WO2004003170A2 - Agents capable of inhibiting ras and uses thereof - Google Patents

Abstract

This invention provides an agent capable of blocking the interaction between GTP-Ras and ERI1, wherein ERI1 is a protein comprising amino acids, the sequence of which amino acids is set forth in SEQ ID NO:7. In one embodiment, the agent comprises at least a portion of the ERI1 sequence. This invention provides a nucleic acid which encodes such agent. This invention also provides uses of the agent for treating cancer.

Description

AGENTS CAPABLE OF INfflBITNG RAS AND USES THEREOF

The invention described herein was made in part with Government support under grant numbers GM48533 and 5T32CA09110 from the National Institutes of Health.

Accordingly, the United States Government has certain rights in this invention.

Reference to Related Applications

This application claims the benefit ofthe filing date of U.S. Provisional Application No. 60/392,355, filed June 28, 2002, the contents of which are incorporated by reference into this application.

Throughout this application, various publications are referenced, either by Arabic numerals or directly in the text. Full citations for those publications refereced by Arabic numerals may be found at the end ofthe specification immediately preceding the claims. The disclosure of all referenced publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.

Background ofthe Invention

1. Ras as an oncoprotein. The Ras family of small GTPases comprises a group of molecular switches that link receptors on the plasma membrane to signaling pathways that regulate cell proliferation and differentiation. In the GTP-bound active state, Ras interacts with effectors molecules that transmit signal to the next downstream component. The hydrolysis of GTP to GDP switches Ras to the inactive state. Guanine nucleotide exchange factors (GEFs) stimulate the dissociation of GDP from Ras and promote the loading of GTP to regenerate the active state. GTPase-activating proteins (GAPs) negatively regulate Ras by increasing the slow instrinsic rate of GTP hydrolysis.

Mutants of Ras are found in 30% of human tumors (Ohkanda et al., 2002). In cases of colorectal or pancreatic cancers, this incidence is as high as 50% or 90%, respectively (Gibbs and Marshall, 1989). Mutational activation of Ras can occur by two mechanisms, both of which have the net result of increasing the fraction of GTP-bound Ras. Most frequently found are dominant mutations that cause amino acid substitutions at residues 12 or 61, resulting in impaired GTPase activity (Gibbs and Marshall, 1989). An interesting property ofthese mutants is their insensitivity to the negative regulatory action of GAPs. Less common are substitutions at residues 16, 116, 119, and 144, which are required for nucleotide binding. Such mutations increase the off rate of bound GDP, thereby facilitating nucleotide exchange. Because cells contain approximately 10 times more GTP than GDP, the more rapid nucleotide exchange ofthese mutants results in an increased fraction of GTP-Ras.

There are three forms of mammalian Ras (H-Ras, K-Ras, and N-Ras). They are identical through their N-terminal 85 residues, which possess the structural features necessary for guanine nucleotide bonding, GTPase activity, and effector function (Barbacid, 1987; Gibbs and Marshall, 1989). Moreover, they all interact with the same set of effectors. The best studied of these are the Raf protein kinases, whose stimulation by Ras activates the ERK MAP kinase cascade, the core of the major proliferative pathway in metazoan cells (Wittinghofer and Nasser, 1996). Other Ras effectors include phosphinositide 3-kinase (PI-3K; Wittinghofer and Herman, 1995), which prevents apoptosis through the Akt protein kinase (Kennedy et al., 1997), and

Ral-GEF (Feig et al., 1996), the guanine nucleotide exchange factor for Ral, another member of the Ras family. The importance of more-recently discovered Ras interacting proteins is less clear (Han et al., 1997). However, in all cases, the association ofthese putative effectors is dependent not only on the nucleotide-bound state of Ras, but also on an intact effector-binding loop. The Ras effector-binding loop undergoes a conformational shift upon GTP binding that allows it to form specific contacts with effector proteins (Wittinghofer and Nasser, 1996). Interestingly, although all known effectors have essentially the same Ras interaction characteristics, they do not share significant sequence similarity.

The only known negative regulators of Ras proteins are the Ras-GAPs, which are ineffective inhibitors of GTPase-defective oncogenic forms of Ras. Therefore, recent efforts to develop cancer therapies that target Ras signaling have focused on its posttranslational lipid modification. Addition of farnesyl and palmitoyl moieties near the C-terminus of Ras proteins is required for membrane association and biological function (Willumsen et al., 1984; Deschenes and Broach, 1987; Hancock et al., 1989). Inhibitors of protein farnesyl transferase (PFTase), the enzyme responsible for the first step in the posttranslational modification of Ras (Ohkanda et al., 2002) block Ras function. Although there are hopeful signs that PFTase inhibitors may ultimately be useful in the treatment of cancer (Ohkanda et al., 2002; Nakamura et al., 2001), a natural protein inhibitor of GTP-Ras could display greater specificity, and thus fewer side effects than PFTase inhibitors. Problems historically associated with bioavailability of protein-based therapeutics are currently being addressed by with the use of protein transduction domains. These are short peptide tags that have the property of transporting fused proteins across animal cell membranes, and have the capability of delivering proteins to all tissues, even crossing the blood-brain barrier (Schwarze et al., 2000).

2. Yeast Ras as a model. The Ras genes are ubiquitous and highly conserved among eukaryotic species. The budding yeast Saccharomyces cerevisiae possesses two functionally overlapping RAS genes. They share 84% sequence identity with their mammalian counterparts through their N-terminal 82 residues (Barbacid,

1987). Although the essential effector of yeast Ras is adenylate cyclase (Toda et al., 1985), an enzyme not regulated by Ras in animal cells, the growth defect associated with loss of yeast Ras function can be complemented by expression of mammalian Ras genes (DeFeo-Jones et al., 1985; Kataoka et al., 1985). Conversely, an activated allele of yeast RASl is capable of causing malignant transformation of mouse fibroblasts (DeFeo-Jones et al, 1985). The conservation of biological properties among these members of the Ras gene family is reflected in the observation that the effector loops of yeast Ras are identical to those of mammalian Ras (DeFeo-Jones et al., 1983).

Yeast has proven to be an excellent model system for studying the regulation and posttranslational modification of Ras proteins. Much of what has been learned from studies of yeast Ras has been directly applicable to understanding mammalian Ras processing, regulation and localization. We have identified a novel yeast protein, designated Eril (for Ras inhibitor 1), which is only 68 amino acids in length and associates in vivo with Ras in an effector-like manner. However, Eril behaves genetically as an inhibitor of Ras. In addition to our excitement at having identified a novel class of Ras regulatory proteins that is of general significance, we regard Eril as having genuine potential for development as an anticancer agent.

3. Sorting of secreted proteins in the endoplasmic reticulum. The field of protein secretion is currently undergoing a revolution in thinking about mechanisms and sites of protein sorting in the secretory pathway. Until very recently, it was thought that all secretory proteins travel together from the ER to the Golgi, where they are sorted to different destinations. However, new evidence from Howard

Riezman's laboratory indicates that sorting of glycosyl-phosphatidylinositol (GPI)- anchored proteins occurs at the ER (Muniz et al., 2001; Morsomme and Riezman,

2002). Specifically, this group has demonstrated that GPI-anchored proteins are sorted from non-GPI-anchored proteins into distinct vesicles upon exit from the ER.

The factors involved in cargo sorting into different ER-derived vesicles are only now being uncovered. For reasons that will be discussed in the next section, it seems likely that, Eril serves an important role in the sorting of GPI-anchored proteins in the ER that is separate from its function as a Ras inhibitor. Our studies on Eril contribute to this area.

Summary of the Invention

This invention provides an agent capable of blocking the interaction between GTP- Ras and ERIl, wherein ERIl is a protein comprising amino acids, the sequence of which amino acids is set forth in SEQ ID NO:7. In one embodiment, the agent comprises at least a portion ofthe ERIl sequence.

This invention provides a nucleic acid which encodes an agent capable of blocking the interaction between GTP-Ras and ERIl, wherein ERIl is a protein comprising amino acids, the sequence of which amino acids is set forth in SEQ ID NO:7. This invention provides a vector which comprises any ofthe nucleic acids described herein. This invention provides a cell which comprises such vector. This invention provides a host-vector system for the production of a polypeptide which comprises a vector described herein and a suitable host cell. This invention provides a method for producing a polypeptide which comprises growing the host vector system under suitable conditions and recovering the polypeptide so produced.

This invention provides a method of treating a subject afflicted with cancer which comprises administering to the subject an amount of an agent described herein effective to treat the subject.

This invention provides a method of preventing a subject from becoming afflicted with cancer which comprises administering to the subject an amount of an agent described herein effective to prevent the subject from becoming afflicted with cancer.

This invention provides a method of inhibiting growth of a cancer cell which comprises contacting the cancer cell with an amount of an agent described herein effective to inhibit growth ofthe cancer cell.

This invention provides a method of inhibiting GTP-Ras signaling which comprises contacting a GTP-Ras containing cell with an amount of an agent described herein effective to inhibit GTP-Ras signaling.

This invention provides a recombinant transfection system, comprising

(i) a first gene construct comprising a coding sequence encoding a inhibitory polypeptide comprising at least one GTP-Ras-binding motif for binding and inhibiting Ras, which coding sequence is operably linked to a transcriptional regulatory sequence for causing expression ofthe first polypeptide in eukaryotic cells; and (ii) a gene delivery composition for delivering the gene construct to a cell and causing the cell to be transfected with the gene construct.

This invention provides a method for treating a subject afflicted with cancer which comprises administering to the subject a recombinant transfection system described herein.

This invention provides a method for preventing a subject from becoming afflicted with cancer which comprises administering to the subject a recombinant transfection system described herein.

Brief Description of Drawings

Figure 1. Predicted amino acid sequence of Eril. Potential transmembrane regions are underlined.

Figure 2. The Eril A mutant invades the agar in a i 4S2-dependent manner (top plates). The indicated mutants were grown for 2 days on rich medium (YPD) at 34°C Plates were then washed with water to remove non-adherent cells. Hyperactive Ras signaling induces invasive growth in the EG 123 strain background

(lower plate). Deletion of the genes encoding the redundant Ras-GAPs, IRA1 and IRA2, results in an increased fraction of GTP-Ras.

Figure 3. The Eril A. mutant undergoes pseudohyphal development in a RAS2- and temperature-dependent manner. Cells were grown on YPD at the indicated temperature for 24h. (a) Eril A/A at 23°C; (b) Eril A/A at 34°C; (c) Eril A/A, ras2A/A at 34°C; (d) Wild type at 34°C

Figure 4. Suppression of the heat shock sensitivities of the Eril A mutant and a hyperactive. Ras pathway mutant (iralA). Cells bearing the indicated plasmids were grown at 30°C for 24h to saturation. Cultures were heat-shocked at 50°C for 30 min. and plated at 23 °C for viability. A. Plasmids were YEp352 (vector), 2μ RAS2- 764 (D.N. RAS2), 2μ IRA2, 2μ RPI1, and ERIl (centromeric). B. Plasmids were YEp352, IμERIl, 2μ RPI1, or centromeric IRA1.

Figure 5. HA-Eril associates specifically with GTP-Ras in an effector loop dependent manner. A. GST fusions of wild-type Ras2, or mutationally activated (V19) Ras2, or GST alone were co-expressed with HA-Eril (both under the inducible control of GAL1) in either wild-type or Ras-GAP-defective (iralA ira2A) cells. GST fusions were isolated from extracts with glutathione affinity resin and eluted with glutathione. Eluate proteins were separated by SDS-PAGE, transferred to PVDF membranes, and subjected to immunoblot analysis using anti-HA antibodies (12CA5) or anti-GST antibodies (Amersham). B. GST fusions of the indicated Ras2 mutants were co-expressed with HA-Eril in wild-type cells and treated as in "A".

Figure 6. HA-Eril resides in the ER membrane. A. Extracts of wild type diploid yeast cells expressing HA-Eril from its own promoter on a 2μ plasmid were treated as indicated prior to ultracentrifugation for lh at 100K x g. Total extract (T), supernatant fraction (S), or pellet (P) corresponding to equivalent cell fractions were subjected to immunoblot analysis with anti-HA antibodies (12CA5). B. Indirect immunofluoresence of HA-Eril in the same cells. Nuclear DNA was stained with DAPI.

Figure 7. Cultures of wild type and Eril A cells, grown either to mid-log phase (filled symbols) or to stationary phase (a 24h saturated culture; open symbols) at 23°C were washed, resuspended in water, and treated with zymolyase 20T (5μg/ml). The reduction in Aδoo over time resulting from cell lysis is indicated as a percentage of the initial absorbance. B. A Eril A strain, transformed with either a centromeric ERIl plasmid, or vector only was streaked onto rich medium with or without 10% sorbitol for osmotic support, and incubated for 3 days at 37°C.

Figure 8. The Eril A mutant growth defect is suppressed either by overexpression of

GFA1, or addition of gfucosamine to the growth medium. A. A Eril A strain, transformed with a centromeric ERIl plasmid, 2μ GFAI, or vector alone was streaked onto YPD and incubated for 3 days at 37°C. B. The pathway for UDP- GlcNAc production. This sugar nucleotide is used for the production of three classes of macromolecules in yeast. C. Crystals of glucosamine (left) or glucose (right) were sprinkled onto a YPD plate spread with Eril A cells. The plate was incubated for 2 days at 37°C.

Figure 9. Cells were grown on YΕPD stained for 30 min. with the chitin-binding dye, calcoflour white (CFW; 40μg/ml) for fluorescence or bright field microscopy. The chitin ring at the bud neck is visible in all but the Eril A cells.

Figure 10. A Eril A, chs3A double mutant is suppressed by GFAI overexpression. The Eril A, chs3A strain, transformed with a centromeric ERIl plasmid, a 2μ GFAI plasmid, or YΕp352 (vector), was streaked onto a YEPD plate and incubated for 3 days at 36°C.

Figure 11. A Eril A mutant accumulates the GaslGPI-protein in the ER-modified form. Cells were grown to mid-log phase in YPD at 23°C in the absence or presence of 25mM glucosamine (GlcN) .Extracts from the indicated mutants were subjected to immunoblot analysis using anti-CPY antisera (A), or anti-Gas 1 antisera (B)

Figure 12. Wild-type or Eril A, cells, transformed with a UPRE- lacZ reporter plasmid (Cox and Walter, 1996; gift of P. Walter), were grown to mid-log phase in the presence or absence of glucosamine (GlcN; 25mM) at the indicated temperatures. The wild-type strain was treated with tunicamycin (1 μg/ml) for 3 h as a UPR-inducing control, β-galacto-sidase activity was measured incrude extracts.

Figure 13. Alignment of two homologs of Eril from Candida tropicalis and Candida albicans

Figure 14. ERIl is a bona fide gene. A. Predicted amino acid sequence of Eril (YPL096c-A) (SEQ ID NO:7). Two potential transmembrane domains are underlined. B. Alignment of S. cerevisiae Eril with homologs from C. tropicalis (SEQ ID NO: 15) and C. albicans (SEQ TD NO: 16). Identical residues are boxed. C. Detection of ERIl RNA. Total RNA was prepared from wild type (1788) and erilΔ (DL2524) cells in log-phase growth, or from saturated cultures (2 days growth). RNA (10 μg) was probed for ERIl and ACT1 (encoding actin) as a loading control.

D. Yeast strain DL2524 was transformed with centromeric plasmid pRS316[ERH] (pCEN[ERH]), which contains the 498 bp intergenic region between YPL096w and YPL097w, or with pRS316 (vector). Transformants were streaked onto YEPD plates and incubated at the indicated temperatures for three days.

Figure 15. Hyperactivation of Ras pathway signaling in an eril A mutant. A. An eril A mutant displays filamentous growth. Single colonies of diploid yeast strains were photographed after 24h on YEPD plates grown at the indicated temperatures. Strains are: wild type (1788), eril A (DL2524), and eril A ras2A (DL2570). B. An erz7Δ mutant displays invasive growth. Haploid yeast strains were streaked onto

YEPD plates and allowed to grow at 34°C for three days (total growth). Non- adherent cells were washed from the plates with distilled water to reveal invasive growth. Strains are: wild type (1783), eril A (DL2522), ras2A (DL838), and eril A ras2A (DL2566). C Hyperactivation of Ras signaling in the EG123 background results in invasive growth. Haploid yeast strains were treated as in "B", except that growth was at 30°C Mutants in the redundant Ras-GAPs encoded by IRA1 and IRA2 were used to activate Ras signaling. Strains are: wild type (1783), iralA (DL2297), and iralA ira2A (DL2709).

Figure 16. Suppression of heat shock sensitivities of erilΔ and iralΔ yeast strains.

A. Strain DL2524 (eril A) was transformed with centromeric vector pRS316, episomal plasmid YEp24[RAS2-Y64] (D.N. RAS2), YEp24[/i^2], or ΌRS316[ERH]. Transformants were subjected to a 50-min heat shock at 50°C, and allowed to recover at room temperature for 36h prior to scoring colony forming units (CFU). B. Strain DL2297 (iralA) was transformed with YΕp24 (vector), pRS416- MET25-ERI1, or YEρ24[ZR4i]. Transformants were subjected to a 30-min heat shock at 50°C, allowed to recover for 36h, and CFU were scored.

Figure 17. Eril associates with Ras2 in a GTP- and effector loop-dependent manner. A. Extracts were prepared from diploid yeast strains 1788 (wild type) and

DL2725 (iralA ira2A) coexpressing ^Eril (from pYeF :TRPl[ERIl]) with GST (from pEGKG), GST-Ras2 (WT), or GST-Ras2^V19 (V19). GST and GST-Ras2 fusions were precipitated with glutathione Sepharose beads and detected by immunoblot (upper panel). Associated ^Eril was also detected by immunoblot (lower panel). B. Extracts were prepared from yeast strain 1788 coexpressing

^HAEril and the indicated forms of GST-Ras2, and treated as in "A". Ras2 forms were: GST-Ras2 (Wild type), GST-Ras2^V19 (V19), GST-Ras2^{v19 A42} (V19 A42), and GST-Ras2^{vl9 N45} (V19 N45). The lower GST-Ras2 band does not reflect differential Ras modification (MJR, unpublished results), and is therefore presumed to be a proteolysis product. C. The N-terminal domain of Eril associates with GTP-Ras.

The indicated trucations of ^Eril were co-expressed with GST-Ras2^V19 in wild type yeast cells and treated as in "A".

Figure 18. Eril is an ER membrane protein. A. HAEril fractionates as an integral membrane protein. The indicated agent was added to lysates of wild type haploid

(1783) yeast cells expressing ^Eril from pRS426- E [HA-ER/i] prior to centrifugation at 100,000g for lh. ^HAΕril from equivalent cell fractions of total lysate (T), supernatant (S), or pellet (P) was detected by immunoblot. B. Immunofluorescence micrographs of wild type diploid (1788) yeast cells expressing ^Eril from 2μ plasmid pRS426[UA-ERIl].

Figure 19. Εril engages Ras at the ΕR. A. Subcellular fractionation of ^HΛΕril and GST-Ras2^vl9-containing membranes. Total membranes from yeast strain 1788 coexpressing ^HAEril with GST-Ras2^v19 (as in Figure 17) were sedimented on a step sucrose/EDTA density gradient. Fractions were subjected to immunoblot analysis to detect GST-Ras2^v19, ^Eril, Gasl (PM marker), Dpml (ER marker), and CPY (vacuolar marker). The mature (m) and immature (i) forms of Gasl are labeled. B. ^Eril associates with Ras2^V19 at the ER. GST-Ras2^V19 was precipitated with glutathione Sepharose beads from the fractions in "A". Precipitated GST-Ras2^V19 and associated ^HAEril were detected by immunoblot.

Figure 20. S. cerevisiae strains used.

Detailed Description of he Invention

This invention provides an agent capable of blocking the interaction between GTP- Ras and ERIl, wherein ERIl is a protein comprising amino acids, the sequence of which amino acids is set forth in SEQ ID NO: 7. (SEQ ID NO:7 sets forth the ERIl amino acid sequence).

The Eril (also referred to as ERIl) (for "Endoplasmic reticulum-associated Ras Inhibitor 1") described herein is synonymous with and may be used interchangeably with the Rin2 described in U.S. Provisional Application 60/392,355 (of which this application claims priority) since Eril and Rin2 are the same protein. The Eril nomenclature was adopted instead ofthe Rin nomenclature so as to avoid confusion with other proteins referred to as Rin.

With respect to the RIN1 or Rinl used herein, RIN is an abbreviation for "Ras Inhibitor." This protein identified in the budding yeast Saccharyomyces cerevisiae has been found to associate with active Ras (GTP-Ras) and inhibit its ability to signal. While the RLN2 described herein is sometimes also referred to as RLNl, such protein is not the RTNl protein described in Han et al, Proc. Natl. Acad. Sci., USA,

94:4954-4959 (May 1997).

Eril is a 68 amino acid protein encoded by a previously unrecognized, non- annotated open reading frame (nORF). A null mutant in ERIl displays a variety of phenotypes that result from hyperactive Ras signaling. These include constitutive filamentous and invasive growth, and failure of quiescent cells to acquire resistance to heat shock. Genetic evidence indicates that ERIl is a negative regulator of RAS function. Specifically, each ofthe above phenotypes of a Eril mutant is suppressed by downregulation of Ras signaling, suggesting that these defects result from an excess of Ras activity. Conversely, overexpression of Eril suppresses the heat shock sensitivity associated with constitutive Ras signaling, indicating that Eril can inhibit Ras pathway activity. We have shown, through in vivo binding experiments, that Eril specifically associates with the active, GTP-bound form of Ras, and not with the inactive GDP-bound form. Moreover, this protein associates with the effector loop of active Ras, which is exposed upon GTP binding, and normally interacts with targets of Ras signaling. Eril behaves, in biochemical fractionation experiments, as an integral membrane protein. Immunofluorescence microscopy images suggest that Eril resides in the endoplasmic reticulum (ER). Although mature Ras is localized to the plasma membrane, it is thought to transit the ER as its C-terminus is processed. We propose that the function of Eril is to prevent Ras from interacting with its effector while being processed in the ER. A utility of such a protein is the inhibition of the mutationally activated Ras (which is locked in the

GTP-bound state) implicated in approximately 30% of human cancers.

In one embodiment, the Ras-inhibitory region of Eril, or its human homolog, is fused to a small protein transduction sequence to facilitate bioavailability (entry into human cells). An example of such sequences is an amino acid sequence from the

HIV Tat protein. Fusion peptides could be used directly (such as by injection or other routes of administration) to treat malignancies in which activated Ras has been implicated.

Eril, or its mammalian homologs, may be exploited to inhibit constitutively active

Ras in human cancers. Oncogenic forms of Ras have been implicated in approximately 30% of human malignancies. Their contribution to tumorigenesis is widely understood to involve aberrant signaling through the Raf protein kinase (the main effector of Ras in mammalian cells), which results in inappropriate cell proliferation. The only Ras inhibitor proteins described previously are the Ras-GAPs, which stimulate the intrinsic GTPase activity of Ras, thereby allowing Ras to turn itself off. However, Ras-GAPs are ineffective inhibitors of oncogenic Ras proteins, because these mutationally activated forms of Ras have lost their ability to hydrolyze GTP. We propose that Eril can inhibit mutationally activated Ras through interaction with its exposed effector domain.

We have identified homologs of Eril from seven additional fungal species. Figure 13 shows an alignment ofthe two closest homologs of Eril from Candida tropicalis and Candida albicans. Eril (as well as its homologs) possesses two highly hydrophobic domains, either of which could serve as transmembrane regions. Biochemical fractionation experiments combined with immunolocalization images indicate that Eril is an integral endoplasmic reticulum (ER) membrane protein. The fractionation experiment reveals that HA-Eril (epitope tagged Eril) sediments with the membrane fraction of cell extracts, and can be solubilized by a membrane- disrupting detergent (TX-100), but not by treatments that would solubilize peripheral membrane proteins (i.e. urea or high pH).

Deletion ofthe ERIl gene results in three phenotypes suggestive of hyperactive Ras signaling. First, when cultivated at 35°C, Eril A mutants are capable of invasive growth in agar medium. This is a phenotype associated with hyperactive Ras signaling in yeast. Moreover, the invasive growth behavior of a Eril A mutant is suppressed by deletion of RAS2, which is required for bona fide invasive growth.

Second, when cultivated at 35°C, the Eril A mutant cells form extended chains of elongated cells similar to those observed during pseudohyphal development, a process that is also driven by hyperactive Ras signaling. Like the invasive growth behavior of Eril A, the elongated morphology is suppressed by deletion of RAS2.

Third, Eril A mutants display a sensitivity to heat shock, similar to hyperactive Ras pathway mutants. Significantly, this heat shock sensitivity is suppressed by downregulation of Ras signaling through overexpression of either a dominant inhibitory form of Ras2, or either of the Ras-GAPs (Iral or Ira2). We have also shown that overexpression of Eril can suppress the heat shock sensitivity associated with deletion of the. Ras-GAP gene, IRA1, which results in hyperactive Ras signaling. Taken in the aggregate, these results indicate that Eril functions as an inhibitor of Ras pathway signaling.

Eril associates specifically with GTP-Ras. We purified wild type and mutationally activated Ras2 (Ras2-V19) expressed in yeast as GST fusion proteins. We found that HA-Eril, coexpressed in these cells, was associated only with the Ras2-V19 mutant, which is locked in the GTP-bound state. By contrast, wild type Ras2 exists predominantly (approximately 95%) in the GDP-bound state. To determine if the differential association observed between the mutant and wild type Ras reflected the nucleotide bound state of these proteins, we repeated the experiment using cells devoid of Ras-GAP (iral A iral A). In these cells, even wild type Ras2 exists only in the GTP-bound state. In this experiment, we found that HA-Eril associated equally well with both wild type and mutant Ras. Therefore, Eril associates specifically with GTP-bound Ras2. This is the first of two criteria generally used in the identification of novel Ras effector proteins.

The second criterion for a novel Ras effector is its association through the Ras effector loop, which includes residues 37-47 of yeast Ras (corresponding to residues 30-40 of human Ras). We examined association of HA-Eril with Ras2-V19 in which we ablated the effector loop with point mutations (T42A or D45N). These mutations, known to eliminate association of Ras with its effectors (Raf in humans, or adenylate cyclase in yeast), eliminated or greatly reduced association of GTP-Ras to Eril. Therefore, Eril association with GTP-Ras requires the Ras effector loop to be intact.

Because Eril behaves in genetic experiments like an inhibitor of Ras, and in biochemical experiments, Eril associates with GTP-Ras through the Ras effector loop, we propose that Eril functions to inhibit Ras by preventing access to Ras effectors. Put simply, we view Eril as a molecular shield that denies access of GTP-

Ras to its effectors. Because Eril resides in the ER, and Ras resides at the plasma membrane, but is thought to associate transiently with the ER as its C-terminus is processed, we further propose that Eril functions to inhibit Ras during its maturation in the ER. If this is true, then the ability of Eril to inhibit Ras is limited by its intracellular location. Therefore, a preferred embodiment of an Eril -based Ras inhibitor would involve mislocalization of Eril to the cell surface. There exist a variety of means to accomplish this. One approach to mislocalization of Eril is to add a myristoylation signal to its N-terminus. Another is to add a prenylation signal to its C-terminus. A third is to fuse it to the cytoplasmic domain of a known plasma membrane protein.

The nucleic acid sequence which encodes ERIl is set forth in SEQ ID NOS: 1 and 6.

