WO2022170919A1 - Recombinant oncolytic adenovirus and application thereof - Google Patents

Abstract

Provided are a recombinant oncolytic adenovirus and an application thereof. E1 and E3 regions of a genome of the recombinant oncolytic adenovirus are deleted; the deletion regions are stably inserted into exogenous nucleic acid sequences of a cholesterol regulatory element and an immunomodulatory element, wherein the cholesterol regulatory element is a functional element comprising an APOA1 gene or a degenerate sequence thereof; the immunomodulatory element is a functional element comprising a gene selected from GM-CSF, IFN-γ, IL2, IL7, IL12, IL15, IL16, IL18, IL21, IL22, IL27, IL28, and IL29 or a degenerate sequence thereof; the genes of the cholesterol regulatory element and the immunomodulatory element are expressed in a non-fusion manner; and the cholesterol regulatory element and the immunomodulatory element are operably connected to exogenous regulatory sequences, comprising a promoter sequence, an enhancer sequence, and a PA sequence. The functional elements carried by the recombinant oncolytic adenovirus can synergistically stabilize T cell functions in a tumor microenvironment.

Description

A kind of recombinant oncolytic adenovirus and its application technical field

The present invention relates to the field of tumor biotherapy; in particular, it relates to the preparation of an oncolytic adenovirus vector that can effectively inhibit tumor growth, invasion and metastasis. The vector can carry and express genes of cholesterol metabolism regulator molecules and immune costimulatory molecules.

Background technique

Some wild-type attenuated or genetically modified virus strains are widely used in the field of anti-tumor therapy because of their ability to replicate preferentially in tumors, and to exert direct oncolytic effects and induce immune activation. However, treatment with oncolytic viruses (OVs) alone has been ineffective. In order to solve this technical problem, the main technical solutions at this stage are: 1) Optimizing the skeleton structure of OVs (such as infection and replication-related genes) or material packaging to reduce their clearance by neutralizing antibodies and increase their impact on tumor tissue infection, Virological manifestations of replication and transmission; 2) Using OVs as a vector for gene therapy, expressing functional genes, and exerting the synergistic effect of OVs and functional genes (such as OVs expressing GM-CFS, IL-2, IL-12, IL-15, etc. Immune costimulatory molecules, or/and PD1, PD-1L, CTLA-4, TIGIT and other immune checkpoint modulators); 3) OVs combined with radiotherapy, chemotherapy and other immunotherapy and other second therapies (Nat Rev Cancer, 2018, 18 (7):419-432; Curr Opin Biotechnol, 2020, 65:25-36).

In recent years, studies have found that the dominant competition of tumor cells for certain metabolic substrates in the tumor microenvironment (TME), as well as the excretion and accumulation of metabolites, can inhibit the normal function of the immune system (Cell, 2015, 162(6): 1229- 41). As a result, a series of new immunotherapy targets have been extended: the metabolism-immunoregulation axis. Especially in immunotherapy mediated by OVs, after the live virus infects tumor cells, the replication of progeny virus and the expression of functional genes accelerate the consumption of nutrients and the accumulation of metabolic by-products in the TME. The deterioration of the metabolic environment induced by tumor development and the intervention of OVs weakens the anti-tumor immune response mediated by OVs and the immunomodulators they carry (Mol Ther, 2020, 28(6) 1417-1421; Front Cell Infect Microbiol, 2019, 9:95). Therefore, the correction of metabolic homeostasis in the abnormal TME has become an urgent problem for OVs therapy.

At present, the technical solutions for targeting metabolic reprogramming to improve the efficacy of OVs mainly focus on correcting the abnormality of the TCA cycle of glucose metabolism, such as lactate accumulation, OVs combined with PDK inhibitor DCA, recombinant oncolytic vaccinia virus (oVV) expressing leptin (oVV-leptin) ) (Cancer Res, 2019, 79(15): 3824-3836; Immunity, 2019, 51(3): 548-560). In 2019, a study in the journal Cell Metabolism reported that cholesterol accumulation in the TME microenvironment can induce the up-regulation of immune checkpoints on anti-tumor cytotoxic T lymphocytes (CTL), thereby accelerating the induction of CTL functional exhaustion (Cell Metab, 2019, 30 (Cell Metab, 2019, 30). 1): 143-156). Based on this research, the applicant creatively integrated the cholesterol metabolism regulatory element APOA1 into the oncolytic adenovirus Ad5 to obtain the recombinant APOA1 oncolytic adenovirus Ad5 (Ad5-APOA1). Compared with Ad5-con, the Ad5-APOA1 corrected the enriched cholesterol in the TME through APOA1, and synergistically inhibited the growth and metastasis of breast cancer and other tumors (PCT/CN2020/078360). However, the Ad5-APOA1 regimen had an incomplete response in some tumors. In order to solve this problem, the applicant has adopted a further optimized technical solution, aiming to further integrate immune regulatory factors on the Ad5-APOA1 backbone, that is, co-expression of cholesterol regulatory elements and immune regulatory elements.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the purpose of the present invention is to provide a recombinant oncolytic adenovirus and its application. The technical solution of the present invention has a mild immune response effect, which is beneficial to the early replication of oncolytic cells in tumor cells and the expression of functional genes, and cholesterol regulatory elements and immune regulatory elements can synergistically promote the formation of long-term immune memory.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following contents of the invention:

A recombinant oncolytic adenovirus whose genome E1 and E3 regions are deleted, and the deletion regions are stably inserted into exogenous nucleic acid sequences of cholesterol regulatory elements and immune regulatory elements; wherein,

The cholesterol regulatory element is a functional element comprising the APOA1 gene or its degenerate sequence;

The immunoregulatory element is a functional element comprising a gene selected from the group consisting of GM-CSF, IFN-γ, IL2, IL7, IL12, IL15, IL16, IL18, IL21, IL22, IL27, IL28 and IL29 or their degenerate sequences ;

The genes of the cholesterol regulatory element and the immune regulatory element are expressed in a non-fusion manner;

The cholesterol regulatory elements and immunomodulatory elements are operably linked to exogenous regulatory sequences, including promoter sequences, enhancer sequences and PA sequences.

As a preferred technical solution of the present application, the adenovirus is selected from the C serotype subgroup, including human adenovirus types 2 and 5.

As a preferred technical solution of the present application, the promoter sequence is selected from constitutive, tissue-specific or inducible promoters; preferably, the constitutive promoter is selected from CMV, SV40 or EF1α promoter.

