WO2021012195A1 - 可结合sftsv的纳米抗体及其应用 - Google Patents

可结合sftsv的纳米抗体及其应用 Download PDF

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WO2021012195A1
WO2021012195A1 PCT/CN2019/097350 CN2019097350W WO2021012195A1 WO 2021012195 A1 WO2021012195 A1 WO 2021012195A1 CN 2019097350 W CN2019097350 W CN 2019097350W WO 2021012195 A1 WO2021012195 A1 WO 2021012195A1
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sequence
seq
antibody
cdr1
sftsv
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PCT/CN2019/097350
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English (en)
French (fr)
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吴喜林
吴稚伟
李彦磊
潘逸
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源道隆（苏州）医学科技有限公司
盼展生物技术公司
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Priority to PCT/CN2019/097350 priority Critical patent/WO2021012195A1/zh
Priority to JP2020538725A priority patent/JP7171737B2/ja
Priority to CN201980001738.0A priority patent/CN112105637B/zh
Priority to KR1020207022152A priority patent/KR102504884B1/ko
Priority to CN201910885757.6A priority patent/CN110684102B/zh
Priority to CN201910887917.0A priority patent/CN110713536B/zh
Publication of WO2021012195A1 publication Critical patent/WO2021012195A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses

Definitions

  • the invention relates to the field of biomedicine. More particularly, it relates to a polypeptide that can bind to SFTSV, the application of the polypeptide in the preparation of SFTSV detection agents or SFTSV therapeutic drugs, and a SFTSV detection kit.
  • Severe fever with thrombocytopenia syndrome is an acute infectious disease caused by a new type of Bunia virus (Severe fever with thrombocytopenia syndrome virus, SFTSV), mainly through tick bites or the blood of acute patients , Mucosal contact transmission, clinical manifestations are mainly fever, thrombocytopenia, leukopenia, and abnormal function of multiple organs including the gastrointestinal tract, liver, and kidneys. Since SFTS was first reported in Hubei and Henan in 2009, 16 provinces in my country have issued case reports.
  • the known anti-GN SFTS neutralizing antibody is Mab4-5 obtained through the screening of phage display SFTS patient antibody library in 2013, and its IC50 is 2 ⁇ g/ml-44.2 ⁇ g/ml.
  • the invention obtains camel-derived nano monoclonal antibodies and their VHH by immunizing camels with antigens, and is used for diagnosing and treating acutely infected SFTS patients. Based on these studies, the present invention provides a polypeptide that can bind to SFTSV, including three complementarity determining regions CDR1-3, CDR1 sequence is or includes one of the sequences shown in SEQ ID NO: 1-74, and CDR2 sequence is or includes SEQ One of the sequences shown in ID NO: 75-151, and the CDR3 sequence is or includes one of the sequences shown in SEQ ID NO: 152-232.
  • the polypeptide further includes four framework regions FR1-4, which are arranged staggered with the CDR1-3.
  • the FR1-4 sequence can be designed as shown in SEQ ID NO: 235-237, but the scope of the present invention is not limited to this.
  • the specific recognition and binding ability of an antibody is mainly determined by the sequence of the CDR region.
  • the FR sequence has little effect and can be designed according to the species, which is well known in the art.
  • a human, murine, or camel-derived FR region sequence can be designed to link the above-mentioned CDRs, thereby a polypeptide or domain that can bind to SFTSV.
  • the polypeptide is a monoclonal antibody.
  • polypeptide is VHH.
  • the polypeptide is a camel-derived VHH or a humanized VHH.
  • the CDR sequence of the polypeptide is as follows:
  • 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.
  • the CDR sequence of the polypeptide is as follows:
  • the sequence of CDR1 is XXXSTAYY (SEQ ID NO: 233), where X is any amino acid, for example, the sequence of CDR1 is selected from SEQ ID NO: 4, 25, 26, 62, 73; and
  • the sequence of CDR2 is selected from SEQ ID NO: 78, 80, 84, 90, 103;
  • the sequence of CDR3 is selected from SEQ ID NO: 155, 157, 161, 169, 172, 179, 180, 181, 210, 218, 229, 230, 231.
  • the CDR sequence of the polypeptide is 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, 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, 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, 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, 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, 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, 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: 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: 25
  • the sequence of CDR2 is SEQ ID NO: 103
  • the sequence of CDR3 is SEQ ID NO: 179; or
  • the sequence of CDR1 is SEQ ID NO: 26
  • the sequence of CDR2 is SEQ ID NO: 80
  • the sequence of CDR3 is SEQ ID NO: 87; or
  • the sequence of CDR1 is SEQ ID NO: 26
  • the sequence of CDR2 is SEQ ID NO: 84
  • 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
  • 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
  • the sequence of CDR3 is SEQ ID NO: 157.
  • the CDR sequence of the polypeptide is as follows:
  • CDR2 is SEQ ID NO: 79
  • sequence of CDR3 is SEQ ID NO: 156; or
  • the sequence of CDR2 is SEQ ID NO: 79
  • the sequence of CDR3 is SEQ ID NO: 157.
  • the CDR sequence of the polypeptide is as follows:
  • CDR3 is SEQ ID NO: 158, 213 or 228.
  • the CDR sequence of the polypeptide is as follows:
  • CDR1 is SEQ ID NO: 24, and the sequence of CDR2 is SEQ ID NO: 102;
  • CDR3 is SEQ ID NO: 178, 189 or 225.
  • the CDR sequence of the polypeptide is as follows:
  • the sequence of CDR3 is SEQ ID NO: 170;
  • CDR1 is SEQ ID NO: 66
  • 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.
  • the CDR sequence of the polypeptide is 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
  • 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: 99
  • 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
  • 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
  • the sequence of CDR2 is SEQ ID NO: 95
  • the sequence of CDR3 is SEQ ID NO: 173.
  • the CDR sequence of the polypeptide is as follows:
  • the sequence of CDR1 is SEQ ID NO: 47
  • the sequence of CDR2 is SEQ ID NO: 125
  • 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
  • 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: 138
  • the sequence of CDR3 is SEQ ID NO: 216.
  • the invention also provides the application of the above-mentioned polypeptides in the preparation of SFTSV detection agents or SFTSV therapeutic drugs.
  • the present invention also provides the nucleic acid coding sequence of the above-mentioned polypeptide.
  • the nucleic acid coding sequence is a DNA coding sequence or an RNA coding sequence.
  • the nucleic acid coding sequence is present in a gene expression frame.
