WO1994004196A1 - Tumour therapy - Google Patents

Abstract

A DNA construct comprising (i) means of expression of a coding sequence in a tumour cell and (ii) a said coding sequence encoding a cytokine. The said means for expression may provide for specific expression selectively in tumour cells, particularly melanoma cells, and pancreatic, breast, colonic and prostatic tumour cells and the cytokine is at least one of interleukin-2, interleukin-4, macrophage colony stimulating factor, interferon-η, tumour necrosis factor and interleukin-7.

Description

TUMOUR THERAPY

The present invention relates to the therapy of tumours, particularly melanomas.

Biological therapy of cancer, based upon the adoptive transfer of modified immune cells, seeks to exploit in vivo specificity to deliver recombinant proteins directly to the tumour mass (Parmiani et al (1992) Trends Exp. Clin. Med. 2, 412-419; Rosenberg (1992) J. Clin. Oncol. 10, 180-100). However, this approach involves removal of cells from the patient followed by their in vitro manipulation and replacement in vivo. Proposed vaccination experiments using genetically modified tumour cells also require a similar period of passage in vitro during which time the neoplastic cells may significantly alter their immunological properties or growth characteristics (Rosenberg (1992) loc. cit.; Roemer & Friedmann (1992) Eur. J. Biochem. 208, 211-225; Pardoll (1992) Curr. Opin. Immunol. 4, 619-623); Fearon et al (1990) Cell 60, 397-403.

There is experimental evidence that the expression of cytokines in tumour cells (following transfection with cytokine cDNA in vitro) leads to rejection of otherwise tumourigenic doses of tumour cells and, in some cases, can immunise animals against established diseases when the transfected cells are injected into the animal. Cytokines shown to have this effect include interleukin-2, interleukin-4, interferon-7, tumour necrosis factor and interleukin-7. This information is summarised in Pardoll (1992) Curr. Opinion Immunol 4, 619-623.

CD28-positive T cell responses, and immune responses mediated by T cells, may be regulated by the B7 antigen as described in WO 92/00092. Also, tumour rejection after direct costimulation of CD8⁺ T cells by B7- transfected melanoma cells is described in Townsend & Allison (1993) Science 259, 368-370.

Malignant melanoma represents a cancer the growth and dissemination of which may be altered significantly by immunological manipulation. Many melanomas synthesise the pigment melanin, which is otherwise produced almost exclusively by melanocytes (Hearing & Tsukamoto (1991) FASEB J. 5, 2902-2909) and indeed several workers have proposed utilising the melanin synthetic pathway for chemotherapeutic intervention (Riley (1991) Eur. J. Cancer 27, 1172-1179; Link & Carpenter (1992) Cancer Res. 52, 4385-4390).

The tyrosinase and TRP-1 genes both encode proteins which play key roles in the synthesis of the pigment melanin, a specific product of melanocytic cells. Our aim has been to utilise the 5' ends of the tyrosinase and tyrosinase-related protein (TRP-1) genes to confer tissue specificity of expression on genes cloned downstream of these promoter elements for therapeutic purposes.

A number of other groups already have shown that tissue specificity of expression resides within the 5' sequences of these genes (eg Bradl, M. et al (1991) Proc. Natl. Acad. Sci. USA 88, 164-168; Jackson, I.J. et al (1991) Nucleic Acids Res. 19, 3799-3804). However we have confirmed and expanded these findings and used the promoters of these genes for therapeutic purposes.

Prostate-specific antigen (PSA) is one of the major protein constituents of the human prostate secretion. It has become a useful marker for the detection and monitoring of prostate cancer. Other groups have characterised the gene encoding PSA and have identified the promoter region which directs the prostate-specific expression of PSA (Lundwall (1989) Biochem. Biophys. Res. Comm. 161, 1151-1159; Riegman et al (1989) Biochem. Biophys. Res. Comm. 159, 95-102; Brawer (1991) Acta Oncol. 30, 161-168).

Carcinoembryonic antigen (CEA) is a widely used tumour marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. The complete gene encoding CEA has been cloned and its promoter region analysed. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the adenocarcinoma cell line SW303, compared with the HeLa cell line. This indicates that c«-acting sequences which convey cell type specific expression are contained within this region (Schrewe et al (1990) Mol. Cell. Biol. 10, 2738-2748).

The c-erb -2 gene and promoter have been characterised previously and the gene product has been shown to be over-expressed in tumour cell lines (Kraus et al (1987) EMBO J. 6, 605-610).

The mucin gene, MUCl, contains 5' flanking sequences which are able to direct expression selectively in breast and pancreatic cell lines, but not in non-epithelial cell lines as taught in WO 91/09867.

Summary of the Invention

One aspect of the invention provides a DNA construct comprising (i) means for expression of a coding sequence in a tumour cell and (ii) a said coding sequence encoding a cytokine. Expression of the cytokine in the tumour cells is believed to stimulate attack by T cells, especially LAK cells. Such T cells will then destroy not only the primary tumour but also any secondary (metastatic) growths.

The tumour may be a melanoma, or a tumour of the breast, colon, brain, pancreas, bladder, skin, prostate, stomach, oesophagus or liver, for example. Preferably, it is a melanoma.

Advantageously, the said means for expression provides for specific expression selectively in tumour cells. Otherwise, the T cells may attack normal cells and/or the germ line may be altered.

By "specific expression selectively in tumour cells" we mean that the expression is usefully higher (for example 2X, 5X, 10X or at least 20X higher) in tumour cells compared to the expression in non-tumour cells. It will be appreciated by those skilled in the art that tumour selective expression may be derived from tissue-specific expression where the tumour rapidly grows from a specific tissue type. Alternatively, highly specific delivery of a non-specific expression construct may be adequate. Known means such as targeted liposomes (carrying anti-tumour-marker antibodies) and viruses, including retroviruses, may be employed.

The constructs of the invention may be introduced into the tumour cells by any convenient method, for example methods involving retroviruses, so that the construct is inserted into the genome of the tumour cell. For example, in Kuriyama et al (1991) Cell Struc. and Func. 16, 503-510 purified retroviruses are administered. Retroviruses provide a potential means of selectively infecting cancer cells because they can only integrate into the genome of dividing cells; most normal cells surrounding cancers are in a quiescent, non-receptive stage of cell growth. Retroviral DNA constructs which contain a promoter segment and a cytokine coding sequence may be made using methods well known in the art. To produce active retrovirus from such a construct it is usual to use an ecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS). Transfection of the cell line is conveniently by calcium phosphate co-precipitation, and stable transformants are selected by addition of G418 to a final concentration of 1 mg/ml (assuming the retroviral construct contains a neo^R gene). Independent colonies are isolated and expanded and the culture supernatant removed, filtered through a 0.45 μm pore-size filter and stored at -70°. For the introduction of the retrovirus into the tumour cells, it is convenient to inject directly retroviral supernatant to which 10 g/ml Polybrene has been added. For tumours exceeding 10 mm in diameter it is appropriate to inject between 0.1 ml and 1 ml of retroviral supernatant; preferably 0.5 ml. Alternatively, as described in Culver et al (1992) Science 256, 1550- 1552, cells which produce retroviruses are injected into the tumour. The retrovirus-producing cells so introduced are engineered to actively produce retroviral vector particles so that continuous productions of the vector occurred within the tumour mass in situ. Thus, proliferating tumour cells can be successfully transduced in vivo if mixed with retroviral vector- producing cells. Other methods involve simple delivery of the construct into the cell for expression therein either for a limited time or, following integration into the genome, for a longer time. An example of the latter approach includes (preferably tumour-cell-targeted) liposomes (Nassander et al (1992) Cancer Res. 52, 646-653).

Immunoliposomes (antibody-directed liposomes) are especially useful in targeting to cancer cell types which over-express a cell surface protein for which antibodies are available. In relation to the present invention, antibodies directed towards tumour cell antigens such as CEA and PSA are preferred. For the preparation of immuno-liposomes MPB-PE (N-[4- (p-maleimidophenyl)butyryl]-phosphatidylethanolamine) is synthesised according to the method of Martin & Papahadjopoulos (1982) /. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface. The liposome is conveniently loaded with the DNA construct of the invention for delivery to the target cells, for example, by forming the said liposomes in a solution of the DNA construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 μm and 0.2 μm pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min. Freshly prepared MPB- PE-liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight. The immunoliposomes are separated from unconjugated antibodies by ultracentrifugation at 80 000 x g for 45 min. Immunoliposomes may be injected intraperitoneally or directly into the tumour.

It will be appreciated that monoclonal antibodies or other molecules that bind to tumour cell surface antigens are useful in targeting the DNA - construct of the invention.

Monoclonal antibodies which will bind to many of these antigens are already known but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclotwl Hybridoma Antibodies: Techniques and Applications'', J G R Hurrell (CRC Press, 1982).

Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).

Suitably prepared non-human antibodies can be "humanized" in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies. Such "humanized" antibodies, or fragments thereof, are preferred as they may give rise to a lower anti- antibody reaction than rodent antibodies.

The variable heavy (V_H) and variable light (V- domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V_H and V_L partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated N domains (Ward et al (1989) Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293- 299.

By "ScFv molecules" we mean molecules wherein the V_H and V_L partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

Whole antibodies, and F(ab')₂ fragments are "bivalent". By "bivalent" we mean that the said antibodies and F(ab')₂ fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites.

Other molecules immunologically reactive with the target cell surface molecule are also useful in this aspect of the invention and include, for example minimal recognition units (MRU) and complementarity determining regions.

Other methods of delivery include adenoviruses carrying external DΝA via an antibody-polylysine bridge (see Curiel Prog. Med. Virol. 40, 1-18) and transfeπϊn-polycation conjugates as carriers (Wagner et al (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414). In the first of these methods a polycation-antibody complex is formed with the DNA construct of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody. The polycation moiety binds the DNA via electrostatic interactions with the phosphate backbone. The adenovirus, because it contains unaltered fibre and pentos proteins, is internalized into the cell and carries into the cell with it the DNA construct of the invention. It is preferred if the polycation is polylysine.

In the second of these methods, a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells is employed. This is accomplished by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with DNA constructs of the invention independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and the DNA constructs of the invention are supplied to the tumour cells, a high level of expression from the construct in the cells is expected.

High-efficiency receptor-mediated delivery of the DNA constructs of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used. This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the DNA construct of the invention, the DNA construct is taken up by the cell by the same route as the adenovirus particle.

It may be desirable to locally perfuse a tumour with the delivery vehicle (for example the retrovirus) for a period of time.

In one embodiment of the invention the said means for expression provides for specific expression selectively in melanoma cells or in melanoma cells and melanocytes. In this embodiment the said means for expression is a promoter or an analogue or part thereof forming part of a gene expressed substantially exclusively in the melanin synthesis pathway. Examples of such promoters include the tyrosinase gene promoter and the tyrosinase-related protein (TRP-1) gene promoter.

By "promoter" we mean that region of DNA which controls, at least to a substantial extent, the transcription of the coding region associated with that region of DNA.

In a further embodiment of the invention the said means for expression provides for specific expression selectively in prostate cancer cells or prostate cancer cells and prostate cells. In this embodiment the said means for expression is a promoter or an analogue or part thereof forming part of a gene expressed substantially exclusively in prostate cancer or prostate cells. An example of such a promoter is the prostate-specific antigen (PSA) gene promoter.

In a still further embodiment of the invention the said means for expression provides for specific expression selectively in colonic cancer cells, or colonic cancer cells and colon cells. In this embodiment the said means for expression is a promoter or an analogue or part thereof forming part of a gene expressed substantially exclusively in colon cancer or colon cells. An example of such a promoter is the carcinoembryonic antigen (CEA) gene promoter.

In another embodiment of the invention the said means for expression is provided by the promoter region of the c-erbB2-gcn&.

In this embodiment the constructs comprising the c-erbB2 gene promoter fused to the cytokine coding sequence may be usefully delivered to breast tumours. The c-erbB3 gene promoter may also be used.

In yet another embodiment the said means for expression is provided by the promoter region of the MUCl gene.

In this embodiment pancreatic or breast tumours may usefully receive the constructs comprising MUCl gene promoter fused to the cytokine coding sequence.

DNA sequences encompassing the promoter sequences useful in the invention are given in the sequence listing.

The cytokine is preferably interleukin-2 or interleukin-4 or macrophage colony stimulating factor. Other cytokines may, however, be used, for example interferon-γ, tumour necrosis factor, and interleukin-7. Nucleotide coding sequences for these are known and are given in the sequence listing. The promoter is joined to the cytokine coding region and placed in a suitable vector system for propagation. The skilled person can use the information given below containing the promoter DNA sequences and coding sequences of some of the cytokines useful in the invention to make suitable constructs. For example, a knowledge of the DNA sequences provides information on where restriction enzyme will cleave the said DNA molecules and allows oligonucleotide primers to be designed for PCR amplification and site-directed mutagenesis.

The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA construct of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the propagation of the DNA construct, which can then be recovered.

The vectors usually include a procaryotic replicon, such as the ColEl ori, for propagation in a procaryote, even if the vector is to be used for expression in other, non-procaryotic, cell types.

It is preferred if the host cell is E. coli. A variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.

DNA fragments with complementary cohesive termini are readily joined together by ligation using methods known in the art and described in Sambrook et al (1989) Molecular Cloning, A laboratory manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

A desirable way to modify the promoter fragment, vector or coding region to be fused in the DNA construct is to use the polymerase chain reaction as disclosed by SaiM et al (1988) Science 239, 487-491.

In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.

The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host- cell for propagating the DNA construct can be either procaryotic or eucaryotic. Bacterial cells are preferred host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343).

Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of bacterial, especially E. coli host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208 or by isolating the plasmid vector DNA and then digesting the said plasmid appropriate restriction enzymes that give diagnostic DNA fragments that can be separated and sized by gel electrophoresis.

The DNA construct of the invention is purified from the host cell using well known methods.

