CA2288967A1 - Selective induction of cell death by delivery of amino-terminal interleukin-1-alpha pro-piece polypeptide - Google Patents

Abstract

The present invention is directed to compositions and methods for selective induction of apoptosis in cancer cells, particularly malignant cancer cells, by delivery of an IL-1.alpha. propiece polypeptide (e.g., a native IL-1.alpha. propiece polypeptide, including IL-1.alpha. propiece polypeptide variant) to a cancer cell.

Description

SELECTIVE INDUCTION OF CELL DEATH BY DELIVERY OF

POLYPEPTIDE
CROSS- ~ ~ NCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. provisional application serial no. 60/048,137, filed May 30,1997, which application is incorporated herein by reference.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made with United States Government support under Grant Number DK31398 and Grant Number DK39776, awarded by the National institutes of Health. The United States Government may have certain rights in this invention.
BACKGROUND OF TfiE INVENTION
The cytokine interleukin-1 (IL-1) was initially described as a low molecular weight protein that acted in conjunction with plant lectins to stimulate the proliferation of murine thymocytes. The spectrum of IL-1-mediated biologic properties is diverse, and includes modulation of the cellular immune response as well as induction of acute inflammatory mediators, such as prostaglandins. Produced primarily by activated monocytes, macrophages and polymorphonuclear leukocytes, IL-1 exists as two distinct genetic forms, termed Interleukin-1a (IL-1a) and Interleukin-1(3 (IL-1(3). Both IL-la and IL-1 (3 are synthesized as precursor proteins of 31-33 kDa size and are subsequently cleaved to mature proteins of 15-17 kDa. The C-terminal halves of IL-la and IL-1 ~i are often referred to as "mature" IL-1 (i and °mature" IL-a," respectively, while the N-terminal halves are referred to as the N-terminal IL-la (or IL-1~) propiece. The C-terminal halves of the precursor molecules of IL-lei and IL-la are not found within cells, suggesting that processing occurs concurrent with release. Notably, it is the C-terminal halves of the IL-1 a and IL-1 ~i molecules that specifically interact with the Types I and II
plasma membrane IL-1 receptors, and it is this interaction, prior to the current invention, to which all reported activities of IL-1 have been ascribed.
SUBSTITUTE SHEET (RULE 26) There are distinct differences between the IL-1a and IL-1(i molecules in terms of sequence homology, intracellular distribution, kinetics of synthesis and mechanisms of proteolytic processing to the mature, C-terminal forms.
For example, the precursor form of IL-Ia is processed between Phe118 and Leu119 by calpain, a calcium-dependent protease. The precursor form of IL-~i is processed by the specific IL-1 (3 converting enzyme (caspase) between Asp116 and A1a117.
Research has focused almost exclusively upon the activities of the processed C-terminal, membrane receptor-binding components of the IL-1 molecules. No specific biologic function has been ascribed to the 16 kDa N-terminal components of the precursors generated by proteolytic processing.
Generation of polyclonal rabbit antibodies to synthetic peptides encoding epitopes for the N-terminal and C-terminal propieces of the IL-la precursor molecule led to the histochemical observation that the N-terminal IL-Ia propiece is present within the cell nucleus (Stevenson et al. (1992) J Cell Physiol 152:223-231). Subsequent studies using radioimmunoprecipitation of activated human monocyte lysates specifically recovered a 16 kDa protein with pI of 4.45, consistent with the predicted physicochemical properties of the IL-la N-terminal propiece (Stevenson, et al. (1993) Proc Natl Acad Sci USA 90:7245-7249), thus demonstrating the existence of the native N-terminal IL-la propiece within cells for the first time.
Examination of the cDNA sequence of the N-terminal IL-1a propiece revealed a polybasic region, T76-NGKVLKKRRL (SEQ ID N0:28), which had characteristics of a nuclear localization sequence (NLS) and could mediate nuclear localization of the propiece (Stevenson et al. (1997) Proc. Natl.
Acad.
Sci. USA 94:508-13). Introduction of the cDNA encoding the N-terminal IL-a propiece into cultured mesangial cells resulted in nuclear accumulation (Stevenson et al. id). Stable expression of the N-terminal IL-1 propiece results in apoptotic death of cultured mesanglial cells and immortalized Rat-1 fibroblasts, which was reported to suggest a role for the polypeptide in the removal of excessive cell populations during the resolution of glomerular inflammation (Turck et al. (1995) J. Am. Soc. Nephrol. 6:779, abstract no.
2052).
SUBSTITUTE SHEET (RULE 26) Apoptosis, or "programmed cell death," is the process by which a cell actively self destructs in response to certain developmental or environmental . stimuli. Apoptosis functions to control cell populations during embryogenesis, immune responses, hormone withdrawal from dependent tissues, normal tissue homeostasis, and tumor regression, as described in Duvall et al.
(198fi) Immunol. Today 7:115-119; Walker et al. (1988) Meth. Achieu. Exp. Pathol.
13:18-54; and Gerschenson et al. (1992) FASEB J. 6:2450-2455.
Apoptosis may be induced by immunologically mediated methods, such as antibody dependent cell cytotoxicity (K cell attack), viral infection, and attack by cytotoxic T lymphocyte effector cells, lymphotoxins, or natural killer (NK) cells. Further, apoptosis may be induced in tumor cells by a variety of physical, chemical, and biochemical agents, such as gamma radiation, UV
Iight, heat shock, cold shock, cisplatin, etoposide, teniposides, DNA
alkylating agents, macromolecular synthesis inhibitors, and the like. The apoptotic death process is associated with profound, well-defined morphological changes in the cell. Cohen et al., (1984) J. Immunol. 132, 38-42 (1984).
Current approaches to chemotherapy could benefit from selective induction of apoptosis in cancer cells. For example, while combination chemotherapy is often the treatment of choice, it often involves the use of ill-tolerated drugs and onerous side effects, which are largely associated with the non-specific nature of the therapy (Kruit et al. (1996) Br. J. Cancer 74 (fi):951). Thus, selective induction of apoptosis would be an attractive tool for cancer therapy. Unfortunately, development of apoptotic-based chemotherapy has met with many obstacles, include the identification of apoptosis-inducing agents that trigger cell death in cancerous cells, but do not substantially effect apoptosis in normal, non-cancerous cells. For recent reviews on the apoptosis in cancer chemotherapy see, e.g., Dixon et acl. (1997) Ann. Pharmczcother.
31:76-82; Guchelaar et czl. (1997) Pharm. World Sci. 19:119-125; Mayer et al.
(1997) Eur. J. Cancer Preu. 6:323-329; Tang et czl. (1998) Prostate 32:284-293.
Thus, there is an urgent need for compositions and methods for use in chemotherapy that are both effective and selectively kill cancerous cells. The present invention addresses this problem.
SUBSTITUTE SHEET (RULE 26) ShfMMARY OF THE INVENTION
The present invention is directed to compositions and methods for selective induction of apoptosis in cancer cells, particularly malignant cancer cells, by delivery of a IL-la propiece polypeptide (e.g., a native IL-la propiece polypeptide, including IL-la propiece polypeptide variants) to a cancer cell.
Thus, in one aspect, the invention features an isolated polypeptide characterized by: a) a nuclear localization sequence, b) amino acid residues 75-108 of SEQ ID N0:2 and equivalents thereof retaining selective apoptosis-inducing activity, and c) an affinity ligand, wherein the polypeptide is characterized by activity in selective induction of apoptosis in a cancer cell.
In one embodiment, the nuclear localization sequence and the apoptosis-inducing amino acid sequence are provided by amino acid residues 55-108 or 1-118 of an N-terminal IL-1a propiece polypeptide. In further related aspects, the invention features an isolated nucleic acid molecule encoding such apoptosis-inducing polypeptides.
In related aspects, the invention features an isolated polypeptide characterized by activity in selective induction of apoptosis in a cancer cell, the polypeptide having: a) amino acid residues 1-108, 11-118, 34-118, 55-118, or 55-108 of SEQ ID N0:2 (or of SEQ ID N0:2 having an amino acid other than serine at position 87); b) amino acid residues 75-108 of SEQ ID N0:2 (or of SEQ ID N0:2 having an amino acid other than serine at position 87), where the amino acid sequence is coupled to a heterologous nuclear localization sequence; c) amino acid residues 1-118, 1-108, or 55-108 of SEQ
ID N0:2 (or of SEQ ID N0:2 having an amino acid other than serine at position 87) coupled to a leucine zipper domain; and d) equivalents thereof that retain selective apoptosis-inducing activity. In further related aspects, the invention features an isolated nucleic acid molecule encoding such apoptosis-inducing polypeptides.
In another aspect the invention features a method for selectively inducing apoptosis in a cancer cell, wherein the method comprises delivering an apoptosis-inducing polypeptide to a malignant cell in an amount effective to induce apoptosis. Delivery may be accomplished by introduction of an apoptosis-inducing polypeptide-encoding nucleic acid molecule into a cancer SUBSTITUTE SHEET (RULE 26) cell, extracellular delivery of the polypeptide to the cancer cell, or direct intracellular delivery of the polypeptide into the cancer cell. In preferred embodiments, the cancer cell is cell of a human tumor such as a coionic tumor, a central nervous system tumor, a leukemia cell, a lung tumor, a . 5 mammary tumor, a melanoma, an ovarian tumor, a prostate tumor, and a kidney tumor.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic showing the amino acid sequence of native human IL-la propiece (1-118) (SEQ ID N0:2), the positions of domains of interest, and the position of serine 87. The following residues of the amino acid sequence are noted in the figure: anionic residues 1-11 (which are not necessary for apoptosis induction), residues 1-34 (helix #1), residues of 34-74 (helix #2), residues 60-74 (region necessary for nuclear localization which can be substituted with a heterologous nuclear localization sequence), residues 81-84 (residues KKRR (SE~,I ID N0:29), which are necessary but not sufficient for nuclear localization, and can be substituted by SV40 NLS), residue 87 (known phosphorylation site that when mutated does not substantially affect apoptotic activity), residues 90-108 (acidic domain essential for apoptosis induction), and residues 108-118 (not necessary for apoptosis) Fig. 2 is a schematic showing the nucleotide and amino acid sequences of native human IL-la propiece (1-118) and the portions relevant in production of exemplary iL-la propiece (1-118) variants Fig. 3 is a graph showing the volume of tumors formed at day 13 after implantation of IL-la propiece (1-118)-treated and EGFP-treated melanoma cells in nude mice.
Fig. 4 is a graph showing the volume of tumors formed at day 16 after implantation of IL-I~c propiece (1-118)-treated and EGFP-treated melanoma cells in nude mice.
Fig. 5 is a graph showing the volume of tumors formed at day 19 after implantation of IL-la propiece (I-118)-treated and EGFP-treated melanoma cells in nude mice.
SUBSTITUTE SHEET (RULE 26) DESCR~'TION OF THE SPECIFIC EMBODIMENTS
Before the present method and compositions for induction of apoptosis in cancer cells are described, it is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, and reagents described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a malignant cell"
includes a plurality of such cells and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.
All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the compositions and methodologies which are described in the publications which might be used in connection with the presently described invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such a disclosure by virtue of prior invention.
DEI~'~TioNs "Native N-terminal IL-1a propiece" refers to a polypeptide that is synthesized as an N-terminal portion of an IL-la precursor polypeptide, and is subsequently released from the precursor polypeptide by proteolytic cleavage. "Native N-terminal IL-Ia-propiece" is meant to encompass SUBSTITUTE SHEET (RULE 26) polypeptides derived from human, murine, bovine, and other eukaryotic sources, where the polypeptide is present at the N-terminus of an IL-la precursor polypeptide. Exemplary IL-la precursor polypeptides that encompass native N-terminal IL-la propiece polypeptides of interest in the present invention are provided in the Sequence Listing as SEQ ID NOS:7-20).
Preferably, native N-terminal IL-la propiece is a native human IL-la propiece, more preferably the polypeptide of SEQ ID N0:2 (encoded by SEfa ID NO:1 or an equivalent thereof). Native N-terminal IL-1a propiece polypeptides are normally characterized by a molecular weight of about I6 kDa and selective induction of apoptosis in a cancer cell, particularly a malignant cancer cell, as described herein. Native human N-terminal IL-1a propiece generally contains amino acids 1-118.
"IL-la propiece polypeptide" or "IL-la propiece" is meant to encompass both a naturally occurring N-terminal product resulting from cleavage of a eukaryotic IL-la precursor polypeptide (e.g., a mammalian IL-la propiece polypeptide, e.g., a human IL-la propiece polypeptide (IL-la propiece (1-118) polypeptide)) as well as biologically active variants of an N-terminal IL-la propiece polypeptide, unless specifically noted otherwise. IL-la propiece polypeptides of the invention may be of any appropriate eukaryotic origin (e.g., vertebrate or non-vertebrate), preferably mammalian, more preferably human origin. Exemplary IL-la propiece polypeptides of interest also include, but are not necessarily limited to, IL-la precursor polypeptides of non-human primate, canine, feline, rodent (e.g., rabbit, guinea pig, rat, mouse, etc.), equine, ruminants (e.g., ovine (e.g., sheep), goat, bovine, etc.), metathrial (e.g., opossum), swine (e.g., pig), and invertebrate (e.g., drosophila, sea urchin, etc.) origin. SEQ ID NOS: 7 20 provide the amino acid sequences of several exemplary IL-1 a precursor polypeptides from which IL-1 a propiece polypeptides and variants thereof can be derived and which are suitable for use in the present invention.
"IL-la propiece (1-118) polypeptide" and "IL-la(1-118)" refers to a polypeptide composed of amino acid residues 1-118 of an IL-la precursor polypeptide. "IL-la(1-118) polypeptide" or "IL-la(1-118)" is also meant to encompass biologically active IL-Ia propiece (1-118) variants unless noted SUBSTfTUTE SHEET (RULE 26) otherwise (e.g., by the phrase "native IL-la propiece (1-118)" or " IL-la propiece (1-118) (SEQ ID N0:2)").
By "IL-la propiece variant" or "IL-1a propiece biologically active variant" (which terms are generally used interchangeably unless specifically noted otherwise) is meant a polypeptide that is modified (e.g., having amino acids) insertion(s), deletion(s), substitution(s), and/or inversions) (e.g., fusion polypeptides, chimeric polypeptides, fragments, conservative and non-conservative amino acid substitutions, and the like), post-translational modification (e.g., myristoylation, phosphorylation, glycosylation, and the like), and/or chemical or biochemical modifications (e.g., PEGylation, conjugation to a detectable label or non-IL-la propiece polypeptide (e.g., as in a fusion protein), and the like) relative to native N-terminal IL-la propiece.
Variants also encompass polypeptides that are dimerized, e.g., by chemical cross-linking or introduction of a dimerization domain, and/or are modified to contain a hormone binding domain (HBD) or other activity-regulatable sequence. Exemplary dimerization domain of interest in the IL-a propiece polypeptide variants of the invention include a synthetic (e.g., heterologous) leucine zipper domain, e.g., based on the Ieucine zipper domain of c-myc (Conseiller (1998) J. Clin. Invest. 101:120-7). For a review of leucine zipper domain structure and function, see, e.g., Alber (1992) Curr Opin Genet Dev.
2:205-210; Vinson, et al. (1989) Science 246(4932):911-91; and Struhl (1989) Trends Biochem Sci.l4(4):137-140. Exemplary hormone binding domains include estrogen binding domains and variants thereof (e.g., an HBD that is mutated so that it is no longer responsive to its natural ligand, but is responsive to binding of a chemical analog of its natural ligand.
The nomenclature used herein for polypeptides and their encoding nucleic acids first indicates the polypeptide from which the variant is derived (e.g., an IL-la N-terminal propiece polypeptide is indicated by IL-la) followed by the amino acid residues of the polypeptide from which the variant is derived in parentheses (e.g., IL-la(11-118) indicates that the polypeptide is derived from amino acid residues 11-118 of IL-la N-terminal propiece polypeptide). Where the variant encompasses an amino acid change relative to native IL-la propiece polypeptide, the change indicated by the one-letter code for the amino acid residue of native IL-la propiece (1-118), the residue SUBSTITUTE SHEET (RULE 26) number, and the amino acid change (e.g., IL-1a propiece (1-118)(S87A) indicates a substitution of alanine for serine at residue 87 of IL-Ia propiece (1-118)). Fusion proteins and other chimeras are indicated by abbreviated names for each amino acid sequence separated by dashes (e.g., IL-a propiece (1-118) -EGFP indicates a fusion protein between IL-1a propiece (1-118) and enhanced green fluorescent protein). The amino acid residue numbering used herein is that provided in Figs. 1 and 2 (SEQ ID N0:2).
The term "biologically active IL-la propiece variant" (e.g., as used in the context of "biologically active fragment of IL-la propiece polypeptide,"
etc.) refers to a polypeptide having an amino acid sequence or modified amino acid sequence of IL-la propiece polypeptide that, when delivered to a cancerous mammalian cell (particularly a malignant cancerous mammalian cell) induces apoptosis or otherwise inhibits cell growth.
"Apoptosis" is a process of programmed cell death that is defined by a number of characteristic phenomena, summarized in Cohen (1993) Immunol.
Today 14:126-30. For example, in apoptotic cells, the cytoplasm condenses, and the endoplasmic reticulum dilates to form vesicles which fuse with the cell membrane, producing characteristic cellular morphology. The earliest changes in apoptosis appear in the mitochondria and are characterized by collapse in electrochemical gradients. Other early changes include an increase in the concentration of phosphatidyl serine on the outer leaflet of the plasma membrane in many cells. More advanced stages of apoptosis include changes in the nuclei including nuclear condensation, the formation of dense crescent-shaped aggregates of chromatin, nucleolus fragmentation, and formation of vesicles at or on the nuclear membrane. A classic signature of apoptosis is the cleavage of nuclear DNA into nucleosomal subunits. During apoptosis endonucleases present in the cell cut the DNA in the linker regions between nucleosomes to release DNA fragments in integer multiples of 180-190 base pairs, giving rise to the appearance of a ladder on gel electrophoresis (see, e.g., Gavrieli et al. (1992) J. Cell Biol. 119:493).
There are also a variety of other assays available for apoptosis such as terminal deoxynucleotidyl transferase-mediated biotinylated dUTP (TUNEL) assays (see White et czl. (1984) J. Virol. 52:410). Growth inhibition may be assessed using a number of commonly used assays, such as the methylcellulose assay SUBSTITUTE SHEET (RULE 26) (Lunardi-Iskandar et al. {1985) Clin. Exp. Immunol. 60:285-293).
"Normal cell" is meant to describe a cell that is not immortalized, transformed, cancerous or malignant.
5 "Immortalized cell" means a cell that is characterized by its capacity for continuous culture and/or cells whose potential to proliferate is not limited to that approximately 50 cell divisions characteristic of normal cells as defined above, e.g., cells whose potential for cell division exceeds the Hayflick limit. Cells can be immortalized as a result of manipulation in in vitro culture 10 (e.g., viral transfection, selection by continuous culture, etc.) or as a result of events that occurred in uivo in the host of origin. As used herein, "culture-immortalized cells" are those immortalized cells produced by manipulation in in vitro culture, while "tumor-derived immortalized cells" are those cells that were immortalized as a result of an in viuo event in the host of origin.
"Malignant cell" is meant to describe a transformed cell, generally an immortalized mammalian cell, that is characterized by loss of both growth control (e.g., rate of cell division) and positional control (e.g., infiltration of the cell to other sites in the host). The malignancy of a cell can be tested by injection of the cell into a host animal; development of tumors or cancers that are anaplastic, invasive, and/or metastatic is indicative of the malignant nature of the injected cell. In general, "malignant cell" as used herein is meant to refer to cells associated with (in uivo) or derived directly from (in vitro) a bona fide metastatic tumor in a mammal (especially a metastatic human tumor), e.g., as distinct from a cell immortalized in culture in vitro (i.e., culture-immortalized cell) or cells derived from such in vitro immortalized cells.
A "human" tumor is a tumor comprising cells that have human chromosomes. Such tumors include those in a Ixuman patient, and tumors resulting from the introduction of a human malignant cell line into a non-human host animal i f cells from such tumors have human chromosomes.
"Apoptosis" refers to a process of programmed cell death, which is defined by a number of characteristic phenomena, such as nuclear changes (e.g., nuclear condensation, formation of dense crescent-shaped aggregates of SUBSTITUTE SHEET (RULE 26) chromatin, nucleolus fragmentation, formation of vesicles at or on the nuclear membrane, etc.), condensation of the cytoplasm, and dilation of the endoplasmic reticulum to form vesicles that fuse with the cell membrane, producing characteristic cellular morphology (see, e.g., Cohen, Immunol.
Today 14:126-30 (1993).
The terms "treatment," "therapy," and the like generally refer to a regimen associated with any improvement in the condition of a mammal afflicted with a disease or condition (e.g., having cancer or a tumor, particularly a malignant cancer or tumor). The improvement can be either subjective or objective. For example, if the mammal is human, the patient may note improved vigor or vitality or decreased pain as subjective symptoms of improvement or response to therapy. Alternatively, the clinician may observe a decrease in tumor size or tumor burden based on physical exam, laboratory parameters, tumor markers, or radiographic findings.
The term "effective amount" means a dosage of a given substance that is sufficient to produce a desired result. The desired result can be subjective or objective improvement in the recipient of the dosage, a decrease in tumor size, a decrease in the rate of growth of cancer cells, or a decrease in metastasis.
The terms "nucleic acid" and 'polynucleotide" refer to deoxyribonucleotides (e.g., DNA and cDNA) or ribonucleotides (e.g., mRNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, reference to a particular nucleic acid sequence is meant to encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences in addition to the specific sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);
Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; and Rassolini et al.
(1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used SUBSTITUTE SHEET (RULE 26) WO 98/54205 PCTlUS98/10839 interchangeably with gene, cDNA, and mRNA encoded by a gene. Unless otherwise indicated, a particular nucleic acid sequence includes the perfect complementary sequence thereo f.
The phrase "a nucleic acid sequence encoding" or "a polynucleotide encoding" refers to a nucleic acid that contains sequence information for a structural RNA such as rRNA, a tRNA, or a primary amino acid sequence of a specific polypeptide, or a binding site for a traps-acting regulatory agent.
This phrase specifically encompasses degenerate variants o f the native sequence (i.e., different codons which encode a single amino acid) or sequences that may be introduced to conform with codon preference in a specific host cell.
"Substantial identity", when referring to the polynucleotides of this invention, means polynucleotides hawing at least 80%, typically at least 90%
and pre ferably at least 95% sequence identity to the nucleotide sequence o f SEA ID N0:1. Sequence identity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al.
(1990) J Mol Biol 215:403-10. For the purposes of the present application, percent identity for the polynucleotides of the invention is determined using the BLASTN program with the default settings (including default gap weights) as described at http://www.ncbi.nlm. nih.gov/cgi-bin/
BLAST/nph-newblast?Jform=0 with the DUST filter selected. The DUST
filter is described at http://www.ncbi.nlm.nih.gov/BLAST/ filtered.html.
Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50~C and 6XSSC (0.9 M
saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55z,-C in IXSSC (0.15 M sodium chloride/0.015 M sodium citrate).
Sequence identity may be determined by hybridization under stringent conditions, for example, at 50~C or higher and 0.1XSSC (15 mM sodium chloridel0.l5 mM sodium citrate). By using probes, particularly labeled SUBSTITUTE SHEET (RULE 26) probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes may be any species, e.g. primate species, particularly human; rodents, such as rats and mice, canines, felines, bovines, ovines a vines q , yeast, Drosophila, Caenhorabditis, etc.

