WO2006063242A1 - Methods and compositions for induction of tumor regression - Google Patents

Abstract

The invention provides methods and compositions for treating tumors and tumor cells using suicide gene/prodrug therapy. In particular, the methods and compositions involve enhanced delivery of the suicide factor to the tumor cells.

Description

METHODS AND COMPOSITIONS FOR INDUCTION OF TUMOR REGRESSION

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 60/634,895, titled "METHODS AND COMPOSITIONS FOR INDUCTION OF TUMOR REGRESSION", filed December 10, 2004, which is herein incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates to the field of tumor therapy, particularly to treating tumors and tumor cells through suicide gene/prodrug therapy.

BACKGROUND OF THE INVENTION

[0003] Conventional treatments for cancer, such as pancreatic cancer, are scarcely effective, so the development of new therapeutic approaches is of major interest. The thymidine kinase/ganciclovir (TK/GCV) enzyme -prodrug therapy has been shown to have some potential efficacy for pancreatic tumors. Rosenfeld et al. (1997) Ann Surg 225:609- 618; DiMaio et al. (1994) Surgery 116:205-213; Carrio et al. (1999) Gene Ther 6:547-553; Carrio et al. (2002) J Gene Med 4:141-149. However the effectiveness of the treatment is in part impeded by the low efficacy of current vectors to transfer genes into all the cells of the tumor.

[0004] Using current gene therapy vectors, tumor transduction generally is limited to a small area of the tumor mass around the injection point, and this is one of the key elements that limits the antitumor effects of many cancer gene therapy strategies. There remains a need for effective therapies and agents for killing cancer cells in a tumor and for inducing tumor regression.

[0005] All publications and patent applications cited herein are hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0006] Translocatory proteins can efficiently translocate across the membrane of mammalian cells and are able to mediate in the intracellular delivery of heterologous proteins iusea io mem. some examples oi sucn proteins are tne numan immunodeliciency virus (HIV) Tat, the Drosophila antennapedia, and the Herpes simplex virus VP22. See, for example, Fawell et al (1994) Proc. Natl. Acad. ScL USA, 91 :664-668; Miot et al. (1991) Proc. Natl. Acad. ScL USA 88:1864-1868; Elliot et al. (1997), Cell, 88:223-233. The intercellular transfer function has been mapped to short peptides of highly basic residues that have been termed protein transduction domains (PTDs) (Leifert et al. (2003) MoI Ther 8:13- 20; Beerens et al. (2003) Curr Gene Ther 3:486-494). As shown in vitro, cells expressing an 11 amino acid peptide from HIV Tat fused to the TK (thymidine kinase) gene (Tat 11-TK) have increased sensitivity to acyclovir through a mechanism involving apoptosis (Tasciotti et al (2003) Cancer Gene Ther 10:64-74).

[0007] Several studies have sought the optimal and the minimal sequence responsible for the cellular uptake. Up to now a sequence of 9 amino acids (RKKRRQRRR) has been reported as the minimal Tat sequence with PTD features (Wender et al. (2000) Proc Natl AcadSci USA 97:13003-13008). The basic characteristics of the sequence have been claimed to be responsible for the transduction properties of the peptides, however a synthetic PTD has been identified that contains only 3 arginine residues also with enhanced transductional potential (Ho et al (2001) Cancer Res 61 :474-477). This PTD was characterized for having several alanine residues that, when properly placed, conferred alpha- helix strength indicating that what was enhancing protein transduction potential was not the number of arginine residues, but the combination with alpha-helical promoting agents. Thus, factors other than the content of basic residues also appear to play a role.

[0008] The current invention includes an 8 amino acid Tat sequence YGRKKRRQ that has protein transduction capability. Accordingly, the invention includes a protein comprising a suicide polypeptide sequence and a protein transduction domain sequence (PTD), wherein the PTD comprises a sequence YGRKKRRQ. It does not comprise a sequence RKKRRQRRR, or a sequence YGRKKRRQRRR, or an entire HIV Tat sequence. The invention also includes a protein comprising a protein transduction domain sequence (PTD) and a suicide polypeptide sequence, wherein the PTD consists essentially of a sequence YGRKKRRQ The invention also encompasses a protein comprising a protein transduction domain sequence (PTD) and a suicide polypeptide sequence, wherein the PTD consists of a sequence YGRKKRRQ. [0009] The invention also contemplates that the suicide polypeptide can be a protein or protein fragment derived from the group consisting of thymidine kinase (e.g., from herpes simplex virus-1 or varicella zoster virus), cytochrome P450 2Bl, cytosine deaminase, the A chain of diphteria toxin, ricin, abrin, caspases, Fas-Ligand, Bax, and TRAIL. Preferably, the suicide polypeptide is a protein or protein fragment derived from herpes simplex virus-1 thymidine kinase or from cytochrome P450 2Bl

[0010] The invention also encompasses a nucleic acid encoding the above-described proteins of the invention, and vectors and cells comprising such a nucleic acid. Preferably, the vector is a phage, virus, plasmid, phagemid, cosmid, YAC, or episome. The recombinant cell is preferably a bacterium, yeast, insect cell, or a mammalian cell.

[0011] In addition, the invention encompasses compositions comprising a protein or a nucleic acid or a cell of the invention. The invention also includes kits comprising a protein or a nucleic acid or a cell of the invention.

[0012] The invention is also directed to methods of treating an individual with a tumor, the treatment comprising administering the nucleic acids or proteins or vectors or cells of the invention to the individual resulting in the introduction of the protein or nucleic acid into tumor cells or into other cells nearby, ultimately resulting in a decrease in tumor burden, for example by tumor cell death or injury.

[0013] Accordingly, the invention includes a method of treating an individual with a tumor to decrease tumor burden by administering a protein or a nucleic acid or a cell of the invention. In one aspect, this method further comprises administering a prodrug.

[0014] Nucleic acids can be administered by techniques known to those of skill in the art. In a preferred aspect, they are preferably administered by electrotransfer. If cells are to be administered, such cells may be attached to a support matrix that is suitable for implantation into the individual. In such cases, the support matrix is preferably in the form of a microcarrier. The microcarrier preferably has a diameter from about 80 μm to about 330 μm. The support matrix may be made of glass, polystyrene, polypropylene, polyethylene, polycarbonate, polypentane, acrylonitrile polymer, nylon, magnetite, natural polysaccharide, a modified polysaccharide, collagen, gelatin or modified gelatin such as crosslinked gelatin. The support matrix may be coated on its external surface with factors known in the art to promote cell adhesion, growth or survival. [UU 15] i he invention also encompasses a method ot preparing the protein ot the invention by incubating a cell containing a nucleic acid encoding the protein under conditions that allow the cell to express the protein. Such a method may further comprise isolating the protein from the host cell. In another aspect, the invention also includes a method of attaching a cell of the invention to a support matrix suitable for implantation into an individual, by placing the cell in contact with the support matrix for a time sufficient to allow the cell to attach to the support matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig. 1 depicts the effect of Tat peptides on the cytotoxicity of the TK/GCV and CYP2B1/CPA suicide systems. Fig. Ia is a schematic illustration of the expression vectors encoding TK, Tat8-TK, Tatl 1-TK. Fig. Ib is a graph depicting cell viability of NIH3T3 cells transfected with the indicated constructs after 5 days of GCV treatment. Values are expressed ± SEM. Fig. Ic is a schematic illustration of the expression vectors encoding CYP2B1, Tat8-CYP2B1, Tatll-CYP,2B1. Fig. Id is a graph depicting cell viability of NIH3T3 cells transfected with the indicated constructs after 3 days of CPA treatment. Values are expressed ± SEM.

