Recombinant chimpanzee oncolytic adenovirus Adsimian-delta24-IL21 as well as construction method and application thereof

Abstract

The invention discloses a recombinant chimpanzee oncolytic adenovirus Adsimian-delta24-IL21 and a construction method and application thereof, the vector is based on serotype 25 chimpanzee adenovirus, the immunogenicity is reduced by deleting an E3 region, and the integrin targeting is enhanced by inserting an RGD peptide fragment; a skeleton plasmid pAdsimian-deltaE3 is in seamless cloning connection with a shuttle plasmid pSRK17-delta24-CMV carrying delta24 mutation E1A, a recombinant oncolytic adenovirus Adsimian-delta24-IL21 and a control virus Adsimian-delta24-EGFP are constructed, the delta24 mutation enables the virus specificity to be copied in Rb defect tumor cells, the IL-21 gene remodels a tumor immune microenvironment by activating CD3 + / CD8 + T cells, and the tumor immune microenvironment of the Rb defect tumor cells is improved. The invention provides an efficient carrier and a method for oncolytic-immune combined treatment of colorectal cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to a chimpanzee oncolytic adenovirus and its method and application, more specifically, to a recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 and its construction method and application, and belongs to the technical field of genetic engineering and viral vector, and is especially suitable for the fields of vaccine development, gene therapy and immunotherapy. BACKGROUND

[0002] Traditional treatment of colorectal cancer is facing a bottleneck due to drug resistance and high recurrence rate, and oncolytic viruses have potential tumor-specific lysis, but human adenovirus is easily affected by pre-existing neutralizing antibodies, and the therapeutic effect is limited. Chimpanzee adenovirus can avoid pre-existing immunity due to significant differences in serotypes with human adenovirus, but the construction of the vector needs to optimize tumor targeting and immune regulation functions.

[0003] Interleukin-21 (IL-21) can activate CD8+ T cells and NK cells, but its short half-life and insufficient local concentration restrict the therapeutic effect of systemic administration. Existing oncolytic virus vectors (such as human adenovirus type 5) are difficult to efficiently deliver IL-21 to tumor sites due to pre-existing immunity and insufficient targeting, and it is difficult to achieve efficient delivery and synergistic effect of IL-21. Chimpanzee adenovirus can avoid pre-existing antibodies due to significant differences in serotypes with human adenovirus, but the construction of the vector and the efficiency of exogenous gene expression have not been optimized. Therefore, how to break through the limitations of traditional oncolytic viruses and combine IL-21 immunity is a trend in the research and development of genetic engineering and viral vector technology. SUMMARY

[0004] The present application aims to provide a recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 and its construction method, which has the technical characteristics of significantly reducing the interference of pre-existing neutralizing antibodies in the human population, improving the in vivo delivery efficiency of oncolytic viruses, targeting the infection of integrin-high-expressing tumor cells, reducing off-target toxicity, enabling the virus to replicate only in Rb-deficient tumor cells, and high safety of normal cells.

[0005] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme:

[0006] The present application is a recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21, which is based on chimpanzee adenovirus of serotype 25, and its genome accession number is GenBank AC_000011.1. The E3 region in the genome is deleted, and an integrin-targeting RGD peptide segment is inserted. The virus carries a human IL-21 gene or a murine IL-21 gene, and the expression is regulated by a CMV promoter. The virus contains a Δ24 mutant E1A domain, which is driven by a human adenovirus type 5 E1A promoter and specifically replicates in tumor cells with defective Rb pathway.

[0007] Preferably, the virus is constructed by the backbone plasmid pAdsimian-ΔE3 and the shuttle plasmid pSRK17-Δ24-CMV; the backbone plasmid pAdsimian-ΔE3 is deleted of the E3 region, and retains the partial immune regulatory genes 12.5K and ADP in the E3 region; the shuttle plasmid pSRK17-Δ24-CMV comprises an adenovirus packaging signal, a kanamycin resistance gene, a Δ24 mutant E1A domain, and a multi-cloning site Spe I, Nhe I and Hind III.

[0008] Preferably, the virus remodels the immune microenvironment by up-regulating the proportion of CD3+ and CD8+ T cells in the tumor microenvironment, inhibits the migration of colorectal cancer cells, and reverses the process of epithelial-mesenchymal transition.

[0009] The application also provides a method for constructing the recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21.

[0010] Step 1) Synthesizing a full genome of chimpanzee adenovirus of serotype 25, which is divided into four fragments Simian25-1 to Simian25-4, and then connecting the four fragments into pAdsimian-E3 by high-fidelity PCR amplification and the seamless cloning technology.

[0011] Step 2) Deleting the E3 region by overlapping PCR to construct the backbone plasmid pAdsimian-ΔE3.

[0012] Step 3) Constructing the shuttle plasmid pSRK17-Δ24-CMV, and inserting the Δ24 mutant E1A domain and the IL-21 gene regulated by the CMV promoter.

[0013] Step 4) After the backbone plasmid pAdsimian-ΔE3 and the shuttle plasmid pSRK17-Δ24-CMV are digested by Pac I and Xba I, connecting them by the seamless cloning technology to obtain the recombinant adenovirus plasmid pAdsimian-Δ24-IL21.

[0014] Step 5) Transfecting HEK293 cells with the recombinant plasmid to package the chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 carrying the IL-21 gene.

[0015] Preferably, the IL-21 gene is amplified from a storage plasmid by PCR, and then inserted downstream of the CMV promoter in the shuttle plasmid after digestion by Cla I.

[0016] The application of a recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 in the preparation of a colorectal cancer treatment drug, the virus inhibits tumor growth through direct oncolysis and the dual mechanism of activating CD3+ and CD8+ T cells in the tumor microenvironment, and has no significant toxicity to human normal lung epithelial cells BEAS-2B and mouse embryonic fibroblasts NIH / 3T3.

[0017] Preferably, the virus enhances the killing ability of colorectal cancer cells SW480 through co-culture with peripheral blood mononuclear cells, and significantly inhibits tumor growth in the MC38 tumor-bearing mouse model without causing body weight loss or organ toxicity.

[0018] The application provides a method for verifying a recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21, which comprises the following steps:

[0019] Step 1) Verify the characteristic bands of pAdsimian-Δ24-IL21 by Hind III enzyme digestion, including 20744 bp, 8672 bp and 4736 bp;

[0020] Step 2) Detect the proportion of CD3+ and CD8+ T cells in tumor tissues by flow cytometry;

[0021] Step 3) Verify the inhibition of colorectal cancer cell migration and the reversal of epithelial-mesenchymal transition marker expression by cell scratch test and qRT-PCR.

[0022] Beneficial effects: The application provides an efficient vector and method for the research and development of oncolytic-immune combined treatment drugs for colorectal cancer, and specifically comprises the following aspects:

[0023] 1. Avoiding pre-existing immunity: precise tumor targeting: inserting an RGD peptide segment enhances the targeted infection of tumor cells with high integrin expression and reduces off-target toxicity.

[0024] 2. Selective replication and killing: Δ24 mutation of the E1A domain allows the virus to replicate only in Rb-deficient tumor cells, and normal cells are highly safe (verified in BEAS-2B and NIH / 3T3 cells).

[0025] 3. Immune microenvironment remodeling: CD8+ T / NK cells are activated by IL-21 expression, the proportion of CD3+ / CD8+ in the tumor microenvironment is up-regulated, and the immunosuppressive state is reversed.

[0026] 4. Inhibition of metastasis and drug resistance: the migration and epithelial-mesenchymal transition (EMT) of colorectal cancer cells (SW480 and MC38) can be significantly inhibited, and the risk of recurrence is reduced.

