AU4435002A - Modified retinoblastoma tumor suppressor proteins - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Board of Regents, The University of Texas System and Baylor College of Medicine Actual Inventor(s): Hong-Ji Xu, Shi-Xue Hu, William F Benedict, Yunli Zhou Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: MODIFIED RETINOBLASTOMA TUMOR SUPPRESSOR PROTEINS Our Ref: 669585 POF Code: 1443/240397, 295734 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 1A

DESCRIPTION

MODIFIED RETINOBLASTOMA TUMOR SUPPRESSOR PROTEINS BACKGROUND OF THE INVENTION This application is a divisional of Australian Patent Application 66573/98, the entire content of which is incorporated herein by reference.

1. Field of the Invention The present invention relates generally to the field of molecular and cellular biology. More particularly, it concerns modifications of the retinoblastoma tumor suppressor. The present invention further relates to the use of the instant modified retinoblastoma tumor suppressors in situations where providing a tumor suppressor or normal cell growth suppressor is indicated.

2. Description of Related Art Cancers and tumors are the second most prevalent cause of death in the United States, causing approximately 450,000 deaths per year. One in three Americans will develop cancer, and one in five will die of cancer (Scientific American Medicine, part 12 I, 1, section dated 1987). While substantial progress has been made in identifying some of the likely environmental and hereditary causes of cancer, the statistics for the cancer death rate indicates a need for substantial improvement in the therapy for cancer and related diseases and disorders.

A number of genes have been implicated in the etiology of cancer. These genes have been identified in connection with hereditary forms of cancer, and in a large number of well-studied tumor cells. Study of cancer genes has helped provide some understanding of the process of tumorigenesis. While a great deal more remains to be learned about cancer genes, the presently known cancer genes serve as useful models for understanding tumorigenesis. Cancer genes are broadly classified into "oncogenes" which, when activated, promote tumorigenesis, and "tumor suppressor genes" which, when damaged, fail to suppress tumorigenesis. While these W:\cisk\nkipeaes\DDiv siof 66573-98 (May2002).doc WO 98/37091 PCT/US98/03041 2 classifications provide a useful method for conceptualizing tumorigenesis, it is also possible that a particular gene may play differing roles depending upon the particular allelic form of that gene, its regulatory elements, the genetic background and the tissue environment in which it is operating.

The oncogenes are somatic cell genes that are mutated from their wild-type alleles (the art refers to these wild-type alleles as protooncogenes) into forms which are able to induce tumorigenesis under certain conditions. There is presently a substantial literature on known and putative oncogenes and the various alleles of these oncogenes. For example, the oncogenes ras and myc are considered as models for understanding oncogenic processes in general. The ras oncogene is believed to encode a cytoplasmic protein, and the myc oncogene is believed to encode a nuclear protein. Neither the ras oncogene nor the myc oncogene alone is able to induce full transformation of a normal cell into a tumor cell, but full tumorigenesis usually occurs when both the ras and myc oncogenes are present and expressed together in the same cell (Weinberg, 1989). Such collaborative effects have been observed between a number of other studied oncogenes.

The collaborative model of oncogene tumorigenesis must be qualified by the observation that a cell expressing the ras oncogene that is surrounded by normal cells does not undergo full transformation. However, if most of the surrounding cells are also ras-expressing, then the ras oncogene alone is sufficient to induce tumorigenesis in a ras-expressing cell. This observation validates the multiple hit theory of tumorigenesis because a change in the tissue environment of the cell hosting the oncogene may be considered a second hit. An alternative and equally valid hypothesis is that events that collaborate with the activation of an oncogene such as ras or myc may include the inactivation of a negative regulatory factor or factors, a tumor suppressor protein (Weinberg, 1989; Goodrich et al., 1992a).

Tumor suppressor genes are genes that, in their wild-type alleles, express proteins that suppress abnormal cellular proliferation. When the gene coding for a tumor suppressor protein is mutated or deleted, the resulting mutant protein or the complete lack of a tumor suppressor protein may fail to correctly regulate cellular proliferation. This can lead to abnormal cellular proliferation, particularly if there is already existing damage to the cellular regulatory WO 98/37091 PCT/US98/03041 3 mechanism. The lack of control of cellular proliferation has been linked to the development of a wide variety of human cancers (Weinberg, 1991). A number of well-studied human tumors and tumor cell lines have been shown to have missing or nonfunctional tumor suppressor genes.

Examples of tumor suppressor genes and candidate tumor suppressor genes include, but are not limited to, the retinoblastoma (RB) gene (Friend et al., 1986; Fung et al., 1987; Lee et al., 1987a), the wild-type p53 gene (Finlay et al., 1989; Baker et al., 1990), the deleted in colon carcinoma (DCC) gene (Fearon et al., 1990a; 1990b), the neurofibromatosis type 1 (NF-1) gene (Wallace etal., 1990; Viskochil et al., 1990; Cawthon et al., 1990), the Wilms tumor (WT-1) gene (Call et al., 1990; Gessler et al., 1990; Pritchard-Jones et al., 1990), the von Hippel-Lindau (VHL) disease tumor suppressor gene (Duan el al., 1995), the Maspin (Zou et al., 1994), Brush-1 (Schott et al., 1994) and BRCA 1 genes (Miki et al., 1994; Futreal et al., 1994) for breast cancer.

and the multiple tumor suppressor (MTS) or pl6 gene (Serrano et al., 1993; Kamb et al.. 1994).

The list of putative tumor suppressor genes is large and growing, with the total number of tumor suppressor genes expected to be well beyond 50 (Knudson, 1993).

The first tumor suppressor gene identified was the retinoblastoma (RB) gene, which causes the hereditary retinoblastoma (Knudson, 1971; Murphree and Benedict, 1984; Knudson, 1985). The retinoblastoma (RB) gene, which was cloned in the middle 1980s, is one of the best studied tumor suppressor genes. The size of the RB gene complementary DNA (cDNA), about 4.7 kb, permits ready manipulation of the gene, and has led to the insertion of the RB gene into a Snumber of cell lines. The RB gene has been shown to be missing or defective in a majority of retinoblastomas, sarcomas of the soft tissues and bones, and in approximately 20 to 40 percent of breast, lung, prostate and bladder carcinomas (Lee et al., WO 90/05180; Bookstein et al., 1991; Benedict et al., 1990).

The most direct proof that the cloned RB gene is indeed a tumor suppressor gene is the observed recovery of tumor suppression function in RB-minus tumor cells from the introduction of a cloned intact copy of the RB gene. A number of reports have indicated that replacement of the normal RB gene in RB-defective tumor cells from disparate types of human cancers could suppress their tumorigenic activity in nude mice (Huang et al., 1988; Goodrich and Lee, 1993; Zhou et al., 1994b). The tumor cell lines studied were derived from widely disparate types of WO 98/37091 PCT/US98/03041 4 human cancers such as the retinoblastoma, osteosarcoma, carcinomas of the bladder, prostate, breast and lung.

While it was observed that introduction of a functional wild-type, full-length retinoblastoma gene into an RB-minus tumor cell "normalizes" the cell, it was not expected that tumor cells which already have normal RB" 1 gene expression would respond to RB" 0 gene therapy, because it was presumed that adding additional RB expression could not correct a non-RB genetic defect. This has in fact been shown for the case of the RB osteosarcoma cell line U-2 OS, where the introduction of an extra pi 10 coding gene did not change the neoplastic phenotype (Huang et al., 1988). Thus, there remains a need for a broadspectrum tumor suppressor gene for treating abnormally proliferating cells having any type of genetic defect.

The RB 1 o cDNA open reading frame sequence (McGee et al., 1989) contains a second in-frame AUG codon located in exon 3, at nucleotides 355-357. The protein initiated from this second AUG codon lacks the N-terminal 112 amino acid residues of the full-length RB protein, 94 and is termed pRB (Xu et al., 1994b). In U.S. Patent 5,496,731 (incorporated herein by 94 reference), the inventors showed that RB-defective tumor cells expressing exogenous pRB9 did 3 not progress through the cell cycle, as evidenced by their failure to incorporate H]-thymidine into DNA. In contrast, the percent of tumor cells undergoing DNA replication were only slightly lower in cells producing the exogenous pRB (the wild-type pRB protein) than in cells that 94 were RB-. Even more striking was that the pRB expression also significantly reduced colony formation of two RB+ (with normal RB alleles) tumor cell lines examined, namely the fibrosarcoma cell line, HT1080, and the cervical carcinoma cell line, HeLa (Xu et al., 1994b), 110 while no such effects were observed when an additional pRB -coding gene(s) was introduced by transfection using plasmid vectors (Fung et al., 1993) or by microcell fusion (Anderson et al., 1994).

However, there is a paucity of tumor suppressor proteins in the art which have all of the properties necessary to facilitate their use in the treatment of diseases, particularly cancer.

WO 98/37091 PCT/US98/03041 SUMMARY OF THE INVENTION The modified retinoblastoma tumor suppressors of the present invention overcome the shortcomings of those described in the art, providing a broad spectrum tumor suppressor with surprising beneficial effects.

The present invention provides broad-spectrum modified retinoblastoma tumor suppressor proteins that are suprisingly at least as effective, and in most cases more effective, than the corresponding wild-type retinoblastoma tumor suppressor proteins in inhibiting cell growth. In particular embodiments, the invention provides retinoblastoma tumor suppressor proteins that have a modified N-terminal region. The invention further provides methods of making and using the modified retinoblastoma tumor suppressor proteins, particularly in circumstances wherein cell growth inhibition is desired. Thus the present invention provides methods for treating diseases, as exemplified by, but not limited to cancer, that are characterized by abnormal cellular proliferation.

A broad-spectrum tumor suppressor gene is a genetic sequence coding for a protein that, when inserted into and expressed in an abnormally proliferating host cell, a tumor cell, suppresses abnormal proliferation of that cell irrespective of the cause of the abnormal proliferation.

Thus, the invention provides an isolated DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB 94 or pRB 56 the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification. The terms "pRB 94 and "pRB 56 refer to retinoblastoma proteins that have a molecular weight of 94 kDa and 56 kDa, respectively. As understood in the art, the pRB 94 and pRB 56 retinoblastoma proteins are fragments of the full length wild-type retinoblastoma protein that have 112 and 379 contiguous amino acids deleted from the N-terminus, respectively.

WO 98/37091 PCT/US98/03041 6 The term "N-terminal", or "N-terminal region", as used herein, will be. understood to refer to the region of a protein corresponding to as much as the first approximately 40% of the amino acid sequence. Thus, these terms will be understood to include up to about the first the first the first 15%, the first 20%, the first-25%, the first 30% or the first 35% of the amino acid sequence of a protein. However, these values are only approximations, and therefore will be understood to include intermediate values, such as 11%, 13%, 17%, 18%, 22%, 26%, 33%, 37%, 38%, 41%, 42% and the like.

The term "modified", as used herein, refers to deletions and/or mutations of the wild-type protein sequence. In certain embodiments, it may also refer to insertion of a heterologous amino acid or amino acids into the wild-type protein sequence. In yet other aspects, the term may refer to post-translational alteration of the wild-type amino acid sequence.

In a further embodiment of the invention, the gene encodes a modified retinoblastoma tumor suppressor protein comprising an N-terminal region that comprises a first sequence region from which at least one amino acid has been deleted. The deletion may produce a modified retinoblastoma tumor suppressor protein with a biological activity equal to, or in certain embodiments, greater than the biological activity of the corresponding wild-type retinoblastoma tumor suppressor protein.

In a particular embodiment of the invention the gene encodes a modified retinoblastoma tumor suppressor protein wherein at least two amino acids have been deleted from the first sequence region. In other embodiments of the invention at least about five amino acids, at least about ten amino acids, at least about 25 amino acids, at least about 50 amino acids, at least about 75 amino acids or at least about 100 amino acids have been deleted from the first sequence region. It will be understood that intermediate deletion sizes are also contemplated, such as, but not limited to, 3, 4, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 amino acids and the like.

PCT/US98/03041 WO 98/37091 7 In other aspects of the invention, the gene encodes a modified retinoblastoma tumor suppressor protein wherein at least about 150 amino acids, at least about 200 amino acids, at lest about 250 amino acids, at least about 300 amino acids or at least about 370 amino acids have been deleted from the first sequence region. However, intermediate 'sized deletions are also provided, exemplified by, but not limited to, 101, 102, 103, 104, 105, 106, 107, 108, 109, .110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 149, 151, 152, 153, 154, 169, 170, 171, 172, 173, 188, 189, 190, 191, 192, 207, 208, 209, 210, 211, 226, 227,228, 229, 230, 245, 246.247, 248, 249, 265, 266,267, 268, 269, 284. 285,286, 287, 288, 304, 305.306, 307, 308, 135, 136, 155, 156, 174, 175, 193, 194.

212,213.

231,232.

251, 252, 270.271, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 195, 196, 197, 198, 199, 200, 201, 202, 203. 204, 205, 214, 215, 216, 217, 218, 219, 220, 221. 222. 223. 224, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 253, 254, 255. 256, 257, 258, 259, 260, 261, 262, 263.

272. 273, 274, 275, 276, 277, 278, 279, 280. 281, 282.

148, 168, 187, 206.

225, 244, 264.

283.

289, 290, 291, 309,310, 311, 292, 293, 312, 313, 294, 314, 295, 315, 296. 297,298, 316,317, 318, 299.

319, 301, 320, 302, 303, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 371, 372, 373, 374, 375, 376, 377 or 378 amino acid deletions. Other intermediate values are disclosed throughout the specification.

In one embodiment of the invention the gene encodes a modified retinoblastoma tumor suppressor protein comprising an N-terminal region that comprises at least a first sequence region located between about amino acid 1 and about amino acid 50 from which at least one amino acid has been deleted. It will be understood that "between about amino acid 1 and about amino acid 50" includes amino acid 1 and amino acid 50, and it is thus so with other deletions described herein. Amino acid 1 is the N-terminal amino acid, and the numbers increase toward the C-terminus.

In further embodiments of the invention, the first sequence region is located between about amino acid 51 and about amino acid 100, between about amino acid 101 and about amino WO 918/37091 PCT/US98/03041 8 acid 150, between about amino acid 151 and about amino acid 200, between about amino acid 201 and about amino acid 250 or between about amino acid 251 and about amino acid 300.

In other embodiments of the present invention, the gene encodes a modified retinoblastoma tumor suppressor protein wherein the first sequence region is located between about amino acid 1 and about amino acid 100, between about amino acid 51 and about amino acid 150, between about amino acid 101 and about amino acid 200, between about amino acid 151 and about amino acid 250 or between about amino acid 201 and about amino acid 300.

In a particular aspect of the invention the gene encodes a modified retinoblastoma tumor suppressor protein wherein the first sequence region is located between about amino acid 1 and about amino acid 150. In additional aspects of the invention the first sequence region is located between about amino acid 51 and about amino acid 200, between about amino acid 101 and about amino acid 250 or between about amino acid 151 and about amino acid 300.

In further embodiments of the invention the gene encodes a modified retinoblastoma tumor suppressor protein wherein the first sequence region is located between about amino acid 1 and about amino acid 200, between about amino acid 51 and about amino acid 250, between about amino acid 101 and about amino acid 300, between about amino acid 1 and about amino acid 250, between about amino acid 51 and about amino acid 300, between about amino acid 1 and about amino acid 300 or between about amino acid 1 and about amino acid 370.

In yet another aspect of the invention the modified retinoblastoma tumor suppressor protein is a modified retinoblastoma protein wherein about amino acid 2 through about amino acid 34 have been deleted from the first sequence region. The location of these particular amino acids is in reference to the human wild-type retinoblastoma protein, but will be understood to correspond to analogous regions of homologous retinoblastoma proteins. In yet another aspect of the invention about amino acid 2 through about amino acid 55 have been deleted from the first sequence region. In still another aspect of the invention about amino acid 2 through about amino acid 78 have been deleted from the first sequence region. In a particular aspect of the invention about amino acid 2 through about amino acid 97 have been deleted from the first sequence WO 98/37091 PCT/US98/03041 9 region. In an additional aspect of the invention about amino acid 2 through about amino acid 148 have been deleted from the first sequence region.

In another embodiment of the invention the modified retinoblastoma tumor suppressor protein is a modified retinoblastoma protein wherein about amino acid 31 through about amino acid 107 have been deleted from the first sequence region. In another embodiment of the invention about amino acid 77 through about amino acid 107 have been deleted from the first sequence region. In a further embodiment of the invention about amino acid 111 through about amino acid 181 have been deleted from the first sequence region. In yet another embodiment of the invention about amino acid 111 through about amino acid 241 have been deleted from the first sequence region. In still another embodiment of the invention about amino acid 181 through about amino acid 241 have been deleted from the first sequence region. In a particular embodiment of the invention about amino acid 242 through about amino acid 300 have been deleted from the first sequence region.

In one aspect of the invention the N-terminal region of the modified retinoblastoma tumor suppressor protein further comprises at least a second sequence region from which at least one amino acid has been deleted. In a particular aspect of the invention, about amino acid 2 through about amino acid 34, and about amino acid 76 through about amino acid 112 have been deleted.

In a further aspect of the invention about amino acid 2 through about amino acid 55, and about amino acid 76 through about amino acid 112 have been deleted.

Another embodiment of the invention provides a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB 94 the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification wherein the gene encodes a modified retinoblastoma tumor suppressor protein comprising at least a first N-terminal mutation, and wherein the modified retinoblastoma tumor suppressor protein has an increased biological activity in comparison to the biological activity of the corresponding wild type retinoblastoma tumor suppressor protein. In one embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a mutation at position 111. In another embodiment of the invention the modified retinoblastoma protein comprises glycine at position 111 in place of aspartic acid.

WO 98/37091 PCT/US98/03041 In a further embodiment of the invention the modified retinoblastoma tumor suppressor protein comprises at least a second N-terminal mutation. In yet another embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a mutation at position 111 and a mutation at position 112. In still another embodiment of the invention the modified retinoblastoma protein comprises glycine at position 111 in place of aspartic acid, and aspartic acid at position 112 in place of glutamic acid. In a particular embodiment of the invention the gene encodes a modified retinoblastoma tumor suppressor protein comprising an N-terminal region from which at least one amino acid has been deleted, and which contains at least one amino acid mutation.

In one aspect of the invention the gene encodes a modified retinoblastoma tumor suppressor protein that comprises a contiguous amino acid sequence from at least about position 370 to about position 928 of SEQ ID NO:2. In another aspect of the invention the gene encodes a modified retinoblastoma tumor suppressor protein that comprises a contiguous amino acid sequence from at least about position 3 to about position 928 of SEQ ID NO:2. When used in this context, "a contiguous amino acid sequence" will be understood to be a contiguous amino acid sequence of at least about 8, about 10, about 12, about 15, about 20, about 25, about 50 or about 100 amino acids and so on up to the full length amino acid sequence.

In a further aspect of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:29. In yet another aspect of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2691 of SEQ ID NO:28. When used herein in this context, "a contiguous nucleic acid sequence" will be understood to be a contiguous nucleic acid sequence of at least about 8, about about 12, about 15, about 17, about 20, about 25, about 50 or about 100 nucleotides and so on up to the full length nucleotide sequence.

In still another aspect of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:31. In a particular aspect of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2628 of SEQ ID NO:30. In an additional aspect of the invention the gene encodes a WO 98/37091 PCT/US98/03041 11 modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:33.

In another embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2559 of SEQ ID NO:32. In a further embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:35. In yet another embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2502 of SEQ ID NO:34. In still another embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:37. In a particular embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2349 of SEQ ID NO:36. In an additional embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:39.

In one aspect of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2559 of SEQ ID NO:38. In another aspect of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:41. In a further aspect of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2697 of SEQ ID NO:40. In yet another aspect of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:43. In still another aspect of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2583 of SEQ ID NO:42. In a particular aspect of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:45. In an additional aspect of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2397 of SEQ ID NO:44.

In one embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:47. In another embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2613 of SEQ ID NO:46. In a further embodiment of the invention the gene encodes a WO 98/37091 PCT/US98/03041 12 modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:49. In yet another embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2619 of SEQ ID NO:48. In still another embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:51. In a particular embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2790 of SEQ ID The invention thus provides a gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43. SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 or SEQ ID NO:51. In one aspect of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2691 of SEQ ID NO:28, from between position 7 and position 2628 of SEQ ID NO:30, from between position 7 and position 2559 of SEQ ID NO:32, from between position 7 and position 2502 of SEQ ID NO:34, from between position 7 and position 2349 of SEQ ID NO:36, from between position 7 and position 2559 of SEQ ID NO:38, from between position 7 and position 2697 of SEQ ID NO:40, from between position 7 and position 2583 of SEQ ID NO:42, from between position 7 and position 2397 of SEQ ID NO:44, from between position 7 and position 2613 of SEQ ID NO:46, from between position 7 and position 2619 of SEQ ID NO:48 or from between position 7 and position 2790 of SEQ ID Another embodiment of the invention provides a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB 94 or pRB 56 the mnodified retinoblastoma tumor suppressor protein comprising an N-terminal modification, where the DNA segment is operationally positioned under the control of a promoter. In one embodiment of the invention this DNA segment is operationally positioned under the control of a recombinant promoter. In another embodiment of the invention the DNA segment is further defined as a recombinant vector. In a particular aspect of the present invention, the recombinant vector is an adenoviral vector. In another aspect, the recombinant vector is a retroviral vector.

WO 98/37091 PCT/US98/03041 13 In a further embodiment of the invention the DNA segment is further defined as a component of a tetracycline responsive expression system. In yet another embodiment of the invention the DNA segment is operatively positioned downstream of a promoter comprising a tetracycline operator nucleic acid sequence; the tetracycline responsive expression system further 5 comprising a second sequence region comprising an isolated gene encoding a fusion protein comprising a transcriptional transactivation domain operatively attached to a tetracycline repressor protein, the second sequence region operatively positioned downstream of a minimal promoter.

In yet another embodiment of the invention the tetracycline responsive expression system is comprised within an adenoviral vector. In still another embodiment of the invention the adenoviral vector is comprised within a recombinant adenovirus.

The invention also provides a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB 94 the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification, which is comprised within a host cell. In one embodiment of the invention the host cell is a prokaryotic cell. In another embodiment of the invention the host cell is a eukaryotic cell. In a further embodiment of the invention the host cell is a human cell. In yet another embodiment of the invention the host cell is a tumor cell. In still another embodiment of the invention the host cell is comprised within an animal. In a particular embodiment of the invention the animal is a human subject.

Another embodiment of the invention provides a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB 94 the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification, which is dispersed in a pharmaceutically acceptable excipient.

Yet another embodiment of the invention provides an isolated DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB 94 the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification, wherein the modified retinoblastoma tumor suppressor protein is characterized as: comprising an N-terminal region that comprises at least a first sequence region from which at least one amino WO 98/37091 PCT/US98/03041 14 acid has been deleted, and wherein the modified retinoblastoma tumor suppressor protein has a biological activity at least about equivalent to the biological activity of the corresponding wild-type retinoblastoma tumor suppressor protein; or comprising an N-terminal region that comprises a first sequence region comprising at least one mutation, and wherein the modified retinoblastoma tumor suppressor protein has an increased biological activity in comparison to the biological activity of the corresponding wild-type retinoblastoma tumor suppressor protein.

In certain aspects of the invention, the DNA segments as described above are contemplated for use in expressing a modified retinoblastoma tumor suppressor protein, for example in a host cell. In other aspects, the DNA segments are contemplated for use in inhibiting cellular proliferation, or in the preparation of a medicament for inhibiting cellular proliferation or treating cancer, for example in a human patient. Thus, the use of the instant DNA segments in the preparation of a modified retinoblastoma tumor suppressor protein, in inhibiting cellular proliferation, and in the preparation of a medicament for inhibiting cellular proliferation or treating cancer is provided. In certain uses, the medicament is intended for administration to a human patient, or formulated for parenteral administration.

The invention further provides a modified retinoblastoma tumor suppressor protein other than pRB 9 4 the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification.

The invention also provides a recombinant host cell comprising a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification. In one aspect of the invention the host cell is a prokaryotic host cell. In another aspect of the invention the host cell is E. coli. In a further aspect of the invention the host cell is a eukaryotic host cell. In yet another aspect of the invention the host cell is a tumor cell. In still another aspect of the invention the DNA segment is introduced into the cell by means of a recombinant vector.

The invention further provides a method of inhibiting cellular proliferation, comprising contacting a cell with an effective inhibitory amount of a first modified retinoblastoma tumor WO 98/37091 PCT/US98/03041 suppressor protein other than pRB 94 the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification. In one embodiment of the invention the first modified retinoblastoma tumor suppressor protein comprises a modified retinoblastoma protein from which amino acids 111 through 241 have been deleted. In another embodiment of the invention the first modified retinoblastoma tumor suppressor protein comprises a modified retinoblastoma protein that comprises a mutation at position 111 and position 112. In a further embodiment of the invention the first modified retinoblastoma tumor suppressor protein is prepared by expressing a DNA segment encoding the modified retinoblastoma tumor suppressor protein in a recombinant host cell and collecting the modified retinoblastoma tumor suppressor protein expressed by the cell. In yet another embodiment of the invention the cell is contacted with the first modified retinoblastoma tumor suppressor protein by providing to the cell a DNA segment that expresses the first modified retinoblastoma tumor suppressor protein in the cell. In still another embodiment of the invention the cell is provided with a tetracycline responsive expression vector system that expresses the first modified retinoblastoma tumor suppressor protein in the cell. In a particular embodiment of the invention the vector system is an adenoviral vector system.

Another aspect of the invention provides a method of inhibiting cellular proliferation, comprising contacting a tumor cell with an effective inhibitory amount of a first modified retinoblastoma tumor suppressor protein other than pRB 94 the protein comprising an N-terminal modification. In one aspect of the invention the cell is located within an animal and the first modified retinoblastoma tumor suppressor protein, or a gene encoding the modified retinoblastoma tumor suppressor protein, is administered to the animal in a pharmaceutically acceptable vehicle. As used herein, the term "gene" is defined as an isolated DNA segment that includes the coding region of the protein, or a portion thereof. Thus the term "gene" includes genomic DNA, cDNA or RNA encoding the protein.

In another aspect of the invention the animal is a human subject. In a further aspect of the invention the cell is further contacted with a second tumor suppressor protein. In yet another aspect of the invention the cell is contacted with a modified retinoblastoma protein and a wild-type retinoblastoma, p53 or other tumor suppressor protein.

WO 98/37091 PCT/US98/03041 16 The invention further provides a method of inhibiting cellular proliferation, comprising contacting a cell with a retinoblastoma protein and a p53 protein in a combined amount effective to inhibit cellular proliferation in the cell.

The invention also provides a method of treating cancer, comprising administering to an animal with cancer a pharmaceutically acceptable composition comprising a biologically effective inhibitory amount of a first modified retinoblastoma tumor suppressor protein, other than pRB 9 4 that comprises an N-terminal modification.

The terms "cancer" or "tumor" are clinically descriptive terms which encompass a myriad of diseases characterized by cells that exhibit unchecked and abnormal cellular proliferation.

The term "tumor", when applied to tissue, generally refers to any abnormal tissue growth, i.e., excessive and abnormal cellular proliferation. A tumor may be "benign" and unable to spread from its original focus, or "malignant" and capable of spreading beyond its anatomical site to other areas throughout the hostbody. The term "cancer" is an older term which is generally used to describe a malignant tumor or the disease state arising therefrom. Alternatively, the art refers to an abnormal growth as a neoplasm, and to a malignant abnormal growth as a malignant neoplasm.

Irrespective of whether the growth is classified as malignant or benign, the causes of excessive or abnormal cellular proliferation of tumor or cancer cells are not completely clear.

Nevertheless, there is persuasive evidence that abnormal cellular proliferation is the result of a failure of one or more of the mechanisms controlling cell growth and division. It is also now believed that the mechanisms controlling cell growth and division include the genetic and tissuemediated regulation of cell growth, mitosis and differentiation. These mechanisms are thought to act at the cell nucleus, the cell cytoplasm, the cell membrane and the tissue-specific environment of each cell. The process of transformation of a cell from a normal state to a condition of excessive or abnormal cellular proliferation is called tumorigenesis.

It has been observed that tumorigenesis is usually a multistep progression from a normal cellular state to, in some instances, a full malignancy. It is therefore believed that multiple "hits" upon the cell regulatory mechanisms are required for full malignancy to develop. Thus, in most WO 98/37091 PCT/US98/03041 17 instances, it is believed that there is no single cause of excessive proliferation, but that these disorders are the end result of a series of cumulative events.

While a malignant tumor or cancer capable of unchecked and rapid spread throughout the body is the most feared and usually the deadliest type of tumor, even so-called benign tumors or growths can cause significant morbidity and mortality by their inappropriate growth. A benign tumor can cause significant damage and disfigurement by inappropriate growth in cosmetically sensitive areas, or by exerting pressure on central or peripheral nervous tissue, blood vessels and other critical anatomical structures.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Relative activities of the modified hCMV promoters. The 5637 bladder carcinoma cells (lanes 1-5) and Saos2 osteocarcinoma cells (lanes 6-10) were transfected with reporter plasmids in which CAT gene expression was driven by the various modified (mhCMVp3, lanes 2 and 7; mhCMVp2, lanes 3 and 8; mhCMVpl, lanes 4 and 9) or full-length hCMV promoters (lanes 5 and 10). The CAT activity is shown on the vertical axis. The CAT activity of the cells transfected with the plasmid carrying the full-length hCMV promoter (lanes and 10) is defined as 100 percent.

FIG. 2. Expression of tTA from the modified mCMVp-tTA cassette has no squelching effects on the 5637 cell growth. A method of staining cells with crystal violet followed by measuring OD 550 was used for quantification of relative cell numbers (OD 55 0 shown on vertical axis; Gillies et al., 1986). Shown is the growth parent cells with and without (0) tetracycline, and the mCMVp-tTA transfected cells with and without tetracycline. Days after transfection are shown on the horizontal axis.

WO 98/37091 PCT/US98/03041 18 FIG. 3A, FIG. 3B and FIG. 3C. The effects of tetracycline-regulatable pRB expression on tumor cell growth (OD 55 0 vertical axis). FIG. 3A. Representative long-term clone from the RB-reconstituted osteosarcoma cell line (Saos-2, clone 11). FIG. 3B. Representative long-term clone from the RB-reconstituted breast carcinoma cell line (MDA-MB-468, clone 19-4).

FIG. 3C. Representative long-term clone from the RB-reconstituted bladder carcinoma cell line (5637, clone 34-6). The cells were grown in the presence of 0.5 pIg/ml of Tc versus absence of Tc Cell growth of the tumor cells stopped 1 to 2 days after pRB expression was turned on in Tc-free medium (days shown on horizontal axis). The growth cessation was irreversible at day 4 (arrows) after stimulation with fresh medium containing 15% serum (Saos-2), 10% serum plus 2 ug/ml phytohemagglutinin (PHA; MDA-468) or 10% serum plus 4 4g/ml of concanavalin A (Con A; 5637).

FIG. 4A, FIG. 4B and FIG. 4C. The effects of tetracycline-regulatable pRB expression on soft agar colony formation. FIG. 4A. Percent colony formation (vertical axis) for three independent Saos2 osteosarcoma cell line clones (RBII0 C14, lane 2; RB110 Cl11, lane 3; RB 10 C113, lane 4) and the Saos2 parent strain (lane FIG. 4B. Percent colony formation (vertical axis) for two independent MDA-MB-468 breast carcinoma cell line clones (Rbll0 C119-4, lane 2; Rb110C120-1, lane 3) and the MDA-MB-468 parent strain (lane FIG. 4C.

Percent colony formation (vertical axis) for two independent 5637 bladder carcinoma cell line clones (Rbll0 C134-6, lane 2; Rbl10 C136-9, lane 3) and the 5637 parent strain (lane Soft agar colony formation of tumor cells with tetracycline-regulatable pRB expression was completely abrogated by induction of pRB in tetracycline-free medium. Colony formation is shown in the presence (open bar) and the absence (hatched bar) of tetracycline.

FIG. 5. Time course analysis of the pRB 94 and pRB" 1 0 expression in representative, Tcregulatable Saos-2 cell clones in Tc-free media and its effects on DNA synthesis, using a 3 H-thymidine incorporation assay. Lack of DNA synthesis as determined by failure of the tumor cells to incorporate thymidine implies growth cessation. The non-synchronized parental Saos-2 cell population maintained steady DNA synthesis; Representative pRB" 0 -reconstituted and pRB 94 -reconstituted Saos-2 clones are illustrated. :Percent 3 H-labeled cells is shown on the vertical axis, and the hours after removal of tetracycline is shown on the horizontal axis.

WO 98/37091 PCT/US98/03041 19 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS A. Tumor Suppressor Proteins 1. Retinoblastoma Based upon study of the isolated RB cDNA clone, the predicted RB gene product has 928 amino acids and an expected molecular weight of 106 kDa (Lee et al., 1987a; 1987b). The natural factor corresponding to the predicted RB gene expression product has been identified as a nuclear phosphoprotein having an apparent relative molecular mass of between 105 and 114 kDa (Lee et al., 1987b; Xu et al., 1989b; Yokota et al., 1988; Whyte et al., 1988). The literature generally refers to the protein encoded by the RB gene as pll10' On SDS-PAGE normal human cells show an RB protein pattern consisting of a lower sharp band with an Mr of 110 kD and a broader, more variable region above this band with an Mr ranging from 110 kD to 116 kD.

The 110 kD band is the underphosphorylated RB protein, whereas the broader region represents the phosphorylated RB protein. The heterogeneity of the molecular mass results from a varying degree of phosphorylation (Xu et al., 1989b).

After years of intense scrutiny, the biological functions of the RB. gene are beginning to be understood (reviewed in Cooper and Whyte, 1989; Hamel et al., 1993; Horowitz, 1993; Riley et al., 1994; Wang et al., 1994; Weinberg, 1995). The RB protein shows cyclical changes in phosphorylation during the cell cycle. Most RB protein is unphosphorylated during G1 phase, but most (perhaps all) RB molecules are phosphorylated in S and G2 phases (Xu et al., 1989b; DeCaprio et al., 1989; Buchkovich et al., 1989; Chen et al., 1989; Mihara et al., 1989). The established components of the pRB pathway include the E2F transcription factors, which are involved in transcriptional control of numerous cellular genes responsible for the advances of cells through the cell cycle (Nevins, 1992; La Thangue, 1994). The pRB also interacts with certain GI phase cyclins (Koff et al., 1992; Resnitzky and Reed, 1995; Geng et al., 1996).

Therefore, the RB gene apparently plays a key role in cell growth regulation being involved in the major decisions during the G1 phase of the cell cycle which govern cell proliferation, quiescence and differentiation (Weinberg, 1995). Furthermore, only the underphosphorylated RB protein binds to SV40 large T antigen. Given that RB protein binding by large T antigen is probably important for the growth promoting effects of large T antigen, this suggests that the WO 98/37091 PCT/US98/03041 underphosphorylated RB protein is the active form of the RB protein, and the phosphorylated RB protein in S and G2 phases is inactive (Ludlow et al., 1989).

It was reported that there was a striking difference in the ratio of underphosphorylated to phosphorylated pRB forms between normal fibroblasts growing exponentially and those arrested in G1 phase. More underphosphorylated pRB was observed in GI arrested cells, suggesting the change in ratio of phosphorylated to underphosphorylated RB proteins was related to the fluctuation of cell cycle (Xu et al., 1989b). Four subsequent papers have described the cell cycle-dependent phosphorylation of RB protein in detail (DeCaprio et al., 1989: Buchkovich et al.; 1989; Chen et al., 1989; Mihara et al., 1989). It is now widely accepted that the product of the RB gene has a key role in the cell cycle control.

Cell proliferation depends on transcriptional activation of genes that are responsible for the onset of DNA synthesis as well as other critical events in the G phase of the cell cycle. As demonstrated by Pardee, transition of cells from a serum mitogens-dependent to serum mitogensindependent state is separated by a distinct time point at several hours before the onset of S phase, namely the R (restriction) point (Pardee, 1989). By passing through the R point, the cell commits itself to complete the remainder of the cell cycle through M phase. Therefore, the R point between the middle Gl and late GI phases of the cell cycle represents a transition in the life of the cell that is as important as the G1/S boundary.

The phosphorylation status of pRB undergoes a readily distinguishable alteration at a time close to and perhaps contemporaneous with the R point transition of the cell cycle (Weinberg, 1995). During middle GI phase, the only pRB species detected is an underphosphorylated form. When cells progress through the cell cycle, the pRB content increases gradually. However, the majority of pRB synthesized after middle G1 phase is hyperphosphorylated. In other words, pRB hyperphosphorylation occurs in late GI, preceding the G1/S boundary (Xu etal., 1991a; Mittnacht etal., 1994). pRB maintains this hyperphosphorylated status throughout the remainder of the cell cycle, becoming dephosphorylated only upon evolution from M/early Gl (Ludlow et al., 1990; Xu et al., 1991a; Mittnacht et al., 1994).

WO 98/37091 PCT/US98/03041 21 The underphosphorylated form of pRB is able to form complexes with the transcription factor E2Fs or directly interact with the E2F site, and switches the E2F site from a positive to negative element in transcriptional control. The E2F site is present in the promoters of diverse cellular genes that are responsible for the advances of cells through the cell cycle, including c-myc, B-myb, cdc2, dihydrofolate reductase, thymidine kinase, and the RB as well as the E2F-1 gene itself (Chellappan et al., 1991; Nevins, 1992; Weintraub et al., 1992; La Thangue, 1994; Shan etal., 1994; Sardet etal., 1995; Shan etal., 1996). Since hyperphosphorylated pRB appears to have lost the ability to interact with E2Fs, the inhibitory function of pRB on cell growth can be abrogated by hyperphosphorylation.

The timing of pRB phosphorylation led to an attractive functional model (Weinberg, 1995). This model suggests that pRB is an R point guardian. pRB exerts most of its growth inhibitory effects in the first two thirds of the G phase. A cell that has progressed through early and middle G1 encounters the R point gate. Should conditions be ready for advance into the remainder of the cell cycle, pRB will undergo phosphorylation and functional inactivation, causing it to open the gate and to permit the cell to proceed into late Gl. Cells that lack normal pRB function for various reasons will proceed freely into late G1. Without pRB, the upstream components of the cell cycle clock that regulate pRB phosphorylation, such as cyclin D, cyclin E and their corresponding cyclin-dependent kinases (CDKs) (Kato et al., 1993; Ewen et al., 1993) lose much of their influences in the decision of the cell to pass through the R point gate. Taken together, pRB allows the cell cycle clock to control the expression of numerous genes that mediate advance of the cell through a critical phase of its growth cycle being involved in the major decisions concurrent with the R point transition. Functional loss of pRB deprives the cell of this clock and thus of an important mechanism for braking cell proliferation.

Various mutations of the RB gene are known, and these are generally inactive. Mutations in RB are seen in virtually all cases of retinoblastoma; additionally, the RB gene products could potentially be inactivated by hyperphosphorylation, and by viral oncoprotein-like cellular protein binding. Although the RB gene was initially named because deletions or mutations within the gene caused the rare childhood ocular tumor, retinoblastoma, loss of pRB function is not only causally related to the retinoblastoma, but is also linked to the progression of many 'common human cancers. Additionally, there is growing evidence suggesting that the RB protein status is WO 98/37091 PCT/US98/03041 22 potentially a prognostic marker in urothelial carcinoma, non-small cell lung carcinoma, and perhaps also in some other types of human neoplasms (Xu, 1995).

In addition, with the revolutionary antigen retrieval technique and the available specific anti-pRB antibodies, immunohistochemistry has recently become one of the highly sensitive and reliable methods for detection of pRB inactivation in routinely processed pathological specimens (Xu, 1995). Altered pRB expression as determined by immunohistochemical analysis appears to signal a poor prognosis in a subset of human malignancies. It was initially reported that loss of functional pRB was a statistically significant negative prognostic factor in high-grade adult soft tissue sarcomas (Cance et al., 1990). Subsequently, two independent studies done concurrently concluded that altered pRB expression was a prognostic factor among patients with transitional cell carcinoma of the bladder (Cordon-Cardo et al.. 1992; Logothetis et al., 1992).

For lung cancer patients, the initial pilot studies have also been promising, implying that altered RB and p53 protein status could be a synergistic prognostic factor in early stage nonsmall cell lung carcinomas (Xu et al., 1994a). A much worse survival pattern has been reported as well for patients with acute myelogenous leukemia who have low or absent levels of pRB protein in their peripheral blood leukemic cells (Kornblau et al., 1994). Since all studies done so far to investigate association between the pRB status in human cancer and the clinical outcome of the patients have been retrospective, and the number of cases in each cohort was fairly small, definitive retrospective and prospective studies with an adequate sample size for statistical calculations are now underway to determine whether or not loss of pRB function can be considered as a prognostic factor in clinical practice.

The most direct proof that the cloned RB gene is indeed a tumor suppressor gene comes from introduction of a cloned intact copy of the gene into cancer cells with observed tumor suppression function. A number of reports have indicated that replacement of the normal RB gene in RB-defective tumor cells from disparate types of human cancers could suppress their tumorigenic activity in nude mice (Huang etal., 1988; Goodrich and Lee, 1993; Zhou etal., 1994b). The tumor cell lines studied were derived from widely disparate types of human cancers such as the retinoblastoma, osteosarcoma, carcinomas of the bladder, prostate, breast and lung (Table 2).

WO 98/37091 PCTIS98/03041 23 Of note, there has been a tendency in the literature to separate the inhibition of cell growth by RB replacement in RB-defective tumor cells from its tumor suppression function (Takahashi et al., 1991; Chen et al., 1992; Goodrich et al., 1992b; Zhou et al., 1994b). After transient transduction with a wild-type pRB-expressing retrovirus or plasmid, as documented in several early studies, the RB-deficient retinoblastoma and osteosarcoma tumor cells in culture displayed striking changes, including cell enlargement, senescent phenotype and lower growth rate (Huang etal., 1988; Templeton etal., 1991). Subsequently, it was found that long-term stable clones of the RB-reconstituted tumor cells can be isolated that grew just as rapidly as the parental or matched RB- revertant clones. The majority of RB+ clones obtained, however, were non-tumorigenic or with significantly reduced tumorigenicity in nude mice. The mechanisms for the dissociation of suppression of tumorigenicity in nude mice from inhibition of tumor cell growth in culture by RB-replacement are unclear. It is certainly possible that RB replacement restores sensitivity to a variety of physiologic growth inhibitory signals which may be present and supplied to cells when tumorigenicity assay is done in nude mice. Such external growth inhibitory agents would be absent under regular cell culture conditions, leading to rapid cell growth (Chen et al., 1992).

Although the molecular mechanism of the RB-mediated tumor suppression have remained unclear, suppression of tumorigenicity of RB- tumor cells in vivo by re-expressing the wild-type pRB implies that the RB gene could be a potential therapeutic target for human cancer.

S In addition, recent reports suggest that RB may also play a role in elicitation of immunogenicity of tumor cells (Lu et al., 1994; Lu et al., 1996), anti-angiogenesis (Dawson et al., 1995) and suppression of tumor invasiveness (Li et al., 1996), which make the emerging RB gene therapy even more attractive. In this regard, preclinical studies have recently demonstrated that treatment of established human xenograft tumors in nude mice by recombinant adenovirus vectors expressing either wild-type or an N-terminal truncated retinoblastoma protein resulted in regression of the treated tumors (Xu et al., 1996). In addition, a constitutively active form of the pRB protein has been tested in a rat artery model of restenosis to inhibit vascular proliferative disorders following balloon angioplasty (Chang et al., 1995).

WO 98/37091 PCT/US98/03041 24 The RB gene expressing the first in-frame AUG codon-initiated RB protein is also referred to herein as the intact RB gene, the RB 11 0 gene or the pl10RB coding gene. It has also been observed that lower molecular weight (<100 kD, 98 kD, or 98-104 kD) bands of unknown origin which are immunoreactive to various anti-RB antibodies can be detected in immunoprecipitation and Western blots (Xu et al., 1989b; Furukawa et al., 1990; Stein et al., 1990).

The RB l 0 o cDNA open reading frame sequence (McGee etal., 1989) contains a second in-frame AUG codon located in exon 3, at nucleotides 355-357. The deduced second AUG codon-initiated RB protein would be 98 kD, or 12 kD smaller than the pl10 RB protein. It has been proposed that the lower molecular weight bands are the underphosphorylated (98 kD) and phosphorylated (98-104 kD) RB protein translated from the second AUG codon of the RB mRNA (Xu etal., 1989b), and this was later shown conclusively (Xu etal., U.S. Patent 5,496,731). This protein is referred to as the p94RB protein.

It has been proposed that introduction of a functional RB 1 o gene into an RB-minus tumor cell will likely "normalize" the cell. Of course, it was not expected that tumor cells which already have normal RB" 10 gene expression would respond to RB" 11 gene therapy, because it was presumed that adding additional RB expression could not correct a non-RB genetic defect. In fact, it has been shown that in the case of RB tumor cell lines, such as the

RB

osteosarcoma cell line U-2 OS, which expresses the normal pl10 introduction of an extr pllO coding gene did not change the neoplastic phenotype of such tumor lines (Huang et al., 1988).

0 In the only reported exception, introduction of a pl 10R coding vector into normal human fibroblasts, WS 1, which have no known RB or any other genetic defects, led to the cessation of cell growth (Fung et al., WO 91/15580, 1991). However, it is believed that these findings were misinterpreted since a plasmid, ppVUO-Neo, producing SV40 T antigen with a well-known growth-promoting effect on host cells was used improperly to provide a comparison with the effect of RB 11 0 expression on cell growth of transfected WS1 fibroblasts (Fung etal.

WO 91/15580, 1991). This view is confirmed by the extensive literature, clearly characterizing RB+ tumor cells as "incurable" by treatment with wild-type RB 11 0 gene. In addition, it is WO 98/37091 PCT/US98/03041 noteworthy that the WS1 cell line per se is a generally recognized non-tumorigenic human diploid fibroblast cell line with limited cell division potential in culture. Therefore, W091/15580 simply does not provide any method for effectively treating RB tumors with an

RB

11 gene. Thus, there remains a need foria broad-spectrum tumor suppressor gene for treating abnormally proliferating cells having any type of genetic defect.

2. p 5 3 Somatic cell mutations of the p53 gene are said to be the most frequently mutated gene in human cancer (Weinberg, 1991). The normal or wild-type p53 gene is a negative regulator of cell growth, which, when damaged, favors cell transformation (Weinberg, 1991). As noted for the RB protein, the p53 expression product is found in the nucleus, where it may act in parallel with or cooperatively with p 10'

R

This is suggested by a number of observations, for example, both p53 and pl1lO proteins are targeted for binding or destruction by the oncoproteins of adenovirus and human papillomavirus. Tumor cell lines deleted for p53 have been successfully treated with wild-type p53 vector to reduce tumorigenicity (Baker et al., 1990).

However, the introduction of either p53 or RB 1 0 into cells that have not undergone lesions at these loci does not affect cell proliferation (Marshall, 1991; Baker et al., 1990; Huang et al., 1988). Such experiments suggest that sensitivity of cells to the suppression of their growth by a tumor suppressor gene is dependent on the genetic alterations that have taken place in the cells.

Such a dependency would be further complicated by the observation in certain cancers that alterations in the p53 tumor suppressor or gene locus appear after mutational activation of the ras oncogene (Marshall, 1991; Fearon et al., 1990a). Therefore, there remains a need for a broadspectrum tumor suppressor gene that does not depend on the specific identification of each mutated gene causing abnormal cellular proliferation.

3. Neurofibromatosis Type 1 Neurofibromatosis type 1 or von Recklinghausen neurofibromatosis results from the inheritance of a predisposing mutant allele or from alleles created through new germline mutations (Marshall, 1991). The neurofibromatosis type 1 gene, referred to as the NF1 gene, is a relatively large locus exhibiting a mutation rate of around 10 4 Defects in the NF1 gene result in a spectrum of clinical syndromes ranging from cafe-au-lait spots to neurofibromas of the skin and peripheral nerves to Schwannomas and neurofibrosarcomas. The NF1 gene encodes a WO 98/37091 PCT/US98/03041 26 protein of about 2485 amino acids that shares structural similarity with three proteins that interact with the products of the ras protooncogene (Weinberg, 1991). For example, the NF1 amino acid sequence shows sequence homology to the catalytic domain of ras GAP, a GTPaseactivating protein for p21 ras (Marshall, 1991).

The role of NF1 in cell cycle regulation is apparently a complex one that is not yet fully elucidated. For example, it has been hypothesized that it is a suppressor of oncogenically activated p21 ras in yeast (Marshall, 1991 citing Ballester et al, 1990). On the other hand, other possible pathways for NF1 interaction are suggested by the available data (Marshall, 1991; Weinberg, 1991). At present, no attempts to treat NF1 cells with a wild-type NF1 gene have been undertaken due to the size and complexity of the NF1 locus. Therefore, it would be highly desirable to have a broad-spectrum tumor suppressor gene able to treat NF 1 and any other type of cancer or tumor.

4. DCC The multiple steps in the tumorigenesis of colon cancer are readily monitored during development by colonoscopy. The combination of colonoscopy with the biopsy of the involved tissue has uncovered a number of degenerative genetic pathways leading to the result of a malignant tumor. One well studied pathway begins with large polyps in which 60% of the cells carry a mutated, activated allele of K-ras. A majority of these tumors then proceed to the inactivation-mutation of the gene referred to as the deleted in colon carcinoma (DCC) gene, followed by the inactivation of the p53 tumor suppressor gene.

The DCC gene is a more than approximately one million base pair gene coding for a 190kD transmembrane phosphoprotein which is hypothesized to be a receptor (Weinberg, 1991), the loss of which allows the affected cell a growth advantage. It has also been noted that the DCC has partial sequence homology to the neural cell adhesion molecule (Marshall, 1991) which might suggest a role for the DCC protogene in regulating cell to cell interactions. As can be appreciated, the large size and complexity of the DCC gene, together with the complexity of the K-ras, p53 and possibly other genes involved in colon cancer tumorigenesis demonstrates a need for a broad-spectrum tumor suppressor gene and methods of treating colon carcinoma cells WO 98/37091 PCT/US98/03041 27 which do not depend upon manipulation of the DCC gene or on the identification of other specific damaged genes in colon carcinoma cells.

Other Tumor Suppressor Proteins Examples of additional tumor suppressor genes and candidate tumor suppressor genes contemplated for use in combination with the tumor suppressor genes of the present invention include, but are not limited to; the Wilms tumor (WT-1) gene (Call et al., 1990; Gessler et al., 1990; Pritchard-Jones et al., 1990), the von Hippel-Lindau (VHL) disease tumor suppressor gene (Duan et al., 1995), the Maspin (Zou et al., 1994), Brush-I (Schott et al., 1994) and BRCA 1 genes (Miki et al., 1994; Futreal et al., 1994) for breast cancer, and the multiple tumor I suppressor (MTS) or p16 gene (Serrano et al., 1993; Kamb et al., 1994).

B. DNA Delivery via Infection with Viral Vectors In certain embodiments of the invention, the tumor suppressor genes may be stably integrated into the genome of the cell. In yet further embodiments, the genes may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance or replication independent of or in synchronization with the host cell cycle. How the tumor suppressor gene is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression vector employed.

1. Adenoviral Vectors Preferred for use in the present invention are adenovirus vectors, and particularly tetracycline-controlled adenovirus vectors. These vectors may be employed to deliver and express a wide variety of genes, including, but not limited to, tumor suppressor genes such as the retinoblastoma and p53 genes, in addition to cytokine genes such as tumor necrosis factor cc, the interferon gene family and the interleukin gene family.

A preferred method for delivery of the expression constructs involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include those WO 98/37091 PCT/US98/03041 28 constructs containing adenovirus sequences sufficient to support packaging of the construct in host cells with complementary packaging functions and to ultimately express a heterologous gene of interest that has been cloned therein.

The expression vector comprises a genetically engineered form of adenovirus.

Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because wild-type adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early and late regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (EIA and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.

In a current system, recombinant adenovirus is generated from homologous recombination between a shuttle vector and a master plasmid which contains the backbone of the adenovirus genome. Due to the possible recombination between the backbone of the adenovirus genome, and the cellular DNA of the helper cells which contain the missing portion of the viral WO 98/37091 PCT/US98/03041 29 genome, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.

Generation and propagation of most adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (E1A and E1B; Graham el al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the E3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of most adenovirus vectors is at least 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone.

Gene transfer in vivo using recombinant El-deficient adenoviruses results in early and late viral gene expression that may elicit a host immune response, thereby limiting the duration of transgene expression and the use of adenoviruses for gene therapy. In order to circumvent these potential problems, the prokaryotic Cre-loxP recombination system has been adapted to generate recombinant adenoviruses with extended deletions in the viral genome in order to minimize expression of immunogenic and/or cytotoxic viral proteins (Lieber et al., 1996).

I

Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100- 200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.

WO 98/37091 PCT/US98/03041 In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 mi) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh meditiu and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.

In some cases, adenovirus mediated gene delivery to multiple cell types has been found to be much less efficient compared to epithelial derived cells. A new adenovirus, AdPK. has been constructed to overcome this inefficiency (Wickham et al., 1996). AdPK contains a heparinbinding domain that targets the virus to heparan-containing cellular receptors, which are broadly expressed in many cell types. Therefore, AdPK delivers genes to multiple cell types at higher efficiencies than unmodified adenovirus, thus improving gene transfer efficiency and expanding the tissues amenable to efficient adenovirus mediated gene therapy.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce the foreign gene expression cassette at the position from which the El-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as WO 98/37091 PCT/US98/03041 31 described by Karlsson et al. (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect (Brough et al., 1996).

Adenovirus growth and manipulation is known to those of skillin the art, and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers,.e.g., 9 to 10" plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No severe side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in S vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al.. 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1991; Rich et al., 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld etal., 1991; 1992), muscle injection (Ragot etal., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). Recombinant adenovirus and adeno-associated virus (see below) can both infect and transduce non-dividing human primary cells.

2. AAV Vectors Adeno-associated virus (AAV) is also an attractive system for use in construction of vectors for delivery of and expression of tumor suppressor genes as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin etal., 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Patent No. 5,139,941 and U.S. Patent No. 4,797,368, each incorporated herein by reference.

WO 98/37091 PCT/US98/03041 32 Studies demonstrating the use of AAV in gene delivery include LaFace et al. (1988); Zhou et al. (1993); Flotte et al. (1993); and Walsh et al. (1994). Recombinant AAV vectors have been used successfully for in vitro and in vivo transduction of marker genes (Kaplitt et al., 1994; Lebkowski et al., 1988; Samulski et al., 1989; Yoder et al., 1994; Zhou et al., 1994a; Hermonat and Muzyczka, 1984; Tratschin et al., 1985; McLaughlin et al., 1988) and genes involved in human diseases (Flotte et al., 1992; Luo et al., 1994; Ohi et al., 1990; Walsh et al., 1994; Wei et al., 1994). Recently, an AAV vector has been approved for phase I human trials for the treatment of cystic fibrosis.

AAV is a dependent parvovirus in that it requires coinfection with another virus (either adenovirus or a member of the herpes virus family) to undergo a productive infection in cultured cells (Muzyczka, 1992). In the absence of coinfection with helper virus, the wild type AAV genome integrates through its ends into human chromosome 19 where it resides in a latent state as a provirus (Kotin et al., 1990; Samulski et al., 1991). rAAV, however, is not restricted to chromosome 19 for integration unless the AAV Rep protein is also expressed (Shelling and Smith, 1994). When a cell carrying an AAV provirus is superinfected with a helper virus, the AAV genome is "rescued" from the chromosome or from a recombinant plasmid, and a normal productive infection is established (Samulski et al., 1989; McLaughlin et al., 1988; Kotin et al., 1990; Muzyczka, 1992).

Typically, recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al., 1988; Samulski et al., 1989; each incorporated herein by reference) and an expression plasmid containing the wild type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al., 1991; incorporated herein by reference). The cells are also infected or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function. rAAV virus stocks made in such fashion are contaminated with adenovirus which must be inactivated by heat shock or physically separated from the rAAV particles (for example, by cesium chloride density centrifugation). Alternatively, adenovirus vectors containing the AAV coding regions or cell lines containing the AAV coding regions and some or all of the adenovirus helper genes could be used (Yang et al., 1994; Clark et al., 1995). Cell lines carrying the rAAV DNA as an integrated provirus can also be used (Flotte et al., 1995).

WO 98/37091 PCT/US98/03041 33 3. Retroviral Vectors In particular aspects of the present invention, delivery of selected genes to target cells through the use of retrovirus infection will be desired. The retroviruses are a group of singlestranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.

The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol. and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et a., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

Concern with the use of defective retrovirus vectors is the potential appearance of wildtype replication-competent virus in the packaging cells. This can result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are WO 98/37091 PCT/US98/03041 34 now available that should greatly decrease the likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

In some cases, the restricted host-cell range and low titer of retroviral vectors can limit their use for stable gene transfer in eukaryotic cells. To overcome these potential difficulties, a murine leukemia virus-derived vector has been developed in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus (Bums etal., 1993). These vectors can be concentrated to extremely high titers (10 9 colony forming units/ml), and can infect cells that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. These vectors may facilitate gene therapy model studies and other gene transfer studies that require direct delivery of vectors in vivo.

4. Baculoviral Vectors Baculovirus expression vectors are useful tools for the production of proteins for a variety of applications (Summers and Smith, 1987; O'Reilly et al., 1992; also U.S. Patent Nos., 4,745,051 (Smith and Summers), 4,879,236 (Smith and Summers), 5,077.214 (Guarino and Jarvis), 5,155,037 (Summers), 5,162,222, (Guarino and Jarvis), 5,169,784 (Summers and Oker- Blom) and 5,278,050 (Summers), each incorporated herein by reference). The inventors contemplate the construction of baculoviral expression vectors wherein gene expression is regulated by tetracycline. These vectors might be particularly useful, for example, where the desired protein is toxic to the insect cells. In these instances, production of the protein can be turned off until the cells have reached a very high density, thereby still allowing for the production of large quantities of the desired protein.

Baculovirus expression vectors are recombinant insect vectors in which the coding region of a particular gene of interest is placed behind a promoter in place of a nonessential baculoviral gene. The classic approach used to isolate a recombinant baculovirus expression vector is to construct a plasmid in which the foreign gene of interest is positioned downstream of the polyhedrin promoter. Then, via homologous recombination, that plasmid can be used to transfer the new gene into the viral genome in place of the wild-type polyhedrin gene (Summers and Smith, 1987; O'Reilly etal., 1992).

WO 98/37091 PCT/US98/03041 The resulting recombinant virus can infect cultured lepidopteran insect cells or larvae and express the foreign gene under the control of the polyhedrin promoter, which is strong and provides very high levels of transcription during the very late phase of infection. The strength of the polyhedrin promoter is an advantage of the use of recombinant baculoviruses as expression 5 vectors because it usually leads to the synthesis of large amounts of the foreign gene product during infection.

Other viral vectors Other viral vectors may be employed for construction of expression vectors in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and SSugden, 1986; Coupar et al., 1988), sindbis virus and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich el al., 1990).

With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al., 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. Chang etal. (1991) recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al., 1991).

6. Modified Viruses In still further embodiments of the present invention, particularly wherein delivery of a selected gene to a specific cell type is desired, the expression constructs to be delivered are housed within an infective virus that has also been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by WO 98/37091 PCT/US98/03041 36 the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).

C. Other Methods of DNA Delivery As well as the viral mediated methods of DNA delivery via infection of cells described above, other methods of introducing the tumor suppressor genes of the present invention into both prokaryotic and eukaryotic cells are contemplated.

1. Transfection and Transformation In order to effect expression of a gene construct, the expression construct must be delivered into a cell. As described herein, a preferred mechanism for delivery is via viral infection, where the expression construct is encapsidated in an infectious viral particle.

However, several non-viral methods for the transfer of expression constructs into eukaryotic and prokaryotic cells also are contemplated by the present invention. In one embodiment of the present invention, the expression construct may consist only of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned which physically or chemically permeabilize the cell membrane.

a. Liposome-Mediated Transfection and Transformation In a further embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and WO 98/37091 PCT/US98/03041 37 Bachhawat, 1991). Also contemplated is an expression construct complexed with Lipofectamine (Gibco BRL).

Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has 5 been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.

In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane Sand promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.

b. Electroporation In certain embodiments of the present invention, the expression construct is introduced into the cell via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.

Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter etal., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.

c. Calcium Phosphate Precipitation or DEAE-Dextran Treatment In other embodiments of the present invention, the expression construct is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).

WO 98/37091 PCT/US98/03041 38 In another embodiment, the expression construct is delivered into the cell using DEAEdextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

d. Particle Bombardment Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

e. Direct Microinjection or Sonication Loading Further embodiments of the present invention include the introduction of the expression construct by direct microinjection or sonication loading. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985), and LTK- fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., 1987).

f. Adenoviral Assisted Transfection In certain embodiments of the present invention, the expression construct is introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Gotten et al., 1992; Curiel, 1994).

g. Receptor Mediated Transfection Still further expression constructs that may be employed to deliver the construct to the target cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in the target cells. In WO 98/37091 PCTUS98/03041 39 view of the cell type-specific distribution of various receptors, this delivery method adds a degree of specificity to the present invention. Specific delivery in the context of another mammalian cell type is described by Wu and Wu (1993; incorporated herein by reference).

Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a DNA-binding agent. Others comprise a cell receptor-specific ligand to which the DNA construct to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishes the operability of the technique. In the context of the present invention, the ligand will be chosen to correspond to a receptor specifically expressed on the neuroendocrine target cell population.

In other embodiments, the DNA delivery vehicle component of a cell-specific gene targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acids to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptors of the target cell and deliver the contents to the cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.

SIn still further embodiments, the DNA delivery vehicle component of the targeted delivery vehicles may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialoganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into the target cells in a similar manner.

D. Marker Genes In certain aspects of the present invention, specific cells are tagged with specific genetic markers to provide information about the fate of the tagged cells. Therefore, the present WO 98/37091 PCT/US98/03041 invention also provides recombinant candidate screening and selection methods which are based upon whole cell assays and which, preferably, employ a reporter gene that confers on its recombinant hosts a readily detectable phenotype that emerges only under conditions where a general DNA promoter positioned upstream of the reporter gene is functional. Generally, reporter genes encode a polypeptide (marker protein) not otherwise produced by the host cell which is detectable by analysis of the cell culture, by fluorometric, radioisotopic or spectrophotometric analysis of the cell culture.

In other aspects of the present invention, a genetic marker is provided which is detectable by standard genetic analysis techniques, such as DNA or RNA amplification by PCRTM or hybridization using fluorometric, radioisotopic or spectrophotometric probes.

1. Screening Exemplary enzymes include esterases, phosphatases, proteases (tissue plasminogen activator or urokinase) and other enzymes capable of being detected by their activity, as will be known to those skilled in the art. Contemplated for use in the present invention is green fluorescent protein (GFP) as a marker for transgene expression (Chalfie et al., 1994). The use of GFP does not need exogenously added substrates, only irradiation by near UV or blue light, and thus has significant potential for use in monitoring gene expression in living cells.

Other particular examples are the enzyme chloramphenicol acetyltransferase (CAT) which may be employed with a radiolabelled substrate, firefly and bacterial luciferase, and the bacterial enzymes P-galactosidase and p-glucuronidase. Other marker genes within this class are well known to those of skill in the art, and are suitable for use in the present invention.

2. Selection Another class of reporter genes which confer detectable characteristics on a host cell are those which encode polypeptides, generally enzymes, which render their transformants resistant against toxins. Examples of this class of reporter genes are the neo gene (Colberre-Garapin etal., 1981) which protects host cells against toxic levels of the antibiotic G418, the gene conferring streptomycin resistance S. Patent 4,430,434), the gene conferring hygromycin B resistance (Santerre etal., 1984; U. S. Patents 4,727,028, 4,960,704 and 4,559,302), a gene WO 98/37091 PCT/US98/03041 41 encoding dihydrofolate reductase, which confers resistance to methotrexate (Alt et al., 1978), the enzyme HPRT, along with many others well known in the art (Kaufman, 1990).

E. Biological Functional Equivalents While the present invention contemplates the use of tumor suppressor proteins, exemplified by the retinoblastoma protein, which contain modifications within the N-terminal region which confer equal or greater tumor suppression activity on the resultant protein, alteration of the unmodified C-terminal portion of the protein such that biological activity is maintained also falls within the scope of the present invention.

SAs mentioned above, modification and changes may be made in the structure of, for example, the retinoblastoma protein, and still obtain a molecule having like or otherwise desirable characteristics. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of tumor suppression activity. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties. Equally, the same considerations may be employed to create a protein or polypeptide with countervailing antagonistic) properties. It is thus contemplated by the inventors that various changes may be made in the sequence of tumor suppressor proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.

In terms of functional equivalents, It is also well understood by the skilled artisan that, inherent in the definition of a biologically functional equivalent protein or peptide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity.

Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted. Of course, a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.

WO 98/37091 PCT/US98/03041 42 It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a protein or peptide, residues in active sites, such residues may not generally be exchanged.

Conservative substitutions well known in the art include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; dspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine:to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

In making such changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine valine leucine phenylalanine cysteine/cystine methionine alanine glycine threonine serine tryptophan tyrosine proline histidine glutamate glutamine aspartate asparagine lysine and arginine The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein. use this shorter portion for non-immunological WO 98/37091 PCT/US98/03041 43 stuff It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.

As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine lysine aspartate glutamate serine asparagine glutamine glycine threonine proline alanine histidine cysteine methionine valine leucine isoleucine tyrosine phenylalanine tryptophan In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid. Two tables of amino acids and their codons is presented below for use in such embodiments, as well as for other uses, such as in the design of probes and primers and the like.

WO 98/37091 PCT/US98/03041 44 Table 1 Preferred Human DNA Codons Amino Acids Codons Alanine Ala A GCC GCT GCA GCG Cysteine Cys C TGC TGT Aspartic acid Asp D GAC GAT Glutamic acid Glu E GAG GAA Phenylalanine Phe F TTC TTT Glycine Gly G GGC GGG GGA GGT Histidine His H CAC CAT Isoleucine Ile I ATC ATT ATA Lysine Lys K AAG AAA Leucine Leu L CTG CTC TTG CTT CTA TTA Methionine Met M ATG Asparagine Asn N AAC AAT Proline Pro P CCC CCT CCA CCG Glutamine Gin Q CAG CAA Arginine Arg R CGC AGG CGG AGA CGA CGT Serine Ser S AGC TCC TCT AGT TCA TCG Threonine Thr T ACC ACA ACT ACG Valine Val V GTG GTC GTT GTA Tryptophan Trp W TGG Tyrosine Tyr Y TAC TAT Codon prevalence shown as decreasing from left (most prevalent) to right (least prevalent).

Underlined codons are those used less than 5 times per one thousand codons.

WO 9.8137091 PCT/US98/03041 Table 2 Preferred Human RNA Codons Amino Acids Codons Alanine Ala A GCC GGU GCA GCG Cysteine Gys C UGG UGU Aspartic acid Asp D GAG GAU Glutanic acid Glu E GAG GAA Phenylalanine Phe F UUG UUU Glycine Gly G GGC GGG GGA GGU Histidine His H CAC CAU Isoleucine lie I AUG AU AUA Lysine Lys K AAG AAA Leucine Leu L GUG CUC UUG CUU GUA UUA Methionine Met M AUG Asparagine Asn N AAG AAU Proline Pro P CCC CCU GCA CCG Glutamine Gin Q GAG CAA Arginine Arg R CGC AGG GGG AGA CGA GGU Serine Ser S AGG UCC UCU AGU UGA UCG Threonine Thr T ACC ACA ACU ACG Valine Val V GUG GUC GUU GUA Tryptophan Trp W UGG Tyrosine Tyr Y UAG UAU Codon prevalence shown as decreasing from left (most prevalent) to right (least prevalent).

Underlined codons are those used less than 5 times per one thousand codons.

WO 98/37091 PCT/US98/03041 46 F. Mutagenesis Mutagenesis may be performed in accordance with any of the techniques known in the art such as and not limited to synthesizing an oligonucleotide having one or more mutations within the sequence of a particular tumor suppressor or cytokine protein. In particular, site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.

The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.

Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred.

with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art.

Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically.

This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing WO 98/37091 PCT/US98/03041 47 enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement. A genetic selection scheme was devised by Kunkel et al. (1987) to enrich for clones incorporating the mutagenic oligonucleotide.

Alternatively, the use of PCRTM with commercially available thermostable enzymes such as Taq polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector. The PCR'M-mediated mutagenesis procedures of Tomic et al. (1990) and Upender et al.

(1995) provide two examples of such protocols. A PCRTM employing a thermostable ligase in addition to a thermostable polymerase may also be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector. The mutagenesis procedure described by Michael (1994) provides an example of one such protocol.

The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides Sand the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of WO 98/37091 PCT/US98/03041 48 the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment.

Examples of such methodologies are provided by U.S. Patent 4,237,224, specifically incorporated herein by reference in its entirety.

G. Pharmaceutically Acceptable Compositions and Routes of Administration Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions of the proteins, nucleic acids, including vectors such as tetracycline-regulated vectors, recombinant viruses and cells in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers to render the compositions suitable for introduction into a patient. Aqueous compositions of the present invention comprise an effective amount of the therapeutic agent dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium, and preferably encapsulated. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients, such as other anticancer agents, can also be incorporated into the compositions.

Solutions of the active ingredients as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with surfactant, such as hydroxypropylcellulose.

Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to WO 98/37091 PCT/US98/03041 49 prevent growth of microorganisms. Intravenous vehicles include fluid and nutrient replenishers.

Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well-known parameters.

An effective amount of the viruses or cells is determined based on the intended goal. The term "unit dose" refers to a physically discrete unit suitable for use in:a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, p depends on the subject to be treated, the state of the subject, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.

1. Parenteral Administration The active compositions of the present invention will often be formulated for parenteral administration, formulated for injection via the intravenous, intramuscular, sub-cutaneous, intratumoral, peritumoral or even intraperitoneal routes. The preparation of an aqueous composition that contains a second agent(s) as active ingredients will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and WO 98/37091 PCT/US98/03041 sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.

It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The active compounds may be formulated into a composition in a neutral or salt form.

Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water.

ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the particular methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the WO 98/37091 PCT/US98/03041 51 active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, peritumoral and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" Edition. pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

2. Other Routes of Administration In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including cremes, lotions, mouthwashes, inhalants and the like.

The expression vectors and delivery vehicles of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route.

This includes oral, nasal; buccal, rectal;vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.

The injection can be general, regional, local or direct injection, for example, of a tumor. Also contemplated is injection of a resected tumor bed, and continuous perfusion via catheter. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.

WO 98/37091 PCTIUS98/03041 52 The vectors of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection also may be prepared. These preparations also may be emulsified. A typical compositions for such purposes comprises a 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as theyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers.

Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well known parameters.

Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray.

An effective amount of the therapeutic agent is determined based on the intended goal.

The term "unit dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.

WO 98/37091 PCTIUS98/3041 53 In certain cases, the therapeutic formulations of the invention could also be prepared in forms suitable for topical administration, such as in cremes and lotions. These forms may be used for treating skin-associated diseases, such as various sarcomas.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable.

H. Chemotherapeutic Agents The methods of the present invention may be combined with any other methods generally employed in the treatment of the particular disease or disorder that the patient exhibits. For example, in connection with the treatment of solid tumors, the methods of the present invention may be used in combination with classical approaches, such as surgery, radiotherapy and the like. So long as a particular therapeutic approach is not known to be detrimental in itself, or counteracts the effectiveness of the tumor suppressor therapy, its combination with the present invention is contemplated. When one or more agents are used in combination with cytokine gene therapy and/or tumor suppressor gene therapy, there is no requirement for the combined results to be additive of the effects observed when each treatment is conducted separately, although this is evidently desirable, and there is no particular requirement for the combined treatment to exhibit synergistic effects, although this is certainly possible and advantageous.

In terms of surgery, any surgical intervention may be practiced in combination with the present invention. In connection with radiotherapy, any mechanism for inducing DNA damage -locally within tumor cells is -contemipiated, such as y-irradiation, X-rays, UV-irradiation, microwaves and even electronic emissions and the like. The directed delivery of radioisotopes to tumor cells is also contemplated, and this may be used in connection with a targeting antibody or other targeting means. Cytokine therapy also has proven to be an effective partner for combined therapeutic regimens. Various cytokines may be employed in such combined approaches.

Examples of cytokines include IL-a IL-1p, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, TGF-P, GM-CSF, M-CSF, G-CSF, TNFa, TNFP, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-a, IFN-P, IFN-y. Cytokines are WO 98/37091 PCT/US98/03041 54 administered according to standard regimens, consistent with clinical indications such as the condition of the patient and relative toxicity of the cytokine. Below is an exemplary, but in no way limiting, table of cytokine genes contemplated for use in certain embodiments of the present invention.

Table 3 Cytokine Reference human IL-la March et al., Nature, 315:641, 1985 murine IL-la Lomedico et al., Nature, 312:458, 1984 human IL-1 3 March et al., Nature. 315:641, 1985; Auron et al., Proc. Natl. Acad Sci. USA, 81:7907, 1984 murine IL-13 Gray, J. Immunol.. 137:3644, 1986: Telford, Nucl. Acids Res., 14:9955, 1986 human IL-Ira human IL-2 human IL-2 human IL-3 murine IL-3 human IL-4 murine IL-4 human IL-5 murine IL-5 human IL-6 murine IL-6 human IL-7 murine IL-7 Eisenberg et al.. Nature, 343:341, 1990 Taniguchi et al., Nature, 302:305, 1983; Maeda et al., Biochem.

Biophys. Res. Commun., 115:1040, 1983 Taniguchi et al., Nature, 302:305, 1983 Yang et al., Cell, 47:3, 1986 Yokota et al., Proc. Natl. Acad. Sci. USA, 81:1070, 1984; Fung et al., Nature, 307:233, 1984; Miyatake et al., Proc. Natl. Acad. Sci.

USA, 82:316, 1985 Yokota et al., Proc. Natl. Acad. Sci. USA, 83:5894, 1986 Norma et al., Nature, 319:640, 1986; Lee et al., Proc. Natl. Acad.

Sci. USA, 83:2061, 1986 Azuma et al., Nucl. Acids Res., 14:9149, 1986 Kinashi et al., Nature, 324:70, 1986; Mizuta et al., Growth Factors, 1:51, 1988 Hirano et al., Nature, 324:73, 1986 Van Snick et al., Eur. J. Immunol., 18:193, 1988 Goodwin et al., Proc. Natl. Acad. Sci. USA, 86:302, 1989 Namen et al., Nature, 333:571, 1988 WO 98/37091 WO 98/709 1PCT[US98/03041 Table 3 (Continued) CYtokine Reference human IL-8 human IL-9 murine IL-9 human Angiogenin human GROct murine MIP- I a murine MIP- I human MIF human G-CSF human GM-CSF murine GM-CSF human M-CSF human EGF human TGF-cx human FGF acidic human f3-ECGF human FGF basic murine IFN-3 Schmid et al., J. Immunol., 139:250, 1987; Matsushima et Exp. Med, 167:1883, 1988; Lindley et Proc. Nat. Acad Sci.

USA, 85:9199, 1988 Renauld et Immunol., 144:4235, 1990 Renauld et JImmuno!., 144:4235, 1990 Kurachi et al., Biochemistry, 24:5494, 1985 Richmond et al., EMBO 1J, 7:2025, 1988 Davatelis et Exp. Med. 167:1939. 1988 Sherry et Exp. Med. 168:2251, 1988 Weiser et al.. Proc. Nail. A cad Sci. USA. 86:7522, 1989 Nagata et al.. Nature, 319:415, 1986; Souza et al.. Science. 232:61, 1986 Cantrell et Proc. Nail. Acad Sci. USA, 82:6250. 1985; Lee et al., Proc. Nat. A cad. Sci. USA, 82:4360, 1985; Wong et Science, 228:810, 1985 Gough et EMBO]J, 4:645, 1985 Wong, Science, 235:1504, 1987; Kawasaki, Science, 230;291, 1985; Ladner, EMBO 6:2693, 1987 Smith et Nuc. Acids Res., 10:4467, 1982; Bell et Nuc!.

Acids Res., 14:8427, 1986 Derynck et Cell, 3 8:287, 1984 Jaye et a.,_Science, 233:541, 1986; Gimenez-Gallego et Biochem. Biophys. Res. Commun., 138:611, 1986; Harper et al., Biochem., 25:4097, 1986 Jaye eta!., Science, 233:541, 1986 Abraham et al., EMBO.IJ, 5:2523, 1986; Sommer et Biochem.

Biophys. Res. Comm., 144:543, 1987 Higashi et Biol. Chem., 25 8:9522, 19 83; Kuga, Nuci. Acids Res., 17:3291, 1989 WO 98/37091 PCT/US98/03041 56 Table 3 (Continued) Cvtokine Reference human IFN-y Gray et al., Nature, 295:503, 1982; Devos et al., Nucl. Acids Res., 10:2487, 1982; Rinderknecht, J. Biol. Chem., 259:6790, 1984 human IGF-I Jansen et al., Nature, 306:609, 1983; Rotwein et al., J. Biol. Chem., 261:4828, 1986 human IGF-II Bell et al., Nature, 310:775, 1984 human p-NGF chain Ullrich et al., Nature, 303:821, 1983 human PDGF A chain Betsholtz et al., Nature, 320:695, 1986 human PDGF B chain Johnsson et al., EMBO 3:921, 1984; Collins et al., Nature, 316:748, 1985 human TGF-P 1 Derynck et al., Nature, 316:701, 1985 human TNF-a Pennica et al., Nature, 312:724, 1984: Fransen et al., Nucl. Acids Res., 13:4417, 1985 human TNF-P Gray et al., Nature, 312:721, 1984 murine TNF-P Gray et al., Nucl. Acids Res., 15:3937, 1987 Compositions of the present invention can have an effective amount of an engineered virus or cell for therapeutic administration in combination with an effective amount of a compound (second agent) that is a chemotherapeutic agent as exemplified below. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. A wide variety of chemotherapeutic agents may be used in combination with the therapeutic genes of the present invention. These can be, for example, agents that directly cross-link DNA, agents that intercalate into DNA, and agents that lead to chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Irrespective of the mechanisms by which the enhanced tumor destruction is achieved, the combined treatment aspects of the present invention have evident utility in the effective treatment of disease. To use the compositions of the present invention in combination with the administration of a chemotherapeutic agent, one would simply administer to an animal at least a first modified retinoblastoma tumor suppressor as disclosed herein in combination with the WO 98/37091 PCT/US98/03041 57 chemotherapeutic agent in a manner effective to result in their combined anti-tumor actions within the animal. These agents would therefore be provided in an amount effective and for a period of time effective to result in their combined presence and their combined actions in the tumor environment. To achieve this goal, the modified retinoblastoma tumor suppressor and chemotherapeutic agents may be administered to the animal simultaneously, either in a single composition or as two distinct compositions using different administration routes.

Alternatively, the modified retinoblastoma tumor suppressor treatment may precede or follow the chemotherapeutic agent treatment by intervals ranging from minutes to weeks. In embodiments where the chemotherapeutic factor and modified retinoblastoma tumor suppressor Sare applied separately to the animal, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the chemotherapeutic agent and modified retinoblastoma tumor suppressor composition would still be able to exert an.

advantageously combined effect on the tumor. In such instances, it is contemplated that one would contact the tumor with both agents within about 5 minutes to about one week of each other and, more preferably, within about 12-72 hours of each other, with a delay time of only about 12-48 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, where several days 3, 4, 5, 6 or 7) or even several weeks 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. It also is conceivable that more than one administrations of either the modified retinoblastoma tumor suppressor or the chemotherapeutic agent will be desired. To achieve tumor regression, both agents are delivered in a combined amount effective to inhibit its growth, irrespective of the times for administration.

A variety of chemotherapeutic agents are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated as exemplary include, e.g., etoposide (VP-16), adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.

As will be understood by those of ordinary skill in the art, the appropriate doses of chemotherapeutic agents will be generally around those already employed in clinical therapies wherein the chemotherapeutics are administered alone or in combination with other chemotherapeutics. By way of example only, agents such as cisplatin, and other DNA alkylating WO 98/37091 PCT/US98/03041 58 may be used. Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m 2 for 5 days every three weeks for a total of three courses.

Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.

Agents that directly cross-link nucleic acids, specifically DNA, are envisaged and are shown herein, to eventuate DNA damage leading to a synergistic antineoplastic combination.

Agents such as cisplatin, and other DNA alkylating agents may be used.

Further useful agents include compounds that interfere with DNA replication, mitosis and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m 2 at 21 day intervals for adriamycin.

to 35-50 mg/m 2 for etoposide intravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of polynucleotide precursors may also be used. Particularly useful are agents that have undergone extensive testing and are readily available. As such, agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells. Although quite toxic, 5-FU, is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used.

Plant alkaloids such as taxol are also contemplated for use in certain aspects of the present invention. Taxol is an experimental antimitotic agent, isolated from the bark of the ash tree, Taxus brevifolia. It binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules. Taxol is currently being evaluated clinically; it has activity against malignant melanoma and carcinoma of the ovary. Maximal doses are 30 mg/m 2 per day for 5 days or 210 to 250 mg/m 2 given once every 3 weeks. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.

WO 98/37091 PCT/US98/03041 59 Exemplary chemotherapeutic agents that are useful in connection with combined therapy are listed in Table 4. Each of the agents listed therein are exemplary and by no means limiting.

The skilled artisan is directed to "Remington's Pharmaceutical Sciences" 15th Edition, chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

Table 4 Chemotherapeutic Agents Useful In Neoplastic Disease Nonproprietary Names Class Type Of Agent Disease (Other Names) Mechlorethamine

(HN

2 Hodgkin's disease, non-Hodgkin's lymphomas Acute and chronic lymphocytic leukemias, Hodgkin's disease, non-Hodgkin's lymphomas, Cyclophosphamide multiple myeloma, neuroblastoma, breast, ovary, Ifosfamide lung, Wilms' tumor, cervix, testis, soft-tissue Nitrogen Mustards sarcomas Melphalan (L-sarcolysin) Multiple myeloma, breast, ovary Chronic lymphocytic leukemia, primary Chlorambucil macroglobulinemia, Hodgkin's disease, non- Hodgkin's lymphomas A/kylating Agents Ethylenimenes and Hexamethylmelamine Ovary Methylmelamines Thiotepa Bladder, breast, ovary Alkyl Sulforates Busulfan Chronic granulocytic leukemia Hodgkin's disease, non-Hodgkin's lymphomas, Carmustine (BCNU) primary brain tumors, multiple myeloma, malignant melanoma Hodgkin's disease, non-Hodgkin's lymphomas, Nitrosoureas Lomustine (CCNU) primary brain tumors, small-cell lung Semustine (methyl-CCNU) Primary brain tumors, stomach, colon WO 98/37091 PCT/US98/03041 Table 4 (Continued) Nonproprietary Names Class Type Of Agent Disease (Other Names) Triazines Antimetabolites Folic Acid Analogs Pyrimidine Analogs Streptozocin (streptozotocin) Dacarbazine (DTIC; dimethyltriazenoimidaz olecarboxamide) Methotrexate (amethopterin) Fluouracil Floxuridine (fluorodeoxyuridine; FUdR) Cytarabine (cytosine arabinoside) Mercaptopurine (6-mercaptopurine; 6-MP) Thioguanine (6-thioguanine; TG) Pentostatin (2-deoxycoformycin) Antimetabolies, continued Malignant pancreatic insulinoma, malignant carcinoid Malignant melanoma, Hodgkin's disease, softtissue sarcomas Acute lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast, head and neck, lung, osteogenic sarcoma Breast, colon, stomach, pancreas, ovary, head and neck, urinary bladder, premalignant skin lesions (topical) Acute granulocytic and acute lymphocytic leukemias Acute lymphocytic, acute granulocytic and chronic granulocytic leukemias Acute granulocytic, acute lymphocytic and chronic granulocytic leukemias Hairy cell leukemia, mycosis fungoides, chronic lymphocytic leukemia Hodgkin's disease, non-Hodgkin's lymphomas, breast, testis Acute lymphocytic leukemia, neuroblastoma, Wilms' tumor, rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's lymphomas, small-cell lung Testis, small-cell lung and other lung, breast, Hodgkin's disease, non-Hodgkin's lymphomas, acute granulocytic leukemia, Kaposi's sarcoma Purine Analogs and Related Inhibitors Vinblastine (VLB) Vinca Alkaloids Vincristine Epipodophyllotoxins Etoposide (VP1 6) Tertiposide WO 98/37091 PCT/US98/03041 Table 4 (Continued) Nonproprietary Names Class Type Of Agent Disease (Other, Names) Natura/ Products nlnrtinnmvrin Choriocarcinoma, Wilms' tumor, (actinomycin D) Daunorubicin (daunomycin; rubidomycin) Antibiotics Doxorubicin Bleomycin Antibiotics, continued Enzymes Biological Response Modifiers Platinum Coordination Complexes Anthracenedione Substituted Urea Methyl Hydrazine Derivative Plicamycin (mithramycin) Mitomycin (mitomycin C) L-Asparaginase Interferon alfa Cisplatin (cis-DDP) Carboplatin Mitoxantrone Hydroxyurea Procarbazine (N-methylhydrazine,

MIH)

Mitotane (o,p'-DDD) rhabdomyosarcoma, testis, Kaposi's sarcoma Acute granulocytic and acute lymphocytic leukemias Soft-tissue, osteogenic and other sarcomas; Hodgkin's disease, non-Hodgkin's lymphomas, acute leukemias, breast, genitourinary, thyroid, lung, stomach, neuroblastoma Testis, head and neck, skin, esophagus, lung and genitourinary tract; Hodgkin's disease, non- Hodgkin's lymphomas Testis, malignant hypercalcemia Stomach, cervix, colon, breast, pancreas, bladder, head and neck Acute lymphocytic leukemia Hairy cell leukemia., Kaposi's sarcoma, melanoma, carcinoid, renal cell, ovary, bladder, non-Hodgkin's lymphomas, mycosis fungoides, multiple myeloma, chronic granulocytic leukemia Testis, ovary, bladder, head and neck, lung, thyroid, cervix, endometrium, neuroblastoma, osteogenic sarcoma Acute granulocytic leukemia, breast Chronic granulocytic leukemia, polycythemia vera, essental thrombocytosis, malignant melanoma Hodgkin's disease Miscellaneous Agents Adrenocortical Adrenal cortex WO 98/37091 PCT/US98/03041 Table 4 (Continued) Nonproprietary Names Class Type Of Agent Disease (Other Names) Suppressant Aminoglutethimide Breast Prednisone (several other Acute and chronic lymphocytic leukemias, non- Adrenocorticosteroids equivalent preparations Hodgkin's lymphomas, Hodgkin's disease, breast available) Hydroxyprogesterone caproate Progestins Medroxyprogesterone Endometrium, breast Hormones and acetate Antagonists Megestrol acetate Diethylstilbestrol Estrogens Ethinyl estradiol (other Breast, prostate preparations available) Antiestrogen Tamoxifen Breast Testosterone propionate Androgens Fluoxymesterone (other Breast preparations available) Antiandrogen Flutamide Prostate Gonadotropin-releasing Leuprolide Prostate hormone analog I. Protein Purification Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The term "purified protein or peptide as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturallyobtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.

Generally, "purified" will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition WO 98/37091 PCT/US98/03041 63 substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific Sactivity of the initial extract, and to thus calculate the degree of purity, herein assessed by a fold purification number". The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification Ssteps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater -fold purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower WO 98/37091 PCT/US98/03041 64 degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain and adequate flow rate. Separation can be accomplished in a matter of minutes, or a most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.

Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to WO 98/37091 PCT/US98/03041 specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; Nacetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is h immunoaffinity chromatography.

L. Use of Cells in Bioreactors The ability to produce biologically active polypeptides is increasingly important to the pharmaceutical industry. The present invention discloses compositions and methods for the efficient regulated expression of, for example, tumor suppressor genes in cells, allowing for the production of these proteins in vitro from previously refractory cell types.

Over the last decade, advances in biotechnology have led to the production of important proteins and factors from bacteria, yeast, insect cells and from mammalian cell culture.

Mammalian cultures have advantages over cultures derived from the less advanced lifeforms in WO 98/37091 PCT/US98/03041 66 their ability to post-translationally process complex protein structures such as disulfidedependent folding and glycosylation. Indeed, mammalian cell culture is now the preferred source of a number of important proteins for use in human and animal medicine, especially those which are relatively large, complex or glycosylated.

Development of mammalian cell culture for production of pharrhaceuticals has been greatly aided by the development in molecular biology of techniques for design and construction of vector systems highly efficient in mammalian cell cultures, a battery of useful selection markers, gene amplification schemes and a more comprehensive understanding of the biochemical and cellular mechanisms involved in procuring the final biologically-active molecule from the introduced vector.

However, the traditional selection of cell types for expressing heterologous proteins has generally been limited to the more "common" cell types such as CHO cells, BHK cells, C127 cells and myeloma cells. In many cases, these cell types were selected because there was a great deal of preexisting literature on the cell type or the cell was simply being carried in the laboratory at the time the effort was made to express a peptide product. Frequently, factors which affect the downstream beyond the T-75 flask) side of manufacturing scale-up were not considered before selecting the cell line as the host for the expression system.

Aspects of the present invention take advantage of the biochemical and cellular capacities of mammalian cells as well as of recently available bioreactor technology. Growing cells according to the present invention in a bioreactor allows for large scale production and secretion of complex, fully biologically-active polypeptides into the growth media. In particular embodiments, by designing a defined media with low contents of complex proteins and using a scheme of timed-stimulation of the secretion into the media for increased titer, the purification strategy can be greatly simplified, thus lowering production cost.

1. Anchorage-dependent and non-anchorage-dependent cultures.

Animal and human cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing freely in suspension throughout the bulk of the culture; or as anchorage- WO 98/37091 PCT/US98/03041 67 dependent cells requiring attachment to a solid substrate for their propagation a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuouis- established cell lines are the most widely used means of large scale production of cells and cell products. Large scale suspension culture based on microbial (bacterial and yeast) fermentation technology has clear advantages for the manufacturing of mammalian cell products. The processes are relatively straightforward to operate and scale up. Homogeneous conditions can be provided in the reactor which allows for precise monitoring and control of temperature, dissolved oxygen, and pH, and ensure that representative samples of the culture can be taken.

However, suspension cultured cells cannot always be used in the production of biologicals. Suspension cultures are still considered to have tumorigenic potential and thus their use as substrates for production put limits on the use of the resulting products in human and veterinary applications (Petricciani, 1985; Larsson and Litwin, 1987). Viruses propagated in suspension cultures as opposed to anchorage-dependent cultures can sometimes cause rapid changes in viral markers, leading to reduced immunogenicity (Bahnemann, 1980). Finally, sometimes even recombinant cell lines can secrete considerably higher amounts of products when propagated as anchorage-dependent cultures as compared with the same cell line in suspension (Nilsson and Mosbach, 1987). For these reasons, different types of anchoragedependent cells are used extensively in the production of different biological products.

The current invention includes cells which are anchorage-dependent of nature.

Anchorage-dependent cells, when grown in suspension, will attach to each other and grow in clumps, eventually suffocating cells-in-the inner core of each clump as they reach a size that leaves the core cells unsustainable by the culture conditions. Therefore, an efficient means of large-scale culture of anchorage-dependent cells is also provided in order to effectively take advantage of the cells' capacity to secrete heterologous proteins.

2. Reactors and processes for suspension.

Large scale suspension culture of mammalian cultures in stirred tanks is contemplated.

The instrumentation and controls for bioreactors have been adapted, along with the design of the WO 98/37091 PCT/US98/03041 68 fermentors, from related microbial applications. However, acknowledging the increased demand for contamination control in the slower growing mammalian cultures, improved aseptic designs have been implemented, improving dependability of these reactors. Instrumentation and controls include agitation, temperature, dissolved oxygen, and pH controls. More advanced probes and autoanalyzers for on-line and off-line measurements of turbidity (a function of particles present), capacitance (a function of viable cells present), glucose/lactate, carbonate/bicarbonate and carbon dioxide are also available. Maximum cell densities obtainable in suspension cultures are relatively low at about 2-4 x 106 cells/mi of medium (which is less than 1 mg dry cell weight per ml), well below the numbers achieved in microbial fermentation.

Two suspension culture reactor designs are most widely used in the industry due to their simplicity and robustness of operation the stirred reactor and the airlift reactor. The stirred reactor design has successfully been used on a scale of 8000 liter capacity for the production of interferon (Phillips et al., 1985; Mizrahi, 1983). Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1. The culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.

The airlift reactor, also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor. The main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1. The airlift reactor scales up relatively readily, has good mass transfer of gasses and generates relatively low shear forces.

Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. However, continuous processes based on chemostat or perfusion principles are available.

WO 98/37091 PCT/US98/03041 69 A batch process is a closed system in which a typical growth profile is seen. A lag phase is followed by exponential, stationary and decline phases. In such a system, the environment is continuously changing as nutrients are depleted and metabolites accumulate. This makes analysis of factors influencing cell growth and productivity, and hence optimization of the process, a complex task. Productivity of a batch process may be increased by controlled feeding of key nutrients to prolong the growth cycle. Such a fed-batch process is still a closed system because cells, products and waste products are not removed.

In what is still a closed system, perfusion of fresh medium through the culture can be achieved by retaining the cells with a fine mesh spin filter and spinning to prevent clogging.

Spin filter cultures can produce cell densities of approximately 5 x 10 7 cells/ml. A true open system and the most basic perfusion process is the chemostat in which there is an inflow of medium and an outflow of cells and products. Culture medium is fed to the reactor at a predetermined and constant rate which maintains the dilution rate of the culture at a value less than the maximum specific growth rate of the cells (to prevent washout of the cell mass from the reactor). Culture fluid containing cells, cell products and byproducts is removed at the same rate.

These perfused systems are not in commercial use for production from mammalian cell culture.

3. Non-perfused attachment systems.

Traditionally, anchorage-dependent cell cultures are propagated on the bottom of small glass or plastic vessels. The restricted surface-to-volume ratio offered by classical and traditional Stechniques, suitable for the laboratory scale, has created a bottleneck in the production of cells and cell products on a large scale. To provide systems that offer large accessible surfaces for cell growth in small culture volume, a number of techniques have been proposed: the roller bottle system, the stack plates propagator, the-spiral film bottles, the hollow fiber system, the packed bed, the plate exchanger system, and the membrane tubing reel. Since these systems are nonhomogeneous in their nature, and are sometimes based on multiple processes, they can sometimes have limited potential for scale-up, difficulties in taking cell samples, limited potential for measuring and controlling the system and difficulty in maintaining homogeneous environmental conditions throughout the culture.

WO 9.8/37091 PCT/US98/03041 A commonly used process of these systems is the roller bottle. Being little more than a large, differently shaped T-flask, simplicity of the system makes it very dependable and, hence, attractive. Fully automated robots are available that can handle thousands of roller bottles per day, thus eliminating the risk of contamination and inconsistency associated with the otherwise required intense human handling. With frequent media changes, roller bottle cultures can achieve cell densities of close to 0.5 x 106 cells/cm 2 (corresponding to 109 cells/bottle or 10 7 cells/mi of culture media).

4. Cultures on microcarriers Van Wezel (1967) developed the concept of the microcarrier culturing systems. In this system, cells are propagated on the surface of small solid particles suspended in the growth medium by slow agitation. Cells attach to the microcarriers and grow gradually to confluency of the microcarrier surface. In fact, this large scale culture system upgrades the attachment dependent culture from a single disc process to a unit process in which both monolayer and suspension culture have been brought together. Thus, combining the necessary surface for the cells to grow with the advantages of the homogeneous suspension culture increases production.

The advantages of microcarrier cultures over most other anchorage-dependent, largescale cultivation methods are several fold. First, microcarrier cultures offer a high surface-tovolume ratio (variable by changing the carrier concentration) which leads to high cell density yields and a potential for obtaining highly concentrated cell products. Cell yields are up to 1-2 x 107 cells/ml when cultures are propagated in a perfused reactor mode. Second, cells can be propagated in one unit process vessels instead of using many small low-productivity vessels flasks or dishes). This results in far better utilization and a considerable saving of culture medium. Moreover, propagation in a single reactor leads to reduction in need for facility space and in the number of handling steps required per cell, thus reducing labor cost and risk of contamination.

Third, the well-mixed and homogeneous microcarrier suspension culture makes it possible to monitor and control environmental conditions pH, pO2, and concentration of medium components), thus leading to more reproducible cell propagation and product recovery.

Fourth, it is possible to take a representative sample for microscopic observation, chemical WO 98/37091 PCT/US98/03041 71 testing, or enumeration. Fifth, since microcarriers settle out of suspension easily, use of a fedbatch process or harvesting of cells can be done relatively easily. Sixth, the mode of the anchorage-dependent culture propagation on the microcarriers makes it possible to use this system for other cellular manipulations, such as cell transfer without the use of proteolytic enzymes, cocultivation of cells, transplantation into animals, and perfusion of the culture using decanters, columns, fluidized beds, or hollow fibers for microcarrier retainment. Seventh, microcarrier cultures are relatively easily scaled up using conventional equipment used for cultivation of microbial and animal cells in suspension.

5. Microencapsulation of mammalian cells I One method which has shown to be particularly useful for culturing mammalian cells is microencapsulation. The mammalian cells are retained inside a semipermeable hydrogel membrane. A porous membrane is formed around the cells permitting the exchange of nutrients, gases, and metabolic products with the bulk medium surrounding the capsule. Several methods have been developed that are gentle, rapid and non-toxic and where the resulting membrane is sufficiently porous and strong to sustain the growing cell mass throughout the term of the culture. These methods are all based on soluble alginate gelled by droplet contact with a calcium-containing solution. Lim Patent 4,321,883) describes cells concentrated in an approximately 1% solution of sodium alginate which are forced through a small orifice, forming droplets, and breaking free into an approximately 1% calcium chloride solution. The droplets are then cast in a layer of polyamino acid that ionically bonds to the surface alginate. Finally the Salginate is reliquefied by treating the droplet in a chelating agent to remove the calcium ions.

Other methods use cells in a calcium solution to be dropped into a alginate solution, thus creating a hollow alginate sphere. A similar approach involves cells in a chitosan solution dropped into algihate,-als creating hollow spheres.

Microencapsulated cells are easily propagated in stirred tank reactors and, with beads sizes in the range of 150-1500 mm in diameter, are easily retained in a perfused reactor using a fine-meshed screen. The ratio of capsule volume to total media volume can kept from as dense as 1:2 to 1:10. With intracapsular cell densities of up to 10 8 the effective cell density in the culture is 1-5 x 107.

WO 98/37091 PCT/US98/03041 72 The advantages of microencapsulation over other processes include the protection from the deleterious effects of shear stresses which occur from sparging and agitation, the ability to easily retain beads for the purpose of using perfused systems, scale up is relatively straightforward and the ability to use the beads for implantation.

6. Perfused attachment systems Perfusion refers to continuous flow at a steady rate, through or over a population of cells (of a physiological nutrient solution). It implies the retention of the cells within the culture unit as opposed to continuous-flow culture which washes the cells out with the withdrawn media chemostat). The idea of perfusion has been known since the beginning of the century, and has been applied to keep small pieces of tissue viable for extended microscopic observation. The technique was initiated to mimic the cells milieu in vivo where cells are continuously supplied with blood, lymph, or other body fluids. Without perfusion, cells in culture go through alternating phases of being fed and starved, thus limiting full expression of their growth and metabolic potential. The current use of perfused culture is to grow cells at high densities 0.1-5 x 108 cells/ml). In order to increase densities beyond 2-4 x 106 cells/ml (or 2 x 105 cells/cm 2 the medium has to be constantly replaced with a fresh supply in order to make up for nutritional deficiencies and to remove toxic products. Perfusion allows for a far better control of the culture environment (pH, pO- 2 nutrient levels, etc.) and is a means of significantly increasing the utilization of the surface area within a culture for cell attachment.

Microcarrier and microencapsulated cultures are readily adapted to perfused reactors but, as noted above, these culture methods lack the capacity to meet the demand for cell densities above 108 cells/ml. Such densities will provide for the advantage of high product titer in the medium (facilitating downstream processing), a smaller culture system (lowering facility needs), and a better medium utilization (yielding savings in serum and other expensive additives).

Supporting cells at high density requires efficient perfusion techniques to prevent the development of non-homogeneity.

The cells of the present invention may, irrespective of the culture method chosen, be used in protein production and as cells for in vitro cellular assays and screens as part of drug development protocols.

WO 98/37091 PCT/US98/03041 73 J. Kits All the essential materials and reagents required for the various aspects of the present invention may be assembled together in a kit. When the components of the kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.

For in vivo use, the instant compositions may be formulated into a single or separate pharmaceutically acceptable syringeable composition. In this case, the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit.

The components of the kit may also be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. The kits of the invention may also include an instruction sheet defining administration of the gene therapy and/or the chemotherapeutic drug.

The kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, injection or blow-molded plastic containers into which the desired vials are retained. Irrespective of the number or type of containers, the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle. Additionally, instructions for use of the kit components is typically included.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its WO 98/37091 PCT/US98/03041 74 practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scoje of the invention.

EXAMPLE 1 Modification of the RB Protein A. Construction of RB cDNAs Expressing N-terminal Truncated pRB Proteins For construction of modified RB cDNAs with various N-terminal deletions, a series of PCRTM primers were designed and synthesized according to the sequences of RB cDNA. The sense primers were determined by the RB cDNA sequences downstream of the deleted N-terminal sequence. All primers contain a HindIII restriction site (underlined) at the 5'-end and the consensus Kozak cassette (GCCGCC) followed by an ATG (italics).

nucleotide sequences of the sense primers are as follows: TGGAGCAGGACAGCGGCCCGGAC-3' SEQ ID NO:14); TGGATTTTACTGCATTATGTCAG-3' SEQ ID TGGAGAAAGTTTCATCTTGTGAT-3' SEQ ID NO:16); 5'-CCCAAGCTTGCCGCCA TGCTGTGGGGAATCTGTATCTTT-3' The complete (OMRbSd2-34; (OMRbSd2-55; (OMRbSd2-78; (OMRbSd2-97; 0 SEQ ID NO:17); 5'-CCCAAGCTTGCCGCCATGTCAAGACTGTTGAAGAAG-3' (OMRbSdl-147, SEQ ID NO:18).

The anti-sense primer 5'-GTCCAAGAGAATTCATAAAAGG-3' (OMRbAS300; SEQ ID NO:13) overlaps with the EcoRI site (underlined) at the nucleotide +900 of the RB cDNA (the A of the first in-frame ATG is designated as position The anti-sense primer was paired with each sense primer described above to amplify various modified 5'-RB cDNA fragments using plasmid F7 as template (which contains the full-length RB cDNA).

After amplification by PCR T M with each pair of primers, the DNA fragments were digested with HindIII and EcoRI and subcloned into plasmid pCMVRB" 1 which had been cut with the same enzymes. The resultant expression plasmids carrying the modified RB cDNAs WO 98/37091 PCT/US98/03041 with N-terminal deletions corresponding to amino acids 2-34 (SEQ ID NO:28 (nucleic acid sequence) and SEQ ID NO:29 (amino acid sequence)), 2-55 (SEQ ID NO:30 (nucleic acid sequence) and SEQ ID NO:31 (amino acid sequence)), 2-78 (SEQ ID NO:32 (nucleic acid sequence) and SEQ ID NO:33 (amino acid sequence)), 2-97 (SEQ ID NO:34 (nucleic acid sequence) and SEQ ID NO:35 (amino acid sequence)) and 1-147 (SEQ ID NO:36 (nucleic acid sequence) and SEQ ID NO:37 (amino acid sequence)) were named as pCMVRBd 2 34 (a deletion of amino acids 2 to 34 of the wild type RB protein), pCMVRBd2 55 (a deletion of amino acids 2 to 55 of the wild type RB protein), pCMVRBd 2 (a deletion of amino acids 2 to 78 of the wild type RB protein), pCMVRBd 2 97 (a deletion of amino acids 2 to 97 of the wild type RB protein) and pCMVRBdi-1 47 (a deletion of amino acids 1 to 147 of the wild type RB protein; amino acid I 148 is a methionine) respectively.

B. Construction of RB cDNAs with Internal Deletions or Mutations A total of seven pRB expression plasmids carrying RB cDNAs with varying internal deletions or mutations have been constructed, namely pCMVRBd 3 i.

1 i 7 (a deletion of amino acids 31 to 107 of the wild type RB protein), pCMVRBd 77 107 (a deletion of amino acids 77 to 107 of the wild type RB protein), pCMVRBml 11 1 12 (a mutation of amino acid 111 of the wild type RB protein from aspartic acid to glycine and a mutation of amino acid 112 from glutamic acid to aspartic acid), pCMVRBdlin.i 8 (a deletion of amino acids 111 to 181 of the wild type RB protein), pCMVRBd 111 241 (a deletion of amino acids 111 to 241 of the wild type RB protein), pCMVRBd 8 1 241 (a deletion of amino acids 181 to 241 of the wild type RB protein) and pCMVRBd 242 30 (a deletion of amino acids 242 to 300 of the wild type RB protein).

For the construction of pCMVRBd 3 1 1 07 an RB cDNA fragment from nucleotide position +325 to +910 was amplified from the plasmid F7 by PCR T M using the primers 5'-GCGCCTGAGGACCTAGATGAGATGTCGTTC-3' (SEQ ID NO:19) and OMRbAS300 (SEQ ID NO:13). This RB cDNA fragment was digested with Bsu36I (underlined) and EcoRI (from OMRbAS300), and inserted into plasmid pCMVRB 11 0 digested with the same enzymes, to replace the original RB cDNA fragment from nucleotides +91 to +900. The nucleic acid sequence of pRBA31-107 is SEQ ID NO:38, and the corresponding amino acid sequence is SEQ ID NO:39.

WO 98/37091 PCT/US98/03041 76 For the construction of pCMVRBd 77 1 07 an RB cDNA fragment (nucleotides +328 to +910) was amplified from the plasmid F7 by PCR T M using the oligonucleotides 5'-GCGGTTAACCCTAGATGAGATGTCGTTCACT-3' (SEQ ID NO:20) and OMRbAS300 (SEQ ID NO:13), followed by digestion with HpaI (underlined) and EcoRI. The amplified, digested fragment was inserted into plasmid pCMVRB" 1 0 digested with the same enzymes, to replace the RB cDNA fragment from nucleotides +230 to +900. The nucleic acid sequence of pRBA77-107 is SEQ ID NO:40, and the corresponding amino acid sequence is SEQ ID NO:41.

For the construction of pCMVRBm 1 ll/ 2 two pairs of primers were used to change nucleotide A (position +332 of the wild-type RB cDNA) to G, in order to change the codon for aspartic acid (GAT) to glycine (GGT), thus creating a new restriction enzyme site, AvrII, and nucleotide G (position +336 of the wild-type RB cDNA) to T, in order to change the codon for glutamic acid (GAG) to aspartic acid (GAT). The first pair of primers are 5'-CCCAAGCTTGCCGTCATGCCGCCCAAAACCCCCCGA-3' (OMRBS1; SEQ ID NO:21) and 5'-CTCACCTAGGTCAACTGCTGCAAT-3' (OMRbAS332; SEQ ID NO:22; the mutated base is in bold). The second pair of primers are 5'-GTTGACCTAGGTGATATGTCGTTC-3' (OMRbS332; SEQ ID NO:23; the mutated bases are in bold) and OMRbAS300 (SEQ ID NO:13). The PCR T M products amplified with OMRBSI and OMRbAS332 were digested with Hind III and AvrII (underlined), and those amplified with OMRbS332 and OMRbAS300 were digested with AvrII and EcoRI. These fragments were ligated together into plasmid pCMVRB" 10 digested with HindIII and EcoRI to replace the corresponding wild-type RB cDNA sequences.

The nucleic acid sequence of pRBmlll11/112 is SEQ ID NO:50, and the corresponding amino acid sequence is SEQ ID NO:51.

For the construction of pCMVRBdiis 8 the RB cDNA fragment (nucleotides +543 to +910) was amplified from plasmid F7 by PCR T M using the oligonucleotides 5'-GCGCCTAGGATCTACTGAAATAAATTCTGCA-3' (SEQ ID NO:24) and OMRbAS300 (SEQ ID NO:13), followed by digestion with AvrII (underlined) and EcoRI. This fragment was then ligated into pCMVRBmll/ 1 1 2 (above) digested with the same enzymes to replace the RB cDNA fragment from nucleotides +331 to +900. The nucleic acid sequence of pRBA 1 1-181 is SEQ ID NO:42, and the corresponding amino acid sequence is SEQ ID NO:43.

WO 98/37091 PCT/US98/03041 77 For the construction of pCMVRBdi 11 241 a 5' RB cDNA fragment containing nucleotides +1 to +331 was obtained by digestion of pCMVRBmII with HindIII and AvrII. The 3' RB cDNA fragment beginning from nucleotide +722 was isolated from the same plasmid digested with Pvull and BamHI. Then the two DNA fragments (in-frame) were ligated into pCMV-G digested with HindIII and BamHI. The nucleic acid sequence of pRBAll 1-241 is SEQ ID NO:44, and the corresponding amino acid sequence is SEQ ID For the construction of pCMVRBdl 81241 a 5'-RB cDNA fragment containing nucleotides +1 to +538 was amplified from plasmid F7 by PCR T M with primers OMRBSI (SEQ ID NO:21) and 5'-CCCGATATCAACTGCTGGGTTGTGTCAAATA-3' (SEQ ID NO:25) using plasmid F7 as a template. The obtained RB cDNA fragment was cut with HindIII and EcoRV (underlined), and inserted into pCMVRB 1 0 to replace the original 5' RB cDNA fragment between the HindIII and PvuII sites. The nucleic acid sequence of pRBA181-241 is SEQ ID NO:46, and the corresponding amino acid sequence is SEQ ID NO:47.

For the construction of pCMVRBd 242 300 primers OMRBS1 (SEQ ID NO:21) and CCCGAATTCGTTTTATATGGTTCTTTGAGCAA-3' (SEQ ID NO:26) were used to amplify the 5' RB cDNA fragment containing nucleotides +1 to +722 using plasmid F7 as a template.

The amplified product was digested with HindIII and EcoRI (underlined), and inserted into pCMVRB" 1 0 digested with the same enzymes to replace the original 5' RB cDNA sequences from nucleotides +1 to +900. The nucleic acid sequence of pRBA242-300 is SEQ ID NO:48, Sand the corresponding amino acid sequence is SEQ ID NO:49.

C. Characterization of N-terminal Modified RB Proteins An RB-defective bladder carcinoma cell line, 5637 was transfected with the expression plasmids carrying the modified RB cDNAs driven by a CMV promoter. The biological function of the mutant pRBs was evaluated by a combined technique involving immunocytochemical staining and 3 H]-thymidine in situ labeling of the tumor cells after transfection (Xu et al., 1994a; 1994b).

Tumor cells were seeded onto coverslips in medium containing tetracycline and transfected with plasmids expressing pRB 94 pRB 11 0 or other mutant RB proteins. At specified WO 98/37091 PCTIUS98/03041 78 time point after removal of tetracycline from the culture medium, the cells were incubated with 1 ml of fresh medium containing 10 tCi 3 H]-methyl thymidine (Amersham, Arlington Heights, IL) for 2 hours at 37 0 C, then fixed and immunochemically stained for expression of RB protein as described previously (Xu et al., 1991a; 1991b). Stained slides were subsequently coated with a thin layer of gelatin and dried at 37 0 C overnight. The slides were then overlaid with autoradiographic emulsion (Type NTB2, Eastman Kodak, Rochester, NY) and exposed for 2 days. After development, slides were examined under a light microscope. Twenty-four hours after transfection, cells were processed for immunocytochemical staining of RB protein and H]thymidine incorporation assay as described above.

The results are illustrated in Table 5. When up to 55 amino acid residues were deleted from the N-terminal of pRB, the DNA synthesis was not significantly reduced in the cells transfected with the mutant pRB expression plasmids compared to cells expressing the fulllength RB protein. However, when another 23 amino acids were removed from the N-terminal, the cellular DNA synthesis was dramatically suppressed by expression of the truncated pRB.

RB Construct Wild-Type d2-34 d2-55 d2-78 d2-97 dl-112 dl-147 d31-107 d77-107 dlll-112 dlll-181 dl11-241 dil -414 Table Cells Incorporating H]-Thymidine RB RB: 14 41 12 42 11 43 3 41 3 42 2 42 4 42 3 41 2 6 3 38 2 24 42 WO 98/37091 PCT/US98/03041 79 Table 5 (Continued) Cells Incorporating ['H]-Thymidine RB Construct .RB mRB d181-241 8 43 d242-300 17 43 As demonstrated in Table 5, the pRB mutants with any deletions between amino acid and 181 significantly inhibit DNA synthesis after being introduced into the tumor cells. Of note, cells transfected with pRBs containing deletions only between amino acid 181 and 241 showed weaker inhibition of DNA synthesis than those transfected with plasmids expressing pRBs carrying deletions between amino acid 55 and 181, although these were still more effective than cells transfected with the full-length pRB expression plasmid. Thus, in view of this data.

modifications that combine certain of the above deletions, for example a deletion between amino acid 1 and amino acid 241, would be expected to have similar significant DNA synthesis inhibitory activity.

Additionally, two pRB mutants with two deletions each, either between amino acid 2 and 34 and between amino acids 76 and 112, or between amino acids 2 and 55 and between amino acids 76 and 112 significantly inhibited DNA synthesis as compared to the wild-type RB. The results indicated the boundary of the putative N-terminal domain probably located between amino acid 182 and 300, most probably between amino acid 182 and 241. In addition, a pRB carrying a point mutation at amino acid position 111 converting aspartic acid to glycine significantly suppressed DNA synthesis, further suggesting that this region is vital for regulating pRB function.

EXAMPLE 2 Modification of the CMV Promoter/Enhancer Controlling Expression of the VP16 Transactivating Domain in the Tetracycline-Responsive Gene Expression System The modified retinoblastoma genes and proteins described above have a number of practical utilities, including, but not limited to, gene therapy. For these aspects, expression systems are needed. While systems such as those described above are appropriate for certain WO 98/37091 PCT/US98/03041 embodiments, they have certain shortcomings in relation to gene therapy using cytotoxic constructs. The original tetracycline-responsive gene expression system of Gossen and Bujard (1992) is an attractive system, but has certain drawbacks, such as squelching effects on cell growth (Gill and Ptashne, 1988). To overcome these and other drawbacks, the inventors have improved the tetracycline-responsive gene expression system.

The original tetracycline repressor/operator-based regulatory system consists of two plasmids, pUHD15-1 and pUHC13-3 S. Patent 5,464,758, incorporated in its entirety herein by reference; Gossen and Bujard 1992). pUHC13-3 is a tetracycline (Tc; tet) sensitive expression vector containing a hybrid minimal human CMV promoter, in which tet operator sequences had been inserted upstream of the TATA box. pUHDI5-1 contains sequences encoding a tetracycline responsive transactivator (tTA), with expression driven by a wild-type CMV promoter. In transient experiments using this system, the inventors found that efficiently reversible transgene expression was observed in many tumor cell lines studied. However, attempts to isolate long-term clones expressing the reporter gene in a tetracycline-responsive manner were unsuccessful. This was most likely caused by the high intracellular levels of the tTA transactivator, whose expression was driven by the strong CMV promoter/enhancer sequence in the plasmid pUHD15-1. The tTA transactivator contains the VP-16 activating domain, which is known to have squelching effects on cell growth (Gill and Ptashne, 1988).

Therefore, to resolve this problem and to further improve the system, the tTA expression cassette was first modified by replacing the strong CMVp enhancer (Boshart et al., 1985) in the original pUHD15-1 plasmid with a pair of 19 bp imperfect direct repeat sequence (a portion of the CMVp enhancer; SEQ ID NO:5). The modification of the hCMV promoter/enhancer was done by removal of a portion of the 5' enhancer sequences from the hCMV promoter.

Three pairs of oligonucleotide primers were designed based on the published sequence of the hCMV promoter (Boshart et al., 1985). A XhoI and an EcoRI restriction enzyme site (underlined) was added to the 5' end of each sense and the anti-sense oligo, respectively. The sense oligos are: 5'-CCGCTCGAGCAATGGGCGTGATAGCGG-3' (OMCMVsl; SEQ ID NO:6); 5'-CCGCTCGAGCACCAAAATCAACGGGA-3' (OMCMVs2; SEQ ID NO:7) and CCGCTCGAGCAACTCCGCCCCATTGAC-3' (OMCMVs3; SEQ ID NO:8), respectively, and WO 98/37091 PCT/US98/03041 81 they shared the same anti-sense primer, 5'-TAGACATATGAATTCGCGGCC-3' (OMCMVas; SEQ ID NO:9).

The template used in PCR T M amplification was plasmid pUHD15-1. PCR T M amplification with primer pairs of OMCMVsl OMCMVas; OMCMVs2 OMCMVas and OMCMVs3 OMCMVas, generated three shorter versions of CMV promoter with lengths of 282bp (namely mhCMVpl), 203bp (mhCMVp2) and 168bp (mhCMVp3) respectively. The purified shortened CMV promoter/enhancer fragments were double digested with XhoI and EcoRI, and inserted into pUHDI5-1 to replace the original hCMV promoter. This produced three new tTA expressing plasmids, namely pmCMVI-tTA, pmCMV2-tTA and pmCMV3-tTA.

I

To determine the relative strength of these promoters, the tTA in these newly constructed plasmids, as well as plasmid pUHDI5-1, was replaced by a chloramphenicol acetyltransferase (CAT) gene from plasmid pRc/CMV-CAT (Invitrogen. San Diego, CA), thus generating four CAT expression plasmids, pmCMVI-CAT, pmCMV2-CAT, pmCMV3-CAT and pCMV-CAT.

In these plasmids. CAT expression is driven by mhCMVpl, mhCMVp2, mhCMVp3 and the full-length hCMVp, respectively. To evaluate the relative activity of the modified CMV promoters, the CAT expression plasmids were introduced into three cell lines, the tumor cell lines 5637 and Saos2, and the embryonal kidney cell line 293, via the Lipofectin method (Life Technologies, Gaithersburg, MD). Forty-eight hours after transfection, cell lysates were prepared and CAT activity was measured by a CAT FLASH assay kit from Stratagene (Stratagene, La Jolla, CA).

As shown in FIG. 1, after enhancer sequences were partially removed, the activity of the promoter was dramatically reduced in all three transfected cell lines. FIG. 1 is a graphical representation of the CAT activity in the 5637 and Saos-2 cell lines. The more enhancer sequences that were deleted, the weaker was the promoter that remained. The order of promoter activity from strongest to weakest is hCMV, mhCMVpl, mhCMVp2 and mhCMVp3. The activity of mhCMVpl is 17.7% of the full-length hCMV promoter, while the mhCMVp3 activity is only 3.3% of the hCMV promoter in 5637 cells (FIG. After comparing the relative promoter activity of the modified promoters, mhCMVpl (SEQ ID NO:5) was chosen for the modified tetracycline regulatable gene expression system. mhCMVpl showed optimal WO 98/37091 PCTIUS98/03041 82 tetracycline-controlled transactivator (tTA) expression with no squelching effects on host cell growth (FIG. an important characteristic for potential use in human gene therapy.

EXAMPLE 3 Construction of Single Plasmid, Tetracycline-Regulated Vector A single plasmid vector named EC1214A was constructed. This plasmid contains: 1) the modified tetracycline-responsive transactivator (tTA) expression cassette to eliminate the squelching effects of tTA on host cell growth; 2) the tTA-dependent promoter from plasmid pUHC13-3; 3) a generic intron sequence; 4) a multiple cloning site downstream of the promoter and intron; and 5) a neoR expression cassette to allow G418 selection. Expression in this system is regulated by tetracycline, or a tetracycline analog. A "tetracycline analog" will be understood to be any one of a number of compounds that are closely related to tetracycline, and which bind to the tet repressor with at least an affinity (Ka) of at least 10 6 preferably with a K, of 109/M, and more preferably with a Ka of 10 Exemplary, but in no way limiting, of such tetracycline analogs are those disclosed by Hlavka and Boothe (1985), Mitschef (1978), the Noyee Development Corporation (1969), Evans (1968) and Dowling (1955), each of which is incorporated herein in its entirety.

