Patent Application
20080032283
The present invention relates to a reagent for detecting cancer cells or diagnosing cancers, and a cell death-inducing agent. Also provided is a method of treating or preventing cancer in a subject.
Telomerase activity is often enhanced in malignantly transformed cells or immortalized cell strains, whereas telomerase activity is hardly detected in normal somatic cells excluding such as germ line cells, blood lineage cells and epithelial cells. Therefore, attempts to detect cancer using telomerase activity as an indicator have been made (Shay J W, Zou Y, Hiyama E, Wright W E. Telomerase and Cancer. Hum Mol Genet. 10 (7): 677-85, 2001).
On the other hand, detection of cancer tissues and metastatic lymph nodes in vivo has been studied eagerly in the field of diagnostic imaging. For example, biological diagnosis with PET and image analysis fully utilizing neural network have been reported. Further, investigations into the anti-tumor activity and safety of replication-selective viruses have been reported (DeWeese T L, van der Poel H, Li S, Mikhak B, Drew R, Goemann M, Hamper U, DeJong R, Detorie N, Rodriguez R, Haulk T, DeMarzo A M, Piantadosi S, Yu D C, Chen Y, Henderson D R, Carducci M A, Nelson W G, Simons J W. A phase I trial of CV706, “A replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy”, Cancer Res 61(20):7464-72, 2001). The present inventors have also found that infecting cancer cells with a virus having a telomerase promoter and replication ability can kill the cancer cells by viral replication (Kawashima T, Kagawa S, Kobayashi N, Shirakiya Y, Umeoka T, Teraishi F, Taki M, Kyo S, Tanaka N, and Fujiwara T., Related Articles, Links Abstract “Telomerase-specific replication-selective virotherapy for human cancer”, Clin Cancer Res 10(1):285-92, 2004).
However, in situ cancer detection system during surgical operation has not yet been developed because of the difficulty in targeting cancer cells. Further, no research has been known to date in which the living body is infected with a virus and the viral kinetics within cancer cells is actually applied to visualization of cancer tissues.
It is an object of the present invention to provide a reagent for detecting cancer cells or diagnosing cancers, and a cell death-inducing agent, which are capable of visualizing cancer cells not only in vitro but also in vivo. It is another object of the present invention to provide a method of treating or preventing cancer in a subject.
As a result of intensive and extensive researches toward the solution of the above problems, the present inventors have found that it is possible to detect cancer cells with extremely high sensitivity and even in vivo, by integrating a gene encoding a fluorescence labeling protein in E3 region of a viral genome and integrating a replication cassette comprising a human telomerase promoter, an E1A gene, an IRES sequence and an E1B gene in this order in E1 region, and then expressing both cassettes. Also, the present inventor have found that a recombinant virus comprising the replication cassette and the labeling cassette above has an anti-cancer effect. Thus, the present invention has been achieved.
The present invention relates to the following.
(1) A reagent for cancer cell detection, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.
(2) A reagent for cancer diagnosis, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.
In (1) and (2) above, these reagents may be used in in vivo detection, diagnosis or navigation surgery. As a specific example of the promoter from human telomerase, hTERT promoter may be given. As a specific example of the labeling protein, GFP may be given. As the promoter capable of regulating the expression of a gene encoding this labeling protein, a cytomegalovirus promoter or hTERT promoter may be used, for example. As the virus, an adenovirus may be used, for example.
(3) A cell death-inducing agent, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a cell death-inducing cassette comprising a gene encoding a protein associated with cell death induction and a promoter capable of regulating the expression of the gene encoding the protein associated with cell death induction is integrated in E3 region of the viral genome.
In this cell death-inducing agent, the promoter from human telomerase may be hTERT promoter. Examples of proteins associated with cell death induction include immunity-associated proteins, apoptosis-inducing proteins and telomerase-associated proteins. More specifically, PA28 may be given as an immunity-associated protein; TRAIL may be given as an apoptosis-inducing protein; and AU5 may be given as a telomerase-associated protein. The promoter capable of regulating the expression of a protein associated with cell death induction may be a cytomegalovirus promoter or hTERT promoter; and the virus may be an adenovirus. As a cell of the present invention, a cancer cell may be used.
(4) A pharmaceutical composition for the treatment of cancer in a subject, comprising a therapeutically effective amount of a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome, and a pharmaceutically acceptable carrier.
In the pharmaceutical composition of the present invention, the promoter from human telomerase may be hTERT promoter. As a specific example of the labeling protein, GFP may be given. As the promoter capable of regulating the expression of a gene encoding this labeling protein, a cytomegalovirus promoter may be used, for example. As the virus, an adenovirus may be used, for example.
(5) A method of detecting cancer cells, comprising infecting cancer cells with the reagent of (1) above and detecting the fluorescence emitted by the cancer cells.
(6) A method of cancer diagnosis, comprising infecting cancer cells with the reagent of (2) above and detecting the fluorescence emitted by the cancer cells.
(7) A method of inducing cell death in a target cell, comprising infecting the target cell with the cell death-inducing agent of (3) above.
According to the present invention, a reagent for detecting cancer cells or for diagnosing cancers, and a cell death-inducing agent are provided. Since the reagent of the present invention is capable of detecting cancer cells with extremely high sensitivity even in vivo, the reagent is useful in the so-called navigation surgery or the like.
(8) A method of treating or preventing cancer in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.
In the method of the present invention, the promoter from human telomerase may be hTERT promoter. As a specific example of the labeling protein, GFP may be given. As the promoter capable of regulating the expression of a gene encoding this labeling protein, a cytomegalovirus promoter may be used, for example. As the virus, an adenovirus may be used, for example.
The morphological changes indicate a cell death of H1299 cells induced by OBP-401.
Hereinbelow, the present invention will be described in detail. The present invention will now be described with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
1. Reagent for Cancer Cell Detection and Detection Method
The present invention relates to a reagent for detecting cancer cells, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome. Further, the present invention relates to a method of detecting cancer cells, comprising infecting cancer cells with the reagent and detecting the fluorescence emitted by the cancer cells. In the present invention, the term “recombinant virus” means a virus in which the replication cassette and the labeling cassette described later are integrated in the genome. The virus used in the present invention is not particularly limited, but an adenovirus is preferable from the viewpoint of safety. Among adenovirus species, type 5 adenovirus is especially preferable mainly because it is easy to handle.
The recombinant virus used in the present invention has a replication cassette integrated in a region corresponding to E1 region of adenovirus genome and a labeling cassette integrated in a region corresponding to E3 region of adenovirus genome. The replication cassette comprises a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order. The E1A gene, IRES sequence and E1B gene are driven by the human telomerase promoter, which results in cancer cell-specific and telomerase-specific proliferation/replication of the virus. The labeling cassette comprises a promoter and a gene encoding a labeling protein. For example, the gene encoding the labeling protein is driven by a CMV (cytomegalovirus) promoter or hTERT promoter (
The “telomerase promoter” determines the transcription initiation site for telomerase and directly regulates the frequency of transcription. Telomerase is an enzyme that maintains the length of telomeres, standing against the shortening of telomeres at the time of replication of eukaryotic chromosomes. The type of such telomerase promoter is not particularly limited, and any suitable telomerase promoter compatible with the virus to be used for the expression of the gene of interest may be used. For example, the promoter for human telomerase reverse transcriptase (hTERT) is preferable. A number of transcription factor-binding sequences are confirmed in a 1.4 kbp region upstream of the 5′ end of hTERT gene. This region is believed to be hTERT promoter. In particular, a 181 bp sequence located upstream of the translation initiation site is a core region important for the expression of the downstream gene. In the present invention, any sequence comprising this core region may be used. Preferably, an upstream sequence of approximately 378 bp containing the entire core region is used as hTERT promoter. It has been confirmed that this sequence of approximately 378 bp is equivalent to the 181 bp core region alone in gene expression efficiency. The nucleotide sequence of an hTERT promoter of 455 bp is shown in SEQ ID NO: 1.
