Use of antibody having nuclear RNA export factor 2 in preparation of drugs for treating hepatocellular carcinoma

Abstract

Provided is a use of an antibody having nuclear RNA export factor 2 in the preparation of drugs for treating hepatocellular carcinoma. For nuclear RNA export factor 2, a single-chain antibody having high affinity and specificity for the nuclear RNA export factor 2 is prepared by using phage display technology, and the single-chain antibody can be used for preparing a detection reagent for the nuclear RNA export factor 2. In addition, treatment of a hepatocellular carcinoma mouse model using the single-chain antibody has shown that the single-chain antibody can effectively enhance the activity of immune cells in the tumor microenvironment, significantly increase the number of CD8+ T cells inside the tumor, and significantly inhibit the growth of the tumor.

Description

Application of antibodies against nuclear RNA export factor 2 in the preparation of drugs for treating liver cancer Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of antibodies against nuclear RNA export factor 2 in the preparation of drugs for treating liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor originating from liver cells and is the most common type of primary liver cancer. The incidence of HCC is particularly high in areas with a high prevalence of hepatitis. For early-stage HCC patients, surgical resection is the preferred treatment, but most patients are diagnosed at an inoperable, advanced stage. Therefore, targeted therapy and immunotherapy have gradually become cutting-edge approaches to treating HCC. In recent years, immune checkpoint inhibitors have made significant progress in liver cancer treatment, providing patients with new treatment options. Unfortunately, not all patients respond to these treatments, and some patients have developed drug resistance. Therefore, further exploration of new molecular targets and immunotherapy strategies has become an important direction in current HCC research.

[0003] Single-chain antibodies (scFv) are small-molecule genetically engineered antibodies composed of the variable region (VH) of the antibody heavy chain and the variable region (VL) of the light chain linked by a peptide linker. They are the smallest functional structural units that possess the full antigen-binding specificity of the parent antibody, and have advantages such as small molecular weight, low immunogenicity, expression in prokaryotic cell systems, and ease of genetic engineering. However, single-chain antibodies often have drawbacks such as low affinity, limited function, and poor stability, which restricts their widespread application.

[0004] Nuclear RNA export factor 2 (NXF2) is a member of the nuclear RNA export factor family and is primarily involved in the transport of mRNA from the nucleus to the cytoplasm. The biological functions of NXF2 have been relatively little studied, with past research focusing primarily on its role in model organisms such as Drosophila, particularly its function in reproductive development. In addition, there are reports that NXF2 could serve as an immunotherapeutic target for acute leukemia (DOI: 10.3892 / mmr.2013.1659). Other researchers found that overexpression of NXF2 in Huh-7 HCC cells led to a downregulation of Huh-7 cell migration and invasion (https: / / doi.org / 10.1158 / 1078-0432.CCR-08-2099). Currently, there are no reports on single-chain antibodies against NXF2 and their potential therapeutic applications in liver cancer.

[0005] Summary of the Invention

[0006] This invention addresses the lack of existing single-chain antibodies against NXF2 by preparing a single-chain antibody with high affinity and specificity for NXF2. Furthermore, it was discovered that this single-chain antibody can be used to treat liver cancer, as single-chain antibodies against NXF2 are functional inhibitors of NXF2. Therefore, the application of NXF2 functional inhibitors in the preparation of liver cancer therapeutic drugs is proposed.

[0007] The first objective of this invention is to provide the application of a functional inhibitor of nuclear RNA export factor 2 in the preparation of drugs for treating liver cancer.

[0008] A second objective of this invention is to provide a single-chain antibody against nuclear RNA export factor 2.

[0009] A third object of the present invention is to provide a formulation containing the single-chain antibody.

[0010] A fourth object of the present invention is to provide the use of the single-chain antibody or the preparation in the preparation of a detection reagent for nuclear RNA export factor 2.

