JP2016160249A - Oncolytic modified adenovirus, modified virus for disease treatment, and virus preparation containing these - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide modified viruses for treating diseases that selectively exert cell-killing activity on diseases associated with cancer cells or the cells accelerating stabilization of ARE-mRNA, such as osteoporosis, rheumatism, viral disease and inflammatory disease.SOLUTION: The invention provides an antitumor adenovirus formulation (described as dl355 in Fig. 1) containing a modified adenovirus with E4orf6 encoding an E4orf6 protein in which C-terminal region is deleted or a modified E4orf6 protein consisting of an amino acid sequence represented by a particular sequence.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a modified adenovirus having a specific cell-killing activity against cancer cells, a disease-modified virus having a specific cell-killing activity against a specific host cell, and an AU-rich element incorporated in the genome. It relates to virus preparations containing these.

  In the treatment of cancer, the development of pharmaceutical therapies that selectively attack cancer cells and do not attack normal cells or cause severe side effects to patients remains a need and difficult challenge . Much of the previous approaches to this problem has been to provide low molecular weight compounds or specific antibodies that target proteins that are characteristically expressed in cancer cells.

  In recent years, therapeutic methods different from such molecular targeting approaches have been studied for practical use. One of them is cancer treatment using so-called oncolytic virus. This treatment method can be expressed as a method of degenerating a tumor by selectively killing cancer cells using a modified pathogenic virus modified to grow only in cancer cells.

  A typical example of an oncolytic virus is an adenovirus vector lacking a gene encoding E1B55K called Onyx-015 (Non-patent Document 1). This virus cannot express the 55 kilodalton protein (E1B55K) encoded by the E1B gene, and includes a host cell in which the tumor suppressor gene p53 does not function normally (including cells expressing the mutant p53) ) Shows a cell killing effect.

  The mechanism by which Onyx-015 exhibits a cytocidal effect on host cells in which p53 does not function normally is currently considered as follows. While cells infected with adenovirus fall into apoptosis through the activation of p53 and die to suppress virus growth, adenovirus E1B55K binds to p53 and suppresses apoptosis. Cells enter S phase without undergoing apoptosis, resulting in adenovirus growth. However, Onyx-015, an adenovirus that cannot express E1B55K, cannot suppress apoptosis due to p53 in normal cells and cannot proliferate, but in cells where p53 does not function normally, Since it can proliferate without change, the cell killing effect is finally exerted on the infected cells.

  Thus, Onyx-015 is theoretically expected to have a cytocidal effect on cells in which p53 does not function normally, typically cancer cells, and has been confirmed empirically. In recent years, clinical trials for Onyx-015 for head and neck squamous cell carcinoma have also been promoted (Non-patent Document 2).

  However, cancer cells in which p53 does not function normally are said to be about 50% of all cancer types, and a problem is pointed out that the application range of Onyx-015 is limited to these cancer types. Regardless of the theoretical mechanism as described above, Onyx-015 can be observed to grow even in some cells in which p53 is wild-type, while it grows in some cancer cells in which p53 does not function normally. Phenomena such as not being recognized are reported. For this reason, a virus that specifically propagates in cancer cells and exhibits a cell-killing effect has been developed by a mechanism different from p53.

  Patent Document 1 discloses an oncolytic virus that is a modified adenovirus containing an E1A gene in which a heterologous destabilizing factor is inserted in the 3 'untranslated region. A modified adenovirus having an E1A gene introduced with a heterologous destabilizing factor cannot proliferate because mRNA transcribed from the gene is destabilized in a normal host cell, but P-MAPK activity is not improved. Since the destabilization is resolved and proliferates in the enhanced cancer cell, it can exhibit a cell-killing activity. This modified adenovirus is one of the oncolytic viruses independent of p53 mutation.

  Patent Document 2 discloses a modified adenovirus in which mutations are introduced into both the Rb binding site of E1A protein and E4orf6 / 7. This modified adenovirus can selectively grow in cancer cells lacking control of E2F and cell cycle checkpoints or cancer cells in which the Rb / p16 tumor suppressor pathway is inactivated.

