Recombinant oncolytic adenovirus having dual tumor targeting properties, preparation method therefor, and use thereof

Abstract

Disclosed in the present invention is a recombinant oncolytic adenovirus having dual tumor targeting properties. Also disclosed in the present invention are a preparation method for and use of the recombinant oncolytic adenovirus. The recombinant oncolytic adenovirus of the present invention is constructed using a chimpanzee adenoviral vector AdC68XY-R1. Experiments show that the recombinant oncolytic adenovirus of the present invention has a higher tumor targeting effect, higher safety, and stronger tumor killing activity.

Description

Recombinant oncolytic adenovirus with dual tumor targeting, preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an oncolytic adenovirus with dual tumor targeting. The present invention also provides a method for preparing the oncolytic adenovirus with dual tumor targeting and its use in the treatment of hypoxic tumors. Background Art

[0002] In the early 20th century, clinicians observed several cases of cancer patients experiencing symptom remission accompanied by viral infections, sparking the search for viral therapies. Many viruses, such as reovirus (RV), coxsackievirus (CV), and Newcastle disease virus (NDV), were found to possess natural oncolytic properties. Oncolytic viruses (OVs) are a class of replication-competent viruses that can directly lyse tumor cells. The emergence of OVs has provided a new therapeutic strategy for cancer treatment. Currently, many viruses have been genetically modified to enhance their tumor toxicity and targeting. OVs selectively infect and kill tumor cells by exploiting genetic differences between tumor cells and normal cells, including activation of oncogenes, inactivation of tumor suppressor genes, overexpression of certain receptors on the tumor cell surface, the hypoxic and acidic tumor microenvironment, and abnormal signaling pathways. OVs can achieve their tumor-killing effects through various pathways, including direct oncolysis, induction of innate and adaptive immune responses, alteration of the tumor microenvironment, and disruption of tumor blood supply.

[0003] Currently, five OVs have received regulatory approval, primarily from herpes simplex virus (HSV) and adenovirus (AdV). AdV is one of the most widely studied and applied OVs due to its advantages, including simple genome, ease of amplification, high tumor selectivity, good safety, and strong ability to express exogenous therapeutic genes. Oncolytic adenovirus, as a novel tumor treatment, has shown promising application prospects in preclinical and clinical studies. However, OVs still have certain limitations, including oncolytic efficacy, tumor targeting, and biosafety. H101, an oncolytic adenovirus derived from Ad serotype 5 (Ad5), was approved for marketing by the National Medical Products Administration (NMPA) in 2005 for the treatment of head and neck squamous cell carcinoma. However, its therapeutic efficacy and application are challenged by the prevalence of preexisting neutralizing antibodies against Ad5 in 75-80% of the population. Furthermore, the hypervariable region of the Ad5 hexon protein binds to coagulation factor X, leading to the adenovirus's easy accumulation in the liver, potentially causing toxic side effects. Therefore, researchers have explored the use of rare human serotypes or adenoviruses from other species as OVs vectors. Chimpanzee adenoviruses are only prevalent in chimpanzees in Africa, and humans generally do not have neutralizing antibodies against chimpanzee adenoviruses. Therefore, the present invention chooses chimpanzee adenovirus vector (AdC68) as the OVs vector.

[0004] The safety and efficacy of OVs are not only related to the oncolytic properties of the virus itself, but also closely related to the optimization and modification strategy of the viral genome. OV genome modification strategies include deleting non-essential genes, adding co-stimulatory molecule genes, adding chemokine genes, adding cytokine genes, adding immune checkpoint inhibitor genes, adding tumor target-specific killer molecule genes, and using tumor-specific promoters.

[0005] Furthermore, the tumor microenvironment (TME) plays a crucial role in tumor development, growth, and metastasis, and its immunosuppressive state is a key factor in the failure of various anti-tumor therapies, including immunotherapy. Hypoxia is a common feature of most solid tumors. Abnormal vascular structures within solid tumors cause insufficient blood supply, and excessive tumor cell proliferation leads to increased oxygen consumption. Oxygen levels within tumor tissue are often below 2%. Therefore, hypoxia can be exploited to target and reshape the TME to develop improved oncolytic adenoviruses, enhancing therapeutic efficacy and clinical applicability.

[0006] Currently, there are few studies on hypoxic replicating OVs developed for AdC68 vectors with dual tumor targeting and immunomodulatory functions. Therefore, there is still a need to further develop oncolytic adenoviruses with better effects. Summary of the Invention

[0007] The purpose of the present invention is to address the deficiencies of the existing technology and provide a recombinant oncolytic adenovirus with dual tumor targeting.

[0008] The concept of the present invention is as follows: using AdC68XY-R1 as a vector, by adding a tumor-specific targeting promoter and inserting a hypoxia response element, the oncolytic virus has the activity of dual targeting tumor tissue; at the same time, the oncolytic virus can efficiently express exogenous genes that enhance T cell effects and tumor killing factors, ultimately obtaining a new oncolytic virus with higher targeting effect, higher safety and higher killing effect.

[0009] Specifically, the present invention uses genetic engineering to transform the AdC68XY-R1 chimpanzee adenovirus vector, and controls the expression of genes necessary for viral proliferation through a tumor-specific promoter, so that it replicates only in tumor cells and is in a low expression or non-expression state in normal cells. In addition, by inserting hypoxia regulatory elements into OVs, exogenous genes that can recruit endogenous effector cells can be expressed under the hypoxic microenvironment conditions of solid tumors. Ultimately, dual tumor targeting is achieved on OVs, and its killing effect is enhanced, thereby strengthening the effect of anti-tumor treatment.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] The first aspect of the present invention provides a recombinant oncolytic adenovirus with dual tumor targeting, wherein the recombinant oncolytic adenovirus is derived from the replicating chimpanzee adenovirus vector AdC68XY-R1, and wherein the promoter is constructed as a tumor-specific and hypoxia-responsive promoter; further, the recombinant oncolytic adenovirus is also constructed to contain an insertion element, and the insertion element recruits endogenous effector cells through expression.

