TW200533752A - Pan cancer oncolytic vectors and methods of use thereof - Google Patents

Abstract

Replication-competent adenoviral vectors which selectively replicate in cancer cells are provided. The replication-competent viral vectors comprise an E2F responsive promoter and/or a telomerase promoter operatively linked to an adenoviral coding region. The replication-competent adenoviral vectors effectively replicate in a variety of types of cancer cells and find broad utility in the treatment of cancer.

Description

-200533752 IX. Description of the Invention: This application claims the benefit of US Application No. 60 / 556,549 (the application date is March 25, titled "Pan Cancer Oncolytic Carrier and Method of Use,"). This application The case is incorporated herein by reference. [Technical Field to which the Invention belongs] The present invention relates to a replication-capable viral vector construct and its use in the treatment of cancer. ^ [Prior art] Adenoviruses are some of the most innovative and most innovative The foundation of potential tools to fight disease. One of these tools is gene therapy, which provides an exogenous nuclear acid sequence to cells. This strategy can be used not only for cancer treatment, but also for other diseases, including Cystic fibrosis, anemia, hemophilia, diabetes, 'Huntington's disease, AIDS, abnormally high serum cholesterol concentration, specific immune deficiency, and many forms of cancer. Gene therapy generally relies on a delivery vector, such as a viral vector to Send the exogenous sequence to the cell. Recombinant adenovirus has been shown to have some therapeutic effect in combating these diseases. For a review, see Jin et al. (1996) Financial System Γ0 ~ 12: 519-527 and Smith et al. (1996) exhaustion therapy 3: 496-502. Adenoviruses that can be selectively replicated in target cells are being developed as therapeutic agents for treating cancer.

In another proposal that can be applied to the treatment of cancer, a specific attenuated replication-competent viral vector has been developed that preferentially destroys these cells by its selective replication in cancer cells. Various cell-specific replication-competent adenoviral architectures that preferentially replicate (and therefore destroy) cells of a particular type are described in, for example, WO 95/19434, WO • 200533752 96/1 7053, WO 98/39464 ' WO 98/39465 'WO 98/39467, WO 98/39466, WO 99/06576, WO 99/25860, WO 00/1 5820, WO 00/46355, WO 02/067861, WO 02/06862, U.S. Patent Application Publication Case No. US20010053352 and US Patent Nos. 5,698,443, 5,871,726, 5,998,205, and 6,432,700. Replicating adenovirus vectors have been designed to selectively replicate in tumor cells.

Although replication-capable adenoviruses can achieve selective targeting and amplification for the treatment of local and disseminated cancers, there is still a need to improve the adenoviral vector itself and its use methods depending on the type of cancer being treated. [Summary of the Invention] [Summary of the Invention] The present invention provides a sequence comprising: a left ITR, an adenovirus packaging signal, an E2F reactive promoter or a TERT promoter operatively linked to an E1a coding region, and an operative Recombinant adenoviral vector linked to an E2F-reactive promoter or TERT promoter of the e 1 b coding region, and the adenoviral backbone of the right ITR. In another aspect, the transcriptional regulatory element operably linked to the E 1 b coding region is an E2F-responsive promoter such as the human E2F-1 promoter comprising SEQ ID NO: 1 or a TERT promoter such as human teRT A promoter, such as a human TERT promoter comprising SEQ ID NO: 2 or SEQ ID NO. 3. In another aspect, the recombinant viral vector comprises a mutation located in the Elb 19k coding region. In a further aspect, the transcription 6-200533752 operably linked to the E1 b coding region is an IRES, TERT promoter such as a human phase π j such as a human cheek TERT promoter, such as a human emblem containing SEQIDN0: 2 or SEQID A D promoter, or an E2F-reactive promoter, for example, it comprises a human promoter called ιΝΝΟ ·]. The recombinant adenoviral vector of the present invention may also include a region located in the E3 coding region, such as E3-6.7 KDa, gpl9 KDa,! !! 6 coffee (ADp), 104%

(㈣ ♦ 14.5 KDa (R, and E3 · 147, such as mutations or deletions, or deletions in the Elb gene, such as the deletion in the gene encoding the i9kD protein, such as the deletion represented by SEQ ID NO: 12 The recombinant adenoviral vector may further comprise a transgene. Exemplary recombinant adenoviral vectors comprise a nucleotide sequence as represented by SEQ id NO: 4 or SEQ ID no: 5, SEQIDN0: 6 or SEQIDN0: 7. The present invention further provides The medicinal composition of the adenovirus vector of the present invention and its use for treating host organisms with neoplastic conditions, such as lung cancer, breast cancer, prostate cancer, or rectal cancer. The present invention also provides a method for selective cell lysis of cancer cells It comprises contacting a cell population with an effective amount of an adenoviral vector of the invention (as described above) under conditions in which the adenovirus vector infects the cell population, resulting in selective lysis of cancer cells in the cell population. [Embodiment] [Detailed Description of the Invention] Unless otherwise indicated, all terms used herein have the same definitions as those skilled in the art recognize, and the present invention The implementation will use conventional microbiology technology and recombinant DNA technology. 200553752 The public documents and other documents cited in this specification include all patents, patent applications, publications (including published patent applications), and The lean library entry number is used to explain the background of the present invention, and -111 'is not used for other details such as implementation. The public documents and other documents cited in this specification include all patent cases, patent applications, publications f (Including published patent applications), and database access codes are incorporated herein. By way of description. In describing the Nichiji of the present invention, the following terms are used and are intended to be defined as follows. "Condition Pfu" stands for plaque formation The terms "virus", "viral particle", "body", "viral vector particle", and "viral particle", are used interchangeably and are broadly understood to mean, for example, that the viral vector of the present invention is transduced Infectious granules formed in a suitable cell or cell line for producing semi-active particles. Viral particles according to the present invention can be used in grandfathers "For the purpose of transplanting into cells in the body or in the tongue. For the present invention ..." refers to an adenovirus, including when the D & A vector of the present invention is encapsulated in an adenovirus Recombinant adenovirus that is formed in the body. "Adenovirus vector" or "Adenoviral adenovirus 1 King war body (can be used interchangeably). As used herein, it refers to a replication-capable, present in cancer cells. Polynucleotide constructs that function well and contain tissue-specific transcriptional regulatory sequences that are linked to the adenoviral gene. In a certain form, the main body of the present invention is: Adenopathies such as cytokine gene sequences. The examples of this adenoviral adenoviral vector include (, kernel is not limited to) DNA, DNA encapsulated 8-200533752 outside adenovirus, and packaged in another virus or virus-like form (Such as herpes simplex virus, and AAV), adenovirus DNA encapsulated in microlipids, adenovirus DNa complexed with polylysine, complexed with synthetic polycation molecules, and transferred to iron Co-protein, or compound with compounds such as PEG In immunologically ,, ,, covering antigenic and / or increased half-life, or conjugated with a non-viral protein of adenovirus DNA. Accordingly, the terms "adenovirus vector" or "adenoviral vector" as used herein include adenovirus or

Adenovirus particles. The term adenovirus, and "adenovirus particles," as used herein, includes any and all diseases that can be classified as adenoviruses, including any adenovirus that infects humans or animals, including all herds, subgroups, and serotypes Therefore, as used herein, 'adeno # A "adenovirus particle" means the virus itself or its derivatives and includes all serotypes and subgroups, as well as natural and recombinant forms, unless otherwise stated (for example, see Male 矣 1 \). In a specific aspect, such glandular infection does not infect human cells. And _ joint member adenopathy may not be wild-type or may be known in various skills or as disclosed herein. m grains "Hai Jinjie" modified way. These modifiers modify the adenoviral gene group of the virus, which is packaged in the particles, and the sensational virus. The lack of soil in the field includes the known pinching effects in this technique, such as the lack of in Yuyi or E4, lb 'E2a' E2b 'E3'-乂 t4 ·, ′ flat code area. By system is meant the second preferred particle used in the adenovirus-containing cells of the present invention with respect to the present invention. In the adenoviral vector and x of the present invention made by Jg ^, the adenoviral vector and / or particle 9-200533752 are selectively in tumor cells and / or abnormally proliferating tissues, such as solid In tumors and other tumors. These include the viruses disclosed in U.S. Patent Nos. 5,677,178, 5,698,443, 5,871,726, 5,801, 〇29, 5,998,205, and 6,432,7GG (their disclosures are hereby incorporated by reference in their entirety). Such viruses can be referred to as "oncolytic viruses, or" oncolytic vectors, "and can be considered lines," cytolytic, "or" cytopathogenic, and cause "selective cells of standard cells" Dissolve. "

Pan-cancer means that the replication-capable adenoviral vector of the present invention generally replicates selectively in tumor cells and / or abnormally proliferating tissues and is not necessarily limited to specific types of cancer for replication. ,, A >,-The definition of the pulse points 笤 r / r 路 疋 is used. This means that the sentence is 5 w, .. _, + to a nuclear-derived element that can be derived from a virus and can be packaged into a virus vector particle. Adenoviral vectors and / or plasmids can be used to transfer DNA, RNA, Glycosides, ☆, or, other nuclear, and long-lived derivatives in a test tube or in vivo to the head of the cell. Many viral vector forms, including adenopathies, are known in the art. The terms "carrier", "taken" and "synthetic carbamate", "',", "glycolic acid carrier,", "polynucleotide carrier xylic acid carrier construct" & "carrier construct," as used herein = For the purpose of use, it means any construct used for gene transfection, as understood by those skilled in the art. 乂 The term "for replication must be used in the evening I m, and the gene thinks its transcription The action vector is a nucleotide sequence that is required for target cells to be incorporated into the target. For example, in the adenoviral vector of Fang and Ben Sheming, the genes necessary for making 仏, 叫, E2a, H'm can be selected from the group consisting of E4 genes. 10 .200533752

As used herein, "packaging cell" is a cell capable of packaging an adenovirus genome or a modified genome to make a virus. Pots can provide missing gene products or their equivalents. Therefore, 'packaging cells can provide The complementary functions of the missing genes in the viral genome and the ability to package adenoviral genomes into adenoviral particles. The production of such particles requires the replication of the genome and the production of the proteins needed to assemble infectious viruses. The particles also Specific proteins may be required for virions to mature. Such proteins may be provided by a carrier or by packaging cells. HeLa-S3 means available from the American Type Culture Collection (ATCC, Manassas, vA) and A human cervical tumor-derived cell line named ATCC number ccl_22. HeLa_S3 is a pure derivative of the female He; u cell line (ATCC CCL-2). HeLa_S3 was developed by TT · Puck et al. 103: 273_284 (1956)). Regarding adenoviral vectors, the term "5," can be used interchangeably with "upstream," and means reverse weight at the left end. Direction of the sequence (the ITR) of. With regard to adenoviral vectors, the term "3," can be used interchangeably with "downstream," and means in the direction of the right ITR. The term "nucleic acid" means deoxyribonucleotides or ribonucleosides in single or double stranded form Acids and their polymers ("Polynucleotides,"). Unless specifically limited, the term 'nucleic acid' encompasses nucleic acids of known analogs of natural nucleotides which contain similar binding properties to reference nucleic acids and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, special nucleic acid molecules / polyglycolic acids are also implied to include variants that have been conservatively modified (eg, degenerate codon access 11 200533752 generation) with complementary sequences and explicitly designated sequences. In particular, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue ( Batzer et al. Nucleic Acid Research 19: 5081 (1991); Otsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol.

Cell. Probes 8: 91-98 (1994)). Nucleotides are based on their bases by

The column standard abbreviations represent: adenine, cytosine (c), thymine (τ), and guanine (G). A nucleotide sequence is "operably linked" when it has a functional relationship with another nucleotide sequence ". For example, a promoter or regulatory DNA sequence is" operably linked "to an encoding RNA and / or protein The DNA sequence means Ru. This sequence is operably linked, or is in a condition that causes the promoter or withered DNA sequence to affect the expression of the coding or structural DNA sequence. The operably linked sequences are typically (but not necessarily) contiguous.

^ Coding sequence and "coding region" means transcribed into RNA

Nucleic acid sequence of mRNA rRNA, tRNA, snRNA, sense RNA or antisense RNA. In a specific aspect, the RNA is then translated into cells to produce a protein. The term "ORF" means an open reading frame. The term "gene" means a region located in the genome and (in addition to the aforementioned coding sequence, is responsible for regulating expression, that is, a defined region of the transcription and translation material (mainly regulatory) of the encoded protein. Genes can also contain other 5 'And 3' non-translated sequences and termination sequences. Depending on the source of the gene, 12 200533752 determines that other elements that may exist are, for example, introns. The terms "heterologous" and "exogenous" are used herein in terms such as When referring to nucleic acid molecules such as promoters and gene coding sequences, they refer to sequences originating from sources other than the diseased host cell or, if they are from the same source, they are also modified to appear in their original form. Therefore, heterogeneity in a virus or cell Genes that include the specific virus or cell that are endogenous but have been modified by the d. The term also includes an unnaturally large copy of the natural nucleic acid sequence. Therefore, the term 咅, T 彳 9 is foreign or heterologous to a virus or cell, or to a virus or cell, and 田 cells are 冋 -derived but located in the host virus or cell genome. The nucleic acid fragments are unknown by location. Complementary techniques, and "Stones: ί," refers to a two-nucleotide sequence that contains complementary bases that can be aligned in the anti-parallel nucleotide sequence, Antiparallel nucleotide sequences that form% hydrogen bonds between residues that are paired with each other. The term "natural" means a gene that exists in a wild-type virus or cell. I U Ί The terms "naturally occurring," and "prebiotic" Describe proteins that can be found in nature and are produced by humans and individuals. For example, there are proteins in natural organisms that can be isolated from natural sources and that have not been artificially altered — '%% to diurnal changes in the leaves (including viruses). Or the nucleotide sequence is naturally occurring. The term "recombinant" Xiangu \; when used herein in connection with nucleic acid molecules, refers to the use of recombinant DNA technology to divide nucleic acids, A, and knives together into progeny nucleic acid molecules

Of, and a form. As used herein "P" and in relation to disease, cells and organisms, the term "recombinant," transformed "B" > transgenic and transgenic "means the host in which the heterologous nucleic acid molecule has been introduced. 扃No, cells and organisms. The nucleic acid molecule can be stably integrated into the host's genome, or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such extrachromosomal molecules can replicate on their own. Recombinant viruses, cells, and organisms are understood to include not only the end products of the transformation process, but also their recombinant progeny. A "non-transformed," "non-transgenic," or "non-recombinant" host means a wild-type virus, cell or organism that does not contain a heterologous nucleic acid molecule.

"Regulatory elements" are nucleotide sequences that are involved in regulating the expression of a nucleic acid molecule. Withering elements include promoters, synergists, and termination signals. They also typically include sequences required for moderate translation of the nucleic acid sequence. The term "priming "Proton" means an untranslated DNA sequence, usually located upstream of the coding region, which contains the binding site of RNA polymerase π and initiates DNA transcription. The promoter region may also include other elements that act as regulators of gene expression The term "minimum promoter" means a promoter element that is incompetent or has a large amount of reduced motility activity in the absence of upstream activation, especially a TATA element. The term 'promoter', in the definition of the present invention, may be any genetic element, for example, a promoter which increases the transcription of a coding sequence operably linked to a promoter compared to a promoter which is operably linked to the coding sequence. The degree of transcriptional activation that is affected by itself is high, and the sequence of Igami I, that is, it increases the transcription induced by this promoter. Taking a human face termination signal sequence, 'in the definition of the present invention may be any genetic element that causes termination of transcription, such as a polymerized signal sequence. Poly = "recognition region" whose sequence is the RNA transcription product followed by polyadenylation (% AATAAA Q NO. 13) for endonuclease cleavage 14 200533752. The polyadenylation signal sequence provides a "poly A site," which is a site on the RNA transcript that can be added to the gland voice residues by post-translational polyadenylation. The polyadenylation signal sequence is useful for true The transcription units in nuclear cells and eukaryotic viruses are useful insulating sequences. Generally, the polyadenylation signal sequence includes two recognition elements flanking the cleavage and polyadenylation sites, and the nucleus ~ poly signal (E.g., WO 2/067861 and WO 2 / 0686627® 1). Representatively, the almost unchanged AAUAAA (SEQ ID NO: 14) hexamer system is located in the u or Gu residue rich 20 to 5G upstream of the more variable element. These two cleavages are usually mediated at the 3 'end of residue A and (in the test tube) are mediated by large' multi-component protein complexes. 。 Suitable for correcting the signal sequence and selecting the amount of strength of the polyadenylation signal sequence. Potential rabbits take 晌 几, 因为 because the completion of the adenylation process and poly (A) position.

Dot intensity correlation (Chao et al.'S Molecular and Cell Biology, 1999, 19: 5588-5600). For example, the strong_late poly-site is weaker: the poly-site is faster to lyse. If the special vector structure needs to substantially reduce non-specific transcription, then those skilled in the art will choose a strong polymerized material from Kao Dan. Any polyglandular bitterness =: sequences can be used in the present invention Purpose. However, in the preferred embodiment of the present invention, the 'stop signal sequence is an SV40 late polyadenylation signal sequence or an SV40 early polyadenylation signal sequence. In a specific aspect of the present invention, the 'stop signal sequence' is isolated from its gene source and inserted into the virus 2 at a suitable position upstream of the E2F or TERT promoter. The term "expression" means the transcription and / or translation of an endogenous gene, transgene or coding region within a knife. In the case of antisense constructs, expression 15 200533752 can refer to the transcription of only antisense DNA.

