Vasn-lrr monoclonal antibodies, methods of making, compositions, and uses

Abstract

This invention relates to VASN-LRR monoclonal antibodies, their preparation methods, compositions, and applications, belonging to the field of biomedicine. It addresses the problems of existing anti-VASN antibodies primarily targeting the EGF domain, resulting in unsatisfactory anti-tumor effects and a lack of high affinity and high specificity for recognizing the LRR domain. The invention provides an antibody comprising the CDR1, CDR2, and CDR3 amino acid sequences of the light and heavy chains, as shown in SEQ ID NO. 3 to SEQ ID NO. 8. This antibody exhibits extremely high affinity and titer, specifically binding to natural VASN protein. Used alone or in combination with lenvatinib, it significantly inhibits the growth of subcutaneous xenografts of liver cancer cells and xenografts of malignant liver cells induced by aflatoxin B1 exposure. It can be used to prepare drugs for treating liver cancer, especially in combination with lenvatinib for the treatment of liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, this invention relates to an isolated VASN-LRR monoclonal antibody against the LRR domain of the VASN protein, its preparation method, composition, and application. Background Technology

[0002] Vasorin protein (VASN) is a type I transmembrane glycoprotein whose extracellular domain contains multiple domains, including a tandemly linked leucine-rich repeat motif (LRR), an epidermal growth factor-like motif (EGF), and a type III fibronectin-like motif (FN3). VASN is abnormally highly expressed in tumors such as hepatocellular carcinoma (HCC) and can promote the proliferation and migration of HCC cells by activating signaling pathways such as STAT3. It is considered a potential serum biomarker and therapeutic target for HCC. Therefore, developing functional monoclonal antibodies targeting VASN is of great significance for understanding the mechanisms of VASN-related diseases and for targeted therapy.

[0003] However, the research and application of existing anti-VASN monoclonal antibodies still face the following problems.

[0004] First, existing antibodies, such as those described in the literature (Mo Wanling et al. Preparation of monoclonal antibodies against VASN-EGFFN3 recombinant protein [J]. Journal of Guangxi Medical University, 2023, 40(4): 690-696), mainly target the EGF domain or its combination domains of VASN. However, the antitumor effect of these antibodies is not ideal. Although these antibodies can be used for the detection and preliminary functional study of VASN protein, there is still a lack of sufficient in vivo experimental evidence on whether they can effectively inhibit tumor growth, which is a significant shortcoming in therapeutic applications.

[0005] Second, although there is literature (e.g., Yin Moli et al. Preparation of mouse anti-human vasorin (VASN) monoclonal antibody using high-efficiency electrofusion technology [J]. Journal of Cellular and Molecular Immunology, 2021), reporting that monoclonal antibodies targeting VASN have inhibitory effects on the proliferation and migration of liver cancer cells, there is a lack of monoclonal antibodies that can specifically recognize the LRR domain of the VASN protein and have high affinity. Existing VASN monoclonal antibodies mostly use the full-length protein or extracellular domain as immunogens, and the binding specificity of different antibodies to the LRR domain is difficult to distinguish. Some studies, such as the Chinese patent document CN113403320A, which discloses a recombinant human VASN protein LRR domain protein and its monoclonal antibody, have preliminarily explored the preparation of monoclonal antibodies using the VASN protein LRR domain as an immunogen, but mainly focused on the expression of the recombinant protein and the establishment of antibody detection methods, without verifying the functional value of this domain antibody in tumor treatment.

[0006] Third, aflatoxin B1 (AFB1) exposure is one of the important risk factors for liver cancer; however, existing studies on VASN-related antibodies have not included intervention models for AFB1-induced liver cancer. For AFB1-related liver cancer, there is still a lack of specific treatment strategies based on targeting VASN.

[0007] Furthermore, existing studies have shown that lenvatinib is a first-line treatment for advanced liver cancer, but most patients eventually develop drug resistance. How to further improve the therapeutic effect of lenvatinib and delay the onset of drug resistance is a pressing clinical problem in the field of liver cancer treatment. Currently, there are no reports on the combined use of anti-VASN antibodies and lenvatinib for liver cancer treatment. Summary of the Invention

[0008] One objective of this invention is to address the following technical problems: Although there are antibodies against VASN proteins in the prior art, most of them target the EGF domain or the full-length protein, and there is a lack of monoclonal antibodies that specifically recognize the LRR domain; at the same time, the existing anti-VASN antibodies have not disclosed the amino acid sequences of their heavy and light chain variable regions, and there is an urgent need for a monoclonal antibody with a clearly defined amino acid sequence that can specifically bind to the LRR domain of the VASN protein and its application.

[0009] To achieve the above objectives, the present invention provides an isolated VASN-LRR monoclonal antibody against the LRR domain of the VASN protein. The monoclonal antibody comprises: light chains CDR1, CDR2, and CDR3, and heavy chains CDR1, CDR2, and CDR3; wherein the amino acid sequence of light chain CDR1 is shown in SEQ ID NO.3, the amino acid sequence of light chain CDR2 is shown in SEQ ID NO.4, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO.5; the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO.6, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO.8.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0011] The present invention provides a pharmaceutical composition comprising any of the monoclonal antibodies described in any one of the claims.

[0012] The present invention provides an antitumor combination drug composition comprising any one of the monoclonal antibodies and lenvatinib.

[0013] The use of any of the monoclonal antibodies provided by this invention in the preparation of medicaments for treating liver cancer.

[0014] The use of any of the monoclonal antibodies provided by this invention in the preparation of a medicament for the combined treatment of liver cancer with lenvatinib.

[0015] Preferably, the liver cancer is aflatoxin B1-induced liver cancer.

[0016] The present invention provides a method for preparing the aforementioned monoclonal antibody, comprising the following steps: Step 1: Obtain the VASN-LRR target gene sequence, ligate it into a vector, and construct a recombinant expression vector; Step 2: The recombinant expression vector was transformed into E. coli, and after induction of expression, recombinant VASN-LRR protein was obtained; Step 3: Purify recombinant VASN-LRR protein using a gel extraction method; Step 4: Immunize BALB / c mice with purified recombinant VASN-LRR protein as an antigen; Step 5: Fuse spleen cells from immunized mice with SP2 / 0 myeloma cells and screen for positive hybridoma cells; Step 6: Culture hybridoma cells, collect and purify antibodies.

[0017] Preferably, in step 2, IPTG is used to induce expression at a concentration of 0.2-1.0 mM, an induction temperature of 15-37℃, and an induction time of 3-6 hours; in step 3, the gel recovery and purification includes: after electrophoresis, staining and cutting the gel, incubation in buffer overnight, and ultrafiltration concentration.

[0018] The present invention has at least the following beneficial effects: 1. A VASN-LRR monoclonal antibody with a defined amino acid sequence against the LRR domain of the VASN protein is provided. The heavy chain variable region and light chain variable region sequences of the antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The monoclonal antibody contains the light chain CDR1 amino acid sequence as shown in SEQ ID NO.3, the light chain CDR2 amino acid sequence as shown in SEQ ID NO.4, and the light chain CDR3 amino acid sequence as shown in SEQ ID NO.5; the heavy chain CDR1 amino acid sequence as shown in SEQ ID NO.6, the heavy chain CDR2 amino acid sequence as shown in SEQ ID NO.7, and the heavy chain CDR3 amino acid sequence as shown in SEQ ID NO.8. The study also provides the hybridoma cell line 6B10 that produces the antibody. This hybridoma cell line is classified as mouse hybridoma cell, with the Latin scientific name *Mus musculus*. It was deposited on June 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46360, enabling those skilled in the art to reproduce the antibody.