As a preferred technical solution of the present application, the cholesterol regulatory element and the immune regulatory element are humanized.

Preferably, the adenovirus is human adenovirus type 5, the cholesterol regulatory element is APOA1 gene, and the immune regulatory element is IL15 gene.

As a preferred technical solution of the present application, the nucleic acid sequence of the cholesterol regulatory element APOA1 is selected from (a) or (b):

(a) as shown in SEQ ID NO: 1;

(b) a nucleic acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology to SEQ ID NO:1.

As a preferred technical solution of the present application, the nucleic acid sequence of the immunoregulatory element IL15 is selected from (c) or (d):

(c) as shown in SEQ ID NO: 2;

(d) a nucleic acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology to SEQ ID NO:2.

A recombinant oncolytic Ad5 comprising a modified Ad5 genome; the modification comprising (i) deletion of the E1, E3 region sequences of the wild-type Ad5 genome, but including the E1A sequence required for early replication; (ii) encoding a cholesterol metabolism regulator and/or an exogenous nucleic acid sequence of an immunomodulatory agent, wherein said exogenous nucleic acid sequence is stably added to at least the deleted region of said modified Ad5 genome.

Preferably, the exogenous nucleic acid sequence encoding a cholesterol metabolism regulator is shown in SEQ ID NO: 1; the exogenous nucleic acid sequence encoding an immunomodulatory agent is shown in SEQ ID NO: 2.

The applicant further integrated mild immunoregulatory factors (such as IL-15, etc.) on the Ad5-APOA1 backbone, namely, a novel oncolytic Ad5-APOA1-IL15 that co-expresses APOA1 and IL-15. First, the technical solution has a mild immune response effect, which is beneficial to the early replication of OVs in tumor cells and the expression of functional genes; second, APOA1 and IL-15 can synergistically promote the formation of long-term immune memory; third, with Compared with short-term potent OVs technical solutions such as arming IL-2, IL-6, GM-CFS or immune checkpoint inhibitors, the long-term mild immune activation effect of this solution can be applied to the immunotherapy of tumors of some important functional organs (such as liver, pancreas, intracranial, etc.).

The present invention also protects the application of any of the aforementioned recombinant oncolytic adenoviruses and the aforementioned recombinant oncolytic Ad5 in the preparation of medicines for treating or alleviating cancer-related diseases.

As a preferred technical solution of the present application, the present invention also protects any combination of the aforementioned recombinant oncolytic adenovirus, the aforementioned recombinant oncolytic Ad5, and the second therapeutic drug in the preparation of a drug for treating or alleviating cancer-related diseases. application.

More preferably, the second therapeutic drug is PD1 antibody.

As a preferred technical solution of the present application, the cancer is selected from the group consisting of gallbladder cancer, basal cell tumor, extrahepatic cholangiocarcinoma, colon cancer, endometrial cancer, uterine fibroids, esophageal cancer, Ewing sarcoma, prostate cancer, gastric cancer, and liver cancer , Hepatocellular carcinoma, Hodgkin lymphoma, laryngeal cancer, lung cancer, melanoma, mesothelioma, pancreatic cancer, rectal cancer, kidney cancer, thyroid cancer, glioma, malignant peripheral nerve cell tumor, malignant peripheral nerve sheath tumor, skin and plexiform neurofibromas, leiomyoma, leiomyosarcoma, fibroma, papillary adenoma, anaplastic thyroid carcinoma, medullary thyroid carcinoma, follicular thyroid carcinoma, hurthle cell carcinoma, thyroid carcinoma, ascites, malignant Ascites, salivary gland tumors, salivary gland mucoepidermoid carcinoma, salivary gland acinar cell carcinoma, gastrointestinal stromal tumors, tumors causing fluid accumulation in the underlying spaces of the body, pleural effusion, pericardial effusion, peritoneal effusion, giant cell tumor, giant bone Any of cell tumor, pigmented villonodular synovitis, giant cell tumor of the tendon sheath, and other sarcomas.

Preferably, the cancer is selected from glioma, pancreatic cancer or liver cancer.

The present invention also claims a pharmaceutical composition comprising the recombinant oncolytic adenovirus described in any one of the above, the recombinant oncolytic Ad5 described above, and a pharmaceutically acceptable carrier or excipient.

Preferably, the composition can be formulated for intratumoral injection.

The present invention also relates to a method for treating cancer, which comprises injecting an effective amount of the recombinant oncolytic Ad5 of the present invention or the pharmaceutical composition into a suitable subject.

In addition, the present invention also relates to the use of the recombinant oncolytic Ad5 in a method for treating cancer.

The present invention also relates to the application of the aforementioned recombinant oncolytic Ad5 or the pharmaceutical composition in the preparation of anticancer drugs.

beneficial effect

Compared with the prior art, the recombinant oncolytic adenovirus and its application provided by the present invention have the following beneficial effects:

(1) The technical solution of the present invention has a mild immune response effect, which is beneficial to the early replication of oncolytic cells in tumor cells and the expression of functional genes;

(2) The cholesterol regulatory element and the immune regulatory element of the present invention can synergistically promote the formation of long-term immune memory;

3) Compared with short-term and powerful OVs technical solutions such as arming IL-2, IL-6, GM-CFS or immune checkpoint inhibitors, the long-term mild immune activation effect of this solution can be applied to tumors of some important functional organs immunotherapy (such as liver, pancreas, intracranial, etc.).

Description of drawings

Various aspects and advantages of the present invention can be obtained from the following detailed description with reference to the accompanying drawings.

Figure 1. Schematic representation of recombinant oncolytic Ad5 virus. In the examples, the target gene sequence was inserted into the adenovirus E1 region to express human and murine APOA1, GM-CSF, IL7, and IL15 functional elements.

Figure 2. Expression of recombinant oncolytic Ad5 virus APOA1, GM-CSF, IL7 and IL15. ELISA was used to detect the expression of target protein in the supernatant of 293T cells infected with recombinant oncolytic Ad5 virus, **** indicates P<0.0001.

Figure 3. In vitro oncolysis assay. Example Oncolysis of recombinant oncolytic Ad-APOA1-IL15 after infection of human and murine tumor cell lines, MOI=0, 1, 10, 100.