Substantial identity, when referring to the polypeptides of the invention are polypeptides having at least 80%, typically at least 90% and preferably at least 95% identity to the amino acid sequence of SEA ID
NO: 2, or that are encoded by polynucleotides which will hybridize under stringent conditions to a polynucleotide having the nucleotide sequence of SEQ ID
NO:1. Percent identity for the polypeptides o f the invention is determined using the BLASTP

program with the default settings (including defaultgap weights) as described at http://www.ncbi.nlm.nih.gov/ cgi-bin/BLAST/nph-newblast?
Jform=0 with the DUST filter selected. The DUST filter is described at http://www.ncbi.nlm.nih.gov/BLAST/frltered.html.

When percentage of sequence identity is used in reference to polypeptides it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art.
Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score o f zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).

A "conservative substitution refers to a change in the amino acid composition of a polypeptide that does not substantially alter a selected biological activity of the polypeptide. Thus, conservatively modified SUBSTITUTE SHEET (RULE 26) variations" of a particular amino acid sequence refers to substitutions of those amino acids that are not critical for a biological activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non polar, etc.) such that the substitutions of even critical amino acids do not substantially after activity.
Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (~); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also, Creighton, Proteins W.H. Freeman and Company (1984). In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be°conservatively modified varzahons" .
"Biological sample" as used herein is a sample of tissue or fluid from an organism that includes, but is not limited to, sputum, amniotic fluid, blood, blood cells (e.g., white cells), or tissue. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. Examples of biological samples include a cell sample from nervous, muscular, glandular or epithelial tissue or from the immune system (e.g., T
cells). A biological sample is typically obtained from a eukaryotic organism, preferably a multicellular eukaryotes such as insect, protozoa, birds, fish, reptiles, and preferably a mammal such as rat, mice, cow, dog, guinea pig, or rabbit, and most preferably a primate such as macaques, chimpanzees, or humans.
The term "antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene, or fragments thereof that specifically bind and recognize an analyte (antigen). "Antibody" encompasses immunoglobulin molecules o f the classes IgG, IgM, IgA, IgD, IgE, and subclasses thereo f (e.g., IgG1), and includes both monoclonal and polyclonal antibodies. The term "antibody" also encompasses any antigen binding antibody form (e.g., Fab, Fab', F(ab)v F(ab)'v single chain Fv, chimeric antibodies (i.e., comprising constant and variable regions from different species), humanized antibodies SUBSTITUTE SHEET (RULE 2fi) (i.e., comprising a complementarity determining region (CDR) from a non-human source) and heteroconjugate antibodies (e.g., bispecific antibodies) and the like). Various antibody fragments can be produced by digestion of an intact antibody or by de novo synthesis (e.g., chemical synthesis or using 5 recombinant DNA methodology).
The phrase "specifically binds to an antibody" or "specifically immunoreactive with", when referring to a polypeptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of polypeptides and other biologics. Thus, 10 under designated immunoassay conditions, the specified antibodies bind to a particular polypeptide and do not significantly bind to other polypeptides present in the sample.
OVERVIEW

15 The present invention is based on the discovery that delivery of a IL-1 a propiece polypeptide facilitates selective induction of apoptosis in cancerous cells, particularly malignant cancerous cells. Delivery of the apoptosis-inducing polypeptides of the invention can be accomplished in a variety of ways including, but not necessarily limited to, DNA-based polypeptide delivery (e.g., by introduction o f an IL-1 a propiece polypeptide-encoding polynucleotide and expression within the host cell), direct intracellular polypeptide introduction (e.g., by fusion of a polypeptide-comprising liposome with a target cell), and polypeptide delivery to the surface of that target cell (i.e., extracellular delivery). Furthermore, the inventors have discovered that while IL-1 a propiece polypeptides facilitate induction of apoptosis in cancer cells (especially in malignant cancerous cells), the IL-1a propiece polypeptides generally do not substantially induce apoptosis in normal cells, making IL-1 a pmpiece polypeptides of interest in chemotherapy. Induction of apoptosis in cancer cells decreases tumor growth and/or tumor mass, and facilitates tumor regression.

