Human ICE LAP-6 polypeptides and DNA (RNA) encoding such ICE LAP-6 and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such ICE LAP-6 for the treatment of a susceptibility to viral infection, tumorogenesis and to diseases and defects in the control embryogenesis and tissue homeostasis, and the nucleic acid sequences described above may be employed in an assay for ascertaining such susceptibility. Antagonists against such ICE LAP-6 and their use as a therapeutic to treat Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, septic shock, sepsis, stroke, chronic inflammation, acute inflammation, CNS inflammation, osteoporosis, ischemia reperfusion injury, cell death associated with cardiovascular disease, polycystic kidney disease, apoptosis of endothelial cells in cardiovascular disease, degenerative liver disease, MS, ALS, cererbellar degeneration, ischemic injury, myocardial infarction, AIDS, myelodysplastic syndromes, aplastic anemia, male pattern baldness, and head injury damage are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Also disclosed are diagnostic assays for detecting mutations in the polynucleotides encoding the interleukin-1 beta converting enzyme apoptosis proteases and for detecting altered levels of the polypeptide in a host.

FIELD OF INVENTION 
This invention relates, in part, to newly identified polynucleotides and 
polypeptides; variants and derivatives of the polynucleotides and 
polypeptides; processes for making the polynucleotides and the 
polypeptides, and their variants and derivatives; agonists and antagonists 
of the polypeptides; and uses of the polynucleotides, polypeptides, 
variants, derivatives, agonists and antagonists. In particular, in these 
and in other regards, the invention relates to polynucleotides and 
polypeptides of human interleukin-1 beta converting enzyme apoptosis 
protease-6, hereinafter referred to as "ICE LAP-6". 
BACKGROUND OF THE INVENTION 
It has recently been discovered that an interleukin-1.beta. converting 
enzyme (ICE) is responsible for cleaving pro-IL-1.beta. into mature and 
active IL-1.beta. and is also responsible for programmed cell death (or 
apoptosis), which is a process through which organisms get rid of unwanted 
cells. 
Apoptosis, or programmed cell death, is a physiologic process important in 
the normal development and homeostasis of metazoans 
In the nematode Caenorhabditis elegans, a genetic pathway of programmed 
cell death has been identified (Ellis, R. E., et al. Annu. Rev. Cell 
Biol., 7:663-698 (1991)). Two genes, ced-3 and ced-4, are essential for 
cells to undergo programmed cell death in C. elegans (Ellis, H. M., and 
Horvitz, H. R., Cell, 44:817-829 (1986)). It is becoming apparent that a 
class of cysteine proteases homologous to Caenorhabditis elegans Ced-3 
play the role of "executioner" in the apoptotic mechanism (Martin, S. J., 
and Green, D. R. (1995) Cell 82, 349-352; Chinnaiyan, A. a. D., VM. (1996) 
Current Biology 6; Henkart, P. (1996) Immunity 4, 195-201). Recessive 
mutations that eliminate the function of these two genes prevent normal 
programmed cell death during the development of C. elegans. The known 
vertebrate counterpart to ced-3 protein is ICE. The overall amino acid 
identity between ced-3 and ICE is 28%, with a region of 115 amino acids 
(residues 246-360 of ced-3 and 164-278 of ICE) that shows the highest 
identity (43%). This region contains a conserved pentapeptide, QACRG (SEQ 
ID NO:10) (residues 356-360 of ced-3), which contains a cysteine known to 
be essential for ICE function. 
The similarity between ced-3 and ICE suggests not only that ced-3 might 
function as a cysteine protease but also that ICE might act as a 
vertebrate programmed cell death gene. ced-3 and the vertebrate 
counterpart, ICE, control programmed cell death during embryonic 
development, (Gagliamini, V. et al., Science, 263:826:828 (1994). 
Mutations of ced-3 and ced-4 abolish the apoptotic capability of cells that 
normally die during C. elegans embryogenesis (Yuan, J. Y., and Horvitz, H. 
R. (1990) Dev Biol 138, 33-41). While no mammalian homologs of ced-4 have 
been identified, ced-3 shares sequence similarity with interleukin-1b 
converting enzyme (ICE) (Yuan, J. et al(1993) Cell 75, 641-652), a 
cysteine protease involved in the processing and activation of pro-IL-1b 
to an active cytokine (Cerretti, D. P., et al (1992) Science 256, 97-100, 
Thornberry, N. A.,et al (1992) Nature 356, 768-774). Recently, numerous 
homologs of ICE/Ced-3 have been characterized, comprising a new gene 
family of cysteine proteases. To date, seven members of the ICE/Ced-3 
family have been identified and include ICE (Cerretti, D. P., et al (1992) 
Science 256, 97-100), TX/ICH2/ICE rel-II (Munday, N. A., et al (1995) J 
Biol Chem 270, 15870-15876; Faucheu, C. et al. (1995) Embo J 14, 
1914-1922; Kamens, J et al.(1995) J Biol Chem 270, 15250-1525612), ICE 
rel-III (Munday, N. A., et al (1995) J Biol Chem 270, 15870-15876), 
ICH1/Nedd-2 (Kumar, S., et al. (1994) Genes and Development 8, 1613-1626; 
Wang, L., et al. (1994) Cell 78, 739-750), Yama/CPP32/Apopain (Tewari, M., 
et al. (1995) Cell 81, 801-809; Fernandes-Alnemri, T., et al. (1994) J. 
Biol. Chem. 269, 30761-30764; Nicholson, D. Wet al. (1995) Nature 376, 
37-43), Mch2 Fernandes-Alnemri, T., et al. (1994) J. Biol. Chem. 269, 
30761-30764) and ICE-LAP3/Mch3/CMH-1 (Duan, H., et al. (1996) J. Biol. 
Chem. 271, 35013-35035; Fernandes-Alnemri, T., et al. (1995) Cancer 
Research 55, 6045-6052; Lippke, J. A., et al. (1996) The Journal of 
Biological Chemistry 271, 1825-1828). All family members share sequence 
homology with ICE/Ced-3 and contain an active site QACRG (SEQ ID NO:10) 
pentapeptide in which the cysteine residue is catalytic. Ectopic 
expression of these proteases in a variety of cells causes apoptosis. 
Phylogenetic analysis of the ICE/ced-3 gene family revealed three 
subfamilies (Chinnaiyan, A. a. D., VM. (1996) Current Biology 6; uan, H., 
et al. (1996) J. Biol. Chem. 271, 35013-35035). Yama, ICE-LAP3, and Mch2 
are closely related to C.elegans Ced-3 and comprise the Ced-3 subfamily. 
ICE and the ICE-related genes, ICE rel II, and ICE rel III form the ICE 
subfamily, while ICH1 and its mouse homologue, NEDD-2 form the NEDD-2 
subfamily. Based on similarities with the structural prototype 
interleukin-1b converting enzyme, ICE/Ced-3 family members are synthesized 
as zymogens that are capable of being processed to form active 
heterodimeric enzymes (Thomberry, N. A.,et al (1992) Nature 356, 768-774). 
It will be important to determine which family members are in fact 
activated in response to apoptotic stimuli. Previous studies have 
demonstrated that pro-Yama and pro-ICE-LAP3 are processed into active 
subunits in response to various death stimuli including engagement of 
Fas/APO-1 or treatment with staurosporine (Duan, H., et al. (1996) J. 
Biol. Chem. 271, 35013-35035; Chinnaiyan, A. M., et al. 1996) Journal of 
Biological Chemistry 271, 4573-4576). Further, the serine protease 
granzyme B, one of the major effectors of cytotoxic T cell-mediated 
apoptosis, was shown to directly activate Yama (but not ICE), in vitro 
(Quan, L. T., et al. (1996) PNAS 93, In Press; Darmon, A. J., et al. 
(1995) Nature 377, 446-448). 
ICE mRNA has been detected in a variety of tissues, including peripheral 
blood monocytes, peripheral blood lymphocytes, peripheral blood 
neutrophils, resting and activated peripheral blood T lymphocytes, 
placenta, the B lymphoblastoid line CB23, and monocytic leukemia cell line 
THP-1 cells (Cerretti, D. P., et al., Science, 256:97-100 (1992)), 
indicating that ICE may have an additional substrate in addition to 
pro-IL-1. The substrate that ICE acts upon to cause cell death is 
presently unknown. One possibility is that it may be a vertebrate homolog 
of the C. elegans cell death gene ced-4. Alternatively, ICE might directly 
cause cell death by proteolytically cleaving proteins that are essential 
for cell viability. 
The mammalian gene bcl-2, has been found to protect immune cells called 
lymphocytes from cell suicide. Also, crmA, a cow pox virus gene protein 
product inhibits ICE's protein splitting activity. 
Clearly, there is a need for factors that are useful for inducing apoptosis 
for therapeutic purposes, for example, as an antiviral agent, an 
anti-tumor agent and to control embryonic development and tissue 
homeostasis, and the roles of such factors in dysfunction and disease. 
Further, there is clear a need for factors that are useful for reducing or 
halting apoptosis for therapeutic purposes, for example, to treat diseases 
caused or associated with apoptosis, such as, particularly Alzheimer's 
disease, Parkinson's disease, rheumatoid arthritis, septic shock, sepsis, 
stroke, chronic inflammation, acute inflammation, CNS inflammation, 
osteoporosis, ischemia reperfusion injury, cell death associated with 
cardiovascular disease, polycystic kidney disease, apoptosis of 
endothelial cells in cardiovascular disease, degenerative liver disease, 
MS, ALS, cererbellar degeneration, ischemic injury, myocardial infarction, 
AIDS, myelodysplastic syndromes, brain damage, aplastic anemia, male 
pattern baldness, and head injury damage. There is a need, therefore, for 
identification and characterization of such factors that are interleukin-1 
beta converting enzyme apoptosis proteases, and which can play a role in 
preventing, ameliorating or correcting dysfunctions or diseases. 
SUMMARY OF THE INVENTION 
Toward these ends, and others, it is an object of the present invention to 
provide polypeptides, inter alia, that have been identified as novel ICE 
LAP-6 by homology between the amino acid sequence set out in FIG. 1 or the 
polypeptide encoded by the deposited clone and known amino acid sequences 
of other proteins of the mammalian ICE/ced-3 family and the C. elegans 
Ced-3 protein. 
It is a further object of the invention, moreover, to provide 
polynucleotides that encode ICE LAP-6, particularly polynucleotides that 
encode the polypeptide herein designated ICE LAP-6 and the polynucleotide 
of the deposited clone. 
In a particularly preferred embodiment of this aspect of the invention the 
polynucleotide comprises the region encoding human ICE LAP-6 set forth in 
FIGS. 2A, 2B, and 2C. 
In accordance with this aspect of the present invention there is provided 
an isolated nucleic acid molecule encoding a mature polypeptide expressed 
by the human cDNA in FIGS.2A, 2B, and 2C or derived using the primers set 
forth in Example 1, or a polynucleotide encoding the polypeptide in FIG. 1 
or derived from the polypeptide encoded by the deposited clone. 
In accordance with this aspect of the invention there are provided isolated 
nucleic acid molecules encoding human ICE LAP-6, including mRNAs, cDNAs, 
genomic DNAs and, in further embodiments of this aspect of the invention, 
biologically, diagnostically, clinically or therapeutically useful 
variants, analogs or derivatives thereof, or fragments thereof, including 
fragments of the variants, analogs and derivatives. 
Among the particularly preferred embodiments of this aspect of the 
invention are naturally occurring allelic variants of human ICE LAP-6. 
It also is an object of the invention to provide ICE LAP-6 polypeptides, 
particularly human ICE LAP-6 polypeptides, that may be employed for 
therapeutic purposes, for example, to treat viral infection, as an 
anti-tumor agent and to control embryonic development and tissue 
homeostasis. 
In accordance with this aspect of the invention there are provided novel 
polypeptides of human origin referred to herein as ICE LAP-6 as well as 
biologically, diagnostically or therapeutically useful fragments, variants 
and derivatives thereof, variants and derivatives of the fragments, and 
analogs of the foregoing. 
Among the particularly preferred embodiments of this aspect of the 
invention are variants of human ICE LAP-6 encoded by naturally occurring 
alleles of the human ICE LAP-6 gene. 
It is another object of the invention to provide a process for producing 
the aforementioned polypeptides, polypeptide fragments, variants and 
derivatives, fragments of the variants and derivatives, and analogs of the 
foregoing. 
In a preferred embodiment of this aspect of the invention there are 
provided methods for producing the aforementioned ICE LAP-6 polypeptides 
comprising culturing host cells having expressibly incorporated therein an 
exogenously-derived human ICE LAP-6-encoding polynucleotide under 
conditions for expression of human ICE LAP-6 in the host and then 
recovering the expressed polypeptide. ICE LAP-6 may also be purified from 
natural sources using any of many well known techniques. 
In accordance with another object the invention there are provided 
products, compositions, processes and methods that utilize the 
aforementioned polypeptides and polynucleotides for research, biological, 
clinical, diagnostic and therapeutic purposes, inter alia. 
In accordance with certain preferred embodiments of this aspect of the 
invention, there are provided products, compositions and methods, inter 
alia, for, among other things: assessing ICE LAP-6 expression in cells by 
determining ICE LAP-6 polypeptides or ICE LAP-6-encoding mRNA; as an 
antiviral agent, an anti-tumor agent and to control embryonic development 
and tissue homeostasis in vitro, ex vivo or in vivo by exposing cells to 
ICE LAP-6 polypeptides or polynucleotides as disclosed herein; assaying 
genetic variation and aberrations, such as defects, in ICE LAP-6 genes; 
and administering an ICE LAP-6 polypeptide or polynucleotide to an 
organism to augment ICE LAP-6 function or remediate ICE LAP-6 dysfunction. 
Agonists targeted to defective cellular proliferation, including, for 
example, cancer cell and solid tumor cell proliferation, may be used for 
the treatment of these diseases. Such targeting may be achieved via gene 
therapy using antibody fusions. Agonists may also be used to treat 
follicular lymphomas, carcinomas associated with p53 mutations, autoimmune 
disorders, such as, for example, SLE, immune-mediated glomerulonephritis; 
and hormone-dependent tumors, such as, for example, breast cancer, 
prostate cancer and ovary cancer; and viral infections, such as, for 
example, herpesviruses, poxviruses and adenoviruses. 
In accordance with certain preferred embodiments of this and other aspects 
of the invention there are provided probes that hybridize to human ICE 
LAP-6 sequences. 
In certain additional preferred embodiments of this aspect of the invention 
there are provided antibodies against ICE LAP-6 polypeptides. In certain 
particularly preferred embodiments in this regard, the antibodies are 
highly selective for human ICE LAP-6. 
In accordance with another aspect of the present invention, there are 
provided ICE LAP-6 agonists. Among preferred agonists are molecules that 
mimic ICE LAP-6, that bind to ICE LAP-6-binding molecules or receptor 
molecules, and that elicit or augment ICE LAP-6-induced responses. Also 
among preferred agonists are molecules that interact with ICE LAP-6 or ICE 
LAP-6 polypeptides, or with other modulators of ICE LAP-6 activities 
and/or gene expression, and thereby potentiate or augment an effect of ICE 
LAP-6 or more than one effect of ICE LAP-6. 
In accordance with yet another aspect of the present invention, there are 
provided ICE LAP-6 antagonists. Among preferred antagonists are those 
which mimic ICE LAP-6 so as to bind to ICE LAP-6 receptor or binding 
molecules but not elicit an ICE LAP-6-induced response or more than one 
ICE LAP-6-induced response. Also among preferred antagonists are molecules 
that bind to or interact with ICE LAP-6 so as to inhibit an effect of ICE 
LAP-6 or more than one effect of ICE LAP-6 or which prevent expression of 
ICE LAP-6. 
In a further aspect of the invention there are provided compositions 
comprising an ICE LAP-6 polynucleotide or an ICE LAP-6 polypeptide for 
administration to cells in vitro, to cells ex vivo and to cells in vivo, 
or to a multicellular organism. In certain particularly preferred 
embodiments of this aspect of the invention, the compositions comprise an 
ICE LAP-6 polynucleotide for expression of an ICE LAP-6 polypeptide in a 
host organism for treatment of disease. Particularly preferred in this 
regard is expression in a human patient for treatment of a dysfunction 
associated with aberrant endogenous activity of ICE LAP-6. 
Other objects, features, advantages and aspects of the present invention 
will become apparent to those of skill in the art from the following 
description. It should be understood, however, that the following 
description and the specific examples, while indicating preferred 
embodiments of the invention, are given by way of illustration only. 
Various changes and modifications within the spirit and scope of the 
disclosed invention will become readily apparent to those skilled in the 
art from reading the following description and from reading the other 
parts of the present disclosure.

GLOSSARY 
The following illustrative explanations are provided to facilitate 
understanding of certain terms used frequently herein, particularly in the 
examples. The explanations are provided as a convenience and are not 
limitative of the invention. 
DIGESTION of DNA refers to catalytic cleavage of the DNA with a restriction 
enzyme that acts only at certain sequences in the DNA. The various 
restriction enzymes referred to herein are commercially available and 
their reaction conditions, cofactors and other requirements for use are 
known and routine to the skilled artisan. 