[0019] 2. This monoclonal antibody exhibits a high affinity for the LRR domain of the VASN protein, at 2.43 × 10⁻⁶. 9 The antibody has a concentration of L / mol and a titer of 1:2187000. This monoclonal antibody specifically binds to the VASN protein and exhibits no cross-reactivity with unrelated proteins such as SHBG and BSA, making it suitable for the detection and functional studies of the VASN protein.

[0020] 3. In vivo animal experiments showed that this monoclonal antibody had an inhibitory effect on the subcutaneous xenograft model of Hep3B liver cancer; when used in combination with lenvatinib, the tumor inhibition rate was 83.6%, which was higher than the 59.2% in the lenvatinib monotherapy group. Under the experimental conditions, the combined treatment was more effective than monotherapy.

[0021] 4. In an aflatoxin B1-induced L02 malignant cell xenograft model, the antibody showed a tumor inhibition rate of approximately 48.0%; when used in combination with lenvatinib, the tumor inhibition rate was 80.6%, higher than the 56.1% in the lenvatinib monotherapy group. These results suggest that this monoclonal antibody has potential application value in the treatment of AFB1-related liver cancer.

[0022] 5. A complete preparation method from antigen preparation, animal immunization, cell fusion to antibody purification is provided, and the induction expression conditions are optimized to IPTG concentration of 0.2-1.0 mM, induction temperature of 15-37℃, induction time of 3-6 hours, and gel recovery purification process, which is convenient for subsequent production applications.

[0023] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0024] Figure 1 These are the PCR amplification analysis results of the LRR gene in this invention; Figure 2 This is the result of PCR identification of monoclonal colonies in this invention; Figure 3 The results of SDS-PAGE for the induced expression of the LRR recombinant protein of this invention; Figure 4 The results show the effect of different induction temperatures on the expression of LRR recombinant protein in this invention; Figure 5 The results show the effect of different IPTG concentrations on the expression of LRR recombinant protein in this invention; Figure 6 The above are the SDS-PAGE results of the LRR recombinant protein after gel extraction and purification according to the present invention. Figure 7 The results of Western blot identification of LRR recombinant protein according to the present invention; Figure 8 The results of the mouse serum titer assay of this invention; Figure 9 This is a cloned image of a hybridoma cell on day 9 after cell fusion according to the present invention; Figure 10 A photograph of a single hybridoma cell clone taken 7 days after subcloning according to the present invention; Figure 11 ELISA results showing the binding activity of different hybridoma clones to VASN protein according to the present invention; Figure 12 The images show the ELISA binding curves and affinity assay results of different antibodies 6B10, 6F6, and 9C9 of this invention; where A corresponds to antibody 6B10, B corresponds to antibody 6F6, and C corresponds to antibody 9C9. Figure 13 The results of SDS-PAGE analysis of the purified antibody of this invention are shown. Figure 14 This is the result of the monoclonal antibody subtype identification in this invention; Figure 15 The indirect ELISA method of this invention is used to detect antibody specificity results; Figure 16 The results of antibody specificity detection using the Western blot method of this invention; Figure 17The results of the immunofluorescence method for detecting the subcellular localization of VASN protein in this invention are shown below; column A corresponds to the Hoechst nuclear staining image, column B corresponds to the FITC-labeled secondary antibody Fluor-488 image, and column C corresponds to the merged image of the Hoechst nuclear staining image and the secondary antibody Fluor-488 image. Figure 18 The appearance of the Hep3B subcutaneous hepatocellular carcinoma xenograft of the present invention; Figure 19 The above are the pathological results of the Hep3B subcutaneous xenograft tumor of liver cancer in this invention; where A is the control group (NC), B is the 6B10 antibody group (6B10), C is the lenvatinib group (Len), D is an enlarged view of the circled red blood cell area in the control group, E is the 6B10+lenvatinib group (6B10+Len), and F is the VASN-KO group. Figure 20 The appearance of the AFB1 malignant L02 hepatocyte xenograft tumor in nude mice according to the present invention; Figure 21 The above are the HE pathological results of AFB1 malignant L02 hepatocellular xenograft tumors in nude mice, where A is the control group (NC), B is the 6B10 antibody group (6B10), C is the lenvatinib group (Len), and D is the 6B10 + lenvatinib group (6B10 + Len).

[0025] Preservation Instructions The hybridoma cell line 6B10, which contains a monoclonal antibody against the LRR domain of the VASN protein, provided by this invention, is classified as mouse hybridoma cell (Mus musculus). It was deposited on June 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo. 46360, enabling those skilled in the art to reproduce it. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it based on the description. It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the reagents and materials described are commercially available unless otherwise specified.

[0027] sequence list SEQ ID NO.1 (Amino acid sequence of the heavy chain variable region) QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVRQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATFFCARMAYYYGSSYGGFFDYWGQGTTLTVSS SEQ ID NO.2 (Amino acid sequence of the light chain variable region) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMFWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTLRRMETEDAATYYCQQWSSFPLTFGAGTKLELK SEQ ID NO.3 (Light chain CDR1): SSVSY SEQ ID NO.4 (Light chain CDR2): DTS SEQ ID NO.5 (light chain CDR3): QQWSSFPLT SEQ ID NO.6 (Heavy chain CDR1): GYTFTDYS SEQ ID NO.7 (Heavy chain CDR2): INTETGEP SEQ ID NO.8 (heavy chain CDR3): ARMAYYYGSSYGGFFDY SEQ ID NO.9 (nucleotide sequence encoding the variable region of the heavy chain) CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTGAAGATCTCCTGCAAGGCTTCTGGGTTATAACCTTCACAGACTATTCAATGCACTGGGTGAGGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAGAT GACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATTTTTCTGTGCTAGAATGGCTTATTACTACGGTAGTAGCTACGGGGGGTTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO.10 (nucleotide sequence encoding the variable region of the light chain) CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTTCTGGTACCAGCAGAAGCCAGGATCCTCCCCAGACTCCTGATTTATGACACATCCAACCTG GCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACACTCAGACGAATGGAGACTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTTTCCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0028] Example 1: Cloning and Recombination of the VASN-LRR Gene (1) Extract total RNA from cells: Collect HL-7702 cells with a culture density of 70%-80%, extract total RNA according to the instructions of the total RNA extraction kit, and reverse transcribe it into cDNA. The sample can be stored at -80℃ for a long time.

[0029] (2) Determining the VASN-LRR gene amplification sequence: The human VASN protein gene sequence NM_138440.3 was obtained from the NCBI database, and the start position of the LRR domain of the VASN protein, approximately amino acids 322-567, was determined using the UniProt database. Amplification primers for VASN-LRR were designed using Vector NTI software.

[0030] Upstream primer: AAA[GGATCC]TGCCCATCCGGCTGCCAGTGCA; where [GGATCC] is the BamHI restriction site.

[0031] Downstream primer: GGG[AAGCTT]TTAGGTGGGCACTGTGGCTGTGG; where [AAGCTT] is the Hind III restriction site.

[0032] (3) Cloning and Identification of the VASN-LRR Gene: PCR amplification was performed using cDNA as a template. Reaction system: 1 μL each of forward and reverse primers, 1.0 μL cDNA template, 12.5 μL 2× Phanta Max Buffer, 0.5 μL dNTPMix, 0.5 μL PhantaMax Super-Fidelity DNA Polymerase, and 8.5 μL ddH2O. Reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 61℃ annealing for 15 s, and 72℃ extension for 70 s, for a total of 35 cycles; final extension at 72℃ for 5 min; storage at 4℃. PCR products were electrophoresed on a 1% (w / v) agarose gel and then visualized using a gel imaging system. Electrophoresis conditions were constant voltage 120 V for 40 min. Figure 1 As shown, lane M is the DNA marker, and lanes 1 and 2 are LRR amplification products. A specific amplification band appears at approximately 1000 bp, consistent with the expected theoretical size of the LRR gene.

