Patent Grant
10071126
This application is a National Stage application of PCT/JP2015/065004, filed May 26, 2015, which claims priority from Japanese application JP 2014-110672, filed May 28, 2014.
The present invention relates to a conditionally replicating adenovirus to highly express REIC (REIC/Dkk-3) protein.
Clinical trials on drugs for cancer with various conditionally replicating (genetically engineered to replicate specifically in a cancer cell) viruses have been previously reported (Non Patent Literature 1). Although treatments of cancer with these drugs have achieved certain results, the effect is limited in most cases, and development of more effective drugs is desired.
Representative examples of drugs for cancer with a conditionally replicating virus for which results of a clinical trial have been reported include Telomelysin (Non Patent Literature 2), which contains an adenovirus type 5 as a skeleton, and Talimogene laherparepvec (T-VEC, old name: Oncovex) (Non Patent Literature 3), which contains a herpes simplex virus type 1 as a skeleton.
In recent years, it has been considered important to allow drugs for cancer with a virus to have a function of activating anticancer immunity. From this viewpoint, although Telomelysin (Non Patent Literature 2) can induce cancer cell death through replication of the adenovirus at a location of administration, it does not encode a cytokine gene or the like which activates anticancer immunity, and thus a strong, systemic anticancer immunity-activating effect is not expected. This may limit the therapeutic effect of Telomelysin.
T-VEC is expected to provide cancer cell death and impart antigenicity to a cancer cell through replication of the herpes virus at a location of administration and to activate anticancer immunity through expression of the cytokine GM-CSF. Although the cytokine GM-CSF has an anticancer immunity-activating effect to induce a dendritic cell as a cancer antigen-presenting cell, to differentiate (Non Patent Literature 1), it has been also reported that the cytokine GM-CSF in a high dosage may induce an immunosuppressive cell to reduce the anticancer immune function to worsen disease condition (Non Patent Literature 4), which may limit the therapeutic effect of T-VEC.
Thus, development of more effective drugs for cancer is desired in light of the above problems of existing drugs for cancer with a conditionally replicating virus. As a conditionally replicating adenovirus to solve the above problems, conditionally replicating adenoviruses having various mutations have been reported (Patent Literatures 1 and 2 and Non Patent Literatures 5 to 10).
On the other hand, an REIC (REIC/Dkk-3) gene is known as a gene associated with immortalization of a cell, and it has been reported that expression of this gene is suppressed in cancer cells, and that the REIC gene is used for treatment of cancers (Patent Literature 3). The REIC has an anticancer immunity-activating effect and an effect of inducing cancer cell death through endoplasmic reticulum stress in gene expression. It is further reported that a partial fragment of the REIC gene has the same effect as the full length REIC (Patent Literature 4), and furthermore an adenovirus to express the REIC/Dkk-3 gene has been reported (Patent Literature 5 and Non Patent Literature 11).
An object of the present invention is to provide a conditionally replicating adenovirus having a strong anticancer effect.
The present inventors effectively combined previously reported techniques relating to conditionally replicating adenoviruses (Patent Literatures 1 and 2 and Non Patent Literatures 5 to 10) to produce a unique conditionally replicating adenovirus. In addition, the adenovirus was newly allowed to encode the REIC gene which expresses REIC protein, which has a unique anticancer immunity-activating effect (Non Patent Literature 11). By virtue of this, the present inventors succeeded in development of an anticancer virus formulation of high inventiveness having uniqueness and novelty in combination and being expected to be far superior to existing drugs for cancer with a conditionally replicating virus, and thus completed the present invention.
Specifically, the present invention is as follows:
[1] A conditionally replicating adenovirus to replicate specifically in a cancer cell and express REIC protein or REIC C domain protein, wherein the conditionally replicating adenovirus is obtained by inserting full-length REIC DNA or REIC C domain DNA into a conditionally replicating adenovirus comprising an ITR (inverted terminal repeat) sequence of an adenovirus type 5 genome and insertion of an HRE sequence, an hTERT promoter, a decorin-encoding DNA, and a DNA encoding a peptide comprising an RGD sequence.
[2] The conditionally replicating adenovirus according to [1], wherein a DNA construct consisting of a promoter sequence, the decorin-encoding DNA, and a poly A addition sequence is inserted into an E3 region of the adenovirus type 5.
[3] The conditionally replicating adenovirus according to [1] or [2], wherein
(i) the hTERT promoter is an hTERT promoter modified through addition of a c-Myc binding site and an Sp1 binding site;
(ii) six HRE sequences each consisting of a nucleotide sequence as set forth in SEQ ID NO: 3 are inserted into upstream of the hTERT promoter;
(iii) an Rb binding region (Retinoblastoma gene binding region), being a part of an E1A region, in the adenovirus type 5 genome sequence as set forth in as SEQ ID NO: 4 is deleted;
(iv) nucleotides of a portion encoding E1B-19 kDa, being a part of an E1B region, in the adenovirus type 5 genome sequence as set forth in as SEQ ID NO: 4 are deleted;
(v) an E3 region is partially deleted;
(vi) a DNA construct consisting of a promoter sequence, the decorin-encoding DNA, and a poly A addition sequence is inserted into the E3 region;
(vii) the DNA encoding a peptide comprising an RGD sequence is inserted into the E3 region; and
(viii) a DNA construct consisting of a CMV promoter sequence, a DNA encoding REIC DNA or REIC C domain DNA, and a poly A addition sequence is inserted into an E1 region.
