Nanometer antibody NbG7-4 capable of being combined with SFTSV and application thereof

Abstract

The invention relates to a polypeptide NbG7-4 capable of identifying and neutralizing SFTSV, which comprises three complementary determining regions CDR1-3, and the sequences of the three complementary determining regions CDR1-3 are respectively shown as SEQ ID NO: 1-3. Nanometer antibody drug development and diagnostic kit research and development are carried out on SFTSV which is high in fatality rate but lacks effective vaccines and specific antiviral drugs, the nanometer antibody VHH which is specifically combined with GN is screened through the platform technology of preparing GN protein, inactivating viruses, immunizing alpaca, displaying nanometer monoclonal antibodies through a phage library and the like, the CDR sequence of the nanometer antibody VHH is identified, and the SFTSV with the high fatality rate is obtained. A humanized antibody NbG7-4 is constructed; meanwhile, the curative effect of the NbG7-4 in treating the SFTSV infection is evaluated at the in-vitro cell level. The invention provides a potential nano antibody new drug for clinical treatment of SFTSV, and also provides a corresponding detection kit for diagnosis of SFTSV.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly to a nano antibody NbG7-4 capable of binding to SFTSV and its application. Background Art

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by a novel Bunyavirus (SFTSV). It is primarily transmitted through tick bites or contact with the blood or mucous membranes of acutely ill patients. Clinical manifestations include fever, thrombocytopenia, leukopenia, and multiple organ dysfunction, including those in the gastrointestinal tract, liver, and kidneys. Since SFTS was first reported in Hubei and Henan in 2009, cases have been reported in 16 provinces in my country, and the incidence has been increasing in recent years. Furthermore, SFTS cases have been reported in South Korea, Japan, the Arab world, and the United States, suggesting that the disease is spreading. The reported mortality rate for SFTS ranges from 6.3% to 30%, with mortality rates as high as 35.4% and 50% reported in South Korea and Japan in 2016, respectively. However, the lack of specific treatments for SFTS has limited therapies to broad-spectrum antiviral therapy and targeted treatment, making the prevention and control of SFTS extremely challenging.

[0003] Specific neutralizing antibodies, such as palivizumab, a monoclonal antibody against respiratory syncytial virus, and rabies virus antiserum, have demonstrated excellent clinical efficacy in antiviral treatment. Studies have shown that neutralizing antibodies against the surface glycoprotein N (GN) of SFTSV play a significant role in patient survival. Our previous research also showed that SFTSV patients whose serum contained anti-GN antibodies recovered well, while the mortality rate among those without GN antibodies was as high as 66.7%. These studies suggest that neutralizing antibody therapy targeting GN may be an effective approach to combat SFTSV.

[0004] In 1993, a new type of natural antibody from the camelid family was discovered. This antibody naturally lacks light chains and is composed only of heavy chains. Its heavy chain contains two constant regions (CH2 and CH3), a hinge region, and a variable heavy chain domain (VHH, i.e., the antigen-binding site). The relative molecular mass of this heavy chain variable region is approximately 13 kDa, only 1 / 10 of that of a conventional antibody. Its molecular height and diameter are both at the nanometer level, making it the smallest functional antibody fragment currently available. Therefore, it is also called a nanobody (Nb). Nanobodies have the characteristics of high stability (no degradation at 90°C), high affinity, over 80% homology with human antibodies, and low toxicity and immunogenicity. Therefore, they have recently been widely used in the development of immunodiagnostic kits, imaging research, and antibody drug development for tumors, inflammation, infectious diseases, and neurological diseases.

[0005] Our previous testing of antibody titers in SFTS patient sera revealed that antisera targeting GN protein were only available at a dilution factor of approximately 100. This suggests that using low-titer sera from SFTSV patients to construct an antibody library may have limited the selection of highly effective neutralizing antibodies. Therefore, we aim to utilize novel technologies to obtain more effective and stable SFTSV-specific neutralizing antibodies with lower IC50 values. Summary of the Invention

[0006] The present invention uses alpaca antigen immunization to obtain alpaca-derived nanomonoclonal antibodies and their VHHs for the diagnosis and treatment of patients with acute SFTSV infection. Based on these studies, the present invention provides a VHH nanoantibody that binds to SFTSV, comprising three complementary determining regions (CDRs) 1-3, the sequences of which are shown in SEQ ID NOs: 1-3, respectively.