The nucleotide sequence of hTERT promoter is not limited to the sequence as shown in SEQ ID NO: 1. Nucleotide sequences of nucleotides which hybridize under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of SEQ ID NO: 1 and have hTERT promoter activity may also be included as a sequence for hTERT promoter. Such nucleotides may be obtained from cDNA libraries or genomic libraries by known hybridization methods such as colony hybridization, plaque hybridization, Southern blotting, etc. using the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or a part thereof as a probe. cDNA libraries may be prepared according to the method described in Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)). Alternatively, commercial cDNA libraries or genomic libraries may be used.
In the above hybridization, examples of stringent conditions include 1×SSC to 2×SSC, 0.1% to 0.5% SDS and 42° C. to 68° C. More specifically, an example may be given where prehybridization is performed at 60-68° C. for more than 30 minutes, and then washing is performed in 2×SSC, 0.1% SDS at room temperature for 5-15 minutes 4 to 6 times.
For detailed procedures of hybridization, see, for example, Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)), in particular, Section 9.47-9.58.
The reason why an E1A gene, an IRES sequence and an E1B gene are located in this order in the present invention is that insertion of the IRES sequence between the E1A gene and E1B gene will results in higher replication ability of the virus when a host cell has been infected with it. E1A gene and E1B gene are genes included in E1 gene. This is one of early genes of viruses, which have early (E) genes and late (L) genes involved in their DNA replication, and encodes a protein involved in the regulation of transcription of viral genome. E1A protein encoded by E1A gene activates the transcription of a group of genes (E1B, E2, E4, etc.) necessary for the production of infectious virus. E1B protein encoded by E1B gene assists the accumulation of late gene (L gene) mRNA in the cytoplasm of the infected host cell to thereby inhibit the protein synthesis in the host cell. Thus, E1B protein promotes viral replication. The nucleotide sequences of E1A gene and E1B gene are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
E1A and E1B may have, other than those nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, nucleotide sequences which hybridize under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of SEQ ID NO: 2 or SEQ ID NO: 3 and encode a protein having E1A or E1B activity. Such nucleotide sequences may be obtained from cDNA libraries or genomic libraries by known hybridization methods such as colony hybridization, plaque hybridization, Southern blotting, etc. using the polynucleotide, or a part thereof, consisting of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 as a probe. cDNA libraries may be prepared according to the method described in Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)). Alternatively, commercial cDNA libraries or genomic libraries may be used. In the above hybridization, examples of stringent conditions include 1×SSC to 2×SSC, 0.1% to 0.5% SDS and 42° C. to 68° C. More specifically, an example may be given where prehybridization is performed at 60-68° C. for more than 30 minutes, and then washing is performed in 2×SSC, 0.1% SDS at room temperature for 5-15 minutes 4 to 6 times. For detailed procedures of hybridization, see, for example, Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)), in particular, Section 9.47-9.58.
IRES (Internal Ribosome Entry Site) is a protein synthesis initiation signal specific to picornavirus. It is believed that this site serves as a ribosome-binding site because it has a complementary sequence to the 3′ terminal sequence of 18 S ribosomal RNA. It is known that mRNA derived from a virus which belongs to picornaviridae is translated via this sequence. Translation efficiency from IRES sequence is high. Even from the middle of mRNA, protein synthesis is performed in a cap structure non-dependent manner. Therefore, in the virus of the present invention, both E1A gene and E1B gene (which is located downstream of the IRES sequence) are translated independently by a human telomerase promoter. By using IRES, the control of expression by a telomerase promoter is exerted on E1A gene and E1B gene independently. Therefore, compared to cases where either E1A gene or E1B gene is controlled by a telomerase promoter, viral replication can be more strictly limited to those cells having telomerase activity. An IRES sequence is shown in SEQ ID NO: 4.
IRES may have, other than the nucleotide sequence as shown in SEQ ID NO: 4, nucleotide sequences which hybridize under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of SEQ ID NO: 4 and encode a protein having IRES activity. Such nucleotide sequences may be obtained from cDNA libraries or genomic libraries by known hybridization methods such as colony hybridization, plaque hybridization, Southern blotting, etc. using the polynucleotide, or a part thereof, consisting of the nucleotide sequence of SEQ ID NO: 4 as a probe. cDNA libraries may be prepared according to the method described in Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)). Alternatively, commercial cDNA libraries or genomic libraries may be used. In the above hybridization, examples of stringent conditions include 1×SSC to 2×SSC, 0.1% to 0.5% SDS and 42° C. to 68° C. More specifically, an example may be given where prehybridization is performed at 60-68° C. for more than 30 minutes, and then washing is performed in 2×SSC, 0.1% SDS at room temperature for 5-15 minutes 4 to 6 times. For detailed procedures of hybridization, see, for example, Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)), in particular, Section 9.47-9.58.
In the present invention, a promoter from human telomerase is located upstream of the E1 gene because such a promoter is capable of promoting the replication in cells having telomerase activity.
The genes contained in the replication cassette of the present invention may be obtained by conventional genetic engineering techniques. For example, as a genetic engineering technique, a method of nucleic acid synthesis with a commonly used DNA synthesizer may be used. Alternatively, after a genetic sequence to be used as a template is isolated or synthesized, primers specific to each gene may be designed and the genetic sequence may be amplified with a PCR apparatus (PCR method; Current Protocols in Molecular Biology, John Wiley & Sons (1987), Section 6.1-6.4); or a gene amplification method using a cloning vector may be used. One of ordinary skill in the art can readily carry out the above methods according to, for example, Moleculer Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989). Purification of resultant PCR products may be performed by known methods such as a method using ethidium bromide, a method using SYBR Green I (Molecular Probes), a method with GENECLEAN (Funakoshi), QIAGEN (QIAGEN), etc. using agarose gel, a method using DEAE-cellulose filter, freeze & squeeze method or a method using a dialysis tube. When agarose gel is used, PCT products are electrophoresed on agarose gel and resultant DNA fragments are cut out from the gel and purified. If necessary, it is possible to confirm by conventional sequencing methods that an expected gene has been obtained. For example, dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) or the like may be used for this purpose. Alternatively, it is also possible to analyze the sequence with an appropriate DNA sequencer (e.g., ABI PRISM; Applied Biosystems).
