JP2002529068A - Methods of treating tumors using FAS-induced apoptosis - Google Patents

Abstract

(57) Abstract: The present invention relates to a method for killing Fas ⁺ tumor cells, which comprises introducing a nucleic acid encoding a Fas ligand (FasL) into a second tumor cell, whereby the second tumor cell is killed. produce FasL and express the nucleic acid, whereby the Fas ⁺ tumor the cells and expressing Fas second tumor cells and interaction to cause apoptosis in Fas ⁺ tumor cells, thereby Fas ⁺ tumor cells Providing a method comprising killing.

Description

DETAILED DESCRIPTION OF THE INVENTION

Description: BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a method for killing Fas ⁺ tumor cells, wherein a second tumor cell has Fas ligand (
Comprising introducing a nucleic acid encoding FasL), thereby produce the second FasL tumor cells expressing said nucleic acid, Fas ⁺ tumor cells and FasL expressing by which second
Interaction with tumor cells causes apoptosis in the Fas ⁺ tumor cells, thereby providing a method of killing the Fas ⁺ tumor cells.

BACKGROUND OF THE INVENTION At present, the main treatment for cancerous tumors is the surgical removal of affected areas of tissues, organs or glands. However, high relapse rates have been a major obstacle to the complete eradication of cancerous cells. One reason for the high frequency of tumor recurrence is that it is extremely difficult to completely remove all cancer cells by surgical methods during primary tumor development. The remaining cancer cells remain stationary for an extended period of time,
ormancy) (Meltzer et al., 1990. A Dormancy and breast cancer. @ J.S.
urg. Oncol. 43: 181-188). Once the primary tissue has been surgically removed, the surgical injury can stimulate rapid tissue and revascularization at the site of the injury. This regeneration process sends a positive signal to the surrounding tissue, for example, by the tissue and vascular growth factors. These factors and the rapid proliferative environment cause the surviving tumor cells to transition from dormancy to rapid growth, thereby causing the cancer to recur.

[0003] All cancer cells share two basic features: the uncontrolled cell cycle;
And the inability to enter the pathway to programmed cell death, apoptosis. Apoptosis is an intrinsic property of all normal cells. The apoptotic process plays an important role in controlling tissue development, organ size and morphology, and cell longevity. Apoptosis acts as a defense to prevent cell and tissue overgrowth. Fas-mediated apoptosis is the best studied pathway of programmed cell death. Fas (APO-1, CD95), or Fas ligand, is a 45 kDa type I membrane protein that belongs to the TNF / nerve growth factor receptor family (Ba
jorath, J. and A. Aruffo. 1997 Prediction of the three-dimensional structur
e of the human Fas receptor by comparative molecular modeling J Comput
Aided Mol Des 11: 3-8 and Watanabe-Fukunaga, RCI Brannan, N. Itoh,
S. Yonehara, NG Copeland, NA Jenkins and S. Nagata, 1992 The cDNA struc
ture, expression, and chromosomal assignment of the mouse Fas antigen.J
Immunol 148: 1274-9. ). FasL, a Fas ligand, is a 40 kDa type II membrane protein belonging to the tumor necrosis factor family (Takahashi, T., M. Tanaka, J. Inazawa).
, T. Abe, T. Suda and S. Nagata, 1994 Human Fas ligand: gene structure, chr
omosomal location and species specificity, Int Immunol 6: 1567-74).

[0004] Binding of FasL (and certain anti-Fas antibodies) to Fas results in the polymerization of the receptor, signaling through the caspase pathway, and rapidly killing the cells bearing the receptor through apoptosis (Larsen, CP , DZ Alexander, R. H
endrix, SC Ritchie and TC Pearson, 1995, Fas-mediated cytotoxicity. A
n immunoeffector or immunoregulatory pathway in T cell-mediated immune r
esponses? Trasnpantaion 60: 221-4; Longthorne, VL and GT Williams, 1
997). Caspase activity is required for commitment to Fas-mediated apoptosis (Embo J 16: 3805-12; Nagata, S. and P. Golstein, 1995, The Fas death fact
or, Science 267: 1449-56; Ogasawara, J., R. Watanabe-Fukunaga, M. Adachi
, A. Matsuzawa, T. Kasugai, Y. Kitamura, N. Itoh, T. Suda and S. Nagata, 19
93, Lethal effect of the ani-Fas antibody in mice [Nature 1993, Oct 7: 36
5 (6446): 568 has a typographical error] Nature 364: 806-9). Fas is expressed on almost all types of cells. Binding of Fas to FasL activates genetically programmed cell death by cascade expression of interleukin-coupled enzyme (ICE) or caspase (Chandler et al., 1998, "Different subcellular distribution of caspase-
3 and caspase-7 following Fas-induced apoptosis in mouse liver ", J. Biol
Chem. 273: 10815-10818; Jones et al., 1998, "Fas-mediated apoptosis in mous.
e hepatocytes involves the processing and activation of caspases "Hepato
logy 27: 1623-1642).

Since both ligands and receptors are membrane proteins, Fas-induced apoptosis is usually mediated by cell-cell contact. However, a soluble form of FasL has also been produced by some cells and has been shown to have some altered activity depending on the target cell (Tanaka, M., T. Itai, M. Adachi and S. Nagata). ,
1998, Downregulation of Fas ligand by shedding (see comments) Nat Med 4
: 31-6; Tanaka, M., T. Suda, T. Takahashi and S. Nagata, 1995, Expression o
f the functional soluble form of human fas ligand in activated lymphocyt
es, Embo J 14: 1129-35).

[0006] The present invention provides a novel method, such as by vector-mediated gene transfer of Fas ligand to cells, to destroy primary tumors and prevent cancer recurrence by activating apoptosis of cancer cells. Provide a good strategy. In this method, cells expressing Fas ligand cause Fas ⁺ tumor cells to undergo apoptosis and die. The vector can be injected with a syringe or micropump into the tumor cells, and therefore does not require conventional surgery to remove the tumor. In the present invention, cancer cell death is induced in several ways: 1) FasL binds to Fas receptors on adjacent tumor cells and induces their apoptosis; 2) FasL induces endothelial cell apoptosis. Induces and destroys blood vessels supplied to tumors; 3)
FasL on tumor cells induces apoptosis of surrounding tissues, depriving tumor cells of any nursery assistance; and 4) a positive factor that apoptosis may re-activate quiescent tumor cells involved in cancer recurrence Prevent release.

A major advantage of this approach is that the Fas-FasL interaction is a major signaling event that activates several p53-dependent and -independent apoptotic pathways (Callera et al. , 1998, "Fas-mediated apoptosis with normal exp
ression bcl-2 and p53 in lymphocytes from aplastic anemia ", Br. J. Haema
tol. 100: 698-703). Thus, apoptotic signaling is amplified by expression of one or more enzymes, and apoptosis is not dependent on p53 or other cell-cycle checkpoint proteins. For example, gene therapy with the p53 gene has been shown to be very promising for the treatment of cancer (Boulikas 1997 "Gene Therapy of prostate canc
er: p53, suicidal gene, and other targets. "Anticancer Res. 17: 1471-150
5), p53 gene therapy works only in about 50-60% of tumor cells with p53 mutation (I
waya et al., 1997, A Histologic grade and p53 immunoreaction as indicators of
early recurrence of node-negative breast cancer.Jpn J Clin Oncol 27: 6
-12).

Another advantage is that FasL is generally a membrane-bound signaling protein rather than an intracellular protein like p53 and caspase. FasL expression on the cell surface transmits an apoptotic signal to surrounding cells by a bystander effect,
It does not require delivery of the therapeutic gene to all cancer cells. Thus, the present invention fulfills the need for non-surgical cancer therapies, which provides a significant improvement over current gene therapies, avoids the use of toxic drugs, and helps prevent tumor recurrence. The present invention provides a means for delivering FasL to a wide range of cell types both in vitro and in vivo, tight control of FasL expression, and easy and reliable quantification of exogenous FasL levels and subcellular localization. A method for delivering FasL for the purpose of destroying tumor cells by providing a means for:

SUMMARY OF THE INVENTION The present invention is a method of killing a Fas ⁺ tumor cell, wherein the second tumor cell has a Fas ligand (Fa
sL) is introduced, whereby the second tumor cell expresses the nucleic acid to produce FasL, whereby Fas ⁺ tumor cells and the second tumor cell expressing FasL Interaction causes Fas ⁺ tumor cells to undergo apoptosis,
Methods are provided that include killing Fas ⁺ tumor cells. In another embodiment, the present invention relates to a method of killing Fas ⁺ tumor cells, comprising introducing the vector Ad / FasL-GFP _TET into a second tumor cell, whereby the second tumor cell is killed.
Tumor cells express FasL, that interaction with a second tumor cells expressing Fas ⁺ tumor cells and FasL is to cause apoptosis in Fas ⁺ tumor cells by the, so by Fas ⁺ tumor cells Provide a way to kill. In yet another embodiment, the invention provides a vector Ad / FasL-GFP _TET . The present invention also provides a controllable expression vector comprising a nucleic acid encoding a transactivator protein that binds to a tet-responsive transactivation expression element, and a control element comprising the tet-responsive transactivation expression element. Thus, there is provided a vector into which a nucleic acid encoding a protein may be inserted downstream of its regulatory region. One such vector provided by the present invention is pAd _TET .

DETAILED DESCRIPTION OF THE INVENTION The present invention may be better understood with reference to the following detailed description of the preferred embodiments and the examples contained therein. Before the present methods are disclosed and described, it is to be understood that this invention is not limited to particular compounds and methods, which can, of course, vary. Also,
The terms used in the specification are intended to describe certain embodiments only, and are not intended to be limiting. As used in this specification and the appended claims, the singular encompasses the plural reference unless the context clearly dictates otherwise. For example, a cell means a single cell and one or more cells.

The present invention is a method of treating a tumor, including a Fas ⁺ tumor cell, comprising introducing a nucleic acid encoding a Fas ligand (FasL) into a second tumor cell, whereby the second tumor cell comprises a second tumor cell. Tumor cells express that nucleic acid to produce FasL, thereby producing Fas ⁺ tumor cells
Interaction with the second tumor cell expressing FasL causes the Fas ⁺ tumor cell to undergo apoptosis, thereby providing a method of treating a tumor comprising Fas ⁺ tumor cell. One skilled in the art will appreciate that there are a number of techniques by which nucleic acid sequences encoding a Fas ligand and, optionally, additional sequences, such as one or more regulatory sequences, may be obtained. One way to obtain this nucleic acid is by constructing the nucleic acid molecule by synthesizing a recombinant DNA molecule. For example, oligonucleotide synthesis procedures are routine in the art, and oligonucleotides encoding particular proteins or regulatory regions are readily available through automated DNA synthesis.

