An antibody that specifically binds to a Gc antigen of a severe fever with thrombocytopenia syndrome virus

Abstract

This invention discloses an antibody or antigen-binding fragment thereof that specifically binds to the Gc antigen of the fever with thrombocytopenia syndrome virus, solving the problem of treating and preventing fever with thrombocytopenia syndrome. The antibody exhibits highly efficient neutralizing ability in viral models and can maintain high purity after humanization.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antibody that specifically binds to the Gc antigen of fever with thrombocytopenia syndrome virus. Background Technology

[0002] Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerging tick-borne virus belonging to the genus Leukopivirus of the order Bunyavirales. Its primary vector is the Haemaphysalis longhorn tick, and it also carries the risk of human-to-human transmission through blood contact. It is widespread in several provinces in central and eastern my country, as well as in East Asian countries such as Japan, South Korea, and Vietnam, posing a serious threat to public health. Clinical manifestations of SFTSV infection initially include high fever, fatigue, and muscle aches, which rapidly progress to thrombocytopenia and leukopenia. Severe cases can lead to multiple organ failure, with a mortality rate as high as 12%–30%. It has been listed as a priority pathogen by the World Health Organization. Currently, prevention and control of SFTSV infection mainly involve symptomatic and supportive treatment. There is still a lack of safe and effective vaccines and specific antiviral drugs, and no monoclonal antibodies against SFTSV have entered clinical trials or are available for commercial use.

[0003] SFTSV's cell membrane proteins include two envelope proteins, Gn and Gc. The Gc protein is responsible for mediating the fusion of the virus with the host cell membrane. It is a key protein for the virus to adsorb and enter the host cell to complete replication and proliferation. It is also an important target antigen that induces the body to produce a specific immune protective response. The protective effect of the vaccine is positively correlated with the concentration of antibodies against this protein.

[0004] Antibody-mediated humoral immune responses are a crucial means for the body to combat viral infections. Highly effective neutralizing and protective antibodies can not only be used for emergency treatment of emerging viral infections and control of epidemic spread, but also help susceptible and high-risk populations prevent viral infection and facilitate the rapid development of serological diagnostic kits. Monoclonal antibodies can effectively inhibit viral replication; passive transfer of polyclonal serum and monoclonal antibodies to laboratory animals can effectively prevent and protect them from corresponding viral infections. Currently, antibody drugs targeting respiratory syncytial virus (RSV) and HIV are available on the market, and several other antibody drugs targeting different viral infections are in the research and clinical trial stages.

[0005] Current treatments for SFTSV rely solely on symptomatic supportive measures such as antipyretics, plasma or platelet transfusions, lacking specific therapies and effective antibody treatments. Therefore, developing an antibody that specifically binds to the Gc protein of SFTSV and possesses highly efficient neutralizing activity to address this urgent clinical need is of great significance for the prevention and control of SFTSV. Summary of the Invention

[0006] To achieve the above objectives, this invention has developed an antibody that specifically binds to the Gc protein of SFTSV (Fever with Thrombocytopenia Syndrome) virus, resulting in superior clinical therapeutic effects. This specific antibody provides a new and effective means for the prevention and treatment of diseases caused by SFTSV infection.

[0007] Specifically, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the Gc antigen of fever with thrombocytopenia syndrome virus, characterized in that the antibody or antigen-binding fragment thereof comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; the antibody or antigen-binding fragment thereof comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0008] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region, the amino acid sequence of which is shown in any one of SEQ ID NO: 13, 15, 17 or 19.

[0009] In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NO: 14, 16, 18 or 20.

[0010] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain variable region, the amino acid sequence of which is shown in any one of SEQ ID NO: 7, 9 or 11.

[0011] In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NO: 8, 10 or 12.

[0012] In some embodiments, the antibody or its antigen-binding fragment further comprises an Fc region.

[0013] In some embodiments, the Fc region is selected from IgG, IgA, IgD, IgE and / or IgM.