Subsequently, individual genes thus obtained are ligated in a specific order. First, above genes are digested with known restriction enzymes, and the resultant DNA fragments are inserted into a known vector according to a known method for ligation. Specific examples of known vectors include pIRES vector which comprises the IRES (internal ribosome entry site in mRNA) of encephalomyocarditis virus (ECMV) and is capable of translating two open reading frames (ORFs) from one mRNA; Escherichia coli-derived plasmids (such as pCR4, pCR2, pCR2.1, pBR322, pBR325, pUC12 and pUC13); Bacillus subtilis-derived plasmids (such as pUB110, pTP5 and pC194); yeast-derived plasmids (such as pSH19 and pSH15); bacteriophages such as λ phage; animal viruses such as retrovirus, vaccinia virus and baculovirus; pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo and so forth. In the present invention, use of pIRES vector is preferable. With this vector, it is possible to prepare a replication cassette containing “a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene” in this order by inserting necessary genes into the multi-cloning site in this order. DNA ligase may be used for ligation of DNAs. Hereinbelow, an example where hTERT is used as human telomerase in the present invention will be described specifically.
E1A gene and E1B gene may be amplified in E1 gene-expressing cells, such as 293 cells, by carrying out RT-PCR and/or DNA-PCR using primers such as E1A-S, E1A-AS, E1B-S and E1B-AS. If necessary, their sequences are confirmed using a known method such as TA cloning. Then, DNA fragments of E1A and E1B may be cut out using a known restriction enzyme.
Subsequently, a replication cassette consisting of hTERT-E1A-IRES-E1B to be used in the present invention may be prepared by inserting individual genes into a multi-cloning site or the like of a known vector (such as pIRES vector) to give the following order: “hTERT promoter sequence-E1A-IRES-E1B”. Alternatively, if necessary, it is also possible to remove a cytomegalovirus (CMV) promoter from a known vector such as pShuttle with known restriction enzymes, and to insert into that site a sequence cut out from phTERT-E1A-IRES-E1B with appropriate restriction enzymes. The adenovirus in which only the replication cassette consisting of hTERT-E1A-IRES-E1B to be used in the present invention has been integrated is designated “Telomelysin”. By expressing E1 gene necessary for proliferation of adenovirus under the control of hTERT promoter, it is possible to proliferate the virus in a cancer cell-specific manner.
In the recombinant virus used as a reagent of the present invention, a labeling cassette is also included together with the replication cassette. The “labeling cassette” is integrated in E3 region of the viral genome.
Here, it should be noted that the primary function of the virus vector used in the present invention is cytotoxicity by viral replication. Therefore, in order to use the reagent of the present invention for the purpose of diagnosing microcancer tissues, occurrence of the cytotoxicity is preferably as late as possible. This is because the emission of fluorescence caused by the replication of the recombinant virus of the present invention disappears when cells are destroyed, and it becomes difficult to identify the site of the microcancer tissue.
On the other hand, E3A and E3B exist in E3 region of adenovirus, and 11.6 kDa ADP (adenovirus death protein) in E3A region has a function to promote cytotoxicity and viral dispersion.
Therefore, in the recombinant virus used in the present invention, viral genomic regions encoding proteins with a function to promote cytotoxicity and viral dispersion (such ADP-encoding E3 region) are deleted to thereby delay the timing of cell death and facilitate identification of cancer cells by emission of GFP fluorescence or the like.
The labeling protein which constitutes the labeling cassette is a protein which emits light in those cells where the above-described virus has replicated, and is visualized. Preferably, a substance which emits fluorescence is used. Examples of such substances include, but are not limited to, green fluorescent protein (GFP) derived from luminescent jellyfish such as Aequorea victoria, enhanced-humanized GFP (EGFP) or red-shift GFP (rsGFP) which are modified variants of GFP (GFP variants). It is also possible to use yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), or Renilla reniformis-derived GFP. A gene encoding any of these proteins may be used in the present invention.
A promoter capable of regulating the expression of the above-described gene may be any promoter as long as it is compatible with the virus to be used for the expression of the above gene of interest. Specific examples of such promoters include, but are not limited to, cytomegalovirus (CMV) promoter, hTERT promoter, SV40 late promoter, MMTV LTR promoter, RSV LTR promoter and SRα promoter. Preferably, CMV promoter or hTERT promoter may be used.
The recombinant gene contained in the labeling cassette of the present invention may be obtained by conventional genetic engineering techniques. For example, as a genetic engineering technique, a method of nucleic acid synthesis with a commonly used DNA synthesizer may be used. Alternatively, after a genetic sequence to be used as a template is isolated or synthesized, primers specific to each gene may be designed and the genetic sequence may be amplified with a PCR apparatus (PCR method; Current Protocols in Molecular Biology, John Wiley & Sons (1987), Section 6.1-6.4); or a gene amplification method using a cloning vector may be used. One of ordinary skill in the art can readily carry out the above methods according to, for example, Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989). Purification of the resultant PCR product may be performed by known methods such as a method using ethidium bromide, a method using SYBR Green I (Molecular Probes), a method with GENECLEAN (Funakoshi), QIAGEN (QIAGEN), etc. using agarose gel, a method using DEAE-cellulose filter, freeze & squeeze method or a method using a dialysis tube. When agarose gel is used, the PCT production is electrophoresed on agarose gel and resultant DNA fragments are cut out from the gel and purified. If necessary, it is possible to confirm by conventional sequencing methods that an expected gene has been obtained. For example, dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) or the like may be used for this purpose. Alternatively, it is also possible to analyze the sequence with an appropriate DNA sequencer (e.g., ABI PRISM; Applied Biosystems). Subsequently, the gene thus obtained is digested with known restriction enzymes. The recombinant gene is so designed that the resultant, digested DNA fragment (encoding the labeling protein) is located downstream of a gene fragment encoding the above-described promoter. Here, shuttle plasmid pHM11 or the like may be used as a plasmid. The two genes (for the labeling protein and the promoter) are ligated with DNA ligase and inserted into a vector to thereby prepare a recombinant gene for the labeling cassette.
As a known vector, pShuttle vector, Escherichia coli-derived plasmid (such as pCR4, pCR2, pCR2.1, pBR322, pBR325, pUC12 or pUC13); Bacillus subtilis-derived plasmid (such as pUB110, pTP5 or pC194); yeast-derived plasmid (such as pSH19 or pSH15); bacteriophage such as λ phage; animal viruse such as retrovirus, vaccinia virus or baculovirus; pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo or the like may be used.
Subsequently, a recombinant gene comprising the above described replication cassette and labeling cassette is cut out with appropriate restriction enzymes and inserted into an appropriate virus expression vector, to thereby prepare a recombinant virus. Examples of virus expression vectors include adenovirus, retrovirus, vaccinia virus and baculovirus. As described above, adenovirus (in particular, type 5 adenovirus) is preferably used. For integration of the cassettes into virus, methods such as electroporation, the liposome method, the spheroplast method or the lithium acetate method may be used.
In the present invention, specifically, the recombinant gene may be prepared by inserting CMV-EGFP-SV40P (A) from pEGFP-N1 (CLONTECH) into shuttle plasmid pHM11, and inserting Csp45I fragment of this plasmid into ClaI site of pShuttle vector in which phTERT-E1A-IRES-E1B has been integrated.