A nucleic acid molecule for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand. These oligonucleotides can be designed such that the resulting double-stranded molecule has appropriate 5 'or 3' overhangs at internal restriction sites or ends for cloning into an appropriate vector. A double-stranded molecule that encodes a relatively large protein or control region first constructs several different double-stranded molecules that encode a particular region of this protein or control region, and then combines those DNA molecules together. Can be synthesized by bonding to For example, Cunningham et al., A Receptor and Antibody Epitopes in Human Grow
th Hormone Identified by Homolog-Scanning Mutagenesis, Science, Vol. 243
pp1330-1336 (1989), a synthetic gene encoding human growth hormone was constructed by first constructing overlapping complementary synthetic oligonucleotides and ligating these fragments together. Ferretti et al., Proc. Nat. Acad. S.
See also ci. 82: 599-603 (1986). This includes synthesizing oligonucleotides
The synthesis of a 1057 base pair synthetic bovine rhodopsin gene is disclosed. Once the appropriate DNA molecule has been synthesized, this DNA can be cloned downstream of an appropriate promoter. Such techniques are common in the art and are well described.

Another method of obtaining a nucleic acid encoding a Fas ligand is to isolate the corresponding wild-type nucleic acid of the organism in which it is found and clone it into a suitable vector.
For example, a DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest. Methods for constructing and screening such libraries are well known in the art, and kits for performing the construction and screening steps are commercially available (eg, Stratagene Cloning Systems, La Jolla, CA). ). Once isolated, the nucleic acid can be cloned directly into an appropriate vector, or can be modified, if necessary, to facilitate subsequent cloning steps. Such modification steps are routine, such as the addition of an oligonucleotide linker that contains a restriction site at the end of the nucleic acid. Common methods are Sambrook et al.
A Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory
Press (1989). Once isolated, standard laboratory techniques,
For example, selected codons can be modified using PCR.

Yet another method of obtaining a nucleic acid encoding a Fas ligand is to amplify a corresponding wild-type nucleic acid from a nucleic acid in a host organism having the wild-type nucleic acid and clone the amplified nucleic acid into a suitable vector. One skilled in the art will appreciate that the amplification step may be combined with a mutagenesis step, e.g., using a primer that is not completely complementary to the target nucleic acid, to simultaneously amplify the nucleic acid and modify certain portions of the nucleic acid. You will understand. The nucleic acid encoding FasL may be DNA, RNA, or any combination thereof, and may contain only those bases typically found or may contain modified bases. These modified nucleotides are well known in the art,
Includes thio-modified deoxynucleotide triphosphates and borano-modified deoxynucleotide triphosphates (Eckstein and Gish, Trends in Biochem. Sci.
., 14: 97-100 (1989) and Porter Nucleic Acids Research, 25: 1611-1617
(1997)).

[0015] Alternatively, the nucleic acid is expressed by the cell when introduced into the cell, thereby producing a ligand and / or receptor that causes the ligand and / or receptor to cause tumor cells to undergo apoptosis. , And may encode other types of signaling ligands and / or receptors so that the tumor is treated. Examples of other signaling molecules that can be used in the methods of the invention include Bax, Bad, Bak and Bik (Adams et al., "Control of cell death" WEH
I Annual Report 1996/1997).

In another embodiment of the invention, the nucleic acid encoding the Fas ligand may also encode other proteins, such as regulatory proteins, that can be used to control the expression of the Fas ligand. For example, a regulatory protein can cause tissue-specific localization of Fas ligand on the cell membrane, or it can also cause precocious turnover of Fas ligand in non-target cells, or induce transcription and / or translation. The expression of FasL can be controlled by the control. The control protein may be encoded by another nucleic acid that is delivered simultaneously or sequentially with the nucleic acid encoding the protein to be expressed or with the protein delivered to the cell. In this embodiment, the two nucleic acids may have different sequences, such as different promoters, thereby controlling the administration of an agent capable of selectively controlling one or both promoters, eg, controlling an inducible promoter. Their sequence can be independently controlled by administration of agents that can selectively control the expression of one or both promoters, such as by the use of steroid hormones such as glucocorticoid hormones.

[0017] The nucleic acid encoding the Fas ligand may include a fusion protein. One skilled in the art will appreciate that fusion proteins can be used to localize proteins, activate or inactivate proteins,
It will be appreciated that it is often used for such purposes as monitoring the location of proteins, isolating proteins and quantifying the amount of proteins. In one embodiment, the fusion protein includes a Fas ligand and a green fluorescent protein. Other examples of fusion proteins containing Fas ligand include GFP gene, CAT gene, neo gene, hygromycin gene and others. An example of a FasL-GFP fusion protein expression construct is shown in FIG. 1 and is described in detail herein. The nucleic acid encoding the Fas ligand may include a sequence capable of controlling the Fas ligand. For example, the nucleic acid may have a glucocorticoid regulatory element (GRE) so that the glucocorticoid hormone can be used to control the expression of a Fas ligand. Other examples of regulatory sequences that can control the expression of adjacent genes include:
This is due to the cloning of RNA aptamers such as H10 and H19 into the promoter region, whereby administration of a drug such as Hoechst dye 33258 inhibits the expression of that gene in vivo (Werstuck et al., "Controlling gene expression"). in
living cells through small molecule-RNA interaction "Science 282: 296-29
8 (1998)). In another embodiment of the invention, the control sequence is a Tet-operon or la
Includes the c operon or any other sequence that can function as a regulatory sequence in eukaryotic cells.

In a preferred embodiment, the FasL protein is under the control of a tetracycline-regulated gene expression system, wherein the expression of FasL is regulated by a tet-response element,
FasL expression requires the interaction of the tet-response element with the tet transactivator.
In a more preferred embodiment, tight control of FasL expression is achieved using an Ad vector in which the tet-response element and the transactivator are constructed at opposite ends in the same vector to avoid enhancer interference. You. Expression is conveniently controlled in a dose-dependent manner by tetracycline or any derivative thereof. Tetracycline derivatives include, but are not limited to, doxycycline. This vector delivers the FasL-GFP gene efficiently in vitro and the expression level of the fusion protein will be regulated by the doxycycline concentration in the medium. In particular,
Examples of such control systems are described below.

[0019] The methods described herein include introducing a nucleic acid encoding a Fas ligand into a cell. One skilled in the art will recognize that this aspect of the method can include either stable or transient introduction of the nucleic acid into the cell. In addition, stably or transiently introduced nucleic acids may or may not integrate into the host genome. One of skill in the art will also recognize that the exact procedure for introducing nucleic acids into cells can vary, and may depend on the particular type or identity of the cell. Examples of methods for introducing nucleic acids into cells include electroporation,
Cell fusion, DEAE-dextran mediated transfection, calcium phosphate-
A variety of "naked DNA" delivery techniques including, but not limited to, mediated transfection, viral vector infection, microinjection, lipofectin-mediated transfection, liposome delivery, and particle bombardment techniques. Cells into which a nucleic acid encoding FasL has been introduced may be Fas-expressing cells or cells that do not express Fas.

In one embodiment of the invention, the promoter is a promoter that confers tissue specificity on the expression of a nucleic acid encoding FasL, as will be appreciated by those skilled in the art. For example, this tissue-specific promoter may be prostate-specific, breast-specific, colon tissue-specific, brain-specific, kidney-specific, liver-specific, bladder-specific,
It may be a lung-specific, thymus-specific, stomach-specific, ovarian-specific, or cervical-specific promoter. When the tissue-specific promoter is a prostate-specific promoter, the promoter includes a PSA promoter, a ΔPSA promoter, ARR2
Includes, but is not limited to, PB promoter and PB promoter. Where the tissue-specific promoter is a breast-specific promoter, this includes, but is not limited to, MMTV and whey-producing protein promoter. Where the tissue-specific promoter is a liver-specific promoter, this includes, but is not limited to, the albumin and alpha-fetoprotein promoters.

When the tissue-specific promoter is a brain-specific promoter, this includes, but is not limited to, the JC virus early promoter, tyrosine hydroxylase and dopamine promoter. If the tissue-specific promoter is a brain-specific promoter, this includes the JC virus early promoter, tyrosine hydroxylase and dopamine hydroxylase, neuron-specific enolase, and glial fibrillary acidic protein promoter, It is not limited to these. Where the tissue-specific promoter is a colon-specific promoter, this promoter includes the MUC1 promoter,
It is not limited to this. Of course, other tissue-specific promoters will be revealed by the Human Genome Project. These promoters could be suitably used to direct tissue-specific expression from the vectors of the invention.

In addition, those skilled in the art will immediately know how to identify a promoter specific for a particular cell type. For example, a gene expressed in one specific tissue type can be identified by comparing expression differences in different tissue types using a gene chip technique or the like. These genes can then be isolated and sequenced, and their promoters isolated and tested in animal models for the ability to drive tissue-specific expression of the heterologous gene. Such methods are within the ability of those skilled in the art. Examples of methods by which tissue-specific promoters can be identified can be found in Greenberg et al., Molecular Endocrinology 8: 230-239, 1994. Tissue specificity can also be achieved by selecting a vector with a high degree of tissue specificity. For example, one can select a vector that selectively infects mucosal cells, such as cells associated with colorectal cancer, optionally combining the nucleic acid encoding FasL with their specific delivery means, such as a suppository. It can be selectively delivered to desired cells.

