Recombinant oncolytic adenovirus vector for expressing human IL-2, 4-1BBL and membrane type IL-21 protein as well as construction method and application of recombinant oncolytic adenovirus vector

Abstract

The invention provides a recombinant oncolytic adenovirus vector for expressing human IL-2, 4-1BBL and membrane type IL-21 proteins as well as a construction method and application thereof, and the method comprises the following steps: inserting two expression cassettes into an E1 region of an adenovirus, namely expression cassettes of IL-2, 4-1BBL and membrane type IL-21 genes regulated by a human cytomegalovirus broad-spectrum enhancer / promoter respectively, and an expression cassette of an E1 gene regulated by a tumor tissue or cell specific telomerase promoter. And co-transfecting HEK 293 cells by using the constructed double-expression cassette shuttle plasmid and an adenovirus skeleton plasmid to obtain the recombinant oncolytic adenovirus vector. The recombinant oncolytic adenovirus vector provided by the invention can efficiently express IL-2, 4-1BBL and membrane type IL-21, and after intratumor injection, the infiltration and activity of natural killer cells in a tumor microenvironment can be effectively improved, the tumor immunosuppression microenvironment is improved, and the antitumor activity is enhanced.

Description

Technical Field

[0001] The present invention belongs to the fields of life science, biotechnology and tumor immune gene therapy, and relates to a recombinant oncolytic adenovirus vector and a preparation method and application thereof, and in particular to a recombinant oncolytic adenovirus vector expressing human IL-2, 4-1BBL and membrane-type IL-21 proteins and a construction method and application thereof. Background Art

[0002] Tumor is one of the major diseases that threaten human health. In my country, it has become the leading cause of death from disease, and the incidence and mortality rates are still rising. With the continuous deepening of tumor biology research, the level of tumor diagnosis and treatment has been significantly improved, but the high metastasis and high recurrence after tumor treatment are still an urgent problem to be solved in the clinical treatment of tumors. Therefore, it is imperative to find new treatment methods. In recent years, tumor immunotherapy has made breakthrough progress. For example, chimeric antigen receptor T cell technology (CAR-T) and immune checkpoint blockers have achieved significant results in the treatment of certain tumors, but have not shown obvious advantages in most tumors. Therefore, seeking different treatment methods, including the combined application of different immunotherapies, is a key direction in the future field of tumor treatment.

[0003] Natural killer (NK) cells are the most important cells of the body's natural immunity and have many potential advantages in anti-tumor. Fully exerting and utilizing the anti-tumor effect of NK cells is one of the powerful means of immunotherapy for anti-tumor. At present, adoptive transfusion of in vitro expanded NK cells is used clinically to enhance anti-tumor immunity. However, the number of infiltrated NK cells in solid tumors is limited, and the infiltrated NK cells are functionally inactivated. Therefore, increasing the infiltration of NK cells in tumors and their functional activity is of great significance for tumor treatment.

[0004] Oncolytic adenovirus (OA) or conditionally replicating adenovirus (CRA) is modified from adenovirus. The most commonly used method is to regulate the expression of the E1A gene required for adenovirus replication through a certain tumor-specific promoter to become an oncolytic adenovirus that replicates in a certain type of tumor cells. As a new type of targeted anti-tumor strategy, oncolytic adenovirus has several advantages: (1) It is a double-stranded DNA non-integrating virus that can infect dividing and non-dividing cells and does not integrate into the cell genome, so it is highly safe; (2) The genome is clear, easy to operate, and has a large packaging capacity. It can insert 8kb of the target gene, which is very conducive to the expression of genes with anti-tumor activity. Therefore, further modification of oncolytic adenovirus will be of great significance to the treatment of tumors in terms of improving the tumor microenvironment and promoting the anti-tumor activity of various immune cells. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a recombinant oncolytic adenovirus vector that can efficiently express IL-2, 4-1BBL and membrane-type IL-21, and its construction method and application, and to further transform the oncolytic adenovirus to increase the number and activity of NK cells in the tumor microenvironment and enhance its anti-tumor immunity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for constructing a recombinant oncolytic adenovirus vector expressing human IL-2, 4-1BBL and membrane-type IL-21 proteins, wherein the construction method comprises: step 1, inserting two expression cassettes into the E1 region of the adenovirus, which are expression cassettes of IL-2, 4-1BBL and membrane-type IL-21 genes regulated by a broad-spectrum enhancer / promoter of human cytomegalovirus, and an expression cassette of the E1 gene regulated by a telomerase promoter specific to tumor tissue or cells; step 2, co-transfecting HEK 293 cells with a double expression cassette shuttle plasmid containing the E1A gene and the IL-2, 4-1BBL and membrane-type IL-21 genes constructed in step 1 and an adenovirus backbone plasmid to obtain a recombinant oncolytic adenovirus vector co-expressing IL-2, 4-1BBL and membrane-type IL-21.

[0008] Furthermore, the IL-2, 4-1BBL and membrane-type IL-21 genes are connected by any of the following means: viral 2A peptide or internal nucleic acid entry site.

[0009] Furthermore, the expression genes of IL-2, 4-1BBL and membrane-type IL-21 are connected in series through several 2A peptide sequences to form an open reading frame.

[0010] Furthermore, the adenovirus described in step 1 comprises any one or more of type 5, type 2, type 3, type 7, type 11, type 35 and type 5 / F35 adenovirus.

[0011] Furthermore, the tumor tissue or cell-specific telomerase promoter described in step 1 comprises any one or more of a telomerase promoter, an alpha-fetoprotein promoter, a prostate-specific antigen promoter, a transcription factor E2F-1 promoter, a survivin promoter, a hypoxia-inducible factor-1 promoter, a nestin promoter, a carcinoembryonic antigen promoter, a p53 promoter, and a Mucin 1 promoter.

[0012] The present invention also provides a recombinant oncolytic adenovirus vector expressing IL-2, 4-1BBL and membrane-type IL-21, which is constructed by the above method.

[0013] The present invention also provides an application of the above-mentioned recombinant oncolytic adenovirus vector expressing IL-2, 4-1BBL and membrane-type IL-21, wherein the recombinant oncolytic adenovirus vector is used to prepare a drug or immune adjuvant for treating cancer.

[0014] Furthermore, the cancer includes: lung cancer, liver cancer, bile duct cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, kidney cancer, bladder cancer, malignant glioma, head and neck malignant tumors, and various malignant sarcomas.

