NbE7 single-domain antibody, NbE7-TAT fusion single-domain antibody and application of NbE7-TAT fusion single-domain antibody

Abstract

The invention discloses an NbE7 single-domain antibody, an NbE7-TAT fusion single-domain antibody and application of the NbE7 single-domain antibody and the NbE7-TAT fusion single-domain antibody, the NbE7 single-domain antibody is used for being specifically combined with an EB virus nuclear antigen, the NbE7 single-domain antibody is composed of a framework region FR and a complementary determining region CDR, and the complementary determining region CDR comprises a CDR1 shown in SEQ ID NO.1, a CDR2 shown in SEQ ID NO.2 and a CDR3 shown in SEQ ID NO.3. The NbE7 single-domain antibody is used for being specifically combined with an EB virus nuclear antigen. The NbE7-TAT fusion single-domain antibody is a fusion protein of an NbE7 single-domain antibody and a TAT peptide fragment of an HIV-1 virus. The NbE7 single-domain antibody and the NbE7-TAT fusion single-domain antibody provided by the invention provide a new thought for the development of drugs for treating EBV latent infection and EB virus related tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an NbE7 single-domain antibody, an NbE7-TAT fusion single-domain antibody, and their uses. Background Technology

[0002] Epstein-Barr virus (EBV) is a DNA virus, also known as human herpesvirus 4 (HHV-4). Most EBV infections are asymptomatic, thus often going unnoticed. Under the influence of the host's immune system, EBV establishes latent infection during long-term infection. Latent EBV infection is a significant risk factor for human cancer. During latent infection, the EBNA1 protein encoded by EBV enables the viral genome to be stably propagated during host cell mitosis. EBNA1 is closely related to the occurrence and development of tumors associated with latent EBV infection. The DNA-binding domain (DBD) on EBNA1 maintains latent EBV infection by binding to EBV episomes. Therefore, EBNA1 is an effective target for the treatment of latent EBV infection and related tumors.

[0003] Antibody therapies with high target specificity are increasingly being used for various human diseases. In recent years, some research has also focused on the development of intracellular targeted antibodies, including intracellular antibodies and intracellular delivery of exogenous antibodies. Among these, the trans-activator (TAT) derived from human immunodeficiency virus type 1 (HIV-1) has been shown to deliver biomolecules into cells. In the bodies of camels and sharks in nature, there exists a type of antibody that is different from but similar to human antibodies, called heavy-chain antibody (HcAb), with a molecular weight of approximately 95 kDa. Although this antibody lacks the light chain portion of ordinary monoclonal antibodies, it still exhibits a high affinity interaction with antigens. In HcAb, the antigen-binding site is formed by the heavy variable domain (VHHs). VHHs typically have a molecular weight of 12-15 kDa and are the smallest antigen-binding fragments found in nature; they are also known as single-domain antibodies (sdAb). Single-domain antibodies have been applied in various fields of biomedicine due to their advantages such as small molecular weight, high antigen affinity, low immunogenicity, and good tissue permeability, including disease diagnosis and treatment, affinity purification reagents, assistance in protein structure analysis, and biosensors.

[0004] Current research on targeted drugs for EBNA1 mainly focuses on small molecule compounds, nucleic acids, and peptides. These all suffer from problems such as low selectivity and poor stability to varying degrees. In recent years, antibody therapies based on biological macromolecules have been increasingly used for various human diseases, representing a promising new drug screening direction that can replace small molecule drugs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide an NbE7 single-domain antibody, an NbE7-TAT fusion single-domain antibody, and their applications.

[0006] The specific technical solution of this invention is as follows:

[0007] The first aspect of the present invention provides an NbE7 single-domain antibody for specifically binding to EB virus nuclear antigen. The NbE7 single-domain antibody is composed of a backbone region FR and a complementarity-determining region CDR, wherein 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] Furthermore, the amino acid sequence of the NbE7 single-domain antibody is shown in SEQ ID NO.4.

[0009] A second aspect of the present invention provides an NbE7-TAT fusion single-domain antibody, wherein the fusion single-domain antibody is a fusion protein of the NbE7 single-domain antibody and the TAT peptide of HIV-1 virus.

[0010] Furthermore, the C-terminus of the NbE7 single-domain antibody is linked to the TAT peptide of the HIV-1 virus;

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

[0012] Preferably, the amino acid sequence of the NbE7-TAT fusion single-domain antibody is shown in SEQ ID NO.6.

