Aminopeptidase N nano antibody, preparation method thereof and application of aminopeptidase N nano antibody in overcoming gastric cancer chemotherapy drug resistance
By preparing neutralizing nanobodies that specifically target aminopeptidase N, the problem of chemotherapy resistance in gastric cancer has been solved, and the efficacy of chemotherapy has been improved both in vitro and in vivo, providing a new treatment strategy.
Patent Information
- Application Number
- CN202511977175.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-25
- Publication Date
- 2026-03-20
AI Technical Summary
Chemotherapy resistance in gastric cancer limits the effectiveness of chemotherapy. There is limited research on aminopeptidase N (APN) neutralizing nanobodies in the current technology, resulting in poor chemotherapy efficacy.
Neutralizing nanobodies that specifically target aminopeptidase N (APN) were prepared. Through nanobodies library screening, recombinant plasmid construction, expression and purification, their high affinity and neutralizing activity for APN protein were verified. They were then used in combination with chemotherapeutic drugs to block cellular drug resistance.
In vitro and in vivo experiments have demonstrated that this antibody can effectively target and inhibit APN, significantly enhance the efficacy of chemotherapy, reduce chemotherapy resistance in gastric cancer, and improve patient prognosis.
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Figure CN121699015A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of nanobodies, in particular to an aminopeptidase N nanobody, a preparation method thereof and application thereof in overcoming gastric cancer chemotherapy resistance. BACKGROUND
[0002] Gastric cancer is a global malignancy, and the first-line comprehensive treatment for clinical gastric cancer is still mainly chemotherapy. However, gastric cancer resistance limits the effectiveness of chemotherapy, and chemotherapy resistance is an important factor that seriously affects the prognosis of gastric cancer and leads to treatment failure.
[0003] Recent studies have shown that the expression of aminopeptidase N (APN) protein is related to poor prognosis of patients, and inhibition of APN protein activity combined with chemotherapy drugs can significantly promote tumor cell apoptosis and inhibit multidrug resistance. In addition, nanobodies are currently the smallest active antigen-binding proteins known, and have many unique advantages that conventional antibodies do not have: small size, strong tissue penetration ability, ability to bind to hidden antigen epitopes of antigen molecules, low-cost mass production, high sensitivity and specificity, weak immunogenicity in the human body, high stability, etc. They have achieved remarkable results in many cancer treatments. However, current research on APN inhibitors has focused on compounds and biologically active peptides, and there has been less systematic research on APN antibodies, especially neutralizing nanobodies of APN. SUMMARY
[0004] The purpose of the present application is to provide an aminopeptidase N nanobody, a preparation method thereof and application thereof in overcoming gastric cancer chemotherapy resistance, in order to solve the problems existing in the prior art. The present application prepares a neutralizing nanobody that specifically targets aminopeptidase N (APN). First, it is verified in vitro that the antibody has high affinity and neutralizing activity to APN protein, and it is confirmed in a gastric cancer chemotherapy-resistant cell line that it can synergistically block cell resistance when used in combination with gastric cancer chemotherapy drugs; subsequently, through a gastric cancer orthotopic drug-resistant tumor model at the animal in vivo level, it is further proved that the antibody can effectively target and inhibit APN in vivo, and significantly enhance the efficacy of chemotherapy under the condition of combined use of drugs. In summary, the APN neutralizing nanobody prepared by the present application provides a new solution for overcoming gastric cancer chemotherapy resistance and improving patient prognosis.
[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:
[0006] The present application provides an aminopeptidase N nanobody, the amino acid sequence of the heavy chain variable region of which is shown in SEQ ID NO. 6.
[0007] The present application also provides the use of the above-mentioned aminopeptidase N nanobody in the preparation of a drug for treating chemotherapy-resistant gastric cancer.
[0008] Optionally, the chemotherapy-resistant gastric cancer includes gastric cancer resistant to cisplatin.
[0009] The present invention also provides the application of the above-mentioned aminopeptidase N nanobody in the preparation of a drug for treating gastric cancer.