Those skilled in the art will recognize that various vectors are somewhat adaptable, depending on the particular host. One example of a particular technique for introducing nucleic acids into a particular host is the use of a retroviral vector system that can package the recombinant retroviral genome (Pastan et al., "A retrovirus carrying an MDR1 cDNA confers mu.
ltidrug resistance and polarized expression of P-glycoprotein in MDCK ce
lls ", Proc. Nat. Acad. Sci. 85: 4486 (1988) and Miller et al.," Redesign of
retrovirus packaging cell lines to avoid recombination leading to helper
virus production "Mol. Cell. Biol. 6: 2895 (1986). The resulting recombinant retrovirus can then be used to infect cells and deliver the nucleic acid sequence encoding Fas ligand to infected cells. The exact method of introducing the nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors.Other techniques are widely used for this purpose, including: Adenovirus vectors (Mitani et al., "Transduction of human bone marrow by ad
enoviral vector ", Human Gene Therapy 5: 941-948 (1994)), adeno-associated virus vector (Goodman et al.," Recombinant adenoassociated virus-mediated gen
e transfer into hematopoietic progenitor cells "Blood 84: 1492-1500 (199
4)), lentiviral vectors (Naidini et al., "In vivo gene delivery and stab
le transduction of nondividing cells by a lentiviral vector ", Science 27
2: 263-257 (1996)), pseudoretroviral vectors (Agrawal et al., "Cell-cy
cle kinetics and VSV-G pseudotyped retrovirus mediated gene transfer in
blood-derived CD34 ⁺ cells ", Exp. Hematol. 24: 738-747 (1996)), vaccinia vectors and physical transfection techniques (Schwarzenberger et al.," Ta
rgeted gene transfer to human hematopoieteic progenitor cell lines throu
gh the c-kit receptor ", Blood 87: 472-478 (1996)).

The present invention can be used in conjunction with these or other methods commonly used for gene transfer methods. In a preferred embodiment of the invention, the specific vector for delivering a nucleic acid encoding a Fas ligand comprises an adenovirus vector. Since it is desirable to be able to control the expression of FasL or FasL fusion, the present invention relates to a vector for controllable expression of FasL or FasL fusion (of course, a plasmid vector, a viral vector, a baculovirus vector, or the like). Which is operably linked to a transcription control sequence used in the method of the invention.
A vector comprising a nucleic acid encoding a FasL or FasL fusion is provided. In one embodiment, the transcription control sequence may be inducible, ie, expression of FasL or a FasL fusion does not occur unless an appropriate activator for that particular transcription control sequence is present. In another embodiment, the transcription control sequence may be repressible, ie, expression of the FasL or Fas fusion occurs in the absence of an appropriate repressor for that particular transcription control sequence.

[0025] In yet another embodiment, the vector may additionally comprise a nucleic acid encoding a trans-acting factor that interacts with a transcription control sequence that affects the transcription of FasL or a FasL fusion. If the transcription control sequence is inducible, the trans-acting factor will be an activator. If the transcription control sequence is repressible, the trans-acting factor will be a repressor. In a more preferred embodiment, the transcription control sequence is a tet response element (TRE) and the trans-acting factor is a tet-responsive trans-acting expression element (tTA). In a most preferred embodiment, the present invention uses the vector Ad / FasL-GFP _TET . This is a replication defective adenovirus vector expressing a fusion of mouse FasL and green fluorescent protein (GFP). FasL-GFP retains the full activity of wild-type FasL, while allowing easy visualization and quantification in both live and fixed cells. The fusion protein is under the control of a tetracycline-regulated gene expression system. Tight control is achieved by creating this novel "double recombinant" Ad vector.
In this vector, a tet-response element and a transactivation element are constructed at opposite ends of the same vector to avoid enhancer interference.

Expression can be conveniently controlled in a dose-dependent manner by tetracycline or any derivative thereof, including but not limited to doxycycline. This vector delivers the FasL-GFP gene to cells in vivo and in vitro, and expression of the fusion protein may be regulated by the concentration of doxycycline added to the medium or administered to the patient. As can be seen from the examples below, Ad / Fas
L-GFP _TET can be delivered to FasL-GFP transformed and primary cell lines, and expression of this fusion protein in those cells is controlled by varying the concentration of doxycycline in the medium. The amount of FasL-GFP can be easily detected and quantified by the fluorescence of the GFP portion, and its amount correlates with the level of apoptosis of the target cell and neighboring cells.

Although this vector design delivers a complete tet-regulated gene expression system, it is more efficient and economical than a multiple vector strategy and can be used in any situation where control of protein expression is desired. Also applicable to: Accordingly, in another embodiment, the invention comprises a nucleic acid encoding a transactivator protein that binds to a tet-responsive transactivation expression element, and a control element comprising the tet-responsive transactivation expression element. It relates to a controllable expression vector, wherein a nucleic acid encoding the protein to be expressed may be inserted downstream of its control element. In a preferred embodiment, the vector is a viral vector.
In a more preferred embodiment, the viral vector is an adenovirus vector, and the nucleic acid encoding the transactivator protein and the nucleic acid encoding the control element are oriented in opposite ends of the vector.

Of course, the vector may be any other type of viral vector, including a vaccinia vector or a retroviral vector. In another embodiment of the vector, the expressed protein is fused to a reporter.
Reporters include, but are not limited to, green fluorescent protein. For example, a preferred vector for FasL-GFP expression is pLAd-C.tTA and pRAd-TGFsL in Ad5
Ligation to part of the genome (snb360), as described below, and FIG.
As shown in -C, the vector Ad / FasL-GFP _TET was prepared. As in a most preferred embodiment, the vector is a pAd _TET, removing the FasL-GFP from the vector pRAd-TGFsL, the resulting vector was described in Figure 1A-C for the production of a vector Ad / GasL-GFP _TET Alternatively, it can be synthesized by ligation to pLAd-C.tTA. vector
pAd _TET can be used for the expression of various unrestricted heterologous proteins for which strict control is desired.

[0029] The nucleic acid or vector encoding FasL contains the nucleic acid or vector and may contain a selectable marker that can be used to screen for cells that express the selectable marker. In this way, cells containing the nucleic acid or vector and expressing the selectable marker can be readily converted from cells containing the nucleic acid or vector but not expressing the selectable marker, and cells not containing the nucleic acid or vector. Can be separated. The specific selectable marker used is, of course, any selectable marker that can be used for selection on eukaryotic cells that do not contain and contain the selectable marker. Selection can be based on the death of cells that do not have and do not express the selectable marker, as in the case where the selectable marker is a gene encoding a drug resistance protein. An example of such a drug resistance gene for eukaryotic cells is the neomycin resistance gene. Cells expressing the neomycin resistance gene can survive in the presence of the antibiotic G418 or Geneticin7, while eukaryotic cells that do not or do not express the neomycin resistance gene will undergo negative selection in the presence of G418. receive. Those skilled in the art will appreciate that there are other examples of selectable markers such as the hph gene selectable with the antibiotic hygromycin B, the E. coli Ecogpt selectable with the antibiotic mycophenolic acid. Thus, the specific selection marker used can be varied.

[0030] The selectable marker can be used to separate cells expressing and containing the selectable marker from cells not containing and / or not expressing the selectable marker by means other than the ability to proliferate in the presence of the antibiotic. Can also be used to release. For example, the selectable marker may encode a protein that, when expressed, allows the cells expressing the selectable marker to be identified to be identified. For example, the selectable marker may encode a photoprotein such as a luciferase protein or green fluorescent protein, and cells expressing the selectable marker encoding the photoprotein do not express the selectable marker encoding the photoprotein. Can be identified from the cells. Alternatively, the selectable marker may be a sequence encoding a protein such as chloramphenicol acetyltransferase (CAT). By methods well known in the art, cells producing CAT can be easily identified and distinguished from cells not producing CAT.

Various vectors and hosts used to express the nucleic acid encoding the Fas ligand can be used to express the nucleic acid in culture or in vitro. For example, a vector containing a nucleic acid encoding a Fas ligand may be introduced into a tissue culture cell line, such as a COS cell, whereby the nucleic acid is expressed in culture.
In this way, one of skill in the art can select a cell type that will have a limited life span in the host organism, such that the host can replace cells expressing FasL with non-target peripheral cells or tissues. Can be effectively wiped out in a short period of time, so that any possible apoptotic effects on can be minimized. Alternatively, cells from the patient may be removed from the patient, administered with a nucleic acid encoding a Fas ligand, and then returned to the patient. In this ex vivo treatment procedure, the cells can be manipulated without imparting unnecessary adverse effects to the patient to facilitate the uptake of the nucleic acid encoding the Fas ligand.

Various vectors and hosts for expressing the nucleic acids of the invention may be used to express the nucleic acids in vivo. For example, a vector containing a nucleic acid encoding a Fas ligand may be introduced into cells of a eukaryotic host, preferably a tumor cell, to treat Fas ⁺ tumor cells in situ. As discussed briefly above, those skilled in the art
It will be appreciated that certain tissues can be treated by selectively administering the vector to a host. For example, the vector can be selectively administered to the lung by administering the adnoviral vector via an aerosol, such as through the use of an inhaler. Alternatively, the use of suppositories can be employed to selectively administer the vector to colon cells. Alternatively, the vector or nucleic acid may be delivered to skin cells or cervix (c) by local delivery of the vector as in a cream.
ervix). One skilled in the art will recognize the various methods commonly used to selectively deliver a vector, or a nucleic acid encoding a Fas ligand, to a particular organ or cell. When the vector of the present invention expresses a protein for treating cancer or other diseases, it can be administered together with (before, during, or after) other therapeutic agents for the cancer or disease to be treated. it can. These agents can be administered at doses known or determined to be effective, and may be administered at reduced doses due to the presence of the vector-expressed protein.

The skilled artisan will also understand that delivery can be facilitated manually by such methods as injection of a vector or nucleic acid into a selected site. For example, direct injection can be used to deliver vectors or nucleic acids to specific brain and / or thoracic sites. In one embodiment of the invention, direct injection of a nucleic acid or vector encoding a Fas ligand is used to deliver to a breast tumor mass. By using the methods and vectors of the present invention, it is expected that the nucleic acid encoding FasL can be administered to cells or patients, most preferably humans, to treat a disease state, preferably cancer. The nucleic acids of the present invention, alone or together with other compounds or compositions (eg, chemotherapeutic agents), or as part of a vector-based delivery system, can be administered intramuscularly. Parenteral (eg, intravenous), topical, transdermal, etc. administration may be by injection, intraperitoneal injection, although topical administration is typically preferred. The exact amount of such nucleic acid, composition, vector, etc. required will depend on the species, age, weight and general condition of the patient, the severity of the disease or condition to be treated, the particular compound or composition used. It will vary from patient to patient depending on the product, mode of administration and the like. Therefore, it is neither possible nor necessary to specify the exact amount. However, an appropriate amount will be determinable by one of skill in the art using methods well known in the art (eg, Martin et al.,
See 1989)

For topical administration, the nucleic acid encoding FasL, the composition thereof, and / or the vector containing the nucleic acid may be a solid, such as, for example, a powder, liquid, suspension, lotion, cream, gel, or the like. It may be a pharmaceutical composition in semi-solid or liquid dosage form, preferably in a unit dosage form suitable for single administration of a precise dose. The composition will typically comprise an effective amount of the selected nucleic acid, composition or vector together with a pharmaceutically acceptable carrier, as well as other pharmaceutical agents, therapeutic agents, carriers, adjuvants, diluents and the like. May be included. "Pharmaceutically acceptable" refers to a material that is not biologically or otherwise harmful, i.e., that the material does not produce any deleterious biological effects or contains any other pharmaceutical composition. It means that it may be administered to an individual with a selected nucleic acid, its composition or vector without causing deleterious interactions with the components.