[0017] Fig. 2 depicts an in vitro bystander effect of TK/GCV and Tat8-TK/GCV on cell viability. Different ratios of NIH3T3TK or NIH3T3Tat8-TK-expressing cells were plated with (a) NIH3T3 wild type cells or (b) NPl 8 wild type. The graphs depict cell viability of the cells after GCV treatment. Values are expressed ± SEM.

[0018] Fig. 3 depicts the effects of TK/GCV and Tat8-TK/GCV therapies on pancreatic tumor growth after microcarrier cell delivery. Fig. 3 a depicts microcarriers in the tumor after hematoxilin-eosin staining (20Ox). Fig. 3b is a graph depicting growth of the tumors in the presence of the indicated MC-cell composition. Significance referred to comparisons between control and treated groups at each time point studied. Codes are ***p<0.001, **p<0.01, *p<0.05.

[0019] Fig. 4 depicts EGFP expression in mouse subcutaneous tumors. Representative EGFP fluorescent images of tumors injected with 50 μg of the plasmid Tat8-TK with (top) or without (bottom) in vivo electrotransfer. Fluorescent images (left), light images (right). Magnification (40 x).

[0020] Fig. 5 depicts effects of TK/GCV and Tat8-TK/GCV therapies on pancreatic tumor growth after DNA electrotransfer at high dose of GCV. Fig. 5a is a graph depicting tumor volume during treatment ot tour groups: Control 1 are tumors injected with IK or Tat8-TK plasmid followed by electrotransfer; Control 2 are tumors injected with saline solution followed by electrotransfer; TK are tumors injected with the TK plasmid followed by electrotransfer; Tatδ-TK are tumors injected with the Tat8-TK plasmid followed by electrotransfer. Control 2, TK and Tat8-TK groups received GCV 100 mg/Kg for 6 days. Significance referred to comparisons between control and treated groups at each time point studied. Codes are ***p<0.001, **p<0.01, *p<0.05. Fig. 5b depicts TUNEL-positive cells in cryosections at days 2 and 4 after electrotransfer with a FITC-conjugated antibody (green) and counterstained with 4',6-diamino-2-phenylindole (DAPI) (blue). Magnification (20Ox).

[0021] Fig. 6 is a graph depicting effects of TK/GCV and Tat8-TK/GCV therapies on pancreatic tumor growth after DNA electrotransfer at moderate dose of GCV. Three different groups were established: Control are tumors injected with TK or Tat8-TK plasmid followed by electrotransfer; TK are tumors injected with the TK plasmid followed by electrotransfer; and Tat8-TK are tumors injected with the Tat8-TK plasmid followed by electrotransfer. TK and Tat8-TK groups received GCV 50 mg/Kg for 14 days. Significance referred to comparisons between control and treated groups at each time point studied. Codes are ***p<0.001, **p<0.01, *p<0.05.

[0022] Fig. 7 shows the cytotoxic effects of the Tat8-TK released protein. Wild type cells were incubated in the presence or absence of GCV with conditioned media from (a) NIH3T3 wild type, NIH3T3TK or NIH3T3Tat8-TK expressing cells or (c) from Tat8-TK or TK transfected COS-7 cells and cultured for three days in the presence (+) or absence (-) of GCV. Cell viability was measured after 2 days of treatment. Values are expressed ± SEM. Statistical analysis was performed using the Mann- Whitney U test with significance reported when P was less than 0.05. Comparisons were performed between (-) and (+) groups. Supernatants from GCV treated (+) or untreated (-) Tat8-TK (Figs. 7b and 7d) or TK (Fig. 7d) expressing cells were immunoprecipitated with an anti-EGFP antibody. Tat8-TK and TK cell lysates and the immunoprecipitates were immunobotted with an anti-EGFP antibody.

DETAILED DESCRIPTION

[0023] According to the present invention, a protein transduction domain (PTD) is used to enhance delivery of a suicide polypeptide to tumor cells and thus elicit enhanced killing of the tumor cells, for example in the presence of a prodrug. Methods and compositions of the invention are used tor Rilling tumor cells and/or in inducing tumor regression. As illustrated herein, cell killing was enhanced and tumor volume reduced when an 8 amino acid PTD fused to a suicide enzyme and the suicide enzyme's respective prodrug are delivered to the cells. The amount of cell killing appears to be well beyond that of the cells initially transduced with the gene expression vector. Thus, the methods and agents of the invention are effective in killing more cells than just those transfected with the suicide gene construct and lead to a greater reduction in tumor size as compared to the suicide gene constructs without the PTD.

[0024] The use of the minimal efficient PTD with translocatory properties is desirable in order to minimize any putative side effects of the PTD. As demonstrated herein, the Tat 8 amino acid peptide is effective as a PTD.

[0025] The terms "protein", "polypeptide", and "peptide" are used interchangeably herein to refer to polymers of amino acids of any length. Techniques for isolating proteins are well known in the art. See, e.g., Protein Analysis and Purification: Benchtop Techniques by Ian M. Rosenberg (2nd ed., 2005).

[0026] The term " individual" refers to any mammal, including humans.

[0027] The term "tumor" as used herein broadly includes any malignant or pre-malignant tissue exhibiting abnormal cell growth that is exemplified, for instance, by hyperplasia, metaplasia, or dysplasia (for a review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d. Ed., W.B. Saunders Co., Philadelphia, pp. 68-79). As used herein, the term "tumor" also includes cells or tissues that are predisposed to, or at risk of, progressing into a malignant or premalignant state.

[0028] As used herein, "treating" an individual with a tumor includes any procedure that results in the alleviation, amelioration, and/or stabilization of a symptom associated with the tumor, and/or a delay in progression of a tumor. More specifically, treatment refers to the administration of the nucleic acids or proteins or compositions or vectors or cells of the invention to a subject after a tumor burden has been determined in that subject using any method known in the art, with a resultant decrease or elimination of the tumor burden. Treatment can involve slowing the growth of the tumor, halting growth of the tumor, causing reduction or regression of the tumor, inhibiting tumor invasion, causing tumor cell death, or causing reduction or regression of metastases. Treatment can also include decreasing the likelihood of tumor development in a subject who may be predisposed or at high risk of developing tumors. A treatment method ot the invention thus reduces probability of developing a tumor in a given time frame and/or reduces the extent of tumor growth in a given time frame, when compared to not using the method. The effects of the treatment methods of the invention can be monitored through the assessment of tumor burden by well- established methods. For example, some clinical criteria (RECIST criteria) for such evaluation have been promulgated by Response Evaluation Criteria in Solid Tumors Working Group, a group of international experts in cancer. One commonly used criterion, for example, is tumor volume, and reduction in tumor burden can be expressed as percentage changed in tumor volume from baseline. James et al., J. Nat. Cancer. Inst. 91(6):523-528 (1999). Preferably, treatment results in a significant reduction in tumor burden. Preferably, treatment reduces the tumor burden by at least 10%, more preferably by at least 25% and most preferably by at least 50%.

[0029] In general, a protein transduction domain sequence (or "PTD") may comprise any synthetic or naturally-occurring amino acid sequence that can mediate or assist in the intracellular delivery of a protein containing the PTD, including heterologous protein sequences that have been attached to the PTD. For example, transduction can be achieved in accordance with the invention by use of a protein sequence, particularly the HIV Tat protein or any fragment thereof with transducing activity. The PTD can also be derived from the Antennapedia homeodomain or the HSV VP22 sequence, or suitable transducing fragments and/or variants thereof.

