WO2015182574A1 - Reic遺伝子を発現する制限増殖型アデノウイルス - Google Patents
Reic遺伝子を発現する制限増殖型アデノウイルス Download PDFInfo
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Definitions
- the present invention relates to a restricted propagation adenovirus that highly expresses REIC (REIC / Dkk-3) protein.
- Non-patent Document 1 Non-patent Document 1
- Non-Patent Document 3 Representative examples of drugs for cancer using restricted-proliferating viruses that have been reported in clinical trials to date are Telomelysin (non-patent document 2) with adenovirus type 5 as the skeleton and herpes simplex virus type 1 as the skeleton There is Talimogene laherparepvec (T-VEC, former name Oncovex) (Non-Patent Document 3).
- Non-Patent Document 1 it has come to be considered that it is important to have a function to activate anti-cancer immunity for drugs against cancer using various viruses.
- Non-Patent Document 2 genes such as cytokines that activate anti-cancer immunity are not encoded, and cancer cell death due to the proliferation of the adenovirus is local in the region where Telomelysin is administered.
- cytokines that activate anti-cancer immunity
- T-VEC activation of anti-cancer immunity can be expected by cancer cell death / antigenization of cancer cells by proliferation of the herpesvirus in the administered region and expression of cytokine GM-CSF.
- the cytokine GM-CSF has an anti-cancer immune activation effect that induces differentiation of dendritic cells, which are cancer antigen-presenting cells (Non-patent Document 1), and induces immunosuppressive cells at high doses to prevent cancer. It has also been reported that there is a possibility of attenuating the immune function and worsening the disease state (Non-patent Document 4), which may limit the therapeutic effect of T-VEC.
- REIC REIC / Dkk-3 gene
- REIC has an effect of activating anticancer immunity and an effect of inducing cell death in cancer cells by endoplasmic reticulum stress during gene expression.
- Patent Document 4 it has been reported that a partial fragment of the REIC gene has the same effect as full-length REIC (Patent Document 4), and an adenovirus expressing the REIC / Dkk-3 gene has also been reported (Patent Documents 5 and 5).
- Non-patent document 11 Non-patent document 11).
- An object of the present invention is to provide a restricted growth type adenovirus having a strong anticancer activity.
- the inventors of the present invention have created their own restricted growth type adenovirus by effectively combining the previously reported technology groups of restricted growth type adenovirus (Patent Documents 1 and 2 and Non-Patent Documents 5 to 10). . Furthermore, a REIC gene that expresses a REIC protein having the unique anti-cancer immunity activation effect (Non-patent Document 11) was newly encoded in this adenovirus. As a result, a highly innovative anticancer virus preparation that has both uniqueness and novelty, and that is expected to have an anticancer action that surpasses the group of drugs against cancer using existing restricted-proliferation viruses. The development was successful and the present invention was completed.
- the present invention is as follows. [1] Restricted growth type adenovirus containing the ITR (inverted terminal repeat) sequence of the type 5 adenovirus genome, and inserted with HRE sequence, hTERT promoter, decorin-encoding DNA and RGD sequence-encoding DNA. In addition, a restricted growth type adenovirus into which full-length REIC DNA or REIC C domain DNA is further inserted and which specifically propagates in cancer cells and expresses REIC protein or REIC C domain protein.
- ITR inverted terminal repeat
- hTERT promoter is a hTERT promoter modified by addition of c-Myc binding site and Sp1 binding site,
- Six HRE sequences consisting of the base sequence represented by SEQ ID NO: 3 are inserted upstream of the hTERT promoter, (iii) a part of the E1A region, lacking the Rb binding region (Retinoblastoma gene binding region) of the type 5 adenovirus genome sequence shown in SEQ ID NO: 4;
- a part of the E1B region wherein the portion encoding E1B-19kDa of the type 5 adenovirus genome sequence shown in SEQ ID NO: 4 has been deleted;
- a part of the E3 region is deleted,
- a DNA construct consisting of a promoter sequence, DNA encoding decorin and a polyA addition sequence is inserted into the E3 region;
- DNA encoding a peptide comprising an RGD sequence is inserted into the E
- REIC is full-length REIC.
- a cancer therapeutic agent comprising the restricted growth type adenovirus according to any one of [1] to [6] as an active ingredient.
- Restricted growth type adenovirus grows specifically in cancer cells, expresses REIC protein, and the expressed REIC protein induces cancer cell death by endoplasmic reticulum stress.
- the cancer therapeutic agent according to [7] which induces cancer immune activity.
- the restricted growth type adenovirus of the present invention is an improvement over the conventional restricted growth type adenovirus and has a stronger anticancer effect than the conventional one. Furthermore, the restricted growth type adenovirus inserted with REIC DNA is not only the anticancer effect of the restricted growth type adenovirus itself, but also the action that activates the anticancer immunity of REIC, and the endoplasmic reticulum stress during gene expression causes the cancer cell to Together with the action of inducing death, these actions work together to exert a synergistic and powerful therapeutic effect against cancer.
- oncolic adenovirus It is a figure which shows the structure of oncolic adenovirus. It is a figure which shows the arrangement
- the present invention is a restriction-proliferating adenovirus that contains REIC (REIC / Dkk-3) DNA and can be used for expression of REIC protein.
- Restricted growth type adenovirus is an adenovirus that is genetically modified and grows only in cancer cells. Although it does not act on normal cells, it can grow only in cancer cells, lyse the cancer cells, and effectively kill the cancer cells. Restricted growth adenovirus is also called oncolytic adenovirus or lytic adenovirus.
- the restricted growth type of the present invention can be used by inserting a foreign gene, it can also be referred to as a restricted growth type adenovirus vector.
- the full-length REIC DNA or REIC DNA fragment is introduced into the restricted growth adenovirus, and not only the cancer-killing cell effect of the restricted growth adenovirus itself, but also the effect of activating anticancer immunity, Synergistic cancer-killing cell effects can be achieved by the effects of REIC on cancer cells, such as the effect of inducing cell death in cancer cells by endoplasmic reticulum stress during gene expression.
