Replication type oncolytic adenovirus capable of blocking TNF-TNFR2 signal channel as well as preparation method and application of replication type oncolytic adenovirus

Abstract

The invention discloses a replicative oncolytic adenovirus capable of blocking a TNF-TNFR2 signal channel as well as a preparation method and application of the replicative oncolytic adenovirus. The invention relates to a replicating oncolytic adenovirus for blocking a TNF-TNFR2 signal channel. The replicating oncolytic adenovirus for blocking the TNF-TNFR2 signal channel comprises a coding gene nucleotide sequence of a TNF antagonist, a TNFR2 antagonist or a combined molecule of the TNFR2 antagonist and a CD47 antagonist. The invention also discloses the application of the replicative oncolytic adenovirus for blocking the TNF-TNFR2 signal channel in preparation of anti-tumor drugs. According to the invention, by blocking a TNF-TNFR2 signal channel and utilizing the replicative oncolytic adenovirus to specifically replicate and dissolve cells in tumor cells, the pertinence and effectiveness of tumor treatment are enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a replicative oncolytic adenovirus that blocks a TNF-TNFR2 signaling pathway, and a preparation method and application thereof. Background Art

[0002] Immunotherapy is considered the fourth pillar of cancer treatment (along with surgery, radiotherapy, and chemotherapy), and works by enhancing the immune system's ability to target and kill tumor cells. Oncolytic virotherapy is a new type of immunotherapy that specifically kills tumor cells and spares normal cells by direct oncolysis and inducing anti-tumor immune responses. It uses natural viruses or genetically modified viruses to achieve selective infection of tumor cells and selective replication in tumor cells through genetic and epigenetic differences between tumor cells and normal cells. Although oncolytic viruses have made great progress in tumor research in recent years. However, clinical results and conclusions still show that many patients cannot benefit from oncolytic virotherapy. Tumor microenvironment heterogeneity and mediated immunosuppression may be the bottleneck problem limiting oncolytic virotherapy.

[0003] Tumor necrosis factor (TNF) is a major inflammatory cytokine and has long been considered a marker of immune activation for tumor immunotherapy. However, in recent years, increasing evidence has shown that TNF also plays a role in promoting tumor occurrence and progression. TNF in the tumor microenvironment (TME) can maintain the expression of inhibitory receptors such as PDL1 and CD47 on tumor cells and lead to low response and immune-related adverse reactions to immune checkpoint blockers (ICBs). Antagonizing TNF not only enhances the antitumor effect of anti-CTLA-4 or anti-PD-1 antibodies, but also alleviates ICB-induced colitis.

[0004] The TNF / TNFR / NF-κB signaling pathway plays a key role in the occurrence, development and metastasis of tumors. TNFR1 and TNFR2 are two natural receptors for TNF, both of which can bind transmembrane TNF (tmTNF) and soluble TNF (sTNF). TNFR1 is widely expressed in mammalian cells, while TNFR2 is usually induced to express on immune cells such as Tregs, macrophages and MDSCs. TNFR2 plays a key role in the differentiation of Tregs and the immunosuppressive function of MDSCs. Elevated TNFR2 expression in the TME is associated with poor prognosis, while TNFR2 deficiency can inhibit tumor growth. Antibodies that antagonize TNFR2 are currently in the clinical research stage. Preclinical research results have shown that antibodies that antagonize TNFR2 have shown good anti-tumor activity, whether as a monotherapy or in combination with PD-1 inhibitors. However, due to the pleiotropic roles of nuclear factor κB (NF-κB) in innate and adaptive immune responses, systemic blockade of TNF / NF-κB also induces some side effects, such as increased infectious diseases and vasculitis in patients treated with systemic TNFα antibodies.

[0005] Currently, there is a lack of a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway, as well as a preparation method and application thereof. Summary of the invention

[0006] The purpose of the present invention is to solve the problems of the prior art and to provide a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway in local tumor expression, as well as a preparation method and application thereof.

[0007] In order to achieve the above-mentioned objectives, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a replicating oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway. In the second aspect, the present invention provides an application of a replicating oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway in the preparation of anti-tumor drugs. In the third aspect, the present invention provides a method for preparing a replicating oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway. In the fourth aspect, the present invention provides a pharmaceutical composition. In the fifth aspect, the present invention provides a method for screening TNFR2 antagonists. In the sixth aspect, the present invention provides an anti-human TNFR2 mouse monoclonal antibody ahTR2. In the seventh aspect, the present invention provides a kit containing a replicating oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway.

[0008] The present invention discloses a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway. The replicative oncolytic adenovirus of the TNF-TNFR2 signaling pathway comprises a nucleotide sequence of a gene encoding a TNF antagonist, a TNFR2 antagonist, or a combination molecule of a TNFR2 antagonist and a CD47 antagonist. The nucleotide sequence of the gene encoding the TNF antagonist is shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding the TNFR2 antagonist is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the combination molecule of a TNFR2 antagonist and a CD47 antagonist is shown in SEQ ID NO.3.

[0009] Furthermore, the amino acid sequence of the TNF antagonist is shown in SEQ ID NO.4, the amino acid sequence of the TNFR2 antagonist is shown in SEQ ID NO.5, and the amino acid sequence of the combination molecule of the TNFR2 antagonist and the CD47 antagonist is shown in SEQ ID NO.6.

