CA2189032A1 - Enhancing the sensitivity of tumor cells to therapies - Google Patents

Abstract

A method for enhancing the effect of a cancer the introducing wild-type therapy sensitizing gene activity into tumor cells having mutant therapy sensitizing gene activity and subjecting the tumor cells to a cancer therapy such as chemotherapy, radiotherapy, biological therapy including immunotherapy, cryotherapy and hyperthermia.

Description

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DESCRIPTION
ENHANCING T~E ~ N~l'l'LVl'l ~' OF Tl~MOR CELLS TO THERAPIES
BACKGROUND OF THE INVENTION
This application is a = continuation- in-part application of IJ.S. Application No. 08/236,221, entitled "ENHANCING THE SENSITIVITY OF TUMOR CELLS TO THERAPIES, "
5 filed May 24, 1994, which is a continuation-in-part application of U.S. Application No. 08/236,221, entitled "ENHANCING THE SENSITIVITY OF T~MOR CELLS TO THERAPIES, "
filed April 29, 1994; the disclosures of the above two parent applications are incorporated herein by reference.
This invention relates to cancer therapies. In particular, this invention relates to a method of enhancing the ef f ect of cancer therapies .
The mainstays of cancer therapy have been surgery, radiation, chemotherapy and biological therapy (see 15 generally, Com~rehensive Textbook of Oncoloq~r, ed. A.R.
Moossor, et al. (Williams & Wilkins, 1991); Cancer:
~rinciples An~l ~ractice of oncoloqY, ed. Vincent T.
DeVita, Jr., Samuel EIellman, Steven A. Rosenberg 4th ed.
(philAt11'1~1~;A: J.B. Lippincott Company, 1993)) . Radiation 20 therapy, which is also called radiotherapy, uses high energy x-rays, electron beams, radioactive isotopes and other forms of radiation known to those skilled in the art to kill cancer cells without ,~ ee~l; r~ tolerable doses to normal tissue.
Chemotherapy refers to the use of drugs to kill cancer cells. There are several classes of chemotherapeutic agents with different modes of action.
For example, many anti-metabolites share structural similarities with normal cellular, , ^ntC and they wo 9~/30002 21~ ~ 0 3 ~ r~
.J ~ --exert their effects by inhibiting - normal cellular processes. Many alkylating agents are effectiYe against proliferating and non-proliferating cancer cell populations. In general, these drugs bind with the cell's 5 DNA in various ways to prevent accurate replication and/or transcription. Many anti-tumor antibiotics insert themselves into DNA where they induce breaks in the DNA or inhibit transcription. In general, alkaloids inhibit the function of chromosome spindles necessary for cell 10 duplication. Hormone agents such as tamoxifen and f lutamide inhibit the growth of some cancers, although their mechanism of action is not completely understood.
In general, biological therapy utilizes agents which are derived ~rom or which benef icially modulate hosi~
15 biological processes. Interferon-alpha and interleukin-2 are two examples of biological therapy agents currently utilized in cancer therapeutics.
Some cancer therapies use modifying agents to enhance the effect of standard treatment methods (see generali~y, 20 Coleman CN, Glover D,J, Turrisi AT. ~Radiation and chemotherapy sensitizers and protectors. " Chemothera~Y:
Pr;n~-;nles and T~ractice. Phil~ lrhi~: W3 Saunders, 225-252, 1989). Chemical modifiers are usually not cytotoxic by themselves but modify or enhance the response of tumor 25 tissue to a standard therapy, e.g., radiation therapy, The eifectiveness of a sensiti2er is generally expressed as the sensitizer ,~rh~n~ ~nt ratio (SER) . The SER is the dose of therapy reS~uired to produce a defined level ~of killing without sensitizer divided by the dose of therapy 30 re~uired for the same level of cell killing wit~ the sensitizer Two examples of clinical approaches to radiation and chemotherapy modification are hypoxic-cell sensitization and thiol depletion. The aamage produced by radiation and 35 alkylating agents is in part~ related -to free radical formation in DNA and other critical cellular macromolecule . Thiol compounds prevent DNA f ree radicals .. . , .. , .. , _ . _ _ _ _ _ _ _ _ _ . . _ ~ w0ssl30002 2~89~32 .~ iC5 /~
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or repair them. If the DNA free radical is exposed to oxygen or an oxygen-mimetic hypoxic cell sensitizer, such as a nitroimidazole, the damage to DNA is fixed, i.e., made irreversible by oxidation. Depletion of thiols by 5 drugs such as b~tll-~ni nf~ sulfoximine (sSo) also increases the toxicity from radiation and radiomimetic chemotherapeutic agents such as alkylating agents.
SrJMMARY OF TH~ INVENTION
This invention features a method for treating cancers 10 which are characterized by loss of wild-type therapy-sensitizing gene activity. The method ' nr~ c in-troducing into tumor cells a source of wild-type therapy-sensitizing gene activity and subj eçting the cells to a cancer therapy. The cancer therapies whose effect may be 15 enhanced by thic invention include, but are not limited to, radiotherapy, chemotherapy, biological therapy including immunotherapy, cryotherapy and hyperthermia.
The cancers that can be treated by this invention include, but are not limited to, carcinoma, sarcoma, central 20 nervous system tumor, r~l~n~ tumor, leukemia, lymphoma, hematopoietic cancer, ovarian carcinoma, osteogenic sarcoma, lung carcinoma, colorectal carcinoma, hepatocellular carcinoma, glioblastoma, prostate cancer, breast cancer, bladder cancer, kidney canc~r, pancreatic 25 cancer, gastric cancer, esophageal cancer, anal cancer, biliary cancer, urogenital cancer, and head and neck cancer.
Thus, this invention features a method of ~nll~nr;ng the effect of a cancer therapy by delivering a source of 30 wild-type therapy-sensitizing gene activity into a tumor cell characterized by 10FS of wild-type therapy-sensi-tizing gene activity and subj ecting the tumor cell to the cancer therapy.
By "delivering" is meant the use of methods known to 35 those skilled in the art for administering drugs to a mammal. These methods include, but are not limited to, ~18~32 Wo 9s/30002 , r~
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delivering a gene or cDD~A of the gene to a tumor cell in a vector, delivering a gene or cD~A oi the gene to a tumor cell by coupling with a vixus capsid, delivering a gene-or cDNA of the gene to a tumor cell by coupling with a ligand or by encapsulation in a liposome, correcting a tumor cell gene point mutation or in~ertion mutation or deletion mutation by recombination techni~ue~, or delivering protein to cells either directiy or in hybrid molecules or by encapsulation methods. Other materialq and methods that result in the presenc-e o~ wild-type therapy-sensitizing gene activity within a tumor cell, such as those described in J. Sambrook, E. F. Fritsch, and T. Maniatis, Moleculax Clnn;n~T: A T~hnrator~r ~nllAl, 2 Ed., Cold Spring Harbor=Laboratory Pres6, Cold Spring Harbor, New York, 1989, and Ausubel et al , Current Protocols i n Moleclll Ar Biolo~, 1994, incorporated by re~erence herein, may also be utilized.
By ~therapy-sensitizing gene" is meant a gene or gene product whose loæs of normal function or regulation 2C renders cancer cells more~ res~stant to ~ therapy.
Restoration of therapy-sensiti2ing gene function results in increased sensitivity of cancer cells to therapy. In particular, it is meant a gene which may promote apoptosis or whose altered function or:-regulation contributes to tumorigenesis and therapy resistance~ including, but not limited to, tumor suppressor genes such as p53; cell cycle regulatory genes such as cyclins, cyclin rlPrPn-lPnt kinases (Steel, M., Lancet 343:931-932~, I994), mitogen activated protein kinases (Blenis, J., Proc. Natl. Acad. Sci.
90:5889-5892, 1993: Marshall, C.J., Nature, 36~:68-6, 1994), inhibitors of cell cycle genes such as pl6 (multiple tumor suppressor 1) (~amb et al., Science 264 :436-490, 1994); and apoptosis genes such as~ fas.
A prospective therapy-sensitizing gene may be identified by the method disclosed in the detailed description of the invention for the therapy-sensitizing gene p53, by substituting p53 with the prospective .. _ .. _ : . . . _ . . .. . , . _ : : . _ _ _ _ _ _ _ _ _ _ _ wo gs/30002 ~ 1 8 ~ ~ ~ 2 P~

candidate gene. For example, tumor cellg are first char~rt,-~1 7~rl by routine serluence analysis or other diagnostic assays known to those skilled in the art to contain a mutated gene or mutated messenger RNA encoding 5 the r~ntli ~te therapy-sensitizing gene to be te~ted. The normal wild-type coding sequence for such a gene is then subcloned by standard methods known to those skilled in the art into a suitable eukaryotic expression vector:
containing a selectable marker gene such as the neomycin 10 resistance gene. For example, the normal coding ser~uence can be amplified by polymerase chain reaction (PCR) from the cDNA of the messenger RNA poplllAtirn of normal fibro-blasts, using appropriate primers to the 3 ~ and 5 ~ ends of the codiIlg ser~uence. Following subcloning into an 15 appropriate eukaryotic expression vector, the vector rr,ntil1n1ng the normal candidate therapy-gensitizing gene of interest can be transf ected into the tumor cells expres~ing the mutated form of the gene. Transfection can be performed by a number of methods known to those skilled 20 in the art, including but not limited to calcium phosphate transfection, lipofection (which uses cationic liposomes), electroporation, and DEAE-dextran facilitated transfection. The transfected cells are expanded in the presence of the appropriate selection agent, such as 25 neomycin. Once the clones have been expanded and se-lected, they are: 1) characterized to document expression of the candidate therapy-s~nc1t171nrJ gene by routine methods known to those skilled in the art and 2 ) tested for sensitivity to chemotherapeutic drugs and/or radiation 3 0 therapy in standard growth assays or clonogenic assays as described in the detailed description of the invention for p53. Increased sensitivity to therapy in multiple clones expressing the candidate therapy-sensitizing gene compared to parental tumor cells indicates that the transfected 35 gene is a therapy-sensitizing gene.
By "wild-type therapy-sensitizing gene activity~ is meant the activity of a therapy-sensitizing gene in a Woss/30002 2l8~n32 P~ J~