Alternatively, or in addition, if parenteral administration is used, it is generally characterized by injection, for example, intravenous injection, including local perfusion through blood vessels, to supply the tissue or organ containing the target cells. . Injectables can be prepared in conventional forms and may be liquid solutions or suspensions, solid forms or emulsions suitable for solution or suspension in liquid prior to injection. Parenteral administration can also employ the use of sustained or sustained release systems, thereby maintaining constant dosage levels (eg, US Pat. No. 3,710,795). The compounds can be injected directly into cells or tissue sites expressing the Fas ⁺ phenotype, or the compounds can be injected such that they diffuse or circulate to the site of the Fas ⁺ expressing transcellular cells.

The dosage will depend on the dosage form, the disease or condition to be treated, and the condition of the individual patient. The dose will also depend on the substance to be administered, eg, a nucleic acid, a vector containing a nucleic acid, or other type of compound or composition. Such doses are known in the art. Further, the dose can be adjusted according to typical doses for the particular disease or condition to be treated. In addition, cultured cells of the target cell type
It can be used to optimize the dose to target cells in vivo, and the transformation from different doses achieved in cultured cells of the same type as target cells can be monitored. Often, a single dose may be sufficient; however, administration can be repeated if desired. Dosage should not be so large as to cause adverse side effects. Generally, dosage will vary with age, condition, sex, and the degree of illness in the patient, and can be determined by one of ordinary skill in the art. The dose can also be adjusted by the individual physician in case of any complication. Examples of effective doses in non-human animals are provided in the examples. Based on the technically acceptable dosage forms, the effective dose in humans can be routinely calculated from the dose given and the dose shown to be effective.

For administration to a patient's cells, the compound or composition, once in the patient, will be adjusted to the patient's body temperature. For ex vivo administration, a compound or composition can be administered by any standard method that will maintain the viability of the cells, for example, by adding it to a medium (suitable for the target cell) and It may be administered by direct addition to the cells. As is known in the art, any media used in this method may be aqueous and non-toxic so as not to render the cells non-viable. In addition, if desired, the medium may contain standard nutrients to maintain cell viability. For in vivo administration, the conjugate can be added to a blood or tissue sample, for example, from a patient, or can be added to a pharmaceutically acceptable carrier, such as saline and buffered saline. And can be administered by any of several methods known in the art. Other examples of administration include inhalation of an aerosol, subcutaneous or intramuscular injection, direct transfection of a nucleic acid encoding a compound, wherein the compound is a nucleic acid or protein, for example,
Includes transfection into bone marrow cells prepared for transplantation, followed by transplantation into a patient, followed by direct transfection into the organ to be transplanted into the patient. Further modes of administration include oral administration, especially when the composition is encapsulated, or rectal administration, especially when the composition is in suppository form. Pharmaceutically acceptable carriers include any material that is not biologically or otherwise detrimental, i.e., the material does not produce any detrimental biological effects, and does not produce other harmful biological effects. The individual may be administered with the selected conjugate without causing deleterious interactions with the components.

In particular, if a particular cell type is targeted in vivo, cells from the target tissue may be biopsied, for example, by local perfusion of an organ or tumor, as described herein and known in the art. As noted, the optimal dose for uptake of the complex into the tissue can be determined in vitro, and the in vivo dose can be optimized, including concentration and length of time. Alternatively, cultured cells of the same cell type can be used to optimize the in vivo dose for the target cells. For example, intratumoral injection volumes and rates can be controlled with controllable pumps, such as computer-controlled pumps or microthermal pumps, to control the rate and distribution of nucleic acids or vectors within the tumor or tissue. Example 4 shows an effective dose of AdFasL-GFP _TET used for in vivo treatment of both breast and brain tumors in mice. One skilled in the art will readily know how to extrapolate these numbers to an effective human dose.

[0039] For either ex vivo or in vivo use, the nucleic acids, vectors or compositions can be administered at any effective concentration. Effective concentrations are cell killing,
An amount that causes a reduction, inhibition or prevention of the transformed phenotype of the cell. The nucleic acids or vectors can be administered as a composition. For example, the composition can include other pharmaceutical agents, pharmaceuticals, carriers, adjuvants, diluents, and the like.
In addition, the composition can include, in addition to the nucleic acid or vector, lipids such as liposomes, liposomes such as cationic liposomes (eg, DOTMA, DOPE, DC-cholesterol) and anionic liposomes. If desired, the liposomes may include proteins to facilitate targeting of particular cells.
The administration of the composition comprising the nucleic acid or vector and the cationic liposome may be administered to a blood vessel that is introduced into the target organ, or may be inhaled during inhalation of airway target cells. For liposomes, see, for example, Brigham et al., Am. J. Resp.
Cell. Mol. Biol. 1: 95-100 (1989); Felgner et al., Proc. Natl. Acad. Sci USA 8
4: 7413-7417 (1987); see U.S. Patent No. 4,897,355. Further, the nucleic acid or vector can be targeted as a component of a microcapsule that can target a particular cell type, e.g., macrophage, or a microparticle in which the delivery of the compound nucleic acid or compound from the microcapsule is designed for a particular rate or dose. It can be administered as a component of a capsule.

[0040] Any cells expressing Fas, especially tumor cells, can be treated by the methods of the present invention. Fas is mainly a surface protein, and cells expressing Fas ligand can be used to treat Fas-expressing cells by Fas-Fas ligand-induced apoptosis. Cells expressing Fas ligand by the interaction of Fas ligand with cell surface Fas can interact with Fas-expressing cells, thus treating Fas-expressing cells that can be contacted by Fas ligand-expressing cells. However, Fas ligand-producing cells can also control Fas-expressing cells by producing soluble Fas ligand that interacts with Fas and also induces apoptosis. The interaction of Fas and the Fas ligand is typically a ligand-receptor binding, but this interaction may not be itself bound and any cell resulting from the result of any interaction of Fas and the Fas ligand. Sexual reactions are also included. Thus, this contemplates any cellular apoptosis by Fas resulting from the expression of Fas ligand by the same cell or a second cell that expresses Fas ligand.

Although any cells expressing Fas can be used to induce and cause apoptosis using the methods of the present invention, preferred embodiments use these methods to induce Fas ⁺ tumor cells to undergo apoptosis. Is to guide. In this embodiment, these tumor cells are selectively induced to undergo apoptosis and die, thereby treating the tumor. In another preferred embodiment, the tumor is a solid tumor,
A recombinant virus that can infect the tumor cells is injected into the tumor, thereby
It causes the expression of the s ligand, whereby the interaction of the FasL-expressing cells with the Fas-expressing cells causes apoptosis of the Fas ⁺ cells. A Fas-expressing cell that is acted upon by a FasL-expressing cell is typically a cell adjacent to the FasL-expressing cell. This is because cell-cell contact is typically required for an apoptotic signal to occur. However, the affected Fas cells can be removed from the immediate surrounding of the FasL-expressing cells, such as when the FasL-expressing cells are mobile and / or the FasL-expressing cells express soluble FasL. Fas ligand-expressing cells also cause their own death if they are Fas ⁺ cells. In this approach, the method of the invention
Although it can cause death of Fas ⁺ cells, tumor cells expressing the Fas ligand also die, thereby eliminating tumor cells that may cause tumor reversion.

The present invention is more specifically described in the following examples, which are intended to be merely illustrative, as numerous modifications and variations will be apparent to those skilled in the art. It is.

EXAMPLES Example 1 In the example of the method described above and depicted in FIG. 1, a recombinant virus containing a nucleic acid encoding a human Fas ligand was constructed. In addition, a recombinant adenovirus was constructed that contained a nucleic acid encoding a human Fas ligand and a nucleic acid encoding a jellyfish green fluorescent protein (GFP) such that the fusion protein was ultimately translated. This fusion protein is used to monitor expression and localization of the protein in cultured cells and animal tissues following transduction by adenovirus. Three different tumor cell lines were isolated from breast cancer patients, all of which were highly sensitive to Fas ligand treatment with adenovirus vectors. This demonstrates that the method can efficiently treat and kill tumor cells. By parallel experiments, several prostate cancer cells were found to have achieved complete death of these cells using adenovirus-mediated transfer of nucleic acid encoding Fas ligand, resulting in
It was shown to be very sensitive to as-mediated apoptosis. Furthermore, it was implanted with 10 ⁵ breast cancer cells or prostate cancer cells on each side of the six nude mice. When the tumor reached about 5 mm in diameter, all tumors on one side of the animal received one adenovirus vector (AdFasL) containing nucleic acid encoding Fas ligand.
0 and ⁸ pfu injection. All tumors on the other side of the animal received one control adenovirus (AdlacZ).
0 and ⁸ pfu injection. Three weeks after injection, all tumors injected with AdFasL showed significant tumor regression compared to control treated tumors. Histological analysis of the residual tumors of some mice showed no residual cancer cells and only infiltrating immune cells and fibroblasts.