[0033] (4) Extraction of pET30a plasmid: The strain containing pET30a was inoculated into 10 mL of LB liquid medium containing kanamycin at 37℃ and 200 rpm and cultured overnight. The next day, an appropriate amount of bacterial culture was taken and the plasmid was extracted according to the instructions of the OMEGA plasmid extraction kit.

[0034] (5) Double digestion of pET30a plasmid and LRR clone gene: pET30a plasmid and purified LRR PCR product were double digested with BamHI and HindIII respectively, and incubated at 37℃ for 4 h. The digested products were recovered by purification kit.

[0035] (6) Ligation of pET30a-LRR recombinant vector: The pET30a plasmid and LRR gene fragment purified by enzyme digestion were ligated at 25°C for 4 h using T4 ligase.

[0036] (7) Transformation and transfer of pET30a-LRR recombinant vector: In a clean bench, 50 μL of trans5α competent cells were mixed with 10 μL of ligation product, incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, and then incubated on ice for 3 min. 600 μL of kanamycin-free LB liquid medium was added, and the cells were cultured at 37℃ and 200 rpm for 1 h with shaking. Then, the cells were evenly spread onto LB solid culture plates containing kanamycin and incubated upside down at 37℃ for 16 h. Twenty-four plump single colonies were selected, transferred to new LB culture plates, and cultured at 37℃ for 6 h for identification.

[0037] (8) Identification of positive monoclonal strains: ① PCR Identification: A small amount of bacterial cells was picked up with a toothpick and transferred to a PCR tube. Colony PCR was performed in a 20 μL reaction system using the following program: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 30 s, 61℃ annealing for 30 s, 72℃ extension for 45 s, for 35 cycles; final extension at 72℃ for 5 min. 2 μL of the PCR product was then subjected to 1% (w / v) agarose gel electrophoresis at 120 V for 40 min. Figure 2 As shown, lane M is a DNA marker, and positive colonies can amplify a band that is consistent with the theoretical size of the LRR gene.

[0038] ② Double enzyme digestion identification: PCR-positive colonies were selected, plasmids were extracted, and double digestion was performed using Hind III and BamHI. After electrophoresis of the digestion products, two bands appeared. Lane M was the DNA marker, and lane 1 was the pET30a-LRR double digestion product. One band corresponded to the pET30a vector, and the other band corresponded to the LRR target gene, proving that the recombinant plasmid was successfully constructed.

[0039] ③ Sequencing and identification: Single clones that were positive for both double enzyme digestion and PCR were selected for sequencing. The sequencing results were compared with the NCBI reference sequence using Vector NTI software and found to be completely consistent, indicating that the pET30a-LRR recombinant vector was successfully obtained.

[0040] Example 2: Expression and purification of LRR recombinant protein (1) Induced expression of LRR recombinant protein Extract the pET30a-LRR plasmid and transform it into *E. coli* BL21 expressing the recombinant protein according to the transformation steps described above. Measure 10 mL of LB broth containing kanamycin into a clear vial. Use a toothpick to pick up a small amount of the pET30a-LRR recombinant strain. Incubate the vial overnight at 37°C and 200 rpm with shaking. The next day, inoculate the strain at a 1 / 10 ratio into new clear vials containing 10 mL of LB broth containing kanamycin. Incubate at 200 rpm and 37°C for 2 h. Then, set up two groups: an uninduced group and an isopropyl-β-D-1-thiogalactopyranoside (IPTG) induced group, containing the pET30a empty vector plasmid and the pET30a-LRR plasmid, respectively. 1. IPTG inducer was added to the IPTG induction group to make a final concentration of 1 mM, while the non-inducible control group was given an equal amount of PBS without IPTG inducer. Protein expression was induced at 200 rpm and 37℃ for 4-5 h.

[0041] After induction time, centrifuge for 5 min at 12000 rpm at 4℃, collect 5 mL of bacterial culture from each group, discard the supernatant, and resuspend the cells in 1 mL of PBS. Disrupt the cells by sonication on ice for 2 min under the following conditions: 30%, on for 10 s, off for 5 s. Then centrifuge at 12000 rpm for 5 min at 4℃. Collect the supernatant and resuspend the precipitate in 100 μL of PBS. The supernatant and precipitate are then subjected to 12% (w / v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for analysis. The specific steps are as follows: ① Take 4 μL of 4×Loading Buffer and add 12 μL of supernatant and precipitate respectively. Mix thoroughly and heat at 95℃ for 5 min.

[0042] ② Add 2.5 μL of marker and 20 μL of sample to an SDS-PAGE protein gel with a mass-volume percentage concentration of 12%.

[0043] ③ First, electrophoresis the sample at 80 V for 30-40 minutes to allow the sample to reach the separation gel. When the marker bands are clearly separated, increase the voltage to 120 V and stop electrophoresis when the sample reaches the bottom.

[0044] ④ Place the protein gel into Coomassie Brilliant Blue solution and stain for about 60 minutes, shaking slowly on a shaker.

[0045] ⑤ Recover the Coomassie Brilliant Blue solution, add the pre-prepared decolorizing solution, and change it every 1-2 hours until the bands are clear and the background is transparent. Finally, scan the image using a gel scanner.

[0046] The results are as follows Figure 3 As shown: Lane M represents the protein marker. Lane 1 is the supernatant induced by pET30a empty vector, and lane 3 is the supernatant without pET30a empty vector induction; neither lane showed the target band. Lane 2 is the supernatant induced by pET30a-LRR, showing a weak target protein band. Lane 6 is the precipitation induced by pET30a-LRR, showing a strong band of approximately 40 kDa; lane 8 is the precipitation induced by pET30a-LRR, showing no such band. Lanes 1 to 4 are the supernatant fraction, and lanes 5 to 8 are the precipitation fraction. Lanes 1 and 5 are the pET30a-induced group, lanes 2 and 6 are the pET30a-LRR-induced group, lanes 3 and 7 are the pET30a-uninduced group, and lanes 4 and 8 are the pET30a-LRR-uninduced group. The results indicate that the LRR recombinant protein is mainly expressed in inclusion body form.

[0047] (2) Condition optimization of LRR recombinant protein expression Consistent with the protein expression steps described above, LRR recombinant protein expression was induced for 4 h at different temperatures (15℃, 24℃, 37℃, 42℃) to determine the optimal temperature. At the optimal temperature, LRR recombinant protein expression was induced for 4 h at different IPTG concentrations: 1 mM, 0.8 mM, 0.6 mM, 0.4 mM, 0.2 mM, and 0 mM to determine the optimal IPTG concentration.

[0048] The results are as follows Figure 4 As shown: Lane M represents the protein marker. Lane 1 corresponds to induction at 15℃, and lane 2 corresponds to induction at 24℃, both showing low expression levels of the target protein. Lane 3 corresponds to induction at 37℃, resulting in the highest expression level. Lane 4 corresponds to induction at 42℃, where the expression level decreases. Therefore, 37℃ was chosen as the optimal induction temperature.

[0049] The results are as follows Figure 5 As shown: Lane M represents the protein marker. Lane 1 corresponds to an IPTG concentration of 0 mM, with no target band; Lane 2 corresponds to 0.2 mM, showing significant expression; Lanes 3 to 6 correspond to 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM, respectively, with no significant increase in expression levels. To reduce costs and minimize the potential toxicity of high-concentration IPTG, 0.2 mM was chosen as the optimal IPTG concentration.

