MXPA99010224A - Chemical compounds - Google Patents

Abstract

The invention provides a gene construct encoding a cell targeting moiety and a heterologous prodrug activating enzyme for use as a medicament in a mammalian host wherein the gene construct is capable of expressing the cell targeting moiety and enzyme as a conjugate within a target cell in the mammalian host and wherein the conjugate is directed to leave the cell thereafter for selective localisation at a cell surface antigen recognised by the cell targeting moiety.

Description

CHEMICAL COMPOUNDS Description of the invention This invention is concerned in particular with gene directed enzyme-directed prodrug therapy (GDEPT) which utilizes the in situ generation of antibodies to provide improved selectivity, particularly for use in cancer therapy. Therapeutic methods of promedication based on known gene therapy include viral directed enzyme-mediated therapy (VDEPT) and gene-directed enzyme-linked therapy (GDEPT), the latter term encompassing VDEPT delivery systems and systems. of non-viral supply. The VDEPT system involves cells that point to the tumor with a viral vector carrying a gene that encodes an enzyme capable of activating a prodrug. The viral vector enters the tumor cell and the enzyme is expressed from the enzyme gene into the cell. In the GDEPT system, alternative methods such as microinjection, liposomal administration and DNA uptake mediated by the receptor, can also be used, as well as viruses to administer the gene encoding the enzyme. In the VDEPT and GDEPT systems the enzyme gene can be regulated transcriptionally by DNA sequences capable of being selectively activated in mammalian cells, for example tumor cells "(EP 415 731 (Wellcome); Huber et al., Proc. Nati, Acad. Sci. OSA, 88, 8039-8043, 1991.) While it provides a degree of selectivity, gene expression can also occur in non-target cells and this is clearly undesirable when the procedure is used to activate In addition, these regulatory sequences will generally lead to reduced expression of the enzyme compared to the use of viral promoters and this will lead to a reduced capacity to convert the prodrug to the target tissue.Expression and localization of the enzyme activating the promedicamento within the cell has disadvantages.The design of the promedicamento is severely limited by the fact that the drug must be able to cross the cell membrane and enter the cell but not be toxic until it is converted to the drug inside the cell by the activating enzyme. Most of the promedicamentos use hydrophilic groups to prevent entry to the cells and thus reduce cytotoxicity. The change of the prodrug by the activating enzyme produces a less hydrophilic drug that can enter the cells to produce anti-cancer effects. This procedure can not be used when the activating enzyme is expressed inside the cell. Another disadvantage is that the target cells lacking intracellular activating enzyme will be difficult to attack because they are unable to generate the active drug. To obtain this "surrounding activity { or pending or latent.}." (or "surrounding cell extermination") desirable, the active drug will have to be able to diffuse out of the cell containing the activating cell to reach the target cells lacking enzyme expression. Many active drugs when they are produced inside a cell will be unable to escape from the cell to achieve this surrounding (or latent) effect. Modifications of the GDEPT have been proposed to overcome some of the problems described above. In ppmer place have been described vectors that are said to express the activating enzyme on the surface of the target cell (WO 96/03515) by attaching a signal peptide and transmembrane domain to the activating enzyme. The procedure, if feasible, would overcome the problems of the activating enzyme located within the cell but would still have to depend on transcriptionally regulated sequences capable of being selectively expressed in target cells to restrict cell expression. As described above there are disadvantages of the use of such sequences. Secondly, vectors have been described that result in the secretion of the enzyme from the target cell (WO 96/16179). In this procedure, the enzyme would be able to diffuse from its generation site since it is extracellular and is not bound to the surface of the cell. The enzyme that has diffused from the target site would be able to activate the promedication in non-target sites, which would lead to undesirable toxicity. To obtain some selectivity it is suggested that enzyme precursors that are cleaved by proteases associated with the pathology to form active enzyme could be used. It is likely that some selectivity is obtained by this procedure but activation is not likely to occur at the target sites. In addition, once activated, the enzyme will still be free to diffuse from the target site and thus have the same disadvantage described above. For the GDEPT procedures, three levels of selectivity can be observed. First, there is selectivity in the infection stage of the cell, such that it targets only specific cell types. For example, cellular selectivity can be provided by the gene delivery system per se. An example of this type of selectivity is summariin the international patent application WO 95/26412 (ÜAB Research Foundation) which describes the use of modified adenovirus fiber proteins incorporating cell-specific ligands. Other examples of targeting systems specific to the cell include the transfer of ex vivo gene to specific cell populations such as lymphocytes and direct injection of DNA into muscle tissue. The second level of selectivity is the control of gene expression after cell infection, such as for example by the use of cell-specific promoters or tissue. If the gene has been delivered to a cell type in a selective manner, then it is important that you choose a promoter that is compatible with the activity in the cell type. The third level of selectivity can be considered as the selectivity of the construct or construct of the expressed gene. Selectivity at this level has received scant attention to date. In the international patent application WO 96/16179 (Wellcome Foundation) it is suggested that enzyme precursors that are cleaved by proteases associated with the pathology to form active enzyme could be selected. It is likely that some selectivity is obtained by this procedure but activation is unlikely to occur at the target sites. In addition, once activated, the enzyme will still be free to diffuse from the target site and thus has the same disadvantage of the activating prodrug in non-target sites leading to undesirable toxicity.

There is a need for more selective GDEPT systems to reduce the undesirable effects in normal tissues that arise from erroneous activation of the prodrug. The present invention is based on the discovery that constructs or constructs of heterologous antibody-enzyme gene can be expressed intracellularly and used in GDEPT systems (or other systems such as AMIRACS - see below) for a targeting system. the cell that arises from the specificity of antibody to supply enzyme available on the surface of the cell in a selective manner. This method can optionally be used in combination with any other technique (s) that improve specificity, such as infection of the target cell and / or tissue-specific expression. According to one aspect of the present invention there is provided a gene construct or construct that encodes an antibody that targets the cell and a heterologous enzyme for use as a medicament in a mammalian host, wherein the gene construct or construct is capable of of expressing the antibody and enzyme as a conjugate within a target cell in the mammalian host and wherein the conjugate can leave the cell thereafter for selective localization to the antigen on the surface of the cell recognized by the antibody. According to another aspect of the present invention there is provided a gene construct or construct that encodes an antibody a targeting portion to the cell and a heterologous enzyme activating the prodrug for use as a medicament in a mammalian host, wherein the construct or construct The gene is capable of expressing the portion that points to the cell and the activating enzyme of the drug, heterologous, I sew a conjugate inside a cell in the mammalian host and where the conjugate is instructed to leave the cell after that for its location or selective location on an antigen on the surface of the cell recognized by the portion that points to the cell. The portion "pointing to the cell" is defined as any polypeptide or fragment thereof that sticks or selectively adheres to a particular cell type in a host by means of recognition of a cell surface antigen. Preferably, the portion that points to the cell is an antibody. Portions pointing to the cell other than the antibodies include ligands, as described for use in Ligand-Directed Enzyme Promedication Therapy as described in the international patent application WO 97/26918, Cancer Research Campaign Technology Limited, such as for example, - the epidermal growth factor, heregulin, c-erbB2 and vascular endothelial growth factor, the latter being preferred. An "antibody pointing to the cell" is defined as an antibody or fragment thereof that sticks or selectively adheres to a particular cell type in a host by recognition of an antigen on the surface of the cell. Preferred cell-targeting antibodies are specific for solid tumors, more preferably colorectal tumors, more preferably an anti-CEA antibody, more preferably antibody A5B7 or antibody 806. 077, wherein antibody 806.077 is especially preferred. The 8O6.077 hybridoma antibody was deposited in the European Collection of Animal Cell Cultures (ECACC), PHLS Center for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom, on February 29, 1996 with accession number 96022936 in accordance with the Budapest Treaty. The A5B7 antibody binds to human carcinoembryonic antigen (CEA) and is particularly appropriate for targeting colorectal carcinoma. The A5B7 antibody is available from DAKO Ltd., 16 Manar Coartyard, Hughenden Avenae, High Wycombe, Bucks HP13 5RE, England, United Kingdom. In general, the antibody (or antibody fragment) -enzyme conjugate must be at least divalent, ie it must be capable of sticking or binding to at least 2 antigens associated with the tumor (which may be the same or different).

The antibody molecules can be converted to human molecules by known methods such as for example by "CDR grafting" as described in the document.

EP239400 or by complete ingestion of variable regions of for example a murine antibody over human constant regions ("chimeric antibodies") as described in US Pat. No. 4,816,567. Human antibodies may be useful for reducing the immunogenicity of an antibody (or fragment). of antibody). A human version of the antibody A5B7 has been described in the international patent application WO 92/01059 (Celltech). The hybridoma that produces the monoclonal antibody A5B7 was deposited with the European Collection of Cultures of Animal Cells, Biological Division, PHLS Center by Applied Microbiology and Research, Portón Down, Salisbury, Wiltshire SP4 OJG, United Kingdom. The date of deposit was July 14, 1993 and the accession number is 93071411. The antibody A5B7 can be obtained from the deposited hybridoma by using standard techniques known in the art such as are documented in Fenge C, Fraune E & Schuegerl K in "Production of Biologicals from Animal Cells in Culture" (Spier RE, Griffiths JR &Meignier B, eds) Butterworth-Heinemann, 1991, 262-265 and Anderson BL & Gruenberg ML in "Co commercial Production of Monoclonal Antibodies" (Seaver S, ed), Marcel Dekker, 1987, 175-195. The cells may require recloning from time to time by limiting the dilution in order to maintain good levels of antibody production. A "heterologous enzyme" is defined as an enzyme for converting a substrate that has been administered to the host and the enzyme is not stably present in nature in the relevant compartment of the host. The enzyme may be foreign to the mammalian host (eg, a bacterial enzyme such as CPG2) or it may not be present in a stable manner in nature within the relevant host compartment (eg, the use of lysosim as an enzyme of ADEPT (for an explanation of ADEPT see later herein) is possible because lysosimus does not occur stably in nature in the circulation, see US 5433955, Akzo NV). The relevant host compartment is that part of the mammalian host in which the substrate is distributed. Preferred enzymes are suitable enzymes for ADEPT or AMIRACS fAntimetabolite with Inactivation of Rescue Agents at Cancer Sites; see Bagshawe (1994) in Cell Biophysics 24 / 25F 83-91) but the enzymes of ADEPT are preferred. Antibody-directed enzyme prodrug therapy (ADEPT) is a known therapeutic method of cancer. ADEPT uses a selective antibody to the tumor conjugated to an enzyme. The conjugate is administered to the patient (usually intravenously), it is allowed to be located in the tumor site (s) and cleared of blood and other normal tissues. Then a prodrug is administered to the patient who is converted by the enzyme (located at the tumor site) to a cytotoxic drug that kills the tumor cells. In patent application WO 96/20011 published the July 4, 1996, a system of ADEPT of "Reverse polarity" based on mutant human enzymes having the advantage of low immunogenicity compared to eg bacterial enzymes. A particular host enzyme consists of human pancreatic CPB (see for example, example 15 [D253K] human CPB &16 [D253R1 therein) and promedications thereto (see examples 18 and 19 therein). The host enzyme is mutated to give a change in. the mode of interaction between the enzyme and the prodrug and in terms of substrate recognition in comparison with the original host enzyme. In the patent application Subsequent International No. PCT / GB96 / 01975 (published March 6, 1997), as WO 97/07796) an additional work with respect to enzyme combinations of mutant CPB / prodrug for ADEPT is described. Preferred preferred enzymes for ADEPT are any of the CPG2 or reversed-polarity CPB enzyme, for example any of [D253K] HCPB, [G251T, D253K] HCPB or [A248S, G251T, D253K1HCPB. A preferred form of CPG2 is one in which "glycosylation sites of the polypeptide have been mutated to prevent or reduce glycosylation on expression in cells, mammals (see for example WO 56/03515, Cancer Research Campaign Technology); this provides improved enzyme activity. Additional considerations arise for enzymes such as CPB that require a prodomain to facilitate correct folding; here, the prodomain could be expressed separately (in trans) or expressed as part of the fusion protein and subsequently eliminated. Large scale purification of CPG2 from Pseudomonas of RS-16 was described in Sherwood et al (1985), Eur, J. Biochem., 148, 447-453. CPG2 can be obtained from the Center for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom. CPG2 can also be obtained by recombinant techniques. The nucleotide coding sequence for CPG2 has been published by Minton, N.P. et al., Gene, 31 (1984) and 31-38. The expression of the coding sequence has been reported in E.coli (Chambers, SP et al., Appi. Microbiol, Biotechnol. (1988), 2_9, 572-578 and in Saccharomyces cerevisiae (Clarke, LE et al., J Gene Microbiol, (1985) 131, S97-904) Total gene synthesis has been described by M. Edwards in Am. Biotech, Lab (1987), 5_, 38-44.Expression of heterologous proteins in E.coli has been reviewed by FAO Marston in DNA Cloning Vol.

III, Practical Approach Series, IRL Press (Editor D M Glover.}., 1987, 59-88. The expression of proteins in yeast has been reviewed in Methods in Enzymology, Volume 194, Academic Press 1991, edited by C. Guthrie and G. R. Fink. While cancer therapeutic methods are preferred, the invention can also be applied to other therapeutic areas as long as a target antigen can be selected and an appropriate combination of enzyme / prodrug can be prepared. For example, inflammatory diseases such as rheumatoid arthritis can be treated for example by using a selective antibody for synovial cells fused to an enzyme capable of converting an anti-inflammatory drug in the form of a prodrug to an anti-inflammatory drug. The use of antibodies to target rheumatoid arthritis disease has been described in Blakey et al, 1988, Scand. J.

Rheu atology, Suppl.76, 279-287. A "conjugate" between antibody and enzyme can be a fusion protein (covalent bond) or the conjugate can be formed by non-covalent linkage between the antibody and the enzyme formed in situ. Preferably, the conjugate is in the form of a fusion protein, more preferably the antibody component of the fusion is at least divalent (for improved avidity of binding compared to the monovalent antibody). Antibody constructs or constructs lacking a portion of Fc are preferred, especially the Fab or F (ab ') 2 fragments.

For CPG2 fusions (or fusions with any non-monomeric enzyme) special considerations apply because CPG2 is a dimeric enzyme and the antibody is preferably divalent and thus there is the potential for an undesirable competent dimerization between two molecular species. Accordingly, a preferred CPG2 fusion is one in which the fusion protein is formed by linking a C terminus of an antibody Fab heavy chain (ie, lacking a pivot region) to a terminus. N of a CPG2 molecule, * then two of these Fab-CPG2 molecules are digested via the dimerization domain of CPG2 to form a conjugate of (Fab-CPG2) 2.

For constructs or antibody constructs with monomeric enzymes F (ab '.sub.2) fragments are preferred, especially F (ab') 2 fragments having a human IgG3 pivot region.The fusions between antibody and enzyme can be made optionally by means of a short peptide linker such as for example (GS) 3. Preferred constructs or fusion constructs are those in which the enzyme is fused to the C-terminus of the antibody, by means of the heavy or light chain thereof wherein fusion by the heavy chain of the antibody is preferred, Thus, a preferred gene construct or construct is a gene construct or construct for use as a medicament as described herein, wherein the antibody conjugate CPG2 enzyme is a fusion protein in which the enzyme is fused to the C-terminus of the antibody by means of the heavy or light chain thereof, whereby dimerization of the conjugate Coding, when expressed, can be carried out by means of a dimerization domain over CPG2.

A more preferred gene construct is a gene construct for use as a medicament wherein the fusion protein is formed by linking a C terminus of an antibody Fab heavy chain to a N terminus of a molecule of CPG2 to form a Fab-CPG2, whereby two molecules of Fab-CPG2, when expressed, are dimerized by means of CPG2 to form a conjugate of (Fab-CPG2) 2. In another embodiment of the invention, a preferred gene construct for use as a medicament is one in which carboxypeptidase is selected from [D253KJHCPB, [G251T, D253KIHCPB or [A248S, G251T, D253K] HCPB]. It is contemplated that it should be possible to obtain a natural multimeric enzyme in monomeric form while retaining substantially the enzymatic activity, then the monomeric form of the enzyme could be used to form a conjugate of the invention. Similarly, it is contemplated that it should "be possible to obtain a natural monomeric enzyme in multimeric form while substantially retaining the enzymatic activity, then the multimeric form of the enzyme could be used to form a conjugate of the invention." The conjugate is instructed to exit the cell after its expression therein through the use of or of a secretory leader sequence which is cleaved as the conjugate passes through the cell membrane.Preferably, the secretory conductor is the driver of secretion that occurs naturally with the antibody According to another aspect of the present invention there is provided the use of a construct or construct of a gene encoding an antibody that targets the cell and a heterologous enzyme for use in the preparation of a medicament for cancer therapy in a mammalian host, wherein the gene construct or construct is capable of expressing The antibody and enzyme as a conjugate within a target cell in the mammalian host and wherein the conjugate can leave the cell thereafter for selective localization to an antigen on the surface of the cell recognized by the antibody. Any suitable administration system can be applied to deliver the gene construct of the present invention, in which viral and non-viral systems are included. Viral systems include retroviral vectors, adenoviral vectors, adeno-associated viruses, -vaccinia, herpes simplex virus, HIV, mouse minute virus, hepatitis B virus and influenza virus. Non-viral systems include unencomplexed DNA, DNA-liposome complexes, DNA-protein complexes and AD-coated gold particles Retroviral vectors lack immunogenic proteins and there is no pre-existing host immunity, but are limited to infecting dividing cells Retroviruses have been used in clinical trials (Rosenberg et al., N. Engl. J. Med., 1990, 323: 570-578) Retroviruses are composed of an RNA genome that is packaged in a Envelope derived from the host cell membrane and viral proteins For the expression of the gene, you must first reverse-reverse your genome of positive-strand RNA to a double-stranded DNA, which is then integrated into the host cell's DNA when using reverse transcriptase and integrase protein contained in the retrovirus particle.The integrated proviruses are capable of using the cellular machinery of the host for the expression of the gene. Uraine is widely used (Miller et al., Methods Enzymol., 1993,217: 581-599). Retroviral vectors are constructed by removing the gag, pol and env genes to make room for the relevant load and eliminate the replication functions of the virus. The virally encoded mRNAs are eliminated and this eliminates any potential immune response to the transduced cells. Genes that code resistance - antibiotic are often included as means of selection. Promoter and enhancer functions may also be included for example to provide tissue-specific information after in vivo administration. Promoter and reinforcer functions contained in the long terminal repeat can also be used. These viruses can only be produced in viral packaging cell lines. The packaging cell line can be constructed by stably inserting the canceled viral genes (gag, pol, and env) into the cell, such that they reside on different chromosomes to prevent recombination. The packaging cell line is used to construct a producer cell line that will generate replication-defective retroviruses that contain the relevant loading gene by inserting the recombinant proviral DNA. Plasmid DNA containing the long terminal repeat sequences flanking a small portion of the gag gene containing the encapsidation sequence and the genes of interest is transfected into the packaging cell line by using standard techniques for DNA transfer and DNA absorption (electroporation, calcium precipitation, etc). Variants of this procedure have been used to decrease the likelihood of production of replication-competent viruses (Jolly, D., Cancer Gene Therapy, 1994, 1.51-64). The host cell range of the virus is determined by the envelope gene (env) and env gene replacement, different cellular specificities can be used. The incorporation of appropriate ligands to the envelope protein can also be used for targeting. The administration can be obtained by any appropriate technique, for example, ex vivo transduction of the patient's cells by direct injection of the virus into the tissue and by administration of the retroviral producer cells. The ex vivo procedure has the disadvantage that it requires isolation and maintenance in tissue culture of the patient's cells, but has the advantage that the extent of gene transfer can be easily quantified and targeted to a specific population. of cells. In addition, a high proportion of viral particles can be obtained to target cells and thus improve transduction efficiency (Anderson et al., Hum.

Gene Ther., 1990, 1: 331-341; Rosenberg et al., N. Engl. J. Med., 1990, 323: 570-578; Culver et al., Hum. Gene Ther., 1991, 2: 107-109 Nienhuis et al., Cancer, 1991, 67: 2700-2704, Anderson et al., Hum Gene Ther., 1990, 1: 331-341, Grossman et al., Nat. Genet., 1994, 6: 335-341, Lotze et al., Hum. Gene Ther., 1992, 3: 167-177; Lotze, M.T., Cell Transplant., 1993, 2: 33-47; Lotze et al., Hum. Gene Ther., 1994, 5: 41-55 and US patent 5399346 (Anderson). In some cases - direct introduction of the virus in vivo is necessary. Retroviruses have been used to treat brain tumors wherein the ability of a retrovirus to infect only diisola cells (tumor cells) can be particularly advantageous. To increase efficiency Oldfield et al., In Hum. Gene Ther., 1993, 4: 39-69 proposed the administration of a retrovirus-producing cell line directly to tumors of the patient's brain. The murine-producing cell would survive in the brain tumor for a period of days and secrete retroviruses capable of transducing the tumor from the surrounding brain. Viruses carrying the thymidine kinase gene of the herpes virus make the cells susceptible to extermination by glanciclovir, which is metabolized to a cytotoxic compound by thymidine kinase. Patent references regarding retroviruses are EP 334301, WO 91/02805 & WO 92/05266 (Viagene) and; US 4650764 (University of Wisconsin). Human adenoviral infections have been described (See Horwitz, M.S., in Virology, 2nd ed. Raven Press, New York, 1990, pp. 1723-1740). Most adults have prior exposure to adenovirus and have antiadenovirus antibodies. These viruses possess a double-stranded DNA phenomenon and replicate independently of the cell division of the host. Adenoviral vectors have advantageous properties. They are able to effect the transduction of a broad spectrum of human tissues and high levels of gene expression can be obtained in dividing and non-dividing cells. Various routes of administration can be used, in which intravenous, intrabiliary, intraperitoneal, intravesicular, intracranial and intrathecal injection and direct injection of the target organ are included. Thus, pointing based on anatomical boundaries is feasible. The adenoviral genome encodes approximately 15 proteins and the infection involves a fiber protein to adhere to or stick to a cell surface receptor. The basic penton of the capsid is coupled to the integrin receptor domains (a3ß3, or 3ß5) on the cell surface to result in the virus being internalized. Viral DNA enters the nucleus and transcription begins without cell division. Expression and replication is under the control of the E1A and E1B genes (see Horwitz, M.S., In Virology, 2nd ed., 1990, pp. 1723-1740). The ellipnation of genes The virus returns incompetent for replication. The expression of adenoviral proteins leads to an immune response that can limit the effectiveness particularly in repeated administration. However, recent procedures in which other adenoviral genes such as the E2a gene (which controls the expression of fiber projection and a variety of other viral proteins) are also eliminated from the viral phenomenon can abolish or greatly reduce the expression of many of these viral proteins in target cells. Adenoviral serotypes 2 and 5 have been used extensively for the construction of the vector. Bett et al., Proc. Nat. Acad. Sci. USA, 1994, 91: 8802-8806 have used an adenoviral type 5 vector system with deletions of the adenoviral genes El and E3- The human embryonic kidney cell line 293 has been designed to express El proteins and can thus transcomplement the deficient viral genome of El. The virus can be isolated from the cell medium 293 and purified by limited dilution plate studies (Graham, F.L. and Prevek, L. in Methods in Molecular Biology: Gene Transfer and Expression Protocols, Humana Press 1991, pp. 109-128). The recombinant viruses can be cultured in 293 cell line cultures and isolated by lysis of the infected cells and purified by cesium chloride density centrifugation. A problem of 293 cells for the production of recombinant adenoviruses is that due to the additional flanking regions of the El genes replication competent adenoviruses (RCA) can arise during the production of the viral particle. Although this material consists only of free-form adenovirus and non-replicating competent recombinant virus, it can have significant effects on the eventual yield of the desired adenoviral material and lead to increased manufacturing costs, quality control issues for production runs and acceptance of lots for clinical use.

Alternative cell lines have been developed such as PER C6 that have gene integration The more defined than the 293 cells (that is, they do not contain flanking viral sequence) that do not allow recombination events that produce RCA and thus have the potential to overcome previous viral production issues. Adenoviral vectors have the disadvantage of a relatively short duration of transgene expression due to the separation of the immune system and loss of dilution during target cell division but improvements in vector design are anticipated. Patent references as adenovirus are: WO 96/03517 (Boehr inger); WO 96/13596 (Rhone Poulenc Rorer); WO 95/29993 (University of Michigan) and WO 96/34969 (Canji). Recent advances in adenoviral vectors for cancer gene therapy include the development of strategies to reduce in-onegenecity, chimeric adenoviral / retroviral vectors and conventional (or restricted) replicative recombinant systems are reviewed in Bilbao et al. , Exp. Opin. Ther.- Patents, 1997, 7 (12): 1427-1446. Adeno-associated viruses (AAV) (Kotin, RM, Hum Gene Ther., 1994, 5: 793-801) consist of single-stranded DNA, non-autonomous parvoviruses capable of integrating into the genome of non-dividing cells of a range of Very spacious guest. The AAV has not been shown to be associated with the human disease and does not produce an immune response. The AAV has two distinct life cycle phases.

The free virus will infect a host cell, integrate and remain dormant. In the presence of adenovirus, the lytic phase of the virus is induced, which is dependent on the expression of the first adenoviral genes and leads to an active replication of the virus. The AAV genome is composed of two open reading frames (called rep and cap) flanked by inverted terminal repeat sequences (ITR). The rep region encodes four proteins that moderate AAV replication, viral MM transcription and endonuclease sanctions used in the integration of the host genome. The rep genes are the only AAV sequences required for viral replication. The cap sequence encodes structural proteins, which form the capsid. viral The ITRs contain the viral origins of replication, provide packaging signals and participate in the integration of viral DNA. Replicating replication defective viruses that have been developed for gene therapy lack rep and cap sequences. The defective replication AAV can be produced by co-transfection of the separate elements necessary for replication of the AAV to an allowable 293 cell line. Patent references regarding the AAV include: WO 94 / 1378-8 (University of Pittsburgh) and US 4797368 (US Department of Health). Pox virus gene therapy vectors have been described (Moss, B. and Flexner, C, Annu, Rev. Immunol., 1987, 5: 305-324; Moss, B., In Virology, 1990, pp. 2079- 2111). Vaccinia viruses are ABN virus enveloped, large, which replicate in the cytoplasm of infected cells. Dividers and non-dividing cells from many different tissues are infected and expression of the gene from a non-integrated genome is observed. The recombinant virus can be produced by inserting the transgene into a plasmid derived from vaccinia and transfecting this MM in cells infected with vaccinia, where homologous recombination leads to the production of the virus. A significant disadvantage is that it produces a host immune response to 150 to 200 virally-coded proteins which makes repeated administration problematic. The herpes simplex virus is a large double-stranded DNA virus that replicates in the nucleus of infected cells appropriate for the administration of the gene (see Kennedy, P.G.E. and Steiner, I., Q.J. Med., 1993, 86: 697-702).

Advantages include a wide range of host cells, infection of dividing and non-dividing cells, and large sequences of "foreign DNAs can be infected to the viral genome by homologous recombination.The disadvantages are the difficulty of rendering the viral preparations free of competent replication virus. and a potent immune response The cancellation of the viral thymidine kinase gene returns to the defective replication of the virus in cells of low levels of thymidine kinase Cells undergoing active cell division (eg, tumor cells) possess sufficient thymidine activity kinase to allow replication.