normal, non-neoplastic cell. Speciically, it means the ability of the protein or a portion of the protein encoded by the therapy-sensitizing ge~e to sensitize a tumor cell to a cancer therapy. A therapy-sensitizing protein having 5 one or more "mutations" that does not affect the therapy-sensitizing ability thereof is still considered "wild-type~ for the purpose of this inventio~. The activity is embodied in the protein expressed from the wild-type therapy-sensitizing gene coding se~nce~ o~=- portions l o thereo .
By "loss of wild-type therapy-6ensitizing gene activity" is meant the absence or alteration of normal therapy-sensitizing gene activity such as the presence~ of a mutant therapy-sensitizing protein, the absence of a 15 wild-type therapy-seneitizing protein or an inhibited wild-type therapy-sensitizing - protein in a cell. The difference from normal in therapy-sensitizing activity may be caused by a genetic difference~at one or more gene~tic loci. The genetic differences may be of several~different 20 types, including, but not limited to, a point mutation where a single base pair is changed to another base pair, an insertion of one or more base pairs, a deletion of one or more base pairs up to the full length of the therapy-sensitizing gene, fusion~ of one gene to another, 25 introduction of additional copies of an existing therapy-sensitizing gene, introduction of one or more copies of a non-therapy-sensitizing gene ~ot formerly present, other alterations of gene transcription, translation and protein function known to those skilled in the art~, or any 3 0 combination of the above .
By "tumor cell" is meant a cell arising in an animal in vivo which is capable o~: undesired proliferation ~-or abnormal persistence or abnormal invasion of tissues.
In a pref erred embodiment, this in:vention introduces 35 a therapy-sensitizing portion of a wild-type therapy-sensitizing protein into a tumor cell, and subjects said tumor cell to a cancer therapy.

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By "therapy-sensltizing portion of a wild-type therapy-sensitizing protein~ is meant the portion of a wild-type therapy-sensitizing protein that has the ability to sensitize a tumor cell expressing mutant therapy-sensi-tizing activity to a cancer therapy. The therapy-sensi-tizing portion of a wild-type therapy-sensitizing protein may be delineated by routine sequence analysis known to those ekilled in the art, including, but not limited to, deletion mutations, point mutations and such as described in Unger et al., "Functional domains of wild-type and mutant therapy-sensitizing proteins involved in transcriptional regulation, transdominant ; nh; hi t i on, and transformation suppression,~ Molec. Cell. Biol. 1~:5186-94, 1994 and J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloninq: A Laboratory Manual, 2 Ed., Cold Spring Harbor Laboratory Press, Cold Spring ~arbor, New York, 1939, and Ausubel et al., Cuxrent Protocols in MQlecular Bioloqy, 1994, incorporated by referenc~ herein.
In another pref erred embodiment, this invention introduces a wild type therapy-sensitizing gene, its cDNA, or a portion thereof encoding the therapy-sensitizing gene activity into a tumor cell, expresses the therapy-sensitizing gene, and subjects the tumor cell to a cancer therapy .
In a further preferred embodiment, the therapy-sensitizing gene, its cDNA, or a portion thereof is intro-duced into the tumor cell by a viral vector selected from the group ;n~ ri;n~, but not limited to, adenovirus vector, retroviral vector, adeno-associated virus vector, herpes virus vector, vaccinia virus vector and papilloma virus vector. The therapy-sensitizing gene, its cDNA, or a portion thereof can also be introduced into the tumor cell by coupling to a virus capsid or particle through polylysine: bridge, conjugating to a ligand such as an asialoglycoprotein or encapsulation in a liposome. The means of introduction into an animal include, but are not limited to, direct injection or aerosolized preparation, .

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intra-arterial infusion, intracavitary illfusion and intravenous infusion.
In some instances, the mutated or abnormal therapy-sensitizing activity may reflect ilhnnrr=lly=increased gene expression~ or gene product activity which may be down regulated by transdominant-negative mutants or other down regulation~ methods known to thDse skilled in the art .
other features and advantages of the.invention will be apparent from the following detailed description of the invention, and from the claims.
RRTr~r DE~CRIPTION OF FIGURES
Figure 1 shows cisplatin sensitivity of T~8G
glioblastoma cells (closed circles) and the same cells with wild-type p53 expressed therein, T98Gp53 cells (open circles ) .
Figure 2 show6 radiation sensitivity of T98G
glioblastoma cells (upper curve~ and T38Gp53 cells (lower curve ) .
D~T~Tr,r n DESCRIPTION OF THE INVENTION
This invention features a new method of ~-nhAnr1ng the ef ect o cancer therapy by introducing into a tumor cell a source o therapy-sensitizing activity (through the introduction of a gene, a cDNA or a protein), which has been lost f rom the tumor cell . Examples of such activitïes include but are no~ limited to the~~fas gene, the retinnhl;~tnm~ gene, the p53 tumor suppress~r gene and other tumor suppressor genes, cell cycle regulatory genes and apoptosis genes.
The ~ S~nsitizin~ Gene r~S3's Relevance to l~uman Cancer JJoss of :normal p53 function, either thrDugh mutation, deletion or inactivation, is one of the most frequently encountered alterations in human cancer, occurring in some 50~ of human cancers (Nigro et al., "Mutations in the p53 gene occur in diverse human tumor type6," Nature, 342:705-~W095130002 2~8~n3~
g 708 (1989); T~k~hA~h; et al., ~p53: A frequent target for genetic abnormalities in lung cancer, ~ Science, 246:491-194 (1989) ) . In addition, some studies suggest that individuals with inherited mutations of p53 are 5 predisposed to a variety of cancers (Malkin et al., "Germ line p53 mutations in a familial syndrome of brea6t cancer, ~ , and other neoplasms, " Science, 250 :1233-1238 (1990); Srivastava et al., "Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-:
10 F~ ; syndrome,~ ~, 348:747-749 (1990); ~i et al., "A cancer family syndrome in twenty-four kindreds, " Cancer Res., 48 :s358-5362 (1988) ) . It has been ehown that the tumors of the6e individuals have lost the wild-type p53 allele which is reminiscent of the 108s of heterozygosity 15 of the rPt;n~-hl~:toma tumor suppressor gene in retinoblastoma and other tumors (Knudson, A.G. ~Mutation and Cancer: Statistical study of retinoblastoma, " Proc.
Natl. Acad. Sci.. rTc~ 68:820-823 (1971); Comings, D.E~. "A
general theory of carrinr~on~ Proc. Natl. Acad. Sci., 20 ~, 70:3324-3328 (1973)).
Some studies disclose that in vitro introduction of the wild-type p53 gene into a variety of different tumor lines results in down regu:Lation of cell proliferation in culture or suppression of the tumorigenic phenotype upon 25 reimplantation of the cells in vivo. These studies include tumor cells derived from glioblastomas (Mercer et al., "Negative growth regulation in a glioblastoma tumor cell line that c~7nr~1t;~n~11y expresses human wild-type p53," Proc. Natl. Acad. 2ci., ~SA, 87:6166-6170 (1990)), 30 colon carcinoma (Baker et al., "Suppression of human colorectal carcinoma cell growth E~y wild-type p53, Science, 249:912-915 (1990)), osteosarcoma (Diller et al., ~p53 functions as a cell cycle control protein in osteosarcomas, ~ Mol . Cell . Biol., 10 :5772-5781 (1990);
35 Chen et al., "Genetic mechanisms of tumor suppression by the human p53 gene,~ Science, 2s0:1576-1580 (1990)), leukemia (Cheng et al., "Suppression of acute lympho-W09s/30002 2l8~n3~
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blastic leukemia by the human wild-type p53 gene, " Cance~
Res., 53:222-226 (1992)), and lung carcinoma (TAkAhA~:h- et al., "Wild-type but not mutant p53 suppresses the growth of human lung cancer cells bearing multiple genetic 5 lesions, n Cancer ReG., 52:2340-2343 (1992~ ) . However, in vitro introduction of the wild-type p53 gene ~=into non-malignant cells does not result in the reduced cell growth as seen in tumor cell lines ~Baker et al., supra).
Not alI tumor cells with p53 mutations display 10 significant down regulation of proliferation by wild-type p53 expression. ~Iinds et al. Cell Growth and Differen-tiation 1:571-80, (1990) disclosed that not all p53 mutants result in equivalent phenotypes. Michalovit~ et al . Cell 62: 671-68~, (1990) disslosed that some mutants of 15 p53 may be 1( nAnt to wild-type p53 with regard~ to growth regulation. Expression of wild-type p53 does not affect growth properties of some tumor cell~ lines, including human papillomavirus-expressing cell=lines, ~and ~673 rhabdomyosarcoma cells (Chen et al., Oncogene 6:1799-1805, 20 lggll. In those cases wheæ p53 was reported to suppress cell proliferation, the effect was sometimes small (Cheng et al ., 1 9 9 2 , supra ) .
Furthermore, the method of using wild-type~53 aIo-ne to down-regulate tumor cells requires stable wild-type ~53 25 expression in tumor cells. In studies o a temperature sensitive mutant of p53, it has been ~ observed that the suppressive effect of wild-type p53 on the proliferation of transformed cells was lost when wild-type p53 expression ceased (Michalovitz et al., l99o~ supra).
30 Since the most efficient gene transfer approaches pres-ently available provide onIy transient expressi--on of p53, this limits the efficacy of therapy with p53 alone.
p53 function is highly complex and has been implicated in a variety of celIular processes includlng 35 proliferation (Baker et al., Science ~: 912-915,199-Michalovitz et al., Cell 62: 671-680,~ 1990), differentiation (Shaulsky et al., Proc. Natl. Asad. Sci.