[0013] A third aspect of the present invention provides a polynucleotide sequence that encodes the NbE7 single-domain antibody or the NbE7-TAT fusion single-domain antibody.

[0014] Furthermore, the nucleotide sequence encoding the NbE7 single-domain antibody is shown in SEQ ID NO.7;

[0015] Preferably, the nucleotide sequence encoding the NbE7-TAT fusion single-domain antibody is shown in SEQ ID NO.8.

[0016] A fourth aspect of the present invention provides a biomaterial expressing the NbE7 single-domain antibody, or expressing the NbE7-TAT fusion single-domain antibody, or containing the polynucleotide sequence described above, wherein the biomaterial comprises at least one of an expression cassette, a vector, a recombinant microorganism, and a cell line.

[0017] The fifth aspect of the present invention provides a method for preparing the NbE7-TAT fusion single-domain antibody, characterized in that the multinucleotide sequence encoding the NbE7-TAT fusion single-domain antibody is expressed in an Escherichia coli prokaryotic expression system.

[0018] The sixth aspect of the present invention provides the use of the NbE7 single-domain antibody, or the NbE7-TAT fusion single-domain antibody, in any of the following:

[0019] (1) Prepare reagents for detecting and / or diagnosing EB virus-related diseases;

[0020] (2) Prepare reagents for recognizing and / or binding to the EBNA1 protein of EB virus;

[0021] (3) Prepare drugs for the prevention and / or treatment of latent EB virus infection;

[0022] (4) To prepare drugs for the prevention and / or treatment of EB virus-related tumors;

[0023] Preferably, the EB virus-associated tumor is an EB virus-positive tumor;

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

[0025] A seventh aspect of the present invention provides a pharmaceutical composition, characterized in that the active ingredient of the pharmaceutical composition comprises the NbE7 single-domain antibody or the NbE7-TAT fusion single-domain antibody.

[0026] The beneficial effects of this invention are:

[0027] This invention provides an NbE7 fusion single-domain antibody and demonstrates its applicability for recognizing and binding to the EBV EBNA1 antigen. Furthermore, this invention provides an NbE7-TAT fusion single-domain antibody and demonstrates its applicability as a tumor growth inhibitor for EBV-related tumors. The NbE7-TAT fusion single-domain antibody significantly inhibited the EBV copy number under latent infection in EBV-positive nasopharyngeal carcinoma cells and EBV-positive Burkitt lymphoma cells, and exhibited good tumor cell proliferation inhibition. Simultaneously, in a cell line-derived nude mouse xenograft model, the NbE7-TAT fusion single-domain antibody significantly inhibited the growth of EBV-positive xenografts, and showed no toxic side effects on mouse organs after administration. The development of NbE7-TAT provides experimental evidence for targeted drug research and personalized tumor treatment of latent EBV infection and related tumors. Specifically:

[0028] This invention first evaluated the affinity of the NbE7 single-domain antibody for the EBNA1 antigen. Furthermore, this invention selected various human-derived tumor cell lines and cell line-derived xenograft (CDX) models to reveal the therapeutic effect of the NbE7-TAT fusion single-domain antibody on latent EBV infection and related tumors. These include the human nasopharyngeal carcinoma cell line C666-1 containing the EBV genome, the human Burkitt lymphoma cell line Raji containing the EBV genome, as well as the human nasopharyngeal carcinoma cell line CNE-2 without the EBV genome and the human lung cancer cell line A549 without the EBV genome.

[0029] In the affinity evaluation of EBNA1, surface ion resonance experiments showed that the dissociation constant of the NbE7 single-domain antibody for the in vitro recombinant EBNA1-DBD protein was 1.42 × 10⁻⁶. -6 M. This indicates that the NbE7 single-domain antibody has good affinity for EBNA1-DBD. In in vitro cell experiments, the NbE7-TAT fusion single-domain antibody significantly inhibited the proliferation of EBV-positive C666-1 cells and Raji cells, but had no significant effect on the proliferation of EBV-negative CNE-2 cells.

[0030] In mouse xenograft models, the NbE7-TAT fusion single-domain antibody significantly slowed the growth of EBV-positive nasopharyngeal carcinoma xenografts and EBV-positive lymphoma xenografts. Compared to cisplatin treatment, the NbE7-TAT fusion single-domain antibody showed a more significant inhibitory effect on the growth of EBV-positive nasopharyngeal carcinoma xenografts. Furthermore, histopathological sections of mouse organs showed no significant inflammatory infiltration or tissue damage, indicating that the NbE7-TAT fusion single-domain antibody has no toxic side effects. Attached Figure Description

[0031] Figure 1This is an expression diagram of NbE7 single-domain antibody and NbE7-TAT fusion single-domain antibody.