In the present invention, specifically, a sequence of a necessary region may be cut out with restriction enzymes from the recombinant gene as prepared above, and inserted into a viral DNA such as Adeno-X Viral DNA using a commercial kit such as Adeno-X Expression System (CLONTECH) (the resultant product is designated “AdenoX-hAIB”).
This AdenoX-hAIB is linearized with a known restriction enzyme and then transfected into cultured cells, such as 293 cells, to thereby prepare an infectious recombinant virus.
The target cancer cells to be detected, treated or prevented in the present invention are not limited. Cancer cells of any kind may be used. For example, solid cancers in the head and neck, stomach, large colon, lung, liver, prostate, pancreas, esophagus, bladder, gallbladder/bile duct, breast, uterus, thyroid, ovary, etc.; or leukemia, lymphoma, sarcoma, mesenchymal tumor or the like may be used. Most of cancer cells derived from human tissues show increase in telomerase activity. The present invention is capable of detecting those cancer cells in general where proliferation has been activated by such telomerase activity.
Since telomerase expression is extremely high in cancer cells compared to normal cells, hTERT is expressed in telomerase-containing cancer cells and the replication cassette functions therein. As a result, the virus replicates, which in turn increases the replication of the labeling protein. Thus, the labeling protein is expressed and visualized.
Therefore, when the reagent of the present invention does not emit fluorescence in normal cells, but whereas emits fluorescence in cancer cells. Thus, it is possible to observe cancer cells visually.
For infecting cells with a recombinant virus, the following method may be used, for example. First, cells such as human large colon cancer cell SW620, human lung cancer cells A549 and H1299 are plated in culture plates containing an appropriate culture broth and cultured in the presence of CO2 gas at 37° C. As the culture broth, one which is conventionally used for culturing animal cells may be used, e.g. DMEM, MEM, or RPMI-1640. If necessary, serum, antibiotics, vitamins, or the like may be added thereto. By inoculating a specific amount (0.1-10 MOI (multiplicity of infection), preferably 1 MOI) of the recombinant virus of the present invention, the cultured cells are infected. MOI means a ratio between the viral quantity (infective unit) and the number of cells when a specific amount of cultured cells are infected with a specific amount of viral particles. MOI is used as an indicator when cells are infected with virus.
In order to confirm viral replication, cells infected with virus are recovered and DNA is extracted therefrom. Then, the DNA is subjected to real time-PCR using primers targeting an appropriate gene contained by the virus of the present invention. Thus, quantitative analysis is possible.
With respect to the detection of labeled cells, cancer cells can be visualized because cells where viral replication is observed emit a specific fluorescence (e.g. green fluorescence when GFP is used) by exposing to excitation light. For example, when cells infected with virus are observed under fluorescence microscope, emission of GFP fluorescence in the cells can be observed. For the observation of infected cells with the passage of time, emission of GFP fluorescence may be observed with a CCD camera.
For the real time labeling and detection of cells of interest in vivo, the recombinant virus of the present invention may be administered into the living body.
The reagent of the present invention may be applied to the diseased site as it is. Alternatively, the reagent of the present invention may be introduced into the living body (target cell or organ) by any known method, e.g. intravenous, intramuscular, intra-abdominal or subcutaneous injection; inhalation through the nasal cavity, oral cavity or lung; oral administration; intravascular administration using catheter or the like. Dose levels are selected appropriately depending on the kind of active ingredient, the administration route, the target of administration, and the age, body weight, sex, symptoms and other conditions of the patient. Usually, dose levels may be selected so that the virus of the present invention (the active ingredient) is administered at a daily dose of about 106-1011 PFU (plaque forming units), preferably about 109-1011 PFU. This amount may be administered once a day, or may be divided into several portions and administered at several times a day.
The reagent of the present invention makes it possible to observe the label in vivo in real time. Thus, the reagent of the present invention is advantageous for use as an in vivo diagnosis agent. This is useful in the so-called navigation surgery.
If excision is performed in a wide range including the diseased organ in a surgical operation, the patient who survived this surgical operation can enjoy a long survival. However, the rate of occurrence of complications caused by the surgical operation itself becomes high. Further, loss of the function of the excised organ inevitably influences on the daily life after the surgical operation. It is important in cancer treatment to introduce a low-invasive treatment to reduce the burden of patients while maintaining the remote result of long survival.
When a low-invasive operation is pursued by minimizing the area of excision, one of the information pieces wanted is the presence or absence of metastatic lymph nodes. As a method for obtaining that information, sentinel node (SN) is attracting attention. SN is the lymph node which first receives the lymph flow from tumors, and there is a hypothesis that the first micro-metastasis occurs in this lymph node. This hypothesis is called the SN theory. Although large scale clinical tests in breast cancer have already been started in primarily Europe and the United States, whether or not this theory is applicable to other solid tumors is still unknown. Examination has just been started.
In vivo cancer diagnosis system using the reagent of the present invention is capable of establishing the technology of allowing direct expression of a fluorescent protein in cancer cells and identifying tumor tissues or metastasis-positive lymph nodes by a highly sensitive, fluorescence detection system during surgical operation. In other words, the technology of “navigation surgery” can be established as a method that is more effective than SN. The recombinant virus of the present invention replicates in a great number of cancer cells having telomerase activity, and those cells can emit, for example, a strong green fluorescence of GFP.
From the analysis of mono-lymph node metastasis sites, about 10% of skip metastasis, i.e., incipient metastasis to the second group or more remote lymph nodes skipping over the first group lymph nodes has been reported. Based on this report, there are a large number of researchers who have pointed out the danger of SN navigation. However, the in vivo cancer diagnosis system using the reagent of the present invention identifies tumor tissues or metastasis-positive lymph nodes directly during the surgical operation in real time and the excision range is navigated. This system is original and epoch-making, and further it is extremely practical for smooth progress of surgical operation. Specifically, the reagent of the present invention is endoscopically injected into the site of tumor (e.g., gastric or large colon mucosa around gastric cancer or large colon cancer; internal region of tumors such as gastric cancer, large colon cancer, lung cancer, pancreatic cancer) several days prior to the surgical operation with the same manual technique used in SN navigation. Then, sufficient time is provided so that the virus is distributed into tumor-infiltrated tissues, metastatic tumor tissues or attending lymph nodes to replicate in tumor sites or metastasis-positive sites.
At the time of surgery, excitation light for GFP fluorescence is projected from the light source onto the surgery field after ventrotomy, and images from a special 3CCD camera are projected on a face mount display. By using a transmissible lens, the visual field of the actual surgical field can also be secured, and it is possible to detect metastasis-positive lymph nodes from overlapped GFP images. Further, by mounting a special filter, it becomes possible to recognize fluorescence with the eyes without using the camera.
2. Reagent for ex vivo Diagnosis
The reagent of the present invention is also applicable to an ex vivo diagnosis agent for the purpose of screening. Currently, quantitative determination of tumor markers is the most common method to know the presence of cancer which cannot be detected with the eyes or its primary focus cannot be identified. However, tumor markers are not necessarily satisfactory in their cancer specificity. Besides, it is extremely difficult to detect every cancer species with a single marker.
It has been confirmed that telomerase activity increases in 85% or more of human malignant tumors, and thus its cancer specificity is believed to be extremely high.
Ex vivo cancer diagnosis using the reagent of the present invention may be performed, for example, as described below.
