Broad-spectrum double-epitope nano antibody BA2-1A10 for neutralizing Ebola virus and application of broad-spectrum double-epitope nano antibody BA2-1A10

Abstract

The invention discloses a double-epitope nano antibody BA2-1A10 for neutralizing Ebola virus in a broad spectrum, which is characterized by comprising a nano antibody BA2, a flexible connecting peptide and a nano antibody 1A10. The invention also discloses a fusion protein antibody. The invention also discloses a polynucleotide, an expression vector, a host cell and an antibody conjugate. The invention further discloses application of the double-epitope nano antibody BA2-1A10, the fusion protein antibody and the antibody conjugate. The invention further discloses application of the double-epitope nano antibody BA2-1A10, the fusion protein antibody and the antibody conjugate. The invention also discloses a pharmaceutical composition. According to the present invention, the double-epitope nanometer antibody BA2-1A10 can be combined with two different epitopes on the Ebola virus GP protein; better broad spectrum and neutralization efficiency are achieved, and three Ebola viruses can be efficiently neutralized at the same time; the compound can be used for preparing reagents and kits for detecting Ebola viruses, and can be used for preparing vaccines or drugs for treating and / or preventing Ebola virus infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a broad-spectrum neutralizing Ebola virus dual-epitope nanobody BA2-1A10 and its applications. Background Technology

[0002] Ebola virus (EBOV) is a highly contagious viral disease that causes hemorrhagic fever in humans and primates. It is one of the most dangerous level 4 viruses to humans. Its three subtypes—Zaire (ZEBOV), Sudan (SUDV), and Bundibugyo (BDBV)—can cause disease in humans with a mortality rate as high as 90%. EBOV invades cells through its unique surface envelope glycoprotein (GP). Blocking the binding of the GP protein to cell receptors with antibodies is one effective way to combat EBOV infection.

[0003] Currently, the FDA-approved drugs for treating Ebola virus infection, Ebanga and Inmazeb, are full-length antibodies targeting the surface glycoprotein of the Ebola virus. However, full-length antibodies have a large molecular weight, poor tissue penetration, and cannot cross the blood-brain barrier, limiting their effectiveness in treating patients in the later stages of viral infection. The mortality rate remains around 30% even after drug treatment, and there are still no broad-spectrum anti-Ebola virus antibody drugs available.

[0004] Nanobodies (VHHs) possess advantages such as small molecular weight, strong permeability, strong antigen recognition ability, and low cost, and are expected to become a specific drug for treating Ebola virus infection. In previous studies, our research group prepared two nanobodies, BA2 and 1A10, and disclosed two nanobodies in CN118515753A and CN118515752A, respectively. These two nanobodies all showed certain inhibition rates against VSV-GFP-ZEBOV, VSV-GFP-SUDV pseudoviruses, and HIV-luc-BDBV pseudoviruses; however, their affinity was not high, and antibodies with higher affinity are still needed. Summary of the Invention

[0005] Based on the technical problems existing in the background art, this invention proposes a broad-spectrum neutralizing Ebola virus dual-epitope nanobody BA2-1A10 and its applications. The dual-epitope nanobody BA2-1A10 of this invention can bind to two different epitopes on the Ebola virus GP protein. Compared with nanobodies BA2 and 1A10, the dual-epitope nanobody BA2-1A10 has significantly improved broad-spectrum neutralization and neutralization efficiency, and can simultaneously and efficiently neutralize three Ebola viruses. It can be used to prepare reagents and kits for detecting Ebola virus, and can be used to prepare vaccines or drugs for treating and / or preventing Ebola virus infection.

[0006] This invention proposes a broad-spectrum neutralizing Ebola virus dual-epitope nanobody BA2-1A10, comprising: nanobody BA2, a flexible linker peptide, and nanobody 1A10; The amino acid sequence of nanobody BA2 includes any of the following: 1) As shown in SEQ ID NO:1; 2) An amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO:1 and still has the function of targeting Ebola virus; 3) An amino acid sequence that still has the function of targeting Ebola virus after adding, deleting, modifying and / or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO:1; The amino acid sequence of nanobody 1A10 includes any of the following: 4) As shown in SEQ ID NO:2; 5) An amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO:2 and still has the function of targeting Ebola virus; 6) An amino acid sequence that still has the function of targeting Ebola virus after adding, deleting, modifying and / or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO:2.

