JP7171737B2 - Nanobodies capable of binding to SFTSV and uses thereof - Google Patents

Description

The present invention relates to the biopharmaceutical field. More particularly, it relates to polypeptides capable of binding to SFTSV, the use of said polypeptides in the manufacture of SFTSV detection agents or SFTSV therapeutic agents, and SFTSV detection kits.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by the Severe fever with thrombocytopenia syndrome virus (SFTSV), mainly caused by tick bites or acute patient It is transmitted by blood and mucosal contact, and the major clinical manifestations are fever, thrombocytopenia, leukopenia, and dysfunction of multiple organs, including the gastrointestinal tract, liver, and kidneys. Except for SFTS being first reported in Hubei and Henan in 2009, in China, 16 provinces have issued case reports, with more than 3500 confirmed cases as of 2014; The incidence has been steadily increasing in recent years, and SFTS cases have all been reported in South Korea, Japan, the Arab world, and the United States, indicating that the epidemic area of the disease is expanding. Currently reported mortality rates for SFTS range from 6.3% to 30%, but the mortality rates promulgated by Korea and Japan in 2016 reached 35.4% and 50%, respectively. However, until now, there are no specific therapeutic drugs for SFTS in clinical practice, and only broad-spectrum antiviral therapy and symptomatic therapy can be implemented, which brings great difficulties in the prevention and control of SFTS.

Specific neutralizing antibodies have very good clinical efficacy in antiviral therapy, such as respiratory syncytial virus monoclonal antibody Palivizumab and rabies virus antiserum. Studies have shown that neutralizing antibodies to the SFTSV surface glycoprotein (Glycoprotein N, GN) have important effects on patient survival. The results of previous studies showed that SFTS patients with serum anti-GN antibodies all recovered well, whereas the mortality rate of SFTS patients without serum GN antibodies reached 66.7%. These studies indicate that neutralizing antibody therapy targeting GN is an effective way to combat SFTSV.

In 1993, a new type of natural antibody from Camelidae was discovered. The antibody naturally lacks light chains and is composed only of heavy chains, which consist of two constant regions (CH2 and CH3), one hinge region, and one variable heavy chain domain. , VHH, that is, the antigen-binding site), the relative molecular weight of the heavy chain variable region is about 13 KDa, only 1/10 that of a normal antibody, and both the height and diameter of the molecule are nano-level. , is the smallest functional antibody fragment currently available, hence also called Nanobody (Nb). Nanobodies have features such as high stability (does not decompose at 90 ° C), high affinity, more than 80% homology with human-derived antibodies, and low toxicity and immunogenicity. Recently, it is widely used in the research and development of immunodiagnostic kits, the research and development of iconography, and the research and development of antibody drugs in the fields of tumors, inflammations, infectious diseases, nervous system diseases and so on.

Currently, the known anti-GN SFTS neutralizing antibody is Mab4-5, obtained by phage display SFTS patient antibody library screening in 2013, with an IC50 between 2 μg/ml and 44.2 μg/ml. The lower the IC50 of a neutralizing antibody, the better, which can significantly reduce the amount of antibody used, saving production costs and increasing the time the antibody maintains effective concentrations in vivo. We have previously detected antibody titers in the sera of SFTS patients, and antisera against GN proteins were only diluted about 100 times. , which may limit screening for neutralizing antibodies. Therefore, it is expected to obtain more effective and stable SFTSV-specific neutralizing antibodies with lower IC50 by new technical means.

The present invention obtains camelid-derived Nanobodies and their VHHs by immunizing camels with antigens and uses them for the diagnosis and treatment of acutely infected SFTS patients. Based on these studies, the present invention comprises three complementarity determining regions CDR1-3, wherein the CDR1 sequence is or comprises one of the sequences shown in SEQ ID NOs: 1-74, and the CDR2 sequence is or comprises one of the sequences set forth in SEQ ID NO: 75-151 and the CDR3 sequence is or comprises one of the sequences set forth in SEQ ID NO: 152-232 A conjugateable polypeptide is provided.

In one particular embodiment, said polypeptide further comprises four frame regions FR1-4 that intersect said CDR1-3. For example, FR1-4 sequences can be designed as shown in SEQ ID NOs: 235-237, although the scope of the invention is not so limited. It is well known in the art that the specific recognition and binding ability of an antibody is determined primarily by the CDR region sequences, with less influence from the FR sequences, and can be designed by species. For example, polypeptides or domains that are capable of binding to SFTSV by designing FR region sequences of human, murine or camelid origin to connect the above CDRs.