Figure 4. Test for the detection of cholesterol content in tumor tissue after Ad5-APOA1 treatment. To detect the cholesterol enrichment in tumor tissue after Ad5-APOA1 treatment, * means P<0.05, ** means P<0.01.

Figure 5. Ad5-GM-CSF, Ad5-IL7 and Ad5-IL15 mediated immune activation assay. The expression of IFN-γ and TNF-α in the supernatant after treatment was detected by ELISA, reflecting the immune activation function of immune regulatory element molecules, ** means P<0.01, *** means P<0.001.

Figure 6. Ad5-APOA1 combined with Ad5-GM-CSF, Ad5-IL7 or Ad5-IL15 mediated immune activation assay. Simulated APOA1 in vitro to correct the depletion effect of cholesterol enrichment on CTL and restore the effect of immunomodulatory factors to activate CTL cells, * means P<0.05, ** means P<0.01, NS means no statistical significance.

Figure 7. In vivo antitumor activity assay of GL261 model. Orthotopic GL261 model to test the combined effect between recombinant oncolytic virus Ad5-mAPOA1 and Ad5-mGM-CSF or Ad5-mIL15, CR means complete cure, 5 C57bl/6 mice per group, virus at 2×10 ⁸ PFU The dose was injected once intratumorally on day 7 of tumor transplantation. In the GL261 subcutaneous tumor model, the virus was injected intratumorally every other day at a dose of 2×10 ⁸ PFU when the tumor volume was >50 mm ³ , three times in a row to verify the anti-tumor effect of Ad5-mAPOA1-mIL15.

Figure 8. In vivo antitumor activity assay of H22 model. H22 subcutaneous tumor model, to verify the anti-tumor effect of Ad5-mAPOA1-mIL15, the virus was injected intratumorally every other day at a dose of 2 × 10 ⁸ PFU when the tumor volume was >50 mm ³ for 3 consecutive times, * means P<0.05, ** Indicates P<0.01.

Figure 9. Panc02 model in vivo antitumor activity test. Panc02 subcutaneous tumor model, to verify the anti-tumor effect of Ad5-mAPOA1-mIL15, the virus was injected intratumorally every other day at a dose of 2×10 ⁸ PFU when the tumor volume was >50 mm ³ , three consecutive times, * means P<0.05, NS means Not statistically significant.

Figure 10. Antitumor rechallenge immune memory test. The immune memory intensity of T cells was measured 7 days after rechallenge in mice cured by GL261. CD44+CD62L+ means central memory T cells, * means P<0.05, ** means P<0.01, and *** means P<0.001.

Detailed ways

Detailed description of the invention

The term "a" or "an" entity refers to one or more of the entities; for example, "recombinant oncolytic Ad5" may be understood to mean one or more recombinant oncolytic Ad5 viruses. Thus, the terms "a," "one or more," and "at least one" may be used interchangeably herein.

"Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acids.

A polynucleotide or polynucleotide region has a specified percentage of another sequence, such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the "sequence" "Identity" means that the percentage of bases (or amino acids) of two sequences are identical when the sequences are aligned. This comparison and percent homology or sequence identity can be determined using software known in the art.

The term "cancer" or "tumor" refers to a group of diseases treatable in accordance with the present disclosure and involving abnormal cell growth, which may invade or spread to the body. Not all tumors are cancerous; benign tumors do not invade and metastasize to other parts of the body. The present disclosure is preferably applicable to solid tumors. Non-limiting examples of tumors or cancers include gallbladder cancer, basal cell tumor, extrahepatic cholangiocarcinoma, colon cancer, endometrial cancer, uterine fibroids, esophageal cancer, Ewing's sarcoma, prostate cancer, gastric cancer, liver cancer, hepatocytes carcinoma, Hodgkin lymphoma, laryngeal cancer, lung cancer, melanoma, mesothelioma, pancreatic cancer, rectal cancer, kidney cancer, thyroid cancer, glioma, malignant peripheral nerve cell tumor, malignant peripheral nerve sheath tumor (MPNST) ), skin and plexiform neurofibromas, leiomyoma, leiomyosarcoma, fibroma, papillary adenoma, anaplastic thyroid carcinoma, medullary thyroid carcinoma, follicular thyroid carcinoma, hurthle cell carcinoma, thyroid carcinoma, ascites, malignant Ascites, salivary gland tumor, salivary gland mucoepidermoid carcinoma, salivary gland acinar cell carcinoma, gastrointestinal stromal tumor (GIST), tumor causing fluid accumulation in the underlying space of the body, pleural effusion, pericardial effusion, peritoneal effusion, giant cell tumor (GCT), bone GCT, pigmented villonodular synovitis (PVNS), giant cell tumor of the tendon sheath (TGCT), and other sarcomas. In a preferred embodiment, the present invention is used in the preparation of medicaments for the treatment of glioma, pancreatic cancer and liver cancer.

The term "treatment" refers to both therapeutic treatment and prophylactic measures aimed at preventing or slowing down (lessening) abnormal physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction in disease severity, stabilization (ie, not worsening) disease state, delay or slowdown in disease progression, improvement or remission of disease state, and resolution of symptoms (partial or complete), Either detectable or undetectable. "Treatment" also means prolonging survival compared to expected survival if not receiving treatment. Patients in need of treatment include those already suffering from the disease or disorder, as well as those susceptible to the disease or disorder, or those for preventing the disease or disorder.

The terms "subject", "individual", "animal", "patient" or "mammal" refer to any subject, particularly mammalian subjects, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, livestock, farm animals, zoo animals, sports animals or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle and the like.

One of ordinary skill in the art will also see and understand that modified genomes as disclosed herein can be modified such that they differ in nucleotide sequence from the modified polynucleotides from which they are derived. For example, a polynucleotide or nucleotide sequence derived from a given DNA sequence may be similar, eg, have a certain percentage identity to the starting sequence, eg, it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% the same.

In addition, nucleotide or amino acid substitutions, deletions or insertions can be made to make conservative substitutions or changes in "non-essential" regions. For example, a polypeptide or amino acid sequence derived from a specified protein, except for one or more individual amino acid substitutions, insertions, or deletions (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 The remainder may be identical to the starting sequence except for one or more amino acid substitutions, insertions or deletions). In certain embodiments, the polypeptide or amino acid sequence derived from a given protein has 1 to 5, 1 to 10, 1 to 15, 1 to 20 individual amino acid substitutions, insertions or deletions relative to the starting sequence.