[0007] A dual-epitope nanobody is a tandem array of two nanobodies that recognize different epitopes of the same antigen, simultaneously binding to two non-overlapping epitopes of the same antigen.

[0008] Preferably, nanobody BA2 is linked to nanobody 1A10 via a flexible linker peptide.

[0009] Preferably, the amino acid sequence of the flexible linker peptide is as shown in SEQ ID NO: 3; or an amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO: 3.

[0010] Preferably, the amino acid sequence of the dual epitope nanobody BA2-1A10 includes any one of the following: 1) As shown in SEQ ID NO:4; 2) An amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO:4 and still has the function of targeting Ebola virus; 3) An amino acid sequence that still has the function of targeting Ebola virus after adding, deleting, modifying and / or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO:4.

[0011] Preferably, the GP protein targets the Ebola virus.

[0012] Preferably, the Ebola virus is one or more of the following: SUDV type Ebola virus, BDBV type Ebola virus, and ZEBOV type Ebola virus.

[0013] The present invention also proposes a fusion protein antibody comprising: the above-mentioned dual epitope nanobody BA2-1A10.

[0014] Preferably, the fusion protein antibody further includes an Fc domain.

[0015] Preferably, the Fc domain is the human IgG1 Fc domain.

[0016] Preferably, the fusion protein antibody also includes a linker.

[0017] Preferably, the Fc domain is connected to the dual epitope nanobody BA2-1A10 via a linker.

[0018] The aforementioned Fc domain is linked to the C-terminus of the amino acid sequence of the dual epitope nanobody BA2-1A10 via a linker.

[0019] The above-mentioned adapter is a GS adapter, and a TEV restriction site is inserted into the GS adapter sequence.

[0020] Preferably, the amino acid sequence of the Fc domain is as shown in SEQ ID NO: 6; or an amino acid sequence with greater than or equal to 80% homology to the amino acid sequence shown in SEQ ID NO: 6.

[0021] Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 7; or an amino acid sequence with greater than or equal to 80% homology to the amino acid sequence shown in SEQ ID NO: 7.

[0022] The aforementioned fusion protein antibody is a dual-epitope heavy chain antibody.

[0023] Preferably, the amino acid sequence of the fusion protein antibody includes any of the following: 1) As shown in SEQ ID NO: 8; 2) An amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO: 8 and still has the function of targeting Ebola virus; 3) An amino acid sequence that still has the function of targeting Ebola virus after adding, deleting, modifying and / or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO: 8.

[0024] The fusion protein antibody with the above amino acid sequence as shown in SEQ ID NO:8 was named BA2-1A10-Fc.

[0025] The present invention also proposes a polynucleotide that encodes the above-mentioned dual epitope nanobody BA2-1A10 or the above-mentioned fusion protein antibody.

[0026] The aforementioned polynucleotides refer to chain-like compounds formed by the polymerization of nucleotides; the aforementioned polynucleotides include DNA or RNA.

[0027] The DNA sequence encoding the above-mentioned dual epitope nanobody BA2-1A10 is shown in SEQ ID NO: 5.

[0028] The above homology is greater than or equal to 80%, and its homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0029] The present invention also proposes an expression vector containing the above-mentioned polynucleotides.

[0030] The present invention also proposes a host cell containing the above-mentioned expression vector or the above-mentioned polynucleotides integrated into the host cell genome.

[0031] Preferably, the host cell is a cell capable of expressing exogenous proteins.

[0032] Preferably, the host cell is a bacterium, yeast, insect cell, or mammalian cell.

[0033] The preparation methods of the above-mentioned dual epitope nanobody BA2-1A10 and the above-mentioned fusion protein antibody include: culturing the above-mentioned host cells, and collecting the dual epitope nanobody BA2-1A10 or the fusion protein antibody from the cell culture.