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88: 8982-8986, 1991), programmed cell death (i.e., apoptosi6) (Yonish-Rouach et al., Nature 352: 345-347, 1991), cellular senescence (Shay et al., Exp. Cell Re6earch. 196: 33-39, 1991), DNA binding (Kern et al., Sclence 252: 1708-1711, 1991; Bargonetti et al., Cell 65:
1083-1091, 1991), and DNA damage-induced Gl arrest (Kastan et al, Cancer Research 51: 6304-6311, 1991; Kuerbitz et al., Proc. Natl. Acad. Sci. USA 8~: 7491-7495, 1992) .
With regard to cancer therapy, the involvement of p53 in DNA damage-induced Gl arrest -i6 one of its most provocative roles.
Wild-type and mutant p53 genes have been transferred into tumor=cells lacking endogenous p53. When these cells were exposed to gamma irradlation, the expression of wild-type p53 led to transient cell cycle arrest at the Gl/S
phase boundary (Kastan et al., "Participation of p53 protein in the cellular response to DNA damage, " Cancer Res., 51.6304-6311 (1991); Kuerbitz et al., "Wild-type p53 i8 a cell cycle checkpoint determinant following irradiation," Proc. Natl. Acad. Sci., USA, 89:7491-7495 (1992); Yoni6h-~ouach et al., "Wild-type p53 induce6 apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6," Na~ure, 352:345-347 (1991). Cells which lacked p53 or which expressed mutant p53 did not arrest (Kastan et al., 6upra; Kuerbitz et al., f~upra) .
It has been proposed that p53 plays an important checkpoint function by preventing entry into S phase until DNA damage is repaired (Vogelstein et al., Cell 70: 523-526, 1992) . Thus, the outcome of DNA damaging radiation or chemotherapy on cancer cells may be affected by the expression of mutant or ~oild-type p53.
In this regard, several lines of evidence suggest that cancer cells which have lost wild-type p53 function are more sensitive to DNA damaging drugs and radiation. By analogy to similar checkpoints in yeast, failure of p53 induced Gl arre6t could enhance cell destruction ~y ~8~)3~
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preventing repair of potentially lethal DNA damage prior to cell division (Vogelstein et al., .su~ra).
Vogelstein et al, su~7ra,~ stated: _ tumor cells are often more sensitive to ~DNA-damaging agents such as those used in radiation and chemotherapy; this sensitivity may be a beneficial side effect of the loss of~ p53:
function, which would otherwise limit cell _ death. p53 mutation6 may therefore constitute one of the few oncogenic alterations that increase rather than decrease the sensitivity of cells to antitumor agents.
This view is supported by studies demonstrating increased sensitivity of tumor cells to radiation and chemotherapy following mutated p53 gene transfer~(Petty et al ., "Expression of - the p53 tumor suppressor gen-e product is a determinant of chemosensltivity, " Biochem. Biophys.
Res. Comm. 199:264-270, I994, not admltted to~ be pr1or art ) .
~owever, other ~ etudies performed with normal hematopoietic cell6, fibroblasts, and gastrointestinal cells from p53 null transgenic mice indicated ~ a re~uirement for p53 in apoptosis (~owe et al., Nature 362:
847-849, 1993; Clarke et al., Nature 362: 849-852, 1993;
Lotem J and Sachs L, Blood 82- 1092-1096, 1993; 3.owe-et al, Cell 74: 957-967, 1993; and Merritt et ai, Cancer Research 54:614-617, 1994, not admitted to be prïor arF).
In the6e etudie6, normal ceIls lacking p53 were more resi6tant to apopto6is following exposure to ra~iation~or DNA damaging d~gs. Similarly, a study of Burkitt's lymphoma cell lines revealed that some but not all cell lines with wild-type p53 gene configuration6 were more sensitive to radiation (O'Connor et al., Cancer Research 53: 4776-4780, 1993, not admitted to be prior art~ .
~owever, evaluation of head and neck cancer cell line6 6howed no correlation between radiation 6ensitivity and expre6sion of either endogenous wild-type or mutant p53 (Brachman et al, Cancer Research 53: 3667-3669,-1993, not admitted to be prior art).

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Lowe, et al., "p~3-dependent apoptosis modulates the cytotoxicity of anticancer agents, " Cell, 74:957-967, 1993 (not admitted to be prior art) stated:
p53 -def icient mouse embryonic iibroblasts were used to examine systematically the requirement for p53 in cellular sensitivity and resistance to a diverse group of anticancer agents. These re6ults demonstrate that an oncogene specifically the adenovirus ElA gene, can sensitize f ibroblasts to apoptosis induced by ionizing radiation, 5-fluorouracil, etoposide, and adriamycin. Furthermore, the p53 tumor suppressor is required for efficient execution of the death program.
Lotem and Sachs, "Hematopoietic cells from mice deficient in wild-type p53 are more resistant to induction of apoptosis by some agents, n Blood, 82:1Q92-1096 ~1993) ~not admitted to be prior art) stated:
In normal f ibroblasts, irrad$ation and other DNA-damaging agents induce the expression of wild-type p53 and this induction of wild-type p53 arrests cells at a control point in G1. It was suggested that this G1 arrest is re~uired for DNA repair before the onset of DNA
replication to prevent the propagation of DNA
damage. Fibroblasts from p53-deficient mice lost this G1 control, continued the cell cycle after irradiation, and thus propagated the DNA
damage. Our results show that, under conditions of high concentration of viability factors, there was no difference in the number of myeloid colony-forming cells in mice with or without wild-type p53. However, when myeloid progenitor cells had only a low concentration of viability factors such as GM-CSF, IL-l~ ~IL-3, IL-6, or SCF, or when apoptosis was induced in these cells by irradiation or heat shock, cells from p53-deficient mice had a higher viability. The comparison of mice homozygous and heterozygous ior p53 deficiency showed that the loss of one allele of wild-type p53 was sufficient for increased resistance to the induction of apoptosis. The higher resistance to induction of apoptosis in p53 -def icient ~ mice was also found in irradiated thymocytes, but not in thymocytes treated with the glucocorticoid dexamethasone or in mature peritoneal granuloc-ytes. The degree of resistance in irradiated myeloid progenitors and thymocytes was related to the dose of wild-type p53.

W0 95/30002 ~ 1 8 ~ 0 3 2 1'4 Hence, the effects of mutant ~and wild type p53 ~on chemotherapy and radiation: sensitivity are unclear from these previous inve6tigations and none of these earlier studies addressea the effects of wild-type ~p53 gene 5 transfer~ on treatment sensitivity in tumor ceils expressing endogenous =mutant p53. ~ =
~nh~nrin~T the Effect of a Cancer Thera~
Thiæ invention features a new method for~enhancing the effect of a cancer therapy by introducing into tumor 10 cells a source of wild-type therapy-sensitizing gene activity before subjecting the tumor cellg to therapy.
Using p53 as an example of therapy-sensitizing gene, this invention can be carried out as follows:
First, a patient ' 8 tumor is determined to contain a 15 p53 mutation by standard diagnostic methods. Wild-type p53 activity such as a portion of p53 proteln having therapy-sensitizing activity or a gene expression vector encoding said portion of p53 protein is then introduced into the tumor cells. This renders the tumor cells with 20 the p53 mutation more sensitive to a cancer therapy administered during the period of wild-type p53 activity.
The cancer therapies whose effect may be enhanced by this method include, but are not limited to, radiotherapy, chemotherapy, biological therapy such as immunothera~y, 25 cryotherapy and hyperthermia. ~
In a cell with mutated~ therapy-sensitizing gene activity such as mutant p53 protein, unrepaired DNA damage may not block entry into S phase or trigger apoptosis.
Without being bound by any theory, applicant believes that 30 tumor cells which have endogenous mutant therapy-sensitizing gene ~activity and which have been restored with wild-type therapy-sensitizing actiyity such as wild-type p53 gene or ~protein would~be particularly sensitive to induction of ~apoptosis by therapeutic modalities given 35 the ;ntr;n~::;c susceptibility of tumor cells to genomic damage and an overloaded or impaired repair~ process . The ~ WO95130002 2~ ~n32 P~

presence of wild-type therapy-sensitizing gene activity in the tumor cells would sensitize such cells to these DNA
rl~ in~ agentæ, and probably also to a variety of other therapeutic modalities which may induce apoptosis.
- 5 The method of ~, ' ;n;n~ p53 sensit;7~t;nn therapy with other therapy is more effecti~re than either therapy alone. When exogenous wild-type p53 activity i8 introduced into a tumor cell, lower doseæ of drugs or radiation are needed to kill the cell, and the therapeutic window of concentrations over which drugs or radiation can be administered without toxicity is increased. In contrast to p53 gene therapy alone, which requires sustained p53 gene expression for tumor suppression, the combined effects of p53 sensitization therapy with other treatments requires only transient existence of a therapy-sensitizing portion of a wild-type p53 protein in the tumor cell during the treatment period to kill the tumor cell . Thi6 method also improves the ef f icacy of biological therapies, including, but not limited to, immunotherapies, such as passive immunotherapies (e.g., i~nt;hnrl; es); adoptive immunotherapies involving the administration of activated immune system effector cells;
active immunotherapies involving ; ; 7~tion to induce anti-tumor immunity; therapies mediated by various cytokines, including, but not limited to, interleukins such as I~-2, I~-6, II.-7, Il,-12, tumor necrosis factors, tumor growth factors, interferons, growth factors such as GM-CSF ana G-CSF by increasing tumor cells~ sensitivity to these cytokines or to the efiector mechanisms of the immune system activated by these cytokines. Furthermore, the claimed p53-mediated sensitization therapy makes tumor cells better targets for the immune system by restoring the apoptotic pathways required for killing by cytotoxic immune cells, including, but not limited to, cytotoxic T
cells, lyl ~nk;n~ activated killer cells, natural killer cells, macrophages, monocytes, and granulocytes.