For analytical purposes, typically, 1 .mu.g of plasmid or DNA fragment is 
digested with about 2 units of enzyme in about 20 .mu.l of reaction 
buffer. For the purpose of isolating DNA fragments for plasmid 
construction, typically 5 to 50 .mu.g of DNA are digested with 20 to 250 
units of enzyme in proportionately larger volumes. 
Appropriate buffers and substrate amounts for particular restriction 
enzymes are described in standard laboratory manuals, such as those 
referenced below, and they are specified by commercial suppliers. 
Incubation times of about 1 hour at 37.degree. C. are ordinarily used, but 
conditions may vary in accordance with standard procedures, the supplier's 
instructions and the particulars of the reaction. After digestion, 
reactions may be analyzed, and fragments may be purified by 
electrophoresis through an agarose or polyacrylamide gel, using well known 
methods that are routine for those skilled in the art. 
GENETIC ELEMENT generally means a polynucleotide comprising a region that 
encodes a polypeptide or a region that regulates transcription or 
translation or other processes important to expression of the polypeptide 
in a host cell, or a polynucleotide comprising both a region that encodes 
a polypeptide and a region operably linked thereto that regulates 
expression. 
Genetic elements may be comprised within a vector that replicates as an 
episomal element; that is, as a molecule physically independent of the 
host cell genome. They may be comprised within mini-chromosomes, such as 
those that arise during amplification of transfected DNA by methotrexate 
selection in eukaryotic cells. Genetic elements also may be comprised 
within a host cell genome; not in their natural state but, rather, 
following manipulation such as isolation, cloning and introduction into a 
host cell in the form of purified DNA or in a vector, among others. 
IDENTITY or SIMILARITY, as known in the art, are relationships between two 
polypeptides as determined by comparing the amino acid sequence and its 
conserved amino acid substitutes of one polypeptide to the sequence of a 
second polypeptide. Moreover, also known in the art is "identity" which 
means the degree of sequence relatedness between two polypptide or two 
polynucleotides sequences as determined by the identity of the match 
between two strings of such sequences. Both identity and similarity can be 
readily calculated (Computational Molecular Biology, Lesk, A.M., ed., 
Oxford University Press, New York, 1988; Biocomputing: Informatics and 
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; 
Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, 
H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in 
Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence 
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, 
New York, 1991). While there exist a number of methods to measure identity 
and similarity between two polynucleotide or polypeptide sequences, the 
terms "identity" and "similarity" are well known to skilled artisans 
(Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 
1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M 
Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. 
Applied Math., 48: 1073 (1988). Methods commonly employed to determine 
identity or similarity between two sequences include, but are not limited 
to disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 
1073 (1988). Preferred methods to determine identity are designed to give 
the largest match between the two sequences tested. Methods to determine 
identity and similarity are codified in computer programs. Preferred 
computer program methods to determine identity and similarity between two 
sequences include, but are not limited to, GCG program package (Devereux, 
J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, 
FASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403 (1990)). 
ISOLATED means altered "by the hand of man" from its natural state; i.e., 
that, if it occurs in nature, it has been changed or removed from its 
original environment, or both. 
For example, a naturally occurring polynucleotide or a polypeptide 
naturally present in a living animal in its natural state is not 
"isolated," but the same polynucleotide or polypeptide separated from the 
coexisting materials of its natural state is "isolated", as the term is 
employed herein. For example, with respect to polynucleotides, the term 
isolated means that it is separated from the chromosome and cell in which 
it naturally occurs. 
As part of or following isolation, such polynucleotides can be joined to 
other polynucleotides, such as DNAs, for mutagenesis, to form fusion 
proteins, and for propagation or expression in a host, for instance. The 
isolated polynucleotides, alone or joined to other polynucleotides such as 
vectors, can be introduced into host cells, in culture or in whole 
organisms. Introduced into host cells in culture or in whole organisms, 
such DNAs still would be isolated, as the term is used herein, because 
they would not be in their naturally occurring form or environment. 
Similarly, the polynucleotides and polypeptides may occur in a 
composition, such as a media formulations, solutions for introduction of 
polynucleotides or polypeptides, for example, into cells, compositions or 
solutions for chemical or enzymatic reactions, for instance, which are not 
naturally occurring compositions, and, therein remain isolated 
polynucleotides or polypeptides within the meaning of that term as it is 
employed herein. 
LIGATION refers to the process of forming phosphodiester bonds between two 
or more polynucleotides, which most often are double stranded DNAs. 
Techniques for ligation are well known to the art and protocols for 
ligation are described in standard laboratory manuals and references, such 
as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 
(1989) ("Sambrook") and Maniatis et al., pg. 146, as cited below. 
OLIGONUCLEOTIDE(S) refers to relatively short polynucleotides. Often the 
term refers to single-stranded deoxyribonucleotides, but it can refer as 
well to single-or double-stranded ribonucleotides, RNA:DNA hybrids and 
double-stranded DNAs, among others. 
Oligonucleotides, such as single-stranded DNA probe oligonucleotides, often 
are synthesized by chemical methods, such as those implemented on 
automated oligonucleotide synthesizers. However, oligonucleotides can be 
made by a variety of other methods, including in vitro recombinant 
DNA-mediated techniques and by expression of DNAs in cells and organisms. 
Initially, chemically synthesized DNAs typically are obtained without a 5' 
phosphate. The 5' ends of such oligonucleotides are not substrates for 
phosphodiester bond formation by ligation reactions that employ DNA 
ligases typically used to form recombinant DNA molecules. Where ligation 
of such oligonucleotides is desired, a phosphate can be added by standard 
techniques, such as those that employ a kinase and ATP. 
The 3' end of a chemically synthesized oligonucleotide generally has a free 
hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, 
readily will form a phosphodiester bond with a 5' phosphate of another 
polynucleotide, such as another oligonucleotide. As is well known, this 
reaction can be prevented selectively, where desired, by removing the 5' 
phosphates of the other polynucleotide(s) prior to ligation. 
PLASMIDS generally are designated herein by a lower case p preceded and/or 
followed by capital letters and/or numbers, in accordance with standard 
naming conventions that are familiar to those of skill in the art. 
Starting plasmids disclosed herein are either commercially available, 
publicly available on an unrestricted basis, or can be constructed from 
available plasmids by routine application of well known, published 
procedures. Many plasmids and other cloning and expression vectors that 
can be used in accordance with the present invention are well known and 
readily available to those of skill in the art. Moreover, those of skill 
readily may construct any number of other plasmids suitable for use in the 
invention. The properties, construction and use of such plasmids, as well 
as other vectors, in the present invention will be readily apparent to 
those of skill from the present disclosure. 
POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide or 
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA 
or DNA. Thus, for instance, polynucleotides as used herein refers to, 
among others, single-and double-stranded DNA, DNA that is a mixture of 
single-and double-stranded regions, single- and double-stranded RNA, and 
RNA that is mixture of single- and double-stranded regions, hybrid 
molecules comprising DNA and RNA that may be single-stranded or, more 
typically, double-stranded or a mixture of single- and double-stranded 
regions. 
In addition, polynucleotide as used herein refers to triple-stranded 
regions comprising RNA or DNA or both RNA and DNA. The strands in such 
regions may be from the same molecule or from different molecules. The 
regions may include all of one or more of the molecules, but more 
typically involve only a region of some of the molecules. One of the 
molecules of a triple-helical region often is an oligonucleotide. 
As used herein, the term polynucleotide includes DNAs or RNAs as described 
above that contain one or more modified bases. Thus, DNAs or RNAs with 
backbones modified for stability or for other reasons are 
"polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs 
comprising unusual bases, such as inosine, or modified bases, such as 
tritylated bases, to name just two examples, are polynucleotides as the 
term is used herein. 
It will be appreciated that a great variety of modifications have been made 
to DNA and RNA that serve many useful purposes known to those of skill in 
the art. The term polynucleotide as it is employed herein embraces such 
chemically, enzymatically or metabolically modified forms of 
polynucleotides, as well as the chemical forms of DNA and RNA 
characteristic of viruses and cells, including simple and complex cells, 
inter alia. 
POLYPEPTIDES, as used herein, includes all polypeptides as described below. 
The basic structure of polypeptides is well known and has been described 
in innumerable textbooks and other publications in the art. In this 
context, the term is used herein to refer to any peptide or protein 
comprising two or more amino acids joined to each other in a linear chain 
by peptide bonds. As used herein, the term refers to both short chains, 
which also commonly are referred to in the art as peptides, oligopeptides 
and oligomers, for example, and to longer chains, which generally are 
referred to in the art as proteins, of which there are many types. 
It will be appreciated that polypeptides often contain amino acids other 
than the 20 amino acids commonly referred to as the 20 naturally occurring 
amino acids, and that many amino acids, including the terminal amino 
acids, may be modified in a given polypeptide, either by natural 
processes, such as processing and other post-translational modifications, 
but also by chemical modification techniques which are well known to the 
art. Even the common modifications that occur naturally in polypeptides 
are too numerous to list exhaustively here, but they are well described in 
basic texts and in more detailed monographs, as well as in a voluminous 
research literature, and they are well known to those of skill in the art. 
Among the known modifications which may be present in polypeptides of the 
present are, to name an illustrative few, acetylation, acylation, 
ADP-ribosylation, amidation, covalent attachment of flavin, covalent 
attachment of a heme moiety, covalent attachment of a nucleotide or 
nucleotide derivative, covalent attachment of a lipid or lipid derivative, 
covalent attachment of phosphotidylinositol, cross-linking, cyclization, 
disulfide bond formation, demethylation, formation of covalent 
cross-links, formation of cystine, formation of pyroglutamate, 
formylation, gamma-carboxylation, glycosylation, GPI anchor formation, 
hydroxylation, iodination, methylation, myristoylation, oxidation, 
proteolytic processing, phosphorylation, prenylation, racemization, 
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to 
proteins such as arginylation, and ubiquitination. 
Such modifications are well known to those of skill and have been described 
in great detail in the scientific literature. Several particularly common 
modifications, glycosylation, lipid attachment, sulfation, 
gamma-carboxylation of glutamic acid residues, hydroxylation and 
ADP-ribosylation, for instance, are described in most basic texts, such 
as, for instance PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. 
E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed 
reviews are available on this subject, such as, for example, those 
provided by Wold, F., Posttranslational Protein Modifications: 
Perspectives and Prospects, pgs. 1-12 in POSTRANSLATIONAL COVALENT 
MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York 
(1983); Seifter et al., Analysis for protein modifications and nonprotein 
cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Protein 
Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 
663: 48-62 (1992). 
It will be appreciated, as is well known and as noted above, that 
polypeptides are not always entirely linear. For instance, polypeptides 
may be branched as a result of ubiquitination, and they may be circular, 
with or without branching, generally as a result of posttranslation 
events, including natural processing event and events brought about by 
human manipulation which do not occur naturally. Circular, branched and 
branched circular polypeptides may be synthesized by non-translation 
natural process and by entirely synthetic methods, as well. 
Modifications can occur anywhere in a polypeptide, including the peptide 
backbone, the amino acid side-chains and the amino or carboxyl termini. In 
fact, blockage of the amino or carboxyl group in a polypeptide, or both, 
by a covalent modification, is common in naturally occurring and synthetic 
polypeptides and such modifications may be present in polypeptides of the 
present invention, as well. For instance, the amino terminal residue of 
polypeptides made in E. coli, prior to proteolytic processing, almost 
invariably will be N-formylmethionine. 
The modifications that occur in a polypeptide often will be a function of 
how it is made. For polypeptides made by expressing a cloned gene in a 
host, for instance, the nature and extent of the modifications in large 
part will be determined by the host cell posttranslational modification 
capacity and the modification signals present in the polypeptide amino 
acid sequence. For instance, as is well known, glycosylation often does 
not occur in bacterial hosts such as E. coli. Accordingly, when 
glycosylation is desired, a polypeptide should be expressed in a 
glycosylating host, generally a eukaryotic cell. Insect cell often carry 
out the same posttranslational glycosylations as mammalian cells and, for 
this reason, insect cell expression systems have been developed to express 
efficiently mammalian proteins having native patterns of glycosylation, 
inter alia. Similar considerations apply to other modifications. 
It will be appreciated that the same type of modification may be present in 
the same or varying degree at several sites in a given polypeptide. Also, 
a given polypeptide may contain many types of modifications. 
In general, as used herein, the term polypeptide encompasses all such 
modifications, particularly those that are present in polypeptides 
synthesized by expressing a polynucleotide in a host cell. 
VARIANT(S) of polynucleotides or polypeptides, as the term is used herein, 
are polynucleotides or polypeptides that differ from a reference 
polynucleotide or polypeptide, respectively. Variants in this sense are 
described below and elsewhere in the present disclosure in greater detail. 
(1) A polynucleotide that differs in nucleotide sequence from another, 
reference polynucleotide. Generally, differences are limited so that the 
nucleotide sequences of the reference and the variant are closely similar 
overall and, in many regions, identical. 
As noted below, changes in the nucleotide sequence of the variant may be 
silent. That is, they may not alter the amino acids encoded by the 
polynucleotide. Where alterations are limited to silent changes of this 
type a variant will encode a polypeptide with the same amino acid sequence 
as the reference. Also as noted below, changes in the nucleotide sequence 
of the variant may alter the amino acid sequence of a polypeptide encoded 
by the reference polynucleotide. Such nucleotide changes may result in 
amino acid substitutions, additions, deletions, fusions and truncations in 
the polypeptide encoded by the reference sequence, as discussed below. 
(2) A polypeptide that differs in amino acid sequence from another, 
reference polypeptide. Generally, differences are limited so that the 
sequences of the reference and the variant are closely similar overall 
and, in many region, identical. 
A variant and reference polypeptide may differ in amino acid sequence by 
one or more substitutions, additions, deletions, fusions and truncations, 
which may be present in any combination. 
RECEPTOR MOLECULE, as used herein, refers to molecules which bind or 
interact specifically with ICE LAP-6 polypeptides of the present 
invention, including not only classic receptors, which are preferred, but 
also other molecules that specifically bind to or interact with 
polypeptides of the invention (which also may be referred to as "binding 
molecules" and "interaction molecules," respectively and as "ICE LAP-6 
binding molecules" and "ICE LAP-6 interaction molecules." Binding between 
polypeptides of the invention and such molecules, including receptor or 
binding or interaction molecules may be exclusive to polypeptides of the 
invention, which is very highly preferred, or it may be highly specific 
for polypeptides of the invention, which is highly preferred, or it may be 
highly specific to a group of proteins that includes polypeptides of the 
invention, which is preferred, or it may be specific to several groups of 
proteins at least one of which includes polypeptides of the invention. 
Receptors also may be non-naturally occurring, such as antibodies and 
antibody-derived reagents that bind specifically to polypeptides of the 
invention. 
Description of the Invention 
The present invention relates to novel ICE LAP-6 polypeptides and 
polynucleotides, among other things, as described in greater detail below. 
In particular, the invention relates to polypeptides and polynucleotides 
of a novel human ICE LAP-6, which is related by amino acid sequence 
homology to human interleukin-1 beta converting enzyme apoptosis protease 
polypeptides. The invention relates especially to ICE LAP-6 having the 
nucleotide and amino acid sequences set out in FIGS. 1 and 2A, 2B and 2C 
respectively. It will be appreciated that the nucleotide and amino acid 
sequences set out in FIGS. 2A, 2B and 2C respectively, were obtained by 
sequencing the cDNA obtained from a human K562 (erythroleukemia) cell line 
cDNA library. 
Polynucleotides 
In accordance with one aspect of the present invention, there are provided 
isolated polynucleotides which encode the ICE LAP-6 polypeptide having the 
deduced amino acid sequence of FIG. 1 or the polypeptide encoded by the 
deposited clone. 
In accordance with another aspect of the present invention, there are 
provided isolated polynucleotides which encode the ICE LAP-6 polypeptide 
having the deduced amino acid sequence of FIG. 1 or the polypeptide 
encoded by the deposited clone. 
Using the information provided herein, such as the polynucleotide primer 
sequences set out in Example 1, a polynucleotide of the present invention 
encoding human ICE LAP-6 polypeptide may be obtained using standard 
cloning and screening procedures, such as those for cloning cDNAs using 
mRNA from cells from human neutrophils and kidney tissue as starting 
material. Illustrative of the invention, the polynucleotide of the 
invention was discovered as described in Example 1. ICE LAP 7 can also be 
obtained from other tissues and cDNA libraries, for example, libraries 
derived from cells of human cells, tissue and cell lines such as, 
activated human neutrophil, erythroleukemia and kidney cells. 
Human ICE LAP-6 of the invention is structurally related to other proteins 
of the human interleukin-1 beta converting enzyme apoptosis protease 
family, as shown by the results of sequencing the cDNA encoding human ICE 
LAP-6 in FIG. 1. The cDNA of FIGS. 2A, 2B and 2C was obtained as described 
in Example 1. The polypeptide of FIG. 1 and the polypeptide encoded by the 
deposited clone each are proteins which have a deduced molecular weight of 
about 45.8 kDa. 