Example 2: Controlled delivery of FasL-GFP fusion protein by complex adenovirus vector Fas ligand (FasL) induces apoptosis in cells expressing Fas receptor,
It plays an important role in the immune response, degenerative and lymphoproliferative diseases and tumorigenesis. It has also been implicated in the generation of immunoprivileged sites and is therefore of interest in the field of gene therapy. Here, mouse FasL and green fluorescent protein (GFP
) Describes the construction and characterization of a replication-defective adenovirus vector expressing the fusion of FasL-GFP retains the full activity of wild-type FasL, while facilitating visualization and quantification in live and fixed cells. The fusion protein is under the control of a tetracycline-controlled gene expression system. Tight control is achieved by creating a novel double recombinant Ad vector. In this vector, a tet-response element and a transactivation element are constructed at opposite ends of the same vector to avoid enhancer interference. Expression can be conveniently controlled in a dose-dependent manner by tetracycline or a derivative thereof. This vector can efficiently deliver the FasL-GFP gene to cells in vitro, and the expression level of the fusion protein can be regulated by doxycycline in the medium. This control is associated with FmA in CrmA-expressing cell lines.
Inhibiting asL expression allows for the production of high titers of the vector. Induction of apoptosis was shown in all cells tested. These results indicate that our vector is a potential valuable tool for FasL-based gene therapy of cancer and for the study of FasL / Fas-mediated apoptosis and immune privilege.

Materials and Methods Cells: HeLa and 293 cells were obtained from the American Type Culture Collection (ATCC CCL-2.1 and ATCC CRL-1573, respectively), 10% fetal calf serum (BCS; Hyclone) and 1% penicillin / streptomycin ( Cellgro) addition of Dulbecco's modified Eagle's medium (DMEM: in Gibco BRL), were maintained as a monolayer under 5% O ₂ at 37 · C. Cultured rat myogenic cells were H-21 (Cel) supplemented with 20% fetal bovine serum (FBS: HyClone) and 1% penicillin / streptomycin and fungizone, respectively.
lgro) medium.

For DNA transfection, 5-10 ⁵ cells per well were plated in a 6-well plate (G
reiner) and transfected 24 hours later using LipofectAMINE (Gibco BRL) according to the manufacturer's instructions. To generate cytokine response regulator A (CrmA) -expressing 293 cell line, pCrmA-
I-Neo was transfected into HEK293 cells. Neo-positive clones were selected by adding G418 to the medium at 0.4 g / L for 4 weeks. At the end of this period, the clones were picked, expanded and assayed for CrmA expression by resistance to FasL-induced apoptosis.

Construction of plasmids and recombinant adenovirus vectors: Vectors pEGFP-1 and pEGFP1-C1 were obtained from Clontech. They are wild-type green fluorescent proteins (w
t GFP) gene, contains redshift mutants, has brighter fluorescence and has "humanized" codon usage (Zhang, GV Gurtu and SR Kain. 1996 "An enhanc
ed green fluorescent protein allows sensitive detection of gene transfer
in mammalian cells ", Biochem Biophys Res Commun 227: 707-11). This protein is referred to as" GFP "in the examples. Mouse FasL cDNA sequence is available at Genbank and is in the Bluescript (Invitrogen) vector. Vector pUHD10-3 and
pUHD15-1 (Gossen, M and H. Bujard, "Tight control of gene expression in mamm
alian cells by tetracycline-responsive promoters "Proc Natl Acad Sci USA
89: 5547-51, 1992) is available from Clontech. Mouse Fas ligand aa 11
The GFP-FasL fusion gene was prepared by inserting a DNA encoding aa 279 into the downstream of the GFP sequence of pEGFP-C1 in frame, and pC.GFsl was prepared. The fusion gene from pC.GFsl was inserted into pUHD10-3 to create p10-3.GFsl. Cowpox virus (vertebrate poxvirus Chordopox virina) in pcDNA3 vector
e)) Cytokine response regulator A (crmA; CPV-W2) cDNA was obtained from Genentech. The CrmA gene was excised from pcDNA3 and inserted into the pIRES-Neo vector (Clontech) to generate pCrmA-I-Neo.

The GFP, FasL, FasL-GFP and lacZ genes were cloned into the E1 shuttle vector pLAd-CMV to construct pLAd-C.Gf, pLAd-C.Fsl, pLAd-C.GFsl and pLAd-C.Lz constructs, respectively. (Figure 1A). Tet-OFF fusion activator protein expression cassette is pUH
It was extracted from D15-1 and inserted into pLAd-CMVie to produce pLAd-C.tTa. The GFP-FasL fusion gene expression cassette was excised from p10-3.GFsl and inserted into the shuttle vector pRAd.mcs for transgene insertion between E4 of Ad5 and the right ITR. PRAd the resulting construct
-Called T.GFsl (Figure 1). The assembly of the rAd / FasL-GFP _TET vector is shown in FIG. 1C. Other rAd genomes used in this study were constructed using a similar strategy. All vectors are Ad5su
It is based on b360 (ΔE3) and also includes all E4 ORF deletions except ORF6 (Huang, MM and P. Hearing, 1989). The adenovirus early region 4 open reading frame 6/7 protein controls the DNA binding activity of the cellular transcription factor E2F directly through the complex (Genes Dev 3: 1699-710).

Viral vector propagation: 293 cells supply Ad5 E1a and E1b functions in trans (Graham, FL, J. Smiley, WC Russell and R. Nairn; "Characteristics
of a human cell line transformed by DNA from human adenovirus type 5 ", J
Gen Virol 36: 59-74, 1977), which was transfected with the ligation mixture containing the rAd vector using the LipofectAMINE method. Keep the transfected cells until adenovirus-associated cytopathic effect (CPE) is observed (
Cells were harvested at that time, usually 7 to 14 days). Vector propagation and amplification were then performed by standard techniques. Stocks were titered on 293 and 293CrmA cells, plaques were counted and vector yield was determined as PFU / ml. GFP vector
Appropriate titration was performed using either fluorescence or X-gal staining. In both cases,
The titer evaluation agreed well with PFU / ml.

Western blot analysis: 10 ⁶ primary rat myoblasts were seeded on 10 cm plates (Greiner). Twenty-four hours later, plates were infected with rAd / FasL-GFP _TET or the vector of interest at a multiplicity of infection (MOI) of 2. At 24 hours post-infection, plates were washed twice with PBS. Cells were collected and 50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 2% SDS, 0.1% bromophenol blue, 1 mM PMSF (Sigma), 50 μg / ml leupeptin (Sigma), 2 μg / ml aprotinin (Sigma) and 1 ng Lysed in 200_l cell lysis buffer containing / ml pepstatin (Sigma). The sample was boiled for 5 minutes and the original volume per lane (10 ⁶ cells
) Was loaded on an 8% SDS-PAGE mini gel (BioRad) and electrophoresed at 20 mA for 3 hours. Human recombinant FasL (C-terminal) was obtained from Santa Cruz laboratory. Semi-dry gel transfer device
The protein was transferred to a nitrocellulose membrane (Pharmacia Biotech) using (BioRad). 10 mM Tris-HCl (pH 7.5), 140 mM NaCl, 3% (w / v) BSA, 5% (w / v) powdered milk, 0.2% (v / v) Tween-20 (Amresco, Solon, OH) and By incubation in a solution containing 0.02% (w / v) sodium azide (Sigma) (2 ° C. at 37 ° C.)
Time) The membrane was blocked. Polyclonal rabbit anti-FasL antibody (Santa Cruz)
Was diluted 1: 100 with blocking solution and incubated with the membrane for 2 hours at ambient temperature. The blot was washed twice with 10 mM Tris-HCl (pH 7.5) and 140 mM NaCl solution,
Next, goat anti-rabbit IgG conjugated to HRPO diluted 1: 10000 (Caltag, Burllingam
e, CA). This blot was prepared using the ECL reagent (Amersham Lif
e Science) overnight and visualized on Kodak X-ray film.

Detection of Apoptosis: Early Detection of Apoptosis in Cultured Adherent Cells In Situ Ce
This was performed using the II Death Detection Kit, AP (Boehringer Mannheim) according to the manufacturer's instructions. This kit contains terminal nucleotidyl transferase-mediated
Fluorescein is incorporated into free 3'-OH DNA ends using the UTP nick end label (TUNEL) method, and fluorescein is detected with an anti-fluorescein antibody conjugated to alkaline phosphatase. After the substrate reaction, the stained cells can be visualized with a light microscope.

Results: Functional analysis of FasL and FasL-GFP proteins: FasL and FasL-G show that the FasLigand-GFP (FasL-GFP) fusion protein retains full FasL activity.
The function of the FP protein was analyzed and compared using transient DNA transfection into Fas-mediated apoptosis-sensitive cells. HeLa cell wells in triplicate with FasL
, GFP-FasL or, as a control, a vector expressing galactosidase. Twenty-four hours after transfection, cells were fixed and analyzed for apoptosis using the TUNEL kit. Transfection efficiencies of 10-25% were typically achieved, as determined by X-Gal staining of cells transfected with pcDNA3-LacZ. Numerous HeLa cells transfected with vectors expressing FasL or FasL-GFP typically showed apoptotic morphology (membrane swelling and loss of adhesion) and stained positive in the TUNEL assay. Cells transfected with the control plasmid showed little apoptosis.
Apoptotic cells in wells transfected with FasL-GFP vector
It is reproducibly similar to cells transfected with the FasL vector, indicating that the wild-type and fusion proteins have comparable activities.

Construction and characterization of adenovirus: Our goal is to produce large quantities of adenovirus vectors with controllable FasL expression. This control is achieved by controlling FasL in target cells.
It allows to control the level of expression, thus facilitating the study of its biological effects. In addition, amplification of an rAd vector that constitutively expresses FasL or FasL-GFP in 293 cells is expected to produce low titers because FasL expression causes apoptosis of virus-producing cells (Muruve DA, AG Nicolson , RC Manfro, TB
Strom, VP Sukhatme and TA Libermann, 1997 Adenovirus-mediated expressi
on of Fas ligand induces hepatic apoptosis after Systemic administration
and apoptosis of ex vivo-infected pancreatic islet allogragts and isogr
agts, Hum Gene Ther 8: 955-63). To achieve controlled FasL-GFP expression,
An rAd / FasL-GFP _TET vector in which FasL-GFP is expressed from the TRE promoter was designed (Gossen, M. and H. Bujard, 1992, Tight control of gene expression in mamma
lian cells by tetracycline-responsive promoters, Proc Natl Acad Sci USA
89: 5547-51). CMVie promoter-driven tTA gene ("tet-off" element)
The TRE-regulated FasL-GFP fusion gene was inserted near the right ITR. This strategy is based on the following considerations.