Cantab Pharmaceuticals have published a patent application regarding herpes virus (WO 92/05263). A variety of other viruses including HIV, mouse miniature virus, hepatitis B virus and influenza virus have been considered as possible vectors for gene transfer (see Jolly, D., Cancer Gene Therapy, 1994, 1: 51-64). The use of attenuated Salmonella Tyhphimurium bacteria that specifically target and replicate hypoxic environments (such as are found in necrotic centers of tumors) as gene delivery vehicles for the therapy based on enzyme promedicamento (Tumour Amplified Prodrug Enzyme Therapy known as TAPET ™) - have also been proposed and are being developed by Vion Pharmaceuticals. This system offers an additional gene administration alternative to the viral and non-viral procedures discussed later herein. ADS administration strategies? Non-viral are also applicable. These systems of administration of DNA includes uncomplexed plasmid DNA, DNA-liposome complexes, ADM-protein complexes and DNA particles coated with gold. The purified nucleic acid can be injected directly into tissues and results in transient or temporal gene expression for example in muscle tissue, particularly effective in muscle regeneration (Wolff et al., Science, 1990,247: 1465-1468) . Davis et al., In Hum. Gene Ther., 1993,4: 733-740 has published regarding the direct injection of DNA in relation to the mature muscle. Skeletal and cardiac muscle is generally preferred. The patent references are: WO 90/11092, US 5589466 (Vical) and WO 97/05185 (hydrogels impregnated with DNA biodegradable for injection, Focal). Plasma DNA on gold particles can be "fired" into cells (e.g., epidermis or melanoma) by using a gene gun. The DNA is coprecipitated on the gold particle and then fired by using an electric spark or pressurized gas as a propellant (Fynan et al., Proc. Nati, Acad Sci. U.S.A., 1993, 90 ^: 11478-11482). Electroporation has also been used to allow the transfer of DNA to solid tumors by using electroporation samples that employ arrays of multiple needles and pulsed, rotating electric fields (Nishi et al., In Cancer Res., 1996, 56: 1050-1055 ). The transfer of high efficiency gene to subcutaneous tumors has also been claimed with a significant improvement in cell transfection and better distribution characteristics with respect to intra-tumor injection procedures. Liposomes work by surrounding hydrophilic molecules with hydrophilic molecules to facilitate entry into the cell. Liposomes are unilamellar or multilamellar spheres composed of lipids. Lipid composition and manufacturing processes affect the structure of the - - < liposome Other molecules can be incorporated into the lipid membranes. The liposomes can be anionic or cationic. Nicolau et al., Proc. Nati Acad. Sci. U.S. ., 1983, 8_0: 1068-1072 have published regarding insulin expression of anionic liposomes injected into rats. Anionic liposomes target mainly reticuloendothelial cells of the liver unless otherwise noted. The molecules can be incorporated to the surface of the liposomes to alter their behavior, for example, selective administration of the cell (Wu, G.Y. and Wu, C.H., J. Biol. Chem., 1987, 262: 4429-4432). Felgner et al., Proc. Nat. Acad. Sci. Ü.S.A., 1987, 84 ^: 7413-7417 have published regarding cationic liposomes, demonstrated their nucleic acid binding through electrostatic interactions and show entry into the cell. Intravenous injection of cationic liposomes leads to the expression of the transgene in most organs upon injection into the blood supply afferent to the organ. Cationic liposomes can be administered by aerosol to the target lung epithelium (Brigha et al., Am. J. Med. Sci., 1989, 298: 278-281). The patent references for liposomes are: WO 90/11092, WO 91/17424, WO 91/16024, W0-93 / 14788 (Vical) and WO 09/01543 (Intracel). In vivo studies with transgene administration of cationic liposome have been published by: (Nabel et al., Rev. Hum. Gene Ther., 1994, 5: 79-92.; Hyde et al., Nature, 1993, 362: 250-255 and Conary et al., J. Clin. Invest., 1994, 93: 1834-1840). We have studied p-particles as systems for the delivery of DNA to phagocytic cells, such procedures have been carried out by Pangea Pharmaceutics in their ENDOSHERE ™ DNA microencapsulation delivery system which has been used for the most efficient transduction of phagocytic cells such as marcrofagos that effect the ingestion of the microspheres. The microspheres encapsulate the plasmid DNA encoding potentially immunogenic peptides which when expressed lead to the display of peptide via MHC molecules on the cell surface which can stimulate the immune response against such peptides and protein sequences containing the same epitopes. This procedure is currently directed towards a potential function in antitumor and pathogen vaccine development but may have other applications of possible gene therapies. In the same way that synthetic polymers have been used to package natural viral coat proteins from DNA that have the ability to homogenous self-assembly to virus-like particles (VLPs) they have been used to package ADM. The major structural coat protein VP1 of the human polyoma virus can be expressed as a recombinant protein and is capable of packaging the plasmid DNA during self-assembly of a VLP. The resulting particles can subsequently be used to effect the transduction of several cell lines, while preliminary studies show little immunogenic response to such VPI-based VPI. Such systems can offer an attractive intermediary between synthetic non-viral polymeric vectors and alternative viral delivery systems since they can offer combined advantages, for example simplicity or production and high level transduction efficiency. In order to improve the specificity of gene administration and expression of the therapeutic gene, the inclusion of targeting elements for delivery vehicles and the use of regulatory expression elements individually and in combination in many of the previously described delivery systems have been investigated. . Improvements have also been made to DNA vectors and are likely to be applicable to all non-viral delivery systems. These include the use of over-coiled minicircles reported by RPR Gencell (which do not have bacterial origins of replication or antibiotic resistance genes and thus are potentially safer since they exhibit a high level of biological containment), episomal expression vectors, as they are developed by Copernicus Gene Systems Inc. (replicating episomal expression systems in which the plasmid is amplified within the nucleus but outside the chromosome and thus avoids genome integration events) and T7 systems as developed by Progenitor (an expression vector, strictly cytoplasmic in which the vector itself expresses the RNA polymerase of phage T7 and the therapeutic gene is driven from a second T7 promoter, by using the polymerase generated by the first promoter). Other more general improvements to the DNA vector technology include the use of cis-acting elements to effect high levels of expression (Vical), derived from alfoid repeat DNA to provide a replication of a cycle once per cell and sequences of nuclear targeting (of the EBNA-1 gene (Calos in Stanford, with Megabios), SV40 premature promoter / enhancer or peptide sequences linked to DNA). Targeting systems based on recognition of the cell receptor by ligand linked to DNA have been described by Michael, S.I. and Curiel, D.T., Gene Therapy, 1994, 1: 223-232. By using the ligand recognized by such a receptor the DNA is selectively linked and internalized to the target cell (Wu, G.Y. and Wu, C.H., J. Biol.

Chem., 1987, 262: 4429-4432). A poly-L-lysine (PLL) polycation has been used to couple a variety of protein ligands to DNA by chemical cross-linking methods. He DNA is electrostatically linked to PLL-ligand molecules. Targeting systems have been published by Zenke et al., Proc. Nat. Acad. Sci. U.S.A., 1990, 7: 3655-3659 using the transferrin receptor; Wu G.Y. and Wu C.H., J.

Biol. Chem., 1987, 262: 4429-4432 using the receptor asialoorosomucoid and Batra et al., Gene Therapy, 1994,1: 255-260, using carbohydrates from the cell surface.

Agents such as chloroquine or co-localized adenoviruses can be used to reduce the degradation of DNA in the asbestos (see Fisher, K.J. and Wilson, J.M., Biochem. J., 1994, 299, 49-58). Cristiano et al., Proc. Nati Acad Sci U.S.A., 1993, 90: 11548-11552 has constructed adenovirus-DNA-ligand complexes. The patent references for receptor-mediated endocytosis are: WO 52/05250 (asialoglycoproteins, University of Connecticut) and US 5354844 (transferrin receptor, Boehringer). The DNA and the ligand can be coated on the surface of the adenovirus to create a coated adenovirus (Fisher, K.J. and Wilson, J., Biochem J., 1994, 299, 49-58).

However, the presence of two receptor trajectories for DNA entry (ligand receptor and adenovirus receptor) reduces the specificity of this delivery system, but the trajectory of the adenovirus receptor can be eliminated by using an antibody against the fiber protein. of adenovirus as the means of binding to DNA (Michael, S.I. and Curiel, D.T., Gene Therapy, 1994, 1: 223-232).

The use of purified endo somalistic proteins in place of intact adenovirus particles is another option (Seth, P., J. Virol., 1994, 68: 1024-1206). The expression of a gene construct or construct of the invention at its target site is preferably under the control of a transcriptional regulatory sequence (TRS). A TRS is a promoter optionally combined with a reinforcer and / or a control element such as a genetic switch (or switch) described hereinafter. An example of a TRS is a "genetic switch" that can be used to control the expression of a gene construct or construct of the invention once it has been delivered to a target cell. The control of gene expression in higher eukaryotic cells by prokaryotic regulatory elements that are preferred for the present invention) has been reviewed by Gossen et al in TIBS, December 18, 1993, 471-475. Suitable systems include the E. coli lac operon and the especially preferred tetracycline E. coli resistance operon. References regarding the tetracycline system include Gossen et al (1 95) Science 268.1766; Damke et al (1995) Methods in Enzymology 257, Academic Press; Yin et al (1996) Anal. Biochem. 235,195 and patents US 5464758, US 5589362, WO 96/01313 and WO 94/29442 (Bujard). A commutator based on ecdysone (International patent application No. PCT / GB96 / 01195, Publication No.

WO 96/37609, Zeneca) is another option. Other options are listed below in the present. Connaught Laboratories (WO93 / 20218) describes a synthetic inducible eukaryotic promoter comprising at least two different classes of inducible elements. Rhone-Poulenc Rorer (WO 96/30512.) Describes an application concerning tetracycline for a conditional gene expression system Ariad (WO 94/18317.) Describes a protein dimerization based system for which shown in vivo activity Bert O'Malley of the Baylor College of Medicine (WO 93/23431, US 5364791, WO 97/10337) describes a molecular switch based on the use of a modified spheroid receptor. NF-KB inducible gene expression (WO 88/05083) Batelle Memorial has described a strain inducible promoter (European patent EP 263908) Examples of TRS that are independent of the cell type include the following: cytomegalovirus promoter / enhancer, promoter of SV40 / retroviral long terminal repeat enhancer. Examples of TRS that are dependent on the cell type (to give an additional degree of targeting) include the following promoters: carcinoembryonic antigen (CEA) to target colorectal, lung and breast cells; alpha-photoprotein (AFP) to target the transformed liver cells; tyrosine hydroxylase, choline acetyl transferase or neuron-specific enolase to target the neuroblast; insulin to target the pancreas and glial fibro acid protein to target glioblastomas. Some oncogenes can also be used, which are selectively expressed in some tumors for example HER-2 / neu or c-erbB2 in the chest and N-myc in neuroblastoma.

Thus, a preferred gene construct or construct for use as a medicament is a construct comprising a transcriptional regulatory sequence comprising a promoter and a control element that is a genetic switch to control the expression of the gene construct. A preferred genetic switching control element is regulated by the presence of tetracycline or ecdysone. A preferred promoter is dependent on the cell type and is selected from the following promoters: carcinoembryonic antigen (CEA); alpha-photoprotein (AFP); tyrosine hydroxylase; choline acetyl transferase; neuron-specific endolase; insulin; glial acid protein fxbro; HER-2 / neu; c-erbB2; and N-myc. Preferably, the gene construct for use as a medicament described herein is packaged within an adenovirus for administration to the mammalian host.A general review of therapy directed to the gene is provided by Douglas et al., Tumor Targeting, 1995, 1: 67-84 The antibody encoded by the gene construct of the invention can be any form of antibody construct such as for example F (ab ') 2; F (ab'). Fab, Fv, Fv chain simple and V-min Any suitable antibody construct is contemplated for example a recently described antibody fragment is "LF (ab.) 2", as described by Zapata (1995) in Protein Engineering, 8, 1057-1062 Disulfide-linked Fv are also contemplated For constructs based on CPG2 enzyme, constructs of Fab fragments digested by means of enzyme dimerization are preferred.The non-human antibodies can be made human for use in humans to reduce the r immune responses of the host. A human-derived antibody, related fragment or antibody binding structure is a polypeptide composed primarily of a structural framework of immunoglobulin sequences derived from humans that support amino acid sequences derived from non-human in and around the antigen binding site (regions of complementary determination or CDR). The appropriate methodology has been described for example in detail WO 91/09967, EP 0328404 and Queen et al. Proc Nati Acad Sci 86,10029, Mountani and Adair (1989) Biotechnology and Genetic Engineering Reviews 10.1 (1992) although alternative humanization methods are also contemplated such as antibody coating of surface residues (EP 519596, Merck / HIN, Padlan et al). According to another aspect of the present invention there is provided a system of two paired components designed for use in a mammalian host in which the components comprise: (i) a first component comprising a gene construct that encodes an antibody that targets the cell and a heterologous enzyme activating the prodrug, wherein the gene construct is capable of expressing the antibody and enzyme as a conjugate within a target cell in the mammalian host- and wherein the conjugate can exit the cell after this for selective localization on a cell surface antigen recognized by the antibody and; (ii) a second component comprising a prodrug that can be converted to an active drug by the enzyme. Antibody-directed enzyme prodrug therapy (ADEPT) is a known cancer therapeutic procedure. ADEPT uses a select s © antibody from the tumor conjugated to an enzyme. The conjugate is administered to the patient (usually intravenously), it is allowed to be located in the tumor site (s) and cleared of blood and other normal tissues. A prodrug is then administered to the patient who is converted by the enzyme (located at the tumor site) to a "cytotoxic drug that kills tumor cells." The present invention can be applied to any ADEPT system. include those based on any of the following enzymes: carboxypeptidase G2; carboxypeptidase A; aminopeptidase; alkaline phosphatase; glucosidases; β-glucuronidase; penicillin amidase; β-lactamase; cytosine deaminase; Nitroreductase or mutant host enzymes in which carboxypeptidase A, carboxypeptidase B and ribonuclease are included. Appropriate references regarding ADEPT systems include Melton RG (1996) in J. National Cancer Institute 88, 1; Niculescu-Duvaz I (1995) in Current Medicinal Chemistry 2,687; Knox RJ (1995) in Clin. Immunother.3,136; WO 88/07378 (CRCT); Blakey et al, Cancer Res, 56.3287-92.1996; US 5587161 (CRCT and Zeneca); WO 97/07769 (Zeneca); and WO 95/13095 (Wellcome). The heterologous enzyme may be in the form of a catalytic antibody; see for example EP 745673. { Zeneca). A review of articles regarding ADEPT systems include Hay & Denny (1996), Drugs of the Future, 21 (9.)., 917-931 and Blakey (1997), Exp. Opin. Ther.Patents, 7 (9), 965- 977. A system of two paired components preferred is one in which: the first component comprises a gene encoding the heterologous enzyme CPG2 and the prodrug of the second component is selected from N- (4- [N, N-bis (2-iodethyl) amino] -phenoxycarbonyl) - L-glutamic, N- (4- [N, K-bis (2-chloroethyl) amino] -phenoxycarbonyl] -L-glutamic-gamma- (3, 5-dicarboxy) anuide or N- (4) acid - J, N-bis (2-chloramethyl) am_b? O] phenoxycarbonyl) -L-glutamic acid or a pharmaceutically acceptable salt thereof Preferred prodrugs for use with CPG2 are described in the following North American patents of Zeneca Limited and Cancer Research Campaign Technology Limited: US 5714148, US 5405990, 5587161 & 5660829.

In another aspect of the invention there is provided a method for administering a cytotoxic drug to a site comprising administering to a host a first component comprising a gene construct as described herein; followed by administration to the host of a second component comprising a prodrug that can be converted to a cytotoxic drug by the heterologous enzyme encoded by the first component. A preferred method for administering a cytotoxic drug to a site is one in which the first component comprises a gene encoding the heterologous enzyme CPG2 and the prodrug of the second component is selected from N- (4- [N, N- bis (2-iodoethyl) amino] fensxycarbonyl) -L-glutamic, N- (4- [K, N-bis (2-chloroethyl) amino] -phenoxycarbonyl) -L-glutamic-gamma- (3,5- dicarboxy) or N- (4- [N, N-bis (2-chloroethyl) -animo] -phenoxycarbonyl) -L-glutamic acid or a pharmaceutically acceptable salt thereof. The abbreviations used herein include: Adenovirus-associated AAV virus ADEPT antibody-directed enzyme-targeting therapy AFP alpha-photoprotein AMIRACS antimetabolite with inactivation of rescue agents at cancer sites APS Ammonium persulphate b.p. base pair BPB blue bromophenol CDR regions of complementary determination CEA embryonic antigen of CL carcinoma light chain constant domain of antibody CPB carboxypeptidase B CPG2 carboxypeptidase G2 CPG2 R6 carboxypeptidase G2 mutated to prevent glycosylation on expression in eukaryotic cells, see example Id DAB 3.3 'diaminobenzidine tetrahydrochloride substrate DEPC diethylpyrocarbonate DMEM Dulbecco's Modified Eagle Medium ECACC European Crops Collection of Animal Cells EIA immunological enzyme test ELISA assay with enzyme-linked immunosorbent FAS folinic acid supplemented FCS fetal bovine serum Fd heavy chain of Fab, Fab 'or F (ab') 2 optionally containing a GDEPT pivot enzyme-directed prodrug therapy HAMA gene Human anti-mouse antibody HCPB human carboxypeptidase B, preferably pancreatic Hinge (from an IgG) a short proline-rich peptide containing the cysteines that bind the two heavy chains HRPO or HRP horseradish peroxidase IRES entry site to the internal ribosome MTX methotrexate NCA non-specific cross-linking antigen NCIMB National Collections of Industrial and Marine OPD bacteria ortho-phenylenediamine PBS phosphate salt pH-regulated PCR polymerase chain reaction PGP N-acid (4 [N, N-bis (2-chloroethyl) amino] -phenoxycarbonyl) -L-glutamic prepro CPB proCPB with a directing sequence N-tepninal pro CPB CPB with its prodomain N-terminal scFv a single chain of Fv SDS-PAGE sodium dodecyl sulfate electrophoresis - polyacrylamide gel SSC sodium citrate salt TBS solution. pH-regulated saline-Tris Temed N, N, N ', N' -tetramethylethylenediamine TFA trifluoroacetic acid TRS transcriptional regulatory sequence VDEPT VHEP virus-directed enzyme-mediated therapy variable region of the VK antibody heavy chain variable region of the light chain of the antibody This specification contemplates conservative amino acid sequences of specific amino acid sequences that retain the relevant biological properties of the component of the invention but differ in sequence by one or more conservative amino acid substitutions, deletions or additions. However, the amino acid sequences listed • specifically are preferred. Typical conservative amino acid substitutions are tabulated below.

Original Substitutions Preferred example substitutions Ala (A) Val, Leu; lie Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn; Gln; Lys; Arg Arg He (I) Leu; Val; Met; Ala, Leu Phe; norleucine Leu (L) Norleucine; He; Val; He Met; To; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Fen; He Leu Fen (F) Leu; Val; He; Ala Leu Pro (p) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) He; Leu; Met; Phe; Leu Ala, Norleucina The amino acid nomenclature is summarized below: Alanine Ala A Arginine Arg R Asparagine. Asn N Aspartic Acid Asp D Cisterine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine He I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr and Valina Val V Any amino acid Xaa X In this specification, nucleic acid variations (deletions, substitutions and additions) of specific nucleic acid sequences that retain the ability to hybridize under severe conditions to the specific sequence in question are contemplated. Severe conditions are defined as 6xSSC, 0.1% SDS at 60 ° C for 5 minutes. However, the nucleic acid sequences listed specifically are preferred. It is contemplated that chemical analogs of natural nucleic acid structures such as "peptide nucleic acid" (PNA) may be an acceptable equivalent, particularly for purposes that do not require protein translation (Wittung (1994) Nature 368,561). The invention will now be illustrated by reference to the following non-limiting examples. The temperatures are in degrees Ceisius. Figure 1 shows a representation of the fusion gene construct comprising the heavy chain Fd fragment of the A5B7 antibody linked at its C terminus via a flexible (G4S) 3 peptide linker to the N terminus of the CPG2 polypeptide. SS represents the signal sequence. L represents a linker sequence. CPG2 / R6 represents CPG2 with its glycosylation sites nullified by means of mutation as explained in the text. Figure 2a shows a representation of the fusion protein (Fab-CPG2) 2 with the dimerization that is carried out by means of non-covalent link between two CPG2 molecules. Figure 2b shows a representation of an antibody fragment of F (ab ') 2. Figure 3 shows a cell-based ELISA analysis of the secreted fusion protein material. Only the positive line of CEA has increased the levels of binding with increased amounts of aggregated fusion protein, while the CEA negative cell line has only constant background levels of end-to-end. The vertical axis represents the optical density readings measured at 490 nm and the horizontal axis the amount of aggregated fusion protein mediated in nanograms ng of protein. The graph shows the data obtained from an experiment where a diversity of cell lines and a negative control (without cell) were incubated with increased amounts with fusion protein using the cell analysis described in example 6. The results show that only the LoVo cell line (CEA positive) shows a corresponding increased OD490 reading with increased amounts of aggregated fusion protein. All other cell lines (negative CEA) and control or reference (no cells) showed only a background OD 0 reading that does not increase with the addition of fusion protein. These results provide evidence that the amount of fusion protein that binds or binds specifically to a positive CEA cell line in a dose-dependent manner and does not bind or bind to negative CEA lines. Figure 4 shows the retention of the secreted fusion protein to recombinant LoVo tumor cells. The vertical axis represents the optical density readings measured at 490 nm and the horizontal axis the amount of added anti-CEA antibody (IIE6) measured in ng / ml protein. The experiment was carried out as described in Example 7 using three different cell lines, recombinant LoVo and Colo320DM lines (which themselves secrete fusion protein) and a control or reference parental LoVo line that does not secrete protein of fusion. Eto. First the cell lines were fixed and washed to separate the existing supernatant and any non-stick material, after which increased concentrations of the anti-CEA antibody (IIE6) were added to the fixed cells. The analysis was developed as described in the text to determine the level of retention of any secreted material and whether the additional added antibody would increase the signal. The results showed that without the added anti-CEA antibody, the control or reference parental LoVo line exhibits only a reading of OD490 nm background (as expected) while the line of Recombinant LoVo provides a very strong OD490 nm reading which indicates that the fusion protein material was retained on the positive LoVo CEA cells. The negative recombinant Colo320DM line of CEA provides a much weaker reading than the LoVo cells, but the signal was higher than the background (possibly due to the non-binding of the antibody secreted prematurely in the method of analysis). The increase in the concentrations of anti-CEA antibody (IIE6) added to the fixed cells shows a dose-related response in the case of parental LoVo cells, which indicates that they are positive for CEA and can bind to the material of CEA binding (such as the fusion protein if present or added). The recombinant Colo320DM and Lo ¥ o cells show little increase in the overall OD490 signal with increased amounts of added antibody with the exception of the LoVo cells that appear to show a slight response at the highest antibody dose. Since the recombinant Colo320DM cell lines are negative to CEA they do not increase in signal due to the anti-CEA antibody, the results for these cells would be expected. In the case of recombinant LoVo cells, the addition signal due to the amounts of antibody added in this analysis can be flooded except at the highest dose due to the relative intensity of the original signal. Figure 5 shows the retention of the secreted fusion protein to the recombinant LoVo tumor cells. The vertical axis represents the average tumor volume (cm3) and the horizontal axis the time in days after the dosage of the prodrug. The experiment was carried out as described in example 12 using a dose of 60 mg / kg of prodrug. The results showed that control GAD (c) tumors (none treated with promedicamento) grow up to 6 times their initial size for 11 days (post-dose day) at which time the tumors were harvested. Tumors treated with GAD prodrug (d) showed a significantly slower growth rate and by day 16 (post-dose day) they had only reached 3 times their initial size. These data indicate a delay in tumor growth of at least 11 days. In the examples below, unless stated otherwise, the following methodology and the following materials have been applied. The DNA is recovered and purified by the use of a GÉNECLEAN ™ II device or set (Stratech Scientific Ltd. or Bio 101 Inc.). The equipment contains: 1.}. 6 M sodium iodide; 2) a concentrated solution of sodium chloride. Tris and EDTA to make a solution of sodium chloride / ethanol / washing water; 3) Glassmilk - a 1.5 ml bottle "containing 1. 25 ml of a suspension of a silica matrix formulated especially in water. This is a purification technique of DNA based on the method of Vogelstein and Gillespie published in Proceedings of the National Academy of Sciences USA (1979) Vol 76, p 615. Briefly, the procedure of the set or equipment is as follows. To 1 volume of silica gel are added 3 • volumes of sodium iodide solution from the equipment. The agarose is melted by heating the mixture at a temperature of 55 ° C for 10 minutes, then Glassmilk (5-10 ml) is added, mixed well and allowed to stand for 10 minutes at room temperature. The glassmilk is centrifuged and washed 3 times with NEW WASH ™ (0.5 ml) of the equipment. The washing buffer is separated from the Glassmilk and the DNA is eluted by incubating the Glassmilk with water (5-10 ml) at a temperature of 55 ° C for 5-10 minutes. The aqueous supernatant "eg contains the eluted DNA is recovered by centrifugation. The elution stage can be repeated and the supernatants are collected. Competent DH5a E. coli cells were obtained from Life Technologies Ltd (DH5a competent cells of MAX ™ efficiency). Double-stranded plasmid DNA mini-preparations are made by using the RPM ™ DNA preparation kit from Bio 101 Inc. (catalog number 2070-400) or a similar product - the kit contains alkaline lysis solution to release the Plasmid DNA from bacterial cells and glassmilk in a centrifugation filter to adsorb the DNA released, which is then eluted with sterile water or pH buffer tris-HCl, 10 mM, 1 mM EDTA, pH 7.5. The standard PCR reaction contains 100 ng of the plasmid DNA (except where stated), 5 μl of dNTPs (2.5 M), 5 μl of pH oxidizing buffer lOx Enzyme (500 mM KCl, 100 mM Tris, pfl 8.3), 15 M MgCl2 and 0.1% gelatin), 1 μl of a concentrated solution of 25 pM / μl of each primer, 0.5 μl of thermostable DNA polymerase and water to obtain a volume of 50 μl »The standard PCR conditions were: 15 cycles of PCR at 94 ° for 90 seconds; 55 ° for 60; 72 ° for 120 seconds, to finish the last cycle with an additional 72 ° for a 10 minute incubation. AMPLITAQ ™, available from Perin-Simer Cetus, is used as the tesmo-stable source of ADM polystyrene. The general molecular biology procedures described in "Molecular Cloning - A Laboratory Manual" second edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989) can be followed. The serum-free medium consists of OPTIMEM ™ I Reduced Serum Medium, GibcoBRL catalog number 31985. This consists of a modification of Eagle's Minimum Essential Medium of pH regulated with Hepes and sodium bicarbonate supplemented with hypoxanthine, thymidine, pyruvate. sodium, L-glutamine, elements in traces and growth factors. The LIPOFECT'IN ™ reagent (GibcoBKE, catalog number 18292-011) is a 1: 1 (w / w) formulation of the liposome of the cationic lipid N- [l-. { 2,3-dioleyloxy) propyl] -n, n, n-tris? Ethylammonium (DOTM &) and dioleyl phosphatidylethanolamine (DOPE) in membrane filtered water. It binds or adheres spontaneously with DNA to form a lipid-DNA complex - see Felgner et al, in Proc. Nati, Acad. Sci. USA (1987) 84, 7431. G418 (sulfate) consists of GENETICIN ™, GibcoBRL catalog number 11811, an aminoglycoside antibiotic related to gentamicin used as a selection agent in molecular genetic experiments. For CEA ELISA analysis each csw ± d & of a 96-well immunoplate (NUNC MAXJSORB ™.) was coated with 50 ng of CEA in pH buffer solution of 50 mM carbonate / bicarbonate coating, pH 9.6 (pH regulated capsules - Sigma C3041) and incubated at 4 ° C overnight The plate was washed three times with PBS-TWEEN ™ (PBS + TWEEN ™ 20 at 0.05%) and then blocked with 150 μl per 1% BSA cavity in PBS-TWEEN ™ for 1 hour at room temperature. The plate was washed three times with PBS-TWEEN ™, 100 μl of the test sample were added per well and incubated at room temperature for 2 hours. The plate was washed three times with PBS-T1EEN ™, 100 μl per cavity of a 1/500 dilution of goat anti-human Kappa antibody, labeled with HRPO (Sigma A 7164) was added in 1% BSA in PBS-TWEEN ™ and incubated at room temperature on an oscillating platform for at least 1 hour. The plate was washed three times with PBS-TWEEN ™ and then once again with PBS.

To detect adhesion, 100 μl is added per reagent solution cavity (one phosphate-citrate pH regulator solution capsule - Sigma P4922 - dissolved in 100 ml of H20 to which is added one 30 mg tablet of dihydrochloride or phenylenediamine - Sigma P8412) and incubated for up to 15 minutes. The reaction was stopped by adding 75 μl of 2M H2SO4 and the absorbance was measured at 490 nm. The CEA ELISA test using an anti-CPG2 reporter antibody was essentially as above but instead of the goat anti-human kappa antibody labeled with HRPO is added a 1/1000 dilution of a polyclonal rabbit anti-CPG2 serum, in 1% BSA in PBS-TWEEN ™ and incubated at room temperature on an oscillating platform during 2 hours . The plate was washed three times with PBS-TWEEN ™. Then a 1/2000 dilution of a labeled antibody is added Goat HRPO anti-rabbit (Sigma A-6154) and is incubated at room temperature on an oscillating platform during 1 hour, the plate was washed three times with PBS-TWEEN ™ and once with PBS. To detect the adhesion or link are added 100 μl per reagent solution cavity (one phosphate-citrate-phosphate pH-revealing solution capsule of P4922 dissolved in 100 ml of H20 to which is added one 30 mg tablet of o-phenylenediamine dihydrochloride - Sigma P 8421 and incubated for up to 15 minutes. The reaction was stopped by adding 75 μl of 2M H2SO4 and the absorbance was read at 490 nm.