Polynucleotides of the present invention may be in the form of RNA, such as 
mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA 
obtained by cloning or produced by chemical synthetic techniques or by a 
combination thereof. The DNA may be double-stranded or single-stranded. 
Single-stranded DNA may be the coding strand, also known as the sense 
strand, or it may be the non-coding strand, also referred to as the 
anti-sense strand. 
The coding sequence which encodes the polypeptide may be identical to the 
coding sequence of the polynucleotide derived using the primers set forth 
in Example 1, or the polynucleotide of FIGS. 2A, 2B, and 2C. It also may 
be a polynucleotide with a different sequence, which, as a result of the 
redundancy (degeneracy) of the genetic code, encodes the polypeptide of 
the cDNA of FIG. 1 or the polypeptide encoded by the deposited clone. 
Polynucleotides of the present invention which encode the polypeptide of 
FIG. 1 or the polypeptide encoded by the deposited clone may include, but 
are not limited to the coding sequence for the mature polypeptide, by 
itself; the coding sequence for the mature polypeptide and additional 
coding sequences, such as those encoding a leader or secretory sequence, 
such as a pre-, or pro- or prepro- protein sequence; the coding sequence 
of the mature polypeptide, with or without the aforementioned additional 
coding sequences, together with additional, non-coding sequences, 
including for example, but not limited to introns and non-coding 5' and 3' 
sequences, such as the transcribed, non-translated sequences that play a 
role in transcription, mRNA processing--including splicing and 
polyadenylation signals, for example--ribosome binding and stability of 
mRNA; additional coding sequence which codes for additional amino acids, 
such as those which provide additional functionalities. Thus, for 
instance, the polypeptide may be fused to a marker sequence, such as a 
peptide, which facilitates purification of the fused polypeptide. In 
certain preferred embodiments of this aspect of the invention, the marker 
sequence is a hexa-histidine peptide, such as the tag provided in the pQE 
vector (Qiagen, Inc.), among others, many of which are commercially 
available. As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 
821-824 (1989), for instance, hexa-histidine provides for convenient 
purification of the fusion protein. The HA tag corresponds to an epitope 
derived of influenza hemagglutinin protein, which has been described by 
Wilson et al., Cell 37: 767 (1984), for instance. 
Also provided is mature ICE LAP-6 processed from its precursor molecule, 
via autocatalysis or by other enzymes, to produce two subunits, which form 
an active heterodimer (both subunits) or tetramer (two sets of such 
heterodimers). 
In accordance with the foregoing, the term "polynucleotide encoding a 
polypeptide" as used herein encompasses polynucleotides which include a 
sequence encoding a polypeptide of the present invention, particularly the 
human ICE LAP-6 having the amino acid sequence set out in FIG. 1 or the 
amino acid sequence of the polypeptide encoded by the deposited clone. The 
term encompasses polynucleotides that include a single continuous region 
or discontinuous regions encoding the polypeptide (for example, 
interrupted by introns) together with additional regions, that also may 
contain coding and/or non-coding sequences. 
The present invention further relates to variants of the herein above 
described polynucleotides which encode for fragments, analogs and 
derivatives of the polypeptide having the deduced amino acid sequence of 
FIG. 1 or the amino acid sequence of the polypeptide encoded by the 
deposited clone. A variant of the polynucleotide may be a naturally 
occurring variant such as a naturally occurring allelic variant, or it may 
be a variant that is not known to occur naturally. Such non-naturally 
occurring variants of the polynucleotide may be made by mutagenesis 
techniques, including those applied to polynucleotides, cells or 
organisms. 
Among variants in this regard are variants that differ from the 
aforementioned polynucleotides by nucleotide substitutions, deletions or 
additions. The substitutions, deletions or additions may involve one or 
more nucleotides. The variants may be altered in coding or non-coding 
regions or both. Alterations in the coding regions may produce 
conservative or non-conservative amino acid substitutions, deletions or 
additions. 
Among the particularly preferred embodiments of the invention in this 
regard are polynucleotides encoding polypeptides having the amino acid 
sequence of ICE LAP-6 set out in FIG. 1 or the amino acid sequence of the 
polypeptide encoded by the deposited clone; variants, analogs, derivatives 
and fragments thereof, and fragments of the variants, analogs and 
derivatives. 
Further particularly preferred in this regard are polynucleotides encoding 
ICE LAP-6 variants, analogs, derivatives and fragments, and variants, 
analogs and derivatives of the fragments, which have the amino acid 
sequence of the ICE LAP-6 polypeptide of FIG. 1 or the polypeptide encoded 
by the deposited clone in which several, a few, 5 to 10, 1 to 5, 1 to 3, 
2, 1 or no amino acid residues are substituted, deleted or added, in any 
combination. Especially preferred among these are substitutions, additions 
and deletions, which do not alter the properties and activities of the ICE 
LAP-6. Also especially preferred in this regard are conservative 
substitutions. Most highly preferred are polynucleotides encoding 
polypeptides having the amino acid sequence of FIG. 1 or the amino acid 
sequence of the polypeptide encoded by the deposited clone, without 
substitutions. 
Further preferred embodiments of the invention are polynucleotides that are 
at least 70% identical to a polynucleotide encoding the ICE LAP-6 
polypeptide having the amino acid sequence set out in FIG. 1 or the amino 
acid sequence of the polypeptide encoded by the deposited clone, and 
polynucleotides which are complementary to such polynucleotides. 
Alternatively, most highly preferred are polynucleotides that comprise a 
region that is at least 80% identical to a polynucleotide encoding the ICE 
LAP-6 polypeptide of the human cDNA and polynucleotides complementary 
thereto. In this regard, polynucleotides at least 90% identical to the 
same are particularly preferred, and among these particularly preferred 
polynucleotides, those with at least 95% are especially preferred. 
Furthermore, those with at least 97% are highly preferred among those with 
at least 95%, and among these those with at least 98% and at least 99% are 
particularly highly preferred, with at least 99% being the more preferred. 
Particularly preferred embodiments in this respect, moreover, are 
polynucleotides which encode polypeptides which retain substantially the 
same biological function or activity as the mature polypeptide encoded by 
the cDNA of 
FIGS. 2A, 2B, and 2C encoded by the polynucleotide sequence of the 
deposited clone, or derived using the primers set forth in Example 1. 
The present invention further relates to polynucleotides that hybridize to 
the herein above-described sequences. In this regard, the present 
invention especially relates to polynucleotides which hybridize under 
stringent conditions to the herein above-described polynucleotides. As 
herein used, the term "stringent conditions" means hybridization will 
occur only if there is at least 95% and preferably at least 97% identity 
between the sequences. 
As discussed additionally herein regarding polynucleotide assays of the 
invention, for instance, polynucleotides of the invention as discussed 
above, may be used as a hybridization probe for cDNA and genornic DNA to 
isolate full-length cDNAs and genomic clones encoding ICE LAP-6 and to 
isolate cDNA and genomic clones of other genes that have a high sequence 
similarity to the human ICE LAP-6 gene. Such probes generally will 
comprise at least 15 bases. Preferably, such probes will have at least 30 
bases and may have at least 50 bases. Particularly preferred probes will 
have at least 30 bases and will have 50 bases or less. 
For example, the coding region of the ICE LAP-6 gene may be isolated by 
screening using the known DNA sequence to synthesize an oligonucleotide 
probe. A labeled oligonucleotide having a sequence complementary to that 
of a gene of the present invention is then used to screen a library of 
human cDNA, genomic DNA or mRNA to determine which members of the library 
the probe hybridizes to. 
The polynucleotides and polypeptides of the present invention may be 
employed as research reagents and materials for discovery of treatments 
and diagnostics to human disease, as further discussed herein relating to 
polynucleotide assays, inter alia. 
The polynucleotides may encode a polypeptide which is the mature protein 
plus additional amino or carboxyl-terminal amino acids, or amino acids 
interior to the mature polypeptide (when the mature form has more than one 
polypeptide chain, for instance). Such sequences may play a role in 
processing of a protein from precursor to a mature form, may facilitate 
protein trafficking, may prolong or shorten protein half-life or may 
facilitate manipulation of a protein for assay or production, among other 
things. As generally is the case in situ, the additional amino acids may 
be processed away from the mature protein by cellular enzymes. 
A precursor protein, having the mature form of the polypeptide fused to one 
or more prosequences may be an inactive form of the polypeptide. When 
prosequences are removed such inactive precursors generally are activated. 
Some or all of the prosequences may be removed before activation. 
Generally, such precursors are called proproteins. 
In sum, a polynucleotide of the present invention may encode a mature 
protein, a mature protein plus a leader sequence (which may be referred to 
as a preprotein), a precursor of a mature protein having one or more 
prosequences which are not the leader sequences of a preprotein, or a 
preproprotein, which is a precursor to a proprotein, having a leader 
sequence and one or more prosequences, which generally are removed during 
processing steps that produce active and mature forms of the polypeptide. 
Deposited Materials 
A deposit containing a human ICE LAP-6 cDNA has been deposited with the 
American Type Culture Collection, as noted above. Also as noted above, the 
human cDNA deposit is referred to herein as "the deposited clone" or as 
"the cDNA of the deposited clone." 
The deposited clone was deposited with the American Type Culture 
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 USA, on 
May 30, 1996. 
The deposited material is a pBluescript SK (-) plasmid (Stratagene, La 
Jolla, Calif.) that contains the full length ICE LAP-6 cDNA, referred to 
as "1095150" upon deposit. 
The deposit has been made under the terms of the Budapest Treaty on the 
international recognition of the deposit of micro-organisms for purposes 
of patent procedure. The strain will be irrevocably and without 
restriction or condition released to the public upon the issuance of a 
patent. The deposit is provided merely as convenience to those of skill in 
the art and is not an admission that a deposit is required for enablement, 
such as that required under 35 U.S.C. .sctn.112. 
The sequence of the polynucleotides contained in the deposited material, as 
well as the amino acid sequence of the polypeptide encoded thereby, are 
controlling in the event of any conflict with any description of sequences 
herein. 
A license may be required to make, use or sell the deposited materials, and 
no such license is hereby granted. 
Polypeptides 
The present invention further relates to a human ICE LAP-6 polypeptide 
which has the deduced amino acid sequence of FIG. 1 and the amino acid 
sequence of the the polypeptide encoded by the deposited clone. 
The invention also relates to fragments, analogs and derivatives of these 
polypeptides. The terms "fragment," "derivative" and "analog" when 
referring to the polypeptide of FIG. 1 or the polypeptide encoded by the 
deposited clone, means a polypeptide which retains essentially the same 
biological function or activity as such polypeptide. Thus, an analog 
includes a proprotein which can be activated by cleavage of the proprotein 
portion to produce an active mature polypeptide. 
The polypeptide of the present invention may be a recombinant polypeptide, 
a natural polypeptide or a synthetic polypeptide. In certain preferred 
embodiments it is a recombinant polypeptide. 
The fragment, derivative or analog of the polypeptide of FIG. 1 or the 
polypeptide encoded by the deposited clone each may be (i) one in which 
one or more of the amino acid residues are substituted with a conserved or 
non-conserved amino acid residue (preferably a conserved amino acid 
residue) and such substituted amino acid residue may or may not be one 
encoded by the genetic code, or (ii) one in which one or more of the amino 
acid residues includes a substituent group, or (iii) one in which the 
mature polypeptide is fused with another compound, such as a compound to 
increase the half-life of the polypeptide (for example, polyethylene 
glycol), or (iv) one in which the additional amino acids are fused to the 
mature polypeptide, such as a leader or secretory sequence or a sequence 
which is employed for purification of the mature polypeptide or a 
proprotein sequence. Such fragments, derivatives and analogs are deemed to 
be within the scope of those skilled in the art from the teachings herein. 
Among the particularly preferred embodiments of the invention in this 
regard are polypeptides having the amino acid sequence of ICE LAP-6 set 
out in FIG. 1 or the amino acid sequence of the polypeptide encoded by the 
deposited clone, variants, analogs, derivatives and fragments thereof, and 
variants, analogs and derivatives of the fragments. Alternatively, 
particularly preferred embodiments of the invention in this regard are 
polypeptides having the amino acid sequence of the ICE LAP-6, variants, 
analogs, derivatives and fragments thereof, and variants, analogs and 
derivatives of the fragments. 
Among preferred variants are those that vary from a reference by 
conservative amino acid substitutions. Such substitutions are those that 
substitute a given amino acid in a polypeptide by another amino acid of 
like characteristics. Typically seen as conservative substitutions are the 
replacements, one for another, among the aliphatic amino acids Ala, Val, 
Leu and Lle; interchange of the hydroxyl residues Ser and Thr, exchange of 
the acidic residues Asp and Glu, substitution between the amide residues 
Asn and Gln, exchange of the basic residues Lys and Arg and replacements 
among the aromatic residues Phe, Tyr. 
Further particularly preferred in this regard are variants, analogs, 
derivatives and fragments, and variants, analogs and derivatives of the 
fragments, having the amino acid sequence of the ICE LAP-6 polypeptide of 
FIG. 1 or the amino acid sequence of the polypeptide encoded by the 
deposited clone, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or 
no amino acid residues are substituted, deleted or added, in any 
combination. Especially preferred among these are substitutions, additions 
and deletions, which do not alter the properties and activities of the ICE 
LAP-6. Also especially preferred in this regard are conservative 
substitutions. Most highly preferred are polypeptides having the amino 
acid sequence of FIG. 1 or the amino acid sequence of the polypeptide 
encoded by the deposited clone without substitutions. 
The polypeptides and polynucleotides of the present invention are 
preferably provided in an isolated form, and preferably are purified to 
homogeneity. 
The polypeptides of the present invention include the polypeptide of FIG. 1 
or the polypeptide encoded by the deposited clone (in particular the 
mature polypeptide) as well as polypeptides which have at least 80% 
identity to the polypeptide of FIG. 1 or the polypeptide encoded by the 
deposited clone and more preferably at least 90% similarity (more 
preferably at least 90% identity) to the polypeptide of FIG. 1 or the 
polypeptide encoded by the deposited clone and still more preferably at 
least 95% similarity (still more preferably at least 95% identity) to the 
polypeptide of FIG. 1 or the polypeptide encoded by the deposited clone 
and also include portions of such polypeptides with such portion of the 
polypeptide generally containing at least 30 amino acids and more 
preferably at least 50 amino acids. 
Fragments or portions of the polypeptides of the present invention may be 
employed for producing the corresponding full-length polypeptide by 
peptide synthesis; therefore, the fragments may be employed as 
intermediates for producing the full-length polypeptides. Fragments or 
portions of the polynucleotides of the present invention may be used to 
synthesize full-length polynucleotides of the present invention. 
Fragments 
Also among preferred embodiments of this aspect of the present invention 
are polypeptides comprising fragments of ICE LAP-6, most particularly 
fragments of the ICE LAP-6 having the amino acid set out in FIG. 1 or the 
amino acid sequence of the polypeptide encoded by the deposited clone, and 
fragments of variants and derivatives of the ICE LAP-6 of FIG. 1 or the 
polypeptide encoded by the deposited clone, such as, for example the amino 
acid sequence of FIG. 4. 
In this regard a fragment is a polypeptide having an amino acid sequence 
that entirely is the same as part but not all of the amino acid sequence 
of the aforementioned ICE LAP-6 polypeptides and variants or derivatives 
thereof. 
Such fragments may be "free-standing," i.e., not part of or fused to other 
amino acids or polypeptides, or they may be comprised within a larger 
polypeptide of which they form a part or region. When comprised within a 
larger polypeptide, the presently discussed fragments most preferably form 
a single continuous region. However, several fragments may be comprised 
within a single larger polypeptide. For instance, certain preferred 
embodiments relate to a fragment of an ICE LAP-6 polypeptide of the 
present comprised within a precursor polypeptide designed for expression 
in a host and having heterologous pre and pro-polypeptide regions fused to 
the amino terminus of the ICE LAP-6 fragment and an additional region 
fused to the carboxyl terminus of the fragment. Therefore, fragments in 
one aspect of the meaning intended herein, refers to the portion or 
portions of a fusion polypeptide or fusion protein derived from ICE LAP-6. 
As representative examples of polypeptide fragments of the invention, there 
may be mentioned those which have from about 5-15, 10-20, 15-40, 30-55, 
41-65, 41-80, 41-90, 50-100, 75-100, 90-115, 100-125, and 110-113 amino 
acids long. 
In this context about includes the particularly recited range and ranges 
larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid at either 
extreme or at both extremes. For instance, about 40-90 amino acids in this 
context means a polypeptide fragment of 40 plus or minus several, a few, 
5, 4, 3, 2 or 1 amino acids to 90 plus or minus several a few, 5, 4, 3, 2 
or 1 amino acid residues, i.e., ranges as broad as 40 minus several amino 
acids to 90 plus several amino acids to as narrow as 40 plus several amino 
acids to 90 minus several amino acids. 
