KR20210013000A - Nano antibody binding to SFTSV and its application - Google Patents

Abstract

The present invention relates to a polypeptide capable of binding to SFTSV, which includes three complementarity determining regions CDR1-3, the CDR1 sequence is one of the sequences represented by SEQ ID NO: 1-74, or comprises a CDR2 sequence. Is one or comprises one of the sequences represented by SEQ ID NO: 75-151, and the CDR3 sequence is one or comprises one of the sequences represented by SEQ ID NO: 152-232. The present invention is a platform technology that develops nano-antibody drugs and studies diagnostic kits for SFTS, which has a high mortality rate but does not have effective vaccines and specific antiviral drugs, and displays nano-bodies using GN proteins, immunization of double-headed camels, and phage libraries. The nano-antibody VHH specifically binding to GN was screened through, for example, the CDR sequences thereof, and the humanized antibody SNB was constructed, and the therapeutic effect of SNB in the treatment of SFTSV infection in vivo was evaluated using a humanized mouse model. The present invention provides a potential new nano antibody drug for clinical treatment of SFTS, and at the same time provides a corresponding detection kit for diagnosis of SFTS.

Description

Nano antibody binding to SFTSV and its application

The present invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding to SFTSV, an application of the polypeptide in the preparation of an SFTSV detection agent or SFTSV therapeutic drug, and an SFTSV detection kit.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by Severe fever with thrombocytopenia syndrome virus (SFTSV).It is mainly caused by tick bites or contact with blood or mucous membranes in acute patients. It is transmitted, and the main clinical symptoms include fever, platelet loss, leukocytosis, and multi-organ dysfunction including gastrointestinal tract, liver and kidney. Since the first report of SFTS in Hubei and Henan in China in 2009, cases have been reported in 16 provinces in China.As of 2014, the number of confirmed cases exceeded 3,500, and the recent incidence rate has been increasing continuously. have. In addition, cases of SFTS have been reported in Korea, Japan, the Arabs and the United States, suggesting that the spread of the disease is expanding. SFTS mortality rates currently reported are 6.3% to 30%, and mortality rates reported by Korea and Japan in 2016 were high at 35.4% and 50%, respectively. However, since there is no specific therapeutic drug for SFTS in clinical practice, only extensive antiviral therapy and symptomatic treatment can be performed, which poses great difficulty in preventing and controlling SFTS.

Specific neutralizing antibodies show excellent clinical efficacy in terms of antiviral therapy, such as the respiratory cell fusion virus monoclonal antibody Palivizumab and rabies virus antisera. Studies have shown that neutralizing antibodies against SFTSV surface glycoprotein (Glycoprotein N, GN) play an important role in patient survival. According to our previous study, all SFTS patients with anti-GN antibodies in their serum recovered well, but SFTS patients without GN antibodies in their serum had a high mortality rate of 66.7%. These studies suggest that GN target neutralizing antibody therapy is an effective method against SFTSV.

In 1993, a new natural antibody from Camelaceae was discovered. This antibody is naturally composed of only a heavy chain due to the deletion of the light chain, and the heavy chain contains two constant regions (CH2 and CH3), a hinge region and a variable heavy chain domain (VHH, that is, an antigen binding site), The relative molecular mass of the heavy chain variable region is about 13 KDa, which is only 1/10 of that of a conventional antibody, and the molecular height and diameter are both at the nano level, and because it is the smallest functional antibody fragment currently available, a Nanobody (Nb) It is also called. Nanobody has high stability (does not decompose even under 90°C conditions), has excellent affinity, has more than 80% homology with human antibodies, and has low toxicity and immunogenicity. It is widely used in research and development and research and development of antibody drugs in the fields of tumors, inflammation, infectious diseases and nervous system diseases.

The currently known SFTS neutralizing antibody of anti-GN is Mab4-5 obtained by phage display SFTS patient antibody library screening in 2013, and its IC50 is 2 μg/ml to 44.2 μg/ml. The lower the IC50 of the neutralizing antibody is, the better, and by significantly reducing the amount of antibody used, not only can the production cost be saved, but also the time for the antibody to maintain an effective concentration in the body can be extended. We previously detected antibody titers in the serum of SFTS patients and found that the antisera against GN protein was only a dilution factor of about 100, which is why the antibody library constructed from the serum of SFTS patients with low antibody titers was used for screening of highly efficient neutralizing antibodies. Suggests that you can limit Therefore, we expect that the novel technical means will be able to obtain SFTSV-specific neutralizing antibodies that are more efficient, stable and have a lower IC50.

The present invention uses an antigen to immunize a camel to obtain a camel-derived nanobody and its VHH, and is used for diagnosis and treatment of acutely infected SFTS patients. Based on these studies, the present invention provides a polypeptide capable of binding to SFTSV, which includes three complementarity determining regions CDR1-3, and the CDR1 sequence is one of the sequences represented by SEQ ID NO: 1-74, or And the CDR2 sequence is one or includes one of the sequences represented by SEQ ID NO: 75-151, and the CDR3 sequence is one or includes one of the sequences represented by SEQ ID NO: 152-232.

In a specific embodiment, the polypeptide further comprises four framework regions FR1-4, and the FR1-4 and CDR1-3 are cross-arranged. For example, the FR1-4 sequence can be designed to appear as SEQ ID NO: 235-237, but the scope of the present invention is not limited thereto. The specific identification and binding ability of the antibody is mainly determined by the CDR region sequence, and the FR sequence is not significantly affected, and can be designed according to species as known in the art. For example, a human, murine or camel-derived FR region sequence can be designed as a polypeptide or domain capable of binding to SFTSV by linking to the CDRs.

In a preferred embodiment, the polypeptide is a monoclonal antibody.

In a preferred embodiment, the polypeptide is VHH.

In a preferred embodiment, the polypeptide is camel derived VHH or humanized VHH.

In a specific embodiment, the CDR sequences of the 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 the polypeptide are as follows.

I) the sequence of CDR1 is XXXSTAYY (SEQ ID NO: 233), where X is an arbitrary 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 sequence of CDR3 is selected from SEQ ID NO: 155, 157, 161, 169, 172, 179, 180, 181, 210, 218, 229, 230, 231.

In a specific embodiment, the CDR sequences of the 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, 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, and 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, and 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, 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 the 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.

In another specific embodiment, the CDR sequences of the 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 the 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 the 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 the 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, 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, 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 the 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, the sequence of CDR2 is SEQ ID NO: 138, and the sequence of CDR3 is SEQ ID NO: 216.

The present invention further provides the application of the polypeptide in the manufacture of an SFTSV detection agent or an SFTSV therapeutic drug.

The present invention further provides a nucleic acid coding sequence for the polypeptide.

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

In a specific embodiment, the nucleic acid coding sequence is present in a gene expression cassette.

The present invention further provides an expression vector comprising the expression cassette of the nucleic acid coding sequence.

In a preferred embodiment, the expression vector is a viral vector.

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

The present invention further provides the application of the nucleic acid coding sequence and expression vector in an SFTSV therapeutic drug.

The present invention further provides an SFTSV detection kit, which includes a detection antibody, a solid substrate, and a coated antibody coated on the solid substrate, wherein the detection antibody and the coated antibody are each one of the polypeptides.

In a specific embodiment, 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: 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.

