Pharmaceutical composition based on recombinant chimpanzee oncolytic adenovirus and application of pharmaceutical composition in colorectal cancer treatment

Abstract

The invention discloses a pharmaceutical composition based on recombinant chimpanzee oncolytic adenovirus and application of the pharmaceutical composition in colorectal cancer treatment, the composition comprises the chimpanzee oncolytic adenovirus carrying IL-21 gene, immunogenicity is reduced by constructing a novel chimpanzee adenovirus vector and deleting an E3 region, and tumor targeting is enhanced by integrating RGD peptide fragment. Meanwhile, shuttle plasmids carrying delta24bpE1A are designed, so that the virus is only copied in Rb defective tumor cells, IL-21 genes are armed, the anti-tumor effect is enhanced through oncolysis and immune activation dual mechanisms, a new strategy is provided for colorectal cancer treatment, and by directionally modifying a chimpanzee adenovirus genome skeleton and inserting an exogenous gene expression cassette, the anti-tumor effect is enhanced. According to the invention, a high-efficiency and low-toxicity innovative medicine scheme is provided for colorectal cancer treatment through dual mechanisms of targeting oncolysis and immune activation.

Description

Technical Field

[0001] This invention relates to a chimpanzee oncolytic adenovirus, its methods and applications, and more specifically, to a pharmaceutical composition based on recombinant chimpanzee oncolytic adenovirus and its application in the treatment of colorectal cancer. This invention belongs to the field of genetic engineering and viral vector technology, and is particularly applicable to the fields of vaccine development, gene therapy and immunotherapy. Background Technology

[0002] Traditional treatments for colorectal cancer (CRC) face significant challenges due to drug resistance and high recurrence rates. Oncolytic virus (OV) therapy, which selectively lyses tumor cells and releases antigens to activate the immune response, has become a hot research topic. However, existing oncolytic viruses (such as human adenovirus type 5) have limited efficacy due to pre-existing neutralizing antibodies in the body, and a single oncolytic mechanism is insufficient to overcome the immunosuppression of the tumor microenvironment. Although interleukin-21 (IL-21) can activate CD8+ T cells and NK cells, its systemic administration suffers from problems such as short half-life and high toxicity.

[0003] Chimpanzee adenovirus (ChAd) has become an ideal candidate for novel oncolytic virus vectors due to its low pre-existing neutralizing antibody rate in humans. However, current technologies for chimpanzee adenovirus vectors lack specific optimization for colorectal cancer and have not been used in combination with immunomodulatory factors such as IL-21. Furthermore, traditional viral preparations suffer from low purity and poor stability, hindering their clinical translation.

[0004] Traditional treatments for colorectal cancer face significant challenges due to drug resistance and high recurrence rates. Oncolytic virus therapy, which selectively lyses tumor cells and activates the immune response, has become a hot research topic. However, existing oncolytic viruses, such as human adenovirus type 5, have limited efficacy due to pre-existing neutralizing antibodies in the body, and a single oncolytic mechanism is insufficient to overcome the immunosuppression of the tumor microenvironment. Although interleukin-21 can enhance anti-tumor immunity by activating CD8-positive T cells and natural killer cells, its systemic administration suffers from problems such as short half-life and high toxicity.

[0005] Chimpanzee adenoviruses have become ideal candidates for novel oncolytic virus vectors due to their low pre-existing neutralizing antibody rates in humans. However, current chimpanzee adenovirus vectors lack specific optimization for colorectal cancer and have not been used in combination with immunomodulatory factors such as IL-21. Furthermore, traditional viral preparations suffer from low purity and poor stability, hindering their clinical translation. Summary of the Invention

[0006] The purpose of this invention is to provide a pharmaceutical composition based on recombinant chimpanzee oncolytic adenovirus and its application in the treatment of colorectal cancer, which has the technical characteristics of significantly reducing interference from pre-existing neutralizing antibodies in the human population, improving the in vivo delivery efficiency of oncolytic virus, targeting infection of tumor cells with high integrin expression, reducing off-target toxicity, enabling the virus to replicate only in Rb-deficient tumor cells, and having high safety for normal cells.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] The present invention discloses a pharmaceutical composition comprising a recombinant chimpanzee oncolytic adenovirus and a pharmaceutically acceptable vector, wherein the virus comprises:

[0009] The chimpanzee adenovirus backbone of serotype 25, with the E3 region missing;

[0010] Δ24 mutation in the E1A region: 24bp deletion of the E1A-CR2 domain;

[0011] The IL-21 gene is regulated by the CMV promoter.

[0012] Preferably, the recombinant chimpanzee oncolytic adenovirus is Adsimian-Δ24-IL21, a chimpanzee oncolytic adenovirus carrying the IL-21 gene; the viral titer of the composition is 1×10⁻⁶. 9 Up to 1×10 12 It has a TCID of 50 / mL and is less than 10% toxic to normal cells.

[0013] Preferably, the IL-21 gene is human IL-21 (SEQ ID NO:1) or mouse IL-21 (SEQ ID NO:2), and the composition is suitable for inhibiting the activity of colorectal cancer cells in vitro, with an IC50 ≤ 5 MOI.

[0014] The present invention provides a method for detecting viral function in the pharmaceutical composition, comprising:

[0015] Step 1) Verify the integrity of the viral genome by PCR using primer pair CMV-F (SEQ ID NO:3) and SV40-R (SEQ ID NO:4);

[0016] Step 2) Detect the secretion level of IL-21 in the supernatant of infected cells by ELISA, with a sensitivity ≥10 pg / mL;

[0017] Step 3) Determine the viral titer using the TCID50 method. The calculation formula is: Titer TCID50 / mL=10^(A+B×C), where A is the number of positive wells at the highest dilution, B is the dilution factor, and C is the proportion of positive wells.

[0018] This invention relates to a drug application for inhibiting the migration of colorectal cancer cells. The drug is the composition described in claims 1-3, which achieves a cancer cell migration inhibition rate of ≥40% by downregulating EMT markers (N-cadherin, Vimentin) and upregulating E-cadherin.

[0019] The present invention relates to an application of a drug that enhances anti-tumor immune killing, characterized in that the drug is the composition described in claims 1-3, and when co-cultured with peripheral blood mononuclear cells (PBMCs), the killing rate against cancer cells is increased by more than 50%, and the proportion of CD8+ T cells increases by ≥3 times, with a significance value of less than 0.001.

[0020] The present invention provides a method for evaluating the oncolytic effect of the pharmaceutical composition, comprising:

[0021] The half-maximal inhibitory concentration (IC50) against colorectal cancer cells SW480 and MC38 was determined by MTT assay.

[0022] The inhibition rate of cancer cell colony formation was assessed by crystal violet staining, and the inhibition rate was not less than 70%.

[0023] The viral progeny replication capacity was detected by quantitative PCR, with an amplification factor of no less than 100-fold.

[0024] The present invention discloses a method for detecting the stability of the pharmaceutical composition according to claim 1, characterized in that it comprises:

[0025] The survival rate of the virus in a simulated human immune environment was determined by detecting the titer of neutralizing antibodies.

[0026] The viral titer reduction rate was verified to be no more than 20% through accelerated stability experiments, which were performed under the condition of storage at 37 degrees Celsius for 7 days.

[0027] The present invention provides a kit for the treatment of colorectal cancer, characterized in that it comprises the pharmaceutical composition described in claims 1-3, and a reagent kit for detecting viral function; the reagent kit includes PCR primer pairs and an enzyme-linked immunosorbent assay kit.

[0028] Beneficial effects: It provides a highly efficient carrier and method for combined oncolytic-immunotherapy of colorectal cancer, specifically including:

[0029] 1. Avoiding pre-existing immunity: Precise tumor targeting: Inserting RGD peptides enhances targeted infection of tumor cells with high integrin expression and reduces off-target toxicity.

[0030] 2. Selective replication and killing: The Δ24 mutation in the E1A domain enables the virus to replicate only in Rb-deficient tumor cells, with high safety in normal cells (verified in BEAS-2B and NIH / 3T3 cells).

[0031] 3. Immune microenvironment remodeling: Activation of CD8+ T / NK cells through IL-21 expression upregulates the CD3+ / CD8+ ratio in the tumor microenvironment, reversing the immunosuppressive state.

[0032] 4. Inhibition of metastasis and drug resistance: It can significantly inhibit the migration and epithelial-mesenchymal transition (EMT) of colorectal cancer cells (SW480, MC38), reducing the risk of recurrence.

[0033] 5. High efficiency and low toxicity: The tumor growth inhibition rate in the MC38 tumor-bearing model reached ≥70%, with no weight loss or organ toxicity, achieving synergistic oncolytic-immunotherapy.

[0034] In summary, the aforementioned beneficial effects demonstrate that the recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 provides feasibility for drug development. The vector constructed from chimpanzee adenovirus (serotype 25) based on the technical solution can significantly reduce interference from pre-existing neutralizing antibodies in the human population and improve the in vivo delivery efficiency of oncolytic virus. Attached Figure Description

[0035] Figure 1 This is a diagram of the packaging and pathogenesis process of Adsimian-Δ24-EGFP according to the present invention.

[0036] Figure 2 This is a gene expression detection diagram of the purified product of this invention.

[0037] Figure 3 This is a bar chart showing the serum neutralization titer results of the rabbits tested in this invention after three immunizations.

[0038] Figure 4 This is a diagram showing the effect of chimpanzee adenovirus infection on the migration of SW480 cells according to the present invention.