[0010] The present invention also provides the application of the above-mentioned aminopeptidase N nanobody combined with chemotherapy drugs in the preparation of drugs for treating gastric cancer.
[0011] Optionally, the chemotherapy drug includes cisplatin.
[0012] The present invention also provides a drug for treating chemotherapy-resistant gastric cancer, wherein the drug uses the above-mentioned aminopeptidase N nanobody as an active ingredient.
[0013] The present invention also provides a drug for treating gastric cancer, wherein the drug uses the above-mentioned aminopeptidase N nanobody and chemotherapy drugs as active ingredients.
[0014] Optionally, the drug may also contain pharmaceutically acceptable excipients.
[0015] Optionally, the pharmaceutically acceptable excipients include one or more of the following: pharmaceutically acceptable solvents, releasing agents, antioxidants, and preservatives.
[0016] The present invention discloses the following technical effects:
[0017] This invention utilizes nanobody technology to prepare aminopeptidase N (APN)-specific nanobodies through nanobody library screening, construction, identification, expression, and purification of recombinant nanobody plasmids. First, the high affinity and neutralizing activity of this antibody for APN protein were verified in vitro, and its synergistic effect in blocking chemotherapeutic resistance in gastric cancer cell lines was demonstrated when used in combination with chemotherapeutic drugs. Subsequently, further evidence was obtained using an in vivo gastric cancer orthotopic drug-resistant xenograft model, demonstrating that the antibody effectively targets and inhibits APN in vivo and significantly enhances the efficacy of chemotherapy under combined treatment. The APN-targeting neutralizing nanobodies prepared in this invention can effectively reduce chemotherapeutic resistance in gastric cancer, improve the efficacy of chemotherapeutic treatment, and improve the prognosis of gastric cancer patients, providing a new treatment strategy for chemotherapeutic-resistant gastric cancer. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1The results show the in vitro affinity of 24 APN nanobodies for APN protein.
[0020] Figure 2 The results show the ability of APN nanobodies to neutralize the enzyme activity of APN protein.
[0021] Figure 3 The results of the detection of APN nanobody combined with chemotherapy drugs for gastric cancer synergistically blocking drug resistance in gastric cancer cells;
[0022] Figure 4 This is a representative in vivo image of the effect of APN nanobodies on the efficacy of chemotherapy in a mouse model of orthotopic drug-resistant gastric cancer xenografts;
[0023] Figure 5 This is a statistical result showing the effect of APN nanobodies on the efficacy of chemotherapy in a mouse model of orthotopic drug-resistant gastric cancer xenografts. Detailed Implementation
[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0029] The technical concept of this invention is as follows:
[0030] This research will explore APN nanobodies at various levels, including molecular mechanisms, cellular function, mouse models, and clinical samples. The proposed basic experimental techniques include: artificial protein synthesis, striped shark immunization, nested PCR amplification, enzyme digestion and ligation transformation, phage display, positive nanobodily clone screening and enrichment, prokaryotic cell monoclonal screening, prokaryotic protein induction and purification, enzyme-linked immunoprecipitation (ELISA), Reed-Muench assay for antibody titer determination, subcutaneous tumor formation in tumor-bearing mice, and tail vein injection in tumor-bearing mice. The specific implementation process is as follows:
[0031] Example 1
[0032] 1. Construction of a natural shark immune APN nanobody library
[0033] The full-length protein of aminopeptidase N (APN) (SEQ ID NO. 1, from Uniprot website), synthesized artificially at Suzhou Genewiz Biotechnology Co., Ltd., was purified by high-performance liquid chromatography (HPLC), and the protein concentration was determined using the BCA method. The purified APN protein was emulsified with adjuvant at a 1:1 (v / v) ratio: complete Freund's adjuvant (CFA) was used for the initial immunization, and incomplete Freund's adjuvant (IFA) was used for booster immunizations. Healthy adult striped bamboo sharks (Chiloscyllium plagiosum) were selected as immunization animals, and immunization was carried out via multiple subcutaneous injections in the back. The immunization dose was 200 μg protein per animal, with a total of 4 immunizations administered at 2-week intervals.