- a restriction-propagating adenovirus is referred to as an oncolytic adenovirus (oncolytic Ad)
- an oncolytic Ad-REIC a restriction-proliferating adenovirus that contains full-length REIC DNA and can express full-length REIC
- an oncolytic Ad-REIC a restriction-proliferating adenovirus that contains full-length REIC DNA and can express full-length REIC
- an oncolytic Ad-REIC a restriction-proliferating adenovirus that contains full-length REIC DNA and can express full-length REIC
- an oncolytic Ad-REIC the restriction-proliferating adenovirus that contains DNA and can express the REIC C domain
- an oncolytic Ad-REIC domain a restriction-proliferating adenovirus that contains DNA and can express the REIC C domain
- the restricted growth type adenovirus used in the present invention has a type 5 adenovirus backbone whose growth is restricted by a human telomerase reverse transcriptase (hTERT) promoter.
- the restricted-proliferation adenovirus of the present invention includes an ITR (inverted terminal repeat) of type 5 adenovirus, and further comprises DNA encoding decorin, a protein that suppresses tumor formation and growth, Modifications (insertion and deletion of specific sequences) have been made.
- Decorin DNA is expressed by the CMV promoter.
- the genome sequence of type 5 adenovirus is described in Virology, 186 (1), 1992, pp. 280-285, and is registered under GenBank Accession No. M73260.
- the genome sequence of type 5 adenovirus is shown in SEQ ID NO: 4.
- the adenovirus genome has ITR (inverted terminal repeat) at both ends, and has an E1A region, an E1B region, an E2 region, an E3 region, and an E4 region in order from the 5 ′ side as an initial transcription region.
- ITR inverted terminal repeat
- hTERT human telomerase reverse transcriptase
- the modified hTERT promoter has one or more c-Myc binding sites (cacgtg, cacgcg or catgcg) and / or one or more Sp1 binding sites (gggcgg, ccgccc, ctccgcctc, cccagcccc, gggcgg, ggggcgg or cccccgccc (SEQ ID NO:
- the wild-type hTERT promoter contains two c-Myc binding sites and five Sp1 binding sites
- the hTERT promoter of the restricted growth adenovirus of the present invention is, for example, one more c- A Myc binding site and 5 Sp1 binding sites are added, including a total of 3 c-Myc binding sites and 10 Sp1 binding sites, which are located at the 3 ′ end of the hTERT promoter.
- a modified hTERT promoter sequence may be included in the hTERT promoter sequence as shown in FIG.
- “E-box” indicates c-Myc binding sequence.
- the hTERT promoter further includes one c-Myc binding site and five Sp1 binding sites, for example, two c-Myc binding sites and five
- a wild-type hTERT promoter having one Sp1 binding site may be bound to one c-Myc binding site and an hTERT promoter containing five Sp1 binding sites.
- a pGL2-hTERT vector containing a binding site and five Sp1 binding sites may be cut with EcoRI and HindIII and then inserted into pSEAP-TERT treated with the same restriction enzymes to produce pSEAP-mTERT.
- the modified hTERT promoter is described in Japanese Patent No. 4327844 and EUNHEE KIM et al., Human Gene Therapy 14: 1415-1428 (October 10, 2003).
- HRE Hypoxia responsive region
- HRE is a DNA element that responds to hypoxia possessed by a gene activated in hypoxia (hypoxia), and includes ACGTG as a consensus sequence.
- the restriction-propagating adenovirus used in the present invention contains a sequence of 5 to 40 bases including the consensus sequence. In normal tissues, the oxygen concentration is about 2 to 9%, but cancer cells are in a hypoxic state of about 1.3%. For this reason, the restricted-proliferating adenovirus containing HRE is promoted in cancer cells.
- HRE sequence examples include a sequence containing the above consensus sequence of a human vascular endothelial growth factor (hVEGF) gene (GenBank Accession No. M63971), and specifically, the 1379th position of the hVEGF gene Examples include the base sequence of the 1412th base from the base (SEQ ID NO: 3). Two or more HREs may be used in combination, and 3 to 12 HREs can be used together. For example, 6 connected (HRE ⁇ 6) or 12 connected (HRE ⁇ 12) (Oh-Joon Kwon et al., Clin Cancer Res; 16 (24) December 15, 2010, pp.60716082). Preferably, six connected (HRE ⁇ 6) are used. HRE may be linked upstream of the hTERT promoter, for example immediately upstream.
- the E1A region is partially deleted.
- the E1A region is present at the 342th to 1545th positions of the type 5 adenovirus genome (SEQ ID NO: 4), and the E1A protein binds to the RB (Retinoblastoma) gene product.
- the E1A region is an indispensable region for adenovirus replication, and the restriction-proliferating adenovirus of the present invention lacks the Rb binding region (Retinoblastoma gene binding region) of the E1A region and retains the replication ability itself.
- a partial deletion of the E1A region in the restricted growth adenovirus of the present invention is referred to as including a mutated active E1A gene.
- the mutated active E1A gene consists of a nucleotide sequence coding for the Rb (retinoblastoma protein) binding site, a mutation in which the 45th Glu residue is replaced with Gly, and the 121-127th amino acid sequence entirely. It has a mutation substituted for Gly.
- the adenovirus that has lost its ability to bind to Rb is active in normal cells. Can suppress adenovirus replication, but tumor cells with suppressed Rb function can be actively replicated to selectively kill cancer cells.
- the recombinant adenovirus of the present invention containing a mutation at the Rb binding site described above has a greatly increased cancer cell specificity.
- the mutation in the Rb binding site is, for example, a part of the E1A region, which is 24 Rb binding regions (Retinoblastoma gene binding region) of 923 to 946 in the type 5 adenovirus genome sequence shown in SEQ ID NO: 4. ( ⁇ E1A (24 bp)) (Candelaria Gomez-Manzano et al., Oncogene (2004) 23, pp.1821-1828).
- the E1B region is partially deleted.
- the E1B region is present in the 1714th to 3509th region of the type 5 adenovirus genome (SEQ ID NO: 4), and the E1B-55kDa protein, which is the gene product of the E1B region, is involved in virus replication by binding to the p53 protein. .
- a partial deletion of the E1B region is also referred to as having an inactive portion in the E1B region, and the restricted-proliferating adenovirus of the present invention is an inactivated E1B 19 kDa gene, E1B 55 kDa gene, or E1B 19 kDa / E1B It has a 55 kDa gene, preferably an inactivated E1B 19 kDa and E1B 55 kDa gene.