[0011] The fifth objective of this invention is to provide an immunotherapy drug for liver cancer.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] This invention utilizes phage display technology to prepare a single-chain antibody with high affinity and specificity for nuclear RNA export factor 2. Furthermore, this invention, using the single-chain antibody to treat a mouse model of liver cancer, revealed that the antibody effectively enhances the activity of immune cells in the tumor microenvironment and significantly increases CD8+ within the tumor. + The number of T cells significantly inhibits tumor growth. Single-chain antibodies against NXF2 are functional inhibitors of NXF2. Therefore, this invention seeks protection for the use of functional inhibitors of nuclear RNA export factor 2 in the preparation of therapeutic drugs for liver cancer.

[0014] Specifically, the inhibitor of nuclear RNA export factor 2 can enhance the function of CD8 within tumors. + The number of T cells inhibits tumor growth.

[0015] Specifically, the liver cancer treatment drug is an immunotherapy drug for liver cancer.

[0016] Specifically, the liver cancer mentioned is primary hepatocellular carcinoma.

[0017] Specifically, the functional inhibitor is a functional inhibitor of human nuclear RNA export factor 2.

[0018] Specifically, the functional inhibitor is an antibody against human nuclear RNA export factor 2 or a formulation containing an antibody against human nuclear RNA export factor 2.

[0019] Optionally, the formulation containing the antibody of human nuclear RNA export factor 2 includes, but is not limited to, a fusion protein containing an antibody of human nuclear RNA export factor 2, a liposome containing an antibody of human nuclear RNA export factor 2, or a liposome containing the fusion protein.

[0020] Optionally, the antibody is a single-chain antibody.

[0021] Specifically, the single-chain antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region contains three complementarity-determining regions (CDRs), namely VH CDR1, VH CDR2, and VH CDR3, with amino acid sequences as shown in SEQ ID NO. 1–3, which play a key role in the binding of the antibody to the antigen. The light chain variable region also contains three CDRs, namely VL CDR1, VL CDR2, and VL CDR3, with amino acid sequences of VL CDR1 and VL CDR3 as shown in SEQ ID NO. 4–5, and the amino acid sequence of VL CDR2 being RAS, which works synergistically with the CDRs of the heavy chain variable region to improve the antibody's recognition ability and affinity for the antigen.

[0022] Specifically, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the present invention is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

[0023] The present invention also seeks protection for a single-chain antibody against said nuclear RNA export factor 2.

[0024] Specifically, the single-chain antibody of nuclear RNA export factor 2 is a single-chain antibody of human nuclear RNA export factor 2; the single-chain antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity-determining regions, namely VH CDR1, VH CDR2 and VH CDR3, whose amino acid sequences are shown in SEQ ID NO. 1 to 3; the light chain variable region comprises three complementarity-determining regions, namely VL CDR1, VL CDR2 and VL CDR3, whose amino acid sequences are shown in SEQ ID NO. 4 to 5, and the amino acid sequence of VL CDR2 is RAS.

[0025] Specifically, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

[0026] The present invention also provides a formulation containing a single-chain antibody of the human nuclear RNA export factor 2.

[0027] Optionally, the formulation containing the antibody of human nuclear RNA export factor 2 includes, but is not limited to, a fusion protein containing an antibody of human nuclear RNA export factor 2, a liposome containing an antibody of human nuclear RNA export factor 2, or a liposome containing the fusion protein.

[0028] Optionally, the fusion protein is a fusion protein of a single-chain antibody and a cell-penetrating peptide.

[0029] Specifically, a cell-penetrating peptide is attached to either end of the single-chain antibody.

[0030] Specifically, the single-chain antibody has a cell-penetrating peptide attached to its C-terminus.

[0031] Optionally, the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.15.

[0032] Specifically, the cell-penetrating peptide is linked to the single-chain antibody using a linker.

[0033] Optionally, the Linker is GGGGSGGGGSGGGGS.

[0034] Specifically, the liposomes are prepared using a thin-film hydration method, which involves mixing dioleoyl lecithin (DOPC), cholesterol, and dioleoyl phosphatidylethanolamine (DOPE) in a molar ratio of 3:1:1 and evaporating the mixture under vacuum to form a lipid membrane. The single-chain antibody or fusion protein described in this invention is then added to a hydration solution (phosphate-buffered saline (PBS) or HEPES buffer), and the lipid membrane is hydrated at 35–39°C. A uniform liposome suspension is obtained by ultrasonic treatment. The particle size of the liposomes is controlled to be 90–110 nm using an extrusion method.