  • the present invention also provides an expression vector containing the expression cassette of the aforementioned nucleic acid coding sequence.
  • the expression vector is a viral vector.
  • the expression vector is an adeno-associated virus expression vector (AAV vector).
  • AAV vector adeno-associated virus expression vector
  • the invention also provides the application of the above-mentioned nucleic acid coding sequence and expression vector in SFTSV therapeutic drugs.
  • the present invention also provides a SFTSV detection kit, comprising a detection antibody, a solid substrate, and a coating antibody coated on the solid substrate.
  • the detection antibody and the coating antibody are respectively one of the above polypeptides .
  • the CDR sequence of the detection antibody is 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
  • the sequence of CDR3 is SEQ ID NO: 176; or
  • the sequence of CDR1 is SEQ ID NO: 13
  • the sequence of CDR2 is SEQ ID NO: 99
  • the sequence of CDR3 is SEQ ID NO: 166.
  • the CDR sequence of the coated antibody is 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
  • the sequence of CDR3 is SEQ ID NO: 176; or
  • the sequence of CDR1 is SEQ ID NO: 6, 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
  • the sequence of CDR3 is SEQ ID NO: 166.
  • the CDR sequence of the detection antibody is 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;
  • the CDR sequence of the coated antibody is 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, 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 sequence of the detection antibody CDR1 is SEQ ID NO: 4
  • the sequence of CDR2 is SEQ ID NO: 84
  • the sequence of CDR3 is SEQ ID NO: 181;
  • the CDR sequence of the coated antibody is 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 of CDR2 is SEQ ID NO: 99, and the sequence of CDR3 is SEQ ID NO: 166.
  • the sequence of the detection antibody CDR1 is SEQ ID NO: 13
  • the sequence of CDR2 is SEQ ID NO: 99
  • the sequence of CDR3 is SEQ ID NO: 166;
  • the CDR sequence of the coated antibody is 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 and 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, 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 is aimed at SFTS with high fatality rate but lack of effective vaccines and specific antiviral drugs for nano antibody drug development and diagnostic kit development, through the preparation of GN protein, immunization of Bactrian camels, the use of phage library to display nano monoclonal antibody platform technology, etc.
  • the present invention provides potential new nano antibody drugs for the clinical treatment of SFTS, and at the same time provides corresponding detection kits for the diagnosis of SFTS.
  • Figure 1 is the detection curve of antiserum titer one week after the third and fourth immunization of camels with sGN;
  • Figure 2 shows the curves of antisera from different dilutions of camels one week after the fourth immunization against SFTSV virus infection of Vero cells in vitro, with the serum before immunization as a control;
  • Figure 3 is an electrophoresis diagram of PCR products amplified by the sGN-VHH phage antibody library as a template
  • Figure 4 shows the panning and identification of the sGN-VHH phage antibody library, where A is the ELISA detection statistics of the phage library for sGN protein after panning; B is the panning from the second round (2 nd ) and the third round (3 rd ) The selected phage antibody library selects 96 clones each for phage ELISA detection statistics;
  • Figure 5 is a statistical chart of ELISA detection of VHH antibodies expressed in prokaryotic cells. Each point represents a clone. The ordinate is OD450 for sGN/OD450 of blank control. A ratio greater than 5.0 is defined as positive;
  • Figure 6 is a statistical diagram of the positive VHH antibody neutralization SFTSV virus infection experiment, a dot represents a clone, and the Y axis is the relative inhibition rate against different viruses;
  • Figure 7 shows the ELISA to detect the binding of SNB and sGN protein at different purified concentrations. Different colors represent different clone numbers.
  • FIG. 8 shows the fluorescent staining photos obtained by virus infection in the presence of different concentrations of antibodies.
  • the fluorescent dots represent the SFTSV virus. Cells without virus infection are used as No infection control, and virus infected cells without antibodies are Infection control;
  • Fig. 9 is a graph showing the inhibition rate of antibodies against SFTSV virus obtained from the fluorescence points of Fig. 7;
  • Figure 10 is a statistical graph of the relative inhibition rate of different mice after challenge.
  • the relative inhibition rate is 1 to the viral load on day 9/viral load on day 6, with human immunoglobulin as the control (Hu-IgG);
  • Figure 10 is the SNB double antibody sandwich ELISA to detect sGN protein statistics, in which, SNB01 (A), SNB02 (B) and SNB37 (C) are used as the detection antibody; D is the coating antibody SNB01, the detection antibody SNB37 double antibody sandwich ELISA detects the OD450 statistical curve of different concentrations of sGN.
  • Figure 11 shows the OD450 statistical curve of the double-antibody sandwich ELISA coated with antibody SNB01 and detection antibody SNB37 to detect different concentrations of SFTSV virus.
  • the emulsified mixture of 250 ⁇ g sGN-rFc protein and 250 ⁇ l Freund's complete adjuvant was used to immunize Bactrian camels. On the 14, 28 and 42 days, sGN-rFc protein and 250 ⁇ l Freund's incomplete adjuvant were used to boost the immunization 3
  • blood was collected to detect the antiserum titer; one week after the fourth immunization, 200ml of blood was collected for the construction of the phage antibody library.
  • Antiserum titer was detected by ELISA, the test plate was coated with GN protein at a concentration of 0.5 ⁇ g/ml, 100 ⁇ l of serially diluted antiserum or purified antibody was added to each well (control is camel serum before immunization), and incubated at 37°C for 1.5h , Wash twice, add 1:10000 diluted horseradish peroxidase labeled Goat anti-Llamma IgG (H+L) secondary antibody to each well, incubate at 37°C for 1h, wash 4-6 times, add 100 ⁇ l TMB bottom Incubate at 37°C for 10 minutes, stop the reaction with 50 ⁇ l 0.2M H 2 SO 4 , and measure OD 450nm.
  • the ELISA test serum titer is defined as the highest dilution factor when the OD450 is 2.1 times or more of the blank control and greater than 0.2.
  • a neutralization experiment of virus infection was performed.
  • the antiserum and pre-immune serum of different dilutions were incubated with SFTSV virus for 60 minutes, and then transferred to Vero cells. After 48 hours, the sGN protein was stained by immunofluorescence to determine the SFTSV infection.
  • the results of the neutralization experiment showed that the ID90 of the sGN-induced antiserum inhibited 90% of SFTSV infection at a 540-fold dilution ( Figure 2).
  • sGN induced a high-titer antiserum, and the antiserum has the ability to effectively inhibit SFTSV virus infection.
