Oncolytic adenovirus vector for expressing VEGF (vascular endothelial growth factor) and TGF (transforming growth factor) beta trapping agent as well as construction method and application thereof

Abstract

The invention provides an oncolytic adenovirus vector for expressing VEGF and TGF beta trapping agents and a construction method and application of the oncolytic adenovirus vector, in the oncolytic adenovirus vector, oncolytic adenovirus is formed by inserting two head-to-head expression cassettes into an E1 region of type-5 adenovirus, one expression cassette is expression of type-5 adenovirus-derived E1A gene regulated by a human telomerase promoter, and the other expression cassette is expression of type-5 adenovirus-derived E1A gene regulated by a human telomerase promoter. The other expression cassette is expression of VEGF and / or TGF beta trapping agent genes regulated by a human cytomegalovirus promoter / enhancer; the vector is a type 5 adenovirus vector and a type 5 / 35 adenovirus vector. The invention also provides a construction method and application of the oncolytic adenovirus vector. After the oncolytic adenovirus of the oncolytic adenovirus vector infects a plurality of human and mouse tumor cells, VEGF and TGF beta generated by the tumor cells can be effectively neutralized, and the oncolytic adenovirus vector can be used for preparing various drugs for treating solid tumors with high expression of VEGF and / or TGF beta.

Description

Technical Field

[0001] The present invention belongs to the fields of life science and biotechnology, and specifically relates to an oncolytic adenovirus vector expressing VEGF and TGFβ trapping agents, and a construction method and application thereof. Background Art

[0002] Malignant tumors have become the number one killer of human health, and the mortality rate has risen to the top of all diseases. Although great progress has been made in the diagnosis and treatment of tumors, the efficacy of traditional treatments is still very limited for patients in the middle and late stages, especially those with distant metastasis and recurrence. Therefore, it is imperative to find new treatments. In the past decade, tumor immunotherapy has made breakthrough progress, and a series of new immunotherapies have been introduced in the clinic, such as therapeutic tumor vaccines, immune checkpoint blockers, chimeric antigen receptor T cell technology (CAR-T), and oncolytic viruses T-Vec, etc., which have achieved better efficacy than traditional treatments for certain tumors, with fewer toxic side effects and relatively high safety. However, the above-mentioned new immunotherapies have only shown significant efficacy in a few types of tumors, and have not shown obvious advantages in most tumors. This is obviously related to the biological diversity of different tumors and the complexity of the body's immune system.

[0003] Even the current new immunotherapy agents have limited effectiveness in treating tumors when used alone. For example, the immune checkpoint blocker PD-1 has an efficacy of 20-40% in treating various tumors. The reasons why most solid tumors are ineffective against tumor immunotherapy are complex. One important reason is that solid tumors can form a strong tumor immunosuppressive microenvironment. This tumor immunosuppressive microenvironment is composed of tumor cells, tumor support cells (such as tumor-associated fibroblasts), tumor-associated immune cells, tumor vascular endothelial cells, and various soluble immunosuppressive factors, including VEGF, TGFβ, IL-10, and IDO1. These cells and soluble immunosuppressive factors form a powerful immunosuppressive microenvironment that can prevent the entry of various immune effector cells (such as T cells, NK cells, etc.), inhibit the function of the immune effector cells that enter, and cause them to lose their anti-tumor activity. Based on the infiltration of T cells inside the tumor, tumors are usually divided into two categories: "cold" tumors and "hot" tumors. Current research shows that "hot" tumors often have a good effect on immunotherapy, while "cold" tumors are often ineffective or have little response to immunotherapy. How to convert "cold" tumors into "hot" tumors is a current research hotspot, and oncolytic viruses have great potential and advantages in this regard.

[0004] Currently, many viruses, such as adenoviruses, retroviruses, herpes simplex virus, vaccinia virus, measles virus, myxoma virus, and reovirus, can be engineered into oncolytic viruses. A common characteristic of oncolytic viruses is their ability to specifically lyse tumors while sparing normal cells. The most studied are oncolytic adenoviruses (OAs), also known as conditionally replicating adenoviruses (CRAs). The most common approach to engineering adenoviruses into oncolytic viruses is to control the expression of the E1A gene, essential for adenoviral replication, through specific tumor-specific promoters, thereby creating oncolytic adenoviruses that replicate in specific tumor cells. Theoretically, these oncolytic adenoviruses can replicate extensively in tumor cells, leading to tumor cell lysis and the release of numerous progeny viruses that can infect adjacent tumor cells for a second round of replication and lysis, ultimately eliminating the tumor. As a novel targeted anti-tumor strategy, current research focuses on enhancing the anti-tumor activity of oncolytic adenoviruses. Oncolytic adenovirus has several major 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 for research, easy to operate, and has a large packaging capacity. It can insert 8kb of target genes, which is very conducive to expressing the corresponding products of genes with anti-tumor activity to exert anti-tumor activity. For example, it can express various cytokines that activate lymphocytes, such as IL-2, IL-7, IL-12 and / or IL-15, and can also express shRNA, Traps, etc. to inhibit various immunosuppressive cytokines such as VEGF, TGFβ, and IDO1, as well as immune checkpoint molecules such as PD-L1 / 2 and TIM-3 expressed on the surface of tumor and inhibitory immune cells.

[0005] Rapid tumor growth relies on the formation of new blood vessels, which is facilitated by the secretion of various angiogenic factors, such as VEGF, by tumor cells and tumor-supporting cells. VEGF not only triggers tumor angiogenesis but also leads to abnormal proliferation of tumor vessels and disruption of the tumor vascular network, severely impairing T cell infiltration into the tumor. Anti-angiogenic therapy is currently an important approach to cancer treatment. The use of anti-VEGF monoclonal antibodies (such as bevacizumab) and VEGF-trapping agents can significantly reduce tumor vascular proliferation, decrease tumor blood supply, normalize tumor vasculature, and promote T cell infiltration into the tumor. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an oncolytic adenovirus vector that efficiently expresses VEGF and / or TGFβ capture agents, as well as a method for constructing and using the vector. After infecting a variety of human and mouse tumor cells, the oncolytic adenovirus can effectively neutralize VEGF and TGFβ produced by tumor cells.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an oncolytic adenovirus vector expressing VEGF and TGFβ trap, wherein the oncolytic adenovirus vector comprises two head-to-head expression cassettes inserted into the E1 region of adenovirus type 5, one of which is regulated by a human telomerase promoter for the expression of the E1A gene derived from adenovirus type 5, and the other is regulated by a human cytomegalovirus promoter / enhancer for the expression of VEGF and / or TGFβ trap genes; the vectors are adenovirus type 5 vectors and adenovirus type 5 / 35 vectors.

[0009] Furthermore, the type 5 adenovirus vector is a replication-deficient adenovirus vector with E1 and E3 regions deleted, and the packaging adenovirus system is Ad Max TM The adenoviral vector system consists of a series of shuttle plasmids and the pBHGfrtΔE1,3FLP backbone plasmid. After the shuttle plasmid with the target gene inserted and the backbone plasmid are co-transfected into HER293 cells, they are recombined in the cells by FLP recombinase and obtained after 10-14 days.

[0010] Furthermore, the human telomerase promoter, the expression cassette that regulates E1A gene expression comprises the human telomerase promoter, E1A and poly A components; the human cytomegalovirus promoter / enhancer, the VEGF and / or TGFβ trap expression cassette that regulates comprises the human cytomegalovirus promoter / enhancer, a series of fusion genes of VEGF and / or TGFβ trap and poly A components; the VEGF trap and TGFβ trap each comprise a series of fusion genes.

[0011] Furthermore, the human telomerase promoter can be replaced by other tumor-specific promoters, including: alpha-fetoprotein promoter, prostate-specific antigen promoter, transcription factor E2F-1 promoter, survivin promoter, hypoxia-inducible factor-1α promoter, nestin promoter, carcinoembryonic antigen promoter, p53 promoter and Mucin 1 promoter; the human cytomegalovirus promoter / enhancer can be replaced by other viral promoters with broad-spectrum promoter activity, as well as human elongation factor 1α promoter.

[0012] Furthermore, in the VEGF and / or TGFβ trap, a series of fusion genes constituting the VEGF trap include: fusion of VEGFR1 (Ig1-7), VEGFR1 (Ig1-6) (amino acids 1 to 300), VEGFR1 (Ig1-5) (amino acids 1 to 558), VEGFR1 (Ig1-4) (amino acids 1 to 431), VEGFR1 (Ig1-3) (amino acids 1 to 339) and IgG1 Fc; fusion of VEGFR2 (Ig1-7), VEGFR2 (Ig1-6) (amino acids 1 to 300), VEGFR2 (Ig1-5) (amino acids 1 to 300), VEGFR2 (Ig1-4) (amino acids 1 to 300), VEGFR2 (Ig1-3) (amino acids 1 to 300) and IgG1 Fc; fusion of VEGFR1(Ig2)(amino acids 1-300), VEGFR2(Ig1-3)(amino acids 1-300) and IgG1 Fc; fusion of VEGFR1(Ig2)(amino acids 129-229), VEGFR2(Ig3)(amino acids 1-300) and IgG1 Fc; the nucleotide sequence of the fusion gene of VEGFR1(Ig1-3), VEGFR2(Ig3) and IgG1 Fc is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the fusion gene of VEGFR1(Ig2)(amino acids 1-300), VEGFR2(Ig1-3) and IgG1 Fc is shown in SEQ ID NO.9, the amino acid sequence is shown in SEQ ID NO.10, and the amino acid sequence is shown in SEQ ID NO.4; VEGFR1(Ig2), VEGFR2(Ig3) and IgG1 The nucleotide sequence of the Fc fusion gene is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6;

[0013] Furthermore, in the VEGF and / or TGFβ trap, a series of fusion genes constituting the TGFβ trap include: a fusion of a TGFβRII extracellular segment sequence and IgG1 Fc, the nucleotide sequence of which is shown in SEQ ID NO.13, and the amino acid sequence of which is shown in SEQ ID NO.14; a fusion of two TGFβRII extracellular segment sequences and IgG1 Fc, the nucleotide sequence of which is shown in SEQ ID NO.15, and the amino acid sequence of which is shown in SEQ ID NO.16; a fusion of a TGFβRII extracellular segment sequence, a TGFβRI extracellular segment sequence and IgG1 Fc, the nucleotide sequence of which is shown in SEQ ID NO.17, and the amino acid sequence of which is shown in SEQ ID NO.18.

