Immunoevasive 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 that can avoid the immune system in vivo.

Background Art

[0002] Cancer is one of the diseases that cause the most deaths worldwide. The development of innovative cancer therapeutics can reduce the medical costs incurred during treatment for this disease, and at the same time, create high added value. Also, according to the statistics for 2008, molecular therapeutics that can overcome conventional anticancer drug resistance accounted for $17.5 billion in the seven major countries (US, Japan, France, Germany, Italy, Spain, UK), and are predicted to occupy a market size of approximately $45 billion in 2018, showing a growth rate of 9.5% compared to 2008. The treatment of cancer is classified into surgery, radiotherapy, chemotherapy, and biological therapy. Among these, chemotherapy is a treatment method that suppresses or kills the growth of cancer cells with chemical substances. The toxicity exhibited by anticancer drugs is such that a significant portion also appears in normal cells, so it exhibits a certain degree of toxicity. Even if an antitumor agent is effective, after a certain period of use, resistance occurs where the effect is impaired. Therefore, there is an urgent need to develop an anticancer drug that selectively acts on cancer cells and does not develop resistance. In recent years, the development of new anticancer drugs targeting the molecular characteristics of cancer through the acquisition of molecular genetic information for cancer has been underway, and there are also reports that drug resistance occurs even in anticancer drugs that target characteristic molecular targets (molecular targets) possessed only by cancer cells. Therefore, the development of new concept anticancer drugs is required.

[0003] On the other hand, there have been experiments using viruses for cancer treatment, based on unsubstantiated reports related to temporary cancer remission after infection with natural viruses or viral vaccination. The first report was in 1912, stemming from a reduction in cervical cancer in patients vaccinated against rabies, and similar results were observed in cancer patients following smallpox vaccination or infection with natural viruses (e.g., mumps, measles). Based on these reports and animal data, live viruses were inoculated into patients for cancer treatment in the late 1940s and early 1950s. However, this often resulted in the problem of tumor regrowth and patient death after a temporary reduction in tumor size. These inoculations also did not result in long-term, sustained remission. In 1957, Albert B. Sabin, MD, who developed the oral live polio vaccine, stated that even when oncolytic viruses kill tumors, the biggest problem was that the individual's immune response to the virus was too rapid, causing the effect to disappear quickly.

[0004] Currently, many oncolytic adenoviruses have been identified, but to date, the only virus approved for clinical use anywhere in the world is Oncorine (H101), a subgroup C adenovirus modified with an E1B-55KD deletion that enables conditional replication in p53-deficient cancer cells (H101 is a close analogue of ONYX015, described by Bischoff et al. in 1996). Oncorine is administered by intratumoral injection for head and neck cancers. Adenoviruses have been widely used not only as gene delivery vectors for gene therapy but also as oncolytic agents for cancer treatment. Adenoviruses exhibit several characteristics that make them suitable for such applications. Specifically, 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. Adenoviruses are safe in that they do not cause life-threatening diseases in humans, and their viral genomes are non-integrative, preventing insertion mutations. Clinical trials using adenovirus vectors have reported that, when administered systemically, this virus has a favorable toxicity and safety profile, although there is still a need for improvement in efficacy.

[0005] Thus, in the field of gene therapy, systemic administration, i.e., injection into the bloodstream via venous or arterial means, is sometimes necessary to reach multiple organs and disseminated cells. For example, in cancer treatment using adenovirus vectors and oncolytic adenoviruses, systemic administration is essential to treat advanced or metastatic disseminated tumors. However, adenoviruses have significant limitations when injected into the bloodstream, which reduces their therapeutic effect. Adenovirus type 5 (Ad5) undergoes various neutralizing interactions that drastically reduce the bioavailability of the virus in the blood. Since >90% of the injected dose remains in the liver, mainly in liver macrophages called Cooper cells, as well as liver sinusoidal endothelial cells (LSECs) and liver cells, the biggest problem with this treatment is liver isolation. Direct interactions with blood cells and proteins are also major obstacles. Adenovirus (Ad5) can bind directly to blood cells such as red blood cells via CAR receptors and to platelets via integrins. Antibodies can not only directly neutralize the virus but also trigger an innate immune response by activating complement and docking viral particles to Fc receptors on mononuclear cells and neutrophils. Furthermore, viral re-administration increases the concentration of anti-Ad neutralizing antibodies (NAbs), thus enhancing viral neutralization. Opsonization of adenovirus by antibodies and complement can similarly enhance clearance by Cooper cells. Overall, these interactions result in a significant reduction in the half-life of Ad in the blood in mice and humans to approximately a few minutes. Although considerable efforts have been made to avoid neutralization by antibodies and immune cells when adenovirus is administered systemically, systemic transmission remains limited, temporary, and generally ineffective (Ferguson et al. 2012).

[0006] Therefore, research into adenoviruses as cancer treatments suitable for systemic administration and capable of evading neutralizing antibodies remains necessary. [Overview of the project] [Problems that the invention aims to solve]

[0007] The objective of the present invention is to provide an antitumor adenovirus.

[0008] Another object of the present invention is to provide a pharmaceutical composition for cancer treatment.

[0009] Furthermore, an object of the present invention is to provide the adenovirus for tumor prevention or treatment.

