1. Cross Reference To Related Applications

This application claims priority under 35 U.S.C. § 119 from U.S. Provisional Application Serial No.60/003,665, filed September 11 , 1995.

2. Statement as to Federally Sponsored Research

This invention was made at least in part with funds from the Federal Government under National Institutes of Health Grant No. 5 RO1 Al 15722. The Government therefore has certain rights in the invention.

3. Background of the Invention

Cell-mediated killing of target cells can be undesirable in vivo. For example, cell- mediated killing of grafts is thought to be responsible for rejection of a graft by a host. Similarly, autoimmune diseases, such as diabetes, are thought to be arise from the undesirable induction of cell-mediated killing of an individual's cells (e.g., pancreatic islet cells). Cell-mediated killing has also hampered progress in the field of gene therapy. Many gene therapy methods entail the use of a viral vector to express a therapeutic gene in a target cell. Generally, such viral vectors also encode viral antigens that induce the patient's immune system to effectuate cell-mediated killing of target cells that are transfected with the virus and therapeutic gene. Thus, these examples illustrate that the induction of an immune response can be undesirable.

Cell-mediated killing by cytotoxic T lymphocytes (CTL) is thought to occur by secretion of cytoplasmic granules containing perforin and granzymes, or by signaling via the Fas pathway (Berke, 1994, Ann. Rev. Immun. 12:735-773 and Nagata et al., 1995, Science 267:1449-1456).

Summary of the Invention

Applicants have discovered that cell-mediated killing of a target cell can be inhibited by expressing in the target cell a proteinase inhibitor of an orthopoxvirus (e.g., SPI-1).

Accordingly, the invention features a method for inhibiting cell-mediated cell death of a mammalian target cell by expressing in the target cell an orthopoxvirus proteinase inhibitor, thereby inhibiting cell-mediated cell death. In particular, the invention can be used to inhibit CTL-mediated cell death and apoptosis. The

invention is thus useful for suppressing the immune system of a patient, e.g., for treating an autoimmune disease, inhibiting graft rejection, or inhibiting the death of cells that are targeted in gene therapy methods.

Brief Description of the Drawings

Figs. 1A and 1B are histograms representing the inhibition of granule-meditated cytolysis of cells infected with CPV or RPV. L1210 (Fig. 1A) or EL4 (Fig. 1B) cells were either mock-infected or infected with CPV or RPV, and cytolysis was assayed by incubation with stimulated CTL21.9 effector cells in a standard 4 hour chromium release assay at an effector to target ratio of 5:1. To facilitate comparison of multiple experiments, the values for % specific lysis for mock-infected cells were set to 100%, and other data are shown relative to mock-infected cells. Each bar represents the mean and standard deviation calculated from 3-5 independent experiments, each performed in triplicate.

Effector cells (CTL21.9 - Havele, et al., 1986, Journal of Immunology 137: 1448- 1454) were stimulated for 24 hours prior to assay at 37°C with a 1/250 dilution of

2C1 1 hybridoma supernatant (anti-CD3 monoclonal antibody - Leo, et al., 1987, Proceedings of the National Academy of Sciences USA 84:1374-4274). L1210 cells (a murine T lymphoma derived from DBA/2 mice; obtained from Dr. Pierre Goldstein, CNRS, Marseille, France or EL4 cells (a murine T lymphoma cell line) were infected at a multiplicity of 10 plaque forming units per cell with either CPV (Brighton Red strain) or RPV (Utrecht strain), or were mock-infected in RPMI (RPM1 1640 containing 5% fetal bovine serum (Gibco) and 10"* M β-mercaptoethanol) for 12 hours at 37 ^C C.

Cells were labeled with ⁵¹ Cr (100μCi per 2 x 10 ⁶ cells; Dupont NEN) at 37°C for 90 minutes, washed three times, and added to V-bottomed 96-well plates (1 x 10" cells per well) with stimulated CTL21.9 effector cells (5 x 10" cells per wall). Plates were centrifuged at 500 rpm for 5 minutes to promote cell contact, and then incubated for 4 hours at 37°C in RPMI. Supernatants were then removed and counted in a gamma counter. Each assay was performed in triplicate. Specific lysis and relative % Specific Lysis were calculated as follows: % Specific Lysis = (sample - spontaneous release)/(total - spontaneous release)x100 Relative % Specific Lysis =

% Specific Lysis (virus infected cells)/% Specific Lysis (mock infected cells).

Figs. 2A - 2D are a series of histograms representing inhibition of Fas-mediated cytolysis of virus-infected L1210-Fas (Fig. 2A), EL4 (Fig. 2B) and YAC-1 (Fig. 2C)