[0012] In a specific embodiment, the AdC68XY-R1 chimpanzee adenovirus vector was purchased from Suzhou Xiangyi Biotechnology Co., Ltd.

[0013] In a specific embodiment, the insertion element is selected from one or more of the following:

[0014] (1) an insertion element that simultaneously expresses one or more of GM-CSF, IFN-α / β / γ, IL-2, IL-3, IL-7, IL-12, IL-15, IL-21, IL-33, IL-35, IL-37, CCL4, CCL20, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL3, CCL4, and αCTLA4;

[0015] and / or

[0016] (2) Expression of TNF-α, T cell engagers (bispecific T cell engagers, BiTE), chimeric antigen receptor (CAR), apoptosis genes, pyroptosis genes, toxins and their combinations.

[0017] Optionally, the insertion element is further coupled to an oxygen sensitive element.

[0018] Preferably, the insertion element is an insertion element that simultaneously expresses two, three, four or five of CXCL9, CXCL10, CXCL11, αCTLA4 and IL-21;

[0019] Preferably, the insertion element simultaneously expresses CXCL9, CXCL10, CXCL11, αCTLA4 and IL-21; preferably, the insertion element comprises exogenous genes encoding and expressing CXCL9, CXCL10, CXCL11, αCTLA4 and IL-21; in a preferred embodiment, the exogenous genes are connected by T2A cleavage peptide, P2A or IRES;

[0020] Preferably, the sequence of the front and back connection of the exogenous gene nucleic acid sequence is interchangeable;

[0021] Preferably, the expression products of the inserted element are CXCL9, CXCL10, CXCL11, αCTLA4 and IL-21;

[0022] Preferably, any one of the expression products of the inserted elements is a fusion protein having at least 70% homology to the amino acid sequence shown in SEQ ID NO: 5, 7, 9, 11 or 13, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology.

[0023] Optionally, the coding nucleotide sequence of the insertion element is shown in 4, 6, 8, 10 or 12 respectively.

[0024] In a preferred embodiment, the insertion element is an insertion element expressing a T cell engager;

[0025] Preferably, the tumor antigen that binds to the T cell engager is selected from one or more of CD19, BCMA, CD20, CD22, CD30, CD33, CD38, CD47, CD73, CD70, CD117, CD123, CD133, CD138, CD147, CD171, CD276, NKG2DL, HER2, MUC1, MUC16, CEA, EpCAM, IL-13Rα2, EGFR, EGFRvIII, GD2, DR5, EphA2, FRα, PSCA, PSMA, TARP, cMet, VEGFR2, BCMA, CTLA-4, PD-L1, AFP, GPC3, AXL, ROR1, ROR2, FAP, Mesothelin, DLL3 and CLD18;

[0026] Preferably, the T cell engager is a bispecific T cell engager;

[0027] More preferably, the bispecific T cell engager is composed of αCD276 or αCD73 or αCD47 connected to αCD3, that is, it is formed by the fusion of scFv segments of two types of antibodies, which can respectively bind to tumor cell surface antigen molecules such as CD276, CD73 or CD47 and CD3 molecules on the surface of T cells, thereby inducing T cells to kill tumor cells without being restricted by MHC.

[0028] Preferably, the expression product of the inserted element is: VLαCD276-L-VHαCD276-L-VHαCD3-L-VLαCD3, or VLαCD73-L-VHαCD73-L-VHαCD3-L-VLαCD3, or VHαCD47-L-VHαCD3-L-VLαCD3-L-VLαCD47, for details, see Chinese patent application No. 202111615701.2.

[0029] More preferably, the bispecific T cell engager further comprises an ODD18 oxygen-sensing element;

[0030] In an optional embodiment, the sequence of the ODD18 oxygen-sensing element is shown as SEQ ID NO: 16; and the amino acid sequence thereof is shown as SEQ ID NO: 25.

[0031] In an optional embodiment, the amino acid sequence of the bispecific T cell engager has at least 70% homology to the amino acid sequences shown in SEQ ID NO: 15, 18 and 20, respectively.

[0032] Among them, SEQ ID NO: 15 exemplifies the amino acid sequence of CD276-CD3-ODD18, and its corresponding nucleotide sequence is SEQ ID NO: 14; SEQ ID NO: 18 exemplifies the amino acid sequence of CD73-CD3-ODD18, and its corresponding nucleotide sequence is SEQ ID NO: 17; SEQ ID NO: 20 exemplifies the amino acid sequence of CD47-CD3-ODD18, and its corresponding nucleotide sequence is SEQ ID NO: 19.

[0033] In a preferred embodiment, the tumor-specific promoter is selected from the human telomerase reverse transcriptase (hTERT) promoter, whose sequence is shown in SEQ ID NO: 1.

[0034] Tumor-specific promoters can also be selected from carcinoembryonic antigen (CEA) promoter, alpha-fetoprotein (AFP) promoter, human prostate-specific antigen (PSA) promoter, cyclooxygenase-2 (COX-2) promoter, apoptosis inhibitor protein (survivin) promoter or human intestinal tissue-specific antigen (A33) promoter, etc.

[0035] In a preferred embodiment, the hypoxia-responsive promoter is a promoter of a transcriptional control system that responds to hypoxia triggering, and comprises a hypoxia regulatory element HRE.

[0036] Preferably, its sequence is as shown in SEQ ID NO: 2, which is a tandem element of 5 HREs

[0037] It will be understood by those skilled in the art that the above-mentioned target genes such as chemokines, cytokines, and killer factors are regulated by the hypoxia-responsive promoter with HRE as the core: under normoxia, the activity of this promoter is extremely low and cannot effectively initiate the transcription and translation of the target gene; under hypoxic conditions, the HIF-1 complex enters the nucleus and binds to the HRE element, effectively activating the promoter activity and promoting the efficient expression of the target gene.