Erythrocytes are removed from a total blood sample taken from a subject. To the remaining cell suspension liquid, the reagent of the present invention is added at a specific ratio (0.1-10 MOI, preferably 1 MOI) and mixed in a test tube. The mixture is left for a specific period of time (e.g., 12-48 hr) to promote infection of cancer cells with the virus and the resultant viral replication. Then, the GFP expression in the cell fraction is analyzed quantitatively by flow cytometry. With the use of this system, it becomes possible to detect free cancer cells present in the peripheral blood with high sensitivity. This method can be used for detecting free cancer cells present in the peripheral blood only in an extremely small quantity.
3. Cell Death-Inducing Agent and Method of Inducing Cell Death
The present invention provides a cell death-inducing agent, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a cell death-inducing cassette comprising a gene encoding a protein associated with cell death induction and a promoter capable of regulating the expression of the gene encoding the protein is integrated in E3 region of the viral genome. Preferably, the cell death-inducing agent of the present invention is used in gene therapy for cancers as an agent for inducing cell death in cancer cells, and may also be used for prevention of recurrence, inhibition and/or prevention of metastasis after surgical operation of cancers.
In the cell death-inducing cassette of the recombinant virus contained in the cell death-inducing agent of the present invention, a gene is integrated which is operated by a promoter and encodes a protein capable of inducing cell death.
In this cell death-inducing cassette used in the recombinant virus, a gene encoding a protein associated with cell death induction and a promoter capable of regulating the expression of the gene are contained. Therefore, when the cell death-inducing agent of the present invention is introduced into cancer cells, the virus replicates specifically in the cancer cells. As a result, intracellular expression level of the cell death-inducing protein increases, enabling induction of cell death only in the cancer cells without damaging normal cells.
A gene encoding a protein associated with cell death induction refers to a gene encoding a protein associated with the induction of cell death in a specific cell.
Specific examples of proteins associated with cell death induction include the following proteins, but are not limited to them. In the present invention, a gene encoding any of these proteins may be integrated.
As a specific example of immunity-associated protein, PA28 may be given. PA28 is a protein which activates intracellular proteasomes. When overexpressed, this protein causes immunological reactions and at the same time induces cell death. As a specific example of apoptosis-inducing protein, TRAIL may be given. TRAIL is a molecule which induces apoptotic cell death by binding to the receptor on cell surfaces. As a specific example of telomerase-associated protein, AU5 may be given. AU5 has a sequence capable of inducing cell death in cells having telomerase activity.
The genes for these proteins associated with cell death induction may be obtained by conventional genetic engineering techniques. For example, as a genetic engineering technique, a method of nucleic acid synthesis with a commonly used DNA synthesizer may be used. Alternatively, after a genetic sequence to be used as a template is isolated or synthesized, primers specific to each gene may be designed and the genetic sequence may be amplified with a PCR apparatus (PCR method; Current Protocols in Molecular Biology, John Wiley & Sons (1987), Section 6.1-6.4); or a gene amplification method using a cloning vector may be used. One of ordinary skill in the art can readily carry out the above methods according to, for example, Moleculer Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989). Purification of the resultant PCR product may be performed by known methods such as a method using ethidium bromide, a method using SYBR Green I (Molecular Probes), a method with GENECLEAN (Funakoshi), QIAGEN (QIAGEN), etc. using agarose gel, a method using DEAE-cellulose filter, freeze & squeeze method or a method using a dialysis tube. When agarose gel is used, the PCT production is electrophoresed on agarose gel and resultant DNA fragments are cut out from the gel and purified. If necessary, it is possible to confirm by conventional sequencing methods that an expected gene has been obtained. For example, dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) or the like may be used for this purpose. Alternatively, it is also possible to analyze the sequence with an appropriate DNA sequencer (e.g., ABI PRISM; Applied Biosystems).
Antioncogenes are also included in cell death-inducing substances for cancer cells, because antioncogenes have a function of inhibiting the replication of cancer cells. For this purpose, the following antioncogenes used in conventional gene therapy may be enumerated.
p53 (SEQ ID NO: 11; Accession No. M14694): various kinds of cancers
p15 (SEQ ID NO: 12; Accession No. L36844): various kinds of cancers,
p16 (SEQ ID NO: 13; Accession No. L27211): various kinds of cancers
APC (SEQ ID NO: 14; Accession No. M74088): large colon cancer, gastric cancer, pancreatic cancer
BRCA-1 (SEQ ID NO: 15; Accession No. U14680): ovarian cancer, breast cancer
DPC-4 (SEQ ID NO: 16; Accession No. U44378): large colon cancer, pancreatic cancer
FHIT (SEQ ID NO: 17; Accession No. NM 112012): gastric cancer, lung cancer, uterus cancer
p73 (SEQ ID NO: 18; Accession No. Y11416): neuroblastoma
PATCHED (SEQ ID NO: 19; Accession No. U59464): basal cell carcinoma
Rbp 110 (SEQ ID NO: 20; Accession No. M15400): lung cancer, osteosarcoma
DCC (SEQ ID NO: 21; Accession No. X76132): large colon cancer
NF1 (SEQ ID NO: 22; Accession No. NM 000267): neurofibromatosis type 1
NF2 (SEQ ID NO: 23; Accession No. L11353): neurofibromatosis type 2
WT-1 (SEQ ID NO: 24; Accession No. NM 000378): Wilms' tumor
These antioncogenes may be obtained by conventional genetic engineering techniques. For example, as a genetic engineering technique, a method of nucleic acid synthesis with a commonly used DNA synthesizer may be used. Alternatively, after a genetic sequence to be used as a template is isolated or synthesized, primers specific to each gene may be designed and the genetic sequence may be amplified with a PCR apparatus (PCR method); or a gene amplification method using a cloning vector may be used. If necessary, it is possible to confirm by conventional sequencing methods that an expected gene has been obtained.
As a promoter capable of regulating the expression of the above gene, any promoter may be used as long as it is an appropriate promoter compatible with the virus to be used for the expression of the gene of interest. Preferably, a CMV promoter or hTERT promoter may be used. However, other promoters such as SV40 late promoter, MMTV LTR promoter, RSV LTR promoter and SRα promoter may also be used.
The cell death-inducing agent of the present invention may be applied to the diseased site as it is. Alternatively, the agent of the present invention may be introduced into the living body (target cell or organ) by any known method, e.g. intravenous, intramuscular, intra-abdominal, intranasal, intradermal or subcutaneous injection; inhalation through the nasal cavity, oral cavity or lung; oral administration; intravascular administration using catheter or the like; implantation (e.g., using slow release technology). Depending on the route of administration, the agent of the present invention may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate it, such as those in the digestive tract.
The cell death-inducing agent of the present invention may be treated, for example, by the method such as freezing to enable easy handling and then used alone, or prepared into pharmaceutical compositions by mixing with known pharmaceutically acceptable carriers such as excipients, dispersions, fillers, binders, lubricants; or known additives (including such as buffers, isotonic agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, and sweetening agents). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, or in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. Alternatively, the agent can be stored in lyophilized form to be rehydrated with an appropriate vehicle or carrier prior to use.