[0010] Furthermore, an object of the present invention is to provide a cancer treatment method comprising administering the adenovirus to an individual suffering from cancer. [Means for solving the problem]

[0011] To solve the aforementioned problems, the present invention provides an adenovirus comprising a nucleic acid encoding a transferrin-binding portion.

[0012] Furthermore, the present invention provides a pharmaceutical composition for cancer treatment that contains the antitumor adenovirus.

[0013] Furthermore, the present invention provides the use of the adenovirus for tumor prevention or treatment.

[0014] Furthermore, the present invention provides a method for treating cancer, comprising administering the adenovirus to an individual suffering from cancer. [Effects of the Invention]

[0015] According to the present invention, an adenovirus containing nucleic acid encoding the transferrin-binding domain of the present invention exhibits significantly increased tumor cell infection and death effects, evades the body's immune response due to increased binding to transferrin, has an increased plasma half-life, is specifically delivered to cancer cells, has systemic therapeutic effects, can be delivered locally, exhibits excellent selectivity, and shows remarkable antitumor efficacy. Therefore, it can be usefully used as an anticancer composition or anticancer adjuvant for various cancers. [Brief explanation of the drawing]

[0016] [Figure 1] This figure shows the antibody evasion mechanism of an antitumor adenovirus containing the transferrin-binding domain of the present invention. [Figure 2] This figure shows the manufacturing process of an antitumor adenovirus containing the transferrin-binding domain (part) of the present invention. [Figure 3] This figure shows the vector map of the adenovirus envelope-associated plasmid pAd1128, in which the transferrin-binding domain of the present invention is contained at the HVR1 position of the hexon. [Figure 4] This figure (CA10G-A) shows a vector map of an adenovirus containing the albumin-binding domain (ABD). [Figure 5] This figure shows the vector map of an adenovirus in which the hexon of the adenovirus vector according to the present invention contains the transferrin binding moiety Tbp29aa (HVR1-Tbp29aa). [Figure 6] This figure shows a vector map of an adenovirus containing a fusion protein in which the hexon of the adenovirus vector according to the present invention contains linkers at the N-terminus and C-terminus of the transferrin binding portion Tbp55aa (HVR1-Tbp(G4S1)35aa). [Figure 7] and [Figure 8]A diagram showing the immune evasion ability of adenovirus containing a transferrin-binding domain: CA10G: control group 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 adenovirus 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 adenovirus vector. [Figure 9] A diagram showing the immune evasion ability according to the insertion position of the transferrin-binding moiety into adenovirus: CA10G: control group adenovirus; CA10GT: HVR1-(G4S1)3-Tbp55aa adenovirus in which the transferrin-binding moiety is inserted at the HVR1 position of the hexon; HVR229aa: HVR2-Tbp29aa adenovirus in which the transferrin-binding moiety is inserted at the HVR2 position of the hexon; and HVR2L355aa: HVR2-(G4S1)3-Tbp55aa adenovirus.

Mode for Carrying Out the Invention

[0017] Hereinafter, the present invention will be described in detail with reference to the examples of the present invention. However, the following examples are presented as illustrations of the present invention, and the present invention is not limited thereby. The present invention can be variously modified and applied within the scope described in the claims below and the equivalent scope interpreted therefrom.

[0018] Unless otherwise specified, nucleic acids are recorded in the 5'→3' direction from left to right. The numerical ranges recited herein include 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 present invention relates to an adenovirus that includes a nucleic acid encoding a transferrin-binding moiety within the coding region of the hypervariable region (HVR) of the hexon protein.

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

[0022] In one embodiment, the adenovirus can include the nucleic acid sequence of hexon containing a transferrin-binding moiety, the nucleic acid sequence of hexon containing a transferrin-binding moiety can include the nucleotide sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 10, and the hexon containing a transferrin-binding moiety can include the 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, the HVR1 can include the amino acid sequence of SEQ ID NO: 7, and the nucleic acid sequence encoding the HVR1 can include the nucleotide sequence of SEQ ID NO: 6.

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

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

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

[0027] In one embodiment, the transferrin-binding moiety can directly attach to the hexon protein; that is, 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. Therefore, 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 adenovirus hexon protein is assembled into a capsid, the transferrin binding site can be located on the outer surface of the hexon protein.

[0029] The adenovirus in this invention can be coated with a transferrin-binding domain by including a transferrin-binding moiety on the outer surface of its hexone protein, thereby protecting itself 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 chimeric adenovirus of human adenovirus serotype 5 / 3.

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

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

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

[0034] In one embodiment, the endogenous gene of the adenovirus has the structure 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 E3 or includes E3; C4 includes L5; and C5 does not include E4 or includes E4.

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

[0036] In one embodiment, E1A may include the base sequence of Sequence ID No. 18.

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

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

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

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

[0041] In one embodiment, the adenovirus may further include an expression cassette that expresses an exogenous gene, and the expression cassette may be contained in the E3 region of the adenovirus's endogenous gene.

[0042] In one embodiment, the adenovirus of the present invention may further include a CMV promoter and an exogenous gene, and the CMV promoter and the exogenous gene operably linked thereto may be included in the E3 region of the endogenous gene of the adenovirus.

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

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

[0045] In one embodiment, the adenovirus in the present invention is a replicable adenovirus, an antitumor or oncolytic adenovirus.