In a specific embodiment, the CDR sequences of the coated 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, 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 a preferred embodiment,

I) The CDR sequences of the detection antibody are as follows. That is, the CDR1 sequence is SEQ ID NO: 4, the CDR2 sequence is SEQ ID NO: 84, the CDR3 sequence is SEQ ID NO: 181, or the CDR1 sequence is SEQ ID NO: 13, and the CDR2 sequence is SEQ ID NO: 99, the sequence of CDR3 is SEQ ID NO: 166,

II) The CDR sequences of the coated antibody are as follows. That is, the CDR1 sequence is SEQ ID NO: 4, the CDR2 sequence is SEQ ID NO: 84, the CDR3 sequence is SEQ ID NO: 181, or the CDR1 sequence is SEQ ID NO: 54, and the CDR2 SEQ ID NO: 132. , The CDR3 sequence is SEQ ID NO: 176, or the CDR1 sequence is SEQ ID NO: 6, the CDR2 sequence is SEQ ID NO: 81, the CDR3 sequence is SEQ ID NO: 158, or the sequence of CDR1 is SEQ ID NO: : 13, the CDR2 sequence is SEQ ID NO: 99, and the CDR3 sequence is SEQ ID NO: 166.

In a preferred embodiment,

I) 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,

II) The CDR sequences of the coated antibody are as follows. That is, the CDR1 sequence is SEQ ID NO: 54, the CDR2 sequence is SEQ ID NO: 132, the CDR3 sequence is SEQ ID NO: 176, or the CDR1 sequence is SEQ ID NO: 13, and the CDR2 sequence is SEQ ID NO: 99, and the sequence of CDR3 is SEQ ID NO: 166.

In another preferred embodiment,

I) 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 sequences of the coated antibody are as follows.

The CDR1 sequence is SEQ ID NO: 4, the CDR2 sequence is SEQ ID NO: 84, the CDR3 sequence is SEQ ID NO: 181, or the CDR1 sequence is SEQ ID NO: 54, the CDR2 SEQ ID NO: 132, and the CDR3 The sequence is SEQ ID NO: 176, or the CDR1 sequence is SEQ ID NO: 6, the CDR2 sequence is SEQ ID NO: 81, the CDR3 sequence is SEQ ID NO: 158, or the sequence of CDR1 is SEQ ID NO: 13 , The CDR2 sequence is SEQ ID NO: 99, and the CDR3 sequence is SEQ ID NO: 166.

The present invention is a platform technology that develops nano-antibody drugs and studies diagnostic kits for SFTS, which has a high mortality rate but does not have effective vaccines and specific antiviral drugs, and displays nano-bodies using GN proteins, immunization of double-headed camels, and phage libraries. Screening for nano-antibody VHH that specifically binds to GN through the like, confirming its CDR sequence, constructing humanized VHH-huFc1 (SNB), and treatment of SNB in the treatment of SFTSV infection in vivo using a humanized mouse model The effect was evaluated. The present invention provides a potential new nano antibody drug for clinical treatment of SFTS, and at the same time provides a corresponding detection kit for diagnosis of SFTS.

1 is an antiserum titer detection curve after 1 week of sGN 3rd and 4th camel immunization.
FIG. 2 is a curve in which antisera after 1 week of immunization of the fourth camel with different dilutions inhibits the in vitro infection of Vero cells of the SFTSV virus, and serum before immunization was used as a control.
3 is an electrophoresis diagram of a PCR product obtained by amplifying a sGN-VHH phage antibody library as a template.
4 is a diagram showing panning and confirmation of the sGN-VHH phage antibody library, where A is an ELISA detection statistics of the phage library after panning of the sGN protein, and B is the second (2 ^nd ) and third order (3 ^rd ) Phage ELISA detection statistics were performed by selecting each of 96 clones from the phage antibody library after panning.
5 is a statistical diagram of ELISA detection of prokaryotic VHH antibodies, each point represents one clone, the ordinate is OD450 for sGN/OD450 of the blank control, and a ratio greater than 5.0 is defined as positive.
6 is an experimental statistical diagram in which a positive VHH antibody neutralizes SFTSV virus infection, one dot represents one clone, and the Y axis represents the relative inhibition rate against different viruses.
Fig. 7 is a diagram showing the binding of SNB and sGN proteins of different purification concentrations detected by ELISA, and different colors indicate different clone numbers.
Figure 8 is a fluorescence staining picture obtained by performing viral infection in an environment in which antibodies of different concentrations are present, fluorescence dots represent SFTSV virus, cells without viral infection are no infection control, antibody Virus-infected cells that were not added were used as an infection control.
9 is a diagram showing the inhibition rate of the antibody against the SFTSV virus obtained based on the fluorescence dots of the statistics of FIG. 7.
Figure 10 is a statistical diagram of the relative inhibition rate after virus attack of different mice, the relative inhibition rate is the virus load on days 1 to 9 / virus load on the 6th day, and human immunoglobulin was used as a control (Hu-IgG).
11 is a statistical diagram of sGN protein detected by SNB DAS-ELISA (double antibody sandwich ELISA), where SNB01 (A), SNB02 (B) and SNB37 (C) were used as detection antibodies, respectively, and D is a coating antibody. SNB01, the OD450 statistical curve of different concentrations of sGN detected by DAS-ELISA of the detection antibody SNB37.
FIG. 12 is a OD450 statistical curve of SFTSV virus of different concentrations detected by DAS-ELISA (double antibody sandwich ELISA) of coating antibody SNB01 and 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 present inventors analyzed and designed a polypeptide sGN that can effectively induce camels to generate a specific antibody for the GN protein. tag (sGN-his) or rabbit Fc (sGN-rFc) was ligated and used for subsequent purification and detection.

2. Acquisition of camel immunity and antisera

First, the first immunization was performed on the double-headed camel using an emulsified mixture of 250 μg sGN-rFc protein and 250 μl complete Freund's adjuvant, and on the 14th, 28th, and 42nd days. sGN-rFc protein and 250 µl incomplete Freund's adjuvant were used to perform booster immunization 3 times, blood was collected 1 week after the 2nd and 3rd immunization to detect antisera titers, and 200 after 1 week of 4th immunization. Blood was collected and used to construct a phage antibody library.

Antiserum titer was detected by ELISA, coated with a detection plate using GN protein with a concentration of 0.5µg/ml, and gradient diluted antiserum or 100µl of purified antibody was added to each well (control group was camel serum before immunization. ), incubated at 37°C for 1 hour and 30 minutes, washed twice, and diluted 1:10000 in each well, labeled Goat anti-Llamma IgG (H+L) 2 of horseradish peroxidase. Secondary antibody is added, incubated at 37°C for 1 hour, washed 4 to 6 times, 100µl TMB substrate is added and incubated at 37°C for 10 minutes, and 50µl 0.2M H ₂ SO ₄ is used for reaction. Was stopped and OD 450nm was measured. Serum titers detected by ELISA are defined to be at least 2.1 times the blank control at OD450 and the highest dilution fold greater than 0.2.

As a result, as shown in Fig. 1, the antisera titers of 3 immunity and 4 immunity are 2.19 × 10 ⁶ and 4.61 × 10 ^6, respectively. This shows that the antigen can induce camels to produce high titer antisera specific for GN protein.