  • the target band is 500bp and the size is in line with expectations ( Figure 3), indicating that the sGN-VHH phage antibody library contains Contains VHH gene. Fifty clones were selected for sequencing. The sequencing results showed that the tested sequences did not have identical repeating sequences; the comparison results showed that most of the different sequences were in the CDR binding region. After testing, the constructed sGN-VHH phage antibody library has a capacity of 2.0 ⁇ 10 9 , a positive rate of 100%, a sequence diversity of 100%, and an effective insertion rate (In frame rate) of greater than 95%.
  • the bacteria transformed with VHH-phagemid were recovered from the phage antibody library and precipitated with PEG/NaCl.
  • the phage antibody library was enriched three times with sGN-His protein coated with 50 ⁇ g/ml.
  • the enriched phage was eluted, transformed, plated, and a single clone was selected for the ELISA binding identification of phage and sGN protein.
  • the clone with a binding reading value> 1.0 was sequenced and cloned into the expression vector pVAX1 and transfected into 293F cells Express the production of Nano monoclonal antibodies.
  • the library after panning was tested for binding to GN protein.
  • the results of phage ELISA showed that the binding reading of the sGN-VHH phage library and sGN protein before enrichment was 0.57, and the readings of the phage library after one, two and three rounds of enrichment were 0.98, 2.2, 3.0 ( Figure 4A).
  • 96 clones were selected from the second and third rounds of the enriched library for single phage ELISA detection.
  • the bacterial supernatant of 122 VHH antibodies was processed and incubated with SFTSV virus to test whether the antibodies can inhibit SFTSV virus infection.
  • the results of the neutralization experiment ( Figure 6) showed that 23 antibodies can achieve an inhibitory effect of more than 50%.
  • the antibody numbers are G77, G78, G79, G87, G95, G103, G109, G111, G112, G113, G114, G116, G117.1, G121, G124, G125, G126, G131, G133, G145, G170 and G171.
  • the above 23 nano monoclonal antibody VHH genes were fused with human Fc genes 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, expressed and produced Nb-huFc (SNB), and purified by ProteinG.
  • the purified VHH-huFc1 (SNB) antibody was collected and tested by ELISA. Some of the antibodies have good binding ability (Figure 7).
  • the corresponding sequence numbers of the constructed humanized antibodies are shown in Table 2.
  • Bioevaluation version 4.0 software sets the detection program: antibody concentration is detected from low concentration to high concentration, and each concentration is tested twice; the binding time is set to 180 seconds, the flow rate is 30 ⁇ l/min; the dissociation time is set to 180 seconds, and the flow rate is 30 ⁇ l/min; pH2.5 10mM glycine set the flow rate to 30 ⁇ l/min for 30 seconds to activate and regenerate the chip surface; PBS equilibrate for 5 seconds, and the flow rate is 30 ⁇ l/min.
  • ka is the binding constant
  • kd is the dissociation constant
  • KD is the affinity
  • SNB02 and SNB16 in VHH-huFc1 were selected for in vitro neutralization experiments.
  • the antibody was diluted to different concentrations and incubated with SFTSV virus at 5% CO 2 37°C for 1 hour. After adding 1.5 ⁇ 10 4 Vero cells and 5% CO 2 37°C incubator for 48 hours, Remove the cell supernatant, add 4% paraformaldehyde to fix for 15 minutes, after washing and blocking, add anti-GN rabbit polyclonal serum (1:1000 dilution), 4°C overnight, after washing with PBST, add 50 ⁇ l anti-rabbit bis Anti-(Alexa Fluor 488Anti-Rabbit IgG(H+L), Code:111-545-144, Jackson ImmunoResearch Laboratories, 1:1000 dilution) incubated for 40min at room temperature, then stained with DAPI for 10min.
  • inhibition rate [1-(average fluorescence intensity of sample group-average fluorescence intensity of cell control group CC)/(average fluorescence intensity of control treatment group-cell The average value of the fluorescence intensity of the control group CC)] ⁇ 100%.
  • the neutralization titer (ID 50 or ND 50 ) is expressed as the multiple of the dilution at 50% inhibition rate.
  • SNB02 has a good neutralizing activity. When the antibody concentration is 9 ⁇ g/ml, the inhibition rate can reach 83.5%.
  • a humanized mouse model was used to evaluate the efficacy of SNB02.
  • ImmunodeficientNOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NCG) mice were purchased from the Model Institute of Animal Science of Nanjing University. Similar to NSG mice, the mice lack the IL2 receptor gene on the basis of SCID mice, resulting in no mouse T cells in the body. B cells and a very small amount of NK cells. 1.0-15x 10 7 PBMC were injected intraperitoneally into NCG mice at 4-6 weeks; after three weeks, blood was collected to detect human T cells by flow cytometry, and human CD45 + , CD3 + , CD4 + and CD8 + were stained. The proportion of human CD45 positive cells reached 5% or more, and it was judged that the mouse humanization was successful.
  • Adeno-associated virus vector (AAV) is derived from non-pathogenic wild-type adeno-associated virus. Due to its safety, wide range of host cells (dividing and non-dividing cells), low immunogenicity, it takes a long time to express foreign genes in vivo It is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide.
  • AAV Helper-Free virus packaging system was purchased from Cell Biolabs, San Diego USA. Insert the above-mentioned DNA coding sequence of SNB02 into the pAAV-MCS plasmid by molecular cloning technology; after the successful construction is proved by sequencing, the constructed plasmid pAAV-Ab and pHelper and pAAV-DJ plasmids are in a 1:1:1 mass ratio AAV-293T cells were co-transfected with PEI transfection reagent. After transfection, the supernatant was collected at 48, 72, 96 and 120 hours, and concentrated with 5xPEG8000 (sigma), and finally purified with 1.37g/ml cesium chloride. The purified AAV was dissolved in PBS and stored at -80°C after identification and aliquots.
  • mice were inoculated with 2x 10 7 TCID50 SFTSV virus, blood was collected on the 3rd day after infection and AAV-SNB02 (1x10 11 gc/100 ⁇ l) intramuscular injection, and blood was collected on 6, 9 and 12 days to detect the virus Load, with AAV-GFP as the control group.
  • the results showed that the SFTSV viral load in the mice injected with AAV-SNB02 was very low, while the SFTSV virus in the control group multiplied in large numbers.
  • the SNB antibody can be applied to the development of a double-antibody sandwich ELISA to detect sGN kits, where the detection sensitivity of the double antibody combination of SNB01 and SNB37 is 3.9ng/ml ( Figure 11D).