Apoptosis-inducing polypeptides o f the invention encompass N terminal IL-1 a propiece and variants thereof having biological activity in apoptosis SUBSTITUTE SHEET (RULE 26) induction in cancer cells, preferably in malignant cancer cells. Preferably, the apoptosis-inducing polypeptides of the invention facilitate apoptosis of malignant cells, but do not significantly or substantially facilitate apoptosis in normal cells. A degree of apoptosis induction in normal cells is tolerable, but preferably the apoptosis-inducing polypeptides of the invention facilitate apoptosis in malignant cancer cells more efficiently than in normal cells. For example, the apoptosis-inducing polypeptides of the invention may induce apoptosis of malignant cells in a shorted post-delivery time period than in normal cells, may facilitate apoptosis in malignant cells at lower polypeptide levels (e.g., with a smaller amount, lower concentration, lower expression level, etc.) than in normal cells, etc.
Apoptosis-inducing polypeptides of particular interest include, but are not necessarily limited to, native IL-1 a propiece polypeptide, more particularly the IL-1 a propiece (1-118) defined by SEA ID N0:2 and equivalents thereof.
Also of particular interest are IL-1 a propiece polypeptide variants, especially variants of SEQ ID N0:2, that retain apoptosis-inducing activity in malignant cells. Also of interest are IL-1 a propiece polypeptides derived from human and non-human origins, e.g., human, non-human primate, canine, feline, rodent, equine, ruminant, metathrial (e.g., opossum), swine, and invertebrate origin. SEA ID NOS:7 20 provide the amino acid sequences of several exemplary IL-1 a precursor polypeptides from which IL-1 a propiece polypeptides and variants thereof can be derived and which are suitable for use in the present invention (human (SEA ID NO: 7), bovine (SEQ ID N0:8), canine (SEA ID N0:9), goat (SEQ ID N0:10), guinea pig (SEQ ID NO:11), Cercoceus troquatus atys (SEA ID N0:12), equine (SEA ID N0:13), feline (SEQ ID N0:14), macaca (SEA ID N0:15), murine (SEQ ID N0:16), sheep (SEQ ID N0:17), rabbit (SEA ID N0:18), rat (SEQ ID N0:19), and pig (SEQ
ID N0:20)).
Native human IZ<1 a propiece polypeptide The amino acid sequence of human IL-1 a propiece (1-118), and features of the polypeptide, are presented in Fig.1 (SEQ ID N0:2). Native human IL-1 a propiece (1-118) is composed of 118 amino acids and has now been characterized as containing: an anionic region (residues 1-11); three a-helices (a-helix #1: residues 1-34; a-helix #2: residues 34-70; and a-helix #3:
SUBSTITUTE SHEET (RULE 26) residues 75-118); a basic sequence associated with nuclear localization (residues 81-84); a phosphorylation site at serine residue 87 (which site is not required for apoptotic activity), and an acidic domain which is required for the induction of apoptosis (from about residue 90 to about residue 108 or 118).
In addition the basic region from about residue 60 to about residue 75 may be necessary for nuclear localization.
I~1 a propiece polypeptide variants IL-la propiece polypeptide variants of the invention encompass polypeptides modified relative to a native N-terminal IL-la propiece (e.g., having amino acids) insertion(s), deletion(s), substitution(s), and/or inversions) (e.g., fusion polypeptides, chimeric polypeptides, fragments, conservative and non-conservative amino acid substitutions, and the like), post-translational modification (e.g., myristoylation, phosphorylation, glycosylation, and the like), and/or chemical or biochemical modifications (e.g., PEGylation, conjugation to a detectable label or non-IL-1 a propiece polypeptide (e.g., as in a fusion protein), and the like). IL-1 a propiece polypeptide variants of the invention are biologically active, e.g.,when delivered to a malignant mammalian cell, induce apoptosis or otherwise inhibit cell growth.
Apoptotic activity involves at least two separable biological activities:
1) nuclear localization; and 2) induction of cell death. These activities of a biologically active IL-1 a propiece polypeptide variant can be provided by naturally-occurring or synthetic amino acid sequences. For example, nuclear localization activity can be provided by a synthetic nuclear localization sequence (e.g., the nuclear localization sequence PKKKRKV (SEA ID N0:30) from SV40) joined to a fragment of IL-1 a(1-118) that has activity in inducing apoptosis.
I~1 a propiece polypeptide variants of particular interest include fragments of native IL-1 a propiece (1-118) having apoptotic actiaity. In genernl, and without being held to theory, IL-1 a propiece (1-118) variants comprise an amino acid sequence from about amino acid residue 50 or 55 to about amino acid residue 110 of SEQ ID N0:2 and equivalents thereof (e.g., the serine at residue 87 may be an amino acid other than serine). Specific exemplary IL-1 a propiece polypeptide variants o f the invention include, but are SUBSTITUTE SHEET (RULE 2fi) notlimited to, IL-1 a(11-118), IL-1 a(34-118), andlL-1 a(55-118), IL-loc propiece (1-118)(S87A), IL-1 a propiece (1-118)-EGFP, and equivalents thereof.
Production of I~1 a pnopiece polypeptide-encoding nucleic acids Nucleic acids encoding a native N terminal IL-1 a propiece may be obtained by any of a variety of methods known in the art (e.g., isolation from natural sources, obtained from ATCC or GenBank libraries, chemical synthesis, etc.) (see, e.g., Sambrook et al., (1989) Molecular Cloning: A
Laboratory Manual (2°d Ed.), vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). For example, DNA encoding a native N terminal IL-1 a propiece can be identified in either cDNA or genomic libraries by its ability to hybridize with nucleic acid probes designed to complement a portion of the nucleotide sequence of SEA ID NO:1, and these polynucleotides isolated by standard methods familiar to those of skill in the art. Suitable probes can be prepared by methods well known in the art (e.g., chemical synthesis followed by purification by gel electrophoresis or anion-exchange HPLC
(Pearson et al., (1983) J. Chrom., 255:137-149)). In general, sense and anti-sense probes will be of approximately 18 nucleotides to the full length of the gene, more typically approximately 30 nucleotides in length. Low stringency conditions, e.g., 50 ° C and 6XSSC (0.9 M salinel0.09 M sodium citrate) are used to identify and isolate polynucleotides having nucleotide sequences similar to that o f the probe. High stringency conditions, e.g., 50 ° C
or higher and O.IXSSC (15 mM salinell.5 mM sodium citrate) are used to identify and polynucleotides having nucleotides essentially identical to that o f the probe.
The isolated polynucleotides may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome.
The polynucleotides of the invention may be obtained as double or single stranded fragments by conventional means, e.g., chemical synthesis, restriction enzyme digestion, PCR ampliftcation, and the like. For the most part, small DNA fragments, such as those useful as primers for PCR, hybridization screening probes, etc., will be of at least 15 nt, usually at least 18 nt or 25 nt, and may be at least about 50 nt. PCR ampli ficatzon requires a pair of primers, typically a pair which will genernted an amplification product of at least 50 nucleotides, preferably at least 100 nucleotides in length.
Suitable primers hybridize to the target polynucleotide under stringent SUBSTITUTE SHEET (RULE 2fi) conditions. The exact composition of the primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art. It is preferable to choose a pair o f primers ttcat will generate an amplification product of at least about 50 nt, preferably at least about 100 nt. Software designed for selecting suitable sequences for primers are commercially available. Larger DNA
fragments, e.g., usually greater than about 100 nt are useful for production of the encoded polypeptide.
Polynucleotides of particular interest are polynucleotides encoding native IL-1 a propiece (1-118) and IL-1 a propiece (1-118) variants. The polynucleotide and amino acid sequences of native human IL-1 a precursor, which sequences include native human IL-1 a propiece (1-118), are provided in GenBank at accession number X02851, and are described in Furutani et al. (1985) Nucleic Acids Res. 13:5869. The nucleotide sequence of native human IL-1 a propiece (1-118) is provided herein as SEA ID NO:1.
Production o f IL-1 a prnpiece polypeptide variants Polynucleotides encoding variants of a native N-terminal IL-1 a propiece can be prepared by methods known in the art. For example, techniques for site specific in vitro mutagenesis are found in Gustin et al. (1993), Biotechniques 14:22; Barany (1985), Gene 37.111-23; Colicelli et al. (1985), Mol. Gen. Genet. 199:537 9; and Prentki et al., (1984), Gene 29:303-13;
Sambrook, et al., supra, pp 15.3-108; Weiner et al. (1993), Gene 126'35-41;
Sayers et al. (1992), Biotechniques 13:592-6; Jones and Winistorfer (1992), Biotechniques 12:528-30; Burton et al. (1990), Nucleic Acids Res 18.7349-55;
Marotti and Tomich (1989), Gene Anal. Tech. 667 70; Zhu (1989), Anal Biochem 177.'120-4; Taylor et al., (1985) Nucl. Acids Res. 13:8749-8764; and Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:482-492. Methods o f using computer modeling for the design of variants of a selected polypeptide are also well known in the art.
The resulting variants may, for example, contain mutations in a native N terminal IL-1 a propiece sequence. The sequence changes may be substitutions, insertions, deletions, or inversions, or a combination thereof.
For example, nucleotide changes may be associated with a degenerate variant of the parent nucleic acid molecule (i.e., the mutation does not change the SUBSTITUTE SHEET (RULE 26) WO 98/54205 PCTlUS98/10839 amino acid sequence encoded by the mutated codon) or non-degenerate (i.e., the mutation changes the amino acid sequence encoded by the mutated codon).
Deletions may further include larger changes, such as deletions of a domain associated with a feature or characteristic of the native polypeptide (e.g., 5 deletion of helix #1 of native IL-1a propiece (1-118)). Other modifications of interest include epitope tagging, e.g. with the FLAG system, HA, etc., as well as production of fusion proteins, e.g., using green fluorescent proteins (GFP, EGFP, etc.), GST (Glutathione S-Transferase), and the like. Both degenerate and non-degenerate mutations may be advantageous in producing or using 10 the polypeptides of the present invention. For example, these mutations may permit higher levels of production, easier purification, or provide additional restriction endonuclease recognition sites. All such modified DNAs (and the encoded polypeptide molecules) are included within the scope of the present invention.
15 Screening for biological actiaity of candidate IL-1 a p~piece polypeptide UarlQntS
IL-1 a propiece polypeptide variants having biological activity in inducing apoptosis of a desired target cell (e.g., a malignant cancerous cell) can be identified by screening variant polypeptide candidates using a cell-20 based in vivo assay and/or using an animal model to assess the ability of the candidate variants to induce apoptosis in a targeted tumor as determined by, e.g., inhibition of tumor growth. Such screening assays may involve delivery o f a variant-encoding polynucleotide to a target cell for expression within the target cell, direct intracellular delivery of the variant polypeptide (e.g., by microinjection), and/or extracellular delivery of the candidate variant polypeptide to a target cell.
Apoptosis can be measured by a variety of techniques, reviewed in Cohen (1993) Immunol. Today 14: 126-30. For example, apoptosis can be measured by determination of cell phenotype. Phenotype refers to how the cell looks, typically microscopically, but gross or macroscopic appearance can be observed. Cell phenotype changes are dependent upon cellular growth rate.
For instance, the microscopic morphology of cells that are rapidly dividing and growing is different than that of cells undergoing cell death by apoptosis.
Determination of cell phenotype is well within the ability of one with ordinary SUBSTITUTE SHEET (RULE 26) skill in the art.
Apoptosis can also be evaluated using a number of biochemical assays, such as "laddering" of the cellular DNA. When testing compounds for the ability to induce apoptosis, cell death (not cytostasis) is an endpoint of compound application to the cell. A classic signature of apoptosis is the cleavage of nuclear DNA into nucleosomal subunits. When subjected to agarose gel electrophoresis, this phenomenon results in the appearance of a DNA ladder as nucleosomal units are sequentially cleaved from the DNA.
The appearance of the DNA ladder is thus indicative of cell death (see, e.g., Gavrieli et al. (1992) J. Cell Biol. 119:493).
In a preferred embodiment, candidate variant polypeptides are assayed for activity in a cell-based assay by expression of candidate variant polypeptide-encoding DNA in a target cell or by extracellular delivery of a candidate polypeptide to a target cell. Apoptosis can be evaluated by the formation of DNA ladders after agarose gel electrophoresis of isolated chromosomal DNA, by the TUNEL method (which involves 3' end-labeling of cleaned nuclear DNA) (Cohen et al. (1984) J. Immunol. 132:38-42; White et al. (1984) J. Virol. 52:410), and/or morphological criteria (Cohen et al., supra). The TUNEL method can also be used to biologically evaluate implanted tumors far apoptosis. Growth inhibition may be assessed using a number of commonly used assays, such as the methylcellulose assay (Lunardi Iskandar et al., Clin. Exp. Immunol. 60:285-293 (1985)). Growth inhibition may be assessed as a measurement of apoptosis using a number of commonly used assays, such as the methylcellulose assay (Lunardi-Iskandar et al.(1985) Clin. Exp. Immunol. 60:285-293).
Rapid screening of IL-1 a(1-118) polypeptide variant candidates can be accomplished by the use of a reporter(e.g., EGFP) fused to the IL-1 a propiece polypeptide candidate variant. For example, the IL-1 a propiece (1-118) variant candidate can be fused to EGFP to provide an IL-1 a propiece (1-118)-EGFP fusion polypeptide, the fusion polypeptide delivered to the target cells, and apoptosis evaluated by distribution of IL-1 a(1-118)-EGFP-associated fluorescence. Distribution of reporter polypeptides such as IL-1 a(1-118)-EGFP
in apoptotic cells is identical to the distributzon of DAPI or Hoechst 33342 dyes, which are conventionally used to detect the nuclear DNA changes SUBSTITUTE SHEET (RULE 26) associated with apoptosis (Cohen et al., supra). Moreover, the presence of the reporter polypeptide does not substantially affect the ability of the polypeptide to induce apoptosis. Thus, the presence of IL-1a propiece (1-118)-EGFP-associated fluorescence in fragmented nuclear bodies or apoptotic bodies is indicative of induction of apoptosis in the target cell.
Activity in apoptosis induction can also be assess by delivering the 1~
1 a propiece polypeptide variant candidate polypeptide to target cancer cells, and then implanting the treated cancer cells into a non-human animal model.
Inhibition of the ability of the treated cells to facilitate tumor production, growth, or metastasis relative to untreated cells is indicative of the apoptotic-activity of the candidate variant polypeptide. Similarly, apoptotic activity of candidate variant polypeptides can be assessed by delivery of the candidate variant directly to a tumor in an in vivo animal model.
Constructs and vectors useful for IL-1 a propiece polypeptide expression Once a nucleic acid encoding a polypeptide of the invention is isolated and cloned, the nucleic acid may be expressed in any of a variety of recombinantly engineered cells. Expression o f natural or synthetic nucleic acids is typically achieued by operably linking a nucleic acid of interest to a promoter (which is either constitutive or inducible), incorporating the construct into an expression vector, and introducing the vector into a suitable host cell.
Typical expression vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors may be suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. In general, the nucleic acids (e.g., promoters and vectors) useful in the composition and methods of the invention can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods.
SUBSTITUTE SHEET (RULE 26) Any suitable promoter can be used in expression constructs for production of IL-1 a propiece polypeptides. For example, the promoter may be a non-specific strong promoter (e.g., CMV promoter, human tyrosinase promoter (nt 26-2293 of GenBanh accession no. U03039) or a tissue-specific promoter, e.g., a promoter that facilitates enhanced transcriptional activity in the target cell but substantially no transcription or relatively lower transcription in non-target cells). Preferably, the expression construct is prepared so that the promoter-IL-1 a propiece polypeptide-encoding polynucleotide is positioned within a sequence containing convenient restriction sites, thus providing an expression cassette that can be easily moved between various expression constructs.
The promoter in the expression construct can be modified to increase its specificity and transcription levels (e.g., potency) in a target cell or target cell type. For example, the tyrosinase promoter can be modified to increase its specificity and potency in melanoma cells, e.g., by including 209 base pairs 5' to the transcription start site along with two additional melanoma-specific promoter fragments placed 5' to the 209 by fragment (Siders et al. (1996) Cancer Res. 56(24):5638-46).
A number of vector systems can be used to express the polypeptides of the invention. These include plasmids, cosmids, and a number of viral vectors, including retroviral vectors, vaccinia vectors, lentiviral vectors, herpes virus vectors (e.g., HSV), Sindbis/semliki forest viruses, adenoviral vectors, and adeno-associated viral (AAV) vectors. Each vector system has advantages and disadvantages, which relate to host cell range, intracellular location, level and duration of transgene expression and ease of scale-up/purification.
In a preferred embodiment, an expression cassette for expressing Ilr1 a propiece polypeptides comprises (from 5' to 3'): 1) a strong promoter or tissue-specific promoter; 2) optionally, a splice signal, as might be found in the first intron of the human growth hormone gene (nucleotides 565-831 of GenBank M13438); 3) an IL-1 a propiece polypeptide coding sequence; and 4) an SV40 or other polyadenylation signal. For in vivo expression, this entire cassette is preferably cloned into an adenovirus E1 type shuttle vector, such as pAC, which contains map units 0-1.5 and map units 9-17 of SUBSTITUTE SHEET (RULE 26) Adenovirus type 5. This construct can be used to make replication-defective adenoviruses by standard techniques.
Suitable constructs for IL-1 a propiece polypeptide expression can be selected according to the ability of such construct to facilitate a desired level of polypeptide production in a selected target cell. For example, the same expression construct may provide for varying expression levels in different cancer cell types; likewise, different expression constructs may provide for varying expression levels in the same cancer cell type. Thus, it is desirable to select an expression construct that provides an optimal level o f IL-1 a propiece polypeptide expression in the selected target cancer cell type. Methods and compositions for preparation of various constructs and modification of such construct to optimize expression, are well known in the art.
IL-1 a propiece polypeptide expression can be detected and quantif-zed by any of a number of means well known to those of skill in the art involving detection of transcription or translation products of the IL-1a propiece polypeptide-encoding polynucleotide. For example, expression can be detected by analytic biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitation reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
The detection of nucleic acids proceeds by well known methods such as Southern analysis, northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and aff "-cnity chromatography (see, e.g., Sambrook, et al.;
supra).
Constructs suitable for delivery to and expression in target cells in vivo Where the polynucleotide is to be prepared for delivery to and expression in a target cell (e.g., a malignant cell) in a mammalian host, the polynucleotide may be prepared as either a viral or non-viral delivery and expression system. In general, the optimal delivery systems are characterized by: I) a suitable host range (e.g., ability to selectively deliver the polynucleotide SUBSTITUTE SHEET (RULE 26) to a target cell (e.g., a malignant cell) or, where the polypeptide to be expressed does not substantially elicit apoptosis in normal cells, a broad host range may be permissible or desirable); 2) stable expression or high levels of transient expression; and 3) non-toxic to normal cells (e.g., to diminish undesirable 5 side-effects). W)xere the delivery system is virally-based, the delivery system is optimally further characterized by: 1) high titer/~g DNA; 2) substantially no replication in host cells; 3) ideally substantially no viral gene expression;
4) stable transmission to daughter cells (e.g., where apoptosis is effected in cells following one or more rounds of cell division); 5) high rescue yield;
and 10 6) substantially no or relatively little replication-competent virus.
The constructs and delivery system used will vary according to a variety of factors, such as the intended application, the target cell, etc.
Non-viral delivery systems Non-viral delivery systems include naked nucleic acid (e.g., as per i5 USPNs 5,693,622 and 5,580,859), transfection facilitatingproteins (e.g., DNA
protein formulations), liposomal formulations (e.g., USPNs 4,394,438;
5,459,127), charged lipids, calcium phosphate precipitating agents, DNA
targeting ligand formulations (e.g., to facilitate receptor-mediated endocytosis), polycationic substances such as poly-L-lysine or DEAC-dextran(Felgner et al., 20 (1987) Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417), and the like. Methods and compositions for production of such non-viral delivery systems are well known in the art.
SUBSTITUTE SHEET (RULE 26) Vial expression and deliaery systems Viral vectors suitable for use in the present invention include any viral vector suitable for delivery of a IL-1 a propiece polypeptide-encoding polynucleotide to a cancer target cell and expression in the target cell at a level sufficient to induce apoptosis in the target cancer cell. Viral vectors of particular interest include, but are not necessarily limited to, those vectors based on retroviruses (including pseudotyped retrouiruses, and Lentiviruses, such as HIV based vectors, which may not require cell division), Sindbis virus, adeno-associated virus (AAV), adenovirus, poxvirus, semliki-forest virus (SFV), and herpesuirus (e.g., CMV, HSY, etc.). Exemplary viral vectors suitable for use in the in vivo delivery methods of the invention are described below.
Ret~uirnl vectors Retroviral vectors may also be used in certain applications. The design of retrouiral vectors is well known to one of skill in the art. In brief, if the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis acting defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins.
Retroviral genomes from which these sequences have been deleted, as well as cell lines containing the mutant genome stably integrated into the chromosome are well known in the art and are commonly used to construct retroviral vectors. Preparation of retroviral vectors and their uses are described in many publications including EP- A 0178 220, U.S. Patent 4,405,712; Gilboa (1986) Biotechniques 4:504-512; Mann et al. (1983) Cell 33:153-159 ; Cone et al.
(1984) Proc. Natl. Acid. Sci. USA 81:6349-6353; Eglitis et al. (1988) Biotechniques 6:608-614; Miller et al. (1989) Biotechniques 7:981-990; and W4 92/07943.
The retroviral vector particles are prepared by recombinantly inserting a nucleic acid encoding a gene product of interest into a retrovirus vector and packaging the vector with retroviral capsid proteins by use of a packaging cell line. The resultant retroviral vector particle is generally incapable of replication in the host cell and is capable of integrating into the host cell genome as a proviral sequence containing the nucleic acid of interest. As a SUBSTITUTE SHEET (RULE 26) result, the host cell produces the gene product encoded by the nucleic acid of interest.
Packaging cell lines are generally used to prepare the retroviral vector particles. Apackaging cell line is a genetically constructed mammalian tissue culture cell line that produces the necessary viral structural proteins required for packaging, but which is incapable of producing infectious virions.
Retroviral vectors, on the other hand, lack the structural genes but have the nucleic acid sequences necessary for packaging. To prepare a packaging cell line, an infectious clone of a desired retrouirus, in which the packaging site has been deleted, is constructed. Cells comprising this construct will express all structural proteins but the introduced DNA will be incapable of being packaged. Alternatively, packaging cell lines can be produced by transducing a cell line with one or more expression plasmids encoding the appropriate core and envelope proteins. In these cells, the gag, pol, and env genes can be derived from the same or different retroviruses.
A number of packaging cell lines suitable for the present invention are available in the prior art. Examples of these cell lines include Crip, GPE86, PA317 and PG13 (see, e.g., Miller et al. (1991) J. Virol. 65:2220-2224).
Examples of other packaging cell lines are described in Cone et al., (1984) Proc. Natl. Acad. Sci. USA 81:6349-6353; Danos et al. (1988) Proc. Natl.
Acad. Sci. USA 85:6460-6464; Eglitis et al. (1988) Biotechniques 6:608-614;
Miller et al. (1989) Biotechniques 7:981-990. Amphotropic or xenotropic envelope proteins, such as those produced by PA317 and GPX packaging cell lines may also be used to package the retroviral vectors. Also of interest is the use of packaging cell lines that produce pseudotyped viral particles having vesicular stomatitis viral coat protein (VSV G), which may be obtained either by transient transfection of a packaging cell line (e.g., a cell expressing a retroviral gag and pol) or through use of a packaging cell line that stably expresses VSV G.
Sindbis/Semliki Forest Viruses Sindbis/semliki forest viruses (Berglund et al. (1993) Biotechnol 11:916-920) are positive-strand RNA viruses that replicate in the cytoplasm, are stably maintained, and can yield very high levels of antisense RNA.
Sindbis vectors are thus a third type of vector useful for maximal utility.
See, SUBSTITUTE SHEET (RULE 26) e.g., Altman-Hamamdzic et al. (1997) Gene Therap 4:815-22.
Adeno-a~,ssociated virus (AAV) vectors Because o f their demonstrated ease o f use, broad host range, stable transmission to daughter cells, high titer/microgram DNA, and stable expression, (Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996), AAV
vectors are a preferred viral vector for delivery of IL-1 a propiece polypeptide-encoding polynucleotides to target cells (see, e.g., Goeddel (ed.) (1990) Methods in Enxymology, Vol. 185, Academic Press, Inc., San Diego, CA; Krieger, (1990) Gene Transfer and Expression -- A Laboratory Manual, Stockton Press, New York, NY and references cited therein). AAV requires helper viruses such as adenovirus or herpes virus to achieve productive infection.
In the absence of helper virus functions, AAV integrates (site-specifically) into a host cell's genome. The integrated AAV genome has no pathogenic effect. The integration step allows the AAV genome to remain genetically intact until the host is exposed to the appropriate environmental conditions (e.g., a lytic helper virus), whereupon it re-enters the lytic life-cycle.
For discussion of AAV, AAV vectors, and uses thereof, see, e.g., Samulski (1993) Curr. Opin. Genet. Dev. 3:74-80 (references cited therein for an overview of the AAV life cycle); West et al. (1987) Virology 160:38-47; U.S.
Patent No. 4,797,368; WO 93/24641; Kotin (1994) Hum. Gene Therap.
5:793-801; Muzyczka (1994) J. Clin. Incest. 94:1351; and Samulski, supra.
AAV displays a very broad range of hosts including chicken, rodent, monkey and human cells (Muxycka, N., Curr. Top. Microbiol. Immunol. 158, 97129 (1992); Tratschin et al., (1985) Mol. Cell. Biol. 5:3251-3260; Lebhowski et al. (1988) Mol. Cell. Biol. 8:3988-3996). They eff~CCiently transduce a wide variety o f dividing and non-dividing cell types in vitro (Flotte et al.
(1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356); Podsakoff et al. (1994) J. Virol.