Western blot analysis of the transfection supernatants was carried out as follows. Minigeles at 10% for the analysis of fusion protein transections were prepared by using an appropriate minigel system (HOEFER MIGHTY S ALL ™). The 10% operating gel consists of: 20 ml of acrylamide, 6 ml of 10 x operating gel regulator solution; 34 ml of H20; 300 ml of 20% SDS; 600 μl of APS; 30 μl of Temed. Regulatory solution of the order of operation gel lOx consists of Tris 3.75 M, pE 8.6. The 6% stacking gel consists of: 9 ml of acrylamide; 4.5 ml of 10 x pH buffer gel stacked; 31.5 ml of H20; 225 μl of 20% SDS; 450 μl of 10% APS; 24 μl of Temed). The buffer solution of pH stacking gel lOx consists of Tris 1.25 M, pS 6.8. The 5x electrophoresis regulatory solution for SDS / PAGE consists of Tris 249 mM, glycine 799 mM, SDS at 0.6% w / v (pH not adjusted) Sample preparation: The pH regulation solution Laemmli 2X consists of Tris 0.125 M; 4% SDS, 30% glycerol, 4 M urea, 0.002% BPB optionally containing 5% β-mercaptoethanol, Supernatants: 25 μl sample + 25 μl buffer solution Laemmli 2X, 40 μl loaded. Standards F (ab ') 2 and CPG2: 2 pl of 10 ng / ml standard, 8 μl of H20, 10 μl of buffer solution Laemmli 2x (-mercaptoethanol), 20 μl loaded, molecular weight markers (Amersham RAINBOW ™: 8 μl of sample, 8 μl of buffer solution Laemmli 2x (+ mercaptoethanol): charged 16 μl Operating conditions: 30 milliamps until the front part is stained on the bottom of the gel, (approximately 1 hour) Staining or staining: a semi-dry expander (LKB) is used on nitrocellulose membrane Miliamperes = 0.7 x cm, for 45 minutes. Blocking: 5% dry skim milk in PBS-TWEEN ™ for 40 minutes. Detection of F (ab ') 2: goat antihuman kappa light chain HRPO labeled antibody, 1/2500 in 0.5% dried skim milk in PBS-TWEEN ™ and incubated overnight. Detection of CPG2: mouse monoclonal anti-CPG2 (1/2000 in 0.5% dried skim milk in PBS-TWEEN ™ incubated overnight; antibody marked with goat anti-mouse Kappa light chain HRPO - Sigma 674301 - (1/10000 in 0.5% dry skimmed milk in PBS -TWEEN ™) incubated for at least 2 hours.Development of stain: Chemiluminescent detection of HRPO based on a luminol substrate in the presence of enhancer (Pierce SUPERSIGNAL ™ substrate.). The substrate working solution was used. prepared as follows: Recommended volume: 0.125 ml / c 2 spot surface Equal volumes of luminol / reinforcer solution and stable peroxide solution, staining incubated with working solution for 5-10 minutes, separating the solution and the stain is placed on a membrane protector and exposed to auto-radiographic film (usually between 30 seconds and 5 minutes) Deposits of microorganisms: Plasmid pNG3-Vkss-HuCk was deposited in the National Collection s of Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen AB2 IRY, Scotland, United Kingdom on April 11, 1996 under the deposit reference number NCIMB 40798 in accordance with the Budapest treaty. Plasmid pNG4-VHss-HulgG2CH1 'was deposited at the National Collections of Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland; United Kingdom on April 11, 1996 under the reference number of deposit NCIMB 40797 in accordance with the Budapest treaty. Plasmid pNG3-Vkss-HuCk-NEO was deposited in the National Collections of Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland, United United on April 11, 1996 under the deposit reference number NCIMB 40799 in accordance with the Budapest treaty. Plasmid pICI266 was deposited under accession number NCIMB 40589 on October 11, 1993 under the Budapest treaty at the National Collections of Industrial and Marine Bacteria Limited (NICMB), 23 St. Machar Drive, Aberdeen, AB2 1RY, Scotland, U.K. Tipsinization: Trypsin EDTA (Gibco BRL 45300-019) and Hanks balanced salt solution were pre-heated in a 37 ° C water bath. The existing media were removed or separated from the cultures and replaced with a volume of HBSS (consisting of half the previous volume of the medium) and the cell layer was washed by carefully oscillating the plate or flask to separate any medium that contains residual serum. The HBSS was separated and a volume of trypsin solution (consisting of a quarter of original average volume) is added with moderate shaking of the flask to ensure that the cell layer is completely covered and allowed to stand for 5 minutes. The trypsin was inactivated by the addition of the appropriate normal culture medium (2 times the volume of the trypsin solution). Then the cell suspension was either counted as cells or diluted further to continue the culture depending on the procedure that will be carried out. Thermal inactivation of fetal bovine serum (FCS): FCS (Viralex A15-651 accredited lot - non-European.) Was stored at -20 ° C. For use, the serum was completely thawed at 4 ° C overnight. The next day the serum was incubated for 15 minutes in a water bath at 37 ° C and then transferred to a water bath at 56 ° C for 15 minutes.The serum was separated and allowed to cool to room temperature before being distributed in aliquots of 50 ml and stored at -20 ° C.

Normal DMEM medium (Gibco components are used BRL): To 500 ml of DMEM (41966-086) 12.5 ml of Hepes are added (15630-056); 5 ml of NEAA (11140-035); 5 ml pen / strep (10378-016) and 50 ml of FCS thermally inactivated. FAS medium (Gibco BRL components are used unless stated otherwise): 490 ml of DMEM (41966- 086); 12.5 ml Hepes (15630-056); 5 ml of non-essential amino acids (11140-035); 5 ml pen / strep (10378-016); 5 ml of vitamins (11120-037); 5 ml of basal amino acids (51051-019); folinic acid (Sigma F8259) a final medium concentration of 10 μg / ml; 50 ml of FCS thermally inactivated; 5 ml of dNTP mixture and concentrated G418 50 mg / ml solution (to produce the concentration of the appropriate selection). DNTP mixture: 35 mg of G (Sigma G6264), 35 mg of C (Sigma C4654), 35 mg of A (Sigma A4036), 35 mg of ü (Sigma U3003), 125 mg of T (Sigma T1895) were dissolved in 100 ml of water, sterilized in a filter and stored at a temperature of -20 ° C. Selection of G418: for LoVo cells (ATCC CCL 229) the selection is carried out at 1.25 mg / ml, for HCT116 cells (ATCC CCL 247) and for Colo320DM cells (ATCC CCL 220) the selection was carried out at 1.5 mg / ml unless stated otherwise. BLÜESCRIPT ™ were obtained from Stratagene Cloning Systems.

Tet-On gene expression vectors were obtained from Clontech (Palo Alto, California) catalog number Kl621-1. Unless otherwise stated or evident from the context employed, the antibody-CPG2 fusion constructs referred to in the examples utilize mutated CPG2 to prevent glycosylation.

Example 1 Construction of ana fusion protein (2_5B7 Fab-CPG2) 2 The construction of an enzyme fusion (A5b7 Fab-CPG2) 2 was planned with the aim of obtaining a binding molecule of bivalent human carcinoembryonic antigen (CEA) which also exhibits Enzyme activity of CPG2. For this purpose, the initial construct was designed to contain an antibody heavy chain Fd fragment of A5B7 linked at its C terminus via a flexible (G4S) 3 peptide linker to the N terminus of the CPG2 polypeptide (Figure 1). The A5B7 antibody binds to the human Brionic Carcinoe Antigen (CEA) and is particularly appropriate for targeting colorectal carcinoma or other cells that carry CEA antigen (the importance of CEA as an antigen associated with cancer is reviewed by Shively, JE and Beatty, JD in "CRC Critical Reviews in Oncology / He atology", vol 2 p.355-399, 1994). The CPG2 enzyme is dimeric in nature consisting of two associated identical polypeptide subunits. Each subunit of this molecular dimer consists of a larger catalytic domain and a smaller domain that forms the interface of the dimer. In general, the conjugate of antibody (or antibody fragment) -enzyme or fusion proteins must be at least divalent, ie able to bind at least two antigens associated with the tumor (which may be the same or different). In the case of the fusion protein (A5B7 Fab-CPG2) 2, the dimerization of the enzyme component is carried out after the expression, similarly to the original enzyme, thus forming an enzyme molecule containing two antibody fragments. Fab (and is thus "divalent with respect to the antibody binding sites) and two molecules of CPG2 (Figure 2a). a) Cloning of the antibody genes &5B7 Methods for the preparation, purification and characterization of the recombinant murine A5B7 F (ab ') 2 antibody have been published (International patent application, Zeneca Limited, WO 96 / 200-11 , see reference example 5 therein). In Example 5, reference section f thereof, the A5B7 antibody genes were cloned into GS-SYSTEM ™ vectors (Celltech), see International patent applications WO 87/04462, WO 89/01036, WO 86 / 05807 and WO 89/10404, with the A5B7 cloned to pEE6 and the light chain to pEE12. These vectors were the source of the A5B7 antibody genes for the construction of the A5B7 Fab-CPG2 fusion protein. b) qAmeric A5B7 vector constructs The variable regions of the urine antibody A5B7 were amplified by PCR of plasmid vectors pEE6 and pEE12 by using appropriate PCR primers that included the restriction sites necessary for direct cloning under the heavy and light chain variable regions to the vectors pNG4-VHss-HulgG2CHl '(deposit number of NCIMB 40797) and pNG3-Vkss-HuCk-NEO (NCIMB deposit number 40799.), respectively The resulting vectors were designated pNG4 / A5B7VH-IgG2CH1 (Fd 'of chimeric heavy chain A5B7) and pNG3 / A5B7VK-HuC &-NEO (chimeric light chain of A5B7). c) Cloning of the CPG2 gene The CPG2 encoding the gene can be obtained from the Center for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom. CPG2 can also be obtained by recombinant techniques. The nucleic acid coding sequence for CPG2 has been published by Minton, N.P. et al., Gene, (1984) 31, 31-38. The expression of the coding sequence has been reported in E, coli (Chambers, SP et al., Appl. Microbiol, Biotechnol. (1988), 29, 572-578) and in Saccharomyces cerevisiae (Clarke, LE et al., J. Gen Microbiol, (1985) 131, 897-904). In addition, the CPG2 gene can be produced as a synthetic DNA construct by a variety of methods and can be used as a source for further experiments. The total synthesis of the gene has been described by M. Edwards in Am. Biotech. Lab (1987), 5, 38-44, Jayaraman et al. (1991) Proc. Nati Acad. Sci. USA 88, 4084-4088, Foguet and Lubbert (1992) Biotechniques 13, 674-675 and Pierce (1994) Biotechniques 16, 708. In preparation for the cloning of the CPG2 gene the vector pNG3-Vkss was constructed, consisting of a simple derivative of pNG3-Vkss-HuCk-NE0 (deposit number of NCIMB 40799). This vector was constructed by first separating the Neomycin gene (since it contains an EcoRI restriction enzyme site) by digestion with the restriction enzyme Xbal, after which the vector fragment was isolated and then re-ligated to form the plasmid pNG3 / Vkss-HuCk. This intermediate vector was digested with SacII and EcsRI enzymes, which removed the HuCk gene fragment. Then the digested product was loaded on a 1% agarose gel and the excised fragment was separated from the remaining vector, after which the vector DNA was cut out of the gel and purified. Two oligonucleotides CME 00261 and CME 00262 (SEQ ID NO: 1 and 2) were designated and synthesized .. These two oligonucleotides were hybridized by the addition of 200 pmoles of each oligonucleotide to a total of 30 μl of H2Or heating at 95 ° and Allow the solution to cool slowly to 30 ° C.

Then 100 pmoles of the annealed DNA product were ligated directly to the previously prepared vector and the ligation mixture was transformed to E. coli. In the obtained clones the introduction of the DNA "cassette" produces a new polylinker sequence in preparation for the subsequent cloning of the CPG2 gene to produce the vector pNG3-Vkss. The structural gene of CPG2 that encodes amino acid residues Q26-K415 inclusive was amplified by PCR using appropriate DNA oligonucleotide primers and standard PCR reaction conditions. The reaction product was analyzed by using a 1% agarose gene, a band of the expected size (approximately 12,000 bp.) Was excised, purified and eluted in 20 μl of H20.This material was then digested using the restriction enzyme SacII, after which the reaction was loaded on a 1% agarose gel and a band of the expected size (approximately 250 bp) was excised and subsequently purified.This fragment was ligated to the plasmid vector pNG3VKss, which had previously been digested with the SacII, dephosphorylated restriction enzyme, run on a 1% agarose gel, the band of the excised, purified linear vector and the ligation mixture was transformed to E. coli. The resulting clones were analyzed for the presence and orientation of the SaclI fragment of CPG2 by DNA restriction analysis using the enzymes BglII and Fsel. Clones that appeared to have a fragment of the correct size and orientation were confirmed by DNA sequencing. This intermediate plasmid was called pNG3-Vkss-SacIICPG2frag. This plasmid was subjected to digestion with the restriction enzymes Agel and EcoRI, dephosphorylated and the fragment of the vector was isolated. The PCR product of the original CPG2 gene was also subjected to digestion with Agel and EcoRI, a fragment of approximately 1000 bp. isolated, ligated and transformed to E. Coli. The resulting clones were analyzed for a full length CPG2 gene (approximately 1200 bp.) by digestion with the restriction enzymes HindIII and EcoRI; the clones with the insert of correct size were subjected to sequencing to confirm the identity. Finally, this plasmid (pNG3 / Vkss-CPG2) was digested with Xbal, dephosphorylated, a fragment of the isolated vector and the Xbal gene fragment and the Neomicin gene fragment (approximately 1000 bp.) Which had also been isolated in the steps premature) was ligated back to the plasmid and transformed to E. coli. The resulting clones were inspected for the presence and orientation of the neomycin gene by individual digests with the Xbal and EcoRI enzymes. This vector was called pNG3-Vss-CPG2-NEO. d) Construction of the variant Rβ and CPG2 The plasmid pNG3-Vkss / CPG2-NEO was used as a template for the PCR mutagenesis of the CPG2 gene in order to identify 3 potential glycosylation sites that had been identified in the enzyme sequence bacterial natural. The glycosylation sites of supposed amino acids (N-X-T / S) were observed at positions "222 (N-I-T), 264 (N-W-T) and 272 (N-V-S) using the positional numeration published by Minton, N.P. et al., in Gene, (1984) 31, 31-38.

The asparagine residue (N) of the 3 glycosylation sites was mutated to glutamine (Q) to thereby cancel the glycosylation sites to avoid any glycosylation event that affects the expression of CPG2 or the activity of the enzyme. A PCR mutagenesis technique in which all 3 sites were mutated in a single series of reactions was used to create the R6 gene variant of CPG2. The vector pNG3 / Vkss / CPG2-NEO was used as the template for three initial PCR reactions. Reaction Rl used synthetic oligonucleotide sequence primers CME 00395 and CME 00397 (SEQ ID NOS: 3 and 4), reaction R2 used synthetic oligonucleotide sequence primers CME 00395 and CME 00399 (SEQ ID NOS: 3 and 5) and Reaction R3 used synthetic oligonucleotide sequence primers CME 00396 and CME 00400 (SEQ ID NOS: 6 and 7). The products of the PCR, Rl and R2 reactions contained the glycosylation sites 222 and 264 + 272 mutated respectively, the product of R3 consists of a copy of the C-terminal segment of the CPG2 gene. The R2 and R3 products (R2 of approximately 750 bp .; R3 of approximately 360 bp), after separation of agarose gel and purification, were joined in an additional PCR reaction. Mixtures of varying amounts of the R2 and R3 products were made and PCR reactions were carried out using the synthetic oligonucleotides CME 00395 and CME 00396. { SEQ ID NOS: 3 and 6). The resulting product R4 (approximately 1200 bps) was again amplified by PCR using the oligonucleotides CME 00398 and CEM 00396 (SEQ ID NOS: 8 and 6). The resulting R5 product (approximately 600 bp) was bound to the product R1 (approximately 620 b.p.) in a final PCR reaction carried out using the oligonucleotides CME 00395 and CME 00396 (SEQ ID NOS: 3 and 6). The resulting PCR product R6 (approximately 1200 bp), which now contained all three mutated glycosylation sites, could be cloned (after digestion with the Agel and BsrGI retention enzymes and isolation of the resulting fragment) to the vector pNG3 / Vkss-CPG2-Neo (which had previously been cut with the retention enzymes of Agel and Bsr Gl and subsequently isolated). This creates the protein sequence of CPG2 / R6 (SEQ ID NO: 9.} which encodes the desired DNA (SEQ ID NO: 10) within the expression vector pNG3 / Vkss-CPG2 R6-NEO. e) Construction of the heavy chain Fd-CPG2 fusion protein gene of A5B7 The heavy chain antibody fragment and the CPG2 enzyme genes were obtained by PCR amplification of plasmid templates. The plasmid pNG4 / A5B7VH-IgG2CH1 'was amplified with primers CME 00966 (SEQ ID NO: 11) and CME 00969 (SEQ ID NO: 12) to obtain the Fd A5B7 component (approximately 30β.p.) And the plasmid pNG3 / Vkss / CPG2 R6-NEO was amplified with the primers CME 00967 ( SEQ ID NO: 13) and CME 00968 (SEQ ID NO: 14) to obtain the enzyme component (approximately 1350 bp). In each case the PCR reaction product was loaded and separated on a 1% agarose gel, a band of the correct product size was excised, subsequently purified and eluted in 20 μl of H20.

An additional PCR reaction was carried out to join (or splice) together the two purified PCR reaction products. Standard PCR reaction conditions were used with varying amounts (between 0.5 to 2 μl) of each PCR product but using 25 cycles (instead of the usual 15 cycles). The reaction product was analyzed using a 1% agarose gel and a band of the expected size (approximately 1650 b.p.) was excised, purified and eluted in 20 μl of E20. This material was then digested using the restriction enzymes Nhel and BamHl, after which a band of the expected size (approximately 1600 b.p.) was recovered and purified. The vector pNG4 / A5B7VH-IgG2CH1 'was prepared to receive the above PCR product by digestion with the restriction enzymes Nhel and BamHI, after which the DNA was dephosphorylated and the band of the larger vector was separated from the H1 fragment of Nhel / Bam smaller. The vector band was recovered, purified and subsequently the similarly restricted PCR product was ligated to the prepared vector and the ligation mixture transformed to E. coli. The DNA was prepared from the clones obtained and subjected to sequencing subsequently to confirm the sequence of the fusion gene. A variety of clones were found to be correct and one of these clones (designated as R2.8) was designated pNG4 / A5B7VH-lgG2CH1 / CPG2 RS (SEQ ID-NO: 15 and SEQ ID NO: 16). f) Co-transfection, transient expression. { or temporal) Plasmids PNG4 / A5B7VH-IgG2CH1 / CPG2 R6 (which encode the chimeric antibody Fd-CPG2 fusion protein) and PNG3 / A5B7VK-HuCK-NEO (which codes for the chimeric light chain of antibody; SEQ ID NO: 17 and SEQ ID NO: 18) were co-transfected into COS-7 cells using a procedure based on LIPOFECTIN ™ as described hereinafter. The C0S7 cells are seeded in a plate of 6 cavities at 2xl05 cells / 2 ml / well, from a subconfluent culture and incubated overnight at a temperature of 37 ° C, 5% C02. A mixture of LIP0FECTIN ™ / serum-free medium is made as follows: 12 ml of LIPOFECTIN ™ plus 200 ml of serum-free medium are incubated at room temperature for 30 minutes. A DNA / serum-free medium mixture is made as follows: 4 mg of DNA (2 mg of each construct) plus 200 ml of serum-free medium. 200 ml of the LIPOFECTIN ™ mixture / serum-free medium is then added to the DNA mixture and incubated for 15 minutes at room temperature. Then 600 ml of the serum-free medium is added to each sample. The cells were washed once with 2 ml of serum free medium and then 1 ml of LIPOFECTIN ™ / DNA mixture was added to the cells and incubated for 5 hours, 37 ° C, - 5% C02. The LIPOFECTIN ™ / DNA mixture is separated from the cells and normal growth medium is added, after which the cells were incubated for 72 hours, 37 ° C, 5% C02. The supernatants of the cell were collected. g) Assay protein analysis apicibody-enz? tpa The supernatant was analyzed for the presence of fusion proteins and antibody when using a CEA binding ELISA analysis using an anti-human kappa light chain reporter antibody (as in the presence of antibody), an ELISA analysis of CEA binding using an anti-CPG2 reporter antibody (as regards the presence of CPG2 fusion protein bound to CEA), an analysis of the activity of the CPG2 enzyme based on HPLC (to measure the activity of specific CPG2) and SDS / PAGE-followed by Western staining (using either anti-human kappa light chain reporter antibodies or anti-human anti-reporter antibodies). CPG2) to detect the expressed material. Analysis of the activity of the HPLC-based enzyme clearly showed that the activity of the CPG2 enzyme was present in the supernatant of the cells and the anti-CEA ELISA assays showed protein binding at levels commensurate with an antibody molecule of Divalent A5B7. The fact that the anti-CEA ELISA analysis detected with an anti-CPG2 reporter antibody also exhibits a clear CEA binding indicates not only that the antibody but also the antibody fusion protein-CPG2 was linked to CEA. Western blot analysis with reporter antibody analysis clearly shows a fusion protein subunit of approximately 90 KDa size expected without any degradation of the smaller products (such as Fab or enzyme) observable. Since it is known that CPG2 exhibits only enzyme activity when in its dimeric state and since only the enzyme antibody fusion protein is present, this indicates that the fusion protein of 90. KDa (seen under SDS / PAGE conditions) is dimerized via the natural CPG2 dimerization mechanism to form an antibody fusion protein molecule - "180 kDa dimeric enzyme (figure 2a) under" original "regulated pH conditions. , this molecule exhibits CPG2 enzymatic activity and CEA antigen binding properties that do not appear to be significantly different in the fusion protein compared to the enzyme or antibody alone. h) Use of the expressed fusion protein and CPG2 prodrug in an in vitro citotosicity analysis An in vitro cell extermination assay was carried out in which the fusion protein (A5B7-CPG2 R6) was compared with a conjugate of A5B7 F (ab ') 2-CPG2"conventional" formed by linking the A5B7 F (ab'.} 2 to CPG2 with a heterobifunctional chemical reagent In each case the material exhibiting equal amounts of CPG2 enzyme activity or Equal amounts of antibody-CPG2 protein were incubated with LoVo tumor cells, which carry CEA, then the cells were washed to separate the unbound protein material and subsequently resuspended in a medium containing CPG2 phenol (PGP, see example 2 below) for a period of 1 hour, after which the cells were washed, resuspended in freshly prepared medium and allowed to proliferate during 4 days. Finally, the cells were treated with dyeing SRB and their numbers are determined. The results obtained clearly showed that the fusion protein (A5B7-CPG2 R6) 2 (together with the prodrug) caused a cell death at least equivalent and resulted in lower numbers of cells at the end of the study period than the equivalent levels of the conjugate of A5B7 F (ab) 2-CPG2 (with the same prodrug). Cell killing (above baseline control levels!) Can occur only if the prodrug is converted to active drug by the CPG2 enzyme (and since the cells are washed to separate the unbound protein, only the linked enzyme the cell will remain in the stage where the prodrug is added.) Thus, this experiment shows that at least as much of the fusion protein A5B7-CPG2 R6 remains bound compared to the conjugate of A5B7 F (abr) 2-CPG2 conventional since a greater degree of cellular extermination occurs (supposedly due to the higher conversion of medication to medication). i) Construction of a coexpression fusion protein vector for use in transient and stable cell line expression For a simpler transfection methodology and direct pairing of both expression cassettes to a single selection marker, a vector of cs -Expression for the expression of the fusion protein was constructed using the existing vectors pNG4 / A5B7VH-IgG2CH1 / CPG2 R6 (which encodes the antibody Fd-CPG2 fusion protein) and pNG3 / A5B7VK-HuCK-NEO (coding the chain light of the antibody). The plasmid pNG4 / A5B7VH-IgG2CH1 / CPG2 R6 was first subjected to digestion with the Seal retention enzyme, the reaction was loaded on a 1% agarose gel and the band of the linear vector was excised from the gel and parked. Then this DNA vector was digested with the restriction enzymes BglII and BamHI, the reaction was loaded on a 1% agarose gel and the desired band (approximately 2700 bp) was recovered and purified. The plasmid pNG3 / A5B7VK- HuCK-NEO was digested with the restriction enzyme BamHI after which the DNA was dephosphorylated, then loaded on a 1% agarose gel and the vector band excised from the gel and purified. The fragment of the heavy chain expression cassette was ligated to the prepared vector and the ligation mixture transformed to E. coli The orientation was inspected by a variety of restriction digests and selected clones that had the heavy chain cassette in the same direction as that of the light chain. These plasmids were designated pNG3-A5B7-CPG2 / R6-coexp. -NEO. j) Gene switches for protein expression It is contemplated that the in vitro expression of CPG2 and CPG2 fusion proteins in mammalian cells can degrade foliate media to lead to slow cell growth or cell death. It is probable that the high activity of the CPG2 enzyme makes so much of foliate deficiency difficult to overcome by means of the complementation of the medium. However, it is thought that in the case of CPG2 or expression of the CPG2 fusion protein of the cells in vivo, it is unlikely that such problems would arise, since the cells would be constantly replenished with all the requirements by the circulatory and normal cell phones A variety of options have been considered to avoid the possible problems of depletion of phobic acid in vitro. One of these solutions involves the use of highly controlled but inducible gene switching systems such as the "TET on" or "TET off" switches (Grossen, M. et al (1995) Science 268: 1766-1679) or the switch ecdysone / muristerone A (No, D. et al (1996) PNAS 33: 3346-3351.}.

Such systems allow precisely controlled expression of a gene of interest and allow stable transformation of mammalian cells with genes encoding toxic or potentially deleterious expression products. A gene switch would allow stable cell lines recom binants incorporating CPG2 fusion genes to be established, maintained and potentially potentially expanded for protein expression and culture seeding for tumor growth studies in vivo.

EXAMPLE 2 HCT116 tumor cells expressing the antibody-enzyme fusion protein are selectively killed in vitro by a prodrug. HCT11 colorectal tumor cells, (ATCC CC1 247) transfected with the antibody gene-fusion protein CPG2 of example 1 can to be selectively exterminated by means of a prodrug that is converted by the enzyme to an active drug. To demonstrate this, untransfected, control or reference HCT116 cells or HCT116 cells transfected with the antibody gene-CPG2 fusion protein are incubated with either the prodrug, acid, 4- [N, N-bis (2- chloroethyl) amino] -phenoxycarbonyl-L-glutamic acid (PGP; Blakey et al, Br. J. cancer 72, 1083, 1995) or the corresponding drug released by CPG2, 4- [N, H-bis (2-chloroethyl) ) amino] phenol The average of PGP and medication in the range of 5 x 10 ~ 4 to 5 x 10"8 M are added to 96-well microtiter plates containing 1000-2,500 cells of HCT116 / well for 1 hour at 37 ° C. The cells are then washed and incubated for an additional three days at 37 ° C. After washing to separate the dead cells, TCA is then added and the amount of cellular protein that adheres to the plates is determined by addition of the SRB dye, as described by Skehan et al (J. Nati. Cancer Inst. 82, 1107, 1990). The medication and medication is determined by the concentration required to inhibit cell growth at 50% (IC50). Treatment of HCT116 cells not transfected or transfected with the drug results in an IC50 of approximately 1 μM. In contrast, the PGP prodrug results in an IC 50 of approximately 200 μM on untransfected cells of approximately 1 μM on transfected cells. These results demonstrate that the transfected cells expressing the antibody-CPG2 fusion protein can convert the PGP prodrug to the more potent active drug, while the non-transfected HCT116 cells are unable to convert the prodrug. Consequently, the transfected HCT116 cells are more than 100 times more sensitive to the PGP prodrug in terms of cell killing as compared to non-transfected HCT116 cells. (See example lj) for questions that involve the possible depletion of folic acid in the cell. These studies demonstrate that the transfection of tumor cells with a gene for an antibody-pristaaa enzyme fusion can lead to selective killing of the tumor cell with a prodrug.

Example 3 Anti-tumor activity of PGP promedicamento in tumors of HCT116 expressing the antibody-CPG2 fusion protein The anti-tumor activity in vivo of the PGP prodrug in HCT116 tumors expressing the antibody-CPG2 fusion protein can be demonstrated as follows.

HCT116 tumor cells transfected with the antibody gene - CPG2 fusion protein or tumor cells from HCT116 untransfected, control or reference, are injected subcutaneously nude athymic mice (107 tumor cells per mouse). When the tumors are 5-7 mm in diameter, the PGP average is administered i.p. to mice (3 doses at intervals per hour for 2 hours in dose ranges of 5-25 mg Kg-1). The anti-tumor effects are determined by measuring the length of the tumors in two directions and calculating the volume of the tumor using the formula: Volume = p / 6 x D2 X d where D is the largest diameter and d is the smallest diameter of the tumor. The volume of the tumor is expressed in relation to the volume of the tumor at the time when the PGP prodrug is administered. Anti-tumor activity is compared to a control group (or reference group) that receives either transfected or non-transfected tumor cells and PBS (170 mM NaCl, 3.4 mM KCl, 12 mM Na2HP0 and KH2P04, 1.8 mM, pH 7.2) in place of the PGP promedicamento. Administration of PGP to established HCT116 tumors from transfected HCT116 cells results in a significant anti-tumor effect, as determined by PGP-treated tumors that decrease in size compared to tumors treated with PBS and by taking a significantly longer time for tumors treated with PBS to reach 4 times their initial tumor volume compared to tumors treated with PBS. In contrast, administration of PGP to established HCT116 tumors of non-transfected cells does not result in any significant anti-tumor activity. Similar studies can be used to demonstrate that the antibody-enzyme gene administered in a vector appropriate to established HCT116 tumors produced from the non-transfected HCT116 cell when used in combination with other PGP prodrugs can result in anti-HIV activity. -significant noise. Thus, non-transfected HCT116 cells are injected into athymic nude mice (1 X 10 7 tumor cells per mouse) and once the tumors are 5-7 mm in diameter the vector containing the antibody-fusion protein of enzyme is injected intra-tumorally. After 1-3 days for YES Allowing the antibody-enzyme fusion protein to be expressed by and binding to the CHT116 tumor cells, the PGP Promedication is administered as described above. This results in a significant anti-tumor activity compared to the control or reference mice receiving PBS instead of the PGP prodrug.

Example 4 Perfected transfection of adherent cell lines using supplemented EUS medium and / or V-79 feeder cells It is contemplated that in vitro expression of CPG2 proteins and CPG2 fusion proteins in branching cells can degrade foliate media to lead to slow cell growth upon cell death.