- the term “deactivation” as used in connection with a gene means that the transcription and / or decoding of the gene is not normal and the function of the normal protein protein encoded by the gene is It means not appearing.
- an inactivated E1B 19 kDa gene is a gene in which a mutation (substitution, addition, partial deletion, or total deletion) is generated in the gene and an active E1B 19 kDa protein cannot be produced.
- the E1B 19kDa gene is deleted, the cell apoptotic ability can be increased, and when the E1B 55kDa gene is deleted, it has tumor cell specificity (Korea Patent Application No. 100528727).
- This deletion of the nucleotide sequence results in the deletion of the 19 kDa E1B-19 kDa, which is the trans-splicing product of the 55 kDa E1B protein, and this deletion is called ⁇ E1B (19 kDa) (Jaesung Kim et al., Cancer Gene Therapy (2002) 9, pp.725-736).
- a stop codon may be introduced so that only E1B (19 kDa) in the E1B region is not expressed.
- the E3 region is deleted. All or part of the DNA encoding the E3 protein may be deleted.
- the E3 region is present at the 27858th to 30839th positions of the type 5 adenovirus genome (SEQ ID NO: 4).
- the E3 region is not necessary for adenovirus growth, and foreign genes can be inserted into the E3 region.
- the E3 region may be partially deleted, and a foreign gene may be inserted into that portion.
- DNA encoding decorin described below can be inserted into this portion.
- Decorin is a protein belonging to SLRP (small leucin rich proteoglycan) and is composed of 10-12 leucine-rich repeats. The core part is arched, and there are several types of extracellular matrix. Binds to growth factors or decorin receptors.
- Decorin inhibits the activity of tumor growth factor (TGF- ⁇ ), prevents collagen fibrosis, participates in the matrix matrix assembly, suppresses tumor cell growth, Acts as a natural antagonist on formation and growth.
- a promoter is linked upstream of the DNA encoding decorin, and a poly A addition sequence (polyadenylation sequence, polyA) is linked downstream of the DNA encoding decorin.
- the promoter is preferably one that operates in animal cells, more preferably mammalian cells, and can regulate the transcription of the decorin gene, and is a promoter derived from a mammalian virus and a promoter derived from the genome of a mammalian cell.
- U6 promoter H1 promoter, CMV (Cytomegalovirus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionein promoter, ⁇ -actin Promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF gene promoter, inducible promoter, cancer ⁇ different promoters (e.g., TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter and AFP promoter) and a tissue-specific promoter (e.g., albumin promoter) including, but not limited thereto.
- tissue-specific promoter e.g., albumin promoter
- a CMV promoter or a cancer cell specific promoter is used.
- a cancer cell specific promoter it is preferable to use the TERT promoter or the E2F promoter.
- TERT telomere reverse transcriptase
- the wild type human hTERT human reverse transcriptase
- the m-hTERT promoter described in (2) above may be used.
- polyadenylation sequence polyA
- the origin of the poly A addition sequence is not limited, and a poly A addition sequence derived from a growth hormone gene, such as a poly A addition sequence derived from a bovine growth hormone gene (BGH polyA) or a poly derived from a human growth hormone gene A addition sequence, SV40 virus-derived poly A addition sequence, human and rabbit ⁇ -globin gene-derived poly A addition sequence, and the like.
- BGH polyA bovine growth hormone gene
- poly A addition sequence derived from a human growth hormone gene A addition sequence
- SV40 virus-derived poly A addition sequence SV40 virus-derived poly A addition sequence
- human and rabbit ⁇ -globin gene-derived poly A addition sequence and the like.
- Adenoviruses containing DNA encoding decorin are described in Japanese Patent Application Laid-Open No. 2008-531010, I-K Choi et al., “Gene Therapy” (2010) 17, 190-201.
- SEQ ID NO: 5 shows the base sequence of DNA encoding decorin (GenBank Accession No. NM_001920.3).
- the base sequence of the CMV promoter (GenBank Accession No. X17403) is shown in SEQ ID NO: 6
- the base sequence of the BGH polyA additional sequence is shown in SEQ ID NO: 7.
- a DNA construct comprising a promoter sequence, a DNA encoding decorin, and a polyA addition sequence containing a DNA encoding decorin may be inserted into the E1A region, E1B region or E3 region, and preferably inserted into the E3 region.
- the E1A region, E1B region and E3 region of the type 5 adenovirus genome are partially deleted.
- a DNA construct in which a CMV promoter, a DNA encoding decorin, and a polyA addition sequence are linked in this order may be inserted into the deletion portion.
- the DNA construct can be inserted into the type 5 adenovirus genome simultaneously with partial deletion of the E1A region, E1B region and E3 region by homologous recombination.
- it may be inserted into the part of the type 5 adenovirus genome sequence shown in SEQ ID NO: 4 in (6) above where the nucleotides 2859 to 30479 are deleted.
- DNA encoding a peptide containing an RGD (Arg-Gly-Asp) sequence is inserted.
- the peptide containing the RGD sequence include peptides consisting of 4 (GRGDS (SEQ ID NO: 8)) to 15 amino acids including RGD, such as CDCRGDCFC (SEQ ID NO: 9) and GSCDCRGDCFCSG (SEQ ID NO: 10).
- GGSDS SEQ ID NO: 8
- DNA encoding a peptide containing the RGD sequence is inserted into, for example, the E3 region, and specifically, inserted between the 32676th base and the 32676th base of the type 5 adenovirus genome E3 region. .
- restricted-proliferation adenovirus By containing DNA encoding a peptide containing the RGD sequence, restricted-proliferation adenovirus can be easily introduced into cancer cells.
- Adenoviruses containing RGD sequences are described, for example, in Hao Wu et al., J Gene Med 2011; 13: 658-669.
- the restricted-proliferation adenovirus of the present invention having the above characteristics (1) to (8) includes an ITR (inverted terminal repeat) sequence of the genome of type 5 adenovirus, DNA encoding HRE sequence, hTERT promoter, decorin and It has a structure in which a DNA encoding a peptide containing an RGD sequence is inserted.
- ITR inverted terminal repeat
- FIG. 1A An example of the structure of the restricted propagation adenovirus of the present invention is shown in FIG. 1A.