The ERIl amino acid sequence is set forth in SEQ ID NOS:l and 7. Peptide fragments within Eril are set forth in SEQ ID NOS: 2-5. ERIl is a 68 amino acid protein which comprises two hydrophobic regions. The presumption is that one is a transmembrane region and the other is involved in binding to and inhibiting Ras. As used herein, the terms "agent" and "compound" include both protein and non-protein moieties.

In one embodiment ofthe agent, the protein is encoded by a nucleic acid comprising nucleotides, the sequence of which nucleotides is set forth in SEQ ID NO: 6 (full length ERIl DNA sequence).

In one embodiment, the agent is capable of binding to GTP-Ras. In one embodiment, the agent inhibits the ability of GTP-Ras to signal.

In one embodiment, wherein the agent is a polypeptide or portion thereof. In one embodiment, the polypeptide is ERIl or a portion thereof. In one embodiment, the polypeptide comprises amino acids, the sequence of which is set forth in SEQ ID

NO: 7 (full length ERIl amino acid sequence). In one embodiment, the polypeptide is encoded by a nucleic acid, the sequence of which is set forth in SEQ ID NO: 6

(full length ERIl DNA sequence)

In one embodiment, the portion is an amino terminal portion of ERIl. Such amino terminal portion may comprise the sequence set forth at residues 1-39 of SEQ ID NO:7, or a portion thereof. Such portion may be truncated at either the amino terminal or carboxy terminal or both to generate a fragment which is a portion ofthe sequence set forth at resdidues 1-39 of SEQ ID NO:7. Such fragment may be a length which from 1 to 39 amino acids in length as long as it retains the activity of ERIl, such as its ability to bind to Ras.

In one embodiment, the polypeptide is encoded by a nucleic acid capable of hybridizing under stringent wash conditions to a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:6. One skilled in the art would be able to determine highly stringent conditions, such as low salt and high temperatures. An example of low salt is 0.1 X SSC. An example of high temperature is 65-68 °C. In one embodiment, .this polypeptide would have the activity of ERIl.

In one embodiment, the agent is a portion of ERIl, and the portion is a GTP-Ras binding motif. In one embodiment, the agent comprises the sequence set forth in

SEQ ID NO: 9. In another embodiment, the agent comprises the sequence set forth in SEQ ID NO: 11. In one embodiment, the Ras binding motif is a hydrophobic domain. In one embodiment, the Ras binding motif comprises the amino acid sequence GFLVLGFTYSVLLISLATFYWL (SEQ ID NO:9). In one embodiment, the Ras binding motif comprises the amino acid sequence

FLHYWCVLLLCPATLWLWALIAW (SEQ ID NO: 11). In one embodiment, the agent comprises amino acid residues 1-39 of Eril (i.e. 1-39 of SEQ ID NO:7).

In one embodiment, the agent is a fusion protein which comprises (1) a peptide which comprises at least one GTP-Ras binding motif, and (2) a transcellular polypeptide for promoting transcytosis of the fusion protein across a cell surface membrane.

In one embodiment of the fusion protein, the peptide corresponds to at least a portion of the sequence set forth in SEQ IN NO: 7 (full length ERIl amino acid sequence). In one embodiment, the portion is a GTP-Ras binding motif. In one embodiment of the fusion protein, the transcellular polypeptide is an internalizing peptide. The transcellular peptides include but are not limited to those derived from a polypeptide selected from the group consisting of antepennepedia protein (Antp), HIV transactivating (TAT) protein, mastoparan, melittin, bombolittin, delta hemolysin, pardaxin, Pseudomonas exotoxin A, clathrin,

Diphtheria toxin and C9 complement protein. The transcellular peptide may also be derived from antepennepedia protein. The transcellular peptide may also be derived from HIV transactivating (TAT) protein, he transcellular peptide may also be derived from herpes-simplex-virus- 1 DNA binding domain (HSV VP22).

The HIV-1 TAT peptide may comprise the following amino acid sequence: Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg (SEQ ID NO: 12). The HSV VP22 peptide may comprise the following amino acid sequence: Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro Val Glu (SEQ ID NO:13). The Antp peptide may comprise the following amino acid sequence: Arg Gin Ile Lys Ile Tip Phe Gin Asn Arg Arg Met Lys Trp Lys Lys (SEQ ID NO: 14). Such sequences may also be found in Schwarze et al Trends in Cell Biology 10:290-295 (2000).

In one embodiment of the fusion protein, the transcellular polypeptide sequence is an accessory peptide sequence which enhances interaction of the fusion protein with a cell surface membrane. In one embodiment the accessory peptide comprises an RGD sequence.

Another aspect of the subject invention relates to chimeric polypeptides which includes a heterologous peptide sequence ("internalizing peptide") which drives the translocation of an extracellular form of a thereapeutic polypeptide sequence across a cell membrane in order to facilitate intracellular localization of the thereapeutic polypeptide. In this regard, the therapeutic polypeptide sequence is one which is active intracellularly, such as a tumor suppressor polypeptide, transcription factor or the like. The internalizing peptide, by itself, is capable of crossing a cellular membrane by, e.g., transcytosis, at a relatively high rate. The internalizing peptide is conjugated, e.g., as a fusion protein, to a therapeutic polypeptide. The resulting chimeric polypeptide is transported into cells at a higher rate relative to the polypeptide alone to thereby provide an means for enhancing the introduction of inhibitory polypeptides into surrounding cells, e.g., to enhance gene therapy and/or topical applications of the therapeutic polypeptide. For convenience, the transcellular therapeutic polypeptides are described below as fusion proteins including CKI polypeptide sequences, though as also described below (section v), many other protein domains can be used in place ofthe CKI polypeptide.

In one embodiment, the internalizing peptide is derived from the drosopholia antepennepedia protein, or homologs thereof. The 60 amino acid long long homeodomain of the homeo-protein antepennepedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is couples. See for example Derossi et al.

(1994) J Biol Chem 269:10444-10450; and Perez et al. (1992) J Cell Sci 102:717- 722. Recently, it has been demonstrated that fragments as small as 16 amino acids long of this protein are sufficient to drive internalization. See Derossi et al. (1996) J

Biol Chem 271:18188-18193. The present invention contemplates a chimeric protein comprising at least one CDK binding motif and at least a portion of the antepennepedia protein (or homolog thereof) sufficient to increase the transmembrane transport ofthe chimeric protein, relative to the CDK binding motif alone, by a statistically significant amount.

Another example of an internalizing peptide is the HIV transactivator (TAT) protein. This protein appears to be divided into four domains (Kuppuswamy et al. (1989) Nucl Acids Res. 17:3551-3561). Purified TAT protein is taken up by cells in tissue culture (Frankel and Pabo, (1989) Cell 55:1189-1193), and peptides, such as the fragment corresponding to residues 37 -62 of TAT, are rapidly taken up by cell in vitro (Green and Loewenstein, (1989) Cell 55:1179-1188). The highly basic region mediates internalization and targeting of the internalizing moiety to the nucleus (Ruben et al., (1989) J. Virol. 63:1-8). Peptides or analogs that include a sequence present in the highly basic region, such as CFITK^GISYGRKXJ^QRRRPPQGS (SEQ ID NO: 17), are conjugated to CKI polypeptides (or CDK binding motifs thereof) to aid in internalization and targeting those proteins to the intracellular milleau.

Another exemplary transcellular CKI polypeptide can be generated to include a sufficient portion of mastoparan (T. Higashijima et al., (1990) J. Biol. Chem. 265:14176) to increase the transmembrane transport ofthe chimeric protein.

While not wishing to be bound by any particular theory, it is noted that hydrophilic polypeptides may be also be physiologically transported across the membrane barriers by coupling or conjugating the polypeptide to a transportable peptide which is capable of crossing the membrane by receptor-mediated transcytosis. Suitable internalizing peptides of this type can be generated using all or a portion of, e.g., a histone, insulin, transferrin, basic albumin, prolactin and insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II) or other growth factors. For instance, it has been found that an insulin fragment, showing affinity for the insulin receptor on capillary cells, and being less effective than insulin in blood sugar reduction, is capable of transmembrane transport by receptor-mediated transcytosis and can therefor serve as an internalizing peptide for the subject transcellular CKI polypeptides. Preferred growth factor-derived internalizing peptides include EGF

(epidermal growth factor)-derived peptides, such as CMHIESLDSYTC (SEQ ID NO: 18) and CMYIEALDKYAC (SEQ ID NO: 19); TGF- beta (transforming growth factor beta )-derived peptides; peptides derived from PDGF (platelet-derived growth factor) or PDGF-2; peptides derived from IGF-I (insulin-like growth factor) or IGF- II; and FGF (fibroblast growth factor)-derived peptides.

Another class of translocating/internalizing peptides exhibits pH-dependent membrane binding. For an internalizing peptide that assumes a helical conformation at an acidic pH, the internalizing peptide acquires the property of amphiphilicity, e.g., it has both hydrophobic and hydrophilic interfaces. More specifically, within a pH range of approximately 5.0-5.5, an internalizing peptide forms an alpha-helical, amphiphilic structure that facilitates insertion ofthe moiety into a target membrane. An alpha-helix-inducing acidic pH environment may be found, for example, in the low pH environment present within cellular endosomes. Such internalizing peptides can be used to facilitate transport of CKI polypeptides, taken up by an endocytic mechanism, from endosomal compartments to the cytoplasm.

A preferred pH-dependent membrane-binding internalizing peptide includes a high percentage of helix-forming residues, such as glutamate, methionine, alanine and leucine. In addition, a preferred internalizing peptide sequence includes ionizable residues having pKa's within the range of pH 5-7, so that a sufficient uncharged membrane-binding domain will be present within the peptide at pH 5 to allow insertion into the target cell membrane.

A particularly preferred pH-dependent membrane-binding internalizing peptide in this regard is aal-aa2-aa3-EAALA(EALA)4-EALEALAA-amide (SEQ ID NO:20), which represents a modification of the peptide sequence of Subbarao et al. (Biochemistry 26:2964, 1987). Within this peptide sequence, the first amino acid residue (aal) is preferably a unique residue, such as cysteine or lysine, that facilitates chemical conjugation of the internalizing peptide to a targeting protein conjugate. Amino acid residues 2-3 may be selected to modulate the affinity of the internalizing peptide for different membranes. For instance, if both residues 2 and 3 are lys or arg, the internalizing peptide will have the capacity to bind to membranes or patches of lipids having a negative surface charge. If residues 2-3 are neutral amino acids, the internalizing peptide will insert into neutral membranes.

Yet other preferred internalizing peptides include peptides of apo-lipoprotein A-l and B; peptide toxins, such as melittin, bombolittin, delta hemolysin and the pardaxins; antibiotic peptides, such as alamethicin; peptide hormones, such as calcitonin, corticotrophin releasing factor, beta endorphin, glucagon, parathyroid hormone, pancreatic polypeptide; and peptides corresponding to signal sequences of numerous secreted proteins. In addition, exemplary internalizing peptides may be modified through attachment of substituents that enhance the alpha-helical character ofthe internalizing peptide at acidic pH.

Yet another class of internalizing peptides suitable for use within the present invention include hydrophobic domains that are "hidden" at physiological pH, but are exposed in the low pH environment of the target cell endosome. Upon pH- induced unfolding and exposure of the hydrophobic domain, the moiety binds to lipid bilayers and effects translocation ofthe covalently linked CKI polypeptide into the cell cytoplasm. Such internalizing peptides may be modeled after sequences identified in, e.g., Pseudomonas exotoxin A, clathrin, or Diphtheria toxin.

Pore-forming proteins or peptides may also serve as internalizing peptides herein. Pore forming proteins or peptides may be obtained or derived from, for example, C9 complement protein, cytolytic T-cell molecules or NK-cell molecules. These moieties are capable of forming ring-like structures in membranes, thereby allowing transport of attached CKI polypeptide through the membrane and into the cell interior.

Mere membrane intercalation of an internalizing peptide may be sufficient for translocation ofthe CKI polypeptide across cell membranes. However, translocation may be improved by attaching to the internalizing peptide a substrate for intracellular enzymes (i.e., an "accessory peptide"). It is preferred that an accessory peptide be attached to a portion(s) ofthe internalizing peptide that protrudes through the cell membrane to the cytoplasmic face. The accessory peptide may be advantageously attached to one terminus of a translocating/internalizing moiety or anchoring peptide. An accessory moiety ofthe present invention may contain one or more amino acid residues. In one embodiment, an accessory moiety may provide a substrate for cellular phosphorylation (for instance, the accessory peptide may contain a tyrosine residue). An exemplary accessory moiety in this regard would be a peptide substrate for N- myristoyl transferase, such as GNAAAARR (SEQ ID NO:21) (Eubanks et al., in Peptides. Chemistry and Biology, Garland Marshall (ed.), ESCOM, Leiden, 1988, pp. 566-69) In this construct, an internalizing, peptide would be attached to the C- terminus of the accessory peptide, since the N-terminal glycine is critical for the accessory moiety's activity. This hybrid peptide, upon attachment to a CKI polypeptide at its C-terminus, is N-myristylated and further anchored to the target cell membrane, e.g., it serves to increase the local concentration of the CKI polypeptide at the cell membrane.

To further illustrate use of an accessory peptide, a phosphorylatable accessory peptide is first covalently attached to the C-terminus of an internalizing peptide and then incorporated into a fusion protein with a CKI polypeptide. The peptide component of the fusion protein intercalates into the target cell plasma membrane and, as a result, the accessory peptide is translocated across the membrane and protrudes into the cytoplasm ofthe target cell. On the cytoplasmic side ofthe plasma membrane, the accessory peptide is phosphorylated by cellular kinases at neutral pH. Once phosphorylated, the accessory peptide acts to irreversibly anchor the fusion protein into the membrane. Localization to the cell surface membrane can enhance the translocation ofthe CKI polypeptide into the cell cytoplasm.

Suitable accessory peptides include peptides that are kinase substrates, peptides that possess a single positive charge, and peptides that contain sequences which are glycosylated by membrane-bound glycotransferases. Accessory peptides that are glycosylated by membrane-bound glycotransferases may include the sequence x-

NLT-x, where "x" may be another peptide, an amino acid, coupling agent or hydrophobic molecule, for example. When this hydrophobic tripeptide is incubated with microsomal vesicles, it crosses vesicular membranes, is glycosylated on the luminal side, and is entrapped within the vesicles due to its hydrophilicity (C. Hirschberg et al., (1987) Ann. Rev. Biochem. 56:63-87). Accessory peptides that contain the sequence x-NLT-x thus will enhance target cell retention of corresponding CKI polypeptide.

In another embodiment of this aspect of the invention, an accessory peptide can be used to enhance interaction of the CKI polypeptide with the target cell. Exemplary accessory peptides in this regard include peptides derived from cell adhesion proteins containing the sequence "RGD", or peptides derived from laminin containing the sequence CDPGYIGSRC (SEQ ID NO:22). Extracellular matrix glycoproteins, such as fibronectin and laminin, bind to cell surfaces through receptor-mediated processes. A tripeptide sequence, RGD, has been identified as necessary for binding to cell surface receptors. This sequence is present in fibronectin, vitronectin, C3bi of complement, von-Willebrand factor, EGF receptor, transforming growth factor beta , collagen type I, lambda receptor of E. coli, fibrinogen and Sindbis coat protein (E. Ruoslahti, Ann. Rev. Biochem. 57:375-413, 1988). Cell surface receptors that recognize RGD sequences have been grouped into a superfamily of related proteins designated "integrins". Binding of "RGD peptides" to cell surface integrins will promote cell-surface retention, and ultimately translocation, ofthe CKI fusion protein.

As described for the poly-CBM proteins above, the internalizing and accessory peptides can each, independently, be added to a CKI polypeptide by either chemical cross-linking or in the form of a fusion protein. In the instance of fusion proteins, unstructured polypeptide linkers can be included between each of the peptide moieties.

The CKI polypeptide can consist of as little as a CDK-binding moitey of a CKI protein, or can include a full length CKI protein and/or poly-CBM protein.

In general, the internalization peptide will be sufficient to also direct export of the CKI polypeptide. However, where an accessory peptide is provided, such as an RGD sequence, it may be necessary to include a secretion signal sequence to direct export of the fusion protein from its host cell. In preferred embodiments, the secretion signal sequence is located at the extreme N-terminus, and is (optionally) flanked by a proteolytic site between the secretion signal and the rest of the fusion protein.

In an exemplary embodiment, the CKI polypeptides is engineered to include an integrin-binding RGD peptide/SV40 nuclear localization signal (see, for example Hart SL et al., 1994; J. Biol. Chem.,269:12468-12474), such as encoded by the nucleotide sequence provided in the Ndel-EcoRl fragment: catatgggtggctgccgtggcgatatgttcggttgcggtgctcctccaaaaaagaagagaaag-gtagctggattc

(SEQ ID NO:23), which encodes the RGD/SV40 nucleotide sequence: MGGCRGDMFGCGAPP-KKKRKVAGF (SEQ ID NO:24). In another embodiment, the protein can be engineered with the HIV-1 tat(l-72) polypeptide, e.g., as provided by the Ndel-EcoRl fragment: Catatggagccagtagatcctagactagagccctggaagcatccaggaagtcagcctaaaactgcttgtaccaattgcta ttgtaaaaagtgttgctttcattgccaagtttgtttcataacaaaagcccttggcatctcctatggcaggaagaagcggagac agcgacgaagacctcctcaaggcagtcagactcatcaagtttctctaagtaagcaaggatt (SEQ ID NO:25), which encodes the HIV-1 tat(l-72) peptide sequence:

MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKK RRQRRRPPQGSQTHQVSLSKQ (SEQ ID NO:26). In still another embodiment, the fusion protein includes the HSV-1 VP22 polypeptide (Elliott G., O'Hare P (1997) Cell, 88l223-233) provided by the Ndel-EcoRl fragment:

cat atg aec tct cgc cgc tec gtg aag teg ggt ccg egg gag gtt ccg cgc gat gag tac gag gat ctg tac tac aec ccg tct tea ggt atg gcg agt ccc gat agt ccg cct gac aec tec cgc cgt ggc gcc eta cag aca cgc teg cgc cag agg ggc gag gtc cgt ttc gtc cag tac gac gag teg gat tat gcc etc tac ggg ggc teg tea tec gaa gac gac gaa cac ccg gag gtc ccc egg acg egg cgt ccc gtt tec ggg gcg gtt ttg tec ggc ccg ggg cct gcg egg gcg cct ccg cca ccc get ggg tec gga ggg gcc gga cgc aca ccc ace ace gcc ccc egg gcc ccc cga aec cag egg gtg gcg act aag gcc ccc gcg gcc ccg gcg gcg gag aec ace cgc ggc agg aaa teg gcc cag cca gaa tec gcc gca etc cca gac gcc ccc gcg teg acg gcg cca aec cga tec aag aca ccc gcg cag ggg ctg gcc aga aag ctg cac ttt age ace gcc ccc cca aac ccc gac gcg cca tgg aec ccc egg gtg gcc ggc ttt aac aag cgc gtc ttc tgc gcc gcg gtc ggg cgc ctg gcg gcc atg cat gcc egg atg gcg gcg gtc cag etc tgg gac atg teg cgt ccg cgc aca gac gaa gac etc aac gaa etc ctt ggc ate aec aec ate cgc gtg acg gtc tgc gag ggc aaa aac ctg ctt cag cgc gcc aac gag ttg gtg aat cca gac gtg gtg cag gac gtc gac gcg gcc acg gcg act cga ggg cgt tct gcg gcg teg cgc ccc ace gag cga cct cga gcc cca gcc cgc tec get tct cgc ccc aga egg ccc gtc gag gaa ttc (SEQ ID NO:27) which encodes the HSV-1 VP22 peptide having the sequence:

MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQR GEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPP PAGSGGAGRTPTTAPRAPRTGRVATKAPAAPAAETTRGRKSAQPESAALPD APASTAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVG

RLAAMHARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRAN ELVNPDVVQDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:28)

In still another embodiment, the fusion protein includes the C-terminal domain of the VP22 protein from, e.g., the nucleotide sequence (Ndel-EcoRl fragment):

cat atg gac gtc gac gcg gcc acg gcg act cga ggg cgt tct gcg gcg teg cgc ccc ace gag cga cct cga gcc cca gcc cgc tec get tct cgc ccc aga egg ccc gtc gag gaa ttc (SEQ ID NO:29) which encodes the VP22 (C-terminal domain) peptide sequence:

MDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE

(SEQ ID NO:30)

Another aspect ofthe present invention pertains generally to the use of internalizing peptides as part of a strategy to deliver therapeutic proteins by gene therapy techniques. In addition to the subject CKI polypeptides, other therapeutic proteins such as various tumor suppressors, transcription factors, signal transduction proteins, antiviral polpeptides, and therapeutic peptides, e.g., which are otherwise localized intracellularly, can be engineered to include an internalizing peptide which drives the translocation of the therapeutic polypeptide into surrounding cells. Thus, the transcytosis system can be used to increase the efficacy of gene therapy by delivering the therapeutic protein not only to cells actually transfected with the expression vector, but also to the surrounding cells. Such constructs can be generated with other therapeutic polypeptides using such of the general teachings herein as applicable and the general knowledge in the art.

In one illustrative embodiment, the therapeutic polypeptide sequence is derived from a tumor suppressor. In addition to the CKI proteins described above, exemplary tumor suppressors from which the subject transcellular proteins can be generated include the Rb protein (and related proteins) and the p53 protein. For instance, the subject constructs can be generated with a therapeutic polypeptide domain comprising at least a functional domain of Rb, pi 07 or other Rb-like protein. Such tumor suppressor domainss have been mapped through both genetic and biochemical means. (Hu et al. (1990) EMBO J. 9:1147-1155; Ewen et al. (1991) Cell 66:1155- 1164; Ewen et al. (1992) Science 255:85-87). An approximately 400 amino acid fragment of Rb and i 07, termed the Rb pocket, is responsible for association of these proteins with the DNA tumor virus oncoproteins and cellular ligands. Within this domain are six regions of extensive sequence similarity between Rb and pi 07.

(Ewen et al. (1991), supra).

Another illustrative example of the subject transcellular proteins include fusion proteins generated with Bcl-2 or Bcl-x polypeptide sequences. The protein Bcl-2 plays a central role in the process of programmed cell death by blocking apoptosis.

For example, when Bcl-2 levels in a cell are elevated, apoptosis is blocked. Conversely, when Bcl-2 levels in a cell are lowered, the rate of cell death is accelerated. The protein encoded by the bcl-2 proto-oncogene has been reported to be capable of inhibiting apoptosis in many hematopoietic cell systems. The bcl-2 protein is a 26 kD membrane-associated cytoplasmic protein (Tsujimoto et al.

(1987) Oncogene 2: 3; USSN 5,202,429 and USSN 5,015,568; Hockenbery et al. (1991) PNAS 88:6961; Monaghan et al. (1992) J. Histochem. Cytochem. 40:1819; Nguyen et al. (1993) J. Biol Chem. 268: 25265; and Nguyen et al. (1994) J. Biol Chem. 269:16521). The capacity of bcl-2 to enhance cell survival is related to its ability to inhibit apoptosis initiated by several factors, such as cytokine deprivation, radiation exposure, glucocorticoid treatment, and administration of anti-CD-3 antibody. Thus, all or a portion of bcl-2 sufficient to inhibit apoptosis can be used to generate the subject transcellular proteins.

Likewise, the transcellular protein can be generated using all or a portion of a protein which interacts with and/or is structurally related to the bcl-2 gene product have also been identified, such as for example bcl-χL and bcl-x^ [Boise et al. (1993) Cell 74: 597; Gonzalez-Garcia et al. (1994) Development 120: 3033; Gottschalk et al. (1994) PNAS 91: 7350], Bax [Oltvai et al. (1993) Cell 74: 609], Mcl-1 [Kozopas et al. (1993) PNAS 90: 3516], and Al [Lin et al. (1993) J. Immunol. 151: 179]

An example of a signal transduction protein which can be used to generate the subject transcellular proteins is the product the mammalian tubby (tub) genes, e.g., which are involved in the control of mammalian body weight. See, for example, USSN 5,646,040. Tubby, an autosomal recessive mutation recently found to be the result of a splicing defect in the tubby gene. Thus, agonist and antagonist forms of tubby proteins can be used to control weight gain in animals. Moreover, these proteins are also excellent candidates for use in the treatment of ocular diseases as mutations in the tubby gene are known to lead to early progressive retinal degeneration.

Still another family of proteins which can be used to generate a transcellular protein of the present invention are the I B proteins. The nuclear factor-kB (NF-κB) is an inducible transcription factor which participates in the regulation of multiple cellular genes after treatment of cells with a variety of factors. These genes are involved in the immediate early processes of immune, acute phase, and inflammatory responses.

NF-κB has also been implicated in the transcriptional activation of several viruses, most notably the type 1 human immunodeficiency virus (HIV-1) and cytomegalovirus (CMV) (Nabel et al. (1987) Nature 326:711; Kaufman et al. (1987) Mol. Cell. Biol. 7:3759; and Sambucetti et al. (1989) EMBO J 8:4251). Activation of the NF-kB transcription factor and various related forms can be initiated by a variety of agents, including TNF-α, phorbol 12-myristate 13 -acetate (PMA), interleukin-1 (IL-1) and interleukin-2 (IL-2). Activation proceeds through a post-translational event in which preformed cytoplasmic NF-κB in the Rel complex is released from a cytoplasmic inhibitory protein. A common feature of the regulation of transcription factors which belong to the Rel-family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as IκBs (Baeuerle et al. (1988) Cell 53:211; Beg et al. (1993) Genes Dev. 7:2064; and Gilmore et al. (1993) Trends in Genetics 9:427). Treatment of cells with different inducers, e.g., IL-1, TNF-α, LPS, dsRNA or PMA, results in dissociation of the cytoplasmic complexes and translocation of free NF-κB to the nucleus (Grilli et al. (1993) International Rev. of Cytology 143:1-62; Baeuerle et al. (1994) Annu. Rev. Immunol. 12:141). The dissociation ofthe cytoplasmic complexes is understood to be triggered by the phosphorylation and subsequent degradation of the IκB protein (Palombella et al. (1994) Cell 78:773; Ghosh et al. (1990) Nature

344:678).

Thus, the IκB proteins provide a therapeutic target for being upregulated, such as be ectopic expression through gene therapy. Accordingly, the subject transcellular protein can be generated with an IκB polypeptide sufficient to bind to and prevent nuclear localization of NF-κB. In preferred embodiments, the IκB polypeptide has been altered, e.g., by point mutagenesis or truncation, to increase the intracellular half-life ofthe fusion protein. For instance, Lys-25 and Lys-26 of human IicBα can be mutated to remove the ability ofthe protein to be ubiquitinated.

In still other embodiments, the trancellular protein can be generated with a transcription factor polypeptide, as well as with a transcription repressor polypeptide, e.g., to potentiate or inhibit the expression of a gene. In other embodiments, the therapeutic portion of the trancellular protein can be provided from a metal binding protein, e.g., for use in inhibiting the action of intracellular

DNA damaging agents such as cisplatin. In yet other embodiments, the therapeutic polypeptide of the trancellular fusion protein can be derived from small peptides/polypeptides which are artificial in sequence, or which are truncated forms of any of such proteins as described above. In an illustrative embodiment, the therapeutic polypeptide can be a peptide inhibitor of cyclin dependent kinases, such described in USSN 5,625,031.