[0034] SEQ ID NO.1:
[0035] MAKGFYISKSLGILGILLGVAAVCTIIALSVVYSQEKNKNANSSPVASTTPSASATTNPASATTLDQSKAWNRYRLPNTLKPDSYRVTLRPYLTPNDRGLYVFKGSSTVRFTCKEATDVIIIHSKKLNYTLSQGHRVVLRGVGGSQPPDIDKTELVEPTEYLVVHLKGSLVKDSQYEMDSEFEGELADDLAGFYRSEYMEGNVRKVVATTQMQAADARKSFPCFDEPAMKAEFNITLIHPKDLTALSNMLPKGPSTPLPEDPNWNVTEFHTTPKMSTYLLAFIVSEFDYVEKQASNGVLIRIWARPSAIAAGHGDYALNVTGPILNFFAGHYDTPYPLPKSDQIGLPDFNAGAMENWGLVTYRENSLLFDPLSSSSSNKERVVTVIAHELAHQWFGNLVTIEWWNDLWLNEGFASYVEYLGADYAEPTWNLKDLMVLNDVYRVMAVDALASSHPLSTPASEINTPAQISELFDAISYSKGASVLRMLSSFLSEDVFKQGLASYLHTFAYQNTIYLNLWDHLQEAVNNRSIQLPTTVRDIMNRWTLQMGFPVITVDTSTGTLSQEHFLLDPDSNVTRPSEFNYVWIVPITSIRDGRQQQDYWLIDVRAQNDLFSTSGNEWVLLNLNVTGYYRVNYDEENWRKIQTQLQRDHSAIPVINRAQIINDAFNLASAHKVPVTLALNNTLFLIEERQYMPWEAALSSLSYFKLMFDRSEVYGPMKNYLKKQVTPLFIHFRNNTNNWREIPENLMDQYSEVNAISTACSNGVPECEEMVSGLFKQWMENPNNNPIHPNLRSTVYCNAIAQGGEEEWDFAWEQFRNATLVNEADKLRAALACSKELWILNRYLSYTLNPDLIRKQDATSTIISITNNVIGQGLVWDFVQSNWKKLFNDYGGGSFSFSNLIQAVTRRFSTEYELQQLEQFKKDNEETGFGSGTRALEQALEKTKANIKWVKENKEVVLQWFTENSK。
[0036] Seven days after the last immunization, shark blood was collected, and the serum APN antibody titer was detected by indirect ELISA: APN protein was coated onto a 96-well microplate (1 μg / well) and incubated overnight at 4°C; after blocking, serially diluted serum was added and incubated at 37°C for 1 hour; horseradish peroxidase-labeled goat anti-shark IgG secondary antibody (1:5000 dilution) was added and reacted at 37°C for 1 hour; finally, TMB substrate was added for color development, the reaction was terminated with 2 M H2SO4, and the absorbance was measured at 450 nm.
[0037] Peripheral blood was collected from immunized sharks, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation using Ficoll-Paque PLUS. Total RNA was extracted using the TRIzol method, and after detecting RNA purity and concentration, first-strand cDNA was synthesized using Oligo(dT) primers and reverse transcriptase.
[0038] Using this cDNA as a template, the VHH gene fragment was amplified in two steps using nested PCR: First round of PCR: Amplification was performed using primers targeting the variable region (VHH) of shark heavy chain antibody (GTCCTGGCTGCTCTTCTACAAGG, SEQ ID NO.2; GGTACGTGCTGTTGAACTGTTCC, SEQ ID NO.3), with the following amplification program: 94℃ for 5 min; 30 cycles of 94℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s; 72℃ for 10 min; Second round of PCR: Using the product from the first round as a template, amplification was performed using nested primers introducing restriction sites (CATGCCATGGCTGAGGTGCAGCTGGTGGAGTCT, SEQ ID NO.4; CATGCGGCCGCTGAGGAGACGGTGACCTGGGT, SEQ ID NO.5), with the same program as the first round.