cells L1210-Fas, EL4 and YAC-1 cells were either mock-infected or infected with the indicated virus as described in the legend to figure 1 Cytolysis of target cells was assayed by incubation with stimulated PMM-1 effector cells in a standard 4 hour chromium release assay at an effector to target ratio of 5 1 To facilitate comparison of multiple experiments, the values for % specific lysis for mock-infected cells were set to 100%, and other data are shown relative to mock infected cells Each bar represents the mean and standard deviation calculated from 3-5 independent experiments, each performed in triplicate Fig 2D is the histogram obtained after L1210-FAS and L1210 cells were either mock-infected or infected with CPV or RPV and incubated with stimulated PMM-1 effector cells at an effector to target ratio of 5 1 and the % specific chromium release was determined Representative data from a single experiment performed in triplicate are shown Virus infections, chromium release assays, and calculations were performed as described in the legend to Figs 1A and 1B PMM-1 effector cells (Kaufmann, et al , 1981 , Proceedings of the National Academy of Sciences USA 78 2502) (a BALB/c derived peritoneal exudate lymphocyte CTL hybridoma, obtained from Dr G Berke, Weizmann Institute of Science, Rehovot, Israel, were stimulated prior at assay by incubating with PMA (10 ng/ml, Sigma) and lonomycin (3#μg/ml, Sigma) at 37 ^β C for three hours L1210-FAS is an L1210 cell transfectant expressing the murine Fas antigen (Rouvier, et al , 1993, Journal of Experimental Medicine 177 195-200) (obtained from Dr Pierre Goldstein,

CNRS, Marseille, France), and YAC-1 is murine Lymphoma cell line

Figs. 3A-3F are a series of histograms representing Fas-mediated cytolysis of target cells infected with virus mutants in the SPI-1 or SPI-2 genes L1210-FAS (Figs 3A and 3D), EL4 (Figs 3B and 3E), or YAC-1 (Figs 3C and 3F) were either mock- infected or infected with either wild type CPV, a CPV mutant in the SPI-1 gene

(Thompson, et al., 1993, Virology 197.328-338) (CPVΔSPI-1), a CPV mutant in the SPI-2 gene (All, et al , 1994, Virology 202.305-314) (CPVΔSPI-2), wild type RPV, RPV mutants in the SPI-1 , SPI-2 (A , et al , 1994, Virology 202 305-314), or both SPI- 1 and SPI-2 genes (RPVΔSPI-1 , RPVΔSPI-2, RPVΔSPI-1/2, respectively) Data are shown as the % specific lysis relative to mock infected cells and are the mean and standard deviations of three independent experiments Virus infections, chromium release assays, and calculations were performed as described in the legends to Figs 1A, 1B, and 2A-2D RPV mutant in both SPI-1 and SPI-2 was constructed from RPVΔSPI-1 by homologous recombination leading to replacement of a 447 bp region of the SPI-2 open reading frame with the E colt lacZ gene driven by the vaccinia virus p11 promoter

Figs 4A-4D are a series of histograms representing Granule-mediated cytolysis of target cells infected with virus mutants in the SPI-1 or SPI-2 genes L1210 (Figs 4A and 4C) or EL4 (Figs 4B and 4D) cells were infected with wild type viruses or virus mutants in either the SPI-1 or SPI-2 genes, or with RPV containing mutations in both the SPI-1 and the SPI-2 genes (RPVΔSPI-1/2, described above) Data are shown as the % specific lysis relative to mock infected cells and represent the mean and standard deviations of three independent experiments Virus infections, chromium release assays, and calculations were performed as described in the legends to Figs 1A, 1 B, and 2A-2D Fig 5 is a listing of the DNA and ammo acid sequences for SPI-1 (SEQ ID Nos

1 and 2, respectively)

Fig 6 is a listing of the DNA and ammo acid sequences for SPI-2 (SEQ ID NOs 3 and 4, respectively)

Detailed Description of the Invention

The invention provides a method for inhibiting cell-mediated killing of a target cell

"Cell-mediated" killing refers to ability of a cell (e g , a cytotoxic T lymphocyte (CTL)) to effectuate the death of a target cell, e g , by cytolysis or apoptosis In practicing the invention, cell death is inhibited by expressing in the target cell a proteinase inhibitor of a pox virus, such as an orthopoxvirus, thereby inhibiting cell-mediated (e g , CTL- mediated) killing of the target cell

If desired, the method can include identifying the target cell as a target of cell- mediated killing Numerous cells are known to be targets of cell-mediated cell death Preferably, the cell is a human cell For example, cells of heterologous or autologous grafts are targets for cell-mediated killing, and such cells can be used in the invention Bone marrow cells are particularly suitable for use in the invention, as such cells can readily be obtained, manipulated in vitro, and then introduced into a patient Other suitable cells include cells that are associated as tissues (e g , liver tissue) or organs (e g , hearts) that can be grafted in methods of transplantation Likewise, cells that are destroyed due to autoimmune diseases, e g , pancreatic islet cells of diabetes patients, also are targets for cell-mediated killing In addition, CD4 ⁺ lymphocytes of patients infected with Human Immunodeficiency Virus (HIV) are considered target cells in the invention Such CD4 ⁺ lymphocytes are thought to be killed by apoptosis that is induced by stimulation of the CD4 molecule on the lymphocytes by a complex of gp120 and antibody The Fas antigen/Fas ligand system is thought to mediate the

death of CD4 ⁺ cells even though the cells are not thought to be infected with HIV Thus, cell-mediated killing of these cells results in the massive depletion of CD4 cells that is observed in AIDS patients When the invention is used in a method of treating a patient, the cell can be autologous or heterologous to the patient In a preferred embodiment, the invention can be used to inhibit cell-mediated killing of cells that are the targets of conventional gene therapy methods Many conventional gene therapy methods employ viral vectors to express a therapeutic gene in a cell of a mammal A "therapeutic" gene is any gene that, when expressed, confers a beneficial effect on a cell In vivo, such a gene is one that ameliorates a sign or symptom of a disorder, or confers a desired phenotype on a cell or another cell of the patient Such a therapeutic gene can be, for example, a gene that corrects a deficiency in gene expression (e g , an insulin gene for correcting a deficiency in insulin expression) Traditional gene therapy methods are hindered because viral vectors carrying therapeutic genes also express viral antigens that induce cell- mediated killing of target cells that are transfected with the viral vectors The invention thus provides a method for inhibiting cell-mediated death of these target cells, with inhibition being accomplished by expressing a poxvirus proteinase inhibitor in the target cell If desired, the proteinase inhibitor can be expressed from the same vector, or a different vector, as the vector used to express the therapeutic gene Additional cells that are targeted for cell-mediated cell killing can readily be identified in conventional in vitro CTL cytotoxiαty assays, such as chromium release assays