Plasmid pMLSIS.CAT (Choi et al., 1991) contains an generic intron sequence which consists of a portion of the 5'-untranslated leader from the adenovirus-major-late region, which contains part of the first exon of the tripartite and the first intervening sequence, as well as a synthetic splice donor/acceptor sequence derived from an IgG variable region. A pair of oligonucleotides, 5'-CTAGAATTCGCTGTCTGCG-3' (SEQ ID NO:10) and GCTCTAGATGCAGTTGGACCTGGGAG-3' (SEQ ID NO:11), flanking the intron sequence in plasmid pMLSIS.CAT and containing an EcoRI and XbaI site, respectively (underlined), were synthesized. After amplification by PCR T M the intron fragment was digested with EcoRI and XbaI, and inserted into the corresponding enzyme sites in plasmid pUHD 15-1.

Subsequently, a small DNA fragment containing Clal, HindIII, EcoRV, EcoRI, PstI, SmaI and BamHI cloning sites (obtained from plasmid pBluescriptSK) was inserted into the new plasmid downstream of the intron to produce an expression vector containing the hCMV promoter, a generic intron, multiple cloning sites and a polyadenylation signal from the WO 98/37091 PCT/US98/03041 83 virus. This intermediate vector was given the name of pCMV-G. The SV40 polyadenylation signal of pCMV-G was then replaced by a HSV thymidine kinase (TK) gene polyadenylation signal sequence to generate a plasmid, named pCMV*-G-TKpA.

Plasmid pRc/CMV (Invitrogen, San Diego, CA) was double digested with restriction enzymes NruI and Xbal. The 5' overhang from the XbaI digest was filled in by Klenow fragment of DNA polymerase (Life Technologies, Gaithersburg, MD), and the blunt-ended insert was ligated to a DNA fragment containing mhCMVI-tTA obtained from plasmid pmCMVl-tTA (Example The new plasmid was named pmCMVl-tTA.neo.

Finally, a DNA fragment containing the tTA-dependent promoter, the generic intron and the TK polyadenylation signal was isolated from plasmid pCMV*-G-TKpA, and inserted into the Bg!Il site of plasmid pmCMV -tTA.neo to produce a vector named EC1214A, which carries both the tTA expression cassette and the tTA-dependent promoter as well as a selection marker, the neomycin resistance gene.

EXAMPLE 4 Construction of a Single Plasmid Tetracycline Positively-Induced (Tet-on) Vector The original tetracycline repressor/operator-based tet-on system also consists of two plasmids, pUHD17-lneo (or pUHD172-lneo) and pUHC13-3 (Gossen et al., 1995). pUHC13-3 is a tetracycline sensitive expression vector containing a hybrid minimal human CMV promoter, in which tet operator sequences had been inserted upstream of the TATA box. pUHD17-lneo or pUHD172-lneo contains sequences encoding a reverse tetracycline responsive transactivator (rtTA), with expression driven by a wild-type CMV promoter. In transient experiments using -this system, it was found that efficiently reversible transgene expression was observed in many tumor cell lines studied. As opposed to the original tetracycline system, expression is turned on in the presence of tetracycline or a tetracycline analog, such as doxycycline, while expression is turned off in the absence of tetracycline. However, the rtTA transactivator contains the VP-16 activating domain, which is known to have squelching effects on cell growth (Gill and Ptashne, 1988).

WO 98/37091 PCT/US98/03041 84 Therefore, to resolve this problem and to further improve the system, the rtTA expression cassette was first modified by replacing the strong CMVp enhancer (Boshart et al., 1985) in the pUHD17-lneo or pUHDI72-1neo plasmid with a pair of 19 bp imperfect direct repeat sequence (SEQ ID NO:5). The modification of the hCMV promoter/enhancer was done by removal of a portion of the 5' enhancer sequences from the hCMV promoter (Example The new rtTA expressing plasmid was named pmCMV 1-rtTA.

A single plasmid vector named EC1214B was constructed using pmCMV1-rtTA. This plasmid contains: 1) the modified reverse tetracycline-responsive transactivator (rtTA) expression cassette to eliminate the squelching effects of rtTA on host cell growth; 2) the rtTA-dependent promoter from plasmid pUHC13-3; 3) a generic intron sequence; 4) a multiple cloning site downstream of the promoter and intron; and 5) a neoR expression cassette to allow G418 selection. The construction was performed as outlined in Example 3.

EXAMPLE Construction of Retinoblastoma (RB) and p53 Tetracycline-Controlled Vectors A. Construction of Inducible pRB 110 Expression Vector To construct an inducible pRB 11 0 expression plasmid, plasmid F7 (Takahashi et al., 1991) or p4.95BT (Friend et al., 1987), containing the full-length RB 1 0 gene cDNA, was digested with the restriction enzymes AcyI at nucleotide -322 and ScaI at +3230 (the A of the second in-frame ATG start codon was designated nucleotide The 5' overhangs generated by the AcyI digest were treated with E. coli DNA polymerase I in the presence of all four dNTPs to generate blunt ends. BamHI linkers were ligated onto the fragment, and the fragment was then digested with BamHI to remove excess linkers and generate BamHI ends (Maniatis et al., 1989; Ausubel et al., 199-2). The resultant RB cDNA fragment of 3552 bp was inserted into the unique BamHI site of EC1214A to generate pCMV*-tTA-RB 10 B. Construction of Inducible pRB 94 Expression Vector It is known that the primary sequence surrounding the AUG codon GCC(AG)CCAUGG (SEQ ID NO:27) is the optimal context for initiation of translation in higher eukaryotes (Kozak 1991). A surprising realization is that, although nearly all vertebrate mRNAs have features that ensure the fidelity of initiation, many mRNAs that encode critical regulatory proteins do not WO 98/37091 PCT/US98/03041 appear to be designed for efficient translation (Kozak 1991). In reviewing the RB cDNA sequence, it was found that the AUG start codon for both the full length pRB i and the Nterminal truncated pRB 94 are in a suboptimal context for initiation of translation in higher eukaryotes. For example, there is an out-of-frame AUG codon at the nucleotide -5 position (the 5 A of the ATG start codon for the pRB 94 cDNA is designated nucleotide and the leading sequence of the ATG codon for pRB 94 is suboptimal as compared to the consensus initiator context shown above. To improve the translation efficiency of the pRB 94 cDNA, site-directed mutagenesis was used to optimize the DNA sequence upstream of the second internal in-frame ATG codon of RB 94 for optimal translational initiation.

I The modified 5'-RB 94 cDNA fragment was obtained by PCR T M using plasmid F7 carrying the full-length RB 11 0 cDNA as the template. The sense primer used for the PCR T M reaction CCCAAGCTTGCCGCCATGTCGTTCACTTTTAC-3'; SEQ ID NO:12) contained a HindIIl restriction site (underlined) and a Kozak cassette (italics; Kozak, 1987). The antisense primer GTCCAAGAGAATTCATAAAAGG-3' (OMRbAS300; SEQ ID NO:13) overlapped with the EcoRI site (underlined) at nucleotide +900 of the RB cDNA (the A of the first in-frame ATG is designated as position The PCR T M product was digested with HindIII and EcoRI, then ligated with a DNA fragment containing the 3'-RB cDNA fragment between EcoRI (position +900) and BamHI (+3548) isolated from plasmid F7. The entire RB 94 cDNA fragment was inserted into the HindIII and BamHI sites of EC1214A to produce the inducible pRB 94 t expression plasmid, pCMV*-tTA-RB 94 C. Construction of Inducible p53 Expression Vector A plasmid, pC53-SN3 (Baker etal., 1990), containing the full length p53 gene cDNA was digested with BamHI, and the fragment containing the full length p53 gene was inserted into the unique BamHI site of EC1214A to generate pCMV*-tTA-p53.

WO 98/37091 PCT/US98/03041 86 EXAMPLE 6 Preparation of Long-Term Tumor Cell Clones with Tetracycline-Regulated pRB110, pRB94 or p53 Expression The modified, single-plasmid tetracycline-responsive mammalian gene expression system has been used to obtain various stable tumor cell lines in which expression of the wild-type or the N-terminal truncated retinoblastoma (RB) tumor suppressor gene, or the p53 tumor suppressor gene can be reversibly turned on and off without detectable leakage.

A. Cell Culture A breast carcinoma cell line, MDA-468 (HTB132) was obtained from ATCC and cultured in Leibovitz's L-15 (Life Technologies, Gaithersburg, MD) with 10% FBS (Life Technologies, Gaithersburg, MD). An osteosarcoma cell line, Saos2 was cultured in medium McCoy's 5A (Life Technologies, Gaithersburg, MD) with 15% FBS (Zhou el al., 1994b). A bladder carcinoma cell line, 5637 (HTB9) obtained from ATCC was cultured with RPMI 1640 medium (Life Technologies, Gaithersburg. MD) containing 10% FBS. All cell culture media were supplemented with 0.5% penicillin/streptomycin. Saos2 and 5367 cells were incubated at 37 0 C in a 5% CO 2 incubator, while MDA-468 cells were cultured at 37 0 C without CO 2 B. Stable Transfection Tumor cells were transfected with the pRB" 10 and pRB 94 expression plasmids, pCMV*tTA-RB 1 1 and pCMV*-tTA-RB 94 via the Lipofectin method according to the manufacturer's instruction manual (Life Technologies, Gaithersburg, MD). During transfection and the subsequent procedures except where specified, 0.5 g/ml of tetracycline (Sigma, St. Louis, MO) was added to the transfection and culture media. Forty-eight hours after transfection, G418 (Life Technologies, Gaithersburg, MD.) was added to the culture media at a concentration of 300 p.g/l.

Two to three weeks later, single colonies were isolated by cloning rings. A duplicate culture was made for each isolated colony. While the original clone was kept in media containing 0.5 pg/ml tetracycline, the duplicate clone was cultured in the absence of tetracycline. The latter was immunochemically stained with a specific anti-RB antibody, RB-WL-1 (Xu et al., 1989a). The matched RB-positive clones were subsequently maintained in medium containing tetracycline and G418 and expended for further analyses.

WO 98/37091 PCT/US98/03041 87 C. Transient Transfection Tumor cells were seeded into 60-mm culture dishes or onto sterile coverslips at concentrations that would reach about 40% confluent next day. Twenty hours later, proper amount of plasmid DNA was mixed with Lipofectin reagent in Opti-MEM medium according to the manufacture's instruction manual (Life Technologies, Gaithersburg, MD). Cells were overlaid with the DNA-Lipofectin complex and incubated in a CO 2 incubator at 37 0 C overnight.

Next day, fresh medium was added to replace the DNA-Lipofectin. Twenty-four or forty-eight hours later, cells were fixed for immunochemical staining or lysed for preparation of cell lysates.

D. Immunocytochemical Staining of RB Protein Immunocytochemical staining was performed as described previously (Xu et al., 1989a).

For detection of RB expression, cells grown on coverslips were fixed in 45% (vol/vol) (wt vol) formaldehyde/0.1 M phosphate buffer for 5 min. After being washed six times with phosphate-buffered saline, cells were blocked with 1% non-fat milk/1.5% goat serum or horse serum in phosphate buffer for 4 hours at room temperature. The RB-WL-1 anti-RB antibody or Canji's monoclonal anti-RB antibody (QED, San Diego, CA) was diluted to 2 g/ml or 0.5 utg/ml respectively in the same solution plus 0.02% Triton X-100, and was incubated with the cell overnight. After being washed, the coverslips were processed for immunostaining with the avidin biotinylated peroxidase complex (ABC) method according to the technical manual (Vector Laboratories, Burlingame, CA).

D E. Immunoblotting for pRB Cell lysate was prepared as previously described (Xu etal., 1991a; 1991b). Briefly, cultured cells in 60 mm dishes were lysed with 0.6 ml of ice-cold lysis buffer containing 100 mM NaC1, 0.2% NP-40, 0.2% sodium deoxycholate, 0.1% SDS and 50 mM Tris-HCl with 50 pg/ml aprotinin and 1 mM PMSF. The cell lysate was passed through 21 gauge needle several times and clarified by centrifugation.

Direct Western immunoblotting was done as described previously (Xu etal., 1991a; 1991b). Sixty micrograms of total cellular protein as determined by the Bradford protein assay (BioRad, Richmond, CA) was electrophoresed in an 8% SDS/polyacrylamide gel and electroblotted to Immobilon polyvinylidene difluoride membranes (PVDF) (Millipore, Bedford, WO 98/37091 PCT/US98/03041 88 MA). After being blocked with 4% bovine serum albumin/1% normal goat serum in Trisbuffered saline, membranes were incubated overnight with RB-WL-1 antibody at a final concentration of 0.4 pg/ml for RB detection. The blots were then probed by the ProtoBlot Western blot alkaline phosphatase system (Promega, Madison, WI).

F. Growth Curve Measurement A crystal violet staining method was used to measure the cell growth changes in the presence or absence of tetracycline (Gillies et al., 1986). Briefly, cells were seeded into 24-well plates in duplicate. In one set of the plates, cells were grown in medium containing 0.5 Hg/ml tetracycline, while in duplicate plates, the same cells were cultured in non-tetracycline media. At each time point, cells were fixed with 1% glutaraldehyde in PBS and stained using 0.5% of crystal violet. After cells at all desired time points were collected, the crystal violet dye was extracted from the stained cells by incubating cells with Sorenson's solution containing 0.9% trisodium citrate, 0.02 N chloric acid and 45% ethanol (vol/vol). The extracted dyes were diluted properly with the Sorenson's solution and optical absorbencies at s550 were measured. Growth curves were obtained by plotting the OD 550 against the time.

G. Soft Agar Assay For soft agar assay, appropriate number of cells were mixed with 0.3% of agarose in complete medium containing 15% FBS and overlaid onto 0.7% base agar in a 35 mm tissue culture dish. Duplicate dishes were prepared for each individual cell clones. Cells in one dish were cultured in the medium containing 0.5 (g/ml of tetracycline and the other cultured in nontetracycline medium. The medium was replenished every 3 days, and colonies (>50 cells) were counted after 3 weeks. Results were calculated as the average of three dishes per cell clone.

H. Tumorigenicity Test in Nude Mice The tumorigenicity test has been described previously (Takahashi et al., 1991). Two groups of athymus nude mice were set up for each cell clone to be tested. One group of mice were given regular water, while the other group was given water containing 5 mg/ml of tetracycline. A total of 5 x 106 cells from each RB 1 o- or RB 94 -reconstituted clone were injected subcutaneously in 0.2 ml of phosphate buffered saline into the right flank of nude mice. RBnegative parental controls including Saos2, 5637 and MDA-468 cells were injected at the WO 98/37091 PCT/US98/03041 89 identical concentration into the left flank of the same mice. Tumors were scored 4 weeks after injection.

I. Time Course Study of 3 H]-Thymidine Incorporation Cells from inducible RB-reconstituted clones were grown on sterile coverslips in medium containing tetracycline. At specified time point after removal of tetracycline from the culture medium, the cells were incubated with 1 ml of fresh medium containing 10 pCi 3 H]-methyl thymidine (Amersham, Arlington Heights, IL) for 2 hours at 37 0 C, then fixed and immunochemically stained for expression of RB protein as described previously (Xu et al., 1991a; 1991b). Stained slides were subsequently coated with a thin layer of gelatin and dried at 37 0 C overnight. The slides were then overlaid with autoradiographic emulsion (Type NTB2, Eastman Kodak, Rochester, NY) and exposed for 2 days. After development, slides were examined under a light microscope.

J. 3 H]-Thymidine Incorporation of Transiently Transfected Cell Cultures Tumor cells were seeded onto coverslips and transfected with plasmids expressing pRB 94 pRB 1 0 or other mutant RB proteins. Twenty-four hours after transfection, cells were processed for immunocytochemical staining of RB protein and 3 H]-thymidine incorporation assay as described in Xu et al. (1991b; 1991c).

K. Characterization of Long-Term Inducible RB Expression Clones f The cell growth suppression and morphological changes after RB replacement that have been reported in the literature are inconsistent. Studies done by the inventors and others indicated that replacement of the normal RB gene into RB-defective tumor cells could suppress their tumorigenic activity in nude mice (Goodrich and Lee 1993, Bookstein et al., 1990a; 1990b; Chen et al., 1992; Goodrich et al., 1992b; Huang et al., 1988 Kratzke et al., 1993; Madreperla et al., 1991; Muncaster et al., 1992; Ookawa et al., 1993; Sumegi et al., 1990; Takahashi et al., 1991; Wang et al., 1993; Xu et al., 1996; Xu et al., 1991c; Zhou et al., 1994b; Xu, 1996; Xu, 1995; Li et al., 1996; Xu et al., 1994b). The tumor cell lines studied were derived from widely disparate types of human cancers such as the retinoblastoma, osteosarcoma, carcinomas of the bladder, prostate, breast and lung (Goodrich and Lee, 1993; Xu, 1996; Xu, 1995 for review).

Although it has been well documented that correction of the RB gene defect alone in tumor cells WO 98/37091 PCT/US98/03041 carrying multiple genetic alterations was sufficient to revert their malignant phenotype, it was more puzzling than it appeared at first sight (Klein, 1990).

As was shown in several early studies, after transient transfection' with pRB-expressing plasmids, some types of the RB-defective tumor cells in culture displayed striking changes, including cell enlargement, senescent-like phenotype and growth cessatioh (Templeton et al., 1991; Qin et al., 1992). Subsequently, it was found that, however, long-term stable clones of the RB-reconstituted tumor cells can be isolated that grew just as rapidly as the parental lines.

Therefore, there has been a tendency in the literature to separate the inhibition of cell growth by RB replacement in RB-defective tumor cells from its tumor suppression function (Chen et al., 1992; Goodrich et al., 1992b; Takahashi et al., 1991; Xu et al., 1991b; Zhou et al., 1994b; Li et al., 1996).

Three RB-defective tumor cell lines were used to establish long-term inducible RB expression clones. They were the osteosarcoma cell line, Saos2, the bladder cancer cell line, 5637 and the breast cancer cell line, MDA-468. The rationale for choosing Saos2, 5637 and MDA-468 as recipient cells was that they are the RB-defective tumor cells most in use for RBreplacement studies. The tumor cells were transfected with the inducible RB" 1 expression plasmid, pCMV*-tTA-RB 1 and the pRB 94 expression plasmid, pCMV*-tTA-RB 94 in the presence of tetracycline. After selection in 400 tg/ml of G418 for approximately 2 to 4 weeks, well separated single colonies were isolated and maintained in tetracycline containing media. A small portion of the isolated clones were cultured separately in the absence of tetracycline (Tc) for 24 to 48 hours and stained with an anti-RB antibody, RB-WL-1. Tight control of pRB protein expression in the stable clones of Tc-responsive RB-reconstituted 5637 bladder carcinoma and MDA-MB-468 breast carcinoma cells is seen.

The RB-reconstituted 5637 cells grown in the presence of 0.5 pg/ml of Tc in the culture medium are RB- by immunocytochemical staining, while after removal of Tc, the pRB expression was turned on in the RB-reconstituted 5637 cells as shown by RB+ immunocytochemical staining. The MDA-MB-468 breast carcinoma tumor cells were also RBby immunocytochemical staining in the presence of 0.5 ug/ml of Tc in culture medium, whereas WO 98/37091 PCT/US98/03041 91 after removal of Tc, the pRB expression was turned on in the RB-reconstituted MDA-MB-468 breast carcinoma cells as shown by RB+ immunocytochemical staining. Note that tetracycline is an inhibitor, rather than an inducer, in this tetracycline-responsive expression system.

S 5 The minimal concentration of tetracycline required to shut off RB expression was also tested. It was found that as little as 0.1 tg/ml of tetracycline can inhibit RB. expression to nondetectable level by immunostaining, indicating that the tetracycline-regulated expression system is very sensitive to tetracycline.

Additionally, it was surprisingly found that, unlike the non-regulatable, long-term RBreconstituted tumor cell lines previously reported, all the long-term tumor cell clones examined irreversibly ceased growing after pRB expression was turned on in Tc-free medium (FIG. 3A, FIG. 3B and FIG. 3C). It is known in the literature that the half-life of pRB in normal and tumor cells is only 4 to 6 hours (Mihara et al., 1989; Xu el al., 1994b; Xu et al., 1989a), and as was illustrated in FIG. 2, using the modified tetracycline-regulatable system, expression of tTA transactivator per se in the presence or absence of low concentration of Tc had no effect on cell growth.

The Saos2 and 5637 clones also failed to synthesize DNA, which were followed by noticeable morphological changes and finally, by cell death. The cellular morphology was markedly altered after pRB expression was induced in Tc-free medium, including cell enlargement, flattening, and lower nucleocytoplasmic ratio than cycling G1/S cells. In the case of the bladder carcinoma cell line, 5637, changes in morphology and growth rate after either transient or stable RB-replacement with a non-regulatable system have not been well documented in the literature (Goodrich et al., 1992b; Takahashi et al., 1991; Zhou et al., 1994b).

In general, the phenotypes of the established Tc-regulatable RB+ tumor lines in Tc-free medium were quite similar to those documented previously for RB plasmid-transfected (or RB retrovirus vector-infected) tumor cell mass cultures (Huang et al., 1988; Templeton et 1991; Qin et al., 1992). All tumor cell clones under permissive condition for pRB expression were WO 98/37091 PCT/US98/03041 92 unable to form colonies in soft agar (FIG. 4A, FIG. 4B and FIG. 4C), and were non-tumorigenic in nude mice.

To compare RB with another common tumor suppressor gene, p53, several long-term stable tumor cell clones with Tc-regulatable wild-type p53 expression have been established from the osteosarcoma cell line, Saos-2. A similar approach as described above was used to establish the p53-reconstituted Saos-2 tumor cell clones. In brief, the parental Saos-2 tumor cells were transfected with the wild-type p53-expressing plasmid, pCMV*-tTA-p53 (Example 5) and selected in geneticin-containing media. The initial G418-resistant mass cultures were subjected to at least two rounds of subcloning in order to obtain stable wild-type p53-reconstituted clones.

Because of complete deletion of the p53 gene, the parental Saos-2 cells have no endogenous p53.

With this model system, it was found that induction of wild-type p53 expression in p53reconstituted Saos-2 clones did result in growth arrest of the RB~/p53 n ull tumor cells. When the Tc-regulated p53-reconstituted Saos-2 clones were grown in the absence of Tc, many tumor cells shrank and detached. Furthermore, as measured by DNA fragmentation assay, abundant low molecular weight DNAs were detected only in samples extracted from p53-reconstituted Saos-2 tumor cells under permissive condition for p53 expression. These observations indicate that the wild-type p53-induced growth arrest of the RB-/p53 nu Saos-2 tumor cells was the result of apoptotic cell death rather than replicative senescence.

Dimri et al. recently reported a biomarker that identifies senescent human cells in culture and in aging skin in vivo. It was show that several human senescent cells expressed a pgalactosidase, histochemically detectable at pH 6 (Dimri et al., 1995). This marker, termed senescence-associated p-galactosidase (SA-p-gal), is expressed by senescent, but not presenescent fibroblasts. SA-p-gal was also absent from immortal cells, but was induced by genetic manipulations that reversed immortality (Dimri et al., 1995). Of note, some cells, such as adult melanocytes, expressed the SA-p-gal (pH 6 activity) independent of senescence or age. Thus, SA-P-gal is not a universal marker of replicative senescence, which is not surprising.

Nevertheless, by utilizing the instant long-term tumor cell clones with tetracyclineregulatable pRB or p53 expression, the SA-p-gal (pH 6 activity) provides a simple assay WO 98/37091 PCT/US98/03041 93 allowing the further characterization the RB-mediated tumor cell growth cessation. The majority of young (early passage) human WI-38 fibroblasts are SA-p-gal negative. In contrast, the senescent (at population doubling level greater than 52) WI-38 cells were strongly SA-p-gal positive. All tetracycline-responsive tumor cell clones examined so far were SA-p-gal negative 5 in the presence of tetracycline and were SA-P-gal positive in tetracycline-free medium The intensity of SA-P-gal staining of tumor cells in RB+ status, however, was variable depending on tumor cell types.

Of note, although p53 reconstitution in Saos-2 p53" ull tumor cells with either noninducible (Chen et al., 1990; Li et al., 1996) or inducible system did suppress their neoplastic phenotype, the p53 reconstituted Saos-2 clones with the tetracycline-regulatable promoter were SA-P-gal negative in either presence or absence of tetracycline. Of great interest, when the p53reconstituted Saos-2 cells were infected with recombinant adenovirus vectors expressing the wild-type pRB"o in Tc-free medium, the tumor cells with both wild-type p53 and pRB 110 expression displayed more intense SA-P-gal positive staining as compared to tumor cells only expressing pRB 1 The results imply that the mechanisms for tumor suppression by pRB and p53 were different from each other, but expression of pRB and p53 together had synergistic effects on RB-mediated tumor cell senescence.

In consideration of its potential therapeutic use, another important finding was the fact that the pRB-mediated replicative senescence (irreversible growth cessation) was tumor-specific.

The young WI-38 fibroblasts at early passage infected with recombinant adenovirus vector, AdCMVpRB 110 at multiplicity of infection (MOI) of 100 remained SA-P-gal negative, and they resumed a normal growth pattern about one week post-infection. Therefore pRB is a relatively safe reagents for anticancer gene therapy. In addition to therapy of advanced malignancies, the emerging RB gene therapy also may be beneficial in treating post-surgery residue tumors, superficial cancers, or premalignancies, as well as non-malignant, hyperproliferative disorders in certain circumstances (Chang et al., 1995; Xu et al., 1996).

WO 98/37091 PCT/US98/03041 94 L. The broad biological basis of the RB-mediated tumor suppression.

In addition to tumor cell-specific senescence and the well-known antiproliferative effects, pRB may also play a role in inhibition of angiogenesis and in elicitation of immunogenicity of tumor cells. The inventors have shown that serum-free conditioned media (CM) collected from the tetracycline-responsive, RB-reconstituted osteosarcoma and non-small cell lung carcinoma cell lines switched from angiogenic to anti-angiogenic after removal of Tc from the cell cultures.

This switch corresponded with the onset of pRB expression as determined by Western blotting and immunohistochemistry (Dawson et al., 1996). The inventors have also reported that HLA class II induction by IFN-y in the RB-defective non-small cell lung carcinoma cell line, H2009, requires reconstitution of the wild-type RB gene expression (Lu et al., 1996). The class II proteins present peptides derived from proteolytically processed antigens to CD4+ T lymphocytes as part of the immune response. Therefore, pRB likely has a role in mediating tumor immunogenicity as well.

To determine if replacement of the retinoblastoma (RB) tumor suppressor gene could inhibit invasion of RB-defective tumor cells, studies were conducted using the Boyden chamber assay (Li et al., 1996). The studies were done in a diverse group of stable RB-reconstituted human tumor cell lines, including those derived from the osteosarcoma and carcinomas of the bladder, breast and lung. The expression of the exogenous wild-type RB protein in these tumor cell lines was driven by either a constitutively active promoter or an inducible promoter. It was found that significantly more tumor cells from the parental RB-defective cell lines and the RBrevertants than from the RB-reconstituted RB cell lines penetrated through the Matrigel in the Boyden chamber assay (p 0.001, two-tailed t-test). Of note, the inhibition of invasiveness of various RB-defective tumor cells by RB replacement was apparently well correlated with suppression of their tumorigenicity in vivo. In contrast, although either functional RB or p53 reexpression effectively suppressed tumor formation in nude mice of the RB-/p53 null osteosarcoma cell line, Saos-2, replacement of the wild-type p53 gene had much less impact on their invasiveness as compared to the RB gene.

Normal human diploid cells senesce in vitro and in vivo after a limited number of cell divisions. This process known as cellular senescence is an underlying cause of aging and a WO 98/37091 PCT/US98/03041 critical barrier for development of human cancers. It has also been demonstrated that RB/p53defective tumor cells reexpressing functional pRB alone via a modified tetracycline-regulated gene expression system were irreversibly growth-arrested at GO/G1 phase of the cell cycle.

These cells displayed multiple morphological changes consistent with cellular senescence and also expressed a senescence-associated p-galactosidase biomarker.

Further studies indicated that telomerase activity, which was presumably essential for an extended proliferative life-span of neoplastic cells, was repressed in the tumor cell lines after induction of pRB (but not p53) expression. These observations suggest that pRB plays a critical role in the intrinsic cellular senescence program. From a practical standpoint, findings imply that cytostatic gene therapy using RB (or RB and p53 together) may result in differential elimination of tumor cells through cellular senescence and crisis. At the same time the replicative lifespan of normal cells in vivo may not be affected. This could provide a potential basis for designing tumor-specific tumor suppressor gene therapy and anti-telomerase gene therapy.

These findings, taken together, may intimate that the RB-mediated tumor suppression has a broad biological basis, which certainly makes the emerging RB tumor suppressor gene therapy for human cancer even more attractive.

M. Enhanced Tumor Suppression by an N-terminal Truncated pRB.

Long-term stable clones of the RB-reconstituted tumor cells can be isolated with noninducible gene expression systems, and most of these clones grow just as rapidly as the parental lines. The inventors have also found that, although the RB-mediated tumor suppression was substantial and had a broad biological basis, it was often incomplete and a portion of the RB-reconstituted tumor cells were able to survive and form RB+ xenograft tumors in nude mice after a prolonged latency period (Takahashi et al., 1991; Xu et al., 1991b; Zhou et al., 1994b; Li et al., 1996). Similar observations have been reported by other investigators (Bookstein et al., 1990b; Goodrich et al., 1992b; Kratzke et al., 1993; Ookawa et al., 1993; Wang et al., 1993).

This phenomenon is referred to by the inventors as tumor suppressor resistance (TSR; Zhou et al., 1994b), which is an equivalent of multiple drug resistance (MDR) in chemotherapeutics. In the latter scenario, low-dose chemotherapy may risk the selection of metastatic tumor cells due to their often inherently higher resistance to cytotoxic agents.

WO 98/37091 PCT/US98/03041 96 The inventors subsequently reported that an N-terminal truncated RB protein of -94 kDa (pRB 94 exerted surprisingly more potent cell growth suppression as compared to the full-length pRB protein in a diversity of tumor cell lines examined, including those having a normal endogenous RB gene. Tumor cells transfected with the pRB 9 4 -expressing plasmids displayed multiple morphological changes frequently associated with cellular senescence. They failed to enter S phase and rapidly died (Xu et al., 1994b; Resnitzky and Reed, 1995).

The inventors recent studies in ectopic animal models demonstrated that treatment of established human RB- and RB bladder xenograft cancers in nude mice by AdCMVpRB94, a replication-deficient adenovirus vector expressing the N-terminal truncated RB protein, resulted in regression of the treated tumors (Xu et al., 1996). Of note, although both the full-length and the truncated forms of the RB protein, when over-expressed in tumor cells via adenovirus vectors, were capable of suppression of tumor growth, the pRB 94 was much more potent than the full-length RB protein. The mechanism for the enhanced tumor suppression by the N-terminal truncated RB protein is not clear yet.

To better understand the functional difference between the N-terminal truncated pRB 94 and the full-length pRB 11 the inventors have also established stable tumor cell lines with Tcresponsive pRB 94 expression. By time course analysis, it was found that as early as 6 hours after 94 removal of tetracycline from the cell culture medium, the pRB9-reconstituted tumor cells accumulated the maximum of both underphosphorylated and phosphorylated pRB 9 followed by failure of the vast majority of the tumor cells to incorporate 3 H-thymidine, an indicator of growth cessation. The pRB 94 protein was completely dephosphorylated within -18 to 24 hours. Most of the pRB 11 0 -reconstituted tumor cells, however, remained immuno-histochemically RB- at the 6 or 8 hr-time points and had normal DNA synthesis (FIG. The pRB 11 0 reached the highest level at the 24 hr-time point as determined by western blotting, and became mostly unphosphorylated from 24 to 48 hours after removal of tetracycline, in which period the pRB reconstituted tumor cells finally ceased DNA synthesis (FIG. Using the SA-P-gal biomarker assay for human senescent cells, it was shown that the Saos-2 cells with pRB 94 expression WO 98/37091 PCT/US98/03041 97 showed more intense SA-P-gal positive staining as compared to the pRB 1 o-expressing cells at 48 hr after removal of Tc. Since pRB 94 has a longer half-life than pRBo and tends to remain in an active, underphosphorylated form S. Patent 5,496,731; Xu et al., 1994b), rapid accumulation of mostly the active forms (underphosphorylated form) of RB protein in the tumor cells may account for the enhanced tumor cell growth suppression by pRB 94 In this regard, another truncated version of pRB, named pRB 56 beginning at amino acid 379, has also been reported as a more potent inhibitor of cell cycle progression compared to the full-length pRB (Wills et al., 1995).

The advantages of the modified system are threefold: 1) it is suitable for establishing long-term stable cell lines with inducible gene expression because of lower constitutive expression of the tTA peptide; 2) the system is now contained within a single plasmid so that only one round of transfection and selection is required; and 3) of importance, the single-plasmid tetracycline-responsive mammalian gene expression system is readily convertible to tetracycline-controlled viral vectors (Examples 7-12 below).

EXAMPLE 7 Construction of Tetracycline-Controlled Adenoviral Vectors The desired cDNA fragment of a gene of interest is first inserted into the single-plasmid tetracycline-regulatable plasmid vector, EC1214A (Example 3) or EC1214B (Example The 9 tetracycline-responsive foreign gene expression cassette and the modified tTA (or rtTA) expression cassette from the corresponding EC1214A or EC1214B plasmid vectors are then recovered using standard methods in the art for DNA manipulation (Maniatis et al., 1989; Ausubel et al., 1992), and inserted into the shuttle plasmid, pAElsplA (Microbix Biosystems, Inc.). The resultant recombinant shuttle plasmids are then co-transfected with the master adenovirus type 5 (Ad5) plasmid, pBHG11, which contains the backbone of the adenovirus Ad5dl309 genome and E1/E3 deletion mutation (Microbix Biosystems, Inc.) into 293 cells using the LIPOFECTIN reagent (GIBCO/BRL Life Technologies). The co-transfection of 293 cells is performed in the presence (for tet-off system) or absence (for tet-on system) of 0.5 Ig/ml of tetracycline.

WO 98/37091 PCT/US98/03041 98 Alternatively, a fragment containing a gene of interest is first inserted into the singleplasmid tetracycline-regulatable plasmid vector, EC1214A or EC1214B. The tetracyclineresponsive foreign gene expression cassette and the modified tTA (or rtTA) expression cassette from the corresponding EC1214A or EC1214B plasmid vectors are then recovered and inserted, respectively, into the shuttle plasmid, pAElsplA and the master adenovirus plasmid, pBHG 11.

The resultant recombinant shuttle plasmids and the recombinant master adenovirus plasmid are co-transfected into 293 cells.

Co-transfection of 293 cells with the recombinant shuttle plasmid and the recombinant master adenovirus plasmid produce infectious virions by in vivo recombination, in which the minigene cassette expressing the gene of interest and the modified tTA (or rtTA) expression cassette are replaced the AEl region or AE and AE3 regions of the Ad5dl309 genome, respectively. Presence of recombinant adenoviruses in the transfected 293 cells is initially identified by cytopathic effect (CPE). Cell culture supernatants are collected from the transfected 293 cells in which CPE has occurred. Recombinant viruses are then isolated by screening adenovirus plaques from 293 cell monolayers after infection with the virus supernatants, and further characterized by restriction enzyme digestion mapping, PCRTM, or by expression of the gene of interest in virus-infected host cells in a tetracycline-regulatable manner.

The recombinant adenoviruses containing the desired foreign gene as well as the modified tTA (or rtTA) expression cassettes are subjected to at least three rounds of plaque purification.

High-titer stocks of the tetracycline-controlled recombinant adenoviruses are prepared by methods modified from Graham and Prevec, (1991). The CsCl ultracentrifugation-purified adenoviruses contain -103 viral particles per ml as measured by OD at 260 nm (1 OD 26 0 1 x 1012 viral particles per ml). The concentrated viral suspension is desalted by gel filtration through Sephadex G50 to generate a final purified virus stock about 1011 plaque-forming units (pfu) per ml in PBS.

EXAMPLE 8 Preparation of Tetracycline-Responsive RB Adenovirus Vector A replication-deficient adenovirus vectors expressing N-terminal truncated pRB 9 4 protein S. Patent No. 5,496,731) has been used in in vivo animal studies of human cancer WO 98/37091 PCT/US98/03041 99 gene therapy (Xu et al., 1996). Unfortunately, the ratio of viral particles to plaque-forming units of the AdCMVpRB94 virus supernatants increased dramatically with passage, making it difficult for large-scale preparation of high-titer stocks of the AdCMVpRB94 virus for human cancer gene therapy clinical trials. This was probably caused by the super cell growth suppression effects of pRB94 protein on the 293 virus-producing cell line.

The modified tetracycline-responsive mammalian gene expression system has been used in a similar manner as described above to generate a tetracycline-controlled pRB 9 4 -containing adenovirus vector, AdVtTA.RB94, which is designed for delivery of high-dose pRB 94 gene therapy. The entire tetracycline regulation cassette can be inserted into the El region of the adenovirus genome. or the RB 94 expression cassette can be inserted into the El region of the adenovirus genome, while the transcriptional transactivation fusion protein expression cassette is inserted into the E3 region of the adenovirus genome. Over-expression of pRB 94 in tumor cells will cause tumor cell-specific senescence and cell death. The pRB 94 cDNA has a modified optimal initiator context sequence. Expression of the pRB94 protein in transduced human tumor cells by AdVtTA.RB94 can be reversibly turned off and on. The novel AdVtTA.RB94 recombinant adenovirus vector can be propagated efficiently in 293 cells with increased yield and quality.

EXAMPLE 9 Preparation of Tetracycline-Responsive RB/p53 Coexpression Vector As described in Example 6 above, although p53 reconstitution in Saos-2 p53 null tumor cells with either non-inducible (Chen et al., 1990; Li et al., 1996) or inducible system did suppress their neoplastic phenotype, the p53 reconstituted Saos-2 clones with the tetracyclineregulatable promoter were SA-p-gal negative in either presence or absence of tetracycline.

However, when the p53-reconstituted Saos-2 cells were infected with recombinant adenovirus vectors expressing the wild-type pRB 110 in Tc-free medium, the tumor cells with both wild-type p53 and pRB 11 expression displayed more intense SA-p-gal positive staining as compared to tumor cells only expressing pRB The results imply that the mechanisms for tumor suppression by pRB and p53 were different from each other, but expression of pRB and p53 together had synergistic effects on RB-mediated tumor cell senescence.

WO 98/37091 PCT/US98/03041 100 Since co-expression of pRB and p53 has synergistic effects on pRB-mediated, tumorspecific senescence (Example and it has been suggested that altered RB and p53 protein status could be a synergistic prognostic factor in non-small cell lung carcinomas, as well as a subset of other human malignancies, including transitional cell carcinomas of the bladder (Xu, 1995; Xu et al., 1994a; Xu et al., 1996), combination pRB and p53 gene therapy is also contemplated as an alternative strategy to surmount possible tumor suppressor resistance.

Insertion of both the modified tetracycline-responsive transactivator (tTA) expression cassette and the tTA-dependent pRBo 0 expression cassette into the El region of the genome facilitates construction of an adenovirus vector simultaneously expressing two tumor suppressor genes, named AdVtTA.RB110/p53. In this vector, the smaller p53 expression cassette is inserted into the E3 region of the 34 kb master plasmid, pBHGl 1, through ligation reaction. Since attempts to replace both RB and p53 genes in the same cell have never been successful (Wang et al., 1993), the inventors reasoned that adenovirus vectors simultaneously expressing the two tumor suppressor genes should be built in the regulatable gene expression system.

EXAMPLE Construction of Tetracycline-Controlled Retroviral Vectors The kat retrovirus production system produces high titer retrovirus supernatant capable of transducing efficiently hematopoietic cell types refractory to conventional retrovirus transduction (Finer et al., 1994). The kat retrovirus plasmid vector with a hybrid LTR with will be combined with EC1214A (Example 3) to generate a retrovirus with Tc-regulatable expression. Since some success using standard retroviral vectors have been reported in the literature, the Tc-controlled retroviral vector may work better than the Tc-controlled adenoviral vector for transduction of certain cell types, such as hematopoietic stem cells.

WO 98/37091 PCT/US98/03041 101 EXAMPLE 11 Therapeutic Administration of Modified RB Constructs A. Treatment of Human Bladder Cancers in vivo.

The human bladder cancer represents an ideal model for practicing tumor suppressor gene 5 therapy of solid tumors by infusing the instant modified RB protein expression retroviral vectors into the bladder. The original experimental model of human bladder cancer was established by Jones and colleagues (Ahlering el al., 1987). It has been shown that human bladder tumor cells of RT4 cell line established from a superficial papillary tumor, which usually does not metastasize, produced tumors only locally when injected by a 22-gauge catheter into the bladder of female nude mice. In contrast, the EJ bladder carcinoma cells which were originally isolated from a more aggressive human bladder cancer produced invasive tumors in the nude mouse bladders which metastasized to the lung spontaneously. Therefore, this model can be used for treatment of experimental bladder cancer by in vivo gene transfer with retroviral vectors.

Tumor cells from RB minus human bladder carcinoma cell line, 5637 (ATCC HTB9) and RB human bladder carcinoma cell line, SCaBER (ATCC HTB3) will be injected directly into the bladders of female athymic (nu/nu) nude mice (6 to 8 weeks of age) by a catheter as initially reported by Jones and colleagues (Ahlering et al., 1987). Development and progression of the nude mouse bladder tumors will be monitored using a fiber-optical system to which a TV monitor is attached. The experimental tumors will subsequently be treated with retrovirus vectors expressing the modified RB proteins of the present invention.

Supematants with high virus titers will be obtained from tissue culture media of selected clones expressing high level of human modified RB protein and confirmed as free of replicationcompetent virus prior to use. The retroviral vector suspension at high titers ranging from 4 x 104 to greater than 1 x 107 colony-forming unit (cfu)/ml, and more preferably at a titer greater than 1 x 106 cfu/ml will then be infused directly into the mouse bladders via a catheter to treat the tumors. The skilled artisan will understand that such treatments may be repeated as many times as necessary via a catheter inserted into the bladder. The tumor regression following transferring the modified RB gene will be monitored frequently via the fiber-optic system mentioned above.

WO 98/37091 PCT/US98/03041 102 The same procedure as described above may be used for treating the human bladder cancer except that the retroviral vector suspension is infused into a human bladder bearing cancer.

B. in vivo Studies Using an Orthotopic Lung Cancer Model Human large cell lung carcinoma, NCI-H460 (ATCC HTB177) cells which have normal pRB1 0 expression will be injected into the right mainstream bronchus of athymic (nu/nu) nude mice (105 cells per mouse). Three days later the mice will be inoculated endobronchically with supernatant from the modified RB, or wild-type RB retrovirus producer cells daily for three consecutive days. Tumor formation suppression in the group of mice treated with the modified RB retrovirus supernatant, in contrast, to the group which is treated with wild-type RB retrovirus supernatant, will indicate that the modified RB-expressing retrovirus inhibits growth of RB+ non-small cell lung carcinoma (NSCLC) cells, whereas the wild-type RB-expressing retrovirus does not.

C. Treatment of Human Non-Small Cell Lung Cancers in vivo.

Non-small cell lung cancer patients having an endobronchial tumor accessible to a bronchoscope, and also having a bronchial obstruction, will be initially selected for modified RB gene therapy. Treatment will be administered by bronchoscopy under topical or general anesthesia. To begin the procedure, as much gross tumor as possible will be resected endoscopically. A transbronchial aspiration needle (21G) will be passed through the biopsy channel of the bronchoscope. The residual tumor site will then be injected with the appropriate modified RB retroviral vector supernatant, modified RB adenovirus suspension or modified RBexpressing plasmid vector-liposome complexes at a volume of 5 ml to 10 ml. Protamine may be added to a concentration of 5 pg/ml. The injections of therapeutic viral or plasmid supernatant comprising one or more of the vectors will be administered around and within the tumor or tumors and into the submucosa adjacent to the tumor. The injections will be repeated daily for five consecutive days and monthly thereafter. The treatment may be continued as long as there is no tumor progression. After one year the patients will be evaluated to determine whether it is appropriate to continue therapy.

WO 98/37091 PCT/US98/03041 103 In addition, as a precaution, the patients will wear a surgical mask for 24 hours following injection of the viral supernatant. All medical personnel will wear masks routinely during bronchoscopy and injection of the viral supernatant. Anti-tussive will be prescribed as necessary. D. Treatment or Prevention of Human Lung Carcinomas With Liposome- Encapsulated Purified Modified RB Protein In yet another alternative, target tumor or cancer cells will be treated by introducing the instant modified RB proteins into cells in need of such treatment by any known method. For example, liposomes are artificial membrane vesicles that have- been extensively studied for their usefulness as delivery vehicles of drugs, proteins and plasmid vectors both in vitro or in vivo (Mannino et al., 1988). Proteins such as erythrocyte anion transporter (Newton el al.. 1988), superoxide dismutase and catalase (Tanswell et al., 1990), and UV-DNA repair enzyme (Ceccoli et al.. 1989) have been encapsulated at high efficiency with liposome vesicles and delivered into mammalian cells in vitro or in vivo. Further, small-particle aerosols provide a method for the delivery of drugs for treatment of respiratory diseases. For example, it has been reported that drugs can be administered in small-particle aerosols by using liposomes as a vehicle.

Administered via aerosols, the drugs are deposited rather uniformly on the surface of the nasopharynx, the tracheobronchial tree and in the pulmonary area (Knight et al., 1988).

To treat or prevent lung cancers, the therapeutic modified RB proteins will be purified, F for example, from recombinant baculovirus AcMNPV-modified RB infected insect cells by immunoaffinity chromatography or any other convenient source. The modified RB protein will then be mixed with liposomes and incorporated into the liposome vesicles at high efficiency.

The encapsulated modified RB will still be active. Since the aerosol delivery method is mild and well-tolerated by normal volunteers and patients, the modified RB-containing liposomes can be administered to treat patients suffering from lung cancers of any stage and/or to prevent lung cancers in high-risk population. The modified RB protein-containing liposomes may administered by nasal inhalation or by a endotracheal tube via small-particle aerosols at a dose sufficient to suppress abnormal cell proliferation. Aerosolization treatments will be administered to a patient for 30 minutes, three times daily for two weeks, with repetition as needed. The modified RB protein will thereby be delivered throughout the respiratory tract and the pulmonary WO 98/37091 PCT/US98/03041 104 area. The treatment may be continued as long as necessary. After one year, the overall condition of the patient will be evaluated to determine if continued therapy is appropriate.

EXAMPLE 12 Induction of Senescence and Telomerase Inhibition by Reexpression of RB Normal human diploid cells senesce in vitro and in vivo after a limited number of cell divisions. This process, known as cellular senescence, is an underlying cause of aging and a critical barrier for development of human cancers. This Example presents studies that demonstrate that reexpression of functional pRB alone in RB/p53-defective tumor cells via a modified tetracycline-regulated gene expression system resulted in a stable growth arrest at the GO/G1 phase of the cell cycle, preventing tumor cells from entering S phase in response to a variety of mitogenic stimuli. These cells displayed multiple morphological changes consistent with cellular senescence and expressed a senescence-associated P-galactosidase biomarker.

Additionally, telomerase activity, which is believed to be essential for an extended proliferative life-span of neoplastic cells, was abrogated or repressed in the tumor cell lines after induction of pRB (but not p 5 3 expression. Strikingly, when returned to an non-permissive medium for pRB expression, the pRB-induced senescent tumor cells resumed DNA synthesis and attempted to divide. However, most cells died in the process, a phenomenon similar to postsenescent crisis of SV40 T-antigen-transformed human diploid fibroblasts in late passage.

These observations provide direct evidence that overexpression of pRB alone in RB/p53defective tumor cells is sufficient to reverse their immortality and cause a phenotype that is, by all generally accepted criteria, indistinguishable from replicative senescence. The results indicate that pRB may play a causal role in the intrinsic cellular senescence program.

A. Materials and Methods Establishing tumor cell lines with Tc-regulatable pRB expression SThe original multiple-plasmid tetracycline repressor/operator-based regulatory system was improved as described in detail above. All RB-reconstituted tumor cell lines used in this Example were subjected to at least two rounds of subcloning following the initial plasmid transfection and are considered pure clones. The homogeneity of these clones was verified by pRB nuclear staining. In addition, a panel assay (Zhou et al., 1994) was used to ensure stable WO 98/37091 PCT/US98/03041 105 expression of the functional pRB under permissive conditions. The RB-reconstituted tumor cells were all RB" in the presence of 0.5 pg/ml of Tc in culture medium; while the great majority of the cells became RB' at 24 hours after removal of Tc as shown by immunocytochemical staining.

Flow cytometric analysis Single cell suspensions collected at each time point were fixed with paraformaldehyde and ethanol before propidium iodide (PI) (Sigma) staining. All profiles were generated using a FACScan flow cytometer (Becton-Dickinson). The first peak (Ml) contains cells with diploid DNA in GO/G1, the second peak (M3) with twice the PI-fluorescence intensity contains S tetraploid G2/M cells, and the area between the two peaks (M2) represents the total number of cells in S phase (Nicoletti et al., 1991).

SA-P-gal assay The assay was performed essentially as previously described (Dimri et al.. 1995).

Briefly, the cells were fixed in 2% formaldehyde/0.2% glutaraldehyde for 5 min and stained with 5-bromo-4-chloro-3-indolyl P-D-galactoside (X-Gal) at pH 6.0 for 6 hours. The staining solution contained 1 mg/ml X-Gal, 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCI and 2 mM MgCl 2 Telomeric repeat amplification protocol (TRAP) assay I The methodology, according to the technical manual, was modified from the original TRAP assay as described by Kim et al. (Kim et al., 1994). In short, -106 cells grown in a 100-mm Petri dish were harvested and resuspended in 200 ul of ice-cold lysis buffer for 30 min on-ice; followed-by-centrifugation at 100;000 x g for 30 min at 4°C. The supernatant was diluted to 0.5 pg protein/l, of which 2 pl was used for each TRAP assay. The telomerase reaction was carried out at 30 0 C for 30 min, which was followed by a 2-step PCR T M amplification with [y-32P]-labeled TS primer (94°C, 30 s and 60 0 C, 30 s for 33 cycles). The PCR

T

-amplified telomerase extension products were subjected to electrophoresis on a 12.5% polyacrylamide gel.

WO 98/37091 PCT/US98/03041 106 B. Results pRB-mediated irreversible growth cessation of tumor cells Using the modified tetracycline (Tc)-regulatable gene expression system as described in detail above, dozens of long-term stable tumor cell clones were established, in which expression of the wild-type pRB can be reversibly turned on and off without significant leakage. The RBreconstituted tumor cell clones were obtained, respectively, from the breast carcinoma cell line, MDA-MB-468, the osteosarcoma cell line Saos-2, and the bladder carcinoma cell line, 5637.

These tumor cell lines were chosen as host cells since they were known to contain both RB and p53 gene mutations (Wang et al., 1993; Chen et al., 1990; Berry et al., 1996; Masuda et al., 1987).

As measured by western blotting, pRB protein induced in the tumor cells reached the highest level about 24 hours after removal of tetracycline from the cell culture medium, and then became completely dephosphorylated within 24 to 40 hours. The effects of induction of pRB expression on tumor cell growth were subsequently examined in representative clones by measuring growth curves and (3H) thymidine incorporation (Xu et al., 1994b), and by flow cytometric analysis (Nicoletti et al., 1991). Cell growth and DNA synthesis of all the long-term tumor cell clones studied ceased 24 to 48 hours after pRB expression was induced (FIG. 3A, FIG. 3B and FIG. 3C). The great majority of the tumor cells were arrested at GO/GI phase of the cell cycle.

After a 4-day induction of pRB expression in Tc-free medium, the growth cessation of the tumor cells was irreversible by stimulation with a variety of mitogens, such as serum growth factors, phytohemagglutinin (PHA) and concanavalin A (Con This was determined by continuous flat growth curves as shown in FIG. 3A, FIG. 3B and FIG. 3C and failure of the tumor cells to incorporate 3 H) thymidine in response to mitogenic stimulation. In the meantime, the tumor cells displayed striking morphological changes consistent with cellular senescence, including cell enlargement, flattening, and lower nucleocytoplasmic ratio than cycling cells.

Furthermore, as measured by DNA fragmentation assay, a small amount of lower molecular weight DNAs were often observed in DNA samples prepared from RB-reconstituted Saos-2 tumor cells grown in non-permissive but not permissive conditions for pRB expression.

WO 98/37091 PCT/US98/03041 107 This finding suggested a low level of spontaneous apoptosis of the RB-defective tumor cell culture, which was inhibited by induction of pRB expression. In addition, switching on pRB expression in the RB-reconstituted 5637 and MDA-MB-468 tumor cell lines also inhibited IFN-y-induced apoptotic cell death.

Expression ofsenescence-associated P-galactosidase A biomarker that identifies senescent human cells in culture and in aging skin in vivo has recently been reported. This marker, termed senescence-associated p-galactosidase (SA-P-gal), is expressed by senescent, but not pre-senescent fibroblasts. SA-P-gal was also absent from immortal cells but was induced by genetic manipulations that reversed immortality (Dimri et al., i 1995). Young (early passage) human WI-38 fibroblasts were SA-p-gal negative, whereas the senescent (at population doubling level greater than 52) WI-38 cells were strongly SA-P-gal positive, which provided a valid control for the SA-P-gal assay. The Tc-responsive RBreconstituted tumor cell clones were totally SA-P-gal negative in the presence of Tc in RB" status), and the majority of the tumor cells became SA-P-gal positive after induction of pRB expression for four to five days in Tc-free medium. The detection of this senescence-associated biomarker in the tumor cells was coincident with the irreversible growth cessation of the tumor cell populations (FIG. 3A, FIG. 3B and FIG. 3C). The intensity of the SA-P-gal staining of the induced RB tumor cells, however, was variable depending on the tumor cell types.

Reexpression ofpRB (but not p53) in tumor cells inhibited telomerase activity k Since telomerase has recently emerged as an attractive candidate for a regulator in cellular senescence (Linskens et al., 1995; Klingelhutz et al., 1996), the effects of pRB and p53 replacement on the telomerase activity of the host tumor cells were determined. In this connection, several long-term stable tumor cell clones with Tc-regulatable wild-type p53 expression from the osteosarcoma cell line, Saos-2 were established. A telomeric repeat amplification protocol (TRAP) assay as recently described (Kim et al., 1994) was used to measure telomerase activity in tumor cells before and after induction of pRB (or p5 3 expression.

Prior to induction of pRB expression, the RB-reconstituted tumor cell clones from all three RB/p53-defective tumor types examined were positive for telomerase activity, whereas the relative telomerase activity was -15 to >100 times lower in the tumor cells after turning on the WO 98/37091 PCT/US98/03041 108 pRB expression as estimated by densitometry of the digitized image. In fact, the telomerase activity was nearly non-detectable in the pRB-expressing MDA-MB-468 and Saos-2 tumor cells.

In contrast, although induction of wild-type p53 expression in Saos-2 did result in growth arrest of the RB/p53""" tumor cells, the p53-reconstituted Saos-2 tumor clones persistently exhibited positive telomerase activity, which was not affected by their p53 status. Thus the differences in telomerase activity cannot be explained simply as a difference in cell proliferation.

Postsenescent crisis of pRB-induced senescent tumor cells after withdrawal ofpRB The pRB-induced tumor cell senescence was stringently dependent on the continued expression of the functional pRB. As shown above, after induction of pRB expression in Tc-free medium for four or more days, the RB-reconstituted MDA-MB-468, Saos-2. and 5637 tumor cells became senescent. When these tumor cells returned to an non-permissive medium for pRB expression, however, a large number of tumor cells were observed that lost cell-cell adherence, detached from the Petri dishes and died. To further characterize this phenomenon, a combined method was employed involving pRB immunocytochemical staining and H) thymidine in situ labeling of the tumor cells.

It was found that after adding 0.5 upg/ml of Tc back to the RB-reconstituted Saos-2 tumor cell cultures that had been maintained in Tc-free medium for 4 to 5 days, nearly all tumor cells were depleted of the exogenous pRB and became RB- at day 6. Subsequently, at day 9 to 10, the tumor cells resumed DNA synthesis, the majority of which however had strikingly aberrant nuclei. They attempted to divide but most died in the process. These tumor cells displayed a phenotype, showing remarkable similarity to postsenescent crisis of the T-antigen-transformed human cells in late passage (Stein, 1985).

In summary, reexpression of functional pRB in RB-defective tumor cells induced growth cessation concurrently with inhibition of telomerase activity. The tumor cells irreversibly lost mitogen responsiveness, entering a viable G1-arrested state. They also exhibited pRB-dependent SA-P-gal positivity (a senescence-associated biomarker) and resistance to apoptotic cell death.

Of note, replacement of either wild-type pRB or p53 in the RB/p53nul Saos-2 was able to block tumor cell growth at the population level, but only pRB induced inhibition of telomerase.

Furthermore, withdrawal of pRB in pRB-induced senescent tumor cells led to a crisis-like WO 98/37091 PCTIUS98/03041 109 phenotype. These observations, taken together, suggest pRB is causally involved in the cellular senescence program. These results are the first direct evidence that overexpression of pRB alone in a variety of RB-defective tumor cells was sufficient to reverse their immortality and cause bona fide replicative senescence. Since all three RB-defective tumor cll- lines examined also have p53 mutations, the pRB-mediated tumor cell senescence apparently do not require wild-type p53 function.

Thus a new link between pRB and telomerase is shown. It is demonstrated, by a telomeric repeat amplification protocol (TRAP) assay, that reexpression of pRB in RB-defective tumor cells inhibits telomerase activity. Because of the high sensitivity of the polymerase chain reaction (PCR

TM

)-based TRAP assay, which detects the enzyme activity in a very small number of telomerase positive cells, and the difficulty in obtaining absolutely pure RB-reconstituted cell clones, the effectiveness of pRB reexpression on inhibition of telomerase activity in RB-defective tumor cells was likely even greater than it had been detected by the in vitro assay.