The term "regulatory element" is intended to include promoters, enhancers and other expression control elements. A promoter is a DNA sequence that directs RNA polymerase to bind DNA and initiate RNA synthesis. A strong promoter can cause high frequency initiation of mRNA. A suitable element for processing in eukaryotic cells is a polyadenylation signal. Regulatory element sequences include those that direct the expression of nucleotide sequences in many cells (eg, tissue-specific regulatory element sequences). Regulatory elements also include insulators, which include a class of DNA elements found on a cell's chromosomes that protect genes in one region of a chromosome from regulation in another region, such as the CTCF motif.

One of ordinary skill in the art can select appropriate regulatory elements based on, for example, the desired tissue specificity and expression level. For example, cell-type-specific or tumor-specific promoters can be used to restrict expression of a gene product to specific cell types. Examples of tissue-specific promoters that can be used in the present technology include the prostate-specific antigen (PSA) promoter, which is specific for prostate cells; the desmin promoter, which is specific for muscle cells; enolated Enzyme promoter, which is specific for neurons; β-globin promoter, which is specific for erythrocytes; tau-globin promoter, which is also specific for erythrocytes; growth hormone promoter, which is specific for pituitary cells are specific; the insulin promoter, which is specific for pancreatic beta cells; the glial fibrillary acidic protein promoter, which is specific for astrocytes; the tyrosine hydroxylase promoter, which is specific for catecholamines neuron specific; amyloid precursor protein promoter, which is specific for neurons; dopamine beta-hydroxylase promoter, which is specific for noradrenergic and adrenergic neurons ; tryptophan hydroxylase promoter, specific for serotonin/pineal cells; reg (pancreatolithin) promoter, specific for colon and rectal tumors and pancreatic and kidney cells; and the parathyroid hormone-related peptide (PTHrP) promoter, which is specific for liver and cecal tumors as well as schwannoma, renal, pancreatic and adrenal cells.

Examples of promoters that function specifically in tumor cells include the stromelysin 3 promoter specific for breast cancer cells; the surfactin A promoter specific for non-small cell lung cancer cells; cancer-specific SLPI expressing Secreted leukocyte protease inhibitor (SLPI) promoter; tyrosinase promoter specific for melanocytes; stress-inducible grp78/Bip promoter specific for fibrosarcoma/tumorigenic cells; AP2 fat specific for adipocytes Enhancer; alpha-1 antitrypsin transthyretin promoter specific for hepatocytes; interleukin-10 promoter specific for glioblastoma multiforme; Small cell lung cell specific c-erbB-2/3/4 promoter; brain tumor cell specific α-B-crystallin/heat shock protein 27 promoter; glioma and meningioma cell specific Basic fibroblast growth factor promoter; epidermal growth factor receptor promoter specific for squamous cell carcinoma, glioma and breast tumor cells; mucin-like glycoprotein (DF3, MUC1) promoter; mts1 promoter specific for metastatic tumors; thyroglobulin promoter specific for thyroid cancer cells; alpha-fetoprotein (AFP) promoter specific for liver cancer cells; villin promoter specific for gastric cancer cells; and the albumin promoter specific for hepatoma cells. In a preferred embodiment, the present invention selects glioma, liver cancer, pancreatic cancer tissue-specific promoters.

In addition to the use of tissue-specific promoters, local administration of the virus can achieve localized expression and effect. Non-tissue specific promoters can be used, such as the early cytomegalovirus (CMV) promoter, human elongation factor 1α (EF1α) promoter. For example, in some embodiments, the exogenous nucleotide sequence is operably linked to a promoter, such as a CMV or EF1α promoter.

The term "APOA1" is in NCBI Gene ID: 335. This gene encodes apolipoprotein A-I, the major protein component of high-density lipoprotein (HDL) in plasma. The encoded precursor protein is proteolytically processed to generate a mature protein that promotes efflux of cholesterol from tissues to the liver and is a cofactor for lecithin cholesterol acyltransferase (LCAT), a protein responsible for the formation of most plasma cholesterol esters. enzymes. This gene is closely related to two other apolipoprotein genes on chromosome 11. Defects in this gene are associated with high-density lipoprotein deficiency, including Tangier disease, as well as systemic nonneural amyloidosis. Alternative splicing results in multiple transcript variants, at least one of which encodes a preprotein. APOA1 gene has multiple transcripts, which are NM_000039.2/NP_000030.1 (cDNA sequence/protein sequence), NM_001318017.2/NP_001304946.1, NM_001318018.2/NP_011304947.1, NM_001318021.1/NP_001304950.1.

The term "IL15" is in NCBI Gene ID: 3600. This gene encodes interleukin 15, a pleiotropic cytokine that activates T cells, B cells and NK cells and mediates the proliferation and survival of these cells. Furthermore, IL15 activates, maintains and expands CD8+ memory T cells without activating regulatory T lymphocytes (Tregs, which have immunosuppressive functions). The role of IL15 is mainly to activate intracellular JAK and STAT by binding to the high-affinity α receptor (IL15Rα) expressed on antigen-presenting cells, thereby presenting IL15 to the IL2/15Rβγ dimer to form a ternary complex. Model pathway, which ultimately promotes the proliferation and activation of target cells, the increase in the secretion of IFN-γ and TNF-α, the up-regulation of Bcl-2 and Bcl-XL (anti-apoptotic proteins), and the down-regulation of Bim and Puma (pro-apoptotic proteins). -- Attenuate apoptosis signal.

Term "E1A". The E1 region genes of the virus can be further divided into E1A and E1B. E1A is mainly composed of two components, 289R (or 13S) and 243R (or 12S). After the adenovirus genome enters the nucleus, the cellular transcription factor first binds to the enhancer upstream of the E1A region to express the E1A protein, which regulates cellular metabolism and makes the viral DNA easier to replicate in the cell. E1A protein can also activate the promoters of other early genes (E1B, E2A, E2B, E3 and E4), wherein E2B drives three other early gene transcription unit terminal protein precursors (pTP, precursor terminal protein) related to viral replication, Expression of single-stranded DNA binding proteins (ssDBP, single-stranded DNA binding proteins) and DNA polymerases (DNA pol, DNA polymerase), the expression products of these three genes are tightly bound into a complex that binds to at least three cellular proteins interact to initiate replication of the viral genome. In the present invention, an independent CMV promoter is included before E1A. GFP and E1A are connected by the 2A linker sequence.

