessing the molecular weight and structural integrity of the bispecific antibodies using SDS-PAGE; detecting their binding activity to the G and F proteins using ELISA; and evaluating their neutralizing efficacy against different Hennipa viruses using a pseudovirus neutralization experiment, demonstrated that all bispecific antibodies exhibited good expression efficiency and correct assembly in mammalian expression systems, simultaneously retaining high affinity binding to both the G and F proteins. Among them, three bispecific antibodies (1E5scFv-5C8, 5C8-1E5scFv, and 5C8V-1E5) showed superior cross-neutralizing activity compared to their parental monoclonal antibodies in the pseudovirus neutralization experiment. Therefore, the bispecific antibody that simultaneously targets the G and F glycoproteins of Hennipa virus provided by this invention can serve as a key active ingredient in the preparation of drugs for the prevention and / or treatment of Hennipa virus infection. This not only provides a new solution for dealing with viral escape mutations, but also lays the technical foundation for the development of highly effective and broad-spectrum antibody cocktail therapies and next-generation antiviral strategies. Attached Figure Description

[0022] Figure 1 Graph showing the results of neutralizing activity assay for parental antibodies at a fixed 1:1 ratio; Figure 2 The SynergyFinder synergistic scoring model performs statistical analysis and visualization of the results data; Figure 3 Schematic diagram of bispecific antibody design; Figure 4 Electrophoresis images of parental antibodies and bispecific antibodies; Figure 5 Binding curves of parental antibodies, bispecific antibodies, and Nipah virus G and F antibodies; Figure 6 Neutralization curves of parental antibodies, bispecific antibodies, and pseudoviruses. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. 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.

[0024] Example 1. Potential analysis of parental antibody combination therapy 1. Neutralization activity assay of parental antibodies at a fixed 1:1 ratio Antibodies targeting Hennipa virus G protein (1E5, 1B6, CN113968908A) and F protein (1D6, CN117487005A; 5C8, CN117402238A) that were independently developed by the inventors in the early stage were selected in pairs and mixed in a fixed concentration ratio (1:1) to conduct a dose gradient experiment to preliminarily determine whether there is a synergistic effect.

[0025] 1) In 96-well cell culture plates, single or paired (1:1) G-targeting antibodies 1E5 and 1B6 and F-targeting antibodies 5C8 and 1D6 were serially diluted three-fold in DMEM medium containing 10% FBS. The initial antibody concentration in each well was 10 μg / mL (10 μg / mL for a single parent antibody; 5 μg / mL for each parent antibody in the antibody combination). A total of 9 dilutions were performed, with 3 replicates for each dilution. The final culture volume of each well was 50 μL.

[0026] 2) Dilute rHIV-NiV and rHIV-HeV pseudoviruses to 2×10⁻⁶ using culture medium. 4 TCID 50 / mL, then pipette 50μL and add it to the cell culture plate to mix with the antibody. Add only culture medium to the blank control wells without antibody and pseudovirus, and add only pseudovirus to the pseudovirus control wells without antibody; bring the volume of the control wells to 100 μL with culture medium.

[0027] 3) Place the culture plate in a 37℃, 5% CO2 incubator and incubate for 1 hour.

[0028] 4) Digest HEK293T cells with 0.25% trypsin, count them, and seed them in 100 μL (containing 3 × 10⁻⁶ cells). 5 (1 cell) to a culture plate. Incubate the culture plate at 37°C in a 5% CO2 incubator for 48 hours.

[0029] 5) Remove the culture plate, carefully aspirate the cell supernatant, add 50 μL of diluted cell lysis buffer to each well, and shake at 300 rpm for 10 minutes at room temperature.

[0030] 6) Take 20 μL of lysis supernatant from each well and transfer it to a 96-well white detection plate.

[0031] 7) Add 50 μL of luciferase assay reagent to each well and use the GloMax™ microplate analyzer to detect the fluorescence intensity.

[0032] 8) Calculate the neutralization rate of the antibody: Neutralization rate % = [1 - (reading value of experimental well - reading value of blank control group) / (reading value of virus control group - reading value of blank control group)] × 100%.

[0033] 9) Fit the dose-response curve using a four-parameter nonlinear regression model ( Figure 1 And calculate the half-maximal inhibitory concentration of the antibody or antibody combination (Table 1).