[0035] The present invention also claims protection for the use of the single-chain antibody or a formulation containing the single-chain antibody of the human nuclear RNA export factor 2 in the preparation of a detection reagent for nuclear RNA export factor 2.

[0036] Specifically, the detection reagents include quantitative and / or qualitative detection reagents.

[0037] Specifically, the detection reagent is a detection reagent for human nuclear RNA export factor 2.

[0038] Optionally, the detection reagent is an ELISA detection reagent for human nuclear RNA export factor 2, or an immunoblotting detection reagent for human nuclear RNA export factor 2.

[0039] The present invention also claims protection for an immunotherapy drug for liver cancer, said drug containing the single-chain antibody described in this invention.

[0040] Specifically, the drug also contains pharmaceutically acceptable excipients for the single-chain antibody.

[0041] Optionally, the single-chain antibody can be used alone as the sole active ingredient or in combination with other tumor immunotherapy drugs.

[0042] The present invention has the following beneficial effects:

[0043] This invention targets nuclear RNA export factor 2 (RNA export factor 2) and utilizes phage display technology to prepare a single-chain antibody with high affinity and specificity for it. This single-chain antibody can be used to prepare detection reagents for RNA export factor 2. Furthermore, this invention, using the single-chain antibody to treat a mouse model of liver cancer, revealed that the single-chain antibody can effectively enhance the activity of immune cells in the tumor microenvironment and significantly increase the activity of CD8+ within the tumor. + The increased number of T cells significantly inhibits tumor growth. Therefore, this invention proposes the application of antibodies against nuclear RNA export factor 2 in the preparation of drugs for treating liver cancer. This invention is beneficial for the development of immunotherapeutic drugs for liver cancer and also for the formulation of new immunotherapy strategies. Attached Figure Description

[0044] Figure 1 is a schematic diagram of the structure of the prokaryotic recombinant expression vector pT7-NXF2(Human)-6×His.

[0045] Figure 2 shows the SDS-PAGE results of IPTG-induced expression of recombinant NXF2 protein; in the figure, sup corresponds to the supernatant sample; pellet corresponds to the precipitate sample.

[0046] Figure 3 shows the affinity screening results of recombinant phages.

[0047] Figure 4 shows the results of bacterial culture PCR identification of the scFv insert fragment.

[0048] Figure 5 is a schematic diagram of the structure of the recombinant prokaryotic expression vector pET28a-GW001.

[0049] Figure 6 shows the SDS-PAGE results of the supernatant and precipitate sample after lysis of bacteria expressing recombinant scFv protein.

[0050] Figure 7 shows the affinity test results of the NXF2-GW001 single-chain antibody.

[0051] Figure 8 shows the sensitivity detection results of the NXF2-GW001 single-chain antibody.

[0052] Figure 9 shows the growth of Hepa1-6 subcutaneous tumors in mice in the IgG control group and the GW001-CPP liposome treatment group; A in the figure represents the size of the Hepa1-6 subcutaneous tumors in mice from day 0 to day 21; B in the figure represents the weight of the Hepa1-6 subcutaneous tumors in mice on day 21; ***p<0.001 in the figure.

[0053] Figure 10 shows the CD8+ levels in tumor tissues of mice in the IgG control group and the GW001-CPP liposome treatment group. + Results of T cell count detection; p < 0.01 in the figure.

[0054] Figure 11 shows the changes in body weight of mice in the IgG control group and the GW001-CPP liposome treatment group. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0056] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0057] Example 1: Preparation of a single-chain antibody against human nuclear RNA export factor 2 (NXF2)

[0058] 1. Construction of the prokaryotic recombinant expression vector pT7-NXF2(Human)-6×His

[0059] The full-length gene sequence of human nuclear RNA export factor NXF2 (NCBI Reference Sequence: NM_022053.4) was obtained from the Gene Bank database. Miaoling Biotechnology Co., Ltd. was commissioned to synthesize the full-length gene sequence of NXF2, and a 6×His tag was introduced at the C-terminus of this sequence. The synthesized NXF2(Human)-6×His sequence was cloned into the pT7 expression vector, then transformed into DH5α competent cells for amplification. The plasmid was extracted and identified, yielding the prokaryotic recombinant expression vector pT7-NXF2(Human)-6×His, which was then used for subsequent experiments. A schematic diagram of the structure of the prokaryotic recombinant expression vector pT7-NXF2(Human)-6×His is shown in Figure 1.