[4] The conditionally replicating adenovirus according to [3], wherein
(iii) 24 nucleotides at positions 923 to 946 as the Rb binding region (Retinoblastoma gene binding region), being a part of the E1A region, in the adenovirus type 5 genome sequence as set forth in SEQ ID NO: 4 are deleted;
(iv) nucleotides at positions 1722 to 1986 as the portion encoding E1B-19 kDa, being a part of the E1B region, in the adenovirus type 5 genome sequence as set forth in as SEQ ID NO: 4 are deleted; and
(v) nucleotides at positions 28592 to 30479, being a part of the E3 region, in the adenovirus type 5 genome sequence as set forth in as SEQ ID NO: 4 are deleted.
[5] The conditionally replicating adenovirus according to any one of [1] to [4], wherein REIC is full length REIC.
[6] The conditionally replicating adenovirus according to any one of [1] to [4], wherein REIC is REIC C domain.
[7] A cancer therapeutic agent comprising the conditionally replicating adenovirus according to any one of [1] to [6] as an active ingredient.
[8] The cancer therapeutic agent according to [7], wherein the conditionally replicating adenovirus replicates specifically in a cancer cell and expresses REIC protein, and the REIC protein expressed induces cancer cell death through endoplasmic reticulum stress, and the REIC protein further induces a systemic anticancer immune activity.
The present specification encompasses the contents described in the specification and/or drawings of JP Patent Application No. 2014-110672, on which the priority of the present application is based.
The conditionally replicating adenovirus according to the present invention results from improvement of a conventional conditionally replicating adenovirus, and has a stronger anticancer effect than conventional conditionally replicating adenoviruses. In addition, the conditionally replicating adenovirus into which REIC DNA has been inserted, in addition to an anticancer effect of the conditionally replicating adenovirus itself, an anticancer immunity-activating effect and an effect of inducing cancer cell death through endoplasmic reticulum stress in gene expression each provided by REIC in combination, and these effects cooperate to exert a synergistic, strong therapeutic effect against cancer.
Now, the present invention will be described in detail.
The present invention is a conditionally replicating adenovirus which comprises REIC (REIC/Dkk-3) DNA and can be used for expression of REIC protein.
A conditionally replicating adenovirus, which refers to an adenovirus genetically engineered to replicate only in cancer cells, does not act on normal cells, and replicates only in cancer cells and lyses a cancer cell to effectively kill it. Conditionally replicating adenoviruses are also called oncolytic adenoviruses or tumorlytic adenoviruses. The conditionally replicating adenovirus according to the present invention can be used with a foreign gene inserted thereinto, and thus can be regarded as a conditionally replicating adenovirus vector.
The conditionally replicating adenovirus according to the present invention, into which full-length REIC DNA or an REIC DNA fragment has been introduced, has, in addition to a cancer cell-killing effect of the conditionally replicating adenovirus itself, effects on cancer cells provided by REIC such as an anticancer immunity-activating effect and an effect of inducing cancer cell death through endoplasmic reticulum stress in gene expression, and thus can exert a synergistic cancer cell-killing effect.
In the present invention a conditionally replicating adenovirus is called an oncolytic adenovirus (oncolytic Ad), a conditionally replicating adenovirus which comprises full-length REIC DNA and can express full-length REIC is called an oncolytic Ad-REIC, and a conditionally replicating adenovirus which comprises REIC C domain DNA and can express an REIC C domain is called an oncolytic Ad-REIC domain.
Each of the conditionally replicating adenoviruses used in the present invention has a skeleton of an adenovirus type 5 whose replication is limited by a human telomerase reverse transcriptase (hTERT) promoter. The conditionally replicating adenovirus according to the present invention comprises an ITR (inverted terminal repeat) of an adenovirus type 5 and further comprises a decorin-encoding DNA, where decorin is a protein which suppresses the formation and growth of a tumor, and contains other modifications (insertion and deletion of specific sequences). The decorin DNA is expressed by a CMV promoter. The genome sequence of the adenovirus type 5 is set forth in Virology, 186 (1), 1992, pp. 280-285, and registered as GenBank Accession No. M73260. The genome sequence of the adenovirus type 5 is set forth in SEQ ID NO: 4. The genome of the adenovirus contains an ITR (inverted terminal repeat) at each end, and contains an E1A region, E1B region, E2 region, E3 region, and E4 region in the order from the 5′ side, as initially transcribed regions.
Features of the conditionally replicating adenovirus according to the present invention are as follows.
(1) An ITR (inverted terminal repeat) of the adenovirus type 5 is comprised. The ITR consists of 100 to 200 nucleotides and is an element essential for replication and packaging of the adenovirus DNA.