[0038] At the same time, the characteristics of the killing factor-coupled oxygen-sensitive element that it degrades under normoxia but is stable under hypoxic conditions can further enhance the specificity of hypoxic expression of the target gene.

[0039] The second aspect of the present invention provides a polynucleotide encoding the aforementioned recombinant oncolytic adenovirus.

[0040] The present invention also provides a polynucleotide for constructing the recombinant oncolytic adenovirus;

[0041] Preferably, the polynucleotide comprises one or more of the promoter, insertion element, hypoxia regulatory element, transcription start and / or termination signal mentioned in the aforementioned recombinant oncolytic adenovirus;

[0042] Preferably, the polynucleotide comprises a polynucleotide sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 6, 8, 10, 12, 14, 17, 19, 21, 22, 23, and 24.

[0043] The third aspect of the present invention provides a recombinant oncolytic adenovirus vector, wherein the vector is prepared using the following method:

[0044] Using the genome of chimpanzee adenovirus vector AdC68XY-R1 as the basic framework, the aforementioned insertion elements are cloned into the replicative chimpanzee adenovirus vector AdC68XY; and / or

[0045] Replacing the chimpanzee adenovirus AdC68XY promoter with a tumor-specific and hypoxia-responsive promoter; and / or

[0046] and / or inserting an expression framework of a hypoxia regulatory element downstream of the E1A gene of the chimpanzee adenovirus AdC68XY-R1.

[0047] The present invention also provides a method for preparing the recombinant oncolytic adenovirus, which is obtained by packaging the vector.

[0048] In an exemplary embodiment, the carrier is prepared using the following method:

[0049] (1) Construction of pAdC68XY-R1-HRE-CXCL9-CXCL10-CXCL11-αCTLA4-IL-21 (SEQ ID NO: 21) and pAdC68XY-R1-HRE-αCD276-αCD3-BiTE (SEQ ID NO: 22), pAdC68XY-R1-EF1α-αCD276-αCD3-BiTE, pAdC68XY-R1-αCD276-αCD3-BiTE, pAdC68XY-R1-HRE-αCD73-αCD3-BiTE (SEQ ID NO: 23), pAdC68XY-R1-EF1α-αCD73-αCD3-BiTE, pAdC68XY-R1-αCD73-αCD3-BiTE, pAdC68XY-R1-HRE-αCD47-αCD3-BiTE (SEQ ID NO: 24);

[0050] (2) The above sequences were cloned into the AdC68XY-R1 vector respectively;

[0051] (3) The recombinant adenoviral vector was transfected into 293A cells, and the recombinant adenoviruses AdC68XY-R1-HRE-CXCL9-CXCL10-CXCL11-αCTLA4-IL-21, AdC68XY-R1-HRE-αCD276-αCD3-BiTE, AdC68XY-R1-EF1α-αCD276-αCD3-BiTE, AdC68XY-R1-αCD276-αCD3-BiTE, AdC68XY-R1-HRE-αCD73-αCD3-BiTE, AdC68XY-R1-EF1α-αCD73-αCD3-BiTE, AdC68XY-R1-αCD73-αCD3-BiTE, and AdC68XY-R1-HRE-αCD47-αCD3-BiTE were packaged and obtained;

[0052] (4) Purification and identification of recombinant adenovirus plaques;

[0053] (5) Verification of recombinant adenovirus function.

[0054] The above-mentioned oncolytic adenovirus engineered to express functional exogenous genes or a combination thereof can be used to prepare biological preparations for treating different types of cancer.

[0055] The fourth aspect of the present invention provides a pharmaceutical composition comprising the aforementioned recombinant oncolytic adenovirus.

[0056] The fifth aspect of the present invention provides use of the aforementioned recombinant oncolytic adenovirus or the aforementioned pharmaceutical composition in the preparation of a medicament for treating tumors;

[0057] Preferably, the tumor is selected from one or more of B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high-grade glioma, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, large intestine cancer, hematopoietic system cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or blood oncogenic diseases.

[0058] In a preferred embodiment, the recombinant oncolytic adenovirus is used alone or in combination with CAR-T.

[0059] A sixth aspect of the present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the aforementioned recombinant oncolytic adenovirus or the aforementioned pharmaceutical composition to a patient in need thereof;

[0060] Preferably, the tumor is selected from one or more of B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high-grade glioma, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, large intestine cancer, hematopoietic system cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or blood oncogenic diseases.

[0061] The seventh aspect of the present invention provides the aforementioned recombinant oncolytic adenovirus kit, wherein the kit comprises:

[0062] The aforementioned recombinant oncolytic adenoviral vector;

[0063] Virus-producing cells.

[0064] The beneficial effect of the present invention is that it constructs an oncolytic adenovirus with dual tumor targeting, which is different from traditional oncolytic adenoviruses. Chemokines or highly specific killer molecules are added to the hypoxia-regulating "switch" and carried on the tumor-specific replication AdC68XY-R1 chimpanzee virus vector, so that it is highly expressed only in hypoxic tumor tissues, exerting anti-tumor effects and reducing off-target side effects.

[0065] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0066] BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, in which:

[0068] FIG1 is a schematic diagram of the construction of eight recombinant adenoviral vectors including pAdC68XY-R1-HRE-CXCL9-CXCL10-CXCL11-αCTLA4-IL-21 according to an embodiment of the present invention.

[0069] FIG2 illustrates the construction of a recombinant plasmid integrating an oncolytic adenovirus with dual tumor targeting according to an embodiment of the present invention.