Highly preferred in this regard are the recited ranges plus or minus as 
many as 5 amino acids at either or at both extremes. Particularly highly 
preferred are the recited ranges plus or minus as many as 3 amino acids at 
either or at both the recited extremes. Especially particularly highly 
preferred are ranges plus or minus 1 amino acid at either or at both 
extremes or the recited ranges with no additions or deletions. Most highly 
preferred of all in this regard are fragments from about 5-15, 10-20, 
15-40, 30-55, 41-65, 41-80, 41-90, 50-100, 75-100, 90-115, 100-125, and 
110-113 amino acids long. 
Among especially preferred fragments of the invention are truncation 
mutants of ICE LAP-6. Truncation mutants include ICE LAP-6 polypeptides 
having the amino acid sequence of FIG. 1 or the amino acid sequence of the 
polypeptide encoded by the deposited clone, or of variants or derivatives 
thereof, except for deletion of a continuous series of residues (that is, 
a continuous region, part or portion) that includes the amino terminus, or 
a continuous series of residues that includes the carboxyl terminus or, as 
in double truncation mutants, deletion of two continuous series of 
residues, one including the amino terminus and one including the carboxyl 
terminus. Fragments having the size ranges set out about also are 
preferred embodiments of truncation fragments, which are especially 
preferred among fragments generally. 
Also preferred in this aspect of the invention are fragments characterized 
by structural or functional attributes of ICE LAP-6. Preferred embodiments 
of the invention in this regard include fragments that comprise 
alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet 
and beta-sheet-forming regions ("beta-regions"), turn and turn-forming 
regions ("turn-regions"), coil and coil-forming regions ("coil-regions"), 
hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta 
amphipathic regions, flexible regions, surface-forming regions and high 
antigenic index regions of ICE LAP-6. 
Among highly preferred fragments in this regard are those that comprise 
regions of ICE LAP-6 that combine several structural features, such as 
several of the features set out above. In this regard, the regions defined 
by the residues about 10 to about 20, about 40 to about 50, about 70 to 
about 90 and about 100 to about 113 of FIG. 1 or the polypeptide encoded 
by the deposited clone, which all are characterized by amino acid 
compositions highly characteristic of turn-regions, hydrophilic regions, 
flexible-regions, surface-forming regions, and high antigenic 
index-regions, are especially highly preferred regions. Such regions may 
be comprised within a larger polypeptide or may be by themselves a 
preferred fragment of the present invention, as discussed above. It will 
be appreciated that the term "about" as used in this paragraph has the 
meaning set out above regarding fragments in general. 
Further preferred regions are those that mediate activities of ICE LAP-6. 
Most highly preferred in this regard are fragments that have a chemical, 
biological or other activity of ICE LAP-6, including those with a similar 
activity or an improved activity, or with a decreased undesirable 
activity. Highly preferred in this regard are fragments that contain 
regions that are homologs in sequence, or in position, or in both sequence 
and to active regions of related polypeptides, which include human 
interleukin-1 beta converting enzyme apoptosis proteases. Among 
particularly preferred fragments in these regards are truncation mutants, 
as discussed above. 
It will be appreciated that the invention also relates to, among others, 
polynucleotides encoding the aforementioned fragments, polynucleotides 
that hybridize to polynucleotides encoding the fragments, particularly 
those that hybridize under stringent conditions, and polynucleotides, such 
as PCR primers, for amplifying polynucleotides that encode the fragments. 
In these regards, preferred polynucleotides are those that correspondent 
to the preferred fragments, as discussed above. Preferred polynucleotides 
fragments may be derived from the sequences of FIGS. 2A, 2B, 2C and 3. 
Vectors, Host Cells, Expression 
The present invention also relates to vectors which include polynucleotides 
of the present invention, host cells which are genetically engineered with 
vectors of the invention and the production of polypeptides of the 
invention by recombinant techniques. 
Host cells can be genetically engineered to incorporate polynucleotides and 
express polypeptides of the present invention. For instance, 
polynucleotides may be introduced into host cells using well known 
techniques of infection, transduction, transfection, transvection and 
transformation. The polynucleotides may be introduced alone or with other 
polynucleotides. Such other polynucleotides may be introduced 
independently, co-introduced or introduced joined to the polynucleotides 
of the invention. 
Thus, for instance, polynucleotides of the invention may be transfected 
into host cells with another, separate, polynucleotide encoding a 
selectable marker, using standard techniques for co-transfection and 
selection in, for instance, mammalian cells. In this case the 
polynucleotides generally will be stably incorporated into the host cell 
genome. 
Alternatively, the polynucleotides may be joined to a vector containing a 
selectable marker for propagation in a host. The vector construct may be 
introduced into host cells by the aforementioned techniques. Generally, a 
plasmid vector is introduced as DNA in a precipitate, such as a calcium 
phosphate precipitate, or in a complex with a charged lipid. 
Electroporation also may be used to introduce polynucleotides into a host. 
If the vector is a virus, it may be packaged in vitro or introduced into a 
packaging cell and the packaged virus may be transduced into cells. A wide 
variety of techniques suitable for making polynucleotides and for 
introducing polynucleotides into cells in accordance with this aspect of 
the invention are well known and routine to those of skill in the art. 
Such techniques are reviewed at length in Sambrook et al. cited above, 
which is illustrative of the many laboratory manuals that detail these 
techniques. 
In accordance with this aspect of the invention the vector may be, for 
example, a plasmid vector, a single or double-stranded phage vector, a 
single or double-stranded RNA or DNA viral vector. Such vectors may be 
introduced into cells as polynucleotides, preferably DNA, by well known 
techniques for introducing DNA and RNA into cells. The vectors, in the 
case of phage and viral vectors also may be and preferably are introduced 
into cells as packaged or encapsidated virus by well known techniques for 
infection and transduction. Viral vectors may be replication competent or 
replication defective. In the latter case viral propagation generally will 
occur only in complementing host cells. 
Preferred among vectors, in certain respects, are those for expression of 
polynucleotides and polypeptides of the present invention. Generally, such 
vectors comprise cis-acting control regions effective for expression in a 
host operatively linked to the polynucleotide to be expressed. Appropriate 
trans-acting factors either are supplied by the host, supplied by a 
complementing vector or supplied by the vector itself upon introduction 
into the host. 
In certain preferred embodiments in this regard, the vectors provide for 
specific expression. Such specific expression may be inducible expression 
or expression only in certain types of cells or both inducible and 
cell-specific. Particularly preferred among inducible vectors are vectors 
that can be induced for expression by environmental factors that are easy 
to manipulate, such as temperature and nutrient additives. A variety of 
vectors suitable to this aspect of the invention, including constitutive 
and inducible expression vectors for use in prokaryotic and eukaryotic 
hosts, are well known and employed routinely by those of skill in the art. 
The engineered host cells can be cultured in conventional nutrient media, 
which may be modified as appropriate for, inter alia, activating 
promoters, selecting transformants or amplifying genes. Culture 
conditions, such as temperature, pH and the like, previously used with the 
host cell selected for expression generally will be suitable for 
expression of polypeptides of the present invention as will be apparent to 
those of skill in the art. 
A great variety of expression vectors can be used to express a polypeptide 
of the invention. Such vectors include chromosomal, episomal and 
virus-derived vectors e.g., vectors derived from bacterial plasmids, from 
bacteriophage, from yeast episomes, from yeast chromosomal elements, from 
viruses such as baculoviruses, papova viruses, such as simian virus 40 
("SV40"), vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies 
viruses and retroviruses, and vectors derived from combinations thereof, 
such as those derived from plasmid and bacteriophage genetic elements, 
such as cosmids and phagemids, all may be used for expression in 
accordance with this aspect of the present invention. Generally, any 
vector suitable to maintain, propagate or express polynucleotides to 
express a polypeptide in a host may be used for expression in this regard. 
The appropriate DNA sequence may be inserted into the vector by any of a 
variety of well-known and routine techniques. In general, a DNA sequence 
for expression is joined to an expression vector by cleaving the DNA 
sequence and the expression vector with one or more restriction 
endonucleases and then joining the restriction fragments together using T4 
DNA ligase. Procedures for restriction and ligation that can be used to 
this end are well known and routine to those of skill. Suitable procedures 
in this regard, and for constructing expression vectors using alternative 
techniques, which also are well known and routine to those skill, are set 
forth in great detail in Sambrook et al. cited elsewhere herein. 
The DNA sequence in the expression vector is operatively linked to 
appropriate expression control sequence(s), including, for instance, a 
promoter to direct mRNA transcription. Representatives of such promoters 
include the phage lambda PL promoter, the E. coli lac, trp and tac 
promoters, the SV40 early and late promoters and promoters of retroviral 
LTRs, to name just a few of the well-known promoters. It will be 
understood that numerous promoters not mentioned are suitable for use in 
this aspect of the invention are well known and readily may be employed by 
those of skill in the manner illustrated by the discussion and the 
examples herein. 
In general, expression constructs will contain sites for transcription 
initiation and termination, and, in the transcribed region, a ribosome 
binding site for translation. The coding portion of the mature transcripts 
expressed by the constructs will include a translation initiating AUG at 
the beginning and a termination codon appropriately positioned at the end 
of the polypeptide to be translated. 
In addition, the constructs may contain control regions that regulate as 
well as engender expression. Generally, in accordance with many commonly 
practiced procedures, such regions will operate by controlling 
transcription, such as repressor binding sites and enhancers, among 
others. 
Vectors for propagation and expression generally will include selectable 
markers. Such markers also may be suitable for amplification or the 
vectors may contain additional markers for this purpose. In this regard, 
the expression vectors preferably contain one or more selectable marker 
genes to provide a phenotypic trait for selection of transformed host 
cells. Preferred markers include dihydrofolate reductase or neomycin 
resistance for eukaryotic cell culture, and tetracycline or ampicillin 
resistance genes for culturing E. coli and other bacteria. 
The vector containing the appropriate DNA sequence as described elsewhere 
herein, as well as an appropriate promoter, and other appropriate control 
sequences, may be introduced into an appropriate host using a variety of 
well known techniques suitable to expression therein of a desired 
polypeptide. Representative examples of appropriate hosts include 
bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium 
cells; fungal cells, such as yeast cells; insect cells such as Drosophila 
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes 
melanoma cells; and plant cells. Hosts for a great variety of expression 
constructs are well known, and those of skill will be enabled by the 
present disclosure readily to select a host for expressing a polypeptides 
in accordance with this aspect of the present invention. 
More particularly, the present invention also includes recombinant 
constructs, such as expression constructs, comprising one or more of the 
sequences described above. The constructs comprise a vector, such as a 
plasmid or viral vector, into which such a sequence of the invention has 
been inserted. The sequence may be inserted in a forward or reverse 
orientation. In certain preferred embodiments in this regard, the 
construct further comprises regulatory sequences, including, for example, 
a promoter, operably linked to the sequence. Large numbers of suitable 
vectors and promoters are known to those of skill in the art, and there 
are many commercially available vectors suitable for use in the present 
invention. 
The following vectors, which are commercially available, are provided by 
way of example. Among vectors preferred for use in bacteria are pQE70, 
pQE60 and pQE-9 , available from Qiagen; pBS vectors, Phagescript vectors, 
Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from 
Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from 
Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, 
pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL 
available from Pharmacia. These vectors are listed solely by way of 
illustration of the many commercially available and well known vectors 
that are available to those of skill in the art for use in accordance with 
this aspect of the present invention. It will be appreciated that any 
other plasmid or vector suitable for, for example, introduction, 
maintenance, propagation or expression of a polynucleotide or polypeptide 
of the invention in a host may be used in this aspect of the invention. 
Promoter regions can be selected from any desired gene using vectors that 
contain a reporter transcription unit lacking a promoter region, such as a 
chloramphenicol acetyl transferase ("CAT") transcription unit, downstream 
of restriction site or sites for introducing a candidate promoter 
fragment; i.e., a fragment that may contain a promoter. As is well known, 
introduction into the vector of a promoter-containing fragment at the 
restriction site upstream of the cat gene engenders production of CAT 
activity, which can be detected by standard CAT assays. Vectors suitable 
to this end are well known and readily available. Two such vectors are 
pKK232-8 and pCM7. Thus, promoters for expression of polynucleotides of 
the present invention include not only well known and readily available 
promoters, but also promoters that readily may be obtained by the 
foregoing technique, using a reporter gene. 
Among known bacterial promoters suitable for expression of polynucleotides 
and polypeptides in accordance with the present invention are the E. coli 
lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the 
lambda PR, PL promoters and the trp promoter. 
Among known eukaryotic promoters suitable in this regard are the 
cytomegalovirus ("CMV") immediate early promoter, the HSV thymidine kinase 
promoter, the early and late SV40 promoters, the promoters of retroviral 
LTRs, such as those of the Rous sarcoma virus ("RoSV"), and 
metallothionein promoters, such as the mouse metallothionein-I promoter. 
Selection of appropriate vectors and promoters for expression in a host 
cell is a well known procedure and the requisite techniques for expression 
vector construction, introduction of the vector into the host and 
expression in the host are routine skills in the art. 
The present invention also relates to host cells containing the 
above-described constructs discussed above. The host cell can be a higher 
eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, 
such as a yeast cell, or the host cell can be a prokaryotic cell, such as 
a bacterial cell. 
Introduction of the construct into the host cell can be effected by calcium 
phosphate transfection, DEAE-dextran mediated transfection, cationic 
lipid-mediated transfection, electroporation, transduction, infection or 
other methods. Such methods are described in many standard laboratory 
manuals, such as SAMBROOK. 
Constructs in host cells can be used in a conventional manner to produce 
the gene product encoded by the recombinant sequence. Alternatively, the 
polypeptides of the invention can be synthetically produced by 
conventional peptide synthesizers. 
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or 
other cells under the control of appropriate promoters. Cell-free 
translation systems can also be employed to produce such proteins using 
RNAs derived from the DNA constructs of the present invention. Appropriate 
cloning and expression vectors for use with prokaryotic and eukaryotic 
hosts are described by Sambrook et al., cited above. 
Generally, recombinant expression vectors will include origins of 
replication, a promoter derived from a highly-expressed gene to direct 
transcription of a downstream structural sequence, and a selectable marker 
to permit isolation of vector containing cells after exposure to the 
vector. Among suitable promoters are those derived from the genes that 
encode glycolytic enzymes such as 3-phosphoglycerate kinase ("PGK"), 
a-factor, acid phosphatase, and heat shock proteins, among others. 
Selectable markers include the ampicillin resistance gene of E. coli and 
the trp 1 gene of S. cerevisiae. 
Transcription of the DNA encoding the polypeptides of the present invention 
by higher eukaryotes may be increased by inserting an enhancer sequence 
into the vector. Enhancers are cis-acting elements of DNA, usually about 
from 10 to 300 bp that act to increase transcriptional activity of a 
promoter in a given host cell-type. Examples of enhancers include the SV40 
enhancer, which is located on the late side of the replication origin at 
bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma 
enhancer on the late side of the replication origin, and adenovirus 
enhancers. 
Polynucleotides of the invention, encoding the heterologous structural 
sequence of a polypeptide of the invention generally will be inserted into 
the vector using standard techniques so that it is operably linked to the 
promoter for expression. The polynucleotide will be positioned so that the 
transcription start site is located appropriately 5' to a ribosome binding 
site. The ribosome binding site will be 5' to the AUG that initiates 
translation of the polypeptide to be expressed. Generally, there will be 
no other open reading frames that begin with an initiation codon, usually 
AUG, and lie between the ribosome binding site and the initiating AUG. 
Also, generally, there will be a translation stop codon at the end of the 
polypeptide and there will be a polyadenylation signal and a transcription 
termination signal appropriately disposed at the 3' end of the transcribed 
region. 
For secretion of the translated protein into the lumen of the endoplasmic 
reticulum, into the periplasmic space or into the extracellular 
environment, appropriate secretion signals may be incorporated into the 
expressed polypeptide. The signals may be endogenous to the polypeptide or 
they may be heterologous signals. 
The polypeptide may be expressed in a modified form, such as a fusion 
protein, and may include not only secretion signals but also additional 
heterologous functional regions. Thus, for instance, a region of 
additional amino acids, particularly charged amino acids, may be added to 
the N-terminus of the polypeptide to improve stability and persistence in 
the host cell, during purification or during subsequent handling and 
storage. Also, a region may be added to the polypeptide to facilitate 
purification. Such regions may be removed prior to final preparation of 
the polypeptide. The addition of peptide moieties to polypeptides to 
engender secretion or excretion, to improve stability and to facilitate 
purification, among others, are familiar and routine techniques in the 
art. 
Suitable prokaryotic hosts for propagation, maintenance or expression of 
polynucleotides and polypeptides in accordance with the invention include 
Escherichia coli, Bacillus subtilis and Salmonella typhimurium. Various 
species of Pseudomonas, Streptomyces, and Staphylococcus are suitable 
hosts in this regard. Moreover, many other hosts also known to those of 
skill may be employed in this regard. 