[0032] Figure 2 The graph shows the affinity of NbE7 single-domain antibody for EBNA1 as detected by surface plasmon resonance.

[0033] Figure 3 This figure shows the results of NbE7-TAT fusion single-domain antibody inhibiting tumor cell proliferation. The groups are EBV-positive nasopharyngeal carcinoma (C666-1), EBV-negative nasopharyngeal carcinoma (CNE-2), and EBV-positive Burkitt lymphoma (Raji) groups, respectively.

[0034] Figure 4 The figure shows the results of reducing EBV copy number in EBV-positive Raji cells using the NbE7-TAT fusion single-domain antibody. Detection was performed using real-time quantitative PCR.

[0035] Figure 5 The figure shows the results of using the NbE7-TAT fusion single-domain antibody to reduce the transcriptional levels of EBV BKRF1, LMP1, BZLF1, and BHLF1 in EBV-positive Raji cells. Real-time quantitative PCR was used for detection.

[0036] Figure 6 This is a graph showing the growth of EBV-positive nasopharyngeal carcinoma xenografts after administration of the NbE7-TAT fusion single-domain antibody. Patients were divided into a negative control group (Vehicle), a group receiving the fusion single-domain antibody (NbE7-TAT), and a group receiving the positive drug cisplatin.

[0037] Figure 7 Images of solid tumors of EBV-positive nasopharyngeal carcinoma after administration of the NbE7-TAT fusion single-domain antibody. Patients were divided into a negative control group (Vehicle), a group receiving the fusion single-domain antibody (NbE7-TAT), and a group receiving the positive drug cisplatin.

[0038] Figure 8 This is a graph showing the growth of EBV-positive lymphoma xenografts after administration of the NbE7-TAT fusion single-domain antibody. Patients were divided into a negative control group (Vehicle) and a group administered the fusion single-domain antibody (NbE7-TAT).

[0039] Figure 9 HE staining images of mouse organ pathological sections after administration of the NbE7-TAT fusion single-domain antibody. These include the heart, liver, spleen, lungs, and kidneys. The mice were divided into a negative control group (Vehicle) and a group administered the fusion single-domain antibody (NbE7-TAT). Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention.

[0041] Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0042] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0043] Example 1: Preparation of NbE7 single-domain antibody and NbE7-TAT fusion single-domain antibody

[0044] I. NbE7 single-domain antibody, NbE7-TAT fusion single-domain antibody

[0045] The applicant screened for NbE7 single-domain antibodies using EBNA1-DBD as the antigen. These NbE7 single-domain antibodies are used to specifically bind to the Epstein-Barr virus nuclear antigen (EBNA1). The NbE7 single-domain antibody consists of a backbone region (FR) and a complementarity-determining region (CDR). The CDRs include 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 NbE7 single-domain antibody is shown in SEQ ID NO.4. In this embodiment, the nucleotide sequence encoding the NbE7 single-domain antibody is shown in SEQ ID NO.7.

[0046] The NbE7-TAT fusion single-domain antibody is a fusion protein of an NbE7 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 in the NbE7-TAT fusion single-domain antibody is shown in SEQ ID NO. 5. In this embodiment, the amino acid sequence of the NbE7-TAT fusion single-domain antibody is shown in SEQ ID NO. 6. In this embodiment, the nucleotide sequence encoding the NbE7-TAT fusion single-domain antibody is shown in SEQ ID NO. 8.

[0047] The amino acid sequence of the NbE7 single-domain antibody complementarity-determining region CDR1 is SEQ ID NO.1:

[0048] GTISGYEGM

[0049] The amino acid sequence of the NbE7 single-domain antibody complementarity-determining region CDR2 is SEQ ID NO.2:

[0050] ELVASISYGATTNY

[0051] The amino acid sequence of the NbE7 single-domain antibody complementarity-determining region CDR3 is SEQ ID NO.3:

[0052] AASVGGQAPHDY

[0053] The amino acid sequence of the NbE7 single-domain antibody is SEQ ID NO.4:

[0054] QVQLQESGGGLVQAGGSLRLSCAASGTISGYEGMGWYRQAPGKERELVASISYGATTNYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAASVGGQAPHDYWGQGTQVTVSS