[0014] Furthermore, the VEGF and / or TGFβ trap, the fusion gene of the VEGF and TGFβ trap maintains the same open reading frame through the viral 2A peptide, linker and GCN4-plC leucine zipper; the 2A peptide comprises E2A, F2A, P2A and T2A; the E2A amino acid sequence is shown in SEQ ID NO.43, the F2A amino acid sequence is shown in SEQ ID NO.44, the P2A amino acid sequence is shown in SEQ ID NO.45, and the T2A amino acid sequence is shown in SEQ ID NO.46; the linker comprises (G4S)3 and A(EAAAK)4A; the (G4S)3 amino acid sequence is shown in SEQ ID NO.47, and the A(EAAAK)4A amino acid sequence is shown in SEQ ID NO.48; the amino acid sequence of the GCN4-plC leucine zipper is shown in SEQ ID NO.49.

[0015] Furthermore, in the VEGF and / or TGFβ trap, the nucleotide sequence of a fusion gene of VEGF and TGFβ trap is shown in SEQ ID NO.50, and the amino acid sequence is shown in SEQ ID NO.51; a fusion gene of VEGF and TGFβ trap comprises: VEGFR1 (Ig1-3) sequence (nucleotides 1 to 996 bp), VEGFR2 (Ig2) sequence (nucleotides 673 to 981 bp), (G4S)3 sequence, TGFβRII extracellular segment sequence, A(EAAAK)4A, TGFβRI extracellular segment sequence, IgG1 Fc sequence and C-myc sequence, the nucleotide sequence of which is shown in SEQ ID NO.52, and the amino acid sequence of which is shown in SEQ ID NO.53; a fusion gene of VEGF and TGFβ trap comprises: VEGFR1 (Ig1-3) sequence (nucleotides 1 to 996 bp), VEGFR2 (Ig2) sequence (nucleotides 673 to 981 bp), (G4S)3 sequence, TGFβRII extracellular segment sequence, A(EAAAK)4A, TGFβRI extracellular segment sequence, GCN4-plC leucine zipper sequence, IgG1 Fc sequence and C-myc sequence, the nucleotide sequence of which is shown in SEQ ID NO.54, and the amino acid sequence is shown in SEQ ID NO.55.

[0016] The present invention also provides a method for constructing the above-mentioned oncolytic adenoviral vector expressing VEGF and TGFβ trap, wherein, in the method, the adenoviral vector adopts AdMAX TM The system consists of two parts: a shuttle plasmid and a backbone plasmid; the target gene is inserted into the shuttle plasmid, and then the shuttle plasmid with the inserted target gene and the backbone plasmid are co-transfected into HEK293 cells. Homologous recombination is carried out in HEK293 cells through Cre recombinase, and the shuttle plasmid containing the target gene expression cassette is recombined into the E1 deletion region of the backbone plasmid to form a complete adenovirus vector containing the target gene. With the assistance of E1A expressed by HEK293 cells, the adenovirus vector replicates in HEK293 cells, causing cell lysis and release of adenovirus.

[0017] The present invention also provides an application of an oncolytic adenoviral vector expressing VEGF and TGFβ trap constructed by a method, wherein the oncolytic adenoviral vector is used to prepare a broad-spectrum anticancer preparation or immune adjuvant.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides an oncolytic adenovirus vector expressing a VEGF and TGFβ trap, and a method for constructing and applying the vector, which can be used for the preparation of therapeutic drugs for various solid tumors that highly express VEGF and / or TGFβ. The oncolytic adenovirus in the resulting drug can be injected into the tumor of a solid tumor patient, or into the abdominal or thoracic cavity of a tumor patient with metastasis to the abdominal or thoracic cavity. By producing direct oncolytic activity, it reduces the concentration of vascular endothelial growth factor (VEGF) and the like in the tumor microenvironment, inducing normalization of tumor blood vessels; neutralizes the concentration of TGFβ1 and TGFβ3 in the tumor microenvironment, inhibits EMT transformation and the generation of tumor stem cells, thereby inhibiting tumor growth and metastasis and inducing specific immune responses against tumor patients, thereby achieving the purpose of treating various types of tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is a schematic diagram of the construction of the shuttle plasmid containing the E1A expression cassette regulated by the telomerase promoter.

[0021] FIG2 is a schematic diagram of the construction of a dual expression cassette shuttle plasmid.

[0022] FIG3 is a schematic diagram of the structures of various VEGF Traps.

[0023] Figure 4 shows the VEGFR1(Ig2) / VEGFR2(Ig3) / IgG1 Fc( Figure 4A ), VEGFR1(Ig1~3) / VEGFR2(Ig3) / IgG1 Fc( Figure 4B ), VEGFR1(Ig2) / VEGFR2(Ig1~3) / IgG1 Fc( Figure 4C ) and VEGFR1(Ig1~3) / VEGFR2(Ig1~3) / IgG1 Fc( Figure 4D ) Schematic diagram of the dual expression cassette shuttle plasmid.

[0024] FIG5 is a schematic diagram of the structures of various TGFβTraps.

[0025] Figure 6 encodes TGFβRII / IgG1 Fc ( Figure 6A )、(TGFβRII)2 / IgG1 Fc( Figure 6B ) and TGFβRII / TGFβRI / IgG1 Fc( Figure 6C ) Schematic diagram of the dual expression cassette shuttle plasmid.

[0026] FIG7 is a schematic diagram showing a fusion gene in which different VEGF traps and TGFβ traps are connected by a linker [(G4S)3] to form an open reading frame.

[0027] Figure 8 is a diagram encoding VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc( Figure 8A ), VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc( Figure 8B ) and VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / TGFβRI / IgG1 Fc( Figure 8C ); VEGFR1(Ig1~3) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc( Figure 8D ), VEGFR1(Ig1~3) / VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc( Figure 8E ) and VEGFR1(Ig1~3) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / TGFβRI / IgG1 Fc( Figure 8F ) Schematic diagram of the dual expression cassette shuttle plasmid.

[0028] FIG9 is a schematic diagram of a fusion gene formed by connecting different VEGF traps and TGFβ traps with P2A (or E2A, T2A, F2A, only P2A is listed in the present invention) to form an open reading frame.

[0029] Figure 10 is a diagram encoding VEGFR1(Ig2) / VEGFR2(Ig3) / P2A / TGFβRII / IgG1 Fc( Figure 10A ), VEGFR1(Ig2) / VEGFR2(Ig3) / P2A / (TGFβRII)2 / IgG1 Fc( Figure 10B ) and VEGFR1(Ig2) / VEGFR2(Ig3) / P2A / TGFβRII / TGFβRI / IgG1 Fc( Figure 10C ); VEGFR1(Ig1~3) / VEGFR2(Ig3) / P2A / TGFβRII / IgG1 Fc( Figure 10D ), VEGFR1(Ig1~3) / VEGFR2(Ig3) / P2A / (TGFβRII)2 / IgG1 Fc( Figure 10E ) and VEGFR1(Ig1~3) / VEGFR2(Ig3) / P2A / TGFβRII / TGFβRI / IgG1 Fc( Figure 10F ) Schematic diagram of the dual expression cassette shuttle plasmid.

[0030] FIG11 is a graph showing the results of the neutralization ability of oncolytic adenovirus encoding single or dual capture agents against human and mouse VEGF-A and TGFβ1.

[0031] FIG. 12 is a graph showing that the replication of oncolytic adenovirus expressing VEGF and / or TGFβ trap is telomere-dependent.

[0032] FIG13 is a graph showing the inhibitory effects of oncolytic adenovirus expressing VEGF and / or TGFβ trap on different tumor cells.

[0033] Figure 14 The graph shows the results of oncolytic adenovirus expressing VEGF and / or TGFβ trap inhibiting the proliferation of human and mouse tumor cells.

[0034] FIG15 is a graph showing the results of oncolytic adenovirus expressing VEGF and / or TGFβ trapping agents inducing apoptosis and necrosis of human and mouse tumor cells. DETAILED DESCRIPTION

[0035] The following further describes the specific embodiments of the present invention in conjunction with the accompanying drawings. These drawings are only for illustrating the embodiments of the present disclosure, and are not intended to limit the scope of the present disclosure.

[0036] The present invention provides an oncolytic adenovirus vector expressing VEGF and TGFβ trap. In the oncolytic adenovirus vector, the oncolytic adenovirus is a type 5 adenovirus with two head-to-head expression cassettes inserted into the E1 region of the adenovirus. One of the expression cassettes is for the expression of the E1A gene derived from the adenovirus type 5 regulated by the human telomerase promoter, and the other expression cassette is for the expression of the VEGF and / or TGFβ trap gene regulated by the human cytomegalovirus promoter / enhancer. The vectors are type 5 adenovirus vectors and type 5 / 35 adenovirus vectors.

[0037] This oncolytic adenovirus can be injected into the tumor of patients with solid tumors, or into the abdominal or thoracic cavity of patients with tumor metastasis. It can produce direct oncolytic activity, reduce the concentration of vascular endothelial growth factor (VEGF) and other factors in the tumor microenvironment, and induce tumor vascular normalization; neutralize the concentration of TGFβ1 and TGFβ3 in the tumor microenvironment, inhibit EMT transformation and the production of tumor stem cells, thereby inhibiting tumor growth and metastasis, and inducing specific immune responses against tumor patients, etc., to achieve the purpose of treating various types of tumor patients.

[0038] Preferably, the type 5 adenovirus vector is a replication-deficient adenovirus vector with E1 and E3 regions deleted, and the packaging adenovirus system is Ad Max TMThe adenoviral vector system consists of a series of shuttle plasmids and the pBHGfrtΔE1,3FLP backbone plasmid. After the shuttle plasmid with the target gene inserted and the backbone plasmid are co-transfected into HER293 cells, they are recombined in the cells by FLP recombinase and can be obtained after 10-14 days.

[0039] The expression cassette for human telomerase promoter-regulated E1A gene expression contains human telomerase promoter, E1A and poly A (pA) components.

[0040] In addition to the human telomerase promoter, other tumor-specific promoters may also be used as replacements, including (but not limited to): 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, etc.