5655-5666), Alexander et al (1994) J. Virol. 68:8282-8287). AAV vectors have been demonstrated to successfully transduce hematopoietic progenitor cells of rodent or human origin (Nahreini et al. (1991) Blood 78:2079). It is believed that AAV may infect virtually any mammalian cell type.
Production of AAV vectors for delivery of IL-1 a propiece polynucleotides can be accomplished according to methods well known in the art. Once a cell or cells have been selected and shown to contain an IL-1 a propiece SUBSTITUTE SHEET (RULE 26) polynucleotide o f interest, the entire AAV IL-1 a propiece polypeptide expression cassette can be easily "rescued" from the host cell genome and amplified by introduction of the AAV viral proteins and wild type adenovirus (Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6466-6470; Tratschin et al.
(1985) Mol. Cell. Biol. 5:3251-3260; Samulski et al. (1982) Proc. Natl. Acad. Sci.
USA 79:2077 2081; Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260).
The copy number of genes introduced by the AAV vector is about 2 orders of magnitude higher or more than that of retrovirally- transduced human tumor-infcltrating lymphocyte (TIL) cell cultures. In vivo expression of AAV delivered genes is relatively long, and may last up to 6 months or more (Conrad et al. (1996) Gene Therapy 3:658-668). Persistence of an AAV
delivered gene has been observed in humans for at least 70 days (10th Annual North American Cystic Fibrosis Conference, Orlando, Florida, Oct. 25-27, 1996).
The promoters used to control the gene expression from AAV include:
(a) viral promoters such as SV40, CMV, retroviral LTRs, herpes virus TK
promoter, paruovirus B-19 promoter (Muzycka (1992) Curr. Top. Microbiol.
Immunol. 158:97-129), AAV p5 and p40 promoters (Tratschin et al. (1993) Am. J. Respir. Cell. Mol. Biol. 7, 349-356); (b) human gene promoters such as the gamma-globin promoter (Walsh et al. (1992) Proc. Nat. Acad. Sci., USA
89: 7257 7261) or the Q-actin promoter; and (c) RNA pol III promoters such as cellular tRNA promoters or the promoter from the adenovirus VAI gene.
An IL-1 a propiece polypeptide expression construct is co-transfected, along with Adenovirus 5 d1309NA (cut with Xbal and CIaI) into HEK 293 cells. Recombinant adenovirus plaques are isolated and expanded by standard techniques. In brief, an adenovirus fraction is collect on a CsCI
gradient, purified and dialyzed against physiologic saline with 10 mM Tris pH 8 and 1 mM MgCI~ A concentrated adenovirus preparation (e.g., containing about 1012 or more viruses in about 100 ~d of a carrier solution such as PBS) is administered, for example by injection into and/or around a defined tumor site (e.g., into and around melanoma lesions).
SUBSTITUTE SHEET (RULE 26) Purification of I~1 a p~piece polypeptides I~1 a propiece polypeptides (which polypeptides include native IL-1 a propiece (1-118) as well as IL-1 a propiece polypeptide variants) may be purified to substantial purity by standard techniques, including selective 5 precipitation with such substances as ammonium sul fate, column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Puri fzcation: Principles and Practice, (1982) Springer-Verlag: New York). For example, IL-1 a propiece polypeptides produced by recombinant DNA technology may be purifted by a combination of cell lysis (e.g., 10 sonication) and affinity chromatography or immunoprecipitation with a specific antibody to IL-1 a propiece polypeptide (e.g., to a native N-terminal I~1 a pro piece or a biologically active portion thereo f). Where a purred naturally-occurring N terminal IL-1 a propiece is desired, the polypeptide can be purified from any source that produces levels of the polypeptide adequate 15 for purification of a desired amount (e.g., from HL-60 cells).
For fusion products, techniques appropriate to the particular fusion protein may be used to release the polypeptide of interest from the non-IL-Ia propiece polypeptide amino acid sequences (e.g., digestion of the fusion protein with an appropriate proteolytic enzyme), with further puri f cation by standard 20 protein chemistry techniques. A puri~Zed protein preferably exhibits a single band on an electrophoretic gel. For example, purified human IL-1 a propiece (1-118) appears as a single band on reducing SDS-PAGE (0.1% SDS, 0.4%
stacking gel, 15% acrylamide resolving gel; molecular mass was determined with reference to the migration of standard protein of known molecular mass 25 and which do not display anomalous migration.
Antibody production using I~a propiece polypeptides of the invention A number of immunogens may be used to produce antibodies specifically reactive with IL-a propiece polypeptides of the invention.
Recombinantprotein is the preferred immunogen forproduction of monoclonal 30 or polyclonal antibodies. Naturally occurring protein may also be used either in pure or impure form. Synthetic peptides based on an amino acid sequence associated with an epitope o f a I~ a propiece polypeptide described herein may also used as an immunogen.
SUBSTITUTE SHEET (RULE 26) Methods of production of polyclonal and monoclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified IL-a propiece polypeptide, is mixed with an adjuvant and injected into an animal of choice (e.g., a mouse, rat, rabbit, pig, goat, cow, horse, chicken, etc .) at intervals of 1-4 weeks. The immunogen may be conjugated to a carrier protein can be used an immunogen. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the IL-a propiece polypeptide. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation o f the antisera to enrich for antibodies reactive to the protein can be done i f desired.
Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, e.g., a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 104 or greater are selected and tested for cross-reactivity against non-IL-a propiece polypeptide, e.g., using a competitive binding immunoassay.
Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell, or by transformation with Epstein Barr Virus, oncogenes, or retroviruses, or by other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells (see, e.g., Huse, et al. (1989) Science 246:1275-1281).
Anti-Ira propiece polypeptide antibodies can be used in a variety of ways, including, but not necessarily limited to, isolation andlor purification of eukaryotic IL-apropiece polypeptides that share antigenic epitopes with, for SUBSTITUTE SHEET (RULE 26) WO 98!54205 PCT/US98/10839 example, human IL-a propiece (1-118) polypeptide, detection of IL-a propiece polypeptide in during therapy, etc.
Detection of IL-1 a propiece-encoding nucleic acids IL-a propiece polypeptide expression in a candidate cell can be assessed either qualitatively or quantitatively by any of a variety of means well known in the art. For example, detection o f nucleic acid (e.g., mRNA, cDNA) can accomplished by northern analysis, Southern analysis (e.g., mRNA isolation, followed by PCR for cDNA production, followed by Southern analysis), sandwich assays, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. See, e.g., Sambrook, et al.; Nucleic Acid Hybridization, A Practical Approach, Ed. Names, B.D., et al., IRL Press, (1985); Gall and Pardue, Proc. Natl. Acad. Sci., U.S.A., 63:378-383 (1969);
and John et al., Nature, 223:582-587 (1969). The selection of a hybridization format is not critical.
Assays involving hybridization can be carried out using nucleic acid probes based on the nucleic acid sequences encoding an IL-1 a propiece polypeptide (e.g., SEQ ID NOS:1 and 2) The probes can be full length or less than the full length of the nucleic acid sequence encoding the IL-1 a propiece polypeptide. Shorter probes are empirically tested for specificity. Preferably nucleic acid probes are 20 bases or longer in length. Detection of a hybridization can be accomplished by virtue of a detectable label, according to methods well known in the art. The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification systems, such as PCR.
An alternative means for determining the level of expression of an IL-1 a propiece polypeptide is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer, et al., Methods Enzymol., 152:649-660 (1987).
Detection of IL-a propiece polypeptides IL-1 a propiece polypeptide may be detected or quantified by a variety of methods. Preferred methods involve the use of specific antibodies. For example, IL-a propiece polypeptide can be isolated from the candidate cell, and the amount or relative amount of IL-a propiece polypeptide determined by ELISA. Alternatively, the amount of IL-a propiece polypeptide in the sample SUBSTITUTE SHEET (RULE 26) can be determined by competitive binding assay, or other immunoassay well known in the art. For a description of different formats for binding assays, including competitive binding assays and direct binding assays, see Basic and Clinical Immunology 7th Edition (D. Stites and A. Terr ed.) (1991); Enzyme Immunoassay, E.T. Maggio, ed., CRC Press, Boca Raton, Florida (1980); and "Prnctice and Theory of Enzyme Immunoassays," P. Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V. Amsterdam (1985), each of which is incorporated herein by reference.
IDENTIFICATION OF TARGET CELLS SUSCEPTIBLE TO IL-1 a PROPIECE
POLYPEPTIDE-FACILITATED APOPTOSIS BY DELIVERY OF IL-a PROPIECE
POLYPEPTlDE TO A CANDIDATE CELL
Identification of target cells susceptible to IL-1 a propiece polypeptide-mediated apoptosis, as well as identif~-CCation of subjects having a cancer amenable to IL-1 a propiece polypeptide-based therapy, can also be accomplished by using a cell-based assay involving administration of an IL-a propiece polypeptide to a candidate target cells. Candidate target cells can be proxy candidate target cells, which cells are representative of an actual in vivo candidate target cell. Proxy candidate target cells can be commercially available cells, such as those available through the National Cancer Institute (NCI) or the American Type Culture Collection. Susceptibility of the proxy candidate target cell is indicative of susceptibility of a candidate target cell having characteristics similar to the proxy candidate target cell. For example, induction o f apoptosis in a commercially-available human melanoma cell (e.g., A2058 human melanoma ATCC CRL 11147), is indicative of the susceptibility of a similar melanoma in a human subject.
Alternatively, candidate target cells can be directly derived from a biopsy of a tumor in a subject who is a candidate for IL-1 a propiece polypeptide-based therapy. Methods for culturing cells from a biopsy sample are well known in the art.
The candidate target cells can be tested for susceptibility to IL-1 a pmpiece polypeptide-induced apoptosis by introduction and expression oflL-1 a propiecepolypeptide-encodingpolynucleotides, directintracellularintroduction SUBSTITUTE SHEET (RULE 26) of an IL-1 a propiece polypeptide, and/or extracellular delivery of an IL_I a propiece polypeptide, each of which can be accomplished using methods well known in the art. Preferably, the target cells are screened for susceptibility to apoptosis associated with delivery of extracellular IL-1 a propiece polypeptide and/or expression of IL-1 a propiece polypeptide-encoding polynucleotides.
For example, candidate target cells can be screened for susceptibility to apoptosis following exposure to a viral vector comprising an IL-1 a propiece polypeptide-encoding nucleic acid. Candidate target cells are grown in culture or plated at 60-80% confluency, or are grown or prepared in suspension at a cell density of from about 103 to about 105 cellslml, more preferably about 2 x 10" cells/ml. The viral vector is then added to the cells at a suitable multiplicity of infection (MOI), e.g., about 10 infectious particles per cell.
Apoptosis can be evaluated as described above for the candidate IL-1 a propiece polypeptide variant assay. In short, apoptosis can be evaluated by the TUNEL method (which involves 3' end-labeling of cleaned nuclear DNA) (Cohen et al. (1984) J. Immunol. 132:38-42) and/or morphological criteria (Cohen et al., supra). Wlxere the screen uses a fusion polypeptide comprising an IL-1 a(1-118) polypeptide and a reporter polypeptide (e.g., EGFP), apoptosis can be evaluated by detection of nuclear localization of the reporterpolypeptide in fragmented nuclear bodies or apoptotic bodies. For example, where an IL
1 a propiece (1-118)-EGFP fusion polypeptide is used, distribution of IL-1 a propiece (1-118)-EGFP-associated fluorescence in apoptotic cells was identical to the distribution of DAPI or Hoechst 33342 dyes, which are conventionally used to detect the nuclear DNA changes associated with apoptosis (Cohen et al., supra).
Alternatively or in addition, susceptibility of candidate target cells can be assessed by delivery of an IL-1 a propiece polypeptide or polynucleotide to a candidate target cell, and subsequent implantation of the treated cell into an animal model. Inhibition of the ability of treated cells to facilitate tumor growth, production, or metastasis (e.g., relative to tumor production by untreated cells) is indicative of susceptibility of the candidate target cell to IL-1 a propiece polypeptide-mediated apoptosis.
Induction of apoptosis in a candidate target cell in the cell-based assay or animal model-based assay described above can be indicative of the SUBSTITUTE SHEET (RULE 26) susceptibility of the target cell in a subject in vivo. Likewise, the candidate target cell screening assay can also aid in the identification of subjects having a cancer amenable to IL-1 a propiece polypeptide-based therapy.
Cancers susceptible to IL-1 a propiece polypeptide-based chemotherapy 5 to date include, but are not limited to, skin cancer (e.g., melanoma), colon cancer (e.g., adenocarcinoma), cancers of the central nervous system (e.g., brain tumors, such as grade II and IV glioblastomas, glioblastoma multiforme, astrocytomas, etc.), leukemia (e.g., acute myelogenous leukemia, acute lymphocytic leukemia, acute myelo-monocytic leukemia, pro-myelocytic 10 leukemia, etc.), prostate cancer (e.g., adenocarcinoma), kidney cancer (e.g., renal cell cancer (e.g., clear cell carcinoma, etc.)), lung cancer (e.g., squamous cell, small cell, large cell undifferentiated, adenocarcinoma, etc.), breast cancer (e.g., adenocarcinoma), and ovarian cancer (e.g., adenocarcinoma, etc.).

The polynucleotides and polypeptides of the invention can be used to facilitate induction of apoptosis in cancer cells in a subject having a cancer cell (e.g., a tumor, especially a malignant tumor) susceptible to IL-1 a propiece polypeptide-facilitated apoptosis. The formulations of polynucleotides and 20 polypeptides described above can be administered by any route suitable to accomplish expression of introduced IL-la propiece polypeptide-encoding polynucleotides(e.g., DNA-based polypeptide delivery), direct intracellular polypeptide delivery, and/or extracellular polypeptide delivery.
Formulations suitable for administration of the polynucleotides and 25 polypeptides of the invention include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and 30 preservatives. The formulations of packaged nucleic acid can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The precise formulation used and/or the formulation preferred will vary with a variety of SUBSTITUTE SHEET (RULE 26) factors including, but not limited to, the mode of IL-Ia propiece polypeptide delivery (e.g., DNA-based IL-1 a propiece polypeptide delivery or IL-1 a propiece polypeptide delivery), the route of administration, and other factors that will be readily apparent to the ordinarily skilled artisan.
Administration of polynucleotides and polypeptides of IL-Za propiece polyPeP~
The pharmaceutical compositions of the invention comprising IL-1a propiece polynucleotides and/or polypeptides are preferably administered topicallyorparenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. More preferably, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection (see, e.g., U.S. Patent No. 5,286,634; Straubringer et al., (1983) Meth Enzymol 101:512-527; Mannion et al. (1988) Biotechniques 6: 682-690; Nicolau et al. (1989) Crit. Rev. Ther. Drug Carrier Syst. 6:239-271, and Behr (1993) Acc. Chem. Res. 26:274-278. Still other methods of administering therapeutics are described in, for example, U.S. Patent Nos. 3,993,754;
4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578.
In preferred embodiments, the pharmaceutical preparations may be contacted with the target tissue by direct application of the preparation to the tissue. The application may accomplished by topical administration or by "open" or "closed" procedures. By "topical", it is meant the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like. "Open"
procedures are those procedures which include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical preparations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdomincxl laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue. "Closed" procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin. For example, the preparations may be administered to the peritoneum by needle lavage. Likewise, the preparations may be administered through endoscopic deuices. Preferably, where a closed procedure is used, the formulation is injected directly into the SUBSTITUTE SHEET (RULE 26) WO 98/54205 PCT/US9$/10839 tumor site.
The pharmaceutical compositions of the invention can also be administered in an aerosol inhaled into the lungs (see, Brigham, et al. (1989) Am. J. Sci., 298(4):278-281) or by direct injection at the site of disease (Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)).
Effective doses of the pharmaceutical compositions of the present invention will vary depending upon many different factors, including the form of the composition administered (e.g., polypeptide or polynucleotide, viral vector or non-viral vector, etc.), the means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages will need to be titrated to optimize safety and efrzcacy;
such can be readily determined and are routine to the ordinarily skilled artisan.
In determining the effective amount of polypeptide or polynucleotide to be administered, the physician evaluates, for example, the particular composition used, the disease state being diagnosed; the age, weight, and condition of the patient, formulation toxicities, disease progression, production of anti-vector antibodies, and the like. The dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular formulation. Doses ranging from about 10 ng to 1 g, 100 ng to 100 mg, 1 ~cg to 10 mg, or 30-300 ~g polypeptide or polynucleotide per patient are typical. Doses generally range between about 0.01 and about 50 mg polypeptide or polynucleotide per kilogram of body weight; preferably between about 0.1 and about 5 mg/kg polypeptide or polynucleotide of body weight or, where a viral uector is used, about 108-10'~
or 1012 particles per injection. In general, where a polynucleotide is delivered, the dose equivalent of naked nucleic acid is from about 1 tcg to 100 ~g for a typical 70 kilogrnm patient, and doses of vectors which include a retroviral particle are calculated to yield an equivalent amount o f inhibitor nucleic acid.
The precise route of administration, formulation, delivery system, and dosage will vary according to the IL-1 a propiece polypeptide-based therapy selected, e.g., DNA-based IL-1 a propiece polypeptide delivery, direct intracellular IL-1 a propiece polypeptide delivery, or extracellular IL-1 a propiece polypeptide delivery. For example, in the case of virus-based delivery SUBSTITUTE SHEET (RULE 2fi) the clinician may choose to administer a quantity of viral particles sufficient to provide an MOI of about or greater than 10 infectious particles per cell estimated to be associated with a target tumor. Where IL-a propiece polypeptide is delivered using a liposome of vesicle, the clinician may choose to administer a quantity of the formulation sufficient to provide a concentration equal to or greater than about 1-3 ~g/ml in the region of the tumor.
DNA based delivery of 1~1 a prnpiece polypeptide Delivery of IL-1 a propiece polypeptide to a target cell can be accomplished by introducing IL-1 a propiece polypeptide-encoding polynucleotides into a target cell in vivo, using methods known to those of ordinary skill in the art as discussed above (see, e.g., Zhu, et al. (1993) Science 261:209-211 (intravenous delivery o f DNA-containing DOTMA-DOPE
complexes), Hyde et al. (1993) Nature 362:250-256 (delivery of cystic fcbrosis transmembrane conductance regulator (CFTR) gene to the lung using liposomes); Brigham et al. (1989) Am. J. Med. Sci. 298:278-281 (in uiuo lung transfection)). In vivo gene transfer may be practiced in a variety of hosts.
Preferred hosts include mammalian species, such as humans, non-human primates, dogs, cats, cattle, horses, sheep, and the like. DNA-based IL-1 a propiece polypeptide delivery is advantageous in that, unlike soluble chemotherapeutic agents, the amount of IL-1 a propiece polypeptide delivered to a target cell can be controlled through regulation of expression of the I~1 a propiece polypeptide-encoding polynucleotide.
Direct intrace11u1ar delivery of I~Ia p~piece polypeptide I~1 a propiece polypeptides o f the invention can be delivered to a target cell by direct intracellular delivery, e.g., by introduction of a IL-Ia propiece polypeptide into the target cell cytoplasm. Methods for accomplishing direct intracellular deliver include contacting a target cell with a liposomal formulation of an IL-I a propiece polypeptide (see, e.g., USPNs 4,394,438;
5,459,127), by microinjection (e.g., stereotactic injection, as into a brain tumor (see e.g., Zlokouic et al. (1997) Neurosurgery 40:805-12)), and other methods well known in the art.
SUBSTITUTE SHEET (RUIN 26) WO 98/54205 PCT/US98l10839 Delivery of IL-1 a pmpiece polypeptide by extracellular coni~act IL-1 a propiece polypeptides of the invention can also be delivered to target cells by contacting the IL-1 a propiece polypeptides with the surface of a target cell, i.e., by delivering the IL-1 a propiece polypeptide extracellularly.
Methods for extracellular delivery suitable for IL-1 a propiece polypeptide delivery include delivery methods suitable for administration of polypeptides to a subject, e.g., parenteral injection and topical administration. Of particular interest is the administration of an IL-1 a propiece polypeptide into and/or around the site of a tumor comprising target cells susceptible to IL-1 a propiece polypeptide-induced apoptosis.
IL-a propiece polypeptides of the invention may be incorporated into liposomes. Liposomes suitable for use in the invention may be of conventional phospholipid:cholesterol composition, or may contain incorporated proteins serving as affinity ligands for the tumor target. Affinity ligands can include, but are not necessarily limited to, polypeptide ligands (e.g., peptide hormones (e.g., EGFP), and fragments thereof retaining receptor binding activity, etc.), antibodies (or fragments thereof) having binding specificity for surface determinants of a targeted cell (e.g., a tumor antigen), etc. The liposome-propiece material may be delivered in any of a variety of ways, e.g., by inftltration into the tumor by local injection or by introduction into a branch of an artery supplying blood to the tumor.
Alternatively, affinity ligands for delivery to a selected target cell may be coupled directly to the IL-a propiece polypeptide, e.g., through a covalent linkage or through a high affinity association such as that obtained when biotin and avidin are coupled to separate polypeptides which then associate by virtue of the avidin-biotin association.
ASSESSMENT OF IL-1 a PROPIECE POLYPEPTIDE-BASED CHEMOTHERAPY
Following delivery o f an IL-1 a propiece polypeptide to a subject having cancer, the subject's progress and the efficacy of IL-1 a propiece polypeptide-based chemotherapy can be assessed by parameters and methods that will be readily apparent to the ordinarily skilled artisan. For example, IL-1 a propiece polypeptide-based chemotherapy can be assessed by observation of the subject of signs of tumor regression using, for example and where appropriate, SUBSTITUTE SHEET (RULE 26) imaging techniques that are capable of visualizing cancerous tissues (e.g., MRI), biopsies, methods for assessing metabolites produced by the cancer tissue or affected tissue in question, the subjective well-being of the patient, etc.
5 For example, the pharmaceutical compositions of the invention can be provided in the form of a viral vector and administered by infusion. Prior to infusion, blood samples are obtained and saved for analysis. Approximately 108 and 1012 viral particles carrying an I~1 a propiece polypeptide-encoding polynucleotide are infused intra-arterially over 60-200 minutes. Vital signs 10 and oxygen saturation by pulse oximetry are closely monitored. Blood samples are obtained 5 minutes and 1 hour following infusion and saved for subsequent analysis. At the physician's discretion, reinfusion can be repeated every month (since IL-1 a propiece polypeptide is non-immunogenic) for a total of 4 to 6 treatments in a one year period. After the first treatment, infusions 15 can be performed on a outpatient basis at the discretion of the clinician.
If the reinfusion is given as an outpatient, the participant is monitored for at least 4, and preferably 8 hours following the therapy.
I f a patient undergoing infusion develops fevers, chills, or muscle aches, he/she receives the appropriate dose of aspirin, ibuprofen or acetaminophen.
20 Patients who experience reactions to the infusion such as fever, muscle aches, and chills are premeditated 30 minutes prior to the future infusions with either aspirin, acetaminophen, or diphenhydramine. Meperidine is used for more severe chills and muscle aches that do not quickly respond to antipyretics and antihistamines. Vector infusion is slowed or discontinued 25 depending upon the severity of the reaction.
Detection of apoptosis by detection of cell growth inhibition Efficacy of IL-1 a propiece polypeptide-based chemotherapy can also be assessed by detection o f apoptosis-associated cell growth inhibition.
"Inhibiting cell growth" or "inhibiting tumor growth" generally means that the 30 rate of increase in mass, size, number and/or the metabolism of treated cells and/or tumors is slower relative to untreated cells and/or tumors. The growth of a cancerous cell or tumor is said to be "inhibited" by IL-1 a propiece polypeptide-based treatment i f, when assayed by means such as radioisotope incorporation into the cells, the treated cells increase in number at a rate that SUBSTITUTE SHEET (RULE 26) is less than the proliferation rate of untreated control cells, and preferably less than about 50% of the untreated cell proliferation rate. More preferably, the proliferation rnte is inhibited by at least 80%. If growth is assayed by a means such as plating in methylcellulose, cell growth is "inhibited" if treated cells give rise to a number o f colonies less titan a number o f colonies that result from plating approximately the same number of untreated cells.
Preferably, the number of colonies that arise from treated cells is less than about 70% of the number from untreated cells. More preferably, the number of colonies is decreased by at least 50%.
"Inhibition of cell growth" also encompasses zero growth and death of tumor cells and/or tumor eradication. When measured in uiuo, "inhibition of tumor growth" means that a treated tumor is smaller (e.g., smaller in diameter and/or mass) or, when tumor growth is associated with metastasis, gives rise to fewer tumors growths as compared to untreated controls.
Inhibition can be evaluated by any accepted method of measuring whether growth or size of the tumor and/or increase in the number of cancerous or tumor cells has been slowed, stopped, or reversed. This includes direct observation and indirect evaluation such as subjective symptoms or objective signs. The clinician may notice a decrease in tumor size or tumor burden (number of tumors) based on physical exam, laboratory parameters, tumor markers, or radiographic findings. Alternatively, if the mammal is human, the patient may note improved vigor or vitality or decreased pain as subjective symptoms of improvement or response to therapy. Some laboratory signs that the clinician may observe for response to therapy include normalization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rnte, and various enzyme levels such as transaminases and hydrogenases. Additionally, the clinician may observe a decrease in a detectable tumor marker such as prostatic specific antigen (PSA) or chorio embryonic antigen (CEA). Alternatively, other tests can be used to evaluate objective improvement such as sonograms, computerized axial tomography scans, nuclear magnetic resonance scans and positron emission testing.
SUBSTITUTE SHEET (RULE 26) EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to carry out the invention and is not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1: Production of DNA encoding native IL-1 aft-118) IL-1 a cDNA was obtained by first treating human HL-60 cells with 0.1 ~M phorbol myristate acetate for 24 hrs to induce IL-1 production. Total mRNA was prepared using standard techniques, and IL-1 a cDNAs obtained from poly-A mRNA by reverse transcription using oligo-dT and MLV reverse transcriptase according to standard protocols and a commercially available kit(SuperscriptReverse Transcriptase, BRL/Life Sciences, Gaithersberg, MD).
The nucleic acid sequence (SEQ ID NO: l) encoding human IL-1 a pro piece (1-118) (IL-1 a propiece (1-118) (SEA ID N0:2)) was specifically isolated through 38 cycles of PCR using Taq polymerase under standard conditions as described by the manufacturer, and using a Perkin Elmer 9600 PCR
machine. The annealing temperature was 56 °C. The 5' primer used in PCR, 5'cagtaCTCGAGCGGCCGCCACCatggccaaagttccagacatg (SEQ ID N0:3), contains (from 5' to 3') a Xhol and a Notl site, a consensus Kozak sequence (GCCACC), a start codon (ATG), and the first 19 nucleotides of human IL-1 a cDNA that follow the start codon. The Kozak sequences incorporated into the 5' primer served to place the primer immediately 5' of the ATG translation initiation codon. The 3' primer used in PCR, 5'gactaAGATCTCTAGATTAgaagctaaaaggtgctgac (SEQ ID N0:4), contains (from 5' to 3') an Xbal site, the inverse complement of an introduced stop codon (TTA), and the inverse complement of the cDNA of the IL-1 a pro piece.
The restriction sites incorporated into the primers allowed for ligation of the resulting amplified PCR product into convenient expression constructs.
SUBSTITUTE SHEET (RULE 26) WO 98/54205 PCTlUS98/I0839 The PCR reaction products were precipitated and resolved on a 1.5%
low melting temperature agarose gel. The amplif-ced human IL-1 a pro piece-containing PCR product was excised from the gel, digested with Xhol and Xbal and cloned into commercially available expression vectors, such as pCDNAI-AMP (Invitrogen).
Example 2: Production oaf IL-1 a_p~»niece <I-1181-EGFP ~ficsionprntein In order to facilitate detection of IL-1 a propiece (1-118) expression and induction of apoptosis, a fusion protein between IL-1 a propiece (1-118) and a fluorescent reporter molecule, green fluorescent protein (GFP), was produced.
Specifccally, a variant of the Aequoria victoria GFP variant, enhanced green fluorescent protein (EGFP), was used in fusion protein production (CLONTECH Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA
94303). The IL-1 a propiece (1-118) cDNA sequence was fused in-frame by insertion of the IL-1 a propiece (1-118)-encoding cDNA into the SaII BamHI
site of plasmid pEGFP-NI (GenBank Accession # U55762). The resulting cDNA insert is presented in the Sequence Listing as SEQ ID N0:5, where nucleotides 1-354 encode IL-1 a propiece (1-118), nucleotides 355-375 encode a linker polypeptide, and nucleotides 376-1092 encode EGFP. The translated fusion protein is presented in the Sequence Listing as SEQ ID NO: 6. The IL-1 a propiece (1-118) portion is present as amino acid residues 1-118, the linker polypeptide is positioned at amino acid residues 119-125, and the EGFP
portion is present as amino acid residues 126 364. To demonstrate that the fusion protein consisting of IL-Ia (1-118) fused to the coding sequence of EGFP possessed apoptosis-inducing properties substantially similar to the IL-1 aparent molecule, 293 cells were transfected with plasmid vectors expressing either IL-1 a or the IL-1 a-EGFP fusion protein. Approximately 5x106 293 cells in 100 mm dishes were transfected with 10 ~g of plasmid DNA using the calcium phosphate method. The plasmids used were pCDNA-IL-1 a (CMV
enhancer/promoter, IL-1 a coding sequence) or pEGFP-NI-IL-1 a (CMV
enhancer/promoter, IL-1 a-EGFP coding sequence). Apoptosis was evaluated 24 hrs after traps fection.
In the IL-1 transfected cells, TUNEL and DAP/ staining revealed approximately 90% apoptosis. The IL-1 a-EGFP vector trnnsfected cells were SUBSTITUTE SHEET (RULE 26) evaluated by fluorescence microscopy with observation of typical nuclear aggregation of the EGFP marker as an indication of apoptosis. Expression of the IL-1 a(1-118)-EGFP also produced apoptosis in approximately 90%
apoptosis. Therefore, presence of the reporter potypeptzde does not substantially affect the ability of the polypeptide to induce apoptosis. The distribution of EGFP signal in cells expressing IL-1 a(1-118)-EGFP was identical to the distribution of DAPI or Hoechst 33342 dyes, which are conventionally used to detect the nuclear DNA changes associated with apoptosis (Cohen et al., supra). This observation suggests that the I~1 a propiece (1-118) polypeptide is associated with DNA fragments generated during apoptosis. Thus, the presence o f I~1 a propiece (1-118)-EGFP-associated fluorescence in fragmented nuclear bodies or apoptotic bodies is indicative of induction of apoptosis in the target cell.
Example 3: Production o~F IL-1 a p~piece ~1-118) variants Variants of the IL-1 a propiece (1-118)-EGFP fusion were produced by site-specific mutagenesis according to methods well known in the art. The IL-1 a propiece (1-118)-EGFP construct described in Example 2 above served as the parent molecule. Where the I~1 a propiece (1-118) variant comprises the first N-terminal amino acids of IL-1a propiece (1-118) (SEQ ID N0:2), the normal methionine initiation codon of IL-1a propiece (1-118) was used.
Where the I~1 a propiece (1-118) variant were N-terminally truncated relative to 1~1 a propiece (1-118) (SEQ ID N0:2) (i.e., the variant polypeptide began with at an amino acid residue other than residue 1 of SEA ID N0:2), an ATG initiation methionine codon was inserted so as to reconstruct a coding sequence. The last codon o f the I~1 a variants were followed by either a termination codon or, in the case of fusion proteins with EGFP, the last codon was placed in the correct reading frame so as to produce a complete fusion protein.
A summary o f the IL-1 a propiece (1-118) variants produced is provided in Table 1. The I~1 a(1-118) variants indicated by an asterisk were produced both as EGFP fusions proteins and as independent polypeptides (i.e., without the EGFP polypeptide). Fig. 2 is a schematic showing the positions of the Ilr 1 a propiece (1-118) variants relative to native I~1 a propiece (1-118).
SUBSTITUTE SHEET (RULE 26) Table 1: Ilrla(1-118) Variants ame ascription -la(1-11 )- la(1-118) fuse ~to~~--EGFP