[0039] 2. Construction of an M13 phage display APN antibody library
[0040] 2.1 Vector preparation and enzyme digestion and ligation: The VHH gene fragment purified by nested PCR amplification and pBSD phage vector was double-digested with restriction endonucleases (PstⅠ and NotⅠ). The digestion products were ligated overnight at 16℃ using T4 DNA ligase at a molar ratio of vector to insert fragment = 1:3.
[0041] 2.2 Escherichia coli transformation and initial library construction: The ligation product was introduced into competent Escherichia coli DH5α cells by electroporation. After transformation, SOC medium was added, and the cells were incubated at 37°C for 1 hour. The cells were then plated on LB agar plates containing ampicillin (100 μg / mL) and incubated overnight at 30°C. Colonies were scraped off and stored in LB medium containing 20% glycerol as the initial antibody library.
[0042] 2.3 Phage Display and Rescue: The initial library bacterial culture was inoculated at a 1:100 ratio into LB medium containing ampicillin and cultured at 37°C with shaking until OD. 600 =0.6, add helper phage M13K07 (10) at a multiplicity of infection (MOI) of 20:1. 12 PFU phage corresponds to 5×10 10 After mixing with CFU bacteria, the mixture was incubated at 37°C for 30 minutes for infection. The supernatant was removed by centrifugation, and the bacterial cells were resuspended in LB medium containing kanamycin and ampicillin. The mixture was then incubated overnight at 30°C with shaking for phage amplification.
[0043] 2.4 Collection and purification of phage library: Centrifuge the overnight culture, collect the supernatant, add 4% (w / v) PEG-8000 and 0.5 M NaCl, precipitate on ice for 1 hour, centrifuge and discard the supernatant, resuspend the phage precipitate with PBS to obtain the APN-specific recombinant phage display library, and determine the phage titer.
[0044] 2.5 Panning and Enrichment: Solid-phase panning was used to coat APN protein (5-10 μg / mL) onto immunotubes or ELISA plates and incubate overnight at 4°C. After blocking, phage libraries were added and bound at room temperature for 1 hour. The cells were washed 10 times with PBST (PBS + 0.1% Tween-20) to remove unbound phages. Finally, the specifically bound phages were eluted with 0.1 M Glycine-HCl (pH 2.2) and immediately neutralized with 1 M Tris-HCl (pH 9.0). The elution buffer was used to infect DH5α bacteria in the logarithmic growth phase, and after amplification, three rounds of panning were performed.
[0045] 2.6 Single clone screening and identification: After the final round of panning, DH5α bacteria were infected with eluted phages, spread on ampicillin plates, and single clones were randomly selected and inoculated into 96-well plates for culture. After induction of expression, positive clones were detected by phage ELISA (using APN as the coating antigen and HRP-labeled anti-M13 antibody as the secondary antibody) to screen for high-affinity candidate strains.
[0046] 3. Prokaryotic expression of APN nanobodies
[0047] The VHH sequence of positive clones obtained from phage panning was amplified by PCR and subcloned into the prokaryotic expression vector pET-27b(+). The ligation product was transformed into DH5α competent cells, plated on kanamycin (50 μg / mL) plates, and single clones were picked and cultured. The plasmid was extracted and verified for correctness by double enzyme digestion and sequencing. The verified recombinant plasmid was heat-transformed into the expression strain, and single clones were inoculated into LB medium containing kanamycin and cultured at 37°C with shaking until OD. 600 =0.6. Add IPTG to a final concentration of 0.2 mM and induce expression at 16℃ for 16-20 hours. Collect bacterial cells by centrifugation, resuspend in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, pH 8.0), and sonicate. For soluble His-tagged nanobodies, purify using nickel column affinity chromatography: pass the supernatant through the column, wash with buffer containing 20 mM imidazole, then elute the target protein with 500 mM imidazole buffer, concentrate using ultrafiltration tube, and replace with PBS buffer. The purity and molecular weight of the purified product were determined by SDS-PAGE, and the protein concentration was determined using the BCA method. After aliquoting, store at -80℃ for later use.