In one preferred embodiment, said polypeptide is a monoclonal antibody.

In one preferred embodiment, said polypeptide is a VHH.

In one preferred embodiment, the polypeptide is a camelid-derived VHH or a humanized VHH.

In one particular embodiment, the CDR sequences of said polypeptide are as follows.
I) the sequence of CDR3 is SEQ ID NO: 176;
II) the sequence of CDR1 is SEQ ID NO: 54 and the sequence of CDR2 is SEQ ID NO: 132, or the sequence of CDR1 is SEQ ID NO: 22 and the sequence of CDR2 is SEQ ID NO: 100 .

In another specific embodiment, the CDR sequences of said polypeptide are as follows.
I) the sequence of CDR1 is XXXSTAYY (SEQ ID NO: 233) and X is any amino acid, for example the sequence of CDR1 is selected from SEQ ID NO: 4, 25, 26, 62, 73,
II) the sequence of CDR2 is selected from SEQ ID NO: 78, 80, 84, 90, 103;
III) the CDR3 sequence is selected from SEQ ID NO: 155, 157, 161, 169, 172, 179, 180, 181, 210, 218, 229, 230, 231;

In one particular embodiment, the CDR sequences of said polypeptide are as follows.
the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 78, and the sequence of CDR3 is SEQ ID NO: 155, or the sequence of CDR1 is SEQ ID NO: 4; the sequence of CDR2 is SEQ ID NO: 78 and the sequence of CDR3 is SEQ ID NO: 157, or the sequence of CDR1 is SEQ ID NO: 4 and the sequence of CDR2 is SEQ ID NO: 80; and the sequence of CDR3 is SEQ ID NO: 157, or the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 157 or the sequence of CDR1 is SEQ ID NO:4, the sequence of CDR2 is SEQ ID NO:84, and the sequence of CDR3 is SEQ ID NO:169, or the sequence of CDR1 is SEQ ID NO:4 and the sequence of CDR2 is SEQ ID NO: 84 and the sequence of CDR3 is SEQ ID NO: 172, or the sequence of CDR1 is SEQ ID NO: 4 and the sequence of CDR2 is SEQ ID NO: 84 and the sequence of CDR3 is SEQ ID NO: 180, or the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 181 or the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 210, or the sequence of CDR1 is SEQ ID NO: 4 and the sequence of CDR2 is SEQ ID NO:84 and the sequence of CDR3 is SEQ ID NO:229, or the sequence of CDR1 is SEQ ID NO:4 and the sequence of CDR2 is SEQ ID NO: 84 and the sequence of CDR3 is SEQ ID NO: 230 or the sequence of CDR1 is SEQ ID NO: 4 and the sequence of CDR2 is SEQ ID NO: 84 and the sequence of CDR3 is SEQ ID NO: : 231, or the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 90, and the sequence of CDR3 is SEQ ID NO: 90 NO: 161, or the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 90, and the sequence of CDR3 is SEQ ID NO: 179, or the sequence of CDR1 is SEQ ID NO: 179 ID NO: 25 and the sequence of CDR2 is SEQ ID NO: 103 and the sequence of CDR3 is SEQ ID NO: 179 or the sequence of CDR1 is SEQ ID NO: 26 and the sequence of CDR2 is SEQ ID NO: 26 ID NO: 80 and the sequence of CDR3 is SEQ ID NO: 87 or the sequence of CDR1 is SEQ ID NO: 26 and the sequence of CDR2 is SEQ ID NO: 84 and the sequence of CDR3 is SEQ ID NO: 157, or the sequence of CDR1 is SEQ ID NO: 62, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 218, or the sequence of CDR1 is SEQ ID NO:73, the sequence of CDR2 is SEQ ID NO:80, and the sequence of CDR3 is SEQ ID NO:157.

In another specific embodiment, the CDR sequences of said polypeptide are as follows.
I) the sequence of CDR1 is SEQ ID NO:5;
II) the sequence of CDR2 is SEQ ID NO: 79 and the sequence of CDR3 is SEQ ID NO: 156 or the sequence of CDR2 is SEQ ID NO: 79 and the sequence of CDR3 is SEQ ID NO: 157 is.

In another specific embodiment, the CDR sequences of said polypeptide are as follows.
I) the sequence of CDR1 is SEQ ID NO: 6 and the sequence of CDR2 is SEQ ID NO: 81;
II) The sequence of CDR3 is SEQ ID NO: 158, 213 or 228.