In general, the criteria for selecting the therapeutic polypeptide portion of the transcellular protein is reasonably straightforward. As with the CKI polypeptides, the therapeutic polypeptide sequence included in the fusion protein should from an intracellular protein which modulates a biological process in the cell, e.g., by either mimicing or antagonizing the wild-type form ofthe protein from which it is derived. The therapeutic polypeptide sequence included in the fusion protein preferably does not includes any membrane association sequences, such as transmembrane domains, myristolation sequences, etc. The therapeutic polypeptide sequence also preferably does not includes any disulfide bonds. It is preferably no larger than about lOOkD, and is even more preferably no larger than 75, 50 or even 30 kd. It is also preferably no smaller than about 20 amino acids residues.

In one embodiment of the agent described herein, the agent is a peptidomimetic. In one embodiment of the agent described herein, the agent is a nonpeptidyl agent. In one embodiment ofthe agent described herein, the agent is a small molecule. In one embodiment of the agent described herein, the agent has a molecular weight less than 500 daltons. As used herein, "nonpeptidyl agent" means an agent that does not consist in its entirety of a linear sequence of amino acids linked by peptide bonds. A nonpeptidyl molecule may, however, contain one or more peptide bonds. In one embodiment, the nonpeptidyl agent is a compound having a molecular weight less than 500 daltons. As used herein, a "small molecule" or small molecular weight molecule is one having a molecular weight less than 500 daltons.

The compounds and/or agents described herein may be made by any means known to one skilled in the art. For example, a protein may be made by recombinant expression from a nucleic acid, such as a plasmid or vector comprising the encoding nucleic acid, wherein the plasmid or vector is in a suitable host cell, i.e. a host- vector system for the production of the polypeptide of interest. A suitable vector may be made which comprises suitable regulatory sequences, such as enhancers and promotors. The host cell may be of any type, including but not limited to mammalian, bacteria and yeast cells. Suitable bacterial cells include E.coli cells. Suitable mammalian cells include but are not limited to human embryonic kidney (HEK) 293T cells, HeLa cells, NIH 3T3 cells Chinese hamster ovary (CHO) cells and Cos cells.

If the protein is produced recombinantly, it may be expressed from a plasmid containing a synthetic nucleic acid insert. Such insertion site in the plasmid may allow linking the protein to a tag, such as a poly-Histidine tag. Such a tag facilitates later protein purification.

A nucleic acid encoding the polypeptide, protein or functional equivalent thereof may be cloned under the control of an inducible promoter, thereby allowing regulation of protein expression. Suitable inducible systems are known to those of skill in the art.

Vectors for expressing the protein or functional equivalents described herein may be selected from commercial sources or constructed for a particular expression system. Such vectors may contain appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences and marker genes. Vectors may be plasmids, or viral-based. One skilled may consult Molecular Cloning: a laboratory manual (Sambrook et al, 1989). Many known techniques and protocols for the manipulation of nucleic acids and analysis of proteins are described in detail in "Short protocols in molecular biology", second addition, Ausubel et al. (John Wiley & Sons 1992).

Methods for the isolation and purification of recombinant proteins are known to those of skill in the art and described in various sources such as in Sambrook et α/.(1989). In bacteria such as E.Coli, the recombinant protein may form inclusion bodies within the bacterial cell, thus facilitating its preparation. If produced in inclusion bodies, the carrier protein may require refolding to a natural conformation.

Additionally, in order to tailor the properties ofthe protein or functional equivalent thereof, one skilled appreciates that alterations may be made at the nucleic acid level from known protein sequences, such as by adding, substituting, deleting or inserting one or more nucleotides. Site-directed mutagenesis is the method of preference that may be employed to make mutated proteins. There are many site-directed mutagenesis techniques known to those skill in the art, including but not limited to oligonucleotide-directed mutagenesis using PCR, such as is described in Sambrook, or using commercially available kits.

Suitable vectors may be selected or constructed, containing appropriate regulatory sequences, including promoter sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. The vectors include but are not limited to plasmids, such as viral e.g. phage, or phagemid, and as described in Sambrook. Techniques and protocols for manipulating nucleic acids, such as in preparing nucleic acid constructs, mutagenesis, sequencing, introducing nucleic acids into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al.

Eds, John Wiley & Sons, 1992, which is incorporated by reference.

This invention also provides soluble forms of the polypeptides described herein. Accordingly, for example, a transmembrane domain for a polypeptide expressed on a cell surface may be removed such that the polypeptide would become soluble.

After having determined which amino acid residues contribute to the receptor- binding domain (supra), it is possible for the skilled artisan to design synthetic peptides having amino acid sequences that define a pre-selected receptor-binding motif. A computer program useful in designing potentially bioactive peptidomimetics is described in U.S. Pat. No. 5,331,573, the disclosure of which is incorporated by reference herein. In addition to choosing a desirable amino acid sequence, the skilled artisan using standard molecular modeling software packages, infra, can design specific peptides having, for example, additional cysteine amino acids located at pre-selected positions to facilitate cyclization of the peptide of interest. Oxidation of the additional cysteine residues results in cyclization ofthe peptide thereby constraining the peptide in a conformation that mimics the conformation of the corresponding amino acid sequence in native Eril. It is contemplated, that any standard covalent linkage, for example, disulfide bonds, typically used to cyclize synthetic peptides maybe useful in the practice of the instant invention. Alternative cyclization chemistries are discussed in International Application PCT/WO 95/01800, the disclosure of which is incorporated herein by reference.

In addition, it is contemplated that a single peptide containing amino acid sequences derived from separate Eril subunit domains may be created, for example two hydrophobic domains.

Eril analogs, including peptides, peptidomimetics, non-peptide small molecules, genes and recombinant polypeptides, may be generated using combinatorial techniques using techniques which are available in the art for generating combinatorial libraries of small organic/peptide libraries. See, for example, Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Patents 5,359,115 and 5,362,899; the Ellman U.S. Patent 5,288,514; the Still et al. PCT publication WO 94/08051; Chen et al. (1994) JACS 116:2661; Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCT publication WO93/20242).

In a preferred embodiment, the combinatorial peptide library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential Eril analog peptide sequences. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential Eril analog nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of Eril analog peptide sequences therein. There are many ways by which the gene library of potential Eril homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes can then be ligated into an appropriate gene for expression. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential Eril analog sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp. 273- 289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science

198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815).

A wide range of techniques are Icnown in the art for screening gene products of combinatorial libraries made by point mutations. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of Eril analog sequences. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Such illustrative assays are amenable to high throughput analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.

In yet another screening assay, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to associate with a Eril receptor via this gene product is detected in a "panning assay". Such panning steps can be carried out on cells cultured from embryos. For instance, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140). In a similar fashion, fluorescently labeled molecules which bind a Eril protein can be used to score for potentially functional peptides. Cells can be visually inspected and separated under a fluorescence microscope, or, where the morphology ofthe cell permits, separated by a fluorescence-activated cell sorter.

In an alternate embodiment, the gene library is expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at very high concentrations, large numbers of phage can be screened at one time. Second, since each infectious phage displays the combinatorial gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages Ml 3, fd, and fl are most often used in phage display libraries, as either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010;

Griffths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624- 628; and Barbas et al. (1992) PNAS 89:4457-4461).

In an illustrative embodiment, the recombinant phage antibody system (RPAS, Pharamacia Catalog number 27-9400-01) can be easily modified for use in expressing and screening peptide combinatorial libraries. For instance, the pCANTAB 5 phagemid ofthe RPAS kit contains the gene which encodes the phage gill coat protein. The peptide combinatorial gene library can be cloned into the phagemid adjacent to the gill signal sequence such that it will be expressed as a gill fusion protein. After ligation, the phagemid is used to transform competent E. coli TGI cells. Transformed cells are subsequently infected with M13KO7 helper phage to rescue the phagemid and its candidate peptide gene insert. The resulting recombinant phage contain phagemid DNA encoding a specific candidate peptide, and display one or more copies ofthe corresponding fusion coat protein. The phage- displayed candidate peptides which are capable of binding a Eril receptor are selected or enriched by panning. For instance, the phage library can be applied to cells which express a Eril receptor and unbound phage washed away from the cells.

The bound phage are then isolated, and if the recombinant phage express at least one copy ofthe wild type gill coat protein, they will retain their ability to infect E. coli. Thus, successive rounds of reinfection of E. coli, and panning will greatly enrich for Eril homologs, which can then be screened for further biological activities in order to differentiate agonists and antagonists.

These techniques have been successfully employed in the art to identify peptide mimetics. For example, potent agonists of erythropoietin (Epo) were identified using random phage display to isolate small peptides which bind to and activate the Epo receptor (Wrighton et al. (1996) Science 273: 458). More recently, Kaushansky has reviewed a large body of work wherein both peptide and small molecule mimetics were identified using several different experimental approaches (Kaushansky (2001) Annals ofthe NY Academy of Sciences 938: 131).

Combinatorial mutagenesis has a potential to generate very large libraries of different peptides, e.g., in the order of 10^ molecules. Combinatorial libraries of this size may be technically challenging to screen even with high-throughput screening assays such as phage display. To overcome this problem, a new technique has been developed recently, recursive ensemble mutagenesis (REM), which allows one to avoid the very high proportion of non-functional proteins in a random library and simply enhances the frequency of functional proteins, thus decreasing the complexity required to achieve a useful sampling of sequence space. REM is an algorithm which enhances the frequency of functional mutants in a library when an appropriate selection or screening method is employed (Arkin and Yourvan, 1992,

PNAS USA 89:7811-7815; Yourvan et al, 1992, Parallel Problem Solving from

Nature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering 6(3):327-331). Recombinantly produced forms of the subject peptides can be produced using, for example, expression vectors containing a nucleic acid encoding a subject peptide, operably linked to at least one transcriptional regulatory sequence. Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of a subject polypeptide. Accordingly, the term transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences, sequences that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding subject polypeptide. Such useful expression control sequences, include, for example, a viral LTR, such as the LTR of the Moloney murine leukemia virus, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage 8 , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design ofthe expression vector may depend on such factors as the choice ofthe host cell to be transformed and/or the type of protein desired to be expressed.

Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

Such peptides may be synthesized and screened for BMP-like activity using any of the standard protocols described below. As discussed above, upon determination ofthe receptor-binding domain of Eril, it is contemplated that the skilled artisan can design non-peptidyl based small molecules, for example, small organic molecules, whose structural and chemical features mimic the same features displayed on at least part of the surface of the receptor-binding domain of Eril.

Because a major contribution to the receptor-binding surface is the spatial arrangement of chemically interactive moieties present within the sidechains of amino acids which together define the receptor-binding surface, a preferred embodiment of the present invention relates to designing and producing a synthetic organic molecule having a framework that carries chemically interactive moieties in a spatial relationship that mimics the spatial relationship of the chemical moieties disposed on the amino acid sidechains which constitute the receptor-binding site of Eril. Preferred chemical moieties, include but are not limited to, the chemical moieties defined by the amino acid side chains of amino acids believed to constitute the receptor-binding domain of Eril. It is understood, therefore, that the receptor- binding surface of the Eril analog need not comprise amino acid residues but the chemical moieties disposed thereon.

For example, upon identification of relevant chemical groups, the skilled artisan using a conventional computer program can design a small molecule having the receptor interactive chemical moieties disposed upon a suitable carrier framework.

Useful computer programs are described in, for example, Dixon (1992) Tibtech 10: 357-363; Tschinke et al. (1993) J. Med. Chem 36: 3863-3870; and Eisen el al. (1994) Proteins: Structure, Function, and Genetics 19: 199-221, the disclosures of which are incorporated herein by reference.

One particular computer program entitled "CAVEAT" searches a database, for example, the Cambridge Structural Database, for structures which have desired spatial orientations of chemical moieties (Bartlett et al. (1989) in "Molecular Recognition: Chemical and Biological Problems" (Roberts, S. M., ed.) pp. 182-196). The CAVEAT program has been used to design analogs of tendamistat, a 74 residue inhibitor of α-amylase, based on the orientation of selected amino acid side chains in the three-dimensional structure of tendamistat (Bartlett et al. (1989) supra). Alternatively, upon identification of a series of analogs which mimic the biological activity of Eril, as determined by in vivo or in vitro assays, the skilled artisan may use a variety of computer programs which assist the skilled artisan to develop quantitative structure activity relationships (QSAR) and further to assist in the de novo design of additional morphogen analogs. Other useful computer programs are described in, for example, Connolly Martin (1991) Methods in Enzymology 203:587-613; Dixon (1992) supra; and Waszkowycz et al. (1994) J. Med. Chenm. 37: 3994-4002.

Thus, for example, one can begin with a portion ofthe three dimensional structure of Eril, corresponding to a region of known or suspected biological importance. One such region may be the Ras binding portion. . Thus, one of ordinary skill in the art, when choosing or designing a Eril analog, can choose or design a molecule having the same or substantially equivalent (e.g., thiol v. hydroxyl) functional groups in substantially the same (e.g., ±1-3 A) three-dimensional conformation.

The molecular framework or backbone of the morphogen analog can be freely chosen by one of ordinary skill in the art so that it (1) joins the functional groups which mimic the portion of the morphogen's contiguous three-dimensional surface, including charge distribution and hydrophobicity/hydrophilicity characteristics, and (2) maintains or, at least, allows the functional groups to maintain the appropriate three-dimensional surface interaction and spatial relationships, including any hydrogen bonding and electrostatic interactions. As described above, peptides are obvious choices for the production of such morphogen analogs because they can provide all of the necessary functional groups and can assume appropriate three- dimensional structures. Biologically active peptides such as F2, F3 or others, then can be used as is or, more preferably, become lead compounds for iterative modification to create a compound that is more stable or more active in vivo. For example, the peptide backbone can be reduced or replaced to reduce hydrolysis in vivo. Alternatively, structural modifications can be introduced to the backbone or by amino acid substitutions that more accurately mimic the protein's structure when bound to the receptor. These second generation structures then can be tested for enhanced binding. In addition, iterative amino acid replacements with alanines, ("alanine scan") can be used to determine the minimum residue contacts required for binding.

Once these minimum functional groups are known, a fully synthetic molecule can be created which mimics the charge or electrostatic distribution of the minimum required functional groups, and provides the appropriate bulk and structure to functionally mimic a second-generation molecule having the desired binding affinity.

In other embodiments, the subject analogs are peptidomimetics of Eril, analog peptides. Peptidomimetics are compounds based on, or derived from, peptides and proteins. The analog peptidomimetics of the present invention typically can be obtained by structural modification of a known analog peptide sequence using unnatural amino acids, conformational restraints, isosteric replacement, and the like. The subject peptidomimetics constitute the continum of structural space between peptides and non-peptide synthetic structures; analog peptidomimetics may be useful, therefore, in delineating pharmacophores and in helping to translate peptides into nonpeptide compounds with the activity ofthe parent analog peptides.

Moreover, as is apparent from the present disclosure, mimetopes of the subject analog peptides can be provided. Such peptidomimetics can have such attributes as being non-hydrolyzable (e.g., increased stability against proteases or other physiological conditions which degrade the corresponding peptide), increased specificity and/or potency, and increased cell permeability for intracellular localization of the peptidomimetic. For illustrative purposes, peptide analogs of the present invention can be generated using, for example, benzodiazepines (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, pl23), C-7 mimics (Huffman et al. in Peptides: Chemistry and Biologyy, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p. 105), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med. Chem 29:295; and Ewenson et al. in Peptides: Structure and Function

(Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), D-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), D-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Communl26:4l9; and Dann et al. (1986) Biochem Biophys Res Commun 134:71), diaminoketones (Natarajan et al. (1984) Biochem Biophys Res Commun 124:141), and methyleneamino-modifed

(Roark et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, pl34). Also, see generally, Session III: Analytic and synthetic methods, in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988)

In addition to a variety of sidechain replacements that can be carried out to generate the subject Eril analog peptidomimetics, the present invention specifically contemplates the use of conformationally restrained mimics of peptide secondary structure. Numerous surrogates have been developed for the amide bond of peptides. Frequently exploited surrogates for the amide bond include the following groups (i) trans-olefins, (ii) fluoroalkene, (iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.

Examples of Surrogates

trans olefin fluoroalkene methyleneamino

phosphonamide sulfonamide

Additionally, peptidomimetics based on more substantial modifications of the backbone ofthe OP-1 analog peptide can be used. Peptidomimetics which fall in this category include (i) retro-inverso analogs, and (ii) N-alkyl glycine analogs (so- called peptoids).

Examples of analogs

retro-inverso N-alkyl glycine

Furthermore, the methods of combinatorial chemistry are being brought to bear, e.g., by G.L. Verdine at Harvard University, on the development of new peptidomimetics. For example, one embodiment of a so-called "peptide morphing" strategy focuses on the random generation of a library of peptide analogs that comprise a wide range of peptide bond substitutes.

dipeptide

peptide morphing

In an exemplary embodiment, the peptidomimetic can be derived as a retro-inverso analog ofthe peptide ,

Retro-inverso analogs can be made according to the methods known in the art, such as that described by the Sisto et al. U.S. Patent 4,522,752. As a general guide, sites which are most susceptible to proteolysis are typically altered, with less susceptible amide linkages being optional for mimetic switching. The final product, or intermediates thereof, can be purified by HPLC.

In another illustrative embodiment, the peptidomimetic can be derived as a retro- enantio analog of a particular analog peptide sequence. Retro-enantio analogs such as this can be synthesized commercially available D-amino acids (or analogs thereof) and standard solid- or solution-phase peptide-synthesis techniques.

In still another illustrative embodiment, trans-olefin derivatives can be made for any of the subject polypeptides. A trans-olefin analog of a BMP analog peptide can be synthesized according to the method of Y.K. Shue et al. (1987) Tetrahedron Letters 28:3225 and also according to other methods known in the art. It will be appreciated that variations in the cited procedure, or other procedures available, may be necessary according to the nature ofthe reagent used.

It is further possible couple the pseudodipeptides synthesized by the above method to other pseudodipeptides, to make peptide analogs with several olefϊnic functionalities in place of amide functionalities. For example, pseudodipeptides corresponding to certain dipeptide sequences could be made and then coupled together by standard techniques to yield an analog ofthe OP-1 analog peptide which has alternating olefinic bonds between residues.

Still another class of peptidomimetic derivatives include phosphonate derivatives.

The synthesis of such phosphonate derivatives can be adapted from known synthesis schemes. See, for example, Loots et al. in Peptides: Chemistry and Biology, (Escom Science Publishers, Leiden, 1988, p. 118); PetriUo et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium, Pierce Chemical Co. Rockland, IL, 1985).

Many other peptidomimetic structures are known in the art and can be readily adapted for use in the subject Eril analog peptidomimetics. To illustrate, the analog peptidomimetic may incorporate the l-azabicyclo[4.3.0]nonane surrogate ( see Kim et al. (1997) J. Org. Chem. .52:2847), or an N-acyl piperazic acid (see Xi et al. (1998) J. Am. Chem. Soc. 120:80), or a 2-substituted piperazine moiety as a constrained amino acid analogue (see Williams et al. (1996) J. Med. Chem. 3.9:1345- 1348). In still other embodiments, certain amino acid residues can be replaced with aryl and biaryl moieties, e.g., monocyclic or bicyclic aromatic or heteroaromatic nucleus, or a biaromatic, aromatic-heteroaromatic, or biheteroaromatic nucleus.

The subject analog peptidomimetics can be optimized by, e.g., combinatorial synthesis techniques combined with such high throughput screening as described above using affinity maturation ofthe library on bladder tumor cells and selection of specific binding moieties by counterscreening using normal bladder cells. Moreover, other examples of mimetopes include, but are not limited to, protein- based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof. A mimetope can be obtained by, for example, screening libraries of natural and synthetic compounds for compounds capable of binding to the Eril binding domain or inhibiting the interaction between the Eril binding domain and the natural ligand. A mimetope can also be obtained, for example, from libraries of natural and synthetic compounds, in particular, chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the same building blocks).

A mimetope can also be obtained by, for example, rational drug design. In a rational drug design procedure, the three-dimensional structure of a compound ofthe present invention can be analyzed by, for example, nuclear magnetic resonance (NMR) or x- ray crystallography. The three-dimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modelling. The predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source (e.g., plants, animals, bacteria and fungi).

Chimeric Eril analog peptides and peptidomimetics: In one aspect, the invention provides chimeric proteins that include one or more analog peptides fused to one or more additional protein domains. In one embodiment, the chimeric protein includes one analog peptide. In other embodiments, the chimeric activator comprises two or more analog peptides, three or more, five or more, or ten or more analog peptides that are covalently linked. When referring to a polypeptide comprising an analog peptide it is meant that the polypeptide comprises the amino acid sequence of a analog peptide covalently linked to other amino acids or peptides to form one polypeptide. The order ofthe analog peptide(s) relative to each other and relative to the other domains ofthe fusion protein can be as desired.

Techniques for making the subject fusion proteins are adapted from well-known procedures. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. Alternatively, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. In another method,

PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments. Amplification products can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).

In certain embodiments, polyanionic or polycatonic binding agents such as oligonucleotides, heparin, lentinan and similar polysaccharide chains, polyamino peptides such as polyaspartate, polyglutamate, polylysine and polyarginine, or other binding agents that maintain a number of either negative or positive charges over their structure at physiological pH's, can be used to specifically bind the subject analog peptides or peptidomimetics. In certain preferred embodiments, a polyanionic component is used, such as heparin, pentosan polysulfate, polyaspartate, polyglutamate, chondroitin sulfate, heparan sulfate, citrate, nephrocalcin, or osteopontin, to name but a few.

Additional domains may be included in the subject fusion proteins of this invention.

For example, the fusion proteins may include domains that facilitate their purification, e.g., "histidine tags" or a glutathione-S-transferase domain. They may include "epitope tags" encoding peptides recognized by known monoclonal antibodies for the detection of proteins within cells or the capture of proteins by antibodies in vitro.

It may be necessary in some instances to introduce an unstructured polypeptide linker region between an analog peptide and other portions of the chimeric protein. The linker can facilitate enhanced flexibility of the fusion protein. The linker can also reduce steric hindrance between any two fragments of the fusion protein. The linker can also facilitate the appropriate folding of each fragment to occur. The linker can be of natural origin, such as a sequence determined to exist in random coil between two domains of a protein. An exemplary linker sequence is the linker found between the C-terminal and N-terminal domains of the RNA polymerase a subunit. Other examples of naturally occurring linkers include linkers found in the lcl and LexA proteins. Alternatively, the linker can be of synthetic origin. For instance, the sequence (Gly Ser)₃ can be used as a synthetic unstructured linker. Linkers of this type are described in Huston et al. (1988) PNAS 85:4879; and U.S. Patent No. 5,091,513.

In some embodiments it is preferable that the design of a linker involve an arrangement of domains which requires the linker to span a relatively short distance, preferably less than about 10 A. However, in certain embodiments, depending, e.g., upon the selected domains and the configuration, the linker may span a distance of up to about 50 A.

Within the linker, the amino acid sequence may be varied based on the preferred characteristics of the linker as determined empirically or as revealed by modeling. For instance, in addition to a desired length, modeling studies may show that side groups of certain amino acids may interfere with the biological activity ofthe fusion protein. Considerations in choosing a linker include flexibility of the linker, charge of the linker, and presence of some amino acids of the linker in the naturally occurring subunits. The linker can also be designed such that residues in the linker contact DNA, thereby influencing binding affinity or specificity, or to interact with other proteins. For example, a linker may contain an amino acid sequence that is recognized by a protease so that the activity of the chimeric protein could be regulated by cleavage. In some cases, particularly when it is necessary to span a longer distance between subunits or when the domains must be held in a particular configuration, the linker may optionally contain an additional folded domain.

In one embodiment of the agent described herein, the agent is an antibody or fragment thereof.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all ofthe amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. patents 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and

5,859,205, which are hereby incorporated by reference. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al, J. Mol. Biol. 294:151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci.

See, e.g., U.S. patents 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization ofthese mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.

In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. patents 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. patents 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')₂, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')₂ fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non- human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non- human sequences. The present invention also includes so-called single chain antibodies.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.

Monoclonal antibodies may be produced by mammalian cell culture in hydridoma or recombinant cell lines such as Chinese hamster ovary cells or murine myeloma cell lines. Such methods are well-known to those skilled in the art. Bacterial, yeast, and insect cell lines can also be used to produce monoclonal antibodies or fragments thereof. In addition, methods exist to produce monoclonal antibodies in transgenic animals or plants (Pollock et al., J. Immunol. Methods, 231:147, 1999; Russell, Curr.Top. Microbiol. Immunol. 240:119, 1999).

In one embodiment of the agents described herein, the agent is an antibody or portion of an antibody. As used herein, "antibody" means an immunoglobulin molecule comprising two heavy chains and two light chains and which recognizes an antigen. The immunoglobulin molecule may derive from any of the commonly

Icnown classes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. It includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. Optionally, an antibody can be labeled with a detectable marker. Detectable markers include, for example, radioactive or fluorescent markers. The antibody may be a human or nonhuman antibody. The nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man. Methods for humanizing antibodies are known to those skilled in the art. As used herein, "monoclonal antibody," also designated as mAb, is used to describe antibody molecules whose primary sequences are essentially identical and which exhibit the same antigenic specificity. Monoclonal antibodies may be produced by hybridoma, recombinant, transgenic or other techniques known to one skilled in the art. The term "antibody" includes, but is not limited to, both naturally occurring and non-naturally occurring antibodies. Specifically, the term "antibody" includes polyclonal and monoclonal antibodies, and antigen-binding fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies, wholly synthetic antibodies, and antigen-binding fragments thereof. Accordingly, in one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a polyclonal antibody. In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody is a chimeric antibody. Such chimeric antibodies may comprise a portion of an antibody from one source and a portion of an antibody from another source.

In one embodiment, the portion of the antibody comprises a light chain of the antibody. As used herein, "light chain" means the smaller polypeptide of an antibody molecule composed of one variable domain (VL) and one constant domain (CL), or fragments thereof. In one embodiment, the portion of the antibody comprises a heavy chain ofthe antibody. As used herein, "heavy chain" means the larger polypeptide of an antibody molecule composed of one variable domain (VH) and three or four constant domains (CHI, CH2, CH3, and CH4), or fragments thereof. In one embodiment, the portion ofthe antibody comprises a Fab portion of the antibody. As used herein, "Fab" means a monovalent antigen binding fragment of an immunoglobulin that consists of one light chain and part of a heavy chain. It can be obtained by brief papain digestion or by recombinant methods. In one embodiment, the portion ofthe antibody comprises a F(ab')₂ portion ofthe antibody.

As used herein, "F(ab')2 fragment" means a bivalent antigen binding fragment of an immunoglobulin that consists of both light chains and part of both heavy chains. It cen be obtained by brief pepsin digestion or recombinant methods. In one embodiment, the portion of the antibody comprises a Fd portion ofthe antibody. In one embodiment, the portion ofthe antibody comprises a Fv portion ofthe antibody.

In one embodiment, the portion of the antibody comprises a variable domain of the antibody. In one embodiment, the portion of the antibody comprises a constant domain of the antibody. In one embodiment, the portion of the antibody comprises one or more CDR domains of the antibody. As used herein, "CDR" or "complementarity determining region" means a highly variable sequence of amino acids in the variable domain of an antibody.

This invention provides humanized forms of the antibodies described herein. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. In one embodiment of the humanized forms ofthe antibodies, some, most or all ofthe amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody would retain a similar antigenic specificity as the original antibody.

