AU737683B2 - Bioactive fusion proteins and pre-existing tumor therapy - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: oeo• 4 4.

t 4 4 4 4 oO44" 4 ••o f 6 Name of Applicant: Whitehead Institute for Biomedical Research Actual Inventor(s): Graham J. Lieschke Richard C. Mulligan Address for Service: IP Australia

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Documents received on: 1 0 MAY 1999 Batch No: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: BIOACTIVE FUSION PROTEINS AND PRE-EXISTING TUMOR THERAPY Our Ref 583055 POF Code: 894/296910 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): la BIOACTIVE FUSION PROTEINS AND PRE-EXISTING TUMOR THERAPY The present application is a divisional application from Australian patent application number 49198/96, the entire disclosure of which is incorporated herein by reference.

Background of the Invention Production of therapeutic proteins, such as those which are dimeric, is often difficult, inefficient and expensive. Production of a dimer may require separate expression of the two components, followed by joining of those components to form a functional dimer. Alternative methods of producing functional dimeric proteins would be useful.

Summary of the Invention 15 The present invention provides a method of treating a disorder characterised by an established tumor comprising administering a therapeutically effective dose of IL-12secreting tumor cells to a subject having a disorder characterised by an established tumor.

:i The present invention relates to fusion proteins which comprise at least two polypeptide monomers (chains of amino acids) joined through a polypeptide linker and are bioactive, as well as to their production. In one embodiment, the bioactive fusion proteins of the present invention comprise two or more polypeptides which occur as subunits or monomers in a corresponding bioactive native dimeric protein and are linked through heterologous amino acid residues (amino acid residues which are not 25 present between two subunits in the native protein). Accordingly, this invention provides DNA encoding a bioactive protein, wherein the bioactive protein comprises two subunits present in a corresponding native dimeric protein and a polypeptide linker, wherein the two subunits are joined in the bioactive protein by a polypeptide linker. As it occurs in nature, the cytokine IL-12 is a heterodimer made up of a 40 kDa subunit (p40) linked by a disulfide bond to a 35 kDa subunit (p35). Gillessen. S. et al., Eur.J.

Immunology, 25 200-206 (1995); Ozmen et al., J. Exp. Med., 180 907-915 (1995); Heinsel et al., Inf. Immun., 62 4224-4249 W:\Elisabeth\PJC\NODELETE\28034-99.doc 2 (1994). For example, the fusion protein is a bioactive interleukin-12 (IL-12) fusion protein which comprises two subunits, designated p35 and p40, joined by a polypeptide linker. In another example, the fusion protein may comprise the subunits of other dimeric hematopoietic growth factors joined by a polypeptide linker, or the subunits of other dimeric cytokine proteins joined by a polypeptide linker. The polypeptide linker may be selected from group (Gly 4 Ser) 3 (Gly 4 Ser) 3 Ser; Gly 6 Ser; and (Gly 4 Ser) 2 Ser. In another example, the bioactive fusion protein may comprise two subunits which are bioactive monomers interleukin-2, GMCSF) in their native form and are joined through a polypeptide linker to produce a fusion protein which is chimeric or hybrid in nature in that it comprises at least two components or subunits which do not occur together in a native protein an interleukin-2/GMCSF fusion protein).

The present invention may relate to methods of producing the subject 15 fusion proteins, constructs useful in their production and host cells containing the constructs from which the encoded fusion proteins are expressed. The subject fusion proteins may be expressed in an appropriate expression system, such as by a retrovirus vector which contains and expresses DNA encoding the subunits or monomers and the polypeptide linker of the desired fusion protein in an appropriate host cell, such as in mammalian cells. The present invention may also relate to cells which have been transduced to secrete IL-12 fusion proteins of the present invention, and particularly to tumor cells which have been transduced to secrete an IL-12 fusion protein. The invention may also relate to the use of the transduced tumor cells, particularly in the treatment of tumors.

Fusion proteins of the present invention are useful for the same purposes therapeutic or diagnostic uses) as the corresponding native protein. For example, IL-12 fusion proteins can be used to enhance the lytic C:\WNWORDUOANNE\PJCSPECI49198DIV.DOC activity of NK/lymphokine activated killer cells, act as a growth factor for activated human T and NK cells and stimulate production of IFN-y by resting peripheral blood mononuclear cells (PBMC). IL-12 is also useful in treating a variety of cancers. For instance, IL-12 is useful for the enhancement of antitumor immunity and, as described herein, tumor cells which secrete either native IL-12 or an IL-12 fusion protein of the present invention can be used to treat established tumors, such as to prevent the further development of a tumor, cause established tumors to regress, prolong survival, or a combination thereof. The fusion proteins have certain advantages over the corresponding native proteins in that they can be made efficiently and reproducibly by the methods described herein. Furthermore, the fusion proteins of the present invention may also have advantages over the corresponding native proteins in terms of modified or enhanced activity, more favourable bioavailability and improved pharmacokinetic properties.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

Brief Description of the Drawings Figure 1 shows the structures of SFG-based retroviral constructs for interleukin-12 production (SD=splice donor; IRES=internal ribosome entry site; SA=splice acceptor; LTR=long terminal repeat).

Figure 2 shows the nucleic acid sequences encoding the linker sequences in interleukin-12 fusion proteins and flanking IL-12 p35 and IL-12 sequences (SEQ ID NO: 1 to 4 and 35), as well as the encoded amino acid sequences (SEQ ID NO: 5 to 7 and 36).

Figures 3A-3U show the full restriction map and the nucleic acid sequence (SEQ ID NO:8 and 9) of pUC19-SFG.

Figures 4A-4C show the nucleic acid sequence (SEQ ID NO: 10 and 11) encoding the murine IL-12 p35 subunit and C:\WNWORDUOANNE\PJC\SPECIg g198DIV.DOC -4the amino acid sequence of the murine IL-12 p35 subunit (SEQ ID NO: 12).

Figures 5A-5D show the nucleic acid sequence (SEQ ID NO: 13 and 14) encoding murine IL-12 p40 subunit and the amino acid sequence (SEQ ID NO: 15) of the murine IL-12 subunit.

Figure 6 shows a standard curve generated using recombinant murine IL-12.

Figures 7A-7D show graphic representations of the effect of immunotherapy of CMS-5 tumor-bearing mice with wild-type, GM-CSF- and IL-12-secreting CMS-5 cells.

Treatment was started either on day 7 (7A and 7B) or day 14 (7C and 7D) after tumor challenge. Endpoints are either survival (7A and 7C) or tumor-free survival (7B and 7D).

Tumors were either untreated or treated with GM-CSFsecreting CMS-5 cells IL-12-secreting CMS-5 cells (c) or wild type CMS-5 cells Figure 8 is a graphic representation of the incidence of regression of established CMS-5 tumors by type of immunotherapy. Tumors were treated as follows: column 1 received no immunotherapy; column 2 was treated with wildtype tumor cells; column 3 was treated with GM-CSFsecreting tumor cells; and column 4 was treated with IL-12- .secreting tumor cells.

25 Figure 9 is a graphic representation of tumor regression in mice treated with systemic IL-12. The open square and closed square, triangle, circle and diamond represent 5 individual mice treated with systemic IL-12 at 0.1 pg/d given 5 days per week for 4 weeks.

Figures 10A-10B show graphic representationsof the superior survival resulting from immunotherapy with IL-12secreting CMS-5 cells compared to systemic IL-12 administration or no treatment (nil). Figure 10A depicts results using a tumor inoculum.of 2 x.10 s cells. Figure I L depicts results using a tumor inoculum of 4 x 10 s cells.

Figures 11A-11B show graphic representations of the comparison of efficacy (proportion of mice surviving) of CMS-5. cells secreting different forms of IL-12 as immunotherapy for established CMS-5 tumors. Figure 11A shows results using tumors initiated by 2 x 10 s CMS-5 cells with treatment starting on day 14 (20 mice per group pooled from two experiments). Figure 11B shows results using tumors initiated by 4 x 10 s CMS-5 cells with treatment starting on day 14 (1 group of 10 mice). The tumors were either untreated or treated with wild type CMS-5 cells GM-CSF-secreting CMS-5 cells native IL-12secreting CMS-5 cells or IL-12 fusion protein-secreting CMS-5 cells Figures 12A-12C are graphic illustrations of the results of immunotherapy of B16 (melanoma) tumors with cytokine-secreting tumor cells. For the pre-existing tumor model, tumors were initiated with 4 x 10 s B16 cells and immunotherapy commenced on day 7 (Figure 12A) or day 14 (Figure 12B). For the challenge model (Figure 12C), 5 x 10 irradiated cells were administered as a vaccine 14 days before tumor challenge with 1 x 106 B16 cells. Tumors were either untreated or treated with wild type B16 cells GM-CSF-secreting B16 cells native IL-12-secreting B16 cells or IL-12 fusion protein-secreting B16 cells Figures 13A-13B are graphic illustrations of the effects on immunotherapy of IL-12 delivery by different cell types in mice with pre-existing renal cell carcinoma;, (RENCA) tumors. Figure 13A shows results when RENCA tumors were treated with either irradiated wild-type CMS-5 tumor cells (C-wt) or CMS-5 tumor cells transduced to secret either native IL-12 (C-nIL-12) or the IL-12 fusion protein (C-scIL-12). Figure 13B shows results when RENCA tumors -6were treated with either a combination of wild-type and RENCA cells (C-wt R-wt), a combination of IL-12 fusion protein-secreting RENCA cells and wild type cells (C-wt R-IL-12) or a combination of IL-12 fusion protein-secreting CMS-5 cells and wild type RENCA cells (C- IL-12 R-wt).

Figures 14A-14B are a graphic representation of the effects on immunotherapy of pre-existing CMS-5 tumors with IL-12 fusion protein-secreting RENCA tumor cells. Figure 14A shows the results when CMS-5 tumors were treated with either wild type RENCA cells or RENCA cells transduced to secrete the IL-12 fusion protein. Figure 14B shows the results when CMS-5 tumors were treated with either a combination of wild type RENCA and wild type CMS-5 cells S 15 (C-wt R-wt), a combination of IL-12 fusion proteinsecreting RENCA cells and wild type CMS-5 cells (C-wt R- IL-12) or a combination of IL-12 fusion protein-secreting cells and wild type RENCA cells (C-IL-12 R-wt).

Detailed Description of the Invention Described herein are bioactive fusion proteins which comprise at least two subunits linked or joined by an intervening amino acid linker, a method of producing the bioactive fusion proteins, constructs useful for producing the fusion proteins which can be expressed in host cells, and host cells containing the constructs.

In one embodiment, the bioactive fusion proteins of Sthe present invention comprise: 1) at least two polypeptide submnits or monomers which correspond to polypeptide subunits present in a native dimeric protein which has a specified bioactivity and 2) at least one polypeptide linker which joins the subunits in such a manner that the resulting fusion protein is bioactive. If the resulting fusion-protein is dimeric (includes two subunits or monomers), the-two components-can be subunits which occur -7in the same native dimeric protein two IL-12 subunits); subunits which occur in two different native dimeric proteins one subunit from IL-12 and one subunit from IL-3) or monomers which are individually bioactive IL-2, GMCSF). Multimeric fusion proteins, which comprise three or more subunits joined by polypeptide linkers, can comprise, for example, three or more of the subunits which occur in the same native dimeric protein three or more IL-12 subunits), three or more 10 subunits which occur in different native dimeric proteins two IL-12 subunits and one IL-3 subunit), three or more bioactive monomers three IL-2 monomers, two IL- 2 monomers and one GMCSF monomer) or a combination of subunits from native dimeric proteins and bioactive monomers two IL-12 subunits and a GMCSF monomer).

In each case, a polypeptide linker is present between two subunits the order is subunit-linker-subunit-linkersubunit). As used herein, the terms subunit and monomer are used interchangeably to refer to the components of a dimeric or multimeric protein and the single component of a monomeric protein. The order of subunits in the fusion protein of the present invention can be p35-linker-p40 or p40-linker-p35. In either case, the polypeptide linker is positioned between the two subunits. A bioactive fusion protein of the present invention which includes subunits which occur in the same native dimeric protein "mimics" or is similar to what is referred to herein as a corresponding native dimeric protein in terms of its bioactivity, but differs from the corresponding native dimeric protein in that the fusion protein includes linker amino acid residues which do not occur in the corresponding native protein (heterologous amino acid residues) between each pair of polypeptide subunits. A corresponding native protein is one which includes the subunits present in the fusion protein and exhibits biological activity also exhibited by the fusion protein.

For example, in the case of a bioactive IL-12 fusion protein, the two subunits, designated p35 and p40, of a mammalian native IL-12 protein human, mouse, rat, dog, cat, monkey, chimpanzee or pig IL-12 protein) are joined through a polypeptide linker. Here, the corresponding native protein is the mammalian native IL-12 protein. Similarly, in the case of another bioactive fusion protein, such as IL-3, the corresponding native protein is IL-3. The amino acid residues of the subunits of the bioactive fusion protein can be the same as those of the subunits of the corresponding native protein or can be different, provided that the resulting fusion protein 15 exhibits the desired bioactivity. For example, the subunit(s) can have a different amino acid sequence from that of the corresponding subunit of a native protein the sequence of the native subunit can differ in that one or more amino acid residues has been deleted or replaced by a naturally-occurring or non-naturallyoccurring amino acid residue, additional amino acid residues have been incorporated, or an amino acid residue has been modified). The desired bioactivity is activity like that of the corresponding native protein it 25 produces a physiological response which also results from the activity of the corresponding native protein). The bioactivity of a fusion protein the duration of its :effect, extent of the resulting response) may be greater or lesser than that of the corresponding native protein.

'The polypeptide linker present in the fusion protein can be of any length and composition appropriate to join two subunits in such a manner that the resulting fusion protein has the desired biological activity and retains its integrity as a dimer or multimer. -The appropriate length and composition of a linker can be determined empirically -9for the specific fusion protein to be produced. Generally, the polypeptide linker will be at least 7 amino acid residues, although it can be shorter 2 to 6 amino acid residues). Typically the linker will be less than amino acid residues in length, such as 7 to 25 amino acid residues or 7 to 20 amino acid residues in length. In one embodiment, the polypeptide linker is 7 to 16 amino acid residues and in specific embodiments is 7, 11, 15 or 16 amino acid residues. Specific linkers used in producing bioactive IL-12 fusion proteins are represented in Figure 2 and described in Example 4. In specific embodiments, the polypeptide linkers are exemplified by the sequences (GlySer) 3 (GlySer) 3 Ser; (Gly 4 Ser) 2 Ser and Gly,Ser, and these linkers can also be used to join subunits of other 15 fusion proteins in addition to the IL-12 fusion proteins of the present invention. Alternatively, other polypeptide linkers can be used to join two IL-12 subunits to produce a bioactive IL-12 fusion protein.