[0030] Generally, a suicide polypeptide sequence or suicide gene product is any protein sequence or protein fragment that causes cell cytotoxicity or cell death (for example by apoptosis), either by itself or in the presence of other compounds. Similarly, a suicide gene is a polynucleotide sequence encoding a suicide polypeptide. Examples of a suicide polypeptide include but are not limited to protein sequences or protein fragments and/or variant sequences derived from thymidine kinase of herpes simplex virus- 1 or varicella zoster virus, cytochrome P450 2Bl, cytosine deaminase, the A chain of diphteria toxin, ricin, and abrin or any other desired cytotoxic protein sequence; or caspases, Fas-Ligand, Bax, and TRAIL. A "prodrug" is a substance that is not cytotoxic in itself, but which can be converted to a cytotoxic product through the action of the suicide polypeptide. [UUJiJ une aspect ot tne invention encompasses a tusion protein suitable tor treating an individual with a tumor comprising a suicide polypeptide sequence and a protein transduction domain sequence (PTD), wherein the PTD comprises a sequence YGRKKRRQ. In addition, the PTD does not comprise a sequence RKKRRQRRR or a sequence YGRKKRRQRRR or an entire HIV Tat sequence. In another aspect of the invention, the PTD consists essentially of a sequence YGRKKRRQ. In a third aspect of the invention the PTD consists of a sequence YGRKKRRQ.

[0032] In accordance with the invention, such proteins can be administered to an individual with a tumor, being introduced for instance into the vicinity of a tumor. Because these sequences impart transduction capability to the protein, these proteins can enter tumor cells. This ultimately results in a decrease in tumor burden by virtue of tumor cell death or injury effected through the suicide polypeptide portion of the protein.

[0033] The invention further encompasses a nucleic acid construct encoding the protein of the invention. The nucleic acid may also be included in a vector designed to facilitate manipulation and/or expression of a nucleic acid. A vector as contemplated by the present invention is at least capable of directing the expression of the nucleic acids encoding the fusion proteins. "Shuttle vectors" comprise the attributes of more than one type of vector. Suitable vectors in the invention also include constructs such as a phage, virus, plasmid, phagemid, cosmid, YAC, or episome. The circular plasmid form, supercoiled, and the linear form also fall within the scope of this invention. The vectors of the invention generally include a transcription unit comprising a polynucleotide sequence comprising the sequence of a fusion protein, which sequence is comprised of a suicide polypeptide fused in frame with the PTD, wherein the PTD comprises or consists essentially of or consists of the sequence YGRKKRRQ. The transcription unit also includes the elements necessary for the expression of the fusion protein in vivo. For instance, the transcription unit may comprise a constitutive or inducible promoter capable of ensuring, in the host cells, the expression of the gene inserted under its control. Examples of suitable promoters include the cytomegalovirus early promoter CMV-IE, of human or murine origin, or optionally of other origin such as rat or guinea pig. More generally, the promoter can be of viral origin or of cellular origin. As a viral promoter other than CMV-IE, there may be mentioned the SV40 virus early or late promoter or the Rous Sarcoma virus LTR promoter. Cellular promoters include those for cytoskeleton genes, such as for example the desmin promoter, or alternatively the actin promoter, i ne vector may also include downstream transcription termination sequences, and remaining vector sequences, for instance, control regions. Suitable origins of replication include, for example, the SV40 viral origin of replication. Suitable termination sequences include, for example, SV40 polyadenylation signal.

[0034] In one embodiment, the PTD-suicide gene product is delivered to the tumor cells through the use of a vector or expression construct encoding the PTD-suicide gene product, wherein the vector or expression construct allows for expression of the PTD-suicide gene in the tumor cells. In one embodiment, the PTD-suicide gene vector or expression construct is introduced to the target cells in the form of a nucleic acid vector in a manner that facilitates cell uptake of the vector. In one embodiment, an electrotransfer procedure is used to enhance cell uptake of the vector or expression construct. If needed, a prodrug is administered after the expression construct is delivered.

[0035] As used herein, "electrotransfer," also called "electroporation," involves the subjecting cells to a high- voltage electric field, and is well established in the art. Transfection efficiencies can be tightly regulated by altering such parameters as frequency, pulse duration and pulse number. In a preferred embodiment, DNA is administered (especially by injection) into tissue and voltage pulses are applied between electrodes disposed in the tissue, thus applying electric fields to cells of the tissue. The electrically- mediated enhancement covers administration using either iontophoresis or electroporation in vivo. Suitable techniques of electroporation and iontophoresis are provided by Singh et al. (1989) Drug Des. Deliv. 4:1-12; Theiss U et al. (1991) Methods Find. Exp. Clin. Pharmacol. 13:353-359; Singh and Maibach (1993) Dermatology. 187:235-238; Singh and Maibach (1994) Crit. Rev. Ther. Drug Carrier Syst. 161-213; Su et al. (1994) J. Pharm. Sci. 83:12-17; Costello et al. (1995) Phys. Ther. 75:554- 563; Howard et al. (1995) Arch. Phys. Med. Rehabil. 76:463-466; Kassan et al. (1996) J. Amer. Acad. Dermatol. 34:657-666; Riviere et al. (1997) Pharm. Res. 14:687- 697; Zempsky et al. (1998) Amer. J. Anesthesiol. 25:158-162; Muramatsu et al. (1998) Int. J. MoI. Med. 1 :55-62; Garrison J. (1998) Med. Device Technol. 9:32-36; Banga et al. (1998) Trends Biotechnol. 16:408-412; Banga et al. (1999) Int. J. Pharm. 179:1-19; Singh et al. (1999) Anticancer Drugs. 10: 139-146; Neumann et al. (1999) Bioelectrochem. Bioenerg. 48:3-16; and Heiser (2000) Methods MoI. Biol. 130:117-134.

[0036] In another embodiment, cells containing the PTD-suicide gene expression construct are delivered to the tumor and the prodrug is subsequently administered. The PTD- suicide gene product is spread trom the introduced cells to the tumor cells. In one embodiment, cells containing the PTD-suicide gene expression construct are attached to a support matrix, such as a microcarrier, prior to delivery to the tumor cells.

[0037] Generally, the microcarrier (MC) is made of material which is preferably nontoxic, for example, glass, polystyrene, polypropylene, polyethylene, polycarbonate, polypentane, acrylonitrile polymer, nylon, magnetite, natural polysaccharide, a modified polysaccharide, collagen, gelatin and modified gelatin such as crosslinked gelatin.

[0038] In one embodiment, a microcarrier of gelatin is a preferred support matrix, as described for example in U.S. Patent Nos. 4,935,365; 6,060,048; and 6,210,664. Suitable gelatin microcarriers are commercially available as Cultispher® porous microcarriers. These gelatin microcarriers generally have diameters ranging from about 80 μm to about 330 μm. In some instances, in the practice of the claimed invention are Cultispher-S®, porous microcarriers of crosslinked gelatin having a diameter between about 80 μm to about 170 μm (mean of about 120 μm). The material and size of the microcarrier will depend on the particular type 'of cells that are attached, the size of the attached cells, the number of cells that can be attached to the microcarrier based on size and/or material of the microcarrier, and the like.