Conventional gene delivery and gene expression methods can be used to express a proteinase inhibitor in a cell For example, vectors derived from mammalian viruses, such as retrovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors, are well known in the art and can be used in the invention Other art-known gene delivery and expression techniques can also be used in the invention For example, the proteinase inhibitor can be expressed from a genetic construct (i e., any nucleic acid, such as a plasmid or cosmid, engineered to express a gene) If desired, the vector can be engineered to contain a "suicide" gene Such genes are known in the art for their ability to encode a factor that renders the cell sensitive to a substance that can be administered to the cell in the event that subsequent killing of the target cell should be desired

The various genetic constructs can be introduced into a cell by conventional methods, such as posome-based methods, direct uptake, electroporation, CaCI- based methods, and the like When hposome-based methods are used, the

liposomes can be engineered to have on their surface desired markers, e g receptors, antibodies, lectins, or carbohydrates

Typically, the nucleic acid encoding the proteinase inhibitor is operably linked to a promoter that is active in a mammalian cell (e g , a promoter that naturally drives expression of a gene in a mammalian cell or a promoter of a virus that infects a mammalian cell) Preferably, the promoter is a promoter of an poxvirus, such as a promoter that naturally drives expression of a proteinase inhibitor If desired, the promoter can be a cell-specific promoter, a tissue-specific promoter, or a stage- specific promoter Such promoters are known in the art and can be used to confer specificity in the practicing the invention When tissues or organs are used as the target cells in the invention, conventional organ perfusion methods are well suited for delivering the nucleic acid encoding the proteinase inhibitor to the cells of the tissue or organ

Preferably, the proteinase inhibitor is a §erpιn βroteinase inhibitor, such as SPI-1 (SEQ ID NO 2, GenBank Accession No UO7766, A et al , supra), SPI-2 (SEQ ID

NO 4, GenBank Accession No UO7763, Ah et al , supra (SPI-2 is also known as crmA in CPV), or SPI-3 (Turner et al , 1995, Viroceptors, Virok es, and Related Immune Modulators Encoded by DNA Viruses, McFadden ed, R G Landes Company, Austin, TX) The proteinase inhibitor can be derived from any pox virus, preferably, the virus is an orthopox virus, such as a cowpox virus, rabbitpox virus, smallpox virus, or vaccinia virus Such virus can be obtained from ATCC Each of these viruses naturally encodes members of the serpin family of proteinase inhibitors, which can be used in the assay Mutant strains of these viruses that have decreased pathogenicity while nonetheless expressing a proteinase inhibitor can used In addition, variants (e g , conservative variants) and mutants of these proteinase inhibitors can be used, provided that the resulting polypeptide retains a detectable ability to inhibit CTL- mediated cell death (e g , as determined in a CTL cytotoxicity assay as described herein)

By "conservative variant" is meant a polypeptide having an ammo acid substitution where the native ammo acid and the substituted ammo acid are of approximately the same charge and polarity Such substitutions typically include, e g , substitutions within the following groups glycine, alanine, va ne, isoleucme, leuc e, methionine, aspartic acid, glutamic acid, asparagme, glutamine, senne, threonme, lysme, arginine, and phenylalanine, tyros e In general, such conservative ammo acid substitutions do not significantly affect the function of the polypeptide If desired, the ability of a variant or mutant to function as a proteinase inhibitor can be measured

in a CTL cytotoxicity assay as described herein, using the wild-type proteinase inhibitor as a standard.

The invention also presumes the use of nucleic acids encoding a protease inhibitor where the nucleic acid is a degenerate variant of the nucleic acid sequences expressly described herein. By "degenerate variants" of a nucleotide sequence is meant nucleotide sequences that encode the same amino acid sequence as a given nucleotide sequence, but in which at least one codon in the nucleotide sequence is different. Degenerate variants occur due to the degeneracy of the genetic code, whereby two or more different codons can encode the same amino acid. Applicants have discovered that the presence of nucleotide encoding SP-1 polypeptide and nucleotide encoding SP-2 polypeptide provide enhanced inhibition of cell-mediated killing.

Included within the invention is an isolated target cell that is resistant to cell- mediated killing. Such cells contain a nucleic acid that consists essentially of a nucleic acid that expresses a poxvirus SPI-1 polypeptide, as described herein. Such cells can also include a nucleic acid that consists essentially of a nucleic acid that expresses an SPI-2 polypeptide. If desired, these cells can also express a therapeutic gene. The term "isolated" target cell means that a gene is delivered to a cell and expressed non-systemically in a population of cells (e.g., in liver cells or pancreatic islet cells). Such cells can be in the form of a cell suspension (e.g., bone marrow cells) or they can be in the form of a tissue or organ (e.g., a liver) for use in organ transplantation methods. Encompassed by this term are cell populations of a mammal that are targeted for SPI-1 expression, without systemic expression of SPI-1 in the mammal. For example, liver cells of a mammal that are targeted for SPI-1 expression are considered "isolated," even when the cells are contained within the mammal.