The term 'upregulated' is used herein to mean that a larger amount of RNA corresponding to a particular gene can be detected in a target cell compared to another cell. For example, if a tumor cell is compared to a non-tumor Cells produce more telomerase RNA, then the telomerase of the tumor cell is subject to increased regulation. When the amount of specific RNA in the target cell (such as a tumor cell) is at least 3 times higher than that of another 2 cell (non-tumor cell) In another specific aspect, the amount of specific RNA is increased at least 5 times. In another specific aspect, the amount of specific RNA is increased at least IO times. Those skilled in the art know how to measure the RNA concentration corresponding to a specific RNA sequence (for example, Northern blot analysis).

Use the date; internal ribosome entry site, or "IRES", means the start codon (such as ATG) that promotes the direct internal ribosome to enter the cistron (protein coding region), thereby guiding the gene The element that performs the transformation # that has nothing to do with the cap structure. Jackson rj, Harwell Mτ, Kamischi VIII (199〇) Trends Biochem Sci 15 (12): 477_83; and Jackson Han] and Kamiski, A (1995) RNA 1 (10): 985-1000. The present invention includes the use of any ires element that facilitates the initiation of a direct internal ribosome into the cistron. Under the translational control of 1RES "is used herein to mean Translation is associated with IRES and is performed in a manner independent of the cap structure. Examples of "IRES" known in the art include (but are not limited to) IRES available from microRNA viruses (Jackson et al., 1990, Trends Biochem Sci 15 (12): 4 7 7-483) and can IRES from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (Bip), vascular endothelial cell growth factor 16 200533752 (VEGP) (Schutz et al. (1998) MoI · Cell BlO1 18 ( 11): 6178 to 6 1 90), fibroblast growth factor 2, and insulin-like growth factor,

Translation initiation factor eIF4G, yeast transcription factor TFIID and hap4. IRES has been reported in different viruses, such as heart virus, rhinovirus, foot-and-mouth disease virus, HCV, and Friend mouse leukemia virus (FrMLV: ^ Moonie gas leukemia virus (MoMLV). As used herein, "IRES" includes ires A functional variant form of the sequence, as long as the mutation can promote the direct internal ribose moon beans to enter the initial bifurcation of cistron. In a preferred embodiment, ires is a source of ruminants. In other embodiments, IRES is of viral or protozoal origin. In the exemplary embodiment described herein, IRES is derived from encephalomyocarditis virus (ECMV) (commercially available from Novovogen, Duke et al. (1992) j irol 66 (3) 1 602-1 609). In another exemplary embodiment described herein, IRES is derived from VEGF. An example of an IRES sequence is described in US Patent No. 6,692,736. "Self-processing cleavage site" or "self-processing cleavage sequence" as used herein refers to a nucleotide or amino acid sequence that undergoes rapid intramolecular (cis) formation of a polypeptide containing a self-processing cleavage site during translation. Cleavage, while $ causes discontinuity A list of mature protein or peptide products. Such self-processing cleavage sites can also be referred to as post-translational or co-translational processing cleavage: for example, 2A site, sequence or functional domain. 2A site, sequence or functional domain The effect of translation presented is to change the activity of the ribosome to promote the hydrolysis of 鐽 like 鐽, so that the synthesis of discontinuous downstream translation products can proceed 'to release polymorphisms from the translation complex (Donnelly, 2001). Or the '2A site, sequence or domain is "self-proteolytic" or lysed by generating a primary cleavage product by cleaving its own end 17 200533752 cis (Fühler; palmenberg, Ann Rev 44: 603- 623 (1990)) ο The term "E2F promoter," as used herein, means the natural coffee promoter and its functional fragments, mutations, and derivatives. The coffee promoter need not be a full-length or wild-type promoter. Xi Yubai Du Sibai and those skilled in the art know how to derive fragments from the E2ρ promoter and test their desired selectivity. The EM promoter fragment of the present invention has selective promoter activity against tumor cells. The term "tumor-selective promoter activity" as used herein means that the promoter activity of the promoter fragment of the present invention is more favorable in tumor cells than in non-tumor cells. High. The term "telomerase promoter, 'or" TERT promoter, "is used herein to refer to the natural TERT promoter and its functional fragments, mutations, and derivatives. The DERT promoter does not require rabbits to enter the sense + meaning To be a king or wild type promoter. Those skilled in the art know how to derive fragments from the TERT promoter and test their desired selectivity. The DERT ERT promoter fragment of the present invention has promoter activity that is selective for tumor cells, that is, it drives a tumor to selectively express an operably linked coding sequence. In a specific aspect, the caffeine promoter of the present invention is the mammalian TERT Kai. Λ 0 cut first 1LK1 promoter. In another specific aspect, the mammalian TERT promoter is a human promoter. In a specific aspect, the E2F promoter according to the present invention has full length complementarity with the SEQ iD NO: 1 nucleotide sequence under severe conditions. In another specific aspect, Yin ’s TERT promoter according to the present invention has a full length that can hybridize with the nucleotide sequence of SEQ ID 2 under the severe conditions. The phrase "Hybrid to" means that when a nucleotide sequence is present—species complex 2: (eg, in a total cell) in DNA 4 RNA, a molecule binds to a specific pyrene sequence, forms a double helix, under severe conditions, or hybrid."

^, J JL LH, Babe-L two-mouth probe probe nucleic acid complementary to the target nucleic acid and the hybrid hybrid medium, including the reduction of the hybrid medium and the stringency to achieve the desired target nucleotide detection Preface 歹 J and Gu Wan's secondary mismatch. on. Nucleic acid hybrids, such as SOUthern and N (mh), may have hybrid conditions, and "stringent rinsing conditions" are sequences that differ under different environmental parameters. Longer sequences are ：::: Acid ^ Laboratory Technology in Biology of Knife Knife-Hybrid Heterogeneity No. 1 Qiu Yan 9 Li "~ Overview of the principle of 1-Hybrid Needle and Analysis Strategy of Nucleic Acid Probes", 4 ^ Mixing and rinsing conditions #in is more specific than the nucleotide sequence. It is a choice of ㈣ ~ B n materials. T w is lower than the ionic strength and pH melting point (but m) at the pH value of about 5t: to 2〇t. (Compared with enthalpy) The probe will be κ under severe conditions. Typically, the sequence is hybridized with high stringency. The dry sequence is hybridized with no relation to other

Tm is (at the specified ionic strength and the temperature at which it is hybridized with a perfectly paired probe. The 2 ^^ Geba sequence is equal to the τ of a specific probe. —1§ Tian's optional non-rigid conditions are complementary nucleic acids in On the filter membrane, Sq th, wo complementary nuclear picric acid hybrids, or Northern, break through the severe heterozygous conditions of 蚯 φ, 隹 仃, and heterozygous ^^ 丨 & 〇p ^ An example is 50% ammonium with 1 milligram of liver and hybridization at 2 C overnight.

An example of severe rinsing conditions for workers is i5M 19 200533752

NaC1 is performed at 72t for about 15 minutes ... An example of a high and severe rinsing condition is rinsing under the machine with 0.2XSSC; first 15 minutes (see Sam Bukebe as described above for the description of SSC buffer). Often, low-severity rinsing is performed to remove background probe signals before high-severity rinsing. One item ^ in (for example) contains more than! An example of a moderately stringent wash of a double helix of 00 nucleotides is lxSSC for 15 minutes at 45t. An example of a low degree of harsh rinsing for ° (two such) double helixes with more than 100 nucleotides is 4-6XSSC at 40. (: For 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), harsh conditions typically include less than about ^

Na ion salt concentration, typically about 0.001 to 10M Na ion concentration * (or other salts) at pH 7.0 · to 8.3, and the temperature is typically to about 2 (TC. Can also be added Destabilizing agents, such as amidine, are used to meet severe conditions. Generally, detection is detected when the signal-to-noise ratio is 2 times (or higher) than that observed in a specific hybrid analysis with an unrelated probe. Specific heterozygosity. The term "homology," as used herein with reference to a nucleic acid molecule, refers to a nucleotide sequence that is naturally associated with a host virus or cell. The same or a percentage "identity" with respect to two or more In the case of a nucleotide or protein sequence, two or more sequences or subsequences are measured using a sequence alignment algorithm (such as the Smith-Watman method 1) described herein or by visual inspection. Perform the largest corresponding alignment and the same amino acid residues or nucleic acids that are the same or have a specific percentage when the two columns are in sequence. / 丨 Between sequence alignments two, M ,, one ~ ... Ten and two Hunmai sequences make the test Sequence alignment. When using sequence alignment calculus It will test 20 200533752 H win test = list the people computer, # f can specify the secondary sequence coordinates and set the sequence calculation program parameters. Then the sequence comparison algorithm calculates the test sequence relative to the reference sequence based on the set program parameters The percent sequence identity can be obtained, for example, by the local homology algorithm of Smith & Watman, Wv. But M Man 482 (1981), and by Nedman & Wang Xu, j. 443 (1970) homology alignment algorithm, using the similar 1 ± k cable method of P. Mori & Lipman, Pr0Cm "CW · & Z. · _ 85: 2444 (1988) 'Using such algorithms (gap, BESTFI, Ding, FASTA,;, FAY Ding A in the Wisconsin Genetics Software Suite, Genetics Computer Group, 575 Scientific Dr., Madison, Wis) computer execution, Yashur et al., J. Mo /. 5 / 〇 /. 2 15: 403-410 (1990) through national biotechnology resources. Fl China ~ (http · // www.nebi.nlm.nih.gov / ) BLAST algorithm performed on publicly obtained carcasses, or by visual inspection (generally see Osbel et al., Supra) Optimal alignment of sequences. For the purpose of the present invention, the optimal alignment of aligned sequences is performed by the local homology algorithm of Smith & Watman MW / 7 · 2: 482 (1 98 1). Alignment. With regard to the present invention, the term "isolated" means a nucleic acid molecule, polymorph, virus, or cell that exists artificially separate from its natural environment and is therefore not a natural product. An isolated nucleic acid or polypeptide can be purified The form exists or may exist in a non-native environment such as, for example, a recombinant host cell. An isolated virus or cell may exist in a purified form (eg, in a cell culture) or may exist in a non-natural environment such as, for example, a recombinant plastic or heterologous organism. 21 200533752 "Normal cell state" or "normal physiological condition" is _ Xihu exists under normal physiological conditions and is non-positive or divides in a regulated manner, that is, the state of cells under normal physiological conditions. Abnormal cell state It is about the same kind of cells, which is in a non-positive division / regulated division state and under normal physiological conditions. The same kind of cells are defined. 'Used in this article' cancer, cancer cells ',' tumorous cells ,,,, "Tumor formation", "tumor", and "tumor cell" (can be used interchangeably) 咅 refers to a relatively spontaneous growth, so that it appears to be characterized by: abnormal growth phenotype or abnormal cell state regulated by acellular proliferation cell. Tumor cells may be _ ^ ^ already "this heart is healthy or benign. It is understood that cancer cells are considered to have an abnormal cellular state. "The buckle basically consists of ..." when used herein to quote a specific nucleotide sequence means that the specific sequence can be at 5, or 3, with either or both ends

The additional residues, such as 忒 and 绞, do not substantially affect the basic and novel characteristics of the cited sequences. Oral bioactive agents, "drug candidates," "combined or grammatically equivalent" are used herein to describe any / to be tested for / whether to directly or indirectly alter the cancer phenotype or cancer sequence (including Nucleic acid ^ _ Molecules of biologically active agents, such as protein shellfish, storms, small organic tillers ^ knives, sugars, polynucleotides, etc.

The present invention provides novel adenoviral vectors, examples of which are described in US20010053352, WO96 / 17〇53, and WO99 / 2586〇. In particular, it is revealed that the expression of the adenovirus gene (required for replication) is an oncolytic adenovirus vector that is regulated by a regulatory region that is selectively activated by trans in cancer cells. The adenoviral vector of the present invention is therefore regarded as a "pan-cancer vector. According to the present invention, a pan-cancer vector contains a cancer-selective regulatory region, such as the E2F or TERT promoter described in detail herein. The adenoviral vector of the present invention is borrowed Prepared by standard techniques known to those skilled in the art. The adenoviral vector of the present invention is transferred into packaging cells to produce virus particles using standard techniques known to those skilled in the art. The packaging cells typically subsidize any wild Functions missing from the adenovirus genome. The production of such particles requires the vector to be replicated and the proteins required for assembling infectious viruses to be produced. The packaging cell line is cultured under conditions that allow the production of the desired viral vector particles Diseased particles are recovered by standard techniques. Preferred packaging cells are those that have been designed to limit the occurrence of homologous recombination that may cause wild-type adenovirus particles. Examples of cells that can be used to produce adenovirus particles include human embryonic kidneys Cell line 293 (Graham et al., Yi G Luan⑽ ·· 36: 59_72 (1977)), human embryo becomes retina Cell line PER.C6 (U.S. Pat. Nos. 5,994,128 and 6,033,900); Fallas et al., Gen. Gui 9: 1909-1917 (1998)) and human cervical tumors The cell line HeLa-S3 (US Patent Application No. 60 / 463,143; ATCC # CCL-2.2). The invention anticipates that all adenovirus serotypes can be used to construct oncolytic adenoviral vectors and adenovirus particles according to the invention. Example腺 " Aden, viral nucleic acid backbone is derived from adenovirus serotypes 2 (Ad2), 5 (Ad5) 4 35 (Ad35), although 23 200533752 of course, other serotypes can be used to broaden, virus, and include any adenovirus ::: Body: The disease collection station "TCC, Manassas," which can be used according to the present invention; can be obtained from the American type culture type, and the present invention includes any adenovirus sera that can be scoring / serving, including these columns Shown in the table! Other serotypes originating from Yu Renhe include: Xiha can be used according to the present invention > It can be used by humans or non-humans to remove the master of the adenovirus used by Zhinai. For example, 'Adenoviruses can belong to subgroups, such as serotypes, 2, I8, " grapevine subgroup A (eg, M ^ 〇 丨), subgroup B (eg, serotypes 3, 7, u, 14, 16, m, 35), subgroup c (e.g., blood subgroup D (e.g., serotype 8 5, 6), "" 10 13, 15, ", 19, 20, 22_30, Γ,] 36-39, 仏 47) , Subgroup E (serotype 4), subgroup F (blood 4 40, 41), or any other adenovirus serotype. Table 1 Examples of major human and animal adenoviruses, including representative viruses corresponding to individual classifications U.S. Type Culture Collection Catalog Number Avian Poison Type 4 Poison 5 ^ Poultry Adenovirus Type 7 Poultry Month! Poison Type 8 Poultry Adenovirus Type 9 Poultry Poison Type 1 Poultry Type 0 Poultry% j Color Poison Type 2 Adenovirus Type 4 5 Adenovirus type 3 adenovirus type 4 6

Adenovirus type Adenoid type 2 1 (monkey adenovirus 1 8) iAIU monkey gland 17) Adenine type 4 7 adenovirus type 4 4 24 ^ 200533752

Adenovirus ATCC VR-541 SA 7 (Monkey Adenovirus 16) ATCC VR-941 Frog Adenovirus (FAV-1) ATCC VR-896 Adenovirus 4 Type 8 (candidate) ATCC VR-1406 Adenovirus 4 Type 2 ATCC VR-1304 Adenovirus Type 49 (candidate) ATCC VR-1407 Adenovirus Type 43 ATCC VR-1305 Avian Adenovirus Type 6 ATCC VR-831 Avian Adenovirus Type 3 Bovine Adenovirus Type 3 ATCC VR-639 Bovine Adenovirus Type 6 ATCC VR-642 Canine Adenovirus ATCC VR-800 Bovine Adenovirus Type 5 ATCC VR-641 Adenovirus 3 Type 6 ATCC VR-913 Sheep Adenovirus Type 5 ATCC VR-1343 Adenovirus 2 Type 9 ATCC VR-272 Pig Adenovirus ATCC VR-359 Bovine Adenovirus Type 4 ATCC VR-640 Bovine Adenovirus Type 8 ATCC VR-769 Bovine Adenovirus Type 7 ATCC VR-768 Adeno-associated Virus Type 2 (AAV-2H) ATCC VR-680 Adenovirus 4 ATCC VR-4 adeno-associated virus type 3 (AAV-3H) ATCC VR-681 Peromyscus adenovirus ATCC VR-528 adenovirus 1 ATCC VR-661 adenovirus 2 ATCC VR-662 chimpanzee adenovirus ATCC VR -593 Adenovirus 3 Type 1 ATCC VR-357 Adenovirus 2 Type 5 ATCC VR-223 Chimpanzee Adenovirus ATCC VR-592 Chimpanzee Adenovirus ATCC VR-591 Adenovirus 2 Type 6 ATCC VR-2 24 Adenovirus 1 Type 9 ATCC VR-254 Adenovirus 2 Type 3 ATCC VR-258 Adenovirus 2 Type 8 ATCC VR-226 Adenovirus Type 6 ATCC VR-6 Adenovirus Type 2 Antiserum: ATCC VR-1079 Adenovirus 6 ATCC VR-1083 Sheep Adenovirus Type 6 ATCC VR-1304 Adenovirus Type 3 ATCC VR-847 Adenovirus Type 7 ATCC VR-7 Adenovirus Type 3 ATCC VR-932 Adenovirus Type 3 ATCC VR-3 Bovine Adenovirus Type 1-ATCC VR-313 25 200533752