[0014] In some embodiments, the Fc region is selected from IgG1, IgG2, IgG3 and / or IgG4, preferably, the Fc region is selected from IgG1.

[0015] In some implementations, the Fc region includes natural Fc fragments and Fc mutants.

[0016] In some implementations, the amino acid mutation is a substitution or deletion of the amino acid.

[0017] In some embodiments, the Fc mutant comprises the substitution and / or truncation of amino acid residues based on the native Fc fragment. For example, mutations can be made at sites such as M428L+N434S, L234A+L235A, etc.

[0018] The present invention also provides a nucleic acid molecule that encodes the antibody or an antigen-binding fragment thereof.

[0019] The present invention also provides the use of the antibody or its antigen-binding fragment, or the nucleic acid molecule, in the preparation of a medicament for treating or preventing diseases caused by viral infection with fever accompanied by thrombocytopenia syndrome.

[0020] Experimental verification has shown that the antibody provided by this invention can specifically bind to the Gc protein of fever with thrombocytopenia syndrome virus (FSH). This antibody exhibits highly efficient neutralizing ability in viral models and maintains high purity even after humanization. This achievement provides new insights for the development of therapeutic and preventative drugs against FSH infection and opens up new and effective avenues for the treatment and control of related diseases.

[0021] Abbreviations and Terminology Definitions

[0022] The following abbreviations are used in this article. CDR: Complementarity-determining region in the immunoglobulin variable region.

[0023] The term "amino acid" refers to twenty common, naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In some embodiments, the term "amino acid" also includes non-natural amino acids. Any suitable non-natural amino acid may be used. In some embodiments, the non-natural amino acid contains a reactive moiety for conjugating the agent with MIAC.

[0024] The term "complementarity-determining region" (CDR) refers to a discontinuous antigenic combination site found within the variable region of a heavy chain polypeptide. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immune interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes overlaps or subsets of amino acid residues when compared with each other. Several methods exist for defining or describing CDRs, including Kabat, Chothia, IMGT, AbM, and Contact, with Kabat being the most commonly used. LCDR and HCDR represent the complementarity-determining regions of the light and heavy chains, respectively.

[0025] The term "specific" means that one of the molecules involved in specific binding does not exhibit significant binding to any molecules other than one or more of its binding partner molecules. Furthermore, the term is also used when a domain containing an antibody-variable region is specific to a particular epitope among multiple epitopes in an antigen. When the epitope bound by the domain containing the antibody-variable region is contained in several different antigens, an antigen-binding molecule containing the domain containing the antibody-variable region can bind to various antigens having said epitope.

[0026] The term "Fc" refers to a sequence of non-antigen-binding fragments (whether in monomeric or multimeric form) produced by antibody digestion or other means, and may contain a hinge region. The original fusion protein source of native Fc is preferably human, but can be any fusion protein. Fc molecules consist of monomeric polypeptides that can be covalently (i.e., disulfide bonds) and non-covalently associated to form dimers or multimers. Depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4), the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4.

[0027] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain originates from one source or species, while the remainder of the heavy chain originates from a different source or species. It refers to an antibody molecule in which a constant region or a portion thereof is altered, replaced, or exchanged, thereby linking the antigen-binding site to a different or altered constant region of a class and / or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug), which imparts new properties to the chimeric antibody. For example, camel-derived antibodies can form chimeric antibodies by replacing their constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing antigens while exhibiting reduced immunogenicity in humans, as compared to the original camel-derived antibody.

[0028] The term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology with the human variable domain. This can be achieved by transplanting the six non-human antibody complementarity-determining regions (CDRs) that together form the antigen-binding site onto the homologous human receptor frame region (FR). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace frame residues of the parent antibody (i.e., the non-human antibody) into the human frame region (reversion mutation). Structural homology modeling can help identify amino acid residues in the frame region that are important for the antibody's binding properties. Therefore, a humanized antibody may contain a non-human CDR sequence, an essentially human frame region optionally containing one or more amino acid reversion mutations mutated into a non-human amino acid sequence, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily reversion mutations, may be applied to obtain humanized antibodies with preferred characteristics, such as affinity and biochemical properties.