The invention also provides a method for determining whether a nucleic acid (i.e. , a "test" nucleic acid) encodes a polypeptide that inhibits cell-mediated killing of a target cell. Thus, this aspect of the invention provides a method for identifying additional proteinase inhibitors that can be used in the methods described above.

One method entails introducing into a target cell a mutant poxvirus that does not express a functional SPI-2 polypeptide; expressing in the target cell the "test" nucleic acid; and detecting inhibition of cell-mediated killing of the target cell as an indication that the test nucleic acid encodes a polypeptide that inhibits cell-mediated killing. In this sense, the "test" nucleic acid is tested for its ability to complement the SPI-2 deletion. Mutant viruses that fail to encode functional SPI-2 are known in the art (Ali

et al., infra), and additional viruses can be produced using conventional mutagenesis methods. It is expected that the majority of such polypeptides will be proteinase inhibitors that, like SPI-2, belong to the serpin family of proteinase inhibitors. Typically, the polypeptide encoded by the "test" nucleic acid is further characterized by testing its ability to confer resistance to cell-mediated killing in the presence of a poxvirus that does not encode a functional SPI-1 polypeptide. Such an SPI-1 mutant has been described (Thompson et al., infra), and additional mutant viruses can be produced using conventional gene manipulation techniques. A "test" nucleic acid encoding a polypeptide that confers resistance to cell-mediated killing in both of the aforementioned genetic settings is considered a new inhibitor of cell-mediated killing.

The nucleic acid encoding such a polypeptide can then be used in the invention to confer on a target cell resistance to cell-mediated killing.

Examples

The following examples and meant to illustrate, not limit, the invention, the metes and bounds of which are determined by the claims. While the methods described in these examples are typical of those that can be used, other procedures known to those skilled in the art may alternatively be used.

Example I: Cowpox Virus and Rabbitpox Virus Inhibit CTL-Mediated Cytolysis

This example shows that cells infected with an orthopoxvirus are resistant to

CTL-mediated cytolysis. In this example, target cells were infected with wild-type cowpox virus (CPV (Brighton Red strain) or rabbbitpox virus (RPV (Utrecht strain)), (American Type Culture Collection, Rockville, MD.), at a multiplicity of infection (moi) of ten plaque forming units (pfu)/cell for twelve hours. Each of these viruses expresses a serpin proteinase inhibitor in the infected cells. The target cells were then assayed for cytolysis by CTL21.9 effector cells in a conventional chromium release assay. In this example, the target cells were L1210 (ATCC) and EL4 cells (thymoma cells). These target cells are known to be efficiently lysed by CTL21.9, and lysis occurs in a calcium-dependent reaction that involves the secretion of cytoplasmic granules (Garner et al., 1994, J. Immun. 153:5413-5421).

Infection of L1210 cells with CPV resulted in a dramatic and reproducible inhibition of cytolysis by CTL21.0 (Fig. 1A), as compared with mock-infected cells. Infection with RPV also resulted in significant inhibition of CTL-mediated cytolysis

(Fig 1A) Infection of EL4 cells with either CPV or RPV resulted in an approximately 50% reduction in the level of cytolysis by CTL21 9 (Fig 1 B) The calcium-dependent nature of cytolysis of these cells, was confirmed by carrying out the assays in the presence of EGTA (data not shown) In sum, this example illustrates that infection by CPV and RPV each can inhibit the ability of CTL to lyse target cells

Example II: CPV and RPV Each Inhibit the Fas Pathway of Cytolysis

This example demonstrates that cytolysis that occurs via the Fas pathway can be inhibited by infecting a target cell with CPV or RPV In this example, PMM-1 cells were used as the cytolytic effectors PMM-1 is a cytolytic hybridoma cell line that does not express perform or granzymes (Kaufmann et al , 1981 , PNAS 78 2502 and

Kelgason et al , 1992, Eur J Immunol 22 3187-3190) These CTL lyse target cells via the Fas pathway (Garner et al , 1994, 153 5413-5421) Each of the target cells in this experiment expressed Fas These cells were L1210-Fas (an L1210 cell transfected with sequences expressing muπne Fas (Rouvier et al , 1993, J Exp Med 177 195-200)), EL4 cells, and YAC-1 cells (ATCC)

Infection of the Fas-expressing target cells with CPV or RPV inhibited the ability of PMM-1 cells to lyse the target cells via the Fas pathway (Figs 2A-2C) As a control, L1210 cells that do not express Fas were used in the experiment L1210 cells, unlike L1210-Fas cells, are not lysed efficiently, indicating that cytolysis by PMM-1 cells is dependent upon expression of Fas (Fig 2D) In addition, the mechanism of killing is independent of calcium concentration, as determined by the observation that killing is unaffected by the presence of EGTA In sum, this example illustrates that expression of CPV of RPV in a target cell can inhibit CTL-mediated cell death the occurs by the Fas pathway of cytolysis

