Immune-evading antitumor adenovirus

Abstract

The present invention relates to an anti-tumor adenovirus capable of evading the in vivo immune system. According to the present invention, an adenovirus comprising a nucleic acid encoding the transferrin-binding domain of the present invention has a significantly increased tumor cell infection and killing effect, and due to increased binding to transferrin, it evades the body's immune response and has an increased plasma half-life. It is specifically delivered to cancer cells, has a systemic therapeutic effect, is capable of local delivery, has excellent selectivity, and shows remarkable anti-tumor efficacy, and therefore can be usefully used as an anti-cancer composition or anti-cancer adjuvant for various cancers.

Description

[Technical field]

[0001] The present invention relates to an antitumor adenovirus capable of evading the immune system in the body. [Background technology]

[0002] Cancer is one of the diseases that causes the most deaths worldwide, and the development of innovative cancer treatments can reduce medical costs incurred during treatment while creating high added value. According to statistics from 2008, molecular treatments that can overcome resistance to conventional anticancer drugs accounted for $17.5 billion in the seven major countries (US, Japan, France, Germany, Italy, Spain, UK), and are expected to account for a market size of about $45 billion in 2018, showing a growth rate of 9.5% compared to 2008. Cancer treatments are classified into surgery, radiation therapy, chemotherapy, and biological therapy. Among these, chemotherapy is a treatment that uses chemicals to suppress or kill the proliferation of cancer cells. The toxicity of anticancer drugs is also seen in normal cells to a certain extent, and even if an antitumor drug is effective, resistance occurs after a certain period of use, which reduces its effectiveness. Therefore, there is an urgent need to develop anticancer drugs that act selectively on cancer cells and do not cause resistance. In recent years, development of new anticancer drugs that target the molecular characteristics of cancer through the acquisition of molecular genetic information for cancer has been progressing, and it has been reported that drug resistance occurs even in anticancer drugs that target molecular targets that only cancer cells have. Therefore, there is a need to develop anticancer drugs with a new concept.

[0003] On the other hand, there are experiments with viruses for cancer treatment based on unproven reports related to temporary cancer remission after infection with natural viruses or vaccination with viruses. The first report was in 1912, which was attributed to a reduction in cervical cancer in patients vaccinated for rabies, and similar results were seen in cancer patients following vaccination for smallpox or infection with natural viruses (e.g., mumps, measles). Based on these reports and animal data, it was in the late 1940s and early 1950s that live viruses were inoculated into patients for cancer treatment. However, problems arose in which tumors would regrow and patients would die, often after a temporary tumor reduction. These inoculations also did not result in long-term sustained remission. In 1957, Albert B. Sabin, MD, who developed the oral live polio vaccine, noted that even when oncolytic viruses kill tumors, the biggest problem is that the individual's immune response to the virus is too rapid, causing the effect to disappear quickly.

[0004] At present, many oncolytic adenoviruses have been identified, but the only virus approved for clinical use anywhere in the world to date is the Oncorine (H101) subgroup C adenovirus modified with an E1B-55KD deletion that allows conditional replication in p53-deficient cancer cells (H101 is a close analog of ONYX015 described by Bischoff et al. in 1996). Oncorine is administered by intratumoral injection for head and neck cancer. Adenoviruses have been widely used as oncolytic agents for cancer therapy as well as gene delivery vectors for gene therapy. Adenoviruses exhibit several characteristics that make them suitable for such applications: the structure and biological properties of adenoviruses have been extensively studied, their genomes can be easily modified, these viruses can infect all replicating and non-replicating cells, and they can be easily produced at high titers for clinical use. In terms of safety, adenoviruses do not cause life-threatening diseases in humans, and their viral genome is non-integrative, which prevents insertional mutagenesis. Clinical trials with adenovirus-based vectors have reported that the virus has a favorable toxicity and safety profile when administered systemically, although there is still a need for improved efficacy.

[0005] Thus, in the field of gene therapy, systemic administration, i.e. injection into the bloodstream intravenously or intraarterially, may be required to reach multiple organs and disseminated cells. For example, in cancer therapy with adenoviral vectors and oncolytic adenoviruses, systemic administration is mandatory to treat disseminated tumors in advanced or metastatic states. However, adenoviruses have significant limitations that reduce therapeutic efficacy when injected into the bloodstream. Adenovirus type 5 (Ad5) undergoes multiple neutralizing interactions that sharply reduce the bioavailability of the virus in the blood. The biggest problem with this therapy is hepatic sequestration, since >90% of the injected dose is retained in the liver, mainly in liver macrophages called Cooper cells, but also in liver sinusoidal endothelial cells (LSECs) and hepatocytes. Direct interactions with blood cells and proteins are major obstacles as well. Ad5 can directly bind to blood cells such as red blood cells via the CAR receptor and to platelets via integrins. Antibodies can not only directly neutralize the virus, but also trigger innate immune responses by complement activation and docking of viral particles to Fc receptors on monocytes and neutrophils. Virus re-administration also increases the concentration of anti-Ad neutralizing antibodies (NAb), thus enhancing virus neutralization. Opsonization of adenovirus by antibodies and complement can also enhance clearance by Cooper cells. Overall, these interactions result in a significant shortening of the half-life of blood Ad in mice and humans to about several minutes. Although considerable efforts have been made to circumvent neutralization by antibodies and immune cells when adenovirus is administered systemically, it has been reported that systemic spread is still limited, temporary, and largely ineffective (Ferguson et al. 2012).

[0006] Therefore, there remains a need for research into adenoviruses as cancer therapeutic agents that are suitable for systemic administration and can circumvent neutralizing antibodies. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an antitumor adenovirus.

[0008] It is also an object of the present invention to provide a pharmaceutical composition for treating cancer.

[0009] Another object of the present invention is to provide a use of the adenovirus for preventing or treating tumors.

[0010] Another object of the present invention is to provide a method for treating cancer, which comprises administering the adenovirus to an individual suffering from cancer. [Means for solving the problem]

[0011] To achieve the above object, the present invention provides an adenovirus comprising a nucleic acid encoding a transferrin-binding moiety.

[0012] The present invention also provides a pharmaceutical composition for treating cancer, comprising the antitumor adenovirus.

[0013] The present invention also provides a use of the adenovirus for preventing or treating tumors.