[0046] In other concrete examples, the adenovirus is a non-replicating adenovirus or a replicating-deficient adenovirus. A replicating-deficient adenovirus or a non-replicating adenovirus is an adenovirus that cannot replicate in target cells and is used in gene therapy as a gene carrier to target cells with the aim of expressing therapeutic genes within the cells without the cells degrading them.

[0047] In one embodiment, the adenovirus may 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 inserting the RGD motif into the H1 loop of the adenovirus fiber protein.

[0049] In one embodiment, the capsid modification may involve substituting a fibril gene or a part thereof with a homologous portion derived from another serotype of adenovirus to form a chimeric adenovirus. It can be constructed to include a capsid that has been substituted with a fibril gene or a part thereof derived from serotype 3 adenovirus, and the portion excluding the fibril gene contains a gene derived from serotype 5 adenovirus.

[0050] In one embodiment, the adenovirus may contain one or more non-adenoviral genes, and the non-adenoviral genes 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 immunostimulatory genes.

[0051] The adenovirus in this invention can be selectively dispersed in desired tissues within the body, and its expression in non-target or non-tumor tissues can be avoided or significantly reduced.

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

[0053] In this invention, the term "transferrin" refers to a glycoprotein that binds to iron very strongly and reversibly. 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 is present in the blood and plays a role in iron absorption. In this invention, blood transferrin binds to the transferrin binding site of this invention, allowing adenoviruses to evade antibodies in the body, thereby increasing the blood half-life of adenoviruses when administered systemically.

[0054] In the present invention, the term "transferrin-binding moiety" refers to any amino acid sequence that can bind to transferrin, i.e., has binding affinity. Preferably, it can bind to serum transferrin, more preferably to human serum transferrin. The term "transferrin-binding moiety" includes natural transferrin-binding domains (e.g., transferrin present in bacterial proteins) and transferrin-binding sequences derived from synthetic peptides. In preferred embodiments, the transferrin-binding moiety is selected from transferrin-binding domains derived from Neisseria Meningitidis, or their functionally equivalent variants. 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 in the present invention, it can be used in the same language as the transferrin-binding moiety. In the present invention, a transferrin-binding moiety, a transferrin-binding domain, and a transferrin-binding peptide / protein are used in combination. The transferrin-binding moiety includes all of the transferrin-binding domain and the transferrin-binding peptide / protein, and more preferably, the transferrin-binding moiety refers to a synthetic peptide that includes the amino acid sequence of a portion of the transferrin-binding domain of the transferrin-binding peptide / protein.

[0055] Furthermore, the present invention includes functional equivalent variants of the transferrin binding moiety described above. In this application, the term “functional equivalent variant” refers to any polypeptide derived from the transferrin binding moiety by the insertion, deletion, or substitution of one or more residues and substantially maintaining the ability to interact with transferrin as determined above. In a preferred embodiment, a polypeptide is considered a functional equivalent variant of the transferrin binding moiety if it exhibits a transferrin binding ability of 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 a functionally equivalent variant of the transferrin-binding domain if it can neutralize the antibody with the transferrin-binding domain of SEQ ID NO: 2 or 4 with an efficiency of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of identity between two polypeptides is determined using computer algorithms and methods widely known to those skilled 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)], but other similar algorithms may also be used. The sequence identity percentage is determined using BLAST and BLAST2.0 with the parameters described herein applied. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information in the United States.

[0056] In the present invention, the types of transferrin binding sites include, but are not limited to, a variety of peptides, antibodies, etc., in addition to the sites described in the examples of the present invention. The transferrin-binding portion is a diverse collection of transferrin-binding peptides (Tbp1, Tbp2, TbpA, TbpB, etc.), iron-regulated proteins, iron-regulated outer membrane proteins, transferrin receptor proteins, and Moraxella, derived from various bacteria as described in US5912336, US7582730, US6437096, US6090576, US7258989, US6004562, US6391316, US10149900, US6610506, US20030186848A1, US20200030428A1, US20080206260A1, US6015688, and US20040258695A1, iron-regulated proteins, iron-regulated outer membrane proteins, transferrin receptor proteins, and Moraxella. The present invention is applicable to any chimeric fusion protein comprising catarrhalis outer membrane protein B1, a transferrin-binding peptide, a recombinant transferrin-binding peptide, a fragment or analog thereof of the said peptide or protein, a transferrin-binding molecule, or an anti-transferrin antibody or a fragment having immunological activity thereof, provided that the transferrin-binding portion bound to the adenovirus binds to transferrin in the blood and can evade the immune response in the body.

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

[0058] In this invention, the term "linker sequence" refers to an amino acid sequence that acts as a hinge between a hexone protein and a transferrin binding site, providing space between the two factors, so that the secondary structure of the hexone protein is unaffected by the presence of the transferrin binding site, 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 structural form of the individual factors. In a preferred embodiment, the linker sequence is a flexible linker peptide with a length of less than 31 amino acids. 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 a linker sequence, it may be represented as Sequence ID No. 5 in this invention. Other linkers can also 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” means a functional linkage between a gene expression regulatory sequence (e.g., a promoter, signal sequence, or array of transcription factor binding sites) and another gene sequence, so that the regulatory sequence modulates the transcription and / or translation of the other gene sequence.