As a representative but non-limiting example, useful expression vectors for 
bacterial use can comprise a selectable marker and bacterial origin of 
replication derived from commercially available plasmids comprising 
genetic elements of the well known cloning vector pBR322 (ATCC 37017). 
Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine 
Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wisc., 
USA). These pBR322 "backbone" sections are combined with an appropriate 
promoter and the structural sequence to be expressed. 
Following transformation of a suitable host strain and growth of the host 
strain to an appropriate cell density, where the selected promoter is 
inducible it is induced by appropriate means (e.g., temperature shift or 
exposure to chemical inducer) and cells are cultured for an additional 
period. 
Cells typically then are harvested by centrifugation, disrupted by physical 
or chemical means, and the resulting crude extract retained for further 
purification. 
Microbial cells employed in expression of proteins can be disrupted by any 
convenient method, including freeze-thaw cycling, sonication, mechanical 
disruption, or use of cell lysing agents, such methods are well know to 
those skilled in the art. 
Various mammalian cell culture systems can be employed for expression, as 
well. Examples of mammalian expression systems include the COS-6 lines of 
monkey kidney fibroblast, described in Gluzman et al., Cell 23: 175 
(1981). Other cell lines capable of expressing a compatible vector include 
for example, the C 127, 3T3, CHO, HeLa, human kidney 293 and BHK cell 
lines. 
Mammalian expression vectors will comprise an origin of replication, a 
suitable promoter and enhancer, and also any necessary ribosome binding 
sites, polyadenylation sites, splice donor and acceptor sites, 
transcriptional termination sequences, and 5' flanking non-transcribed 
sequences that are necessary for expression. In certain preferred 
embodiments in this regard DNA sequences derived from the SV40 splice 
sites, and the SV40 polyadenylation sites are used for required 
non-transcribed genetic elements of these types. 
The ICE LAP-6 polypeptide can be recovered and purified from recombinant 
cell cultures by well-known methods including ammonium sulfate or ethanol 
precipitation, acid extraction, anion or cation exchange chromatography, 
phosphocellulose chromatography, hydrophobic interaction chromatography, 
affinity chromatography, hydroxylapatite chromatography and lectin 
chromatography. Most preferably, high performance liquid chromatography 
("HPLC") is employed for purification. Well known techniques for refolding 
protein may be employed to regenerate active conformation when the 
polypeptide is denatured during isolation and or purification. 
Polypeptides of the present invention include naturally purified products, 
products of chemical synthetic procedures, and products produced by 
recombinant techniques from a prokaryotic or eukaryotic host, including, 
for example, bacterial, yeast, higher plant, insect and mammalian cells. 
Depending upon the host employed in a recombinant production procedure, 
the polypeptides of the present invention may be glycosylated or may be 
non-glycosylated. In addition, polypeptides of the invention may also 
include an initial modified methionine residue, in some cases as a result 
of host-mediated processes. 
ICE LAP-6 polynucleotides and polypeptides may be used in accordance with 
the present invention for a variety of applications, particularly those 
that make use of the chemical and biological properties of ICE LAP-6. 
Additional applications relate to diagnosis and to treatment of disorders 
of cells, tissues and organisms. These aspects of the invention are 
illustrated further by the following discussion. 
Polynucleotide Assays 
This invention is also related to the use of the ICE LAP-6 polynucleotides 
to detect complementary polynucleotides such as, for example, as a 
diagnostic reagent. Detection of a mutated form of ICE LAP-6 associated 
with a dysfunction will provide a diagnostic tool that can add or define a 
diagnosis of a disease or susceptibility to a disease which results from 
under-expression over-expression or altered expression of ICE LAP-6. 
Individuals carrying mutations in the human ICE LAP-6 gene may be detected 
at the DNA level by a variety of techniques. Nucleic acids for diagnosis 
may be obtained from a patient's cells, such as from blood, urine, saliva, 
tissue biopsy and autopsy material. The genomic DNA may be used directly 
for detection or may be amplified enzymatically by using PCR prior to 
analysis. PCR (Saiki et al., Nature, 324:163-166 (1986)). RNA or cDNA may 
also be used in the same ways. As an example, PCR primers complementary to 
the nucleic acid encoding ICE LAP-6 can be used to identify and analyze 
ICE LAP-6 expression and mutations. For example, deletions and insertions 
can be detected by a change in size of the amplified product in comparison 
to the normal genotype. Point mutations can be identified by hybridizing 
amplified DNA to radiolabeled ICE LAP-6 RNA or alternatively, radiolabeled 
ICE LAP-6 antisense DNA sequences. Perfectly matched sequences can be 
distinguished from mismatched duplexes by RNase A digestion or by 
differences in melting temperatures. 
Sequence differences between a reference gene and genes having mutations 
also may be revealed by direct DNA sequencing. In addition, cloned DNA 
segments may be employed as probes to detect specific DNA segments. The 
sensitivity of such methods can be greatly enhanced by appropriate use of 
PCR or another amplification method. For example, a sequencing primer is 
used with double-stranded PCR product or a single-stranded template 
molecule generated by a modified PCR. The sequence determination is 
performed by conventional procedures with radiolabeled nucleotide or by 
automatic sequencing procedures with fluorescent-tags. 
Genetic testing based on DNA sequence differences may be achieved by 
detection of alteration in electrophoretic mobility of DNA fragments in 
gels, with or without denaturing agents. Small sequence deletions and 
insertions can be visualized by high resolution gel electrophoresis. DNA 
fragments of different sequences may be distinguished on denaturing 
formamide gradient gels in which the mobilities of different DNA fragments 
are retarded in the gel at different positions according to their specific 
melting or partial melting temperatures (see, e.g., Myers et al., Science, 
230: 1242 (1985)). 
Sequence changes at specific locations also may be revealed by nuclease 
protection assays, such as RNase and S1 protection or the chemical 
cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 
4397-4401 (1985)). 
Thus, the detection of a specific DNA sequence may be achieved by methods 
such as hybridization, RNase protection, chemical cleavage, direct DNA 
sequencing or the use of restriction enzymes, (e.g., restriction fragment 
length polymorphisms ("RFLP") and Southern blotting of genomic DNA. 
In addition to more conventional gel-electrophoresis and DNA sequencing, 
mutations also can be detected by in situ analysis. 
In accordance with a further aspect of the invention, there is provided a 
process for determining disease associated with viral infection, 
tumorogenesis and to control embryogenesis and tissue homeostasis. 
Diseases associated with viral infection, tumorogenesis and to control 
embryogenesis and tissue homeostasis, or a susceptibility to viral 
infection, tumorogenesis and to diseases and defects in the control of 
control of embryogenesis and tissue homeostasis. Thus, a mutation in ICE 
LAP-6 indicates a susceptibility to viral infection, tumorogenesis and to 
diseases and defects in the control embryogenesis and tissue homeostasis, 
and the nucleic acid sequences described above may be employed in an assay 
for ascertaining such susceptibility. Thus, for example, the assay may be 
employed to determine a mutation in a human ICE LAP-6 protein as herein 
described, such as a deletion, truncation, insertion, frame shift, etc., 
with such mutation being indicative of a susceptibility to viral 
infection, tumorogenesis and to diseases and defects in the control of 
embryogenesis and tissue homeostasis. 
A mutation may be ascertained for example, by a DNA sequencing assay. 
Tissue samples, including but not limited to blood samples are obtained 
from a human patient. The samples are processed by methods known in the 
art to capture the RNA. First strand cDNA is synthesized from the RNA 
samples by adding an oligonucleotide primer consisting of polythymidine 
residues which hybridize to the polyadenosine stretch present on the 
mRNA's. Reverse transcriptase and deoxynucleotides are added to allow 
synthesis of the first strand cDNA. Primer sequences are synthesized based 
on the DNA sequence of the ICE LAP-6 protein of the invention. The primer 
sequence is generally comprised of at least 15 consecutive bases, and may 
contain at least 30 or even 50 consecutive bases. 
Individuals carrying mutations in the gene of the present invention may 
also be detected at the DNA level by a variety of techniques. Nucleic 
acids for diagnosis may be obtained from a patient's cells, including but 
not limited to blood, urine, saliva, tissue biopsy and autopsy material. 
The genomic DNA may be used directly for detection or may be amplified 
enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) 
prior to analysis. RT-PCR can also be used to detect mutations. It is 
particularly preferred to used RT-PCR in conjunction with automated 
detection systems, such as, for example, GeneScan. RNA or cDNA may also be 
used for the same purpose, PCR or RT-PCR. As an example, PCR primers 
complementary to the nucleic acid encoding ICE LAP-6 can be used to 
identify and analyze mutations. For example, deletions and insertions can 
be detected by a change in size of the amplified product in comparison to 
the normal genotype. Point mutations can be identified by hybridizing 
amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense 
DNA sequences. Perfectly matched sequences can be distinguished from 
mismatched duplexes by RNase A digestion or by differences in melting 
temperatures. 
Primers, selected by well known methods, may be used for amplifying ICE 
LAP-6 cDNA isolated from a sample derived from a patient. The invention 
also provides the primers selected with 1, 2, 3 or 4 nucleotides removed 
from the 5' and/or the 3' end. The primers may be used to amplify the gene 
isolated from the patient such that the gene may then be subject to 
various techniques for elucidation of the DNA sequence. In this way, 
mutations in the DNA sequence may be diagnosed. 
Sequence differences between the reference gene and genes having mutations 
may be revealed by the direct DNA sequencing method. In addition, cloned 
DNA segments may be employed as probes to detect specific DNA segments. 
The sensitivity of this method is greatly enhanced when combined with PCR. 
For example, a sequencing primer is used with double-stranded PCR product 
or a single-stranded template molecule generated by a modified PCR. The 
sequence determination is performed by conventional procedures with 
radiolabeled nucleotide or by automatic sequencing procedures with 
fluorescent-tags. 
Genetic testing based on DNA sequence differences may be achieved by 
detection of alteration in electrophoretic mobility of DNA fragments in 
gels with or without denaturing agents. Small sequence deletions and 
insertions can be visualized by high resolution gel electrophoresis. DNA 
fragments of different sequences may be distinguished on denaturing 
formamide gradient gels in which the mobilities of different DNA fragments 
are retarded in the gel at different positions according to their specific 
melting or partial melting temperatures (see, e.g., Myers et al., Science, 
230:1242 (1985)). 
Sequence changes at specific locations may also be revealed by nuclease 
protection assays, such as RNase and S1 protection or the chemical 
cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)). 
Thus, the detection of a specific DNA sequence and/or quantitation of the 
level of the sequence may be achieved by methods such as hybridization, 
RNase protection, chemical cleavage, direct DNA sequencing or the use of 
restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms 
(RFLP)) and Southern blotting of genomic DNA. 
The invention provides a process for diagnosing or detecting, disease, 
particularly Alzheimer's disease, Parkinson's disease, rheumatoid 
arthritis, septic shock, sepsis, stroke, chronic inflammation, acute 
inflammation, CNS inflammation, osteoporosis, ischemia reperfusion injury, 
cell death associated with cardiovascular disease, polycystic kidney 
disease, apoptosis of endothelial cells in cardiovascular disease, 
degenerative liver disease, MS, ALS, cererbellar degeneration, ischemic 
injury, myocardial infarction, AIDS, myelodysplastic syndromes, aplastic 
anemia, male pattern baldness, and head injury damage, as well as a 
susceptibility to viral infection and cancer, an to detect aberrant 
control of embryonic development and tissue homeostasis, comprising 
determining from a sample derived from a patient altered expression of 
polynucleotide having the sequence of FIG. 1 or the polynucleotide 
sequence of the deposited clone as compared to normal control samples. 
Expression of polynucleotide can be measured using any one of the methods 
well known in the art for the quantation of polynucleotides, such as, for 
example, PCR, RT-PCR, RNase protection, Northern blotting and other 
hybridization methods. 
In addition to more conventional gel-electrophoresis and DNA sequencing, 
mutations can also be detected by in situ analysis. 
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase 
chromosomal spread can be used to provide a precise chromosomal location. 
As an example of how this is performed, ICE LAP-6 DNA is digested and 
purified with QIAEX II DNA purification kit (QIAGEN, Inc., Chatsworth, 
Calif.) and ligated to Super Cos 1 cosmid vector (STRATAGENE, La Jolla, 
Calif.). DNA is purified using Qiagen Plasmid Purification Kit (QIAGEN 
Inc., Chatsworth, Calif.) and 1 mg is labeled by nick translation in the 
presence of Biotin-dATP using BioNick Labeling Kit (GibcoBRL, Life 
Technologies Inc., Gaithersburg, Md.). Biotinilation is detected with 
GENE-TECT Detection System (CLONTECH Laboratories, Inc. Palo Alto, 
Calif.). In situ Hybridization is performed on slides using ONCOR Light 
Hybridization Kit (ONCOR, Gaithersberg, Md.) to detect single copy 
sequences on metaphase chromosomes. Peripheral blood of normal donors is 
cultured for three days in RPMI 1640 supplemented with 20% FCS, 3% PHA and 
penicillin/streptomycin, synchronized with 10.sup.-6 M methotrexate for 17 
hours and washed twice with unsupplemented RPMI. Cells are incubated with 
10.sup.-3 M thymidine for 7 hours. The cells are arrested in metaphase 
after 20 minutes incubation with colcemid (0.5 .mu.g/ml) followed by 
hypotonic lysis in 75 mM KCI for 15 minutes at 37.degree. C. Cell pellets 
are then spun out and fixed in Carnoy's fixative (3:1 methanouacetic 
acid). 
Metaphase spreads are prepared by adding a drop of the suspension onto 
slides and aid dried. Hybridization is performed by adding 100 ng of probe 
suspended in 10 ml of hybridization mix (50% formamide, 2.times.SSC, 1% 
dextran sulfate) with blocking human placental DNA 1 .mu.g/ml), Probe 
mixture is denatured for 10 minutes in 70.degree. C. water bath and 
incubated for 1 hour at 37.degree. C., before placing on a prewarmed 
(37.degree. C.) slide, which is previously denatured in 70% 
formamide/2.times.SSC at 70.degree. C., and dehydrated in ethanol series, 
chilled to 4.degree. C. 
Slides are incubated for 16 hours at 37.degree. C. in a humidified chamber. 
Slides are washed in 50% formamide/2.times.SSC for 10 minutes at 
41.degree. C. and 2xSSC for 7 minutes at 37.degree. C. Hybridization probe 
is detected by incubation of the slides with FITC-Avidin (ONCOR, 
Gaithersberg, Md.), according to the manufacturer protocol. Chromosomes 
are counterstained with propridium iodine suspended in mounting medium. 
Slides are visualized using a Leitz ORTHOPLAN 2-epifluorescence microscope 
and five computer images are taken using Imagenetics Computer and 
Macintosh printer. 
Once a sequence has been mapped to a precise chromosomal location, the 
physical position of the sequence on the chromosome can be correlated with 
genetic map data. Such data are found, for example, in V. McKusick, 
Mendelian Inheritance in Man (publicly available on-line via computer 
(Internet)). The relationship between genes and diseases that have been 
mapped to the same chromosomal region are then identified through linkage 
analysis using well known methods. 
Unless otherwise stated, transformation was performed as described in the 
method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973). 
Chromosome Assays 
The sequences of the present invention are also valuable for chromosome 
identification. The sequence is specifically targeted to and can hybridize 
with a particular location on an individual human chromosome. Moreover, 
there is a current need for identifying particular sites on the 
chromosome. Few chromosome marking reagents based on actual sequence data 
(repeat polymorphisms) are presently available for marking chromosomal 
location. The mapping of DNAs to chromosomes according to the present 
invention is an important first step in correlating those sequences with 
genes associated with disease. 
In certain preferred embodiments in this regard, the cDNA herein disclosed 
is used to clone genomic DNA of an ICE LAP-6 gene. This can be 
accomplished using a variety of well known techniques and libraries, which 
generally are available commercially. The genomic DNA is used for in situ 
chromosome mapping using well known techniques for this purpose. 
Typically, in accordance with routine procedures for chromosome mapping, 
some trial and error may be necessary to identify a genomic probe that 
gives a good in situ hybridization signal. 
In some cases, in addition, sequences can be mapped to chromosomes by 
preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer 
analysis of the 3' untranslated region of the gene is used to rapidly 
select primers that do not span more than one exon in the genomic DNA, 
thus complicating the amplification process. These primers are then used 
for PCR screening of somatic cell hybrids containing individual human 
chromosomes. Only those hybrids containing the human gene corresponding to 
the primer will yield an amplified fragment. 
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a 
particular DNA to a particular chromosome. Using the present invention 
with the same oligonucleotide primers, sublocalization can be achieved 
with panels of fragments from specific chromosomes or pools of large 
genomic clones in an analogous manner. Other mapping strategies that can 
similarly be used to map to its chromosome include in situ hybridization, 
prescreening with labeled flow-sorted chromosomes and preselection by 
hybridization to construct chromosome specific-cDNA libraries. 
Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphase 
chromosomal spread can be used to provide a precise chromosomal location 
in one step. This technique can be used with cDNA as short as 50 or 60. 
For a review of this technique, see Verma et al., HUMAN CHROMOSOMES: A 
MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York (1988). 