  • the sensitivity of the combination to detect SFTSV true virus was 3.75x 10 6 gc/ml ( Figure 12), indicating that the kit can not only detect SFTSV true virus, but also detect SFTSV true virus with a copy number as low as 10 ⁇ 6 . It can be seen that the Nanobody Double Antibody Sandwich Detection Kit can be applied to the detection of SFTSV virus infection.

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Abstract

一种可结合SFTSV的多肽，包括3个互补决定区CDR1-3，CDR1序列为或包括SEQ ID NO:1-74所示序列之一，CDR2序列为或包括SEQ ID NO:75-151所示序列之一，CDR3序列为或包括SEQ ID NO:152-232所示序列之一。通过制备GN蛋白、免疫双峰骆驼、利用噬菌体库展示纳米单抗的平台技术等，筛选到特异性结合GN的纳米抗体VHH，鉴定了其CDR序列，并构建了人源化抗体SNB；同时利用人源化小鼠模型体内评估SNB在治疗SFTSV感染的疗效。其为SFTS的临床治疗提供潜在的纳米抗体新药,同时为SFTS的诊断提供相应的检测试剂盒。

Description

可结合SFTSV的纳米抗体及其应用 技术领域
本发明涉及生物医药领域。更特别地，涉及一种可结合SFTSV的多肽，所述的多肽在制备SFTSV检测剂或SFTSV治疗药物中的应用，以及一种SFTSV检测试剂盒。
背景技术
发热伴血小板减少综合征(Severe fever with thrombocytopenia syndrome,SFTS)是一种由新型布尼亚病毒(Severe fever with thrombocytopenia syndrome virus，SFTSV)引起的急性传染病，主要通过蜱虫叮咬或急性病人的血液、黏膜接触传播，临床主要表现为发热、血小板减少、白细胞减少以及包括胃肠道、肝脏、肾脏在内的多器官功能异常。自2009年首次在湖北和河南报道SFTS以来，我国已有16个省份发布了病例报告，截止2014年的确诊病例数已超过3500例，且近年来发病率不断升高；同时，在韩国、日本、阿拉伯和美国均有SFTS病例报道，提示该病的流行区域正在扩大。目前报道的SFTS死亡率在6.3％-30％之间；而韩国和日本在2016年公布的死亡率分别高达35.4％和50％。然而，临床至今缺乏针对SFTS的特异性治疗药物，只能进行广谱抗病毒治疗和对症下药，这为SFTS的预防及控制带来极大困难。
特异性中和抗体在抗病毒治疗方面有着很好的临床疗效，如呼吸道合胞病毒单抗Palivizumab和狂犬病毒抗血清等。已有研究指出，针对SFTSV表面糖蛋白(Glycoprotein N，GN)的中和抗体对病人生存率起着重要作用。而我们前期的研究结果也显示，血清含抗GN抗体的SFTS病人均康复良好，而血清中不含GN抗体的SFTS病人其死亡率高达66.7％。这些研究提示，靶向GN的中和抗体治疗将是对抗SFTSV行之有效的方法。
1993年，一种来源于骆驼科的新型天然抗体被发现。该抗体天然缺失轻链而只由重链组成，其重链包含两个恒定区(CH2和CH3)、一个铰链区和一个重链可变区(Variable  heavy chain domain，VHH，即抗原结合位点)，该重链可变区的相对分子质量约为13KDa，仅为常规抗体的1/10，且分子高度和直径均在纳米级别，是目前可获得的最小的功能性抗体片段，因此又被称为纳米单抗(Nanobody，Nb)。因纳米单抗稳定性高(90℃条件下仍不会降解)、亲和力高、与人源抗体同源性超过80％、毒性和免疫原性均较低等特点，最近纳米单抗被广泛用于免疫诊断试剂盒研发、影像学研发以及针对肿瘤、炎症、传染病和神经系统疾病等领域的抗体药物研发。
目前已知抗GN的SFTS中和抗体是2013年通过噬菌体展示SFTS病人抗体库筛选得到的Mab4-5，其IC50为2μg/ml-44.2μg/ml。中和抗体的IC50越低越好，能极大节约抗体的使用量从而节省生产成本，同时也能延长抗体在体内维持有效浓度的时间。我们前期对SFTS病人血清的抗体效价做了检测，发现针对GN蛋白的抗血清仅为100左右的稀释倍数，这提示我们，以低抗体滴度的SFTS病人血清构建抗体库可能限制了高效中和抗体的筛选。因此我们期望通过新的技术手段，获得更为高效稳定的、IC50更低的SFTSV特异性中和抗体。
发明内容
本发明通过用抗原免疫骆驼，获取骆驼源纳米单抗及其VHH，用于诊断和治疗急性感染的SFTS病人。基于这些研究，本发明提供了一种可结合SFTSV的多肽，包括3个互补决定区CDR1-3，CDR1序列为或包括SEQ ID NO:1-74所示序列之一，CDR2序列为或包括SEQ ID NO:75-151所示序列之一，CDR3序列为或包括SEQ ID NO:152-232所示序列之一。
在一个具体实施方案中，所述多肽还包括4个框架区FR1-4，所述FR1-4与所述CDR1-3交错排列。例如，可将FR1-4序列设计为如SEQ ID NO:235-237所示，但本发明的范围不限于此。抗体的特异性识别和结合能力主要由CDR区序列决定，FR序列影响不大，可根据物种来设计，这是本领域公知的。例如，可设计人源、鼠源或骆驼源的FR区序列来连接上述CDR，从而一个可结合SFTSV的多肽或结构域。
在一个优选实施方案中，所述多肽为单克隆抗体。
在一个优选实施方案中，所述多肽为VHH。
在一个优选实施方案中，所述多肽为骆驼源的VHH或人源化的VHH。
在一个具体实施方案中，所述多肽的CDR序列如下：
I)CDR3的序列为SEQ ID NO:176；并且
II)CDR1的序列为SEQ ID NO:54并且CDR2的序列为SEQ ID NO:132；或者
CDR1的序列为SEQ ID NO:22并且CDR2的序列为SEQ ID NO:100。
在另一个具体实施方案中，所述多肽的CDR序列如下：
I)CDR1的序列为XXXSTAYY(SEQ ID NO:233)，其中X为任意氨基酸，例如CDR1的序列选自SEQ ID NO:4、25、26、62、73；并且
II)CDR2的序列选自SEQ ID NO:78、80、84、90、103；并且
III)CDR3的序列选自SEQ ID NO：155、157、161、169、172、179、180、181、210、218、229、230、231。
在一个具体实施方案中，所述多肽的CDR序列如下：