[0010] Application of the replicative oncolytic adenovirus for blocking the TNF-TNFR2 signaling pathway in the preparation of anti-tumor drugs.

[0011] Furthermore, the tumor includes any one of pancreatic cancer, colon cancer, lung cancer or kidney cancer.

[0012] A method for preparing a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway of the present invention comprises the following steps:

[0013] (1) constructing a shuttle vector containing a first promoter, an E1A early activation replication element, a second promoter, and a nucleotide sequence encoding an antagonist;

[0014] (2) co-transfecting the shuttle vector and human adenovirus type 5 backbone plasmid into HEK 293 cells;

[0015] (3) Collecting and purifying transfected viral particles;

[0016] (4) Amplify viral particles to the desired titer.

[0017] Furthermore, the nucleotide sequence of the first promoter is shown in SEQ ID NO.7, the nucleotide sequence of the E1A early activation replication element is shown in SEQ ID NO.8, and the nucleotide sequence of the second promoter is shown in SEQ ID NO.9.

[0018] A pharmaceutical composition of the present invention comprises a replication-competent oncolytic adenovirus and a pharmaceutically acceptable excipient.

[0019] Furthermore, the pharmaceutically acceptable excipient is one or a combination of a buffer solution, a preservative or a stabilizer.

[0020] A method for evaluating the anti-tumor effect of a replicative oncolytic adenovirus in vivo of the present invention comprises the following steps:

[0021] (1) Inoculate tumor cells into experimental animals to form a tumor model;

[0022] (2) After the tumor reaches a certain volume, injecting the virus into the tumor tissue;

[0023] (3) Regularly measure tumor volume and animal body weight, and record survival rate.

[0024] A method for screening a TNFR2 antagonist of the present invention comprises the following steps:

[0025] (1) Immunizing mice with recombinant human TNFR2FC protein;

[0026] (2) fusing spleen cells from immunized mice with myeloma cells to form hybridoma cells;

[0027] (3) screening monoclonal antibodies that can specifically bind to human TNFR2;

[0028] (4) The specific binding ability of the screened monoclonal antibodies was verified by ELISA and flow cytometry analysis techniques.

[0029] The anti-human TNFR2 mouse monoclonal antibody ahTR2 of the present invention can specifically bind to human TNFR2 and inhibit the binding of TNFα to TNFR2.

[0030] The invention discloses a kit containing a replicative oncolytic adenovirus capable of blocking a TNF-TNFR2 signaling pathway.

[0031] Beneficial effects: The present invention blocks the TNF-TNFR2 signaling pathway and utilizes a replicative oncolytic adenovirus to specifically replicate and dissolve cells in tumor cells, thereby enhancing the pertinence and effectiveness of tumor treatment.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] (1) Immunomodulatory effect: By locally overexpressing TNF- / TNFR2 pathway antagonists, the present invention can regulate chronic inflammation and immune tolerance in the tumor microenvironment, thereby improving the therapeutic effect and helping to activate and enhance the body's anti-tumor immune response.

[0034] (2) Enhanced targeting: By utilizing the differences in genetic characteristics and epigenetics between tumor cells and normal cells, the oncolytic adenovirus of the present invention can selectively replicate in tumor cells, reducing damage to normal cells and improving the safety of treatment.

[0035] (3) Multi-target therapy: The oncolytic adenovirus involved in the present invention can express TNF antagonists, TNFR2 antagonists, and combination molecules of TNFR2 antagonists and CD47 antagonists, thereby achieving multi-target therapy and increasing the breadth and depth of the therapeutic effect.

[0036] (4) Development of pharmaceutical compositions: The present invention also relates to pharmaceutical compositions comprising the recombinant adenovirus or adenovirus vector and a pharmaceutically acceptable excipient, which provides a new form and option for clinical medication.

[0037] (5) Innovation in preparation method: The present invention provides a new method for preparing replicative oncolytic adenovirus, including the steps of constructing a recombinant plasmid, co-transfection, virus collection, amplification and purification, providing a new technical path for the preparation of viral vectors.

[0038] (6) Verification of anti-tumor activity: The anti-tumor activity of oncolytic adenovirus was verified through in vivo and in vitro experiments, including tumor growth inhibition and improved survival rate, providing a scientific basis for preclinical research and clinical application.

[0039] (7) Reduced side effects: Compared with the side effects that may be caused by systemic blocking of the TNF / NF-κB signaling pathway, the present invention may reduce the patient's systemic side effects and improve the safety of treatment by locally overexpressing antagonists.

[0040] (8) In-depth understanding of therapeutic mechanisms: The research results of the present invention help to deeply understand the role of the TNF-TNFR2 signaling pathway in tumor immunity and provide an important theoretical basis for the development of new tumor immunotherapy strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 is a screening diagram for the TNFR2 antagonist of the present invention; Figure 1 A represents the binding of ahTR2 of the present invention to the extracellular segment protein of recombinant human TNFR2; Figure 1 B is the binding of ahTR2 of the present invention to CHO cells overexpressing human TNFR2 molecules; Figure 1 C is the ability of ahTR2 of the present invention to inhibit the binding of TNFα to human TNFR2; Figure 1D is the tumor growth curve of ahTR2 of the present invention in treating MC38 tumor-bearing human TNFR2 transgenic C57BL / 6 model; Figure 1 E is the heavy chain and light chain variable region sequence of ahTR2 of the present invention.

