Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 96/07739 1 ~ PCIIUS9S/12037
- 2199609
,
Description
INTERLEUKrN-1 TYPE 3 RECEPTORS
5 Technical Field
The present invention relates generally to cell surface receptors, and
more specifically, to Interleukin-l Type 3 receptors.
Back~round Of The Invention
Interleukin-1 ("IL-l") is a cytokine which is known to be a key mediator
of immunological and pathological responses to stress, infection and antigenic challenge
(Oppenheimetal.,Immunol. Today 7:45-46, 1986;Dinarello,FASEBJ. 2:108-115,
1988; and Mizel, FASEB J. 3:2379-2388, 1989). In addition, IL-l is known to have a
variety of effects on the brain and central nervous system. For example, IL-l has been
15 post~ ted to be involved in the induction of fever (Kluger, Physiol. Rev. 71:93-127,
1991), increased duration of slow wave sleep (Opp et al., Am. J. Physiol. 260:R52-R58,
1991), decreased appetite (McCarthyetal., Am. J. Clin. Nu~r. 42:1179-1182, 1985),
activation of the hypothalamic-pituitary-adrenal ("~PA") axis (Woloski et al., Science
230:1035-1037, 1985), and inhibition ofthe hypothalamic-pituitary-gonadal axis (River
20 and Vale, Endocrinology 124:2105-2109, 1989).
In light of the above-noted effects of IL-l (as well as many others),
substantial effort has been undertaken in order to identify receptors for IL-l. Briefly, at
least two types of receptors are known to be expressed on the surface of certain immune
cells in both human and murine derived lines. Type I receptors bind both IL-la and
25 IL-1,~, and can be found on T cells, fibroblasts, keratinocytes, endothelial cells, synovial
lining cells, chondrocytes and hepatocytes (U.S. Patent Nos. 4,968,607, 5,081,228, and
5,180,812; Cl~i~o~ e et al., PNAS 86:8029-8033, 1989; Dinarello et al., Blood
7~:1627-1652, 1991). Type II receptors can be found on various B cell lines, incluriin~
the Raji human B-cell Iymphoma line (Bomsztyk et al., PNAS 86:8034-8038, 1989;
30 Horuk et al., J. Biol. Chem. 262:16275-16278, 1987, Horuk and McCubrey, Biochem.
J. 260:657-663, 1989).
The present invention provides new, previously llni~P.n~ified Interleukin
receptors, desi~n~te~ Interleukin-l Type 3 receptors ("IL-1-3R"). In addition, the
present invention provides compositions and methods which utilize such receptors, as
35 well as other, related advdnlages.
WO 96/07739 21 9 9 6 0 9 PCT/US95/12037
Summary of the Invention
Briefly statèd, the present invention provides compositions and methods
which comprise Interleukin-l Typ~3 receptors. Within one aspect of the present
invention isolated nucleic acjd~molècules are provided which encode Interleukin-l Type
5 3 receptors. Within one embodiment, the isolated nucleic acid molecules comprise the
sequence of nucleotides in Sequence I.D. No. 1, from nucleotide number 129 to
nucleotide number 1814. Within another embodiment, the isolated nucleic acid
molecules encode a protein having the amino acid sequence of Sequence I.D. No. 2,
from amino acid number 1 to amino acid number 562. Within other embodiments,
10 isolated nucleic acid molecules are provided in Sequence I.D. No. 3, from nucleotide
number 89 to nucleotide number 1771. Within another embodiment, the nucleic acidmolecules encode a protein having the amino acid sequence of Sequence I.D. No. 4,
from amino acid number 1 to amino acid number 561. Nucleic acid molecules which
encode IL-1 Type 3 receptors ofthe present invention may be isolated from virtually any
15 warm-blooded animal, inchl-ling for example, humans, macaques, horses, cattle, sheep,
pigs, dogs, cats, rats and mice.
Within related aspects of the present invention, isolated nucleic acid
molecules are provided which encode soluble Interleukin-1 Type 3 receptors. Within
one embodiment, the isolated nucleic acid molecules comprise the sequence of
20 nucleotides in Sequence I.D. No. 1, from nucleotide number 129 to nucleotide
number 1136. Within other embodiments, the isolated nucleic acid molecules encode a
protein having the amino acid sequence of Sequence I.D. No. 2, from amino acid
number 1 to amino acid number 336. Within another embodiment, the nucleic acid
molecules comprise the sequence of nucleotides in Sequence I.D. No. 3, from nucleotide
25 number 89 to nucleotide number 1102. Within yet another embodiment, the nucleic acid
molecules encode a protein having the amino acid sequence of Sequence I.D. No. 4,
from amino acid number 1 to amino acid number 338. As above, nucleic acid molecules
which encode soluble IL-1 Type 3 receptors of the present invention may be isolated
from virtually any warm-blooded animal, including for example, hllm~n~7 macaques,
30 horses, cattle, sheep, pigs, dogs, cats, rats and mice.
Within other aspects of the present invention, eAl lession vectors are
provided which are capable of t,.p-essing the above-described nucleic acid molecules.
Within one embodim~nt, such vectors comprise a promoter operably linked to one of the
above-described nucleic acid molecules. Within other embotlim~nt~, reco~l~bina.ll viral
35 vectors are provided which are capable of directing the ~ eSSiOn of one of the above
described nucleic acid molecules. Representative examples of such viral vectors include
retroviral vectors, adenoviral vectors, and herpes simplex virus vectors. Also provided
WO 96/07739 2 1 9 9 6 0 9 PCT/US95/12037
by the present invention are host cells co"lain,ng one of the above-described
rcco",bina"l vectors.
Within other aspects of the present invention, isolated Interleukin-1 Type
3 receptors are provided. Within one embodiment, such receptors have the amino acid
5 sequence of Sequence I.D. No. 2, from amino acid number 1 to amino acid number 562.
Within another embodiment, the receptors have the sequence of Sequence I.D. No. 4,
from amino acid number 1 to amino acid number 561. Within yet further aspects ofthe
invention, isolated soluble Interleukin-1 Type 3 receptors are provided. Within one
embodiment, the isolated soluble Interleukin-l Type 3 receptors have the amino acid
10 sequence of Sequence I.D. No. 2, from amino acid number I to amino acid number 336.
Within another embodiment, the soluble receptors have the sequence of Sequence I.D.
No. 4, from amino acid number I to amino acid number 338.
Within other aspects of the invention, isolated antibodies capable of
specifically binding to an Interleukin-l Type 3 receptor are provided. Within one
15 embodiment, the antibody may be selected from the group consisting of polyclonal
antibodies, monoclonal antibodies, and antibody fragments. Within other embodiments,
antibodies are provided which are capable of blocking the binding of IL-1 to an
Interleukin-1 Type 3 receptor. Within plerellcd embodiments, the antibody is selected
from the group consisting of murine and human antibodies. In addition to antibodies,
20 the present invention also provides hybridomas which produces an antibody as described
above.
Within yet another aspect of the present invention, nucleic acid molecules
are provided which are capable of specifically hybridizing to a nucleic acid molecule
çnco-ling any of the Interleukin-l Type 3 receptors described above. Such molecules
25 may be between at least "y" nucleotides long, wherein "y" is any integer between 14 and
2044, and furthe"llGle, may be selected suitable for use as probes or primers described
below. Particularly prere" cd probes of the present invention are at least 18 nucleotides
in length.
These and other aspects of the present invention will become evident
30 upon l crel ence to the following detailed description and ~ttac~ed ~ wi"gs. In addition,
various rcÇclences are set forth below which describe in more detail certain procedures
or compositions (e.g., pl~mids7 etc.), and are therefore incorporated by reference in
their entirety.
35 Brief Des~ ,lion of the Drawin~s
Figure 1 schematically illustrates a rat IL-1 type 3 receptor.
WO 96/07739 ' PCT/US95/12037
2199609,. ~
Figure 2 is a table which lists the homology of a human IL-l type 3
receptor with its rat homologue, and other interleukin r ecel)lol~.
Figure 3 is a graph which shows stimulation of a reporter product via a
human IL-1 type 3 receptor.
Figure 4 is a graph which shows the ~ es~ion pattern of the IL-l Type
3 receptor based upon RNA prote~;lion assays.
Figures 5A and B are two graphs wh ich show inhibition of thymocyte
proliferation by soluble IL-1 receptors.z
10 Detailed Description of the Invention
Definitions
Prior to setting forth the invention, it may be helpful to an understanding
thereof to set forth definitions of certain terms to be used hereinafter.
"Interleukin-l Type 3 Receptors" ("IL-1-3R") refers to receptor proteins
15 which bind Interleukin-l (a or ,~), and, when expressed on a cell surface, tr~n.cduce the
signal provided by Interleukin-l to the cell, thereby rne~ ting a biological effect within
the cell. In their native configuration, IL-1 Type 3 receptors exist as membrane bound
proteins, consisting of an extracellular domain, transmembrane domain, and intr~cPllul~r
domain (see Figure 1). IL-1-3R may be di~ting~ hed from other Interleukin-1 receptors
20 based upon criteria such as affinity of substrate binding, tissue distribution, and sequence
homology. For example, IL-1-3R of the present invention should be greater than 50%
homologous, preferably greater than 75% to 80% homologous, more preferably greater
than 85% to 90% homologous, and most preferably greater than 92%, 95%, or 97%
homologous to the IL-1-3R disclosed herein (e.g, Sequence I.D. No. 1). As utilized
25 within the context ofthe present invention, IL1-3R should be understood to include not
only the proteins which are disclosed herein, but su~s~ y similar derivatives and
analogs as di.~cu~sed below.
"Soluble Interleukin-l Type 3 Receptor" ("sIL1-3R") refers to a protein
which has an amino acid sequence corresponding to the extracelll.l~r region of an
30 Interleukin-l Type 3 receptor. ~he extracellular region of IL-1-3R may be readily
deterrnined by a hydrophobicity analysis utili7ing a computer program such as
PROTEAN (DNASTAR, Madison, WI), or by an ~lignmPnt analysis with other known
type 1 and type 2 Interleukin-l . ecepto, ~.
"Nucleic acid molecule" refers to a nucleic acid polymer or nucleic acid
35 sequence, which exists in the form of a separate fragment or as a component of a larger
nucleic acid construct. The nucleic acid molecule must have been derived from nucleic
acids isolated at least once in subslal~lially pure form, (i.e., subslal~lially free of
- 2199609 '
wo 96/07739 PCr/USg5/12037
con~..;n~l;n~ endogenous materials), and in a quantity or concentration enabling- identification and recovery. Such sequences are pre~el~bly provided in the form of an
open reading frame uninterrupted by internal no~ cl~tecl sequences, or introns. As
utilized herein, nucleic acid molecules should be understood to include deoxyribonucleic
5 acid ("DNA") molecules (in~lutling genomic and cDNA molecules), ribonucleic acid
("RNA") molecules, hybrid or chimeric nucleic acid molecules (e.g., DNA-RNA
hybrids), and where app~up.iate, nucleic acid molecule analogs and derivatives (e.g.,
peptide nucleic acids ("PNA")). Nucleic acid molecules of the present invention may
also comprise sequences of non-translated nucleic acids where such additional sequences
10 do not interfere with manipulation or eAplession of the open reading frame (e.g.,
sequences which are 5' or 3' from the open reading frame).
"Reco~..binant eA~I es~ion vector" refers to a replicable nucleic acid
construct used either to amplify or to express nucleic acid sequences which encode IL-I
Type 3, or sIL-l Type 3 receptors. This construct comprises an assembly of (1) a15 genetic element or elements having a regulatory role in gene ~A~,ression, for example,
promoters, and (2) the structural or coding sequence of interest. The recon.bi~
eA~,t;ssion vector may also comprise approp.iate transcription and l-~nslalion initiation
and te--.~h~alion sequences.
As noted above, the present invention provides isolated nucleic acid
molecules encoding Interleukin-l Type 3 ~eceplols. One representative IL-l Type 3
receptor which may be obtained utilizing the methods described herein (see, e.g.,
Example 1) is schematically illustrated in Figure 1. Briefly, this IL-1 Type 3 receptor
(see Sequence I.D. Nos. 1 and 2) is composed of an EAtracellul~r N-terminal Domain
25 (amino-acids 1 - 336), a T-~ns.l.~b.ane Domain (amino acids 337 - 357), and a C-terminal Intracell~ r Domain (35~ - 562).
Although the above IL-1 Type 3 receptor has been provided for purposes
of illustration (see also Sequence I.D. Nos. 3 and 4), the present invention should not be
so limited. In particular, "IL-1-3R" and "sIL-1-3R" as utilized herein should beunderstood to include a wide variety of IL-1 Type 3 receptors which are encoded by
nucleic acid molecules that have substantial similarity to the sequences disclosed in
Sequences I.D. Nos. 1 and 3. As utilized within the context of the present invention,
nucleic acid molecules which encode IL-1 Type 3 receptors are de~omed to be
sllb~ lly similar to those disclosed herein if: (a) the nucleic acid sequence is derived
from the coding region of a native IL-1 Type 3 receptor gene (incl~ ing, for cA~,--ple,
allelic variations of the sequences disclosed herein); (b) the nucleic acid sequçnce is
capable of hybridization to nucleic acid sequences of the present inventionunder
WO 96/07739 PCT/US95/12037
2199609 6
,.., ~; ~
conditions of either moderat~ e.g, 50% fo~ a~ide, 5 x SSPE, 5 x Denhardt's, 0.1%SDS, 100 ug/ml Salmon Sperm DNA, and a temperature of 42~C) or high ~ling~ncy
(~e Sambrook et al., Moleczllar Cloning: A Labora1 ~ry Manual, 2d Ed., Cold Spring
Harbor Laboratory Press, NY, 1989); or (c) nucleic acid sequPnces are degenerate as a
5 result of the genetic code to the nucleic acid sequences defined in (a) or (b).
Furthermore, as noted above, although DNA molecules are primarily referred to herein,
as should be evident to one of skill in the art given the disclosure provided herein, a
wide variety of related nucleic acid molecules may also be utilized in various
embodiments described herein, including for example, RNA, nucleic acid analogues, as
10 well as chimeric nucleic acid molecules which may be composed of more than one type
of nucleic acid.
In addition, as noted above, within the context of the present invention
"IL-l Type 3 receptors" and "soluble IL-I Type 3 receptors" should be understood to
include derivatives and analogs of the IL-1 Type 3 receptors described above. Such
derivatives include allelic variants and genetically engineered variants that contain
conservative amino acid substitutions and/or minor additions, substitutions or deletions
of amino acids, the net effect of which does not substantially change the biological
activity (e.g., signal transduction) or function of the IL-1 Type 3 receptor. Such
derivatives are generally greater than about 50% homologous, preferably greater than
75% to 80% homologous, more preferably greater than 85% to 90% homologous, and
most ple~rably greater than 92%, 95% or 97% homologous. Homology may be
dete~ined, for example, by compa~ing sequence il,ro""alion using the GAP computer
program, version 6.0, available from the University of Wisconsin Genetics Computer
Group (UWGCG).
The primary amino acid structure of IL-1 Type 3 receptors may also be
modified by derivatizing amino acid side chains, and/or the amino or carboxy termus
~,vith various functional groups, in order to allow for the formation of various conjugates
(e.g., protein-IL-1-3R conjugates). Alternatively, conjugates of IL-1-3R (and sIL-1-3R)
may be constructed by reco"lbinanlly producing fusion proteins. Such fusion proteins
may colll~,lise, for example, IL-1-3R-protein Z wherein protein Z is another cytokine
receptor (e.g., L-2R, IL-3R, IL-4R, IL-5R, lL-6R, IL-7R, IL-8R, IL-9R, IL-lOR, IL-
11R, IL-12R, IL-13R, IL-14R, IL-15R or TNF (a or ~) receptor; see W091/03553); abinding portion of an antibody; a toxin (as di~cussed below); or a protein or peptide
which facilit~tes purification or identification of IL-1-3R (e.g., poly-His). For e,.a",plc,
a fusion protein such as human IL-1-3R (His)n or sIL-1-3R (His)n may be constructed in
order to allow purification of the protein via the poly-His residue, for example, on a
Nl A nickel-r~ ing column. The amino acid sequence of a IL- 1 Type 3 receptor may
WO 96t07739 2 1 9 9 6 0 9 PCT/US95/12037
also be linked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK)
(Sequence I.D. No. 5) (Hopp et al., Bio/Technology 6:1204, 1988) in order to f~çilit~te
purification of expressed reco...bin~.lt protein.