Example 111: CPV SPI-2 and SPI-1 Inhibit Fas-mediated Cytolysis

This example demonstrates that expression of SPI-2 and SPI-1 are responsible for the ability of CPV and RPV to inhibit CTL-mediated cytolysis Mutated versions of SPI-1 and SPI-2 were used in these experiments to demonstrate the role of SPI-1 and SPI-2 The SPI-1 mutant, CPVΔSPI-1 , has been described previously (Thompson et al , 1993, Virology 197 328-338) Likewise, the SPI-2 mutant

CPVΔSPI-2, has been described (A et al , 1994, Virology 202 305-314) Cells infected with CPV containing a mutation in the SPI-2 gene were lysed by PMM-1

effectors at a level comparable to the level of lysis obtained with mock-infected cells (Figs. 3A-3C). Cells infected with a CPV mutant having a mutation in the SPI01 gene showed comparable inhibition to wild-type CPV-mfected cells Similarly, mutation of the RPV SPI-2 gene almost completely relieved virus-mediated inhibition of cytolysis of infected L1210-FAS cells. In addition, mutation of SPI-2 partially relieved virus- mediated inhibition of EL4 cells and YAC-1 cells (Figs 3D-3F) In these expeπments, mutation of both the SPI-1 and SPI-2 genes completely abrogated RPV-mediated inhibition of cytolysis in EL4 and YAC-1 cells (Figs 3E and 3F) These data indicate that both SPI-1 and SPI-2 confer resistance to CTL-mediated cytolysis that occurs via the Fas pathway

Example IV: Use of SPI-1 and SPI-2 in Combination to Inhibit Granzyme- Mediated Cytolysis

In contrast to the results obtained with PMM-1 effector cells, mutations in either the SPI-1 or SPI-2 genes alone for both CPV and RPV did not completely relieve virus-mediated inhibition of granule-mediated cytolysis by CTL21 9 effector cells

(Figs 4A-4D) However, an RPV strain that contained a mutation in both the SPI-1 and SPI-2 genes completely lacked the ability to inhibit cytolysis by CTL21 9 cells (Figs. 4C and 4D) These data indicate that, while SPI-1 and SPI-2 individually are able to inhibit cytolysis via the Fas pathway, complete inhibition of granule-mediated cytolysis is best achieved by expression of both SPI-1 and SPI-2

The description provided herein is meant to illustrate, but not limit, the scope of the invention Indeed, following the guidance provided herein, those of ordinary skill in the art can readily practice various additional embodiments of the invention

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT Bleackley et al , R Chris

(ul TITLE OF INVENTION METHOD FOR INHIBITING CELL MEDIATED KILLING OF TARGET CELLS

(ill) NUMBER OF SEQUENCES 4

(IV) CORRESPONDENCE ADDRESS

(A) ADDRESSEE Fish _ Richardson P C (B) STREET 4225 Executive Square Suite 1400

(C) CITY La Jolla

(D) STATE CA (E> COUNTRY USA (F) ZIP 92037 (v) COMPUTER READABLE FORM

(A) MEDIUM TYPE Floppy disk

(B) COMPUTER IBM PC compatible

<C) OPERATING SYSTEM PC-DOS/MS-DOΞ

(D) SOFTWARE Patentln Release #1 0, Version #1 30 (vi) CURRENT APPLICATION DATA

(A) APPLICATION NUMBER US

(B) FILING DATE

(C) CLASSIFICATION

(V i) PRIOR APPLICATION DATA (A) APPLICATION NUMBER US 60/003,665

(B) FILING DATE 11-SEP-199S

(vill) ATTORNEY/AGENT INFORMATION

(A) NAME Wetherell, Jr , John R

(B) REGISTRATION NUMBER 31,678 (C) REFERENCE/DOCKET NUMBER 07254/019001

(IX) TELECOMMUNICATION INFORMATION

(A) TELEPHONE 619/678-5070

(B) TELEFAX 619/678-5099

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(XI) SEQUENCE DESCRIPTION SEQ ID NO 1

ATG GAT ATC TTT AAA GAA CTA ATC TTA AAA CAC CCT GAT GAA AAT GTT 48 Met Asp lie Phe Lys Glu Leu lie Leu Lys His Pro Asp Glu Asn Val 1 5 10 15

TTG ATT TCT CCA GTT TCT ATT TTA TCT ACT TTA TCT ATT CCA AAT CAT 96 Leu He Ser Pro Val Ser He Leu Ser Thr Leu Ser He Pro Asn His 20 25 30 GGA GCA GCT GGT TCT ACA GCT GAA CAA CTA TCA AAA TAT ATA GAG AAT 144 Gly Ala Ala Gly Ser Thr Ala Glu Gin Leu Ser Lys Tyr He Glu Asn 35 40 45

GTG AAT GAG AAT ACC CCC GAT GAT AAG AAG GAT GAC AAT AAT GAC ATG 192 Val Asn Glu Asn Thr Pro Asp Asp Lys Lys Asp Asp Asn Asn Asp Met 50 55 60

GAC GTA GAT ATT CCG TAT TGT GCG ACA CTA GCT ACC GCA AAT AAA ATA 240 Asp Val Asp He Pro Tyr Cys Ala Thr Leu Ala Thr Ala Asn Lys He 65 70 75 80

