NbES9 single-domain antibody, NbES9-TAT fusion single-domain antibody and application thereof

Abstract

The invention discloses an NbES9 single-domain antibody, an NbES9-TAT fusion single-domain antibody and application thereof, the NbES9 single-domain antibody is used for specific binding with an EB virus nuclear antigen, the NbES9 single-domain antibody is composed of a skeleton region FR and a complementary determining region CDR, and the complementary determining region CDR comprises a CDR1 as shown in SEQ ID NO.1, a CDR2 as shown in SEQ ID NO.2 and a CDR3 as shown in SEQ ID NO.3. The NbES9 single-domain antibody, the NbES9-TAT fusion single-domain antibody, the NbES9 single-domain antibody, the NbES9-TAT fusion single-domain antibody and the application of the NbES9 single-domain antibody and the NbES9-TAT fusion The NbES9-TAT fusion single-domain antibody is a fusion protein of a NbES9 single-domain antibody and a TAT peptide fragment of an HIV-1 virus. The NbES9 single-domain antibody and the NbES9-TAT fusion single-domain antibody provide potential choices for research and development of clinical drugs for treating EBV latent infection and EB virus related tumors.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a NbES9 single-domain antibody, a NbES9-TAT fusion single-domain antibody and uses thereof. BACKGROUND

[0002] Epstein-Barr virus (EBV) is a DNA virus and a member of the herpes virus family. Human herpes viruses are good at establishing latent infection, and EBV is also good at establishing latent infection. EBV latent infection is an important risk factor for causing human cancer. During latent infection, the EBNA1 protein can fix the EBV latent genome on the host cell chromosome. Long-term chromosome anchoring is a major inducement to trigger various cell-derived human cancers. The DNA binding domain (DBD) of EBNA1 can bind to the EBV latent genome and the host chromosome to maintain EBV latent infection. Therefore, EBNA1 is an effective target for treating EBV latent infection and related tumors.

[0003] Antibody therapy with high target specificity has been increasingly applied to various human diseases. In recent years, some studies have also begun to focus on the development of intracellular targeting antibodies, including intracellular antibodies and intracellular delivery of exogenous antibodies. Among them, the trans-activator (TAT) derived from human immunodeficiency virus type 1 (HIV-1) has been shown to deliver biological macromolecules into cells. In nature, there is a type of antibody similar to human antibodies in camels and sharks, called heavy-chain antibody (HcAb), with a molecular weight of about 95 kDa. Although this antibody lacks the light chain part in ordinary monoclonal antibodies, it still has a high affinity interaction with antigens. In HcAb, the binding site with the antigen is completed by the heavy variable domain (VHHs), also known as single-domain antibody (sdAb). Single-domain antibodies have been applied to various fields of biomedicine due to their small molecular weight, high antigen affinity, low immunogenicity, good tissue penetration, and other advantages, including disease diagnosis and treatment, affinity purification reagents, auxiliary protein structure analysis, biosensors, etc.

[0004] Currently, the development of EBNA1 targeting drugs mainly focuses on small molecule compounds, nucleic acids and peptides. These have more or less problems such as low selectivity and poor stability. In recent years, antibody therapy of biological macromolecules has been increasingly applied to various human diseases, which is a promising new drug screening direction instead of small molecule drugs. SUMMARY

[0005] In order to solve the problems in the prior art, the present application aims to provide an NbES9 single-domain antibody, an NbES9-TAT fusion single-domain antibody and uses thereof.

[0006] The specific technical solutions of the present application are as follows:

[0007] The first aspect of the present application provides an NbES9 single-domain antibody for specifically binding to an Epstein-Barr virus nuclear antigen, which is composed of a framework region FR and a complementarity determining region CDR, and the complementarity determining region CDR includes CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.

[0008] Further, the amino acid sequence of the NbES9 single-domain antibody is shown in SEQ ID NO. 4.

[0009] The second aspect of the present application provides an NbES9-TAT fusion single-domain antibody, which is a fusion protein of the NbES9 single-domain antibody and a TAT peptide segment of HIV-1 virus.

[0010] Further, the C-terminal of the NbES9 single-domain antibody is connected with the TAT peptide segment of HIV-1 virus.