The DNA encoding the bioactive fusion protein can be 20 cDNA or genomic DNA and can be from a variety of animals, particularly mammals. For example, the DNA can be human, mouse, rat, dog, cat, monkey, chimpanzee, pig or ferret DNA. The DNA can encode a complete or entire subunit a complete IL-12 p35 subunit and a complete IL-12 p40 subunit) or a fragment or portion of a subunit(s), provided that the encoded fusion protein has the desired biological activity when it is expressed. The nucleic acid sequences of DNA encoding mouse IL-12 p35 and p40 subunits are'represented in Figures 4 and 5, respectively. The nucleic acid sequences of DNA encoding human IL-12 p35 and subunits have been published (Gubler et al. in Proceedings of the National Academy of Sciences, USA, 88:4143 (1991); Figure 4A-4C and 5A-5D). All or a portion of IL-12 DNA can be used to produce the subject IL-12 fusion protein, provided that the encoded fusion protein is bioactive (has IL-12 activity).

Any expression system appropriate for expressing a protein such as a mammalian, bacterial, yeast or insect expression system, can be used to express the fusion proteins of the present invention. For example, as described herein, a viral a retroviral) vector which expresses DNA cDNA) encoding the desired fusion protein in a mammalian host cell has been used. As also described herein, retroviruses containing cDNA encoding the and p40 subunits of IL-12 and an intervening polypeptide linker (an IL-12 fusion protein) have been constructed and transfected into packaging cells BOSC23 packaging cells). Target cells 15 fibrosarcoma cell line) were infected with virus-containing supernatants and cultured; media conditioned by infected cells was assayed for IL-12 activity using an interleukin-2 and concanavalin-A primed splenocyte proliferation bioassay. Packaging or producer cell lines other than BOSC23 cells can be used to produce infectious retroviruses containing the fusion protein-encoding DNA. In addition, target cells other than a fibrosarcoma cell line, such as B16 melanoma or renal cell carcinoma cell lines, can be used to produce the fusion protein. IL-12 bioactivity was 25 demonstrable in cells infected with the retroviruses, as described in Example 4.

Specific retroviruses have been constructed for .expression of an IL-12 fusion protein (Example 1 and Figure 1) and cells infected with the retroviruses have been shown to produce bioactive IL-12 fusion proteins (see Example 4).

The retroviruses used all included the SFG retroviral backbone whose sequence is shown in Figure 3. The vectors designated pSFG.IL-12.p35 and pSFG.IL-12.p40 include, respectively, the cDNA for the IL-12 p35 subunit or the cDNA for the IL-12 p40 subunit. The vector designated

I

-11pSFG.IL-12p35-IRES-p40 includes cDNA encoding the IL-12 subunit and cDNA encoding the IL-12 p40 subunit, separated by an internal ribosome entry site sequence. The vector designated pSFG.IL-12p40-IRES-p35 includes the same components as plasmid pSFG.IL-12p35-IRES-p40 but the dimers are in the reverse order, as indicated. The vectors.

designated pSFG.IL-12.p35-linker-p40 and pSFG.IL-12.p40include cDNAS encoding each IL-12 subunit linked by the (Gly 4 Ser) 2 Ser and (Gly 4 Ser) 3 Ser linker respectively.

The vectors designated pSFG.IL-12.p35-linker-Ap40 and pSFG.IL-12.p40-linker-Ap35 include linked cDNAs in which sequences encoding a putative 22 amino acid leader sequence were deleted from the second cDNA. The vector designated pSFG.hIL-12.p40.1inker.Ap35 is a human form of the IL-12 fusion protein and is analogous to the murine form pSFG.IL- 12.p40.1inker.Ap35 except that the linker is shorter due to a deletion which occurred during the cloning (see Figure 2, construct As described in Example 4, IL-12 bioactivity was shown in conditioned medium from cells infected with 20 the retroviruses.

Prokaryotic and eukaryotic host cells transfected by the described vectors are also provided by this invention.

For instance, cells which can be transfected with the vectors of the present invention include, but are not limited to bacterial cells such as E. coli, insect cells (baculovirus), yeast or mammalian cells such as Chinese hamster ovary cells (CHO). Tumor cells which are transduced to secrete the'IL-12 fusion proteins of the present invention are particularly useful in the present invention.

Thus, expression vectors described herein can be used to transform, transfect or transduce host cells, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), using standard procedures -12used in producing other well known proteins. Similar procedures, or modifications thereof, can be employed to prepare recombinant proteins according to the present invention by microbial means or tissue-culture technology.

For example, fibroblast-derived 3T3 cells can be transduced with the vectors of the present invention to express and secrete the IL-12 fusion proteins of the present invention.

Tumor cells and 3T3 cells are useful in the context of the.

present invention, as cells transduced to secrete IL-12 or IL-12 fusion proteins of the present invention are a useful source of the protein or fusion protein for purification). Tumor cells transduced to secrete IL-12 or IL-12 fusion proteins also have particular utility as antitumor agents as described herein.

15 The tumor cells to be transduced can be selected from the individual to be treated or from another individual; furthermore, the tumor cells to be transduced can be the same type as the tumor cells of the tumor to be treated or the tumor cells can be of a different type from the tumor to be treated. For example, a CMS-5 tumor can be treated with CMS-5 tumor cells, renal cell carcinoma (RENCA) tumor cells, or B16 tumor cells which secrete IL-12 or an IL-12 fusion protein of the present invention. Alternatively, the tumor can be treated with a combination of IL-12- 25 secreting, IL-12 fusion protein-secreting and wild type cells of the same or different cell type. For example, a RENCA tumor can be treated with a combination of wild type :RENCA cells and IL-12 fusion protein-secreting CMS-5 cells or with a combination of native IL-12-secreting CMS-5 cells and -L-12 fusion protein-secreting RENCA tumor cells' The present invention also relates to transduced tumor cells which express native IL-12 or IL-12 fusion proteins of the present invention and their use in treating tumors.

That is, transduced tumor.cells which express and secrete IL-12 or IL-12 fusion proteins are useful as therapeutic .L -13agents for the treatment of cancer or to treat established tumors, and provide a means for reversing tumors (reducing their size or causing their complete regression) or preventing further growth of an established tumor. As described herein, transduced tumor cells expressing IL-12 or IL-12 fusion proteins of the present invention cause the regression of established tumors, prevent the establishment of tumors, prolong survival or a combination thereof in animals to which they are administered in a therapeutically appropriate dose.

For instance, the tumor cells secreting IL-12 or the IL-12 fusion protein of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition. The particular 15 physiological medium may include, but is not limited to, water, buffered saline, polyols glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.

The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists, and will depend on the ultimate pharmaceutical formulation desired.

Methods of introduction of the IL-12-secreting or IL-12 fusion protein-secreting tumor cells include, but are not limited to, intradermal, intramuscular, intraperitoneal, 25 intravenous, subcutaneous, oral and intranasal. The tumor cells secreting native IL-12 or an IL-12 fusion protein can be administered at or near the site of a tumor to be treated or at any other site on the body, provided that the IL-12 or IL-12 fusion protein produces the desired therapeutic effect (regression of established tumors, prevention of tumor establishment or prolonged survival)..

SAs described herein, proximity of tumor site and administration site is not necessary for the efficacy of the treatment. Other suitable methods of introduction can also- include rechargeable-or-biodegradable devices and slow -14release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents. Treatment regimens will depend upon the dose, route of delivery, frequency with which the composition is administered, type, size and stage of the tumor to be treated, and the age, health and other physical characteristics of the individual to be treated.

In one method of the invention, a therapeutically effective quantity (dose) of the transduced tumor cells (optionally formulated with a physiologically appropriate medium) is administered to an individual having a tumor to be treated decreased in size or prevented from increasing in size). The transduced tumors cells can also 15 be administered in a therapeutically appropriate dose to an individual to prevent the establishment of a tumor. For example, the transduced tumor cells can be administered to an individual in need of anticancer therapy an individual with an established tumor or an individual in whom establishment of a tumor is to be prevented).

Alternatively, the IL-12 fusion protein of the present invention can be administered directly to the individual in a therapeutically effective dose; the IL-12 fusion protein can be optionally combined with a physiologically 25 acceptable medium as described above.

As described herein, the efficacy of IL-12-secreting tumor cells as antitumor immunotherapy was assessed in mice 'with established tumor burdens. The immunogenic (fibrosarcoma) and non-immunogenic B16 (melanoma) tumors were used; RENCA tumors were also utilized as described herein.

As shown in Examples 6-8, work described herein demonstrates that for the immunotherapeutic treatment of S14-day established palpable CMS-5 tumors, immunotherapy with IL-12-secreting and IL-12 fusion protein-secreting tumor cells prolonged survival by inducing the regression of tumors. Furthermore, immunotherapy with IL-12-secreting tumor cells induced tumor regression even when the palpable tumor burden averaged more than 5% of body mass. Although IL-12 has antitumor activity against CMS-5 tumors when administered systemically, for mice with larger tumor burdens at the onset of therapy there was a significant survival advantage of IL-12-secreting tumor cell immunotherapy over systemic IL-12 therapy. This shows that there is an advantage in delivering the IL-12 by transduced tumor cells rather than just as systemic cytokine therapy.

Data from tumor cells transduced to express an IL-12 fusion S: protein (SFG.IL-12.p40.1inker.Ap35) indicate that the murine and human forms of the fusion protein have a 15 specific activity at least equal to the native molecule in an in vitro bioassay.

Results described herein show that IL-12-secreting B16 cell vaccination altered the natural history of the growth of later established B16 tumors, and they also appear to be able to enhance immunological mechanisms capable of modulating tumor growth. IL-12-secreting B16 cells are useful as immunotherapy for established B16 tumors, as they effectively prolong survival. These results show that there exist inducible innate mechanisms able to modulate 25 the natural history of established tumors in the mouse, and that IL-12-secreting cells are more potent at inducing them than GM-CSF-secreting cells. These results, which are more fully described in the Examples below, show that IL-12secreting tumor cells have efficacy as immunotherapy for established tumors.

The present invention is illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLE 1 Construction of Plasmids -16- The general structure of the plasmids used in these studies is shown schematically in Figure 1. The confirmed sequences of the linkers in each of the fusion proteins are given in Figure 2.

Source of plasmids The plasmids containing cDNAs for the murine IL-12 and p40 subunits (pBS.IL-12.p35 and pBS.IL-12.p40) were provided by Hoffmann-La Roche (Nutley, NJ). The numbering of base pairs in this document corresponds to the maps of the inserts of these two plasmids (Figures 4 and The plasmid containing the SFG retroviral backbone was provided ."by Dr. Dan Ory (Whitehead Institute, Cambridge, MA) as pSFG-TPA, a pUC plasmid containing the SFG retroviral backbone between the HindIII and EcoR1 sites with a tissue 15 plasminogen activator cDNA between the unique Ncol and BamH1 sites in the SFG retrovirus. A nucleotide sequence map of the SFG retroviral backbone is shown in Figure 3.

o Plasmid pSFG.IL-12.p35 The IL-12p35 cDNA was provided in pBluescript with the sequences surrounding the translational initiation ATG optimized to ACCATGG according to the rules of Kozak. The IL-12p35 cDNA fragment was excised as a Ncol-EcoRl fragment, the EcoR1 overhang having been filled using the Klenow fragment of E. colil DNA polymerase 1. This fragment was ligated using T4 DNA ligase into the Ncol-BamHl sites -of pSFG, the BamH1 overhang having been filled using the Klenow fragment of E. coli DNA polymerase 1. The resulting plasmid is designated pSFG.IL-12.p35.

Plasmid pSFG.IL-12.40 The IL-12p40 cDNA was provided in pBluescript. The Ncol-BamHl- fragment containing the IL-12p40 cDNA was -17excised and ligated into the Ncol-BamHl sites of pSFG to make pSFG.IL-12.p40.

General Strategy for Construction of SFG-based Vectors The general strategy for constructing the SFG-based retroviral vectors for IL-12 fusion protein expression is as follows: Two oligonucleotides encoding the sense and antisense strand of a (Gly 4 Ser) 3 linker fragment and contiguous IL-12 cDNA sequences to be linked (with terminal sequences for the creation of cohesive ligatable overhangs) were synthesized using a "PCR-mate" 391 DNA synthesizer (Applied Biosystems, Foster City, CA). The sequence of the (Gly 4 Ser) 3 linker was that of Huston et al. (Proc. Natl.

Acad. Sci. USA, 85:5879-5883(1988)).

For the two fusion proteins using complete IL-12 15 cDNAs, the oligonucleotides were designed to be cloned into a unique restriction enzyme site at the 3' end of the first cDNA, reconstructing the 3' end of the first cDNA and Senabling a Ncol-Ncol fragment encompassing the full cDNA and linker sequence to be cloned into the Ncol site of the SFG plasmid containing the other cDNA.

The cloning strategy was similar for the two fusion proteins with a deletion of 66 bp coding the first 22 amino acids of the second cDNA. Linker oligonucleotides were S" designed to be cloned into unique restriction enzyme sites that lay 3' of bp 66 of the translated bases of the second cDNA in the fusion protein construct. This enabled a fragment to be excised for cloning that reconstructed the 3' end of the first cDNA joined to the linker and contained the linker joined to codon 23 of the second cDNA.

The sequence of the linker and contiguous cDNA regions in plasmids was determined using a "Sequenase" kit (Amersham, Cleveland, OH).

-18- Plasmid--DSFG.IL-12 .D35-linker-D4o The oligonucleotides were: sense,

TCGGTCGGGTGGC.GG

C.GGA.TCT.TCCATGGAGCT-39 (SEQ ID NO: 16); and antisense, 5'-CCATGGAAGA.TCC.GCC.GCC.ACC.C

CCCACCACCGCCCGA.GCC.

ACC.GCC.ACC.GGCGGGCT31 (SEQ ID NO: 17).

These two oligonucleotides were annealed, phosphorylated using T4 polynucleotide kinase, and ligated into the Sac. site of pBS.IL-12.p35 which had been dephosphorylated using calf intestinal phosphatase. The Ncol-Ncol fragment of the resulting plasmid containing the IL-12p35 cDNA and correctly orientated linker was excised and ligated into the dephosphorylated Ncol site of pSFG.IL-12p40 to create pSFG.IL-12.p35-linker.p40 (the correct orientation of this ligated fragment was demon- :e :'strated by a Sadl digest).