[0043] FIG2 is a schematic diagram of the construction of a recombinant oncolytic adenovirus expressing a TNF antagonist, a TNFR2 antagonist, and a combination molecule of a TNFR2 antagonist and a CD47 antagonist according to the present invention; Figure 2A This is a schematic diagram of a shuttle vector of an embodiment of the present invention for preparing a recombinant adenovirus expressing a TNF antagonist (AdV-TNFi). The structure is a shuttle vector comprising two promoters, one of which is used to express the E1A protein and the other is used to express the TNF antagonist (SEQ ID1). Figure 2B This is a schematic diagram of a shuttle vector of an embodiment of the present invention for preparing a recombinant adenovirus expressing a TNFR2 antagonist (AdV-ahTR2). The structure is a shuttle vector comprising two promoters, one of which is used to express the E1A protein and the other is used to express the TNFR2 antagonist (SEQ ID2). Figure 2C This is a schematic diagram of a shuttle vector of an embodiment of the present invention for preparing a recombinant adenovirus expressing a TNFR2 antagonist and a CD47 antagonist combination molecule (AdV-ATA47). The structure is a shuttle vector comprising two promoters, one of which is used to express the E1A protein, and the other promoter is used to express the TNFR2 antagonist and a CD47 antagonist combination molecule (SEQ ID3).

[0044] Figure 3 It is a diagram showing the expression and binding detection of the inserted genes of AdV-TNFi, AdV-ahTR2 and AdV-ATA47 of the present invention; Figure 3 A shows the expression of TNFi and its binding with human TNFα after AdV-TNFi of the present invention infects 293T cells; Figure 3 B shows the expression of TNFi and its binding with mouse TNFα after AdV-TNFi of the present invention infects 293T cells; Figure 3 C shows the expression of ahTR2 and its binding to human TNFR2 after AdV-ahTR2 of the present invention infects 293T cells; Figure 3 D shows the expression of ATA47 and its binding with human TNFR2 and CD47 after AdV-ATA47 of the present invention infects 293T cells.

[0045] Figure 4 This is a diagram showing the therapeutic effect of AdV-TNFi of the present invention in a human pancreatic cancer PANC-1 tumor-bearing NCG mouse model after human PBMC immune reconstitution; Figure 4 A is a schematic diagram of drug administration of the present invention; Figure 4 B. Figure 4 C is a tumor growth curve diagram of the present invention.

[0046] Figure 5 The therapeutic effect of AdV-TNFi of the present invention in a mouse colon cancer tumor-bearing mouse model; Figure 5 A is a schematic diagram of drug administration of the present invention; Figure 5 B is a tumor growth curve diagram of the present invention; Figure 5 C is a survival curve diagram of the mice of the present invention.

[0047] Figure 6 This is a diagram showing the therapeutic effect of AdV-TNFi of the present invention in a mouse lung cancer tumor-bearing mouse model; Figure 6 A is a tumor growth curve diagram of the present invention; Figure 6 B is a survival curve diagram of the mice of the present invention.

[0048] Figure 7 This is a diagram showing the therapeutic effect of AdV-TNFi of the present invention in a mouse renal cancer tumor-bearing mouse model; Figure 7 A is a tumor growth curve diagram of the present invention; Figure 7 B is a survival curve diagram of the mice of the present invention.

[0049] Figure 8 This is a diagram showing the therapeutic effect of AdV-ahTR2 of the present invention in a mouse colon cancer tumor-bearing human TNFR2 transgenic C57 BL / 6 mouse model: Figure 8 A is the tumor growth curve of the present invention; Figure 8 B is the weight change curve of the mice of the present invention; Figure 8 C is the survival curve of the mice of the present invention.

[0050] Fig. 9 This is a diagram showing the therapeutic effect of AdV-ATA47 of the present invention in a human colon cancer HCT116 tumor-bearing NCG mouse model after human PBMC immune reconstitution: Fig. 9 A. Fig. 9 B is a tumor growth curve diagram of the present invention. DETAILED DESCRIPTION

[0051] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0052] The following examples are provided to help further understand the present invention, but the present invention is not limited thereto.

[0053] The first aspect of the present invention provides a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway. The replicative oncolytic adenovirus of the TNF-TNFR2 signaling pathway comprises a nucleotide sequence of a gene encoding a TNF antagonist, a TNFR2 antagonist, or a combination molecule of a TNFR2 antagonist and a CD47 antagonist. The nucleotide sequence of the gene encoding the TNF antagonist is shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding the TNFR2 antagonist is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the combination molecule of a TNFR2 antagonist and a CD47 antagonist is shown in SEQ ID NO.3.

[0054] In some embodiments, the nucleotide sequence of the first promoter is shown in SEQ ID NO.7, the nucleotide sequence of the E1A early activation replication element is shown in SEQ ID NO.8, and the nucleotide sequence of the second promoter is shown in SEQ ID NO.9.

[0055] A second aspect of the present invention provides a use of a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway in the preparation of an anti-tumor drug.

[0056] In some embodiments, the tumor is pancreatic cancer.

[0057] In some embodiments, the tumor is pancreatic and colon cancer.

[0058] In some embodiments, the tumor is colon cancer.

[0059] In some embodiments, the tumor is renal cancer.