The cell death-inducing agent of the present invention may be administered orally or parenterally depending on the form of the agent, e.g. oral administration agents such as tablets, capsules, powders, granules, pills, liquids, syrups, sustained release formulations, etc. and parenteral administration agents such as injections, external medicines, suppositories, eye drops, etc. Preferably, local injection into muscle or abdominal cavity, or intravenous injection may be enumerated.
Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be fluid to the extent that easy syringeability exists, unless the pharmaceutical form is a solid or semi-solid such as when slow release technology is employed. In any event, it must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms.
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents such as, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter-sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients selected from those enumerated above.
Dose levels are selected appropriately depending on the kind of active ingredient, the administration route, the target of administration, and the age, body weight, sex, symptoms and other conditions of the patient. Usually, dose levels may be selected so that the virus of the present invention (the active ingredient) is administered at a daily dose of about 106-1011 PFU (plaque forming units), preferably about 109-1011 PFU. This amount may be administered once a day, or may be divided into several portions and administered at several times a day.
When the virus of the present invention is administered, it is also possible to use a known immunosuppressant or the like to suppress the immunity of the living body to thereby make the viral infection easy.
Further, the virus of the present invention may be used jointly with at least one anticancer agent selected from the group consisting of known anticancer agents and radiation. Specific examples of anticancer agents include, but are not limited to, the following agents.
(1) Alkylating agents: These agents have an effect of causing cytotoxicity by introducing alkyl groups into the nucleic acid/protein of cancer cells. Examples include carboquone, busulphan (mustard drugs) and nimustine (nitrosoureas).
(2) Antimetabolic agents: These agents have an effect of inhibiting cell synthesis by antagonizing enzymes in metabolic processes. Examples include methotorexate (folates), mercaptopurine (purines), cytarabine (pyrimidines), fluorouracil, tegafur and carmofur.
(3) Antibiotics: These agents have an anti-cancer effect. Examples include actinomycin D, bleomycin, adriamycin and mitomycin C.
(4) Antimicrotubule agents: These agents act on microtubules and exhibit an anticancer effect. Examples include docetaxel, paclitaxel (taxanes) and vinorelbine, vincristine, vinblastine (alkaloids).
(5) Platinum preparations: These preparations have an effect of inhibiting DNA synthesis by forming intra- or inter-DNA strand crosslinks or DNA-protein crosslinks. Examples include cisplatin, carboplatin and nedaplatin.
(6) Topoisomerase inhibitors: Iinotecan (topoisomerase I inhibitor), podophyllotoxin derivatives (topoisomerase II inhibitor) and the like may be enumerated. Topoisomerase is an enzyme that catalyzes a reaction of changing the linking number of DNA by transiently cutting one or both strands of DNA.
It is believed that there is an extremely low possibility that the cell death-inducing agent of the present invention will produce side effects for the reasons described below. Thus, the cell death-inducing agent of the present invention can be a very safe preparation.
(1) There is little telomerase activity in normal somatic cells, and yet the virus of the present invention is hard to be infectious in suspending cells such as hematopoietic cells.
(2) Since the virus of the present invention has replication ability, it is possible to use this virus at a lower concentration than that of conventional non-replication competent virus used in conventional gene therapy.
(3) Even when the virus of the present invention has been administered in excess, antiviral action works through ordinary immune reaction in the living body.
It is possible to induce cell death in a target cell by infecting the target cell with the recombinant virus of the present invention. The kinds of target cells are not particularly limited. For example, tumor cells, cells with active replication, cells whose telomerase activity has been increased, or the like may be used.
The expressing “infecting cells with the recombinant virus” means as described above. In order to confirm whether cell death has been induced or not, morphological observation may be performed as described below. Briefly, cells adhering to the bottom of a culture dish are infected with the recombinant virus of the present invention. After a specific time period, the form of the cells becomes circular and they suspend in the culture broth as glossy cells peeled off from the bottom. At this point, the life maintaining mechanism in these cells has been broken up, and it can be measured that cell death has been induced. Alternatively, it is also possible to confirm cell death with a commercial viable cell assay kit using tetrazorium salts (e.g., MTT, XTT).
4. Pharmaceutical composition for the treatment of cancer in a subject and Method of treating or preventing cancer in a subject
The present invention provides a pharmaceutical composition for the treatment of cancer in a subject, comprising a therapeutically effective amount of a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome, and a pharmaceutically acceptable carrier.
Further, the present invention provides a method of treating or preventing cancer in a subject, comprising administering to the subject a therapeutically effective amount of the cell death-inducing agent or the pharmaceutical composition described above.
The term “cancer” has its understood meaning in the art, for example, an uncontrolled growth of tissue that has the potential to spread to distant sites of the body (i.e., metastasize). The term “tumor” is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. Preferably, the inventive methods disclosed herein are used to prevent and treat malignant tumors. The term “cancer”, as used herein, includes but not limited to solid cancers in the head and neck, brain, kidney, stomach, large bowel, small intestine, colorectum, lung, liver, prostate, pancreas, esophagus, bladder, gallbladder/bile duct, squamous cell, breast, uterus, thyroid, ovary, bone, skin etc.; or leukemia, lymphoma, sarcoma, melanoma, carcinoma, mesenchymal tumor, neoplasm of the central nervous system (e.g., spinal axis tumors) or the like. Preferred are methods of treating and preventing tumor-forming cancers.
The term “subject”, as used herein, includes humans, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
The pharmaceutical composition of the present invention may be applied to the diseased site as it is. Alternatively, the pharmaceutical composition of the present invention may be introduced into the living body (target cell or organ) by any known method, e.g. intravenous, intramuscular, intra-abdominal, intranasal, intradermal or subcutaneous injection; inhalation through the nasal cavity, oral cavity or lung; oral administration; intravascular administration using catheter or the like.
The virus included in the pharmaceutical composition of the present invention may be treated, for example, by the method such as freezing to enable easy handling and then used alone, or prepared into pharmaceutical compositions by mixing with known pharmaceutically acceptable carriers such as excipients, dispersions, fillers, binders; or known additives (including such as buffers, isotonic agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, and sweetening agents). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, or in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. Alternatively, the agent can be stored in lyophilized form to be rehydrated with an appropriate vehicle or carrier prior to use.
The pharmaceutical composition of the present invention may be administered orally or parenterally depending on the form of the agent, e.g. oral administration agents such as tablets, capsules, powders, granules, pills, liquids, syrups, sustained release formulations, etc. and parenteral administration agents such as injections, external medicines, suppositories, eye drops, etc. Preferably, intratumoral injection, local injection into muscle or abdominal cavity, or intravenous injection may be employed.
Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be fluid to the extent that easy syringeability exists, unless the pharmaceutical form is a solid or semi-solid such as when slow release technology is employed. In any event, it must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms.
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents such as, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter-sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients selected from those enumerated above.
Dose levels are selected appropriately depending on the kind of active ingredient, the administration route, the target of administration, and the age, body weight, sex, symptoms and other conditions of the patient. Usually, dose levels may be selected so that the virus of the present invention (the active ingredient) is administered at a daily dose of about 106-1011 PFU (plaque forming units), preferably about 109-1011 PFU. This amount may be administered once a day, or may be divided into several portions and administered at several times a day.
