5E2-12 monoclonal antibody and application thereof

Abstract

The invention discloses a 5E2-12 monoclonal antibody and application thereof, the 5E2-12 monoclonal antibody is specifically combined with EB virus nuclear antigen and comprises VHCDR1-3 of a heavy chain variable region and VLCDR1-3 of a light chain variable region, the amino acid sequences of VHCDR1, VHCDR2 and VHCDR3 are respectively shown as SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5, and the amino acid sequences of VLCDR1, VLCDR2 and VLCDR3 are respectively shown as SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6. The invention further discloses a preparation method of the 5E2-12 monoclonal antibody. The invention also provides an antibody fragment of the 5E2-12 monoclonal antibody. The 5E2-12 monoclonal antibody provides a potential choice for research and development of clinical medicines for diagnosing and 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 a 5E2-12 monoclonal antibody and its uses. Background Technology

[0002] Epstein-Barr virus (EBV) is a DNA virus and a member of the herpesvirus family. Human herpesviruses are adept at establishing latent infection, and EBV is no exception. Latent EBV infection is a significant risk factor for human cancer. During latent infection, the EBV nuclear antigen (EBNA1) can anchor the latent EBV genome to the host cell chromosome. Long-term chromosomal anchoring is a major contributing factor to various cell-derived human cancers. The DNA-binding domain (DBD) on EBNA1 can bind to both the latent EBV genome and the host chromosome to maintain latent EBV infection. Therefore, EBNA1 is an effective target for treating 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. Monoclonal antibody (mAb) drugs, with their good targeting, stability, and low toxicity, have had a significant impact on clinical treatment. Since the advent of hybridoma cell technology in 1975, the development of monoclonal antibody drugs has been rapid; in 2023 alone, more than 175 antibody therapies have received clinical approval or are under regulatory review. However, conventional antibody therapies are not suitable for many intracellular proteins, while a recent study demonstrated that monoclonal antibodies targeting intracellular oncogenic Ras mutants have significant anti-tumor effects. This highlights the feasibility of developing antibody drugs targeting intracellular proteins.

[0004] Currently, the development of 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 development direction that can replace small molecule drugs. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a 5E2-12 monoclonal antibody and its uses.

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

[0007] The first aspect of this invention provides a 5E2-12 monoclonal antibody or an antibody fragment thereof, said 5E2-12 monoclonal antibody for specifically binding to EB virus nuclear antigen, comprising a V-region of the heavy chain variable region. H CDR1-3 and V of the light chain variable region L CDR1-3, wherein V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. The V L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6, respectively;

[0008] The antibody fragment is selected from Fab, single-chain antibody, single-domain antibody, and V. H and V L One or more of the Fv regions formed by non-covalent interactions.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO.7.

[0010] Furthermore, the amino acid sequence of the light chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO.8.

[0011] A second aspect of the present invention provides a polynucleotide sequence encoding the 5E2-12 monoclonal antibody or an antibody fragment thereof.

[0012] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO. 9;

[0013] Furthermore, the nucleotide sequence encoding the light chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO. 10.

[0014] A third aspect of the present invention provides a biological material expressing the 5E2-12 monoclonal antibody or an antibody fragment of the monoclonal antibody, or containing the polynucleotide sequence described above, wherein the biological material comprises at least one of an expression cassette, a vector, a recombinant microorganism, and a cell line.

[0015] The fourth aspect of the present invention provides the use of the 5E2-12 monoclonal antibody or an antibody fragment thereof in any of the following:

[0016] (1) Prepare reagents for detecting and / or diagnosing EB virus-related diseases;

[0017] (2) Preparation of reagents that specifically bind to the EBNA1 protein of EB virus;

[0018] (3) Prepare drugs for the prevention and / or treatment of latent EB virus infection;

[0019] (4) To prepare drugs for the prevention and / or treatment of EB virus-related tumors;

[0020] Preferably, the EB virus-associated tumor is an EB virus-positive tumor;

[0021] Preferably, the EBV-related tumor is EBV-positive nasopharyngeal carcinoma or EBV-positive lymphoma.

[0022] A fifth aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the 5E2-12 monoclonal antibody or an antibody fragment thereof.

