Note: Descriptions are shown in the official language in which they were submitted.
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HER4 ~ N R~ 'UK TYR08INE RINASE
This application is a continuation-in-part of
United States Application Serial No. 08/150,704, filed
November 10, 1993, which is a continuation-in-part of
United States Application Serial No. 07/981,165, filed
November 24, 1992, each of which applications are
incorporated herein in their entireties.
1. Introduction
The present invention is generally directed to a
novel receptor tyrosine kinase related to the
epidermal growth factor receptor, termed HER4/pl80
("HER4"), and to novel diagnostic and therapeutic
1~ compositions comprising HER4-derived or HER4-related
biological components. The invention is based in part
upon applicants discovery of human HER4, its complete
nucleotide coding sequence, and functional properties
of the H~R4 receptor protein. More specifically, the
invention is directed to HER4 biologics comprising,
for example, polynucleotide molecules encoding HER4,
HER4 polypeptides, anti-HE~4 antibodies which
recognize epitopes of HER4 polypeptides, ligands which
interact with HER4, and diagnostic and therapeutic
compositions and methods based fundamentally upon such
molecules. In view of the expression of HER4 in
several human cancers and in certain tissues of
neuronal and muscular origin, the present invention
provides a framework upon which effective biological
therapies may be designed. The invention is
hereinafter described in detail, in part by way of
experimental examples specifically illustrating
various aspects of the invention and particular
embodiments thereof.
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2. Background of the Invention
Cells of virtually all tissue types express
transmembrane receptor molecules with intrinsic
tyrosine kinase activity through which various growth
and differentiation factors mediate a range of
biological effects (reviewed in Aaronson, 1991,
Science 254:1146-52). Included in this group of
receptor tyrosine kinases (RTKs) are the receptors for
polypeptide growth factors such as epidermal growth
factor (EGF), insulin, platelet-derived growth factor
(PDGF), neurotrophins (i.e., NGF), and fibroblast
growth factor (FGF). Recently, the ligands for
several previously-characterized receptors have been
identified, including ligands for c-kit (steel
factor), met ~hepatocyte growth factor), trk (nerve
growth factor) (see, respectively, Zsebo et al., 1990,
Cell 63:195-201; Bottardo et al ., 1991, Science
251:80Z-04; Kaplan et al., 1991, Nature 350:158-160).
In addition, the soluble factor NDF, or heregulin-
alpha (HRG-~), has been identified as the ligand for
HER2, a receptor which is highly related to HER4 (Wen
et al ., l99Z, Cell 69:559-72; Holmes et al ., 1992,
Science 256:1205-10).
The heregulins are a family of molecules that
were first isolated as specific ligands for HER2 (Wen,
et al ., 1992, Cell, 69:559-572; Holmes et al ., 1992,
Science 256:1205-1210; Falls et al ., 1993, Cell
72:801-815; and Marchionni et al ., 1993, Nature
362:312-318). A rat homologue was termed Neu
differentiation factor (NDF) based on its ability to
induce differentiation of breast cancer cells through
its interaction with HERZ/Neu (Wen et al., supra).
Heregulin also appears to play an important role in
development and maintenance of the nervous system
based on its abundant expression in cells of neuronal
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origin and on the recognition that alternatively
spli~ed forms of the heregulin gene encode for two
recently characterized neurotrophic activities. one
r neural-derived factor is termed acetylcholine receptor
5 inducing activity (ARIA)(Falls et al., supra). This
heregulin isoform is responsible for stimulation of
neurotransmitter receptor synthesis during formation
of the neuromuscular junction. A second factor is
called glial growth factor (GGF) reflecting the
10 proliferative affect this molecule has on glial cells
in the central and peripheral nervous system
(Marchionni et al ., supra ) . Additional, less well
characterized molecules that appear to be isoforms of
heregulin, include p45, gp30, and p75 (Lupu et al.,
l99o, Science 249:1552-1555; and Lupu et al., 1992,
Proc. Natl. Acad. Sci. U.S.A. 89:2287-2291).
Several HER2-neutralizing antibodies fail to
block heregulin activation of human breast cancer
cells. Heregulin only activates tyrosine
phosphorylation of HER2 in cells o~ breast, colon, and
neuronal origin, and not in fibroblasts or ovarian
cell lines that overexpress recombinant HER2 (Peles et
al., 1993, EMBO J. 12:961-971).