[0050] (3) Purification of LRR recombinant protein ① Recombinant protein pretreatment: Measure 20 mL of LB liquid medium containing kanamycin and aliquot it into two clear vials. A small amount of pET30a-LRR recombinant engineered strain is picked up with a toothpick. Incubate the two vials overnight at 37°C and 200 rpm with shaking. The next day, inoculate the culture into two new conical flasks containing 150 mL of LB liquid medium containing kanamycin at a 1:10 ratio. Incubate at 200 rpm and 37°C for 2 h, then add IPTG to a final concentration of 0.2 mM. Induce protein expression at 200 rpm and 37°C for 3-6 h. Aliquot the bacterial culture into 50 mL centrifuge tubes and centrifuge at 4°C and 2500×g for 20 min. Discard the supernatant and resuspend the bacterial pellet in 6 mL of PBS. Disrupt the cell resuspension on ice and sonicate for 60 min under the following conditions: 30%, on for 10 s, off for 5 s. Then centrifuge at 4°C and 12000 rpm for 5 min. Discard the supernatant, wash the precipitate inclusion bodies with washing buffer, centrifuge at 12000×g for 5 min at 4℃, retain the precipitate, incubate on ice with equilibration buffer for 1 h to dissolve the bacterial precipitate, centrifuge at 12000×g for 5 min at 4℃, and retain the supernatant protein solution.

[0051] ② Gel purification of LRR recombinant protein: Add an appropriate amount of 4× Loading Buffer to the collected supernatant protein solution, vortex thoroughly to mix, and heat at 95℃ for 5 min. Perform electrophoresis using a 12% (w / v) SDS-PAGE protein gel, leaving more wells in the upper gel without a comb to increase the sample loading volume. After electrophoresis, remove the protein gel and place it in a glass culture dish, pour in 250 mM KCl solution at low temperature, and gently shake the glass culture dish until a clear milky white band appears. Cut off the milky white protein band area and cut it into small pieces, pour in PBS buffer, and gently shake for 3-5 min until the milky white color fades to colorless and transparent. Cut the gel strip into small pieces and place it in a 15 mL centrifuge tube, add an appropriate amount of PBS buffer, and incubate at 4℃ on a shaker for 16 h. The next day, centrifuge at 12000×g for 5 min at 4℃, collect the supernatant, concentrate the protein through a 10 kDa ultrafiltration tube, and filter to obtain approximately 2 mL of purified LRR recombinant protein. The purity of LRR recombinant protein was analyzed using the Coomassie Brilliant Blue method.

[0052] The results are as follows Figure 6 As shown: Lane M is the protein marker, and lane 1 is the purified LRR recombinant protein obtained through gel extraction. A single, clear band is visible at approximately 40 kDa, indicating high purity and suitability for subsequent immunization. Western blot analysis of the purified protein, using anti-His antibody as the primary antibody, revealed a specific band at approximately 40 kDa, confirming that the recombinant protein carries a His tag and is correctly expressed. Mass spectrometry analysis confirmed that the obtained peptide fingerprint matched the theoretical amino acid sequence of the LRR domain of the VASN protein, further confirming the presence of the LRR domain of the human VASN protein in the purified recombinant protein.

[0053] Example 3 Identification of LRR recombinant protein (1) Western blot method LRR recombinant protein was subjected to electrophoresis at concentrations of 10 ng, 20 ng, and 50 ng. Transfer conditions were 200 mA constant current for 70 min. Anti-His antibody was diluted 1:10000 to prepare the primary antibody, and the presence of a His tag in the LRR recombinant protein was used for specific identification.

[0054] The results are as follows Figure 7 As shown, at loading amounts of 10 ng, 20 ng, and 50 ng, a specific band appeared at approximately 40 kDa, and the signal intensity increased with increasing loading amount, indicating that the purified LRR recombinant protein carries a His tag and is the correctly expressed target protein.

[0055] (2) Mass spectrometry identification The purified LRR recombinant protein was subjected to 12% (w / v) SDS-PAGE electrophoresis, followed by Coomassie brilliant blue staining. The LRR recombinant protein gel was excised and sent to a mass spectrometry platform for analysis using an AB Sciex 4800 Plus MALDI-TOF / TOF mass spectrometer. The obtained peptide mass fingerprint was searched in a database and showed a high degree of match with the theoretical amino acid sequence of the LRR domain of the VASN protein, further confirming that the purified recombinant protein contained the LRR domain of the human VASN protein.

[0056] Example 4: Preparation of LRR monoclonal antibody (1) Immunization of BALB / c mice Five-week-old mice were immunized using a multi-site subcutaneous, long-term, low-dose approach. The LRR recombinant protein concentration was adjusted to 1 μg / μL. An equal volume of Freund's complete adjuvant was used for the first immunization; an equal volume of Freund's incomplete adjuvant was used for the subsequent three immunizations. The LRR dose was 150 μg in week 1, and 80 μg in weeks 3, 5, and 7. Six days after the fourth immunization, blood was collected from immunized mice, centrifuged at 10,000 rpm for 6 min, and the serum was retained and stored at 4°C for later use. Serum titers were detected by indirect ELISA.

[0057] (2) Mouse serum titer detection The LRR recombinant protein was diluted to 1.5 μg / mL with PBS and coated onto an ELISA plate at a volume of 100 μL / well, with three replicates. The plate was incubated overnight at 4°C. Blocking was performed with 1% BSA at 37°C for 30 min. A 1% BSA solution was used as a blank well. Mouse serum samples were diluted from 1:1000 to 1:2187000 and incubated at 37°C for 1 h. Goat anti-mouse secondary antibody (1:10000 dilution) was added, and the plate was incubated at 37°C for 30 min. After each incubation, the plate was washed three times with 300 μL / well of PBST. TMB chromogenic solution was added under light, and the plate was incubated at 37°C for 15 min. The reaction was terminated with 2 M sulfuric acid, and the OD was measured. 450 nm value. A P / N value ≥ 2.1 was used as the cutoff value for determining the serum titer of immunized mice. Mice with a serum dilution ratio greater than 1:10000 underwent a pulse immunization. Cell fusion occurred three days after the pulse immunization, where N represents the OD value of the negative serum. 450 nm value, P is positive serum OD 450 nm value.

[0058] The results are as follows Figure 8As shown: The negative control was serum from unimmunized mice diluted 1:1000. Serum from five immunized mice was serially diluted starting at 1:1000, and the OD values ​​at each dilution were measured. 450 Value. OD of immunized mouse serum. 450 Values ​​and negative control OD 450 The ratio of the values ​​was used as the P / N value. When the dilution factor was 1:2187000, the P / N values ​​of the serum from the five immunized mice were all greater than 2.1, indicating that the serum titer reached 1:2187000, which met the fusion requirements.

[0059] (3) Culture SP2 / 0 myeloma cells Resuscitated SP2 / 0 myeloma cells were cultured and passaged 5-6 times. The SP2 / 0 myeloma cells were in good condition with a moderate proliferation rate and were suitable for cell fusion. Nine culture flasks of SP2 / 0 myeloma cells were required for a single culture session, with the medium replaced with fresh medium 24 hours before cell fusion. The next day, the SP2 / 0 myeloma cells were gently washed with PBS, and 5 mL of serum-free medium was added. Cells were then detached from the bottom of the flask using a 3 mL Pasteur pipette. Finally, the SP2 / 0 myeloma cells were resuspended in 10 mL of RPMI 1640 medium containing 30% serum and cultured at 37°C for 1 hour.