Bischoff et al., Science, 1996, 274, 373-376. Ganly et al., Clin. Cancer Res. 2000, Volume 6, Pages 798-806

International Publication No. 2004/042015 Pamphlet International Publication No. 2014/153204 Pamphlet

  The present invention relates to an oncolytic virus that does not depend on p53, which selectively shows a killing effect on cancer cells, and a modified virus that shows a selective killing activity on cells associated with a specific disease. It is intended to provide.

  The present inventors focused on the fact that an adenovirus having E4orf6 encoding an E4orf6 protein lacking the C-terminal region exhibits a cell-killing activity against cancer cells, and has a cell-killing activity against normal cells. We worked on the construction of more suppressed viruses and completed the following inventions.

(1) An antitumor adenovirus preparation comprising an E4orf6 protein deleted from the C-terminal region or a modified adenovirus having E4orf6 encoding a modified E4orf6 protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
(2) The antitumor adenovirus preparation according to (1), wherein the E4orf6 protein from which the C-terminal region has been deleted is a protein in which amino acids from the 158th to the C-terminal of the amino acid sequence of the E4orf6 protein have been deleted.
(3) The antitumor according to (1) or (2), wherein an AU-rich element sequence is introduced into the 3 ′ untranslated region of the E1A gene or E1B gene or at a position adjacent to the 3 ′ untranslated region Adenovirus preparation.
(4) AU-rich containing a disease-treating modified virus having an AU-rich element sequence introduced in the 3 ′ untranslated region of a viral gene essential for self growth or in a position adjacent to the 3 ′ untranslated region Virus preparations for treatment of diseases for treating diseases involving cells with enhanced stabilization of mRNA containing element sequences (except for anti-tumor virus preparations in which the virus is an adenovirus).
(5) The virus preparation for disease treatment according to (4), wherein the disease is a malignant tumor, osteoporosis, rheumatism, viral disease or inflammatory disease.
(6) The virus is selected from the group consisting of adenovirus, herpes virus, measles virus, mumps virus, influenza virus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, reovirus, poxvirus, Sendai virus and enterovirus. The virus preparation for disease treatment according to (4) or (5), which is a virus.

  According to the present invention, an anti-tumor virus preparation comprising an oncolytic virus that is applicable not only to cancers having p53 mutations but also to a wider variety of cancers and that exhibits increased specificity for cancer cells. Can be provided. Moreover, the viral preparation for disease treatment of the present invention can be a therapeutic drug having an unprecedented mechanism of action against cancer, osteoporosis, rheumatism, viral disease or inflammatory disease.

It is a graph which shows selective cell-killing activity with respect to HeLa cell of dl355 which is one of the modified adenoviruses of this invention. It is a graph which shows the cell-killing activity with respect to a human non-small cell lung cancer cell (H1299 cell) of dl355 which is one of the modified adenoviruses of this invention. It is a graph which shows the inhibitory effect with respect to the growth of HeLa cell transplanted to the nude mouse of dl355 which is one of the modified adenoviruses of this invention. It is a graph which shows the virus production amount in each cancer cell line of dl355 which is one of the modified adenoviruses of this invention. It is a graph which shows the cell killing activity with respect to a human non-small cell lung cancer cell (H1299 cell) of ARET which is one of the modified adenoviruses. It is a graph which shows the inhibitory effect with respect to the growth of HeLa cell transplanted to the nude mouse of ARET which is one of the modified adenoviruses.

<ΔE4orf6>
The present invention provides, as a first aspect, an antitumor virus preparation comprising a modified adenovirus having E4orf6 encoding E4orf6 protein having a deleted C-terminal region or a modified E4orf6 protein consisting of the amino acid sequence represented by SEQ ID NO: 1. provide. Hereinafter, the modified adenovirus according to the first embodiment is represented as ΔE4orf6.

  The present inventor confirmed that the gene product of E4orf6 (E4orf6 protein), which is one of the early gene products of adenovirus, interacts with pp32 protein in its C-terminal region, and that the α helix present in the C-terminal region And through this interaction, E4orf6 protein and pp32 bind to mRNA containing an AU-rich element in the 3 ′ untranslated region via a HuR protein that binds directly to the AU-rich element, We found that mRNA containing AU-rich elements was forced and constitutively transported and stabilized outside the nucleus in a CRM1-independent manner (Higasino et al., J. Cell Biol., 2005, vol. 170, No. 15).

