Human neutralizing antibodies and bispecific antibodies targeting the envelope protein of fever with thrombocytopenia syndrome virus

Abstract

The present invention relates to the field of antibody technology, and in particular to humanized neutralizing antibodies and bispecific antibodies targeting the envelope protein of the fever with thrombocytopenia syndrome virus. The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment of the present invention are shown in SEQ ID NO.1-3, the amino acid sequences of the light chain complementary determining regions CDR1 and CDR3 are shown in SEQ ID NO.4 and 6, and the amino acid sequence of CDR2 is AAS. The antibody or its antigen-binding fragment and the bispecific antibody provided by the present invention can specifically bind to the envelope protein of the fever with thrombocytopenia syndrome virus, have high affinity and neutralizing activity, and have good preventive and therapeutic effects on the infection of the fever with thrombocytopenia syndrome virus.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to humanized neutralizing antibodies and bispecific antibodies targeting the envelope protein of the HFTS virus. Background Art

[0002] Fever with thrombocytopenia syndrome virus (SFTSV) is a member of the Bunyaviridae family, which includes the Rift Valley fever virus (RVFV). SFTSV is transmitted through tick bites. Infected people experience acute fever, accompanied by thrombocytopenia and leukocytopenia, gastrointestinal dysfunction and other symptoms. Severe patients will develop multiple organ failure or even death, with a mortality rate of 12-30%. The World Health Organization has listed SFTSV as one of the viruses that need the most attention. There is currently no clinically available specific drug for SFTSV infection.

[0003] Therapeutic antibody drugs not only play an important role in tumors and autoimmune diseases, but are also effective in the treatment of infectious diseases. Currently available drugs for the treatment and prevention of viral infections include Synagis for the prevention of respiratory syncytial virus (RSV) infection in children, REGN-COV for the treatment of new coronavirus infection, Mab114 and REGN-EB3 for the treatment of Ebola virus infection, Trogarzo for the treatment of HIV infection, and Rabishield for post-exposure prevention of rabies virus.

[0004] The M gene of SFTSV encodes two envelope proteins, Gn and Gc. The virus binds to the surface of the host through its surface envelope proteins to initiate infection. Studies have shown that the surface proteins of SFTSV and RVFV are important immunogens and important targets for neutralizing antibodies. CN114736291A discloses an antibody SF5 targeting the SFTSV Gn protein, and proves that the antibody has neutralizing activity and can prevent and treat mice infected with fever with thrombocytopenia syndrome virus. Summary of the invention

[0005] The present invention provides antibodies targeting the envelope protein of the fever with thrombocytopenia syndrome virus.

[0006] The present invention first uses the Gc protein of SFTSV as an antigen, and through flow sorting, screens and obtains memory B cells that can specifically bind to Gc from PBMCs of discharged personnel who have recovered from SFTSV infection, and then performs RT-PCR on the screened single B cells to obtain the variable region sequence and fragment of the antibody, and further connects it to the expression vector with the constant region. After mammalian cell expression and purification, a series of functional tests are performed, including the ability to bind to the antigen, the ability to neutralize the virus, etc., to obtain a human monoclonal antibody SF83 with neutralizing and protective activity. SF83 is then constructed into a bispecific antibody with the antibody SF5 (see patent application CN114736291A) targeting Gn obtained in the previous screening, and two bispecific antibodies bsAb1 and bsAb3 with better neutralizing activity than the parent single antibody are expressed using a mammalian cell expression system, and it is proved that it can prevent or treat mice infected with SFTSV.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] The present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof specifically binds to the envelope protein of the fever with thrombocytopenia syndrome virus;

[0009] The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO.1-3, respectively; the amino acid sequence of the light chain complementary determining region CDR1 is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is AAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.6.

[0010] The above-mentioned antibody or its antigen-binding fragment can specifically bind to the envelope protein of the fever with thrombocytopenia syndrome virus and has high affinity and neutralizing activity for the fever with thrombocytopenia syndrome virus.

[0011] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.7 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.8.