[0034] The present invention also proposes an antibody conjugate comprising: 1) The above-mentioned dual epitope nanobody BA2-1A10 or the above-mentioned fusion protein antibody; and 2) A conjugate selected from any one or a combination of the following: detectable markers, enzymes, drugs, solid-phase carriers, and antibodies.

[0035] The aforementioned dual-epitope nanobody BA2-1A10 or fusion protein antibody can be labeled and detected, for example, by using detectable markers or enzymes that produce detectable substances. These detectable markers include: radioactive isotopes for detection, luminescent substances, colored substances, colloidal gold markers, polyethylene glycol, streptavidin, or biotin, etc.; the luminescent substances can be fluorescent agents, etc.

[0036] The radioactive isotopes used for the above-mentioned detection include: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, etc.

[0037] The aforementioned solid support can be polystyrene plates, beads, etc.

[0038] The antibodies in the above-mentioned conjugation portion can be antibodies that target specific proteins or specific tissues.

[0039] This invention also proposes applications of the above-mentioned dual epitope nanobody BA2-1A10, the above-mentioned fusion protein antibody, and the above-mentioned antibody conjugate, characterized in that the applications include: 1) Application in the preparation of reagents and / or kits for the detection of Ebola virus; 2) Use in the preparation of vaccines or medicines for the treatment and / or prevention of Ebola virus infection.

[0040] The Ebola virus mentioned above is one or more of the following: SUDV, BDBV, and ZEBOV.

[0041] The present invention also proposes a pharmaceutical composition comprising: the above-mentioned dual epitope nanobody BA2-1A10, the above-mentioned fusion protein antibody, and one of the above-mentioned antibody conjugates.

[0042] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0043] The above-mentioned drug composition is suitable for administration via subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

[0044] The pharmaceutically acceptable excipients mentioned above include at least one of the following: excipients, stabilizers, antioxidants, colorants, diluents, sustained-release agents, pH adjusters, etc.; such as starch, lipids, waxes, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc.

[0045] The dual-epitope nanobody BA2-1A10 of this invention is formed by genetically engineering two nanobodies, BA2 and 1A10, tandemly linked at a specific length. It binds to two different epitopes on the Ebola virus GP protein. Compared to nanobodies BA2 and 1A10, the dual-epitope nanobody BA2-1A10 exhibits significantly improved broad-spectrum neutralization efficiency, simultaneously and efficiently neutralizing three Ebola viruses—SUDV, BDBV, and EBOV—which have a mortality rate of up to 90% in humans. Its neutralization capacity is particularly enhanced against SUDV Ebola virus, increasing by several tens of times. The dual-epitope nanobody BA2-1A10 provided by this invention can be used to prepare vaccines or drugs for the prevention and / or treatment of Ebola virus infection, as well as diagnostic kits for Ebola virus infection.

[0046] The dual-epitope nanobody BA2-1A10 of this invention is genetically engineered from camel-derived nanobodies BA2 and 1A10. Its simple structure and small molecular weight facilitate expression and use; it is easily and efficiently expressed in large quantities in *E. coli* and various eukaryotic systems; it possesses superior affinity, specificity, and broad-spectrum activity compared to single nanobodies, making it more advantageous for targeted drug development and directed delivery to cellular targets; as a drug, it exhibits low immunogenicity in humans and is less likely to induce immune rejection. The dual-epitope nanobody provided by this invention shows promise for use in Ebola virus drug development and has high application value in the prevention and treatment of Ebola virus. Attached Figure Description

[0047] Figure 1 The images show the purification results of the dual epitope fusion protein antibody BA2-1A10-Fc. In the images, A is the chromatogram of protein A column purification, B is the SDS-PAGE result, and M is the marker.

[0048] Figure 2 AC represents the ELISA results of the binding affinity of the single-epitope fusion protein antibodies BA2-Fc, 1A10-Fc, and the double-epitope fusion protein antibody BA2-1A10-Fc to SUDV-GP, BDBV-GP, and ZEBOV-GP proteins.