FAS medium (supplemented with folinic acid) described in Present was developed for CPG2 and fusion proteins of CPG2 expressing cell lines in order to better support the growth of such cell lines. In preparation for transfection, the adherent cell lines were cultured in a normal DMEM medium and passaged at least three times before transfection. V-79 feeder cells (hamster ulmon fibroblast, obtained from MRC Radiobiology Unit, Harwell, Oxford, UK) were cultured in a normal DMEM medium and passed three times before use. For transfection, a viable count is made (using a hemocytometer / trypan blue stain) of the adherent cells and the cells were deposited at 2 X 105 cells per cavity in a 6-well plate (Costar 3516.) and allowed to stand for 18 hours. -24 hours for the cells to re-adhere For each individual transfection, 20 μl of LIPOFECTIN ™ is added to 80 μl of serum-free medium and allowed to stand at room temperature for 30 minutes Plasmid DNA (2 μg ) of interest was added to 100 μl of serum-free medium and subsequently added to the LIPOFECTIN ™ mixture and allowed to stand for an additional 15 minutes.The individual 6-well plates were washed with 2 ml of serum-free medium per well. Separate any serum and replace it with 800 μl of freshly prepared serum-free medium, then mixtures of 200 μl of DNA / LIPOFECTIN ™ / serum-free medium that had previously been prepared were added to each well. The serum was incubated at 37 ° C for 5 hours, the medium was prepared and 2 μl of fresh medium was added and incubated for an additional 48 hours. The transfected cells in the 6-well plate were scraped, the cell suspension was separated and centrifuged. All of the supernatant was removed and the cell pellet was resuspended in 20 ml of an appropriate fresh growth medium (eg, DMEM FAS medium) containing the appropriate selective agent for the transfected DNA (eg, G418). Aliquots (200 μl.) Are deposited per cavity in a 96-well plate (1.25 X 10 4 cells per well) To enhance the expansion of the clone, fibroblast feeder cells can be added to the transfected cells. 79 semi-confluents were trypsinized and a viable count was carried out.The cells were resuspended at 1 X 10d cells / ml in a sterile glass vessel and irradiated using a cesium source by exposure to 5000 rads during 12 minutes Cells can then be stored at 4 ° C for 24-48 hours (irradiated cells are metabolically active but will not divide and can "act as" feeders "for other cells without contaminating the culture.) Aliquatizing cells must be deposited to 4 X 104 cells per well in a 96-well plate to produce a confluent plate for emerging recombinant clones. The feeder cells adhere initially to the plate but over time they separate and float to the medium to leave any recombinant clone still attached to the cavity. The changes of the medium (200 μl in time) are carried out twice a week to separate the floating cells and replenish the medium. Colonies are allowed to develop for 10-14 days, then the supernatant is filtered by standard ELISA analysis for secretion of fusion proteins. To measure the expression rate in the case of the fusion gene constructs (A5B7-CPG2) 2, recombinant cells are seeded at 1 X 10d in 10 ml of freshly prepared normal culture medium for exactly 24 hours. Then the supernatant is separated, centrifuged to separate cell debris and subjected to analysis for fusion protein and enzyme activity by the ELISA and HPLC methods described above. The results for a variety of recombinant fusion protein (A5B7-CPG2) 2 cell lines are shown below: Cell line Clone ng / 106 Cells / 24 h HCT 116 F7 6550 C12 3210 HCT 116 F6 15560 Cl 6151 B3 4502 A8 4650 D5 630 H9 610 Gil 2081 H4 2380 A4 1634 LoVo B9 S370 Cl 7350 F12 2,983 C7 10-770 G10 414Q Coló 320DM B3 10540 G4 1720 B9 885 B10 3090 F12 35660 Example 5 Construction of a stable inducible fusion protein (A5B7-CPG2) 2 expressing the tumor cell line a) Construction of an inducible fusion protein expression vector. To facilitate the expression of a single inducible mammalian cell promoter, an IRES-based version was constructed (Internal! Ribosome Entry Site; see Y. Sugimoto et al., Biotechnology (1994), 12, © 94-8) of the fusion protein (A5B7-CPG2) 2. The pNG3 construct pNG3 / A5B7VK-HuCK-NEO (chimeric light chain A5B7, described in Example Ib above) was used as a template for the amplification of the light chain gene. The gene was amplified "using the oligonucleotides CME 3153 and CME 3231 (SEQ ID NOS: 19 and 20.) .. A PCR product of the expected size (approximately 700 b.p.) was purified. This product was then digested using the restriction enzymes EcoRI and BamHI and subsequently purified. The fragment was then cloned into the Bluescript ™ + KS vector (prepared to receive the fragment by digestion with the same restriction enzymes EcoRI and BamH1.) After which the DNA was dephosphorylated and the band of the larger vector purified. of restricted PCR similarly bound to the prepared vector and the ligation mixture was transformed to E. coli.

The DNA was prepared from the clones obtained and analyzed by restriction digestion to inspect the insertion of the PCR fragment. The appropriate clones were subjected to sequencing to confirm the genetic sequence. A number of the clones with the correct sequence were obtained and one of these clones was given the designation of plasmid A5B7 Bluescript ™. In a similar manner, the chimeric heavy chain A5B7 was amplified by PCR of the plasmid pNG4 / A5B7VH-IgG2CH1 / CPG2 R6 (described in the previous example) using the oligonucleotides CME 3151 and CME 3152 (SEQ ID NOS: 21 and 22). A PCR reaction product of the expected size (approximately 1800 b.p.) was purified.

This product was then digested * using the restriction enzymes BamHI and Xba I after which the fragment band was purified. The fragment was also cloned into the Bluescript ™ + KS vector which had been prepared to receive the above fragment by digestion with the same restriction enzymes, BamHl and Xbal, after which the DNA was dephosphorylated and the band of the larger vector was purified. The similarly restricted PCR fragment was ligated to the prepared vector and the ligation mixture was transformed to E. coli. The DNA was prepared from the obtained clones and analyzed by restriction digestion to inspect the insertion of the PCR fragment. The appropriate clones were subjected to sequencing to confirm the correct sequence. A variety of the clones with the correct sequence were obtained and one of these clones received the designation of the plasmid Bluescript ™ Fd-CPG2 R6. The IRES sequence was the source of the pSXLC vector (described in Y. Sugi oto et al. Biotechnology (1994), 12, 694-8 and obtained from the authors). The IRES sequence was excised by digestion with the restriction enzymes.

BamHl and Ncol. A band of the expected size (approximately 500 b.p.) was purified and ligated to the Bluescript ™ Fd-CPG2 R6 plasmid (which had previously been prepared by restriction with the same enzymes). The ligation mixture was transformed into E. coli and the DNA was prepared from the obtained clones. The DNA was analyzed by restriction digestion to inspect the insertion of the fragments and the appropriate clones were subsequently sequenced to confirm the genetic sequence. A variety of the clones with the correct sequence were obtained and one of these clones is given the designation of plasmid Bluescript ™ IBES Fd-CPG2 B £. To facilitate the subsequent cloning steps, it was necessary to cancel the Xba I site that had been carried out on the IRES fragment. This was carried out by PCR mutagenesis with the CME oligonucleotide primers 3322 and CME 3306 (SEQ ID NOS 23 and 24) and the Bluescxipt ™ USES Fd-CPG2 R6 as template DNA. A PCR reaction product of the expected size (approximately 500 b.p.) was purified, subjected to digestion with the restriction enzymes BamHl and Ncol and bound to the plasmid Bluescrípt ™ IRES Fd-CPG2 R6 (which had previously been prepared by restriction with the same restriction enzymes). The ligation mixture was transformed into E. coli and the DNA was prepared from the obtained clones. The DNA was analyzed by restriction digestion to inspect the insertion of the fragment and the appropriate clones were subsequently sequenced to confirm the genetic sequence. A variety of the clones with the correct sequence were obtained and one of these clones was given the designation of the Bluescript ™ IEES Fd-CPG2 R6-Xba plasmid. The chimeric light chain fragment of A5B7 was excised from plasmid A5B7 Bluescript ™ by digestion with the restriction enzymes EcoRl and BamHl. A band of the expected size (approximately 700 b.p.) was purified, ligated to the plasmid Bluescript ™ IRES Fd-CPG2 R6-Xbadel prepared appropriately and the ligation mixture was transformed to E. coli The DNA was prepared from the obtained clones and analyzed by restriction digestion to inspect the insertion of the fragment. The appropriate clones were subsequently sequenced to confirm the genetic sequence. A variety of clones with the correct sequence were obtained and one of these clones is given the designation of the plasmid Bluescript ™ A5B7 IRES Fd-CPG2 R6-Xba del. The genetic sequence of the chimeric fusion protein of A5B7 based on complete IRES is shown in SEQ ID NOS 52. Then the chimeric fusion protein gene of A5B7 based on IRES was transferred to an expression vector regulated by tetracycline. The vectors for the Tet On gene expression system were obtained from Clontech. The tetracycline switchable expression vector pTRE (otherwise known as pHUD10-3, see Gossen et al (1992), PNAS, 89, 5547-51) was prepared to accept the IRES-based fusion protein cassette. by digestion with the EcoRI and Xbal restriction enzymes, dephosphorylated and the band of the largest purified vector. The IRES gene cassette was excised from the Bluescript ™ A5B7 IRES Fd-CPG2 R6-Xba plasmid using the same restriction enzymes. The fragment of approximately 3000 b.p. was ligated to the prepared vector and the ligation sample was transformed to E. coli. The DNA was prepared from the clones obtained and analyzed by restriction digestion to inspect the insertion of the PCR fragment. The appropriate clones were subsequently sequenced to confirm the genetic sequence. A variety of the clones with the correct sequence were obtained and one of these clones is given the designation of plasmid pHUD10-3 / A5B7 IRES Fd-CPG2 R6. b) Construction of a stable inducible fusion protein that expresses the cell line. The standard lipofection transfection methodology (as previously described but without the use of feeder cells) was used to produce recombinant HCT116 tumor cell lines. A co-transfection using 1 μg of the plasmid pHUD10-3 / A5B7 / IRES Fd-CPG2 R6 and 1 μg of the plasmid expressing the transactivator pTet-On (from the Clontech team) was carried out and the positive clones were selected using the medium of FAS containing 750 μg of G418 / ml. c) Induction studies of recombinant HCT116 inducible cell lines. The obtained clone cultures were divided into plates of 48 duplicated cavities, each containing 1 x 10 6 cells. The cells were cultured for 48 hours, with one of the plates induced with 2 μg / ml of doxycycline and the other acting as a control (or reference) not induced. Expression of the fusion protein (A5B7-CPG2) 2 in the supernatant of the cell was tested using the ELISA / Western blot analyzes described in the Ig example. The results indicate that the induction of the fusion proteins of the inducible cell line by the use of doxycycline could be clearly demonstrated, for example one of the obtained clones (Fll), the induced cells produce 120 ng / ml of fusion protein in the supernatant whereas the non-induced cells produce only background levels of fusion protein (less than 1 ng / ml).

Example 6 Cell-based ELISA analysis of secreted fusion protein material - Cells were seeded in 96-well plates (Becton Dickinson Biocat ™ poly-DHQisin, 35-6461.) At a density of 1 x 104 cells per cell. cavity in 100 μl of normal culture medium and allowed to stand for approximately 40 hours at 37 ° C. 100 μl of 6% formaldehyde is diluted in DMEM and allowed to stand for 1 hour at 4 ° C. The plates were centrifuged and washed 3 times in PBS containing 0. 05% Tween by immersion rinse (first 2 washes for 2 minutes and the final wash for 5 minutes). 100 μl of duplicate dilutions of the cell culture supernatant containing fusion protein or anti-CEA Chimeric A5B7 were added to each well as appropriate and the plates were incubated overnight at 4 ° C. Plates were washed as described above and in the case of chimeric fusion proteins, 100 μl of a 1: 1000 dilution of HRP-labeled anti-human kappa antibody (Sigma A-7164) was added and incubated for 2 hours at room temperature (Erna anti-CPG2 detection methodology can be used in the case of the murine scFv fusion protein). The plates were washed as described above and the HRP was detected using the OPD substrate (Sigma P-8412). "Color is allowed to develop for approximately 5 minutes, stopped with 75 μl per 2 M H2SO4 cavity and OD reading at 490 nm In the case of the fusion protein (A5B7-CPG2.) .2 the material was produced in the supernatant of the recombinant Colo320DM tumor cells (CEA-ve) .The content of fusion protein was measured by using CEA ELISA analyzes described above Increased amounts of fusion protein were added to a variety of negative CEA cell lines and the positive LoVo line of CEA The results shown in Figure 3 clearly show that only the CEA positive line shows increased levels of adhesion or binding with the increased properties of aggregated fusion proteins while the negative CEA cell lines show only constant background link or adhesion levels from start to finish. This clearly demonstrates that the fusion protein adheres or sticks specifically and is retained on the positive LoVo cells of CEA.

Example 7 Cells LoVo tumor cells expressing the antibody-enzyme fusion protein exhibit retention of the fusion protein on the cell surface LoVo colorectal tumor cells transfected with the fusion protein gene (A5B7-CPG2) 2 secrete and retain the fusion protein on its cell surface. This can be demonstrated by comparing the original fusion protein and recombinant expressing LoVo cells under the conditions summarized in the cell-based ELISA analysis of the secreted fusion protein (Figure 4). In the development of reaction color it could be seen that the recombinant LoVo cells had retained the expressed fusion protein (by showing a high level of color). In control or reference experiments using cells expressing the Colo320DM fusion protein, the analysis showed some retention of the expressed fusion protein (probably non-specific) and the original LoVo cells exhibit only background activity. Positive controls in which the CEA binding antibody was added to test the tumor cells expressing re-fusion fusion proteins and the original LoVo controls resulted in a signal that is obtained from the original LoVo (thus demonstrating that the CEA is present in the original cells) but no increased signal of the Colo320DM (negative CEA). The recombinant LoVo cells still give a strong initial signal that the added antibody made a difference to the overall signal obtained, which was considerably higher than any of the control experiments. It is thus appreciated that the antibody of anti-CEA fusion protein of CPG2 enzyme secreted from CEA-positive tumor cell lines are bound to the cell surface (via CEA) while the same protein expressed from negative CEA tumors does not show such a link Example 8 LoVo tumor cells expressing the antibody-enzyme fusion protein are selectively killed in vitro by a prodrug LoVo colorectal tumor cells transfected with the fusion protein gene (A5B7-CPG) 2 can be selectively killed by a drug that is converted by the enzyme CPG2 to an active drug. To demonstrate this, non-transfected LoVo cells or LoVo calialas transfected with antibody gene-CPG2 fusion protein are incubated with either the prodrug, 4- [N, N-bis (2-chloroethyl) ami-no. ] phenoxycarbonyl-L-glutamic (PGP? Blakey et al, (1995) Br.

J. Cancer 72, PL083) or the corresponding medicament released by CPG2, 4- [N, N-bis (2-chloroethyl) amino} phenol as described in example 2 with HCT116 cells. Transfected cells expressing the CPG2 fusion protein antibody can convert the PGP prodrug to the most potent active drug while untransfected LoVo cells are unable to convert the prodrug. These studies demonstrate that transfection of tumor cells with a gene for an antibody / enzyme fusion protein can lead to selective killing of tumor cells with a prodrug.

EXAMPLE 9 Establishment of fusion protein expressing LoVo tumor heteroplast grafts in athymic mice. Tumor cells expressing recombinant LoVo fusion protein (A5B7-CPG2) 2 or mixtures of recombinant and original LoVo cells were injected subcutaneously into athymic nude mice. (107 tumor cells per mouse). Tumor growth rates for mixtures of 100% LoVo and 20%: 80% recombinant LoVo: original cell mixtures were compared to those of parental cell tumors alone. No significant difference was observed in the obtained growth curves that do not show required corrections, during the comparisons between the cell lines. The rates of tumor growth observed showed that in each case for heteroplastic graft tumors to reach a size of 10 X 10 mm approximately 12 days are needed.

Example 10 Determination of enzyme activity in tumor heteroplast graft samples To act as a standard for analysis, a standard CPG2 enzyme curve was prepared in 20% homogenized normal tumor product (parental cell tumor). Subsequent dilutions of samples were made in the same homogenized product at 20% normal tumor. The excised tumor tissue is removed from the storage at -80 ° C (previously frozen instantly in liquid nitrogen) and allowed to thaw. Any residual skin tissue is removed before the tumor is cut into small fragments with a scalpel. The tumor tissue was transferred to a pre-weighted tube and the weight of the tumor tissue was measured. PBS containing 0.2 mM ZnCl2 solution is added to each tumor sample to give a 20% (w / v) mixture, homogenized and placed on the ice. Dilutions of the sample tumors (in 20% normal tumor homogenate) are prepared by net, 1/10, 1/2, and 1/40. For the standard curve, dilutions of the CPG2 enzyme were made at the following concentrations to a final volume of 400 μl. Similarly, 400 μl of each of the recombinant tumor sample dilutions were also prepared. After equilibration at 30 ° C, they are added 4 μl of 10 mM methotrexate solution (fX). The reaction was stopped exactly after 10 minutes by the addition of 600 μl of ice-cold methanol + 0.2% TFA, centrifuged and the supernatant is collected. Then the substrate and the product in the supernatant were separated by HPLC (using an Exchange Column cation exchange column, HICRO ™ S5SCX-100A, mobile phase = 60% methanol, 40% formamide d ammonium 60 mM / TFA at 0. 1%, detection 3O0 n. ). To calculate the activity of the enzyme in the tumor tissue, the standard curve was plotted as methotrexate metabolite area units (the standards are such that only the -30% of the substrate is metabolized to ensure that it is not limiting the speed). The test samples were analyzed by comparing the unit area of the metabolite against the standard curve and then multiplying them with the dilution factor. Finally, making the working assumption that 1 ml = 1 g, the results were multiplied by 5 (since the samples were originally diluted to a 20% homogenized product). The results obtained with 20% recombinant LoVo: 80% parental cells expressing the fusion protein (A5B7 Fab-CPG2) 2 showed the following results: the tumors taken on the fifth day had an average enzyme activity = 0.26 U / g ( range between 0.18-0.36 ü / g) and at day 12 had the average enzyme activity = 0.65 D / g (range between 0.19-1.1 U / g).

Example 11 Determination of Enzyme Activity in Plasma Samples To act as a standard for analysis, a standard CPG2 enzyme curve was prepared in normal 20% plasma at the following concentrations: 0.2, 0.4, 0.6, 0. 8 and 1.0 U / ml. Similarly, all test plasma samples were also diluted to 20% normal plasma.

Additional dilutions of these samples were also made, such as net 1/10, 1/20 and 1/50 when using also 20% normal serum. Aliquots of 200 μl of each standard of CPG2 and dilutions of test sample were equilibrated at 30 ° C. 2 μl of 10 mM MTX were added to each of the tubes and mixed well at a temperature of 3 ° C. The reaction was stopped after exactly 10 minutes (to increase the sensitivity of the analysis, the incubation time can be increased by 30 minutes) by adding 500 μl of ice-cold methanol + 0.2% TFA and analyzing the product using detection HPLC as described above in Example 10. No activity is seen in the plasma except in the rare cases when the level of enzyme activity in the tumor was greater than 2.0 U / g, in which case the enzyme levels in the plasma were measured in the range of 0.013 to 0.045 U / ml.

Example 12 Anti-tumor activity of PGP-promedicamento in tumors of LoVo expressing antibody - CPG2 fusion protein Tumor cells expressing fusion protein (A5B7-CPG2) LoVo recombinants or mixtures of recombinant and parental LoVo cells were injected subcutaneously into nude nude mice as described in example 9.

When the tumors are 5-7 mm in diameter, the PGP averaging is administered i .p. to mice (3 doses in DMSO pH buffer / 0. 015 M sodium bicarbonate at intervals per hour for 2 hours at dose ranges of 40-80 mg Kg "1).

The anti-tumor effects were determined by measuring the length of the tumors in two directions and calculating the volume of the tumor using the formula: Volume = p / 6 x D? X d where D is the largest diameter and d is the smallest diameter of the tumor. The volume of the tumor can be expressed in relation to the volume of the tumor at the time when the PGP prodrug is administered or alternatively the average volumes of the tumor can be calculated. Anti-tumor activity was compared to control or reference groups receiving either transfected or non-transfected tumor cells and pH-regulating solution without PGP promedication. Administration of PGP to established LoVo tumors from recombinant LoVo cells or mixtures of recombinant LoVo cells / parental LoVo cells results in a significant anti-tumor effect as shown by tumors treated by PGP that decrease in size compared with controls and they take a significantly longer time for tumors treated with PGP reach 4 times their initial tumor volume compared to controls or reference (figure 5). The administration of PGP to LoVo tumors established with the non-transfected cell does not result in any significant anti-tumor activity.

Similar studies can be used to demonstrate that the antibody-enzyme gene delivered in an appropriate gene delivery vector to established LoVo tumors produced from untransfected parental LoVo cells when used in combination with the PGP prodrug can result in a significant anti-tumor activity. Thus, untransfected LoVo cells are injected into mice with athymic knots (1 X 10 7 tumor cells per mouse) and once the tumors are 5-7 mm in diameter, the vector containing the fusion protein gene of antibody-enzyme is injected intra-tumorally. After 1-3 days to allow the antibody-enzyme fusion protein to be expressed and attached to the LoVo tumor cell the PGP prodrug is administered as described above. This results in significant activity compared to controls or references.

Example 13 Construction of a fusion protein (806.077 Fab-CPG2) 2 The construction of an enzyme fusion (806.077 Fab-CPG2) 2 was planned with the aim of obtaining a bivalent human carcinoembryonic antibody (CEA) binding molecule that also exhibits CPG2 enzyme activity. For this purpose, the initial construct was designed to contain a fragment of heavy chain Fb of antibody 806.077 bound at its C terminus via a peptide linker (G4S.) 3 flexible to the N terminus of the CPG2 polypeptide (as shown in Figure 1 but replacing 806.077 in place of A5B7.) Antibody 806.077 (described in International Patent Application WO 97/42329, Zeneca Limited) sticks or binds to a very high degree of specificity to human CEA. the 806.077 antibody is particularly suitable for targeting colorectal carcinoma and other CEA antigen-bearing cells In general, the conjugate of antibody (or antibody fragment) enzyme or fusion proteins must be at least divalent, ie capable of binding to at least two tumor associated antigens (which may be the same or different.) In the case of the fusion protein of (806,077 Fab-CPG2) the dimerization of the compound is carried out. of the enzyme (after expression, similar to the original enzyme, to form an enzyme molecule that contains two Fab antibody fragments (and is thus bivalent with respect to antibody binding sites) and two molecules of CPG2 (figure 2a). a) Cloning of the antibody genes 806.077 The methods for the cloning and characterization of the recombinant murine antibody 806.077 F (ab ') 2 have been published (international patent application WO 97/42329, example 7). Reference example 7. 5 describes the cloning of variable region genes from antibody 806.077 to Bluescript ™ KS vectors. These vectors were subsequently used as the variable region source of 806,077 for the construction of chimeric chain and heavy chain Fd genes of 806. 077. b) Chimeric 806.077 antibody vector constructions International patent application WO 97/42329, eg 8 describes the cloning of light chain and heavy chain Fd genes 806. 077 in the picfG3-Vkss-HuCk-NEO vectors ( deposit of NCIMB number 40799) and pNG4-VBss-HulgG2CH1 '(deposit of NCIMB number 40797) respectively. The resulting vectors were assigned pNG4 / VHss806.077VH-IgG2CH1 '(chimeric heavy chain fd' 806.077) and pNG3 / vKss8.06.077VK-HuCK-NEO (chimeric light chain of 806.077). These vectors were the source of the 80S.077 antibody genes for the construction of the Fab-CPG2 fusion protein 806,077. c) Construction of the heavy chain fd-CPG2 fusion protein gene of 806.077 The cloning and construction of the CPG2 used is described in example 1, sections c and d. Similarly, the construction of the vector, pNG4 / A5B7VH-IgG2CH1 / CPG2 R6 which is used for the construction of the variable heavy chain Fb-CPG2 of 806.077 is described in Example 1, section e. The variable heavy chain gene 806.077 was removed from the vector pNG4 / VHss806.077VH-IgG2CH1 by digestion with restriction enzymes HindIII and Nhel and a band of the expected size (approximately 300 b.p.) containing a variable region gene was purified. The same restriction enzymes (HindIII / Nhel) were used to digest the pNG4 / A5B7VH-IgG2CH1 / CPG2 R6 vector in preparation by replacing the variable region 806.077 with that of the A5B7 antibody. After digestion, the DNA was then dephosphorylated, the band of the larger vector was separated and purified. Then the similarly restricted variable region gene fragment was ligated to this prepared vector and the ligation mixture transformed to E. coli. The DNA was prepared from the obtained clones and analyzed by restriction digestion analysis and subsequently subjected to sequence to confirm the genetic fusion sequence. We found that a diversity of the clones were correct and one of these clones pNG4 / VHss806vH-IgG2CH1 / CPG2 R6, was chosen for additional work. The heavy chain Fd-CPG2 fusion protein gene sequence of 806,077 created or shown in SEQ ID NOS 25 and 26. d) Co-transfection, transient expression - and fusion protein analysis Plasmids pNG4 VHss806.077VH-IgG2CH1 / CPG2 R6 (which encodes the chimeric Ftγ-PG2 fusion protein of the antibody) and pNG3 / VHss806.077VK-HuCK-NEO (which encodes the light chain of the antibody) were co-transfected into COS-7 cells using a procedure based on LIPOFECTIN ™ described in the above example If. The analysis of the fusion protein was carried out as described in the example lg. The analysis of enzyme activity on the basis of HCPLC clearly shows that the amount of CPG2 enzyme is present in the cell supernatant and anti-CEA ELISA assays show binding or binding of protein at levels commensurate with a molecule * antibody 806,077 bivalent. The fact that the anti-CEA ELISA analysis detected with an anti-CPG2 reporter antibody also exhibits a clear binding or binding of CEA indicates that not only the antibody but also the antibody-CPG2 fusion protein that bound to CEA. Western blot analysis with reporter antibody analysis clearly exhibits a fusion protein subunit (806,017 Fab-CPG2) 2 of the approximate expected 90 KDa size with only a small degradation or smaller products (such as Fab or enzyme) observable . Since it is known that only CPG2 exhibits enzymatic activity when it is in a dimeric state and since only the enzyme antibody fusion protein is present, this indicates that the 90 kDa fusion protein (see under SDS conditions) / PAGE) is dimerized via the natural CPG2 dimerization mechanism to form an antibody-dimeric 180-kDa fusion protein molecule (figure 2a) under "original- '' 'regulated pH conditions.In addition, this molecule exhibits activity Enzymatic CPG2 and binding or antigen binding properties of CEA do not appear to differ significantly in the fusion protein compared to the enzyme and antibody alone. e) Construction of a fusion protein coexpression vector (806,077 Fab-CPG2) for the use of transient and stable cell line expression For a simpler methodology for the direct pairing of both expression cassettes to an individual selection marker a Coexpression vector for expression of the fusion protein was constructed using the existing vectors pNG4 / VHss8O6.077VH-IgG2CH1 / CPG2 (which encodes the antibody CPG2 fusion protein) and? NG3 / VKss806.077VK-HuCK-NEO (which encodes the light chain of the antibody). The plasmid pNG4 / V? Ss806.077VH-IgG2CH1 / CPG2 was first restricted with the restriction enzyme Seal, the band of the linear vector was purified, subjected to digestion with the restriction enzymes BglII and BamHI and a desired band ( approximately 2700 bp.} -fue purified.The plasmid was subjected to digestion with the enzyme restriction pNg3 / VKss806.077VK-HuCK-NEO after which the DNA was dephosphorylated and the band of the vector was purified. The heavy chain expression cassette fragment was ligated to the prepared vector and the ligation mixture was transformed to E. coli. The orientation was inspected by a variety of restriction digests and the selected clones that had the heavy chain cassette in the same direction as that of the light chain. This plasmid was designated as pNG3-806,077-CPG2 / R6-coexp.-NEO.