[0070] Among them, eight recombinant adenoviral vectors, including pAdC68XY-R1-HRE-CXCL9-CXCL10-CXCL11-αCTLA4-IL-21, were identified by digestion with BglII, Mfe1, and Xho1, and the recombinant plasmid bands were all correct;

[0071] FIG3 shows plaques of eight recombinant adenoviruses, including AdC68XY-R1-HRE-CXCL9-CXCL10-CXCL11-αCTLA4-IL-21, according to an embodiment of the present invention. All eight recombinant adenoviruses can produce obvious plaques in 293A cells.

[0072] Figure 4 shows the expression of the AdC68XY-R1-HRE-CXCL9-CXCL10-CXCL11-αCTLA4-IL-21 recombinant adenovirus according to an embodiment of the present invention in human ovarian cancer cells (SKOV3) under normoxic and hypoxic conditions. As shown in the figure, in human ovarian cancer cells, the target genes CXCL9, CXCL10, CXCL11, αCTLA4, and IL-21 all showed high expression under hypoxic conditions and low expression under normoxic conditions.

[0073] Figure 5 illustrates the cytotoxic effects of supernatants collected from the AdC68XY-R1-HRE-αCD47-αCD3-BiTE recombinant adenovirus of the present invention on human ovarian cancer cells (SKOV3) under normoxic and hypoxic conditions. As shown in the figure, this dual-tumor-targeting oncolytic adenovirus can achieve low-level cytotoxicity under normoxic conditions and highly effective cytotoxicity under hypoxic conditions.

[0074] Figure 6 illustrates the cytotoxic effects of supernatants collected from the AdC68XY-R1-HRE-αCD276-αCD3-BiTE recombinant adenovirus of the present invention on human ovarian cancer cells (SKOV3) under normoxic and hypoxic conditions. As shown in the figure, this dual-tumor-targeting oncolytic adenovirus can achieve low-level cytotoxicity under normoxic conditions and highly effective cytotoxicity under hypoxic conditions.

[0075] Figure 7 illustrates the cytotoxic effects of supernatants collected from the AdC68XY-R1-HRE-αCD73-αCD3-BiTE recombinant adenovirus of the present invention on human ovarian cancer cells (SKOV3) under normoxic and hypoxic conditions. As shown in the figure, this dual-tumor-targeting oncolytic adenovirus can achieve low-level cytotoxicity under normoxic conditions and highly effective cytotoxicity under hypoxic conditions.

[0076] Best Mode for Carrying Out the Invention

[0077] The following examples are only used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0078] The following description of the present application is only for the purpose of illustrating various embodiments of the present application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. A person skilled in the art can easily derive multiple equivalents, variations, and modifications without departing from the scope of the present application, and it should be understood that such equivalent embodiments are included within the scope of the present invention. All documents cited in this application, including publications, patents, and patent applications, are incorporated by reference in their entirety.

[0079] Definitions and Explanations of Terms

[0080] In order that the present invention may be more readily understood, selected terms are defined below.

[0081] The term "hypoxia inducible factor (HIF)" as used herein refers to a central regulatory factor that allows cells to sense and adapt to changes in oxygen levels. As a heterodimeric nuclear transcription factor, it is expressed in almost all biological cells. The currently discovered HIF family includes three members: HIF-1, HIF-2, and HIF-3. Each is composed of the oxygen-sensitive α subunit HIF-α (which has three members: HIF-1α, HIF-2α, and HIF-3α) and the oxygen-insensitive β subunit HIF-1β. Under oxygen-rich conditions, HIF-α binds to pVHL and is degraded. HIF-α is hydroxylated at conserved proline sites by prolyl hydroxylases (PHDs), a prerequisite for its recognition by pVHL. Under hypoxic conditions, PHD is inactivated and the hydroxylation of HIF-α is inhibited, preventing it from being recognized and degraded by pVHL. It then forms heterodimers with HIF-1β, which can bind to the hypoxia response element (HRE) on the DNA sequence and activate the expression of a series of downstream genes, thereby triggering the oxygen tolerance adaptive response of tissue cells.

[0082] The term "hypoxia regulatory element" used in this article has the same meaning as "hypoxia response element (HRE)", which is a regulatory sequence that mediates cellular hypoxia response. It is composed of a 5'-TACGTG-3' site that binds to activated HIF-1 and some variable flanking sequences, and is also called a hypoxia response element. In the process of hypoxia transcriptional activation, HRE is the smallest cis-regulatory element. Within the HRE region, there are 5'-ATCGTGGG-37 for transcriptional activation of vascular endothelial growth factor (VEGF) and 5'-TACGTGCT-3' for transcriptional activation of EPO. HRE is a hypoxia-sensitive inducible regulatory element with high activity in solid tumors. It plays an important role in the growth, invasion and metastasis of tumor cells.

[0083] The term "bispecific T cell engager (BiTE)" used in this article is a bispecific antibody, which is formed by the fusion of scFv segments of two types of antibodies, which can bind to CD3 molecules or other activating factors on the surface of T cells and tumor cell surface antigen molecules, respectively, thereby inducing T cells to kill tumor cells, and this process is not restricted by MHC. Specifically, BiTE consists of two single-chain variable fragments (scFv) connected in series by a flexible linker. One scFv recognizes the T cell surface protein CD3εH, while the other scFv recognizes specific tumor cell surface antigens. This structure of BiTE and its ability to specifically bind to proteins allow it to physically bridge T cells to tumor cells to form a T cell-BiTE-tumor cell complex, inducing immune synapse formation, stimulating T cell activation, and producing cytokines that kill tumors.

[0084] For example, the preparation method of this type of BiTE can be specifically referred to Example 1 of the Chinese patent application with application number 202111615701.2, wherein different scFvs are selected according to needs to specifically bind to tumor cell surface antigen molecules such as CD276, CD73 or CD47.