[0014] In addition, the present invention provides a method for treating cancer, which comprises administering the adenovirus to an individual suffering from cancer. Effect of the Invention

[0015] According to the present invention, an adenovirus containing a nucleic acid encoding the transferrin-binding domain of the present invention has significantly increased tumor cell infection and killing effects, avoids immune responses in the body due to increased binding to transferrin, has an increased plasma half-life, is specifically delivered to cancer cells, has a systemic therapeutic effect, is capable of local delivery, has excellent selectivity, and shows remarkable anti-tumor efficacy, and therefore can be usefully used as an anti-cancer composition or anti-cancer adjuvant for various cancers. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows the antibody evasion mechanism of an anti-tumor adenovirus containing the transferrin-binding domain of the present invention. [Diagram 2] FIG. 1 shows a process for producing an antitumor adenovirus containing the transferrin-binding domain (portion) of the present invention. [Diagram 3] FIG. 1 shows a vector map of the adenovirus envelope-related plasmid pAd1128, in which the transferrin-binding domain of the present invention is contained at the HVR1 position of hexon. [Figure 4] FIG. 1 shows a map of an adenovirus vector containing an albumin binding domain (ABD) (CA10G-A). [Diagram 5] FIG. 1 shows a vector map of an adenovirus of the present invention, which contains the transferrin-binding moiety Tbp29aa in the hexon of the adenovirus vector (HVR1-Tbp29aa). [Figure 6] FIG. 1 shows a vector map of an adenovirus containing a fusion protein in which the hexon of the adenovirus vector of the present invention contains linkers at the N-terminus and C-terminus of the transferrin binding moiety Tbp55aa (HVR1-Tbp(G4S1)35aa). [Figure 7] and [Figure 8]1 shows the immune evasion ability of adenoviruses containing a transferrin-binding domain: CA10G: control adenovirus; CA10G-A (CA10G-a): adenovirus containing an albumin-binding domain; HVR1-Tbp29aa (Tbp-29aa): adenovirus containing the transferrin-binding moiety Tbp29aa in the hexon of the adenoviral vector; and HVR1-Tbp(G4S1)3 55aa (Tbp-(G4S1)3-55aa): adenovirus containing the transferrin-binding moiety Tbp55aa and a linker in the hexon of the adenoviral vector. [Figure 9] Figure showing immune evasion ability depending on the insertion position of the transferrin-binding moiety into the adenovirus: CA10G: control adenovirus; CA10GT: HVR1-(G4S1)3-Tbp55aa adenovirus in which the transferrin-binding moiety has been inserted into the HVR1 position of hexon; HVR229aa: HVR2-Tbp29aa adenovirus in which the transferrin-binding moiety has been inserted into the HVR2 position of hexon; and HVR2L355aa: HVR2-(G4S1)3-Tbp55aa adenovirus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention will be described in detail below with reference to the following examples. However, the following examples are presented as examples of the present invention and are not intended to limit the present invention, and the present invention can be modified and applied in various ways within the scope of the claims and the equivalent scope interpreted therefrom.

[0018] Unless otherwise specified, nucleic acids are written from left to right in 5' to 3' orientation. Numeric ranges recited herein are inclusive of the numbers defining the range, and include each integer or any non-integer fraction within the defined range.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are described herein.

[0020] In one aspect, the invention relates to an adenovirus that contains a nucleic acid encoding a transferrin binding moiety in the coding region of the hypervariable region (HVR) of the hexon protein.

[0021] In one embodiment, the adenovirus may comprise the nucleic acid sequence of SEQ ID NO: 1 or 3.

[0022] In one embodiment, the adenovirus may include a nucleic acid sequence of a hexon including a transferrin binding portion, the nucleic acid sequence of the hexon including the transferrin binding portion may include a base sequence of SEQ ID NO: 8 or a base sequence of SEQ ID NO: 10, and the hexon including the transferrin binding portion may include an amino acid sequence of SEQ ID NO: 9 or 11.

[0023] In one embodiment, the hypervariable region of the adenovirus hexon protein may be HVR1, which may include the amino acid sequence of SEQ ID NO: 7, and the nucleic acid sequence encoding the HVR1 may include the base sequence of SEQ ID NO: 6.

[0024] In one embodiment, the nucleic acid encoding the transferrin binding moiety may be included between codons expressing the 154th and 155th amino acids of the base sequence (SEQ ID NO: 6) encoding adenovirus hexon.

[0025] In one embodiment, the transferrin binding moiety may include the amino acid sequence of SEQ ID NO: 2 or 4, and the nucleic acid sequence encoding the transferrin binding moiety may include the base sequence of SEQ ID NO: 1 or 3.

[0026] In one embodiment, the N-terminus, C-terminus, or N-terminus and C-terminus of the transferrin binding moiety can be linked to the hexon protein via a linker, and the linker can include the amino acid sequence of SEQ ID NO:5.

[0027] In one embodiment, the transferrin-binding moiety can be attached directly to the hexon protein, i.e., the N-terminus and C-terminus of the transferrin-binding moiety are directly linked to the hexon protein. However, the transferrin-binding moiety can also be linked to the hexon protein via a linker sequence. Thus, in other embodiments, the N-terminus and / or C-terminus of the transferrin-binding moiety are linked to the hexon protein by a linker sequence.

[0028] In one embodiment, when the adenoviral hexon protein is assembled into a capsid, the transferrin binding moiety can be located on the outer surface of the hexon protein.

[0029] The adenovirus of the present invention can be coated with a transferrin-binding domain by including a transferrin-binding portion on the outer surface of the hexon protein, thereby protecting the adenovirus from neutralizing antibodies present in the bloodstream.

[0030] In one embodiment, the adenovirus may be a human adenovirus selected from the group consisting of human adenovirus serotypes 1 to 57, may be human adenovirus serotype 5 (GenBank: AY339865.1), or may be a human adenovirus serotype 5 / 3 chimeric adenovirus.

[0031] In one embodiment, the adenovirus may further comprise a tissue-specific promoter or a tumor-specific promoter, which may be operably linked to an endogenous gene of the adenovirus.

[0032] In one embodiment, the promoter may be selected from the group consisting of E2F promoter, telomerase hTERT promoter, tyrosinase promoter, prostate-specific antigen promoter, alpha-fetoprotein promoter, and COX-2 promoter.

[0033] In one embodiment, the telomerase hTERT promoter can include the nucleotide sequence of SEQ ID NO: 17 and can be operably linked to the E1A and E1B endogenous genes of the adenovirus.

[0034] In one embodiment, the endogenous gene of the adenovirus has a structure of 5'-ITR-C1-C2-C3-C4-C5 3'ITR; C1 includes E1A, E1B or E1A-E1B; C2 includes E2B-L1-L2-L3-E2A-L4; C3 does not include or includes E3; C4 includes L5; and C5 does not include or includes E4.