For example, plasmid vector DNA can be prepared on a large scale from cleaved lysates by banding in a CsCl gradient according to the methods of Clewell & Helinski (1970) Biochemistry 9, 4428-4440 and Clewell (1972) J. Bacteriol. 110, 667-676. Plasmid DNA extracted in this way can be freed from CsCl by dialysis against sterile, pyrogen-free buffer through Visking tubing or by size-exclusion chromatography.

Alternatively, plasmid DNA may be purified from cleared lysates using ion-exchange chromatography, for example those supplied by Qiagen. Hydroxyapatite column chromatography may also be used.

Preferably, naked DNA is injected in the tumour, for example at a dose of 0.1 ng to 1.0 mg vector DNA cm^"3 of tumour, preferably about 0.1-10 μg cm^"3 vector DNA. The DNA may be circular or linear. Linear DNA may be obtained from circular DNA by cleavage with an appropriate restriction enzyme. By "appropriate restriction enzyme" we mean one that does not cleave the DNA within the promoter region or cytokine coding region.

At present, it is most preferable to use 1.0 μg of DNA per cm³ of tumour in a volume of 100 μl. The DNA may be dissolved in phosphate-buffered saline (PBS), or it may be used as a precipitate with calcium phosphate. Of course, other suitable buffers or carriers may usefully be employed. The expression of the said DNA in the tumour may be analysed by reverse transcriptase-PCR (that is, the messenger RNA expressed from the DNA in the tumour is isolated, converted into complementary DNA (cDNA) using the enzyme reverse transcriptase, and the resultant cDNA is amplified using the polymerase chain reaction and may be detected radiolabelling or staining), or by northern blot analysis or by RNase protection assays.

Such injection may be repeated at hourly, daily or weekly intervals.

Uptake of naked DNA may depend on the three-dimensional growing mass of tumour so, although it is preferred that the tumour to be treated is melanoma, a prostate tumour, or a colon tumour or a pancreatic tumour, or a breast tumour, it may be any solid tumour.

It is most preferred if substantially all cells in the tumour take up DNA and express the cytokine, but it is not essential for a useful clinical effect, as the antitumour effect of the cytokine is not limited to the tumour cell expressing the cytokine but will occur in non-transfected cells within the tumour and at secondary (metastatic) sites. Thus, if 5 % , preferably 25 % , more preferably 50% and most preferably substantially 100% of the tumour cells express the cytokine a clinically useful effect may be seen. It is desirable to express a plurality of cytokine coding sequences in a tumour cell, or to express a plurality of cytokine coding sequences in a tumour wherein each cytokine coding sequence is present in a separate DNA construct. It is preferable if the different cytokines, expressed by the plurality of coding sequences, stimulate different effector cells of the immune system.

In one embodiment, each of the coding sequences of the plurality are directly joined to a means for expression in a tumour cell but are contained within the same DNA construct. Thus, once the DNA is introduced into the tumour, every cell that takes up the DNA may express all of the cytokine coding sequences in the plurality.

In a further embodiment, a plurality of DNA constructs is introduced into the tumour, each construct of the plurality comprises a means for expression of a coding sequence in a tumour cell and a coding sequence encoding a different cytokine. In this embodiment it is possible to vary the proportion of cytokine coding sequences in the plurality.

The components of the plurality comprise two or more of coding sequences encoding interleukin-2, interleukin-4, macrophage colony stimulating factor, interferon-7, tumour necrosis factor and interleukin-7. The ratio of any two of the said coding sequences in the plurality may be, one to another, 100:1, 10:1 or 1:1.

Thus, a particular plurality of coding sequences useful in the invention is interleukin-2:interleukin-4:macrophage colony stimulating factor in a molar ratio of 1 : 1 : 1. This particular combination of coding sequences will express a plurality of cytokines useful in attracting cytotoxic T cells, eosinophils and macrophages to the tumour, and to secondary (metastatic) sites. All of these cell types have been shown to have anti-tumour activity.

It is preferred that the means of expressing each coding sequence in the plurality is a tumour specific promoter.

It is preferred that the plurality of DNA constructs is injected directly into the tumour.

It is further preferred that the tumour into which the DNA construct is injected directly is a melanoma, breast cancer, prostate cancer or colon cancer.

It is desirable to treat melanoma with a DNA construct wherein the means of expressing is the tyrosinase promoter.

In a further embodiment it is preferred if the B-cell accessory molecule B7 antigen is co-expressed with the cytokine in the tumour or tumour cell. B7 binds CD28 on T-cells and stimulates the activity of T-cells against tumours as is described in WO 92/00092.

The cDNA encoding the B7 antigen molecule can be obtained using the method described by Freeman et al (1989) J. Immunol. 143, 2714-2722 incorporated herein by reference and the nucleotide and predicted amino acid sequence can be obtained therefrom. The nucleotide sequence of B7 cDNA is given as SEQ ID No 23.

The term "fragment" as used herein means a portion of the amino acid sequence corresponding to the B7 antigen. For example, a fragment of the B7 antigen useful in the method of the present invention is a polypeptide containing a portion of the amino acid sequence corresponding to the extracellular portion of the B7 antigen, ie the DNA encloding amino acid residues from position 1 to 215 of the sequence corresponding to the B7 antigen described by Freeman et al, supra.

Complementary cDNA sequences encoding the amino acid sequence corresponding to the B7 antigen or fragments or derivatives thereof can be synthesised by the polymerase chain reaction (see US Patent No 4,683,202) using primers derived from the published sequence of the antigen (Freeman et al, supra). These cDNA sequences can then be assembled into a vector so that the expression of the B7 antigen is driven by a means for expression in the tumour cell.

It is preferred if the means for expression is a tumour-specific promoter.

It is further preferred if the promoter is the tyrosinase or TRP-1 promoter.

It is preferred if the tumour is melanoma.

The techniques for assembling and expressing DNA encoding the amino acid sequences corresponding to B7 antigen and the cytokines useful in the invention, eg synthesis of oligonucleotides, PCR, transforming cells, constructing vectors and the like are well-established in the art, and most practitioners are familiar with the standard resource materials for specific conditions and procedures. However, the following paragraphs are provided for convenience and notation of modifications where necessary, and may serve as a guideline.

Complementary cDNA clones containing DNA encoding B7 proteins are obtained to provide DNA for assembling into the DNA constructs for use in the methods of the invention. Alternatively, cDNA clones may be prepared from RNA obtained from cells expressing B7 antigen or the cytokines based on knowledge of the published sequences for these proteins using standard procedures. Published sequences for the cDNAs are given as SEQ ID Nos.

The cDNA is amplified using the polymerase chain reaction ("PCR") technique (see US Patent Nos. 4,683,195 and 4,683,202 to Mullis et al and Mullis & Faloona (1987) Methods Enzymol. 154, 335-350) using synthetic oligonucleotides encoding the sequences of the proteins as primers. PCR is then used to adapt the fragments for ligation to the DNA encoding the promoter fragments and to expression plasmid DNA to form cloning and expression plasmids.

It is desirable to express a single cytokine coding sequence or a plurality of cytokine coding sequences in a tumour cell, in combination with the B7 coding sequence, or to express a cytokine coding sequence in a tumour in combination with a B7 coding sequence wherein the cytokine coding sequence and the B7 coding sequence are present in a separate DNA construct. It is preferable if the different cytokines, expressed by the plurality of coding sequences, stimulate different effector cells of the immune system.

In one embodiment, each of the coding sequences of the plurality of cytokines or B7 coding sequence are directly joined to a means for expression in a tumour cell but are contained within the same DNA construct. Thus, once the DNA is introduced into the tumour, every cell that takes up the DNA may express all of the cytokine coding sequences in the plurality and the B7 coding sequence. In a further embodiment, a plurality of DNA constructs is introduced into the tumour, each construct of the plurality comprises a means for expression of a coding sequence in a tumour cell or a coding sequence encoding a different cytokine or B7 molecule. In this embodiment it is possible to vary the proportion of cytokine coding sequences and B7 molecules introduced into the tumour.

It will be appreciated by one skilled in the art that the same or different cytokine or B7 coding sequence may be expressed in the tumour cell from separate DNA constructs or that the said coding sequences may be expressed in the tumour cell from the same DNA construct wherein each coding sequence has an independent means for expression or that the said coding sequences may be expressed in the tumour cell from the same DNA construct wherein each coding sequence has the same means for expression. In the latter case the coding sequences for a cytokine or a B7 may be fused such that a fusion polypeptide is made; it is preferred if a linker joins the polypeptides in the fusion that is cleaved in the environment of the tumour cell to release the active cytokine or B7.

When melanoma is to be treated by the DNA constructs comprising a gene promoter from a melanin synthesis pathway gene such as tyrosinase, it is desirable if the patient to be treated is not black.

It is further preferred if the patient to be so treated is fair-skinned.

In a further aspect of the invention the DNA constructs are used in conjunction with chemotherapy. Thus, the DNA construct, or a plurality of such constructs, may be administered at the same time as, preceding or after treatment with chemotherapeutic agents. Chemotherapeutic agents useful in this aspect of the invention include cisplatin, dacarbazine, tamoxifen, nitrosoureas including carmusine (BCNU), vinca alkaloids, melphalan, doxorubicin, adriamycin, etoposide, 5-fluorouracil and other generally used agents.

These are listed in the table:

It is preferred if the DNA construct or the plurality of constructs expresses interleukin-2 which will facilitate the substantial destruction of the vasculature and promote the action of the chemotherapeutic agent.

Further aspects of the invention provide a composition comprising a construct of the invention and means for selectively delivering it to a tumour and a method of treating a tumour and/or ameliorating metastasis therefrom comprising delivering into cells of the tumour a construct of the invention.

The invention will now be described with reference to the following Examples and Figures wherein:

Figure 1 shows the tissue specific expression cassettes using the tyrosinase and the TRP-1 gene promoters;

Figure 2 shows the relative activity of tyrosinase and TRP-1 promoters in murine B16.F1 melanoma and NIH 3T3 cells;

Figure 3 shows the retroviral vector pBabe Puro (Tyr-/S-Gal).

Figure 4 shows the c-erbB-2/CAT construct of Example 5.

Figure 5 shows the result of a comparison of activity of the construct of Example 5 in two cell lines: T47D, which is a breast carcinoma cell line with base line c-erbB-2 expression, and ZR75-1, which is a breast carcinoma cell line with elevated c-erbB-2 expression.

SEQ ID No 1 shows the nucleotide sequence of the CEA gene including the promoter region. SEQ ID No 2 shows the sequence of the PSA gene including the promoter region.

Figure 6 shows the 5' flanking sequence with 71 bp of transcribed sequence of the human MUCl gene (SEQ ED No 3). The TATA box (boxed) and transcriptional start site (+1) are indicated. The sequence (- 787 to +71) covers the region required for maximum transcription of the reporter gene (-743 to +33).

Figure 7 shows the DNA sequence of the human c-erbB-2 5' region as determined by Hudson et al (1990) J. Biol. Chem. 265, 4389-4393 (SEQ ID No 4).

Figure 8 shows the DNA sequence of the human c-erbB-3 5' region (SEQ ID No 5) and the predicted amino acid sequence of the first exon (SEQ ID No 6).

SEQ ID No 7 shows the DNA sequence of the tyrosinase promoter.

SEQ ID No 8 shows the DNA sequence of the TRP-1 promoter.

SEQ ID No 9 shows the DNA gene sequence encoding interleukin-2 (DL- 2); the cDNA sequence is readily derived from the positions of the exons.

SEQ ID No 10 shows the cDNA sequence encoding interleukin-4 (IL-4).

SEQ ID No 11 shows the cDNA sequence encoding interleukin-7 (IL-7).

SEQ ID No 12 shows the cDNA sequence encoding tumour necrosis factor (TNF). SEQ ID No 21 shows the cDNA sequence encoding interferon-gamma (IFN-7).

SEQ ID No 22 shows the cDNA sequence encoding human granulocyte macrophage colony stimulating factor GM-CSF.

SEQ ID No 23 shows the B7 cDNA sequence.

The following information is useful to the person skilled in the art to identify coding regions and promoter sequences for use in the invention. Journal references and EMBL database accession numbers are given.

SEQ ID No 1

ID HSCEAOl standard; DNA; PRI; 3281 BP; AC M59255; M31966; DE Human carcinoembryonic antigen (CEA) gene, complete eds; KW carcinoembryonic antigen; OS Homo sapiens (human); OC Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia; OC Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae; RN [1]; RP 1-3281; RA. Schrewe H., Thompson J., Bona M., Hefta L.J., Maruya A.,; RA Hassauer M., Shively J.E., von Kleist S., Zimmermann W; RT "Cloning of the complete gene for carcinoembryonic antigen:; RT Analysis of its promoter indicates a region conveying cell; RT type-specific expression"; RL Mol. Cell. Biol. 10:2738-2748(1990); FH Ke y Loc ati o n/Q u al ifi e rs ; FH ; FT s i g_pepti d e join(1769..1832,2725..2762); FT /gene="CEA"; FT exon 1659..1832; FT /number=l /gene="CEA" /codon_start= 1659; FT exon 2725..3084; FT /number=2 /gene="CEA" /codon_start=2725; SQ Sequence 3281 BP; 847 A; 953 C; 871 G; 610 T; 0 other; CC SEQ ID No 21

ID HSIFNGAMM standard; RNA; PRI; 1011 BP; AC M26683; DT 23-NOV-1989 (Rel. 21, Created); DT 26-MAY-1992 (Rel. 32, Last updated, Version 5); DE Human interferon gamma (IFN-gamma) mRNA, complete eds; KW interferon gamma; type II; OS Homo sapiens (human); OC Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia; OC Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae; RN. [1]; RP 1-1011; RA Fan X., Stark G.R., Bloom B.R; RT "molecular cloning of a gene selectively induced by gamma; RT interferon from human macrophage cell line u937ⁿ; RL Mol. CeU. Biol. 9:1922-1928(1989); FH Key Location Qualifiers; FH; FT CDS 15..131; FT /product = "interferon gamma" /gene = "IFN-gamma"; FT /codon_start=l; FT polyA_signal 971..976; FT /gene = "IFN-gamma"; SQ Sequence 1011 BP; 301 A; 236 C; 184 G; 290 T; 0 other;

SEQ ID No 2

ID HSPSAA standard; DNA; PRI; 7130 BP; AC M27274; DT 23-APR-1990 (Rel. 23, Last updated, Version 1); DT 02-FEB-1990 (Rel.