[0085] As used herein, the term "cytokine" is a generic term for proteins that are released by one cell population and act as intercellular mediators on another cell population. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines are growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factors, such as tumor necrosis factor-α (TNF-α) and tumor necrosis factor-β (TNF-β); Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-α (NGF-α); platelet-derived growth factor; placental growth factor; transforming growth factors (TGFs), such as TGF-α (TGF-α) and TGF-β ( erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α (IFN-α), interferon-β (IFN-β), and interferon-γ (IFN-γ); colony-stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-33; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term "cytokine" includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0086] The term "chemokines" as used herein refers to a class of small cytokines or signaling proteins secreted by cells. They are named chemokines because they have the ability to induce directional chemotaxis of nearby reactive cells. Chemokines are divided into four major subfamilies: CXC, CC, CX3C, and XC. All of these proteins exert their biological effects by interacting with G protein-linked transmembrane receptors (called chemokine receptors). These proteins act by binding to chemokine receptors, which are G protein-coupled transmembrane receptors that are selectively expressed on the surface of target cells. Chemotactic cytokines can be divided into four subfamilies according to the arrangement of cysteine ​​at their amino terminal (N-terminal): CXC, CC, XC and CX3C: CC chemokine subfamily: CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28; CXC chemokine subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17; XC chemokine subfamily: XCL1, XCL2; CX3C chemokine subfamily: CX3CL1.

[0087] The term "αCTLA4" used in this article is an antibody against the immunosuppressive signal CTLA-4 on T cells. CTLA-4 is homologous to the second signal molecule of the immune response, CD28, and competes with CD28 for binding to the B7 molecule (CD80 and CD86), so it can directly inhibit the proliferation and activation of T cells. CTLA-4 antibodies can inhibit the activation of T cells by blocking the binding of CTLA-4 to the B7 molecule, thereby activating the immune system to treat tumors. In addition, the expression level of CTLA-4 on the surface of tumor-infiltrating Treg cells is significantly higher than that of peripheral Treg cells and effector T cells in the periphery and tumors. Therefore, in the tumor microenvironment, Treg cells can compete to capture CTLA-4 antibodies, and the Fc part of the antibody will be recognized by the Fc receptor, activating NK cells, macrophages, etc., and clearing Treg cells through the ADCC effect to exert a therapeutic effect.

[0088] As used herein, the term "tumor antigen" includes, but is not limited to, targets listed in publicly available databases, including those available on the internet and incorporated herein by reference. These target databases include:

[0089] Therapeutic Target (http: / / xin.cz3.nus.edu.sg / group / cjttd / ttd.asp);

[0090] Cytokines and cytokine receptors (http: / / www.cytokinewebfacts.com / , http: / / www.copewithcytokines.de / cope.cgi, and http: / / cmbi.bjmu.edu.cn / cmbidata / cgf / CGF_Database / cytokine.medic.kumamoto-u.ac.jp / CFC / indexR.html);

[0091] Chemokine (http: / / cytokine.medic.kumamoto-u.ac.jp / CFC / CK / Chemokine.html);

[0092] Chemokine receptors and GPCRs (http: / / csp.medic.kumamoto-u.ac.jp / CSP / Receptor.html, http: / / www.gpcr.org / 7tm / );

[0093] Olfactory receptors (http: / / senselab.med.yale.edu / senselab / ORDB / default.asp);

[0094] Receptors (http: / / www.iuphar-db.org / iuphar-rd / list / index.htm); Cancer targets (http: / / cged.hgc.jp / cgi-bin / input.cgi);

[0095] Secreted proteins as potential antibody targets (http: / / spd.cbi.pku.edu.cn / );

[0096] Protein kinase (http: / / spd.cbi.pku.edu.cn / ), and

[0097] Human CD marker (http: / / content.labvelocity.com / tools / 6 / 1226 / CD_table_final_locked.pdf) and (Zola et al., "CD molecules 2005: human cell differentiation molecules," Blood, 106: 3123-3126 (2005)).

[0098] The term "polynucleotide" refers to a polymeric form of two or more nucleotides (ribonucleotides or deoxynucleotides or modified forms of either type of nucleotide). The term includes single- and double-stranded forms of DNA.

[0099] As used herein, the term "effective amount" refers to an amount of a therapy sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof; prevent the disorder from developing; cause regression of the disorder; prevent the recurrence, development, onset, or progression of one or more symptoms associated with the disorder; detect the disorder; or enhance or improve the prophylactic or therapeutic effect of another therapy (e.g., a prophylactic agent or a therapeutic agent).

[0100] "Patient" and "subject" can be used interchangeably herein to refer to animals, such as mammals, including primates (e.g., humans, monkeys, and chimpanzees), non-primates (e.g., cows, pigs, camels, alpacas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, whales), birds (e.g., ducks or geese), and sharks. Preferably, the patient or subject is a human, e.g., a human being treated or evaluated for a disease, disorder, or condition; a human at risk for a disease, disorder, or condition; a human suffering from a disease, disorder, or condition; and / or a human being being treated for a disease, disorder, or condition. Example

[0101] Example 1: Construction of oncolytic adenoviral vector with dual tumor targeting

[0102] 1.1 Exogenous gene amplification: 8 plasmid exogenous genes were cloned and constructed in the laboratory. After sequencing to confirm the correct sequence, PCR amplification was performed, and after gel excision and recovery, they were recombined and ligated with the adenovirus backbone plasmid. Among them, the exogenous gene HRE-CXCL9-CXCL10-CXCL11-αCTLA4-IL21 expresses five factors under the control of HRE and CMV mini promoter sequence (as shown in SEQ ID NO: 3); HRE-αCD276-αCD3-BiTE, HRE-αCD73-αCD3-BiTE and HRE-αCD47-αCD3-BiTE express αCD276-αCD3-BiTE, αCD73-αCD3-BiTE and αCD47-αCD3-BiTE respectively under the control of HRE and CMV mini promoter sequence; EF1α-αCD276-αCD3-BiTE and EF1α-αCD73-αCD3-BiTE are controls without HRE regulation and under the control of EF1α promoter (as shown in SEQ ID NO: 3). NO: 26) were used to express αCD276-αCD3-BiTE and αCD73-αCD3-BiTE, respectively; αCD276-αCD3-BiTE and αCD73-αCD3-BiTE were also controls without HRE regulation, and αCD276-αCD3-BiTE and αCD73-αCD3-BiTE were expressed under the CMV mini promoter, respectively.