TAC TGT AGC GAT AGT ATC GAG TTC CAC GCC TCC TTC CTA CAA AAA ATA 288 Tyr Cys Ser Asp Ser He Glu Phe His Ala Ser Phe Leu Gin Lys He 85 90 95 AAA GAC GGT TTT CAA ACT GTA AAC TTT AAT AAT GCT AAC CAA ACA AAG 336 Lys Asp Gly Phe Gin Thr Val Asn Phe Asn Asn Ala Asn Gin Thr Lys 100 105 110

GAA CTA ATC AAC GAA TGG GTT AAG ACG ATG ACA AAT GGT AAA ATT AAT 384 Glu Leu He Asn Glu Trp Val Lys Thr Met Thr Asn Gly Lys He Asn 115 120 125

TCC TTA TTG ACT AGT CCG CTA TCC ATT AAT ACT CGT ATG ACA GTT GTT 432 Ser Leu Leu Thr Ser Pro Leu Ser He Asn Thr Arg Met Thr Val Val 130 135 140

AGC GCC GTC CAT TTT AAA GCA ATG TGG AAA TAT CCA TTT TCT AAA CAT 480 Ser Ala Val His Phe Lys Ala Met Trp Lys Tyr Pro Phe Ser Lys His 145 150 155 160

CTT ACA TAT ACA GAC AAG TTT TAT ATT TCT AAG AAT ATA GTT ACC AGC 528 Leu Thr Tyr Thr Asp Lys Phe Tyr He Ser Lys Asn He Val Thr Ser 165 170 175 GTT GAT ATG ATG GTG AGC ACT GAG AAC GAC TTA CAA TAT GTA CAT ATT 576 Val Asp Met Met Val Ser Thr Glu Asn Asp Leu Gin Tyr Val His He 180 185 190

AAT GAA TTA TTC GGA GGA TTC TCT ATT ATC GAT ATT CCA TAC GAG GGA 624 Asn Glu Leu Phe Gly Gly Phe Ser He He Asp He Pro Tyr Glu Gly 195 200 205

AAC TCT AGT ATG GTA ATT ATA CTA CCG GAC GAC ATA GAA GGT ATA TAT 672 Asn Ser Ser Met Val He He Leu Pro Asp Asp He Glu Gly He Tyr 210 215 220

AAC ATA GAA AAA AAT ATA ACA GAT GAA AAA TTT AAA AAA TGG TGT GGT 720 Asn He Glu Lys Asn He Thr Asp Glu Lys Phe Lys Lys Trp Cys Gly 225 230 235 240

ATG TTA TCT ACT AAA AGT ATA GAC TTG TAT ATG CCA AAG TTT AAA GTG 768 Met Leu Ser Thr Lys Ser He Asp Leu Tyr Met Pro Lys Phe Lys Val 245 250 255 GAA ATG ACA GAA CCG TAT AAT CTG GTA CCG ATT TTA GAA AAT TTA GGA 816 Glu Met Thr Glu Pro Tyr Asn Leu Val Pro He Leu Glu Asn Leu Gly 260 265 270

CTT ACT AAT ATA TTC GGA TAT TAT GCA GAT TTT AGC AAG ATG TGT AAT 864 Leu Thr Asn He Phe Gly Tyr Tyr Ala Asp Phe Ser Lys Met Cys Asn 275 280 285

GAA ACT ATC ACT GTA GAA AAA TTT CTA CAT ACG ACG TTT ATA GAT GTT 912 Glu Thr He Thr Val Glu Lys Phe Leu His Thr Thr Phe He Asp Val 290 295 300

AAT GAG GAG TAT ACA GAA GCA TCG GCC GTT ACA GGA GTA TTT ATG ACT 960 Asn Glu Glu Tyr Thr Glu Ala Ser Ala Val Thr Gly Val Phe Met Thr 305 310 315 320

AAC TTT TCG ATG GTA TAT CGT ACG AAG GTC TAC ATA AAC CAT CCA TTC 1008 Asn Phe Ser Met Val Tyr Arg Thr Lys Val Tyr He Asn His Pro Phe 325 330 335

ATG TAC ATG ATT AAA GAC AAC ACA GGA CGT ATA CTT TTT ATA GGG AAA 1056 Met Tyr Met He Lys Asp Asn Thr Gly Arg He Leu Phe He Gly Lys 340 345 350

TAC TGC TAT CCG CAA TAA 1074

Tyr Cys Tyr Pro Gin * 355

(2) INFORMATION FOR SEQ ID NO 2

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 358 amino acids

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(xi) SEQUENCE DESCRIPTION SEQ ID NO 2

Met Asp He Phe Lys Glu Leu He Leu Lys His Pro Asp Glu Asn Val 1 5 10 15

Leu He Ser Pro Val Ser He Leu Ser Thr Leu Ser He Pro Asn His 20 25 30

Gly Ala Ala Gly Ser Thr Ala Glu Gin Leu Ser Lys Tyr He Glu Asn 35 40 45

Val Asn Glu Asn Thr Pro Asp Asp Lys Lys Asp Asp Asn Asn Asp Met 50 55 60 Asp Val Asp He Pro Tyr Cys Ala Thr Leu Ala Thr Ala Asn Lys He 65 70 75 80