[0015] Furthermore, the oncolytic adenovirus activates anti-tumor activity by selectively replicating in telomerase-positive tumor cells and directly dissolving tumor cells.

[0016] Furthermore, after the oncolytic adenovirus is injected into the tumor, the adenovirus vector itself acts as an immune adjuvant and cooperates with the oncolytic adenovirus to express its encoded IL-2, membrane-type IL-21 and 4-1BBL gene products, thereby exerting anti-tumor activity by attracting, chemotactic and amplifying natural killer cells in the tumor microenvironment.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a recombinant oncolytic adenovirus vector that can efficiently express IL-2, 4-1BBL and membrane-type IL-21, and a construction method and application thereof. The construction method inserts the coding sequence expressing IL-2, 4-1BBL and membrane-type IL-21 into the shuttle plasmid by carrying two expression cassette plasmid vectors, and obtains a recombinant oncolytic adenovirus vector that can efficiently express IL-2, 4-1BBL and membrane-type IL-21. By intratumoral injection of the recombinant oncolytic adenovirus vector, tumor cells are lysed on the one hand, and IL-2, 4-1BBL and membrane-type IL-21 that are efficiently expressed on the other hand can effectively increase the infiltration and activity of natural killer (NK) cells in the tumor microenvironment, improve the tumor immunosuppressive microenvironment, and enhance its anti-tumor activity. Therefore, the present invention is suitable for the preparation of therapeutic drugs or immune adjuvants for various solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1This is a schematic diagram of the structure of the open reading frame in the Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 plasmid, which is formed by the tandem connection of three genes IL-2, 4-1BBL and membrane-type IL-21 sequences and the 2A peptide sequence.

[0021] FIG2 shows the identification results of the Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 plasmid, including gene expression and functional identification diagrams after infection of HEK293 cells.

[0022] Figure 3 This is a graph showing the expression levels of the early viral replication gene E1A and the late genes Hexon and Penton after tumor cells were infected with the oncolytic adenovirus Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 for different periods of time.

[0023] FIG4 is a graph showing the survival of tumor cells 48 hours after infection with oncolytic adenoviral vectors Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 of different titers.

[0024] FIG5 shows the results that tumor cells infected with oncolytic adenovirus Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 can effectively expand and activate NK cells.

[0025] Figure 6 shows the intratumoral activation of NK cells by oncolytic adenovirus Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (Ad5-1416). DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are further described below.

[0027] It should be understood that in this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0028] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0029] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0030] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0031] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass in the embodiment description of the present application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0032] The present invention provides a method for constructing a recombinant oncolytic adenovirus vector expressing human IL-2, 4-1BBL and membrane-type IL-21 proteins. The construction method comprises: step 1, inserting two expression cassettes into the E1 region of the adenovirus, which are expression cassettes of IL-2, 4-1BBL and membrane-type IL-21 genes regulated by a broad-spectrum enhancer / promoter of human cytomegalovirus (hCMV e / p), and an expression cassette of the E1 gene regulated by a telomerase promoter specific to tumor tissue or cells; step 2, co-transfecting HEK 293 cells with a double expression cassette shuttle plasmid containing the E1A gene and the IL-2, 4-1BBL and membrane-type IL-21 genes constructed in step 1 and an adenovirus backbone plasmid to obtain a recombinant oncolytic adenovirus vector co-expressing IL-2, 4-1BBL and membrane-type IL-21.

[0033] Preferably, the IL-2, 4-1BBL and membrane-type IL-21 genes are linked via any of the following means: viral 2A peptide or internal nucleic acid entry site (IRES).

[0034] The expression genes of IL-2, 4-1BBL and membrane-type IL-21 are connected in series to form an open reading frame through several (two or more, preferably two) 2A peptide sequences.

[0035] The adenovirus in step 1 includes (including but not limited to): any one or more of type 5, type 2, type 3, type 7, type 11, type 35 and type 5 / F35 adenovirus.

[0036] The tumor tissue or cell-specific telomerase promoter of step 1 includes (including but not limited to): any one or more of the telomerase promoter, alpha-fetoprotein (AFP) promoter, prostate-specific antigen (PSA) promoter, transcription factor E2F-1 promoter, survivin promoter, hypoxia-inducible factor-1 (HIF-1) promoter, Nestin promoter, carcinoembryonic antigen (CEA) promoter, p53 promoter and Mucin 1 promoter.

[0037] The present invention also provides a recombinant oncolytic adenovirus vector expressing IL-2, 4-1BBL and membrane-type IL-21 constructed by the method.

[0038] The present invention also provides the use of the recombinant oncolytic adenovirus vector expressing IL-2, 4-1BBL and membrane-type IL-21. The recombinant oncolytic adenovirus vector is used to prepare a drug or immune adjuvant for treating cancer.

[0039] Preferably, cancers include (including but not limited to): lung cancer, liver cancer, bile duct cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, kidney cancer, bladder cancer, malignant glioma, head and neck malignancies, and various types of malignant sarcomas.

[0040] Oncolytic adenoviruses can directly lyse tumor cells and activate antitumor activity by selectively replicating in telomerase-positive tumor cells.

[0041] After the oncolytic adenovirus is injected into the tumor, the adenovirus vector itself acts as an immune adjuvant and cooperates with the oncolytic adenovirus to express its encoded IL-2, membrane-bound IL-21, and 4-1BBL gene products, exerting anti-tumor activity by attracting, chemotactic, and amplifying natural killer cells in the tumor microenvironment.

[0042] In summary, the present invention further modifies the oncolytic adenovirus to increase the number and activity of NK cells in the tumor microenvironment and enhance their anti-tumor immunity. The technical solution of the present invention fully considers the following contents:

[0043] NK cells, as the most important cells of the body's natural immunity, kill target cells quickly without the need for pre-sensitization, and have many potential advantages in anti-tumor. Fully exerting and making good use of the anti-tumor effect of NK cells is one of the powerful means of immunotherapy for anti-tumor. At present, NK cells can be directly or indirectly amplified clinically through known cytokines such as IL-2, IL-15, IL-18, IL-21, and then adoptively infuse the in vitro amplified NK cells to enhance anti-tumor immunity. However, the infiltration number of the infused NK cells in solid tumors is limited, and their functions are inactivated. Therefore, the present invention aims to increase the infiltration and functional activity of NK cells in tumors and enhance their anti-tumor ability.