Adenovirus 1 Type 4 ATCC VR-15 Adenovirus Type 1 ATCC VR-1078 Adenovirus 2 Type 1 ATCC VR-256 Adenovirus 1 Type 8 ATCC VR-1 095 Baboon Adenovirus ATCC VR-275 Adenovirus 1 Type 0 ATCC VR -1 1 Adenovirus 3 Type 3 ATCC VR-626 Adenovirus 3 Type 4 ATCC VR-716 Adenovirus 1 Type 5 ATCC VR-16 Adenovirus 2 Type 2 ATCC VR-257 Adenovirus 2 Type 4 ATCC VR-259 Adenovirus 1 Type 7 ATCC VR-1094 Adenovirus Type 4 ATCC VR-1081 Adenovirus 1 Type 6 ATCC VR-17 Adenovirus 1 Type 7 ATCC VR-18 Adenovirus 1 Type 6 ATCC VR-1093 Bovine Adenovirus Type 2 ATCC VR- 3 14 S V-30 ATCC VR-203 Adenovirus 3 Type ATCC VR-625 Adenovirus 2 Type 0 ATCC VR-255 Adenovirus 1 Type 3 ATCC VR-14 Adenovirus 1 Type 4 ATCC VR-1091 Adenovirus 1 8 ATCC VR-19 S V-39 ATCC VR-353 Adenovirus 1 Type 1 ATCC VR-849 Duck Adenovirus (Egg Reduction Syndrome) ATCC VR-921 Adenovirus Type 1 ATCC VR-1 Chimpanzee Adenovirus ATCC VR-594 Adenovirus 1 Type 5 ATCC VR-1092 Adenovirus 1 Type 3 ATCC VR-1090 Adenovirus Type 8 ATCC VR-1368 S V-3 1 ATCC VR-204 Adenovirus Type 9 ATCC VR-1086 Mouse Adenovirus ATCC VR- 550 Adenovirus Type 9 ATCC VR-10 Adenovirus 4 Type 1 ATCC VR-930 C1 ATCC VR-20 Adenovirus 4 Type 0 ATCC VR-931 Adenovirus 3 Type 7 ATCC VR-929 Marble Spleen Disease Virus Adenovirus 3 Type 5 ATCC VR-71 8 SV-32 (M3) ATCC VR-205 Adenovirus 2 Type 8 ATCC VR -1 1 06 Adenovirus 1 Type 0-ATCC VR-1087 | Adenovirus Type 20-ATCC VR-1097 26 200533752

Adenovirus 2 Type 1 ATCC VR-1098 Adenovirus 2 Type 5 ATCC VR-1 103 Adenovirus 2 Type 6 ATCC VR-1 104 Adenovirus 3 Type 1 ATCC VR-1 109 Adenovirus 1 Type 9 ATCC VR-1096 S V -36 ATCC VR-208 S V-38 ATCC VR-355 SV-25 (M8) ATCC VR-201 SV-15 (M4) ATCC VR-197 Adenovirus 2 Type 2 ATCC VR-1100 SV-23 (M2) ATCC VR-200 Adenovirus 1 Type 1 ATCC VR-12 Adenovirus 2 Type 4 ATCC VR-1102 Avian Adenovirus Type 1 S V-1 1 (M5) ATCC VR-196 Adenovirus Type 5 ATCC VR-5 Adenovirus 2 3 ATCC VR-1 101 SV-27 (M9) ATCC VR-202 Avian Adenovirus Type 2 (GAL) ATCC VR-280 SV-1 (Ml) ATCC VR-195 SV-17 (M6) ATCC VR-198 Adenovirus 2 Type 9 ATCC VR-1107 Adenovirus Type 2 ATCC VR-846 SV-34 ATCC VR-207 SV-20 (M7) ATCC VR-199 S ^ V-3 7 " ATCC VR-209 " SV-33 ( M10) ATCC VR-206 ~~ Avian adeno-associated virus ATCC VR-865 Adeno-associated (satellite) virus type 4 ATCC VR-646 Adenovirus 3 Type 0 A—TCC VR-273 Adeno-associated (satellite) virus type 1 ATCC VR- 645 Infectious canine hepatitis (Rubarth's disease) Adenovirus 2 Type 7 ATCC VR-1 105 Adenovirus 1 Type 2 ATCC VR-863 Adeno-associated virus type 2 k virus 7 type ATCC VR-848 The recombinant adenovirus vector of the present invention can be used as a therapeutic agent for preventing and / or treating cancer. The vector of the present invention preferentially replicates in tumor cells and undergoes selective lysis. In a specific aspect, the vector of the present invention (containing the E2F promoter operably linked to a gene necessary for replication) is compared to 27 200533752.

Non-defective cells in the Rb-pathway will preferentially kill tumor cells with defective Rb-pathway. In another specific aspect, the vector of the present invention (containing the TERT promoter operably linked to genes necessary for replication) preferentially kills tumors with increased expression of telomerase over non-tumor cells cell. In another specific aspect, the vector of the present invention (E2F promoter containing genes operably linked to recoating and TERT promoter operably linked to genes necessary for replication) will be preferentially killed. Tumor cells with Rb pathway defects and increased expression of telomerase. Unintentionally limited by theoretical considerations, in a specific aspect, virus replication that is specifically regulated by the E2F or TERT promoter is prevented from being read-through transcripts that terminate the signal sequence upstream. In some specific aspects, the recombinant viral vector of the present invention is further provided with a 3 14 E4 gene-linked selective promoter. In other specific aspects, the recombinant viral vector of the invention comprises a deletion in the Elb gene of the adenovirus. In another specific aspect, the recombinant viral vector of the invention comprises an IRES or a self-processing cleavage site (eg, 2A) sequence. In yet another example, the recombinant viral vector of the present invention comprises a heterologous coding sequence or a carbinol base that is operatively linked to a natural or heterologous promoter. In yet another specific aspect, the recombinant viral vector of the invention comprises a targeting ligand. Therefore, the combination and sequential position of the genetic elements used in the vector of the present invention can provide and enhance the selectivity of the vector while minimizing the toxicity and side effects in animals. In a specific evil, the recombinant viral vector of the present invention comprises a terminating sequence (as described in Patent Application No. § §3003, 0624). Insertion of the + adenylation signal sequence can reduce oncolytic adenovirus vector replication in non-target cells 28 200533752 and thus reduce toxicity. The termination signal sequence can be placed before any promoter in this vector (5,). In a specific aspect, the termination sequence is placed at the 5 'end of the E2F promoter (operably linked to the Ela or Eib coding sequence). In a specific aspect, the termination signal sequence is placed at the 5 'end of the TERT promoter, which is operably linked to the Ela or Elb coding sequence. In an alternative embodiment, the invention further comprises a mutation or deletion in the Elb gene. In a specific aspect, the mutation or deletion in the Elb gene department causes the Elb_19kD protein to become non-functional. The modification in the Elb region can be related to the presence of the full-length or partial region of E3.

Vector combination. In a specific aspect, a deletion is made in Elb (represented by SEQ ID NO: 4 or SEQ ID NO: ό). In a specific aspect, the first 261 nucleotides of the Elb open reading frame were deleted. In a specific aspect, the nucleotide sequence in SEQ ID 12 is deleted from the vector. In a specific case, the Elb deletion is the same as the Elb deletion in the virus CG5757 or OV945. The oncolytic adenovirus vector of the present invention may optionally contain a heterologous coding sequence 歹 j or a gene. For example, the heterologous coding sequence can encode immunostimulatory proteins including (but not limited to) cytokines (GM_CSF, IU, IL, IL4, IL5, IFNa, IF々, TNFa, IU2, IU8, and ⑽), stimulation and Proteins interacting with immune cells (B7, CD28, MHCI class, MHC class II, TAPs), tumor-associated antigens (from MART), gP100 (PmeM7), tyrosinase, tyrosinase · related proteins Catalase-related protein f 2, melanocyte-stimulating hormone receptor, magei, 29 200533752 MAGE2, MAGE3, MAGE12, BAGE, GAGE, NY-ESO-1, / 5-catenin, MUM-1, CDK -4, caspase 8, KIA 0205, HLA-A2R1701, alpha-fetoprotein, telomerase catalytic protein, 0-25 0, MUC-1, oncofetal protein, p53, Her2 / neu, triose Phosphoisomerase, CDC-27, LDLR-FUT, telomerase reverse transcriptase, immunogenic sequences with PSMA), antibodies that block inhibitory signals (CTLA4 blockers), chemokines (MIPIα, MIP3a, CCR7 ligand, and calreticulin) and other proteins.

In another specific aspect, the heterologous coding sequence encodes an anti-angiogenic protein. Anti-angiogenic proteins include (but are not limited to) MET-1, MET-2, TrpRS fragments, proliferating protein-related proteins, prolactin fragments, PEDF, vascular inhibitors, various fragments of extracellular interstitial proteins, and growth factors / Cytokine inhibitor. Various fragments of extracellular interstitial protein include (but are not limited to) angiostatin, endostatin, cytokinin, fibrinogen-E fragment, coagulation factor, tumor suppressor, cancer suppressor, and resting Vegetarian. In another specific aspect, the heterologous coding sequence encodes a growth factor / cytokine inhibitor. Growth factor / cytokine inhibitors include (but are not limited to) VEGF / VEGFR antagonists, sFlt-1, sFlk, sNRPl, vascular protein / tie antagonists, sTie-2, chemokines (IP-10, PF-4 , GroJ, FGF / FGFR antagonists (sFGFR), Ephrin / Eph antagonists (sEphB4 and sephinB2), inhibitors including IFN-Y (Mig), IFN-α, PDGF, TGF /? And IGF-1; And its analogs. In another specific aspect, the heterologous coding sequence encodes a suicide group 30 200533752

because. A "suicide gene" encodes a protein that itself may cause cell death (eg, when diphtheria toxin A is expressed), or the expression of this protein makes the cell selectively sensitive to specific drugs, such as the herpes simplex thymidine kinase gene (HSV-TK) The performance of the cell makes anti-viral compounds such as asilox, more silox, and FIAU (1_ (2-deoxy_2_fluoro-heart D-arabinofuranosyl) -5-carbonuria, bite) and other antiviral compounds sensitive. Other suicide genes include, but are not limited to, carboxypeptidase G2 (CPG2), carboxyesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytosine kinase ( dcK), nitro reductase (NR), purine nuclear phosphatase (pNp), thymic bitter phosphatase (τρ), varicella zoster virus thymidine kinase (VZV-TK), and xanthine-guanine phosphate ribose Transferase (XGPRT). Heterologous genes can exhibit their effects at the RNA level 'for example by encoding antisense information or ribozymes, acting on splicing or 3, processing (such as polyadenylation) proteins, or, for example, by mediating changes in mRNA f products Rate, altered mRNA delivery, and / or altered translational regulation: a protein that expresses the extent of another gene in the cell. Adding a heterologous gene to a disease does not necessarily lead to the virus having an additional antitumor mechanism of action. In another specific aspect, the adenoviral vector of the present invention further includes a < > targeting ligand in the protein of the shell protein. In a specific example, A A is a fibrous protein and the targeting system is located in the HI carcass of fibrous protein. In another specific aspect, the shell protein is a fibrin protein nTv. In yet another specific aspect, the guide system is located in the fibrous egg ^ in the received HI ring. In another specific aspect, look! The ligand is added to the basal end of the adenovirus fiber protein. In other specific 7 beta disease matrilineal errors, the 'fibrillar segments were replaced by fibroblasts derived from another-adenovirus serum 31 200533752 type for targeting. For examples of targeting adenoviruses, see, for example, WO 00/67576, WO 99/39734, US 6,683,170, US 6,555,368, US 5,922,315, US 5,543,328, and US 5,846,782. Adenoviral vector particles may also include other mutations to the fibrin. Examples of such mutations include, but are not limited to, those described in U.S. Patent Application Nos. 10 / 403,337, WO 98/07877, WO 01/92299, and U.S. Patent Nos. 5,962,3 1 1, 6,153,435, 6,455 3, 14 and Wu et al. (J. Virol. 2003 Jul 1; 77 (13): 7225-7235). These include (without limitation) the reduction of the binding of the viral vector particle to a specific cell type or more than one cell type, the promotion of the binding of the viral vector particle to a specific cell type or more than one cell type, and / or a reduction in the animal body Mutations that produce an immune response to the viral vector particle. In addition, the adenoviral vector of the present invention may contain mutations to other viral capsid proteins. Examples of such mutations include, but are not limited to, those described in U.S. Patent Nos. 5,731,190, 6,127,525, and 5,922,315. Other mutated adenovirus lines are described in U.S. Patent Nos. 6,057,155, 5,543,328, and 5,756,086. In Yu, another aspect of the present invention provides a selective cytolysis in a cell population, that is, killing tumors A method for sex cells, which comprises applying an effective amount of the virus vector and / or virus particle of the present invention to enable the virus vector and / or virus particle to infect tumorous cells existing in the cell population, selectively replicate therein and Contact the cell population under conditions that kill the tumorous cells. The population of cells can be formulated in vivo, in a test tube, or from a living body. The invention further comprises an adenoviral vector particle, wherein the targeting protein 32 200533752 is included in the capsid protein of the particle, and the capsid protein is a fibrin; The adenoviral vector with protein complexes and fibrin = is known by those skilled in the art = :. Using standard techniques known to those skilled in the art, dagger coat cells provide supplements to the adenoviral genome to be packaged into adenoviral particles. The manufacturing needs of such particles

The vector is copied and the proteins required for the virus production in Huanmu are produced. The packaging cell line was cultured under a strip that allowed the dream ^ # 卞 I & sosh virus vector particles. Viral particles are recovered by standard techniques. Examples of packaging cells include (but are not limited to) Xiao et al., Which have been designed to limit the "recombination of packaging cells" that may cause wild-type adenoviruses, for example, as disclosed in U.S. Patent #u 5,994,128 (Faurus et al. ) And M33, _ (Baut et al.) Cells. Also, 'the carrier particles of the present invention can be produced, for example, in PerC6 or H.-S3, cells (for example, see US Patent Application No. 60 / 463,143) oE2F The promoter is not intended to be limited by theory, and the selectivity of the E2F_reactive promoter (hereinafter sometimes referred to as the E2F promoter) is reported to be based on suppressing the E2F promoter / trans-activating protein in tumor cells deficient in the Rb pathway. Resting In cells, coffee and tumor-associated protein pRB bind to form a ternary complex. In its complex form, E2F functions to suppress the transcriptional activity of promoters (including the E2F-1 promoter itself) with E2f binding sites ( Cui Kelu and Mu Le Ur〇g.⑽Cycle Res] 995; 1: 91 · 99). E2F] promoter is transcriptionally inactive in resting cells 33 -200533752. In normal periodic cells, the pRB_E2F complex system Solution in a regulated way Isolation, enabling the depressive effect of E2F and subsequent cell cycle cycling (Dyson, N., Genes & Development 1 998; 12: 2245-2262) 〇 In most tumor types, the Rb cell cycle regulatory pathway is disrupted , Implying that the Rb pathway is necessary to regulate tumorigenesis (Strauss M, Lucas J and Batik J., Nat Med 1995; 121: 1245-1246).