[0060] 2. Expression, purification, and determination of recombinant human nuclear RNA export factor 2 protein

[0061] (1) Transformation: The prokaryotic recombinant expression vector pT7-NXF2(Human)-6×His was transformed into BL21 competent cells (Novozymes). After heat shock at 42°C for 90 seconds, the cells were placed on ice for 2 minutes and spread on LB solid plates containing a final concentration of 50 μg / mL kanamycin. The cells were cultured overnight at 37°C to screen and identify NXF2 recombinant protein expression strains.

[0062] (2) IPTG-induced expression of NXF2 recombinant protein: Single colonies of the NXF2 recombinant protein expression strain were picked and cultured overnight at 37°C with shaking at 220 rpm in 10 mL LB medium (containing 50 μg / mL kanamycin); the overnight cultured bacterial solutions were inoculated into 10 mL LB medium (containing 50 μg / mL kanamycin) at a volume ratio of 1:100 and cultured overnight at 37°C with shaking at 220 rpm; when OD600 nm=0.6, IPTG was added to a final concentration of 0.5 mM / 0.2 mM, cultured with shaking at 180 rpm, and induced overnight at 16°C, with the untreated samples serving as negative controls; the bacterial cells were collected by centrifugation, and the cells were sonicated. The supernatant and precipitate were collected by centrifugation to obtain supernatant and precipitate samples respectively; 25 μL of each sample were analyzed by 12% SDS-PAGE, and the results are shown in Figure 2.

[0063] As shown in Figure 2, differential bands were obtained by induction with 0.5 mM / 0.2 mM IPTG compared to the negative control, indicating successful induction of expression. Among them, the most obvious band was observed in the 70–100 kDa region induced by bacterial lysis and precipitation under the 0.2 mM IPTG condition. This induction condition was used for subsequent large-scale induction of expression.

[0064] (3) Large-scale induction of NXF2 recombinant protein expression: The bacterial culture of the NXF2 recombinant protein expression strain was inoculated into 600 mL of LB kanamycin resistant medium at a volume ratio of 1:100 for large-scale induction of expression. The induction conditions were: IPTG at a final concentration of 0.2 mM, cultured overnight at 16°C and 180 rpm with shaking.

[0065] (4) Purification of NXF2 recombinant protein: The bacterial cells were collected by centrifugation, sonicated in an ice bath, and the lysate was collected after centrifugation. The protein was then denatured and renatured, concentrated and centrifuged multiple times using a 30kD ultrafiltration tube, and then aliquoted and lyophilized for storage. Testing showed that the NXF2 recombinant protein purified using this method could meet the needs of subsequent immunization.

[0066] 3. Preparation of single-chain antibodies against human nuclear RNA export factor 2

[0067] (1) Immunization of New Zealand rabbits: The purified NXF2 recombinant protein was diluted with PBS to a final concentration of 250 μg / mL; it was then mixed with the corresponding adjuvant at a volume ratio of 1:1; Freund's adjuvant was used for the first injection, followed by Freund's incomplete adjuvant; after the antigen and adjuvant were completely mixed to form a stable emulsion, the emulsion was injected subcutaneously at multiple points on the back of the New Zealand rabbits. Before the first immunization, blood was collected from the ear vein to separate blank control serum; immunizations were performed every 3 weeks, and samples were collected after 4 rounds to separate post-immunization serum and spleen. Serum and Trizol-treated spleen tissue were stored at 80°C.

[0068] (2) Construction of scfv phage particle: RNA was extracted from the spleen of immunized New Zealand rabbits and reverse transcribed to obtain B lymphocyte cDNA; using the obtained cDNA as a template, specific primers were designed to amplify the variable regions (VL and VH) of the antibody light chain and heavy chain, respectively. The amplified VL and VH were ligated into scFv fragments using a linker (GGGGSGGGGSGGGGS), digested with enzymes, and then ligated into the insertion vector (pCANTAB 5E) to construct the phage particle pCANTAB 5E-scFv.