[0034] Table 1. Neutralizing activity of antibody combinations or parental antibodies

[0035] 2. Analysis of parental antibody co-neutralization matrix On a two-dimensional plane, the effects of two antibodies at different concentration combinations were systematically tested, depicting a panoramic view of their interactions across all possible concentration combinations. Eight concentration gradients were set for each antibody, with an initial well concentration of 5 μg / mL and three-fold serial dilutions to form a complete concentration matrix. The neutralization experiment procedure was the same as above. The results were statistically analyzed and visualized using the SynergyFinder (https: / / synergyfinder.fimm.fi) synergy scoring model. Figure 2 ).

[0036] result: When parental antibodies are used in a 1:1 ratio, the three antibody combinations targeting different proteins, 1E5+1D6, 1E5+5C8, and 1B6+1D6, showed cross-neutralizing activity against rHIV-HNV pseudoviruses that was superior to both parental antibodies. Figure 1 (See Table 1). Among them, antibody 1E5 targeting the G protein, combined with antibodies 1D6 or 5C8 targeting the F protein, exhibited comparable optimal cross-neutralizing activity. Furthermore, the neutralizing activities of the 1E5+1D6 and 1E5+5C8 combinations at different concentrations were systematically tested, confirming that the two antibody combinations had a significant synergistic neutralizing enhancement effect at low concentrations. Figure 2 ).

[0037] Example 2. Design and preparation of bispecific antibodies 1. Design of bispecific antibodies This invention employs a modular strategy based on genetic engineering to construct 1E5+1D6 or 1E5+5C8 bispecific antibodies. Its core method involves gene fusion of the variable region or single-chain variable fragment (scFv) of one parental antibody (named the second monoclonal antibody) with the full length of another parental antibody (named the first monoclonal antibody). Figure 3The amino acid sequences and their coding sequences of the heavy chain variable region of monoclonal antibody 1E5 are shown in SEQ ID NO. 3 and 10, and the amino acid sequences and their coding sequences of the light chain variable region are shown in SEQ ID NO. 6 and 13, respectively. The amino acid sequences and their coding sequences of the heavy chain variable region of monoclonal antibody 5C8 are shown in SEQ ID NO. 1 and 8, and the amino acid sequences and their coding sequences of the light chain variable region are shown in SEQ ID NO. 5 and 12, respectively. The amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1D6 are shown in SEQ ID NO. 15 and 16, respectively. The amino acid sequences and their coding sequences of the heavy chain constant region are shown in SEQ ID NO. 4 and 11, respectively, and the amino acid sequences and their coding sequences of the light chain constant region are shown in SEQ ID NO. 7 and 14, respectively. The amino acid sequences and their coding sequences of the linker that plays a flexible linking role in the bispecific antibody are shown in SEQ ID NO. 2 and 9, respectively. The construction of bispecific antibodies can be achieved through the following two main approaches.

[0038] (1) Fusion of variable regions: The heavy chain variable region (VH) and light chain variable region (VL) of the second monoclonal antibody are respectively connected to the N-terminus of the full-length genes of the heavy chain (H) and light chain (L) of the first monoclonal antibody through flexible peptide linkers. Figure 3 Among them, those belonging to this fusion type include: 1) Using 1E5 as the first monoclonal antibody, The heavy chain variable region (VH) and light chain variable region (VL) of 1D6, which serves as the second monoclonal antibody, are linked to the N-terminus of the full-length heavy chain (H) and light chain (L) genes of 1E5, respectively, via flexible peptide linkers to 1D6V-1E5; or, The heavy chain variable region (VH) and light chain variable region (VL) of 5C8, which serves as the second monoclonal antibody, are linked to the N-terminus of the full-length heavy chain (H) and light chain (L) genes of 1E5, respectively, via flexible peptide linkers, forming 5C8V-1E5.

[0039] 2) Using 1D6 as the first monoclonal antibody, The heavy chain variable region (VH) and light chain variable region (VL) of 1E5, which serves as the second monoclonal antibody, are linked to the N-terminus of the full-length 1D6 heavy chain (H) and light chain (L) genes, respectively, via flexible peptide linkers to 1E5V-1D6.

[0040] 3) Using 5C8 as the first monoclonal antibody, The heavy chain variable region (VH) and light chain variable region (VL) of 1E5, which serves as the second monoclonal antibody, are linked to the N-terminus of the full-length 1D6 heavy chain (H) and light chain (L) genes, respectively, via flexible peptide linkers to 1E5V-5C8.