[0103] 1.2 Recombinant Adenovirus Construction: The AdC68XY-R1 plasmid was digested with PI-SceI (NEB, Catalog No. R0696S) and inactivated. The gene fragment was ligated with the linearized vector using homologous recombination (Novagen, Catalog No. c112-02). The ligation product was transformed into Escherichia coli Stbl2 (Video Biotech, Catalog No. DL1045) and grown overnight on ampicillin-containing plates. Single colonies were randomly picked for sequencing. After verifying that all sequences were correct, pAdC68XY-R1-HRE-CXCL9-CXCL10-CXCL11-αCTLA4-IL21, pAdC68XY-R1-HRE-αCD276-αCD3-BiTE, pAdC68XY-R1-EF1α-αCD276-αCD3-BiTE, pAdC68XY-R1-αCD276-αCD3-BiTE, pAdC68XY-R1-HRE-αCD73-αCD3-BiTE, pAdC68XY-R1-EF1α-αCD73-αCD3-BiTE, pAdC68XY-R1-αCD73-αCD3-BiTE, and pAdC68XY-R1-HRE-αCD47-αCD3-BiTE were successfully cloned. There are 8 plasmids. The plasmid construction map is shown in Figure 1.

[0104] Example 2: Identification of recombinant adenovirus molecular clones

[0105] The cloned recombinant adenovirus plasmids were identified by PCR and sequencing using specific primers. They were also digested with multiple enzymes (MfeI, BglII, XhoI) and then electrophoresed on a 1% agarose gel to verify that the molecular clones were complete and correct, as evidenced by DNA bands. The enzyme digestion maps of the eight plasmids are shown in Figure 2.

[0106] Example 3: Packaging and amplification of oncolytic adenovirus with dual tumor targeting

[0107] 1.1 Viral Packaging: The recombinant adenoviral molecular clone (plasmid DNA) obtained above was linearized using PacI. Eight recombinant plasmid DNAs were transfected into 293A cells at 60-70% confluency in a 6-well plate according to the instructions of the Lipofectamine 3000 transfection kit. DNA / lipofectamine complexes were added for transfection. Six hours after transfection, the culture medium was changed to DMEM supplemented with 5% FBS and 1% double-antibody. Cytopathic effects were observed daily under a microscope until numerous plaques appeared on the 293A cells after 5-7 days. Viruses were harvested at passage 0 and stored at -80°C. Figure 3 shows that cytopathic effects occurred around day 5-7 after infection.

[0108] 2.2 Virus amplification: The P0 virus was harvested and frozen and thawed three times. Part of the frozen and thawed virus supernatant was then used to infect the virus production cells HEK293 cells, and the virus TCID was measured in the HEK293 cells. 50 The tissue infectious dose (TID) was determined and calculated using the Reed-Muench method.

[0109] Example 4: Detection of protein expression of oncolytic adenovirus with dual tumor targeting under normoxic and hypoxic conditions

[0110] 1.1 SKOV3 cells were plated in two 12-well plates with a cell number of approximately 3 × 10 5 After culturing for about 24 hours, when the cells adhered to the wall and covered about 80%-90% of the entire bottom surface, proceed to the next step. Infect the cells with AdC68XY-R1-HRE-CXCL9-CXCL10-CXCL11-αCTLA4-IL21 virus at an MOI of 10 and 100. Perform the same operation on another plate. After 2 hours of infection, place the two 12-well plates in normoxia (21% O2) and hypoxia (1% O2) incubators, collect the 48-hour cell supernatant, centrifuge at 12000g for 5 minutes, transfer the supernatant into new EP tubes, and place in a -80℃ refrigerator for testing.

[0111] 1.2 ELISA kits for CXCL9 (ABclone, Catalog No. RK04210), CXCL10 (ABclone, Catalog No. RK00054), CXCL11 (RayBiotech, Catalog No. ELH-ITAC), and IL21 (BioLegend, Catalog No. 433804) were used to detect the supernatants collected under normoxia and hypoxia. Specific detection methods were referred to the kit instructions.

[0112] 1.3 αCTLA4 ELISA detection steps are as follows: recombinant human CTLA4 protein (CP33, novoprotein) was diluted with coating solution to a concentration of 0.5 μg / ml, coated in a 96-well ELISA plate, incubated at 4°C overnight, blocked with 5% skim milk at room temperature for 1 hour, rinsed four times with PBST, added with 100 μL of the supernatant to be tested and the positive and negative controls, incubated at room temperature for 3 hours, rinsed four times with PBST, added with goat anti-human HRP enzyme-labeled secondary antibody (1:5000) and incubated at room temperature for 1 hour, rinsed four times with PBST, added with TMB substrate solution, protected from light for 15 minutes, and finally added with 50 μL of 2M H2SO4 stop solution and color development solution, added with stop solution, and read the OD490.

[0113] The results showed (see Figure 4) that the expression of CXCL9, CXCL10, CXCL11, IL21, and CTLA4 was higher under hypoxia than under normoxia. For CXCL9 expression, when MOI = 10, the hypoxia OD value was 10 times that of normoxia. When MOI = 100, the maximum value was not detected due to overexposure to hypoxia, making comparison impossible. For CXCL10 expression, when MOI = 10, the hypoxia OD value was 7.2 times that of normoxia. When MOI = 100, the maximum value was not detected due to overexposure to hypoxia, making comparison impossible. For CXCL11 expression, when MOI = 100, the hypoxia OD value was 88.8 times that of normoxia. For αCTLA4 expression, when MOI = 100, the hypoxia OD value was 2.1 times that of normoxia. For IL-21 expression, when MOI = 100, the hypoxia OD value was 10.7 times that of normoxia.