[0029] The term "frame region" or "FR" refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0030] The term "amino acid mutation" refers to a mutation or change in amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one or more amino acids based on the original protein or polypeptide.

[0031] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence.

[0032] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivering bound molecules. Carriers can be anti-adhesives, adhesives, coating agents, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, humectants, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0033] The term "neutralizing activity" refers to antibodies or their antigen-binding fragments that have antiviral activity, which can specifically recognize viral antigens and effectively bind to and neutralize viral activity, preventing viral invasion of target cells and blocking viral replication in target cells, thus playing an important role in antiviral activity. Attached Figure Description

[0034] Figure 1 The results show the neutralizing activity of the antibody against the pseudovirus in this invention. Detailed Implementation

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of this application, suitable methods and materials are described below. In case of conflict, the patent specification shall prevail.

[0036] Example 1: Immunization of mice

[0037] SFTSV-Gc-his protein (SFTSV_AFB82725.1-2017 PNAS; Gc Extracellular Domain (Gc_ECD): 563-996) was used as an immunogen, and mice were immunized with Freund's adjuvant. A second immunization was performed 2 weeks after the first immunization, and a third immunization was performed 3 weeks later. Negative serum was collected from mice 3 days before the first immunization, and 50 µL of blood was collected 7 days after each immunization by tail clipping. Negative and immune sera were diluted proportionally (1:0.1K, 1:0.3K, 1:0.9K, 1:2.7K, 1:8.1K, 1:24.3K), and the serum titer of SFTSV-Gc-his protein was detected using ELISA. When the titer met the requirements and anti-SFTSV-Gc antibodies were detected at dilutions >1:10K, the mouse spleen and lymph nodes were harvested.

[0038] Example 2 Cell Fusion

[0039] B lymphocytes used in the experiment were obtained from Balb / c mice that had undergone four immunizations. The spleen and lymph nodes were placed in a cell strainer, which was then placed in a 50 mL centrifuge tube. DMEM was added to the spleen, and the cells were ground to prepare a spleen cell suspension. The suspension was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. The B cells were resuspended in 5 mL of erythrocyte lysis buffer, lysed at room temperature for 5 min, and then 30 mL of DMEM was added. After mixing, the cells were centrifuged at 2000 rpm for 5 min, and the cells were counted.

[0040] Myeloma cells SP2 / 0 were passaged one day before fusion to ensure they were in logarithmic growth phase at the time of the experiment. Spleen cells and SP2 / 0 were mixed at a 2:1 ratio and centrifuged at 2000 rpm for 5 min. The mixed cells were washed twice with fusion buffer and centrifuged at 2000 rpm for 5 min. The final cell density was 1 × 10⁻⁶ cells / mL. 7 Add fusion buffer to suspend the cells. Within 5 minutes, transfer the cell suspension to the fusion chamber of an electrofusion apparatus (BTX; ECM 2001) for fusion. After fusion, transfer the cells from the fusion chamber to complete culture medium containing HAT and incubate at 37°C for 30 minutes. After incubation, plate the cells in 96-well plates containing feeder cells and incubate at 37°C with 5% CO2.