  The AU-rich element (also referred to as AU-Rich Element, AU-rich sequence, hereinafter referred to as ARE) is a base sequence or region rich in adenine (A) and uracil (U) in the 3 ′ end untranslated region of mRNA. is there. It is known that ARE is frequently present in the 3 'untranslated region of mRNA transcribed from a gene encoding a growth factor or cytokine. Hereinafter, mRNA containing ARE is referred to as ARE-mRNA.

  It is known that ARE-mRNA is rapidly degraded by recognizing and binding to ARE by Tristetraprolin (TTP), Zfp36L1, Zfp36L2, AUF1, KSRP, and the like. In addition, binding of HuR protein, whose expression is induced by various stresses such as heat shock, to ARE-mRNA causes ARE-mRNA to escape from the rapid degradation and is stabilized.

  Although not bound by the following inferences, ΔE4orf6 cannot stabilize ARE-mRNA by E4orf6 protein, so it cannot grow in normal host cells, but ARE-mRNA stabilization is enhanced. Cells, such as tumor cells, do not require stabilization of ARE-mRNA with E4orf6 protein, and therefore it is speculated that the modified adenovirus can grow and exhibit cytocidal activity against tumor cells. .

  Thus, ΔE4orf6 according to the first aspect of the present invention can be understood as one of the oncolytic viruses independent of p53 mutation.

  The stabilization of ARE-mRNA in the present invention means that ARE-mRNA is not transiently stabilized by heat shock or other various stresses, for example, cancer cells, It means a state in which ARE-mRNA can be constantly stabilized in cells without the need for such stimulation.

  One specific example of ΔE4orf6 is dl355 (Halbert et al., J. Virol., 1985), an adenovirus in which the region coding from the 158th to the C terminus of the amino acid sequence encoded by E4orf6 has been deleted. 56, No. 1). As shown in the following examples, dl355 exhibits a cell-killing activity against cancer cells, but exhibits little cell-killing activity against normal cells in which ARE-mRNA stabilization is not enhanced.

  Another example of ΔE4orf6 is a modified adenovirus having E4orf6 encoding a modified E4orf6 protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (Kuroshima et al., Oncogene, 2011, Vol. 30, pages 2912-2920. ). The amino acid sequence shown in SEQ ID NO: 1 corresponds to an amino acid sequence in which leucine at position 245 of the wild type E4orf6 protein is replaced with proline. By this substitution, the α-helix structure existing on the C-terminal side of E4orf6 is disrupted, and the function of E4orf6 relating to the interaction with pp32 protein is substantially lost. Therefore, like dl355, such a modified adenovirus exhibits a cell-killing activity against cancer cells, but exhibits little cell-killing activity against normal cells in which ARE-mRNA stabilization is not enhanced. Inferred.

  ΔE4orf6, including the above-mentioned dl355, can be prepared and used by an ordinary genetic engineering technique based on, for example, a commercially available adenovirus vector pAxcwit provided by Takara Bio Inc. Moreover, the use of a virus preparation containing ΔE4orf6, particularly the virus preparation as an antitumor agent, is based on the method described in Non-Patent Document 1 or 2 or by a method in which these are modified by the ordinary ability of a person skilled in the art. It can be carried out.

<AdARET>
The second aspect of the present invention includes an anti-tumor adenovirus comprising a modified adenovirus having an ARE introduced in the 3 ′ untranslated region of the E1A gene or E1B gene or at a position adjacent to the 3 ′ untranslated region. A formulation is provided. Hereinafter, the modified adenovirus of the second embodiment is referred to as AdARET.

  As described above, ARE has a function of promptly decomposing mRNA containing it together with TTP and the like. Therefore, by introducing ARE into the 3 'untranslated region of the E1A gene or E1B gene or at a position adjacent to the 3' untranslated region, mRNA transcribed from such gene can be destabilized. Although not bound by the following inferences, AdARET is unable to proliferate in normal cells where ARE-mRNA is rapidly degraded because E1A protein or E1B protein is not expressed, but stabilization of ARE-mRNA. It is inferred that cells in which E1 is enhanced, such as tumor cells, can stably proliferate because E1A protein or E1B protein is stably expressed, and exhibit cell-killing activity against tumor cells.

  Thus, AdARET according to the second aspect of the present invention can be understood as one of oncolytic viruses that do not depend on the mutation of p53, like ΔE4orf6.