[0060] (4) Spleen cell preparation Mice with serum dilutions higher than 1:10000 were selected for pulse immunization. 100-200 μg of LRR recombinant protein was injected intraperitoneally. Three days after the pulse immunization, blood was collected from the mice via the orbital rim. After standing at room temperature for 1 hour, the blood was centrifuged at 10000×g for 10 minutes. The supernatant was used as a positive control for ELISA and stored at -80℃. The immunized mice were sacrificed and disinfected by immersion in 100 mL of 75% alcohol for 5-9 minutes. After UV disinfection in a laminar flow hood for 30 minutes, the mice were fixed on a foam board, back down, and their abdomens were sprayed with alcohol for disinfection. The abdominal skin was cut open with scissors while simultaneously pulling the skin with forceps to fully expose the peritoneum. Alcohol-soaked cotton balls were used to wipe and disinfect the peritoneum. The disinfected peritoneum was then cut open with new scissors to expose the organs, including the left spleen, which was then dissected with forceps. Clean the spleen, place it on a copper grid, and squeeze the spleen with the push handle to separate it into individual spleen cells. Rinse twice with serum-free RPMI 1640 medium and collect the individual spleen cells in a 10 mL round-bottom centrifuge tube.

[0061] (5) Cell fusion Wash SP2 / 0 myeloma cells and spleen cells separately, centrifuge for 3 min at 1000 rpm, retain the pellet, and repeat three times. Before fusion, preheat 50% PEG solution and 1 / 3 hybridoma cell reconstitution solution in a 37°C incubator. Add the resuspended SP2 / 0 myeloma cells to the spleen cells and mix thoroughly. Centrifuge for 3 min at 1000 rpm, discard the supernatant, and repeat three times. Gently tap the round-bottom centrifuge tube on the table to loosen the two cell types, incubate at 37°C for 30 s, and slowly add 1 mL of 50% PEG solution along the tube wall, gently mixing while adding. The entire process is completed in a 37°C water bath. After incubating at 37°C for 1 min, add 2 mL of serum-free 1640 medium, invert the tube to thoroughly mix the 1640 medium with the PEG solution, thus terminating the PEG solution reaction. Continue adding 4 mL of serum-free 1640 medium and incubate at 37°C for 10 min. Centrifuge at 1050 rpm for 4 min, retain the precipitate, add 2 mL of preheated 1 / 3 hybridoma cell reconstitution medium and mix thoroughly. Then transfer to 90 mL of 1 / 3 hybridoma cell reconstitution medium and mix thoroughly. Finally, evenly distribute the fused cells into nine 96-well plates, add PBS to the outermost wells to prevent medium evaporation, and incubate at 37°C in a 5% CO2 cell culture incubator. After 24 hours of culture, aspirate the old medium from the wells, and add 200 μL of complete medium containing HAT selector additive to each well to bring the final concentration of HAT additive to the standard working concentration. The standard working concentrations are: hypoxanthine 100 μM, aminopterin 0.4 μM, and thymidine 16 μM. Replace the complete medium containing HAT selector additive every 2 to 3 days, and continue culturing for approximately 10 to 14 days. Finally, screen for positive hybridoma lines using an indirect ELISA method.

[0062] Nine days after fusion, hybridoma cell clones were visible under a microscope, such as... Figure 9 As shown, the cells grow in colonies and have a good morphology.

[0063] (6) Screening of positive hybridoma strains When the hybridoma cells reach 10-13 days of growth, the number of hybridoma cell lines in the 96-well plate is confirmed by microscopy and labeled. Cell supernatant from the labeled wells is collected and hybridomas are screened using ELISA. Using the hybridoma cell supernatant as a sample, hybridoma cells secreting anti-LRR antibodies are determined based on a P / N ratio > 2.1. Positive hybridoma cell lines are then cultured in 24-well plates and retested using an indirect ELISA method. Only two positive results are required to accurately identify positive hybridoma cell lines and avoid selecting false positives.

[0064] (7) Subcloning of positive hybridoma strains Strongly positive and rapidly growing hybridoma cell lines were selected as subcloning targets. During subcloning, cells were gently blown off with a pipette and collected in centrifuge tubes. Counting was performed using a CountStar automated cell counter, and the positive hybridoma cells were uniformly diluted to a concentration of 40 cells / mL. Following a limiting dilution method, serial dilutions were performed to achieve cell counts of approximately 2 cells / well, 1 cell / well, and 0.5 cells / well. After 7-9 days, when the hybridoma cells filled one-quarter of the well volume, a positive result was confirmed by indirect ELISA. Subcloning was repeated three times using the above method. Finally, the monoclonal hybridoma cell line was preserved and expanded, and used for the large-scale production of anti-LRR monoclonal antibodies.

[0065] On the 7th day after subcloning, as Figure 10 As shown, a single hybridoma cell clone with uniform morphology is visible, indicating that a stable monoclonal cell line secreting antibodies has been obtained.

[0066] After limiting dilution subcloning, several hybridoma cell lines stably secreting anti-LRR monoclonal antibodies were obtained and named 9C9, 6E7, 6B10, and 6F6, respectively. The binding ability of the culture supernatant of each clone to VASN proteins from different sources was detected using an indirect ELISA method: full-length eukaryotic VASN protein and prokaryotic LRR recombinant protein were coated onto ELISA plates, respectively. The full-length eukaryotic VASN protein was expressed by HEK293 cells in its native conformation; the prokaryotic LRR recombinant protein was expressed by *E. coli* in its denatured conformation. The supernatant of each clone was added, and HRP-labeled goat anti-mouse IgG was used as a secondary antibody. OD was detected after TMB staining. 450 value.

[0067] The results are as follows Figure 11 As shown: the positive control is serum from immunized mice, the negative control is serum from unimmunized mice, and the positive control OD... 450 The OD value was significantly higher than that of the negative control, validating the effectiveness of the experimental system. Among the screened clones, 6B10 showed a significantly higher OD value for eukaryotic VASN protein expression. 450 The 6B10 antibody showed the highest binding activity and also exhibited high binding activity to the prokaryotically expressed LRR recombinant protein. These results indicate that the 6B10 antibody can effectively recognize the native conformation of the VASN protein, demonstrating greater potential for biological applications, and therefore it was selected as the optimal monoclonal antibody strain for further research.

[0068] (8) Large-scale preparation of monoclonal antibodies Seven-week-old female BALB / c mice were selected and injected intraperitoneally with 500 μL of paraffin oil per mouse. One week later, 1×10⁻⁶ mice were collected. 6Monoclonal hybridoma cells dissolved in 500 μL of 1640 medium were injected intraperitoneally into mice. The mice's abdominal and mental state were observed 6-9 days later. After the mice's abdomens became distended and their fur became disordered, they were euthanized by cervical dislocation, and ascites fluid was collected. The ascites fluid was centrifuged at 12000×g for 5 min at 4℃, and the middle layer was extracted and stored at -80℃ for later use.

[0069] (9) Monoclonal antibody purification ① Sample preparation: Dilute the ascites fluid with binding / washing buffer to ensure that the sample solution has appropriate ionic strength and pH value.