One skilled in the art would know how to make the humanized antibodies of the subject invention. Various publications, several of which are hereby incorporated by reference into this application, also describe how to make humanized antibodies. For example, the methods described in United States Patent No. 4,816,567 comprise the production of chimeric antibodies having a variable region of one antibody and a constant region of another antibody.

United States Patent No. 5,225,539 describes another approach for the production of a humanized antibody. This patent describes the use of recombinant DNA technology to produce a humanized antibody wherein the CDRs of a variable region of one immunoglobulin are replaced with the CDRs from an immunoglobulin with a different specificity such that the humanized antibody would recognize the desired target but would not be recognized in a significant way by the human subject's immune system. Specifically, site directed mutagenesis is used to graft the CDRs onto the framework. Other approaches for humanizing an antibody are described in United States Patent

Nos. 5,585,089 and 5,693,761 and WO 90/07861 which describe methods for producing humanized immunoglobulins. These have one or more CDRs and possible additional amino acids from a donor immunoglobulin and a framework region from an accepting human immunoglobulin. These patents describe a method to increase the affinity of an antibody for the desired antigen. Some amino acids in the framework are chosen to be the same as the amino acids at those positions in the donor rather than in the acceptor. Specifically, these patents describe the preparation of a humanized antibody that binds to a receptor by combining the CDRs of a mouse monoclonal antibody with human immunoglobulin framework and constant regions.

Human framework regions can be chosen to maximize homology with the mouse sequence. A computer model can be used to identify amino acids in the framework region which are likely to interact with the CDRs or the specific antigen and then mouse amino acids can be used at these positions to create the humanized antibody.

The above patents 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 D ofthe CDRs in a three dimensional model ofthe antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some ofthe methods that one skilled in the art could employ to make humanized antibodies. This invention provides a nucleic acid which encodes any of the agents described herein.

In one embodiment of the nucleic acid, which nucleic acid comprises nucleotides, the sequence of which nucleotides is set forth in SEQ ID NO: 6 (full length ERIl

DNA sequence)

This invention provides a polypeptide encoded by any ofthe nucleic acids described herein.

In one embodiment ofthe nucleic acid, the nucleic acid encodes an agent which is a polypeptide or portion thereof, which polypeptide comprises amino acids, the sequence of which amino acids is set forth in SEQ ID NO: 7 (full length ERIl amino acid sequence). In one embodiment, the agent is a portion ofthe polypeptide, and the portion is a GTP-Ras binding motif.

In one embodiment ofthe nucleic acid, the nucleic acid encodes an agent which is a fusion protein which comprises (1) a peptide which comprises at least one GTP-Ras binding motif, and (2) a transcellular polypeptide for promoting transcytosis of the fusion protein across a cell surface membrane.

In one embodiment of the peptide which comprises at least one GTP-Ras binding motif, the peptide corresponds to at least a portion of the sequence set forth in SEQ ID NO: 7 (full length ERIl amino acid sequence). In one embodiment, the portion is a GTP-Ras binding motif.

In one embodiment of the transcellular polypeptide, the transcellular polypeptide is an internalizing peptide. The internalizing peptides include but are not limited to those derived from a polypeptide selected from the group consisting of antepennepedia protein, HIV transactivating (TAT) protein, mastoparan, melittin, bombolittin, delta hemolysin, pardaxin, Pseudomonas exotoxin A, clathrin, Diphtheria toxin and C9 complement protein. In one embodiment, the internalizing peptide is derived from antepennepedia protein. In one embodiment, the internalizing peptide is derived from HIV transactivating (TAT) protein. In another, it is derived from HSV vP22.

In one embodiment ofthe transcellular polypeptide, the transcellular polypeptide is an accessory peptide sequence which enhances interaction of the fusion protein with a cell surface membrane. In one embodiment, the accessory peptide includes an RGD sequence.

The nucleic acids described herein include but are not limited to DNA, RNA, cDNA and genomic DNA. In one embodiment, the nucleic acid comprises nucleotides, the sequence of which nucleotides is set forth in SEQ ID NO 6 (full length ERIl DNA sequence).

This invention provides the nucleic acids described herein, wherein the nucleic acids may be altered by the insertion, deletion and/or substitution of one or more nucleotides, which could result in an alteration of the nucleic acid sequence. In one embodiment, the nucleotide changes do not result in a mutation at the amino acid level. One embodiment, the nucleotide change may result in an amino acid change.

Such amino acid change could be one which does not affect the protein's function.

This invention provides nucleic acids capable of hybridizing to the described nucleic acids. This invention provides nucleic acids which are complementary to the described nucleic acids. This invention also provides fragments of the described nucleic acids and polypeptides. This invention provides a vector which comprises any of the nucleic acids described herein. In one embodiment, the vector is a replicable vector. The vectors induce but are not limited to plasmids, cosmids, λ phages and YACs. This invention provides a cell which comprises any ofthe vectors described herein. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a prokaryotic cell. This invention provides a host-vector system for the production of a polypeptide which comprises any of the vectors described herein and a suitable host cell. This invention provides a method for producing a polypeptide which comprises growing a host vector system described herein under suitable conditions and recovering the polypeptide so produced. As used herein, the agents and/or compounds described herein may be admixed with a carrier.

For certain of the therapeutic uses of the subject chimeric proteins, particularly cutaneous uses such as for the control of keratinocyte proliferation, direct administration of the protein will be appropriate (rather than use of a gene therapy construct). Accordingly, the subject CKI polypeptide formulations may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. In preferred embodiments, the chimeric protein is dispersed in lipid formulations, such as miscelles, which closely resemble the lipid composition of natural cell membranes to which the chimeric protein is to be delivered.

The optimum concentration ofthe active ingredient(s) in the chosen medium can be determined empirically, according to procedures well Icnown to medicinal chemists. As used herein, "biologically acceptable medium" includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the CKI polypeptide (or internalization peptide or endothelialization polypeptide as the case may be), its use in the pharmaceutical preparation ofthe invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's

Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985).

In an exemplary embodiment, the subject CKI protein mixture is provided for transmucosal or transdermal delivery. For such administration, penetrants appropriate to the barrier to be permeated are used in the formulation with the polypeptide. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the proteins of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.

In accordance with the subject method, expression constructs of the subject CKI polypeptides (and endothelialization polypeptide as appropriate) may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively transfecting cells in vivo with a recombinant fusion gene. Approaches include insertion ofthe subject fusion gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection ofthe gene construct or CaPO4 precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g. locally or systemically.

A preferred approach for in vivo introduction of nucleic acid encoding one of the subject CKI polypeptides into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the gene product. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA ofthe host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding a CKI polypeptide, rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology. Ausubel, F.M. et al. (eds.) Greene

Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ψCrip, ψCre, ψ2 and ψAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc.

Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

In choosing retroviral vectors as a gene delivery system for the subject fusion proteins, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction of the recombinant gene, is that the target cells must be dividing. In general, this requirement will not be a hindrance to use of retroviral vectors to deliver the subject fusion gene constructs. In fact, such limitation on infection can be beneficial in circumstances where the tissue (e.g. nontransformed cells) surrounding the target cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors.

Furthermore, it has been shown that it is possible to limit the infection spectrum of refroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol

73:3251-3255; and Goud et al. (1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins (Neda et al. (1991) J Biol Chem 266:14143- 14146). Coupling can be in the form ofthe chemical cross-linking with a protein or other variety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single-chain antibody/env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression ofthe fusion gene ofthe retroviral vector.

Another viral gene delivery system useful in the present invention utilitizes adeno virus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivate in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berlcner et al. (1988)

BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3,

Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482- 6486), and smooth muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity ofthe adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts ofthe viral El and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology, E.J. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127). Expression ofthe inserted fusion gene can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.

Yet another viral vector system useful for delivery ofthe subject CKI polypeptides is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). It is also one ofthe few viruses that may integrate its DNA into non- dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790). Other viral vector systems that may have application in gene therapy have been derived from herpes virus, vaccinia virus, and several RNA viruses. In particular, heφes virus vectors may provide a unique strategy for persistent expression ofthe subject fusion proteins in cells of the central nervous system and ocular tissue (Pepose et al. (1994) Invest Ophthalmol Vis Sci 35:2662-2666)

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a the subject proteins in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems ofthe present invention rely on endocytic pathways for the uptake of the gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.

In a representative embodiment, a gene encoding one ofthe subject proteins can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075). For example, lipofection of neuroglioma cells can be carried out using liposomes tagged with monoclonal antibodies against glioma- associated antigen (Mizuno et al. (1992) Neurol. Med. Chir. 32:873-876).

In yet another illustrative embodiment, the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene bindmg agent such as poly-lysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180). For example, the subject gene construct can be used to transfect hepatocytic cells in vivo using a soluble polynucleotide carrier comprising an asialoglycoprotein conjugated to a polycation, e.g. poly-lysine (see U.S. Patent 5,166,320). It will also be appreciated that effective delivery of the subject nucleic acid constructs via receptor-mediated endocytosis can be improved using agents which enhance escape ofthe gene from the endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part ofthe delivery system to induce efficient disruption of DNA-containing endosomes (Mulligan et al. (1993) Science 260-926; Wagner et al. (1992) PNAS 89:7934; and Christiano et al. (1993) PNAS 90:2122).

In clinical settings, the gene delivery systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof. In other embodiments, initial delivery ofthe recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g. Chen et al. (1994) PNAS 91 : 3054-3057).

Moreover, the pharmaceutical preparation can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced in tact from recombinant cells, e.g. retroviral packages, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system. In the case ofthe latter, methods of introducing the viral packaging cells may be provided by, for example, rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals, and can be adapted for release of viral particles through the manipulation ofthe polymer composition and form. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of an the viral particles by cells implanted at a particular target site. Such embodiments of the present invention can be used for the delivery of an exogenously purified virus, which has been incorporated in the polymeric device, or for the delivery of viral particles produced by a cell encapsulated in the polymeric device. By choice of monomer composition or polymerization technique, the amount of water, porosity and consequent permeability characteristics can be controlled. The selection of the shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder to be treated and the individual patient response. The generation of such implants is generally known in the art. See, for example, Concise Encyclopedia of Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, MA, 1990); and the Sabel et al. U.S. Patent No. 4,883,666. In another embodiment of an implant, a source of cells producing the recombinant virus is encapsulated in implantable hollow fibers. Such fibers can be pre-spun and subsequently loaded with the viral source (Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et al. U.S. Patent No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res. 82:41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183), or can be co- extruded with a polymer which acts to form a polymeric coat about the viral packaging cells (Lim U.S. Patent No. 4,391,909; Sefton U.S. Patent No. 4,353,888;

Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs 35:791-799; Sefton et al. (1987) Biotechnol. Bioeng. 29:1135-1143; and Aebischer et al. (1991) Biomaterials 12:50-55). Again, manipulation of the polymer can be carried out to provide for optimal release of viral particles.

This invention provides a method of treating a subject afflicted with cancer which comprises administering to the subject an amount of an agent described herein effective to treat the subject.

These cancers include but are not limited to the following: prostate cancer; biliary tract cancer; brain cancer, including glioblastomas and medelloblastomes; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms, including acute lymphocytic and myelogenous leukemia, multiple myeloma, AIDS associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowens' disease and Paget's disease; liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphozytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma, basocellular cancer and squamous cell cancer; testicular cancer, including terminal tumors (seminoma, non- seminoma (teratomas, choriocarcinomas)), stromal tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor.

As used herein, "treating" means either slowing, stopping or reversing the progression ofthe disorder. In the preferred embodiment, "treating" means reversing the progression to the point of eliminating the disorder.

This invention provides a method of preventing a subject from becoming afflicted with cancer which comprises administering to the subject an amount of an agent described herein effective to prevent the subject from becoming afflicted with cancer.

This invention provides a method of inhibiting growth of a cancer cell which comprises contacting the cancer cell with an amount of an agent described herein effective to inhibit growth ofthe cancer cell.

As used herein, "inhibits" means that the amount is reduced as compared with the amount that would occur in a control sample. In a preferred embodiment, inhibits means that the amount is reduced 100%.

This invention provides a method of inhibiting GTP-Ras signaling which comprises contacting a GTP-Ras containing cell with an amount of an agent described herein effective to inhibit GTP-Ras signaling.

This invention provides a method of inliibiting GTP-Ras from interacting with its effector which comprises contacting a GTP-Ras containing cell with an amount of an agent described herein effective to inhibit GTP-Ras from interacting with its effector.

In one embodiment, the effector is Raf. Raf is a protein kinase that transmits signal from Ras to the ERK MAP kinase cascade. In one embodiment, the effector is phosphatidylinositol-3-kinase (PI3K), whose catalytic activity produces 3- phospoinositides that activate the Akt protein kinase, which has been implicated in the negative control of apoptosis. In one embodiment, the effector is Ral-GEF, which is a nucleotide exchange protein that activates Ral GTPase.

In one embodiment of the methods described herein, the cell is present in a subject and the contacting is effected by administering the agent to the subject.

In one embodiment of the methods described herein, the agent is administered orally, intravenously, subcutaneously, intramuscularly, topically or by liposome- mediated delivery.

In one embodiment ofthe methods described herein, the subject is a human being, a primate, an equine, an opine, an avian, a bovine, a porcine, a canine, a feline or a murine subject.

In one embodiment of the methods described herein, the effective amount of the agent is between about lmg and about 50mg per kg body weight of the subject. In one embodiment, the effective amount ofthe agent is between about 2mg and about 40mg per kg body weight ofthe subject. In one embodiment, the effective amount of the agent is between about 3mg and about 30mg per kg body weight ofthe subject. In one embodiment, the effective amount of the agent is between about 4mg and about 20mg per kg body weight of the subject. In one embodiment, the effective amount of the agent is between about 5mg and about lOmg per kg body weight of the subject. In one embodiment of the methods described herein, the agent is administered at least once per day. In one embodiment, the agent is administered daily. In one embodiment, the agent is administered every other day. In one embodiment, the agent is administered every 6 to 8 days. In one embodiment, the agent is administered weekly.

As for the amount of the compound and/or agent for administration to the subject, one skilled in the art would know how to determine the appropriate amount. As used herein, a dose or amount would be one in sufficient quantities to either inhibit the disorder, treat the disorder, treat the subject or prevent the subject from becoming afflicted with the disorder. This amount may be considered an effective amount. A person of ordinary skill in the art can perform simple titration experiments to determine what amount is required to treat the subject. The dose of the composition of the invention will vary depending on the subject and upon the particular route of administration used. In one embodiment, the dosage can range from about 0.1 to about 100,000 Dg/kg body weight ofthe subject. Based upon the composition, the dose can be delivered continuously, such as by continuous pump, or at periodic intervals. For example, on one or more separate occasions. Desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by one skilled in the art.

The effective amount may be based upon, among other things, the size of the compound, the biodegradability of the compound, the bioactivity of the compound and the bioavailability ofthe compound. If the compound does not degrade quickly, is bioavailable and highly active, a smaller amount will be required to be effective.

The effective amount will be known to one of skill in the art; it will also be dependent upon the form of the compound, the size of the compound and the bioactivity of the compound. One of skill in the art could routinely perform empirical activity tests for a compound to determine the bioactivity in bioassays and thus determine the effective amount. In one embodiment ofthe above methods, the effective amount of the compound comprises from about 1.0 ng/kg to about 100 mg/kg body weight ofthe subject. In another embodiment ofthe above methods, the effective amount of the compound comprises from about 100 ng/kg to about 50 mg/kg body weight ofthe subject. In another embodiment ofthe above methods, the effective amount ofthe compound comprises from about 1 Dg/kg to about 10 mg/kg body weight of the subject. In another embodiment of the above methods, the effective amount of the compound comprises from about 100 Dg/kg to about 1 mg/kg body weight of the subject.

As for when the compound and/or agent is to be administered, one skilled in the art can determine when to administer such compound and/or agent. The administration may be constant for a certain period of time or periodic and at specific intervals. The compound may be delivered hourly, daily, weekly, monthly, yearly (e.g. in a time release form) or as a one time delivery. The delivery may be continuous delivery for a period of time, e.g. intravenous delivery. In one embodiment of the methods described herein, the agent is administered at least once per day. In one embodiment ofthe methods described herein, the agent is administered daily. In one embodiment of the methods described herein, the agent is administered every other day. In one embodiment ofthe methods described herein, the agent is administered every 6 to 8 days. In one embodiment ofthe methods described herein, the agent is administered weekly.

This invention provides a composition which comprises a carrier and an agent or compound described herein.

This invention provides the agents, compounds and/or compositions described herein and carrier. Such carrier may be a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known to those skilled in the art. Such pharmaceutically acceptable carriers may include but are not limited to aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

As used herein, "composition" may mean a mixture. The compositions include but are not limited to those suitable for oral, rectal, intravaginal, topical, nasal, opthalmic, or parenteral administration to a subject. As used herein, "parenteral" includes but is not limited to subcutaneous, intravenous, intramuscular, or intrasternal injections or infusion techniques. As used herein, "administering" may be effected or performed using any of the methods Icnown to one skilled in the art. The methods for administration to the subject include but are not limited to oral, rectal, intravaginal, topical, nasal, opthalmic, parenteral subcutaneous, intravenous, intramuscular, or intrasternal injections or infusion techniques.

As used herein, "subject" means any animal or artificially modified animal capable of becoming afflicted with the disorder. The subjects include but are mot limited to a human being, a primate, an equine, an opine, an avian, a bovine, a porcine, a canine, a feline or a mouse. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. The animals include but are not limited to mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates. In the preferred embodiment, the subject is a human being.

As used herein, "administering" may be effected or performed using any of the methods Icnown to one skilled in the art. The compound may be administered by various routes including but not limited to aerosol, intravenous, oral or topical route. The administration may comprise intralesional, intraperitoneal, subcutaneous, intramuscular or intravenous injection; infusion; liposome-mediated delivery; topical, intrathecal, gingival pocket, per rectum, intrabronchial, nasal, transmucosal, intestinal, oral, ocular or otic delivery. In a further embodiment, the administration includes intrabronchial administration, anal, intrathecal administration or transdermal delivery. The compounds and/or agents ofthe subject invention may be delivered locally via a capsule which allows sustained release of the agent or the peptide over a period of time. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines) and the agent coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors. Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.

The carrier may be a diluent, an aerosol, a topical carrier, an aqueous solution, a nonaqueous solution or a solid carrier.

This invention provides a recombinant transfection system, comprising

(i) a first gene construct comprising a coding sequence encoding a inhibitory polypeptide comprising at least one GTP-Ras-binding motif for binding and inhibiting Ras, which coding sequence is operably linked to a transcriptional regulatory sequence for causing expression ofthe first polypeptide in eukaryotic cells; and

(ii) a gene delivery composition for delivering the gene construct to a cell and causing the cell to be transfected with the gene construct.

In one embodiment ofthe recombinant transfection system described herein, the recombinant transfection system further comprises a second gene construct comprising a coding sequence encoding a transcellular polypeptide which promotes transcytosis across a cell surface membrane, wherein the gene delivery composition also delivers the second gene construct to the cell.

In one embodiment ofthe recombinant transfection system described herein, the gene construct is provided in a vector.

In one embodiment ofthe recombinant transfection system described herein, the first and second gene constructs are provided as part of a single vector. In one embodiment ofthe recombinant transfection system described herein, the first and second gene constructs are provided in a polycistronic message.

In one embodiment ofthe recombinant transfection system described herein, the first and second gene constructs are provided in separate vectors.

In one embodiment ofthe recombinant transfection system described herein, the vector is a viral vector. The viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

In one embodiment ofthe recombinant transfection system described herein, the gene delivery composition comprises a recombinant viral particle.

In one embodiment ofthe recombinant transfection system described herein, the gene delivery composition is selected from the group consisting of a liposome and a poly-cationic nucleic acid binding agent.

In one embodiment ofthe recombinant transfection system described herein, the gene delivery composition further comprises a pharmaceutically acceptable carrier for administration to an subject.

In one embodiment ofthe recombinant transfection system described herein, the subject is a human being, a primate, an equine, an opine, an avian, a bovine, a porcine, a canine, a feline or a murine subject.

This invention provides a method for treating a subject afflicted with cancer which comprises administering to the subject a recombinant transfection system described herein.

As mentioned above, the Eril analogs of the invention may comprise modified proteins or small molecules, for example, peptides or small organic molecules. It is contemplated that any appropriate methods can be used for producing a preselected morphogen analog. For example, such methods may include, but are not limited to, methods of biological production from suitable host cells or synthetic production using synthetic organic chemistries. For example, modified Eril proteins or Eril -based peptides may be produced using conventional recombinant DNA technologies, well known and thoroughly documented in the art. Under these circumstances, the proteins or peptides may be produced by the preparation of nucleic acid sequences encoding the respective protein or peptide sequences, after which, the resulting nucleic acid can be expressed in an appropriate host cell. By way of example, the proteins and peptides may be manufactured by the assembly of synthetic nucleotide sequences and/or joining DNA restriction fragments to produce a synthetic DNA molecule. The DNA molecules then are ligated into an expression vehicle, for example an expression plasmid, and transfected into an appropriate host cell, for example E. coli. The protein encoded by the DNA molecule then is expressed, purified, folded if necessary, tested in vitro for binding activity with a Eril receptor, and subsequently tested to assess whether the morphogen analog induces or stimulates Eril -like biological activity.

The processes for manipulating, amplifying, and recombining DNA which encode amino acid sequences of interest generally are well known in the art, and therefore, are not described in detail herein. Methods of identifying and isolating genes encoding Eril and its cognate receptors also are well understood, and are described in the patent and other literature.

Briefly, the construction of DNAs encoding the biosynthetic constructs disclosed herein is performed using known techniques involving the use of various restriction enzymes which make sequence specific cuts in DNA to produce blunt ends or cohesive ends, DNA ligases, techniques enabling enzymatic addition of sticky ends to blunt-ended DNA, construction of synthetic DNAs by assembly of short or medium length oligonucleotides, cDNA synthesis techniques, polymerase chain reaction (PCR) techniques for amplifying appropriate nucleic acid sequences from libraries, and synthetic probes for isolating Eril genes as well as their cognate receptors. Various promoter sequences from bacteria, mammals, or insects to name a few, and other regulatory DNA sequences used in achieving expression, and various types of host cells are also known and available. Conventional transfection techniques, and equally conventional techniques for cloning and subcloning DNA are useful in the practice of this invention and known to those skilled in the art. Various types of vectors may be used such as plasmids and viruses including animal viruses and bacteriophages. The vectors may exploit various marker genes that impart to a successfully transfected cell a detectable phenotypic property that can be used to identify which of a family of clones has successfully incorporated the recombinant DNA ofthe vector.

One method for obtaining DNA encoding the biosynthetic constructs disclosed herein is by assembly of synthetic oligonucleotides produced in a conventional, automated, oligonucleotide synthesizer followed by ligation with appropriate ligases. For example, overlapping, complementary DNA fragments may be synthesized using phosphoramidite chemistry, with end segments left unphosphorylated to prevent polymerization during ligation. One end of the synthetic DNA is left with a "sticky end" corresponding to the site of action of a particular restriction endonuclease, and the other end is left with an end corresponding to the site of action of another restriction endonuclease. The complementary DNA fragments are ligated together to produce a synthetic DNA construct.

After the appropriate DNA molecule has been synthesized, it may be integrated into an expression vector and transfected into an appropriate host cell for protein expression. Useful prokaryotic host cells include, but are not limited to, E. coli and B. subtilis. Useful eukaryotic host cells include, but are not limited to, yeast cells, insect cells, myeloma cells, fibroblast 3T3 cells, monkey kidney or COS cells, Chinese hamster ovary (CHO) cells, mink-lung epithelial cells, human foreskin fibroblast cells, human glioblastoma cells, and teratocarcinoma cells. Alternatively, the genes may be expressed in a cell-free system such as the rabbit reticulocyte lysate system.

The vector additionally may include various sequences to promote correct expression of the recombinant protein, including transcriptional promoter and termination sequences, enhancer sequences, preferred ribosome binding site sequences, preferred mRNA leader sequences, preferred protein processing sequences, preferred signal sequences for protein secretion, and the like. The DNA sequence encoding the gene of interest also may be manipulated to remove potentially inhibiting sequences or to minimize unwanted secondary structure formation. The morphogenic protein analogs proteins also may be expressed as fusion proteins. After being translated, the protein may be purified from the cells themselves or recovered from the culture medium and then cleaved at a specific protease site if so desired.

For example, if the gene is to be expressed in E. coli, it is cloned into an appropriate expression vector. This can be accomplished by positioning the engineered gene downstream of a promoter sequence such as Trp or Tac, and/or a gene coding for a leader peptide such as fragment B of protein A (FB). During expression, the resulting fusion proteins accumulate in refractile bodies in the cytoplasm ofthe cells, and may be harvested after disruption ofthe cells by French press or sonication. The isolated refractile bodies then are solubilized, and the expressed proteins folded and the leader sequence cleaved, if necessary, by methods already established with many other recombinant proteins.

Expression of the engineered genes in eukaryotic cells requires cells and cell lines that are easy to transfect, are capable of stably maintaining foreign DNA with an unrearranged sequence, and which have the necessary cellular components for efficient transcription, translation, post-translation modification, and secretion ofthe protein. In addition, a suitable vector carrying the gene of interest also is necessary.

DNA vector design for transfection into mammalian cells should include appropriate sequences to promote expression of the gene of interest as described herein, including appropriate transcription initiation, termination, and enhancer sequences, as well as sequences that enhance translation efficiency, such as the Kozak consensus sequence. Preferred DNA vectors also include a marker gene and means for amplifying the copy number ofthe gene of interest. A detailed review ofthe state ofthe art ofthe production of foreign proteins in mammalian cells, including useful cells, protein expression-promoting sequences, marker genes, and gene amplification methods, is disclosed in Bendig (1988) Genetic Engineering 7:91-127.

The best characterized transcription promoters useful for expressing a foreign gene in a particular mammalian cell are the SV40 early promoter, the adenovirus promoter (AdMLP), the mouse metallothionein-I promoter (mMT-I), the Rous sarcoma virus (RSV) long tenninal repeat (LTR), the mouse mammary tumor virus long terminal repeat (MMTV-LTR), and the human cytomegalovirus major intermediate-early promoter (hCMV). The DNA sequences for all of these promoters are known in the art and are available commercially.

The use of a selectable DHFR gene in a dhfr^" cell line is a well characterized method useful in the amplification of genes in mammalian cell systems. Briefly, the DHFR gene is provided on the vector carrying the gene of interest, and addition of increasing concentrations of the cytotoxic drug methotrexate, which is metabolized by DHFR, leads to amplification of the DHFR gene copy number, as well as that of the associated gene of interest. DHFR as a selectable, amplifiable marker gene in transfected Chinese hamster ovary cell lines (CHO cells) is particularly well characterized in the art. Other useful amplifiable marker genes include the adenosine deaminase (ADA) and glutamine synthetase (GS) genes.

The choice of cells/cell lines is also important and depends on the needs of the experimenter. COS cells provide high levels of transient gene expression, providing a useful means for rapidly screening the biosynthetic constructs of the invention. COS cells typically are transfected with a simian virus 40 (SV40) vector carrying the gene of interest. The transfected COS cells eventually die, thus preventing the long term production of the desired protein product but provide a useful technique for testing preliminary analogs for binding activity.

The various cells, cell lines and DNA sequences that can be used for mammalian cell expression ofthe single-chain constructs ofthe invention are well characterized in the art and are readily available. Other promoters, selectable markers, gene amplification methods and cells also may be used to express the proteins of this invention. Particular details of the transfection, expression, and purification of recombinant proteins are well documented in the art and are understood by those having ordinary skill in the art. Further details on the various technical aspects of each ofthe steps used in recombinant production of foreign genes in mammalian cell expression systems can be found in a number of texts and laboratory manuals in the art, such as, for example, Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, (1989).