[0044] The present invention utilizes oncolytic adenovirus itself as an excellent gene carrier. First, several immunomodulatory genes that can induce NK cell proliferation are constructed into oncolytic adenovirus to obtain a recombinant oncolytic adenovirus vector, and then the recombinant oncolytic adenovirus vector is injected into the tumor so that it can efficiently express IL-2, 4-1BBL and membrane-type IL-21 in the tumor microenvironment, thereby promoting the proliferation and activation of NK cells in the tumor. IL-2, 4-1BBL and membrane-type IL-21 were selected as immunomodulatory genes. Among them, IL-2 efficiently expressed by recombinant oncolytic adenovirus is the first cytokine discovered to stimulate the proliferation and activation of T cells and NK cells, and has been applied clinically. 4-1BBL (CD137L) efficiently expressed by recombinant oncolytic adenovirus and its receptor 4-1BB (CD137) are a co-stimulatory molecule, belonging to the tumor necrosis factor superfamily member, which can functionally promote NK cells to release cytokines such as IFN-γ and induce ADCC. IL-21 is an important immunomodulatory cytokine that promotes the proliferation and activity of NK cells. Studies have shown that the use of one or two of the above-mentioned immunomodulatory factors alone can promote the proliferation and activity of NK cells in vitro, but the combined effect of the three is still unclear. In addition, the number of NK cells that are adopted and transferred to tumor tissue is limited. Therefore, the present invention makes full use of the oncolytic properties of oncolytic adenovirus vectors. While dissolving tumor cells, the immunomodulatory factors are highly expressed to promote NK cell proliferation to enhance anti-tumor immunity.

[0045] Therefore, the present invention provides a recombinant oncolytic adenovirus vector that can efficiently express IL-2, 4-1BBL and membrane-type IL-21, and its preparation method and application. Specifically, the oncolytic adenovirus vector used in the present invention adopts the AdMAXTM system (Mixrobix, Canada) and is divided into two parts, namely, a shuttle plasmid and a backbone plasmid. First, the shuttle plasmid is modified accordingly. The human telomerase promoter hTERTp (GenBank: AF098956) is first amplified from the DNA of human A549 lung cancer cells and inserted into the pDC515 (AdMAXTM) shuttle plasmid, named pST plasmid, and the adenovirus E1A gene is PCR amplified from pXC1 (AdMAXTM) and inserted into the pST plasmid, named pST-E1A (1) plasmid. Then, the hTERTp-E1A-pA expression cassette is amplified by PCR and reversely inserted into the pDC511 (AdMAXTM) shuttle plasmid, named pST-E1A (2) plasmid. The human CMVp promoter expression cassette was amplified from the pcDNA3.1(-)-myc-His B plasmid (InvitrogenTM) and inserted into the pST-E1A(2) shuttle plasmid, and a polypeptide sequence containing four 2A peptides and restriction sites was synthesized, such as Figure 1 As shown, the multiple cloning site in the hCMVp expression cassette is replaced, and the constructed shuttle plasmid is named pST-E1A(R) / CMVp plasmid. Then, the target genes of IL-2, 4-1BBL and membrane-type IL-21 (mIL-21) are inserted into the pST-E1A(R) / CMVp shuttle plasmid in sequence, and pST-E1A(R) / CMVp-IL-2, pST-E1A(R) / CMVp-IL-2 -4-1BBL and pST-E1A(R) / CMVp-IL-2 -4-1BBL-mIL-21 can be constructed respectively. Two or more immunomodulatory genes are connected in series by different 2A peptide sequences, and multiple target genes are connected in series by multiple 2A peptide sequences to form an open reading frame, such as Figure 3 shown.

[0046] The nucleic acid and amino acid sequences involved in the present invention are as follows:

[0047] SEQ ID NO: 1 Upstream primer for constructing plasmid

[0048] 5'-gcg cta gct agctca ata ttg gcc att agc c-3'

[0049] SEQ ID NO:2 Downstream primer for constructing plasmid

[0050] 5'-gaa gat cta taa ctt cgt ata atg tat gct ata cga agt tat cga tcgagc cat aga gcc c-3'

[0051] SEQ ID NO:3 Synthetic oligonucleotide fragment

[0052] 5'-gtcgacggtacccgagcaaaaaggagcggcagtggcgtgaagcagacattgaattttgaccttttgaaactg gcaggtgatgtagagtccaaccctgggccactcgagagagcaaaacggtctggaagtgggcagtgcaccaactacgcgttgctgaaattggcaggggatgtggaatctaatccaggccctgaattccgagccaaacgaagcggctctgga gaaggccgtggatctctgctgacctgcggagacgtagaggagaatccagggcccggatccagagctaagaggtcgggttccggcgccaccaatttcagtttgctcaagcaggctggtgatgtggaggagaatcccggtccgaagctt-3'

[0053] SEQ ID NO: 4 Human telomerase promoter upstream primer

[0054] 5'-gct cta gat gcg ctg tcg ggg cca ggc-3'

[0055] SEQ ID NO: 5 Human telomerase promoter downstream primer

[0056] 5'-ccg aat tcc acg tgc gcc cac gtg cgc c-3'

[0057] SEQ ID NO: 6 Human telomerase promoter sequence inserted into plasmid

[0058] 5’-ctcgagagctgcgctgtcggggccaggccgggctcccagtggattcgcgggcacagacgcccaggaccg cgcttcccacgtggcggagggactggggacccgggcacccgtcctgccccttcaccttccagctccgcctcctccgcgcggaccccgccccgtcccgacccctcccgggtccccggcccagccccctccgggccctcccagcccctccccttcctttccgcggccccgccctctcctcgcggcgcgagtttcaggcagcgctgcgtcctgctgcgcacgtgggaagccctggccgatgggctcgacgcacgtgggcgcacgtgggcgcacgtgggagcttggcattccggtacagatccacc-3’

[0059] SEQ ID NO:7 Forward primer for amplifying IL-2 gene

[0060] 5’-ccg ctc gag atg tac agg atg caa ctc c-3’

[0061] SEQ ID NO:8 Reverse primer for amplifying IL-2 gene

[0062] 5’-cgg aat tca gtc agt gtt gag atg atg-3’