[0114] Example 5: Effects of Dual Tumor-Targeting Adenovirus on Ovarian Cancer Cells under Normoxic and Hypoxic Conditions

[0115] 1.1 Preparation of viral supernatants expressing different BITEs: SKOV3 (stored in Shanghai Xinwan Biotechnology Co., Ltd.) is a Luciferase-expressing cell line. Cells were cultured at 1×10 5Cells were plated with 1000 cells / well in two 24-well plates and cultured for about 24 hours. When the cells adhered to the wall and covered about 80%-90% of the entire bottom surface, 7 adenoviruses expressing different BITEs were infected with the cells at a virus amount of MOI of 10 and 100. After 2 hours, the two 24-well plates were placed in normoxia (21% O2) and hypoxia (1% O2) incubators, respectively. The 48-hour cell supernatant was collected and centrifuged at 12000g for 5 minutes. The supernatant was transferred and aliquoted into new EP tubes and placed in a -80℃ refrigerator for detection.

[0116] 1.2 Target cell plating: SKOV3 cells were plated at 1×10 4 The cells were plated at a density of 1000 cells / well in a 96-well plate and cultured at 37°C overnight until they adhered to the wall.

[0117] 1.3 Killing experiment: After 24 hours, supernatants of different BITEs with 21% O2 and 1% O2 were added, and then activated T cells were added with an effector-target ratio of 4:1. Controls with supernatant and T cells alone and without T cells were set up, and the cells were cultured at 37°C overnight for killing.

[0118] 1.4 Killing Assay: Killing assay was performed around 18 hours after the cells were plated. The supernatant was removed, and 50 μL of cell lysis buffer (Promega, Cat. No. E1531) was added to each well. The cells were incubated at room temperature with shaking for 30 minutes. 30 μL of luciferase substrate (Promega, Cat. No. E151A) was added to each well. The cells were assayed using a GloMax Navigator Microplate Luminometer (Promega, Steady-Glo protocol).

[0119] The calculation formula of cell killing rate is as follows: Cell killing rate (%) = (luciferase activity value of control virus supernatant + target cells + T cell group) - (luciferase activity value of experimental group supernatant + target cells + T cell group) / (luciferase activity value of control virus supernatant + target cells + T cell group) × 100

[0120] The results, as shown in Figure 5, show that the AdC68XY-R1-HRE-αCD47-αCD3-BiTE recombinant adenovirus, regulated by the hypoxia-responsive promoter HRE, not only exhibits strong cytotoxicity but also exhibits hypoxia responsiveness, with low expression in normoxia and high expression in hypoxia. Under normoxia, no cytotoxicity was detected at an MOI of 10, and the cell killing rate was less than 2% at an MOI of 100. However, under hypoxia, the killing rate reached 30% at an MOI of 10 and 56% at an MOI of 100, demonstrating significant differences.

[0121] The αCD276-αCD3-BiTE supernatant, expressed by the recombinant adenovirus AdC68XY-R1-HRE-αCD276-αCD3-BiTE, which is regulated by the hypoxia-responsive promoter HRE, not only exhibited strong killing ability but also displayed hypoxia-responsiveness, with low expression in normoxia and high expression in hypoxia. Under normoxia, no killing was detected at MOIs of 10 and 100. However, under hypoxia, the killing rate was 11% at an MOI of 10 and reached 93% at an MOI of 100, a significant difference. Control viruses without the HRE sequence, expressing either the CMV mini promoter or the EF1α promoter, showed no hypoxia-responsive killing in the supernatant of the αCD276-αCD3-BiTE harvested under both normoxia and hypoxia. Under normoxia, the killing rates were 7% and 17% at an MOI of 10, and 62% and 92% at an MOI of 100. Under hypoxia, the killing rates were 1% and 10% at an MOI of 10, and 63% and 75% at an MOI of 100. This indicates that the control virus without HRE sequence regulation had similar killing abilities in hypoxia and normoxia, or the killing ability in hypoxia was lower than that in normoxia, indicating no hypoxia responsiveness (Figure 6).

[0122] The αCD73-αCD3-BiTE supernatant, expressed by the AdC68XY-R1-HRE-αCD73-αCD3-BiTE recombinant adenovirus under the control of the hypoxia-responsive promoter HRE, not only exhibited strong killing ability but also displayed hypoxia responsiveness, with low expression under normoxia and high expression under hypoxia, with significant differences. Under normoxia, the killing rate was 5% at an MOI of 10, and 27% at an MOI of 100. In hypoxia, however, the killing rate was 38% at an MOI of 10 and reached 95% at an MOI of 100, demonstrating a significant difference. In contrast, control viruses without HRE sequence regulation, whether driven by the CMV mini promoter or the EF1α promoter, showed no hypoxia-responsive killing in the supernatant of αCD73-αCD3-BiTE harvested under normoxic and hypoxic conditions. Under normoxia, the killing rates were 5% and 14% at an MOI of 10, and 22% and 79% at an MOI of 100, respectively. Under hypoxia, the killing rates were 2% and 15% at an MOI of 10, and 24% and 61% at an MOI of 100, respectively. This indicates that the control viruses without HRE sequence regulation had similar killing abilities under hypoxia and normoxia, or the killing under hypoxia was lower than that under normoxia, indicating no hypoxia-responsiveness (Figure 7).

[0123] The present invention applies the hypoxia control system to chimpanzee adenovirus AdC68-XY-R1, which exhibits good tumor tropism and hypoxic environment responsiveness to inserted cytokines, chemokines or killer factors.