22, Created); DE Human prostate-specific antigen gene, complete eds;

KW Alu repetitive element; kallikrein; prostate specific antigen; OS Homo sapiens (human); OC Eukaryota; Animalia; Metazoa; Chordata;

Vertebrata; Mammalia; OC Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae; RN [1]; RP 1-7130; RA Lundwall A; RT.

"Characterization of the gene for prostate-specific antigen, a; RT human glandular kallikrein"; RL Biochem. Biophys. Res. Commun.

161:1151-1159(1989); DR SWISS-PROT; P07288; PROS$HUMAN; FH

Key Location/Qualifiers; FH; FT CDS 675..720; FT /note = "prostate-specific antigen, exon 1; FT /nomgen="APS" /map="19ql3.3-qter"; FT /hgml_locus_uid="LN0098S""; FT intron 721..1958; FT /note="PSA intron A"; FT CDS 1959..2118; FT /note = "prostate-specific antigen, exon 2"; FT intron 2119..3755; FT /note = "PSA intron B"; FT repeat region 2583..2935; FT /note="Alu repeat"; FT CDS 3756..4042; FT /note= "prostate-specific antigen, exon 3"; FT intron 4043..4185; FT /note="PSA intron C"; FT CDS 4186..4322; FT /note = "prostate-specific antigen, exon 4"; FT intron 4323..5698; FT /note="PSA intron D"; FT CDS 5699.-5854; FT /note = "prostate-specific antigen, exon 5"; SQ Sequence 7130 BP; 1530 A; 2024 C; 1867 G; 1709 T; 0 other;

SEQ ID No 8

ID MMTRP15 standard; DNA; ROD; 1236 BP; AC X59513; DT 26-JUL-1991 (Rel. 28, Created); DT 26-JUL-1991 (Rel. 28, Last updated, Version 2); DE Mouse 5' end of TRP1 gene for tyrosinase-related protein-1; KW TRP1 gene; tyrosinase; tyrosinase-related protein-1; OS Mus musculus (mouse); OC Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia; OC Theria; Eutheria; Rodentia; Myomorpha; Muridae; Murinae; RN. [1]; RA Jackson I.J., Chambers D.M., Budd P.S., Johnson R; "The tyrosinase-related protein-1 gene has a structure and promoter sequence very different from tyrosinase. ";Nucleic Acids Res. 19:3799-3804(1991) SQ Sequence 1236 BP; 357 A; 234 C; 282 G; 363 T; 0 other;

SEQ ID No 22

ID HSCSFGMA standard; DNA; PRI; 3194 BP; AC M13207; DT

07-JUN-1987 (Rel. 12, Created); DT 24-DEC-1990 (Rel. 26, Last updated, Version 2); DE Human granulocyte-macrophage colony-stimulating factor (hGM-CSF); DE gene, complete eds; KW granulocyte-macrophage colony stimulating factor; OS Homo sapiens (human); OC Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia; OC Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae; RN [1]; RP 1-3194; RA Kaushansky K., O'Hara P.J., Berkner K., Segal G.M., Hagen F.S.,; RA Adamson J.W; RT "Genomic cloning, characterization, and multilineage; RT growth-promoting activity of human granulocyte-macrophage; RT colony-stimulating factor"; RL Proc. Natl. Acad. Sci. U.S.A. 83:3101-3105(1986); RN [2]; RP 1-3194; RA Kaushansky K; RT; RL Unpublished; DR CPGISLE; HSCSFGMA; Release pre-1.0; DR SWISS-PROT; P04141; CSF2_HUMAN; SQ Sequence 3194 BP; 700 A; 859 C; 945 G; 690 T; 0 other; CC

SEQ ID No 9

ID HSIL21 standard; DNA; PRI; 5737 BP; AC J00264; DT 29-JUL-1991 (Rel. 28, Created); DT 29-JUL-1991 (Rel. 28, Last updated, Version 1); DE Human interleukin 2 (11-2) gene, complete coding sequence; KW immune response gene; interleukin; interleukin 2; lymphokine; KW T-cell; T-cell growth factor; OS Homo sapiens (human); OC Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia; OC Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae; RN [1]; RP 431-624, 715-774, 3068-3211, 5057-5443; RA Maeda S., Nishino N., Obaru K., Mita S., Nomiyama H., Shimada K.,; RA Fujimoto K., Teranishi T., Hirano T., Onoue K; RT "Cloning of interleukin 2 mRNAs from human tonsils"; RL Biochem. Biophys. Res. Commun. 115:1040-1047(1983); RN CC Key Location/Qualifiers; FH; FT CDS join (478..624,715..774,3068..3211,5057..5167); SQSequence5737BP; 1995 A; 932 C; 922 G; 1888 T; 0 other; CC; ID HSIL4 standard; RNA; PRI; 614 BP; AC M13982; DT 07-JUN-1987 (Rel. 12, Created); DT 03-SEP-1992 (Rel. 33, Last updated, Version 2);

SEQ ID No 10

KW interleukin; OS Homo sapiens (human); OC Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia; OC Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae; RN [1]; RP 1-614; RA Yokota T., Otsuka T., Mosmann T., Banchereau J., DeFrance T.,; RA Blanchard D., De Vries J.E., Lee F., Arai K.i. "Isolation and characterization of a human interleukin cDNA clone homologous to mouse B-cell stimulatory factor 1, that expresses B-cell- and T-cell-stimulating activities"Proc. Natl. Acad. Sci. U.S.A. 83:5894-5898(1986). ; DR SWISS-PROT; P05112; IL4_HUMAN; FHKey Location/Qualifiers; FH; FT mRNA < 1..614; FT /note="IL-4 mRNA"; FT CDS 64..524; FT /note = "interleukin 4" /gene="IL4" /partial; FT sig_peρtide 64..135; FT /note = "interleukin 4 signal peptide"; T mat_ρeptide 136..522; FT /note = "interleukin 4 mature peptide"; SQ Sequence 614 BP; 174. A; 150 C; 129 G; 161 T; 0 other;

SEQ ID No 11

ID HSEL7A standard; RNA; PRI; 1589 BP; AC J04156; DT 22-APR-1989 (Rel. 19, Created); DT 06-JUL-1989 (Rel. 20, Last updated, Version 1); DE Human interleukin 7 (IL-7) mRNA, complete eds; KW interleukin; interleukin 7; OS Homo sapiens (human); OC Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia; OC Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae; RN [1]; RP 1-1589; RA Goodwin R.G., Lupton S., Schmierer A., Hjerrild K.J., Jerzy R.,; RA Clevenger W., Gillis S., Cosman D., Namen A.E; RT "Human interleukin 7: Molecular cloning and growth factor activity; RT on human and murine B-lineage cells"; RL Proc. Natl. Acad. Sci. U.S.A. 86:302-306(1989); DR SWISS-PROT; P13232; IL7_HUMAN; CC Draft entry and computer-readable sequence [1] kindly submitted by; CC R.Goodwin, 05-JAN-1989; FH Key Location/Qualifiers; FH; FT mRNA < 1..1589; FT /note = "interleukin 7 mRNA"; FT CDS 385..918; FT /note = "interleukin 7 precursor"; FT CDS 385..459; FT /note = "interleukin 7 signal peptide"; FT CDS 460..915; FT /note= "interleukin 7"; SQ Sequence 1589 BP; 532 A; 284 C; 339 G; 434 T; 0 other;

SEQ ID No 12

Human tumour necrosis factor mRNA; ID HSTNFAA standard; RNA; PRI; 1585 BP; AC M10988; DT 16-JUL-1988 (Rel. 16, Created); DT 02-SEP-1992 (Rel. 33, Last updated, Version 2); DE Human tumor necrosis factor (TNF) mRNA; KW ; OS Homo sapiens (human); OC Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia; OC Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae; RN [1]; RP 1-1585; RA Wang A.M., Creasey A.A., Ladner M.B., Lin L.S., Strickler J.,; RA Van Arsdell J.N., Yamamoto R., Mark D.F; RT "Molecular cloning of the complementary DNA for human tumor; RT necrosis factor"; RL Science 228:149-154(1985); DR SWISS-PROT; P01375; TNFA_HUMAN; FH Key Location/Qualifiers; FH; FT CDS 86..787; FT /note= "tumor necrosis factor" /gene="TNFA"; FT /codon_start= 1; SQ Sequence 1585 BP; 352 A; 473 C; 389 G; 371 T; 0 other; CC

SEQ ID No 7

ID MMTYRl standard; DNA; ROD; 4758 BP; AC D00439; DT 14-FEB-1991 (Rel. 27, Created); DT 14-FEB-1991 (Rel. 27, Last updated, Version 1); DE Mouse tyrosinase gene, 5'flank and exon 1; KW melanin; melanocyte; monooxygenase; tyrosinase; OS Mus musculus (mouse); OC Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia; OC Theria; Eutheria; Rodentia; Myomorpha; Muridae; Murinae; RN [1]; RP 2481-3363; RA ; RN [2]; RP 1-4758; RA Yamamoto H., Takeuchi S., Kudo T., Sato C, Takeuchi T; RT "Melanin production in cultured albino melanocytes transfected; RT with mouse tyrosinase cDNA"; RL Jpn. J. Genet. 64:121-135(1989); FH Key Location/Qualifiers; FH; FT misc signal 2004..2008; FT /note- "putative CAT box"; FT misc_signal 2128..2133; FT /note = "putative CAT box"; FT misc_signal 2140..2146; FT /note = "putative TATA box"; FT misc_signal 2264..2268; FT /note = "putative CAT box"; FT misc_signal 2272..2279; FT /note = "putative TATA box"; FT misc signal 2286..2289; FT /note = "putative CAT box" ; FT misc signal 2434..2440; FT /note = "putative TATA box"; FT misc feature 2465.-2466; FT /note="CAP sites"; FT CDS 2545.. > 3363; FT /note = "tyrosinase gene, exon 1" /partial; SQ Sequence 4758 BP; 1550 A; 859 C; 878 G; 1465 T; 6 other; CC

SEQ ID No 23

; ID HSIGB7 standard; RNA; PRI; 1491 BP. ; AC M27533; ; DT

23-APR-1990 (Rel. 23, Created) ; DT 23-APR-1990 (Rel. 23, Last updated, Version 1) ; DE Human Ig rearranged B7 protein mRNA

VCl-region, complete eds. ; KW constant region; immunoglobulin; variable region. ; OS Homo sapiens (human) ; OC Eukaryota; Animalia;

Metazoa; Chordata; Vertebrata; Mammalia; ; OC Theria; Eutheria;

Primates; Haplorhini; Catarrhini; Hominidae. ; RN [1] ; RP 1-1491 ; RA Freeman G.J., Freedman A.S., Segil J.M., Lee G., Whitman J.F., ; RA Nadler L.M.; ; RT "B7, a new member of the Ig superfamily with unique expression on ; RT activated and neoplastic B cells"; ; RL J. Immunol. 143:2714-2722(1989). ; CC Draft entry and computer readable copy of sequence [1] kindly ; CC provided by G.J. Freeman, 08-SEP-1989. ; FH Key Location/Qualifiers ; FH ; FT CDS 318..1184 ; FT /note =" transmembrane protein Bl precursor" ; FT CDS 318..395 ; FT /note =" transmembrane protein Bl signal ; FT peptide" ; FT CDS 396..1181 ; FT /note = "transmembrane protein Bl" ; SQ Sequence 1491 BP; 419 A; 343 C; 311 G; 418 T; 0 other; ; CC

Example 1: Demonstration of tissue specificity of 5' sequences of murine tyrosinase and TRP-1 genes.

A 2.5kb fragment from the 5' end of the tyrosinase gene was generated by PCR from genomic DNA of the B16 melanoma line. The oligonucleotides used (Pair 1: 5'-CGGAATTTCATGCCCCAGTTGAC- AACATAG-3', SEQ ID No 13; 5'-CACTCGAGAACTTTTTCTCCT- TTAGATCATACAA-3', SEQ ID No 14) were derived from the murine sequence published by Yamamoto etal (1989) Jpn. J. Genet. 64, 121-135. Shorter 5' sequences were generated also using oligonucleotides matched f r o m t h e Y a m a m o t o p a p e r ( P a i r 2 : 5'CGGGAATTCATGCCCCAGTTGACAACATAG-3', SEQ ID No 15; 5'-GAGCTCGAGTGTCACAGACTTCTTTTCCA-3,SEQIDNol6;Pair 3: 5'-AAACGAATTCCATCCAGTAAGTCCATTACT-3', SEQ ID No 17; 5'-GAGCTCGAGTGTCACAGACTTCTTTC-3', SEQ ID No 18). The 769bp fragment of the tyrosinase gene extends from position -815 to position -46 in the promoter. A 4.0kb fragment of 5' sequence of the TRP-1 gene was provided by Dr I.J. Jackson, MRC Genetics Unit, Edinburgh and from this a 1.4kb fragment was derived by Xbal-Sall digestion. The promoter sequence at the 5' of TRP-1 gene may be obtained following the methods described in Jackson et al (1991) Nucl. Acids Res. 19, 3799-3804.

These 5' sequences, and the SV40 promoter as a control, were inserted upstream of the j8-galactosidase gene in the vector pNASS (obtained from Clontech Ltd) as indicated in Figure 1.