[0039] Figure 5 This is a diagram showing the effect of the present invention on the migration of MC38 cells after infection with chimpanzee adenovirus.

[0040] Figure 6 This is a graph showing the effect of the replicating and non-replicating chimpanzee adenoviruses of this invention on the viability of SW480 cells.

[0041] Figure 7 This is a graph showing the effect of the replicating and non-replicating chimpanzee adenoviruses of this invention on the viability of SW620 cells.

[0042] Figure 8 This is a graph showing the effect of the recombinant chimpanzee oncolytic adenovirus of the present invention on the viability of SW480, SW620, and HCT116 cells.

[0043] Figure 9This is a graph showing the effect of the recombinant chimpanzee oncolytic adenovirus of the present invention on the activity of colorectal cancer cells MC38 and CT26WT.

[0044] Figure 10 This figure shows the effect of the recombinant chimpanzee oncolytic adenovirus of the present invention on the activity of normal human lung epithelial cells BEAS-2B and mouse embryonic fibroblasts NIH / 3T3.

[0045] Figure 11 This is a comparative diagram of the progeny replication ability of the chimpanzee adenovirus of this invention in different colorectal cancer and normal cells.

[0046] Figure 12 This is a graph showing the effect of co-culturing chimpanzee oncolytic adenovirus and PBMC on the viability of SW480 cells.

[0047] Figure 13 This is a graph showing the tumor volume and body weight in mice according to the present invention.

[0048] Figure 14 This is a diagram showing the results of HE staining of various organs according to the present invention.

[0049] Figure 15 This is a flow cytometry analysis of spleen tissue cells from mice in each treatment group of this invention.

[0050] Figure 16 These are flow cytometry analyses of mouse tumor tissue cells in each treatment group of this invention.

[0051] Figure 17 These are flow cytometry analyses of mouse lymph node cells in each treatment group of this invention.

[0052] Figure 18 This is a graph showing the expression level of IL-21 in mouse serum according to the present invention.

[0053] Figure 2 a: PCR electrophoresis image of the purified product Adsimian-Δ24-hIL21 carrying the target gene hIL21. M: DL2000 DNA Marker. Figure 2 b: The target genes mIL21 (732bp) and EGFP (717bp) of Adsimian-Δ24-mIL21 and Adsimian-Δ24-EGFP. M: DL2000 DNA Marker. Figure 2 c: Expression of hIL21 in cell culture supernatant. Figure 2 d: Changes in mIL21 translation levels after Adsimian-Δ24-mIL21 infection of colorectal cancer cells. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0054] Figure 4 a: Scratch patterns of SW480 cells after different treatments. Figure 4 b: Migration rate analysis of SW480 cells after 24 hours. Figure 4 c: qPCR detection results of SW480 mRNA expression. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0055] Figure 5 Image a: Scratch patterns on MC38 cells after different treatments. Figure 5 b: Migration rate analysis of MC38 cells after 24 hours. Figure 5 c: qPCR detection results of MC38 mRNA expression. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0056] Figure 6 a: Cell viability of SW480 cells at four time points when infected with different MOIs. Figure 6 b: Cell viability measured 48 h after SW480 infection with different MOI viruses. Figure 6 c: SW480 cells were infected with 1 MOI, 5 MOI, 10 MOI, and 20 MOI of virus, respectively, and cell viability was detected by crystal violet staining. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0057] Figure 7 a: Cell viability of SW620 cells at four time points when infected with different MOIs. Figure 7 b: Cell viability measured 48 h after SW620 infection with different MOI viruses. Figure 7 c: SW620 cells were infected with 1 MOI, 5 MOI, 10 MOI, and 20 MOI of virus, respectively, and cell viability was detected by crystal violet staining. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0058] Figure 8 a: Cell viability of SW480 cells at four time points when infected with different MOIs. Figure 8 b: Cell viability of SW620 cells at four time points when infected with different MOIs. Figure 8 c: Cell viability of HCT116 at four time points when infected with different MOIs. Figure 8At d: 48h, the viability levels of SW480, SW620, and HCT116 cells under different MOI infection levels. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0059] Figure 9 a: Cell viability of MC38 cells at four time points when infected with different MOIs. Figure 9 b: Cell viability of CT26WT cells at four time points when infected with different MOIs. Figure 9 Mid-day (d): Viability levels of MC38 and CT26WT cells at different MOI infection levels at 48 h. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0060] Figure 10 a: Cell viability of BEAS-2B at four time points when infected with different MOIs. Figure 10 b: Cell viability of NIH / 3T3 cells at four time points when infected with different MOIs. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0061] Figure 11 a: Comparison of progeny replication capacity of Adsimian-Δ24-EGFP and Adsimian-Δ24-hIL21 in human colorectal cancer cells SW480, SW620, HCT116 and normal human lung epithelial cells BEAS-2B. Figure 11 b: Comparison of progeny replication capacity of Adsimian-Δ24-EGFP and Adsimian-Δ24-mIL21 in mouse colorectal cancer cells MC38, CT26WT and mouse embryonic fibroblasts NIH / 3T3. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0062] Figure 12 a: Viability assay of SW480 cells infected with chimpanzee adenovirus at 24h after co-culturing with PBMC. Figure 12 b: Viability assay of SW480 cells infected with chimpanzee adenovirus at 48h after co-culturing with PBMCs. ns: p>0.05, *: p<0.1, **: p<0.01, ***: p<0.001.

[0063] Figure 13 a: Timeline of gene therapy in mice with colon cancer xenografts. Figure 6 b: Schematic diagram of mouse weight changes. Figure 6c: Changes in tumor volume in mice in the PBS group, Adsimian-Δ24-EGFP group, and Adsimian-Δ24-mIL21 group. Figure 6 d: Changes in tumor volume in mice treated with PBS. Figure 6 Image of tumor volume changes in mice in the Adsimian-Δ24-EGFP group (e): Figure 6 f: Tumor volume changes in Adsimian-Δ24-mIL21 mice. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0064] Figure 15 a: CD3 levels in spleen tissue of the PBS group, Adsimian-Δ24-EGFP group, and Adsimian-Δ24-mIL21 group on day 15 + Flow cytometry results. Figure 8 b: CD4 count in spleen tissue of PBS group, Adsimian-Δ24-EGFP group, and Adsimian-Δ24-mIL21 group on day 15 + Figure 1 shows the results of flow cytometry analysis of cells and CD8+ cells. Figure 8 c: On day 15 after treatment, CD3 in the spleen + The proportion of cells. Figure 8 d: On day 15 after treatment, CD4 in the spleen + The proportion of cells. Figure 8 Zhong e: On the 15th day after treatment, CD8 in the spleen + The proportion of cells. Figure 8 f: PBS group, Adsimian-Δ24-EGFP group, and Adsimian-Δ24-mIL21 group on day 30 CD3 + Flow cytometry results. Figure 8 In the PBS group, Adsimian-Δ24-EGFP group, and Adsimian-Δ24-mIL21 group, CD4 counts on day 30 were... + Cells and CD8 + Flow cytometry results. Figure 8 h: On day 30 after treatment, CD3 in the spleen + Percentage of cells. Figure 8 Zhong i: On the 30th day after treatment, CD4 in the spleen + Percentage of cells. Figure 8 J: On the 30th day after treatment, CD8 in the spleen + Percentage of cells. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0065] Figure 16 a: CD3 levels in tumor tissue of the PBS group, Adsimian-Δ24-EGFP group, and Adsimian-Δ24-mIL21 group on day 15 + Flow cytometry results. Figure 9 b: CD4 count in tumor tissue of the PBS group, Adsimian-Δ24-EGFP group, and Adsimian-Δ24-mIL21 group on day 15 + Cells and CD8 + Flow cytometry results. Figure 9 c: CD3 on day 15 after treatment + The proportion of cells in tumor tissue. Figure 9 d: CD4 count on day 15 after treatment + The proportion of cells in tumor tissue. Figure 9 e: CD8 on day 15 after treatment + The proportion of cells in tumor tissue. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0066] Figure 17 In the Adsimian-Δ24-EGFP group and the Adsimian-Δ24-mIL21 group, CD3 levels in lymph nodes were observed on day 30. + Flow cytometry results. Figure 10 b: CD4+ levels in lymph nodes of the Adsimian-Δ24-EGFP group and the Adsimian-Δ24-mIL21 group on day 30 + Cells and CD8 + Flow cytometry results. Figure 10 c: CD3 on day 30 after treatment + The proportion of cells in lymph nodes. Figure 10 d: CD4 count on day 30 after treatment + The proportion of cells in lymph nodes. Figure 10 e: CD8 on day 30 after treatment + The proportion of cells in lymph nodes. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0067] Figure 18 In the Chinese dictionary: ns: p>0.05, ****: p<0.0001. Detailed Implementation

[0068] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0069] 1.1 Technical solution / principle of the invention:

[0070] This technology constructs a novel chimpanzee adenovirus vector (serotype 25) by deleting the E3 region to reduce immunogenicity and integrating the RGD peptide to enhance tumor targeting. Simultaneously, a shuttle plasmid carrying the Δ24 mutant E1A is designed, allowing the virus to replicate only in Rb-deficient tumor cells. Further enhancement of the IL-21 gene enhances anti-tumor effects through a dual mechanism of oncolysis and immune activation, providing a new strategy for colorectal cancer treatment. By directionally modifying the chimpanzee adenovirus genome backbone (e.g., by deleting the E1 / E3 region) and inserting exogenous gene expression cassettes, the limitations of traditional human adenovirus vectors due to pre-existing immunity are overcome. Simultaneously, vector capacity and gene expression efficiency are optimized, providing a safer and more efficient technical tool for infectious disease control, tumor immunotherapy, and genetic disease gene repair.