[0007] In a specific embodiment, the Nanobody comprises the amino acid sequence shown in SEQ ID NO:8.

[0008] In a specific embodiment, the Nanobody is a camelid Nanobody or a humanized Nanobody.

[0009] The present invention also provides a heavy chain antibody or antigen-binding fragment that can bind to SFTSV, which comprises the above-mentioned nanobody as a structural domain.

[0010] The present invention also provides nucleic acids encoding the above-mentioned nanobodies, heavy chain antibodies or antigen-binding fragments.

[0011] The present invention also provides an expression vector comprising the above nucleic acid.

[0012] The present invention also provides a host cell comprising the above nucleic acid or expression vector.

[0013] The present invention also provides the use of the above-mentioned nanobody or heavy chain antibody or antigen-binding fragment or nucleic acid in the preparation of a detection agent for SFTSV or a detection agent for the surface glycoprotein of SFTSV.

[0014] The present invention also provides the use of the above-mentioned nanobody or heavy chain antibody or antigen-binding fragment or nucleic acid in the preparation of a therapeutic drug for SFTSV infection.

[0015] This study targets SFTSV, a highly lethal virus lacking effective vaccines and specific antiviral drugs, by developing a nanoantibody drug and diagnostic kit. By preparing GN protein, inactivated viruses, and alpacas, and utilizing a phage library platform for displaying nanomonoclonal antibodies, the team screened for a nanoantibody VHH that specifically binds to GN, identified its CDR sequence, and constructed a humanized version of NbG7-4. The efficacy of NbG7-4 in treating SFTSV infection was also evaluated at the cellular level. This study provides a potential new nanoantibody drug for the clinical treatment of SFTSV and a corresponding detection kit for the diagnosis of SFTSV. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the antiserum titer detection curve one week after the third immunization of alpacas with sGN and the fourth and fifth immunizations with inactivated viruses.

[0017] Figure 2 The curves of the inhibition of AE five subtypes of SFTSV virus infection of Vero cells in vitro by antisera of different dilutions one week after the 3rd, 4th and 5th immunization of alpacas, with the serum before immunization as the control.

[0018] Figure 3 For the first round (1 st ) and the second round (2 nd ) Statistical chart of phage ELISA detection of 48 clones selected from each phage antibody library after panning.

[0019] Figure 4 Figure 1 is a statistical graph of ELISA detection of prokaryotically expressed VHH antibodies. Each point represents a clone. The vertical axis is the OD450 against sGN / OD450 of the blank control. A ratio greater than 5.0 is defined as positive.

[0020] Figure 5 ELISA was used to detect the binding of different purified concentrations of NbG7-4 to sGN protein, and Nb15 was used as the isotype control antibody.

[0021] Figure 6Fluorescent staining photos obtained by virus infection in the presence of different concentrations of antibodies. Fluorescent spots represent SFTSV virus. Taking SFTSV-B (HB29) as an example, cells without virus infection serve as the no infection control (Noinfection control), and virus-infected cells without antibody are the infection control (Infection control).

[0022] Figure 7 Based on Figure 6 The curve of the antibody inhibition rate against SFTSV virus obtained by counting the fluorescent points. DETAILED DESCRIPTION

[0023] 1. Preparation of Immunogen

[0024] Based on the GN protein and gene sequence information of SFTSV, we analyzed and designed a peptide, sGN, that effectively induces the production of specific antibodies against GN in alpacas. A His-tag (sGN-his) or rabbit Fc (sGN-rFc) was attached to the C-terminus for subsequent purification and detection. The inactivated virus used for immunization was SFTSV-E (JS2013-24).

[0025] 2. Alpaca immunization and acquisition of antiserum

[0026] Alpacas were primed with an emulsified mixture of 250 μg of sGN-rFc protein and 250 μl of complete Freund's adjuvant. Booster immunizations were administered twice on days 14 and 21 with sGN-rFc protein and 250 μl of incomplete Freund's adjuvant. Booster immunizations were also administered twice with inactivated virus on days 28 and 35. Blood was collected one week after the third and fourth immunizations for antiserum titer determination. One week after the fifth immunization, 200 ml of blood was collected for phage antibody library construction.