Example 14 Construction of a fusion protein (55.1 scFv-CFG2) 2 Antibody 55.1, described in U.S. Patent 5,665,357, recognizes the antigen associated with the CA55.1 tumor that is expressed in most colorectal tumors and is only weakly expressed or it is absent in normal colon tissue. The determination of the heavy and light chain cDNA sequence of 35.1 is described in Example 3 of the aforementioned patent. A plasmid expression vector allowing the secretion of the antibody fragment to the periplasm of E. coli using a single pelB leader sequence (pICI266) has been deposited as accession number NCIMB 40589 on October 11, 1993 under the Treaty of Budapest in the National Collections of- Industrial and Marine Bacteria Limited (NCIMB), 23 St. Machar Drive, Aberdeen, AB2 1RY, Scotland, U.K. This vector was modified as described in example 3.3a of the US Pat. No. 5,665,357 to create the plasmid pICI1646; this plasmid was used for the cloning of several 55.1 antibody fragments as described in the additional subsections of Example 3, in which the production of a scFv 55.1 construct that was designated as pICI1657 is included. Plasmid pICI1657 (otherwise known as pICI-55.1 scFv) was used as a starting point for the construction of the fusion protein (55.1 scFv-CPG2) 2. The scFv 55.1 gene was amplified using the oligonucleotides CME 3270 and CME 3272 (SEQ ID NOS 27 and 28 respectively) and the plasmid pICI1657 with the template DNA. The resulting PCR product band of approximately 790 b.p. It was purified. Similarly, the plasmid pNG4 / A5B7 ¥ H-IgG2CH1 / CPG2 R6 described in the example was used as the template DNA in a standard PCR reaction to amplify the CPG2 gene using the CME oligonucleotide primers 3274 and CME 3275 (SEQ ID NOS 29 and 30 respectively). The expected PCR product band of approximately 1200 b.p. It was purified. An original PCR reaction was carried out to join (or splice) two purified PCR reaction products together. Standard PCR reaction conditions were used using variable quantities (from 0.5 to 2 μl.) Of each PCR product but using 25 cycles (instead of usual cycles) with the oligonucleotides CME 3270 and CME 3275 (SEQ ID NOS: 27 &30). A reaction product of the expected size (approximately 200Q b.p.). it was excised, purified and diluted in 20 μl H20, subjected to dilution using the EcoRI restriction enzyme and purified. The vector pNG4 / VHss806.077VH-IgG2CH1 / CPG2 was prepared to receive the above PCR product by digestion with. the EcoRI restriction enzyme, dephosphorylated, the band of the larger vector was separated from the smaller and purified fragment. The similarly restricted PCR product was ligated to the prepared vector and the ligation mixture was transformed to E. coli. The DNA was prepared from the clones obtained and analyzed by restriction digestion of HindIII / NotI to inspect the correct orientation of the fragment and the appropriate clones subjected to sequence subsequently to confirm the genetic fusion sequence. A variety of the clones with the correct sequence were obtained and one of these clones is given the designation of the plasmid pNG4 / 55. lscFv / CPG2 R6. The DNA and amino acid sequence of the fusion protein are shown in SEQ ID NOS: 31 and 32.

Example 15 Modification of plasmid pNG4 / 55. lscFv / CPG2 Rβ to facilitate the exchange of the scFv gene During the construction of pNG4 / 55. lscFv / CPG2 R6 a single BspEI (AccIII isosquisomer) was introduced into the coding sequence of the flexible linker (G4S) 3, located between the antibody and the CPG2 genes. To facilitate the cloning of the alternative scFv constructs the 3 'EcoRI site of the CPG2 gene in the plasmid pNG4 / 55. 1scFv / CPG2 R6 was canceled in order to allow the insertion of alternative antibody genes in the table, both behind the plasmid signal sequence and 5 'of the CPG2 gene via a cloning of the EcoRI / BpsEI fragment. This modification was obtained by PCR mutagenesis in which first the plasmid pNG4 / 55.1scFv / COPG2 R6 was amplified using the oligonucleotides CME 3903 and CME 3906 (SEQ ID NOS: 33 and 34 respectively). Second, the plasmid pNG4 / 55. lscFv / CPG2 R6 was again amplified but using the oligonucleotides CME 4040 and CME 3905 (SEQ ID NOS: 35 and 36 respectively). The first band of the expected PCR product of approximately 420 b.p. It was purified. The second ECR reaction was similarly treated and the expected PCR product band of approximately 450 b .p. purified.

An additional PCR reaction was carried out to join (or splice) the two PCR products purified together. Standard PCR reaction conditions were used using varying amounts (between 0.5 and 2 μl) of each PCR product but using between 15 and 25 cycles with the oligonucleotides CME 39G5 7 CME 3906 (SEQ ID NOS: 36 and 34). A reaction product of the expected size (approximately 840 bp.) Was purified, digested using the restriction enzymes Notl and Xabl and the expected fragment band of approximately 469 bp was purified.The plasmid pNG4 / 55.1scFv / CPG2 The original R6 was prepared to receive the above PCR product by digestion with the restriction enzymes of Notl and Xbal, dephosphorylated and the band of the longest vector separated from the smaller fragment.The vector band was purified and similarly the PCR product restricted similarly it was ligated to the separated vector and the ligation sample was transformed to E. coli DNA was prepared from the obtained clones and analyzed by restriction digestion of EcoRI to inspect the insertion of the modified fragment and the appropriate clones were subjected to sequences subsequently to confirm the sequence change. A variety of clones with the correct sequence were obtained and one of these clones is given the designation of plasmid pNG3 / 55.lscFv / CPG2 R6 / EcoRI. This mutation removes the EcoRI site consisting of the 3f site of the CPG gene "and simultaneously introduces an additional restriction codon.The DNA sequence of the fusion protein gene up to and including the two restriction codons shown in FIG. SEQ ID NO: 37 Example 16 Construction of an 806.077 scFv antibody gene ScFv 806.077 was created using the vectors pNG4 / VHss806.077VH-IgG2CH1 'and pNH3 / VKss806.077VK-HuCK-NEO which are the variable reaction sources of VH and VK of 806.077. The VH gene of 806.077 was amplified from the plasmid pNG4 / VHss806.077VH-IgG2CH1 'using standard PCR conditions with the oligonucleotides CME 3260 and CME 3266 (SEQ ID NOS: 39 and 40 respectively). The VK of 806.077 was amplified from the plasmid pNG3 / VKss806.077VK-HuCK-NEO using the oligonucleotides CME 3262 and CME 3267 (SEQ ID NOS: 41 and 43 respectively). The PCR reaction products of VH and VK were purified. An additional PCR reaction was carried out to join (or splice) the two additional purified PCR reaction products. Standard PCR reaction conditions were used using varying amounts (between 0.5 to 2 μl) of each PCR product but using between 15 and 25 cycles with the flanking oligonucleotides (SEQ ID NOS: 39 and 41 respectively). A reaction product of the expected size (approximately 730 b.p.) was purified, subjected to digestion, using the restriction enzymes Noc1 and Xhol and an expected fragment band of approximately 720 b.p. It was purified. The plasmid pICII657 (otherwise known as PICI-55.1 scFv) had been further modified by inserting a double-stranded DNA cassette produced from the two oligonucleotides CME 3143 and CME 3145 (SEQ ID NOS: 45 and 46) between the existing restriction sites Xhol and EcoR using standard cloning techniques to create the vector pICI266-55.1 scFv tag / his (the DNA sequence of the tag / his gene of scFv 55.1 is shown in SEQ ID NOS: 47). This vector was prepared to receive the above PCR product by digestion with the restriction enzymes Ncol and Xhol, dephosphorylated and the band of the largest vector separated from the smallest fragment. The vector band was purified and subsequently the similarly restricted PCR product was ligated to the vector prepared in the transformed ligation mixture to E. coli. The DNA was prepared from the clones obtained and analyzed by EcoRI restriction digestion to inspect the insertion of the modified fragment and the appropriate clones were subsequently subjected to frequency to confirm the sequence change. A variety of the clones with the correct sequence were obtained and one of these clones is given the designation of the plasmid pICI266 / 806IscFvtag / his (alternatively known as pICI266-80d H / VLscFv / his). The DNA and protein sequence of the scFv / his 806S gene are shown in (SEQ ID NOS: 25 and 26).

Example 17 Construction of a fusion protein (806,077 scFv-CPG2) 2 Plasmid pICI266 / 806IscFvtag / his was used as the source for 806scFv. The gene was amplified using the oligonucleotides CME 3907 and CME 3908 (SEQ ID NOS: 48 and 49) and one band of the expected size was purified. This fragment was then digested using the restriction enzymes EcoRI and BspEI after which an expected fragment band of approximately 760 b.p. The purified plasmid pNG4 / 55. lscFv / CPG2 R6 / EcoRI was prepared to receive the above fragment by digestion with the EcoRI and BspEI, dephosphorylated restriction enzymes and the band of the larger vector separated from the smaller fragment. The vector band was purified and subsequently the similarly restricted fragment was ligated to the prepared vector and the ligation sample was transformed to E. coli DNA was prepared from the obtained clones and analyzed by EcoRI restriction digestion to inspect the insertion of the modified fragment The appropriate clones were subsequently sequenced to confirm the genetic sequence A variety of the clones with the correct sequence were obtained and one of these clones is given the designation of the pNG4 / 806IscFv / CPG2 R6 / EcoRI plasmid. DNA and protein sequence of the 806 fusion protein gene. 077scFv / CPG2 R6 are shown in (SEQ. ID NOS: 50 and 51).

Example 18 Co-transfection, transient expression of the antibody-CPG2 fusion proteins As described in the If example, the plasmids encoding other fusion protein variants can be transfected using the standard conditions given in order to obtain the expression transient of its coded fusion protein of COS7 cells. In the case of fusion proteins of (Fab-CPG2) 2, the co-transfection of appropriate plasmids or transfection of fusion proteins can be carried out. Similary, the individual expression plasmids of fusion proteins (scFv-CPG2) 2 can also be transfected by the same protocol. In each case a maximum total of 4 mg of DNA is used in an individual transfection.

Example 19 Genetic switches for protein expression As described in the example. Ij, the use of genetically controlled but strongly inducible switch systems such as "TET on" or "TET off" (Grossen, M. Et al (1995) Science 268: 1766-179) or ecdysone / uristerone A (No, D. et al (1996) PNAS 93: 3346-3351) can be used for the expression of fusion proteins. Appropriate methodology and cloning strategies as described in Example 5 can be used for antibody Fab-enzyme fusions that require an IRES sequence for expression. The insertion of the appropriate gene cassette into switchable expression vectors can be used if the fusion protein product is a single polypeptide chain such as the scFv-enzyme constructs.

Example 20 Determination of the properties of antibody-enzyme binding proteins secreted from the COS7 cell The material supernatant of COS7 cells can be analyzed for the presence of antibody fusion proteins as described in the example » you. Similatly, the use of expressed fusion protein and CPG2 prodrug and an in vitro cytotoxicity assay can be carried out as previously described in example lh- The analysis of HTLP-based enzymatic activity can show that the enzymatic activity of CPG2 is present in the supernatant of the cells and anti-CEA ELISA analysis can be detected with an anti-CPG2 reporter antibody to confirm the binding or binding of the protein to commensurate levels with a bivalent antibody molecule A5B7 and also to demonstrate that the antibody-CPG2 fusion protein (not only the antibody component) is linked to CEA. Western blot analysis with reporter antibody assays clearly exhibits a fusion protein subunit of the expected size. Since it is known that CPG2 only exhibits enzymatic activity when it is in a di eric state and since only the antibody enzyme fusion protein is present, this indicates that the fusion protein (see under the conditions of SDS / PAGE) is dimerized via the natural CPG2 dimerization mechanism to form a dimeric anti-enzyme fusion protein molecule under "original" regulated pH conditions. In addition this molecule exhibits CPG2 enzymatic activity and CEA antigen binding properties that do not appear to be significantly different in the fusion protein compared to the enzyme or antibody alone. The results obtained from the cytotoxicity analysis can demonstrate that the antibody-enzyme fusion protein (together with the prodrug) causes at least equivalent cell killing and results in a lower number of cells at the end of the period of analysis of the levels equivalents of the conjugate of A5B7 F (ab-) 2-CPG2 (with the same prodrug). Since cell killing (at levels greater than the basal control levels), it can occur only if the prodrug is converted to the active drug by the CPG2 enzyme (and since the cells are washed to separate the unbound protein, only the enzyme bound to the cell will persist in the stage where the prodrug is added.) Thus, this experiment can show that at least as much of the fusion protein (A5B7-CPG2 R6) 2 remains bound compared to the conjugate of A5B7 F (ab) 2-CPG2 since a greater degree of cellular extermination occurs (presumably due to the higher conversion of the medication to medication).

EXAMPLE 21 In Vitro and In Vivo Determination of Expressed Fusion Enzyme-antibody Properties of Recombinant Tumor Cells The construction of tumor cell lines expressing fusion protein can be carried out as described in example 4.

Retention of the fusion protein on the cell surface of recombinant LoVo tumor cells expressing the antibody-enzyme fusion protein can be shown using the techniques described in example 7. The selective killing of LoVo tumor cells cultured Transfected with an antibody-CPG2 fusion protein gene by a prodrug that is converted by the enzyme to an active drug can be demonstrated as described in example 8. The establishment of LoVo tumor xenografts that express the antibody-protein Enzyme fusion in athymic mice can be carried out as described in example 9. The determination of the activity of the enzyme in tumor xenograft samples can also be determined as described in example 10. The determination of the Enzymatic activity in plasma samples is carried out as described in example 11. The anti-tum activity of the PGP averaging in LoVo tumors expressing the antibody-CPG2 fusion protein can be evaluated using the method described in example 12. The results of these experiments can be used to demonstrate that the antibody - CPG2 fusion protein secreted from CEA positive tumor cell lines are bound to the cell surface (via CEA) while the same protein expressed from CEA-negative tumors shows no such binding. These results can demonstrate that the transfected cells expressing the antibody-CPG2 fusion protein can convert the PGP prodrug to the most potent active drug while the LoVo not transfected are unable to convert the promedicamento. Consequently, the transfected LoVo cells will be more than 100 times more sensitive to PGP promedication in terms of cell death compared to untransfected LoVo cells, thus demonstrating that the transfection of tumor cells with a gene for a protein antibody-enzyme fusion can lead to selective killing of tumor cells with a prodrug. Administration of PGP to established LoVo tumors of recombinant LoVo cells or mixtures of parental LoVo recombinant / LoVo cells may result in a significant anti-tumor effect, as determined by PGP-treated tumors that decrease in size compared to tumors treated only with the pSS regulatory solution and that take only significantly longer for tumors treated with PGP to reach 4 times its initial tumor volume, compared to tumors treated with the pH-regulating formulation. In contrast, administration of PGP to established LoVo tumors from non-transfected cells would not result in any significant antitumor activity. Similar studies can be used to demonstrate that the antibody-enzyme gene delivered to an appropriate gene vector to the established LoVo tumors produced from untransfected parental LoVo cells when used in combination with the PGP drug can result in a significant anti-tumor activity. Thus, non-transfected LoVo cells are injected to athymic nude mice (1 X 107 tumor cells per mouse) and once the tumors are 5-7 m in diameter the vector containing the antibody fusion protein gene- enzyme is injected intra-tumorally. After 1-7 days to allow the antibody-enzyme fusion protein to be expressed by and bound to the LoVo tumor cells, the PGP prodrug is administered as previously described. This results in a significant anti-tumor activity as compared to the control or reference mice receiving the pH-regulating formulation in. place of the prompting of PGP.

Example 22 Preparation of fusion protein (murine A5B7 Fab-CPG2) 2 (Murine Fab-CPG2 A5B7) 2 is expressed from the COS-7 and CHO cells essentially as described in part (d) of example 48 of International patent application WO 97/42329 (Zeneca Limited, published on November 13, 1997) by cloning the genes for the light chain of A5B7 and Fd A5B7 linked in their C terminus via a flexible peptide (G4S) 3 linker to CPG2 in the co-expression vector pEE14. The murine A5B7 light chain is isolated from pAF8 (described in part g of the refer of example 5 of the international patent application WO 96/20011 Zeneca Limited). Plasmid pAF8 is cut with EcoRI and the resulting 732 bp fragment is isolated by electrophoresis on a 1% agarose gel. This fragment is cloned into plasmid pEE14 (described by Bebbington in METHODS: A Campanion to Methods in Enzymology (1991) 2,136-145) if larmente cut by EcoRI and the resulting plasmid is used to transform the strain E. coli DH5a. The transformed cells are deposited on L agar plus ampicillin (100 ag / ml). A clone containing a plasmid with the correct sequ and orientation is confirmed by DNA sequ analysis (SEQ ID NO: 57) and the plasmid is referred to as pEE14 / A5B7muVkmuCK. The amino acid sequ of the purified signal sequ (amino acid residues 1 to 22) and the murine light chain (amino acid residues 23 to 235) is shown in SEQ ID NO: 58. The murine fd CPG2 gene is prepared of the var6ent R6 of the CPG2 gene (d of ej 1) and the fd sequ A5B7 of murine in pAFl (described in part d of example 5 of refer in the international patent application WO) 96/20011, Zeneca Limited). A PCR reaction with the oligonucleotides SEQ ID NOS: 53 and 54 in pAF1 provides a fragment of 247 bp. This is cut with HindIII and Ba Hl and cloned to the pUC19 cut similarly. The resulting plasmid is used to transform E. coli strain DH5'a. The transformed cells are deposited on L agar plus ampicillin (100 μg / ml). A clone containing a plasmid with the correct sequ is designated pUC19 / muCH1 / NnoI-AccIII (fd). A second PCR with oligonucleotides SEQ ID NOS: ' 55 and 56 on pNG / VKss / CPG2 / R6-neo (example 1) provides a 265 bp fragment that is cut with HindIII and Eco RI and cloned into pUC19 cut similarly as above to give the plasmid pUC19 / muCH1-linker-CPG2 / AccIII-SacII. The plasmid is cut with pUC19 / muCH1 / NcoI-AccIII (Fd) and HindIII and AccIII the 258 bp fragment isolated by electrophoresis on a 1% agarose gel. This fragment is cloned into pUC19 / muCH1-linker-CPG2 / AccIII-SacIII cut with HidlII and AccIII to give the plasmid pUC19 / muCH1-linker-CPG2 / NcoI-Scall. A fragment of 956 bp is isolated pNG / VKss / CEG2 / R6-neo when cut with SacII and EcoRI. This is cloned into püC19 / muCH1-linker-CPG2 / NcoI-SacII cut with SacII and EcoRI to give plasmid püC19 / muCH1-linker-RC7CPG2 (R6). The construct Complete gene is prepared by isolating a HindIII to Ncol fragment of 498 bp to pAF1 and cloned into pUC19 / muCH1-linker-RC / CPG2 (R6) cut with HindIII and Ncol. The resulting plasmid is used to transform the E. coli strain DH5a. The transformed cells are deposited on L agar and more ampicillin (100 μg / ml). A clone containing a plasmid with the correct sequ and orientation is confirmed by DNA sequ analysis (SEQ ID NO: 59) and the plasmid is designated pUC19 / muA5B7-RC / CPG2 (R6). The amino acid sequ of the ded signal sequ (amino acid residue 1 to 19) and the murine Fd-linker-CPG2 (amino acid residues 20 to 647) is shown in SE IB NO: 60. ' Alternatively the genetic sequ of CPG2 described in example 1 can be obtained by total gene synthesis and converted to the variant R6 as described in d of example 1. In this case, the basic residue C at position 933 in SEQ ID NO: 59 is changed to G. the amino acid sequence of the SEQ ID NO: 60 remains without alteration. For expression in the vector pEE14 the gene is first cloned into pEE6 (this is a derivative of pEEd.hCMV -Stephens and Cockett, 1989, Nucleic Acids Research 17.7110, in which a HindIII site upstream of the promoter of hCMV has been converted to a BglII site). Plasmid pUC19 / muA5B7-RC / CPG2 (R6) is cut with HindIII and EcoRI and the 1974 bp fragment isolated by electroporosis on a 1% agar gel. This is cloned to pEE6 cut with HindIII and EcoRI strain E. coli DH5μ. to give the plasmid pEE6 / muA5B7-RC / CPG2 (R6). The pEE14 co-expression vector is made by first contracting the plasmid pEE6 / muA5B7-RC / CPG2 (R6) with BglII and Ba Hl and isolating the 4320 bp fragment on a 1% agarose gel. This fragment is cloned into pEEl4 / A5B7muVkmuCK cut with BglII and BamHI. The resulting plasmid is used to transform the E. coli strain DH5a.

The transformed cells are deposited on L agar plus ampicillin (100 μg / ml). A clone containing a plasmid with the correct sequence and orientation is confirmed by DNA sequence analysis and the plasmid is designated pEE14 / muA5B7-RC / CPG2 (R6). For the expression of (murine A5B7 Fab-CPG2.) 2, the plasmid pEE14 / muA5B7-RC7CPG2 (R6) is used to transfect the C0S7 or CHO cells as described in example 48 of the international patent application WO 97 / 42329, Zeneca Limited, published on November 13, 1997. The supernatants of COS cells and the CHO clone supernatants are analyzed in their activity as described in example 1 and demonstrate that they have CEA binding and enzyme activity. of CPG2.