[0023] Furthermore, the pharmaceutical composition further includes cell-penetrating peptides;

[0024] Preferably, the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.11.

[0025] The beneficial effects of this invention are:

[0026] This invention provides a 5E2-12 monoclonal antibody and demonstrates its application in recognizing and binding to the EBV EBNA1 antigen epitopes 461-471. Furthermore, this invention provides the combined use of the 5E2-12 monoclonal antibody and the S10 transmembrane peptide, demonstrating its application as a tumor growth inhibitor for EBV-related tumors. The 5E2-12 monoclonal 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 5E2-12 monoclonal antibody significantly inhibited the growth of EBV-positive xenografts, and showed no toxic side effects on mouse organs after administration. The development of the 5E2-12 monoclonal antibody provides experimental evidence for targeted drug research and personalized tumor treatment for latent EBV infection and related tumors in clinical practice. Specifically:

[0027] This invention first evaluated the affinity of the 5E2-12 monoclonal antibody for the EBNA1 antigen and identified epitopes. Furthermore, this invention selected various human-derived tumor cell lines and cell line-derived xenograft (CDX) models to reveal the therapeutic effect of the 5E2-12 monoclonal antibody against 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 HK1 without the EBV genome and the human lung cancer cell line A549 without the EBV genome.

[0028] In the affinity evaluation of EBNA1, surface ion resonance experiments showed that the dissociation constant of the 5E2-12 monoclonal antibody for recombinant EBNA1-DBD protein in vitro was 196 nM. This indicates that the 5E2-12 monoclonal antibody has good affinity for EBNA1-DBD. In in vitro cell experiments, the 5E2-12 monoclonal 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 HK1 cells.

[0029] In mouse xenograft models, the 5E2-12 monoclonal antibody significantly slowed the growth of EBV-positive nasopharyngeal carcinoma xenografts and EBV-positive lymphoma xenografts. The inhibitory effect of the 5E2-12 monoclonal antibody on the growth of EBV-positive nasopharyngeal carcinoma xenografts was similar to that of cisplatin. Furthermore, histopathological sections of mouse organs showed no significant inflammatory infiltration or tissue damage, indicating that the 5E2-12 monoclonal antibody has no toxic side effects. Attached Figure Description

[0030] Figure 1 This is an SDS-PAGE electrophoresis image of the 5E2-12 monoclonal antibody.

[0031] Figure 2 A graph showing the affinity of the 5E2-12 monoclonal antibody for EBNA1-DBD as detected by surface plasmon resonance.

[0032] Figure 3 The image shows the results of ELISA identification of the binding of the 5E2-12 monoclonal antibody to the EBNA1-DBD epitope. EBNA1 DBDmut1 and mut2 are the truncated and mutant clones of epitopes 461-471, respectively.

[0033] Figure 4 This figure shows the results of the 5E2-12 monoclonal antibody combined with S10 inhibiting tumor cell proliferation. The groups are EBV-positive nasopharyngeal carcinoma (C666-1), EBV-negative nasopharyngeal carcinoma (HK1), and EBV-positive Burkitt lymphoma (Raji).

[0034] Figure 5 The image shows the results of blocking latent EBV infection in EBV-positive Raji cells with the combination of 5E2-12 monoclonal antibody and S10. The EBER of EBV BKRF1, BZLF1, and BYRF1 transcription levels and EBV copy number were detected by real-time quantitative PCR.

[0035] Figure 6 This is a graph showing the growth curves of EBV-positive and EBV-negative xenografts after administration of the 5E2-12 monoclonal antibody in combination with S10. For EBV-positive nasopharyngeal carcinoma xenografts, they were divided into a negative control group (Vehicle and S10), a monoclonal antibody combined with S10 administration (5E2-12+S10), and a positive control group (cisplatin). For EBV-positive lymphomas, they were divided into a negative control group (Vehicle and S10), and groups receiving different doses of monoclonal antibody combined with S10 administration (5E2-12+S10). For EBV-negative A549 xenografts, they were divided into a negative control group (Vehicle) and a monoclonal antibody combined with S10 administration (5E2-12+S10).