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:78，并且CDR3的序列为SEQ ID NO:155；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:78，并且CDR3的序列为SEQ ID NO:157；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:80，并且CDR3的序列为SEQ ID NO:157；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:157；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:169；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:172；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:180；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:181；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:210；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:229；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:230；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:231；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:90，并且CDR3的序列为SEQ ID NO:161；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:90，并且CDR3的序列为SEQ ID NO:179；或
CDR1的序列为SEQ ID NO:25，CDR2的序列为SEQ ID NO:103，并且CDR3的序列为SEQ ID NO:179；或
CDR1的序列为SEQ ID NO:26，CDR2的序列为SEQ ID NO:80，并且CDR3的序列为SEQ ID NO:87；或
CDR1的序列为SEQ ID NO:26，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:157；或
CDR1的序列为SEQ ID NO:62，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:218；或
CDR1的序列为SEQ ID NO:73，CDR2的序列为SEQ ID NO:80，并且CDR3的序列为SEQ  ID NO:157。
在另一个具体实施方案中，所述多肽的CDR序列如下：
I)CDR1的序列为SEQ ID NO:5；并且
II)CDR2的序列为SEQ ID NO:79，并且CDR3的序列为SEQ ID NO:156；或者
CDR2的序列为SEQ ID NO:79，并且CDR3的序列为SEQ ID NO:157。
在另一个具体实施方案中，所述多肽的CDR序列如下：
I)CDR1的序列为SEQ ID NO:6，CDR2的序列为SEQ ID NO:81；并且
II)CDR3的序列为SEQ ID NO:158、213或228。
在另一个具体实施方案中，所述多肽的CDR序列如下：
I)CDR1的序列为SEQ ID NO:24，CDR2的序列为SEQ ID NO:102；并且
II)CDR3的序列为SEQ ID NO:178、189或225。
在另一个具体实施方案中，所述多肽的CDR序列如下：
I)CDR3的序列为SEQ ID NO:170；并且
II)CDR1的序列为SEQ ID NO:66，并且CDR2的序列为SEQ ID NO:93；或者
CDR1的序列为SEQ ID NO:65，并且CDR2的序列为SEQ ID NO:143；或者
CDR1的序列为SEQ ID NO:16，并且CDR2的序列为SEQ ID NO:93；或者
CDR1的序列为SEQ ID NO:68，并且CDR2的序列为SEQ ID NO:93。
在另一个具体实施方案中，所述多肽的CDR序列如下：
CDR1的序列为SEQ ID NO:21，CDR2的序列为SEQ ID NO:98，并且CDR3的序列为SEQ ID NO:166；或
CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:89，并且CDR3的序列为SEQ ID NO:166；或
CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:99，并且CDR3的序列为SEQ ID NO:166；或
CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:106，并且CDR3的序列为 SEQ ID NO:166；或
CDR1的序列为SEQ ID NO:27，CDR2的序列为SEQ ID NO:104，并且CDR3的序列为SEQ ID NO:166；或
CDR1的序列为SEQ ID NO:18，CDR2的序列为SEQ ID NO:95，并且CDR3的序列为SEQ ID NO:173。
在另一个具体实施方案中，所述多肽的CDR序列如下：
CDR1的序列为SEQ ID NO:47，CDR2的序列为SEQ ID NO:125，并且CDR3的序列为SEQ ID NO:202；或
CDR1的序列为SEQ ID NO:60，CDR2的序列为SEQ ID NO:142，并且CDR3的序列为SEQ ID NO:202；或
CDR1的序列为SEQ ID NO:60，CDR2的序列为SEQ ID NO:138，并且CDR3的序列为SEQ ID NO:216。
本发明还提供了上述多肽在制备SFTSV检测剂或SFTSV治疗药物中的应用。
本发明还提供了上述多肽的核酸编码序列。
在一个实施方案中，所述核酸编码序列为DNA编码序列或RNA编码序列。
在一个具体实施方案中，所述核酸编码序列存在于基因表达框中。
本发明还提供了包含上述核酸编码序列的表达框的表达载体。
在一个优选实施方案中，所述表达载体为病毒载体。
在一个优选实施方案中，所述表达载体为腺相关病毒表达载体(AAV载体)。
本发明还提供了上述核酸编码序列和表达载体在SFTSV治疗药物中的应用。
本发明还提供了一种SFTSV检测试剂盒，包括检测抗体、固体基质以及包被于所述固体基质上的包被抗体，所述检测抗体和所述包被抗体分别为上述多肽中的一种。
在一个具体实施方案中，所述检测抗体的CDR序列如下：
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:181；或
CDR1的序列为SEQ ID NO:54，CDR2的序列为SEQ ID NO:132，并且CDR3的序列为SEQ ID NO:176；或
CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:99，并且CDR3的序列为SEQ ID NO:166。