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The therapy-sensitizing activity may be embodied in a portion or portions of wild-type p53 gene~protein. A
therapy-sensitizing portion may be delineated by routine mutation analysis, such as point mutations and deletion 5 mutations, known to those skilled in the art.
Small molecules which mimic the wild type therapy sensitizing gene product activity may also be employed to enhance cancer therapy, including, but not limited to, peptide6, modi~ied peptides or organic~chemical compounds.
10 Other useful agents include small molecules which bind to mutated therapy sensitizing gene products and serve as allosteric regulators i~ducing a conformational change which establishes the wild-type therapy-sensitizing activity of that gene product.
- Because p53 or other therapy-sensitizing gene mutations have been observed in virtually every cancer mi n~, this invention has very broad application. In a preferred embodiment, tumors that are localized can be treated by direct delivery of a porti~n of the~wild-type 2 o p53 gene encoding the therapy- sensitizing activity to the tumor cells, using presently available gene delivery vehicles, including, but not limited to, infection by p53 adenovirus vector, implantation of a p53 retrovirus vector packaging line, or transfectioIl~of p53 cDN~ ~acilitated by 25 adenovirus capsids in a linked complex. With the development of targeting approaches which permit accumulation of gene transfer Yectors at the tumor site, this approach can be ~ t~n~ to di seminated cancers.
Other gene-expresslon vector systems may also be utilized, 30 including, but not limited to, lipofection or direct DNA
injection_: Other methods of gene transfer and expression known to those skilled in the art may also be utilized.
The examples provided below for the therapy sensitizing gene p53 may also be adapted by one skilled in the art to 35 other therapy-sensitizing genes for the treatment of cancer. =~

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Exam~le 1. Transfe~rinq a 1~53 Gene into a Tumor Cell The wild-type p53 gene or a part of the gene may be introduced into a tumor cell in a vector, such that the gene remains extrachromosomal. Wild-type p~3 protein is expressed from the extrachromosomal wild-type p53 gene or a part of the gene.
Alternatively, the wild-type p53 gene may be introduced into a tumor cell ~ in such a way that it replaces the endogenous mutant p53 gene present in the lo cell. This approach would result in the correction of the p53 gene mutation (Revet et al., ~Homologous DNA targeting with RecA protein-coated short DNA probeg and electron microscope mapping on linear duplex molecules, ~' Journal of Molecular Biology, 232(3) :779-91, 1993; Thomas et al., "High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors, R Molecular and C,o~ r Biology, 12(7) :2919-23, 1992; Mansour et al., "Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination, " Proc. Natl. Acad. Sci.
87 (19) :7688-92, 1990; Capecchi, RAltering the genome by homologous recombination, ~ Science, 2a~4 (D.910) :1288-92, 1989; Sedivy and Joyner, ~'Gene targeting, ~ published by W.H. Freeman, 1992; incorporated by reference herein) .
A preferred vector for p53 gene transfer has the ability to transfer the gene to all or most of the cells in the target cell population, and to achieve sufficiently long expression and sufficiently high expression levels to promote the desired effect. Possible vector designs and gene transfer approaches include but are not limited to the following:
1). Adenovirus vectors. Adenoviral vectors can be obtained in higher titer than retroviral vectors, enabling a potentially higher efficiency of ~gene delivery. They are particularly attractive in being able to infect a broad range of cell types, both dividing and non-dividing (Graham FL, Prevec L. Manipulation of adenovirus vectors.
In Murray EJ, ed. "Methods in Molecular Biology" vol. 7, W095/30002 2~n32 p~"~
~- / 18 Gene Transfer and Expression~ Protocols~ Clifton, New ~ersey; The Humana Press, Inc. (1991). pp. 109-128, incorporated by reference herein~. ~hese vectors replace part of the early region -gene required for viral 5 replication with the transgene (i.e., an exogenous gene~to be transferred to a cell) of interest. Virus particles are obtained by transfecting the DNA into an appropriate packaging cell line which supplies the missing replication functions. Examples of such vectors have been described 10 (Berner KL. "Development of Adenovirus vectors for the expression of heterologous genes, ~ Biotechniques . (19B8) 6:6616-629, incorporated by reference herein). Irtra-arterial infusion of adenovirus vectors would be suitable for, but not limited to, liver cancer a~d head and neck 15 cancers.
2 ) . Retroviral vectors . These vector6 are the best characterized or human gene tr~nsfer,- and have been used in gene therapy protocols (~u et al., J. of Biochemistry, Z66:1433~-14342, 1991, incorporated by reference:herein).
20 Retroviral vectors consist of a modified retroviral genome ~nnt~;n;n~ the gene of interest to be transferred (i.e.
transgene), and often a selectable marker gene. The vector itself provides the viral LTR (Long ~ Terminal Repeat) sequences necessary for stable i~tegration of the 25 gene, but is defective for- replication and require6 ~a packaging cell li~e to provide the transacting replication factors. Examples of retroviral vectors and packagi~g cell lines have=been described (Kriegler, M. (1990) "Gene Transfer and Expression- -A Laboratory Manual, 'l Stockton 30 Press, New York, and Jolly, D., Cancer Ge~e Therapy, 1:51-64, 1994, incorporated by reference herein). A p~3-retroviral vector has been described (Cheng et al., (1992) ~Suppression of acute lyphoblastic leukemia by the human wild-type p53 gene, " Cancer Res. 52:222-22~ inc'orporated 35 by reference herein) Retroviral vectors have a broad range of infectivity with respect to cell type. ~ Transgene expres6ion ls _ _ _ . . . . _ . . ... , , . :,,,,, .,, , , , , . _ ~ wo ss/30002 ~ ~ 8 9 ~1 3 2 r~

u3ually driven from a strong viral promoter which has broad tissue specif icity . Examples are the viral LTR
(l:ong Terminal Repeat), Cytomegalovirus (CMV) promoter, Simianvirus 40 ~SV40) promoter (Miller AD and Rosman GJ.
~ Improved retroviral vectors for gene transfer and expression, " ~1989) BioTechniques 7:9B0-990, incorporated by ref erence herein) .
A packing cell line secreting the p53 retroviral vector can be implanted at the tumor site to increase the efficiency of retroviral gene transfer. The cell line provides a c~ nt;n~ us source of vector and improves the efficiency of gene transfer (Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM. In vivo gene transfer with retroviral vector-producer cells for treatment of expl~; t~l brain tumors. (1992) Soience 256: 1550-2).
3) . Adeno-associated virus. Vectors based on adeno-associated virus have the range of inf~ct~h; 1; ty of adenovirus. In addition, these vectors provide the potential f or stable integration of exogenous DNA at preferred sites in the host genome. A d~scussion of such vectors can be found in "Current Topics in Microbiology and Immunology" vol. 158, (Muzyczka N, ed), Springer-Verlag, pp. 97-129, (1992), herein incorporated by ref erence .

4). Other Yiral vectors. Vectors based on herpes, vaccinia, papilloma virus can also be used to transfer gene to tumor cells. A discussion of these vectors can be found in Kriegler, M. "Gene Transfer and Expression. A
~aboratory Manual.~ Stockton Press, New York, (1990); and Jolly D. "Viral Vectors for Gene Therapy, " Cancer Gene Therapy vol. 1:51-64 (1994), herein incorporated by ref erence .

5 ) . Coupled adenovirus capsids . Exogenous DNA may be transferred to a tumor cell by an adenovirus capsid.
In this approach, the DNA to be transferred is coupled to the outside of the virus capsid through a polylysine bridge (Curiel, et al., "High efficiency gene transfer Wo ss/300,02 2 ~ 8 ~ ~ 3 ~ r~

mediated by adenoviru6 coupled to DNA-polylysine complexes, " (1992) ~uman Gene Therapy, .-i:147-154, incorporated by reference herein~ . Entry of DNA intolthe cell is achieved through the natural pathways of virus int~rn~l; 7ation, but gene tranefer and expression is independent of the viral genome. For example, p53 gene can be coupled to an adeno virus cap6id which in turn i6 delivered into a lung r~ i n~ cell by receptor-mediated endocytosis. Thus in this approach the virus particle is used as a carrier for transfection of DNA rather than as a vehicle for infection. ~igh efficiencies of gene transfer can be achieved with thi6 approach, particularly when the complex of virus and DNA incorporates an additional ligand 6uch as but not li~ited to transferrin (Wagner et al., "Coupling of ~adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes, " ~1992) Proc. Natl. Acad. sci., USA, 89:6099-6:LD3, incorporated by reference herein) Tissue and cell type specific ligands can al60 be=incorporated to facilitate ~c~ tion of the complex in the target tissue.