The present invention also includes IL-1-3R (and sIL-1-3R) proteins
5 which may be produced either with or without associated native-pattern glycosylation.
For example, eAples~ion of IL-1-3R DNAs in bacteria such as E. coli provides non-
glycosylated molecules. In contrast, IL-1-3R expressed in yeast or ~anll~alian
~AI,Ies~ion systems (as diQcl~ssed below) may vary in both glycosylation pattern and
molecular weight from native IL-1-3R, depending on the amino acid sequence and
10 ~Apression system which is utilized. In addition, functional mut~nhQ of m~mm~ n IL-I-
3R having inactivated glycosylation sites may also be produced in a homogeneous,reduced-carbohydrate form, utilizing oligonucleotide synthesis, site-directed
mutagenesis, or random mutagenesis techniques. Briefly, N-glycosylation sites ineukaryotic proteins are generally characterized by the amino acid triplet Asn-Al-Z,
15 where Al is any amino acid except Pro, and Z is Ser or Thr. In this triplet, asparagine
provides a side chain amino group for covalent attachment of carbohydrate. Such sites
may be el;...;n~ed by deleting Asn or Z, substituting another amino acid for Asn or for
residue Z, or inserting a non-Z amino acid between Al and Z, or an amino acid other
than Asn between Asn and Al.
Proteins which are substantially similar to IL-1-3R proteins may also be
constructed by, for example, substihuting or deleting various amino acid residues which
are not required for biological activity. For example, cysteine residues may be deleted
or replaced with other amino acids to prevent formation of incorrect intramolecular
riiQ~ fide bridges upon renaturation. Similarly, adjacent dibasic amino acid residues may
be modified for CA~UI es~ion in yeast systems in which KEX2 protease activity is present.
Not all mutations in the nucleotide sequence which encodes IL-1-3R will
be eApl essed in the final product. For cAalll?le, nucleotide substitutions may be made in
order to avoid secondary structure loops in the transcribed mRNA, or to provide codons
that are more readily tr~nQl~ted by the selected host, and thereby ~nh~nce eAI,res~ion
within a selected host.
Generally, substitutions àt the amino acid level should be made
conservatively, i.e., the most ple~lred substihlte amino acids are those which have
characteristics resembling those of the residue to be replaced. When a substitution,
deletion, or insertion strategy is adopted, the potential effect of the deletion or insertion
on biological activity should be considered utili7.ing for example, the sign~lling assay
disclosed within the Examples.
WO 96/07739 PCT/US9~/12037
2199609~ 8
. . ,~ i,
?
Mutations which are made to the sequence of the nucleic acid molecules
of the present invention should generally preserve the reading frame phase of the coding
sequences. Furthermore, the mutations should pl~r~lably not create complç...~ ..y
regions that could hybridize to produce secondaly mRNA structures, such as loops or
5 ha,ll,;ns, which would adversely affect llall~lalion of the receptor mRNA. Although a
mutation site may be predetermined, it is not necessdly that the nature of the mutation
per se be predetermined. For example, in order to select for optimum characteristics of
mut~nt~ at a given site, random mutagenesis may be conducted at the target codon, and
the expressed IL-1-3R mnt~ntS screened for the biological activity. Representative
10 methods for random mutagenesis include those described by Ladner et al. in U.S. Patent
Nos. 5,096,815; 5,198,346; and 5,223,409.
As noted above, mutations may be introduced at particular loci by
synthesizing oligonucleotides col,lai~ing a mutant sequence, flanked by restriction sites
enabling ligation to fr~gm~nts of the native sequence. Following ligation, the resulting
reconstructed sequence encodes an analog having the desired anino acid insertion,
substitution, or deletion.
Alternatively, site-directed mutagenesis procedures may be employed to
provide an altered gene having particular codons altered according to the substitution,
deletion, or insertion required. Exemplary methods of making the alterations set forth
above are disclosed by Walder et al. (Gene ~2:133, 1986); Bauer et al. (Gene 37:73,
1985); Craik, Bio Techniques, January 1985, 12-19); Smith et al. (Genetic Engineering
Principles and Methods, Plenum Press, 1981); Sambrook et al. (Molecular cloning: A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989); and U.S.
Patent Nos. 4,518,584 and 4,737,462, which are incorporated by rererence herein.IL-l Type 3 receptors, as well as substantially similar derivatives or
analogs may be used as therapeutic reag~r ts, immunogens, reagents in receptor-based
immuno~cs~ys, or as binding agents for affinity purification procedures. Moreover, IL-1
Type 3 receptors of the present invention may be utilized to screen compounds for IL-1
Type 3 receplor agonist or antagonistic activity. IL-1 Type 3 receptor proteins may also
be covalently bound through reactive side groups to various insoluble substrates, such as
cyanogen bromine-activated, biso~i~ ane-activated, carbonyldiimid~701e-activated, or
tosyl-activated, agarose structures, or by adsorbing to polyolefin surfaces (with or
without glutaraldehyde cross-linking). Once bound to a substrate, IL-1-3R may be used
to selectively bind (for purposes of assay or purification) anti-IL-1-3R antibodies or l:L-
1.
WO 96/07739 2 1 ~ g 6 ~ 9 PCT/US95/12037
.,_ 9
ISOLATION OF IL-1 TYrF 3 RECErr-)R cDNA CLONES
- As noted above, the present invention provides isolated nucleic acid
molecules which encode IL-1 Type 3 receptG,s. Briefly, nucleic acid molecules which
encode IL-l Type 3 receptors of the present invention may be readily isolated from a
5 variety of warm-blooded ~nimAlc, including for example, h-lmAnc, macaques, horses,
cattle, sheep, pigs, dogs, cats, rats and mice. Particularly prefelled tissues from which
nucleic acid molecules which encode IL-1 Type 3 receptors may be isolated include
brain, kidney and lung. Nucleic acid molecules which encode IL-l Type 3 receptors of
the present invention may be readily isolated from conventionally prepared cDNA
10 libraries (see, e.g., Sambrook et al., Molecular C10~1ing: A ~aboratory Manual, 2d Ed.,
Cold Spring Harbor Laboratory Press, NY, 1989) or from commercially obtained
libraries (e.g., Stratagene, LaJolla, Calif.) utilizing the disclosure provided herein.
Particularly p.efelled methods for obtaining isolated DNA molecules which encode IL-l
Type 3 receptors of the present invention are described in more detail below in Example
15 1 (see also Sequence I.D. Nos. 1 and 3).
As noted above, within particularly prert- I ed embo-~imentc of the
invention, isolated nucleic acid molecules are provided which encode human IL-1 Type
3 receptors. Briefly, such nucleic acid molecules may be readily obtained by probing a
human cDNA library either with a specific sequence as described below in Example 1, or
20 with a rat sequence (e.g, Sequence I.D. Nos. 2 or 4) under conditions of highstringency (e.g., 50% forrnamide, 5 x SSC, 5x Denharts, 0.1% SDS, 100 ug/ml salmon
sperrn DNA, at 42~C for 12 hours). This may be followed by extensive washing with 2x
SSC col-tAi~ g 0.2% SDS at 50~C. Suitable cDNA libraries may be obtained from
commercial sources (e.g., Stratagene, LaJolla, Calif.; or Clontech, Palo Alto, Calif., or
25 prepa,t:d utili7ing standard techniques (see, e.g,. Sambrook et al., supra).
PRODUCTION OF RECOMBINANT IL- I TYPE 3 RECEPTORS
As noted above, the present invention also provides reco",binan~
eA~"ession vectors which include synthetic or cDNA-derived DNA fragm~nts encoding
30 IL-I Type 3 receptors or substantially similar proteins, which are operably linked to
suitable transcriptional or l,~nslalion regulatory rl~..e~lls derived from ~ ,Ali~n
microbial, viral or insect genes. Such regulatory ~le...e~.lc include a transcriptional
promoter, an optional operator sequence to control l~nsc~iplion, a sequence encoding
suitable mRNA ribosomal binding sites, and, within plefe,led embo~irn~ntc, sequences
35 which control the te~ h-alion of llanscliplion and translation. The ability to replicate in
a host, usually cGnrelled by an origin of replication, and a selection gene to f~c-ilitA~te
recognition of l~a,~ a~ may additionally be incorporated. DNA regions are
WO 96/07739 21 9 9 6 0 9 PCTIUS95/12037
~ ~10
3 ~
operably linked when they are functionally related to each other. For example, DNA for
a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is
l A~lessed as a precursor which participates in the secretion of the polypeptide; a
promoter is operably linked to a coding sequence if it controls the llallscl;plion of the
sequence; or a ribosome binding site is operably linked to a coding sequence if it is
positioned so as to permit translation. Generally, operably linked means contiguous and,
in the case of secretory leaders, contiguous and in reading frame.
Expression vectors may also contain DNA sequences necessary to direct
the secretion of a polypeptide of interest. Such DNA sequences may include at least one
10 secretory signal sequence. Representative secretory signals include the alpha factor
signal sequence (pre-pro sequence; Kurjan and Herskowitz, Cell 30:933-943, 1982;Kurjan et al., U.S. Patent No. 4,546,082; Brake, EP 116,201), the PHO5 signal
sequence (Beck et al., WO 86/00637), the BARI secretory signal sequence (MacKay
et al., U.S. Patent No. 4,613,572; MacKay, WO 87/002670), the SUC2 signal sequence
15 (Carlson et al., Mol. CelL Biol. 3:439-447, 1983), the a-l-antitrypsin signal sequence
(Kurachi etal., Proc. NatL Acad. Sci. U~A 78:6826-6830, 1981), the ~-2 plasmin
inhibitor signal sequence (Tone et al., J. Biochem. (Tokyo) 102: 1033-1042, 1987), the
tissue plasminogen activator signal sequence (Pennica et al., Nature 301:214-221,
1983), the E. coli PhoA signal sequence (Yuan et al., J. Biol. Chem. 265:13528-13552,
20 1990) or any of the bacterial signal sequences reviewed, for e,.a.,~ple, by Oliver (Ann.
Rev. Microbiol. 39:615-649, 1985). Alternatively, a secretory signal sequence may be
synthesized accolding to the rules established, for example, by von Heinje (Eur. J.
Biochem. 133:17-21, 1983; J. Mol. Biol. 18~:99-105, 1985; Nuc. AcidsRes. 14:4683-
4690, 1986).
For eAI,lession, a nucleic acid molecule encoding a IL-1 Type 3 receptor
is inserted into a suitable e,.l)-e~sion vector, which in turn is used to transforrn or
lla~lsre~l applopliate host cells for ~Ap.e~ion. Host cells for use in practicing the
present invention include "~A"""~ n, avian, plant, insect, bacterial and fungal cells.
Pler~lled eukaryotic cells include cultured ~ n cell lines (e.g, rodent or humancell lines) and filngal cells, inclurlinE~ species of yeast (e.g, Saccharomyces spp.,
particularly S. cerevisiae, Schizos~qccharomyces spp., or Kluyveromyces spp.) orfil~mçntQus fungi (e.g, Aspergillus spp., Neurospora spp.). Strains of the yeastSacc~., o,~,yces cerevisiae are particularly pl ef~. - ed. Methods for producingrecoml)inalll proleins in a variety of prokaryotic and eukaryotic host cells are generally
known in the art (see "Gene Expression Technology," Methods in Enzymology, Vol.
185, Goeddel (ed.), ~c~dçmic Press, San Diego, Calif., 1990; see also, "Guide to Yeast
Genetics and Molecular Biology," Melhods in Enzymology, Guthrie and Fink (eds.)
WO 96/07739 2 1 9 9 6 ~ 9 PCI~/US95/12037
riemic Press, San Diego, Cali~, 1991). In general, a host cell will be selected on the
basis of its ability to produce the protein of interest at a high level or its ability to carry
out at least some of the processing steps ~-ece~C~,-y for the biological activity of the
protein. In this way, the number of cloned DNA sequences which must be llansrecle
5 into the host cell may be ;;;~ed and overall yield of biologically active protein may
be ,naAi.";zed.
Suitable yeast vectors for use in the present invention include YRp7
(Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1978), YEpl3 (Broach et al.,
Gene 8:121-133, 1979), POT vectors (Kawasaki etal., U.S. Patent No. 4,931,373,
which is incorporated by reference herein), pJDB249 and pJDB219 (Beggs, Nature
275:104-108, 1978) and derivatives thereof. Such vectors will generally include a
selectable marker, which may be one of any number of genes that exhibit a dominant
phenotype for which a phenotypic assay exists to enable transro~nanls to be selected.
Plefe"ed selectable Illalkel~ are those that complement host cell auxotrophy, provide
antibiotic resi~t~nce or enable a cell to utilize specific carbon sources, and include rlr-u2
(Broach et al., ibid.), URA3 (Botstein et al., Gene 8:17, 1979), HI53 (Struhl et al., ibid.)
or POTl (Kawasaki et al., ibid.). Another suitable select~ble marker is the CAT gene,
which confers chloramphenicol resistance on yeast cells.
Preferred promoters for use in yeast include promoters from yeast
glycolytic genes (Hitzeman etal., J. Biol. Chem. 255:12073-12080, 1980; Alber and
Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982; Kawasaki, U.S. Patent No.
4,599,311) or alcohol dehydrogenase genes (Young et al., in Gene~icEr~ c~ir~ of
Microorganisms for Chemicals, Hollaender et al. (eds.), p. 355, Plenum, New York,
1982; A,rlllleler, Me~h En~ymol. 101:192-201, 1983). In this regard, particularly
plerelled promoters are the TPII promoter (Kawasaki, U.S. Patent No. 4,599,311,
1986) and the ADH2-4C promoter (Russell et al., Na~ure 304:652-654, 1983; Irani and
Kilgore, U.S. Patent Application Serial No. 07/784,653, which is incorporated herein by
reference). The ~A~ ssion units may also include a ~Idllsc~il,Lional terminator, such as
the lPII te-lllindlor (Alber and Kawasaki, ibid.).
In addition to yeast, proteins of the present invention can be cApressed in
fil~m~ntous fungi, for example, strains of the fungi Aspergillus (McKnight et al., U.S.
Patent No. 4,935,349, which is incorporated herein by lerelence). Examples of useful
promoters include those derived from Aspergill1ls nidulans glycolytic genes, such as the
ADH3 promoter (McKnight et al., EMBO J. ~:2093-2099, 1985) and the ~7iA promoter.
An ~,Aalllple of a suitable lellllillaLor is the ADH3 terminator (McKnight et al., ibid.,
1985). The t..pression units utilizing such collll)ollents are cloned into vectors that are
capable of insertion into the chromosomal DNA of Aspergillus.
WO 96/07739 P~ u:,9~/12037
2 1 9 9 6sO~
12
Techniques for transforming fungi are well known in the literature, and
have been described, for instance, by Beggs (ibid.), Hinnen et al. (Proc. NatL Acad. Sci.
USA 75:1929-1933, 1978), Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1740-1747,1984), and Russell (Nature 301:167-169, 1983). The gen~lyl~e of the host cell will
5 generally contain a genetic defect that is complemented by the selectable marker present
on the e ,~,.ession vector. Choice of a particular host and select~ble marker is well
within the level of ordinary skill in the art. To opl;.,.i~e production of the heterologous
proteins in yeast, for example, it is prel~l, ed that the host strain carries a mutation, such
as the yeast pep4 mutation (Jones, Genetics 85:23-33, 1977), which results in reduced
10 proteolytic activity.
In addition to fi~ngal cells, cultured lllallllmalian cells may be used as host
cells within the present invention. Preferred cultured l~ n cells for use in the
present invention include the COS-I (ATCC No. CRL 1650), COS-7 (ATCC No. CRL
1651), BHK (ATCC No. CRL 1632), and 293 (ATCC No. CRL 1573; Graham et al., J.