在一个具体实施方案中，所述包被抗体的CDR序列如下：
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:181；或
CDR1的序列为SEQ ID NO:54，CDR2的序列为SEQ ID NO:132，并且CDR3的序列为SEQ ID NO:176；或
CDR1的序列为SEQ ID NO:6，CDR2的序列为SEQ ID NO:81，并且CDR3的序列为SEQ ID NO:158；或
CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:99，并且CDR3的序列为SEQ ID NO:166。
在一个优选实施方案中，
I)所述检测抗体的CDR序列如下：CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:181；或CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:99，并且CDR3的序列为SEQ ID NO:166；
并且，
II)所述包被抗体的CDR序列如下：CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:181；或CDR1的序列为SEQ ID NO:54，CDR2的序列为SEQ ID NO:132，并且CDR3的序列为SEQ ID NO:176；或CDR1的序列为SEQ ID NO:6，CDR2的序列为SEQ ID NO:81，并且CDR3的序列为SEQ ID NO:158；或CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:99，并且CDR3的序列为SEQ ID NO:166。
在一个优选实施方案中，
I)所述检测抗体CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且 CDR3的序列为SEQ ID NO:181；并且
II)所述包被抗体的CDR序列如下：CDR1的序列为SEQ ID NO:54，CDR2的序列为SEQ ID NO:132，并且CDR3的序列为SEQ ID NO:176；或CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:99，并且CDR3的序列为SEQ ID NO:166。
在另一个优选实施方案中，
I)所述检测抗体CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:99，并且CDR3的序列为SEQ ID NO:166；并且
所述包被抗体的CDR序列如下：
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:181；或CDR1的序列为SEQ ID NO:54，CDR2的序列为SEQ ID NO:132，并且CDR3的序列为SEQ ID NO:176；或CDR1的序列为SEQ ID NO:6，CDR2的序列为SEQ ID NO:81，并且CDR3的序列为SEQ ID NO:158；或CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:99，并且CDR3的序列为SEQ ID NO:166。
本发明针对高致死率但缺乏有效疫苗和特异性抗病毒药物的SFTS进行纳米抗体药物开发和诊断试剂盒研发，通过制备GN蛋白、免疫双峰骆驼、利用噬菌体库展示纳米单抗的平台技术等，筛选到特异性结合GN的纳米抗体VHH，鉴定了其CDR序列，并构建了人源化的VHH-huFc1(SNB)；同时利用人源化小鼠模型体内评估SNB在治疗SFTSV感染的疗效。本发明为SFTS的临床治疗提供潜在的纳米抗体新药,同时为SFTS的诊断提供相应的检测试剂盒。
附图说明
图1为sGN第3和4次免疫骆驼一周后的抗血清效价检测曲线；
图2为不同稀释度的第4次免疫骆驼一周后的抗血清抑制SFTSV病毒体外感染Vero细胞的曲线，以免疫前的血清为对照；
图3为sGN-VHH噬菌体抗体文库为模板扩增的PCR产物的电泳图；
图4为sGN-VHH噬菌体抗体文库的淘选鉴定，其中，A为噬菌体文库针对sGN蛋白淘选后ELISA检测统计图；B为从第二轮(2 nd)和第三轮(3 rd)淘选后的噬菌体抗体文库各挑选96个克隆进行噬菌体ELISA检测统计图；
图5为原核表达的VHH抗体的ELISA检测统计图，每个点代表一个克隆，纵坐标为针对sGN的OD450/空白对照的OD450，比值大于5.0定义为阳性；
图6为阳性VHH抗体中和SFTSV病毒感染实验统计图，一个点代表一个克隆，Y轴为针对不同病毒的相对抑制率；
图7为ELISA检测不同纯化浓度的SNB与sGN蛋白的结合，不同的颜色代表不同的克隆号。
图8为在不同浓度的抗体存在的环境下进行病毒感染得到的荧光染色照片，荧光点代表SFTSV病毒，无病毒感染的细胞作为无感染对照(No infection control)，不加抗体的病毒感染细胞为感染对照(infection control)；
图9为根据图7统计的荧光点得到的抗体对SFTSV病毒抑制率图；
图10为不同小鼠攻毒后的相对抑制率统计图，相对抑制率为1-第9天病毒载量/第6天病毒载量，以人免疫球蛋白为对照(Hu-IgG)；
图10为SNB双抗夹心ELISA检测sGN蛋白统计图，其中，分别用SNB01(A)、SNB02(B)和SNB37(C)为检测抗体；D为包被抗体SNB01，检测抗体SNB37的双抗体夹心ELISA检测不同浓度的sGN的OD450统计曲线。
图11为包被抗体SNB01，检测抗体SNB37的双抗体夹心ELISA检测不同浓度的SFTSV病毒的OD450统计曲线。
具体实施方式
1.免疫原的制备
我们依据NCBI网站上HB29 SFTSV的GN蛋白序列和基因序列信息，分析并设计了可有效诱导骆驼产生针对GN蛋白的特异性抗体的多肽sGN，在C端连接His-tag(sGN-his) 或兔Fc(sGN-rFc)用于后续纯化及检测。
2.骆驼免疫与抗血清的获得
用250μg sGN-rFc蛋白与250μl弗氏完全佐剂的乳化混合物对双峰骆驼进行初免，在第14天、28天、42天用sGN-rFc蛋白与250μl弗氏不完全佐剂加强免疫3次，第2次和第3次免疫1周后，采血检测抗血清滴度；第4次免疫1周后，采血200ml用于噬菌体抗体库的构建。
抗血清效价通过ELISA检测，用浓度为0.5μg/ml的GN蛋白包被检测板，每孔加入梯度稀释的抗血清或者纯化的抗体100μl(对照为免疫前骆驼血清)，37℃孵育1.5h，洗涤2次，每孔加入1：10000稀释的辣根过氧化物酶标记的Goat anti-Llamma IgG(H+L)二抗，37℃孵育1h，洗涤4-6次后，加100μl TMB底物，37℃孵育10min，50μl 0.2M的H 2SO 4中止反应，测定OD 450nm。ELISA检测血清效价规定为在OD450是空白对照的2.1倍以上并且大于0.2的最高稀释倍数。
结果如图1所示，3免和4免的抗血清效价分别为2.19×10 6和4.61×10 6。由此可见，该抗原可诱导骆驼产生特异性针对GN蛋白的高滴度抗血清。