[0035] In one embodiment, the adenovirus endogenous gene may further include an IRES sequence between E1A and E1B.

[0036] In one embodiment, the E1A may comprise the base sequence of SEQ ID NO:18.

[0037] In one embodiment, the E1B may comprise the nucleotide sequence of SEQ ID NO:19.

[0038] In one embodiment, the IRES may comprise the base sequence of SEQ ID NO:20.

[0039] In one embodiment, the promoter can be operably linked to the endogenous adenovirus genes E1A and E1B.

[0040] In one embodiment, the adenovirus may comprise hTERT promoter-E1A-IRES-EIB comprising the nucleotide sequence of SEQ ID NO:15.

[0041] In one embodiment, the adenovirus may further comprise an expression cassette for expressing a foreign gene, and the expression cassette may be contained in the E3 site of an endogenous gene of the adenovirus.

[0042] In one embodiment, the adenovirus of the present invention may further comprise a CMV promoter and a foreign gene, and the CMV promoter and the foreign gene operably linked thereto may be contained in the E3 site of an endogenous gene of the adenovirus.

[0043] In one embodiment, the adenovirus may be an anti-tumor adenovirus, which is an oncolytic adenovirus.

[0044] In one embodiment, the adenovirus of the present invention may have a higher tumor killing ability than a wild-type adenovirus and may be an oncolytic adenovirus.

[0045] In one embodiment, the adenovirus of the present invention is a replication-competent adenovirus, an anti-tumor or an oncolytic adenovirus.

[0046] In another embodiment, the adenovirus is a replication-incompetent or replication-defective adenovirus. Replication-defective or replication-defective adenoviruses are adenoviruses that cannot replicate in target cells and are used in gene therapy as gene carriers to target cells in order to express therapeutic genes in the cells and not to degrade the cells.

[0047] In one embodiment, the adenovirus can further include capsid modifications to enhance the infectivity of the adenovirus or to target it with receptors present on tumor cells.

[0048] In one embodiment, the capsid modification may involve the insertion of an RGD motif within the H1 loop of the adenoviral fiber protein.

[0049] In one embodiment, the capsid modification may involve replacing the fiber gene or a portion thereof with a homologous portion derived from an adenovirus of another serotype to form a chimeric adenovirus, which may include a capsid having a fiber gene or a portion thereof replaced from a serotype 3 adenovirus, and the portion excluding the fiber gene may include a gene from a serotype 5 adenovirus.

[0050] In one embodiment, the adenovirus may contain one or more non-adenoviral genes, which may be genes used in cancer gene therapy, and the genes used in cancer gene therapy may be selected from the group consisting of tumor suppressor genes, genes encoding anti-tumor interfering RNA, and immune stimulatory genes.

[0051] The adenovirus of the present invention can selectively distribute to desired tissues in vivo, and can avoid or significantly reduce expression in non-target or non-tumor tissues.

[0052] In one embodiment, the antitumor adenovirus of the present invention can be used in cancer gene therapy.

[0053] In the present invention, the term "transferrin" refers to a glycoprotein that binds very strongly and reversibly to iron. Although only about 0.1% of the iron present in the body is bound to transferrin, it plays a very important role in regulating the amount of iron in the body. Transferrin exists in the blood and plays a role in absorbing iron. In the present invention, blood transferrin binds to the transferrin-binding portion of the present invention, allowing the adenovirus to evade antibodies in the body, thereby playing a role in increasing the half-life in the blood when the adenovirus is administered systemically.

[0054] In the present invention, the term "transferrin-binding moiety" refers to any amino acid sequence capable of binding to transferrin, i.e., having binding affinity. Preferably, it is capable of binding to serum transferrin, more preferably to human serum transferrin. The term "transferrin-binding moiety" includes transferrin-binding sequences derived from natural transferrin-binding domains (e.g., transferrin present in bacterial proteins) and synthetic peptides. In a preferred embodiment, the transferrin-binding moiety is selected from the transferrin-binding domain from Neisseria meningitidis, functionally equivalent variants thereof. The term "transferrin-binding domain" refers to any region derived from a natural protein that can bind to transferrin with sufficient specificity to ensure protection from neutralizing antibodies, but can be used in the present invention as a similar term to transferrin-binding moiety. In the present invention, a transferrin binding / binding moiety, a transferrin binding domain, and a transferrin binding peptide / protein are used interchangeably, and the transferrin binding / binding moiety includes the entire transferrin binding domain and the transferrin binding peptide / protein, and more preferably, the transferrin binding / binding moiety can refer to a synthetic peptide that includes a partial amino acid sequence of the transferrin binding domain of the transferrin binding / binding peptide / protein.

[0055] The present invention also includes functionally equivalent variants of the transferrin-binding moiety described above. As used herein, the term "functionally equivalent variant" refers to any polypeptide derived from the transferrin-binding moiety by the insertion, deletion, or substitution of one or more residues, which substantially maintains the ability to interact with transferrin as determined above. In preferred embodiments, a polypeptide is considered a functionally equivalent variant of a transferrin-binding moiety if it exhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the transferrin-binding ability of the transferrin-binding domain of SEQ ID NO: 2 or 4. Preferably, a polypeptide is considered to be a functionally equivalent variant of the transferrin-binding portion if it can neutralize the antibody with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% efficiency with the transferrin-binding domain of SEQ ID NO: 2 or 4. The identity level between two polypeptides is determined using computer algorithms and methods well known to those of ordinary skill in the art. The identity between two amino acid sequences is preferably determined by the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)], although other similar algorithms can also be used. BLAST and BLAST 2.0 are used to determine the percent sequence identity, applying the parameters described herein. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0056] In the present invention, the types of transferrin binding moieties include, but are not limited to, various peptides, antibodies, and the like in addition to the moieties described in the examples of the present invention. The transferrin-binding moiety may be any of the transferrin binding peptides (Tbp1, Tbp2, TbpA, TbpB, etc.) derived from various bacteria, iron-regulated proteins, iron-regulated outer membrane proteins, transferrin receptor proteins, Moraxella The present invention is applicable to any moiety that can bind to transferrin in the blood and avoid an immune response in the body, including the outer membrane protein B1 of N. catarrhalis, a chimeric fusion protein containing a transferrin-binding peptide, a recombinant transferrin-binding peptide, a fragment of said peptide or protein or an analog thereof, a transferrin-binding molecule, or an anti-transferrin antibody or a fragment thereof having immunological activity, and that is conjugated to an adenovirus.

