Note: Descriptions are shown in the official language in which they were submitted.
CA 022~906 1998-11-19
WO 97/44468 PCT/US97/07282
HUMAN rNlERLEUKTN-lj AND ANTAGON~STS THEREOF
FIELD OF THE INVENTION
The present invention relates to compositions and
~ methods for affecting the human immune system. In
particular, it provides nucleic acids, proteins, and
antibodies and other antagonists which regulate immune
system response and development. Diagnostic and
therapeutic uses of these materials are also disclosed.
BACKGROUND OF THE INVENTION
Recombinant DNA technology refers generally to the
techni~ue of integrating genetic information from a donor
source into vectors for subsequent processing, such as
through introduction into a host, whereby the transferred
genetic information is copied and/or expressed in the new
environment. Commonly, the genetic information exists in
the form of complementary DNA (cDNA) derived from
messenger RNA (mRNA) coding for a desired protein
product. The carrier is frequently a plasmid having the
capacity to incorporate cDNA for later replication in a
host and, in some cases, actually to control expression
of the cDNA and thereby direct synthesis of the encoded
product in the host.
For some time, it has been known that the mammalian
immune response is based on a series of complex cellular
interactions, called the "immune network". Recent
research has provided new insights into the inner
workings of this network. While it remains clear that
much of the response does, in fact, revolve around the
~ network-like interactions of lymphocytes, macrophages,
granulocytes, and other cells, immunologists now
generally hold the opinion that soluble proteins, known
as lymphokines, cytokines, or monokines, play a critical
role in controlling these cellular interactions.
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Thus, there is considerable interest in the
isolation, characterization, and mechanisms of action of
cell modulatory factors, an understanding of which will
lead to significant advancements in the diagnosis and
therapy of numerous medical abnormalities, e.g., immune
system disorders.
Lymphokines apparently mediate cellular activities
in a variety of ways. They have been shown to support
the proliferation, growth, and differentiation of
pluripotential hematopoietic stem cells into vast numbers
of progenitors comprising diverse cellular lineages
making up a complex immune system. Proper and balanced
interactions between the cellular components are
necessary for a healthy immune response. The different
cellular lineages often respond in a different manner
when lymphokines are administered in coniunction with
other agents.
Cell lineages especially important to the immune
response include two classes of lymphocytes: B-cells,
which can produce and secrete immunoglobulins (proteins
with the capability of recognizing and binding to foreign
matter to effect its removal), and T-cells of various
subsets that secrete lymphokines and induce or suppress
the B-cells and various other cells (including other
T-cells) making up the immune network. These lymphocytes
interact with many other cell types.
Another important cell lineage is the mast cell
(which has not been positively identified in all
m~mm~lian species), which is a granule-containing
connective tissue cell located proximal to capillaries
throughout the body. These cells are found in especially
high concentrations in the lungs, skin, and
gastrointestinal and genitourinary tracts. Mast cells
play a central role in allergy-related disorders,
particularly anaphylaxis as follows: when selected
antigens crosslink one class of immunoglobulins bound to
receptors on the mast cell surface, the mast cell
degranulates and releases mediators,e.g., histamine,
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serotonin, heparin, and prostaglandins, which cause
allergic reactions, e.g., anaphylaxis.
Research to better understand and treat various
immune disorders has been hampered by the general
inability to maintain cells of the immune system
in vitro. Immunologists have discovered that culturing
these cells can be accomplished through the use of T-cell
and other cell supernatants, which contain various growth
factors, including many of the lymphokines.
Okamura, et al . (1995) Nature 378:88-91 describes a
new cytokine that induces certain T cells to produce
interferon gamma (IFN-~), the cytokine being designated
IGIF. The factor has been identified in mouse Kupffer
cells and activated macrophages. No human equivalent had
been described until now.
From the foregoing, it is evident that the discovery
and development of new lymphokines could contribute to
new therapies for a wide range of degenerative or
abnormal conditions which directly or indirectly involve
the immune system and/or hematopoietic cells. In
particular, the discovery and development of lymphokines
which enhance or potentiate the beneficial activities of
known lymphokines would be highly advantageous. The
present invention provides new interleukin compositions
and related compounds, and methods for their use.
SUMMARY OF THE INVENTION
The present invention is directed to primate, e.g.,
human, interleukin-1~ (IL-1~) and its biological
activities. It includes nucleic acids coding for
polypeptides themselves and methods for their production
and use. The nucleic acids of the invention are
characterized, in part, by their homology to cloned
complementary DNA (cDNA) se~uences enclosed herein,
and/or by functional assays for IL-1~ activity applied to
the polypeptides, which are typically encoded by these
nucleic acids. Methods for modulating or intervening in
~ the control of an immune response are provided.
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The invention is based, in part, on the discovery
and cloning of human cDNAs which are capable of
expressing proteins having IL-l~ activity. Equivalent
vectors may be constructed by using polymerase chain
reaction (PCR) techniques and the sequences of the
inserts.
The invention provides inter alia a substantially
pure primate IL-l~, a fusion protein comprising primate
IL-l~ sequence, an antibody specific for binding to a
primate IL-l~, and a nucleic acid encoding a human IL-l~
or fusion protein thereof.
In the substantially pure IL-l~ embodiment, the
IL-l~ may comprise a mature sequence contained within
the amino acid sequence defined by SEQ ID NO: 2. The
latter sequence contains a signal sequence which is not a
typical signal sequence but which, like a prosequence
analogous to the IL-lb prodomain that is cleaved by a
convertase-like enzyme, is likely to run from the amino
acid position 1 (met) to about 36 (asp). See Dinarello
(1994) FASEB J. 1314-1325. The mature protein should
begin at about 37 (tyr). Alternativley, the IL-l~ may
exhibit a post-translational modification pattern
distinct from that of natural IL-l~. In another
embodiment, the composition will comprise the primate
IL-l~ and a pharmaceutically acceptable carrier.
Generally, the IL-l~ can induce production of IFN-~ by a
T cell or NK cell, alone or in combination with IL-12 or
IL-2.
Fusion proteins of the invention comprise IL-l~ or a
substantial fragment thereof covalently linked to a
binding partner. In one fusion protein embodiment, the
protein may comprise a sequence of SEQ ID NO: 2 and/or
sequence of another cytokine or chemokine. Such protein
may comprise a modification in sequence corresponding to
3~ an amino acid residue of SEQ ID NO: 2 at positions 88
(tyr) to 96 (met); or may exhibit IL-l~ agonist activity,
and comprise a substitution in sequence corresponding to
an amino acid residue of SEQ ID NO: 2 at positions
37 (tyr) to 82 (ile) or 102 (val) to 191 (asn).
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W097/44468 5 PCT~S97/07282
In another fusion protein embodiment the
pharmacokinetic half-life of the IL-l~ or fragment
thereof is increased by conjugation to another
polypeptide, e.g., to part of an immunoglobulin (Ig)
chain, preferably a constant region ~Fc), or to a
polyethylene glycol (PEG) molecule(s), sometimes referred
to as pegylation. Such fusion proteins may be referred
to as IL-l~-Ig and PEG-IL-l~, respectively. Methods for
linking polyethylene glycol (PEG) groups and Ig chains,
parts thereof or other polypeptides to proteins are well
known in the art.
See, e.g., International Application Publication
No. WO 96/18412, which describes the fusion of
polypeptides derived from immunoglobulin chains to a
variety of cytokines to increase the circulating
half-life of the cytokines. Methods for conjugating PEG
to proteins have been described, e.g., by Davis et al .
~U.S. Patent No. 4,179,337), Nakagawa et al . (U. S . Patent
No. 4,791,192), and Nitecki et al . (U . S . Patent No.
4,902,502).
In certain antibody embodiments, the IL-ly is a
human protein; the antibody is raised against a peptide
sequence of SEQ ID NO: 2; the antibody is raised to a
purified primate IL-l~; the antibody is a monoclonal
antibody; or the antibody is labeled.
In various nucleic acid embodiemnts, the IL-l~ is
from a human; the nucleic acid (e.g., SEQ ID NO: 1)
encodes a peptide sequence of SEQ ID NO: 2; the nucleic
acid is an expression vector; or the nucleic acid
comprises a deoxyribonucleic acid nucleotide.
The present invention also embraces a kit
comprising: a substantially pure primate IL-l~, or
fragment thereof; an antibody which specifically binds a
primate IL-l~; or a nucleic acid encoding a human I~ or
peptide. Various of these kits will be capable of making
a qualitative or quantitative analysis.
Another embodiment of the invention includes a
method of modulating physiology or development of a cell
comprising contacting the cell, or an immune system
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W097/44468 6 PCT~S97/07282
containing the cell, with an agonist or antagonist of a
primate IL-1~. This includes methods where the
contacting is in combination with IL-2 and/or IL-12.
Alternatively, the invention provides a method where the
contacting is with an antagonist, e.g., an antibody
against a primate IL-1~, and may be combined with
antagonists to IL-2 and/or IL-12. Often, the modulating
is regulation of IFN-~ production; and includes
contacting is with an agonist of a human IL-1~. In other
embodiments, the modulating is regulation of: an
infectious disease; a vaccine response; an allergic
reaction; a T helper mediated response; or a cancer.
DESCRIPTION OF THE INVENTION
All references cited herein are hereby incorporated
in their entirety by reference.
The present invention provides the amino acid
sequence and DNA sequence of human interleukin molecules
having particular defined properties, both structural and
biological, designated herein as human interleukin-1~
(IL-1~). A cDNA encoding this molecule was obtained from
an activated human monocyte cDNA library, designated M1.
Some of the standard methods are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular
Cloninq, A Laboratorv Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor Press; Sambrook, et al.
(1989) Molecular Cloninq: A Laboratorv Manual, (2d ed.),
vols 1-3, CSH Press, NY; Ausubel, et al., Bioloav, Greene
Publishing Associates, Brooklyn, NY; or Ausubel, et al.
(1987 and periodic supplements) Current Protocols in
Molecular BioloqY, Greene/Wiley, New York; all of which
are each incorporated herein by reference.
Isolation of the human gene solved uncertainties
which make its isolation far from certain. These
uncertainties include: (1) whether a human counterpart of
the mouse protein exists; (2) the level of similarity of
the encoding nucleic acid sequences; (3) where in the
sequence homology exists, useful in a PCR approach;
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(4) the level of expression, thereby providing a useful
source for isolating a natural cDNA gene portioni
(5) cross-species biological activity, useful, e.g., in a
context of expression cloning using mouse cells; and
~ 5 (6) immunological antibody cross reaction, important in
an antibody binding based approach. Many other problems
exist in successfully deriving a cross-species isolation.
A complete nucleotide sequence ( SEQ ID NO: 1 ) and
corresponding amino acid sequence ( SEQ ID NO: 2) of the
human IL-1~ coding segment is provided in the Sequence
Listing. The human isolate shows homology to the mouse
counterpart, about 71% identity at the nucleotide level;
and about 65% at the amino acid level.
As used herein, the term IL-1~ shall be used to
describe a protein comprising a protein or peptide
segment having the mature amino acid sequence shown in
SEQ ID NO: 2, or a substantial fragment thereof. The
invention also includes a mutein agonist or antagonist.
Typically, such agonists exhibit less than about 10%
sequence differences, and thus will often have between
1- and 11-fold substitutions. It also encompasses
allelic and other variants, e.g., natural polymorphic, of
the protein described. Typically, it will bind to its
corresponding biological receptor with high affinity,
e.g., at least about 100 nM, usually better than about
30 nM, preferably better than about 10 nM, and more
preferably at better than about 3 nM. The term shall
also be used herein to refer to related naturally
occurring forms, e.g., alleles, polymorphic variants, and
metabolic variants of the human protein.
This invention also encompasses proteins or peptides
having substantial amino acid se~uence homology with the
amino acid sequence in SEQ ID NO: 2, but excluding any
protein or peptide which exhibits substantially the same
or lesser amino acid sequence homology than does the
corresponding IGIF protein found in the mouse. It will
include sequence variants with relatively few
substitutions, e.g., preferably less than about 3-5.