15 Gen. Virol. 36:59-72, 1977) cell lines. A plefelled BHK cell line is the BE~ 570 cell
line (deposited with the American Type Culture Collection under accession number CRL
10314). In addition, a number of other mammalian cell lines may be used within the
present invention, including Rat Hep I (ATCC No. CRL 1600), Rat Hep II (ATCC No.CRL 1548), TCMK (ATCC No. CCL 139), Human lung (ATCC No. CCL 75.1),
20 Human hepatoma (ATCC No. HTB-52), Hep G2 (ATCC No. HB 8065), Mouse liver
(ATCC No. CCL 29.1), NCTC 1469 (ATCC No. CCL 9.1), SP2/0-Agl4 (ATCC No.
1581), HIT-T15 (ATCC No. CRL 1777), Ltk- (ATCC) No. CCL 1.3) and RlNm
5AHT2B (Orskov and Nielson, FEBS 229(1):175-178, 1988).
Mammalian ~ - es~ion vectors for use in carrying out the present
25 invention should include a promoter capable of directing the transcription of a cloned
gene or cDNA. Preferred promoters include viral promoters and cellular promoters.
Viral promoters include the immecli~te early cytomegalovirus promoter (Boshart et al.,
Cell 41:521-530, 1985) and the SV40 promoter (Sub.~uani et al., Mol. Cell. Biol.1:854-864, 1981). Cellular plo-..otel~ include the mouse metallothionein-1 promoter
30 (Paln~iter et al., U.S. Patent No. 4,579,~21), a mouse Vj promoter (Bergman et al.,
Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983; Grant et al., Nuc. Acids Res. 15:5496,
1987) and a mouse VH promoter (Loh et al., Cell 33:85-93, 1983). A particularly
prere..t;d promoter is the major late promoter from Adenovirus 2 (~ n and Sharp,Mol. Cell. Biol. 2:1304-13199, 1982). Such e,.~,~tssion vectors may also contain a set
35 of RNA splice sites located dow--sl-~ from the promoter and upstream from the DNA
sequence encoding the peptide or protein of interest. Pl el~--ed RNA splice sites may be
obtained from SV40, adenovirus and/or immunoglobulin genes. Alternatively, within
W096/07739 2I9~6:09 PCTrUS95/12037
- 13
certain embo~im~ntc RNA splice sites may be located downstream from the DNA
sequence encoding the peptide or protein of interest. Also co~ ined in the eAI,res~ion
vectors is a polyadenylation signal located dow.,sl.ea... of the coding sequence of
interest. Suitable polyadenylation signals include the early or late polyadenylation
5 signals from SV40 (~ n and Sharp, ibid.), the polyadenylation signal from the
Adenovirus S ElB region and the human growth holl,.one gene tel--unalor (DeNoto
et al., Nuc. Acids Res. 9:3719-3730, 1981). The eApl es~ion vectors may include a
noncoding viral leader sequence, such as the Adenovirus 2 tripartite leader, located
between the promoter and the RNA splice sites. P~fe~ed vectors may also include
10 enhancer sequences, such as the SV40 enhancer and the mouse l enhancer (Gillies, Cell
33:717-728, 1983). Expression vectors may also include sequences encoding the
adenovirus VA RNAs. Suitable vectors can be obtained from commercial sources (e.g.,
Invitrogen, San Diego, CA; Stratagene, La Jolla, CA).
Cloned DNA sequences may be introduced into cultured .nA.~....~li~n cells
15 by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725,
1978; Corsaro and Pearson, Somatic Ce~l Gene~ics 7:603, 1981; Graham and Van derEb, Virolo~y 52:456, 1973), electroporation ~eumann et al., E~BO J. 1:841-845,
1982), or DEAE-dextran mediated transfection (Ausubel et al. (eds.), Current Protocols
in Molecular Biolof~y, John Wiley and Sons, Inc., NY, 1987), which are incorporated
20 herein by reference. To identify cells that have stably integrated the cloned DNA, a
select~kle marker is generally introduced into the cells along with the gene or cDNA of
interest. Pler~ ed selectable markers for use in cultured m~nm~ n cells include genes
that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The
selectable marker may be an amplifiable sçlect~ble marker. Pler~.,ed amplifiable25 sçlect~hle n~a~ke~ are the DHFR gene and the neomycin recict~nce gene. S~lect,ble
".a-kers are reviewed by Thilly (Mammalian Cell Technolo0!, Butte~wo~lh Publishers,
Stoneham, MA, which is incorporated herein by reference). The choice of sçlect~ble
markers is well within the level of ordinary skill in the art.
Selectable markers may be introduced into the cell on a separate vector
30 at the same time as the IL-l Type 3 receptor seq~çnce, or they may be introduced on the
same vector. If on the same vector, the selectable marker and the IL-1 Type 3 rece~,lor
sequence may be under the control of di~ele..l p-o...ole-~ or the same promoter, the
latter arrangement producing a dicistronic message. Constructs of this type are known
in the art (for example, Levinson and Simonsen, U.S. Patent No. 4,713,339). It may
35 also be advantageous to add additional DNA, known as "carrier DNA" to the mixture
which is introduced into the cells.
WO 96/07739 PCT/US95/12037
21996b~ 14
Tlansr~;led ,~ç~."",Ali~n cells are allowed to grow for a period of time,
typically 1-2 days, to begin cA~lcs~;ng the DNA sequence(s) of interest. Drug selectio
is then applied to select for growth of cells that are c,.,~ - essing the select~kle marker in a
stable fashion. For cells that have been l~ ~n~ ;led with an amplifiable selectable marker
5 the drug conc~l,l-alion may be increased in a stepwise manner to select for increased
copy number of the cloned sequences, thereby increasing e,.l,.ession levels. Cells
cA~"essii1g the introduced sequences are selected and screened for production of the
protein of interest in the desired form or at the desired level. Cells which satisfy these
criteria may then be cloned and scaled up for production.
~lere--ed prokaryotic host cells for use in carrying out the present
invention are strains of the bacteria Escherichia coli, although Bacillus and other genera
are also useful. Techniques for transforming these hosts and expressing foreign DNA
sequences cloned therein are well known in the art (see, e.g, Maniatis et al., Molecular
Cloning A LaboratoryManual, Cold Spring Harbor Laboratoly, 1982; or Sambrook
15 et al., supra). Vectors used for eApl essing cloned DNA sequences in bacterial hosts will
generally contain a selectable marker, such as a gene for antibiotic re~i~t~nce, and a
promoter that functions in the host cell. Appropriate promoters include the trp (Nichols
and Yanofsky, Mefh Enzymol. 101:155-164, 1983), lac (Cac~d~ban et al., J. Bacteriol.
143:971-980, 1980), and phage k (Queen, J. Mol. Appl. Genet. 2:1-10, 1983) promoter
20 systems. Plasmids useful for transro,l,.ig bacteria include pBR322 (Bolivar et al., Gene
2:95-113, 1977), the pUC plasmids (Messing, Meth. En~ymol. 101:20-78, 1983; Vieira
and Messing, Gene 19:259-268, 1982), pCQV2 (Queen, ibid.), pMAL-2 (New F.ngl~nd
Biolabs, Beverly, MA) and derivatives thereof. Plasmids may contain both viral and
bacterial elements
Given the teachin~ provided herein, promoters, terminators and methods
for introducing e~yres~ion vectors encoding IL-1 Type 3 receptors of the presentinvention into plant, avian and insect cells would be evident to those of skill in the art.
The use of baculoviruses, for example, as vectors for e,.p,essing heterologous DNA
sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28:215-
224,1990). In addition, the use of Agrohac~erium rhizogenes as vectors for e,.p,ess"lg
genes in plant cells has been reviewed by Sinkar et al. (J. Biosci. (Bangalore) 11 :47-58,
1987).
Host cells co~ -g DNA molecules of the present invention are then
cultured to express a DNA molecule encoding a IL-1 Type 3 receptor. The cells are
35 cultured according to ~landald methods in a culture medium col.l~il-;g nutrients
required for growth of the chosen host cells. A variety of suitable media are known in
the art and generally include a carbon source, a nitrogen source, ess.onti~l amino acids,
WO 96/07739 2 1 9 g 6 ~ 9 . PCT/US95/12037
vitamins and minerals, as well as other components, e.g, growth factors or serum, that
- may be required by the particular host cells. The growth medillm will generally select
for cells co..l~ g the DNA molecules by, for example, drug selection or deficiency in
an essenti~l nutrient which is colnl)lemented by the select~ble marker on the DNA
S construct or co-ll ~Isr~;led with the DNA construct.
Suitable growth conditions for yeast cells, for example, include culturing
in a chemically defined medium, comprising a nitrogen source, which may be a non-
amino acid nitrogen source or a yeast extract, inorganic salts, vitamins and e.~enti~l
amino acid supplements at a temperature between 4~C and 37~C, with 30~C being
10 particularly prerelled. The pH of the medium is preferably m~int~ined at a pH greater
than 2 and less than 8, more preferably pH 5-6. Methods for rn~int~ining a stable pH
include buffering and con~Lal~l pH control. Plerel-ed agents for pH control include
sodium hydroxide. Plerell~d buffering agents include succinic acid and Bis-Tris (Sigrna
Chemical Co., St. Louis, MO). Due to the tendency of yeast host cells to
15 hyperglycosylate heterologous proteins, it may be preferable to express the IL-l Type 3
receptors of the present invention in yeast cells having a defect in a gene required for
asparagine-linked glycosylation. Such cells are pl efel ~bly grown in a metlium co.~ ng
an osmotic stabilizer. A prefelled osmotic stabilizer is sorbitol supplemented into the
medium at a concentration between O.l M and 1.5 M, preferably at 0.5 M or 1.0 M.20 Cultured m~mm~ n cells are generally cultured in commercially available serum-
co..l~ g or serum-free media. Selection of a me~ and growth conditions
appro~,liate for the particular cell line used is within the level of ordhlai y skill in the art.
IL-l Type 3 receptors may also be eAI)Iessed in non-human ~ sgel~ic
anim~lc, particularly ll ansgenic warm-blooded ~nim~l~ Methods for producing
25 transgenic ~nim~ , inçluding mice, rats, rabbits, sheep and pigs, are known in the art and
are disclosed, for c,~lnple, by Hammer et al. (Nature 315:680-683, 1985), Palmiter
et al. (Science 222:809-814, 1983), Brinster et al. (Proc. Na~l. Acad. Sci. USA 82:4438-
4442, 1985), Palmiter and Brinster (Cell 41:343-345, 1985) and U.S. Patent No.
4,736,866, which are incorporated herein by rel~rellce. Briefly, an eA~ ssion unit,
30 inr,lu~1in~ a DNA seqll~?nr,e to be ~AI~Iessed together with applopl;alely positioned
c.~pl-ession control sequences, is introduced into pronuclei of fertilized eggs.Introduction of DNA is commonly done by microinjection. Integration of the injected
DNA is detected by blot analysis of DNA from tissue samples, typically samples of tail
tissue. It is generally prerel~d that the introduced DNA be incorporated into the germ
35 line ofthe animal so that it is passed on to the animal's progeny.
Within particularly pl efel l ed embodiments of the invention, "knockout"
animals may be developed from embryonic stem cells through the use of homologous
WO 96/07739 2 1 9 9 6 0 9 PCT/US95/12037
16
reco",l~ dlion (Capecchi, Science 24~:1288-1292, 1989) or ~ntic~nce oligonucleotide
(Stein and Chen, Science 261(5124):1004-1012, 1993; Milligan etal., Semin. Conc.Biol. 3(6):391-398, 1992).
Within a ple;relled embodiment of the invention, a ~l~nsgenic animal,
5 such as a mouse, is developed by targeting a mutation to disrupt a IL- 1 Type 3 receptor
sequence (see Mansour et al., "Disruption of the proto-oncogene int-2 in mouse
embryo-derived stem cells: A general strategy for targeting mutations to non-selectable
genes," Na~1~re 336:348-352, 1988). Such animals may readily be utilized as a model to
study the role ofthe IL-1 Type 3 receptor in metabolism.
SOLUBLE IL I TYPE 3 R~c~rrroR~ ANr~ RFCEPTOR PEPTIDES
As noted above, the present invention also provides soluble IL-l Type 3
receptors and receptor peptides. Within the context of the present invention, IL-1 Type
3 receptor peptides should be understood to include portions of a IL-I Type 3 receptor
15 or derivatives thereof ~liccucced above, which do not contain transmembrane domains,
and which are at least 8, and more preferably 10 or greater amino acids in length.
Briefly, the structure of the IL-1 Type 3 receptor as well as putative transmembrane
domains may be predicted from the primary translation products using the
hydrophopicity plot function of, for example, PROTEAN (DNA STAR, Madison, WI),
20 or according to the methods described by Kyte and Doolittle (J. Mol. Biol. I57:105-
132, 1982). While not wishing to be bound by a graphical representation, based upon
this hydrophopicity analysis, IL-I Type 3 receptors are believed to have the general
structure shown in Figure 1. In particular, these receptors are believed to comprise an
extracçll~ r amino-terminal domain, a ~ nl~ )lane domain, and an intrac~ll..l~r
25 domain.
Within one aspect of the invention, isolated IL-1 Type 3 receptor
peptides are provided comprising the extracell~ r amino-terminal domain of a IL-1
Type 3 receptor. Within a prerell~d embodiment, an isolated IL-I Type 3 receptorpeptide is provided comprising the sequence of amino acids shown in Sequence I.D.
30 No.2, ~om amino acid number 1 to amino acid number 336. Within other embodimentQ
isolated IL-1 Type 3 receptor peptides are provided cG.llplis;.lg the ceq~ence of amino
acids shown in Sequence I.D. No. 4, from amino acid number I to amino acid
number 338.
IL-I Type 3 receptor peptides may be prepaled by, among other
35 methods, culturing suitable host/vector systems to produce the recG--.bina..l translation
products ofthe present invention. Supe-nala.lts from such cell lines may then be treated
by a variety of purification procedures in order to isolate the IL-I Type 3 receplor
WO 96/07739 PCr/US95/12037
21199609
peptide. For example, the supclllalalll may be first concentrated using co".l"ercially
available protein conc~ lion filters, such as an Amicon or Millipore Pellicon
ultrafiltration unit. Following concel~l-alion, the concentrate may be applied to a
suitable purification matrix such as, for e,~a".ple, IL-l or an anti-IL-l Type 3 receptor
S antibody bound to a suitable support. Alternatively, anion or cation exchange resins
may be employed in order to purify the receptor or peptide. Finally, one or morereversed-phase high pclrollnal~ce liquid ch,c"--atography (RP-HPLC) steps may beemployed to further purify the IL-I Type 3 receptor peptide.
Alternatively, IL-I Type 3 receptor peptides may also be plcpared
10 utili7ing standard polypeptide synthesis protocols, and purified utili7:ing the above-
described procedures.
A IL-l Type 3 receptor peptide is deemed to be "isolated" or purified
within the context of the present invention, if only a single band is detected subsequent
to SDS-polyacrylamide gel analysis followed by staining with Coomassie Brilliant Blue.
ANrIsoDE~ TO IL- 1 TYPE 3 RECEPTORS
Within one aspect of the present invention, IL-l Type 3 receptors,
inl~lurling derivatives thereof, as well as portions or fragments of these proteins such as
the IL-l Type 3 receptor peptides discussed above, may be utilized to prepare antibodies
20 which specifically bind to IL-l Type 3 receptors. Within the context of the present
invention the term "antibodies" includes polyclonal antibodies, monoclonal antibodies,
fr~&m~nts thereof such as F(ab')2 and Fab fragments, as well as reco,nbilla~lly produced
binding partners. These binding partners incorporate the variable regions from a gene
which encodes a specifically binding monoclonal antibody. Antibodies are defined to be
25 specifically binding if they bind to the IL- 1 Type 3 receptor with a KA of greater than or
equal to 107 M-1 and prcre~ably greater than or equal to 108M-l, and bind to IL-l Type
I or Type II receptors with an affinity of less than KA 107 M-l, and plerclably less than
105M-1 or 103M-1. The affinity of a monoclonal antibody or binding partner may be
readily detc"",ned by one of ordinary skill in the art (~e Scalclla,d, Ann. N.Y. AcaG~.