[0057] The antibody includes not only the form of a whole antibody but also a functional fragment of an antibody molecule. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule means a fragment that retains an antigen-binding function. Examples of antibody fragments include: (i) a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and a first heavy chain constant region (CH1); (ii) an Fd fragment consisting of a VH and CH1 domain; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)]; (v) an isolated CDR region; (vi) an F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments; and (vii) a single chain Fv fragment in which the VH and VL domains are linked by a peptide linker that connects them to form an antigen-binding site. (viii) bispecific single chain Fv dimers (PCT / US92 / 09965) and (ix) diabodies, which are multivalent or multispecific fragments produced by gene fusion (WO94 / 13804).

[0058] In the present invention, the term "linker sequence" refers to an amino acid sequence that acts as a hinge between the hexon protein and the transferrin binding moiety, providing space between the two factors, and allowing the secondary structure of the hexon protein to be unaffected by the presence of the transferrin binding moiety, and vice versa. The linker sequence can have any length that allows the two factors to move independently of each other while maintaining the tertiary structure of the individual factors. In a preferred embodiment, the linker sequence is a flexible linker peptide less than 31 amino acids in length. More preferably, the linker sequence contains less than 10, less than 5, less than 4, or less than 2 amino acids. In one embodiment, the linker sequence contains two or more amino acids selected from the group consisting of glycine, serine, alanine, and threonine. In another embodiment, the linker is a polyglycine linker. In the case of the linker sequence, it can be represented as SEQ ID NO: 5 in the present invention. Other linkers can be used as alternatives (Reddy Chichili, VP., Kumar, V., and Sivaraman, J. (2013). Linkers in the structural biology of protein-protein interactions. Protein Science 22(2):153-67).

[0059] In the present invention, the term "operably linked" refers to a functional connection between a gene expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another genetic sequence, such that the regulatory sequence regulates the transcription and / or translation of the other genetic sequence.

[0060] In the present invention, the term "promoter" refers to an untranslated nucleic acid sequence upstream of a coding region, which contains a binding site for RNA polymerase and has transcription initiation activity to mRNA of a gene downstream of the promoter. In the expression cassette of the present invention, the promoter can be any promoter that can initiate the expression of shRNA. Specifically, the promoter of the present invention may be a constitutive promoter that constantly induces the expression of a gene of interest at all times, or an inducible promoter that induces the expression of a gene of interest at a specific position or time. Examples of such promoters include the U6 promoter, the H1 promoter, the CMV (cytomegalovirus) promoter, the SV40 promoter, the CAG promoter (Hitoshi Niwa et al., Gene, 108:193-199, 1991), the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985), the Rsyn7 promoter (U.S. Patent Application Serial No. 08 / 991,601), the rice actin promoter (McElroy et al., Plant Cell 2:163-171, 1990), the ubiquitin promoter (Christensen et al., Plant Mol. Biol. 12:619-632, 1989), ALS promoter (U.S. Patent Application No. 08 / 409,297), etc. In addition, any promoter known to those skilled in the art, such as promoters disclosed in U.S. Patent Nos. 5,608,149; 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, and 5,608,142, etc., may be used, but is not limited thereto. Preferably, the promoter of the present invention may be a U6 promoter, a HI promoter, or a CMV promoter, and according to a preferred embodiment of the present invention, a CMV promoter may be used.

[0061] In the present invention, the term "tissue-specific" is intended to mean that a promoter operably linked to a gene essential for replication functions in a tissue-specific manner to drive replication in the tissue. This can occur due to the presence of a positive transcription factor that activates the promoter in the tissue and is absent in non-target tissues. This can also occur due to the absence of a transcription inhibitor that normally forms in the non-target tissue and prevents transcription as a result of the promoter. Thus, when transcription occurs, it is directed toward the gene essential for replication so that replication of the vector and its associated functions occur in the target tissue.

[0062] Tissue specificity is particularly relevant for targeting the abnormal counterpart of a particular tissue while avoiding the normal counterpart of the tissue, or for treating the abnormal tissue while avoiding other types of surrounding tissues other than the abnormal tissue. In certain embodiments, the promoter is "tumor-specific", meaning that the promoter functions specifically in tumor tissue. For example, the recombinant adenovirus of the present invention is useful for treating metastasis to the liver. A specific example is colon cancer, which often metastasizes to the liver. It has been confirmed that when colon cancer metastasizes to the liver, the CEA promoter is active in the metastatic cells, but not in normal liver cells. Thus, normal human adult liver cannot support the replication of a virus that has a viral gene essential for replication linked to the colon cancer CEA-specific promoter. Replication must occur in the cells of the primary cancer. Another example is the alpha fetoprotein promoter, which is only active in hepatocellular carcinoma. Another example is the tyrosinase promoter, which is only active in melanoma, but not in normal skin. In each case, replication is expected in abnormal cells but not in normal cells.

[0063] Examples of tissue-specific promoters include, but are not limited to, the alpha fetoprotein promoter, the DE3 promoter, the tyrosinase promoter, the carcinoembryonic antigen (CEA) promoter, the surfactant protein promoter, the E2F promoter, the telomerase hTERT promoter, the prostate-specific antigen promoter, the COX-2 promoter, the albumin gene promoter, the hepatitis virus core promoter, the promoter of globulin-binding protein that binds thyroxine and the ErbB2 promoter.

[0064] In the present invention, the term "adenovirus" refers to any virus that can be classified as an adenovirus, i.e., any virus belonging to the family Adenoviridae, which is identified as a non-enveloped virus with an icosahedral nucleocapsid containing a double-stranded DNA genome. This term includes any adenovirus capable of infecting humans or animals, including all groups, subgroups and serotypes that use CAR as a receptor to infect target cells. Adenoviruses in the present invention include, by way of non-limiting example, avian, canine, equine, bovine, ovine, porcine, human or frog adenoviruses. In a preferred embodiment, the adenovirus in the present invention is a human adenovirus, i.e., an adenovirus capable of infecting humans. According to the present invention, a "serotype" is each immunologically distinct type of adenovirus. In the case of human adenoviruses, there are at least 57 serotypes divided into several subgroups (A to G).

[0065] The adenovirus in the present invention is a recombinant adenovirus. In the present invention, the term "recombinant" refers to an adenovirus that is not naturally occurring. Such recombinant adenoviruses contain one or more modifications compared to the wild type. Such modifications include, but are not limited to, modifications to the adenoviral genome that is packaged within the particle to produce an infectious virus. Other modifications include removing genes important for replication from the viral genome to obtain a replication-deficient virus (i.e., a virus that cannot reproduce). Examples of modifications include deletions known in the art, such as deletions of one or more of the E1a, E1b, E2a, E2b, E3, or E4 coding regions. Other modifications include deletions of the entire coding region of the adenoviral genome. Such adenoviruses are referred to as "gutless" adenoviruses. Also included are chimeric adenoviruses produced by combining elements from different serotypes. Adenoviral particles are composed of a capsid that encloses viral DNA. In this application, the term "capsid" refers to a viral protein shell formed by subunits called capsomeres, which may be pentagonal or hexagonal. Adenovirus capsids are shaped like regular icosahedrons with 20 equilateral triangles. Most of the capsid is formed by hexon proteins, and each vertex is a complex formed by a penton base and a fiber protein.