-
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WO 97/44468 8 rCT/US97107282
A substantial polypeptide "fragment", or "segment",
is a stretch of amino acid residues of at least about
8 amino acids, generally at least 10 amino acids, more
generally at least 12 amino acids, often at least
14 amino acids, more often at least 16 amino acids,
typically at least 18 amino acids, more typically at
least 20 amino acids, usually at least 22 amino acids,
more usually at least 24 amino acids, preferably at least
26 amino acids, more preferably at least 28 amino acids,
and, in particularly preferred embodiments, at least
about 30 or more amino acids. Sequences of segments of
different proteins can be compared to one another over
appropriate length stretches.
Amino acid sequence homology, or sequence identity,
is determined by optimizing residue matches, if
necessary, by introducing gaps as required. See, e.g.,
Needleham, et al., (1970) J. Mol. Biol. 48:443-453;
Sankoff, et al ., (1983) chapter one in Time WarPs, Strinq
Edits, and Macromolecules: The Theorv and Practice of
Sequence Comparsion, Addison-Wesley, Reading, MA; and
software packages from IntelliGenetics, Mountain View,
CA; and the University of Wisconsin Genetics Computer
Group (GCG), Madison, WI; each of which is incorporated
herein by reference. This changes when considering
conservative substitutions as matches. Conservative
substitutions typically include substitutions within the
following groups: glycine, alanine; valine, isoleucine,
leucine; aspartic acid, glutamic acidi asparagine,
glutamine; serine, threonine; lysine, argininei and
phenylalanine, tyrosine.
Homologous amino acid sequences are intended to
include natural allelic and interspecies variations in
the cytokine sequence. Typical homologous proteins or
peptides will have from 50-100% homology (if gaps can be
introduced), to 60-100% homology (if conservative
substitutions are included) with an amino acid sequence
segment of SEQ ID NO: 2. Homology measures will be at
least about 70%, generally at least 76%, more generally
at least 81%, often at least 85%, more often at least
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W097/44468 9 PCT~S97/07282
88%, typically at least 90%, more typically at least 92%,
usually at least 94%, more usually at least 95%,
preferably at least 96%, and more preferably at least
97%, and in particularly preferred embodiments, at least
98% or more. The degree of homology will vary with the
length of the compared segments. Homologous proteins or
peptides, such as the allelic variants, will share most
biological activities with the embodiment described in
SEQ ID N0: 2. Preferably a related polypeptide will
contain a plurality of such matching fragments, e.g., at
least 2, preferably 3, 4, 5, or more of specific, or
assorted, lengths.
As used herein, the term "biological activity" is
used to describe, without limitation, synergistic
induction by splenocytes of IFN-y in combination with
IL-12 or IL-2, with or without anti-type I or anti-type
II IL-l receptor antibodies, or more structural
properties as receptor binding and cross-reactivity with
antibodies raised against the same or a polymorphic
2 0 variant of the described human IL~
The terms ligand, agonist, antagonist, and analog
include molecules that modulate the characteristic
cellular responses to IL-1~ or IL-1~-like proteins, as
well as molecules possessing the more standard structural
binding competition features of ligand-receptor
interactions, e.g., where the receptor is a natural
receptor or an antibody. The cellular responses likely
are mediated through binding of IL-l~ to cellular
receptors distinct from the type I or type II IL-l
receptors. Also, a ligand is a molecule which serves
either as a natural ligand to which said receptor, or an
analog thereof, binds, or a molecule which is a
functional analog of the natural ligand. The functional
analog may be a ligand with structural modifications, or
may be a wholly unrelated molecule which has a molecular
shape which interacts with the appropriate ligand binding
determinants. The ligands may serve as agonists or
antagonists, see, e.g., Goodman, et al. (eds) (1990)
, _
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Goodman & Gilman's: The Pharmacoloqical Bases of
Thera~eutics, Pergamon Press, New York.
Rational drug design may also be based upon
structural studies of the molecular shapes of a receptor
or antibody and other effectors or ligands. Ef~ectors
may be other proteins which mediate other functions in
response to ligand binding, or other proteins which
normally interact with the receptor. One means for
determining which sites interact with specific other
proteins is a physical structure determination, e.g.,
x-ray crystallography or 2 dimensional NMR techniques.
These will provide guidance as to which amino acid
residues form the molecular contact regions. For a
detailed description of protein structural determination,
see, e.g., Blundell and Johnson ~1976) Protein
Crystalloara~hY, Academic Press, New York.
The human IL-ly protein has a number of different
biological activities. The human IL-l~ is homologous to
the mouse IGIF protein, but has structural differences.
For example, the human IL-l~ gene coding sequence has
only about 71% homology with the nucleotide coding
se~uence of mouse IGIF. At the amino acid level, there
is about 64% identity. This level of similarity suggests
that the new IL-l~ protein is related to the other IL-la
and IL-1~.
The mouse IGIF molecule has rather m;n;m~lly defined
biological activities. In particular, it has the ability
to stimulate IFN-~ production which augments NK activity
in spleen cells. See Okamura, et al . ( 1995) Nature
378:88-91.
The activities of the mouse IL-la~ IL-l~, and IGIF
have been compared as to their ability to induce IFN-~,
alone or in combination with IL-2 or IL-12 in SCID
splenocytes and purified NK cells. ~ee Hunter, et al.
~1995) J. Immunol. 155:4347-4354; and Bancroft, et al.
~1991) Immunol. Revs. 124:5-xxx. The IGIF was found to
be much more potent in stimulating IFN-1~ than IL-l~ or
IL~
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In I~~2 activated NK cells, IFN-~ production is
blocked by the addition of anti-IL-l~ antibodies. See
Hunter, et al. (1995). However, mouse IGIF can overcome
this block and induce IFN-~. This is the only cytokine
known to be able to do this. In addition, in vivo,
administration of mouse IGIF to mice infected with the
parasite T. Cruzi significantly decreases parasitemia.
The present disclosure also describes new activities
which have been discovered using the mouse IGIF molecule.
Applicants have confirmed that the mouse IGIF molecule
produced by similar recombinant means to the human IL-l~
protein characterized herein, exhibit the biological
activity of inducing T cells to produce IFN-~. See
assays described, e.g., in de Waal Malefyt, et al ., in de
Vries and de Waal Malefyt (eds. 1995) "Interleukin-10"
Landes Co., Austin, TX. It also modestly stimulates
IFN-~ by NK cells. But, as with the human described
above, there is substantial synergy with IL-12.
Cross-species biological activity has not yet been
detected with these assays, e.g., the biological activity
of mouse IGIF on human cells has not been yet
demonstrated, nor has the activity of human IL-l~ been
demonstrated on mouse cells. This suggests that the
receptors, which are expected to include multiple
different polypeptide chains, exhibit species specificity
for their corresponding ligands. The IL-la and IL-l~
ligands both signal through heterodimeric receptors.
This invention further contemplates use of isolated
nucleic acid or fragments, e.g., which encode this or a
closely related protein, or fragments thereof, to encode
a biologically active corresponding polypeptide. In
addition, this invention covers isolated or recombinant
DNA which encodes a biologically active protein or
polypeptide having characteristic IL-l~ activity.
Typically, the nucleic acid is capable of hybridizing,
under appropriate conditions, with a nucleic acid
sequence segment of SEQ ID NO: 1. Said biologically
active protein or polypeptide can be a full length
protein, or fragment, and will typically have a segment
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of amino acid sequence highly homologous to one shown in
SEQ ID NO: 2. Further, this invention covers the use of
isolated or recombinant nucleic acid, or fragments
thereof, which encode proteins having fragments which are
homologous to the disclosed IL-l~ protein. The isolated
nucleic acids can have the respective regulatory
sequences in the 5' and 3' flanks, e.g., promoters,
enhancers, poly-A addition signals, and others from the
natural gene.
An "isolated" nucleic acid is defined herein to mean
a nucleic acid, e.g., an RNA, DNA, or a mixed polymer,
which is substantially pure, e.g., separated from other
components which naturally accompany a native sequence,
such as ribosomes, polymerases, and flanking genomic
sequences from the originating species. The term
embraces a nucleic acid sequence which has been removed
from its naturally occurring environment, and includes
recombinant or cloned DNA isolates, which are thereby
distinguishable from naturally occurring compositions,
and chemically synthesized analogs or analogs
biologically synthesized by heterologous systems. A
substantially pure molecule includes isolated forms of
the molecule, either completely or substantially pure.
An isolated nucleic acid will generally be a
homogeneous composition of molecules but will in some
embodiments contain heterogeneity, preferably minor.
This heterogeneity is typically found at the polymer ends
or portions not critical to a desired biological function
or activity.
A "recombinant" nucleic acid is defined herein
either by its method of production or its structure. In
reference to its method of production, e.g., a product
made by a process, the process is use of recombinant
nucleic acid techniques, e.g., involving human
intervention in the nucleotide sequence. Typically this
intervention involves in vitro manipulation, although
under certain circumstances it may involve more classical
~n;m~l breeding techniques. Alternatively, it can be a
nucleic acid made by generating a sequence comprising
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fusion of two fragments which are not naturally
contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring mutants as
found in their natural state. Thus, for example,
products made by transforming cells with any unnaturally
occurring vector is encompassed, as are nucleic -acids
comprising sequence derived using any synthetic
oligonucleotide process. Such a process is often done to
replace a codon with a redundant codon encoding the same
or a conservative amino acid, while typically introducing
or removing a restriction enzyme sequence recognition
site.
Alternatively, the process is performed to join
together nucleic acid segments of desired functions to
generate a single genetic entity comprising a desired
combination of functions not found in the commonly
available natural forms, e.g., encoding a fusion protein.
Restriction enzyme recognition sites are often the target
of such artificial manipulations, but other site specific
targets, e.g., promoters, DNA replication sites,
regulation sequences, control sequences, or other useful
features may be incorporated by design. A similar
concept is intended for a recombinant, e.g., fusion,
polypeptide. This will include a dimeric repeat.
Specifically included are synthetic nucleic acids which,
by genetic code redundancy, encode similar polypeptides
to fragments of the IL-l~, and fusions of sequences from
various different interleukin or related molecules, e.g.,
growth factors.
A "fragment" in a nucleic acid context is defined
herein to mean a contiguous segment of at least about
17 nucleotides, generally at least 21 nucleotides, more
generally at least 25 nucleotides, ordinarily at least
30 nucleotides, more ordinarily at least 35 nucleotides,
often at least 39 nucleotides, more often at least
45 nucleotides, typically at least 50 nucleotides, more
typically at least 55 nucleotides, usually at least
60 nucleotides, more usually at least 66 nucleotides,
preferably at least 72 nucleotides, more preferably at
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least 79 nucleotides, and in particularly preferred
embodiments will be at least 85 or more nucleotides.
Typically, fragments of different genetic sequences can
be compared to one another over appropriate length
stretches.
A nucleic acid which codes ~or an IL-1~ will be
particularly useful to identify genes, mRNA, and cDNA
species which code for itself or closely related
proteins, as well as DNAs which code for polymorphic,
allelic, or other genetic variants, e.g., from different
individuals. Preferred probes for such screens are those
regions of the interleukin which are conserved between
different polymorphic variants or which contain
nucleotides which lack specificity, and will pre~erably
be full length or nearly so. In other situations,
polymorphic variant specific sequences will be more
useful.
This invention further covers recombinant nucleic
acid molecules and fragments having a nucleic acid
sequence identical to or highly homologous to the
isolated DNA set forth herein. In particular, the
sequences will often be operably linked to DNA segments
which control transcription, translation, and DNA
replication. These additional segments typically assist
in expression of the desired nucleic acid segment.
Homologous nucleic acid sequences, when compared to
one another or SEQ ID NO: 1 sequences, exhibit
significant similarity. The standards for homology in
nucleic acids are either measures for homology generally
used in the art by sequence comparison or based upon
hybridization conditions. Comparative hybridization
conditions are described in greater detail below.