[0041] The human cytomegalovirus promoter / enhancer-regulated VEGF and / or TGFβ trap (Traps) expression cassette contains the human cytomegalovirus promoter / enhancer, a series of fusion genes of VEGF and / or TGFβ trap, and poly A (pA) components; VEGF trap and TGFβ trap each contain a series of fusion genes.

[0042] The promoter required for regulation can be the human cytomegalovirus promoter / enhancer, or can be replaced by other viral promoters with broad-spectrum promoter activity, as well as the human elongation factor 1 (EF1) promoter, etc.

[0043] In the VEGF and / or TGFβ trap, a series of fusion genes constituting the VEGF trap include (but are not limited to): fusion of VEGFR1 (Ig1-7), VEGFR1 (Ig1-6) (amino acids 1 to 300), VEGFR1 (Ig1-5) (amino acids 1 to 558), VEGFR1 (Ig1-4) (amino acids 1 to 431), VEGFR1 (Ig1-3) (amino acids 1 to 339) and IgG1 Fc; fusion of VEGFR2 (Ig1-7), VEGFR2 (Ig1-6) (amino acids 1 to 300), VEGFR2 (Ig1-5) (amino acids 1 to 300), VEGFR2 (Ig1-4) (amino acids 1 to 300), VEGFR2 (Ig1-3) (amino acids 1 to 300) and IgG1 Fc; fusion of VEGFR1 (Ig1-3) (amino acids 1 to 339), VEGFR2 (Ig1-7), VEGFR2 (Ig1-6) (amino acids 1 to 300), VEGFR2 (Ig1-5) (amino acids 1 to 300), VEGFR2 (Ig1-4) (amino acids 1 to 300), VEGFR2 (Ig1-3) (amino acids 1 to 300) and IgG1 Fc; 3) (amino acids 1-300) and IgG1 Fc; fusion of VEGFR1 (Ig 2) (amino acids 1-300), VEGFR2 (Ig1-3) (amino acids 1-300) and IgG1 Fc. Fusion of VEGFR1 (Ig2) (amino acids 129 to 229), VEGFR2 (Ig 3) (amino acids 1 to 300), and IgG1 Fc; the nucleotide sequence of the fusion gene of VEGFR1 (Ig1-3), VEGFR2 (Ig 3), and IgG1 Fc is shown in SEQ ID NO. 7, and the amino acid sequence is shown in SEQ ID NO. 8; the nucleotide sequence of the fusion gene of VEGFR1 (Ig2) (amino acids 1 to 300), VEGFR2 (Ig1-3), and IgG1 Fc is shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO. 10; the nucleotide sequence of the fusion gene of VEGFR1 (Ig2), VEGFR2 (Ig 3), and IgG1 Fc is shown in SEQ ID NO. 5, and the amino acid sequence is shown in SEQ ID NO. 6;

[0044] A series of fusion genes constituting the TGFβ trap, whose sequence features include (but are not limited to): a fusion of a TGFβRII extracellular segment sequence and an IgG1 Fc, the nucleotide sequence of which is shown in SEQ ID NO.13, and the amino acid sequence of which is shown in SEQ ID NO.14; a fusion of two TGFβRII extracellular segment sequences and an IgG1 Fc, the nucleotide sequence of which is shown in SEQ ID NO.15, and the amino acid sequence of which is shown in SEQ ID NO.16; a fusion of a TGFβRII extracellular segment sequence, a TGFβRI extracellular segment sequence and an IgG1 Fc, the nucleotide sequence of which is shown in SEQ ID NO.17, and the amino acid sequence of which is shown in SEQ ID NO.18.

[0045] The fusion gene of VEGF and TGFβ trap maintains the same open reading frame through the viral 2A peptide, linker and GCN4-plC leucine zipper.

[0046] 2A peptides include (including but not limited to): E2A, F2A, P2A and T2A; the E2A amino acid sequence is shown in SEQ ID NO.43 (QCTNYALLKLAGDVESNPGP), the F2A amino acid sequence is shown in SEQ ID NO.44 (VKQTLNFDLLKLAGDVESNPGP), the P2A amino acid sequence is shown in SEQ ID NO.45 (ATNFSLLKQAGDVEENPGP), and the T2A amino acid sequence is shown in SEQ ID NO.46 (ATNFSLLKQAGDVEENPGP).

[0047] Linker comprises (including but not limited to): (G4S)3 and A(EAAAK)4A; the amino acid sequence of (G4S)3 is shown in SEQ ID NO.47 (GGGGSGGGGSGGGGS), and the amino acid sequence of A(EAAAK)4A is shown in SEQ ID NO.48 (LAEAAAKEAAAKEAAAKEAAAKAAA).

[0048] The amino acid sequence of the GCN4-plC leucine zipper is shown in SEQ ID NO. 49 (RMKQLEDKVEELLSKNYHLENEVARLKKLVGERCGG).

[0049] The VEGF and / or TGFβ trap, wherein the nucleotide sequence of a fusion gene of VEGF and TGFβ trap is shown as SEQ ID NO.50, and the amino acid sequence is shown as SEQ ID NO.51.

[0050] A VEGF and TGFβ trap fusion gene comprises: VEGFR1 (Ig1-3) sequence (nucleotides 1 to 996 bp), VEGFR2 (Ig2) sequence (nucleotides 673 to 981 bp), (G4S)3 sequence, TGFβRII extracellular segment sequence, A(EAAAK)4A, TGFβRI extracellular segment sequence, IgG1 Fc sequence and C-myc sequence, the nucleotide sequence of which is shown in SEQ ID NO.52, and the amino acid sequence is shown in SEQ ID NO.53.

[0051] A fusion gene of a VEGF and TGFβ trap comprises: a VEGFR1 (Ig1-3) sequence (nucleotides 1 to 996 bp), a VEGFR2 (Ig2) sequence (nucleotides 673 to 981 bp), a (G4S)3 sequence, a TGFβRII extracellular segment sequence, A(EAAAK)4A, a TGFβRI extracellular segment sequence, a GCN4-plC leucine zipper sequence, an IgG1 Fc sequence, and a C-myc sequence. The nucleotide sequence is shown in SEQ ID NO. 54, and the amino acid sequence is shown in SEQ ID NO. 55.

[0052] The present invention also provides a method for constructing an oncolytic adenoviral vector expressing VEGF and TGFβ trapping agent, wherein the adenoviral vector adopts AdMAX TM The system is divided into two parts: shuttle plasmid and backbone plasmid. That is, the present invention provides a series of oncolytic adenovirus construction methods, and the adenovirus vector used adopts AdMAX TM The system consists of two parts: a shuttle plasmid and a backbone plasmid. The target gene is inserted into the shuttle plasmid, which is then co-transfected with the backbone plasmid into HEK293 cells. Cre recombinase undergoes homologous recombination in HEK293 cells, integrating the target gene expression cassette in the shuttle plasmid into the E1 deletion region of the backbone plasmid. This creates a complete adenoviral vector containing the target gene. With the assistance of E1A expressed in HEK293 cells, the adenoviral vector replicates in the HEK293 cells, causing cell lysis and the release of adenovirus.

[0053] The present invention constructs a series of different shuttle plasmids, in which one or two expression cassettes are inserted into the multiple cloning site of the shuttle plasmid. If two expression cassettes are used, the expression cassettes can be connected in a head-to-head, tail-to-tail, or head-to-tail manner. One expression cassette is a human telomerase promoter driving the E1A gene sequence, enabling selective expression of E1A in telomerase-positive cells (such as approximately 90% of tumor cells), while never driving the expression of adenovirus late genes, causing the adenovirus to selectively replicate in telomerase-positive cells, leading to cell lysis. The other expression cassette is a human cytomegalovirus enhancer / promoter regulating different lengths of extracellular domain sequences of human vascular endothelial growth factor receptor 1 (VEGFR1 or Flt-1) and / or vascular endothelial growth factor receptor 2 (VEGFR2 or KDR), different lengths of extracellular domain sequences of TGFβRI and / or TGFβRII, and a human IgG1 Fc domain. The fusion gene product formed by these combined sequences is a trap for VEGF and TGFβ. The expression cassette for expressing VEGF and TGFβ capture agents can express VEGF or TGFβ capture agents individually, or can further express the two capture agents simultaneously in the form of fusion genes (through various linkers such as (G4S)3, A(EAAAK)4A, etc.), viral 2A peptides (P2A, F2A, E2A and T2A, etc.) or internal nucleic acid entry sites (IRES).

[0054] In the present invention, the constructed shuttle plasmid and the adenovirus backbone plasmid pBHGfrt(del)E1,3FLP are co-transfected into HEK293 cells via calcium phosphate transfection. The shuttle plasmid and the backbone plasmid undergo homologous recombination in the HEK293 cells. After successful homologous recombination, the HEK293 cells undergo pathological changes and form plaques. This process usually takes 10 to 14 days. After forming plaques, the cells can be selected and further infected with HEK293 cells. After several rounds, the desired oncolytic adenovirus can be selected and amplified.

[0055] The present invention also provides an application of an oncolytic adenoviral vector expressing VEGF and TGFβ trap constructed by the method, wherein the oncolytic adenoviral vector is used to prepare a broad-spectrum anticancer preparation or immune adjuvant.

[0056] The oncolytic adenovirus preparation of the present invention is a broad-spectrum anticancer preparation with broad anticancer activity, particularly against solid tumors, 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 malignant sarcomas. Among existing malignant tumors, 85-90% are telomerase-positive. The oncolytic adenovirus of the present invention is a selectively replicating adenovirus regulated by telomerase and can replicate in telomerase-positive tumors, causing tumor cell lysis and thus exerting antitumor activity. The present invention has the advantage of killing tumor stem cells. Tumor stem cells have stronger telomerase activity than ordinary tumor cells. Therefore, the oncolytic adenovirus of the present invention has a stronger replication advantage in tumor stem cells, making it more likely to cause tumor stem cell lysis.