[0041] (2) scFv module fusion: The VH and VL of the second monoclonal antibody are connected by a flexible peptide linker to form an scFv fragment, and the scFv module is fused to the N-terminus or C-terminus of the full-length heavy chain gene of the first monoclonal antibody. Figure 3 Among them, those belonging to this fusion type include: 1) Using 1E5 as the first monoclonal antibody, The VH and VL segments of 1D6, serving as the second monoclonal antibody, are linked via a flexible peptide linker to form an scFv fragment. This scFv module is then fused to the N-terminus of the full-length heavy chain gene of the first monoclonal antibody, 1E5, into the 1D6scFv-1E5 fragment; or The VH and VL segments of 1D6, serving as the second monoclonal antibody, are linked via a flexible peptide linker to form an scFv fragment. This scFv module is then fused to the C-terminus of the full-length 1E5 heavy chain gene of the first monoclonal antibody, 1E5-1D6scFv; or The VH and VL segments of 5C8, serving as the second monoclonal antibody, are linked via a flexible peptide linker to form an scFv fragment. This scFv module is then fused to the N-terminus of the full-length heavy chain gene of the first monoclonal antibody, 1E5, into the 5C8scFv-1E5 fragment; or The VH and VL of 5C8, which serves as the second monoclonal antibody, are linked together via a flexible peptide linker to form an scFv fragment. This scFv module is then fused into the C-terminus of the full-length gene of the first monoclonal antibody 1E5 heavy chain, specifically the 1E5-5C8scFv.

[0042] 2) Using 1D6 as the first monoclonal antibody, The VH and VL segments of 1E5, serving as the second monoclonal antibody, are linked via a flexible peptide linker to form an scFv fragment. This scFv module is then fused to the N-terminus of the full-length heavy chain gene of the first monoclonal antibody, 1D6, into 1E5scFv-1D6; or The VH and VL of 1E5, which serves as the second monoclonal antibody, are linked together via a flexible peptide linker to form an scFv fragment. This scFv module is then fused into the C-terminus of the full-length gene of the first monoclonal antibody, 1D6 heavy chain, as 1D6-1E5scFv.

[0043] 3) Using 5C8 as the first monoclonal antibody, The VH and VL of 1E5, which serves as the second monoclonal antibody, are linked together via a flexible peptide linker to form an scFv fragment. This scFv module is then fused to the N-terminus of the full-length gene of the first monoclonal antibody 1D6 heavy chain, 1E5scFv-5C8. The VH and VL of 1E5, which serves as the second monoclonal antibody, are linked together via a flexible peptide linker to form an scFv fragment. This scFv module is then fused into the 5C8-1E5scFv at the C-terminus of the full-length heavy chain gene of the first monoclonal antibody, 1D6.

[0044] The construction strategy of this invention is symmetrical, allowing the variable region or scFv of the first monoclonal antibody to be interactively fused into the framework of the second monoclonal antibody in the same experimental design, thereby obtaining bispecific antibodies with different arrangements and spatial conformations for subsequent systematic comparison.

[0045] 2. Preparation of bispecific antibodies The full-length genes of the bispecific antibody light and heavy chains were constructed into the pcDNA3.1 expression plasmid. SEQ ID NO. 17 and 19 represent the signal peptides and their encoding genes for heavy chain expression, and SEQ ID NO. 18 and 20 represent the signal peptides and their encoding genes for light chain expression. 15 μg each of the antibody heavy and light chain plasmids were transfected into 30 mL of Expi293 mammalian cell expression system and cultured at 125 rpm in 5% CO2 for 96 h. (3000×) g Centrifuge at 4℃ for 15 min to collect the expression supernatant. Filter through a 0.22 μm syringe filter and purify the antibody using an rProtein A affinity chromatography column (equilibration buffer: PBS; elution buffer: 0.1 M glycine, pH 3.0; neutralization buffer: 1 M Tris, pH 9.0). Concentrate the collected antibody and displace it into PBS solution to determine the concentration. Take 5 μg and analyze the purity and molecular form of the bispecific antibody by SDS-PAGE. Figure 4 (), then repackage and store at -80℃.

[0046] result: For either the 1E5+1D6 or 1E5+5C8 combination, six forms of bispecific antibodies were designed for each combination. Figure 3 A total of 12 species were prepared. Among them, 5 species were successfully prepared by the 1E5+1D6 combination: 1D6V-1E5, 1E5V-1D6, 1E5-1D6scFv, 1D6-1E5scFv, and 1E5scFv-1D6; 4 species were successfully prepared by the 1E5+5C8 combination: 5C8V-1E5, 1E5V-5C8, 5C8-1E5scFv, and 1E5scFv-5C8. Figure 4 The molecular weights of the heavy chain, light chain, VH, VL, and scFv of the antibody are approximately 50 kDa, 25 kDa, 12.5 kDa, 12.5 kDa, and 25 kDa, respectively. Under reducing conditions, the molecular weights of the heavy and light chain bands of the bispecific antibody and the parent antibody are as expected (due to the presence of 1–2 linkers in the heavy or light chain of the bispecific antibody, the actual molecular weight is slightly larger than the theoretical molecular weight), and the purity of all bands is >90%.