[0060] The third aspect of the present invention provides a method for preparing a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway, comprising the following steps:

[0061] (1) constructing a shuttle vector containing a first promoter, an E1A early activation replication element, a second promoter and a nucleotide sequence encoding the antagonist;

[0062] (2) co-transfecting the shuttle vector and human adenovirus type 5 backbone plasmid into HEK 293 cells;

[0063] (3) Collecting and purifying transfected viral particles;

[0064] (4) Amplify viral particles to the desired titer.

[0065] The fourth aspect of the present invention provides a pharmaceutical composition comprising a replication-competent oncolytic adenovirus and a pharmaceutically acceptable excipient.

[0066] In some embodiments, the pharmaceutically acceptable excipient is a buffered solution.

[0067] In some embodiments, the pharmaceutically acceptable excipient is a preservative.

[0068] In some embodiments, the pharmaceutically acceptable excipient is a stabilizer.

[0069] In some embodiments, the pharmaceutically acceptable excipient is a combination of two of a buffer solution and a preservative.

[0070] In some embodiments, the pharmaceutically acceptable excipient is a combination of three of a buffer solution, a preservative, and a stabilizer.

[0071] The fifth aspect of the present invention provides a method for screening a TNFR2 antagonist, comprising the following steps:

[0072] (1) Immunizing mice with recombinant human TNFR2FC protein;

[0073] (2) fusing spleen cells from immunized mice with myeloma cells to form hybridoma cells;

[0074] (3) screening monoclonal antibodies that can specifically bind to human TNFR2;

[0075] (4) The specific binding ability of the screened monoclonal antibodies was verified by ELISA and flow cytometry analysis techniques.

[0076] The sixth aspect of the present invention provides an anti-human TNFR2 mouse monoclonal antibody ahTR2, which can specifically bind to human TNFR2 and inhibit the binding of TNFα to TNFR2.

[0077] A seventh aspect of the present invention provides a kit containing a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway.

[0078] Example 1

[0079] Screening of anti-human TNFR2 hybridoma antibodies

[0080] 1.1 Immunity

[0081] Balb / c mice (6-8 weeks old, female) were emulsified with 50 μg of recombinant human TNFR2-FC (Enbrel) in the presence of complete Freund's adjuvant (CFA), with 100 μl of protein and 100 μl of CFA per mouse, and multiple immunizations were performed subcutaneously (50 μl per injection). In the second and third weeks, 100 μl of protein and 100 μl of IFA were also emulsified and immunized per mouse in the presence of incomplete Freund's adjuvant (IFA). In the fourth week, 100 μg of protein was directly injected intraperitoneally for booster immunization.

[0082] 1.2 Fusion

[0083] The spleen cells of the immunized mice were fused with the mouse myeloma cells SP20. The specific steps are as follows:

[0084] Remove the spleen under sterile conditions, remove the surrounding connective tissue, and wash it clean. Chop the spleen cells, homogenize, filter, centrifuge, resuspend and count; remove the mouse myeloma cells SP20 that were passaged 24 hours in advance, mix them thoroughly with the spleen cells and centrifuge them, and discard the supernatant. Preheat the cell pellet in a 37°C water bath; within 60 seconds, slowly add 1ml PEG1450 preheated to 37°C to the centrifuge tube, stir gently for 1 minute, and add culture medium. After 10 minutes of incubation, centrifuge the mixture, discard the supernatant; resuspend the cells in BMDM culture medium containing 20% ​​FBS, 1×HAT (Sigma, H0262-10VL) and 1× double antibody. Then distribute the resuspended cells to 15 96-well cell culture plates for incubation, 200μl per well, 5% CO2, 37°C.

[0085] 1.3 Hybridoma Screening

[0086] On the 7th day after fusion, a complete medium change was performed. The medium was BMDM medium containing 15% FBS. On the 10th day after fusion, ELISA was performed. Cells in the wells with positive test were subjected to the first and second rounds of secondary sorting.

[0087] The steps of ELISA detection are as follows: First, use TNFR2-His (Beijing Yiqiao Shenzhou Technology Co., Ltd., 10417-H08H) to coat the ELISA plate and block it. Next, incubate 100μl of hybridoma supernatant in the ELISA plate, then seal the plate with a sealing film and incubate at 37°C for 1 hour. Then, incubate with goat anti-mouse HRP secondary antibody, add 100μl to each well, and incubate at 37°C for 1 hour. Finally, perform TMB color development and use an ELISA reader for detection: use a wavelength of 450nm for detection.

[0088] After three rounds of screening, we finally obtained a mouse monoclonal antibody that can specifically bind to human TNFR2 and named it ahTR2.

[0089] 1.4 Binding of anti-human TNFR2 mouse monoclonal antibody ahTR2 to recombinant human TNFR2 antigen

[0090] The binding of monoclonal antibody ahTR2 to recombinant human TNFR2 antigen was demonstrated by ELISA using standard methods and procedures as follows:

[0091] TNFR2-his antigen (1 μg / ml, 100 μl / well) was coated, the ELISA plate was blocked, and the serially diluted TNFR2 monoclonal antibody to be tested was added for incubation (37°C, 1 h), and then goat anti-mouse HRP secondary antibody was added for incubation, TMB was used for color development, and the ELISA plate was detected at a wavelength of 450 nm.

[0092] Experimental results and analysis: ELISA test results are as follows Figure 1 As shown in A, the EC of ahTR2 50 It is 16ng / mL.