It is also noteworthy that, when maintained in non-permissive conditions for pRB (or p 5 3 expression, the pRB-reconstituted Saos-2 clone apparently had much lower telomerase activity than the p53-reconstituted Saos-2 clone. The difference implies that, even before switching-on of the pRB expression in Tc-free medium, there must be low baseline expression of pRB from the Tc-responsive promoter in Saos-2 cells (Gossen and Bujard, 1995). The leakiness of pRB in pRB-reconstituted tumor cells under non-permissive conditions is below the S immunodetection threshold for pRB protein (Xu et al., 1991b), but it might be sufficient to inhibit the most telomerase activity. Since the tumor cells lacking telomerase activity likely resume telomere decline, this would eventually trigger the intrinsic cellular senescence program if it remains intact in the tumor cells.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the WO 98/37091 PCT/US98/03041 110 compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Page(s) 234 240are claims pages They appear after the sequence listing(s) WO 98/37091 PCT/US98/03041 121 SEOUENCE LISTING SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Board of Regents, The University of Texas System STREET: 201 W. 7th Street CITY: Austin STATE: Texas COUNTRY: USA POSTAL CODE (ZIP): 78701 TELEPHONE: (512)418-3000 TELEFAX: (512)474-7577 (ii) TITLE OF INVENTION: MODIFIED RETINOBLASTOMA TUMOR SUPPRESSOR

PROTEINS

(iii) NUMBER OF SEQUENCES: 51 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/038,118 FILING DATE: 02-FEB-1997 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 3555 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2790 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CGCGTC ATG CCG CCC AAA ACC CCC CGA AAA ACG GCC GCC ACC GCC GCC Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala 1 5 GCT GCC GCC GCG GAA CCC CCG GCA CCG CCG CCG CCG CCC CCT CCT GAG Ala Ala Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Glu 20 25 SUBSTITUTE SHEET (RULE 26) WO 98/37091 122 GAG GAC CCA GAG CAG GAC AGC GGC CCG GAG GAC CTG CCT CTC Glu Asp Pro Glu Gin Asp Ser Gly Pro Glu Asp Leu Pro Leu PCT/US98/03041 GTC AGG Val Arg 144 CTT GAG TTT Leu Glu Phe AAA TTA AAG Lys Leu Lys GAA ACA GAA GAA CCT GAT TTT ACT GCA Glu Thr Glu Glu Pro Asp Phe Thr Ala TTA TGT CAG Leu Cys Gin TTA ACT TGG Leu Thr Trp 192 ATA CCA GAT CAT Ile Pro Asp His GTC AGA Val Arg 70 GAG AGA GCT Glu Arg Ala

TGG

Trp 240 GAG AAA Glu Lys GTT TCA TCT GTG Val Ser Ser Val GGA GTA TTG GGA Gly Val Leu Gly TAT ATT CAA AAG Tyr Ile Gin Lys

AAA

Lys AAG GAA CTG TGG Lys Glu Leu Trp ATC TGT ATC TTT Ile Cys Ile Phe GCA GCA GTT GAC Ala Ala Val Asp GAT GAG ATG TCG Asp Glu Met Ser ACT TTT ACT GAG Thr Phe Thr Glu

CTA

Leu 120 CAG AAA AAC ATA Gin Lys Asn Ile GAA ATC Glu Ile 125 AGT GTC CAT Ser Val His AAA GTT GAT Lys Val Asp 145

AAA

Lys 130 TTC TTT AAC TTA CTA AAA GAA ATT GAT Phe Phe Asn Leu Leu Lys Glu Ile Asp 135 ACC AGT ACC Thr Ser Thr 140 GAT GTA TTG Asp Val Leu AAT GCT ATG TCA Asn Ala Met Ser

AGA

Arg 150 CTG TTG AAG AAG Leu Leu Lys Lys

TAT

Tyr 155 TTT GCA Phe Ala 160 CTC TTC AGC AAA Leu Phe Ser Lys GAA AGG ACA TGT Glu Arg Thr Cys CTT ATA TAT TTG Leu Ile Tyr Leu

ACA

Thr 175 CAA CCC AGC AGT Gin Pro Ser Ser

TCG

Ser 180 ATA TCT ACT GAA Ile Ser Thr Glu

ATA

Ile 185 AAT TCT GCA TTG Asn Ser Ala Leu CTA AAA.GTT TCT Leu Lys Val Ser

TGG

Trp 195 ATC ACA TTT TTA Ile Thr Phe Leu GCT AAA GGG GAA Ala Lys Gly Glu GTA TTA Val Leu 205 CAA ATG GAA Gin Met Glu CTT GAC TAT Leu Asp Tyr 225

GAT

Asp 210 GAT CTG GTG ATT Asp Leu Val Ile TTT CAG TTA ATG Phe Gin Leu Met CTA TGT GTC Leu Cys Val 220 AAA GAA CCA Lys Glu Pro TTT ATT AAA CTC Phe Ile Lys Leu CCT CCC ATG Pro Pro Met TTG CTC Leu Leu 235 TAT AAA Tyr Lys 240 ACA GCT GTT Thr Ala Val ATA CCC Ile Pro 245 ATT AAT GGT TCA Ile Asn Gly Ser CCT CGA ACA CCC AGG Pro Arg Thr Pro Arg 250 SUBSTITUTE SHEET (RULE 26) PCTIUS98/03041 WO 98/37091 CGA GGT CAG AAC AGG AGT GCA CGG ATA GCA AAA Arg 255 Gly Gin Asn Arg Ala Arg Ile Ala CAA CTA GAA AAT GAT Gin Leu Giu Asn Asp 270 GAA TGT AAT ATA GAT ACA AGA ATT ATT Thr Arg Ile Ilie GTT CTC TGT AAA Val Leu Cys Lys GAA CAT GiU His 280 864 Giu Cys Asn Ile Asp 285 GAG GTG AAA Giu Val Lys CTT GGA CTT Leu Giy Leu 305

AAT

Asn 290 GTT TAT TTC AAA Val Tyr Phe Lys TTT ATA CCT TTT Phe Ile Pro Phe ATG AAT TCT Met Asn Ser 300 AAT CTT TCT Asn Leu Ser GTA ACA TCT AAT Val Thr Ser Asn CTT CCA GAG GTT Leu Pro Giu Val

GAA

Giu 315 960 AAA CGA Lys Arg 320 TAC GAA GAA ATT Tyr Giu Giu Ile CTT AAA AAT AAA Leu Lys Asn Lys

GAT

Asp 330 CTA GAT GCA AGA Leu Asp Ala Arg

TTA

Leu 335 TTT TTG GAT CAT Phe Leu Asp His

GAT

Asp 340 AAA ACT CTT CAG Lys Thr Leu Gin GAT TCT ATA GAC Asp Ser le Asp 1008 1056 1104 TTT GAA ACA CAG Phe Giu Thr Gin ACA CCA CGA AAA Thr Pro Arg Lys

AGT

Ser 360 AAC CTT GAT GAA Asn Leu Asp Giu GAG GTG Giu Val 365 AAT GTA ATT Asn Val Ilie CAA CAA TTA Gin Gin Leu 385 AAT CTG ATT Asn Leu Ile 400 CCA CAC ACT CCA Pro His Thr Pro AGG ACT GTT ATG Arg Thr Val Met AAC ACT ATC Asn Thr Ile 380 CCT TCA GIAA Pro Ser Giu ATG ATG ATT TTA Met Met Ile Leu TCA GCA AGT GAT Ser Ala Ser Asp TGC ACA GTG AAT Cys Thr Val Asn 410

CAA

Gin 395 1152 1200 1248 TCC TAT TTT AAC AAC Ser Tyr Phe Asn Asn 405 CCA AAA GAA AGT Pro Lys Giu Ser ATA CTG AAA AGA GTG Ile Leu Lys Arg Val GAT ATA GGA TAC Asp Ile Gly Tyr TTT AAA GAG AAA Phe Lys Giu Lys

TTT

Phe 430 1296 1344 GCT AAA GCT GTG Ala Lys Ala Val CAG GGT TGT GTC Gin Gly Cys Val ATT GGA TCA CAG CGA TAC Ile Gly Ser Gin Arg Tyr 445 AAA CTT GGA GTT CGC TTG TAT TAC Lys Leu Gly Val Arg Leu Tyr Tyr 450 GTA ATG GAA TCC Val Met Giu Ser ATG CTT AAA Met Leu Lys 460 CTT CTG AAT Leu Leu Asn 1392 TCA GAA GAA GAA Ser Giu Giu GiU 465.

CGA TTA TCC Arg Leu Ser ATT CAA AAT TTT AGC AAA Ile Gin Asn Phe Ser Lys 470 475 1440 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PTU9/34 PCTIUS98/03041 GAC AAC Asp Asn 480 ATT TTT CAT ATG Ile Phe His Met

TCT

Ser 485 TTA TTG GCG TGC GCT CTT GAG GTT GTA Leu Leu Ala Cys Ala Leu Glu Val Vai 490 1488

ATG

Met 495 GCC ACA TAT AGC Ala Thr Tyr Ser AGT ACA TCT CAG Ser Thr Ser Gin CTT GAT TCT GGA Leu Asp Ser Giy 1536 1584 GAT TTG TCT TTC Asp Leu Ser Phe CCA TGG Pro Trp 515 ATT CTG AAT Ile Leu Asn CTT AAT TTA AAA Leu Asn Leu Lys GCC TTT Ala Phe 525 GAT TTT TAC Asp Phe Tyr ACA AGA GAA Thr Arg Giu 545

AAA

Lys 530 GTG ATC GAA AGT Val Ile Glu Ser

TTT

Phe 535 ATC AAA GC.A GAA Ile Lys Ala Giu GGC AAC TTG Gly Asn Leu 540 CGA ATC ATG Arg Ile Met 1632 1680 ATG ATA AAA CAT Met Ile Lys His

TTA

Leu 550 GAA CGA TGT GAA Giu Arg Cys Glu GAA TCC Glu Ser 560 CTT GCA TGG CTC Leu Ala Trp Leu

TCA

Ser 565 GAT TCA CCT TTA Asp Ser Pro Leu

TTT

Phe 570 GAT CTT ATT AAA Asp Leu Ile Lys 1728 TCA AAG GAC CGA Ser Lys Asp Arg GGA CCA ACT GAT CAC CTT GAA TCT GCT Gly Pro Thr Asp His Leu Giu Ser Ala 585 1776 CCT CTT AAT CTT Pro Leu Asn Leu CTC CAG AAT AAT Leu Gin Asn Asn ACT GCA GCA GAT Thr Ala Ala Asp ATG TAT Met Tyr 605 1824 CTT TCT CCT Leu Ser Pro AAT TCT ACT Asn Ser Thr 625

GTA

Val 610 AGA TCT CCA AAG Arg Ser Pro Lys AA.A GGT TCA ACT Lys Gly Ser Thr ACG CGT GTA Thr Arg Val 620 TTC CAG ACC Phe Gin Thr 1872 1920 GCA AAT GCA GAG Ala Asn Ala Glu

ACA

Thr 630 CAA GCA ACC TCA Gin Ala Thr Ser CAG AAG 4Em Lys 640 CCA TTG AAA TCT Pro Leu Lys Ser

ACC

Thr 645 TCT CTT TCA CTG Ser Leu Ser Leu TAT AAA AAA GTG Tyr Lys Lys Val

TAT

Tyr 655 CGG CTA GCC TAT Arg Leu Ala Tyr CGG CTA AAT ACA Arg Leu Asn Thr TGT GAA CGC CTT Cys Giu Arg Leu 1968 2016 2064 2112 TCT GAG CAC CCA Ser Glu His Pro

GAA

Giu 675 TTA GAA CAT ATC Leu Giu His Ile

ATC

Ile 680 TGG ACC CTT TTC Trp Thr Leu Phe CAG CAC Gin His 685 ACC CTG CAG Thr Leu.Gin.

GAG TAT GAA CTC ATG AGA GAC AGG CAT Giu Tyr Giu Leu Met Arg Asp Arg His 695 TTG GAC CAA Leu Asp Gin 700 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98103041 ATT ATG ATG Ile Met Met 705 TGT TCC ATG TAT Cys Ser Met Tyr ATA TGC AAA GTG Ile Cys Lys Val AAG AAT ATA GAC Lys Asn Ile Asp 715 CTT CCT CAT GCT Leu Pro His Ala 2160 CTT AAA.

Leu Lys 720 TTC AAA ATC ATT Phe Lys Ile Ile

GTA

Val 725 ACA GCA TAC AAG Thr Ala Tyr Lys

GAT

Asp 730 CAG GAG ACA TTC Gin Giu Thr Phe CGT GTT TTG ATC Arg Val Leu Ile GAA GAG GAG TAT Giu Giu Giu Tyr 2208 2256 2304 TCT ATT ATA GTA Ser Ile Ile Val TAT AAC TCG GTC Tyr Asn Ser Val ATG CAG AGA CTG Met Gin Arg Leu AA.A ACA Lys Thr 765 AAT ATT TTG Asn Ilie Leu CCT CAC ATT Pro His Ilie 785 TAT GCT TCC ACC Tyr Ala Ser Thr

AGG

Arg 775 CCC CCT ACC TTG Pro Pro Thr Leu TCA CCA ATA Ser Pro le 780 CCC TTA CGG Pro Leu Arg 2352 2400 CCT CGA AGC CCT Pro Arg Ser Pro AAG TTT CCT AGT Lys Phe Pro Ser

TCA

Ser 795 ATT CCT Ile Pro 800 GGA GGG AAC ATC Gly Gly Asn Ile ATT TCA CCC CTG Ile Ser Pro Leu

AAG

Lys 810 AGT CCA TAT AAA Ser Pro Tyr Lys

ATT

Ile 815 TCA GAA GGT CTG Ser Giu Gly Leu ACA CCA ACA AAA Thr Pro Thr Lys ACT CCA AGA TCA Thr Pro Arg Ser

AGA

Arg 830 2448 2496 2544 ATC TTA GTA TCA Ile Leu Val Ser GGT GAA TCA TTC Giy Giu Ser Phe

GGG

Gly 840 ACT TCT GAG AAG Thr Ser Giu Lys TTC CAG Phe Gin 845 AAA ATA AAT Lys Ile Asn GCT GAA GGA Ala Giu Gly 865 ATT GAA GGA Ile Giu Gly 880 ATG GTA TGT AAC Met Vai Cys Asn

AGC

Ser 855 GAC CGT GTG CTC Asp Arg Val Leu AAA AGA AGT Lys Arg Ser 860 CGC TTT GAT Arg Phe Asp AGC AAC CCT CCT Ser Asn Pro Pro

AAA

Lys 870 CCA CTG AAA AAA Pro Leu Lys Lys 2592 2640 2688 TCA GAT GAA Ser Asp Giu

GCA

Ala 885 GAT GGA AGT AAA Asp Gly Ser Lys CTC CCA GGA GAG Leu Pro Gly Giu

TCC

Ser 895 AAA TTT CAG CAG Lys Phe Gin Gin CTG GCA GAA ATG Leu Ala Glu Met TCT ACT CGA ACA Ser Thr Arg Thr 2736 ATG CAA AAG CAG Met Gin Lys Gin ATG AAT GAT AGC Met Asn Asp Ser ATG GAT ACC TCA AAC Met Asp Thr Ser Asn 920 AAG GAA Lys Giu 925 2784 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTIUS98/03041 126 GAG AAA TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT GGATTCATTG Glu Lys 2840

TCTCTCACAG

TTCAGCTCTT

ACTTGAAATG

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

ATGTGACTGT

TTTGTGGATA

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA

TACTATCATA

TTTCTTCATC

ATAACTTTCC

TAAAATGTGC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

CAGGTTCTGT

AGATGCAATT

AAGATTGAAA

TCCAAAGTAA

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

TTTTAAATGA

TTATGGCCAC

GTTTGGGTGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAA.A

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

CCTCAGAATG

GGATTATTGA.

ATTTAATATC

TTCCTAAGCC

AATCCTGCCA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA.

AATCTGATAT

AGCAAAGTAT

TAAAAGAACT

TAGTC

2900 2960 3020 3080 3140 3200 3260 3320 3380 3440 3500 3555 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 928 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr 1 5 10 Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro 25 Pro Glu Gln Asp Ser Gly Pro Glu Asp Leu 40 Phe Glu Glu Thr Glu Glu Pro Asp Phe Thr 55 Lys Ile Pro Asp His Val Arg Glu Arg Ala 70 Pro Pro Pro Leu Ala Leu Trp Leu 75 Ala Ala Ala Ala Pro Glu Glu Asp Val Arg Leu Glu Cys Gln Lys Leu Thr Trp Glu Lys SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/XJS98/03041 127 Val Ser Ser Val Asp Giy Val Leu Gly Gly Tyr Ile Gin Lys Lys Lys 90 Glu Leu Trp Gly Ile Cys Ile Phe Ile Ala Ala Val Asp LeuAsp Giu 100 05 110' Met Ser Phe Thr Phe Thr Giu Leu Gin Lys Asn Ile Giu le Ser Val 115 120 125 His Lys Phe Phe Asn Leu Leu Lys Glu Ile Asp Thr Ser Thr Lys Val 130 135 140 Asp Asn Ala Met Ser Arg Leu Leu Lys Lys Tyr Asp Val Leu Phe Ala 145 1S0 155 160 Leu Phe Ser Lys Leu Glu Arg Thr Cys Giu Leu Ile Tyr Leu Thr Gin 165 170 175 Pro Ser Ser Ser Ilie Ser Thr Giu Ile Asn Ser Ala Leu Val Leu Lys 180 185 190 Val Ser Trp Ile Thr Phe Leu Leu Ala Lys Giy Glu Val Leu Gin Met 195 200 205 Glu Asp Asp Leu Val Ile Ser Phe Gin Leu Met Leu Cys Val Leu Asp 210 2.15 220 Tyr Phe Ile Lys Leu Ser Pro Pro Met Leu Leu Lys Giu Pro Tyr Lys 225 230 235 240 Thr Ala Val Ile Pro Ile Asn Gly Ser Pro Arg Thr Pro Arg Arg Gly 245 250 255 Gin Asn Arg Ser Ala Arg Ile Ala Lys Gin Leu Glu Asn Asp Thr Arg 260 265 270 Ile Ile Giu Val Leu Cys Lys Glu His Glu Cys Asn Ile Asp Glu Val 275 280 285 Lys Asn Val Tyr Phe Lys Asn Phe Ile Pro Phe Met Asn Ser Leu Gly 290 295 300 Leu Val Thr Ser Asn Gly Leu Pro Giu Val Glu Asn Leu Ser Lys Arg 305 310 315 320 Tyr Giu Glu Ile Tyr Leu Lys Asn Lys Asp Leu Asp Ala Arg Leu Phe 325 330 335 Leu Asp His Asp Lys Thr Leu Gin Thr Asp Ser Ile Asp Ser Phe Giu 340 345 350 Thr Gin Arg Thr Pro Arg Lys Ser Asn Leu Asp Glu Glu Val Asn Val 355 360 365 Ile Pro Pro His Thr Pro Val Arg Thr Vai Met Asn Thr Ile Gin Gin 370 375 380 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/03041 Leu 385 Ile Lys Ala Gly Glu 465 Ile Thr Ser Tyr Glu 545 Leu Lys Asn Pro Thr 625 Pro Leu Met Met Ile Leu Ser Ala Ser Asp Ser Arg Val Val 450 Giu Phe Tyr Phe Lys 530 Met Ala Asp Leu Val1 610 Al a Leu Al a Tyr Val Gly 435 Arg Arg His Ser Pro 515 Val Ile Trp Arg Pro 595 Arg Asn Lys Tyr Thr Tyr Glu 440 Val Asn Al a Gin Val 520 Ile Arg Pro Asp His 600 Lys Al a Ser Thr Val Ile 425 Ile Met Phe Cys Asn 505 Leu Lys Cys Leu His 585 Thr Gly Thr Leu Leu 665 Asn 410 Phe Gly Glu Ser Ala 490 Leu Asn Ala Giu Phe 570 Leu Ala Ser Ser Phe 650 Cys Gln 395 Pro Lys Ser Ser Lys 475 Leu Asp Leu Giu His 555 Asp Glu Ala Thr Ala 635 Tyr Glu Pro Ser Giu Asn Ser Phe 430 Tyr Lys Asn Val Thr 510 Phe Leu Met Lys Cys 590 Tyr Val Thr Val Leu 670 Leu 400 Leu Lys Leu Glu Asn 480 Ala Leu Phe Arg Ser 560 Ser Leu Ser Ser Lys 640 Arg Glu SUBSTITUTE SHEET (RULE 26) WO 98/37091 His Gin ~1 Met 705 Phe Glu Ile Leu Ile 785 Gly Glu Val Asn Gly 865 Gly Phe Lys Pro Asn 690 Cys Lys Thr Val Gin 770 Pro Gly Gly Ser Gin 850 Ser Ser Gin Gin Glu 675 Glu Ser Ile Phe Phe 755 Tyr Arg Asn Leu Ile 835 Met Asn Asp Gin Lys 915 PCT/US98/03041 129 Leu Glu His Ile Ile Trp Thr Leu Phe Gin His Thr Leu 680 685 Tyr Glu Leu Met Arg Asp Arg His Leu Asp Gin Ile Met 695 700 Met Tyr Gly Ile Cys Lys Val Lys Asn Ile Asp Leu Lys 710 715 720 Ile Val Thr Ala Tyr Lys Asp Leu Pro His Ala Val Gin 725 730 735 Lys Arg Val Leu Ile Lys Glu Glu Glu Tyr Asp Ser Ile 740 745 750 Tyr Asn Ser Val Phe Met Gin Arg Leu Lys Thr Asn Ile 760 765 Ala Ser Thr Arg Pro Pro Thr Leu Ser Pro Ile Pro His 775 780 Ser Pro Tyr Lys Phe Pro Ser Ser Pro Leu Arg Ile Pro 790 795 800 Ile Tyr Ile Ser Pro Leu Lys Ser Pro Tyr Lys Ile Ser 805 810 815 Pro Thr Pro Thr Lys Met Thr Pro Arg Ser Arg Ile Leu 820 825 830 Gly Glu Ser Phe Gly Thr Ser Glu Lys Phe Gin Lys Ile 840 845 Val Cys Asn Ser Asp Arg Val Leu Lys Arg Ser Ala Glu 855 860 Pro Pro Lys Pro Leu Lys Lys Leu Arg Phe Asp Ile Glu 870 875 880 Glu Ala Asp Gly Ser Lys His Leu Pro Gly Glu Ser Lys 885 890 895 Lys Leu Ala Glu Met Thr Ser Thr Arg Thr Arg Met Gin 900 905 910 Met Asn Asp Ser Met Asp Thr Ser Asn Lys Glu Glu Lys 920 925 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 3218 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 130 (ix) FEATURE: NAI4E/KEY: CDS LOCATION:7. .2454 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCCGTC ATG TCG TTC ACT TTT ACT GAG CTA CAG AAA AAC ATA GAA ATC Met Ser Phe Thr Phe Thr Giu Leu Gin Lys Asn Ile Glu Ile

AGT

Ser GTC CAT AAA TTC TTT AAC TTA CTA Val His Lys Phe Phe Asn Leu Leu AAA GAA Lys Giu 25 ATT GAT ACC AGT ACC Ile Asp Thr Ser Thr AAA GTT GAT AAT GCT ATG TCA AGA CTG TTG AAG AAG TAT GAT GTA TTG Lys Vai Asp Asn Aia Met Ser Arg Leu Leu Lys Lys Tyr Asp Val Leu TTT GCA CTC TTC AGC AAA TTG GAA Phe Ala Leu Phe Ser Lys Leu Giu ACA TGT GAA CTT Thr Cys Giu Leu ATA TAT TTG Ile Tyr Leu GCA TTG GTG Ala Leu Val ACA CAA CCC Thr Gin Pro AGC AGT TCG ATA Ser Ser Ser Ile

TCT

Ser ACT GAA ATA AAT Thr Giu Ile Asn CTA AAA Leu Lys GTT TCT TGG ATC Val Ser Trp Ile TTT TTA TTA OCT Phe Leu Leu Ala GGG GAA GTA TTA Gly Giu Val Leu 288

CAA

Gin ATG GAA GAT GAT Met Giu Asp Asp GTG ATT TCA TTT CAG TTA ATG CTA TGT Vai Ile Ser Phe Gin Leu Met Leu Cys 105 CTT GAC TAT TTT Leu Asp Tyr Phe AAA CTC TCA CCT Lys Leu Ser Pro

CCC

Pro 120 ATG TTG CTC AAA Met Leu Leu Lys GAA CCA Oiu Pro 125 TAT AAA ACA Tyr Lys Thr CGA GOT CAG Arg Giy Gin 145 OTT ATA CCC ATT Val Ile Pro Ile GOT TCA CCT CGA Gly Ser Pro Arg AAC AGO AGT GCA Asn Arg Ser Ala ATA GCA AAA CAA Ile Ala Lys Gin

CTA

Leu 155 ACA CCC AGO Thr Pro Arg 14 0 GAA AAT GAT Giu Asn Asp AAT ATA GAT Asn Ile Asp ACA AGA Thr Arg 160 ATT ATT GAA OTT Ile Ile Giu Val TGT AAA GAA CAT Cys Lys Glu His GAA TOT Oiu Cys 170

GAG

Giu 175 OTO AAA AAT OTT Val Lys Asn Val TTC AAA AAT Phe Lys Asn TTT ATA Phe Ile 185 CCA GAG Pro Oiu 200 CCT TTT ATG AAT Pro Phe Met Asn CTT GGA CTT GTA Leu Giy Leu .Vai TCT AAT OGA CTT Ser Asn Gly Leu GTT OAA AAT Val Giu Asn CTT TCT Leu Ser 205 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/0304 1 AAA CGA TAC Lys Arg Tyr TTA TTT TTG Leu Phe Leu 225

GAA

Glu 210 GAA ATT TAT CTT AAA AAT AAA GAT CTA Glu Ile Tyr Leu Lys Asn Lys Asp Leu 215 GAT GCA AGA Asp Ala Arg 220 ATA GAC AGT Ile Asp Ser GAT CAT GAT AAA Asp His Asp Lys CTT CAG ACT GAT Leu Gin Thr Asp) TTT GAA ACA CAG AGA ACA CCA CGA AAA AGT Phe Glu Thr Gin Arg Thr Pro Arg Lys Ser 240 245 AAT GTA ATT CCT CCA CAC ACT CCA GTT AGG Asn Val Ilie Pro Pro His Thr Pro Val Arg AAC CTT Asn Leu 250 GAT GAA GAG GTG Asp Giu Giu Val

ACT

Thr 265 GTT ATG AAC ACT Val Met Asn Thr CAA CAA TTA ATG Gin Gin Leu Met

ATG

Met 275 ATT TTA AAT TCA le Leu Asn Ser

GCA

Al a 280 AGT GAT CAA CCT Ser Asp Gin Pro TCA GAA Ser Giu 285 AAT CTG ATT Asn Leu Ilie ATA CTG AAA Ile Leu Lys 305 TAT TTT AAC AAC Tyr Phe Asn Asn ACA GTG AAT CCA Thr Val Asn Pro AAA GAA AGT Lys Giu Ser 300 GAG AAA TTT Giu Lys Phe AGA GTG AAG GAT Arg Val Lys Asp GGA TAC ATC TTT" Gly Tyr Ile Phe

AAA

Lys 315 GCT AAA Ala Lys 320 GCT GTG GGA CAG Ala Val Gly Gin TGT GTC GAA Cys Val Glu ATT GGA Ilie Gly 330 TCA CAG CGA TAC Ser Gin Arg Tyr

AAA

Lys 335 CTT GGA GTT CGC Leu Gly Val Arg TAT TAC CGA GTA Tyr Tyr Arg Vai

ATG

Met 345 GAA TCC ATG CTT Glu Ser Met Leu

AAA

Lys 350 1008 1056 1104 TCA GAA GAA GAA Ser Giu Glu Glu

CGA

Arg 355 TTA TCC ATT CAA Leu Ser Ile Gin TTT AGC AAA CTT Phe Ser Lys Leu CTG AAT Leu Asn 365 GAC AAC ATT Asp Asn Ile ATG GCC ACA Met Ala Thr 385

TTT

Phe 370 CAT ATG TCT TTA His Met Ser Leu GCG TGC GCT CTT Ala Cys Ala Leu GAG GTT GTA Giu Val Val 380 TCT GGA ACA Ser Gly Thr 1152 1200 TAT AGC AGA AGT Tyr Ser Arg Ser

ACA

Thr 390 TCT CAG AAT CTT Ser Gin Asn Leu GAT TTG Asp Leu 400 TCT TTC CCA TGG Ser Phe Pro Trp

ATT

Ile 405 CTG AAT GTG CTT Leu Asn Val Leu AAT TTA Asn Leu 410 AAA GCC TTT Lys Ala Phe 1248 TTT TAC AAA GTG ATC GAA AGT TTT ATC AAA GCA GAA GGC AAC Phe Tyr Lys Val Ile Glu Ser Phe Ile Lys Ala Glu Gly Asn 1296 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 ACA AGA GAA ATG Thr Arg Glu Met AAA CAT TTA GAA CGA TGT Lys His Leu Glu Arg Cys 440 GAA CAT CGA ATC ATG 1344 Glu His Arg lie Met 445 GAA TCC CTT Glu Ser Leu CAA TCA AAG Gin Ser Lys 465 TGG CTC TCA GAT Trp Leu Ser Asp CCT TTA TTT GAT Pro Leu Phe Asp CTT ATT AAA Leu Ile Lys 460 TCT GCT TGT Ser Ala Cys 1392 1440 GAC CGA GAA GGA Asp Arg Glu Gly

CCA

Pro 470 ACT GAT CAC CTT Thr Asp His Leu

GAA

Glu 475 CCT CTT Pro Leu 480 AAT CTT CCT CTC Asn Leu Pro Leu

CAG

Gin 485 AAT AAT CAC ACT Asn Asn His Thr

GCA

Ala 490 GCA GAT ATG TAT Ala Asp Met Tyr 1488

CTT

Leu 495 TCT CCT GTA AGA Ser Pro Val Arg

TCT

Ser 500 CCA AAG AAA AAA GGT TCA ACT ACG CGT Pro Lys Lys Lys Gly Ser Thr Thr Arg 1536 AAT TCT ACT GCA AAT GCA GAG ACA CAA Asn Ser Thr Ala Asn Ala Glu Thr Gin 515 ACC TCA GCC TTC Thr Ser Ala Phe CAG ACC Gin Thr 525 1584 CAG AAG CCA Gin Lys Pro TAT CGG CTA Tyr Arg Leu 545 AAA TCT ACC TCT Lys Ser Thr Ser TCA CTG TTT TAT Ser Leu Phe Tyr AAA AAA GTG Lys Lys Val 540 CGC CTT CTG Arg Leu Leu 1632 1680 GCC TAT CTC CGG Ala Tyr Leu Arg

CTA

Leu 550 AAT ACA CTT TGT Asn Thr Leu Cys

GAA

Glu 555 TCT GAG Ser Glu 560 CAC CCA GAA TTA His Pro Glu Leu

GAA

Glu 565 CAT ATC ATC TGG ACC CTT TTC CAG CAC His Ile Ile Trp Thr Leu Phe Gin His 570 1728

ACC

Thr 575

ATT

Ile 6 CTG CAG AAT GAG Leu Gin Asn Glu GAA CTC ATG AGA Glu Leu Met Arg

GAC

Asp 585 AGG CAT TTG GAC Arg His Leu Asp

CAA

Gin 590 1776 1824 ATG ATG TGT Met Met Cys ATG TAT GGC ATA Met Tyr Gly Ile AAA GTG AAG AAT Lys Val Lys Asn ATA GAC Ile Asp 605 CTT AAA TTC Leu Lys Phe GTT CAG GAG Val Gin Glu 625

AAA

Lys 610 ATC ATT GTA ACA Ile Ile Val Thr

GCA

Ala 615 TAC AAG GAT CTT Tyr Lys Asp Leu CCT CAT GCT Pro His Ala 620 GAG TAT GAT Glu Tyr Asp 1872 1920 ACA TTC AAA CGT Thr Phe Lys Arg TTG ATC AAA GAA Leu Ile Lys Glu TCT ATT Ser Ile 640 ATA GTA TTC TAT Ile Val Phe Tyr AAC TCG GTC TTC ATG Asn Ser Val Phe Met 645 CAG AGA CTG AAA ACA Gin Arg Leu Lys Thr 650 1968 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/U598/03041

AAT

Asn 655 ATT TTG CAG TAT Ile Leu Gin Tyr TCC ACC AGG CCC CCT Ser Thr Arg Pro Pro ACC TTG TCA CCA Thr Leu Ser Pro 2016 2064 CCT CAC ATT CCT Pro His Ile Pro

CGA

Arg 675 AGC CCT TAC-AAG Ser Pro Tyr l~ys CCT AGT TCA CCC Pro Ser Ser Pro TTA CGG Leu Arg 685 ATT CCT GGA Ile Pro Gly ATT TCA GAA Ile Ser Glu 705

GGG

Gly 690 AAC ATC TAT ATT Asn Ile Tyr Ile CCC CTG AAG AGT Pro Leu Lys Ser CCA TAT. AAA Pro Tyr Lys 700 AGA TCA AGA Arg Ser Arg 2112 2160 GGT CTG CCA ACA Gly Leu Pro Thr

CCA

Pro 710 ACA AAA ATG ACT Thr Lys Met Thr

CCA

Pro 715 ATC TTA Ile Leu 720 GTA TCA ATT GGT Vai Ser Ile Gly TCA TTC GGG ACT Ser Phe Giy Thr GAG AAG TTC CAG Glu Lys Phe Gin

AAA

Lys 735 ATA AAT CAG ATG Ile Asn Gin Met TGT AAC AGC GAC Cys Asn Ser Asp

CGT

Arg 745 GTG CTC AAA AGA Val Leu Lys Arg 2208 2256 2304 GCT GAA GGA AGC Ala Giu Giy Ser CCT CCT AAA CCA Pro Pro Lys Pro AAA AAA CTA CGC Lys Lys Leu Arg TTT GAT Phe Asp 765 ATT GAA GGA Ile Giu Gly TCC AAA TTT Ser Lys Phe 785

TCA

Ser 770 GAT GAA GCA GAT Asp Giu Aia Asp AGT AAA CAT CTC Ser Lys His Leu CCA GGA GAG Pro Gly Glu 780 CGA ACA CGA Arg Thr Arg 2352 2400 CAG CAG AAA CTG Gin Gin Lys Leu GAA ATG ACT TCT Glu Met Thr Ser

ACT

Thr 795 ATG CAA Met Gin 800 AAG CAG AAA ATG Lys Gin Lys Met GAT AGC ATG GAT ACC TCA AAC AAG GAA Asp Ser Met Asp Thr Ser Asn Lys Glu GAG AAA TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT GGATTCATTG Glu Lys 815 TCTCTCACAG ATGTGACTGT ATAACTTTCC CAGGTTCTGT TTATGGCCAC ATTTAATATC TTCAGCTCTT TTTGTGGATA TAAAATGTGC AGATGCAATT GTTTGGGTGA. TTCCTAAGCC ACTTGAAATG TTAGTCATTG TTATTTATAC AAGATTGAAA ATCTTGTGTA AATCCTGCCA TTTAAAAAGT TGTAGCAGAT TGTTTCCTCT TCCAAAGTAA AATTGCTGTG CTTTATGGAT AGTAAGAATG GCCCTAGAGT GGGAGTCCTG ATAACCCAGG CCTGTCTGAC TACTTTGCCT TCTTTTGTAG CATATAGGTG ATGTTTGCTC TTGTTTTAT TAATTTATAT GTATATTTTT TTAATTTAAC ATGAACACCC TTAGAAAATG TGTCCTATCT ATCTTCCAAA TGCAATTTGA 2448 2504 2564 2624 2684 2744 2804 2864 2924 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTIUS98/03041 134 TTGACTGCCC ATTCACCAAA ATTATCCTGA ACTCTTCTGC AAAAATGGAT ATTATTAGAA 2984 ATTAGAAAAA AATTACTAAT TTTACACATT AGATTTTATT TTACTATTGG AATCTGATAT 3044 ACTGTGTGCT TGTTTTATAA AATTTTGCTT TTAATTAAAT AAAAGCTGGA AGCAAAGTAT 3104 AACCATATGA TACTATCATA CTACTGA.AAC AGATTTCATA CCTCAGAATG TAAAAGAACT 3164 TACTGATTAT TTTCTTCATC CAACTTATGT TTTTAAATGA GGATTATTGA TAGT 3218 INFORMATION FOR SEQ ID NO: 4: Wi SEQUENCE CHARACTERISTICS: LENGTH: 816 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Ser Phe Thr Phe Thr Glu Leu Gin Lys Asn Ile Giu Ile Ser Val 1 5 10 His Lys Phe Phe Asn Leu Leu Lys Giu Ile Asp Thr Ser Thr Lys Val 25 Asp Asn Ala Met Ser Arg Leu Leu Lys Lys Tyr Asp Val Leu Phe Ala 40 Leu Phe Ser Lys Leu Giu Arg Thr Cys Glu Leu Ile Tyr Leu Thr Gin 55 Pro Ser Ser Ser Ile Ser Thr Giu Ile Asn Ser Ala Leu Val Leu Lys 70 75 Val Ser Trp Ile Thr Phe Leu Leu Ala Lys Gly Giu Val Leu Gin Met 90 Giu Asp Asp Leu Val Ile Ser Phe Gin Leu Met Leu Cys Vai Leu Asp 100 105 110 Tyr Phe Ile Lys Leu Ser Pro Pro Met Leu Leu Lys Giu Pro Tyr Lys 115 120 125 Thr Ala Val Ile Pro Ile Asn Gly Ser Pro Arg Thr Pro Arg Arg Gly 130 135 140 Gin Asn Arg Ser Ala Arg Ile Ala Lys Gin Leu Giu Asn Asp Thr Arg 145 150 155 160 Ile Ile Giu Val Leu Cys Lys Giu His Giu Cys Asn Ile Asp Giu Val 165 170 175 Lys Asn Val Tyr Phe Lys Asn Phe Ile Pro Phe Met Asn Ser Leu Gly 180 185 190 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 Leu Tyr Leu 225 Thr Ile Leu Ile Lys 305 Ala Gly Glu Ile Thr 385 Ser Tyr Glu Leu Lys 465 Asn Val Glu 210 Asp Gin Pro Met Ser 290 Arg Val Val Glu Phe 370 Tyr Phe Lys Met Ala 450 Asp Leu Thr 195 Glu His Arg Pro Met 275 Tyr Val Gly Arg Arg 355 His Ser Pro Val Ile 435 Trp Arg Pro Ser Ile Asp Thr His 260 Ile Phe Lys Gin Leu 340 Leu Met Arg Trp Ile 420 Lys Leu Glu Leu Asn Tyr Lys Pro 245 Thr Leu Asn Asp Gly 325 Tyr Ser Ser Ser Ile 405 Glu His Ser Gly Gin 485 Leu Lys 215 Leu Lys Val Ser Cys 295 Gly Val Arg Gin Leu 375 Ser Asn Phe Glu Ser 455 Thr Asn Pro 200 Asn Gin Ser Arg Ala 280 Thr Tyr Glu Val Asn 360 Ala Gin Val Ile Arg 440 Pro Asp His Glu Lys Thr Asn Thr 265 Ser Val Ile Ile Met 345 Phe Cys Asn Leu Lys 425 Cys Leu His Thr Val Asp Asp Leu 250 Val Asp Asn Phe Gly 330 Glu Ser Ala Leu Asn 410 Ala Glu Phe Leu Ala 490 Glu Leu Ser 235 Asp Met Gin Pro Lys 315 Ser Ser Lys Leu Asp 395 Leu Glu His Asp Glu 475 Ala Asn Asp 220 Ile Glu Asn Pro Lys 300 Glu Gin Met Leu Glu 380 Ser Lys Gly Arg Leu 460 Ser Asp Leu 205 Ala Asp Glu Thr Ser 285 Glu Lys Arg Leu Leu 365 Val Gly Ala Asn Ile 445 Ile Ala Met Ser Arg Ser Val Ile 270 Glu Ser Phe Tyr Lys 350 Asn Val Thr Phe Leu 430 Met Lys Cys Tyr Lys Leu Phe Asn 255 Gin Asn Ile Ala Lys 335 Ser Asp Met Asp Asp 415 Thr Glu Gin Pro Leu 495 Arg Phe Glu 240 Val Gin Leu Leu Lys 320 Leu Glu Asn Ala Leu 400 Phe Arg Ser Ser Leu 480 Ser SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/03041 Pro Val Thr Ala Pro Leu 530 Leu Ala 545 His Pro Gln Asn Met Cys Phe Lys 610 Glu Thr 625 Ile Val Len Gin Ile Pro Gly Gly 690 Giu Gly 705 t- Val Ser Asn Gin Gly Ser Gly Ser 770 Arg Asn 515 Lys Tyr Gin Gin Ser 595 Ile Phe Phe Tyr Arg 675 Asn Len Ile Met Asn 755 Asp Ser 500 Ala Ser Leu Leu Tyr 580 Met Ile Lys Tyr Ala 660 Ser Ile Pro Gly Val 740 Pro Giu Pro Giu Thr Arg Giu 565 Gin Tyr Val Arg Asn 645 Ser Pro Tyr Thr Gin 725 Cys Pro Ala Lys Gin Len 535 Asn Ile Met Ile Ala 615 Leu Vai Arg Lys Ser 695 Thr Phe Ser Pro Giy 775 Lys Ala 520 Ser Thr Ile Arg Cys 600 Tyr Ile Phe Pro Phe 680 Pro Lys Gly Asp Leu 760 Ser Gly 505 Thr Len Leu Trp Asp 585 Lys Lys Lys Met Pro 665 Pro Len Met Thr Arg 745 Lys Lys Ser Ser Phe Cys Thr 570 Arg Val1 Asp Giu Gin 650 Thr S er Lys Thr Ser 730 Val Lys His Thr Al a Tyr Gin 555 Leu His Lys Leu Gin 635 Arg Leu Ser Ser Pro 715 Gin Len Leu Len Thr Phe Lys 540 Arg Phe Leu Asn Pro 620 Gin Len Ser Pro Pro 700 Arg Lys Lys Arg Pro 780 Arg Gin 525 Lys Leu Gin Asp Ile 605 His Tyr Lys Pro Len 685 Tyr Ser Phe Arg Phe 765 Gly Vai 510 Thr Val Leu His Gin 590 Asp Al a Asp Thr Ile 670 Arg Lys Arg Gin Ser 750 Asp Gin Asn Gin Tyr Ser Thr 575 Ile Len Val Ser Asn 655 Pro Ile Ile Ile Lys 735 Ala Ile Ser Ser Lys Arg Gin 560 Len Met Lys Gin Ile 640 Ile His Pro Ser Len 720 Ile Gin Gin Lys SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 137 Gin Gin Lys Leu Ala Glu Met Thr Ser Thr Arg Thr Arg Met Gin 790 795 800 Gin Lys Met Asn Asp Ser Met Asp Thr Ser Asn Lys Glu Glu Lys 805 810 815 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 285 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTCGAGCAAT GGGCGTGATA GCGGTTTGAC TCACGGGGAT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTAC AGAGCTCGTT TAGTGAACCG TCAGATCGCC TGGAGACGCC CATAGAAGAC ACCGGGACCG ATCCAGCCTC CGCGGCCGCG TTCCAAGTCT CCACCCCATT ACTTTCCAAA ATGTCGTAAC GGTGGGAGGT CTATATAAGC ATCCACGCTG TTTTGACCTC

AATTC

120 180 240 285 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CCGCTCGAGC AATGGGCGTG GATAGCGG INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCGCTCGAGC ACCAAAATCA ACGGGA INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 138 LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CCGCTCGAGC AACTCCGCCC CATTGAC 27 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TAGACATATG AATTCGCGGC C 21 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTAGAATTCG CTGTCTGCG 19 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCTCTAGATG CAGTTGGACC TGGGAG 26 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 139 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CCCAAGCTTG CCGCCATGTC GTTCACTTTT AC 32 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GTCCAAGAGA ATTCATAAAA GG 22 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CCCAAGCTTG CCGCCATGGA GCAGGACAGC GGCCCGGAC 39 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCCAAGCTTG CCGCCATGGA TTTTACTGCA TTATGTCAG 39 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CCCAAGCTTG CCGCCATGGA GAAAGTTTCA TCTTGTGAT 39 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 140 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CCCAAGCTTG CCGCCATGCT GTGGGGAATC TGTATCTTT 39 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CCCAAGCTTG CCGCCATGTC AAGACTGTTG AAGAAG 36 INFORMATION FOR SEQ ID NO: 19: .SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GCGCCTGAGG ACCTAGATGA GATGTCGTTC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCGGTTAACC CTAGATGAGA TGTCGTTCAC T 31 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 141.

STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CCCAAGCTTG CCGTCATGCC GCCCAAAACC CCCCGA 36 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: CTCACCTAGG TCAACTGCTG CAAT 24 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GTTGACCTAG GTGATATGTC GTTC 24 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GCGCCTAGGA TCTACTGAAA TAAATTCTGC A 31 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) .SEQUENCE DESCRIPTION: SEQ ID NO: SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 142 CCCGATATCA ACTGCTGGGT TGTGTCAAAT A 31 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: CCCGAATTCG TTTTATATGG TTCTTTGAGC AA 32 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 10 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: modified base OTHER INFORMATION:/note= "R=A or G" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GCCRCCAUGG INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 3455 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2691 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GCCGTC ATG CAG GAC AGC GGC CCG GAG GAC CTG CCT CTC GTC AGG CTT 48 Met Gln Asp Ser Gly Pro Glu Asp Leu Pro Leu Val Arg Leu 1 5 GAG TTT GAA GAA ACA GAA GAA CCT GAT TTT ACT GCA TTA TGT CAG AAA 96 Glu Phe Glu Glu Thr Glu Glu Pro Asp Phe Thr Ala Leu Cys Gin Lys 20 25 SUBSTITUTE SHEET (RULE 26) WO: 98/37091 PCT/US98/03041 TTA AAG ATA CCA Leu Lys Ile Pro CAT GTC AGA GAG His Val Arg Glu AGA GCT Arg Ala TGG TTA ACT Trp Leu Thr TGG GAG Trp Glu AAA GTT TCA TCT GTG GAT GGA GTA Lys Val Ser Ser Val Asp Gly Val GGA GGT TAT ATT Gly Gly Tyr Ile CAA AAG AAA Gin Lys Lys AAG GAA CTG Lys Glu Leu TGG GGA ATC TGT Trp Gly Ile Cys TTT ATT GCA GCA GTT GAC CTA GAT Phe Ile Ala Ala Val Asp Leu Asp GAG ATG Glu Met TCG TTC ACT TTT Ser Phe Thr Phe GAG CTA CAG AAA Glu Leu Gin Lys ATA GAA ATC AGT Ile Glu Ile Ser

GTC

Val CAT AAA TTC TTT His Lys Phe Phe TTA CTA AAA GAA Leu Leu Lys Glu

ATT

Ile 105 GAT ACC AGT ACC AAA Asp Thr Ser Thr Lys GTT GAT AAT GCT Val Asp Asn Ala TCA AGA CTG TTG Ser Arg Leu Leu

AAG

Lys 120 AAG TAT GAT GTA Lys Tyr Asp Val TTG TTT Leu Phe 125 GCA CTC TTC Ala Leu Phe CAA CCC AGC Gin Pro Ser 145 AAA TTG GAA AGG Lys Leu Glu Arg

ACA

Thr 135 TGT GAA CTT ATA Cys Glu Leu Ile TAT TTG ACA Tyr Leu Thr 140 TTG GTG CTA Leu Val Leu 432 AGT TCG ATA TCT Ser Ser Ile Ser

ACT

Thr 150 GAA ATA AAT TCT Glu Ile Asn Ser

GCA

Ala 155 AAA GTT Lys Val 160 TCT TGG ATC ACA Ser Trp Ile Thr TTA TTA GCT AAA Leu Leu Ala Lys

GGG

Gly 170 GAA GTA TTA CAA Glu Val Leu Gin

ATG

Met 175 GAA GAT GAT CTG Glu Asp Asp Leu ATT TCA TTT CAG Ile Ser Phe Gin

TTA

Leu 185 ATG CTA TGT GTC Met Leu Cys Val GAC TAT TTT ATT Asp Tyr Phe Ile CTC TCA CCT CCC Leu Ser Pro Pro

ATG

Met 200 TTG CTC AAA GAA Leu Leu Lys Glu CCA TAT Pro Tyr 205 AAA ACA GCT Lys Thr Ala GGT CAG AAC Gly Gin Asn 225 ATA CCC ATT AAT Ile Pro Ile Asn TCA CCT CGA ACA Ser Pro Arg Thr CCC AGG CGA Pro Arg Arg 220 AGG AGT GCA CGG Arg Ser Ala Arg

ATA

Ile 230 GCA AAA CAA CTA GAA AAT GAT ACA Ala Lys Gin Leu Glu Asn Asp Thr 235 AGA ATT Arg Ile 240 ATT GAA GTT CTC Ile Glu Val Leu

TGT

Cys 245 AAA GAA CAT GAA Lys Glu His Glu TGT AAT Cys Asn 250 ATA GAT GAG Ile Asp Glu SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/0304 1 AAA AAT GTT TAT Lys Asn Val Tyr AAA AAT TTT Lys Asn Phe ATA CCT Ile Pro 265 TTT ATG AAT TCT Phe Met Asn Ser

CTT

Leu 270 GGA CTT GTA ACA Gly Leu Val Thr AAT GGA CTT CCA Asn Giy Leu Pro

GAG

Giu 280 GTT GAA AAT CTT Vai Giu Asn Leu TCT AAA Ser Lys 285 CGA TAC GAA Arg Tyr Giu TTT TTG GAT Phe Leu Asp 305

GAA

Giu 290 ATT TAT CTT AAA Ile Tyr Leu Lys

A-AT

Asn 295 AAA GAT CTA GAT Lys Asp Leu Asp GCA AGA TTA Ala Arg Leu 300 GAC AGT TTT Asp Ser Phe CAT GAT AAA ACT His Asp Lys Thr

CTT

Leu 310 CAG ACT GAT TCT Gin Thr Asp Ser

ATA

Ile 315 GAA ACA Giu Thr 320 CAG AGA ACA CCA Gin Arg Thr Pro AAA AGT AAC CTT Lys Ser Asn Leu GAA GAG GTG AAT Giu Giu Val Asn ATT CCT CCA CAC Ile Pro Pro His CCA GTT AGG ACT Pro Val Arg Thr ATG AAC ACT ATC Met Asn Thr Ile

CAA

Gin 350 1008 1056 1104 CAA TTA ATG ATG Gin Leu Met Met TTA AAT TCA GCA Leu Asn Ser Ala GAT CAA CCT TCA Asp Gin Pro Ser GAA AAT Giu Asn 365 CTG ATT TCC Leu Ile Ser CTG AAA AGA Leu Lys Arg 385

TAT

Tyr 370 TTT AAC AAC TGC Phe Asn Asn Cys

ACA

Thr 375 GTG AAT CCA AAA Vai Asn Pro Lys GAA AGT ATA Giu Ser Ile 380 1152 GTG AAG GAT ATA Val Lys Asp Ile

GGA

Giy 390 TAC ATC TTT AAA GAG AAA TTT GCT Tyr Ile Phe Lys Giu Lys Phe Ala 395 1200 AAA GCT Lys Ala 400 GTG GGA CAG GGT Vai Gly Gin Gly GTC GAA ATT GGA Vai Gu Ile Gly

TCA

Ser 410 CAG CGA TAC AAA Gin Arg Tyr Lys 1248 1296

CTT

Leu 415

GAA

Giu GGA OTT CGC TTG Giy Vai Arg Leu GAA GA-A CGA TTA Glu Giu Arg Leu 435

TAT

Tyr 420 TAC CGA GTA A-TO Tyr Arg Val Met

GAA

G1u 425 TCC ATO CTT AAA Ser Met Leu Lys

TCA

Ser 430 TCC ATT CAA AAT Ser Ile Gin Asn

TTT

Phe 440 AGC AAA CTT CTO AAT GAC Ser Lys Leu Leu Asn Asp 445 1344 AAC ATT TTT CAT ATG TCT TTA TTG Asn Ile Phe His Met Ser Leu Leu 450 TGC GCT CTT GAG Cys Ala Leu Giu GTT GTA ATG Vai Vai Met 460 GGA ACA GAT Gly Thr Asp 1392 GCC ACA TAT Ala Thr Tyr 465 AGC AGA AGT ACA Ser Arg Ser Thr CAG A-AT CTT GAT Gin Asn Leu Asp 1440 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 TTG TCT Leu Ser 480 TTC CCA TGG ATT CTG AAT GTG CTT AAT Phe Pro Trp Ile Leu Asn Val Leu Asn 485 AAA GCC TTT GAT Lys Ala Phe Asp TAC AAA GTG ATC Tyr Lys Val Ile AGT TTT ATC, AAA Ser Phe IA~ Lys GAA GGC AAC TTG Giu Gly Asn Leii

ACA

Thr 510 1488 1536 1584 AGA GAA ATG ATA Arg Giu Met Ile CAT TTA GAA CGA His Leu Giu Arg GAA CAT CGA ATC Giu His Arg Ile ATG ,GAA Met Giu 52.5 TCC CTT GCA Ser Leu Ala TCA AAG GAC Ser Lys Asp 545 CTC TCA GAT TCA Leu' Ser Asp Ser TTA TTT GAT CTT Leu Phe Asp Leu ATT AAA CAA Ile Lys Gin 540 GCT TGT CCT Ala Cys Pro 1632 1680 CGA GAA GGA CCA Arg Giu Gly Pro

ACT

Thr 550 GAT CAC CTT GAA Asp His Leu Giu CTT AAT Leu Asn 560 CTT CCT CTC CAG Leu Pro Leu Gin

AAT

Asn 565 AAT CAC ACT GCA Asn His Thr Aia

GCA

Aia 570 GAT ATG TAT CTT Asp Met Tyr Leu 1728 1776

TCT

Ser 575 CC-T GTA AGA TCT CCA AAG AAA AAA GGT Pro Val Arg Ser Pro Lys Lys Lys Giy 580 ACT ACG CGT GTA Thr Thr Arg Val

AAT

Asn 590 TCT ACT GCA AAT Ser Thr Ala Asn GAG ACA CAA GCA Giu Thr Gin Ala TCA GCC TTC CAG Ser Ala Phe Gin ACC CAG Thr Gin 605 1824 AAG CCA TTG Lys Pro Leu CGG CTA GCC Arg Leu Ala 625 TCT ACC TCT CTT Ser Thr Ser Leu

TCA

Ser 615 CTG TTT TAT AAA Leu Phe Tyr Lys AAA GTG TAT Lys Val Tyr 620 CTT CTG TCT Leu Leu Ser 1872 1920 TAT CTC CGG CTA Tyr Leu Arg Leu

AAT

Asn 630 ACA CTT TGT GAA Thr Leu Cys Giu GAG CAC Giu His 640 CCA GAA TTA GAA Pro Giu Leu Giu ATC ATC TGG ACC Ile Ile Trp Thr TTC CAG CAC ACC Phe Gin His Thr

CTG

Leu 655 CAG AAT GAG TAT Gin Asn Giu Tyr CTC ATG AGA GAC Leu Met Arg Asp CAT TTG GAC CAA His Leu Asp Gin 1968 2016 2064 ATG ATG TGT TCC Met Met Cys Ser AAA TTC AAA ATC Lys Phe Lys Ile 690 TAT GGC ATA TGC Tyr Gly Ile Cys GTG AAG AAT ATA Vai.Lys Asn Ile GAC CTT Asp Leu 685 ATT GTA ACA GCA Ile Val Thr Ala AAG GAT CTT CCT Lys Asp Leu Pro CAT GCT GTT His Ala Val 700 2112 SUBSTITUTE SHEET (RULE 26) WO 98/37091 CAG GAG ACA Gin Giu Thr 705 PCTIJS98/03041I TTC AAA CGT GTT Phe Lys Arg Vai ATC AAA GAA GAG Ile Lys Giu Giu TAT GAT TCT Tyr Asp Ser 2160 ATT ATA Ile Ile 720 GTA TTC TAT AAC Vai Phe Tyr Asn

TCG

Ser 725 GTC TTC ATG CAG Val Phe Met Gin

AGA

Arg 730 CTG AAA ACA AAT Leu Lys Thr Asn TTG CAG TAT GCT Leu Gin Tyr Ala

TCC

Ser 740 ACC AGG CCC CCT Thr Arg Pro Pro TTG TCA CCA ATA Leu Ser Pro Ile

CCT

Pro 750 2208 2256 2304 CAC ATT CCT CGA His Ile Pro Arg

AGC

Ser 755 CCT TAC AAG TTT Pro Tyr Lys Phe AGT TCA CCC TTA Ser Ser Pro Leu CGG ATT Arg Ile 765 CCT GGA GGG Pro Giy Giy TCA GAA GGT Ser Giu Gly 785 ATC TAT ATT TCA Ile Tyr Ile Ser CTG AAG AGT CCA Leu Lys Ser Pro TAT AAA ATT Tyr Lys Ile 780 TCA AGA ATC Ser Arg Ile 2352 2400 CTG CCA ACA CCA Leu Pro Thr Pro AAA ATG ACT CCA Lys Met Thr Pro TTA GTA Leu Vai 800 TCA ATT GGT GAA Ser Ile Gly Giu

TCA

Ser 805 TTC GGG ACT TCT Phe Gly Thr Ser

GAG

Giu 810 AAG TTC CAG AAA Lys Phe Gin Lys

ATA

Ile 815 AAT CAG ATG GTA Asn Gin Met Val

TGT

Cys 820 AAC AGC GAC CGT Asn Ser Asp Arg CTC AAA AGA AGT Leu Lys Arg Ser

GCT

Ala 830 2448 2496 2544 GAA GGA AGC AAC Giu Giy Ser Asn

CCT

Pro 835 CCT AAA CCA CTG Pro Lys Pro Leu

AAA

Lys 840 AAA CTA CGC TTT Lys Leu Arg Phe GAT ATT Asp Ile 845 GAA GGA TCA Giu Gly Ser AAA TTT CAG Lys Phe Gin 865

GAT

Asp 850 GAA GCA GAT GGA Giu Ala Asp Gly

AGT

Ser 855 AAA CAT CTC CCA Lys His Leu Pro GGA GAG TCC Gly Giu Ser 860 ACA CGA ATG Thr Arg Met 2592 2640 CAG AAA CTG GCA Gin Lys Leu Ala

GAA

Giu 870 ATG ACT TCT ACT Met Thr Ser Thr

CGA

Arg 875 CAA AAG Gin Lys 880 CAG AAA ATG AAT Gin Lys Met Asn AGC ATG GAT ACC TCA AAC AAG GAA GAG Ser Met Asp Thr Ser Asn Lys Glu Glu AAA TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT GGATTCATTG Lys 895 TCTCTCACAG ATGTGACTGT ATAACTTTCC CAGGTTCTGT TTATGGCCAC ATTTAATATC TTCAGCTCTT TTTGTGGATA TAAAATGTGC AGATGCAATT GTTGGGTGA TTCCTAAGCC ACTTGAAATG TTAGTCATTG TTATTTATAC AAGATTGAAA ATCTTGTGTA AATCCTGCCA 2688 2741 2801 2861 2921 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAPA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA

TACTATCATA

TTTCTTCATC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

TCCAAAGTAA

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

AC!TCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

TTTTAAATGA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

CCTCAGAATG

GGATTATTGA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA.