[0063] SEQ ID NO:9 Human IL-2 sequence inserted into plasmid

[0064] 5'-atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagtgcacctacttcaagttctacaaagaaaacacagctacaactggagcatttactg ctggatttacagatgattttgaatggaattaataattacaagaatcccaaactcaccaggatgctcacatttaagttttacatgcccaagaaggccacagaactgaaacatcttcag tgtctagaagaagaactcaaacctctggaggaagtgctaaatttagctcaaagcaaaaactttcacttaagacccagggacttaatcagcaatatcaacgtaatagttctggaacta aagggatctgaaacaacattcatgtgtgaatatgctgatgagacagcaaccattgtagaatttctgaacagatggattaccttttgtcaaagcatcatctcaacactgacttga-3'

[0065] SEQ ID NO:10 Amino acid sequence corresponding to the human IL-2 sequence inserted into the plasmid

[0066] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0067] SEQ ID NO: 11 Upstream primer for amplifying 4-1BBL gene

[0068] 5'-gcg cga aga tct atg gaa tac gcc tct gac gc-3'

[0069] SEQ ID NO: 12 Downstream primers for amplifying 4-1BBL gene

[0070] 5'-cgg gat cct tcc gac ctc ggt gaa gg-3'

[0071] Human 4-1BBL sequence inserted into the plasmid, SEQ ID NO:13

[0072] 5’-atggaatacgcctctgacgcttcactggaccccgaagccccgtggcctcccgcgccccgcgctcgcgcctgccgcgtactgccttgggccctggtcgcggggctgctgctgctgctgctgctcgctgccgcctgcgccgtcttcctcgcctgcccctgggccgtgtccggggctcgcgcctcgcccggctccgcggccagcccgagactccgcgagggtcccgagctttcgcccgacgatcccgccggcctcttggacctgcggcagggcatgtttgcgcagctggtggcccaaaatgttctgctgatcgatgggcccctgagctggtacagtgacccaggcctggcaggcgtgtccctgacggggggcctgagctacaaagaggacacgaaggagctggtggtggccaaggctggagtctactatgtcttctttcaactagagctgcggcgcgtggtggccggcgagggctcaggctccgtttcacttgcgctgcacctgcagccactgcgctctgctgctggggccgccgccctggctttgaccgtggacctgccacccgcctcctccgaggctcggaactcggccttcggtttccagggccgcttgctgcacctgagtgccggccagcgcctgggcgtccatcttcacactgaggccagggcacgccatgcctggcagcttacccagggcgccacagtcttgggactcttccgggtgacccccgaaatcccagccggactcccttcaccgaggtcggaataa-3’

[0073] Amino acid sequence corresponding to the human 4-1BBL sequence inserted into the plasmid, SEQ ID NO:14

[0074] MEYASDASLDPEAPWPPARARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE

[0075] SEQ ID NO: 15 Upstream primer for amplifying membrane-type IL-21 gene

[0076] 5'-cgg gat cca tga gat cca gtc ctg gca ac-3'

[0077] SEQ ID NO: 16 Downstream primers for amplifying membrane-type IL-21 gene

[0078] 5'-ccc aag ctt acc tgt ggt tgc agt aaa gg-3'

[0079] SEQ ID NO: 17 Human IL-21 sequence inserted into plasmid

[0080] 5’-atgagatccagtcctggcaacatggagaggattgtcatctgtctgatggtcatcttcttggggacactggtccacaaatcaagctcccaaggtcaagatcgccacatgattagaatgcgtcaacttatagatattgttgatcagctgaaaaattatgtgaatgacttggtccctgaatttctgccagctccagaagatgtagagacaaactgtgagtggtcagctttttcctgttttcagaaggcccaactaaagtcagcaaatacaggaaacaatgaaaggataatcaatgtatcaattaaaaagctgaagaggaaaccaccttccacaaatgcagggagaagacagaaacacagactaacatgcccttcatgtgattcttatgagaaaaaaccacccaaagaattcctagaaagattcaaatcacttctccaaaagatgattcatcagcatctgtcctctagaacacacggaagtgaagattcccaattcgcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaccacaggtaa-3’

[0081] Amino acid sequence corresponding to the human IL-21 sequence inserted into the plasmid of SEQ ID NO:18

[0082] MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSQFAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHR

[0083] SEQ ID NO:19 pDC523 IL-2 / 4-1BBL / mIL-21 shuttle plasmid

[0084]

[0085] SEQ ID NO:20 Amino acid sequence corresponding to pDC523 IL-2 / 4-1BBL / mIL-21 shuttle plasmid

[0086] MEYASDASLDPEAPWPPARARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSELERAKRSGSGQCTNYALLKLAGDVESNPGPGSYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTRSRAKRSGSGATNFSLLKQAGDVEENPGPKLRSRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFL PAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSQFAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHR

[0087] 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 specific 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.

[0088] If no specific techniques or conditions are specified in the examples, they are all conventional methods, carried out according to the techniques or conditions described in the literature in the field, or according to the product instructions. The reagents or instruments used are conventional products that can be purchased through regular channels.

[0089] The primers, DNA sequence synthesis and DNA sequence determination used in the examples were all completed by Sangon Biotechnology Co., Ltd.

[0090] The acquisition methods of various biological materials described in the examples are only to provide an experimental acquisition method to achieve the specific disclosed purpose, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples; in industrial implementation, various cells derived from mammals such as rats, mice, pigs or humans are in vitro, and include those obtained from cell banks or commercially purchased, as well as those prepared according to the introduction of existing literature, and those induced by various commercially available stem cells using known methods.

[0091] The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. The embodiments will help to understand the present invention, but the content of the present invention is not limited to the following embodiments.

[0092] Example 1: Transformation of shuttle plasmid.

[0093] The adenovirus shuttle plasmid suitable for encoding multiple genes was constructed, and another shuttle plasmid pDC52342A was constructed based on the pDC511 shuttle plasmid in the AdMAXTM system. The specific process includes the following:

[0094] 1.1. A pair of primers were synthesized, wherein the upstream primer was 5'-gcg cta gct agctca ata ttg gcc att agcc-3' (SEQ ID NO: 1, with an Nhe I site introduced at the 5' end) and the downstream primer was 5'-gaa gat cta taa ctt cgt ataatg tat gct ata cga agt tat cga tcg agc cat aga gcc c-3' (SEQ ID NO: 2, with a BglII site and a LoxP sequence introduced at the 5' end). PCR amplification was performed using pcDNA3.1(-)-myc-His B plasmid (InvitrogenTM) as a template. High-fidelity Q5 PCR enzyme (NEB) was used for amplification. The amplification conditions were 98°C for 10 s, 72°C for 20 s, and 72°C for 60 s for 30 cycles. A PCR fragment of 1180 bp (i.e., CMVp expression cassette) was obtained. The fragment was amplified using Nhe After double digestion with I and BglII, the digested fragment (digested fragment 1) was recovered from the gel, and the pDC511 shuttle plasmid was double digested with Xba I and BamH I, and the digested large fragment (digested fragment 2) was recovered from the gel. Digested fragment 1 and digested fragment 2 were ligated in the presence of ligase, and then transformed into DH5 competent medium. The cloned PCR fragment for digestion identification was inserted into the pDC511 plasmid and then sequenced to confirm that the CMVp expression cassette sequence obtained by PCR was correct. The obtained plasmid was named pDC523.