30 Sci. 51:660-672, 1949).
Polyclonal antibodies may be readily generated by one of ordinary skill in
the art from a variety of warm-blooded animals such as horses, cows, goats, sheep,
dogs, chickens, rabbits, mice, or rats. Briefly, the IL-l Type 3 receptor is utilized to
immllni7e the animal through illllape~iloneal~ intram~lscul~r~ intraocular, or subcut~neous
35 injections. The immllnogenicity of a IL-l Type 3 receptor or IL-1 Type 3 receplor
peptide may be increased through the use of an adjuvant such as Freund's complete or
inco~..pl~te adjuvant. Following several booster immunizations, small samples of serum
WO g6/07739 PCTnUS95/12037
21996~9 18
. ~
't .
are collected and tested for reactivity to the IL-l Type 3 receptor. A variety of assays
may be utilized in order to detect antibodies which specifically bind to a IL-l Type 3
receptor. Exemplary assays are described in detail in An~ibodies: A Laboratory Mar~ual,
Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988. Representative
S examples of such assays include: Countercurrent Immuno-Electrophoresis (CIEP),
Radioimmunoassays, Radioimmunopl eci~;lalions, Enzyme-Linked Immuno-Sorbent
Assays (ELISA), Dot Blot assays, Inhibition or Competition assays, and sandwich
assays (see U.S. Patent Nos. 4,376,110 and 4,486,530; see also An~ibodies: A
Laboratory Manual, supra). Particularly preferred polyclonal antisera will give a signal
10 that is at least three times greater than background. Once the titer of the animal has
reached a plateau in terms of its reactivity to the IL-l Type 3 receptor, larger quantities
of polyclonal antisera may be readily obtained either by weekly blee-ling~, or by
ex~n~-in~ting the animal.
Monoclonal antibodies may also be readily generated using well-known
15 techniques (see U.S. Patent Nos. PUE 32,011,4,902,614,4,543,439, and 4,411,993; see
also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses,
Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and An~ibodies: A
Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,
1988). Briefly, within one embodiment a subject animal such as a rat or mouse is
20 injected with a form of IL-l Type 3 receptor suitable for generating an immllne response
against the IL-l Type 3 receptor. Rel)lesenlali~e examples of suitable forms inc.lude,
among others, cells which express the IL- 1 Type 3 receptor, or peptides which are based
upon the IL-l Type 3 receptor sequence. Additionally, many techniques are known in
the art for incleas;ng the result~nt immune response, for example, by coupling the
25 receptor or receptor peptides to another protein such as ovalbumin or keyhole limpet
hemocyanin (KLH), or through the use of adjuvants such as Freund's complete or
incomplete adjuvant. The initial immunization may be through i~ ?eliloneal,
intr~mllscul~r~ intraocular, or subcutaneous routes.
Between one and three weeks after the initial immllni7~tion the animal
30 may be reimm--ni7~d with another booster immunization. The animal may then be test
bled and the serum tested for binding to the IL-l Type 3 receptor using assays as
described above. Additional immuni7~tions may also be accomplished until the animal
has pl~te~--ed in its reactivity to the IL-I Type 3 receptor. The animal may then be
given a final boost of IL-l Type 3 receptor or IL-l Type 3 receptor peptide, and three
35 to four days later sacrificed. At this time, the spleen and Iymph nodes may be harvested
and disrupted into a single cell suspension by passing the organs through a mesh screen
or by rupturing the spleen or Iymph node l"~".b,a,-es which encapsidate the cells.
21996~9
WO 96/07739 PCI/US95112037
,_ 19
Within one embodiment the red cells are subsequently Iysed by the addition of a
hypotonic solution, followed by immediate return to isotonicity.
Within another embodiment, suitable cells for pl epa. ing monoclonal
antibodies are obtained through the use of in vi~ro imm~ ;on techniques. Briefly, an
5 animal is sacrificed, and the spleen and Iymph node cells are removed as described
above. A single cell suspension is prepared, and the cells are placed into a culture
co..~ g a form of the IL-1 Type 3 receptor that is suitable for generating an immune
response as described above. Subsequently, the Iymphocytes are harvested and fused as
described below.
Cells which are obtained through the use of in vitro imm-lni7~tion or
from an imrnunized animal as described above may be immortalized by transfection with
a virus such as the Epstein-Barr virus (EBV) (see Glasky and Re~ding, Hybridoma
8(4):377-389, 1989). Alternatively, within a ptefe~ed embodiment, the harvested
spleen and/or Iyrnph node cell suspensions are fused with a suitable myeloma cell in
15 order to create a "hybridoma" which secretes monoclonal antibodies. Suitable myeloma
lines are preferably defective in the construction or ~AI,ression of antibodies, and are
additionally syngeneic with the cells from the immunized animal. Many such myeloma
cell lines are well known in the art and may be obtained from sources such as the
American Type Culture Collection (ATCC), Rockville, Maryland (see Catalogue of Cell
20 Lines & Hybridomas, 6th ed., ATCC, 1988). Repl ese,llali.~e myeloma lines include: for
hllm~n~ UC 729-6 (ATCC No. CRL 8061), MC/CAR-Z2 (ATCC No. CRL 8147), and
SKO-007 (ATCC No. CRL 8033); for mice, SP2/0-Agl4 (ATCC No. CRL 1581), and
P3X63Ag8 (ATCC No. TIB 9); and for rats, Y3-Agl.2.3 (ATCC No. CRL 1631), and
YB2/0 (ATCC No. CRL 1662). Particularly plerel~èd fusion lines include NS-l (ATCC
25 No. TIB 18) and P3X63 - Ag 8.653 (ATCC No. CRL 1580), which may be utilized for
fusions with either mouse, rat, or human cell lines. Fusion between the myeloma cell
line and the cells from the imml-ni7ed animal may be accompli.~hed by a variety of
metho~, inclu-ling the use of polyethylene glycol (PEG) (~e Anfibodies: A Laboratory
Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988) or
30 electrofusion (see ~ e~ a~ and Vienken, J. Membrane Biol. 67: 165-182, 1982).
Following the fusion, the cells are placed into culture plates co..l~ ;ng a
suitable metlil~m, such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium)(JRH Biosciences, T çn~Y~ KS). The medium may also contain additional ingredients,
such as Fetal Bovine Serum ("FBS," i.e., from Hyclone, Logan, Utah, or JRH
35 Biosciences), thymocytes which were harvested from a baby animal of the same species
as was used for immuni7~tion, or agar to solidify the me-lium Additionally, the mer1illm
should contain a reagent which selectively allows for the growth of fused spleen and
WO 96/07739 2 1 9 9 6 0 ~ ~ PCI/US95/12037
myeloma cells. Particularly pref~.led is the use of HAT (hy~,o~ Lh;ne, aminopterin,
and thymidine) (Sigma Chemical Co., St. Louis, MO). After about seven days, the
resl-hin~ fused cells or hybridomas may be sc~eened in order to determine the presence
of antibodies which recognize the IL-1 Type 3 receptor. Following several clonal5 dilutions and reassays, a hybridoma producing antibodies which bind to IL-1 Type 3
eceplor may be isolated.
Other techniques may also be utilized to construct monoclonal antibodies
(see Huse et al., "Generation of a Large Co...bina~ional Library of the lmml~noglobulin
Repertoire in Phage Lambda," Science 2~6:1275-1281, December 1989; see also Sastry
10 et al., "Cloning of the Immunological Repertoire in E.scherichia coli for Generation of
Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-
Specific cDNA Library," Proc. Na~l. Acad. .~ci. U~A 86:5728-5732, August 1989; see
also Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid
Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9, January 1990; these
15 references describe a commercial system available from Stratacyte, La Jolla, California,
which enables the production of antibodies through recombillalll techniques). Briefly,
mRNA is isolated from a B cell population and utilized to create heavy and light chain
immllnoglobulin cDNA t;,.~le~sion libraries in the klMMUNOZAP(H) and
klMMUNOZAP(L) vectors. These vectors may be screened individually or co-
20 expressed to form Fab fr~gm~nt~ or antibodies (see Huse et al., supra; see also Sastry etal., supra). Positive plaques may subsequently be converted to a non-lytic plasmid
which allows high level e,.~- es~ion of monoclonal antibody fragments from E coli.
Similarly, binding partners may also be constructed utili7ing recon.bina~l
DNA techniques to incorporate the variable regions of a gene which encodes a
25 specifically binding antibody. The construction of these proteins may be readily
accomplished by one of o-din&-y skill in the art (see Larrick et al., "Polymerase Chain
Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable
Region Genes From Single Hybridoma Cells," Biotechnolo~y 7:934-938, September
1989; Rieçl~ nl- et al., "Reshaping Human Antibodies for Therapy," Na~ure 332:323-
30 327, 1988; Roberts et al., "Generation of an Antibody with F.nh~nced Affinity andSpecificity for its Antigen by Protein Engine~,.ing," Nature 328:731-734, 1987;
Verhoeyen et al., "Resl.apil1g Human Antibodies: Grafting an Antilysozyme Activity,"
Science 239:1534-1536, 1988; Chaudhary et al., "A RecG~llbinalll lmmllnotoxin
Consisting of Two Antibody Variable Domains Fused to Pseudomonas Exotoxin,"
35 Nature 339:394-397, 1989; see also, U.S. Patent No. 5,132,405 entitled "Biosynthetic
Antibody Binding Sites"), given the disclosure provided herein. Briefly, within one
embodiment, DNA molecules encoding IL-1 Type 3 receptor-specific antigen binding
W0 96/07739 2 1 ; ' ~ ~ PCIIUS95/12037
-- 21
domains are amplified from hybridomas which produce a speçific~lly binding monoclonal
antibody, and inserted directly into the geno..~c of a cell which produces humanantibodies (see Verhoeyen et al., stlpra; see a~so Reiçllm~nn et al., supra). This
technique allows the antigen-binding site of a specifically binding mouse or rat5 monoclonal antibody to be llan~relled into a human antibody. Such antibodies are
preferable for therapeutic use in humans because they are not as antigenic as rat or
mouse antibodies.
Alternatively, the antigen-binding sites (variable region) may be either
linked to, or inserted into, another completely different protein (see Ch~udh~ry et al.,
10 supra), res~lting in a new protein with antigen-binding sites of the antibody as well as
the functional activity of the completely different protein. As one of ordinary skill in the
art will recognize, the antigen-binding sites or IL-I Type 3 receptor binding domain of
the antibody may be found in the variable region of the antibody. Furthermore, DNA
sequences which encode smaller portions of the antibody or variable regions which
15 specifically bind to "~,.,...~li~n IL-1 Type 3 receptor may also be utilized within the
context of the present invention. These portions may be readily tested for binding
specificity to the IL-1 Type 3 receptor utilizing assays described below.
Within a p.ere~l~d embodiment, genes which encode the variable region
from a hybridoma producing a monoclonal antibody of interest are amplified using20 oligonucleotide primers for the variable region. These primers may be synthesi7ed by
one of-ordinary skill in the art, or may be purchased from commercially available
sources. Stratacyte (La Jolla, CA) sells primers for mouse and human variable regions
including among others, primers for VHa, VHb, VHC, VHd, CH1~ VL and CL regions
These primers may be utilized to amplify heavy or light chain variable regions, which
25 may then be inserted into vectors such as IMMUNOZAP*(H) or IMMUNOZAP*(L)
(Stratacyte), respectively. These vectors may then be introduced into E. coli for
e"~ ion. Utilizing these techniques, large amounts of a single-chain protein
CO..~ g a fusion ofthe VH and VL domams may be produced (see Bird et al., Science
242:423-426, 1988).
Other "antibodies" which may also be ~rtl)a,ed ~Itili7ing the disclosure
provided herein, and thus which are also deemed to fall within the scope of the present
invention include h--..,~n;,ed antibodies (e.g., U.S. Patent No. 4,816,567 and
WO94/10332), micobodies (e.g, W094/09817) and llansgelic antibodies (e.g,
GB 2 272 440).
Once suitable antibodies have been obtained, they may be isolated or
purified by many techniques well known to those of ordinary skill in the art (see
Antibodies: A Laboratory Manual, .supra). Suitable techniques include peptide or
W0 96/07739 ~ '~ ;. PCT/US95tl2037
2 1 9 9 6 0 9
protein affinity columns, HPLC or RP-~LC, purification on protein A or protein Gcolumns, or any co,~binalion of these techniques. Within the context of the present
invention, the term "isolated" as used to define antibodies or binding pa,ll,e,~ means
"subst~nti~lly free of other blood components."
Antibodies of the present invention have many uses. For example,
antibodies may be utilized in flow cytometry to sort IL-l Type 3 receptor-bearing cells,
or to histochemically stain IL-l Type 3 receptor-bearing tissues. Briefly, in order to
detect IL-I Type 3 receptors on cells, the cells (or tissue) are incubated with a labeled
antibody which specifically binds to IL-I Type 3 receptors, followed by detection of the
presence of bound antibody. These steps may also be accomplished with additionalsteps such as washings to remove unbound antibody. Representative examples of
suitable labels, as well as methods for conjugating or coupling antibodies to such labels
are described in more detail below.
In addition, purified antibodies may also be utilized therapeutically to
block the binding of IL-l or other IL-I Type 3 receptor substrates to the IL-I Type 3
receptor i~7 vit~o or in vivo. As noted above, a variety of assays may be utilized to
detect antibodies which block or inhibit the binding of IL-I to the IL-l Type 3 receptor,
including in~er alia, inhibition and competition assays noted above. Within one
embodiment, monoclonal antibodies (plepared as described above) are assayed for
binding to the IL-I Type 3 receptor in the absence of IL-l, as well as in the presence of
varying concentrations of L-l. Blocking antibodies are identified as those which, for
example, bind to IL-I Type 3 receptors and, in the plesence of IL-l, block or inhibit the
binding of IL-I to the IL-l Type 3 receptor.
Antibodies of the present invention may also be coupled or conjug~ted to
a variety of other compounds (or labels) for either diagnostic or therapeutic use. Such
compounds include, for cxa",l)le, toxic molecules, molecules which are nontoxic but
which become toxic upon exposure to a second compound, and radion.-c.lides
Represe,llalive examples of such molecules are described in more detail below.
Antibodies which are to be utilized ther~pelltic.~lly are pleferably
provided in a therapeutic composition comprising the antibody or binding partner and a
physiologically acceptable carrier or diluent. Suitable carriers or dihl~nt~ inclllde, among
others, neutral buffered saline or saline, and may also include additional excipients or
stabilizers such as buffers, sugars such as glucose, sucrose, or dextrose, c~ el~ting agents
such as EDTA, and various preservatives.
219960Y
WO g6/07739 PCI/US95/12037
_ 23
LABELS
The nucleic acid molecules, antibodies, and IL-1 Type 3 receptors
(inr,lu~ling sIL-l 3R) of the present invention may be labeled or conju~ted (either
through covalent or non-covalent means) to a variety of labels or other moleculç~,
5 in~ din~ for example, fluorescc,ll markers, enzyme ~--a-kel~, toxic molecules, molecules
which are nontoxic but which become toxic upon exposure to a second compound, and
radionl.çlide~.
Represe..lali~re examples of fluorescent labels suitable for use within the
present invention include, for example, Fluorescein Isothiocyanate (FITC), Rhodamine,
10 Texas Red, Luciferase and Phycoerythrin (PE). Particularly l)lere,led for use in flow
cytometry is FITC which may be conjugated to purified antibody according to the
method of Keltkamp in "Conjugation of Fluo~escein Isothiocyanate to Antibodies.
I. Experiments on the Conditions of Conjugation," Immunology 18:865-873, 1970. (See
also Kellk~ ), "Conjugation of Fluo~escein Isothiocyanate to Antibodies. II. A
15 Reproducible Method," Immunology 18:875-881, 1970; and Goding, "Conjugation of
Antibodies with Fluorochromes: Modification to the Standard Methods," J. Immunol.
Me~hods 13:215-226, 1970.) For histochemical staining, ~P, which is preftlled, may
be conjugated to the purified antibody according to the method of Nakane and Kawaoi
("Peroxidase-Labeled Antibody: A New Method of Conjugation," J. Histochem.