[0011] Preferably, the amino acid sequence of the TAT peptide segment is shown in SEQ ID NO. 5.

[0012] Preferably, the amino acid sequence of the NbES9-TAT fusion single-domain antibody is shown in SEQ ID NO. 6.

[0013] The third aspect of the present application provides a polynucleotide sequence encoding the NbES9 single-domain antibody or encoding the NbES9-TAT fusion single-domain antibody.

[0014] Further, the nucleotide sequence encoding the NbES9 single-domain antibody is shown in SEQ ID NO. 7.

[0015] Preferably, the nucleotide sequence encoding the NbES9-TAT fusion single-domain antibody of claim 3 or 4 is shown in SEQ ID NO. 8.

[0016] The fourth aspect of the present application provides a biological material expressing the NbES9 single-domain antibody or expressing the NbES9-TAT fusion single-domain antibody or containing the polynucleotide sequence, and the biological material includes at least one of an expression cassette, a vector, a recombinant microorganism and a cell line.

[0017] The fifth aspect of the present application provides a preparation method of the NbES9-TAT fusion single-domain antibody, wherein a polynucleotide sequence encoding the NbES9-TAT fusion single-domain antibody is expressed in an E. coli prokaryotic expression system.

[0018] The sixth aspect of the present application provides the use of the NbES9 single-domain antibody or the NbES9-TAT fusion single-domain antibody in any one of the following:

[0019] (1) preparation of a reagent for detecting and / or diagnosing an EBV-related disease;

[0020] (2) preparation of a reagent specifically binding to EBV EBNA1 protein;

[0021] (3) preparation of a drug for preventing and / or treating latent infection of EBV;

[0022] (4) preparation of a drug for preventing and / or treating an EBV-related tumor;

[0023] Preferably, the EBV-related tumor is an EBV-positive tumor.

[0024] Preferably, the EBV-related tumor is an EBV-positive nasopharyngeal carcinoma or an EBV-positive lymphoma.

[0025] The seventh aspect of the present application provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the NbES9 single-domain antibody or the NbES9-TAT fusion single-domain antibody.

[0026] The present application has the following beneficial effects:

[0027] The present application provides a NbES9 fusion single-domain antibody, and proves that it can be used for recognizing and binding to EBV EBNA1 antigen. Further, the present application provides a NbES9-TAT fusion single-domain antibody, and proves that it can be used as a tumor growth inhibitor for EBV-related tumors. The NbES9-TAT fusion single-domain antibody significantly inhibits the EBV copy number under latent infection in EBV-positive nasopharyngeal carcinoma cells and EBV-positive Burkitt's lymphoma cells, and has a good tumor cell proliferation inhibition effect. Meanwhile, in a nude mouse tumor model derived from a cell line, the NbES9-TAT fusion single-domain antibody significantly inhibits the growth of EBV-positive transplanted tumors, and has no toxic side effects on the organs of mice after administration. The development of NbES9-TAT provides an experimental basis for the research of targeted drugs for the clinical treatment of EBV latent infection and related tumors and individualized treatment of tumors.

[0028] Specifically:

[0029] The present application first evaluates the affinity of the NbES9 single-domain antibody to the EBNA1 antigen. Further, the present application selects a plurality of human-derived tumor cell lines and cell line-derived xenograft (CDX) models to reveal the therapeutic effect of the NbES9-TAT fusion single-domain antibody on EBV latent infection and its related tumors. The models include human nasopharyngeal carcinoma cell line C666-1 cells containing the EBV genome, human Burkitt's lymphoma cell line Raji cells containing the EBV genome, and human nasopharyngeal carcinoma cell line CNE-2 cells and human lung cancer cell line A549 cells without the EBV genome.

[0030] In the affinity evaluation of EBNA1, surface plasmon resonance experiments show that the dissociation constant of the NbES9 single-domain antibody to the recombinant EBNA1-DBD protein in vitro is 6.4 x 10 -7 M. This indicates that the NbES9 single-domain antibody has good affinity to EBNA1-DBD. In in vitro cell experiments, the NbES9-TAT fusion single-domain antibody significantly inhibits the proliferation activity of EBV-positive C666-1 cells and Raji cells, but has no obvious effect on the proliferation ability of EBV-negative CNE-2 cells.