One of these mutations results in disrupted E2F_pRB binding and increased free E2F in tumor cells. Rb itself is mutated in some types of tumors, while factors upstream of Rb are deregulated in other types of tumors (Winberger, ra. Cell 1995; 81: 323-330). One of the changes in these milk pathways in tumors is the loss of binding to E2F, which significantly increases free E2F in tumor cells. Abundant free E2F will then cause high expression of E2F-reactive genes (including E2F "genes) in tumor cells. Thus, the term "Rb pathway defective cells" can be functionally defined as when analyzed by changes in agglutination shift or by chromatin immunoprecipitation (Takahashi Y, et al.,

Dev. 2000 Apr 1; 14 (7): 804-16) When measured, it showed cells that were rich in "free state" E2F. The E2F4 promoter has been shown in rodents to up-regulate markers and genes in adenovirus in vivo. But not in normal proliferating cells (Pall MJ et al. 'Nature Med 1997; Oct; 3 (iO): ii45-1149). T2F binding site. In the specific aspect of the E2F-reactive promoter, the 'E2F-reactive promoter is a mammalian E2p promoter. In another specific aspect, it is human E2F enlightenment 7

Respect the mover. For example, the E2F 34 200533752 promoter can be a human E2F-1 promoter. Further, the human E2F-1 promoter may be, for example, an E2F-1 promoter having a sequence as described in SEQ ID NO: 1. Many examples of E2F promoters are known in the art (e.g. Parr et al. 'Natural Medicine 1997; 3 (10): 1 145-1 149, WO 02/067861, US20010053352 and WO 98/1 3508). E2F reactive promoters typically share common traits such as the SP I and / or ATT7 sites located near their E2 site (usually near the transcription start site) and lack a discernable TATA box. E2F-reactive promoters include E2F promoters such as the E2F-1 promoter, the dihydrofolate reductase (dhfr) promoter, the dna-nickelase A (DPA) promoter, the c-myc promoter, and the B-myb promoter. The force of XT O X? 1 contains four E2F sites that act as transcriptional repression elements in serum-starved cells. In a specific aspect, the E2F-reactive promoter has at least two E2F sites. In another specific aspect, the promoter is operably linked to the adenovirus Ela region. In yet another specific μ sample, the E2F promoter is operably linked to the adenovirus Eb region. In another specific aspect, the E2F promoter is operably linked to the adenovirus E4 region. In a specific aspect of this month, the recombinant viral vector of the present invention selectively replicates and lyses Rb pathway-deficient cells. In a specific aspect, the E2F promoter of the present invention is a mammalian caiqi; in another specific aspect, the mammalian E2F promoter is a human E2F th ^ -7 Qiu Ding is a human being Γ / For example, It contains or is essentially a large, 2F promoter grouped by seqidn0: i. Specific aspects of the invention include an adenoviral vector comprising an E2F promoter, wherein the 豸 e2f promoter comprises a nucleotide sequence selected from: 35 200533752 (a) a nucleotide sequence listed in SEQ ID NO: 1; (b) a A fragment of the nucleotide sequence of seq⑴ NO: 1, wherein the fragment has tumor-selective promoter activity; (c) its full-length pair has a nucleotide sequence of at least 90, 92, 92 listed in SEQ ID NO: 1; A nucleotide sequence of 93, 94, 95, 96, 97,%, or higher identity, wherein the nucleotide sequence has tumor-selective promoter activity; and (d) has a stringent and listed under severe conditions A full-length complementary nucleotide sequence that is hybridized to the nucleotide sequence of seq ID NO. · 1, wherein the nucleotide sequence has tumor-selective promoter activity and is a group of nucleotide sequences. In another specific aspect of the invention, the E2f promoter comprises nucleotides 7 to 27 of SEQ m NO: 1. In another specific aspect of the present invention, the E2F promoter comprises nucleotides SEQ to NO: 1 to 270, wherein nucleotide 75 of SEQ ID NO: 1 is τ instead of c. In other specific aspects, the E2F promoter according to the present invention, when using the following sequence alignment algorithm or by visual inspection measurement for maximum correspondence alignment and permutation, is listed in SEQ m NO: ι Nucleic acid sequences have at least 8G, 85, 87, 89, 9G, 91, 92, 93, 94, 95 96, 97-98 "% or more sequence identity. In a particular aspect, a given percentage of sequence identity is present in a region of a sequence having a length of at least about 50 nucleotides. In another specific aspect, the given sequence has the same sequence, and there is a product having a length of at least about j 00 nucleotides in the sequence 'Chinese prescription, 3 j member specific aspect, the given A defined percentage of sequence identity is present in a region of a sequence that is at least about 200 nucleotides in length. In another specific aspect, the given percent sequence identity is stored in the full length of the sequence. 36 200533752 E2F-reactive promoter does not need to be full-length or wild-type promoter, but it should have at least 3 times, at least times, at least 1G times, at least 2 (M tone, at least 30-fold, at least 50-fold, Tumor selectivity of at least 100-fold or even at least 300-fold. Tumor selectivity can be analyzed by many techniques using known techniques, such as those used in WO 02/067861, Example 4, such as Determined by RT-PCR or by comparison to replication of selected cell types. Tumor selectivity of adenoviral vectors can also be quantified by El A RNA levels (as further described in WO2 / 067861, Example 4), And for the purpose of the present invention, the level of El A RNA obtained in H460 (ATCC, catalog number HTB-177) cells can be compared with that obtained in PERC (clonics catalog number CC2555) to determine tumor selection. The conditions of the experiment may be changed, but representatively still as described in WO02 / 067861. Telomere pickling (TERT) starts early

Ran Si is limited by theory. The understanding of selective TERT expression in cancer is based on the current known TERT as telomerase (a type that has also been shown to be active in ~ 85% of human cancers but not active in normal somatic cells). Rate-limiting catalytic subunits (kilion A et al.,

Hum Mol Genet. 1997 Nov; 6 (12): 2011-9; Kim NW et al., Science 1994 Dec 23; 266 (5 193): 201 1-5; Shay JW et al., European Journal of Cancer 1997,5,787- 791, 8 people by Shi Duhua, etc., 86111111〇811 ^ 16 丨 01 Dec 2000; 10 (6): 399-406). Cancer cells seem to require immortalization for tumorigenesis and telomerase activity is most often required for immortalization. (Jin NW et al., Science 1994 Dec 23; 266 (5193): 2011-5; Kiyano T et al. 37 200533752, Nature 1998; 396: 84). Therefore, most tumor cells have an uncontrolled telomerase pathway. Such tumors are specifically identified by the present invention using a TERT promoter that is operably linked to genes and / or coding regions necessary for replication (e.g. Ela, Elb or E4).

The term TERT promoter is used herein to mean the full-length TERT promoter and its functional fragments, mutations and derivatives. The TERT promoter need not be a full-length or wild-type promoter. Those skilled in the art know how to derive fragments from the TERT promoter and test their desired specificity. In a specific aspect, the TERT promoter of the present invention is a mammalian TERT promoter. In another specific aspect, the mammalian TERT promoter is a human TERT promoter (hTERT). In a specific aspect of the invention, the TERT promoter comprises or essentially consists of SEQ ID NO: 2 (which is a 23 9 bp fragment of the hTERT promoter). In another specific aspect of the invention, the TERT promoter comprises or essentially consists of SEQ ID NO: 3 (which is a 245 bp fragment of the hTERT promoter). In a specific aspect, the TERT promoter is operably linked to the adenovirus El a region. In another specific aspect, the TERT promoter is operably linked to the Elb region of the adenovirus. In yet another specific aspect, the TERT promoter is operably linked to the adenovirus E4 region. A specific aspect of the present invention includes an adenoviral vector comprising a TERT promoter, wherein the TERT promoter comprises a segment selected from: (a) a nucleotide sequence listed in SEQ ID NO. · 2; J; (b) A fragment listed in the nucleotide sequence of SEQ ID NO: 2, wherein the fragment has tumor selective promoter activity; (c) its full-length pair has a nucleotide sequence of 38 200533752 listed in SEQ ID NO. · 2 A nucleotide sequence that is at least 90% identical, wherein the nucleotide sequence has tumor-selective promoter activity; and ⑷ has a full-length hybrid under stringent conditions with the acid sequence listed in SEQ ID NO: 2 Complementary nucleotide sequences, in which the nucleotide sequence has tumor-selective promoter activity, and constitutes a group of nucleotide sequences. Other examples of TERT promoters are known to those skilled in the art (e.g. wo 98/14593). In other specific aspects, according to the TERT promoter of the present invention, the following sequence alignment method Λ or the largest corresponding alignment and arrangement by visual inspection measurement are listed in SEQ ID: 2 or The sequence of ㈤ has at least 80, 85, δ 7, 89, 90, 91, 92, 93, 94 ·, =, 96, 97, 98, 99% or higher sequence homology. In the specific case, the percentage of sequence identity given by 'is present in a region having a length of at least about 5 ^ 0 nucleotides. In another specific aspect, the given sequence of 疋 J homology is present in a region whose length is at least about 100 nucleotides = sequence. In another specific aspect, the given sequence exists in a region having a sequence length of at least 'approximately fan nucleotides with the same percentage. In another aspect, the given one exists in the full length of the sequence. The composition and method of the present invention, and another aspect of the present invention, is to provide a pharmaceutical composition comprising the recombinant virus and / or particles of the present invention and a pharmaceutically acceptable carrier. These adenoviral vector suspensions of the present invention in a pharmaceutically acceptable carrier are suitable for use in unit dosage form (sterile parenteral solution or 4 sub-liquids, sterile parenteral solution or oral Solutions or suspensions, oil-in-water or 39 200533752 water-in-oil emulsions and the like) are known for the topical or systemic administration of mixtures to an individual for parenteral and enteral drug delivery. The composition also includes the bodies and granules of the present invention in a cool-dried and / or reconstituted form. Acceptable pharmaceutical carriers are, for example, saline: white sulfate (Elkins-Slnn Co., Ltd., Cherry Hills, egg water, aqueous buffers (such as phosphonate buffer and Tris buffer) Polybrene (Sigma Chemicals) Company, Shenglu ^ n Renxing, Louis City, M0) and saline and recommended sugar washed with phosphoric acid. The choice of suitable medicine for the medicine through the teachings in this article is considered to be obvious to those who are skilled in the art It is easy to know. These solutions are sterile and generally do not contain any substance other than the desired adenoviral particles. The composition may contain the medically necessary conditions to achieve approximate health conditions. Auxiliary substances, such as pH regulators, lavenders, and ^ and 绫 granules, toxicity regulators and the like, such as sodium acetate, sodium chloride, / eight Sheng. Emulsification evaluation of calcium, sodium lactate, ', May include the ability to increase cell adenovirus reduction, such as lidocaine. The endogenous and / or anesthetic virus vectors are effective to inhibit # & 主 # a line replication and inhibit, Prevent the two Guang Ganzai ... the tumor cells stop or break The tumor cell growth amount is administered to the host. This administration can be performed * 1. »', 9 by systemic administration as described herein, or by directly injecting the carrier into the rash. Primary tumor. In one proposal, the carrier was made at least 5 × 10 viral particles per kilogram of body weight / generally not more than 1 × 10,3 viral particles per # or even small? D Systemic administration. In the other- In the proposal, the heart is loaded with a weight of at least 2χΐ〇 丨 〇 kg weight—usually no more than 1x1013 virus particles per carrier is injected into the patient tr for administration. In yet another proposal, the system will In this case, transduction can be pre-treated with 40 200533752 as a transduction enhancer as described in USSN 10/327869. The exact dose to be administered depends on various factors including the age and weight of the patient , And sex, and the size and severity of the tumor being treated. The carrier can be administered one or more times. The treating physician can choose the dosage level and form of the composition for single- or multiple administrations. If necessary, 'a variety of Immunosuppressive or Eliminating pre-existing antibodies reduces the immune response so that repeated administration and / or enhanced replication can be achieved by reducing the immune response to the virus. The administration of the adenoviral vector of the invention can be combined with other antitumor proposals (many examples of which The technology is known) combination. Anti-tumor proposals such as & will vary depending on the type of cancer being treated. Delivery can be achieved in a variety of ways using microlipids, direct injection, total injection, catheter, topical application, inhalation, etc. The prescription and the prescription provide a method for treating a patient suffering from a tumor condition, which comprises administering a therapeutically effective amount of the adenovirus vector of the present invention to the patient ^ (typically, a cancer patient). Although the mechanism of action is not part of the present invention ', the viral vectors described herein are believed to be selectively distributed to tumor cells and distributed on tumor stroma due to their ability to selectively replicate in tumor tissues. It is possible that all tumor conditions have the potential to be treated by the method of the invention. Tumor types include (but are not limited to) hematopoietic, vasculature, pancreas, god: system, liver, gastrointestinal tract, biliary tract, treasure lung, head and neck, soft tissue sarcoma and cancer, skin f, genital tract, Respiratory system, and similar tumors. In a specific aspect, the tumor being treated is one having a high mitotic index relative to normal tissue. In another—a specific towel, the tumor 41 200533752 is a solid tumor. In the specific aspect, the patient is a human patient. For a human patient, if the heterologous coding sequence is included in the vector, the heterologous coding sequence can be of human origin, but if the heterogeneous code is stepped on, the flat code sequence will not be in the recipient's body. To produce / cause a harmful immune response, genes from closely related species that exhibit high homology to humans and the same or equivalent function on organism 2 can be used. In a specific heart-like towel 4 the heterologous coding sequence encodes a therapeutic protein or a therapeutic construct. A therapeutically active amount of a nucleic acid sequence or a therapeutic gene is an amount effective for 1 to and for a period of time to achieve a desired result. This amount may vary depending on the patient's gender, age, weight, and the like. The present invention also provides a drug candidate for screening to identify agents that can be used to modulate: F: / or TERT performance 'and thus can be used to treat cancer. Appropriate host cells are those in which the regulation region of E2F and / or TERT can be H%. In a specific aspect, E2F and / or TERT Taxi Week

Section areas are used to make a variety of performance vectors that can be subsequently used for screening analysis. In the Aw egg and the limb form of the first hall, the marker protein is Ela, Elb, virus replication, or 1 la Λ ^ 5, all of which can be measured using techniques that are already familiar with this technique. . The expression vector may be a self-replicating vector; a vector in vitro or a vector integrated into the genome of the host. Generally, these tables include transcription and transduction-regulating nucleic acid sequences that are operably linked to a nucleic acid encoding a marker 'protein and / or E2F. Peptone can be any egg sugar that can be easily detected by Moon Valley (#,, 7 enzymes or FITC), color eggs can be based on delayed radiation (such as camphorin " i such as galactose: yt enzyme), enzyme Activity (Example 42

200533752 Such as the experimental "ϋ" or antibody reaction (for example, the protein of which antibodies are already produced. In addition, the label "white" may be shellfish particles of the present invention. Private version 'In a specific aspect, the virus of the present invention The vector is used to analyze the anti-cancer efficacy of the candidate therapeutic agent. According to this specific aspect, an effective amount of the viral vector is used to infect the virus vector in the cell population: cells, which are selectively replicated and killed Contact the cell population under the condition of dying the tumorous cells. Compare the viral vectors with and without the candidate Chinese agent. The value is to identify the regulatory cells that regulate E2F and / or TERU. Candidates for the efficacy of lysing cancer cells :: Secondary :: Lack of E2F and, or similar viral vectors of the TERT promoter .... n, the degree of expression is different, the candidate agent can regulate E2F and / 1. T, ER two It is a candidate for the treatment of cancer and for the further development of active pharmaceutical agents based on the identified succinylpyridine. 1st = In a specific aspect, the candidate pharmaceutical agent is added to the owner of the expression vector. , ", Tian cells' and compare the performance of the target | 4 protein with the control group. If the performance is different, then the candidate agent can regulate the performance of E2F or coffee, :::: for the treatment of cancers involving these genes and the use of Candidates for the further development of active agents based on the 1′k leucine agents identified here: The active agents obtained from this post can be used, for example, in combination with the oncolytic adenopathy of the present invention. In a specific embodiment, in the expression morphology provided by the bioactive agent invention, in a nucleic acid or protein aspect, the candidate agent suppresses the cancer phenotype, regulates the phenotype, or is more particularly preferred, such as relative to normal tissue 43 200533752 phenotype · 八 ；; also 丄 丄 Ω is typically a branched knife, such as a small organic compound with a molecular weight greater than 100 and small. The preferred small molecular system is _, Doulton or less than _ or less than 5 ... Doulton. Candidates: Functional Groups Required for Structural Interactions of 或 or Less

Apparently includes at least one amine, a few bases, and a radical bond) ': Substitute a functional chemical beauty g ^ Ertu Lie to 乂 two r beauty to ... to include meridian-or more of the above official: == ΓΓ 冓And / or aromatic or polyaromatic structures. Candidates _ Γ include peptides, carbohydrates, fatty acids, sterols, ^ t t 疋, their derivatives, and similar structures: Medium. Particularly preferred are peptides. ... and. The biomolecular compound library of the second line is transmitted from a variety of different sources, including synthetic or natural compounds and biodiesel 4 :: 子 可: random and targeted synthetic species in many ways, organically available or easily including randomized oligos. Bitter. Alternatively,... May also serve as a library of bacterial, fungal, plant and animal extracts. In addition, natural or synthetic one or two: = Γ chemical, physical, and biochemical methods, t known Leche for direct or random chemistry ", such as ^ s and chemical amidation to produce structural analogs The person's implementation department uses (unless otherwise specified) the genetics that are accustomed to the technology: two chemistry, molecular biology, microbiology, recombinant DNA, two technical skills ^ example physical science, cell culture and transgenic organisms (for example) Manatis et al., 1982, Molecular Selective Production 44 200533752 (published by Cold Spring Harbor Laboratory, Cold Spring Harbor, 'deleted, split m version (cold spring harbor real); / Mbuke et al., New York)' Sam Booker With Russell, Kab AU publishes' Cold Spring Harbor ', Spring Harbor Laboratory Publishing, Cold Spring Harbor, New York) .: Subselection' 3rd Edition (Revealing of Cold Molecular Science, Spear et al., 1992,- Reed A case (Joseph Hamley New); Glover, 1985, Election ceremony (irl = son & including regular more Nat '1992, ❹class # cutting skills., Learn: out: Jin: Yoag Siri and Fink '1991, Yankan :: Two' Academy Publishing 'Buchlo and Lane, New York, 198: ❹: " Published by the Laboratory, Cold Spring Harbor, New York). Jacobi / Li, (Cold Spring Harbor Reality), Jacoby and Bastam, 丨 ## （BD · 黑 母 斯 洗 8 J 希 今 浙 绝 # 'n 、 & J. Higgins, 1984); # 莩 （BD Hei Sisi 8 j Xi Lingzhe — — ，, 丄 希 金 " Jin, Bian Zhe, 1984) \ Animal Cell Mouth Race (RJ · Ferghini 'Yak R. Reese Co., Ltd., 1987); Definitely: Jia Yingyu (IRL Publishing, 1986); B. Piper, Election of the Shoulders of Lu / Yao (1984), Thesis, Mo Daddy Xuecai (College Publishing Limited,