- FIG. 1D shows the mutation of the restriction-proliferating adenovirus of the present invention from the wild type 5 adenovirus, and further shows the insertion positions of decorin DNA and REIC DNA.
- FIG. 1B and FIG. 1C show the structure of a restriction-propagating adenovirus into which REIC DNA is inserted.
- the structure of the restriction-proliferating adenovirus shown in FIG. 1A is the structure shown in FIG. 1D, in which a DNA construct in which a CMV promoter, a DNA encoding decorin, and a polyA addition sequence are linked in this order is inserted into the E3 region.
- a part of the E1A region of the type 5 adenovirus genome is deleted, a part of the E1B region is deleted, and a part of the E3 region is further deleted.
- the HTERT promoter modified with the HRE sequence is included upstream of the E1A region, the DNA encoding the peptide containing the RGD sequence is included downstream of the E3 region, and further, the promoter, DNA encoding decorin, and polyA are included in the E3 region. Constructs consisting of sequences are included.
- the restricted propagation adenovirus of the present invention shown in FIG. 1A has the following structural features.
- the hTERT promoter is modified by adding a c-Myc binding site and an Sp1 binding site.
- Six HRE sequences consisting of the base sequence represented by SEQ ID NO: 3 are inserted upstream of the hTERT promoter.
- a DNA construct comprising a promoter sequence, a DNA encoding decorin and a polyA addition sequence is inserted into the E3 region.
- DNA encoding a peptide containing the RGD sequence is inserted into the E3 region.
- a multicloning site (insertion site) for inserting a foreign gene may be included in the E1A region, E1B region, or E3 region.
- a foreign gene such as REIC DNA described later can be inserted into the multicloning site.
- the restriction-proliferating adenovirus of the present invention has a structure represented by ITR- ⁇ E1A- ⁇ E1B-promoter-decorin DNA-polyA addition sequence-RGD sequence-ITR, and “promoter-decorin DNA-poly The construct consisting of “A additional sequence” is inserted into the deleted E3 region.
- the structure (gene map) of such a restricted growth adenovirus is shown in FIG.
- Oncolytic Ad-REIC or oncolytic Ad-REIC domain can be produced by inserting full-length REIC DNA or REIC DNA domain into the above-mentioned restricted-proliferative adenovirus (Oncolic Ad).
- REIC DNA The base sequence of REIC DNA is represented by SEQ ID NO: 11.
- the amino acid sequence of the REIC protein encoded by REICREDNA is represented by SEQ ID NO: 12.
- REIC is sometimes referred to as REIC / Dkk-3.
- the base sequence of REIC ⁇ C domain DNA is represented by SEQ ID NO: 13
- the amino acid sequence of REIC C domain protein encoded by the domain is represented by SEQ ID NO: 14.
- REIC DNA or REIC C domain DNA contained in the restricted growth adenovirus of the present invention hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 11 or 13. Soy DNA, base sequence represented by SEQ ID NO: 11 or 13, BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information), etc.
- DNA encoded or the protein encoded by the DNA e.g., Having a sequence identity of at least 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more when calculated using default or default parameters
- one or more or several (1 to 10 preferably the amino acid sequence of the DNA encoded or the protein encoded by the DNA
- “stringent conditions” are, for example, “1XSSC, 0.1% SDS, 37 ° C.” conditions, and more severe conditions are “0.5 XSSC, 0.1% SDS, 42 ° C.” conditions. There are more severe conditions such as “0.2XSSC, 0.1% SDS, 65 ° C.”. Thus, isolation of DNA having high homology with the probe sequence can be expected as the hybridization conditions become more severe.
- the above combinations of SSC, SDS, and temperature conditions are examples, and the necessary stringency can be realized by appropriately combining the probe concentration, the probe length, the hybridization reaction time, and the like.
- “Stringent conditions” can be appropriately determined by those skilled in the art as conditions under which DNA having high sequence identity hybridizes.
- the REIC DNA contained in the DNA construct of the present invention is a DNA encoding the protein represented by SEQ ID NO: 2.
- REIC® DNA or REIC® C domain DNA can be obtained from human cells, human tissues, etc. based on the sequence information of SEQ ID NOs: 11-14.
- a CMV (cytomegarovirus) promoter is linked upstream of the full-length REIC DNA or REIC C domain DNA, and a poly A addition sequence (polyadenylation sequence, polyA) is linked downstream.
- the origin of the poly A addition sequence (polyadenylation sequence, polyA) is not limited, and a poly A addition sequence derived from a growth hormone gene, such as a poly A addition sequence derived from a bovine growth hormone gene (BGA polyA) or a poly derived from a human growth hormone gene A addition sequence, SV40 virus-derived poly A addition sequence, human and rabbit ⁇ -globin gene-derived poly A addition sequence, and the like. Inclusion of the poly A addition sequence in the DNA construct increases transcription efficiency.
- the base sequence of the CMV promoter (GenBank Accession No. X17403) is shown in SEQ ID NO: 6, and the base sequence of the BGH polyA addition sequence (GenBank Accession No. M57764) is shown in SEQ ID NO: 7.
- a DNA construct comprising a CMV promoter sequence, a DNA encoding REIC DNA or REIC DNA domain, and a polyA addition sequence, including DNA encoding REIC DNA or REIC DNA domain, is inserted into the E1A region, E1B region or E3 region. Preferably, it is inserted into the E1 region (E1A or E1B region).
- the above DNA construct can be inserted into the E1A region, E1B region or E3 region of the type 5 adenovirus genome by homologous recombination.
- it may be inserted into the E1 region of the above-mentioned restricted propagation adenovirus.
- homologous recombination it is possible to insert only a DNA construct consisting of a CMV promoter sequence, a DNA encoding a REIC DNA, or a REIC C domain DNA, and a polyA addition sequence, including DNA encoding REIC DNA or REIC C domain DNA.
- it is a construct that contains the E1 region, and is a homologous construct that contains a DNA construct containing REIC DNA or REIC C domain DNA in the E1 region, and has deleted part of the E1A region and part of the E1B region. It may be inserted by recombination.
- E1A and E1B regions of the type 5 adenovirus are partially deleted ( ⁇ E1A (24 bp) and ⁇ E1B (19 kDa)), and the type 5 adenovirus contains REIC DNA or REIC C domain DNA.