[0049] Figure 2 DF represents the plaque reduction and neutralization results of the single-episode fusion protein antibody BA2-Fc, 1A10-Fc and the double-episode fusion protein antibody BA2-1A10-Fc against true SUDV, BDBV and ZEBOV Ebola virus strains. Detailed Implementation

[0050] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0051] Example 1: Expression and purification of single-episode fusion protein antibodies BA2-Fc, 1A10-Fc and dual-episode fusion protein antibody BA2-1A10-Fc The extracellular domain (30-650 aa) of the GP protein from three Ebola virus strains—SUDV (GenBank: ACR33190.1), BDBV (GenBank: YP_003815435.1), and ZEBOV (GenBank: AIW47462.1)—was synthesized at Anhui General Biotechnology Co., Ltd. and cloned into the pTT5 plasmid expression vector. The highly glycosylated mucin-like domain (313-464 aa) was truncated. The plasmid was extracted and transfected into human embryonic kidney cells (HEK 293F cells, ATCC, CBP60437). Cell supernatant was collected after 3 days and purified by affinity chromatography and size exclusion chromatography (refer to Ma H, et al., 2021. Potent Neutralization of SARS-CoV-2 by Hetero-Bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain. J Virol). 95:10.1128 / jvi.02438-20), three antigenic GP proteins were obtained, denoted as SUDV-GP, BDBV-GP, and ZEBOV-GP, respectively; Nanobodies BA2 and 1A10 were prepared according to the methods described in CN118515753A and CN118515752A, respectively. We used a flexible linker peptide (the amino acid sequence of which is shown in SEQ ID NO: 3) to connect the C-terminus of the amino acid sequence of nanobody BA2 (the amino acid sequence of which is shown in SEQ ID NO: 1) to the N-terminus of the amino acid sequence of nanobody 1A10 (the amino acid sequence of which is shown in SEQ ID NO: 2), to obtain the dual epitope nanobody BA2-1A10 (the amino acid sequence of which is shown in SEQ ID NO: 4, and the DNA sequence of which is shown in SEQ ID NO: 5).

[0052] The secretion-inducing peptide gene sequence was fused to the N-terminus of the amino acid sequences of nanobodies BA2, 1A10, and the dual-epitope nanobodies BA2-1A10, respectively, to ensure secretion after expression. The human IgG1 Fc domain was fused to the C-terminus of the amino acid sequences of nanobodies BA2, 1A10, and BA2-1A10, respectively, using a linker (the amino acid sequence of which is shown in SEQ ID NO: 7). These were then cloned into the mammalian expression vector pTT5, and the constructed pTT5 vectors were transfected into HEK 293F cells (ATCC, CBP60437, density approximately 2.5 × 10⁻⁶) using polyethyleneimine. 6 In Freestyle (cells / mL) TM Human embryonic kidney cells HEK 293F transfected in 293 expression medium (purchased from Yonglian Biotechnology) were cultured in a CO2 incubator (the incubator should not be excessively dry) at 37°C and 150 rpm for 5 days. Then, the cells were centrifuged at 2000 rpm for 10 min, and the supernatant was collected. The supernatant was purified by protein A column to obtain single epitope fusion protein antibody BA2-Fc, single epitope fusion protein antibody 1A10-Fc, and double epitope fusion protein antibody BA2-1A10-Fc, respectively.

[0053] SDS-PAGE electrophoresis analysis was performed on the single-episode fusion protein antibody BA2-Fc, the single-episode fusion protein antibody 1A10-Fc, and the dual-episode fusion protein antibody BA2-1A10-Fc. Typical results are shown below. Figure 1 As shown.

[0054] Figure 1 The images show the purification results of the dual epitope fusion protein antibody BA2-1A10-Fc. In the images, A is the chromatogram of protein A column purification, B is the SDS-PAGE result, and M is the marker.

[0055] Depend on Figure 1 It can be seen that a high-purity dual epitope fusion protein antibody BA2-1A10-Fc was obtained, with a molecular weight of approximately 55 kD.

[0056] The amino acid sequence of the dual epitope fusion protein antibody BA2-1A10-Fc was determined and is shown in SEQ ID NO: 8.