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TAAAAGAACT

TAGT

2981 3041 3101 3161 3221 3281 3341 3401 3455 INFORMATION FOR SEQ ID NO: 29: Wi SEQUENCE CHARACTERISTICS: LENGTH: 895 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: Met 1 Giu Gin Asp Ser Gly Pro Glu Asp Leu Pro Leu Val Arg Leu Giu Phe Giu Thr Glu Glu Pro Asp Phe Thr Ala 25 Arg Glu Arg Ala Trp 40 Leu Cys Gin Lys Leu Lys Giu Lys Val Ile Pro Asp His Val Ser Ser Val Asp Gly Leu Thr Trp Leu Trp Val Leu Ile Phe Gly Gly Tyr Ilie Lys Lys Lys Glu Gly Ilie Cys Ile Ala Ala Asp Leu Asp Giu Glu Met Ser Phe Thr Phe Leu Gln Lys Giu Ile Ser Val His Lys Phe Phe Asn Ala Met 115 Asn 100 Leu Leu Lys Glu Ile 105 Lys Lys 120 Asp Thr Ser Tyr Asp Val Ser Arg Leu Leu Thr Lys Val Asp 110 Leii Phe Ala Leu 125 Leu Thr Gln Pro Phe Ser 130 Lys Leu Glu Arg Thr 135 Cys Glu Leu Ilie Tyr 140 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTIUS98/03041 148 Ser Ser Ser Ile Ser Thr Giu Ile Asn Ser Ala Leu Vai Leu Lys Val 145 150 155 160 Ser Trp Ile Thr Phe Leu Leu Ala Lys Gly Giu Val Leu Gin Met Giu 165 170 175 Asp Asp Leu Vai Ile Ser Phe Gin Leu Met Leu Cys Val Leu Asp Tyr 180 185 190 Phe Ile Lys Leu Ser Pro Pro Met Leu Leu Lys Giu Pro Tyr Lys Thr 195 200 205 Ala Val Ile Pro Ile Asn Giy Ser Pro Arg Thr Pro Arg Arg Gly Gin 210 215 220 Asn Arg Ser Aia Arg Ile Ala Lys Gin Leu Giu Asn Asp Thr Arg Ile 225 230 235 240 Ile Giu Val Leu Cys Lys Giu His Giu Cys Asn Ile Asp Giu Vai Lys 245 250 255 Asn Val Tyr Phe Lys Asn Phe Ile Pro Phe Met Asn Ser Leu Gly Leu 260 265 270 Val Thr Ser Asn Gly Leu Pro Giu Val Giu Asn Leu Ser Lys Arg Tyr 275 280 285 Giu Giu Ile Tyr Leu Lys Asn Lys Asp Leu Asp Aia Arg Leu Phe Leu 290 295 300 Asp His Asp Lys Thr Leu Gin Thr Asp Ser Ile Asp Ser Phe Giu Thr 305 310 315 320 Gin Arg Thr Pro Arg Lys Ser Asn Leu Asp Giu Giu Val Asn Val Ile 325 330 335 Pro Pro His Thr Pro Val Arg Thr Vai Met Asn Thr Ile Gin Gin Leu 340 345 350 Met Met Ile Leu Asn Ser Ala Ser Asp Gin Pro Ser Giu Asn Leu Ile 355 360 365 Ser *Tyr Phe Asn Asn Cys Thr Val Asn Pro Lys Giu Ser Ile Leu Lys 370 375 380 Arg Val Lys Asp Ile Giy Tyr Ile Phe Lys Glu Lys Phe Ala Lys Ala 385 390 395 400 Vai Gly Gin Giy Cys Val Giu Ile Giy Ser Gin Arg Tyr Lys Leu Gly 405 410 415 Vai Arg Leu Tyr Tyr Arg Val Met Glu Ser Met Leu Lys Ser Giu Giu 420 425 430 Giu Arg Leu Ser Ile Gin Asn Phe Ser Lys Leu Leu Asn Asp Asn Ile 435 440 445 SUBSTITUTE SHEET (RULE W6 98/37091 PCT/US98/03041 Phe Tyr 465 Phe Lys Met Ala Asp 545 Leu Val Ala Leu Ala 625 Pro Asn Cys Lys Thr 705 Val Gln His 450 Ser Pro Val Ile Trp 530 Arg Pro Arg Asn Lys 610 Tyr Glu Glu Ser Ile 690 Phe Phe Tyr Met Arg Trp Ile Lys 515 Leu Glu Leu Ser Ala 595 Ser Leu Leu Tyr Met 675 Ile Lys Tyr Ala Ser Ser Ile Glu 500 His Ser Gly Gin Pro 580 Glu Thr Arg Glu Glu 660 Tyr Val Arg Asn Ser 740 Leu Thr Leu 485 Ser Leu Asp Pro Asn 565 Lys Thr Ser Leu His 645 Leu Gly Thr Val Ser 725 Thr Leu Ser 470 Asn Phe Glu Ser Thr 550 Asn Lys Gin Leu Asn 630 Ile Met Ile Ala Leu 710 Val Arg Ala 455 Gin Val Ile Arg Pro 535 Asp His Lys Ala Ser 615 Thr Ile Arg Cys Tyr 695 Ile Phe Pro Cys Asn Leu Lys Cys 520 Leu His Thr Gly Thr 600 Leu Leu Trp Asp Lys 680 Lys Lys Met Pro Ala Leu Asn Ala 505 Glu Phe Leu Ala Ser 585 Ser Phe Cys Thr Arg 665 Val Asp Glu Gln Thr 745 Leu Asp Leu 490 Glu His Asp Glu Ala 570 Thr Ala Tyr Glu Leu 650 His Lys Leu Glu Arg 730 Leu Glu Ser 475 Lys Gly Arg Leu Ser 555 Asp Thr Phe Lys Arg 635 Phe Leu Asn Pro Glu 715 Leu Ser Val 460 Gly Ala Asn Ile Ile 540 Ala Met Arg Gin Lys 620 Leu Gin Asp Ile His 700 Tyr Lys Pro Va1 Thr Phe Leu Met 525 Lys Cys Tyr Val Thr 605 Val Leu His Gin Asp 685 Ala Asp Thr Ile Met Ala Asp Leu Asp Phe 495 Thr Arg 510 Glu Ser Gin Ser Pro Leu Leu Ser 575 Asn Ser 590 Gin Lys Tyr Arg Ser Glu Thr Leu 655 Ile Met 670 Leu Lys Val. Gln Ser Ile Asn Ile 735 Pro His 750 Thr Ser 480 Tyr Glu Leu Lys Asn 560 Pro Thr Pro Leu His 640 Gin Met Phe Glu Ile 720 Leu Ile SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 150 Pro Arg Ser Pro Tyr Lys Phe Pro Ser Ser Pro Leu Arg Ile Pro Gly 755 760 765 Gly Asn Ile Tyr Ile Ser Pro Leu Lys Ser Pro Tyr Lys Ile Ser Glu 770 775 780 Gly Leu Pro Thr Pro Thr Lys Met Thr Pro Arg Ser Arg Ile Leu Val 785 790 795 800 Ser Ile Gly Glu Ser Phe Gly Thr Ser Glu Lys Phe Gin Lys Ile Asn 805 810 815 Gin Met Val Cys Asn Ser Asp Arg Val Leu Lys Arg Ser Ala Glu Gly 820 825 830 Ser Asn Pro Pro Lys Pro Leu Lys Lys Leu Arg Phe Asp Ile Glu Gly 835 840 845 Ser Asp Glu Ala Asp Gly Ser Lys His Leu Pro Gly Glu Ser Lys Phe 850 855 860 Gin Gin Lys Leu Ala Glu Met Thr Ser Thr Arg Thr Arg Met Gin Lys 865 870 875 880 Gin Lys Met Asn Asp Ser Met Asp Thr Ser Asn Lys Glu Glu Lys 885 890 895 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 3392 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2628 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCCATC ATG GAT TTT ACT GCA TTA TGT CAG AAA TTA AAG ATA CCA GAT 48 Met Asp Phe Thr Ala Leu Cys Gin Lys Leu Lys Ile Pro Asp 1 5 CAT GTC AGA GAG AGA GCT TGG TTA ACT TGG GAG AAA GTT TCA TCT GTG 96 His Val Arg Glu Arg Ala Trp Leu Thr Trp Glu Lys Val Ser Ser Val 20 25 GAT GGA GTA TTG GGA GGT TAT ATT CAA AAG AAA AAG GAA CTG TGG GGA 144 Asp Gly Val Leu Gly Gly Tyr Ile Gin Lys Lys Lys Glu Leu Trp Gly 40 ATC TGT ATC TTT ATT GCA GCA GTT GAC CTA GAT GAG ATG TCG TTC ACT 192 Ile Cys Ile Phe Ile Ala Ala Val Asp Leu Asp Glu Met Ser Phe Thr 55 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTILJS98/03041 TTT ACT GAG Phe Thr Glu CTA CAG AAA AAC Leu Gin Lys Asn

ATA

Ile GAA ATC AGT GTC Glu Ile Ser Val AAA TTC TTT Lys Phe Phe AAC TTA Asn Leu CTA AAA GAA ATT GAT ACC AGT ACC AAA Leu Lys Giu Ile Asp Thr Ser Thr Lys 85

GTT

Vai GAT AAT GCT ATG Asp Asn Aia Met

TCA

Ser AGA CTG TTG, AAG Arg Leu Leu Lys TAT GAT GTA TTG Tyr Asp Vai Leu GCA CTC TTC AGC Ala Leu Phe Ser.

AAA

Lys 110 TTG GAA AGG ACA Leu Giu Arg Thr

TGT

Cys 115 GAA CTT ATA TAT Giu Leu Ile Tyr ACA CAA CCC AGC Thr Gin Pro Ser AGT TCG Ser Ser 125 ATA TCT ACT GAA ATA AAT TCT GCA Ile Ser Thr Giu Ile Asn Ser Aia 130

TTG

Leu 135 GTG CTA AAA GTT Vai Leu Lys Vai TCT TGG ATC Ser Trp Ile 140 GAT GAT CTG Asp Asp Leu ACA TTT TTA TTA GCT AAA GGG Thr Phe Leu Leu Aia Lys Giy 145

GAA

Giu 150 GTA TTA CAA ATG Val Leu Gin Met GTG ATT TCA TTT CAG TTA ATG CTA TGT GTC CTT Val Ile Ser Phe Gin Leu Met Leu Cys Vai Leu 160 16; C

GAC

Asp 170 TAT TTT ATT AAA Tyr Phe Ile Lys S28

CTC

Leu 175 TCA CCT CCC ATG Ser Pro Pro Met CTC AAA GAA CCA Leu Lys Giu Pro AAA ACA GCT GTT Lys Thr Ala Val CCC ATT AAT GGT Pro Ile Asn Giy CCT CGA ACA CCC Pro Arg Thr Pro

AGG

Arg 200 CGA GGT CAG AAC Arg Gly Gin Asn AGG AGT Arg Ser 205 GCA CGG ATA Ala Arg Ile CTC TGT AAA Leu Cys Lys 225 AAA CAA CTA GAA AAT GAT ACA AGA ATT Lys Gin Leu Giu Asn Asp Thr Arg Ile 215 ATT GAA GTT Ile Giu Val 220 AAT GTT TAT Asn Val Tyr GAA CAT GAA TGT Giu His Giu Cys

AAT

Asn 230 ATA GAT GAG GTG Ile Asp Giu Vai TTC AAA Phe Lys 240 AAT TTT ATA CCT TTT ATG AAT TCT CTT Asn Phe Ile Pro Phe Met Asn Ser Leu 245

GGA

Gly 250 CTT GTA ACA TCT Leu Vai Thr Ser

AAT

Asn 255 GGA CTT CCA GAG Giy Leu Pro Giu

GTT

Vai 260 GAA XAT CTT TCT Giu Asn Leu Ser CGA TAC GAA GAA Arg Tyr Giu Giu

ATT

Ile 270 TAT CTT AAA AAT Tyr Leu Lys Asn AAA GAT Lys Asp 275 CTA GAT GCA Leu Asp Ala TTA TTT TTG GAT Leu Phe Leu Asp CAT GAT His Asp 285 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 AAA ACT CTT Lys Thr Leu CCA CGA AAA Pro Arg Lys 305 ACT CCA GTT Thr Pro Val 320

CAG

Gin 290 ACT GAT TCT ATA Thr Asp Ser Ile AGT TTT GAA ACA Ser Phe Glu Thr CAG AGA ACA Gin Arg Thr 300 AGT AAC CTT GAT GAA GAG GTG AAT GTA ATT CCT CCA CAC Ser Asn Leu Asp Glu Glu Val Asn Val Ile Pro Pro His AGG ACT GTT Arg Thr Val

ATG

Met 325 AAC ACT ATC CAA Asn Thr Ile Gin

CAA

Gin 330 TTA ATG ATG ATT Leu Met Met Ile 1008

TTA

Leu 335 AAT TCA GCA AGT Asn Ser Ala Ser

GAT

Asp 340 CAA CCT TCA GAA AAT CTG ATT TCC TAT Gin Pro Ser Glu Asn Leu Ile Ser Tyr 345 1056 AAC AAC TGC ACA Asn Asn Cys Thr

GTG

Val 355 AAT CCA AAA GAA Asn Pro Lys Glu ATA CTG AAA AGA Ile Leu Lys Arg GTG AAG Val Lys 365 1104 GAT ATA GGA Asp Ile Gly GGT TGT GTC Gly Cys Val 385

TAC

Tyr 370 ATC TTT AAA GAG Ile Phe Lys Glu TTT GCT AAA GCT Phe Ala Lys Ala GTG GGA CAG Val Gly Gin 380 GTT CGC TTG Val Arg Leu 1152 1200 GAA ATT GGA TCA Glu Ile Gly Ser

CAG

Gin 390 CGA TAC AAA CTT Arg Tyr Lys Leu

GGA

Gly 395 TAT TAC Tyr Tyr 400 CGA GTA ATG GAA Arg Val Met Glu

TCC

Ser 405 ATG CTT AAA TCA Met Leu Lys Ser

GAA

Glu 410 GAA GAA CGA TTA Glu Glu Arg Leu ATT CAA AAT TTT Ile Gin Asn Phe

AGC

Ser 420 AAA CTT CTG AAT Lys Leu Leu Asn AAC ATT TTT CAT Asn Ile Phe His

ATG

Met 430 1248 1296 1344 TCT TTA TTG GCG Ser Leu Leu Ala

TGC

Cys 435 GCT CTT GAG GTT Ala Leu Glu Val

GTA

Val 440 ATG GCC ACA TAT Met Ala Thr Tyr AGC AGA Ser Arg 445 AGT ACA TCT CAG AAT CTT GAT TCT Ser Thr Ser Gin Asn Leu Asp Ser 450 ACA GAT TTG TCT Thr Asp Leu Ser TTC CCA TGG Phe Pro Trp 460 1392 ATT CTG AAT Ile Leu Asn 465 GTG CTT AAT TTA Val Leu Asn Leu GCC TTT GAT TTT TAC AAA GTG ATC Ala Phe Asp Phe Tyr Lys Val Ile 475 1440 GAA AGT Glu Ser 480 TTT ATC AAA GCA Phe Ile Lys Ala

GAA

Glu 485 GGC AAC TTG ACA Gly Asn Leu Thr

AGA

Arg 490 GAA ATG ATA AAA Glu Met Ile Lys 1488 1536

CAT

His 495 TTA GAA CGA TGT Leu Glu Arg Cys

GAA

Glu 500 CAT CGA ATC ATG His Arg Ile Met

GAA

Glu 505 TCC CTT GCA TGG Ser Leu Ala Trp SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 TCA GAT TCA CCT Ser Asp Ser Pro TTT GAT CTT ATT Phe Asp Leu Ile

AAA

Lys 520 CAA TCA AAG GAC Gin Ser Lys Asp CGA GAA Arg Glu 525 1584 GGA CCA ACT Gly Pro Thr CAG AAT AAT Gin Asn Asn 545 CAC CTT GAA TCT His Leu Glu Ser

GCT

Ala 535 TGT CCT CTT AAT Cys Pro Leu Asn CTT CCT CTC Leu Pro Leu 540 1632 CAC ACT GCA GCA His Thr Ala Ala

GAT

Asp 550 ATG TAT CTT TCT CCT GTA AGA TCT Met Tyr Leu Ser Pro Val Arg Ser 555 1680 CCA AAG Pro Lys 560 AAA AAA GGT TCA Lys Lys Gly Ser ACG CGT GTA AAT Thr Arg Val Asn ACT GCA AAT GCA Thr Ala Asn Ala 1728

GAG

Glu 575 ACA CAA GCA ACC Thr Gin Ala Thr GCC TTC CAG ACC CAG AAG CCA TTG AAA Ala Phe Gin Thr Gin Lys Pro Leu Lys 585 1776 ACC TCT CTT TCA Thr Ser Leu Ser TTT TAT AAA AAA Phe Tyr Lys Lys

GTG

Val 600 TAT CGG CTA GCC Tyr Arg Leu Ala TAT CTC Tyr Leu 605 1824 1872 CGG CTA AAT Arg Leu Asn GAA CAT ATC Glu His Ile 625 CTT TGT GAA CGC CTT CTG TCT GAG CAC Leu Cys Glu Arg Leu Leu Ser Glu His 615 CCA GAA TTA Pro Glu Leu 620 AAT GAG TAT Asn Glu Tyr ATC TGG ACC CTT Ile Trp Thr Leu CAG CAC ACC CTG Gin His Thr Leu

CAG

Gin 635 1920 GAA CTC Glu Leu 640 ATG AGA GAC AGG Met Arg Asp Arg TTG GAC CAA ATT Leu Asp Gin Ile

ATG

Met 650 ATG TGT TCC ATG Met Cys Ser Met GGC ATA TGC AAA Gly Ile Cys Lys

GTG

Val 660 AAG AAT ATA GAC Lys Asn Ile Asp

CTT

Leu 665 AAA TTC AAA ATC Lys Phe Lys Ile

ATT

Ile 670 1968 2016 2064 GTA ACA GCA TAC Val Thr Ala Tyr

AAG

Lys 675 GAT CTT CCT CAT Asp Leu Pro His

GCT

Ala 680 GTT CAG GAG ACA Val Gin Glu Thr TTC AAA Phe Lys 685 CGT GTT TTG Arg Val Leu AAC TCG GTC Asn Ser Val 705 AAA GAA GAG GAG Lys Glu Glu Glu

TAT

Tyr 695 GAT TCT ATT ATA Asp Ser Ile Ile GTA TTC TAT Val Phe Tyr 700 CAG TAT GCT Gin Tyr Ala 2112 2160 TTC ATG CAG AGA Phe Met Gin Arg

CTG

Leu 710 AAA ACA AAT ATT Lys Thr Asn Ile

TTG

Leu 715 TCC ACC Ser Thr 720 AGG CCC CCT ACC Arg Pro Pro Thr TCA CCA ATA CCT Ser Pro Ile Pro

CAC

His 730 ATT CCT CGA AGC Ile Pro Arg Ser 2208 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTIUS98/03041 CCT TAC AAG TTT CCT AGT TCA CCC TTA CGG ATT CCT GGA GGG AAC ATC Pro Tyr Lys Phe Pro Ser Ser Pro Leu Arg Ile Pro Gly Gly Asn Ile 735 740 745 750 2256 2304 TAT ATT TCA CCC CTG AAG AGT CCA TAT AAA ATT TCA GAA GGT CTG Tyr Ile Ser Pro Leu Lys Ser Pro Tyr Lys Ile Ser Giu Gly Leu

CCA

Pro ACA CCA ACA Thr Pro Thr GAA TCA TTC Giu Ser Phe 785 TGT AAC AGC Cys Asn Ser AAA ATG Lys Met 770 ACT CCA AGA TCA AGA Thr Pro Arg Ser Arg 775 GGG ACT TCT GAG Gly Thr Ser Giu TTC CAG Phe Gin ATC TTA GTA TCA ATT GGT Ile Leu Val Ser Ile Gly 780 AAA ATA AAT CAG ATG GTA Lys Ile Asn Gin Met Vai 795 GCT GAA GGA AGC AAC CCT Aia Giu Gly Ser Asn Pro

CCA

Pro GAC CGT GTG Asp Arg Vai AAA AGA AGT Lys Arg Ser CGC TTT GAT Arg Phe Asp

CCT

Pro 815

GCA

Aila

ATT

Ile 810

GAA

Giu 2352 2400 2448 2496 2544 2592 GAT GGA Asp Gly CTG AAA AAA CTA Leu Lys Lys Leu 820 AGT AAA CAT CTC Ser Lys His Leu 835 ATG ACT TCT ACT Met Thr Ser Thr 850 GGA TCA GAT Gly Ser Asp

GAA

Giu 830 CCA GGA GAG Pro Giy Giu 840 CGA ACA CGA Arg Thr Arg 855 TCC AAA TTT CAG CAG AAA Ser Lys Phe Gin Gin Lys 845 ATG CAA AAG CAG AAA ATG Met Gin Lys Gin Lys Met 860 CTG GCA GAA Leu Ala Giu AAT GAT AGC ATG GAT ACC TCA AAC AAG GAA GAG AAA TGAGGATCTC Asn Asp Ser Met Asp Thr Ser Asn Lys Giu Giu Lys 2638

AGGACCTTGG

ATAACTTTCC

TAAAATGTGC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

TGGACACTGT

CAGGTTCTGT

AGATGCAATT

AAGATTGAAA

TCCAAAGTAA

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

GTACACCTCT

TTATGGCCAC

GTTTGGGTGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

GGATTCATTG

ATTTAATATC

TTCCTAAGCC

AATCCTGCCA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TCTCTCACAG

TTCAGCTCTT

ACTTGAAATG

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

AC!TGTGTGCT

AACCATATGA

ATGTGACTGT

TTGTGGATA

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA

TACTATCATA

2698 2758 2818 2878 2938 2998 3058 3118 3178 3238 3298 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 155 CTACTGAAAC AGATTTCATA CCTCAGAATG TAAAAGAACT TACTGATTAT TTTCTTCATC 3358 CAACTTATGT TTTTAAATGA GGATTATTGA TAGT 3392 INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 874 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Met Asp Phe Thr"Ala Leu Cys Gin Lys Leu Lys Ile Pro Asp His Val 1 5 10 Arg Giu Arg Ala Trp Leu Thr Trp Glu Lys Val Ser Ser Val Asp Gly 25 Val Leu Gly Gly Tyr Ile Gln Lys Lys Lys Glu Leu Trp Gly Ile Cys 40 Ile Phe Ile Ala Ala Val Asp Leu Asp Glu Met Ser Phe Thr Phe Thr 55 Giu Leu Gin Lys Asn Ile Giu Ile Ser Val His Lys Phe Phe Asn Leu 70 75 Leu Lys Giu Ile Asp Thr Ser Thr Lys Val Asp Asn Ala Met Ser Arg 90 Leu Leu Lys Lys Tyr Asp Val Leu Phe Ala Leu Phe Ser Lys Leu Giu 100 105 110 Arg Thr Cys Giu Leu Ile Tyr Leu Thr Gin Pro Ser Ser Ser Ile Ser 115 120 125 Thr Glu Ile Asn Ser Ala Leu Vai Leu Lys Val Ser Trp Ile Thr Phe 130 135 140 Leu Leu Aia Lys Gly Glu Vai Leu Gin Met Giu Asp Asp Leu Val Ile 145 150 155 160 Ser Phe Gin Leu Met Leu Cys Vai Leu Asp Tyr Phe Ile Lys Leu Ser 165 170 175 Pro Pro Met Leu Leu Lys Giu Pro Tyr Lys Thr Ala Vai Ile Pro Ile 180 185 190 Asn Gly Ser Pro Arg Thr Pro Arg Arg Giy Gin Asn Arg Ser Ala Arg 195 200 205 Ile Ala Lys Gin Leu Giu Asn Asp Thr Arg Ile Ile Giu Val Leu Cys 210 215 220 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 Lys 225 Asn Leu Lys Leu Lys 305 Vai Ser Cys Giy Vai 385 Arg Gin Leu Se:; Asn 465 Phe Giu Ser Giu Phe Pro Asn Gin 290 Ser Arg Ala Thr Tyr 370 Giu Vali Asn Ala Gin 450 Val Ile Arg Pro His Ile Glu Lys 275 Thr Asn Thr Ser Vai 355 Ile Ile Met Phe Cys 435 Asn Leu Lys Cys Leu 515 Giu Pro Val 260 Asp Asp Leu Val Asp 340 Asn Phe Giy Giu Ser 420 Aia Leu Asn Aila Giu 500 Phe Cys Phe 245 Giu Leu Ser Asp Met 325 Gin Pro Lys Ser Ser 405 Lys Leu Asp Leu Giu 485 His Asp Asn 230 Met Asn Asp Ile Giu 310 Asn Pro Lys Giu Gin 390 Met Leu Giu Ser Lys 470 Gly Arg Leu Giu Leu Lys 265 Leu Phe Asn Gin Asn 345 Ile Ala Lys Ser Asp 425 Met Asp Asp Thr Glu 505 Gin Val Gly 250 Arg Phe Giu Val Gin 330 Leu Leu Lys Leu Giu 410 Asn Ala Leu Phe Arg 490 Ser Ser Lys 235 Leu Tyr Leu Thr Ile 315 Leu Ile Lys Ala Giy 395 Giu Ile Thr Ser Tyr 475 Giu Leu Lys Asn Val Vai Thr Giu Giu Asp His 285 Gin Arg 300 Pro Pro Met Met Ser Tyr Arg Val 365 Val Gly 380 Val Arg Glu Arg Phe His Tyr Ser 445 Phe Pro 460 Lys Vai Met Ile Ala Trp Asp Arg 525 Tyr Ser Ile 270 Asp Thr His Ile Phe 350 Lys Gin Leu Leu Met 430 Arg Trp Ile Lys Leu 510 Giu Phe Asn 255 Tyr Lys Pro Thr Leu 335 Asn Asp Gly Tyr Ser 415 Ser Ser Ile Giu His 495 Ser Giy Lys 240 Giy Leu Thr Arg Pro 320 Asn Asn Ile Cys Tyr 400 Ile Leu Thr Leu Ser 480 Leu Asp Pro SUBSTITUTE SHEET (RULE 26) W~O 98/37091 PCT/US98/03041 Thr Asn 545 Lys Gin Leu Asn Ile 625 Met Ile Ala Leu Vai 705 Arg Lys Ser Thr Phe 785 AsD 530 His Lys Ala Ser Thr 610 Ile Arg Cys Tyr Ile 690 Phe Pro Phe Pro Lys 770 Gly His Thr Gly Thr Leu 595 Leu Trp Asp Lys Lys 675 Lys Met Pro Pro Leu 755 Met Thr Leu Ala Ser Ser 580 Phe Cys Thr Arg Val 660 Asp Glu Gin Thr Ser 740 Lys Thr Ser Glu Ala Thr 565 Ala Tyr Glu Leu His 645 Lys Leu Glu Arg Leu 725 Ser Ser Pro Glu Ser Asp 550 Thr Phe Lys Arg Phe 630 Leu Asn Pro Glu Leu 710 Ser Pro Pro Arg Lys 790 Ala 535 Met Arg Gin Lys Leu 615 Gin Asp Ile His Tyr 695 Lys Pro Leu Tyr Ser 775 Phe Cys Tyr Val Thr Vai 600 Leu His Gin Asp Ala 680 Asp Thr Ile Arg Lys 760 Arg Gin Pro Leu~ Asn Gin 585 Tyr Ser Thr Ile Leu 665 Val Ser Asn Pro Ile 745 Ile Ile Lys Leu Ser Ser 570 Lys Arg Glu Leu Met 650 Lys Gin Ile Ile His 730 Pro Ser Leu Ile Asn Pro 555 Thr Pro Leu His Gin 635 Met Phe Glu Ile Leu 715 Ile Gly Glu Vai Asn 795 Leu 540 Val Ala Leu Ala Pro 620 Asn Cys Lys Thr Vai 700 Gin Pro Gly Gly Ser 780 Gin Pro Arg Asn Lys Tyr 605 Glu Glu Ser Ile Phe 685 Phe Tyr Arg Asn Leu 765 Ile Met Leu Ser Ala Ser 590 Leu Leu Tyr Met Ile 670 Lys Tyr Ala Ser Ile 750 Pro Gly Val Gin Pro Glu 575- Thr Arg Glu Glu Tyr 655 Vai Arg Asn Ser Pro 735 Tyr Thr Glu Cys Asn Lys 560 Thr Ser Leu His Leu 640 Gly Thr Val Ser Thr 720 Tyr Ile Pro Ser Asn 800 Ser Asp Arg Val Lys Arg Ser Ala Gly Ser Asn Pro Pro Lys 815 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 Pro Leu Lys Lys 820 Gly Ser Lys His 835 Leu Arg Phe Asp Ile 825 Leu Pro Gly Glu Ser 840 Glu Gly Ser Asp Glu Ala Asp 830 LysLeu Ala Lys Phe Gin Gin 845 Glu Met Thr Ser Thr Arg 850 Thr Arg Met Gin Lys Gin 855 860 Lys Met Asn Asp Ser 865 Met Asp Thr Ser Lys Glu Glu Lys INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 3323 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2559 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GCCATC ATG GAG AAA GTT TCA TCT GTG GAT GGA GTA TTG GGA GGT TAT Met Glu Lys Val Ser Ser Val Asp Gly Val Leu Gly Gly Tyr 1 5 ATT CAA AAG AAA AAG GAA CTG TGG GGA ATC TGT ATC TTT ATT GCA GC1 Ile Gin Lys Lys Lys Glu Leu Trp Gly Ile Cys Ile Phe Ile Ala Ali GTT GAC CTA GAT Val Asp Leu Asp ATG TCG TTC ACT TTT ACT GAG CTA CAG AAA AAC Met Ser Phe Thr Phe Thr Glu Leu Gin Lys Asn ATA GAA ATC Ile Glu Ile ACC AGT ACC Thr Ser Thr GTC CAT AAA TTC Val His Lys Phe AAC TTA CTA AAA Asn Leu Leu Lys GAA ATT GAT Glu Ile Asp AAG AAG TAT Lys Lys Tyr AAA GTT GAT AAT Lys Val Asp Asn ATG TCA AGA CTG Met Ser Arg Leu GAT GTA Asp Val TTG TTT GCA CTC Leu Phe Ala Leu

TTC

Phe AGC AAA TTG GAA Ser Lys Leu Glu ACA TGT GAA CTT Thr Cys Glu Leu TAT TTG ACA CAA Tyr Leu Thr Gin AGC AGT TCG Ser Ser Ser ATA TCT Ile Ser 105 ACA TTT Thr Phe 120 ACT GAA ATA AAT Thr Glu Ile Asn GCA TTG GTG CTA AAA GTT TCT TGG ATC Ala Leu Val Leu Lys Val Ser Trp Ile 115 TTA TTA GCT Leu Leu Ala AAA GGG Lys Gly 125 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 GAA GTA TTA Glu Val Leu CTA TGT GTC Leu Cys Vai 145 ATG GAA GAT GAT Met Giu Asp Asp CTG GTG ATT TCA TTT CAG TTA ATG Leu Val Ile Ser Phe Gin Leu Met 135 140 CTT GAC TAT TTT Leu Asp Tyr Phe AAA CTC TCA CCT Lys Leu Ser Pro

CCC

Pro 155 ATG TTG CTC Met Leu Leu AAA GAA Lys Glu 160 CCA TAT AAA ACA Pro Tyr Lys Thr GTT ATA CCC ATT Val Ile Pro Ile

AAT

Asn 170 GGT TCA CCT CGA Gly Ser Pro Arg

ACA

Thr 175 CCC AGG CGA GGT Pro Arg Arg Gly AAC AGG AGT GCA Asn Arg Ser Ala ATA GCA AAA CAA Ile Ala Lys Gin

CTA

Leu 190 GAA AAT GAT ACA Glu Asn Asp Thr ATT ATT GAA GTT Ile Ile Giu Val

CTC

Leu 200 TGT AAA GAA CAT Cys Lys Giu His GAA TGT Glu Cys 205 AAT ATA GAT Asn Ile Asp ATG AAT TCT Met Asn Ser 225 GTG AAA AAT GTT Val Lys Asn Val

TAT

Tyr 215 TTC AAA AAT TTT Phe Lys Asn Phe ATA CCT TTT Ile Pro Phe 220 GAG GTT GAA Glu Val Glu 672 720 CTT GGA CTT GTA Leu Gly Leu Vai

ACA

Thr 230 TCT AAT GGA CTT Ser Asn Gly Leu AAT CTT Asn Leu 240 TCT AAA CGA TAC Ser Lys Arg Tyr GAA ATT TAT CTT Glu Ile Tyr Leu

AAA

Lys 250 AAT AAA GAT CTA Asn Lys Asp Leu

GAT

Asp 255 GCA AGA TTA TTT Ala Arg Leu Phe GAT CAT GAT AAA Asp His Asp Lys

ACT

Thr 265 CTT CAG ACT GAT Leu Gin Thr Asp

TCT

Ser 270 ATA GAC AGT TTT Ile Asp Ser Phe

GAA

Glu 275 ACA CAG AGA ACA Thr Gin Arg Thr CGA AAA AGT AAC Arg Lys Ser Asn CTT GAT Leu Asp 285 GAA GAG GTG Glu Giu Vai AAC ACT ATC Asn Thr Ile 305

AAT

Asn 290 GTA ATT CCT CCA Val Ile Pro Pro ACT CCA GTT AGG Thr Pro Val Arg ACT GTT ATG Thr Val Met 300 CAA CAA TTA ATG Gin Gin Leu Met ATT TTA AAT TCA GCA AGT GAT CAA Ile Leu Asn Ser Ala Ser Asp Gin 315 CCT TCA Pro Ser 320 GAA AAT CTG ATT Glu Asn Leu Ile TAT TTT AAC AAC Tyr Phe Asn Asn ACA GTG AAT CCA Thr Val Asn Pro 1008 1056 AAA GAA AGT ATA Lys Glu Ser Ile 335 CTG AAA AGA GTG AAG GAT Leu Lys Arg Val Lys Asp 340 ATA GGA TAC ATC TTT Ile Gly Tyr Ile Phe 345 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 GAG AAA TTT GCT Giu Lys Phe Ala

AAA

Lys 355 GCT GTG GGA CAG Ala Vai Gly Gin TGT GTC GAA ATT Cys Val Giu Ile GGA TCA Gly Ser 365 1104 CAG CGA TAC Gin Arg Tyr ATG CTT AAA Met Leu Lys 385

AAA

Lys 370 CTT GGA GTT CGC Leu Gly Val Arg

TTG

Leu 375 TAT TAC CGA GTA Tyr Tyr Arg Vai ATG GAA TCC Met Giu Ser 380 TTT AGC AAA Phe Ser Lys 1152 1200 TCA GAA GAA GAA Ser Giu Giu Glu

CGA

Arg 390 TTA TCC ATT CAA Leu Ser Ile Gin CTT CTG Leu Leu 400 AAT GAC AAC ATT Asn Asp Asn Ile

TTT

Phe 405 CAT ATG TCT TTA His Met Ser Leu

TTG

Leu 410 GCG TGC GCT CTT Ala Cys Ala Leu GTT GTA ATG GCC Val Val Met Ala

ACA

Thr 420 TAT AGC AGA AGT Tyr Ser Arg Ser

ACA

Thr 425 TCT CAG AAT CTT Ser Gin Asn Leu 1248 1296 1344 TCT GGA ACA GAT Ser Giy Thr Asp

TTG

Leu 435 TCT TTC CCA TGG Ser Phe Pro Trp

ATT

Ile 440 CTG AAT GTG CTT Leu Asn Val Leu AAT TTA Asn Leu 445 AAA GCC TTT GAT TTT TAC AAA GTG Lys Ala Phe Asp Phe Tyr Lys Val 450

ATC

Ile 455 GAA AGT TTT ATC Giu Ser Phe Ile AAA GCA GAA Lys Ala Giu 460 TGT GAA CAT Cys Glu His 1392 GGC AAC TTG Gly Asn Leu 465 ACA AGA GAA ATG Thr Arg Glu Met

ATA

Ile 470 AAA CAT TTA GAA Lys His Leu Giu 1440 CGA ATC Arg Ile 480 ATG GAA TCC CTT Met Glu Ser Leu

GCA

Ala 485 TGG CTC TCA GAT Trp Leu Ser Asp CCT TTA TTT GAT Pro Leu Phe Asp

CTT

Leu 495 ATT AAA CAA TCA Ile Lys Gin Ser

AAG

Lys 500 GAC CGA GAA GGA Asp Arg Giu Gly ACT GAT CAC CTT Thr Asp His Leu

GAA

Giu 510 1488 1536 1584 TCT GCT TGT CCT Ser Ala Cys Pro

CTT

Leu 515 AAT CTT CCT CTC Asn Leu Pro Leu AAT AAT CAC ACT Asn Asn His Thr GCA GCA Aia Ala 525 GAT ATG TAT Asp Met Tyr ACG CGT GTA Thr Arg Val 545

CTT

Leu 530 TCT CCT GTA AGA Ser Pro Val Arg CCA AAG AAA AAA Pro Lys Lys Lys GGT TCA ACT Gly Ser Thr 540 ACC TCA GCC Thr Ser Ala 1632 1680 AAT TCT ACT GCA Asn Ser Thr Ala GCA GAG ACA CAA Ala Giu Thr Gin

GCA

Ala 555 TTC CAG Phe Gin 560 ACC CAG AAG CCA Thr Gin Lys Pro AAA TCT ACC Lys Ser Thr TCT CTT Ser Leu 570 TCA CTG TTT TAT Ser Leu Phe Tyr 1728 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041

AAA

Lys 575 AAA GTG TAT CGG Lys Val Tyr Arg GCC TAT CTC CGG Ala Tyr Leu Arg

CTA

Leu 585 AAT ACA CTT TGT Asn Thr Leu Cys 1776 1824 CGC CTT CTG TCT Arg Leu Leu Ser CAC CCA GAA TTA His Pro Glu Leu

GAA

Glu 600 CAT ATC ATC TGG His Ile Ile Trp ACC CTT Thr Leu 605 TTC CAG CAC Phe Gin His TTG GAC CAA Leu Asp Gin 625

ACC

Thr 610 CTG CAG AAT GAG Leu Gin Asn Glu

TAT

Tyr 615 GAA CTC ATG AGA Glu Leu Met Arg GAC AGG CAT Asp Arg His 620 AAA GTG AAG Lys Val Lys 1872 1920 ATT ATG ATG TGT Ile Met Met Cys ATG TAT GGC ATA Met Tyr Gly Ile AAT ATA Asn Ile 640 GAC CTT AAA TTC Asp Leu Lys Phe ATC ATT GTA ACA Ile Ile Val Thr TAC AAG GAT CTT Tyr Lys Asp Leu 1968 2016 CAT GCT GTT CAG His Ala Val Gin ACA TTC AAA CGT Thr Phe Lys Arg

GTT

Val 665 TTG ATC AAA GAA Leu Ile Lys Glu GAG TAT GAT TCT ATT ATA GTA TTC TAT Glu Tyr Asp Ser Ile Ile Val Phe Tyr 675

AAC

Asn 680 TCG GTC TTC ATG Ser Val Phe Met CAG AGA Gin Arg 685 2064 CTG AAA ACA Leu Lys Thr TCA CCA ATA Ser Pro Ile 705

AAT

Asn 690 ATT TTG CAG TAT Ile Leu Gin Tyr

GCT

Ala 695 TCC ACC AGG CCC Ser Thr Arg Pro CCT ACC TTG Pro Thr Leu 700 CCT AGT TCA Pro Ser Ser 2112 2160 CCT CAC ATT CCT Pro His Ile Pro

CGA

Arg 710 AGC CCT TAC AAG Ser Pro Tyr Lys

TTT

Phe 715 CCC TTA Pro Leu 720 CGG ATT CCT GGA Arg Ile Pro Gly

GGG

Gly 725 AAC ATC TAT ATT Asn Ile Tyr Ile

TCA

Ser 730 CCC CTG AAG AGT Pro Leu Lys Ser

CCA

Pro 735 TAT AAA ATT TCA Tyr Lys Ile Ser GGT CTG CCA ACA Gly Leu Pro Thr

CCA

Pro 745 ACA AAA ATG ACT Thr Lys Met Thr 2208 2256 2304 AGA TCA AGA ATC Arg Ser Arg Ile

TTA

Leu 755 GTA TCA ATT GGT Val Ser Ile Gly TCA TTC GGG ACT Ser Phe Gly Thr TCT GAG Ser Glu 765 AAG TTC CAG Lys Phe Gin AAA AGA AGT Lys Arg Ser 785

AAA

Lys 770 ATA AAT CAG ATG Ile Asn Gin Met

GTA

Val 775 TGT AAC AGC GAC Cys Asn Ser Asp CGT GTG CTC Arg Val Leu 780 AAA AAA CTA Lys Lys Leu 2352 2400 GCT GAA GGA AGC Ala Glu Gly Ser

AAC

Asn 790 CCT CCT AAA CCA Pro Pro Lys Pro

CTG

Leu 795 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTIUS98/03041 162 CGC TTT GAT ATT GAA GGA TCA GAT GAA GCA GAT GGA AGT AAA CAT CTC Arg Phe Asp Ile Glu Gly Ser Asp Glu Ala Asp Gly Ser Lys His Leu 800 805 810 2448 GGA GAG TCC AAA Gly Glu Ser Lys ACA CGA ATG Thr Arg Met

CAA

Gin 835

AAA

TTT CAG CAG AAA CTG GCA GAA ATG ACT TCT ACT Phe Gin Gin Lys Leu Ala Giu Met Thr Ser Thr 820 825 830 AAG CAG AAA ATG AAT GAT AGC ATG GAT ACC TCA Lys Gin Lys Met Asn Asp Ser Met Asp Thr Ser 840 845 TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT AAC AAG GAA GAG Asn Lys Glu Glu Lys

GGATTCATTG

ATTTAATATC

TTCCTAAGCC

AATCCTGCCA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TAAAAGAACT

TAGT

TCTCTCACAG

TTCAGCTCTT

ACTTGAAATG

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

ATGTGACTGT

TTTGTGGATA

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA

TACTATCATA

TTTCTTCATC

ATAACTTTCC

TAAAATGTGC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

CAGGTTCTGT

AGATGCAATT

AAGATTGAAA

TCCAAAGTA-A

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

TTTTAAATGA

TTATGGCCAC

GTTTGGGTGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

CCTCAGAATG

GGATTATTGA

2496 2544 2599 2659 2719 2779 2839 2899 2959 3019 3079 3139 3199 3259 3319 3323 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 851 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: Met Giu Lys Val Ser Ser Val Asp Gly Val Leu Gly Giy Tyr Ile Gln 1 5 10 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/03041 Lys Leu Ile Thr Leu Val Leu Val 145 Pro Arg Asp Asp Ser 225 Ser Arg Ser Val Ile 305 Lys Giu Giu Met Vai His Val Asp Ala Leu Gin Pro 100 Lys Vai 115 Met Giu Asp Tyr Lys Thr Giy Gin 180 Arg Ile 195 Vai Lys Giy Leu Arg Tyr Phe Leu 260 Giu Thr 275 Val Ile Gin Leu Leu Trp Gly Ile Phe Asn Ser Leu Ile Thr 120 Vai Leu Pro Ala Leu 200 Phe Asn Tyr Lys Pro 280 Thr Leu Cys Ile Thr Giu Leu Leu Arg Leu Giu Arg Ser Thr 105 Phe Leu Ile Ser Ser Pro Ile Asn 170 Arg Ile 185 Cys Lys Lys Asn Gly Leu Leu Lys 250 Thr. Leu 265 Arg Lys Pro Vai Asn Ser Ile Gin Giu Lys Cys Ile Aia Gin 140 Met Ser Lys His Ile 220 Giu Lys Thr Asn Thr 300 Ser Asp Giu Ser Vai Tyr Leu Vai Cys Giu 160 Pro Asn Ile Asn Leu 240 Ala Asp Giu Thr Ser 320 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTJUS98/03041 164 Giu Asn Leu Ile Ser Tyr Phe Asn Asn Cys Thr Val Asn Pro Lys Giu 325 330 335 Ser Ile Leu Lys Arg Vai Lys Asp Ile Gly Tyr Ile Phe Lys Giu Lys 340 *34*5 350 Phe Ala Lys Ala Vai Gly Gin Gly Cys Val Giu Ile Gly Ser Gin Arg 355 360 365 Tyr Lys Leu Gly Val Arg Leu Tyr Tyr Arg Val Met Giu Ser Met Leu 370 375 380 Lys Ser Giu Giu Giu Arg Leu Ser Ile Gin Asn Phe Ser Lys Leu Leu 385 390 395 400 Asn Asp Asn Ile Phe His Met Ser Leu Leu Ala Cys Ala Leu Glu Vai 405 410 41-5 Val Met Ala Thr Tyr Ser Arg Ser Thr Ser Gin Asn Leu Asp Ser Gly 420 425 430 Thr Asp Leu Ser Phe Pro Trp Ile Leu Asn Val Leu Asn Leu Lys Ala 435 440 445 Phe Asp Phe Tyr Lys Val Ile Glu Ser Phe Ile Lys Ala Glu Gly Asn 450 455 460 Leu Thr Arg Giu Met Ile Lys His Leu Giu Arg Cys Giu His Arg Ile 465 470 475 480 Met Glu Ser Leu Ala Trp Leu Ser Asp Ser Pro Leu Phe Asp Leu Ile 485 490 495 Lys Gin Ser Lys Asp Arg Giu Gly Pro Thr Asp His Leu Giu Ser Ala 500 505 510 Cys Pro Leu Asn Leu Pro Leu Gin Asn Asn His Thr Ala Ala Asp Met 515 520 525 Tyr Leu Ser Pro Val Arg Ser Pro Lys Lys Lys Gly Ser Thr Thr Arg 530 535 540 Val Asn Ser Thr Ala Asn Ala Giu Thr Gin Ala Thr Ser Ala Phe Gin 545 550 555 560 Thr Gin Lys Pro Leu Lys Ser Thr Ser Leu Ser Leu Phe Tyr Lys Lys 565 570 575 Val Tyr Arg Leu Ala Tyr Leu Arg Leu Asn Thr Leu Cys Giu Arg Leu 580 585 590 Leu Ser Giu His Pro Giu Leu Giu His Ile Ile Trp, Thr Leu Phe Gin 595 600 605 His Thr Leu Gin Asn Giu Tyr Giu Leu Met Arg Asp Arg His Leu Asp 610 615 620 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PTU9/34 PCT[tJS98/03041 Gin 625 Asp Ala Asp Thr Ile 705 Arg Lys Arg Gin Ser 785 Asp Giu Arg G~bu Ile Leu Vai Ser Asn 690 Pro Ile Ile Ile Lys 770 Ala Ile Ser Met Glu 850 Met Lys Gin le 675 Ile His Pro Ser Leu 755 Ile Glu Glu Lys Gin 835 Lys Met Phe Glu 660 Ile Leu Ile Gly Glu 740 Val Asn Giy Gly Phe 820 Lys Cys Lys 645 Thr Val Gin Pro Giy 725 Gly Ser Gin S er Ser 805 Gin Ser 630 Ile Phe Phe Tyr Arg 710 Asn Leu Ile Met Asn 790 Asp Gin Met Ile Lys Tyr Al a 695 Ser Ile Pro Gly Val 775 Pro Glu Lys Tyr Val Arg Asn 680 Ser Pro Tyr Thr Giu 760 Cys Pro Ala Leu Gly Thr Val 665 Ser Thr Tyr Ile Pro 745 Ser Asn Lys Asp Ala 825 Ile Cys 635 Ala Tyr 650 Leu Ile Val Phe Arg Pro Lys Phe 715 Ser Pro 730 Thr Lys Phe Gly Ser Asp Pro Leu 795 Gly Ser 810 Giu Met Lys Lys Lys Met Pro 700 Pro Leu Met Thr Arg 780 Lys Lys Thr Val Asp Giu Gln 685 Thr Ser Lys Thr S er 765 Val Lys His Ser Lys Leu Glu 670 Arg Leu Ser Ser Pro 750 Giu Leu Leu Leu Thr 830 Asn Pro 655 Glu Leu Ser Pro Pro 735 Arg Lys Lys Arg Pro 815 Arg Ile 640 His Tyr Lys Pro Leu 720 Tyr Ser Phe Arg Phe 800 Gly Thr Gin Lys Met Asn Asp Ser Met Asp Thr Ser Asn Lys INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 3266 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT[US98/03041 166 LOCATION:7. .2502 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: GCCATC ATG CTG TGG GGA ATC TGT ATC TTT ATT GCA GCA GTT GAC CTA Met Leu Trp Gly Ile Cys Ile Phe Ile Ala Ala Val Asp Leu GAG ATG TCG TTC ACT TTT ACT GAG CTA Giu Met Ser Phe Thr Phe Thr Giu Leu AAA AAC ATA GAA Lys Asn Ile Giu AGT GTC CAT AAA Ser Vai His Lys TTT AAC TTA CTA Phe Asn Leu Leu GAA ATT GAT ACC AGT ACC Giu Ile Asp Thr Ser Thr AAA GTT GAT Lys Val Asp TTT GCA CTC Phe Ala Leu

AAT

Asn so GGT ATG TCA AGA CTG TTG AAG AAG TAT Ala Met Ser Arg Leu Leu Lys Lys Tyr 55 GAT GTA TTG Asp Val Leu ATA TAT TTG Ile Tyr Leu TTC AGC AAA TTG Phe Ser Lys Leu AGG ACA TGT GAA Arg Thr Cys Giu 240 ACA CAA Thr Gin CCC AGC AGT TCG Pro Ser Ser Ser

ATA

Ile TCT ACT GAA ATA Ser Thr Glu Ile TCT GCA TTG GTG Ser Ala Leu Val 288

CTA

Leu AAA GTT TCT TGG Lys Val Ser Trp, ACA TTT TTA TTA Thr Phe Leu Leu

GCT

Ala 105 AAA GGG GAA GTA Lys Gly Glu Val

TTA

Leu 110 CAA ATG GAA GAT Gin Met Giu Asp CTG GTG ATT TCA Leu Vai Ile Ser CAG, TTA ATG CTA Gin Leu Met Leu TGT GTC Cys Vai 125S CTT GAC TAT TTT ATT AAA CTC TCA Leu Asp Tyr Phe Ile Lys Leu Ser CCC ATG TTG CTC Pro Met Leu Leu AAA GAA CCA Lys Giu Pro 140 ACA CCC AGG Thr Pro Arg TAT AAA ACA GCT GTT ATA CCC Tyr Lys Thr Aia Val Ile Pro

ATT

Ile 0 AAT GGT TCA CCT Asn Gly Ser Pro CGA GGT Arg Gly 160 CAG AAC AGG AGT Gin Asn Arg Ser CGG ATA GCA AAA Arg Ile Aia Lys

CAA

Gin 17 0 CTA GAA AAT GAT Leu Giu Asn Asp

ACA

Thr 175 AGA ATT ATT GAA Arg Ile Ile Giu CTC TGT AAA GAA Leu Cys Lys Giu

CAT

His 185 GAA TGT AAT ATA Glu Cys Asn Ile

GAT

Asp 190 GAG GTG AAA AAT Giu Vai Lys Asn TAT TTC AAA AAT Tyr Phe Lys Asn ATA CCT TTT ATG Ile Pro Phe Met AAT TCT Asn Ser 205 SUBSTITUTE SHEET (RULE 26) WO 98/37091 CTT GGA CTT Leu Gly Leu AAA CGA TAC Lys Arg Tyr 225 PCT/US98/03041

GTA

Val 210 ACA TCT AAT GGA Thr Ser Asn Gly CCA GAG GTT GAA Pro Glu Val Glu AAT CTT TCT Asn Leu Ser 220 GAT GCA AGA Asp Ala Arg GAA GAA ATT TAT Glu Glu Ile Tyr

CTT

Leu 230 AAA AAT AAA GAT Lys Asn Lys Asp TTA TTT Leu Phe 240 TTG GAT CAT GAT Leu Asp His Asp

AAA

Lys 245 ACT CTT CAG ACT Thr Leu Gin Thr

GAT

Asp 250 TCT ATA GAC AGT Ser Ile Asp Ser GAA ACA CAG AGA Glu Thr Gin Arg

ACA

Thr 260 CCA CGA AAA AGT Pro Arg Lys Ser CTT GAT GAA GAG Leu Asp Glu Glu

GTG

Val 270 AAT GTA ATT CCT CCA CAC ACT CCA GTT Asn Val Ile Pro Pro His Thr Pro Val 275

AGG

Arg 280 ACT GTT ATG AAC Thr Val Met Asn ACT ATC Thr Ile 285 CAA CAA TTA Gin Gin Leu AAT CTG ATT Asn Leu Ile 305 ATG ATT TTA AAT TCA GCA AGT GAT CAA Met Ile Leu Asn Ser Ala Ser Asp Gln CCT TCA GAA Pro Ser Glu 300 TCC TAT TTT AAC Ser Tyr Phe Asn TGC ACA GTG AAT CCA AAA GAA AGT Cys Thr Val Asn Pro Lys Glu Ser 315 ATA CTG Ile Leu 320 AAA AGA GTG AAG Lys Arg Val Lys ATA GGA TAC ATC Ile Gly Tyr Ile AAA GAG AAA TTT Lys Glu Lys Phe

GCT

Ala 335 AAA GCT GTG GGA Lys Ala Val Gly GGT TGT GTC GAA Gly Cys Val Glu GGA TCA CAG CGA Gly Ser Gin Arg

TAC

Tyr 350 1008 1056 1104 AAA CTT GGA GTT Lys Leu Gly Val TTG TAT TAC CGA Leu Tyr Tyr Arg

GTA

Val 360 ATG GAA TCC ATG Met Glu Ser Met CTT AAA Leu Lys 365 TCA GAA GAA Ser Glu Glu GAC AAC ATT Asp Asn Ile 385

GAA

Glu 370 CGA TTA TCC ATT Arg Leu Ser Ile

CAA

Gin 375 AAT TTT AGC AAA Asn Phe Ser Lys CTT CTG AAT Leu Leu Asn 380 GAG GTT GTA Glu Val Val 1152 TTT CAT ATG TCT TTA TTG GCG TGC GCT Phe His Met Ser Leu Leu Ala Cys Ala 1200 1248 ATG GCC Met Ala 400 ACA TAT AGC AGA Thr Tyr Ser Arg ACA TCT CAG AAT Thr Ser Gin Asn GAT TCT GGA ACA Asp Ser Gly Thr GAT TTG TCT TTC CCA Asp Leu Ser Phe Pro 415 ATT CTG AAT GTG CTT AAT TTA AAA GCC Ile Leu Asn Val Leu Asn Leu Lys Ala 425

TTT

Phe 430 1296 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 GAT TTT TAC AAA GTG ATC GAA AGT TTT ATC Asp Phe Tyr Lys Val Ile 435 Glu Ser Phe AAA GCA GAA GGC Lys Ala Glu Gly Ile 440 AAC TTG Asn Leu 445 1344 ACA AGA GAA Thr Arg Glu GAA TCC CTT Glu Ser Leu 465 ATA AAA CAT TTA Ile Lys His Leu

GAA

Glu 455 CGA TGT GAA CAT Arg Cys Glu His CGA ATC ATG Arg Ile Met 460 CTT ATT AAA Leu Ile Lys 1392 1440 GCA TGG CTC TCA Ala Trp Leu Ser TCA CCT TTA TTT Ser Pro Leu Phe

GAT

Asp 475 CAA TCA Gin Ser 480 AAG GAC CGA GAA Lys Asp Arg Glu

GGA

Gly 485 CCA ACT GAT CAC Pro Thr Asp His

CTT

Leu 490 GAA TCT GCT TGT Glu Ser Ala Cys 1488 1536 CTT AAT CTT CCT Leu Asn Leu Pro

CTC

Leu 500 CAG AAT AAT CAC Gin Asn Asn His

ACT

Thr 505 GCA GCA GAT ATG Ala Ala Asp Met

TAT

Tyr 510 CTT TCT CCT GTA Leu Ser Pro Val TCT CCA AAG AAA Ser Pro Lys Lys

AAA

Lys 520 GGT TCA ACT ACG Gly Ser Thr Thr CGT GTA Arg Val 525 1584 AAT TCT ACT Asn Ser Thr CAG AAG CCA Gin Lys Pro 545 AAT GCA GAG ACA Asn Ala Glu Thr

CAA

Gin 535 GCA ACC TCA GCC Ala Thr Ser Ala TTC CAG ACC Phe Gin Thr 540 AAA AAA GTG Lys Lys Val 1632 1680 TTG AAA TCT ACC Leu Lys Ser Thr CTT TCA CTG TTT Leu Ser Leu Phe

TAT

Tyr 555 TAT CGG Tyr Arg 560 CTA GCC TAT CTC Leu Ala Tyr Leu CTA AAT ACA CTT Leu Asn Thr Leu GAA CGC CTT CTG Glu Arg Leu Leu 1728 1776

TCT

Ser 575 GAG CAC CCA GAA Glu His Pro Glu

TTA

Leu 580 GAA CAT ATC ATC Glu His Ile Ile

TGG

Trp 585 ACC CTT TTC CAG Thr Leu Phe Gin

CAC.