[0071] 1.2 Experimental Methods (Packaging, Amplification, and Purification of Recombinant Chimpanzee Oncolytic Adenovirus)

[0072] 1.2.1.1 Adenovirus Packaging

[0073] Prepare HEK293 cells for virus packaging, linearized endotoxin-free plasmids, and pre-digest with Pac I. Use PEI transfection reagent. The specific steps are as follows:

[0074] (1) One day before transfection, 3×10 5 HEK293 cells were seeded on 6-well plates, and the transfection time was optimal when the cells reached 70% confluence.

[0075] (2) Add 3 μg of linearized plasmid and 10.5 μg / μL PEI (1:3.5) to 250 μL of serum-free DMEM medium and mix. Slowly add PEI mixture to the DNA mixture, shake to mix evenly, and incubate at room temperature for 15 min.

[0076] (4) Take a 6-well plate, slowly inject the PEI-DNA mixture into the 6-well plate, then place it in a cell culture incubator, and after culturing for 6 hours, discard the supernatant and replace it with 2% FBS+DMEM medium.

[0077] (5) During this process, viral cavities will form in about 7-14 days. When obvious pathological changes occur in the cells, collect the cells and supernatant. During the process, the culture medium will turn yellow due to nutrient consumption. To avoid cell detachment, the medium can be changed by replacing half of the medium. However, if a large number of floating cells appear, the medium should not be changed. Instead, the culture medium should be added in appropriate amounts until the cells become diseased.

[0078] 1.2.1.2 Massive adenovirus amplification

[0079] The initial (P0) adenovirus has a low titer. To increase its titer and viral particle size, multiple amplifications are necessary. The specific steps are as follows:

[0080] (1) In a 6-well plate (10% FBS + DMEM), 3 × 10 5 HEK293 cells per cell / well were placed on 6-well plates with 70% cell confluence as the optimal concentration.

[0081] (2) Inoculate 200 μL of the above P1 generation virus into the above 6-well plate. After 2 to 3 days, collect cells and supernatant, and repeat the freeze-thaw cycle 3 times to obtain the virus of the P2 generation.

[0082] (3) When the confluence of HEK293 cells cultured in a 10cm dish reaches 80%, HEK293 cells are infected with 200μL of P2 generation bacterial solution. After 2-3 days, the cells and supernatant are separated, and 2mL of supernatant is retained. This process is repeated 3 times to obtain the P3 generation strain.

[0083] (4) After extracting the viral genome and identifying adenoviruses containing the target gene, large-scale amplification can be performed. Large-scale amplification of adenoviruses is achieved by repeating step 3.

[0084] 1.2.1.3 Adenovirus density gradient centrifugation purification

[0085] (1) On this basis, the amplified chimpanzee oncolytic adenovirus is repeatedly thawed until it is completely dissolved, and then the virus can be purified.

[0086] (2) Place the dissolved chimpanzee adenovirus on ice, then aliquot it into 50mL centrifuge tubes, balance the mixture, place the centrifuge tubes in a centrifuge, centrifuge at 12000rpm for 10min, and collect the supernatant in a clean bottle.

[0087] (3) Mix the virus supernatant and PEG8000 solution at a ratio of 2:1 and place on ice overnight.

[0088] (4) On the second day, centrifuge at 12,000 rpm for 20 minutes, pour out the supernatant and keep the precipitate.

[0089] (5) Add 1.1 g / mL cesium chloride to the centrifuge tubes, and then resuspend the virus precipitate in each centrifuge tube by refluxing with cesium chloride solution. Then, refluxing the solutions in all centrifuge tubes with 2 mL and 1 mL of 1.1 g / mL cesium chloride solution in sequence, and finally collect all the solutions in the same centrifuge tube.

[0090] (6) Place the centrifuge tubes on a centrifuge at 4°C and centrifuge at 12,000 rpm for 10 minutes. Collect the supernatant for further testing.

[0091] (7) Add 2 mL of 1.4 g / mL CsCl solution to an ultracentrifuge tube, then slowly add 3 mL of 1.3 g / mL CsCl solution using a pipette. Finally, slowly add 5 mL of 1.1 g / mL CsCl solution containing oncolytic adenovirus along the wall and observe whether a CsCl gradient is formed. If a gradient is formed, place the tube in a high-speed vacuum centrifuge at 12,000 rpm.

[0092] (8) In an ultracentrifuge tube, use a pipette to remove the liquid at the top, and then aspirate the adenovirus below the subviral band into a 1.5 mL Ep tube. The purified chimpanzee adenovirus band is mainly distributed at the interface of the 1.3-1.4 g / mL cesium chloride solution.

[0093] (9) Cut open the dialysis bag to a suitable length and place the 10mM Na2EDTA·2H2O solution in a magnetic stirrer at 100℃ and boil for 10 minutes.

[0094] (10) Transfer the adenovirus collected by centrifugation to a dialysis bag, clamp both sides with clamps, and then place it in a beaker containing dialysis solution. Place the beaker on a magnetic stirrer and stir evenly. Dialyze overnight in a 4°C refrigerator. In order to better remove the CsCl solution in the oncolytic adenovirus, perform a second dialysis after the first overnight dialysis. The second dialysis time is about 4 hours or more.

[0095] (11) After two dialysis sessions, collect the oncolytic adenovirus from the dialysis bag and place it in a 1.5 mL Ep tube in a -80°C freezer.

[0096] 1.2.1.4 PCR detection (detection of purified products)

[0097] Based on the differences in virus-specific genes, a pair of gene-specific primers was designed between the promoter and terminator to display gene length, thereby verifying the length of the purified viral genome by PCR. The primer sequences are shown in Table 3.3.2.1 below.

[0098] Table 3.3.2.1 Universal primers for validating the viral genome

[0099]

[0100] 1.2.1.5 Western Blot detection of mIL21 protein expression

[0101] Using GAPDH protein as an internal control, the changes in total cellular protein after different treatments were detected by Western blotting. The specific steps are as follows:

[0102] (1) Protein extraction

[0103] Seeds from the same batch at the same cell density in 6-well plates. After culture according to the experimental treatment, remove the cells and wash them twice with PBS. If the virus treatment group is present, wash the non-adherent cells again. Add 200 μL of 1× Loading buffer to each well to cover the bottom of the culture medium. After the cells are completely dissolved, transfer them to 1.5 mL EP tubes, incubate at 100 °C for 15 min, and store at -80 °C for later use.

[0104] (2) Preparation of SDS-polyacrylamide gel

[0105] First, select an appropriate amount of gel based on the relative molecular mass of the target protein; carefully inject the separating gel (avoiding air bubbles), seal it with anhydrous alcohol, and after the gel solidifies and polymerizes, try to remove the liquid from the gel. Then carefully inject the building block gel and place it into the pre-designed comb teeth.

[0106] (3) Electrophoresis

[0107] Add the denatured protein sample into the sample well, connect the power supply, perform electrophoresis at 80V for 30 minutes, and then perform electrophoresis at 120V for 60 minutes to allow the bromophenol blue to migrate to the bottom of the gel by 0.5 cm. Then turn off the power supply.

[0108] (4) Immunoblot-transfer

[0109] Remove the glass slide from the electrophoresis apparatus and rinse it with deionized water. Immerse the cut PVDF in methanol for 2 minutes. Completely immerse the sponge pad and filter paper in the transfer buffer. With the black side down, place the sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad in that order and clamp them together. Be extremely careful to avoid air bubbles throughout the process. Carefully place the clamping plate in the transfer tank, pour the pre-cooled transfer solution (4°C) into the transfer chamber, connect the power supply, and run at 120V for 120 minutes.

[0110] (5) Closed

[0111] Place the PVDF membrane in 5% skim milk powder (TBST), with one side facing up, and shake on a shaker at room temperature for 2 hours or at 4°C overnight in a sealed container.

[0112] (6) Immunological testing

[0113] Primary antibody binding to target protein: After washing with 15-20 mL TBST for 10 min, the prepared PVDF membrane was cut according to its size and the size of the target protein, and placed in a protein cassette to incubate with the primary antibody overnight.

[0114] Binding of enzyme-labeled secondary antibody to primary antibody: Discard the primary antibody, wash 10 times with 10 mL TBST for 10 min each time, take the enzyme-labeled secondary antibody corresponding to the primary antibody, and incubate on a shaker at room temperature for 2 h. Recover the secondary antibody and wash 3 times with 10 mL TBST for 10 min each time.

[0115] (7) Chemiluminescence detection

[0116] Liquids A and B are mixed in a 1:1 ratio to prepare a working solution. The PVDF membrane is placed on the detection stage, and the working developing solution is added to completely cover the membrane. After incubation for about 2 minutes, development is performed in a high-sensitivity chemiluminescence imager.

[0117] 1.2.1.6TCID 50 Detection of infection titer of purified product

[0118] (1) Inoculate 3 × 10⁶ cells per well in a 96-well plate. 3 HEK293 cells in the logarithmic growth phase were placed in a cell culture incubator and cultured overnight until all cells adhered.

[0119] (2) Add 10 to each of the 96-well plates. -4 , 10 -5 ...10 -11 Different concentration gradients of oncolytic adenovirus were prepared, with 10 replicates for each concentration gradient. 80 μL of virus solution was added to each well. The remaining wells in the 96-well plate were filled with only 80 μL of DMEM culture medium, serving as a blank control group. After mixing, the plates were placed in the incubator for further incubation.