20 Cytochem. 22:1084-1091, 1974; see a~so, Tijssen and Kurstak, "Highly Efficient and
Simple Methods for Plepa.~tion of Peroxidase and Active Peroxidase Antibody
Conjugates for Enzyme Tmmuno~ss~ys," A~7al. Biochem. 136:451-457, 1984).
Representative examples of enzyme markers or labels include alkaline
phosphatase, horse radish peroxidase, and ,~-galactosidase. Representative examples of
25 toxic molecules include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed
antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A. R~plesel.lali~e
examples of molecules which are nontoxic, but which become toxic upon exposure to a
second compound include thymidine kinases such as HSVTK and VZVTK.
Represenlali~e examples of radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97,
30 Tc-99m, Rh-105, Pd-109, In-111, 1-123, 1-125, 1-131, Re-186, Re-188, Au-198, Au-
199, Pb-203, At-211, Pb-212 and Bi-212.
As will be evident to one of skill in the art given the disclosure provided
herein, the above described nucleic acid moleculçc, antibodies, and IL- 1 Type 3receptors may also be labeled with other molecules such as colloidal gold, as well either
35 member of a high affinity binding pair (e.g, avidin-biotin).
WO 96/07739 2 1 ~ 9 6 Q g ; . 1~ P~I;U~ 3~I12O37
24
DLAGNOSTIC USE OF IL 1 TYPE 3 REcEproR SEQUENCES
Within another aspect of the present invention, probes and primers are
provided for detectin~ IL-l Type 3 receptors. Within one embodiment of the invention,
probes are provided which are capable of hybridizing to IL-1 Type 3 receptor DNA or
RNA. For purposes of the present invention, probes are "capable of hybridizing" to IL-
1 Type 3 receptor DNA if they hybridize to Sequence I.D. Nos. 1 or 3 under conditions
of moderate or high stringency (see Sambrook et al., supra); but not to IL-l Type I or
Type II receptor nucleic acid sequences. Preferably, the probe may be utilized to
hybridize to suitable nucleotide sequences in the presence of 50% formamide, 5x SSPE,
5x Denhardt's, 0.1% SDS and 100 ug/ml Salmon Sperm DNA at 42~C, followed by a
first wash with 2x SSC at 42~C, and a second wash with 0.2x SSC at 55 to 60~C.
Probes of the present invention may be composed of either
deoxyribonucleic acids (DNA) ribonucleic acids (RNA), nucleic acid analogues, or any
combination of these, and may be as few as about 12 nucleotides in length, usually about
14 to 18 nucleotides in length, and possibly as large as the entire sequence of the IL-l
Type 3 receptor. Selection of probe size is somewhat dependent upon the use of the
probe. For example, in order to determine the presence of various polymorphic forms of
the IL-l Type 3 receptor within an individual, a probe comprising virtually the entire
length of the IL-l Type 3 receptor coding sequence is prefe,led. IL-l Type 3 receptor
probes may be utilized to identify polymo~l,his~s linked to the IL-1 Type 3 receptor
gene (see, for example, Weber, Genomics 7:524-530, 1990; and Weber and May, Amer.
J. Hum. Gen. 44:388-396, 1989). Such polymorphisms may be associated with
inherited tlice~ces such as diabetes.
Probes may be constructed and labeled using techniques which are well
known in the art. Shorter probes of, for example, 12 or 14 bases may be generated
synthetically. Longer probes of about 75 bases to less than 1.5 kb are preferably
generated by, for example, PCR amplification in the presence of labeled precursors such
as 32P-dCTP, digoxigenin-dUTP, or biotin-dATP. Probes of more than 1.5 kb are
generally most easily amplified by transfecting a cell with a plasmid co.l~ -g the
relevant probe, growing the llan~re~led cell into large quantities, and purifying the
relevant sequence from the ll~n!7~cled cells (see Sambrook et al., supra).
Probes may be labeled by a variety of l.,alkel~., inclu~ , for cAa"~le,
radioactive ",alkel ., flu~"escelll markers, enzymatic markers, and chromogenic ",a,ke,~
The use of 32p is particularly plefe"ed for ",a,king or labeling a particular probe.
Probes of the present invention may also be utilized to detect the
presence of a IL-l Type 3 receptor mRNA or DNA within a sample. However, if IL-lType 3 receptors are present in only a limited number, or if it is desired to detect a
21 9960g; ' '
WO 96107739 P~ 3~1l2037
._ 25
se1ected mutant sequence which is present in only a limited number, or if it is desired to
clone a IL-1 Type 3 receptor from a selected warm-blooded animal, then it may bebeneficial to amplif~ the relevant sequence such that it may be more readily detected or
obtained.
A variety of methods may be utilized in order to amplify a selected
sequence, inrlutiing, for example, RNA amplification (see Lizardi et al., Bio/Technology
6:1197-1202, 1988; Kramer et al., Na~1~re 339:401-402, 1989; Lomeli et al., Clinical
Chem. 35(9):1826-1831, 1989; U.S. Patent No. 4,786,600), and DNA amplification
ntili~ing Polymerase Chain Reaction ("PCR") (.see U.S. Patent Nos. 4,683,195,
4,683,202, and 4,800,159) (see also, U S. Patent Nos. 4,876,187, and 5,011,769, which
describe an alternative detection/amplification system comprising the use of scissile
linkages).
Within a particularly preferred embodiment, PCR amplification is utilized
to detect or obtain a IL-l Type 3 receptor DNA. Briefly, as described in greater detail
below, a DNA sample is denatured at 95~C in order to generate single stranded DNA.
Specific primers, as cli~c-lssed below, are then annealed at 37~C to 70~C, depending on
the propol ~ion of AT/GC in the primers. The primers are extended at 72~C with Taq
polymerase in order to generate the opposite strand to the template. These stepsc~ r.~ le one cycle, which may be repeated in order to amplify the s~lected sequence.
Primers for the amplification of a selected sequence should be sPlected
from sequences which are highly specific and forrn stable duplexes with the target
sequence. The primers should also be non-complen~e~ y, especially at the 3' end,should not form dimers with themselves or other primers, and should not form
secondary structures or duplexes with other regions of DNA. In general, primers of
about 18 to 20 nucleotides are p~efe,lèd, and may be easily synthesi~ed using terhniques
well known in the art.
PHARMACEU rICAL COMPOS1T~ONS AND THERAPEI~TIC USES
As noted above, the present invention provides pharrn~ce~ltic~l
COIJIPOS;l;OnS~ as well as methods for using the same (for either prophylactic or
thela~eulic use). Briefly, the phaln.~ce~ltical compositions ofthe present invention may
colllplise an IL-l 3R, sIL-l 3R, antibody which is capable of specifically binding IL-
1 3R, IL-l 3R antagonists or agonists, in con.l.inalion with a pharm~ce~lti~lly
acceplable carrier, diluent, or excipient. Such compositions may colll~lise buffers such
as neutral buffêred saline, phosphate buffered saline and the like, carbohydrates such as
glucose, mannose, sucrose or dextrose, proteins, polypeptides or amino acids,
antioxidants, chelating agents such as EDTA or glutathione, and preservatives.
W O 96107739 2 1 ~ 9 6 0 9 PC~rnUS95/12037
~ ?~ ~ - 26
Compositions of the present invention may be form~ ted for the manner
of a~ n~ on indicated, inr,lu~lin~ for e Aa",ple, for oral, nasal, venous, vaginal or
rectal ~minictration. Within other embodiments, the compositions may be ~dminict~ed
as part of a sust~ined release implant (e.g, intra-articularly). Within yet other
5 embo-lim~nts/ the compositions may be formuli7ed as a Iyophilizate, ~Itili~in~ app, op, iale
excipients which provide stability as a Iyophilizate, and subsequent to rehydration.
Pharm~ceutical compositions of the present invention may be utilized in
order to treat a wide variety of diseases including, for example, immune-associated
~ice~ces such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis,
10 myacth~mi~ gravis, scleritis, scleroderma, septic shock, allogra~ rejection, and graft
versus host (GVH) disease. In particular, pharmaceutical compositions of the present
invention may be administered in a manner appropriate to the disease to be treated (or
prevented). Although approp,iate dosages may be determined by clinical trials, the
quantity and frequency of administration will be detell.,h-ed by such factors as the
15 condition of the patient, and the type and severity of the patient's disease.Within other aspects of the present invention, viral vectors are provided
which may be utilized to treat dice~ces wherein either the IL-l Type 3 receptor (or a
mutant IL-l Type 3 receptor) is over-expressed, or where no IL-l Type 3 receptor is
expressed. Briefly, within one embodiment of the invention, viral vectors are provided
20 which direct the production of ~ ;cence IL-l Type 3 receptor RNA, in order to prohibit
the over eA~Ies~ion of IL-l Type 3 receptors, or the cA~.~ssion of mutant IL-l Type 3
receptors. Within another embodiment, viral vectors are provided which direct the
CAIJI es~;on of IL-l Type 3 receptor cDNA. Viral vectors suitable for use in the present
invention include, among others, reco~bina~l vaccinia vectors (U.S. Patent Nos.
25 4,603,112 and 4,769,330), reco~ )inal~l pox virus vectors (PCT Publication No. WO
89/01973), and plerelably, reco",binalll retroviral vectors (~Reconll~ina,,l Retroviruses
with Amphotropic and Ecoplropic Host Ranges," PCT Publication No. WO 90/02806;
"Retroviral Par~ n~ Cell Lines and Processes of Using Same," PCT Publication No.WO 89/07150; and ''~ntic~nce RNA for Treal...e..l of Retroviral Disease States," PCT
30 Publication No. WO/03451), and herpesvirus vectors (~it, Adv. Exp. Med. Biol. 215:219-236, 1989; U.S. Patent No. 5,288,641).
Within various embodiments of the invention, the above-desc, ibed
compositions may be ?Idminictered in vivo, or ex vil~O. Replese"lali~re routes for in vivo
a(lminictration include intradermally ("i.d."), intracranially ("i.c."), i~ oneally
35 ("i.p."), intrathecally ("i.t."), intravenously ("i.v."), subcutaneously ("s.c.") or
intr~muscul~rly ("i.m.").
~ s - ~
W096/07739 ~ G~ PCT/USgS/12037
_ 27
Within other embodi-,.e.,ls of the invention, the vectors which contain or
express nucleic acid molecules of the present invention, or even the nudeic acidmoleculçs themselves, may be adminictered by a variety of alternative techniques,
inc~ 1in~ for ~ ."ple direct DNA injection (Acsadi et al., Nature 352:815-818, 1991);
5 microprojectile bo."bald,nel~l (Williams et al., PNAS 88:2726-2730, 1991); liposomes
(Pickering et al., Circ. 89(1):13-21, 1994; and Wang et al., PNAS8~:7851-7855, 1987);
lipofection (Felgner et al., Proc. Na~l. Acad. Sci. USA 84:7413-7417, 1989); DNAligand (Wu et al., J. of Biol. Chem. 26~:16985-16987, 1989); ~dministration of DNA
linked to killed adenovirus (Michael et al., J. Biol. Chem. 268(10):6866-6869, 1993; and
10 Curiel et al., Hum. Gene Ther. 3(2):147-154, 1992), r~tlol,ans~osons, cytofectin-
medi~tçd introduction (DMRIE-DOPE, Vical, Calif.) and transferrin-DNA complexes
(Zenke).
The following examples are offered by way of illustration, and not by
15 way of limitation.
WO96/07739 21'9960{9~ PCrlUS95/12037
EXAMPLES
EXAMPLE 1
ISOLATION OF ~RLEUK~ TYPE 3 RECEproR cDNA
A. Isolation of Interleukin-1 Type 3 Receptor cDNA From a Rat
Lun~ cDNA Library
Male Sprague-Dawley rats (Madison, WI) weighing between 175-250
10 gm are decapitated, and the lungs excised. Total RNA is then isolated from the lung
utili7ing a Promega RNAgents Total RNA Kit (catalog #Z5110, Promega, Wisc.)
according to the m~mlf~cturers instructions, followed by the isolation of poly A+ RNA
utili7ing a Promega PolyATract kit (catalog # Z5420). A cDNA phage library is then
prepared utili7ing a Giga-Pack Gold library construction kit according to the
15 m~mlf~ctllrers' instructions (catalog #237611, Stratagene, LaJolla, Cali~), which is in
turn plated and screened es.cçnti~lly as described by Sambrook et al., (Molecular
Cloning) ~vith oligonucleotide (5'-CTTCAACTGC ACATACCCTC CAGTAACAAA
CGGGGCAGTG AATCTGACAT-3') (Sequence I.D. No. 6). This oligonucleotide is
complementary to nucleotides 211-260 of the rat IL-l Type 3 receptor cDNA sequence
20 shown in Sequence I.D. No. 3.
The phage library is rescreened until a single pure phage isolate is
obtained. The phage is then grown on bacterial host XL1-Blue (Stratagene, LaJolla,
Calif.), and plasmid DNA is excised with ExAssist helper phage (Stratagene) in SOLR
cells. The SOLR cells are then plated, and plasmid DNA is isolated and sequçnced25 utili7.ing the Sanger dideoxy protocol.
A rat IL-1 Type 3 receptor cDNA sequence that may be obtained
utili7ing this procedure is set forth in Sequence I.D. No. 3.
B. Isolation of Interleukin-l Type 3 Receptor cDNA From a
30 Co~ lically Available Rat cDNA Library
IL-l Type 3 receptor cDNA can also be isolated from col.llllerc;ally
available rat cDNA libraries. For example, two million plaques from a rat phage library
(Clontech, catalog # RL1048a) may be plated according to the m~mlf~ctllrer's
instructions, and screened with oligonucleotide Sequence I.D. No. 6 essenti~lly as
35 described above.
A rat IL-l Type 3 receptor cDNA sequence that may be obtained
utili7.ing this procedure is set forth in Sequence I.D. No.3.
WO 96/07739 2 1 9 9 6 0 9 PCT/US95/12037
-~ 29
C. Isolation of IL-1 Type 3 Receptor cDNA From a Human cDNA Library
IL-l Type 3 receptor cDNA can also be i~ol~ted from cG..~.,.erc;ally
available human cDNA libraries. Briefly, applu~inlalely two million plaques from a
5 human phage library (Clontech, catalog # HL1158a) are plated according to the
m~mlf~cturers instructions, and s~- eel~ed with oligonucleotide (5'-CCTCCCATAA
CATCTGGGGA AGTCAGTGTA ACATGGTATA AAAATTCTAG C-3') (Sequence
I.D. No. 7) çssenti~lly as described above. This oligonucleotide is complementary to
nucleotides 260-310 of the human IL-I Type 3 receptor cDNA sequence shown in
10 Sequence I.D. No. I .
The phage library is lescleened and isolated as described above. The
human sequence that is obtained utilizing this procedure is appl uxlmately 89.1 %
identical at the nucleotide level and 89.2% identical at the amino acid level to that of the
common region ofthe above-described rat L-l Type 3 receptors.
EXAMPLE 2
EXPRESSION OF IL I TYPE 3 RECEPTOR cDNA
A. Expression of Rat Interleukin-l Type 3 Receptor
In order to express IL-I Type 3 Iecel)lor cDNA, a ~ ------Ali~n cell
cA~Ie~ion vector (pCDM7amp) is first constructed. Briefly, pCDM7amp is a DNA
plasmid which contains 1) an ampicillin resistance gene that provides for selection in
prokaryotic cells, 2) a bacterial origin of replication which allows propagation and
amplification in host bacterial cells, 3) a CMV (cytomegalovirus) promoter whichsponsors transcription in ~ n cells, 4) a multiple cloning site (MCS), which is a
series of adjacent restriction sites in the DNA sequence that are useful for the insertion
of approp-idle DNA fragments, and 5) a SV 40 T-antigen splice and polyadenylation
site.
pCDM7-Amp is constructed from pCDM8 (Seed, Nature 329:840-842,
1987; Seed and Aruffo, Proc. Natl. Acad. Sci. 84:3365-3369, 1987; Thomsen et al., Cell
63:485-493, 1990; Bernot and Auffray, Proc. Natl. Acad. Sci 88:2550-2554, 1991; Han
et al., Nature 349:697-700, 1991) by deletion of the adeno origin of replication, M13
origin of replication and sup F selection marker. An ampicillin re~i~ance marker is then
added in orderto f~r.ilit~te selection ofthe plasmid.