[0027] Antiserum titers were determined by ELISA. Plates were coated with 0.5 μg / ml GN protein. 100 μl of serially diluted antiserum or purified antibody (pre-immune alpaca serum was used as a control) was added to each well. The plates were incubated at 37°C for 1.5 hours and washed twice. A 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-Llamma IgG (H+L) secondary antibody was added to each well. The plates were incubated at 37°C for 1 hour. After washing 4-6 times, 100 μl of TMB substrate was added and incubated at 37°C for 10 minutes. The reaction was terminated with 50 μl of 0.2 M H₂SO₄, and the OD₄450 nm was measured. The ELISA titer was determined as the highest dilution at which the OD₄450 was at least 2.1 times that of the blank control and greater than 0.2.

[0028] The results are as follows Figure 1 As shown, the titer of antiserum from immunizations 3, 4, and 5 reached 2.88×10 5It can be seen that this antigen can induce alpacas to produce high-titer antiserum specific for GN protein.

[0029] In order to further verify whether the high titer alpaca antiserum can effectively prevent SFTSV virus infection, a neutralization experiment of virus infection was performed. Antiserum and pre-immune serum of different dilution concentrations were incubated with SFTSV virus for 60 minutes, and then transferred to Vero cells. After 48 hours, the infection of SFTSV was judged by cell immunofluorescence staining of sGN protein. The results of the neutralization experiment showed that the ID90 of the antiserum inhibiting 90% AE infection of the five subtypes of SFTSV was more than 540 times the dilution ( Figure 2 In summary, the sequential immunization of sGN plus inactivated virus induced high-titer antiserum, and the antiserum had the ability to effectively inhibit SFTSV virus infection.

[0030] 3. VHH phage library construction and panning

[0031] 200 ml of peripheral blood was collected from the immunized alpaca and PBMCs were isolated using lymphocyte separation medium (GE Ficoll-Paque Plus). RNA was extracted according to the TRIzol operating manual and converted into cDNA using oligo(dT). The VHH gene of the alpaca was cloned into a phagemid plasmid using molecular cloning technology and transformed into TG1 bacteria to obtain a VHH phage library.

[0032] Fifty clones were selected for sequencing. The sequencing results showed that there were no identical repeat sequences in the sequences tested; the alignment results showed that the difference sequences were mostly in the CDR binding region. The capacity of the constructed sGN-VHH phage antibody library was 1.2×10 9 The positive rate was 99%, the sequence diversity was 100%, and the effective insertion rate (In frame rate) was greater than 95%.

[0033] With the help of M13KO7 helper phage, the phage antibody library was recovered with bacteria transformed with VHH-phagemid and precipitated with PEG / NaCl. The phage antibody library was enriched twice with 50μg / ml of sGN-His protein. In order to further verify the positive phage rate of sGN-VHH protein binding in the enriched library, 48 clones were selected from the libraries after the first and second rounds of enrichment for single phage ELISA detection. The results showed that in the first round of the library, 45.8% of the single phage clones were positive, and 85.4% of the phage clones in the second round of the library were positive, and the average binding read value was around 3.0 ( Figure 3), clones with binding read values ​​> 1.0 were sequenced and cloned into the expression vector pVAX1, and transfected into 293F cells to express and produce nano-mAb.

[0034] 4. Construction of VHH Prokaryotic Expression Library and VHH Expression

[0035] The 1st-sGN-VHH and 2nd-sGN-VHH phage antibody libraries enriched after the first and second rounds of panning were amplified by PCR; all VHH gene fragments in the antibody library were obtained and purified, and the VHH gene fragments were cloned into a prokaryotic expression vector and transformed into the SS320 strain to construct a prokaryotic expression antibody library of VHH; the prokaryotic expression antibody library was spread on a plate and cultured overnight. The next day, 190 monoclonal colonies were randomly selected and the antibody expression supernatant was induced using IPTG. The antibody supernatant was then tested for binding to sGN protein by ELISA.