Example 23: Armaceutical composition The following illustrates one. Representative pharmaceutical dosage form containing a gene construct of the invention that can be used for therapy in combination with an appropriate prodrug. A sterile aqueous solution, for injection either parenterally or directly to the tumor tissue containing 10-10 adenovirus particles containing a gene construct as described in example 1. After 3-7 days, three doses of 1 g of promedicamento are administered as sterile solutions at hourly intervals. The prodrug is selected from N- (4- [N, N-bis (-iodoethyl) amino] -phenoxycarbonyl) -L-glutamic acids, N- (4- [N, N-bis (2-chloroethyl) amino]. .-phenoxycarbonyl) -L-glutamic-gampta- (3, 5-dicarboxy) anuide or N- (4- [N, N-bis (2-chloroethyl) amino] phenoxycarbonyl] -L-glutamic acid or a salt pharmaceutically acceptable thereof.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Zeneca Limited (B) STREET: 15 Stanhope Gate (C) CITY: London (D) STATE: England (E) COUNTRY: United Kingdom of the Great Britain (F) ZIP CODE (ZIP): W1Y 6LN (G) TELEPHONE: 0171 304 5000 (H) TELEFAX: 0171 304 5151 (I) TELEX: 0171 304 2042 (ii) TITLE OF THE INVENTION: CHEMICAL COMPOUNDS (iii) SEQUENCE NUMBER: 60 (iv) FORMS THAT CAN BE READ BY COMPUTER (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMMING ELEMENTS: Patentin # 1.0, version # 1.30 (EPO) (vi) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBERS: GB 9709421.3 (B) SUBMISSION DATE: May 10, 1997 (2) INFORMATION FOR SEQ ID NOS: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other. Nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1 GGGAATTCCT CGAGGAGCTC C 21 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 CCGGGGAGCT CCTCGAGGAA TTCCCGC 27 (2) INFORMATION FOR SEQ ID MO: 3 .: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 CAGAAGCGCG ACAACGTG 18 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 CGAGGCCTTG CCGGTGATCT GGACCTGCAC G? AGGCGAT 39 (2) INFORMATION FOR SEQ ID NO: 5-: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5 GGGGATGATG TTCGAGACCT GGCCGGCCTT GGCGATGGTC CACTGGAAGC GCAGGTTCTT 60-CGC 63 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6 CTTGCCGGCG CCCAGATC 18 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 GTCTCGAACA TCATCCCC 18 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8 ATCACCGGCA AGGCCTCG 18 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1236 base pairs (B) TYPE: nucleic acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9 ATGGATTTTC AAGTGCAGAT TTTCAGCTTC CTGCTAATCA GTGCTTCAGT CATAATGTCC 60 CGCGGGCAGA AGCGCGACAA CGTGCTGTTC CAGGCAGCTA CCGACGAGC & GCCGGCCGTG 120 ATCAAGACGC TGGAGAAGCT GGTCAACATC GAGACCGGCA CCGGTGACGC CGAGGGCATC 180 GCCGCTGCGG GCAACTTCCT CGAGGCCGAG CTCAAGAACC TCGGCTTCAC GGTCACGCGA 240 AGCAAGTCGG CCGGCCTGGT GGTGGGCGAC AACATCGTGG GCAAGATCAA GGGCCGCGGC 300 GGCAAGAACC TGCTGCTGAT GTCGCACATG GACACCGTCT ACCTCAAGGG CATTCTCGCG 360 AAGGCCCCGT TCCGCGTCGA AGGCGACAAG GCCTACGGCC CGGGCATCGC. CGACGACAAG 420 GGCGGCAACG CGGTCATCCT GCACACGCTC AAGCTGCTGA AGGAATACGG CGTGCGCGAC 480 TACGGCACCA TCACCGTGCT GTTCAACACC GACGAGGAAA AGGGTTCCTT CGGCTCGCGC 540 GACCTGATCC AGGAAGAAGC CAAGCTGGCC GACTACGTGC TCTCCTTCGA GCCCACCAGC 600 GCAGGCGACG AAAAACTCTC GCTGGGCACC TCGGGCATCG CCTACGTGCA GGTCCAGATC 660 ACCGGCAAGG CCTCGCATGC CGGCGCCGCG CCCGAGCTGG GCGTGñACGC GCTGSTCGAG 720 GCTTCCGACC TCGTGCTGCG CACGATGAAC ATCGACGACA AGGCGAAGAA.CCTGCGCTTC 780 CAGTGGACCA TCGCCAAGGC CGGCCAGGTC TCGAACATCA TCCCCGCCAG CGCC &CGCTG 840 AACGCCGACG TGCGCTACGC GCGCAACGAG GACTTCGACG CCGCCATGAA GACGC? SGAA 900 GAGCGCGCGC AGCAGAAGAA GCTGCCCGAG GCCGACGTGA AGGTGATCGT C & CGCSCGGC 960 CGCCCGGCCT TCAATGCCGG CGAAGGCGGC AAGAAGCTGG TCGACAAGGC GGTGGCCTAC 1020 TACAAGGAAG CCGGCGGCAC GCTGGGCGTG GAAGAGCGCA CCGGCGGCGG CACCGACGCG 1080 GCCTACGCCG CGCTCTCAGG CAAGCCAGTG ATCGAGAGCC TGGGCCTGCe GGGCSTCGGC 1140 TACCACAGCG ACAAGGCCGA GTACGTGGAC ATCAGCGCGñ TTCCGCGCCG CCTGTACATG 1200 GCTGCGCGCC TGATCATGGA TCTGGGCGCC GGCAAG '1236 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 412 amino acids (B) TYPE: amino acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10 Met Asp Phe Gln Val Gln lie Phe Ser Phe Leu Leu lie Wing Being 1 5 10 15 Val lie Met Being Arg Gly Gln Lys Arg Asp Asn Val Leu Sha Gln Wing 20. 25 30 Wing Thr Asp Glu Gln Pro Wing Val lie Lys Thr Leu Glu Lys Leu Val 35 40 45 Asn He Glu Thr Gly Thr Gly Asp Wing Glu Gly lie Wing Wing Wing Gly 50 55 - 60 Asn Phe Leu Glu Wing Glu Leu Lys Asn Leu Gly Phe T r V «l Thr Arg 65 70 75 80 Ser Lys Ser Wing Gly Leu Val Val Gly Asp Asi * lie Val Gly Lys He 85 90 95 Lys Gly Arg Gly Gly Lys Asn Leu Leu Met Ser Met His Met Asp Thr 100 105 110 Val Tyr Leu Lys Gly He Leu Wing Lys Wing Pro Phe Arg Val Slu Gly 115 120 125"Asp Lys Wing Tyr Gly Pro Gly He Wing Asp Asp Lys Gly Gly Asn Wing 130 135 140 Val He Leu His Thr Leu Lys Leu Leu Lys Glu Tyr Gly Val Arg Asp 145 150 155 160 Tyr Gly Thr He Thr Val Leu Phe Asn Thr Asp Glu Glu Lys Gly Ser 165 170 175 Phe Gly Ser Arg Asp Leu He Gln Glu Glu Wing Lys Leu Wing Asp Tyr 180 185 190 Val Leu Ser Phe Glu Pro Titr Ser Wing Gly Asp Glu Lys Leu Ser Leu 195 200 205 Gly Thr Ser Gly He Ala Tyr Val Gln Val. Gln He Thr Gly Lys Ala 210 215 220 Ser His Wing Gly Ala Wing Pro Glu Leu Gly Val Asn Wing Leu Val Glu 225 230 235 240 Wing Being Asp Leu Val Leu Arg Thr Met As As Asp Asp Lys Wing Lys 245 '250 255 Asn Leu Arg Phe Gln Trp Thr He Wing Lys Wing Wing Gln Gl Val Val Asn 260 265 270 He He Pro Wing Wing Wing Thr Leu Asn Wing Asp Val Arg Tyr Ala Arg 275 280 285 Asn Glu Asp Phe Asp Wing Wing Met Lys Thr Leu Glu Glu Arg Wing Gln 290 295 300 Gln Lys Lys Leu Pro Glu Wing Asp Val Lys Val He Val Thr Arg Gly 305 310 315 320 Arg Pro Wing Phe Asn Wing Gly Glu Gly Gly Lys Lys Leu Val Asp Lys 325 330 335 Wing Val Wing Tyr Tyr Lys Glu Wing Gly Gly Thr Leu Gly Val Glu Glu 340 345 350 Arg Thr Gly Gly Gly Thr Asp wing Wing Tyr Wing Ala Leu Se_rr Gly Lys 355 360 365 Pro Val He Glu Ser Leu Gly Leu Pro Gly Phe Gly Tyr His Ser Asp 370 375 380 Lys Ala Glu Tyr Val Asp lie Be Ala He Pro Arg Arg Leu Tyr Met 385 390 395 400 Ala Ala Arg Leu He Met Asp Leu Gly Ala Gly Lys 405 410 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11 CCACTCTCAC AGTGAGCTCG G 21 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12 ACCGCTACCG CCACCACCAG AGCCACCACC GCC &CTGTC TTGTCCACCT TGGTG 55 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13 ACCCCCTCTA GAGTCGAC 18 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14 TCTGGTGGTG GCGGTAGCGG TGGCGGGGGT TCCCAGAAGC GCGACAACGT GCTG 54 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1929 base pairs (B) TYPE: nucleic acid (C) STRING: individual, (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO-: 15 ATGGAGTTGT GGCTGAACTG GATTTTCCTT GTAACACTTT TAAATGGTAT CCAGTGTGAG 60 GTGAAGCTGG TGGAGTCTGG AGGAGGCTTG GTACAGCCTG GGGGTTCTCT GAGACTCTCC 120 TGTGCAACTT CTGGGTTCAC CTTCACTGAT TAC? ACATGA ACTGGGTCCG CCAGCCTCCA 180 GGAAAGGCAC TTGAGTGGTT GGGTTTTATT GGAAACAAAG CTAATGGTTA CACAACAGAG 240 TACAGTGCAT CTGTGAAGGG TCGGTTCACC ATCTCCAGAG ATAAATCCCA AAGCATCCTC 300 TATCTTCAAA TGAACACCCT GAGAGCTGAG GACAGTGCCA CTTATTACTG TACAAGAGAT 360 AGGGGGCTAC GGTTCTACTT TGACTACTGG GGCCAAGGCA CCACTCTCAC AGTGAGCTCG 420 GCTAGCACCA AGGGACCATC GGTCTTCCCC CTGGCCCCCT GCTCCAGGAG CACCTCCGAG 480 AGCACAGCCG CCCTGGGCTG CCTGGTCAAG GñCTACTTCC CCGAACCGGT GACGGTGTCG 540 TGGAACTCAG GCGCTCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA 600 GGACTCTACT CCCTCAGCAG CGTCGTGACG G GCCCTCCA GCAACTTCGG CACCCSG CC 660 TACACCTGCA ACGTAGATCA CAAGCCCAGC AACACCAAGG TGGACAAGAC AGTTGGCGGT 720 GGTGGCTCTG GTGGTGGCGG TAGCGGTGGC GGGGGTTCCC AGññGCGCGA CñACG? GCTG 780 TTCCAGGCAG CTACCGACGA GCAGCCGGCC GTGATCAAGA CGCTGGAGAA GCTGGTCAAC 840 ATCGAGACCG GCACCGGTGA CGCCGAGGGC ATCGCCGCTG CCGECAACTT CCTCG &GGCC 900 GAGCTCAAGA ACCTCGGCTT CACGGTCACG CGAAGCAAGT CGGCCGGCCT GGTGGTGGGC 960 GACAACATCG TGGGCAAGAT CAAGGGCCGC GGCGGCAAGA ACCTGCTGCT GATGTCGCAC 1020 ATGGACACCG TCTACCTCAA GGGCATTCTC GCGAAGGCCC CGSTCCGCGT CGAAGGCGñC 1080 AAGGCCTACG GCCCGGGCAT CGCCGACGAC ñ & GGGCGGCA ACGCGGTCAT CCTGCACaCG 1140 CTCAAGCTGC TGAAGGAATA CGGCGTGCGC GñCTACGGCA CCATCACCGT GCTGTTCñAC 1200 ACCGACGAGG AAAAGGGTTC CTTCGGCTCG CGCGACCTGA TCCSGGAAGA AGCCAAGCTG 1260 GCCGACTACG TGCTCTCCTT CGAGCCCACC AGCGCAGGCG ACGAññAACT CTCGCTGGGC 1320 ACCTCGGGCA TCGCCTACGT GCAGGTCCAG ATCACCGGCS. SGGCOTCGCa TGCCGGCSCC 1380 GCGCCCGAGC TGGGCGTGAA CGCGCTGGTC GAGGCTTCCG ACCTCGTGCT GCGCACG &TG 1440 AACATCGACG ACAAGGCGAA GAACCTGCGC TTCCAGTGGA CCATCGCCAA GGCCGGCCAG 1500 GTCTCGAACA TCATCCCCGC CAGCGCCACG CTGAACGCCG ACGTGCGCTA CGCGCGCSAC 1560 GAGGACTTCG ACGCCGCCAT GAAGACGCTG GññGAGCGCG CGCAGCAGAA GAAGCTGCCC 1620 GAGGCCGACG TGAAGGTGAT CGTCACGCGC GGCCGCCCGG CCTTCAATGC CGGCGAAGGC 1680 GGCAAGAAGC TGGTCGACAA GGCGGTGGCC TACTACAAGG AAGCCGGCGG CACGCTGGGC 1740 GTGGAAGAGC GCACCGGCGG CGGCACCGAC GCGGCCTACG CCGCGCTCTC AGGCñAGCCA 1800 GTGATCGAGA GCCTGGGCCT GCCGGGCTTC GGCTACCACA GCGACAAGGC CGAGTACGTG 1860 GACATCAGCG CGATTCCGCG CCGCCTGTAC ATGGCTGCGC GCCTGATCAT GGATCTGGGC 1920 GCCGGCAAG 1929 (2) INFORMATION FOR SEQ ID O: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 16 Met Glu Leu Trp Leu Asn Trp He Phe Leu Val Thr Leu Leu Asn Gly 1 5 10 15 He Gln Cys Glu Val Lys Leu-Val Glu Ser Gly Gly Gly Leu Vsl Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leut Ser Cys Wing Thr Ser Gly Phe Tfer Phe 35 40 45 Thr Asp Tyr Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala. Leu 50 55 60 Glu Trp Leu Gly Phe He Gly Asn Lys Wing Asn Gly Tyr Thr Bar Glu 65 70 75 80 Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Lys Ser 85 90 95 Gln Ser He Leu Tyr Leu Tour Kfet Asn Thr Leu Arg Wing Glu Asp Ser 100 105 110 Wing Thr Tyr Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Pile Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Wing Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Pro Pro Cys Ser Arg Ser Tbr Ser Glu 145 150 155 160 Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Pne Pro Ma Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn 210 215 220 Val Asp His Lys Pro Ser Asa Thr Lys Val Asp Lys Thr Val Gly Gly 225 230 235 24Q Gly Gly Be Gly Gly Gly Gly Be Gly Gly Gly Gly Ser Gln Lys Arg 245 250 255 Asp Asn Val Leu Phe Gln Wing Wing Thr Asp Glu Without Pro Wing Val lie 260 265 270 Lys Thr Leu Glu Lys Leu Val Asn He Glu Thr Sly Thr Gly Asp Ala 275 280 285 Glu Gly He Ala Ala Ala Gly Asn Phe Leu Glu Ala Glu Leu Lys Asn 290 295 3SQ Leu Gly Phe Thr Val Thr Arg Ser Lys Ser Wing Gly Leu Val Val Gly 305 310 315 _ 320 Asp Asn He Val Gly Lys He Lys Gly Arg Gly Gly Lys Asn eu Leu 325 330 335 Leu Met Ser Met Met Asp Thr Val Tyr Leu Lys Gly He Leu Ala Lys 340 345 350 Wing Pro Phe Arg Val Glu Gly Asp Lys Wing Tyr Gly Pro Gly He Wing 355 360 365 Asp Asp Lys Gly Gly Asn Wing Val He Leu His Thr Leu Lys Leu Leu 370 375 380 Lys Glu Tyr Gly Val Arg Asp Tyr Gly Thr He I & Val Leu Phe Asit 385 390 395 400 Thr Asp Glu Glu Lya Gly Ser. Phe Gly Ser Arg Asp Leu He Gla Glu 405 410 415 Glu Ala Lys Leu Ala Asp Tyr Val Leu Ser Phe Glu Pro Thr Ser Ala 420 425 430 Gly Asp Glu Lys Leu Ser Leu Gly Thr Ser Gly lie Wing Tyr Val Gln 435 440 44S Val Gln He Thr Gly Lys Ala Ser His Wing Gly Ma Wing Pro Glu Leu 450 455 460 Gly Val Asn Ala Leu Val Glu Ala Ser Asp Leu Val Leu Arg Thr Mfet 465 470 475 480 Asn He Asp Asp Lys Wing Lys Asn Leu Arg Phe Gln Trp Thr He Wing 485 490 495 Lys Ala Gly Gln Val Ser Asa He He Pro Wing Ala Wing Thr Leu AS? 500 505 510 Wing Asp Val Arg Tyr Wing Arg Asn Glu Asp Phe Asp Wing Wing Met Lys 515 520 525 Thr Leu Glu Glu Arg Wing Gln Gln Lys Lys Leu Pro Glu Wing Asp Val 530 535 540 Lys Val He Val Thr Arg Gly Arg Pro Ala Phe Asp Ala Gly Glu Gly 545 550 555 560 Gly Lys Lys Leu Val Asp Lys Wing Val Wing Tyr Tyr Lys Glu Wing Gly 565 570 575 Gly Thr Leu Gly Val Glu Glu Arg Thr Gly Gly Gly Thr Asp Wing Wing 580 585 590 Tyr Ala Ala Leu Ser Gly Lys Pro Val He Glu Ser Leu Gly Leu Pro 595 600 60S "Gly Phe Gly Tyr His Ser Asp Lys Ala Glu Tyr Val Asp He Ser Wing 610 615 620 He Pro Arg Arg Leu Tyr Met Wing Wing Arg Leu He Met Asp Leu Gly 625 630 635 640 Wing Gly Lys (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 705 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17 ATGGATTTTC AAGTGCAGAT TTTCAGCTTC CTGCTAATCA GTGCTTCAGT CATAATGTCC 60 AGAGGACAAA CTGTTCTCTC CCAGTCTCCA GCAATCCTGT CTGCATCTCC AGGGGAGAAG 120 GTCACAATGA CTTGCAGGGC CAGCTCAAGT GTAACTTACA TTCACTGGTA CCAGCñGAAG 180 CCAGGTTCCT CCCCCAAATC CTGGATTTAT GCCACATCCA SCCTGGCTTC TGGAGTOCCT 240 GCTCGCTTCA GTGGCAGTGG GTCTGGGACC TCTTACTCTC TCACAATCAG CAGAGTGGAG 300 GCTGAAGATG CTGCCACTTA TTACTGCCAA CATTGGAGTA GTAAACCACC GACGTTCGGT 360 GGAGGCACCA AGCTCGAGAT CAAACGGACT GTGGCTGCAC CATCTGTCTT CATCTTCCCG 420 CCATCTGATG AGCAGTTGAA ATCTGGAACT GCCTCTGTTG TGTGCCTGCT GAATAACTTC 480 TATCCCAGAG AGGCCAAAGT ACAGTGGAAG GTGGATAACG CCCTCCAATC GGGTAACTCC 540 CAGGAGAGTG TCACAGAGCA GGACAGCAAG GACAGCACCT ACAGCCTCAG CAGCACCCTG 600 ACGCTGAGCA AAGCAGACTA CGAGAAACAC AARGTCTACG CCTGCGAAGT CACCCATCAG 660 GGCCTGAGTT CGCCCGTCAC AAAGAGCTTC AACAGGGGAG AGTGT 705 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 235 amino acids (B) TYPE: amino acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 18 Met Asp Phe Gln Val Gln He Phe Ser Phe Leu Leu I Be Wing Being 1 5 10 15 Val He Met Ser Arg Gly Gln Thr Val Leu Ser Gln Ser Pro Wing He 20 25 30 Leu Ser Wing Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 40 45 Ser Ser Val Thr Tyr He His Trp Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Ser Trp He Tyr Wing Thr Ser Asn Leu Wing Ser Gly Val Pro 65 70 75 80 Wing Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He 85 90 95 Ser Arg Val Glu Wing Glu Asp Wing Wing Thr Tyr Tyr Cys Gln His Trp 100 105 110 Ser Ser Lys Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu? Le Lys 115 120 125 Arg Thr Val Ala Wing Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155. 160 Tyr Pro Arg Glu Wing Lys Val Gln Trp Lys Val Asp Asn Wing Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: acid nucleic (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 19 AAGCTTGAAT TCGCCGCCAC TATGGATTTT CAAGTGCAG 39 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20 TTAATTGGAT CCGAGCTCCT ATTAACACTC TCCCCTGTTG AAGC 44 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21 AAGCTTCCGG ATCCCTGCAG CCATGGAGTT GTGGCTGAAC TGGATTTTCC 50 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22 AAGCTTAGTC TAGATTATCA CTTGCCGGCG CCCAGATC 38 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23 CGGGGGATCC AGATCTGAGC TCCTGTAGAC GTCGACATTA ATTCCG 46 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 24 GGAAAATCCA GTTCAGCCAC AACTCCATGG 30 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1926 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25 ATGAAGTTGT GGCTGAACTG GATTTTCCTT GTA2 SCTTT TAAATGGAAT TCAGTGTSAG 60 GTGCAGCTGC AGCAGTCTGG GGCAGAGCTT GTGitGGTCAG GGGCCTCAGT CAAGTTGTCC 120 TGCACAGCTT CTGGCTTCAA CATTAAAGAC AACTATATGC ACTGGGTGAA GCAGAGGCCT 180 GAACAGGGCC TGGAGTGGAT TGCATGGATT GATCCTGAGA ATGGTGATAC TGAATATGCC 240 CCGAAGTTCC GGGGCAAGGC CACTTTGACT GCAGACTCAT CCTCCAACAC AGCCTACCTG 300 CACCTCAGCA GCCTGACATC TGAGGACACT GCCGTCTATT ACTGTCATGT CCTGATCTAT 360 GCTGGTTATT TGGCTATGGA CTACTGGGGT CAAGGñACCT CAGTCGCCGT GAGCTCGGCT 420 AGCACCAAGG GACCATCGGT CTTCCCCCTG GCCCCCTGCT CCAGGAGCAC CTCCGAGAGC 480 ACAGCCGCCC TGGGCTGCCT GGTCAAGGAC TACTTCCCCG AACCGGTGAC GGTGTCGTGG 540 AACTCAGGCG CTCTGACCAG CGGCGTGCAC ACCTTCCCGG CTGTCCTACA GTCCTCAGGA 600 CTCTACTCCC TCAGCAGCGT CGTGACGGTG CCCTCCAGCA ACTTCGGCAC CCAGACCTAC 660 ACCTGCAACG TAGATCACAA GCCCAGCAAC ACCAAGGTGG ACAAGACAGT TGGCGGTGGT 720 GGCTCTGGTG GTGGCGGTAG CGGTGGCGGG GGTTCCCAGA AGCGCGACAA CSTGCTGTTC 780 CAGGCAGCTA CCGACGAGCA GCCGGCCGTG ATGAAGACGC TGGAGAAGCT GGTCAACATC 840 GAGACCGGCA CCGGTGACGC CGAGGGCATC GCCGCTGCGG GCAACTTCCT CGAGGCCGAG 900 CTCAAGAACC TCGGCTTCAC GGTCACGCGA AGCAAGTCGG CCGGCCTGGT GGTGGGCGAC 960 AACATCGTGG GCAAGATCAA GGGCCGCGGC GGCAAGAACC TGCTGCTGAT GTCGCACATG 1020 GACACCGTCT ACCTCAAGGG CATTCTCGCG AAGGCCCCGT TCCGCGTCGA AGGCGACAAG 1080 GCCTACGGCC CGGGCATCGC CGACGACAAG GGCGGCAACG CGGTCATCCT GCACACGCTC 1140 AAGCTGCTGA AGGAATACGG CGTGCGCGAC TACGGCACCA TCACCGTGCT GXTCAACACC 1200 GACGAGGAAA AGGGTTCCTT CGGCTCGCGC GACCSGATCC AGGAAGAAGC C & AGCTGGCC 1260 GACTACGTGC TCTCCTTCGA GCCCACCAGC GCAGGCGACG AAAAACTCTC GCTGGGCACC 1320 TCGGGCATCG CCTACGTGCA GGTCCAGATC ACCGGCAAGG CCTCGCATGC CGGCGCCGCG 1380 CCCGAGCTGG GCGTGAACGC GCTGGTCGAG GCTTCCGACC TCGTGCTGCG CACGATGAAC 1440 ATCGACGACA AGGCGAAGAA CCTGCGCTTC CACTGGACCA TCGCCAAGGC CSGCCAGGTC 1500 TCGAACATCA TCCCCGCCAG CGCCACGCTG AACGCCGACG TGCGCTACGC GCGCAACGAG 1560 GACTTCGACG CCGCCATGAA GACGCTGGAA GAGCGCGCGC AGCAGAAGAA GCTGCCCGAG 1620 GCCGACGTGA AGGTGATCGT CACGCGCGGC CGCCCGGCCT TCAATGCCGG CGAAGGCGGC 1680 AAGAAGCTGG TCGACAAGGC GGTGGCCTAC TACAAGGAAG CCGGCGGCAC GCTGGGCGTG 1740 GAAGAGCGCA CCGGCGGCGG CACCGACGCG GCCTACGCCG CGCTCTCAGG CAAGCCAGTG 1800 ATCGAGAGCC TGGGCCTGCC GGGCTTCGGC TACCACAGCG ACAAGGCCGA GTACGTGGAC 1860 ATCAGCGCGA TTCCGCGCCG CCTGTACATG GCTGCGCGCC TGATCATGGA TCTGGGCGCC 1920 GGCAAG 1926 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 642 amino acids (B) TYPE: amino acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID O: 26 Met Lys Leu Trp Leu Asn Trp He Phe Leu Val Thr Leu Lett Asn Gly 1 5 10 15 He Gln Cys Glu Val Gln Leu Gln Gln Ser Gly Wing Glu Leu Val Arg 20 25 30 Ser Gly Wing Ser Val Lys Leu Ser Cys Thr Wing Ser Gly Pfae Asn He 35 40 45 Lys Asp Asn Tyr Met His Trp Val Lys Gis », Arg Pro Glu Gin Gly Leu 50 55 60 Glu Trp He Wing Trp He Asp Pro Glu Asn Gly Asp Thr Glu Tyr.Ala 65 70 75 80 Pro Lys Phe Arg Gly Lys Wing Thr Leu Thr Wing Asp Being Ser Asn 85 90 9 & Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys His Val Leu He Tyr Wing Gly Tyr Leu Wing Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Ser Val Wing Val Ser Wing Being Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 '155 160 Thr Ala Ala Leu Gly Cys Leu 'Basi Lys Asp Tyr Phe Pro Gln Pio Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His T & Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr < _ys Asn Val 210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gln Lys Arg Asp 245 250 255 Asn Val Leu Phe Gln Ala Alai Thr Asp Glu Gln Pro Ala Val lie Lys 260 265"270 Thr Leu Glu Lys Leu Val Asa He Glu Thr Gly Thr Gly Asp Ala Glu 275 280 285 Gly He Ala Ala Ala Gly Asft Phe Leu Glu Ala Glu Leu Lys Asit Leu 290 29S 300 Gly Phe Thr Val Thr Arg Ser Lys Ser Wing Gly Leu Val Val Gly Asp 305 310 315 320 Asn He Val Gly Lys He Lys Gly Arg Gly Gly Lys Asn Leu Leu Leu 325 330 335 Met Ser His Met Asp Thr Val Tyr Leu Lys Gly He Leu Ala Lys Wing 340 345 350 Pro Phe Arg Val Glu Gly Asp Lys Wing Tyr Gly Pro Gly He Wing Asp 355 360 365 Asp Lys Gly Gly Asn Ala Val He Leu His Thr Leu Lys Leu Leu Lys 370 375 380 Glu Tyr Gly Val Arg Asp Tyr Gly Thr He Thr Val Leu Phe Asn Thr 385 390 395 400 Asp Glu Glu Lys Gly Ser Phe Gly Ser Arg Asp Leu He Gln Glu Glu 405 410 415 Ala Lys Leu Ala Asp Tyr Val Leu Ser Phe Glu Pro Thr Ser Ala Gly 420 425 430 Asp Glu Lys Leu Ser Leu Gly Shr Ser Gly lie Wing Tyr Val Gln Val, 435 440 445 Gln He Thr Gly Lys Ala Ser Sis Ala Gly Ala Ala Pro Glu Leu Gly 450 455 460 Val Asn Ala Leu Val Glu Ala Ser Asp Leu Val Leu Arg. Thr Met Asn 465 470 475 480 He Asp Asp Lys Ala Lys Asn Leu Arg Phe Gln Trp Thr He Ala Lys 485 490 495 Wing Gly Gln Val Ser Asn He He Pro Wing "Be Wing Thr Leu Wing Handle 500 505 510 Asp Val Arg Tyr Wing Arg Asn Glu Asp Phe Asp Wing Wing Met Lys Thr 515 520 525 Leu Glu Glu Arg Wing Gln Gln Lys Lys Leu Pro Glu Wing Asp Val Lys 530 535 540 Val He Val Thr Arg Gly Arg Pro Wing Phe Asn Wing Gly Glu Gly Gly 545 550 555 560 Lys Lys Leu Val Asp Lys Wing Val Wing Tyr Tyr Lys Glu Wing Gly Gly 565 570 575 Thr Leu Gly Val Glu Glu Arg Thr Gly Gly Gly Thr Asp Ala Ala Tyr 580 585 590 Ala Ala Leu Ser Gly Lys Pro Val He Glu Ser Leu Gly Leu Pro Gly 595 600 605 Phe Gly Tyr His Ser Asp Lys Wing Glu Tyr Val Asp He Ser Wing He 610 615 620 Pro Arg Arg Leu Tyr Met Ala Ala Arg Leu He Met Asp Leu Gly Ala (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE : nucleic acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 27 AAGCTTGGAA TTCAGTGTCA GGTCCAACTG CAGCAGCCT 39 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 28 GCTACCGCCA CCTCCGGAGC CACCACCGCC CCGTTTGATC TCGAGCTTGG TGCC 54 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 58 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29 TCCGGAGGTG GCGGTAGCGG TGGCGGGGGT TCCCAGAAGC GCGACAACGT GCTGTTCC 58 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH; 24 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 30 CCTCGAGGAA TTCTTTCACT TGCC 24 ' (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2019 base pairs (B) TYPE: nucleic acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID M5: 31 ATGAAGTTGT GGCTGAACTG GATTTTCCTT GTAACACTTT TAAATGGAAT TCAGTGTCAG 60 GTCCAACTGC AGCAGCCTGG GGCTGAACTG GTGSAGCCTG GGGC? TCAGT GCAGCTGTCC 120 TGCAAGGCTT CTGGCTACAC CTTCACCGGC TACTGGATAC ACTGGGTGAA GCAGAGGCCT 180 GGACAAGGCC TTGAGTGGAT TGGAGAGGTT AATCCTAGTA CCGGTCGTTC TGACTACAAT 240 GAGAAGTTCA AGAACAAGGC CACACTGACT & T_M_AC & CCTCCACCAC AGCCTACATG 300 CAACTCAGCA GCCTGACATC TGAGGACTCT GCGGTCTATT ACTGTGCAAG AGAGAGGGCC 360 TATGGTTACG ACGATGCTAT GGACTACTGG GGCCSAGGGA CCACGGTCAC CGTCTCCTCA 420 GGTGGCGGTG GCTCGGGCGG TGGTGGGTCG GGTGGCGGCG GATCTGACAT TGAGCTCTCA 480 CAGTCTCCAT CCTCCCTGGC TGTGTCAGCA GGAEAGñAGG TCACCATGAG CTGCAAATCC 540 AGTCAGAGTC TCCTCAACAG TAGAACCCGA AAGSACTACT TGGCTTGGTA CCAGCAGAGA 600 CCAGGGCAGT CTCCTAAACT GCTGATCTAT TGGGCATCCA CTAGGACATC TGGGGTCCCT 660 GATCGCTTCA CAGGCAGTGG ATCTGGGACA GATTTCACTC TCACCATCAG CAGTGTGCAG 720 GCTGAAGACC TGGCAATTTA TTACTGCAAG CAATCTTATA CTCTTCGGAC GTTCGGTGGA 780 GGCACCAAGC TCGAGATCAA ACGGGGCGGT GGTGSCTCCG GAGGTGGCGG TAGCGGTGGC 840 GGGGGTTCCC AGAAGCGCGA CAACGTGCTG TTCCSGGCAG CTACCGACGA GCAGCCGGCC 900 GTGATCAAGA CGCTGGAGAA GCTGGTCAAC ATCG5GACCG GCACCGGTGA CGCCGAGGGC 960 ATCGCCGCTG CGGGCAACTT CCTCGAGGCC GSGCTCAAGA ACCTCGGCTT CACGGTCACG 1020 CGAAGCAAGT CGGCCGGCCT GGTGGTGGGC GACAACATCG TGGGCAAGAT CAAGGGCCGC 1080 GGCGGCAAGA ACCTGCTGCT GATGTCGCAC ATGESCSCCG TCTACCTCAA GGGCATTCTC 1140 GCGAAGGCCC CGTTCCGCGT CGAAGGCGAC AAGGCCTACG GCCCGGGCAT CGCCGACGAC 1200 AAGGGCGGCA ACGCGGTCAT CCTGCACACG CTCAAGCTGC TGAAGGAATA CGGCGTGCGC 1260 GACTACGGCA CCATCACCGT GCTGTTCAAC ACCTCGAGG AAAdGGS? TC CTTCGGCTCG 1320 CGCGACCTGA TCCAGGAAGA AGCCAAGCTG GCCS &CTACG TGCTCTCCTT CGAGCCCACC 1380 AGCGCAGGCG ACGAAAAACT CTCGCTGGGC ACCTCGGGCA TCGCCTACGT GCAGGTCCAG 1440 ATCACCGGCA AGGCCTCGCA TGCCGGCGCC GCGCCCGAGC TGGGCGTGAA CGCGCTGGTC 1500 GAGGCTTCCG ACCTCGTGCT GCGCACGATG AACATCGACG ACAAGGCGAA GAACCTGCGC 1560 TTCCAGTGGA CCATCGCCAA GGCCGGCCAG GTCTCGAACA TCATCCCCGC CAGCGCCACG 1620 CTGAACGCCG ACGTGCGCTA CGCGCGC? AC GAGG &CTTCG ACGCCGCCAT GAAGACGCTG 1680 Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Het 165 170 175 Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn 180 185 190 Tyr Leu Wing Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu 195 200 205 He Tyr Trp Wing Ser Thr Arg Titr Ser Gly Val Pro Asp Arg Phe Thr 210 215 220 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Be Ser Ser Val Gln 225 230 235 240 Wing Glu Asp Leu Wing He Tyr Tyr Cys Lys Gla Ser Tyr Thr Leu Arg 245 250 255 Thr Phe Gly Gly Gly Thr Lys Leu. Glu Heys Arg Gly Gly Gly Gly 260 26S 270 Be Gly Gly Gly Gly Be Gly Gly Gly Gly Ser Glxt Lys Arg Asp Asn 275 280 28S Val Leu Phe Gln Wing Wing Thr Asp Glu Gln Pro Wing Val He Lys Thr 290 295 300 Leu Glu Lys Leu Val Asn He Glu Thr Gly Thr Gly Asp Ala Glu Gly 305 310 315 320 He Wing Wing Wing Wing Gly Asn Pbe Leu Glu Wing Glu Leu Lys Asn Leu Gly 325 330 335 Phe Thr Val Thr Arg Ser Lys Ser Ala Giy Leu Val Val Gly Asp Asn 340 345 350 He Val Gly Lys He Lys Gly Arg Gly Gly Lys Asn Leu Leu Leu Met 355 360 365 Ser His Met Asp Thr Val Tyr Leu. Lys Gly Lie Leu Wing Lys Ala Pro 370 375 380 Phe Arg Val Glu Gly Asp Lys Wing Tyr Gly Pro Gly He Wing Asp Asp 385 390 395 400 Lys Gly Gly Asn Wing Val He Leu Bis Thr Leu Lys Leu Leu Lys Gla 405 410 415 Tyr Gly Val Arg Asp Tyr Gly Thr He Thr Val Leu Phe Asn Thr Asp 420 425 430 Glu Glu Lys Gly Ser Phe Gly Ser Arg Asp Leu He Gln Glu Glu Wing 435 440. 445 Lys Leu Wing Asp Tyr Val Leu Ser Phe Glu Pro Thr Ser Wing Gly Asp 450 455 460 Glu Lys Leu Ser Leu Gly Thr Ser Gly He Wing Tyr Val Gln Val Gln 465 470 475 480 He Thr Gly Lys Ala Ser His Wing Gly Ala Wing Pro Glu Leu Gly Val 485 490 495 Asn Ala Leu Val Glu Ala Be Asp Leu Val Leu Arg Thr Met Asn He 500 505 510 Asp Asp Lys Wing Lys Asn Leu Arg Phe Gln Trp Thr He Wing Lys Wing 515 520 525 GAAGAGCGCG CGCAGCAGAA GAAGCTGCCC GAGGCCGACG TGAAGGTGAT CGTCACGCGC 1740 GGCCGCCCGG CCTTCAATGC CGGCGAAGGC GGCAAGAAGC TGGTCGACAA GGCGGTGGCC 1800 TACTACAAGG AAGCCGGCGG CACGCTGGGC GTGGAAGAGC GCACCGGCGG CGGCACCGAC 1860 GCGGCCTACG CCGCGCTCTC AGGCAAGCCA GTGATCGAGA GCCTGGGCCT GCCGGGCTTC 1920 GGCTACCACA GCGACAAGGC CGAGTACGTG GACATCAGCG CGATTCCGCG CCGCCTGTAC 1980 ATGGCTGCGC GCCTGATCAT GGATCTGGGC GCCGGCAAG 2019 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 673 amino acids (B) TYPE: amino acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 32 Met Lys Leu Trp Leu Asn Trp He Phe Leu Val Thr Leu Leu Asn Gly 1 5 10 15 He Gln Cys Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Gln Leu Ser Cys ls Ma Ser Gly Tyr Thr Phe 35 40 45 Thr Gly Tyr Trp He His Trp Val Lys Gla Arg Pro Gly Gln Gly Leu 5 ° 55 ° Glu Trp He Gly Glu Val Asn Pro Ser Thr Gly Arg Ser ñsp Tyr Asn 65 70 75 80 Glu Lys Phe Lys Asn Lys Wing Thr Leu Thr Val Asp Lys Ser Ser Thr 85 SO 95 Thr Wing Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Wing Val 100 105 110 Tyr Tyr Cys Wing Arg Glu Arg Wing Tyr Gly Tyr Asp Asp Wing Met Asp 115 120 125 Yr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Giv Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp He Glu Leu Ser 145 150 155 160 Gly Gln Val Ser Asn He He Pro Wing Wing Thr Leu Asn Wing Asp 530 535 540 Val Arg Tyr Wing Arg Asn Glu Asp Phe Asp Wing Wing Met Lys Thr Leu 545 550 555 560 Glu Glu Arg Wing Gln Gln Lys Lys Leu Pro Giu Wing Asp Val Lys Val 565 570 575 He Val Thr Arg Gly Arg Pro Ma Phe Asn Wing Giy Glu Gly Gly Lys 580 585 S90 Lys Leu Val Asp Lys Wing Val. Ala Tyr Tyr Lys Glu Ala Gly Gly Thr 595 &Q 605 Leu Gly Val Glu Glu Arg Thr GXy Gly Gly Thr Asp Ala Ala Tyr Ala 610 615 620 Ala Leu Ser Gly Lys Pro Val He Glu Ser Leu Gly Leu Pro Gly Phe 625 630 635 640 Gly Tyr His Ser Asp Lys Ala Glu Tyr Val Asp He Ser Ala He Pro 645 650 655 Arg Arg Leu Tyr Met Ala Ala Arg Leu He Met Asp Leu Gly Ala Gly 660 665 670 Lys (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 33 GGGCGCCGGC AAGTGATAAA ATTCCTCGAG GAGCTCC 37 (2) INFORMATION FOR SEQ ID NO-: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34 CGCCACCTCT GACTTGAGC 19 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35 GGAGCTCCTC GAGGAATTTT ATCACTTGCC GGCGCCC 37 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 36 GCTGAACGCC GACGTGCGC 19 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2025 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 37 ATGAAGTTGT GGCTGAACTG GATTTTCCTT GTAACACTTT TAAATGGAAT TCAGTGTCAG 60 GTCCAACTGC AGCAGCCTGG GGCTGAACTG GTGAAGCCTG GGGCTTCAGT GCAGCTGTCC 120 TGCAAGGCTT CTGGCTACAC CTTCACCGGC TACTGGATAC ACTGGGTGAA GCAGAGGCCT 180 GGACAAGGCC TTGAGTGGAT TGGAGAGGTT AATCCTAGTA CCGGTCGTTC TGACTACAAT 240 GAGAAGTTCA AGAACAAGGC CACACTGACT GTAGACAAAT CCTCCACCAC AGCCTACATG 300 CAACTCAGCA GCCTGACATC TGAGGACTCT GCGGTCTATT ACTGTGCAAG AGAGAGGGCC 360 TATGGTTACG ACGATGCTAT GGACTACTGG GGCCAAGGGA CCACGGTCAC CGTCTCCTCA 420 GGTGGCGGTG GCTCGGGCGG TGGTGGGTCG GGTGGCGGCG GATCTGACAT TGAGCTCTCA 480 CAGTCTCCAT CCTCCCTGGC TGTGTCAGCA GGAGAGAAGG TCACCATGAG CTGCAAATCC 540 AGTCAGAGTC TCCTCAACAG TAGAACCCGA AñGAACTACT TGGCTTGGTA CCAGCAGAGA 600 CCAGGGCAGT CTCCTAAACT GCTGATCTAT TGGGCATCCA CTAGGACATC TGGGGTCCCT 660 GATCGCTTCA CAGGCAGTGG ATCTGGGACA GATTTCACTC TCACCATCAG CAGTGTGCAG 720 GCTGAAGACC TGGCAATTTA TTACTGCAAG CSATCTTATA CTCTTCGGAC GTTCGGTGGA. 78G GGCACCAAGC TCGAGATCAA ACGGGGCGGT GGTGGCTCCG GAGGTGGCGG TAGCGGTGGC 840 GGGGGTTCCC AGAAGCGCGA CAACGTGCTG TTCCAGGCAG CTACCGACGA GCAGCCGGCC. 900 GTGATCAAGA CGCTGGAGAA GCTGGTCAAC ATCGAGACCG GCACCGGTGA CGCCGAGGGC 960 ATCGCCGCTG CGGGCAACTT CCTCGAGGCC GAGCTCAAGA ACCTCGGCTT CACGGTCACG 1020 CGAAGCAAGT CGGCCGGCCT GGTGGTGGGC GACAACATCG TGGGCAAGAT CAAGGGCCGC 1080 GGCGGCAAGA ACCTGCTGCT GATGTCGCAC ATGGACACCG TCTACCTCAA GGGCATTCTC 1140 , GCGAAGGCCC CGTTCCGCGT CGAAGGCGAC AAGGC € TACG GCCCGGGCAT CGCCGACGAC 12Q0 AAGGGCGGCA ACGCGGTCAT CCTGCACACG CTCASGCTGC TGAAGGAATA CGGCGTGCGC 1260 GACTACGGCA CCATCACCGT GCTGTTCAAC ACCG__CGAGG AAAAGGGTTC CTTCGGCTCG 132.0 CGCGACCTGA TCCAGGAAGA AGCCAAGCTG GCCGñCTACG TGCTCTCCTT CGAGCCCACC 1380 AGCGCAGGCG ACGAAAAACT CTCGCTGGGC ACCTCGGGCA TCGCCTACGT GCAGGTCCAG 1440 ATCACCGGCA AGGCCTCGCA TGCCGGCGCC GCGCCCGAGC TGGGCGTGAA CGCGCTGGTC 1500 GAGGCTTCCG ACCTCGTGCT GCGCACGATG AACATCGACG ACAAGGCGAA GAACCTGCGC 1560 TTCCAGTGGA CCATCGCCAA GGCCGGCCAG GTCTCGAACA TCATCCCCGC CAGCGCCACG 1620 CTGAACGCCG ACGTGCGCTA CGCGCGCAAC GAGGACTTCG ACGCCGCCAT GAAGACGCTG 1680 GAAGAGCGCG CGCAGCAGAA GAAGCTGCCC GAGGCeGACG TGAAGGTGAT CGTCACGCGC 1740.