First, this strategy uses two Ad vectors as previously described (Harding, TC, BJ Geddes, D. Murphy, D. Knight and JB Uney, 1998,
Switching transgene expression in the brain using an adenoviral tetracyc
Use a single vector instead of the line-regulatable system (see comments, Nat Biotechnol 16: 553-5) to deliver the entire tet-regulated expression system. The use of a single vector allows for more efficient delivery to target cells and allows for more uniform control of protein expression. This strategy also achieves the maximum possible separation between the CMVie promoter and the enhancer element of the TRE promoter, minimizing the (unregulated) background of expression of the FasL-GFP protein (FIGS. 1B and 1C). Placing the TRE promoter at the right end of the Ad5 genome gives similar results for the E1A enhancer element located within the Ad5 packaging signal (Hearing, P. and T. Shenk, 1983, The adenovirus type 5 E1A
transcriptional control region contains a duplicated enhancer element, C
ell 33: 695-703). These elements have been reported to interact with certain promoters cloned into the E1 region (Shi, Q., Y. Wang and R, Worton, 1997,
Modulation of the specificity and activity of a cellular promoter in an
adenoviral vector, Hum Gene Ther 8: 403-10).

[0055] The recombinant adenovirus vector used in this study was assembled in an in vitro large-scale ligation reaction as schematically illustrated in FIG. 1C. These genomes were then gel-purified, transfected into 293 cells, and the resulting culture was grown until virus-induced CPE was observed. In the case of vectors expressing β-galactosidase or GFP, CPE occurs at a significantly earlier time point than for FasL or FasL-GFP expression vectors, which indicates that adenovirus vector replication may be adversely affected by FasL activity. It shows what it is. The primary vector stock was amplified by established techniques, rAd DNA was extracted and checked for structural integrity by restriction enzyme digestion.

The titers of rAd / FasL and rAd / FasL-GFP _TET in 293 cells are typically rAd / LacZ
Or 30-100 times lower than the titer of rAd / GFP. Comparing the titers of the rAd vectors with the FasL activity, a considerable improvement (8- to 12-fold) in the yield of these vectors when produced in 293CrmA cells was seen (Figure 2). Amplification of the control vector rAd / LacZ in 293 or 293CrmA cells resulted in essentially the same yield (FIG. 2). Subsequently, purification and amplification of all vectors having FasL activity were performed on 293CrmA cells.

Apoptosis Induction by Adenovirus-Mediated FasL Expression: Adenovirus-Mediated
To functionally demonstrate FasL expression, HeLa cells were transduced with rAd / FasL-GFP _TET at different MOIs. Twenty-four hours after transduction, cells were analyzed for apoptosis. Cells infected with rAd / FasL-GFP _TET showed a typical apoptotic morphology. The number of apoptotic cells increased with the vector titer. In contrast, plates transduced with the control vector rAd / LacZ at the same MOI did not generate apoptotic cells above the untransduced control. The overall efficiency of the transduction was measured by X-gal staining, with increasing MOI
An increase in galactosidase positive cells was shown. It was observed that the number of apoptotic cells was significantly greater than the number of cells with detectable GFP fluorescence or co-transduced X-gal stained cells. Thus, the interaction of FasL on the surface of infected cells with the Fas receptor of its neighboring cells results in apoptosis of cells not infected with the vector but adjacent to the infected cells.

Detection and cell localization of FasL-GFP fusion protein: Wild type FasL is a type II membrane protein. To prove that the FasL-GFP fusion protein is also suitable for cell membranes,
The fluorescence of the GFP part was used. This fluorescence can be detected in living cells using a fluorescence microscope with a FITC filter set. This technique was used to observe the cellular localization of the FasL-GFP fusion protein when expressed from the rAd vector. In HeLa cells, FasL-G
Expression of FP causes apoptosis at protein levels near the detection threshold of GFP.
Therefore, FasL-GFP expression was analyzed in rat primary myoblasts that were found to be relatively resistant to Fas-L induced apoptosis. High levels of FasL-GFP expression were detected in myoblasts 24 hours after rAd / FasL-GFP _TET infection at MOI 10. Membrane-associated expression of FasL-GFP is prominent in most of the transduced cells. Conversely, the fluorescence pattern of GFP itself is evenly distributed in the cytoplasm of the cell, while often being excluded from the nucleus. These localization differences also indicate that transduced 293CrmA
It is evident that the cells have been expanded even more strongly. These results indicate that the FasL-GFP fusion protein is directed to the cell surface, and that it can interact with the Fas receptor in a manner similar to wt FasL.

Control of FasL-GFP expression from rAd vector: To show that the vector of the present invention can control the amount of FasL activity produced in target cells by our rAd vector,
Fa under inducible or non-inducible conditions at both protein synthesis and functional levels
An experiment was performed to determine the level of sL expression. rAd / FasL-GFP F in _TET vector
Expression of the asL-GFP fusion protein is activated by binding of the tetR-VP16 fusion protein (expressed constitutively from the same vector; see FIG. 1C) to the heptamer of the tet-operator upstream of the minimal CMVie promoter. (Gossen, M. and H. Buj
ard, 1992, Tight control of gene expression in mammalina cells by tetracy
cline-responsive promoters, Proc Natl Acad Sci USA 89: 5547-51). The presence of doxycycline in the cell inhibits this binding—and thus FasL-GFP expression—in a concentration-dependent manner.

First, the amount of FasL-GFP produced in the transduced cells was measured using Western blot analysis. Rat primary myogenic cells were infected at MOI2 with rAd / FasL-GFP _{TE T,} and the cells were cultured in the absence or presence of doxycycline tetracycline derivative. A low MOI was chosen to maximize the number of cells transduced with the single copy vector. After 48 hours, the cells were lysed and the lysate was analyzed by Western blotting using a polyclonal antibody against the extracellular domain of FasL. A single specific band larger than the expected size of wt FasL was detected. Band intensity decreased with increasing doxycycline concentration, and no band was detected in cell lysates cultured in the presence of doxycycline at 0.5 mg / L or higher. No FasL-specific bands were observed in cells transduced with the control vector. No smaller sized bands, corresponding to degradation or cleavage products, were detected in either the cell lysate or the culture supernatant. These results are rAd / Fa
The amount of GFP-FasL protein in cells produced from the sL-GFP _TET vector can be controlled by the concentration of doxycycline in the medium, and this protein is stable and does not undergo any measurable degradation once it reaches the cell surface Is shown.

We also analyzed FasL activity, a control of the induction of apoptosis in Fas-positive target cells. HeLa cell wells were transduced with rAd / FasL-GFP _{TET at} MOI2 and cultured in the presence of various concentrations of doxycycline. Cells were analyzed for an apoptotic phenotype 24 hours after transduction. The results confirmed that the induction of apoptosis in cells transduced with rAd / FasL-GFP _TET could be controlled by doxycycline. In a controlled protein expression system of our choice, the presence of doxycycline inhibits the binding of tTA to the TRE and terminates FasL-GFP transcription in a dose-dependent manner. A constitutively expressed activator was inserted into the E1 region and the FasL-GFP expression cassette was inserted into a novel cloning site between the E4 promoter and Ad5 right ITR.
This arrangement minimizes the effect of the E1A enhancer present in the adenovirus packaging region and the CMVie enhancer in the tTA promoter on the TRE, thereby reducing background expression of this fusion protein in the presence of the inhibitor It was because it was inferred. This system worked well in the context of adenovirus vectors, and expression of FasL-GFP could be efficiently controlled by varying the concentration of doxycycline in the cell culture medium.

During the course of this experiment, we noticed that 293 cells were susceptible to FasL-induced apoptosis. This effect significantly limits the titer of the FasL-expressing rAd vector. This is especially true when regulated or tissue specific promoters have been used to express the FasL protein. This is because high levels of protein expression are unavoidable during vector replication in 293 cells. To solve this problem, we created a 293 cell line that constitutively expresses CrmA.
This protein acts to specifically inhibit the activity of regulatory caspases essential for the Fas apoptotic pathway. By producing our FasL-containing vector in these cells, a significant improvement in vector titer was achieved.

In summary, we have developed and tested an rAd vector that expresses a novel FasL-GFP fusion protein under the control of a tetracycline-regulated gene expression system. This vector combines high titer and efficient delivery to multiple types of dividing and non-dividing cells with easy control of protein expression and easy detection of fusion proteins in both live and fixed cells It was made. This vector is a valuable tool for the treatment of disease through immunization, transplantation and cancer treatment.

Embodiment 3 Bystanders using adenovirus delivery of Fas ligand fusion gene (
This example describes one mode of bystander gene therapy using the Fas ligand-fusion gene approach to induce apoptosis (programmed cell death) in prostate adenocarcinoma through a paracrine / autocrine mechanism. I do. This study offers a new and powerful therapy for the treatment of prostate cancer (PCa). In addition, specificity for the prostate or any other tissue can be achieved by using a tissue-specific promoter to allow parenteral delivery of the virus for the treatment of metastatic disease. Our therapeutic approach is to deliver and express the Fas ligand (CD95L-fusion gene) by a second generation adenovirus lacking E1A, E3 and E4. CD95L expression is controlled by the Tet operator, which allows for in vitro and in vivo doxycycline control. The CD95L used in this project
Mouse CD95L cDNA fused in frame with a C-terminal 4 amino acid linker of enhanced GFP with N-terminal shortened by 10 amino acids. Table 1 shows our data using five PCa cell lines, but the PCa cell lines
Confirms the general literature report of being resistant to CH-11 agonist activity (Hedlund et al., The prostate 36: 92-101, 1998 and Rokhlin et al., Can. Res. 57:
1758-1768, 1997). In contrast, we demonstrated sensitivity to AdGFP-FasL and C2-ceramide in the five PCa cell lines tested so far.

[0065] Percent cytotoxicity was determined using the MTS assay. Briefly, cells were seeded in 12-well plates containing 1 ml of medium. Prior to treatment, cells were grown to 75% confluence and treated with 500 ng / ml CH-11 anti-Fas antibody, 500 ng / ml normal mouse serum or 30.MC2-ceramide. For adenovirus transduction, approximately 1 × 10 ⁵ cells / well were treated with AdCMVGFP or AdGFPFasL _TET for a MOI of 10-1.
000. For each cell line, the positive control was left untreated and 1 ml of medium was used as a negative control. Cells were incubated at 37_C for 48 hours for maximum cell killing. Aspirate medium and add 0.5 ml fresh medium + 100_l CellTite per well
Replaced with r96 ⁷ AQueous One Solution reagent. Cells were further incubated at 37_C for 1-3 hours. After the incubation, 120 1 of the medium was placed in a 96-well plate, and the absorbance was read at 490 nm using a Vmax kinetic microplate reader. Percent cytotoxicity was calculated as follows:% cytotoxicity = [1- (OD of experimental well / OD of positive control well)] × 100 For the ceramide assay, 1 × 10 ⁴ cells / well were 96 Seeded in a well plate. The next morning, cells were washed and incubated in serum-free RPMI 1640 with 100 l of 300 M dihydro- or C2-ceramide (diluted from a 10 mM stock in ethanol). Twenty-four hours later, 20 l Celltier 967 Aqueous One Solution reagent was added to each well and the plates were incubated for another 1-4 hours. Absorbance and% cytotoxicity were measured as described above. In each experiment, data points were collected in triplicate.