This plasmid was sequenced using the following two primers: 5'-CAGAGTGAAAATGAAGCT-3' (SEQ ID NO: 18) and -GAAGCTCTGCATCCTGCT-3' (SEQ ID NO: 19), corresponding to bp 601-618 and 613-630 of the IL-12p35 cDNA. Sequencing demonstrated that a deletion had occurred during cloning resulting in a loss of 15 bp from the linker sequences, but maintaining an intact reading frame. The sequence of the linker in this plasmid is-given in Figur~e 2.

P-lasmid _DSFG -IL-12,.D40. linker, The oligonucleotides were: sense, GGC.GGC.GGA.TCT.TCCATG-3' (SEQ ID NO: 20); and antisense-, 5'9.GATCCATGGA.AGA.TCC.GCC.GCC.ACC.CGACCCACCACCCCCGA.G CC.ACC.GCC.ACC-GGATCGGACCCTGCA-3' (SEQ ID NO: 21.).

These two oligonucleotides were annealed and ligated' into the SseS3871 and BaT1l sites Of PBS.IL-12.p40. The Ncol-Ncol fragment of the resulting plasmid containing th e IL-12p40 cDNA and correctly orientated linker was excised -19and ligated into the dephosphorylated Ncol site of pSFG.IL-12p35 to create pSFG.IL-12.p40.linker.p35 (the correct orientation of this ligated fragment was demonstrated by a Xcml digest).

This plasmid was sequenced using the following two primers: 5'-CTATTACAATTCCTCATG-3' (SEQ ID NO: 22) .and 5'-GAGGGCAAGGGTGGCCAA-3' (SEQ ID NO: 23), corresponding to base pairs 997-1014 of the IL-12 p40 cDNA and base pairs 91-74 of the IL-12 p35 cDNA (an antisense primer).

Sequencing confirmed that the sequence of the linker and contiguous IL-12 cDNA sequences were as expected.

4. Subsequent restriction enzyme mapping of pSFG.IL-12.p40.1inker.p35 after the transfection and expression studies were completed revealed that it probably 15 contained a concatamer of Ncol-Ncol-fragments from the final cloning step.

Plasmid DSFG.IL-12.35 linker.oAp4 The oligonucleotides were: sense, 20 G.TCG.GGT.GGC.GGC.GGA.TCT.ATG.TGG-3' (SEQ ID NO: 24) and antisense, C.GCC.ACC.GGCGGAGCTCCAGCAAA-3' (SEQ ID NO: These two oligonucleotides were annealed, phosphorylated using T4 polynucleotide kinase, and ligated into pBS.IL-12.p40 from which the 30 base pair 5' Xcml-Xcml fragment had been excised. The Sacl-Sacl fragment from the resultant plasmid was excised and ligated into the Sad site of pBS.IL-12.p35 which had been dephosphorylated using calf intestinal phosphatase (the correct orientation of the ligated fragment was demonstrated by a Ncol-EcoR1 digest).

The Ncol-EcoR1 fragment of the resultant vector was excised, the EcoR1 overhang having been filled using the Klenow fragment of E. coli DNA polymerase 1, and ligated into the Ncol and Kienow-filled BamHl sites of pSFG to create pSFG. IL-12 .p35 .linker.Adp4O.

This plasmid was sequenced using the following primers: 5'-CAGAGTGAAAATGAAGCT-3' (SEQ ID NO: 18) and 5'-GAAGCTCTGCA.TCCTGCT-3 I (SEQ ID NO: 19), corresponding to base pairs 601-618 and 613-630 of the IL-12p35 cDNA, and 5'-GTCATCTTCTTCAGGCGT-3' (SEQ ID NO: 34), an antisense primer corresponding to base pairs 217-200 of the IL-i2 cDNA. Sequencing confirmed that the sequence of the linker and contiguous IL-12 cDNA sequences were as expected.

Plasmid r)SFG. IL-12 .D40-linker.Ar,35 The oligonucleotides were: sense, .GGToGGG.TCGGGT.GGC.GGC.GGA.TCT.AGG.GTCATTCGTCT- 3 (SEQ ID 1NO: 26) and antisense, :00 A.GCC.ACC. GCCoACC.GGATCGGACCCTGCAGGCCAGAGA.3o (SEQ ID NO: 27).

:.o~oThese two oligonucleotides were annealed, phosphorylated using T4 polynucleotide kinase, and ligated into the PflM1 site in pBS.IL-12.p35 which had been dephosphorylated using calf intestinal phosphatase. The orientation of this ligated fragment was, confirmed by an 5se83871/EcoR1 digest. The Sse83871-EcoRl fragment from the resultant plasmid was excised, the EcoRi overhang having been filled using the Klenow fragment of E. coi DNA polymerase 1, and ligated'into the Sse8387l and \Klenow-filled EamHI sites of pSFG.IL-l2.p40 to create pSFG. IL-12 .p40 This plasmid was sequenced using the primer 5'-GCAAAGGCGGGAATGTCT-3' (SEQ ID NO: 28), corresponding to base pairs 960-977 of the IL-12.p40 CDNA. The sequence of the second linker codon was difficult-to read, but its sequence was-determined-by sequencing the cloned linker in -21the intermediate plasmid using the antisense primers 5'-AGGAATAATGTTTCAGTT-3' (SEQ ID NO: 29) and 5'-CAGCAGTGCAGGAATAAT-3' (SEQ ID NO: 30) corresponding to base pairs 224-207 and 233-216 of the IL-12 p35 cDNA respectively. Sequencing confirmed that the sequence of the linker and contiguous IL-12 cDNA sequences were as expected.

Plasmids pSFG.IL-12.p35.IRES.p40 and pSFG.IL- 12.p40.IRES.p35 The encephalomyelocarditis virus (ECMV) internal ribosome entry site (IRES) fragment was provided by Dr.

Michael Sadelain (Whitehead Institute, Cambridge, MA), and was as previously described (Ghattas et l. Mol. Cell.

Biol., 11:5848-5859 (1991)).

15 EXAMPLE 2 Cells and Tissue Culture BOSC23 packaging cells (Pear et al., Proc. Natl. Acad.

Sci. USA, 90:8382-8396(1993)) were obtained from Dr. Dirk Lindemann (Whitehead Institute, Cambridge, MA). They were passaged in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% calf serum, 50 U/ml penicillin and g/ml streptomycin.

CMS-5 tumor cells (DeLeo et Exp. Med., 146:720-734 (1977)) were obtained from Jason Salter (Whitehead Institute, Cambridge, MA). They were passaged in DMEM supplemented with 10% foetal calf serum, 50 U/ml penicillin and 50 pg/ml streptomycin. The same medium was used for the collection of CMS-5 conditioned medium.

C57BL/6 splenocytes for IL-12 assays were obtained by mincing a spleen through a sieve (Falcon 2350, Becton 30 Dickinson, Franklin Lakes, NJ) and collecting the cells in IL-12 medium (as detailed in Schoenhaut al. (.L Immunol,, 148:3433-3440 (1992)) supplemented with 2% foetal calf serum.

-22- EXAMPLE a Generation of BOSC23-derived Producer Cells and Collection of Conditioned Media BOSC23 cells were plated at 2 x 106cells per 6 cm tissue culture dish and transfected by CaPO 4 transfection with the various constructs as previously described (Pear .t al., Proc. Natl. Acad. Sci. USA, 90:8382-8396 (1993)).

Twenty-four hours after transfection, the medium was replaced with 5 ml fresh medium. Virus-containing supernatants were collected 24 hours later, filtered through a 0.45 pm filter and polybrene added to a final concentration of 8 pg/ml. 2.5 ml of virus-containing supernatant was used to infect CMS-5 cells immediately for 4 hours (in preparation for this infection, CMS-5 cells had been plated at 5x10' cells/6 cm tissue culture dish the '15 previous day) and the remaining 2.5 ml frozen at -70 *C.

The following day, the frozen 2.5 ml of virus-containing supernatant was thawed and used for a second 4 hour infection of the CMS-5 cells. To collect IL-12-containing conditioned medium, the medium was replaced the following day with 5 ml fresh medium which was harvested 24 hours later. These conditioned media were filtered through a 0.2 pm filter and frozen at -70°C for later assay for IL-12 bioactivity. 5 ml of fresh medium was added to the cells and a second set of conditioned media collected 24 25 hours later which were also filtered and frozen for later assay. The infected CMS-5 cells were then lysed, and genomic DNA prepared for later analysis.

SEXAMPLE 4 Bioassav for Murine Interleukin-12 Levels of bioactive interleukin-12 were determined using a concanavalin-A and interleukin-2 primed splenocyte proliferation assay, as described in Schoenhaut e al. (J.

Immunol.,148:3433-3440 (1992)). The concanavalin A was obtained commercially from Boehringer (Mannheim, Germany) -23and the recombinant human interleukin-2 commercially from Chiron Therapeutics (Emeryville, CA). To harvest cells for the measurement of 3 H]thymidine incorporation into cellular DNA, a Skatron (Sterling, VA) cell harvester and filtermats (#7031) were used. To assay for inhibitory activity in conditioned media, the 50 sl sample volume comprised 25 p1 of 1000 pg/ml recombinant murine IL-12 and 1l of the test sample. Samples of conditioned media were assayed in duplicate at several dilutions in the range 1:1 to 1:1000. A standard curve was constructed for each bioassay using recombinant murine IL-12 in the range 20-10,000 pg/ml. The recombinant murine IL-12 was obtained S: from Hoffmann-La Roche (Nutley, NJ). To calculate the bioactive IL-12 concentration in test samples in pg/ml, the 15 linear part of the standard curve was approximated using the curve-fit function of "KaleidaGraph 2.1.1" software and the resultant formula used for calculations. Conditioned media were verified to have hIL-12 immunoreactivity by hIL- 12 ELISA assay (commercial kit, R D Systems).

The following constructs (Figure 1) were assessed for their ability to express a bioactive IL-12 fusion protein: A. pSFG.IL-12.p35.1inker.p40; B. pSFG.IL-12.p40.linker.p35; C. pSFG.IL12-p35.linker.Ap40; D. pSFG.IL12-p40.1inker.Ap35; and E. pSFG.hIL-12.p40.1inker.Ap35.

The sequences for the linkers in each construct were as follows, as confirmed by sequencing (some adjacent confirmed IL-12 sequences are given for orientation): -24- A. 5'->>>IL-2p35.AGC.TCC.GCC-GGT.GGT.GGT.GGG.TCGGGTGGC .GGC.GGA.TCT.TCC.ATG.GGT.CCTCAG. >IL12p 4

O

3 9 (SEQ ID NO: 1); B. S'->,,IL-12p4.CCC.TGC.AGG.GTC.CGA.TCCG.GGC.GGTGGC

.TCG.GGC.GGT.GGT.G.TCG.GGT.CC.GGATTCCATGG

GT.CAA.u>>IL-2p35-3' (SEQ ID NO: 31); C. 5' >>IL-12p35.S'

TAT.CTG.AGC.TCC.GCC-GGT.GGC.GGT.GGC.

TCG.GGC.GGT.GGT.GGG.TCG.GGT.GGCGGCGGATCTATGTGGGA

G.CTG.GAG.AAA.>>>IL-12p40 3' (SEQ ID NO: 32); D. 5'->>>IL-12p40.TGT.GTT.CCC.TGC.AGG.GTC.CGATCC-GGTGGC

.GGT.GGC.TCG.GGC.GGT.GGT.GGG.TCG.GGT.CGGCGGATCTA

GG.GTC.ATT.CCA.GTC.TCT.GGA.CCT.GCC. >>>IL-1 2 p 3 5-3' (SEQ ID NO: 33); and E. S'->>hIL-12p4O.TGC.AGT.GGT.GGC.GGT.GGC.GGC GGA.TCT.AGA.AAC.>>>hIL-12p35-3' (SEQ ID NO: No IL-12 bioactivity was detectable in media conditioned by mock-transfected CMS-5 cells, and cells infected with the SFG retrovirus alone, or by a related retrovirus (MFG) carrying the -lac-z gene. However, media conditioned by these cells contained significant inhibitory activity at 1:2 and 1:10 dilutions, inhibiting as much as 95t of the bioactivity of 500 pg/ml of rmIL-12 (Table 1, and other data not shown). Despite this 'background of inhibitory activity in the conditioned media, bioactive IL-12 production proved to be still demonstrable.

Constructs for the expression of single subunits of the IL-12 protein (pSFG.Il-12.p35 and pSFG.IL-l2.p40) resulted in no detectable bioactivity on their own.

However, cotransfection of BOSC2i cells with these constructs together resulted in bioactive IL-12 secretion by infected CMS-5 cells. Similarly, CMS-5 cells infected with the SFG.IL-12.p35 retrovirus and 24 hours later with the SFG.IL-12.p40 retrovirus also produced bioactive IL-12 (Table 1).

The dicistronic constructs designed to express both IL-12 subunits using the IRES sequence resulted in similar levels of bioactive IL-12 production (despite an undetectable level of viral infection as determined by Southern hybridization analysis (see below)) (Table 1).

The ability of IRES-containing retroviruses to result in bioactive IL-12 production has been confirmed by generating stable clonal retrovirus producing cell lines using both these constructs.

All IL-12 fusion protein constructs resulted in S 15 significant bioactive IL-12 production by infected cells. Of particular note was the SFG.IL-12.p40 linker.Ap35 construct, for which IL-12 bioactivity was demonstrable in undiluted conditioned medium (despite the background of substantial inhibitory activity) and for which a 1:1000 dilution of conditioned medium contained bioactivity equivalent to 301 pg/ml of rmIL-12 (Table 1).

All constructs resulted in titratable IL-12 bioactivity 'despite significant non-specific inhibitory activity in the conditioned media as well. Bioactivity of hIL-12 was confirmed at Hoffman-LaRoche (Nutley, NJ, laboratory of Dr.

M. Gately), and the specific activity of the hIL-12 fusion protein was determined to be approximately equivalent to the specific activity of recombinant native human IL-12.