[0031] In the mouse xenograft model, the NbES9-TAT fusion single-domain antibody significantly slows down the growth of EBV-positive nasopharyngeal carcinoma xenografts and EBV-positive lymphoma transplants. Compared with the positive drug cisplatin treatment, the NbES9-TAT fusion single-domain antibody has a more obvious effect on inhibiting the growth of EBV-positive nasopharyngeal carcinoma xenografts. In addition, pathological sections of mouse organs show no obvious inflammatory infiltration and tissue damage, indicating that the NbES9-TAT fusion single-domain antibody has no toxic side effects. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The figure is an expression diagram of the NbES9 single-domain antibody and the NbES9-TAT fusion single-domain antibody.

[0033] Figure 2 The figure is a curve graph of surface plasmon resonance for detecting the affinity of the NbES9 single-domain antibody to EBNA1.

[0034] Figure 3 The figure is a result graph of the NbES9-TAT fusion single-domain antibody inhibiting tumor cell proliferation. It includes the EBV-positive nasopharyngeal carcinoma cell (C666-1) group, the EBV-negative nasopharyngeal carcinoma cell (CNE-2) group, and the EBV-positive Burkitt's lymphoma (Raji) group.

[0035] Figure 4 The figure is a result graph of the NbES9-TAT fusion single-domain antibody reducing the EB virus copy number in EBV-positive Raji cells. Real-time quantitative PCR method is used for detection.

[0036] Figure 5 Graph of the results of the NbES9-TAT fusion single-domain antibody in reducing the EBV BKRF1, LMP1, BZLF1 and BHLF1 transcription levels in EBV-positive Raji cells. Detected by real-time quantitative PCR.

[0037] Figure 6 Graph of the growth of EBV-positive nasopharyngeal carcinoma transplanted tumors after administration of the NbES9-TAT fusion single-domain antibody. Divided into negative control (Vehicle) group, fusion single-domain antibody administration (NbES9-TAT) group, and positive drug cisplatin (Cisplatin) group.

[0038] Figure 7 Graph of the growth of EBV-positive nasopharyngeal carcinoma transplanted tumors after administration of the NbES9-TAT fusion single-domain antibody. Divided into negative control (Vehicle) group, fusion single-domain antibody administration (NbES9-TAT) group, and positive drug cisplatin (Cisplatin) group.

[0039] Figure 8 Graph of the growth of EBV-positive nasopharyngeal carcinoma transplanted tumors after administration of the NbES9-TAT fusion single-domain antibody. Divided into negative control (Vehicle) group, fusion single-domain antibody administration (NbES9-TAT) group, and positive drug cisplatin (Cisplatin) group.

[0040] Figure 9 HE staining of mouse organ pathological sections after administration of the NbES9-TAT fusion single-domain antibody. Includes heart, liver, spleen, lung and kidney. Divided into negative control (Vehicle) group and fusion single-domain antibody administration (NbES9-TAT) group. DETAILED DESCRIPTION

[0041] The present application will be further described below in connection with specific embodiments, and the advantages and features of the present application will become more apparent from the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present application.

[0042] The reagents and consumables used in the following examples, unless otherwise specified, can be obtained commercially.

[0043] Unless otherwise specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions can be used.

[0044] Example 1: Preparation of NbES9 single-domain antibody and NbES9-TAT fusion single-domain antibody

[0045] I. NbES9 single-domain antibody and NbES9-TAT fusion single-domain antibody

[0046] The NbES9 single-domain antibody is screened by using EBNA1-DBD as antigen, and the NbES9 single-domain antibody specifically binds to Epstein-Barr virus nuclear antigen (EBNA1). The NbES9 single-domain antibody is composed of a framework region FR and a complementarity determining region CDR, and the complementarity determining region CDR includes CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3. In this embodiment, the amino acid sequence of the NbES9 single-domain antibody is shown in SEQ ID NO. 4. In this embodiment, the nucleotide sequence encoding the NbES9 single-domain antibody is shown in SEQ ID NO. 7.