[0055] The amino acid sequence of the TAT peptide in the NbE7-TAT fusion single-domain antibody is SEQ ID NO. 5:

[0056] YGRKKRRQRRR

[0057] The amino acid sequence of the NbE7-TAT fusion single-domain antibody is SEQ ID NO. 6:

[0058] QVQLQESGGGLVQAGGSLRLSCAASGTISGYEGMGWYRQAPGKERELVASISYGATTNYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAASVGGQAPHDYWGQGTQVTVSSLEHHHHHHYGRKKRRQRRR

[0059] The nucleotide sequence encoding the NbE7 single-domain antibody is SEQ ID NO.7:

[0060] CAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCACTATTTCTGGTTATGAAGGTATGGGCTGGTATCGCCAGGCGCCGGGCAAAGAACGCGAACTTGTTGCCAGTATTAGTTATGGTGCTACTACCAATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCTTCTGTTGGTGGTCAAGCTCCACATGATTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC

[0061] Nucleotide sequence encoding the NbE7-TAT fusion single-domain antibody SEQ ID NO.8:

[0062] CAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCACTATTTCTGGTTATGAAGGTATGGGCTGGTATCGCCAGGCGCCGGGCAAAGAACGCGAACTTGTTGCCAGTATTAGTTATGGTGCTACTACCAATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCTTCTGTTGGTGGTCAAGCTCCACATGATTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCCTCGAGCACCACCACCACCACCACTATGGCCGCAAAAAACGCCGCCAGCGCCGTCGC

[0063] II. Expression and purification of the NbE7 single-domain antibody and the NbE7-TAT fusion single-domain antibody

[0064] In this embodiment, the nucleotide sequence gene of the NbE7-TAT fusion single-domain antibody was inserted into the pET22b prokaryotic expression vector, and protein expression was performed using the mature *E. coli* BL21(DE3) prokaryotic cell expression system. The plasmid was transformed into BL21(DE3) cells using a heat shock transformation method. The expression conditions for the NbE7-TAT fusion single-domain antibody were: 16℃, 220 rpm, induced for 12 hours with 0.5 mM IPTG. After expression, the bacterial cells were collected by centrifugation at 4℃, 6700 g for 20 minutes and 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 lysed at low temperature under high pressure (800 bar) using a high-pressure cell disruptor, and the supernatant was collected by ultracentrifugation. The NbE7-TAT fusion single-domain antibody in the supernatant was purified using Ni-NTA affinity chromatography. After purification, protein purity was observed by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Finally, the protein was concentrated using ultrafiltration tubes and transferred to sterile PBS, then stored at -80°C. The expression methods for NbE7 single-domain antibodies and TAT fusion NbE7 single-domain antibodies were the same.

[0065] Figure 1 The images show Coomassie brilliant blue staining of the purified NbE7 single-domain antibody and the NbE7-TAT fusion single-domain antibody. The molecular weights of the NbE7 single-domain antibody and the TAT fusion NbE7 single-domain antibody are 13 kDa and 15 kDa, respectively.

[0066] Example 2: Affinity evaluation of NbE7 single-domain antibody to EBNA1-DBD protein

[0067] This embodiment uses surface ion resonance experiments to analyze the recognition of EBNA1 by NbE7 single-domain antibodies. Specifically, it includes the following steps:

[0068] First, a 3D dextran sensor chip conjugated with recombinant EBNA1-DBD was bound to mobile phase NbE7 single-domain antibody at different concentrations. The concentrations were set to 5, 2.5, 1.25, 0.63, 0.31, 0.16, and 0.08 μM, respectively. The mobile phase was loaded at a rate of 2 μL / s and flowed through the chip placed on a biomolecular interaction analyzer. The sensor chip then captured the binding interaction between the immobilized EBNA1-DBD and the 5E2-12 monoclonal antibody.

[0069] Then, the sensor data representing the binding reaction were analyzed using BIEvaluation software. The dissociation constant (K) was calculated by fitting data using this software. D ), K D This represents the affinity index between the NbE7 single-domain antibody and EBNA1.

[0070] Experimental results are as follows Figure 2 As shown. Figure 2 The graph shows the affinity of the NbE7 single-domain antibody for EBNA1-DBD as detected by surface plasmon resonance. K was calculated after fitting. D 1.42×10 -6 M indicates that the NbE7 single-domain antibody has a strong affinity for EBNA1 encoded by EB virus.