6 ) . Other methods . Liposome -mediated gene transf er is effective for in vivo gene delivery (Zhu et al., ~ Systemic gene expression af ter intravenous DNA delivery into adult mice, " (1993) Science ~;L:209-11; Yoshimura et al., (1992) Nucleic Acids Research 20:3233-32~0;
incorporated by reference herein) A DNA-liposome complex can be administered locally or systemically. The advantage of this approach is low toxicity and absence of viral genomes. ~ith the choice of an appropriate promoter (e.g., CMV promoter), an extended ~eriod of=expre66ion can be achieved (Zhu et al., "Systemic gene expression after intravenous DNA delivery into adult mice, ~ (1993) Science 261: 209-11, incorporated by reference herein).
In addition, ligand-D~A conjugates have been utilized to target transgene-expre~ssion to specific cell types.
For example, asialoglycoproteIn-DNA conjugates have been ~ WO95/3000~ 8~3~ P~v~

used to target exogenouæ genes specifically to hepatocyte~
via the asialoglycoprotein receptor. Dire-ct gene transfer of naked DNA may be effective for some tissues as well, such as, but not limited to, muscle. These methods of 5 gene transfer may be applied singly or in, ' inAt;nn by those skilled in the art to achieve the expression in the tumor of a portion of a wild-type p53 gene or other therapy-sensitizing gene encoding the therapy-sensitizing activity .
10 r le 2. Introduction of ~53 Protein to a Tumor Cell Wild-type p53 protein or a portion of the wild-type p53 protein which has therapy-sensitizing activity may be supplied to cells which carry mutant p53 alleles. This may be achieved in vivo by several methods including but 15 not limited to intravenous, intra-tumoral, intra-arterial, intra-cavitary, or intrathecal infusionE;. Aerosolized preparations may be employed for delivery to the respiratory tract and topical preparations may also be utilized. The active molecules can algo be introduced 20 into the cells by microinjection, by liposomes, or by electroporation methods. The p53 prO~ein can also be introduced into tumor cells by receptor-mediated endocytosis. Alternatively, p53 protein may be actively taken up by the cells, or taken up by diffusion, to 25 restore p53 activity to the cells.
A chimeric protein comprising p53 and a targeting sequence can be used to introduce wild type p53 activity into a cell bearing a receptor for the targeting sequence.
For example, the targeting specificity of insulin-like-3 0 growth - f ac tor - I ( IGF - I ) o r Int er leukin - 2 ( IL - 2 ) can be used to deliver p53 protein to IGF-I receptor or Il.-2 receptor bearing cells. The chimeric protein can be obtained by constructing rh; ~~r~r cDNAs through r~( '; n~nt techniques and expressing them in either 35 procaryotic or eucaryotic systems.

Wo g~/30002 ~ 1 8 ~ n 3 2 r~

Thus, when p53 iB chimerized to growth factor IGF-I, which binds to specific~celi surface receptors on lung carcinoma cells, the chimeric protein can be targeted to lung carcinoma cells by receptor mediated endocytosis.
E le 3 . ~1lm; n; qtration of Aqents In practicing the methods of the invention, the compositions, such as those discussed in Examples 1 and 2 akove, can be used alone or in comkination with one another, or in comkination with other therapeutic or diagnostic agents. These compositions can ke utilized in vivo to a human patient, or in vltro. In employing them in vivo, the compositio~s can be administered to the patient in a variety of ways, including but not limited to parenterally, intravenously, subcutaneousLy, intramuscularly, colonicaIly, rectally, vaginally, nasally, orally, transdermally, topically, ocularIy, intraperitoneally, intracavitarily, intrathecally or as suitakly formulated surgical implants employing=a variety of dosage forms.
The dosage for the compositions of the present invention can range broadly depending upon the desired effects and the therapeutic .indication. As will be readily apparent to one skilled in the art, the useful~in vivo do6age to be administered and the particular mode of administration will vary depending uponr the condition of the patient, the cancer tre~ted and the particul~ar composition employed. The determination of effective dosage levels, i.e. the dosage levels nf~rPqq~ry to achie~e the desired result, will be within the amkit of one skilled in the art. Typically, applications of compositions are commenced at =lower dosage levels, with dosage level being increased until the desired effect is achieved ~
Ef f ective delivery requires the agent to =enter i =nto the tumor cells. Chemical modificatiorl of the agent may be all that is required for penetration. However, in the ~ Wo 9s/30002 2 18 ~ fl 3. 2 r~
event that such modification is insufficient, the modified agent can be co-formulated with permeability enhancers, auch as but not limited to Azone or oleic acid, in a liposome. The liposomes can either represent a slow 5 release pres,-nti~tinn vehicle in which the modified agent and ~e~ -~h; l; ty enhancer transfer from the liposome into the transfected cell, or the liposome phospholipids can participate directly with the modified agent and p~:L --h; l; ty enhancer in facilitating cellular delivery.
Drug delivery vehicles may be employed for systemic or topical administration. Topical administration of agents is advantageous since it allows localized cnn~ntration at the site of administration with minimal systemic absorption. This simE~lifies the delivery 15 strategy of the agent to the disease site and reduces the extent of toxicological characterization. Furthermore, the amount of material to be administered is far less than that required for other administration routes.
Agents may also be systemically administered.
20 Systemic absorption refers to the accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include but are not limited to: oral, intravenous, intra-arterial, intralymphtic, subcutaneous, 25 intraperitoneal, intranasal, intramuscular, intrathecal and ocular. Each of these administration routes exposes the agent to an accessible diseased tissue. Subcutaneous administration drains into a localized lymph node which proceeds through the lymphatic network into the 3 o circulation . The rate of entry into the circulation has been shown to be a function of molecular weight or size.
Intraperitoneal administration may also lead to entry into the circulation with the molecular weight or size of the agentdelivery vehicle complex controlling the rate of 3 5 entry .
Drug delivery vehicles can be designed to serve as a slow release reservoir, or to deliver their contents Wo 9s/30002 2 ~ 8 ~ ~ 3 ~ r~ J~

directly to the target cell. An advantage of using dirçct delivery drug vehicles is th~a;i: ~ multiple molecules are delivered per vehicle uptake èvent. Such vehicles have been shown to also increase the circulation half-life~of drugs which would otherwise be rapidly cleared f rom the blood stream. Some examples of such specialized drug delivery vehicles which fall into this category include but are not limited to liposomes, hydrogels, cyclodextrins, biodegradable polymers (surgical implants or nAnnr~rRules), and h; n~lh~:ive microspheres .
~iposomes offer several advantages: They are generally nontoxic and biodegradablç in composition; they may display long circulation half lives; and recognition molecules can be readily attached t~ their surface ~or targeting to tissues. Finally, cost effective manufdctùre of liposome-based pharmaceuticals, either in ~ a liriuid suspension or lyophilized product, has demonstrated the viability of this technology as an acceptable drug delivexy system.
Orally-administered fo~ tinnq can be prepared in several forms, including but not limited to capsules, chewable tablets, enteric-coated tablets, syrups, emulsions, su6pensions, or as solid forms suitable for solution or susFension in liquid prior to administration.
Suitable excipients are, for~ exdmple, ~Tater, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride or the like. In addition, if desired, the pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like.
If desired, absorption ~nhi;nr1 ng ,UL _~dlatiOnS (e .g ., liposomes) may be utilized.
~cAmnle 4- Incr,~ ;nr Tumox ~ells' Senqitivi~Y to Chemothera~v 1). Cells. ~98~ glioblastoma cells (Mercer et al., "Negative growth regulation in a glioblastoma tumor cell ~ W0 95/30002 2 ~ 3; 2 line that conditionally expresse6 human wild-type p53 . ~' (1990) Proc. Natl. Acad. Sci., USA, 87:6166-6170) were obtained i-rom ATCC and cultured at 3iC in 10% Co2 in Dulbecco' 8 Modified Eagles Medium supplemented with 10%
- 5 heat inactivated fetal bovine serum, gentamycin, nonessential amino acids, and sodium pyruvate. These cells are derived from a biopsy of a patie~t with glioblastoma mulitforme and: have been shown to have a homozygous mutation in the p53 gene at codon 237 (from met to ile, ATG to ATA) (Ullrich et al r ~Human wild-type p53 adopts a unique conformational and phosphorylation state vivo during growth arrest of glioblastoma cells . "
(1992) Oncogene, 7(8) :1635-43) .
2). Plasmids. A plasmid (pLp53RNL) cc~n~;n;n~ the wild-type p53 gene and the neomycin (G418) resistance gene wae used. The plasmid pLp53RNL was kindly provided by Dr.
Martin Haas (University of California, San Diego), and has been previously described (Cheng et al., "Suppression of acute lymphoblastic leukemia by the human wild-type p53 gene, " (1992) Cancer Res., 53 :222-226) . This plasmid carries the retroviral sequence Lp53RNL in which wild-type p53 expression is driven from the Moloney murine leukemia virus (MoMLV) LTR. The neomycin resistance gene is driven from the Rous Sarcoma Virus (RSV) promoter.
3) . Transfections. The plasmid was introduced into T98G cel~s using cationic liposomes. T98G cells were plated in 10 cm culture dishes at about 5 x 105 cells per plate. The following day cells were transfected with 15 llg D~A using I,ipofectamine (BRL) ~ and following the manufacturer's instructions. Five days ~ollowing transfection, cultures were selected in 100 llg/ml G418.
Clones were picked about three weeks later and ~ n~lF~.l Prior to det.-rm;n-n~ growth kinetics and plati~g ef f iciencies, cultures were adapted to growth in the absence of G418 for 7-10 days. One colony is denoted T98Gp53 because it contains the exo~enous wild type p53 gene .