In order to further confirm whether the high titer of camel antisera can effectively prevent SFTSV virus infection, a virus infection neutralization experiment was performed. Different dilution concentrations of antisera and pre-immunization serum were incubated with SFTSV virus for 60 minutes, respectively, and transferred to Vero cells, 48 hours later, to determine SFTSV infection by cellular immunofluorescence staining for sGN protein. According to the results of the neutralization experiment, it was found that antisera induced by sGN inhibited 90% of SFTSV infections with ID90 540-fold or higher dilution (FIG. 2). In summary, sGN induces high titers of antisera, while the antisera has the ability to effectively inhibit SFTSV virus infection.

3. Construction and panning of VHH phage library

After 200 ml of immunization, peripheral blood of camels was collected, PBMCs of camels were obtained using lymphocyte isolate (GE Ficoll-Paque Plus), RNA was extracted according to the TRIzol operation manual, and cDNA was used using oligo (dT). The camel's VHH gene was cloned into a phagemid plasmid through techniques such as primer amplification and molecular cloning, and transformed with TG1 bacteria to obtain a VHH phage library. In order to confirm whether the sGN-VHH phage library was successfully constructed, as a result of amplifying the VHH target gene of the immune sGN camel by PCR, it was found that the target band was 500 bp and the size was consistent with the expected (Fig. 3), which is the above. It means that the VHH gene was included in the sGN-VHH phage antibody library. Sequence analysis was performed by selecting 50 clones, and according to the sequencing results, it was found that there were no repeat sequences with completely identical sequences. The comparison results showed that most of the difference sequences were in the CDR binding region. One sGN-VHH phage antibody library constructed above through detection has a storage capacity of 2.0×10 ⁹ , a positivity rate of 100%, a sequence diversity of 100%, and an effective insertion rate of 95%. That's it.

Phage antibody libraries were recovered using VHH-phagemid transformed bacteria with the aid of M13KO7 auxiliary phage and precipitated with PEG/NaCl. The phage antibody library was concentrated three times on the sGN-His protein coated with 50 μg/ml. The concentrated phage was eluted, transformed, plated, and a single clone was selected to confirm ELISA binding of the phage to the sGN protein, and the clone was sequenced with a binding reading >1.0 to be cloned into the expression vector pVAX1, and 293F cells Is transfected to express and produce a nanobody.

The panned library and the GN protein perform binding detection. According to the phage ELISA results, the binding readings of the sGN-VHH phage library and sGN protein before concentration were 0.57, and the readings of the phage libraries after the first, second and third enrichment were 0.98, 2.2 and 3.0, respectively (Fig. 4A. ). After concentration, in order to further confirm the phage-positive rate of sGN-VHH protein binding in the library, 96 clones were selected from the library after the second and third concentrations, and single phage ELISA detection was performed. According to the results, it was found that 24.5% of single phage clones were positive in the secondary library, 67% of phage clones were positive in the tertiary library, and the average reading of binding was about 3.0 (Fig. 4B). The sGN-VHH phage library with high binding ability was successfully concentrated through sGN protein panning.

4. Construction of VHH prokaryotic expression library and VHH expression

PCR amplification was performed on the second and third panning and concentrated 2nd-sGN-VHH and 3rd-sGN-VHH phage antibody libraries. Gene fragments of all VHHs in the antibody library were obtained and purified, and the gene fragments of VHH were cloned into a prokaryotic expression vector to transform the SS320 strain, and a prokaryotic antibody library of VHH was constructed. The prokaryotic antibody library was applied to the plate and incubated overnight, 1000 monoclonal colonies were randomly selected the next day, antibody supernatant expression was induced using IPTG, and ELISA binding detection was performed on the antibody supernatant and sGN protein.

According to the results, the bacterial supernatant bound with the sGN protein but not with the blank control, and the reading of sGN binding/read of the blank control was greater than 5.0 (Fig. 5 and Table 1). Sequencing was performed on these sequences to remove repeat sequences for comparison, and finally 142 VHH antibody sequences (SEQ ID NO: 1-142) were obtained. As verified by further experiments, it was found that all of these 142 VHH antibodies and CDRs obtained from VHH antibodies were able to specifically bind to the SFTSV virus.

Table 1 142 VHH antibodies and sGN protein binding values and their sequence

In order to further confirm whether the sequence could well inhibit SFTSV virus infection, the bacterial supernatant of 122 VHH antibodies was treated and then incubated with the SFTSV virus, thereby testing whether the antibody could inhibit the SFTSV virus infection. According to the results of the neutralization experiment (Fig. 6), 23 antibodies can implement 50% or more inhibitory effect. Antibody serial 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, respectively. to be.

5. VHH-huFc(SNB) eukaryotic expression

Through molecular cloning technology, the 23 nanobody VHH genes were fused with a human Fc gene and inserted into a pVAX1 eukaryotic expression vector to construct an Nb-huFc-pVAX1 expression plasmid. Constructed Nb-huFc-pVAX1 was transfected into 293F cells, and Nb-huFc (SNB) was expressed and produced, and purified using Protein G. Purified VHH-huFc1 (SNB) antibodies were collected and ELISA measurement was performed, and some antibodies had excellent binding ability (FIG. 7). The corresponding sequence numbers of the constructed humanized antibodies are shown in Table 2.

Table 2 Original antibody serial number and CDR sequence corresponding to humanized antibody SNB

4000RU of sGN protein is coupled to the CM5 chip according to the instructions of the amino coupling kit through a BIAcore ×100 instrument, and ethanolamine blocks the coupled chip. The antibody gradient dilution is done at different concentrations. Bioevaluation version 4.0 software sets up the detection program. Antibody concentration is detected from low to high concentration, and each concentration is detected twice. The binding time is set to 180 seconds, and the flow rate is 30 μl/min. The dissociation time was set to 180 seconds, and the flow rate was 30 μl/min. pH 2.5 10mM glycine (glycin) set the flow rate to 30 μl/min, the time to 30 seconds, and activates and regenerates the chip surface. The PBS was equilibrated for 5 seconds and the flow rate was 30 μl/min. Analyze the experimental data to obtain binding, dissociation and affinity constants. As shown in Table 3, the affinity of most antibodies reached 10 ^-9 (Nano mole grade), where the two antibodies SNB02 and SNB07 reached 10 ^-10 (Pico mole grade). Here we can see that a high affinity VHH-huFc1 (SNB) antibody was obtained.

Table 3 SNB affinity summary

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

6. SNB Neutralizing SFTSV Infected Vero Cells

In vitro neutralization experiments were performed by selecting SNB02 and SNB16 in VHH-huFc1. Antibody gradient dilution is at different concentrations, incubated with SFTSV virus in 5% CO ₂ 37°C for 1 hour, 1.5×10 ⁴ Vero cells are added, and incubated in 5% CO ₂ 37°C incubator for 48 hours After removal, the cell supernatant was removed and fixed with 4% paraformaldehyde for 15 minutes. After washing and blocking, anti-GN rabbit polyclonal serum (1:1000 dilution) was added to spend the night at 4°C, followed by PBST. After washing 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 dilution) was added at room temperature. Incubate for 40 minutes, followed by DAPI staining for 10 minutes, and observed by photographing with a fluorescence microscope after washing, and the neutralization inhibition rate and neutralization titer are calculated based on statistics of green spots and fluorescence intensity. Inhibition rate = [1-(average fluorescence intensity of the sample group-average fluorescence intensity of the cell control group CC) / (average fluorescence intensity of the control treatment group-the average fluorescence intensity of the cell control CC)] × 100%. Neutralization titer (ID ₅₀ or ND ₅₀ ) is expressed as a multiple of the dilution at 50% inhibition.