- DNA constructs can be inserted.
- a DNA construct comprising a CMV promoter sequence, a DNA encoding a REIC DNA or REIC C domain DNA, and a polyA addition sequence, including a DNA encoding a REIC DNA or REIC C domain DNA is, for example, pE1sp1B-HmT which is an E1 shuttle vector It may be inserted into the E1 region by homologous recombination using -Rd19 / CMV-REIC-polA ((left homology part: 22-341) (right homology part: 3523-5790)). By using this shuttle vector, the following DNA construct is inserted into the 342th to 3522th positions of the type 5 adenovirus genome sequence.
- a DNA construct consisting of a CMV promoter sequence, DNA encoding REIC DNA or REIC C domain DNA, and a polyA addition sequence is inserted into the E1 region, and the Rb binding site of the E1A region is deleted, and E1B- A construct lacking the 19kDa coding part.
- E1A region and E1B region of type 5 adenovirus were partially deleted ( ⁇ E1A (24 bp) and ⁇ E1B (19 kDa)), and type 5 adenovirus was transferred to CMV promoter sequence, REIC DNA or REIC DNA domain DNA. And a DNA construct comprising a polyA addition sequence can be inserted.
- a DNA construct consisting of a CMV promoter sequence, a DNA encoding REIC DNA or REIC C domain DNA, and a polyA addition sequence is obtained between the 3524th and 3525th positions of the type 5 adenovirus genome sequence, or the 3523rd and 3524th positions. It may be inserted between the th.
- FIGS. 1B and 1C The structures of restricted-proliferating adenovirus inserted with DNA encoding REIC DNA or REIC C domain DNA are shown in FIGS. 1B (Oncolytic Ad-REIC) and C (Oncolytic AD-REIC domain).
- the structures shown in FIGS. 1B and 1C are derived from a DNA encoding a CMV promoter sequence, REIC DNA or REIC C domain DNA, and a polyA addition sequence in the E1 region of the restriction-proliferating adenovirus (Oncortic Ad) shown in FIG. 1A.
- the DNA construct is inserted at position 3524.
- Restricted growth type adenovirus of the present invention (Oncolic Ad), restricted growth type adenovirus containing full-length REIC (DNA (Oncolytic Ad-REIC), or restricted growth type adenovirus containing REIC C domain DNA (Ad-REIC C domain) can be prepared according to the description above and the cited references.
- Oncolic Ad restricted growth adenovirus of the present invention
- restriction-propagating adenovirus containing the full-length REIC DNA of the present invention can be used in humans and other mammals.
- Administration to a subject results in delivery to the subject's cancer cells.
- Cancer cells are killed by the action of oncolytic adenovirus, full-length REIC protein or REIC C domain protein is expressed in cancer cells, and cell death is selectively induced in cancer cells by endoplasmic reticulum stress at the time of expression and cancer It activates immunity, suppresses tumor cell growth, and exerts a therapeutic effect on cancer.
- the anti-cancer immune activity by REIC not only acts locally on cancer cells, but also leads to strong anti-cancer immune activation throughout the body.
- Restricted growth adenovirus containing full-length REIC DNA Oncolic Ad-REIC
- restricted growth adenovirus containing REIC C domain DNA Oncortic Ad-REC domain
- the anti-cancer effect by anti-cancer immunity activity etc. acts synergistically, and a stronger anti-cancer effect can be obtained.
- the present invention includes a virus preparation for cancer treatment containing such a restricted-proliferating adenovirus (oncolytic Ad, oncolytic Ad-REIC, oncolytic Ad-REIC domain).
- cancer to be treated examples include, but are not limited to, for example, brain / nerve tumor, skin cancer, stomach cancer, lung cancer, liver cancer, lymphoma / leukemia, colon cancer, pancreatic cancer, anal / rectal cancer, esophageal cancer, uterine cancer, breast cancer, Examples include adrenal cancer, renal cancer, renal pelvic and ureteral cancer, bladder cancer, prostate cancer, urethral cancer, penile cancer, testicular cancer, bone and osteosarcoma, leiomyoma, rhabdomyosarcoma, mesothelioma and the like.
- the restricted growth type adenovirus (oncolytic Ad, oncolytic Ad-REIC, oncolytic Ad-REIC domain) of the present invention can be used for the treatment of primary cancer and metastatic cancer.
- the restricted-proliferation adenovirus of the present invention is a method that can be used in the field of gene therapy, for example, blood vessels such as intravenous administration and intraarterial administration. It can be administered by internal administration, oral administration, intraperitoneal administration, intrathoracic administration, intratracheal administration, intrabronchial administration, subcutaneous administration, transdermal administration, and the like.
- the therapeutically effective dose of the restricted growth adenovirus of the present invention may be administered.
- a therapeutically effective amount can be readily determined by one skilled in the art of gene therapy.
- the dose can be appropriately changed depending on the severity of the disease state, sex, age, weight, habits, etc. of the subject. Contains carriers, diluents and excipients commonly used in the pharmaceutical field. For example, lactose and magnesium stearate are used as carriers and excipients for tablets.
- aqueous solution for injection isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as polyalcohols such as alcohol and propylene glycol, nonionic surfactants, etc. You may use together.
- suitable solubilizers such as polyalcohols such as alcohol and propylene glycol, nonionic surfactants, etc. You may use together.
- oily liquid sesame oil, soybean oil and the like are used, and as a solubilizing agent, benzyl benzoate, benzyl alcohol and the like may be used in combination.
- Oncolytic Ad Oncolytic Ad
- REIC-C REIC C domain
- DCN decorin
- pCA14 / REIC and pCA14 / REIC-C vectors were digested with BglII, and then the CMV-REIC-polA and AMV-REIC-C-polA expression cassettes were previously digested with BglII p ⁇ E1sp1B-HmT-Rd19 shuttle vector (Kim E et al., Hum Gene Ther 2003; 14: 1415-1428; Kim JH et al., J Natl Cancer Inst 2006; 98: 1482-1493) and cloned into p ⁇ E1sp1B-HmT-Rd19 / REIC and p ⁇ E1sp1B-HmT- Rd19 / REIC-C adenovirus E1 shuttle vector was obtained.