9 4, so 6. 4* 4 *4 4. 9 TABLE

I

Construct ConsrucAgonist assay CIL-12 bioactivity, pg/mi Antagonist assay (V inhibition of .500 pg/mi IL-12 in assay) Dilution 1:1 of CM in 1:100 assay 1:1000 Dilution 1:2 of CM in assay 1:10 1:1000 N4o DNA <50 <50 <50 56 62 8.6 SFG-empty <50 <50 <50 12 47 -91 MFG-lac-z <50 <50 <50 66 56 64 SFG.IL-12p35 <50 <50 <50 65 76 SFG.IL-12p40 <50 <50 <50 94 84 38 2X infection^ 199.7 234.2 137.2 1 1 -84 2X transfection b 244.7 118.8 <50 12 -3 -2 A 86.5 <50 <50 44 60 46 B 253.8 <50 <50 41 12 -14 C 189.2 57.0 <50 43 42 47 D 297.8 600.1 301.2 -48 -143 -93 These data are from one of three separate assays.

a. Target cells infected sequentially with pSFG.IL-12.p35 and then pSFG.IL-12.p40 viruses..(each containing only the respective cDNA between the Ncol and BamI sites) b. BOSC23 cells were transfected with a mixture of pSFG.IL-12p35 and pSFG.IL,-12.p4o constructs I I -27- These data indicate IL-12 agonist activity was present in media conditioned by cells infected with the fusion protein retroviral constructs. It is presumed that this results from bioactivity of secreted respective fusion proteins.

The fusion proteins were demonstrated to be present using Western blotting. Serum-free CM from cells or CMS-5 cells expressing native IL-12 or the IL-12 fusion protein (SFG.IL-12.p35.IRES.p40 or SFG.IL- 12.p40.1inker.Ap35) were collected, filtered (0.2 Am) and stored at -70 0 C. The CMs were concentrated 20-30-fold, and 20 pg total protein sample was run on 10% polyacrylamide gels with or without 10% -mercaptoethanol. The primary antibody was a polyclonal goat anti-rmIL-12 antibody (gift 15 of D. Presky, Hoffman-La Roche, NJ). The "Renaissance" detection system (NEN Dupont) was used. A preliminary analysis indicated a 4-fold greater signal resulted from CM containing the single chain IL-12 (SFG.IL- 12.p40.1inker.Ap35) fusion protein, and hence a 5 Ag total 20 protein sample from this CM was loaded. The control lanes were CM from wild-type cells without or spiked with 50 ng rmIL-12.

EXAMPLE 5 Southern Hybridization Analysis of Genomic DNA from Infected CMS-5 Cells Southern hybridization analysis of genomic DNA from the populations of infected CMS-5 cells was performed to demonstrate the presence of a hybridizing band consistent with infection of these cells by retroviruses of the expected structure, and to determine the efficiency of viral infection (by determination of retroviral copy number by genome).

From these Nhel digests of genomic DNA, a hybridizing retrovirus-derived band of 985 bp plus the size of the insert cloned into the Ncol-BamHl sites of SFG was -28predicted (See Figure The size of the various cloned fragments were: IL-12.p35 cDNA, 0.6 kb; IL-12.p40 cDNA, kb; IRES, 0.7 kb; linker, 0.05 kb; the putative leader sequence deleted in two constructs was 0.066 bp.

The BOSC23 cell supernatants resulted in viral copy numbers of between 0.1 and 1.4 copies/genome (mostly ,0.1- 0.3 copies/genome) for all constructs except for the IREScontaining constructs, where no hybridizing band of the expected size (3.2 kb) was seen (Table 2).

Of particular note are the comparative results for the IL-12 fusion proteins retrovirus constructs in these populations of infected cells. Although the pSFG.IL-12.p35.1inker.p40 retrovirus was present at 1.4 copies/genome, this corresponded with a relatively low 15 level of bioactive IL-12 production (Table However, the SFG.IL-12.p40 linker.Ap35 retrovirus resulted in a relatively high level of IL-12 bioactivity, although it was S'present at 0.2 copies/genome.

9 r -29- TABLE 2: Retrovirus Cony Number in CMS-S Cells infected by SFG. IL-12 Retroviruses SFG.IL-12 construct Retrovirus containingT: cony number" Nil 0 IL-12 .p35 0.1 IL-12 .p40 0.3 Sequential infection (p35/p40) 0.3/0.3 Co-transfection (p351p4O) 0.1/0.1 IL-12.p35-IRES-p4o <Ol IL-12.p40.IRES.p35 <Ol IL-12.p35.linker.p40 IL-12.p40.linker.p35 0 1 b/ IL-12.p35.linker.Ap4O0.b IL-12.p40.linker.Ap350.b a a a.

a a a 1 copy control 0.1 copy control 1. O 0 .l a Relative to a plasmid copy number control of 13.5 pg of pSFG.IL- 12.p35 linker.p40, calculated to be equimolar to 1 copy/genome for pg genomic DNA.

Mean of results from one Southern blot probed first with a p35 and then with a p40 radiolabelled probe. Relative intensity of signals was quantitated using a Fuji BAS-II phosphoimager.

EXAMPLE 6 Comparison of Immunotherapy of Established Immunogenic CMS-5 Tumors with GM-CSF-secreting and IL-12-secreting Tumor Cells Cvtokine-Secreting Tumor Cells SFG retroviruses generated by CRE or CRIP packaging cell lines were used for the transduction of tumor cells.

The amount of cytokine secreted in vitro by the tumor cells used in these studies were (in ng/ml/48h/lO irradiated cells [all collected in 10 cells infected with CRIP- 10 packaged SFG.GM-CSF, B16>250, CMS-5>250; cells infected with CRE-packaged SFG.p35.IRES.p40.IL-12, B16 1-3, 60-400; cells infected with CRE-packaged

SFG.IL-

12.p40.1inker.Ap35, CMS-5 490-950; cells infected with CRIP-packaged SFG.IL-12.p35.IRES.p40, Bl6 90; and cells infected with CRIP-packaged SFG.IL-12.p40.1inker.Ap35, B16 170 and RENCA 45. The tumor cells were irradiated to prevent the formation of additional tumors therefrom after injection into mice, and cytokine secretion was characterized for the same irradiated cells. GM-CSF concentrations of conditioned media (CM) were determined by ELISA (Endogen, Cambridge) and IL-12 levels by a bioassay based on the proliferation of concanavalin-A and interleukin-2 primed splenocytes (Schoenhaut e al. j Immunol 148:3433 (1992)).

In an initial procedure, fibrosarcoma tumors were initiated with 2 x 10 s CMS-5 tumor cells injected subcutaneously on the back of syngeneic BALB/C mice, and immunotherapy (irradiated wild-type, GM-CSF-secreting or IL-12-secreting tumor cells) commenced either 7 or 14 days later. In this experiment, mice were stratified into multiple groups of 5 to 10 mice that received either 1, 2.

or 3 weekly doses of immunotherapy at either 1 x 1 0 6 or 5 x cells/dose. However, the primary analysis was -31stratified only by the type of cells used as immunotherapy and the day on which treatment began, regardless of other scheduling variables.

Mice treated with irradiated IL-12-secreting tumor cells showed significantly better long-term tumor-free survival compared to untreated mice or mice treated with wild-type or GM-CSF-secreting tumor cells, for therapy schedules starting either 7 or 14 days after tumor challenge (Figures 7A and 7B, p<0.05 for all comparisons with IL-12-secreting tumor cell immunotherapy). When 0 immunotherapy was commenced 7 days after tumor transplantation, much of the survival advantage of mice treated with IL-12-secreting tumor cells was due to prevention of the development of late tumors (Figure 7C, 15 p<0.05 for all comparisons by the log rank test). When immunotherapy commenced 14 days after tumor transplantation, much of the survival advantage was due to the regression of established tumors (Figures 7C, 7D and 8, p<0.005 compared to mice receiving wild-type or GM-CSF- 20 secreting tumor cell immunotherapy). The palpable tumors that regressed in mice receiving IL-12-secreting tumor cell immunotherapy range from 1 to 8.5 mm in average diameter, and became impalpable 20-43 days (median 30 days) after tumor transplantation. The number of animals per group were respectively: 7A and 7B, 137, 40, 40, 38; and 7C and 7D, 18, 39, 40, 40. P-values given are for the least significant difference between treatment with IL-12secreting cells and other groups. To determine the overall effect, data were pooled from groups that received likeimmunotherapy cells by several schedules.

Subgroup analyses of therapies commencing on day 14 suggested that superior survival from immunotherapy with IL-12-secreting tumor cells resulted from schedules with more than one weekly dose of immunotherapy doses of 5 x 10' rather than 1 x 10' IL-12-secreting cells -32- Hence, in all subsequent experiments utilizing transduced tumor cells, immunotherapy regimens comprised the higher cell dose administered weekly for 4 weeks.

Statistical Analyses All analyses were conducted on the basis of the intention to treat at the time of the random allocation of mice to groups. Descriptive statistics were calculated for major endpoints. Except where otherwise stated, differences in the survival endpoint were evaluated by the Wilcoxon rank-sum test. For survival analyses, occasional deaths immediately after anaesthesia and treatment were treated as censored events. The chi-square test was used to measure the association of categorical variables. Where p-values summarize the comparisons between multiple groups, 15 only the largest p-value is given. Analyses were conducted using JMP software on a Power Macintosch 6100/60 computer.

o* EXAMPLE 7 Study of Mechanisms of IL-12-induced Tumor Regression and Improved Survival In order to determine whether immunotherapy with IL- 12-secreting tumor cells was effective against larger tumor burdens, tumors were established with 4 x 10' tumor cells, which, compared with 2 x 10 s cells, resulted in higher tumor incidence (day 14 palpability rates of 98/100 vs.

83/100, respectively), larger mean tumor size (6.7±3.0 vs.

3.7±2.4 mm diameter at day 14, respectively) and shorter median survival without treatment (31 vs. 37 days).

Following establishment of these larger tumors, 70% (7/10) of" tumor-bearing mice treated with IL-12-secreting tumor cell immunotherapy from day 14 survived with complete tumor regression, compared with 0/10 mice treated with wild-type tumor cells (ps0.001).

The administration of IL-12 systemically (intraperitoneally) to mice bearing tumors initiated by 2 x -33s CMS-5 cells also resulted in the regression of established tumors (Figure 9) and improved survival (4/5 at days for mice treated with 0.1 pg/d, compared to 0/5 for placebo-treated mice). For mice with tumors established from 2 x 10 s CMS-5 cells, an IL-12 dose of 0.1 g/d survival) was superior to 1 (3/5 survival), 0.01 survival) and 0.001 pg/d, for regimens starting either, 7 or 14 days after tumor transplantation.

It was therefore possible that the regression of tumors in mice receiving immunotherapy with IL-12-secreting cells was not dependent on the local release of IL-12 at the site of the irradiated tumor cells administered as immunotherapy, but rather on a systemic effect of IL-12.

This was evaluated by comparing different schedules :15 starting on day 14 which combined either wild-type or IL- 12-secreting tumor cell immunotherapy and systemic therapy with either IL-12, placebo or nothing. In mice with tumors initiated by 2 x 10 s CMS-5 cells, there was a tendency for median and overall survival to be better for immunotherapy 20 with IL-12-secreting tumor cells than for systemic IL-12 therapy (Figure 10A). This tendency was statistically significant for mice with tumors initiated by 4 x 10 5 cells (Figure 10B, p=0.006 comparing groups receiving systemic IL-12 (either alone or in combination with wild-type cells) vs. mice receiving IL-12-secreting tumor cell immunotherapy (either alone or with systemic therapy with diluent)).

Comparisons of smaller, uniformly-treated groups tumors initiated by 4 x 10 s cells) indicated.

that. combining administration of wild type cells with systemic IL-12 was not different to systemic IL-12 therapy alone (p=0.85) and appeared inferior to vaccination with IL-12-secreting tumor cells alone (p=0.04) or given with placebo systemic therapy (p=0.19).

-34- EXAMPLE 8 Antitumor Effect of IL-12 Fusion Protein In the pre-existing CMS-5 tumor model, immunotherapy with CMS-5 tumor cells expressing the IL-12 fusion protein SFG.IL-12.p40.1inker.Ap35 was as effective as therapy with tumor cells making native IL-12 (Figures 11A and 11B). For mice with tumors initiated by either 2 x 10 s or 4 x 1' s survival was greater than 90% for groups of mice treated with CMS-5 cells secreting either form of IL-12, compared to less than 40% for mice that received no treatment, or treatment with wild-type or GM-CSF-secreting cells (ps0.02).

EXAMPLE 9 Comparison of ImmunotheraDy of Established Nonimmunogenic B16 Tumors with GM-CSF-secretina and IL-12-secretinq Tumor Cells In order to assess the efficacy of immunotherapy with IL-12-secreting tumor cells in another tumor model, the non-immunogenic B16 melanoma was studied. B16 tumor cells were transduced to make native IL-12 at 90 ng/ml/48hr/10 6 irradiated cells or the single chain IL-12 (SFG.IL- 12.p40.1inker.Ap35) at 170 ng/ml/48hr/10' irradiated cells.

B16 tumors were initiated with 4 x 10 s cells and S. immunotherapy of established tumors commenced either on day 7 (25% tumor palpability) or day 14 (93% tumor palpability, mean tumor diameter 5.74±3.23, This procedure was analyzed after 31 days of follow up, when only 1/60 of mice that were treated with wild-type cells, CM-CSFsecreting cells or nothing as immunotherapy survived.

Although mice treated with IL-12-secreting cells had comparably poor overall survival, their median survival was significantly prolonged compared to that of control mice treated with wild-type cells when treatment commenced on day 7 (Figure 12A, 24 vs. 18 days p=0.01) and day 14 (Figure 12B, 28 vs. 18 days, p=0.0005). Similarly, median survival was prolonged with therapy with IL-12 fusion protein-secreting tumor cells when treatment commenced on day 7 (21 vs. 18 days, p=0.08) and day 14 (24 vs. 18 days, p=0.006). In 3/4 scenarios, IL-12-secreting tumor cells were superior to GM-CSF-secreting cells (respective pvalues 0.01, 0.14, 0.003, 0.02).

Given the potent effect of GM-CSF-secreting B16 cells to induce antitumor immunity when used as a vaccine prior to tumor challenge, but their lack of effect on tumor growth when administered after tumor establishment, the effects of IL-12-secreting and GM-CSF-secreting B16 cells were compared as vaccines in a B16 tumor challenge model.

The IL-12-secreting B16 cells used in initial studies secreted native IL-12 at 1-3 ng/ml/48hr/10' irradiated cells. GM-CSF-secreting B16 cells induced antitumor 15 immunity when used as vaccines before tumor transplantation (Figure 12C, 80% 100-day survival).

EXAMPLE 10 Immunotherapeutic Effect of IL-12 Delivery by Tumor Cells of Different Oriqin from Tumor to be Treated The effect of the delivery of IL-12 by tumor cells of different origin from the tumor to be treated on survival was assessed in renal cell carcinoma (RENCA) tumors.