[0047] Example 3. Evaluation of the functional activity of bispecific antibodies 1. ELISA validation of the dual-targeting ability of bispecific antibodies G and F proteins. The day before the experiment, 100 µL of 1 µg / mL NiV G or F protein was coated onto each well of a 96-well ELISA plate and incubated overnight at 4°C in a humidified chamber. On the day of the experiment, the plates were washed three times with a plate washer, and 100 µL of blocking buffer was added to each well, incubating at 37°C for 1 h. After washing three times again, 150 µL of 5 µg / mL antibody was added to the first well, and 100 µL of dilution buffer was added to the remaining wells. 50 µL of the antibody was added to each subsequent well, and this was repeated 1:3 serially to a final volume of 100 µL per well, incubating at 37°C for 1 h. After three washes, HRP-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) was diluted 1:10000, and 100 µL was added to each well, incubating at 37°C for 1 h. After washing the plate three times, add 100 µL of TMB single-component chromogenic solution to each well and incubate at room temperature in the dark for 6 min. Then, add 50 µL of stop solution to each well to terminate the reaction. Place the ELISA plate in a microplate reader to detect OD values ​​at 450–630 nm. Import the data into GraphPad Prism software and fit the dose-response curve using four parameters. Figure 5 ) Calculate EC 50 Values ​​(Table 1).

[0048] 2. Detection of antibody neutralizing activity using a pseudovirus neutralization assay. Antibody cross-neutralizing activity was evaluated using HNV pseudoviruses (HIV virus particles lacking the Env protein, replication-defective, displaying Hennipa virus G and F proteins on the surface, capable of only single-round infection and unable to produce progeny viruses) with a pre-packaged HIV backbone. First, antibodies were diluted with DMEM in 96-well cell culture plates. 75 μL of antibody dilution (2 μg / mL) was added to the first well, and 50 μL of DMEM was added to the remaining wells. Then, 25 μL of the solution was transferred from the first well to the next well and mixed. This was repeated 1:3 serially, with a final volume of 50 μL per well. After diluting the pseudoviruses with DMEM, 50 μL was added to each well, mixed, and incubated at 37°C for 1 h. HEK 293T cells were then counted at a rate of 3 × 10⁻⁶ cells / well. 5 Add 100 μL of the culture medium to each well at a concentration of cells / mL, and incubate the cell culture plate at 37°C for 48 h. Remove the cell culture plate, carefully aspirate 100 µL of culture medium from each well, add 100 µL of luciferase assay reagent (Vazyme, catalog number DD1204-01), and lyse by shaking at 350 rpm for 10 min. Transfer 150 μL of supernatant to a white 96-well plate and read the luminescence value using a GloMax™ luminescence detector. Calculate the antibody's protective effect on cells, import the data into GraphPad Prism software, and fit a dose-response curve using four parameters. Figure 6 ), calculate IC 50Values ​​(Table 1).

[0049] result: This invention, through systematic evaluation of antibody binding and neutralization activities, found that bispecific antibodies exhibited significantly enhanced overall function compared to their parental monoclonal antibodies (Table 2). Although bispecific antibodies were generally slightly weaker than parental antibodies in binding activity, except for 1E5V-5C8, the other eight bispecific antibodies all acquired the ability to bind both G and F proteins simultaneously (EC). 50 The range was 5.2–86.1 ng / mL, expanding the binding spectrum. Compared to the limited or unbalanced neutralizing activity of parental monoclonal antibodies, most bispecific antibodies showed significantly superior neutralizing efficacy, and exhibited more balanced inhibition against the two pseudoviruses (rHIV-HeV and rHIV-NiV). The bispecific antibodies 5C8V-1E5, 5C8-1E5scFv, and 1E5scFv-5C8 showed the most outstanding performance, demonstrating highly efficient binding to both G and F proteins (EC50). 50 The effective concentrations ranged from 5.2–25.3 ng / mL and 9.3–19.6 ng / mL, respectively, and both exhibited nanogram-level, potent cross-neutralizing activity (IC50) against both rHIV-HeV and rHIV-NiV pseudoviruses, superior to either parent antibody. 50 The effective ranges were 1.5–2.3 ng / mL and 0.9–2.7 ng / mL, respectively. Among them, the bispecific antibody 5C8-1E5scFv exhibited particularly strong neutralizing activity (IC50). 50 The concentrations were 1.5 and 0.9 ng / mL, respectively.