[0095] 1.2. Synthesize an oligonucleotide fragment comprising four 2A peptide sequences (E2A, F2A, T2A and P2A) and a multiple cloning site (the nucleotide sequence of the oligonucleotide fragment is shown in SEQ ID NO: 3 in the sequence table). The oligonucleotide fragment is double-digested with Nhe I and Hind III and a small fragment (enzyme-digested fragment 3) is recovered from gel. The pDC523 plasmid obtained in 1.1 is also double-digested with Nhe I and Hind III and a large fragment (enzyme-digested fragment 4) is recovered from gel. In the presence of ligase, the enzyme-digested fragment 3 and the enzyme-digested fragment 4 are ligated and subsequently transformed into DH5 competent cells. A clone is selected for enzyme digestion and identification. The oligonucleotide fragment is inserted into the pDC523 plasmid and then sequenced. The shuttle plasmid with the correct sequence is named pDC523 42A plasmid.

[0096] The spectrum of the pDC523 42A plasmid is as follows Figure 1 As shown in the figure, the structure of the open reading frame in the Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 plasmid is formed by the series connection of three genes IL-2, 4-1BBL and membrane-type IL-21 sequence and 2A peptide sequence. The pDC523 42A plasmid is used to insert the target gene in the following examples.

[0097] Example 2: Cloning of telomerase promoter.

[0098] For obtaining the telomerase promoter, the specific process includes:

[0099] A pair of primers for human telomerase promoter was designed, the upstream primer was 5'-gct cta gat gcg ctg tcg gggcca ggc-3' (SEQ ID NO: 4, Xba I site introduced at the 5' end), and the downstream primer was 5'-ccg aat tcc acg tgc gcccac gtg cgc c-3' (SEQ ID NO: 5, EcoR I site introduced at the 5' end). Human lung cancer A549 cell genomic DNA was used as a template for PCR amplification. High-fidelity Q5 PCR enzyme (NEB) was used for amplification. The amplification conditions were 98°C for 10 s, 72°C for 20 s, and 72°C for 20 s for 30 cycles. A PCR fragment of 297 bp was obtained. After double digestion with Xba I and EcoR I sites, the digested fragment (3') was recovered from gel. pDC315 plasmid (AdMAXTM) was also digested with Xba I and EcoR I sites. After double restriction digestion at site I, the large restriction fragment (restriction fragment 4') was recovered from the gel, and the restriction fragment 3' and the restriction fragment 4' were connected, and then transformed into DH5 competent cells. The cloned restriction fragment was selected and identified, and the PCR fragment was inserted into the pDC315 plasmid and sequenced. It was confirmed that the fragment obtained by PCR was completely consistent with the human telomerase promoter sequence (GenBank: AF098956), and three E-box (CACGTG) boxes from the C-myc gene regulatory sequence were inserted at the 3 ends of the telomerase promoter (the sequence is shown in SEQ ID NO: 6 in the sequence list).

[0100] Example 3: Cloning of IL-2, 4-1BBL and membrane-type IL-21 (mIL-21) genes.

[0101] The target genes IL-2, 4-1BBL and membrane-type IL-21 (mIL-21) were inserted into the pDC523 42A plasmid obtained in Example 1. The specific process includes the following steps:

[0102] 3.1. 5 ml of peripheral blood was drawn from healthy subjects, and peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation medium. After stimulation with LPS (10 ng / ml) for 12 hours, total cellular RNA was extracted using an RNA extraction kit (Qiagen). Using this RNA as a template, cNDA was synthesized using a reverse transcription kit (Takara) and named PBMCs cNDA. 1×10 6Total RNA from personal K562 leukemia cells was used as a template to synthesize cNDA using a reverse transcription kit (Takara) and was named K562 cNDA.

[0103] 3.2. This step designs 6 pairs of primers for amplifying the target gene and constructing related plasmids, which specifically includes the following steps:

[0104] 3.2.1. Primers for amplifying IL-2 gene were designed. The upstream primer was 5'-ccg ctc gag atg tac aggatg caa ctc c-3' (SEQ ID NO: 7) and the downstream primer was 5'-cgg aat tca gtc agt gtt gag atgatg-3' (SEQ ID NO: 8). The PBMCs cDNA obtained in 3.1 was used as a template for PCR amplification. High-fidelity Q5PCR enzyme (NEB) was used for amplification. The amplification conditions were 98°C for 10 s, 72°C for 20 s, and 72°C for 20 s for 30 cycles. The PCR fragment was amplified and the digested fragment (enzyme digested fragment 5) was recovered from gel after double enzyme digestion. The large fragment (enzyme digested fragment 6) of pDC523 42A plasmid was also recovered from gel after double enzyme digestion. The PCR fragment and the pDC523 42A shuttle plasmid fragment were ligated and transformed into DH5 competent cells. The PCR fragment was identified by restriction digestion and inserted into pDC523 The 42A plasmid was then sequenced, confirming that the fragment obtained by PCR was completely consistent with the human IL-2 sequence (the nucleotide sequence is shown in SEQ ID NO:9 in the sequence table, and the corresponding amino acid sequence is shown in SEQ ID NO:10), and the obtained plasmid was named pDC523 IL-2;

[0105] 3.2.2. Primers for amplifying 4-1BBL gene were designed. The upstream primer was 5'-gcg cga aga tct atggaa tac gcc tct gac gc-3' (SEQ ID NO: 11, with BglII site introduced at the 5' end) and the downstream primer was 5'-cgg gatcct tcc gac ctc ggt gaa gg-3' (SEQ ID NO: 12, with BamH I site introduced at the 5' end). PCR amplification was performed using PBMC scDNA of 3.1 as template. High-fidelity Q5 PCR enzyme (NEB) was used for amplification. The amplification conditions were 98°C for 10s, 72°C for 20s, and 72°C for 30 cycles. The PCR fragment was amplified and double-digested with Bgl II and BamH I, and the digested fragment (digested fragment 7) was recovered from the gel. The pDC523 IL-2 plasmid obtained in 3.2.1 was cloned and cloned with BamH I. After restriction digestion at site I and dephosphorylation treatment, the large fragment (restriction fragment 8) was recovered from the gel. Restriction fragment 7 and restriction fragment 8 were ligated in the presence of ligase, and then transformed into DH5 competent medium. The cloned restriction digestion and identification PCR fragment was inserted into the pDC523 IL-2 plasmid and then sequenced. It was confirmed that the fragment obtained by PCR was completely consistent with the human 4-1BBL sequence (GenBank: NM_003811, whose nucleotide sequence is shown in SEQ ID NO: 13, and the corresponding amino acid sequence is shown in SEQ ID NO: 14). The obtained plasmid was named pDC523 IL-2 / 4-1BBL.