RENCA

tumors were initiated with 4 x 10 s cells, and immunotherapy of established tumors commenced on day 14. In one procedure, groups of mice were treated with either irradiated wild type CMS-5 cells or CMS-5 tumor cells transduced to secrete either native IL-12 or the fusion protein SFG.IL-12.p40.linker.Ap35 (Figure 13A). In. another procedure, additional groups of mice were treated with a S 30 combination of irradiated CMS-5 and RENCA wild type cells, a combination of irradiated wild type CMS-5 and IL-12secreting RENCA tumor cells or a combination of irradiated wild type RENCA tumor cells and IL-12-secreting CMS-5 tumor cells (Figure 13B).

-36- In the first procedure, immunotherapy with both tumor cells secreting native IL-12 and the IL-12 fusion protein prolonged the median survival (p-values of p-0.02 and p-0.06, respectively). In the second procedure, mice treated with a combination of irradiated RENCA tumor cells and IL-12-secreting CMS-5 tumor cells exhibited a trend toward increased survival.

Additionally, CMS-5 tumors were initiated with 4 x 10 s cells, and immunotherapy of established tumors commenced on day 14. In one procedure, the established CMS-5 tumors were treated with either irradiated wild type RENCA tumor cells or RENCA tumor cells transduced to secrete either native IL-12 or the IL-12 fusion protein SFG.IL- 12.p40.1inker.Ap35 (Figure 14A). In another procedure, 15 additional groups of mice were treated with a combination of irradiated CMS-5 and RENCA wild type cells, a combination of irradiated wild type CMS-5 and IL-12secreting RENCA tumor cells or a combination of irradiated wild type RENCA tumor cells and IL-12-secreting CMS-5 tumor 20 cells (Figure 14B).

In the first procedure, immunotherapy with RENCA cells secreting the IL-12 fusion protein moderately prolonged the survival of the mice compared to mice treated with wild type RENCA cells; the effect was consistent with the lower dose of IL-12 delivered by the transduced RENCA cells. In the second procedure, both mice treated with a combination of IL-12-secreting CMS-5 cells and wild type RENCA cells Sand mice treated with a combination of IL-12-secreting

CMS-

cells and wild type RENCA cells showed significantly prolonged survival (p-values of p=0.004'and p-0.04)" compared with mice treated with wild type RENCA and wild.

type CMS-5 cells.

-37- Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

-38- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Whitehead Institute for Biomedical Research STREET: 9 Cambridge Center CITY: Cambridge STATE/PROVINCE: MA COUNTRY: US POSTAL CODE/ZIP: 02142 TELEPHONE: 617-258-5104 TELEFAX: 617-258-6294

APPLICANT/INVENTOR:

NAME: Graham J. Lieschke STREET: 5 Rollins Court CITY: Cambridge STATE/PROVINCE: MA COUNTRY: US POSTAL CODE/ZIP: 02139

APPLICANT/INVENTOR:

NAME: Richard C. Mulligan STREET: 2 Sandy Pond Road CITY: Lincoln STATE/PROVINCE: MA COUNTRY: US POSTAL CODE/ZIP: 01773 (ii) TITLE OF INVENTION: Bioactive Fusion Proteins and Pre-existing Tumor Therapy (iii)' NUMBER OF SEQUENCES: 36 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Hamilton, Brook, Smith Reynolds, P.C.

STREET:.Two Militia Drive -39- CITY: Lexington STATE: Massachusetts COUNTRY: USA ZIP: 02173 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/385,335 FILING DATE: 08-FEB-1995 (viii) ATTORNEY/AGENT INFORMATION: e NAME: Granahan, Patricia REGISTRATION NUMBER: 32,227 REFERENCE/DOCKET NUMBER: WHI95-01A.PCT (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 617-861-6240 TELEFAX: 617-861-9540 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 54 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID KO:l: AGCTCCGCCG GTGGTGGTGG GTCGGGTGGC GGCGGATCTT CCATGGGTCC TCAG S4 INFORMATION FOR SEQ ID NO:2: (i)SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRAZUDEDZNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genemic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GTCCGATCCG GTGGCGGTGG CTCGGGCGGT GGTGGGTCGG GTGGCGGCGG ATCTTCCATG GGTCAA 66 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID K0:3: AGCTCCGCCG GTGGCGGTGG CTCGGGCGGT GGTGGGTCGG GTGGCGGCGG ATCTATGTGG GAGCTG .66 -41- INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GTCCGATCCG GTGGCGGTGG CTCGGGCGGT GGTGGGTCGG GTGGCGGCGG ATCTAGGGTC

ATTCCA

66 C..o INFORMATION FOR SEQ ID

PC

SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 1 5 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown -42ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly (fly Ser Ser 1 10 is INFORMATION FOR SEQ ID NO:7: Ci) SEQUENCE CHARACTERISTICS: LENGTH: i5 amino acids CB) TYPE: amino acid STRA2NDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 8cr I 5 10 INFORMATION FOR SEQ ID NO:S: Ui) SEQUENCE CHARACTERISTICS: CA) LENGTH: 63S0 base pairs TYPE: nucleic acid STRA1NDEDNESS: unknown CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) lxi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AAGCTTTGCT CTTAGGAGTT TCCTAATACA TCCCAAACTC AAATATATAA AGCATTTGAC TTGTTCTATG CCCTAGGGGG CGGGGGGAAG CTAAGCCAGC TTTTTTTAAC ATTTAAAATG 1.20 TTAATTCCAT TTTAAATGCA CAGATGTTTT TATTTCATAA GGGTTTcAAT GTGTGAAT GCTGCAATAT TCCTGTTACC

AAAGCTAGTA

TTAGAGTTTC TGTCATTAAC

GTTTCCTTCC

AAGCCAGTTT GCATCTGTCA

GGATCAATTT

CAATTAGTTG ATTTTTATTT

TTGACATATA

TGGCAAGCTA GCTTAAGTAA,

CGCCATTTTG

AGAAAAGTTC AGATCAAGQT

CAGGAACAGA

ATCTGTGGTA AGCAGTTCCT

GCCCCGGCTC

TGGGCCAAAC AGGATATCTG

TGGTAAGCAG

TGGTCCCCAG ATGCGGTCCA

GCCCTCAGCA

G;TGCCCCAAG GACCTGAAAT

GACCCTGTGC

TCGCTTCTGT TCGCGCGCTT

ATGCTCCCCQ

TCGGGGCGCC AGTCCTCCQA

TTGACTGAGTC

TCTTGCAGTT GCATCCGACT

TGTGGTCTCGC

TTGACTACCC GTCAGCGGGG

GTCTTTCATT

GCCCAGGGAC CACCGACCCA CCACCGGGAG

G

TCCGATTGTC TAGTGTCTAT

GACTGATTTTA

TAGCTCTGTA TCTGQCGGAC CCGTGGTGGA

A

CCTGGGAGAC GTCCCAGGGA

CTTCGGGGGC

TCCCGATCGT TTAGGACTCT TTGGTGCACC

CE

AGGAQACQAG AACCTAAAAC AGTTCCCGCC

T(

CCGAAGCCGC GCCGCGCGTC TTGTCTQCTG

CJ

-43- TAAATAAAA TAGATAAACG TGGAAATTAC TCAGTTGACA ACATAAATGC

GCTGCTGAGC

CCCATTATGC CAGTCATATT

AATTACTAGT

CATGTQAATG AAAGACCCCA

CCTGTAGGTT

CAAGGCATGG AAAAATACAT

AACTGAGAAT

TGGAACAGCT GAATATGGGC

CAAACAGGAT

AGGGCCAAGA ACAGATGGAA

CAGCTGAATA

TTCCTGCCCC GGCTCAGGGC

CAAGAACAGA

GTTTCTAGAG AACCATCAGA

TGTTTCCAGG;

TTATTTGAA CTAACCAATC

AGTTCGCTTC

=GTCAMTA AAGAGCCCAC

AACCCCTCAC

'GCCCGGGTA CCCGTGTATC

CAATAAACCC

*TGTTCCTTQ GGAGGGTCTC

CTCTGAGTGA

'GGGGGCTCG TCCGGGATCG

GGAGACCCCT

TAAGCTGGC CAGCAACTTA

TCTGTGTCTG

TGCGCCTGC GTCGGTACTA

GTTAGCTAAC

CTGACGAGT TCGGAACACC cG;GCCGCAAC ~TTTTTG1'G GCCCGACCTG AGTCCTAA

.AA

CCCTTAGAG GAGGGATATG

TGGTTCTG;GT

:CGTCTGAA TTTTTGCTT

CGGTTTGGGA

GCATCGTT CTGTGTTGTC TCTGTCTGAC *480 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 240 300 360 420 -44- AAAATATGGG CCCGGGCTAG TGTGTTTCTG TATTTGTCTG CCTGTTACCA CTCCCTTAAG 1500 9

TTTGACCTTA

CAAGAAGAGA

GCCGCGAGAC

ACCTGGCCCG

TTTTGACCCC

TCCATCCGCC

TTATCCAGCC

GGCACCCCCG

CTCTCTCCAA

GGCGGCAGCC

CGACACAGTG

TTACACAGTC

ACACGCCGCC

GCGCGGATCC

ITTGACTCAA

GATTTTATTT

GCTAGCTTAA

GTTCAGATCA

GGTAAGCAGT

GGTCACTGGA

CGTTGGGTrA

GGCACCTTTA

CATGGACACC

CCTCCCTGGG

CCGTCTCTCC

CTCACTCCTT

CCCCTTGTAA

GCTCACTTAC

TACCAAGAAC

TGGGTCCGCC

CTGCTGACCA

CACGTGAAGG

GGATTAGTCC

CAATATCACC

AGTCTCCAGA

GTAACGCCAT

AGGTCAGGAA

TCCTGCCCCG

CCTTCTGCTC TGCAGQAATGG

*ACCGAGACCT

CAGACCAGGT

TCAAGCCCTT

CCCTTGAACC

CTCTAGGCGC

ACTTCCCTGA

AGGCTCTCTA

AACTGGACCG

GACACCAGAC

CCCCCACCGC

CTGCCGACCC

AATTTGTTAA

AGCTGAAGCC

AAAAGGWGG

TTTGCAAGGC

CAGATGGAAC,

GCTCAGGGCC

CATCACCCAG

GGGGTACATC

TGTACACCCT

TCCTCGTTCG

CCCCATATGG

CCCTGACATG

CTTAGTCCAG

ACCGGTGGTA

TAAGAACCTA

CCTCAAAGTA

CGGGGGTGGA

AGACAGGATA

TATAGAGTAC

AATGAAAGAC

ATGGAAAAAT

AGCTGAATAT

A.AGAACAGAT

CCAACCTTTA

GTTAAGATCA

GTGACCTGGG

AAGCCTCCGC

ACCCCGCCTC

CCALTATGAGA

ACAAGAGTTA

CACGAAGTCT

CCTCACCCTT

GAACCTCGCT

GACGGCATCG

CCATCCTCTA

TCAGTGGTCC

GAGCCATAGA

CCCACCTGTA

ACATAACTGA

GGGCCAAACA

ACGTCGGATG

AGGTCTTTTC

AAGCCTTGGC

CTCCTCTTCC

GATCCTCCCT

TCTTATATGG

CTAACAGCCC

GGAGACCTCT

ACCGAGTCGG

G-GAAAGGACC

CAGCTTGGAT

GACTGCCATG

AGGCTCTAGT

TAAAATAAAA

GGTTTGGCAA

CAATAGAGAA

CGATATCTGT

AAGATGTCGA GCGGATCGCT CACAACCAGT CGGTAGATGT 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2590 2640 GGAACAGCTG AATATGGGCC AAACAGGATA*TCTGTGGTAA GCAGrTCCT CCCCGGCTCA GGGCCAAWA cAGATGGTCC 2700 9 .9 9* 9* 99 9. 9 9 9 9*S 9 CCAGATGCGG TCCAGCCCTc AGCAGTTTCT CAAGGACCTG AAATGACCCT

GTGCCTTATT

CTGTTCGCGC GCTTCTGCTC

CCCGAGCTCA

CGCCAGTCCT CCGATTGACT

GAGTCGCCCG

AGTTGCATCC GACTTGTGGT

CTCGCTGTTC

ACCCGTCAGC GGGGGTCTTT

CACACATGCA

CTTAAGTATT TACATTAAAT GGccATAG.TA ACATGGAGTA TTCAQAATGT

GTCATAAATA

CTTTCTACTT TTTCTTTTAT

TTTTTTTTGT

TTGTTTGTTT GTTTGTTGGT

TGGTTGGTTA

CAAGCTAGAC TATTAGCTAC TCTGTAACCC TGTTTTAGCC TTCCCACATC

TAAGATTACA(

TGATTGATTG ATTGATGTGT

GTGTGTGTGA

GTGTATGGGT GTGTGTGAAT

GTGTGTATGT

GTGCATGTGT GTGTGTGTGA CTGTG;TCTAT

C

GTGTGTGTGT GTGTGTQTGT

GTGTGTTGTG

GCTCCGGCTC AGGTGTCAGG

TTGGTTTTTG

ACTGGCCGTC GTTTTACAAC GTCGTGACTG G CCTTGCAGCA CATCCCCCTT TCGCCAGCTG G CCCT'TCCCAA CAGTTGCGCA

GCCTGAATGGC

TACGI ATCTG TGCGQTATTT CACACCGCAT AGAGAACCAT

CAGATGTTTC

TGAACTAACC

AATCAGTTCG

ATAAAAGAGC

CCACAACCCC

GGTACCCGTG

TATCCAATAA

CTTGGGAGGG

TCTCCTCTGA

GCATGTATCA AAATTAATTT CTTAAAGTTA

CATTGGCTTC

TTTCTAATTT

TAAGATAGTA

CCTCTGTCTT

CCATTTGTTG

ITTTTTTTT AAAGATCCTA4 kGGGTGACCT

TGAAGTCATGC

;GTATGAGCT ATCATTTTTG rTGTGTTTGT GTGTGTGACT

C

=TTGTGTGT GTGAGTGTGT

C

TGTATGACT GTGTGTGTGT

G

LAAAAkTATT

CTATGGTAGTG

,GACAGAGTC

TTTCACTTAGC

CGAAACCCT

GGCGTTACCCA

CGTAXTAGC GAAGAGGCCC

G

GAATGGCGC

CTGATGCGGTA

TGGTGCACT

CTCAGTACAAT

CAGGGTGCCC

CTTCTCGCT2'

TCACTCGGGG

ACCCTCTTGC

GTGATTGACT

GGTTTTTTTT

CTTGAAATAA

TCTCCATTGG

rTGTTGTTGT

CACTATAGTT

;GTAGCCTGC

.TATATTGAT

TGAAAATGT

TGTGTGTGT

FTGTGTGTGT

'AGAGCCAAC

TTGGAATTC

ACTTAATCG

CACCGATCG

TTTTCTCCT

CTGCTCTGA

2760 2820 2880 2940 -3000 3060 3120 3180 3240 3300 3360 3420 3480 3S40 3600 3660.