[0070] ② Sample purification: Place 2 mL of rProtein G Beads into the chromatography column. Equilibrate the column with 5 times the volume of rProtein G Beads binding buffer to ensure the anti-LRR antibody and the packing material are in the same buffer system, thus protecting the anti-LRR antibody. Add the sample to the equilibrated rProtein G Beads and incubate overnight at 4°C with shaking to allow the anti-LRR antibody to fully bind to the rProtein G Beads. The next day, wash the column with 10-15 times the volume of rProtein G Beads washing buffer to remove non-specifically adsorbed proteins. Collect the eluent containing 5-10 times the volume of rProtein G Beads elution buffer, which contains the purified antibody. The eluent was equilibrated sequentially with 3 times the volume of rProtein G Beads in binding buffer and 5 times the volume of rProtein G Beads in double-distilled water, and finally equilibrated with 5 times the volume of rProtein G Beads in 20% ethanol. The eluent was then stored in an equal volume of 20% ethanol at 4°C. The collected eluent was further concentrated by centrifugation using an ultrafiltration tube.

[0071] The purified antibody was identified by SDS-PAGE, and the results are shown in the figure. Figure 13 Lane M is the protein marker, lane 1 is ascites fluid, and lane 2 is the purified antibody, where antibody bands are visible. Thus, a high-purity anti-VASN-LRR monoclonal antibody was obtained and named 6B10.

[0072] The purified 6B10 antibody was sequenced, and the N-terminal amino acid sequences of its heavy and light chains were determined by Edman degradation or mass spectrometry. The encoding gene of the antibody was then amplified from hybridoma cells using RACE-PCR. Sequencing results showed that the amino acid sequence of the heavy chain variable region of the 6B10 antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2. The complementarity-determining region (CDR) sequence, as defined by the Kabat numbering system, is as follows: The amino acid sequences of the light chains CDR1, CDR2, and CDR3 are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively; the amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively. The nucleotide sequences encoding the variable regions of the heavy and light chains are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0073] Example 5 Identification of anti-LRR monoclonal antibodies (1) Identification of monoclonal antibody subtypes Identification was performed according to the Sigma-Aldrich monoclonal antibody subtype kit procedure, as follows: Various subtype antibodies, including IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA, were diluted 1:1000 to their working concentrations and coated onto an ELISA plate. The plates were incubated at 37°C for 1 h. An equal volume of PBS was added to the negative control instead of the test antibody. The test sample, purified 6B10 antibody, was added to the ELISA plate at 100 μL / well and incubated at 24°C for 1 h. Goat anti-mouse IgG secondary antibody was diluted 1:10000 and added to the ELISA plate at 100 μL / well, and incubated at 24°C for 0.5 h. After each incubation step, the ELISA plate was washed three times with 1×PBST. TMB chromogenic buffer was added, and the plates were incubated at 37°C in the dark for 15 min. Stop solution was added, and OD was measured. 450 nm value.

[0074] The results are as follows Figure 14 As shown, the detection well OD of anti-IgG2b antibody 450 The highest value, significantly higher than other subtype detection wells and the negative control, indicates that this anti-LRR monoclonal antibody is IgG2b type with a κ light chain. Negative control OD 450 The value is close to the background value, proving that the experimental system is effective.

[0075] (2) Monoclonal antibody affinity Samples were distributed using a checkerboard matrix method, and the LRR recombinant protein was diluted to 2 μg / mL and 1 μg / mL. Anti-LRR monoclonal antibodies from three clones (6B10, 6F6, and 9C9) were serially diluted 2-fold from 2 μg / mL to 0.03125 μg / mL (2 to the power of negative 6 micrograms per milliliter). Three replicates were set up, and 1% (w / v) BSA solution was used as a blank well. The remaining steps were the same as for indirect ELISA. The IC50 was calculated using GraphPad Prism 5 software. 50The affinity constant Ka is calculated by the following formula: Ka=(n-1) / 2×(n[Ab']t-[Ab]t), n=[Ab]t / [Ab']t, where[Ab]t and[Ab']t correspond to the antibody concentrations (mol / L) matching the coating concentrations of two different LRR recombinant proteins, thus obtaining the affinity constant.

[0076] The results are as follows Figure 12 The figure shows the ELISA binding curves of three monoclonal antibodies: 6B10, 6F6, and 9C9. In the figure, A corresponds to antibody 6B10, B to antibody 6F6, and C to antibody 9C9. The horizontal axis represents antibody concentration, and the vertical axis represents OD. 450 The affinity constant Ka of 6B10 is calculated using the formula as 2.43 × 10⁻⁶. 9 The L / mol ratio indicates that the antibody has extremely high affinity, and therefore it was selected as a representative antibody for further research.

[0077] (3) Monoclonal antibody specificity identification ① Indirect ELISA: The following proteins were coated onto the ELISA plate at a concentration of 2 μg / mL. The first protein was LRR-His recombinant protein, expressed by *E. coli* and tagged with His. The second protein was SHBG-His recombinant protein, a His-tagged sex hormone-binding globulin, used as an irrelevant protein control. The third protein was bovine serum albumin (BSA), also used as an irrelevant protein control. Positive and negative controls were also included. The positive control wells were coated with full-length eukaryotic VASN protein, expressed by HEK293 cells in its native conformation; the negative control wells were uncoated, containing only blocking buffer. The plates were incubated overnight at 4°C. Anti-LRR monoclonal antibody was added at a dilution of 1:10000 as the primary antibody for detection. Subsequent steps followed the indirect ELISA procedure.

[0078] The results are as follows Figure 15 As shown: The anti-LRR monoclonal antibody reacted only with the LRR-His recombinant protein and the eukaryotic VASN protein. The OD values ​​of these two wells were... 450 The value was significantly higher than that of the negative control well; compared with the OD values ​​of SHBG-His recombinant protein and BSA 450 The value is close to the level of the negative control well, indicating that the antibody has good specificity.

[0079] ② Western blot method: The following four protein samples were subjected to 12% (w / v) SDS-PAGE gel electrophoresis, with the following loading amounts: bovine serum albumin 50 ng, SHBG-His recombinant protein 10 ng, RBP4-His recombinant protein 10 ng, and LRR-His recombinant protein 10 ng. SHBG-His is a His-tagged sex hormone-binding globulin, and RBP4-His is a His-tagged retinol-binding protein 4; both served as irrelevant protein controls. LRR-His is a His-tagged LRR recombinant protein. After electrophoresis, the sample was blocked at 24°C with 5% (w / v) skim milk for 2 hours. Anti-LRR monoclonal antibody was added at a dilution of 1:10000 as the primary antibody, and the sample was incubated overnight at 4°C. The sample was washed three times with TBST for 10 minutes each time. Goat anti-mouse secondary antibody was added at a dilution of 1:10000, and the sample was incubated at 24°C for 1 hour. Rinse three times with TBST for 10 minutes each time, and finally develop with ECL chemiluminescent solution.

[0080] The results are as follows Figure 16 As shown, lane M is the protein marker, lane 1 is bovine serum albumin (BSA), lane 2 is SHBG-His recombinant protein, lane 3 is RBP4-His recombinant protein, and lane 4 is LRR-His recombinant protein. A specific band appeared only in lane 4, with a molecular weight of approximately 40 kDa. No bands were observed in the other lanes, further confirming that the antibody specifically recognizes the LRR domain and shows no cross-reactivity with SHBG, RBP4, or BSA.