[0120] (3) The 96-well plate was observed with a microscope every day and its pathological changes were recorded for a total of 7 days.

[0121] (4) According to TCID 50 The calculation formula in the figure is used to calculate the titer of oncolytic adenovirus, which is then used as the basis for subsequent experiments.

[0122] 1.2.1.7 Animal Immunization Methods

[0123] Detection of neutralizing titer of novel chimpanzee adenovirus

[0124] (1) Preparation before immunization

[0125] Two healthy SPF-grade New Zealand white rabbits were selected and named "Rabbit No. 1" and "Rabbit No. 2" for this experiment. They weighed approximately 1000-2000g. Blood was collected from them before immunization as negative serum controls.

[0126] (2) Immune process

[0127] The immunization was administered via intramuscular injection of the replicating chimpanzee adenovirus Adsimian-Δ24-EGFP, with a dose of 1 × 10⁻⁶ per immunization. 10 VP was administered to each rabbit via two injections in the groin area, for a total of four injections, with each injection containing 250 μL of virus dilution. A total of five immunizations were given, with intervals of 0, 3, 2, 2, and 2 weeks. Two weeks after the third immunization, approximately 1 mL of blood was collected from the marginal ear vein of each rabbit. The serum was separated and frozen at -15°C (the collection time for the third and fourth immunizations was marked) for neutralizing antibody testing. Two weeks after the fifth immunization, whole blood was collected, serum was separated, and after aseptic filtration, 25 mL of serum was obtained from each New Zealand White rabbit and frozen at -15°C (the collection time for the five immunizations was marked) for neutralizing antibody testing.

[0128] 1.2.1.8 Experimental Procedure for Neutralizing Antibody Assay

[0129] (1) With 1×10 3 HEK293 cells from cells / well were seeded into 96-well plates and cultured overnight in a cell culture incubator.

[0130] (2) The collected serum was diluted 10 times with PBS, filtered to remove bacteria, and incubated at 56°C for 30 min to inactivate complement.

[0131] (3) Dilute the inactivated serum samples with culture medium at ratios of 1:2, 1:4, ... 1:256, and dilute to 1000 TCID. 50 Mix equal volumes of Adsimian-Δ24-EGFP viral solution ( / mL) and incubate at 37°C for 2 hours.

[0132] (4) Each sample was repeated 3 times, with antibody control, cell control and virus control. The samples were placed in a 37°C, 5% CO2 incubator and cultured for about 10 days. The results were determined when 70% of the virus control showed lesions.

[0133] (5) Observe the cell pathogenesis in the cell plate under a microscope, record the pathogenesis, and calculate the neutralizing antibody titer.

[0134] Cell scratch assay

[0135] (1) Inoculate 6 × 10⁶ cells per well of a 6-well plate. 5The number of cells / well should be such that they can completely fill the bottom of the well after adhering to the wall.

[0136] (2) When the cells are fully grown at the bottom of the culture dish, draw two lines on the bottom surface with a medium standard gun and then measure with a ruler; rinse twice with PBS to remove the detached cells, add 2 mL of serum-free culture medium to each well, take pictures under an optical microscope, and mark and record the results. Record 0 h.

[0137] (3) Place the 6-well plate into the cell culture incubator, take the same part, take the same part for 24 hours, set 3 synchronous samples for each group, and use Image J software to calculate the moving distance.

[0138] The effect of replicating and non-replicating chimpanzee adenoviruses on the viability of colorectal cancer cells.

[0139] (1) Take cells in the logarithmic growth phase, 3 × 10⁶ cells per well. 3 Colorectal cancer cells SW480 and SW620 were seeded at 100 μL per well in a 96-well plate and incubated overnight at 37°C.

[0140] (2) The next day, 1 MOI, 5 MOI, 10 MOI and 20 MOI of different MOI numbers of replicating chimpanzee adenovirus Adsimian-Δ24-EGFP and non-replicating chimpanzee adenovirus Adsimian-EGFP were added to the 96-well plate, and experimental groups were set up in duplicate and blank control groups.

[0141] (3) Add 20 μL of MTT solution containing PMSF to a 96-well plate and continue to incubate at 37°C for 4 h.

[0142] (4) Place the 96-well plate containing MTT onto the microplate, measure the absorbance at a wavelength of 490 nm, and calculate the cell viability of each group.

[0143] Detecting the effect of recombinant chimpanzee adenovirus on colon cancer cell viability

[0144] (1) Human colorectal cancer cells SW480, SW620, and HCT116 in the logarithmic growth phase, mouse colorectal cancer cells MC38 and CT26WT, as well as normal human lung epithelial cells BEAS-2B and mouse embryonic fibroblasts NIH / 3T3 were pre-cultured in 96-well plates and cultured overnight.

[0145] (2) Add Adsimian-Δ24-mIL21 or Adsimian-Δ24-hIL21 and the control virus Adsimian-Δ24-EGFP to a 96-well plate. The specific operation steps are the same as in 3.2.3.

[0146] Detection of the replication capacity of recombinant chimpanzee adenovirus

[0147] (1) Colorectal cancer infection

[0148] 1) Human colorectal cancer cells SW480, SW620, and HCT116 in logarithmic growth phase, mouse colorectal cancer cells MC38 and CT26WT, as well as normal human lung epithelial cells BEAS-2B and mouse embryonic fibroblasts NIH / 3T3, were collected at a concentration of 5 × 10⁻⁶ cells / mL. 4 Cells / wells were seeded in 12-well plates and cultured overnight in a 37°C cell culture incubator.

[0149] 2) The next day, add 2 MOI (i.e., 1×10⁻⁶) to the 12-well plate. 5 The study used chimpanzee oncolytic adenovirus (PFU) and included replicates for the experimental group.

[0150] 3) After 24h, 48h, 72h and 96h of infection with colorectal cancer cells, the supernatant and bottom adherent cells were blown off, and the virus particles were released by repeated freeze-thaw cycles three times.

[0151] (2) Extraction of adenovirus genomic DNA

[0152] 1) Place the collected virus solution in a centrifuge and centrifuge at 2000 rpm for 5 minutes. At this time, the upper layer of solution contains a lot of virus, while the precipitate is basically some cell fragments.

[0153] 2) Place a portion of the supernatant containing the virus into a centrifuge at 4°C and centrifuge at 12,000 rpm for 5 minutes. Collect the supernatant and remove the precipitate.

[0154] 3) Add 20 μL of proteinase K to the collected virus solution and invert the Ep tube to mix thoroughly.

[0155] 4) Add 500 μL of the solution and 400 μL of anhydrous ethanol to the Ep tube, stir thoroughly, then add the mixed solution to the adsorption tower, cover it, and let it stand at room temperature for 2 minutes.

[0156] 5) Centrifuge at 12,000 rpm for 1 minute, pour out the supernatant in the collection tube, and then place the absorption tower back into the collection tube.

[0157] 6) Pour the rinsing liquid into the absorption tower, centrifuge at 12000 rpm for 1 minute, drain the liquid from the collection tube, and then place the absorption tower back into the collection tube.

[0158] 7) Repeat step (6);

[0159] (3) qPCR on-machine detection

[0160] 1) Prepare standard solutions (using plasmids containing the target gene, linear DNA digested with enzymes, or PCR products as standards).

[0161] 2) The concentration of the standard DNA was determined using Nanodrop 2000, the copy number of the standard DNA was calculated according to the formula, and then diluted with ddH2O to 1×10⁻⁶. 9 1×10 8 1×10 7 1×10 6 1×10 5 1×10 4 1×10 3 copies / μL

[0162] 3) qPCR primer design

[0163] qPCR primers were designed using the HEXON fraction of chimpanzee adenovirus. The primers are shown in the table below:

[0164] Table 3.3.7 qPCR primers for the chimpanzee adenovirus HEXON gene

[0165]

[0166] Co-culture of recombinant oncolytic adenovirus with PBMC cells

[0167] 1.2.1.9 Isolation and activation of PBMCs from peripheral blood

[0168] (1) Healthy blood samples were collected. Peripheral blood mononuclear cells (PBMCs) were separated by Solarbio and density gradient centrifugation in this experiment.

[0169] (2) Fresh PBMCs (2×10 6 Cells were proliferated in Roswell Park Memorial Institute (RPMI) 1640 intact medium containing 3 μg / mL anti-CD3 antibody, 1 μg / mL anti-CD28 antibody, 10% FBS, 100 U / mL IL-2 and 1% penicillin / streptomycin.

[0170] (3) Change the culture medium every other day until the PBMC count reaches the level required for subsequent experiments, up to a maximum of 5 times. Place SW480 cells in 6-well plates (5 × 10⁻⁶ cells / well). 5 After incubation for 6 hours on cells / well, the cells were treated with mitomycin C (10 μg / mL) for 2 hours, and then washed with PBS for 2 hours.

[0171] (4) The generated cells were mixed with PBMCs (5×10⁻⁶). 6 The cells / well were combined and co-cultured at a PBMC:tumor cell ratio of 10:1. After 3 days, PBMCs with activated tumor cells were obtained.

[0172] 1.2.1.10 Co-culture of PBMCs and SW480 cells in vitro

[0173] (1) Lay out 96-hole boards (3×10) one day in advance. 3 Cells / wells were cultured overnight in a cell culture incubator at 37°C with 5% CO2.