[0057] The mechanism of the anti-tumor activity of the oncolytic adenovirus of the present invention, in addition to its direct oncolytic effect, can also effectively neutralize various VEGFs secreted by tumor cells, tumor-associated immune cells such as macrophages, tumor cell fibroblasts, etc., including VEGF-A and VEGF-B, thereby eliminating or reducing the stimulation of tumor blood vessels, reducing the formation of tumor neovascularization, and normalizing tumor blood vessels; by neutralizing TGFβ1 and TGFβ3, inhibiting tumor growth and metastasis caused by TGFβ signals. The TGFβ signaling pathway plays an important role in the growth and metastasis of tumors. In addition to causing tumor metastasis through epithelial-mesenchymal transition, TGFβ has a strong immunosuppressive effect and is one of the key molecules in the formation of tumor immunosuppressive microenvironment, inhibiting the activity of various immune effector cells. The present invention helps to improve the tumor immunosuppressive microenvironment and restore or activate the anti-tumor and anti-tumor activity of immune effector cells by neutralizing TGFβ molecules secreted by tumor cells, tumor-associated immune cells and supporting cells.

[0058] The methods of using the oncolytic adenovirus preparation of the present invention include local application and systemic administration. Local application is mainly intratumoral injection, including local injection in solid tumors, intrathoracic injection after the tumor metastasizes to the thoracic cavity, and intraperitoneal injection after the tumor metastasizes to the abdominal cavity. For distant metastatic tumors, the virus can be targeted and delivered to the metastatic site through cell carriers or cell-derived extracellular vesicles (including exosomes and microparticles). The cell carrier can be the patient's own bone marrow mesenchymal stem cells, mesenchymal stem cells derived from the umbilical cord or umbilical cord blood of a healthy fetus, autologous or allogeneic adipose mesenchymal stem cells, etc. The extracellular vesicles can be released from the above after infection with oncolytic adenovirus, or can be released from tumor cell lines after infection with oncolytic adenovirus.

[0059] The oncolytic adenovirus preparation of the present invention can be used as an immune adjuvant to change the tumor immune microenvironment, and can convert "cold" tumors into "hot" tumors, thereby enhancing the effects of other tumor immunotherapies, such as immune checkpoint blockers PD-1 / PD-L1, adoptive immunotherapy including enhanced antigen receptor-T adoptive immunotherapy, TCR-T adoptive immunotherapy, and the efficacy of therapeutic tumor vaccines and neoantigen tumor vaccines. The tumor immune microenvironment is generally divided into two categories: "cold" tumors and "hot" tumors, based on whether there is infiltration of immune effector cells, especially T cells, in the tumor microenvironment. "Cold" tumors often have little or no response to immunotherapy. However, through some immune adjuvants, especially oncolytic viruses, that can cause acute inflammatory changes in the local tumor microenvironment, "cold" tumors can often be converted into "hot" tumors, thereby improving the tumor's response to immunotherapy.

[0060] The nucleic acid and amino acid sequences involved in the present invention are as follows:

[0061] SEQ ID NO.1 human telomerase promoter sequence:

[0062] tagagctgcgctgtcggggccaggccgggctcccagtggattcgcgggcacagacgcccaggaccgcgcttcccacgtggcggagggactggggacccgggcacccgtcctgccccttcaccttccagctccgcctcctccgcgcggaccccgccccgtcccgacccctcccgggtccc cggcccagccccctccgggccctcccagcccctccccttcctttccgcggccccgccctctcctcgcggcgcgagtttcaggcagcgctg cgtcctgctgcgcacgtgggaagccctggccgatgggctcgacgcacgtgggcgcacgtgggcgcacgtggggagcttggcattccggtac

[0063] SEQ ID NO.2 Adenovirus type 5E1A sequence:

[0064] atgagacatattatctgccacggaggtgttattaccgaagaaatggccgccagtcttttggaccagctgatcgaagaggtactggctgataatcttccacctcctagccattttgaaccacctacccttcacgaactgtatgatttagacgtgacggcccccgaagatcccaacgaggaggcggtttcgcagatttttcccgactctgtaatgttggcggtgcaggaagggattgacttactcacttttccgccggcgcccggttctccggagccgcctcacctttcccggcagcccgagcagccggagcagagagccttgggtccggtttctatgccaaaccttgtaccggaggtgatcgatcttacctgccacgaggctggctttccacccagtgacgacgaggatgaagagggtgaggagtttgtgttagattatgtggagcaccccgggcacggttgcaggtcttgtcattatcaccggaggaatacgggggacccagatattatgtgttcgctttgctatatgaggacctgtggcatgtttgtctacagtcctgtgtctgaacctgagcctgagcccgagccagaaccggagcctgcaagacctacccgccgtcctaaaatggcgcctgctatcctgagacgcccgacatcacctgtgtctagagaatgcaatagtagtacggatagctgtgactccggtccttctaacacacctcctgagatacacccggtggtcccgctgtgccccattaaaccagttgccgtgagagttggtgggcgtcgccaggctgtggaatgtatcgaggacttgcttaacgagcctgggcaacctttggacttgagctgtaaacgccccaggccataa

[0065] SEQ ID NO.3 E1A expression cassette sequence:

[0066]

[0067] SEQ ID NO.4 Peptide sequence of 42A:

[0068] ggcgcgccggtacccgagcaaaaaggagcggcagtggcgtgaagcagacattgaattttgaccttttgaaactggcaggtgatgtagagtccaaccctgggccagtcgacagagcaaaacggtctggaagtgggcagtgcaccaactacgcgttgctgaaattggcaggggatgtggaatctaatccaggccctgaattccgagccaaacgaagcggctctggagaaggccgtggatctctgctgacctgcggagacgtagaggagaatccagggcccggatccagagctaagaggtcgggttccggcgccaccaatttcagtttgctcaagcaggctggtgatgtggaggagaatcccggtccgaagctt

[0069] SEQ ID NO.5 Nucleotide sequence of VEGFR1(Ig2) / VEGFR2(Ig3) / IgG1 Fc:

[0070]

[0071] SEQ ID NO. 6 Amino acid sequence of VEGFR1(Ig2) / VEGFR2(Ig3) / IgG1 Fc:

[0072] MVSYWDTGVLLCALLSCLLLTGSSSGDYKDDDDKSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0073] SEQ ID NO. 7 Nucleotide sequence of VEGFR1(Ig1 - 3) / VEGFR2(Ig3) / IgG1 Fc:

[0074]

[0075] SEQ ID NO. 8 Amino acid sequence of VEGFR1(Ig1-3) / VEGFR2(Ig3) / IgG1 Fc:

[0076] MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0077] SEQ ID NO. 9 Nucleotide sequence of VEGFR1(Ig2) / VEGFR2(Ig1-3) / IgG1 Fc:

[0078]

[0079] SEQ ID NO. 10 VEGFR1(Ig2) / VEGFR2(Ig1-3) / IgG1 Fc amino acid sequence:

[0080] MQSKVLLAVALWLCVETRAASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0081] SEQ ID NO. 11 VEGFR1(Ig1-3) / VEGFR2(Ig1-3) / IgG1 Fc nucleotide sequence:

[0082]

[0083] SEQ ID NO. 12 VEGFR1(Ig1 - 3) / VEGFR2(Ig1 - 3) / IgG1 Fc amino acid sequence:

[0084] MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0085] SEQ ID NO.13 TGFβRII / IgG1 Fc nucleotide sequence:

[0086]

[0087] SEQ ID NO. 14 TGFβRII / IgG1 Fc amino acid sequence:

[0088] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0089] SEQ ID NO. 15 (TGFβRII)2 / IgG1 Fc nucleotide sequence:

[0090]

[0091] Amino acid sequence of SEQ ID NO.16 (TGFβRII)2 / IgG1 Fc:

[0092] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0093] Nucleotide sequence of SEQ ID NO.17 TGFβRII / TGFβRI / IgG1 Fc:

[0094]

[0095] SEQ ID NO. 18 TGFβRII / TGFβRI / IgG1 Fc amino acid sequence:

[0096] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELAEAAAKEAAAKEAAAKEAAAKAAAGLLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0097] SEQ ID NO. 19 VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc nucleotide sequence:

[0098]

[0099] Amino acid sequence of SEQ ID NO.20 VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc:

[0100] MVSYWDTGVLLCALLSCLLLTGSSSGDYKDDDDKSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0101] Nucleotide sequence of SEQ ID NO.21 VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc:

[0102]

[0103] SEQ ID NO.22 VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc amino acid sequence:

[0104] MVSYWDTGVLLCALLSCLLLTGSSSGDYKDDDDKSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0105] SEQ ID NO.23 VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / TGFβRI / IgG1 Fc nucleotide sequence:

[0106]

[0107] SEQ ID NO. 24 VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / TGFβRI / IgG1 Fc Amino Acid Sequence:

[0108] MVSYWDTGVLLCALLSCLLLTGSSSGDYKDDDDKSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELAEAAAKEAAAKEAAAKEAAAKAAAGLLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0109] SEQ ID NO. 25 VEGFR1(Ig1~3) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc Nucleotide Sequence:

[0110]

[0111] SEQ ID NO. 26 VEGFR1(Ig1 - 3) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc amino acid sequence:

[0112] MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0113] SEQ ID NO. 27 VEGFR1(Ig1-3) / VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc nucleotide sequence:

[0114]

[0115] SEQ ID NO. 28 VEGFR1(Ig1-3) / VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc amino acid sequence:

[0116] MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0117] SEQ ID NO. 29 VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / (G4S) 3 / TGFβRII / TGFβRI / IgG1 Fc nucleotide sequence:

[0118]

[0119] SEQ ID NO.30 VEGFR1(Ig1-3) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / TGFβRI / IgG1 Fc amino acid sequence:

[0120] MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELAEAAAKEAAAKEAAAKEAAAKAAAGLLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0121] SEQ ID NO.31VEGFR1(Ig2) / VEGFR2(Ig3) / IgG1 Fc / P2A / TGFβRII / IgG1

[0122] Fc nucleotide sequence:

[0123]

[0124] Amino acid sequence of SEQ ID NO. 32 VEGFR1(Ig2) / VEGFR2(Ig3) / IgG1 Fc / P2A / TGFβRII / IgG1 Fc:

[0125] MVSYWDTGVLLCALLSCLLLTGSSSGDYKDDDDKSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKATNFSLLKQAGDVEENPGPTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0126] SEQ ID NO.33 VEGFR1(Ig2) / VEGFR2(Ig3) / IgG1 Fc / P2A / (TGFβRII)2 / IgG1 Fc nucleotide sequence:

[0127]

[0128] SEQ ID NO.34 VEGFR1(Ig2) / VEGFR2(Ig3) / IgG1 Fc / P2A / (TGFβRII)2 / IgG1 Fc amino acid sequence:

[0129]

[0130] SEQ ID NO.35 VEGFR1(Ig2) / VEGFR2(Ig3) / IgG1 Fc / P2A / TGFβRII / TGFβRI / IgG1Fc nucleotide sequence:

[0131]

[0132] SEQ ID NO.36 VEGFR1(Ig2) / VEGFR2(Ig3) / IgG1 Fc / P2A / TGFβRII / TGFβRI / IgG1Fc amino acid sequence:

[0133]

[0134] SEQ ID NO. 37 VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / IgG1 Fc / P2A / TGFβRII / IgG1 Fc nucleotide sequence:

[0135]

[0136] SEQ ID NO. 38 VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / IgG1 Fc / P2A / TGFβRII / IgG1 Fc amino acid sequence:

[0137]

[0138] SEQ ID NO.39 VEGFR1(Ig1-3) / VEGFR2(Ig3) / IgG1 Fc / P2A / (TGFβRII)2 / IgG1 Fc nucleotide sequence:

[0139]

[0140] SEQ ID NO.40 VEGFR1(Ig1-3) / VEGFR2(Ig3) / IgG1 Fc / P2A / (TGFβRII)2 / IgG1 Fc amino acid sequence:

[0141]

[0142] SEQ ID NO.41 VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / IgG1 Fc / P2A / TGFβRII / TGFβRI / IgG1 Fc nucleotide sequence:

[0143]

[0144] SEQ ID NO.42 VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / IgG1 Fc / P2A / TGFβRII / TGFβRI / IgG1 Fc amino acid sequence:

[0145]

[0146] SEQ ID NO.43E2A amino acid sequence:

[0147] QCTNYALLKLAGDVESNPGP

[0148] SEQ ID NO.44F2A amino acid sequence:

[0149] VKQTLNFDLLKLAGDVESNPGP

[0150] SEQ ID NO.45P2A amino acid sequence:

[0151] ATNFSLLKQAGDVEENPGP

[0152] SEQ ID NO.46T2A amino acid sequence:

[0153] ATNFSLLKQAGDVEENPGP

[0154] SEQ ID NO.47(G4S)3 amino acid sequence:

[0155] GGGGSGGGGSGGGGS

[0156] SEQ ID NO.48A (EAAAK) 4A amino acid sequence:

[0157] LAEAAAKEAAAKEAAAKEAAAKAAA

[0158] SEQ ID NO.49 Amino acid sequence of GCN4-plC leucine zipper:

[0159] RMKQLEDKVEELLSKNYHLENEVARLKKLVGERCGG

[0160] SEQ ID NO.50 Nucleotide sequence of a fusion gene of VEGF and TGFβ trap:

[0161]

[0162] SEQ ID NO.51 amino acid sequence of a fusion gene of VEGF and TGFβ trap:

[0163]

[0164] SEQ ID NO.52 VEGFR1(Ig1-3) / VEGFR2(Ig2) / (G4S)3 / TGFβRII / A(EAAAK)4A / TGFβRI / IgG1 Fc / C-myc nucleotide sequence:

[0165]

[0166] SEQ ID NO.53 VEGFR1(Ig1-3) / VEGFR2(Ig2) / (G4S)3 / TGFβRII / A(EAAAK)4A / TGFβRI / IgG1 Fc / C-myc amino acid sequence:

[0167] MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKADVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKVDGGGGSGGGGSGGGGSRSTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDELAEAAAKEAAAKEAAAKEAAAKAAAGLLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEFDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEQKLISEEDL

[0168] SEQ ID NO.54 VEGFR1(Ig1-3) / VEGFR2(Ig2) / (G4S)3 / TGFβRII / A(EAAAK)4A / TGFβRI / GCN4-plC / IgG1 Fc / C-myc nucleotide sequence:

[0169]

[0170] SEQ ID NO.55 VEGFR1(Ig1-3) / VEGFR2(Ig2) / (G4S)3 / TGFβRII / A(EAAAK)4A / TGFβRI / GCN4-plC / IgG1 Fc / C-myc amino acid sequence:

[0171]

[0172] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the scope of the present invention.

[0173] If no specific techniques or conditions are specified in the examples, all are conventional methods, which are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. All reagents and instruments used are conventional products that can be purchased through regular channels.

[0174] Example 1: Cloning of the telomerase promoter.

[0175] A pair of primers for the human telomerase promoter were designed. The upstream primer was 5'-gct cta gat gcg ctg tcg gggcca ggc-3' (with an Xba I site introduced at the 5' end) and the downstream primer was 5'-ccg aat tcc acg tgc gcc cac gtgcgc c-3' (with an EcoR I site introduced at the 5' end). PCR amplification was performed using the genomic DNA of human lung cancer A549 cells 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 20 s for 30 cycles. A 297 bp fragment was amplified and digested with Xba I and EcoR I sites, followed by gel recovery of the digested fragment. The pDC315 plasmid (AdMAX TM ) was also double-digested with Xba I and EcoR I sites, and the large enzyme-digested fragment was recovered from the gel. The PCR fragment and the shuttle plasmid fragment were ligated and transformed into DH5α competent cells. The clone was selected, the PCR fragment was identified by enzyme digestion, and 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) cassettes (SEQ ID NO. 1) from the C-myc gene regulatory sequence were inserted at the 3′ end of the telomerase promoter.

[0176] Example 2: Construction of a shuttle plasmid containing the E1A expression cassette regulated by the telomerase promoter.

[0177] A pair of E1A primers were designed, with the upstream primer being 5'-ccg caa ttg cca tga gac ata tta tct gcc-3' (with an Mfe I site introduced at the 5' end) and the downstream primer being 5'-ccg ctc gag tta tgg cct ggg gcg ttt a-3' (with an Xho I site introduced at the 5' end). PCR amplification was performed using the pcDNA3.1 E1A plasmid 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 30 s for 30 cycles. An 870 bp fragment was amplified and digested with Mfe I and Xho I sites, followed by gel recovery of the digested fragment. The telomerase promoter pDC315hTERTp plasmid obtained above was cloned and cloned with EcoR I and Sal I. After double enzyme digestion at site I, the large enzyme-digested fragment was recovered from the gel, and the PCR fragment and the shuttle plasmid fragment were connected. The fragment was transformed into DH5α competent medium, and the clone was selected for enzyme digestion and identification. The PCR fragment was inserted into the pDC315 hTERTp plasmid and sequenced. It was confirmed that the sequence of the E1A fragment obtained by PCR was completely correct (SEQ ID NO.2). The obtained plasmid was the E1A expression cassette regulated by the telomerase promoter (SEQ ID NO.3). Figure 1A shown.

[0178] Another pair of primers was designed to amplify the E1A expression cassette. The upstream primer was 5'-gga att ccc ccg aca gcgcag ctc ta-3' (with an EcoR I site introduced at the 5' end) and the downstream primer was 5'-gga cta gtg gat ctt cga tgctag acg tcc-3' (with an Spe I site introduced at the 5' end). PCR amplification was performed using the pDC315 hTERTp plasmid 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 30 s for 30 cycles. A 1400 bp fragment was amplified and digested with EcoR I and Spe I sites, followed by gel recovery of the digested fragment. The pDC311 shuttle plasmid (AdMAX TM ) After double digestion with EcoR I and Xba I sites, the large fragment was recovered from the gel, and the PCR fragment and the pDC311 shuttle plasmid fragment were ligated and transformed into DH5α competent cells. The clone was selected and the PCR fragment was identified by restriction digestion. The PCR fragment was inserted into the pDC311 plasmid and sequenced to confirm that the E1A expression cassette sequence obtained by PCR was completely correct. The obtained plasmid pDC hTERTp E1A is the shuttle plasmid for the E1A expression cassette regulated by the telomerase promoter, as shown in FIG. Figure 1B shown.

[0179] Example 3: Construction of a dual expression cassette shuttle plasmid.

[0180] An oligonucleotide fragment containing four viral 2A peptide sequences and restriction enzyme cleavage sites (SEQ ID NO. 4) was synthesized and digested with Xho I and Hind III and then recovered from the gel. The pcDNA3.1(-)-myc-His B plasmid (Invitrogen TM ) After double digestion with Xho I and Hind III, the large fragment was recovered from the gel, and the 2A oligonucleotide fragment and the pcDNA3.1(-) plasmid fragment were ligated. The fragment was transformed into DH5α competent cells, and clones were selected for restriction enzyme digestion and identification to confirm that the 2A oligonucleotide fragment was inserted into the pcDNA3.1(-) plasmid, thus obtaining pcDNA3.1(-)4×2A. Figure 2A shown.

[0181] A pair of primers was designed to amplify the CMVp-2A-pA expression cassette from pcDNA3.1(-)4×2A. The upstream primer was 5'-gcc cgc aat tgg aca ttg att att gac tag-3' (with an Mfe I site introduced at the 5' end), and the downstream primer was 5'-gaa gat ctc cat aga gcc cac cgc atc-3' (with a Bgl II site introduced at the 5' end). PCR amplification was performed using the pcDNA3.1(-)4×2A plasmid as a template. High-fidelity Q5 PCR enzyme (NEB) was used for amplification. Amplification conditions were 98°C for 10 s, 72°C for 20 s, and 72°C for 30 s. A 1395 bp CMVp-4×2A-pA fragment was amplified. The digested fragment was recovered from gel after double digestion with Mfe I and Bgl II. The pDC hTERTp E1A shuttle plasmid was cloned with EcoRI and BglII. After the enzyme digestion, the large fragment was recovered from the gel and the PCR fragment was connected to the pDC hTERTp E1A shuttle plasmid fragment. The fragment was transformed into DH5α competent medium. The clone was selected and the PCR fragment was inserted into the pDC hTERTp E1A plasmid for identification and sequencing. The sequence of the CMVp-4×2A-pA fragment expression cassette obtained by PCR was confirmed to be completely correct. The obtained plasmid pdShuttle was the double expression cassette shuttle plasmid. Figure 2B shown.

[0182] Example 4: Construction of shuttle plasmid expressing VEGF trap.