[0047] The NbES9-TAT fusion single-domain antibody is a fusion protein of the NbES9 single-domain antibody and a trans-activator (TAT) derived from human immunodeficiency virus type 1 (HIV-1). In this embodiment, the amino acid sequence of the TAT peptide segment in the NbES9-TAT fusion single-domain antibody is shown in SEQ ID NO. 5. In this embodiment, the amino acid sequence of the NbES9-TAT fusion single-domain antibody is shown in SEQ ID NO. 6. In this embodiment, the nucleotide sequence encoding the NbES9-TAT fusion single-domain antibody is shown in SEQ ID NO. 8.

[0048] The amino acid sequence of the complementarity determining region CDR1 of the NbES9 single-domain antibody is SEQ ID NO. 1:

[0049] GTIFQSDFM

[0050] The amino acid sequence of the complementarity determining region CDR2 of the NbES9 single-domain antibody is SEQ ID NO. 2:

[0051] EFVAGIDYGTNTYY

[0052] The amino acid sequence of the complementarity determining region CDR3 of the NbES9 single-domain antibody is SEQ ID NO. 3:

[0053] AATVWSDTDFAY

[0054] The amino acid sequence of the NbES9 single-domain antibody is SEQ ID NO. 4:

[0055] QVQLQESGGGLVQAGGSLRLSCAASGTIFQSDFMGWYRQAPGKEREFVAGIDYGTNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATVWSDTDFAYWGQGTQVTVSS

[0056] Amino acid sequence of TAT peptide segment in NbES9-TAT fusion single-domain antibody SEQ ID NO. 5:

[0057] YGRKKRRQRRR

[0058] Amino acid sequence of NbES9-TAT fusion single-domain antibody SEQ ID NO. 6:

[0059] QVQLQESGGGLVQAGGSLRLSCAASGTIFQSDFMGWYRQAPGKEREFVAGIDYGTNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATVWSDTDFAYWGQGTQVTVSSLEHHHHHHYGRKKRRQRRR

[0060] Nucleic acid sequence encoding NbES9 single-domain antibody SEQ ID NO. 7:

[0061] CAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCACTATTTTTCAATCTGATTTTATGGGCTGGTATCGCCAGGCGCCGGGCAAAGAACGCGAATTTGTTGCCGGTATTGATTATGGTACTAATACCTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCTACTGTTTGGTCTGATACTGATTTTGCTTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC

[0062] Nucleotide sequence encoding NbES9-TAT fusion single-domain antibody SEQ ID NO. 8:

[0063] CAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCACTATTTTTCAATCTGATTTTATGGGCTGGTATCGCCAGGCGCCGGGCAAAGAACGCGAATTTGTTGCCGGTATTGATTATGGTACTAATACCTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCTACTGTTTGGTCTGATACTGATTTTGCTTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCCTCGAGCACCACCACCACCACCACTATGGCCGCAAAAAACGCCGCCAGCGCCGTCGC

[0064] II. Expression and purification of NbES9-TAT fusion single-domain antibody

[0065] The nucleotide sequence of the NbES9-TAT fusion single-domain antibody in this example was inserted into the pET22b prokaryotic expression vector, and the mature Escherichia coli BL21(DE3) prokaryotic cell expression system was used for protein expression. The plasmid was transformed into BL21(DE3) using heat shock transformation. The expression conditions of the NbES9-TAT fusion single-domain antibody were as follows: 16°C, 220 rpm, 0.5 mM IPTG induction for 12 hours. After expression, the bacterial cells were collected by centrifugation at 4°C and 6700 g for 20 minutes, resuspended in 200 mL of pyrogen-free buffer containing 20 mM Tris-HCl pH 8.0, 1 M NaCl, 5 mM β-ME, and 5 mM imidazole pH 8.0. The bacterial cells were broken at a pressure of 800 bar using a high-pressure cell disruptor, and the supernatant was collected by ultracentrifugation. The NbES9-TAT fusion single-domain antibody in the supernatant was purified using Ni-NTA affinity chromatography. After purification, the protein purity was observed by SDS-PAGE electrophoresis and Coomassie blue staining. Finally, the protein was concentrated using an ultrafiltration tube and replaced into sterile PBS, and stored at -80°C. The expression method of the NbES9 single-domain antibody and the TAT fusion NbES9 single-domain antibody was the same.