3720 3790 3840 3900 3960 j.

C. 4

TGCCGCATAG

TTGTCTGCTC

TCAGAGGTTT

TATTTTTATA

GGGAUAATGT

CGCTCATGAG

GTATTCAACA

TTGCTCACCC.

TGGGTTACAT

AACGTTTTCC

TTGACGCCGG

AGTACTCACC

GTGCTGCCAT

GACCGAAGGA

GTTGGCGALCC

TAGCAATGGC

GGCAACAATT

CCCTTCCGGC

GTATCATTGC

CGGGGAGTCA

TTAAGCCAGC

CCGGCATCCG

TCACCGTCAT

GGTTAATGTC

GCGCGCIAACC

ACAATAACCC

TTTCCGTGTC

AGAAACGCTG

CGAACTGGAT

AATGATGAGC

GCAAGACAA

AGTCACAGAA

AACCATGAGT

GCTAACCGCT

GGAGCTGAAT

AACAACGTTG

AATAGACTGG

TGGCTZGGTTT

AGCACTGGGG

GGCAACTATG

CCCGACACCC

CTTACAGACA

CACCGAAACG

ATATATAA

CCTATTTGTT

TGATAAATGC

GCCCTTATTC

GTGAAAGTAA

CTCAACAGCG

ACTTTTAAG

CTCGGTCGCC

AAGCATCTTA

GATAACACTG

TTTTTGCACA.

GAAGCCkTAC.

CGCAAACTAT

ATGGAGGCGG.

ATTGCTGATA

CCAGATGGTA

-46-

GCCAACACCC

AGCTGTGACC

CGCGATGACG

TGG "TTTA

TATTTTTCTA

'TTCAATAATA

CCTTTTTTGC

AAGATGCTGA

GTAAGATCCT

TTCTGCTATG

GCATACACTA

CGGATGGCAT

CGGCCAACTT

AECATGGGGGA

CAAACGACGA

TAACTGGCGA

ATAAAGTTGC

ftATCTGGAGC

AGCCCTCCCG

GCTGACGCGC

GTCTCCGGGA

AAAGGGCCTC

GACGTCAGGT

AATACATTCA

TTGAAAAAGG

GGCA.TTTTGC

AGATCAGTTG

TGAGAGTTT

TGGCGCGGTA

TTCTCAGAAT

GACAGTAAGA

ACTTCTGACA

TCATGTAACT

GCGTGACACC,

ACTACTTACT

AGGACCACTT

CGGTGAGCGT

CCTGACGGGC

GCTGCATGTG

GTGATACGCC

GGCACTTTTC

AATATGTATC

AAGAGTATGA

CTTCCTGTTT

GGTGCACGAG

CGCCCCGAAG

TTATCCCGTA

GACTTGGTTG

GAATTATGCA

ACGATCGGAG

CGCCTTGATC

ACGATGCCTG

CTAGCTTCCC

CTGCGCTCGG

GGGTCTCGCG

4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 S 040 5100 5160 S220 C C TATCGTA= ATCTACACGA CGCTGAGATA GGTGCCTCAC GATGAACGAA~ ATAGACAGAT TGATTAAGCA TTGGTAACTG TCAGACCAAG* -lTAT=TA TATACTTTAG ATTGATTTAA -47- TGAAGATCCT TTT77GATAAT cTcATGACCA ACTATTT TTAATTTAAA

AGGATCTAGG

5280 AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAG;

GATCTTCTTG

CGCTACCAGC

AGATCCTTTT

GGTGGTTTGT

CTGGCTTCAG CAGAGCGCAG

ACCACTTCAA

TGGCTGCTGC

CGGATAAGGC

GAACGACCTA

CCGAAGGGAG

CGAGGGAGCT

TCTGACTTGA

CCAGCAACGC

TTCCTGCGTT

CCGCTCGCCG

GCCCAATACG

ACAGGTTTCC

CTCAiTACGGC

TGAGCGGATA

GAACTCTGTA

CAGTGGCGAT

GCAGCGGTCG

CACCGAACTG

AAAGGCGGAC

TCCAGGGGGA

GCGTCGATTT

GGCCTTTTTA

ATCCCCTGAT

CAGCCGAACG

CAAACCGCCT

CGACTGGAAA

ACCCCAGGCT

ACAATTTCAC

TTTCTGCGCG

TTGCCGGATC

ATACCAA-ATA

GCACCGCCTA

AAGTCGTGTC

GGCTGAACGG

AGATACCTAC

AGGTATCCGG

AACGCCTGGT

TTGTGATGCT

CGGTTCCTGG

rCTGTGGATA kCCGAGCGCA

'TCCCCGCGC

TAALTCTGCTG

AAGAGCTACC

CTGTCCTTCT

CATACCTCGC

TTACCGGGTT

GGGGTTCGTG

AGCGTGAGCA

TAAGCGGCAG

ATCTTTATAG

CGTCAGWGGQ

CCTTTTGCTG

ACCGTATTAC

GCGAGTCkGT

GTTGGCCGAT

CCCCGTAGAA

;CTTGCAAACA

AACTCTTTTT7

AGTGTAGCCG

TCTGCTAATC

GGACTCAAGA

CACACAGCCC

TTGAGAAAGC

GGTCGGAACA

TCCTGTCGGG

GCGGAGCCTA

GCCTTTTGCT

CGCCTTTGAG

GAGCGAGGAA

TCATTAATGC2 AATTAATGTG3

TCGTATGTTG

TGATTACGCC

AAGATCAAAG

AAAAAACCAC

CCGAAGGTAA

TAGTTAQGCC

CTGTTACCAG

CGATAGTTAC

AGCTTGGAGC

GCCACGCTTC

GGAGAGCGCA

TTTCGCCACC

TQGAAAAACG

CACATGTTCT

TGAGCTGATA

;CGGAAGAGC

kGCT'GGCACG

LGTTAGCTCA

MQTGGAATTG

5340 5400 S460 5520 5580 5640 5700 5760 5820 5850 5940 6000 6060 6120 6180 6240 6300 6350 GCGGGCAGTG -AGCGCAACGC TTACACTTTA

TGCTTCCGGC

ACAGGAAACA

GCTATGACCA

-48- INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 6350 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLODGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID K0:9: TTCGAAACGA GAATCCTCAA AGGATTATGT AGGGTTTGAG AACAAGATAC GGGATCCCCC GCCCCCCTTC GATTCGGTCG AATTAAGGTA AAATTTACGT GTCTACAAAA ATAAAGTATT

TTTATATATT

AAAAAAATTG

CCCAAAGTTA

TCGTAA.ACTG;

TAAATTTTAC

CACGTACTTA

120

ISO

9**b

CGACGTTATA

AATCTCAAAG

TTCGGTCAAA

GTTAATCAAC

ACCGTTCGAT

WTCTTTTCAAG;

TAGACACCAT

ACCCGGTPTG

ACCAGGGGTC

CACGGGGTTC

AGCGAAGACA.

AGCCCCGCGG

AGGACAATGG

ACAGTAATTG

CGTAGACAGT

TAAAAATAAA

CGAATTCATT

TCTAGTTCCA

TCGTCAAGGA

TCCTATAGAC

TACGCCAGOT

TTTCGATCAT ATT T =TT ATCTATTTGC ACCTTTAATr CAAAGGAAGG AGTCAACTGT CCTAGTTAAA

GGGTAATACG

AACTGTATAT

GTACACTTAC

GCGGTAAAAC

GTTCCGTAC!C

GTCCTTGTCT

ACCTTGTCGA

CGGGGCCGAG

TCCCGGTTCT

ACCATTCGTC AAGGCGGGG CGGGAGTCGT

CAAAGATCTC

TGTATTTACG

GTCAGTATAA

TTTCTGGGGT

TTTTTATGTA

CTTATACCCG

TGTCTACCTT

CCGAGTCCCG

TTGGTAGTCT

CGACGACTC!G

TTAATGATCA

GGACATCCAA

TTGACTCTTA

GTTTGTCCTA

GTCGACTTAT

GTTCTTGTCT

ACAAAGGTCC

240 300 360 420 480 540 600 660 720 780 CTGGACTTTA CTGGGACAcG GAATAAACTT GATTGMTAG

TCAAGCGAAG

AGCGCGCGA.A

TACGAGGGGC

TCAGGAGGCT AACTGACTCA TCGAGTTATT

TTCTCGGGTG

GCGGGCCCAT GGGCACATAG

TTGGGGAGTG

GTTATTTGGG

CS

C.

C

AGAACGTCAA

AACTGATGGG

CGGGTCCCTG

AGGCTAACAG

ATCGAGACAT

GGACCCTCTG

AGGGCTAGCA

TCCTCTGCTC

GGCTTCGGCG

ACACAA.AGAC

AAPACTGGAAT

GTTCTTCTCT

CGGCGCTCTG

TGGACCGGGC

AAAACTGGGG

CGTAGGCTGA

CAGTCGCCCC

GTGGCTGGGT

ATCACAGATA

AGACCGCCTG

CAGGGTCCCT

AATCCTGAGA

TTGGATTTTG

CGGCGCGCAG

ATAAACAGAC

CCAGTGACCT

GCAACCCAAT

CCGTGGAAAT

GTACCTGTGG

GGAGGGACCC

GGCAGAGAGG

GAGTGAGGAA

GGGGAACATT

CGAGTGAATQ

P.TGGTTCTTG

CTGACTAAAA

GGCACCACCT

GAAGCCCCCG

AACCACGTGG

TCAAGGGCGG

AA6CAGACGAC

TTTTATACCC

TTCTACAGCT

GGAA6GACGAG

TGGCTCTGGA

GTCTGGTCCA

AGTTCGGGAA

GGGAACTTGG

GAGATCCGCG

TGAAGGGACT

TCCGAGAGAT

T7TGACCTGGC

TACGCGGACG

TGACTGCTCA

GCAAAAACAC

GGGGAATCTC

AGGCAGACTT

GTCGTAGCAA

GGGCCCGATC

CGCCTAGCGA

ACGTCTTACC

GTAGTGGGTC

CCCCATGTAG

AC .TGTGGGA

AGGAGCAAGC

GGGGTATACC

GMGCTGTAC

GAATCAGGTC

TGGCCALCCA6T -49- ACACCAGAGC GACAAGGAAC CAGAAAGTAA ACCCCCGAGC GGTGGCCCTC CATTCGACCG

CCTCCCAGAG

AGGCCCTAGC

GTCGTTGAAT

CAGCCATGAT

AGCCTTGTGG

CGGGCTGGAC

CTCCCTATAC

AAAAACGAAA

GACACAACAG

GGACAATGGT

GTGTTGGTCA

GGTTGGAAAT

CAATTCTAGT

CACTGGACCC

TTCGGAGGCG

TGGGGCGGAG

GG'iATACTCT

TTCTCAAT

GTGCTTCAGA

GAGACTCACT

CCTCTGGGGA

AGACACAGAC

CAATCGATTG

GCCGGCGTTG

TCAGGATTTT

ACCAAGACCA

GCCAAACCCT

AGACAGACTG

GAGGGAATTC

GCCATCTACA

TGCAGCCTAC

TCCAGAAAAG

TTCGGAACCG

GAGGAGAAGG

CTAGGAGGGA

AGAATATACC

GATTGTCGG

:CTCTGGAGA

960 1020 1080 1140 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160

C

AGGTAGGCGG

AATAGGTCGG

CCGTGGGGGC

GAGAGAGGTT

CCGCCGTCGG

.4 GGAGTGGGAA TGGCTCAGCC GCTGTGTCAC ACCCAGGCGG CTTGTT ATTCTTGGAT Cfl'GGAGCGA cC~CTCTGG -so- GGAGTTTCAT CTGCCGTAGC AATGTGTCAG GACGACTGGT GGGGGTGGCG

GTCGAACCTA

2220 a a

TQTGCGGCGG

CGCGCCTAGG

AAACTGAGTT

CTAAAATAAA

CGATCGAATT

CAAGTCTAGT

CCATTCGTCA

TTTGTCCTAT

GGTCTACGCC

GTTCCTGGAC

GACAAGCGCG

GCGGTCAGGA

TCAACGTAGG

TGGGCAGTCG

GAATTCPATAA

GTGCACTTCC

VCCTAXTCAGG

TCAGAGGTCT

CATTGCGGTA

TCCAGTCCTT

AGGACGGGGC

AGACACCATT

AGGTCGGGAG

TTTACTGGGA

CGAAGACGAG

GGCTAACTGA

CTGAACACCA

CCCCCAGAAA

ATGTAATTTA

GACGGCTGGG

TTAAA.CAATT

TCGACTTCGG

TTTTCCCCCC

AAACGTTCCG

GTCTACCTTG

CGAGTCCCGG

CGTCAAGGAC

TCGTCAAAGA

CACGGAATAA

GGGCTCGAGT

CTCAGCG(;GC

GAGCGACAAG

GTGTGTACGT

CCGGTATCAT

CAGTATTTAT.