[0066] The adenovirus of the present invention may have a modification in the genome sequence that confers selective replication in cells. To authorize the expression of the adenovirus in tissues requiring expression or tumor tissues to be treated, the adenovirus of the present invention may include a tissue-specific promoter or a tumor-specific promoter. Thus, in one embodiment, the adenovirus further includes a tissue-specific promoter or a tumor-specific promoter.

[0067] In the present invention, the term "recombinant" also includes replication-conditional adenoviruses that replicate preferentially in certain types of cells or tissues, but replicate poorly or not at all in others. For example, among the adenoviruses presented in the present invention are those that replicate in abnormally proliferating tissues such as solid tumors and other neoplasms. These include the viruses described in U.S. Pat. Nos. 5,998,205 and 5,801,029. Such viruses are often referred to as "cytolytic" or "cytopathic" viruses (or vectors) and, if they have such an effect on neoplastic cells, as "oncolytic" viruses (or vectors).

[0068] As used herein, the term "adenovirus hexon protein" or "hexon protein" (previously referred to as "protein II") refers to a major capsid structural protein found in adenovirus that self-assembles to form trimers, each of which is hexagonal in shape. 240 hexon trimers assemble into the adenovirus capsid. The hexon protein is essential for viral capsid assembly, the icosahedral crystal symmetry of the capsid, and the integrity of the capsid. The major structural features of the hexon protein are common among adenovirus serotypes, although the size and immunological properties of the hexon protein vary among the serotypes.

[0069] In the present invention, the term "hexon protein" encompasses any adenovirus hexon protein, such as, but not limited to, the protein defined in the UniProt database sequence with accession number P04133, deposited on February 19, 2014, which corresponds to the hexon protein of human adenovirus C serotype 5; the protein defined in the UniProt database sequence with accession number P03277, deposited on February 19, 2014, which corresponds to the hexon protein of human adenovirus C serotype 2; the protein defined in the UniProt database sequence with accession number P42671, deposited on February 19, 2014, which corresponds to the hexon protein of avian adenovirus gal1 (strain Phelps); and the protein defined in the UniProt database sequence with accession number P11819, deposited on February 19, 2014, which corresponds to the hexon protein of human adenovirus F serotype 40. This term is inclusive of all naturally occurring variants of the hexon protein that occur naturally in other subgroups or serotypes.

[0070] In the present invention, the expression "outer surface of hexon protein" refers to the region exposed on the surface of the capsid of hexon protein. To confirm whether the transferrin binding moiety of the present invention is introduced into the inner part or outer surface of the adenovirus hexon protein, an assay to detect binding to human serum transferrin can be performed as described in the experimental part of this patent application (e.g., ELISA assay) or by in vitro neutralization analysis. If human serum transferrin can bind to adenovirus, it means that the transferrin binding moiety is introduced into the outer surface of the adenovirus hexon protein.

[0071] It has been reported that Loop1 (L1) and Loop2 (L2) of the hexon protein are exposed on the outer surface of the viral capsomere structure. L1 contains six hypervariable regions (HVRs), i.e., HVR1 contains HVR6, and L2 contains the seventh hypervariable region (HVR7). In this application, the term "hypervariable region" or "HVR" refers to a region of surface-exposed loops with differences in length and sequence that determine the serotype of the adenovirus. There are seven hypervariable regions of the adenovirus hexon in each subunit of the trimer. In the present invention, the nomenclature used for the HVRs is as described in Crawford-Miksza and Schnurr (Crawford-Miksza and Schnurr. 1996. Virology, 224(2):357-367). In a preferred embodiment of the present invention, the HVR is HVR1, but is not limited thereto. The sequence encoding the transferrin-binding moiety is inserted such that the fusion protein produced contains the transferrin-binding moiety next to amino acid D154 of the hexon protein, according to the numbering of the hexon protein, GenBank Accession No. BAG48782.1, dated June 14, 2008, which corresponds to the hexon protein from human adenovirus serotype 5.

[0072] The adenovirus of the present invention is an oncolytic adenovirus, preferably an adenovirus whose genome further comprises a mutation in one or more genes selected from the group consisting of E1a, E1b, E4 and VA-RNA to achieve selective replication in tumors.

[0073] In another embodiment, the adenoviral genome further comprises a capsid modification to enhance adenoviral infectivity or target it as a receptor present on tumor cells. Preferably, the capsid modification is the insertion of an RGD motif into the H1 loop of the adenoviral fiber protein. In another embodiment, the adenoviral genome is a chimeric adenovirus genome derived from one given serotype, with a fragment or region of the genome replaced with a homologous region of the genome from another serotype. Preferably, such a chimeric adenovirus is a human adenovirus derived from serotype 5, with a portion of the fiber gene replaced with a homologous region from another serotype, preferably human adenovirus 3 or human adenovirus 35. In a preferred embodiment, the capsid modification replaces a portion of the fiber gene with a homologous portion from another adenovirus serotype, forming a chimeric adenovirus. In the present invention, type 5 / 3 adenovirus refers to a virus that contains a part of the fiber gene derived from type 3 adenovirus, and the rest of the genome is derived from type 5 adenovirus.

[0074] In another embodiment, the adenovirus genome includes an additional gene inserted into the genome. In one embodiment, the gene is used for gene therapy or vaccine immunization. Preferably, the gene is a gene used for cancer gene therapy, more preferably a gene selected from the group consisting of at least a prodrug-activating gene, a tumor-suppressor gene, a gene encoding an anti-tumor interfering RNA, and an immunostimulatory gene.

[0075] In one aspect, the present invention relates to a pharmaceutical composition for treating cancer, comprising the adenovirus of the present invention as an active ingredient.

[0076] In a preferred embodiment, the adenovirus of the present invention is an oncolytic adenovirus. In the present application, the term "oncolytic adenovirus" refers to any adenovirus that is replication-competent or capable of replicating in tumors without any preference. The therapeutic effect of oncolytic adenoviruses is based on their ability to cytolyze tumor cells that are to be replicated and eliminated. The death of tumor cells can be detected by any modern method, such as measuring the number of viable cells, cytopathic effect, apoptosis of tumor cells, synthesis of viral proteins in tumor cells (e.g., metabolic labeling, Western blot of viral proteins or PCR with reverse transcription of viral genes required for replication) or reduction in tumor size.