[0069] (3) Amplification and Screening of Recombinant Phages: *E. coli* TG1 was co-infected with scFv phage particles and helper phage M13KO7 to obtain infectious recombinant phages, i.e., a phage library expressing the NXF2 single-chain antibody. Recombinant phages with relatively high affinity were selected through a three-round Phage Elisa screening. The results of the recombinant phage affinity screening are shown in Figure 3. In the upper figure, the bars of the same color as NXF2 represent the binding strength (OD450 value) between the phage and NXF2, and the bars of the same color as BSA represent the binding strength with BSA. As shown in the figure, the OD450 value of most phages was significantly higher when binding to NXF2 than when binding to BSA, indicating that these phages exhibited a strong target binding ability. The lower figure in Figure 3 shows the ratio of OD450 values ​​(NXF2 / BSA); the higher the ratio, the stronger the affinity of the phage for NXF2. Observations revealed that phages in groups 5, 19, and 21 exhibited high binding capacity at the NXF2 / BSA ratio, indicating that these phages had a relatively strong affinity for the target protein NXF2 and were selected for subsequent experiments.

[0070] Single clones were isolated and amplified in small quantities. Using primer pairs (F: AGCCGGCCATGGGGCCCA; R: CGCACCTGCGGCCGCCC), the scFv insert fragment was identified by bacterial PCR, and the results are shown in Figure 4. As shown in Figure 4, the PCR bands corresponding to samples 5, 19, and 21 were approximately 750 bp, consistent with the expected scFv fragment size, indicating that these three groups of phages successfully inserted the target scFv fragment. Those with superior binding ability were then sequenced. Sequencing results show that the heavy chain variable region of the scFv insert fragment (named GW001) in the recombinant phage with superior binding ability includes VH CDR1 shown in SEQ ID NO.1, VH CDR2 shown in SEQ ID NO.2, and VH CDR3 shown in SEQ ID NO.3. The light chain variable region of this scFv insert fragment includes VL CDR1 shown in SEQ ID NO.4, VL CDR2 with the amino acid sequence RAS, and VL CDR3 shown in SEQ ID NO.5. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7. The nucleotide sequences encoding each of the above CDRs, as well as the heavy chain variable region and the light chain variable region, are shown in SEQ ID NO.8 to 14 respectively (among which, the nucleotide sequence encoding VL CDR2 is not included in the sequence listing because the number of bases is less than 10, and the nucleotide sequence encoding VL CDR2 is AGGGCATCC).

[0071] (4) Construction of a recombinant expression vector for scFv recombinant protein (GW001 antibody): A 6×His Tag (synthesized by Miaoling Biotechnology) was introduced into the C-terminus of the nucleotide sequence of GW001. The synthesized sequence was inserted into the prokaryotic expression vector (pET28a), transformed into DH5α competent cells for amplification, and the plasmid was extracted to obtain the recombinant prokaryotic expression vector pET28a-GW001. The structural schematic diagram of the recombinant prokaryotic expression vector is shown in Figure 5. The inserted scFv fragment is denoted as APR-1 in the recombinant expression vector.

[0072] (5) Expression and purification of scFv recombinant protein (GW001 antibody): The recombinant expression vector pET28a-GW001 was transformed into BL21 competent cells. Following the expression and purification steps for human nuclear RNA export factor 2 recombinant protein, low-temperature induction and purification concentration were performed, followed by SDS-PAGE detection. The SDS-PAGE results of the supernatant and precipitate samples after bacterial lysis expressing scFv recombinant protein are shown in Figure 6. As shown in Figure 6, under non-induction conditions (sup and pellet columns), there was almost no obvious protein expression, indicating that the recombinant protein was not significantly expressed without IPTG induction. However, after IPTG induction, the sup (supernatant) column showed fewer protein bands, indicating that the GW001 antibody was mostly expressed in an insoluble form. A distinct protein band, approximately 25 kDa, was observed in the pellet column, consistent with the expected molecular weight of the recombinant GW001 antibody (marked in the box), indicating that the target protein was mainly present in the insoluble component and may form inclusion bodies. The purified recombinant GW001 antibody is designated as NXF2-GW001 single-chain antibody.