In another specific embodiment, the CDR sequences of said polypeptide are as follows.
I) the sequence of CDR1 is SEQ ID NO: 24 and the sequence of CDR2 is SEQ ID NO: 102;
II) The sequence of CDR3 is SEQ ID NO: 178, 189 or 225.

In another specific embodiment, the CDR sequences of said polypeptide are as follows.
I) the sequence of CDR3 is SEQ ID NO: 170;
II) The sequence of CDR1 is SEQ ID NO:66 and the sequence of CDR2 is SEQ ID NO:93 or The sequence of CDR1 is SEQ ID NO:65 and the sequence of CDR2 is SEQ ID NO:143 or the sequence of CDR1 is SEQ ID NO: 16 and the sequence of CDR2 is SEQ ID NO: 93 or the sequence of CDR1 is SEQ ID NO: 68 and the sequence of CDR2 is SEQ ID NO :93.

In another specific embodiment, the CDR sequences of said polypeptide are as follows.
the sequence of CDR1 is SEQ ID NO: 21, the sequence of CDR2 is SEQ ID NO: 98, and the sequence of CDR3 is SEQ ID NO: 166, or the sequence of CDR1 is SEQ ID NO: 13; the sequence of CDR2 is SEQ ID NO: 89 and the sequence of CDR3 is SEQ ID NO: 166; or the sequence of CDR1 is SEQ ID NO: 13 and the sequence of CDR2 is SEQ ID NO: 99; and the sequence of CDR3 is SEQ ID NO: 166, or the sequence of CDR1 is SEQ ID NO: 13, the sequence of CDR2 is SEQ ID NO: 106, and the sequence of CDR3 is SEQ ID NO: 166 or the sequence of CDR1 is SEQ ID NO: 27, the sequence of CDR2 is SEQ ID NO: 104, and the sequence of CDR3 is SEQ ID NO: 166, or the sequence of CDR1 is SEQ ID NO: 18 and the sequence of CDR2 is SEQ ID NO:95 and the sequence of CDR3 is SEQ ID NO:173.

In another specific embodiment, the CDR sequences of said polypeptide are as follows.
the sequence of CDR1 is SEQ ID NO: 47, the sequence of CDR2 is SEQ ID NO: 125, and the sequence of CDR3 is SEQ ID NO: 202, or the sequence of CDR1 is SEQ ID NO: 60; the sequence of CDR2 is SEQ ID NO: 142 and the sequence of CDR3 is SEQ ID NO: 202, or the sequence of CDR1 is SEQ ID NO: 60 and the sequence of CDR2 is SEQ ID NO: 138; And the sequence of CDR3 is SEQ ID NO:216.

The present invention further provides use of the above polypeptides in the manufacture of SFTSV detection agents or SFTSV therapeutic agents.

The invention further provides nucleic acid coding sequences for the above polypeptides.

In one embodiment, said nucleic acid coding sequence is a DNA coding sequence or an RNA coding sequence.

In one particular embodiment, said nucleic acid coding sequence is present in a gene expression cassette.

The invention further provides an expression vector comprising an expression cassette of the above nucleic acid coding sequences.

In one preferred embodiment, said expression vector is a viral vector.

In one preferred embodiment, said expression vector is an adeno-associated virus vector (AAV vector).

The present invention further provides the use of the above nucleic acid coding sequences and expression vectors in the manufacture of SFTSV therapeutic agents.

The present invention further provides an SFTSV detection kit comprising a detection antibody, a solid matrix, and a coating antibody coated on the solid matrix, wherein the detection antibody and the coating antibody are each one of the above polypeptides. offer.

In one particular embodiment, the CDR sequences of said detection antibody are as follows.
the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 181, or the sequence of CDR1 is SEQ ID NO: 54; the sequence of CDR2 is SEQ ID NO: 132 and the sequence of CDR3 is SEQ ID NO: 176; or the sequence of CDR1 is SEQ ID NO: 13 and the sequence of CDR2 is SEQ ID NO: 99; And the sequence of CDR3 is SEQ ID NO:166.

In one specific embodiment, the CDR sequences of said coating antibody are as follows.
the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 181, or the sequence of CDR1 is SEQ ID NO: 54; the sequence of CDR2 is SEQ ID NO: 132 and the sequence of CDR3 is SEQ ID NO: 176; or the sequence of CDR1 is SEQ ID NO: 6 and the sequence of CDR2 is SEQ ID NO: 81; and the sequence of CDR3 is SEQ ID NO: 158, or the sequence of CDR1 is SEQ ID NO: 13, the sequence of CDR2 is SEQ ID NO: 99, and the sequence of CDR3 is SEQ ID NO: 166 .