  In the second aspect of the present invention, the ARE is the stability of the ARE-mRNA in the cell in relation to the function mentioned above, ie, the protein that promotes the degradation of the ARE-mRNA or the protein that stabilizes the ARE-mRNA. It is sufficient if it has a function of controlling ARE exists in 3 'untranslated region of c-fos gene, c-myc gene, TNF-α gene, cox-2 gene and other various genes involved in animal cell proliferation, differentiation induction or immune response It is known that many kinds of base sequences have been reported. In the present invention, any such ARE can be used, but the use of ARE present in a human gene is particularly preferable. The ARE to be introduced may be a single, a plurality of or a plurality of types of AREs.

  The ARE in the second aspect of the present invention includes a base sequence rich in A and T (thymine) corresponding to the ARE in the DNA molecule in addition to the base sequence or region rich in A and U in the RNA molecule. Or a region. Therefore, “ARE is contained” is used when the RNA molecule contains ARE, and the DNA molecule contains a base sequence or region rich in A and T (thymine) corresponding to ARE. . Further, “ARE is introduced” means that the genome sequence or DNA molecule encoding a gene has a base sequence rich in A and T (thymine) corresponding to ARE, so that ARE is included in the mRNA molecule that is a transcription product. It means to incorporate the area.

  In the second embodiment of the present invention, ARE is introduced into the 3 'untranslated region of the E1A gene and / or E1B gene or at a position adjacent to the 3' untranslated region. In particular, ARE is preferably introduced at a position from the end of the ORF of the E1A gene to the start of the poly A sequence. The position adjacent to the 3 ′ end untranslated region of the gene includes ARE at the 3 ′ end of the mRNA due to RNA polymerase read-through when the mRNA is transcribed from the E1A gene or E1B gene by RNA polymerase. It means such a position.

  One specific example of AdARET according to the second aspect of the present invention is an ARE (5′-gtattattattattattattattattattattattag-3, sequence contained in the human TNF-α gene in the 3 ′ untranslated region of the E1A gene. No. 2) is an adenovirus introduced (hereinafter referred to as ARET). As shown in the examples below, ARET exhibits cell-killing activity against cancer cell lines, whereas it exhibits little cell-killing activity against normal cells in which ARE-mRNA stabilization is not enhanced.

  AdARET can be prepared and used by an ordinary genetic engineering technique based on, for example, a commercially available adenovirus vector pAxcwit provided by Takara Bio Inc.

<AdARET / ΔE4orf6>
A third aspect of the present invention relates to E4orf6 encoding an E4orf6 protein from which the C-terminal region has been deleted and the 3 ′ untranslated region or the 3 ′ untranslated region in relation to the first and second aspects described above. An antitumor adenovirus preparation comprising a modified adenovirus having an E1A gene and / or an E1B gene into which ARE is introduced at a position adjacent to the region is provided. The ARE in the third aspect of the present invention is as described in the second aspect. Hereinafter, the modified adenovirus according to the third aspect of the present invention is referred to as AdARET / ΔE4orf6.

  AdARET / ΔE4orf6 according to the third aspect of the present invention is a cell in which stabilization of ARE-mRNA is not enhanced by having both selectivity for cancer cells in ΔE4orf6 and selectivity for cancer cells in AdARET. This is advantageous in that the cell-killing activity against the cell is more surely suppressed, and as a result, the selectivity of the cell-killing activity against cells in which the stabilization of ARE-mRNA is enhanced is enhanced.

  AdARET / ΔE4orf6 can be prepared by an ordinary genetic engineering technique based on, for example, a commercially available adenovirus vector pAxcwit provided by Takara Bio Inc. or based on dl355, which is an example of ΔE4orf6. Specifically, it is prepared by synthesizing DNA having a base sequence corresponding to an arbitrary ARE, and recombining the DNA at a desired position on an adenovirus vector using various genetic engineering techniques. Can do.

<CRV-ARE>
The fourth aspect of the present invention includes a disease-treating modified virus having an ARE introduced in the 3 ′ untranslated region of a viral gene essential for self-growth or at a position adjacent to the 3 ′ untranslated region. The present invention relates to a virus preparation (except for an anti-tumor virus preparation in which the virus is an adenovirus) for treating a disease involving cells in which stabilization of ARE-mRNA is enhanced. Hereinafter, the modified virus for treatment of diseases according to the fourth aspect of the present invention is represented as Conditionally replicated viral AU-rich element (CRV-ARE).