(2) A human telomerase reverse transcriptase (hTERT) promoter is comprised. The hTERT promoter is comprised in the upstream of the E1 region of the adenovirus type 5 genome, and is linked, for example, to the immediate upstream of the E1 region. Preferably, the hTERT promoter has been modified. A modified hTERT promoter is called an m-hTERT promoter. A modified hTERT promoter comprises 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 cccccgcccc) (SEQ ID NO: 1). A wild-type hTERT promoter comprises two c-Myc binding sites and five Sp1 binding sites. The hTERT promoter of the conditionally replicating adenovirus according to the present invention comprises, for example, three c-Myc binding sites and 10 Sp1 binding sites in total with further addition of one c-Myc binding site and five Sp1 binding sites. The c-Myc binding site and Sp1 binding site may be comprised at the 3′ end or 5′ end of the hTERT promoter, or may be comprised in the interior of the hTERT promoter sequence. One exemplary sequence of modified hTERT promoters is set forth in
(3) A hypoxia responsive element (HRE) is comprised. The HRE, which is a DNA element having a gene to be activated by hypoxia and responds to hypoxia, comprises ACGTG as a consensus sequence. Conditionally replicating adenoviruses used in the present invention comprise a sequence consisting of 5 to 40 nucleotides comprising the consensus sequence. Oxygen concentration is approximately 2 to 9% in a normal tissue. In contrast, oxygen concentration is approximately 1.3% in a cancer cell, which indicates hypoxia. As a result, replication of a conditionally replicating adenovirus comprising an HRE is accelerated in a cancer cell. Examples of HRE sequences include a sequence comprising the consensus sequence in a human vascular endothelial growth factor (hVEGF) gene (GenBank Accession No. M63971), specifically, the nucleotide sequence from the 1379th nucleotide to the 1412th nucleotide in the hVEGF gene (SEQ ID NO: 3). A plurality of HREs linked may be used, and for example, 3 to 12 HREs linked may be used, or six HREs linked (HRE×6) or 12 HREs linked (HRE×12) may be used (Oh-Joon Kwon et al., Clin Cancer Res; 16(24) Dec. 15, 2010, pp. 60716082). Preferably, six HREs linked (HRE×6) is used. The HRE is suitably linked to the upstream of the hTERT promoter, for example, to the immediate upstream of the hTERT promoter.
(4) An E1A region is partially deleted. The E1A region is a region at positions 342 to 1545 in the adenovirus type 5 genome (SEQ ID NO: 4), and E1A protein binds to an RB (Retinoblastoma) gene product. The E1A region is a region essential for replication of adenoviruses, and the conditionally replicating adenovirus according to the present invention contains deletion of an Rb binding region (Retinoblastoma gene binding region) in the E1A and possesses replication capability itself. The partial deletion of the E1A region in the conditionally replicating adenovirus according to the present invention is also described as “comprising a modified and active E1A gene”. Here, the modified and active E1A gene contains a mutation causing substitution of Glu residue at position 45 with Gly and a mutation causing total substitution of the amino acid sequence at positions 121 to 127 with Gly in a nucleotide sequence encoding the Rb (retinoblastoma protein) binding site. In a tumor cell, not only p53 protein is mutated, but also Rb is mutated or Rb-related signaling mechanism is significantly damaged. Thus, an adenovirus with the binding ability to Rb deleted is suppressed from replication due to the activity of Rb in a normal cell, and in contrast, actively replicates in a tumor cell, in which the function of Rb is suppressed, and thus a cancer cell can be selectively killed. Accordingly, the recombinant adenovirus according to the present invention, which comprises the above-described mutation in the Rb binding site, has highly enhanced specificity to cancer cells. For a mutation in the Rb binding site, for example, 24 nucleotides at positions 923 to 946, being a part of the E1A region, as the Rb binding region (Retinoblastoma gene binding region) in the adenovirus type 5 genome sequence as set forth in SEQ ID NO: 4 may be deleted (ΔE1A (24 bp)) (Candelaria Gomez-Manzano et al., Oncogene (2004) 23, pp. 1821-1828).
(5) An E1B region is partially deleted. The E1B region is a region at positions 1714 to 3509 in the adenovirus type 5 genome (SEQ ID NO: 4), and E1B-55 kDa protein, a gene product of the E1B region, is involved in replication of a virus through binding to p53 protein. The partial deletion of the E1B region is also described as “containing an inactivated portion in the E1B region”, and the conditionally replicating adenovirus according to the present invention contains an inactivated E1B 19 kDa gene, E1B 55 kDa gene, or E1B 19 kDa/E1B 55 kDa gene, and preferably contains inactivated E1B 19 kDa and E1B 55 kDa genes. In the present specification, “inactivation” used in relation to a gene means that a gene is not transcribed and/or decoded normally, and the protein encoded by the gene lacks its normal function. For example, an inactivated E1B 19 kDa gene is a gene in which a mutation (substitution, addition, partial deletion, or total deletion) has been generated and cannot produce active E1B 19 kDa protein. In the case that the E1B 19 kDa gene is deleted, the apoptotic capability of a cell can be enhanced, and if the E1B 55 kDa gene is deleted, specificity to tumor cells is provided (KR Patent Application No. 100528727). For example, nucleotides at positions 1722 to 1986, being a part of the E1B region, in the adenovirus type 5 genome sequence as set forth in SEQ ID NO: 4 are suitably deleted. Deletion of this nucleotide sequence leads to deletion of a portion encoding E1B-19 kDa of 19 kDa, as a trans-splicing product of E1B protein of 55 kDa, and thus this deletion is called ΔE1B (19 kDa) (Jaesung Kim et al., Cancer Gene Therapy (2002) 9, pp. 725-736). Alternatively, a stop codon may be introduced so that only the E1B (19 kDa) is not expressed in the E1B region.