As a result, as shown in FIGS. 8 and 9, SNB02 has excellent neutralizing activity, and when the antibody concentration is 9 μg/ml, the inhibition rate may reach 83.5%. The humanized mouse model is used to evaluate the therapeutic efficacy of SNB02.

7. SNB treatment of SFTS infection in vivo

ImmunodeficientNOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NCG) mice were purchased from Nanjing University Model Animal Place, similar to NSG mice, and the mice were based on SCID mice, and the IL2 receptor gene was deleted, and thus, in vivo There are no mouse T cells or B cells within, and there are very few NK cells. 1.0-15×10 ⁷ PBMC was injected intraperitoneally into the body of NCG mice of 4 to 6 weeks, blood was collected after 3 weeks, and flow cytometric analysis of human T cells, human CD45 ⁺ , CD3 ⁺ , CD4 ⁺ and CD8 ⁺ were stained, The proportion of human CD45 positive cells reached 5% or more, and it was determined that the mice were successfully humanized. 2×10 ⁷ TCID50 of SFTSV virus was inoculated, and the first antibody treatment was performed on the 3rd and 6th days after infection, respectively. That is, 400 µg SNB02 antibody/mouse (the amount of antibody is about 20 mg/kg mouse) is injected intraperitoneally. By detecting the viral load in the mouse blood by collecting blood each time before and on the 9th day of antibody treatment, it was determined whether the mouse was successfully infected with the SFTS virus and whether the SNB antibody could control the SFTSV-infected mouse, and human IgG was used as a control. Used. As shown in Fig. 11, according to the results of detecting the virus load in the mouse body 3 days after treatment completion (day 9), the virus load in the body of 9 mice and 7 mice treated with SNB02 was very good. And the control mice receiving Hu-IgG treatment showed massive proliferation of SFTS virus in the body. According to the comparative analysis of virus inhibition rate of the two groups, SNB02 significantly controls SFTS virus infection (Fig. 10).

8. In vivo experiment using SNB02 loaded with AAV virus vector

Adeno-associated virus vector (AAV) is derived from a non-pathogenic wild-type adeno-associated virus, has excellent safety, has a wide range of host cells (dividing and non-dividing cells), has low immunogenicity, and has a long time to express foreign genes in vivo. As such, it is considered one of the most promising gene transfer vectors and is widely used in gene therapy and vaccine research worldwide.

The AAV Helper-Free virus packaging system was purchased from Cell Biolabs, San Diego, USA. The DNA coding sequence of the SNB02 was inserted into the pAAV-MCS plasmid through molecular cloning technology, and the construction was verified to be successful through sequence analysis, and then the constructed plasmid pAAV-Ab and pHelper and pAAV-DJ plasmids were 1:1: AAV-293T cells were co-transfected using a PEI transfection reagent at a mass ratio of 1. After transfection, the supernatant was collected at 48 hours, 72 hours, 96 hours and 120 hours, respectively, concentrated using 5×PEG8000 (sigma), and finally purified with 1.37 g/ml cesium chloride. The purified AAV was dissolved in PBS, checked and aliquoted, and stored at -80°C.

The humanized mice were inoculated with 2×10 ⁷ TCID50 SFTSV virus, blood was collected on the 3rd day after infection, and AAV-SNB02 (1×10 ¹¹ gc/100 μl) was injected intramuscularly, on the 6th, 9th, and 12th days. Blood was collected to detect viral load, and AAV-GFP was used as a control. According to the results, the SFTSV virus load in the mice injected with AAV-SNB02 was all very low, but the SFTSV virus in the control mice was found to proliferate in large quantities.

9. DAS-ELISA (double antibody sandwich ELISA)

The detection plate was coated with different concentrations of SNB antibody, incubated at 37°C for 2 hours at 100 µl per well, washed 2 to 4 times, blocked with 10% bovine serum, and 200 µl per well at 37°C for 1 hour. After incubation, washing 2 to 4 times, 100 μl of gradient diluted protein, virus or sample was added to each well, incubated at 37° C. for 1 hour and 30 minutes, washed twice, and 1:200 to each well Add 100 μl of 1:10000 diluted horseradish peroxidase labeled SNB antibody, incubate at 37° C. for 1 hour, wash 4 to 6 times, add 100 μl of TMB substrate, and add 10 at 37° C. After incubation for minutes, the reaction was stopped with 50 µl of 0.2M H ₂ SO ₄ , and OD450nm was measured. ELISA-detected positive samples are defined as having OD450 equal to or greater than 2.1 times the blank control (i.e., not coated with the detection source and denoted by Neg in the figure) and the highest dilution factor with an optical density value greater than 0.2.

According to the results, when the coating antibody is SNB02 or SNB07 and the detection antibody is SNB01, the combination can recognize the sGN protein and the negative control protein (Figure 11A), and when the detection antibody is SNB02, the ELISA detection background is very high. (Fig. 11B), when the detection antibody is SNB37 and the coating antibodies are SNB01, SNB02 and SNB07, ELISA was found to be able to recognize sGN protein and negative control protein (Fig. 11C). The SNB antibody can be applied to the development of a DAS-ELISA detection sGN kit, where the dual antibody combination of SNB01 and SNB37 has a detection sensitivity of 3.9 ng/ml (Fig. 11D). The sensitivity of the SFTSV real virus detected by the combination was detected as 3.75×10 ⁶ gc/ml (Fig. 12), which means that the kit can detect not only the real virus of SFTSV, but also the SFTSV real virus up to as low as 10^6 copies. Means you can. As can be seen here, the nanobody DAS detection kit can be applied to detect SFTSV virus infection.

The above is only a relatively preferred embodiment of the present invention and does not limit the present invention. All modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should all be included within the protection scope of the present invention.