[0012] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0013] On the basis of the heavy chain complementary determining regions CDR1, CDR2, CDR3 and the light chain complementary determining regions CDR1, CDR2, CDR3 described above, the amino acid sequence of the heavy chain variable region is an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO. The antibodies or antigen-binding fragments thereof corresponding to the amino acid sequences having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in NO.8 are also within the scope of protection of the present invention.

[0014] Preferably, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.9 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.9, and the amino acid sequence of the light chain is as shown in SEQ ID NO.10 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.10.

[0015] In the present invention, the antigen-binding fragment is selected from any one of Fab, Fab', F(ab')2, Fab'-SH, Fd, Fv, and scFv.

[0016] In some embodiments of the present invention, the heavy chain and / or light chain of the antibody or antigen-binding fragment thereof further comprises a leader sequence.

[0017] In some embodiments of the present invention, the heavy chain of the antibody or antigen-binding fragment thereof further comprises a leader sequence as shown in SEQ ID NO.23, and the light chain further comprises a leader sequence as shown in SEQ ID NO.24.

[0018] The present invention provides a bispecific antibody, which comprises the above-mentioned antibody or antigen-binding fragment thereof.

[0019] Preferably, the bispecific antibody comprises the above-mentioned antibody or antigen-binding fragment thereof, and an antibody that specifically binds to the envelope protein Gn of the FET virus.

[0020] In some embodiments of the present invention, the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the antibody that specifically binds to the envelope protein Gn of the fever and thrombocytopenia syndrome virus are shown as SEQ ID NO.11-13, respectively, the amino acid sequence of the light chain complementary determining region CDR1 is shown as SEQ ID NO.14, the amino acid sequence of CDR2 is GKN, and the amino acid sequence of CDR3 is shown as SEQ ID NO.16.

[0021] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody that specifically binds to the FERR virus envelope protein Gn is as shown in SEQ ID NO.17 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.18 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.18.

[0022] In some embodiments of the present invention, the amino acid sequence of the heavy chain of the bispecific antibody is shown as SEQ ID NO.19, and the amino acid sequence of the light chain is shown as SEQ ID NO.20.

[0023] In some embodiments of the present invention, the amino acid sequence of the heavy chain of the bispecific antibody is shown as SEQ ID NO.21, and the amino acid sequence of the light chain is shown as SEQ ID NO.22.

[0024] In some embodiments of the present invention, the heavy chain and / or light chain of the bispecific antibody further comprises a leader sequence.

[0025] In some embodiments of the present invention, the heavy chain of the bispecific antibody further comprises a leader sequence as shown in SEQ ID NO.23, and the light chain further comprises a leader sequence as shown in SEQ ID NO.24 or 25.

[0026] The present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof; or a nucleic acid molecule encoding the above-mentioned bispecific antibody.

[0027] According to the amino acid sequence and codon rules of the antibody or its antigen-binding fragment or bispecific antibody described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment or bispecific antibody. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment or bispecific antibody is not unique, and all nucleic acid molecules capable of encoding the above-mentioned antibody or its antigen-binding fragment or bispecific antibody are within the scope of protection of the present invention.

[0028] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the antibody or its antigen-binding fragment is shown as SEQ ID NO.26, and the nucleotide sequence of the nucleic acid molecule encoding the light chain of the antibody or its antigen-binding fragment is shown as SEQ ID NO.27.

[0029] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the bispecific antibody is shown as SEQ ID NO.28, and the nucleotide sequence of the nucleic acid molecule encoding the light chain of the bispecific antibody is shown as SEQ ID NO.29.

[0030] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the bispecific antibody is shown as SEQ ID NO.30, and the nucleotide sequence of the nucleic acid molecule encoding the light chain of the bispecific antibody is shown as SEQ ID NO.31.

[0031] In some embodiments of the present invention, the nucleotide sequences of the nucleic acid molecules encoding the leading sequences shown in SEQ ID NOs. 23, 24, and 25 are shown in SEQ ID NOs. 32, 33, and 34, respectively.