[0048] 4. Detecting the affinity of nanobodies for APN proteins
[0049] 4.1 Nanobody Coating: The purified APN nanobodies were serially diluted with carbonate coating buffer (0.05 M, pH 9.6) (typically ranging from 0.5 to 10 μg / mL), and 100 μL was added to each well of a 96-well high-binding microplate. A commercially available APN monoclonal antibody (catalog number: ab7417, 1 μg / mL) manufactured by Abcam was used as a positive control.
[0050] 4.2 Blocking and washing: Discard the liquid in the wells, add 300 μL of PBST (PBS + 0.1% Tween-20) blocking buffer containing 3% bovine serum albumin (BSA) to each well, block at 37°C for 2 hours, wash the microplate 3 times with PBST washing buffer, soaking for 5 minutes each time, and pat dry on absorbent paper.
[0051] 4.3 Antigen binding: Dilute the soluble full-length APN protein solution with PBST (containing 1% BSA) to a working concentration of 1 μg / mL, and add 100 μL to each well. Add an equal volume of PBS to the blank control wells, and incubate the ELISA plate at 37°C for 1.5 hours to allow the antigen to fully bind to the coated nanobody. After incubation, wash three times with PBST and pat dry.
[0052] 4.4 Primary and secondary antibody incubation: Add the primary antibody against APN diluted with blocking buffer, incubate at 37°C for 1 hour, wash the plate, add goat anti-mouse IgG-HRP (1:5000), and incubate at 37°C in the dark for 1 hour.
[0053] 4.5 Color Development and Detection: After thorough washing, add 100 μL of freshly prepared TMB colorimetric solution to each well and incubate at room temperature in the dark for 10-15 minutes. Once a clear blue reaction appears in the positive control wells, add 50 μL of 2 M H2SO4 to each well to terminate the reaction. Immediately measure the absorbance (OD value) of each well using a microplate reader at a wavelength of 450 nm.
[0054] The affinity of 24 positive clone APN nanobodies was tested, and the results are as follows: Figure 1 As shown, the top 18 APN nanobodies with strong affinity were finally selected for subsequent experiments.
[0055] 5. Detection of the neutralizing ability of nanobodies on APN protease activity.
[0056] 5.1 Cell Culture and Seeding: Human gastric cancer cell line SGC-7901 was seeded in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured in a humidified incubator at 37℃ and 5% CO2. Cells in the logarithmic growth phase were harvested, digested and resuspended with 0.25% trypsin, and cultured at 1 × 10⁶ cells per well. 4 One cell was seeded into a 96-well culture plate with a final volume of 100 μL per well, and the cells were allowed to adhere and grow to a confluence of approximately 80%.
[0057] 5.2 Nanobody Treatment and Neutralization Reaction: The purified APN nanobody to be tested was serially diluted with complete culture medium to create 10 concentration gradients. The old culture medium was discarded, and 100 μL of different concentrations of nanobody diluent was added to each well. APN monoclonal antibody (Abcam, catalog number: ab7417) was used as a positive control well with the same serial dilutions. Wells containing only 100 μL of complete culture medium served as a negative control (representing the background of maximum APN enzyme activity). Eight replicates were set up for each dilution, and the entire experiment was independently repeated three times. Cells were then returned to the incubator for another 24 hours.
[0058] 5.3 Cell lysis and enzyme activity assay: After incubation, the supernatant was discarded, and the cells were gently washed twice with pre-chilled PBS. 50 μL of cell lysis buffer containing 0.5% NP-40 was added to each well, and the cells were lysed on ice for 30 minutes. Subsequently, 50 μL of enzyme reaction working solution (containing 200 μM Ala-MCA substrate, 50 mM Tris-HCl, pH 7.5) was added to each well. The 96-well plate was incubated at 37°C in the dark for 60 minutes.
[0059] 5.4 Fluorescence Measurement and Data Acquisition: Immediately after the reaction, the fluorescence intensity of each well was detected using a fluorescence microplate reader (excitation wavelength: 360 nm, emission wavelength: 460 nm). The relative fluorescence units (RFU) of each replicate well were recorded. The neutralization titer was calculated using the Reed-Muench method, as shown in the following formula:
[0060] Inhibition rate (%) = [1 - (RFU of nanobody pores / RFU of negative control pores)] ×100%.