[0081] (4) Subcellular localization of VASN protein Huh7, HepG2, and HL-7702 cells were seeded in 24-well plates at a density of 2 × 10⁶ cells per well. 4Cells were cultured overnight at 37°C, washed three times with pre-chilled PBS, and incubated for 10 min at room temperature with 200 μL of 4% paraformaldehyde. After washing three times with pre-chilled PBS, 200 μL of 0.1% Triton X-100 was added, and incubated for 10 min at room temperature. After washing three times with pre-chilled PBS, 200 μL of 1% BSA solution containing glycine was added, and incubated for 30 min at room temperature. After washing three times with pre-chilled PBS, 200 μL of LRR monoclonal antibody diluted 1:1000 was added as primary antibody, and incubated overnight at 4°C on a shaker. After washing three times with pre-chilled PBS, FITC-labeled goat anti-mouse IgG diluted 1:1000 was added as secondary antibody, and incubated for 1 hour at 24°C in the dark on a shaker. After washing three times with pre-chilled PBS, Hoechst nuclear staining solution diluted 1:10000 was added, and incubated for 1 min at room temperature. Wash three times with pre-cooled PBS and take pictures using the EVOS FL fully automated cell imaging system.

[0082] The results are as follows Figure 17 As shown, column A represents the Hoechst nuclear staining images for Huh7, HepG2, and HL-7702 cells; column B represents the FITC-labeled secondary antibody Fluor-488 image, which is green fluorescent and represents the localization of the VASN protein; column C is a merged image combining the Hoechst nuclear staining image and the Fluor-488 image. In Huh7, HepG2, and HL-7702 cells, the FITC-labeled secondary antibody, which is green fluorescent, is mainly distributed on the cell membrane and does not overlap with the Hoechst-stained nuclear location, indicating that the VASN protein is mainly located on the cell membrane, consistent with theory.

[0083] Example 6: Anti-VASN-LRR antibody inhibits Hep3B liver cancer cell subcutaneous xenografts in nude mice Thirty 4-week-old male BALB / c-nu nude mice were selected and raised under standard specific pathogen-free conditions.

[0084] ① Model construction: Hep3B-NC and Hep3B-VASN-KO liver cancer cells in logarithmic growth phase were digested with trypsin, resuspended in serum-free MEM medium, and the cell density was adjusted to 4×10⁶ cells / year. 7 Cells / mL. The cell suspension was mixed with Matrigel at a 1:1 volume ratio, and then 0.2 mL of the mixture was subcutaneously injected into the right axilla of each nude mouse. The mixture contained 4 × 10⁶ cells / mL. 6 A total of 24 nude mice were inoculated with Hep3B-NC cells, and 6 nude mice were inoculated with Hep3B-VASN-KO cells.

[0085] Hep3B-VASN-KO cells were constructed using CRISPR-Cas9 technology. hVASN gRNA-A1:CATGGTCACTGCACGTTCAG; hVASN gRNA-A2:GCCTCGGACAAGAACGGGTC. Sequencing confirmed successful knockout to ensure that VASN protein is not expressed.

[0086] ② Grouping and drug administration: One week after subcutaneous cell injection, tumor volume was monitored regularly. Tumors in each group were targeted until the average tumor volume reached approximately 100 mm. 3 Twenty-four nude mice inoculated with Hep3B-NC cells were randomly divided into four groups (n=6 per group) according to tumor volume: control group (NC), 6B10 antibody group (6B10), lenvatinib group (Len), and 6B10+lenvatinib group (6B10+Len). Six nude mice inoculated with Hep3B-VASN-KO cells were designated as the VASN-KO group and received no intervention. The specific intervention protocol was as follows: intratumoral injection of saline in the NC group, intratumoral injection of 70 μg 6B10 antibody solution in the 6B10 antibody group, intraperitoneal injection of 10 mg / kg lenvatinib in the Len group, and simultaneous administration of the above-mentioned doses of 6B10 and lenvatinib in the 6B10+lenvatinib group. All administration procedures were performed every 3 days.

[0087] ③ Indicator Observation and Sample Collection: During the experiment, the weight, mental state, and tumor volume of the nude mice were recorded every 3 days. The tumor volume was calculated using the formula: V = (major axis × minor axis) 2 2. After three weeks of experimentation, all nude mice were euthanized using carbon dioxide asphyxiation. The subcutaneous transplanted tumors were dissected and completely removed, weighed, photographed, and then the tumor tissue was divided into two parts: one part was immediately frozen and stored in an ultra-low temperature freezer at -80°C for subsequent protein analysis; the other part was fixed in 10 times its volume of 4% paraformaldehyde solution for subsequent paraffin embedding, HE staining, and immunohistochemical experiments.

[0088] Experimental results Tumor appearance: Subcutaneous xenografts removed after the experiment, as shown in the image. Figure 18 As shown in the figure, the tumor volume of the control group (NC) was the largest; the tumor volume of the 6B10 antibody group (6B10) and the lenvatinib group (Len) was significantly smaller than that of the control group; the tumor volume of the 6B10+lenvatinib group (6B10+Len) was the smallest, similar to that of the VASN-KO group which had VASN gene knockout and did not express VASN protein. This indicates that the anti-VASN-LRR antibody can effectively inhibit the growth of Hep3B liver cancer and has a synergistic effect with lenvatinib.

[0089] HE pathology: HE staining results of transplanted tumor tissue are as follows Figure 19As shown in the figure, A is the control group (NC), B is the 6B10 antibody group (6B10), C is the lenvatinib group (Len), D is a magnified view of the circled red blood cell area in the control group, E is the 6B10 + lenvatinib group (6B10 + Len), and F is the VASN-KO group. In the figure, the green area represents the tissue necrosis area, the blue area represents red blood cells, and the red area represents the cancer nest area. The control group showed dense cancer nests and little necrosis; the 6B10 antibody group and the lenvatinib group showed an expanded necrosis area; the 6B10 + lenvatinib group had the largest necrosis area and the fewest residual cancer nests, further confirming the superiority of combination therapy.

[0090] Example 7: Effect of anti-VASN-LRR antibody on subcutaneous xenografts of malignant cells in nude mice exposed to aflatoxin B1 (AFB1). Twenty-four 5-week-old male BALB / c-nu nude mice were selected and raised under standard specific pathogen-free conditions.

[0091] ① Model construction: Malignant L02 hepatocytes exposed to AFB1 in the logarithmic growth phase at passage 36 were named L02-AFB1-P36. After trypsin digestion, the cells were resuspended in serum-free 1640 medium, and the cell density was adjusted to 2×10⁻⁶. 7 0.1 mL of the mixture was injected subcutaneously into the right axilla of each nude mouse using a 1 mL insulin syringe. The mixture contained 2 × 10⁻⁶ insulin molecules per mL. 6 Each cell.

[0092] L02-AFB1-P36 hepatocytes were obtained by continuously exposing L02 normal human hepatocytes to 0.1 μg / mL aflatoxin B1, passaged every 7 days, and cultured for 36 generations. The cells were verified to have tumorigenic ability in nude mice.

[0093] ② Grouping and drug administration: One week after subcutaneous cell injection, tumor volume was monitored regularly. Tumors in each group were targeted until the average tumor volume reached approximately 100 mm. 3 Tumors were randomly divided into four groups (n=6 per group) based on tumor volume: control group (NC), 6B10 antibody group (6B10), lenvatinib group (Len), and 6B10+lenvatinib group (6B10+Len). The specific dosing regimens were as follows: the control group received intratumoral injection of normal saline; the 6B10 antibody group received intratumoral injection of 70 μg of 6B10 antibody solution; the lenvatinib group received intraperitoneal injection of 10 mg / kg lenvatinib; and the 6B10+lenvatinib group received both the aforementioned doses of 6B10 and lenvatinib simultaneously. All dosing procedures were performed every 3 days.

[0094] ③ Indicator Observation and Sample Collection: During the experiment, the weight, mental state, and tumor volume of the nude mice were recorded every 3 days. The tumor volume was calculated using the formula: V = (major axis × minor axis) 22. After 16 days of experimentation, all nude mice were euthanized using carbon dioxide asphyxiation. The subcutaneous transplanted tumors were dissected and completely removed, and then weighed, photographed, recorded, and preserved.