[0032] The present invention provides a biological material comprising the above-mentioned nucleic acid molecule; the biological material is an expression cassette, a vector or a host cell.

[0033] The above expression cassette can be obtained by operably linking the nucleic acid molecule to a promoter and / or a terminator.

[0034] The above-mentioned vectors include but are not limited to plasmid vectors, viral vectors and the like.

[0035] The host cells include microbial cells or animal cells, wherein microbial cells include but are not limited to Escherichia coli, yeast, etc., and animal cells include but are not limited to insect cells, CHO cells, 293T cells, etc.

[0036] The antibodies or antigen-binding fragments thereof or bispecific antibodies provided by the present invention can be prepared by conventional methods in the art, including host expression, chemical synthesis, etc.

[0037] In some embodiments of the present invention, a nucleic acid molecule encoding the antibody or its antigen-binding fragment or the bispecific antibody is introduced into a host cell to obtain a recombinant cell, and the recombinant cell is cultured to obtain the antibody or its antigen-binding fragment or the bispecific antibody through separation and purification.

[0038] The present invention provides any of the following uses of the above-mentioned antibody or antigen-binding fragment thereof, the bispecific antibody, the nucleic acid molecule or the biomaterial:

[0039] (1) Use in the preparation of a drug for preventing and / or treating HFTS virus infection or a disease caused by HFTS virus infection;

[0040] (2) Use in the preparation of a product for detecting the presence or level of fever with thrombocytopenia syndrome virus or its envelope protein in a sample;

[0041] (3) Use in the preparation of products for diagnosing HFTS virus infection or diseases caused by HFTS virus infection.

[0042] In the above (2) and (3), the product includes a detection reagent or a diagnostic reagent.

[0043] In the above (2), the sample can be a sample from a living human or animal (including blood, excrement, oral and nasal secretions, etc.), or it can be a sample not from a living human or animal, such as cells cultured in vitro or cell culture fluid.

[0044] The present invention provides an antibody conjugate, which is obtained by conjugating the above-mentioned antibody or its antigen-binding fragment or the bispecific antibody with a marker; the marker is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0045] The present invention provides a pharmaceutical composition, which comprises the above-mentioned antibody or antigen-binding fragment thereof or the bispecific antibody.

[0046] The present invention provides a detection reagent, which comprises the above-mentioned antibody or antigen-binding fragment thereof or the bispecific antibody.

[0047] The beneficial effects of the present invention include at least: the antibodies or antigen-binding fragments thereof and the bispecific antibodies provided by the present invention can specifically bind to the envelope protein of the HFTS virus, have high affinity and neutralizing activity, have a good preventive effect on infection with the HFTS virus, and have a good therapeutic effect on animals infected with the HFTS virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 The diagrams are the results of molecular sieve purification and SDS-PAGE identification of the extracellular segment protein of SFTSV Gc in Example 1 of the present invention.

[0050] Figure 2 These are the antibody molecular sieve purification result diagram and SDS-PAGE identification result diagram in Example 4 and Example 5 of the present invention, wherein A, B, and C are SF83 antibody, bispecific antibody bsAb1, and bsAb3, respectively.

[0051] Figure 3 This is a kinetic curve diagram of the SF83 antibody binding to the extracellular segment of SFTSV Gc in Example 6 of the present invention.

[0052] Figure 4 This is a kinetic curve diagram of the binding of the bispecific antibodies bsAb1 and bsAb3 to SFTSV Gn and Gc in Example 6 of the present invention.

[0053] Figure 5 The results of neutralization of SFTSV infection by SF83 antibody, bispecific antibody bsAb1 and bsAb3 in Example 7 of the present invention are shown, wherein A, B and C are the results of pseudovirus neutralization experiments of SF83 antibody, bispecific antibody bsAb1 and bsAb3, respectively, and D, E and F are the results of live virus neutralization experiments of SF83 antibody, bispecific antibody bsAb1 and bsAb3, respectively.