Results: Clearly, the analyzed PCa cell lines in Table 1 are largely insensitive to CH-11. C2-
Sensitivity to ceramide was relatively uniform at the 30 μM dose, suggesting that the apoptotic pathway was intact. Most importantly, all cell lines are responsive to AdGFP-FasL administration, with DU145 being the least sensitive. These experiments show some important points. First, we used FACS analysis to identify all candidate PCa for all cell lines that used CD95 (Fas receptor).
It shows that it is expressed on the cell line. Second, we show that the fas receptor inhibitory antibody (ZB4) does not inhibit the induction of apoptosis by AdGFP-FasL. We performed this experiment several times with different doses of ZB4 and always obtained that the virus induced apoptosis to the same extent in the presence or absence of the antibody. This suggests that newly synthesized CD95-CD95L may interact in the Golgi on its way to the plasma membrane (Bennett et al., Science 282: 290-293, 1998),
Alternatively, it may interact as a pre-formed functional apoptotic signal-generating complex when it reaches the cell surface.

Third, our results indicate that there are no adenovirus-specific properties that facilitate apoptosis induction in PCa. This means that PCa can be used as a control virus (AdCM
VGFP) plus CH-11 demonstrated by infection with 500 ng / ml. The result is CH-
11 still failed to induce apoptosis. These results indicate that apoptosis occurs only in the CD95 ⁺ -CH-11 resistant PCa cell line when virus-directed intracellular CD95L expression occurs, indicating that it was not virus-dependent. . One piece of the last and most relevant information is that AdGFP-Fa
It is about whether or not sL _TET can be administered. 2x1 when administered parenterally
Since the dose as low as 0 ⁸ pfu of virus kill mice, this is very important. To solve this problem, xenografts of PPC1 were performed in Balbc nu / nu mice and treated with various doses of AdCMVGFP control or AdGFP-FasL virus. These single-dose administration studies provided evidence that tumor cell growth was slowed or stopped. In addition, out of 14 animals treated with the virus, none died with this virus. In summary, we conclude that the GFP-FasL fusion protein in the present Ad5 delivery system has strong therapeutic potential for PCa treatment.

Development of AdGFP-FasL Variants Upregulated by Doxycycline Our current viruses are designed for orthotopically administration to PCa. If the virus escapes the tumor and enters the body, it will be lethal if enough virus reaches the reticuloendothelial system (often the liver).
Administration of doxycycline (dox) can down-regulate the expression of CD95L from AdGFP-FasL and avoid this risk. A viral vector showing "very low" basal activity induced by doxycycline is described in Example 1
It is constructed using the Tet control element described in (1). This vector is in the absence of dox
Completely repressed for GFP-FasL expression, induction started at 10 ng / ml and max induction 100-5
Induced at 00ng / ml. These are doses that are easily achievable in humans (
Typical dosage levels, 1-3 g / ml). Terminate dox if adverse effects are noted. However, doxycycline has a serum half-life of 16 hours, which means that the addition of dox to down-regulate the expression of Fas ligand rapidly increases the effective doxycycline dose within minutes by parenteral administration. Believe that it would be good to address adverse effects in patients. However, the half-life of GFP-FasL is also a factor in this and is determined experimentally in experimental groups B and C. If necessary,
The addition of a PEST signal can promote degradation (see Clontech catalog).

Method: From a technical point of view, this is purely molecular biology. We have replaced the current Tet repressor and operator system with the rTS ^kid B / C and rtTA systems (Freundlieb et al., J. Gene Med. 1: 4-12, 1999). It has already been shown that our prostate-specific promoters (PSA, PSADBam, PB and ARRPB2, Appendix) can be placed in the virus (substituting CMVie) to achieve tissue specificity such that only prostate epithelial cells can control rtTA. It is shown. All viruses were grown by standard techniques from 3x plaque purified samples that were evaluated as negative for wild-type adenovirus by PCR. All viruses were grown in the presence of 1 μg / ml doxycycline in the HKE293 packaging cell line, which constitutively expresses the cowpox virus cytokine response regulator crmA (Rubinchik et al.). This is the G
Required to prevent FP-FasL-induced apoptosis. Virus was always purified by isopycnic centrifugation of CsCl, desalted by chromatography, concentrated by filtration, aliquoted in PBS 10% glycerol and stored frozen at -80 ° C. Virus was thawed only once and administered to anesthetized animals at 15 ul / min by infusion as described above or by tail vein with a tuberculin syringe. Tumors and animal tissues are collected, as appropriate, for frozen sections or for fixation and embedding.
Apoptosis was analyzed by tunel assay and immunostaining by & E to determine neutrophil infiltration and associated GFP expression.

Testing of Original (dox Downregulated) AdGFP-FasL _{Tetd on} Prostate Cancer Xenografts in Balbc nu / nu Mice These experiments are performed to determine both toxicological and efficiency parameters. Specifically, increasing amounts of ¹ × 10 ⁹ to 5 × ^{10 10} pfu of AdGFP-FasL _Tetd were injected into 75-100 mm ³ tumors to measure: A) 1 dose unit or 3 dose units of the virus every 4 days The lowest dose that, after orthotopic administration, is successful for reducing tumor volume by more than 75%. Tumors were grown from CD95L sensitive PPC1, intermediately sensitive LnCAP C2-4, and the more resistant Du145 cell line. Other parameters of dosing were developed based on the results of endpoints that always resulted in tumor regression. B) Highest tolerated virus dose by upright administration (up to 5 × ^{10 10} pfu). C) At the same site or distally (6-1
2 months) To determine if the tumor reappears (C4-2). D) Highest iv (tail vein) dose at which 50% of mice survive. E) Using data from D, test the effect of doxycycline administration on animal survival curves and the duration of doxycycline protection (Balbc nu / nu mice do not have a CTL response, so adenovirus may survive long term). To do. F) Dosing of 1 μg / ml using FACS analysis
Long-term monitoring of GFP (as a GFP-FasL fusion) in the presence of x
Determine the half-life of K562 cells (CD95L resistant, see Table 1). The same set of experiments as in B1 above are performed with the Tet-inducible virus constructed as described above.

AdGFP-FasL administered (up-regulated) to normal experimental Beagle dogs _Tetu And (down-regulated) AdGFP-FasL_TetdToxicological studies of animal There are a number of animal models for PCa, but pathological and anatomical
The disease is not well represented. Human AdRSVbgal (serotype 5) adenovirus
Will infect dog epithelial cells, including prostate cancer, both in vitro and in vivo
(Andrawiss et al., Prostatic Can. Prostatic Dis. 2:
25-35, 1999). AdGFP-Fas_TetA dog (immune competent) versus an immune-deficient mouse (Balbc nu
/ nu), the human phase I of this gene therapy approach
Additional support for testing is provided.

In the following section, we will examine whether orthotopic delivery of AdGFP-FasL to the hormonal prostate is safe without minimal or collateral damage in sexually mature normal dogs. do an experiment. Purified and concentrated adenovirus (AdGFP-FasL up- or down-regulated and reporter virus AdCMVLacZ, all serotype 5) is injected by abdominal surgical means into one lobe of the dog's prostate. This approach is preferred over transrectal introduction. This is because having a direct view of the prostate may help in more accurate virus transfer in this first series of experiments. Second, due to the highly vascular nature of the dog's prostate, direct visibility allows the needle entry path to be sealed with local tissue glue and finger compression to prevent virus leakage from the injection site. . Based on these results,
3D ultrasound-guided transrectal transfer is used to mimic the proposed approach to humans.

Use virus doses of 5 × ^{10 9} , 1 × ^{10 10} and 5 × ^{10 10 in} a constant volume of 400 μl: One set of 2 dogs is given 5 × ^{10 10} pfu of AdCMV-LacZ and the spread of the virus is determined histologically. Monitoring was enabled. The dog was monitored for any signs of distress throughout the first 72 hours. Feces were collected and analyzed for virus shedding by PCR. Urine was also collected by Foley catheter and assayed for 293 cells and virus shed by PCR. On day 7, (2 for each virus dose)
Dogs) were euthanized with sodium pentobarbitur and processed as described (Andrawiss et al., Prostatic Can. Prostatic Dis. 2: 25-35, 1999). Samples of all tissues are frozen in OCT, while the rest are fixed and processed for histology (tunel, immunochemistry), or PCR using DNA extraction and virus-specific primers
And stored frozen at -80.C. LacZ expression was examined in the AdCMV-LacZ group to monitor systemic virus shedding.

Example 4: AdGFP-FasL_TETIntratumoral Inhibition of Breast and Brain Tumors in Mice
In this experiment, 10⁶MCF-7 cells were implanted on both sides of Balbc nu / nu mice. Tumor
When the tumor size reaches 5 mm in diameter, 2 x 10⁹AdGFP-F of pfu
asL_TETTo the right of the mouse or 2x10⁹pfu AdLacZ on the left, barbird
Infusion was performed for 10 minutes using a perfusion pump. In treated mice as compared to untreated mice
The tumor suppression was 80-100%. In contrast, in all tumors injected with AdLacZ
Three weeks after transplantation, the cells grew to about 2 cm in diameter. This indicates that FasL-induced apoptosis
Can be used as a new treatment for breast cancer. Similarly, 10⁶SF767 cells were implanted on both sides of Balbc nu / nu mice. Large tumor
When the size reaches 5mm in diameter, 2 x 10⁹AdGFP-FasL of pfu _TET To the right of the mouse or 2x10⁹pfu AdLacZ on left side
Infusion was performed for 10 minutes using a flow pump. Compared to untreated mice, treated mice
Tumor suppression was 80-100%. In contrast, in all tumors injected with AdLacZ,
Three weeks after planting, they grew to about 2 cm in diameter. This indicates that FasL-induced apoptosis
It shows that it can be used as a new therapy for brain tumors.