[0071] Example 3: Evaluation of the inhibitory effect of NbE7-TAT fusion single-domain antibody on in vitro tumor cell proliferation

[0072] This embodiment uses the CCK-8 assay to analyze cell proliferation viability. The Cell Counting Kit (CCK-8) is used to detect cell proliferation viability. Before the assay, 5 × 10⁶ cells were counted per well. 4 100 μL of C666-1, CNE-2, and Raji cells were passaged into 96-well cell culture plates and cultured for 12 hours. Then, NbE7-TAT fusion single-domain antibody was added to the cell culture plates to a final concentration of 30 μM, and the same concentration of NbE7 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 culture incubator for another hour. Subsequently, the absorbance (OD) at 450 nm was measured using a microplate reader. 450 ).

[0073] Experimental results are as follows Figure 3 As shown. Figure 3 The figures show the proliferation detection results on EBV-positive C666-1, Raji, and EBV-negative CNE-2 cells. Compared with NbE7 single-domain antibody administration, intervention with 30 μM NbE7-TAT fusion single-domain antibody significantly inhibited the proliferation of EBV-positive nasopharyngeal carcinoma cells and lymphoma cells, but had no significant effect on EBV-negative nasopharyngeal carcinoma cell lines.

[0074] Example 4: Effect of NbE7-TAT fusion single-domain antibody on latent EBV infection

[0075] This embodiment uses real-time quantitative PCR to detect the effect of NbE7-TAT fusion single-domain antibody on latent EBV infection. The specific steps include:

[0076] C666-1 and Raji cells were added at a ratio of 1×10⁻⁶ per well. 6Cells were seeded into six-well cell culture plates and cultured for 12 hours. Then, NbE7-TAT fusion single-domain antibody (30 μM) was added for intervention, while the negative control group received the same volume of PBS. After 24 hours of further culture, total RNA was extracted and dissolved in 20 μL of enzyme-free water. The total RNA was then reverse transcribed into cDNA. The mRNA expression level was detected using quantitative real-time PCR.

[0077] Experimental results are as follows Figure 4 and 5 As shown.

[0078] Figure 4 This is a plot showing the transcriptional levels of EBER1 and EBER2 after intervention with the NbE7-TAT fusion single-domain antibody. EBERs are small RNAs transcribed from EBV, which occur in high-copy forms during latent EBV infection, and their transcriptional levels reflect the EBV genome copy number. Intervention with the NbE7-TAT fusion single-domain antibody significantly reduced the copy numbers of EBER1 and EBER2 in EBV-positive Raji cells.

[0079] Figure 5 This study investigated the transcriptional levels of BKRF1, LMP1, BZLF1, and BHLF1 in the latent EBV genome after intervention with the NbE7-TAT fusion single-domain antibody. The intervention significantly reduced the transcriptional levels of BKRF1, LMP1, BZLF1, and BHLF1 in EBV-positive Raji cells, indicating EBV genome loss following the intervention.

[0080] Example 5: Evaluation of the inhibitory effect of NbE7-TAT fusion single-domain antibody on EBV-positive xenograft tumors

[0081] I. Establishment of BALB / c nude mouse xenograft tumor model and NbE7-TAT fusion single-domain antibody therapy

[0082] Five-week-old male BALB / c nude mice were used to establish a mouse xenograft tumor model. First, 3 × 10⁸ mice were used per mouse. 6 C666-1 cells or 3×10 6 Raji cells were collected from in vitro cultures and resuspended in pre-chilled sterile PBS on ice. They were then mixed with 50% cold matrix gel and kept on ice. The cell mixture was then rapidly injected subcutaneously into the vascularized right side of BALB / c nude mice. The mice were fed and observed daily. When the tumor diameter reached approximately 7 mm, they were grouped.

[0083] This experiment used two xenograft models. The first was an EBV-positive nasopharyngeal carcinoma xenograft model (EBV... +The C666-1 Xenograft tumor was divided into three groups: a negative control (Vehicle) group, a positive drug cisplatin (Cisplatin) group, and a treatment (NbE7-TAT) group. The second type is an EBV-positive Burkitt lymphoma xenograft model (EBV...). + The Raji Xenografttumor was divided into two groups: the Vehicle group and the drug intervention (NbE7-TAT) group. Each group consisted of 3 animals.