In one preferred embodiment,
I) The CDR sequences of the detection antibody are as follows. the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 181, or the sequence of CDR1 is SEQ ID NO: 13; the sequence of CDR2 is SEQ ID NO: 99 and the sequence of CDR3 is SEQ ID NO: 166;
II) The CDR sequences of the coating antibody are as follows. the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 181, or the sequence of CDR1 is SEQ ID NO: 54; the sequence of CDR2 is SEQ ID NO: 132 and the sequence of CDR3 is SEQ ID NO: 176, or the sequence of CDR1 is SEQ ID NO: 6 and the sequence of CDR2 is SEQ ID NO: 81; and the sequence of CDR3 is SEQ ID NO: 158, or the sequence of CDR1 is SEQ ID NO: 13, the sequence of CDR2 is SEQ ID NO: 99, and the sequence of CDR3 is SEQ ID NO: 166 .

In one preferred embodiment,
I) the sequence of said detection antibody CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 181;
II) The CDR sequences of the coating antibody are as follows. the sequence of CDR1 is SEQ ID NO: 54, the sequence of CDR2 is SEQ ID NO: 132, and the sequence of CDR3 is SEQ ID NO: 176, or the sequence of CDR1 is SEQ ID NO: 13; The sequence for CDR2 is SEQ ID NO:99 and the sequence for CDR3 is SEQ ID NO:166.

In another preferred embodiment,
I) the sequence of said detection antibody CDR1 is SEQ ID NO: 13, the sequence of CDR2 is SEQ ID NO: 99 and the sequence of CDR3 is SEQ ID NO: 166;
The CDR sequences of the coating antibody are as follows.
the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 181, or the sequence of CDR1 is SEQ ID NO: 54; the sequence of CDR2 is SEQ ID NO: 132 and the sequence of CDR3 is SEQ ID NO: 176, or the sequence of CDR1 is SEQ ID NO: 6 and the sequence of CDR2 is SEQ ID NO: 81; and the sequence of CDR3 is SEQ ID NO: 158, or the sequence of CDR1 is SEQ ID NO: 13, the sequence of CDR2 is SEQ ID NO: 99, and the sequence of CDR3 is SEQ ID NO: 166 .

The present invention develops nano-antibody drugs and researches and develops diagnostic kits for SFTS, which has a high fatality rate but no effective vaccines and specific antiviral drugs, produces GN proteins, and immunizes Bactrian camels. Then, we screen the nanobody VHH that specifically binds to GN, identify its CDR sequences, and construct a humanized VHH-huFc1 (SNB) by platform technology that displays nanobodies using a phage library. together to assess in vivo the therapeutic efficacy of SNB in treating SFTSV infection in a humanized mouse model. The present invention provides a potential new drug of nano-antibodies for clinical treatment of SFTS, as well as detection kits for diagnosing SFTS.

Detection curves of antiserum titers one week after 3rd and 4th immunization of camels with sGN. Figure 10. Curves of different dilutions of antiserum inhibiting in vitro SFTSV virus infection of Vero cells one week after the fourth immunization of camels, with pre-immune sera used as controls. FIG. 2 is an electropherogram of PCR products amplified using the sGN-VHH phage antibody library as a template. Panning assay of the sGN-VHH phage antibody library, where A is the ELISA detection statistics of the phage library after panning against the sGN protein, B is the 2nd (2 ^nd ) and 3rd round. (3 ^rd ) Statistical chart of 96 clones each selected from the panned phage antibody library for phage ELISA detection. ELISA detection statistics of prokaryotic expression of VHH antibodies, where each dot represents one clone and the ordinate is OD450 of sGN/OD450 of blank control, defined as positive if the ratio is greater than 5.0. . Statistical chart of experiments in which positive VHH antibodies neutralize SFTSV virus infection, one dot represents one clone and the Y-axis is the relative percentage inhibition against different viruses. ELISA detection of binding of different purified concentrations of SNB and sGN protein is shown, different colors represent different clone numbers. Photographs of fluorescent staining obtained by viral infection in the presence of different concentrations of antibody, where fluorescent dots represent SFTSV virus, virus-uninfected cells were used as a No infection control, and antibodies were added. Unspiked virus-infected cells are used as an infection control. Figure 8 shows the inhibition rate of antibodies against SFTSV virus obtained based on the fluorescent dots statistically shown in Figure 7; Statistical chart of the relative inhibition rate of different mice after challenge, relative inhibition rate is viral load on day 1-9/viral load on day 6 with human immunoglobulin (Hu-IgG) as control. use. Statistical plot of sGN protein detected by SNB double antibody sandwich ELISA, where SNB01 (A), SNB02 (B) and SNB37 (C) were used as detection antibodies, D is the coating antibody SNB01, respectively; OD450 statistical curves of different concentrations of sGN detected by double antibody sandwich ELISA with detection antibody SNB37 are shown. Statistical curve of OD450 of different concentrations of SFTSV virus detected in double antibody sandwich ELISA with coating antibody SNB01, detection antibody SNB37.