[0054] Figure 6 This is the effect of SF83 antibody, bispecific antibodies bsAb1 and bsAb3 in protecting mice in Example 8 of the present invention. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] Example 1: Expression and purification of the extracellular segment of SFTSV Gc

[0057] The coding sequence of 6×his tag and translation termination codon were connected to the 3' end of the SFTSV Gc protein extracellular segment coding region (amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.15), and constructed into PCAGGS vector (purchased from Invitrogen) through EcoRI and XhoI. The ligation product was then transformed into Top10 competent cells (purchased from Tiangen) for transformation. The successfully connected plasmid was extracted and transfected into 293F cells (purchased from Invitrogen) to express the SFTSV Gc extracellular segment protein.

[0058] The cell culture medium containing the target protein was purified by nickel ion affinity chromatography (HisTrap TM HP (GE)) and gel filtration chromatography (Superdex 200Increase 10 / 300GL) to obtain a relatively pure target protein. SDS-PAGE identified the size of the target protein as 49KD (theoretical size). Figure 1 shown.

[0059] Example 2: Isolation of SFTSV Gc protein-specific memory B cells

[0060] With the informed consent of a patient who recovered from SFTSV infection and was discharged from the hospital (one patient from Beijing Ditan Hospital affiliated to Capital Medical University), 15 mL of blood was collected and PBMCs were isolated. 7 / mL density and the purified SFTSV Gc extracellular segment protein in Example 1 with a final concentration of 400nM were incubated on ice for half an hour, then washed twice with PBS, and then incubated with the following antibodies (all purchased from BD, with a concentration of 10μg / mL): anti-human CD3 / PE-Cy5, anti-humanCD16 / PE-Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human CD38 / APC, anti-human IgG / FITC, and anti-His / PE. After the antibody was incubated on ice for half an hour, the PBMCs were washed twice with PBS.

[0061] PBMCs washed with PBS were sorted by FACSAria III and PE - Cy5 - APC - APC-Cy7 + Pacific Blue+ FITC + PE + The cells (i.e., B cells) were directly collected into a 96-well plate, 1 cell / well.

[0062] Example 3: Single B cell PCR, sequence analysis and humanized antibody design

[0063] According to the method described in Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus published by Qihui Wang et al. in Science Translational Medicine, Vol. 8, Issue 369 in December 2016, the B cells obtained in Example 2 were reverse transcribed by Superscript III reverse transcriptase (Invitrogen), the reverse transcription primers were as shown in Table 1, and the reaction was carried out at 55° C. for 60 minutes.

[0064] Table 1. Primers for reverse transcription reaction

[0065]

[0066] The reverse transcription product was used as a template and PCR was performed using HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa). The corresponding primers were designed and the reaction conditions were as follows: 95°C, 5min; 95°C 30s, 55°C (heavy chain / κ chain) 30s, 72°C 90s, 35 cycles; 72°C, 7min. The product was used as a template for another round of PCR (PCRb) under the following conditions: 95°C, 5min; 95°C 30s, 58°C (heavy chain) / 60°C (κ chain) 30s, 72°C 90s, 35 cycles; 72°C, 7min to obtain the antibody variable region. For specific operation steps, please refer to the article "High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies" published by Hua-Xin Liao et al. in the Journal of Virological Methods in 2009.

[0067] PCR products were separated by 1.2% agarose gel electrophoresis. The bands between 400-500 bp were excised and sent to a sequencing company for sequencing. The sequencing results were analyzed using IMGT online software.

[0068] The correct variable region sequence obtained by IMGT online software analysis was connected to the corresponding heavy chain / κ chain constant region by bridge PCR and cloned into the expression vector pCAGGS (purchased from Addgene). The heavy chain was connected by EcoRI and XhoI, and the κ chain was connected by SacI and XhoI. B cell sequencing and expression plasmid construction are as follows:

[0069] The human antibody design strategy is as follows:

[0070] Heavy chain: CMV promoter-EcoR I-Leader sequences-heavy chain variable region-CH-Xho I;

[0071] Light chain (κ): CMV promoter-Sac I-Leader sequences-light chain variable region-CL (κ) -Xho I.