GGCCGCCCGG CCTTCAATGC CGGCGAAGGC GGCAAGaAGC TGGTCGAC &A GGCGGTGGCC 1800 TACTACAAGG AAGCCGGCGG CACGCTGGGC GTGGASGAGC GCACCGGCGG CGGCACCGAC 1860 GCGGCCTACG CCGCGCTCTC AGGCAAGCCA GTGATCGAGA GCCTGGGCCT GCCGGGCTTC 1920 GGCTACCACA GCGACAAGGC CGAGTACGTG GACATCAGCG CGATTCCGCG CCGCCTGTAC 1980 ATGGCTGCGC GCCTGATCAT GGATCTGGGC GCCGGe &__ ST GATAA 2025 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 288 amino acids (B) TYPE: amino acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 38 Met Lys Tyr Leu Leu Pro Thr Wing Wing Wing Gly Leu Leu Leu Wing Leu 1 5 10 * 15 Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Gln Pro Gly Ala Glu 20 25 30 Leu Val Lys Pro Gly Ala Ser Val Gln Leu Ser Cys Lys Ala Ser Sly 40 45 Tyr Thr Phe Thr Gly Tyr Trp He His Trp Val Lys Gln Arg Pro Sly 50 55 € 0 Gln Gly Leu Glu Trp He Gly Gla Val Asn Pro Ser Thr Gly Arg Ser 65 70 75 80 Asp Tyr Asn Glu Lys Phe Lys Asn Lys Wing Thr Leu Thr Val Asp Lys 85 90 95 Ser Ser Thr Thr Ala Tyr Met Gla. Leu Ser Ser Leu Thr Ser Glu Asp 100 105 110 Ser Wing Val Tyr Tyr Cys Wing Arg Glu Arg Wing Tyr Gly Tyr Asp Asp 115 120 125 Wing Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp He 145 150 155 160 Glu Leu Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Wing Gly Glu Lys 165 170 175 Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr 180 185 190 Arg Lys Asn Tyr Leu Wing Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro 195 200 205 Lys Leu Leu He Tyr Trp Wing Ser Thr Arg Thr Ser Gly Val Pro Asp 210 215 220 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser 225 230 235 240 Ser Val Gln Ala Glu Asp Leu Ala He Tyr Tyr C s Lys Gln Ser 1? 245 250 255 Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Arg Glu 260 265 270 Gln Lys Leu He Ser Glu Glu Asp Leu Asn His His His His His His 275 280 285 (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 39 GCCCAACCAG CCATGGCCGA GGTGCAGCTG CAGCAG 36 (2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 40 CGACCCACCA CCGCCCGAGC CACCGCCACC CGAGCTCACG GCGACTGAGG TTCC 54 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 41 TCGGGCGGTG GTGGGTCGGG TGGCGGCGGA TCTCAGATTG TGCTCACCCA GTCT 54 (2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear, (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 42 CCGTTTGATC TCGAGCTTGG TCCC 24 (2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 843 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other acid, nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 43 ATGAAATACC TATTGCCTAC GGCAGCCGCT GGATTGTTAT TACTCGCTGC CCAACCAGCC 60 ATGGCCGAGG TGCAGCTGCA GCAGTCTGGG GCAGAGCTTG TGAGGTCAGG GGCCTCAGTC 120 AAGTTGTCCT GCACAGCTTC TGGCTTCAAC ATTAAAGACA ACTATATGCA CTGGGTGAAG 180 CAGAGGCCTG AACAGGGCCT GGAGTGGATT GCATGGATTG ATCCTGSGAA TGGTGATACT 240 GAATATGCCC CGAAGTTCCG GGGCAAGGCC ACTTTGACTG CAGACTC & TC CTCCAACACA 300 GCCTACCTGC ACCTCAGCAG CCTGACATCT GAGGACACTG CCGTCTATTA CTGTCATGTC 360 CTGATCTATG CTGGTTATTT GGCTATGGAC TACTGGGGTC AAGGAACCTC AGTCGCCGTG 420 60 AGCTCGGGTG GCGGTGGCTC GGGCGGTGGT GGGTCGGGTG GCGGCGGATC TCAGATTGTG 480 CTCACCCAGT CTCCAGCAAT CATGTCTGCA TCTCCAGGGG AGAAGGTCAC CñTAACCTGC 540 AGTGCCAGCT CAAGTGTAAC TTACATGCAC TGGTTCCAGC AGAAGCCSGG CACTTCTCCC 600 AAACTCTGGA TTTATAGCAC ATCCAACCTG GCTTCTGGAG TCCCTGCTCG CTTCAGTGGC 660 AGTGGATCTG GGACCTCTTA CTCTCTCACA ATCAGCCGAA TGGAGGCTGA AGATGCTGCC 720 ACTTATTACT GCCAGCAAAG GAGTACTTAC CCGCTCACGT TCGGTGCTGG GACCAAGCTC 780 GAGATCAAAC GGGAACAAAA ACTCATCTCA GAAGAAGATC TGAATCACCA CCATCACCAC 840 CAT 843 (2) INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 281 amino acids (B) TYPE: amino acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 44 Met Lys Tyr Leu Leu Pro Thr Wing Wing Wing Gly S_eu Leu Leu% &Wing 1 5 10 15 Wing Gln Pro Wing Wing Wing Glu Val Gln Leu Gln Gln Ser Wing Wing Glu 20 25 30 Leu Val Arg Ser Gly Wing Ser Val Lys Leu Ser Cys Thr Wing Ser Gly 35 40 45 Phe Asn He Lys Asp Asn Tyr Met His Trp Val Lys Gln Arg Pro Glu 50 55 60 Gln Gly Leu Glu Trp He Wing Trp He Asp Pro Glu Asn Gly Asp Thr 65 70 ~ 75 80 Glu Tyr Ala Pro Lys Phe Arg.Gly Lys Ala Tbr Leu Thr Ala fisp Ser 85 90 95 Ser Ser Asn Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp 100 105 110 Thr Wing Val Tyr Tyr Cys His Val Leu He Tyr Wing Gly Tyr Leu Wing 115 120 125 Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Wing Val Ser "Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln He Val 145 150 155 160 Leu Thr Gln Ser Pro Wing He Met Wing Ser Wing Pro Gly Glu Lys Val 165 170 175 Thr He Thr Cys Ser Wing Being Ser Val Thr Tyr Met His Trp Phe 180 185 190 Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp He Tyr Ser Thr Ser 195 200 205 Asn Leu Wing Ser Gly Val Pro Wing Arg Phe Ser Gly Ser Gly Ser Gly 210 215 220 Thr Ser Tyr Ser Leu Thr He S r Arg Met Glu Wing Sla Asp Wing Wing 225 230 235 240 Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Leu Thr Phe Gly Wing 245 250 255 Gly Thr Lys Leu Glu He Lys Arg Glu Gln Lys Leu lie Ser Glu Glu 260 265 270 Asp Leu Asn His His His His His His His 275 280 (2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 45 TCGAGATCAA ACGGGAACAA AAACTCATCT CAGAAGAñGA TCTGAATCAC CACCATCACC 6Q ACCATTAATG AG 72 (2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 46 AATTCTCATT AATGGTGGTG ATGGTGGTGA TTCAGATCTT CTTCTGAGAT GAGTTTTTGT 60 TCCCGTTTGA TC 72 (2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: "(A) LENGTH: 864 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 47 ATGAAATACC TATTGCCTAC GGCAGCCGCT GGATTGTTAT TACTCGCTGC CCAACCAGCC 60 ATGGCCCAGG TCCAACTGCA GCAGCCTGGG GCTGAACTGG TGAAGCCTGG GGCTTCAGTG 120 CAGCTGTCCT GCAAGGCTTC TGGCTACACC TTCACCGGCT ACTGGATACA CTGGGTGAAG 180 CAGAGGCCTG GACAAGGCCT TGAGTGGATT GGAGAGGTTA ATCCTAGTAC CGGTCGTTCT 240 GACTACAATG AGAAGTTCAA GAACAAGGCC ACACTGACTG TAGACAAATC CTCCACCACA 300 GCCTACATGC AACTCAGCAG CCTGACATCT GAGGACTCTG CGGTCTATTA CTGTGCAAGA 360 GAGAGGGCCT ATGGTTACGA CGATGCTATG GACTACTGGG GCCAAGGGAC CACGGTCACC 420 GTCTCCTCAG GTGGCGGTGG CTCGGGCGGT GGTGGGTCGG GTGGCGGCGG ATCTGACATT 480 GAGCTCTCAC AGTCTCCATC CTCCCTGGCT GTGTCAGCAG GAGAG__AGGT CACCATGAGC 540 TGCAAATCCA GTCAGAGTCT CCTCAACAGT AGAACCCGAA AGAACTACTT GGCTTGGTAC 600 CAGCAGAGAC CAGGGCAGTC TCCTAAACTG CTGATCTATT GGGCATCCAC TAGGACATCT 660 GGGGTCCCTG ATCGCTTCAC AGGCAGTGGA TCTGGGACAG ATTTCACTCT CACCATCAGC 720 AGTGTGCAGG CTGAAGACCT GGCAATTTAT TACTGCAAGC AATCTTSTAC TCTTCGGACG 780 TTCGGTGGAG GCACCAAGCT CGAGATCAAA CGGGAACAAA AACTCATCTC AGAAGAAGAT 840 CTGAATCACC ACCATCACCA CCAT 864 (2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 48 AAGCTTGGAA TTCAGTGTGA GGTGCAGCTG CñGC 34 (2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 49 CGCCACCTCC GGAGCCACCA CCGCCCCGTT TGATCTCGAG CTTGG 45 [2) INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1998 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 50 ATGAAGTTGT GGCTGAACTG GATTTTCCTT GTAACACTTT TAAATGGAAT TCAGTGTGAG 60 GTGCAGCTGC AGCAGTCTGG GGCAGAGCTT GTGAGGTCAG, GGGCCTCAGT CAAGTTGTCC 120 TGCACAGCTT CTGGCTTCAA CATTAAAGAC AACTATATGC ACTGGGTGAA GCAGAGGCCT 180 GAACAGGGCC TGGAGTGGAT TGCATGGATT GATCCTGAGA ATGGTGATAC TGAATATGCC 240 CCGAAGTTCC GGGGCAAGGC CACTTTGACT GCAGACTCAT CCTCCAACAC AGCCTACCTG 300 CACCTCAGCA GCCTGACATC TGAGGACACT GCCGTCTATT ACTGTCATGT CCTGATCTAT 360 GCTGGTTATT TGGCTATGGA CTACTGGGGT CAAGGAACCT CAGTCGCCGT (SSCTCGGGT 420 GGCGGTGGCT CGGGCGGTGG TGGGTCGGGT GGCGGCGGAT CTCAGATTGT GC CACCCAG 480 TCTCCAGCAA TCATGTCTGC ATCTCCAGGG GAGAAGGTCA CCATAACCTG CAGTGCCAGC 540 TCAAGTGTAA CTTACATGCA CTGGTTCCAG CAGAAGCCAG GCACTTCTCC CSSACTCTGG 600 ATTTATAGCA CATCCAACCT GGCTTCTGGA GTCCCTGCTC GCTTCAGTGG CAGTGGATCT 660 GGGACCTCTT ACTCTCTCAC AATCAGCCGA ATGGAGGCTG AAGATGCTGC CACTTATTAC 720 TGCCAGCAAA GGAGTACTTA CCCGCTCACG TTCGGTGCTG GGACCAAGCT CGAGATCAAA 780 CGGGGCGGTG GTGGCTCCGG AGGTGGCGGT AGCGGTGGCG GGGGTTCCCA GAAGCGCGAC 840 AACGTGCTGT TCCAGGCAGC TACCGACGAG CAGCCGGCCG TGATCAAGAC GCTGGAGAAG 900 CTGGTCAACA TCGAGACCGG CACCGGTGAC GCCGAGGGCA TCGCCGCTGC GGGCAACTTC 960 CTCGAGGCCG AGCTCAAGAA CCTCGGCTTC ACGGTCACGC GñAGCAAGTC GGCOGGCCTG 1020 GTGGTGGGCG ACAACATCGT GGGCAAGATC AAGGGCCGCG GCGGCAAGAA CGXGCTGCTG 1080 ATGTCGCACA TGGACACCGT CTACCTCAAG GGCATTCTCG CGAAGGCCCC GTTCCGCGTC 1140 GAAGGCGACA AGGCCTACGG CCCGGGCATC GCCGACGACA AGGGCGGCñA CGCGGTCATC 1200 CTGCACACGC TCAAGCTGCT GAAGGAATAC GGCGTGCGCG ACTACGGCAC CASCACCGTG 1260 CTGTTCAACA CCGACGAGGA AAAGGGTTCC TTCGGCTCGC GCGACCTGAT CCAGGAAGAA 1320 GCCAAGCTGG CCGACTACGT GCTCTCCTTC GAGCCCACCA GCGCAGGCGA CGa &AAACTC 1380 TCGCTGGGCA CCTCGGGCAT CGCCTACGTG CBGGTCCAGA TCACCGGCAA GGCCTCGCAT 1440 GCCGGCGCCG CGCCCGAGCT GGGCGTGAAC GCGCTGGTCG AGGCTTCCGA CCTCGTGCTG 1500 CGCACGATGA ACATCGACGA CAAGGCGAAG ABCCTGCGCT TCCAGTGGAC CATCGCCAAG 1560 GCCGGCCAGG TCTCGAACAT CATCCCCGCC AGCGCCACGC TGAACGCCGA CGTGCGCTAC 1620 GCGCGCAACG AGGACTTCGA CGCCGCCATG AAGACGCTGG AAGAGCGCGC GCAGCAGAAG 1680 AAGCTGCCCG AGGCCGACGT GAAGGTGATC GTCACGCGCG GCCGCCCGGC CTTCAATGCC 1740 GGCGAAGGCG GCAAGAAGCT GGTCGACAAG GCGGTGGCCT SCTACAAGGA AGCCGGCGGC 1800 ACGCTGGGCG TGGAAGAGCG CACCGGCGGC GGCACCGACG CGGCCTACGC CGCGCTCTCA 1860 GGCAAGCCAG TGATCGAGAG CCTGGGCCTG CCGGGCTTCG GCTACCACAG CGACAAGGCC 1920 GAGTACGTGG ACATCAGCGC GATTCCGCGC CGCCTGTACA TGGCTGCGCG CCTGñTCATG 1980 GATCTGGGCG CCGGCAAG 1938? (2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 666 amino acids (B) TYPE: amino acid (C) CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 51 Met Lys Leu Trp Leu Asn Trp He Phe Leu Val Thr Leu Leut Asn Gly 1 5 10"15 He Gln Cys Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Lea Val Arg 25 30 Ser Gly Wing Ser Val Lys Leu Ser Cys Thr Wing Ser Gly Phe Asn He 40 45 Lys Asp Asn Tyr Met His Txp Val Lys Gs Arg Pro Glu Gln Gly Leu 50 55 60 Glu Trp He Wing Trp He Asp Pro Glu Asm Gly Asp Thr Glu Tyr Wing 65 70 75 80 Pro Lys Phe Arg Gly Lys Wing Thr Leu Thr Wing sp Ser Ser Ser Asn 85 9ß 95 Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Aso Thr Ala Val 100 -. 100 - 105 110 Tyr Tyr Cys His Val Leu He Tyr Wing Gly Tyr Leu Wing Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Ser Val Wing Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln He Val Leu Thr Gln 145 150 155 160 Ser Pro Ala He Met Ser Ala Be Pro Gly Glu Lys Val Thr He Thr 165 170 175 Cys Ser Ala Ser Ser Ser Val Thr Tyr Met His Trp Phe Gla Gl »Lys 180 185 190 Pro Gly Thr Ser Pro Lya Leu Trp He Tyr Ser Thr Ser Asn Leu Ala 195 200 205 Ser Gly Val Pro Wing Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr 210 215 220 Ser Leu Thr He Ser Arg Met Glu Wing Glu Asp Wing Wing Thr Tyr Tyr 225 230 235 240 Cys Gn Gln Arg Ser Thr Tyr Fro Leu Thr Pise Gly Wing Gly _Bfr Lys 245 250 255 Leu Glu He Lys Arg Gly Gly Gly Gly. Be Gly Gly Gly Gly Be Gly 260 265 - 270 Gly Gly Gly Ser Gln Lys Arg Asp Asn Val Leu She Gln Ala Wing Thr 275 280 285 Asp Glu Gln Pro Wing Val He Lys Thr Leu Glu Lys Leu Val Asn He 290 295 300 Glu Thr Gly Thr Gly Asp Wing Glu Gly lie Wing Wing Wing Gly Asn Phe 305 310 315 320 Leu Glu Ala Glu Leu Lys Asn Leu Gly Phß Thr Val Thr Arg Ser Lys 325 330: 335 Be Ala Gly Leu Val Val Gly Asp Asn He Val Gly Lys He Lys. Gly 340 345 350 Arg Gly Gly Lys Asn Leu Leu Leu Met Ser His Ket Asp Thr Val Tyr 355 360 36S Leu Lya Gly He Leu Wing Lys Wing Pro Phe Arg to Glu Gly Asp Lys 370 375 380 Wing Tyr Gly Pro Gly He Wing Asp Asp Lys Gly Gly Wing Wing Val He 385 390 395 400 Leu His Thr Leu Lys Leu Leu Leu Lys Glu Tyr Gly Val Arg Asp Tyr Gly 405 410 415 Thr He Thr Val Leu Phe Asn Tter Aap jSlu Glu Lys Gly Ser She dly 420 425 430 Ser Arg Asp Leu He Gln Glu Glu Ala Lys Leu Ala Asp Tyr Val Leu 435 440 445 Ser Phe Glu Pro Thr Ser Wing Gly Asp Glu Lys Leu Ser Leu Gly Thr 450 455 460 Ser Gly He Wing Tyr Val Gin Val Without He Thr Gly Lys Wing His His 465 470 475 480 Ala Gly Ala Ala Pro Glu Leu Gly Val Asn Ala Leu Val Glu Ala Ser 485 490 495 Asp Leu Val Leu Arg Thr Met Asn He Asp Asp Lys Ala Lys Asa Leu 500 505 510 Arg Phe Gln Trp Thr He Ala Lys Ala Giy Gln Val Being Asn He He 515 520 525 Pro Wing Being Wing Thr Leu Asn Wing Asp Val Axg Tyr Wing Arg ASE. Glu 530 S35 540 Asp Phe Asp Wing Wing Met Lys Thr Leu Glu Glu Arg Wing Gln Gln Lys 545 550 555 560 Lys Leu Pro Glu Wing Asp Val Lys Val He Val Thr Arg Gly Arg Pro 565 570 575 _Ala Phe Asn Wing Gly Glu Gly Gly Lys Lys Leu Val Asp Lys Wing Val 580 585 590 Wing Tyr Tyr Lys Glu Wing Gly Gly Thr Leu Gly Val Glu Glu Arg Thr S95 600 605 Gly Gly Gly Thr Asp Ala Ala Ty_r Ala Ala Le ». Ser Gly Lys Pro V & 610 615 620 He Glu Ser Leu Gly Leu Pro Gly Phe Gly Tyr His Ser Asp Lys Wing 625 630 635 640 Glu Tyr Val Asp He Ser Wing Pro Pro Arg Arg Leu Tyr Met Ala Wing 645 650 655 Arg Leu He Met Asp Leu Gly Wing Gly Lys 660 665 (2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3217 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: S2 GAATTCGCCG CCACTATGGA TTTTCAAGTG CAGATTTTCA GCTTCCTGCT MTCAGTGCT 60 TCAGTCATAA TGTCCAGAGG ACAAACTGTT CTCTCCCAGT CTCCAGCAAT CCTGTCTGCA 120 TCTCCAGGGG AGAAGGTCAC AATGACTTGC AGGGCCAGCT CAAGTGTAAC TEACATTCAC 180 TGGTACCAGC AGAAGCCAGG TTCCTCCCCC AAATCCTGGA TTTATGCC-IC a? CCAACCTG 240 1-J? GCTTCTGGAG TCCCTGCTCG CTTCAGTGGC AGTGGGTCTG GGACCTCTTA CTCTCTCACA 300 ATCAGCAGAG TGGAGGCTGA AGATGCTGCC ACTTATTACT GCCAACATTG GAGTAGTAAA 360 CCACCGACGT TCGGTGGAGG CACCAAGCTC GAGATCAAAC GGACTGTGGC TGCACCATCT 420 GTCTTCATCT TCCCGCCATC TGATGAGCAG TTGAAATCTG GAACTGCCTC TGTTGTGTGC 480 CTGCTGAATA ACTTCTATCC CAGAGAGGCC AAAGTACAGT GGAAGGTGGA TSACGCCCTC 540 115 > CAATCGGGTA ACTCCCAGGA GAGTGTCACA GAGCAGGACA GCAAGGACAG CACCTACAGC 600 CTCAGCAGCA CCCTGACGCT GAGCAAAGCA GACTACGAGA AACACAAAGT CTACGCCTGC 660 GAAGTCACCC ATCAGGGCCT GAGTTCGCCC GTCACAAAGA GCTTCAACAG GGGAGAGTGT 720 TAATAGGAGC TCGGATCCAG ATCTGAGCTC CTGTAGACGT CGñCAT? SAT TCOTSTTATT 780 TTCCACCATA TTGCCGTCTT TTGGCAATGT GAGGGCCCGG AAACCTGGCC CTSTCTTCTT 840 2d) GACGAGCATT CCTAGGGGTC TTTCCCCTCT CGCCAAAGGA ATGCAAGGTC TGTTGAATGT 900 CGTGAAGGAA GCAGTTCCTC TGGAAGCTTC TYGAAGACAA ACAACGTCTG TAGCGACCCT 960 TTGCAGGCAG CGGAACCCCC CACCTGGCGA CAGGTGCCTC TGCGGCCAAA AGCCACGTGT 1020 ATAAGATACA CCTGCAAAGG CGGCACAACC CCAGTGCCAC GTTGTGAGTT GGSTAGTTGT 1080 GGAAAGAGTC AAATGGCTCT CCTCAAGCGT ATTCAACAAG GGGCTGAAGG ATGCCCAGAA 1140 255 GGTACCCCAT TGTATGGGAT CTGATCTGGG GCCTCGGTGC ACATGCTTTA CATETGTTTA 1200 GTCGAGGTTA AAAAACGTCT AGGCCCCCCG AACCACGGGG ACGTGGTTTT CCTTTGAAAA 1260 ACACGATGAT AATACCATGG AGTTGTGGCT GAACTGGATT TTCCTTGTAA CACTTTTAAA 1320 TGGTATCCAG TGTGAGGTGA AGCTGGTGGA GTCTGGAGGA GGCTTGGTAC AGCCTGGGGG 1380 TTCTCTGAGA CTCTCCTGTG CAACTTCTGG GTTCACCTTC ACTGATTACT ACATGAACTG 1440 GGTCCGCCAG CCTCCAGGAA AGGCACTTGA GTGGTTGGGT TTTATTGGAA ACAAAGCTAA 1500 TGGTTACACA ACAGAGTACA GTGCATCTGT GSAGGGTCGG TTCACCATCT CCAGAGATAA 1560 ATCCCAAAGC ATCCTCTATC TTCAAATGAA CACCCTGAGA GCTGAGGACA GTGCCACTTA 1620 TTACTGTACA AGAGATAGGG GGCTACGGTT CTACTTTGAC TACTGGGGCC AAGGCACCAC 1680 TCTCACAGTG AGCTCGGCTA GCACCAAGGG ACCATCGGTC TTCCCCCTGG CCCCCTGCTC 1740 CAGGAGCACC TCCGAGAGCA CAGCCGCCCT GGGCTGCCTG GTCAAGGACT ACTTCCCCGA 1800 ACCGGTGACG GTGTCGTGGA ACTCAGGCGC TCTGACCAGC GGCGTGCACA CCTTCCCGGC 1860 TGTCCTACAG TCCTCAGGAC TCTACTCCCT CAGCAGCGTC GTGACGGTGC CCTCCAGCAA 1920 CTTCGGCACC CAGACCTACA CCTGCAACGT AGATCACAAG CCCAGCAACA CCAAGGTGGA 1980 CAAGACAGTT GGCGGTGGTG GCTCTGGTGG TGSCGGTAGC GGTGGCGGGG GTTCCCAGSA 2040 GCGCGACAAC GTGCTGTTCC AGGCAGCTAC CGSCGAGCAG CCGGCCGTGA TCAAGACGCT 2100 GGAGAAGCTG GTCAACATCG AGACCGGCAC CGSTGACGCC GAGGGCATCG CCGCTGCGGG 2160 CAACTTCCTC GAGGCCGAGC TCAAGAACCT CGGCTTCACG GTCACGCGAA GCAAGTCGGC 2220 CGGCCTGGTG GTGGGCGACA ACATCGTGGG CAAGATCAAG GGCCGCGGCG GCAAGAACCT 2280 GCTGCTGATG TCGCACATGG ACACCGTCTA CCTCfiAGGGC ATTCTCGCGA AGGCCCCGTT 2340 CCGCGTCGAA GGCGACAAGG CCTACGGCCC GGSCSTCGCC GACGACSAGG GCGGCAACGC 2400 GGTCATCCTG CACACGCTCñ AGCTGCTGAA GGAATACGGC GTGCGCGACT ACGGCACCAT 2460 CACCGTGCTG TTCAACACCG ACGAGGAAAA GGGTTCCTTC GGCTCGCGCG ACCTGATCCA 2520 GGAAGAAGCC AAGCTGGCCG ACTACGTGCT CTCCTTCGAG CCCACCAGCG CAGGCGACG & 2580 AAAACTCTCG CTGGGCACCT CGGGCATCGC CTACGTGCAG GTCCAGATCA CCGGCAAGGC 2640 CTCGCATGCC GGCGCCGCGC CCGAGCTGGG CGTGAACGCG CTGGTCGAGG CTTCCGACCT 2700 CGTGCTGCGC ACGATGAACA TCGACGACAA GGCGAAGAAC CTGCGCTTCC AGTGGACCAT 2760 CGCCAAGGCC GGCCAGGTCT CGAACATCAT CCCCGCCAGC GCCACGCTGA ACGCCGACGT 2820 GCGCTACGCG CGCAACGAGG ACTTCGACGC CGCCATGAAG ACGCTGGAAG AGCGCGCGCA 2880 GCAGAAGAAG CTGCCCGAGG CCGACGTGAA GGTGATCGTC ACGCGCGGCC GCCCGGCCTT 2940 CAATGCCGGC GAAGGCGGCA AGAAGCTGGT CGACAAGGCG GTGGCCTACT ACAAGGñAGC 3000 CGGCGGCACG CTGGGCGTGG AAGAGCGCAC CGGCGGCGGC ACCGACGCGG CCTACGCCGC 3060 GCTCTCAGGC AAGCCAGTGA TCGAGAGCCT GGGCCTGCCG GGCTTCGGCT ACCACAGCGA 3120 CAAGGCCGAG TACGTGGACA TCAGCGCGAT TCCGCGCCGC CTGTACATGG CTGCGCGCCT 3180 GATCATGGAT CTGGGCGCCG GCAAGTGATA ATCTAGA 3217 (2) INFORMATION FOR SEQ ID NO: 53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 54 TGGATCTGAA GCTTAAACTA ACTCCATGGT GACCC 35 (2) INFORMATION FOR SEQ ID SO: 54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 54 GCCACGGATC CCGCCACCTC CGGAGCCACC ACCGCCñCAA TCCCTGGGCA CAATTTTCTT 60 G 61 (2) INFORMATION FOR SEQ ID NO: 55 > : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 55 GCCCAGGAAG CTTGGCGGTG GTGGCTCCGG AGGTGGCSGT AGCGGTGGCG GGGGTTCCCA 60 GAAGCGCGAC AACGTGCTGT TCCAGGCAGC TACC 94 (2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: another nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 56 ATGTGCGAAT TCAGCAGCAG GTTCTTGCCG CCGO? GCCCT TGATCTTGCC C 51 (2) INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 732 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear • (ii) TYPE OF MOLECULE: other nucleic acid (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 16..720 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID O: 57 GAATTCGCCG CCACC ATG GAT TTT CAA GTG CAG ATT TTC AGC TTC CTG CTA 51 Met Asp Phe Gln Val Gln He Phe Ser Phe Leu Leu 1 5 10 ATC AGT GCT TCA GTC ATA ATG TCC AGA GGA CA ACT GTT CTC TCC CRG 99 Wing Being Val He Met Being Arg Gly Gln Thr Val Leu Being Gln 15 20 25 TCT CCA GCA ATC CTG TCT GCA TCT CCA GGG GAG AAG GTC ACA ATG ACT 147 Ser Pro Wing He Leu Ser Wing Being Pro Gly Glu Lys Val Thr Me £ . Thr 30 35 «or TGC AGG GCC AGC TCA AGT GTA ACT TAC ATT CAC TGG TAC CAG CAG AAS 195 Cys Arg Wing Ser Ser Val Thr Tyr He His Trp Tyr Gln Gln Lys 45 50 55 63 CCA GGT TCC TCC TCC TCC TCC TGG ATT TAT GCC ACA TCC AAC CTG GCf 243 Pro Gly Ser Pro Lys Ser Trp He Tyr Ala Thr Ser Asn Leu Ma 65 70 - 75 TCT GGA GTC CCT GCT CGC TTC AGT GGC AGT GGG TCT GGG ACC TCT TAC 291 Ser Gly Val Pro Wing Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr 80 85 90 TCT CTC ACA ATC AGC AGA GTG GAG GCT GAA GAT GCT GCC ACT TAT TAC_ 339 Ser Leu Thr He Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr 95 100 105 TGC CAA CAT TGG AGT AGT AAA CCA CCG ACG TTC GGT GGA GGC ACC AAS 387 Cys Gln His Trp Ser Ser Lys Pro Pro Thr Phe Gly Gly Gly Thr Lys 110 115 120 CTG GAA ATC AAA CGG GCT GAT GCT GCA CCA ACT GTA TCC ATC TTC CCA 435 Leu Glu He Lys Arg Wing Asp Wing Wing Pro Thr Val Ser He Phe Pro 125 130 135 140 CCA TCC AGT GAG CAG TTA ACA TCT GGT GGT GCC TCA GTC GTG TGC TTC 483 Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala S Val Val Cys Pfae 145 150 155 TTG AAC AAC TTC TAC CCC AAA GAC ATC AAT GTC AAG TGG AAG ATT GAT 531 Leu Asn Asn Phe Tyr Pro Lys Asp He Asn Val Lys Trp Lys He Asp 160 165 170 GGC AGT GAA CGA CAA AAT GGC GTC CTG AAC AGT TGG ACT GAT CAG GAC 579 Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Tr Thr Asp Gln Asp 175 180 185 AGC AAA GAC AGC ACC TAC AGC ATG AGC AGC ACC CTC ACG TTG ACC AAG 627 Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys 190 195 200 GAC GAT TAT GAA CGA CAT AAC AGC TAT ACC TGT GAG GCC ACT CAC AAG 675 Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys 205 210 215 220 ACA TCA ACT TCA CCC ATT GTC AAG AGC TTC AAC AGG AAT GAG TGT 720 Thr Ser Thr Ser Pro He Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 TAATAAGAAT TC 732 (2) INFORMATION FOR SEQ ID NO: 58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 235 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 58 Met Asp Phe Gln Val Gln He Phe Ser Phe Leu Leu He Ser Wing Ser 1 5 10 15 Val He Met Ser Arg Gly Gln Thr Val Leu Ser Gln Ser Pro Wing He 20 25 30 Leu Ser Ala Be Pro Gly Glu Lys Val Thr Met Thr Cys Arg Wing Ser 35 40 45 Ser Ser Val Thr Tyr He His Trp Tyr Gln Gln Lys Pro Gly Ser Ser 55 60 Pro Lys Ser Trp He Tyr Wing Thr Ser Asn Read Wing Ser Gly Val Pro 65 70 75 80 Wing Arg Phe Being Gly Being Gly Being Gly Thr Being Tyr Being Leu Thr He 85 90 95 Being Arg Val Glu Wing Glu Asp Wing Wing Thr Tyr Tyr Cys Gln Bis Trp 100 IOS "Not Being Lys Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 115 120 125 Arg Ala Asp Ala Ala Pro Thr Val Ser He Phe Pro Pro Ser Ser Glu 130 135 140 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 145 150 155 160 Tyr Pro Lys Aap He Asn Val Lya Trp Lys He Asp Gly Ser Glu Arg 165 170 15 Gln Aan ßly Val Leu Aan Ser Tzp hr Asp Gl »Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 195 200 205 Arg His Asn Ser Tyr Thr Cys Glu Wing Thr His Lys Thr Ser Thr Ser 210 215 220 Pro He Val Lys Ser Phe Aan Arg Asn Glu Cys 225 230 235 (2) INFORMATION FOR SEQ ID NO: 59: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1974 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 16..1956 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 59 AAGCTTGCCG CCACC ATG AAG TTG TGG CIG AAC TGG ATT TTC CTT GTA ACA 51 Met Lys Leu Trp Leu Asn Trp He Phe Leu Val Thr 5 10 CTT TTA AAT GGT ATC CAG TGT GAG GTG AAG CTG GTG GAG TCT GGA GGA 99 Leu Leu Asn Gly He Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly! 5 20 25 GGC TTG GTA CAG CCT GGG GGT TCT CTG AGA CTC TCC TGT GCA ACT TCT 147 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser 30 35 40 GGG TTC ACC TTC ACT GAT TAC TAC ATG AAC TGG GTC CGC CAS CCT CCA 195 Gly Phe Thr Phe Thr Asp Tyr Tyr Met Asn Trp- to Arg Gla Pro Pro 4S 50 55 60 GGA AAG GCA CTT GAG TGG TTG GGT TTT ATT GGA AAC MR. GCT ñ &T GGT 243 Gly Lys Wing Leu Glu Trp Leu Gly Phe He € l Asn Lys Wing Aan Gly 65 70 75 TAC ACA ACA GAG TAC AGT GCA TCT GTG AAG GGT CGG TTC ACC ATC TCC 291 Tyr Thr Thr Glu Tyr Ser Wing Ser Val Lys Gly Arg Phe Thr He Ser 80 85 SO AGA GAT AAA TCC CAA AGC ATC CTC TAT CTT CAA ATG AAC ACC CTG AGA 339 Arg Asp Lys Ser Gln Ser He Leu Ty Leu Gln Met Asn Thr Leu Arg 95 100 OS GCT GAG GAC AGT GCC ACT TAT TAC TGT ACA AGA GAT AGG GGG CTA CGG 387 Wing Glu Asp Ser Wing Thr Tyr Tyr Cys Thr Arg Asp Arg Gly Leu Arg "O H5 120 TTC TAC TTT GAC TAC TGG GGC CA GGC 8CC ACT CTC ACA GTC TCC TCA 435 Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 125 130 135 140 GCC AAA ACG ACA CCC CCA TCT GTC TAT CCA CTG GCC CCT GSA TCT GCT 483 Wing Lya Thr Thr Pro Pro Ser Val Tyr Era Leu Ala Pro Gl $ Ser Ala 145 150 '155 GCC CAA ACT AAC TCC ATG GTG ACC CTG GGA TGC CTG GTC AAS GGC TAT 531 Wing Gln Thr Asn Ser Met Val Thr to Gly Cys Leu Val Lya Gly Tyr 160 165 10 TTC CCT GAG CCA GTG ACA GTG ACC TGS A2C TCT GGA TCT CTS TCC AdC 579 Phe Pro Glu Pro Val Thr Val Thr Trp Handle Ser Gly Ser Le »Ser Ser 175 180 185 GGT GTG CAC ACC TTC CCA GCT GTC CTG CAG TCT GAC CTC TAC ACT CTG 627 Gly Val His Thr Phe Pro Wing Val Leu Gln Ser Asp Leu Tyr Thr Leu 190 195 200 AGC AGC TCA GTG ACT GTC CCC TCC AGC ACC TGG CCC AGC GAS ACC GTC 675 Ser Ser Val Val Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val 205 210 215 220 ACC TGC AAC GTT GCC CAC CCG GCC AGC AGC ACC AAG GTG GAC AAG AAA 723 Thr Cys Asn Val Wing His Pro Wing Ser Ser Thr Lys Val Aap Lys Lys 225 230 235 ATT GTG CCC AGG GAT TGT GGC GGT GGT GGC TCC GGA GGT GGC GGT GGT AGC 771 He Val Pro Arg Asp Cys Gly Gly Gly Gly Gly Gly Gly Gly Gly 240 245 250 GGT GGC GGG GGG TCC CAG AAG CGC GAC AAC GTG CTG TTC CAG GCA GCT 819 Gly Gly Gly Gly Ser Gln Lys Arg Asp Asn Val Leu Phe Gln Ala Ma 255 260 265 ACC GAC GAG CAG CCG GCC GTG ATC AAG ACG CTG GAS AAS CTG GTC'A & C 867 Thr Asp Glu ßln Pro Ala Val He Lys Thr Leu Glu Lys Leu Val Asa 270 275 280 ATC GAG ACC GGC ACC GGT GAC GCC GAG GGC ATC GCC GCT GCG GGC AAC 91S He Glu Thr Gly Thr Gly Aap Wing Glu Gly He Wing wing Wing Gly Aan 28S 290 295 300 TTC CTC GAG GCC GAG CTC AAG AAC CTC GGC TTCACG GTC ACG CGA AGC 963 Phe Leu Glu Wing Glu Leu Lys Aan Leu Gly Phe Thr Val Thr Arg Ser 305 310 315 AAG TCG GCC GGC CTG GTG GTG GGC GAC AAC ATC GTG GGC AAG ATC AAG 1011 Lya Ser Wing Gly Leu Val Val Gly ñsp Asn He Val Gly Lys He Lys 320 '325 330 GGC CGC GGC GGC AAG AAC CTG CTG'CTG ATG TCG CRC ATG GAC ACC GTC 1059 Gly Arg Gly Gly Lya Aan Leu Leu Leu Met Sez Bis Met Aap Thr Val 335 340 345 TAC CTC AAG GGC ATT CTC GCG AAG GC CCG TTC CGC GTC GAA GGC GAC 1107 Tyr Leu Lya Gly He Leu Wing Lya Ma Pro Phe Arg Val Glu Gly Aap 350 355 360 AAG GCC TAC GGC CCG GGC ATC GCC GAC GAC AAG GGC GGC AAC GCG GTC 1155 Lya Wing Tyr Gly Pro Gly He Wing Aap Aap Lya Gly Gly Asn Wing Val 365 370 375 380 ATC CTG CAC AGC CTC AAG CTG CTG AAG GAA TAC SGC GTG CGC GAC TAC 1203 He Leu Hia Thr Leu Lys Leu Leu Lya Glu Tyr Gly Val Arg Asp Tyr 385 390 395 GGC ACC ATC ACC GTG CTG TTC AAC ACC GAC GAG GBA AAG GGT TTC TTC 1251 Gly Thr He Thr Val Leu Phe Asn Thr Asp Glu Glu Lys Gly Ser Phe 400 405 410 GGC TCG CGC GAC CTG ATC CAG GAA GAA GCC AAG CI © GCC GAC TAC G3TG 1299 Gly Ser Arg Asp Leu He Gln Glu Glu Ala Lys Read Ala Aap Tyr Val 415 420 125 CTC TCC TTC GAG CCC ACC AGC GCA GGC GAC GRA ASA CTC TCG CTG GGC 1347 Leu Ser Phe Glu Pro Thr Ser Wing Gly Aap Glu Lys Leu Ser Leu Gly 430. 435 440 ACC TCG GGC ATC GCC TAC GTG CAG GTC AAC ATC ACC GGC AAG GCC TCS 1395 Thr Ser Gly He Wing Tyr Val Gln Val Asn He Thr Gly Lys Wing Ser 445 450 455 460 CAT GCC GGC GCC GCG CCC GAG CTG GGC GTG AAC GCG CTG GTC GAG GCT 1443 His Ala Gly Ala Ala Pro Glu Leu Gly Val Asn Ala Leu Val Glu Ala 65 470 475 TCC GAC CTC GTG CTG CGC ACG ATG AAC ATC GAC GAC AAG GCG AAG ABC 1491 Ser Aap Leu Val Leu Arg Thr Met Aaa He Asp Asp Lys Wing Lys Asn 480 485 490 CTG CGC TTC AAC TGG ACC ATC GCC AAG GCC GGC AAC GTC TCG AAC ATC 1539 Leu Arg Phe Aan Trp Thr He Ala Lys Wing Giy Asn Val Ser Asn He 95 500 SOS ATC CCC: GCC AGC: GCC ACG CTG AAC GCC GAC GTG CGC TAC GCG CGC AAC 1587 He Pro > Wing Being Wing Thr Leu Aan Wing Asp Val Arg Tyr Wing Arg Aan 510 515 520 GAG GAC TC GAC GCC GCC ATG AAG ACG CTG GAA GAS CGC GCG CAG CAG 1635 Glu Asp Phe Asp Wing Wing Met Lys Thr Leu Glu Glu Arg Wing Gln Gln 525 530 535 540 AAG AAG CTG CCC GAG GCC GAC GTG AAG GTG ATC GTC ACG CGC GGC CGC 1683 Lys Lys Leu Pro Glu Wing Asp Val Lys Val He Val Thr Arg Gly Arg 545 550-555 CCG GCC TC AAT GCC GGC GAA GGC GGC AAG AAG CTG GTC GAC AAG GCG 1731 Pro Wing Phe Asn Wing Gly Glu Gly Gly Lya Lya Leo Val Asp Lys Wing 560 565 570 GTG GCC TAC TAC TGA TGA TGA TAG Tyr Tyr TAG TAG TAG TAG TAG TAG TAG TAG LY Glu GGC GGC ACG CTG GGC GTG Wing Gly Gly Thr Leu Gly Val Glu Glu Glu Arg 575 580 585 ACC GGC GGC GGC ACC GAC GCC GCG GCC TAC GCC GCG CTC TCA GGC AAG CCA 1827 Thr Gly Gly Gly Thr Asp Ala Ala Tyr Ala Ala Leu Ser Gly Lys Pro 590 595 600 GTG ATC GAG AGC CTG GGC CTG CCG GGC TTC GGC TAC CAC AGC GAC AAG 1875 Val He Glu Ser Leu Gly Leu Pro Gly Phe Gly Tyr Sis Ser Aap Lya 605 610 615 620 GCC GAG TAC GTG GAC ATC AGC GCG ATT CCG CG C CGC CTG TAC ATG GST 1923 Wing Glu Tyr Val Aap He Ser Wing Pro Pro Arg Arg Leu Tyr Mee Wing 625 630 635 GCG CGC CTG ATC ATG GAT CTG GGC < GCC GGC AAG TGATABSAAT TCCTCG &ß 1974 Wing Arg Leu He Met Aap Leu Gly Wing Gly Lys 640 645 (2) INFORMATION FOR SEQ ID NO: 60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 647 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 60 Mat Lyß Leu Trp Leu Asn Trp XI »Pfaß Leu Val fa_r Leu Leu Aan Gly 1 S 10 15 lie Gin Cys Glu Val Lys Leu Val Glu Sar Gly Gly Gly Leu Val Gn 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 0 45 Thr Aap Tyr Tyr Mßt Asn Trp Val Arg Glp Pro Pro Gly Lya Ala Leu 50 55 60 Glu Trp Leu Gly Phß He Gly Aaa Lya Wing Aan Gly TS Thr Thr Glu 65 70 75 80 Tyr Ser Ala Ser Val Ly * ßly Arg Sha Thr II * Ser Arg Aap Lys Ser 85 90 95 ßln Sar He Leu Tyr Leu Gln Met Aan Thr Leu Arg Ala Glu Asp Ser 100 105 110 Wing Thr Tyr Tyr Cys Thr Arg Aap Arg Gly Leu Arg Sha Tyr Phe Aap 115 120 125 Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Wing Lya Thr Thr 130 135 140 Pro Pro Ser Val Tyr Pro Leu Wing Pro Gly Ser Wing Ala Gln Thr Asn 145 150 155"160 Ser Mat Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Bhß Pro Glu Pro 165 170 17S Val Thr Val Thr Trp? An Ser Gly Ser Leu Ser Ser Gly Val His Thr 180 185 190 Phß Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Val 195 200 - 205 Thr Val Pro Ser Sßx Thr Trp Pro Ser Glu Thr Val Thr Cys Asa Val 210 215 220 Wing His Pro Wing Being Ser Thr Lys Val Aap Lya Lya? L? Val Pro Arg 225 230 235 240 Aap Cys Gly ßly ßly Gly Ser Gly ßly Gly Gly Ser Bly Gly Gly Gly 245 250 255 Being Gln Lya Arg Aap Aan Val Leu Phe Gln Wing Wing Thr Aap Glu Gln 260 265 270 Pro Ala Val He Lya Thr Lau Glu Lys Leu Val Aaa Ha ßlu «go Gly 275 280 28S Thr Gly Asp Wing Glu Gly Xla Wing Wing Wing Gly Asn Phß Leu ßlu Wing 290 295 300 Glu Leu Lys Aan Leu Gly Phe Thr Val Thr Arg Ser Lys Ser-Ala Gly 305 310 31S 320 Leu Val Val Gly Asp Asn He Val Gly Lya He Lya Gly Arg Gly Gly 325 330 335 Lys Asn Leu Leu Leu Met Being His Met Asp Tbr Val Tyr Leu Lys Gly 340 345 350 He Leu Wing Lys Wing Pro Phe Arg Val Glu Gly Aap Lya Wing Tyr Giy 355 360 365 Pro Gly He Wing Asp Asp Lys Gly Gly Asn Wing Val He Leu His Thr 370 375 380 Leu Lys Leu Leu Lys Glu Tyr Gly Val Arg Asp Tyr Gly Thr He Thr 385 390 395 400 Val Leu Phe Asn Thr Asp Glu Glu Lys Gly Ser Phe Gly Ser Arg Asp 4 5 5 410 415 Leu He Gln Glu Glu Wing Lys Leu Wing Asp Tyr Val Leu Ser Phe Giu 420 425 430 Pro Thr Ser Wing Gly Asp Glu Lys Leu Ser Leu Gly Thr Ser Gly He 35 440 445 Wing Tyr Val Gln Val Asn He Thr Gly Lya Wing Ser Hia Wing Gly Wing 4S0 455 460 Wing Pro Glu Leu Gly Val Aan Wing Leu Val Glu Wing Ser Asp Leu Val 465 470 475 480 Leu Arg Thr Met Aan He Aap Sap Lys Wing Lya Aan Leu Arg Bhé Asn 485 490 495 Trp Thr He Wing Lya Wing Gly Aan Val Ser Aan He He Pro Wing Being 500 505 510 Wing Thr Leu Aan Wing Asp Val Arg Tyr Wing Arg Asn ßlu Asp Ehe Asp 5. S20 525 Wing wing Met Lys Thr Leu Glu Glu Arg Wing Gln Gln Lys Lys Leu Pro 530 535 S40 Glu Wing Asp Val Lya Val He Val Thr Arg Gly Arg Pro Wing Bhe Asn S45 550 S55 5S0 Wing Gly Glu Gly Gly Lys Lys Leu Val Aap Lya Wing Val Wing Tyr Tyr 565 570 575 Lys Glu Wing Gly Gly Thr Leu Gly Val Glu Glu Arg Thr Gly Gly Gly 580 S85 590 Thr Asp Ala Ala Tyr Ala Ala Leu Ser Gly Lya Pro Val He Glu Ser 595 600 605 Leu Gly Leu Pro Gly Phe Gly Tyr His Ser Asp Lys Ala Glu Tyr Val 610 615 620 Asp lie be Ala He Pro Arg Arg Leu Tyr Mßt Ala Ala Arg Leu He 625 630 635 640 Met Asp Leu Gly Ala Gly Lys 645