A full-length rat IL-l Type 3 recel)lor clone in pBluescriptSK- is isolated
from the phage clone described above, and cut with EcoRV and HindIII, relç~sing two
WO 96/07739 PCTtUS95/12037
219961),g
3 0
inserts. The inserts are then ieol?ted and ligated to pCDM7-Amp which had been
similarly cut. The reSulting product is used to lla~ lll E. coli DH5a, and colonies are
e~A.~ d by restriction digests for correct o.i~.ltalion of the two inserts (i.e., proper
formation ofthe IL-l Type 3R coding sequence.)
COS-7 (ATCC No. CRL 1651) cells are then lli.n!~r~ ed with pCDM7-
Amp ~ e IL-l Type 3 ,t;ceplor cDNA (lO ug DNA/lO cm plate of cells) utili~ng
400 ~lg/ml of DEAE-Dextran and lO0 ~lM chlolo.lu,ne. The cells are ~ rt;~;led for 4
hours, then shocked with 10% DMSO for 2 rninl~tes The cells are then washed, andgrown in DMEM CG"I~i";.,g 10% Fetal Bovine Serum for 2 days in a 24-well plate.
B. Expression of Human Interleukin-l Type 3 Receptor
A full-length human IL-l Type 3 receplor clone in pBluescriptSK- is
so!~ted from the phage clone described above, and cut with NotI and ~oI, rele~ing the
insert. The insert is then isolated and ligated to pCDM7-Arnp which had been similarly
cut. The resulting product is used to l,a"~"" E. coli DH5a, from which larger
q"~ntities of plasmid DNA may be isolated.
COS-7 (ATCC No. CRL 1651 ) cells are then transfected with pCDM7-
Amp cG.-Ih;.~ IL-l Type 3 receptor cDNA (lO ug DNA/I0 cm plate of cells) utili7in~
400 llg/ml of DEAE-Dextran and lO0 ~M chloroquine. The cells are ~,~nsr~cled for 4
hours, then shocked with 10% DMSO for 2 minutçs The cells are then washed, and
grown in DMEM co.~ g 10% Fetal Bovine Serum for 2 days in a 24-well plate.
EXAMPLE 3
CONSTRUCTION AND EXPRESSION OF SOLUBLE
HUMAN ~ERLEu~rN-l TYPE 3 REcEProR
A. Plasmid Construction
l . Vector ~1 epa, ~lion
An ~ es~ion vector co.. t~ g the N-terminal portion of the human IL-
1 type 3 ,ecep~or, also It;rel.ed to as the "soluble" form of the receptor, is constructed
çsce~ y as described below. Briefly, pCDM7amp DNA (as desclil.ed above) is
subjected to restriction endonuclease digestion with two enzymes, NotI and X7wI, each
of which have one recoP~ ;on site in this vector, both located in the MCS. The product
35 is a lh~eali,ed DNA fragment with the CMV promoter/PI~h-..cer ;lnl~le~ tely upsll~,am
ofthe cut site, and the polyadenylation signal dowl,sliea,l, ofthe cut site.
WO 96/07739 2 1 9 9 6 0 9 PCT/US95112037
- 31
A~er digestion, the cleaved vector is isolated by agarose gel
electrophoresis and purified using the Gene Clean procedure (Bio 101, San Diego, CA).
The vector is now ready to co~,ll)ine with a DNA fragment encoding the soluble human
IL-1 type 3 receptors.
2. Insert Pl epa, ~lion
Into this prepared vector is ligated a DNA fragment co..l~;..;..p: the
coding region ofthe first 336 amino acids ofthe human IL-l type 3 receptor set forth in
Sequence ID No. I (from nucleotide number 129 to nucleotide number 1136).
Briefly, two oligonucleotides are first synthesi7ed for use as primers in
PCR. These oligonucleotides can be synthesized on a DNA synthesizer. The first
primer consists of the sequence 5'-CCTACTCGAG ATGTGGTCCT TGCTGCTC-3'
(Sequence ID No: 8). The first four nucleotides of this sequence serve as a spacer, and
increase the efficiency of endonuclease cleavage in a subsequent reaction to be
described. Nucleotides 5 through 10 encode a XhoI endonuclease cleavage site, and
nucleotides 11 through 28 are identical to the N-terminal coding region of the human
IL-type 3 receptor (nucleotides 129 to 146 in Sequence ID No: 1)).
The second primer consists of the sequence 5'-ATGCGCGC~CC
GCCTATCGAA AATCCGGAGC TGG-3' (Sequence Id No: 9). The first four
nucleotides of this sequence serve as a spacer, and increase efficiency of endonuclease
cleavage in a subsequent reaction to be described. Nucleotides 5 through 12 encode a
No~I endonucle~ce cleavage site. Nucleotides 13 through 15 encode a translation stop
codon, and nucleotides 16 though 33 are complem~ aly to the coding region of thehuman IL- l type 3 receptor immediately p~ eceding the l~ "l,l ~ne region
(nucleotides 1133 through 1116 in Sequence ID No. 1).
The fragment encoding soluble human IL-l type 3 receptor is then
generated by PCR. Briefly, lOOng of each primer are colllbined in a 0.5ml test tube,
along with lng of the entire human IL-l type 3 receptor DNA sequence conlailled in a
cloning vector, such as Bluescript (Strat~nç, La Jolla, CA). Ten microliters of lOX
PCR buffer, 5ul of 25rnM MgCl, 1ul of 25mM aTP, and 1ul of Taq polyrnerase/Vent
polymerase (16:1 ratio) are also added to the reaction. The complete sample is then
overlayed with lOOul of mineral oil to prevent evaporation, and the sample is placed in a
thermocycler. Reaction conditions are: 94~C for 15 seconds, 55~C for 60 seconds, and
72~C for 60 seconds. These conditions are repeated for 25 cycles.
Product from the reaction is analyzed by agarose gel elec~lophoresis to
verify the size of the fragment (1009 bp) and also to determine the appro~i...ale amount
of DNA generated. The DNA is then isolated by phenol/chlorofo,... extraction and
WO 96/07739 ~ PCI/US95/12037
21g960~ ~
1 ~ ~ . 3 2
purified over a G-50 mini-spin column (Boehringer l~nnh~im, ~ntliAn~rolis, IN).
App~u~alely 10ug of the purified DNA fragment is digested with 20 units each of
XhoI and NotI restriction endonucleases in a standard reaction to generate cohesive
ends on the fragment which are co..lpalible with the pCDM7 vector plepaled as det~iled
5 above. The digested fragment is then agarose gel purified to remove impurities and
co~ ting DNA species.
3. Ligation
One hundred nanograms of vector DNA is combined with 100ng of insert
10 DNA in a 1.5ml mini-tube with lul of 10X ligation buffer, lul of DNA ligase
(Boehringer Mannheim), and water to a total volume of 10ul. This sample is incubated
at 23~C for 2 hours.
4. Transformation
One hundred microliters of competent E. coli bacteria cells are combined
with the ligation product and incubated on ice for 30 minutes. The sample is then
inc~lbated at 42~C for 45 seconds. One milliliter of bacterial medium (Circle Grow, Bio
101, San Diego, CA) is then added, and the sample is shaken at 37~C for 60 minutes.
The sample is then plated on a bacterial growth plate contAining bacterial medium and
20 ampicillin at 100ug/ml (Fisher Scientific), and incubated for 16 hours at 37~C.
5. Construct verification
Ten colonies from the ampicillin plate are s~lected and grown in 1 ml of
bacterial medium for 24 hours. One hundred microliters of each culture is stored by
25 adding an equal volume of 50% glycerol solution and frozen at -70~C in mini-tubes.
Plasmid DNA is then extracted from the re~"A~ g cultures by the mini-prep procedure
essçnti~lly as described by Maniatis et al. (s~pra), and the recovered DNAs are analyzed
by restriction digest with X7~oI and Noll restriction endonucle~es. The products of
restriction digest are vi.cu~li7ed by agarose gel electrophoresis and eth;t~ m bromide
30 st~ini~ Correct pl~mids will yield two bands: a vector band of applo~,~..alely 3
kilobases, and an insert fragment of 1009 bases.
The frozen stock of a colony co,~lA~ g the correct plasmid is used to
inoculate one liter of bacterial growth medium CGIl~ g ampicillin (lOOug/ml). The
culture is shaken at 37~C for 24 hours, and plasmid DNA is isolated by a maxi-prep
35 procedure (Promega). The portion on this plasmid coding for soluble human IL-1 type
3 receptor is analyzed by DNA sequencing (US Biochemical) in order to verify that the
sequence is correct.
i ';
Wo 96/07739 2 1 9 9 6 0 9 Pcrluss5ll2o37
33
B. Transfection Procedure and Expression
COS-7 (ATCC No. CRL 1651) or L-tk- cells (ATCC No. CCL 1.3 ) are
seeded at lxl06 or 3X106 cells on 10 cm tissue culture dishes and inc~lbated over night.
S Cells are then transfected by a standard DEAE dextran method. Briefly, 10~1g of IL-l
type 3 receptor eAplession plasmid DNA are diluted in 3 ml of Dulbecco's modified
Minimum F.ssenti~l Medium (D-MEM) supplemented with ~ t~mine, pyruvate, 25mM
HEPES, 100 microgram/ml DEAE dextran (0.5 Md., Sigma, St. Louis) and 0.1 mM
chloroquine (Sigma). Cells are incubated in this transfection mixture for 4 hours at
10 37~C. After one washing step with D-MEM cells are incubated for 48 hours in D-MEM
supplemented with 10% fetal calf serum. At this stage cells are ready for further
analysis ofthe expressed IL-l type 3 receptor.
EXAMPLE 4
SIGNALING o~ IL- 1 VIA THE IL- I TYPE 3
RECEPTOR IN ~ FUNCTIONAL ASSAY
IL-l type 1 receptor cDNA and type 3 receptor cDNA are separately
20 L,~llsre.;Led into Jurkat cells (ATCC no. TIB 152) together with a reporter plasmid
consisting of the HIV promoter region (HIV-LTR) linked to the bacterial
chloramphenicol acetyltransferase (CAT) gene. Stimulation ofthe L-~nsre~;Led cells with
human IL-l alpha leads through a signaling cascade involving the Ll~nsclipLion factor
NF-kappaB to the production of CAT, which in turn can be measured by co"~,l,ercially
25 available assays (Promega, Madison, Wl) (see also Leungetal., J. Biol. Chem.
269:1579-1582, 1994).
Results are shown in Figure 3. Briefly, a~J~JIuxil~aLely equal stimlllation
of CAT activity for both receptors can be seen over mock transfected control cells. This
indicates that human IL-1 alpha can signal through the IL-1 type 3 receptor.
EXAMPLE S
EXPRESSION, LOCALIZATION, AND ACTIV1TY OF THE IL-l TYPE 3 REcEProR
A. Expression Pattern ofthe IL-l Type 3 Receptor
In order to deLell";ne in which rat tissues and parts of the rat brain the
IL-l Type 3 receptor is expressed, RNA protection assays are performed. Briefly, total
219 9 6 o ~ .; s~ i' {~; PCT/US95/12037
34
RNA is isolated from each tissue or part of the brain and annealed at 65~C to 32p
labeled RNA generated from a plasmid co~ a 600 bp fragment which covers the
entire Llanslllem~ ne region and portions of the extracçll~ r and intracclllll~r domains
of the Type 3 receptor cDNA. Samples are then digested with RNase and fractionated
5 on a denaturing polyacrylamice gel. The gel is then dried and the radioactivity
q~ ntit~ted using a PhosphoImager (Figure 4).
As can be seen in Figure 4, the highest level of cAI)~ession is in the lung,
followed by the epididymus and testis. When various areas of the brain are ~xal~ ed,
the cerebral cortex contains the highest level of the Type 3 receptor, although other
10 areas of the brain were also positive.
B . Localization of the IL- I Tvpe 3 Receptor by In Situ
Hybridization
Utilizing in si~u hybridization histochemistry, the IL-l type 3 receptor
may be found in the thymus and the spleen. In the thymus the signal is most prominent
in the cortical region and not in the medulla. Within the rat brain the IL-l type 3
receptor eAIJI ession is detectable in the hippocampus and the fourth ventricle This is in
contrast to the localization of the IL-l type I receptor which is restricted to the dentate
gyrus granule cells.
Briefly, cli~sected tissue is frozen in isopenlane cooled to -42~C and
subsequently stored at -80~C prior to sectioning on a cryostat. Slide-mounted tissue
sections are then stored at -80~C. Sections are removed from storage and placed
directly into 4% buffered pa~afo~ aldehyde at room temperature. After 60 minutçs,
slides are rinsed in isotonic phosphate buffered saline (10 min.) and treated with
proteinaseK (I ~,lg/ml in 100mM Tris/HCl, pH8.0) for 10 min~tes at 37~C.
Subsequently, sections are successively washed in water (1 n~in.), 0.1 M triethanolamine
(pH 8.0, plus 0.25% acetic anhydride) for lO min~ltçs and 2X SSC (0.3 mM NaCl,
0.03 mM sodium citrate, pH 7.2) for 5 minutes. Sections are then dehydrated through
graded alcohols and air dried. Post-fixed sections are hybridized with 1.0 x 106 dpm
[35S]UTP-labeled riboprobes in hybridization buffer co~ i";.~g 75% ~ lç, 10%
dextran sulphate, 3X SSC, 50 mM sodium phosphate buffer pH 7.4), lX Denhardt's
solution, 0.1 mg/ml yeast tRNA and 10 mM dithiothreitol in a total volume of 30 ~l.
The diluted probe is applied to sections on a glass coverslip and hybridized overnight at
55~C in a humid environ-~enl. Post-hybridization, sections are washed in 2X SSC for
35 5 minte~ and then treated with RNase A (200 llg/ml in 10mM Tris/HCl, pH8.0,
cC-,t~ g 0.5 M NaCl) for 60 minutes at 37~C. Subsequently, sections are washed in
2X SSC for 5 minutçs, lX SSC for 5 minutes, O.IX SSC for 60 min-ltes at 70~C, 0.5X
w0 96/07739 2 1 9 9 6 0 ~ '
SSC at room te"~pe~ re for 5 minutes and then dehydrated in graded alcohols and air
dried. For signal detection, sections are placed on Kodak Bio Max X-ray film andexposed for the required length of time or dipped in photographic emulsion (Amsersham
LM-l) for high resolution analysis. Autoradiograms are analyzed using automated
5 image analysis (DAGE camera/Mac TT) while dipped sections were examined using a
Zeiss Axloscope.
C. Inhibition of Thvmocyte Proliferation by the IL- 1 T,vpe 3
Receptor
Ability of the IL- I type 3 receptor to inhibit mouse thymocyte
proliferation may also be examined. Briefly, the proliferative response of T Iymphocyte
lectins such as phytohemagglutin (PHA) is very low, but is markedly enh~nced by IL-1.
Thus, soluble type human and rat type 3 receptors may be utilized to competitively
inhibit proliferation of mouse thymocytes stimulated by IL-1. Soluble human Type 1
15 receptor produced in baculovirus may be used as a positive control.
Briefly, soluble IL-1 type I or type 3 receptors are added to wells of a 96
well plate and serially diluted IL-l is also added. Thymi are removed from young mice
and a single cell suspension prepared in tissue culture media. Cells are washed 3 times
and resuspended at a concentration of 107 cells/ml. Cells are plated at 100 microliters in
20 a 96 well flat bottom microtiter plate. PHA is added to stim~ te the cells. Plates are
then incubated for 48 hours in a 37~C, 5% CO2 humidified incubator, and [3Hl
thymidine is added to the cells for the last 4 to 6 hours. Cells are then harvested and the
[3Hl thymidine incorporation determined by liquid scintillation counting.