AAAAAAAACA

ACCAACCAAT

AGACATTGGG

ATTCTAATGT

TCTGTCCTAT

ATATCTCATG

TTACTTTCTG

TACCTTTTTA

TCGACTTATA

7TCTTGTCTA

GGGGCC=AT

TCTCTTGGTA

ACTTGATTGG

TATTTTCTCG

CCATGGGCAC

GAACCCTCCC

CGTACATAGT

GAATTTCAAT

AAAGATTAA

GGAGACAGAA

TAAAAAAAAA

TCCCACTGGA

CCATACTCGA

AGTCACCAGG

CTCGGTATCT

GGGTGGACAT

TGTATTGACT

CCCGGTTTGT

CCTTGTCGAC

CCCGGTTCTT

GTCTACAAAG

TTAGTCAAGC

GGTGTTGQGGG

ATAGGTTATT

AGAGGAGACT

TTATTAAX

GTAACCGAAG

ATTCTATCAT

GGTAAACAAC

TTTCTAGGAT

ACTTCAGTAC,

TAGTAMAAAC

GCCCCCACCT GGTAGGAGAT

CTGACGGTAC

TCCGAGATCA

ATT&TAT~Ir1, CCXAkACCGTT

CTTATCTCTT

CCTATAGACA

TTATACCCGG

GTCTACCAGG

GTCCCACGGG

GAAGAGCGAA

AGTGAGCCCC

TGGGAGAACG

CACTAACTGA

CCAAAAAAAA

=ACTTTATT

AGAGGTAACC

AACAACAACA

GTGATATCAA

CCATCGGACG

CATATAACTA

2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 TGT,%CCTCPAT AAGTCTTACA GAAG)ATGA AAAGAAAATA AACAAACAAA CAAACAACCA GTTCGATCTG ATAATCGATG ACAAA;LTCGG AAGGGTGTAG

ACTAACTAAC

CACATACCCA

TAACTACACA

CACACACTTA

CACACACACT

CACACATACA

-51-

AACACAAACA

TACACACACA

CACACACTGA

CACTCACACA

CACTTTTACA

CACACACACA

3480 3540 CACGTACACA CACACACACT GACACAGATA CACATACTGA CACACACACA CACACACACA

CACACACACA

CGAGGCCGAG

TGACCGGCAG

GGAACGTCGT

CACACACACA

TCCACAGTCC

CAAAATGTTG

GTAGGGGGAA

CACACAACAC

AACCAAAAAC

CAGCACTGAC

AGCGGTCGAC

ITTTATAA GATACCATCA

CTCTCGGTTG

5 9 *95 9 GGGAAGGGTT GTCAACGCGT CGGACTTACC

ATGCGTAGAC

ACGGCGTATC

AACAGACGAG

AGTCTCCAAA

ATAAAAATAT

CCCCTTTACA

GCGAGTACTC

CATAAGTTGT

AACGAGTGGG

ACCCAATGTA

TTGCAAAAGG

AACTGCGGCC

ACCCCATAAA

AATTCGGTCG

GGCCGTAGGC

AGTGGCAGTA

CCAATTACAG

CGCGCCTTGG

TGTTATTGGG

AAAGGCACAG

TCTTTGCGAC

GCTTGACCTA

TTACTACTCG

CGTTCTCGTT

GTGTGGCGTA

GGGCTGTGGG

GAATGTCTGT

GTGGCTTTGC

TACTATTATT

GGATAAACAA

ACTATTTACG

CG-GGAATAAG

CACTTTCATT

GAGTTGTCGC

TGAAAATC

GAGCCAGCGG

TCTGTCTCAG

CCTTTTGGGA

CGCATTATCG

GCTTACCGCG

TACCACGTGA

CGGTTGTGGG

TCGAC-ACTGG

GCGCTACTGC

ACCAAAGAAT

PLTAAAAAGAT

FLAGTTATTAT

;GAAAAAACG4 r1TCTACGACT

:ATTCTAGGA

%AGPLCGATAC

"GTATGTGAT3

AAAGTGAATC

CCGCAATGGG

CTTCTCCGGG

GACTACGCCA

GAGTCATGTr

CGACTGCGCG

CAGAGGCCCT

TTTCCCGGAG

CTGCAGTCCA

TVATGTAAGT

PACTTTTTCC

CCGTAAAACG

rCTAGTCAAC kCTCTCAAAA kCCGCGCCAT3

LAGAGTCTTA

GAACCTTAAG

TTGAATTAGC

CGTGGCTAGc

TAAAAGAGGA

AGACGAGACT

GGACTGCCCG

CGACGTACAC

CACTATGCGG

CCGTGAAAAG

TTA7ACATAG rTCTCATACT

=AGGACAAA

:CACGTGCTC

;CGGGGCTTC

MATAGGGCAT.

TGAACCAAC

3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 TCATGAGTGG TCAGTGTCTT TTCGTAGAAT GCCTACCGTA CTGTCATTCT CTTAATACGT 4680 CACGACGGTA TTGGTACTCA CTGGCTTCCT CGATTGGCGA CAACCCTTGG CCTCGACTTA ATCGTTACCG TTGTTGCAALC CCGTTGTTAA TTATCTGACC GGGAA6GGCCG ACCGACCAAA CATAGTAACG TCGTGACCCC GCCCCTCAGT CCGTTGATAC 0* C C

C.

C

C.

C C C

*C.C

C

C C

C

ACTAATTCGT

TTGAAGTAAA

TTTAGGGAAT

CTAGAAGAAC

GCGATGGTCG

GACCGAAGTC

TGGTGAAGTT

ACCGACGACG

GCCTAT1TCCG

CTTGCTGGAT

GGCTTCCCTC

GCTCCCTCGA

AACCATTGAC

AATTAAATTT

TGCACTCAAA

TCTAGGAAAA

CCACCAAACA

GTCTCGCGTC

CTTGAGACAT

GTCACCGCTA

CGTCGCCAGC

GTGGCTTGAC

TTCGCT

AGGTCCCCCT

CTATTGTGAC

AAAAACGTGT

CTTCGGTATG

GCGTTTGATA

TACCTCCGCC

TAACGACTAT

GGTCTACCAT

CTACTTGCTT

AGTCTG=TC

TCCTAGATCC

AGCAAGGTGA

AAAGACGCGC

AACGGCCTAG

TATGGTTTAT

CGTGGCGGAT

TTCAGCACAG

CCGACTTGCC

TCTATGGATG

TCCATAGGCC

TTGCGGACCA

-52- GCCGGTTGAA TGAAGACTGT TGTACCCCCT AGTACATTGA GTTTGCTGCT CGCACTGTGG ATTOACCGCT TGATAATGA TATTTCAACG TCCTGGTGAA TTAGACCTCG GCCACTCGCA TCGGGAGGGC ATAGCATCAA TATCTGTCTA GCGACTCTAT AAATGADTAT ATATGAAATC ACTCTAGGA AAAACTATTA CTCGCAGTCT GGGGCATCTT TGCTAGCCTc

GCGGAACTAG

TGCTACGGAC

GATCGAAGGG

GACGCGAGCC

CCCAGAGCGC

TAGATGTGCT

CCACGGAGTG

TAACTAAATT

GAGTACTGGT

TTCTAGTTTC

TTTTTTGGTG

GGCTTCCATT

ATCAATCCGG

GACAATGGTC

GCTATCAATGz

TCGAACCTCG

CGGTGCGAAG

CCTCTCGCGT

AAAGCGGTGG

4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 .5820 5890 5940

ATTAGACGAC

TTCTCGATGQ

GACAGGAAGA

GTATGGAGCG

AALTGGCCCAAL

CCCCAAIGCAC

TCGCACTCGT

ATTCGCCGTC

TAGAAATATC

GAACGTTTGT

TTGAGAAkAA

TCACATCGGC

AGACGATTAG

CCTGAGTTCT

GTGTGTCGGG

AACTCTTTCG

CCAGCCTTGT

AGGACAGCCC

GCACT CGCAGCTAAA AACACTACGA GCAGTCCCCC CGCCTCGGAT ACCTTTrrGC GGTCGTTGCG CCGGAAAAAT AAGGACGCAA TAGGGGACTA GGCGAGCGGC GTCGGCTTGC CGGGTTATGC GTTTGGCGGA TGTCCAAAGG GCTGACCTTT GAGTAATCCG TGGGGTCCGA kCTCGCCTAT TGTTAAAGTG

GCCAAGGACC

AGACACCTAT

TGGCTCGCGT

GAGGGGCGCG

CGCCCGTCAC!

AATGTGAAAT

TGTCCTTTGT

-S3- GGhAAACGAC

TGGCATAATG

CGCTCAGTCA

CAACCGGCTA

TCGCGTTGCG

ACGAAGGCCG

CGATACTGGT

CGGAA.AACGA

GCGGAAACTC

CTCGCTCCTT

AGTAATTACG

TTAATTACAC

AGCATACAAC

ACTAATGCGG

GTGTACAAGA

ACTCGACTAT

CGCCTTCTCG

TCGACCGTGC

TCAATCGAGT

ACACCTTAAC

0~ INFORMATION FOR SEQ ID Ui) SEQUENCE CHARACTERISTICS: LENGTH: 713 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID K0:10: AAGCTTGGGC TGCAGGTCGA TCGACTCTAG AGGATCGATC CCCACCATGG GTCAATCACG CTACCTCCTC TTTTTGGCCA CCCTTGCCCT CCTAAACCAC CTCAGTTTGG CCAGGGTCAT TCCAGTCTCT GGACCTGCCA GGTGTCTTAG CCAGTC!CCGA AACCTGCTGA AGACCACAGA TGACATGGTG AAGACGGCCA GAGAAAAACT GAAACATTAT TCCTGCACTG CTGAAGACAT CGATCATGAA GACATCACAC GGGACCAAAC! CAGCACATTG AAGACCTGTT TACCACTGGA ACTACACAAG AACGAGAGTT GCCTGGCTAC TAGAGAGACT TCTTCCACAA CAAGAGGGAG CTGCCTGCCC CCACAGAAGA CGTCTTTGAT GATGACC!CTG TGCCTTGGTA GCATCTATGA 6000 6060 6120 6180 6240 6300 6350 120 180 240 :300 360 420 e.g.

ego.

09 Sr

C

S

@050

C

ego.

S.

go S

S.

5* 0

S.

0* C S S. C *000 05 00 0 0@ C S CC. 0

S

0 *005S0 0 -54- GGACTTGAAG ATGTACCAGA CAGAGTTCCA GGCCATCAAC GCAGCACTTC AGAATCACAA CCATCAGCAG ATCATTCTAG ACAAGGGCAT GCTGGTGGCC ATCGATGAGC TGATGCAGTC TCTGAATCAT AATGGCGAGA CTCTGCGCCA GAAACCTCCT GTGGGAQAAG CAGACCCTTA CAGAGTGAAA ATGAAGCTCT GCATCCTGCT TCACGCCTTC AGCACCCGCG TCGTGACCAT CAACAGQGTG ATGGGCTATC TGAGCTCCGC CTGAGAATTC ATTGATCCAC TAG INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 713 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TTCGAACCCG ACGTCCAGCT AGCTGAGATC TCCTAGCTAG GGGTGGTACC CAGTTAGTGC GATGGAGGAG AAA.AACCGGT GGGAACGGGA GGATTTGGTG GAGTCAAACC GGTCCCAGTA AGGTCAGAGA CCTGGACGGT CCACAGAATC GGTCAGGGCT TTGGACGACT TCTGGTGTCT ACTGTACCAC T1TCTGCCGGT CTCTTTTTGA CTTTG-..'TAATA AGGACGTGAC GACTTCTGTA GCTAGTACTT CTGTAGTGTG CCCTGGTTTG GTCGTGTAAC TTCTGGACAA ATGGTGACCT -TGATGTGTTC TTGCTCTCAA CGGACCGATG ATCTCTCTGA AGAAGGTGTT GTTCTCCCTC.

GACOGACGGG GGTTCTTCT GCAGAAACTA CTACTGGGAC ACGGAACCAT CGTAGATACT CCTGAACTTC TACATGGTCT GTCTCAAGGT CCGGTAGTTG CGTCGTGAAG TCTTAGTGTT GGTAGTCGTC TAGTAAGATC TGTTCCCGTA CGACCACCGG TAGCTACTCG ACTACGTCAG 480 600 660 713 2.20

ISO

240 300 360 420 480 AGACTTAGTA TTACCGCTCT GAGACGCGGT CTTTGGAGGA CACCCTCTTC GTCTGGGAAT GTCTCACTTT TACTTCGAGA CGTAGGACGA AGTGCGGAAG TCGTGGGCGC AGCACTGGTA GTTGTCCCAC TACCCGATAG ACTCGAGGCG GACTCTTAAG TAACTAGGTG ATC INFORMATION FOR SEQ ID NO:12: Wi SEQUENCE CHARACTERISTICS: (A),LENGTH: 215 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Gly Gin Ser Arg Tyr Leu Leu Phe Leu Ala Thr Leu Ala Leu Leu 600 660 713 0.

I

Asn 10 Pro His Leu Ser Leu Leu Ser Gin Ser Ala Arg Val Ile 25 Val Ser Gly Pro Ala Arg Asp Met Val Ala Glu Asp Cys 35 Arg Asn Leu Leu Lys 40 Lys Leu Lys His Tyr Thr Thr Ser Cys Asp Thr Lys Thr Ile Asp Cys Leu.

Ala Arg Giu His Glu Asp Pro Leu Glu le Thr 70 LAU His Arg Asp Gin Thr Se .r Thr Leu 75 Lys Asn Glu 8cr Cys Leu Ala Lys Thr s0 Thr Arg Lys Thr Giu Thr Ser Ser Thr 100 Thr Arg Gly Ser 2i05 CYs Leu Pro Pro Gin 110 -56- Ser Lou Met Met Thr Lou Cys Lou Gly Ser le Tyr Giu Asp Lou Lys 115 120 12S Met Tyr Gin Thr Giu Phe Gin Ala Ile Asn Ala Ala Lou Gin Asn His 130 135 140 Asn Hiu Gin Gin Ile Ile Le", hAs. Lyst fl321 T_ i 14S 1.50 r

A.

S

S

S

Giu Lou Met Gin Ser Lou Asn Hi.

165 Pro Pro Vai Gly Giu Ala Asp Pro

ISO

le Lou Lou His Ala Phe Ser Thr 195 200 Met Giy Tyr Lou Ser Ser Ala 210 215 155 Asn Giy Giu Thr 170 Tyr Arg Vai Lys 185 Aig Val Vai Thr va.& rlJ. up~ 160' Lou Arg Gin Lys 1.75 Met Lys Lou Cys 190 le Asn Arg Val 205 INFORMATION FOR SEQ ID NO:13: Ui) SEQUENCE CHARACTERISTICS: LENGTH: 1061 base pairs TYPE: nucleic acid STRPJNDEDNESS: unicnown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) Cxi) SEQUENCE DESCRIPTION: SEQ ID KO:13: AAGCTTGGGC TGCAGGTCGA TCGACTCTAG AGGATCGA= CCCACCATGG GTC!CTCAGAA GCTAACCATC TCCTGGTI'TG CCATCG11wT GCTGGTGTCT CCACTCATGG CCATGTGGQA GCTGGAGAAAL GACGTTTATG TTGTAGAGGT GGACTGGACT CCCGATGCCC CTGGAOAAAC AGTGAACCTC ACCTGTGACA CGCCTGAAGA AGATGACATC ACCTGGACCT CAGACCAGAG3 120 180 240 REaOiFED -SHEET (RULE 91) o

ACATGGAGTC

TGGCCAGTAC

CAAGAAGGAA

CCTGAAGTGT

AAACATGGAC

GACATGTGGA

GAAGTATTCA

CATTGAACTG

CTTCATCAGG

GAACTCACAG

CTTCTCCCTC

GGAGGGGTGT

CAAAGGCGGG

ATAGGCTCTG

ACCTGCCACA

AATGGAATTT

-57- GAAAGACCCT GACCATCACT AAGGAGGCGA GACTC TGAGC GGTCCACTGA AATTTTAA)A GAAGCACCAA ATTACTCCGG ACGGTTCACG TG;CTCATGGC TGGTGCAAAG' TI'GAAGTTCA ACATCAAQAG ATGGCGTCTC TGTCTGCAGA GTGTCCTGCC AGGAGGATGT GCGTTGGAAG CACGGCAGCA GACATCATCA. AACCAGACCC GTGGAGGTCA GCTGGGAGTA AAGTTCTTTG TTCGAATCCA AACCAGAAAG GTGCGTTCCT AATGTCTGCG TGCAAGCTCA

CAGTAGCAGT

GAAGGTCACA

CACCTGCCCA

GAATAAATAT

GCCCAAGAAC

CCCTGACTCC

GCGCAAGAAA

CGTAGAGAAG

GGATCGCTAT

TCCCCTGACT

CTGGACCAAA

ACTGCCGAGG

GAGAACTACA

TTGCAGATGA

TGGAGCACTC

GAAAAGATGA

ACATCTACCG

TACAATTCCT

CTCGGGCAGT

GGGACTATGA

AGACCCTGCC

GCACCAGCTT

,AGCCTTTGAA.