[0041] Example 3: Screening of positive clones using ELISA method

[0042] After 7 days of culture, the fusion supernatant was initially screened. SFTSV-Gc-his protein was diluted to 1 μg / mL with PBS buffer (pH 7.4), and 100 µL was added to each well of a 96-well ELISA plate. The plates were incubated at 37°C for 2 hours (for coating with two different antigens, and for screening hybridoma cell supernatants from mice immunized with the corresponding target proteins). The coating solution was discarded, and the plates were washed three times with PBST, then blocked with 5% skim milk blocking buffer for 2 hours. The blocking solution was discarded, and the plates were washed three times with PBST. 100 μL of the fusion supernatant was added to each well of the coated ELISA plate. Negative controls (blank wells) and positive controls (1000 μL 1*PBS + 1 μL positive serum) were set up, and the plates were incubated at 37°C for 1 hour. The liquid in the wells was discarded. The plates were washed three times with PBST. The secondary antibody, goat anti-mouse IgG-FC-HRP, was diluted 1:20000 with PBS. 100 μL / well, incubated at 37℃ for 30 min. After washing the plate 6 times with PBST, 100 µL of TMB substrate was added to each well, and the plate was incubated at room temperature in the dark for 10 min. The colorimetric reaction was terminated by adding 100 µL of 1M HCl solution to each well. The absorbance of each well in the 96-well plate was measured at a wavelength of 450 nm using a multi-mode microplate reader, and the data were analyzed. Cell lines with an OD>1.0 result in the reaction between the supernatant and the coating protein were selected as candidate positive cell lines for initial screening. The culture supernatant of the positive cell lines was aspirated and discarded, and fresh HT complete medium was added.

[0043] Example 4: Production of small-scale antibody samples from candidate cell lines

[0044] Hybridoma cells were cultured in T75 incubator until cell coverage reached 80-90%. The supernatant from two flasks was discarded, and 60 mL of hybridoma-SFM was added. The cells were then cultured at 37°C with 5% CO2 for 6-7 days. The culture supernatant was collected after low-speed centrifugation and purified.

[0045] Example 5: Gc protein binding activity of the murine antibody of the present invention

[0046] The binding activity of antibodies to Gc protein on SFTSV was determined by ELISA. SFTSV-Gc-coated plates were incubated at 37°C for 2 hours, blocked with 5% skim milk, washed three times with PBST, and then tested with mouse candidate antibodies as primary antibodies (diluted to 0.6, 0.12, and 0.024 μg / mL in PBS) and goat anti-mouse-IgG-FC-HRP as secondary antibodies. After TMB staining, the absorbance of each well was read at 450 nm. The results are shown in Table 1, indicating that most mouse antibodies exhibited good binding activity to the Gc protein.

[0047] Table 1. Binding activity of murine antibodies to Gc protein

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] Example 6 Neutralizing activity of the murine antibody pseudovirus of the present invention

[0054] Vero cells were resuspended in diluent (DMEM + 10% FBS) and then 3 × 10⁻⁶ cells were added. 450 μL / well was seeded onto a 96-well blank plate and incubated overnight. The antibody was serially diluted with diluent, and 30 μL / well was added to each well of the 96-well plate. Then, 30 μL of a 1:25 diluted HB29 pseudovirus strain (Pseudovirus-SFTS-HB29-M-Luciferase-GFP) was added and mixed, and incubated at 37°C for 1 h. Next, 50 μL of the antibody-pseudovirus mixture was added to a 96-well blank plate pre-inoculated with Vero cells. After 22 h of incubation, 50 μL / well of luciferase assay reagent was added, and the fluorescence value was measured using a microplate reader. The pseudovirus neutralization rate of the antibody was calculated. The pseudovirus neutralization activity of the mouse antibody is shown in Table 2. The results showed that the mouse antibodies of this invention all exhibited varying degrees of neutralizing activity against SFTSV pseudovirus (HB29 / 2010 OX=992212).

[0055] Table 2. Pseudovirus neutralizing activity of murine antibodies

[0056]

[0057] Example 7: Live virus neutralizing activity of the murine antibody of the present invention

[0058] Candidate antibodies with excellent neutralizing activity against SFTSV pseudovirus were selected for further testing of live virus neutralizing activity. 100 TCID50 units of SFTSV virus (subtype A) were thoroughly mixed with different concentrations of candidate antibodies and incubated at 37°C for 1 hour. After incubation, Vero cells were infected with this mixture and cultured for approximately 6 days. The cell state was observed under a microscope to determine the neutralizing activity of the antibody against the live virus. The criteria were as follows: complete cytopathic effect indicated ineffectiveness, half-cytopathic effect indicated partial effectiveness, and no cytopathic effect indicated complete neutralization. P represents complete neutralization, M represents partial effectiveness, and N represents ineffectiveness. Specific test results are shown in Table 3. The experimental results show that some of the murine antibodies in this invention have good live virus neutralizing activity against SFTSV virus (subtype A).