[0057] Example 2: Affinity analysis of single-episode fusion protein antibodies BA2-Fc, 1A10-Fc and dual-episode fusion protein antibody BA2-1A10-Fc to ZEBOV-GP, BDBV-GP and SUDV-GP. Immunoplates were coated with 2 µg / mL SUDV-GP, BDBV-GP, and ZEBOV-GP proteins overnight, respectively. Unbound sites were blocked with MPBS at room temperature for 2 h, and then the plates were washed 4 times with PBS-0.1% Tween 20 (PBST). Then, the single-epitope fusion protein antibodies BA2-Fc, 1A10-Fc, and dual-epitope fusion protein antibody BA2-1A10-Fc obtained in Example 1 were serially diluted at a ratio of 1:4 (concentration range: 300-0.0003 nM). 100 µL of the dilution was added to each well of the immunoplate and incubated at room temperature with shaking for 1 h. 100 µL / well of anti-human IG1-FC-HRP secondary antibody (Beijing Yiqiao Shenzhou) was added and incubated at room temperature with shaking for 1 h. Then, 100 µL / well of TMB was added, and after 5 min of development, 50 µL of 1 M H2SO4 was added to stop development. The absorbance at 450 nm was measured, and the obtained data were used for nonlinear curve fitting using GraphPad software. The fitting equation was: Y = Bottom + (Top-Bottom) / (1+10^((LogEC)). 50 -X) HillSlope).

[0058] The results are as follows Figure 2 As shown in AC.

[0059] Figure 2 AC represents the ELISA results of the binding affinity of the single-epitope fusion protein antibodies BA2-Fc, 1A10-Fc, and the double-epitope fusion protein antibody BA2-1A10-Fc to SUDV-GP, BDBV-GP, and ZEBOV-GP proteins.

[0060] Depend on Figure 2 A shows that the ECG binding of the single-epitope fusion protein antibody BA2-Fc, 1A10-Fc, and the dual-epitope fusion protein antibody BA2-1A10-Fc to the ZEBOV-GP protein is significantly reduced. 50 The values ​​are 0.18 nM, 0.24 nM, and 0.11 nM, respectively. Depend on Figure 2 B shows that the ECG binding of the monoepithelial fusion protein antibody BA2-Fc, 1A10-Fc, and the biepithelial fusion protein antibody BA2-1A10-Fc to the BDBV-GP protein is [missing information]. 50 The values ​​are 2.79 nM, 0.26 nM, and 0.11 nM, respectively. Depend on Figure 2 C shows that the EC50 of the single-epitope fusion protein antibodies BA2-Fc, 1A10-Fc, and the dual-epitope fusion protein antibody BA2-1A10-Fc for binding to SUDV-GP protein is significant.50 The values ​​were 0.15 nM, 0.19 nM, and 0.09 nM, respectively. This shows that the dual epitope fusion protein antibody BA2-1A10-Fc has a higher affinity for the three GP proteins.

[0061] The above EC 50 The half-maximal effective concentration (CMC) is the concentration of a drug, antibody, or toxin that, after a specific exposure time, achieves 50% of its maximum biological effect. It is commonly used to measure drug efficacy. 50 The lower the value, the stronger the drug's potency, meaning it produces the same biological effect at a lower concentration. In a quantitative response, EC... 50 It refers to the drug dose that can elicit 50% of the maximum response intensity; in qualitative reactions, it refers to the drug dose that elicits a positive response in only 50% of experimental animals.

[0062] Example 3: Characterization of the neutralizing ability of single-epitope fusion protein antibodies BA2-Fc, 1A10-Fc and dual-epitope fusion protein antibody BA2-1A10-Fc against true Ebola virus. Because pseudoviruses are genetically engineered viruses that only retain viral surface glycoproteins to simulate the infection process, they cannot fully mimic the conformational changes or interactions with other receptors of real viruses. Real viruses, on the other hand, are more infectious and pathogenic, and their infection involves a more complex immune response. Since the two differ in structure, infection mechanism, and immune response, neutralization activity experiments with pseudoviruses using nanobodies alone cannot definitively determine their affinity for real viruses.