[0077] In one embodiment, the compositions of the invention may be administered systemically.

[0078] In one embodiment, the compositions of the present invention may be for intravenous administration.

[0079] In one embodiment, the composition of the present invention may further include an anti-cancer drug, such as acivicin, aclarubicin, acodazole, acronisine, adzelesin, alanosine, aldesleukin, allopurinol sodium, altretamine, aminoglutethimide, amonafide, ampligen, amsacrine, androgen, anguidine, aphidicolin glycinate, asarly, asparaginase, 5-azacytidine, azathioprine, Bacillus Calmette-Guerin (BCG), Baker's Antifol, beta-2-deoxythioguanosine, bisantrene HCl, bleomycin sulfate, busulfan, buthionine sulfoximine, BWA 773U82, BW 502U83 / HCl, BW 7U85 mesylate, cerasemide, carbetimer, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladribine, corticosteroids, Corynebacterium parvum, CPT-11, crisnatol, cyclocytidine, cyclophosphamide, cytarabine, sitembena, davismaleate, dacarbazine, dactinomycin, daunorubicin HCl, deazauridine, dexrazoxane, dianhydrogalactitol, diaziquone, dibromodulcitol, didemnin BB, diethyldithiocarbamic acid, diglycaldehyde, dihydro-5-azacytidine, doxorubicin, echinomycin, edatrexate, edelfosine, eflornithine, Elliot's solution, elsamitrucin, epirubicin, esorubicin, estramustine phosphate, estrogen, etanidazole, etiophos, etoposide, fadrazol, fazarabine, fenretinide, filgrastim, finasteride, flavone acetic acid, floxuridine, fludarabine phosphate, 5'-fluorouracil, Fluozole®, flutamide, nitrile Gallium acetate, gemcitabine, goserelin acetate, hepsulfame, hexamethylene bisacetamide, homoharringtonine, hydrazine sulfate, 4-hydroxyandrostenedione, hydroxyurea, idarubicin HCl, ifosfamide, 4-ipomeanol, iproplatin, isotretinoin, leucovorin calcium, leuprolide acetate, levamisole, liposomal daunorubicin, liposomal encapsulated doxorubicin, lomustine, lonidamine, maytansine, mechlorethamine hydrochloride, melphalan, menogaril, mervalone, 6-mercapto Purine, Mesna, Methanol Extract of Bacillus Calmette-Guerin, Methotrexate, N-Methylformamide, Mifepristone, Mitoguazone, Mitomycin-C, Mitotane, Mitoxantrone Hydrochloride, Monocyte / Macrophage Colony Stimulating Factor, Nabilone, Nafoxidine, Neocarzinostatin, Octreotide Acetate, Ormaplatin, Oxaliplatin, Paclitaxel, Pera, Pentostatin, Piperazinedione, Pipobroman, Pirarubicin, Piritrexim, Piroxantrone Hydrochloride, PIXY-321, Plicamycin, Porfima -sodium, prednimustine, procarbazine, progestin, pyrazofurin, razoxane, sargramostim, semustine, spirogermanium, spiromustine, streptonigrin, streptozocin, sulofenur, suramin sodium, tamoxifen, taxotere, tegafur, teniposide, terephthalamidine, teroxylon, thioguanine, thiotepa, thymidine injection, tiazofurin, topotecan, toremifene, tretinoin, trifluoperazine hydrochloride, trifluridine, trimetrexate, TNF (tumornecrosis factor), uracil mustard, vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinzolidine, Yoshi 864, zorubicin, cytosine arabinoside, etoposide, melphalan, taxol and mixtures thereof.Preferably, it is cisplatin, paclitaxel, 5-FU (5-fluorouracil), methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, melphalan, chlorambucil, cyclophosphamide, vindesine, mitomycin, bleomycin, tamoxifen and taxol, more preferably, it is cisplatin, paclitaxel or 5-FU (5-fluorouracil), but it is not limited thereto as long as it can achieve the purpose of showing a synergistic effect on anticancer effect by treating it in combination with the composition of the present invention.

[0080] In one embodiment, the cancer may be any one selected from the group consisting of colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain cancer, head and neck cancer, melanoma, myeloma, leukemia, lymphoma, gastric cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, esophageal cancer, small intestine cancer, anal cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic cancer, bone cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, glioblastoma multiforme, and pituitary adenoma.

[0081] The composition of the present invention may further include an adjuvant. Any adjuvant known in the art may be used without limitation, and may further include, for example, Freund's complete or incomplete adjuvant to enhance its effect.

[0082] The composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharma- ceutically acceptable carrier. Here, the pharma-ceutically acceptable carrier includes carriers, excipients and diluents that are commonly used in the pharmaceutical field. The pharma-ceutically acceptable carriers that can be used in the composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0083] The composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injectable solutions by the usual methods.

[0084] When preparing a formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant that is commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing the active ingredient with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and can contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-water-soluble solvents, suspensions, emulsions, freeze-dried preparations, and suppositories.

[0085] Examples of non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases include witepsol, tween 61, cacao butter, laurin, and glycerogelatin.

[0086] The compositions according to the invention can be administered to an individual by a variety of routes, any mode of administration can be envisaged, for example by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection, but most preferably by intravenous administration.

[0087] The dosage of the pharmaceutical composition according to the present invention is selected taking into consideration the age, weight, sex, physical condition, etc. of an individual. It is obvious that the concentration of the single domain antibody contained in the pharmaceutical composition can be selected in various ways depending on the subject, and is preferably contained in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmacological activity may not be exhibited, and if it exceeds 5,000 μg / ml, toxicity to the human body may be exhibited.

[0088] The composition of the present invention can be used for the prevention or treatment of cancer and its complications, and can also be used as an anti-cancer adjuvant.

[0089] The composition in the present invention is administered in a therapeutically effective amount or a pharmacologic effective amount.The term "pharmacologic effective amount" means an amount sufficient to treat disease with a reasonable benefit / risk ratio applicable to medical treatment, and effective dose level can be determined according to individual type and severity, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, factors including drugs used simultaneously, and other factors well known in the art.

[0090] In one aspect, the invention relates to the tumor prophylactic or therapeutic use of an adenovirus comprising a nucleic acid encoding a transferrin-binding moiety of the invention in the coding region of the hypervariable region (HVR) of the hexon protein.