5 la(11-118) la 11-118; anionic amino acids in elix 1 remove la(1-90) IL-la(1-90); anionic residues in he ix 3 removed la(11- ) la(11- ) -1 a ( 5-I1 1 a ( -118); contains on y a ix 3 ) -la( 4-11 ) la(34-118); a ix 1 removed in its entirety 10 IL-la(1-118) IL-la(1-118), er 87 to Ala point mutation, 87A removed phosphorylation site la(5 -11 ) la(55-11 ), first he ix an elf of secon elix removed.

-la 66-118) IL-la(6 -118); asic residues etween 55 and 4 removed IL-la(1-108) IL-la(1-108); acidic residues around 95 and 107 restored IL-la(1-118)- Leucine zipper omain (L- inserted between IP- IL-la(1-118) and 15 EGFP EGFP in fusion protein construct via BamHI
and AgeI sites NL5-IL-la(86-118)40 nuclear loca ization sign sequence ( ) added to IL-la(86-lI8) la( 5-11 ) 40 a de to la(75-118) iL-la(55-108) la( -108), first he ix an ha f of secon a ix removed, ast 10 AA removed IL-la(75-108) V40 added to IL-la(75-108) 20 la propiece inserted etween la(1-108) and in fusion protein (1-108)-ZIP-EGFP construct via BamHI and AgeI sites iL-la(55-1 inserte etween -la(5 -108) an in fusion protein 8)-Z
V a EGFP construct via BamHI and AgeI sites NLS-iL-la(75- inserted between NL la( 5-118) and P via Bam I18)-ZIP and AgeI sites NLS-I~-la( inserte etween NL -la(75-108) and P via am 108)-ZIP and AgeI sites pr;C~rr'Y-N1-ILla(1-Tamoxifen-sensitive mouse estrogen receptor ( 525 -> R) ormone 118)-musER binding domain (musER) to C-terminus of IL-la(1-118)-EGFP

fusion. Mutation in musER renders hormone binding domain insensitive to estrogens but sensitive to binding of the estrogen analog 4-OH tamoxifen.

The amino acid sequences of NLS-IL-la(75-118) (SEQ ID N0:21), NLS-IL-la(75-108) (SEQ ID N0:22), NLS-IL-la(75-108)-ZIP (SEQ iD
N0:23), NLS-IL-la(75-118)-ZIP (SEQ ID N0:24), IL-la(1-118)-ZIP (SEQ ID
N0:25), IL-la(1-108)-ZIP (SEQ ID N0:2fi), and IL-Ia(55-108)-ZIP (SEQ ID
35 N0:27) are provided in the Sequence Listing.
SUBSTITUTE SHEET (RULE 26) Example 4: Vectors for expression ~ I~1 a propiece l1-118) and variants thereo Plasmid vectors. All plasmid vectors used in transfection studies were composed o f standard plasmid backbones, a CMV enhancer promoter operably linked to a coding sequence For IL-1 a propiece (1-118) or a variant thereof, followed by a polyadenylation signal derived from the SV40 virus. In some cases an intron and splice sequence were placed between the CMV
enhancer/promoter and the coding sequence. The coding sequence included a Kozak sequence (ccacc) positioned immediately 5' to the ATG initiation codon. Plasmid uectors were introduced into cells by a variety of methods appropriate to a given cell types including calcium phosphate transfection, electroporation, or cationic liposome-mediated transfection (e.g., Lipofection).
Exemplary commercial vectors used herein include pCDNA (Invitrogen) and pEGFP-NI (Clontech).
Retroviral vectors. Retroviral vectors used were as described in Finer et al. (1994) Blood 83(1):43-50. These vectors used a modifted Moloney murine sarcoma virus promoter. Instead of the murine coat proteins, the viruses were pseudotyped with the coat protein of vesicular stomatitis uirus (VSV G) as described in Ory et al. (1996) Proc. Natl. Acad. Sci. USA
93(21):11400-6. Retroviral preparation were applied to proliferating cells n culture medium to allow infection. Cells were evaluated about 24 to 96 hrs later. Polybrene (8 ~g/ml) was added to some transfections to enhance ef~CCiency of retroviral infection.
Sindbis viral vectors. Sindbis viral vectors expressing either the control EGFP construct or IL-1 a propiece (1-118)-EGFP were prepared using standard methods and a hit from Invitrogen. The general method for Sindbis viral vector production is described in Altman-Hamamdzic (1997) Gene Therap. 4(8):815-22. Vector titers of approximately 10'-108 uiral particles/ml were obtained.
SUBSTITUTE SHEET (RULE 26) 4?
Example 5: Induction oaf ap~~tosis by expression of I~1 a propiece fl-118) variants The ability of the IL-1 a propiece (1-118) variants described above to undergo nuclear localization and to induce apoptosis was tested by transient expression in 293 human kidney cells as described in Example 2 above. 293 cells were used since this cell line was convenient and had proved susceptible to IL-1 a propiece (1-118)-induced apoptosis. Thus, the ability of an IL-1 a propiece (1-118-EGFP variant to induce apoptosis in 293 cells served as an initial screen for those variants that will likely also induce apoptosis in cancer cells. Nuclear localization and induction of apoptosis were observed by fluorescence microscopy and scored by direct cell counting under fluorescence microscopy. The results are summarized in Table 2. Results denoted by dashes ( -) indicates that there was no detectable nuclear localization or induction of apoptosis. Results were scored on a semi-quantitative scale, with IL-1 a propiece polypeptide set at 4+.
SUBSTITUTE SHEET (RULE 26) Table 2: Nuclear localization and apoptosis induction by Ilrla propiece (1-118) variants Name Nuclear ApopWsiName Nuclear Apoptosis Localizatios LocalizationInduction n Inductio n IL-la 4+ 4+ IL-la(5~-118) 4+ 3-4+

propiece (1-lI8)-EGFP

-la(11-118)4+ 4+ -la(i-108) 4+ 4+

IL-la(1-90)1+ - IL-la propiece 4+ 4+
(1-118)-ZIP-EGFP

la(11-90) -- - la( 6-118) 4+ --IL-la(75-118)1+ - -la( 75-118) 4+ 4+ "

IL-la(34-118)4+ 4++ L -la(75-108) 4+ 4+"

IL-la 4+-- 3-4+ pEGFP-NI-ILla(1-4+ 4+' propiece 118)-musER
(1-118)S87A

IL-la 4+ 4+ IL-la(55-108) 4+ 4+

propiece (55-108)-ZIP-EGFP

NLS-IL- 4+ -- 4+"' NLS-IL-la( 75-118)-4+ - - 4+**

la(75-108)- ZIP

ZIP

'Observation of apoptosis was dependent upon addition of 10 nM 4-6H
tamoxifen (an estrogen analog that binds the musER portion of the fusion protein).
'~'~Apoptosis is delayed until 72 hours.
These data show that much of the N terminal portion of IL-1 a propiece (1-118) is not necessary to effect apoptosis, since the variants containing only amino acid residues 1-108,11-118, 34-118, 55-118, 55-108, 75-118, and 75-108 effectively induced apoptosis. The anionic portion of the terminal helix of residues 90-108 or 90-118 are apparently required for apoptosis-inducing activity since the IL-1 a(1-90) uariant did not effect detectable apoptosis as measured in the apoptosis assay. The data also show that an intact terminal helix of residues 75-118 is required but not sufficient, and that portions of helix 2 are required.
Moreover, the nuclear localization signal sequence (NLS~ KKRR at 82 is not an independent NLS in this context. The serine residue at position 87 following KKRR is not necessary for killing. This result was particularly surprising since Ser87 is a putative phosphorylation site and was thought important for biological actiuity. Amino acid residues 5b-74 are apparently SUBSTITUTE SHEET (RULE 26) required for nuclear localization and for killing, since IL-1 a(75-118) was not detected in the nucleus and did not kill cells while IL-1 a(55-118) exhibited both nuclear localization and effected killing.
Finally, inclusion of a heterologous NLS was effective in delivering IL-1 a propiece polypeptides to the nucleus and allowing the polypeptide to effect apoptosis. This effect was particularly interesting when the heterologous NLS
was fused to the IL-1 a(75-118) polypeptide. When used alone, the IL-1 a(75-118) polypeptide was not transported to the nucleus in very high amounts, and no apoptotic effect was observed. Addition of the heterologous NLS facilitated transport of the IL-1 a(75-118) polypeptide to the nucleus, where the IL-1 a(75-118) polypeptide efficiently effected apoptosis. This observation suggests that the NLS and apoptotic-inducing activity of the IL-1 a propiece polypeptide are separable, and that heterologous NLS can substitute for the NLS present in IL-1 a propiece polypeptide.
In summary, these data show that the minimum region of the IL-1 a propiece (1-118) required to accomplish both nuclear localization and killing is a region spanning or contained within amino acid residues 55-108, while the amino acid residues spanning 75-108 are necessary for apoptosis induction. Equivalents of either or both of these amino acid sequences that retain activity in either nuclear localization, apoptosis-induction, or both can be readily produced.
To further confirm that activity of the apoptosis-inducing polypeptides, constructs encoding the variants IL-1 a(11-118), IL-1 a(34-118), IL-1 a(55-118), and IL-1 a propiece (1-118)S87A were prepared without the reporter EGFP
fusion polypeptide, and the ability of these variants to induce apoptosis in cells tested. Each of these polypeptides induced apoptosis at levels similar to those observed when the polypeptides were part of the EGFP fusion protein.
Example 6: Induction of ayoptosis in matignant melanoma cells by transient. plasmid-based expression o~F IL-1 aft-1181 The susceptibility of malignant melanoma cells to apoptosis induced by IL-1 a propiece (1-118) was tested in several melanoma cell types: human melanoma WM 266-4 (ATCC CRL-1676); human malignant melanoma A-375 (ATCC CRL-1619); human malignant, melanoma A2058 (ATCC CRL-11147);
SUBSTITUTE SHEET (RULE 26) humanmalignantmelanomaSKMEL-31 (ATCCHTB-73); humanmalignant melanoma RPMI-7591 ATCC HTB-66 (metastasis to lymph node). Two primary melanoma isolates, MZWR (from a metastasis from a right axillary melanoma from a 35 years old Caucasian male) and MZVA (a metastatic 5 melanoma from right arm of a 69 year old Caucasian female) were also tested. In addition, human chronic myelogenous leukemia K 562 cells (ATCC
CCL-243), and 293 human kidney cells (ATCC CRL-1573) (transformed primary embryonal cell) were tested. Normal human primary dermal ~-Cbroblasts and Rat-1 fibroblasts served as controls. All melanoma cell lines 10 were metastatic on the basis of their isolation from metastases or metastatic nodules.
A transient expression strategy was used in order to evaluate induction of IL-1 a propiece (1-118)-mediated apoptosis without artifacts associated with prolonged selection. The expression vector encoding the IL-1 a(1-118)-EGFP
15 fusion protein described in Example 2 above was used in order to facilitate identification of those cells expressing the IL-1 a(1-118). Cells were transiently transfected by the method optimal for the cell being tested (either CczP04 or Lipofectin). Expression of IL-1 a(1-118) and induction of apoptosis were examined using a fluorescence microscope at 24 hrs and 48 hrs post-20 transfection. A minimum of approximately 100 cells, which displayed characteristic EGFP fluorescence, were evaluated by fluorescence microscopy.
Apoptosis was scored as nuclear fragmentation, marked apoptotic bodies, and cytoplasmic boiling. The characteristics of nuclear fragmentation were particularly visible when IL-1 a(1-118)-EGFP condensed in apoptotic bodies.
SU9STtTUTE SHEET (RULE 26) Table 3: Induction of apoptosis by transient, plasmid-based expression in melanoma cells CELL LINE % Apoptotic % Apoptotrc at 24 at 48 hrs post transfectionhrs post tmnsfection Normal Human primary Dermal Fib~6lastsg RPM17951 Human Melanoma (ATCC 61 %
HTB66) 100%

WM266-4 Human Melanoma (ATCC 56%
CRL1676) A2058 Human Melanoma (ATCC CCL 86%
11147) 100%

A375 Human Melanoma (ATCC CRL 67%
1619) 100%

SK MEL31 Human Melanoma (ATCC 75%
HTB 73) Primary Isolate Human Melanoma 75%
(MZVA) Primary Isolate Human Melanoma 75%
(MZWR) 293 Human Kidney Cells (ATTC 71 % 90%
CRL 1573) (transformed) Rat-1 Fibroblasts (immortalized)14%