Without being bound by theory, 2A itself has only a post-translational protein cleavage site as a linker sequence, and the 2A peptide will remain behind the protein before 2A. Linking GFP and E1A using 2A allows separation of early replication elements and GFP-tagged proteins.

Without being bound by theoretical limitations, more than 100 serotypes have been found in the selection of adenovirus serotypes, of which 52 are human adenoviruses, which are divided into six subgroups (subgroups) A, B, C, D, E and F. The tropism for host cells, tumorigenicity, and disease history vary. Human adenovirus types 2 and 5 commonly used in gene therapy belong to subgroup C in serology, and have 95% homology in DNA sequence. The process of adenovirus infection of cells begins with the attachment of the head segment region of adenovirus cilia to specific receptors on the cell surface. Because human adenovirus mainly shares a receptor with coxsackie B virus, this receptor is called coxsackie/adenovirus receptor or CAR (coxsackie/adenovirus receptor). The low-level expression of CAR undoubtedly restricts the transduction efficiency of adenovirus. However, since the C subgroup adenovirus vector has been successfully used clinically, its safety has been extensively tested in humans, and it is highly safe as a vector. The recombinant oncolytic adenovirus of the present invention can replicate in tumor cells.

The medicines of the present invention are used in three aspects: preventive products, therapeutic products and diagnostic products. Preferably, the application is a therapeutic article. Treatment is monotherapy, adjuvant therapy or combination therapy.

The routes of administration of the drugs described in the present invention include but are not limited to oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, Intraperitoneal, intramuscular, subcutaneous, intravenous, in situ tumor administration. The preferred route of administration is intravenous injection, in situ on the tumor.

recombinant human adenovirus type 5

According to the replication-type oncolytic adenovirus vector according to any one of the present invention, the oncolytic adenovirus includes A, B, C, D, E and F subtypes, preferably C subtype adenovirus; more preferably, Human adenovirus type 5 Ad5, recombinant oncolytic Ad5 virus.

The present invention provides a recombinant oncolytic adenovirus capable of secreting cholesterol regulatory elements and immune regulatory elements. Specifically, the construction scheme of the replication-type oncolytic adenovirus vector includes the following steps: inserting the target sequence of the present invention into the adenovirus backbone vector deleted in the E1 and E3 regions. The target sequence can be inserted into one of the E1 or E3 regions or assigned to the E1 and E3 regions; the first constitutive promoter CMV controls the adenovirus early replication element (E1A) and detection tag (EGFP); the second constitutive promoter EF1α Controlled cholesterol regulatory elements (APOA1) and immune regulatory elements (eg IL-15). For example, the regulatory sequences of the first component promoter and the second component promoter of the present invention can be jointly assigned to the E1 region or the E3 region, or can be assigned to the E1 or E3 region respectively, such as the first component promoter is assigned to the E1 region, the second The constitutive promoter regulatory region is assigned to the E3 region. The first component promoter regulatory sequence and/or the second component promoter regulatory sequence in the E1 or E3 region is in no particular order of combination within and between the structures, including two combinations of the first and second regulatory sequences before and after, two promoters Variation combination, variation combination of functional sequences after two promoters. For example, CMV-E1A-EGFP/EF1α-APOA1-IL15 whose first and second regulatory regions are in the same E1 region can also be replaced by CMV-EGFP-E1A/EF1α-IL15-APOA1 and other post-promoter functional regions The method, promoter replacement such as EF1α-E1A-EGFP/CMV-EGFP-E1A, or the replacement of two functional regions such as EF1α-APOA1-IL15/CMV-E1A-EGFP, etc. The first component of the promoter sequence regulatory region and the second component of the promoter regulatory region are subject to the gene manipulation mode of reverse expression or back-to-back expression.

The first and second promoters, in addition to common constitutive promoters (eg, CMV, EF1α, SV40, etc.), can be substituted for condition-specific promoters or tissue-specific promoters common in the field. Interchanges between the first and second promoters do not include promoters of the same type.

Theoretically, the tag sequence (such as EGFP) used to detect virus titer and E1A are under the control of the same promoter, the recombinant oncolytic Ad5 for experimental use includes the detection tag sequence, and the EGFP tag of recombinant oncolytic Ad5 for pharmaceutical use Sequences can be replaced with other tracer labels or deleted. The tag sequence for experimental use is not limited to other tag sequences such as chemiluminescence, autofluorescence or His commonly used in the art.

E1A and EGFP or APOA1 and IL15 are connected by similar functional sequences such as PA2 or IRES2.

In one embodiment, the recombinant oncolytic Ad5 comprises an exogenous nucleic acid sequence encoding cholesterol metabolism regulation, and the cholesterol metabolism regulation sequence is selected from the human APOA1 sequence (in subsequent embodiments, the prefix is with h, which is of human origin). In another embodiment, the cholesterol metabolism regulatory sequence is selected from the murine APOA1 sequence (in the following embodiments, the prefix with m is of murine origin).

In one embodiment, the recombinant oncolytic Ad5 comprises an exogenous nucleic acid sequence encoding an immunomodulatory sequence selected from the group consisting of GM-CSF, IFN-γ, IL2, IL7, IL12, IL15, IL16, IL18, IL21, Cytokines such as IL22, IL27, IL28 and IL29 or chemokines such as MIP-1 and MCP-1. In a preferred embodiment, the immunoregulatory sequence is selected from IL7 or IL15. In a more preferred embodiment, the immunoregulatory sequence is human IL15 or humanized IL15 (in the following embodiments, the prefix is with h, which is of human origin). In a preferred embodiment, the immunoregulatory sequence is murine IL15 (in subsequent embodiments, the prefix with m is of murine origin).

In one embodiment, the recombinant oncolytic Ad5 comprises exogenous nucleic acid sequences encoding cholesterol metabolism regulatory sequences and immune regulation. For example, in one embodiment the cholesterol metabolism regulatory sequence is APOA1 and the immune regulatory sequence is IL15. The introduced foreign sequence is preferably inserted into the E1 region and placed after the E1A or E1A-EGFP sequence. For example, in one embodiment, the recombinant oncolytic Ad5 is Ad5-ΔE1 (CMV-E1A-EGFP/EF1α-APOA1-IL15); a secondary insertion into the E3 region, such as Ad5-ΔE1 (CMV-E1A-EGFP) -ΔE3 (EF1α-APOA1-IL15).