[0084] In the nasopharyngeal carcinoma xenograft model, the cisplatin group served as a positive control, receiving intraperitoneal injections of 4 mg / kg cisplatin twice weekly. In the nasopharyngeal carcinoma and lymphoma xenograft models, the NbE7-TAT treatment group received intratumoral injections of 30 μM of the fusion single-domain antibody NbE7-TAT every 3 days. Intratumoral injections were administered at three points per mouse, with each point containing approximately 30 μL, equivalent to a 40 μg dose of the single-domain antibody. The Vehicle group received intratumoral injections of the same volume of PBS at the same time points.

[0085] After drug administration, the tumor volume of mice was measured every other day. The tumor volume measurement should be completed after the previous drug was absorbed and before the next drug was administered. In the nasopharyngeal carcinoma xenograft model, mice were sacrificed on day 20 after treatment. In the lymphoma model, mice were sacrificed on day 26, and solid tumors were isolated.

[0086] II. Experimental Results

[0087] Figure 6 This is a graph showing the growth curve of EBV-positive nasopharyngeal carcinoma xenografts after administration of the NbE7-TAT fusion single-domain antibody. Patients were divided into a negative control group (Vehicle), a fusion single-domain antibody administration group (NbE7-TAT), and a positive control group (cisplatin). Compared to the Vehicle group, both cisplatin and NbE7-TAT significantly inhibited the growth of nasopharyngeal carcinoma xenografts, with the NbE7-TAT group showing a more pronounced inhibitory effect than the cisplatin group.

[0088] Figure 7 Images of EBV-positive nasopharyngeal carcinoma solid tumors after administration of the NbE7-TAT fusion single-domain antibody. Patients were divided into a negative control group (Vehicle), a fusion single-domain antibody administration group (NbE7-TAT), and a positive drug cisplatin group. Compared to the Vehicle group, both cisplatin and NbE7-TAT significantly inhibited the final volume of nasopharyngeal carcinoma xenografts, with the NbE7-TAT group showing a more pronounced inhibitory effect than the cisplatin group.

[0089] Figure 8This is a graph depicting the growth of EBV-positive lymphoma xenografts after administration of the NbE7-TAT fusion single-domain antibody. Patients were divided into a negative control group (Vehicle) and a group administered the fusion single-domain antibody (NbE7-TAT). Compared to the Vehicle group, the NbE7-TAT group significantly inhibited the growth of lymphoma xenografts.

[0090] Figure 9 HE-stained images of mouse organ sections after administration of the NbE7-TAT fusion single-domain antibody. These include the heart, liver, spleen, lung, and kidney. The mice were divided into a negative control group (Vehicle) and a group treated with the fusion single-domain antibody (NbE7-TAT). No inflammatory cell infiltration or tissue damage was observed in the five major organs of the mice after treatment with the NbE7-TAT fusion single-domain antibody.

[0091] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any equivalent modifications made by those skilled in the art to the technical solutions of the present invention by reading the present invention specification are covered by the claims of the present invention.

Claims

1. An NbE7 single-domain antibody, characterized in that, The NbE7 single-domain antibody is used to specifically bind to EB virus nuclear antigen. The NbE7 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 NbE7 single-domain antibody according to claim 1, characterized in that, The amino acid sequence of the NbE7 single-domain antibody is shown in SEQ ID NO.

4.

3. An NbE7-TAT fusion single-domain antibody, characterized in that, The fusion single-domain antibody is a fusion protein of the NbE7 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 NbE7 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 NbE7-TAT fusion single-domain antibody is shown in SEQ ID NO.

6.

5. A polynucleotide sequence, characterized in that, The polynucleotide sequence encodes the NbE7 single-domain antibody of claim 1 or 2, or the NbE7-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 NbE7 single-domain antibody of claim 1 or 2 is shown in SEQ ID NO.7; Preferably, the nucleotide sequence encoding the NbE7-TAT fusion single-domain antibody according to claim 3 or 4 is shown in SEQ ID NO.

8.

7. A biomaterial, characterized in that, The biomaterial expresses the NbE7 single-domain antibody of claim 1 or 2, or the NbE7-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 NbE7-TAT fusion single-domain antibody according to claim 3 or 4, characterized in that, The multinucleotide sequence encoding the NbE7-TAT fusion single-domain antibody as described in claim 3 or 4 was expressed in an E. coli prokaryotic expression system.

9. Use of the NbE7 single-domain antibody of claim 1 or 2, or the NbE7-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) Prepare reagents for recognizing and / or binding 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 NbE7 single-domain antibody as described in claim 1 or 2, or the NbE7-TAT fusion single-domain antibody as described in claim 3 or 4.

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