[0066] Figure 1The Coomassie blue staining diagram of the purified NbES9 single domain antibody and NbES9-TAT fusion single domain antibody. The molecular weight of the NbES9 single domain antibody and the NbES9-TAT fusion single domain antibody is 13 kDa and 15.5 kDa, respectively.

[0067] Example 2: Evaluation of the affinity of the NbES9 single domain antibody to the EBNA1-DBD protein

[0068] In this example, surface plasmon resonance experiments were used to analyze the recognition of the NbES9 single domain antibody to EBNA1. The specific steps include the following:

[0069] First, the 3D dextran sensor chip coupled with recombinant EBNA1-DBD was combined with different concentrations of flow phase NbES9 single domain antibody. The concentrations were set to 5, 2.5, 1.25, 0.63, 0.31, 0.16 and 0.08 μM, respectively. The flow phase was loaded at a speed of 2 μL / s, and after flowing through the chip placed on the biomolecular interaction instrument, the sensor chip captured the combination between the immobilized EBNA1-DBD and the 5E2-12 monoclonal antibody.

[0070] Then, the BIevaluation software was used to analyze the sensor data obtained representing the binding reaction. Through the software fitting, the dissociation constant (K D ), K D represents the affinity index between the NbES9 single domain antibody and EBNA1.

[0071] The experimental results are shown in Figure 2 . Figure 2 The curve graph of the surface plasmon resonance detection of the affinity of the NbES9 single domain antibody to EBNA1-DBD. The K D is 6.4 x 10 -7 M, indicating that the NbES9 single domain antibody has strong affinity to the EBNA1 encoded by the Epstein-Barr virus.

[0072] Example 3: Evaluation of the inhibitory effect of the NbES9-TAT fusion single domain antibody on the proliferation of tumor cells in vitro

[0073] In this example, CCK-8 experiments were used to analyze the cell proliferation activity. Cell Counting Kit (CCK-8) was used to detect cell proliferation activity. Before detection, 5 x 10 4C666-1, CNE-2 and Raji cells were passaged into 96-well cell culture plates at 100 μL per well, and then cultured in the cell incubator for 12 hours. Then, NbES9-TAT fusion single-domain antibody was added to the cell culture plates to a final concentration of 30 μM, and the same concentration of NbES9 single-domain antibody was added to the negative control wells. After 24 hours of culture, 10 μL of CCK-8 working solution was added to each well, and the plates were returned to the cell incubator for 1 hour. Subsequently, the absorbance (OD) at 450 nm was measured using a microplate reader. 450

[0074] The experimental results are shown in Figure 3 . Figure 3 The figure shows the proliferation detection results on EBV-positive C666-1, Raji and EBV-negative CNE-2 cells. Compared with the administration of simple NbES9 single-domain antibody, the intervention of 30 μM NbES9-TAT fusion single-domain antibody significantly inhibited the proliferation of EBV-positive nasopharyngeal carcinoma cells and lymphoma cells, but had no significant effect on the EBV-negative nasopharyngeal carcinoma cell line.

[0075] Example 4: Effect of NbES9-TAT fusion single-domain antibody on EBV latent infection

[0076] This example uses real-time quantitative PCR to detect the effect of NbES9-TAT fusion single-domain antibody on EBV latent infection. The specific steps include the following:

[0077] C666-1 and Raji cells were inoculated into six-well cell culture plates at 1 x 10 6 cells per well, and then cultured in the cell incubator for 12 hours. Then, NbES9-TAT fusion single-domain antibody was added to a concentration of 30 μM, and the same volume of PBS was added to the negative control group. After 24 hours of culture, total RNA was extracted from the cells and dissolved in 20 μL of enzyme-free water. Then, the total RNA was reverse transcribed into cDNA. The expression level of mRNA was detected by a fluorescence quantitative PCR instrument.