It will be appreciated that the stable integration of one or more exogenous nucleic acid sequences into the modified Ad5 genome does not interfere with the expression of the native Ad5 gene (Ad5-con), and the exogenous nucleic acid sequences are stably integrated into the modified Ad5 genome. Ad5 genome, so that functional expression of exogenous nucleic acid sequences can be expected.

Recombinant genes encoding cholesterol metabolism regulatory elements and/or immune regulatory elements contain nucleic acid encoding proteins and regulatory elements for protein expression. Typically, regulatory elements are present in the recombinant gene operably linked to the nucleic acid sequence to be expressed, and are selected based on the host cell to be used for expression, and may include transcriptional promoters, ribosome binding sites and terminators. Within a recombinant expression vector, "operably linked" is intended to mean a nucleic acid sequence of interest to allow expression of the nucleotide sequence (eg, in an in vitro transcription/translation system, or in a host cell when a virus is introduced into a host cell). expression) is linked to regulatory element sequences.

Oncolytic virus therapy mainly uses virus-induced DAMP pattern molecules and PAMP pattern molecules to break through the "cold" TME in the tumor and increase the infiltration, proliferation and differentiation of anti-tumor effector T cells. The enriched cholesterol in the tumor stroma up-regulates ER stress in CTL cells and induces the up-regulation of immune checkpoints such as PD1, resulting in a depleted phenotype. Removal of intratumorally enriched cholesterol helps to prolong the transient contact time of MHC-I-epitope complexes on CTLs and antigen-presenting cells around the tumor, increasing cytokines such as IFN-γ, TNF-α and Gzm-B release.

combination

OVs can be prepared in suitable pharmaceutically acceptable carriers or excipients. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the formulation must be sterile and must be fluid for easy syringability. It must be stable under the conditions of manufacture and storage and must be free from contamination by microorganisms such as bacteria and fungi.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity is maintained, for example, by the use of coatings such as lecithin, by the maintenance of the desired particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases it is preferred to include isotonic agents such as sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents that prolong absorption, for example, aluminum monostearate or gelatin.

For parenteral administration in aqueous solutions, for example, the solution should be suitably buffered (if necessary) and the liquid diluent first made isotonic with sufficient saline or dextrose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous perfusate, or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject. In addition, for human administration, the formulation responses meet the sterility, pyrogenicity, general safety, and purity requirements required by FDA standards for biologicals.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterile water. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those exemplified above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder containing the active ingredient plus any additional desired ingredient from a sterile-filtered solution.

As used herein, "carrier" includes any and all solvents, dispersion media, carriers, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like . Such media and agents for pharmaceutically active substances are well known in the art. In addition to any conventional media or agents incompatible with the active ingredient, it is contemplated that other media or agents may be used in the therapeutic compositions. Supplementary active ingredients can also be incorporated into the compositions.

The phrase "pharmaceutically acceptable" refers to molecular entities and components that do not produce allergic or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; fixed forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.

therapy

The invention discloses a recombinant oncolytic Ad5 virus. In another aspect, there is also disclosed a method of treating or alleviating cancer therewith, comprising administering to a subject in need thereof an effective amount of a recombinant oncolytic Ad5 virus or a pharmaceutical composition comprising the above-described recombinant oncolytic Ad5 virus.

In one aspect, the application of recombinant oncolytic Ad5 in the preparation of a drug for treating or alleviating cancer-related diseases is disclosed. In another aspect, a pharmaceutical composition of the recombinant oncolytic Ad5 is disclosed.

In some embodiments, the recombinant oncolytic Ad5 virus or pharmaceutical composition is administered intratumorally. In some embodiments, the recombinant oncolytic Ad5 virus or pharmaceutical composition is injected directly into the tumor as an injectable solution.

In some embodiments, recombinant oncolytic Ad5 viruses carrying cholesterol regulatory element and/or immune regulatory element genes can be combined with other cancer therapeutics. For example, the treatment of cancer can be implemented with recombinant oncolytic Ad5 virus and other secondary therapies such as surgery, PD1 antibodies, CAR-T or chemoradiotherapy. This process may involve simultaneous contacting of cells with expression constructs and reagents or multiple factors. This can be accomplished by contacting the cells with a single composition or pharmacological formulation comprising both doses, or by simultaneously contacting the cells with two different compositions or formulations, one comprising the expression construct and the other comprising the expression construct. One contains a second agent.

Viral treatment can be given at intervals ranging from minutes to weeks before or after other drug treatments. In embodiments where other agents and OVs are separately applied to the cells, it will generally be ensured that there is not a significant period of time between each delivery time so that the agent and virus can still beneficially exert a combined effect on the cells. In such a case, it is expected that the cells can be contacted with the two therapies within 12-24 hours of each other. However, in some cases, when administrations are separated by a period of days to weeks, it may be necessary to extend the duration of treatment significantly.

Construction of recombinant oncolytic Ad5 virus

The recombinant oncolytic Ad5 virus used in the present invention is modified on the basis of the ViraPower ^™ Adenoviral system. The E1A gene, a key element of viral replication, was inserted into a non-replicative adenovirus vector, thereby restoring the replication function of the recombinant oncolytic adenovirus. The construction of the replicative recombinant oncolytic Ad5 virus is divided into five steps: 1) construction of the shuttle plasmid pShuttle connecting the target gene; 2) homologous recombination of the shuttle plasmid and the viral vector; 3) rescue of the oncolytic adenovirus; 4) viral Amplification and purification; 5) titer detection of virus.