[0036] Figure 7 HE staining images of mouse organ pathological sections after administration of 5E2-12 monoclonal antibody in combination with S10. These include the heart, liver, spleen, lungs, and kidneys. The mice were divided into a negative control group (Vehicle) and a group receiving the monoclonal antibody in combination with S10 (5E2-12+S10). Detailed Implementation

[0037] 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.

[0038] Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0039] 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.

[0040] Example 1: Preparation of 5E2-12 monoclonal antibody

[0041] I. 5E2-12 Monoclonal Antibody

[0042] This embodiment uses EBNA1 461-471Using this antigen, a 5E2-12 monoclonal antibody was screened and identified as the IgG subtype. Hybridoma cells containing the 5E2-12 monoclonal antibody were then preserved. Sequencing analysis showed that the 5E2-12 monoclonal antibody specifically binds to the Epstein-Barr virus nuclear antigen (EBNA1), with epitopes 461-471. The 5E2-12 monoclonal antibody consists of a heavy chain (H chain) and a light chain (L chain), each including a variable region (V region). H ) and light chain variable region (V L ), heavy chain variable region (V H This includes the heavy chain complementarity determinant region (V). H CDR1, V H CDR2 and V H CDR3), light chain variable region (V L This includes the light chain complementarity determinant region (V). L CDR1, V L CDR2 and V L CDR3). Among them, V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6, respectively. The amino acid sequence of the heavy chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO.7, the nucleic acid sequence encoding the heavy chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO.9, the amino acid sequence of the light chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO.8, and the nucleic acid sequence encoding the light chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO.10.

[0043] The amino acid sequence of the heavy chain complementarity-determining region CDR1 of the 5E2-12 monoclonal antibody, SEQ ID NO.1:

[0044] GFTFNTYT

[0045] The amino acid sequence of the light chain complementarity-determining region CDR1 of the 5E2-12 monoclonal antibody, SEQ ID NO.2:

[0046] QTLLNTINQKNY

[0047] The amino acid sequence of the heavy chain complementarity-determining region CDR2 of the 5E2-12 monoclonal antibody, SEQ ID NO.3:

[0048] ITSGGTYI

[0049] The amino acid sequence of the light chain complementarity-determining region CDR2 of the 5E2-12 monoclonal antibody, SEQ ID NO.4:

[0050] FAS

[0051] The amino acid sequence of the heavy chain complementarity-determining region CDR3 of the 5E2-12 monoclonal antibody, SEQ ID NO. 5:

[0052] TREGYGNYWYFDV

[0053] The amino acid sequence of the light chain complementarity-determining region CDR3 of the 5E2-12 monoclonal antibody, SEQ ID NO. 6:

[0054] QQHYNSPLT

[0055] The amino acid sequence of the variable region of the heavy chain of the 5E2-12 monoclonal antibody, SEQ ID NO.7:

[0056] DVQLVESGGGLVKPGGSLKLSCAASGFTFNTYTMSWVRQTPEKRLEWVATITSGGTYIYYPDNVKGRFTISSRDNAKNSLFLQMSSLKSEDTAMYYCTREGYGNYWYFDVWGAGTTLTVSS

[0057] The amino acid sequence of the variable region of the light chain of the 5E2-12 monoclonal antibody, SEQ ID NO. 8:

[0058] DIVLTQSPSSLPMSVGQKVTMSCKSSQTLLNTINQKNYLAWYQQKPGQSPKLLVFFA STRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYNSPLTFGGGTRLEIK

[0059] The nucleic acid sequence encoding the variable region of the heavy chain of the 5E2-12 monoclonal antibody is SEQ ID NO.9:

[0060] GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAATACCTATACCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTACTAGTGGTGGTACTTACATTTACTAT CCAGACAATGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAATTCCCTGTTCCTGCAGATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTACAAGAGAAGGTTATGGTAATTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACTCTCACAGTCTCCTCA

[0061] The nucleic acid sequence encoding the variable region of the light chain of the 5E2-12 monoclonal antibody is SEQ ID NO.10:

[0062] GACATTTGTGCTGACACAGTCTCCATCCTCCCTGCCTATGTCAGTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGACCCTTTAAATACTATCAATCAAAAGAACTATTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCAAAACTTCTGGTATTTTTTGC ATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGATTACTTCTGTCAGCAGCATTATAACAGTCCTCTCACGTTCGGTGGTGGGACCAGACTGGAAATAAAAC