[0060] In the present invention, the term "promoter" refers to an untranslated nucleic acid sequence upstream of an encrypted region that includes a binding site for RNA polymerase and has transcription initiation activity to the mRNA of a downstream gene of the promoter. In the expression cassette of the present invention, the promoter can be any promoter capable of initiating shRNA expression. Specifically, the promoter of the present invention can be a constitutive promoter that continuously induces the expression of the target gene at all times, or an inducible promoter that induces the expression of the target gene at a specific location or time. Examples include the U6 promoter, H1 promoter, CMV (cytomegalovirus) promoter, SV40 promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108:193-199, 1991), CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985), Rsyn7 promoter (US Patent Application No. 08 / 991, 601), rice actin promoter (McElroy et al., Plant Cell 2:163-171, 1990), and ubiquitin promoter (Christensen et al., Plant Cell 2:163-171, 1990). Examples include the ALS promoter (Mol. Biol. 12:619-632, 1989) and the U.S. Patent Application No. 08 / 409,297). In addition, any promoter known to those skilled in the art can be used, and is not limited to those disclosed in U.S. Patents 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. Preferably, the promoter of the present invention may be the U6 promoter, the HI promoter, or the CMV promoter, and according to a preferred embodiment of the present invention, the CMV promoter can be used.

[0061] In this invention, the term "tissue-specific" means that a promoter, to which genes essential for replication are operably linked, functions in a tissue-specific manner and promotes replication within that tissue. This can occur due to the presence of a positive transcription factor that activates the promoter in that tissue, but not in the non-target tissue. It can also occur due to the absence of transcription inhibitors that are normally formed in the non-target tissue and prevent transcription as a result of the promoter. Therefore, when transcription occurs, it is directed towards the genes essential for replication so that the vector replication and its associated functions take place in the target tissue.

[0062] Tissue specificity is particularly related to targeting abnormal counterparts in a specific tissue while avoiding normal counterparts, or treating abnormal tissue while avoiding other types of surrounding tissue. In certain embodiments, a promoter is "tumor-specific," meaning that the promoter functions specifically in tumor tissue. For example, the recombinant adenovirus of the present invention is useful in treating liver metastases. A specific example is colorectal cancer, which often metastasizes to the liver. When colorectal cancer metastasizes to the liver, the CEA promoter has been observed to be activated in metastatic cells but not in normal liver cells. As a result, the liver of a normal adult human cannot support the replication of a virus that has replication-essential viral genes linked to the colorectal cancer CEA-specific promoter. Replication must occur in the cells of the primary cancer. Another example is the alpha-fetoprotein promoter, which is active only in hepatocellular carcinoma. Another example is the tyrosinase promoter, which shows activation only in melanoma and 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 non-restrictive promoters such as the alpha-fetoprotein promoter, DE3 promoter, tyrosinase promoter, carcinoembryonic antigen (CEA) promoter, surfactant protein promoter, E2F promoter, telomerase hTERT promoter, prostate-specific antigen promoter, COX-2 promoter, albumin gene promoter, hepatitis virus core promoter, and globulin-binding protein promoters that bind to thyroxine and ErbB2 promoters.

[0064] In this invention, the term “adenovirus” refers to any virus that can be classified as an adenovirus, i.e., any virus belonging to the family Adenoviridae that is identified as a non-external membrane virus having an icosahedral nucleocapsid containing a double-stranded DNA genome. This term includes all groups, subgroups, and serotypes that use CAR as a receptor for infecting target cells, as well as any adenovirus capable of infecting humans or animals. In this invention, adenoviruses include, in non-limiting examples, avian, canine, equine, feline, mitten, swine, human, or frog adenoviruses. In a preferred embodiment, adenovirus in this invention is a human adenovirus, i.e., an adenovirus capable of infecting humans. According to this invention, “serotype” refers to each immunologically distinct type of adenovirus. In the case of human adenoviruses, there are at least 57 serotypes, which are classified into several subgroups (A to G).

[0065] The adenovirus in this invention is a recombinant adenovirus. In this invention, the term “recombinant” refers to an adenovirus that does not occur naturally. Such recombinant adenoviruses include one or more mutations compared to the wild type. Such mutations include, without limitation, mutations to the adenovirus genome that are packed into particles to produce infectious viruses. Other mutations include obtaining replication-deficient viruses (i.e., viruses that cannot reproduce) by removing genes important for replication from the viral genome. Examples of mutations include deletions known in the usual art, such as deletions of one or more of the E1a, E1b, E2a, E2b, E3, or E4 coding regions. Other mutations include deletions of entire coding regions of the adenovirus genome. Such adenoviruses are referred to as “gutless” adenoviruses. Chimeric adenoviruses, which are produced by combining factors derived from different serotypes, are also included. Adenovirus particles consist of a capsid that encapsulates the 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. The adenovirus capsid takes the form of an icosahedron having 20 equilateral triangles. Most of the capsid is formed by hexon proteins, and each vertex is a complex formed by penton bases and fibrous proteins.

[0066] The adenovirus in the present invention may have a genomic sequence modification that confers selective replication to cells. To authorize the expression of the adenovirus in a tissue requiring its expression or in a tumor tissue to be treated, the adenovirus in 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 this invention, the term “recombinant” also includes replication-conditional adenoviruses that preferentially replicate in certain types of cells or tissues, but only partially or completely replicate in other types. For example, among the adenoviruses presented in this invention, those that replicate in tissues that proliferate abnormally, such as solid tumors and other neoplasms. Such include the viruses described in U.S. Patents 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, they are referred to as “oncolytic” viruses (or vectors).