When the virus included in the pharmaceutical composition of the present invention is administered, it is also possible to use a known immunosuppressant or the like to suppress the immunity of the living body to thereby make the viral infection easy.
Further, the pharmaceutical composition of the present invention may be used jointly with at least one anticancer agent selected from the group consisting of known anticancer agents and radiation. Specific examples of anticancer agents include, but are not limited to, the following agents.
(1) Alkylating agents: These agents have an effect of causing cytotoxicity by introducing alkyl groups into the nucleic acid/protein of cancer cells. Examples include carboquone, busulphan (mustard drugs) and nimustine (nitrosoureas).
(2) Antimetabolic agents: These agents have an effect of inhibiting cell synthesis by antagonizing enzymes in metabolic processes. Examples include methotorexate (folates), mercaptopurine (purines), cytarabine (pyrimidines), fluorouracil, tegafur and carmofur.
(3) Antibiotics: These agents have an anti-cancer effect. Examples include actinomycin D, bleomycin, adriamycin and mitomycin C.
(4) Antimicrotubule agents: These agents act on microtubules and exhibit an anticancer effect. Examples include docetaxel, paclitaxel (taxanes) and vinorelbine, vincristine, vinblastine (alkaloids).
(5) Platinum preparations: These preparations have an effect of inhibiting DNA synthesis by forming intra- or inter-DNA strand crosslinks or DNA-protein crosslinks. Examples include cisplatin, carboplatin and nedaplatin.
(6) Topoisomerase inhibitors: Iinotecan (topoisomerase I inhibitor), podophyllotoxin derivatives (topoisomerase II inhibitor) and the like may be enumerated. Topoisomerase is an enzyme that catalyzes a reaction of changing the linking number of DNA by transiently cutting one or both strands of DNA.
It is believed that there is an extremely low possibility that the pharmaceutical composition of the present invention will produce side effects for the reasons described below. Thus, the pharmaceutical composition of the present invention can be a very safe preparation.
(1) There is little telomerase activity in normal somatic cells, and yet the virus included in the pharmaceutical composition of the present invention is hard to be infectious in suspending cells such as hematopoietic cells.
(2) Since the virus included in the pharmaceutical composition of the present invention has replication ability, it is possible to use this virus at a lower concentration than that of conventional non-replication competent virus used in conventional gene therapy.
(3) Even when the virus included in the pharmaceutical composition of the present invention has been administered in excess, antiviral action works through ordinary immune reaction in the living body.
Methods of preparing various pharmaceutical compositions with a specific amount of active compound are known, or will be apparent, to those skilled in the art.
Hereinbelow, the present invention will be described in more detail with reference to the following Examples. However, the present invention is not limited to these Examples.
This Example preliminary examined whether or not fluorescence will be emitted in vitro when cancer cells are co-infected with virus Telomelysin comprising the replication cassette and non-replicating virus Ad-GFP comprising the labeling cassette.
Human large colon cancer cell SW620 and human lung cancer cells A549 and H1299 were infected with 0.1 MOI (multiplicity of infection) of Ad-GFP (
As a result, tendency to green color was hardly recognized when human large colon cancer cell SW620 and human lung cancer cells A549 and H1299 were infected with 0.1 MOI of Ad-GFP. However, when 1 MOI of TRAD was used jointly, fluorescence could be detected only in cancer cells, and no fluorescence was detected in normal cells such as human fibroblast cells WI38 and NHLF and human umbilical vascular endothelial cell (HUVEC) (
This Example preliminary examined whether or not fluorescence will be emitted in vivo when cancer tissues are co-infected with virus Telomelysin comprising the replication cassette and non-replicating virus Ad-GFP comprising the labeling cassette.
Ad-GFP (8×105 PFU) and TRAD (8×106 PFU) were intratumorally administered to human large colon cancer SW5620 and human lung cancer A549 tumors transplanted subcutaneously into the dorsal of nude mice. Then, fluorescence was observed with the passage of time.
In any of the tumors, spot-like fluorescence had begun to be detected from day 2 after the administration and disappeared by day 14 (
1. Preparation of GFP-Expressing, Replication-Competent Virus (Telomelysin-GFP) which Comprises the Replication Cassette Comprising Telomerase Promoter and E1 Gene and the Labeling Cassette Comprising Gene Encoding GFP in a Single Virus
The outline of Telomelysin-GFP is shown in
Such a replication-incompetent virus was prepared as described below.
2. Preparation of Recombinant Virus
From RNA extracted from 293 cells, E1A gene of 897 bp was amplified by RT-PCR using the specific primers (E1A-S and E1A-AS) and PCR conditions described below.
Composition of the PCR solution: 1×PCR buffer
From DNA extracted from 293 cells, E1B gene of 1822 bp was amplified by DNA-PCR using the following primers E1B-S and E1B-AS.
The composition of the PCR solution and the reaction conditions (cycles, temperature) used were the same as used for the amplification of E1A gene.
Each PCR product was subjected to TA cloning (TA Cloning Kit Dual Promoter; Invitrogen) to thereby confirm their sequences. Then, DNA fragments of 911 bp (E1A) and 1836 bp (E1B) were cut out, respectively, with restriction enzyme EcoRI.
E1A and E1B were inserted into the MluI site and the SalI site, respectively, of pIRES vector (CLONTECH) in the normal orientation (E1A-IRES-E1B).
A 455 bp hTERT promoter sequence which had been cut out with restriction enzymes MluI and BglII was inserted into the XhoI site located upstream of the E1A of E1A-IRES-E1B in the normal orientation (phTERT-E1A-IRES-E1B).
The cytomegalovirus (CMV) promoter contained in pShuttle vector was removed by treatment with restriction enzymes MfeI and NheI. Then, a 3828 bp sequence cut out from phTERT-E1A-IRES-E1B using restriction enzymes NheI and NotI was inserted into that site (pSh-hAIB).
pEGFP-N1 (CLONTECH) was digested with AgeI/NheI, blunt-ended with Klenow fragment and self-ligated (pEGFP-N2).
This pEGFP-N2 was digested with NsiI/AflII and blunt-ended with T4 DNA polymerase, followed by preparation of a BglII site using BglII linker. This BglII fragment was inserted at the BamHI site of pHM11 (PHM11-EGFP-N2).
Further, Csp45I fragment from pHM11-EGFP-N2 was inserted at the ClaI site of pShuttle vector in which phTERT-E1A-IRES-E1B had been integrated (pSh-hAIB).
A 4381 bp sequence was cut out from the thus prepared recombinant gene (pSh-hAIB) using restriction enzymes I-CeuI and PI-SceI, and inserted into the Adeno-X Viral DNA of Adeno-X Expression System (CLONTECH) (AdenoX-hAIB). This AdenoX-hAIB was treated with restriction enzyme PacI for linearization and then transfected into 293 cells to thereby prepare an infectious recombinant adenovirus (hereinafter, referred to as “Telomelysin-GFP” or “OBP-401”).
1. Morphological Changes in Human Lung Cancer Cells Caused by Infection with Telomelysin-GFP
Human non-small-cell lung cancer-derived H1299 cells cultured in vitro were infected with Telomelysin-GFP at 1 MOI or 10 MOI. Specifically, H1299 cells were plated in 24-well plates at 5×104 cells/well. After 24 hours, cells were counted and the virus was added to the culture broth to give a concentration of 1 MOI or 10 MOI. Subsequently, the morphology of cells was observed under inversed microscope with the passage of time to examine the cytotoxic activity of the virus.