(6) An E3 region is deleted. It is only required that a DNA encoding E3 protein be totally or partially deleted. The E3 region is a region at positions 27858 to 30839 in the adenovirus type 5 genome (SEQ ID NO: 4). The E3 region is not required for replication of adenoviruses and a foreign gene can be inserted into the E3 region. In this case, it is suitable that the E3 region is partially deleted and a foreign gene is inserted into the portion. For example, nucleotides at positions 28592 to 30479, being a part of the E3 region, in the adenovirus type 5 genome sequence as set forth in SEQ ID NO: 4 are suitably deleted (4E3). Into this portion, a decorin-encoding DNA to be described later can be inserted, for example.
(7) A decorin (DCN)-encoding DNA, where decorin is a protein which suppresses the formation and growth of a tumor, is inserted. Decorin is a protein belonging to SLRPs (small leucine-rich proteoglycan) and contains 10 to 12 leucine-rich repeats, and the core site is arch-shaped and binds to several types of growth factors present in the extracellular matrix or a decorin receptor. Decorin acts as a natural antagonist for the formation and growth of a tumor through suppression of the activity of a tumor growth factor (TGF-β) to prevent fibrillation of collagen and association with the matrix assembly to suppress the growth of a tumor cell. Introduction of decorin into a conditionally replicating adenovirus facilitates introduction of the conditionally replicating adenovirus into a cancer cell to enhance the tumor cell-killing activity. In the conditionally replicating adenovirus according to the present invention, a promoter is linked to the upstream of the decorin-encoding DNA, and a poly A addition sequence (polyadenylated sequence, polyA) is linked to the downstream of the decorin-encoding DNA. The promoter is a promoter which operates preferably in an animal cell, more preferably in a mammalian cell and can regulate transcription of the decorin gene, and examples thereof include, but are not limited to, promoters derived from mammalian viruses and promoters derived from mammalian cell genomes, such as a U6 promoter, an H1 promoter, a CMV (Cytomegalovirus) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a tk promoter of an HSV, an RSV promoter, an EF1α promoter, a metallothionein promoter, a β-actin promoter, a promoter of the human IL-2 gene, a promoter of the human IFN gene, a promoter of the human IL-4 gene, a promoter of the human lymphotoxin gene, a promoter of the human GM-CSF gene, inducible promoters, cancer cell-specific promoters (e.g., a TERT promoter, a PSA promoter, a PSMA promoter, a CEA promoter, an E2F promoter and an AFP promoter), and tissue-specific promoters (e.g., an albumin promoter). Preferably, a CMV promoter or a cancer cell-specific promoter is used. In the case that a cancer cell-specific promoter is used, it is preferred to use a TERT promoter or an E2F promoter. For the TERT (telomere reverse transcriptase) promoter, a wild-type hTERT (human telomere reverse transcriptase) promoter or an m-hTERT promoter described in (2) may be used. The origin of the poly A addition sequence (polyadenylated sequence, polyA) is not limited, and examples thereof include a poly A addition sequence derived from a growth hormone gene such as a poly A addition sequence derived from the bovine growth hormone gene (BGH polyA) and a poly A addition sequence derived from the human growth hormone gene, a poly A addition sequence derived from an SV40 virus, and a poly A addition sequence derived from the human or rabbit β-globin gene. The poly A addition sequence comprised in a DNA construct increases the transcription efficiency.
An adenovirus containing the decorin-encoding DNA is described in JP Patent Publication (Kokai) No. 2008-531010 and I-K Choi et al., Gene Therapy (2010)17, 190-201. The nucleotide sequence of the decorin-encoding DNA (GenBank Accession No. NM_001920.3) is set forth in SEQ ID NO: 5. The nucleotide sequence of the CMV promoter (GenBank Accession No. X17403) and the nucleotide sequence of the BGH poly A addition sequence (GenBank Accession No. M57764) are set forth in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
A DNA construct comprising the decorin-encoding DNA and consisting of the promoter sequence, the decorin-encoding DNA, and the poly A addition sequence is suitably inserted into the E1A region, the E1B region, or the E3 region, and is preferably inserted into the E3 region. In the adenovirus vector according to the present invention, the E1A region, E1B region, and E3 region of the adenovirus type 5 genome are partially deleted, as described below. The DNA construct in which the CMV promoter, the decorin-encoding DNA, and the poly A addition sequence are linked in the order presented is suitably inserted into the deleted portion. For example, the DNA construct can be inserted into the adenovirus type 5 genome concomitantly with partial deletion of the E1A region, E1B region, and E3 region through homologous recombination. For example, the DNA construct is suitably inserted into the deleted portion of the 28592nd to 30479th nucleotides in the adenovirus type 5 genome sequence as set forth in SEQ ID NO: 4, described in the above (6).