Tyr Cys Ser Asp Ser He Glu Phe His Ala Ser Phe Leu Gin Lys He 85 90 95

Lys Asp Gly Phe Gin Thr Val Asn Phe Asn Asn Ala Asn Gin Thr Lys 100 105 110

Glu Leu He Asn Glu Trp Val Lys Thr Met Thr Asn Gly Lys He Asn 115 120 125

Ser Leu Leu Thr Ser Pro Leu Ser He Asn Thr Arg Met Thr Val Val 130 135 140 Ser Ala Val His Phe Lys Ala Met Trp Lys Tyr Pro Phe Ser Lys His 145 150 155 160

Leu Thr Tyr Thr Asp Lys Phe Tyr He Ser Lys Asn He Val Thr Ser 165 170 175

Val Asp Met Met Val Ser Thr Glu Asn Asp Leu Gin Tyr Val His He 180 185 190

Asn Glu Leu Phe Gly Gly Phe Ser He He Asp He Pro Tyr Glu Gly 195 200 205

Asn Ser Ser Met Val He He Leu Pro Asp Asp He Glu Gly He Tyr 210 215 220 Asn He Glu Lys Asn He Thr Asp Glu Lys Phe Lys Lys Trp Cys Gly 225 230 235 240

Met Leu Ser Thr Lys Ser He Asp Leu Tyr Met Pro Lys Phe Lys Val 245 250 255

Glu Met Thr Glu Pro Tyr Asn Leu Val Pro He Leu Glu Asn Leu Gly 260 265 270 Leu Thr Asn He Phe Gly Tyr Tyr Ala Asp Phe Ser Lys Met Cys Asn 275 280 285

Glu Thr He Thr Val Glu Lys Phe Leu His Thr Thr Phe He Asp Val 290 295 300

Asn Glu Glu Tyr Thr Glu Ala Ser Ala Val Thr Gly Val Phe Met Thr 305 310 315 320

Asn Phe Ser Met Val Tyr Arg Thr Lys Val Tyr He Asn His Pro Phe 325 330 335

Met Tyr Met He Lys Asp Asn Thr Gly Arg He Leu Phe He Gly Lys 340 345 350 Tyr Cys Tyr Pro Gin * 355

(2) INFORMATION FOR SEQ ID NO: 3 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1038 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(IX) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1..1038

(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 3 :

ATG GAT ATC TTC AGG GAA ATC GCA TCT TCT ATG AAA GGA GAG AAT GTA 48 Met Asp He Phe Arg Glu He Ala Ser Ser Met Lys Gly Glu Asn Val 360 365 370 TTC ATT TCT CCA GCG TCA ATC TCG TCA GTA TTG ACA ATA CTG TAT TAT 96 Phe He Ser Pro Ala Ser He Ser Ser Val Leu Thr He Leu Tyr Tyr 375 380 385 390

GGA GCT AAT GGA TCC ACT GCT GAA CAG CTA TCG AAA TAT GTA GAA AAG 144 Gly Ala Asn Gly Ser Thr Ala Glu Gin Leu Ser Lys Tyr Val Glu Lys 395 400 405

GAG GAG AAC ATG GAT AAG GTT AGC GCT CAA AAT ATC TCA TTC AAA TCC 192 Glu Glu Asn Met Asp Lys Val Ser Ala Gin Asn He Ser Phe Lys Ser 410 415 420

ATA AAT AAA GTA TAT GGG CGA TAT TCT GCC GTG TTT AAA GAT TCC TTT 240 He Asn Lys Val Tyr Gly Arg Tyr Ser Ala Val Phe Lys Asp Ser Phe 425 430 435

TTG AGA AAA ATT GGC GAT AAG TTT CAA ACT GTT GAC TTC ACT GAT TGT 288 Leu Arg Lys He Gly Asp Lys Phe Gin Thr Val Asp Phe Thr Asp Cys 440 445 450 CGC ACT ATA GAT GCA ATC AAC AAG TGT GTA GAT ATC TTT ACT GAG GGG 336 Arg Thr He Asp Ala He Asn Lys Cys Val Asp He Phe Thr Glu Gly 455 460 465 470

AAA ATC AAT CCA CTA TTG GAT GAA CAA TTG TCT CCT GAT ACC TGT CTC 384 Lys He Asn Pro Leu Leu Asp Glu Gin Leu Ser Pro Asp Thr Cys Leu 475 480 485

CTA GCA ATT AGT GCC GTA TAC TTT AAA GCA AAA TGG TTG ACG CCA TTC 432 Leu Ala He Ser Ala Val Tyr Phe Lys Ala Lys Trp Leu Thr Pro Phe 490 495 500

GAA AAG GAA TTT ACC AGT GAT TAT CCC TTT TAC GTA TCT CCG ACG GAA 480 Glu Lys Glu Phe Thr Ser Asp Tyr Pro Phe Tyr Val Ser Pro Thr Glu 505 510 515 ATG GTA GAT GTA AGT ATG ATG TCT ATG TAC GGC AAG GCA TTT AAT CAC 528 Met Val Asp Val Ser Met Met Ser Met Tyr Gly Lys Ala Phe Asn His 520 525 530

GCA TCT GTA AAG GAA TCA TTC GGC AAC TTT TCA ATC ATA GAA CTG CCA 576 Ala Ser Val Lys Glu Ser Phe Gly Asn Phe Ser He He Glu Leu Pro 535 540 545 550