As shown in Figure 5, both human L-1 type 3 and rat IL-1 type 3
25 receptors effectively inhibit thymocyte proliferation in a manner similar to that observed
for soluble human type 1 receptor. This result strongly indicates that the type 3 receptor
inhibits thymocyte proliferation by binding to the exogenously added IL-1.
From the foregoing, it will be appreciated that, although specific
30 embodiments of the invention have been described herein for purposes of illustration,
various modifications may be made without deviating from the spirit and scope of the
invention. Accordingly, the invention is not limited except as by the appended claims.
WO 96/07739 ~ PCT/US95/12037
2 1 9 ~3 6 o~
36
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS: Lovenberg, Timothy W.
Oltersdorf, Tilman
Liaw, Chen W.
Clevenger, William
DeSouza, Errol B.
(ii) TITLE OF INVENTION: INTERLEUKIN-1 TYPE 3 RECEPTORS
(iii) NUMBER OF SEQUENCES: 9
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Seed and Berry
(B) STREET: 6300 Columbia Center, 701 Fifth Avenue
(C) CITY: Seattle
(D) STATE: Washington
(E) COUNTRY: US
(F) ZIP: 98104
(v) COMPUTER READABLE FORM:
.(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McMasters, David D.
(B) REGISTRATION NUMBER: 33,963
WO 96/07739 21 9g 6 0.~, ' PCI/US95tl2037
_ 37
(C) REFERENCE/DOCKET NUMBER: 690068.402PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (206) 622-4900
(B) TELEFAX: (206) 682-6031
(C) TELEX: 3723836
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1965 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 129..1814
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CGCCCGCCCA CGGCGGCGGG GAAATACCTA GGCATGG M G TGGCATGACA GGGCTCGTGT 60
CCCTGTCATA TTTTCCACTC TCCACGAGGT CCTGCGCGCT TCAATCCTGC AGGCAGCCCG 120
GTTTGGGG ATG TGG TCC TTG CTG CTC TGC GGG TTG TCC ATC GCC CTT CCA 170
Met Trp Ser Leu Leu Leu Cys Gly Leu Ser Ile Ala Leu Pro
- 1 5 10
CTG TCT GTC ACA GCA GAT GGA TGC M G GAC ATT TTT ATG MM M T GAG 218
Leu Ser Val Thr Ala Asp Gly Cys Lys Asp Ile Phe Met Lys Asn Glu
15 20 25 30
ATA CTT TCA GCA AGC CAG CCT TTT GCT TTT M T TGT ACA TTC CCT CCC 266
W O 96/07739 PC~rrUS95/12037
2199609 38
~le Leu Ser Ala Ser Gln Pro Phe Ala Phe Asn Cys Thr Phe Pro Pro
ATA ACA TCT GGG GM GTC AGT GTA ACA TGG TAT MM MT TCT AGC MM 314
Ile Thr Ser Gly Glu Val Ser Val Thr Trp Tyr Lys Asn Ser Ser Lys
50 55 60
ATC CCA GTG TCC MM ATC ATA CAG TCT AGA ATT CAC CAG GAC GAG ACT 362
Ile Pro Val Ser Lys Ile Ile Gln Ser Arg Ile His Gln Asp Glu Thr
65 70 75
TGG ATT TTG TTT CTC CCC ATG GM TGG GGG GAC TCA GGA GTC TAC CM 410
Trp Ile Leu Phe Leu Pro Met Glu Trp Gly Asp Ser Gly Val Tyr Gln
80 85 90
TGT GTT ATA MG GGT AGA GAC AGC TGT CAT AGA ATA CAT GTA MC CTA 458
Cys Val Ile Lys Gly Arg Asp Ser Cys His Arg Ile His Val Asn Leu
95 100 105 llo
ACT GTT m GM MM CAT TGG TGT GAC ACT TCC ATA GGT GGT TTA CCA 506
Thr Val Phe Glu Lys His Trp Cys Asp Thr Ser Ile Gly Gly Leu Pro
115 120 125
MT TTA TCA GAT GAG TAC MG CM ATA TTA CAT CTT GGA MM GAT GAT 554
Asn Leu Ser Asp Glu Tyr Lys Gln Ile Leu His Leu Gly Lys Asp Asp
130 135 140
AGT CTC ACA TGT CAT CTG CAC TTC CCG MG AGT TGT GTT TTG GGT CCA 602
Ser Leu Thr Cys His Leu His Phe Pro Lys Ser Cys Val Leu Gly Pro
145 150 155
ATA MG TGG TAT MG GAC TGT MC GAG ATT MM GGG GAG CGG TTC ACT 650
Ile Lys Trp Tyr Lys Asp Cys Asn Glu Ile Lys Gly Glu Arg Phe Thr
160 165 170
GTT TTG GM ACC AGG CTT TTG GTG AGC MT GTC TCG GCA GAG GAC AGA 698
Val Leu Glu Thr Arg Leu Leu Val Ser Asn Val Ser Ala Glu Asp Arg
~O 96/07739 21 9 9 6 0 9 PCT/US95/12037
i ~ ', ' ~ f r ~
39 ~:
175 180 185 190
GGG MC TAC GCG TGT CM GCC ATA CTG ACA CAC TCA GGG MG CAG TAC 746
Gly Asn Tyr Ala Cys Gln Ala Ile Leu Thr His Ser Gly Lys Gln Tyr
195 200 205
GAG GTT TTA MT GGC ATC ACT GTG AGC ATT ACA GM AGA GCT GGA TAT 794
Glu Val Leu Asn Gly Ile Thr Val Ser Ile Thr Glu Arg Ala Gly Tyr
210 215 220
GGA GGA AGT GTC CCT MA ATC ATT TAT CCA MA AAT CAT TCA ATT GM 842
Gly Gly Ser Val Pro Lys Ile Ile Tyr Pro Lys Asn His Ser Ile Glu
225 230 235
GTA CAG CTT GGT ACC ACT CTG ATT GTG GAC TGC MT GTA ACA GAC ACC 890
Val Gln Leu Gly Thr Thr Leu Ile Val Asp Cys Asn Val Thr Asp Thr
240 245 250
MG GAT MT ACA MT CTA CGA TGC TGG AGA GTC MT MC ACT TTG GTG 938
Lys Asp Asn Thr Asn Leu Arg Cys Trp Arg Val Asn Asn Thr Leu Val
255 - 260 265 270
GAT GAT TAC TAT GAT GM TCC MM CGA ATC AGA GM GGG GTG GM ACC 986
Asp Asp Tyr Tyr Asp Glu Ser Lys Arg Ile Arg Glu Gly Val Glu Thr
275 280 285
CAT GTC TCT TTT CGG GM CAT MT TTG TAC ACA GTA MC ATC ACC TTC 1034
His Val Ser Phe Arg Glu His Asn Leu Tyr Thr Val Asn Ile Thr Phe
290 295 300
TTG GM GTG MM ATG GM GAT TAT GGC CTT CCT TTC ATG TGC CAC GCT 1082
Leu Glu Val Lys Met Glu Asp Tyr Gly Leu Pro Phe Met Cys His Ala
305 310 315
GGA GTG TCC ACA GCA TAC ATT ATA TTA CAG CTC CCA GCT CCG GAT TTT 1130
Gly Val Ser Thr Ala Tyr Ile Ile Leu Gln Leu Pro Ala Pro Asp Phe
320 325 330
WO 96/07739 PCT/USg5/12037
;21g~1609 '
CGA GCT TAC TTG ATA GGA GGG CTT ATC GCC TTG GTG GCT GTG GCT GTG 1178
Arg Ala Tyr Leu Ile Gly Gly Leu Ile Ala Leu Val Ala Val Ala Val
335 340 345 350
TCT GTT GTG TAC ATA TAC MC ATT TTT MG ATC GAC ATT GTT CTT TGG 1226
Ser Val Val Tyr Ile Tyr Asn Ile Phe Lys Ile Asp Ile Val Leu Trp
355 360 365
TAT CGA AGT GCC TTC CAT TCT ACA GAG ACC ATA GTA GAT GGG MG CTG 1274
Tyr Arg Ser Ala Phe His Ser Thr Glu Thr Ile Val Asp Gly Lys Leu
370 375 380
TAT GAC GCC TAT GTC TTA TAC CCC MG CCC CAC MG GM AGC CAG AGG 1322
Tyr Asp Ala Tyr Val Leu Tyr Pro Lys Pro His Lys Glu Ser Gln Arg
385 390 395
CAT GCC GTG GAT GCC CTG GTG TTG MT ATC CTG CCC GAG GTG TTG GAG 1370
His Ala Val Asp Ala Leu Val Leu Asn Ile Leu Pro Glu Val Leu Glu
400 405 410
AGA CM TGT GGA TAT MG TTG TTT ATA TTC GGC AGA GAT GM TTC CCT 1418
Arg Gln Cys Gly Tyr Lys Leu Phe Ile Phe Gly Arg Asp Glu Phe Pro
415 420 425 430
GGA CM GCC GTG GCC MT GTC ATC GAT GM MC GTT MG CTG TGC AGG 1466
Gly Gln Ala Val Ala Asn Val Ile Asp Glu Asn Val Lys Leu Cys Arg
435 440 445
AGG CTG ATT GTC ATT GTG GTC CCC GM TCG CTG GGC TTT GGC CTG TTG 1514
Arg Leu Ile Val Ile Val Val Pro Glu Ser Leu Gly Phe Gly Leu Leu
450 455 460
MG MC CTG TCA GM GM CM ATC GCG GTC TAC AGT GCC CTG ATC CAG 1562
Lys Asn Leu Ser Glu Glu Gln Ile Ala Val Tyr Ser Ala Leu Ile Gln
465 470 475
WO 96/07739 2 1 9 9 6 0 9 PCT/US95/12037
._ 41
GAC GGG ATG MG GTT ATT CTC ATT GAG CTG GAG MM ATC GAG GAC TAC 1610
Asp Gly Met Lys Val Ile Leu Ile Glu Leu Glu Lys Ile Glu Asp Tyr
480 485 490
ACA GTC ATG CCA GAG TCA ATT CAG TAC ATC MM CAG MG CAT GGT GCC 1658
Thr Val Met Pro Glu Ser Ile Gln Tyr Ile Lys Gln Lys His Gly Ala
495 500 505 510
ATC CGG TGG CAT GGG GAC TTC ACG GAG CAG TCA CAG TGT ATG MG ACC 1706
Ile Arg Trp His Gly Asp Phe Thr Glu Gln Ser Gln Cys Met Lys Thr
515 520 525
MG TTT TGG MG ACA GTG AGA TAC CAC ATG CCG CCC AGA AGG TGT CGG 1754
Lys Phe Trp Lys Thr Val Arg Tyr His Met Pro Pro Arg Arg Cys Arg
530 535 540
CCG TTT CTC CGG TCC ACG TGC CGC AGC ACA CAC CTC TGT ACC GCA CCG 1802
Pro Phe Leu Arg Ser Thr Cys Arg Ser Thr His Leu Cys Thr Ala Pro
545 550 555
CAG GCC CAG MC TAGGCTCMG MGMMGMG TGTACTCTCA CGACTGGCTA 1854
Gln Ala Gln Asn
560
AGACTTGCTG GACTGACACC TATGGCTGGA AGATGACTTG TTTTGCTCCA TGTCTCCTCA 1914
TTCCTACACC TATTTTCTGC TGCAGGATGA GGCTAGGGTT AGCATTCTAG A 1965
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 562 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
( i i ) MOLECULE TYPE: protei n
WO 96/07739 . PCT/US95/12037
.g I, ' ,~
2199609 42
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Trp Ser Leu Leu Leu Cys Gly Leu Ser Ile Ala Leu Pro Leu Ser