[0174] (2) Tumor cells were infected with recombinant chimpanzee oncolytic adenovirus (MOI = 10); 24 h after viral infection, the cell supernatant was discarded, and 100 U / mL IL-2 was added to activate PBMCs (5 × 10⁻⁶ cells / mL). 3 (cells / well); after incubation for 48 h, remove the supernatant and suspended cells, add fresh culture medium, and determine cell viability using the MTT assay.

[0175] Statistical analysis

[0176] In this experiment, data processing and statistical analysis were performed using software such as Snapgene, ImageJ, and GraphPad Prism. All experiments were conducted in triplicate. Data are expressed as mean ± SD and significance analysis was performed. p > 0.05 indicates no significant difference (labeled ns); p < 0.05 is labeled *; p < 0.01 is labeled **; p < 0.001 is labeled ***.

[0177] 1.3 Experimental Results

[0178] Packaging of chimpanzee oncolytic adenovirus Adsimian-Δ24-EGFP

[0179] like Figure 1 As shown, approximately day 6 after transfection with pAdsimian-Δ24-EGFP, faint empty spots appeared under a microscope, followed by further observation under a fluorescence microscope. Under 100x magnification, the chimpanzee oncolytic adenovirus carrying EGFP expressed strong green fluorescence in HEK293 cells, and the intensity of the green fluorescence increased significantly with increasing transfection time. Furthermore, empty spots appeared in the cells under bright-field vision. This indicates that the transfection was successful, and the constructed chimpanzee oncolytic adenovirus (carrying the EGFP gene) was able to express the exogenous gene in the cells.

[0180] The purified product can efficiently express the target gene.

[0181] 1.3.1.1 Validation of target gene expression

[0182] The amplified chimpanzee adenovirus was purified by CsCl density gradient centrifugation, and the expression of exogenous genes IL-21 and EGFP was verified by PCR, ELISA and Western Blot.

[0183] The purified product was used to extract the viral genome using a kit, and PCR identification was performed using pre-designed universal primers. Results could be obtained as follows: Figure 2 Figures a and b are shown in Figure 1. Figure a shows the PCR electrophoresis results of the target gene hIL21 carried by the purified virus Adsimian-Δ24-hIL21. The band size is consistent with the Snapgene reference size of 496 bp. Figure b shows the PCR verification electrophoresis results of the target genes mIL21 (732 bp) and EGFP (717 bp) of Adsimian-Δ24-mIL21 and Adsimian-Δ24-EGFP. The band sizes are consistent with the PCR electrophoresis results of Adsimian-Δ24-mIL21 and Adsimian-Δ24-EGFP.

[0184] Purified chimpanzee adenovirus was used to infect colorectal cancer cells at an infection titer of 5 MOI, and the culture supernatant was harvested 48 h post-infection. The cell culture supernatant was analyzed by ELISA. According to data from Graphpad software, as shown in Figure c, after 48 h of infection with the Adsimian-Δ24-hIL21 virus, the experimental groups (HCT116, SW620, and SW480 cells) showed significantly higher levels of IL-21 compared to the control group (p<0.001). The level detected in SW480 cells was significantly higher than in the other two cell types, demonstrating that the chimpanzee adenovirus carrying IL-21 successfully expressed hIL21, indicating the successful construction of the Adsimian-Δ24-hIL21 virus.

[0185] MC38 cells were infected with a 5 MOI dose of Adsimian-Δ24-mIL21 virus. After 48 hours, the supernatant was discarded, cellular proteins were extracted, and Western blotting was performed to detect the protein expression of Adsimian-Δ24-mIL21 virus in colon cancer cells. The results showed that colorectal cancer cells infected with Adsimian-Δ24-mIL21 virus could express IL-21 protein, while basal cells and colorectal cancer cells infected with the control virus Adsimian-Δ24-EGFP could not express IL-21 protein.

[0186] In summary, these results demonstrate that large-scale production of adherent HEK293 cells and chimpanzee oncolytic adenovirus obtained by CsCl density gradient centrifugation can drive the expression of exogenous genes IL-21 and EGFP.

[0187] 1.3.1.2 Detection of virus particle number and titer in purified product

[0188] (1) Number of virus particles

[0189] The number of virus particles in the purified viral solution was detected using Nanodrop2000, and the results are shown in the table below.

[0190] Table 3.4.2.2.a Number of virus particles after purification

[0191]

[0192] (2) Infection titer

[0193] After 8 days of viral infection, the 96-well plate was placed under a microscope for spot counting. The results are shown in the table below:

[0194] Table 3.4.2.2.b Virus infection titer after purification

[0195]

[0196]

[0197] The novel chimpanzee adenovirus has the ability to produce neutralizing antibodies.

[0198] Neutralizing antibodies can specifically bind to viruses and prevent them from infecting host cells; high levels of antibodies usually indicate immune protection. In vaccine development, the production of neutralizing antibodies is a key indicator of vaccine effectiveness. Detecting the level of neutralizing antibodies in rabbits after injection of chimpanzee adenovirus can assess whether the newly constructed chimpanzee adenovirus vector can elicit a sufficiently strong immune response, thus providing a basis for subsequent clinical trials.

[0199] This experiment used Adsimian-Δ24-EGFP to detect the titer of the neutralizing antibody, such as... Figure 3 As shown, the two rabbits reached 32,000 IU / mL after the fourth immunization and up to 256,000 IU / mL after the fifth immunization. This indicates that the chimpanzee adenovirus constructed in this application can produce high-titer neutralizing antibodies in animals after multiple immunizations. High antibody levels usually indicate immune protection. This experiment provides an experimental basis for subsequent vaccine development.

[0200] Table 3.4.3. Neutralization titers in experimental rabbits after the last three immunizations.

[0201]

[0202] Chimpanzee adenovirus carrying IL-21 inhibits the migration of colorectal cancer cells.

[0203] We used a cell scratch assay to observe the migration behavior of colorectal cancer cells after infection with Adsimian-Δ24-IL21 and to evaluate the effect of chimpanzee adenovirus carrying IL-21 on the migration ability of colorectal cancer cells.

[0204] Cell scratch assays revealed that the constructed chimpanzee oncolytic adenovirus inhibited the migration of colorectal cancer cells. Colorectal cancer cells SW480 and MC38 infected with the recombinant virus Adsimian-Δ24-IL21 showed significantly weaker migration ability at 24 hours compared to cells treated with PBS. Figure 4 (a / b) indicates that the chimpanzee adenovirus Adsimian-Δ24-IL21 has the ability to inhibit the migration of colorectal cancer cells.

[0205] To further investigate the factors influencing the migration ability of chimpanzee adenovirus on colorectal cancer cells, the effects of Adsimian-Δ24-IL21 treatment on cell migration and EMT process were analyzed by detecting the expression of epithelial-mesenchymal transition (EMT) markers at the mRNA level. Figure 5 As shown, the experimental results indicate that after treatment with chimpanzee adenovirus Adsimian-Δ24-IL21, the expression of the epithelial marker E-cadherin in human colorectal cancer cell line SW480 was significantly upregulated (p<0.01), while the expression of the stromal markers N-cadherin and Vimentin showed a significant downregulation trend (p<0.01). This change at the molecular level indicates that chimpanzee oncolytic adenovirus carrying IL-21 effectively reversed the EMT process. In the mouse colorectal cancer cell line MC38, after treatment with chimpanzee adenovirus, the expression of the epithelial marker E-cadherin was significantly upregulated (p<0.001), while the expression of the stromal markers N-cadherin and Vimentin showed a significant downregulation trend. Figure 5 c in the text.

[0206] Adsimian-Δ24-EGFP has a stronger ability to kill colon cancer cells (e.g., ... Figure 6-7 )

[0207] This experiment investigated the cytotoxic effect of the inserted E1A(Δ24) fragment on colorectal cancer cells by comparing the replication abilities of purified chimpanzee adenoviruses Adsimian-EGFP and Adsimian-Δ24-EGFP in SW480 and SW620 colorectal cancer cells, as well as their time- and dose-dependent replication. An MTT assay was designed, using four different doses of Adsimian-EGFP and Adsimian-Δ24-EGFP (1 MOI, 5 MOI, 10 MOI, and 20 MOI) at four time points: 24h, 48h, 72h, and 96h.

[0208] MTT assay results showed that both non-replicating Adsimian-EGFP and replicating Adsimian-Δ24-EGFP exhibited dose-dependent enhancement of their cytotoxic effects on colorectal cancer cells SW480 and SW620. Furthermore, Adsimian-Δ24-EGFP had a more significant effect on the viability of colorectal cancer cells compared to Adsimian-EGFP. This indicates that E1A(Δ24) enhances the cytotoxicity of chimpanzee adenovirus against cancer cells. Under the same MOI conditions after infection with Adsimian-Δ24-EGFP, the antitumor activity of SW480 and SW620 cells decreased over time.

[0209] Furthermore, the effects of Adsimian-EGFP and Adsimian-Δ24-EGFP on the formation of SW480 and SW620 cell clones were observed by crystal violet staining. It was found that the Adsimian-Δ24-EGFP treatment group significantly inhibited the generation of SW480 and SW620 cells compared with the Adsimian-EGFP treatment group, further indicating that the reproducible Adsimian-Δ24-EGFP with inserted E1A(Δ24) fragment has a stronger ability to inhibit the proliferation of colorectal cancer cells.