The fully synthetic CMV-E1A-P2A-EGFP-BGH polyA gene fragment was connected between attL1 and attL2 on pShuttle (pENTER/D-TOPO) to form Ad5-con;

The fully synthetic CMV-E1A-P2A-EGFP-EF1α-hAPOA1-BGH polyA gene fragment was ligated between attL1 and attL2 on pShuttle (pENTER/D-TOPO) to form Ad5-hAPOA1;

The fully synthetic CMV-E1A-P2A-EGFP-EF1α-mAPOA1-BGH polyA gene fragment was connected between attL1 and attL2 on pShuttle (pENTER/D-TOPO) to form Ad5-mAPOA1;

The fully synthetic CMV-E1A-P2A-EGFP-EF1α-mGM-CSF-BGH polyA gene fragment was connected between attL1 and attL2 on pShuttle (pENTER/D-TOPO) to form Ad5-mGM-CSF;

The fully synthetic CMV-E1A-P2A-EGFP-EF1α-mIL7-BGH polyA gene fragment was connected between attL1 and attL2 on pShuttle (pENTER/D-TOPO) to form Ad5-mIL7;

The fully synthetic CMV-E1A-P2A-EGFP-EF1α-hIL15-BGH polyA gene fragment was connected between attL1 and attL2 on pShuttle (pENTER/D-TOPO) to form Ad5-hIL15;

The fully synthetic CMV-E1A-P2A-EGFP-EF1α-mIL15-BGH polyA gene fragment was connected between attL1 and attL2 on pShuttle (pENTER/D-TOPO) to form Ad5-mIL15;

The fully synthetic CMV-E1A-P2A-EGFP-EF1α-hAPOA1-IRES2-hIL15-BGH polyA gene fragment was ligated between attL1 and attL2 on pShuttle (pENTER/D-TOPO) to form Ad5-hAPOA1-hIL15;

The fully synthetic CMV-E1A-P2A-EGFP-EF1α-mAPOA1-IRES2-mIL15-BGH polyA gene fragment was ligated between attL1 and attL2 on pShuttle (pENTER/D-TOPO) to form Ad5-mAPOA1-mIL15.

DH5α competent cells were transformed, single clones were selected, plasmids were extracted (strain preservation), and plasmid DNA (E1A) was identified by PCR.

The pShuttle plasmid was integrated into the pAd/PL-DEST adenovirus backbone genome vector using the LR Clonase II enzyme mix recombination kit. DH5α competent cells were transformed, single clones were selected, the plasmid was extracted (strain preservation), the length of the plasmid DNA fragment was identified by PacI digestion and PCR, and the sequence was confirmed by sequencing.

Correctly recombined plasmids were subjected to plasmid extraction, PacI digestion and linearization of the viral genome, and transfection into 293T cells to complete virus packaging, amplification and purification. The structure of each recombinant oncolytic Ad5 virus is shown in Figure 1, the nucleic acid sequence of hAPOA1 is shown in SEQ ID NO: 1, and the nucleic acid sequence of hIL15 is shown in SEQ ID NO: 2.

In vitro experiments

1) Expression of APOA1, GM-CSF, IL7 and IL15 of recombinant oncolytic Ad5 virus in vitro

The recombinant oncolytic Ad5 virus constructed in the present invention infects 2×10 ⁶ 293T cells with MOI value=10, and collects the cell culture supernatant 48 hours later to detect the expressions of APOA1, GM-CSF, IL7 and IL15 by ELISA. The results are shown in Figure 2. Each recombinant oncolytic Ad5 virus constructed can express the corresponding functional protein.

2) In vitro oncolysis experiment

Human glioma cell line U87, human glioma cell line U251, human liver cancer cell line HepG2, human pancreatic cancer cell line PANC1, mouse glioma cell line GL261, mouse liver cancer cell line H22, mouse pancreas cell line were cultured in 96-well plates The cancer cell line Panc02 and the mouse colon cancer cell line MC38 were inoculated with 5×10 ³ cells per well, and the recombinant oncolytic Ad5-hAPOA1-hIL15 or Ad5-mAPOA1-mIL15 virus with different MOI was inoculated overnight after the cells adhered to the wall. MOI values = 0, 1, 10, 100. After 24h, 48h and 72h of infection, the cell viability was detected by CCK8 method, and the results are shown in Figure 3.

Functional experiment

1) Functional experiments that APOA1 promotes the reduction of cholesterol enrichment in tumor tissues

GL261, H22 and Panc02 tumor cell lines were subcutaneously inoculated, and when the tumor volume reached more than 100 mm ³ , PBS, 1×10 ⁸ PFU of Ad5-con and Ad5-mAPOA1 were injected into the tumor respectively. After 2 days, the tumor tissues of each group were collected and 10 mg of tumor The tissue was lysed, and total cholesterol was extracted, and Amplex Red (invitrogen) was used to detect the cholesterol content of each group. The results are shown in Figure 4. The recombinant oncolytic Ad5 virus carrying APOA1 can reduce the cholesterol content in tumor tissue.

2) Functional experiment of GM-CSF, IL7 and IL15 to activate immune cells

GL261 cells were seeded in 24-well plates at 5×10 ⁴ cells per well. After the cells adhered, Ad5-con virus and recombinant oncolytic Ad5 virus carrying mGM-CSF, mIL7 and mIL15 respectively infected the cells at MOI=10 24h, the spleens of the immune normal mice were taken, ground, filtered, and the lymphocytes were co-cultured with the infected tumor cells at 1:20. After 48h, the supernatant was taken to detect the expression of IFN-γ and TNF-α by ELISA.

3) Functional experiment of APOA1 combined with GM-CSF, IL7 and IL15 to activate immune cells in a cholesterol-rich environment

Under the condition of 0.75ug/ml cholesterol medium, recombinant oncolytic Ad5 virus carrying mGM-CSF, mIL7 and mIL15 alone (MOI=10) or 1:1 combined recombinant oncolytic Ad5-mAPOA1 virus (MOI=5:MOI= 5) Infected GL261 cells for 24 hours, then took the spleen of immune normal mice, ground, filtered to take the lymphocytes and co-cultured with the infected tumor cells at 1:20, and 48 hours later, the supernatant was taken to detect the expression of IFN-γ and TNF-α by ELISA. The results are as follows shown in Figure 6.

In vivo experiments

1) Anti-tumor experiment of recombinant oncolytic Ad5 virus in vivo

Subcutaneous and intracranial xenografts of GL261, H22 and Panc02 cells were used to evaluate the antitumor activity of recombinant oncolytic Ad5 virus. GL261 adopts intracranial tumor transplantation, and the evaluated recombinant oncolytic Ad5 viruses involve Ad5-con, Ad5-mAPOA1, Ad5-mIL15, Ad5-mGM-CSF, Ad5-mAPOA1+Ad5-mIL15 combination and Ad5-mAPOA1+Ad5-mGM -CSF combination; GL261 was used to subcutaneously transplant the tumor to evaluate the in vivo antitumor activity of Ad5-mAPOA1-mIL15, and the results are shown in Figure 7. The recombinant oncolytic Ad5-mAPOA1-mIL15 was evaluated by subcutaneous tumor transplantation for H22 and Panc02, and the results are shown in Figures 8 and 9. Compared with recombinant oncolytic Ad5 virus carrying mAPOA1 or mIL15 alone, Ad5-mAPOA1-mIL15 has obvious anti-tumor advantages. The technical inspiration from the examples of the present invention makes it easy for those skilled in the art to obtain the anti-tumor effect of Ad5-hAPOA1-hIL15 in a humanized animal model.