As a result, cell death was induced by viral replication in a concentration dependent manner and also a time dependent manner. 120 hours after 10 MOI infection, most of the cells became circular and were suspending under inverted microscope (
2. Emission of GFP Fluorescence in Human Lung Cancer Cells Caused by Infection with Telomelysin-GFP
The inverted microscopic images shown in
3. Verification of Telomelysin-GFP Replication by Quantitative Real Time PCR
Human lung cancer cell H1299 was infected with Telomelysin-GFP at 10 MOI. Cell samples were harvested at 2, 26, 50 and 74 hours after the infection and DNA was extracted therefrom. Real time PCR was performed using the following primers targeting the E1A gene of Telomelysin-GFP, to thereby quantitatively analyze the viral proliferation/replication. The primers and PCR conditions used are as described below.
Composition of PCR Solution: 1×LC FastStart DNA Master SYBR Green I
The results revealed that Telomelysin-GFP had already replicated 1,000,000-fold at 26 hours after the infection (
1. Emission of GFP Fluorescence in Human Large Bowel Cancer Cells Caused by Infection with Telomelysin-GFP
Human large colon cancer-derived SW620 cells were infected with Telomelysin-GFP at 10 MOI. Changes in the cells were observed with the passage of time under inverted microscope and fluorescent microscope.
As a result, GFP green fluorescence indicating viral replication in a time dependent manner was recognized as in the case of H1299 cells (
2. Verification of Telomelysin-GFP Replication by Quantitative Real Time PCR
In the same manner as in H1299 cells, SW620 human large colon cancer cells were infected with Telomelysin-GFP at 10 MOI. Cell samples were harvested at 2, 26, 50, 74 and 98 hours after the infection and DNA was extracted therefrom. Then, viral replication was quantitatively analyzed by real time PCR. Real time PCR was performed using the following primers targeting the E1A gene of Telomelysin-GFP, to thereby quantitatively analyze the viral replication. Conditions of the real time PCR (composition of the reaction solution, cycle, time period, etc.) were the same as in H1299 cells.
The results revealed that Telomelysin-GFP had already replicated 1,000,000-fold at 26 hours after the infection and was almost plateau up to 98 hours after the infection (
Normal human lung fibroblast cells (NHLF) cultured in vitro were infected with Telomelysin-GFP at 1 MOI or 10 MOI. Changes were observed under inverted microscope up to 120 hours after the infection.
As a result, no morphological changes were observed, and cell death was not induced (
When the inverted microscopic images shown in
H1299 human lung cancer cell, SW620 human large colon cancer cell, and normal human lung fibroblast cell (NHLF) were infected with Telomelysin-GFP at 10 MOI as described above. Cell samples were harvested with passage of time, and DNA was extracted therefrom. Then, viral replication was quantitatively analyzed by real time PCR.
The results revealed that Telomelysin-GFP had already replicated about 1,000,000-fold in cancer cells at 24 hours after the infection, and emitted remarkable GFP fluorescence at 72 hours after the infection (
1. Telomelysin-GFP (107 PFU) was administered into the tumor of H1299 human lung cancer transplanted into nude mice. Then, emission of GFP fluorescence was observed with a CCD camera with the passage of time.
The results revealed that emission of GFP fluorescence caused by Telomelysin-GFP replication began to be recognized within 24 hours after the infection, and that the range and luminance were gradually enhanced 3 days and 5 days after the infection (
2. In the same manner as described above, Telomelysin-GFP (107 PFU) was administered into the tumor of H1299 human lung cancer transplanted into nude mice. One week and three weeks after the infection, subcutaneous tumor was removed. Emission of GFP fluorescence was observed on the entire tumor and on a cut surface using a CCD camera.
As a result, even when fluorescence emission was weak on the surface of the removed tumor, replication of Telomelysin-GFP could be confirmed in a wide range on the cut surface (
3. In the same manner as described above, HT29 human large colon cancer cell was transplanted into the rectal wall of nude mice as an orthotopic model, and Telomelysin-GFP (107 PFU) was administered at the time when gross tumor was formed. Emission of GFP fluorescence caused by Telomelysin-GFP replication began to be recognized one week after the infection with a CCD camera (
HT29 human large colon cancer cell was transplanted into the rectal wall of nude mouse. When gross tumor was formed, the tumor was removed and analyzed after hematoxylin-eosin (HE) staining.
As a result, tumor was formed around the rectum, and tumor cell mass could be confirmed in lymph vessels in the rectal wall (
HT29 human large colon cancer cell was transplanted into the rectal wall, and ventrotomy was performed when gross tumor was formed. As a result, swelling was recognized in three lymph nodes (LN) around the aorta (
Emission of GFP fluorescence was recognized by fluorescence imaging with a CCD camera, in the transplanted HT29 rectal tumor and one of the three para-aortic lymph nodes (
Emission of GFP fluorescence was recognized by fluorescence imaging with a CCD camera, in only one of the three para-aortic lymph nodes (
Histological analysis of para-aortic lymph nodes detected metastatic tumor tissue in the only one lymph node which was found GFP fluorescence-positive by fluorescence imaging with a CCD camera. Thus, it was confirmed that Telomelysin-GFP replicates only in metastasis-positive lymph nodes (
Human non-small-cell lung cancer-derived H1299 cells cultured in vitro were infected with OBP-401 at an MOI of 10. Cell morphology was evaluated at indicated time points by phase-contrast photomicrography. Cells were also assessed for GFP expression under fluorescence microscopy (×200 magnification).
As a result, H1299 cells expressed bright GFP fluorescence as early as 20 hours after OBP-401 infection. The fluorescence intensity gradually increased followed by rapid cell death due to the cytopathic effect (CPE) of OBP-401, as evidenced by floating, highly light-refractile cells under phase-contrast photomicrographs (
H1299 tumor cells (1×107 cells/each) were subcutaneously injected into the right flank of mice. OBP-401 (1×107 PFU/body) was administered intratumorally for 3 cycles every 2 days. PBS was used as a control. Six mice were used for each group. Tumor growth was expressed by tumor mean volume±SE. Statistical significance was defined as p<0.05 (*) (Student's t-test).
As a result, administration of OBP-401 resulted in a significant tumor growth suppression compared with mock-treated tumors at 27 days after initiation of treatment (p<0.05) (
According to the present invention, a reagent for detecting cancer cells or diagnosing cancers and a cell death-inducing agent are provided. Since the reagent of the present invention is capable of detecting cancer cells with extremely high sensitivity even in the living body, the reagent is useful for the so-called navigation surgery or the like.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-285383 | Sep 2004 | JP | national |
This application claims the benefit of the filing date of JP application 2004-285383 filed on Sep. 29, 2004. JP application 2004-285383 is herein incorporated by reference in its entirety. This application is also a continuation-in-part application of U.S. application Ser. No. 11/158,479 filed on Jun. 21, 2005, incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11158479 | Jun 2005 | US |
| Child | 11696965 | Apr 2007 | US |