[0027] 5. High efficiency and low toxicity: the tumor growth inhibition rate in the MC38 tumor-bearing model is ≥70%, and there is no body weight decrease or organ toxicity, realizing oncolysis-immune synergistic treatment.

[0028] In summary, the above beneficial effects have shown that the recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 provides feasibility for drug research and development, and the vector constructed based on the chimpanzee adenovirus (serotype 25) of the technical scheme can significantly reduce the interference of pre-existing neutralizing antibodies in the human population and improve the in vivo delivery efficiency of the oncolytic virus. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a schematic diagram of the construction of the pAdsimian-E3 vector of the present application.

[0030] Figure 2 It is a process diagram of the construction of the pAdsimian-E3 of the present application.

[0031] Figure 3 It is an Adsimian-ΔE3 map identified by Pac I and Xba I double enzyme digestion of the present application.

[0032] Figure 4 It is a process diagram of the construction of pSRK17-CMV of the present application.

[0033] Figure 5 It is a verification diagram of pAdsimian-Δ24-mIL21, pAdsimian-Δ24-hIL21, pAdsimian-Δ24-EGFP of the present application.

[0034] Figure 6 It is a tumor volume and body weight curve diagram of mice of the present application.

[0035] Figure 7 It is a HE staining detection diagram of the condition of each organ of the present application.

[0036] Figure 8 It is a flow cytometry analysis diagram of the spleen tissue cells of mice in each treatment group of the present application.

[0037] Figure 9 It is a flow cytometry analysis diagram of the tumor tissue cells of mice in each treatment group of the present application.

[0038] Figure 10 It is a flow cytometry analysis diagram of the lymph node cells of mice in each treatment group of the present application.

[0039] Figure 11 It is an expression content diagram of IL-21 in the serum of mice of the present application.

[0040] Among them, Figure 2Fig. 1a: 1, 2 are Adsimian-5 fragments, total length 8936 bp; 3 is Adsimian-2 fragment, total length 12103 bp; 4 is Adsimian-3 fragment, total length 12125 bp. M1, M2: DL5000 DNA Marker. Figure 2 Fig. 1b: 1-4 are Adeasy-1 Amp fragments, total length 3711 bp from left to right. M: DL5000 DNA Marker. Figure 2 Fig. 1c: Pac I and Xba I double enzyme digestion verification of pAdsimian-E3. M: DL5000 DNA Marker. Figure 2 Fig. 1d: pAdsimian-E3 partial sequencing result.

[0041] Figure 3 Fig. 2: 1-4 are samples to be identified. M1, M2: DL15000 DNA MARKER.

[0042] Figure 4 Fig. 3a: signal fragment and right fragment are obtained. M1: DL2000 Plus DNA Maker; 1: signal fragment PCR product, band size 507 bp; M2: DL5000 DNA Maker; 2: right fragment PCR product, band size 2700 bp. Figure 4 Fig. 3b: CMV-SV40 fragment is obtained. M: DL2000 Plus DNA Maker; 1: CMV-SV40 fragment PCR product, 620 bp. Figure 4 Fig. 3c: pSRK17 PCR verification (primer 1000 bp verification). M: DL5000 DNA Marker; 1-10: pSRK17 detection samples. Figure 4 Fig. 3d: pSRK17-CMV (Sph I enzyme digestion verification). M: DL5000 DNA Marker; 1: pSRK17-CMV No. 1 sample; 2: pSRK17-CMV No. 2 sample; 3: pSRK17 No. 1 sample; 4: pSRK17 No. 2 sample. Figure 4 Fig. 3e: pSRK17-CMV partial sequencing result. Figure 4 Fig. 3f: pSRK17-Δ24-CMV partial sequencing result.

[0043] Figure 5 Fig. 4a: pAdsimian-Δ24-hIL21 Hind III enzyme digestion verification diagram. M1, M2: DL15000 DNA Marker. Figure 5b: pAdsimian-Δ24-mIL21 HindⅢ restriction enzyme digestion verification diagram. M1, M2: DL15000 DNA Marker. Figure 5 c: pAdsimian-Δ24-EGFP HindⅢ restriction enzyme digestion verification image. M1: DL15000 DNA Marker. Figure 5 Partial sequencing results of d: pAdsimian-Δ24-hIL21. Figure 5 Partial sequencing results of e: pAdsimian-Δ24-mIL21.

[0044] Figure 6 a: Timeline of gene therapy for colon cancer xenografts in mice. Figure 6 b: Schematic diagram of mouse weight changes. Figure 6 c: 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.

[0045] Figure 8 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 8In 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.

[0046] Figure 9 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.

[0047] Figure 10 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 Medium d: proportion of CD4 cells in lymph nodes on day 30 after treatment. + Medium d: proportion of CD4 cells in lymph nodes on day 30 after treatment. Figure 10 Medium e: proportion of CD8 cells in lymph nodes on day 30 after treatment. + Medium e: proportion of CD8 cells in lymph nodes on day 30 after treatment. ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.0001.

[0048] Figure 11 Medium: ns: p>0.05, ****: p<0.0001. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present application will be described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0050] Technical solutions / principles of the present application: the present technology constructs a new chimpanzee adenovirus vector (serotype 25), deletes the E3 region to reduce immunogenicity, and integrates the RGD peptide segment to enhance tumor targeting; at the same time, a shuttle plasmid carrying the Δ24 mutant E1A is designed, so that the virus only replicates in Rb-deficient tumor cells. Further, the IL-21 gene is armed, which enhances the anti-tumor effect through the dual mechanisms of oncolysis and immune activation, and provides a new strategy for the treatment of colorectal cancer. By modifying the chimpanzee adenovirus genome skeleton (such as E1 / E3 region deletion modification) and inserting an exogenous gene expression cassette, the problem of limitation caused by pre-existing immunity in traditional human adenovirus vectors is solved, and the vector capacity and gene expression efficiency are optimized, which provides a safer and more efficient technical tool for infectious disease prevention and control, tumor immunotherapy, and genetic disease gene repair.

[0051] Recombinant virus construction method: based on the chimpanzee adenovirus (serotype 25) skeleton, the E3 region is deleted and the Δ24 E1A gene is integrated through genetic engineering modification, realizing tumor-specific replication and reduced immunogenicity.

[0052] IL-21 gene is inserted into the virus genome using seamless cloning technology to form a recombinant virus Adsimian-Δ24-IL21 with oncolysis and immune activation functions.

[0053] Oncolytic effect: the virus targets tumor cells with Rb pathway defects and directly lyses and kills tumors.

[0054] Immune activation: IL-21 synergizes with tumor growth and metastasis inhibition by enhancing CD8+ T cell infiltration and function in the tumor microenvironment.

[0055] Application value: Breakthrough the limitation of traditional oncolytic viruses (such as human adenovirus type 5) caused by pre-existing antibodies, and overcome the immunosuppressive microenvironment of tumor by IL-21, to provide an efficient and safe combined treatment drug development strategy for colorectal cancer.

[0056] 1. Experimental materials

[0057] 1.1 Construction of pAdsimian-E3 and verification

[0058] 1.1.1.1 Design and construct pAdsimian-E3 series primers and reaction system

[0059] The simian adenovirus basic vector pAdsimian-E3 containing the E3 gene was constructed as the basis for knocking out the E3 gene in the next step. The company was commissioned to synthesize Adsimian (GenBank accession number AC_000011.1) in four fragments: Simian25-1 (1-3487bp), Simian25-2 (3391-15493bp), Simian25-3 (15478-27607bp), Simian25-4 (27584-36519bp). After obtaining the sequences of the four fragments, pAdsimian-E3 was obtained by four fragment seamless cloning. Figure 1 The schematic diagram of pAdsimian-E3 vector construction.