Substantial homology in the nucleic acid sequence
comparison context means either that the segments, or
their complementary strands, when compared, are identical
when optimally aligned, with appropriate nucleotide
insertions or deletions, in at least about 60% of the
nucleotides, generally at least 66~, ordinarily at least
71%, often at least 76%, more often at least 80%, usually
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at least 84%, more usually at least 88~, typically at
least 91%, more typically at least about 93%, preferably
at least about 95%, more preferably at least about 96 to
98% or more, and in particular embodiments, as high at
about 99% or more of the nucleotides.
Alternatively, substantial homology exists when the
segments will hybridize under selective hybridization
conditions, to a strand or its complement, typically
using a sequence derived from SEQ ID NO: 1. Typically,
selective hybridization will occur when there is at least
about 55% homology over a stretch of at least about
14 nucleotides, more typically at least about 65%,
preferably at least about 75%, and more preferably at
least about 90%. See, Kanehisa (1984) Nuc. Acids Res.
12:203-213, which is incorporated herein by reference.
The length of homology comparison, as described, may be
over longer stretches, and in certain embodiments will be
over a stretch of at least about 17 nucleotides,
generally at least about 20 nucleotides, ordinarily at
least about 24 nucleotides, usually at least about
28 nucleotides, typically at least about 32 nucleotides,
more typically at least about 40 nucleotides, preferably
at least about 50 nucleotides, and more preferably at
least about 75 to 100 or more nucleotides. Preferably a
related nucleic acid will contain a plurality of such
matching fragments, e.g., at elast 2, preferably 3, 4, 5,
or more of specific, or assorted, lengths.
Stringent conditions, in referring to homology in
the hybridization context, will be stringent combined
conditions of salt, temperature, organic solvents, and
other parameters typically controlled in hybridization
reactions. Stringent temperature conditions will usually
include temperatures in excess of about 30 C, more
usually in excess of about 37- C, typically in excess of
about 45- C, more typically in excess of about 55- C,
preferably in excess of about 65- C, and more preferably
in excess of about 70 C. Stringent salt conditions will
ordinarily be less than about 500 mM, usually less than
about 400 mM, more usually less than about 300 mM,
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W097/~4468 16 PCT~S97/07282
typically less than about 200 mM, preferably less than
about 100 mM, and more preferably less than about 80 mM,
even down to less than about 20 mM. However, the
combination of parameters is much more important than the
measure of any single parameter. See, e.g., Wetmur and
Davidson (1968) J. Mol. Biol. 31:349-370.
The isolated DNA can be readily modified by
nucleotide substitutions, nucleotide deletions,
nucleotide insertions, and inversions of nucleotide
stretches. These modifications result in novel DNA
sequences which encode this protein, its derivatives, or
proteins having IL-lr activity. These modified sequences
can be used to produce mutant proteins (muteins) or to
enhance the expression of variant species. Enhanced
expression may involve gene amplification, increased
transcription, increased translation, and other
mechanisms. Such mutant IL-l~ derivatives include
predetermined or site-specific mutations of the protein
or its fragments, including silent mutations using
genetic code degeneracy.
"Mutant IL-l~" as defined herein encompasses a
polypeptide otherwise falling within the homology
definition of the human IL-ly as set forth.~above, but
having an amino acid sequence which differs from that of
human IL-l~ as found in nature, whether by way of
deletion, substitution, or insertion. In particular,
"site specific mutant IL-l~" encompasses a protein having
substantial homology with the sequence of SEQ ID NO: 2,
and typically shares most of the biological activities of
the form disclosed herein.
Although site specific mutation sites are
predetermined, mutants need not be site specific. Human
IL-l~ mutagenesis can be achieved by making amino acid
insertions or deletions in the gene, coupled with
expression. Substitutions, deletions, insertions, or
any combinations may be generated to arrive at a
final construct. Insertions include amino- or
carboxy-terminal fusions. Random mutagenesis can be
conducted at a target codon and the expressed human IL-l~
CA 022~906 l998-ll-l9
WO 97/44468 17 PCTIUS97/07282
mutants can then be screened for the desired activity.
Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are
well known in the art, e.g., by M13 primer mutagenesis.
See also Sambrook, et al. (1989) and Ausubel, et al.
(1987 and periodic Supplements).
The mutations in the DNA normally should not place
coding sequences out of reading frames and preferably
will not create complementary regions that could
hybridize to produce secondary mRNA structure such as
loops or hairpins.
The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will
produce suitable synthetic DNA fragments. A double
stranded fragment will often be obtained either by
synthesizing the complementary strand and annealing the
strand together under appropriate conditions or by adding
the complementary strand using DNA polymerase with an
appropriate primer sequence.
Polymerase chain reaction (PCR) techniques can often
be applied in mutagenesis. Alternatively, mutagenisis
primers are commonly used methods for generating defined
mutations at predetermined sites. See, e.g, Innis,
et al. (eds. 1990) PCR Protocols: A Guide to Methods and
ApPlications Academic Press, San Diego, CA.
As described above, the present invention
encompasses the human IL-l~ whose sequence is defined in
SEQ ID NO: 2 and described above. Allelic and other
variants are also contemplated.
The present invention also provides recombinant
proteins, e.g., heterologous fusion proteins using
segments from this human protein. A heterologous fusion
protein is a fusion of proteins or segments which are
naturally not normally fused in the same manner. Thus,
the fusion product of a growth factor with an interleukin
is a continuous protein molecule having sequences fused
in a typical peptide linkage, typically made as a single
translation product and exhibiting properties derived
CA 0225~906 l998-ll-l9
W 097/44468 18 PCTnJS97/07282
from each source peptide. A similar concept applies to
heterologous nucleic acid sequences.
In addition, new constructs may be made from
combining similar functional or structural domains from
other related proteins, e.g., growth factors or other
cytokines. For example, receptor-binding or other
segments may be "swapped" between different new fusion
polypeptides or fragments. See, e.g., Cunningham, et al.
(1989) Science 243:1330-1336; and O'Dowd, et al. ~1988)
J. Biol. Chem. 263:15985-15992, each of which is
incorporated herein by reference. Thus, new chimeric
polypeptides exhibiting new combinations of specificities
will result from the functional linkage of receptor
binding specificities. For example, the receptor binding
domains from other related ligand molecules may be added
or substituted for other domains of this or related
proteins. The resulting protein will often have hybrid
function and properties.
For example, a fusion protein may include a
targetting ~om~in which may serve to provide sequestering
of the fusion protein to a particular organ, e.g., a
ligand portions which is specifically bound by spleen
cells and would serve to accumulate in the spleen.
Candidate fusion partners and sequences can be
selected from various sequence data bases, e.g., G~nR~nk,
c/o IntelliGenetics, Mountain View, CA; and BCG,
University of Wisconsin Biotechnology Computing Group,
Madison, WI.
The present invention particularly provides muteins
which act as agonists or antagonists of the IL~
Structural alignment of human and mouse ILl~ with other
members of IL-l family show conserved features/residues,
particularly 12 ~ strands folded into ~-trefoil fold.
See Bazan, et al. (1996) Nature 379:591. Alignment with
the human IL-l~ sequence (using the met initiation
residue of the signal peptide) indicates that the ~1 (leu
41 to val 47), ~2 (val 55 to asp 59), ~3 (pro 64 to asp
68), ~4 (phe 83 to tyr 88), ~5 (met 96 to val 102), ~6
(ser 108 to glu 113), ~7 (lys 115 to lys 120), ~8 (phe
CA 022~906 l998-ll-l9
W097/44468 19 PCT~S97/07282
137 to pro 143), ~9 (asn 147 to ser 153), ~10 (phe 160 to
glu 164), ~11 (phe 170 to lys 175), and ~12 (phe 187 to
asn 191) strands correspond to similar sequence in the
mouse IGIF. See also, Lodi, et al. (1994) Science
263:1762-1766; Sayle and Milner-White (1995) TIBS 20:374-
376; and Gronenberg, et al. (1991) Protein En~ineerinq
4:263-269.
The IL-la and IL-l~ ligands bind an IL-l receptor
type I as the primary receptor and this complex then
forms a high affinity receptor complex with the IL-l
receptor type III. The mouse IL-l~ does not bind to the
known mouse IL-l receptor types I, II (decoy receptor),
or III. In addition, the mouse IGIF biological activity
cannot be blocked with anti-type I, II, or III
antibodies. This suggests that the related mouse IGIF
binds to receptors related to the IL-l receptors already
isolated, but not yet identified as receptors for the
IGIF.
However, the solved structures for IL-l~, the
natural IL-l receptor antagonist (IL-lRa), and a
co-structure of IL-lRa/IL-l receptor type I, suggest how
to make a mouse IGIF or a human IL-l~ antagonist. The
loop between the ~4 strand and the ~5 strand is the
primary binding segment for other IL-l ligands to the
receptor type III. In IL-l~ and IL-l~ ligands, this loop
spans 8 residues, while in IL-lRa, this loop is "cut off"
(only 2 residues remain). Therefore, IL-lRa binds
normally to receptor type I, but can not interact with
receptor type III. This makes IL-lRa into an effective
IL-l antagonist.
This suggests that modifications to the loop between
the ~4 and the ~5 strands will lead to variants with
predictable biological activities. For example,
substitution of KDSEVRGL (residues between ~4 and ~5
strand, with the first residue in the ~5 strand) in the
mouse IGIF (or the corresponding KDSQPRGM of human IL-l~
to EPH) with EPH (corresponding IL-lRa residues) should
generate an IGIF antagonist. Alternatively, substitution
of KDSEVR of mouse IGIF with QGEESND (IL-l~ residues
CA 022~906 l998-ll-lg
W097/44468 20 PCT~S97/07282
between ~4 and ~5 strand) should allow interaction with
the type III receptor (in human IL-l~, substitute KDSQPR
with QGEESND). With mouse IL-lRa, it was shown that
replacement of the mouse IL-lRa residues with these mouse
IL-l~ residues introduced IL-l activity in IL-lRa (IL-lRa
can now bind type III receptor). This will establish
whether type III receptor can be used by mouse IGIF.
In addition, a construct which substitutes KDSEVR of
the mouse IGIF with the Flag epitope DYKDDDDK would
probably bind to the primary IGIF receptor. In human
IL-l~, the substitution would be KDSQPR with DYKDDDDK.
This mutant protein could act as an antagonist (unable to
bind secondary mIL-l~ receptor). This tagged molecule
should be useful to clone the mouse IGIF primary
receptor, which has not been identified as such.
Similar variations in the human IL-l~ ligand
sequence, e.g., in the corresponding region between
residues 88-96, should provide similar interactions with
receptor. Substitutions with either mouse sequences or
human sequences are indicated. Conversely, conservative
substitutions away from the receptor binding interaction
regions will probably preserve most biological
activities.
"Derivatives" of the human IL-l~ include amino acid
sequence mutants, glycosylation variants, metabolic
derivatives and covalent or aggregative conjugates with
other chemical moieties. Covalent derivatives can be
prepared by linkage of functionalities to groups which
are found in the IL-l~ amino acid side chains or at the
N- or C-termini, e.g., by means which are well known in
the art. These derivatives can include, without
limitation, aliphatic esters or amides of the carboxyl
terminus, or of residues containing carboxyl side chains,
O-acyl derivatives of hydroxyl group-containing residues,
and N-acyl derivatives of the amino terminal amino acid
or amino-group containing residues, e.g., lysine or
arginine. Acyl groups are selected from the group of
alkyl-moieties including C3 to C18 normal alkyl, thereby
forming alkanoyl aroyl species.
CA 022~906 l998-ll-l9
W097/44468 21 PCT~S97/07282
In particular, glycosylation alterations are
included, e.g., made by modifying the glycosylation
patterns of a polypeptide during its synthesis and
processing, or in further processing steps. Particularly
- 5 preferred means for accomplishing this are by exposing
the polypeptide to glycosylating enzymes derived from
cells which normally provide such processing, e.g.,
mammalian glycosylation enzymes. Deglycosylation enzymes
are also contemplated. Also embraced are versions of the
same primary amino acid sequence which have other minor
modifications, including phosphorylated amino acid
residues, e.g., phosphotyrosine, phosphoserine, or
phosphothreonine.