[0073] Example 2 Performance Detection of Single-Chain Antibody for Human Nuclear RNA Export Factor 2

[0074] 1. ELISA determination of the affinity of single-chain antibodies

[0075] The purified recombinant protein NXF2 was diluted with coating buffer (C3041, Sigma-Aldrich) to a final concentration of 20 μg / mL. 100 μL of this buffer was used to coat 96-well microplates, and the plates were incubated at 37°C for 2 h. The plates were then washed twice with washing buffer (P3563, Sigma-Aldrich), and 200 μL of blocking buffer (37515, Thermo Fisher) was added to each well, and the plates were blocked at 37°C for 1 h. After washing twice, purified antibody (NXF2-GW001 single-chain antibody) was added to each well, with a final antibody concentration of 10 ng / mL, and the plates were incubated at 37°C for 1 h. After washing three times, 100 μL of horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (1:10000 dilution) was added to each well, and the plates were incubated at 37°C for 1 h. After washing three times, TMB substrate (34021, Thermo Fisher Scientific) was added to each well. Fisher), 100 μL / well, incubate in the dark for 10–15 minutes, then add 50 μL / well of stop solution. Immediately after termination, use a microplate reader to detect OD450 and analyze the affinity of the tested single-chain antibody. The affinity test results of NXF2-GW001 single-chain antibody by ELISA are shown in Figure 7. The results indicate that NXF2-GW001 single-chain antibody has a strong affinity for NXF2.

[0076] 2. Sensitivity of Western blot detection of single-chain antibodies

[0077] Immunoblot detection was performed on antigen (NXF2) with different loading amounts (10, 50, 100, 500, 1000 ng). Rabbit anti-human NXF2 antibody was diluted 1:800 as the primary antibody, and horseradish peroxidase-labeled goat anti-rabbit IgG was diluted 1:10000 as the secondary antibody. Chemiluminescent colorimetric detection was performed. The sensitivity detection results of NXF2-GW001 single-chain antibody by Western blot are shown in Figure 8. A band began to appear when the antigen loading amount was 50 ng, that is, the detection loading amount of NXF2-GW001 single-chain antibody described in this invention is 50 ng.

[0078] Example 3: Effects of a single-chain antibody against human nuclear RNA export factor 2 on liver cancer.

[0079] 1. Preparation of fusion antibody GW001-CPP liposomes

[0080] (1) Fusion of NXF2-GW001 single-chain antibody with cell-penetrating peptide

[0081] The penetrant peptide sequence (RQIKIWFQNRRMKWKK; shown in SEQ ID NO.15) was used as a cell-penetrating peptide and fused with the NXF2-GW001 single-chain antibody using genetic engineering technology (the fusion expression vector used was pET28a) to form the fusion antibody GW001-CPP. The penetrant peptide sequence is located at the C-terminus of the NXF2-GW001 single-chain antibody, and the penetrant peptide and NXF2-GW001 are linked by a linker (GGGGSGGGGSGGGGS).

[0082] (2) Preparation of fusion antibody GW001-CPP liposomes

[0083] Liposomes were prepared using a thin-film hydration method. Dioleoyl lecithin (DOPC, 850375P, Sigma-Aldrich), cholesterol (C8667, Sigma-Aldrich), and dioleoyl phosphatidylethanolamine (DOPE, 850725P, Sigma-Aldrich) were mixed in a 3:1:1 molar ratio and evaporated to dryness under vacuum to form a lipid membrane. GW001-CPP antibody was added to hydration solution PBS (1 mg antibody added to 1 mL of hydration solution (1:1 mg / mL)), and the lipid membrane was hydrated at 37°C. A homogeneous liposome suspension was then obtained by sonication. The particle size of the liposomes was controlled to be 90–110 nm using an extrusion method, and their particle size distribution and zeta potential were detected by dynamic light scattering (DLS).