[0068] In this invention, the term "adenovirus hexon protein" or "hexon protein" (traditionally referred to as "protein II") refers to the major structural protein of the capsid found in adenoviruses, which self-assembles and forms trimers, each with a hexagonal shape. 240 hexon trimers are assembled to form the adenovirus capsid. The hexon protein is essential for viral capsid assembly, the icosahedral symmetry of the capsid crystal, and the integrity of the capsid. While the main structural features of the hexon protein are common among adenovirus serotypes, the size and immunological properties of the hexon protein differ between serotypes.

[0069] In the present invention, the term "hexon protein" includes, as a non-restrictive example, any adenovirus hexon protein, such as the protein defined in the UniProt database sequence of accession number P04133 registered on February 19, 2014, which corresponds to the hexon protein of human adenovirus C serotype 5; the protein defined in the UniProt database sequence of accession number P03277 registered on February 19, 2014, which corresponds to the hexon protein of human adenovirus C serotype 2; the protein defined in the UniProt database sequence of accession number P42671 registered 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 of accession number P11819 registered on February 19, 2014, which corresponds to the hexon protein of human adenovirus F serotype 40. This expression encompasses all naturally occurring variants of hexone proteins that occur spontaneously in other subgroups or serotypes.

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

[0071] The hexon proteins Loop1 (L1) and Loop2 (L2) have been reported to be exposed on the outer surface of the viral capsomere structure. L1 contains six hypervariable regions (HVRs), i.e., HVR6 in HVR1, and L2 contains a seventh hypervariable region (HVR7). In this application, the terms “hypervariable region” or “HVR” refer to regions in the surface-exposed loops that differ in length and sequence to determine the adenovirus serotype. Each subunit of the trimer contains seven hypervariable regions of the adenovirus hexon. In this invention, the names used for HVR are as described in Crawford-Miksza and Schnurr (Crawford-Miksza and Schnurr. 1996. Virology, 224(2):357-367). In a preferred embodiment of this invention, HVR is HVR1, but is not limited thereto. The sequence encoding the transferrin binding portion is inserted so that the transferrin binding portion is located after amino acid D154 of the hexon protein, according to the numbering of the hexon protein with GenBank accession number BAG48782.1 dated June 14, 2008, which corresponds to the hexon protein derived from human adenovirus serotype 5 in the fusion protein being produced.

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

[0073] In other embodiments, the adenovirus genome further includes capsid modifications to enhance adenovirus infectivity or to target it as a receptor present in tumor cells. Preferably, the capsid modification involves inserting an RGD motif into the H1 loop of the adenovirus filament protein. In other embodiments, the adenovirus genome is a chimeric adenovirus genome derived from one given serotype, comprising a fragment or region of genome substituted with a homologous region from another serotype's genome. Preferably, such a chimeric adenovirus is a human adenovirus derived from serotype 5, comprising a portion of filamentous gene substituted with a homologous region from another serotype, preferably human adenovirus 3 or human adenovirus 35. In preferred embodiments, the capsid modification involves substituting a portion of filamentous gene with a homologous portion from another adenovirus serotype to form a chimeric adenovirus. In this invention, type 5 / 3 adenovirus includes a portion of the fibre gene derived from type 3 adenovirus, and the remaining genome represents a virus derived from type 5 adenovirus.

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

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

[0076] In preferred embodiments, the adenovirus in the present invention is an oncolytic adenovirus. In this application, the term “oncolytic adenovirus” refers to any adenovirus that has or can replicate in tumors without selectivity. The therapeutic effect of oncolytic adenoviruses is based on their ability to cytodegrade 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 effects, apoptosis of tumor cells, synthesis of viral proteins in tumor cells (e.g., metabolic labeling, Western blotting of viral proteins, or PCR using reverse transcription of viral genes necessary for replication), or reduction in tumor size.

[0077] In one embodiment, the composition of the present invention can be administered systemically.