[0060] Using the four fragment plasmids of Simian25 synthesized as the initial template, four target fragments were obtained by PCR, namely: Adsimian-2, Adsimian-3, Adsimian-4 and Adeasy-1 Amp, and four pairs of PCR primers were designed (the primers involved in PCR are shown in Table 2.3.3.1)

[0061] Table 2.3.1.1

[0062]

[0063]

[0064] 1.1.1.2 Purification of PCR products and ligation transformation

[0065] (1) Purification of PCR products

[0066] The PCR product amplified by 2.3.1.1 was electrophoresed using a 1% agarose gel, and the target band was cut under a UV lamp and placed in a 1.5 mL EP tube. The target band was recovered according to the gel recovery kit.

[0067] 1) The weight of the gel was weighed on an electronic balance, and 100 μL of Buffer GDP gel was immersed in a metal bath at 55°C for 10 min. During this process, the EP tube was turned over to completely dissolve.

[0068] 2) The EP tube was instantaneously removed, and the solution on the tube wall was not attached to the wall. The adsorption column tube was placed in the collection tube, and the solution in the EP tube was transported to the adsorption tower in two times. After centrifugation at room temperature at 12000 rpm for 1 min, the filtrate was discarded.

[0069] 3) 300 μL Buffer GDP was added to the adsorption column, and centrifugation was performed at room temperature at 12000 rpm for 1 min, and the filtrate was discarded. 700 μL Buffer GW (to which anhydrous ethanol had been added) was added to the adsorption tower, and centrifugation was performed at room temperature at 12000 rpm for 1 min, and the filtrate was removed. This process was repeated twice.

[0070] 4) Centrifugation was performed at room temperature at 12000 rpm for 2 min to completely remove the remaining liquid, and then the adsorption column was placed in a new 1.5 mL EP tube. 30 μL of 55°C pretreated eluent was added to the adsorption column, and placed for 2 min, and centrifugation was performed at 12000 rpm for 1 min. The resulting solution was added to the adsorption tower, and elution was performed twice.

[0071] 5) At this time, the purified Adsimian-2, Adsimian-3, Adsimian-4, and AMP fragment linearized vectors were obtained.

[0072] (2) Four fragment ligation and transformation

[0073] 1) Ligation

[0074] The fragments were ligated using a MultiSOne Step Cloning Kit (C113 Vazyme) polyclonal kit, transformed into XL10 competent cells, and plated on Amp-resistant plates for screening. The gel recovery kit used was FastPure Gel DNA Extraction Mini Kit (Vazyme DC301). 2) Transformation

[0075]

[0076] ​Take the DH5a competent from -80°C refrigerator and thaw it on ice for 5-7 min. In the clean bench, add all the connectors to the just-thawed DH5a competent cells, pat the bottom gently to make it uniform, and place it for 30 min. Set the temperature of the water bath furnace to 42°C in advance, and heat shock for 45 sec. Quickly put the competent cells back on ice and place them for 2 min. In the clean bench, add 700 μL of LB liquid medium without antibiotics to the competent cells, and incubate at 220 rpm at 37°C for 20 min. After 20 min, separate the cells at 5000 rpm, retain 100 μL of supernatant, and then coat it on a solid LB plate with Amp resistance and incubate it in a bacterial incubator at 37°C overnight.

[0077] 3) Small-scale extraction of plasmid

[0078] Add Amp antibiotic to the LB liquid medium at a final concentration of 0.1%; select 6-8 single clones from the LB solid culture dish, place them in LB liquid medium, incubate at 220 rpm at 37°C for 14 h, and the culture solution is turbid; after 14 h, mix 500 μL of bacterial solution and 500 μL of 20% glycerol uniformly, and store them at -80°C.

[0079] 1) Take 4 mL of the above culture medium in a 1.5 mL EP tube, centrifuge at 8000 rpm at room temperature for 1 min, and remove the supernatant as much as possible.

[0080] 2) Add 600 μL of Buffer QLB reagent, shake it well with vortex, shake it completely, and incubate it at room temperature for 3 min.

[0081] 3) Transfer all the above solutions to the adsorption column of the set collection tube, centrifuge at 12000 rpm at room temperature for 1 min, and discard the filtrate.

[0082] 4) Add 600 μL of Buffer QWB reagent along the periphery of the adsorption column, centrifuge at 12000 rpm at room temperature for 1 min, and remove the filtrate.

[0083] 5) Separate the adsorption column, centrifuge at 12000 rpm at room temperature for 1 min, and completely drain the remaining liquid.

[0084] 6) In a 1.5 mL clean EP tube, add 30 μL of buffer QEB, centrifuge at 12000 rpm for 1 min, and obtain the plasmid pAdsimian-E3.

[0085] 7) Sequence the collected plasmid, and retain the sequenced plasmid and culture medium.

[0086] 1.1.1.3 pAdsimian-E3 candidate strain enzyme digestion verification and sequencing verification

[0087] First, Xba I and Pac I enzyme digestion verification of the correct enzyme digestion fragment of the monoclonal plasmid, and then sent to the sequencing company to further confirm by first generation sequencing method.

[0088] 1.2 Construction of pAdsimian-ΔE3 and verification

[0089] 1.1.1.4 Design and construct pAdsimian-ΔE3 series primers and set PCR reaction system

[0090] The construction of pAdsimian-ΔE3 which lacks part of the E3 region is carried out by overlapping PCR method.

[99]

[0091] (1) PCR to obtain DBP-12.5k and 14.7k-E4 fragments

[0092] 2.3.1 Obtain the plasmid pAdsimian-E3 as a template, and obtain two key fragments of Adsimian-ΔE3 by PCR method, named DBP-12.5k fragment and 14.7k-E4 fragment respectively, and splice the two key fragments by fusion PCR method, and the spliced fragment is named DBP-E4. This step can delete part of the E3 region by PCR method. The primers involved in PCR are shown in Table 2.3.2.1.

[0093] Table 2.3.2.1.

[0094]

[0095] (2) Nhe I enzyme digestion to obtain 24014 bp key large fragment

[0096] The enzyme digestion uses pAdsimian-E3 as a template, and Nhe I enzyme digestion is used to obtain a 24014 bp large fragment. The enzyme digestion system is shown in Table 2.3.2.1.d. The gel recovery kit used is FastPure Gel DNA Extraction Mini Kit (Vazyme DC301).

[0097] Table 2.3.2.1.d Nhe I enzyme digestion fragment system

[0098]

[0099] 1.1.1.5 Purification of PCR products and ligation transformation

[0100] (1) Purification of products ​

[0101] The PCR product of 2.3.2.1, i.e., the DBP-12.5k fragment and the 14.7k-E4 fragment, and the Nhe I enzyme were cut to obtain a 24014bp large fragment. The size of the band was observed by 1% agarose gel electrophoresis, and whether the band was consistent with the required band was observed. The target band was cut under the UV lamp and placed in a 1.5mL EP tube. The target band was recovered according to the gel recovery kit. The purification process was the same as that in 2.3.1.2.

[0102] (2) Ligation and transformation

[0103] The fragments obtained by the above purification and recovery were connected by seamless cloning, and the kit MultiSOne Step Cloning Kit(C113 Vazyme) was used to complete the ligation, and the competent XL10 was transformed and plated on Amp-resistant plates for screening.

[0104] 1.1.1.6 pAdsimian-ΔE3 candidate strain enzyme digestion verification

[0105] The colonies were selected for amplification culture, and preliminary enzyme digestion verification (Pac I and Xba I) was performed. The plasmids with correct enzyme digestion bands were selected for sequencing.

[0106] 1.3 Construction of pSRK17-CMV and verification

[0107] 1.1.1.7 Design and construction of pSRK17-CMV series primers and reaction system

[0108] The shuttle plasmid pSRK17-CMV is used to carry foreign target genes, and is convenient for recombination into the adenovirus backbone plasmid pAdsimian-ΔE3 to obtain a recombinant adenovirus vector.