1. Preparation of Immunogen Based on the GN protein sequence and gene sequence information of HB29 SFTSV on the NCBI website, the peptide sGN, which can effectively induce the production of specific antibodies against the GN protein by camels, was analyzed and designed, and His-tag (sGN-his) or rabbit Fc (sGN-rFc) were attached to the C-terminus for subsequent purification and detection.

2. Camelid immunization and antiserum acquisition Bactrian camels were first immunized with an emulsified mixture of 250 μg of sGN-rFc protein and 250 μl of Freund's complete adjuvant. Boosted 3 times with Freund's incomplete adjuvant, one week after the second and third immunizations, blood was collected to detect the antiserum titer, and one week after the fourth immunization, construction of a phage antibody library was performed. 200 ml of blood was collected for the purpose.

Antiserum titers were detected by ELISA, GN protein at a concentration of 0.5 μg/ml was used to coat detection plates, and 100 μl of gradient diluted antisera or purified antibodies were added to each well (preimmune camels). serum was used as a control), incubated at 37° C. for 1.5 h, washed twice, and horseradish peroxidase-labeled Goat anti-Llamma IgG (H+L) secondary antibody diluted 1:10000 was added to each well. Add 100 μl of TMB substrate, incubate at 37° C. for 10 min, stop the reaction with 50 μl 0.2 M H ₂ SO ₄ and measure OD 450 nm. did. Serum titers detected by ELISA were defined as the highest dilution at which the OD450 was greater than or equal to 2.1 times the blank control and greater than 0.2.

The results are shown in Figure 1 and the antiserum titers for the 3rd and 4th immunizations were 2.19 x ¹⁰⁶ and 4.61 x ¹⁰⁶ respectively. As can be seen, the antigen was able to induce the generation of specific high titer antisera against the GN protein by camels.

To further verify whether the high titer camelid antiserum can effectively inhibit SFTSV virus infection, a virus infection neutralization experiment was performed. Different dilutions of antisera and preimmune sera were each incubated with SFTSV virus for 60 min, then transferred to Vero cells, and after 48 h SFTSV infection was determined by cellular immunofluorescent staining for sGN protein. The results of the neutralization experiments showed that the ID90 at which sGN-induced antisera inhibited 90% of SFTSV infection was greater than 540-fold dilution (Fig. 2). Thus, sGN induced high titer antisera, and the antisera had the ability to effectively inhibit SFTSV virus infection.

3. Construction of VHH phage library and panning 200 ml of camel peripheral blood after immunization was collected, and camel PBMC were obtained by separating with lymphocyte separation liquid (GE Ficoll-Paque Plus). RNA was extracted and converted back to cDNA using oligo(dT), and by techniques such as primer amplification and molecular cloning, the camelid VHH gene was cloned into a phagemid plasmid, transformed into TG1 bacteria, and a VHH phage library was prepared. got To further confirm whether the construction of the sGN-VHH phage library was successful, PCR amplification of the VHH target gene from sGN-immunized camels resulted in a target band of 500 bp, consistent with the expected size ( FIG. 3), indicating that the sGN-VHH phage antibody library contains VHH genes. Fifty clones were selected and sequenced, the sequencing results showed that the sequenced sequences did not have exactly the same repeats, and the comparison showed that the differential sequences were mostly in the CDR binding regions. indicated to be located. As a result of detection, the capacity of the constructed sGN-VHH phage antibody library was 2.0×10 ⁹ , the positive rate was 100%, the sequence diversity was 100%, and the frame rate ( In frame rate) was greater than 95%.