[0063] Therefore, in a biosafety level 4 (BSL-4) laboratory, we used the plaque reduction method to conduct neutralization experiments on live Ebola virus. The specific steps are as follows: Vero E6 cells were planted at a density of 1.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells in 24-well plates and incubated overnight. The single-epitope fusion protein antibody BA2-Fc, 1A10-Fc, and the dual-epitope fusion protein antibody BA2-1A10-Fc were serially diluted 4-fold in DMEM medium containing 2.5% fetal bovine serum (FBS) to create seven concentration gradients: 2500 nM, 625 nM, 156.25 nM, 39.06 nM, 9.77 nM, 2.44 nM, and 0.61 nM. Each dilution was replicated in triplicate. Diluted antibodies were mixed with 600 PFU / ml of SUDV, BDBV, and ZEBOV Ebola virus strains, respectively, and incubated at 37°C for 1 h to allow for sufficient antibody-virus interaction. The mixture was then added to Vero E6 monolayer cells and incubated at 37°C for 1 h. The supernatant was then removed, and the cells were covered with 1 mL of DMEM medium containing 2.5% FBS and 0.9% carboxymethyl cellulose. The cells were cultured at 37°C and 5% CO2 for 14 days. Cells were then fixed with 10% formaldehyde for 30 min, thoroughly washed, and the plaques were visualized and manually counted by visual inspection. An S-curve was used to fit the neutralization curve, and the half-maximal inhibitory concentration (IC50) was calculated. 50 To determine the neutralizing efficacy of antibodies, a positive control without antibodies and a negative control with cells were established.

[0064] The results are as follows Figure 2 As shown in DF.

[0065] Figure 2 DF represents the plaque reduction and neutralization results of the single-episode fusion protein antibody BA2-Fc, 1A10-Fc and the double-episode fusion protein antibody BA2-1A10-Fc against true SUDV, BDBV and ZEBOV Ebola virus strains.

[0066] Depend on Figure 2 D shows that the IC50 values ​​of the single-epitope fusion protein antibody BA2-Fc, 1A10-Fc, and the dual-epitope fusion protein antibody BA2-1A10-Fc for neutralizing the ZEBOV strain of Ebola virus are... 50 The values ​​are 6.74 nM, 6.97 nM, and 1.90 nM, respectively.

[0067] Depend on Figure 2 E shows that the IC50 values ​​of the single-epitope fusion protein antibody BA2-Fc, 1A10-Fc, and the dual-epitope fusion protein antibody BA2-1A10-Fc in neutralizing the BDBV type Ebola virus strain are: 50 The values ​​are 6.04 nM, 6.82 nM, and 2.21 nM, respectively.

[0068] Depend on Figure 2 F shows that the IC50 values ​​of the single-epitope fusion protein antibody BA2-Fc, 1A10-Fc, and the dual-epitope fusion protein antibody BA2-1A10-Fc for neutralizing the SUDV type Ebola virus strain are: 50 The values ​​were 17.15 nM, 31.28 nM, and 0.81 nM, respectively.

[0069] This shows that the dual epitope fusion protein antibody BA2-1A10-Fc has a significantly improved ability to neutralize three types of Ebola virus; especially against the SUDV type Ebola virus, its neutralizing ability has increased by several tens of times.

[0070] IC 50 IC50 stands for half maximal inhibitory concentration, which refers to the concentration of a drug or inhibitor required to inhibit a specified biological process (such as enzyme activity, receptor binding, cell proliferation, etc.) by half in in vitro experiments. 50 The lower the IC value, the stronger the inhibitory effect of the drug or inhibitor, meaning that it can achieve a 50% inhibitory effect at a lower concentration. 50 It is mainly used to characterize the antagonistic ability of antagonists in in vitro experiments, such as the inhibitory effect of anticancer drugs on specific targets (such as EGFR kinase). 50 The calculations typically involve fitting experimental data into a dose-response curve and then determining the concentration corresponding to the 50% inhibition point.