- E3 shuttle vector was linearized with XmnI and adenovirus total vector del-RGD linearized with SpeI together with BJ5183 The E. coli strain was cotransformed and homologous recombination was performed. As a result, an adenoviral vector del-RGD / DCN having no replication ability was obtained.
- the newly constructed p ⁇ E1sp1B-HmT-Rd19 / REIC and p ⁇ E1sp1B-HmT-Rd19 / REIC-C adenovirus E1 shuttle vectors were linearized by XmnI digestion and then co-combined with BJ5183 E. coli strain along with de1-RGD / DCN digested with BstBI. Transformation and homologous recombination were performed. As a result, tumor-specific oncolytic adenovirus expressing REIC or REIC-C and decorin was obtained. Plasmid DNA was digested with PacI, introduced into 293A cells and propagated.
- Example 2 Expression method of REIC protein in various cells by addition of oncolytic Ad, oncolytic Ad-REIC and oncolytic Ad-REIC domain
- MOI multiplicity of infection
- mice monoclonal antibody human REIC / Dkk-3 antibody (1: 1000 dilution) (primary antibody) was reacted, thoroughly washed with 0.1% Tween-20-containing TBS (T-TBS), and then labeled with horseradish peroxidase. Reaction with the next antibody. Further, after washing with T-TBS, color was developed using an ECL kit (Amersham Pharmacia Biotech, Chandler, AZ) which is a chemiluminescence detection method kit. A band of REIC protein is observed around 60 kDa by Western blot.
- FIG. 3 shows the expression of Western blot analysis of REIC protein in various cells by adding oncolytic Ad (adenovirus), oncolytic Ad-REIC, and oncolytic Ad-REIC domain.
- oncolytic Ad-REIC By adding oncolytic Ad-REIC in various cells, it is possible to express REIC protein equivalent to or higher than conventional Ad-REIC. Since REIC protein has an effect of activating anticancer immunity in vivo (WO2009 / 119874), oncolytic Ad-REIC is also expected to activate anticancer immunity in vivo. .
- Example 3 Method for Confirming Cell Death Induction Rate in Various Cells by Addition of Oncolytic Ad and Oncolytic Ad-REIC
- MOI multipleplicity of infection
- FIG. 4 shows cell death induction rates in various cells by the addition of oncolytic Ad and oncolytic Ad-REIC. As shown in FIG. 4, oncolic Ad-REIC significantly induced cell death compared to other compounds.
- Example 4 Method for Treatment Effect of Oncolytic Ad and Oncolytic Ad-REIC on Human Prostate Cancer
- PC3 human prostate cancer cells 2 ⁇ 10 6 cells / 0.1 ml PBS were administered by subcutaneous injection to the right thigh of adult male nude mice.
- Ten days after the tumor volume reached 200 to 300 mm 3 adenovirus was administered intratumorally (Day 0 in FIG. 5).
- Tumor volume was calculated using the formula 1/2 (w1 x w2 x w2). In this formula, w1 represents the maximum tumor diameter and w2 represents the minimum tumor diameter.
- Example 5 Method of NK cell induction effect of oncolytic Ad and oncolytic Ad-REIC PC3 human prostate cancer cells 2 ⁇ 10 6 cells / 0.1 ml PBS were administered by subcutaneous injection to the left and right thighs of adult male nude mice. This mouse is a mouse tumor model with at least two tumor sites. Ten days after the bilateral tumor volume was 200-300 mm 3 , adenovirus was administered into the right tumor. Three days after vector injection, natural killer (NK) cells in peripheral lymphocytes were measured by flow cytometry using an anti-NK cell antibody (eBioscience Inc., 10255 Science Center Drive, San Diego, CA 92121, USA).
- NK natural killer
- Example 6 Induction Method of Antigen-Specific Immune Response by Oncolytic Ad and Oncolytic Ad-REIC A cancer-bearing model in an immunocompetent mouse was prepared, and anticancer was performed after oncolytic Ad-REIC or oncolytic Ad was administered into the tumor Studies were conducted to identify cancer-specific CTL cells responsible for immunity. Cancer-bearing mice by subcutaneous transplantation of malignant thymoma cells [EG-7] (1.0 x 10 6 cells) introduced with the foreign antigen OVA (ovalbumin) gene into C57 / BL6 mice with normal immune system A model was created.
- EG-7 malignant thymoma cells
- OVA ovalbumin
- oncolytic Ad-REIC or oncolytic Ad was injected into the tumor (dosage was 1.0 ⁇ 10 6 pfu / tumor).
- tumors were collected, and the kinetics of the proportion of specific CD8-positive CTLs against OVA in the tumor-infiltrating lymphocytes (TIL), CD8 antibody, OVA tetramer (H-2 kb restricted OVA epitope) The antibody was recognized by flow cytometry.
- results The results are shown in FIG. As shown in FIG. 7, the frequency of tetramer-positive CD8 cells was increased in the tumor of mice administered with oncolytic Ad-REIC. That is, administration of oncolytic Ad-REIC induced a stronger OVA antigen-specific immune response than when oncolytic Ad was administered. Based on the results of this experiment, it is considered possible to induce a cancer antigen-specific immune response by administering oncolytic Ad-REIC that encodes the REIC gene and expresses the REIC protein into the tumor.
- the restricted growth adenovirus of the present invention can be used for cancer treatment.