Figure 1 shows (A) pNASS/3, a promoterless mammalian expression vector described by MacGregor & Caskey (1989) Nucl. Acids Res. 17, 2365. Three unique restriction sites allow cloning of promoter sequences upstream of an expression cassette containing the SV40 splice donor/acceptor sequence (sd/sa), the j8-galactosidase gene and the SV40 polyadenylation sequence. SV40 3-Gal contains the SV40 early viral promoter (from the pBabe Puro vector, as described by Morgenstern & Land (1990) Nucl. Acids Res. 18, 3596, cloned into pNASS/S. (B) 2496 bp (Tyr-jS-Gal 1) or 769 bp (Tyr-3-dal 2) fragments of the mouse tyrosinase promoter (Yamamoto et al (1989) Jap. J. Genet. 64, 121-135) were generated by PCR from genomic DNA of the B16.F1 melanoma cell line and cloned into the EcoRI and Xhol restriction sites of pN ASS5. (C) The plasmids TRP-1-β-Gal 1 and 2 were a gift from I. Jackson and contain 4 kbp and 1.4 kbp of the TRP-1 promoter (Jackson et al (1991) Nucl. Acids Res. 19, 3798-3804) upstream of the S-galactosidase gene and the SV40 polyadenylation sequence. The different constructs were transfected into a variety of murine and human cells of melanocytic and non-melanocytic origin, including B16 melanoma cells or NIH 3T3 fibroblasts and subsequent /3-galactosidase activity was measured 72-96 hours after transfection both by fluorometric assay, using 4- methylumbelliferyl-/3-D-galactoside (MUG) as substrate, and by histochemical analysis using X-gal as substrate. By both assays the various tyrosinase and TRP-1 promoter containing 5' sequences were shown to drive /5-galactosidase activity in a murine melanocyte (Mel-ab) line and the B16 melanoma and the human melanoma lines SK23, HMB-2, Mel 8, TXM13, T8 and SS3. No activity was observed in the murine 3T3 or L cell tines or the human HeLa, LS174T, HT29, HOS, SW620 and HUVEC lines, none of which are of melanocytic origin (see Figure 2 and Table 1).

Figure 2 shows the relative activity of tyrosinase and TRP-1 promoters in murine B16.F1 melanoma and NIH 3T3 cells. Cells were transfected with 10 μg of the appropriate plasmid DNA using the calcium phosphate method. 72-96 hours after the calcium phosphate precipitate had been washed away the cells were analysed for expression of /3-galactosidase using the quantitative MUG assay. Data are expressed as mean of triplicate values ± SD. The data presented are representative of four similar experiments.

In contrast, the SV40 promoter was able to direct expression of the reporter gene to high levels in both cell types.

Table 1

galactosidase from Tyrosinase and TRP-1 promoters. Each cell line indicated was transfected with 10 μg of plasmid DNA of Tyr-/S-Gal 1 and 2, TRP-l-/3-Gal 1 and 2. pNASS-β and SV40-/3-Gal were used in each case as a negative and positive control for transfection. Expression of β- galactosidase was scored as positive (+) if several cells stained blue 96 hours after transfection; a cell line was scored as negative (-) if no blue cells were observed after transfection and if the quantitative MUG assay showed no expression above background levels (transfection with pNASS- β)-

These results confirm and extend the reports of other groups showing excellent tissue specificity of gene expression in melanocytic cells of either murine or human origin when the 5' promoter regions of either the tyrosinase or TRP-1 gene are utilised.

Example 2: Materials and methods pertaining to the other Examples.

Construction of Expression Plasmids and Retroviral Vectors.

Subcloning was carried out via standard recombination DNA techniques (Sambrook et al (1989) Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press, NY, USA). Restriction endonuclease enzymes were supplied by Northumbria Biologicals (NBL, Cramlington, UK) and Taq polymerase was supplied by Stratech (Luton, UK). Oligonucleotides, synthesised on an Applied Biosystems 380B and purified by denaturing acrylamide electrophoresis, were provided -by the Oligonucleotide Synthesis Laboratory, ICRF Clare Hall, South Mimms, UK. Polymerase chain reaction (PCR) amplification of DNA fragments was carried out on a Techne PHC-2 Thermocycler and reaction mixes were prepared in a hood separate from normal areas of DNA handling. Amplified DNA sequences were subcloned into the PCR II vector (Invitrogen; British Biotechnology Products Ltd, Oxford, UK) and their identities were confirmed by restriction endonuclease mapping. The correct fragments were then shuttled from PCR II into the appropriate expression plasmid.

Cell Culture. All cell lines used in this study were checked routinely and found to be free of mycoplasma infection. Apart from Melab cells which were cultured in medium supplemented as described previously (Burrows et al (1991) Cancer Res. 51, 4768-4775) the lines were grown in Eagle's minimal essential medium supplemented with 10% (v/v) fetal calf serum and 4 mM L-glutamine. HUVEC (Human umbilical vein endothelial cells) were maintained in Medium 199 (Gibco-Biocult Ltd, Paisley, Scotland) supplemented with Earle's salts, 20% (v/v) fetal calf serum, endothelial cell growth supplement (0.12 mg/ml) 0.09 mg.ml heparin and glutamine. Cultures were maintained at 37° C in a humidified atmosphere of 90% air/10% C0₂.

DNA Transfection. IO⁶ adherent cells were transfected with 10 μg of plasmid DNA by calcium phosphate co-precipitation using the Profection method (Promega, Madison, WI) according to the manufacturer's instructions. 24 hours after the application of the precipitate to the tissue culture medium, cells were washed three times in serum-free medium and incubated in normal medium for 72-96 hours when they were stained for /3-galactosidase expression.

Intra-Tumoral Injection of DNA. 1-1.5 x IO⁵ tumour cells of either the B16 Fl murine melanoma or the Colo 26 colon carcinoma were injected subcutaneously in 100 μl inoculum volumes into the flank region of syngeneic mice (C57 for B16 Fl, Balb/C for Colo 26). Ten days later the animals were anaesthetised by halothane inhalation (ICI Pharmaceuticals, Macclesfield, UK), the tumours, approximately 4 mm in diameter, were located by palpation and injected with 1 μg DNA in 100 μl volumes of either PBS or as calcium phosphate precipitates via a 27-gauge needle.

Quantitative Assay for /S-Galactosidase Expression. Transfected cells were assayed for enzyme activity by the technique of MacGregor et al

(1991) Methods in Molecular Biology 7, 217-235 (Ed., E.J. Murray) Humana Press Inc, Clifton, NJ, USA. Briefly cells were resuspended in Z buffer (60 mM Na^O^R_jO, 40 mM NaH₂P0₄.H₂0, 10 mM KC1, 1 mM MgS0₄.7H₂0) at 10⁷ cells per ml. 105 μl of this cell suspension were dispensed per well of a microtiter plate and 15 μl of 1 % Triton X- 100 were added to each well o give a final concentration of 0.1 % . After 10 minutes at room temperature, 30 μl of 3 mM methylumbelliferyl-/5-D- galactoside (MUG) (Sigma, Poole, UK) in Z buffer were added to each well and the reaction was allowed to proceed for 90 minutes at 37°C. 75 μl of 300 mM glycine, 15 mM EDTA, pH 11.2 were added to stop the reaction. Fluorescence was measured on a microtiter dish fluorescence reader (excitation at 350 nm and emission read at 450 nm).

Cells expressing /3-galactosidase convert the MUG substrate, a non- fluorescent galactoside analogue, to the fluorescent molecule 4- methylumbelliferone.

Histochemical Detection of /3-Galactosidase-expressing Cells. 72-96 hours following DNA transfection, adherent cells were washed once in phosphate buffered saline (PBS) and fixed for 10 minutes at 4°C with 3.8% formaldehyde in PBS. The fixative was removed by three washes with PBS and the cells were then incubated with X-gal solution [5-bromo- 4-chloro-3-indoyl-/8-galactopyranoside (Sigma) at 40 mg/ml in dimethylformamide was diluted to 1 mg/ml in 5 mM K₃Fe(CN)₆; 5 mM K₄Fe(CN)₆.3H₂0; 2 mM MgCl₂; 0.01 % sodium deoxycholate; 0.2% NP40. All solutions were prepared using glass] at 37°C for at least 4 hours according to published techniques (Bondi et al (1982) Histochem. 76, 153-158). After staining the X-gal solution was removed, the cells were washed three times in PBS and the cells were inspected under a light microscope. Cells expressing the /3-galactosidase gene hydrolyse the chromogenic substrate X-gal to give the blue dye bromochloroindole. Control untransfected cells also were stained to assess the background endogenous /3-gal staining.

Detecting of 3-Galactosidase-expressing Tumour Cells. 2, 4, 6 or 10 days after injection of DNA into the tumours, animals were killed by C0₂ inhalation, their tumours were excised, minced to 1 mm cubes with scalpels and pushed through a stainless steel sieve with a 5 ml syringe plunger, into culture medium. An aliquot of the resulting cell suspension was spun onto a glass microscope slide using a cytospin centrifuge. Slides were air-dried then fixed for 5 minutes in 3.8% formaldehyde in PBS. The cells were rinsed in PBS and incubated overnight in X-gal stain before being inspected under a light microscope for the presence of blue cells.

Generation of Recombinant Retrovirus Stocks. The AM 12 packaging cell line (Markowitz et al (1988) Virol. 167, 400-406) containing the packaging constructs for Moloney Leukaemia Virus was transfected with 10 μg of retroviral plasmid DNA using the calcium phosphate co- precipitation method. 48 hours following transfection the cells were split into puromycin (Sigma) selection medium (1 μg/ml) and surviving colonies were selected and pooled two weeks later. Virus was harvested from these producer cells by exposing fresh medium to 5 x 10⁶ cells on a 90 mm plate and harvesting the medium 16 hours later. The medium was filtered through a 0.45 μm filter (Nalge (UK) Ltd, Rotherwas, England) to remove cell debris and was then used to infect target cells. The target cells were split 24 hours earlier to a density of IO⁵ cells per 90 mm plate. Polybrene (Aldrich, Gillingham, Dorset) was added to the viral supernatant to 4 μg/ml to enhance virus-cell surface interactions and the target cells were exposed to 1 ml of viral supernatant for 2.5 hours at 37°C. 8 ml of normal growth medium were added to the plate and the infected cells were grown for a further 72-96 hours before being stained for expression of /3-galactosidase. Example 3: Preparation of tyrosinase promoter- or TRP-1 promoter- driven expression vectors containing cvtokine cDNA's.

The pBCMGNeo-mIL-2 vector was provided by Dr P. Frost, University of Texas, Houston and is described in Eur. J. Immunol. 18, 97-194 (1988), although other vectors are suitable. This vector had been used to transfect B16 melanoma cells (a non-cell-type-specific approach) and IL-2 producing cells had been selected (Fearon et al (1990) Cell 60, 397-403). The HCMV promoter of this vector was removed by Xbal-Sall digestion and replaced with the 1.4kb Xbal-Sall fragment of TRP-1 5' sequences or the 780bρ tyrosinase 5' sequence fragment generated by Pair 3 oligonucleotides. These constructs were transfected into murine B16 melanoma cells or 3T3 fibroblasts. For the TRP-1 - IL2 construct a total of 60 puromycin-resistant clones were isolated and screened by ELISA for IL-2 production (Genzyme Ltd). Clones were characterised as high (> 960 pg/ml), intermediate (150-960 pg/ml) and low (≤ 150 pg/ml) expressers. Of the 60 clones, 13 clones were found to be producing and secreting measurable quantities of IL-2 while ten clones of 3T3 cells and four pooled bulk populations of 3T3 did not contain any cells expressing detectable amounts of IL-2 activity. These results show that the tissue- specific promoter, TRP-1, is able to drive expression of a cytokine cDNA in an appropriate cell type. Repeated analysis over a 6-8 week culture period showed that the observed phenotype is stable.

Alternatively, the IL-2 coding sequence can be incorporated into a tyrosinase promoter vector as follows:

The murine IL-2 cDNA is PCR amplified from pBCMGNeo mIL-2 using the primers GCGGCCGCGC ATGTACAGC ATGC AGCTCGCA (SEQ ID No 19) and GCGGCCGCTAAATAAATAGAGAGCCTTATG (SEQ ID No 20).

The PCR fragment is cloned into the vector PCRII (available from Invitrogen) and then excised from the PCRII vector using Notl digestion. The Notl fragment is cloned into the Notl site of Tyr-/3-Gal-l (described in Example 1) in place of the /S-galactosidase gene. This produces Tyr IL- 2 with a 2494 bp promoter from the tyrosinase gene driving expression of IL-2.

B16 clones have been injected into groups of syngeneic C57 mice. To date only the cell clone selected for drug resistance, ie lacking IL-2 expression, is forming progressively growing tumours in these animals. The IL-2 secreting B16 cells are not forming palpable tumours and, if they do develop, are clearly growing at a slower rate in vivo.

In addition to the cells secreting IL-2, IL-2 expression is assessed using RT-PCR wherein RNA is isolated, primers such as oligo dT used to prime synthesis of cDNA from the mRNA using reverse transcriptase and the level of IL-2 RNA estimated by amplifying with IL-2-specific oligonucleotides.

We have placed cDNA for IL-4 (bought from British Biotechnology Ltd) downstream of both promoter sequence but the construct may utilise any cytokine gene (eg GM-CSF, TNF, IFN), be combined with the HSV tk gene for ganciclovir selection, or may utilise cDNAs encoding for genes which might stimulate the immune response (eg MHC antigens, MAGE (melanoma antigens) etc). This procedure allows targeted expression of the requisite gene to the cell type of interest, ie melanocyte-derived cells. Replacement of the tyrosinase or TRP-1 promoter sequences with sequences which are expressed by other tumour types in a specific fashion (eg 5' promoter sequences of the CEA gene for colorectal tumours, 5' sequences of prostate secreted antigen for prostatic tumours) permits targeted expression of similar genes to other tumour types.

Example 4: Introduction of tissue specific promoter-driven genes into target cells in vivo.