  As described above, ARE has a function of promptly decomposing mRNA containing it together with TTP and the like. Therefore, by introducing ARE into the 3 ′ untranslated region of a gene essential for virus growth or at a position adjacent to the 3 ′ untranslated region, mRNA transcribed from such gene can be destabilized. it can. Although not restricted by the following reasoning, the CRV-ARE according to the fourth aspect of the present invention does not express the protein encoded by the essential gene in normal cells in which ARE-mRNA is rapidly degraded. Therefore, in the cells where the stabilization of ARE-mRNA is enhanced, for example, tumor cells, the protein is stably expressed, so that the protein can proliferate and the stabilization of ARE-mRNA is enhanced. It is presumed that the virus inherently has toxicity, specifically, cell killing activity against certain cells.

  From the above, the CRV-ARE according to the fourth aspect of the present invention is a modified virus capable of growing in a host cell in which ARE-mRNA stabilization is enhanced and selectively killing the host cell. It can be used as a virus preparation for treating diseases involving cells in which stabilization of ARE-mRNA is enhanced. In the present invention, virus propagation is used interchangeably with virus replication.

  The CRV-ARE according to this embodiment is based on a virus capable of lysing or killing human cells if it normally proliferates, or within the 3 ′ untranslated region of a gene essential for the growth of the virus. 'Can be prepared by introducing an ARE at a position adjacent to the untranslated region.

  The CRV-ARE may be a virus that can kill human host cells as long as it properly grows. Examples of such viruses include adenovirus, herpes virus, measles virus, mumps virus, influenza virus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, reovirus, poxvirus, Sendai virus and enterovirus. However, it is not limited to these. In particular, use of adenovirus is preferable.

  The genome structure of the virus has already been elucidated, and a number of genes essential for growth in each virus have been identified in studies on each virus, along with specific nucleotide sequences. The gene into which ARE in this embodiment is to be introduced can be appropriately selected from such genes.

  Examples of preferable genes into which ARE is introduced include E1A gene, E1B gene in adenovirus, ICP34.5 gene, ICP47 gene, ICP6 gene in herpes virus, M protein gene in measles virus, P protein gene in mumps virus, influenza Examples of the virus include NS1 protein gene, vaccinia virus includes thymidine kinase gene, and other polymerase genes necessary for replication of each virus itself.

  An example of a host cell in which stabilization of ARE-mRNA is enhanced is a cancer cell (Halbert et al., 1985, J. Viol., 56, 250-257). Therefore, a virus preparation containing CRV-ARE according to the fourth aspect of the present invention (except for an antitumor virus preparation in which the virus is an adenovirus) can be used as an antitumor virus preparation.

  Another example of a host cell with increased ARE-mRNA stabilization is osteoclasts. In osteoclasts, an osteoclast differentiation inducing factor (ODF / Rank1) essential for its activation is abnormally increased, but its mRNA is an ARE-mRNA containing ARE in the 3'-terminal untranslated region. Therefore, by infecting osteoclasts with abnormally enhanced ODF / Rank1 with the CRV-ARE of the present invention, the osteoclasts can be selectively killed and the progression or worsening of osteoporosis can be suppressed. Expected. Thus, the virus preparation containing CRV-ARE according to the fourth aspect of the present invention can be used as a virus preparation for the treatment of osteoporosis.

  Another example of host cells with enhanced ARE-mRNA stabilization is peripheral mononuclear cells and synovial cells in rheumatic patients (Thiel et al., Exp. Cell Res., 2006, 312, 12). No.). In the peripheral mononuclear cells of rheumatic patients, the expression of TTP that promotes the degradation of ARE-mRNA is greatly reduced as compared with that in healthy individuals, while the expression of TNF-α translated from ARE-mRNA is abnormally increased. It is presumed that stabilization of ARE-mRNA is enhanced. Therefore, by infecting peripheral mononuclear cells and synovial cells with the CRV-ARE of the present invention, the peripheral mononuclear cells are selectively killed to induce the expression of TNF-α and to induce osteoclasts by inflammation. It is expected that the progression or deterioration of rheumatism can be suppressed by suppressing the proliferation of synovial cells that are targets of inflammation. Thus, the virus preparation containing CRV-ARE according to the fourth aspect of the present invention can be used as a virus preparation for treating rheumatism.