With the help of M13KO7 helper phage, VHH-phagemid transformed bacteria were used to revive the phage antibody library and precipitated with PEG/NaCl. The phage antibody library was enriched three times with 50 μg/ml coated sGN-His protein. Enriched phage were eluted, transformed, plate-coated, monoclonals were selected for phage and sGN protein ELISA binding assays, clones with binding values >1.0 were sequenced, and the expression vector pVAX1 was used. and transfected into 293F cells to express and generate nanomonoclonal antibodies.

Binding detection between the panned library and the GN protein was performed. Phage ELISA results showed a binding value of 0.57 between the sGN-VHH phage library and the sGN protein before enrichment, and a phage library value of 0.98 after the first, second and third enrichments, respectively. , 2.2, 3.0 (Fig. 4A). To further validate the proportion of positive phage that bound to sGN-VHH proteins in the enriched library, 96 clones were selected from each of the second and third enrichment libraries and subjected to single Phage ELISA detection was performed. The results showed 24.5% single phage clones positive in the second round library, 67% positive phage clones in the third round library, and an average binding value of about 3 0 (FIG. 4B), indicating that panning of sGN proteins successfully enriched the sGN-VHH phage library with high binding capacity.

4. Construction of Prokaryotic Expression Library of VHHs and Expression of VHHs The 2nd-sGN-VHH and 3rd-sGN-VHH phage antibody libraries after the second and third rounds of panning enrichment were PCR amplified and all The VHH gene fragment is obtained and purified, the VHH gene fragment is cloned into a prokaryotic expression vector, the SS320 strain is transformed to construct a VHH prokaryotic expression antibody library, and a prokaryotic expression antibody library is constructed. was coated on a plate, cultured overnight, and the next day, 1000 monoclonal colonies were randomly selected, induced with IPTG to express antibody supernatants, and subjected to ELISA binding detection of antibody supernatants and sGN protein. gone.

The results showed that the bacterial supernatant bound sGN protein but not the blank control, with an sGN binding value/blank control value greater than 5.0 (Figure 5 and Table 1). These sequences were sequenced and compared to remove repetitive sequences and finally 142 VHH antibody sequences (SEQ ID NO: 1-142) were obtained. Further experiments demonstrated that these 142 VHH antibodies and the CDRs derived from the VHH antibodies were all capable of binding specifically to the SFTSV virus.

Table 1 Binding values of 142 VHH antibodies and sGN proteins and their sequences

To further verify whether the sequences can successfully inhibit SFTSV virus infection, bacterial supernatants of 122 VHH antibodies were incubated with SFTSV virus after treatment to determine whether the antibodies were able to inhibit SFTSV virus infection. tested. Results of neutralization experiments showed that 23 antibodies were able to achieve an inhibitory effect of 50% or more (Figure 6). Antibody numbers are respectively G77, G78, G79, G87, G95, G103, G109, G111, G112, G113, G114, G116, G117.1, G121, G124, G125, G126, G131, G133, G145, G170 and G171 Met.

5. Eukaryotic expression of VHH-huFc (SNB) By molecular cloning technology, the above 23 nanomonoclonal antibody VHH genes were fused with human Fc genes and inserted into the pVAX1 eukaryotic expression vector for Nb-huFc-pVAX1 expression. A plasmid was constructed and formed. The constructed Nb-huFc-pVAX1 was transfected into 293F cells to express and produce Nb-huFc (SNB) and purified using Protein G. Purified VHH-huFc1 (SNB) antibodies were collected and subjected to ELISA measurements, showing that some antibodies had very good binding capacity (Fig. 7). The corresponding SEQ ID NOs of the constructed humanized antibodies are shown in Table 2.

Table 2 Corresponding original antibody numbers and CDR sequences of humanized antibody SNB

4000 RU of sGN protein was coupled to the CM5 chip based on the Amino Coupling Kit manual via a BIAcore x100 instrument, and the coupled chip was sealed with ethanolamine. Antibodies were gradient diluted to each concentration. The detection program was set up with Bioevaluation version 4.0 software. The concentration of antibody was detected from low to high concentration, each concentration was detected twice, binding time was set to 180 seconds, flow rate was set to 30 μl/min, dissociation time was set to 180 seconds, flow rate was set to 30 μl/min. set, pH 2.5 10 mM glycine, set flow rate to 30 μl/min, time is 30 seconds, activate surface of chip, regenerate, balance with PBS for 5 seconds, flow rate is 30 μl /min. Experimental data were analyzed to obtain association, dissociation and affinity constants. The results are shown in Table 3, the affinity of most antibodies can reach 10 ⁻⁹ (Nano mole level), among which two antibodies SNB02 and SNB07 can reach 10 ⁻¹⁰ (Pico mole level). rice field. As can be seen, high affinity VHH-huFc1 (SNB) antibodies were obtained.