[0093] 1.5ahTR2 binding to TNFR2-CHO cells

[0094] The anti-human TNFR2 mouse monoclonal antibody ahTR2 was co-incubated with a TNFR2-overexpressing CHO-K1 cell line (CHO-K1 cells were infected with a lentivirus expressing human TNFR2, and a cell line with stable high expression of human TNFR2 was obtained through drug screening) and stained, and the antibody binding strength was analyzed by flow cytometry analysis technology. The specific steps are as follows:

[0095] TNFR2-CHO was counted, centrifuged, resuspended, and added to an EP tube. The anti-TNFR2 antibody to be tested was added at a final concentration of 10ug / mL, incubated and washed, and then Anti-mouse-IgG-AF488 was added. The sample was detected using Beckman CytoFLEX.

[0096] Experimental results and analysis: Figure 1As shown in B, the anti-human TNFR2 mouse monoclonal antibody ahTR2 can bind to TNFR2 on the cell membrane. Isotype in the figure is a negative control antibody that does not bind to TNFR2.

[0097] 1.6 Inhibition of binding between recombinant human TNFR2 and TNFα by ahTR2

[0098] In order to determine whether the TNFR2 monoclonal antibody of the present invention can inhibit the binding of TNFα to TNFR2, we used a competitive ELISA method to perform the test:

[0099] TNFα antigen (GenScript, Z02682) was coated, the ELISA plate was blocked, and gradient dilutions of recombinant human TNFR2 monoclonal antibody and fixed concentration of TNFR2-hFc (Enbrel) protein were added for incubation, and then mouse anti-human HRP secondary antibody was added for incubation, color was developed, and the plate was detected by ELISA instrument.

[0100] Experimental results and analysis: The results are as follows Figure 1 As shown in C, ahTR2 can specifically block the binding of TNFα to soluble TNFR2.

[0101] 1.7ahTR2 inhibits the growth of mouse colon tumors in vivo

[0102] In order to determine whether the TNFR2 monoclonal antibody of the present invention can inhibit the growth of mouse colon tumors, we conducted efficacy verification in MC38 tumor-bearing C57BL / 6-hTNFR2 transgenic mice (Shanghai Model Organisms Science Co., Ltd.):

[0103] Mouse colon cancer cells MC-38 were cultured in DMEM medium containing 10% FBS and passaged 2-3 times a week. On the day of inoculation, the cells were treated with trypsin, centrifuged and precipitated, the supernatant was discarded, washed, and finally suspended in PBS at a cell density of 2×10 7 cells / ml.

[0104] Next, mouse MC-38 cells were subcutaneously transplanted into C57BL / 6-hTNFR2 transgenic mice, and 0.1 ml of cell suspension containing 2×10 6 cells was subcutaneously injected into the back of each mouse. On the 7th day after inoculation, the mice were randomly divided into groups according to the tumor volume and injected into the tail vein once every three days for a total of 3 times, with each injection volume of 0.2 ml (ahTR2 monoclonal antibody injection solution 1 mg / ml obtained by ascites purification) per mouse. The tumor volume of the mice was monitored during the period.

[0105] Experimental results and analysis: The results are as follows Figure 1 As shown in D, ahTR2 can effectively inhibit the growth rate of mouse colon tumor MC38 in mice.

[0106] 1.8ahTR2 heavy chain and light chain variable region sequencing

[0107] The ahTR2 hybridoma cells were sequenced and the sequence information of the V region (SEQ ID2) was successfully obtained. The specific sequence information is as follows Figure 1 As shown in E.

[0108] Example 2

[0109] Construction of recombinant oncolytic adenovirus expressing TNF antagonist, TNFR2 antagonist, and combination of TNFR2 antagonist and CD47 antagonist

[0110] 2.1 Construction of recombinant oncolytic adenovirus expressing TNF antagonist

[0111] Virus E1A (GenBank: MH629744.1) and the extracellular domain of human TNFR1 (SEQ ID1, GenBank: NC_000012.12) were inserted into the pShuttle plasmid pDC316 (Genscript, China). In order to promote the secretion of TNFR1, the signal peptide (MGLSTVPDLLLPLVLLELLVGIYPSGVIG) was retained at the front end of the TNFR1 sequence, and a 6×His tag was added to the rear end. The virus was named AdV-TNFi.

[0112] 2.2 Construction of recombinant oncolytic adenovirus expressing TNFR2 antagonist

[0113] The virus E1A (GenBank: MH629744.1) and ahTR2 scfv sequence (SEQ ID2) were inserted into the pShuttle plasmid pDC316. The ahTR2 scfv sequence was obtained by connecting the VH sequence and VL sequence of the ahTR2 antibody sequence through a linker (GGGGSGGGGSGGGGS). The C-terminus of the scfv was connected to the Fc segment of the antibody (mouse IgG2a subtype), and the N-terminus of the scfv was a signal peptide (MDWTWRILFLVAAATGAHS). The virus was named AdV-ahTR2.