[0095] Experimental results Tumor appearance: as shown Figure 20 As shown, the tumor volume was the largest in the control group; the tumor volume in the 6B10 antibody group and the lenvatinib group was significantly smaller than that in the control group; the tumor volume in the 6B10 + lenvatinib group was the smallest, indicating that the anti-VASN-LRR antibody can significantly inhibit the growth of AFB1-induced malignant L02 hepatocellular carcinoma xenografts and has a synergistic effect with lenvatinib.

[0096] HE pathology: HE staining results of transplanted tumor tissue are as follows Figure 21 As shown in the figure, A is the control group (NC), B is the 6B10 antibody group (6B10), C is the lenvatinib group (Len), and D is the 6B10 + lenvatinib group (6B10 + Len). The red area in the figure represents the cancer nest area, and the green area represents the necrotic area. The control group has dense cancer nests and little necrosis; the 6B10 antibody group and the lenvatinib group show an expansion of the necrotic area and a reduction in cancer nests; the 6B10 + lenvatinib group has the largest necrotic area and the fewest residual cancer nests, further confirming that the combination of anti-VASN-LRR antibody and lenvatinib has an excellent synergistic inhibitory effect on AFB1-induced liver cancer.

[0097] Comparative Example 1: Preparation and efficacy comparison of anti-VASN-EGF monoclonal antibodies 1. Preparation of anti-VASN-EGF antibody Using the EGF domain of human VASN protein as an immunogen, with the amino acid sequence CPPGWEGGQCHCLPGLRGPSCQ, an anti-VASN-EGF monoclonal antibody was obtained using the same immunization, fusion, and screening methods as in Example 4, and named 3E5.

[0098] 2. Comparison of antibody affinity The affinity of the 3E5 antibody was determined using the same ELISA checkerboard method as in Example 5. The results showed that the affinity of the 3E5 antibody was 2.1 × 10⁻⁶. 7 L / mol, while the affinity of the 6B10 antibody of this invention is 2.43 × 10⁻⁶. 9 L / mol, the latter being approximately 115 times that of the former, indicates that antibodies targeting the LRR domain have significantly higher affinity.

[0099] 3. Comparison of anti-tumor effects A Hep3B subcutaneous xenograft model in nude mice was established according to the method in Example 6. The following groups were set up, with 6 mice in each group: control group (NC), 6B10 antibody group (6B10), anti-VASN-EGF monoclonal antibody group (3E5), lenvatinib group (Len), 6B10 + lenvatinib group (6B10 + Len), and 3E5 + lenvatinib group (3E5 + Len). The 6B10 antibody group and the anti-VASN-EGF monoclonal antibody group received intratumoral injections of 70 μg of their respective antibodies. The lenvatinib group received intraperitoneal injections of 10 mg / kg body weight of lenvatinib. The 6B10 + lenvatinib group and the 3E5 + lenvatinib group received both the corresponding antibody and lenvatinib simultaneously. The drug administration regimen was the same as in Example 6. After 3 weeks of experimentation, the animals were sacrificed, the tumors were dissected, their volume was measured, and the tumor inhibition rate was calculated.

[0100] Experimental results: Table 1. Inhibitory effects of different treatment groups on Hep3B subcutaneous hepatocellular carcinoma xenografts The above results indicate that the 6B10 antibody targeting the LRR domain alone achieved a tumor inhibition rate of 50.0%, significantly higher than the 19.0% of the anti-VASN-EGF monoclonal antibody group targeting the EGF domain. When combined with lenvatinib, the tumor inhibition rate of the 6B10 + lenvatinib group was 83.6%, also significantly higher than the 66.1% of the E5 + lenvatinib group. This demonstrates that the LRR domain selected in this invention has unexpected technical effects, generating a stronger anti-tumor immune response, and exhibits superior synergistic effects with lenvatinib.

[0101] Comparative Example 2: Effects of antibodies with different domains on AFB1-induced malignant cell xenografts 1. Experimental Model A subcutaneous xenograft model of L02-AFB1-P36 in nude mice was established according to the method in Example 7. Five-week-old male BALB / c-nu nude mice were used, and each mouse was subcutaneously injected with 2×10⁻⁶ tumor cells. 6 1 L02-AFB1-P36 cell. When the tumor volume reaches approximately 100 mm... 3 At the time of the experiment, participants were randomly divided into three groups, with n=6 in each group: the control group (NC) received intratumoral injection of saline, the 6B10 antibody group (6B10) received intratumoral injection of 70 μg of 6B10 antibody, and the anti-VASN-EGF monoclonal antibody group (3E5) received intratumoral injection of 70 μg of 3E5 antibody. Administered the drugs every 3 days for 16 days. After the experiment, the tumors were removed, weighed, and the tumor inhibition rate was calculated.

[0102] 2. Experimental Results Table 2. Inhibitory effects of antibodies with different structural domains on AFB1-induced malignant cell xenografts The results showed that the 6B10 antibody significantly inhibited AFB1-induced malignant transformed L02 cell xenografts, with a tumor inhibition rate of 48.0%, while the 3E5 antibody only showed a weak inhibitory effect, with a tumor inhibition rate of 12.2%. This indicates that antibodies targeting the LRR domain of the VASN protein have a unique therapeutic advantage in AFB1-related hepatocellular carcinoma, while antibodies targeting the EGF domain are less effective. These results further confirm the inventiveness and practicality of this invention in selecting the LRR domain as a target.

[0103] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A VASN-LRR monoclonal antibody, characterized in that, The monoclonal antibody contains the following amino acid sequences: light chain CDR1 as shown in SEQ ID NO.3, light chain CDR2 as shown in SEQ ID NO.4, and light chain CDR3 as shown in SEQ ID NO.5; heavy chain CDR1 as shown in SEQ ID NO.6, heavy chain CDR2 as shown in SEQ ID NO.7, and heavy chain CDR3 as shown in SEQ ID NO.

8.

2. The monoclonal antibody of claim 1, wherein, The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

3. A pharmaceutical composition, characterized by, It includes the monoclonal antibody according to any one of claims 1-2.

4. An antitumor combination drug composition, characterized in that, It comprises the monoclonal antibody as described in any one of claims 1-2 and lenvatinib.

5. The use of the monoclonal antibody according to any one of claims 1-2 in the preparation of a medicament for treating liver cancer.

6. The use of the monoclonal antibody according to any one of claims 1-2 in the preparation of a medicament for use in combination with lenvatinib for the treatment of liver cancer.

7. A method of producing the monoclonal antibody of claim 1, characterized by, Includes the following steps: Step 1: Obtain the VASN-LRR target gene sequence, ligate it into a vector, and construct a recombinant expression vector; Step 2: The recombinant expression vector was transformed into E. coli, and after induction of expression, recombinant VASN-LRR protein was obtained; Step 3: Purify recombinant VASN-LRR protein using a gel extraction method; Step 4: Immunize BALB / c mice with purified recombinant VASN-LRR protein as an antigen; Step 5: Fuse spleen cells from immunized mice with SP2 / 0 myeloma cells and screen for positive hybridoma cells; Step 6: Culture hybridoma cells, collect and purify antibodies.

8. The preparation method according to claim 7, characterized in that, In step 2, expression is induced using IPTG at a concentration of 0.2-1.0 mM, at an induction temperature of 15-37℃, and for 3-6 hours. In step 3, gel recovery and purification includes: after electrophoresis, staining and gel cutting, incubation in buffer overnight, and ultrafiltration concentration.

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