His 590 ACC CTG CAG AAT Thr Leu Gin Asn TAT GAA CTC ATG Tyr Glu Leu Met GAC AGG CAT TTG Asp Arg His Leu GAC CAA Asp Gin 605 1824 ATG ATG TGT Met Met Cys 610 TCC ATG TAT GGC Ser Met Tyr Gly

ATA

Ile 615 TGC AAA GTG AAG Cys Lys Val Lys AAT ATA GAC Asn Ile Asp 620 CCT CAT GCT Pro His Ala 1872 1920 CTT AAA TTC Leu Lys Phe 625 AAA ATC ATT GTA Lys Ile Ile Val

ACA

Thr 630 GCA TAC AAG GAT Ala Tyr Lys Asp

CTT

Leu 635 GTT CAG Val Gin 640 GAG ACA TTC AAA Glu Thr Phe Lys GTT TTG ATC AAA Val Leu Ile Lys

GAA

Glu 650 GAG GAG TAT GAT Glu Glu Tyr Asp 1968 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 TCT ATT ATA GTA Ser Ile Ile Val 655 AAT ATT TTG CAG Asn Ile Leu Gin TTC TAT AAC TCG GTC TTC ATG Phe Tyr Asn Ser Vai Phe Met 660 665 CAG AGA CTG AAA Gin Arg Leu Lys

ACA

Thr 670 2016 2064

TAT

Tyr 675 GCT TCC ACC AGG Ala Ser Thr Arg

CCC

Pro 680 CCT ACC TTG TCA Pro Thr Leu Ser CCA ATA Pro Ile 685 CCT CAC ATT Pro His Ile ATT CCT GGA Ile Pro Gly 705

CCT

Pro 690 CGA AGC CCT TAC Arg Ser Pro Tyr TTT CCT AGT TCA Phe Pro Ser Ser CCC TTA CGG Pro Leu Arg 700 CCA TAT AAA Pro Tyr Lys 2112 2160 GGG AAC ATC TAT Gly Asn Ile Tyr TCA CCC CTG AAG Ser Pro Leu Lys ATT TCA Ile Ser 720 GAA GGT CTG CCA Glu Gly Leu Pro CCA ACA AAA ATG Pro Thr Lys Met

ACT

Thr 730 CCA AGA TCA AGA Pro Arg Ser Arg 2208

ATC

Ile 735 TTA GTA TCA ATT GGT GAA TCA TTC GGG Leu Val Ser Ile Gly Giu Ser Phe Gly TCT GAG AAG TTC Ser Giu Lys Phe 2256 2304 AAA ATA AAT CAG ATG GTA TGT AAC AGC Lys Ile Asn Gin Met Vai Cys Asn Ser 755 CGT GTG CTC AAA Arg Val Leu Lys AGA AGT Arg Ser 765 GCT GAA GGA Ala Giu Gly ATT GAA GGA Ile Giu Gly 785

AGC

Ser 770 AAC CCT CCT AAA Asn Pro Pro Lys

CCA

Pro 775 CTG AAA AAA CTA Leu Lys Lys Leu CGC TTT GAT Arg Phe Asp 780 CCA GGA GAG Pro Giy Glu 2352 2400 TCA GAT GAA GCA Ser Asp Giu Aia

GAT

Asp 790 GGA AGT AAA CAT Gly Ser Lys His TCC AAA Ser Lys 800 TTT CAG CAG AAA Phe Gin Gin Lys

CTG

Leu 805 GCA GAA ATG ACT Ala Giu Met Thr

TCT

Ser 810 ACT CGA ACA CGA Thr Arg Thr Arg 2448

ATG

Met 815 CAA AAG CAG AAA Gin Lys Gin Lys

ATG

Met 820 AAT GAT AGC ATG GAT ACC TCA AAC AAG Asn Asp Ser Met Asp Thr Ser Asn Lys

GAA

Glu 830 GAG AAA Glu Lys TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT GGATTCATTG TCTCTCACAG ATGTGACTGT ATAACTTTCC CAGGTTCTGT TTATGGCCAC ATTTAATATC TTCAGCTCTT TTTGTGGATA TAAAATGTGC AGATGCAATT GTTTGGGTGA TTCCTAAGCC ACTTGAAATG TTAGTCATTG TTATTTATAC AAGATTGAAA ATCTTGTGTA AATCCTGCCA TTTAAAAAGT TGTAGCAGAT TGTTTCCTCT TCCAAAGTAA AATTGCTGTG CTTTATGGAT AGTAAGAATG GCCCTAGAGT GGGAGTCCTG ATAACCCAGG CCTGTCTGAC TACTTTGCCT 2496 2552 2612 2672 2732 2792 2852 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/03041

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA

TACTATCATA

TTTCTTCATC

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

TTGTTTTTAT TAATTTATAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

TTTTAAATGA

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

CCTCAGAATG

GGATTATTGA

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TAAAAGAACT

TAGT

2912 2972 3032 3092 3152 3212 3266 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 832 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Leu Trp Gly Ile Cys Ile Phe Ile Ala Ala Val Asp Leu Asp Giu Met Ser Phe His Lys Phe Asp Asn Ala Phe Thr Giu Leu Gin Lys Asn Ile Giu Ile Ser Val Thr Lys Vai Asn Leu Leu Giu Ile Asp Thr Met Ser Arg Leu Phe Leu Leu Lys Lys Tyr Leu Phe Ala Ser Lys Leu Arg Thr Cys Giu Leu Ile Tyr Leu Thr Pro Ser Ser Ser Ser Thr Glu Ile Ser Ala Leu Val Val Ser Trp Giu Asp Asp 115 Ile 100 Thr Phe Leu Leu Lys Giy Giu Val Leu Lys Gin Met Leu Asp Leu Vai Ile Ser Leu Met Leu Tyr Phe 130 Thr Ala i4 5 Ile Lys Leu Ser Pro 135 Asn Pro Met Leu Leu Giu Pro Tyr Lys Val Ile Pro Ile Gly Ser Pro Thr Pro Arg Arg SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 Gin Ile Lys Leu Tyr 225 Leu Thr lie Leu Ile 305 Lys Ala Giy Glu Ile 3 t Thr S er Tyr Giu Asn Ile Asn Val 210 Glu Asp Gin Pro Met 290 Ser Arg Val Vai Giu 370 Phe Tyr Phe Lys Met 450 Arg Giu Val 195 Thr Giu p~is Arg Pro 275 Met Tyr Vai Gly Arg 355 Arg His Ser Pro Vai 435 Ile Ser Vai 180 Tyr Ser Ile Asp Thr 260 His Ile Phe Lys Gin 340 Leu Leu Met Arg Trp 420 Ile Lys Ala 165 Leu Phe Asn Tyr Lys 245 Pro Thr Leu Asn Asp 325 Giy Tyr Ser Ser Ser 405 Ile Giu His Arg Cys Lys Giy Leu 230 Thr Arg Pro Asn Asn 310 Ile Cys Tyr Ile Leu 390 Thr Leu Ser Leu Ile Aia Lys Gin Leu Giu Asn Lys Asn Leu 215 Lys Leu Lys Vali Ser 295 Cys Giy Vai Arg Gin 375 Leu Ser Asn Phe Giu 455 Giu Phe 200 Pro Asn Gin Ser Arg 280 Ala Thr Tyr Giu Vai 360 Asn Aia Gin Vai Ile 440 Arg His 185 Ile Glu Lys Thr Asn 265 Thr Ser Vai Ile Ile 345 Met Phe Cys Asn Leu 425 Lys Cys 170 Giu Pro Val Asp Asp 250 Leu Val1 Asp Asn Phe 330 Gly Giu Ser Ala Leu 410 Asn Ala Glu Cys Phe Glu Leu 235 Ser Asp Met Gin Pro 315 Lys Ser Ser Lys Leu 395 Asp Leu Giu His Asn Met Asn 220 Asp le Giu jAsn Pro 300 Lys Giu Gin Met Leu 380 Glu Ser Lys Giy Arg 460 Ile Asn 205 Leu Ala Asp Giu Thr 285 Ser Giu Lys Arg Leu 365 Leu Vai Gly Al a Asn 445 Ile Asp Asp 190 Ser Ser Arg Ser Val1 270 Ile Glu Ser Phe Tyr 350 Lys Asn Val Thr Phe 430 Leu Met Thr 175 Giu Leu Lys Leu Phe 255 Asn Gin Asn Ile Al a 335 Lys Ser Asp Met Asp 415 Asp Thr Glu Arg Vai Gly Arg Phe 240 Giu Val1 Gin Leu Leu 320 Lys Leu Glu Asn Ala 400 Leu Phe Arg Ser SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 Leu 465 Lys Asn Pro Thr Pro 545 Leu His Gin Met Phe 625 Glu Ile Leu Ile Giy 705 Glu Val Asn Ala Trp, Asp Arg Leu Pro Val Arg 515 Ala Asn 530 Leu Lys Ala Tyr Pro Giu Asn Giu 595 Cys Ser 610 Lys Ile Thr Phe Val Phe Gin Tyr 6.75 Pro Arg 690 Giy Asn Gly Leu Ser Ile Gin Met 7.55 Leu Ser Asp Ser Pro Leu Phe Asp Leu Ile 470 Giu Leu 500 Ser Ala Ser Leu Leu 580 Tyr Met Ile Lys Tyr 660 Ala Ser Ile Pro Giy 740 Val Gly 485 Gin Pro Giu Thr Arg 565 Giu Giu Tyr Val Arg 645 Asn Ser Pro Tyr Thr 725 Giu Cys Pro Asn Lys Thr Ser 550 Leu His Leu Gly Thr 630 Val Ser Thr Tyr Ile 710 Pro Ser As n Thr Asn Lys Gin 535 Leu Asn Ile Met Ile 615 Ala Leu Val Arg Lys 695 Ser Thr Phe Ser Asp His Lys 520 Ala Ser Thr Ile Arg 600 Cys Tyr Ile Phe Pro 680 Phe Pro Lys Gly Asp 760 His Thr 505 Gly Thr Leu Leu Trp 585 Asp Lys Lys Lys Met 665 Pro Pro Leu Met Thr 745 Axg Leu 490 Al a Ser Ser Phe Cys 570 Thr Arg Val Asp Giu 650 Gin Thr Ser Lys Thr 730 Ser Val 475 Giu Ala Thr Ala Tyr 555 Giu Leu His Lys Leu 635 Glu Arg Leu Ser Ser 715 Pro Giu Leu Ser Asp Thr Phe 540 Lys Arg Phe Leu Asn 620 Pro Giu Leu Ser Pro 700 Pro Arg Lys Lys Ala Met Arg 525 Gin Lys Leu Gin Asp 605 Ile His Tyr Lys Pro 685 Leu Tyr Ser Phe Arg 765 Lys Cys Tyr 510 Val Thr Val Leu His 590 Gin Asp Aia Asp Thr 670 Ile Arg Lys Arg Gin 750 Ser Gin Pro 495 Leu Asn Gin Tyr Ser 575 Thr Ile Leu Val Ser 655 Asn Pro Ile Ile Ile 735 Lys Ala Ser 480 Leu Ser Ser Lys Arg 560 Giu Leu Met Lys Gin 640 Ile Ile His Pro Ser 720 Leu Ile Glu SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 Gly Ser Asn Pro Pro 770 Gly Ser Asp Glu Ala 785 Lys Pro Leu Lys Lys Leu Arg 775 780 Phe Asp Ile Glu Gly Ser Lys His Pro Gly Glu Ser Phe Gin Gin Lys Leu Ala Glu Met Thr Ser 805 810 Lys Gin Lys Met Asn Asp Ser Met Asp Thr 820 825 Thr Arg Thr Arg Met Gin 815 Ser Asn Lys Glu Glu Lys 830 INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 3113 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2349 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: GCCGTC ATG TCA AGA CTG TTG AAG AAG TAT GAT GTA TTG TTT GCA CTC Met Ser Arg Leu Leu Lys Lys Tyr Asp Val Leu Phe Ala Leu

TTC

Phe AGC AAA TTG GAA AGG ACA TGT GAA CTT ATA TAT TTG ACA CAA CCC Ser Lys Leu Glu Arg Thr Cys Glu Leu Ile Tyr Leu Thr Gin Pro AGC AGT TCG ATA TCT ACT GAA ATA Ser Ser Ser Ile Ser Thr Glu Ile TCT TGG ATC ACA TTT TTA TTA GCT Ser Trp Ile Thr Phe Leu Leu Ala AAT TCT GCA Asn Ser Ala TTG GTG CTA AAA GTT Leu Val Leu Lys Val 144

AAA

Lys 55 GGG GAA GTA Gly Glu Val TTA CAA ATG GAA Leu Gln Met Glu GTC CTT GAC TAT Val Leu Asp Tyr 192 GAT GAT CTG GTG ATT TCA TTT CAG TTA ATG CTA TGT Asp Asp Leu Val Ile Ser Phe Gln Leu Met Leu Cys TTT ATT Phe Ile AAA CTC TCA CCT Lys Leu Ser Pro CCC ATG TTG CTC AAA GAA CCA TAT AAA ACA Pro Met Leu Leu Lys Glu Pro Tyr Lys Thr 85 GGT TCA CCT CGA ACA CCC AGG CGA GGT CAG Gly Ser Pro Arg Thr Pro Arg Arg Gly Gin GTT ATA CCC ATT Val Ile Pro Ile AAC AGG AGT GCA CGG ATA GCA AAA CAA CTA GAA AAT Asn Arg Ser Ala Arg Ile Ala Lys Gin Leu Glu Asn GAT ACA AGA ATT Asp Thr Arg Ile 125 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/IJS98/03041 ATT GAA GTT Ile Giu Val AAT GTT TAT Asn Val Tyr 145

CTC

Leu 130 TGT AAA GAA CAT Cys Lys Giu His TGT AAT ATA GAT Cys Asn Ile Asp GAG GTG AAA Giu Val Lys 140 CTT GGA CTT Leu Giy Leu TTC AAA AAT TTT Phe Lys Asn Phe CCT TTT ATG AAT Pro Phe Met Asn

TCT

Ser 155 GTA ACA Vai Thr 160 TCT AAT GGA CTT Ser Asn Gly Leu GAG GTT GAA AAT CTT TCT AAA CGA TAC Giu Val Giu Asn Leu Ser Lys Arg Tyr 170

GAA

Giu 175 GAA ATT TAT CTT Giu Ile Tyr Leu

AAA

Lys 180 AAT AAA GAT Asn Lys Asp CTA GAT Leu Asp 185 GCA AGA TTA TTT Ala Arg Leu Phe

TTG

Leu 190 GAT CAT GAT AAA Asp His Asp Lys

ACT

Thr 195 CTT CAG ACT GAT Leu Gin Thr Asp ATA GAC AGT TTT Ile Asp Ser Phe GAA ACA Giu Thr 205 CAG AGA ACA Gin Arg Thr CCT CCA CAC Pro Pro His 225

CCA

Pro 210 CGA AAA AGT AAC Arg Lys Ser Asn GAT GAA GAG GTG Asp Giu Giu Val AAT GTA ATT Asn Val Ile 220 CAA CAA TTA Gin Gin Leu ACT CCA GTT AGG Thr Pro Val Arg GTT ATG AAC ACT Val Met Asn Thr ATG ATG Met Met 240 ATT TTA AAT TCA Ile Leu Asn Ser

GCA

Aia 245 AGT GAT CAA CCT Ser Asp Gin Pro GAA AAT CTG ATT Giu Asn Leu Ile

TCC

Ser 255 TAT TTT AAC AAC Tyr Phe Asn Asn

TGC

Cys 260 ACA GTG AAT Thr Val Asn CCA AAA Pro Lys 265 GAA AGT ATA CTG Giu Ser.le Leu

AAA

Lys 270 AGA GTG AAG GAT Arg Vai Lys Asp

ATA

Ile 275 GGA TAC ATC TTT Gly Tyr Ile Phe

AAA

Lys 280 GAG AAA TT GCT Giu Lys Phe Ala AAA GCT Lys Ala 285 GTG GGA CAG Val Gly Gin GTT CGC TTG Val Arg Leu 305

GGT

Gly 290 TGT GTC GAA ATT Cys Val Glu Ile TCA CAG CGA TAC Ser Gin Arg Tyr AAA CTT GGA Lys Leu Gly 300 TCA GAA GAA Ser Giu Giu TAT TAC CGA GTA Tyr Tyr Arg Val GAA TCC ATG CTT Glu Ser Met Leu

AAA

Lys 315 GAA CGA Giu Arg 320 TTA TCC ATT CAA Leu Ser Ile Gin TTT AGC AAA CTT Phe Ser Lys Leu AAT GAC AAC ATT Asn Asp Asn Ile 1008

TTT

Phe 335 CAT ATG TCT TTA TTG GCG TGC GCT CTT His Met Ser Leu Leu Ala Cys Ala Leu 340 GAG GTT Giu Val 345 GTA ATG GCC Val Met Ala 1056 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041I TAT AGC AGA AGT ACA TCT CAG AAT CTT Tyr Ser Arg Ser Thr Ser Gin Asn Leu 355 GAT TCT GGA ACA GAT TTG TCT Asp Ser Gly Thr Asp Leu Ser 360 365 1104 TTC CCA TGG Phe Pro Trp AAA GTG ATC Lys Val Ile 385

ATT

Ile 370 CTG AAT GTG CTT AAT TTA AAA GCC TTT Leu Asn Val Leu Asn Leu Lys Ala Phe 375 GAT TTT TAC Asp Phe Tyr 380 ACA AGA GAA Thr Arg Giu 1152 GAA AGT TTT ATC Giu Ser Phe Ile

AAA

Lys 390 GCA GAA GGC AAC Ala Giu Gly Asn

TTG

Leu 395 1200 ATG ATA Met Ile 400 AAA CAT TTA GAA Lys His Leu Glu

CGA

Arg 405 TGT GAA CAT CGA Cys Giu His Arg

ATC

Ile 410 ATG GAA TCC CTT Met Glu Ser Leu 1248 1296 TGG CTC TCA GAT Trp Leu Ser Asp CCT TTA TTT GAT Pro Leu Phe Asp ATT AA.A CAA TCA IlieLys Gin Ser GAC CGA GAA GGA CCA ACT GAT CAC CTT Asp Arg Giu Gly Pro Thr Asp His Leu 435 TCT GCT TGT CCT Ser Ala Cys Pro CTT AAT Leu Asn 445 1344 CTT CCT CTC Leu Pro Leu GTA AGA TCT Val Arg Ser 465

CAG

Gin 450 AAT AAT CAC ACT Asn Asn His Thr GCA GAT ATG TAT Ala Asp met Tyr CTT TCT CCT Leu Ser Pro 460 AAT TCT ACT Asn Ser Thr 1392 CCA AAG AAA AAA GGT TCA ACT ACG CGT Pro Lys Lys Lys Gly Ser Thr Thr Arg

GTA

Val 475 GCA AAT Ala Asn 480 GCA GAG ACA CAA Ala Giu Thr Gin

GCA

Aia 485 ACC TCA GCC TTC Thr Ser Ala Phe ACC CAG AAG CCA Thr Gin Lys Pro

TTG

Leu 495 AAA TCT ACC TCT Lys Ser Thr Ser

CTT

Leu 500 TCA CTG TTT TAT Ser Leu Phe Tyr AAA GTG TAT CGG Lys Val Tyr Arg

CTA

Leu 510 1440 1488 1536 1584 1632 GCC TAT CTC CGG Ala Tyr Leu Arg

CTA

Leu 515 AAT ACA CTT TGT Asn Thr Leu Cys CGC CTT CTG TCT Arg Leu Leu Ser GAG CAC Glu His 525 CCA GAA TTA Pro Giu Leu AAT GAG TAT Asn Giu Tyr 545 CAT ATC ATC TGG ACC CTT TTC CAG CAC His Ile Ile Trp Thr Leu Phe Gin His 535 ACC CTG CAG Thr Leu Gin 540 ATT ATG ATG Ile Met Met GAA CTC ATG AGA Giu Leu Met Arg

GAC

Asp 550 AGG CAT TTG GAC Arg His Leu Asp 1680 TGT TCC Cys Ser 560 ATG TAT GGC Met Tyr Gly ATA TGC Ile Cys 565 AAA GTG AAG AAT Lys Val Lys Asn GAC CTT AAA TTC Asp Leu Lys Phe 1728 SUBSTITUTE SHEET (RULE WO 98/37091 PCT/US98/03041 AAA ATC ATT GTA ACA GCA TAC AAG GAT CTT CCT Ile Ile Val Thr Ala Tyr Lys Asp Leu CAT GCT GTT His Ala Val Pro 585 CAG GAG Gin Glu 590 177-6 1824 ACA TTC AAA CGT Thr Phe Lys Arg GTT TTG ATC AAA GAA-GAG GAG TAT GAT TCT Val Leu Ile Lys Gli Glu Glu Tyr Asp Ser 595 600 ATT ATA lie lle 605 GTA TTC TAT Val Phe Tyr CAG TAT GCT Gin Tyr Ala 625 TCG GTC TTC ATG Ser Val Phe Met

CAG

Gin 615 AGA CTG AAA Arg Leu Lys ACA AAT ATT TTG Thr Asn Ile Leu 620 1872 1920 TCC ACC AGG CCC Ser Thr Arg Pro

CCT

Pro 630 ACC TTG TCA CCA Thr Leu Ser Pro

ATA

Ile 635 CCT CAC ATT Pro His Ile CCT CGA Pro Arg 640 AGC CCT TAC AAG Ser Pro Tyr Lys CCT AGT TCA CCC Pro Ser Ser Pro

TTA

Leu 650 CGG ATT CCT GGA Arg Ile Pro Gly

GGG

Gly 655 AAC ATC TAT ATT Asn Ile Tyr Ile CCC CTG AAG AGT Pro Leu Lys Ser TAT AAA ATT TCA Tyr Lys Ile Ser

GAA

Glu 670 1968 2016 2064 GGT CTG CCA ACA Gly Leu Pro Thr

CCA

Pro 675 ACA AAA ATG ACT Thr Lys Met Thr

CCA

Pro 680 AGA TCA AGA ATC Arg Ser Arg Ile TTA GTA Leu Val 685 TCA ATT GGT Ser Ile Gly CAG ATG GTA Gin Met Val 705

GAA

Glu 690 TCA TTC GGG ACT Ser Phe Gly Thr

TCT

Ser 695 GAG AAG TTC CAG Glu Lys Phe Gin AAA ATA AAT Lys Ile Asn 700 GCT GAA GGA Ala Glu Gly 2112 2160 TGT AAC AGC GAC Cys Asn Ser Asp

CGT

Arg 710 GTG CTC AAA AGA Val Leu Lys Arg

AGT

Ser 715 AGC AAC Ser Asn 720 CCT CCT AAA CCA Pro Pro Lys Pro AAA AAA CTA CGC Lys Lys Leu Arg

TTT

Phe 730 GAT ATT GAA GGA Asp Ile Glu Gly TCA GAT GAA GCA GAT Ser Asp Glu Ala Asp AGT AAA CAT CTC Ser Lys His Leu GGA GAG TCC AAA Gly Glu Ser Lys

TTT

Phe 750 2208 2256 2304 2349 CAG CAG AAA CTG Gin Gin Lys Leu

GCA

Ala 755 GAA ATG ACT TCT Glu Met Thr Ser

ACT

Thr 760 CGA ACA CGA ATG Arg Thr Arg Met CAA AAG Gin Lys 765 CAG AAA ATG Gin Lys Met GAT AGC ATG GAT Asp Ser Met Asp TCA AAC AAG GAA Ser Asn Lys Glu GAG AAA Glu Lys 780 TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT GGATTCATTG TCTCTCACAG ATGTGACTGT ATAACTTTCC CAGGTTCTGT TTATGGCCAC ATTTAATATC TTCAGCTCTT TTTGTGGATA TAAAATGTGC AGATGCAATT GTTTGGGTGA TTCCTAAGCC ACTTGAAATG 2409 2469 2529 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PTU9/34 PCT[US98/03041

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAA.A

AATTACTAAT

TGTTTTATAA

TACTATCATA

TTTCTTCATC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

AAGATTGAAA

TCCAAAGTAA

ATAACCCAGG

TTGTTT'rrAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

TTTTAAATGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

CCTCAGAATG

GGATTATTGA.

AATCCTGCCA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TAAAAGAACT

TAGT

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

2589 2649 2709 2769 2829 2889 2949 3009 3069 3113 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 781 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: Met Ser Arg Leu Leu Lys Lys Tyr Asp Val Leu Phe Ala Leu Phe Ser Lys Leu Glu Ser Ile Ser Ile Thr Phe Thr Cys Glu Leu Giu Ile Asn Ile Tyr Leu Ala Leu Val Glu Val Leu Thr Gin Leu Lys Ser Ser Ser Trp Leu Leu Ala Leu Val Met Glu Asp Asp Asp Tyr Phe Ile Ile Ser Phe Leu Met Leu Cys Val Leu Pro Tyr Lys Lys Leu Ser Pro Pro Giy Ile Pro Ile Ser Ala Arg 115 Met Leu Leu Lys Ser Pro Arg Thr 105 Lys Gin Leu Giu Thr Ala Val Gin Asn Arg 110 Ile Ile Giu Pro Arg Arg Gly Ile Ala Asn Asp Thr SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIIJS98/03041 Val Tyr 145 Ser Ile Asp Thr His 225 Ile Phe Lys Gin Leu 305 Leu Met Arg Trp Ile 385 Lys Leu Leu 130 Phe Asn Tyr Lys Pro 210 Thr Leu Asn Asp Gly 290 Tyr Ser Ser Ser Ile 370 Giu His Ser Cys Lys Gly Leu Thr 195 Arg Pro Asn *Asn Ile 275 Cys Tyr Ile Leu Thr 355 Leu Ser Leu Asp Lys Asn Leu Lys 180 Leu Lys Val Ser Cys 260 Gly Vai Arg Gin Leu 340 Ser Asn Phe Giu Ser 420 Giu Phe Pro Asn Gin S er Arg Ala 245 Thr Tyr Giu Vai Asn 325 Ala Gin Val Ile Arg 405 Pro His Ile I50 Giu Lys Thr Asn Thr 230 Ser Val Ile Ile Met 310 Phe Cys Asn Leu Lys 390 Cys Leu Glu 140 Leu Lys Leu Phe Asn 220 Gin Asn Ile Ala Lys 300 Ser Asp Met Asp Asp 380 Thr Giu Gin Val Gly Arg Phe Giu 205 Vai Gin Leu Leu Lys 285 Leu Giu Asn Ala Leu 365 Phe Arg Ser Ser Lys Leu Tyr Leu 190 Thr Ile Leu Ile Lys 270 Ala Gly Giu Ile Thr 350 Ser Tyr Glu Leu Lys 430 Val Thr 160 Giu His Arg Pro Met 240 Tyr Val Gly Arg Arg 320 His Ser Pro Val Ile 400 Trp, Arg SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 Glu Leu Ser 465 Ala Ser Leu Leu Tyr 545 Met Ile Lys Tyr Ala 625 Ser Ile Pro Gly Val 705 Pro Pro 435 Asn Lys Thr Ser Leu 515 His Leu Gly Thr Val 595 Ser Thr Tyr Ile Pro 675 Ser Asn Lys Thr Asn Lys Gin Leu 500 Asn Ile Met Ile Ala 580 Leu Val Arg Lys Ser 660 Thr Phe Ser Pro Asp His Leu Glu Ser Ala Cys Pro Leu Asn Leu Pro His Lys Ala 485 Ser Thr Ile Arg Cys 565 Tyr Ile Phe Pro Phe 645 Pro Lys Gly Asp Leu 725 Thr Gly 470 Thr Leu Leu Trp Asp 550 Lys Lys Lys Met Pro 630 Pro Leu Met Thr Arg 710 Lys Ala 455 Ser Ser Phe Cys Thr 535 Arg Val Asp Glu Gin 615 Thr Ser Lys Thr Ser 695 Val Lys 440 Ala Thr Ala Tyr Glu 520 Leu His Lys Leu Glu 600 Arg Leu Ser Ser Pro 680 Glu Leu Leu Asp Thr Phe Lys 505 Arg Phe Leu Asn Pro 585 Glu Leu Ser Pro Pro 665 Arg Lys Lys Arg Met Arg Gin 490 Lys Leu Gin Asp Ile 570 His Tyr Lys Pro Leu 650 Tyr Ser Phe Arg Phe 730 Tyr Val 475 Thr Val Leu His Gin 555 Asp Ala Asp Thr Ile 635 Arg Lys Arg Gin Ser 715 Asp Leu 460 Asn Gin Tyr Ser Thr 540 Ile Leu Val Ser Asn 620 Pro Ile Ile Ile Lys 700 Ala Ile Pro Thr Pro Leu 510 His Gin Met Phe Glu 590 Ile Leu Ile Gly Glu 670 Val Asn Gly Gly Val Ala Leu 495 Ala Pro Asn Cys Lys 575 Thr Val Gin Pro Gly 655 Gly Ser Gin Ser Ser 735 Arg Asn 480 Lys Tyr Glu Glu Ser 560 Ile Phe Phe Tyr Arg 640 Asn Leu Ile Met Asn 720 Asp SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 Glu Ala Asp Gly Ser Lys His Leu Pro Gly Glu Ser 740 745 Lys Phe Gin Gin 750 Lys Leu Ala Glu Met Thr Ser Thr Arg Thr 755 760 Arg Met Gin Lys Gin Lys 765 Met Asn Asp Ser Met Asp Thr Ser Asn Lys Glu Glu Lys 770 775 780 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 3323 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2559 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: CGCGTC ATG CCG CCC AAA ACC CCC CGA AAA ACG GCC GCC ACC GCC GCC Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala

GCT

Ala GCC GCC GCG GAA Ala Ala Ala Glu CCG GCA CCG CCG Pro Ala Pro Pro CCG CCC CCT Pro Pro Pro CCT GAG Pro Glu GTC GAC CTA GAT GAG ATG TCG TTC ACT Val Asp Leu Asp Glu Met Ser Phe Thr ATA GAA ATC AGT GTC CAT AAA TTC TTT Ile Glu Ile Ser Val His Lys Phe Phe 55 TTT ACT GAG CTA CAG AAA AAC Phe Thr Glu Leu Gin Lys Asn 40 AAC TTA CTA AAA GAA ATT GAT Asn Leu Leu Lys Glu Ile Asp ACC AGT ACC Thr Ser Thr AAA GTT GAT AAT Lys Val Asp Asn

GCT

Ala 70 ATG TCA AGA CTG Met Ser Arg Leu TTG AAG AAG TAT Leu Lys Lys Tyr ACA TGT GAA CTT Thr Cys Glu Leu GAT GTA Asp Val ATA TAT Ile Tyr TTG TTT GCA CTC Leu Phe Ala Leu AGC AAA TTG GAA Ser Lys Leu Glu

AGG

Arg TTG ACA CAA Leu Thr Gin AGC AGT TCG Ser Ser Ser ATA TCT Ile Ser 105 ACA TTT Thr Phe 120 ACT GAA ATA AAT Thr Glu Ile Asn GCA TTG GTG CTA AAA GTT TCT TGG ATC Ala Leu Val Leu Lys Val Ser Trp Ile 115 TTA TTA GCT Leu Leu Ala AAA GGG Lys Gly 125 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTIUS98/0304 1 GAA GTA TTA CAA ATG GAA GAT GAT Glu Val Leu Gin Met Giu Asp Asp 130 CTG GTG ATT Leu Val Ile 135 TCA TTT CAG TTA ATG Ser Phe Gin Leu Met 140 CTA TGT GTC CTT GAC TAT TTT ATT AAA CTC TCA CCT Leu Cys Vai Leu Asp Tyr Phe Ile Lys Leu Ser Pro CCC ATG TTG CTC Pro Met Leu Leu 155 GGT TCA CCT CGA Gly Ser Pro Arg 480 AAA GAA Lys Glu 160 CCA TAT AAA ACA Pro Tyr Lys Thr GTT ATA CCC ATT Val Ile Pro Ile

AAT

Asn 170 528 576 ACA Thr 175 CCC AGG CGA GGT Pro Arg Arg Gly AAC AGG AGT GCA Asn Arg Ser Ala

CGG

Arg 185 ATA GCA AAA CAA Ile Aia Lys Gin

CTA

Leu 190 GAA AAT GAT ACA Glu Asn Asp Thr

AGA

Arg 195 ATT ATT GAA GTT Ile Ile Giu Val TGT AAA GAA CAT Cys Lys Giu His GAA TGT Glu Cys 205 624 AAT ATA GAT Asn Ile Asp ATG AAT TCT Met Asn Ser 225

GAG

Glu 210 GTG AAA AAT GTT Vai Lys Asn Val TTC AAA AAT TTT Phe Lys Asn Phe ATA CCT TTT Ile Pro Phe 220 GAG GTT GAA Glu Val Glu CTT GGA CTT GTA Leu Gly Leu Val TCT AAT GGA CTT Ser Asn Gly Leu

CCA

Pro 235 720 AAT CTT Asn Leu 240 TCT AAA CGA TAC Ser Lys Arg Tyr GAA ATT TAT CTT Glu Ile Tyr Leu

AAA

Lys 250 AAT AAA GAT CTA Asn Lys Asp Leu

GAT

Asp 255 GCA AGA TTA TTT Ala Arg Leu Phe GAT CAT GAT AAA Asp His Asp Lys

ACT

Thr 265 CTT CAG ACT GAT Leu Gin Thr Asp ATA GAC AGT TTT Ile Asp Ser Phe

GAA

Glu 275 ACA CAG AGA ACA Thr Gin Arg Thr CGA AAA AGT AAC Arg Lys Ser Asn OTT GAT Leu Asp 285 GAA GAG GTG Glu Glu Va1 AAC ACT ATC Asn Thr Ile 305

AAT

Asn 290 GTA ATT CCT CCA Val Ile Pro Pro ACT CCA GTT AGG Thr Pro Val Arg ACT GTT ATG Thr Vai Met 300 AGT GAT CAA Ser Asp Gin CAA CAA TTA ATG Gin Gin Leu Met

ATG

Met 310 ATT TTA AAT TCA Ile -Leu Asn Ser CCT TCA Pro Ser 320 GAA AAT CTG ATT Glu Asn Leu Ile

TCC

Ser 325 TAT TTT AAC AAC Tyr Phe Asn Asn ACA GTG AAT CCA Thr Val Asn Pro 1008 1056 AAA GAA AGT Lys Giu Ser 335 ATA CTG AAA AGA GTG AAG GAT Ile Leu Lys Arg Vai Lys Asp 340 ATA GGA TAC ATC TTT Ile Gly Tyr Ile Phe 345 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT[US98/03041 GAG AAA TTT GCT Glu Lys Phie Ala GCT GTG GGA CAG Ala Val Gly Gin

GGT

Gly 360 TGT GTC GAA ATT GGA TCA CYs Val Giu Ile Gly Ser 1104 1152 CAG CGA TAC Gin Arg Tyr ATG CTT AAA Met Leu Lys 385

AAA

Lys 370 CTT GGA GTT CGC Leu Gly Val Arg TAT TAC CGA GTA ATG GAA TCC Tyr Tyr Arg Val Met Giu Ser 380 TCA GAA GAA GAA Ser Giu Giu Giu

CGA

Arg 390 TTA TCC ATT CAA Leu Ser Ile Gin TTT AGC AAA Phe Ser Lys 1200 CTT CTG Leu Leu 400 AAT GAC AAC ATT Asn Asp Asn Ile CAT ATG TCT TTA His Met Ser Leu GCG TGC GCT CTT Ala Cys Ala Leu GTT GTA ATG GCC Val Val Met Ala TAT AGC AGA AGT Tyr Ser Arg Ser TCT CAG AAT CTT Ser Gin Asn Leu 1248 1296 1344 TCT GGA ACA GAT Ser Gly Thr Asp TCT TTC CCA TGG Ser Phe Pro Trp CTG AAT GTG CTT Leu Asn Val Leu AAT TTA Asri Leu 445 AAA GCC TTT Lys Ala Phe GGC AAC TTG Gly Asn Leu 465 TTT TAC AAA GTG Phe Tyr Lys Val GA.A AGT TTT ATC Giu Ser Phe Ile AAA GCA GAA Lys Ala Giu 460 TGT GAA CAT Cys Glu His 1392 1440 ACA AGA GAA ATG Thr Arg Giu Met

ATA

Ile 470 AAA CAT TTA GAA Lys His Leu Glu CGA ATC Arg Ile 480 ATG GAA TCC CTT Met Giu Ser Leu TGG CTC TCA GAT Trp Leu Ser Asp

TCA

Ser 490 CCT TTA TTT GAT Pro Leu Phe Asp

CTT

Leu 495 ATT AAA CAA TCA Ile Lys Gin Ser GAC CGA GAA GGA Asp Arg Glu Gly

CCA

Pro 505 ACT GAT CAC CTT Thr Asp His Leu

GAA

Glu 510 1488 1536 1584 TCT GCT TGT CCT Ser Ala Cys Pro AAT CTT CCT CTC Asn Leu Pro Leu

CAG

Gin 520 AAT AAT CAC ACT Asn Asn His Thr GCA GCA Ala Ala 525 GAT ATG TAT Asp Met Tyr ACG CGT GTA Thr Arg Val 545 TCT CCT GTA AGA Ser Pro Val Arg

TCT

Ser 535 CCA AAG AAA AAA Pro Lys Lys Lys GGT TCA ACT- Gly Ser Thr 540 ACC TCA GCC Thr Ser Ala 1632 1680 AAT TCT ACT GCA Asn Ser Thr Ala GCA GAG ACA CAA Ala Giu Thr Gin TTC CAG ACC CAG AAG Phe Gin Thr Gin Lys 560 CCA TTG AAA TCT ACC TCT CTT Pro Leu Lys Ser Thr Ser Leu 565 570 TCA CTG TTT TAT Ser Leu Phe Tyr 1728 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041

AAA

Lys 575 AAA GTG TAT CGG Lys Val Tyr Arg CTA GCC Leu Ala 580 TAT CTC CGG Tyr Leu Arg

CTA

Leu 585 AAT ACA CTT TGT Asn Thr Leu Cys 1776 1824 CGC CTT CTG TCT Arg Leu Leu Ser

GAG

Giu 595 CAC CCA GAA TTA His Pro Giu Leu CAT ATC ATC TGG His Ile Ile Trp, ACC CTT Thr Leu 605 TTC CAG CAC Phe Gin His TTG GAC CAA Leu Asp Gin 625

ACC

Thr 610 CTG CAG AAT GAG Leu Gin Asn Giu

TAT

Tyr 615 GAA CTC ATG AGA Giu Leu Met Arg GAC AGG CAT Asp Arg His 620 AAA GTG AAG Lys Vai Lys 1872 1920 ATT ATG ATG TGT Ile Met Met Cys ATG TAT GGC ATA Met Tyr Gly Ile AAT ATA Asn Ilie 640 GAC CTT AAA TTC Asp Leu Lys Phe

AAA

Lys 645 ATC ATT GTA ACA Ile Ile Val Thr

GCA

Al a 650 TAC AAG GAT CTT Tyr Lys Asp Leu CAT GCT GTT CAG His Ala Val Gin ACA TTC AAA CGT Thr Phe Lys Arg

GTT

Val1 665 TTG ATC AAA GAA Leu Ile Lys Giu 1968 2016 2064 GAG TAT GAT TCT Giu Tyr Asp Ser ATA GTA TTC TAT Ile Vai Phe Tyr TCG GTC TTC ATG Ser Val Phe Met CAG AGA Gin Arg 685 CTG AA.A ACA Leu Lys Thr TCA CCA ATA Ser Pro Ile 705 ATT TTG CAG TAT Ile Leu Gin Tyr TCC ACC AGG CCC Ser Thr Arg Pro CCT ACC TTG Pro Thr Leu 700 CCT AGT TCA Pro Ser Ser 2112 2160 CCT CAC ATT CCT Pro His Ile Pro AGC CCT TAC AAG Ser Pro Tyr Lys CCC TTA Pro Leu 720 CGG ATT CCT GGA Arg Ile Pro Giy

GGG

Giy 725 AAC ATC TAT ATT Asn Ile Tyr Ile

TCA

Ser 730 CCC CTG AAG AGT Pro Leu Lys Ser

CCA

Pro 735 TAT AAA ATT TCA Tyr Lys Ile Ser GGT CTG CCA ACA Giy Leu Pro Thr ACA AAA ATG ACT Thr Lys Met Thr 2208 2256 2304 AGA TCA AGA ATC Arg Ser Arg Ile GTA TCA ATT GGT Vai Ser Ile Giy TCA TTC GGG ACT Ser Phe Gly Thr TCT GAG Ser Giu 765 AAG TTC CAG Lys Phe Gin

AAA

Lys 770 ATA AAT CAG ATG Ile Asn Gin Met

GTA

Val 775 TGT AAC AGC GAC Cys Asn Ser Asp CGT GTG CTC Arg Vai Leu 780 AAA AAA CTA Lys Lys Leu 2352 AAA AGA AGT GCT GAA GGA AGC AAC Lys Arg Ser Ala Glu Gly Ser Asn 785 790 CCT CCT AAA CCA Pro Pro Lys Pro

CTG

Leu 795 2400 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 CGC TTT Arg Phe 800 GAT ATT Asp Ile

CCA

Pro 815 GGA GAG TCC.

Gly Glu Ser GAA GGA TCA GAT GAA GCA GAT GGA AGT AAA CAT CTC Giu Gly Ser Asp Glu Ala Asp Gly Ser Lys His Leu 805 810 AAA TTT CAG CAG AAA CTG GCA GAA ATG ACT TCT ACT Lys Phe Gin Gin Lys Leu Ala Glu Met Thr Ser Thr 820 825 830 CAA AAG CAG AAA ATG AAT GAT AGC ATG GAT ACC TCA Gin Lys Gin Lys Met Asn Asp Ser Met Asp Thr Ser 835 840 845 AAA TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT Lys 2448 2496 2544 2599 CGA ACA CGA ATG Arg Thr Arg Met AAC AAG, GAA GAG Asn Lys Glu Giu 850

GGATTCATTG

ATTTAATATC

TTCCTAAGCC

AATCCTGCCA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TAAAAGAACT

TAGT

TCTCTCACAG

TTCAGCTCTT

ACTTGAAATG

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

ATGTGACTGT

TTTGTGGATA

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA

TACTATCATA

TTTCTTCATC

ATAACTTTCC

TAAAATGTGC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

CAGGTTCTGT

AGATGCAATT

AAGATTGAAA

TCCAAAGTAA

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

TTTTAAATGA

TTATGGCCAC

GTTTGGGTGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA.

CCTCAGAATG

GGATTATTGA

2659 2719 2779 2839 2899 2959 3019 3079 3139 3199 3259 3319 3323 INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 851 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala Ala Ala 1 5 10 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 Ala Leu Ile Thr Leu Leu Val Leu Val 145 Pro Arg Asp Asp Ser 225 Ser Arg Ser Val Ile 305 Ala Asp Ser Lys Phe Thr Leu Gin 130 Leu Tyr Arg Thr Glu 210 Leu Lys Leu Phe Asn 290 Gin Glu Glu Val Val Ala Gin Lys 115 Met Asp Lys Gly Arg 195 Val Gly Arg Phe Glu 275 Val Gin Pro Met His Asp Leu Pro 100 Val Glu Tyr Thr Gin 180 Ile Lys Leu Tyr Leu 260 Thr Ile Leu Pro Ser Lys Asn Phe Ser Ser Asp Phe Ala 165 Asn Ile Asn Val Glu 245 Asp Gin Pro Met Ala Phe Phe Ala 70 Ser Ser Trp Asp Ile 150 Val Arg Glu Val Thr 230 Glu His Arg Pro Met 310 Pro Thr Phe 55 Met Lys Ser Ile Leu 135 Lys Ile Ser Val Tyr 215 Ser Ile Asp Thr His 295 Ile Pro Pro 25 Phe Thr 40 Asn Leu Ser Arg Leu Glu Ile Ser 105 Thr Phe 120 Val Ile Leu Ser Pro Ile Ala Arg 185 Leu Cys 200 Phe Lys Asn Gly Tyr Leu Lys Thr 265 Pro Arg 280 Thr Pro Leu Asn Pro Glu Leu Leu Arg 90 Thr Leu Ser Pro Asn 170 Ile Lys Asn Leu Lys 250 Leu Lys Val Ser Pro Pro Leu Gin Lys Glu Leu Lys 75 Thr Cys Glu Ile Leu 'Ala Phe Gin 140 Pro Met 155 Gly Ser Ala Lys Glu His Phe Ile 220 Pro Glu 235 Asn Lys Gin Thr Ser Asn Arg Thr 300 Ala Ser 315 Pro Lys Ile Lys Glu Asn Lys 125 Leu Leu Pro Gin Glu 205 Pro Val Asp Asp Leu 285 Val Asp Glu Asn Asp Tyr Leu Ser 110 Gly Met Leu Arg Leu 190 Cys Phe Glu Leu Ser 270 Asp Met Gin Val Ile Thr Asp Ile Ala Glu Leu Lys Thr 175 Glu Asn Met Asn Asp 255 Ile Glu Asn Pro Asp Glu Ser Val Tyr Leu Val Cys Glu 160 Pro Asn Ile Asn Leu 240 Ala Asp Glu Thr Ser 320 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/03041 Giu Asn Leu Ile Ser Tyr Phe Asn Asn Ser Phe Tyr Lys 385 Asn Val1 Thr Phe Leu 465 Met Lys Cys Tyr Val 545 Thr Val.

Leu His Ile Ala Lys 370 Ser Asp Met Asp Asp 450 Thr Giu Gin Pro Leu 530 Asn Gin Tyr Ser Thr 610 Leu Lys 355 Leu Glu Asn Ala Leu 435 Phe Arg Ser Ser Leu 515s Ser Ser Lys Arg Giu 595 Leu Asp Gly 360 Tyr Ser Ser Ser Ile 440 Giu His Ser Gly Gin 520 Pro Giu Thr Arg Glu 600 Glu Ile 345 Cys Tyr Ile Leu Thr 425 Leu Ser Leu Asp Pro 505 Asn Lys Thr Ser Leu 585 His Leu Cys 330 Giy Vai Arg Gin Leu 410 Ser Asn Phe Giu Ser 490 Thr Asn Lys Gin Leu 570 Asn Ile Met Thr Vai Asn Pro Tyr Giu Vai Asn 395 Ala Gin Vai Ile Arg 475 Pro Asp His Lys Ala 555 Ser Thr Ile Arg Ile Ile Met 380 Phe Cys Asn Leu Lys 460 Cys Leu His Thr Gly 540 Thr Leu Leu Trp Asp 620 Phe Gly 365 Giu Ser Ala Leu Asn 445 Aila Giu Phe Leu Al a 525 Ser Ser Phe Cys Thr 605 Arg Lys 350 Ser Ser Lys Leu Asp 430 Leu Giu His Asp Giu 510 Ala Thr Aila Tyr Giu 590 Leu His Lys 335 Giu Gin Met Leu Glu 415 Ser Lys Gly Arg Leu 495 Ser Asp Thr Phe Lys 575 Arg Phe Leu Giu Lys Arg Leu Leu 400 Val Gly Al a Asn Ile 480 Ile Ala Met Arg Gin 560 Lys Leu Gin Asp SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIIJS98/03041 Gin 625 Asp Al a Asp Thr Ile 705 Arg Lys Arg Gin Ser 785 Asp Giu Arg Giu Ile Leu Vai Ser Asn 690 Pro Ile Ile Ile Lys 770 Aia Ile Ser Met Giu 850 Met Lys Gin Ile 675 Ile His Pro Ser Leu 755 Ile Giu Giu Lys Gin 835 Lys Met Phe Giu 660 Ile Leu Ile Gly Glu 740 Val Asn Giy Giy Phe 820 Lys Cys Lys 645 Thr Vai Gin Pro Gly 725 Giy Ser Gin Ser Ser 805 Gin Gin Ser 630 Ile Phe Phe Tyr Arg 710 Asn Leu Ile Met Asn 790 Asp Gin Lys Tyr Vai Arg Asn 680 Ser Pro Tyr Thr Glu 760 Cys Pro Ala Leu Asn 840 Giy Thr Vai 665 Ser Thr Tyr Ile Pro 745 Ser Asn Lys Asp Ala 825 Asp Ile Ala 650 Leu Vai Arg Lys Ser 730 Thr Phe Ser Pro Giy 810 Glu Ser Cys 635 Tyr Ile Phe Pro Phe 715 Pro Lys Gly Asp Leu 795 Ser Met Met Lys Lys Lys Met Pro 700 Pro Leu Met Thr Arg 780 Lys Lys Thr Asp Val Asp Giu Gin 685 Thr Ser Lys Thr Ser 765 Vai Lys His Ser Thr 845 Lys Leu Giu 670 Arg Leu Ser Ser Pro 750 Giu Leu Leu Leu Thr 830 Ser Ile 640 His Tyr Lys Pro Leu 720 Tyr Ser Phe Arg Phe 800 Gly Thr Lys INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 3461 base pairs TYPE: nucleic acid CC) STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7. .2697 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTfUS98/03041 188 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGCGTC ATG CCG CCC AAA ACC CCC CGA AAA ACG CCC GCC ACC GCC GCC Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala GCT CC Ala Ala GCC CCG GAA Ala Ala Glu CCC GCA CCG CCC Pro Ala Pro Pro

CCG

Pro 25 CCC CCC CCT Pro Pro Pro CCT GAG Pro Glu GTC AGG Va 1 Arg GAG GAC CCA GAG Giu Asp Pro Clu GAC AGC GGC CCC Asp Ser Gly Pro GAC CTG CCT CTC Asp Leu Pro Leu CTT GAG TTT GAA GAA ACA CAA GAA Leu Clu Phe Glu Glu Thr Clu Clu CAT TTT ACT GCA Asp Phe Thr Ala TTA TGT CAG Leu Cys Gin TTG GTC CAC Leu Val Asp AAA TTA AAC ATA CCA CAT CAT Lys Leu Lys Ile Pro Asp His

GTC

Val AGA GAG ACA CCT Arg Ciu Arg Ala CTA CAT Leu Asp GAG ATC TCC TTC Ciu Met Ser Phe TTT ACT GAG CTA Phe Thr Ciu Leu AAA AAC ATA CAA Lys Asn Ile Ciu ATC AGTCGTC CAT AAA Ile Ser Val His Lys

TTC

Phe 100 TTT AAC TTA CTA Phe Asn Leu Leu

AAA

Lys 105 CAA ATT CAT ACC Clu Ile Asp Thr ACC AAA CTT CAT Thr Lys Val Asp CCT ATC TCA ACA Ala Met Ser Arg

CTC

Leu 120 TTG AAC AAC TAT Leu Lys Lys Tyr CAT GTA Asp Val 125 384 432 TTC TTT CCA Leu Phe Ala TTC ACA CAA Leu Thr Gin 145 TTC ACC AAA TTC CAA AGC ACA TGT GAAi Phe Ser Lys Leu Ciu Arg Thr Cys Clu 135 CTT ATA TAT Leu Ile Tyr 140 TCT GCA TTG Ser Ala Leu CCC ACC ACT TCC Pro Ser Ser Ser

ATA

Ile 150 TCT ACT CAA ATA Ser Thr Ciu Ile GTC CTA Val Leu 160 AAA GTT TCT TG Lys Val Ser Trp ACA TTT TTA TTA Thr Phe Leu Leu

GCT

Ala 170 AAA CCC CAA CTA Lys Gly Clu Val

TTA

Leu 175 CAA ATG CAA CAT Gin Met Glu Asp CTC GTG ATT TCA Leu Val Ile Ser

TTT

Phe 185 CAC TTA ATG CTA Gin Leu Met Leu CTC CTT CAC TAT TTT ATT AAA CTC TCA Val Leu Asp Tyr Phe Ile Lys Leu Ser 195

CCT

Pro 200 CCC ATC TTC CTC Pro Met Leu Leu AAA GAA Lys Giu 205 624 CCA TAT AAA Pro Tyr Lys GCT CTT ATA CCC ATT AAT GCT T CA CCT Ala Val Ile Pro Ile Asn Cly Ser Pro 215 SUBSTITUTE SHEET (RULE 26) CCA ACA CCC Arg Thr Pro 220 672 WO 98/37091 WO 9837091PCT/US98/03041 AGG CGA GGT Arg Arg Gly 225 CAG AAC AGG AGT Gin Asn Arg Ser

GCA

Ala 230 CGG ATA GCA AAA Arg Ile Ala Lys

CAA

Gin 235 CTA GAA AAT Leu Giu Asn 720 GAT ACA Asp Thr 240 AGA ATT ATT GAA Arg Ile Ile Giu

GTT

Val 245 CTC TGT AAA GAA Leu Cys Lys Giu GAA TGT AAT ATA Giu Cys Asn Ile GAG GTG AAA AAT Giu Vai Lys Asn

GTT

Vai 260 TAT TTC AAA AAT Tyr Phe Lys Asn ATA CCT TTT ATG Ile Pro Phe Met

AAT

Asn 270 TCT CTT GGA CTT Ser Leu Giy Leu

GTA

Vai 275 ACA TCT AAT GGA Thr Ser Asn Gly CCA GAG GTT GAA Pro Giu Val Giu AAT CTT Asn Leu 285 TCT AAA CGA Ser Lys Arg AGA TTA TTT Arg Leu Phe 305

TAC

Tyr 290 GAA GAA ATT TAT Giu Giu Ile Tyr AAA AAT AAA GAT Lys Asn Lys Asp CTA GAT GCA Leu Asp Ala 300 TCT ATA GAC Ser Ile Asp TTG GAT CAT GAT Leu Asp His Asp

AAA

Lys 310 ACT CTT CAG ACT Thr Leu Gin Thr

GAT

Asp 315 AGT TTT Ser Phe 320 GAA ACA CAG AGA Giu Thr Gin Arg CCA CGA AAA AGT Pro Arg Lys Ser CTT GAT GAA GAG Leu Asp Giu Giu 1008

GTG

Val 335 AAT GTA ATT CCT CCA CAC ACT CCA GTT Asn Vai Ile Pro Pro His Thr Pro Val 340 ACT GTT ATG AAC Thr Val Met Asn 1056 ATC CAA CAA TTA Ile Gin Gin Leu

ATG

Met 355 ATG ATT TTA AAT Met Ile Leu Asn GCA AGT GAT CAA Aia Ser Asp Gin CCT TCA Pro Ser 365 1104 GAA AAT CTG Giu Asn Leu AGT ATA CTG Ser Ile Leu v 385

ATT

Ile 370 TCC TAT TTT AAC Ser Tyr Phe Asn

AAC

Asn 375 TGC ACA GTG AAT Cys Thr Vai Asn CCA AAA GAA Pro Lys Giu 380 AAA GAG AAA Lys Giu Lys 1152 i2 00 AAA AGA GTG AAG Lys Arg Vai Lys

GAT

Asp 390 ATA GGA TAC ATC Ile Giy Tyr Ile TTT GCT Phe Ala 400 AAA GCT GTG GGA CAG GGT TGT GTC GAA ATT GGA TCA CAG CGA Lys Ala Vai Giy Gin Giy Cys Vai Giu Ile Giy Ser Gin Arg 1248 AAA CTT GGA GTT CGC TTG TAT TAC CGA Lys Leu Gly Vai Axg Leu Tyr Tyr Arg

GTA

Vai 425 ATG GAA TCC ATG Met Giu Ser Met

CTT

Leu 430 1296 1344 AAA TCA GAA GAA Lys Ser Giu Giu CGA TTA TCC ATT CAA AAT TTT AGC AAA CTT CTG Arg Leu Ser Ile Gin Asn Phe Ser Lys Leu Leu SUBSTITUTE SHEET (RULE 26) WO 98/37091 AAT GAC AAC Asn Asp Asn GTA ATG GCC Val Met Ala 465 PCTIUS98/03041

ATT

Ile 450 TTT CAT ATG TCT TTA TTG GCG TGC GCT Phe His Met Ser Leu Leu Ala Cys Ala 455 CTT GAG GTT Leu Giu Val 460 GAT TCT GGA Asp Ser Gly 1392 ACA TAT AGC AGA Thr Tyr Ser Arg

AGT

Ser 470 ACA TCT CAG AAT Thr Ser Gin Asn

CTT

Leu 475 1440 ACA GAT Thr Asp 480 TTG TCT TTC CCA TGG ATT CTG AAT GTG Leu Ser Phe Pro Trp Ile Leu Asn Val

CTT

Leu 490 AAT TTA AAA GCC Asn Leu Lys Ala 1488 1536

TTT

Phe 495 GAT TTT TAC AAA GTG ATC GAA AGT TTT Asp Phe Tyr Lys Val Ile Giu Ser Phe 500 AAA GCA GAA GGC Lys Ala Giu Gly TTG ACA AGA GAA Leu Thr Arg Glu

ATG

Met 515 ATA AAA CAT TTA Ile Lys His Leu CGA TGT GAA CAT Arg Cys Glu His CGA ATC Arg Ile 525 1584 ATG GAA TCC Met Giu Ser AAA CAA TCA Lys Gin Ser 545

CTT

Leu 530 GCA TGG CTC TCA GAT TCA CCT TTA TTT GAT CTT ATT Ala Trp Leu Ser Asp Ser Pro Leu Phe Asp Leu Ile 1632 AAG GAC CGA GAA Lys Asp Arg Glu CCA ACT GAT CAC CTT GAA TCT GCT Pro Thr Asp His Leu Giu Ser Ala 555 1680 TGT CCT Cys Pro 560 CTT AAT CTT CCT Leu Asn Leu Pro CAG AAT AAT CAC Gin Asn Asn His

ACT

Thr 570 GCA GCA GAT ATG Ala Ala Asp Met 1728 1776

TAT

Tyr 575 CTT TCT CCT GTA Leu Ser Pro Val TCT CCA AAG AAA Ser Pro Lys Lys

AAA

Lys 585 GGT TCA ACT ACG Gly Ser Thr Thr GTA AAT TCT ACT Val Asn Ser Thr AAT GCA GAG ACA Asn Ala Giu Thr

CAA

Gin 600 GCA ACC TCA GCC Ala Thr Ser Ala TTC CAG Phe Gin 605 1824 ACC CAG AAG Thr Gin Lys GTG TAT CGG Val Tyr Arg 625

CCA

Pro 610 TTG AAA TCT ACC Leu Lys Ser Thr CTT TCA CTG TTT Leu Ser Leu Phe TAT AAA AAA Tyr Lys Lys 620 GAA CGC CTT Glu Arg Leu 1872 1920 CTA GCC TAT CTC Leu Ala Tyr Leu

CGG

Arg 630 CTA AAT ACA CTT Leu Asn Thr Leu

TGT

Cys 635 CTG TCT Leu Ser 640 GAG CAC CCA GAA Glu His Pro Glu

TTA

Leu 645 GAA CAT ATC ATC Glu His Ile Ile

TGG

Trp 650 ACC CTT TTC CAG Thr Leu Phe Gin 1968 ACC CTG CAG AAT Thr Leu Gin Asn

GAG

Glu 660 TAT GAA CTC ATG AGA GAC AGG CAT TTG Tyr Giu Leu Met Arg Asp Arg His Leu 665

GAC

Asp 670 2016 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 CAA ATT ATG ATG Gin Ile Met Met TCC ATG TAT GGC Ser Met Tyr Gly

ATA

Ile 680 TGC AAA GTG AAG Cys Lys Val Lys AAT ATA Asn Ile 685 2064 GAC CTT AAA Asp Leu Lys GCT GTT CAG Ala Val Gin 705

TTC

Phe 690 AAA ATC ATT GTA Lys Ile Ile Val

ACA

Thr 695 GCA TAC AAG GAT Ala Tyr Lys Asp CTT CCT CAT Leu Pro His 700 GAG GAG TAT Glu Glu Tyr 2112 2160 GAG ACA TTC AAA Glu Thr Phe Lys GTT TTG ATC AAA Val Leu Ile Lys GAT TCT Asp Ser 720 ATT ATA GTA TTC Ile Ile Val Phe AAC TCG GTC TTC Asn Ser Val Phe CAG AGA CTG AAA Gin Arg Leu Lys AAT ATT TTG CAG Asn Ile Leu Gin GCT TCC ACC AGG Ala Ser Thr Arg

CCC

Pro 745 CCT ACC TTG TCA Pro Thr Leu Ser

CCA

Pro 750 2208 2256 2304 ATA CCT CAC ATT Ile Pro His Ile CGA AGC CCT TAC Arg Ser Pro Tyr

AAG

Lys 760 TTT CCT AGT TCA Phe Pro Ser Ser CCC TTA Pro Leu 765 CGG ATT CCT Arg Ile Pro AAA ATT TCA Lys Ile Ser 785 GGG AAC ATC TAT Gly Asn Ile Tyr TCA CCC CTG AAG Ser Pro Leu Lys AGT CCA TAT Ser Pro Tyr 780 CCA AGA TCA Pro Arg Ser 2352 2400 GAA GGT CTG CCA Glu Gly Leu Pro CCA ACA AAA ATG Pro Thr Lys Met AGA ATC Arg Ile 800 TTA GTA TCA ATT Leu Val Ser Ile GAA TCA TTC GGG ACT TCT GAG AAG TTC Glu Ser Phe Gly Thr Ser Glu Lys Phe 2448 2496

CAG

Gin 815 AAA ATA AAT CAG ATG GTA TGT AAC AGC Lys Ile Asn Gin Met Val Cys Asn Ser 820

GAC

Asp 825 CGT GTG CTC AAA Arg Val Leu Lys AGT GCT GAA GGA Ser Ala Glu Gly AAC CCT CCT AAA Asn Pro Pro Lys

CCA

Pro 840 CTG AAA AAA CTA Leu Lys Lys Leu CGC TTT Arg Phe 845 2544 GAT ATT GAA Asp Ile Glu GAG TCC AAA Glu Ser Lys 865 TCA GAT GAA GCA Ser Asp Glu Ala GGA AGT AAA CAT Gly Ser Lys His CTC CCA GGA Leu Pro Gly 860 ACT CGA ACA Thr Arg Thr 2592 2640 TTT CAG CAG AAA Phe Gin Gin Lys GCA GAA ATG ACT Ala Glu Met Thr CGA ATG Arg Met 880 CAA AAG CAG AAA Gin Lys Gin Lys AAT GAT AGC ATG Asn Asp Ser Met ACC TCA AAC AAG Thr Ser Asn Lys 2688 SUBSTITUTE SHEET (RULE 26) WO 98/37091 192 GAA GAG AAA TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT Glu Glu Lys 895 PCTJUS98/03041 2737