[0114] 2.3 Construction of recombinant oncolytic adenovirus expressing a combination of TNFR2 antagonist and CD47 antagonist

[0115] The virus E1A (GenBank: MH629744.1) and the TNFR2 antagonist and CD47 antagonist combination molecule (ATA47, SEQ ID3) sequence were inserted into the pShuttle plasmid pDC316. The ATA47 sequence was connected to the Fc segment of the antibody (human IgG1 subtype) at the C-terminus of ahTR2 scfv, and the C-terminus of the Fc segment was connected to the extracellular segment of human Sirpα (CD47 antagonist, EELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSG) through a linker (GGGGSGGGGSGGGGS). The N-terminus of ahTR2 scfv was a signal peptide sequence (MDWTWRILFLVAAATGAHS). The virus was named AdV-ATA47.

[0116] 2.4 Virus packaging and amplification

[0117] Virus packaging: co-transfect HEK 293 cells with pDC316 plasmid and human adenovirus type 5 backbone plasmid pBHGloxdelE13cre (Microbix, Canada). Transfect 293T cells at 1ug / well in a six-well plate and culture at 5% CO2 and 37°C. After 2 days, digest the cells and transfer them to a 10cm dish, and change the medium every 2-3 days. When 80% of the cells die, use 10ml of culture medium to blow off the cells and collect them in a 15ml centrifuge tube, freeze and thaw twice, centrifuge at 3000rpm / min for 15min, collect the virus supernatant and store it at -80°C as virus seed.

[0118] Virus amplification and titer detection: Take 50ul of virus seed solution and add it to 60% 293T cells in a 10cm dish. Culture at 37℃ with 5% CO2 until the cell density reaches above 90%. Passage at a ratio of 1 to 3 until 80% of the cells show pathological changes and there are about 10 dishes of cells. Collect the virus according to the above method and purify the virus using cesium chloride density gradient centrifugation; use the TCID50 method for titer determination.

[0119] Example 3

[0120] Expression and binding detection of genes inserted into AdV-TNFi, AdV-ahTR2 and AdV-ATA47

[0121] 3.1 Expression and binding detection of AdV-TNFi inserted genes

[0122] Virus infection: 5 × 10 5After 293T cells adhered to the wall, AdV-TNFi virus was added at an MOI of 1, and the supernatant was collected for detection after 72 hours. The AdV-Ctrl virus treated in the same way was used as a control.

[0123] Human TNF-α binding assay: 100 ng / well human TNF-α was pre-coated on a polystyrene microplate to detect the expression of soluble TNFi (TNFR1 extracellular segment) and its binding to human TNF-α after AdV-TNFi infection in vitro. 100 uL supernatant or purified protein (gradient dilution) was added, incubated at 37°C for 2 h, and anti-6x-his tag monoclonal antibody [HRP] (Genscript, A00612) was added after washing, and incubated at 37°C for 1 h. TMB was added as a substrate, and the absorbance value was read at 450 nm.

[0124] Mouse TNF-α binding assay: 100 ng / well mouse TNF-α (GenScript, Z02682) was pre-coated on a polystyrene microplate to detect the expression of soluble TNFi after AdV-TNFi infection in vitro and its binding to mouse TNF-α. 100 uL of supernatant or purified protein (gradient dilution) was added, incubated at 37°C for 2 h, and anti-6x-his tag monoclonal antibody [HRP] was added after washing, and incubated at 37°C for 1 h. TMB was added as a substrate, and the absorbance value was read at 450 nm.

[0125] Results and discussion: Figure 3 As shown in A and 3B, AdV-TNFi can secrete TNFi protein after infecting cells, and TNFi protein can specifically bind to human and mouse TNFα.

[0126] 3.2 Expression and binding detection of AdV-ahTR2 inserted genes

[0127] Virus infection: 5 × 10 5 After 293T cells adhered to the wall, AdV-ahTR2 virus was added at an MOI of 1. The supernatant was collected for detection after 72 hours, and the AdV-Ctrl virus treated in the same way was used as a control.

[0128] 300ng / well human TNFR2-his was pre-coated on a polystyrene microplate to detect the expression of ahTR2-Fc and its binding to human TNFR2 after AdV-ahTR2 infection in vitro. 100uL supernatant or purified protein (gradient dilution) was added, incubated at 37℃ for 2h, and anti-mouse Fc antibody [HRP] was added after washing, and incubated at 37℃ for 1h. TMB was added as a substrate, and the absorbance value was read at 450nm.

[0129] Results and discussion: Figure 3As shown in C, AdV-ahTR2 can secrete ahTR2 protein after infecting cells, and ahTR2 protein can specifically bind to human TNFR2 protein.

[0130] 3.3 Expression and binding detection of AdV-ATA47 inserted genes

[0131] Virus infection: 5 × 10 5 After 293T cells adhered to the wall, AdV-ATA47 virus was added at an MOI of 1. The supernatant was collected for detection after 72 hours, and the AdV-Ctrl virus treated in the same way was used as a control.

[0132] TNFR2 end detection: 300ng / well human TNFR2-his was pre-coated on a polystyrene microplate to detect the expression of ATA47 and its binding to human TNFR2 after AdV-ATA47 infection in vitro. 100uL supernatant or purified protein (gradient dilution) was added, incubated at 37℃ for 2h, and anti-human Fc antibody [HRP] was added after washing, and incubated at 37℃ for 1h. TMB was added as a substrate, and the absorbance value was read at 450nm.

[0133] CD47 end detection: 300ng / well human CD47-his was pre-coated on a polystyrene microplate to detect the expression of ATA47 and its binding to human CD47 after AdV-ATA47 infection in vitro. 100uL supernatant or purified protein (gradient dilution) was added, incubated at 37℃ for 2h, and anti-human Fc antibody [HRP] was added after washing, and incubated at 37℃ for 1h. TMB was added as a substrate, and the absorbance value was read at 450nm.