New York, Gene Transgenic Carriers for Mammalian Cells. Edited by Axe, Qin, and M.P. Carlos, 1987, Cold Spring Harbor Laboratory.); Edited by Mel and Walker, Academy Publishing, London, 19 87); f. Handbook of Dragon Epidemiology, Volumes I-IV (DM · Weir and c c. Boudmerville, ed. 1986); Riot, Endemic Epidemiology, 6th Edition, Blakewell Scientific Publishing, Oxford, 1988; Hogan et al., V, Su Qu Zhi Zhi Zuo (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1 986 ). Examples The present invention is described with reference to the following examples, which are provided as examples 45-200533752 and are not intended to limit the present invention in any way. Use standard techniques known to the art or techniques specifically described below. Example 1: The construction and sequencing of OV945 was confirmed based on the E2F-1 promoter sequence (GenBank S74230), and Gong 1 was designed 1 405.77.1 (5'-ataccggtggtactcccatccggacaaagcctgcgcg; SEQ ID NO: 8) and 1405.77.2 (5 '-agaccggtcgagggctcgatcccgctccg; SEQ ID NO: 9) and a 270 nt clone was cloned from human genomic DNA by PCR

E2F-1 promoter (SEQ ID NO: 1). Using the Agel (or PinAI) locus within the primer, CPI 131 (US Patent 6,692,736, Zhang et al. Cancer

Res. 2002 Jul 1; 62 (13): 3743-50). The human uroplakin II promoter was replaced with the E2F-1 promoter to form the plastid CP1493. CP1493 has an E2F-1 promoter operably linked to Ela, an IRES promoter operably linked to the Elb region, and most of the E1B 19-KDa region has been deleted. The CP1493 plastids were sequenced and confirmed to be correct. CP1493 was cotransfected with pBHGE3 (available from Microbix Biosystems, Inc., Dorset, Ontario, Canada and described in U.S. Patent 6,140,087) into 293 cells to obtain recombinant virus OV945. The structure of OV945 was confirmed by PCR amplification and subsequent degrading of the diagnostic enzymes corresponding to specific regions. Further sequencing of the OV945 E1 region also confirmed its genomic structure (SEQ ID NO: 6). Example 2: Construction and sequencing of CG5757. Primers 1244.39.1 (SEQ ID NO: 10; aagtcgaccggtaccgt ggcggagggactggggac) and 1244.39.2 (SEQ ID NO: 11; aagtcgaccggtgcgggggtggccgggggggggggggg) were obtained from plastid pGRN316 (derived 46 200533752).

From Jielong, Menlo Park, California; also described in Jun En 60,21 16-2121, 2000) hTERT promoter was cloned by PCR. The IRES fragment present in CP1493 was replaced with the hTERT promoter using the Sail selection site located in these primers. The derived plastid (CP 1509) has the Ela and E1B 55K genes regulated by the transcriptional regulation of the E2F and hTERT promoters, respectively. CP1 509 and pBHGE3 (available from Microbix Biosystems Co., Ltd., Toronto, Ontario, Canada and described in U.S. Patent 6,140,08 7) were co-transfected into 293 cells to obtain a recombinant virus. The nucleotide sequence of the left end of the CG5757 virus is listed in SEQ ID NO. · 4. Example 3: Construction of OV947 and confirmation of genomic structure OV947 is similar in structure to CG5757 but has no E1B 19k deletion. The E2F-1 and hTERT promoter lines are derived from CP1509, which are 0.27Kb Agel fragment and 0.24Kb Sail fragment, respectively. These promoters were selected into CP686 (U.S. Patent 6,692,736; Li et al., Cancer Res. 2001 Sep 1; 62 (1 7): 6428-36) in the 5 'end of the ElA and E1B genes, respectively. CP 1498. The 0.27Kb Agel fragment (E2F-1 promoter) was selected to replace the AFP promoter, and the 0.24Kb Sail fragment (hTERT promoter) was selected to replace IRES in CP686. Plastid CP 1498 was used to cotransfect pBHGE3 (available from Microbix Biosystems, Inc., Toronto, Ontario, and described in U.S. Patent 6,140,087) into 293 cells to obtain OV947. The structure of OV947 was confirmed by PCR amplification and diagnostic enzyme decomposition for the corresponding specific region (Figure 1). Example 4: Construction of OV1025 and confirmation of genomic structure OV1025 has El A and E1B under the transcriptional regulation of the hTERT promoter. 47 -200533752 Compared to OV945 and CG5757, the E1B 19K region is not missing in OV1025. The presence of the Agel site in the PCR primers 1244 · 3 9 · 1 (SEQ ID NO: 1〇) and 1244.3 9.2 (SEQ ID NO ·· 11) was used to clone the butyr ERt promoter to replace the CP686 (U.S. Patent 6,692,736 Li et al., AFP promoter in Cancer Res. 2001 Sep 1; 62 (17): 642 8-36). The resulting DNA construct CP1429 and PBHGE3 were co-transfected into 293 cells to produce recombinant virus OV1025. The structure of OV1025 was confirmed by PCR amplification and subsequent diagnostic enzyme degradation corresponding to specific regions (Figure 1). Example 5: Cell lines Table 2 lists exemplary cell lines used to characterize the replication effect of the pan-cancer virus of the present invention.

Table 2. Cell lines used for virus assessment. Cell name Cell source ATCC Note Hep3B Hepatocellular carcinoma ΗB-8064 A549 Lung cancer CCL-1 85 Lo Vo Colorectal adenocarcinoma CCL-229 SW 4 8 0 Colorectal adenocarcinoma CCL-228 LNC aP Breast cancer CRL-1740 P anc -1 Pancreatic Cancer CRL-1469 H eLa Cervical Epithelial Adenocarcinoma CCL-2 25 3 JBV Human Bladder Metastatic Cell Carcinoma MD Anderson Cancer Center Dr. Collin Dinai Laboratory 1-3 8 / VA -1 3 via SV40 Transformed human lung fibroblasts CCL-75.1 Η BL -1 0 0 Normal breast cells ΗΤΒ-124 ATCC has discontinued WI-3 8 正常 Normal lung fibroblasts CCL-75 IMR-90 Normal lung fibroblasts CCL-1 86 ARP E -1 9 Normal retinal pigmented epithelial cells CRL-23 02 BSM Primary bladder smooth muscle cells obtained from Cambrex, East 48 200533752 Human primary 原 Lung FB, New Jersey, Lung FB primary lung fibroblasts obtained from Cambrex 'Eastern Laceiver, New Jersey, human primary primary _ HMEC primary breast epithelial cells were obtained from Cambrex' Eastern Laceiver, New Jersey, human primary HMVEC-L primary lung microvascular endothelial cells were obtained from Cambrex, La Serphe, New Jersey, human primary seat__ HRE Primary Renal Epithelial Cells from Cambrex, East La Serve, New Jersey, Human Primary Cells S AEC Primary Small Respiratory Epithelial Cells from Cambrex, Dong La Human primary cells in Sever, New Jersey PrEC Primary prostate epithelial cells were obtained from Cambrex, East La Sever, New Jersey human primary cells

Example 6: Analysis of detection of the selective expression of El The transcriptional regulation of the E1 gene is representatively analyzed by a western transduction method. The E1 A and E1 B performance of the sample virus was compared with wild-type adenovirus type 5 (named OAV802) 24 hours after the infection (the multiplicity of infection (MOI) was 10 pfu / cell). In a representative study, approximately 25 micrograms of the cell lysate obtained from each sample was immunostained with a mouse monoclonal antibody against Ea or Bg and visualized by ECL. Example 7: Cytopathic effects and crystal violet analysis of tumor selective cytotoxicity analysis: In a representative study, the crystal violet analysis was used to test cancer cells (Hep3B, LoVo, A549, 253J BV) and normal cells. (HRE, HMVEC-L and wi_38 cells). In short, the cells were laid flat in a trough plate. After one day, the cells were infected with CG5757 or OV802 with various MOI values of 49-200533752 per cell at 10, Ϊ, 〇 ″, 0.001, 001, and 〇. At selected time points after infection, the cells were observed under a microscope to determine their CPE. Cells were then fixed with 10% formalin and stained with 1% crystal violet using standard procedures and the amount of staining was observed.

MTT analysis ... In a representative study, cells were spread on a 96-slot plate at a concentration of 10,000 cells / slot the day before virus infection. Cells were infected at different MOIs (pfu / cells) for a selected period of time and the cytotoxicity of the virus was determined in a MTT analysis as previously described (Li et al., 2001) as a percentage relative to the control group. GraphPad software was used to analyze the cytotoxicity data to perform S-shaped dose response curve fitting and determine the LD50. The relative LD50 between tumor and normal cells was compared, and the selectivity index (SILD) was used to express the specificity of cytotoxicity of oncolytic carriers. The calculation formula of siLD is ((Ad5 vs. tumor LD5G) / (oncolytic virus vs. tumor LD5.)) / ((Ad5 vs. normal LD5.) / (oncolytic virus vs. normal Ld5G)). Relative LD50 (OV802 / oncolytic vector) value! This indicates that this vector has the same cell lethality as wild type OV802. The SILD value is higher than ,,,,,, and indicates that it has tumor cell selectivity. In vitro analysis of primary tumor cultures: In a representative study, primary human tumor or normal tissue samples were collected from patients who had undergone surgical resection of colorectal or pancreatic cancer. Tissue samples are quickly placed in the appropriate frozen medium solution, dissected on ice, and selected uniform tissue sections. Fragments of each tissue sample in a cube were prepared, rinsed and placed in a medium suitable for virus infection. Add a known amount of wild-type adenovirus or recombinant virus vector. After two hours, the culture medium was replaced with 50 • 200533752 into a fresh medium supplemented with Tengdao Su Pan Λί AY " ρΓ ΑΛ 4- \. *. Tissue samples were placed in squares, and clothes were placed in 6-slot flat plates.

塾 (pore size 0.45 mm, Mllllpore, Billica, MA), and cultured under appropriate conditions. Examine tissue samples by immunohistochemical staining using adenovirus-specific antibodies at 24 hrs after infection using the standard procedure. Or = 5 days after infection after a specific number of cold cycles; the offspring should be tested for disease LD ^. Then, as described above, SILD SILD value greater than "ι" calculated from normal tissue and tumor tissue indicates that it has tumor cell selectivity. Analysis of in vitro primary tissue cultures Example 8 · Virus production and growth kinetics analysis of R. adenovirus In a representative study, cells were clothed at 5E5 cells / slot on the day before virus infection. -Slot on flat plate. Infect the oncolytic virus and OV80 with an MOI of 2 (pfu / cell) for a selected period of time, such as a small day. Cell lysates were collected and plaque potency was determined on 293 cells. For growth kinetics studies, infected cells were harvested at corresponding time points and the force value was determined. Example 9: Antitumor efficacy in vivo A549 xenograft model: In a representative study, athymic BALB / C nu / nu mice were used for six to eight weeks. Generally, 0.1 ml of 5 × 10G A549 cells were mixed with 0.1 ml of Matrigel and injected subcutaneously into each animal. Nude mice producing subcutaneous A549 tumors were injected intratumorally at a dose of approximately 4 x 108 disease particles / mm3. Depending on the course of treatment, the virus can be carried out for several days and the main shot. Tumor volume and weight were monitored and statistical analysis was performed using a Prisni GraphPad. 51 200533752 253JB-V xenograft model: In a representative study, athymic BALB / C nu / nu mice of six to eight weeks of age were used. Approximately 0.01 ml of 2E6 253J B-v cells were combined with 0 ml of Matrige: Us subcutaneously into each animal. Nude mice producing subcutaneous 253J Beta-v tumors were injected intratumorally at a dose of about 4x108 virus particles / mm3 for four consecutive days beginning on day 20. Monitor tumor volume and weight and perform statistical analysis using Prism Graphpad.

Example 10: Selective expression of CG5 757 of E1 The following example demonstrates the specific expression of E1A and E1B genes in CG5757, which results in virus replication and kills cells. Using the procedure described in Example 6, normal lung fibroblasts WI-38 (ATCC # CCL-75) (Rb +) and allotype WI-38 / VA-13 cells (Bading (^ # (3 (: 1 ^ 75.1) for comparison. ^ 1-3 8 / ¥ eight-13 lines are defective via the 3 ¥ 40 transformation and therefore have a defective Rb pathway. During infection, wild-type OV802 showed that for wi-38 / VA The relative E1 expression in -l 3 cells is not selective. In contrast, CG5757 only displays measurable E1 in Rb-deficient VA13 cells. This selective e 1 expression indicates that CG5757 is affected by the E2F-1 promoter Transcriptional regulation. The regulation of the E1B 55K gene in CG5757 has also been shown to be cancer-specific, as it is only detected in infected cancer cells and not in normal HRE cells. Therefore, it was observed that after infection with CG5757 Both E1A and E1B genes are tumor-specific, and virus replication is selective for Rb-deficient and telomerase-positive cancer cells. In order to demonstrate the tumor selectivity of CG5757, a variety of cancer cell lines (Hep3B, LoVo, A549, 253J BV, Panc-1 and Hela) 52 -200533752 and normal cells (HRE and WI-38) were analyzed for virus susceptibility E1A expression after CG5757. E 1 A expression induced by CG5757 was detected only in these cancer cells. CG5757 clearly confirmed that the tumor selectively expressed El A and E1B genes, and its products played an essential role in activating other adenovirus genes Example 11: CG5757 in vitro tumor selective cytotoxicity

The cytopathic effects induced by wild-type adenovirus OV802 and tumor-selective CG5757 virus infection in different cells were compared by microscopy and crystal violet staining. OV802 showed no lethality to cancer cells compared to normal cells, and infection with CG5757 resulted in cytolytic (kill) specificity to cancer cells. CG5757 shows 1,000 to 10,000 times less infectivity in normal cells than OV802. In vitro MTT analysis was also used to quantitatively compare the cytotoxicity of CG5757 and wild-type Ad5 (OV802) on different cells. Among the cancer cells tested, CG5757 performed a dose-response study similar to that of wild-type Ad5 (Figure 3). Under selected (for example) 1 pfu / cell MOI (Figures 3C and D), CG5757 exhibited the same killing kinetics in Hep3B cells as wild-type Ad5. Furthermore, the cytotoxicity data was analyzed using GraphPad software to perform S-shaped dose response curve fitting and determine the LD50 value. Comparing the LD50 values of wild-type adenovirus (OV802) and oncolytic vector (CG5757) shows the strength of the virus's cytotoxicity and can be used to normalize the transduction efficacy of different cells. Table 3 shows the SILD of CG575 7, that is, CG5 757 relative to Ad5 in primary cells (HRE, PrEC, HMEC, HMVEC-L, BSM, lung

FB, SAEC, WI-38) and tumor cells (Hep3B, A549, LoVo, SW480, LNCap, Panc-1, Hela and 253J B-V) are calculated using SILD 53 200533752 formula. A SILD value higher than "1" indicates tumor cell selectivity. Table 3. Selectivity index of CG5757 HRE PrEC HMEC HMVEC-L BSM Lung FB SAEC WI-38 Hep3B 1676.74 6957.82 85.50 154.79 75.76 2.27 62.99 75.76 A549 84.14 349.13 4.29 7.77 3.80 0.11 3.16 3.80 LoVo 340.18 14.11.37.35 SW 35 237.64 29.14 52.76 25.83 0.77 21.47 25.82 LNCap 115.65 479.88 5.90 10.68 5.23 0.16 4.34 5.23 Panc-1 118.55 491.93 6.05 10.94 5.36 0.16 4.45 5.36 Hela 1731.10 7183.40 88.28 159.81 78.22 2

Based on the selectivity index derived from cytotoxicity analysis using a variety of different cells, 57 of the 64 relative cytotoxicity comparisons between CG5 757 and wild-type Ad5 between tumors and normal cell lines yielded choices with values greater than 1 Sex index, proved to have strong tumor selectivity. The in vitro primary tumor culture model of Example 7 was used to determine the tissue-specific virus expression and production of CG5757 in clinically obtained tissue samples. Following the procedure described in Example 7, tissue samples isolated from primary human tumors or normal tissues were quickly placed in frozen supplements supplemented with 10% fetal bovine serum (FCS) and 100 / xg / ml penicillin and chain. Improvement of Mycin by Iscove 54 200533752