为了进一步验证该高滴度的骆驼抗血清是否能有效阻止SFTSV病毒感染，进行病毒感染的中和实验。将不同稀释浓度的抗血清和免疫前血清分别与SFTSV病毒共同孵育60min，随后转移到Vero细胞，48h后通过对sGN蛋白的细胞免疫荧光染色来判断SFTSV的感染。中和实验结果显示，sGN诱导的抗血清抑制90％SFTSV感染的ID90为540倍稀释度以上(图2)。综上所述，sGN诱导了高滴度抗血清，同时该抗血清具有高效抑制SFTSV病毒感染的能力。
3.VHH噬菌体库构建及淘选
收集200ml免疫后骆驼的外周血，利用淋巴细胞分离液(GE Ficoll-Paque Plus)分离获得骆驼的PBMC，根据TRIzol操作手册，提取RNA，并利用oligo(dT)反转为cDNA,通过引物扩增，以及分子克隆等技术，将骆驼的VHH基因克隆至phagemid质粒，转化TG1细菌，得到VHH噬菌体库。为了进一步鉴定sGN-VHH噬菌体库是否构建成功，通过PCR 扩增免疫sGN骆驼的VHH目的基因，可以看出目的条带为500bp，大小符合预期(图3)，说明该sGN-VHH噬菌体抗体文库里含有VHH基因。挑选50个克隆进行测序，测序结果显示，所测序列没有完全一致的重复序列；比对结果显示，差异序列大多在CDR结合区。经检测，该构建了一个sGN-VHH噬菌体抗体文库的库容为2.0×10 9，阳性率为100％，序列多样性(Diversity)为100％，有效插入率(In frame rate)大于95％。
在M13KO7辅助噬菌体的帮助下，用VHH-phagemid转化的细菌，进行噬菌体抗体库的复苏，并用PEG/NaCl进行沉淀。将包被有50μg/ml的sGN-His蛋白进行三次富集噬菌体抗体库。将富集的噬菌体，洗脱、转化、涂板、挑取单克隆进行噬菌体与sGN蛋白ELISA的结合鉴定，将结合读值>1.0的克隆进行测序，并克隆至表达载体pVAX1，转染293F细胞表达生产纳米单抗。
淘选后的文库与GN蛋白进行结合检测。噬菌体ELISA结果显示，没有富集前的sGN-VHH噬菌体文库与sGN蛋白的结合读值为0.57，经过一轮、二轮、三轮富集后的噬菌体文库读值分别为0.98、2.2、3.0(图4A)。为了进一步验证富集后的文库中结合sGN-VHH蛋白的阳性噬菌体率，从第2轮和第3轮富集后的文库里各挑选了96个克隆进行单个噬菌体ELISA检测。结果显示，第2轮文库里，24.5％的单个噬菌体克隆为阳性，第3轮文库里67％的噬菌体克隆克隆为阳性，而且结合的平均读值在3.0左右(图4B)，通过sGN蛋白淘选成功的富集了高结合力的sGN-VHH噬菌体文库。
4.VHH原核表达文库的构建及VHH表达
对上述二轮和三轮淘选富集后的2nd-sGN-VHH和3rd-sGN-VHH噬菌体抗体文库进行PCR扩增；获取并纯化抗体库中所有VHH的基因片段，将VHH的基因片段克隆至原核表达载体，转化SS320菌株，构建VHH的原核表达抗体库；将原核表达抗体库涂布平板，过夜培养，次日随机挑选单克隆菌落1000个，使用IPTG诱导表达抗体上清，对抗体上清与sGN蛋白进行ELISA结合检测。
结果显示，有细菌上清与sGN蛋白结合，同时不与空白对照结合，sGN结合的读值/空白对照的读值大于5.0(图5和表1)。将这些序列进行测序比对，剔除重复序列，最 终获得142个的VHH抗体序列(SEQ ID NO:1-142)。进一步的实验证实，这142个VHH抗体以及从VHH抗体得到的CDR均可特异性地结合SFTSV病毒。
表1 142个VHH抗体与sGN蛋白的结合值及其序列
Figure PCTCN2019097350-appb-000001
Figure PCTCN2019097350-appb-000002
Figure PCTCN2019097350-appb-000003
为了进一步验证该序列是否能很好的抑制SFTSV病毒感染，将122个VHH抗体的细菌上清通过处理后与SFTSV病毒共孵，测试抗体是否能抑制SFTSV病毒感染。中和实验结果显示(图6)，有23个抗体能达到50％以上的抑制效果。抗体编号分别为G77、G78、G79、G87、G95、G103、G109、G111、G112、G113、G114、G116、G117.1、G121、G124、G125、G126、G131、G133、G145、G170和G171。
5.VHH-huFc(SNB)真核表达
通过分子克隆技术，将上述23个纳米单抗VHH基因融合人的Fc基因并插入在pVAX1真核表达载体，构建形成Nb-huFc-pVAX1表达质粒。将构建好的Nb-huFc-pVAX1，转染293F细胞，表达生产Nb-huFc(SNB)，并利用ProteinG纯化。收集纯化的VHH-huFc1(SNB)抗体进行ELISA测定，部分抗体有很好的结合能力(图7)。构建的人源化抗体相应的序列号如表2所示。
表2 人源化抗体SNB对应的原抗体编号及CDR序列
Figure PCTCN2019097350-appb-000004
通过BIAcore x100仪器，根据氨基偶联试剂盒说明书，偶联4000RU的sGN蛋白至CM5芯片上；乙醇胺封闭偶联好的芯片。抗体梯度稀释为不同的浓度。Bioevaluation version 4.0软件设置检测程序：抗体浓度从低浓度到高浓度检测，每个浓度做2个重复检测；结合时间设为180秒，流速为30μl/min；解离时间设为180秒，流速为30μl/min；pH2.5 10mM的甘氨酸将流速设为30μl/min，时间为30秒，活化、重生芯片表面；PBS平衡5秒，流速为30μl/min。分析实验数据，获得结合、解离和亲和常数。结 果如表3所示，大部分的抗体的亲和力能达到10 -9(Nano mole级),其中有两个抗体SNB02和SNB07能达到10 -10(Pico mole级)。由此可见，我们获得了具有高亲和力的VHH-huFc1(SNB)抗体。
表3 SNB亲和力的总结。
Clone ID ka(1/Ms) kd(1/s) KD(M)
SNB01 4.05E+04 6.91E-05 1.70589E-09
SNB02 1.88E+07 3.91E-03 2.08391E-10
SNB07 8.66E+04 3.86E-05 4.45398E-10
SNB28 1.05E+05 2.97E-04 2.8361E-09
SNB29 7.39E+03 5.56E-05 7.51393E-09
SNB37 3.74E+04 1.09E-04 2.92338E-09
注：ka为结合常数，kd为解离常数，KD为亲和力。
6.SNB中和SFTSV感染Vero细胞