[0183] The VEGF family belongs to the platelet-derived growth factor superfamily. Its main members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). The main receptors include VEGFR1 (Flt-1), VEGFR2 (KDR), and VEGFR3 (Flt-4). VEGF-A and VEGF-B primarily bind to VEGFR1 and VEGFR2, respectively, and are associated with angiogenesis; while VEGF-C and VEGF-D primarily bind to VEGFR3, contributing to lymphangiogenesis. VEGF receptors are protein-tyrosine kinases, single transmembrane proteins with an extracellular domain composed of seven immunoglobulin-like (Ig-like) domains. The VEGF-binding region primarily binds to the first, second, and third Ig-binding domains. Compared to VEGFR2, VEGFR1 binds to VEGF with a higher affinity (approximately 10-fold). Soluble extracellular domains of VEGFR1 and / or VEGFR2 can be used to form VEGF traps, similar to VEGF antibodies. Currently, an FDA-approved VEGF trap (Aflibercept) is used in patients with metastatic colorectal cancer. Aflibercept is a fusion of the Ig2 and Ig3 sequences of VEGFR1 and VEGFR2.

[0184] Construct several VEGF traps, including VEGFR1 (Ig2) / VEGFR2 (Ig3) / IgG1 Fc (human IgG1 Fc segment) (SEQ ID NO.5, NO.6, as Figure 3A As shown), VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / IgG1Fc (SEQ ID NO.7, NO.8, as shown Figure 3B As shown), VEGFR1 (Ig2) / VEGFR2 (Ig1-3) / IgG1 Fc (SEQ ID NO.9, NO.10, as shown), Figure 3C ) and VEGFR1 (Ig1-3) / VEGFR2 (Ig1-3) / IgG1 Fc (SEQ ID NO.11, NO.12, as shown) Figure 3D shown).

[0185] All the above sequences were synthesized (Shanghai Biotechnology Co., Ltd.). The synthesized fragments were double-digested with Asc I and Not I and then recovered from gel. The pdShuttle was also double-digested with Asc I and Not I and then recovered from gel. They were ligated and transformed into DH5α competent medium. The cloned PCR fragments identified by enzyme digestion were inserted into the pdShuttle plasmid and sequenced. Thus, VEGFR1 (Ig2) / VEGFR2 (Ig3) / IgG1 Fc (such as Figure 4A As shown), VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / IgG1 Fc (as shown Figure 4B As shown), VEGFR1(Ig2) / VEGFR2(Ig1~3) / IgG1 Fc(as shown Figure 4C ) and VEGFR1(Ig1~3) / VEGFR2(Ig1~3) / IgG1Fc(as shown) Figure 4D (shown) Dual expression cassette shuttle plasmid.

[0186] Example 5: Construction of shuttle plasmid expressing TGFβ trap.

[0187] The VEGF family belongs to the platelet-derived growth factor superfamily. Its main members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). The main receptors include VEGFR1 (Flt-1), VEGFR2 (KDR), and VEGFR3 (Flt-4). VEGF-A and VEGF-B primarily bind to VEGFR1 and VEGFR2, respectively, and are associated with angiogenesis; while VEGF-C and VEGF-D primarily bind to VEGFR3, contributing to lymphangiogenesis. VEGF receptors are protein-tyrosine kinases, single transmembrane proteins with an extracellular domain composed of seven immunoglobulin-like (Ig-like) domains. The VEGF-binding region primarily binds to the first, second, and third Ig-binding domains. Compared to VEGFR2, VEGFR1 binds to VEGF with a higher affinity (approximately 10-fold). Soluble extracellular domains of VEGFR1 and / or VEGFR2 can be used to form VEGF traps, similar to VEGF antibodies. Currently, an FDA-approved VEGF trap (Aflibercept) is used in patients with metastatic colorectal cancer. Aflibercept is a fusion of the Ig2 and Ig3 sequences of VEGFR1 and VEGFR2.

[0188] Several TGFβtraps were constructed, including TGFβRII / IgG1 Fc (SEQ ID NO.13, NO.14, as shown in FIG. Figure 5A As shown), (TGFβRII)2 / IgG1 Fc (SEQ ID NO.15, NO.16, as shown Figure 5B as shown) and TGFβRII / TGFβRI / IgG1 Fc (SEQ ID NO.17, NO.18, as shown) Figure 5C As shown), the TGFβRII / TGFβRI / IgG1 Fc fusion gene TGFβRII and TGFβRI are connected by a linker (A(EAAAK)4A).

[0189] All the above sequences were synthesized (Shanghai Biotechnology Co., Ltd.). The synthesized fragments were digested with Asc I and Not I and then recovered from the gel. The pdShuttle was also digested with Asc I and Not I and then recovered from the gel. They were ligated and transformed into DH5α competent cells. The PCR fragments identified by enzyme digestion were selected and inserted into the pdShuttle plasmid and sequenced. The fragments encoding TGFβRII / IgG1 Fc (such as Figure 6A As shown), (TGFβRII)2 / IgG1 Fc (as shown Figure 6B ) and TGFβRII / TGFβRI / IgG1 Fc (as Figure 6C (shown) Dual expression cassette shuttle plasmid.

[0190] Example 6: Construction of a dual-capture shuttle plasmid expressing VEGF and TGFβ.

[0191] VEGF and TGFβ play a crucial role in the development, progression, and metastasis of tumors. In addition to directly promoting tumor growth and metastasis, VEGF and TGFβ can also significantly impact tumors by influencing the immune system. VEGF and TGFβ are two key negative regulators of the tumor's immunosuppressive microenvironment. Animal studies and human trials have fully demonstrated their significant anti-tumor effects. However, there are currently few reports on their combined anti-tumor effects.

[0192] Several oncolytic adenoviral shuttle plasmids encoding both VEGF and TGFβ traps were constructed. Specifically, the above VEGF and TGFβ trap coding sequences can be further combined to form a fusion gene via a linker, or connected via different 2A peptides to form a single open reading frame, or even connected via internal nucleic acid entry sites (IRES) to form independent open reading frames. This invention only lists the construction of VEGF and TGFβ dual-trap shuttle plasmids in the form of linkers and 2A peptides.

[0193] Different VEGF traps and TGFβ traps were connected with a linker [(G4S)3] to form an open reading frame fusion gene including VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc (SEQ ID NO.19, NO.20, as shown in FIG. Figure 7A As shown), VEGFR1(Ig2) /

[0194] VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc (SEQ ID NO.21, NO.22, such as Figure 7B ) and VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / TGFβRI / IgG1 Fc (SEQ ID NO.23, NO.24, as shown) Figure 7C VEGFR1(Ig1-3) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc (SEQ ID NO.25, NO.26, as shown); Figure 7D As shown), VEGFR1(Ig1-3) / VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc (SEQ ID NO.27, NO.28, as shown Figure 7E ) and VEGFR1(Ig1-3) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / TGFβRI / IgG1 Fc (SEQ ID NO.29, NO.30, as shown) Figure 7F shown).

[0195] All the above sequences were synthesized (Shanghai Bioengineering Co., Ltd.). The synthesized fragments were double-digested with Asc I and Not I and then recovered from gel. The pdShuttle was also double-digested with Asc I and Not I and then recovered from gel. They were ligated and transformed into DH5α competent medium. The cloned PCR fragments identified by enzyme digestion were inserted into the pdShuttle plasmid and sequenced. The genes encoding VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc (such as Figure 8A As shown), VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc (as shown Figure 8B ) and VEGFR1(Ig2) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / TGFβRI / IgG1Fc (as shown) Figure 8CVEGFR1(Ig1~3) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / IgG1 Fc (as shown); Figure 8D As shown), VEGFR1(Ig1~3) / VEGFR2(Ig3) / (G4S)3 / (TGFβRII)2 / IgG1 Fc (as shown Figure 8E ) and VEGFR1(Ig1~3) / VEGFR2(Ig3) / (G4S)3 / TGFβRII / TGFβRI / IgG1 Fc (as shown) Figure 8F (shown) Dual expression cassette shuttle plasmid.

[0196] Different VEGF traps and TGFβ traps were linked with P2A (or E2A, T2A, F2A, only P2A is listed in the present invention) to form an open reading frame fusion gene including VEGFR1(Ig2) / VEGFR2(Ig3) / P2A / TGFβRII / IgG1 Fc (SEQ ID NO.31, NO.32, as shown in FIG. Figure 9A As shown), VEGFR1(Ig2) / VEGFR2(Ig3) / P2A / (TGFβRII)2 / IgG1Fc (SEQ ID NO.33, NO.34, as shown Figure 9B ) and VEGFR1(Ig2) / VEGFR2(Ig3) / P2A / TGFβRII / TGFβRI / IgG1 Fc (SEQ ID NO.35, NO.36, as shown) Figure 9C VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / P2A / TGFβRII / IgG1 Fc (SEQ ID NO.37, NO.38, as shown); Figure 9D As shown), VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / P2A / (TGFβRII) 2 / IgG1 Fc (SEQ ID NO. 30, NO. 40, as shown Figure 9E ) and VEGFR1 (Ig1-3) / VEGFR2 (Ig3) / P2A / TGFβRII / TGFβRI / IgG1 Fc (SEQ ID NO.41, NO.42, as shown) Figure 9F shown).

[0197] All the above sequences were synthesized (Shanghai Biotechnology Co., Ltd.). The synthesized fragments were double-digested with Asc I and Not I and then recovered from gel. The pdShuttle was also double-digested with Asc I and Not I and then recovered from gel. They were ligated and transformed into DH5α competent cells. The cloned PCR fragments identified by enzyme digestion were inserted into the pdShuttle plasmid and sequenced. The genes encoding VEGFR1 (Ig2) / VEGFR2 (Ig3) / P2A / TGFβRII / IgG1 Fc (such as Figure 10A As shown), VEGFR1(Ig2) / VEGFR2(Ig3) / P2A / (TGFβRII)2 / IgG1 Fc (as shown Figure 10B ) and VEGFR1(Ig2) / VEGFR2(Ig3) / P2A / TGFβRII / TGFβRI / IgG1 Fc (as shown) Figure 10C VEGFR1(Ig1~3) / VEGFR2(Ig3) / P2A / TGFβRII / IgG1 Fc(as shown); Figure 10D As shown), VEGFR1(Ig1~3) / VEGFR2(Ig3) / P2A / (TGFβRII)2 / IgG1 Fc (as shown Figure 10E ) and VEGFR1(Ig1~3) / VEGFR2(Ig3) / P2A / TGFβRII / TGFβRI / IgG1 Fc (as shown) Figure 10F (shown) Dual expression cassette shuttle plasmid.