[0039] The configuration of the support is preferably spherical, as in a bead, but may be cylindrical, elliptical a flat sheet or strip, a needle or pin shape, and the like. Bead sizes may range from about 10 μm to about 1000 μm in diameter, for example from about 90 to about 150 μm, or for example around 100 μm. For a description of various microcarrier beads, see, for example. Fisher Biotech Source 87-88. Fisher Scientific, Co., 1987, pp. 72-75; Sigma Cell Culture Catalog, Sigma Chemical Co., St. Louis, 1991, pp. 162-163; Ventrex Product Catalog, Ventrex Laboratories, 1989.

[0040] To improve cell adhesion, survival and function, the solid matrix may optionally be coated on its external surface with factors known in the art to promote cell adhesion, growth or survival. Such factors include cell adhesion molecules, extracellular matrix, such as, for example, fibronectin, laminin, collagen, elastin, glycosaminoglycans, or proteoglycans or growth factors. Alternatively, if the solid matrix to which the implanted cells are attached is constructed of porous material, the growth- or survival-promoting factor or factors may be incorporated into the matrix material, from which they would be slowly released after implantation in vivo. The use of any survival or growth promoting material would depend of its eπect on me αesireα outcome, i.e., me reduction ot tumor growth and/or tumor cell number.

[0041 J Determining the amount of nucleic acid, proteins, or cells of the invention that is suitable for administration to a particular individual is routine to one of skill in the art. The exact amount required will depend on a number of variables, as will be apparent to one of skill in the art. For instance, the severity and type of the tumor, and the efficacy of the suicide polypeptide must be considered when selecting the amount administer to the subject, as well as the patient's age, weight, condition, and the exact method of administration selected. For polypeptide therapeutics, for example, the dosage can be in the range of about 5 μg to about 50 mg/kg of patient body weight. For polynucleotide therapeutics, depending on its expression in the patient, nucleic acid can be administered for instance in a range of about 100 ng to about 200 mg.

[0042] In one aspect, the invention relates to methods of making the nucleic acids, proteins and cells of the invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, virology, animal cell culture and biochemistry which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook, Fritsch & Maniatis, 1989); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al, eds., 1987); "Current Protocols in Protein Science" (John E Coligan, et al. eds. Wiley and Sons, 1995); and "Protein Purification: Principles and Practice" (Robert K. Scopes, Springer- Verlag, 1994).

[0043] Further in accordance with the present invention is a method of preparing a protein of the invention by maintaining a cell containing a nucleic acid encoding the protein under conditions that allow the cell to express the protein. For example, this method can include the steps of (1) inserting a nucleic acid sequence encoding a PTD into an expression vector in-frame with nucleic acid sequence encoding a suicide polypeptide or visa versa, wherein the resulting open reading frame is operatively linked to a promoter; (2) introducing the vector into a host cell; and (3) maintaining the host cell under conditions allowing expression of the vector, resulting in expression of a fusion protein. The nucleic acids or vectors may be introduced into the host cell by any one of established methods, such as transformation, transfection (e.g., with calcium phosphate or DEAE dextran), lipofection, infection and electroporation.

[0044] In another aspect, the invention includes a method of attaching a cell to an implantable support matrix by placing the cell in contact with the support matrix for a sufficient time to allow the cell to attach to the support matrix. For example, the cell may be suspended in growth medium and preincubated for hours with support matrix prior to implantation. Alternatively, the cells may be allowed to grow over days on the support matrix.

[0045] In yet another aspect, the invention provides kits for use in any of the methods described herein. In some aspects, the kit comprises any of the proteins, nucleic acids, vectors and cells described herein in combination with a pharmaceutically acceptable carrier, and instructions for their use in any of the methods described herein.

[0046] An aspect of the invention is the use of the proteins and/or nucleic acids of the invention as a medicament. The products of this invention can be used as a medicament for treatment of the human or animal body. The medicament contains a clinically effective amount for treatment of a disease such as cancer. These compositions can be used for administration to a subject suspected of having or being at risk for the disease, optionally in combination with other forms of treatment appropriate for their condition.

[0047] The examples provided herein are to illustrate, but not limit, the invention.

EXAMPLES

GENERAL TECHNIQUES

Expression Constructs

[0048] Expression constructs and vectors were made using standard PCR and cloning methods known in the art. Primer 1 (CCCAAGCTTGTTAGCCTCCCCCATCTC) and primer 2 (CCGCTCGAGATGGGAGGTGGAGGTTATGGCAGGAAGAAGCGGAGACAG GCTTCGTACCCCTGCCATC) were used to create the Tat8-TK fusion protein. Primer 1 and primer 3

(CCGCTCGAGATGGGAGGTGGAGGTTATGGCAGGAAGAAGCGGAGAC AGCGACGAAGAGCTTCGTACCCCTGCCATC) were used to make the Tatl 1-TK fusion protein. Primer 1 and primer 4 (CCGCTCGAGATGGCTTCGTACCCCTGCC) were used to ampiny me i K gene. ruK. proαucts were cloned m ptαblvi- 1 vector (Promega, Madison, Wl, USA). Confirmation of the correct sequence was performed by direct sequencing of the recombinant plasmids with universal primers T7 and SP6. The cloned sequences were then digested with Xhol and HindIII and inserted into the same restriction sites of the pEGFP-Nl plasmid (Clontech). The resultant vectors were designated as pTat8-TK-EGFP, pTatl 1-TK- EGFP and pTK-EGFP.

[0049] Primer 5 (CCGCTCGAGTCACCGAGCTGAGAAGCAG) and primer 6 (CCGGCTAGCCTCGAGATGGGAGGTGGAGGTTATGGCAGGAAGAAGCGGAGACA GGAGCCCAGTATCTTGCTCC) were used to make the Tat8-CYP2B1 fusion protein. Primer 5 and primer 7

(CCGGCTAGCCTCGAGATGGGAGGTGGAGGTTATGGCAGGAAGAAG CGGAGACAGCGACGAAGAGAGCCCAGTATCTTGCTCC) were used to create the Tatl 1-CYP2B1 fusion protein. Primer 5 and primer 8

(CCGGCTAGCCTCGAGATGGAGCCCAG TATCTTGCTCC) were used to amplify the CYP2B1 gene. PCR products were cloned in pGEM-T vector (Promega). The correct sequence was confirmed by direct sequencing of the recombinant plasmids using universal primers T7 and SP6. The cloned sequences were digested with Nhel and Xhol and inserted into the same restriction sites of the pSecTag2/Hygro plasmid (Invitrogen). The resultant vectors were designated as pSecTat8-CYP2Bl, pSecTatl 1-CYP2B1 and pSecCYP2Bl.

[0050] For the electrotransfer experiments, plasmids were expanded into E.coli strain JMl 09 and purified with the EndoFree plasmid Giga kit (Qiagen GmbH, Hilden, Germany) in accordance with the supplier's protocol. DNA was dissolved in Endofree TE buffer and kept frozen at a concentration of 5 μg/μl.