[0210] Recombinant chimpanzee oncolytic adenovirus has targeted killing ability against colon cancer cells (e.g. Figure 8-10 )

[0211] To investigate whether the chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 carrying IL-21 has the ability to inhibit the proliferation of colon cancer cells and induce colon cancer cell death at the cellular level, different MOI viral infection doses were set up to infect human colorectal cancer cells SW480, HCT116, SW620 and normal human lung epithelial cells BEAS-2B, as well as mouse colorectal cancer cells MC38, CT26WT and mouse embryonic fibroblast cells NIH / 3T3, and comparisons were set at different time points of 24h, 48h, 72h and 96h.

[0212] The results are as follows Figure 8As shown, during this process, the mortality rate of colon cancer cells increased and cell viability decreased, and this phenomenon became more pronounced over time, with the killing effect being directly proportional to viral infection. However, there was no significant difference between chimpanzee oncolytic adenovirus carrying IL-21 and the control virus Adsimian-Δ24-EGFP, indicating that other conditions are required for IL-21 to exert its significant anti-tumor effect, which needs further investigation. Furthermore, compared to human colorectal cancer cells SW480, HCT116, and SW620, Adsimian-Δ24-hIL21 showed the lowest cell survival rate (approximately 50%) in SW480 cells after 96 hours of infection, demonstrating the best infection effect compared to the other two cell types. This suggests that Adsimian-Δ24-hIL21 may have a more effective inhibitory effect on SW480 cells. In a comparison of MC38 and CT26WT murine colorectal cancer cells, Adsimian-Δ24-mIL21 was found to have a stronger inhibitory effect on the growth of MC38 cells. After 96 hours of infection with a 20 MOI viral dose, MC38 cells showed the lowest cell survival rate, approximately 60%. Therefore, MC38 cells will be the preferred choice for in vivo treatment of colorectal cancer tumors.

[0213] Progeny replication capacity of recombinant chimpanzee oncolytic adenovirus in different cells

[0214] Colorectal cancer cells SW480, SW620, HCT116, MC38, and CT26WT, as well as normal human lung epithelial cells BEAS-2B and mouse embryonic fibroblasts NIH / 3T3, were infected with a viral dose of 2 MOI. Virus and supernatant were harvested at four time points: 24h, 48h, 72h, and 96h. Viral genome was extracted, and viral titer was determined using an absolute quantification method. Results are as follows: Figure 11 As shown.

[0215] The ability of colorectal cancer cells to produce progeny viruses increased with culture time, but showed no significant increase after 72 hours, possibly because the cell number and viral production capacity in the culture dish had reached their peak. However, viral progeny replication in normal cells (BEAS-2B, NIH / 3T3) was weak, with almost no proliferation. Furthermore, human colorectal cancer cells showed the highest expression level and best viral production in SW480 cells, followed by SW620, both peaking within 72 hours. HCT116 cells exhibited the worst replication among the three types of colorectal cancer cells. Subsequent experiments selected SW480 cells as the target for human colorectal cancer cell research. In mouse colorectal cancer cells, the recombinant virus showed higher viral production capacity in MC38 cells compared to CT26WT. These results indicate that the novel chimpanzee adenovirus constructed in this study has a high replication capacity in colorectal cancer cells without affecting normal cells.

[0216] Co-culturing chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 with PBMCs enhances its ability to kill colorectal cancer cells. IL-21 is an immune gene that can inhibit tumor cell growth and has potential in cancer treatment. However, in previous experiments comparing the IL-21-carrying chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 with the control virus Adsimian-Δ24-EGFP, the results showed almost no difference in the killing power of the IL-21-carrying virus against cancer cells. This may be because the expression and function of IL-21 may require specific immune environmental conditions to be manifested.

[0217] To further investigate the immunomodulatory effects of the chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 carrying IL-21, human peripheral monocytes (PBMCs) were extracted from blood and co-cultured with SW480 tumor cells. Figure 12 As shown, the results indicated that the cytotoxic effect on tumor cells was significantly different only when PBMC was added compared to when PBMC was not added (p<0.01), demonstrating the crucial role of PBMC in immune activation. At 48 h, the antitumor activity of Adsimian-Δ24-IL21 was significantly enhanced (p<0.001), with a significant effect on killing SW480 tumor cells. This suggests that co-culturing Adsimian-Δ24-IL21 and PBMC exhibits a stronger antitumor effect.

[0218] 1.4 Conclusion

[0219] This application investigates the virulence of the recombinant chimpanzee virus Adsimian-Δ24-IL21 expressing the IL-21 gene and its inhibitory effect on colorectal cancer cells.

[0220] (1) In this experiment, the constructed chimpanzee adenovirus was transfected into HEK293 cells using transfection reagents to package novel chimpanzee adenoviruses including Adsimian-EGFP, Adsimian-Δ24-EGFP, Adsimian-Δ24-mIL21, and Adsimian-Δ24-hIL21. After large-scale amplification and purification, the integrity of gene expression of the purified virus was identified and analyzed by PCR, enzyme-linked immunosorbent assay (ELISA) and Western blotting.

[0221] (2) In the cell scratch assay, chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 can inhibit the migration of colorectal cancer cells SW480 and MC38. Furthermore, by detecting the expression of EMT pathway-related genes at the mRNA level, chimpanzee adenovirus Adsimian-Δ24-IL21 has the effect of reversing the EMT process.

[0222] (3) Observation by MTT and crystal violet staining revealed that compared with the non-replicating chimpanzee adenovirus Adsimian-EGFP, the replicating chimpanzee adenovirus Adsimian-Δ24-EGFP had a stronger killing ability against colorectal cancer cells SW480 and SW620, indicating that the replicating Adsimian-Δ24-EGFP with inserted E1A(Δ24) fragment has a stronger ability to inhibit the proliferation of colorectal cancer cells.

[0223] (4) Progeny replication verified that chimpanzee oncolytic adenoviruses Adsimian-Δ24-IL21 and Adsimian-Δ24-EGFP can be amplified in large quantities in human colorectal cancer cells SW480, SW620, and HCT116, and mouse colorectal cancer cells MC38 and CT26WT, with no significant difference between the two, and cannot be replicated in normal human lung epithelial cells BEAS-2B and mouse embryonic fibroblast cells NIH / 3T3.

[0224] (5) Human colorectal cancer cells SW480 infected with chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 were co-cultured with human peripheral monocytes (PBMCs), and the activity of SW480 cells was measured, further verifying that the recombinant virus Adsimian-Δ24-IL21 has a significant killing effect on colorectal cancer cells. This confirms that chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 has a significant effect on killing colorectal cancer cells.

[0225] 2. In vivo study on the inhibition of colorectal cancer xenograft growth in MC38 mice by chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21.

[0226] This study aimed to explore the efficacy of recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 in treating colorectal cancer in MC38 mice through in vivo experiments. First, a xenograft mouse model was established by subcutaneously inoculating MC38 cells into four-week-old C57BL / 6 mice, followed by intratumoral injection of recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21. By dynamically monitoring tumor growth curves, histopathological analysis, and flow cytometry analysis of multiple organ immune cell subsets (including spleen single cells, tumor tissue, and draining lymph nodes), the study sought to investigate whether chimpanzee adenovirus Adsimian-Δ24-IL21 could effectively deliver IL-21 and achieve a therapeutic effect in the MC38 colorectal cancer mouse model; and whether chimpanzee adenovirus Adsimian-Δ24-IL21 could enhance its anti-tumor effect by remodeling the tumor immune microenvironment. The results will provide a reference for the application of novel chimpanzee oncolytic virus-cytokine combination therapies.

[0227] 2.1 Experimental Materials

[0228] 2.11 Establishment of a mouse MC38 cell line subcutaneous tumor animal model

[0229] 1) MC38 cells were subcutaneously injected into four-week-old C57BL / 6 mice. Ten days later, when the tumor grew to 80–120 mm, 3 When it was time, they were divided into 3 groups of 8 each.

[0230] 2) Mice were grouped according to the table below and given intratumoral injections. The first day of injection was designated as day 1. Each injection contained 1.0 × 10⁻⁶ mg / L. 9 PFU, injected every other day for a total of 2 doses, for a total of 2.0 × 10⁻⁶. 9 PFU was collected from various organs and subcutaneous tumors of mice on day 15 after treatment and on day 30, the last day of observation, for further analysis.

[0231] Table 4.3.1.1 Mouse Grouping

[0232]

[0233] 2.12 Sample Collection

[0234] (1) Collect peripheral blood from mice

[0235] After catching the mouse, gently press its left eyelid until the eyeball protrudes. Immediately remove the eyeball with forceps and collect the blood in a 1.5 mL EP tube. Then, allow the serum to stand at room temperature for at least 30 minutes before centrifugation. Centrifuge at 3000 rpm for 20 minutes, then place the sample in the centrifuge. After centrifugation, collect the supernatant and aliquot it for storage at -80°C. After blood collection, mice were euthanized by cervical spinal cord dislocation, soaked in 75% alcohol, and various tissues were collected under sterile conditions.

[0236] (2) Collect subcutaneous tumors from mice

[0237] After euthanizing the mice, they were fixed on a dissection table, and the skin on their sides was carefully incised with scissors to separate them from the skin and subcutaneous tumors to prevent infection. After removing the subcutaneous tumors, the mice were rinsed twice with PBS and divided into two groups: one group was placed in fixative, and the other group was used for flow cytometry analysis.

[0238] (3) Collect organs such as the spleen of the mouse.

[0239] The mouse's skin was cut along the center line, and the skin was carefully lifted upwards. After the skin was cut, a new set of sterile dissection instruments was used to dissect the mouse, and the spleen was removed with forceps. The spleen was rinsed twice with PBS, placed in a culture dish, and kept on ice for flow cytometry experiments.