<Virus preparation>
The virus preparation of the present invention contains at least one modified virus according to the present invention. Furthermore, in addition to the modified virus according to the present invention, the virus preparation of the present invention includes any other virus, therapeutically effective drug, pharmaceutically acceptable ingredient, buffer solution, excipient, adjuvant, preservative. It can contain any ingredient normally used in drugs, fillers, stabilizers, thickeners and / or formulations.

  Pharmaceutically acceptable ingredients are well known to those skilled in the art, and those skilled in the art can use the ingredients from the ingredients described in the 16th revised Japanese Pharmacopoeia and other standards within the scope of their normal performance. Can be selected and used as appropriate. In addition, it is preferable to use various components that are used in preparations containing modified viruses.

  The viral preparations of the present invention can be in any form suitable for administration, eg solid, semi-solid or liquid form such as solution, lyophilized powder, emulsion, suspension, tablet, pellet, capsule and the like. However, it is not limited to these. Usually, virus preparations are used in the form of parenteral preparations such as injections and drops. Examples of carriers that can be used in parenteral preparations include aqueous carriers that are usually used in preparations, such as isotonic solutions containing physiological saline, glucose, D-sorbitol, and the like.

  The above-mentioned form of the virus preparation is obtained by, for example, a method of producing a conventional virus preparation containing the oncolytic virus or other virus vectors described in Non-Patent Document 1 or 2, or a method in which these are modified by the ordinary ability of a person skilled in the art. Can be manufactured.

  The virus preparation of the present invention is administered as a subject to animals selected from the group consisting of pets, livestock, production animals and humans, particularly humans. Administration of a virus preparation to a subject can be performed following a conventional virus preparation containing, for example, the oncolytic virus or other virus vectors described in Non-Patent Document 1 or 2. The route of administration is determined by those skilled in the art in consideration of the form of the formulation, the disease, the disease site, and various factors considered when administering other drugs.

  A preferable administration route of the virus preparation of the present invention is parenteral administration such as intravascular administration (preferably intravenous administration), intraperitoneal administration, intestinal administration, and local administration in or near a tumor. In one of the preferred embodiments, the virus preparation of the present invention is administered to a subject by intravenous administration or local administration in or near a tumor. Administration may be a single dose or repeated doses.

When the virus preparation of the present invention is administered to a subject, particularly a human, the dosage range is, for example, 1 × 10 ³ to 1 × 10 ¹⁴ , preferably 1 × 10 ⁵ to 1 × 10 ⁶ per human subject. ¹² , more preferably 1 × 10 ⁶ to 1 × 10 ¹¹ , most preferably 1 × 10 ⁷ to 1 × 10 ¹⁰ plaque forming units (pf).

  Although the disease treatment using the virus preparation of the present invention is effective alone, any other treatment, for example, the conventional treatment and the treatment using the virus preparation of the present invention may be used in combination. For example, in the case of cancer treatment, chemotherapy using other anticancer agents, cancer immunotherapy, or radiation treatment may be used in combination.

  The invention will now be described in more detail by way of non-limiting examples. In the examples, operations using commercially available kits were performed according to the protocol of the kit manufacturer. It should be noted that the present invention is not limited to the specific methodologies, protocols, cell lines, animal species and genera, constructs and reagents described in the present specification, and it should be understood that these can be changed as appropriate. It is easily understood by the contractor. Also, dl355, which is ΔE4orf6, is Princeton University, T.W. From Dr. Shenk and Onyx-015 (dl1520), an oncolytic virus, is available from the University of California, A.M. Each strain distributed by Dr. Berk was used.

<Example 1> Cell killing activity of dl355 against HeLa cell line 1) Preparation of virus solution W162 cells / DMED (10% FBS) medium was seeded in a 10 cm dish at 5 × 10 ⁶ cells / well and cultured for 24 hours. And then infected with 10 MOI of dl355. 48 hours after infection, the cells were collected together with the medium, and freeze-thawing was repeated three times to release the virus from the infected cells. The supernatant liquid from which cell debris and the like were removed by centrifugation was collected as a virus stock solution, stored at −80 ° C., and used for the following experiments.