Cell Culture and Transfections

[0051] NIH3T3 (Swiss mouse embryo fibroblasts) and COS-7 cells (Monkey African green kidney, SV40 transformed) were purchased from the American Type Cell collection (ATCC). NP-18 cells were derived from a poorly differentiated liver metastasis from a human adenocarcinoma of the pancreas that had been perpetuated as a xenograft in nude mice. NP-18 cells have an epithelial morphology with a doubling time of 36 ±2 hours. This cell line is tumorogenic and develops distant metastasis when injected intrapancreatically (Reyes et al. (1996) Cancer Res 56:5713-5719). IUU52] (Jell culture and transfections were pertormed using standard methods, techniques and reagents known in the art. NIH3T3 cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 mg/ml), streptomycin (100 mg/ml), and glutamine (2mM) (Gibco BRL, Life Technologies, Paisley, UK). NP-18 cells were mantained in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 mg/ml) and streptomycin (100 mg/ml). Transient transfections were performed by seeding 50,000 NIH3T3 cells in 60 mm² cell culture dishes. Transfections were performed 24 hours later using Superfect transfection reagent (Qiagen, GmbH, Hilden, Germany) according to the manufacturer's protocol employing 5 μg of plasmid DNA. When stated, 1 μg of the pCMV β-galactosidase plasmid was cotransfected to normalize for transfection efficiency. 24 hours later, either GCV (10 μg/ml) or CPA (1 mM) treatment started and cell viability was measured 5 or 3 days later, respectively. Stable cell lines expressing the TK or the Tat-8TK genes were obtained after transfection of 10 μg of either pTat8 -TK-EGFP or pTK-EGFP plasmids with Superfect transfection reagent. After transfection, cells were seeded in 100 mm² cell culture dishes and subjected to G418 (Sigma, Poole,UK) selection (800 μg/ml) for 14 days. G418-resistant clones were analyzed for GCV sensitivity and GFP expression.

Coplating Assay

[0053] To evaluate Tat mediated spread, cell viability was determined for mixed cell populations composed of different percentages of NIH3T3/Tat8-TK or NIH3T3/TK stable cell lines and NIH3T3 wild type cells or NP-18 wild type cells, following GCV exposure. 1x10⁴ or 2x10⁴ cells were plated at different ratios in quadruplicate in 24-well plates. 24 hours later, cells were incubated with 10 μg/ml of GCV and cell viability was measured 5 days later using standard methods.

Conditioned media assay

[0054] 1.5x10⁵ NIH3T3/Tat8-TK cells, NIH3T3/TK cells and NIH3T3 wild type cells were lated in duplicate and treated with GCV when stated. In the transient transfection experiments d0⁵ COS-7 cells were cotransfected with 5 μg of the Tat8-TK or TK constructs and 1 μg of te pCMV -galactosidase to normalize for transfection efficiency and treated with GCV when ated. Seventy two hours later, supernatants were removed and centrifuged at 600 g for 20 min. ne ml of these media were mixed with an equal volume of fresh DMEM + 10% FBS medium and GCV was added when stated. 5x104 NIH3T3 wild type cells were cultured in the presence of these conditioned media for two days and then cell viability was measured.

Immunoprecipitation

[0055] 1.5x10⁵ NIH3T3/Tat8-TK cells and NIH3T3 wild type cells were plated in duplicate in 60 mm³ plates in the presence or absence of GCV. In the transient transfection experiments IxIO⁵ NIH3T3 cells were transfected with 5 μg of the Tat8-TK or TK constructs and treated with GCV when stated. Seventy two hours later, supernatants were centrifuged at 600 g for 20 min and pre-incubated for 1 hour at 4⁰C with protein-G beads (Amersham Biosciences, Wikstrδms, Sweden). After centrifugation supernatants were incubated overnight with protein-G beads pre-bound with anti-EGFP antibody (Molecular Probes, Leiden, The Netherlands). After several washes with PBS, protein complexes and protein lysates from NIH3T3/Tat8-TK cells and NIH3T3 wild type cells were analysed by electrophoresis. Western blotting was done by standard methods and visualized by the enhanced chemiluminescence detection system (Amersham Biosciences). EGFP was detected with an anti-EGFP monoclonal antibody (Chemicon International, Temecula, CA, USA).

Animals

[0056] Male BALB/c nude mice were used in all the in vivo experiments. Mice were fed ad libitum and maintained under a 12 hour light/dark cycle. AU the animal procedures were previously approved by the Animal Ethics Committee of the Autonomous Government of Catalonia, and performed in accordance with recommendations for the proper care and use of laboratory animals. When stated, animals were anaesthetized intraperitoneally with a combination of 2,2,2-Tribromethanol 97% and 2,2,2-Tribromoethanol 97% (Avertin, Aldrich) at a working solution of 20 mg/mL.

[0057] Tumor xenografts were developed after subcutaneous inj ection of 1 x 10⁷ NP- 18 into the flanks of BALB/c nude mice. Tumor volume was measured every other day and was calculated according to the formula V(mm³ ) = longest diameter (mm) x smallest diameter² (mm²)/2. ivxicrocarriers

[0058] Dry gelatin microcarriers (MC) were rehydrated in PBS for one hour at room temperature, followed by washing twice with PBS and once with DMEM. The final MC suspension contained 500 μg of MC per ml of serum-supplemented DMEM.

[0059] Cell attachment: 1x106 of either NIH3T3/TK or NIH3T3/Tat8-TK cells were incubated with 250 μg of the above pretreated microcarriers in 1 ml of DMEM in a siliconized polypropylene tube (Sigma) at 37°C. After 24 hours of attachment, the medium was removed and the MC-cells were washed twice with PBS and re-suspended in 200 μl of PBS for injection. In vivo: 5μl of MC-cells were injected into subcutaneous tumors developed in nude mice. GCV treatment was initiated two days after injection and lasted for 14 days. Monitoring of tumor growth was performed every other day.

In vivo DNA electrotransfer

[0060] DNA electrotransfer was performed on anaesthetized animals. 50 μg of plasmid DNA were injected into the subcutaneous tumors using a 33-gauge needle. The injected volume was 10 μl. Following the intratumoral injection of the plasmid DNA, an electrical field was applied to the area surrounding the injection site. Tumors were held by tweezer style in vivo electrodes and electric pulses were delivered using a square-wave electric pulse generator (BTX 820 electroporator; Genetronics Inc., San Diego). In all the experiments, eight 20 ms pulses were delivered at a frequency of 1 Hz and an output voltage of 500V/cm (Slack et al. (2002) J Gene M?<i4:381-389). A conductive gel applied to the tumor ensured electrical contact with the skin.

Detection of EGFP expression in electrotransferred tumor

[0061] Mice were sacrificed and perfused with PBS and 4% paraformaldehyde in PBS 48 hours after the gene transfer procedure. Tumors were then excised and fixed with 4% paraformaldehyde in PBS overnight. After washing with PBS, the tumors were sliced (20 μm) with a vibratome (Leica VTlOOOS) and sections mounted onto slides. Enhanced green fluorescent protein (EGFP) expression was viewed directly under a fluorescent microscope (Leica DMR). Images were captured using a digital camera (Spot RT Colour, Diagnostic Instruments) with SPOT Advanced version 3.2.4. Apoptosis determination by TUNEL assay

[0062] Tunel analysis was performed using an in situ death detection kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Briefly, frozen tissue sections (5 μm) were fixed in 4% paraformaldehyde for 20 min at room temperature, incubated with blocking solution (3% H₂O₂ in methanol), and then permeabilized for 2 minutes on ice with 0.1% Triton X-IOO in 0.1% sodium citrate. The TUNEL reaction mixture was prepared using a 9:1 buffer-to-enzyme ratio, and sections were incubated in a humidified chamber for 1 hour at 37⁰C. After rinsing three times for 5 minutes with PBS, sections were mounted using diaminophenylindole in the mounting medium, and apoptotic nuclei were viewed under a fluorescent microscope (Leica). Images were captured using a digital camera (Spot RT Colour, Diagnostic Instruments) with SPOT Advanced version 3.2.4. A negative control was established using the labeling solution without terminal transferase on sections as described above.