GGATTCATTG

ATTTAATATC

TTCCTAAGCC

AATCCTGCCA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TAAAAGAACT

TAGT

TCTCTCACAG

TTCAGCTCTT

ACTTGAAATG

T'rrAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

ATGTGACTGT

TTTGTGGATA

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATA.A

TACTATCATA

TTTC.TTCATC

ATAACTTTCC

TAAA.ATGTGC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

CAGGTTCTGT

AGATGCAATT

AAGATTGAAA

TCCAAAGTAA

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

TTTTAAATGA

TTATGGCCAC

GTTTGGGTGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

CCTCAGAATG

GGATTATTGA

2797 2857 2917 2977 3037 3097 3157 3217 3277 3337 3397 3457 3461 INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS: LENGTH: 897 amino acids TYPE: amino acid TOPOLOGY- linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala 1 5 10 Ala Ala Ala Ala Glu Pro Glu Gln Phe Glu Glu Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro 25 Asp Ser Gly Pro Glu Asp Leu Pro Leu Val 40 Thr Glu Glu Pro Asp Phe Thr Ala Leu Cys 55 Glu Glu Asp Arg Leu Glu Gln Lys Leu Lys Ile Pro Asp His Val Arg Glu Arg Ala Trp Leu 70 Val Asp Leu SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/03041 Giu Met Ser Phe Thr Phe Thr Giu Leu Gin Lys Asn Ile Glu Ile Ser Val Val Ala Gin 145 Lys Met Asp Lys Giy 225 Arg Vai Giy Arg Phe 305 Giu Vai Gin Leu His Asp Leu 130 Pro Vai Glu Tyr Thr 210 Gin Ile Lys Leu Tyr 290 Leu Thr Ile Leu Ile 370 Lys Asn 115 Phe Ser Ser Asp Phe 195 Aia Asn Ile Asn Val 275 Giu Asp Gin Pro Met 355 Ser Phe 100 Ala Ser Ser Trp Asp 180 Ile Val Arg Glu Val 260 Thr Giu His Arg Pro 340 Met Tyr Phe Met Lys Ser Ile 165 Leu Lys Ile Ser Val 245 Tyr Ser Ile Asp Thr 325 His Ile Phe Asn Ser Leu Ile 150 Thr Val Leu Pro Ala 230 Leu Phe Asn Tyr Lys 310 Pro Thr Leu Asn Leu Leu Arg Leu 120 Giu Arg 135 Ser Thr Phe Leu Ile Ser Ser Pro 200 Ile Asn 215 Arg Ile Cys Lys Lys Asn Gly Leu 280 Leu Lys 295 Thr Leu Arg Lys Pro Val Asn Ser 360 Asn Cys 375 Lys 105 Leu Thr Giu Leu Phe 185 Pro Gly Ala Giu Phe 265 Pro Asn Gin Ser Arg 345 Ala Thr 90 Giu Lys Cys Ile Ala 170 Gin Met Ser Lys His 250 Ile Giu Lys Thr Asn 330 Thr S er Val Ile Lys Giu Asn 155 Lys Leu Leu Pro Gin 235 Giu Pro Val Asp Asp 315 Leu Val Asp Asn Asp Tyr Leu 140 Ser Gly Met Leu Arg 220 Leu Cys Phe Giu Leu 300 Ser Asp Met Gin Pro 380 Thr Asp 125 Ile Ala Giu Leu Lys 205 Thr Giu Asn Met Asn 285 Asp Ile Giu Asn Pro 365 Lys Ser 110 Val Tyr Leu Val Cys 190 Giu Pro Asn Ile Asn 270 Leu Ala Asp Giu Thr 350 Ser Giu Thr Leu Leu Val Leu 175 Val Pro Arg Asp Asp 255 Ser Ser Arg Ser Val 335 Ile Giu Ser Lys Phe Thr Leu 160 Gin Leu Tyr Arg Thr 240 Glu Leu Lys Leu Phe 320 Asn Gin Asn Ile SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 Leu 385 Lys Leu Glu Asn Ala 465 Leu Phe Arg Ser Ser 545 Leu Ser Ser Lys Arg 625 Giu Leu Met Lys Ala Gly Glu Ile 450 Thr Ser Tyr Glu Leu 530 Lys Asn Pro Thr Pro 610 Leu His Gin Met Arg Val Val Giu 435 Phe Tyr Phe Lys Met 515 Ala Asp Leu Val Ala 595 Leu Ala Pro Asn Cys 6.75 Val Lys Asp Ile Gly Tyr Ile Phe Lys Giu Lys Phe Ala Gly Arg 420 Arg His Ser Pro Val 500 Ile Trp Arg Pro Arg 580 Asn Lys Tyr Giu Giu 660 Ser Gin 405 Leu Leu Met Arg Trp 485 Ile Lys Leu Giu Leu 565 Ser Ala Ser Leu Leu 645 Tyr Met 390 Gly Tyr Ser Ser Ser 470 Ile Giu His Ser Gly 550 Gin Pro Giu Thr Arg 630 Giu Giu Tyr Cys Val Tyr Arg Ile Gin 440 Leu Leu 455 Thr Ser Leu Asn Ser Phe Leu Giu 520 Asp Ser 535 Pro Thr Asn Asn Lys Lys Thr Gin 600 Ser Leu 615 Leu Asn His Ile Leu Met Gly Ile 680 Giu Val 425 Asn Ala Gin Val Ile 505 Arg Pro Asp His Lys 585 Ala Ser Thr Ile Arg 665 Cys Ile 410 Met Phe Cys Asn Leu 490 Lys Cys Leu His Thr 570 Gly Thr Leu Leu Trp 650 Asp Lys 395 Gly Giu Ser Ala Leu 475 Asn Al a Giu Phe Leu 555 Ala Ser Ser Phe Cys 635 Thr Arg Val Ser Ser Lys Leu 460 Asp Leu Glu His Asp 540 Giu Ala Thr Al a Tyr 620 Glu Leu His Lys Gin Met Leu 445 Giu Ser Lys Gly Arg 525 Leu Ser Asp Thr Phe 605 Lys Arg Phe Leu Asn 685 Arg Leu 430 Leu Val Gly Ala Asn 510 Ile Ile Ala Met Arg 590 Gin Lys Leu Gin Asp 670 Ile Tyr 415 Lys Asn Val Thr Phe 495 Leu Met Lys Cys Tyr 575 Val Thr Val Leu His 655 Gin Asp 400 Lys Ser Asp Met Asp 480 Asp Thr Giu Gin Pro 560 Leu Asn Gin Tyr Ser 640 Thr Ile Leu SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 195 Lys Phe Lys Ile Ile Val Thr Ala Tyr Lys Asp Leu Pro His Ala Val 690 695 700 Gin Glu Thr Phe Lys Arg Val Leu Ile Lys Glu Glu Glu Tyr Asp Ser 705 710 715 720 Ile Ile Val Phe Tyr Asn Ser Val Phe Met Gin Arg Leu Lys Thr Asn 725 730 735 Ile Leu Gin Tyr Ala Ser Thr Arg Pro Pro Thr Leu Ser Pro Ile Pro 740 745 750 His Ile Pro Arg Ser Pro Tyr Lys Phe Pro Ser Ser Pro Leu Arg Ile 755 760 765 Pro Gly Gly Asn Ile Tyr Ile Ser Pro Leu Lys Ser Pro Tyr Lys Ile 770 775 780 Ser Glu Gly Leu Pro Thr Pro Thr Lys Met Thr Pro Arg Ser Arg Ile 785 790 795 800 Leu Val Ser Ile Gly Glu Ser Phe Gly Thr Ser Glu Lys Phe Gin Lys 805 810 815 Ile Asn Gin Met Val Cys Asn Ser Asp Arg Val Leu Lys Arg Ser Ala 820 825 830 Glu Gly Ser Asn Pro Pro Lys Pro Leu Lys Lys Leu Arg Phe Asp Ile 835 840 845 Glu Gly Ser Asp Glu Ala Asp Gly Ser Lys His Leu Pro Gly Glu Ser 850 855 860 Lys Phe Gin Gin Lys Leu Ala Glu Met Thr Ser Thr Arg Thr Arg Met 865 870 875 880 Gin Lys Gin Lys Met Asn Asp Ser Met Asp Thr Ser Asn Lys Glu Glu 885 890 895 Lys INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: 3347 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2583 (xi).SEQUENCE DESCRIPTION: SEQ ID NO: 42: SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 196 CGCGTC ATG CCG CCC AAA ACC CCC CGA AAA ACG GCC GCC ACC GCC GCC Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala

GCT

Ala GCC GCC GCG GAA CCC CCG GCA CCG CCG Ala Ala Ala Glu Pro Pro Ala Pro Pro CCG CCC CCT CCT Pro Pro Pro Pro GAG GAC CCA GAG Glu Asp Pro Glu GAC AGC GGC CCG Asp Ser Gly Pro GAC CTG CCT CTC Asp Leu Pro Leu GTC AGG Val Arg CTT GAG TTT Leu Giu Phe AAA TTA AAG Lys Leu Lys GAA ACA GAA GAA CCT GAT TTT ACT GCA Glu Thr Glu Giu Pro Asp Phe Thr Ala TTA TGT CAG Leu Cys Gin TTA ACT TGG Leu Thr Trp ATA CCA GAT CAT Ile Pro Asp His

GTC

Val AGA GAG AGA GCT Arg Glu Arg Ala 240 GAG AAA Glu Lys GTT TCA TCT GTG Val Ser Ser Val GGA GTA TTG GGA Gly Val Leu Gly

GGT

Gly TAT ATT CAA AAG Tyr Ile Gin Lys AAG GAA CTG TGG Lys Glu Leu Trp ATC TGT ATC TTT ATT GCA GCA GTT GAC CTA Ile Cys Ile Phe Ile Ala Ala Val Asp Leu 336 GTC GAA TCT ACT Val Glu Ser Thr ATA AAT TCT GCA Ile Asn Ser Ala GTG CTA AAA GTT Val Leu Lys Vai TCT TGG Ser Trp 125 384 ATC ACA TTT Ile Thr Phe CTG GTG ATT Leu Val Ile 145 TTA GCT AAA GGG GAA GTA TTA CAA ATG GAA GAT GAT Leu Ala Lys Gly Giu Vai Leu Gin Met Giu Asp Asp 135 140 432 TCA TTT CAG TTA Ser Phe Gin Leu

ATG

Met 150 CTA TGT GTC CTT Leu Cys Val Leu TAT TTT ATT Tyr Phe Ile 480 AAA CTC Lys Leu 160 TCA CCT CCC ATG Ser Pro Pro Met

TTG

Leu 165 CTC AAA GAA CCA Leu Lys Giu Pro

TAT

Tyr 170 AAA ACA GCT GTT Lys Thr Ala Val

ATA

Ile 175 CCC ATT PAT GGT Pro Ile Asn Gly

TCA

Ser 180 CCT CGA ACA CCC Pro Arg Thr Pro

AGG

Arg 185 CGA GGT CAG AAC Arg Gly Gin Asn

AGG

Arg 190 AGT GCA CGG ATA Ser Ala Arg Ile

GCA

Ala 195 AAA CPA CTA GAA Lys Gin Leu Glu

PAT

Asn 200 GAT ACA AGA ATT Asp Thr Arg Ile ATT GAA Ile Glu 205 GTT CTC TGT Vai Leu Cys GAA CAT GAA TGT Glu His Giu Cys

PAT

Asn 215 ATA GAT GAG GTG Ile Asp Glu Val AAA PAT GTT Lys Asn Val 220 SUBSTITUTE SHEET (RULE 26) WO 98/37091 TAT TTC AAA Tyr Phe Lys 225 PCTIUS98/03041 AAT TTT ATA CCT TTT ATG AAT TCT CTT Asn Phe Ile Pro Phe Met Asn Ser Leu

GGA

Gly 235 CTT GTA ACA Leu Val Thr TCT AAT Ser Asn 240 GGA CTT CCA GAG Gly Leu Pro Glu

GTT

Val 245 GAA AAT CTT TCT Glu Asn Leu Ser CGA TAC GAA GAA Arg Tyr Giu Glu

ATT

Ile 255 TAT CTT AAA AAT Tyr Leu Lys Asn

AAA

Lys 260 GAT CTA GAT GCA Asp Leu Asp Ala

AGA

Arg 265 TTA TTT TTG GAT Leu Phe Leu Asp

CAT

His 270 GAT AAA ACT CTT Asp Lys Thr Leu

CAG

Gin 275 ACT GAT TCT ATA Thr Asp Ser Ile AGT TTT GAA ACA Ser Phe Giu Thr CAG AGA Gin Arg 285 ACA CCA CGA Thr Pro Arg CAC ACT CCA His Thr Pro 305

AAA

Lys 290 AGT AAC CTT GAT Ser Asn Leu Asp GAG GTG AAT GTA Glu Val Asn Val ATT CCT CCA Ile Pro Pro 300 TTA ATG ATG Leu Met Met GTT AGG ACT GTT ATG AAC ACT ATC CAA Val Arg Thr Val Met Asn Thr Ile Gin

CAA

Gin 315 960 ATT TTA AAT TCA GCA AGT Ile Leu Asn Ser Ala Ser 320

GAT

Asp 325 CAA CCT TCA GAA Gin Pro Ser Glu

AAT

Asn 330 CTG ATT TCC TAT Leu Ile Ser Tyr 1008 AAC AAC TGC ACA GTG AAT CCA AAA GAA Asn Asn Cys Thr Vai Asn Pro Lys Glu 340

AGT

Ser 345 ATA CTG AAA AGA Ile Leu Lys Arg 1056 AAG GAT ATA GGA Lys Asp Ile Gly

TAC

Tyr 355 ATC TTT AAA GAG Ile Phe Lys Glu TTT GCT AAA GCT Phe Ala Lys Ala GTG GGA Vai Gly 365 1104 CAG GGT TGT Gin Gly Cys TTG TAT TAC Leu Tyr Tyr 385 GAA ATT GGA TCA Glu Ile Gly Ser

CAG

Gin 375 CGA TAC AAA CTT Arg Tyr Lys Leu GGA GTT CGC Gly Val Arg 380 GAA GAA CGA Glu Giu Arg 1152 1200 CGA GTA ATG GAA Arg Vai Met Glu

TCC

Ser 390 ATG CTT AAA TCA Met Leu Lys Ser TTA TCC Leu Ser 400 ATT CAA AAT TTT Ile Gin Asn Phe AAA CTT CTG AAT Lys Leu Leu Asn AAC ATT TTT CAT Asn Ile Phe His

ATG

Met 415 TCT TTA TTG GCG Ser Leu Leu Ala GCT CTT GAG GTT Ala Leu Giu Val GTA ATG Val Met 425 GCC ACA TAT Ala Thr Tyr 1248 1296 1344 AGA AGT ACA TCT Arg Ser Thr Ser

CAG

Gin 435 AAT CTT GAT Asn Leu Asp TCT GGA Ser Gly 440 ACA GAT TTG TCT TTC CCA Thr Asp Leu Ser Phe Pro 445 SUBSTITUTE SHEET (RULE 26) WO 98137091 TGG ATT CTG Trp Ile Leu ATC GAA AGT Ile Glu Ser 465 PCT/US98/03041

AAT

Asn 450 GTG CTT AAT TTA Val Leu Asn Leu GCC TTT GAT TTT Ala Phe Asp Phe TAC AAA GTG Tyr Lys Val 460 GAA ATG ATA Glu Met Ile 1392 TTT ATC AAA GCA GAA GGC AAC TTG ACA Phe Ile Lys Ala Glu Gly Asn Leu Thr 470 AAA CAT Lys His 480 TTA GAA CGA TGT Leu Glu Arg Cys CAT CGA ATC ATG His Arg Ile Met

GAA

Giu 490 TCC CTT GCA TGG Ser Leu Ala Trp 1440 1488 1536 1584

CTC

Leu 495 TCA GAT TCA CCT Ser Asp Ser Pro TTT GAT CTT ATT Phe Asp Leu Ile

AAA

Lys 505 CAA TCA AAG GAC Gin Ser Lys Asp

CGA

Arg 510 GAA GGA CCA ACT Glu Gly Pro Thr CAC CTT GAA TCT His Leu Giu Ser TGT CCT CTT AAT Cys Pro Leu Asn CTT CCT Leu Pro 525 CTC CAG AAT AAT CAC ACT GCA GCA Leu Gin Asn Asn His Thr Ala Ala 530 GAT ATG TAT CTT TCT CCT GTA AGA Asp Met Tyr Leu Ser Pro Val Arg 535 .540 ACG CGT GTA AAT TCT ACT GCA AAT Thr Arg Val Asn Ser Thr Ala Asn 555 1632 TCT CCA AAG Ser Pro Lys 545 AAA AAA GGT TCA Lys Lys Gly Ser 1680 GCA GAG Ala Giu 560 ACA CAA GCA ACC Thr Gin Ala Thr

TCA

Ser 565 GCC TTC CAG ACC Ala Phe Gin Thr AAG CCA TTG AAA Lys Pro Leu Lys

TCT

Ser 575 ACC TCT CTT TCA Thr Ser Leu Ser

CTG

Leu 580 TTT TAT AAA AAA Phe Tyr Lys Lys

GTG

Val 585 TAT CGG CTA GCC Tyr Arg Leu Ala 1728 1776 1824 CTC CGG CTA AAT Leu Arg Leu Asn

ACA

Thr 595 CTT TGT GAA CGC Leu Cys Giu Arg CTG TCT GAG CAC Leu Ser Giu His CCA GAA Pro Glu 605 TTA GAA CAT Leu Glu His TAT GAA CTC Tyr Glu Leu 625

ATC

Ile 610 ATC TGG ACC CTT Ile Trp Thr Leu CAG CAC ACC CTG Gin His Thr Leu CAG AAT GAG Gin Asn Glu 620 ATG TGT TCC Met Cys Ser 1872 1920 ATG AGA GAC AGG Met Arg Asp Arg TTG GAC CAA ATT Leu Asp Gin Ile

ATG

Met 635 ATG TAT Met Tyr 640 GGC ATA TGC AAA Gly Ile Cys Lys AAG AAT ATA GAC Lys Asn Ile Asp AAA TTC AAA ATC Lys Phe Lys Ile 1968 2016 ATT Ile 655 GTA ACA GCA TAC Val Thr Ala Tyr GAT CTT CCT CAT Asp Leu Pro His GTT CAG GAG ACA Val Gin Giu Thr SUBSTITUTE SHEET (RULE 26) W 098/37091 PCTIUS98/03041 AAA CGT GTT TTG Lys Arg Vai Leu AAA GAA GAG GAG Lys Giu Glu Glu

TAT

Tyr 680 GAT TCT ATT ATA Asp Ser Ile Ile GTA TTC Val Phe 685 2064 TAT AAC TCG Tyr Asn Ser GCT TCC ACC Ala Ser Thr 705 TTC ATG CAG AGA Phe Met Gin Arg

CTG

Leu 695 AAA ACA AAT ATT Lys Thr Asn Ile TTG CAG TAT Leu Gin Tyr 700 2112 AGG CCC CCT ACC Arg Pro Pro Thr

TTG

Leu 710 TCA CCA ATA CCT CAC ATT CCT CGA Ser Pro Ile Pro His Ile Pro Arg 715 2160 AGC CCT Ser Pro 720 TAC AAG TTT CCT Tyr Lys Phe Pro TCA CCC TTA CGG Ser Pro Leu Arg CCT GGA GGG AAC Pro Gly Gly Asn

ATC

Ile 735 TAT ATT TCA CCC Tyr lie Ser Pro AAG AGT CCA TAT Lys Ser Pro Tyr ATT TCA GAA GGT Ile Ser Giu Gly 2208 2256 2304 CCA ACA CCA ACA Pro Thr Pro Thr ATG ACT CCA AGA Met Thr Pro Arg

TCA

Ser 760 AGA ATC TTA GTA Arg Ile Leu Vai TCA ATT Ser Ile 765 GGT GAA TCA Gly Giu Ser GTA TGT AAC Val Cys Asn 785 GGG ACT TCT GAG Gly Thr Ser Glu

AAG

Lys 775 TTC CAG AAA ATA Phe Gin Lys Ile AAT CAG ATG Asn Gin Met 780 GGA AGC AAC Gly Ser Asn 2352 2400 AGC GAC CGT GTG Ser Asp Arg Vai

CTC

Leu 790 AAA AGA AGT GCT Lys Arg Ser Aia CCT CCT Pro Pro 800 GAA GCA Glu Ala 815 AAA CCA CTG AAA Lys Pro Leu Lys

AAA

Lys 805 CTA CGC TTT CAT Leu Arg Phe Asp GAA GGA TCA GAT Glu Gly Ser Asp GAT GGA AGT Asp Giy Ser

AAA

Lys 820 CAT CTC CCA GGA His Leu Pro Gly TCC AAA TTT CAG Ser Lys Phe Gin 2448 2496 2544 AAA CTG GCA GAA Lys Leu Ala Glu

ATG

Met 835 ACT TCT ACT CGA Thr Ser Thr Arg CGA ATG CAA AAG Arg Met Gin Lys CAG AAA Gin Lys 845 ATG AAT GAT Met Asn Asp ATG GAT ACC TCA Met Asp Thr Ser AAG GAA GAG AAA Lys Giu Glu Lys TGAGGATCTC 2593 AGGACCTTCG TGGACACTGT GTACACCTCT GGATTCATTG TCTCTCACAG ATGTGACTGT ATAACTTTCC CAGGTTCTGT TTATGGCCAC ATTTAATATC TTCAGCTCTT TTTGTGGATA TAAAATGTGC AGATGCAATT GTTTGGGTGA TTCCTAAGCC ACTTGAAATG TTAGTCATTG TTATTTATAC AAGATTGAAA ATCTTGTGTA A.ATCCTGCCA TTTAAAAAGT TGTAGCAGAT TGTTTCCTCT TCCAAAGTAA AATTGCTGTG CTTTATGGAT AGTAAGAATG GCCCTAGAGT 2653 2713 2773 2833 2893 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

ATAACCCAGG CCTGTCTGAC TACTTTGCCT TTGTTTTTAT TAATTTATAT GTATATTTTT TGTCCTATCT ATCTTCCAAA TGCA.ATTTGA ACTCTTCTGC AAAAATGGAT ATTATTAGA.A AGATTTTATT TTACTATTGG AATCTGATAT TTAATTAAAT AAAAGCTGGA AGCAAAGTAT AGATTTCATA CCTCAGAATG TAAAAGAACT TTTTAAATGA GGATTATTGA TAGT

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA.

TACTATCATA

TTTCTTCATC

2953 3013 3073 3133 3193 3253 3313 3347 INFORMATION FOR SEQ ID NO: 43: Wi SEQUENCE CHARACTERISTICS: LENGTH: 859 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala 1 Al a Ala Ala Glu Asp Ala Glu Pro Pro Ala Pro Pro Pro Pro Pro Pro Glu Pro Glu Gin Phe Glu Glu so Lys Ile Pro Asp Ser Gly Pro Glu Asp Leu Pro Asp Phe Thr Ala Leu Leu Arg Leu Glu Thr Glu Giu Asp His Val Val Asp Gly Cys Gln Lys Leu Arg Glu Arg Ala Trp, Leu Thr Trp, Giu Ser Ser Val Leu Gly Tyr Ile Gln Lys Lys Lys Glu Leu Trp Ser Thr Glu 115 Gly 100 Cys Ile Phe Ile 105 Ala Ala Val Asp Leu Val Glu 110 Trp Ile Thr Ile Asn Ser Ala Leu 120 Val Leu Lys Val Phe Leu 130 Ile Ser 145 Leu Ala Lys Gly Val Leu Gln Met Glu 140 Tyr Asp Asp Leu Val Phe Gin Leu Leu Cys Val Leu Asp 155 Phe Ile Lys SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 98/709 1PCT[US98/03041 Ser Ile Arg Cys Lys 225 Gly Leu Thr Arg Pro 305 Asn Asn Ile Cys &ryr 385 Ile Leu Thr Leu Pro Gly Al a 195 Glu Phe Pro Asn Gin 275 S er Arg Ala Thr Tyr 355 Glu Val Asn Ala Gin 435 Val Met Ser 180 Lys His Ile Giu Lys 260 Thr Asn Thr Ser Val 340 Ile Ile Met Phe Cys 420 Asn Leu Leu 165 Pro Gin Glu Pro Val 245 Asp Asp Leu Val Asp 325 Asn Phe Gly Giu Ser 405 Ala Leu Asn Leu Arg Leu Cys Phe 230 Giu Leu Ser Asp Met 310 Gin Pro Lys Ser Ser 390 Lys Leu Asp Leu Lys Thr Glu Asn 215 Met Asn Asp Ile Giu 295 Asn Pro Lys Glu Gin 375 Met Leu Glu Ser Lys 455 Glu Pro Asn 200 Ile Asn Leu Ala Asp 280 Giu Thr Ser Giu Lys 360 Arg Leu Leu Val Gly 440 Ala Pro Arg 185 Asp Asp Ser Ser Arg 265 Ser Val Ile Glu Ser 345 Phe Tyr Lys Asn Val 425 Thr Phe Tyr 170 Arg Thr Giu Leu Lys 250 Leu Phe Asn Gin Asn 330 Ile Ala Lys S er Asp 410 Met Asp Asp Lys Gly Arg Val Gly 235 Arg Phe Giu Vai Gin Leu Leu Lys Leu Glu 395 Asn Al a Leu Phe Thr Gin Ile Lys 220 Leu Tyr Leu Thr Ile 300 Leu Ile Lys Ala Gly 380 Glu Ile Thr Ser Tyr 460 Ala Asn Ile 205 Asn Val Glu Asp Gin 285 Pro Met Ser Arg Val 365 Val Glu Phe Tyr Phe 445 Lys Val Arg 190 Glu Val Thr Glu His 270 Arg Pro Met Tyr Val 350 Gly Arg Arg His Ser 430 Pro Val Ile 175 Ser Val Tyr Ser Ile 255 Asp Thr His Ile Phe 335 Lys Gin Leu Leu Met 415 Arg Trp Ile Pro Ala Leu Phe Asn 240 Tyr Lys Pro Thr Leu 320 Asn Asp Gly Tyr Ser 400 Ser Ser Ile Giu SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTIUS98/03041 Ser 465 Leu Asp Pro Asn Lys 545 Thr Ser Leu His Leu 625 Gly.

Thr Val Ser Thr 705 Tyr Ile Pro Phe Glu Ser Thr Asn 530 Lys Gin Leu Asn Ile 610 Met Ile Ala Leu Val 690 Arg Lys Ser Thr Ile Arg Pro Asp 515 His Lys Ala Ser Thr 595 Ile Arg Cys Tyr Ile 675 Phe Pro Phe Pro Lys 755 Lys ys Leu 500 His Thr Gly Thr Leu 580 Leu Trp Asp Lys Lys 660 Lys Met Pro Pro Leu 740 Met Ala Glu 485 Phe Leu Ala Ser Ser 565 Phe Cys Thr Arg Val 645 Asp Glu Gin Thr Ser 725 Lys Thr Glu 470 His Asp Glu Ala Thr 550 Ala Tyr Glu Leu His 630 Lys Leu Glu Arg Leu 710 Ser Ser Pro Gly Arg Leu Ser Asp 535 Thr Phe Lys Arg Phe 615 Leu Asn Pro Glu Leu 695 Ser Pro Pro Arg Asn Ile Ile Ala 520 Met Arg Gin Lys Leu 600 Gin Asp Ile His Tyr 680 Lys Pro Leu Tyr Ser 760 Leu Met Lys 505 Cys Tyr Val Thr Val 585 Leu His Gin Asp Ala 665 Asp Thr Ile Arg Lys 745 Arg Thr Glu 490 Gin Pro Leu Asn Gin 570 Tyr Ser Thr Ile Leu 650 Val Ser Asn Pro Ile 730 Ile Ile Arg 475 Ser Ser Leu Ser Ser 555 Lys Arg Glu Leu Met 635 Lys Gin Ile Ile His 715 Pro Ser Leu 31u ,eu Lys %sn Pro 540 rhr Pro Leu His Gin 620 Met Phe Glu Ile Leu 700 Ile Gly Glu Val Met Ala Asp Leu 525 Vai Ala Leu Ala Pro 605 Asn Cys Lys Thr Val 685 Gin Pro Gly Gly Ser 765 Ile Trp Arg 510 Pro Arg Asn Lys Tyr 590 Glu Glu Ser Ile Phe 670 Phe Tyr Arg Asn Leu 750 Ile Lys I Leu 495 Glu Leu Ser Ala Ser 575 Leu Leu Tyr Met Ile 655 Lys Tyr Ala Ser Ile 735 Pro Gly fis 180 'er ;iy 31n Pro Glu 560 rhr Arg Glu Glu Tyr 640 Val Arg Asn Ser Pro 720 Tyr Thr Glu SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 Ser Phe 770 Gly Thr Ser Glu Phe Gin Lys Ile Gin Met Val Cys Asn 785 Ser Asp Arg Val Lys Arg Ser Ala Glu 795 Gly Ser Asn Pro Lys Pro Leu Lys Leu Arg Phe Asp Glu Gly Ser Asp Glu Ala 815 Asp Gly Ser Ala Glu Met 835 His Leu Pro Gly Glu 825 Ser Lys Phe Gin Gin Lys Leu 830 Lys Met Asn Thr Ser Thr Arg Arg Met Gin Lys Asp Ser 850 Met Asp Thr Ser Lys Glu Glu Lys INFORMATION FOR SEQ ID NO: 44: SEQUENCE CHARACTERISTICS: LENGTH: 3161 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2397 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: CGCGTC ATG CCG CCC AAA ACC CCC CGA AAA ACG GCC GCC ACC GCC GCC Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala

GCT

Ala GCC GCC GCG GAA CCC CCG GCA CCG CCG CCG CCG CCC CCT CCT GAG Ala Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Glu GAG GAC CCA GAG CAG GAC AGC GGC CCG Glu Asp Pro Glu Gin Asp Ser Gly Pro CTT GAG TTT GAA GAA ACA GAA GAA CCT Leu Glu Phe Glu Glu Thr Glu Glu Pro 55 GAC CTG CCT CTC GTC AGG Asp Leu Pro Leu Val Arg GAT TTT ACT GCA Asp Phe Thr Ala TTA TGT CAG Leu Cys Gin TTA ACT TGG Leu Thr Trp AAA TTA AAG Lys Leu Lys ATA CCA GAT CAT GTC AGA GAG AGA Ile Pro Asp His Val Arg Glu Arg 70 GCT TGG Ala Trp 240 GAG AAA Glu Lys GTT TCA TCT GTG GAT GGA GTA TTG GGA GGT TAT ATT CAA AAG Val Ser Ser Val Asp Gly Val Leu Gly Gly Tyr Ile Gin Lys SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/0304 1

AAA

Lys AAG GAA CTG TGG Lys Glu Leu Trp ATC TGT ATC TTT ATT GCA GCA GTT GAC CTA Ile Cys Ile Phe Ile Ala Ala Val Asp Leu GCT GTT ATA CCC ATT AAT GGT TCA CCT Ala Val Ile Pro Ile Asn Gly Ser Pro 115 ACA CCC AGG CGA Thr Pro Arg Arg GGT CAG Giy Gin 125 AAC AGG AGT Asn Arg Ser ATT GAA GTT Ile Giu Val 145

GCA

Ala 130 CGG ATA GCA AAA Arg Ile Ala Lys

CAA

Gin 135 CTA GAA AAT GAT Leu Giu Asn Asp ACA AGA ATT Thr Arg Ile 140 GAG GTG AAA Giu Val Lys CTC TGT AAA GAA Leu Cys Lys Giu

CAT

His 150 GAA TGT AAT ATA Giu Cys Asn Ile AAT GTT Asn Val 160 TAT TTC AAA AAT Tyr Phe Lys Asn

TTT

Phe 165 ATA CCT TTT ATG Ile Pro Phe Met TCT CTT GGA CTT Ser Leu Gly Leu ACA TCT AAT GGA Thr Ser Asn Gly CCA GAG GTT GAA Pro Giu Val Giu CTT TCT AAA CGA Leu Ser Lys Arg GAA GAA ATT TAT Giu Giu Ile Tyr AAA AAT AAA GAT Lys Asn Lys Asp GAT GCA AGA TTA Asp Ala Arg Leu TTT TTG Phe Leu 205 GAT CAT GAT Asp His Asp CAG AGA ACA Gin Arg Thr 225

AAA

Lys 210 ACT CTT CAG ACT Thr Leu Gin Thr TCT ATA GAC AGT Ser Ile Asp Ser TTT GAA ACA Phe Glu Thr 220 AAT GTA ATT Asn Val Ile CCA CGA AAA AGT Pro Arg Lys Ser

AAC

Asn 230 CTT GAT GAA GAG Leu Asp Glu Giu

GTG

Val 235 CCT CCA CAC ACT CCA GTT Pro Pro His Thr Pro Val

AGG

Arg 245 ACT GTT ATG AAC Thr Val Met Asn

ACT

Thr 250 ATC CAA CAA TTA Ile Gin Gin Leu

ATG

Met 255 ATG ATT TTA AAT Met Ile Leu Asn

TCA

Ser 260 GCA AGT GAT CAA Ala Ser Asp Gin

CCT

Pro 265 TCA GAA AA.T CTG Ser Glu Asn Leu

ATT

Ile 270 TCC TAT TTT AAC AAC TGC ACA GTG AAT Ser Tyr Phe Asn Asn Cys Thr Val Asn 275

CCA

Pro 280 AAA GAA AGT ATA Lys Glu Ser Ile CTG AAA Leu Lys 285 AGA GTG AAG Arg Val Lys GTG GGA CAG Vai Gly Gin 305 ATA GGA TAC ATC Ile Gly Tyr Ile

TTT

Phe 295 AAA GAG AAA TTT Lys Giu Lys Phe GCT AAA GCT Ala Lys Ala 300 AAA CTT GGA Lys Leu Gly GGT TGT GTC GAA ATT GGA TCA CAG CGA Gly Cys Val Giu Ile Gly Ser Gin Arg 310

TAC

Tyr 315 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTJUS98/0304 1 205 GTT CGC Val Arg 320 TTG TAT TAC CGA GTA ATG GAA TOO ATG Leu Tyr Tyr Arg Val Met Glu Ser Met AAA TCA GAA GAA Lys Ser Giu Giu 1008 1056 OGA TTA TCC ATT CAA AAT TTT AGC AAA Arg Leu Ser Ile Gin Asn Phe Ser Lys 340 OTG AAT GAO AAO Leu Asn Asp Asn

ATT

Ile 350 TTT CAT ATG TOT Phe His Met Ser TTG GCG TGC GCT Leu Aia Cys Ala GAG GTT GTA ATG Giu Val Val Met GCC ACA Ala Thr 365 1104 1152 TAT AGC AGA Tyr Ser Arg TTC CCA TGG Phe Pro Trp 385 ACA TCT CAG AAT Thr Ser Gin Asn GAT TCT GGA ACA GAT TTG TOT Asp Ser Gly Thr Asp Leu Ser 380 ATT CTG AAT GTG Ile Leu Asn Val AAT TTA AAA GCC Asn Leu Lys Ala GAT TTT TAO Asp Phe Tyr 1200 AAA GTG Lys Val 400 ATO GAA AGT TTT Ile Giu Ser Phe

ATO

Ile 405 AAA GCA GAA GGO Lys Ala Giu Gly TTG ACA AGA GAA Leu Thr Arg Glu 1248 '1296 ATG Met 415 ATA AAA OAT TTA Ile Lvs His Leu

GAA

Giu 420 OGA TGT GAA OAT Arg Cys Giu His ATC ATG GAA TOO Ile Met Giu Ser GOA TGG OTO TOA GAT TOA OCT TTA TTT Ala Trp Leu Ser Asp Ser Pro Leu Phe 435

GAT

Asp 440 OTT ATT AAA CAA Leu Ile Lys Gin TOA AAG, Ser Lys 445 1344 GAO OGA GAA Asp Arg Giu OTT OCT OTO Leu Pro Leu 465 OCA ACT GAT CAC Pro Thr Asp His GAA TOT GOT TGT Giu Ser Ala Cys OCT OTT AAT Pro Leu Asn 460 OTT TOT COT Leu Ser Pro 1392 1440 CAG AAT AAT CAC Gin Asn Asn His

ACT

Thr 470 GOA GOA GAT ATG Ala Ala Asp Met GTA AGA Val Arg 480 TOT OCA AAG AAA Ser Pro Lys Lys

AAA

Lys 485 GGT TOA ACT AOG Giy Ser Thr Thr GTA AAT TOT ACT Vai Asn Ser Thr 1488 IS719 AAT GOA GAG ACA Asn Ala Giu Thr

CAA

Gin 500 GOA ACC TOA GC Ala Thr Ser Ala CAG ACC CAG AAG Gin Thr Gin Lys TTG AAA TOT ACC TOT OTT TCA OTG TTT Leu Lys Ser Thr Ser Leu Ser Leu Phe 515

TAT

Tyr 520 AAA AAA GTG TAT Lys Lys Vai Tyr OGG OTA Arg Leu 525 1584 GOC TAT OTO Ala Tyr Leu OTA AAT ACA OTT TGT GAA OGO OTT OTG Leu Asn Thr Leu Cys Giu Arg Leu Leu 535 TOT GAG CAC Ser Glu His 540 1632 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 CCA GAA TTA GAA CAT ATC ATC TGG ACC Pro Glu Leu 545 Glu His Ile Ile Trp 550 Thr CTT TTC CAG Leu Phe Gin

CAC

His 555 ACC CTG CAG Thr Leu Gin 1680 AAT GAG Asn Glu 560 TAT GAA CTC ATG Tyr Glu Leu Met

AGA

Arg 565 GAC AGG CAT TTG Asp Arg His Leu CAA ATT ATG ATG Gin Ile Met Met

TGT

Cys 575 TCC ATG TAT GGC Ser Met Tyr Gly TGC AAA GTG AAG Cys Lys Val Lys

AAT

Asn 585 ATA GAC CTT AAA Ile Asp Leu Lys 1728 1776 1824 AAA ATC ATT GTA Lys Ile Ile Val GCA TAC AAG GAT Ala Tyr Lys Asp

CTT

Leu 600 CCT CAT GCT GTT Pro His Ala Val CAG GAG Gin Glu 605 ACA TTC AAA Thr Phe Lys GTA TTC TAT Val Phe Tyr 625 GTT TTG ATC AAA Val Leu Ile Lys

GAA

Glu 615 GAG GAG TAT GAT Glu Glu Tyr Asp TCT ATT ATA Ser Ile Ile 620 AAT ATT TTG Asn Ile Leu 1872 1920 AAC TCG GTC TTC Asn Ser Val Phe CAG AGA CTG AAA Gin Arg Leu Lys

ACA

Thr 635 CAG TAT Gin Tyr 640 GCT TCC ACC AGG Ala Ser Thr Arg CCT ACC TTG TCA Pro Thr Leu Ser

CCA

Pro 650 ATA CCT CAC ATT Ile Pro His Ile CCT CGA AGC CCT TAC Pro Arg Ser Pro Tyr TTT CCT AGT TCA Phe Pro Ser Ser TTA CGG ATT CCT Leu Arg Ile Pro 1968 2016 2064 GGG AAC ATC TAT Gly Asn Ile Tyr TCA CCC CTG AAG Ser Pro Leu Lys

AGT

Ser 680 CCA TAT AAA ATT Pro Tyr Lys Ile TCA GAA Ser Glu 685 GGT CTG CCA Gly Leu Pro TCA ATT GGT Ser Ile Gly 705 CCA ACA AAA Pro Thr Lys ATG ACT Met Thr 695 CCA AGA TCA AGA Pro Arg Ser Arg ATC TTA GTA Ile Leu Val 700 AAA ATA AAT Lys Ile Asn 2112 2160 GAA TCA TTC Glu Ser Phe GGG ACT Gly Thr 710 TCT GAG AAG TTC Ser Glu Lys Phe

CAG

Gin 715 CAG ATG Gin Met 720 GTA TGT AAC AGC Val Cys Asn Ser CGT GTG CTC AAA Arg Val Leu Lys

AGA

Arg 730 AGT GCT GAA GGA Ser Ala Glu Gly AAC CCT CCT AAA Asn Pro Pro Lys

CCA

Pro 740 CTG AAA AAA CTA Leu Lys Lys Leu

CGC

Arg 745 TTT GAT ATT GAA Phe Asp Ile Glu 2208 2256 2304 TCA GAT GAA GCA Ser Asp Glu Ala GGA AGT AAA CAT Gly Ser Lys His CCA GGA GAG TCC Pro Gly Glu Ser AAA TTT Lys Phe 765 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/0304 1 CAG CAG AAA CTG GCA GAA ATG ACT TCT ACT CGA Gin Gin Lys Leu Ala Glu Met Thr Ser Thr Arg 770 775 ACA CGA ATG CAA AAG Thr Arg Met Gin Lys 780 2352 2397 CAG AAA ATG AAT GAT AGC ATG GAT ACC TCA AAC AAG GAA GAG AAA Gin Lys Met Asn Asp Ser Met Asp Thr Ser Asn Lys Glu Giu Lys

TGAGGATCTC

ATGTGACTGT

TTTGTGGATA

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA

TACTATCATA

TTTCTTCATC

AGGACCTTGG

ATAACTTTCC

TAAAATGTGC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

TGGACACTGT

CAGGTTCTGT

AGATGCAATT

AAGATTGAAA

TCCAAAGTAA

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

TTTTAAATGA

GTACACCTCT

TTATGGCCAC

GTTTGGGTGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

CCTCAGAATG

GGATTATTGA

GGATTCATTG

ATTTAATATC

TTCCTAAGCC

AATCCTGCCA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TAAAAGAACT

TAGT

TCTCTCACAG

TTCAGCTCTT

ACTTGAAATG

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

2457 2517 2577 2637 2697 2757 2817 2877 2937 2997 3057 3117 3161 INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 797 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO: Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala T 1 10 hr Ala Ala Ala Ala Ala Ala Glu Pro Pro Ala Pro Pro Pro 25 Pro Pro Pro Pro Pro Glu Gin Asp Ser Gly Pro Glu Asp Leu Pro Leu Val 40 Phe Glu Glu Thr Giu Glu Pro Asp Phe Thr Ala Leu Cys .55 Glu Glu Asp Arg Leu Glu Gln Lys Leu SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/03041 208 Ile Ser Leu Pro Ala 130 Leu Phe Asn Tyr Lys 210 Pro Thr Leu Asn Asp 290 Gly Tyr Ser Ser Pro Ser Trp Ile Arg Cys Lys Gly Leu 195 Thr Arg Pro Asn Asn 275 Ile Cys Tyr Ile Leu 3.55 Asp Val Gly 100 Asn Ile Lys Asn Leu 180 Lys Leu Lys Vai Ser 260 Cys Giy Val Arg Gin 340 Leu His Asp Ile Gly Al a Glu Phe 165 Pro Asn Gin Ser Arg 245 Ala Thr Tyr Giu Val 325 Asn Al a Val Gly Cys Ser Lys His Ile Glu Lys Thr Asn 230 Thr Ser Vai Ile Ile 310 Met Phe Cys Arg Val Ile Pro Gin 135 Glu Pro Val1 Asp Asp 215 Leu Val Asp Asn Phe 295 Gly Giu Se r Al a Giu Leu Phe Arg 120 Leu Cys Phe Giu Leu 200 Ser Asp Met Gin Pro 280 Lys Ser Ser Lys Leu 360 Arg Gly Ile 105 Thr Glu Asn Met Asn 185 Asp Ile Giu Asn Pro 265 Lys Glu Gin Met Leu 345 Glu Ala Gly 90 Al a Pro Asn Ile Asn 170 Leu Al a Asp Giu Thr 250 Ser Giu Lys Arg Leu 330 Leu Val Trp 75 Tyr Al a Arg Asp Asp i55 Ser Ser Arg Ser Val 235 Ile Glu Ser Phe Tyr 315 Lys Asn Val Leu Ile Val Arg Thr 140 Giu Leu Lvs Leu Phe 220 Asti Gin Asn Ile Ala 300 Lys Ser Asp Met Thr Gin Asp Gly 125 Arg Val Gly Arg Phe 205 Glu Val Gin Leu Leu 285 Lys Leu Giu Asn Ala 365 Trp Lys Leu Gin Ile Lys Leu Tyr 190 Leu Thr Ile Leu Ile 270 Lys Ala Gly Giu Ile 350 Thr Giu Lys Ala Asn Ile Asn Val 175 Giu Asp Gin Pro Met 255 Ser Arg Vai Val Giu 335 Phe Tyr Lys Lys Val Arg Giu Val 160 Thr Giu His Arg Pro 240 Met Tyr Val Gly Arg 320 Arg His Ser SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 Arg Trp, 385 Ile Lys Leu Glu Leu 465 Ser Ala Ser Leu Leu 545 Tyr Met Ile L~ys Tyr 625 Ala Ser Ser Thr Ser 370 Ilie Leu Asn Giu Ser Phe His Leu Giu 420 Ser Asp. Ser 435 Gly Pro Thr 450 Gin Asn Asn Pro Lys Lys Glu Thr Gin 500 Thr Ser Leu 515 Arg Leu Asn 530 Giu His Ile Giu Leu Met Tyr Gly Ile 580 Val Thr Ala 5-9-5 Arg Val Leu 610 Asn Ser Val Ser Thr Arg Pro Tyr Lys 660 Gin Val Ile 405 Arg Pro Asp His Lys 485 Al a Ser Thr Ile Arg 565 Cys Tyr Ile Phe Pro 645 Phe Asn Leu 390 Lys Cys Leu His Thr 470 Gly Thr Leu Leu Trp 550 Asp Lys Lys Lys Met 630 Pro Pro Leu 375 Asn Ala Giu Phe Leu 455 Al a Ser Ser Phe Cys 535 Thr Arg Val Asp Glu 615 Gin Thr Ser Asp Leu Giu His Asp 440 Giu Ala Thr Aia Tyr 520 Giu Leu His Lys Leu 600 Giu Arg Leu Ser Ser Lys Gly Arg 425 Leu Ser Asp Thr Phe 505 Lys Arg Phe Leu Asn 585 Pro Giu Leu Ser Pro 665 Gly Thr Aia Phe 395 Asn Leu 410 Ile Met Ile Lys Ala Cys Met Tyr 475 Arg Val 490 Gin Thr Lys Val Leu Leu Gin His 555 Asp Gin 570 Ile Asp His Ala Tyr Asp Lys Thr 635 Pro Ile 650 Leu Arg Asp Leu 380 Asp Phe Thr Arg Giu Ser Gin Ser 445 Pro Leu 460 Leu Ser Asn Ser Gin Lys Tyr Arg 525 Ser Giu 540 Thr Leu Ile Met Leu Lys Val Gin 605 Ser Ile 620 Asn Ile Pro His Ile Pro Ser Tyr Glu Leu 430 Lys Asn Pro Thr Pro 510 Leu His Gin Met Phe 590 Giu Ile Leu Ile Gly 670 Phe Lys Met 415 Al a Asp Leu Val Ala 495 Leu Ala Pro Asn Cys 575 Lys Thr Val Gin Pro 655 Gly Pro Val 400 Ile Trp, Arg Pro Arg 480 Asn Lys Tyr Giu Glu 560 Ser Ile Phe Phe Tyr 640 Arg Asn SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 Ile Pro Gly 705 Val Pro Glu Lys Met 785 Ile 675 Pro Ser Asn Lys Asp 755 Ala Asp Ser Thr Phe Ser Pro 740 Gly Glu Ser Pro Lys Gly Asp 725 Leu Ser Met Met Leu Met Thr 710 Arg Lys Lys Thr Asp 790 Lys Thr 695 Ser Val Lys His Ser 775 Thr Ser 680 Pro Glu Leu Leu Leu 760 Thr Ser Lys Arg Gin 715 Ser Asp Glu Arg Glu 795 Ile Ile 700 Lys Ala Ile Ser Met 780 Glu Ser 685 Leu Ile Glu Glu Lys 765 Gin Lys INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: 3377 base pai TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2613 (xi) SEQUENCE DESCRIPTION: SEQ CGCGTC ATG CCG CCC AAA ACC CCC CGA Met Pro Pro Lys Thr Pro Arg 1 5 GCT GCC GCC GCG GAA CCC CCG GCA CCC Ala Ala Ala Ala Glu Pro Pro Ala Prc 20 GAG GAC CCA GAG CAG GAC AGC GGC CCC Glu Asp Pro Glu Gln Asp Ser Gly Prc CTT GAG TTT GAA GAA ACA GAA GAA CC Leu Glu Phe Glu Glu Thr Glu Glu Prc 5 ID NO: 46: AAA ACG GCC GCC ACC GCC GCC Lys Thr Ala Ala Thr Ala Ala CCG CCG CCG CCC CCT CCT GAG SPro Pro Pro Pro Pro Pro Glu 25 SGAG GAC CTG CCT CTC GTC AGG SGlu Asp Leu Pro Leu Val Arg 40 GAT TTT ACT GCA TTA TGT CAG Asp Phe Thr Ala Leu Cys Gin SUBSTITUTE SHEET (RULE 26) WO 98/37091 AAA TTA AAG Lys Leu Lys GAG AAA GTT Glu Lys Val PCT/US98/03041 ATA CCA GAT CAT GTC AGA GAG AGA Ile Pro Asp His Val Arg Glu Arg 70 GCT TGG Ala Trp TTA ACT TGG Leu Thr Trp TCA TCT GTG GAT Ser Ser Val Asp GGA GTA TTG GGA Gly Val Leu Gly TAT ATT CAA AAG Tyr Ile Gin Lys

AAA

Lys AAG GAA CTG TGG Lys Glu Leu Trp GGA ATC Gly Ile 100 TGT ATC TTT Cys Ile Phe GCA GCA GTT GAC Ala Ala Val Asp 336 384 GAT GAG ATG TCG Asp Glu Met Ser ACT TTT ACT GAG Thr Phe Thr Glu

CTA

Leu 120 CAG AAA AAC ATA Gin Lys Asn Ile GAA ATC Glu Ile 125 AGT GTC CAT AAA TTC TTT AAC TTA CTA AAA GAA ATT GAT Ser Val His Lys Phe Phe Asn Leu Leu Lys Glu Ile Asp ACC AGT ACC Thr Ser Thr 140 GAT GTA TTG Asp Val Leu AAA GTT GAT AAT GCT ATG TCA Lys Val Asp Asn Ala Met Ser

AGA

Arg 150 CTG TTG AAG AAG Leu Leu Lys Lys

TAT

Tyr 155 TTT GCA Phe Ala 160 CTC TTC AGC AAA Leu Phe Ser Lys GAA AGG ACA TGT Glu Arg Thr Cys CTT ATA TAT TTG Leu Ile Tyr Leu

ACA

Thr 175 CAA CCC AGC AGT Gin Pro Ser Ser ATG GTC GCT GTT Met Val Ala Val

ATA

Ile 185 CCC ATT AAT GGT Pro Ile Asn Gly

TCA

Ser 190 CCT CGA ACA CCC Pro Arg Thr Pro CGA GGT CAG AAC Arg Gly Gin Asn

AGG

Arg 200 AGT GCA CGG ATA Ser Ala Arg Ile GCA AAA Ala Lys 205 CAA CTA GAA Gin Leu Glu GAA TGT AAT Glu Cys Asn 225 GAT ACA AGA ATT Asp Thr Arg Ile GAA GTT CTC TGT Glu Val Leu Cys AAA GAA CAT Lys Glu His 220 AAT TTT ATA Asn Phe Ile 672 720 ATA GAT GAG GTG Ile Asp Glu Val

AAA

Lys 230 AAT GTT TAT TTC Asn Val Tyr Phe

AAA

Lys 235 CCT TTT Pro Phe 240 ATG AAT TCT CTT Met Asn Ser Leu

GGA

Gly 245 CTT GTA ACA TCT Leu Val Thr Ser

AAT

Asn 250 GGA CTT CCA GAG Gly Leu Pro Glu

GTT

Val 255 GAA AAT CTT TCT Glu Asn Leu Ser CGA TAC GAA GAA Arg Tyr Glu Glu TAT CTT AAA AAT Tyr Leu Lys Asn

AAA

Lys 270 GAT CTA GAT GCA Asp Leu Asp Ala

AGA

Arg 275 TTA TTT TTG GAT Leu Phe Leu Asp GAT AAA ACT CTT Asp Lys Thr Leu CAG ACT Gin Thr 285 SUBSTITUTE SHEET (RULE 26) WO 98/37091 GAT TCT ATA Asp Ser Ile CTT GAT GAA Leu Asp Glu 305 PCTIUS98/03041 212 AGT TTT GAA ACA Ser Phe Glu Thr

CAG

Gin 295 AGA ACA CCA CGA Arg Thr Pro Arg AAA AGT AAC Lys Ser Asn 300 GTT AG ACT Val Arg Thr GAG GTG AAT GTA Giu Val Asn Vai CCA CAC ACT Pro Pro His Thr

CCA

Pro 315 GTT ATG Val Met 320 AAC ACT ATC CAA Asn Thr Ile Gin

CAA

Gin 325 TTA ATG ATG ATT Leu Met Met Ile

TTA

Leu 330 AAT TCA GCA-AGT Asn Ser Aia Ser

GAT

Asp 335 CAA CCT TCA GAA Gin Pro Ser Glu

AAT

Asn 340 CTG ATT TCC TAT Leu Ile Ser Tyr

TTT

Phe 345 AAC AAC TGC ACA Asn Asn Cys Thr

GTG

Val 350 1008 1056 1104 AAT CCA AAA GAA Asn Pro Lys Giu

AGT

Ser 355 ATA CTG AAA AGA Ile Leu-Lys Arg

GTG

Vai 360 AAG GAT ATA GGA Lys Asp Ile Gly TAC ATC Tyr Ile 365 TTT AAA GAG Phe Lys Giu GGA TCA CAG Gly Ser Gin 385 TTT GCT AAA GCT Phe Aia Lys Ala GGA CAG GGT TGT Gly Gin Gly Cys GTC GAA ATT Val Giu Ile 380 CGA GTA ATG Arg Val Met 1152 1200 CGA TAC AAA CTT Arg Tyr Lys Leu GTT CGC TTG TAT Val Arg Leu Tyr GAA TCC Giu Ser 400 ATG CTT AAA TCA Met Leu Lys Ser

GAA

Glu 405; GAA GAA CGA TTA Glu Giu Arg Leu ATT CAA AAT TTT Ile Gin Asn Phe

AGC

Ser 415 AAA CTT CTG AAT Lys Leu Leu Asn AAC ATT TTT CAT Asn Ile Phe His TCT TTA TTG GCG Ser Leu Leu Ala

TGC

Cys 430 1248 1296 1344 GCT CTT GAG GTT Ala Leu Giu Val

GTA

Val 435 ATG GCC ACA TAT Met Ala Thr Tyr

AGC

Ser 440 AGA AGT ACA TCT Arg Ser Thr Ser CAG AAT Gin Asn 445 CTT GAT TCT Leu Asp Ser ACA GAT TTG, TCT Thr Asp Leu Ser

TTC

Phe 455 CCA TGG ATT CTG Pro Trp Ile Leu AAT GTG CTT Asn Val Leu 460 1392 AAT TTA Asn Leu GCA GAA Ala Glu 480 AAA GCC Lys Ala 465 TTT GAT TTT Phe Asp Phe

TAC

Tyr 470 AAA GTG ATC GAA AGT TTT ATC AAA Lys Val Ile Glu Ser Phe Ile Lys 475 1440) GGC AAC TTG ACA Gly Asn Leu Thr

AGA

Arg 485 GAA ATG ATA AAA Glu Met Ile Lys

CAT

His 490 TTA GAA CGA TGT Leu Giu Arg Cys 1488 1536

GAA

Glu 495 CAT CGA ATC ATG His Arg Ile Met

GAA

Giu 500 TCC CTT GCA TGG Ser Leu Ala Trp TCA GAT TCA CCT Ser Asp Ser Pro SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 TTT GAT CTT ATT Phe Asp Leu Ile

AAA

Lys 515 CAA TCA AAG GAC CGA GAA GGA CCA ACT Gin Ser Lys Asp Arg Glu Gly Pro Thr 520 GAT CAC Asp His 525 1584 CTT GAA TCT Leu Glu Ser GCA GCA GAT Ala Ala Asp 545

GCT

Ala 530 TGT CCT CTT AAT Cys Pro Leu Asn CCT CTC CAG AAT Pro Leu Gin Asn AAT CAC ACT Asn His Thr 540 AAA AAA GGT Lys Lys Gly 1632 ATG TAT CTT TCT CCT GTA AGA TCT CCA Met Tyr Leu Ser Pro Val Arg Ser Pro TCA ACT Ser Thr 560 ACG CGT GTA AAT Thr Arg Val Asn ACT GCA AAT GCA Thr Ala Asn Ala ACA CAA GCA ACC Thr Gin Ala Thr 1680 1728 1776 1824 GCC TTC CAG ACC Ala Phe Gin Thr AAG CCA TTG AAA Lys Pro Leu Lys ACC TCT CTT TCA Thr Ser Leu Ser TTT TAT AAA AAA Phe Tyr Lys Lys TAT CGG CTA GCC Tyr Arg Leu Ala CTC CGG CTA AAT Leu Arg Leu Asn ACA CTT Thr Leu 605 TGT GAA CGC Cys Glu Arg ACC CTT TTC Thr Leu Phe 625 CTG TCT GAG CAC Leu Ser Glu His GAA TTA GAA CAT Glu Leu Glu His ATC ATC TGG Ile Ile Trp 620 ATG AGA GAC Met Arg Asp 1872 1920 CAG CAC ACC CTG Gin His Thr Leu AAT GAG TAT GAA Asn Glu Tyr Glu AGG CAT Arg His 640 TTG GAC CAA ATT Leu Asp Gin Ile

ATG

Met 645 ATG TGT'TCC ATG Met Cys Ser Met

TAT

Tyr 650 GGC ATA TGC AAA Gly Ile Cys Lys AAG AAT ATA GAC Lys Asn Ile Asp AAA TTC AAA ATC Lys Phe Lys Ile

ATT

Ile 665 GTA ACA GCA TAC Val Thr Ala Tyr 1968 2016 2064 GAT CTT CCT CAT Asp Leu Pro His GTT CAG GAG ACA Val Gin Glu Thr

TTC

Phe 680 AAA CGT GTT TTG Lys Arg Val Leu ATC AAA Ile Lys 685 GAA GAG GAG Glu Glu Glu CAG AGA CTG Gin Arg Leu 705

TAT

Tyr 690 GAT TCT ATT ATA Asp Ser Ile Ile TTC TAT AAC TCG Phe Tyr Asn Ser GTC TTC ATG Val Phe Met 700 AGG CCC CCT Arg Pro Pro 2112 2160 AAA ACA AAT ATT Lys Thr Asn Ile CAG TAT GCT TCC Gin Tyr Ala Ser ACC TTG Thr Leu 720 TCA CCA ATA CCT Ser Pro Ile Pro ATT CCT CGA AGC Ile Pro Arg Ser TAC AAG TTT CCT Tyr Lys Phe Pro 2208 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98O3O4 1 214 TCA CCC TTA CGG Ser Pro Leu Arg CCT GGA GGG AAC Pro Gly Gly Asn TAT ATT Tyr Ile ACA CCA Thr Pro AGT CCA TAT Ser Pro Tyr

AAA

Lys 755 ATT TCA GAA GGT Ile Ser Giu Gly ACT CCA AGA Thr Pro Arg TCT GAG AAG Ser Giu Lys 785 GTG CTC AAA Val Leu Lys

TCA

Ser 770 AGA ATC TTA GTA Arg Ile Leu Vai ATT GGT GAA TCA Ile Giy Giu Ser TCA CCC CTG Ser Pro Leu 750 ACA AAA ATG Thr Lys Met 765 TTC GGG ACT Phe Gly Thr 780 AGC GAC CGT Ser Asp Arg CCA CTG AAA Pro Leu Lys TTC CAG AAA ATA Phe Gin Lys Ile

AAT

Asn 790 CAG ATG GTA TGT Gin Met Val Cys

AAC

Asn 795

AAA

Lys 2256 2304 2352 2400 2448 2496 2544 2592 AGA AGT GCT Arg Ser Aia

GAA

Giu 805 GGA AGC AAC CCT Giy Ser Asn Pro B00 AAA CTA Lys Leu

CCT

Pro 810

GCA

Aila CGC TTT GAT Arg Phe Asp

ATT

Ile 820

TCC

Ser GAA GGA TCA GA Giu Giy Ser As AAA TTT CAG CA Lys Phe Gin Gi

TGAA

p Giu 825

GAAA

n Lys GAT GGA AGT Asp Gly Ser

AAA

Lys 830 CTC CCA GGA Leu Pro Giy

GAG

Giu 835

CGA

Arg CTG GCA GAA Leu Aia Giu ATG ACT Met Thr 845 ATG GAT Met Asp TCT ACT CGA Ser Thr Arg

ACA

Thr 850 ATG CAA AAG Met Gin Lys ATG AAT GAT AGC Met Asn Asp Ser 860 ACC TCA AAC AAG GAA GAG AAA Thr Ser Asn Lys Giu Giu Lys TGAGGATCTC AGGACCTTGG TGGACACTGT 2643 GTACACCTCT GGATTCATTG TCTCTCACAG ATGTGACTGT ATAACTTTCC

TTATGGCCAC

GTTTGGGTGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC!