[0109] The operation is as follows: the signal fragment is obtained by PCR with the synthesized plasmid pSignal as the template; the right fragment is obtained by PCR with Adsimian-E3 as the template. The purified and recovered signal fragment and right fragment are assembled by fusion PCR to obtain the signal+right fragment. The KAN fragment is obtained by PCR with the synthesized plasmid containing the KAN fragment. The PCR reagents and PCR program are referred to 2xPhanta Flash Master Mix(Vazyme, P510). The gel recovery kit is FastPure Gel DNA Extraction Mini Kit(Vazyme, DC301).

[0110] 1.1.1.8 Purification of PCR product and ligation and transformation

[0111] (1) Purification of PCR products

[0112] The PCR product of 2.3.3.1, i.e., the signal+right fragment and the KAN fragment, were subjected to 1% agarose gel electrophoresis, and the target band of the expected size was cut off.

[0113] (2) Connection and transformation of the signal+right fragment and the KAN fragment

[0114] After recovering the signal+right fragment and the KAN fragment, the concentration and mass of the recovered product were calculated using NanoDrop2000, and the dose was calculated according to the Seamless Cloning Kit reagent MultiSOne Step Cloning Kit (Vazyme, C113). Under the action of the seamless cloning enzyme, the signal+right fragment and the KAN fragment were connected by seamless cloning, and the competent cells were DH5a, which were coated on a Kanamycin-resistant plate for screening.

[0115] 1.1.1.9 Enzymatic digestion verification

[0116] The plasmids pSRK17 and pSRK17-CMV to be verified were subjected to Sph I enzyme digestion verification, and the correctly verified plasmids were subjected to sequencing verification.

[0117] 1.4 Construction and verification of pSRK17-Δ24-CMV

[0118] 1.1.1.10 Design and construction of pSRK17-Δ24-CMV series primers and PCR reaction system

[0119] In order to insert the E1A(Δ24) fragment into pSRK17-CMV, a pair of primers containing Cla I enzyme digestion sites at both ends were designed on the existing plasmid pSE1A(Δ24) in the laboratory, and the E1A(Δ24) fragment was obtained by PCR, and then purified and subjected to Cla I enzyme digestion. By taking Cla I as the enzyme digestion site, linearize pSRK17-CMV. After agarose gel electrophoresis, the fragment was purified and recovered, and the linearized vector and the linearized fragment were connected by enzyme ligation, and then amplified in DH5a competent cells, and single colony spots were screened on a kanamycin-resistant plate, and sequenced.

[0120] Table 2.3.4.1. a Series of primers for obtaining E1A(Δ24) fragment

[0121]

[0122] E1 is A or R to get E1A (Δ24) fragment A, TG is added below AP TCA RAG tube AT AC plus AT, see Table 2.3.4.1.b, and set the procedure as Table 2.3.4.1.c.

[0123] Table 2.3.4.1.b PCR reaction system for obtaining E1A (Δ24) fragment

[0124]

[0125] Table 2.3.4.1.c PCR reaction procedure for obtaining E1A (Δ24) fragment

[0126]

[0127] 1.1.1.11 Purification of PCR products and ligation transformation

[0128] (1) Purification of PCR products

[0129] The PCR product of 2.3.4.1, i.e. the E1A (Δ24) fragment and the linearized pSRK17-CMV fragment, was subjected to 1% agarose gel electrophoresis. After electrophoresis, the target band was cut under ultraviolet light and placed in a 1.5 mL EP tube. The target band was recovered according to the gel recovery kit.

[0130] (2) Ligation and transformation

[0131] The purified and recovered E1A (Δ24) fragment and the linearized pSRK17-CMV fragment were seamlessly cloned and ligated using the kit MultiSOne Step Cloning Kit (C113 Vazyme). The competent DH5α was used for amplification, and the kanamycin-resistant plate was used for resistance screening. After overnight culture in LB liquid medium (with a final concentration of 1% kanamycin), the full and spherical single colony spots were taken out for expansion culture.

[0132] (3) Sequencing verification

[0133] After 12 hours of bacterial liquid amplification, the single colony liquid from the previous overnight culture was preserved in 20% glycerol, labeled and stored in a -80°C refrigerator. Part of it was subjected to sequencing.

[0134] 1.5 Construction of recombinant chimpanzee oncolytic adenovirus plasmid pAdsimian-Δ24-IL21, pAdsimian-Δ24-EGFP and verification

[0135] 1.1.1.12 Design of sequence primers and PCR reaction system for obtaining mIL21, hIL21 and EGFP fragments

[0136] The primer for synthesizing hIL21 was obtained by PCR from the plasmid stored by the research group. The mIL21 genome was synthesized by commissioning Kang Company, and was constructed into the shuttle plasmid pSRK17-Δ24-CMV, named pSRK17-Δ24-mIL21. The specific steps for constructing pSRK17-Δ24-hIL21 are as follows:

[0137] Table 2.3.5.1.a Primers for obtaining hIL21 fragment

[0138]

[0139] According to the above sequence, to obtain the hIL21 fragment, the following ingredients were added to the PCR tube, and the PCR program was set as follows:

[0140] Table 2.3.5.1.b Reaction system for obtaining hIL21 fragment by PCR

[0141]

[0142] Table 2.3.5.1.c Reaction program for obtaining hIL21 fragment by PCR

[0143]

[0144] 1.1.1.13 Purification of PCR product and ligation transformation

[0145] (1) Purification of PCR product

[0146] The PCR products of 2.3.5.1, i.e. mIL21, hIL21, EGFP fragment and linearized pSRK17-CMV fragment, were subjected to 1% agarose gel electrophoresis, and the target band was recovered.

[0147] (2) Ligation and transformation

[0148] The mIL21, hIL21 and EGFP fragments obtained by purification and recovery in the previous step were ligated by seamless cloning reagent, and the ligation was completed using the kit MultiSOne Step Cloning Kit(C113 Vazyme), and competent DH5α was used for amplification, and kanamycin-resistant plates were used for resistance screening. After overnight culture in LB liquid medium (with a final concentration of 1% kanamycin), the full and spherical monoclonal spots were taken out for expansion culture.

[0149] (3) Sequencing verification

[0150] The selected strains pSRK17-Δ24-mIL21 and pSRK17-Δ24-hIL21, pSRK17-Δ24-EGFP were sent to the company for sequencing.

[0151] 1.1.1.14 enzyme digestion and ligation transformation

[0152] (1) Backbone plasmid and shuttle plasmid enzyme digestion

[0153] The backbone plasmid pAdsimian-ΔE3, shuttle plasmid pSRK17-Δ24-mIL21, pSRK17-Δ24-hIL21, pSRK17-Δ24-EGFP were linearized by enzyme digestion according to Table 2.3.5.4.a, 2.3.5.4.b in a 37°C water bath overnight.

[0154] Table 2.3.5.4.a pAdsimian-ΔE3 double enzyme digestion reaction system

[0155]

[0156] Table 2.3.5.4.b pSRK17-Δ24-mIL21, pSRK17-Δ24-hIL21, pSRK17-Δ24-EGFP single enzyme digestion system

[0157]

[0158] (2) Linearized vector electrophoresis gel purification and recovery

[0159] The enzyme digestion product fragments of the previous step were subjected to 1% agarose gel electrophoresis, and after electrophoresis, the target band was cut under ultraviolet light and placed in a 1.5 mL EP tube. The target band was recovered according to the gel recovery kit. The specific steps of purification operation were the same as 2.3.1.2

[0160] (3) Ligation transformation

[0161] The components were added in the EP tube according to Table 2.3.5.4.c, and the seamless cloning kit was used for ligation, and the program was set to 50°C for 15 min in the PCR instrument.