选取VHH-huFc1中的SNB02和SNB16进行体外中和实验。将抗体梯度稀释为不同的浓度，与SFTSV病毒一起，于5％CO 2 37℃下共孵育1个小时，添加1.5x 10 4个Vero细胞，5％CO 2 37℃温箱培养48小时后，移除细胞上清，添加4％多聚甲醛固定15min后，经过洗涤封闭后，添加抗GN的兔多克隆血清(1:1000稀释)，4℃过夜，PBST洗涤后，添加50μl抗兔的二抗(Alexa Fluor 488Anti-Rabbit IgG(H+L),Code:111-545-144,Jackson ImmunoResearch Laboratories，1:1000稀释)室温孵育40min，接着进行DAPI 10min染色，洗涤后，荧光显微镜拍照观察，统计绿色斑点和荧光强度计算中和抑制率与中和滴度，抑制率＝[1-(样品组的荧光强度均值-细胞对照组CC的荧光强度均值)/(对照处理组的荧光强度均值-细胞对照组CC的荧光强度均值)]×100％。中和滴度(ID 50或ND 50)表述为50％抑制率时的稀释度的倍数。
结果如图8和9所示，SNB02具有很好的中和活性，当抗体浓度在9μg/ml时，抑制率能达到83.5％。利用人源化小鼠模型来评估SNB02的疗效。
7.SNB治疗SFTS体内感染
ImmunodeficientNOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NCG)小鼠购自南京大学模式动物所，与NSG小鼠类似，该小鼠在SCID小鼠基础上缺失了IL2受体基因，导致体内没 有小鼠T细胞，B细胞以及极少量的NK细胞。1.0-15x 10 7PBMC腹腔注射进4-6周的NCG小鼠体内；三周后，采血流式检测人T细胞，通过染色人的CD45 +、CD3 +、CD4 +和CD8 +。人CD45阳性细胞的比例到达5％以上，判定为小鼠人源化成功。接种2x 10 7TCID50的SFTSV病毒，感染后第3和6天，分别进行一次抗体治疗，即，腹腔注射400μg SNB02抗体/小鼠(抗体的量为大约20mg/kg小鼠)。每次抗体治疗前和第9天进行采血，检测小鼠血液中的病毒载量，从而判定小鼠是否成功的感染了SFTS病毒以及SNB抗体是否能控制SFTSV感染小鼠，以人IgG为对照。结果如图11所示，完成治疗三天后(第9天)，检测小鼠体内的病毒载量，接受SNB02治疗的9只小鼠，7只小鼠体内的病毒载量均得到了很好的抑制，而接受Hu-IgG治疗的对照组小鼠，体内SFTS病毒在大量繁殖，两组病毒抑制率对比分析，SNB02有显著性的的控制SFTS病毒感染(图10)。
8.使用AAV病毒载体装载的SNB02进行体内实验
腺相关病毒载体(AAV)源于非致病的野生型腺相关病毒，由于其安全性好、宿主细胞范围广(分裂和非分裂细胞)、免疫源性低，在体内表达外源基因时间长等特点，被视为最有前途的基因转移载体之一，在世界范围内的基因治疗和疫苗研究中得到广泛应用。
AAV Helper-Free病毒包装系统购于Cell Biolabs,San Diego USA。将上述SNB02的DNA编码序列通过分子克隆技术插入到pAAV-MCS质粒；通过测序证明构建成功后，将构建好的质粒pAAV-Ab与pHelper和pAAV-DJ质粒按照质量比1:1:1的方式使用PEI转染试剂共转染AAV-293T细胞。转染后分别于48、72、96和120小时收集上清，并用5xPEG8000(sigma)进行浓缩，最后用1.37g/ml氯化铯进行纯化。纯化的AAV溶解于PBS里，进行鉴定和分装后保存于-80℃。
将上述人源化小鼠接种2x 10 7TCID50的SFTSV病毒，感染后第3天采血以及接受AAV-SNB02(1x10 11gc/100μl)肌肉注射，并在第6、9和12天采血，检测病毒载量，以AAV-GFP为对照组。结果显示，注射了AAV-SNB02的小鼠体内的SFTSV病毒载量均很低，而对照组小鼠体内的SFTSV病毒大量繁殖。
9.双抗夹心ELISA
用不同浓度的SNB抗体包被检测板，每孔100μl，37℃孵育2h，洗涤2-4次，10％牛血清封闭，每孔200μl，37℃孵育1h，洗涤2-4次，每孔加入梯度稀释的蛋白，病毒或者样品100μl，37℃孵育1.5h，洗涤2次，每孔加入1：200-1：10000稀释的辣根过氧化物酶标记的SNB抗体100μl，37℃孵育1h，洗涤4-6次后，加100μl TMB底物，37℃孵育10min，50μl 0.2M的H 2SO 4中止反应，测定OD450nm。ELISA检测的阳性样品规定为在OD450是空白对照(即不包被检测原，图中标记为Neg)的2.1倍以上并且其光密度值大于0.2的最高稀释倍数。
结果显示，当包被抗体为SNB02或SNB07，检测抗体为SNB01时，该组合能够识别sGN蛋白和阴性对照蛋白(图11A)；当检测抗体为SNB02时，ELISA检测背景很高(图11B)；当检测抗体为SNB37，包被抗体为SNB01、SNB02和SNB07时，ELISA能识别sGN蛋白和阴性对照蛋白(图11C)。SNB抗体可以应用于双抗体夹心ELISA检测sGN试剂盒的开发，其中SNB01与SNB37的双抗组合其检测灵敏度为3.9ng/ml(图11D)。测量该组合检测SFTSV真病毒的灵敏度为3.75x 10 6gc/ml(图12),说明该试剂盒不仅能检出SFTSV的真病毒，还能检出低至10^6拷贝数的SFTSV真病毒。由此可见，该纳米抗体双抗夹心检测试剂盒能应用在SFTSV病毒感染检测。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种可结合SFTSV的多肽，其特征在于，包括3个互补决定区CDR1-3，CDR1序列为或包括SEQ ID NO:1-74所示序列之一，CDR2序列为或包括SEQ ID NO:75-151所示序列之一，CDR3序列为或包括SEQ ID NO:152-232所示序列之一。
  2. 根据权利要求1所述的多肽，其特征在于，所述多肽还包括4个框架区FR1-4，所述FR1-4与所述CDR1-3按顺序交错排列。
  3. 根据权利要求2所述的多肽，其特征在于，所述多肽为单克隆抗体。
  4. 根据权利要求2所述的多肽，其特征在于，所述多肽为VHH。
  5. 根据权利要求4所述的多肽，其特征在于，所述多肽为骆驼源的VHH或人源化的VHH。
  6. 权利要求1-5中任一项所述的多肽在制备SFTSV检测剂或SFTSV治疗药物中的应用。
  7. 一种核酸编码序列，其特征在于，编码权利要求1-5中任一项所述的多肽。
  8. 权利要求7所述的核酸编码序列在制备SFTSV治疗药物中的应用。
  9. 一种SFTSV检测试剂盒，其特征在于，包括检测抗体、固体基质以及包被于所述固体基质上的包被抗体，所述检测抗体和所述包被抗体分别为权利要求1-5中任一项所述的多肽中的一种。
  10. 根据权利要求9所述的试剂盒，其特征在于，所述检测抗体的CDR序列如下：
    CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:84，并且CDR3的序列为SEQ ID NO:181；或CDR1的序列为SEQ ID NO:54，CDR2的序列为SEQ ID NO:132，并且CDR3的序列为SEQ ID NO:176；或CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:99，并且CDR3的序列为SEQ ID NO:166。
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