[0240] (4) Collect mouse lymph nodes

[0241] The skin of the mice was completely cut open to expose the groin, armpit and other areas. The groin and axillary lymph nodes on the side inoculated with the subcutaneous tumor were carefully collected and placed in a culture dish containing appropriate culture medium and placed on ice to prepare for subsequent flow cytometry experiments.

[0242] 2.13 Preparation of Single-Cell Suspension

[0243] The components were pulverized and screened to prepare single-cell suspensions for flow cytometry analysis. The preparation process for single-cell suspensions of each tissue is as follows:

[0244] 2.14 Spleen cell preparation

[0245] (1) Crush the spleen obtained in the above steps, shake well, and place it in a 50mL centrifuge tube. Set the centrifuge to 1600rpm and centrifuge for 8min.

[0246] (2) After centrifugation, discard the supernatant, add 5 mL of red blood cell lysis buffer to each test tube, and react at room temperature for 5 min.

[0247] (3) Add a large amount of PBS to stop the red lysis, centrifuge the sample at 1600 rpm for 5 min, and remove the supernatant.

[0248] (4) Resuspend the precipitate with PBS, pass it through a 200-mesh nylon filter, dispense it, centrifuge and discard the supernatant, add 1 mL of PBS to resuspend it, and obtain a mouse spleen single cell suspension.

[0249] (5) Take 10 μL of mouse spleen single cell suspension and 190 μL of PBS for counting, to be carried out in subsequent experiments.

[0250] 2.15 Lymph node cell preparation

[0251] (1) Rinse the collected PBS, grind it into a homogenate with surgical scissors, add 1 mL of PBS to wash, and stir well.

[0252] (2) Sieve the tissue through a 200-mesh nylon filter, place it in a centrifuge tube, set the speed to 3000 rpm, and run for 5 minutes.

[0253] (3) After centrifugation, discard the supernatant, resuspend the turbid liquid with PBS, and perform a second centrifugation.

[0254] (4) Discard the supernatant, resuspend it with 500 μL of PBS, and repackage it into 3 small tubes for the next step of the experiment.

[0255] 2.16 Preparation of single-cell suspension for subcutaneous tumors

[0256] (1) The mice were euthanized, disinfected with 75% ethanol, and the subcutaneous tumors were removed. Adipose tissue, necrotic tumor tissue and mouse skin were removed. The mice were then transferred to a 6cm 10-dish container (containing 2mL RPMI-1640 basic culture medium) and completely cut into 1mm pieces with sterile surgical scissors. 3 size.

[0257] (2) Transfer the above tissue suspension to a 15 mL centrifuge tube, add 8 mL of RPMI-1640 basic culture medium, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend in 10 mL of the prepared tissue digestion solution, transfer to a new 50 mL sterile centrifuge tube and place in a 37℃ constant temperature shaker, shake at 220 rpm for 45 min.

[0258] (3) After incubation, the digested tissue was filtered through a 200-mesh nylon filter and 15 mL of sterile centrifuge tube was collected to obtain a single-cell suspension. After centrifugation, the supernatant was removed and the cells were washed once with 10 mL of RPMI-1640 cell culture medium containing 10% FBS. The cells were then resuspended in 5 mL of cell culture medium.

[0259] (4) At room temperature, add 3 mL of lymphocyte separation medium to a 15 mL sterile centrifuge tube, and then carefully overlap the single cell suspension obtained in (3) onto the lymphocyte separation medium.

[0260] (5) Centrifuge at 2000 rpm for 20 min, then reduce the speed to 0.

[0261] (6) The white blood cells (mouse TILs) were carefully and gently resuspended by blowing and washing before subsequent activation tests were performed; other interstitial cells were deposited at the bottom of the tube.

[0262] 2.17 Flow cytometry analysis of the proportion of immune cells in different mouse tissues

[0263] (1) Divide the antibody into 4 portions. According to the manufacturer's instructions, add 1 μL of antibody to each sample. The staining method for each tube is as follows:

[0264] Table 4.3.3 Flow cytometry staining protocols

[0265]

[0266] (2) Add the corresponding antibody to each of the single staining tube A, single staining tube B, single staining tube C and the test tube to be tested, stir well, and place in a dark place for 30 minutes.

[0267] (3) After the culture is completed, set the centrifuge to 3000 rpm and centrifuge the sample for 5 min. After centrifugation, discard the supernatant and adjust the volume to 1 mL with PBS. Resuspend the cells by pipetting and repeat twice.

[0268] (4) Place on ice and protect from light. Resuspend the cells using a pipette and then send them to a flow cytometer for analysis.

[0269] 2.18 Data Analysis

[0270] Statistical analysis was performed using CytExpert and GraphPad Prism 9.0. Results are expressed as mean ± SD. Intergroup comparisons were performed using t-tests, one-way ANOVA, two-way ANOVA, and survival analysis. A p-value < 0.05 was considered statistically significant.

[0271] 2.2 Experimental Results

[0272] 2.21 Tumor volume and body weight curves in mice

[0273] Day 0 was the day of treatment. Mouse body weight and tumor volume were measured every other day to dynamically observe changes in tumor volume and mouse body weight after drug injection. Based on the development of the mouse tumor, this application sets an upper limit of 2000 mm² for tumor volume observation. 3 The observation period was set at 30 days.

[0274] Observations revealed that, compared with the PBS group and the Adsimian-Δ24-EGFP control virus group, the tumor growth in mice treated with the recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 gene was significantly slowed, and the tumor volume was relatively smaller. Figure 13 In addition, Adsimian-Δ24-mIL21 gene therapy can significantly reduce tumor volume and even show regression, see [reference needed]. Figure 13 Furthermore, throughout the observation and measurement period, there were no significant differences in the activity level and body weight of the xenograft mice (p>0.05). Figure 13 In summary, Adsimian-Δ24-mIL21 effectively inhibited the growth of colon cancer tumors in mice and significantly suppressed tumor development.

[0275] 2.22 Detection of toxicity of chimpanzee oncolytic adenovirus in mice

[0276] This experiment aimed to evaluate the safety of injecting PBS, Adsimian-Δ24-EGFP, and Adsimian-Δ24-mIL21 into mice. Fifteen days after injection, organs such as the heart, liver, spleen, lungs, and kidneys were harvested for observation and HE staining. Figure 14The study found that the Adsimian-Δ24-EGFP and Adsimian-Δ24-mIL21 genes did not cause significant damage to the heart, liver, spleen, lungs, kidneys, and other organs of mice, indicating that the chimpanzee oncolytic adenovirus has low toxicity to mouse organs and has a certain degree of safety.

[0277] 2.23 Chimpanzee oncolytic adenovirus carrying IL-21 enhances anti-tumor immunity.

[0278] like Figure 15 As shown, the changes in the proportion of immune cells in spleen tissue: the tumor intervention model injected with chimpanzee oncolytic adenovirus showed a significant therapeutic effect on day 14: mice in the PBS control group showed rapid tumor growth (median volume of 1500 mm²) on day 14 after intervention. 3 The Adsimian-Δ24-EGFP empty vector group and the Adsimian-Δ24-mIL21 treatment group both showed significant tumor regression (p<0.01). To analyze early immune status, on day 15 of treatment, three tumor-bearing mice from each of the three groups were randomly selected to prepare spleen tissue single-cell suspensions.

[0279] Flow cytometry analysis revealed that the Adsimian-Δ24-EGFP group had significantly higher CD4 counts than the PBS group. + The percentage of cells increased, ranging from 23.03% to 38.88%, CD8 + Cell percentage increased from 23.02% to 28.97%; while the Adsimian-Δ24-mIL21 group showed CD4+. + The decreasing cell percentage (0.48% vs 16.94% in the PBS group, p = 0.0055) may be related to IL-21-induced Th cell subset redistribution or regulatory T cell exhaustion.

[106] This can be verified through FoxP3 / CTLA-4 co-staining experiments.

[0280] At the treatment endpoint (day 30), flow cytometry analysis was performed on individuals in the Adsimian-Δ24-mIL21 group who experienced complete tumor regression. The results showed that CD3+ cells were present in spleen cells. + The proportion of cells was 1.68-fold higher than that of the control group (AdsimianΔ24-EGFP) (37.00% ± 2.00% vs 22.00% ± 2.00%, p < 0.05), of which CD8+... + Cell percentage reached 35.00% ± 5.00% (a 1.42-fold increase compared to day 14), CD4 + Cell recovery to 44.00% ± 2.00% indicates that IL-21 may achieve a long-term anti-tumor effect by dynamically regulating the balance of T cell subsets, with early preferential expansion of CD8. +Effector cells rapidly control tumor burden and subsequently reconstruct CD4. + Help cells maintain immune memory.

[0281] 2.24 Changes in the proportion of immune cells in tumor tissue

[0282] like Figure 16 As shown, in this experiment, primary cells of tumor tissue from tumor-bearing mice (n=3) were isolated by enzymatic digestion and detected by multicolor flow cytometry.

[0283] The results showed that, compared with the PBS group (CD3) + Compared to the Adsimian-Δ24-EGFP group (cell percentage 26.00% ± 2.00%), the CD3 concentration in the Adsimian-Δ24-EGFP group was significantly lower. + Cell infiltration was significantly increased, approximately 1.76 times that of the control group (PBS group) (46.00% ± 2.00%, p < 0.0001, one-way ANOVA), while the Adsimian-Δ24-mIL21 treatment group further increased it to 2.00 times (52.00% ± 2.00%, p < 0.0001), indicating that the viral vector backbone itself has immune-activating properties, and the co-expression of the IL-21 gene can produce a synergistic effect.