W09s/30002 2~ n3~ J~/~

3) . Plating efficiencfy~ Cells were =plated in triplicate at low densit~ 100-50D cells per 6 cm plate, and allowed to grow for two weeks. Plates were stained in 0.5~ methylene blue in, thAnrl and colonies were counted.
5 Plating eficiency of transfected cells was 20!'6. Parental cells had a plating ~ffiri~nry of 509~.
4) . Control parertal T98G cells and T98Gp53 cals which had been adapted 2 weeks to culture in the absence of the antibiotics G418 were plated in 24 well plates at 10 about 2 x 104 cells per well. The next day they were exposed for one hDur to varying concentrations of cisplatin (a chemotherapeutic agent) from 10 to 40 ~lM in increments of 10~M. The cisplatin was removed after one hour and replaced with complete medium (DMEM + 109~ Fetal 15 Bovine Serum) and cells were allowed to grow for 7 days.
After 7 days, cells were counted or stained with crystal violet . ~n the latter case ,~ absorbance at 54 0 nm is proportional to cell viability. For ~clonogenic assays, cells were replated following treatment in 6 well plates 20 at 500-1000 cells per well. ~lones were counted 7 to 10 days later by staining in o . 596 methylene blue, 70~ ETOH.
Colony counts from p53 transfectas and parental T98G
glioblastoma cells were compared. As shown in figure 1, T98Gp53 cells were conslderably more sensitive to ~the 25 efects o: cisplatin than were the parental Tg8G cells.
Subsequent assays conf irmed this increased sensitivity .
The cDncentration of cisplatin needed to achieve a 50~6 reduction in colony count was reduced from about 30 IlM in the case of T98G parental ~cells= and empty vector-30 trAnr~lllr~d cells to 15-20 IIM cisplatin in the case of cells transduced with wild-type p53 gene.
r le 5. Inc~eas;nr Tumor Cells' SensitivitY to RA~1~ OtheraPY
Control parental T98G cells and T98G~53 cells were 35 grown for two weeks without G4l8 and then plated at about 5, 000 cells per T25 flask. The next day, cells were _ . _ ., . . , .. . . . . . . _ _ _ _ _ _ ~woss/30oo2 2~8~n32 subjected to gamma radiation from a Cobalt 60 source in doses ranging from 100 rads td 1500 xads in increments of lO0 rads. Cells were then incubated for an additional 5-12 days and colonies were stained i~ 0 5% methylene blue-methanol, counted, and compared to control untreated cells. As shown in figure 2, wild-type p53 transduced T98Gp53 cells show enhanced sensitivity to radiation, with 50g6 reduction in colony counts occurring at about 200 rads as compared to 400 rads for the parental cells.
ExamDle 6. ~53 Gene S~nf:itization TheraDY
The treatment described below applies to tumors with mutant p53 activity.
1. I~l~ont;~ication of Tumors with D53 ~hn~rm~l;ties Routine molecular biolDgy diagnostic techniques can be used to identify tumors that have p53 abnormalities, including, but not limited to, single-strand conformation polymorphism (SSCP~, PCR , sequencing and related molecular biology methods to detect gene abnormalities known to those skilled in the axt ( "Goneral Molecular Biology Methods Current Protocols in Molecular Biology, "
John Wiley and Sons, 1994; and J.Sambrook, E.F. Fritsch, and T.Maniatis, Molec~ r Clon;n~: A LaboratorY Manual, 2 Ed., Cold Spring Harbor= Laboratory Press, Cold Spring Harbor, New York, 1989, incorporated by reference herein) .
2. Sensitization of ~ ~rs with D53 Vectoxs BY
Direct In~ection or Aerosol;7~d Pre~7arations In this application, a suitable wild-type p53 vector and/or producer cell line is injected into a tumor or into a former tumor site following surgical resection or ablation ~to treat residual tumor cells) to permit expression by the tumor cell Df a portion of a wild-type p53 gene encoding the therapy-sensltizing activity.
Aerosolized vector preparations may also be utilized to deliver wild-type p53 to resection sites or tumors in the Xespiratory tract. Subsequently, the patient is treated with chemotherapy, radiotherapy, biological therapy, Wo 95/30002 ~ 1 g 9 ~

cryotherapy or hyperthermia appropriate for the treatment of said tumor known to those skilled in the art as described in "Cancer:Principles and Practice of Oncology, "
Devita, ~Iellman, Rosenberg Eds., L?E~pencott, 1993; "Manual of oncologic Therapeutics, " Wittes Ed., Lippencott, 1993;
and "Biologic Therapy of Cancer, ~ Devita et al., eds., Lippencott, 1991, incorporated by reference herein.
This approach may be employed to treat locali~ed primary tumors including but not limited to central nervous system tumors, sarcomas, and early stage carcinomas (lung, prostate, breast, bladder, kidney, hepatocellular, pancreatic, gastric, esophageal, colorectal, anal, head and neck, biliary, and urogenital) .
This approach may also be utilized to treat metastatic lesions of these and other tumors. ~ In these applications, a 3uitable wild-type p53 vector and/or producer cell line is injected into a metagtatic tumor or into the metastatic tumor site following surgical resection or ablation to permit expression by the tumor cell of a portion of a wild-type p53 gene encoding the therapy-sensitizing activity. Aerosolized vector preparations may also be utilized to deliver wild-type p53 to resection sites or tumors ~ in the respiratory tract .
Subsequently, the patient is treated with chemotherapy, radiotherapy, biological therapy, cryotherapy, or hyper~h~rm;~;lrrropriate for ~ the treatment of ~said metastatic tumor known to those skilled in the art as described in Cancer:Principles and Practice of: Oncology, Devita, Hellman, Rosenberg Eds., Lippencott, 1993; and Manual of Oncologic Therapeutics, Wittes Ed., Lippencott;
and "Biologic Therapy of Cancer, ~ Devita et al., eds., Lippencott, 1991, incorporatecI by refererce her--ein 3. Sensitization of Tumors with P53 Vectors bY
Intra-~rter.i~l Infusion ~ ~
Intra-arterial infusion shemotherapeutic drugs and other agents has been 11~; 1; Zl~i in the treatment of numerous f orms of primary~ and metastatic~ cancers ~ Wo ss/30002 2 1 ~ r~
(Cancer:~rir,ciples and PracticP o Oncology, Devita, Hellman, Rosenberg Eds., Lippencott; and Manual of Oncologic Therapeutics, Wittes Ed., Lippencott. ) . In this application of p53 therapy-sensitization, these intra-arterial infusion methods are employed to deliver a suitable wild-type p53 vector and/or producer cell line to permit expression by the tumor cell of a portion of the wild-type p53 gene encoding thle therapy-sensitizing activity. Sub3equently, the patient is treated with chemotherapy, radiotherapy, biological therapy, cryotherapy or hyperthermia appropriate for the treatment of said primary or metastatic tumor known to those skilled in the art as de6cribed in Cancer_~rinciples and Practice of Oncology, Devita, Hellman, Rosenberg Eds., Lippencott;
and Manual of Oncologic Therapeutics , Witteq Ed ., Lippencott; and "siologic Therapy of Cancer, ~ Devita et al., eds., Lippencott, 1591, incorporated by reference herein. This approach may be applied to the treatment of tumors such as but not limited to primary hepatocellular carcinoma, liver metastases and head and neck tumors.
This approach may be adapted by those skilled in the art of arterial infusion to treat any tumor with an accessible arterial vasculature for infusion.
4. Sensitization of Tumors with ~53 Vectors b~f IntracavitarY Infll.qion In these applications, body cavities containing tumor cells are first infused with a suitable wild-type p53 vector and/or producer cell line to permit expression by the tumor cells of a portion of a wild-type p53 gene encoding the therapy-sensitizing activity. Subse~uently, the patient is treated with chemotherapy, radiotherapy, biological therapy, cryotherapy, or hyperthermia appropriate f or the treatment of said primary or metastatic cavitary tumor known to those skilled in the art as described in Cancer:Principles and Practice of Oncology, Devita, Hellman, Rosenberg Eds., Lippencott; and Manual of Oncologic Therapeutics , Wittes Ed., Lippencott ;

2~ 3~
Wo 95/30002 ~ , ~ P~

and "Biologic Therapy of Cancer, ~ Devita et al., eds., Lippencott, 199~, incorporate~ y reference herein.
This approach may be applied but is not limited to the treatment of malignant pleural effusions (pleural 5 cavity), ascites (~h~lt 1 n:ll /peritoneal cavity), leptomeningeal tumors (cerebrospinal/ventricular system), pericardial effusions (pericardial cavity) and bladder:
carcinomas (bladder infusions).
5. Tumor Purqlnq of HematoPoietic Stem/Proqenitor ~ells bY ~53 Sensitization In this application, autologous hematopoietic stem/progenitor cells are purged of residual tumor cells by p53 sensitization befoæ' they are utilized~ to rescue patients from the effects of myelosuppressive/ablative 15 cancer therapies. Hematopoietic stem/progenitor cell preparations are harYested from the patient by standard methods (Cancer:Principles and Practice of= Oncology, Devita, Hellman, Rosenberg Eds., Lippencott; and Manual of Oncologic Therapeutics, Wittes Ed., Lippencott; and ~30ne 20 Marrow Transplantation, " Forman et al. Eds, 1993, incorporated by reference=herein) and transduced ex vivo with a suitable wild-type p53 ~vector and/or producer cell line to permit expression of a portiolI of a wild-type~p53 gene encoding the thera~y-sensitizing activity.
25 Subse~uently, the transduced cell preparation is subjected to cytotoxic purging techniques known to those skilled in the art (Cancer:Principles and Practice of Oncology, Devita, Hellman, Rosenberg Eds., Lippencott; and Manual of Oncologic Therapeutics, Wittes Ed., Lippe~cot~;~'and n'~3One 30 Marrow Transplantation, " Forman et al. Eds., 1993 , incorporated by reference herein).
The patients are then treated with myelo-suppressive/ablative cancer therapy and the hematopoietic stem pr4genitor cells purged_of residual tumor cells by 35 p53 sensitization are then infused into patients to rescue them from the myelosuppressive effects o~ very aggressive cancer treatment.