17%

While normal human primary dermal fibroblasts did not exhibit elevated levels of apoptosis, all the melanoma cell lines tested were susceptible to apoptosis following IL-1 a(1-118) expression. R.at-1 fibroblasts (an immortalized non-malignant cell line) were only minimally susceptible to IL-la (1-lI8) apoptosis. In contrast, 293 human kidney cells (which are capable of forming tumors when introduced into mice and are thus malignant by this criteria), were highly susceptible to apoptosis induced by IL-la(1-118).
These data show that the IL-1 a propiece (1-118) polypeptide induces apoptosis in human melanoma cells, but does not substantially induce apoptosis in normal human cells.
Example 7.~ Inhibition of melanoma tumor ~rourth by expression o f I~1 a proniece (1-118 A375 melanoma cells were grown to 80% confluency in RPM/ 1640 medium containing 5% fetal bovine serum. The cells were treated with retroviruses carrying either an EGFP-encoding polynucleotide (control) or native human IL~1 a propiece (1-118) polypeptide-encoding polynucleotide fused in one continuous open reading frame to the coding sequence of EGFP.
SUBSTITUTE SHEET (RULE 26) The retroviruses were as described in Example 4 and were pseudotyped with VSV G. Approximately 5 x 108 viruses were applied to approximately 1 x 10' dividing A3?5 cells in a 150 cmz tissue culture flask in a final volume of 18 ml. At 24 hrs post-infection, the cells were trypsinized to remove them from the culture flask, centrifuged, and suspended in phosphate buffered saline (PBS) at approximately 106 cells/ml. A volume of 0.1 ml of the cell suspension was injected subcutaneously into the flanks of female, ? week-old, immunodefzcient mice (nude mice). Tumor formation in the flanks was measured with calipers and tumor volume calculated by the formula:
Volume = (longest dimension x shortest dimension2) / 2.
Measurements of mean tumor volume in each of 8 mice per construct were taken at days 13, 16, and 19 post-injection. The results are presented in Figs.
3-5 (tumor volume at days 13, 16, and 19, respectively) and are summarized in Table 4 below.
Table 4: Tumor volume associated with melanoma cells expressing IL-1 a prnpiece (1-118~EGFP or EGFP
Construct Day 13 volume Day 16 volume Day 19 volume EGFP (control)22 mm' 20 mm' 32 mm' IL-1 a propiece2.3 mm' 3.7 mm' 4.1 mm' (1-118)-EGFP

The tumor volume in mice treated with Ilr1 a propiece (1-118) was significantly smaller than in control mice. In 3 of the 8 mice that received IL-1 a propiece (1-118), there was zero or negligible tumor growth. These data show that pre-treatment of A3?5 melanoma cells with retroviral vectors for expression of Il<-1 a propiece (1-118) prevents andlor severely diminishes tumor growth in the nude mouse model. These data suggest that Ilr1 a propiece (1-118) can be used to facilitate inhibition of tumor growth in vivo.
SUBSTITUTE SHEET (RULE 26) Example 8: Induction of apoptosis in cells of the standard National Cancer Institute ~lVCl) panel Cells from the NCI panel of tumor cells were tested for their susceptibility to IL-1 a propiece (1-118) by delivery of the IL-1 a propiece (1 118)-EGFP fusion protein. Cells were treated with the expression vectors indicated below so as to express either a control sequence (EGFP), IL-1 a propiece (1-118)-EGFP, or IL-1 a propiece (1-118) (without EGFP). The choice of vectors was based solely on the vector most efficient for a given cell type.
After the period indicated, cells were evaluated for morphological signs of apoptosis, including aggregation oflL-1 a propiece (1-118)-EGFP into nuclear apoptotic bodies. Cells were counted under a fluorescence microscope and scored as to the presence or absence of apoptotic signs. In some cases, cells were scored by flourescent TUNEL assay and counted in a flow cytometer.
Apoptosis is expressed as a percent of cells displaying typical advanced changes of apoptosis. In each cell line tested, expression of EGFP alone or transfection alone (viral vector only was not associated with any detectable apoptosis.
a) Colon cancer cells IL-1 a propiece (1-118) was expressed in the following cells, which are derived from malignant human colon cancers (adenocarcinomas~, by infection with the retrovirus vector described above. Apoptosis was evaluated at 96 hrs post-infection.
Table 5: Anoatosis in colon caacer_ Cell No. Total % Cell No. Total %
ApoptoticCells ApoptoticCells These data suggest that most coon cancers are susceptible to IL-1 a propiece (1-118)-based chemotherapy.
SUBSTITUTE SHEET (RULE 26) b) Cells of cent~l nervous system tumors IL-1 a propiece (1-118) was expressed in the following cells, which are derived from malignant human central nervous system tumors, by infection with the retrouirus vector described above. Apoptosis was evaluated at 96 hrs post infection. The specific cancer types of each cell are as follows: SF 268, astrocytoma; SF 539, glioblastoma; SNB-19, gliblastoma; SNB-75, astrocytoma; and U251, glioblastoma.
Table 6: Apoptosis in cancers of the central nervous system..
Cell No. Total % Cell No. Total ApoptoticCells ApoptoticCells l aese aorta suggest tnat most tumors oT the centrac nervous system are susceptible to IL-1 a propiece (1-118)-based chemotherapy.
c) Leukemia cells IL-1 a propiece (1-118) was expressed in the following cells, which are derived from malignant human leukemias. Expression was accomplished using the retrouiral vector and protocol described above. Apoptosis was evaluated at 96 hrs post-infection. The specific leukemia subtypes of the cells are as follows: CCRF CEM, acute lymphocytic leukemia (ALL); I~562, acute myelogenous leukemia (AML); MOLT 4, ALL; SR, immunoblastoma large cell; and RPMI 8226, myeloblastoma.
Table 7.~ Apoptosis in leukemias.
Cell No. Total% Cell No. Total %
ApopioticCells Apoptotic Cells CEM

SUBSTITUTE SHEET (RULE 26) These data suggest that most leukemias are susceptible to I~1 a propiece (1-118)-based chemotherapy.
d) Prostate cancer cetls IL-1 a propiece (1-118) was expressed in the prostate cancer cell PC 3, 5 which is derived from a malignant human prostate tumor. Expression was accomplished using the retrovirus vector and protocol described above.
Apoptosis was evaluated at 96 hrs post-infection.
Table 8: Apoptosis in prostate cancer.
Celt No. Total %

ApoptoticCells This experzment suggests t atprostate cancers may be susceptible to IL-1 a propiece (1-118)-based chemotherapy.

e) Kidney cancer cells IL-1 a propiece (1-118) was expressed in the following cells, which are 15 derived from malignant human kidney tumors (renal tumors). Expression was accomplished using the retrovirus vector and protocol described above.
Apoptosis was evaluated at 96 hrs post-infection.
Table 9: .Apoptosis in kidney cancers (renal).
Cel1 No. Total % Cell No. Total ApoptoticCells Apoptotic Cells These data suggest that most kidney cancers are susceptible to IL-1 a propiece (1-118)-based chemotherapy.
25 fj Skin cancer cells (melanoma) IL-1 a propiece (1-118) was expressed in the following human melanoma cells. Expression was accomplished using the retrovirus vector and protocol described above. Apoptosis was evaluated at 96 hrs post infection.
SU8ST1TUTE SHEET (RULE 26) Table 10: Apoptosis in melanomas.
Cell No. Total % Cell No. Total lo ApoptoticCells ApoptoticCells SKMEL- 93 100 93 Ml4 45 81 56 SI~IE~S 73 107 68 SKMEI<28 65 101 64 l ~.ese aaaa suggesa an,at mezanomas are suscepztoze to 11.-1 a propzece ( 1-118)-based chemotherapy.
g) Lung cancer cells IL-1 a propiece (1-118) was expressed in the following cells, which are derived from malignant human lung cancers. Expression was accomplished using the retrovirus vector and protocol described above.
Table 11: Apoptosis in lung cancer.
Cell No. Total olo Cell No. Total !o ApoptoticCells Apoptotic Cells !'hese data suggest that lung cancers are susceptible to IL-1 a propiece (1-118)-based chemotherapy.
h) Breast cancer cells I~1 a propiece (1-118) was expressed in the following cells, which are derived from malignant human breast cancer. Expression was accomplished using the retrovirus vector and protocol described above.
SUBSTITUTE SHEET (RULE 26) Table 12: Apoptosis in cancers of the mammary gland.
Cell No. Total% Cell No. Total %
ApoptoticCells ApoptoticCells ~ ~~e~e uuca suggesa areas oreast cancers are susceptible to 1L-1 a propiece (1-118)-based chemotherapy.
I) Ovary cancer cells lfl I~1 a propiece (1-118) was expressed in the following cells, which are derived from malignant human ovarian cancers. Expression was accomplished using either the retrouiral expression vector and protocol described above, or the Sindbis viral expression vector and protocol described above. Apoptosis was evaluated at 96 hrs post-infection with retrovirus or at 24 hrs post-infection with Sindbis viral vectors.
Table 13: Apoptosis in ovarian cancers using Sindbis viral vector-mediated exp~ssion (including floating cells).
Cell No. Total % Cell No. Total %
ApoptoticCells Apoptotic Cells This particular cell line floats off the plate when it undergoes apoptosis;
the percentages reported are the percent of total cells apoptotic. These data show that ovarian cancers are susceptible to IZr1 a propiece (1-118)-based chemotherapy. Further, vectors other than retroviral vectors can be used to accomplish IL-1 a(1-118) delivery.
SUBSTITUTE SHEET (RULE 26) Summary Delivery of IL-1 a propiece (1-118) to cancers of the colon, central nervous system, lung, mammary bland, skin (e.g., melanoma), ovary, prostate, and kidney (e.g., renal cancer), as well as leukemia, are susceptible to I~1 a propiece (1-118)-based chemotherapy.
Example 9: Induction ofanoptosis by cellular deliven~ ofl~1 a n~piece The ability o f I~1 a propiece (1-118) to induce apoptosis by extracellular delivery to cells was tested in vitro. Purified IL-1 a propiece (1-118) polypeptide (without EGI'P) was expressed in E. coli using an inducible Trp vector system. IL-1 a propiece (1-118) was isolated as inclusion bodies, dissolved in urea, and gradually renatured by reduction of urea concentration.
Purified I~1 a(1-118) was applied to lymphoma cells, ovarian cancer cells, and Rat-1 immortalized fibroblasts grown in culture medium with reduced serum at a concentration of 1.5 pg/ml (100 nM). Apoptosis was evaluated after 48 hrs. The results are summarized in Table 14.
Table 14: Induction o f apoptosis by extracellular delivery of I~1 a p~piece (1-118) Cell Name (cell type) % apoptotic cells as measured by TUNEL

P388D1 (lymphoma) 55%

Raw 264.7 (lymphoma) >90%

MI (lymphoma) 30%

CHD (ovarian cancer) 45%

Rat-1 (immortalized fibroblast)<2-5%

These data show that tumor cells are particularly susceptible to apoptosis induced by extracellular delivery of IL-1 a propiece (1-118) polypeptide.
SUBSTITUTE SHEET (RULE 26) Example 10: Purification of IL-1 a propiece f1-118) Recombinant IL-Ia propiece (1-118) polypeptide was purified by expression of the polypeptide in bacteria. cDNA encoding the N-terminal IL-1 a propiece was subcloned into the Ncol/Pstl site of the bacterial expression vector, pTRC99A. This plasmid was used to transform E. coli, followed by induction with IPTG. The recombinant protein was recovered in the insoluble inclusion bodies by first pelleting the bacteria in chilled de-ionized water at 3000g for 20 minutes at 4 °C. After addition of protease inhibitors (PMSF, EDTA, pepstatin. Leupeptin), the cells were subjected to three cycles of IO freeze/thawing in the presence of 1% Triton X 100. The material was centrifuged at IO,DOOg for 20 minutes at 4 °C and the resulting pellets (inclusion bodies) incubated for 10 min at 37°C with 1 mM EDTA, 0.2% NP-40, 2 mM MgClv 1 mM CaClv 0.2 mglml lysozyme and 10 ug/ml DNAse. Following centrifugation at 5000 g for 20 min at 4 °C, the purified inclusion bodies were dissolved in 6 M urea, 50 mM Tris/HCI, pH 8.0 with protease inhibitors. Analysis of the recombinant propiece protein was performed with reducing 15% SDS-PAGE gels and Coomassie Blue staining..
Purification of the recombinant protein to homogeneity was performed by two sequential gel chromatography sizing steps with G75 in the same 6M
urea, Tris/HCI, pH 8.0 buffer. Purred recombinant propiece protein was refolded by step-wise dialysis in 50 mM Tris/HCI, pH 8 buffer containing decreasing concentrations o f urea. This recombinant propiece preparation has been used to prepare goat polyclonal affinity puri fxed anti-IL-1 a propiece IgG.
SUBSTITUTE SHEET (RULE 26) SEQUENCE LISTING
(1) GENERAL INFORMATION

~J (i) APPLICANT: The Regents of the University of California (ii) TITLE OF THE INVENTION: Selective Induction of Apoptosis in Malignant Cancer Cells by Delivery of N-Terminal 10 Interleukin-1-Alpha Pro-Piece Polypeptide (iii) NUMBER OF SEQUENCES: 30 (iv) CORRESPONDENCE ADDRESS:

1 (A) ADDRESSEE: Fulbright 8 Jaworski L.L.P.
~J

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~JO (2) INFORMATION FOR SEO ID N0:1:

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60 (A) NAME/KEY: Coding Sequence (B) LOCATION: 1...354 (D) OTHER INFORMATION:

s~J (xi) SEQUENCE DESCRIPTION: SEfl ID N0:1:

Met Ala Lys Val Pro Asp Met Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Glu Asp Ser Ser Ser Ile Asp His Leu Ser Leu Asn Gln SUBSTITUTE SHEET (RULE 26) TCC CTC GGC
TTC
TAT
CAT
GTA
AGC
TAT
GGC

LysSer Phe Tyr His Pro HisGluGlyCysMet Vat Ser Tyr Gly Leu CTG AGT ATC TCT ACC

AspGln Ser Val Ser Glu SerLysThrSerLys Leu Ser Ite Ser Thr AGC ATG GTG GTA GCA

LeuThr Phe Lys Glu Val ThrAsnGlyLysVal Ser Met Val Val Ala TTG AGT TTA AGC TCC

LeuLys Lys Arg Arg Gln IleThrAspAspAsp Leu Ser Leu Ser Ser AAT GAC TCA GAG GAA

LeuGlu Ala Ile Ala Glu IleIleLysProArg Asn Asp Ser Glu Glu TTC

SerAla Pro Phe Ser Phe (2) INFORMATION H0:2:
FOR SEa 1D

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids 3O (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (v) FRAGMENT TYPE:
internal (xi) SEQUENCE DESCRIPTION:
SEO ID N0:2:

MetAla Lys Val Pro Asp LysAsnCysTyrSer Asp Met Phe Glu Leu GluAsn Glu Glu Asp Asp LeuSerLeuAsnGln Ser Ser Ser Ile His LysSer Phe Tyr His Pro HisGluGlyCysMet Val Ser Tyr Gly Leu AspGln Ser Vai Ser Glu SerLysThrSerLys Leu Ser Ile Ser Thr LeuThr Phe Lys Glu Val ThrAsnGlyLysVal Ser Met Val Val ALa LeuLys Lys Arg Arg Gln IleThrAspAspAsp Leu Ser Leu Ser Ser LeuGtu Ala Ile Ala Glu IleIleLysProArg Asn Asp Ser Glu Glu 100 105 i10 SerAla Pro Phe Ser Phe (2) INFORMATION N0:3:
FOR SEfi ID

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
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SEA ID N0:

GCGGCCGCCA
CCATGGCCAA
AGTTCCAGAC
ATG

(2) INFORMATION
FOR SEC ID N0:4:

(i) SEDUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single SUBSTITUTE SHEET (RULE 26) (D) TOPOLOGY:
linear (xi) SEQUENCE SEAID
DESCRIPTION: N0:4:

TCTAGATTAG
AAGCTAAAAG

(2) INFORMATION IDN0:5:
FOR SEO

1O (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1095 irs base pa (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY:
linear (ii) MOLECULE
TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY:
Coding Sequence 2O (B) LOCATION:
1...1092 (D) OTHER INFORMATION:

(xi) SEQUENCE SEOID :
DESCRIPTION: N0:5 GAC ATG TTT CTG

MetAla Lys Val Pro GluAsp LysAsnCysTyrSer Asp Met Phe Leu a3O GAAAAT GAA GAA GAC ATTGAT CTGTCTCTGAATCAG 96 AGT TCC TCC CAT

GluAsn Glu Glu Asp IleAsp LeuSerLeuAsnGln Ser Ser Ser His GTA AGC TAT CTC

LysSer Phe Tyr His GlyPro HisGluGlyCysMet Vat Ser Tyr Leu CTG AGT ATC ACC

AspGln Ser Val Ser SerGlu SerLysThrSerLys Leu Ser Ile Thr AGC ATG GTG GCA

LeuThr Phe Lys Glu ValVal ThrAsnGlyLysVal Ser Met Val Ala TTG AGT TTA TCC

LeuLys Lys Arg Arg SerGln IleThrAspAspAsp Leu Ser Leu Ser AAT GAC TCA GAA

LeuGlu Ala Ile Ala GluGlu IleIleLysProArg Asn Asp Ser Glu TTC TTG GAT GTC

SerAla Pro Phe Ser ProPro AlaThrMetValSer Phe Leu Asp Val TTC ACC GGG CCC

LysGty Glu Glu Leu ValVal lleLeuValGluLeu Phe Thr Gly Pro GGC CAC AAG GTG

AspGly Asp Val Asn PheSer SerGlyGluGlyGlu Gty His Lys Val 145150 155 1b0 GGC AAG CTG AAG

GlyAsp Ala Thr Tyr ThrLeu PheIleCysThrThr Gly Lys Leu Lys rIO GGCAAG CTG CCC GTG ACCCTC ACCACCCTGACCTAC 576 CCC TGG CCC GTG

GlyLys Leu Pro Val ThrLeu ThrThrleuThrTyr Pro Trp Pro Val AGC CGC TAC CAC

SUBSTITUTE SHEET (RULE 26) Gly Vat Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp GTC CAG GAG CGC ACC ATC

~J Phe Phe Lys Ser Ala Met Pro Glu Gly 1yr Val Gln Gtu Arg Thr Ile CGC GCC GAG GTG AAG TTC

Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe CTG AAG GGC ATC GAC TTC

Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe CTG GAG TAC AAC TAC AAC

Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn CAG AAG AAC GGC ATC AAG

Ser His Asn Val Tyr lle Met Ala Asp Lys Gln Lys Asn Gly lle Lys GAC GGC AGC GTG CAG CTC

25 Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu GGC GAC GGC CCC GTG CTG

Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu 3~ 305 310 315 320 TCC GCC CTG AGC AAA GAC

Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp CTG GAG TTC GTG ACC GCC

Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala TAC AAG TAA

Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 45 (2) INFORMATION FOR SEO ID N0:6:

(i) SEQUENCE CHARACTERISTICS:

tA) LENGTH: 3b4 amino acids (B) TYPE: amino acid ~J~(C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SECUENCE DESCRIPTION: SED ID N0:6:
Met Ala Lys Val Pro Asp Met Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Glu Asp Ser Ser Ser !le Asp His Leu Ser Leu Asn Gln 60 Lys Ser Phe Tyr His Val Ser Tyr Gly Pro Leu His Glu Gty Cys Met Asp Gln Ser Val Ser Leu Ser lle Ser Glu Thr Ser Lys Thr Ser Lys Leu Thr Phe Lys Glu Ser Met Val Val Val Ala Thr Asn Gly Lys Val srJ b5 70 75 80 Leu Lys Lys Arg Arg Leu Ser Leu Ser Gln Ser Ile Thr Asp Asp Asp Leu Glu Ala Ile Ala Asn Asp Ser Glu Glu Glu Ile Ile Lys Pro Arg Ser Ala Pro Phe Ser Phe Leu Asp Pro Pro Val Ala Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Vat Asn Gly His Lys Phe Ser Vat Ser Gly Glu Gly Glu SUBSTITUTE SHEET (RULE 25) Gly Asp Ala Thr Tyr Gly Lys leu Thr Leu Lys Phe Ile Cys Thr Thr 1b5 170 175 Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr rJ 180 185 190 Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu GLy Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Gtu Vat Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gty Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn 1 ~J 260 265 270 Ser His Asn Val Tyr ile Met Ala Asp Lys Gln Lys Asn Gly lle Lys Vat Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ata Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Vat Leu Leu Glu Phe Val Thr Ala 2rJ 340 345 350 Ala Gty Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys a3O (2) INFORMATION FOR 5E0 ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single 35 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None 4O (xt) SECUENCE DESCRIPTION: SEA ID N0:7:
Met Ala Lys Val Pro Asp Met Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Gtu Asp Ser Ser Ser Ile Asp His Leu Ser Leu Asn Gln 45 Lys Ser Phe Tyr His Val Ser Tyr Gly Pro Leu His Glu Gty Cys Met Asp Gln Ser Val Ser Leu Ser Ile Ser Glu Thr Ser Lys Thr Ser Lys Leu Thr Phe Lys Glu Ser Met Vat Val Val Ala Thr Asn Gly Lys Val ~J~ b5 70 75 80 Leu Lys Lys Arg Arg Leu Ser Leu Ser Gln Ser Ite Thr Asp Asp Asp Leu Glu Ata tle Ala Asn Asp Ser Glu Glu Glu Ile Ile Lys Pro Arg 55 Ser Ala Pro Phe Ser Phe Leu Ser Asn Val Lys Tyr Asn Phe Met Arg Ile Ile Lys Tyr Glu Phe Iie Leu Asn Asp Ala Leu Asn Gtn Ser Ile Ile Arg Ala Asn Asp Gln Tyr Leu Thr Ala Ala Ala Leu His Asn Leu Asp Glu Ala Val Lys Phe Asp Met Gly Ata Tyr Lys Ser Ser Lys Asp Asp Ala Lys Ile Thr Val Ile Leu Arg Ile Ser Lys Thr Gln Leu Tyr 6~J Val Thr Ata Gtn Asp Glu Asp Gln Pro Val Leu Leu Lys Glu Met Pro i95 200 205 Gtu Ite Pro Lys Thr Ile Thr Gly Ser Glu Thr Asn Leu Leu Phe Phe Trp Gtu Thr His Gly Thr Lys Asn Tyr Phe Thr Ser Val Ala His Pro Asn Leu Phe Ile Ala Thr Lys Gln Asp Tyr Trp Val Cys Leu Ala Gly Gly Pro Pro Ser Ile Thr Asp Phe Gln Ile Leu Glu Asn Gln Ala 260 2b5 270 SUBSTITUTE SHEET (RULE 26) (2) INFORMATION FOR SEC ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids ~J (B> TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ZO (~~) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEO ID N0:8:
Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser 1 rJ Glu Asn Glu Asp Tyr Ser Ser Glu Ile Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr Asp Ala Ser Tyr Glu Pro Leu Arg Glu Asp Gln Met Asn Lys Phe Met Ser Leu Asp Thr Ser Glu Thr Ser Lys Thr Ser Lys Leu Ser Phe Lys Glu Asn Val Val Met Val Ala Ala Ser Gly Lys Ile Leu Lys Lys Arg Arg Leu Ser Leu Asn Gln Phe Ile Thr Asp Asp Asp 25 Leu Glu Ala I le Ala Asn Asn Thr Glu Glu Glu I le I to Lys Pro Arg Ser Ala His Tyr Ser Phe Gln Ser Asn Val Lys Tyr Asn Phe Met Arg Val Ile His Gln Glu Cys Ile Leu Asn Asp Ala Leu Asn Gln Ser Ile a~O 130 135 140 Ile Arg Asp Met Ser Gly Pro Tyr Leu Thr Ala Thr Thr Leu Asn Asn Leu Glu Glu Ala Val Lys Phe Asp Met Val Ala Tyr Val Ser Glu Glu 3rJ Asp Ser Gln Leu Pro Val Thr Leu Arg Ile Ser Lys Thr Gln Leu Phe Val Ser Ala Gln Asn Glu Asp Gtu Pro Val Leu Leu Lys Glu Met Pro Glu Thr Pro Lys Ile Ile Lys Asp Glu Thr Asn Leu Leu Phe Phe Trp Glu Lys His Gly Ser Met Asp Tyr Phe Lys Ser Val Ala His Pro Lys Leu Phe Ile Ala Thr Lys Gln Glu Lys Leu Val His Met Ala 5er Gly 45 Pro Pro Ser I to Thr Asp Phe Gln I le Leu Glu Lys (2) INFORMATION FOR SEA ID N0:9:
rJO (i) SEflUENCE CHARACTERISTICS:
(A) LENGTH: 265 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (0) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEO ID N0:9:
sO Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Glu Tyr Ser Ser Glu lle Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr Asp Met Ser Cys Asp Pro Leu His Glu Asp Cys Net 6rJ 35 40 45 Ser Leu Ser Thr Ser Glu Ile Ser Lys Thr Ser Gln Leu Thr Phe Lys Glu Asn Val Vat Val Val Ata Ala Asn Gly Lys ile Leu Lys Lys Arg 7O Arg Leu Ser Leu Ser Gln Phe Ile Thr Asp Asp Asp Leu Glu Gly Ile Ala Asn Asp Thr Glu Gtu Vat Ile Met Lys Pro Arg Ser Val Ala Tyr Asn Phe His Asn Asn Glu Lys Tyr Asn Tyr ile Arg Ite Ile Lys Ser SUBSTITUTE SHEET (RULE 26) Gln Phe Ile Leu Asn Asp Asn Leu Asn Gln Ser Ile Val Arg Gln Thr Gly Gly Asn Tyr Leu Met Thr Ala Ala Leu Gln Asn Leu Asp Asp Ala rJ 145 150 155 160 Vat Lys Phe Asp Met Gly Ala Tyr Thr Ser Glu Asp Ser Lys Leu Pro Val Thr Leu Arg Ile Ser Lys Thr Arg Leu Phe Yal Ser Ala Gln Asn 1~ Glu Asp Glu Pro Val Leu Leu Lys Glu Met Pro Glu Thr Pro Lys Thr 1le Arg Asp Glu Thr Asn Leu Leu Phe Phe Trp Glu Arg His Gly Ser Lys His Tyr Phe Lys Ser Val Ala Gln Pro Lys Leu Phe Ile Ala Thr 1 rJ 225 230 235 240 Gtn Glu Arg Lys Leu Val His Met Ala Arg Gly Gln Pro Ser Ile Thr Asp Phe Arg Leu Leu Glu Thr Gln Pro (2) INFORMATION FOR SEA ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids 25 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 30 ( ») MOLECULE TYPE: None (xi) SEDUENCE DESCRIPTION: SEa ID N0:10:
Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser 35 Glu Asn Gtu Asp Tyr Ser Ser Glu lle Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr Asp Ala Ser Tyr Gly Pro Leu Arg Glu Asp His Met Asn Lys Phe Met Ser Leu Asp Thr Ser Glu Thr Ser Lys Thr Ser Arg 50 55 b0 Leu Ser Phe Lys Glu Asn Val Val Met Val Thr Aia Asn Gly Lys Ile Leu Lys Lys Arg Arg Leu Ser Leu Asn Gln Phe lle Thr Asp Asp Asp 4rJ Leu Gtu Ata lle Ala Asn Asp Thr Glu Glu Glu lle Ile Lys Pro Arg Ser Ala His Tyr Ser Phe Gln Ser Asn Vat Lys Tyr Asn Phe Met Arg Val Ile His Gln Glu Cys Ile Leu Asn Asp Ala Leu Asn Gln Ser Ile ~JD 130 135 140 Ite Arg Asp Met Ser Gly Pro Tyr Leu Met Ata Ala Thr Leu Asn Asn Leu Glu Glu Ala VaL Lys Phe Asp Met Val Ala Tyr Yal Ser Glu Glu 1b5 170 i75 55 Asp Ser Gln Leu Pro Val Thr Leu Arg Ile Ser Lys Thr Gln Leu Phe 180 185 i90 Val Ser Ala Gln Asn Glu Asp Glu Pro Val Leu Leu Lys Glu Met Pro Glu Thr Pro Lys Ile Ile Lys Asp Glu Thr Asn Leu Leu Phe Phe Trp Glu Lys His Gly Ser Met Asp Tyr Phe Lys Ser Val Ala His Pro Lys Leu Phe Ile Ala Thr Lys Gln Glu Lys Leu Val His Met Ala Ser Gly srJ Pro Pro Ser Ile Thr Asp Phe Gln Ile Leu Gtu Lys 2b0 265 (2) INFORMATION FOR SEC ID N0:11:
O (i) SEQUENCE CHARACTERISTICS:
<A) LENGTH: 204 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSTITUTE SHEET (RULE 2fr~

(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEO ID N0:11:
Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Glu Tyr Ala Ser Ala lle Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr Asp Thr Asn Tyr Asp Pro Leu His Glu Asn Arg Val Asp Glu Pro Val Ser Pro Asn 1~ 35 40 45 Pro Tyr Glu Asn Ser Glu Glu 5er Asn Phe Thr Leu Glu Asp Ser Ser 50 55 b0 Asp Ser Ser Ala Val Val Leu Thr Ser Ala His Gly Glu Val Leu Lys Lys Arg Arg Leu Ser Leu Asn Gln Thr Met Ser Asn Glu Asp Leu Glu Ala Ile Ala Asn Asp Ser Glu Glu Glu Ile Ile Glu Pro Trp Ser Val Pro 1yr Ser Phe Gln Ser Asn Leu Lys Phe Lys Tyr Gln Arg Ser Ile Lys Lys Gly Ala Val Ile Thr Asp Ala Met His Gln Ser Leu Ile Arg Glu Ser Asn Gly Gln His Leu Lys Ala Met His Val Val Asp Arg Lys His Glu Val Lys Phe Asp Ile Asp Gly 1yr Vat Ser Thr Ala Thr Arg Ile Arg Pro Val Thr Leu Lys Ile Ser Lys Thr Gln Leu Tyr Val Cys Ala Gln Glu Glu GLy Gln Pro Val Leu Leu Lys Glu (2) INFORMAT10N FOR SEO ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: Linear 4O (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEA 1D N0:12:
Met Ala Lys Val Pro Asp Met Phe Glu Asp Leu Lys Asn Cys Tyr Ser 1 5 10 1s Glu Asn Glu Glu Asp Ser Ser Ser lle Asp His Leu Phe Leu Asn Gln Lys Ser Phe Tyr Asp Yal Ser Tyr Gly Pro Leu His Glu Ser Cys Met 5~ Asp Gln Ser Val Ser Leu Ser Ile Ser Glu lle Ser Lys Thr Ser Lys Leu Ser Phe Lys Gln Ser Met Val Val Val Ser Thr Asn Gly Lys Val Leu Lys Lys Arg Arg Leu Ser Leu Ser Gln Ser Ite Ala Asp Asp Asn Leu Glu Ala Ile Ala Asn Asp Ser Glu Glu Glu Ile Ile Lys Pro Arg Ser Ala Pro Phe Ser Phe Leu Ser Asn Met Thr Tyr His lle Ile Arg Ile Ile Lys His Glu Ser Ile Leu Asn Asp Thr Leu Asn Gln Thr lle Ile Arg Ala Asn Asp Gln Tyr Leu Thr Ala Ala Ala Ile His Asn Leu Asp Glu Ala Val Lys Phe Asp Met Gly Ala Tyr Thr Ser Ser Lys Asp tb5 170 175 Asp Thr Lys Val Pro Val Ile Leu Arg 1le Ser Lys Thr Gln Leu Tyr Val Ser Ala Gln Asp Glu Asp Gln Pro Val Leu Leu Lys Glu Met Pro Gtu ile Pro Lys Thr Thr Thr Gly Gly Glu Thr Asn Ser Leu Ser Ser Trp Glu Thr Arg Gly Thr Lys Asn Tyr Phe Ile Ser Val Ala His Pro Asn Leu Phe Ile Ala Thr Lys His Asp Asn Trp Val Cys Leu Ala Lys SUBSTITUTE SHEET (RULE 26) Gly Leu Pro Ser Ile Thr Asp Phe Gln Ile Leu Glu Asn Gln Ala 2b0 265 270 (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None i5 (xi ) SEWUENCE DESCRIPTION: SEQ ID N0:13:
Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Asp Tyr Ser Ser Glu Ile Asp His Leu Ser Leu Thr Gln 'Z0 20 25 30 Lys Ser Phe Tyr Asp Ala Ser Tyr Asp Pro Leu Pro Glu Asp Cys Met Asp Thr Phe Met Ser Leu Ser Thr Ser Glu Thr Ser Lys Thr Ser Lys 50 55 b0 25 Leu Asn Phe Lys Glu Ser Val Val Leu Val Ala Ala Asn Gly Lys Thr b5 70 75 80 Leu Lys Lys Arg Arg Leu Ser Leu Asn Gln Phe Ile Thr Asn Asp Asp Leu Glu Ala Ile Ala Asn Asp Pro Glu Gtu Gly Ile Ile Arg Pro Arg Ser Val His Tyr Asn Phe Gln Ser Asn Thr Lys Tyr Asn Phe Met Arg Ile Val Asn His Gln Cys 7hr Leu Asn Asp Ala Leu Asn Gln Ser Val 35 Ile Arg Asp Thr Ser Gly Gln Tyr Leu Ala Thr Ala Ala Leu Asn Asn 145 150 155 1b0 Leu Asp Asp Ala Val Lys Phe Asp Met Gly Ata Tyr Thr Ser Glu Glu 1b5 170 175 Asp Ser Gln Leu Pro Val Thr Leu Arg Ile Ser Lys Thr Arg Leu Phe Val Ser Ala Gln Asn Glu Asp Glu Pro Val Leu Leu Lys Glu Met Pro Asp Thr Pro Lys Thr Ile Lys Asp Glu Thr Asn Leu Leu Phe Phe Trp 45 Glu Arg His Gly Ser Lys Asn Tyr Phe Lys Ser Val Ala His Pro Lys Leu Phe Ile Ala Thr Lys Gln Gly Lys Leu Val His Met Ala Arg Gly Gln Pro Ser Ile Thr Asp Phe Gln tle Leu Asp Asn Gln Phe ~J0 2b0 265 270 (2) INFORMATION FOR SEA ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
'rJrJ (A) LENGTH: 270 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 6O (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEA ID N0:14:
Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser 6rJ 1 S 10 15 Glu Asn Glu Glu Tyr Ser Ser Glu Ile Asp His Leu Thr Leu Asn Gln Lys Ser Phe Tyr Asp Ala Ser Tyr Asp Pro Leu His Glu Asp Cys Thr 70 Asp Lys Phe Met Ser Pro Ser Thr Ser Glu Thr Ser Lys Thr Pro Gln Leu Thr Leu Lys Lys Ser Val Val Met Val Ala Ala Asn Gly Lys 1le b5 70 75 80 Leu Lys Lys Arg Arg Leu Ser Leu Asn Gln Phe Leu Thr Ala Asp Asp SUBSTITUTE SHEET (RULE 26) Leu Glu Ala Ile Ala Asn Glu Val Glu Glu Glu Ile Met Lys Pro Arg Ser Vat Ala Pro Asn Phe Tyr Ser Ser Glu Lys Tyr Asn Tyr Gln Lys lle Ile Lys Ser Gln Phe Ile Leu Asn Asp Asn Leu Ser Gln Ser Val Ile Arg Lys Ala Gly Gly Lys Tyr Leu Ala Ala Ala Ala Leu Gln Asn 145 150 155 1b0 1~ Leu Asp Asp Ala Val Lys Phe Asp Met Gly Ala Tyr Thr Ser Lys Glu Asp Ser Lys Leu Pro Val Thr Leu Arg Ile Ser Lys Thr Arg Leu Phe Val Ser Ala Gln Asn Glu Asp Glu Pro Val Leu Leu Lys Glu Met Pro 19s zoo zos Glu Thr Pro Lys Thr Ile Arg Asp Glu Thr Asn Leu Leu Phe Phe Trp Glu Arg His Gly Ser Lys Asn Tyr Phe Lys Ser Val Ala His Pro lys Leu Phe Ile Ala Thr Gln Glu Glu Gln Leu Val His Met Ala Arg Gty Leu Pro Ser Val Thr Asp Phe Gln Ile Leu Glu 1hr Gln Ser (2) INFORMATION FOR SEA ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 amino acids (8) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi ) SECUENCE DESCRIPTION: SEA ID N0:15:
Met Ala Lys Val Pro Asp Met Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Glu Val Ser Ser Ser Ile Asp Nis Leu Ser Leu Asn Gln Lys Ser Phe Tyr Asp Val Ser Tyr Gly Pro Leu His Glu Gly Cys Met Asp Gln Ser Val Ser Leu Ser Ile Ser Glu Ile Ser Lys Thr Ser Lys Leu Thr Phe Lys Gln Ser Met Val Val Val Ser Thr Asn Gly Lys Val Leu Lys Lys Arg Arg Leu Ser Leu Ser Gln Ser Ile Thr Asp Asn Asn Leu Glu Ala Ile Ala Asn Asp Ser Glu Glu Glu Ile Ile Lys Pro Arg 5~ 1 OD 105 110 Ser Ala Pro Phe Ser Phe Leu Ser Asn Met Thr Tyr His Phe Ile Arg lle Ile Lys Nis Glu Phe Ile Leu Asn Asp Thr Leu Asn Gln Thr lle Ile Arg Ala Asn Asp Gln Tyr Leu Thr Ala Ala Ala ILe His Asn Leu Asp Glu Ala Vel Lys Phe Asp Met Gly Ala Tyr Thr Ser Ser Lys Asp Asp Thr Lys Val Pro Val Ile Leu Arg Ile Ser Lys Thr Gln Leu Tyr Val Ser Ala Gln Asp Glu Asp Gln Pro Val Leu Leu Lys Glu Met Pro Glu Ile Pro Lys Thr Ile Thr Gly Ser Glu Thr Asn Phe Leu Phe Phe s5 Trp Glu Thr His Gly Thr Lys Asn Tyr Phe Ile Ser Val Ata His Pro Asn Leu Phe Ile Ala Thr Lys His Asp Asn Trp Val Cys Leu Ala Lys Gly Leu Pro Ser lle Thr Asp Phe Gln Ile Leu Glu Asn Gln Ala (2) INFORMATION FOR SEG 1D N0:16:
(i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26) (A) LENGTH: 270 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEO ID N0:16:
1~ Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Asp Tyr Ser Ser Ala Ile Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr Asp Ala Ser Tyr Gly Ser Leu His Glu Thr Cys Thr Z~J 35 40 45 Asp Gln Phe Val Ser Leu Arg Thr Ser Glu Thr Ser Lys Met Ser Asn Phe Thr Phe Lys Glu Ser Arg Val 7hr Val Ser Ala Thr Ser Ser Asn b5 70 75 80 Gly Lys Ile Leu Lys Lys Arg Arg Leu Ser Phe Ser Glu Thr Phe Thr Glu Asp Asp Leu Gln Ser Ile Thr His Asp Leu Glu Glu Thr Ile Gln Pro Arg Ser Ata Pro Tyr Thr Tyr Gln Ser Asp Leu Arg Tyr Lys Leu 2rJ 11s 120 125 Met Lys Leu Val Arg Gtn Lys Phe Val Met Asn Asp Ser Leu Asn Gln Thr ile Tyr Gln Asp Val Asp Lys His Tyr Leu Ser Thr Thr Trp Leu Asn Asp Leu Gln Gln Glu Val Lys Phe Asp Met Tyr Ala Tyr Ser Ser 1b5 170 175 Gty Gly Asp Asp 5er Lys Tyr Pro Val Thr Leu Lys Ile Ser Asp Ser Gln Leu Phe Val Ser Ala Gln Gly Glu Asp Gln Pro Vat Leu Leu Lys Glu Leu Pro Glu Thr Pro Lys Leu Ile Thr Gly Ser Glu Thr Asp Leu Ite Phe Phe Trp Lys Ser Ile Asn Ser Lys Asn Tyr Phe Thr Ser Ala Ala Tyr Pro Glu Leu Phe Ile Ala Thr Lys Glu Gln Ser Arg Val His Leu Ala Arg Gly Leu Pro Ser Met Thr Asp Phe Gtn Ile Ser (2) INFORMATION FOR SEA ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids (B) TYPE: amino acid ~J~ (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None 55 (xi ) SECUENCE DESCRIPTION: SEQ ID N0:17:
Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Gtu Asp Tyr Ser Ser Glu Ile Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr Asp Ala Ser Tyr Glu Pro Leu Arg Glu Asp His Met Asn Lys Phe Met Ser Leu Asp Thr Ser Glu Thr Ser Lys Thr Ser Arg 6~J Leu Ser Phe Lys Glu Asn Val Val Met Val Thr Ala Asn Gly Lys 1le Leu Lys Lys Arg Arg Leu Ser Leu Asn Gln Phe ile Thr Asp Asp Asp Leu Glu Ata Ile Ala Asn Asp Thr Glu Glu Glu lle Ile Lys Pro Arg Ser Ala His Tyr Ser Phe Gln Ser Asn Val Lys Tyr Asn Phe Met Arg Val Ile His Gln Glu Cys Ile Leu Asn Asp Ala Leu Asn Gln Ser Ile t30 135 140 SUBSTITUTE SHEET (RULE 26) ?1 Ile Arg Asp Met Ser Gly Pro Tyr Met Thr Ala Ala Thr Leu Asn Asn Leu Glu Glu Ala Val Lys Phe Asp Met Val Ala Tyr Vat Ser Glu Glu 1b5 170 175 ~J Asp Ser Gln Leu Pro Val Thr Leu Arg Ile Ser Lys Thr Gln Leu Phe Val Ser Ala Gln Asn Glu Asp Glu Pro Val Leu His Lys Glu Met Pro 195 zoo zo5 Glu Thr Pro Lys Ile Ile Lys Asp Glu Thr Asn Leu Leu Phe Phe Trp 1~ 210 215 220 Glu Lys His Gly Ser Met Asp Tyr Phe Lys Ser Val Ala His Pro Lys Leu Phe Ile Ala Thr Lys Gln Glu Lys Leu Val His Met Ala Ser Gly IJ~ Pro Pro Ser Ile Thr Asp Phe Leu Ile Leu Glu Lys 2b0 2b5 (2) INFORMATION FOR SEA ID N0:18:
2O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single 25 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEO ID N0:18:
Met Ata Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Phe Ser Glu Asn Glu Glu Tyr Ser Ser Ala Ile Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr Asp Ala Ser Tyr Glu Pro Leu His Glu Asp Cys Met Asn Lys Yal Val Ser Leu Ser Thr Ser Glu Thr Ser Val Ser Pro Asn 50 55 b0 Leu Thr Phe Gln Glu Asn Val Val Ala Val Thr Ala Ser Gly Lys Ile b5 70 75 80 Leu Lys Lys Arg Arg Leu Ser Leu Asn Gln Pro Ile Thr Asp Yal Asp Leu Glu Thr Asn Val Ser Asp Pro Giu Glu Gly Ile Ile Lys Pro Arg Ser Val Pro Tyr Thr Phe Gln Arg Asn Met Arg Tyr Lys Tyr Leu Arg 4rJ 115 120 125 Ile Ile Lys Gln Glu Phe Thr Leu Asn Asp Ala Leu Asn Gln Ser Leu Val Arg Asp Thr Ser Asp Gln Tyr Leu Arg Ala Ala Pro Leu Gln Asn 145 150 155 1b0 ~J~ Leu Gly Asp Ala Val Lys Phe Asp Met Gly Val Tyr Met Thr Ser Lys Glu Asp Ser lle Leu Pro Val Thr Leu Arg Ite Ser Gln Thr Pro Leu Phe Vat Ser Ala Gln Asn Glu Asp Glu Pro Val Leu Leu Lys Glu Met rJ~J 195 200 205 Pro Glu Thr Pro Arg Ile Ile Thr Asp Ser Glu Ser Asp Ile Leu Phe Phe Trp Glu Thr Gln Gty Asn Lys Asn Tyr Phe Lys Ser Ala Ala Asn Pro Gtn Leu Phe Ile Ala Thr Lys Pro Glu His Leu Val His Met Ala Arg Gly Leu Pro Ser Met Thr Asp Phe Gln Ile Ser (2) INFORMATION FOR S~~ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 amino acids (B) TYPE: amino acid ?~ (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None SUBSTITUTE SHEET (RULE 26) (xi) SEQUENCE DESCRIPTION: SEO ID N0:19:
Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser rJ Glu Asn Glu Glu Tyr Ser Ser Ala Ile Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr Asp Ala Ser Tyr Gly Ser Leu His Glu Asn Cys Thr Asp Lys Phe Val Ser Leu Arg Thr Ser Glu Thr Sec Lys Met Ser Thr 1~ 50 55 60 Phe Thr Phe Lys Glu 5er Arg Val Val Val Ser Ala Thr Ser Asn Lys b5 70 75 80 Gly Lys Ile Leu Lys Lys Arg Arg Leu Ser Phe Asn Gtn Pro Phe Thr 15 Glu Asp Asp Leu Glu Ala Ile Ala His Asp Leu Glu Glu Thr Ile Gln Pro Arg Ser Ata Pro His Ser Phe Gln Asn Asn Leu Arg Tyr Lys Leu Ite Arg lle Val Lys Gln Glu Phe Ile Met Asn Asp Ser Leu Asn Gln 2~ 130 135 140 Asn Ile Tyr Val Asp Met Asp Arg Ile His Leu Lys Ata Ala Ser Leu Asn Asp Leu Gtn Leu Glu Val Lys Phe Asp Met Tyr Ala Tyr Ser Ser 25 Gty GEy Asp Asp Ser Lys Tyr Pro Val Thr Leu Lys Val Ser Asn Thr GLn Leu Phe Val Ser Ala Gln Gly Glu Asp Lys Pro Val Leu Leu Lys Glu Ile Pro Glu Thr Pro Lys Leu Ile Thr Gly Ser Glu Thr Asp Leu Ile Phe Phe Trp Glu Lys Ile Asn Ser Lys Asn Tyr Phe Thr Ser Ala Ala Phe Pro Glu Leu Leu lle Ala Thr Lys Glu Gln Ser Gln Val His 35 Leu Ala Arg Gly Leu Pro Ser Met Ile Asp Phe Gln Ite Ser (2) INFORMATION FOR SEG ID N0:20:
(i) SE~UEHCE CHARACTERISTICS:
(A) LENGTH: 270 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single 45 (D) TOPOLOGY: linear (xi) SEDUENCE DESCRIPTION: SEO ID N0:20:
Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser rJ~ 1 5 10 15 Glu Asn Glu Gtu Tyr Ser Ser Asp Ile Asp His Leu Ser leu Asn Gln Lys Ser Phe Tyr Asp Ala Ser Tyr Glu Pro Leu Pro Gty Asp Gly Met 55 Asp Lys Phe Met Pro Leu Ser Thr Ser Lys Thr Ser Lys Thr Ser Arg Leu Asn Phe Lys Asp Ser Vat Vat Met Ala Ala Ala Asn Gly Lys Ile Leu Lys Lys Arg Arg Leu Ser Leu Ile Gln Phe Ile Thr Asp Asp Asp Leu Glu Ala Ile Ala Asn Asp Thr Gtu Glu Glu Ile Ile Lys Pro Arg Ser Ala Thr Tyr Ser Phe Gln Ser Asn Met Lys Tyr Asn Phe Met Arg 6~J Val Ile Asn His Gln Cys Ile Leu Asn Asp Ala Arg Asn Gln Ser Ile Ile Arg Asp Pro Ser Gly Gln Tyr Leu Met Ala Ala Val Leu Asn Asn Leu Asp Glu Ala Val Lys Phe Asp Met Ala Ala Tyr Thr Ser Asn Asp 7~ 1b5 170 175 Asp Ser Gln Leu Pro Vat Thr Leu Arg ile Ser Glu Thr Arg Leu Phe Val Ser Ala Gln Asn Glu Asp Glu Pro Vat Leu Leu Lys Gtu Leu Pro SUBSTITUTE SHEET (RULE 2~) Glu ThrProLys Thr Ile ThrSerLeuLeuPhePhe Lys Asp Glu Trp Glu LysHisGly Asn Met LysSerAlaAlaHisPro Asp Tyr Phe Lys rJ Leu LeuIleAla Thr Arg LeuValHisMetAlaArg Gln Glu Lys Gly Leu ProSerVat Thr Asp LeuGluAsnGinSer Phe Gln lle 260 2b5 270 ZO (2)INFORMATION N0:21:
FOR SEQ IO