[0072] The SF83 antibody was obtained through sequencing analysis and screening, and the comparison results of its heavy chain and light chain with germline genes are shown in Tables 2 and 3. The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the SF83 antibody are shown in SEQ ID NO.1-3, respectively, the amino acid sequence of the light chain complementary determining region CDR1 is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is AAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.6; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8; the amino acid sequence of the heavy chain is shown in SEQ ID NO.9, and the amino acid sequence of the light chain is shown in SEQ ID NO.10. The nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the SF83 antibody is shown in SEQ ID NO.26, and the nucleotide sequence of the nucleic acid molecule encoding the light chain is shown in SEQ ID NO.27.

[0073] Table 2. Comparison of SF83 antibody heavy chain and germline genes

[0074] VH alleles DH allele JH allele Consistency (VH) SF83 IGHV3-30*04 IGHD2-15*01 IGHJ4*02 94.90％

[0075] Table 3. Comparison of SF83 antibody light chain and germline genes

[0076] VL allele JL allele Consistency (VL) SF83 IGLV1-17*01 IGKJ4*01 95.8％

[0077] Example 4: Expression of SF83 antibody

[0078] 293F cells were cultured in 293 cell serum-free medium (purchased from Sino Biological, catalog number: M293TII-1L). 293F was co-transfected with a plasmid containing the light and heavy chain encoding genes of the specific antibody obtained in Example 3. 24 hours after transfection, a feed solution (purchased from Sino Biological, catalog number: M293-SUPI-100ML) was added to the cells, and the culture was continued for 2 days, and the feed solution was added again, and the supernatant was collected after continuing to culture for 2 days.

[0079] The collected supernatant was centrifuged at 6000 rpm for 30 min, mixed with an equal volume of buffer containing 20 mM sodium phosphate (pH 8.0), filtered through a 0.22 μm filter membrane, and bound to a protein A pre-loaded column (5 mL, GE Healthcare). The bound protein was eluted with 10 mM glycine (pH 3.0). The protein was collected and concentrated and then subjected to molecular sieve chromatography. The target peak was determined by SDS-PAGE (reducing and non-reducing), and the results are shown in Figure 2. Figure 2 As shown in A, purified SF83 antibody was obtained.

[0080] Example 5: Construction and expression of bispecific antibodies

[0081] The SF83 antibody obtained above and the Gn-targeting antibody SF5 obtained in the previous screening (see CN114736291A) were constructed into a bispecific antibody, and two bispecific antibodies bsAb1 and bsAb3 were expressed using a mammalian cell expression system. The amino acid sequence of the heavy chain of bsAb1 is shown in SEQ ID NO.19, and the amino acid sequence of the light chain is shown in SEQ ID NO.20. The amino acid sequence of the heavy chain of bsAb3 is shown in SEQ ID NO.21, and the amino acid sequence of the light chain is shown in SEQ ID NO.22. The nucleotide sequence of the nucleic acid molecule encoding the heavy chain of bsAb1 is shown in SEQ ID NO.28, and the nucleotide sequence of the nucleic acid molecule of the light chain is shown in SEQ ID NO.29. The nucleotide sequence of the nucleic acid molecule encoding the heavy chain of bsAb3 is shown in SEQ ID NO.30, and the nucleotide sequence of the nucleic acid molecule of the light chain is shown in SEQ ID NO.31. The heavy chains of bsAb1 and bsAb3 may further comprise the leader sequence shown in SEQ ID NO.23, the light chain of bsAb1 may further comprise the leader sequence shown in SEQ ID NO.24, and the light chain of bsAb3 may further comprise the leader sequence shown in SEQ ID NO.25.

[0082] 293F cells were cultured in 293 cell serum-free medium (purchased from Sino Biological, catalog number: M293TII-1L). 293F were co-transfected with plasmids containing the light and heavy chain encoding genes of bispecific antibodies bsAb1 and bsAb3, respectively. Feed solution (purchased from Sino Biological, catalog number: M293-SUPI-100ML) was added to the cells 24 hours after transfection, and culture was continued for 2 days, and the feed solution was added again, and the supernatant was collected after culturing for another 2 days.