注：ｋａは結合定数であり、ｋｄは解離定数であり、ＫＤは親和力である。 Table 3 Summary of SNB affinity

Note: ka is the association constant, kd is the dissociation constant and KD is the affinity.

6. Neutralization of SNB in SFTSV infection of Vero cells SNB02 and SNB16 in VHH-huFc1 were selected for in vitro neutralization experiments. Antibodies were gradient diluted to different concentrations and incubated with SFTSV virus for 1 hour at 37°C with 5% CO ₂ , 1.5 x 10 ⁴ Vero cells were added and incubated at 37°C with 5% CO ₂ . , after 48 hours of culture in an incubator, remove the cell supernatant, add 4% paraformaldehyde to fix for 15 min, wash and seal, and anti-GN rabbit polyclonal serum (diluted to 1:1000). was added, left overnight at 4° C., washed with PBST, and added with 50 μl of anti-rabbit secondary antibody (Alexa Fluor 488 Anti-Rabbit IgG (H+L), Code: 111-545-144, Jackson ImmunoResearch Laboratories, 1:1000) was added, incubated at room temperature for 40 min, then stained with DAPI for 10 min, washed, photographed and observed under a fluorescence microscope, and the green spots and fluorescence intensity were statistically analyzed to determine neutralization inhibition. Inhibition rate = [1 - (average fluorescence intensity of sample group - average fluorescence intensity of cell control group CC) / (average fluorescence intensity of control treatment group - cells mean fluorescence intensity of control group CC)]×100%. Neutralization titers (ID ₅₀ or ND ₅₀ ) were expressed as fold dilutions where inhibition is 50%.

The results are shown in Figures 8 and 9, SNB02 had very good neutralizing activity and the inhibition rate could reach 83.5% when the antibody concentration was 9 μg/ml. The effects of SNB02 were evaluated in a humanized mouse model.

7. Treatment of SFTS In Vivo Infection with SNB ImmunodeficientNOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NCG) mice were purchased from the Nanjing University Model Animal Institute and are similar to NSG mice, which lack the IL2 receptor gene on SCID mice and therefore generate murine T, B and SzJ cells in vivo. There are very few NK cells. 1.0-15×10 ⁷ PBMCs were injected intraperitoneally into 4-6 week old NCG mice and 3 weeks later bled and stained for human CD45 ⁺ , CD3 ⁺ , CD4 ⁺ and CD8 ⁺ . , detected human T cells. When the ratio of human CD45-positive cells reached 5% or more, it was judged that the humanization of the mouse was successful. 2×10 ⁷ TCID50 of SFTSV virus was inoculated, and antibody treatment was given once on days 3 and 6 after infection, i.e., 400 μg of SNB02 antibody/mouse (the amount of antibody was approximately 20 mg/mouse). kg mouse) were injected intraperitoneally. Blood was collected before each antibody treatment and on the 9th day to detect the viral load in the blood of mice, thereby whether mice are normally infected with SFTS virus and SNB antibody can control SFTSV infection in mice. human IgG was used as a control. Results are shown in FIG. 11, where 3 days after completing treatment (day 9), the in vivo viral load of mice was detected and the in vivo viral load in 7 of the 9 mice receiving SNB02 treatment. Both doses were very well inhibited, the control group of mice receiving Hu-IgG treatment grew large amounts of SFTS virus in vivo, and a comparative analysis of the virus inhibition rates of the two groups showed that SNB02 It showed that SFTS virus infection could be significantly controlled (Fig. 10).

8. In vivo experiments with SNB02 carried in AAV viral vectors Adeno-associated viral vectors (AAV) are derived from non-pathogenic wild-type adeno-associated virus, have high safety and broad host cell range (dividing and non-dividing cells). cells), has characteristics such as low immunogenicity and long time to express foreign genes in vivo, it is regarded as one of the most promising gene transfer vectors, and is widely used in gene therapy and vaccine research worldwide. Widely applied.