In addition, vectors suitable for mammalian cell expression can be used in gene therapy treatment protocols, whereby the peptide is produced in the cells of the patient being treated. In yet other embodiments, the subject expression constructs are derived by insertion of the subject gene into viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. As described in greater detail below, such embodiments ofthe subject expression constructs are specifically contemplated for use in various in vivo and ex vivo gene therapy protocols.

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA ofthe host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility ofthe spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding a fusion protein of the present invention, rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis et al., (1985) Science 230:1395-1398; Danos and Mulligan, (1988) PNAS USA 85:6460-6464; Wilson et al., (1988) PNAS USA 85:3014-3018; Armentano et al, (1990) PNAS USA 87:6141-6145; Huber et al., (1991) PNAS USA 88:8039-8043; Ferry et al., (1991) PNAS USA 88:8377- 8381; Chowdhury et al., (1991) Science 254:1802-1805; van Beusechem et al., (1992) PNAS USA 89:7640-7644; Kay et al., (1992) Human Gene Therapy 3:641- 647; Dai et al., (1992) PNAS USA 89:10892-10895; Hwu et al., (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT

Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification ofthe infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al., (1989) PNAS USA 86:9079-9083; Julan et al., (1992) J. Gen Virol 73:3251- 3255; and Goud et al., (1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins (Neda et al., (1991) J. Biol. Chem. 266:14143- 14146). Coupling can be in the form ofthe chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g., single-chain antibody/env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.

Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivate in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al., (1988) BioTechniques 6:616; Rosenfeld et al., (1991) Science 252:431-434; and Rosenfeld et al., (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d!324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including aiiway epithelium (Rosenfeld et al., (1992) cited supra), endothelial cells (Lemarchand et al, (1992) PNAS USA 89:6482-6486), hepatocytes (Herz and Gerard, (1993) PNAS USA 90:2812-2816) and muscle cells (Quantin et al., (1992) PNAS USA 89:2581-2584). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts ofthe viral El and E3 genes but retain as much as 80% ofthe adenoviral genetic material (see, e.g., Jones et al., (1979) Cell 16:683; Berkner et al., supra; and Graham et al., in Methods in Molecular Biology, E.J. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109- 127). Expression ofthe inserted chimeric gene can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the viral E3 promoter, or exogenously added promoter sequences.

Yet another viral vector system useful for delivery of the subject chimeric genes is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review, see Muzyczka et al., Curr. Topics in Micro, and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al., (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al, (1989) J. Virol. 63:3822- 3828; and McLaughlin et al., (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in

Tratschin et al., (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., (1984) PNAS USA 81:6466- 6470; Tratschin et al., (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol. 2:32-39; Tratschin et al., (1984) J. Virol. 51:611-619; and

Flotte et al, (1993) J. Biol. Chem. 268:3781-3790).

Other viral vector systems that may have application in gene therapy have been derived from herpes virus, vaccinia virus, and several RNA viruses. In particular, herpes virus vectors may provide a unique strategy for persistence of the recombinant gene in cells ofthe central nervous system and ocular tissue (Pepose et al., (1994) Invest Ophthalmol Vis Sci 35:2662-2666)

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a protein in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly lysine conjugates, and artificial viral envelopes.

In a representative embodiment, a gene encoding a polypeptide can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al., (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075). For example, lipofection of neuroglioma cells can be carried out using liposomes tagged with monoclonal antibodies against glioma- associated antigen (Mizuno et al, (1992) Neurol. Med. Chir. 32:873-876).

In yet another illustrative embodiment, the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as poly lysine (see, for example, PCT publications WO93/04701, W092/22635,

WO92/20316, W092/19749, and WO92/06180). For example, any of the subject gene constructs can be used to transfect specific cells in vivo using a soluble polynucleotide carrier comprising an antibody conjugated to a polycation, e.g., poly lysine (see U.S. Patent 5,166,320). It will also be appreciated that effective delivery of the subject nucleic acid constructs via -mediated endocytosis can be improved using agents which enhance escape of the gene from the endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA- containing endosomes (Mulligan et al., (1993) Science 260-926; Wagner et al., (1992) PNAS USA 89:7934; and Christiano et al., (1993) PNAS USA 90:2122).

In clinical settings, the gene delivery systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the construct in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof. In other embodiments, initial delivery ofthe recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g., Chen et al., (1994) PNAS USA 91: 3054-3057).

Alternatively, morphogen analogs which are small peptides, usually up to 50 amino acids in length, may be synthesized using standard solid-phase peptide synthesis procedures, for example, procedures similar to those described in Merrifield (1963) J. Am. Chem. Soc, 85:2149. For example, during synthesis, N-α-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal end to an insoluble polymeric support, e.g., polystyrene beads. The peptides are synthesized by linking an amino group of an N- α-deprotected amino acid to an α-carboxy group of an N-α-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation. Commonly used N-α-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.

Briefly, the C-terminal N-α-protected amino acid is first attached to the polystyrene beads. Then, the N-α-protecting group is removed. The deprotected α-amino group is coupled to the activated α-carboxylate group of the next N-α-protected amino acid. The process is repeated until the desired peptide is synthesized. The resulting peptides are cleaved from the insoluble polymer support and the amino acid side chains deprotected. Longer peptides, for example greater than about 50 amino acids in length, typically are derived by condensation of protected peptide fragments. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art and so are not discussed in detail herein. See for example, Atherton et al. (1963) Solid Phase Peptide Synthesis: A Practical Approach (IRL Press,), and Bodanszlcy (1993) Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer- Verlag, and Fields et al. (1990) Int. J. Peptide Protein Res. 35 : 161 -214, the disclosures of which are incorporated herein by reference.

Purification ofthe resulting peptide is accomplished using conventional procedures, such as preparative HPLC, e.g., gel permeation, partition and/or ion exchange chromatography. The choice of appropriate matrices and buffers are well Icnown in the art and so are not described in detail herein.

With regard to the production of non-peptide small organic molecules that induce

Eril like biological activities, these molecules can be synthesized using standard organic chemistries well known and thoroughly documented in the patent and other literatures.

This invention provides Eril proteins, or functional equivalents thereof, for use in the therapy ofthe disorders described herein. Preferably, the functional equivalent is a peptide or protein. The term "functional equivalent" includes fragments, mutants, and muteins of Eril.

The functional equivalents include soluble forms of the Eril proteins. A suitable soluble form ofthese proteins, or functional equivalents thereof, might comprise, for example, a truncated form ofthe protein from which the transmembrane domain has been removed by chemical, proteolytic or recombinant methods.

Preferably, the functional equivalent is at least 80% homologous to the corresponding protein. In a preferred embodiment, the functional equivalent is at least 90% homologous as assessed by any conventional analysis algorithm such as for example, the Pileup sequence analysis software (Program Manual for the Wisconsin Package, 1996). Amino acid numbering is as provided in GenBank Protein Accession Number AAK20997 for DC-SIGN and AAG13848 for DC- SIGNR.

Proteinaceous, functionally equivalent fragments or analogues may belong to the same protein family as the Eril proteins identified herein. By "protein family" is meant a group of proteins that share a common function and exhibit common sequence homology. Homologous proteins may be derived from non-human species. Preferably, the homology between functionally equivalent protein sequences is at least 25% across the whole of amino acid sequence ofthe complete protein or of the Ras binding fragment. More preferably, the homology is at least 50%, even more preferably 75% across the whole of amino acid sequence of the protein or protein fragment. More preferably, homology is greater than 80% across the whole of the sequence. More preferably, homology is greater than 90% across the whole of the sequence. More preferably, homology is greater than 95% across the whole ofthe sequence.

The term "a functionally equivalent analogue" is used to describe those compounds that possess an analogous function to an activity of the Eril proteins and may, for example comprise a peptide, cyclic peptide, polypeptide, antibody or antibody fragment. These compounds may be proteins, or may be synthetic agents designed so as to mimic certain structures or epitopes on the inhibitor protein. Preferably, the compound is an antibody or antibody fragment.

The term "functionally equivalent analogue" also includes any analogue of Eril obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to Ras. Amino acid substitutions may be made, for example, by point mutation ofthe DNA encoding the amino acid sequence.

The functional equivalent of Eril may be an analogue of a fragment ofthe Eril. The Eril or functional equivalent may be chemically modified, provided it retains its ability to bind to Ras.

This invention also provides functional equivalents of such polypeptides and fragments thereof. Such functional equivalents may be at least 75 % homologous to the native sequence. Such functional equivalents may also be at least 80%, at least 85%>, at least 90%, at least 95% or at least 100% homologous to the native sequence. A functionally equivalent fragment may be a fragment of the polypeptide that still binds to its target ligand.

This invention also provides functionally equivalent analogs of such polypeptides and polypeptide fragments. Such analogs would have an activity which is analogous to the polypeptide or fragment. Such analogs may be obtained by changing the amino acid sequence, such as by an insertion, deletion or substitution of at least one amino acid. Such an analog would still bind to its ligand. Amino acid substitutions may be conservative substitutions. Such conservative substitutions may ones within the following groups: (1) glycine and alanine; (2) valine, isoleucine, and leucine; (3) aspartic acid and glutamic acid; (4) asparagine and glutamine; (5)serine and threonine; (6) lysine and arginine; (7) phenylalanine and tyrosine. Such substitutions may also be homologous substitutions such as within the following groups: (a) glycine, alanine, valine, leucine, and isoleucine; (b) phenylalanine, tyrosine, and tryptophan; (c) lysine, arginine, and histidine; (d) aspartic Acid, and glutamic Acid; (e) asparagine and glutamine; (f) serine and threonine; (g) cysteine and methionine.

The functional equivalent may also be modified such as by a chemical modification, yet wherein it still binds to its respective ligand.

The nucleic acids, polyepeptides and antibodies or any other agent or compound described herein may be isolated and/or purified. One skilled in the art would know how to isolate and/or purify them. Methods are provided in any laboratory manual such as "Molecular Cloning" by Samrook, Fritsch and Maniatis.

As used herein, the following standard abbreviations are used throughout the specification to indicate specific amino acids: A=ala=alanine; R=arg=arginine; N=asn=asparagine D=asp=aspartic acid; C=cys=cysteine; Q=gln=glutamine; E=glu=glutamic acid; G=gly=glycme; H=his=histidine;

I=ile=isoleucine;L=leu=leucine; K=lys=lysine; M=met=methionine;

F=phe=phenylalanine; P=pro=proline; S=ser=serine; T=thr=threonine; W=trp=tryptophan; Y=tyr=tyrosine; and V=val=valine.

This mvention provides a transgenic nonhuman animal which comprises a transgene encoding the polypeptide of interest or a functional equivalent thereof. The following U.S. Patents are hereby incorporated by reference: U.S. Patent No. 6,025,539, IL-5 transgenic mouse; U.S. Patent No. 6,023,010, Transgenic non- human animals depleted in a mature lymphocytic cell-type; U.S. Patent No. 6,018,098, In vivo and in vitro model of cutaneous photoaging; U.S. Patent No.

6,018,097, Transgenic mice expressing human insulin; U.S. Patent No. 6,008,434, Growth differentiation factor-11 transgenic mice; U.S. Patent No. 6,002,066; H2-M modified transgenic mice; U.S. Patent No. 5,994,618, Growth differentiation factor- 8 transgenic mice; U.S. Patent No. 5,986,171, Method for examining neurovirulence of polio virus, U.S. Patent No. 5,981,830, Knockout mice and their progeny with a disrupted hepsin gene; U.S. Patent No. 5,981,829, .DELTA.Nur77 transgenic mouse; U.S. Patent No. 5,936,138; Gene encoding mutant L3T4 protein which facilitates HJN infection and transgenic mouse expressing such protein; U.S. Patent No. 5,912,411, Mice transgenic for a tetracycline-inducible transcriptional activator; U.S. Patent No. 5,894,078, Transgenic mouse expressing C-100 app.

The methods used for generating transgenic mice are well Icnown to one of skill in the art. For example, one may use the manual entitled "Manipulating the Mouse Embryo" by Brigid Hogan et al. (Ed. Cold Spring Harbor Laboratory) 1986. See for example, Leder and Stewart, U.S. Patent No. 4,736,866 for methods for the production of a transgenic mouse.

For sometime it has been known that it is possible to carry out the genetic transformation of a zygote (and the embryo and mature organism which result therefrom) by the placing or insertion of exogenous genetic material into the nucleus of the zygote or to any nucleic genetic material which ultimately forms a part ofthe nucleus of the zygote. The genotype of the zygote and the organism which results from a zygote will include the genotype of the exogenous genetic material. Additionally, the inclusion of exogenous genetic material in the zygote will result in a phenotype expression ofthe exogenous genetic material.

The genotype of the exogenous genetic material is expressed upon the cellular division ofthe zygote. However, the phenotype expression, e.g., the production of a protein product or products of the exogenous genetic material, or alterations of the zygote's or organism's natural phenotype, will occur at that point of the zygote's or organism's development during which the particular exogenous genetic material is active. Alterations of the expression of the phenotype include an enhancement or diminution in the expression of a phenotype or an alteration in the promotion and/or control of a phenotype, including the addition of a new promoter and/or controller or supplementation of an existing promoter and/or controller ofthe phenotype.

The genetic transformation of various types of organisms is disclosed and described in detail in U.S. Pat. No. 4,873,191, issued Oct. 10, 1989, which is incorporated herein by reference to disclose methods of producing transgenic organisms. The genetic transformation of organisms can be used as an in vivo analysis of gene expression during differentiation and in the elimination or diminution of genetic diseases by either gene therapy or by using a transgenic non-human mammal as a model system of a human disease. This model system can be used to test putative drugs for their potential therapeutic value in humans.

The exogenous genetic material can be placed in the nucleus of a mature egg. It is preferred that the egg be in a fertilized or activated (by parthenogenesis) state. After the addition of the exogenous genetic material, a complementary haploid set of chromosomes (e.g., a sperm cell or polar body) is added to enable the formation of a zygote. The zygote is allowed to develop into an organism such as by implanting it in a pseudopregnant female. The resulting organism is analyzed for the integration ofthe exogenous genetic material. If positive integration is determined, the organism can be used for the in vivo analysis of the gene expression, which expression is believed to be related to a particular genetic disease.

The "transgenic non-human animals" of the invention are produced by introducing "transgenes" into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 pi of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster, et al. (1985) Proc.

Natl. Acad. Sci. U.S.A. 82, 4438-4442). As a consequence, all cells ofthe fransgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention. Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad. Sci U.S.A. 73, 1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida

(Hogan, et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner, et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6927-6931; Van der Putten, et al. (1985) Proc. Natl. Acad. Sci U.S.A. 82, 6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al. (1987) EMBO J. 6, 383- 388). Alternatively, infection can be performed at a later stage. Virus or virus- producing cells can be injected into the blastocoele (Jahner, D., et al. (1982) Nature 298, 623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non- human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (Jahner, D. et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans, M. J., et al. (1981) Nature 292, 154-156; Bradley, M. O., et al.

(1984) Nature 309, 255-258; Gossler, et al. (1986) Proc. Natl. Acad. Sci U.S.A. 83, 9065-9069; and Robertson, et al. (1986) Nature 322, 445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus- mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240, 1468-1474. As used herein, a "transgene" is a DNA sequence introduced into the germline of a non-human animal by way of human intervention such as by way of the above described methods.

One skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

First Series of Experiments

Determination of the membrane topology of Eril, and to identification of its Ras-interacting region.

We have identified a novel gene in budding yeast, Eril, which behaves genetically as an inhibitor of Ras. We have shown that Eril is an integral ER membrane protein that associates specifically with GTP-bound Ras in an effector-like manner. Its short sequence (68 amino acids) indicates that it possesses two hydrophobic regions, one of which is a transmembrane domain and the other is presumed to interact with Ras. Protease protection assays are conducted on epitope-tagged Eril to determine its orientation in the membrane.

Truncated forms of Eril are also tested for association with GTP-bound GST-Ras2 to determine which region is the Ras-binding domain. Additionally, alanine scanning and site-directed mutagenesis are used to identify residues important for

Ras interaction. In addition, the Eril-Ras2 interaction is reconstituted in vitro to compare the affinity of Eril for GTP-Ras to that of Ras effectors.

Understanding the function of Eril in GPI-protein anchoring or secretion. It has been shown that Eril serves a second function in the transport of GPI-proteins from the ER to the Golgi. This function involves the production/attachment of anchors to target proteins, and/or sorting of GPI-anchored proteins into ER-derived vesicles. It is determined which of these processes is deficient in Eπ7Δ cells by measuring GPI-anchor attachment. The point in the anchor biosynthesis pathway that is blocked in Erz7Δ cells is determined upon detecting a deficiency in anchoring. The ΕR-to-Golgi protein transport is focused on upon finding that anchoring is normal. Specifically, two Ras-related G-proteins involved in this process (Yptl and Sari) are tested for association with Εril. Finally, selection is performed for suppressors of the Eril A growth defect, which results from a deficiency in GPI-protein transport. This provides an unbiased approach to the identification ofthe second Εril target.

Determination whether yeast Ras can signal from the ΕR, and creating a more effective Εril-based Ras inhibitor. In one embodiment, Εril functions as an inhibitor of Ras, for example while the GTPase is in the ΕR. It is believed that Εril may be used for development as a therapeutic agent for Ras-involved neoplasias. However, because the majority of Ras resides on the plasma membrane, and we find that Εril localizes to the ΕR, it seems likely that the ability of Εril to inhibit Ras is restricted by its intracellular location. To test this model, the ability of ΕR- restricted Ras to function by fusing it to a transmembrane domain with an ΕR- localization signal is examined. A more effective Εril-based Ras inhibitor is created by directing it to the plasma membrane. To accomplish this, prenylation or myristoylation signals are fused to the Ras-binding domain of Εril. Εril may also be attached to the cytoplasmic tail of a known integral plasma membrane protein.

PRELIMINARY STUDIES

A novel gene was idnetified in Saccharomyces cerevisiae that appears to have two separate roles. We have designated this gene ERIl, for Ras Inhibitor I, based on our characterization of one of its functions. The ERIl gene (YPL096c-A) encodes a previously non-annotated open reading frame (nORF) of only 68 amino acids (Figure 1). Although we have not yet identified any metazoan ERIl homologs, we have uncovered many fungal genes related to ERIl. The closest homologs identified are from C. albicans and C. tropicalis. ERIl was isolated through a genetic screen for mutants with growth defects that were additive with that of a conditional mutant in PKC1 (sttl-1; Yoshida et al., 1992). The details of that screen are presented elsewhere (Romeo et al., submitted). Because pkcl mutants are compromised for cell wall integrity signaling, we anticipated that mutants answering the screen would display cell wall defects on their own. Indeed, among the phenotypes displayed by Erz7 mutants is a weakened cell wall (discussed in section 4 below).

Several lines of evidence support the conclusion that ERIl is a bona fide gene. First, ERIl is expressed as an mRNA of approximately 300 nucleotides. Second, we can detect an epitope-tagged form of the Εril protein expressed from its own promoter. Third, the original Eril mutant was recessive, resulting from a frameshift mutation after the eighth codon. Fourth, the phenotypic defects of a precise Erz7Δ mutant are complemented by a centromeric plasmid bearing 500 bp of sequence corresponding to ERIl and its regulatory elements only.

The Erz7Δ phenotypes can be divided into two categories that we believe reflect two distinct functions of this small protein. One set of phenotypes is associated with hyperactive Ras pathway signaling, and the other phenotypes are associated with a failure to transport cell wall proteins, specifically GPI-anchor proteins, to the cell surface. Our understanding ofthese two functions is detailed below.

1. A ErilA mutant displays hyperactive Ras phenotypes. Some strain backgrounds of Saccharomyces cerevisiae (including that used in these studies) have the capacity to undergo a dimorphic shift in response to nutrient limitation from ovoid, budding cells to a multicellular form consisting of filaments of elongated cells (Gimeno et al., 1992; Mosch and Fink; 1997). Filamentous growth, also known as pseudohyphal growth, is associated with an ability to invade the agar substratum, and is thought to provide a means by which cells can forage for nutrients at a distance from the colony. The Ras/cAMP pathway is a major controlling element of this shift (Ward et al., 1995; Kubler et al., 1997; Lorenz and Heitman, 1997; Mosch et al., 1999). Hyperactive Ras pathway signaling induces precocious pseudohyphal/invasive growth (Gimeno et al., 1992; Figure 2, lower plate). Invasive growth can be detected simply by washing the cells from an agar plate after two days of growth (Roberts and Fink,1994).

We found that a Erz7Δ mutant undergoes invasive growth when cultivated at 34°C

(Figure 2). Significantly, this behavior was suppressed by deletion of RAS2, which is required for pseudohyphal/invasive growth (Mosch et al., 1996, 1999).

Microscopic examination of Eril A cells grown at 34°C (Figure 3) revealed that they become elongated and grow as connected chains of cells, both features characteristic of pseudohyphal development. This phenotype was also suppressed by a ras2A mutation, indicating that RAS2 is required for pseudohyphal/invasive growth of Erz7Δ cells. These results suggested that Erz7Δ cells are hyperactivated for Ras signaling.

Another phenotype associated with hyperactive Ras signaling in yeast is the failure to acquire thermotolerance in preparation for stationary phase (Nikawa et al., 1987; Kim and Powers, 1991). A saturated Erz7Δ culture lost two logs of viability relative to wild type in response to a brief heat shock at 50°C (Figure 4A). This defect was suppressed by downregulation of the Ras/cAMP pathway through expression of a dominant negative form of RAS2 (Jung et al., 1994), or overexpression of either the Ras-GAP IRA2 (Tanaka et al., 1990), or a downstream inhibitor of Ras pathway output, RPI1 (Sobering et al., 2002). Conversely, the heat shock sensitivity of a mutant in the Ras-GAP, IRA1 was suppressed by overexpression of ERIl (Figure 4B). These results, taken in the aggregate, suggest that Εril is an inhibitor of Ras pathway activity.

2. Εril specifically associates with GTP-bound Ras through the effector loop.

We were interested to determine the level at which Εril acts on the Ras pathway to inhibit signaling. An additional heat shock experiment suggested that Εril acts at, or above the level of Ras. Specifically, overexpression of ERIl failed to suppress the heat shock sensitivity of a constitutive allele of CYR1 (SRA4-6; Cannon et al., 1986; data not shown), which encodes adenylate cyclase, the direct effector of Ras in S. cerevisiae. Therefore, we tested for association of Eril with Ras2 in vivo. For this experiment, GST-tagged Ras2 was co-expressed in yeast cells with a fully functional

HA-tagged Eril (HA-Eril). We used either wild-type RAS2 or a constitutively active allele (RAS2-V19; both gifts of R. Deschenes). Affinity purified glutathione- S-transferase (GST)-Ras2 was tested for association with HA-Eril by immunoblot analysis. Although HA-Eril failed to associate with wild-type GST-Ras2, it reproducibly copurified with the constitutive form of Ras2 (Figure 5A). Wild-type Ras exists predominantly in the GDP-bound state in vivo (Barbacid, 1987; Wittinghofer and Herrmann, 1995). Constitutively active RAS mutants have lost their intrinsic ability to hydrolyze GTP, and are thus "locked" in the GTP-bound state. To determine if Eril association with Ras2 was dependent on the nucleotide bound state ofthe G-protein, we isolated GST-Ras2 from cells devoid of Ras-GAPs

(iral A iral A). In this setting, Ras is virtually 100% GTP-bound. In the absence of the Ras-GAPs HA-Eril associated with wild-type GST-Ras2 just as well as with the constitutive form (Figure 5 A), indicating that Eril specifically associates with GTP- bound Ras2. Another indication of the specificity of the interaction is that we have not been able to detect association of Eril with GTP-Rhol, the yeast GTPase most closely related to Ras.

To identify the region of Eril that associates with GTP-Ras, we tested a C-terminal HA-Eril truncation (containing residues 1-39) and an N-terminal HA-Eril truncation (containing residues 28-68) for association with GST-Ras2-V19. Figure 17C shows that the Ras-interaction region of Eril resides within the N-terminal 39 amino acids.

GTP-dependent association of a protein with Ras is one of two criteria used in the identification of novel Ras effectors (Wittinghoffer and Herman, 1995). The other criterion is the requirement of an intact Ras-effector loop for interaction. The Ras- effector loop is a highly conserved region that is exposed through a conformational change induced by GTP-binding (Wittinghofer and Nassar, 1996). Mutations in the Ras effector loop, comprised of residues 39-47 in yeast Ras (corresponding to residues 32-40 in mammalian Ras), disrupt interaction with effector proteins (Farnworth et al., 1991; Akasaka et al., 1996; Marshall et al., 1988; Sun et al., 1994). Therefore, we introduced either of two effector loop mutations into a constitutive Ras2 mutant (creating RAS2-V19, A42 and RAS2-V19, N45), and tested them for association with Eril. Figure 5B shows that both effector loop mutations block the GTP-Ras interaction with Eril, indicating that Eril associates with Ras in an effector loop-dependent manner.

To summarize, Eril fits the operational definition of a novel Ras effector, but it behaves genetically as a Ras inhibitor. We propose that Eril acts as a competitive inhibitor of Ras signaling by shielding the effector loop of GTP-Ras from interaction with its effector. This is exciting because the only known inhibitors of Ras proteins are the Ras-GAPs, which act by stimulating the intrinsic GTPase activity of Ras

(Barbacid, 1987, Bourne et al., 1991). Because oncogenic (constitutively GTP- bound) Ras mutants have lost their ability to hydrolyze GTP, the Ras-GAPs are ineffective as inhibitors ofthese oncoproteins.

3. Eril resides in the endoplasmic reticulum (ER) membrane. The primary sequence of Eril possesses two highly hydrophobic regions (residues 7-28 and residues 34-56; Figure 1), either of which is long enough to constitute a transmembrane domain. To determine if Eril is associated with a membrane, we examined the fractionation pattern of HA-Eril in yeast cell extracts. Figure 6A shows that HA-Eril sediments with the pellet from a 100k x g centrifugation. The protein is liberated by addition of Triton X-100 (non-ionic detergent) to disrupt membranes, but not by other treatments that would liberate peripherally associated membrane proteins (eg. urea or high pH). Therefore, we conclude that Eril is an integral membrane protein. To determine with which intracellular membrane Eril associates, we conducted indirect immunofluorescence microscopy on cells expressing HA-Eril from its own promoter on a multicopy plasmid. Figure 6B shows a pattern of perinuclear fluorescence with swirls out to the plasma membrane, which is characteristic of ER resident proteins (Rose et al., 1989; Bartels et al., 1999; Romano et al., 1998; Schmidt et al., 1998). The punctate fluorescence at the cell surface is regarded to be peripheral ER. However, we cannot exclude the possibility that a small pool of Eril resides at the plasma membrane. This pattern was consistently observed in cells across the entire cell cycle. We were barely able to detect HA-Eril expressed from a centromeric plasmid, but the pattern of localization appeared to be the same (not shown). The Eril sequence does not possess a dilysine motif for retrieval of ER membrane proteins. However, it is not uncommon that such a motif is missing, and has led to speculation that unrecognized ER-retention signals exist within the transmembrane domains of ER-resident proteins (Sato et al., 1996; Schmidt et al., 1998).