Dulbecco's Medium (IMDM) solution. Tissue samples were taken on ice in the afternoon at noon to take uniform tissue sections and prepare 1 mm3 cubes of each, and, and my samples. Rinse them and place them in a 6-slot flat plate supplemented with pcs IMDM in. Add aliquots containing 1x109 pfu CG5757 or Ad5 (also known as OV802) to a well containing appropriate tissue. After two hours, the culture medium was replaced with fresh IMDM supplemented with 5% FCS, 10 ^ M insulin, and 10 " M hydrocortisone. Tissue samples were placed on Millicell thin-film culture inserts (apertures: 0,45 mm, Mllllpore, Billica, MA) installed in each slot of a flat pan and incubated under a pit in a 5% atmosphere. At 5 & post-infection, the culture medium and the infected, and, and were combined and conjugated for a second freeze-thaw cycle. The valence of cell lysates was measured on 293 cells and the TCIDw valence of each sample was calculated. The results confirmed that CG5757 was detected in tumor colonic tissues on average more than 10 times higher than that detected in adjacent normal tissues. For example, the primary tumour isolated from a patient whose infectious CG5757 in tumor tissue is present is about 100,000 times higher than that in normal tissue. In contrast, there was no significant difference in virus production between tumors or normal tissues after infection with wild-type Ad5. To evaluate the tumor selectivity of virus production, compare the tciD5G value of CG5757 with the TCID5 of Ad5 in the same tissue (to control the difference in infectivity and virus production). Value to generate a selectivity index (as calculated above, the selectivity index obtained from each different primary tumor ranges from about 4 to more than 3000, and most samples have a selectivity index greater than or less than that. Tissue-specific virus Header; fR τ4η 1 was only confirmed by immunohistochemical staining. According to the procedure in Example 7, a part of the primary tumor samples infected with cg5757 or wild 55 200533752 Ad5 were used 24 hours after infection. Anti-E j A antibodies analyzed the performance of Ad E1A. The results showed that El A was detected only in colon tumor tissues, but not in normal tissues such as colon, pancreas and spleen. These results prove that E2F- 丨 is activated The daughter is active and selective in primary colon tumors. CG5 757 is preferentially produced in tumor tissues from tissue samples collected from most patients. These scriptures use clinical primary tissues The results obtained from in vitro experiments provide further evidence that CG5757 has a high degree of tumor selectivity. Results in vitro model Example 12: In vitro tumor selection of OV945 and OV947 Cytotoxicity

MTT assay was used to evaluate tumor-selective cytotoxicity in vitro for OV945 and OV947 in Hep3B and BSM cells. The results are shown in Table 8. The cytopathic potency index is the relative LD50 value expressed by the ratio of LD50 of OV80 obtained in the selected cells divided by LD50 of the test viral vector (Johnson et al., 2002, Cancer cells, 1: 325-337) . As shown in Table 4, these two test viruses have virtually the same potency as the OV802 tumor cell line Hep3B. On the other hand, the sample viruses were about 333 times worse than normal cell lines (BSM). Table 4 · Cytopathogenicity efficacy index "------- 〇V802 OV945 OV947 Hep3B thin month pack 1 1 1 BS Μ cells 1 0.003 0.003 56 -200533752 Example 13: In-tube tumors for CG5757, OV945 and OV947 Selective cytotoxicity

Crystal violet analysis was used to evaluate OV945 and OV947. OV802 was used as a control group. Tumor cells (Hep3B, A549, and LoVo) were infected with MOI of 1 pfu / cell. Normal cells (lung FB, IMR90, and WI-38) were infected with an MOI of 10 pfu / cell. All infections were performed in triplicate. Six days after infection with OV945, OV947, or OV802, most, if not all, cells exhibited CPE for each tumor cell line. In contrast, normal cells infected with OV945 and OV947 exhibited little (if any) CPE, but normal cells infected with OV802 exhibited complete CPE. The cells were then stained with crystal violet. Tumor cells infected with any of these three viruses showed very little staining. Except for those infected with OV802 (which shows very little staining), normal cells are stained on most surfaces. In-tube cytotoxicity MTT assay was used to compare oncolytic vectors with wild-type OV802. Table 5 summarizes the LD50 values obtained after MTT analysis of different cells. Table 5. Comparison of LD50 (pfu / cell) from different cells

Hep3B A549 LoVo SW480 LNCap Panc-1 HRE PrEC OV802 0.0003 0.0008 0.0124 0.0146 0.0018 0.0030 0.0006 0.0184 CG5757 0.0011 0.0577 0.2275 0.1601 0.0948 0.1590 3.6946 477.0356 OV945 0.0017 0.0135 0.0412 1.0753 0.8655 0.0806 0.9781 0.5058 OV947 0.0311 0.1923 0.4903 1.1968 0.7 299 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 1.3509 implementation Example 14. Selective tumor production of CG5 757. The procedure described in Example 8 was used to compare CG5 757 with wild-type virus by analysis of the amount of lysis (viral yield) and growth kinetics performed by a group of tumors and normal cells. Selective replication in test tubes (Figures 4 and 5). In the analysis of lysing amount, all cells were infected with MOI of 2 pfu / cell for 72 hours and the virus was harvested on 293 cells for plaque potency determination (Figure 4). For the study of growth kinetics, infected cells were harvested at the corresponding time points and the valence was determined (Figure 5). CG5757 shows that compared to wild

Type OV802 has a certain degree of selective replication in tumor cells. In normal cells, the amount of lysis is about 1,000 to 100,000 times lower than that of wild-type OV802. In addition, the virus growth curves of Hep3B and HRE cells (Figure 5) also indicate that CG5757 replication is similar to wild-type OV802 in cancer cells, but not in normal cells. The same analysis was performed on the viruses OV945 and OV947. In normal cells, the amount of lysis of OV945, OV947, and CG5757 was about 1,000 to 100,000 times lower than that of wild-type OV802 (Figure 6). Example 15: Selective production of tumors of OV945 and OV947. The procedure described in Example 8 was used to compare OV945 and OV947 with wild-type viruses due to the amount of lysis (virus yield) performed by various cells (including HBL 100 cells). Selective replication in test tube. See Figure 7. Example 16: CG5 75 7 and OV945-in vivo antitumor efficacy The procedure described in Example 9 was used to determine the in vivo antitumor efficacy of CG5757 and OV945. CG5757 was tested in A549 xenograft mode. CG5757 was injected on the 20th, 22nd and 22nd days after tumor implantation. 58 200533752 Figure 13 shows the antitumor efficacy of treatment group (^ 〇) than that of control group (η = 1 〇) (p < 〇〇1 by Dunnette, ^ A analysis, using PBS / glycerol as On the day, Li Ling Dao, T, and) the total virus dose was as high as 1.6 × 10 3 of the disease-free particles per kilogram of tongue “quote weight did not change compared to the control group. Α549 mode is also used to prove the anti-tumor effect of & ^ ^ ^ #. After intramuscular administration of Le OV945, the treatment group showed a statistically significant inhibitory effect on tumor growth compared to the control group. The body weight was also not significant, indicating that the toxicity after intratumoral injection had been minimized. Similar studies were performed using nude mice with subcutaneous 2S1Tu + -tumor. CG5757 or OV945 was cut and injected. Various treatments were administered via intratumoral injection of 4x10s vp / mm3. For CG5757, # — 士-% 70% are two different courses and the tumors are injected · lamp, Wang She · course 1: 20, 21, 22, 23, 24 in the following days after tumor implantation; course 2:20, 24, 27, 3Π · 30, Course 3: 20, 27, 34, 41. All CQ57S7 Nan 5 5 7 treated groups in different treatment courses had significant tumor regression compared to the control group (p < 〇. 〇1 was analyzed by Dunnette's method of A] sj Λ and Tian an 〇VA, using PBS / Glycerin is right. Folder m.

J ,,,, and J Ling are shown in Figure 10. After four weeks of treatment, the flat J 接 g volume of 253J BV tumors in animals with five consecutive tumors and large tumors> CG5757 (4xl08 particles / mm3 tumors) was reduced to 72% of baseline, and Control group

It increased to 994% of the baseline. R 0 In addition, about 50% of the animals in each treatment group showed 25 3J B-V tumors, which showed a complete regression from a _ species of spear plant, suggesting that CG5 75 7 has a strong antitumor effect. OVJ945, a 253J B-V tumor model, was tested. The results are shown in Figure 11. 59 200533752 Example 17: CG5 757: In vivo toxicity analysis The in vivo toxicity of CG5757 with wild-type Ad5 and replication-deficient Addl312 in immune-deficient SCID mice after intravenous administration was compared. SCID mice were injected with a single intravenous dose of CG5757, wild-type Ad5, replication-deficient Addl3 12, or PBS supplemented with 10% glycerol (vehicle control group) at a single intravenous dose and closely monitored the animals . No pathology was observed in the vehicle, Addl312 or CG5757 treatment groups. Individual body weight data were collected over 28 days and compared between different treatment groups. Compared with the study day, all treatment groups did not gain weight between days 3 and 8. Ad5 treated animals are here! By the 5th day, they gradually lost weight, at which point they became dying and ended. In contrast, the CG5757 treatment group increased plate weight after the first 3 to 8 days of weight loss, and the average body weight change in this group was not significant compared to the vehicle- or Addi3 j 2 treatment group. : In addition, the selected clinical monitoring was monitored on days 2, 4, 14, and 28 of the study. During the study, the observed creatine kinase (ck) was not different between any treatment groups at any time point, indicating that there was no significant toxicity in skeletal muscle, ~ muscle, thin, or kidney (Figure 4B and Data not shown). Furthermore, the serum levels of amino acid aminotransferase (ALT) and aspartate aminotransferase (ast) were apparent on the second day and were not in any treatment group. OV802) treated mice were observed on the 4th day, compared with the control group or the Addl312 treatment group, the Δ§τ and alt concentrations were significantly increased (P < 0.05). In the CG5757 treatment group, both AST and ALT increased on the 4th day of the study, but compared with those treated with vehicle or Addl312 60, 200533752 showed no significant difference. On the 14th day, all the serum parameters were returned to the positive section and 5 in total. These observations prove that Cg5757 is less toxic than the wild type and therefore has potential as a therapeutic agent.

Example 18: In-tube combination of CG5757 and chemotherapeutic agents. This study has investigated the synergistic effect between existing chemotherapeutic agents and the replication-capable carriers of the present invention. In-tube cell analysis was used to evaluate CG5757 and various known chemical agents Of co-administration. Various known chemotherapeutic agents were combined with CG5 757 to test its in vitro effects on cells in vitro and in cells of LNc ^ p and cells. Optimize the haniness of each chemotherapeutic agent to a specific concentration that is suitable for the individual = moonburst type without generating a wide range of cytotoxic effects from the agent alone. Under these conditions, certain chemotherapeutic agents exhibit a non-force-forming effect with CG5757. Among the tested agents, doxorubicin and taxol showed a potential synergistic cytotoxicity in combination with CG5757. Hydroxorodorubicin is not cytotoxic to Hep3B at 5 ng / ml, while CG5757 destroys only 30% of cells by day 7 at a MOI of 0.005 (pfu / cell); however, oxydanox Erythromycin co-administration with CG5757 produced cytotoxicity to approximately 85% of cells on the 7th day after treatment. Similar results were observed when taxol (1 ng / ml) and CG5 75 7 (moi 0.1) were co-administered to LNCaP cells. These results demonstrate that the recombinant viral vectors of the present invention can provide synergistic benefits to existing chemotherapeutic compositions and treatment methods. [Schematic description] Figure 1 A-F is a diagrammatic illustration of the structure of an exemplary pan-cancer replication-capable virus vector construct. 61 200533752 Figure 1 A-4 illustrates the structure of wild-type adenovirus serotype $. Figure 1B depicts the structure of CG5757, which sequentially contains a left melon, an adenovirus packaging signal, and a human telomerase (hTERT) artificially linked to the i EU coding region. ) Promoter wherein the Elb coding region comprises a deletion located in a muscle-coding region and a right ITR. Main figure ^ describes the structure of 0V947, which in turn contains a left m, an adenopathy without coating, a human promoter operably linked to the Eu coding region, and an Isi operably linked to the Elb coding region. , And right ITR. Main figure 1D cat describes the structure of OV945, which sequentially contains a left itr, an adenovirus packaging signal, a human reactive promoter operably linked to the Ela coding region, and an internal ribose operably linked to the Elb coding region. (IRES) where the e 1 b coding region contains a mutation located in the £ 1 b 19k coding region, and the right ITR.

FIG. 1E depicts the structure of OV948, which in sequence includes a left ITR, an adenovirus packaging signal, a hTERT promoter operably linked to the Ela coding region, a human E2F-1 promoter inserted to the E1 b coding region, And right ITR. Figure 1F depicts the structure of 〇〇〇25, which in sequence contains the left ITR, adenopathy without packaging §fl, hTERT promoter operably linked to the e 1 a coding region, and operably linked to the E 1 b coding region. Internal ribosome entry site (IRES), and right I but r. Figure 2 shows the tumor growth inhibition effect of OV945 infected A549 xenograft model. On the day after the Dinglie tumor implantation, the tumor was injected with OV945 ·· Course # 1: 20, 21, 22, 23 (triangle); Course # 2 · · 20, 23, 26, 29 (star); Course # 3:20, 27, 34, 41 (diamond). The other group of tumors was treated with the PBS-glycerol control group (square) by the course 2 method. Figures 3A-D show CG5757 compared to wild type adenovirus type 5

(OAV802) MTT cytotoxicity analysis results on Hep3B and HRE cells. Cells were infected with different doses of virus and subjected to MTT analysis at selected time points. The cytotoxicity of the virus is expressed as a percentage of the uninfected cell control group. In the figure, OAV802 is represented by a square and CG5757 is represented by a diamond. The graph shows post-infection cytotoxicity measured by MTT analysis, and is plotted as a percentage value relative to uninfected cells. Figures 3a and 3B show the cytotoxicity to Hep3B and HRE cell lines after 8 days of infection with various MOIs. Figures 3C and 3D show the cytotoxicity to Hep3B and HRE cell lines after different days of infection with MOI of 1, respectively. Figure 4 shows the selective production of CG5757 in different cell lines. Various cells were infected with CG5757 (white cylinder) or OV802 (black cylinder) with an MOI of 2 (pfu / cell) for 72 hours. The cell lysate was collected and the plaque viability assay was performed on 293 cells. Figure 5 shows the results of the growth kinetics study. CG5757 (triangle) and OV802 (square) pairs with MoI of 2 (_ / cell) were infected with cells (Figure 5A) or HRE cells (_5B) for a selected period of time. The infected cells were collected and subjected to force analysis at 293 cells at corresponding time points. Figure 6 shows the selective production of OV945 (grey bars), OV947 (white bars) 63 200533752, and CG5757 (oblique stripes bars) compared to OV802 (black bars) in different cells. Figure 7 shows the selective production of OV945 (white cylinder) and 0V947 (grey cylinder) compared to OV802 (black cylinder) in different cells.

Figure 8 shows the results of MTT analysis tested with viruses OV945 (square), OV947 (diamond), and OV802 (triangle) on Hep3B cell line (A) and BSM cell line (B). Cells were harvested on day 10 after infection for MTT analysis. Figure 9 shows the tumor growth inhibitory effect of CG5757-infected A549 xenograft model. On days 20, 21, 22, and 23 after tumor implantation, tumors were injected with CG5757 (round) or PBS-glycerol control (square). Figure 10 shows the tumor growth inhibitory effect of the 253J B-V xenograft model infected with CG5757. The tumors were injected with CG5757 on the following days after tumor implantation: Course # 1: 20, 21, 22, 23, 24 (round); Course # 2: 20, 24, 27, 30 (diamond): Course # 3: 20, 27, 34, 41 (triangle). The other group of tumors were treated with PBS-Glycerol control group (star) according to the course # 2. Figure Π shows the tumor growth inhibitory effect of the 253J B-V xenograft model infected with OV945. The tumors were injected with OV945 on the following days after tumor implantation: Course # 2: 20, 22, 22, 23, 24 (triangle); Course # 2: 20, 24, 27, 30 (round); Course # 3 ·· 20, 27, 34, 41 (diamond) ° Another group of tumors were treated with PBS-glycerol control group (square) according to course # 2. 64 -200533752 [Brief description of the sequence] The following is a description of the sequence provided by the present invention. SEQ ID NO] is a 270 bp fragment containing a sequence derived from a human E2F promoter. SEQ ID NO: 2 contains human telomerase. A 239 bp fragment of the promoter sequence. SEQ ID NO: 3 is a 245 bp fragment containing a sequence derived from a human promoter.