CCCATTCCTA

AGGAGACAGA.

AAGTCCAATG

CATGCAGCA.A

GTCAAAGAGT TTCTAGATGC CACTCACATC TGCTGCTCCA AATTTCAAAA ACAAGACTTT 300 360 420 480 540 600 660 720 780 840 900 960 1020 GTGGGCATGT GTTCCCTGCA GGGTCCGATC CTAGGAATTC C INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH2: 1060 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID KO:14: 1061 RECTIFIED SHEET (RULE 91) -58-

TCCTAGCTAG

TTCGAACCCG ACGTCCAGCT CGATTGGTAG AGGACCAAAC CGACCTC'XTT CTGCAAATAC TCACTTGGAG TGGACACTGT TGTACCTCAG TATCCGAGAC

ACCGGTCATG

GTTCTTCCTT

GGACTTCACA

TTTGTACCTG

CTGTACACCT

CTTCATAAGT

GTAACTTGAC

TGGACGGTGT

TTACCTTAAA

CTTCGTGGTT

AACTTCAAGT

TACCGCAGAG

CACAGGACGG

CGCAACCTTC

AGCTGAGATC

GGTAGCAAAA

AACATCTCCA

GCGGACTTCT

CTTTCTGGGA

TTCCTCCGCT

CCAGGTGACT

TAATGAGGCC

TGTAGTTCTC

ACAGACGTCT

TCCTCCTACA

GTGCCGTCGT

TTGGTCTGGG

CGACCCTCAT

AAGCTTAGGT

CACGCA.AGGA

ACGTTCGAGT

TCTACTGTAG

CTGGTAGTGA

CTGAGACTCG

TTAAAATTTT

TGCCAAGTGC!

GTCATCGTCA.

CTTCCAGTGT

GTGGACGGGT

CTTATTTATA

CGGGTTCTTG

GGGACTGAGG

CGCGTTCTTT

GCATCTCTTC

CGACCACAQA GGTGAGTACC CCTGACCTGA GGGCTACGGG GGGTGGTACC CAGGAGTCTT

TGGACCTGGA

CAGTTTCTCA,

GTGAGTQTAQ

TTAAAGTTT

AC!GAGTACCG;

AGGGGACTGA

GACCTGG7T

TGACGGCTCC

CTCTTGATGT

AACGTCTACT

ACCTCGTGAG

CTTTTCTACT

TGTAGATGGC

GGTACACCCT

GACCTCTTTG

GTCTGGTCTC!~

AAGATCTACG

ACGACGAGGT

TGTTCTGAAA,

ACCACGTTTC

GAGCCCGTCA

CCCTGATACT

TCTGGGACQG

CGTGGTCGAA

TCGGAAACTT

GGGTAAGGAT

TCCTCTGTCT

TTCAGGTTAC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 GAAGTAGTCC CTGTAGTAGT CTTGAGTGTC CACCTCCAGT GAAGAGGGAG TTCAAGAAAC CCTCCCCACA TTGGTCTTTC GTTTCCGCCC! TTACAGACGC CCTAGCGATA ATGTTAAGGA GTACGTCGTT CACCcTACA cAAGGGAcGT CCCAGGCTAG GATCTTAAGG INFORMATION FOR SEQ ID (i)-SEQUENCE CHARACTERISTICS: LENGTH: 33S amino acids 1060 -59- TYPE: amino acid STRAN4DEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi)-SEQUENCE DESCRIPTION: SEQ ID Met Gly Pro Gin Lys Leu Thr Ile Ser Trp Phe Ala1le Val Leu Le Iu 1 5 10 Val Ser Pro Leu Met Ala Met Trp Giu Leu Giu Lys Asp Val Tyr Val Vai Giu Vai Asp Trp Thr Pro Asp 40 Aia Pro Giy Giu Thr Vai Asn Leu Thr Cys so Asp Thr Pro Giu Giu 55 Asp Asp Ile Thr Trp Thr Ser Asp Gin Arg His Gly Vai Ile Giy 70 Ser Giy Lys Thr Leu 75 Thr Ile Thr Val Lys

*SSSSS

Giu Phe Leu Leu 5cr His Ser Thr Giu Asp Ala e5 Giy Gin Tyr Thr Cys 90 His Lys Gly Gly Giu Thr 5cr 100 His Leu Leu Leu His 105 Lys Lys Giu Asn Gly Ile Trp 110 Lou Lys Cys Ile Leu Lys Asn Phe 120 Gly. Arg 135 Lys Asn Lys Thr Phe 125 Glu Ala 130 Arg Asn 145 Pro Asn Tyr Met Asp Leu Ser Phe Thr Cys Ser 140 Trp Lou Val Gin LYS Phe Asn le Thr Cys Gly Met Lys Ser 155 Ser Ser Ser Ser Pro 160 Asp Ser Arg Ala Vai 165 Ala Ser Leu Ser Ala Giu Lys 170 175 Val Thr Leu Asp Gin Arg Asp Tyr

ISO

Giu Asp Val Tkar cys Pro Thr Ala 195 200 Giu Lys Tyr Ser Val Ser Cys Gin 185 190 Giu Giu Thr Leu Pro le Giu Leu 205 Ala Leu Giu Ala Arg Gin 210 Gin Asn 21S Lys Tyr Giu Asn Tyr 220 Phe 225 Phe Ile Arg Asp le Lys Pro Asp Pro Pro Lys 235 Vai Ser Trp Met Lys Pro Leu Lys 245 Asn Ser Gin Val Giu 250 Ser Thr Ser Asn Leu Gin 240 Giu Tyr Pro 255 Phe Phe Val 270 Giu Gly Cys Asp Ser Trp Arg Ile Gin 275 Ser 260 Thr Pro His Ser Tyr 26S Phe Ser Leu Lys Arg Lys Lys Giu

LYS

280 Met Lysn Giu Thr Giu 28S Asn Gin 290 Lys Giy Ala Phe Leu 295 Val Giu Lys Thr Ser 300 Thr Giu Vai Gin Cys 305 Lys Giy Giy Asn Val 310 Lys Trp 325 Cys Vai Gin Ala Asp Arg Tyr Tyr Asn 320 Ser Ser Cys Ser Ala Cys Vai Pro 330 Cys Arg Val Arg Ser 335 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: S(A) LENGTH: 61 base pairs TYPE: nucleic acid STRA2NDEDNESS: unknown CD) TOPOLOGY: Unknown (ii) MOLECULE TYPE: DNA (genomic) -61- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CCGCCGGTGG CGGTGGCTCG GGCGGTGGTG GGTCGGGTGG CGGCGGATCT TCCATGGAGC T 61 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17: CCATGGAAGA TCCGCCGCCA. CCCGACCCAC CACCGCCCGA GCCACCGCCA CCGGCGGAGC *T 61 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID 2;0:18: CAGAGTGAAA ATGAAGCT 1 -62- INFORMATION FOR SEQ ID NO:19: Ci SEQUENCE CHARACTERISTICS: CA) LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID XO:19: GAAGCTCTGC ATCCTGCT 18 0. *o.

INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 62 base pairs CB) TYPE: nucleic acid STRA2NDEDNESS: unknown CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID too.. GGGTCCGATC CGGTGGCGGT GGCTCGGGCG GTGGTGGGTC GGGTGGCGGC GGATCTTCCA 0 go TG 62 INFORMATION FOR SEQ ID NO:21: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: unknown CD) TOPOLOGY: unknown ii) MOLECULE TYPE: DNA (genomic) -63- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GATCCATGGA AGATCCGCCG CCACCCGACC CACCACCGCC CGAGCCACCG CCACCGGATC GGACCCTGCA INFORMATION FOR SEQ ID NO:22: i)SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs .9 TYPE: nucleic acid CC) STRANDEDNESS: unknown CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CTATTACAAT TCCTCATG 1 INFORMATION FOR SEQ ID NO:23: Ci SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs S TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GAGGGCAAGG GTGGCCAA i INFORMATION FOR SEQ ID NO:24: Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 67 base pairs -64- TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TTGCTGGAGC TCCGCCGGTG GCGGTGGCTC GGGCGGTGGT GGGTCGGGTG GCGGCGGATC TATGTGG, 67 INFORMATION FOR SEQ ID 9. Ci)SEQUENCE CHARACTERISTICS: LENGTH: 67 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CACATAGATC CGCCGCCACC CGACCCACCA CCGCCCGAGC CACCGCCACC GGCGGAGCTC CAGCAAA 67 INFORMATION FOR SEQ ID NO:26: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 82 base pairs S(B) TYPE: nucleic acid STRA2IDEDNESS: unknown TOPOLOGY: Unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CTGGCCTGCA GGGTCCGATC CGGTGGCGGT GGCTCGGGCG GTGGTGGGTC GGGTGGCGGC GGATCTAGGG TCATTCCAGT CT 82 INFORMATION FOR SEQ ID NO:27: Wi SEQUENCE CHARACTERISTICS: LENGTH: 82 base pairs TYPE: nucleic acid STR.ANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) SEQUENCE DESCRIPTION: SEQ ID NO:27: CTGGAATGAC CCTAGATCCG CCGCCACCCG ACCCACCACC GCCCGAGCCA CCGCCACCGG ATCGGACCCT GCAGGCCAGA GA 82 INFORMATION FOR SEQ ID N:28: Wi SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STR7ANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID 110:28: GCAAAGGCGG, GAATGTCT 1 INFORMATION FOR SEQ ID N10:29: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs -66- TYPE: nucleic acid STRPJNDEDNESS: unknown TOPOLOGY: unknown (iMOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: AGGAATAATG TTTCAGT' is8 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANqDEDNESS: unknown TOPOLOGY: unknown.

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CAGCAGTGCA GGAATAAT i INFORMATION FOR SEQ ID NO:31: i)SEQUENCE CHARACTERISTICS: LENGTH: 75 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:31: CCCTGCAGGG TCCGATCCGG TGGCGGTGGC TCGGGCGGTG GTGGGTCGGG TGGCGGCGGA TCTTCCATGG GTCAA 7S -67- INFORMATION FOR SEQ ID NO:32: Wi SEQUENCE CHARACTERISTICS: LENGTH: 75 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CCCTGCAGGG TCCGATCCGG TGGCGGTGGC TCGGGCGGTG GTGGGTCGGG TGGCGGCGGA TCTTCCATGG GTCAA INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: TGTGTTCCCT GCAGGGTCCG ATCCGGTGGC GGTGGCTCGG GCGGTGGTGG GTCGGGTGGC GGCGGATCTA GGGTCATTCC AGTCTCTGGA CCTGCC 96 INFORMATION FOR SEQ ID NO: 34: Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 18 base pairs TYPE: nucleic acid CC) STRANDEDNESS: unknown CD) TOPOLOGY: unknown -68- (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GTCATCTTCT TCAGGCGT 1 INFORMATION FOR SEQ ID NO:3S: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRAMDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID *TGCAGTGGTG GCGGTGGCGG CGGATCTAGA AAC 33 INORMATION FOR SEQ ID NO:36: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: G14~ Gly Gly Gly Gly Gly I .16

Claims (9)

1. A method of treating a disorder characterised by an established tumor comprising administering a therapeutically effective dose of IL-12-secreting tumor cells to a subject having a disorder characterised by an established tumor.

2. A method according to claim 1 wherein the treatment results in the reduction of the size of the tumor, prolonged survival of the subject compared with an untreated subject or both.

3. A method according to claims 1 or 2 wherein the IL-12-secreting tumor cells are selected from the group including CMS-5 tumor cells and B16 tumor cells.

4. A method of reducing the size of at least one established tumor in a subject S: 15 comprising administering a therapeutically effective dose of IL-12-secreting tumor cells to a subject having said tumor, thereby reducing the size of said tumor.

A method according to claim 4, wherein the size of the tumor is reduced by :I greater than

6. A method according to any one of claims 1 to 5 wherein the established tumor is a melanoma, a fibrosarcoma or a renal cell carcinoma.

7. A method according to any one of claims 1 to 6 wherein the IL-12-secreting 25 tumor cells secrete a bioactive IL-12 protein which comprises IL-12 p35 subunit and IL- 12 p40 subunit joined by a polypeptide linker.

8. A method of treating a disorder characterized by an established tumor comprising administering a therapeutically effective dose of IL-12-secreting tumor cells, wherein said cells secrete a bioactive IL-12 protein which comprises an IL-12 subunit and an IL-12 p40 subunit joined by a polypeptide linker.

9. A method of reducing the size of at least one established tumor in a subject comprising administering a therapeutically effective dose of IL-12-secreting tumor cells to a subject having said established tumors wherein said cells secrete a bioactive IL-12 ZTR, protein which comprises an IL-12 p35 subunit and an IL-12 p40 subunit joined by a S polypeptide linker, thereby reducing the size of the established tumor. W:\Eisabeth\PJC\NODELETE\28034-99.doc A method according to claim 1 substantially a hereinbefore described with reference to any one of the examples. DATED: 27 June, 2001 PHILLIPS ORMVONDE FITZPATRICK Attorneys for: WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH WA\Elisabeth\PJCV40DELETE\28034.9g~doc