2) Confirmation of cell-killing activity 3 × 10 ³ HeLa cells and human normal BJ cells were inoculated in a 96-well dish containing 100 μL of DMED (10% FBS) medium and cultured at 37 ° C. . The medium was removed, and 100 μL of DMEM medium containing 10% FBS was newly added. Further, 1, 10, 100 MOI of dl355 or 100 μL of DMED (10% FBS) medium (mock) prepared in Example 1 was added, and 37 Incubate at 72 ° C. for 72 hours.

  72 hours after the start of the culture, an XTT assay using Cell Proliferation kit II (XTT) (Roche) was performed to measure cell viability. The result is shown in FIG. From this experiment, it was confirmed that dl355 exhibits a cell-killing activity against HeLa cells, but hardly shows a cell-killing activity on BJ cells, which are normal cells.

<Example 2> Cell killing activity of dl355 against H1299 cells The cell killing activity of dl355 against human non-small cell lung cancer cells (H1299 cells) was the same as in Example 1, except that the culture was started immediately after adding virus solution. The cell viability was measured by performing an XTT assay using Cell Proliferation kit II (XTT) (Roche) 1 day, 3 days, 5 days and 7 days after the start of culture. Moreover, the same experiment was performed using Onyx-015 as a positive control. The result is shown in FIG. It was confirmed that dl355 exhibits a cytocidal activity exceeding Onyx-015 against H1299 cells.

<Example 3> Inhibitory effect on tumor growth in nude mice of dl355 1) Cell culture After washing HeLa cells cultured at 37 ° C for 3 days in 10 mL DMED (10% FBS) medium / 10 cm dish with PBS And released with trypsin. After trypsin was inactivated by adding DMEM containing 10% FBS, the cells were precipitated, and Hanks buffer was added to resuspend the cells. After washing the cells by repeating the precipitation and suspension three times, a cell suspension of 1 × 10 ⁶ cells / 100 μL was prepared.

2) Transplantation and viral drug administration Prepared by the above 1) subcutaneously in the base of the right hind paw of nude mice (Athymic BALB / c nu / nu, n = 5) purchased from Japan Charles River and acclimated for 2 weeks. 100 μL of the cell suspension was injected, and the cells were reared until the tumor had a major axis and a minor axis of about 5 mm.

While the tumor was injected with 100 μL of dl355 (9.0 × 10 ⁹ VP / mL) prepared in Example 1 1) about 2 to 3 times at intervals of 2 days, the breeding was continued for another 28 days. diameter) was measured over time the size of the tumor calculated by ^2/2. In addition, as a control in which 100 μL of PBS was injected instead of dl355, the size of the tumor was measured in the same manner. The results (average value of 5 animals) are shown in FIG.

  As shown in FIG. 3, dl355 suppressed the growth of a tumor mass caused by cancer cells transplanted into nude mice. In addition, when the above experiment was performed twice in total, one mouse was observed in each experiment in which the tumor disappeared completely.

<Example 4>
MCF-7 (human breast adenocarcinoma cells), H1299, U2OS (human osteosarcoma cells), Saos-2 (human osteoblast-like osteosarcoma cells), A549 (human lung) suspended in DMED (10% FBS) medium Alveolar epithelial adenocarcinoma cells), MBA-MD-231 (human breast adenocarcinoma cells), PC3 (human prostate cancer cells), HSC3 (human tongue cancer cell line), C33A (human cervical cancer cells) and Each HeLa cell was seeded in a 12-well dish at 5 × 10 ⁴ cells / well, cultured for 24 hours, and separately infected with 1MOI of dl355 and Onyx-015. 48 hours after infection, each cell was collected together with the medium, and freeze-thawing was repeated three times to release the virus from the infected cell. The supernatant from which cell debris was removed by centrifugation was collected as a virus stock solution, and the amount of virus production in each cancer cell line (Adeno-X Rapid Titer Kit, Clontech) was used (Adeno-X Rapid Titer Kit, Clontech). (Virus Particle) was measured. The result is shown in FIG.