A major group of derivatives are covalent conjugates
of the interleukin or fragments thereof with other
proteins of polypeptides. These derivatives can be
synthesized in recombinant culture such as N- or
C-terminal fusions or by the use of agents known in the
art for their usefulness in cross-linking proteins
through reactive side groups. Preferred derivatization
sites with cross-linking agents are at free amino groups,
carbohydrate moieties, and cysteine residues.
Fusion polypeptides between the interleukin and
other homologous or heterologous proteins are also
provided. Homologous polypeptides may be fusions between
different growth factors, resulting in, for instance, a
hybrid protein exhibiting ligand specificity for multiple
different receptors, or a ligand which may have broadened
or weakened specificity of binding to its receptor.
Likewise, heterologous fusions may be constructed which
would exhibit a combination of properties or activities
of the derivative proteins. Typical examples are fusions
of a reporter polypeptide, e.g., luciferase, with
a segment or domain of a receptor, e.g., a ligand-binding
segment, so that the presence or location of a desired
ligand may be easily determined. See, e.g., Dull,
et al ., U.S. Patent No. 4,859,609. Other gene fusion
partners include glutathione-S-transferase (GST),
bacterial ~-galactosidase, trpE, Protein A, ~-lactamase,
CA 022~906 l998-ll-l9
WO 97/44468 22 PCT/US97/07282
alpha amylase, alcohol dehydrogenase, and yeast alpha
mating factor. See, e.g., Godowski, et al. (1988)
Science 241: 812-816 .
The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22 :1859-1862, will
produce suitable synthetic DNA fragments. A double
stranded fragment will often be obtained either by
synthesizing the complementary strand and annealing the
strand together under appropriate conditions or by adding
the complementary strand using DNA polymerase with an
appropriate primer sequence.
Such polypeptides may also have amino acid residues
which have been chemically modified by phosphorylation,
sulfonation, biotinylation, or the addition or removal of
other moieties, particularly those which have molecular
shapes similar to phosphate groups. In some embodiments,
the modifications will be useful labeling reagents, or
serve as purification targets, e.g., affinity ligands.
Fusion proteins will typically be made by either
recombinant nucleic acid methods or by synthetic
polypeptide methods. Techniques for nucleic acid
manipulation and expression are described generally, for
example, in Sambrook, et al. (1989) Molecular Cloninq: A
Laboratorv Manual (2d ed.), Vols. 1-3, Cold Spring Harbor
Laboratory, and Ausubel, et al. (eds)(1987 and periodic
supplements) Current Protocols in Molecular Bioloqv,
Greene/Wiley, New York, which are each incorporated
herein by reference. Techniques for synthesis of
polypeptides are described, for example, in Merrifield
(1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield
(1986) Science 232: 341-347; and Atherton, et al. (1989)
Solid Phase Pe~tide Synthesis: A Practical A~roach, ~RL
Press, Oxford; each of which is incorporated herein by
reference. See also Dawson, et al. (1994) Science
266:-776-779 for methods to make larger polypeptides.
CA 022~906 l998-ll-l9
W097/44468 23 PCT~S97/07282
This invention also contemplates the use of
derivatives of the human IL-l~ other than variations in
amino acid sequence or glycosylation. Such derivatives
may involve covalent or aggregative association with
chemical moieties. These derivatives generally fall into
three classes: (1) salts, (2) side chain and terminal
residue covalent modifications, and (3) adsorption
complexes, for example with cell membranes. Such
covalent or aggregative derivatives are useful as
immunogens, as reagents in immunoassays, or in
purification methods such as for affinity purification of
a receptor or other binding molecule, e.g., an antibody.
For example, the human IL-l~ ligand can be
immobilized by covalent bonding to a solid support such
as cyanogen bromide-activated Sepharose, by methods which
are well known in the art, or adsorbed onto polyolefin
surfaces, with or without glutaraldehyde cross-linking,
for use in the assay or purification of IL-l~ receptor,
antibodies, or other similar molecules. The IL-l~ can
also be labeled with a detectable group, for example
radioiodinated by the chloramine T procedure, covalently
bound to rare earth chelates, or conjugated to another
fluorescent moiety for use in diagnostic assays.
The human IL-l~ of this invention can be used as an
immunogen for the production of antisera or antibodies
specific, e.g., capable of distinguishing between mouse
IGIF and human IL-l~, for the interleukin or any
fragments thereof. The purified interleukin can be used
to screen monoclonal antibodies or antigen-binding
fragments prepared by immunization with various forms of
impure preparations containing the protein. In
particular, the term "antibodies" also encompasses
antigen blnding fragments of natural antibodies. The
purified interleukin can also be used as a reagent to
detect any antibodies generated in response to the
presence of elevated levels of expression, or
immunological disorders which lead to antibody production
to the endogenous cytokine. Additionally, IL-l~
fragments may also serve as immunogens to produce the
CA 0225~906 1998-11-19
W O 97/44468 24 PCTrUS97/07282
antibodies of the present invention, as described
immediately below. For example, this invention
contemplates antibodies having binding affinity to or
being raised against the amino acid sequence shown in
SEQ ID NO: 2, fragments thereof, or homologus peptides.
In particular, this invention contemplates antibodies
having binding affinity to, or having been raised
against, specific fragments which are predicted to be, or
actually are, exposed at the exterior protein surface of
the native cytokine.
The blocking of physiological response to these
interleukins may result from the inhibition of binding of
the ligand to the receptor, likely through competitive
inhibition. Thus, in vitro assays of the present
invention will o~ten use antibodies or ligand binding
segments of these antibodies, or fragments attached to
solid phase substrates. These assays will also allow for
the diagnostic determination of the effects of either
binding region mutations and modifications, or ligand
mutations and modifications, e.g., ligand analogs.
This invention also contemplates the use of
competitive drug screening assays, e.g., where
neutralizing antibodies to the interleukin or fragments
compete with a test compound for binding to a receptor or
antibody. In this manner, the neutralizing antibodies or
fragments can be used to detect the presence of any
polypeptide which shares one or more binding sites to a
receptor and can also be used to occupy binding sites on
a receptor that might otherwise bind an interleukin.
DNA which encodes the protein or fragments thereof
can be obtained by chemical synthesis, screening cDNA
libraries, or by screening genomic libraries prepared
from a wide variety of cell lines or tissue samples.
Natural sequences can be isolated using standard methods
and the sequences provided herein in SEQ ID NO: 1.
This DNA can be expressed in a wide variety of host
cells for the synthesis of a full-length human
interleukin or fragments which can in turn, for example,
be used to generate polyclonal or monoclonal antibodies;
CA 022~906 l998-ll-l9
W097/44468 25 PCT~S97/07282
for binding studies; for construction and expression of
modified agonist/antagonist molecules; and for
structure/function studies. Each variant or its
fragments can be expressed in host cells that are
transformed or transfected with appropriate expression
vectors. These molecules can be substantially free of
protein or cellular contaminants, other than those
derived from the recombinant host, and therefore are
particularly useful in pharmaceutical compositions when
combined with a pharmaceutically acceptable carrier
and/or diluent. The human protein, or portions thereof,
may be expressed as fusions with other proteins.
Expression vectors are typically self-replicating
DNA or RNA constructs containing the desired receptor
gene or its fragments, usually operably linked to
suitable genetic control elements that are recognized in
a suitable host cell. These control elements are capable
of effecting expression within a suitable host. The
specific type of control elements necessary to effect
expression will depend upon the eventual host cell used.
Generally, the genetic control elements can include a
prokaryotic promoter system or a eukaryotic promoter
expression control system, and typically include a
transcriptional promoter, an optional operator to control
the onset of transcription, transcription enhancers to
elevate the level of mRNA expression, a sequence that
encodes a suitable ribosome binding site, and sequences
that terminate transcription and translation. Expression
vectors also usually contain an origin of replication
that allows the vector to replicate independently of the
host cell.
The vectors of this invention include those which
contain DNA which encodes a protein, as described, or a
fragment thereof encoding a biologically active
ec~ivalent polypeptide. The DNA can be under the control
of a viral promoter and can encode a selection marker.
This invention further contemplates use of such
expression vectors which are capable of expressing
eukaryotic cDNA coding for such a protein in a
CA 0225~906 1998-11-19
W O 97/44468 26 PCTAUS97/07Z82
prokaryotic or eukaryotic host, where the vector is
compatible with the host and where the eukaryotic cDNA
coding for the receptor is inserted into the vector such
that growth of the host containing the vector expresses
the cDNA in question. Usually, expression vectors are
designed for stable replication in their host cells or
for amplification to greatly increase the total number of
copies of the desirable gene per cell. It is no~ always
necessary to require that an expression vector replicate
in a host cell, e.g., it is possible to effect transient
expression of the interleukin protein or its fragments in
various hosts using vectors that do not contain a
replication origin that is recognized by the host cell.
It is also possible to use vectors that cause integration
of the human protein or its fragments into the host DNA
by recombination.
Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other
vehicles which enable the integration of DNA fragments
into the genome of the host. Expression vectors are
specialized vectors which contain genetic control
elements that effect expression of operably linked genes.
Plasmids are the most commonly used form of vector but
all other forms of vectors which serve an equivalent
function and which are, or become, known in the art are
suitable for use herein. See, e.g., Pouwels, et al.
(1985 and Supplements) Cloninq Vectors: A Laboratorv
Manual, Elsevier, N.Y., and Rodriquez, et al . (eds)
Vectors: A SurveY of Molecular Cloninq Vectors and Their
.Uses, Buttersworth, Boston, 1988, which are incorporated
herein by reference.
Transformed cells are cells, preferably mammalian,
that have been transformed or transfected with receptor
vectors constructed using recombinant DNA techniques.
Transformed host cells usually express the desired
protein or its fragments, but for purposes of cloning,
amplifying, and manipulating its DNA, do not need to
express the subject protein. This invention further
contemplates culturing transformed cells in a nutrient
CA 022~906 l998-ll-l9
W097/44468 27 PCT~S97/072~2
medium, thus permitting the interleukin to accumulate in
the culture. The protein can be recovered, either from
the culture or from the culture medium.
For purposes of this invention, nucleic sequences
are operably linked when they are functionally related to
each other. For example, DNA for a presequence or
secretory leader is operably linked to a polypeptide if
it is expressed as a preprotein or participates in
directing the polypeptide to the cell membrane or in
secretion of the polypeptide. A promoter is operably
linked to a coding sequence if it controls the
transcription of the polypeptide; a ribosome binding site
is operably linked to a coding sequence if it is
positioned to permit translation. Usually, operably
linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences
that in turn control expression.
Suitable host cells include prokaryotes, lower
eukaryotes, and higher eukaryotes. Prokaryotes include
both gram negative and gram positive organisms, e.g.,
E. coli and B. subtilis. Lower eukaryotes include
yeasts, e.g., S. cerevisiae and Pichia, and species of
the genus Di c tyo s t e 1 i um. Higher eukaryotes include
established tissue culture cell lines from animal cells,
both of non-mammalian origin, e.g., insect cells, and
birds, and of mammalian oriyin, e.g., human, primates,
and rodents.
Prokaryotic host-vector systems include a wide
variety of vectors for many different species. As used
herein, E. coli and its vectors will be used generically
to include equivalent vectors used in other prokaryotes.
A representative vector for amplifying DNA is pBR322 or
many of its derivatives. Vectors that can be used to
express the receptor or its fragments include, but are
not limited to, such vectors as those containing the lac
promoter (pUC-series); trp promoter (pBR322-trp); Ipp
promoter (the pIN-series); lambda-pP or pR promoters
(pOTS); or hybrid promoters such as ptac (pDR540). See
CA 022~5906 1998-11-19
W 097/44468 28 PCT/US97/07282 --
Brosius, et al. (1988) 'IExpression Vectors Employing
Lambda-, trp-, lac-, and Ipp-derived Promoters", in
Vectors: A Survev of Molecular Clonin~ Vectors and Their
Uses, (eds. Rodriguez and Denhardt), Buttersworth,
Boston, Chapter 10, pp. 205-236, which is incorporated
herein by reference.