W095/30002 2 1 ~ 9 0 3 2 P~llu.~

The administration of myelosuppressive~ablative treatment and rescue by hematopoietic stem/progenitor cell infusion is well described in the prior art and has been utilized to treat a wide variety of solid and 5 hematopoietic malignancies (Cancer:Principles and Practice of Oncology, Devita, Hellman, Rosenberg Eds., Lippencott and Manual of t)~rnl o~i c Therapeutics, Wittes Ed., Lippencott; and "Bone Marrow Transplantation, ~ Forman et al. Eds., 1993, incorporated by reference herein). The 10 p53 sen6itization o~ residual tumor cells to destruction by cytotoxic purging agents will decrease the number o~
tumor cells in the hematopoietic stem progenitor cell infusion 1It;1;7.ori to rescue patients. This will decrease the lik~lih~nd of tumor recurrence which may occur from 15 the infusion of hematopoietic stem/progenitor cell prel~arations which contain residual tumor cell6.
6. Treatme~t of Di6srminr~ted Meta6tatic Tllmnr bY
~53 $~nsitizatiOn In this application, a suitable wild-type p53 vector 20 and/or producer cell line is injected systemically or parenterally to permit expression by tumor cells of a portion of a wild-type p53 gene encoding the therapy-sensitizing acti~rity. Subsequently, the patient is treated with chemotherapy, radiotherapy, biological 25 therapy, cryotherapy or hyperthermia appropriate for the treatment of the metastatic tumor known to those skilled in the art a6 described in Cancer Prirciples and Practice of Oncology, Devita, Hellman, Rosenberg Eds., ~ippencott;
and Manual of Oncologic Therapeutics , Wittes Ed ., 30 ~ippencott; and "Biologic Therapy of Cancer, ~' Devita et al., eds., I,ippencott, 1991, incorporated by reference herein .
The individual application6 of p53-mediated sen6itization therapy outlined above may also be utilized 35 in combinations that may be applied by those skilled in the art of multir~ l;ty cancer therapeutics, ~or example, as described in Cancer Principles ana Practice of =

WO 95/30002 ~ ~ 8 ~ 0 3 ~ I .11.1~ ~ ~, L

oncology, Devita, Hellman, Rosenberg Eds., Lippencott; and Manual of Oncologic TherapeuticR , Wittes Ed ., Lippencott ;
and ~Biologic Therapy of Cancer, ~ Devita et al., eds., Lippencott, 1991, incorporated by rEEèrence herein.

7. Treatment of Glinhl~qt M~lltiforme bY o53-ted S~nqitizatiQ~ ~hera~Y
Glioblastoma multiforme represents the most frequently encountered intracranial brain tumor, with some 20, 000 ~ew cases being diagnosed each year in the U~S.
Although it rarely metastasizes outside of the central nervous system, it is nevertheless the most malignant form of astrocytoma, and presents a therapeutic challenge~to the physician employing presen~t conventio~al approaches.
These approaches include surgery, radiat~ion, and chemotherapy, and while advances have been made in all areas, mean survival time from diagnosis is still only about one year. Glioblastomas are relatively radiation resistant, and respond poorly to most chemotherapeutic drugs. Of those chemothexapeutic agents which have been shown to have some effectiveness initially, including cisplatin, BCNU (carmustine) and PCV ~procarbazine CCNU, vincristine), none shows sustained effectiveness.
Due to their location in the brain, the morbidity of even modest tumor progression in glioblastoma patients ls high. Small decreases in tumor volume are expected~to have a beneficial effect~ to~ patients. Furthermore, glioblastoma rarely metastasi~es outside the central nervous system, making this disease an ideal target =for localized gene transfer, including local infection with p53 bearing adenovirus, or lo~cal transfection= with p53 cDNA facilitated by adenovirus capsids, or implantation of a p53 bearing viral vector packaging line at the tumor site. Similarly, this approach could have bEnefit fDr brain metastases of other cancers in which a decrease :in morbidity may result from even small reductions in tumor volumes .

~wos~/30002 7~'18~n~2 P~"-' ~

8. Treatment of He~atocellular '~rcinoma and Head and Neck Cancers bY ~53 -r'~ at~ed Sensitization Theral~Y
Hepatocellular carcinoma and head and neck cancers are characterized by frequent p53 mutations (up to 30~) and are excellent targets for adenovirus-based p53-mediated sensitization therapy and related forms of p53-~^~l; ~t~tl sensitization therapY. Intra-arterial deliverY
of the p53 vector would enable high efficiency delivery of wild-type p53 therapy-sensitizing activity into the tumor.
However, systemic delivery of p53 gene for clinical benefits may not be required in many cases because hepatocellular carcinomas and head and neck cancers of ten produce localized morbidity as in the case of glioblastoma. ~iver metastases of colorectal carcinoma and other tumors with p53 mutations: could be similarly treated by intra-arterial infusion of a p53 vector followed by appropriate tumor therapy known to those skilled in the art of cancer treatment 9 . Treatment of T lln~ Cancer bv p53 -me~l; ated Sensitization Thera~Y
~ung epithelium is also an excellent target for adenovirus-based p53-mediated sensitization therapy.
Small cell lung carcinoma, which is initially very ~5 sensitive to chemotherapy, acquires resistance with disease progression. Introduction of wild-type p53 can be used to treat this tumor by sensitizing the tumor cells to therapy. Non small cell. Iung carcinoma, also characterized by p53 mutations in some 509~ of cases, is often refractory to chemotherapy. Therefore, p53-mediated sensitization therapy can be l~t; 1; 7F~rl in the treatment of these tumors.
le 7. Screeninq for Small Molecules with The~al)Y
s~nsl~;7;n~ ActivitY
Small molecules with therapy sensitizing are nt; f i ed by their ability to enhance cancer treatment 2~ 89032 w0 95/30002 ef f icacy relative to control solutions that do not con~ain the candidate small molecule. Each candidate molecule ~is tested for its efficacy in sensitizing cancer therapy in cell lines, in animal models,~and in controllea rl inir~l 5 studies using methods known to those skilled in the art and approved by the Food and Drug Administration, such as, but not limited to, those promulgated in The Federal Register 47 (no . 56): 12558-1~2564 , March 23 , 1982 . The small molecules with therapy~ sensitizing or ~enhancing 10 activity may be utilized in cancer therapy employing the approaches described previously for proteins with wild-type therapy sensitizing activity. As small molecules readily diffuse into tissues following administration, this approach may be utilized to treat both localized and 15 metastatic tumors in combinatisn with other therapies.~
Small molecules which mimic or cgnfer ~rild type therapy sensitizing activity can be screened in binding assays with the appropriate target. (See Houghtçn, R A.
"Peptide libraries, criteria = and trends. n Trends in 20 Ger~çtics 9.235-239, 1993). Comoinatorial libraries of peptides, modified peptides or organic chemical compounds are generated by methods known to those skilled ~in the art (Jayarickreme et al, "Creation and functional screening of a multi-use peptide libraryll Proc. Natl. Acad. Sci.
25 USA, 91:1614-1618; ~oughten,: R.A. ~Peptide libraries, criteria and trçnds_" Trends in Genetics 9:235-239, 1gg3;
Phillips et al., "TransitiDn state characterization; a new approach , `; n i nrj inhibitor analogues and variation ~in enzyme structure." Biorh-m;rtrY, 1992, 31(4) :959-63;
30 Eichler and ~oughten, "Identification of substrate analog trypsin inhibitors through the screçning of synthetic peptide combinatorial libraries. '~ Biochemistr~ 32 :11035-11041, 1993; ~uston et al., ~Medical applications ~ o~
single-chain antibodies. ~' ~International Reviews of 35 Immllnrloqv, 1993, 10(2-3~ :195-217; Van de Wateroeemd ~
"Recent progress in QSAR-technology, '~ Druq Desiqn and Discoverv, 1993, 9(3-4) :277-85) .