dl355 was confirmed to grow well for all the cancer cell lines used compared to Onyx-015.
Example 5 Construction of ARET Princeton University, T.W. PXhoIC (Logan et al, Cancer Cells 2, 527-532, 1984), a plasmid in which an E1 region containing an E1A gene and an E1B gene of adenovirus was inserted into an E. coli vector pBR322, provided by Dr. Shenk, was expressed as a restriction enzyme. Ring opening with HpaI. A synthetic double-stranded DNA fragment consisting of a base sequence corresponding to ARE (5′-gtgattattattattattattattattattag-3, SEQ ID NO: 2) contained in the human TNF-α gene and its complementary strand was incorporated into the E1A gene PXhoIC-ARETNF, a plasmid containing the E1 region into which the ARE was introduced in the 3 ′ untranslated region of was constructed.

  Primer set designed using pXhoIC-ARETNF as a template and including a ClaI restriction site at both ends (forward primer: 5′-gcgatacgattgacgtgttagttatttacccc-3 ′, reverse primer: 5′-ggttatcattattcatggtcatgcatactgcacatgcatacatgca PCR was performed using 4) to prepare an amplified fragment of the E1 region into which ARE was introduced. This amplified fragment is incorporated into the ClaI restriction site of pAxcwit (Takara Bio) containing an adenovirus genome lacking the E1A gene and the E1B gene, and contains an adenovirus genome in which ARE is introduced into the 3 ′ untranslated region of the E1A gene. PAx-ARETNF was constructed as a plasmid.

Recombinant adenovirus stock was prepared by infecting 293 cells with chain DNA obtained by cleaving pAx-ARETNF with the restriction enzyme pacI, using Hilymax (Dojindo Laboratories), and further, Fast-Trap A purified virus (9.0 × 10 ⁹ virus particles / mL) was prepared using an adenovirus purification kit (EMD Millpore). Hereinafter, this virus is referred to as ARET.

<Example 6> Cytocidal activity of ARET against H1299 cells The same procedure as in Example 2 was performed except that dl355 was replaced with ARET of Example 5, and the cytocidal activity of ARET against H1299 cells was confirmed by XTT assay. . The result is shown in FIG.

  As shown in FIG. 5, ARET showed superior cytotoxic activity against H1299 cells compared to Onyx-015.

<Example 7> Inhibitory effect on growth of tumor in nude mice of ARET Tumor growth by HeLa cells transplanted into mice in the same manner as in Example 3 except that dl355 was replaced with ARET of Example 5 The suppression effect of ARET was confirmed. The result is shown in FIG.

  As shown in FIG. 6, ARET almost completely suppressed the growth of the tumor mass by cancer cells transplanted into nude mice. One mouse was observed in which the mass disappeared completely.

  The virus preparation of the present invention has applicability as a therapeutic agent for diseases such as cancer, osteoporosis, rheumatism and the like, which involve cells with enhanced ARE-mRNA stabilization.

Claims (6)

  An antitumor adenovirus preparation comprising an E4orf6 protein having a C-terminal region deleted or a modified adenovirus having E4orf6 encoding a modified E4orf6 protein consisting of the amino acid sequence shown in SEQ ID NO: 1.   The antitumor adenovirus preparation according to claim 1, wherein the E4orf6 protein from which the C-terminal region has been deleted is a protein in which the amino acid sequence of the E4orf6 protein has been deleted from the 158th to the C-terminal.   The antitumor adenovirus preparation according to claim 1 or 2, wherein an AU-rich element sequence is introduced into the 3 'untranslated region of the E1A gene or E1B gene or at a position adjacent to the 3' untranslated region.   An AU rich element sequence comprising a disease treatment modified virus introduced with an AU-rich element sequence in a position adjacent to or adjacent to the 3 ′ untranslated region of a viral gene essential for self growth; Virus preparations for treatment of diseases for treating diseases involving cells with enhanced stabilization of mRNA (excluding anti-tumor virus preparations in which the virus is an adenovirus).   The virus preparation for disease treatment according to claim 4, wherein the disease is a malignant tumor, osteoporosis, rheumatism, viral disease or inflammatory disease. The virus is a virus selected from the group consisting of adenovirus, herpes virus, measles virus, mumps virus, influenza virus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, reovirus, poxvirus, Sendai virus and enterovirus. The virus preparation for disease treatment according to claim 4 or 5, wherein

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