[0083] The collected supernatant was centrifuged at 6000rpm for 30min, mixed with an equal volume of buffer containing 20mM sodium phosphate (pH 7.4), filtered through a 0.22μm filter membrane, and bound to a protein A pre-loaded column (5mL, GE Healthcare). The bound protein was eluted with 10mM glycine (pH 3.0). The protein was collected and concentrated and then subjected to molecular sieve chromatography. The target peak was determined by SDS-PAGE (reducing and non-reducing), and the results are shown in Figure 2. Figure 2 As shown in B and C, purified bsAb1 and bsAb3 antibodies were obtained.

[0084] Example 6: Surface plasmon resonance technology to detect the binding ability of antibodies to SFTSV Gn, Gc

[0085] Surface plasmon resonance analysis was performed using Biacore 8K (Biacore Inc.). The specific steps are as follows:

[0086] A protein A chip (purchased from GE Healthcare) was used. First, the purified antibodies obtained in Example 4 and Example 5 were immobilized on the chip. The antibody immobilization amount was about 100 RU. The SFTSV Gn / Gc extracellular segment protein was diluted in multiples with 0.05% PBST solution and loaded one by one from low concentration to high concentration. The kinetic curve of antibody binding to the SFTSV Gn extracellular segment is shown in Figure 3 and Figure 4 The binding kinetic constants were calculated using Biacore 8 Kevaluation software (Biacore, Inc.). The results showed that the SF83 antibody had a high affinity for the extracellular segment of SFTSV Gc, and the bispecific antibodies bsAb1 and bsAb3 also had a high affinity for SFTSV Gn and Gc.

[0087] Example 7: Detection of Antibody Neutralization of SFTSV Virus Infection

[0088] The ability of antibodies to neutralize SFTSV virus infection was detected using pseudovirus neutralization assay and live virus neutralization assay, respectively.

[0089] In the pseudovirus neutralization experiment, the antibody and VSV-SFTSV-EGFP pseudovirus were incubated at 37 degrees for 1 hour at a ratio of 1:1, then transferred to Vero cells and cultured in a 37-degree incubator for 12 hours. The pseudovirus neutralization ability of the antibody was evaluated by reading the number of EGFP fluorescence points under a CQ1 confocal microscope. Figure 5 As shown in A, B, and C, the half-maximal inhibitory concentrations of SF83, bsAb1, and bsAb3 were 3.49±0.38 mg / mL, 0.31±0.08 mg / mL, and 0.16±0.07 mg / mL, respectively.

[0090] In the live virus neutralization experiment, the antibody and HB29 live virus (100TCID 50 ) were incubated at 37°C for 1 hour at a ratio of 1:1, then transferred to Vero cells and incubated for 2 hours before discarding, and then cultured in a 37°C incubator for 48 hours. The cells were first fixed at -20°C (the fixative was a mixture of methanol and ethanol in equal proportions) and the live virus neutralizing ability of the antibody was evaluated by ELISA (primary antibody: Mab4-5 antibody; secondary antibody: Goat-anti-human-IgG HRP). The results are shown in Figure 5 As shown in D, E, and F, the half-maximal inhibitory concentrations of SF83, bsAb1, and bsAb3 were 29.45±12.80 mg / mL, 1.25±0.45 mg / mL, and 1.05±0.09 mg / mL, respectively.

[0091] Example 8: Animal protection experiment

[0092] Animal protection experiments were performed on SF83 antibody and bispecific antibodies bsAb1 and bsAb3.