[0078] The experimental results are shown in Figure 4 and 5 .

[0079] Figure 4 The figure shows the transcription levels of EBER1 and EBER2 after intervention with NbES9-TAT fusion single-domain antibody. EBER is a small RNA transcribed from EBV, which appears in a high copy form during EBV viral latent infection, and its transcription level reflects the copy number of the EB virus genome. After intervention with NbES9-TAT fusion single-domain antibody, the copy number of EBER1 and EBER2 in EBV-positive Raji cells was significantly reduced.

[0080] ​Figure 5 The transcription levels of BKRF1, LMP1, BZLF1 and BHLF1 in the latent genome of EBV were intervened by NbES9-TAT fusion single-domain antibody. After the intervention of fusion single-domain antibody, the transcription levels of BKRF1, LMP1, BZLF1 and BHLF1 in EBV-positive Raji cells were significantly reduced, indicating the loss of EBV genome after intervention.

[0081] Example 5: Evaluation of the inhibitory effect of NbES9-TAT fusion single-domain antibody on EBV-positive xenograft tumor

[0082] I. Establishment of BALB / c nude mouse xenograft tumor model and treatment of NbES9-TAT fusion single-domain antibody

[0083] 5-week-old male BALB / c nude mice were used to establish mouse xenograft tumor models. First, 3x10 6 C666-1 cells or 3x10 6 Raji cells were collected from in vitro culture and resuspended in sterile PBS pre-cooled on ice, then mixed with 50% cold Matrigel and placed on ice for standby. Then the cell mixture was quickly injected into the right subcutaneous of BALB / c nude mice with rich blood vessels, continued to be raised and observed daily, and when the tumor diameter reached about 7mm, the grouping was carried out.

[0084] There were two kinds of xenograft tumor models in this experiment. The first was the EBV-positive nasopharyngeal carcinoma xenograft tumor model (EBV + C666-1 Xenograft tumor), which was divided into three groups, including the negative control (Vehicle) group, the positive drug cisplatin (Cisplatin) group and the treatment (NbES9-TAT) group. The second was the EBV-positive Burkitt's lymphoma xenograft tumor model (EBV + Raji Xenograft tumor), which was divided into two groups, Vehicle group and drug intervention (NbES9-TAT) group. Each group above had 3 mice.

[0085] In the nasopharyngeal carcinoma xenograft tumor model, the cisplatin group was used as the positive control drug, and 4mg / kg of cisplatin was injected intraperitoneally twice a week. In the nasopharyngeal carcinoma and lymphoma xenograft tumor models, the NbES9-TAT treatment group was injected intratumorally with 30μM of fusion single-domain antibody NbES9-TAT every 3 days, and the intratumoral injection followed the three-point injection of each mouse, with each point injection volume of about 30μL, equivalent to a single-domain antibody dosage of 40μg. The Vehicle group was injected intratumorally with the same volume of PBS at the same time points.

[0086] After administration, the tumor volume of the mice was measured every other day, and the tumor volume measurement needed to be completed after the last administration and before the next administration. In the nasopharyngeal carcinoma xenograft model, the mice were sacrificed on the 20th day after treatment, and in the lymphoma model, the mice were sacrificed on the 26th day, and the solid tumors were isolated.

[0087] II. Experimental results

[0088] Figure 6 The growth curve of EBV-positive nasopharyngeal carcinoma xenografts after administration of NbES9-TAT fusion single-domain antibody. Divided into negative control (Vehicle) group, fusion single-domain antibody administration (NbES9-TAT) group, and positive drug cisplatin (Cisplatin) group. Compared with the Vehicle group, the cisplatin or NbES9-TAT group can significantly inhibit the growth of nasopharyngeal carcinoma xenografts, and the inhibition effect of the NbES9-TAT group is more obvious than that of the cisplatin group.

[0089] Figure 7 The graph of EBV-positive nasopharyngeal carcinoma solid tumors after administration of NbES9-TAT fusion single-domain antibody. Divided into negative control (Vehicle) group, fusion single-domain antibody administration (NbES9-TAT) group, and positive drug cisplatin (Cisplatin) group. Compared with the Vehicle group, the cisplatin or NbES9-TAT group can significantly inhibit the final volume of nasopharyngeal carcinoma xenografts, and the inhibition effect of the NbES9-TAT group is more obvious than that of the cisplatin group.