Biological relationships between various human
malignancies and genetic aberrations in growth factor-
receptor tyrosine kinase signal pathways are known to
exist. Among the most notable such relationships
involve the EGF receptor (EGFR) family of receptor
tyrosine kinases (see Aaronson, supra ) . Three human
EGFR-family members have been identified and are known
to those skilled in the art: EGFR, HER2/p185'r~32 and
HER3/p160'r~33 (see, respectively, Ullrich et al., 1984,
Nature 309:418-25; Coussens et al ., 1985, Science
230:1132-39; Plowman et al ., 1990, Proc. Natl. Acad.
3s
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Sci. U.S.A. 87:4905-09). EGFR-related molecules from
other species have also been identified.
The complete nucleotide coding sequence of other
EGFR-family members has also been determined from
other organisms including: the drosophila EGFR ("DER":
Livneh et al ., 1985, Cell 40:599-607), nematode EGFR
("let-23": Aroian et al., 1990, Nature 34~:693-698),
chicken EGFR ("CER": Lax et al ., 1988, Mol. Cell.
Biol. 8:1970-1978), rat EGFR (Petch et al., 1990, Mol.
Cell. Biol. 10:2973-2982), rat HER2/Neu (Bargmann et
al., 1986, Nature, 319:226-230) and a novel member
isolated from the fish and termed Xiphophorus melanoma
related kinase ("Xmrk": Wittbrodt et al., 1989, Nature
342:415-421). In addition, PCR technology has led to
the isolation of other short DNA fragments that may
encode novel receptors or may represent species-
specific homologs of known receptors. One recent
example is the isolation tyro-2 (Lai, C. and Lemke,
G., 1991, Neuron 6:691-704) a fragment encoding 54
amino acids that is most related to the EGFR family.
Overexpression of EGFR-family receptors is
frequently observed in a variety of aggressive human
epithelial carcinomas. In particular, increased
expression of EGFR is associated with more aggressive
carcinomas of the breast, bladder, lung and stomach
(see, for example, Neal et al., 1985, Lancet 1:366-68;
Sainsbury et al., 1987, Lancet 1:1398-1402; Yasui et
al., 1988, Int. J. Cancer 41:211-17; Veale et al.,
1987, Cancer 55:513-16). In addition, amplification
and overexpression of HER2 has been associated with a
wide variety of human malignancies, particularly
breast and ovarian carcinomas, for which a strong
correlation between HER2 overexpression and poor
clinical prognosis and/or increased relapse
probability have been established (see, for example,
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Slamon et al., 1987, Science 235:177-82, and 1989,
Science 244:707-12). Overexpression of ~ER2 has also
been correlated with other human carcinomas, including
carcinoma of the stomach, endometrium, salivary gland,
bladder, and lung ~Yokota et al., 1986, Lancet 1:765-
67; Fukushigi et al ., 1986, Mol. Cell. Biol. 6:955-58;
Yonemura et al., 1991, Cancer Res. 51:1034; Weiner et
al., 1990, Cancer Res. 50:421-25; Geurin et al ., 1988,
oncoqene Res. 3:21-31; Semba et al., 1985, Proc. Natl.
Acad. Sci. U.S.A. 82:6497-6501; Zhau et al., 1990,
Mol. Carcinoa. 3:354-57; McCann et al ., 1990, Cancer
65:88-92). Most recently, a potential link between
HER2 overexpression and gastric carcinoma has been
reported (Jaehne et al., 1992, J. Cancer Res. Clin.
Oncol. 118:474-79). Finally, amplified expression of
the recently described HER3 receptor has been observed
in a wide variety of human adenocarcinomas (Poller et
al., 1992, J. Path 168:275-280; Krause et al., 1989,
Proc. Natl. Acad. Sci. U.S.A. 86:9193-97; European
Patent Application No. 91301737, published 9.4.91, EP
444 961).
Several structurally related soluble polypeptides
capable of specifically binding to EGFR have been
identified and characterized, including EGF,
transforming growth factor-alpha (TGF-~), amphiregulin
(AR), heparin-binding EGF (HB-EGF), and vaccinia virus
growth factor (VGF) (see, respecti~ely, Savage et al.,
1972, J. Biol. Chem. 247:7612-21; Marquardt et al .,
1984, Science 223:1079-82; Shoyab et al ., 1989,
Science 243:1074-76; Higashiyama et al., 1991, Science
251:936-39; Twardzik et al ., 1985, Proc. Natl. Acad.
Sci. U.S.A. 82:5300-04). Despite the close structural
relationships among receptors of the EGFR-family, none
of these ligands has been conclusively shown to
interact with HER2 or HER3.