Although the invention has been described with reference to specific embodiments, such details are not to be construed as limitations on the scope of the invention, which is set forth in the following claims. Various documents are referenced in this application. The entire disclosure of these documents,
It is hereby incorporated by reference into this application to fully describe the state of the art to which this invention pertains.

[0076]

Table 1: Fas-mediated cytotoxicity in prostate cancer cells treated with anti-Fas antibody, C2-ceramide (22) hours or AdGFP-FasL _TET for 48 hours-(expressed as% cytotoxicity_SD )

* MOI 10, ^H MOI 1000. In all experiments, N = 3 (however, TSU and PC-3
N = 2) for ceramide experiments using. Bursent cytotoxicity was determined using the MTS assay. Briefly, cells were seeded in 12-well plates containing 1 ml of medium. Prior to treatment, cells were grown to 75% confluence and 500 mg / ml CH-11 anti-F
Treatment with either the as antibody, 500 ng / ml normal mouse serum or 30 μM C2-ceramide. For adenoviral transduction, approximately 1x10 ⁵ cells / well
Treated with AdCMVGFP or AdGFPFasL _TET at MOI 10-1000. For each cell line,
The positive control was left untreated and 1 ml of medium was used as a negative control. Cells were incubated for 48 hours at 37 ° C. for maximum cell killing. The medium was aspirated and replaced with 0.5 ml of fresh medium + 100 μl of CellTiter 96 AQueous One Solution reagent per well. The cells were incubated for an additional 1-3 hours at 37 ° C. After incubation, 120 μl of medium was placed in a 96-well plate and the absorbance reading at 490 nm was performed on a Vmax kinetic microplate reader. Percent cytotoxicity was calculated as follows:% cytotoxicity = [1- (OD of experimental well / OD of positive control well)] × 100 For ceramide assay, 1 × 10 ⁴ cells were plated in a 96-well plate. Sowed. The next morning, the cells were washed and 100 μl of 30 μM dihydro- or C2-ceramide (10 mM in ethanol).
(Diluted from stock) in serum-free RMPI 1640. Twenty-four hours later, 20 μl of Celltiter 96 AQuesous One Solution reagent was added to each well and the plate was incubated for an additional 1-4 hours. Absorbance and% cytotoxicity were measured as described above. In each experiment, data points were taken in triplicate.

[Brief description of the drawings]

FIG. 1A schematically shows a pLAd-C.tTA vector. This plasmid is
It contains the leftmost 450 bp of Ad5, followed by the CMVie enhancer / promoter inserted into the MCS and the tTA gene from pUHD15-1. The adapter contains restriction sites XbaI, Avr2 and SpeI, which all generate cohesive ends compatible with XbaI. After assembly into the rAd vector, E1A polyA is used for efficient tTA expression. A similar strategy was used to construct pLAd vectors containing other transgenes.

FIG. 1B schematically shows a pRAd.T.GFsL vector. This plasmid contains the Ad5 (sub360) sequence from the unique EcoRI site (27333 bp) to the right ITR (35935 bp), with E3 and E4 deleted (Orf6 of E4 remains). This schematic is TR
Figure 3 shows a controllable FasL-GFP expression cassette consisting of the E promoter, FasL-GFP fusion protein and bovine growth hormone (BGH) polyA. This cassette has 35810bp
Was inserted into the MCS.

FIG. 1C shows rAdFasL-GFP_TETThe vector is shown schematically. rAd / FasL-GFP _TET Is shown in FIG. 1C. Reading frame of GFP and FasL
The joint area was enlarged. Other rAd vectors were made with a similar strategy.

FIG. 2 shows rAd vector titer and F in 293 cells and 293CrmA cells.
4 is a graph showing a comparison of asL activity. 10 ⁴ 293 cells or 293 in a 12-well plate
CrmA cells were plated, and one day later, r-Ad / FasL, rAdFasL-GFP _TET or rAd / LacZ was infected at MOI5. Cells were collected and lysed 48 hours after transduction. Lysate
Titration was performed on 293CrmA cells, and PFU / ml was measured. The results represent the mean and mean error of two independent sets of experiments.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) C12N 5/10 A61K 37/02 (72) Inventor Norris James S United States South Carolina 29464 Mount Pleasant Cascue Province 1010 F term (reference) 4B024 AA01 CA04 CA20 DA02 DA03 EA02 EA04 FA02 GA13 HA17 4B065 AA91Y AA93X AB01 AC14 AC20 BA02 BA05 CA44 CA46 4C084 AA02 AA13 CA53 DC50 NA14 ZB262 4C087 AA02 BC83 CA12 CA50 NA14 Z26

Claims (50)

[Claims] 1. A method for killing Fas ⁺ tumor cells, comprising the steps of:
A nucleic acid encoding a Fas ligand (FasL), whereby said second tumor cell expresses said nucleic acid to produce FasL, thereby said Fas ⁺ tumor cell and said second expressing FasL. The interaction of said Fas ⁺ tumor cells with said tumor cells causes apoptosis in said Fas ⁺ tumor cells, thereby killing said Fas ⁺ tumor cells. 2. The method of claim 1, wherein FasL is membrane-bound. 3. The method of claim 2, wherein the interaction of the Fas ⁺ tumor cell with a second tumor cell expressing FasL comprises a cell-cell interaction. 4. The method according to claim 1, wherein the tumor is a solid tumor. 5. The method of claim 1, wherein FasL is a fusion protein. 6. The method according to claim 5, wherein the fusion protein comprises FasL and green fluorescent protein. 7. The method of claim 5, wherein the fusion protein comprises FasL and a regulatory protein. 8. The method according to claim 1, wherein the nucleic acid encoding FasL also comprises a control region capable of controlling the expression of a sequence encoding FasL. 9. The method of claim 8, wherein the control region comprises a Tet operon. 10. The method of claim 1, wherein the nucleic acid encoding the Fas ligand (FasL) is introduced into the second tumor cell by a vector. 11. The method according to claim 10, wherein the vector is a viral vector. 12. The method according to claim 11, wherein the viral vector is an adenovirus vector. 13. The method of claim 1, wherein the viral vector is a vaccinia vector.
2. The method according to 1. 14. The method according to claim 11, wherein the viral vector is a retroviral vector. 15. The method of claim 1, wherein FasL is expressed using a tissue-specific promoter. 16. The method of claim 15, wherein the tumor cell is a prostate tumor cell and the tissue-specific promoter comprises a prostate-specific promoter. 17. The method of claim 15, wherein the tumor cells are breast cancer cells and the tissue-specific promoter comprises a breast-specific promoter. 18. The method of claim 15, wherein the tumor cell is a colon tumor cell and the tissue-specific promoter comprises a colon-specific promoter. 19. The method of claim 15, wherein the tumor cells are brain tumor cells and the tissue-specific promoter comprises a brain-specific promoter. 20. The method of claim 15, wherein the tumor cell is a kidney tumor cell and the tissue-specific promoter comprises a kidney-specific promoter. 21. The method of claim 15, wherein the tumor cell is a bladder tumor cell and the tissue-specific promoter comprises a bladder-specific promoter. 22. The method of claim 15, wherein the tumor cell is a lung tumor cell and the tissue-specific promoter comprises a lung-specific promoter. 23. The method of claim 15, wherein the tumor cell is a liver tumor cell and the tissue-specific promoter comprises a liver-specific promoter. 24. The method of claim 15, wherein the tumor cells are thymic tumor cells and the tissue-specific promoter comprises a thymus-specific promoter. 25. The method of claim 15, wherein the tumor cell is a gastric tumor cell and the tissue-specific promoter comprises a gastric-specific promoter. 26. The method of claim 15, wherein the tumor cell is an ovarian tumor cell and the tissue-specific promoter comprises an ovary-specific promoter. 27. The method of claim 15, wherein the tumor cell is a cervical tumor cell and the tissue-specific promoter comprises a cervical-specific promoter. 28. The prostate-specific promoter is PSA, ΔPSA, ARR2PB and
16. The method of claim 15, wherein the method is selected from the group consisting of PB. 29. The method of claim 1, which is performed ex vivo. 30. The method of claim 1, which is performed in vivo. 31. The method of claim 1, which is performed in vitro. 32. A control comprising: (A) a transactivator protein that binds to a tet-responsive transactivation expression element; and (B) a nucleic acid encoding a control element comprising a tet-responsive transactivation expression element. A controllable expression vector, wherein a nucleic acid encoding the protein to be expressed may be inserted downstream of the control element. 33. The vector according to claim 32, wherein the vector is a viral vector. 34. The adenovirus vector of claim 33, wherein the nucleic acid encoding the nucleic acid transactivator protein and regulatory elements is directed to opposite ends of the vector. 35. The method of claim 3, wherein the viral vector is a vaccinia vector.
3. The method according to 3. 36. The method according to claim 33, wherein the viral vector is a retroviral vector. 37. The method of claim 32, wherein the expressed protein is fused to a reporter. 38. The method of claim 33, wherein said reporter is a green fluorescent protein. 39. The method of claim 34, which is pAd _TET . 40. A method for killing Fas ⁺ tumor cells, comprising introducing the vector Ad / FasL-GFP _TET into a second tumor cell, whereby the second tumor cell is transformed into Fas.
express sL, whereby interaction of said second tumor cells expressing Fas ⁺ tumor cells and FasL is to cause apoptosis in the Fas ⁺ tumor cells, thereby Fa
How to kill s ⁺ tumor cells. 41. A vector for controlled expression of FasL, comprising a nucleic acid encoding FasL operably linked to a transcription control sequence. 42. The vector according to claim 37, wherein the transcription control sequence is inducible. 43. The vector according to claim 37, wherein the transcription control sequence can be suppressed. 44. The vector according to claim 37, which is a viral vector. 45. The vector of claim 40, which is an adenovirus vector. 46. The vector according to claim 40, which is a vaccinia vector. 47. The vector according to claim 40, which is a retroviral vector. 48. The vector according to claim 37, wherein the transcription control sequence is a tet-responsive transcriptional activator expression element. 49. The vector further comprising a nucleic acid encoding a transactivator protein that interacts with a tet-responsive transactivator expression element.
4. The vector according to 4. 50. The vector according to claim 45, which is Ad / FasL-GFP _TET .