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

[0079] In one embodiment, the composition of the present invention may further contain anticancer agents, for example, asibicin, acralubicin, acodazole, acronisin, adzeresin, alanosine, aldesleukin, allopurinol sodium, altoretamine, aminoglutethimide, amonafide, amprigen, amsacrin, androgen, anguidin, aphydicolinglycinate, asari, asparaginase, 5-azacitidine, azathioprine, Bacillus calmette-Guélain (BCG), Baker's Antifol, beta-2-deoxythioguanosine, bisanthren HCl, bleomycin sulfate, busulfan, butionine sulfoximine, BWA 773U82, BW 502U83 / HCl, BW 7U85 mesylate, cerasemide, carbethymer, carboplatin, carmustine, chlorambucil, chloroquinoxaline sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladribine, corticosteroid, Corynebacterium parvum, CPT-11, cristatol, cyclocytidine, cyclophosphamide, cytarabine, cytembena, davismareate, dacarbazine, dactinomycin, daunorubicin HCl, deazauridine, dexrazoxane, dianhydrogalactitol, diaziquan, dibromodulcitol, didemnin BB, Diethyldithiocarbamic acid, Diglycaldehyde, Dihydro-5-Azacitidine, Doxorubicin, Echinomycin, Edatrexate, Edelfosine, Eflornithine, Elliott's Solution, Ersamitrusine, Epirubicin, Esorbicin, Estramustine Phosphate, Estrogen, Etanidazole, Ethiophos, Etoposide, Fadrazole, Fazarabine, Fenretinide, Filgrastim, Finasteride, Flavone acetate, Furoxuridine, Fludarabine Phosphate, 5'-Fluorouracil, Fluozol (registered trademark), Flutamide, Nitrogen Gallium acid, gemcitabine, goserelin acetate, hepsulfame, hexamethylenebisacetamide, homohalingtonin, hydrazine sulfate, 4-hydroxyandrostenedione, hydroxyurea, idarubicin HCl, ifosfamide, 4-ipomeanol, iproplatin, isotretinoin, leucovorin calcium, leuprolide acetate, levamisol, liposomal daunorubicin, liposomal encapsulated doxorubicin, lomustine, ronidamine, meitansine, mechloretamine hydrochloride, melphalan, menogalyl, melbaron, 6-mercapto Purine, Mesna, Bacillus calmette-guélain methanol extract, methotrexate, N-methylformamide, mifepristone, mitogwazone, mitomycin-C, mitotane, mitoxantrone hydrochloride, monocyte / macrophage colony-stimulating factor, nabilone, naphoxidine, neocarlutinostatin, octreotide acetate, ormaplatin, oxaliplatin, paclitaxel, perla, pentostatin, piperazinedione, pipobromane, pirarubicin, pyritrexime, pyroxantrone hydrochloride, PIXY-321, plicamycin, porfima - Sodium, Prednimustine, Procarbazine, Progestin, Pyrazofulin, Lazoxane, Salglamostim, Semustine, Spirogermanium, Spiromustine, Streptonigrin, Streptozocin, Slofenul, Suramin sodium, Tamoxifen, Taxotere, Tegaflu, Teniposide, Terephthalamidine, Teroxylone, Thioguanine, Thiotepa, Thymidine injection, Thiazofulin, Topotecan, Toremifene, Tretinoin, Trifluoperazine hydrochloride, Trifluridine, Trimethrexate, TNF (tumorThe present invention may include necrosis factor, uracil mustard, vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinzolidine, Yoshi 864, zolubicin, cytosine arabinoside, etoposide, melphalan, taxol, and mixtures thereof. Preferably, the present invention may include cisplatin, paclitaxel, 5-FU (5-fluorouracil), methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, melphalan, chlorambucil, cyclophosphamide, vindesine, mitomycin, bleomycin, tamoxifen, and taxol, and more preferably, cisplatin, paclitaxel, or 5-FU (5-fluorouracil), but is not limited to these as long as it can be used in combination with the composition of the present invention to achieve the objective of exhibiting a synergistic effect in anticancer activity.

[0080] In one example, the cancer may be selected from the group consisting of colorectal cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, 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, perianal cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, genital cancer, Hodgkin's disease, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal-pelvic cancer, bone cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumors, primary CNS lymphoma, myeloma, glioblastoma pleomorphic, and pituitary adenoma.

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

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

[0083] The compositions of the present invention can be used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, or sterile injection solutions, by conventional methods.

[0084] When formulation, the drug can be prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules, and such solid formulations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups, and can contain a variety of excipients in addition to commonly used diluents such as water and liquid paraffin, such as wetting agents, sweeteners, fragrances, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations, and suppositories.

[0085] Non-water-soluble solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, tween 61, cocoa butter, laurindi, and glycerol gelatin.

[0086] The compositions according to the present invention can be administered to individuals via a variety of routes. While any method of administration is conceivable, they can be administered, for example, orally, intravenously, intramuscularly, subcutaneously, or intraperitoneally by injection, but intravenous administration is most preferred.

[0087] The dosage of the pharmaceutical composition according to the present invention is selected considering the individual's age, weight, sex, physical condition, etc. 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 target, and preferably it is 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, no pharmaceutical activity may be shown, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

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

[0089] The compositions of the present invention are administered in a therapeutically effective or pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined depending on factors including the individual's species and severity, age, sex, drug activity, sensitivity to the drug, administration time, route of administration and elimination rate, duration of treatment, drugs used concurrently, and other factors well known in the field.

[0090] In one aspect, the present invention relates to the tumor prevention or therapeutic use of adenoviruses, which include a nucleic acid encoding the transferrin binding portion of the present invention in the coding region of the hypervariable region (HVR) of a hexon protein.

[0091] In one aspect, the present invention relates to a cancer treatment method comprising administering an adenovirus containing a nucleic acid encoding the transferrin binding portion of the present invention in the coding region of the hypervariable region (HVR) of a hexon protein to an individual suffering from cancer. [Modes for carrying out the invention]

[0092] The present invention will be described in more detail through the following embodiments. However, the following embodiments are merely for the purpose of embodying the content of the present invention and do not limit the present invention thereto.