[0059] Table 3. Live virus neutralizing activity of the Gc murine antibody of the present invention

[0060]

[0061] Example 8: Human-centered engineering design

[0062] The variable region of the candidate antibody was humanized, and the modification process followed specific design principles: no protein modification sites such as glycosylation, deamidation, and isomerization were introduced, no integrin binding sites or cysteine ​​residues were introduced, and the reversion mutations of important amino acids in the framework region were required to maintain the original physicochemical and biochemical activities of the antibody. The specific modification methods are as follows.

[0063] Using the IgBLAST tool, the variable region of the murine antibody 47 was compared with the human Germline sequence. The antibody frame region (FR) was then replaced with the human Germline sequence with the highest sequence similarity. Based on this humanization modification, key amino acids affecting antibody affinity were reverse-mutated, i.e., mutated to the corresponding sites of the original camel antibody frame region (FR). The humanization percentage was defined as the similarity ratio between the designed sequence frame region and the human Germline sequence frame region. The designed humanized sequences were compared and screened with human Germline sequences, and sequences with a humanization percentage of 85% or higher were finally selected. Detailed sequence information is shown in Table 4. The heavy and light chain combinations of humanized antibodies 47-1~6 are shown in Table 5.

[0064] Table 4. Antibody sequence numbers of the present invention

[0065]

[0066]

[0067] Table 5. Heavy and light chain combination information of humanized antibodies 47-1~6

[0068]

[0069] Example 9 Construction and expression of humanized antibodies

[0070] The designed antibody sequence was genetically synthesized and embedded within a human IgG antibody framework. Subsequently, molecular cloning techniques were used to insert the antibody fragment into a PVAC vector, thereby constructing a mammalian cell expression plasmid. The expression plasmid was introduced into a CHO host cell line using liposome transfection, and fermentation supernatant was obtained through fed-batch cell culture. This fermentation supernatant was then purified using affinity chromatography to obtain the target humanized antibody.

[0071] The expression levels of the fermentation supernatant before purification were compared and analyzed. Meanwhile, the purity of the antibody purified by one-step affinity chromatography was determined using size exclusion high-performance liquid chromatography (SEC-HPLC), and the specific results are shown in Table 6.

[0072] Table 6. SEC-HPLC Purity Detection Results

[0073]

[0074] Example 10: Pseudovirus Neutralizing Activity of the Antibody of the Present Invention

[0075] The detection method is the same as in Example 6, and the results are shown in [example 6]. Figure 1 The isotype control 1 was Human IgG1, kappa IsotypeControl (supplier: Sino Biological; catalog number: HGEK). The results showed that the antibody of this invention had good neutralizing activity against the pseudovirus.

[0076] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the Gc antigen of the Severe Fever with Thrombocytopenia Syndrome Virus, characterized in that, The antibody or its antigen-binding fragment comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; the antibody or its antigen-binding fragment comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region, the amino acid sequence of which is shown in any one of SEQ ID NO: 13, 15, 17 or 19.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NO: 14, 16, 18 or 20.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises a light chain variable region, the amino acid sequence of which is shown in any one of SEQ ID NO: 7, 9 or 11.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment contains an amino acid sequence as shown in any one of SEQ ID NO: 8, 10 or 12.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment also includes an Fc region.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The Fc region is selected from IgG, IgA, IgD, IgE and / or IgM.

8. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The Fc region is selected from human IgG1, IgG2, IgG3 and / or IgG4, or an Fc region amino acid sequence that has one or more amino acid mutations with human IgG1, IgG2, IgG3, IgG4.

9. The antibody or its antigen-binding fragment according to claim 8, characterized in that, The amino acid mutation is a substitution or deletion of an amino acid.

10. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-9 in the preparation of a medicament for treating or preventing diseases caused by viral infection with fever accompanied by thrombocytopenia syndrome.

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