SEQUENCE LISTING <110> Y-CLONE MEDICAL SCIENCES CO., LTD. <120> Nano-antibody that can be combined with SFTSV and application thereof <130> 1 <160> 237 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 1 Gly Phe Thr Phe Asp Asp Ser Asn 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 2 Gly His Thr Leu Ser Ser Asn Cys 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 3 Gly Pro Phe Tyr Asn Thr His Cys 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 4 Gly Asp Thr Ser Thr Ala Tyr Tyr 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 5 Gly Asp Thr Tyr Ser Ser Ser Cys 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 6 Gly Phe Thr Ser Cys 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 7 Gly Val Thr Leu Asp Asp Phe Leu 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 8 Gly Phe Ser Phe Asn Ala Tyr Cys 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 9 Gly Tyr Thr Phe Ser Asn Thr Cys 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 10 Gly Arg Thr Tyr Arg Thr Tyr Cys 1 5 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 11 Glu Asp Ala His Ser Thr Thr Glu Tyr Ile Val Ser Thr Thr Cys 1 5 10 15 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR <400> 12 Gly Val Thr Tyr Gly Ser Tyr Cys 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 13 Gly Phe Thr Leu Ser Met Tyr Asp 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 14 Glu Tyr Thr Gly Ser Arg Asn Cys 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 15 Gly Tyr Thr Tyr Asn Asn Tyr Arg 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 16 Gly Leu Tyr Tyr Pro Pro Leu Cys 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 17 Arg Tyr Asp Tyr Ser Arg Thr Cys 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 18 Gly Phe Thr Val Ser Arg Tyr Asp 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 19 Gly Ser Ala Tyr Ser Thr Asn Cys 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 20 Gly Tyr Thr Tyr Ser Ser Asn Cys 1 5 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 21 Gly Phe Thr Phe Asn Ala Tyr Asp 1 5 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 22 Val Tyr Thr Tyr Arg Gly Asn Asn 1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 23 Arg Tyr Thr Pro Thr Ile Thr Arg 1 5 <210> 24 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 24 Val Tyr Thr Ser Ser Thr Met Trp 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 25 Gly Tyr Thr Ser Thr Ala Tyr Tyr 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 26 Gly Gly Thr Ser Thr Ala Tyr Tyr 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 27 Gly Phe Thr Ser Ser Asn Tyr Asp 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 28 Gly Phe Thr Ser Ser Ser Cys Gly 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 29 Gly Tyr Thr Tyr Ser Ser Val Cys 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 30 Gly Tyr Thr Tyr Asn Thr Ala Tyr 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 31 Gly Tyr Ala Tyr Ser Thr Tyr Cys 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 32 Gly Tyr Thr Tyr Asn Glu Tyr Ser 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 33 Gly Tyr Asn Phe Asn Asn Val Cys 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 34 Glu Tyr Thr Arg Ser Ser Arg Cys 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 35 Gly Ser Thr Asp Ser Arg Arg Cys 1 5 <210> 36 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 36 Gly Tyr Pro Tyr Ser Ser Lys Thr Tyr Thr Asn Asn Cys 1 5 10 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 37 Arg Tyr Ser Ser Ser Arg Arg Cys 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 38 Gly Tyr Ile Tyr Asn Asp Tyr Phe 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 39 Thr Tyr Asn Tyr Glu Ser Ser Gln 1 5 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 40 Thr Thr Thr Asn Tyr 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 41 Gly Tyr Thr Tyr Asn Tyr Tyr Cys 1 5 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 42 Gly Tyr Thr Tyr Tyr Asn Ser Asn Cys 1 5 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 43 Gly Gly Ser Val Thr Thr Gly Asn Tyr Tyr 1 5 10 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 44 Gly Tyr Thr Phe Asn Thr Arg Cys 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 45 Leu Tyr Thr Tyr Ser Tyr Asn Cys 1 5 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 46 Gly Phe Thr Ser Asn Ser Cys Gly 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 47 Gly Met Met Ser Asn Ala Cys 1 5 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 48 Gly Tyr Thr His Ser Ser Asp Ser 1 5 <210> 49 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 49 Arg Ser Val Asn Arg Asp Thr Cys 1 5 <210> 50 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 50 Ala Phe Ile Ser Asn Asn His Cys 1 5 <210> 51 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 51 Gly Phe Thr Phe Ser Ser Tyr Asp 1 5 <210> 52 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 52 Gly Phe Thr Phe Asp Asp Ser Asp 1 5 <210> 53 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 53 Gly Tyr Arg Cys One <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 54 Ala Tyr Thr Tyr Arg Gly Asn Asn 1 5 <210> 55 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 55 Gly Ala Thr Tyr Asn Ile Asn Phe 1 5 <210> 56 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 56 Gly Tyr Thr Tyr Ser Asn Tyr 1 5 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 57 Gly Phe Thr Phe Ser Ser Tyr Tyr 1 5 <210> 58 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 58 Gly Ser Ile Tyr Ser Ser Asn Ala 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 59 Gly Tyr Thr Tyr Ser Ser Ala Cys 1 5 <210> 60 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 60 Gly Met Tyr Ser Asn Thr Cys 1 5 <210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 61 Gly Gly Ser Ile Thr Thr Asn Tyr Tyr Gly 1 5 10 <210> 62 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 62 Gly Asp Ser Ser Thr Ala Tyr Tyr 1 5 <210> 63 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 63 Gly Asn Thr Tyr Thr Ser Ser Cys 1 5 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 64 Gly Gly Ser Ile Thr Thr Ala Gly Tyr Gly 1 5 10 <210> 65 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 65 Gly Leu Tyr Tyr Leu Pro Leu Cys 1 5 <210> 66 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 66 Gly Leu Trp His Pro Pro Leu Cys 1 5 <210> 67 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 67 Gly Tyr Thr Tyr Gly Ser Tyr Cys 1 5 <210> 68 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 68 Gly Leu Tyr Tyr Ser Pro Leu Cys 1 5 <210> 69 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 69 Arg Tyr Thr Ser Ser 1 5 <210> 70 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 70 Gly Tyr Arg Tyr Asn Ala Cys Ser 1 5 <210> 71 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 71 Gly Asn Pro Ser Gly Arg Lys Phe 1 5 <210> 72 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 72 Gly Ser Ser Gly Leu Ile Phe Ser Gly Ser Ala 1 5 10 <210> 73 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 73 Glu Asp Thr Ser Thr Ala Tyr Tyr 1 5 <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 74 Gly Tyr Thr Tyr Ser Ser His Cys 1 5 <210> 75 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 75 Ile Lys Ser Asp Gly Ser Thr Ser 1 5 <210> 76 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 76 Ile Tyr Thr Gly Gly Gly His Thr Tyr 1 5 <210> 77 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 77 Val Tyr Pro His Leu Thr Tyr 1 5 <210> 78 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 78 Ile Tyr Arg Gly Gly Arg Ala Thr Val 1 5 <210> 79 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 79 Ile Cys Ser Asp Gly Ser Ala Ala 1 5 <210> 80 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 80 Ile Tyr Arg Gly Gly His Ser Thr Val 1 5 <210> 81 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 81 Ile Tyr Thr Arg Asp Gly Arg Pro Tyr 1 5 <210> 82 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 82 Ile Asp Ser Glu Gly Arg Ile Asp 1 5 <210> 83 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 83 Ile Asp Ser Gly Gly Ser Ser Ser 1 5 <210> 84 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 84 Ile Tyr Arg Gly Gly Arg Ser Thr Val 1 5 <210> 85 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 85 Ile Asp Arg Ser Gly Ser Ala Ser 1 5 <210> 86 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 86 Ile Thr Ala Leu Ser Ala Thr Ser 1 5 <210> 87 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 87 Thr Tyr Arg His Gly Gly Thr Thr Val 1 5 <210> 88 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 88 Leu Glu Ser Asp Gly Ser Thr Ser 1 5 <210> 89 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 89 Ile Tyr Thr Ser Gly Arg Arg Pro Trp 1 5 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 90 Ile Phe Arg Gly Gly Arg Ser Thr Val 1 5 <210> 91 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 91 Ile Ser Ile Leu Tyr Ser Gly Ile Thr Val Ser Tyr 1 5 10 <210> 92 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 92 Ile Ser Ala Gly Gly Asp His Thr Tyr 1 5 <210> 93 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 93 Ile Asp Arg Asp Gly Ser Thr Ser 1 5 <210> 94 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 94 Ile Cys Ser Asp Gly Ser Thr Ser 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 95 Ile Phe Val Ser Gly Arg Ser Pro Trp 1 5 <210> 96 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 96 Ile Tyr Arg Asp Asp Gly Thr Thr Tyr 1 5 <210> 97 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 97 Ile Tyr Thr Gly Gly Gly Ser Thr Tyr 1 5 <210> 98 