[0090] Figure 8 The growth curve of EBV-positive lymphoma xenografts after administration of NbES9-TAT fusion single-domain antibody. Divided into negative control (Vehicle) group and fusion single-domain antibody administration (NbES9-TAT) group. Compared with the Vehicle group, the NbES9-TAT group can significantly inhibit the growth of lymphoma xenografts.

[0091] Figure 9 The HE staining of mouse organ pathological sections after administration of NbES9-TAT fusion single-domain antibody. Including heart, liver, spleen, lung and kidney. Divided into negative control (Vehicle) group and fusion single-domain antibody administration (NbES9-TAT) group. After treatment with NbES9-TAT fusion single-domain antibody, there was no inflammatory cell infiltration and tissue damage in the five major organs of the mice.

[0092] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any equivalent transformation of the technical solutions of the present application by a person of ordinary skill in the art through reading the specification of the present application is covered by the claims of the present application.

Claims

1. An NbES9 single-domain antibody, characterized in that, The NbES9 single-domain antibody is used to specifically bind to EB virus nuclear antigen. The NbES9 single-domain antibody consists of a backbone region FR and a complementarity-determining region CDR. The complementarity-determining region CDR includes CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.

3.

2. The NbES9 single-domain antibody according to claim 1, characterized in that, The amino acid sequence of the NbES9 single-domain antibody is shown in SEQ ID NO.

4.

3. An NbES9-TAT fusion single-domain antibody, characterized in that, The fusion single-domain antibody is a fusion protein of the NbES9 single-domain antibody of claim 1 and the TAT peptide of HIV-1 virus.

4. The fusion single-domain antibody according to claim 3, characterized in that, The C-terminus of the NbES9 single-domain antibody is linked to the TAT peptide of the HIV-1 virus. Preferably, the amino acid sequence of the TAT peptide is shown in SEQ ID NO.5; Preferably, the amino acid sequence of the NbES9-TAT fusion single-domain antibody is shown in SEQ ID NO.

6.

5. A polynucleotide sequence, characterized in that, The polynucleotide sequence encodes the NbES9 single-domain antibody of claim 1 or 2, or the NbES9-TAT fusion single-domain antibody of claim 3 or 4.

6. The polynucleotide sequence according to claim 5, characterized in that, The nucleotide sequence encoding the NbES9 single-domain antibody of claim 1 or 2 is shown in SEQ ID NO.7; Preferably, the nucleotide sequence encoding the NbES9-TAT fusion single-domain antibody of claim 3 or 4 is shown in SEQ ID NO.

8.

7. A biomaterial, characterized in that, The biomaterial expresses the NbES9 single-domain antibody of claim 1 or 2, or the NbES9-TAT fusion single-domain antibody of claim 3 or 4, or contains the polynucleotide sequence of claim 5 or 6, and the biomaterial includes at least one of expression cassette, vector, recombinant microorganism and cell line.

8. A method for preparing the NbES9-TAT fusion single-domain antibody according to claim 3 or 4, characterized in that, The multinucleotide sequence encoding the NbES9-TAT fusion single-domain antibody as described in claim 3 or 4 was expressed in an Escherichia coli prokaryotic expression system.

9. Use of the NbES9 single-domain antibody of claim 1 or 2, or the NbES9-TAT fusion single-domain antibody of claim 3 or 4, in any of the following: (1) Prepare reagents for detecting and / or diagnosing EB virus-related diseases; (2) Preparation of reagents that specifically bind to the EBNA1 protein of EB virus; (3) Prepare drugs for the prevention and / or treatment of latent EB virus infection; (4) To prepare drugs for the prevention and / or treatment of EB virus-related tumors; Preferably, the EBV-related tumor is EBV-positive nasopharyngeal carcinoma or EBV-positive lymphoma.

10. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition includes the NbES9 single-domain antibody as described in claim 1 or 2, or the NbES9-TAT fusion single-domain antibody as described in claim 3 or 4.

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