Histology

[0063] Tumors were excised and fixed with 4% paraformaldehyde in PBS overnight. After washing with PBS, they were submitted to standard paraffin processing and sectioned at 5 μm with a microtome (Leica RM2135). Sections were stained with hematoxilyn and eosin.

Statistical analysis

[0064] Due to repeated measurements for each animal and to unbalanced designs, general linear-mixed models were used to estimate the effect of the treatment on the tumor growth. These models allow the analysis of the overall effect, at fixed times and among the different groups (control versus treated). All models included the mice as random effect. Tests for a random intercept and slope were statistically significant. Estimation of coefficients, their standard errors and p-values were based on restricted maximum likelihood. Comparisons of models were based on likelihood ratio tests derived from model fits using maximum likelihood fit. In order to check the models, the residuals were plotted versus fitted values. The variance function structure was used to model heterocedasticity in the day-to-day errors. Expected values derived from the model including interaction term were used to plot the effect of tumoral growth in relation to the group and the day of evaluation. All the analyses were performed with S-PLUS functions using the nlme library (Pinheiro et al. Mixed-Effects Models in S and S-PLUS: New York, 2000). Statistical significance was considered when p<0.05.

EXAMPLE 1

An 8 amino acid peptide from HIV Tat enhances cytotoxicity of suicide gene

[0065] In order to investigate the ability of the Tat peptide to transport suicide proteins from expressing cells to recipient cells, expression vectors were constructed using a previously used 11 amino acid peptide (YGRKKRRQRRR) and a new shorter 8 amino acid peptide (YGRKKRRQ) of the HIV Tat protein fused to the N-terminal of two different suicide genes, the thymidine kinase (TK) gene and the cytochrome P450 2Bl (CYP2B1) gene. The TK chimeric proteins were also fused to the enhanced green fluorescent protein (EGFP) for monitoring expression of the proteins. EGFP protein was not fused to the Tat- CYP2B1 proteins. Constructs with the respective backbone carrying the unmodified TK or CYP2B1 genes were made for control constructs. The various constructs are diagrammed in Fig. Ia and Fig. Ic. NIH3T3 cells were transfected with the indicated constructs and the CYP2B1 constructs were cotransfected with the pCMVβ-galactosidase plasmid in order to normalize for transfection efficiency. The day after transfection, TK or CYP2B1 transfected cells were exposed to 10 μg/ml of ganciclovir (GCV) or ImM of cyclophosphamide (CPA) for five or three days respectively, and cell viability was determined. Extensive cell death was observed in all treatments and an enhanced cytotoxicity was always observed in the Tat expressing cells from both the TK and the CYP2B1 constructs (Fig. Ib and Id). Similar effects were obtained with fusions with the 11 or 8 amino acid peptide Tat, thus indicating that the shorter 8 amino acid sequence was also functional as a PTD.

[0066] This result indicates that the increased effect is not dependent on a specific construct, thus suggesting that at least for suicide gene therapy, Tat domains enhance resulting cytotoxicity. bXAMPLb λ

Release ofTat8-TKfrom cells

[0067] To determine whether the Tat8-TK fusion protein was able to spread from primary expressing cells into neighboring cells, stable NIH3T3 cells expressing the TK or the Tat8- TK proteins were established. To this end, NIH3T3 cells were transfected with the TK or the Tat8-TK expression vectors and subjected to G418 selection. Clones expressing the EGFP and sensitive to ganciclovir treatment were selected for the studies.

[0068] In order to demonstrate whether the enhanced Tat8-TK cytotoxicity could be explained by the intercellular spreading of the Tat8-TK protein, we performed a group of experiments in stable cell lines expressing TK or Tat8-TK and in cultures transiently transfected with the TK or Tat8-TK plasmids. To this end NIH3T3 wild type cells were cultured for two days in the presence or absence of GCV with conditioned media either from NIH3T3 wild type, NIH3T3/TK, NIH3T3/Tat8-TK cultures or from COS-7 cells previously transfected with the DNA plasmids Tat8-TK or TK treated or untreated with GCV for 3 days. As shown in Figure 7a, NIH3T3 wild type cells cultured with conditioned media from NIH3T3/Tat8-TK cells treated with GCV showed a significantly reduced viability (62%). However, no effect was observed in the same type of cultures derived from the NIH3T3/TK cells. Moreover, NIH3T3 wild type cell cultured with conditioned media from COS-7 cells transfected with the Tat8-TK construct and treated with GCV also showed an increased statistically significant cell death (30%). Again, this effect was not observed in the cultures that received TK conditioned media (Figure 7c). These results suggested that the Tat8-TK protein but not the TK protein was able to exert a cytotoxic effect in the non-genetically- transduced cells. Interestingly, this enhanced cytotoxicity was observed only in the cultures in which the conditioned media was derived from Tat8-TK expressing cells previously treated with GCV, suggesting that the release of Tat8-TK protein to the extracellular media required the killing of the Tat8-TK expressing cells.

[0069] We next analysed the extracellular levels of Tat8-TK or TK proteins in GCV- treated and non-treated Tat8-TK- or TK- expressing cells, talcing advantage of the fused EGFP in these constructs. Extracellular media free of dead or detached cells coming from GCV-treated or untreated Tat8-TK- or TK- expressing cells were immunoprecipitated with an anti-EGFP antibody and analysed by Western blot. As shown in figures 7b and 7d, a band corresponding to the Tat8-TK or TK proteins was present only in the media of treated cells, indicating that the release of these proteins required the killing of the expressing cells. No protein was detected in similar experiments carried out with media from NIH3T3 wild type cells (data not shown). Western blot analysis of cell lysates identified a band corresponding to the Tat8-TK or TK proteins fused to EGFP by antibodies directed against EGFP (Figure 7b and 7d).

EXAMPLE 3

Bystander effect ofTat8-TK

[0070] GCV-induced cytotoxicity in neighboring cells was examined in cell mixtures of wild type NIH3T3 cells with NIH3T3TK or NIH3T3Tat8-TK expressing cells. Different ratios of NIH3T3TK or NIH3T3Tat8-TK-expressing cells were plated in quadruplicate in 24- well plates with NIH3T3 wild type cells or NP 18 wild type. Twenty-four hours later, the cells were incubated with 10 μg/ml of GCV. Cell viability was measured after 5 days of treatment. Among the various cocultures studied, the strongest cytotoxicity was observed in the mixtures with NIH3T3 wild type and NIH3T3Tat8-TK. Particularly in the conditions where only 5% of TK or Tat8-TK expressing cells were present, 70% cell death was achieved in the mixtures of the TK expressing cells whereas 90% cell death was obtained in the Tat8- TK mixtures (Fig. 2a). Similarly, when 20% of TK or Tat8-TK expressing cells were present, about 7% cell survival was seen in the mixtures of the TK expressing cells whereas about 1 to 2% cells survived in the Tat8-TK mixtures (data not shown). Similar results were observed with xenogeneic cell cocultures of NIH3T3TK or NIH3T3Tat8-TK expressing cells cultured with NP-18 wild type pancreatic tumor cells (Fig. 2b). Without being bound by any particular theory, the enhanced effect in the Tat8-TK cocultures appears to be related to the ability of Tat8-TK protein to enter into non-transfected cells.