[0063] II. Preparation and Purification of 5E2-12 Monoclonal Antibody

[0064] In this embodiment, the ascites solution was prepared by intraperitoneal injection of 0.5 mL / mouse of Freund's incomplete adjuvant in 8-week-old male BALB / c mice. Seven days later, the mice were intraperitoneally injected with 5 × 10⁻⁶ PBS diluted with PBS. 50.5 mL of 5E2-12 monoclonal hybridoma cell suspension was collected. Mice were continuously observed. After about 10 days, when the mice's abdomens became significantly enlarged and they showed signs of slow movement, the mice were euthanized by cervical dislocation. The ascites was removed, centrifuged to remove insoluble matter, and the supernatant was stored at -80°C for later use.

[0065] 5E2-12 monoclonal antibody purification:

[0066] In this embodiment, the purification of murine antibodies was performed using Protein G affinity chromatography. After thawing the ascites fluid, the floating flocculent insoluble matter was scraped off with a pipette tip, and 5 volumes of Protein G binding buffer (25 mM NaH2PO3 pH 7.5, 150 mL NaCl) were added. Then, 5 mL of Protein G resin equilibrated with binding buffer was added, and the mixture was incubated on ice with a shaker for 2 hours. After incubation, the mixture was added to a gravity chromatography column, the permeate was discarded, and at least 10 volumes of binding buffer were added to wash away non-specifically bound packing material. Finally, 5 volumes of 0.1 M glycine pH 3.0 elution buffer were added, and 1 / 10 elution volume of 1 M Tris-HCl pH 8.5 neutralization buffer was added to the collection tube beforehand. Samples were taken for SDS-PAGE electrophoresis, and Coomassie brilliant blue staining was used to observe the purity of the purified antibody. Concentrate mAb to 0.3 mL using an ultrafiltration tube, replace the buffer with sterile PBS, and detect protein concentration using the A280 method. Store at -80℃ for later use.

[0067] Figure 1 This is a Coomassie brilliant blue staining image of the purified 5E2-12 monoclonal antibody. The molecular weights of the heavy chain (HC) and light chain (LC) of the 5E2-12 monoclonal antibody are 53 kDa and 27 kDa, respectively.

[0068] Example 2: Affinity evaluation of 5E2-12 monoclonal antibody against EBNA1-DBD protein

[0069] This embodiment uses surface ion resonance experiments to analyze the recognition of EBNA1-DBD by the 5E2-12 monoclonal antibody. The specific steps include the following:

[0070] First, a 3D dextran sensor chip coupled with recombinant EBNA1-DBD was bound to mobile phase 5E2-12 monoclonal 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 captured the binding interaction between the immobilized EBNA1-DBD and the 5E2-12 monoclonal antibody.

[0071] 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 5E2-12 monoclonal antibody and the EBNA1 DBD.

[0072] Experimental results are as follows Figure 2 As shown. Figure 2 A curve showing the affinity of the 5E2-12 monoclonal antibody for EBNA1-DBD detected by surface plasmon resonance. K was calculated after fitting. D The value was 196 nM, indicating that the 5E2-12 monoclonal antibody has a strong affinity for EBNA1 encoded by EB virus.

[0073] Example 3: Epitope identification of EBNA1 binding by 5E2-12 monoclonal antibody

[0074] This embodiment uses enzyme-linked immunosorbent assay (ELISA) to analyze antibody epitopes. EBNA1 DBD, EBNA1DBDmut1 and mut2, and SUMO-tag proteins were coated onto 96-well half-well plates. The next day, wells without bound proteins were blocked with 5% skim milk. Then, different concentrations of 5E2-12 monoclonal antibody were added and incubated at 37°C for 1 hour. The binding of 5E2-12 monoclonal antibody was detected using HRP-labeled goat anti-mouse IgG. After color development, the absorbance of the 96-well half-well plate was measured at 450 nm using a microplate reader.