[0061] The results are as follows Figure 2 As shown. The APN nanobody with the strongest neutralizing ability (Nano-α-APN8) was selected for subsequent anti-drug resistance experiments. Sequencing revealed the following heavy chain variable region sequence of this antibody:
[0062] SEQ ID NO.6:
[0063] EVQLQASGGGFVQPGGSLRLSCAASGFFFYNYAMGWFRQAPGKEREFVSAISWGAGMYDYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAVVMAITKKQPVWISVETTYWGQGTQVTVS.
[0064] Among them, FR1: EVQLQASGGGFVQPGGSLRLSC;
[0065] FR2: MGWFRQAPGKEREF;
[0066] FR3: VKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC;
[0067] FR4: TTYWGQGTQVTVS;
[0068] CDR1: AASGFFFYNYA;
[0069] CDR2: VSAISWGAGMYDYYADS;
[0070] CDR3: AVVMAITKKQPVWISVE.
[0071] 6. Cellular level assay of APN nanobody combined with chemotherapy drugs for gastric cancer to block drug resistance in gastric cancer cells.
[0072] 6.1 Cell Culture and Seeding: The human gastric cancer cisplatin-resistant cell line SGC-7901 / DDP was seeded in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured at 37°C in a 5% CO2 incubator. After the cells reached the logarithmic growth phase, they were digested, resuspended, and seeded at 5 × 10⁶ cells per well. 3 - 1×10 4 Cells were seeded at a density of 100 μL in 96-well cell culture plates, with a final volume of 100 μL per well, and allowed to adhere to the plates until the cells reached approximately 80% confluence.
[0073] 6.2 Experimental Groups and Drug Treatment: The experiment was set up with the following groups, each with 8 parallel replicates and 3 independent replicates (i.e., 3 biological replicates):
[0074] Experimental group (DDP+Nano-α-APN): After incubating with nanobody diluents at multiple concentrations around ND50 for 2-4 hours, a series of concentration gradients (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μg / ml) were added, followed by the addition of cisplatin (DDP) working solution at a series of concentration gradients (0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9 μM).
[0075] Cisplatin monotherapy group (DDP): Cisplatin was added only at the same gradient concentration;
[0076] Positive control group (DDP+α-APN): APN monoclonal antibody (catalog number: ab7417) manufactured by Abcam was used, and other treatments were the same as those for the experimental group.
[0077] Negative control group (PBS): Add an equal volume of PBS.
[0078] 6.3 Combined treatment and incubation: Discard the original culture medium, add fresh complete culture medium containing the corresponding drug or PBS according to the above grouping, and put the 96-well plate back into the incubator to continue incubation for 48-72 hours. During this period, observe and record the cytopathic effect (CPE) of the cells in each well under an inverted microscope every day, including but not limited to morphological changes such as cell shrinkage, shedding, fragmentation, and decreased transparency.
[0079] 6.4 Cell viability assay: After incubation, add 10 μL of CCK-8 solution directly to each well, and incubate the culture plate at 37°C in the dark for 1-4 hours. Use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm and calculate the cell viability: Cell viability = (OD experimental group - OD blank group) / (OD negative control group - OD blank group).
[0080] Through experimentation, it was found that when the concentration of the APN nanobody prepared in this invention is 5 μg / ml, or the concentration of the commercially available APN monoclonal antibody is 10 μg / ml, it exhibits a good effect in blocking chemotherapy resistance. The results are as follows... Figure 3 As shown, the APN nanobody (5 μg / ml) of the present invention can effectively block cisplatin resistance in the gastric cancer cisplatin-resistant cell line SGC-7901 / DDP, and is superior to the existing Abcam commercial APN monoclonal antibody (10 μg / ml).
[0081] 7. In vivo determination of whether APN nanobodies enhance the efficacy of chemotherapy in a mouse orthotopic drug-resistant gastric cancer xenograft model.
[0082] 7.1 Laboratory Animals and Ethics: Four-week-old male BALB / c nude mice were used and housed in an SPF-grade animal experimental barrier environment. They had free access to sterile feed and water. All animal handling procedures were reviewed and approved by the Animal Ethics Committee of the Experimental Animal Center of Xiamen University.