[0106] 3.2.3. Primers for amplifying membrane-type IL-21 gene were designed. The upstream primer was 5'-cgg gat cca tgagat cca gtc ctg gca ac-3' (SEQ ID NO: 15, with BamH I site introduced at the 5' end) and the downstream primer was 5'-cccaag ctt acc tgt ggt tgc agt aaa gg-3' (SEQ ID NO: 16). The PBMCs cDNA in 3.1 was used as a template for PCR amplification. High-fidelity Q5 PCR enzyme (NEB) was used for amplification. The amplification conditions were 98°C for 10s, 72°C for 20s, and 72°C for 30 cycles. A PCR fragment of 790 bp was obtained. The fragment was digested with double enzymes and then the digested fragment (digested fragment 9) was recovered from the gel. The pDC523IL-2 / 4-1BBL plasmid obtained in 3.2.2 was cloned and cloned with Bgl II and Xho After double enzyme digestion, the large fragment (enzyme-cut fragment 10) was recovered from the gel. In the presence of ligase, the enzyme-cut fragment 9 and the enzyme-cut fragment 10 were connected, and then transformed into DH5 competent state. The cloned enzyme-cut identification PCR fragment was inserted into the pDC523 IL-2 / 4-1BBL plasmid and then sequenced. It was confirmed that the fragment obtained by PCR was completely consistent with the human IL-21 sequence (GenBank: NM_000074, whose nucleotide sequence is shown in SEQ ID NO: 17, and the corresponding amino acid sequence is shown in SEQ ID NO: 18). The obtained plasmid was named pDC523 IL-2 / 4-1BBL / mIL-21, referred to as shuttle plasmid. The sequences of the three genes IL-2, 4-1BBL and mIL-21 of the plasmid were respectively connected in series by two 2A peptide sequences to form an open reading frame (whose nucleotide sequence is shown in SEQ ID NO: 19, and the corresponding amino acid sequence is shown in SEQ ID NO: 20), as shown in Figure 2, which shows a schematic diagram of the structure of the open reading frame.

[0107] Example 4: Construction of recombinant oncolytic adenoviral vector.

[0108] By AdMax TM Cre recombinant adenovirus system instructions, that is, the construction of adenovirus vectors encoding multiple genes. The specific process is as follows:

[0109] The shuttle plasmid Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (abbreviated as Ad5-1416) obtained in Example 3 was extracted with an EndoFree Plasmid Midi Kit (QIAGEN), and the 260 / 280 ratio was determined to be about 1.9, and the plasmid concentration was adjusted to 1 g / l; pBGHfrt△E1,3Cre (for construction of type 5 adenovirus vector) and pBGHfrt△E1,3Cre (5 / F35) (for construction of type 5 / F35 adenovirus vector) adenovirus backbone plasmids were extracted with an EndoFree Plasmid Maxi Kit (QIAGEN), and the 260 / 280 ratio was determined to be about 1.9, and the plasmid concentration was adjusted to 1 g / l. Low-generation HEK 293T cells were passaged to 60 mm 3 When the cells were 60-70% confluent, the shuttle plasmid Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 and the backbone plasmid pBGHfrt△E1,3Cre or pBGHfrt△E1,3Cre (5 / F35) were added to the culture dish by calcium phosphate precipitation method (CalPhos TM Mammalian Transfection Kit, ClonTech) was used for transfection. The transfection system is shown in Table 1 below. After the transfection system was prepared, it was added to HEK293 cells for transfection after incubation for 10 minutes.

[0110] Table 1. Transfection systems.

[0111]

[0112] After transfection, agarose-containing culture medium was prepared according to the system shown in Table 2.

[0113] Table 2. Culture medium system.

[0114]

[0115] Solution A in Table 2 was boiled and dissolved in a microwave oven, and then placed in a 44°C water bath. After the temperature dropped to 44°C, 30 ml was taken and mixed with solution B placed at 44°C. The culture medium in each culture dish after the transfection plasmid was aspirated, and 15 ml of the A / B mixture was added to each culture dish.

[0116] Each culture dish was placed in a 37°C, 5% CO2 incubator for culture. The agarose-containing culture medium will form a solid state. The condition of the cells in the culture dish was observed regularly. Plagues were formed in about 10 to 14 days. The plaques were picked out and further amplified in HEK293T cells. Viral DNA was extracted from some cells for PCR identification. The correct clone was named Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21. After 2 or 3 rounds of amplification, the HEK 293T cells were collected and lysed by freeze-thawing. The adenovirus purification kit (ViraBind TM After purifying adenovirus using the Adenovirus Purification Kit (Cell Biolabs, Inc), two types of recombinant oncolytic adenoviruses, Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21, were obtained. The two recombinant oncolytic adenoviruses were then expressed and their functions were identified.

[0117] Example 5: Expression and identification of IL-2, 4-1BBL and membrane-type IL-21 in HEK 293T cells infected with Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21.

[0118] The purified recombinant oncolytic adenovirus Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 obtained in Example 4 was used to infect HEK 293T cells for expression and functional identification. The specific process includes the following:

[0119] 5.1. Transduce HEK 293T cells into 6-well plates (two replicate wells) at a cell density of 1×10 6 / 3ml / well, after the cells adhered to the wall, Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 was added at a virus titer of 50MOI and 100MOI. After continued culture for 24 hours, the supernatant was collected and the expression of IL-2 was detected by ELISA, and the cells were collected for flow cytometry detection of 4-1BBL and IL-21.