[0093] Example 1. Production of an anti-cancer (anti-tumor) virus containing a transferrin-binding domain. We created antitumor adenoviruses containing transferrin-binding moieties by inserting nucleotide sequences (SEQ ID NO: 1 and SEQ ID NO: 3) encoding two transferrin-binding moieties, Tbp29aa (SEQ ID NO: 2) and Tbp55aa (SEQ ID NO: 4), respectively, from the transferrin-binding domains (TBDs) of transferrin-binding proteins (TBP) derived from Neisseria Meningitidis strains K454, S3131, and B16B6, into the HVR1 region of the hexon of an adenovirus vector. Specifically, referencing the amino acids of TbpA in Neisseria Meningitidis strains K454, S3131, and B16B6, two transferrin-binding moieties, Tbp29aa (SEQ ID NO: 2) and Tbp55aa (SEQ ID NO: 4), were designed in the transferrin-binding domain. Then, the nucleotide sequence encoding Tbp29aa (SEQ ID NO: 1) and the nucleotide 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 commissioned to be 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 amino acids 154 (gaa, E) and 155 (gac, D) of the hexon-encoding sequence) of the pAd1128 (OD260, adenoviral plasmid) plasmid (SEQ ID NO: 14), respectively, and transformed into E.Coli DH10B. Subsequently, clones in which the insertion of each transferrin binding site was confirmed by colony PCR underwent miniprep, and then the sequence analysis was sent to Macrogen to confirm the base sequence again.Subsequently, to produce adenovirus genomes (comid) used as materials for adenovirus production, four plasmids—pAd1128-tbp (containing each transferrin-binding subcoding sequence), pAd1127 (containing E and PIX sites), pAd1129 (containing E3 and Fiber)-GFP, and pAd1130 (containing E4)—were treated with restriction enzymes (Sfil), followed by ligation to produce cosmids (Figure 2). The cosmids were then packaged into lambda phages and transduced by infecting E. coli (OD101). Several clones were cultured in liquid, miniprep-treated, and then treated with restriction enzymes. The size of the DNA fragments was compared to the expected pattern to obtain clones similar to the expected pattern. Subsequently, the confirmed clones were miniprepped and treated with restriction enzymes to remove the origin of replication, antibiotic resistance genes, etc., to create a linear adenovirus genome. 7 µg of this genome was then transduced into the HE293 cell line to produce a seed virus, which was subsequently used in experiments. A schematic diagram of the seed virus exocophorus vector is shown in Figure 3. The vector structure of the adenovirus containing the transferrin-binding portion 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). In addition, the vector structure of the albumin-binding domain (ABD) used as a positive control group is shown in Figure 4, which is the 5 / 3 adenovirus CA10G-A (an adenovirus containing the sequence of SEQ ID NO: 12) contained in the HVR1 region.

[0094] Example 2. Confirmation of evasion ability of neutralizing antibodies against antitumor viruses. We investigated whether the adenovirus containing the transferrin-binding domain prepared in Example 1 could kill cancer cells by binding to transferrin in vivo, thereby avoiding antibody attack. Specifically, lung cancer cell line A549 was placed in a 96-well plate at a rate of 1 x 10⁶ per well. 4The cells were dispensed in 100 μl portions. The positive control group, CA10G-A (a type 5 / 3 adenovirus containing the albumin-binding portion in the HVR1 region: an adenovirus containing the sequence of SEQ ID NO: 12), was conjugated in a culture medium containing 10 mg / ml of albumin for 4 hours at 4°C before cell treatment to avoid antibody exposure. In the case of the present invention, HVR1-TBP29aa (a type 5 / 3 adenovirus containing the transferrin-binding portion of SEQ ID NO: 1 in the HVR1 region) and HVR1-(G4S1)3-TBP55aa (a type 5 / 3 adenovirus containing the transferrin-binding portion and linker of SEQ ID NO: 3 in the HVR1 region), the cells were conjugated in a culture medium containing 1 mg / ml of transferrin for 4 hours at 4°C before cell treatment. Subsequently, the cells were treated with the virus reacted with albumin or transferrin, along with a control virus (CA10G: 5 / 3 adenovirus) (infectious units, IFUs) at 0, 5, 10, 20, 50, and 100-fold dilutions relative to the cell number, respectively. The culture medium was then treated with adenovirus type 5 antibody (Abcam, ab6982) at a concentration of 0.1 ng / ml. 72 hours after viral treatment, the cell-killing ability of the virus was quantitatively evaluated using a Sigma-Aldrich cell division kit (XTT assay kit), and 96-well plates were stained with crystal violet reagent to visualize the cell-killing ability of the virus.

[0095] As a result, it was confirmed that in a 50 MOI (Multiplicity of Infection) scenario, the HVR1-Tbp(G4S1)3 55aa (Tbp-(G4S1)3-55aa) virus exhibited superior antibody evasion compared to the positive control group CA10G-A (Figures 7 and 8).

[0096] Example 3. Production of a virus containing a transferrin-binding domain at the HVR2 position. Using the method described in Example 1, the nucleotide sequence encoding Tbp29aa (SEQ ID NO: 2) (SEQ ID NO: 1) and the nucleotide 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 inserted into the HVR2 region of the hexon of an adenovirus vector, respectively, to produce adenoviruses (HVR2-Tbp29aa and HVR2-(G4S1)3-Tbp55aa) containing transferrin binding sites.