Lower eukaryotes, e.g., yeasts and Dictyostelium,
may be transformed with IL-1~ sequence containing
vectors. For purposes of this invention, the most com.mon
lower eukaryotic host is the baker's yeast, Saccharomyces
cerevisiae. It will be used to generically represent
lower eukaryotes although a number of other strains and
species are also available. Yeast vectors typically
consist of a replication origin (unless of the
integrating type), a selection gene, a promoter, DNA
encoding the receptor or its fragments, and sequences for
translation termination, polyadenylation, and
transcription termination. Suitable expression vectors
for yeast include such constitutive promoters as
3-phosphoglycerate kinase and various other glycolytic
enzyme gene promoters or such inducible promoters as the
alcohol dehydrogenase 2 promoter or metallothionine
promoter. Suitable vectors include derivat~ves of the
following types: self-replicating low copy number (such
as the YRp-series), self-replicating high copy number
(such as the YEp-series); integrating types (such as the
YIp-series), or mini-chromosomes (such as the
YCp-series).
Higher eukaryotic tissue culture cells are normally
the preferred host cells for expression o~ the
functionally active interleukin protein. In principle,
any hi~her eukaryotic tissue culture cell line is
workable, e.g., insect baculovirus expression systems,
whether from an invertebrate or vertebrate source.
However, m~mm~lian cells are preferred. Transformation
or transfection and propagation of such cells has become
a routine procedure. Examples of useful cell lines
include HeLa cells, Chinese hamster ovary (CHO) cell
lines, baby rat kidney (BRK) cell lines, insect cell
CA 022~906 l998-ll-l9
W097/4~68 29 PCT~S97/07282
lines, bird cell lines, and monkey (COS) cell lines.
Expression vectors for such cell lines usually include an
origin of replication, a promoter, a translation
initiation site, RNA splice sites (if genomic DNA is
S used), a polyadenylation site, and a transcription
termination site. These vectors also usually contain a
selection gene or amplification gene. Suitable
expression vectors may be plasmids, viruses, or
retroviruses carrying promoters derived, e.g., from such
sources as from adenovirus, SV40, parvoviruses, vaccinia
virus, or cytomegalovirus. Representative examples of
suitable expression vectors include pCDNAli pCD, see
Okayama, et al. (lg85) Mol. Cell Biol. 5:1136-1142;
pMClneo PolyA, see Thomas, et al . ( 1987) Cell 51:S03-512;
and a baculovirus vector such as pAC 373 or pAC 610.
For secreted proteins, an open reading frame usually
encodes a polypeptide that consists of a mature or
secreted product covalently linked at ist N-terminus to a
signal peptide. The signal peptide is cleaved prior to
secretion of the mature, or active, polypeptide. The
cleavage site can be predicted with a high degree of
accuracy from empirical rules, e.g., von-Heijne (1986)
Nucleic Acids Research 14:4683-4690, and the precise
amino acid composition of the signal peptide does not
appear to be critical to its function, e.g., Randall,
et al . (1989) Science 243:1156-1159; Kaiser st al. ~1987)
Science 235:312-317.
It will often be desired to express these
polypeptides in a system which provides a specific or
defined glycosylation pattern. In this case, the usual
pattern will be that provided naturally by the expression
system. However, the pattern will be modifiable by
exposing the polypeptide, e.g., an unglycosylated form,
to appropriate glycosylating proteins introduced into a
heterologous expression system. For example, the
interleukin gene may be co-transformed with one or more
genes encoding m~mm~1ian or other glycosylating enzymes.
Using this approach, certain m~mm~lian glycosylation
patterns will be achievable in prokaryote or other cells.
CA 022~5906 1998-ll-l9
W097/44468 30 PCT~S97/07282
The source of human IL-l~ can be a eukaryotic or
prokaryotic host expressing recombinant huIL-l~ DNA, such
as is described above. The source can also be a cell
line such as mouse Swiss 3T3 fibroblasts, but other
m~mm~lian cell lines are also contemplated by this
invention, with the preferred cell line being from the
human species.
Now that the entire se~uence is known, the human
IL~ ragments, or derivatives thereof can be prepared
by conventional processes ~or synthesizing peptides.
These include processes such as are described in Stewart
and Young (1984) Solid Phase PePtide SYnthesis, Pierce
Chemical Co., Rockford, IL; Bodanszky and Bodanszky
(1984) The Practice of Pe~tide Svnthesis,
Springer-Verlag, New York; and Bodanszky (1984) The
Principles of Peptide SYnthesis, Springer-Verlag, New
York; all of each which are incorporated herein by
reference. For example, an azide process, an
acid chloride process, an acid anhydride process, a
mixed anhydride process, an active ester process
(for example, p-nitrophenyl ester, N-hydroxysuccinimide
ester, or cyanomethyl ester), a carbodiimidazole
process, an oxidative-reductive process, or a
dicyclohexylcarbodiimide (DCCD)/additive process can be
used. Solid phase and solution phase syntheses are both
applicable to the foregoing processes.
The IL-l~ protein, fragments, or derivatives are
suitably prepared in accordance with the above processes
as typically employed in peptide synthesis, generally
either by a so-called stepwise process which comprises
condensing an amino acid to the terminal amino acid, one
~y one in se~uence, or by coupling peptide fragments to
the terminal amino acid. Amino groups that are not being
used in the coupling reaction typically must be protected
to prevent coupling at an incorrect location.
If a solid phase synthesis is adopted, the
C-terminal amino acid is bound to an insoluble carrier or
support through its carboxyl group. The insoluble
carrier is not particularly limited as long as it
CA 022~906 l998-ll-l9
W097/44468 31 PCT~S97/07282
has a binding capability to a reactive carboxyl group.
Examples of such insoluble carriers include
halomethyl resins, such as chloromethyl resin or
bromomethyl resin, hydroxymethyl resins, phenol resins,
tert-alkyloxycarbonylhydrazidated resins, and the like.
An amino group-protected amino acid is bound in
sequence through condensation of its activated carboxyl
group and the reactive amino group of the previously
formed peptide or chain, to synthesize the peptide step
by step. After synthesizing the complete sequence, the
peptide is split off from the insoluble carrier to
produce the peptide. This solid-phase approach is
generally described by Merrifield, et al. (1963) in
J. Am. Chem. Soc. 85:2149-2156, which is incorporated
herein by reference.
The prepared protein and fragments thereof can be
isolated and purified from the reaction mixture by means
of peptide separation, for example, by extraction,
precipitation, electrophoresis, various forms of
chromatography, and the like. The interleukin of this
invention can be obtained in varying degrees of purity
depending upon its desired use. Purification can be
accomplished by use of the protein purification
techniques disclosed herein, see below, or by the use of
the antibodies herein described in methods of
immunoabsorbant affinity chromatography. This
immunoabsorbant affinity chromatography is carried out by
first linking the antibodies to a solid support and then
contacting the linked antibodies with solubilized lysates
of appropriate cells, lysates of other cells expressing
the interleukin, or lysates or supernatants of cells
producing the protein as a result of DNA techniques, see
below.
Generally, the purified protein will be at least
about 40% pure, ordinarily at least about 50% pure,
usually at least about 60% pure, typically at least about
70% pure, more typically at least about 80% pure,
preferable at least about 90% pure and more preferably at
least about 95% pure, and in particular embodiments,
CA 022~906 1998-11-19
W097/~4468 32 PCT~S97/07282
97%-99% or more. Purity will usually be on a weight
basis, but can also be on a molar basis. Different
assays will be applied as appropriate.
Antibodies can be raised to the various human IL-l~
proteins and fragments thereof, both in naturally
occurring native forms and in their recombinant forms,
the difference being that antibodies to the active ligand
are more likely to recognize epitopes which are only
present in the native conformations.
Anti-idiotypic antibodies are also contemplated,
which would be useful as agonists or antagonists of a
natural receptor or an antibody.
Antibodies, including binding fragments and single
chain versions, against predetermined fragments of the
protein can be raised by immunization of animals with
conjugates of the fragments with immunogenic proteins.
Monoclonal antibodies are prepared from cells secreting
the desired antibody. These antibodies can be screened
for binding to normal or defective protein, or screened
for agonistic or antagonistic activity. These monoclonal
antibodies will usually bind with at least a KD of about
1 mM, more usually at least about 300 ~M, typically at
least about lOO~M, more typically at least about 30 ~M,
preferably at least about lO ~M, and more preferably at
least about 3 ~M or better.
The antibodies, including antibody antigen binding
fragments, of this invention can have significant
diagnostic or therapeutic value. They can be potent
antagonists that bind to the interleukin and inhibit
binding to the receptor or inhibit the ability of huamn
IL-l~ to elicit a biological response. They also can be
useful as non-neutralizing antibodies and can be coupled
to toxins or radionuclides to bind producing cells, or
cells localized to the source of the interleukin.
Further, these antibodies can be conjugated to drugs or
other therapeutic agents, either directly or indirectly
by means of a linker.
CA 022~906 l998-ll-l9
WO 97/44468 33 PCT/US97107282
As used in this invention, the term "antibody
antigen binding fragment~ includes, e.g., Fab, Fc,
F(ab)2, Fv and scFv fragments, which are employed with
their standard immunological meanings. See, e.g., Klein,
5 IIrlmunology (John Wiley, New York, ~982); Parham, Chapter
14, in Weir, ed. Immunochemistry, 4th Ed. (Blackwell
Scientific Publishers, Oxford, 1986). Such fragments can
be made from intact antibodies by chemical cleavage of
through the use of recombinant DNA methodology. See,
e.g., U.S. Patent No. 4,642,334, describing the
production of recombinant Fv fragments, and WO 93/11236
for scFv.
The antibodies of this invention can also be useful
in diagnostic applications. As capture or
non-neutralizing antibodies, they can bind to the
interleukin without inhibiting receptor binding. As
neutralizing antibodies, they can be useful in
competitive binding assays. They will also be useful in
detecting or ~uantifying II.~
Protein fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. The human IL-l~
and its fragments may be fused or covalently~linked to a
variety of immunogens, such as keyhole limpet hemocyanin,
bovine serum albumin, tetanus toxoid, etc. See
Microbioloqv, Hoeber Medical Division, Harper and Row,
1969; Landsteiner (1962) SpecificitY of Seroloqical
Reactions, Dover Publications, New York; and Williams,
et al. (1967) Methods in Immunoloqv and Immunochemistry~
Vol. 1, Academic Press, New York; each of which are
incorporated herein by reference, for descriptions of
methods of preparing polyclonal antisera. A typical
method involves hyperimmunization of an animal with an
antigen. The blood of the animal is then collected
shortly after the repeated immunizations and the gamma
globulin is isolated.
In some instances, it is desirable to prepare
monoclonal antibodies from various m~mmi~l ian hosts, such
as mice, rodents, primates, humans, etc. Description of
CA 022~5906 1998-11-19
W 097/44468 34 PCT~US97/07282 _
techniques for preparing such monoclonal antibodies may
be found in, e.g., Stites, et al. (eds) Basic and
Clinical Immunoloqv (4th ed.), Lange Medical
Publications, Los Altos, CA, and references cited
therein; Harlow and Lane (1988) Antibodies: A Laboratorv
Manual, CSH Press; Goding (1986) Monoclonal Antibodies:
PrinciPles and Practice (2d ed) Academic Press, New York;
and particularly in Kohler and Milstein (1975) in Nature
256: 495-497, which discusses one method of generating
monoclonal antibodies. Each of these references is
incorporated herein by reference. Summarized briefly,
this method involves injecting an animal with an
immunogen. The animal is then sacrificed and cells taken
from its spleen, which are then fused with myeloma cells.
The result is a hybrid cell or "hybridoma" that is
capable of reproducing in vitro. The population of
hybridomas is then screened to isolate individual clones,
each of which secrete a single antibody species to the
immunogen. In this manner, the individual antibody
species obtained are the products of immortalized and
cloned single B cells from the immune ~n;m~l generated in
response to a specific site recognized on the immunogenic
substance.