[0284] In the analysis of immune cell subsets, CD8+ in the tumor of the Adsimian-Δ24-EGFP group + The proportion of cells in the Adsimian-Δ24-mIL21 group increased 1.61-fold compared to the PBS group (42.00% ± 5.00% vs 26.00% ± 5.00%, p < 0.05), while the increase in the Adsimian-Δ24-mIL21 group reached 2.11-fold (55.00% ± 2.00%, p < 0.01). This indicates that IL-21 not only enhances overall T cell infiltration but also specifically promotes CD8 cell growth. + Cell enrichment in the tumor microenvironment. This effect may be related to the activation of the IL-21-regulated CXCR3 / CXCL10 chemokine axis.

[108] Or enhanced T cell proliferation signal

[109] This is relevant and requires spatial transcriptome analysis.

[110] Further verification was achieved through in vitro migration experiments.

[0285] 2.25 Changes in the proportion of immune cells in draining lymph nodes

[0286] To investigate the immunoregulatory role of Adsimian-Δ24-mIL21 in the tumor microenvironment, this application conducted a longitudinal observation using a tumor-bearing mouse model (n=8). At day 30 post-intervention, inguinal lymph node immune cells were analyzed in surviving individuals (IL-21 group n=3, EGFP group n=3). Single lymph node cells were collected and prepared, and immunophenotypic analysis was performed using multicolor flow cytometry (Note: All PBS control group members died due to tumor progression and were therefore not included in the final analysis).

[0287] like Figure 17 As shown, based on the results of flow cytometry analysis, compared with the Adsimian-Δ24-EGFP group (30.00% ± 5.00%), the CD3 concentration in the Adsimian-Δ24-mIL21 treatment group was significantly higher. + The cell population percentage significantly increased to 65.00% ± 5.00% (p < 0.0001); compared to the EGFP group (30.00% ± 5.00%), CD4... + The proportion of cells increased (EGFP group 30.00% ± 5.00% vs IL-21 group 45.00% ± 5.00%), and CD8... + The cell percentage significantly increased to 45.00% ± 5.00% (p < 0.01). This result indicates that IL-21 may specifically amplify CD4+. + CD8 + Cells enhance the anti-tumor immune response.

[0288] 2.26 Changes in IL-21 levels in mouse serum

[0289] This experiment used quantitative enzyme-linked immunosorbent assay (ELISA) to evaluate the delivery efficiency and functional expression of IL-21 mediated by a novel chimpanzee adenovirus vector in C57BL / 6 mice.

[0290] like Figure 18 As shown in the experimental data, compared with the negative control group (PBS: 202.3±15.6 pg / mL; n=5) and the empty vector control group (EGFP: 498.7±42.1 pg / mL; n=5), the serum IL-21 concentration in the target treatment group was significantly upregulated (2987.4±213.5 pg / mL; n=5); one-way ANOVA, p<0.0001), with differences reaching approximately 15-fold (PBS vs Adsimian-Δ24-mIL21) and 6-fold (EGFP vs Adsimian-Δ24-mIL21). This experiment demonstrates that the chimpanzee adenovirus vector constructed in this application mediates high delivery efficiency of the immune gene IL-21, and the combined analysis with flow cytometry can further prove the anti-tumor effect of IL-21.

[0291] 2.27 Experiment Summary:

[0292] (1) Chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 has significant anti-tumor activity. After intratumoral injection of recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21, the growth of subcutaneously inoculated tumors in mice was significantly inhibited, and the tumors of some mice (3 / 8) completely regressed. Its efficacy was significantly better than that of the empty vector group and the PBS control group, and it had no significant effect on the body weight of mice.

[0293] (2) Histopathological analysis showed that chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 did not harm mouse organs. Hematoxylin-eosin staining (HE staining) was used to analyze the staining of the heart, liver, spleen, lungs and kidneys of mice in three different treatment groups: PBS group, Adsimian-Δ24-EGFP group and Adsimian-Δ24-mIL21 group. The results showed that chimpanzee oncolytic adenovirus did not cause significant toxicity to mouse organs.

[0294] (3) Flow cytometry analysis revealed the characteristics of immune microenvironment remodeling after chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 treatment. Firstly, CD3+ levels in tumor tissue of the IL-21 treatment group were significantly increased after Adsimian-Δ24-mIL21 viral therapy. + CD8 + The significant increase in cells indicates that Adsimian-Δ24-mIL21 exerts its anti-tumor effect by activating cellular immunity and regulating the immune microenvironment. Secondly, CD8+ cells were observed in the spleen and draining lymph nodes. + The increased proportion suggests that local IL-21 expression may induce a systemic immune response.

[0295] (4) Chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 effectively delivers IL-21 and exerts its anti-tumor function. In an enzyme-linked immunosorbent assay (ELISA) experiment, this application found that the IL-21 content in the serum of mice injected with Adsimian-Δ24-mIL21 was 15 times that of the PBS group. Further analysis with flow cytometry results confirmed that the high level of IL-21 expression significantly promoted CD3. + Cell proliferation indicates that Adsimian-Δ24-mIL21 regulates T cell activation.

[0296] Notably, the tumor volume in the empty vector group Adsimian-Δ24-EGFP, which did not express IL-21, was still reduced by approximately 25% compared to the PBS group. This suggests that the chimpanzee adenovirus vector itself may possess a certain oncolytic effect, and the tumor-specific replication caused by Δ24 deficiency may enhance immunogenicity by directly killing tumor cells or releasing tumor-associated antigens. However, the degree of immune cell infiltration in the Adsimian-Δ24-EGFP empty vector group was significantly lower than that in the Adsimian-Δ24-mIL21 treatment group, further demonstrating that IL-21 expression can significantly enhance the anti-tumor immune effect of chimpanzee adenovirus.

[0297] This application confirms that the Adsimian vector system can achieve efficient delivery and functional expression of IL-21. This result provides an experimental basis for the later preclinical application and translation of chimpanzee adenovirus expressing IL-21, and also provides a reference for related immunotherapy strategies such as tumor immunotherapy drugs and drugs for the intervention of autoimmune diseases.

[0298] Finally, it should be noted that the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, It comprises a recombinant chimpanzee oncolytic adenovirus and a pharmaceutically acceptable vector, said virus comprising: The chimpanzee adenovirus backbone of serotype 25, with the E3 region missing; Δ24 mutation in the E1A region: 24bp deletion of the E1A-CR2 domain; The IL-21 gene is regulated by the CMV promoter.

2. The pharmaceutical composition according to claim 1, characterized in that, The recombinant chimpanzee oncolytic adenovirus is Adsimian-Δ24-IL21, a chimpanzee oncolytic adenovirus carrying the IL-21 gene; the viral titer of the composition is 1×10⁻⁶. 9 Up to 1×10 12 It has a TCID of 50 / mL and is less than 10% toxic to normal cells.

3. The pharmaceutical composition according to claim 2, characterized in that, The IL-21 gene is human IL-21 (SEQ ID NO:1) or mouse IL-21 (SEQ ID NO:2), and the composition is suitable for inhibiting the activity of colorectal cancer cells in vitro, with IC50 ≤ 5 MOI.

4. A method for detecting viral function in the pharmaceutical composition of claim 1, characterized in that, include: Step 1) Verify the integrity of the viral genome by PCR using primer pair CMV-F (SEQ ID NO:3) and SV40-R (SEQ ID NO:4); Step 2) Detect the secretion level of IL-21 in the supernatant of infected cells by ELISA, with a sensitivity ≥10 pg / mL; Step 3) Determine the viral titer using the TCID50 method. The calculation formula is: Titer TCID50 / mL=10^(A+B×C), where A is the number of positive wells at the highest dilution, B is the dilution factor, and C is the proportion of positive wells.

5. The application of a drug that inhibits the migration of colorectal cancer cells, characterized in that, The drug is the composition according to claims 1-3, which achieves a cancer cell migration inhibition rate of ≥40% by downregulating EMT markers (N-cadherin, Vimentin) and upregulating E-cadherin.

6. The application of a drug that enhances anti-tumor immune killing, characterized in that, The drug is the composition according to claims 1-3. When co-cultured with peripheral blood mononuclear cells (PBMCs), it increases the killing rate of cancer cells by more than 50% and increases the proportion of CD8+ T cells by ≥3 times, with a significance level of p-value less than 0.

001.

7. A method for evaluating the oncolytic effect of the pharmaceutical composition according to claims 1-3, characterized in that, include: The half-maximal inhibitory concentration (IC50) against colorectal cancer cells SW480 and MC38 was determined by MTT assay. The inhibition rate of cancer cell colony formation was assessed by crystal violet staining, and the inhibition rate was not less than 70%. The viral progeny replication capacity was detected by quantitative PCR, with an amplification factor of no less than 100-fold.

8. A method for detecting the stability of the pharmaceutical composition of claim 1, characterized in that, include: The survival rate of the virus in a simulated human immune environment was determined by detecting the titer of neutralizing antibodies. The viral titer reduction rate was verified to be no more than 20% through accelerated stability experiments, which were performed under the condition of storage at 37 degrees Celsius for 7 days.

9. A kit for the treatment of colorectal cancer, characterized in that, The invention comprises the pharmaceutical composition of claims 1-3, and a reagent kit for detecting viral function; the reagent kit includes PCR primer pairs and an enzyme-linked immunosorbent assay kit.

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