[0120] 5.2. The supernatant collected in 5.1 was tested by ELISA (Dakoway Biotechnology Co., Ltd.). The results were as follows: Figure 2B As shown, it can be seen that 24 hours after HEK 293T cells were infected with Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21, HEK293T cells were able to secrete a large amount of IL-2.

[0121] 5.3. The cells collected in 5.1 were digested with 0.25% trypsin + 0.01% EDTA to make a single cell suspension, washed twice with PBS, stained with human 4-1BBL (Biolegend) and IL-21 (Biolegend), and then detected by flow cytometry. The results are as follows: Figure 2A It was shown that Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 could effectively express human 4-1BBL and IL-21 after infecting HEK 293T cells for 24 hours.

[0122] Figure 2 shows the results of the identification of the Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 plasmid. Gene expression and functional identification diagram after infection of HEK 293 cells. Figure 2A This is the peak graph detected by flow cytometry 24 hours after HEK 293T cells were infected with Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21; Figure 2B These are the ELISA test results for IL-2 in the supernatant of HEK 293T cells 24 hours after infection with Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21.

[0123] Example 6: After tumor cells (K562, HCT116 and RKO) were infected with Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21, the expression of proteins related to viral replication was detected by Western blot.

[0124] The purified recombinant adenovirus Ad5 IL-2 / 4-1BBL / mIL-21 obtained in Example 4 was used to infect human K562 leukemia cells and colorectal cancer HCT116 and RKO cells, and then Western blot was used to detect the expression levels of the early gene E1A and the late genes Hexon and Penton of viral replication. Specifically, the following process was included:

[0125] 1×10 6 Tumor cells were transferred to 6-well plates, and Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 was added to the cells at a virus titer of 100 MOI and cultured for 24, 48, and 72 hours. The cells were collected and Western blot was used to detect the expression of the early viral replication gene E1A and the late genes Hexon and Penton.

[0126] The results showed that the oncolytic adenovirus Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 could infect different pancreatic cancer cells, and the expression levels of the virus's early gene E1A and late genes Hexon and Penton in different tumor cells varied with the extension of infection time, indicating that the virus can replicate in tumor cells and is related to the cell type.

[0127] See also Figure 3 As shown, the figure shows the expression levels of the early viral replication gene E1A and the late genes Hexon and Penton after tumor cells were infected with the oncolytic adenovirus Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 for different periods of time.

[0128] Example 7: Oncolytic effect of Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 on tumor cells (K562, HCT116 and RKO).

[0129] The purified recombinant adenovirus Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 obtained in Example 4 was used to infect tumor cells K562, HCT116 and RKO, and the tumor cell lysis was observed. The specific process includes the following:

[0130] 5×10 3 Tumor cells were transferred to a 96-well plate, and Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 was added to the cells at virus titers of 0, 50, 100, and 500 MOI and cultured for 48 hours. Then, 10 μl of CCK8 (Transgene) reagent was added to the plate. After incubation at 37°C for 2 hours, the absorbance was measured at 450 nm using a microplate reader to analyze cell viability to determine the lytic effect of oncolytic virus on tumor cells.

[0131] The results showed that with the increase of virus titer, the oncolytic effect of Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 on tumor cells (K562, HCT116 and RKO) gradually increased in a concentration-dependent manner; and different tumor cells have different sensitivities to oncolytic adenoviruses, and their oncolytic effects are different.

[0132] See Figure 4, which shows the survival of tumor cells 48 hours after infection with oncolytic adenovirus vector Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 of different titers.

[0133] Example 8: Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 induces expansion and activation of NK cells.

[0134] The purified adenovirus Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 obtained in Example 3 was used to infect tumor cells, and the effects of tumor cells infected with the virus on the proliferation and activity of NK cells were observed. Specifically, the following process was included:

[0135] 8.1. Tumor cells (K562 and HCT116) were infected with Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 virus at an MOI titer of 100 for 24 hours, and then the tumor cells were collected.

[0136] 8.2. NK cells were isolated from healthy human peripheral blood mononuclear cells (PBMCs) using anti-CD56 immunomagnetic beads (Miltenyi Biopharmaceuticals). NK cells were then co-incubated with tumor cells infected with Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (Ad5-1416) oncolytic virus at a ratio of 1:5, and CD56 was detected by flow cytometry. + The proportion of cells.

[0137] 8.3. Incubate NK cells with 2 μM CFSE in a 37°C cell culture incubator for 10 minutes to label the sorted NK cells, and then co-incubate with tumor cells infected with Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (Ad5-1416) virus for 4 days, and detect CFSE by flow cytometry. + At the same time, the cell supernatant was collected and the secretion of IFN-γ in the supernatant was detected by ELISA.

[0138] The results showed that it could significantly promote the proliferation of NK cells. Compared with the control virus (Ad5-1459), co-incubation with tumor cells infected with Ad5-1416 virus could significantly increase the expression of CD56 + NK and CFSE + NK cell count. This indicates that Ad5-1416 virus significantly promotes the proliferation of NK cells. At the same time, the level of INF-γ in the supernatant increased significantly, indicating that Ad5-1416 virus promotes NK cell activation.

[0139] See Figure 5 , which shows that tumor cells infected with oncolytic adenovirus Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 can effectively expand and activate NK cells. Figure 5ANK cells isolated from healthy human PBMCs were co-incubated with tumor cells infected with oncolytic adenovirus Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (Ad5-1416) or control virus Ad5TERTp-E1A (Ad5-1459), and CD56 was detected by flow cytometry. + The proportion of cells; Figure 5B After CFSE-labeled NK cells were co-incubated with tumor cells infected with oncolytic adenovirus (Ad5-1459 or Ad5-1416) for 4 days, CFSE was detected by flow cytometry. + Changes in cell proportions; Figure 5C is the level of IFN-γ in the supernatant.

[0140] Example 9: Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 induces intratumoral expansion and anti-tumor ability of NK cells.

[0141] The purified adenovirus Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 obtained in Example 3 was injected intratumorally, and NK cells were adoptively transferred to observe its anti-tumor effect and the infiltration of NK cells in the tumor. The specific process includes the following:

[0142] 9.1. NOD / SCID mice were subcutaneously inoculated with 1×10 7 A subcutaneous tumor model was constructed in mice with a number of HCT116 cells. On day 12 of inoculation, oncolytic adenovirus Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (Ad5-1416) or control virus (Ad5-1459) was injected intratumorally twice a week for a total of 5 injections. At the same time, 3×10 6 The number of NK cells was injected twice in total. The tumor size of the mice was measured during the period, and the mice were killed 4 days after the last virus injection. The tumor tissue was collected and weighed to analyze its anti-tumor effect.