[0162] Table 2.3.5.4.c Seamless cloning system

[0163]

[0164] (4) Plasmid miniprep and sequencing

[0165] The extracted plasmid was sent to Kang Bi for sequencing, and thus the construction of pAdsimian-Δ24-mIL21, pAdsimian-Δ24-hIL21 and pAdsimian-Δ24-EGFP was completed.

[0166] 1.6 Experimental results

[0167] Successful construction of pAdsimian-E3: First, the fragments for constructing pAdsimian-E3 were obtained by PCR, and after 1% agarose gel electrophoresis, they were named Adsimian-2 fragment, Adsimian-3 fragment, Adsimian-4 fragment and Adeasy-1 Amp fragment, respectively. The electrophoresis results were observed, and the fragments of the expected size were obtained (see Figure 2 Fig. 2a), from left to right, 1, 2 are Adsimian-4 fragments, 8936 bp in total; 3 is Adsimian-2 fragment, 12103 bp in total; 4 is Adsimian-3 fragment, 12125 bp in total. The Adeasy-1 Amp fragment obtained by PCR was electrophoretic, and the band size was consistent with the expected size, i.e. 3711 bp. After ligation and transformation, the plasmid to be verified was obtained, and the Pac I and Xba I double enzyme digestion was used for verification. The enzyme digestion products were electrophoretic on 1% agarose gel, and the electrophoretic bands showed 30496 bp and 6322 bp size, which was consistent with the expectation. The enzyme-digested plasmid was sequenced (see Figure 2 Fig. 2d) The sequencing result is correct, and thus the construction of pAdsimian-E3 is successful.

[0168] Successful construction of pAdsimian-ΔE3: pAdsimian-ΔE3 with E3 region deletion was constructed. After enzyme digestion, PCR and obtaining of key fragments, ligation and transformation were performed, and four strains of bacteria were selected for plasmid extraction and identification by Pac I and Xba I double enzyme digestion. After 1% agarose gel electrophoresis, the electrophoresis results are shown in Figure 3 Fig. 3), Marker shows up to 15000 bp, which can show the large enzyme digestion fragment (30496 bp) of pAdsimian-ΔE3 and the small enzyme digestion fragment (6300 bp) for verification. From left to right, 1, 2, 3, 4, the enzyme digestion results are all correct, and the sequencing is sent for further verification. The sequencing results show that pAdsimian-ΔE3 is correctly constructed.

[0169] Successful construction of shuttle plasmid pSRK17-CMV and pSRK17-Δ24-CMV: With signal plasmid as template and signal F / R as upstream and downstream primers, 1% agarose gel electrophoresis was performed after PCR to obtain the electrophoretogram Figure 4In the middle a), No. 1 is a 507bp band, which is consistent with the designed signal fragment theoretical band size. After 1% agarose gel electrophoresis after PCR, the electrophoretogram (Figure 2) can be obtained. Figure 4 In the middle a), No. 2 is a 2700bp band, which is consistent with the designed right fragment theoretical band size. After 1% agarose gel electrophoresis after PCR, the electrophoretogram (Figure 3) can be obtained. Figure 4 In the middle b, No. 1 is a 620bp band. The electrophoretic results of PCR are consistent with the expectation.

[0170] After the plasmid pSRK17 is constructed, a pair of verification primers pSRK17 verification F / R is designed. If the PCR result shows a 1000bp band, the construction is successful. As shown in the electrophoretogram c (Figure 4), No. 5 and No. 7 samples show a 1000bp band. The full sequence is sent to Kang Company for sequencing, and the sequencing result is consistent with the design expectation (see Figure 5). Figure 4 In the middle c), No. 5 and No. 7 samples show a 1000bp band. The full sequence is sent to Kang Company for sequencing, and the sequencing result is consistent with the design expectation (see Figure 5). Figure 4 In the middle e, f, Figure e is the sequencing result of pSRK17-CMV, and Figure f is the partial sequencing result of pSRK17-Δ24-CMV.

[0171] At the same time, Sph I single enzyme digestion identification is also performed. If the enzyme digestion shows 2600bp and 4000bp, then the pSRK17-CMV is successfully constructed (see Figure 6). Figure 4 In the middle d), the pSRK17-CMV is successfully constructed.

[0172] pAdsimian-Δ24-IL21 and pAdsimian-Δ24-EGFP are successfully constructed: According to the Snapgene simulation Hind III enzyme digestion identification diagram, the characteristic band number of the constructed plasmid pAdsimian-Δ24-gene can be used to preliminarily distinguish whether the construction is successful. After 1% agarose gel electrophoresis of the enzyme digestion product, see the following Figure 5 . Among them, Figure 5 In the middle a, the pAdsimian-Δ24-hIL21 enzyme digestion result shows 20744bp, 8672bp, 4736bp, which is consistent with the theoretical enzyme digestion band size. The No. 1, 3, 4 samples are selected for sequencing detection. Figure 5Figure c is the pAdsimian-Δ24-EGFP enzyme digestion result map, showing 20273bp, 8696bp, 4736bp, which is consistent with the theoretical enzyme digestion band size, select 1, 2, preserved bacterial liquid sample detection. At this point, the construction of recombinant chimpanzee oncolytic adenovirus plasmid is completed.

[0173] 1.7 Technical summary

[0174] The technical scheme of the present application focuses on the development of a new chimpanzee oncolytic adenovirus vector, and the construction of chimpanzee oncolytic adenovirus vector plasmids pAdsimian-Δ24-hIL21, pAdsimian-Δ24-mIL21 and control virus plasmid pAdsimian-Δ24-EGFP carrying IL-21 gene.

[0175] (1) Synthesize the full sequence of simian 25 type adenovirus whole genome simian 25. First, by searching for information and synthesizing the full sequence of simian 25 type adenovirus whole genome simian 25, due to the length of the whole genome (more than 36k bp), the key fragments are synthesized in four segments, which is beneficial to the subsequent modification. (2) Successfully constructed pAdsimian-E3 containing simian adenovirus whole sequence. The synthesized four fragment plasmids were obtained by high fidelity enzyme, then connected by seamless cloning technology, and finally verified by enzyme digestion and sequencing, which confirmed that the plasmid pAdsimian-E3 containing simian 25 type adenovirus whole genome was constructed.

[0176] (2) Successfully constructed pAdsimian-ΔE3 backbone plasmid with E3 deletion. By enzyme digestion and overlapping PCR, pAdsimian-ΔE3 backbone plasmid was successfully constructed. When deleting E3, part of the immune regulatory genes in E3 region (such as 12.5K, ADP) were designed to retain, and the virus replication ability was enhanced.

[0177] (3) Successfully constructed pSRK17-Δ24-CMV shuttle plasmid. In order to express exogenous genes by gene targeting therapy, pSRK17-Δ24-CMV shuttle plasmid was designed and constructed. pSRK17-Δ24-CMV is based on the existing pShuttle-CMV in the laboratory, and the following functional elements are inserted: CMV promoter regulates exogenous gene transcription; multiple cloning site (MCS) contains XhoI, EcoRⅠ, Spe I and other enzyme digestion sites; kanamycin resistance selection marker. Δ24 mutation in E1A region gives specific oncolytic activity

[95] The GC content (45-60%) was optimized by Snapgene software to avoid the interference of inverted repeat sequences, and finally the high-purity plasmid (A260 / A280 = 1.8-2.0) was obtained by amplification of competent cells DH5a.