[0075] Experimental results are as follows Figure 3 As shown. Figure 3 The image shows the results of ELISA identification of the binding of the 5E2-12 monoclonal antibody to the EBNA1-DBD epitope. EBNA1 DBD mut1 is a truncated clone of epitope 461-471, and EBNA1 DBD mu2 is a mutant clone of epitope 461-471 containing the Ala amino acid. The affinity of 5E2-12 for binding to mut1 and mut2 is significantly decreased.

[0076] Example 4: Evaluation of the inhibitory effect of 5E2-12 monoclonal antibody on tumor cell proliferation in vitro

[0077] This embodiment uses lactate dehydrogenase assay to analyze cytotoxicity. Before assay, prepare 5 × 10⁵ samples per well. 4150 μL of C666-1, HK1, and Raji cells were passaged into 96-well cell culture plates and cultured for 12 hours. Then, different concentrations of 5E2-12 monoclonal antibody and 10 μM of cell-penetrating peptide S10 (S10 served as a negative control; the amino acid sequence of S10 is: KWKLARAFARAIKKLGGSGGGSYARALRRQARTG SEQ ID NO.11) were premixed and added to the plates. After incubation for 23 hours, 15 μL of LDH release reagent was added to the control group. After centrifugation at 400×g for 5 minutes, 120 μL of supernatant was removed from each well and added to 60 μL of LDH detection working solution, followed by incubation at room temperature for 1 hour. Subsequently, the absorbance at 490 nm was measured using a microplate reader.

[0078] Experimental results are as follows Figure 4 As shown. Figure 4 The figure shows the toxicity test results on EBV-positive C666-1, Raji, and EBV-negative HK1 cells. Intervention with the 5E2-12 monoclonal antibody in combination with S10 significantly inhibited the proliferation of EBV-positive nasopharyngeal carcinoma and lymphoma cells, but had no significant effect on EBV-negative nasopharyngeal carcinoma lines.

[0079] Example 5: Effect of 5E2-12 monoclonal antibody on latent EBV infection

[0080] This embodiment uses real-time quantitative PCR to detect the effect of 5E2-12 monoclonal antibody on latent EBV infection. The specific steps include:

[0081] Raji cells were applied at a density of 1 × 10⁻⁶ per well. 6 Cells were seeded into six-well cell culture plates and cultured for 12 hours. Then, 5 μM of 5E2-12 monoclonal antibody and 10 μM of S10 were added for intervention. The negative control group received the same volume of PBS. After culturing for another 24 hours, 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.

[0082] Experimental results are as follows Figure 5 As shown, intervention with 5E2-12 monoclonal antibody combined with S10 significantly reduced the transcription levels of BKRF1, BZLF1, and BYRF1 in EBV-positive Raji cells, indicating EBV genome loss after intervention. EBER is a small RNA transcribed from EBV, appearing in high-copy form during latent EBV infection, and its transcription level reflects the EBV genome copy number. Intervention with 5E2-12 monoclonal antibody combined with S10 significantly reduced the copy numbers of EBER1 and EBER2 in EBV-positive Raji cells.

[0083] The primer sequences for real-time quantitative PCR are shown in the table below:

[0084]

[0085] Example 5: Evaluation of the inhibitory effect of 5E2-12 monoclonal antibody on EBV-positive xenograft tumors

[0086] I. Establishment of a BALB / c nude mouse xenograft tumor model and treatment with 5E2-12 monoclonal antibody

[0087] 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, 3×10 6 Raji cells or 3×10 6 A549 collected in vitro cultured cells and resuspended them in pre-chilled sterile PBS on ice. The mixture was 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.

[0088] This experiment used three xenograft models. The first was an EBV-positive nasopharyngeal carcinoma xenograft model (EBV+NPCmodel), divided into four groups: a negative control (Vehicle) group, a positive drug cisplatin group, a negative drug control (S10) group, and a monoclonal antibody combined with S10 administration (5E2-12+S10) group, with 3 n=3 in each group. The second was an EBV-positive Burkitt lymphoma xenograft model, divided into three groups: a Vehicle group, an S10 group, and different doses of monoclonal antibody combined with S10 administration (5E2-12+S10) groups, with 5 n=5 in each group. The third was an EBV-negative A549 xenograft model, with the same grouping as the lymphoma model, serving as a negative control model, with 3 n=3 in each group.