There are two main routes of delivery:-

1) Retroviral delivery

2) Direct delivery

Incorporation into a Retroviral Vector. The ability of the melanocyte- specific promoters to function after delivery via a retroviral vector was examined because retroviral-mediated gene delivery is a promising route for delivery of gene therapy in vivo (Miller (1992) Nature 357, 455-460). The retroviral vector pBabe Tyr-/3-Gal was constnicted from the pBabe Puro vector (Morgenstern & Land (1990) Nucl. Acids Res. 18, 3587-3596) (Figure 3). Here /3-galactosidase is expressed from the 769 bp tyrosinase promoter fragment of Tyr-/S-Gal 2 inserted into pBabe Puro in the opposite orientation to the direction of expression of the viral mRNA driven from the Moloney Leukaemia Virus (MLV) Long Terminal Repeat (LTR).

Following transfection of the vector into the AM12 amphotropic packaging cell line, recombinant retroviral particles were used to infect either B16 or NIH 3T3 cells. 72-96 hours following infection, expression of the β- galactosidase gene was observed preferentially in B16 cells relative to the NIH 3T3 target cells by both histochemical and fluorimetric assays. These results demonstrate that the tyrosinase and TRP-1 promoters can confer tissue specificity of expression upon an heterologous gene in both human and murine melanocyte-derived cell lines when delivered in the context of a retroviral vector.

Experiments on route 2 have yielded interesting results. Syngeneic C57/BL mice were injected s.c. in the flank region with lxlO⁵ B16 cells and the animals were monitored until a tumour of approximately 0.4 x 0.4 cm had developed. Similar Colo tumours were established in Balb-C mice. At this time a single injection of 1.0 μg of the tyrosinase promoter/pNASS DNA was inoculated in 100 μl volumes directly into the centre of the tumour either as 'naked' DNA or as calcium phosphate- coprecipitated material. Similarly, pNASS-/3 and TRP-/3-Gal-2 DNA was inoculated. At varying times thereafter, for example at 2, 4, 6 or 10 days, mice were killed, and the tumours were removed and snap-frozen. Cryostat sections of these tumours were stained for /3-galactosidase activity. Protein expression, manifest by the detection of bright blue cells, was clearly apparent in the majority of the injected tumours. The Tyr-jS- Gal 2 construct caused the gradual accumulation of positive blue cells in the injected B16 tumours over the ten day period of examination; whereas the same construct injected into the non-melanocytic Colo 26 tumours produced no blue staining. Similar results were obtained in three independent replicate experiments and from these it was apparent that:- (1) the promoterless, control pNASS β construct produced no blue cells in either Colo 26 or B16 tumours; (2) there was a gradual increase in the proportion of blue cells in the positive groups over the 10 day period of examination (10 days was the last time-point examined because of increasing tumour burden) up to an estimated 10-15% of cells (3) no qualitative or quantitative difference was obvious between the tyrosinase or TRP-1 promoter elements or between material injected as naked DNA or as a CaP0 -precipitate. Frozen sections of B16 tumours stained 10 days after DNA injection showed similar results. Interestingly the only blue-staining tissue, apart from the neoplastic cells, was confined to the base of the hair follicles and thus, presumably, indicated transduction of normal melanocytes.

These results show that direct gene transfer may be accomplished by intra- tumoural injections. Morphological assessment of the sections indicated that the blue cells were restricted to areas occupied by neoplastic tissue, which is presumed to reflect the tissue specificity conferred by the 5' tyrosinase or TRP-1 gene sequence.

These experiments suggest that direct injections permit good levels of expression of introduced genes. The activity produced may be altered by modification of the introduced DNA (eg incorporation in liposomes, use of different precipitating material, variation in route of delivery). Taken in combination our results indicate that placing therapeutic genes under control of tissue-specific promoter regions may restrict expression to cells of a specific lineage. This could be important both for safety/specificity purposes and would permit the refinement of what otherwise may be a fairly non-specific event. The utilisation of a cytokine gene has been shown to induce modifications in subsequent tumour behaviour. Direct delivery of DNA via an intratumoural injection has been shown to produce high levels of expression of the introduced gene suggesting that such promoter-restricted expression may be further limited to the target cells by the simple expedient of targeting inoculation. The use of genes encoding for proteins capable of eliciting a subsequent systemic response may permit this method to be used for disseminated, rather than localised, neoplastic disease. Example 5; c-βr-?B-2 promoter and reporter enzyme

Reporter enzyme gene. The bacterial chloramphenicol acetyl transferase (CAT) gene was obtained from Promega as the "pCAT-basic" vector.

The CAT reporter system is designed to allow sensitive and rapid testing for eukaryotic transcriptional regulatory sequences. This reporter system relies on the linkage of genomic DNA fragments containing putative regulatory sequences to the chloramphenicol acetyltransferase (CAT) reporter gene. Transcriptional effects upon the CAT reporter gene are detected after transfection into cultured cells. Since CAT is a bacterial gene, levels of CAT enzyme activity in crude cell extracts can be quickly and easily assayed with little or no background from endogenous cellular gene activity. The pCAT-Basic plasmid lacks eukaryotic promoter and enhancer sequences. This allows the researcher maximum flexibility in cloning any putative regulatory sequences into the convenient multiple cloning sites. Expression of CAT activity in cells transfected with this plasmid is dependent on insertion of a functional promoter upstream from the CAT gene. Enhancer elements can be inserted upstream from the promoter or at the BamHI site downstream from the CAT gene. Sequences to be tested for transcriptional activity can be cloned into the following unique sites located immediately upstream from the CAT gene: Xbal, Accl, Sail, Pstl, Sphl and HindUl. Enhancer elements can be cloned separately into the BamHI site downstream from the CAT transcriptional unit. The vector also contains the gene for ampicillin resistance.

Promoter. The human c-erbB-2 promoter has been cloned to -500 by two groups (Ishi et al (1987) Proc Natl Acad Sci USA 84, 4374-4378; Tal et al (1987) Mol Cell Biol 7, 2597-2601) and to -1500 by a third group (Hudson et al (1990a) J Biol Chem 265, 4389-4393). We have taken oligonucleotides to 30b regions around +40 and -500 and, using PCR against human genomic DNA, recovered a 540bp fragment representing the c-erbB-2 proximal promoter. Using oligos to -1000 and -500 we then "PCRed" out a further 500bp representing the c-erbB-2 distal promoter. The two promoter regions were fused at the Smάl site at -500 and the full promoter cloned upstream of the CAT gene to generate a reporter plasmid for assaying c-erbB-2 promoter activity in cell lines in vitro. Further constructs were made by either deleting 5' regions of the promoter using convenient restriction enzyme sites, or using PCR technology, to generate a series of promoter deletion mutants linked to CAT 3' end always +40; 5' ends as follows: -1000, -500, -400, -300, -213, -177, -100; (Figure 1).

Construction of c-erbB-2 plasmid. The c-erbB-2 promoter was incorporated in the pCAT-basic plasmid to give the plasmid shown in Figure 1 by digesting the plasmid with Xbal and then filling the ends with Klenow fragment to create a blunt-ended vector suitable for cloning the blunt-ended PCR products.

The CAT activity from the various promoter constructs was compared to baseline activity from the promoterless CAT parent plasmid by calcium phosphate mediated DNA transfection into a number of different breast cell lines. Immortalised normal and tumour lines which have low endogenous c-erbB-2 expression showed little activity of the c-erbB-2 promoter, ie all the reporter constructs containing c-erbB-2 sequences generated no more CAT activity than the promoterless control plasmid. This result makes it unlikely that c-erbB-2 expression is actively repressed in these cell lines (by a tumour suppressor-like activity). Example 6: Promoter region of the carcinoembryonic antigen gene

The CEA gene is cloned using standard methods as described by Schrewe et al (1990) Mol. Cell. Biol. 10, 2738-2748 and sequenced using the dideoxy chain termination method of Sanger et al (1980) J. Mol. Biol. 143, 161-178.

To define the actual portion of the 5' untranslated region which is required for the promoter activity of the CEA gene, we carried out functional tests by placing restriction endonuclease fragments of various lengths from the putative promoter regions of both genes upstream of the CAT reporter gene and assaying for CAT activity in a transient transfection assay in two different human cell lines. For this purpose, we chose the CEA-producing adenocarcinoma cell line SW403 and, as a negative control, the HeLa cell line. The CEA promoter constructs showed an enhanced expression of the CAT gene in SW403 cells, which was nine times greater than in HeLa cells, when the shortest construct was used. It appears that cis regulatory sequences, which are responsible for this enhancement, along with a functional transcription initiator, are both present within the first 424 nucleotides upstream of the translational start. It is also interesting that longer CEA constructs are approximately 50% less active in HeLa cells than is the shortest construct. A possible explanation for this phenomenon is that a silencer region could exist between nucleotides -424 and -832 upstream from the translational start, which reduces the activities in both cell lines through interaction with common trøn-.-acting regulatory factors. Such silencer sequences have indeed been described for other genes.

Thus, the promoter of the CEA gene is useful for expressing cytokines, according to the methods of the invention, in colon tumours. As found here for CEA, a number of other eucaryotic genes have also been reported which do not contain obvious TATA boxes. The promoters of such genes can be divided into two classes. The members of the first class are G+C rich and are found primarily in housekeeping genes. These promoters usually contain several transcription initiation sites spread over a fairly large region, as well as potential binding sites for Spl. The members of the second class are not G+C rich, are not constitutively active, but are regulated during differentiation or development and initiate transcription at only one or a few tightly clustered start sites. Included in this class are a number of genes that are regulated during mammalian immunodifferentiation, eg the T-cell receptor /S-chain genes and the V_preB gene, as well as some Drosophila homeotic genes. The CEA gene shows a closer resemblance to this latter group, because its promoter is not obviously G+C rich, it contains no identifiable Spl-binding sites, it reveals only a few tightly clustered start sites, and, most importantly, it is not constitutively expressed.

Figure 6 shows the nucleotide sequence from the promoter region of CEA compared with the promoter region of the non-specific cross-reacting antigen gene (NCA) and the CGMl gene. The numbers indicate the distance in nucleotides from the initiation codon for each gene. Gaps have been introduced to aUow optimal alignment. Identical nucleotides are indicated by dots. The cluster of transcriptional start sites determined for CEA and NCA by SI nuclease assays are indicated by arrows.

Example 7; Promoter region of the prostate-specific antigen gene

The PSA gene is cloned using standard methods as described by Riegman et al (1989) Biochem. Biophys. Res. Comm. 159, 95-102 and Lundwall (1989) Biochem. Biophys. Res. Comm. 161, 1151-1159 and sequenced using the dideoxy chain termination method of Sanger et al (1980) J. Mol. Biol. 143, 161-178.

The sequence of the promoter region of PSA gene, compared to that of the hGK-1 gene, is shown in Figure 7. Dots represent identical nucleotides. Putative transcriptional regulatory elements are boxed.

PSA is expressed at a high level in the prostate; hGK-1, a human kallikrein-like gene, is expressed at lower level in the prostate.

The differences in nucleotide sequence between the PSA and hGK-1 promoters are probably important determinants in prostate-specific gene expression.

Thus, the promoter of the PSA gene is useful for expressing cytokines, according to the method of the invention, in prostate tumours.

Example 8: Promoter region of the MUCl gene

The mucin gene, MUCl, is selectively expressed in breast and pancreatic cell lines but not in non-epithelial cell lines. The promoter region for this gene may be obtained by the methods taught in WO 91/09867.

The 5' sequences flanking the human MUCl gene are analyzed for their ability to direct expression of a reporter gene (the chloramphenical transferase gene, CAT) in cell lines which normally express or do not express the MUCl gene. A construct containing 2.9 kb of MUCl 5' flanking sequence shows expression of CAT in breast and pancreatic cell lines but not in the non-epithelial cell lines HT 1080, SK23 and HTB96. Deletion analysis shows that maximum expression was obtained in ZR-75 (breast cancer line) and HPAP (pancreatic cancer line) with only 743 bp of 5' flanking sequence. Sequences within 1.6 kb of the transcriptional start site showed enhancing activity in a vector carrying an enhancerless SV40 promoter. Analysis of proximal 5' sequences in a promoterless CAT vector carrying the SV40 enhancer shows that sequences between - 60 and -150 were crucial for tissue specific expression. An Spl site at - 99/-90 and an E-box (E-MUC1) at -84/-64 in this region are shown by mutational analysis to play a role in the regulation of transcription. Gel shift analysis with oligonucleotides and nuclear extracts of ZR-75 showed protein binding to both of these sites. Spl binding activity is similar in ZR-75 and HT1080 cells whereas binding of factors to the E-MUC1 oligonucleotide reveals quantitative and qualitative differences between epithelial and non-epithelial cells.

Thus, the promoter of the MUCl gene is useful for expressing cytokines, according to the method of the invention, in pancreatic and breast tumours.

Example 9: Treatment of patients

1. Patient selection a) Patients with metastatic malignant melanoma with good performance data (WHO Grade zero 1 or 2) with a life expectancy of at least three months, normal renal and liver function and haematology, normal bilirubin and no evidence of cerebral secondaries are selected. b) Written consent is obtained. c) Patients need not have received prior chemotherapy because of the low activity, toxicity and immunosuppression of such treatments. They can be administered after the gene therapy is completed, if indicated. d) Diagnosis of metastasis is confirmed by fine needle aspiration cytology.

2. Administration of constructs a) The constructs used are composed of a 769 bp fragment or a 2.5 kb fragment of the 5' flanking sequence of the murine tyrosinase gene driving the human IL-2 gene within the promoterless mammalian expression vector pN ASS/3 (Clontech, Ca, USA). The decision to use the murine promoter sequence is based upon our demonstration that this sequence works well in human cells. Initial purification of the bulk grown plasmids DNA is achieved using QIAGEN-tips for plasmid purification (this is an anion exchange resin). The bacterial cells used as recipients for the plasmid constructs are the E. coli strain JM109. Verification of plasmid purity is by agarose gel electrophoresis. It is prepared to the same pyrogen free standards as monoclonal antibodies which are given in much higher amounts. It is administered in sterile saline. b) All injections are given by a qualified medical practitioner with MRCP or equivalent and training in medical oncology. A 27 gauge needle is used and local anaesthetic administered first. c) Patients are admitted for 24 hours following the injection and will be seen at three days and one week and thereafter weekly for one month and then monthly. The injection site is carefully examined and analgesia given as necessary.