[0178] (4) Successfully constructed chimpanzee oncolytic adenovirus plasmids pAdsimian-Δ24-IL21 and pAdsimian-Δ24-EGFP. Linearized pAdsimian-ΔE3 and linearized pSRK17-Δ24-IL21, pSRK17-Δ24-EGFP were connected by seamless cloning. Due to the large number of bases of pAdsimian-ΔE3, the success rate is low when the shuttle plasmid is connected by seamless cloning. In order to improve the connection rate and accuracy, through optimization of enzyme digestion conditions and improvement of product quality, the plasmid cloning efficiency and accuracy were significantly improved in multiple experiments. The specific performance is: the quality of the enzyme digestion product is significantly improved, including the cutting efficiency and the purity of the product; the plasmid cloning failure rate is significantly reduced, and the cloning efficiency is significantly improved; the plasmid genome integrity is verified, and no additional mutations or insertions are introduced.

[0179] 2. In vivo study on chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 inhibiting the growth of MC38 mouse colorectal cancer transplanted tumors

[0180] The effect of recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 on treating MC38 mouse colorectal cancer was explored by in vivo experiments. First, a transplanted tumor mouse model was established by subcutaneously inoculating MC38 cells into four-week-old C57BL / 6 mice, and then intratumorally injecting recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21. By dynamically monitoring the tumor growth curve, histopathological analysis, and flow cytometry detection of immune cell subpopulations in multiple organs (including spleen single cells, tumor tissues, and draining lymph nodes), it was aimed to explore whether chimpanzee adenovirus Adsimian-Δ24-IL21 could effectively deliver IL-21 and achieve therapeutic effect on MC38 colorectal cancer mouse model; whether chimpanzee adenovirus Adsimian-Δ24-IL21 could enhance anti-tumor effect by remodeling tumor immune microenvironment. The research results will provide certain reference for the application of new chimpanzee oncolytic virus-cytokine combined therapy.

[0181] 2.1 Experimental materials

[0182] 2.11 Establishment of mouse MC38 cell line subcutaneous tumor animal model

[0183] 1) MC38 cells were subcutaneously inoculated into four-week-old C57BL / 6 mice, and after 10 days, when the tumor grew to 80-120mm 3At the time of sacrifice, the mice were divided into 3 groups of 8 mice each.

[0184] 2) The mice were injected intratumorally according to the groupings in the table below. The first day of injection was designated day 1, and each injection was 1.0 x 10 9 PFU, every other day for a total of 2 injections for a total of 2.0 x 10 9 PFU. On day 15 after treatment and on the last day of observation, day 30, the mice were sacrificed and their organs and subcutaneous tumors were collected for subsequent analysis.

[0185] Table 4.3.1.1 Mouse Groupings

[0186]

[0187] 2.12 Sample Collection

[0188] (1) Collection of mouse peripheral blood

[0189] After the mouse was caught, the left eyelid was gently pressed with the hand, and after the eyeball was protruded, the eyeball was immediately taken out with tweezers, and the blood was collected in a 1.5 mL EP tube. Then, at least 30 min at room temperature before serum centrifugation. The speed of the centrifuge was set at 3000 rpm for 20 min, and then the sample was placed in the centrifuge. After centrifugation, the supernatant was taken and stored in a -80°C refrigerator. After taking blood, the mouse was sacrificed by cervical spinal cord dislocation, soaked in 75% alcohol, and various tissues were collected on a sterile workbench.

[0190] (2) Collection of mouse subcutaneous tumors

[0191] After the mouse was sacrificed, it was fixed on the dissection table, the skin on the side was cut open with scissors, and it was carefully separated from the skin and subcutaneous tumor to prevent infection. After the subcutaneous tumor was removed, it was washed with PBS twice, and divided into two groups, one group was placed in fixative, and the other group was used for flow cytometry detection.

[0192] (3) Collection of mouse organs such as spleen

[0193] The mouse skin was cut along the center line and carefully lifted upwards. After the skin was cut, a new set of sterile dissection instruments was used to dissect it, and the spleen was taken out with tweezers and washed with PBS twice, then placed in a culture dish and on ice, waiting for the flow cytometry experiment.

[0194] (4) Collection of mouse lymph nodes

[0195] The mouse skin was completely cut open, exposing the inguinal and axillary regions, and the inguinal and axillary lymph nodes inoculated with subcutaneous tumors were carefully collected and placed in a culture dish containing appropriate medium and placed on ice for subsequent flow cytometry experiments.

[0196] 2.13 Preparation of single cell suspension

[0197] The components were ground and sieved to make a single cell suspension for flow cytometry analysis. The preparation of single cell suspension of each tissue was as follows:

[0198] 2.14 Preparation of spleen cells

[0199] (1) The spleen obtained from the above step was ground and repeatedly shaken, and placed in a 50 mL centrifuge tube. The centrifuge was set to 1600 rpm per minute, and centrifuged for 8 minutes.

[0200] (2) After centrifugation, the supernatant was discarded, and 5 mL of red blood cell lysing solution was added to each test tube and reacted at room temperature for 5 minutes.

[0201] (3) A large amount of PBS was added to terminate the red cell lysis, and the measured object was centrifuged at a rotation speed of 1600 rpm for 5 minutes to remove the supernatant.

[0202] (4) The precipitate was resuspended with PBS, passed through a 200-mesh nylon filter, and the supernatant was discarded after centrifugation. 1 mL of PBS was added to resuspend the mouse spleen single cell suspension.

[0203] (5) 10 μL of mouse spleen single cell suspension and 190 μL of PBS were taken for counting, and were used in subsequent experiments.

[0204] 2.15 Preparation of lymph node cells

[0205] (1) The collected PBS was washed and ground into a homogenate with surgical scissors, 1 mL of PBS was added for washing, and the mixture was stirred evenly.

[0206] (2) The homogenized tissue was sieved with a 200-mesh nylon filter and placed in a centrifuge tube, set to 3000 rpm for 5 minutes.

[0207] (3) After centrifugation, the supernatant was discarded, and the turbid mixture was resuspended with PBS and subjected to a second centrifugation.

[0208] (4) The supernatant was discarded, and the mixture was resuspended with 500 μL of PBS and re-dispensed into 3 small tubes for use in the next step of the experiment.

[0209] 2.16 Preparation of single cell suspension of subcutaneous tumor

[0210] (1) The mouse was sacrificed, and after 75% ethanol disinfection, the mouse subcutaneous tumor was removed, and the adipose tissue, necrotic tumor tissue and mouse skin were removed, then transferred to a 6 cm 10 dish (containing 2 mL RPMI-1640 basic medium) and completely cut into 1 mm 3 sized pieces with sterile surgical scissors.

[0211] (2) Transfer the above tissue suspension into a 15 mL centrifuge tube, add 8 mL of RPMI-1640 basic medium, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend with 10 mL of prepared tissue digestion solution, transfer to a new 50 mL sterile centrifuge tube, and place in a 37°C constant temperature shaker, 220 rpm, shake incubate for 45 min.

[0212] (3) After incubation is completed, filter the digested tissue with a 200 mesh nylon filter, collect 15 mL of sterile centrifuge tube to obtain a single cell suspension, centrifuge to remove the supernatant, wash once with 10 mL of RPMI-1640 cell culture medium containing 10% FBS, and resuspend the cells with 5 mL of cell culture medium.

[0213] (4) At room temperature, add 3 mL of lymphocyte separation medium to a 15 mL sterile centrifuge tube, then carefully overlap the single cell suspension obtained in (3) on the lymphocyte separation medium.

[0214] (5) Centrifuge at 2000 rpm for 20 min, and reduce the speed to 0.

[0215] (6) Carefully resuspend the white cells (mouse TILs) by blowing and washing, and then perform subsequent activation tests; other interstitial cells are deposited at the bottom of the tube.