[0091] In one aspect, the invention relates to a method of treating cancer by administering to an individual suffering from cancer an adenovirus that contains a nucleic acid encoding a transferrin binding moiety of the invention in the coding region of the hypervariable region (HVR) of the hexon protein. [Mode for carrying out the invention]

[0092] The present invention will be described in more detail with reference to the following examples, which are merely intended to illustrate the present invention and are not intended to limit the present invention.

[0093] Example 1. Preparation of an anti-cancer (anti-tumor) virus containing a transferrin-binding domain The nucleotide sequences (sequence numbers 1 and 3) encoding two types of transferrin-binding moieties, Tbp29aa (sequence number 2) and Tbp55aa (sequence number 4), from the transferrin binding domain (TBD) of the transferrin binding protein (TBP) derived from Neisseria meningitidis strains K454, S3131, and B16B6, respectively, were inserted into the HVR1 region of the hexon of an adenovirus vector to produce an antitumor adenovirus containing the transferrin-binding moiety. Specifically, two types of transferrin-binding moieties, Tbp29aa (SEQ ID NO: 2) and Tbp55aa (SEQ ID NO: 4), were designed in the transferrin-binding domain with reference to the amino acids of TbpA from Neisseria meningitidis strains K454, S3131, and B16B6, and then a base sequence encoding Tbp29aa (SEQ ID NO: 1) and a base sequence encoding a fusion protein containing linkers (SEQ ID NO: 5) at the N-terminus and C-terminus of Tbp55aa (SEQ ID NO: 4) were synthesized by Bio Basic Inc. After synthesis, the target sequences were amplified by PCR, and then inserted into the HVR1 position (between the codons expressing the 154th amino acid (gaa, E) and the 155th amino acid (gac, D) of the base sequence encoding hexon) of pAd1128 (OD260, adenoviral plasmid) plasmid, and transformed into E. coli DH10B. Thereafter, mini preps were performed on the clones in which the insertion of each transferrin binding moiety was confirmed by colony PCR, and sequence analysis was requested from Macrogen to confirm the base sequence again.Then, to prepare the adenovirus genome (comid) used as a material for producing adenovirus, the four plasmids pAd1128-tbp (containing each transferrin binding site coding sequence), pAd1127 (containing E and PIX sites), pAd1129 (containing E3 and Fiber)-GFP, and pAd1130 (containing E4) were treated with a restriction enzyme (Sfil) and ligated to produce cosmids (Figure 2). The cosmids were then packaged in lambda phage and infected into E. coli (OD101) for transduction. Several clones were cultured in liquid, mini-prepped, treated with restriction enzymes, and the sizes of the DNA fragments were compared with the expected patterns to obtain clones with the expected patterns. The confirmed clones were then miniprepped and treated with restriction enzymes to remove replication origins, antibiotic resistance genes, etc. to create a linear adenovirus genome, and 7ug was then transduced into HE293 cell lines to produce seed viruses, which were then used in experiments. A schematic diagram of the seed virus envelope vector is shown in Figure 3, and the vector structure of the adenovirus containing the transferrin binding moiety Tbp29aa is shown in Figure 5 (adenovirus containing SEQ ID NO: 8; HVR1-TBP29aa), and the vector structure of the adenovirus containing Tbp55aa with linkers at the N-terminus and C-terminus is shown in Figure 6 (adenovirus containing SEQ ID NO: 10; HVR1-(G4S1)3-TBP55aa). Additionally, the vector structure of type 5 / 3 adenovirus CA10G-A (adenovirus containing the sequence of SEQ ID NO: 12) used as a positive control, which contains an albumin binding domain (ABD) in the HVR1 region, is shown in FIG.

[0094] Example 2. Confirmation of anti-tumor virus neutralizing antibody evasion ability It was confirmed whether the adenovirus containing the transferrin-binding domain prepared in Example 1 binds to transferrin in vivo, thereby avoiding the attack of antibodies and killing cancer cells. Specifically, the lung cancer cell line A549 was cultured at 1x10 per well in a 96-well plate. 4The positive control group, CA10G-A (5 / 3 type adenovirus containing an albumin binding portion in the HVR1 region: adenovirus containing the sequence of SEQ ID NO: 12), was subjected to a binding reaction in a culture medium containing 10 mg / ml of albumin for 4 hours at 4°C before cell treatment in order to avoid antibodies, and in the case of HVR1-TBP29aa (5 / 3 type adenovirus containing a transferrin binding portion of SEQ ID NO: 1 in the HVR1 region) and HVR1-(G4S1)3-TBP55aa (5 / 3 type adenovirus containing a transferrin binding portion of SEQ ID NO: 3 and a linker in the HVR1 region) of the present invention, the binding reaction was performed in a culture medium containing 1 mg / ml of transferrin for 4 hours at 4°C before cell treatment. Then, the cells were treated with albumin or transferrin-reacted virus and control virus (CA10G:5 / 3 type adenovirus) (infectious units, IFU) at 0, 5, 10, 20, 50, and 100 times the number of cells, respectively, and the culture medium was treated with adenovirus type 5 antibody (Abcam, ab6982) at a concentration of 0.1ng / ml. 72 hours after virus treatment, the cell killing ability by the virus was quantitatively evaluated using a cell division kit (XTT assay kit) manufactured by Sigma-Aldrich, and the 96-well plate was stained with crystal violet reagent to visualize the cell killing ability by the virus.

[0095] As a result, it was confirmed that at an MOI (multiplicity of infection) of 50, the HVR1-Tbp(G4S1)3 55aa (Tbp-(G4S1)3-55aa) virus had superior antibody evasion ability to the positive control CA10G-A (Figures 7 and 8).

[0096] Example 3. Construction of a virus containing a transferrin-binding domain at the HVR2 position Using the method of Example 1, the base sequence (sequence number 1) encoding Tbp29aa (sequence number 2) and the base sequence encoding a fusion protein containing linkers (sequence number 5) at the N-terminus and C-terminus of Tbp55aa (sequence number 4) were inserted into the HVR2 region of the hexon of an adenovirus vector to prepare adenoviruses containing a transferrin-binding portion (HVR2-Tbp29aa and HVR2-(G4S1)3-Tbp55aa).