[0134] Results and discussion: Figure 3 As shown in D, AdV-ATA47 infected cells can normally secrete ATA47 protein, and ATA46 protein can specifically bind to human TNFR2 and CD47 protein.

[0135] Example 4

[0136] Evaluation of the therapeutic effect of AdV-TNFi in pancreatic cancer

[0137] In vivo antitumor effect of AdV-TNFi (PANC-1 model): In the humanized mouse model, NCG mice were subcutaneously inoculated with 5×10 6 Human PANC-1 cells. When the tumor grows to about 100 mm 3 At the same time, PANC-1 tumor-bearing mice were randomly divided into groups and intravenously injected with 2×10 human PBMCs (Milestone biotech, China). 6 AdV (5×108 PFUs), and the tumor diameter was measured with a vernier caliper, using the formula V = 0.5 × length × width 2 Tumor volume was calculated.

[0138] PANC-1 human pancreatic tumor NCG model experimental plan: 6-8 week old NOD-Prkdc carrying PANC-1 pancreatic cancer scid Iq null The (NCG) mice were randomly divided into PBS control group (5 mice), AdV-Ctrl group (7 mice) and AdV-TNFi group (7 mice). The tumor growth curves of the mice were recorded.

[0139] Results and conclusions: Figure 4 As shown, in human immune-reconstituted NCG mice carrying human PANC-1 pancreatic cancer, the anti-tumor efficacy of the AdV-TNFi group was significantly better than that of the AdV-Ctrl group.

[0140] Example 5

[0141] Evaluation of the therapeutic effect of AdV-TNFi in a colon cancer model

[0142] In vivo antitumor effect of AdV-TNFi (MC38 model): 2×10 6 MC38 tumor cells were inoculated subcutaneously on the right side of C57BL / 6 mice to establish a subcutaneous tumor model. 3 At 4 pm, mice were randomly divided into groups and injected intratumorally with AdV or PBS. The tumor diameter was measured with a vernier caliper and the formula V = 0.5 × length × width was used. 2 Tumor volume was calculated.

[0143] Experimental protocol of MC38 colon cancer model: 6-8 week-old C57BL / 6 mice were subcutaneously transplanted with MC38 colorectal tumors and injected with PBS, 1×10 9 PFU AdV-Ctrl or AdV-TNFi. There were 7 mice in each group, and the tumor growth curve and Kaplan-Meier survival curve of mice were recorded.

[0144] Results and conclusions: Figure 5 As shown in the MC38 colon cancer mouse model, AdV-TNFi significantly enhanced the anti-tumor efficacy of mice. Compared with the AdV-Ctrl control group, the survival rate of the AdV-TNFi group was also significantly improved, with 1 / 7 mice cured.

[0145] Example 6

[0146] Evaluation of the therapeutic effect of AdV-TNFi in lung cancer models

[0147] In vivo antitumor effect of AdV-TNFi (LLC model): 2×10 6 Renca cells were inoculated subcutaneously on the right side of C57BL / 6 mice to establish a mouse subcutaneous tumor model. 3 At 4 pm, mice were randomly divided into groups and injected intratumorally with AdV or PBS. The tumor diameter was measured with a vernier caliper and the formula V = 0.5 × length × width was used. 2 Tumor volume was calculated.

[0148] LLC lung cancer model experimental protocol: 6-8 week-old C57BL / 6 mice were subcutaneously inoculated with LLC lung cancer cells and injected intratumorally with PBS, 5×10 8 PFU AdV-Ctrl or AdV-TNFi, 5 mice in each group, and the tumor growth curve and Kaplan-Meier survival curve of mice were recorded.

[0149] Results and conclusions: Figure 6 As shown, in the LLC lung cancer model, compared with the AdV-Ctrl control group, the tumors in the AdV-TNFi group of mice were significantly reduced, showing a significantly enhanced anti-tumor effect, and the survival rate was also significantly improved.

[0150] Example 7

[0151] Evaluation of the therapeutic effect of AdV-TNFi in a renal cancer model

[0152] In vivo antitumor effect of AdV-TNFi (Renca model): 2×10 6 Renca cells were inoculated subcutaneously on the right side of C57BL / 6 mice to establish a mouse subcutaneous tumor model. 3 At 4 pm, mice were randomly divided into groups and injected intratumorally with AdV or PBS. The tumor diameter was measured with a vernier caliper and the formula V = 0.5 × length × width was used. 2 Tumor volume was calculated.

[0153] Renca renal cancer model experimental plan: 6-8 week-old C57BL / 6 mice were subcutaneously inoculated with Renca renal cancer cells and injected intratumorally with PBS, 5×10 8 PFU AdV-Ctrl or AdV-TNFi. 8 mice in each group, the tumor growth curve and Kaplan-Meier survival curve of mice were recorded.

[0154] Results and conclusions: Figure 7 As shown in the Renca renal cancer model, the AdV-TNFi group showed a significantly enhanced anti-tumor effect compared with the AdV-Ctrl group. At the same time, the survival rate was also significantly improved.