[0216] 2.17 Flow cytometry analysis of the proportion of immune cells in different tissues of mice

[0217] (1) Divide the antibodies into 4 parts, and according to the manufacturer's instructions, add 1 μL of antibody to each sample, and the staining method of each tube is as follows:

[0218] Table 4.3.3 Flow cytometry staining scheme

[0219]

[0220] (2) Add the corresponding antibodies to single staining tube A, single staining tube B, single staining tube C, and the test tube to be detected, stir evenly, and place in the dark for 30 min.

[0221] (3) After incubation, set 3000 rpm, place the sample in the centrifuge for 5 min of centrifugation. After centrifugation, discard the supernatant, adjust the volume to 1 mL with PBS, suspend the cells by blowing, and repeat 2 times.

[0222] (4) Place on ice, avoid light. Resuspend the cells with a pipette, then send to the flow machine for analysis.

[0223] 2.18 Data analysis

[0224] Statistical analysis was performed using CytExpert and GraphPad Prism 9.0. The results were expressed as mean ± SD. The comparisons between groups were made using t-test, one-way ANOVA, two-way ANOVA, survival analysis. Differences were significant at p<0.05.

[0225] 2.2 Experimental results

[0226] 2.21 Tumor volume and body weight curve of mice

[0227] The mice were measured for body weight and tumor volume every other day, and the changes in tumor volume and body weight after injection of the treatment drug were dynamically observed. According to the development of the mouse tumor, the upper limit of the tumor volume observation was set to 2000 mm 3 , and the observation period was set to 30 days.

[0228] It was found that compared with the PBS group and the Adsimian-Δ24-EGFP control virus group, the tumor growth of the mice in the recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 gene therapy group was significantly slowed down, and the tumor volume was relatively small, as shown in Figure 6 , f of the specification. In addition, Adsimian-Δ24-mIL21 gene therapy can significantly reduce tumor volume and even cause regression, as shown in Figure 6 , f of the specification. In addition, there was no significant difference in the activity of the tumor-bearing mice during the entire observation and measurement period, and there was no significant difference in the body weight of the mice (p>0.05), as shown in Fig. b. In summary, Adsimian-Δ24-mIL21 can effectively inhibit the growth of colon cancer tumors in mice and better inhibit the development of tumors.

[0229] 2.22 Toxicity detection of chimpanzee oncolytic adenovirus in mice

[0230] In order to evaluate the safety of injection of PBS, Adsimian-Δ24-EGFP, and Adsimian-Δ24-mIL21 in mice, the heart, liver, spleen, lung, kidney, and other organs were taken out after 15 days of injection, and HE staining was observed. As shown in Figure 7 , it was found that Adsimian-Δ24-EGFP and Adsimian-Δ24-mIL21 genes had no obvious damage to the heart, liver, spleen, lung, and kidney of mice, indicating that chimpanzee oncolytic adenovirus had less toxicity to mouse organs and had certain safety.

[0231] 2.23 Chimpanzee oncolytic adenovirus carrying IL-21 enhances anti-tumor immune ability

[0232] As shown in Figure 8The changes in the proportion of immune cells in the spleen tissue are shown: the tumor intervention model injected with chimpanzee oncolytic adenovirus had a significant therapeutic effect at day 14: the PBS control group of mice had rapid tumor growth (with a median volume of 1500 mm 3 ) on day 14 after intervention, while the Adsimian-Δ24-EGFP empty vector group and the Adsimian-Δ24-mIL21 treatment group both showed significant tumor regression (p<0.01). To analyze the early immune state, on day 15 of treatment, 3 tumor-bearing mice were randomly selected from each of the three groups for preparation of a single-cell suspension of spleen tissue.

[0233] Flow detection found that the proportion of CD4 + cells in the Adsimian-Δ24-EGFP group increased compared with the PBS group, ranging from 23.03% to 38.88%, and the proportion of CD8 + cells increased from 23.02% to 28.97%; while the Adsimian-Δ24-mIL21 group showed a decreasing trend in the proportion of CD4 + cells (0.48% vs 16.94% in the PBS group, p=0.0055), which may be related to IL-21-induced redistribution of Th cell subsets or regulatory T cell exhaustion

[106] , which can be verified by subsequent FoxP3 / CTLA-4 co-staining experiments.

[0234] At the end of treatment (day 30), flow cytometry analysis was performed on the Adsimian-Δ24-mIL21 group of tumor completely regressed individuals, and the results showed that the proportion of CD3 + cells in the spleen cells increased by 1.68 times compared with the control group (AdsimianΔ24-EGFP) (37.00%±2.00% vs 22.00%±2.00%, p<0.05), of which the proportion of CD8 + cells was 35.00%±5.00% (an increase of 1.42 times compared with day 14), and the proportion of CD4 + cells recovered to 44.00%±2.00%, indicating that IL-21 may achieve long-term anti-tumor effect by dynamically regulating the balance of T cell subsets, preferentially expanding CD8 + effector cells in the early stage to rapidly control tumor load, and rebuilding CD4 + helper cells to maintain immune memory in the later stage.

[0235] 2.24 Changes in the proportion of immune cells in tumor tissue

[0236] As shown in Figure 9 , the primary cells of tumor-bearing mice (n=3) were separated by enzyme digestion method, and were detected by multi-color flow cytometry.

[0237] Results showed that compared with PBS group (CD3 + cells ratio 26.00% ± 2.00%), Adsimian-Δ24-EGFP group CD3 + cell infiltration increased significantly, about 1.76 times of the control group (PBS group) (46.00% ± 2.00%, p < 0.0001, one-way ANOVA), and the Adsimian-Δ24-mIL21 treatment group was further increased to 2.00 times (52.00% ± 2.00%, p < 0.0001), indicating that the virus vector backbone itself has immune activation properties, and the co-expression of IL-21 gene can produce a synergistic effect.

[0238] In the analysis of immune cell subgroups, the proportion of intratumoral CD8 + cells in the Adsimian-Δ24-EGFP group was 1.61 times higher than that in the PBS group (42.00% ± 5.00% vs 26.00% ± 5.00%, p < 0.05), and the Adsimian-Δ24-mIL21 group increased by 2.11 times (55.00% ± 2.00%, p < 0.01), indicating that IL-21 not only enhances the infiltration of total T cells, but also specifically promotes the enrichment of CD8 + cells in the tumor microenvironment. This effect may be related to the activation of IL-21-regulated CXCR3 / CXCL10 chemokine axis

[108] or enhanced T cell proliferation signals

[109] , which needs to be further verified by spatial transcriptome analysis

[110] and in vitro migration experiments.

[0239] 2.25 Changes in the proportion of immune cells in the draining lymph nodes

[0240] To explore the immune regulation of Adsimian-Δ24-mIL21 in the tumor microenvironment, the present application carried out longitudinal observation through tumor-bearing mouse models (n = 8). At the 30th day after intervention, the inguinal lymph node immune cells of the surviving individuals (IL-21 group n = 3, EGFP group n = 3) were analyzed, and after collecting and preparing the lymph node single cells, multi-color flow cytometry was used for immune phenotype analysis (Note: all PBS control groups died due to tumor progression, so they were not included in the final analysis).

[0241] As shown in Figure 10 , according to the results of flow cytometry detection and analysis, compared with the Adsimian-Δ24-EGFP group (30.00% ± 5.00%), the CD3 +The proportion of cell population was significantly increased to 65.00% ± 5.00% (p < 0.0001,); compared with the EGFP group (30.00% ± 5.00%), CD4 + The proportion of cell population was significantly increased to 65.00% ± 5.00% (p < 0.0001,); compared with the EGFP group (30.00% ± 5.00%), CD4 + The proportion of cell population was significantly increased to 65.00% ± 5.00% (p < 0.0001,); compared with the EGFP group (30.00% ± 5.00%), CD4 + The proportion of cell population was significantly increased to 65.00% ± 5.00% (p < 0.0001,); compared with the EGFP group (30.00% ± 5.00%), CD4 + The proportion of cell population was significantly increased to 65.00% ± 5.00% (p < 0.0001,); compared with the EGFP group (30.00% ± 5.00%), CD4

[0242] 2.26 Changes in IL-21 content in mouse serum

[0243] In this experiment, the quantitative enzyme-linked immunosorbent assay (ELISA) was used to evaluate the delivery efficiency and functional expression of IL-21 mediated by the novel chimpanzee adenovirus vector in C57BL / 6 mice.