W095/30002 ~18~n32 p~" ~

Putative 6mall molecules can also be analyzed in biological assays for function. In a specific example, a retroviral vector library encoding and expre6sing peptides could be directly screened for t~erapy sensitizing 5 activity using the methods described in examples above and that of Gudkov et al., 1993, "Isolation of ge~etic suppres60r elements, ;n~3llr-;n~ resistance to topoisomerase II interactive cytotoxic drug6, from~human topoisomerase II CD~A, " Proc. Natl. A~A.1. Sci. IJSA, 90 :3231-3235, lO incorporated by reference herein.
Exam~le 8 . Tn~; CitY test; n~ of Putative Thera~Y
Sf~nC;tiz~inf~ Molecule6 Methods are provided for de~r~;nln~ whether an agent active in any of the methods listed above has little or no 15 effect on healt~y cells. Such agents are then formulated in a pharmaceutically acceptable buffer or in buffers useful for standard animal tests.
By "pharmaceutically acceptable buffer'~ is meant any buffer which can be used in a pharmaceutical composition 20 prepared for storage and subsequent administration, which comprise a pharmaceutically effective amount of an agent as degcribed herein in a pharmaceutically acceptable carrier or diluent. Acceptable carr~ers or diluents for therapeutic use are well known in the pharmaceutical art, 25 and are described, for example, in Reminqton~ s Pharmaceutin~l Scie~ces, Mack pl~hliqhin~ Co. (A.R. ~ennaro edit. 1985). Preservatives, stabilizers, dyes and even f lavoring agen~s may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid 30 and esters o p-hydroxybenzoic acid may be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents may be used. Id.
A. A-l~;tion~l 6cre~r~s for ToxicitY: Method 1 Agents identified as having therapy sensitizing 35 activity are assessed for toxicity to cultured human cells. This assessment is based on the ability of living W095,30002 2~n3~ P~ C~

cells to reduce 2,3,-bis[2-methoxy-4-nitro-5-sulpho-nylphenyl] -5- [ (phenylamino) carbonyl] -2H-tetrazolium hydrQxide] otherwise referred ~tq as XTT (Paull et al., J.
Heteroc~l. Chem. _25:763=767:-"(1987); Weislow et al., (1989), J. Natl. (~;ln~, Inst. 81:577) . ~Tiable, 1 ;An cells are capable of reductive cleauage of an ~-~ bond in the tetrazole ring of XTT to form XTT formazan. Dead cells or cells with impaired energy metabolism are ; nr~r~hl e of this cleavage reactior . The ~xtent of the 10 cleavage is directly proportional to the number of living cells tested.
Cells from a human cell line such as HeLa cells are seeded at 103 per well in 0.1 ml of cell culture medium (Dulbecco's modified minimal essential medium supplemented 15 wlth 105. fetal calf serum) in the wells of a 96 =well microtiter plate . Cells are allowed -to~ adhere to ~ the plate by culture at 37 C in an atmosphere of 959~ air, 5~6 CO2. After overright culture, solutions of ~test substances are added in tlllpl ;~A~e to wells at concentrations that 20 represent eight half-decade:14g dilutions. In parallel, the solvent used to dissolve the test substance is added in duplicate to other wells. The culture of the cells is c~-nt;n~ for a period oi time, typically 24 hours. At the end o~ that time, a solution of XCT and a coupler 25 ~methyl~hf~n~7~--;um sulfate) is added to~each of the ~est wells and the ;n~llhF~ n is continued for an additiona~ 4 hours before the optical density in each of the wells is determined at 450 nm in ~ an automated plate reader.
Substances that kill mammalian cells, or impair their 30 energy metabolism, or slow their growth are detected by a reduction in the optical density at 450 nm in a well as compared to a well which receiYed no test substance.
B. ~l;tional screens fo~ ToxicitY: Method 2 Therapy sensitizing molecules are tested for 35 cytotoxic effec~s on cultured human cell l~nes using incorporation of 35S methionine iIIto protein as~- an indicator of cell viability. HeLa cells are grown in 96 __ _ .. : . ... _ _ ~w095/30002 ~lsgn~,2~ r~
well plates in Dulbecco' s minimal essential medium supplemented with 103c fetal calf serum and 50/lg/ml penicillin and streptomycin. Cells are initially seeded at 103 cells/well, 0.1 ml/well. Cells are grown for 48 hrs without exposure to the therapy sensitizing molecule, then medium is removed and varying dilutions of the therapy sensitizing molecule prepared in complete medium are added to each well, with control wells receiving no cytokine modulator. Cells are ;nrllh~tr~ for an additional 48-72 hrs. Medium is changed every 24 hrs and replaced with fresh medium containing the same concentration of the therapy sensitizing molecules. Medium is then removed and replaced with complete medium without antifungal. Cells are incubated f or 24 hr in the absence of therapy sensitizing molecule, then viability is estimated by the incorporation of 35S into protein. Medium is removed, replaced with complete medium without methionine, and incubated for 30 min. Medium is agaln removed, and replaced with complete medium without methionine but ~ nt::;n;n~ 0.1 ,~Ci/ml 35S methionine. Cells are incubated for 3 hrs. Wells are washed 3 times in PBS, then cells are p~ ilh;l;7ed by adding 100~ methanol for 10 min. Ice cold 1096 trichloroacetic acid (TCA) is added to fill wells; plates are incubated on ice ior 5 min. This TCA
wash is repeated two more times. Wells are again washed in methanol, then air dried. 50~11 of s~;n~ ;on cocktail are added to each well and dried onto the wells by centrifugation. Plates are used to expose X ray film.
Densitometer scanning of the autoradiogram, including wells without antifungal, is used to determine the dosage at which 5096 of cells are not viable ~IDso) (Culture of Animal Cells. A manual of basic technir~ue. (1987) . R.
Ian Freshney. John Wiley & Sons, Inc., ~ew York) .
~Y~ mle 9 ~tlm;n;stratio~ Df The~aT~Y SpnF:it;7;nsr Molecules The invention features novel therapy sensitizing molecules discovered by the methods described above. It W0 95/30002 2 i 8 ~ n 3 ~ r~ t ,~

al60 includes novel pharmaeeutical compositions which include therapy sensitizing = molecules discovered ~as de6cribed above formulated i~ pharmaceutically acce~table formulations ~ -By "therapeutically effect~!ve amount" is meant an amount that relieves (to some extent) one or more symptoms of the disease or condition i~ the patient. Additionally, by "therapeutically effective amount" is meant an amount that returns to ~ormal, either partially or completely, physiological or =h; nrl~m; cal parameters associated with or eausative of a myeotie disease or: eondition . Generally, it is an amount between about 1 nmole and 1 llmole of the moleeule, depenaent O~l its ECs~ and on the age, size, and disease assoeiated with the patient.
All publieations referenced are hereby incorporated by referenee herein, ;n=rlll11;nr the nucleic acid se~uences and amino aeid sequenees listed in eaeh publication.
Other embodiments are within the f ollowing claims .

Claims (22)

1. Method of increasing the effect of a cancer therapy, comprising the steps of:
delivering wild-type therapy-sensitizing gene activity to a tumor cell characterized by loss of said wild-type therapy-sensitizing gene activity, and subjecting said tumor cell to said cancer therapy.

2. The method of claim 1, wherein a portion of a therapy-sensitizing protein with said therapy-sensitization gene activity is introduced into the tumor cell.

3. The method of claim 1, wherein a portion of a therapy-sensitizing gene or a portion of a cDNA encoding said therapy-sensitizing gene activity is introduced into the tumor cell.

4. The method of claim 1 wherein said cancer therapy is radiation therapy.

5. The method of claim 1 wherein said cancer therapy is chemotherapy.

6. The method of claim 1, wherein said cancer therapy is biological therapy.

7. The method of claim 1, wherein said cancer therapy is cryotherapy.

8. The method of claim 1, wherein said cancer therapy is hyperthermia.

9. The method of claim 1 wherein said tumor cell is selected from the group consisting of carcinoma cells, sarcoma cells, central nervous system tumor cells, melanoma tumor cells, leukemia cells, lymphoma tumor cells, hematopoietic tumor cells, ovarian carcinoma cells, osteogenic sarcoma cells, lung carcinoma cells, colorectal carcinoma cells, hepatocellular carcinoma cells, glioblastoma cells, prostate cancer cells, breast cancer cells, bladder cancer cells, kidney cancer cells, pancreatic cancer cells, gastric cancer sells, esophageal cancer cells, anal cancer cells, biliary cancer cells, urogenital cancer cells, and head and neck cancer cells.

10. The method of claim 3 wherein said portion of a therapy-sensitizing gene or said portion of a cDNA is in a vector.

11. The method of claim 10, wherein said vector is selected from the group consisting of adenovirus vector, retroviral vector, adeno-associated virus vector, herpes virus vector, vaccinia virus vector and papilloma virus vector.

12. The method of claim 3, wherein said portion of a therapy-sensitizing gene or said portion of a cDNA is coupled to a virus capsid or particle.

13. The method of claim 12, wherein said portion of a therapy-sensitizing gene or said portion of a cDNA is coupled to said capsid or particle through a polylysine bridge.

14. The method of claim 3, wherein said portion of a therapy-sensitizing gene or said portion of a cDNA is encapsulated in a liposome.

15. The method of claim 3, wherein said portion of a therapy-sensitizing gene or said portion of a cDNA is conjugated to a ligand.

16. The method of claim 15, wherein said ligand is an asialoglycoprotein.

17. The method of claim 3, wherein said portion of a therapy-sensitizing gene or said portion of a cDNA is introduced to said tumor cell by direct injection or aerosolized preparation.

18. The method of claim 3, wherein said portion of a therapy-sensitizing gene or said portion of a cDNA is introduced to said tumor cell by intra-arterial infusion.

19. The method of claim 3, wherein said portion of a therapy-sensitizing gene or said portion of a cDNA is introduced to said tumor cell by intracavitary infusion.

20. The method of claim 3, wherein said portion of a therapy-sensitizing gene or said portion of a cDNA is introduced to sald tumor cell by intravenous infusion.

21. The method of claim 1, wherein said therapy-sensitizing gene activity is fas therapy-sensitizing activity.

22. The method of claim 1, wherein said therapy-sensitizing gene activity is p53 therapy-sensitizing activity.