[0036] The results showed that 171 bacterial supernatants bound to sGN protein and did not bind to the blank control. The sGN binding reading / blank control reading was greater than 5.0 ( Figure 4 ).

[0037] To further verify whether this sequence can effectively inhibit SFTSV infection, the bacterial supernatants of these 171 VHH antibodies were treated and co-incubated with SFTSV virus to test whether the antibodies can inhibit SFTSV infection. Among them, the antibody numbered NbG7-4 achieved an inhibition rate of over 90%. The sequences of the complementary determining regions CDR1-3 of antibody NbG7-4 are shown in SEQ ID NOs:1-3, and the framework region sequences FR1-4 are shown in SEQ ID NOs:4-7.

[0038] Through molecular cloning technology, the NbG7-4 nano-monoclonal antibody VHH gene was fused with the human Fc gene and inserted into the pVAX1 eukaryotic expression vector to construct the Nb-huFc-pVAX1 expression plasmid. The constructed Nb-huFc-pVAX1 was transfected into 293F cells for expression and purification using Protein G. The purified NbG7-4 antibody was collected for ELISA assay, and the results were as follows: Figure 5 As shown, the nanobody showed good binding ability to SFTSV virus.

[0039] 5. NbG7-4 neutralizes SFTSV infection in Vero cells

[0040] The NbG7-4 antibody was diluted to different concentrations and incubated with the SFTSV virus at 5% CO2 and 37°C for 1 hour. 4Vero cells were cultured in a 5% CO2 incubator at 37°C for 48 hours. The supernatant was removed and the cells were fixed with 4% paraformaldehyde for 15 minutes. After washing and blocking, anti-GN rabbit polyclonal serum (1:1000 dilution) was added and incubated at 4°C overnight. After washing with PBST, 50 μl of anti-rabbit secondary antibody (Alexa Fluor 488 Anti-Rabbit IgG (H+L), Code: 111-545-144, Jackson ImmunoResearch Laboratories, 1:1000 dilution) was added and incubated at room temperature for 40 minutes. DAPI staining was then performed for 10 minutes. After washing, the cells were photographed under a fluorescence microscope and observed. The green spots and fluorescence intensity were counted to calculate the neutralization inhibition rate and neutralization titer. Inhibition rate = [1-(mean fluorescence intensity of the sample group - mean fluorescence intensity of the cell control group CC) / (mean fluorescence intensity of the control group - mean fluorescence intensity of the cell control group CC)] × 100%. Neutralization titer (ID 50 or ND 50 ) is expressed as the dilution multiple that results in 50% inhibition.

[0041] The results are as follows Figure 6 and 7 As shown, NbG7-4 has good neutralizing activity. When the antibody concentration is 1 μg / ml, the inhibition rate of five subtypes of SFTSV (SFTSV AE) can reach 88.3% or above.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A VHH nanobody that can bind to SFTSV, characterized in that It includes three complementary determining regions CDR1-3, and the sequences are shown in SEQ ID NO: 1-3 respectively.

2. The Nanobody according to claim 1, characterized in that It includes the amino acid sequence shown in SEQ ID NO:

8.

3. The Nanobody according to claim 1, characterized in that The nanobody is a camel-derived nanobody or a humanized nanobody.

4. A heavy chain antibody or antigen-binding fragment that can bind to SFTSV, characterized in that: Comprising the Nanobody of any one of claims 1-3.

5. A nucleic acid, characterized in that Encoding the Nanobody according to any one of claims 1 to 3 or the heavy chain antibody or antigen-binding fragment according to claim 4.

6. An expression vector, characterized in that Comprising the nucleic acid according to claim 5.

7. A host cell, characterized in that Comprising the nucleic acid according to claim 5 or the expression vector according to claim 6.

8. Use of the Nanobody according to any one of claims 1 to 3, or the heavy chain antibody or antigen-binding fragment according to claim 4, or the nucleic acid according to claim 5 in the preparation of a detection agent for SFTSV or a detection agent for the surface glycoprotein of SFTSV.

9. Use of the Nanobody according to any one of claims 1 to 3, or the heavy chain antibody or antigen-binding fragment according to claim 4, or the nucleic acid according to claim 5 in the preparation of a therapeutic drug for SFTSV infection.

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