[0198] Example 7: Construction of oncolytic adenoviral vector expressing VEGF and / or TGFβ dual-capture agents.

[0199] By AdMax TM Cre recombinant adenovirus system instructions. Various constructed shuttle plasmids were extracted using the EndoFree Plasmid Midi Kit (QIAGEN). When the 260 / 280 ratio was around 1.9, the plasmid concentration was adjusted to 1 μg / μl. The pBGHfrt△E1,3Cre adenovirus backbone plasmid was extracted using the EndoFree Plasmid Maxi Kit (QIAGEN). When the 260 / 280 ratio was around 1.9, the plasmid concentration was adjusted to 1 μg / μl. Low-passage HEK 293 cells were passaged to 60 mm 3 When the cells were 60-70% confluent, the shuttle plasmid and backbone plasmid were separated by calcium phosphate precipitation (CalPhos TM Transfection was performed using a Mammalian Transfection Kit (ClonTech). The transfection system is shown in Table 1.

[0200] Table 1. Transfection system.

[0201]

[0202] After the transfection system was prepared, it was incubated for 10 minutes and then added to HEK 293 cells.

[0203] After transfection, prepare the culture medium containing agarose according to Table 2 below.

[0204] Table 2. Culture medium.

[0205]

[0206] After boiling and dissolving liquid A in a microwave oven, place it in a 44°C water bath. After the temperature drops to 44°C, take 30 ml and mix it with liquid B placed at 44°C; aspirate the culture medium in each culture dish after the above plasmid transfection, and add 15 ml of the A / B mixture to each culture dish.

[0207] 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. Plaques were formed in about 10 to 14 days. The plaques were picked out and further amplified in 293 cells. Viral DNA was extracted from some cells for PCR identification. After 2 or 3 rounds of amplification of the correct clone, the 293 cells were collected, frozen, lysed, and purified using adenovirus purification kit (ViraBind). TM Adenovirus was purified using Adenovirus Purification Kit (Cell Biolabs, Inc) and then expressed and functionally characterized.

[0208] Example 8: Neutralization effect of oncolytic adenovirus expressing VEGF and / or TGFβ dual-capture agent on human and mouse VEGF-A and TGFβ1.

[0209] 293T cells were passaged into 6-well plates at a seeding density of 5 × 10 5 / well, and on the second day (24 h) when the cell confluence was about 90%, the control oncolytic virus (Ad on -Control), a single oncolytic adenovirus encoding VEGF trap (Ad on -Flt1(Ig1~3)-KDR(Ig3)-IgG1Fc), single oncolytic adenovirus encoding TGFβtrap (Ad on -TGFβRII / TGFβRI-IgG1Fc) and an oncolytic adenovirus encoding both VEGF and TGFβtrap [Ad on-Flt1(Ig1~3)-KDR(Ig3) / TGFβRII / TGFβRI-IgG1Fc (fusion gene form) and (Ad on -Flt1(Ig2)-KDR(Ig3)-IgG1Fc / P2A / (TGFβRII)2-IgG1Fc) (viral P2A peptide-linked form)], with a viral titer of 100 pfu / cell. After 2 hours of infection, the cells were washed twice with PBS to remove uninfected viruses, and fresh DMEM culture medium (containing 10% fetal bovine serum) was added at 3 ml / well. After further culture for 24 hours, the supernatant was collected and centrifuged at 10,000 rpm at 4°C for 5 minutes. The cells were then aliquoted and frozen or directly tested by ELISA.

[0210] The neutralization ability of various capture agent oncolytic adenoviruses on human and mouse VEGF-A and TGFβ1 was detected using ELISA kits for hVEGF-A, mVEGF-A, hTGFβ1 and mTGFβ1 (Shenzhen Dakoway). The standard substances in the kits were dissolved into 2000 pg (hVEGF-A), 1000 pg (mVEGF-A), 800 pg (hTGFβ1) and 800 pg (mTGFβ1). 50 μl of the above standard substances were added to the corresponding 96-well ELISA wells. In the plate, 50 μl of the supernatant obtained by the above method was added to the corresponding wells, and each supernatant was added in triplicate. After incubation for 2 hours, the neutralization ability of different Traps viruses on human and mouse VEGF-A and TGFβ1 was detected by ELISA. The results showed that oncolytic adenoviruses expressing VEGF and TGFβ traps alone or simultaneously had the ability to specifically neutralize VEGF-A and TGFβ1, among which the ability to neutralize human VEGF-A was the strongest, and it could also significantly neutralize human TGFβ1; oncolytic adenovirus expressing human VEGF trap could also effectively neutralize mouse VEGF, although the neutralization ability was weaker than that of neutralizing human VEGF-A; similarly, oncolytic adenovirus expressing human TGFβtrap could effectively neutralize mouse TGFβ1, and the ability to neutralize human TGFβ1 was stronger than that of neutralizing mouse TGFβ1, as shown in Figure 5. Figure 11A 、 Figure 11B shown.

[0211] The neutralization ability of various capture agent oncolytic adenoviruses on VEGF-A and TGFβ1 secreted by tumor cells was tested after infection of human and mouse tumor cells. Human SK-OV-3 ovarian cancer cells and mouse CT26 colorectal cancer cells were detected at 1×10 5 / well were inoculated into 24-well plates, and single or double capture agent oncolytic adenovirus was added after 24 hours, with a virus titer of 100 pfu / cell. After further culture for 24 hours, the cell supernatant was collected and the content of human and mouse VEGF-A and TGFβ1 was detected by ELISA kit. The results showed that the oncolytic adenovirus containing the corresponding trap could significantly neutralize the VEGF-A and TGFβ1 secreted by tumor cells after infecting human SK-OV-3 ovarian cancer cells and mouse CT26 colorectal cancer, especially the VEGF-A and TGFβ1 produced by human tumor cells, which could basically neutralize the VEGF-A and TGFβ1 produced by tumor cells. Figure 11C 、 Figure 11D shown.

[0212] Example 9: Replication of oncolytic adenovirus expressing VEGF or / and TGFβ trap is telomerase-dependent.

[0213] 80-90% of malignant tumor cells are telomerase-positive, which is the basis for the use of the telomerase promoter to regulate the selective replication of oncolytic adenoviruses in tumor cells, producing an oncolytic effect. To confirm whether the series of oncolytic adenoviruses constructed in this invention are telomerase-dependent, that is, whether the series of oncolytic adenoviruses specifically express E1A after infecting tumor cells, different tumor cells were infected with oncolytic adenoviruses expressing VEGF and / or TGFβ traps, and the expression of E1A protein was detected by Western blot (WB).

[0214] Human colon cancer cell line HCT116 was passaged into 6-well plates at a density of 1×10 6 After cells adhered to the wall, adenovirus encoding VEGF trap was used to

[0215] (Ad on -Flt1(Ig1~3)-KDR(Ig3)-IgG1Fc), single oncolytic adenovirus encoding TGFβtrap (Ad on -TGFβRII / TGFβRI-IgG1Fc) and an oncolytic adenovirus encoding both VEGF and TGFβtrap [Ad on -Flt1(Ig1~3)-KDR(Ig3) / TGFβRII / TGFβRI-IgG1Fc (fusion gene form) and (Ad on-Flt1(Ig2)-KDR(Ig3)-IgG1Fc / P2A / (TGFβRII)2-IgG1Fc)(viral P2A peptide linked form)] infection, a non-oncolytic adenovirus Ad5EGFP was used as a control virus, the titer of each virus was 100 pfu / cell, 2 hours after infection, washed twice with PBS to remove the uninfected virus, added fresh DMEM culture medium (containing 10% fetal bovine serum) 3 ml / well, and cultured for another 24 hours before the cells were collected and lysed for WB detection. The results showed that E1A expression could be detected in HCT116 tumor cells infected with single and dual capture agent oncolytic adenoviruses, while HCT116 tumor cells infected with Ad5 EGFP had no E1A expression, as shown in Figure 2. Figure 12A shown.

[0216] To test whether oncolytic adenovirus expressing E1A is universal in tumor cells, Ad5 EGFP and Ad on -Flt1(Ig1~3)-KDR(Ig3) / TGFβRII / TGFβRI-IgG1Fc infected different tumor cells, including human MDA-MB-231 breast cancer cells, SK-OV-3 ovarian cancer cells, and SGC-7901 gastric cancer cells. The infection method was the same as above, and the cells were collected for WB detection. The results showed that Ad on -Flt1(Ig1~3)-KDR(Ig3) / TGFβRII / TGFβRI-IgG1Fc can express E1A in different human tumor cells, while Ad5 EGFP has no E1A expression, such as Figure 12B shown.

[0217] Example 10: Oncolytic adenovirus expressing VEGF and / or TGFβ dual-capture agents has a significant inhibitory effect on the growth of human and mouse tumor cells.

[0218] Different tumor cells, including mouse colorectal cancer cells CT26, MC38 and breast cancer 4T1 cells, human breast cancer MDA-MB-231 cells and ovarian cancer SK-OV-3 cells, were seeded into 24-well plates and stained with simple oncolytic adenovirus (Adon-Control) and oncolytic adenovirus expressing double Traps (Ad on-Flt1(Ig1~3)-KDR(Ig3) / TGFβRII / TGFβRI-IgG1Fc) was infected with different virus titers, and the proliferation of tumor cells was significantly inhibited under the microscope for 48 hours, and the degree of inhibition showed a relationship between the virus titer effect. Moreover, compared with simple oncolytic adenovirus, Ad-VEGF / TGFβTraps has the effect of inhibiting tumor cell proliferation, but the cell morphological changes of the former are significantly different from those of the latter. It is speculated that this may be because the VEGF and TGFβ secreted by tumor cells are neutralized, blocking or inhibiting the growth of tumor cells stimulated by the signals of the two, as well as affecting the morphological changes and proliferation of tumor cells through epithelial-mesenchymal transition (EMT). Figure 13A 、 Figure 13B 、 Figure 13C 、 Figure 13D and Figure 13E shown.

[0219] CCK8 was used to detect different viral titers of VEGF / TGFβ trap oncolytic adenovirus to infect different human and mouse tumor cells. After 72 hours, the CCK8 kit was used to detect the effect of oncolytic adenovirus on the proliferation of different tumor cells. The results showed that Ad-VEGF / TGFβtrap could inhibit the proliferation of human and mouse tumor cells, and the inhibitory effect showed a viral dose-effect relationship. Figure 14 shown.