(8) A DNA encoding a peptide comprising an RGD (Arg-Gly-Asp) sequence is inserted. Examples of peptides comprising an RGD sequence include RGD-comprising peptides consisting of 4 (e.g., GRGDS (SEQ ID NO: 8)) to 15 amino acids, such as peptides represented by CDCRGDCFC (SEQ ID NO: 9) and GSCDCRGDCFCSG (SEQ ID NO: 10). The DNA encoding a peptide comprising an RGD sequence is inserted, for example, into the E3 region, and is suitably inserted, specifically, between the 32676th nucleotide and the 32677th nucleotide in the E3 region in the adenovirus type 5 genome. The DNA encoding a peptide comprising an RGD sequence comprised in a conditionally replicating adenovirus facilitates introduction of the conditionally replicating adenovirus into a cancer cell. An adenovirus comprising an RGD sequence is described, for example, in Hao Wu et al., J Gene Med 2011; 13: 658-669.
The conditionally replicating adenovirus, with the above features (1) to (8), according to the present invention comprises the ITR (inverted terminal repeat) sequence of the adenovirus type 5 genome and has a structure in which the HRE sequence, the hTERT promoter, the decorin-encoding DNA, and the DNA encoding a peptide comprising an RGD sequence are inserted.
For example, the conditionally replicating adenovirus according to the present invention illustrated in
(i) the hTERT promoter is an hTERT promoter modified through addition of a c-Myc binding site and an Sp1 binding site;
(ii) six HRE sequences each consisting of a nucleotide sequence as set forth in SEQ ID NO: 3 are inserted into upstream of the hTERT promoter;
(iii) an Rb binding region (Retinoblastoma gene binding region), being a part of an E1A region, in the adenovirus type 5 genome sequence as set forth in as SEQ ID NO: 4 is deleted; for example, 24 nucleotides at positions 923 to 946 as the Rb binding region (Retinoblastoma gene binding region) in the adenovirus type 5 genome sequence as set forth in SEQ ID NO: 4 are deleted;
(iv) nucleotides of a portion encoding E1B-19 kDa, being a part of an E1B region, in the adenovirus type 5 genome sequence as set forth in as SEQ ID NO: 4 are deleted; for example, nucleotides at positions 1722 to 1986 as the portion encoding E1B-19 kDa in the adenovirus type 5 genome sequence as set forth in as SEQ ID NO: 4 are deleted;
(v) an E3 region is partially deleted; for example, nucleotides at positions 28592 to 30479 in the adenovirus type 5 genome sequence as set forth in as SEQ ID NO: 4 are deleted;
(vi) a DNA construct consisting of a promoter sequence, the decorin-encoding DNA, and a poly A addition sequence is inserted into the E3 region; and
(vii) the DNA encoding a peptide comprising an RGD sequence is inserted into the E3 region.
A multi-cloning site (insertion site) for insertion of a foreign gene may be contained in the E1A region, E1B region, or E3 region. A foreign gene such as REIC DNA to be described later can be inserted into the multi-cloning site.
The above elements need to be operably linked. Here, “operably linked” means that elements are linked so that each element exerts the function and expression of a gene to be expressed is enhanced.
For example, the conditionally replicating adenovirus according to the present invention has a structure represented by ITR-4E1A-4E1B-promoter-decorin DNA-poly A addition sequence-RGD sequence-ITR, and the construct consisting of “promoter-decorin DNA-poly A addition sequence” is inserted into a deleted portion of the E3 region. The structure (gene map) of such a conditionally replicating adenovirus is illustrated in
An oncolytic Ad-REIC or an oncolytic Ad-REIC domain can be produced by inserting full-length REIC DNA or REIC C domain DNA into the above conditionally replicating adenovirus (oncolytic Ad).
The nucleotide sequence of the REIC DNA is set forth in SEQ ID NO: 11. The amino acid sequence of REIC protein encoded by the REIC DNA is set forth in SEQ ID NO: 12. In the present invention, REIC is occasionally referred to as REIC/Dkk-3.
The nucleotide sequence of the REIC C domain DNA is set forth in SEQ ID NO: 13, and the amino acid sequence of REIC C domain protein encoded by the domain is set forth in SEQ ID NO: 14.
The REIC DNA or REIC C domain DNA comprised in the conditionally replicating adenovirus according to the present invention is a DNA encoding a protein having an anticancer immunity-activating effect and an effect of inducing cancer cell death through endoplasmic reticulum stress in gene expression, among DNAs to hybridize with a DNA containing a nucleotide sequence complementary to a nucleotide sequence as set forth in SEQ ID NO: 11 or 13 under stringent conditions, DNAs having sequence identity of at least 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more to a nucleotide sequence as set forth in SEQ ID NO: 11 or 13 as calculated by using a BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., with default parameters, i.e., initially set parameters), DNAs encoding a protein consisting of an amino acid sequence obtained by providing the amino acid sequence of a protein encoded by the DNA with substitution, deletion, and/or addition of one or several (1 to 10, preferably 1 to 5, more preferably 1 or 2) amino acids, and other DNAs. Here, “stringent conditions” refer to conditions of around “1×SSC, 0.1% SDS, 37° C.”, more stringent conditions refer to conditions of around “0.5×SSC, 0.1% SDS, 42° C.”, and even more stringent conditions refer to conditions of around “0.2×SSC, 0.1% SDS, 65° C.”. It is expected that the more stringent hybridization conditions are as mentioned, the higher homology to a probe sequence a DNA isolated has. It is to be noted that the combinations of SSC, SDS, and temperature are just examples, and stringency required can be achieved by appropriately combining probe concentration, probe length, reaction duration for hybridization, etc. Those skilled in the art could appropriately determine “stringent conditions” for hybridization of a DNA with high sequence identity. Further, the REIC DNA comprised in the DNA construct in the present invention is a DNA encoding a protein as set forth in SEQ ID NO: 2.