[0155] Example 8

[0156] Evaluation of the therapeutic effect of AdV-ahTR2 in a colon cancer model

[0157] In vivo antitumor effect of AdV-ahTR2 (MC38 model): 2×10 6 MC38 tumor cells were inoculated subcutaneously on the right side of C57BL / 6-huTNFR2 mice to establish a mouse subcutaneous tumor model. 3 At 4 pm, mice were randomly divided into groups and injected intratumorally with AdV or PBS. The tumor diameter was measured with a vernier caliper and the formula V = 0.5 × length × width was used. 2 The tumor volume was calculated, and the weight changes and survival rate of the mice were observed.

[0158] MC38 colon cancer model experimental protocol: 6-8 week-old C57BL / 6-huTNFR2 mice were subcutaneously transplanted with MC38 colorectal tumors and injected with PBS, 5×10 8 PFU AdV-Ctrl or AdV-ahTR2, for a total of 3 times. The tumor growth curve and Kaplan-Meier survival curve of the mice were recorded.

[0159] Results and conclusions: Figure 8 As shown, in the C57BL / 6-huTNFR2 tumor-bearing mouse model, the AdV-ahTR2 group can significantly improve the anti-tumor effect of mice. There was no significant difference in the body weight of mice in the control group and the experimental group, and no obvious side effects were observed. AdV-ahTR2 significantly improved the survival rate of mice, and 1 / 8 of the mice had completely eliminated tumors.

[0160] Example 9

[0161] Evaluation of the therapeutic effect of AdV-ATA47 in a colon cancer model

[0162] In vivo antitumor effect of AdV-ATA47 (HCT116 model): In the humanized mouse model, NCG mice were subcutaneously inoculated with 5×10 6 Human HCT116 cells. When the tumor grew to about 100 mm3, HCT116 tumor-bearing mice were randomly divided into groups and intravenously injected with 2×106 human PBMCs (Milestone biotech, China). AdV (5×10 8 PFUs) to monitor tumor volume.

[0163] HCT116 human pancreatic tumor NCG model experimental plan: 6-8 week old NCG mice carrying HCT116 colon cancer were randomly divided into control group, AdV-Ctrl group and AdV-ATA47 group, with 6 mice in each group, and the tumor growth curve was recorded.

[0164] Results and conclusions: Fig. 9 As shown, in human immune-reconstituted NCG mice bearing human HCT116 colon cancer, the anti-tumor efficacy of the AdV-ATA47 group was significantly better than that of the AdV-Ctrl group.

[0165] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway, characterized in that: The replicative oncolytic adenovirus of the TNF-TNFR2 signaling pathway comprises a nucleotide sequence encoding a TNF antagonist, a TNFR2 antagonist or a combination molecule of a TNFR2 antagonist and a CD47 antagonist. The nucleotide sequence encoding the gene of the TNF antagonist is shown in SEQ ID NO.1, the nucleotide sequence encoding the gene of the TNFR2 antagonist is shown in SEQ ID NO.2, and the nucleotide sequence encoding the gene of the combination molecule of the TNFR2 antagonist and the CD47 antagonist is shown in SEQ ID NO.

3.

2. The replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway according to claim 1, characterized in that: The amino acid sequence of the TNF antagonist is shown in SEQ ID NO.4, the amino acid sequence of the TNFR2 antagonist is shown in SEQ ID NO.5, and the amino acid sequence of the combination molecule of the TNFR2 antagonist and CD47 antagonist is shown in SEQ ID NO.

6.

3. Use of the replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway according to claim 1 in the preparation of anti-tumor drugs.

4. The use according to claim 3, characterized in that: The tumor includes any one of pancreatic cancer, colon cancer, lung cancer or kidney cancer.

5. A method for preparing a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway according to claim 1, characterized in that The steps include: (1) constructing a shuttle vector containing a first promoter, an E1A early activation replication element, a second promoter, and a nucleotide sequence encoding the TNF antagonist, TNFR2 antagonist, or a combination molecule of a TNFR2 antagonist and a CD47 antagonist according to claim 1; (2) co-transfecting the shuttle vector and human adenovirus type 5 backbone plasmid into HEK 293 cells; (3) Collecting and purifying transfected viral particles; (4) Amplify viral particles to the desired titer.

6. The method for preparing a replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway according to claim 5, characterized in that: The nucleotide sequence of the first promoter is shown in SEQ ID NO.7, the nucleotide sequence of the E1A early activation replication element is shown in SEQ ID NO.8, and the nucleotide sequence of the second promoter is shown in SEQ ID NO.

9.

7. A pharmaceutical composition, characterized in that: Comprising the replicative oncolytic adenovirus according to any one of claims 1 to 4.

8. A method for screening a TNFR2 antagonist as claimed in claim 1, characterized in that The steps include: (1) Immunizing mice with recombinant human TNFR2FC protein; (2) fusing spleen cells from immunized mice with myeloma cells to form hybridoma cells; (3) screening monoclonal antibodies that can specifically bind to human TNFR2; (4) The specific binding ability of the screened monoclonal antibodies was verified by ELISA and flow cytometry analysis techniques.

9. An anti-human TNFR2 mouse monoclonal antibody ahTR2, characterized in that: The compound can specifically bind to the human TNFR2 as claimed in claim 1 and inhibit the binding of TNFα to TNFR2.

10. A kit containing the replicative oncolytic adenovirus that blocks the TNF-TNFR2 signaling pathway according to claim 1.

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