Once a sequence has been mapped to a precise chromosomal location, the 
physical position of the sequence on the chromosome can be correlated with 
genetic map data. Such data are found, for example, in V. McKusick, 
MENDELIAN INHERITANCE IN MAN (publicly available on line via computer). 
The relationship between genes and diseases that have been mapped to the 
same chromosomal region are then identified through linkage analysis 
(coinheritance of physically adjacent genes). 
Next, it is necessary to determine the differences in the cDNA or genomic 
sequence between affected and unaffected individuals. If a mutation is 
observed in some or all of the affected individuals but not in any normal 
individuals, then the mutation is likely to be the causative agent of the 
disease. 
With current resolution of physical mapping and genetic mapping techniques, 
a cDNA precisely localized to a chromosomal region associated with the 
disease could be one of between 50 and 500 potential causative genes. 
(This assumes 1 megabase mapping resolution and one gene per 20 kb). 
Polypeptide Assays 
The present invention also relates to a quantitative and semi-quantitative 
diagnostic assays for detecting levels of ICE LAP-6 protein in cells and 
tissues, including determination of normal and abnormal levels. Thus, for 
instance, a diagnostic assay in accordance with the invention for 
detecting over-expression of ICE LAP-6 protein compared to normal control 
tissue samples may be used to detect the presence of a tumor, or other 
abnormal cell growth or proliferation, for example. Assay techniques that 
can be used to determine levels of a protein, such as an ICE LAP-6 protein 
of the present invention, in a sample derived from a host are well-known 
to those of skill in the art. Such assay methods include 
radioimmunoassays, competitive-binding assays, Western Blot analysis and 
ELISA assays. Among these ELISAs frequently are preferred. An ELISA assay 
initially comprises preparing an antibody specific to ICE LAP-6, 
preferably a monoclonal antibody. In addition a reporter antibody 
generally is prepared which binds to the monoclonal antibody. The reporter 
antibody is attached a detectable reagent such as radioactive, fluorescent 
or enzymatic reagent, in this example horseradish peroxidase enzyme. 
To carry out an ELISA a sample is removed from a host and incubated on a 
solid support, e.g. a polystyrene dish, that binds the proteins in the 
sample. Any free protein binding sites on the dish are then covered by 
incubating with a non-specific protein such as bovine serum albumin. Next, 
the monoclonal antibody is incubated in the dish during which time the 
monoclonal antibodies attach to any ICE LAP-6 proteins attached to the 
polystyrene dish. Unbound monoclonal antibody is washed out with buffer. 
The reporter antibody linked to horseradish peroxidase is placed in the 
dish resulting in binding of the reporter antibody to any monoclonal 
antibody bound to ICE LAP-6. Unattached reporter antibody is then washed 
out. Reagents for peroxidase activity, including a colorimetric substrate 
are then added to the dish. Immobilized peroxidase, linked to ICE LAP-6 
through the primary and secondary antibodies, produces a colored reaction 
product. The amount of color developed in a given time period indicates 
the amount of ICE LAP-6 protein present in the sample. Quantitative 
results typically are obtained by reference to a standard curve. 
A competition assay may be employed wherein antibodies specific to ICE 
LAP-6 attached to a solid support and labeled ICE LAP-6 and a sample 
derived from the host are passed over the solid support and the amount of 
label detected attached to the solid support can be correlated to a 
quantity of ICE LAP-6 in the sample. 
Antibodies 
The polypeptides, their fragments or other derivatives, or analogs thereof, 
or cells expressing them can be used as an immunogen to produce antibodies 
thereto. These antibodies can be, for example, polyclonal or monoclonal 
antibodies. The present invention also includes chimeric, single chain, 
and humanized antibodies, as well as Fab fragments, or the product of an 
Fab expression library. Various procedures known in the art may be used 
for the production of such antibodies and fragments. 
Antibodies generated against the polypeptides corresponding to a sequence 
of the present invention can be obtained by direct injection of the 
polypeptides into an animal or by administering the polypeptides to an 
animal, preferably a nonhuman. The antibody so obtained will then bind the 
polypeptides itself. In this manner, even a sequence encoding only a 
fragment of the polypeptides can be used to generate antibodies binding 
the whole native polypeptides. Such antibodies can then be used to isolate 
the polypeptide from tissue expressing that polypeptide. 
For preparation of monoclonal antibodies, any technique which provides 
antibodies produced by continuous cell line cultures can be used. Examples 
include the hybridoma technique (Kohler, G. and Milstein, C., Nature 256: 
495-497 (1975), the trioma technique, the human B-cell hybridoma technique 
(Kozbor et al., Immunology Today 4: 72 (1983) and the EBV-hybridoma 
technique to produce human monoclonal antibodies (Cole et al., pg. 77-96 
in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985). 
Techniques described for the production of single chain antibodies (U.S. 
Pat. No. 4,946,778) can be adapted to produce single chain antibodies to 
immunogenic polypeptide products of this invention. Also, transgenic mice, 
or other organisms such as other mammals, may be used to express humanized 
antibodies to immunogenic polypeptide products of this invention. 
The above-described antibodies may be employed to isolate or to identify 
clones expressing the polypeptide or purify the polypeptide of the present 
invention by attachment of the antibody to a solid support for isolation 
and/or purification by affinity chromatography. 
Thus, among others, antibodies against ICE LAP-6 may be employed to inhibit 
the action of such ICE LAP-6 polypeptides, for example, in the treatment 
of Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, septic 
shock, sepsis, stroke, chronic inflammation, acute inflammation, CNS 
inflammation, osteoporosis, ischemnia reperfusion injury, cell death 
associated with cardiovascular disease, polycystic kidney disease, 
apoptosis of endothelial cells in cardiovascular disease, degenerative 
liver disease, MS, ALS, cererbellar degeneration, ischemic injury, 
myocardial infarction, AIDS, myelodysplastic syndromes, aplastic anemia, 
male pattern baldness, and head injury damage. 
ICE LAP-6 Binding Molecules and Assays 
This invention also provides a method for identification of molecules, such 
as receptor molecules, that bind ICE LAP-6. Genes encoding proteins that 
bind ICE LAP-6, such as receptor proteins, can be identified by numerous 
methods known to those of skill in the art, for example, ligand panning 
and FACS sorting. Such methods are described in many laboratory manuals 
such as, for instance, Coligan et al., Current Protocols in Immunology 
1(2): Chapter 5 (1991). 
For instance, expression cloning may be employed for this purpose. To this 
end polyadenylated RNA is prepared from a cell responsive to ICE LAP-6, a 
cDNA library is created from this RNA, the library is divided into pools 
and the pools are transfected individually into cells that are not 
responsive to ICE LAP-6. The transfected cells then are exposed to labeled 
ICE LAP-6. (ICE LAP-6 can be labeled by a variety of well-known techniques 
including standard methods of radio-iodination or inclusion of a 
recognition site for a site-specific protein kinase.) Following exposure, 
the cells are fixed and binding of ICE LAP-6 is determined. These 
procedures conveniently are carried out on glass slides. 
Pools are identified of cDNA that produced ICE LAP-6-binding cells. 
Sub-pools are prepared from these positives, transfected into host cells 
and screened as described above. Using an iterative sub-pooling and 
re-screening process, one or more single clones that encode the putative 
binding molecule, such as a receptor molecule, can be isolated. 
Alternatively a labeled ligand can be photoaffinity linked to a cell 
extract, such as a membrane or a membrane extract, prepared from cells 
that express a molecule that it binds, such as a receptor molecule. 
Cross-linked material is resolved by polyacrylamide gel electrophoresis 
("PAGE") and exposed to X-ray film. The labeled complex containing the 
ligand-receptor can be excised, resolved into peptide fragments, and 
subjected to protein microsequencing. The amino acid sequence obtained 
from microsequencing can be used to design unique or degenerate 
oligonucleotide probes to screen cDNA libraries to identify genes encoding 
the putative receptor molecule. 
Polypeptides of the invention also can be used to assess ICE LAP-6 binding 
capacity of ICE LAP-6 binding molecules, such as receptor molecules, in 
cells or in cell-free preparations. 
Agonists and Antagonists--Assays and Molecules 
The invention also provides a method of screening compounds to identify 
those which enhance or block the action of ICE LAP-6 on cells, such as its 
interaction with ICE LAP-6-binding molecules such as receptor molecules. 
An agonist is a compound which increases the natural biological functions 
of ICE LAP-6 or which functions in a manner similar to ICE LAP-6, while 
antagonists decrease or eliminate such functions. 
For example, a cellular compartment, such as a membrane or a preparation 
thereof, such as a membrane-preparation, may be prepared from a cell that 
expresses a molecule that binds ICE LAP-6, such as a molecule of a 
signaling or regulatory pathway modulated by ICE LAP-6. The preparation is 
incubated with labeled ICE LAP-6 in the absence or the presence of a 
candidate molecule which may be an ICE LAP-6 agonist or antagonist. The 
ability of the candidate molecule to bind the binding molecule is 
reflected in decreased binding of the labeled ligand. Molecules which bind 
gratuitously, i.e., without inducing the effects of ICE LAP-6 on binding 
the ICE LAP-6binding molecule, are most likely to be good antagonists. 
Molecules that bind well and elicit effects that are the same as or 
closely related to ICE LAP-6 are agonists. 
ICE LAP-6-like effects of potential agonists and antagonists may by 
measured, for instance, by determining activity of a second messenger 
system following interaction of the candidate molecule with a cell or 
appropriate cell preparation, and comparing the effect with that of ICE 
LAP-6 or molecules that elicit the same effects as ICE LAP-6. Second 
messenger systems that may be useful in this regard include but are not 
limited to AMP guanylate cyclase, ion channel or phosphoinositide 
hydrolysis second messenger systems. 
Another example of an assay for ICE LAP-6 antagonists is a competitive 
assay that combines ICE LAP-6 and a potential antagonist with 
membrane-bound ICE LAP-6 receptor molecules or recombinant ICE LAP-6 
receptor molecules under appropriate conditions for a competitive 
inhibition assay. ICE LAP-6 can be labeled, such as by radioactivity, such 
that the number of ICE LAP-6 molecules bound to a receptor molecule can be 
determined accurately to assess the effectiveness of the potential 
antagonist. 
Potential antagonists include small organic molecules, peptides, 
polypeptides and antibodies that bind to a polypeptide of the invention 
and thereby inhibit or extinguish its activity. Potential antagonists also 
may be small organic molecules, a peptide, a polypeptide such as a closely 
related protein or antibody that binds the same sites on a binding 
molecule, such as a receptor molecule, without inducing ICE LAP-6-induced 
activities, thereby preventing the action of ICE LAP-6 by excluding ICE 
LAP-6 from binding. 
Potential antagonists include a small molecule which binds to and occupies 
the binding site of the polypeptide thereby preventing binding to cellular 
binding molecules, such as receptor molecules, such that normal biological 
activity is prevented. Examples of small molecules include but are not 
limited to small organic molecules, peptides or peptide-like molecules. 
Antoher antagonist is an oligopeptide comprising the cleavage site 
recognition motif for ICE LAP-6. 
Other potential antagonists include antisense molecules. Antisense 
technology can be used to control gene expression through antisense DNA or 
RNA or through triple-helix formation. Antisense techniques are discussed, 
for example, in--Okano, J. Neurochem. 56: 560 (1991); 
OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC 
Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, 
for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et 
al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). 
The methods are based on binding of a polynucleotide to a complementary 
DNA or RNA. For example, the 5' coding portion of a polynucleotide that 
encodes the mature polypeptide of the present invention may be used to 
design an antisense RNA oligonucleotide from about 10 to 40 base pairs in 
length. A DNA oligonucleotide is designed to be complementary to a region 
of the gene involved in transcription thereby preventing transcription and 
the production of ICE LAP-6. The antisense RNA oligonucleotide hybridizes 
to the mRNA in vivo and blocks translation of the mRNA molecule into ICE 
LAP-6 polypeptide. The oligonucleotides described above can also be 
delivered to cells such that the antisense RNA or DNA may be expressed in 
vivo to inhibit production of ICE LAP-6. 
Agonists targeted to defective cellular proliferation, including, for 
example, cancer cells and solid tumor cells may be used for the treatment 
of these diseases. Such targeting may be achieved via gene therapy of 
using antibody fusions. 
Agonists may also be used to treat follicular lymphomas, carcinomas 
associated with p53 mutations, autoimmune disorders, such as, for example, 
SLE, immune-mediated glomerulonephritis; and hormone-dependent tumors, 
such as, for example, breast cancer, prostate cancer and ovary cancer; and 
viral infections, such as, for example, herpesviruses, poxviruses and 
adenoviruses. 
The antagonists may be employed in a composition with a pharmaceutically 
acceptable carrier, e.g., as hereinafter described. 
The antagonists may be employed for instance to inhibit the action of ICE 
LAP-6 polypeptides, for example, in the treatment of Alzheimer's disease, 
Parkinson's disease, rheumatoid arthritis, septic shock, sepsis, stroke, 
chronic inflammation, acute inflammation, CNS inflammation, osteoporosis, 
ischemia reperfusion injury, cell death associated with cardiovascular 
disease, polycystic kidney disease, apoptosis of endothelial cells in 
cardiovascular disease, degenerative liver disease, MS, ALS, cererbellar 
degeneration, ischemic injury, myocardial infarction, AIDS, 
myelodysplastic syndromes, aplastic anemia, male pattern baldness, and 
head injury damage. 
The antagonists may be employed in a composition with a pharmaceutically 
acceptable carrier, e.g., as hereinafter described. 
Compositions 
The invention also relates to compositions comprising the polynucleotide or 
the polypeptides discussed above or the agonists or antagonists. Thus, the 
polypeptides of the present invention may be employed in combination with 
a non-sterile or sterile carrier or carriers for use with cells, tissues 
or organisms, such as a pharmaceutical carrier suitable for administration 
to a subject. Such compositions comprise, for instance, a media additive 
or a therapeutically effective amount of a polypeptide of the invention 
and a pharmaceutically acceptable carrier or excipient. Such carriers may 
include, but are not limited to, saline, buffered saline, dextrose, water, 
glycerol, ethanol and combinations thereof. The formulation should suit 
the mode of administration. 
Kits 
The invention further relates to pharmaceutical packs and kits comprising 
one or more containers filled with one or more of the ingredients of the 
aforementioned compositions of the invention. Associated with such 
container(s) can be a notice in the form prescribed by a governmental 
agency regulating the manufacture, use or sale of pharmaceuticals or 
biological products, reflecting approval by the agency of the manufacture, 
use or sale of the product for human administration. 
Administration 
Polypeptides and other compounds of the present invention may be employed 
alone or in conjunction with other compounds, such as therapeutic 
compounds. 
The pharmaceutical compositions may be administered in any effective, 
convenient manner including, for instance, administration by topical, 
oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, 
subcutaneous, intranasal or intradermal routes among others. 
The pharmaceutical compositions generally are administered in an amount 
effective for treatment or prophylaxis of a specific indication or 
indications. In general, the compositions are administered in an amount of 
at least about 10 .mu.g/kg body weight. In most cases they will be 
administered in an amount not in excess of about 8 mg/kg body weight per 
day. Preferably, in most cases, dose is from about 10 .mu.g/kg to about 1 
mg/kg body weight, daily. It will be appreciated that optimum dosage will 
be determined by standard methods for each treatment modality and 
indication, taking into account the indication, its severity, route of 
administration, complicating conditions and the like. 
Gene Therapy 
The ICE LAP-6 polynucleotides, polypeptides, agonists and antagonists that 
are polypeptides may be employed in accordance with the present invention 
by expression of such polypeptides in vivo, in treatment modalities often 
referred to as "gene therapy." 
Thus, for example, cells from a patient may be engineered with a 
polynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo, and 
the engineered cells then can be provided to a patient to be treated with 
the polypeptide. For example, cells may be engineered ex vivo by the use 
of a retroviral plasmid vector containing RNA encoding a polypeptide of 
the present invention. Such methods are well-known in the art and their 
use in the present invention will be apparent from the teachings herein. 
Similarly, cells may be engineered in vivo for expression of a polypeptide 
in vivo by procedures known in the art. For example, a polynucleotide of 
the invention may be engineered for expression in a replication defective 
retroviral vector, as discussed above. The retroviral expression construct 
then may be isolated and introduced into a packaging cell is transduced 
with a retroviral plasmid vector containing RNA encoding a polypeptide of 
the present invention such that the packaging cell now produces infectious 
viral particles containing the gene of interest. These producer cells may 
be administered to a patient for engineering cells in vivo and expression 
of the polypeptide in vivo. These and other methods for administering a 
polypeptide of the present invention by such method should be apparent to 
those skilled in the art from the teachings of the present invention. 
Retroviruses from which the retroviral plasmid vectors herein above 
mentioned may be derived include, but are not limited to, Moloney Murine 
Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma 
Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia 
virus, human immunodeficiency virus, adenovirus, Myeloproliferative 
Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral 
plasmid vector is derived from Moloney Murine Leukemia Virus. 