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 98 Ile Phe Arg Ser Gly Arg Thr Ser Trp 1 5 <210> 99 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 99 Ile Phe Thr Ser Gly Arg Arg Pro Trp 1 5 <210> 100 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 100 Ile Thr Ser Thr Gly Thr Arg Gln Tyr 1 5 <210> 101 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 101 Ile Tyr Thr Arg Gly Asp Arg Thr Phe 1 5 <210> 102 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 102 Ile Tyr Arg Gly Asn Gly Ala Thr Gly 1 5 <210> 103 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 103 Ile Trp Arg Gly Gly His Ser Thr Leu 1 5 <210> 104 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 104 Ile Phe Ser Ser Gly Arg Arg Pro Trp 1 5 <210> 105 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 105 Leu His Thr Asp Gly Leu Thr Ser 1 5 <210> 106 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 106 Ile Phe Thr Ser Gly Arg Arg Ser Trp 1 5 <210> 107 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 107 Ile Tyr Thr Ala Thr Gly Arg Thr Tyr 1 5 <210> 108 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 108 Ile Asp Ser Asp Gly Ser Thr Ser 1 5 <210> 109 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 109 Ile Asp Ser Asp Gly Val Thr Asp 1 5 <210> 110 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 110 Ile Asn Ser Val Gly Arg Thr Arg 1 5 <210> 111 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 111 Ile Tyr Thr Ser Ile Gly Arg Thr Tyr 1 5 <210> 112 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 112 Ile Glu His Asp Gly Lys Ile Ile 1 5 <210> 113 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 113 Ile Tyr Arg Gly Ser Gly Thr Thr His 1 5 <210> 114 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 114 Ile Tyr Leu Ala Asn Gly Ala Thr Tyr 1 5 <210> 115 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 115 Ile Tyr His Gly Asp Gly Thr Thr Tyr 1 5 <210> 116 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 116 Ile Gly Ser Asp Gly Thr Thr Lys 1 5 <210> 117 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 117 Val Ser Pro Arg Gly Glu Ser Ile Tyr 1 5 <210> 118 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 118 Ile Phe Ser Ser Arg Asp Tyr Thr Asp 1 5 <210> 119 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 119 Ile Asp Thr Gly Gly Ser Thr Tyr 1 5 <210> 120 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 120 Ile Tyr Thr Arg Asp Ser Arg Thr Tyr 1 5 <210> 121 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 121 Ile Thr Ala Ser Gly Ser Thr Tyr 1 5 <210> 122 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 122 Ile Ser Ala Gly Gly Ile Ser Ile Asp 1 5 <210> 123 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 123 Trp Gly Ser Val Gly Ser Ser Thr Thr Tyr 1 5 10 <210> 124 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 124 Ile Phe Ser Asp Gly Glu Thr Ala 1 5 <210> 125 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR <400> 125 Ile Tyr Thr Gly Ile Gly Thr Thr Tyr 1 5 <210> 126 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 126 Ile Tyr Thr Gly Asp Gly Ser Thr His 1 5 <210> 127 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 127 Ile Ser Ala Gln Gly Val Ile Pro Gly 1 5 <210> 128 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 128 Ile Asp Ser Ala Gly Ser Thr Arg 1 5 <210> 129 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 129 Ile Arg Ser Gly Gly Gly Asn Thr Tyr 1 5 <210> 130 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 130 Ile Ser Arg Ile Asp Asn Ser Thr Tyr 1 5 <210> 131 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 131 Phe Val Thr Gly Ala Gly Ser Thr Tyr 1 5 <210> 132 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 132 Ile Thr Val Thr Gly Thr Arg Gln Tyr 1 5 <210> 133 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 133 Ile Asp Asn Asn Gly Trp Ser Thr 1 5 <210> 134 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 134 Ile Asp Thr Asn Gly Ser Thr Ser 1 5 <210> 135 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 135 Ile Tyr Arg Asp Gly Ser Ala Pro Tyr 1 5 <210> 136 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 136 Ile Tyr Thr Gly Gly Ser Ala Thr Ser 1 5 <210> 137 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 137 Ile Cys Ser Asp Gly Ser Ser Ala 1 5 <210> 138 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 138 Ile Tyr Thr Gly Ile Gly Ser Thr Tyr 1 5 <210> 139 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 139 Ile Gly Tyr Ser Gly Ser Thr Tyr 1 5 <210> 140 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 140 Ile Cys Ser Asp Gly Ser Thr Ala 1 5 <210> 141 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 141 Ile Thr Phe Thr Gly Arg Thr Leu 1 5 <210> 142 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 142 Ile Tyr Thr Asn Val Gly Thr Thr Tyr 1 5 <210> 143 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR <400> 143 Ile Asp Arg Asp Gly Arg Thr Ser 1 5 <210> 144 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 144 Ile His Thr Asp Gly Ser Thr Ser 1 5 <210> 145 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 145 Ser Asn Thr Asn Gly Gly Ser Thr Tyr 1 5 <210> 146 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 146 Thr Asn Arg Asp Gly Met Ser Tyr 1 5 <210> 147 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 147 Val Tyr Asn Asp Gly Gly His Thr Tyr 1 5 <210> 148 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 148 Ile Phe Thr Arg Gly Thr Thr Lys 1 5 <210> 149 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 149 Leu Tyr Leu Gly Gly Ser Ile Thr Tyr 1 5 <210> 150 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 150 Ile Asp Thr Ile Gly Glu Ile Asp 1 5 <210> 151 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 151 Leu Asp Gly Asp Gly Arg Val Arg 1 5 <210> 152 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 152 Ala Ala Ala Trp Arg Pro Pro Cys Val Pro 1 5 10 <210> 153 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 153 Ala Ala Asp Leu Ser Pro Tyr Asp Cys Tyr Thr Gly Ser Leu Asp Met 1 5 10 15 Ala Ser Arg Phe Thr 20 <210> 154 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 154 Ala Ala Val Leu Cys Thr Asp Asp Tyr Lys Met Ala Pro Ala Asn Tyr 1 5 10 15 <210> 155 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 155 Ala Ala Gly Leu Ala Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala 1 5 10 15 Gly Phe Arg Tyr 20 <210> 156 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 156 Ala Ala Arg Arg Thr Trp His Ala Gly Phe Pro Tyr 1 5 10 <210> 157 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 157 Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala 1 5 10 15 Gly Phe Arg Tyr 20 <210> 158 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 158 Ala Ala Asn Arg Arg Ala Tyr Pro Tyr Gly Gly Asp Cys Arg Leu Arg 1 5 10 15 His Ala Glu Phe Asp Tyr 20 <210> 159 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 159 Ala Ala Asp Val Pro Gly Arg Arg Glu Val Arg Gly Leu Gly Pro Cys 1 5 10 15 Asp Arg Met Tyr Asp Tyr 20 <210> 160 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 160 Ala Ala Lys Val Pro Phe Gly Arg Gly Ser Cys Ala Tyr Ser Thr Ala 1 5 10 15 His Trp Phe Pro Tyr 20 <210> 161 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 161 Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Asp 1 5 10 15 Gly Phe Arg Tyr 20 <210> 162 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 162 Val Ala Asp Leu Ser Ala Trp Cys Arg Ala Val Arg Pro Gly Val Ile 1 5 10 15 Thr Tyr Asn Tyr 20 <210> 163 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 163 Ala Ala Asp Pro Arg Asp Pro Asn Gly Ser Arg Thr Asp Cys Thr Val 1 5 10 15 Leu Thr Ser Lys Asp Leu Tyr Asn Ser 20 25 <210> 164 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 164 Ala Ala Gly Pro Gly Cys Ser Trp Ser Ser Phe Ala Tyr 1 5 10 <210> 165 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 165 Val Ala Met Thr Trp Asp Gly Thr Cys His Ile Thr Ser Glu Phe Tyr 1 5 10 15 Tyr <210> 166 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 166 Ala Ala Val Ile Gly Val Asp Ile Arg 1 5 <210> 167 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 167 Ala Ile Arg Trp Gly Asp Cys Asp Ser Ala Ser Trp Ser Arg Arg Thr 1 5 10 15 Trp Tyr Ala Val 20 <210> 168 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 168 Ala Arg Ser Arg Ala Ser Leu Trp Ser Gly Asn Trp Tyr Arg Ser Leu 1 5 10 15 Ser Glu Asp Glu Tyr Asn Ser 20 <210> 169 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 169 Ala Ala Gly His Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Asp 1 5 10 15 Gly Phe Arg Tyr 20 <210> 170 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 170 Ala Ala Ala Asp Ala Gln Arg Gly Arg Thr Cys Phe Phe Gly Ala 1 5 10 15 <210> 171 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 171 Ala Val Arg Trp Tyr Trp Asp Ala Gly Phe Lys Tyr 1 5 10 <210> 172 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 172 Ala Ala Gly His Asp Gly Arg Ser Gly Tyr Leu Pro Leu Asn Trp Ala 1 5 10 15 Gly Phe Arg Tyr 20 <210> 173 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 173 Ala Ala Val Ile Gly Tyr Asp Ile Arg 1 5 <210> 174 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 174 Ala Ala Asp Ser Arg Thr Pro Ser Asp Cys Tyr Ser Gly Ser Trp Leu 1 5 10 15 Glu Lys Tyr Pro Ser Glu Tyr Ser Tyr 20 25 <210> 175 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 175 Ala Ala Asp Val Val Ser Tyr Tyr Ser Asp Tyr Val Cys Thr Asp Ala 1 5 10 15 Ala Asp Phe Gly Tyr 20 <210> 176 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 176 Ala Ala Gly Thr Thr Arg Leu Gly Ser Leu Leu Ala Pro Thr Tyr Arg 1 5 10 15 Tyr <210> 177 