(i) SEQUENCE
CHARACTERISTICS:

(A)LENGTH: 59 amino acids (B)TYPE: amino acid 1 (C)STRANDEDNESS:
rJ single (D)TOPOLOGY: linear (xt) N0:21:
SEQUENCE
DESCRIPTION:
SEQ
ID

Met SerSerVal Vat Gly LysLysArgLysVatAsp Ala Pro Lys Ala Thr AsnGlyLys Val Leu ArgLeuSerLeuSerGln Lys Lys Arg Ser 25 Ile ThrAspAsp Asp Leu AlaAsnAspSerGluGlu Glu Ala Ile Glu Ile IleLysPro Arg Ser SerPhe Ala Pro Phe a~O (2) N0:22:
INFORMATION
FOR
SEQ
ID

( i) SEQUENCE
CHARACTERISTICS:

(A)LENGTH: 49 amino acids (8)TYPE: amino acid 35 (C)STRANDEDNESS:
single (D)TOPOLOGY: linear (xt) N0:22:
SEQUENCE
DESCRIPTION:
SEQ
ID

Met SerSerVal Val Gly LysLysArgLysValAsp Ala Pro Lys Ala Thr AsnGlyLys Val Leu ArgLeuSerLeuSerGln Lys Lys Arg Ser 45 Ile ThrAspAsp Asp Leu AlaAsnAspSerGluGlu Glu Ala lle Glu Ile rJO (2) INFORMATION FOR SEO 1D N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 amino acids (B) TYPE: amino acid 55 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xt) DESCRIPTION: SEQIDN0:23:
SEQUENCE

sO

Met Ser ValGlyAlaProLysLysLysArgLysVatAspAla Ser Val Thr Asn ValLeuLysLysArgArgLeuSerLeuSerGlnSer Gly Lys srJIle Thr AspLeuGluAlalleAlaAsnAspSerGluGluGlu Asp Asp Ile Leu LysAlaLeuLysGluLysLeuLysAlaLeuGluGlu Asp Leu Lys Leu LeuGluGluLysLeuLysAlaLeuValGlyGluArg Lys Ala 7O b5 70 75 80 (2) N0:24:
INFORMATION
FOR
SEO
IO

(i) CHARACTERISTICS:
SEQUENCE

SUBSTITUTE SHEET (RULE 26) (A) LENGTH: 90 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Met Ser Ser Val Val Gly Ala Pro Lys Lys Lys Arg Lys Val Asp Ala Thr Asn Gly Lys Val Leu Lys Lys Arg Arg Leu Ser Leu Ser Gln Ser Ile Thr Asp Asp Asp Leu Glu Ata lle Ala Asn Asp Ser Glu Glu Glu 1 rJ lle Ite Lys Pro Arg Ser Ata Pro Phe Ser Phe Leu Asp Leu Lys Ala Leu Lys Glu Lys Leu Lys Ala Leu Glu Glu Lys Leu lys Ala Leu Glu Gtu Lys Leu Lys Ata Leu Val Gly Glu Arg (2) INFORMATION FOR SEA ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 149 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEA ID N0:25:
Met Ala Lys Val Pro Asp Met Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Glu Asp Ser Ser Ser Ile Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr His Val Ser Tyr Gly Pro Leu His Glu Gly Cys Met Asp Gln Ser Vat Ser Leu Ser Ile Ser Gtu Thr Ser Lys Thr Ser Lys Leu Thr Phe Lys Glu Ser Met Val Val Val Ala Thr Asn Gly Lys Val Leu Lys Lys Arg Arg Leu Ser Leu Ser Gln Ser Ile Thr Asp Asp Asp Leu Gtu Ala Ile Ala Asn Asp Ser Glu Glu Glu Ile lle Lys Pro Arg Ser Ala Pro Phe Ser Phe Leu Asp Leu Lys Ala Leu Lys Glu Lys Leu 115 120 t25 Lys Ala Leu Glu Glu Lys Leu Lys Ala Leu Glu Glu Lys Leu Lys Ala rJ0 130 135 140 Leu Vat Gly Glu Arg (2) INFORMATION FOR SEO ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 139 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single 60 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEG ID N0:26:
srJ Met Ala Lys Val Pro Asp Met Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Glu Asp Ser Ser Ser Ile Asp His Leu Ser Leu Asn Gln Lys Ser Phe Tyr His Val Ser Tyr Gly Pro Leu His Glu Gly Cys Met Asp Gln Ser Val Ser Leu Ser Ile Ser Glu Thr Ser Lys Thr Ser Lys Leu Thr Phe Lys Gtu Ser Met Vat Val Val Ala Thr Asn Gly Lys Val SUBSTITUTE SHEET (RULE 26) LeuLys Arg Arg Leu GlnSerIle Thr Asp Lys Ser Leu Ser Asp Asp LeuGlu lle Ala Asn GluGlu1le Leu Asp Ala Asp Ser Glu Leu Lys 5 AlaLeu Glu Lys Leu GluGluLys Leu Lys Lys Lys Ala Leu Ala Leu GluGlu Leu Lys Ala GluArg Lys Leu Val Gly ZO (2) INFORMATION N0:27:
FOR SEG ID

(i) QUENCE CHARACTERISTICS:
SE

(A) LENGTH: 86 amino acids (B) TYPE: amino acid Z (C) STRANDEDNESS:
5 single (D) TOPOLOGY: linear (x') N0:27:
SEQUENCE
DESCRIPTION:
SEA

O

MetSer Ser Glu Thr SerLysLeu Thr Phe Ile Ser Lys Thr Lys Glu SerMet Val Val Ala LysValLeu Lys Lys Val Thr Asn Gly Arg Arg 25 LeuSer Ser Gln Ser AspAspLeu Glu Ala Leu lle Thr Asp Ile Ala AsnAsp Glu Glu Glu LeuLysAla Leu Lys Ser Ile Leu Asp Glu Lys LeuLys Leu Glu Glu AlaLeuGlu Glu Lys Ala Lys Leu Lys Leu Lys AlaLeu Gly Glu Arg Val (2) N0:28:
INFORMATION
FOR
SEO

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
single 4O (D) TOPOLOGY: linear (xi) N0:28:
SEQUENCE
DESCRIPTION:
SEO
ID

AsnGly Val Leu Lys Leu Lys Lys Arg Arg (2) N0:29:
INFORMATION
FOR
SEU
ID

(1) SEQUENCE
CHARACTERISTICS:

5O (A) LENGTH: 4 amino acids (8) TYPE: amino acid (C) STRANDEDNESS:
single (D) 10POLOGY: linear (xi) SECUENCE DESCRIPTION: SEC ID N0:29:
Lys Lys Arg Arg (2) INFORMATION FOR SEA ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear rIO (xi) SEQUENCE DESCRIPTION: SEO ID N0:30:
Pro Lys Lys Lys Arg Lys Val SUBSTITUTE SHEET (RULE 26)

Claims (12)

76

1. An isolated apoptosis-inducing polypeptide characterized by:
a) an apoptosis-inducing domain consisting essentially of amino acid residues 75-108 of SEQ ID NO:2 and equivalents thereof retaining selective apoptosis-inducing activity; and b) a nuclear localization sequence, which sequence is heterologous to the apoptosis-inducing domain, wherein the heterologous nuclear localization sequence and apoptosis-inducing domain are operably joined and the apoptosis-inducing polypeptide is characterized by activity in selective induction of apoptosis in a cancer cell.

2. An isolated apoptosis-inducing polypeptide characterized by:
a) a nuclear localization sequence, and b) an apoptosis-inducing domain consisting essentially of amino acid residues 75-108 of SEQ ID NO:2 and equivalents thereof retaining selective apoptosis-inducing activity; and c) an affinity ligand;
wherein the nuclear localization sequence, apoptosis-inducing domain, and affinity ligand are operably joined and the apoptosis-inducing polypeptide is characterized by activity in selective induction of apoptosis in a cancer cell.

3. The isolated polypeptide of claim 2, wherein the nuclear localization sequence and the apoptosis-inducing domain are provided by amino acid residues 55-108 of an N-terminal IL-1.alpha. propiece polypeptide.

4. The isolated polypeptide of claim 2, wherein the nuclear localization sequence and the apoptosis-inducing domain are provided by amino acid residues 1-118 of SEQ ID NO:2.

5. An isolated polypeptide characterized by activity in selective induction of apoptosis in a cancer cell, the polypeptide consisting essentially of an amino acid sequence selected from the group consisting of:
a) amino acid residues 1-108 of SEQ ID NO:2;

b) amino acid residues 11-118 of SEQ ID NO:2;
c) amino acid residues 34-118 of SEQ ID NO:2;
d) amino acid residues 55-118 of SEQ ID NO:2;
e) amino acid residues 55-108 of SEQ ID NO:2;
f) amino acid residues 75-108 of SEQ ID NO:2 coupled to a heterologous nuclear localization sequence;
g) amino acid residues 75-118 of SEQ ID NO:2 coupled to a heterologous nuclear localization sequence;
h) amino acid residues 1-118 of SEQ ID NO:2 coupled to a leucine zipper domain;
i) amino acid residues 1-108 of SEQ ID NO:2 coupled to a leucine zipper domain;
j) amino acid residues 55-108 of SEQ ID NO:2 coupled to a leucine zipper domain; and k) equivalents thereof retaining selective apoptosis-inducing activity.

6. An isolated polypeptide characterized by activity in selective induction of apoptosis in a cancer cell, the polypeptide consisting essentially of an amino acid sequence selected from the group consisting of:
a) amino acid residues 1-108 of SEQ ID NO:2;
b) amino acid residues 11-118 of SEQ ID NO:2;
c) amino acid residues 34-118 of SEQ ID NO:2;
d) amino acid residues 55-118 of SEQ ID NO:2;
e) amino acid residues 55-108 of SEQ ID NO:2;
f) amino acid residues 75-108 of SEQ ID NO:2 coupled to a nuclear localization sequence;
g) amino acid residues 75-118 of SEQ ID NO:2 coupled to a nuclear localization sequence;
h) amino acid residues 1-118 of SEQ ID NO:2 coupled to a leucine zipper domain;
i) amino acid residues 1-108 of SEQ ID NO:2 coupled to a leucine zipper domain;
j) amino acid residues 55-108 of SEQ ID NO:2 coupled to a leucine zipper domain; and k) equivalents thereof retaining selective apoptosis-inducing activity:
wherein serine at position 87 of SEQ ID NO:2 is substituted with an amino acid other than serine.

7. An isolated nucleic acid molecule encoding the polypeptide of claims 1, 2, 5 , or 6.

8. A pharmaceutical composition comprising the polypeptide of claims 1, 2, 5, or 6 in an amount effective to selectively induce apoptosis and a biocompatible carrier for use in the induction of apoptosis in a cancer cell.

9. The pharmaceutical composition of claim 8, wherein the composition is suitable for extracellular delivery of the polypeptide to the cancer cell.

10. A pharmaceutical composition comprising the polynucleotide of claim 7 and a biocompatible carrier for use in the induction of apoptosis in a cancer cell.

11. The pharmaceutical composition of claims 8 or 10, wherein the cancer cell is selected from the group consisting of: a colonic tumor cell, a central nervous system tumor cell, a leukemia cell, a lung tumor cell, a mammary tumor cell, a melanoma cell, an ovarian tumor cell, a prostate tumor cell, and a renal tumor cell.

12. The pharmaceutical composition of claim 11, wherein the cancer cell is a malignant cancer cell.