The identification of Eril as an ER-resident protein was somewhat surprising, because until very recently Ras was thought to signal exclusively from the cytoplasmic surface ofthe plasma membrane (Barbacid, 1987; Clarke, 1992; Bartels et al., 1999). However, Ras undergoes a series of posttranslational modifications at its C-terminal CaaX (cysteine, two aliphatic residues, and any residue) motif. These steps include farnesylation ofthe CaaX cysteine, followed by C-terminal proteolysis to remove the aaX sequence, and ultimately methyl esterification of the exposed cysteinyl -carboxyl group. Newly synthesized Ras is farnesylated in the cytosol. However, the latter two modifications are executed by enzymes that reside in the ER both in yeast and mammals (Boyartchuk et al., 1997; Dai et al., 1998; Romano et al., 1998; Schmidt et al., 1998). Farnesylation of the CaaX sequence targets Ras to the cytoplasmic face of ER membranes (Choy et al., 1999; Apolloni et al., 2000). The final steps of Ras CaaX modification are thought to occur at the ER prior to transit ofthe mature form through the classical exocytic pathway (in the case of H-Ras and N-Ras) to the plasma membrane (Prior and Hancock, 2001). However, in contrast to conventional cargo carried in vesicle lumens, Ras must be transported on the cytoplasmic surface of vesicles. This leaves open the possibility that Ras can undergo nucleotide exchange and interact with its effectors while associated with ER and Golgi membranes. A recent study by Chiu et al., (2002) suggests that mammalian H-Ras and N-Ras can indeed recruit Raf to these compartments and propagate signal in response to mitogens even when restricted to endomembranes. Therefore, we tested the possibility that Eril engages Ras at the ER. Total membranes from cells coexpressing GST-Ras2^V19 and ^HAEril were sedimented on a step sucrose-EDTA density gradient. As anticipated from the immunofluorescence localization of ^Eril, the majority ofthe ^HΛEril (56%) co-sedimented with the ER marker Dpml in fraction 1 (Figure 19 A), with the remainder diminishing through fractions 2-6. The PM marker Gasl sedimented in fractions 3-6, with the majority (66%) sedimenting in fractions 4 and 5. However, the immature, ER-modified form of Gasl (26) sedimented in fraction 1. By contrast, GST-Ras2^V19 was evenly distributed across the entire gradient, suggesting its localization at endomembranes as well as at the PM. After disruption of the membranes with detergent, GST- Ras2^V19 was affinity purified from the gradient fractions and tested for association with ^HAEril. ^HAEril was found in association with GST-Ras2^V19 mainly from fraction 1 (Figure 19B), indicating that Eril engages GTP-bound Ras at the ER. Some ^Eril was detected with GST-Ras2^V19 isolated from fractions 3 and 4, suggesting that a pool of Eril may also engage Ras at a heavier membrane. The failure of ^Eril to associate with GST-Ras2^V19 from fraction 2 may be explained by the predominance of the vacuolar marker CPY in that fraction. Because both proteins were overexpressed, some of each may be targeted for destruction in the vacuole, where they are not likely to interact.

We can imagine two possibilities for the Ras-related function of Eril. One is that it maintains Ras in an inactive state while it is being processed in the ER. In this case, Eril would serve as a molecular chaperone for immature Ras, preventing access to effector proteins of GTP-Ras before the G-protein has reached the cell surface. The other possibility is that Eril regulates normal Ras signaling from the ER. Of course, it is not at all clear that yeast Ras has a signaling function in the ER. Moreover, the majority of mature yeast Ras resides on the plasma membrane (Bartels et al., 1999).

4. A Eril A mutant displays growth arrest at 37°C and cell wall defects at permissive temperatures. Eril has a second function that is unrelated to its inhibitory action on Ras. Because we isolated the Eril mutant in a synthetic lethal screen with apkcl^ts, we anticipated that it would display a cell wall defect. Indeed, the Erz7Δ mutant is extremely sensitive to cell lysis by treatment with zymolyase, a wall lytic enzyme (Figure 7A). This was true for both log phase and stationary phase cells. The Erz7Δ mutant is also temperature sensitive for growth at 37°C (Figure 7B). Moreover, this growth defect was suppressed by addition of sorbitol to the medium for osmotic support. However, the failure to grow at restrictive temperature is reversible (not shown), indicating that htllA cells undergo growth arrest, rather than cell lysis at high temperature. None of these phenotypes are characteristic of hyperactive Ras signaling. More importantly, we could not suppress either the zymolyase sensitivity, or the temperature sensitivity ofthe Erz7Δ mutant by down regulation of Ras (data not shown).

To gain an understanding ofthe cause ofthe growth arrest of the Eril A mutant, we screened a 2μ genomic yeast library for multicopy suppressors of the growth defect at 37°C. Four identical plasmids were recovered from this screen (Figure 8A).

Deletion analysis revealed that the responsible gene was GFAI, which encodes glutamine: fructose-6-phosphate amidotransferase (Watzele and Tanner, 1989). Gfal catalyzes the production of glucosamine-6-phosphate, which is the first and rate-limiting step in the production of UDP-N-acetyl glucosamine (UDP-GlcNAc; Orlean, 1997) for the biosynthesis of chitin, glycosyl phosphatidylinositol (GPI)- anchors, and N-glycosylation of proteins (Figure 8B). Although N-glycosylated proteins serve important functions in the vacuole, on the cell surface, and in cell cycle progression (Jones et al., 1997; Orlean, 1997), both chitin and GPI-proteins function principally on the cell surface. Chitin is a carbohydrate component of the cell wall made up of polymers of GlcNAc (Cabib, 1987). GPI-anchored proteins comprise a major proteinaceous component of the cell wall (Caro et al., 1997). UDP-GlcNAc is used in the first step in the production of GPI-anchors, which are attached to target proteins in the ΕR (Popolo et al., 1999). After secretion of anchored proteins to the cell surface, they are tethered to the plasma membrane by their lipid anchors. In most cases (perhaps all), the anchors are ultimately clipped to liberate the proteins for covalent attachment to cell wall carbohyrdrates. An alternative pathway for the production of UDP-GlcNAc is through salvage of exogenous glucosamine, which can be phosphorylated by the hexokinases. Indeed, gfal mutants are glucosamine auxotrophs (Watzele and Tanner, 1989). To confirm that GFAI overexpression was suppressing Erz7Δ by driving UDP-GlcNAc production, we tested the ability of exogenous glucosamine to suppress this growth defect. Figure 8C shows that glucosamine crystals, but not glucose crystals, were capable of suppression.

In our experience, this is a highly unusual phenotype. Mutants with cell wall defects are not generally suppressed by GFAI overexpression or exogenous glucosamine.

Therefore, we had to develop a new framework with which to understand these results.

Because either exogenous glucosamine or GFAI overexpression can increase chitin levels in wild-type cells by elevating endogenous pools of UDP-GlcNAc (C. Specht, personal communication), we explored the possibility that the Eril A mutant was deficient in producing chitin. Toward this end, we examined GFAI expression and cell wall chitin levels in the Erz7Δ mutant, and were surprised to find that both are elevated approximately 4-fold over wild-type levels (not shown). It has been reported that certain types of cell wall mutants (eg. fksl, gasl) hyperaccumulate chitin in the lateral cell wall (Valdivieso et al., 2000; P. Robbins, personal communication). This is thought to be an emergency response to cell wall stress. Our initial interpretation of our results was that Erz7Δ cells are deficient in some aspect of cell wall biogenesis, to which they respond by hyperaccumulating chitin. By overexpressing GFAI, or providing glucosamine in the growth medium we are merely assisting these cells in their effort to produce chitin. In fact, microscopic examination of Erz7Δ cells stained with calcoflour white, a chitin binding fluorescent dye, revealed that they deposit large amounts of chitin in the lateral cell wall (Figure 9). Although oddly, this mutant deposits chitin only in the mother cell wall, apparently excluding it from the bud. This seems rather counterintuitive, because cell wall stress is generally the greatest at the bud tip, the site at which the wall is being remodeled during growth. By contrast, the mother cell wall is a relatively static structure. This point will be revisited below. Chitin synthase 3 (Chs3) was recently shown to be responsible for executing the otherwise poorly characterized "chitin emergency response" (Valdivieso et al., 2000). Therefore, we deleted the normally non-essential CHS3 gene in the context of a Eril A mutant, making three predictions for the behavior of the double mutant.

First, the Eril A, ch$3A mutant should fail to hyperaccumulate chitin. Figure 9 shows that this prediction was borne out. Second, if chitin production is an important adaptive response for survival of Eril A cells, the double mutant should be appreciably less healthy than the Erz7Δ mutant. This prediction was not borne out, because the restrictive temperature ofthe double mutant (36°C) was only one degree lower than that of the Erz7Δ mutant, suggesting that chitin deposition is not important for their survival. Third, and most importantly, if GFA 7/glucosamine suppress the growth defect of Eril A by driving chitin biosynthesis, the Eril A chs3A double mutant should not be suppressible by these treatments. This prediction was also not borne out, as shown in Figure 10.

The finding that GFAI overexpression suppresses the growth defect of the double mutant without hyperaccumulation of chitin (Figure 9) indicates that chitin is not the important product resulting from elevated UDP-GlcNAc pools in this mutant. It seems likely that chitin overproduction in Erz7Δ cells is an unintended consequence ofthe true purpose the cell has in elevating UDP-GlcNAc pools. This conclusion is supported by the earlier observation that Erz7Δ cells accumulate chitin in the walls of mother cells, but not in buds. It seems that they make more chitin than they need in their effort to drive other UDP-GIcNAc-dependent pathways, and "dump" this chitin in the place least affected by its presence, the mother cell wall.

5. The Eril A mutant specifically accumulates GPI-proteins in the ΕR. What is the important product of elevated UDP-GlcNAc pools in the Eril A mutant? The exclusion of chitin leaves N-glycosylation of proteins and GPI-anchors as the only other known products for which UDP-GlcNAc is used. To determine if Eril A cells display a deficiency in the production of either of these types of secreted proteins, we examined the modification state of Gasl and Carboxypeptidase Y (CPY). Gasl is an abundant GPI-anchored protein that undergoes O-linked and N-linked glycosylation in the ER and Golgi (as do virtually all GPI-proteins) in addition to its lipid modification, which is also attached in the ER (Popolo and Vai, 1999). CPY is a vacuolar protein that is N-glycosylated in the ER and Golgi prior to its transport to the vacuole where it is proteolytically processed to its mature form (Jones et al., 1997). The immunoblot shown in Figure 11 (left) shows that Eπ7Δ cells are not appreciably impaired for maturation of CPY, suggesting that modification and secretion of N-glycosylated proteins is normal. Although this experiment reveals only that the steady-state levels of CPY precursor forms in Eril A are not high enough to detect, pulse-labeling experiments confirm that the kinetics of CPY maturation are identical in wild-type and Eril A cells (not shown). By contrast, we found that approximately 50% of Gasl exists in an underglycosylated state (Figure 11, right).

Fully glycosylated Gasl migrates as a 125 kDa band (Conzelmann and Riezman, 1988). In mutants that affect ΕR-to-Golgi protein transport, Gasl accumulates as a

100 kDa ΕR-modified form (Popolo et al., 1988). The Erz7Δ mutant accumulates a 100 lcDa form of Gasl, even when grown under permissive conditions (23°C), suggesting that transport of this protein is retarded in the ΕR. Strikingly, this underglycosylated form of Gasl was chased to the mature form by inclusion of 25 mM glucosamine in the growth medium.

If GPI-anchor proteins are accumulating in the ΕR of Erz7Δ cells, as suggested by the above results, this mutant would be expected to display an unfolded protein response (UPR). The UPR is a stress signal that emanates from the ΕR under conditions in which the capacity of the ΕR is being exceeded (Patil and Walter,

2001). The signal, which is received in the nucleus, activates a transcriptional program mediated by the unfolded protein response element (UPRΕ), resulting in expression of genes that enhance the protein folding (HSPs) and transport capacity of the ΕR (Chapman et al., 1998; Sidrauski et al, 1998). We examined the Erz7Δ mutant for UPR-driven gene expression.

Treatment of wild-type cells with tunicamycin stimulates a strong UPR (Cox and Walter, 1996) by blocking the first step in N-linked glycosylation (catalyzed by Alg7), which occurs in the ER (Orlean 1997; Figure 12). Figure 12 shows that Eril A cells drive strong expression of a UPRE-regulated lacZ reporter in a temperature-dependent manner. Significantly, the prodigious UPR in these cells can be partially suppressed by exogenous glucosamine. This result is consistent with the hypothesis that GPI-proteins accumulate in the ER of Erz7Δ cells, and that this defect is suppressed by driving GPI anchor production through GE 4 i/glucosamine.

We conclude from these results that Eril A cells are specifically deficient in ΕR-to- Golgi transport of GPI-anchor proteins. The mutant attempts to compensate for this transport defect by driving an early step in the modification of GPI-proteins - anchoring to the ΕR membrane through elevated UDP-GlcNAc pools. This conclusion is supported by the dual observation that a further increase in UDP- GlcNAc, driven by exogenous glucosamine, both enhances the maturation of Gasl and suppresses the UPR in Erz7Δ cells. The cell wall defect observed in a Erz7Δ mutant can be explained by a deficiency of GPI-proteins at the cell surface. The growth arrest observed at elevated temperatures is likely to result from an extreme defect in ΕR-to-Golgi protein secretion caused by retardation of GPI-proteins in the

ΕR. This conclusion is supported by the temperature-dependent increase in the UPR of Eril A cells.

6. What is the role of Εril in GPI-anchor protein transport? Further support for the notion that Εril serves a second function that is independent of Ras regulation comes from our failure to suppress the hyperactive Ras phenotypes

(pseudohyphal/invasive growth and heat shock sensitivity) by overexpression of GFAI, or treatment with exogenous glucosamine (not shown). However, the diminutive size of Εril leads us to believe that it is not likely to possess multiple functional domains. If this is true, then its role in the transport of GPI-proteins should be analogous to its role in the regulation of Ras.

Two classes of mutants have been described that display GPI-protein trafficking defects that are similar to that of Eril A. The first group is comprised of mutants in GPI-anchor production or attachment (eg. gpi3, or gaal; Popolo and Vai, 1999). Such mutants specifically accumulate GPI-proteins in the ΕR because anchor attachment, which happens in this compartment, is strictly required for transport of GPI-proteins from the ER to the Golgi in COP Il-coated vesicles (Doering and Schekman, 1996). The second mutant class has only one established member to date. Mutants in the RET1 gene, which encodes the α-subunit of coatomer (also known as COP I), are specifically deficient in ER-to-Golgi transport of GPI- anchored proteins (Sutterlin et al., 1997). The coatomer complex has been implicated in the retrieval of proteins from the Golgi to the ER (Letourner et al., 1994; Lewis and Pelham, 1996). Sutterlin et al. (1997), have proposed that coatomer functions to retrieve specific factors from the Golgi that are required for the transport of GPI-anchored proteins out of the ER.

Perhaps surprisingly, we have found that the growth defect of a retl-1 mutant, like that of Eril A, is suppressed by either GFAI overexpression or exogenous glucosamine (not shown). This is true even though retl-1 cells are not impaired for GPI-anchoring (Sutterlin et al., 1997), and suggests that anchoring may be a rate- limiting step for ER-to-Golgi transit of GPI-proteins. To which class of mutants Eπ7Δ belongs is an open question that can be answered only by measurement of GPI-anchor attachment. Our bias, as presented in the next section, is that Εril is important for GPI-anchored protein transport, most likely through regulation of a small GTPase involved in ΕR-to-Golgi vesicular traffic.

The concept of a specific deficiency in transporting one class of protein from the ΕR to the Golgi may seem odd to the reader. This is because until very recently, it was thought that all secretory proteins travel together from the ΕR to the Golgi, where they are sorted to different destinations. However, new evidence from Howard

Riezman's laboratory indicates that sorting of GPI-anchored proteins occurs at the ΕR (Sutterlin et al., 1997, Muniz et al., 2001). The factors involved in protein sorting into different ΕR-derived vesicles are only now being uncovered. Among these is a Ras-like GTPase named Yptl (Morsomme and Riezman, 2002).

METHODS

1. Determining the membrane topology of Eril, and identifying its Ras- interaction region. We have shown that Eril is an integral ER membrane protein. Its primary sequence indicates that it possesses two highly hydrophobic regions, both of which have the potential to span a membrane. We have also shown that Eril associates with GTP-Ras in a manner that requires an intact Ras-effector loop. It seems most likely that one ofthe hydrophobic regions within Eril serves to tether it to the ER membrane and the other interacts with its G-protein target on the cytoplasmic face ofthe ER. In this section, we describe a structure/function analysis that gives a picture ofthe membrane topology of Eril and identify the region of this tiny protein that interacts with Ras. The Eril-Ras2 interaction is reconstituted in vitro for the purpose of determining the affinity of Eril for GTP-Ras.

A. Determination of the membrane topology of Eril.

To determine if either end or the middle of Eril resides in the lumen of the ER, protease protection assays (Abeliovich et al., 1998) are conducted using various HA- tagged forms of Eril. A detailed membrane topological analysis of a much more complicated ER-membrane protein has recently been reported (Stel4; Romano and Michealis, 2001). A set of myc-tagged Stel4 plasmids may be used as internal controls. The basis for the analysis is that an epitope that resides in the lumen ofthe ER will be protected from digestion by proteinase K.

We have an N-terminally HA-tagged form of Eril that is fully functional. A C- terminally tagged form is constructed, and an HA-epitope is placed at an internal site that resides between the two hydophobic regions (between residues 28 and 34). These new forms are examined for function by complementation of the Er/iΔ growth defect when expressed from a centromeric vertor. They are also tested for proper localization by indirect immunofluoresence. We can detect HA-Εril when expressed from a 2μ plasmid, so the protease protection experiments are conducted under conditions of mild Εril overexpression. The HA-tagged forms of Εril are co- expressed in yeast cells with one of several myc-tagged forms of Stel4 as protected and unprotected controls (Romano and Michealis, 2001).

As an independent test of the orientation of the C-terminus of Εril, a Εril-Suc2 fusion protein is constructed. Suc2 (invertase) can be used as a topology reporter because it becomes highly glycosylated when translocated to the lumen of the ER (Deshaies and Schekman, 1990). This modification is detectable as a mobility shift by SDS-PAGE. To analyze the orientation of the C-terminus of Eril, full-length Eril is fused to Suc2 lacking its native signal sequence. The Eril-Suc2 fusion is expressed in a suc2A sfrain, and the orientation of Suc2 in relation to the ER membrane is examined by the gel migration pattern of the fusion using anti-Suc2 antibodies. Glycosylation is detected by mobility shift after treatment of extracts with endoglycosidase H (Endo H) to remove the sugar chains. If the Eril-Suc2 fusion is found to be glycosylated, this is taken as evidence that the C-terminus of Eril resides in the ER lumen. If not, it is concluded that the C-terminal Eril tail is cytoplasmic.

B. Identification ofthe Ras-interacting region of Eril.

1. GST-pulldowns of truncated forms of Eril.

Determination of what residues within Eril is important for its Ras interaction is important. Unfortunately, Eril does not lend itself to two-hybrid interactions, perhaps because of its highly hydrophobic nature. Therefore, as a first step, HA- tagged versions of the N-terminal and C-terminal halves of Eril are co-expressed under GAL1 control with GTP-bound Ras2 (Ras2-V19). Eril fragments are tested for in vivo association by copurification with GST-Ras2, in the same manner as described in above. If it is found that one of the hydrophobic domains of Eril is capable of Ras association, this complements the results of the protease protection assays. For example, if it is found that the Eril-Suc2 fusion is glycosylated, that the C-terminal, but not the N-terminal HA-tag is protease protected, and that the N- terminal half of Eril associates with GTP-Ras2, this paints a clear picture of Eril with its C-terminal half in the ER membrane and its N-terminal half cytoplasmically exposed for association with Ras.

2. Alanine scanning mutants

A more comprehensive approach to the identification of the Ras-interaction site on

Eril is to mutate each residue, in patches of two or three within a single mutant, to alanyl residues. This is conducted by the PCR overlap extension method (Ho et al., 1989). Because Eril is only 68 amino acids, this is not an onerous task. Mutants are tested initially for the ability to complement the growth defect of the Erz7Δ mutant at 37°C, and its invasive growth phenotype at 34°C. This is done by expression of Eril mutants on a centromeric plasmid under the control of their own promoter. Because these phenotypes reflect two independent Εril functions, this analysis allows separation of those functions. HA-tagged alanyl mutants is also examined for ΕR localization, and the ability to associate with GTP-bound GST- Ras2. It is likely that these initial mutants fall into several categories. First, and probably most frequent, are mutants that fail to complement either phenotype and fail to interact with Ras, but still localize to the ΕR. Second are mutants that complement only one ofthe phenotypes and remain in the ΕR. This is an especially interesting class, because such mutants are likely to be compromised for only one interaction. However, this class may be very rare among alanine patch mutants. Third are mutants that fail to complement one or both phenotypes, and no longer localize primarily to the ΕR. This is also a potentially interesting class, because such mutants may reveal an ΕR retention signal within Εril.

3. Site-directed mutants Of course, one caveat to the mutational approach to identification ofthe Ras-binding site is that some mutants may fail to function simply because they do not fold properly. This may make it difficult to interpret the importance of a mutant that fails to interact with GTP-bound Ras2 if it retains no biological function. For this reason, to identify the G-protein interaction site definitively, it is useful to identify Erz7 mutants that have lost the ability to interact with only one of its targets. To achieve this goal, we use site-directed mutagenesis of individual residues suggested to be functionally important through the alanine scan. For example, if an alanine patch mutant fails to complement either phenotypic defect and fails to interact with GTP- Ras2 (Class 1), we alter the residues within that patch in an effort to identify mutants that can complement one phenotype, but not the other. The coveted mutant is one that complements the growth defect at 37°C, but not the invasive growth phenotype, resides in the ΕR, and fails to interact with GTP-Ras2. Such a mutant may be specifically defective in the Ras interaction. After the second target of Eril function is identified, then the collection of alanyl scan and site-directed Eril mutants are examined for association with the other target.

4. Establishing in vitro association of Eril with GTP-Ras

Once the Ras-binding domain of Eril is identified, then association of this domain with GTP-Ras is detected in vitro. This may be a first step toward measuring the affinity of Eril for GTP-Ras. GST-Ras2 (wild-type and Ras2N19) will be expressed in E. coli, as described previously (Vojtek et al., 1993). To enhance solubility, the Ras-binding domain (RBD) may be expressed free of the transmembrane domain. The Εril RBD may be expressed in E. coli as a fusion to maltose-binding protein (MBP) with an HA-tag. The fusion proteins may be purified using glutathione and maltose resins, respectively. Immobilized Ras (GTP- or GDP-bound) may be incubated with MBP-HA-Εril RBD. Association may be detected by immunoblot. GTP-dependence of the in vitro Εril -Ras association is taken as evidence that the in vivo interaction has been successfully recapitulated. If an in vitro association between Εril and Ras2 is demonstrated, it is then asked if an association can be detected between Εril and human H-Ras expressed and purified in the same way. Vojtek et al, (1993) described the in vitro association of GST-H-

Ras with MBP- Rafl. It is then determined if varying concentrations of Εril can displace Ras-bound Rafl, and vice versa (Raf antibodies are available commercially from several sources). This tells whether Εril acts as a competitive inhibitor.

5. Surface plasmon resonance analysis.

After having the ability to detect the Εril-Ras association in vitro, then surface plasmon resonance is used to measure the affinity of Εril RBD for GTP-Ras (Jonsson et al., 1991). The same E. co/z^'-expressed proteins described above are used for this analysis. Binding affinities for GTP-Ras with its effectors (Herrmann et al.,

1996; Wang et al., 2002) range from high-affinity binding of Raf (K_d = 20 nM) to low-affinity bindmg of Ral-GΕF (K_d = 1 μM). If Εril acts as a competitive inhibitor of Ras-effector interactions in the ER, it is anticipated that its affinity for GTP-Ras is comparable to, or higher than, the affinity ofthe effectors.

Using a BiaCore 2000 (Pharmacia). anti-GST antibodies may be first be coupled to the sensor chip, as described (Jonsson et al., 1991). GST or GST-Ras2-V19, preloaded with either GDP or GTPγS may be immobilized on the chip. For kinetic measurements, Eril RBD may be introduced to the binding surface at varying concentrations. The Ka values may be determined using BIA (BiaCore) evaluation software.

2. Understanding the function of Eril in GPI-protein anchoring or secretion

We have shown that Eril A mutants specifically accumulate the GPI-protein, Gasl, in the ΕR-modified form, suggesting that this class of secreted protein is retarded in the ΕR. The finding that the Erz7Δ mutant displays a strong unfolded protein response (UPR) supports this conclusion. We can think of at least two explanations for the behavior of Erz7Δ cells. They may be deficient in ΕR-to-Golgi transport of GPI-anchored proteins, or in the production or attachment of GPI-anchors, which is required for transport of these proteins out of the ΕR (Doering and Schekman, 1996). We distinguish between these possibilities. If it is found that anchoring is deficient, then biochemical assays are conducted to determine the step at which the block occurs. If it is found that anchoring is not deficient, then both a candidate- based approach and an unbiased genetic approach to identifying the putative G- protein target of Εril in the secretory pathway are taken.

A. Determining the nature of the ErilA defect in GPI- proteins function. The first question addressed is whether Erz7Δ cells are deficient in GPI-anchoring.

GPI-anchored proteins can be separated easily from unanchored proteins on the basis of differential hydrophobicity. We separate anchored from unanchored Gasl by phase separation into Triton X-114 (Sutterlin et al., 1997), followed by immunoblot analysis of Gasl. Unanchored Gasl partitions into the aqueous phase, whereas anchored Gasl partitions into the detergent phase. These experiments are conducted under the same growth conditions that result in 50% of the Gasl in the ER-modified state. Very little unanchored Gasl can be detected in wild-type cells.

Therefore, if the retardation of Gasl in the ER reflects an anchoring problem, this is easily detected by selective partitioning of the mature (125 lcDa) form from the underglycosylated (100 kDa) form. A mutant in GAAl, which is deficient in anchor attachment is used as a control.

If an anchoring defect is not detected by immunoblot analysis of steady-state Gasl, then the kinetics of Gasl anchoring are examined by pulse labeling proteins in vivo. Anchoring efficiency is then measured by phase separation in Triton X-114, as above, except that labeled Gasl is immunoprecipitated from the aqueous and detergent phases (Sutterlin et al., 1997). Although this is a more laborious assay, it is more sensitive than measurements of steady state levels of anchored versus unanchored protein.

B. Anchor biosynthesis

If an anchoring defect is detected, then focus may initially be on the first two steps in GPI-anchor synthesis, in part because these can be measured in cell-free systems (Costello and Orlean, 1992; Stevens, 1993; Orlean, 1997). The first of these reactions is carried out by an ER-localized complex (GPI-N- acetylglucosaminyltranferase or GPI-GnT) that transfers GlcNAc from UDP- GlcNAc to phosphatidylinositol, yielding GlcΝAc-PI. In yeast, the GPI-GnT complex is comprised of at least three subunits (GPI1,2,3). The GPI-GnT complex of mammalian cells has been better characterized and is known to possess at least six components (Watanabe et al., 2000). Intriguingly, an uncharacterized component of the mammalian complex migrates by SDS-PAGE as a 6 kDa protein, very similar in size to Eril. The second reaction is the deacetylation of GlcΝAc-PI to GlcΝ-PI. Although the entity responsible for this reaction has not been identified, the reaction is stimulated by GTP (Stevens, 1993), raising the possibility that it is regulated by a GTPase. These first two reactions can be followed together by thin layer separation of lipids labeled in vitro with UDP-[6-³H]GlcNAc. The effect of a Eril A mutation on the in vitro production of GlcNAc and GlcN-PI is assessed. If the GPI-anchoring step affected in Eπ7Δ cells by this approach is not identified, then the target of Εril action by genetic means (see section 3b, below) is identified.