TAT GTT GGA GAT ACT AGT ATG ATG GTC ATT CTT CCA GAC AAG ATT GAT 624 Tyr Val Gly Asp Thr Ser Met Met Val He Leu Pro Asp Lys He Asp 555 560 565

GGA TTA GAA TCC ATA GAA CAA AAT CTA ACA GAT ACA AAT TTT AAG AAA 672 Gly Leu Glu Ser He Glu Gin Asn Leu Thr Asp Thr Asn Phe Lys Lys 570 575 580

TGG TGT AAC TCT CTG GAA GCT ACG TTT ATC GAT GTT CAC ATT CCC AAG 720 Trp Cys Asn Ser Leu Glu Ala Thr Phe He Asp Val His He Pro Lys 585 590 595 TTT AAG GTA ACA GGC TCG TAT AAT CTG GTG GAT ACT CTA GTA AAG TCA 768 Phe Lys Val Thr Gly Ser Tyr Asn Leu Val Asp Thr Leu Val Lys Ser 600 605 610

GGA CTG ACA GAG GTG TTC GGT TCA ACT GGA GAT TAT AGC AAT ATG TGT 816 Gly Leu Thr Glu Val Phe Gly Ser Thr Gly Asp Tyr Ser Asn Met Cys 615 620 625 630

AAT TTA GAT GTG AGT GTC GAC GCT ATG ATC CAC AAA ACG TAT ATA GAT 864 Asn Leu Asp Val Ser Val Asp Ala Met He His Lys Thr Tyr He Asp 635 640 645

GTC AAT GAA GAG TAT CCA GAA GCA GCT GCA GCA ACT TCT GTA CTA GTG 912 Val Asn Glu Glu Tyr Pro Glu Ala Ala Ala Ala Thr Ser Val Leu Val 650 655 660

GCA GAC TGT GCA TCA ACA GTT ACA AAT GAG TTC TGT GCA GAT CAT CCG 960 Ala Asp Cys Ala Ser Thr Val Thr Asn Glu Phe Cys Ala Asp His Pro 665 670 675 TTC ATC TAT GTG ATT AGG CAT GTT GAT GGC AAA ATT CTT TTC GTT GGT 1008 Phe He Tyr Val He Arg His Val Asp Gly Lys He Leu Phe Val Gly 680 685 690

AGA TAT TGC TCT CCA ACA ACT AAT TGT TAA 1038

Arg Tyr Cys Ser Pro Thr Thr Asn Cys * 695 700

(2) INFORMATION FOR SEQ ID NO:4:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 346 ammo acids

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

(ill MOLECULE TYPE: protein

(xι) SEQUENCE DESCRIPTION. SEQ ID NO•4.

Met Asp He Phe Arg Glu He Ala Ser Ser Met Lys Gly Glu Asn Val 1 5 10 15

Phe He Ser Pro Ala Ser He Ser Ser Val Leu Thr He Leu Tyr Tyr 20 25 30

Gly Ala Asn Gly Ser Thr Ala Glu Gin Leu Ser Lys Tyr Val Glu Lys 35 40 45

Glu Glu Asn Met Asp Lys Val Ser Ala Gin Asn He Ser Phe Lys Ser 50 55 60 He Asn Lys Val Tyr Gly Arg Tyr Ser Ala Val Phe Lys Asp Ser Phe 65 70 75 80

Leu Arg Lys He Gly Asp Lys Phe Gin Thr Val Asp Phe Thr Asp Cys 85 90 95

Arg Thr He Asp Ala He Asn Lys Cys Val Asp He Phe Thr Glu Gly 100 105 110

Lys He Asn Pro Leu Leu Asp Glu Gin Leu Ser Pro Asp Thr Cys Leu 115 120 125

Leu Ala He Ser Ala Val Tyr Phe Lys Ala Lys Trp Leu Thr Pro Phe 130 135 140 Glu Lys Glu Phe Thr Ser Asp Tyr Pro Phe Tyr Val Ser Pro Thr Glu 145 150 155 160

Met Val Asp Val Ser Met Met Ser Met Tyr Gly Lys Ala Phe Asn His 165 170 175

Ala Ser Val Lys Glu Ser Phe Gly Asn Phe Ser He He Glu Leu Pro 180 185 190

Tyr Val Gly Asp Thr Ser Met Met Val He Leu Pro Asp Lys He Asp 195 200 205

Gly Leu Glu Ser He Glu Gin Asn Leu Thr Asp Thr Asn Phe Lys Lys 210 215 220 Trp Cys Asn Ser Leu Glu Ala Thr Phe He Asp Val His He Pro Lys 225 230 235 240

Phe Lys Val Thr Gly Ser Tyr Asn Leu Val Asp Thr Leu Val Lys Ser 245 250 255

Gly Leu Thr Glu Val Phe Gly Ser Thr Gly Asp Tyr Ser Asn Met Cys 260 265 270

Asn Leu Asp Val Ser Val Asp Ala Met He His Lys Thr Tyr He Asp 275 280 285

Val Asn Glu Glu Tyr Pro Glu Ala Ala Ala Ala Thr Ser Val Leu Val 290 295 300 Ala Asp Cys Ala Ser Thr Val Thr Asn Glu Phe Cys Ala Asp His Pro 305 310 315 320

Phe He Tyr Val He Arg His Val Asp Gly Lys He Leu Phe Val Gly 325 330 335

Arg Tyr Cys Ser Pro Thr Thr Asn Cys * 340 345