[0089] In the NPC model, cisplatin was used as a positive control, with 4 mg / kg of cisplatin injected intratumorally every 3 days; S10 was used as a negative control, with 40 μM S10 injected intratumorally every 3 days. In the NPC and A549 xenograft models, the experimental groups received a premixed solution of 20 μM 5E2-12 mAb and 40 μM S10 injected intratumorally every 3 days. In the lymphoma model, the experimental groups received three different doses (20, 10, and 5 μM) of a premixed solution of 5E2-12 mAb and 40 μM S10 injected intratumorally every 3 days. Intratumoral injections were administered at three points per mouse, with a total volume of 30 μL. The maximum dose was equivalent to 90 μg mAb and 5 μg cell-penetrating peptide (CPP). The Vehicle group received the same volume of PBS injected intratumorally at the same time points.

[0090] Following drug administration, tumor volume in mice was measured every other day. Tumor volume measurement should be completed after the previous dose has been absorbed and before the next dose. In the NPC model, mice were sacrificed on day 20 after treatment. In the lymphoma model and the A549 negative control model, mice were sacrificed on day 26, and solid tumors were isolated.

[0091] II. Experimental Results

[0092] Figure 6 Tumor growth curves for three xenograft models after administration of the 5E2-12 monoclonal antibody are shown. The models were divided into a negative control group (Vehicle), a monoclonal antibody combined with S10 administration (5E2-12+S10), and a positive control group (cisplatin). Compared to the Vehicle group, both cisplatin and 5E2-12+S10 significantly inhibited the growth of EBV-positive nasopharyngeal carcinoma xenografts and lymphoma xenografts, but did not inhibit the growth of EBV-negative A549 xenografts.

[0093] Figure 7 HE-stained images of mouse organ sections after administration of the 5E2-12 monoclonal antibody combined with S10 in the NPC experimental group. These include the heart, liver, spleen, lung, and kidney. The mice were divided into a negative control (Vehicle) group and a group receiving the monoclonal antibody combined with S10 (5E2-12+S10). After treatment with 5E2-12+S10, no inflammatory cell infiltration or tissue damage was observed in the five major organs of the mice.

[0094] 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. A 5E2-12 monoclonal antibody or an antibody fragment thereof, characterized in that, The 5E2-12 monoclonal antibody is used to specifically bind to EB virus nuclear antigen, and it contains the V region of the heavy chain variable region. H CDR1-3 and V of the light chain variable region L CDR1-3, wherein V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. The V L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6, respectively; The antibody fragment is selected from Fab, single-chain antibody, single-domain antibody, and V. H and V L One or more of the Fv regions formed by non-covalent interactions.

2. The 5E2-12 monoclonal antibody or an antibody fragment thereof according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO.7; And / or, the amino acid sequence of the light chain variable region of the 5E2-12 monoclonal antibody is shown in SEQ ID NO.

8.

3. A polynucleotide sequence, characterized in that, The polynucleotide sequence encodes the 5E2-12 monoclonal antibody of claim 1 or 2, or an antibody fragment thereof.

4. The polynucleotide sequence according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the 5E2-12 monoclonal antibody of claim 1 is shown in SEQ ID NO. 9; And / or, the nucleotide sequence encoding the light chain variable region of the 5E2-12 monoclonal antibody of claim 1 is shown in SEQ ID NO.

10.

5. A biomaterial, characterized in that, The biological material expresses the 5E2-12 monoclonal antibody of claim 1 or 2 or an antibody fragment of the monoclonal antibody, or contains the polynucleotide sequence of claim 3 or 4, and the biological material includes at least one of expression cassette, vector, recombinant microorganism and cell line.

6. Use of the 5E2-12 monoclonal antibody of claim 1 or 2, or an antibody fragment thereof, 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) Prepare drugs for the prevention and / or treatment of EB virus-related tumors.

7. The use according to claim 6, characterized in that, The EB virus-associated tumors are tumors that are positive for EB virus.

8. The use according to claim 7, characterized in that, The EBV-positive tumors are either EBV-positive nasopharyngeal carcinomas or EBV-positive lymphomas.

9. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition includes the 5E2-12 monoclonal antibody as described in claim 1 or 2, or an antibody fragment thereof.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition also includes cell-penetrating peptides; Preferably, the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.11.

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