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Abstract
Description
[1] 5型アデノウイルスのゲノムのITR(inverted terminal repeat)配列を含み、HRE配列、hTERTプロモーター、デコリンをコードするDNA及びRGD配列を含むペプチドをコードするDNAが挿入された制限増殖型アデノウイルスに、さらに全長REIC DNA又はREIC CドメインDNAが挿入され、癌細胞で特異的に増殖しREICタンパク質又はREIC Cドメインタンパク質を発現する制限増殖型アデノウイルス。
[2] プロモーター配列、デコリンをコードするDNA及びpolyA付加配列からなるDNAコンストラクトが5型アデノウイルスのE3領域中に挿入されている、[1]の制限増殖型アデノウイルス。
[3](i) hTERTプロモーターがc-Myc結合部位及びSp1結合部位の付加により修飾されたhTERTプロモーターであり、
(ii) hTERTプロモーターの上流に配列番号3で表される塩基配列からなるHRE配列が6つ挿入され、
(iii) E1A領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列のRb結合領域(Retinoblastoma遺伝子結合領域)を欠失しており、
(iv) E1B領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列のE1B-19kDaをコードする部分が欠失しており、
(v) E3領域の一部が欠失しており、
(vi) プロモーター配列、デコリンをコードするDNA及びpolyA付加配列からなるDNAコンストラクトがE3領域中に挿入され、
(vii) RGD配列を含むペプチドをコードするDNAが、E3領域中に挿入され、かつ
(viii) CMVプロモーター配列、REIC DNA又はREIC CドメインDNAをコードするDNA、並びにpolyA付加配列からなるDNAコンストラクトがE1領域中に挿入されている[1]又は[2]の制限増殖型アデノウイルス。
[4] (iii) E1A領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列の第923~946のRb結合領域(Retinoblastoma遺伝子結合領域)である24個の塩基を欠失しており、
(iv) E1B領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列の第1722~1986のE1B-19kDaをコードする部分の塩基が欠失しており、
(v) E3領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列の第28592~30479の塩基が欠失している、
[3]の制限増殖型アデノウイルス。
[5] REICが全長REICである、[1]~[4]のいずれかの制限増殖型アデノウイルス。
[6] REICがREICのCドメインである、[1]~[4]のいずれかの制限増殖型アデノウイルス。
[7] [1]~[6]のいずれかの制限増殖型アデノウイルを有効成分として含む、癌治療剤。
[8] 制限増殖型アデノウイルスが癌細胞中で特異的に増殖し、REICタンパク質を発現し、発現したREICタンパク質が小胞体ストレスにより癌細胞の細胞死を誘導し、さらにREICタンパク質が全身的抗癌免疫活性を誘導する、[7]の癌治療剤。
(1)5型アデノウイルスのITR(inverted terminal repeat)を含む。ITRは100~200塩基からなりアデノウイルスDNAのDNA複製及びパッケージングに必須なエレメントである。
(i) hTERTプロモーターがc-Myc結合部位及びSp1結合部位の付加により修飾されたhTERTプロモーターである。
(ii) hTERTプロモーターの上流に配列番号3で表される塩基配列からなるHRE配列が6つ挿入されている。
(iii) E1A領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列のRb結合領域(Retinoblastoma遺伝子結合領域)を欠失している。例えば、配列番号4に示す5型アデノウイルスゲノム配列の第923~946のRb結合領域(Retinoblastoma遺伝子結合領域)である24個の塩基を欠失している。
(iv) E1B領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列のE1B-19kDaをコードする部分の塩基が欠失している。例えば、配列番号4に示す5型アデノウイルスゲノム配列の第1722~1986のE1B-19kDaをコードする部分の塩基が欠失している。
(v) E3領域の一部が欠失している。例えば、配列番号4に示す5型アデノウイルスゲノム配列の第28592~30479の塩基が欠失している。
(vi) プロモーター配列、デコリンをコードするDNA及びpolyA付加配列からなるDNAコンストラクトがE3領域中に挿入されている。
(vii) RGD配列を含むペプチドをコードするDNAが、E3領域中に挿入されている。
E1領域及びE3領域において、それぞれ、REIC若しくはREIC Cドメイン(REIC-C)並びにデコリン(DCN)を発現するオンコリティックアデノウイルスを作製するために、最初にREIC又はREIC Cを発現するpCA14 Ad E1シャトルベクターを作製した。REIC又はREIC C遺伝子をpShuttole/REIC又はREIC-CからNheI-blunt-HindIIIを用いて切り出し、あらかじめXbaI-blunt-HindIII で消化しておいたpCA14 Ad E1シャトルベクターにサブクローニングした。pCA14/REIC及びpCA14/REIC-CベクターをBglIIで消化し、次いでCMV-REIC-polA及びAMV-REIC-C-polA発現カセットをあらかじめBglIIで消化しておいたpΔE1sp1B-HmT-Rd19シャトルベクター(Kim E et al., Hum Gene Ther 2003;14:1415-1428;Kim JH et al., J Natl Cancer Inst 2006;98:1482-1493)にクローニングし、pΔE1sp1B-HmT-Rd19/REIC及びpΔE1sp1B-HmT-Rd19/REIC-CアデノウイルスE1シャトルベクターを得た。pSP72-E3/DCN(I-K Choi et al., Gene Therapy 2010;17:190-201.) E3シャトルベクターをXmnIを用いて線状化し、SpeIで線状化したアデノウイルストータルベクターdel-RGDと共にBJ5183大腸菌株を共形質転換し相同組換えを行った。その結果、複製能力のないアデノウイルスベクターdel-RGD/DCNを得た。新たに構築したpΔE1sp1B-HmT-Rd19/REIC及びpΔE1sp1B-HmT-Rd19/REIC-CアデノウイルスE1シャトルベクターをXmnI消化により線状化し、次いでBstBIで消化したde1-RGD/DCNと共にBJ5183大腸菌株を共形質転換し相同組換えを行った。その結果、REIC若しくはREIC-C並びにデコリンを発現する腫瘍特異的オンコリティックアデノウイルスを得た。プラスミドDNAをPacIで消化し、293A細胞に導入し増殖させた。
方法
Ad-REIC処理後のREICタンパク質発現を測定するため、細胞を平底6ウェルプレートに播き24時間インキュベートした。細胞を図に記載のMOI(multiplicity of infection)でアデノウイルスを完全培地(300μl)中で1時間感染処理し、PBS(phosphate buffered saline)で2回洗浄し、溶解バッファー(50 mM HEPES, pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM PMSF, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 2 mM Na3VO4, 1 mM NaF, 10 mM β-GP)を用いて溶解させ、REICタンパク質を抽出した。