3. Studies on initial needle aspirate for diagnostic purposes a) immunocytochemistry for melanoma cells and assessment of cell cycle distribution, b) PCR to assess cytokine expression - IL-2, interferon-7 and TNFα. 4. Dosage schedule tyrosinase/IL-2

Dose Biopsy

Cohort 1 100 μg DNA/200 μl 1 week 2 100 μg DNA/200 μl 2 weeks

5. Studies of excisional biopsy after construct injection a) immunochemistry for melanoma cells. b) genomic PCR to assess the construct. c) staining for lymphocyte sub-populations and dendritic cells, PCR for L-2 interferon-γ and TNFor. In situ hybridisation for the same cytokines. d) assessment of cytotoxic T cell response to autologous melanoma cells. Cells obtained from the biopsies will be used in chromium release assays, as well as peripheral T cells.

6. Studies on stored DNA preparations a) In order to verify that the prepared DNA has not been degraded, routine examination of an aliquot of the injected material by agarose gel electrophoresis should be carried out.

Assessment of results

The effectiveness of this approach is assessed by three criteria.

1) Assessment of IL-2 expression bv RTPCR in situ hybridisation and immunochemistry

A similar level of expression within 10-15% of tumour cells is found. 2) Assessment of local immune response bv immunocvtochemistrv

Lymphocyte subpopulations and dendritic cells are stained to assess subtypes of cells present after the injections.

3) Assessment of cvtotoxic T cell responses

There is 1-2 weeks of local IL-2 production.

There is a demonstration of a positive T cell response.

Genes that can be expressed include cytokines such as TNFα, GM-CSF, IL-4, interferon-γ or the proteins involved in T cell antigen recognition like class 1 molecules, or B7.

Safety

Considering the life expectancy of these patients who already have metastatic cancer, the risks of insertion of genetic material into the somatic cells of the body would appear to be minimal. Clearly there may be events resulting from positional integration into the genome, eg insertional mutagenesis, inactivation or enhancement of expression, which could theoretically be deleterious. However, these have not manifested themselves in over 200 injections into recipient mice and their importance appears to be more theoretical than practical. Moreover, should adverse immunological reactions occur, they are unlikely to be beyond control with a range of immunosuppressive agents. Again, the short life expectancy of these patients makes long term undesirable sequelae an unlikely event. The risks of chemotherapy with marrow suppression, allergic reactions, Budd-Chiari syndrome and infection would all seem to pose much greater clinical problems than the local injection of DNA.

Example 10: Co-injection of IL-2 expressing and B7-expressing DNA constructs into a melanoma

A TRP-1-B7 construct is made using PCR, the sequence information in the sequence listing and a DNA vector such that expression of the B7 coding sequence is driven by the TRP-1 promoter.

The TRP-1-B7 constiiict and the TRP-l-IL-2 construct of Example 3 are prepared in sterile, pyrogen free water. 100 μg of each DNA construct in 200 μl of water is injected into the melanoma at weekly intervals until the tumour regresses.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Imperial Cancer Research Technology Ltd

(B) STREET: Sardinia House, Sardinia Street

(C) CITY: London

(E) COUNTRY: United Kingdom

(F) POSTAL CODE (ZIP): C2A 3NL

(G) TELEPHONE: 071 242 1136 (H) TELEFAX: 071 831 4991 (I) TELEX: 265107 ICRF G

(ii) TITLE OF INVENTION: Tumour therapy

(iii) NUMBER OF SEQUENCES: 22

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3281 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GAGCTCCTCA CACGGACTCT GTCAGCTCCT CCCTGCAGCC TATCGGCCGC CCACCTGAGG 60

CTTGTCGGCC GCCCACTTGA GGCCTGTCGG CTGCCCTCTG CAGGCAGCTC CTGTCCCCTA 120

CACCCCCTCC TTCCCCGGGC TCAGCTGAAA GGGCGTCTCC CAGGGCAGCT CCCTGTGATC 180

TCCAGGACAG CTCAGTCTCT CACAGGCTCC GACGCCCCCT ATGCTGTCAC CTCACAGCCC 240

TGTCATTACC ATTAACTCCT CAGTCCCATG AAGTTCACTG AGCGCCTGTC TCCCGGTTAC 300

AGGAAAACTC TGTGACAGGG ACCACGTCTG TCCTGCTCTC TGTGGAATCC CAGGGCCCAG 360

CCAGTGCCTG ACACGGAACA GATGCTCCAT AAATACTGGT TAAATGTGTG GGAGATCTCT 420

AAAAAGAAAC ATATCACCTC CGTGTGGCCC CCAGCAGTCA GAGTCTGTTC CATGTGGACA 480

CAGGGGCACT GGCACCAGCA TGGGAGGAGG CCAGCAAGTG CCCGCGGCTG CCCCAGGAAT 540

GAGGCCTCAA CCCCCAGAGC TTCAGAAGGG AGGACAGAGG CCTGCAGGGA ATAGATCCTC 600

CGGCCTGACC CTGCAGCCTA ATCCTGAGTT CAGGGTCAGC TCACACCACG TCGACCCTGG 660

AGACCTGCTC AGGACCCAGG ACCCCATTTT TCCACCCTAA CCGATGCTCT CTGCTCTCTC CTAGCCTCAC TTCTAACCTT CCAAGCTCAC TATTGAATCC ACGCCGTTCA ATGTCGCAGA TTGTCCACAA TCTGCCCCAG CATCTTTTTG GCTACAGCTG ATGGCAACCG TCAAATTATA GGATATGTAA TAGGAACTCA CATACAGTGG TCGAGAGATA ATATACCCCA ATGCATCCCT AGAATGACAC AGGATTCTAC ACCCTACACG TCATAAAGTC CAACTGGCCA GTTCCGGGTA TACCGTGAGT GATTCCCCCA TCAGTTCTAC TTCCCACACA CAGGATTATC AGGCCTGGGC ATTACGAACC ATGTTAGGGT TTGGGCATTT AGTGCAGGAT TCAACAGATC AGAATTCCTT TCCGGCATCC AGACCCTGCA

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7130 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GAATTCCACA TTGTTTGCTG CACGTTGGAT TTTGAAATGC ATATTTCTGG GCTAGAGGAT CTGTGGACCA CAAGATCTTT TATCTGTGGA GCTGGATTCT GGGTTGGGAG TGCAAGGAAA ACATCTATTT CAGGAGCATG AGGAATAAAA GTTCTAGTTT GGATCAGGGA GTCTCACAAT CTCCTGAGTG CTGGTGTCTT GTGCAAAGGA TCTAGGCACG TGAGGCTTTG TATGAAGAAT TGTTTCTGTT TCATCCTGGG CATGTCTCCT CTGCCTTTGT GAGCTACAAG GGCCTGGTGC ATCCAGGGTG ATCTAGTAAT CTCTCCCTCC CCTTCCACAG CTCTGGGTGT GGGAGGGGGT GGGGAGGGCC TTGGTCAGCC TCTGGGTGCC AGCAGGGCAG AAGGTTTTAT AGGGCTCCTG GGGGAGGCTC CCCAGCCCCA AGAGCTGTGT CACCATGTGG GTCCCGGTTG TCTTCCTCAC GTGAGAGGGG CCATGGTTGG GGGGATGCAG GAGAGGGAGC

GTCTGTAGGA TAGGCATGGG GTACTGGAAT AGCTGACCTT CCCAAGAGTT CAAGCAGATA CAGCATGGCC TAGAGCCTCA CATCATGAAT CGCACTGTTA GCATGAATCA TCTGGCACGG AAGGCACTTG GGCCGAATGT TCCAAGGGAT TAAATGTCAT GTGAGCCCTG TACTTGGAAC GTTCAGGCTT TGAGCAGTGC TTACTGTACA GGGGGGTGAG GGAAAGGGAG AAGATGAGGA CTGTCTTGTG GCCGAGTGGA CCATGGGGCT ATCCCAAGAA

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 858 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CGAGCGGCCC CTCAGCTTGC GCGGCCCAGC CCCGCAAGGC CGGGAGGAGC TCCTGGCCAG TGGTGGAGAG TGGCAAGGAA GCCCAGGTTT ACTCCCTTAA GTGGAAATTT CTTCCCCCAC GGAGGGAACC CAGGCTGCTG GAAAGTCCGG CTGGGGGGGG ACGGGGTGTG GAACGGGACA GGGAGCGGTT AGAAGGGTGG GGGGGAGGGA GCCCAAAACT AGCACCTAGT CCACTCATTA GCCGCTCTGC TTCAGTGGAC CCGGGGAGGG CGGGGAAGTG GGCTTCCCGA CCTTGCTGTA CAGGACCTCG ACCTAGCTGG TTAGTTGTTG CCCTGAGGCT AAAACTAGAG CCCAGGGGCC CCCCCCTCCC CCGGAGCCAG GGAGTGGTTG GTGAAAGGGG CGGGTAGTCA GGGGGTTGAG GATTAGAGCC CTTGTACCCT GAGGAGGAAG AGGTAGGAGG TAGGGGAGGG GGCGGGGTTT CTGTGCCTAG GGCGGGCGGG CGGGGAGTGG GGGGACCGGT GCCCGCTCCA CCTCTCAAGC AGCCAGCGCC TGCCTGAATC CATTTCACCA CCACCATG

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1581 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

CATGGTGTCC GACTTATGCC CGAGAAGATG TTGAGCAAAC TTATCGCTTA TCTGCTTCTC 60

ATAGAGTCTT GCAGACAAAC TGCGCAACTC GTGAAAGGTA GGCGGATCTG GGTCGACCTG 120

CAGGTCAACG GATCCCTTCT TGACCAGTAT AGCTGCATTC TTGGCTGGGG CATTCCAACT 180

AGAACTGCCA AATTTAGCAC ATAAAAATAA GGAGGCCCAG TTAAATTTGA ATTTCAGATA 240

AACAATGAAT AATTTGTTAG TATAAATATG TCCCATGCAA TATCTTGTTG AAATTAAAAA 300

AAAAAGTCTT CCTTCCATGC CCCACCCCTA CCACTAGGCC TAAGGAATAG GGTCAGGGGC 360

TCCAAATAGA ATGTGGTTGA GAAGTGGAAT TAAGCAGGCT AATAGAAGGC AAGGGGCAAA 420

GAAGAAACCT TGAATGCATT GGGTGCTGGG TGCCTCCTTA AATAAGCAAG AAGGGTGCAT 480

TTTGAAGAAT TGAGATAGAA GTCTTTTTGG GCTGGGTGCA GTTGCTCGTG GTTGTAATTC 540

CAGCACTTTG GGAGGCTGAG GCGGGAGGAT CACCTGAGGT TGGGAGTTCA AGACCAGCCT 600

CACCAACGTG GAGAACCCTG TCTTTACTAA AAATACAAAA AATTCAGCTG GTCATGGTGG 660

CACATGCCTG TAATCCCAGC TGCTCGGGAG GCTGAGGCAG GAGAATCACT TGAACCAGGG 720

AGGCAGAGGT TGTGGTGAGC AGAGATCGCG CCATTGCTCT CCAGCCTGGG CAACAAGAGC 780

AAAAGTTCGT TTAAAAAAAA AAAAAAGTCC TTTCGATGTG ACTGTCTCCT CCCAAATTTG 840

TAGACCCTCT TAAGATCATG CTTTTCAGAT ACTTCAAAGA TTCCAGAAGA TATGCCCCGG 900

GGGTCCTGGA AGCCACAAGG TAAACACAAC ACATCCCCCT CCTTGACTAT CAATTTTACT 960

AGAGGATGTG GTGGGAAAAC CATTATTTGA TATTAAAACA AATAGGCTTG GGATGGAGTA 1020

GGATGCAAGC TCCCCAGGAA AGTTTAAGAT AAAACCTGAG ACTTAAAAGG GTGTTAAGAG 1080

TGGCAGCCTA GGGAATTTAT CCCGGACTCC GGGGGAGGGG GCAGAGTCAC CAGCCTCTGC 1140

ATTTAGGGAT TCTCCGAGGA AAAGTGTGAG AACGGCTGCA GGCAACCCAG GCGTCCCGGC 1200

GCTAGGAGGG ACGACCCAGG CCTGCGCGAA GAGAGGGAGA AAGTGAAGCT GGGAGTTGCC 1260

GACTCCCAGA CTTCGTTGGA ATGCAGTTGG AGGGGGCGAG CTGGGAGCGC GCTTGCTCCC 1320

AATCACAGGA GAAGGAGGAG GTGGAGGAGG AGGGCTGCTT GAGGAAGTAT AAGAATGAAG 1380

TTGTGAAGCT GAGATTCCCC TCCATTGGGA CCGGAGAAAC CAGGGGAGCC CCCCGGGCAG 1440 CCGCGCGCCC CTTCCCACGG GGCCCTTTAC TGCGCCGCGC GCCCGGCCCC CACCCCTCGC 1500

AGCACCCCGC GCCCCGCGCC CTCCCAGCCG GGTCCAGCCG GAGCCATGGG GCCGGAGCCG 1560

CAGTGAGCAC CATGGAGCTG G 1581 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1305 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1204..1284