The REIC DNA or REIC C domain DNA can be obtained from a human cell, a human tissue, or the like on the basis of sequence information of SEQ ID NOs: 11 to 14.
A CMV (cytomegalovirus) promoter is linked to the upstream of the full length REIC DNA or REIC C domain DNA, and to the downstream thereof a poly A addition sequence (polyadenylated sequence, polyA) is linked. The origin of the poly A addition sequence (polyadenylated sequence, polyA) is not limited, and examples thereof include a poly A addition sequence derived from a growth hormone gene such as a poly A addition sequence derived from the bovine growth hormone gene (BGH polyA) and a poly A addition sequence derived from the human growth hormone gene, a poly A addition sequence derived from an SV40 virus, and a poly A addition sequence derived from the human or rabbit β-globin gene. The poly A addition sequence comprised in a DNA construct increases the transcription efficiency. The nucleotide sequence of the CMV promoter (GenBank Accession No. X17403) and the nucleotide sequence of the BGH poly A addition sequence are set forth in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
A DNA construct comprising the DNA encoding the REIC DNA or REIC C domain DNA and consisting of the CMV promoter sequence, the DNA encoding the REIC DNA or REIC C domain DNA, and the poly A addition sequence is suitably inserted into the E1A region, E1B region, or E3 region, and is preferably inserted into the E1 region (E1A or E1B region). For example, the DNA construct can be inserted into the E1A region, E1B region, or E3 region of the adenovirus type 5 genome through homologous recombination.
For example, the DNA construct is suitably inserted into the E1 region of the above conditionally replicating adenovirus. In homologous recombination, only the DNA construct comprising the DNA encoding the REIC DNA or REIC C domain DNA and consisting of the CMV promoter sequence, the DNA encoding the REIC DNA or REIC C domain DNA, and the poly A addition sequence may be inserted, or a construct containing the E1 region which is obtained by inserting a DNA construct comprising the REIC DNA or REIC C domain DNA into the E1 region and partially deleting each of the E1A region and the E1B may be inserted through homologous recombination. This homologous recombination enables insertion of the DNA construct comprising the REIC DNA or REIC C domain DNA into the adenovirus type 5 concomitant with partial deletion of the E1A region and E1B region (4E1A (24 bp) and ΔE1B (19 kDa)) in the adenovirus type 5.
For example, the DNA construct is suitably inserted, specifically, between the nucleotide at position 342 and the nucleotide at position 3522 in the adenovirus type 5 genome sequence as set forth in SEQ ID NO: 4 through homologous recombination. For example, the DNA construct comprising the DNA encoding the REIC DNA or REIC C domain DNA and consisting of the CMV promoter sequence, the DNA encoding the REIC DNA or REIC C domain DNA, and the poly A addition sequence is suitably inserted into the E1 region through homologous recombination with pE1sp1B-HmT-Rd19/CMV-REIC-polA ((left homology portion: 22-341) (right homology portion: 3523-5790)) as an E1 shuttle vector. With use of this shuttle vector, the following DNA construct is inserted between position 342 and position 3522 in the adenovirus type 5 genome sequence.
A construct in which the DNA construct consisting of the CMV promoter sequence, the DNA encoding the REIC DNA or REIC C domain DNA, and the poly A addition sequence is inserted into the E1 region, and the Rb binding site in the E1A region is deleted, and the portion encoding the E1B-19 kDa in the E1B region is deleted.
Thus, the DNA construct consisting of the CMV promoter sequence, the DNA encoding the REIC DNA or REIC C domain DNA, and the poly A addition sequence can be inserted into the adenovirus type 5 concomitantly with partial deletion of the E1A region and E1B region (ΔE1A (24 bp) and ΔE1B (19 kDa)) in the adenovirus type 5.
Alternatively, the DNA construct consisting of the CMV promoter sequence, the DNA encoding the REIC DNA or REIC C domain DNA, and the poly A addition sequence may be inserted, for example, between position 3524 and position 3525 or between position 3523 and position 3524 in the adenovirus type 5 genome sequence.
The structure of the conditionally replicating adenovirus into which the DNA encoding the REIC DNA or REIC C domain DNA is illustrated in
The above elements need to be operably linked. Here, “operably linked” means that elements are linked so that each element exerts the function and expression of a gene to be expressed is enhanced.
The conditionally replicating adenovirus (oncolytic Ad), conditionally replicating adenovirus comprising the full length REIC DNA (oncolytic Ad-REIC), or conditionally replicating adenovirus comprising the REIC C domain DNA (Ad-REIC C domain) according to the present invention can be produced in accordance with the above description and the descriptions of the cited references.
The conditionally replicating adenovirus (oncolytic Ad) according to the present invention administered to a subject of a human or another mammal is delivered to a cancer cell in the subject and replicates in the cancer cell to kill the cancer cell.