図3に、オンコリティックAd(アデノウイルス)、オンコリティックAd-REIC、オンコリティックAd-REIC domainの添加による各種細胞でのREICタンパク質のウエスタンブロット分析での発現を示す。
方法
Ad-REIC処理後の殺細胞率を調べるために、細胞を平底6ウェルプレートに播き24時間インキュベートした。細胞を図に記載のMOI(multiplicity of infection)でアデノウイルスで完全培地(300μl)中で1時間処理し、新鮮培地1700μlを添加した。図に示した日数経過後、死細胞率(%)を5視野の顕微鏡観察により測定した。なお、図4~6において、データは平均±標準偏差で表す。統計的有意差検定は分散分析又はMann-Whitney Uテストで行った。p<0.05で有意差があると判断した。
オンコリティックAd及びオンコリティックAd-REICの添加による各種細胞での細胞死誘導率を図4に示す。図4に示すように、オンコリティックAd-REICは他の化合物に比べ有意に細胞死を誘導した。
方法
PC3ヒト前立腺癌細胞2 x 106個/0.1ml PBSを成体雄ヌードマウスの右大腿部に皮下注射により投与した。腫瘍体積が200~300mm3になった10日後に、アデノウイルスを腫瘍内投与した(図5のDay 0)。腫瘍体積は式1/2 (w1 x w2 x w2)を用いて計算した。この式において、w1は最大腫瘍直径を、w2は最少腫瘍直径を示す。
結果を図5に示す。図5に示すように、オンコリティックAd-REICを用いた場合、治療効果は用量依存的に認められた。他の治療群に比較して、オンコリティックAd-REIC(107)の効果が大きかった。実験に供したすべてのマウスに対して明確な毒性は認められなかった。
方法
PC3ヒト前立腺癌細胞2 x 106個/0.1ml PBSを成体雄ヌードマウスの左右の大腿部に皮下注射により投与した。このマウスは少なくとも2か所の腫瘍部位を有するマウス腫瘍モデルである。両側の腫瘍体積が200~300mm3になった10日後に、アデノウイルスを右側の腫瘍内に投与した。ベクター注射3日後に、末梢リンパ球中のナチュラルキラー(NK)細胞を抗NK細胞抗体(eBioscience Inc., 10255 Science Center Drive, San Diego, CA 92121, USA)を用いたフローサイトメトリーにより測定した。
結果を図6に示す。図6に示すように、オンコリティックAd-REICを用いた場合、他の治療群に比較して、NK細胞誘導効果は有意に大きかった。
方法
免疫正常マウスでの担癌モデルを作製し、オンコリティックAd-REIC又はオンコリティックAdを腫瘍内投与後に、抗癌免疫を担う癌特異的CTL細胞を同定する研究を行った。正常の免疫系を持つC57/BL6マウスに、外来抗原であるOVA(卵白アルブミン)遺伝子を導入した悪性胸腺腫細胞[EG-7]株(1.0 x 106 cells)を皮下移植して担癌マウスモデルを作成した。腫瘍径が100mm3以上になった時点で、オンコリティックAd-REIC又はオンコリティックAdを腫瘍内へ注入した(投与量は1.0 x 106pfu/tumor)。治療後3日目に腫瘍を回収し、腫瘍内浸潤リンパ球(TIL)において、OVAに対する特異的CD8陽性CTLが占める割合の動態を、CD8抗体、OVAテトラマー(H-2kb拘束性にOVAエピトープを認識する抗体)を用いてフローサイトメトリーで解析した。
結果を図7に示す。図7に示すように、オンコリティックAd-REIC投与のマウスの腫瘍内において、テトラマー陽性のCD8細胞の頻度が上昇していた。すなわち、オンコリティックAd-REIC を投与することにより、オンコリティックAdを投与した場合と比べて、より強いOVA抗原特異的免疫応答が誘導された。本実験の結果を踏まえて、REIC遺伝子をコードしREICタンパク質を発現するオンコリティックAd-REICを腫瘍内に投与することにより、癌抗原特異的免疫応答を誘導することが可能であると考えられる。
Claims (8)
- 5型アデノウイルスのゲノムのITR(inverted terminal repeat)配列を含み、HRE配列、hTERTプロモーター、デコリンをコードするDNA及びRGD配列を含むペプチドをコードするDNAが挿入された制限増殖型アデノウイルスに、さらに全長REIC DNA又はREIC CドメインDNAが挿入され、癌細胞で特異的に増殖しREICタンパク質又はREIC Cドメインタンパク質を発現する制限増殖型アデノウイルス。
- プロモーター配列、デコリンをコードするDNA及びpolyA付加配列からなるDNAコンストラクトが5型アデノウイルスのE3領域中に挿入されている、請求項1記載の制限増殖型アデノウイルス。
- (i) hTERTプロモーターがc-Myc結合部位及びSp1結合部位の付加により修飾されたhTERTプロモーターであり、
(ii) hTERTプロモーターの上流に配列番号3で表される塩基配列からなるHRE配列が6つ挿入され、
(iii) E1A領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列のRb結合領域(Retinoblastoma遺伝子結合領域)を欠失しており、
(iv) E1B領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列のE1B-19kDaをコードする部分の塩基が欠失しており、
(v) E3領域の一部が欠失しており、
(vi) プロモーター配列、デコリンをコードするDNA及びpolyA付加配列からなるDNAコンストラクトがE3領域中に挿入され、
(vii) RGD配列を含むペプチドをコードするDNAが、E3領域中に挿入され、かつ
(viii) CMVプロモーター配列、REIC DNA又はREIC CドメインDNAをコードするDNA、並びにpolyA付加配列からなるDNAコンストラクトがE1領域中に挿入されている請求項1又は2に記載の制限増殖型アデノウイルス。 - (iii) E1A領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列の第923~946のRb結合領域(Retinoblastoma遺伝子結合領域)である24個の塩基を欠失しており、
(iv) E1B領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列の第1722~1986のE1B-19kDaをコードする部分の塩基が欠失しており、
(v) E3領域の一部であって、配列番号4に示す5型アデノウイルスゲノム配列の第28592~30479の塩基が欠失している、
請求項3記載の制限増殖型アデノウイルス。 - REICが全長REICである、請求項1~4のいずれか1項に記載の制限増殖型アデノウイルス。
- REICがREICのCドメインである、請求項1~4のいずれか1項に記載の制限増殖型アデノウイルス。
- 請求項1~6のいずれか1項に記載の制限増殖型アデノウイルを有効成分として含む、癌治療剤。
- 制限増殖型アデノウイルスが癌細胞中で特異的に増殖し、REICタンパク質を発現し、発現したREICタンパク質が小胞体ストレスにより癌細胞の細胞死を誘導し、さらにREICタンパク質が全身的抗癌免疫活性を誘導する、請求項7記載の癌治療剤。
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