[0097] Example 4. Confirmation of neutralizing antibody evasion ability of antitumor viruses containing a transferrin-binding domain at the HVR2 position. The neutralizing antibody evasion ability of adenoviruses HVR2-Tbp29aa and HVR2-(G4S1)3-Tbp55aa, which contain a transferrin binding site in the HVR2 region of the hexone of the adenovirus vector prepared in Example 3, was compared with that of HVR1-(G4S1)3-Tbp55aa. Specifically, lung cancer cell line A549 was placed in a 96-well plate at a rate of 1 x 10⁶ per well. 4 The cells were dispensed in 100 μl portions. CA10GT (HVR1-(G4S1)3-Tbp55aa), HVR2-Tbp29aa (a 5 / 3 adenovirus containing the transferrin binding portion of SEQ ID NO: 1 in the HVR2 region), and HVR2-(G4S1)3-Tbp55aa (a 5 / 3 adenovirus containing the transferrin binding portion of SEQ ID NO: 3 and a linker in the HVR2 region) were bound in a culture medium containing 2 mg / ml of transferrin at 4°C for 6 hours. The dispensed cells were then treated with these solutions at cell number ratios of 0, 5, 10, 20, 50, and 100 times, respectively (CA10G: 3.38 x 10 10 ifu / ml;CA10GT:8.03x10 9 ifu / ml;HVR2-29aa:6.94x10 10 ifu / ml; and HVR2-(G4S1)3-55aa:1.29x10 10Cells were treated with adenovirus type 5 antibody (Abcam, ab6982) at a concentration of 0.1 ng / ml (ifu / ml). 72 hours after viral treatment, the ability of the virus to kill cells was quantitatively evaluated using a Sigma-Aldrich cell division kit (XTT assay kit).

[0098] As a result, it was shown that only CA10GT, in which the transferrin binding portion was inserted into the HVR1 region of the hexon of the adenovirus vector, was able to bind to transferrin in vivo, thereby avoiding antibody attack and killing cancer cells. Viruses in which the transferrin binding portion was inserted into the HVR2 region of the hexon of the adenovirus vector showed almost no antibody evasion ability (Figure 9).

Claims

1. An adenovirus having a nucleic acid encoding a transferrin-binding moiety in the coding region of the hypervariable region (HVR) of a hexon protein, wherein the hypervariable region of the hexon protein is HVR1.

2. The adenovirus according to claim 1, comprising the nucleic acid sequence of sequence number 1, 3, 8, or 10.

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

4. The adenovirus according to claim 1, wherein the N-terminus, C-terminus, or N-terminus and C-terminus of the transferrin binding site are linked to a hexone protein via a linker.

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

5.

6. The adenovirus according to claim 1, wherein the hexone protein HVR1 comprises the amino acid sequence of SEQ ID NO:

7.

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

8. The adenovirus is a human adenovirus, as described in claim 1.

9. The adenovirus according to claim 8, wherein the human adenovirus is selected from the group consisting of human adenovirus serotypes 1 to 57.

10. The adenovirus according to claim 8, wherein the human adenovirus is human adenovirus serotype 5.

11. The adenovirus according to claim 8, wherein the human adenovirus is human adenovirus serotype 5 / 3.

12. The adenovirus according to claim 1, further comprising a tissue-specific promoter or a tumor-specific promoter.

13. The adenovirus according to claim 12, wherein the promoter is operably linked to an endogenous gene of the adenovirus.

14. The adenovirus according to claim 12, wherein the promoter is selected from the group consisting of the E2F promoter, the telomerase hTERT promoter, the tyrosinase promoter, the prostate-specific antigen promoter, the alpha-fetoprotein promoter, and the COX-2 promoter.

15. The endogenous gene of adenovirus has the structure 5'-ITR-C1-C2-C3-C4-C5 3-'ITR; The C1 includes E1A, E1B, or E1A-E1B; The aforementioned C2 includes E2B-L1-L2-L3-E2A-L4; The C3 either does not include E3 or includes E3; The C4 includes L5; and The adenovirus according to claim 13, wherein C5 either does not contain E4 or contains E4.

16. The adenovirus according to claim 15, further comprising an IRES sequence between E1A and E1B.

17. The adenovirus according to claim 15, wherein the promoter is operably linked to the endogenous genes E1A and E1B of the adenovirus.

18. The adenovirus according to claim 1, further comprising an expression cassette for expressing an exogenous gene.

19. The adenovirus according to claim 18, wherein the expression cassette is located at the E3 site of the endogenous gene of the adenovirus.

20. The adenovirus according to claim 1, wherein the adenovirus is an antitumor adenovirus.

21. The adenovirus according to claim 1, which is an oncolytic adenovirus.

22. The adenovirus according to claim 1, further comprising capsid modification for enhancing the infectivity of the adenovirus or for targeting it as a receptor present in tumor cells.

23. The adenovirus according to claim 22, wherein the capsid modification involves inserting an RGD motif into the H1 loop of the adenovirus fibrous protein.

24. The adenovirus according to claim 22, wherein the capsid modification replaces a fibrillary gene or a portion thereof with a homologous portion derived from another serotype of adenovirus to form a chimeric adenovirus.

25. The adenovirus according to claim 24, comprising a capsid in which a fibril gene derived from serotype 3 adenovirus or a portion thereof is substituted, and the portion excluding the fibril gene comprises a gene derived from serotype 5 adenovirus.

26. The adenovirus according to claim 1, comprising one or more non-adenoviral genes.

27. The adenovirus according to claim 26, wherein the non-adenoviral gene is a gene used in cancer gene therapy.

28. The adenovirus according to claim 27, 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 immunostimulatory genes.

29. A pharmaceutical composition for cancer treatment comprising the adenovirus described in claim 1 as an active ingredient.

30. A pharmaceutical composition for cancer treatment according to claim 29, which is administered systemically.

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