Such vectors well include one or more promoters for expressing the 
polypeptide. Suitable promoters which may be employed include, but are not 
limited to, the retroviral LTR; the SV40 promoter; and the CMV promoter 
described in Miller et al., Biotechniques 7:980-990 (1989), or any other 
promoter (e.g., cellular promoters such as eukaryotic cellular promoters 
including, but not limited to, the histone, RNA polymerase III, and 
.beta.-actin promoters). Other viral promoters which may be employed 
include, but are not limited to, adenovirus promoters, thymidine kinase 
(TK) promoters, and B19 parvovirus promoters. The selection of a suitable 
promoter will be apparent to those skilled in the art from the teachings 
contained herein. 
The nucleic acid sequence encoding the polypeptide of the present invention 
will be placed under the control of a suitable promoter. Suitable 
promoters which may be employed include, but are not limited to, 
adenoviral promoters, such as the adenoviral major late promoter; or 
heterologous promoters, such as the CMV promoter; the respiratory 
syncytial virus ("RSV") promoter; inducible promoters, such as the MMT 
promoter, the metallothionein promoter; heat shock promoters; the albumin 
promoter; the ApoAl promoter; human globin promoters; viral thymidine 
kinase promoters, such as the Herpes Simplex thyrmidine kinase promoter; 
retroviral LTRs (including the modified retroviral LTRs herein above 
described); the .beta.-actin promoter; and human growth hormone promoters. 
The promoter also may be the native promoter which controls the gene 
encoding the polypeptide. 
The retroviral plasmid vector is employed to transduce packaging cell lines 
to form producer cell lines. Examples of packaging cells which may be 
transfected include, but are not limited to, the PE501, 17, Y-2, YAM, 
PA 12, T19 14X, VT .multidot.17H2, YCRE, YCRIP, GP.sup.+ E-86, GP.sup.+ 
envAm12, and DAN cell lines as described in Miller, A., Human Gene Therapy 
1: 5-14 (1990). The vector may be transduced into the packaging cells 
through any means known in the art. Such means include, but are not 
limited to, electroporation, the use of liposomes, and CaPO.sub.4 
precipitation. In one alternative, the retroviral plasmid vector may be 
encapsulated into a liposome, or coupled to a lipid, and then administered 
to a host. 
The producer cell line will generate infectious retroviral vector 
particles, which include the nucleic acid sequence(s) encoding the 
polypeptides. Such retroviral vector particles then may be employed to 
transduce eukaryotic cells, either in vitro or in vivo. The transduced 
eukaryotic cells will express the nucleic acid sequence(s) encoding the 
polypeptide. Eukaryotic cells which may be transduced include, but are not 
limited to, embryonic stem cells, embryonic carcinoma cells, as well as 
hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, 
keratinocytes, endothelial cells, and bronchial epithelial cells. 
EXAMPLES 
The present invention is further described by the following examples. The 
examples are provided solely to illustrate the invention by reference to 
specific embodiments. These exemplification's, while illustrating certain 
specific aspects of the invention, do not portray the limitations or 
circumscribe the scope of the disclosed invention. 
Certain terms used herein are explained in the foregoing glossary. 
All examples were carried out using standard techniques, which are well 
known and routine to those of skill in the art, except where otherwise 
described in detail. Routine molecular biology techniques of the following 
examples can be carried out as described in standard laboratory manuals, 
such as Sambrook et al., cited above. 
All parts or amounts set out in the following examples are by weight, 
unless otherwise specified. As used herein, "CTLs" means cytotoxic 
lymphocytes. 
Unless otherwise stated, size separation of fragments in the examples below 
was carried out using standard techniques of agarose and polyacrylamide 
gel electrophoresis ("PAGE") in Sambrook and numerous other references 
such as, for instance, by Goeddel et al., Nucleic Acids Res. 8: 4057 
(1980). 
Unless described otherwise, ligations were accomplished using standard 
buffers, incubation temperatures and times, approximately equimolar 
amounts of the DNA fragments to be ligated and approximately 10 units of 
T4 DNA ligase ("ligase") per 0.5 .mu.g of DNA. 
Example 1 
Cloning, Expression and Purification of Human ICE LAP-6 Summary 
Members of the ICE/ced-3 gene family are belived to be effector components 
of the cell death machinery. Herein this Example, a novel member of this 
family designated ICE LAP-6 is characterized. By phylogenetic analysis, 
ICE LAP-6 is classified into the Ced-3 subfamily which includes Ced-3, 
Yama/CPP32/apopain, Mch2 and ICE LAP-3/ Mch3/CMH-1. ICE LAP-6 contains an 
active site QACGG (SEQ ID NO:11) pentapeptide, rather than the QACRG (SEQ 
ID NO:10) pentapeptide shared by other family members. Overexpression of 
ICE LAP-6 induces apoptosis in MCF7 breast carcinoma cells. ICE LAP-6 is 
also proteolytically processed into an active cysteine protease by 
granzyme B, an important component of cytotoxic T cell-mediated apoptosis. 
Once activated, ICE LAP-6 is able to cleave the death substrate poly 
(ADP-ribose) polymerase (P) into signature apoptotic fragments. 
Overexpression of ICE LAP-6 in MCF7 breast carcinoma cells induces cell 
death and mutation of the putative catalytic cysteine residue abolishes 
its apoptotic potential. Furthermore, granzyme B directly activates ICE 
LAP-6 and Yama in vitro, suggesting that granzyme B may mediate its 
cytotoxic effect via activation of several ICE/Ced-3 family members. Once 
activated, Yama and ICE LAP-6 are both able to cleave the DNA repair 
enzyme poly (ADP-ribose) polymerase (P) into signature apoptotic 
fragments. Taken together, these results indicate that ICE LAP-6, like 
other members of the Ced-3 subfamily, likely plays an important role in 
the apoptotic mechanism. 
Cloning of ICE LAP-6 
A cDNA corresponding to the partial open reading frame of ICE LAP-6 was 
identified as a sequence homologous to ICE LAP-3 (Duan, H., et al. (1996) 
J. Biol. Chem. 271, 35013-35035) on searching database comprising ESTs 
made by established EST methods (Adams, M. D., et al. (1991) Science 252, 
1651-1656; Adams, M. D., et al (1992) Nature 355, 632-634). A novel cDNA 
clone, encoding a partial open reading frame, was identified and showed 
sequence homology with members of the ICE/ced-3 gene family. Of 22 
positive clones, 6 clones yielded a 2.3 kb cDNA containing an 1252-base 
pair open reading frame that encoded a novel protein with a predicted 
molecular weight of 45.8 kD, designated ICE LAP-6 (see FIG. 1). The 
putative initiator methionine (GCCATGG; Met codon underlined) was in 
agreement with the consensus Kozak's sequence for translation initiation 
(Kozak, M. (1989) J Cell Biol 108, 229-241). This clone contains an open 
reading frame encoding the C-terminal 300 amino acids of ICE LAP-6. Full 
length cDNAs were obtained by screening an oligo-d(T) primed cDNA library 
of the human chronic myelogenous leukemia cell line K562. Approximately, 
1.times.10.sup.6 transformants were screened with a .sup.32 P-labeled DNA 
fragment generated by PCR, corresponding to nucleotides 615 to 940 of the 
ICE LAP-6 open reading frame (Sambrook, J., et al (1989) Molecular 
Cloning: A Laboratory Manual, Second Edition, Cold spring Harbor 
Laboratory Press, New York). Double-stranded DNA sequencing was carried 
out by the dideoxy chain termination method using modified T7 DNA 
polymerase (Sequenase, United States Biochemical Corporation). Sequence 
alignments were performed using DNASTAR Megalign software. 
Northern Blot 
Analyses of Adult and fetal human multiple tissue Northern blots (Clontech) 
containing 2 .mu.g/lane poly(A).sup.+ RNA were hybridized, according to 
the manufacturer's instructions, using the same .sup.32 P-labeled ICE 
LAP-6 probe used for library screening. 
Expression Vectors 
The DNA inserts encoding the C-terminal FLAG-tagged (ICE LAP-6 flag) or 
His6-tagged (ICE LAP-6 His) ICE LAP-6 were generated by PCR and subcloned 
into the mammalian expression vector pcDNA3 (Invitrogen). The 5' PCR 
primer (GAACGGGGTACCGCCATGGACGAAGCGGATCGGC) [SEQUENCE ID NO.5] contained a 
Kpn1 restriction site and the two 3' primers 
(TGCTCTAGATTACTTGTCATCGTCGTCCTTGTAGTCTGATGTTTTAAAGT TAAGTTTTTTCCGGAG) 
[SEQUENCE ID NO. 9] or (TGCTCTAGATTAGTGGTGGTGGTGGTGGTGTGATGTTTTAAAGAAAAGT 
TTTTTCCGGAG) [SEQUENCE ID NO. 6] encoded a FLAG epitope tag (DYKDDDDK) or 
a His6 tag, respectively. Alteration of the active site cysteine 286 to an 
alanine was accomplished by site-directed mutagenesis employing a 
four-primer PCR-based method (Higuchi, R., et al (1988) Nucleic Acids 
Research 16, 7351-7367). The mutagenetic oligonucleotides were 
AAGCTCTTTTTCATCCAGGCCGCGGGTGGGGAGCA GAAGAC [SEQUENCE ID NO. 7] and 
GTCTTTCTGCTCCCCACCCGCGGCCTGGATGAAAAAAGC [SEQUENCE ID NO. 8]. The presence 
of the introduced mutation and fidelity of PCR replication were confirmed 
by sequence analysis. 
Apoptosis Assays 
MCF7 breast carcinoma cells were transiently transfected as described 
previously (Chinnaiyan, A. M., et al (1995) Cell 81, 505-512). Briefly, 
2.5.times.10.ident.MCF7 cells were transfected with 0.25 .mu.g of the 
reporter plasmid pCMV .beta.-galactosidase plus 1 .mu.g of test plasmid in 
6-well tissue culture dishes using lipofectamine as per manufacturer's 
instructions. The transfection was carried out in 1 ml of Opti-MEM Minimal 
Media (GIBCO-BRL) and after 5 hours, 1 ml of serum-containing growth media 
was added. Two days later, the cells were fixed with 0.5% glutaraldehyde 
and stained with X-gal for 4 hours. Cells were visualized by 
phase-contrast microscopy. At least 300 .beta.-galactosidase-positive 
cells were counted for each transfection (n=3) and identified as apoptotic 
or nonapoptotic based on morphological alterations typical of adherent 
cells undergoing apoptosis including becoming rounded, condensed, and 
detaching from the dish (Cohen, J. J. (1993) Immunology Today 14, 
126-130). Expression and Purification of His6-Tagged Yama and His6-tagged 
ICE LAP-6 and .sup.35 S-labeled Yama and ICE LAP-6 proteins were generated 
by in vitro transcription/translation using the TNT kit (Promega) 
according to the instructions of the manufacturer; the template plasmids 
were ICE LAP-6 His and Yama His (Tewari, M., et al. (1995) Cell 81, 
801-809). The translated proteins were purified by chromatography as 
described previously (Tewari, M., et al. (1995) Cell 81, 801-809). 
Activation of ICE LAP-6 and Yama by Granzyme B-Purified in 
vitro-translated pro-ICE LAP-6 or pro-Yama was activated by incubation 
with granzyme B as described previously (Quan, L. T., et al (1996) PNAS 
93, In Press). Briefly, 48 ml of .sup.35 S-labeled protein was incubated 
with 20 pmole of purified granzyme B (22) in a total volume of 50 .mu.l. 
After 4 hours, 20 ml of reaction was removed for SDS-PAGE analysis. 520 
pmole of anti-GraB (Quan, L. T., et al (1996) PNAS 93, In Press) was added 
to the rest of the reaction mix to neutralize granzyme B activity. 
Following a 15 min incubation, 1 ml (150 mg) of purified P Tewari, M., 
et al. (1995) Cell 81, 801-809) was added and the reaction was allowed to 
proceed for 2 hours. The control reaction containing P alone or P 
plus granzyme B and anti-GraB was carried out under identical conditions, 
except that Yama or ICE LAP-6 was not added. The reaction buffer contained 
50 mM Hepes (pH 7.4), 0.1 M NaCi, 0.1% CHAPs, and 10% sucrose. All 
incubations were carried out at 37.degree. C. in 10 mM DTT. Samples were 
analyzed by immunoblotting with anti-P monoclonal antibody C-2-10 as 
described previously Tewari, M., et al. (1995) Cell 81, 801-809). 
ICE LAP-6 is a Novel Member of the ICE/ced-3 Gene Family 
A blast search of GenBank protein data base revealed that the predicted 
protein sequence of ICE LAP-6 has significant similarity to the members of 
the ICE/Ced-3 family, particularly in the regions corresponding to the 
active subunits of ICE (Thomberry, N. A.,et al (1992) Nature 356, 
768-774). In this region, ICE LAP-6 shares 31% sequence identity (55% 
sequence similarity) with the C. elegans CED-3 protein, 33% identity (52% 
sequence similarity) with ICE-LAP3, 30% identity (56% similarity) with 
Mch2a and 29% sequence identity (52% similarity) with Yama. ICE LAP-6 also 
has 25% -28% sequence identity with ICE and the ICE-related genes, ICE rel 
II and ICE rel III. Phylogenetic analysis of the ICE/ced-3 gene family 
showed that ICE LAP-6 is a member of the Ced-3 subfamily which includes 
Yama, ICE-LAP3, and Mch2 (FIG. 5). Like Ced-3, ICE LAP-6 contains a long 
N-terminal putative prodomain. Based on the x-ray crystal structure of ICE 
(Walker, N. P. C. et al, (1994) Cell 78, 343-352; Wilson, K. P., et al 
(1994) Nature 370, 270-275), the amino acid residues His237, Gly238, 
Cys285 of ICE are involved in catalysis, while the residues Argl79, Gln283 
and Arg341 form a binding pocket for the carboxylate side chain of the P1 
aspartic acid. These six residues are conserved in all ICE/Ced-3 family 
members thus far cloned as well as in ICE LAP-6. However, residues that 
form the P2-P4 binding pockets are not widely conserved among family 
members, suggesting that they may determine substrate specificity. 
Surprisingly, ICE LAP-6 contains a unique active site pentapeptide QACGG 
(SEQ ID No:11), instead of the QACRG (SEQ ID NO:10) shared by other family 
members. 
Distribution of ICE LAP-6 
Northern blot analysis revealed that ICE LAP-6 is constitutively expressed 
in a variety of human tissues. Two ICE LAP-6 mRNA transcripts were 
detected. The 2.3 kilobase transcript corresponds to the size of the cDNA 
clones isolated from the K562 library. The other transcript, which is 
approximately 3 kb, is believed to represent an alternatively spliced ICE 
LAP-6 isoform. 
Overexpression of ICE LAP-6 in MCF7 Cells Induces Apoptosis 
To study the functional role of ICE LAP-6, MCF7 breast carcinoma cells were 
transiently transfected with an expression vector encoding the full-length 
ICE LAP-6 protein (ICE LAP-6-flag) and subsequently assessed for apoptotic 
features. Like the other ICE/Ced-3 family members, expression of ICE LAP-6 
caused cell death (FIG. 6). The ICE LAP-6-transfected MCF7 cells displayed 
morphological alterations typical of adherent cells undergoing apoptosis, 
becoming rounded, condensed, and detaching from the dish. ICE LAP-6 
induced apoptosis was inhibited by the broad spectrum ICE inhibitor z-VAD 
fmk (Pronk, G. J., (1996) Science 271, 808-810). To determine whether the 
amino acid residue Cys286, corresponding to the catalytic Cys285 of ICE, 
was essential for apoptotic activity, a mutant form of ICE LAP-6 was 
generated in which the cysteine residue was altered to an alanine. MCF7 
breast carcinoma cells were transiently transfected with the reporter gene 
b-galactosidase and either C-terminal flag-tagged ICE LAP-6, the mutant 
version with the catalytic cysteine residue inactivated (ICE LAP-6 mt) or 
ICE as described elsewhere herein. Percent apoptotic cells represents the 
mean value from three independent experiments. As predicted, 
overexpression of the mutant form of ICE LAP-6 did not induce apoptotic 
changes in MCF7 cells (FIG. 7). Furthermore, these results demonstrate 
that an ICE/Ced-3 family member containing an active site QACGG (SEQ ID 
NO:11) pentapeptide (rather than QACRG (SEQ ID NO:10)) may still possess 
apoptosis-inducing potential and presumably enzymatic activity. 