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 177 Ala Ala Gly Phe Met Tyr Gly Glu Thr Arg Ser Pro Asn Trp Val Asn 1 5 10 15 Tyr <210> 178 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 178 Ala Lys Lys Arg Thr Tyr Asp Cys Tyr Ser Gly Thr Cys Ser 1 5 10 <210> 179 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 179 Ala Ala Gly Leu Tyr Gly Gly Ser Pro Tyr Phe Pro Leu Asn Trp Thr 1 5 10 15 Gly Phe Arg Tyr 20 <210> 180 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 180 Ala Ala Gly Leu Arg Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala 1 5 10 15 Gly Phe Arg Tyr 20 <210> 181 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 181 Ala Ala Gly His Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala 1 5 10 15 Gly Phe Arg Tyr 20 <210> 182 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 182 Lys Thr Val Lys Asp Pro Thr Ser Pro Pro Gly Cys Ser Arg Gly Tyr 1 5 10 15 <210> 183 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 183 Ala Ala Asp Leu Leu Ile Gly Ala Cys Ser Gln Met Arg Arg Thr Asn 1 5 10 15 Phe Asp Tyr <210> 184 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 184 Ala Ala Asn Pro Tyr Ser Pro Gly Ala Gly Arg Glu Leu Leu Ser Tyr 1 5 10 15 Pro Tyr Thr Asp 20 <210> 185 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 185 Ala Ala Asn Gln Gly Ser Gly Asp Tyr Cys Tyr Met Ala Met Leu Ile 1 5 10 15 Tyr Gly Met Phe Tyr 20 <210> 186 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 186 Ala Ala Arg Asp Gly Ser Trp Phe Leu Ser Leu Val Pro Ala Thr Tyr 1 5 10 15 Gly Tyr <210> 187 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 187 Ala Ala Asp Leu Gln Ile Gly Ser Cys Ser Gln Met Arg Arg Tyr Asn 1 5 10 15 Tyr Ala Tyr <210> 188 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 188 Gly Ala Asp Pro Gly Glu Gly Ser Tyr Cys Ala Tyr Glu Ala Pro Glu 1 5 10 15 Val Gly Ala Leu Asp Ile 20 <210> 189 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 189 Ala Ala Gly Arg Thr Tyr Asp Cys Tyr Pro Gly Thr Cys Ser 1 5 10 <210> 190 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 190 Ala Ala Ser Leu Arg Ala Arg Trp Val Gln Arg Gly Ala Pro Leu Leu 1 5 10 15 Pro Ser Phe Tyr Gly Tyr 20 <210> 191 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 191 Ala Ala Gly Pro Trp Val Ala Thr Pro Glu Ile Ala Asn Glu Tyr Asn 1 5 10 15 Tyr <210> 192 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 192 Val Ala Gly Ile Trp Thr Cys Gly Arg Ser Ala Leu Thr Asp Pro Asn 1 5 10 15 Asn Tyr <210> 193 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 193 Ala Ala Thr Phe Gly Leu Phe Trp Glu Ser Trp Glu Trp Tyr Lys Asn 1 5 10 15 Trp His Tyr <210> 194 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 194 Ala Ala Asp Pro Gly Val Leu Cys Gly Arg Ser Trp Val Gly Arg Phe 1 5 10 15 Pro Tyr <210> 195 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR <400> 195 Ala Ala His Gly Ala Phe Ala Ala Arg Asn Asp Pro Arg Gln Trp Arg 1 5 10 15 Tyr <210> 196 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 196 Ala Ala Gln Tyr Gly Thr Cys Gln Gly Leu Leu Ser Arg Tyr Phe Ala 1 5 10 15 Tyr <210> 197 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 197 Ala Ala Ala His Glu Pro Gly Ser Trp Thr Asp Ile Glu Ala Arg Gly 1 5 10 15 Lys Ile Ser Asp Asp Pro Phe Gly Tyr 20 25 <210> 198 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 198 Ala Arg Ala Ser Phe Arg Gly Ser Trp Phe Phe Glu Gly 1 5 10 <210> 199 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 199 Ala Ala Ala Arg Arg Trp Tyr Tyr Glu Asn Ser Cys Leu Lys Val Leu 1 5 10 15 Arg Ser Pro Gly Asp Tyr 20 <210> 200 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 200 Ala Ala Gly Pro Pro Ser Gly Pro Leu Arg Ala Cys His Glu Ser Val 1 5 10 15 Phe Gly Tyr <210> 201 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 201 Lys Thr Val Arg Asp Pro Ala Ser Pro Pro Ser Cys Ser Gly Gly Tyr 1 5 10 15 <210> 202 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 202 Ala Ala Asp Arg Val Leu Gly Arg Cys Ser Arg Arg Leu Leu Ser Asp 1 5 10 15 Phe Gly Tyr <210> 203 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 203 Ala Ala Gly Thr Tyr Tyr Ser Asp Tyr Asp Pro Pro Arg Tyr Glu Tyr 1 5 10 15 Lys Tyr <210> 204 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 204 Ala Ala Lys Glu Arg Pro Leu Cys Gly Ser Phe Trp Glu Arg Gly Asp 1 5 10 15 Glu Tyr Ala Ser 20 <210> 205 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 205 Ala Ala Val Ser Trp Ala Cys Trp Arg Leu Ser Gly Thr Gly Phe Asn 1 5 10 15 Tyr <210> 206 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 206 Ala Ala Asp Leu Gly Val Asp Asp Tyr Ser Asp Tyr Leu Asp His Pro 1 5 10 15 Phe Gly Tyr <210> 207 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 207 Ala Ala Asp Ile Gly Gly Ser Trp Pro Arg Lys Pro His Pro Asp Pro 1 5 10 15 Asn Phe Gly Gly Glu Cys Gly Gly Tyr Gly Met Ala Phe 20 25 <210> 208 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 208 Ala Ala Asp Ser Ser Ser Leu Pro Cys Tyr Pro Arg Ala Ala Gln Phe 1 5 10 15 Pro Arg Leu Arg Tyr 20 <210> 209 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 209 Ala Ala Arg Pro Ala Pro Val Ser Gly Ile Thr Arg Phe Arg Leu Asn 1 5 10 15 Arg Ser Leu Leu Pro Asn Glu Tyr Asn Ser 20 25 <210> 210 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 210 Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala 1 5 10 15 Asn Phe Arg Tyr 20 <210> 211 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 211 Ala Ala Glu Gly Gly Trp Arg Asp Tyr Val Arg Ser Trp Gly Arg Asn 1 5 10 15 Phe Gly Tyr <210> 212 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 212 Ala Ala Asp Leu Trp Arg Gly Pro Pro Phe Gly Gly Tyr Trp Ser Pro 1 5 10 15 Thr Lys Ser Glu Phe Ala Tyr 20 <210> 213 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 213 Ala Ala Asn Arg Arg Ala Tyr Pro Tyr Gly Gly Asp Cys Arg Val Arg 1 5 10 15 His Ala Glu Phe Asp Tyr 20 <210> 214 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 214 Ala Ala Arg Phe Arg Ala His Asp Gly Tyr Trp Asn Trp Gln Asn Ala 1 5 10 15 Gly Asn Tyr Asn Tyr 20 <210> 215 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 215 Ala Ala Arg His Tyr Trp Ser Ala Gly Phe Pro Tyr 1 5 10 <210> 216 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 216 Ala Ala Asp Arg Val Leu Gly Arg Cys Ser Arg Arg Ile Leu Ser Asp 1 5 10 15 Phe Gly Tyr <210> 217 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 217 Ala Arg Ser Ser Pro Arg Thr Val Val Ala Gly Phe Gly Asp 1 5 10 <210> 218 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 218 Ala Ala Gly Leu Asp Gly Gly Asn Gly Tyr Leu Pro Leu Asn Trp Ala 1 5 10 15 Gly Phe Arg Tyr 20 <210> 219 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 219 Ala Arg Gly Pro Phe Gly Cys Tyr Ser Val Ser Gly Cys Tyr Arg Lys 1 5 10 15 Gly Gly Val Asp Asn Tyr 20 <210> 220 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 220 Lys Thr Val Arg Asp Pro Thr Ser Pro Arg Ser Cys Ser Gly Gly Tyr 1 5 10 15 <210> 221 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 221 Ala Ala Tyr His Ser Gly Ser Trp Cys Tyr Leu Pro His Leu Gly Ser 1 5 10 15 Tyr Gly Tyr <210> 222 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 222 Ala Ala Gly Trp Arg Leu Ser Leu Arg Val Ser Asp Phe Asn Tyr 1 5 10 15 <210> 223 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 223 Ala Ala Ser Phe Arg Pro Thr Trp Phe Cys Arg Gly Leu Ala Pro Tyr 1 5 10 15 Lys Tyr Asn Leu 20 <210> 224 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 224 Thr Ser Ala Ser Leu Thr Trp Asp Gly Gly Asn Trp Tyr Cys Pro Thr 1 5 10 15 His Gly Tyr <210> 225 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 225 Ala Ala Gly Arg Thr Tyr Asp Cys Tyr Ser Gly Thr Cys Ser 1 5 10 <210> 226 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 226 Ala Ala Gly Ala Tyr Arg Ala Ser Phe Thr Tyr 1 5 10 <210> 227 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 227 Asn Thr Met Trp Gly Ala Arg Gln Asn Lys Asp 1 5 10 <210> 228 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 228 Ala Ala Asn Arg Arg Pro Tyr Pro Tyr Gly Gly Asp Cys Arg Leu Arg 1 5 10 15 His Ala Glu Phe Asp Tyr 20 <210> 229 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 229 Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Ala Trp Ala 1 5 10 15 Gly Phe Arg Tyr 20 <210> 230 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 230 Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala 1 5 10 15 Arg Phe Arg Tyr 20 <210> 231 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 231 Ala Ala Gly Val Ala Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala 1 5 10 15 Gly Phe Arg Tyr 20 <210> 232 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 232 Ala Ala Gly Phe Ala Gly Cys Tyr Gly Ser Ser Trp Tyr Gly Ser Ala 1 5 10 15 Asp Phe Gly Tyr 20 <210> 233 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <220> <221> misc_feature <222> (1)..(3) <223> Xaa can be any naturally occurring amino acid <400> 233 Xaa Xaa Xaa Ser Thr Ala Tyr Tyr 1 5 <210> 234 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 234 Gln Val Arg Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu 1 5 10 15 Thr Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 235 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 235 Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Cys Glu Met Val Ala 1 5 10 15 Tyr <210> 236 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 236 Ala Asp Ser Thr Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys 1 5 10 15 His Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly 20 25 30 Val Tyr Tyr Cys 35 <210> 237 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> FR4 <400> 237 Gly Gln Gly Thr Arg Val Thr Val Ser Ser 1 5 10