[0143] 9.2. The tumor tissues of mice were fixed with 4% paraformaldehyde for 48 hours, and then dehydrated, paraffin-embedded and sectioned. Immunohistochemistry was performed to detect CD56 in the tissues. + The ratio of cells was used to analyze the infiltration of NK cells.

[0144] The results showed that oncolytic adenovirus Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (Ad5-1416) could significantly inhibit the growth of tumors in mice with colon cancer. Immunohistochemical staining results showed that after adoptive transfer of NK cells to mice treated with oncolytic adenovirus Ad5 TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (Ad5-1416), CD56 in tumor tissues + The number of infiltrated cells increased significantly, indicating that the Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (Ad5-1416) virus can increase the infiltration and function of NK cells in tumor tissues.

[0145] See Figure 6, which shows the activation of NK cells in tumors by oncolytic adenovirus Ad5TERTp-E1A-CMVp-IL-2 / 4-1BBL / mIL-21 (Ad5-1416). Fig. 6A Schematic diagram of the construction and treatment of NOD / SCID mouse subcutaneous tumor model; Figure 6B -D Mouse tumor growth curve, tumor size and tumor weight statistics; Fig. 6E Immunohistochemical staining to detect CD56 in tumor tissue + Cell infiltration.

[0146] The present invention provides a recombinant oncolytic adenovirus vector expressing human IL-2, 4-1BBL and membrane-type IL-21 proteins, and a construction method and application thereof, and specifically relates to a method for activating NK cells in a tumor and its application after injecting a recombinant oncolytic adenovirus vector encoding multiple immunomodulatory genes into a tumor. The method constructs a shuttle plasmid carrying two expression cassettes, inserts the coding sequence expressing IL-2, 4-1BBL and membrane-type IL-21 into the shuttle plasmid, and obtains a recombinant oncolytic adenovirus vector that can efficiently express IL-2, 4-1BBL and membrane-type IL-21. The recombinant oncolytic adenovirus vector can efficiently express IL-2, 4-1BBL and membrane-type IL-21, and after intratumoral injection, it can effectively increase the infiltration and activity of natural killer (NK) cells in the tumor microenvironment, improve the tumor immunosuppressive microenvironment, and enhance its anti-tumor activity.

[0147] The construction method and use of the recombinant oncolytic adenovirus vector expressing IL-2, 4-1BBL and membrane-type IL-21 provided by the present invention belong to the fields of life science and biotechnology. The expression genes of IL-2, 4-1BBL and membrane-type IL-21 are connected in series to form an open reading frame through several 2A peptide sequences. On the one hand, the recombinant oncolytic adenovirus vector can replicate in tumor cells, causing the lysis of tumor cells to produce anti-tumor activity; on the other hand, the recombinant oncolytic adenovirus vector can efficiently express IL-2, 4-1BBL and membrane-type IL-21, increase the infiltration and activity of the number of NK cells in the tumor, improve the tumor immunosuppressive microenvironment, restore or activate the anti-tumor and anti-tumor activity of NK cells, and provide an important tool for the immunotherapy of tumors.

[0148] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of this application without departing from the principles of the present invention should be included in the protection scope of this application.

Claims

1. A method for constructing a recombinant oncolytic adenovirus vector expressing human IL-2, 4-1BBL and membrane-bound IL-21 protein, characterized in that: The construction method comprises: Step 1, inserting two expression cassettes into the E1 region of adenovirus, which are expression cassettes of IL-2, 4-1BBL and membrane-type IL-21 genes regulated by the broad-spectrum enhancer / promoter of human cytomegalovirus, and an expression cassette of the E1 gene regulated by the telomerase promoter specific to tumor tissue or cells; Step 2: The double expression cassette shuttle plasmid containing the E1A gene and IL-2, 4-1BBL and membrane-type IL-21 genes constructed in step 1 is co-transfected with the adenovirus backbone plasmid into HEK 293 cells to obtain a recombinant oncolytic adenovirus vector that co-expresses IL-2, 4-1BBL and membrane-type IL-21.

2. The construction method according to claim 1, characterized in that: The IL-2, 4-1BBL and membrane-type IL-21 genes are connected by any of the following means: viral 2A peptide or internal nucleic acid entry site.

3. The construction method according to claim 2, characterized in that: The expression genes of IL-2, 4-1BBL and membrane-type IL-21 are connected in series to form an open reading frame through several 2A peptide sequences.

4. The construction method according to claim 1, characterized in that: The adenovirus described in step 1 comprises any one or more of type 5, type 2, type 3, type 7, type 11, type 35 and type 5 / F35 adenovirus.

5. The construction method according to claim 1, characterized in that: The tumor tissue or cell-specific telomerase promoter described in step 1 comprises any one or more of a telomerase promoter, an alpha-fetoprotein promoter, a prostate-specific antigen promoter, a transcription factor E2F-1 promoter, a survivin promoter, a hypoxia-inducible factor-1 promoter, a nestin promoter, a carcinoembryonic antigen promoter, a p53 promoter, and a Mucin 1 promoter.

6. A recombinant oncolytic adenoviral vector expressing IL-2, 4-1BBL and membrane-bound IL-21 constructed by the method according to any one of claims 1 to 5.

7. A use of a recombinant oncolytic adenoviral vector expressing IL-2, 4-1BBL and membrane-bound IL-21 as claimed in claim 6, characterized in that: The recombinant oncolytic adenovirus vector is used to prepare drugs or immune adjuvants for treating cancer.

8. The use of the recombinant oncolytic adenovirus vector according to claim 7, characterized in that: The cancers include: lung cancer, liver cancer, bile duct cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, kidney cancer, bladder cancer, malignant glioma, head and neck malignant tumors, and various malignant sarcomas.

9. The use of the recombinant oncolytic adenovirus vector according to claim 7, characterized in that: The oncolytic adenovirus directly dissolves tumor cells by selectively replicating in telomerase-positive tumor cells, thereby activating anti-tumor activity.

10. The use of the recombinant oncolytic adenovirus vector according to claim 9, characterized in that: After the oncolytic adenovirus is injected into the tumor, the adenovirus vector itself acts as an immune adjuvant and cooperates with the oncolytic adenovirus to express its encoded IL-2, membrane-type IL-21 and 4-1BBL gene products, thereby exerting anti-tumor activity by attracting, chemotactic and amplifying natural killer cells in the tumor microenvironment.

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