[0093] In the prevention experiment, IFNAR at 6-8 weeks of age - / - Mice (purchased from the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention) were intraperitoneally injected with 200 μl of 20 mg / kg of the purified SF83 obtained in Example 4 or 200 μl of 5 mg / kg of the purified bispecific antibodies bsAb1 and bsAb3 obtained in Example 5. After 24 hours, 100 TCID 50 The weight changes and status of mice were observed every day. All mice in the PBS control group died, the survival rate of the 20mg / kg SF83 group was 20%, and the survival rates of bsAb1 and bsAb3 were 100%. Figure 6 A (survival rate) and B (body weight) are shown. In the treatment experiment, IFNAR - / - 4-5 mice / group, subcutaneously injected with 100 TCID 50SFTSV was administered, and 24 hours later, 200 μl of 20 mg / kg of the purified SF83 obtained in Example 4 or 200 μl of 10 mg / kg, 5 mg / kg, 2.5 mg / kg of the purified bispecific antibodies bsAb1 and bsAb3 obtained in Example 5 or 200 μl of 5 mg / kg of the control antibody SF5 were intraperitoneally injected. The survival rates of the mice in the bsAb1 and bsAb3 antibody groups with doses of 10 mg / kg and 5 mg / kg were 100%, the survival rates of the mice in the bsAb1 and bsAb3 antibody groups with doses of 2.5 mg / kg were 75% and 25%, respectively, the survival rate of the mice in the SF83 group was 20%, the survival rate of the control antibody SF5 was 80%, and all the mice in the PBS control group died. The results are shown in FIG. Figure 6 C (survival rate) and D (body weight) are shown. In the delayed treatment experiment, IFNAR - / - 4-5 mice / group were injected subcutaneously with 100 TCID 50 SFTSV was administered, and 200 μl of 10 mg / kg of the purified bispecific antibodies bsAb1 and bsAb3 obtained in Example 5 were intraperitoneally injected 48 or 72 hours later. The survival rate of the mice in the antibody group was 100% 48 hours after the challenge, while all the mice in the PBS group died ( Figure 6 E and F). 72 hours after the challenge, the survival rates of mice in the bsAb1 and bsAb3 bispecific antibody groups were 75% and 100%, respectively, while all mice in the PBS group died ( Figure 6 G and H).

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that The antibody or antigen-binding fragment thereof specifically binds to the envelope protein of the fever with thrombocytopenia syndrome virus; The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO.1-3, respectively; the amino acid sequence of the light chain complementary determining region CDR1 is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is AAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.7 or has at least 80% similarity to the amino acid sequence as shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8 or has at least 80% similarity to the amino acid sequence as shown in SEQ ID NO.

8.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO.9 or has at least 80% similarity to the amino acid sequence as shown in SEQ ID NO.9, and the amino acid sequence of the light chain is as shown in SEQ ID NO.10 or has at least 80% similarity to the amino acid sequence as shown in SEQ ID NO.

10.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antigen binding fragment is selected from any one of Fab, Fab', F(ab')2, Fab'-SH, Fv, and scFv.

5. A bispecific antibody, characterized in that The bispecific antibody comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and an antibody that specifically binds to the envelope protein Gn of the fever with thrombocytopenia syndrome virus; The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the antibody that specifically binds to the envelope protein Gn of the fever and thrombocytopenia syndrome virus are shown in SEQ ID NO.11-13, respectively, the amino acid sequence of the light chain complementary determining region CDR1 is shown in SEQ ID NO.14, the amino acid sequence of CDR2 is GKN, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

16.

6. The bispecific antibody according to claim 5, characterized in that The amino acid sequence of the heavy chain variable region of the antibody that specifically binds to the fever and thrombocytopenia syndrome virus envelope protein Gn is as shown in SEQ ID NO.17 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.18 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.

18.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; or, the nucleic acid molecule encodes the bispecific antibody according to claim 5 or 6.

8. Biomaterial, characterized in that The biological material comprises the nucleic acid molecule of claim 7; The biological material is an expression cassette, a vector or a host cell.

9. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the bispecific antibody according to claim 5 or 6, the nucleic acid molecule according to claim 7, or the biomaterial according to claim 8: (1) Use in the preparation of a drug for preventing and / or treating fever with thrombocytopenia syndrome virus infection; (2) Use in the preparation of a product for detecting the presence or level of fever with thrombocytopenia syndrome virus or its envelope protein in a sample; (3) Application in the preparation of products for diagnosing fever with thrombocytopenia syndrome virus infection.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the bispecific antibody according to claim 5 or 6.

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