AAV Helper-Free virus packaging system was purchased from Cell Biolabs, San Diego USA. Molecular cloning techniques were used to insert the above SNB02 DNA coding sequence into the pAAV-MCS plasmid, and after verification of successful construction by sequencing, the constructed plasmid pAAV-Ab was transformed into the pHelper and pAAV-DJ plasmids, 1: AAV-293T cells were co-transfected with PEI transfection reagent at a mass ratio of 1:1. Supernatants were collected at 48, 72, 96 and 120 hours respectively after transfection, concentrated with 5xPEG8000 (sigma) and finally purified with 1.37 g/ml cesium chloride. Purified AAV was dissolved in PBS, assayed, aliquoted and stored at -80°C.

The above humanized mice were inoculated with 2×10 ⁷ TCID50 of SFTSV virus, bled on day 3 post-infection and injected intramuscularly with AAV-SNB02 (1×10 ¹¹ gc/100 μl) for 6 days. Blood was collected on day 9, day 9 and day 12 to detect viral load and AAV-GFP was used as a control group. The results showed that the in vivo SFTSV viral load in the AAV-SNB02-injected mice were all very low, while the in vivo SFTSV viral load in the control mice was abundant.

9. Double antibody sandwich ELISA
Each concentration of SNB antibody was used to coat the detection plate, 100 μl each well, incubated for 2 h at 37°C, washed 2-4 times, sealed with 10% calf serum, 200 μl each well, 37°C. 1 h, washed 2-4 times, gradient diluted protein added to each well, 100 μl of virus or sample incubated at 37° C. for 1.5 h, washed 2 times, 1:200-1:10000 100 μl of horseradish peroxidase-labeled SNB antibody diluted to 100 μl was added to each well, incubated at 37° C. for 1 h, washed 4-6 times, then added with 100 μl of TMB substrate, incubated at 37° C. for 10 min, and incubated with 50 μl. The reaction was stopped with 2M _H2SO4 and _OD450nm was measured. A positive sample detected by ELISA has an OD450 greater than or equal to 2.1 times that of the blank control (i.e., the uncoated analyte, marked by Neg in the figure) and an optical density value greater than or equal to 0.2. It was defined as the highest dilution factor when greater.

The results showed that when the coating antibody was SNB02 or SNB07 and the detection antibody was SNB01, the combination was able to distinguish between the sGN protein and the negative control protein (Fig. 11A), and when the detection antibody was SNB02, the ELISA assay back The ELISA was shown to be able to distinguish between the sGN protein and the negative control protein when the ground was very high (Fig. 11B), the detection antibody was SNB37, and the coating antibodies were SNB01, SNB02 and SNB07 (Fig. 11C). The SNB antibody was applicable to the development of a double antibody sandwich ELISA detection sGN kit, and the detection sensitivity of the double antibody combination of SNB01 and SNB37 was 3.9 ng/ml (Fig. 11D). As a result of measurement, the sensitivity of the combination to detect SFTSV bona fide virus was 3.75×10 ⁶ gc/ml (FIG. 12). SFTSV bona fide virus could also be detected. As can be seen, the nano-antibody double antibody sandwich detection kit was applicable to detect SFTSV virus infection.

The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the present invention. should also be included in the protection scope of the present invention.

Claims (10)

An antibody capable of binding to SFTSV, comprising complementarity determining regions CDR1, CDR2 and CDR3, wherein each of CDR1, CDR2 and CDR3 has a sequence shown in the table below.

2. The antibody of claim 1, further comprising four frame regions FRl, FR2, FR3 and FR4 that intersect said CDRl, CDR2 and CDR3 in order. 3. The antibody of claim 2, which is a monoclonal antibody. 3. The antibody of claim 2, which is a VHH. 5. The antibody according to claim 4, which is a camel-derived VHH or a humanized VHH. Use of the antibody according to any one of claims 1-5 in the manufacture of an SFTSV detection agent or SFTSV therapeutic drug. A polynucleotide encoding the antibody of any one of claims 1-5. Use of the polynucleotide of claim 7 in the manufacture of a SFTSV therapeutic drug. A detection antibody, a solid matrix, and a coating antibody coated on the solid matrix, wherein the detection antibody and the coating antibody are each one of the antibodies according to any one of claims 1 to 5. An SFTSV detection kit characterized by: The CDR sequences of the detection antibody are
The sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 84, and the sequence of CDR3 is SEQ ID NO: 181, or the sequence of CDR1 is SEQ ID NO: 54, the sequence of CDR2 is SEQ ID NO: 132 and the sequence of CDR3 is SEQ ID NO: 176, or the sequence of CDR1 is SEQ ID NO: 13 and the sequence of CDR2 is SEQ ID NO: 99; 10. The kit of claim 9, wherein the sequence of CDR3 is SEQ ID NO:166.

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