[0071] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A broad-spectrum neutralizing Ebola virus dual-epitope nanobody BA2-1A10, characterized in that, include: Nanobody BA2, flexible linker peptide and nanobody 1A10; The amino acid sequence of nanobody BA2 includes any of the following: 1) As shown in SEQ ID NO:1; 2) An amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO:1 and still has the function of targeting Ebola virus; 3) An amino acid sequence that still has the function of targeting Ebola virus after adding, deleting, modifying and / or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO:1; The amino acid sequence of nanobody 1A10 includes any of the following: 4) As shown in SEQ ID NO:2; 5) An amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO:2 and still has the function of targeting Ebola virus; 6) An amino acid sequence that still has the function of targeting Ebola virus after adding, deleting, modifying and / or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO:

2.

2. The broad-spectrum neutralizing Ebola virus dual-epitope nanobody BA2-1A10 according to claim 1, characterized in that, Nanobody BA2 is linked to nanobody 1A10 via a flexible linker peptide; preferably, the amino acid sequence of the flexible linker peptide is as shown in SEQ ID NO: 3; or an amino acid sequence with greater than or equal to 80% homology to the amino acid sequence shown in SEQ ID NO:

3. Preferably, the amino acid sequence of the dual epitope nanobody BA2-1A10 includes any one of the following: 1) As shown in SEQ ID NO:4; 2) An amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO:4 and still has the function of targeting Ebola virus; 3) An amino acid sequence that still has the function of targeting Ebola virus after adding, deleting, modifying and / or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO:

4.

3. The broad-spectrum neutralizing Ebola virus dual-epitope nanobody BA2-1A10 according to claim 1 or 2, characterized in that, The GP protein targets the Ebola virus; preferably, the Ebola virus is one or more of the following: SUDV type Ebola virus, BDBV type Ebola virus, and ZEBOV type Ebola virus.

4. A fusion protein antibody, characterized in that, include: The dual epitope nanobody BA2-1A10 as described in any one of claims 1-3; Preferably, the fusion protein antibody further includes an Fc domain; preferably, the Fc domain is a human IgG1 Fc domain; preferably, the fusion protein antibody further includes a linker; preferably, the Fc domain is linked to the dual epitope nanobody BA2-1A10 via the linker. Preferably, the amino acid sequence of the Fc domain is as shown in SEQ ID NO: 6; or an amino acid sequence with greater than or equal to 80% homology to the amino acid sequence shown in SEQ ID NO:

6. Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 7; or an amino acid sequence with greater than or equal to 80% homology to the amino acid sequence shown in SEQ ID NO:

7. Preferably, the amino acid sequence of the fusion protein antibody includes any of the following: 1) As shown in SEQ ID NO: 8; 2) An amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO: 8 and still has the function of targeting Ebola virus; 3) An amino acid sequence that still has the function of targeting Ebola virus after adding, deleting, modifying and / or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO:

8.

5. A polynucleotide, characterized in that, The polynucleotide encodes the dual epitope nanobody BA2-1A10 of any one of claims 1-3, or the fusion protein antibody of claim 4.

6. An expression carrier, characterized in that, The expression vector contains the polynucleotide of claim 5.

7. A host cell, characterized in that, The host cell contains the expression vector of claim 6 or the host cell genome integrates the polynucleotide of claim 5; preferably, the host cell is a cell capable of expressing exogenous proteins; preferably, the host cell is a bacterium, yeast, insect cell or mammalian cell.

8. An antibody conjugate, characterized in that, Antibody conjugates include: 1) The dual epitope nanobody BA2-1A10 according to any one of claims 1-3 or the fusion protein antibody according to claim 4; and 2) A conjugate selected from any one or a combination of the following: detectable markers, enzymes, drugs, solid-phase carriers, and antibodies.

9. The application of the dual epitope nanobody BA2-1A10 as described in any one of claims 1-3, the fusion protein antibody as described in claim 4, and the antibody conjugate as described in claim 8, characterized in that, The applications include: 1) Application in the preparation of reagents and / or kits for the detection of Ebola virus; 2) Use in the preparation of vaccines or medicines for the treatment and / or prevention of Ebola virus infection.

10. A pharmaceutical composition, characterized in that, include: The pharmaceutical composition comprises, preferably, one of the following: the dual epitope nanobody BA2-1A10 as described in any one of claims 1-3, the fusion protein antibody as described in claim 4, and the antibody conjugate as described in claim 8; preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

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