Other suitable techniques involve in vi tro exposure
of lymphocytes to the antigenic polypeptides or
alternatively to selection of libraries of antibodies in
phage or similar vectors. See, Huse, et al. (1989)
"Generation of a Large Combinatorial Library of the
Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-
546. The polypeptides and antibodies of the present
invention may be used with or without modification,
including chimeric or humanized anti~odies. Frequently,
the polypeptides and antibodies will be labeled by
joining, either covalently or non-covalently, a substance
which provides for a detectable signal. A wide variety
of labels and conjugation techniques are known and are
reported extensively in both the scientific and patent
literature. Suitable labels include radionuclides,
CA 022~906 l998-ll-l9
W097/44468 35 PCT~S97/07282
enzymes, substrates, cofactors, inhibitors, fluorescent
moieties, chemiluminescent moieties, magnetic particles,
and the like. Patents, teaching the use of such labels
include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437i 4,275,149; and 4,366,241. Also,
recombinant or chimeric immunoglobulins may be produced,
see Cabilly, U.S. Patent No. 4,816,567.
The antibodies of this invention can also be used
for affinity chromatography in isolating the IL~
Columns can be prepared where the antibodies are linked
to a solid support, e.g., particles, such as agarose,
Sephadex, or the like, where a cell lysate may be passed
through the column, the column washed, followed by
increasing concentrations of a mild denaturant, whereby
the purified protein will be released.
The antibodies may also be used to screen expression
libraries for particular expression products. Usually
the antibodies used in such a procedure will be labeled
with a moiety allowing easy detection of presence of
antigen by antibody binding.
Antibodies raised against each human IL-ly will also
be used to raise anti-idiotypic antibodies. These will
be useful in detecting or diagnosing various
i D unological conditions related to expression of the
protein or cells which express receptors for the protein.
They also will be useful as agonists or antagonists of
the interleukin, which may be competitive inhibitors or
substitutes for naturally occurring ligands.
Both naturally occurring and recombinant forms of
the human IL-1~ molecules of this invention are
particularly useful in kits and assay methods. For
example, these methods would also be applied to screening
for binding activity, e.g., receptors for these proteins.
Several methods of automating assays have been developed
in recent years so as to permit screening of tens of
thousands of compounds per year. See, e.g, a BIOMEK
automated workstation, Beckman Instruments, Palo Alto,
California, and Fodor, et al. (1991) Science 251:767-773,
which is incorporated herein by reference. The latter
CA 0225~906 1998-11-19
W 097/44468 3~ PCT/US97/07282
describes means for testing binding by a plurality of
defined polymers synthesized on a solid substrate. The
development of suitable assays to screen for a receptor
or agonist/antagonist homologous proteins can be greatly
facilitated by the availability of large amounts of
puri~ied, soluble IL-ly in an active state such as is
provided by this invention.
Purified IL-ly can be coated directly onto plates
for use in the aforementioned receptor screening
techniques. However, non-neutralizing antibodies to
these proteins can be used as capture antibodies to
immobilize the respective interleukin on the solid phase,
useful, e.g., in diagnostic uses.
This invention also contemplates use of IL~
fragments thereof, peptides, and their fusion products in
a variety of diagnostic kits and methods for detecting
the presence of the protein or its receptor.
Alternatively, or additionally, antibodies against the
molecules may be incorporated into the kits and methods.
Typically the kit will have a compartment containing
either a defined IL-l~ peptide or gene segment or a
reagent which recognizes one or the other. Typically,
recognition reagents, in the case of peptide, would be a
receptor or antibody, or in the case of a gene segment,
would usually be a hybridization probe.
A preferred kit for determining the concentration
of, for example, IL-ly, a sample would typically comprise
a labeled compound, e.g., receptor or antibody, having
known binding affinity for IL-l~y, a source of IL-l~y
(naturally occurring or recombinant) as a positive
control, and a means for separating the bound from free
labeled compound, for example a solid phase for
immobilizing the IL-l~ in the test sample. Compartments
cont~ln;ng reagents, and instructions, will normally be
provided.
Antibodies, including antigen binding fragments,
specific for human IL-l~ or a peptide fragment, or
receptor fragments are useful in diagnostic applications
to detect the presence of elevated levels of IL-ly and/or
CA 022~906 l998-ll-l9
W097/44468 37 PCT~S97/07282
its fragments. Diagnostic assays may be homogeneous
~without a separation step between free reagent and
antibody-antigen complex) or heterogeneous (with a
separation step). Various commercial assays exist, such
as radioimmunoassay (RIA), enzyme-linked immunosorbent
assay (ELISA), enzyme immunoassay (EIA),
enzyme-multiplied immunoassay technique (EMIT),
substrate-labeled fluorescent immunoassay (SLFIA) and the
like. For example, unlabeled antibodies can be employed
by using a second antibody which is labeled and which
recognizes the antibody to IL-l~ or to a particular
fragment thereof. These assays have also been
extensively discussed in the literature. See, e.g.,
Harlow and Lane (1988) Antibodies: A Laboratorv Manual,
CSH., and Coligan (Ed.) (1991) and periodic supplements,
Current Protocols In Immunolo~v Greene~Wiley, New York.
Anti-idiotypic antibodies may have similar use to
serve as agonists or antagonists of IL-l~. These should
be useful as therapeutic reagents under appropriate
circumstances.
Frequently, the reagents for diagnostic assays are
supplied in kits, so as to optimize the sensitivity of
the assay. For the subject invention, depending upon the
nature of the assay, the protocol, and the label, either
labeled or unlabeled antibody, or labeled receptor is
provided. This is usually in conjunction with other
additives, such as buffers, stabilizers, materials
necessary for signal production such as substrates for
enzymes, and the like. Preferably, the kit will also
contain instructions for proper use and disposal of the
contents after use. Typically the kit has compartments
for each useful reagent, and will contain instructions
for proper use and disposal of reagents. Desirably, the
reagents are provided as a dry lyophilized powder, where
the reagents may be reconstituted in an aqueous medium
having appropriate concentrations for performing the
assay.
CA 022~906 l998-ll-l9
W097t44468 38 PCT~S97/07282
Any of the aforementioned constituents of the
diagnostic assays may be used without modification or may
be modified in a variety of ways. For example, labeling
may be achieved by covalently or non-covalently joining a
moiety which directly or indirectly provides a detectable
signal. In any of these assays, a test compound, IL~
or antibodies thereto can be labeled either directly or
indirectly. Possibilities for direct labeling include
label groups: radiolabels such as 125I, enzymes (U.S.
Pat. No. 3,645,090) such as peroxidase and alkaline
phosphatase, and fluorescent labels (U.S. Pat. No.
3,940,475) capable of monitoring the change in
fluorescence intensity, wavelength shift, or fluorescence
polarization. Both of the patents are incorporated
herein by reference. Possibilities for indirect labeling
include biotinylation of one constituent followed by
binding to avidin coupled to one of the above label
groups.
There are also numerous methods of separating the
bound from the free ligand, or alternatively the bound
from the free test compound. ~he IL-l~ can be
immobilized on various matrixes followed by washing.
Suitable matrices include plastic such as an ELISA plate,
filters, and beads. Methods of immobilizing the receptor
to a matrix include, without limitation, direct adhesion
to plastic, use of a capture antibody, chemical coupling,
and biotin-avidin. The last step in this approach
involves the precipitation of antibody/antigen complex by
any of several methods including those utilizing, e.g.,
an organic solvent such as polyethylene glycol or a salt
such as ammonium sulfate. Other suitable separation
techniques include, without limitation, the fluorescein
antibody magnetizable particle method described in
Rattle, et al. (1984) Clin. Chem. 30(9):1457-1461, and
3S the double antibody magnetic particle separation as
described in U.S. Pat. No. 4,659,678, each of which is
incorporated herein by reference.
CA 022~906 l998-ll-l9
W097/44468 39 PCT~S97/07282
The methods for linking protein or fragments to
various labels have been extensively reported in the
literature and do not require detailed discussion here.
Many of the techni~ues involve the use of activated
carboxyl groups either through the use of carbodiimide or
active esters to form peptide bonds, the formation of
thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated
olefin such as maleimide, for linkage, or the like.
Fusion proteins will also find use in these applications.
Another diagnostic aspect of this invention involves
use of oligonucleotide or polynucleotide sequences taken
from the sequence of an IL-1~. These sequences can be
used as probes for detecting levels of the IL-1~ in
patients suspected of having an immulogoical disorder.
The preparation of both RNA and DNA nucleotide sequences,
the labeling of the sequences, and the preferred size of
the sequences has received ample description and
discussion in the literature. Normally an
oligonucleotide probe should have at least about
14 nucleotides, usually at least about 18 nucleotides,
and the polynucleotide probes may be up to several
kilobases. Various labels may be employed, most commonly
radionuclides, particularly 32p. However, other
techniques may also be employed, such as using biotin
modified nucleotides for introduction into a
polynucleotide. The biotin then serves as the site for
binding to avidin or antibodies, which may be labeled
with a wide variety of labels, such as radionuclides,
fluorescers, enzymes, or the like.
Alternatively, antibodies may be employed which can
recognize specific duplexes, including DNA duplexes, RNA
duplexes, DNA-RNA hybrid duplexes, or DNA-protein
duplexes. The antibodies in turn may be labeled and the
assay carried out where the duplex is bound to a surface,
so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
The use of probes to the novel anti-sense RNA may be
carried out in any conventional techniques such as
CA 022~906 l998-ll-l9
W097/44468 40 pcT~ss7lo7282
nucleic acid hybridization, plus and minus screening,
recombinational probing, hybrid released translation
(HRT), and hybrid arrested translation (HART). This also
includes amplification techniques such as polymerase
chain reaction (PCR).
Diagnostic kits which also test for the qualitative
or ~uantitative presence of other markers are also
contemplated. Diagnosis or prognosis may depend on the
combination of multiple indications used as markers.
Thus, kits may test for combinations of markers. See,
e.g., Viallet, et al. (1989) Pro~ress in Growth ~actor
Res. 1:89-97.
This invention also provides reagents with
significant therapeutic value. The IL-l~ (naturally
occurring or recombinant), fragments thereof, mutein
agonists and antagonists, and antibodies, along with
compounds identified as having binding affinity to the
interleukin or its receptor or antibodies, should be
useful in the treatment of conditions exhibiting abnormal
expression of the interleukin. Such abnormality will
typically be manifested by immunological disorders.
Additionally, this invention should provide therapeutic
value in various diseases or disorders associated with
abnormal expression or abnormal triggering of response to
the interleukin. The mouse IGIF has been suggested to be
involved in tumors, allergies, and infectious diseases,
e.g., pulmonary tuberculosis, leprosy, fulminant
hepatitis, and viral infections, such as HIV.
In addition, the dendritic cell expression profile
shows human IL-l~ primarily expressed in activated
dendritic cells. Activated dendritic cells are also a
major producer of IL-12, and it is thought that this
dendritic cell produced IL-12 plays a major role in
directing a Thl type response. The combination of IL-l~
and IL-12 should be extremely potent in inducing IFN-~.
It is possible that the combination of these two
pro-inflammatory cytokines under certain circumstances
could lead to septic shock. An IL-l~ antagonist, mutein
or antibody, could prove very useful in this situation.
CA 022~906 l998-ll-l9
W097/44468 PCT~S97/07282
41
It is possible that a natural IL-1~ antagonist is
produced by NK cells or activated T-cells to counteract
the effects of IL-1~.
Recombinant IL-1~, mutein agonists or antagonists,
or IL-1~ antibodies can be purified and then administered
to a patient. These reagents can be combined for
therapeutic use with additional active ingredients, e.g.,
in conventional pharmaceutically acceptable carriers or
diluents, along with physiologically innocuous
stabilizers and excipients. These combinations can be
sterile filtered and placed into dosage forms as by
lyophilization in dosage vials or storage in stabilized
aqueous preparations. This invention also contemplates
use of antibodies or binding fragments thereof which are
not complement binding.
Receptor screening using IL-lr or fragments thereof
can be performed to identify molecules having binding
affinity to the interleukin. Subsequent biological
assays can then be utilized to determine if a receptor
can provide competitive binding, which can block
intrinsic stimulating activity. Receptor fragments can
be used as a blocker or antagonist in that it blocks the
activity of IL-1~. Likewise, a compound having intrinsic
stimulating activity can activate the receptor and is
thus an agonist in that it simulates the activity of
IL-1~. This invention further contemplates the
therapeutic use of antibodies to IL-1~ and other
molecules as antagonists.