[0071] Proteins fused to PTDs probably enter cells through a combination of different mechanisms that may slightly differ from one PTD to the other. Lundberg et al. (2003) MoI Ther 8: 143-150. The Tat PTD has been proposed to enter the cell through the heparan sulfate proteglycans receptors, through a caveolar-mediated endocytic pathway, or through lipid raft-dependent macropinocytosis. Tyagi et al. (2001) J Biol Chem 276:3254-3261 ; Ferrari et a!. (2003) MoI Ther 8:284-294; Wadia et al. (2004) Nat Med 10:310-315. [0072] Evidence of intercellular spreading of the Tat8-TK/EGFP protein is provided herein. Protein transduction was not visualized following EGFP expression, immunofluorescence or by flow cytometry analysis. However, a strong enhanced cytotoxic response was observed with the CYP2B1/CPA and the TK/GCV systems. Moreover, the coplating experiments performed with the TK/GCV system indicates the presence of a strong bystander effect. The low confluency of the cultures due to the very early elevated cell mortality suggests that this enhanced bystander is likely independent of the traveling of toxic metabolites. Without being bound by any particular theory, an explanation for the enhanced cytotoxicity achieved with the Tat8-TK constructs is that the killing of Tat8-TK expressing cells by ganciclovir results in the release of the fused proteins, which in turn are internalized by neighboring cells and trigger their killing. This can lead to an amplification mechanism of cell death, not mediated by intracellular trafficking but by enhancing uptake of the post- released TK. Alternatively, or additionally, the Tat motif may introduce a conformational change in the TK or CYP proteins which transforms them into more active enzymes. In any event, the Tat8-suicide gene fusion in combination with the prodrug is effective in killing a population of cells.

EXAMPLE 4

The use of micro carrier as a delivery vehicle for cells expressing Tat8-TK/GCV

[0073] Efficacy of Tat8-TK in combination with GCV in antitumor therapy was tested using two independent gene transfer approaches: cell-microcarrier gene delivery and DNA electrotransfer method.

[0074] Microcarrier cell culture technology has been successful in producing viral vaccines, recombinant proteins, and viral vectors for gene therapy. See, for example, Kistner et al (1998) Vaccine 16:960-968; Goldman et al. (1998) Biotechnol Bioeng 60:596-607; Wu et al. (2002) Biotechnol Prog 18:617-622. The use of gelatin microcarriers attached to human retinal pigment epithelial cells stereotaxically implanted in rodents and in non-human primate models of Parkinson's disease (PD) have produced long term amelioration of motor and behavioral deficits, and similar results were obtained in a pilot clinical study with human patients with advanced PD (Watts et al (2003) J Neural Transm Suppl 215-227; Bakay et al. (2004) Front Biosci 9: 592-602). [0075] NIH3T3 TK or NIH3T3Tat8-TK expressing cells attached to microcarriers were inoculated into NP-18 tumors developed in the subcutaneous tissue of nude mice (Fig. 3a). Three groups of animals were used: MC-control group injected with microcarriers; MC-TK group injected with microcarriers attached to NIH3T3-TK expressing cells; MC-Tat8-TK group injected with microcarriers attached to NIH3T3-Tat8-TK expressing cells. After two days, animals were treated for 14 days with GCV 50 mg/Kg and sacrificed two days later. Statistically significant differences were observed in the growth curves among controls and both TK (p=0.002) and Tat8-TK (pO.OOOl) groups. An inhibition of growth was observed in the group that received TK/GCV until day 13, although tumors started to regrow on days 15 and 17. More importantly, the group injected with cells attached to microcarriers, MC+Tat8-TK, showed a 35.6 % reduction in the initial tumor volume, which was already evident on day 8 after treatment and persisted throughout the entire treatment. Statistically significant differences in tumor growth between the MC+TK and the MC+Tat8-TK groups were detected, with the MC+Tat8-TK group revealing an increased response to GCV treatment (p<0.0001). Moreover 50% of the tumors from this group were completely eradicated (Fig. 3b).

[0076] Direct injection of P A317 cells producing TK retroviral vectors into pancreatic tumors higher than 40 mm³ did not have a significant effect in tumor progression, even though a high number of cells were injected (Carrio et al. (1999), Supra). On the contrary, as shown herein, the implantation of a low number of NIH3T3-TK or NIH3T3-Tat8TK expressing cells attached to gelatin microcarriers enhanced tumor cytotoxicity. A slight increase in tumor volume was observed on day 2 after microcarrier injection in control and treated groups, probably due to the contribution of the microcarriers to the final volume. However, on day 6, tumors went back to the initial volume and from then on the treated groups started to show the therapeutic effect. Both NIH3T3-TK and NIH3T3-Tat8-TK injected tumors treated with GCV for 14 days showed a reduction in tumor progression. However, an enhanced tumor reduction in the Tat8TK group was observed and was maintained throughout the treatment period. The enhanced therapeutic effect could most likely be due to the transduction properties of the released Tat8-TK protein from the dead cells, leading to an increased killing in cells without the expression vector but nearby in the population, the bystander effect. EXAMPLE 5

Delivery by electrotransfer

[0077] Delivery of Tat8-TK by DNA electrotransfer was also tested in the efficacy of the Tat8-TK/GCV system. DNA electrotransfer methodology has been shown to be very powerful for gene delivery not only intramuscularly but also intratumorally. Moreover, electrochemotherapy has been used in clinical studies since 1991, proving that it is feasible, efficient and well tolerated. Mi et al. (1999) Proc Natl Acad Sci USA 96:4262-4267; Ivanov et al. (2003) J Gene Med 5:893-899; Riera et al (2004) J Gene Med 6:111-118; Sersa et al. (2003) Cancer Therapy 1 :133-142; Goto et al. (2000) Proc Natl Acad Sci USA 97:354-359.

[0078] Subcutaneous NP-18 tumors in mice were injected with the Tat8-TK/EGFP fusion construct with or without in vivo electroporation (EP). Two days later, tumors were sliced and EGFP expression was observed under a fluorescent microscope to determine the extent of intratumoral transgene expression. Tumors from the control group, which received DNA injection without EP, expressed no EGFP. In contrast, tumors that received plasmid DNA with EP showed bright and numerous EGFP expressing cells covering a wide area of the tumor (Fig. 4). Expression could also be observed after 7 and 14 days of electroporation, although at lower levels.

EXAMPLE 6

Tumoricidal effect at high doses of GCV

[0079] To examine the anti -tumor activity of Tat8-TK and TK in combination with GCV after electroporation, NP-18 pancreatic cancer cells were injected s.c. into nude mice and tumors were allowed to establish until they reached a mean volume of 80 mm³. Animals were then randomized and tumors were treated with intratumoral injections of Tat8-TK or TK, followed by electroporation. In one series of in vivo experiments, a high dose of GCV (100mg/Kg) was injected into the animals for a short period of time and tumor volume was monitored on days 2, 4 and 6. No toxicity of GCV was observed, as shown by the tumor growth in control animals (no statistically significant differences were observed between the two control groups). Both the TK and the Tat8-TK groups showed an overall reduction in tumor growth when compared to control 1 (p=0.0008; pO.0001) and control 2 (p=0.0188 and p<0.001) respectively. A maximum reduction with respect to the initial tumor volume was observed on day 4, with 59.5% for the Tat8-TK group (p=0.0002) and 17.1% for the TK group (p=ns) (Fig. 5a). In both groups, TK and Tat8-TK, the average tumor volumes at day 6 were significantly smaller when compared to those from the two control groups. Notably, complete tumor regression on day 6 was achieved in 5 out of 10 animals treated with Tat8- TK but only in 2 out of 10 animals treated with TK. TUNEL analysis of tumor xenografts was performed to determine whether any differences in apoptotic cell death could be observed between the TK and the Tat8-TK group. Massive cell death was found within the subcutaneous tumor tissues that were processed 2 and 4 days after the treatment, with a marked increase in the percentage of cells that underwent apoptosis in the Tat8-TK group. (Fig. 5b)

[0080] This clearly superior apoptotic cell death observed in the Tat8-TK group may be due to the contribution of the Tat8 peptide to the bystander effect. In fact, in a tumor mass, cells are heavily in contact with each other, and this situation will favor the transduction capabilities of the released protein.