SEQ ID NO: 4 is a 275 1 bp fragment containing the sequence obtained from the adenoviral vector CG5 757. SEQ ID NO: 5 is a 4022 bp fragment containing the sequence obtained from the adenoviral vector OV947. SEQ ID NO: 6 is a 3207 bp fragment containing the sequence obtained from the adenoviral vector OV945. SEQ ID NO: 7 is a 4304 bp fragment containing the sequence obtained from the adenoviral vector OV1025. SEQ ID NOs: 8 and 9 are primer sequences used to amplify the E2F-1 promoter. SEQ ID NOs: 10 and 11 are primer sequences 歹 | J used to amplify the TERT promoter. SEQ ID NO ·· 12 is the Elb portion deleted in various adenoviral vectors of the invention including CG5757 and OV947. SEQ ID NO: 13 is a polyadenylation consensus DNA sequence. SEQ ID NO: 14 is a polyadenylated consensus RNA sequence. 65 200533752 SEQUENCE LIST < 110 > Yu, De Chao Li, Yuanhao < 120 > Pan-cancer oncolytic carrier and method of use < 130> 3802-210-27 < 150 > US 60 / 556,549 < 151 > 2004 -03-25 • < 160〉 14 < 170〉 Patentln Version 3.3 < 210 > 1 < 211 > 270 < 212 > DNA < 213〉 Human < 400〉 1

tggtaccatc cggacaaagc ctgcgcgcgc cccgccccgc cattggccgt accgccccgc 60 gccgccgccc catcccgccc ctcgccgccg ggtccggcgc gt taaagcca ataggaaccg 120 ccgccgttgt tcccgtcacg gccggggcag ccaattgtgg cggcgctcgg cggctcgtgg 180 ctct t tcgcg gcaaaaagga tttggcgcgt aaaagtggcc gggactttgc aggcagcggc 240 ggccgggggc ggagcgggat cgagccctcg 270 < 210 > 2 < 211> 239 < 212> DNA < 213 > human 1 200533752 < 40〇 > 2 cgtggcggag ggactgggga cccgggcacc cgtcctgccc cttcaccttc cagctccgcc tcctccgcgc ggaccccgcc ccgtcccgac ccctcccggg tccccggccc agccccctcc gggccctccc agcccctccc cttcctttcc gcggccccgc cctctcctcg cggcgcgagt ttcaggcagc gctgcgtcct gctgcgcacg tgggaagccc tggccccggc cacccccgc < 210 > 3 < 211> 245 < 212 > DNA < 213 > human < 400 > 3 ccccacgtgg cggagggact ggggacccgg gcacccgtcc tgccccttea ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc ccgacccctc ccgggtcccc ggcccagccc cctccgggcc ctcccagccc ctccccttee tttccgcggc cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc gcacgtggg a agccctggcc ccggccaccc ccgcg

≪ 210 > 4 < 211 > 2751 < 212> DNA < 213 > oncolytic adenovirus < 400 > 4 catcatcaat aaatatacct tattttggat tgaagccaat atgataatga gggggtggag tttgtgacgt ggcgcggggc gtgggaacgg ggcgggtgac gtagtagtgt ggcggaagtg tgatgttgca agtgtggcgg aacacatgta agegaeggat gtggcaaaag tgacgttttt 200533752 ggtgtgcgcc ggtgtacaca ggaagtgaca attttcgcgc ggttttaggc ggatgttgta 240 gtaaatttgg gcgtaaccga gtaagatttg gccattttcg cgggaaaact gaataagagg 300 aagtgaaatc tgaataattt tgtgttactc atagcgcgta atatttgtct agggccgcgg 360 ggactttgac cgtttacgtg accggtggta ccatccggac aaagcctgcg cgcgccccgc 420 cccgccattg gccgtaccgc cccgcgccgc cgccccatcc cgcccctcgc cgccgggtcc 480 ggcgcgttaa agccaatagg aaccgccgcc gttgttcccg tcacggccgg ggcagccaat 540 tgtggcggcg ctcggcggct cgtggctctt tcgcggcaaa aaggatttgg cgcgtaaaag 600 tggccgggac tttgcaggca gcggcggccg ggggcggagc gggatcgagc cctcgaccgg 660 tgactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga aatggccgcc 720 agtct11tgg accagctgat cgaagaggta ctggctgata atcttccacc tcctagccat 780 11 tgaaccac ctacccttea cgaactgtat gatttagacg tgacggcccc cgaagatccc 840 aaegaggagg cggtttegea gatttttccc gactctgtaa tgttggcggt gcaggaaggg 900 attgacttac tcacttttcc gccggcgccc ggttctccgg aggcgcctca cctttcccgg 960 cagcccgagc ageeggagea gagagccttg ggtccggt11 ctatgccaaa ccttgtaccg 1020 gaggtgatcg atcttacctg ccacgaggct ggetΐtccac ccagtgacga egaggatgaa 1080 gagggtgagg agtttgtgtt agattatgtg gagcaccccg ggcacggttg caggtcttgt 1140 cattatcacc ggaggaatac gggggaccca gatattatgt gttcgctttg ctatatgagg 1200 acctgtggca tgtttgtcta cagtaagtga aaattatggg cagtgggtga tagagtggtg 1260 ggtttggtgt ggtaattttt tacagttttg tggtttaaag aattttgtat 1320 1380 200533752 tgtgattttt ttaaaaggtc ctgtgtctga acctgagcct gagcccgagc cagaaccgga gcctgcaaga cctacccgcc gtcctaaaat ggcgcctgct atcctgagac gcccgacgtc acctgtgtct atagtagtac ggatagctgt gactccggtc cttctaacac acctcctgag atacacccgg tggtcccgct gtgccccatt aaaccagttg ccgtgagagt tggtgggcgt cgccaggctg tggaatgtat cgaggacttg cttaacgagc ctgggcaacc 11tggacttg agctgtaa ttttaatttt agagaatgca ac gccccaggcc ataaggtgta aacctgtgat tgcgtgtgtg 9 gttaacgcct ttgtttgctg aatggtcgac cggtaccgtg gcggagggac tggggacccg ggcacccgtc ctgccccttc accttccagc tccgcctcct ccgcgcggac cccgccccgt cccgacccct cccgggtccc cggcccagcc ccctccgggc cctcccagcc cctccccttc ctttccgcgg ccccgccctc tcctcgcggc gcgagtttca ggcagcgctg cgtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcaccgg tcgacgcgct gcggctgctg t tgctttttt gagttttata aaggataaat ggagcgaaga aacccatctg agcggggggt acctgctgga ttttctggcc atgcatctgt ggagagcggt tgtgagacac aagaatcgcc tgctactgit gtcttccgtc cgcccggcga taataccgac ggaggagcag cagcagcagc aggaggaagc caggcggcgg cggcaggagc agagcccatg gaacccgaga gccggcctgg accctcggga atgaatgttg tacaggtggc tgaactgtat ccagaactga gacgcatttt gacaattaca gaggatgggc aggggctaaa gggggtaaag agggagcggg gggcttgtga ggctacagag gaggctagga atctagcttt tagcttaatg accagacacc gtcctgagtg tattactttt caacagatca aggataattg cgctaatgag cttgatctgc tggcgcagaa 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 4 200533752 gtattccata gagcagctga ccacttactg gctgcagcca ggggatgatt ttgaggaggc tattagggta tatgcaaagg tggcacttag gccagattgc aagtacaaga tcagcaaact tgtaaatatc aggaattgtt gctacatttc tgggaacggg gccgaggtgg agatagatac ggaggatagg gtggccttta gatgtagcat gataaatatg tggccggggg tgcttggcat ggacggggtg gttattatga atgtaaggtt tactggcccc aattttagcg g

• < 210 > 5 < 211> 4020 < 212> DNA < 213 > oncolytic adenovirus < 4〇〇 > 5 caicatcaat aaatatacct tattttggat tgaagccaat atgataatga gggggtggag tttgtgacgt ggcgcggggc gtgggaacgg ggcgggtgac gtagtagtgt ggcggaagtg tgatgttgca agtgtggcgg aacacatgta agcgacggat gtggcaaaag tgacgttttt ggtgtgcgcc ggtgtacaca ggaagtgaca attttcgcgc ggttttaggc ggatgttgta ^^ gtaaatttgg gcgtaaccga gtaagatttg gccattttcg cgggaaaact gaataagagg aagtgaaatc tgaataatΐΐ tgtgttactc atagcgcgta atatttgtct agggccgcgg ggactttgac cgtttacgtg accggtggta ccatccggac aaagcctgcg cgcgccccgc cccgccattg gccgtaccgc cccgcgccgc cgccccatcc cgcccctcgc cgccgggtcc ggcgcgttaa agccaatagg aaccgccgcc gttgttcccg tcacggccgg ggcagccaat tgtggcggcg ctcggcggct cgtggctctt tcgcggcaaa aaggatttgg cgcgtaaaag 2520 2580 2640 2700 2751 60 120 180 240 300 360 420 480 540 600 660 200533752 tggccgggac tttgcaggca gcggcggccg ggggcggagc gggatcgagc cctcgaccgg tgactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga aatggccgcc agtctttttgg acc gta ctggctgata atcttccacc tcctagccat tttgaaccac ctacccttea cgaactgtat gatttagacg tgacggcccc cgaagatccc aaegaggagg cggtttcgca gatttttccc gactctgtaa tgttggcggt gcaggaaggg attgacttac tcacttttcc gccggcgccc ggttctccgg aggcgcctca cctttcccgg cagcccgagc ageeggagea gagageettg ggtccggttt ctatgccaaa ccttgiaccg gaggtgatcg atcttacctg ccacgaggct ggetttccac ccagtgacga egaggatgaa gagggtgagg agtttgtgtt agattatgtg gagcaccccg ggcacggttg caggtcttgt cattatcacc ggaggaatac gggggaccca gatattatgt gttcgctttg ctatatgagg acctgtggca tgtttgtcta cagtaagtga aaattatggg cagtgggtga tagagtggtg ggtttggtgt ggtaattttt ttttaatttt tacagttttg tggtttaaag aattttgtat tgtgat1111 ttaaaaggtc ctgtgtctga acctgagcct gagcccgagc cagaaccgga gcctgcaaga cctacccgcc gtcctaaaat ggcgcctgct atcctgagac gcccgacgtc acctgtgtct agagaatgea atagtagtac ggatagctgt gactccggtc cttctaacac acctcctgag atacacccgg tggtcccgct gtgccccatt aaaccagttg ccgtgagagt tggtgggcgt cgccaggctg egaggaettg ettaaegage ctgggcaacc tttggacttg agctgtaaac gccccaggcc ataagg tggaatgtat tgta aacctgtgat tgcgtgtgtg gttaacgcct ttgtttgctg aatggtcgac cggtaccgtg geggagggae tggggacccg 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 200533752 ggcacccgtc ctgccccttc accttccagc tccgcctcct ccgcgcggac cccgccccgt cccgacccct cccgggtccc cggcccagcc ccctccgggc cctcccagcc cctccccttc ctitccgcgg ccccgccctc tcctcgcggc gcgagtttca ggcagcgctg cgtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcaccgg tcgacatgga ggcttgggag tgtttggaag atttttctgc tgtgcgtaac ttgctggaac agagctctaa cagtacctct tggttttgga ggtttctgtg gggctcatcc caggcaaagt tagtctgcag aattaaggag ^ gattacaagt gggaatttga agagcttttg aaatcctgtg gtgagctgtt tgattctttg aatctgggtc accaggcgct tttccaagag aaggtcatca agactttgga tttttccaca ccggggcgcg ctgcggctgc tgttgctttt ttgagtttta taaaggataa atggagcgaa gaaacccatc tgagcggggg gtacctgctg gattttctgg ccatgcatct gtggagagcg gttgtgagac acaagaatcg cctgctactg ttgtcttccg tccgcccggc gataataccg acggaggagc agcagcagca gcaggaggaa gccaggcggc ggcggcagga gcagagccca tggaacccga gagccgg cct ggaccctcgg gaatgaatgt tgtacaggtg gctgaactgt ^^ atccagaact gagacgcatt ttgacaatta cagaggatgg gcaggggcta aagggggtaa agagggagcg gggggcttgt gaggctacag aggaggctag gaatctagct tttagcttaa tgaccagaca ccgtcctgag tgtattactt ttcaacagat caaggataat tgcgctaatg agcttgatct gctggcgcag aagtattcca tagagcagct gaccacttac tggctgcagc caggggatga ttttgaggag gctattaggg tatatgcaaa ggtggcactt aggccagatt gcaagtacaa gatcagcaaa cttgtaaata tcaggaattg ttgctacatt tctgggaacg 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 200533752 gggccgaggt ggagatagat acggaggata gggtggcctt tagatgtagc atgataaata tgtggccggg ggtgcttggc atggacgggg tggttattat gaatgtaagg tttactggcc ccaattttag cggtacggtt ttcctggcca ataccaacct tatcctacac ggtgtaagct tctatgggtt taacaatacc tgtgtggaag cctggaccga tgtaagggtt cggggctgtg ccttttactg ctgctggaag ggggtggtgt gtcgccccaa aagcagggct tcaattaaga aatgcctctt tgaaaggtgt accttgggta tcctgtctga gggtaactcc agggtgcgcc ^ acaatgtggc ctccgactgt ggttgcttca tgctagtgaa aagcgtg gct gtgattaagc ataacatggt atgtggcaac tgcgaggaca gggcctctca gatgctgacc tgctcggacg gcaactgtca cctgctgaag accattcacg tagccagcca ctctcgcaag gcctggccag tgtttgagca taacatactg acccgctgtt ccttgcattt gggtaacagg aggggggtgt tcctacctta ttgagtcaca ctaagatatt gettgagccc gagageatgt ccaaggtgaa cctgaacggg gtgtttgaca tgaccatgaa gatctggaag gtgctgaggt aegatgagae ccgcaccagg tgcagaccct gcgagtgtgg cggtaaacat gtttggetet agegatgaag atacagattg ccaatgcaat attaggaacc agcctgtgat gctggatgtg accgaggagc tgaggcccga tcacttggtg ctggcctgca cccgcgctga aggtactgaa atgtgtgggc gtggcttaag ggtgggaaag aatatataag gtgggggtct tatgtagttt tgtatctgtt t tgeageage cgccgccgcc atgagcacca actcgtttga tggaageatt gtgagctcat atttgacaac gcgcatgccc ccatgggccg gggtgcgtca gaatgtgatg ggctccagca ttgatggtcg ccccgtcctg cccgcaaact ctactacctt gacctacgag accgtgtctg 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 .200533752

≪ 210> 6 < 211> 3207 < 212> DNA < 213 > oncolytic adenovirus < 400> 6 catcatcaat aaatatacct tattttggat tgaagccaat atgataatga gggggtggag tttgtgacgt ggcgcggggc gtgggaacgg ggcgggtgac gtagtagtgt ggcggaagtg tgatgttgca agtgtggcgg aacacatgta agcgacggat gtggcaaaag tgacgttttt 9 ggtgtgcgcc ggtgtacaca ggaagtgaca attttcgcgc ggttttaggc ggatgttgta gtaaatttgg gcgtaaccga gtaagatttg gccattttcg cgggaaaact gaataagagg aagtgaaatc tgaataat11 tgtgttactc atagcgcgta atatttgtct agggccgcgg ggactttgac cgtttacgtg accggtggta ccatccggac aaagcctgcg cgcgccccgc cccgccattg gccgtaccgc cccgcgccgc cgccccatcc cgcccctcgc cgccgggtcc ggcgcgttaa agccaatagg aaccgccgcc gttgttcccg tcacggccgg ggcagccaat tgtggcggcg ctcggcggct cgtggctctt tcgcggcaaa aaggatttgg cgcgtaaaag tggccgggac tttgcaggca gcggcggccg ggggcggagc gggatcgagc cctcgaccgg tgactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga aatggccgcc agtct11tgg accagctgat cgaagaggta ctggctgata atcttccacc tcctagccat tttgaaccac ctacccttca cgaactgtat gatttagacg tgacggccc c cgaagatccc aacgaggagg cggtttcgca gatttttccc gactctgtaa tgttggcggt gcaggaaggg attgacttac tcacttttcc gccggcgccc ggttctccgg aggcgcctca cctttcccgg 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 9 1020 200533752 cagcccgagc agccggagca gagagccttg ggtccggttt ctatgccaaa ccttgtaccg gaggtgatcg atcttacctg ccacgaggct ggctttccac ccagtgacga cgaggatgaa gagggtgagg agtttgtgtt agattatgtg gagcaccccg ggcacggttg caggtcttgt cattatcacc ggaggaatac gggggaccca gatattatgt gttcgctttg ctatatgagg acctgtggca tgtttgtcta cagtaagtga aaattatggg cagtgggtga tagagtggtg ggtttggtgt ggtaattttt ttttaatttt tacagttttg tggtttaaag aattttgtat tgtgattttt ttaaaaggtc ctgtgtctga acctgagcct gagcccgagc cagaaccgga gcctgcaaga cctacccgcc gtcctaaaat ggcgcctgct atcctgagac gcccgacgtc acctgtgtct agagaatgca atagtagtac ggatagctgt gactccggtc cttctaacac acctcctgag atacacccgg tggtcccgct gtgccccatt aaaccagttg ccgtgagagt tggtgggcgt cgccaggctg tggaatgtat cgaggacttg cttaacgagc ctgggcaacc tttggacttg agctgtaaac gccccaggcc ataaggtgta aacct gtgat tgcgtgtgtg ^^ gttaacgcct ttgtttgctg aatggtcgac taattccggt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 I860 1920 1980 2040 2100 10 .200533752 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgtcgacgc gctgcggctg ctgttgcttt tttgagtttt ataaaggata aatggagcga agaaacccat ctgagcgggg ggtacctgct ggattttctg gccatgcatc tgtggagagc ggttgtgaga cacaagaatc gcctgctact gttgtcttcc gtccgcccgg cgataatacc gacggaggag cagcagcagc agcaggagga agccaggcgg cggcggcagg agcagagccc atggaacccg agagccggcc tggaccct cg ggaatgaatg ttgtacaggt ggctgaactg tatccagaac tgagacgcat tttgacaatt acagaggatg ggcaggggct aaagggggta aagagggagc ggggggcttg tgaggctaca gaggaggcta ggaatctagc ttttagctta atgaccagac accgtcctga gtgtattact tttcaacaga tcaaggataa ttgcgctaat gagcttgatc tgctggcgca gaagtattcc atagagcagc tgaccactta ctggctgcag ccaggggatg at 11 tgagga ggctat tagg gtatatgcaa aggtggcact taggccagat tgcaagtaca agatcagcaa acttgtaaat atcaggaatt gttgctacat ttctgggaac ggggccgagg tggagataga tacggaggat agggtggcct ttagatgtag catgataaat atgtggccgg gggtgcttgg catggacggg gtggttatta tgaatgtaag gtttactggc cccaatttta gcggtacggt tttcctggcc aataccaacc ttatcctaca cggtgtaagc ttctatgggt ttaacaatac ctgtgtggaa gcctggaccg atgtaagggt tcggggctgt gccttttact gctgctggaa gggggtggtg tgtcgcccca aaagcagggc ttcaattaag aaatgcctct ttgaaaggtg taccttgggt atcctgt 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3207 11 200533752