The quantities of reagents necessary for effective
therapy will depend upon many different factors,
including means of administration, target site,
physiological state of the patient, and other medicants
administered. Thus, treatment dosages should be titrated
to optimize safety and efficacy. Typically, dosages used
in vi tro may provide useful guidance in the amounts
useful for in situ administration of these reagents
Animal testing of effective doses for treatment of
particular disorders will provide further predictive
indication of human dosage.
CA 022~906 l998-ll-lg
W097/44468 PCT~S97/07282
42
Various considerations are described, e.g., in
Gilman, et al. (eds~ (1990) Goodman and Gilman's: The
Pharmacoloqical Bases of Therapeutics, 8th Ed., Pergamon
Press; and Remin~ton's Pharmaceutical Sciences, 17th ed.
(1990), Mack Publishing Co., Easton, Penn. Methods for
~m;n;stration are discussed therein and below, e.g., for
oral, intravenous, intraperitoneal, or intramuscular
administration, transdermal diffusion, and others.
Pharmaceutically acceptable carriers will include water,
saline, buffers, and other compounds described, e.g., in
the Merck Index, Merck & Co., Rahway, New Jersey.
Because of the likely high affinity binding between an
IL-1~ and its receptors, low dosages of these reagents
would be initially expected to be effective. Thus,
dosage ranges would ordinarily be expected to be in
amounts lower than 1 mM concentrations, typically less
than about 10 ~M concentrations, usually less than about
100 nM, preferably less than about 10 pM (picomolar), and
most preferably less than about 1 fM (femtomolar), with
an appropriate carrier. Slow release formulations, or
slow release apparatus will often be utilized for
continuous administration.
IL-1~ fragments thereof, and antibodies or its
fragments, antagonists, and agonists, may be administered
directly to the host to be treated or, depending on the
size of the compounds, it may be desirable to conjugate
them to carrier proteins such as ovalbumin or serum
albumin prior to their administration. Therapeutic
formulations may be administered in any conventional
dosage formulation. While it is possible for the active
ingredient to be administered alone, it is preferable to
present it as a pharmaceutical formulation. Formulations
comprise at least one active ingredient, as defined
above, together with one or more acceptable carriers
thereof. Each carrier must be both pharmaceutically and
physiologically acceptable in the sense of being
compatible with the other ingredients and not in~urious
to the patient. Formulations include those suitable for
oral, rectal, nasal, or parenteral (including
CA 022~906 l998-ll-l9
W097/44468 PCT~S97/07282
subcutaneous, intramuscular, intravenous and intradermal)
administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any
methods well known in the art of pharmacy. See, e.g.,
Gilman, et al. (eds) (1990) Goodman and Gilman's: The
Pharmacolo~ical Bases of Thera~eutics, 8th Ed., Pergamon
Press; and Reminqton's Pharmaceutical Sciences, 17th ed.
(1990), Mack Publishing Co., Easton, Penn.; Avis, et al .
(eds.) (1993) Pharmaceutical Dosaae Forms: Parenteral
Medications Dekker, NY; Lieberman, et al . (eds.) (1990)
Pharmaceutical Dosaqe Forms: Tablets Dekker, NY; and
Lieberman, et al . (eds.) ~1990) Pharmaceutical Dosaqe
Forms: Dis~erse S~stems Dekker, NY. The therapy of this
invention may be combined with or used in association
with other therapeutic agents.
The description of the IL-lr ligand herein provides
means to identify a receptor, as described above. Such
receptor should bind specifically to the IL-l~ with
reasonably high affinity. Various constructs are made
available which allow either labeling of the IL-l~ to
detect its receptor. For example, directly labeling
IL-ly, fusing onto it markers for secondary labeling,
e.g., FLAG or other epitope tags, etc., will allow
detection of receptor. This can be histological, as an
affinity method for biochemical purification, or labeling
or selection in an expression cloning approach. A
two-hybrid selection system may also be applied making
appropriate constructs with the available IL-l~
sequences. See, e.g., Fields and Song (1989) Nature
340:245-246.
The broad scope of this invention is best understood
with reference to the following examples, which are not
intended to limit the inventions to the specific
embodiments.
CA 02255906 1998-11-19
W 097/44468 44 PCT~US97/07282
EXAMPLES
I. General Methods
Some of the standard methods are described or
referenced, e.g., in Maniatis, et al . ( 1982) Molecular
Cloninq, A Laboratorv Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor Press; Sambrook, et al.
(1989) Molecular Cloninq: A LaboratorY Manual, (2d ed.),
vols 1-3, CSH Press, NY; Ausubel, et al., RioloqY, Greene
Publishing Associates, Rrooklyn, NY; or Ausu~el, et al.
(1987 and Supplements) Current Protocols in Molecular
Bioloqv, Greene/Wiley, New York. Methods for protein
purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g.,
Ausubel, et ~1. (1987 and periodic supplements);
Deutscher (1990) "Guide to Protein Purification" in
Methods in Enzymoloqv, vol. 182, and other volumes in
this series; and manufacturer's literature on use of
protein purification products, e.g., Pharmacia,
Piscataway, N.J., or Bio-Rad, Richmond, CA. Combination
with recombinant techniques allow fusion to appropriate
segments, e.g., to a FLAG sequence or an equivalent which
can be fused via a protease-removable se~uence. See,
e.g., Hochuli (1989) Chemische Industrie 12:69-70;
Hochuli (1990) "Purification of Recombinant Proteins with
Metal Chelate Absorbent" in Setlow (ed.) Genetic
Enqineerina. Princi~le and Methods 12:87-98, Plenum
Press, N.Y.; and Crowe, et al. (1992) OIAex~ress: The
Hiqh Level Ex~ression & Protein Purification Svstem
QUIAGEN, Inc., Chatsworth, CA.
Computer sequence analysis is performed, e.g., using
available software programs, including those from the GCG
(U. Wisconsin~ and GenBank sources. Public sequence
databases were also used, e.g., from G~nRAnk and others.
CA 022~906 l998-ll-l9
W097/44468 4 PCT~S97/07282 --
Many techniques applicable to IL-4 and IL-10 may be
applied to IL- 1~, as described, e.g., in U.S. Patent No.
5,017,691 (IL-4), U.S.S.N. 07/453,951 (IL-10), and
U.S.S.N. 08/110,683 (IL-10 receptor).
II. Amplification of human IL-l~ fragment by PCR
Two primers are selected (see SEQ ID NO: 1). RT-PCR
is used on an appropriate mRNA sample selected for the
presence of message to produce a cDNA, e.g., an activated
human monocyte cell sample.
III. Tissue distribution of human IL-l~
Message for the gene encoding IL-ly has been
detected in dendritic cells. Expression is high in day 5
dendritic cells, LPS activated dendritic cells, GM-CSF
and IL-4 treated dendritic cells, and in a mixture of
dendritic cells. High expression has not been detected
in B cells or NK cells. Low levels have been detected in
some monocyte cell cDNA libraries. Expression is
generally not detected in fetal tissue, though low levels
have been seen in fetal spleen, lung, and small
intestine. Low levels are also detected in fetal gall
bladder, and adult tonsil.
IV. Production of mouse IGIF protein
A GST fusion construct was engineered for expression
in E. coli. Protein was expressed, and purified using
standard procedures. Similar methods are applicable to
human IL~
A mouse IGIF pGex plasmid was constructed and
transformed into E. coli. Freshly transformed cells were
grown in LB medium containing 50 ~g/ml ampicillin and
induced with IPTG (Sigma, St. Louis, MO). After
overnight induction, the bacteria were harvested and the
pellets cont~;n;ng IGIF were isolated. The pellets were
homogenized in TE buffer (50 mM Tris-base pH 8.0, 10 mM
EDTA and 2 mM pefabloc) in 2 liters. This material was
CA 0225~906 1998-11-19
W O 97/44468 46 PCTrUS97107282 --
passed through a microfluidizer (Microfluidics, Newton,
MA) three times. The fluidized supernatant was spun down
on a Sorvall Gs-3 rotor for 1 h at 13,000 rpm. The
resulting supernatant containing the IGIF was filtered
and passed over a glutathione-SEPHAROSE column
equilibrated in 50 mM Tris-base pH 8Ø The fractions
containing the IGIF-GST fusion protein were pooled and
cleaved with thrombin (Enzyme Research Laboratories,
Inc., South Bend, IN). The cleaved pool was then passed
over a Q-SEPHAROSE column equilibrated in 50 mM
Tris-base. Fractions containing IGIF were pooled and
diluted in cold distilled H20, to lower the conductivity,
and passed back over a fresh Q-Sepharose column. The
fractions containing IGIF were pooled, aliquoted, and
stored in the -70~ C freezer.
Comparision of the CD spectrum with mouse IL-l~
suggests that the protein is correctly folded. See
Hazuda, et al . ~1969) J. Biol. Chem. 264:1689-1693.
V. Biological Assays with mouse IGIF
Biological assays confirmed the IFN-~ inducing
activity on T cells. IGIF stimulates production of IFN-
~by purified NK cells, and that induction is strongly
synergized with IL-12 or IL-2. Similar assays will be
performed with human IL-l~.
Mouse IGIF does not appear to function efficiently
on human cell, e.g., through the human receptor.
Mouse IGIF has been established to induce IFN-
~by splenocytes from SCID mice, as described above.
The splenocytes thus express a receptor for mouse IGIF.
The induction also is synergized with IL-12 or IL-2.
Mouse IGIF, in combination with IL-12 (and TNF-~)
also induced IL-2 activated NK cells to produce IFN-~.
This induction, in the case of IL-2 activated NK cells,
appears to be IL-l~ dependent, as treatment with
anti-IL-l~ blocks the IFN-~ production. The mouse IGIF
can overcome this block and induce IFN-
~
CA 022~906 l998-ll-l9
W097/44468 47 PCT~S97/07282
VI. Preparation of antibodies specific for IL-l~
Inbred Balb/c mice are immunized intraperitoneally
with recombinant forms of the human protein, e.g.,
purified soluble IL-l~ FLAG or stable transfected NIH-3T3
cells. Animals are boosted at appropriate time points
with protein, with or without additional adjuvant, to
further stimulate antibody production. Serum is
collected, or hybridomas produced with harvested spleens.
Alternatively, Balb/c mice are immunized with cells
transformed with the gene or fragments thereof, either
endogenous or exogenous cells, or with isolated membranes
enriched for expression of the antigen. Serum is
collected at the appropriate time, typically after
numerous further administrations. Various gene therapy
techniques may be useful, e.g., in producing protein in
situ, for generating an immune response.
Monoclonal antibodies may be made. For example,
splenocytes are fused with an appropriate fusion partner
and hybridomas are selected in growth medium by standard
procedures. Hybridoma supernatants are screened for the
presence of antibodies which bind to the human IL~
e.g., by ELISA or other assay. Antibodies which
specifically recognize human IL-l~ but not species
variants may also be selected or prepared.
In another method, synthetic peptides or purified
protein are presented to an immune system to generate
monoclonal or polyclonal antibodies. See, e.g., Coligan
(1991) Current Protocols in Immunoloqy Wiley/Greene; and
Harlow and Lane (1989) Antibodies: A Laborator~ Manual
~old Spring Harbor Press. In appropriate situations, the
binding reagent is either labeled as described above,
e.g., fluorescence or otherwise, or immobilized to a
substrate for panning methods. Nucleic acids may also be
introduced into cells in an animal to produce the
antigen, which serves to elicit an immune response. See,
e.g., Wang, et al. (1993) Proc. Nat'l. Acad. Sci.
90:4156-4160; Barry, et al. (1994) BioTechniaues 16:616-
619; and Xiang, et al. (1995) Immunitv 2: 129-135.
CA 0225~906 1998-ll-l9
W O 97/44468 48 PCT/US97/07282
VII. Production of fusion proteins with IL-l~
Various fusion constructs are made with IL-l~. This
portion of the gene is fused to an epitope tag, e.g., a
FLAG tag, or to a two hybrid system construct. See,
e.g., Fields and Song (1989) Nature 340:245-246.