Val Thr Ala Asp Gly Cys Lys Asp Ile Phe Met Lys Asn Glu Ile Leu
Ser Ala Ser Gln Pro Phe Ala Phe Asn Cys Thr Phe Pro Pro Ile Thr
Ser Gly Glu Val Ser Val Thr Trp Tyr Lys Asn Ser Ser Lys Ile Pro
Val Ser Lys Ile Ile Gln Ser Arg Ile His Gln Asp Glu Thr Trp Ile
Leu Phe Leu Pro Met Glu Trp Gly Asp Ser Gly Val Tyr Gln Cys Val
Ile Lys Gly Arg Asp Ser Cys His Arg Ile His Val Asn Leu Thr Val
100 105 110
Phe Glu Lys His Trp Cys Asp Thr Ser Ile Gly Gly Leu Pro Asn Leu
115 120 125
Ser Asp Glu Tyr Lys Gln Ile Leu His Leu Gly Lys Asp Asp Ser Leu
130 135 140
Thr Cys His Leu His Phe Pro Lys Ser Cys Val Leu Gly Pro Ile Lys
145 150 155 160
Trp Tyr Lys Asp Cys Asn Glu Ile Lys Gly Glu Arg Phe Thr Val Leu
165 170 175
Glu Thr Arg Leu Leu Val Ser Asn Val Ser Ala Glu Asp Arg Gly Asn
WO 96t07739 2 1 ~ ~ 6 Q ~ PCTtUS95/12037
__ 43 : .
180 185 190
Tyr Ala Cys Gln Ala Ile Leu Thr His Ser Gly Lys Gln Tyr Glu Val
195 200 205
Leu Asn Gly Ile Thr Val Ser Ile Thr Glu Arg Ala Gly Tyr Gly Gly
210 215 220
Ser Val Pro Lys Ile Ile Tyr Pro Lys Asn His Ser Ile Glu Val Gln
225 230 235 240
Leu Gly Thr Thr Leu Ile Val Asp Cys Asn Val Thr Asp Thr Lys Asp
245 250 255
Asn Thr Asn Leu Arg Cys Trp Arg Val Asn Asn Thr Leu Val Asp Asp
260 265 270
Tyr Tyr Asp Glu Ser Lys Arg Ile Arg Glu Gly Val Glu Thr His Val
275 280 285
Ser Phe Arg Glu His Asn Leu Tyr Thr Val Asn Ile Thr Phe Leu Glu
290 295 300
Val Lys Met Glu Asp Tyr Gly Leu Pro Phe Met Cys His Ala Gly Val
305 310 315 320
Ser Thr Ala Tyr Ile Ile Leu Gln Leu Pro Ala Pro Asp Phe Arg Ala
325 330 335
Tyr Leu Ile Gly Gly Leu Ile Ala Leu Val Ala Val Ala Val Ser Val
340 345 350
Val Tyr Ile Tyr Asn Ile Phe Lys Ile Asp Ile Val Leu Trp Tyr Arg
355 360 365
Ser Ala Phe His Ser Thr Glu Thr Ile Val Asp Gly Lys Leu Tyr Asp
370 375 380
WO 96/07739 P~ 3S/12037
2~996S ' ' ~ 44
Ala Tyr Val Leu Tyr Pro Lys Pro His Lys Glu Ser Gln Arg His Ala
385 390 395 400
Val Asp Ala Leu Val Leu Asn Ile Leu Pro Glu Val Leu Glu Arg Gln
405 410 415
Cys Gly Tyr Lys Leu Phe Ile Phe Gly Arg Asp Glu Phe Pro Gly Gln
420 425 430
Ala Val Ala Asn Val Ile Asp Glu Asn Val Lys Leu Cys Arg Arg Leu
435 440 445
Ile Val Ile Val Val Pro Glu Ser Leu Gly Phe Gly Leu Leu Lys Asn
450 455 460
Leu Ser Glu Glu Gln Ile Ala Val Tyr Ser Ala Leu Ile Gln Asp Gly
465 470 475 480
Met Lys Val Ile Leu Ile Glu Leu Glu Lys Ile Glu Asp Tyr Thr Val
485 490 495
Met Pro Glu Ser Ile Gln Tyr Ile Lys Gln Lys His Gly Ala Ile Arg
500 505 510
Trp His Gly Asp Phe Thr Glu Gln Ser Gln Cys Met Lys Thr Lys Phe
515 520 525
Trp Lys Thr Val Arg Tyr His Met Pro Pro Arg Arg Cys Arg Pro Phe
530 535 ~40
Leu Arg Ser Thr Cys Arg Ser Thr His Leu Cys Thr Ala Pro Gln Ala
545 550 555 560
Gln Asn
WO 96/07739 2 1 9 9 6 0 ~3 PCT/US95/12037
(2) INFORMATION FOR SEQ ID NO:3:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2044 base pairs
(B) TYPE: nucl ei c aci d
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i x ) FEATURE:
( A ) NAME / KEY: CDS
(B) LOCATION: 89. .1771
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CCGGCTGGCC TAGGATCAGG CMGMMGG CTGMCGCCT TTCTMGGAC GGACTCTTTC 60
TGTACAGCTC CACTTGGGGA AGCCCGM ATG GGG ATG CCA CCC TTG CTC TTC 112
Met Gly Met Pro Pro Leu Leu Phe
TGT TGG GTG TCT TTC GTG CTT CCA CTT TTT GTG GCA GCA GGT MC TGT 160
Cys Trp Val Ser Phe Val Leu Pro Leu Phe Val Ala Ala Gly Asn Cys
10 15 20
ACT GAT GTC TAT ATG CAC CAT GAG ATG ATT TCA GAG GGC CAG CCT TTC 208
Thr Asp Val Tyr Met His His Glu Met Ile Ser Glu Gly Gln Pro Phe
25 30 35 40
CCC TTC MC TGC ACA TAC CCT CCA GTA ACA MC GGG GCA GTG MT CTG 256
Pro Phe Asn Cys Thr Tyr Pro Pro Val Thr Asn Gly Ala Val Asn Leu
45 50 55
ACA TGG CAT AGA ACA CCC AGT MG AGC CCA ATC TCC ATC MC AGA CAC 304
Thr Trp His Arg Thr Pro Ser Lys Ser Pro Ile Ser Ile Asn Arg His
W O 96/07739 ~ PC~rrUS95/12037
2199609~ -~
46
GTT AGA ATT CAC CAG GAC CAG TCC TGG ATT TTG TTT CTT CCG TTG GCA 352
Val Arg Ile His Gln Asp Gln Ser Trp Ile Leu Phe Leu Pro Leu Ala
75 80 85
TTG GAG GAC TCA GGC ATC TAT CM TGT GTT ATA MG GAT GCC CAC AGC 400
Leu Glu Asp Ser Gly Ile Tyr Gln Cys Val Ile Lys Asp Ala His Ser
90 95 100
TGT TAC CGA ATA GCT ATA MC CTA ACC GTT TTT AGA MA CAC TGG TGC 448
Cys Tyr Arg Ile Ala Ile Asn Leu Thr Val Phe Arg Lys His Trp Cys
105 110 115 120
GAC TCT TCC MC GM GAG AGT TCC ATA MT TCC TCA GAT GAG TAC CAG 496
Asp Ser Ser Asn Glu Glu Ser Ser Ile Asn Ser Ser Asp Glu Tyr Gln
125 130 135
CM TGG TTA CCC ATA GGA MA TCG GGC AGT CTG ACG TGC CAT CTC TAC 544
Gln Trp Leu Pro Ile Gly Lys Ser Gly Ser Leu Thr Cys His Leu Tyr
140 145 150
TTC CCA GAG AGC TGT GTT TTG GAT TCA ATA MG TGG TAT MG GGT TGT 592
Phe Pro Glu Ser Cys Val Leu Asp Ser Ile Lys Trp Tyr Lys Gly Cys
155 160 165
GM GAG ATT MM GTG AGC MG MG TTT TGC CCT ACA GGA ACA MG CTT 640
Glu Glu Ile Lys Val Ser Lys Lys Phe Cys Pro Thr Gly Thr Lys Leu
170 175 180
CTT GTT MC MC ATC GAC GTG GAG GAT AGT GGG AGC TAT GCA TGC TCA 688
Leu Val Asn Asn Ile Asp Val Glu Asp Ser Gly Ser Tyr Ala Cys Ser
185 190 195 200
GCC AGA CTG ACA CAC TTG GGG AGA ATC TTC ACG GTT AGA MC TAC ATT 736
Ala Arg Leu Thr His Leu Gly Arg Ile Phe Thr Val Arg Asn Tyr Ile
205 210 215
WO 96/07739 1 9 9 6 0 ~ ~ PCT/USg5tl2037
47
GCT GTG MT ACC MG GM GTT GGG TCT GGA GGA AGG ATC CCT MC ATC 784
Ala Val Asn Thr Lys Glu Val Gly Ser Gly Gly Arg Ile Pro Asn Ile
220 225 230
ACG TAT CCA MM MC MC TCC ATT GM GTT CM CTT GGC TCC ACC CTC 832
Thr Tyr Pro Lys Asn Asn Ser Ile Glu Val Gln Leu Gly Ser Thr Leu
235 240 245
ATT GTG GAC TGC MT ATA ACA GAC ACG MG GAG MT ACG MC CTC AGA 880
Ile Val Asp Cys Asn Ile Thr Asp Thr Lys Glu Asn Thr Asn Leu Arg
250 255 260
TGC TGG CGA GTT MC AAC ACC CTG GTG GAC GAT TAC TAC AAC GAC TTC 928
Cys Trp Arg Val Asn Asn Thr Leu Val Asp Asp Tyr Tyr Asn Asp Phe
265 270 275 280
MM CGC ATC CAG GM GGA ATC GM ACC MT CTG TCT CTG AGG MT CAC 976
Lys Arg Ile Gln Glu Gly Ile Glu Thr Asn Leu Ser Leu Arg Asn His
285 290 295
ATT CTG TAC ACA GTG MC ATA ACA TTC TTA GM GTG MM ATG GAG GAC 1024
Ile Leu Tyr Thr Val Asn Ile Thr Phe Leu Glu Val Lys Met Glu Asp
300 305 310
TAC GGC CAT CCT TTC ACA TGC CAC GCT GCG GTG TCC GCA GCC TAC ATC 1072
Tyr Gly His Pro Phe Thr Cys His Ala Ala Val Ser Ala Ala Tyr Ile
315 320 325
ATT CTG MM CGC CCA GCT CCA GAC TTC CGG GCT TAC CTC ATA GGA GGT 1120
Ile Leu Lys Arg Pro Ala Pro Asp Phe Arg Ala Tyr Leu Ile Gly Gly
330 335 340
CTC ATG GCT TTC CTA CTT CTG GCC GTG TCC ATT CTG TAC ATC TAC MC 1168
Leu Met Ala Phe Leu Leu Leu Ala Val Ser Ile Leu Tyr Ile Tyr Asn
345 350 355 360
ACC TTT MG GTC GAC ATC GTG CTT TGG TAT AGG AGT ACC TTC CAC ACT 1216
WO96/07739 ; i ~,'t i~ PCI/US95/12037
219g609 48
~hr Phe Lys Val Asp Ile Val Leu Trp Tyr Arg Ser Thr Phe His Thr
365 370 375
GCC CAG GCT CCA GAT GAC GAG MG CTG TAT GAT GCC TAT GTC TTA TAC 1264
Ala Gln Ala Pro Asp Asp Glu Lys Leu Tyr Asp Ala Tyr Val Leu Tyr
380 385 390
CCC MG TAC CCA AGA GM AGC CAG GGC CAT GAT GTG GAC ACA CTG GTG 1312
Pro Lys Tyr Pro Arg Glu Ser Gln Gly His Asp Val Asp Thr Leu Val
395 400 405
TTG MG ATC TTG CCC GAG GTG CTG GAG MM CAG TGT GGA TAT MG TTA 1360
Leu Lys Ile Leu Pro Glu Val Leu Glu Lys Gln Cys Gly Tyr Lys Leu
410 415 420
TTC ATA TTT GGC AGG GAT GM TTC CCT GGA CM GCT GTG GCC AGC GTC 1408
Phe Ile Phe Gly Arg Asp Glu Phe Pro Gly Gln Ala Val Ala Ser Val
425 430 435 440
ATT GAT GM MC ATT MG CTG TGT AGG AGG CTG ATG GTC CTC GTG GCA 1456
Ile Asp Glu Asn Ile Lys Leu Cys Arg Arg Leu Met Val Leu Val Ala
445 450 455
CCA GAG ACA TCC AGC TTC AGC TTT CTG MG MC TTG ACT GM GM CM 1504
Pro Glu Thr Ser Ser Phe Ser Phe Leu Lys Asn Leu Thr Glu Glu Gln
460 465 470
ATC GCT GTC TAC MT GCC CTC GTC CAG GAC GGC ATG MG GTC ATT CTG 1552
Ile Ala Val Tyr Asn Ala Leu Val Gln Asp Gly Met Lys Val Ile Leu
475 480 485
ATT GM CTG GAG AGA GTC MG GAC TAC AGC ACC ATG CCC GAG TCC ATT 1600
Ile Glu Leu Glu Arg Val Lys Asp Tyr Ser Thr Met Pro Glu Ser Ile
490 495 500
CAG TAC ATC CGA CAG MG CAC GGG GCC ATC CAG TGG GAT GGG GAC TTC 1648
Gln Tyr Ile Arg Gln Lys His Gly Ala Ile Gln Trp Asp Gly Asp Phe
WO 96/07739 ! ' ~,
49
505 510 515 520
ACA GAG CAG GCA CAG TGC GCC MG ACG AAA TTC TGG MG MM GTG AGA 1696
Thr Glu Gln Ala Gln Cys Ala Lys Thr Lys Phe Trp Lys Lys Val Arg
525 530 535
TAT CAT ATG CCA CCC AGG AGG TAC CCG GCA TCT CCC CCC GTC CAG CTG 1744
Tyr His Met Pro Pro Arg Arg Tyr Pro Ala Ser Pro Pro Val Gln Leu
540 545 550
CTA GGA CAC ACA CCC CGC ATA CCA GGC TAGTGCAGTG CCACCGCCAC 1791
Leu Gly His Thr Pro Arg Ile Pro Gly
555 560
GGGGCTCATA ACTCCTTMG AGCGGTTAGT GTGTGGTGGC TCGCACTACA ACCTCTCTGG 1851
ATCATCTACC CCCGTAGCTT GCTCTTTTGT GCTTGTGAGC GACCTCGTCC TTAGCCACGT 1911
CATATTTTGA ~ 1 IGI I I GTTTTGTTTG TTTGTTGTAT GCTTTTAGTC ATAGCTGATT 1971
CGTACTACTC CTGTTTGCTT CATGGTTCCT GMTCCCAGA GACTCCCTGA GCATGGGTGG 2031
CTATCATGTT GGG 2044
(2) I NFORMATION FOR SEQ ID NO:4:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 561 amino acids
(B) TYPE; amino acid
(D) TOPOLOGY: l i near
( i i ) MOLECULE TYPE: protei n
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Gly Met Pro Pro Leu Leu Phe Cys Trp Val Ser Phe Val Leu Pro
WO 96/07739 PCI/US95/12037
2~9g6o~ ' ~
~eu Phe Val Ala Ala Gly Asn Cys Thr Asp Val Tyr Met His His Glu
Met Ile Ser Glu Gly Gln Pro Phe Pro Phe Asn Cys Thr Tyr Pro Pro
Val Thr Asn Gly Ala Val Asn Leu Thr Trp His Arg Thr Pro Ser Lys
Ser Pro Ile Ser Ile Asn Arg His Val Arg Ile His Gln Asp Gln Ser
Trp Ile Leu Phe Leu Pro Leu Ala Leu Glu Asp Ser Gly Ile Tyr Gln
~ys Val Ile Lys Asp Ala His Ser Cys Tyr Arg Ile Ala Ile Asn Leu
100 105 110
Thr Val Phe Arg Lys His Trp Cys Asp Ser Ser Asn Glu Glu Ser Ser
115 120 125
Ile Asn Ser Ser Asp Glu Tyr Gln Gln Trp Leu Pro Ile Gly Lys Ser
130 135 140
Gly Ser Leu Thr Cys His Leu Tyr Phe Pro Glu Ser Cys Val Leu Asp
145 150 155 160
~er Ile Lys Trp Tyr Lys Gly Cys Glu Glu Ile Lys Val Ser Lys Lys
165 170 175
~he Cys Pro Thr Gly Thr Lys Leu Leu Val Asn Asn Ile Asp Val Glu
180 185 190
~sp Ser Gly Ser Tyr Ala Cys Ser Ala Arg Leu Thr His Leu Gly Arg
195 200 205
WO 96/07739 ~ PC'r/US95/12037
~l996o9
51
Ile Phe Thr Val Arg Asn Tyr Ile Ala Val Asn Thr Lys Glu Val Gly
210 215 220
Ser Gly Gly Arg Ile Pro Asn Ile Thr Tyr Pro Lys Asn Asn Ser Ile
225 230 235 240
~lu Val Gln Leu Gly Ser Thr Leu Ile Val Asp Cys Asn Ile Thr Asp
245 250 255
~hr Lys Glu Asn Thr Asn Leu Arg Cys Trp Arg Val Asn Asn Thr Leu
260 265 270
Val Asp Asp Tyr Tyr Asn Asp Phe Lys Arg Ile Gln Glu Gly Ile Glu
275 280 285
Thr Asn Leu Ser Leu Arg Asn His Ile Leu Tyr Thr Val Asn Ile Thr
290 295 300
Phe Leu Glu Val Lys Met Glu Asp Tyr Gly His Pro Phe Thr Cys His
305 310 315 320
~la Ala Val Ser Ala Ala Tyr Ile Ile Leu Lys Arg Pro Ala Pro Asp
325 330 335
~he Arg Ala Tyr Leu Ile Gly Gly Leu Met Ala Phe Leu Leu Leu Ala
340 345 350
Val Ser Ile Leu Tyr Ile Tyr Asn Thr Phe Lys Val Asp Ile Val Leu
355 360 365
Trp Tyr Arg Ser Thr Phe His Thr Ala Gln Ala Pro Asp Asp Glu Lys
370 375 380
Leu Tyr Asp Ala Tyr Val Leu Tyr Pro Lys Tyr Pro Arg Glu Ser Gln
385 390 395 400
WO 96/07739 2 1 ~ 9 6 0 ~ PCT/US95/12037
~ly His Asp Val Asp Thr Leu Val Leu Lys Ile Leu Pro Glu Val Leu
405 410 415
~lu Lys Gln Cys Gly Tyr Lys Leu Phe Ile Phe Gly Arg Asp Glu Phe
420 425 430
Pro Gly Gln Ala Val Ala Ser Val Ile Asp Glu Asn Ile Lys Leu Cys
435 440 445
Arg Arg Leu Met Val Leu Val Ala Pro Glu Thr Ser Ser Phe Ser Phe
450 455 460
Leu Lys Asn Leu Thr Glu Glu Gln Ile Ala Val Tyr Asn Ala Leu Val
465 470 475 480
~ln Asp Gly Met Lys Val Ile Leu Ile Glu Leu Glu Arg Val Lys Asp
485 490 495
~yr Ser Thr Met Pro Glu Ser Ile Gln Tyr Ile Arg Gln Lys His Gly
500 505 510
Ala Ile Gln Trp Asp Gly Asp Phe Thr Glu Gln Ala Gln Cys Ala Lys
515 520 525
Thr Lys Phe Trp Lys Lys Val Arg Tyr His Met Pro Pro Arg Arg Tyr
530 535 540
Pro Ala Ser Pro Pro Val Gln Leu Leu Gly His Thr Pro Arg Ile Pro
545 550 555 560
Gly
t2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
WO 96/07739 2 1 9 9 6 0 Y ~ PCT/US95/12037
_. 53
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Asp Tyr Lys Asp Asp Asp Asp Lys
(2) INFORMATION FOR SEQ ID NO:6:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CTTCMCTGC ACATACCCTC CAGTMCMM CGGGGCAGTG MTCTGACAT 50
(2) INFORMATION FOR SEQ ID NO:7:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
( B ) TYPE: nucleic acid
( C ) STRANDEDNESS: s i ngl e
(D) TOPOLOGY: linear
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCTCCCATM CATCTGGGGA AGTCAGTGTA ACATGGTATA AAMTTCTAG C 51
W O 96/07739 PC~rnUS95/12037
2l996oc~
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CCTACTCGAG ATGTGGTCCT TGCTGCTC 28
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATGCGCGGCC GCCTATCG M M TCCGGAGC TGG 33