C. Identification ofthe Εril target for GPI-protein transport. Our bias is that Εril functions in GPI-anchored protein transport, rather than anchor assembly or attachment. It would seem likely based on its interaction with Ras, that Εril regulates one of the small G-proteins that function in the secretory pathway to mediate ΕR-to-Golgi vesicle traffic. The two most attractive candidates are Sari and Yptl, because both are required for ΕR-to-Golgi protein transport, and because both are known to reside on the cytoplasmic face of the vesicles (Segev, 1991;

Barlowe et al., 1994), where we would anticipate Εril to function. Sari is the GTPase subunit of the COP II vesicle coat (Barlowe et al., 1994). For several reasons, Yptl is an even more attractive candidate than Sari. Yptl is a Rab (Ras from brain) GTPase that is required for tethering of ΕR-derived vesicles to Golgi membranes (Cao et al., 1998). Yptl was also shown recently to be required for sorting of GPI-anchored proteins from non-GPI-anchored proteins upon ΕR exit (Morsomme and Riezman, 2002). Finally, GFAI was isolated as a multicopy suppressor of a mutant that compromises Yptl function by affecting its prenylation state (Bialek-Wyrzykowska et al, 2000). Both a directed and an unbiased approach are taken to the identification ofthe second target of Εril function.

1. The candidate approach - Yptl and Sari

GST fusions of GTP-bound and GDP-bound (or nucleotide free) forms of these

GTPases are constructed for co-expression in yeast with HA-Εril. For Yptl, both GTP-bound (yptl-Q67L; Richardson et al., 1998) and nucleotide free (YPT1-N121I; Jones et al., 1995) forms have been described. For Sari, GTP-bound (SAR1-H77G) and GDP-bound (SAR1-T37N) forms exist (Aridor et al., 2001). These experiments are directly analogous to those described above using GST-Ras fusions.

Detection of a GTP-dependent association of Eril with Yptl or Sari supports a model of Eril as a regulator of small G-proteins, and lead to further investigations of the nature of this regulation. For example, identification of Yptl as a Eril- interacting protein, leads to testing directly the role of Eril in GPI-anchored protein sorting at the ER. Briefly, this involves the use of an in vitro ER budding assay that measures incorporation of cargo proteins into ER-derived vesicles (Muniz et al.,

2000). The assay is conducted by incubation of permeabilized spheroplasts (essentially membranes) with exogenous cytosol and cofactors from the same yeast strain (Muniz et al., 2001). Vesicles formed in this reaction are then separated from total membranes by centrifugation followed by flotation into a Nycodenz gradient. Different classes of vesicles are then separated by immunoisolation using an endogenously expressed cargo protein that has an epitope tag on its cytosolic tail (eg. Gap 1 -HA). The epitope tag is exposed on the surface of the vesicle, which allows its immobilization on beads. Using this assay, at least two classes of ER- derived vesicles can be distinguished ~ those that carry non-GPI-anchored cargo (found in the Gap-HA isolated vesicles) and those that carry GPI-anchored protein cargo (found in the non-Gap-HA vesicles; Muniz et al., 2001). More recently, cytosolic and membrane fractions from a conditional yptl mutant have been used to demonstrate that this G-protein is required for sorting of GPI-anchored proteins into distinct ER-derived vesicles (Morsomme et al., 2002). ErilΔ extracts are tested for the ability to segregate GPI-anchored proteins from non-anchored proteins.

2. The unbiased approach - selection of suppressor mutations

A finding that Erz7Δ cells are deficient in GPI-anchoring, or that they are deficient in GPI-protein transport but Εril does not associate with either Yptl or Sari, results in proceeding with an unbiased approach to the identification of a Εril target associated with GPI-anchor protein function. We have considerable evidence that the growth defect displayed by Eril A cells at 37°C results from a defect in ER-to- Golgi GPI-protein transport. Our multicopy suppressor screen of this growth defect yielded only GFAI, which works by elevating UDP-GlcNAc pools, presumably to drive GPI-anchor production. Therefore, a selection for dominant and recessive mutational suppressors of the Eril A growth defect is conducted. Selection of recessive (loss-of-function) mutants is conducted in haploids, whereas selection of dominant (gain-of-function) mutants is conducted in homozygous Erz7Δ diploids.

A finding that GPI-anchoring is normal (i.e. secretion is deficient), prompts attempts to exclude any suppressor that acts by elevating UDP-GlcNAc pools (eg. dominant GFAI), because such suppressors do not have the potential to illuminate the Εril target in the secretory pathway. Toward this end, we screen the collection of Eril A suppressors for those that also suppress the chitin hyper-accumulation phenotype. Such mutants are not likely to act by driving the anchoring process. This screen is anticipated yield components of the secretion pathway that may be specific for sorting of GPI-anchored proteins in the ΕR. Even if we find that Eril associates with Yptl or Sari, the proposed suppressor screen may provide further insight into the mechanism of protein sorting in the ER.

If it is found that Eπ7Δ cells are deficient in GPI-anchor production or attachment, then no mutants are excluded from the initial "search-and-capture" mission. It is anticipated that the isolation of one or more ofthe known components ofthe anchor assembly/attachment pathway, and perhaps a small G-protein not previously recognized are involved. There are seven genes Icnown in yeast to be directly involved in the nine steps of GPI-anchor assembly and attachment to target proteins (Orlean, 1997). However, many ofthe steps have yet to be associated with genes or even catalytic entities.

Of course, we are very interested in any GTPase that presents itself as a result of these mutant hunts. We proceed with these by constructing GDP- and GTP-bound GST fusions for testing for in vivo association with HA-Εril . However, if a GTPase that interacts with Eril is not identified, GST fusions of other candidate interactors are tested. Additionally, suppressor mutations (dominant and recessive) and null mutations in suppressor genes are examined in ERIl cells for GPI-protein-related defects.

3. Determining whether yeast Ras can signal from the ER and creating a more effective Eril-based

Ras inhibitor

Eril has been shown to associate in vivo with GTP-Ras in an effector loop- dependent manner. However, Eril behaves genetically as an inhibitor of Ras. We have shown that Eril resides in the perinuclear and peripheral ER membrane. By contrast, the vast majority of Ras resides on the plasma membrane (Bartels et al., 1999). Although we cannot exclude the possibility that a small pool of Eril exists in the plasma membrane to act on Ras, even strongly overexpressed Eril appears to be restricted to the ER (data not shown). Therefore, our hypothesis is that Eril acts as a competitive inhibitor of the Ras-effector interaction while Ras is in the ER. Two predictions arise from this model. First, Ras that is restricted to the ER is competent to signal. Such a finding would be consistent with the need for an ER-resident Ras regulator. Second, the ability of Eril to inhibit Ras is restricted by its intracellular segregation from Ras at the cell surface. Indeed, although overexpression of Eril suppresses the heat shock sensitivity of a partial Ras-GAP mutant (iral A), it does not suppress the more severe heat shock sensitivities of either a mutant completely devoid of Ras-GAP (iral A iral A), or of a Ras mutant that is locked in the GTP- bound state (Ras2-V19). If our model is correct, we should be able to improve the ability of Eril to inhibit Ras simply by allowing it access to its target at the plasma membrane.

D. Restricting Ras to the ER

Chiu et al. (2002) recently demonstrated that H-Ras that is stringently restricted to the ER was capable of signaling. This was accomplished by fusing Ras to the C- terminus of a transmembrane domain that possesses an ER localization signal (avian infectious bronchitis virus M protein). The orientation of viral M protein fusions expressed in yeast suggests this concept can be successfully carried over to our system (Carolyn Machamer, JHU, personal communication). Therefore, we create similar fusions of Ras2 with the viral M protein, or alternatively to the transmembrane domain of Sec 12, which is also Icnown to be necessary and sufficient for ER localization of fused proteins (Sato et al., 1996). In the latter case, we fuse the Secl2 domain to the C-terminus of Ras to achieve cytoplasmic orientation. We also determine if the CaaX sequence is required for function in the Sec 12 context, as it is in the M protein fusion (Chiu et al., 2002). ER localization is detected by fluorescence microscopy using a functional form of Ras that is fused at its N- terminus to green fluorescent protein (gift of Susan Michaelis). Fusions that are restricted to the ER are tested for the ability to complement the lethality of raslA ras2A. An ER-localized fusion of an activated Ras allele (Ras2-V19) is tested for complementation of the ras null and for heat shock sensitivity in wild type cells. These experiments are also done in the absence of ERIl to determine the effect of the Ras inhibitor on the function of ER-localized Ras. Ability of ER-restricted Ras to function in any of these contexts, would supports a model in which ER-localized Ras requires regulation.

E. Targeting Eril to the plasma membrane.

Two basic approaches are taken to target Eril, or a portion of Eril to the plasma membrane. One approach is to attach a lipid modification target sequence either to the N-terminus or the C-terminus of Eril. This approach has been used successfully to target cytoplasmic proteins to the plasma membrane, including Ras effectors (Stokoe et al., 1994; Wennstrom and Downward, 1999; Kulik et al., 1997). Another approach is to fuse Eril sequences to the C-terminus of a type-1 plasma membrane protein. In this case, Eril is presented on the cytoplasmic face of the plasma membrane as part of a protein that is normally directed to the cell surface.

1. Addition of lipid modification motifs The most commonly used membrane-targeting sequences are for N-terminal myristoylation, or C-terminal farnesylation (Reuther et al., 2000). Fusion ofthe N- terminal 8 amino acids from the rat sarcoma virus Gag protein (MGQSLTTH) is sufficient for cotranslational myrisoylation of the glycyl residue (Kaplan et al., 1990). Alternatively, N-terminal acylation with both myristate and palmitate has been described for the Src protein kinases (Resh 1994), and can be achieved by including a cysteine residue within a consensus myristoylation sequence (eg. MGCSLTTH; Reuther et al., 2000).

Adding an N-terminal myristoylation sequence to Eril is expected to result in cytoplasmic presentation of the C-terminal region. Because we do not currently know the orientation of Eril in the membrane, we also construct C-terminal fusions of Eril to the farnesylation sequence of Ras. For these experiments, we use the yeast Ras2 CaaX motif (GGCCIIS). Although the CaaX tetrapeptide is sufficient to act as a signal for prenylation (Reuther et al., 2000), Ras can also be palmitoylated at a nearby cysteine, which directly neighbors the farnesylated cysteine in yeast Ras.

This additional modification is known to enhance membrane association of Ras (Hancock et al., 1990; Bhattacharya et al., 1995). Our job is complicated by the fact that Eril is normally directed to the ER membrane. Because Eril possesses no dilysine motif for ER retrieval, it is possible that it possesses an ER localization signal within its transmembrane domain, as is the case for Secl2 (Sato et al., 1996).

Therefore, we create membrane targeting fusions not only to full-length Eril, but also to the N-terminal half and the C-terminal half of Eril, so as to remove one of the potential transmembrane domain sequences. Upon determining which region interacts with Ras, and which resides in the membrane, that information guides the construction of fusions.

All fusions include an HA-epitope so that their intracellular locations can be assessed by indirect immunofluoresence. We know that an N-terminal HA-tag does not impair Eril function. We also determine in the course of our experiments if a C- terminal tag can be used. Additionally, upon success at directing localization of Eril to the plasma membrane, our prediction is that it will be a potent inhibitor of Ras, whose function is essential. Therefore, we place expression of our fusions under the inducible control of the GAL1 promoter. In this way, if we achieve effective inhibition of Ras signaling, growth arrest will be conditional.

2. Fusion of Eril to a type-1 plasma membrane protein

In a parallel approach to the fusions described above, we will fuse Eril (or parts of Eril) to the Mid2 protein. Mid2 is a cell surface sensor for cell wall integrity signaling, which possesses a single transmembrane domain that separates an N- terminal extracellular domain from a C-terminal intracellular domain (Rajavel et al., 1999, Philip and Levin 2001). We have shown that Mid2 is evenly distributed throughout the plasma membrane, making it a good candidate for fusion to Eril. We create fusions that express the Mid2 extracellular domain and transmembrane domain attached to Eril sequences. The replacement of the intracellular domain of Mid2 with Eril sequences can result in the presentation of Eril to Ras on the cytoplasmic face ofthe plasma membrane.

Our alanine scanning and site directed mutagenesis described above can produce a Erz7 mutant with a defective ΕR-retention signal. Upon finding such a mutant that mislocalizes Εril to the cell surface, it is included with other plasma membrane- localized forms of Εril for analysis in the next step. Alternatively, upon finding that Εril possesses an ΕR targeting sequence within its transmembrane domain, we can replace that region with the Mid2 transmembrane domain. As described above, these fusions will include HA-tags and be expressed under GAL1 control.

3. Testing for Ras inhibition

After expression of Εril on the cell surface, then tests for inhibition of Ras are performed in two ways. If cell surface expression of a Εril fusion does not cause growth arrest, we test it for suppression of hyperactive Ras phenotypes.

Specifically, we test for suppression of the heat shock sensitivities and invasive growth phenotypes associated with loss of IRA1, or IRAl and IRA2 together, as well as suppression of constitutive RAS2 (RAS2-V19). However, if surface expression of a Eril fusion results in growth arrest, it must be determine if loss of Ras signaling is the cause. If so, the Eril-induced growth arrest may be suppressed by mutations that activate the cAMP signaling pathway downstream of Ras. For example, a constitutive mutation in adenylate cyclase, which allows cell growth in the absence of Ras (SRA4-6; Cannon et al., 1986) should relieve the growth arrest caused by expression of a strongly Ras-inhibitory form of Eril. Evidence that plasma membrane expression of Eril results in enhanced Ras inhibitory function may be taken as support for the hypothesis that the modest ability of wild-type Eril to inhibit Ras is a consequence of its intracellular location.

We also create a form of Eril that effectively inhibits Ras signaling at the plasma membrane, and create suitable Eril constructs for expression in Ras-fransformed mouse fibroblasts. We also create fusions of Eril -derived sequences to the protein transduction domain of HIV-TAT for the purpose of introducing Eril into animal cells from the medium (Schwarze et al., 2000).

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Ras oncoproteins are monomeric GTPases that link signals from the cell surface to pathways that regulate cell proliferation and differentiation. Constitutively active mutant forms of Ras are found in approximately 30% of human tumors. Here we report the isolation of a novel gene from Saccharomyces cerevisiae, designated ERIl

(for Endoplasmic Reticulum-associated Ras Inhibitor 1), which behaves genetically as an inhibitor of Ras signaling. ERIl encodes a 68-amino acid protein that associates in vivo with GTP-bound Ras in a manner that requires an intact Ras- effector loop, suggesting that Eril competes for the same binding site as Ras target proteins. We show that Eril localizes primarily to the membrane ofthe endoplasmic reticulum (ER), where it engages Ras. The recent demonstration that signaling from mammalian Ras is not restricted to the cell surface, but can also proceed from the cytoplasmic face ofthe ER suggests a regulatory function for Eril at that membrane.

The Ras family of small GTPases comprises a group of molecular switches that, in the GTP-bound active state, transmit signals by interaction with effector proteins (44). Ras proteins play an important role in the transduction of external signals that regulate cell proliferation, differentiation, and metabolism (reviewed in ref. 33). Constitutively GTP-bound mutants of Ras are found in approximately 30% of human tumors (25). In cases of colorectal or pancreatic cancers, this incidence is as high as 50% or 90%, respectively (11). The three forms of mammalian Ras (H-Ras, K-Ras, and N-Ras) are identical through their N-terminal 85 residues, which possess the structural features necessary for guanine nucleotide exchange, GTPase activity, and effector function (11). Moreover, they all interact with the same set of effectors. The best studied of these are the Raf protein kinases, whose stimulation by Ras activates the ERK MAP kinase cascade, the core ofthe major proliferative pathway in metazoan cells (45). Other Ras effectors include phosphoinositide 3-kinase (PI- 3K; 44), which prevents apoptosis through the Akt protein kinase, Ral-GEF, the guanine nucleotide exchange factor for Ral-GTPases (10a), and the 14-3 -3 -binding protein RTN1 (42). In all cases, effector association is dependent not only on the nucleotide-bound state of Ras, but also on an intact effector loop, a highly conserved region that is exposed for effector interaction through a conformational change induced by GTP-binding (45).

The budding yeast Saccharomyces cerevisiae possesses two functionally overlapping RAS genes, which share 84% sequence identity with their mammalian counterparts though their N-terminal domains (17). Although the essential effector of yeast Ras is adenylyl cyclase (39), an enzyme not regulated by Ras in animal cells, loss of yeast Ras function can be complemented by expression of mammalian Ras genes (9, 16). Conversely, a mutationally activated allele of yeast RASl is capable of causing malignant transformation of mouse fibroblasts (9). The conservation of biological properties among these members of the Ras gene family is reflected in the observation that the effector loops of yeast Ras are identical to those of mammalian Ras.

Newly synthesized Ras undergoes a series of evolutionarily conserved posttranslational modifications at its C-terminal CAAX motif that render it more hydrophobic (3, 6, 8, 29, 32). The first modification, prenylation of the CAAX cysteine, targets Ras to endomembranes (6). The next two steps, proteolytic removal ofthe AAX residues and carboxymethylation ofthe prenylated cysteine, occur at the endoplasmic reticulum (ER) prior to transit of the mature form to the plasma membrane (PM; 27). In contrast to conventional cargo carried in vesicle lumens, Ras must be transported on the cytoplasmic surface of vesicles. This leaves open the possibility that Ras can undergo nucleotide exchange and interact with its effectors while associated with endomembranes. Indeed, Chiu et al. (5) demonstrated that signaling from mammalian H-Ras and N-Ras is not restricted to the plasma membrane as previously thought, but can proceed from the ER and Golgi compartments, resulting in differential activation of its various signaling pathways. However, nothing is known about Ras regulation at endomembranes. Here, we describe a novel yeast gene encoding a Ras inhibitor that engages Ras at the ER. MATERIALS AND METHODS

Strains and growth conditions. The S. cerevisiae strains used in this study are listed in Figure 20. Yeast cultures were grown in YEPD (1% Bacto yeast extract, 2% Bacto Peptone, 2% glucose) with or without 10% sorbitol. Synthetic minimal

(SD) medium (31a) supplemented with the appropriate nutrients was used to select for plasmid maintenance and gene replacement. Escherichia coli DH5α was used to propagate all plasmids. E. coli cells were cultured in Luria broth medium (1% Bacto Tryptone, 0.5% Bacto yeast extract, 1% NaCl) and transformed to carbenicillin resistance by standard methods.

Construction of plasmids and genomic deletions. The ERIl gene was isolated from a centromeric yeast library (30) by complementation of the temperature- sensitive growth defect associated with an erz7-7 mutant (DL2698). The 498bp intergenic region between YPL096w and YPL097w (MSY1), which carries the ERIl gene, was amplified by PCR from genomic DNA from strain 1783 and cloned into the EcoRl site of centromeric (pRS316) and 2μ (pRS426) plasmids to yield pRS316[Eic/J] (pl380) and pRS426[Erø] (pl381), respectively.

Construction of a fully functional HA-tagged form of Εril (HA-ERI1) under the transcriptional control of the GALl promoter, the MET25 promoter, or its own promoter, is described below. For construction of GAL-HA-ERH, the ERIl ORF was amplified with 285 bp of 3' sequence and subcloned into pYeFl (URA3; 1) using Notl and EcoRl. This construction (pYeFl[ER7i]; pl461) fused ERIl in- frame at its Ν-terminus with a single copy ofthe HA epitope and places it under the inducible control of the GALl promoter. To switch markers, the URA3 gene of pYeFl[Ei7i] was disrupted with TRP1 by subcloning a Smal fragment bearing TRP1 (from ΌVC18[TRP1]) into the EcoRV site in URA3, resulting in pYeFl ::TRP1[ERI1] (ρl482). ^Εril was capable of complementing the erilA growth defect even when repressed with 2% glucose (not shown). To create HA- ERIl under the control of the ERIl promoter, a Bamiil -EcoRl fragment from pYeFl[ERH] that includes FLA-ERH but not the promoter, was first inserted into pRS316 and pRS426. Then 680bp of sequence 5' to the ERIl start codon was amplified by PCR and inserted into the BamHl site of the resulting plasmids, yielding pRS316[HA-ER/i] (pl740) and pRS426[HA-ER/i] (pi 742). To create ERIl and HA-ERI1 under the constitutive control ofthe MET25 promoter, the ERIl (or HA-ERI1) coding sequence was amplified and inserted into pRS426- Er25

(2μ) or pRS416-MET25 (cen) between the MET25 promoter and the CYCl terminator (23). The pΕGKG[&4S2] (21) and pEGKG[Λ4S2 ] plasmids, which express GST-Ras2 under the control of GALl, were gifts of Bob Deschenes. Effector site mutants in RAS2^VI9 were constructed by the PCR overlap extension method (13) and cloned into pEGKG.

Deletion of the genomic copies of ERIl, RAS2, IRA1 and IRA2 in the 1783 strain background is described below. To delete the genomic copy of ERIl, 1508 bp of sequence 5' to the ERIl start codon and 677 bp of sequence 3' of the ERIl stop codon were amplified in separate PCR reactions from genomic DNA from strain

1783. The 5' fragment was amplified with primers that placed an EcoRl site at end adjacent to the ERIl coding sequence and a BamΑl site at the opposite end. The 3' fragment was amplified with primers that placed a Notl site adjacent to the ERIl coding sequence and a BamHl site at the opposite end. These fragments were ligated in a three-molecule reaction to the EcoRl and Notl sites of the integrative plasmid pRS304 (34) to create a unique BamHl site between the fragments. The resulting plasmid, pRS304[OT7Δ::7KP./] (pl356), was linearized with BamHl and used to transform yeast strains to tryptophan prototrophy. For disruption of RAS2, the τpras2::LEU2 plasmid of Kataoka et al. (17) was used as described (gift of S. Powers). Deletion of IRA1 was described previously (36). For deletion of IRA2, the

LEU2 gene of pRS304 was first replaced with the HIS4 gene (Smal to EcoRV) from pBluescript2 fflS¥] (gift of Susan Michaelis) by insertion of the HIS4 fragment between the Hpal and Aatll sites of pRS304 to create YlpHIS4 (pi 187). Next, 1.1 kb of sequence 5 ' to the IRA2 start codon and 1.8kb of sequence 3 ' of the IRA2 stop codon were amplified in separate PCR reactions from genomic DNA. The 5' fragment was amplified with primers that placed a Notl site at end adjacent to the IRA2 coding sequence and an Spel site at the opposite end. The 3' fragment was amplified with primers that placed a BamHl site adjacent to the IRA2 coding sequence and an Spel site at the opposite end. These fragments were ligated in a three-molecule reaction to the BamHl and Notl sites of YlpHIS4 to create a unique Spel site between the fragments. The resulting plasmid, YIp[z>α2Δ::.fflS ] (pl584), was linearized with Spel and used to transform yeast strains to histidine prototrophy.

All genomic deletions were confirmed by PCR.

Immunodetection of ^HAEril and association of ^HAEril with GST-Ras2. Indirect immunofluorescence microscopy to detect ^HAEril was performed as described previously (15) using a Zeiss Axioskop filled with a FITC filter. For subcellular fractionation experiments, transformants of yeast strain 1783 bearing pRS416[ E7/25-HA-E#7;] were grown to mid-log phase in YΕPD, and lysates were prepared as described previously (15). Lysates were centrifuged at 100,000g in an SW50.1 rotor (Beckman) for lh. Supernatant and pellet fractions (resuspended in lysis buffer to equivalent volume as supernatant fractions) were separated by SDS-

PAGΕ for immunoblot detection of ^HAΕril. For in vivo association experiments, double transformants bearing pYeFl ::TRP1[ERI1] and ΌΕGKG[RAS2] (or a mutant form of RAS2) were precultured in YEP with 2% raffinose, followed by induction with 4% galactose for 5h. Lysate preparation and immunoblotting were done as described previously, except for the addition 0.5% ΝP-40 to the lysis buffer (15).

GST-Ras2 was precipitated from lysates with glutathione Sepharose 4B beads (Amersham Pharmacia) equilibrated in Tris Buffered Saline TBS (50 mM Tris-HCl [pH 7.6], 8% NaCl), with 0.1% NP-40 (TBSN). Briefly, 4 ml of TBSN and 50 μl beads were added to 0.5 ml extract (10-15mg/ml protein) and tumbled for lh at 4°C. Beads were washed three times with 10 ml TBSN and eluted with SDS-PAGE loading dye (130 μl final volume). ^HAEril was detected (from 10-20 μl of sample) with mouse monoclonal antibody 12CA5 (BabCo), and GST-Ras2 was detected (from 2-4 μl sample) with anti-GST (Amersham Pharmacia).

For separation of ER from plasma membrane, ^HAEril and GST-Ras2^V19 were induced as above prior to cell lysis by agitation with glass beads. Membranes were fractionated by sedimentation on a step sucrose/EDTA density gradient by a method modified as follows from Valdivia et al. (40). Unlysed cells were removed by centrifugation (500g for 5 min). Total cell lysates (0.2 ml) were overlaid on a step sucrose/EDTA gradient [0.2 ml 55%, 0.5 ml 45%, 0.4 ml 30% sucrose (w/w) in 20 mM triethanolamine, pH 7.2, 5 mM EDTA] and centrifuged at 46,400 rpm in an SW50.1 rotor for 5h. Fractions (0.2 ml) were collected manually from the top and separated by SDS-PAGE for immunoblot analysis for GST-Ras2^V19, ^Eril, Gasl (with rabbit anti-Gas 1 serum; gift of Laura Popolo), Dpml (with mouse monoclonal anti-Dpml, 5C5; Molecular Probes) and CPY (with mouse monoclonal anti-CPY, 10A5; Molecular Probes). After addition of non-ionic detergent (0.5% NP-40) to the fractions, GST-Ras2^V19 was then precipitated from fractions and subjected to immunoblot analysis for ^Eril. Films from immunoblots were scanned into Adobe Photoshop (v. 5.0) for densitometric analysis using Image Gauge (v. 3.3) software.