[0084] 2. Modeling

[0085] (1) Experimental animals: 5-week-old C57BL / 6 mice, provided by the Animal Center of Sun Yat-sen University.

[0086] (2) Cell culture: The Hepa1-6 liver cancer cell line was cultured in DMEM medium (Kangning) containing 10% fetal bovine serum (Gibco), 100 U / mL sodium pyruvate and 1% penicillin-streptomycin solution (Beyotime) at 37°C and 5% CO2. The cells were passaged every 2 to 3 days, and cells in the logarithmic growth phase were used for in vivo experiments.

[0087] (3) Inoculation and grouping of tumor cells: Hepa1-6 liver cancer cells (5×10⁶ cells) resuspended in PBS were... 7 100 μL / mouse was injected subcutaneously into the right rib area of ​​mice; mice were randomly divided into three groups after inoculation: a non-tumor-bearing control group, an IgG control group (BioXcell, #BE0085; 50 μg IgG injected intraperitoneally every 3 days), and a GW001-CPP liposome treatment group (50 μg GW001-CPP liposomes injected intraperitoneally every 3 days), with administration lasting for 21 days.

[0088] (4) Indicator detection: The mouse's body weight change, tumor growth curve and tumor weight on day 21 were recorded, and CD8 in the tumor tissue was detected. + T cell count.

[0089] Figure 9 shows the growth of Hepa1-6 subcutaneous tumors in mice in the IgG control group and the GW001-CPP liposome treatment group; A in Figure 9 represents the size of the Hepa1-6 subcutaneous tumors in mice from day 0 to day 21; B in Figure 9 represents the weight of the Hepa1-6 subcutaneous tumors in mice on day 21. As shown in Figure 9, the NXF2-GW001 single-chain antibody described in this invention can significantly inhibit tumor growth (p < 0.001).

[0090] CD8 in tumor tissues of mice in the IgG control group and the GW001-CPP liposome treatment group + The results of T cell count detection are shown in Figure 10. As can be seen from Figure 10, the NXF2-GW001 single-chain antibody described in this invention can significantly increase CD8+ levels in tumor tissue. + T cell count (p < 0.01).

[0091] Figure 11 shows the changes in body weight of mice in the IgG control group and the GW001-CPP liposome treatment group. As can be seen from Figure 11, there was no significant change in body weight of the mice after drug administration.

[0092] 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 changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of functional inhibitors of nuclear RNA export factor 2 in the preparation of drugs for treating liver cancer.

2. The application according to claim 1, characterized in that, The liver cancer treatment drug mentioned is an immunotherapy drug for liver cancer.

3. The application according to claim 1, characterized in that, The functional inhibitor is an antibody against nuclear RNA export factor 2 or a formulation containing an antibody against nuclear RNA export factor 2.

4. The application according to claim 3, characterized in that, The antibody is a single-chain antibody, which includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes three complementarity-determining regions, namely VH CDR1, VH CDR2, and VH CDR3, whose amino acid sequences are shown in SEQ ID NO. 1 to 3. The light chain variable region includes three complementarity-determining regions, namely VL CDR1, VL CDR2, and VL CDR3, whose amino acid sequences are shown in SEQ ID NO. 4 to 5, and whose amino acid sequence is RAS.

5. The application according to claim 4, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

7.

6. A single-chain antibody against nuclear RNA export factor 2, characterized in that, The single-chain antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity-determining regions, namely VH CDR1, VH CDR2 and VH CDR3, whose amino acid sequences are shown in SEQ ID NO. 1 to 3; the light chain variable region comprises three complementarity-determining regions, namely VL CDR1, VL CDR2 and VL CDR3, whose amino acid sequences are shown in SEQ ID NO. 4 to 5, and the amino acid sequence of VL CDR2 is RAS.

7. The single-chain antibody according to claim 6, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

7.

8. A formulation comprising the single-chain antibody of claim 6 or 7.

9. The use of the single-chain antibody of claim 6 or 7 or the formulation of claim 8 in the preparation of a detection reagent for nuclear RNA export factor 2.

10. An immunotherapy drug for liver cancer, characterized in that, Contains the single-chain antibody as described in claim 6 or 7.

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