The epitope tag may be used in an expression cloning
procedure with detection with anti-FLAG antibodies to
detect a binding partner, e.g., receptor for the IL-l~.
The two hybrid system may also be used to isolate
proteins which specifically bind to IL-l~.
VIII. Mapping of human IL-l~ by in situ hybridization
Chromosome spreads are prepared. In situ
hybridization is performed on chromosome preparations
obtained from phytohemagglutinin-stimulated human
lymphocytes cultured for 72 h. 5-bromodeoxyuridine is
added for the final seven hours of culture (60 ~g/ml of
medium), to ensure a posthybridization chromosomal
banding of good quality.
An appropriate fragment, e.g., a PCR fragment,
amplified with the help of primers on total B cell cDNA
template, is cloned into an appropriate vector. The
vector is labeled by nick-translation with 3H. The
radiolabeled probe is hybridized to metaphase spreads as
described in Mattei, et al. (1985) Hum. Genet. 69:327-
331.
After coating with nuclear track emulsion (KODAK
NTB2), slides are exposed, e.g., for 18 days at 4~ C.
To avoid any slipping of silver grains during the
banding procedure, chromosome spreads are first stained
with buffered Giemsa solution and metaphase
photographed. R-banding is then performed by the
fluorochrome-photo~ysis-Giemsa (FPG) method and
metaphases rephotographed before analysis.
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W097/44468 49 PCT~S97/07282
IX. Structure activity relationship
Information on the criticality of particular
residues is determined using standard procedures and
analysis. Standard mutagenesis analysis is performed,
e.g., by generating many different variants at determined
positions, e.g., at the positions identified above, and
evaluating biological activities of the variants. This
may be performed to the extent of determining positions
which modify activity, or to focus on specific positions
to determine the residues which can be substituted to
either retain, block, or modulate biological activity.
Alternatively, analysis of natural variants can
indicate what positions tolerate natural mutations. ~his
may result from populational analysis of variation among
individuals, or across strains or species. Samples from
selected individuals are analysed, e.g., by PCR analysis
and sequencing. This allows evaluation of population
polymorphisms.
X. Isolation of a Receptor for Human IL-l~
A human IL-l~ can be used as a specific binding
reagent to identify its binding partner, by taking
advantage of its specificity of binding, much like an
antibody would be used. A binding reagent is either
labeled as described above, e.g., fluorescence or
otherwise, or immobilized to a substrate for panning
methods.
The binding composition is used to screen an
expression library made from a cell line which expresses
a binding partner, i.e. receptor. Standard staining
techniques are used to detect or sort intracellular or
surface expressed receptor, or surface expressing
transformed cells are screened by panning. Screening of
intracellular expression is performed by various st~ining
or immunofluorescence procedures. See also McMahan,
et al. (1991) EMBO J. 10:2821-2832.
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WO 97/44468 PCT~US97/07282
For example, on day 0, precoat 2-chamber permanox
slides with 1 ml per chamber of fibronectin, 10 ng/ml in
PBS, for 30 min at room temperature. Rinse once with
PBS. Then plate COS cells at 2-3 x 105 cells per chamber
in 1.5 ml of growth media. Incubate overnight at 37' C.
On day 1 for each sample, prepare 0.5 ml o~ a
solution of 66 ~g/ml DEAE-dextran, 66 ~M chloro~uine, and
4 ~g DNA in serum free DME. For each set, a positive
control is prepared, e.g., of human IL-1~-FLAG cDNA at
1 and 1/200 dilution, and a negative mock. Rinse cells
with serum free DME. Add the DNA solution and incubate
5 hr at 37' C. Remove the medium and add 0.5 ml 10% DMSO
in DME for 2.5 min. Remove and wash once with DME. Add
1.5 ml growth medium and incubate overnight.
On day 2, change the medium. On days 3 or 4, the
cells are fixed and stained. Rinse the cells twice with
Hank's Buffered Saline Solution (HBSS) and fix in
4~ paraformaldehyde (PFA)/glucose for 5 min. Wash 3X
with HBSS. The slides may be stored at -80- C after all
liquid is removed. For each chamber, 0.5 ml incubations
are performed as follows. Add HBSS/saponin (0.1%) with
32 ~l/ml of 1 M NaN3 for 20 min. Cells are then washed
with HBSS/saponin lX. Add human IL-1~ or IL-l~/antibody
complex to cells and incubate for 30 min. Wash cells
twice with HBSS/saponin. If appropriate, add first
antibody for 30 min. Add second antibody, e.g., Vector
anti-mouse antibody, at 1/200 dilution, and incubate for
30 min. Prepare ELISA solution, e.g., Vector Elite ABC
horseradish peroxidase solution, and preincubate for
30 min. Use, e.g., 1 drop of solution A ~avidin) and
1 drop solution B (biotin) per 2.5 ml HBSS/saponin. Wash
cells twice with HBSS/saponin. Add ABC HRP solution and
incubate for 30 min. Wash cells twice with HBSS, second
wash for 2 min, which closes cells. Then add Vector
diaminobenzoic acid (DAB) for 5 to 10 min. Use 2 drops
of buffer plus 4 drops DAB plus 2 drops of H2O2 per 5 ml
of glass distilled water. Carefully remove chamber and
rinse slide in water. Air dry for a few minutes, then
CA 022~906 1998-11-19
W097/44468 51 rCT/US97/07282
add 1 drop of Crystal Mount and a cover slip. Bake for
5 min at 85-90 C.
Evaluate positive staining of pools and
progressively subclone to isolation of single genes
responsible for the binding.
Alternatively, IL-l~ reagents are used to affinity
purify or sort out cells expressing a receptor. See,
e.g., Sambrook, et al . or Ausubel, et al .
Another strategy is to screen for a membrane bound
receptor by panning. The receptor cDNA is constructed as
described above. The ligand can be immobilized and used
to immobilize expressing cells. Immobilization may be
achieved by use of appropriate antibodies which
recognize, e.g., a FLAG sequence of a IL-l~ fusion
construct, or by use of antibodies raised against the
first antibodies. Recursive cycles of selection and
amplification lead to enrichment of appropriate clones
and eventual isolation of receptor expressing clones.
Phage expression libraries can be screened by human
IL-l~. Appropriate label techniques, e.g., anti-FLAG
antibodies, will allow specific labeling of appropriate
clones.
Many modifications and variations of this invention
can be made without departing from its spirit and scope, as
will be apparent to those skilled in the art. The specific
embodiments described herein are offered by way of example
only, and the invention is to be limited by the terms of
the appended claims, along with the full scope of
equivalents to which such claims are entitledi and the
invention is not to be limited by the specific embodiments
that have been presented herein by way of example.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Schering Corporation
(B) STREET: 2000 Galloping Hill Road
(C) CITY: Kenilworth
(D) STATE: New Jersey
(E) COUNTRY: U.S.A.
(F) POSTAL CODE (ZIP): 07033-0530
(G) TELEPHONE: 908-298-2906
(H) TELEFAX: 908-298-5388
(I) TELEX:
(ii) TITLE OF INVENTION: Human Cytokine
(iii) NUMBER OF SEQUENCES: 2
(iv) CO~l~u'~ K READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: Apple Macintosh
(C) OPERATING SYSTEM: Macintosh 7. 5.3
(D) SOFTWARE: Microsoft Word 5.la
(v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/651,998
(B) FILING DATE: 20-MAY-199 6
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1380 base pairs
t~) TYPE: nucleic acid
tC) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 022~906 1998-ll-l9
W097/44468 53 PCT~S97/07282
(ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 435..1016
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
AAAGAGATAC TCAGAAAGAG GTACAGGTTT TGGAAGGCAC AGAGCCCCAA CTTTTACGGA 60
AGAAAAGATT TCATGAAAAT AGTGATATTA CATTAAAAGA AGTACTCGTA TCCTCTGCCA 120
CTTTATTTCG ACTTCCATTG CCCTAGGAAA GAGCCTGTTT GAAGGCGGGC CCAAGGAGTG 180
CCGACAGCAG TCTCCTCCCT CCACCTTCTT CCTCATTCTC TCCCCAGCTT GCTGAGCCCT 240
TTGCTCCCCT GGCGACTGCC TGGACAGTCA GCAAGGAATT GTCTCCCAGT GCATTTTGCC 300
CTCCTGGCTG CCAACTCTGG CTGCTAAAGC GGCTGCCACC TGCTGCAGTC TACACAGCTT 360
CGGGAAGAAG AAAGGAACCT CAGACCTTCC AGATCGCTTC CTCTCGCAAC AAACTATTTG 420
TCGCCAGAAT AAAG ATG GCT GCT GAA CCA GTA GAA GAC AAT TGC ATC AAC 470
Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn
1 5 10
TTT GTG GCA ATG AAA TTT ATT GAC AAT ACG CTT TAC TTT ATA GCT GAA 518
Phe Val Ala Met Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu
15 20 25
GAT GAT GAA AAC CTG GAA TCA GAT TAC TTT GGC AAG CTT GAA TCT AAA 566
Asp Asp Glu Asn Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys
30 35 40
TTA TCA GTC ATA AGA AAT TTG AAT GAC CAA GTT CTC TTC ATT GAC CAA 614
Leu Ser Val Ile Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln
45 50 55 60
GGA AAT CGG CCT CTA TTT GAA GAT ATG ACT GAT TCT GAC TGT AGA GAT 662
Gly Asn Arg Pro Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp
65 70 75
AAT GCA CCC CGG ACC ATA TTT ATT ATA AGT ATG TAT AAA GAT AGC CAG 710
Asn Ala Pro Arg Thr Ile Phe Ile Ile Ser Net Tyr Lys Asp Ser Gln
80 85 90
CCT AGA GGT ATG GCT GTA ACT ATC TCT GTG AAG TGT GAG AAA ATT TCA 758
Pro Arg Gly Met Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser
g5 100 105
ACT CTC TCC TGT GAG AAC AAA ATT ATT TCC TTT AAG GAA ATG AAT CCT 806
Thr Leu Ser Cys Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro
110 115 120
CCT GAT AAC ATC AAG GAT ACA AAA AGT GAC ATC ATA TTC TTT CAG AGA 854
Pro Asp Asn Ile Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg
125 130 135 140
.
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W O 97/44468 54 PCTrUS97/07282
AGT GTC CCA GGA CAT GA~ AAT AAG ATG CAA TTT GAA TCT TCA TCA TAC 902
Ser Val Pro Gly His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr
145 150 155
GAA GGA TAC TTT CTA GCT TGT GAA AAA GAG AGA GAC CTT TTT AAA CTC 950
Glu Gly Tyr Phe Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu
160 165 170
ATT TTG AAA AAA GAG GAT GAA TTG GGG GAT AGA TCT ATA ATG TTC ACT 998
Ile Leu Lys Lys Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr
175 180 185
GTT CAA AAC GAA GAC TAG CTATTAAAAT TTCATGCCGG GCGCAGTGGC 1046
Val Gln Asn Glu Asp
190
TCACGCCTGT AATCCCAGCC CTTTGGGAGG CTGAGGCGGG CAGATCACCA GAGGTCAGGT 1106
GTTCAAGACC AGCCTGACCA ACATGGTGAA ACCTCATCTC TACTAAAAAT ACAAAAAATT 1166
AGCTGAGTGT AGTGACCCAT GCCCTCAATC CCAGCTACTC AAGAGGCTGA GGCAGGAGAA 1226
TCACTTGCAC TCCGGAGGTG GAGGTTGTGG TGAGCCGAGA TTGCACCATT GCGCTCTAGC 1286
CTGGGCAACA ACAGCAAAAC TCCATCTCAA AAAATAAAAT AAATAAATAA ACAAATAAAA 1346
AATTCATAAT GTGAAAAAAA AAAAAAAAAA AAAG 1380
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met
1 5 10 15
Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn
Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile
Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro
Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg
Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met
Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys
100 105 110
CA 02255906 l998-ll-l9
W 097/44468 55 PCT~US97/07282
Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile
115 120 125
Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly
130 135 140
His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe
145 150 155 160
Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys
165 170 175
Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu
180 185 190
Asp