< 2] 〇 > 7 < 2Π > 4304 < 212 > DNA < 213> tgaataattt tgtgttactc ggactttgac cgtttacgtg accggtaccg tcctgcccct tcaccttcca gctccgcctc ctcccgggtc cccggcccag ccccctccgg, y ggccccgccc tctcctcgcg gcgcgagttt ggaagccctg gccccggcca cccccgcacc acggaggtgt tattaccgaa gaaatggccg tactggctga taatcttcca cctcctagcc atgatΐtaga cgtgacggcc cccgaagatc ccgactctgt aatgttggcg gtgcaggaag tgaagccaat atgataatga gggggtggag 60 ggcgggtgac gtagtagtgt ggcggaagtg 120 agcgacggat gtggcaaaag tgacgtίΐΐΐ 180 attttcgcgc ggttttaggc ggatgttgta 240 gccattttcg cgggaaaact gaataagagg 300 atagcgcgta atatttgtct agggccgcgg 360 tggcggaggg actggggacc cgggcacccg 420 ctccgcgcgg accccgcccc gtcccgaccc 480 gccctcccag cccctcccct tcct t tgggt gtgggtctgggt acat attatctgcc 660 ccagtctttt ggaccagctg atcgaagagg 720 attttgaacc acctaccctt cacgaactgt 780 ccaacgagga ggcggtttcg cagatttttc 840 ggattgactt actcactttt ccgccggcgc 900 12 960 ^ 200533752 ccggttctcc ggagccgcct cacctttccc ggcagcccga gcagccggag cagagagcct tgggtccggt ttctatgcca aaccttgtac cggaggtgat cgatcttacc tgccacgagg ctggctttcc acccagtgac gacgaggatg aagagggtga ggagtttgtg ttagaitatg tggagcaccc cgggcacggt tgcaggtctt gtcattatca ccggaggaat acgggggacc cagatattat gtgttcgctt tgctatatga ggacctgtgg catgtttgtc tacagtaagt gaaaattatg ggcagtgggt gatagagtgg tgggtttggt gtggtaattt tttttttaat ttttacagtt ttgtggttta aagaattttg tattgtgatt tttttaaaag gtccigtgtc tgaacctgag cctgagcccg agccagaacc ggagcctgca agacctaccc gccgtcctaa aatggcgcct gctatcctga gacgcccgac atcacctgtg tctagagaat gcaatagtag tacggatagc tgtgactccg gtccttctaa cacacctcct gagatacacc cggtggtccc gctgtgcccc attaaaccag ttgccgtgag agttggtggg cgtcgccagg ctgtggaatg tatcgaggac ttgcttaacg agcctgggca acctttggac ttgagctgta aacgccccag gccataaggt gtaaacc tgt gattgcgtgt gtggttaacg cctttgtttg ctgaatggtc, gactaattcc ggttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 13 -200533752 ctgaaggatg: cccagaaggt accccat tgt atgggatctg atctggggcc tcggtgcaca 2100 tgct t tacat gtgtttagtc gaggttaaaa aacgtctagg ccccccgaac cacggggacg 2160 tggt 11 tcct ΐtgaaaaaca cgatgtcgac atggaggctt gggagtgttt ggaagatttt 2220 tctgctgtgc gtaact tgct ggaacagagc tctaacagta cctcttggtt ttggaggttt 2280 ctgtggggct catcccaggc aaagttagtc tgcagaatta aggaggatta caagtgggaa 2340 tttgaagagc ttttgaaatc ctgtggtgag ctgtttgatt ctttgaatct gggtcaccag 2400 ^ gcgcttt tcc aagagaaggt catcaagact ttggattttt ccacaccggg gcgcgctgcg 2460 gctgctgttg cttttttgag 111tataaag gataaatgga gcgaagaaac ccatctgagc 2520 ggggggtacc tgctggattt tctggccatg catctgtgga gagcggttgt gagacacaag 2580 aatcgcctgc tactgttgtc t tccgtccgc ccggcgataa taccgacgga ggagcagcag 2640 cagcagcagg aggaagccag gcggcggcgg caggagcaga gcccatggaa cccgagagcc 2700 ggcctggacc ctcgggaatg aatgttgtac aggtggctga actgtatcca gaactgagac 2760 gcat 11 tgac aattacagag gatgggcagg ggctaaaggg ggtaaagagg gagcgggggg 2820 qe cttgtgaggc tacagaggag gctaggaatc tagcttttag cttaatgacc agacaccgtc 2880 ctgagtgtat tacttttcaa cagatcaagg ataattgcgc taatgagctt gatctgctgg 2940 cgcagaagta t tccatagag cagctgacca cttactggct gcagccaggg gatgattttg 3000 aggaggctat tagggtatat gcaaaggtgg cacttaggcc agattgcaag tacaagatca 3060 gcaaact tgt aaatatcagg aattgttgct acatttctgg gaacggggcc gaggtggaga 3120 tagatacgga ggatagggtg gcctttagat gtagcatgat aaatatgtgg ccgggggtgc 3180 14 200533752 ttggcatgga cggggtggtt attatgaatg taaggtttac tggcc ccaat tttagcggta cggttttcct ggccaatacc aaccttatcc tacacggtgt aagcttctat gggtttaaca atacctgtgt ggaagcctgg accgatgtaa gggttcgggg ctgtgccttt tactgctgct ggaagggggt ggtgtgtcgc cccaaaagca gggcttcaat taagaaatgc ctctttgaaa ggtgtacctt gggtatcctg tctgagggta actccagggt gcgccacaat gtggcctccg actgtggttg cttcatgcta gtgaaaagcg tggctgtgat taagcataac atggtatgtg • gcaactgcga ggacagggcc tctcagatgc tgacctgctc ggacggcaac tgtcacctgc tgaagaccat tcacgtagcc agccactctc gcaaggcctg gccagtgttt gagcataaca tactgacccg ctgttccttg catttgggta acaggagggg ggtgttecta ccttaccaat gcaatttgag tcacactaag atattgcttg agcccgagag catgtccaag gtgaacctga acggggtgtt tgacatgacc atgaagatet ggaaggtgct gaggtaegat gagac'ccgca ccaggtgcag accctgcgag tgtggcggta aacatattag gaaccagcct gtgatgctgg atgtgaccga ggagctgagg cccgatcact tggtgctggc ctgcacccgc gctgagtttg f getetagega tgaagataca gattgaggta ctgaaatgtg ttaagggtgg gaaagaatat ataaggtggg ggtettatgt agttttgtat ctgttttgca gcagccgccg ccgccatgag caccaactcg 11tgatggaa gcattgtgag ctcatatt tgggcgtggc tg acaacgcgca tgcccccatg ggccggggtg cgtcagaatg tgatgggctc cagcattgat ggtcgccccg tcctgcccgc aaactctact accttgacct acgagaccgt gtctggaacg ccgttggaga ctgcagcctc cgccgccgct tcagccgctg cagccaccgc ccgc 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4304 200533752 < 210> 8 < 211 > 34 < 212> DNA < 2] 3 > synthetic oligonucleotide < 400 > δ ataccggtgg taccatccgg acaaagcctg cgcg 34 < 210> 9 < 211> 29 < 212 > DNA < 213: > synthetic oligo Nucleotide < 400 > 9 agaccggtcg agggctcgat cccgctccg 29 < 210 > 10 < 211〉 35 < 212> DNA < 213 > Synthetic oligonucleotide < 400 > 10 aagtcgaccg gtaccgtggc ggagggactg gggac 35 &lt210; 11 < 211 > 34 < 212 > DNA synthetic oligonucleotides < 400 > 11 aagtcgaccg gtgcgggggt ggccggggcc aggg < 210 > 12 16 34 .200533752

≪ 211 > 261 < 212 > DNA < 213 > adenovirus < 400> 12 atggaggctt gggagtgttt ggaagatttt tctgctgtgc gtaacttgct ggaacagagc 60 tctaacagta cctcttggtt ttggaggttt Ctgtggggct catcccaggc aaagttagtc 120 tgcagaatta aggaggatta caagtgggaa tttgaagagc ttttgaaatc ctgtggtgag 180 ctgtttgatt ctttgaatct gggtcaccag gcgcttttcc aagagaaggt catcaagact 240 ttggattttt ccacaccggg g 261

< 210 > 13 < 211〉 6 < 212〉 DNA < 213 > synthetic oligonucleotide < 400 > 13 aataaa 6

m < 210 > 14 plants, < 211〉 6 < 212> RNA < 213 > synthetic oligonucleotides < 400 > 14 aauaaa 6 17

Claims (1)

200533752 10. Scope of patent application: 1. A recombinant viral vector comprising an adenovirus nucleic acid backbone, wherein the nucleic acid backbone comprises: a left ITR, an adenovirus packaging signal, and an E2F reactive promoter operably linked to the Ela coding region 2. A transcriptional element and a right ITR operatively linked to the Elb coding region, wherein the transcriptional element is selected from the group consisting of a TERT promoter and Xunbei. 2. According to the scope of patent application! The recombinant viral vector of the item, wherein the E2F-reactive promoter is a human E2F-1 promoter. 3. The recombinant viral vector according to claim 1 in the patent scope, wherein the human E2F-1 promoter comprises seq ID NO: 1. … 4. Recombinant viral vector according to item 丨 of the patent scope, wherein the transcription factor of this work 11 linked to the E1 b coding region is IRES.… 5. Recombinant viral vector according to item 丨 of patent scope Body, which makes the CD operatively linked to the Elb coding region as the TErt promoter. 〇 · According to the scope of the patent application The TERT promoter is a human TERT promoter TERT :: Recombinant viral vector claimed in claim 5, wherein the promoter comprises SEQ ID NO ·· 2 or SEQ ID NO ·· 3. __8 · According to the recombinant virus vector in the scope of the patent application, the middle to the second = free left ITR, adenovirus packaging signal, Ela coding area, 、, 届 [^ domain, and female and right 1TR Institute The group of adenoviral elements was derived from adenopathy maternal serotype 5 (Ad5). 9. According to the recombinant virus vector of the member of the scope of patent application, the further step 66-200533752 includes a mutation or deletion in the E 3 coding region. 10. According to the recombinant virus vector of the scope of the patent application, at least one E3 coding region has been deleted from the backbone. Π. A recombinant viral vector according to item 丨 of the application, wherein the E3 region in the backbone encodes at least one natural E3 protein. 12. Recombinant viral vector according to item u of the patent application scope, wherein the E3 coding region is selected from the group consisting of E3-6 7KDa, gpl9KDa, η · 6ΚΕ ^ (ADP), 10.4 KDa (RID〇〇, 14.5 KDa (RID0) And E3] 4.7Kda group. 13. According to the recombinant virus vector of the scope of the patent application, the E3 region located in the backbone encodes all natural E3 proteins. 14. According to the scope of the patent application Item 15. The recombinant viral vector further comprising a mutation or deletion in the Elb gene. 15. The recombinant viral vector according to item 14 of the application, wherein the mutation or deletion results in an active 19-protein expressed by the wild-type Elb gene Loss. 16. The recombinant viral vector according to item 15 of the scope of the patent application, wherein the deletion in the Elb gene contains SEq ID NO: 12. 1 • The recombinant viral vector according to item 1 of the scope of patent application, which comprises SEQ ID NO: 4 or SEQ ID NO: 5. 18. Recombinant viral vector according to item 丨 of the scope of patent application, which comprises SEQ ID NO: 6. 19. Recombinant viral vector according to item 丨 scope of patent application , Which contains SEQ ID NO: 7 〇67 200533752 2 0. According to the patent application, the recombinant virus vector of item 1 of the target is further included, which further comprises a transgene. 21. A pharmaceutical composition comprising The adenoviral vector of item 17 and a pharmaceutically acceptable carrier. 22 · -A pharmaceutical composition comprising the adenoviral vector of item 18 according to the scope of the patent application and a pharmaceutically acceptable carrier. 23. -Medicine A composition comprising an adenoviral vector and a pharmaceutically acceptable carrier according to item 19 of the scope of application for a patent. 24. A method for treating a host organism with a neoplastic condition by limulus / alpha, comprising: a therapeutically effective amount The composition according to item (1) of the scope of the applied patent is then happy to be given to the host organism. The method containing ▲ 庵 l · right treatment of a host organism with a neoplastic condition includes "" therapeutic effective amount basis Application for exclusive administration to the host organism. Composition 26. —A host for the treatment of tumorous conditions Containing a method for treating an upper-right object, its effective method is to administer the host organism according to the scope of the patent application]. The composition of the item wherein the tumor 27. The therapeutic condition according to item 24 of the scope of the patent application is lung cancer, breast puncture, ^ ^ treatment method Li cancer, gangliocarcinoma, or colon cancer. Among the tumors, the tumors 28. According to the scope of application for patent No. 25 cure pain: Sexual conditions are lung cancer, breast cancer, h treatment method Li cancer, keloid cancer, or colon cancer. 29. Sexually transmitted diseases according to item% of the range of φ; the original method; cancer, breast cancer, prostate cancer, or colon cancer. The investment 30. The treatment method according to item 27 of the patent application 68 200533752 The drug is injected by tumor. 31. The treatment method according to item 28 of the scope of patent application, wherein the administration is via intratumoral injection. 32. The treatment method according to item 29 of the scope of application, wherein the administration is by intratumoral injection. 33. A method for selectively lysing cancer cells, comprising contacting a population of cells with an effective amount of an adenovirus vector comprising an adenovirus nucleic acid backbone, The contact is caused by the adenoviral vector to infect the cells of the cell group, resulting in selective cell lysis of cancer cells in the cell group. The nucleic acid backbone contains a sequence of adenoviruses and is operably linked to the Ela code. The E2f responsive promoter in the region, which is operably linked to the E1 b coding region recording element, is selected from the group consisting of IRES. A transcription-acting element and a right ITR, wherein the TERT promoter, an E2F-reactive promoter, and a method thereof are used, wherein the operationally-reactive promoter is human E2F-34. According to the scope of application for patent, it is operatively linked to E2f 1 promoter in the E1 a coding region. Wherein the human • The method according to item 34 of the scope of patent application The promoter comprises the method of item SEQ ID NO: 1, wherein the operational element is IRES. The method of the above item, wherein the operational element is a TERT promoter. Item method, wherein the TERT 36. Transcribed to the Elb coding region in accordance with the scope of the patent application 37. Transcribed to the Elb coding region in accordance with the scope of the patent application 33. 38 According to the scope of the patent application 37 69- The 200533752 promoter is the human TERT promoter. The method of clause, wherein the human SEQ ID NO. · 3. Method, wherein the operated element is an E2F reactive promoter 39. According to the scope of the patent application, the 38 TERT promoter contains SEQ ID NO. 2 or 40. According to the scope of the patent application, it is linked to the E1 b coding region. Transcription, promoter 0 41. The method according to item 4 ^ U of the scope of patent application, wherein the E2F reactive promoter is a human E2F- promoter. 42. The method of claim 41, wherein the human E2FL promoter comprises SEQ ID NO: 1. 4 3 · According to the method of applying for the patent @ 丁 月 号 ~ item 33, it further includes a mutation or deletion in the E3 coding region. 44. The method according to item% of the patent application scope, wherein at least one E3 coding region has been deleted from the skeleton. 45. The method according to item 3 of the scope of patent application, wherein the E3 region located in the skeleton encodes at least one natural E3 protein. 46. The recombinant viral vector according to item 1 of the scope of patent application, which further includes mutations or deletions in the Elb gene. 4 7 · The method according to item 33 of the scope of patent application, wherein the mutation or deletion results in the activity expressed by the wild-type Elb gene! 9kD protein lost 0 4 8 · According to the method of claim 33 in the scope of the patent application, wherein the deletion of 5x in the E1 b gene includes SEQ ID NO: 12. 4 9 · The method according to item 33 of the scope of patent application, wherein the adenovirus 70 200533752 vector comprises SEQ ID NO: 4 or SEQ ID NO: 5. &Apos; wherein the adenovirus, wherein the adenovirus 50. The method according to item 33 of the patent application, the vector comprises SEQ ID NO: 6. 5 1. Method according to item 33 of the scope of patent application The vector comprises SEQ ID NO: 7. Η ^ 一 、 Schematic: Like the next page. 71