[0124] The above embodiments are exemplary and should not be construed as limiting the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A recombinant oncolytic adenovirus with dual tumor targeting, characterized in that: The recombinant oncolytic adenovirus is derived from the replicative chimpanzee adenovirus vector AdC68XY-R1, and the promoter therein is constructed as a tumor-specific and hypoxia-responsive promoter; further, the recombinant oncolytic adenovirus is also constructed to contain an insertion element, and the insertion element recruits endogenous effector cells through expression.

2. The recombinant oncolytic adenovirus according to claim 1, wherein The insertion element is selected from one or more of the following: (1) an insertion element that simultaneously expresses one or more of GM-CSF, IFN-α / β / γ, IL-2, IL-3, IL-7, IL-12, IL-15, IL-21, IL-33, IL-35, IL-37, CCL4, CCL20, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL3, CCL4, and αCTLA4; and / or (2) an insertion element that expresses one or more of TNF-α, a T cell engager, a chimeric antigen receptor, an apoptosis gene or a pyroptosis gene, or a toxin; Optionally, the insertion element is further coupled to an oxygen sensitive element.

3. The recombinant oncolytic adenovirus according to claim 1 or 2, wherein The insertion element is an insertion element that simultaneously expresses two, three, four or five types of CXCL9, CXCL10, CXCL11, αCTLA4 and IL-21; Preferably, the insertion element simultaneously expresses CXCL9, CXCL10, CXCL11, αCTLA4 and IL-21; Preferably, the insertion element comprises exogenous genes encoding and expressing CXCL9, CXCL10, CXCL11, αCTLA4 and IL-21; more preferably, the exogenous genes are connected by T2A cleavage peptide, P2A or IRES; Preferably, the sequence of the front and back connection of the exogenous gene nucleic acid sequence is interchangeable; Preferably, the expression products of the inserted element are CXCL9, CXCL10, CXCL11, αCTLA4 and IL-21; Preferably, any one of the expression products of the inserted elements has at least 70% homology to the amino acid sequence shown in any one of SEQ ID NOs: 5, 7, 9, 11 or 13.

4. The recombinant oncolytic adenovirus according to claim 1 or 2, wherein The insertion element is an insertion element expressing a T cell adaptor; Preferably, the tumor antigen that binds to the T cell engager is selected from one or more of CD19, BCMA, CD20, CD22, CD30, CD33, CD38, CD47, CD70, CD73, CD117, CD123, CD133, CD138, CD147, CD171, CD276, NKG2DL, HER2, MUC1, MUC16, CEA, EpCAM, IL-13Rα2, EGFR, EGFRvIII, GD2, DR5, EphA2, FRα, PSCA, PSMA, TARP, cMet, VEGFR2, BCMA, CTLA-4, PD-L1, AFP, GPC3, AXL, ROR1, ROR2, FAP, Mesothelin, DLL3 and CLD18; Preferably, the T cell engager is a bispecific T cell engager; More preferably, the bispecific T cell engager comprises αCD3, and any one of αCD276, αCD73, and αCD47; More preferably, the bispecific T cell engager further comprises an ODD18 oxygen-sensing element; Optionally, the expression product of the inserted element is a fusion protein having at least 70% homology to the amino acid sequence shown in SEQ ID NO: 15, 18, 20.

5. The recombinant oncolytic adenovirus according to any one of claims 1 to 4, characterized in that The tumor-specific promoter is selected from human telomerase reverse transcriptase promoter, carcinoembryonic antigen promoter, alpha-fetoprotein promoter, human prostate-specific antigen promoter, cyclooxygenase-2 promoter, apoptosis inhibitory protein promoter or human intestinal tissue-specific antigen promoter.

6. The recombinant oncolytic adenovirus according to any one of claims 1 to 5, characterized in that The hypoxia-responsive promoter is a promoter of a transcriptional control system that responds to hypoxia triggering and comprises a hypoxia regulatory element.

7. A polynucleotide for constructing the recombinant oncolytic adenovirus according to any one of claims 1 to 6; Preferably, the polynucleotide comprises one or more of the promoter, insertion element, hypoxia regulatory element, transcription start and / or termination signal mentioned in the recombinant oncolytic adenovirus according to any one of claims 1 to 6; Preferably, the polynucleotide comprises a polynucleotide sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 6, 8, 10, 12, 14, 17, 19, 21, 22, 23, and 24.

8. A recombinant oncolytic adenoviral vector, wherein the vector is prepared using the following method: Using the genome of chimpanzee adenovirus vector AdC68XY-R1 as the basic framework, the insert is cloned into the replication-competent chimpanzee adenovirus vector AdC68XY-R1; and / or The promoter of chimpanzee adenovirus AdC68XY-R1 is replaced with a tumor-specific and hypoxia-responsive promoter; and / or an expression framework of a hypoxia regulatory element is inserted downstream of the E1A gene of the chimpanzee adenovirus AdC68XY-R1.

9. A method for preparing a recombinant oncolytic adenovirus according to any one of claims 1 to 6, which uses the polynucleotide shown in claim 7 or is obtained by packaging the vector according to claim 8.

10. A pharmaceutical composition comprising the recombinant oncolytic adenovirus according to any one of claims 1 to 6.

11. Use of the recombinant oncolytic adenovirus according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating tumors; Preferably, the tumor is selected from one or more of B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high-grade glioma, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, large intestine cancer, hematopoietic system cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or blood oncogenic diseases.

12. The use according to claim 11, wherein the recombinant oncolytic adenovirus is used alone or in combination with CAR-T.

13. A kit for preparing the recombinant oncolytic adenovirus according to any one of claims 1 to 6, characterized in that: The kit comprises: The recombinant oncolytic adenovirus vector according to claim 8; Virus-producing cells.

Images