Each of the conditionally replicating adenovirus comprising the full length REIC DNA (oncolytic Ad-REIC) and conditionally replicating adenovirus comprising the REIC C domain DNA (oncolytic Ad-REC domain) according to the present invention administered to a subject of a human or another mammal is delivered to a cancer cell in the subject. The cancer cell is killed via the effect of the oncolytic adenovirus, and full-length REIC protein or REIC C domain protein is expressed in the cancer cell, and selectively induces cancer cell death through endoplasmic reticulum stress in its expression, and simultaneously activates cancer immunity and suppresses the tumor cell growth, and thus the therapeutic effect on cancer is exerted. The anticancer immune activity due to REIC not only acts on a cancer cell locally, but also provides strong, systemic activation of anticancer immunity. The conditionally replicating adenovirus comprising the full length REIC DNA (oncolytic Ad-REIC) and conditionally replicating adenovirus comprising the REIC C domain DNA (oncolytic Ad-REC domain) provide a stronger anticancer effect through a synergistic effect of the cancer-killing effect of the conditionally replicating adenovirus itself and the anticancer effect, for example, due to the anticancer immune activity of REIC. The present invention encompasses virus formulations for cancer treatment comprising such a conditionally replicating adenovirus (oncolytic Ad, oncolytic Ad-REIC, oncolytic Ad-REIC domain). Examples of cancer to be treated include, but are not limited to, brain/nerve tumor, skin cancer, gastric cancer, lung cancer, liver cancer, lymphoma/leukemia, colon cancer, pancreatic cancer, anal/rectal cancer, esophageal cancer, uterine cancer, breast cancer, adrenal cancer, kidney cancer, renal pelvis cancer, bladder cancer, prostate cancer, urethral cancer, penis cancer, testicular cancer, osteosarcoma, leiomyoma, rhabdomyoma, and mesothelioma. The conditionally replicating adenovirus (oncolytic Ad, oncolytic Ad-REIC, oncolytic Ad-REIC domain) according to the present invention may be used for treatment of primary cancer or metastatic cancer.
The conditionally replicating adenovirus (oncolytic Ad, oncolytic Ad-REIC, oncolytic Ad-REIC domain) according to the present invention may be administered by using a method available in the field of gene therapy, and examples thereof include intravascular administration such as intravenous administration and intraarterial administration, oral administration, intraperitoneal administration, intrathoracic administration, intratracheal administration, intrabronchial administration, subcutaneous administration, and transdermal administration.
The conditionally replicating adenovirus (oncolytic Ad, oncolytic Ad-REIC, oncolytic Ad-REIC domain) according to the present invention is suitably administered in a therapeutically effective amount. Those skilled in the art of gene therapy could easily determine the therapeutically effective amount. In addition, the dose may be appropriately changed in accordance with the seriousness of pathological condition, sex, age, body weight, habit, etc., of a subject. A carrier, a diluting agent, and a diluent which are commonly used in the field of drug formulation are comprised. Example of carriers and diluents to be used for a tablet include lactose and magnesium stearate. Example of aqueous solutions to be used for injection include saline and isotonic solutions comprising glucose or other adjuvants, which may be used in combination with a suitable solubilizing agent, for example, an alcohol or a polyalcohol such as propylene glycol, an nonionic surfactant, or the like. Examples of oily solutions to be used include sesame oil and soybean oil, which may be used in combination with a solubilizing agent such as benzyl benzoate and benzyl alcohol.
The present invention will be specifically described with reference to the following Examples, but the present invention is never limited to these Examples.
To produce oncolytic adenoviruses to express REIC or REIC C domain (REIC-C) and decorin (DCN) in the E1 region and E3 region, respectively, a pCA14 Ad E1 shuttle vector to express the REIC or REIC C was first produced. The REIC or REIC C gene was cut out of pShuttole/REIC or REIC-C with NheI-blunt-HindIII, and was subcloned into the pCA14 Ad E1 shuttle vector digested in advance with XbaI-blunt-HindIII. The pCA14/REIC and pCA14/REIC-C vectors were digested with BglII, and then were each cloned into a pΔE1sp1B-HmT-Rd19 shuttle vector (Kim E et al., Hum Gene Ther 2003; 14:1415-1428; Kim J H et al., J Natl Cancer Inst 2006; 98:1482-1493) whose CMV-REIC-polA and AMV-REIC-C-polA expression cassettes had been digested in advance with BglII to obtain pΔE1sp1B-HmT-Rd19/REIC and pΔE1sp1B-HmT-Rd19/REIC-C adenovirus E1 shuttle vectors. A pSP72-E3/DCN (I-K Choi et al., Gene Therapy 2010; 17:190-201.) E3 shuttle vector was linearized with XmnI, with which homologous recombination was conducted via cotransformation of a BJ5183 E. coli strain together with an adenovirus total vector del-RGD linearized with SpeI. As a result, an adenovirus vector del-RGD/DCN with no replication capability was obtained. Newly constructed pΔE1sp1B-HmT-Rd19/REIC and pΔE1sp1B-HmT-Rd19/REIC-C adenovirus E1 shuttle vectors were digested to linearize with XmnI, with which homologous recombination was then conducted via cotransformation of a BJ5183 E. coli strain together with the del-RGD/DCN digested with BstBI. As a result, tumor-specific oncolytic adenoviruses to express REIC or REIC-C and decorin were obtained. The plasmid DNA was digested with PacI, and introduced into a 293A cell for replication.