Claims (18)

1. CLAIMS 1. A gene construct or construct that encodes a portion targeting the cell and a heterologo enzyme activating the prodrug for use as a drug in a mammalian host wherein the gene construct is capable of expressing the portion pointing to the cell and the heterologous activating enzyme of the prodrug as a conjugate within a cell in the mammalian host and where the conjugate is instructed to leave the cells after this for its selective localization in a. antigen from the surface of the cell recognized by the portion that points to the cell ,.
2. 2. A gene construct for use as a medicament according to claim 1, characterized in that the portion that points to the cell is an antibody.
3. 3. A gene construct for use as a medicament according to claim 2, characterized in that the antibody is an anti-CEA antibody selected from the antibody A5B7 or the antibody 806.077.
4. 4. A gene construct for use as a medicament according to any of the preceding claims, characterized in that the heterologous enzyme activating the prodrug is a carboxypeptidase.
5. 5. A gene construct for use as a medicament according to claim 4, characterized in that the carboxypeptidase is CPG2.
6. 6. A gene construct for "use as a medicament according to claim 5, characterized in that CPG2 has mutated polypeptide glycosylation sites to prevent or reduce glycosylation on the expression of mammalian cells
7. 7. A gene construct for use as medicament according to any of claims 5-6, characterized in that the conjugate of antibody-enzyme of CPG2 is a fusion protein in which the enzyme is fused to the C-terminus of the antibody through the heavy or light chain thereof by which dimerization of the encoded conjugate when expressed can be carried out by means of a dimerization domain on CPG2
8. 8. A gene construct for use as a medicine according to claim 7, characterized in that the fusion protein is formed by linking a C-terminus of a heavy chain of antibody Fab to a N-terminus of a CPG2 molecule to form a Fab-CPG2 whereby two molecules of fab-CPG2 when expressed are dimerized by means of CPG2 to form a conjugate of (Fab-CPG2) 2.
9. 9. A gene construct for use as a medicament according to claim 4, characterized in that the carboxypeptidase is selected from [D253K1HCPB, [G251T, D253K] HCPB or [A248S, G251T, .D253K} HCPB.
10. 10. A gene construct for co-use or medicament according to any of the preceding claims, characterized in that it comprises a transcriptional regulatory sequence comprising a promoter and a control element comprising a genetic switch to control the expression of the gene construct.
11. 11. A gene construct for use as a medicament according to claim 10, characterized in that the transcriptional regulatory sequence comprises a genetic switch control element regulated by the presence of tetracycline or ecdysone.
12. 12. A gene construct for use as a medicament according to claims 10 or 11, characterized in that the promoter is dependent on the cell type and is selected from the following promoters: carcinoembryonic antigen (CEA); alpha-photoprotein (AFP); Tyrosiria Hydrixylase; acetyl transferase hill; neuron-specific enolase; insulin; acid fiber fibro glial? HER-2 / neu; cerB2; and N-myc.
13. 13. A gene construct for use as a medicament according to any of the preceding claims; characterized in that it is packaged within an adenovirus for administration to the mammalian host.
14. 14. The use of a gene construct. as defined in any of claims 1-12 for the manufacture of a medicament for cancer therapy in a mammalian host.
15. 15. A system of two paired components designed for use in a mammalian host in which components comprise: (i) a first component comprising a. Gene construct as defined in any of claims 1- 13; (ii) A second component comprising a prodrug that can be converted to a cytotoxic medicament by the heterologous enzyme encoded by the first component.
16. 16. A two-component system paired according to claim 15 characterized in that: the first component comprises a gene encoding the heterologous enzyme CPG2 and the second component of prodrug is selected from N- (4- [N, N-bis (2-iodoethyl) amino] -phenoxycarbonyl) -L-glutamic acid, N- (4- [N, N-bis (2-chloroethyl) amino] -f-enoxycarbonyl) -L-glutamic-gamma- (3,5-dicarboxy) ) anuide or N- (4- [N, N-bis (2-chloroethyl) aminoj-phenoxycarbonyl) -L-glutamic acid or a pharmaceutically acceptable salt thereof. 17 A method for administering a cytotoxic drug to a site, characterized in that it comprises administering to a host a first component comprising a gene construct as defined in any of claims 1-13; followed by administration to the host of a second component comprising a prodrug that can be converted to a cytotoxic medicament by the heterologous enzyme encoded by the first component. The method according to claim 17, characterized in that the first component comprises a gene encoding the heterologous enzyme CPG2 and the second component of the prodrug selected from N- (4- [N, N-bis (2-iodoethyl) ) amino] phenoxycarbonyl) -L-glutamic, N- (4- [N, N-bis (2-chloroethyl) amino] phenoxycarbonyl) -L-glutamic-gamma- (3,5-dicarboxy) anuide or N-acid - (4- [N, N-bis (2-chloroethyl) -amino] phenoxycarbonyl) -L-glutamic acid or a pharmaceutically acceptable salt thereof.