Proteolytic Activation of ICE LAP-6 by Granzyme B 
Members of the ICE/ced-3 gene family are synthesized as proenzymes and 
activated by proteolytic cleavage at specific aspartate residues to form 
heterodimeric enzymes. In ICE, this cleavage removes the prodomain and 
produces a heterodimeric complex consisting of p20 and p10subunits 
(Thornberry, N. A.,et al (1992) Nature 356, 768-774). Similarly, activated 
Yama is comprised of two subunits, pl7 and pl2, which are derived from a 
32 kDa proenzyme Nicholson, D. Wet al. (1995) Nature 376, 37-43). The 
mechanism by which death signals activate ICE/Ced-3 family members is 
poorly understood. Recent studies on granzyme B, however, suggest that 
cytotoxic T cells may utilize this secreted serine protease to directly 
activate members of the ICE/Ced-3 family. It has been demonstrated that 
granzyme B can proteolytically activate pro-Yama, generating an active 
enzyme capable of cleaving the death substrate P into characteristic 
fragments (Darmon, A. J., et al (1995) Nature 377, 446-448). By contrast, 
ICE, although cleaved by granzyme B, fails to be activated. Thus, it was 
determined whether ICE LAP-6 can serve as a substrate for granzyme B. His6 
tagged ICE LAP-6 and Yama were generated by in vitro 
transcription/translation, and subsequently purified by Ni-affinity 
chromatography as described elsewhere herein. The purified in 
vitro-translated pro-ICE LAP-6 or pro-Yama was incubated with purified 
granzyme B (Hanna, W. L., et al (1993) Protein Expr Purif 4,398-404; Quan, 
L. T., et al (1995) Journal of Biological Chemistry 270, 10377-10379). 
After 4 hours at 37.degree. C., ICE LAP-6 was proteolytically processed 
into 3 fragments. The two low molecular weight bands represent the active 
subunits of ICE LAP-6 and correspond to the p17 and p12 subunits of active 
Yama. The 32 kDa band is an likely intermediate, in which only the 
pro-domain is removed (a similar intermediate is generated in the 
activation of ICE LAP-3) Duan, H., et al. (1996) J. Biol. Chem. 271, 
35013-35035; Chinnaiyan, A. M., et al. 1996) Journal of Biological 
Chemistry 271, 4573-4576). Next, granzyme B-mediated cleavage of ICE LAP-6 
was assessed for generation of an active enzyme by assaying for P 
cleavage. P is proteolyzed during many forms of apoptosis, and the 
enzyme(s) responsible is likely of the ICE/Ced-3 family. To exclude the 
possibility of direct cleavage of P by granzyme B, granzyme B-processed 
ICE LAP-6 and Yama were incubated with a selective inhibitor of granzyme B 
(anti-GraB). Both granzyme B-processed Yama and ICE LAP-6 were active as 
determined by their ability to cleave P. Unlike ICE, ICE LAP-6 and 
other members of the Ced-3 subfamily are able to cleave the P into 
signature apoptotic fragments (Tewari, M., et al. (1995) Cell 81, 801-809; 
Fernandes-Alnemri, T., et al. (1994) J. Biol. Chem. 269, 30761-30764- 
Nicholson, D. Wet al. (1995) Nature 376, 37-43; Fernandes-Alnemri, T., et 
al. (1995) Cancer Research 55, 6045-6052; Lippke, J. A., et al. (1996) The 
Journal of Biological Chemistry 271, 1825-1828; Fernandes-Alnemri, T., et 
al. (1995) Cancer Res 55, 2737-2742). 
Provided by the present invention is a novel member of the ICE/Ced-3 family 
of cysteine proteases. ICE LAP-6 has a unique active site QACGG (SEQ ID 
NO:11) pentapeptide and is classified in the subfamily most related to 
Ced-3 and Yama. Ectopic expression of ICE LAP-6 in mammalian cells causes 
apoptosis. 
Importantly, ICE LAP-6, like Yama, was directly activated by granzyme B in 
vitro, suggesting that cytotoxic T cells may mediate apoptosis by 
activating more than one ICE/Ced-3 family member in susceptible target 
cells. Yama, ICE-LAP3, and now ICE LAP-6, have been shown to be 
proteolytically activated by apoptotic stimuli. 
Example 2 
Gene Therapeutic Expression of Human ICE LAP-6 
Fibroblasts are obtained from a subject by skin biopsy. The resulting 
tissue is placed in tissue-culture medium and separated into small pieces. 
Small chunks of the tissue are placed on a wet surface of a tissue culture 
flask, approximately ten pieces are placed in each flask. The flask is 
turned upside down, closed tight and left at room temperature overnight. 
After 24 hours at room temperature, the flask is inverted--the chunks of 
tissue remain fixed to the bottom of the flask--and fresh media is added 
(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin). The 
tissue is then incubated at 37.degree. C. for approximately one week. At 
this time, fresh media is added and subsequently changed every several 
days. After an additional two weeks in culture, a monolayer of fibroblasts 
emerges. The monolayer is trypsinized and scaled into larger flasks. 
A vector for gene therapy is digested with restriction enzymes for cloning 
a fragment to be expressed. The digested vector is treated with calf 
intestinal phosphatase to prevent self-ligation. The dephosphorylated, 
linear vector is fractionated on an agarose gel and purified. 
ICE LAP-6 cDNA capable of expressing active ICE LAP-6, is isolated. The 
ends of the fragment are modified, if necessary, for cloning into the 
vector. For instance, 5' overhanging may be treated with DNA polymerase to 
create blunt ends. 3' overhanging ends may be removed using S1 nuclease. 
Linkers may be ligated to blunt ends with T4 DNA ligase. 
Equal quantities of the Moloney murine leukemia virus linear backbone and 
the ICE LAP-6 fragment are mixed together and joined using T4 DNA ligase. 
The ligation mixture is used to transform E. coli and the bacteria are 
then plated onto agar-containing kanamycin. Kanamycin phenotype and 
restriction analysis confirm that the vector has the properly inserted 
gene. 
Packaging cells are grown in tissue culture to confluent density in 
Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), 
penicillin and streptomycin. The vector containing the ICE LAP-6 gene is 
introduced into the packaging cells by standard techniques. Infectious 
viral particles containing the ICE LAP-6 gene are collected from the 
packaging cells, which now are called producer cells. 
Fresh media is added to the producer cells, and after an appropriate 
incubation period media is harvested from the plates of confluent producer 
cells. The media, containing the infectious viral particles, is filtered 
through a Millipore filter to remove detached producer cells. The filtered 
media then is used to infect fibroblast cells. Media is removed from a 
sub-confluent plate of fibroblasts and quickly replaced with the filtered 
media. Polybrene (Aldrich) may be included in the media to facilitate 
transduction. After appropriate incubation, the media is removed and 
replaced with fresh media. If the titer of virus is high, then virtually 
all fibroblasts will be infected and no selection is required. If the 
titer is low, then it is necessary to use a retroviral vector that has a 
selectable marker, such as neo or his, to select out transduced cells for 
expansion. 
Engineered fibroblasts then may be injected into rats, either alone or 
after having been grown to confluence on microcarrier beads, such as 
cytodex 3 beads. The injected fibroblasts produce ICE LAP-6 product, and 
the biological actions of the protein are conveyed to the host. 
It will be clear that the invention may be practiced otherwise than as 
particularly described in the foregoing description and examples. 
Numerous modifications and variations of the present invention are possible 
in light of the above teachings and, therefore, are within the scope of 
the appended claims. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 11 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 416 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
#Arg Cys Arg Leu Arg Leurg Arg Leu Leu Arg 
# 15 
#Asp Val Leu Leu Ser Argal Asp Gln Leu Trp 
# 30 
#Ile Gln Arg Ala Gly Seris Met Ile Glu Asp 
# 45 
#Ile Ile Asp Leu Glu Thrln Ala Arg Gln Leu 
# 60 
#Ser Cys Leu Glu Asp Threu Pro Leu Phe Ile 
# 80 
#Thr Asn Arg Gln Ala Glyla Ser Phe Leu Arg 
# 95 
#Thr Pro Val Val Leu Arghr Leu Glu Asn Leu 
# 110 
#Pro Glu Thr Pro Arg Proro Glu Val Leu Arg 
# 125 
#Val Gly Ala Leu Glu Serly Gly Phe Gly Asp 
# 140 
#Leu Ser Met Glu Pro Cyssp Leu Ala Tyr Ile 
#160 
#Phe Cys Arg Glu Ser Glyle Asn Asn Val Asn 
# 175 
#Cys Glu Lys Leu Arg Argly Ser Asn Ile Asp 
# 190 
#Val Lys Gly Asp Leu Thris Phe Met Val Glu 
# 205 
#Leu Ala Arg Gln Asp Hiseu Ala Leu Leu Glu 
# 220 
#Leu Ser His Gly Cys Glnys Val Val Val Ile 
#240 
#Tyr Gly Thr Asp Gly Cyshe Pro Gly Ala Val 
# 255 
#Phe Asn Gly Thr Ser Cysys Ile Val Asn Ile 
# 270 
#Phe Ile Gln Ala Cys Glyys Pro Lys Leu Phe 
# 285 
#Ala Ser Thr Ser Pro Gluis Gly Phe Glu Val 
# 300 
#Asp Ala Thr Pro Phe Glner Asn Pro Glu Pro 
#320 
#Ala Ile Ser Ser Leu Prohe Asp Gln Leu Asp 
# 335 
#Thr Phe Pro Gly Phe Valhe Val Ser Tyr Ser 
# 350 
#Tyr Val Glu Thr Leu Aspys Ser Gly Ser Trp 
# 365 
#Asp Leu Gln Ser Leu Leurp Ala His Ser Glu 
# 380 
#Gly Ile Tyr Lys Gln Metla Val Ser Val Lys 
#400 
#Leu Phe Phe Lys Thr Serhe Leu Arg Lys Lys 
# 415 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1578 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- GCCATGGACG AAGCGGATCG GCGGCTCCTG CGGCGGTGCC GGCTGCGGCT GG - #TGGAAGAG 
60 
- CTGCAGGTGG ACCAGCTCTG GGACGTCCTG CTGAGCCGCG AGCTGTTCAG GC - #CCCATATG 
120 
- ATCGAGGACA TCCAGCGGGC AGGCTCTGGA TCTCGGCGGG ATCAGGCCAG GC - #AGCTGATC 
180 
- ATAGATCTGG AGACTCGAGG GAGTCAGGCT CTTCCTTTGT TCATCTCCTG CT - #TAGAGGAC 
240 
- ACAGGCCAGG ACATGCTGGC TTCGTTTCTG CGAACTAACA GGCAAGCAGG AA - #AGTTGTCG 
300 
- AAGCCAACCC TAGAAAACCT TACCCCAGTG GTGCTCAGAC CAGAGATTCG CA - #AACCAGAG 
360 
- GTTCTCAGAC CGGAAACACC CAGACCAGTG GACATTGGTT CTGGAGGATT CG - #GTGATGTC 
420 
- GGTGCTCTTG AGAGTTTGAG GGGAAATGCA GATTTGGCTT ACATCCTGAG CA - #TGGAGCCC 
480 
- TGTGGCCACT GCCTCATTAT CAACAATGTG AACTTCTGCC GTGAGTCCGG GC - #TCCGCACC 
540 
- CGCACTGGCT CCAACATCGA CTGTGAGAAG TTGCGGCGTC GCTTCTCCTC GC - #TGCATTTC 
600 
- ATGGTGGAGG TGAAGGGCGA CCTGACTGCC AAGAAAATGG TGCTGGCTTT GC - #TGGAGCTG 
660 
- GCGCGGCAGG ACCACGGTGC TCTGGACTGC TGCGTGGTGG TCATTCTCTC TC - #ACGGCTGT 
720 
- CAGGCCAGCC ACCTGCAGTT CCCAGGGGCT GTCTACGGCA CAGATGGATG CC - #CTGTGTCG 
780 
- GTCGAGAAGA TTGTGAACAT CTTCAATGGG ACCAGCTGCC CCAGCCTGGG AG - #GGAAGCCC 
840 
- AAGCTCTTTT TCATCCAGGC CTGTGGTGGG GAGCAGAAAG ACCATGGGTT TG - #AGGTGGCC 
900 
- TCCACTTCCC CTGAAGACGA GTCCCCTGGC AGTAACCCCG AGCCAGATGC CA - #CCCCGTTC 
960 
- CAGGAAGGTT TGAGGACCTT CGACCAGCTG GACGCCATAT CTAGTTTGCC CA - #CACCCAGT 
1020 
- GACATCTTTG TGTCCTACTC TACTTTCCCA GGTTTTGTTT CCTGGAGGGA CC - #CCAAGAGT 
1080 
- GGCTCCTGGT ACGTTGAGAC CCTGGACGAC ATCTTTGAGC AGTGGGCTCA CT - #CTGAAGAC 
1140 
- CTGCAGTCCC TCCTGCTTAG GGTCGCTAAT GCTGTTTCGG TGAAAGGGAT TT - #ATAAACAG 
1200 
- ATGCCTGGTT GCTTTAATTT CCTCCGGAAA AAACTTTTCT TTAAAACATC AT - #AAGGCCAG 
1260 
- GGCCCCTCAC CCTGCCTTAT CTTGCACCCC AAAGCTTTCC TGCCCCAGGC CT - #GAAAGAGG 
1320 
- CTGAGGCCTG GACTTTCCTG CAACTCAAGG ACTTTGNAGC CGGCACAGGG TC - #TGCTCTTT 
1380 
- CTCTGCCAGT GACAGACAGG CTCTTAGCAG CTTCCAGATT GACGACAAGT GC - #TGAACAGT 
1440 
- GGAGGAAGAG GGACAGATGA ATGCCGTGGA TTGCACGTGG NCTCTTGAGC AG - #TGGCTGGT 
1500 
- CCAGGGCTAG TGACTTGGTG TCCCATGATC CCTGTGTTGG TCTCTAGGAG CA - #GGGATTAA 
1560 
#1578 AT 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 639 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- CTGACTGCCA AGAAAATGGT GCTGGCTTTG CTGGAGCTGG CGCGGCAGGA CC - #ACGGTGCT 
60 
- CTGGACTGCT GCGTGGTGGT CATTCTCTCT CACGGCTGTC AGGCCAGCCA CC - #TGCAGTTC 
120 
- CCAGGGGCTG TCTACGGCAC AGATGGATGC CCTGTGTCGG TCGAAAAGAT TG - #TGAACATC 
180 
- TTCAATGGGA CCAGCTGCCC CAGCCTGGGA GGGAAGCCCA AGCTCTTTTT CA - #TCCAGGCC 
240 
- TGTGGTGGGG AGCAGAAAGA CCATGGGTTT GAGGTGGCCT CCACTTCCCC TG - #AAGACGAG 
300 
- TCCCCTGGCA GTAACCCCGA GCCAGATGCC ACCCCGTTCC AGGAAGGTTT GA - #GGACCTTC 
360 
- GACCAGCTGG ACGCCATATC TAGTTTGCCC ACACCCAGTG ACATCTTTGT GT - #CCTACTCT 
420 
- ACTTTCCCAG GTTTTGTTTC CTGGAGGGAC CCCAAGAGTG GCTCCTGGTA CG - #TTGAGACC 
480 
- CTGGACGACA TCTTTGAGCA GTGGGCTCAC TCTGAAGACC TGCAGTCCCT CC - #TGCTTAGG 
540 
- GTCGCTAATG CTGTTTCGGT GAAAGGGATT TATAAACAGA TGCCTGGTTG CT - #TTAATTTC 
600 
# 639 TCTT TTAAAACATC ATAAGGCAG 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 203 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
#Gln Asp His Gly Ala Leueu Glu Leu Ala Arg 
# 15 
#Gly Cys Gln Ala Ser Hisal Ile Leu Ser His 
# 30 
#Asp Gly Cys Pro Val Serla Val Tyr Gly Thr 
# 45 
#Thr Ser Cys Pro Ser Leusn Ile Phe Asn Gly 
# 60 
#Ala Cys Gly Gly Glu Glneu Phe Phe Ile Gln 
# 80 
#Ser Pro Glu Asp Glu Serlu Val Ala Ser Thr 
# 95 
#Pro Phe Gln Glu Gly Leulu Pro Asp Ala Thr 
# 110 
#Ser Leu Pro Thr Pro Sereu Asp Ala Ile Ser 
# 125 
#Gly Phe Val Ser Trp Argyr Ser Thr Phe Pro 
# 140 
#Thr Leu Asp Asp Ile Pheer Trp Tyr Val Glu 
#160 
#Ser Leu Leu Leu Arg Valer Glu Asp Leu Gln 
# 175 
#Lys Gln Met Pro Gly Cysal Lys Gly Ile Tyr 
# 190 
#Methe Asn Phe Leu Arg Lys Lys Leu Phe Phe 
# 200 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 34 TGGA CGAAGCGGAT CGGC 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 60 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- TGCTCTAGAT TAGTGGTGGT GGTGGTGGTG TGATGTTTTA AAGAAAAGTT TT - #TTCCGGAG 
60 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 41 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
# 41 AGGC CGCGGGTGGG GAGCAGAAGA C 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 39 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
# 39 CCCG CGGCCTGGAT GAAAAAAGC 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 66 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- TGCTCTAGAT TACTTGTCAT CGTCGTCCTT GTAGTCTGAT GTTTTAAAGT TA - #AGTTTTTT 
60 
# 66 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 5 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- Gln Ala Cys Arg Gly 
1 5 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 5 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- Gln Ala Cys Gly Gly 
1 5 
__________________________________________________________________________