Purification of an adenovirus, titer measurement, and qualitative analysis were carried out in accordance with the description of Yoo J Y et al., Mol Ther 2007; 15:295-302.
The oncolytic adenovirus vector comprising no REIC is referred to as oncolytic Ad, the oncolytic adenovirus vector comprising the full length REIC DNA is referred to as oncolytic Ad-REIC, and the oncolytic adenovirus vector comprising the REIC C domain DNA is referred to as oncolytic Ad-REIC domain.
Method
To measure expression of REIC protein after treatment with the Ad-REIC, cells were seeded in a flat-bottomed 6-well plate and incubated for 24 hours. The cells were infected with an adenovirus at an MOI (multiplicity of infection) as set forth in Figures in a complete medium (300 μL) for 1 hour, and washed with PBS (phosphate buffered saline) twice, and lysed with a lysis buffer (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) to extract REIC protein.
After centrifugation, the supernatant was diluted with an identical volume of 4×SDS sample buffer, and heated at 95° C. for 5 minutes. A sample (protein: 5 μg) was separated on a 10% SDS-PAGE gel, and electrophoretically transferred onto a polyvinylidene fluoride (PVDF) membrane. The blot transferred was blocked with a 5% skim milk powder and 0.1% Tween-20-containing TBS (Tris-buffered saline) at room temperature for 1 hour. The resultant was then reacted with a mouse monoclonal anti-human REIC/Dkk-3 antibody (1:1000 dilution) (primary antibody) and thoroughly washed with 0.1% Tween-20-containing TBS (T-TBS), and thereafter reacted with a secondary antibody labeled with horseradish peroxidase. After further washing with T-TBS, the resultant was allowed to develop color by using an ECL kit (Amersham Pharmacia Biotech Inc., Chandler, Ariz.), a kit for chemiluminescence detection. The band for REIC protein is found around 60 kDa in western blotting.
Results
In each cell, addition of the oncolytic Ad-REIC allows for expression of REIC protein at a level comparable to or higher than conventional Ad-REICs. REIC protein is known to have an effect of activating anticancer immunity in vivo (WO2009/119874), and thus the oncolytic Ad-REIC is also expected to have an effect of activating anticancer immunity in vivo.
Method
To investigate the cell-killing rate after treatment with the Ad-REIC, cells were seeded in a flat-bottomed 6-well plate and incubated for 24 hours. The cells were treated with an adenovirus at an MOI (multiplicity of infection) as set forth in Figures in a complete medium (300 μL) for 1 hour, and 1700 μL of a fresh medium was added thereto. On the days as set forth in Figures, the dead cell ratio (%) was measured in microscopic observation for 5 views. In
Results
Method
To the right femur of an adult male nude mouse, 2×106 cells/0.1 mL PBS of PC3 human prostate cancer cells was administered via subcutaneous injection. After 10 days, when the tumor volume reached 200 to 300 mm3, an adenovirus was intratumorally administered (Day 0 in
Results
Method
To the left and right femurs of an adult male nude mouse, 2×106 cells/0.1 mL PBS of PC3 human prostate cancer cells was administered via subcutaneous injection. This mouse was a mouse tumor model having at least two tumor sites. After 10 days, when the tumor volume in both sides reached 200 to 300 mm3, an adenovirus was administered into the right tumor. Three days after the vector injection, natural killer (NK) cells in the peripheral lymphocytes were counted by using flow cytometry with an anti-NK cell antibody (eBioscience Inc., 10255 Science Center Drive, San Diego, Calif. 92121, USA).
Results
Method
A cancer model was produced with an immune-competent mouse, and study was conducted for identification of a cancer-specific CTL cell, which serves for anticancer immunity, after intratumoral administration of the oncolytic Ad-REIC or oncolytic Ad. A malignant thymoma cell [EG-7] strain (1.0×106 cells) into which an OVA (ovalbumin) gene as a foreign antigen had been introduced was subcutaneously transplanted to a C57/BL6 mouse having a normal immune system to produce a cancer mouse model. When the tumor diameter reached 100 mm3 or larger, the oncolytic Ad-REIC or oncolytic Ad was injected into the tumor (dose: 1.0×106 pfu/tumor). Three days after the treatment, the tumor was collected and the fraction of CTLs positive for OVA-specific CD8 in the tumor-infiltrating lymphocytes (TIL) was dynamically analyzed by using flow cytometry with a CD8 antibody and an OVA tetramer (antibody which recognizes H-2kb-restricted OVA epitope).
Results
The conditionally replicating adenovirus according to the present invention can be used for treatment of cancer.
SEQ ID NO: 1, 2, 3, 8, 9, 10 synthetic
All of the publications, patents, and patent applications cited herein are wholly incorporated herein by reference.
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| Filing Document | Filing Date | Country | Kind |
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| PCT/JP2015/065004 | 5/26/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/182574 | 12/3/2015 | WO | A |
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