[0244] As shown in Figure 11 , the experimental data showed that the serum IL-21 concentration of the target treatment group was significantly up-regulated (2987.4 ± 213.5 pg / mL; n = 5) 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); one-way ANOVA, p < 0.0001), and the difference could reach about 15 times (PBS vs Adsimian-Δ24-mIL21) and 6 times (EGFP vs Adsimian-Δ24-mIL21). This experiment showed that the chimpanzee adenovirus vector constructed in this application had high delivery efficiency for immune gene IL-21, and the combination of flow cytometry analysis could further prove the anti-tumor effect of IL-21.

[0245] 2.27 Experiment summary:

[0246] (1) The chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 has significant anti-tumor ability. 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, which was significantly better than the empty vector group and the PBS control group, and had no obvious effect on the body weight of mice.

[0247] (2) Histopathology analysis of chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 showed no harm to mouse organs. Hematoxylin-eosin staining (HE staining) was used to analyze the staining of heart, liver, spleen, lung and kidney of mice in PBS group, Adsimian-Δ24-EGFP group and Adsimian-Δ24-mIL21 group. The chimpanzee oncolytic adenovirus did not cause obvious toxicity to the organs of mice.

[0248] (3) Flow cytometry analysis revealed the characteristics of immune microenvironment remodeling of chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 treatment. First, the CD3 + , CD8 + cells in the tumor tissue of IL-21 treatment group after Adsimian-Δ24-mIL21 virus treatment increased significantly, indicating that Adsimian-Δ24-mIL21 played an anti-tumor role by activating cellular immunity and regulating immune microenvironment. Second, the proportion of CD8 + in the spleen and draining lymph nodes increased, indicating that local IL-21 expression might induce systemic immune response.

[0249] (4) Chimpanzee oncolytic adenovirus Adsimian-Δ24-mIL21 effectively delivered IL-21 and played an anti-tumor function. In enzyme-linked immunosorbent assay (ELISA) experiments, the present 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 of the results of flow cytometry showed that high expression of IL-21 significantly promoted the proliferation of CD3 + cells, indicating that Adsimian-Δ24-mIL21 regulated T cell activation.

[0250] Notably, the empty vector group Adsimian-Δ24-EGFP, which did not express IL-21, still reduced the tumor volume by about 25% compared with the PBS group, indicating that chimpanzee adenovirus vector itself might have a certain oncolytic effect. Tumor-specific replication caused by Δ24 deletion might enhance immunogenicity by directly killing tumor cells or releasing tumor-associated antigens. However, the immune cell infiltration of the Adsimian-Δ24-EGFP empty vector group was much lower than that of the Adsimian-Δ24-mIL21 treatment group, further proving that the expression of IL-21 could significantly enhance the anti-tumor immune effect of chimpanzee adenovirus.

[0251] The present application proves that the Adsimian vector system can achieve efficient delivery and functional expression of IL-21, which provides an experimental basis for the late preclinical application and transformation of chimpanzee adenovirus expressing IL-21, and also provides a reference for related immunotherapy strategies such as tumor immunotherapy drugs and autoimmune disease intervention drugs.

[0252] Finally, it should be noted that the present application is not limited to the above embodiments, but can have many variations. All variations that can be directly derived or inferred from the content disclosed in the present application by those of ordinary skill in the art should be considered within the scope of the present application.

Claims

1. A recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21, characterized in that: The virus is based on a chimpanzee adenovirus of serotype 25, in which the E3 region is deleted from the genome and an integrin-targeting RGD peptide segment is inserted; the virus carries a human or murine IL-21 gene, which is expressed under the control of a CMV promoter; and the virus comprises a Δ24 mutant E1A domain, which is driven by a human adenovirus type 5 E1A promoter and is specific for replication in tumor cells that are deficient in the Rb pathway.

2. The recombinant chimpanzee oncolytic adenovirus Adsimian-A24-IL21 according to claim 1, characterized in that: The virus is constructed by a backbone plasmid pAdsimian-ΔE3 and a shuttle plasmid pSRK17-Δ24-CMV; the backbone plasmid pAdsimian-ΔE3 is deleted of the E3 region and retains part of the immunomodulatory genes 12.5K and ADP of the E3 region; and the shuttle plasmid pSRK17-Δ24-CMV comprises an adenovirus packaging signal, a kanamycin resistance gene, a Δ24 mutant E1A domain, and a multiple cloning site Spe I, Nhe I, and Hind III.

3. The recombinant chimpanzee oncolytic adenovirus Adsimian-A24-IL21 according to claim 1 or 2, characterized in that: The virus remodels the immune microenvironment by upregulating the proportion of CD3+ and CD8+ T cells in the tumor microenvironment, inhibits the migration of colorectal cancer cells, and reverses the process of epithelial-mesenchymal transition.

4. A method of constructing the recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 of claim 1, characterized by The method comprises the following steps: Step 1) Synthesis of the full genome of chimpanzee adenovirus of serotype 25, divided into four fragments Simian25-1 to Simian25-4, amplified by high-fidelity PCR, and connected by seamless cloning to obtain pAdsimian-E3; Step 2) Deletion of the E3 region by overlapping PCR to construct the backbone plasmid pAdsimian-ΔE3; Step 3) Construction of the shuttle plasmid pSRK17-Δ24-CMV, insertion of the Δ24 mutant E1A domain and the IL-21 gene regulated by the CMV promoter; Step 4) After the backbone plasmid pAdsimian-ΔE3 and the shuttle plasmid pSRK17-Δ24-CMV are digested by Pac I and Xba I, they are connected by seamless cloning to obtain the recombinant adenovirus plasmid pAdsimian-Δ24-IL21; Step 5) Transfection of HEK293 cells with the recombinant plasmid to package chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 carrying the IL-21 gene.

5. The method of constructing a recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 according to claim 4, characterized in that: The IL-21 gene is amplified from a storage plasmid by PCR and inserted downstream of the CMV promoter of the shuttle plasmid after digestion by Cla I.

6. Use of a recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 for the preparation of a medicament for the treatment of colorectal cancer, characterized in that: The virus inhibits tumor growth through direct oncolysis and activation of CD3+ and CD8+ T cells in the tumor microenvironment, and has no significant toxicity to human normal lung epithelial cells BEAS-2B and mouse embryonic fibroblasts NIH / 3T3.

7. Use according to claim 6, characterized in that: The virus enhances the killing ability of colorectal cancer cells SW480 through co-culture with peripheral blood mononuclear cells, and significantly inhibits tumor growth in the MC38 tumor-bearing mouse model without causing body weight loss or organ toxicity.

8. A method of verifying the recombinant chimpanzee oncolytic adenovirus Adsimian-Δ24-IL21 according to any one of claims 1 to 3, characterized in that The method comprises the following steps: Step 1) Verification of the characteristic bands of pAdsimian-Δ24-IL21 by Hind III digestion, including 20744 bp, 8672 bp, and 4736 bp; Step 2) Detect the proportion of CD3+ and CD8+ T cells in tumor tissues by flow cytometry; Step 3) Verify the inhibition of colorectal cancer cell migration and the reversal of epithelial-mesenchymal transition marker expression by cell scratch test and qRT-PCR.

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