2) mAPOA1 and mIL15 carried by Ad5 have a synergistic effect of promoting immune memory effect

The cured GL261 mice were used for the challenge experiment. After 7 days, the spleen of the mice was taken for flow cytometry to detect the changes of CD8+ T cell immune memory biomarkers (CD44 and CD62L). The results are shown in Figure 10. In the oncolytic Ad5 group co-expressing APOA1 and IL15, CD62L and CD44 were significantly increased, showing a synergistic effect of promoting immune memory.

Those skilled in the art will readily appreciate that the present invention is well adapted to attain the ends and advantages mentioned as well as those inherent herein. The methods, variations and compositions described herein as presently representative of the preferred embodiments are exemplary only and do not limit the scope of the invention. Modifications and other uses may occur to those skilled in the art, but are also included within the true spirit of the invention as defined by the scope of the claims.

The invention exemplarily described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. The terms and expressions used are intended to be descriptive and not limiting and are not intended to exclude any equivalents of the features shown or described, or parts thereof, Modifications are possible within the scope of the present invention. Therefore, it is to be understood that although this invention has been specifically disclosed in terms of preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention as defined in the appended claims.

Claims (10)

1. A recombinant oncolytic adenovirus, characterized in that the E1 and E3 regions of its genome are deleted, and the deletion regions are stably inserted into exogenous nucleic acid sequences of cholesterol regulatory elements and immune regulatory elements, wherein,

   The cholesterol regulatory element is a functional element comprising the APOA1 gene or its degenerate sequence;

   The immunoregulatory element is a functional element comprising the genes selected from GM-CSF, IFN-γ, IL2, IL7, IL12, IL15, IL16, IL18, IL21, IL22, IL27, IL28 and IL29 or their degenerate sequences ;

   The genes of the cholesterol regulatory element and the immune regulatory element are expressed in a non-fusion manner;

   The cholesterol regulatory elements and immunomodulatory elements are operably linked to exogenous regulatory sequences, including promoter sequences, enhancer sequences and PA sequences.
2. The recombinant oncolytic adenovirus according to claim 1, wherein the adenovirus is selected from the subgroup of serotype C, including human adenovirus types 2 and 5.
3. The recombinant oncolytic adenovirus according to claim 1, wherein the promoter sequence is selected from constitutive, tissue-specific or inducible promoters; preferably, the constitutive promoter is selected from CMV, SV40 or EF1α promoter.
4. The recombinant oncolytic adenovirus according to claim 1, wherein the cholesterol regulatory element and the immune regulatory element are humanized.
5. The recombinant oncolytic adenovirus according to claim 1 or 4, wherein the adenovirus is human adenovirus type 5, the cholesterol regulatory element is APOA1 gene, and the immune regulatory element is IL15 gene.
6. The recombinant oncolytic adenovirus according to claim 5, wherein the nucleic acid sequence of the cholesterol regulatory element APOA1 is selected from (a) or (b):

   (a) as shown in SEQ ID NO: 1;

   (b) a nucleic acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology to SEQ ID NO: 1;

   Preferably, the nucleic acid sequence of the immunoregulatory element IL15 is selected from (c) or (d):

   (c) as shown in SEQ ID NO: 2;

   (d) a nucleic acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology to SEQ ID NO:2.
7. A recombinant oncolytic Ad5 comprising a modified Ad5 genome, characterized in that: the modification comprises (i) deletion of the E1 and E3 region sequences of the wild-type Ad5 genome, but including the E1A sequence required for early replication; (ii) encoding An exogenous nucleic acid sequence of a cholesterol metabolism regulator and/or an immunomodulator, wherein the exogenous nucleic acid sequence is stably added to at least the deleted region of the modified Ad5 genome; preferably, the The exogenous nucleic acid sequence encoding a cholesterol metabolism regulator is shown in SEQ ID NO: 1; the exogenous nucleic acid sequence encoding an immunomodulator is shown in SEQ ID NO: 2.
8. Use of the recombinant oncolytic adenovirus according to any one of claims 1-6 and the recombinant oncolytic Ad5 according to claim 7 in the preparation of a medicine for treating or alleviating cancer-related diseases; preferably, any one of claims 1-6 Use of the recombinant oncolytic adenovirus, the recombinant oncolytic Ad5 according to claim 7, and a second therapeutic drug in the preparation of a drug for treating or alleviating cancer-related diseases; more preferably, the second therapeutic drug is PD1 antibody.
9. The application according to claim 8, wherein the cancer is selected from the group consisting of gallbladder cancer, basal cell tumor, extrahepatic cholangiocarcinoma, colon cancer, endometrial cancer, uterine fibroids, esophageal cancer, Ewing's sarcoma, prostate cancer Cancer, gastric cancer, liver cancer, hepatocellular carcinoma, Hodgkin lymphoma, laryngeal cancer, lung cancer, melanoma, mesothelioma, pancreatic cancer, rectal cancer, kidney cancer, thyroid cancer, glioma, malignant peripheral nerve cell tumor , malignant peripheral nerve sheath tumor, skin and plexiform neurofibroma, leiomyoma, leiomyosarcoma, fibroma, papillary adenoma, anaplastic thyroid carcinoma, medullary thyroid carcinoma, follicular thyroid carcinoma, hurtle cell carcinoma, thyroid Carcinoma, ascites, malignant ascites, salivary gland tumor, salivary gland mucoepidermoid carcinoma, salivary gland acinar cell carcinoma, gastrointestinal stromal tumor, tumor causing fluid accumulation in the latent space of the body, pleural effusion, pericardial effusion, peritoneal effusion, macroscopic Any of cell tumor, giant cell tumor of bone, pigmented villonodular synovitis, giant cell tumor of tendon sheath and other sarcomas, preferably glioma, pancreatic cancer or liver cancer.
10. A pharmaceutical composition comprising the recombinant oncolytic adenovirus according to any one of claims 1-7 or the recombinant oncolytic Ad5 according to claim 7, and a pharmaceutically acceptable carrier or excipient; preferably Yes, the composition is formulated for intratumoral administration.

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