Heat shock sensitivity and FRE: lacZ assays. Heat shock sensitivity of eril A was assayed after 24h growth on a YEPD plate. The iral A strain was tested for heat shock sensitivity after 36h on an SD plate. Cells were collected from plates, resuspended in YEPD at vlδoo =1.0 and serial 10-fold dilutions were made in YEPD. One microliter of each dilution was spotted onto a YEPD plate, which was incubated in a 50°C water bath for either 30 min (iral A), or 50 min (eril A) by submerging the plate in a sealed bag. Colonies were allowed to grow for 2 days at room temperature before counting. Percent survivors were calculated as colony-forming units relative to non-heat shocked controls. Each value represents the mean and standard deviation of at least three experiments. The ability of Eril overproduction to downregulate Ras2^vl9-driven FREv.lacZ expression was tested as described by Mosch et al. (22). Yeast strain YHUM120 was transformed with centromeric plasmids pRS3l6[RAS2] or τpRS3l6[RAS2-V19] and either multicopy plasmid ΌRS424[ERI1] or pRS424. Transformants were patched onto SD plates and allowed to grow at 30°C for 24h. Lysates were made from cells collected from plates and assayed for β-galactosidase activity. Values represent the mean and standard deviation from at least three independent transformants. Results and Discussion

ERIl encodes a novel yeast gene. We isolated ERIl through a genetic screen for mutants with growth defects that were additive with that of a conditional mutant in protein kinase C (pkcl; 30), a regulator of cell wall biogenesis. The ERIl gene (YPL096c-A) encodes a previously non-annotated open reading frame (nORF) of only 68 amino acids (Figure 14A). Several lines of evidence support the conclusion that ERIl is a bonafϊde gene. First, searches of genome databases have revealed several putative ERIl homologs from a variety of fungi, including C. albicans, S. pombe, A. fumagatus, and N. crassa. The closest of these to S. cerevisiae Eril are shown in Figure 14B. Database searches have not revealed any metazoan Eril homologs. Second, a single orphan SAGE-tag (serial analysis of gene expression) corresponding to the ERIl locus was identified previously (41), suggesting that a polyadenylated mRΝA is expressed at a low level from this gene. Indeed, we found that an ERIl probe hybridizes to an mRΝA of approximately 300 nucleotides from both log phase and stationary phase cells (Figure 14C). Third, the original eril mutant was recessive, resulting from a frameshift mutation after the eighth codon. Fourth, a precise deletion of ERIl results in a growth defect at elevated temperature (37° C) that is complemented by a centromeric plasmid bearing 498 bp of sequence corresponding to ERIl and its regulatory elements only (Figure 14D). Fifth, we can detect an epitope-tagged form of the Eril protein expressed from its own promoter

(see Fig. 18B).

The erz7Δ phenotypes can be divided into two categories that reflect two distinct functions of this small protein. One set of phenotypes, described here, is associated with hyperactive Ras pathway signaling. The other phenotypes, including the growth defect at elevated temperature, are associated with a deficiency in anchoring of glycosyl-phosphatidylinositol (GPI)-proteins and their subsequent secretion to the cell surface. The latter phenotypes are responsible for the additive growth defect observed with pkcl mutants and will be described elsewhere.

ERIl behaves genetically as a Ras inhibitor. Some strains of Saccharomyces cerevisiae have the capacity to undergo a dimorphic shift on solid medium in response to nutrient limitation from ovoid, budding cells to a multicellular form consisting of filaments of elongated cells (12, 28). This shift is enhanced by hyperactivation of Ras pathway signaling (12, 19, 43), and specifically requires signaling from Ras2 (22). Microscopic examination of diploid erz7Δ cells grown on solid rich medium (Fig. 15 A) revealed that they display the characteristic features of filamentous growth when cultivated at 34°C. This was unexpected, both because nutrients were not limiting in this setting and because the strain background used in this study (EG123; 35) is not known to be capable of filamentous growth. Therefore, to determine if the observed behavior of erz7Δ cells reflects bona fide filamentous growth, we deleted RAS2 in an erz7Δ mutant. The eril A ras2A mutant displayed a normal budding morphology, indicating that Ras2 is required for filamentation of eril A cells. This result also suggested that Ras pathway signaling is hyperactivated in the eril A mutant. Another behavior associated with filamentous growth is the ability to invade the agar, which can be detected after washing nonadeherent cells from the plate (28). An ez*z7Δ mutant displayed agar invasion

(Fig. 15B) similar to that observed for mutants in the same strain background with hyperactive Ras resulting from loss of Ras-GTPase-activating protein (Ras-GAP) function (Fig. 15C). Moreover, an erz7Δ ras2A mutant was suppressed for agar invasion, supporting the notion that Ras pathway signaling is hyperactive in erz7Δ mutants. By contrast, deletion of RAS2 failed to suppress the growth defect of eril A cells at 37°C (not shown), suggesting that this phenotype is not the result of hyperactive Ras signaling. Additionally, hyperactive Ras pathway mutants are not temperature-sensitive for growth, supporting the conclusion that this phenotype is independent of Ras function.

Hyperactive Ras signaling in yeast also results in the failure to acquire thermotolerance in preparation for stationary phase (18, 24). We reasoned that if the invasive growth phenotype of erz7Δ cells was a consequence of hyperactive Ras pathway signaling, this mutant should also display a defect in the acquisition of thermotolerance. A saturated erziΔ culture lost two logs of viability relative to wild type in response to a brief heat shock at 50°C (Fig. 16A). This defect was suppressed by downregulation ofthe Ras pathway through expression of a dominant negative form ofRAS2 (14), or through overexpression of the Ras-GAP encoded by IRA2 (38). Conversely, the heat shock sensitivity of a mutant defective in one of two redundant Ras-GAPs (iral A), was partially suppressed by overexpression of ERIl under the control of the MET25 promoter (Fig. 16B), further supporting the conclusion that Eril can inhibit Ras pathway signaling. However, we were not able to demonstrate with this assay that the more severe heat shock sensitivity resulting from either a complete loss of Ras-GAP activity (in an iral A iral A mutant), or a constitutively active allele of RAS2 (RAS2-V19) was suppressed by ERIl overexpression (data not shown), suggesting that the ability of Eril to inhibit Ras pathway signaling is limited. We detected no increase in severity ofthe heat shock sensitivity of an iral A ira2A mutant in the presence of an erz7Δ mutation (not shown).

As a final test of the ability of Eril to regulate Ras pathway activity, we examined the expression of a Ras2-regulated reporter gene. Hyperactivation of Ras pathway signaling induces expression of a reporter under the control of the filamentous response element (FREv.lacZ; 22). This reporter has the advantage of providing a more sensitive output for Ras pathway signaling than the heat shock sensitivity assay. Expression of constitutively active RAS2-V19 from a centromeric plasmid induced FREv.lacZ expression 1.7-fold as compared with expression of wild type

RASl (201 + 9 U vs. 121 + 10 U). Overexpression of Eril from a multicopy plasmid reproducibly diminished FREv.lacZ expression in the presence of RAS2-V19 (163 + 18 U). Further overexpression of Eril under the control of the MET25 promoter (yielding approximately 10-fold more Eril than the multicopy plasmid; not shown) did not diminish reporter activity further (161 + 19 U), underscoring the limitation of overexpressed Eril to inhibit Ras pathway signaling. These results, taken in the aggregate, indicate that Eril is an inhibitor of Ras pathway activity that is capable of partially downregulating the output of an oncogenic form of Ras.

Eril associates with Ras2 in a GTP- and effector loop-dependent manner. We were interested to determine the point at which Eril acts to inhibit Ras pathway signaling. An additional heat shock experiment suggested that Eril acts at the level of Ras. Specifically, overexpression of ERIl failed to suppress the modest heat shock sensitivity of a constitutive mutant in adenylyl cyclase (SRA4-6; data not shown; 4), the direct effector of Ras in S. cerevisiae. Therefore, we tested for association of Eril with Ras2 in vivo. Glutathione-S-transferase (GST)-tagged Ras2, or a constitutively active mutant form (GST-Ras2 ¹⁹) was co-expressed in yeast cells with HA-tagged Eril (^Eril). GST-Ras2 was affinity purified from cell extracts made in the presence of non-ionic detergent (0.5% NP-40) to disrupt membranes and tested for association with ^HAEril by immunoblot analysis. Although only a weak ^HAEril signal was detected in association with wild-type Ras2, we reproducibly detected a strong signal associated with the activated form of

Ras2 (Fig. 17A). Because wild-type yeast Ras2 exists almost entirely in the GDP- bound state in vivo (11a), whereas the constitutively active Ras2^VI9 is "locked" in the GTP-bound state (11), the observed difference in association between the two forms could reflect the difference in bound nucleotides. To determine if Eril association with Ras2 is dependent on its nucleotide-bound state, we isolated GST-

Ras2 from cells devoid of Ras-GAPs (iral A iralA). In this setting, Ras is predominantly GTP-bound. In the absence ofthe Ras-GAPs, ^Eril associated with wild-type GST-Ras2 as well as with the activated form (Fig. 17A), indicating that Eril associates specifically with GTP-bound Ras2. Another indication of the specificity of the interaction is that we were not able to detect association of Eril with a constitutive form of Rhol (GST-Rhol^68H), the yeast GTPase most closely related to Ras (data not shown). Section 1.01

GTP-dependent association of a protein with Ras is one of two criteria used in the identification of novel Ras effectors (44). The other criterion is the requirement of an intact Ras-effector loop for interaction. Mutations in the Ras effector loop, comprised of residues 39-47 in yeast Ras (corresponding to residues 32-40 in mammalian Ras), disrupt interaction with effector proteins (1, 10, 20, 37). To determine if Eril associates with GTP-Ras2 through its effector loop, we introduced either of two mutations known to disrupt Ras interaction with adenylyl cyclase (A42 or N45;l, 37) into the constitutive RAS2 mutant (creating RAS2-V19, A42 and RAS2- V19, N45). Figure 17B shows that both effector loop mutations blocked the GTP- Ras interaction with Eril, indicating that Eril associates with Ras in an effector loop-dependent manner. To summarize, Eril fits the operational definition of a Ras effector, but behaves genetically as a Ras inhibitor. Therefore, Eril may act as a competitive inhibitor of Ras signaling by shielding the effector loop of GTP-Ras from interaction with its effector.

Eril engages Ras at the ER membrane. The Eril protein possesses two highly hydrophobic regions (residues 7-28 and residues 34-56; Fig. 14A) either of which is long enough to constitute a transmembrane domain. To determine if Eril is associated with a membrane, we examined the fractionation pattern of ^HAEril in yeast cell extracts. Figure 18A shows that ^HAEril sedimented with the pellet from a 100,000g centrifugation. ^HAEril was liberated by addition of non-ionic detergent (Triton X-100) to disrupt membranes, but not by other treatments that would liberate peripherally associated membrane proteins (i.e. urea or high pH). Therefore, we conclude that Eril is an integral membrane protein. To determine with which intracellular membrane Eril associates, we conducted indirect immunofluorescence microscopy on cells expressing ^Eril from its own promoter on a multicopy plasmid. ^HAEril displayed a pattern of perinuclear fluorescence with swirls out to the cell periphery (Fig. 18B), which is characteristic of proteins that reside in the ER (2, 29, 31, 32). This pattern was observed consistently in cells across the entire cell cycle.

Although the majority of Ras resides on the PM (2), recent studies indicate that it also associates with the ER, at least transiently in both mammals and yeast (5, 6, 8, 27, 29, 32). Therefore, we tested the possibility that Eril engages Ras at the ER.

Total membranes from cells coexpressing GST-Ras2^v19 and ^Eril were sedimented on a step sucrose/EDTA density gradient. As anticipated from the immunofluorescence localization of ^HAEril, the majority of the ^Eril (56%) co- sedimented with the ER marker Dpml in fraction 1 (Fig. 19 A), with the remainder diminishing through fractions 2-6. The PM marker Gasl sedimented in fractions 3-

6, with the majority (66%) sedimenting in fractions 4 and 5. However, the immature, ER-modified form of Gasl (26) sedimented in fraction 1. By contrast, GST-Ras2^V19 was evenly distributed across the entire gradient, suggesting its localization at endomembranes as well as at the PM. After disruption of the membranes with detergent, GST-Ras2^v19 was affinity purified from the gradient fractions and tested for association with ^HAEril. ^Eril was found in association with GST-Ras2^V19 mainly from fraction 1 (Fig. 19B), indicating that Eril engages

GTP-bound Ras at the ER. Some ^Eril was detected with GST-Ras2^V19 isolated from fractions 3 and 4, suggesting that a pool of Eril may also engage Ras at a heavier membrane. The failure of ^HAEril to associate with GST-Ras2^V19 from fraction 2 may be explained by the predominance of the vacuolar marker CPY in that fraction. Because both proteins were overexpressed, some of each may be targeted for destruction in the vacuole, where they are not likely to interact.

Eril and Ras reside largely on separate membranes, with the majority of Eril in the ER, but apparently no more than 20% of the total Ras at that organelle (see Fig. 19A). Therefore, to rule out the possibility that ^Eril and GST-Ras2^V19 associate fortuitously in vitro only after liberation from their respective membranes, we tested for their association after expression in separate cell populations. Cultures of cells expressing GST-Ras2^V19 or ^HAEril were combined and GST-Ras2^v19 was affinity purified from extracts made in the presence of detergent. In contrast to the association detected when the proteins were coexpressed in the same cell population

(see Fig. 17A), we were not able to detect their association in vitro when the proteins were initially in separate membranes (data not shown). This indicates that the observed interaction occurs in vivo between the pools of Eril and Ras2 that reside on the same membrane. Because the GTP-Ras2/Eril association was detected in vivo, we do not know if this interaction is direct or bridged by another protein. Eril could be part of an ER protein complex that regulates Ras signaling.

If yeast Ras normally signals from endomembranes as has been shown for mammalian Ras (5), Eril (or an Eril -containing complex) may function to regulate such signaling at the ER. Alternatively, Eril may function as a molecular chaperone to maintain Ras in an inactive state while the GTPase is being processed at the ER. In either case, the segregation of the majority of these proteins on separate membranes is likely to explain the limited ability of Eril to inhibit GTP-bound Ras. It may be possible to enhance the efficacy of Ras inhibition by targeting Eril to the PM.

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Claims

Claims

1. An agent capable of blocking the interaction between GTP-Ras and ERIl, wherein ERIl is a protein comprising amino acids, the sequence of which amino acids is set forth in SEQ ID NO:7.

2. The agent of claim 1, wherein the protein is encoded by a nucleic acid comprising nucleotides, the sequence of which nucleotides is set forth in SEQ ID NO:6.

3. The agent of claim 1, wherein the agent is capable of binding to GTP-Ras.

4. The agent of claim 3, wherein the agent inhibits the ability of GTP-Ras to signal.

5. The agent of claim 3, wherein the agent is a polypeptide or portion thereof.

6. The agent of claim 5, wherein the polypeptide is ERIl or a portion thereof.

7. The agent of claim 6, wherein the agent is a portion of ERIl , and the portion is a GTP-Ras binding motif.

8. The agent of claim 6, wherein the agent is a portion of ERIl, which portion comprises amino acids, the sequence of which is set forth in SEQ ID NO: 9.

9. The agent of claim 6, wherein the agent is a portion of ERIl, which portion comprises amino acids, the sequence of which is set forth in SEQ ID NO:l 1.

10. The agent of claim 5, wherein the polypeptide comprises amino acids, the sequence of which amino acids is set forth in SEQ ID NO: 7.

11. The agent of claim 3, wherein the agent is a polypeptide comprising the sequence set forth at residues 1-39 of SEQ ID NO:7.

12. The agent of claim 3, wherein the agent is a polypeptide encoded by a nucleic acid which hybridizes under high stringency to a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:6.

13. The agent of claim 3, wherein the agent is a polypeptide encoded by a nucleic acid comprising the nucleotide sequence set forth at nucleotides 1- 117 of SEQ ID NO:6.

14. The agent of claim 5, wherein the polypeptide is encoded by a nucleic acid, the sequence of which nucleic acid is set forth in SEQ ID NO: 6 .

15. The agent of claim 1, wherein the agent is a fusion protein which comprises

(1) a peptide which comprises at least one GTP-Ras binding motif, and (2) a transcellular polypeptide for promoting transcytosis of the fusion protein across a cell surface membrane.

16. The agent of claim 15, wherein the peptide corresponds to at least a portion ofthe sequence set forth in SEQ ID NO: 7.

17. The agent of claim 15, wherein the portion is a GTP-Ras binding motif.

18. The agent of claim 16, wherein the peptide comprises at least a portion of the sequence set forth at residues 1-39 of SEQ ED NO:7.

19. The agent of claim 16, wherein the peptide is encoded by a nucleic acid which hynridizes under high stringency conditions to the nucleic acid set forth at nucleotides 1-117 of SEQ ID NO:6.

20. The agent of claim 16, wherein the portion of ERIl is the sequence set forth in SEQ ID NO:9.

21. The agent of claim 16, wherein the portion of ERIl is the sequence set forth in SEQ ID NO:ll.

22. The agent of claim 16, wherein the agent is encoded by a nucleic acid which hybridizes under high stringency to a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:6.

23. The agent of claim 15, wherein the transcellular polypeptide is an internalizing peptide.

24. The agent of claim 15, wherein the transcellualr peptide is derived from a polypeptide selected from the group consisting of antepennepedia protein,

HIV transactivating (TAT) protein, mastoparan, melittin, bombolittin, delta hemolysin, pardaxin, Pseudomonas exotoxin A, clathrin, Diphtheria toxin, C9 complement protein and Herpes-simplex-virus- 1 DNA binding protein.

25. The agent of claim 15, wherein the transcellular polypeptide is an accessory peptide which enhances interaction ofthe fusion protein with a cell surface membrane.

26. The agent of claim 25, wherein the accessory peptide comprises an RGD sequence.

27. The agent of claim 3, wherein the agent is a peptidomimetic.

28. The agent of claim 3, wherein the agent is a nonpeptidyl agent.

29. The agent of claim 3, wherein the agent is a small molecule.

30. The agent of claim 3, wherein the agent has a molecular weight less than 500 daltons.

31. The agent of claim 3, wherein the agent is an antibody or fragment thereof.

32. The agent of claim 31, wherein the antibody is a monoclonal antibody.

33. The agent of claim 31 , wherein the antibody is a polyclonal antibody.

34. The agent of claim 31 , wherein the antibody is a humanized antibody.

35. The agent of claim 31, wherein the antibody is a chimeric antibody.

36. The agent of claim 31, wherein the portion ofthe antibody comprises a light chain of the antibody.

37. The agent of claim 31, wherein the portion of the antibody comprises a heavy chain ofthe antibody.

38. The agent of claim 31, wherein the portion ofthe antibody comprises a Fab portion ofthe antibody.

39. The agent of claim 31, wherein the portion of the antibody comprises a F(ab')₂ portion ofthe antibody.

40. The agent of claim 31, wherein the portion of the antibody comprises a Fd portion ofthe antibody.

41. The agent of claim 31, wherein the portion of the antibody comprises a Fv portion of the antibody.

42. The agent of claim 31, wherein the portion of the antibody comprises a variable domain ofthe antibody.

43. The agent of claim 31, wherein the portion ofthe antibody comprises one or more CDR domains ofthe antibody.

44. A nucleic acid which encodes the agent of any one of claims 1 -43.

45. The nucleic acid of claim 44, which nucleic acid comprises nucleotides, the sequence of which nucleotides is set forth in SEQ ID NO: 6 (full length ERIl

DNA sequence)

46. A polypeptide encoded by the nucleic acid of claim 44.

47. The nucleic acid of claim 44, wherein the agent is a polypeptide or portion thereof, which polypeptide comprises amino acids, the sequence of which amino acids is set forth in SEQ ID NO:7.

48. The nucleic acid of claim 47, wherein the agent is a portion of the polypeptide, and the portion is a GTP-Ras binding motif.

49. The nucleic acid of claim 47, wherein the agent is a portion of the poljφeptide, and the portion comprises the amino acid sequence set forth in SEQ ID NO:9.

50. The nucleic acid of claim 47, wherein the agent is a portion of the polypeptide, and the portion comprises the amino acid sequence set forth in SEQ ID NO: 11.

51. The nucleic acid of claim 47, wherein the nucleic acid hybridizes under high stringency to a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:6.

52. The nucleic acid of claim 47, wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence set forth at residues 1-39 of SEQ ID NO:7.

53. The nucleic acid of claim 47, wherein the nucleic acid comprises the sequence set forth at nucleotides 1-117 of SEQ ID NO:6.

54. The nucleic acid of claim 44, wherein the agent is a fusion protein which comprises (1) a peptide which comprises at least one GTP-Ras binding motif, and (2) a transcellular polypeptide for promoting transcytosis of the fusion protein across a cell surface membrane.

55. The nucleic acid of claim 54, wherein the peptide corresponds to at least a portion ofthe sequence set forth in SEQ IN NO: 7 .

56. The nucleic acid of clahn 55, wherein the portion is a GTP-Ras binding motif.

57. The nucleic acid of claim 55, wherein the portion comprises the amino acid sequence set forth in SEQ ID NO:9.

58. The nucleic acid of claim 55, wherein the portion comprises the amino acid sequence set forth in SEQ ID NO: 11.

59. The nucleic acid of claim 54, wherein the transcellular polypeptide sequence is an internalizing peptide.

60. The nucleic acid of claim 59, wherein the transcellular polypeptide is derived from a polypeptide selected from the group consisting of antepennepedia protein, HTV transactivating (TAT) protein, mastoparan, melittin, bombolittin, delta hemolysin, pardaxin, Pseudomonas exotoxin A, clathrin, Diphtheria toxin, C9 complement protein, and Herpes-simplex- virus- 1 DNA binding protein.

61. The nucleic acid of claim 54, wherein the transcellular polypeptide sequence is an accessory peptide sequence which enhances interaction of the fusion protein with a cell surface membrane.

62. The nucleic acid of claim 61, wherein the accessory peptide includes an RGD sequence.

63. The nucleic acid of any one of claims 49-62, wherein the nucleic acid is DNA

64. The nucleic acid of any one of claims 49-62, wherem the nucleic acid is RNA.

65. The nucleic acid of claim 63, wherein the DNA is cDNA.

66. The nucleic acid of claim 63, wherein the nucleic acid comprises nucleotides, the sequence of which nucleotides is set forth in SEQ ID NO: 6.

67. A vector which comprises the nucleic acid of any one of claims 44-62.

68. The vector of claim 67, wherein the vector is a replicable vector.

69. The vector of claim 68, wherein the vector is a vector a plasmid, cosmid, λ phage or YAC.

70. A cell which comprises the vector of any one of claims 67-69.

71. The cell of claim 70, wherein the cell is a eukaryotic cell.

72. The cell of claim 70, wherein the cell is a prokaryotic cell.

73. A host-vector system for the production of a polypeptide which comprises the vector of any one of claims 67-69 and a suitable host cell.

74. A method for producing a polypeptide which comprises growing the host vector system of claim 73 under suitable conditions and recovering the polypeptide so produced.

75. A method of treating a subject afflicted with cancer which comprises administering to the subject an amount ofthe agent of any one of claims 1-43 effective to treat the subject.

76. A method of preventing a subject from becoming afflicted with cancer which comprises administering to the subject an amount ofthe agent of any one of claims 1-43 effective to prevent the subject from becoming afflicted with cancer.

77. A method of inhibiting growth of a cancer cell which comprises contacting the cancer cell with an amount of the agent of any one of claims 1-43 effective to inhibit growth ofthe cancer cell.

78. A method of inhibiting GTP-Ras signaling which comprises contacting a GTP-Ras containing cell with an amount ofthe agent of any one of claims 1- 43 effective to inhibit GTP-Ras signaling.

79. A method of inhibiting GTP-Ras from interacting with its effector which comprises contacting a GTP-Ras containing cell with an amount ofthe agent of any one of claims 1-43 effective to inhibit GTP-Ras from interacting with its effector.

80. The method of claim 79, wherein the effector is selected from the group consisting of Raf, PI3K and Ral-GEF.

81. The method of claim 77-79, wherein the cell is present in a subject and the contacting is effected by administering the agent to the subject.

82. The method of any one of claims 75, 76 and 81, wherein the agent is administered orally, intravenously, subcutaneously, intramuscularly, topically or by liposome-mediated delivery.

83. The method of any one of claims 75, 76 and 81, wherein the subject is a human being, a primate, an equine, an opine, an avian, a bovine, a porcine, a canine, a feline or a murine subject.

84. The method of claim 75 or 76, wherein the effective amount of the agent is between about lmg and about 50mg per kg body weight ofthe subject.

85. The method of claim 75 or 76 wherein the effective amount of the agent is between about 2mg and about 40mg per kg body weight ofthe subject.

86. The method of claim 75 or 76, wherein the effective amount of the agent is between about 3mg and about 30mg per kg body weight ofthe subject.

87. The method of claim 75 or 76, wherem the effective amount of the agent is between about 4mg and about 20mg per kg body weight ofthe subject.

88. The method of claim 75 or 76, wherein the effective amount of the agent is between about 5mg and about lOmg per kg body weight ofthe subject.

89. The method of any one of claims 75, 76 and 81, wherein the agent is administered at least once per day.

90. The method of any one of claims 75, 76 and 81, wherein the agent is administered daily.

91. The method of any one of claims 75, 76 and 81, wherein the agent is administered every other day.

92. The method of any one of claims 75, 76 and 81, wherein the agent is administered every 6 to 8 days.

93. The method of any one of claims 75, 76 and 81, wherein the agent is administered weekly.

94. A recombinant transfection system, comprising

(i) a first gene construct comprising a coding sequence encoding a inhibitory polypeptide comprising at least one GTP-Ras-binding motif for binding and inhibiting Ras, which coding sequence is operably linked to a transcriptional regulatory sequence for causing expression ofthe first polypeptide in eukaryotic cells; and

(ii) a gene delivery composition for delivering the gene construct to a cell and causing the cell to be transfected with the gene construct.

95. The recombinant transfection system of claim 94, further comprising a second gene construct comprising a coding sequence encoding a transcellular polypeptide which promotes transcytosis across a cell surface membrane, wherein the gene delivery composition also delivers the second gene construct to the cell.

96. The recombinant transfection system of claim 94, wherein the gene construct is provided in a vector.

97. The recombinant transfection system of claim 95, wherein the first and second gene constructs are provided as part of a single vector.

98. The recombinant transfection system of claim 95, wherein the first and second gene constructs are provided in a polycistronic message.

99. The recombinant transfection system of claim 95, wherein the first and second gene constructs are provided in separate vectors.

100. The recombinant recombinant transfection system of claim 96, 97 or 99, wherein the vector is a viral vector.

101. The recombinant transfection system of claim 100, wherein the viral vector is an adenoviral vector.

102. The recombinant transfection system of claim 100, wherein the viral vector is an adeno-associated viral vector.

103. The recombinant transfection system of claim 100, wherein the viral vector is a retroviral vector.

104. The recombinant transfection system of claim 94 or 95, wherein the gene delivery composition comprises a recombinant viral particle.

105. The recombinant transfection system of claim 94 or 95, wherein the gene delivery composition is selected from the group consisting of a liposome and a poly-cationic nucleic acid binding agent.

106. The recombinant transfection system of claim 94 or 95, wherein the gene delivery composition further comprises a pharmaceutically acceptable carrier for adminstration to an subject.

107. The recombinant transfection system of claim 106, wherein the subject is a human being, a primate, an equine, an opine, an avian, a bovine, a porcine, a canine, a feline or a murine subject.

108. A method for treating a subject afflicted with cancer which comprises administering to the subject the recombinant transfection system of claim 94 or 95.

109. A method for preventing a subject from becoming afflicted with cancer which comprises administering to the subject the recombinant transfection system of claim 94 or 95.

110. Use of the agent of any one of any one of claims 1-43 for the preparation of a medicant for treating cancer.

111. Use of the agent of any one of claims 1-43 for the preparation of a medicant for preventing cancer.

112. Use of the agent of any one of claims 1-43 for the preparation of a medicant for inhibiting growth of a cancer cell.

113. Use of the agent of any one of claims 1-43 for the preparation of a medicant for inhibiting GTP-Ras signaling.

114. Use of the agent of any one of claims 1-43 for the preparation of a medicant for inhibiting GTP-Ras from interacting with its effector.

115. A pharmaceutical package comprising a composition comprising the agent of any one of claims 1-43 with instructions for administering the composition.