Claims (10)

In the polypeptide capable of binding to SFTSV,
Three complementarity determining regions CDR1-3 are included, the CDR1 sequence is or comprises one of the sequences represented by SEQ ID NO: 1-74, and the CDR2 sequence is one of the sequences represented by SEQ ID NO: 75-151 It is or comprises the same, and the CDR3 sequence is one of the sequences represented by SEQ ID NO: 152-232, or a polypeptide capable of binding to SFTSV, characterized in that it comprises the same. The method of claim 1,
The polypeptide further comprises four framework regions FR1-4, wherein the FR1-4 and the CDR1-3 are cross-arranged. A polypeptide capable of binding to SFTSV. The method of claim 2,
The polypeptide capable of binding to SFTSV, characterized in that the monoclonal antibody. The method of claim 2,
The polypeptide capable of binding to SFTSV, characterized in that VHH. The method of claim 4,
The polypeptide capable of binding to SFTSV, characterized in that the camel-derived VHH or humanized VHH. Application of the polypeptide according to any one of claims 1 to 5 in the manufacture of an SFTSV detection agent or an SFTSV therapeutic drug. A nucleic acid coding sequence, characterized in that it encodes the polypeptide according to any one of claims 1 to 5. Application of the nucleic acid coding sequence according to claim 7 in the manufacture of an SFTSV therapeutic drug. In the SFTSV detection kit,
SFTSV, characterized in that it includes a detection antibody, a solid substrate, and a coated antibody coated on the solid substrate, wherein the detection antibody and the coated antibody are one of the polypeptides according to any one of claims 1 to 5, respectively. Detection kit. The method of claim 9,
The CDR sequence of the detection antibody is.
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, the sequence of CDR2 is SEQ ID NO: 99, and the sequence of CDR3 is SEQ ID NO: 166.

Images