[0220] Example 11: Oncolytic adenovirus expressing VEGF and / or TGFβ dual-capture agents can induce cell death in human and mouse tumors.

[0221] Human HCT116 colon cancer cells, SGC7901 gastric cancer cells, SK-OV-3 ovarian cancer cells, A549 lung cancer cells, mouse CT26, MC38 rectal cancer cells, and 4T1 breast cancer cells were seeded into 6-well plates and oncolytic adenovirus expressing double Traps (Ad on -Flt1(Ig1~3)-KDR(Ig3) / TGFβRII / TGFβRI-IgG1Fc) was used to infect tumor cells with different virus titers (0MOI, 10MOI and 100MOI for human tumor cells, 0MOI, 100MOI and 250MOI for mouse tumor cells). After 48 hours, the virus-infected cells were collected and stained with an apoptosis kit (Annexin V / PI) (Biolegend). The apoptosis and necrosis of tumor cells were detected by flow cytometry. The results showed that the double-traps oncolytic adenovirus can induce apoptosis and necrosis in human and tumor cells, and the degree of apoptosis and necrosis showed a dose-effect relationship with the virus titer. The virus dose required to induce similar apoptosis in mouse tumor cells was significantly higher than that in human tumor cells, such as Figure 15A (Induces apoptosis in human tumor cells), Figure 15B(Inducing apoptosis of mouse tumor cells).

[0222] The present invention provides a method for constructing an oncolytic adenoviral vector and its application. The oncolytic adenoviral vector contains two expression cassettes, one of which is an adenoviral E1A gene regulated by a telomerase promoter, and the other is a VEGF and TGFβ trap fusion gene regulated by a cytomegalovirus promoter. The construction method of the oncolytic adenoviral vector includes the following steps: inserting the E1A expression cassette and the VEGF and TGFβ trap expression cassette head-to-head into the multiple cloning site of the shuttle plasmid pDC315, co-transfecting them with the backbone plasmid pBHGloxΔE1, E3 Cre into HER293 cells, and recombining them in the cells to produce the oncolytic adenoviral vector in about 2 weeks. After infecting a variety of human and mouse tumor cells, the oncolytic adenovirus can effectively neutralize the VEGF and TGFβ produced by the tumor cells.

[0223] 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 scope of protection of this application.

Claims

1. An oncolytic adenoviral vector expressing VEGF and TGFβ trap, characterized in that: In the oncolytic adenovirus vector, the oncolytic adenovirus is a type 5 adenovirus with two head-to-head expression cassettes inserted into the E1 region of the type 5 adenovirus, one of which is an expression cassette of the type 5 adenovirus-derived E1A gene regulated by the human telomerase promoter, and the other is an expression cassette of the VEGF and / or TGFβ trap gene regulated by the human cytomegalovirus promoter / enhancer; the vectors are type 5 adenovirus vectors and type 5 / 35 adenovirus vectors.

2. The oncolytic adenoviral vector expressing VEGF and TGFβ trap according to claim 1, characterized in that: The type 5 adenovirus vector is a replication-deficient adenovirus vector with E1 and E3 regions deleted, and the packaging adenovirus system is Ad Max TM The adenovirus vector system consists of a series of shuttle plasmids and pBHGfrtΔE1,3FLP backbone plasmids. After the shuttle plasmids and backbone plasmids with the target gene inserted are co-transfected into HER293 cells, they are recombined in the cells by FLP recombinase and obtained after 10-14 days.

3. The oncolytic adenoviral vector expressing VEGF and TGFβ trap according to claim 1, characterized in that: The human telomerase promoter, the expression cassette regulating the expression of the E1A gene comprises the human telomerase promoter, E1A and poly A components; The human cytomegalovirus promoter / enhancer, VEGF and / or TGFβ trap expression cassette comprises human cytomegalovirus promoter / enhancer, a series of fusion genes of VEGF and / or TGFβ trap and poly A component; VEGF trap and TGFβ trap each comprise a series of fusion genes.

4. The oncolytic adenoviral vector expressing VEGF and TGFβ trap according to claim 3, characterized in that: The human telomerase promoter can be replaced by other tumor-specific promoters, including: alpha-fetoprotein promoter, prostate-specific antigen promoter, transcription factor E2F-1 promoter, survivin promoter, hypoxia-inducible factor-1α promoter, nestin promoter, carcinoembryonic antigen promoter, p53 promoter and Mucin 1 promoter; The human cytomegalovirus promoter / enhancer can be replaced by other viral promoters with broad-spectrum promoter activity, as well as the human elongation factor 1α promoter.

5. The oncolytic adenoviral vector expressing VEGF and TGFβ trap according to claim 3, characterized in that: In the VEGF and / or TGFβ trap, a series of fusion genes constituting the VEGF trap include: Fusion of VEGFR1(Ig1-7), VEGFR1(Ig1-6)(amino acids 1-300), VEGFR1(Ig1-5)(amino acids 1-558), VEGFR1(Ig1-4)(amino acids 1-431), VEGFR1(Ig1-3)(amino acids 1-339) and IgG1 Fc; Fusion of VEGFR2(Ig1-7), VEGFR2(Ig1-6)(amino acids 1-300), VEGFR2(Ig1-5)(amino acids 1-300), VEGFR2(Ig1-4)(amino acids 1-300), VEGFR2(Ig1-3)(amino acids 1-300) and IgG1 Fc; Fusion of VEGFR1(Ig1-3)(amino acids 1-339), VEGFR2(Ig 3)(amino acids 1-300) and IgG1 Fc; 2) (amino acids 1-300), VEGFR2 (Ig1-3) (amino acids 1-300) and IgG1 Fc fusion; VEGFR1 (Ig2) (amino acids 129-229), VEGFR2 (Ig 3) (amino acids 1-300) and IgG1 Fc fusion; The nucleotide sequence of the fusion gene of VEGFR1 (Ig1-3), VEGFR2 (Ig 3) and IgG1 Fc is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the fusion gene of VEGFR1 (Ig 2) (amino acids 1 to 300), VEGFR2 (Ig1-3) and IgG1 Fc is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10; the nucleotide sequence of the fusion gene of VEGFR1 (Ig2), VEGFR2 (Ig 3) and IgG1 Fc is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.

6.

6. The oncolytic adenoviral vector expressing VEGF and TGFβ trap according to claim 5, characterized in that: In the VEGF and / or TGFβ trap, a series of fusion genes constituting the TGFβ trap include: A fusion of a TGFβRII extracellular segment sequence and IgG1 Fc, the nucleotide sequence is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14; a fusion of two TGFβRII extracellular segment sequences and IgG1 Fc, the nucleotide sequence is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.16; a fusion of a TGFβRII extracellular segment sequence, a TGFβRI extracellular segment sequence and IgG1 Fc, the nucleotide sequence is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.

18.

7. The oncolytic adenoviral vector expressing VEGF and TGFβ trap according to claim 6, characterized in that: The VEGF and / or TGFβ trap, wherein the fusion gene of VEGF and TGFβ trap maintains the same open reading frame through the viral 2A peptide, linker and GCN4-plC leucine zipper; The 2A peptide comprises E2A, F2A, P2A and T2A; the amino acid sequence of E2A is shown in SEQ ID NO.43, the amino acid sequence of F2A is shown in SEQ ID NO.44, the amino acid sequence of P2A is shown in SEQ ID NO.45, and the amino acid sequence of T2A is shown in SEQ ID NO.46; Linker comprises (G4S)3 and A(EAAAK)4A; the amino acid sequence of (G4S)3 is shown in SEQ ID NO.47, and the amino acid sequence of A(EAAAK)4A is shown in SEQ ID NO.48; The amino acid sequence of the GCN4-plC leucine zipper is shown in SEQ ID NO.

49.

8. The oncolytic adenoviral vector expressing VEGF and TGFβ trap according to claim 7, characterized in that: In the VEGF and / or TGFβ trap: A fusion gene of VEGF and TGFβ trap has a nucleotide sequence as shown in SEQ ID NO.50, and an amino acid sequence as shown in SEQ ID NO.51; A VEGF and TGFβ trap fusion gene comprises: VEGFR1 (Ig1-3) sequence (nucleotide 1 to 996 bp), VEGFR2 (Ig2) sequence (nucleotide 673 to 981 bp), (G4S)3 sequence, TGFβRII extracellular segment sequence, A (EAAAK)4A, TGFβRI extracellular segment sequence, IgG1 Fc sequence and C-myc sequence, wherein the nucleotide sequence is shown in SEQ ID NO.52, and the amino acid sequence is shown in SEQ ID NO.53; A fusion gene of VEGF and TGFβ capture agent comprises: VEGFR1 (Ig1-3) sequence (nucleotides 1 to 996 bp), VEGFR2 (Ig2) sequence (nucleotides 673 to 981 bp), (G4S)3 sequence, TGFβRII extracellular segment sequence, A(EAAAK)4A, TGFβRI extracellular segment sequence, GCN4-plC leucine zipper sequence, IgG1 Fc sequence and C-myc sequence, and its nucleotide sequence is shown in SEQ ID NO.54, and its amino acid sequence is shown in SEQ ID NO.

55.

9. The method for constructing an oncolytic adenoviral vector expressing VEGF and TGFβ trap according to any one of claims 1 to 8, characterized in that: In the method described, the adenoviral vector adopts AdMAX TM The system consists of two parts: a shuttle plasmid and a backbone plasmid; the target gene is inserted into the shuttle plasmid, and the shuttle plasmid with the inserted target gene and the backbone plasmid are co-transfected into HEK293 cells. The shuttle plasmid containing the target gene expression cassette is recombined into the E1 deletion region of the backbone plasmid by Cre recombinase in HEK293 cells to form a complete adenovirus vector containing the target gene. With the assistance of E1A expressed by HEK293 cells, the adenovirus vector replicates in HEK293 cells and causes cell lysis and release of adenovirus.

10. Use of an oncolytic adenoviral vector expressing VEGF and TGFβ trap constructed according to the method of claim 9, characterized in that: The oncolytic adenovirus vector is used to prepare a broad-spectrum anticancer preparation or immune adjuvant.

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