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGATCCGTCC CGGGACTAGC AGGGCTTTGG GCAGCAACCC GGCCAGGTCC GGGCAGCTGG TGGGGGAGGT TCCAGAGGTC TCTCTAGTGT CCTCTCCGCG TCCCACTTCA CTGCCCCATC GACTTGGAAG GCACCTGGGA GGGTGTAAGC GCCTTGGTGT AAGAGCGGCG GGAACTGCGG CCGCCCGGAC GGTGCGGCCA GGCAGCTGTT CTCCCAGGCG GCCGTGGGGG GCAGCAGAGG GCCCCTCCCG GGGTAGAAGT GGAAAGGCGG GCTCCGGGGT ACCCCCGCCC CCCATCCAAA TCCCCGGGAG AGGCCCGGCC AAGCGGCCAG AGACAGTGCA ATTTCACGCG GTCTCTGTGG GGATGAATTA TGGGGTTTCG AGTCTGGGAG AAACTGAGGT AACACCCTCC CCCTCAAAAA CACACAGAGA GAAATATTCA CAAGTGAACC AACCGGCTAG GGGAGTTGAG TGATTTGGTT AGGGGGCAGG GCTTTGGAGA GCTTTCCACT CCCTCATTCA GGGCTTTCGG AATCTCGACC TCCCCTTGGC CTATCTCCTG CCATCCTCGA TCTGCTCCGC CAAGTTGCGG GACCGCGGGG GCGGTCCGAG GCTCCGCAAT CCCCACTCCA GCCTCGCGCG GACTCACCCC CTTCCCTCTG CGTTCCTCCC TCCCTCTCTC ACCCCTCCCC TGCCATCCCT CCCCGGACTC CGGCTCCGGC

TCCGCTGCCG TCGCCGCAGC AGCCACCAAT TCGCCAGCGG TTCAGGTGGC TCTTGCCTCG 1140

ATGTCCTAGC CTAGGGGCCC CCGGGCCGGA CTTGGCTGGG CTCCCTTCAC CCTCTGCGGA 1200

GTC ATG AGG GCG AAC GAC GCT CTG CAG GTG CTG GGC TTG CTT TTC AGC 1248 Met Arg Ala Asn Asp Ala Leu Gin Val Leu Gly Leu Leu Phe Ser 1 5 10 15

CTG GCC CGG GGC TCC GAG GTG GGC AAC TCT CAG GCA GGTAAGTGCC 1294

Leu Ala Arg Gly Ser Glu Val Gly Asn Ser Gin Ala 20 25

CAGAGAGCAC C 1305

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Arg Ala Asn Asp Ala Leu Gin Val Leu Gly Leu Leu Phe Ser Leu

10 15

Ala Arg Gly Ser Glu Val Gly Asn Ser Gin Ala 20 25

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4752 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AATTCATGCC CCAGTTGACA ACATAGCTGG TTCAAGACAA TGAAATCTTC ACTCTGGACC AATCAACATT CATACATTCC CCTGAGAGCT ATTTCTCTTC TCATCCTAAT TCTCTGCTCA CATATGAAAA TCTTGTACAT TCCCATGAGA TTGCATTGAA TGTCCATATG TATACCTTAC TTCTCATTCC TATTGCTTTG GTCTTTAGAA ATCTTGGGAG TGTTATGCCT CTTCCACTTA ATTCTCTAGA AGTTTAAACA ACTGCTATTC TGCTCTTCTA CAAAATATGT TTTCTCCCAG AAAACTCTCC TCAACTTTCT

GGAAAAAAGA ACATAGGCTA CCCCACACTT GAAATTTTGA AATATGAATG TCCTCTGTCT 4500

CTAGCTGAGT ACTCTGGCGC TTCCAAAATG GAAACCTTTA AAGGGCCACT GTAAATTACA 4560

GCTGCTAATT CCTGGTGCCA ATGGTGATAA GTGTTTACTA AACCTAGTGA GTACTTTATA 4620

GCATGGGTCT GCTGCGAAGT AACATTGCTG TATATTTTCA GTCATTCTAC CTTAATTCAT 4680

GAACTGCAAA ACTCTCATCT AGCTTTTTAC TTCTCTAGCT ATTGCTTTAA GTTCTATCAG 4740

GCTCAGGTGT GG 4752 (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1236 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: AAGCTTTGTA GAGTAATCAT GTATTCCAAA CTCAGGCTTA ATGTATGAGT TTTCAACTTC CAGGAGAAAA CGTCTCTTTA AACAGAAAAT ACAAGTGTGA CATTGGCCTT AGTTCGACCA TCTTCCTTTG TGGTATACAG ATAAGAAAAA TAAAATCACT TCAGCGTCTC TAATACATCT TCCAAATCAG TGTGTCTGAC ATCACAAGGA AACCAGTGGG GAGGGAGTCA TGTGCTGCCT ATTCACTGGT GTGAGAAGGG ATTAGTGAGA GCTGGAAGAG TGAGGAATCT GGCTTGGGAT TTACTGTCTG GCAGAAAATC GGCATCAGGG GAAAAGCAGA CATCCAACAA CACTAGCTCT CCTTCCAGGG ATTCATGGTA CTGGTGAGCA GCTCTGTGGT CTCTAGGAAC ATGAAGGAGA TTTGCTTGCT ATAAACCTGT CCATGGTTAA CTATTACTAT GGTAGTCACC AACTAGTGGA TATGGAAAGT CTTTTTGGAT CAGGGTGATC TTTTTATGTA GGTGCACGAG AGCAGGTGCC CAGATTCTCA AGGAGGGCTT TGATCTGATG TGGTTGCAAG GCACTGAAGT CAGTCTCTCT TTACCACTGT GCCTTCTCCC CAGCCCAAGA ATAGTATTCT AGAAACTCAA AGACCAGGAG AGTGAGTTCT GTCATCTAGC AAAGGTGAAT AATTATTTTG ACTATTGTTT AGAAATGTTG

TTCCGGAGTG GGTTGAAAAG TATGCAAAAG AACTTTTGCA ACTCTGTTTT TGCCTTTCTG 1140 TTTTTCAGCT GTATTTTCAT CTGAGCACCC CTGTCTTCTC CATGCAAAGA GCAGCATAGG 1200 AGACCTGTGT TCTGAACTCT TGCTTCGAGA AGAATG 1236 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5737 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TTAATCAACA AATCTAAACA TTTATTCTTT TCATCTGTTT CAATATGCTA TTCACATGTT CAGTGTAGTT TTATGACAAA TTGTATCCCC ACCCCCTTAA AGAAAGGAGG AAAAACTGTT GCATGAATTA GAGCTATCAC CTAAGTGTGG GCTAATGTAA ATCCATTCAG TCAGTCTTTG GGGGTTTAAA GAATTCCAAA ATGAAGGTAA TGTTTTTTCA GACAGGTAAA GTCTTTGAAA ATTTTGACAC CCCCATAATA TTTTTCCAGA ATTAACAGTA AGAGTTCCCT ATCACTCTCT TTAATCACTA CTCACAGTAA TACAGGATGC AACTCCTGTC TTGCATTGCA CTAAGTCTTG CCTACTTCAA GTTCTACAAA GAAAACACAG CTACAACTGG CAGATGATTT TGAATGGAAT TAATGTAAGT ATATTTCCTT TTAGTAATCT AGCTGGAGAT CATTTCTTAA TAACAATGCA AAGAATCCCA AACTCACCAG GATGCTCACA TTTAAGTTTT ACAATATTTT ATGTTCAATT TCTGTTTTAA TAAAATTCAA AGATGGGACT AATAGCAGCT CATCTGAGGT AAAGAGTAAC ACCCAAGTTT GATAATGAAG CCTCTATTAA AACAGTTTTA TTGTGTGTTG GTGGGGGTGG GAAGAAAACA TAAAAATAAT GACAATTCTA AACAAAAATG TTCATTTATG GTTTCATTTA ATTTGATTAT GTCATTTTAG TATGTAAAAT ACCAAAATCT TAAAAATCTT TTCTTGTTTT AGGAAAGGTT TCTAAGTGAG CACAGAGTCT GGGGCCAGAT ATCTGAAGTG AAATCTCAGC

TTAAATTTTA TATTTATTGT TGAATGTATG GTTTGCTACC ATCTTAAAAC TATAAATATG GATCTTTTAT GATTCTTTTT TGGTTTCACT TATTTATCCC AAAATATTTA TTATTATGTT TGTAGATTGG TTAGTAAAAC TATTTAATAA ATTTGATAAA TGTTATTTTG GAAACAGCAC AGAGTAAGCA TTTAAATATT TGTAGGATGG TTAAAATGCT TACAAAAGTC ACTCTTTCTC AGATGTAGAC TTCTCAAAAG CCCTTGCTTT GTCCTTTCAA TCTGGCATCT CTTAGCAGAT TATATTTTCC TTCTTCTTAA TTGAAACTCT TCATAGATTT GGTGTGGCTA TGAATTC

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 614 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GATCGTTAGC TTCTCCTGAT AAACTAATTG CCTCACATTG TCACTGCAAA TTAATGGGTC TCACCTCCCA ACTGCTTCCC CCTCTGTTCT TCCTGCTAGC AACTTTGTCC ACGGACACAA GTGCGATATC ACCTTACAGG AGATCATCAA AGCCTCACAG AGCAGAAGAC TCTGTGCACC GAGTTGACCG TAACAGACAT TCCAAGAACA CAACTGAGAA GGAAACCTTC TGCAGGGCTG CGACTGTGCT TACAGCCACC ATGAGAAGGA CACTCGCTGC CTGGGTGCGA CTGCACAGCA CACAAGCAGC TGATCCGATT CCTGAAACGG CTCGACAGGA ACCTCTGGGG TTGAATTCCT GTCCTGTGAA GGAAGCCAAC CAGAGTACGT TGGAAAACTT CTAAAGACGA TCATGAGAGA GAAATATTCA AAGTGTTCGA GCTGAATATT AGTTTTTGAT AGCTTTATTT TTTAAGTATT TATATATTTA TAACTCATCA TATATATAGA ATCT

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1589 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GAATTCCTCT GGTCCTCATC CAGGTGCGCG GGAAGCAGGT GCCCAGGAGA TGAAGATTCC ATGCTGATGA TCCCAAAGAT TGAACCTGCA GACCAAGCGC CTGAAAGTAC ACTGCTGGCG GATCCTACGG AAGTTATGGA AAAGGCAAAG CGCCGTAGTG TGTGCCGCCC CCCTTGGGAT GGATGAAACT GCAGTCGCGG AGGAACCAGC TGCAGAGATC ACCCTGCCCA ACACAGACTC GGCAACTCCG GGGTCCTGGG AGTGACTATG GGCGGTGAGA GCTTGCTCCT GCTCCAGTTG GACTACGCCC tϊCCTCCCGCA GACCATGTTC CATGTTTCTT TTAGGTATAT CCTCCCCTGA TCCTTCTTCT GTTGCCAGTA GCATCATCTG ATTGTGATAT GATGGCAAAC AATATGAGAG TGTTCTAATG GTCAGCATCG ATCAATTATT AAAGAAATTG GTAGCAATTG CCTGAATAAT GAATTTAACT TTTTTAAAAG GATGCTAATA AGGAAGGTAT GTTTTTATTC CGTGCTGCTC GCAAGTTGAG AAAATGAATA GCACTGGTGA TTTTGATCTC CACTTATTAA AAGTTTCAGA ATACTGTTGA ACTGCACTGG CCAGGTTAAA GGAAGAAAAC CAGCTGCCCT CAACCAACAA AGAGTTTGGA AGAAAATAAA TCTTTAAAGG AACAGAAAAA TTGTGTTTCC TAAAGAGACT ATTACAAGAG ATAAAAACTT GTTGGAATAA GGCACTAAAG AACACTGAAA AATATGGAGT GGCAATATAG AAACACGAAC TCCTCCAAGA ATCTATCTGC TTATGCAGTT TTTCAGAGTG GAATGCTTCC TGAATGCACC ATGGTCAAAA CGGATTAGGG CATTTGAGAA ATGCATATTG AGATGAATAC AAACAATGGA AACTGAATGC TCCAGTCAAC AAACTATTTC GAACATTTAT CAATCAGTAT AATTCTGTAC TGATTTTTGT AAGACAATCC TCAGTTGCAA TAATACTTCT CAAACCTGTT TAAATATTTC AAGACATTAA TATATAATGG TTTCAAAGAT TCAAAATTGA CATTGCTTTA CTGTCAAAAT CTCACTATGA ATCTATTATA CTGTATTAAG AGTGAAAATT GTCTTCTTCT TGTTTTAGAG TTAACAATGA TATATGGATA ATGCCGGTGA GAATAAGAGA TTAAGTAAGC AACAGCATAA CAAGGTCCAA GATACCTAAA AGAGATTTCA TTAATCATGA ATGTGTAACA CAGTGCCTTC AATAAATGGT ATAGCAAATG AAAAAAGGAC AATTTCAAAA AAATAAAAT

(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1585 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CACACCCTGA CAAGCTGCCA GGCAGGTTCT CTTCCTCTCA CATACTGACC CCCTCTCTCC CCTGGAAAGG ACACCATGAG CACTGAAAGC ATGATCCGGG GGCCGAGGAG GCGCTCCCCA AGAAGACAGG GGGGCCCCAG GGCTCCAGGC CCTCAGCCTC TTCTCCTTCC TGATCGTGGC AGGCGCCACC ACGCTCTTCT CTTTGGAGTG ATCGGCCCCC AGAGGGAAGA GTCCCCCAGG GACCTCTCTC TCTGGCCCAG GCAGTCAGAT CATCTTCTCG AACCCCGAGT GACAAGCCTG TGTAGCAAAC CCTCAAGCTG AGGGGCAGCT CCAGTGGCTG AACCGCCGGG CCTGGCCAAT GGCGTGGAGC TGAGAGATAA CCAGCTGGTG GTGCCATCAG CCTCATCTAC TCCCAGGTCC TCTTCAAGGG CCAAGGCTGC CCCTCCACCC CACCCACACC ATCAGCCGCA TCGCCGTCTC CTACCAGACC AAGGTCAACC CATCAAGAGC CCCTGCCAGA GGGAGACCCC AGAGGGGGCT GAGGCCAAGC GCCCATCTAT CTGGGAGGGG TCTTCCAGCT GGAGAAGGGT GACCGACTCA CAATCGGCCC GACTATCTCG ACTTTGCCGA GTCTGGGCAG GTCTACTTTG CCTGTGAGGA GGACGAACAT CCAACCTTCC CAAACGCCTC CCCTGCCCCA TACCCCCTCC TTCAGACACC CTCAACCTCT TCTGGCTCAA AAAGAGAATT GGTCGGAACC CAAGCTTAGA ACTTTAAGCA ACAAGACCAC CACTTCGAAA AGGAATGTGT GGCCTGCACA GTGAAGTGCT GGCAACCACT AAGAATTCAA CCAGAACTCA CTGGGGCCTA CAGCTTTGAT CCCTGACATC TGGAATCTGG GCCTTTGGTT CTGGCCAGAA TGCTGCAGGA CTTGAGAAGA CCTCACCTAG AAGTGGACCT TAGGCCTTCC TCTCTCCAGA TGTTTCCAGA CTTCCTTGAG CAGCCCTCCC CATGGAGCCA GCTCCCTCTA TTTATGTTTG CACTTGTGAT TTATTTATTA TTTATTTATT TACAGATGAA TGTATTTATT TGGGAGACCG GGGGACCCAA TGTAGGAGCT GCCTTGGCTC AGACATGTTT TCCGTGAAAA ACAATAGGCT GTTCCCATGT AGCCCCCTGG CCTCTGTGCC TTCTTTTGAT AAAATATTAT CTGATTAAGT TGTCTAAACA ATGCTGATTT GGTGACCAAC