EXAMPLE 7

Tumoricidal effect at moderate doses of GCV

[0081] Antitumoral efficacy of Tat8-TK at lower doses of GCV for an extended period, were studied in a second protocol where tumors were injected with TK or Tat8-TK plasmids, submitted to electroporation and treated with GCV for 14 days at a moderate dose (50 mg/Kg). NP- 18 pancreatic tumors were developed in the s.c. tissue of nude mice. Animals were randomized and treatment was initiated when tumors reached a volume of 70- 80 mm³. Three different groups were established: Control: tumors injected with TK or Tat8-TK plasmid followed by electrotransfer; TK: tumors injected with the TK plasmid followed by electrotransfer; and Tat8-TK: tumors injected with the Tat8-TK plasmid followed by electrotransfer. TK and Tat8-TK groups received GCV 50 mg/Kg for 14 days. As shown in Fig. 6, an enhanced antitumor effect was observed in the Tat8-TK group. In fact, statistically significant differences were observed between the Tat8-TK and the control groups (p<0.0001). Although a tendency to a therapeutic effect in the TK group was observed, no statistically significant differences were determined (p=0.1616). In both groups a reduction in the initial tumor volume was observed on day 9, being of 43.24% for the Tat8-TK group and 20.18% for the TK group. However, significance was only achieved with the Tat8-TK group (p=0.0079). On day 14 tumors from the Tat8-TK group stabilized growth to the initial tumor volume. However, tumors from the TK group reach higher volumes than the initial one. Nevertheless, statistically significant differences were observed on day 14, when control tumors are compared to each treated group (Tat8-TK p=0.002; TK p=0.0106). In both groups, 30% of tumors were completely eradicated.

[0082] Thus, the reduction in tumor progression in the Tat8-TK treated group was statistically significant at all the time points studied, whereas in the TK/GCV group significance was only reached at the end of the treatment. It is worthwhile to note that whereas on days 9 and 11 the volume of tumors from the Tat8-TK group was lower than the initial volume, by the end of the treatment the tumors had reached the initial volume, thus showing a tendency to regrow. At 14 days after the Tat8-TK administration together with GCV treatment, no tumor cells with Tat8-TK may remain and the remaining cells may actively contribute to tumor growth.

[0083] The tendency of the tumor to regrow was not observed in the microcarrier experiments, where a persistent effect was seen throughout the entire treatment, probably because the NH3T3 TK or Tat8-TK expressing cells were not completely killed by GCV after 14 days. Thus, a way to achieve a more sustained effect on tumor reduction by the electrotransfer approach would be to readminister the therapy on day 9. Because of low immunogenicity characteristic of plasmid DNA, the repeated use of this therapy at some interval in clinical use could be feasible. In sum, these experiments demonstrate the use of the protein transduction domain Tat8 clearly enhances the efficacy of the TK/GCV therapy in tumor models of pancreatic cancer.

Claims

1. A protein comprising a suicide polypeptide sequence and a protein transduction domain sequence (PTD), wherein the PTD comprises a sequence YGRKKRRQ, the PTD does not comprise a sequence ROLRRQRRR, the PTD does not comprise a sequence YGRKKRRQRRR, and the PTD does not comprise an entire HIV Tat sequence.

2. A protein comprising a protein transduction domain sequence (PTD) and a suicide polypeptide sequence, wherein the PTD consists essentially of a sequence YGRKKRRQ.

3. A protein comprising a protein transduction domain sequence (PTD) and a suicide polypeptide sequence, wherein the PTD consists of a sequence YGRKKRRQ.

4. A nucleic acid encoding the protein of claim 1, 2 or 3.

5. A vector comprising the nucleic acid of claim 4.

6. A cell comprising the nucleic acid of claim 4.

7. The nucleic acid of claim 4, wherein the nucleic acid is in the form of DNA.

8. A composition comprising the protein of claim 1, 2 or 3.

9. A composition comprising the nucleic acid of claim 4.

10. A composition comprising the cell of claim 6.

11. A method of treating an individual with a tumor, by administering the protein of claim 1, 2 or 3.

12. A method of treating an individual with a tumor, by administering the nucleic acid of claim 4.

13. The method of claim 12, wherein the nucleic acid is administered by electrotransfer.

14. A method of treating an individual with a tumor, by administering the cell of claim 6.

15. The method of claim 14, wherein prior to administration, the cell is attached to a support matrix.

16. The method of claim 15, wherein the support matrix is in the form of a microcarrier.

17. The method of claim 16, wherein the microcarrier has a diameter from 80 μm to 330 μm.

18. The method of claim 15, wherein the support matrix is made of a material selected from the group consisting of glass, polystyrene, polypropylene, polyethylene, polycarbonate, polypentane, acrylonitrile polymer, nylon, magnetite, natural polysaccharide, a modified polysaccharide, collagen, gelatin and modified gelatin such as crosslinked gelatin.

19. The method of claim 15, wherein the support matrix is coated with factors known in the art to promote cell adhesion, growth or survival.

20. A method of preparing the protein of claim 1, 2 or 3 by maintaining a cell containing a nucleic acid encoding the protein under conditions that allow the cell to express the protein.

21. A method of attaching the cell of claim 6 to a support matrix, by placing the cell in contact with the support matrix for a time sufficient to allow the cell to attach to the support matrix.

22. A kit containing the protein of claim 1, 2, or 3.

23. A kit containing the nucleic acid of claim 4.

24. A container containing the protein of claim 1 , 2 or 3.

25. A container containing the nucleic acid of claim 4.

26. The protein of claim 1 , 2 or 3, for use in therapy.

27. The nucleic acid of claim 4, for use in therapy.

28. The cell of claim 6, for use in therapy.

29. The protein of claim 1, 2, or 3, wherein the suicide polypeptide sequence is derived from thymidine kinase or a fragment thereof.

30. A nucleic acid encoding the protein of claim 29.

31. A cell comprising the nucleic acid of claim 30.

32. A composition comprising the protein of claim 29.

33. A composition comprising the nucleic acid of claim 30.

34. A method of treating an individual with a tumor, by administering the protein of claim 29.

35. A method of treating an individual with a tumor, by administering the nucleic acid of claim 30.

36. A method of treating an individual with a tumor, by administering the cell of claim 31.

37. The method of claim 36, wherein prior to administration, the cell is attached to a support matrix.

38. A kit containing the protein of claim 29.

39. A kit containing the nucleic acid of claim 30.

40. A container containing the protein of claim 29.

41. A container containing the nucleic acid of claim 30.

42. A method of preparing the protein of claim 29, by maintaining a cell containing a nucleic acid encoding the protein under conditions that allow the cell to express the protein.

43. The protein of claim 29, for use in therapy.

44. The nucleic acid of claim 30, for use in therapy.

45. The protein of claim 1, 2 or 3, wherein the suicide polypeptide sequence is a protein derived from cytochrome P450 2Bl or a fragment thereof.