[0050] In summary, this invention has obtained a candidate molecule with excellent enhanced function by constructing parental antibodies targeting different antigens into bispecific antibodies. This molecule is significantly superior to single parental antibodies in terms of antigen binding spectrum width, cross-neutralization balance and efficacy, and has outstanding potential for application in the prevention and treatment of Hennipa virus disease.

[0051] Table 2. Binding and neutralizing activities of bispecific antibodies or parental antibodies

[0052] Note: ">" indicates EC 50 Value > 1000 ng / mL; " / " indicates no binding.

Claims

1. A bispecific antibody that simultaneously targets the G and F glycoproteins of Hennipa virus, characterized in that, The bispecific antibody comprises a complete first monoclonal antibody and a second monoclonal antibody whose heavy chain variable region and light chain variable region are respectively linked to the N-terminus of its heavy chain variable region and the N-terminus of its light chain variable region; or, the bispecific antibody comprises a complete first monoclonal antibody and a single-chain antibody linked to the N-terminus or C-terminus of its heavy chain variable region, wherein the single-chain antibody is formed by linking the heavy chain variable region and light chain variable region of the second monoclonal antibody. The first monoclonal antibody and the second monoclonal antibody are combinations targeting different proteins. The sequence combination of the heavy chain variable region and the light chain variable region of the first monoclonal antibody and the sequence combination of the heavy chain variable region and the light chain variable region of the second monoclonal antibody are selected from the combination of SEQ ID NO.1 and SEQ ID NO.5 and the combination of SEQ ID NO.3 and SEQ ID NO.

6.

2. The bispecific antibody simultaneously targeting the G and F glycoproteins of Hennipa virus according to claim 1, characterized in that, The sequence combination of the heavy chain variable region and the light chain variable region of the first monoclonal antibody is the combination of SEQ ID NO.1 and SEQ ID NO.5, and the sequence combination of the heavy chain variable region and the light chain variable region of the second monoclonal antibody is the combination of SEQ ID NO.3 and SEQ ID NO.

6.

3. The bispecific antibody simultaneously targeting the G and F glycoproteins of Hennipa virus according to claim 2, characterized in that, In the bispecific antibody that simultaneously targets the G and F glycoproteins of Hennipa virus, the C-terminus of the heavy chain constant region of the first monoclonal antibody contains tandem sequences of peptides as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.2, and SEQ ID NO.

6.

4. The bispecific antibody simultaneously targeting the G and F glycoproteins of Hennipa virus according to claim 2, characterized in that, In the bispecific antibody that simultaneously targets Hennipa virus G and F glycoproteins, the heavy chain variable region of the first monoclonal antibody, from the N-terminus to the C-terminus, is composed of peptides shown in SEQ ID NO.3, SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.2, and SEQ ID NO.1 linked together.

5. The bispecific antibody simultaneously targeting the G and F glycoproteins of Hennipa virus according to claim 1, characterized in that, The sequence combination of the heavy chain variable region and the light chain variable region of the first monoclonal antibody is SEQ ID NO.3 and SEQ ID NO.6, and the sequence combination of the heavy chain variable region and the light chain variable region of the second monoclonal antibody is SEQ ID NO.1 and SEQ ID NO.

5.

6. The bispecific antibody simultaneously targeting the G and F glycoproteins of Hennipa virus according to claim 5, characterized in that, In the bispecific antibody that simultaneously targets the G and F glycoproteins of Hennipa virus, the heavy chain variable region of the first monoclonal antibody, from the N-terminus to the C-terminus, is composed of peptides shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in tandem, and the light chain variable region of the first monoclonal antibody, from the N-terminus to the C-terminus, is composed of peptides shown in SEQ ID NO.5, SEQ ID NO.2, and SEQ ID NO.6 in tandem.

7. A polynucleotide encoding a bispecific antibody that simultaneously targets the G and F glycoproteins of Hennipa virus as described in any one of claims 1–6.

8. An expression vector containing the polynucleotide of claim 7.

9. A host cell containing the expression vector of claim 8.

10. The use of the bispecific antibody targeting both G and F glycoproteins of Hennipa virus as described in any one of claims 1–6 in the preparation of a therapeutic agent for Hennipa virus disease.

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