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

ATTTAATATC

TTCCTAAGCC

AATCCTGCCA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TTCAGCTCTT

ACTTGAAATG

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TTTGTGGATA

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA

TACTATCATA

TAAAATGTGC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG,

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAGGTTCTGT

AGATGCAATT

AAGATTGAAA

TCCAAAGTAA

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

2703 2763 2823 2883 2943 3003 3063 3123 3183 3243 3303 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 215 CCTCAGAATG TAAAAGAACT TACTGATTAT TTTCTTCATC CAACTTATGT TTTTAAATGA 3363 GGATTATTGA TAGT 3377 INFORMATION FOR SEQ ID NO: 47: SEQUENCE CHARACTERISTICS: LENGTH: 869 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala Ala Ala 1 5 10 Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Glu Glu Asp 25 Pro Glu Gin Asp Ser Gly Pro Glu Asp Leu Pro Leu Val Arg Leu Glu 40 Phe Glu Glu Thr Glu Glu Pro Asp Phe Thr Ala Leu Cys Gin Lys Leu 55 Lys Ile Pro Asp His Val Arg Glu Arg Ala Trp Leu Thr Trp Glu Lys 70 75 Val Ser Ser Val Asp Gly Val Leu Gly Gly Tyr Ile Gin Lys Lys Lys 90 Glu Leu Trp Gly Ile Cys Ile Phe Ile Ala Ala Val Asp Leu Asp Glu 100 105 110 Met Ser Phe Thr Phe Thr Glu Leu Gin Lys Asn Ile Glu Ile Ser Val 115 120 125 His Lys Phe Phe Asn Leu Leu Lys Glu Ile Asp Thr Ser Thr Lys Val 130 135 140 Asp Asn Ala Met Ser Arg Leu Leu Lys Lys Tyr Asp Val Leu Phe Ala 145 150 155 160 Leu Phe Ser Lys Leu Glu Arg Thr Cys Glu Leu Ile Tyr Leu Thr Gin 165 170 175 Pro Ser Ser Ser Met Val Ala Val Ile Pro Ile Asn Gly Ser Pro Arg 180 185 190 Thr Pro Arg Arg Gly Gin Asn Arg Ser Ala Arg Ile Ala Lys Gin Leu 195 200 205 Glu Asn Asp Thr Arg Ile Ile Glu Val Leu Cys Lys Glu His Glu Cys 210 215 220 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 216 Asn Ile Asp Glu Val Lys Asn Val Tyr Phe Lys Asn Phe Ile Pro Phe 225 230 235 240 Met Asn Ser Leu Gly Leu Val Thr Ser Asn Gly Leu Pro Glu Val Glu 245 250 255 Asn Leu Ser Lys Arg Tyr Glu Glu Ile Tyr Leu Lys Asn Lys Asp Leu 260 265 270 Asp Ala Arg Leu Phe Leu Asp His Asp Lys Thr Leu Gin Thr Asp Ser 275 280 285 Ile Asp Ser Phe Glu Thr Gin Arg Thr Pro Arg Lys Ser Asn Leu Asp 290 295 300 Glu Glu Val Asn Val Ile Pro Pro His Thr Pro Val Arg Thr Val Met 305 310 315 320 Asn Thr Ile Gin Gin Leu Met Met Ile Leu Asn Ser Ala Ser Asp Gin 325 330 335 Pro Ser Glu Asn Leu Ile Ser Tyr Phe Asn Asn Cys Thr Val Asn Pro 340 345 350 Lys Glu Ser Ile Leu Lys Arg Val Lys Asp Ile Gly Tyr Ile Phe Lys 355 360 365 Glu Lys Phe Ala Lys Ala Val Gly Gin Gly Cys Val Glu Ile Gly Ser 370 375 380 Gin Arg Tyr Lys Leu Gly Val Arg Leu Tyr Tyr Arg Val Met Glu Ser 385 390 395 400 Met Leu Lys Ser Glu Glu Glu Arg Leu Ser Ile Gin Asn Phe Ser Lys 405 410 415 Leu Leu Asn Asp Asn Ile Phe His Met Ser Leu Leu Ala Cys Ala Leu 420 425 430 Glu Val Val Met Ala Thr Tyr Ser Arg Ser Thr Ser Gin Asn Leu Asp 435 440 445 Ser Gly Thr Asp Leu Ser Phe Pro Trp Ile Leu Asn Val Leu Asn Leu 450 455 460 Lys Ala Phe Asp Phe Tyr Lys Val Ile Glu Ser Phe Ile Lys Ala Glu 465 470 475 480 Gly Asn Leu Thr Arg Glu Met Ile Lys His Leu Glu Arg Cys Glu His 485 490 495 Arg Ile Met Glu Ser Leu Ala Trp Leu Ser Asp Ser Pro Leu Phe Asp 500 505 510 Leu Ile Lys Gin Ser Lys Asp Arg Glu Gly Pro Thr Asp His Leu Glu 515 520 525 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 217 Ser Asp 545 Thr Phe Lys Arg Phe 625 Leu Asn Pro Giu Leu 705 Ser Pro Pro Arg Lys 785 Lys Arg Ala 530 Met Arg Gin Lys Leu 610 Gin Asp Ile His Tyr 690 Lys Pro Leu Tyr Ser 770 Phe Arg Phe Cys Tyr Val Thr Val 595 Leu His Gin Asp Al a 675 Asp Thr Ile Arg Lys 755 Arg Gin Ser Asp Pro Leu Asn Gin 580 Tyr Ser Thr Ile Leu 660 Val Ser Asn Pro Ile 740 Ile Ile Lys Ala Ile 820 Leu Ser Ser 565 Lys Arg Giu Leu Met 645 Lys Gin Ile Ile His 725 Pro Ser Leu Ile Glu 805 Glu Asn Pro 550 Thr Pro Leu His Gin 630 Met Phe Giu Ile Leu 710 Ile Gly Giu Val Asn 790 Gly Gly Leu 535 Val Al a Leu Ala Pro 615 Asn Cys Lys Thr Val 695 Gin Pro Giy Gly Ser 775 Gin Ser Ser Pro Arg Asn Lys Tyr 600 Glu Giu Ser Ile Phe 680 Phe Tyr Arg Asn Leu 760 Ile Met Asn Asp Leu Ser Ala Ser 585 Leu Leu Tyr Met Ile 665 Lys Tyr Ala Ser Ile 745 Pro Gly Val Pro Giu 825 Gin Pro Giu 570 Thr Arg Giu Glu Tyr 650 Val Arg Asn Ser Pro 730 Tyr Thr Giu Cys Pro 810 Ala Asn Lys 555 Thr Ser Leu His Leu 635 Giv Thr Val Ser Thr 715 Tyr Ile Pro Ser Asn 795 Lys Asp Asn 540 Lys Gin Leu Asn Ile 620 Met le Ala Leu Val 700 Arg Lys Ser Thr Phe 780 Ser Pro Gly Thr Gly Thr Leu 590 Leu Trp Asp Lys Lys 670 Lys Met Pro Pro Leu 750 Met Thr Arg Lys Lys 830 Al a Ser Ser 575 Phe Cys Thr Arg Val 655 Asp Glu Gin Thr Ser 735 Lys Thr Ser Val Lys 815 His Ala Thr 560 Ala Tyr Glu Leu His 640 Lys Leu Giu Arg Leu 720 Ser Ser Pro Giu Leu 800 Leu Leu SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 218 Pro Gly Glu Ser 835 Arg Thr Arg Met 850 Lys Phe Gin Gin Lys 840 Gin Lys Gin Lys Met 855 Leu Ala Glu Met Thr Ser Thr 845 Asn Asp Ser Met Asp Thr Ser 860 Asn 865 Lys Glu Glu Lys INFORMATION FOR SEQ ID NO: 48: SEQUENCE CHARACTERISTICS: LENGTH: 3383 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2619 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: CGCGTC ATG CCG CCC AAA ACC CCC CGA AAA ACG GCC GCC ACC GCC GCC Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala 1 5 GCT GCC GCC GCG GAA CCC CCG GCA CCG CCG CCG CCG CCC CCT CCT GAC Ala Ala Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Gli 20 25 Ir GAG GAC CCA GAG Glu Asp Pro Glu GAC AGC GGC CCG GAG GAC CTG CCT CTC Asp Ser Gly Pro Glu Asp Leu Pro Leu GTC AGG Val Arg 144 CTT GAG TTT Leu Glu Phe AAA TTA AAG Lys Leu Lys GAA ACA GAA GAA CCT GAT TTT ACT GCA Glu Thr Glu Glu Pro Asp Phe Thr Ala TTA TGT CAG Leu Cys Gin TTA ACT TGG Leu Thr Trp ATA CCA GAT CAT Ile Pro Asp His AGA GAG AGA GCT Arg Glu Arg Ala 240 GAG AAA Glu Lys GTT TCA TCT GTG Val Ser Ser Val GGA GTA TTG GGA Gly Val Leu Gly

GGT

Gly TAT ATT CAA AAG Tyr Ile Gin Lys 288 336

AAA

Lys AAG GAA CTG TGG Lys Glu Leu Trp

GGA

Gly 100 ATC TGT ATC TTT Ile Cys Ile Phe GCA GCA GTT GAC CTA Ala Ala Val Asp Leu GAT GAG ATG TCG Asp Glu Met Ser ACT TTT ACT GAG Thr Phe Thr Glu CTA CAG AAA AAC ATA GAA ATC Leu Gln Lys Asn Ile Glu Ile 120 125 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/0304 1 219 AGT GTC CAT Ser Val His AAA GTT GAT Lys Val Asp 145 TTC TTT AAC TTA CTA AAA GAA ATT GAT Phe Phe Asn Leu Leu Lys Glu Ile Asp ACC AGT ACC Thr Ser Thr 140 GAT GTA TTG Asp Val Leu AAT GCT ATG TCA AGA CTG TTG AAG AAG Asn Ala Met Ser Arg Leu Leu Lys Lys 150

TAT

Tyr 155 TTT GCA Phe Ala 160 CTC TTC AGC AAA TTG GAA AGG ACA TGT Leu Phe Ser Lys Leu Glu Arg Thr Cys

GAA

Giu 170 CTT ATA TAT TTG Leu Ile Tyr Leu

ACA

Thr 175 CAA CCC AGC AGT TCG ATA TCT ACT GAA ATA AAT TCT GCA TTG, Gin Pro Ser Ser Ser Ile Ser Thr Giu Ile Asn Ser Ala Leu

GTG

Val 190 CTA AAA GTT TCT TGG ATC ACA TTT TTA Leu Lys Val Ser Trp Ile Thr Phe Leu 195 GCT AAA GGG GAA Ala Lys Gly Glu GTA TTA Val Leu 205 CAA ATG GAA Gin Met Giu CTT GAC TAT Leu Asp Tyr 225 GAT CTG GTG ATT Asp Leu Val Ile T7T CAG TTA ATG Phe Gin Leu Met CTA TGT GTC Leu Cys Val 220 AAA GAA CCA Lys Glu Pro TTT ATT AAA CTC TCA CCT CCC ATG TTG Phe Ile Lys Leu Ser Pro Pro Met Leu

CTC

Leu 235 TAT AAA Tyr Lys 240 ACA GGG TCG AAT Thr Gly Ser Asn CTT GGA CTT GTA Leu Gly Leu Vai

ACA

Thr 250 TCT AAT GGA CTT Ser Asn Gly Leu

CCA

Pro 255 GAG GTT GAA AAT Glu Val Giu Asn TCT AAA CGA TAC Ser Lys Arg Tyr GAA ATT TAT CTT Giu Ile Tyr Leu

AAA

Lys 270 AAT AAA GAT CTA Asn.Lys Asp Leu GCA AGA TTA TTT Ala Arg Leu Phe GAT CAT GAT AAA Asp His Asp Lys ACT CTT Thr Leu 285 CAG ACT GAT Gin Thr Asp AGT AAC CTT Ser Asn Leu 305 ATA GAC ACT TTT Ile Asp Ser Phe

GAA

Giu 295 ACA CAG, AGA ACA Thr Gin Arg Thr CCA CGA AAA Pro Arg Lys 300 CAT GAA GAG GTG Asp Ciu Giu Val GTA ATT CCT CCA CAC ACT CCA GTT Val Ile Pro Pro His Thr Pro Vai 315 AGG ACT Arg Thr 320 GTT ATG AAC ACT Vai Met Asn Thr CAA CAA TTA ATG Gin Gin Leu Met

ATG

Met 330 ATT TTA AAT TCA Ile Leu Asn Ser 1008 1056 AGT GAT CAA CCT Ser Asp Gin Pro TCA GAA AAT Ser Giu Asn 340 CTG ATT TCC TAT TTT AAC AAC Leu Ile Ser Tyr Phe Asn Asn 345

TGC

Cys 350 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/0304 1 220 ACA GTG AAT CCA Thr Val Asn Pro GAA AGT ATA CTG Glu Ser Ile Leu

AAA

Lys 360 AGA GTG AAG GAT Arg Val Lys Asp ATA GGA Ile Gly 365 1104 TAC ATC TT Tyr Ile Phe GAA ATT GGA Giu Ile Gly 385 GAG AAA TTT GCT Giu Lys Phe Ala

AAA

Lys 375 GCT GTG GGA CAG Ala Val Giy Gin GGT TOT GTC Gly Cys Val 380 TAT TAC CGA Tyr Tyr Arg 1152 1200 TCA CAG CGA TAC Ser Gin Arg Tyr

AAA

Lys 390 CTT GGA GTT CGC Leu Giy Val Arg

TTG

Leu 395 GTA ATG Val. Met 400 GAA TCC ATG CTT Glu Ser Met Leu

AAA

Lys 405 TCA GAA GAA GAA Ser Giu Glu Glu TTA TCC ATT CAA Leu Ser Ile Gin

AAT

Asn 415 TTT AGC AAA CTT Phe Ser Lys Leu AAT GAC AAC ATT Asn Asp Asn Ile CAT ATG TCT TTA His Met Ser Leu 1248 1296 1344 GCG TGC GCT CTT Ala Cys Ala Leu GTT GTA ATG GCC Val. Val Met Ala

ACA

Thr 440 TAT AGC AGA AGT Tyr Ser Arg Ser ACA TCT Thr Ser 445 CAG AAT CTT GAT TCT GGA ACA GAT Gin Asn Leu Asp Ser Gly Thr Asp TCT TTC CCA TGG Ser Phe Pro Trp ATT CTG AAT Ile Leu Asn 460 GAA AGT TTT Giu Ser Phe GTG CTT AAT Val Leu Asn 465 TTA AAA GCC TTT OAT TTT TAC AAA GTG Leu Lys Ala Phe Asp Phe Tyr Lys Val ATC AAA Ile Lys 480 GCA GAA GGC AAC Ala Giu Gly Asn

TTG

Leu 485 ACA AGA GAA ATG Thr Arg Giu Met AAA CAT TTA GAA Lys His Leu Oiu 1392 1440 1488 1536 1584

CGA

Arg 495 TGT GAA CAT CGA Cys Giu His Arg ATO GAA TCC CTT Met Giu Ser Leu TOG CTC TCA GAT Trp Leu Ser Asp

TCA

Ser 510 CCT TTA TTT GAT Pro Leu Phe Asp ATT AAA CAA TCA Ile Lys Gin Ser

AAG

Lys 520 GAC CGA GAA OGA Asp Arg Giu Oly CCA ACT Pro Thr 525 GAT CAC CTT GAA TCT OCT TOT CCT Asp His Leu Giu Ser Ala Cys Pro 530

CTT

Leu 535 AAT CTT CCT CTC Asn Leu Pro Leu CAG AAT AAT Gin Asn Asn 540 CCA AAG AAA Pro Lys Lys 1632 CAC ACT GCA His Thr Ala 545 GCA OAT ATG TAT Ala Asp Met Tyr

CTT

Leu 550 TCT CCT GTA AGA Ser Pro Val Arg

TCT

Ser 555 1680 AAA GOT Lys Gly 560 TCA ACT ACO CGT Ser Thr Thr Arg AAT TCT ACT OCA Asn Ser Thr Ala AAT OCA GAG ACA CAA Asn Ala Giu Thr Gin 570 1728 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041

GCA

Ala 575 ACC TCA GCC Thr Ser Ala TTC CAG ACC CAG AAG CCA TTG Phe Gin 580 Thr Gin Lys Pro Leu 585 AAA TCT ACC TCT CTT Lys Ser Thr Ser Leu 590 1776 1824 TCA CTG TTT TAT Ser Leu Phe Tyr AAA GTG TAT CGG Lys Val Tyr Arg CTA GCC TAT CTC CGG Leu 600 Ala Tyr Leu Arg CTA AAT Leu Asn 605 ACA CTT TGT Thr Leu Cys ATC TGG ACC Ile Trp Thr 625

GAA

Glu 610 CGC CTT CTG TCT Arg Leu Leu Ser CAC CCA GAA TTA His Pro Glu Leu GAA CAT ATC Glu His Ile 620 GAA CTC ATG Glu Leu Met 1872 CTT TTC CAG CAC ACC CTG CAG AAT GAG Leu Phe Gin His Thr Leu Gin Asn Glu 630 1920 AGA GAC Arg Asp 640 AGG CAT TTG GAC Arg His Leu Asp

CAA

Gin 645 ATT ATG ATG TGT Ile Met Met Cys ATG TAT GGC ATA Met Tyr Gly Ile AAA GTG AAG AAT Lys Val Lys Asn GAC CTT AAA TTC Asp Leu Lys Phe

AAA

Lys 665 ATC ATT GTA ACA Ile Ile Val Thr

GCA

Ala 670 1968 2016 2064 TAC AAG GAT CTT Tyr Lys Asp Leu CAT GCT GTT CAG His Ala Val Gin

GAG

Glu 680 ACA TTC AAA CGT Thr Phe Lys Arg GTT TTG Val Leu 685 ATC AAA GAA Ile Lys Glu TTC ATG CAG Phe Met Gin 705 GAG TAT GAT TCT Glu Tyr Asp Ser 1 ATA GTA TTC TAT Ile Val Phe Tyr AAC TCG GTC Asn Ser Val 700 TCC ACC AGG Ser Thr Arg 2112 2160 AGA CTG AAA ACA Arg Leu Lys Thr ATT TTG CAG TAT Ile Leu Gin Tyr

GCT

Ala 715 CCC CCT Pro Pro 720 ACC TTG TCA CCA ATA CCT CAC ATT CCT Thr Leu Ser Pro Ile Pro His Ile Pro 725

CGA

Arg 730 AGC CCT TAC AAG Ser Pro Tyr Lys 2208 CCT AGT TCA CCC Pro Ser Ser Pro

TTA

Leu 740 CGG ATT CCT GGA Arg Ile Pro Gly AAC ATC TAT ATT Asn Ile Tyr Ile

TCA

Ser 750 2256 2304 CCC CTG AAG AGT Pro Leu Lys Ser

CCA

Pro 755 TAT AAA ATT TCA Tyr Lys Ile Ser

GAA

Glu 760 GGT CTG CCA ACA Gly Leu Pro Thr CCA ACA Pro Thr 765 AAA ATG ACT Lys Met Thr GGG ACT TCT Gly Thr Ser 785

CCA

Pro 770 AGA TCA.AGA ATC Arg Ser Arg Ile

TTA

Leu 775 GTA TCA ATT Val Ser Ile GGT GAA TCA TTC Gly Glu Ser Phe 780 GTA TGT AAC AGC Val Cys Asn Ser 795 2352 2400 GAG AAG TTC CAG Glu Lys Phe Gin

AAA

Lys 790 ATA AAT CAG ATG Ile Asn Gin Met SUBSTITUTE SHEET (RULE 26) WO 98/37091 PTU9I34 PCTIUS98/03041 GAC CGT Asp Arg 800 CTG AAA Leu Lys 815 AGT AAA Ser Lys GTG CTC AAA AGA AGT Val. Leu Lys Arg Ser 805 AAA CTA CGC TTT GAT Lys Leu Arg Phe Asp 820 GCT GAA GGA AGC Ala Giu Giy Ser ATT GAA GGA TCA Ile Giu Giy Ser 825

AAC

Asn 810

GAT

Asp CCT CCT AAA CCA Pro Pro Lys Pro GAA GCA GAT Giu Ala Asp CAT CTC His Leu

CCA

Pro 835 GGA GAG Gly Glu TCC AAA Ser Lys ATG ACT TCT Met Thr Ser CGA ACA CGA ATG Arg Thr Arg Met TTT CAG CAG AAA CTG GCA GAA Phe Gin Gin Lys Leu Ala Giu 840 845 AAG CAG AAA ATG AAT GAT AGC Lys Gin Lys Met Asn Asp Ser 860 TGAGGATCTC AGGACCTTGG 2448 2496 2544 2592 2639 ATG GAT ACC TCA AAC AAG GAA GAG AAA Met Asp Thr Ser Asn Lys Giu Giu Lys 865 870

TGGACACTGT

CAGGTTCTGT

AGATGCAATT

AAGATTGAAA

TCCAAAGTAA

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

GTACACCTCT

TTATGGCCAC

GTTTGGGTGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

CCTCAGAATG

GGATTCATTG

ATTTAATATC

TTCCTAAGCC

AATCCTGCCA.

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TCTCTCACAG

TTCAGCTCTT

ACTTGAAATG

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

ATGTGACTGT

TTTGTGGATA

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA.

TACTATCATA

ATAACTTTCC

TAAAATGTGC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

2699 2759 2819 2879 2939 2999 3059 3119 3179 3239 3299 3359 TAAAAGAACT. TACTGATTAT TTTCTTCATC CAACTTATGT TTTTAAATGA. GGATTATTGA TAGT 3383 INFORMATION FOR SEQ ID NO: 49: SEQUENCE CHARACTERISTICS: LENGTH: 871 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTIUS98/03041 Met 1 Ala Pro Phe Lys Val Glu Met His Asp 145 Leu Pro Val Glu Tyr 225 Thr Val Asp Asp Pro Ala Glu Glu Ile Ser Leu Ser Lys 130 Asn Phe Ser Ser Asp 210 Phe Gly Glu Leu Sex 290 Pro Glu Gin Glu Pro Ser Trp Phe 115 Phe Ala Ser Ser Trp 195 Asp Ile Ser Asn Asp 275 Ile Lys Thr 5 Pro Pro Asp Ser Thr Glu Asp His Val Asp Gly Ile 100 Thr Phe Phe Asn Met Ser Lys Leu 165 Ser Ile 180 Ile Thr Leu Val Lys Leu Asn Ser 245 Leu Ser 260 Ala Arg Asp Ser Pro Arg Lys Thr Ala Ala Thr Ala Ala Ala Ala Ala Gly Glu Val 70 Gly Cys Thr Leu Arg 150 Glu Ser Phe Ile Ser 230 Leu Lys Leu Phe Pro Pro Pro 55 Arg Val Ile Glu Leu 135 Leu Arg Thr Leu Ser 215 Pro Gly Arg Phe Glu 295 Pro i 31u 40 Asp Giu Leu Phe Leu 120 Lys Leu Thr Glu Leu 200 Phe Pro Leu Tyr Leu 280 Thr Pro 25 ksp Phe Arg Gly Ile 105 Gin Glu Lys Cys Ile 185 Ala Gin Met Val Glu 265 Asp Gir 10 Pro I Leu Thr Ala Gly 90 Ala Lys Ile Lys Glu 170 Asn Lys Leu Leu Thr 250 Glu His Arg Pro Pro rka rrp 75 Tyr Ala Asn Asp Tyr 155 Leu Ser Gly Met Leu 235 Ser Ile Asp Thr Pro Leu 1 Leu Leu Ile Val Ile Thr 140 Asp Ile Ala Glu Leu 220 Lys Asn Tyr Lys Pro 300 Pro Val Dys Thr Gin Asp Glu 125 Ser Val Tyr Leu Val 205 Cys Glu Gly Leu Thr 285 Arg Glu Arg Gin i Trp Lys Leu 110 le Thr Leu Leu Val 190 Leu Val Pro Leu Lys 270 Leu Lys lu 1eu Lys 3iu Lys Asp Ser Lys Phe Thr 175 Leu Gin Leu Tyr Pro 255 Asn Gin Ser Asp Glu Leu Lys Lys Glu Va1 Val Ala 160 Gin Lys Met Asp Lys 240 Glu Lys Thr Asn SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCT/US98/03041 224 Leu 305 Val Asp Asn Phe Gly 385 Giu Ser Ala Leu Asn 465 Al a Giu Phe -Leu Ala 545 Ser Ser Phe Asp Met Gin Pro Lys 370 Ser Ser Lys Leu Asp 450 Leu Giu His Asp Giu 530 Ala Thr Ala Tyr Glu Asn Pro Lys 355 Giu Gin Met Leu Giu 435 Ser Lys Gly Arg Leu 515 Ser Asp Thr Phe Lys 595 Giu Thr S er 340 Giu Lys Arg Leu Leu 420 Val Gly Ala Asn Ile 500 Ile Ala Met Arg Gin 580 Lys Val Ile 325 Giu Ser Phe Tyr Lys 405 Asn Val1 Thr Phe Leu 485 Met Lys Cys Tyr Val 565 Thr Val Asn 310 Gin Asn Ile Al a Lys 390 Ser Asp Met Asp Asp 470 Thr Giu Gin Pro Leu 550 Asn Gin Tyr Val Gin Leu Leu Lys 375 Leu Giu Asn Aia Leu 455 Phe Arg Ser Ser Leu 535 Ser Ser Lys Arg Ile Leu Ile Lys 360 Ala Gly Giu Ile Thr 440 Ser Tyr Glu Leu Lys 520 -Asn Pro Thr Pro Pro Pro Met Met *330 Ser Tyr 345 Arg Val Val Gly Val Arg Giu Arg 410 Phe His 425 Tyr Ser Phe Pro Lys Val Met Ile 490 Ala Trp 505 Asp Arg Leu Pro Val Arg Ala Asn 570 Leu Lys His 315 Ile Phe Lys Gin Leu 395 Leu Met Arg Trp, Ile 475 Lys Leu Giu Leu Ser 555 Ala Ser Thr Leu Asn Asp Giy 380 Tyr Ser Ser Ser Ile 460 Giu His Ser Gly Gin 540 Pro Giu Thr Pro Asn Asn Ile 365 Cys Tyr Ile Leu Thr 445 Leu Ser Leu Asp Pro 525 Asn Lys Thr Ser Leu 605 Vai Ser Cys 350 Gly Val Arg Gin Leu 430 Ser Asn Phe Giu Ser 510 Thr Asn Lys Gin Leu 590 Asn Arg Thr 320 Ala Ser 335 Thr Val Tyr Ile Giu Ile Val Met 400 Asn Phe 415 Ala Cys Gin Asn Val Leu Ile Lys 480 Arg Cys 495 Pro Leu Asp His His Thr Lys Gly 560 Ala Thr 575 Ser Leu Thr Leu 585 Leu Ala Tyr Leu Arg 600 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 Cys Giu Arg Leu Leu Ser Glu His Pro Giu Leu Giu His Ile Ile Trp 610 615 Thr Leu Phe 625 Arg His Leu Val Lys Asn Asp Leu Pro 675 Giu Giu Giu 690 Gin Arg Leu 705 Thr Leu Ser Ser Ser Pro Lys Ser Pro 755 Thr Pro Arg 770 Ser Giu Lys 785 Vai Leu Lys Lys Leu Arg His Leu Pro 835 Ser Thr Arg 850 Thr Ser Asn 865 Glm Asp Ile 660 Hi s Tyr Lys Pro Leu 740 Tyr Ser Phe Arg Phe 820 Gly Thr His Gin 645 Asp Ala Asp Thr Ile 725 Arg Lys Arg Gin Ser 805 Asp Giu Arg Thr 630 Ile Leu Val Ser Asn 710 Pro Ile Ile Ilie Lys 790 Ala Ile Ser Met Leu Met Lys Gin Ile 695 Ile His Pro Ser Leu 775 Ile Glu Glu Lys Gin 855 Gin Met Phe Giu 680 Ile Leu Ile Gly Giu 760 Val1 Asn Gly Gly Phe 840 Lys A.sn Cys Lys 665 Thr Val1 Gin Pro Gly 745 Gly Ser Gin S er Ser 825 Gin Gin Giu Ser 650 Ile Phe Phe Tyr Arg 730 Asn Leu Ile Met Asn 810 Asp Gin Lys Tyr 635 Met Ile Lys Tyr Al a 715 Ser Ile Pro Giy Val1 795 Pro Giu Lys Met 620 Giu Tyr Val Arg Asn 700 Ser Pro Tyr Thr Giu 780 Cys Pro Ala Leu Asn 860 Leu Giy Thr VTal 685 Ser Thr Tyr Ile Pro 765 S er Asn Lys Asp Ala 845 Asp Met Ile Aia 670 Leu Val.

Arg Lys Ser 750 Thr Phe Ser Pro Gly 830 Giu Ser Arg Cys 655 Tyr Ile Phe Pro Phe 735 Pro Lys Giy Asp Leu 815 Ser Met Met Asp 640 Lys Lys Lys Met Pro 720 Pro Leu Met Thr Arg 800 Lys Lys Thr Asp Lys Giu Giu Lys 870 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 3554 base pairs SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 226 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:7..2790 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGCGTC ATG CCG CCC AAA ACC CCC CGA AAA ACG GCC GCC ACC GCC GCC Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala 48

GCT

Ala GCC GCC GCG GAA CCC CCG GCA CCG CCG Ala Ala Ala Glu Pro Pro Ala Pro Pro CCG CCC CCT CCT Pro Pro Pro Pro GAG GAC CCA GAG CAG GAC AGC GGC CCG GAG GAC CTG CCT CTC GTC AGG Glu Asp Pro Glu Gin Asp Ser Gly Pro Glu Asp Leu Pro Leu Val Arg CTT GAG TTT GAA GAA ACA GAA GAA CCT GAT TTT ACT GCA Leu Glu Phe Glu Glu Thr Glu Glu Pro Asp Phe Thr Ala TTA TGT CAG Leu Cys Gin TTA ACT TGG Leu Thr Trp AAA TTA AAG Lys Leu Lys ATA CCA GAT CAT Ile Pro Asp His AGA GAG AGA GCT Arg Glu Arg Ala

TGG

Trp 240 GAG AAA Glu Lys GTT TCA TCT GTG GAT GGA GTA TTG GGA Val Ser Ser Val Asp Gly Val Leu Gly 85

GGT

Gly TAT ATT CAA AAG Tyr Ile Gin Lys

AAA

Lys AAG GAA CTG TGG Lys Glu Leu Trp

GGA

Gly 100 ATC TGT ATC TTT Ile Cys Ile Phe GCA GCA GTT GAC Ala Ala Val Asp GGT GAT ATG TCG Gly'Asp Met Ser

TTC

Phe 115 ACT TTT ACT GAG Thr Phe Thr Glu CAG AAA AAC ATA Gin Lys Asn Ile GAA ATC Glu Ile 125 AGT GTC CAT Ser Val His AAA GTT GAT Lys Val Asp 145

AAA

Lys 130 TTC TTT AAC TTA CTA AAA GAA ATT GAT ACC AGT ACC Phe Phe Asn Leu Leu Lys Glu Ile Asp Thr Ser Thr 135 140 AAT GCT ATG TCA Asn Ala Met Ser CTG TTG AAG AAG Leu Leu Lys Lys

TAT

Tyr 155 GAT GTA TTG Asp Val Leu TTT GCA Phe Ala 160 ACA CAA Thr Gin 175 CTC TTC AGC AAA Leu Phe Ser Lys GAA AGG ACA TGT Glu Arg Thr Cys

GAA

Glu 170 CTT ATA TAT TTG Leu Ile Tyr Leu CCC AGC AGT Pro Ser Ser ATA TCT ACT GAA ATA AAT TCT GCA TTG Ile Ser Thr Glu Ile Asn Ser Ala Leu 185 SUBSTITUTE SHEET (RULE 26) WC 98/37091 PCT/US98/03041 CTA AAA GTT TCT Leu Lys Val Ser ATC ACA TTT TTA Ile Thr Phe Leu

TTA

Leu 200 GCT AAA GGG GAA Ala Lys Gly Glu GTA TTA Val Leu 205 CAA ATG GAA Gin Met Glu CTT GAC TAT Leu Asp Tyr 225 GAT CTG GTG ATT Asp Leu Val Ile

TCA

Ser 215 TTT CAG TTA ATG Phe Gin Leu Met CTA TGT GTC Leu Cys Val 220 AAA GAA CCA Lys Giu Pro TTT ATT AAA CTC Phe Ile Lys Leu CCT CCC ATG TTG Pro Pro Met Leu TAT AAA Tyr Lys 240 ACA GCT GTT ATA Thr Ala Val Ile ATT AAT GGT TCA Ile Asn Gly Ser CGA ACA CCC AGG Arg Thr Pro Arg GGT CAG AAC AGG Gly Gin Asn Arg GCA CGG ATA GCA Ala Arg Ile Ala CAA CTA GAA AAT Gin Leu Giu Asn ACA AGA ATT ATT Thr Arg Ile Ile GTT CTC TGT AAA Val Leu Cys Lys CAT GAA TGT AAT His Giu Cys Asn ATA GAT Ile Asp 285 GAG GTG AAA Glu Vai Lys CTT GGA CTT Leu Gly Leu 305

AAT

Asn 290 GTT TAT TTC AAA Val Tyr Phe Lys

AAT

Asn 295 TTT ATA CCT TTT Phe le Pro Phe ATG AAT TCT Met Asn Ser 300 AAT CTT TCT Asn Leu Ser GTA ACA TCT AAT Val Thr Ser Asn

GGA

Gly 310 CTT CCA GAG GTT Leu Pro Giu Val

GAA

Glu 315 AAA CGA Lys Arg 320 TAC GAA GAA ATT Tyr Giu Giu Ile

TAT

Tyr 325 CTT AAA AAT AAA Leu Lys Asn Lys

GAT

Asp 330 CTA GAT GCA AGA Leu Asp Ala Arg

TTA

Leu 335 TTT TTG GAT CAT Phe Leu Asp His

GAT

Asp 340 AAA ACT CTT CAG Lys Thr Leu Gin

ACT

Thr 345 GAT TCT ATA GAC Asp Ser Ile Asp 1008 1056 1104 TTT GAA ACA CAG Phe Glu Thr Gin ACA CCA CGA AAA Thr Pro Arg Lys

AGT

Ser 360 AAC CTT GAT GAA Asn Leu Asp Glu GAG GTG Glu Vai 365 AAT GTA ATT Asn Val Ile CAA CAA TTA Gin Gin Leu 385 CCA CAC ACT CCA Pro His Thr Pro

GTT

Val 375 AGG ACT GTT ATG Arg Thr Val Met AAC ACT ATC Asn Thr Ile 380 CCT TCA GAA Pro Ser Glu 1152 1200 ATG ATG ATT TTA Met Met Ile Leu

AAT

Asn 390 TCA GCA AGT GAT Ser Aia Ser Asp AAT CTG Asn Leu 400 ATT TCC TAT TTT Ile Ser Tyr Phe

AAC

Asn 405 AAC TGC ACA GTG Asn Cys Thr Val

AAT

Asn 410 CCA AAA GAA AGT Pro Lys Giu Ser 1248 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041

ATA

Ile 415 CTG AAA AGA GTG Leu Lys Arg Vai

AAG

Lys 420 GAT ATA GGA TAC Asp Ile Gly Tyr TTT AAA GAG AAA Phe Lys Glu Lys

TTT

Phe 430 1296 GCT AAA GCT GTG GGA CAG GGT TGT GTC Ala Lys Aia Val Gly Gin Gly Cys Vai 435

GAA

Giu 440 ATT GGA TCA CAG Ile Gly Ser Gin CGA TAC Arg Tyr 445 1344 AAA CTT GGA Lys Leu Giy TCA GAA GAA Ser Giu Giu 465 CGC TTG TAT TAC Arg Leu Tyr Tyr GTA ATG GAA TCC Val Met Giu Ser ATG CTT AAA Met Leu Lys 460 CTT CTG AAT Leu Leu Asn 1392 1.440 GAA CGA TTA TCC Giu Arg Leu Ser CAA AAT TTr AGC Gin Asn Phe Ser

AAA

Lys 475 GAC AAC Asp Asri 480 ATT TTT CAT ATG Ile Phe His Met

TCT

Ser 485 TTA TTG GCG TGC Leu Leu Ala Cys

GCT

Al a 490 CTT GAG GTT GTA Leu Giu Val Val 1488 1536 ATG Met 495 GCC ACA TAT AGC Ala Thr Tyr Ser AGT ACA TCT CAG Ser Thr Ser Gin CTT GAT TCT GGA Leu Asp Ser Giy

ACA

Thr GAT TTG TCT TTC CCA TGG ATT CTG AAT Asp Leu Ser Phe Pro Trp Ile Leu Asn 515

GTG

Val 520 CTT AAT TTA AAA Leu Asn Leu Lys GCC TTT Ala Phe 525 1584 GAT TTT TAC Asp Phe Tyr ACA AGA GAA Thr Arg Giu 545 GTG ATC GAA AGT Val Ile Glu Ser

TTT

Phe 535 ATC AAA GCA GAA Ile Lys Ala Giu GGC AAC TTG Gly Asn Leu 540 CGA ATC ATG Arg Ile Met 1632 1680 ATG ATA AAA CAT Met Ile Lys His

TTA

Leu 550 GAA CGA TGT GAA Giu Arg Cys Giu

CAT

His 555 GAA TCC Glu Ser 560 CTT GCA TGG CTC Leu Ala Trp Leu

TCA

Ser 565 GAT TCA CCT TTA Asp Ser Pro Leu

TTT

Phe 570 GAT CTT ATT AAA Asp Leu Ile Lys 1728 1776 CAA Gin 575 TCA AAG GAC CGA Ser Lys Asp Arg

GAA

Glu 580 GGA CCA ACT GAT Gly Pro Thr Asp CTT GAA TCT GCT Leu Giu Ser Ala CCT CTT AAT CTT CCT CTC CAG AAT AAT Pro Leu Asn Leu Pro Leu Gin Asn Asn 595

CAC

His 600 ACT GCA GCA GAT Thr Ala Ala Asp ATG TAT Met Tyr 605 1824 CTT TCT CCT Leu Ser Pro AAT TCT ACT Asn Ser Thr 625 AGA TCT CCA AAG Arg Ser Pro Lys

AAA

Lys 615 AAA GGT TCA ACT Lys Gly Ser Thr ACG CGT GTA Thr Arg Val 620 1872 GCA AAT GCA GAG ACA CAA GCA ACC TCA Ala Asn Ala Glu Thr Gin Ala Thr Ser 630 GCC TTC CAG ACC Ala Phe Gin Thr 635 1920 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCT/US98/03041 CAG AAG Gin Lys 640 CCA TTG AAA TCT ACC TCT CTT TCA CTG Pro Leu Lys Ser Thr Ser Leu Ser Leu 645 TAT AAA AAA GTG Tyr Lys Lys Val 1968

TAT

Tyr 655 CGG CTA GCC TAT Arg Leu Ala Tyr CGG CTA AAT ACA Arg Leu Asn Thr TGT GAA CGC CTT Cys Glu Arg Leu 2016 2064 TCT GAG CAC CCA Ser Glu His Pro

GAA

Glu 675 TTA GAA CAT ATC Leu Glu His Ile TGG ACC CTT TTC Trp Thr Leu Phe CAG CAC Gin His 685 ACC CTG CAG Thr Leu Gin ATT ATG ATG Ile Met Met 705

AAT

Asn 690 GAG TAT GAA CTC Glu Tyr Glu Leu AGA GAC AGG CAT Arg Asp Arg His TTG GAC CAA Leu Asp Gin 700 AAT ATA GAC Asn Ile Asp 2112 2160 TGT TCC ATG TAT Cys Ser Met Tyr ATA TGC AAA GTG Ile Cys Lys Val CTT AAA Leu Lys 720 TTC AAA ATC ATT Phe Lys Ile Ile ACA GCA TAC AAG Thr Ala Tyr Lys CTT CCT CAT GCT Leu Pro His Ala 2208 2256

GTT

Val 735 CAG GAG ACA TTC Gin Glu Thr Phe

AAA

Lys 740 CGT GTT TTG ATC Arg Val Leu Ile

AAA

Lys 745 GAA GAG GAG TAT Glu Glu Glu Tyr

GAT

Asp 750 TCT ATT ATA GTA TTC TAT AAC TCG GTC Ser Ile Ile Val Phe Tyr Asn Ser Val 755 ATG CAG AGA CTG Met Gin Arg Leu AAA ACA Lys Thr 765 2304 AAT ATT TTG Asn Ile Leu CCT CAC ATT Pro His Ile 785 TAT GCT TCC ACC Tyr Ala Ser Thr CCC CCT ACC TTG Pro Pro Thr Leu TCA CCA ATA Ser Pro Ile 780 CCC TTA CGG Pro Leu Arg 2352 2400 CCT CGA AGC CCT Pro Arg Ser Pro

TAC

Tyr 790 AAG TTT CCT AGT Lys Phe Pro Ser ATT CCT Ile Pro 800 GGA GGG AAC ATC Gly Gly Asn Ile ATT TCA CCC CTG Ile Ser Pro Leu AGT CCA TAT AAA Ser Pro Tyr Lys TCA GAA GGT CTG Ser Glu Gly Leu ACA CCA ACA AAA Thr Pro Thr Lys

ATG

Met 825 ACT CCA AGA TCA Thr Pro Arg Ser

AGA

Arg 830 2448 2496 2544 ATC TTA GTA TCA Ile Leu Val Ser GGT GAA TCA TTC Gly Glu Ser Phe ACT TCT GAG AAG Thr Ser Glu Lys TTC CAG Phe Gin 845 AAA ATA AAT Lys Ile Asn ATG GTA TGT AAC Met Val Cys Asn GAC CGT GTG CTC Asp Arg Val Leu AAA AGA AGT Lys Arg Ser 860 2592 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTIUS98/03041 230 GCT GAA GGA AGC AAC Ala Glu Gly Ser Asn 865 CCT CCT AAA Pro Pro Lys 870 GAA GCA GAT Glu Ala Asp CCA CTG AAA AAA Pro Leu Lys Lys

CTA

Leu 875 CGC TTT GAT Arg Phe Asp ATT GAA Ile Glu 880 GGA TCA GAT Gly Ser Asp GGA AGT AAA Gly Ser Lys

CAT

His 890 CTC CCA GGA GAG Leu Pro Gly Giu 885

CTG

Leu 2640 2688 2736 2784

TCC

Ser 895 AAA TTT CAG CAG Lys Phe Gin Gin

AAA

Lys 900

ATG

Met GCA GAA ATG Ala Glu Met

ACT

Thr 905 TCT ACT CGA ACA Ser Thr Arg Thr

CGA

Arg 910 ATG CAA AAG CAG Met Gin Lys Gin AAT GAT AGC ATG Asn Asp Ser Met 920 GAT ACC TCA Asp Thr Ser AAC AAG GAJ Asn Lys Gli 925

GGATTCATTG

I

GAG AAA TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT Giu Lys 2840

TCTCTCACAG

TTCAGCTCTT

AC"TTGAAATG

TTTAAAAAGT

AGTAAGAATG

TCTTTTGTAG

TTAATTTAAC

TTGACTGCCC

ATTAGAAAAA

ACTGTGTGCT

AACCATATGA

TACTGATTAT

ATGTGACTGT

TTTGTGGATA

TTAGTCATTG

TGTAGCAGAT

GCCCTAGAGT

CATATAGGTG

ATGAACACCC

ATTCACCAAA

AATTACTAAT

TGTTTTATAA

TACTATCATA

TTTCTTCATC

ATAACTTTCC

TAAAATGTGC

TTATTTATAC

TGTTTCCTCT

GGGAGTCCTG

ATGTTTGCTC

TTAGAAAATG

ATTATCCTGA

TTTACACATT

AATTTTGCTT

CTACTGAAAC

CAACTTATGT

CAGGTTCTGT

AGATGCAATT

AAGATTGAAA

TCCAAAGTAA

ATAACCCAGG

TTGTTTTTAT

TGTCCTATCT

ACTCTTCTGC

AGATTTTATT

TTAATTAAAT

AGATTTCATA

TTTTAAATGA

TTATGGCCAC

GTTTGGGTGA

ATCTTGTGTA

AATTGCTGTG

CCTGTCTGAC

TAATTTATAT

ATCTTCCAAA

AAAAATGGAT

TTACTATTGG

AAAAGCTGGA

CCTCAGAATG

GGATTATTGA

ATTTAATATC

TTCCTAAGCC

AATCCTGCCA

CTTTATGGAT

TACTTTGCCT

GTATATTTTT

TGCAATTTGA

ATTATTAGAA

AATCTGATAT

AGCAAAGTAT

TAAAAGAACT

TAGT

2900 2960 3020 3080 3140 3200 3260 3320 3380 3440 3500 3554 INFORMATION FOR SEQ ID NO: 51: Wi SEQUENCE CHARACTERISTICS: LENGTH: 928 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala Ala Ala 1 5 10 SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 9837091PCTfUS98/0304 I Ala Pro Phe Lys Val Giu Met His Asp 145 Leu Pro Val Giu Tyr 225 Thr, Gin Ile Lys Leu 305 Ala Giu Giu Ile Ser Leu Ser Lys 130 Asn Phe Ser Ser Asp 210 Phe Ala Asn Ile Asn 290 Val Giu Gin Giu Pro Ser Trp Phe 115 Phe Ala Ser Ser Trp 195 Asp Ile Val Arg Giu 275 Val Thr Pro Asp Thr Asp Vai Gly 100 Thr Phe Met Lys Ser 180 Ile Leu Lys Ile Ser 260 Val Tyr Ser Pro Ser Giu His Asp Ile Phe Asn Ser Leu 165 Ile Thr Vai Leu Pro 245 Ala Leu Phe Asn Ala Gly Giu Val 70 Gly Cys Thr Leu Arg 150 Glu S er Phe Ile Ser 230 Ile Arg Cys Lys Gly 310 Pro Pro Pro 55 Arg Val Ile Glu Leu 135 Leu Arg Thr Leu Ser 215 Pro Asn Ile Lys Asn 295 Leu Pro Giu 40 Asp Giu Leu Phe Leu 120 Lys Leu Thr Glu Leu 200 Phe Pro Gly Ala Giu 280 Phe Pro Pro 25 Asp Phe Arg Gly Ile 105 Gin Giu Lys Cys Ile 185 Ala Gin Met Ser Lys 265 His Ile Glu Pro Leu Thr Al a Gly 90 Ala Lys Ile Lys Glu 170 Asn Lys Leu Leu Pro 250 Gin Glu Pro Val Pro Pro Al a Trp 75 Tyr Al a Asn Asp Tyr 155 Leu Ser Gly Met Leu 235 Arg Leu Cys Phe Giu 315 Pro Leu Leu Leu Ile Val Ile Thr 140 Asp Ile Ala Glu Leu 220 Lys Thr Glu Asn Met 300 Asn Pro Val Cys Thr Gin Asp Glu 125 Ser Val Tyr Leu Val 205 Cys Giu Pro Asn Ile 285 Asn Leu Glu Arg Gin Trp Lys Leu 110 Ile Thr Leu Leu Val1 190 Leu Val Pro Arg Asp 270 Asp Ser Ser Glu Leu Lys Glu Lys Gly Ser Lys Phe Thr 175 Leu Gin Leu Tyr Arg 255 Thr Giu Leu Lys Asp Glu Leu Lys Lys Asp Val Val1 Al a 160 Gin Lys Met Asp Lys 240 Gly Arg Val Gly Arg 320 SUBSTITUTE SHEET (RULE 26) WO 98/37091 PCTUS98/03041 Tyr C Leu I Thr Ile I Leu 385 Ile Lys Ala Gly Glu 465 Ile Thr Ser Tyr Glu 545 Leu Lys Asn Pro lu sp 1n ?ro 370 4et Ser Arg Va1 Val 450 Glu Phe Tyr Phe Lys 530 Met Ala Asp Leu Val 610 Glu His Arg 355 Pro Met Tyr Val Gly 435 Arg Arg His Ser Pro 515 Val Ile Trp Arg Pro 595 Arg Ile Asp 340 rhr His Ile Phe Lys 420 Gin Leu Leu Met Arg 500 Trp Ile Lys Leu Glu 580 Leu Ser Tyr 325 Lys Pro Thr Leu Asn 405 Asp Gly Tyr Ser Ser 485 Ser Ile Glu His Ser 565 Gly Gin Pro Leu Thr Arg Pro Asn 390 Asn Ile Cys Tyr Ile 470 Leu Thr Leu Ser Leu 550 Asp Pro Asr LyE Lys Leu C Lys Vai 2 375 Ser Cys Gly Val Arg 455 Gin Leu Ser Asn Phe 535 Glu Ser Thr 1 Asn Lys 615 ~sn ;in 3er 360 krg kia rhr ryr Glu 440 Val Asn Ala Gin Vai 520 Ile Arg Pro Asp His 600 Lys Lys Asp L 330 Thr Asp S 345 Asn Leu Thr Val I Ser Asp C Vai Asn I 410 Ile Phe 425 Ile Gly Met Glu Phe Ser Cys Ala 490 Asn Leu 505 Leu Asn Lys Ala Cys Glu Leu Phe 570 His Leu 585- Thr Ala Gly Ser leu er ~sp let ln 395 ?ro :,ys Ser Ser Lys 475 Leu Asp Leu Glu His 555 Asp Glu Ala Thr Asp Ile I Glu C Asn 380 Pro Lys Glu Gin Met 460 Leu Glu Ser Lys Gly 540 Arg Leu Ser Asp Thr 620 la ~sp lu 365 rhr 3er Glu Lys Arg 445 Leu Leu Vai Gly Ala 525 Asn Ile Ile Ala Met 605 Arc Arg Ser 350 Val Ile Glu Ser Phe 430 Tyr Lys Asn Val Thr 510 Phe Leu Met Lys Cyi 59( Tyl SVa: Leu I 335 Phe Asn Gin Asn Ile 415 Ala Lys Ser Asp Met 495 Asp Asp Thr Glu Gin 575 Pro r Leu 1 Asn ~he ;lu lal 31n Leu 100 Ueu Lys Leu Glu Asn 480 Ala Leu Phe Arg Ser 560 Ser Leu Ser Ser SUBSTITUTE SHEET (RULE 26) WO 98/37091 WO 98/709 1PCTIUS98/03041 Thr Ala Asn Ala Giu Thr Gin Ala Thr Ser Ala Phe Gin Thr Gin Lys 630 Pro Leu His Gin Met 705 Phe Glu le Leu Ile 785 Gly Giu Vai Asn Gly 865 Giy Phe Lys Leu Ala Pro Asn 690 Cys Lys -Thr Val1 Gin 770 Pro Gly Gly Ser Gin 850 Ser Ser Gin Gin Lys Tyr Giu 675 Giu Ser Ile Phe Phe 755 Tyr Arg Asn Leu Ile 835 Met Asn Asp Gin Lys 915 Ser Leu 660 Leu Tyr Met Ile Lys 740 Tyr Ala Ser Ile Pro 820 Giy Vai Pro Giu Lys 900 Met Thr 645 Arg Giu Giu Tyr Vai 725 Arg Asn Ser Pro Tyr 805 Thr Giu Cys Pro Ala 885 Leu Asn Ser Leu His Leu Gly 710 Thr Val1 Ser Thr Tyr 790 Ile Pro Ser Asn Lys 870 Asp Ala Asp Leu Asn Ile Met 695 Ile Aia Leu Val Arg 775 Lys Ser Thr Phe Ser 855 Pro Gly Giu Ser Ser Thr Ile 680 Arg Cys Tyr Ile Phe 760 Pro Phe Pro Lys Giy 840 Asp Leu Ser Met Met 920 Leu Leu 665 Trp Asp Lys Lys Lys 745 Met Pro Pro Leu Met 825 Thr Arg Lys Lys Thr 905 Asp Phe 650 Cys Thr Arg Vai Asp 730 Giu Gin Thr Ser Lys 810 Thr Ser Val Lys His 890 Ser Thr 635 Tyr Giu Leu His Lys 715 Leu Giu Arg Leu Ser 795 Ser Pro Giu Leu Leu 875 Leu Thr Ser Lys Arg Phe Leu 700 Asn Pro Giu Leu Ser 780 Pro Pro Arg Lys Lys 860 Arg Pro Arg Asn Lys Leu Gin 685 Asp Ile His Tyr Lys 765 Pro Leu Tyr Ser Phe 845 Arg Phe Gly Thr Lys 925 Vai Leu 670 H~is Gin Asp Ala Asp 750 Thr Ile Arg Lys Arg 830 Gin Ser Asp Giu Arg 910 Giu Tyr 655 Ser Thr Ile Leu Val1 735 Ser Asn Pro Ile Ile 815 Ile Lys Ala Ile Ser 895 Met Glu 640 Arg Giu Leu Met Lys 720 Gin Ile Ile His Pro 800 Ser Leu Ile Giu Giu 880 Lys Gin Lys SUBSTITUTE SHEET (RULE 26)

Claims (27)

1. A DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB 9 4 or pRB 56 in which said modified protein comprises an insertion, substitution or deletion within the N-terminal 379 amino acids of SEQ ID NO: 2, which modified protein has increased biological activity as compared to the corresponding wild-type retinoblastoma tumor suppressor protein, with the proviso that said modified protein does not consist of a deletion of amino acids 1-378 of SEQ ID NO: 2, does not consist of a deletion of amino acids 37-89 of SEQ ID NO: 2, does not consist of a deletion of amino acids 89-140 of SEQ ID NO: 2, does not consist of a deletion of amino acids 140-202 of SEQ ID NO: 2, does not consist of a deletion of amino acids 249-309 of SEQ ID NO: 2, does not consist of a deletion of amino acids 309-343 of SEQ ID NO: 2, does not consist of a deletion of amino acids 343-389 of SEQ ID NO: 2, does not consist of a deletion of amino acids 56-112 of SEQ ID NO: 2, and does not consist of an amino acid substitution at position 5, 230, 249, 252, 356 and 373 of SEQ ID NO: 2.

2. The DNA segment of claim 1, wherein said gene encodes a modified retinoblastoma tumor suppressor protein in which at least one amino acid has been deleted from a first sequence region in the N-terminal 379 amino acids.

3. The DNA segment of claim 2, wherein at least two amino acids have been deleted from said first sequence region.

4. The DNA segment of claim 3, wherein at least 25 amino acids have been deleted from said first sequence region. The DNA segment of claim 4, wherein at least 100 amino acids have been deleted from said first sequence region.

6. The DNA segment of claim 5, wherein at least 150 amino acids have been deleted from said first sequence region. WAdskankiispecesOMsionaI 68573-98 (May2002).doc 235

7. The DNA segment of claim 6, wherein at least 300 amino acids have been deleted from said first sequence region.

8. The DNA segment of claim 2, wherein said first sequence region is located: a) between amino acid 1 and amino acid b) between amino acid 51 and amino acid 100; c) between amino acid 101 and amino acid 150; d) between amino acid 151 and amino acid 200; e) between amino acid 201 and amino acid 250; f) between amino acid 251 and amino acid 300; g) between amino acid 1 and amino acid 100; h) between amino acid 51 and amino acid 150; i) between amino acid 101 and amino acid 200; j) between amino acid 151 and amino acid 250; k) between amino acid 201 and amino acid 300; I) between amino acid 1 and amino acid 150; m) between amino acid 51 and amino acid 200; n) between amino acid 101 and amino acid 250; o) between amino acid 151 and amino acid 300; p) between amino acid 1 and amino acid 200; q) between amino acid 51 and amino acid 250; r) between amino acid 101 and amino acid 300; s) between amino acid 1 and amino acid 250; t) between amino acid 51 and amino acid 300; or u) between amino acid 1 and amino acid 300.

9. The DNA segment of claim 2, wherein: a) amino acid 2 through amino acid 34 have been deleted from said first sequence region; b) amino acid 2 through amino acid 55 have been deleted from said first sequence region; c) amino acid 2 through amino acid 78 have been deleted from said first sequence region; W:\cska~nkitspecies\DivisionaI 66573-98 (May2002).doc 236 d) amino acid 2 through amino acid 97 have been deleted from said first sequence region; e) amino acid 2 through amino acid 148 have been deleted from said first sequence region; f) amino acid 31 through amino said first sequence region; g) amino acid 77 through amino said first sequence region; h) amino acid 111 through amino said first sequence region; i) amino acid 111 through amino said first sequence region; j) amino acid 181 through amino said first sequence region; or k) amino acid 242 through amino said first sequence region. acid 107 have been deleted from acid 107 have been deleted from acid 181 have been deleted from acid 241 have been deleted from acid 241 have been deleted from acid 300 have been deleted from The DNA segment of claim 2, wherein at least one amino acid has been deleted from said protein in a second sequence region different from the first sequence region.

11. The DNA segment of claim 10, wherein amino acid 2 through amino acid 34, and amino acid 76 through amino acid 112 have been deleted.

12. The DNA segment of claim 10, wherein amino acid 2 through amino acid and amino acid 76 through amino acid 112 have been deleted.

13. The DNA segment of claim 1, wherein said gene encodes a modified retinoblastoma tumor suppressor protein comprising a substitution mutation at position 111. W:Wskaski~speIesDMsionaI 66573-98 (MayZOO2).doc 237

14. The DNA segment of claim 13, wherein said modified retinoblastoma tumor suppressor protein comprises glycine at position 111 in place of aspartic acid.

15. The DNA segment of claim 1, wherein said modified retinoblastoma tumor suppressor protein comprises at least a second insertion, substitution or deletion within the N-terminal 379 amino acids of SEQ ID NO:2.

16. The DNA segment of claim 15, wherein said gene encodes a modified retinoblastoma tumor suppressor protein comprising a substitution mutation at position 111 and a substitution mutation at position 112.

17. The DNA segment of claim 16, wherein said modified retinoblastoma tumor suppressor protein comprises glycine at position 111 in place of aspartic acid, and aspartic acid at position 112 in place of glutamic acid.

18. The DNA segment of claim 1, wherein said gene encodes a modified retinoblastoma tumor suppressor protein in which at least one amino acid has been deleted, and in which at least one amino acid has been substituted.

19. The DNA segment of claim 2, wherein said gene encodes a modified retinoblastoma tumor suppressor protein consisting of the contiguous amino acid sequence of SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:47; or SEQ ID NO:51. The DNA segment of claim 2, wherein said gene consists of the contiguous nucleic acid sequence from position 7 to position 2691 of SEQ ID NO:28; from position 7 to position 2628 of SEQ ID NO:30; from position 7 to position 2559 of SEQ ID NO:32; from position 7 to position 2502 of SEQ ID NO:34; from position 7 to position 2349 of SEQ ID NO:36; from position 7 to position 2559 of SEQ ID NO:38; from position 7 to position 2697 of SEQ ID W:\ciska\nkspecies\Divisional 66573-98 (May2002).doc 238 from position 7 to position 2583 of SEQ ID NO:42; from position 7 to position 2613 of SEQ ID NO:46; or from position 7 to position 2790 of SEQ ID

21. A recombinant nucleic acid comprising the DNA segment of any of claims 1-20 operationally linked to a promoter.

22. A recombinant vector comprising the nucleic acid of claim 21.

23. The recombinant vector of claim 22, which is an adenoviral vector.

24. A recombinant adenovirus comprising the nucleic acid of claim 21. A host cell recombinantly transformed with the DNA segment of any of claims 1-20, the nucleic acid of claim 21, the vector of claim 22 or 23, or the adenovirus of claim 24.

26. The host cell of claim 25, wherein said host cell is a eukaryotic cell.

27. The host cell of claim 26, wherein said eukaryotic cell is a human cell.

28. The host cell of claim 26, wherein said eukaryotic cell is a tumor cell.

29. A composition comprising the DNA segment according to any of claims 1-20, the nucleic acid of claim 21, the vector of claim 22 or 23, or the adenovirus of claim 24; and a pharmaceutically acceptable excipient. A modified retinoblastoma tumor suppressor protein encoded by the DNA segment of any one of claims 1-20.

31. The modified retinoblastoma tumor suppressor protein of claim encapsulated within liposome vesicles.

8573-98 (May2002).doc 239 32. The DNA segment of any one of claims 1-20, the nucleic acid of claim 21, the vector of claim 22 or 23, the adenovirus of claim 24, or the modified protein of claim 30 or 31 for use in inhibiting cellular proliferation. 33. Use of a composition comprising the DNA segment of any one of claims 1-20, the nucleic acid of claim 21, the vector of claim 22 or 23, the adenovirus of claim 24, or the modified protein of claim 30 or 31 for the manufacture of a medicament for inhibiting cellular proliferation. 34. The DNA segment of any one of claims 1-20, the nucleic acid of claim 21, the vector of claim 22 or 23, the adenovirus of claim 24, or the modified protein of claim 30 or 31 for use in treating cancer. Use of a composition comprising the DNA segment of any one of claims 1-20, the nucleic acid of claim 21, the vector of claim 22 or 23, the adenovirus of claim 24, or the modified protein of claim 30 or 31 for the manufacture of a medicament for treating cancer. 36. A method of inhibiting cellular proliferation, comprising contacting a cell with an effective inhibitory amount of a modified retinoblastoma protein according to any one of claim 30 or 31. 37. A method of inhibiting cellular proliferation, comprising contacting a cell with an effective inhibitory amount of a DNA segment according to any one of claims 1-20, the nucleic acid of claim 21, the vector of claim 22 or 23, or the adenovirus of claim 24. 38. The method of claim 36, wherein said cell is located within an animal. 39. The method of claim 30, further comprising contacting said cell with a p53 tumor suppressor protein, or a DNA segment encoding p53 in a combined amount with the modified retinoblastoma tumor suppressor protein effective to inhibit cellular proliferation in said cell. W:;cskaVnkispeclesDivsiona 866573-98 (May2002).doc 240 The method of claim 37, further comprising contacting said cell with a p53 tumor suppressor protein, or a DNA segment encoding p53 in a combined amount with the DNA segment, nucleic acid, vector or adenovirus effective to inhibit cellular proliferation in said cell. 41. A method of treating cancer, comprising administering to an animal with cancer a pharmaceutically acceptable composition comprising the protein according to claim 30 or 31. DATED: 22 May, 2002 PHILLIPS ORMONDE FITZPATRICK Attorneys for: BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM and BAYLOR COLLEGE OF MEDICINE Qei-iB ^dEjs^0 W:\ciska\nki\speces\Divisional 66573-98 (May2002).doc