[0097] Example 4. Confirmation of the ability of anti-tumor viruses containing a transferrin-binding domain at the HVR2 position to evade neutralizing antibodies The neutralizing antibody evasion ability of the adenoviruses HVR2-Tbp29aa and HVR2-(G4S1)3-Tbp55aa, which contain a transferrin-binding moiety in the HVR2 region of the hexon of the adenovirus vector prepared in Example 3, was compared with that of HVR1-(G4S1)3-Tbp55aa. Specifically, the lung cancer cell line A549 was cultured at 1x10 per well in a 96-well plate. 4 CA10GT (HVR1-(G4S1)3-Tbp55aa), HVR2-Tbp29aa (a 5 / 3 type adenovirus containing the transferrin binding moiety of SEQ ID NO: 1 in the HVR2 region) and HVR2-(G4S1)3-Tbp55aa (a 5 / 3 type adenovirus containing the transferrin binding moiety of SEQ ID NO: 3 and a linker in the HVR2 region) were reacted with each other in a culture medium containing 2 mg / ml of transferrin at 4°C for 6 hours, and the dispensed cells were treated at 0, 5, 10, 20, 50 and 100 times the number of cells (CA10G: 3.38x10 10 ifu / ml; CA10GT:8.03x10 9 ifu / ml;HVR2-29aa:6.94x10 10 ifu / ml; and HVR2-(G4S1)3-55aa: 1.29x10 10The cells were treated with 0.1ng / ml of adenovirus type 5 antibody (Abcam, ab6982) at a concentration of 0.1ng / ml. 72 hours after virus treatment, the cell killing ability of the virus was quantitatively evaluated using a cell division kit (XTT assay kit) manufactured by Sigma-Aldrich.

[0098] As a result, it was shown that only CA10GT in which the transferrin-binding moiety was inserted into the HVR1 region of the hexon of the adenovirus vector was able to avoid antibody attack and kill cancer cells by binding to transferrin in the body, and that the virus in which the transferrin-binding moiety was inserted into the HVR2 region of the hexon of the adenovirus vector had almost no ability to evade antibodies (Figure 9).

Claims

1. An adenovirus comprising a nucleic acid encoding a transferrin binding moiety in the coding region of the hypervariable region (HVR) of a hexon protein.

2. The adenovirus of claim 1 comprising the nucleic acid sequence of SEQ ID NO: 1, 3, 8 or 10.

3. The adenovirus of claim 1 , wherein the transferrin binding portion comprises the amino acid sequence of SEQ ID NO: 2 or 4.

4. The adenovirus of claim 1, wherein the N-terminus, C-terminus, or N-terminus and C-terminus of the transferrin binding portion is linked to the hexon protein via a linker.

5. The adenovirus of claim 4 , wherein the linker comprises the amino acid sequence of SEQ ID NO:

5.

6. The adenovirus of claim 1 , wherein the hypervariable region of the hexon protein is HVR1.

7. The adenovirus of claim 6, wherein HVR1 of the hexon protein comprises the amino acid sequence of SEQ ID NO:

7.

8. 2. The adenovirus of claim 1, comprising a nucleic acid encoding a transferrin-binding portion between codons expressing the 154th and 155th amino acids of the base sequence encoding hexon.

9. 2. The adenovirus of claim 1, wherein the transferrin binding portion is located on the outer surface of the hexon protein when the hexon protein is assembled into the adenovirus capsid.

10. The adenovirus of claim 1 , wherein the adenovirus is a human adenovirus.

11. The adenovirus according to claim 10, wherein the human adenovirus is selected from the group consisting of human adenovirus serotypes 1 to 57.

12. The adenovirus of claim 10, wherein the human adenovirus is human adenovirus serotype 5.

13. The adenovirus of claim 10, wherein the human adenovirus is human adenovirus serotype 5 / 3.

14. The adenovirus of claim 1, further comprising a tissue-specific promoter or a tumor-specific promoter.

15. The adenovirus of claim 14 , wherein the promoter is operably linked to an endogenous gene of the adenovirus.

16. The adenovirus according to claim 14, wherein the promoter is selected from the group consisting of E2F promoter, telomerase hTERT promoter, tyrosinase promoter, prostate-specific antigen promoter, alpha-fetoprotein promoter and COX-2 promoter.

17. The endogenous gene of adenovirus has the structure 5'-ITR-C1-C2-C3-C4-C5 3-'ITR; said C1 comprising E1A, E1B or E1A-E1B; said C2 comprising E2B-L1-L2-L3-E2A-L4; said C3 does not include E3 or includes E3; said C4 comprising L5; and The adenovirus of claim 15, wherein the C5 does not include E4 or includes E4.

18. The adenovirus of claim 17, further comprising an IRES sequence between E1A and E1B.

19. 18. The adenovirus of claim 17, wherein the promoter is operably linked to the E1A and E1B endogenous genes of the adenovirus.

20. The adenovirus of claim 1 , further comprising an expression cassette for expressing a foreign gene.

21. The adenovirus of claim 20 , comprising an expression cassette in the E3 region of an endogenous gene of the adenovirus.

22. The adenovirus according to claim 1 , wherein the adenovirus is an anti-tumor adenovirus.

23. The adenovirus of claim 1, which is an oncolytic adenovirus.

24. The adenovirus of claim 1, further comprising a capsid modification to enhance infectivity of the adenovirus or to target it to a receptor present on a tumor cell.

25. 25. The adenovirus of claim 24, wherein the capsid modification is the insertion of an RGD motif into the H1 loop of the adenovirus fiber protein.

26. 25. The adenovirus of claim 24, wherein the capsid modification involves replacing the fiber gene or a portion thereof with a homologous portion derived from an adenovirus of another serotype to form a chimeric adenovirus.

27. 27. The adenovirus of claim 26, comprising a capsid replaced with a fiber gene or a portion thereof from a serotype 3 adenovirus, the portion excluding the fiber gene comprising a gene from a serotype 5 adenovirus.

28. The adenovirus of claim 1, which comprises one or more non-adenoviral genes.

29. The adenovirus of claim 28, wherein the non-adenoviral gene is a gene used in cancer gene therapy.

30. The adenovirus according to claim 29, wherein the gene used in cancer gene therapy is selected from the group consisting of tumor suppressor genes, genes encoding anti-tumor interfering RNA, and immune stimulatory genes.

31. A pharmaceutical composition for treating cancer, comprising the adenovirus according to claim 1 as an active ingredient.

32. The pharmaceutical composition for cancer therapy according to claim 31 , which is administered systemically.

33. 2. Use of an adenovirus for tumor prevention or treatment, comprising a nucleic acid encoding a transferrin-binding moiety in the coding region of the hypervariable region (HVR) of the hexon protein.

34. A method of treating cancer comprising administering to an individual suffering from cancer an adenovirus that contains a nucleic acid encoding a transferrin-binding moiety in the coding region of the hypervariable region (HVR) of the hexon protein.

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