TGCTGAGGCC TCTGCTCCCC AGGGAGTTGT GTCTGTAATC GGCCTACTAT TCAGTGGCGA 1560 GAAATAAAGG TTGCTTAGGA AAGAA 1585

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CGGAATTTCA TGCCCCAGTT GACAACATAG 30

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CACTCGAGAA CTTTTTCTCC TTTAGATCAT ACAA 34

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CGGGAATTCA TGCCCCAGTT GACAACATAG 30

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GAGCTCGAGT GTCACAGACT TCTTTTCCA 29

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: AAACGAATTC CATCCAGTAA GTCCATTACT 30

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAGCTCGAGT GTCACAGACT TCTTTC 26

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GCGGCCGCGC ATGTACAGCA TGCAGCTCGC A 31

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GCGGCCGCTA AATAAATAGA GAGCCTTATG 30

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1011 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

GGTTTATTTT CCAGATGCAA TCAATGCCCC AGTCACCTGC TGTTATAACT TCACCAATAG 60

GAAGATCTCA GTGCAGAGGC TCGCGAGCTA TAGAAGAATC ACCAGCAGCA AGTGTCCCAA 120

ACAAGCTGTG ATGTGAGTTC AGCACACCAA CCTTCCCTGG CCTGAAGTTC TTCCTTGTGG 180

AGCAAGGGAC AAGCCTCATA AACCTAGAGT CAGAGAGTGC ACTATTTAAC TTAATGTACA 240

AAGGTTCCCA ATGGGAAAAC TGAGGCACCA AGGGAAAAAG TGAACCCCAA CATCACTCTC 300

CACCTGGGTG CCTATTCAGA ACACCCAATT TCTTTAGCTT GAAGTCAGGA TGGCTCCACC 360

TGGACACCTA TAGGAGCAGT TTGCCCTGGG TTCCCTCCTT CCACCTGCGT TCCTCCTCTA 420

GCTCCCATGG CAGCCCTTTG GTGCAGAATG GGCTGCACTT CTAGACCAAA ACTGCAAAGG 480 AACTTCATCT AACTCTGTCC TCCCTCCCCA CAGCTTACAG ACCATTGTGG CAAGGAGATC 540

TGTGCTGACC CCAAGCAGAA GTGGGTTCAG GATTCCATGG ACCACCTGGA CAAGCAAACC 600

CAAACTCCGA AGACTTGAAC ACTCACTCCA CAACCCAAGA ATCTGCAGCT AACTTATTTT 660

TCCCTAGCTT TCCCCAGACA CCTTGTTTAT TTTATTATAA TGAATTTTGT TTGTTGATGT 720

GAAACATTAT GCCTTAAGTA ATGTTAATTC TTATTTAAGT TATTGATGTT TTAAGTTTAT 780

CTTTCATGGT ACTAGTGTTT TTTAGATACA GAGACTTGGG GAAATTGCTT TTCCTCTTGA 840

ACCACAGTTC TACCCCTGGG ATGTTTTGAG GGTCTTTGCA AGAATCATTA ATACAAAGAA 900

TTTTTTTTAA CATTCCAATG CATTGCTAAA ATATTATTGT GGAAATGAAT ATTTTGTAAC 960

TATTACACCA AATAAATATA TTTTTGTACA AAAAAAAAAA AAAAAAAAAA A 1011 (2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3194 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

AAGCTTGCTG AGAGTGGCTG CAGTCTCGCT GCTGGATGTG CACATGGTGG TCATTCCCTC 60

TGCTCACAGG GGCAGGGGTC CCCCCTTACT GGACTCAGGT TGCCCCCTGC TCCAGGTCCT 120

GGGTGGGAGC CCATGTGAAC TGTCAGTGGG GCAGGTCTGT GAGAGCTCCC CTCACACTCA 180

AGTCTCTCAC AGTGGCCAGA GAAGAGGAAG GCTGGAGTCA GAATGAGGCA CCAGGGCGGG 240

CATAGCCTGC CCAAAGGCCC CTGGGATTAC AGGCAGGATG GGGAGCCCTA TCTAAGTGTC 300

TCCCACGCCC CACCCCAGCC ATTCCAGGCC AGGAAGTCCA AACTGTGCCC CTCAGAGGGA 360

GGGGGCAGCC TCAGGCCCAT TCAGACTGCC CAGGGAGGGC TGGAGAGCCC TCAGGAAGGC 420

GGGTGGGTGG GCTGTCGGTT CTTGGAAAGG TTCATTAATG AAAACCCCCA AGCCTGACCA 480

CCTAGGGAAA AGGCTCACCG TTCCCATGTG TGGCTGATAA GGGCCAGGAG ATTCCACAGT 540

TCAGGTAGTT CCCCCGCCTC CCTGGCATTT TGTGGTCACC ATTAATCATT TCCTCTGTGT 600

ATTTAAGAGC TCTTTTGCCA GTGAGCCCAG TACACAGAGA GAAAGGCTAA AGTTCTCTGG 660

AGGATGTGGC TGCAGAGCCT GCTGCTCTTG GGCACTGTGG CCTGCAGCAT CTCTGCACCC 720

GCCCGCTCGC CCAGCCCCAG CACGCAGCCC TGGGAGCATG TGAATGCCAT CCAGGAGGCC 780

CGGCGTCTCC TGAACCTGAG TAGAGACACT GCTGCTGAGA TGGTAAGTGA GAGAATGTGG 840

GCCTGTGCCT AGGCCACCCA GCTGGCCCCT GACTGGCCAC GCCTGTCAGC TTGATAACAT 900

CAGAAATCAG TAATATTTAT ATATTTATAT TTTTAAAATA TTTATTTATT AGTTCATATT CCATATTTAT TCAAGATGTT TTACCGTAAT AATTATTATT TTCTACTTGT CCAGTGTTCT AGTTTGTTTT TAACCATGAG CAAATGCCAG CCTTCCCATG AGGCAGGGGA GGGAGGAAAC GGGGAGGTGG AGAGGGGGCG AGGCGTTGGG CACTATCCAA GGGCCAACAC TGTCAGAGCA GAGGGGAGGT CATAGTCGGA ATTC

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1491 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CCAAAGAAAA AGTGATTTGT CATTGCTTTA TAGACTGTAA GAAGAGAACA GGAGTCTTAC CCTGAAATCA AAGGATTTAA AGAAAAAGTG GAATTTTTCT GTGAAACTAA ATCCACAACC TTTGGAGACC CAGGAACACC CTCCAATCTC GTAAACATCA CTGGAGGGTC TTCTACGTGA GCAATTGGAT TGTCATCAGC TTGCACCTGG GAAGTGCCCT GGTCTTACTT GGGTCCAAAT TGTTGGCTTT CCTAAGCATC TGAAGCCATG GGCCACACAC GGAGGCAGGG AACATCACCA CATACCTCAA TTTCTTTCAG CTCTTGGTGC TGGCTGGTCT TTCTCACTTC TTATCCACGT GACCAAGGAA GTGAAAGAAG TGGCAACGCT GTCCTGTGGT CTGTTGAAGA GCTGGCACAA ACTCGCATCT ACTGGCAAAA GGAGAAGAAA CTATGATGTC TGGGGACATG AATATATGGC CCGAGTACAA GAACCGGACC TCACTAATAA CCTCTCCATT GTGATCCTGG CTCTGCGCCC ATCTGACGAG AGTGTGTTGT TCTGAAGTAT GAAAAAGACG CTTTCAAGCG GGAACACCTG CGTTATCAGT CAAAGCTGAC TTCCCTACAC CTAGTATATC TGACTTTGAA CTAATATTAG AAGGATAATT TGCTCAACCT CTGGAGGTTT TCCAGAGCCT GGTTGGAAAA TGGAGAAGAA TTAAATGCCA TCAACACAAC AGTTTCCCAA CTGAGCTCTA TGCTGTTAGC AGCAAACTGG ATTTCAATAT GACAACCAAC TGTGTCTCAT CAAGTATGGA CATTTAAGAG TGAATCAGAC CTTCAACTGG AGCAAGAGCA TTTTCCTGAT AACCTGCTCC CATCCTGGGC CATTACCTTA ATGGAATTTT TGTGATATGC TGCCTGACCT ACTGCTTTGC CCCAAGATGC

GGAGGAATGA GAGATTGAGA AGGGAAAGTG TACGCCCTGT ATAACAGTGT CCGCAGAAGC 1200

AAGGGGCTGA AAAGATCTGA AGGTAGCCTC CGTCATCTCT TCTGGGATAC ATGGATCGTG 1260

GGGATCATGA GGCATTCTTC CCTTAACAAA TTTAAGCTGT TTTACCCACT ACCTCACCTT 1320

CTTAAAAACC TCTTTCAGAT TAAGCTGAAC AGTTACAAGA TGGCTGGCAT CCCTCTCCTT 1380

TCTCCCCATA TGCAATTTGC TTAATGTAAC CTCTTCTTTT GCCATGTTTC CATTCTGCCA 1440

TCTTGAATTG TCTTGTCAGC CAATTCATTA TCTATTAAAC ACTAATTTGA G 1491

Claims

1. A DNA construct comprising (i) means for expression of a coding sequence in a tumour cell and (ii) a said coding sequence encoding a cytokine.

2. A construct according to Claim 1 wherein the said means for expression provides for specific expression selectively in tumour cells.

3. A construct according to Claim 2 wherein the tumour cells are melanoma cells.

4. A construct according to Claim 2 wherein the tumour cells are breast tumour cells.

5. A construct according to Claim 2 wherein the tumour cells are colon tumour cells.

6. A construct according to Claim 2 wherein the tumour cells are pancreatic tumour cells.

7. A construct according to Claim 2 wherein the tumour cells are prostate tumour cells.

8. A construct according to Claim 3 wherein the said means for expression is a promoter or an analogue or part thereof forming part of a gene expressed exclusively in the melanin synthesis pathway.

9. A construct according to Claim 8 wherein the gene is tyrosinase or TRP-1.

10. A construct according to Claim 4 wherein the said means for expression is provided by the c-erb-B2 gene promoter or the

MUCl gene promoter or the c-erb-B3 gene promoter.

11. A construct according to Claim 5 wherein the said means for expression is provided by the CEA gene promoter.

12. A construct according to Claim 6 wherein the said means for expression is provided by the MUCl gene promoter.

13. A construct according to Claim 7 wherein the said means for expression is provided by the PSA gene promoter.

14. A construct according to any one of the preceding claims wherein the cytokine is interleukin-2 or interleukin-4.

15. A construct according to any one of the preceding claims further comprising a B7 coding sequence and means for expression thereof in a tumour cell.

16. A composition comprising a construct according to any one of the preceding claims and means for selectively delivering it to a tumour.

17. A composition according to Claim 16 wherein the selective delivery means is a liposome carrying tumour cell targeting means or a retrovirus or adenovirus specific for the tumour cells.

18. A method of treating a tumour and/or ameliorating metastasis therefrom comprising delivering into cells of the tumour a construct according to any one of Claims 1 to 15.

19. A method of treating a tumour and/or ameliorating metastasis therefrom comprising delivering into cells of the tumour a construct according to any one of Claims 1 to 15 expressing at least two coding sequences encoding respective cytokines wherein the said cytokines may be the same as or different from one another.

20. A method of treating a tumour and/or ameliorating metastasis therefrom comprising delivering into cells of the tumour a plurality of constructs according to any one of Claims 1 to 15 expressing at least two coding sequences encoding respective cytokines wherein the said cytokines may be the same as or different from one another.

21. A method according to Claims 19 or 20 wherein the cytokines are chosen from interleukin-2, interleukin-4, macrophage colony stimulating factor, interferon-7, tumour necrosis factor and interleukin-7.

22. A method according to Claims 19 or 20 wherein the coding sequences encode interleukin-2, interleukin-4 and macrophage colony stimulating factor and are present in 1:1:1 molar ratio.

23. A method according to any one of Claims 18 to 20 wherein the tumour cells are melanoma, breast, pancreas, prostate or colon cells and naked DNA is injected directly into the tumour.

24. A method according to any one of Claims 18 to 23 additionally comprising administering a chemotherapeutic agent.

25. A method according to Claim 24 wherein the chemotherapeutic agent is at least one of cisplatin, dacarbazine, tamoxifen, nitrosourea, vinca alkaloid, melphalan, doxorubicin, adriamycin, etoposide and 5-fluorouracil.

26. A method according to any one of Claims 18 to 25 further comprising delivering into cells of the tumour a construct comprising a B7 coding region and means for expression thereof in a tumour cell.

27. A method according to any one of Claims 18 to 25 comprising delivering into cells of the tumour a construct comprising a B7 coding region and a cytokine coding region and means for expression thereof in a tumour cell.