[0083] 7.2 Establishment of an orthotopic drug-resistant gastric cancer xenograft model: SGC-7901 / DDP drug-resistant cells stably expressing luciferase in the logarithmic growth phase were collected, resuspended in serum-free medium, and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 6 50 μL of cells (resuspended in a 1:1 mixture of Matrigel and serum-free culture medium) were used to anesthetize mice with isoflurane inhalation. The mice were then fixed in a sterile operating table, and the abdominal area was prepared and disinfected. A longitudinal incision of about 1 cm was made below the left costal margin to expose the stomach wall. 50 μL of cell suspension was slowly injected into the subserosa of the stomach wall using a microsyringe. A small transparent vesicle was observed to form. The incision was then sutured. Butorphanol 5 mg / kg was subcutaneously injected for 3 consecutive days after the operation to reduce the animals' pain.
[0084] 7.3 Experimental Grouping and Dosing Regimen: After tumor formation in the mice, the tumor-bearing mice were randomly divided into the following 4 groups (n=5 in each group):
[0085] Experimental group (DPP+Nano-α-APN): 5 mg / kg of APN-targeting nanobody was injected via tail vein, followed by intraperitoneal injection of cisplatin (DDP 3 mg / kg) at a certain interval (4 hours, based on antibody pharmacokinetics). The administration was repeated twice a week for 4 consecutive weeks.
[0086] Negative control group (DDP): The same volume of PBS was injected via the tail vein, and cisplatin was administered in the same manner as the experimental group;
[0087] Positive control group (DDP+α-APN): APN monoclonal antibody (catalog number: ab7417) produced by Abcam was injected via tail vein, and the remaining treatments were the same as those for the experimental group;
[0088] Blank control group (Ctrl): PBS was injected via tail vein, and no DDP treatment was given.
[0089] 7.4 Monitoring of Treatment Efficacy: 72 hours after the last administration, the tumor chemotherapy progress in mice of each group was monitored using the IVIS Spectrum small animal in vivo 3D bioluminescence imaging system. Bioluminescence intensity was quantitatively analyzed as mean radiance (p / sec / cm²). 2 The value is represented as / sr and displayed using a logarithmic scale (log10).
[0090] The results are as follows Figure 4 and Figure 5 As shown, the model carrying orthotopic drug-resistant gastric cancer xenografts exhibited strong resistance to cisplatin, and its tumor-suppressive effect was not statistically different from the blank control group (ns). However, the APN nanobody prepared in this invention can significantly enhance the chemosensitivity of this model to cisplatin, and has a significant effect in reversing chemosuppression resistance in gastric cancer. Its efficacy is significantly better than the existing commercially available Abcam APN monoclonal antibody (**P < 0.01).
[0091] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. An aminopeptidase N nanobody, characterized in that, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.
6.
2. The use of the aminopeptidase N nanobody according to claim 1 in the preparation of a medicament for treating chemotherapy-resistant gastric cancer.
3. The application according to claim 2, characterized in that, The chemotherapy-resistant gastric cancer includes gastric cancer resistant to cisplatin.
4. The use of the aminopeptidase N nanobody according to claim 1 in the preparation of a drug for treating gastric cancer.
5. The use of the aminopeptidase N nanobody combined with chemotherapeutic drugs as described in claim 1 in the preparation of a drug for treating gastric cancer.
6. The application according to claim 5, characterized in that, The chemotherapy drugs include cisplatin.
7. A drug for treating chemotherapy-resistant gastric cancer, characterized in that, The drug uses the aminopeptidase N nanobody as described in claim 1 as its active ingredient.
8. A drug for treating gastric cancer, characterized in that, The drug uses the aminopeptidase N nanobody and chemotherapeutic drugs as active ingredients as described in claim 1.
9. The drug according to claim 7 or 8, characterized in that, The drug also contains pharmaceutically acceptable excipients.
10. The medicament according to claim 9, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: pharmaceutically acceptable solvents, releasing agents, antioxidants, and preservatives.