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Recently, several groups have reported the
identification of specific ligands for HER2. Some of
these ligands, such as gp30 (Lupu et al., 1990,
Science 249:1552-55; Bacus et al., 1992, Cell Growth
and Differentiation 3:401-11) interact with both EGFR
and HER2, while others are reported to bind
specifically to HER2 (Wen et al., 1992, Cell 69:559-
72; Peles et al'., 1992, Cell 69:205-16; Holmes et al.,
1992, Science 256:1205-10; Lupu et al., 1992, Proc.
lo Natl. Acad. Sci. U.S.A. 89:2287-91; Huang et al.,
1992, J. Biol. Chem. 276:11508-121). The best
characterized of these ligands are neu differentiation
factor (NDF) purified and cloned from ras-transformed
Ratl-EJ cells (Wen et al., Peles et al., supra), and
15 the heregulins (HRG-~ 1, -B2, -~3), purified and
cloned from human MDA-MB-231 cells (Holmes et al.,
supra). NDF and HRG-~ share 93~ sequence identity and
appear to be the rat and human homologs of the same
protein. Both of these proteins are similar size (44-
20 45 kDa), increase tyrosine phosphorylation of HER2 in
MDA-MB-453 cells and not the EGF-receptor, and have
been reported to bind to HER2 in cross-linking studies
on human breast cancer cells. In addition, NDF has
been shown to induce differentiation of human m~mrAry
25 tumor cells to milk-producing, growth-arrested cells,
whereas the heregulin family have been reported to
stimulate proliferation of cultured human breast
cancers cell monolayers.
Interestingly, although members of the heregulin
30 family are capable of stimulating tyrosine
phosphorylation of HER2 in many mammary carcinoma cell
lines, they are not able to act on this receptor in
the ovarian carcinoma cell line SKOV3 or in HER2
transfected fibroblasts (Peles et al., 1993, EMB0 J. 4
3~ 12:961-971). These observations indicated the
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existence of other receptors for heregulin responsible
for the activation of HER2. Such cross-activation
between members of the receptor tyrosine kinase family
has been already reported and is believed to arise
5 from a ligand induced receptor heterodimerization
event (Wada et al., 1990, Cell 61:1339-1347).
Recently, it has been reported that HER3 binds
heregulin (Carraway et al., 1994, J. Biol. Chem.
269:14303-14306), and in fact, this receptor seems to
10 be involved in the heregulin-mediated tyrosine kinase
activation of HER2 (carraway et al., supra;
Sliwkowski et al ., 1994 , J. Biol. Chem. 269:14661-
14665).
The means by which receptor polypeptides
15 transduce regulatory signals in response to ligand
binding is not fully understood, and continues to be
the subject of intensive investigation. However,
important components of the process have been
uncovered, including the understanding that
20 phosphorylation of and by cell surface receptors hold
fundamental roles in signal transduction. In addition
to the involvement of phosphorylation in the signal
process, the intracellular phenomena of receptor
dimerization and receptor crosstalk function as
25 primary components of the circuit through which ligand
binding triggers a resulting cellular response.
Ligand binding to transmembrane receptor tyrosine
kinases induces receptor dimerization, leading to
activation of kinase function through the interaction
30 of adjacent cytoplasmic domains. Receptor crosstalk
refers to intracellular communication between two or
more proximate receptor molecules mediated by, for
example, activation of one receptor throuyh a
' mechanism involving the kinase activity of the other.
35 one particularly relevant example of such a phenomenon
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is the binding of EGF to the EGFR, resulting in
activation of the EGFR kinase domain and cross-
phosphorylation of HER2 (Kokai et al., 1989, Cell
58:287-92; Stern et al., 1988, EMBO J. 7:995-1001;
S King et al., 1989, Oncoqene 4:13-18).
3. Summary of the Invention
HER4 is the fourth member of the EGFR-family of
receptor tyrosine kinases and is likely to be involved
not only in regulating normal cellular function but
also in the loss of normal growth control associated
with certain human cancers. In this connection, HER4
appears to be closely connected with certain
carcinomas of epithelial origin, such as
adenocarcinoma of the breast. As such, its discovery,
and the elucidation of the HER4 coding sequence, open
a number of novel approaches to the diagnosis and
treatment of human cancers in which the aberrant
expression and/or function of this cell surface
receptor is involved.
The complete nucleotide sequence encoding the
prototype HER4 polypeptide of the invention is
disclosed herein, and provides the basis for several
general aspects of the invention hereinafter
described. Thus, the invention includes embodiments
directly involving the production and use of HER4
polynucleotide molecules. In addition, the invention
provides HER4 polypeptides, such as the prototype HER4
polypeptide disclosed and characterized in the
sections which follow. Polypeptides sharing nearly
equivalent structural characteristics with the
prototype HER4 molecule are also included within the
scope of this invention. Furthermore, the invention
includes polypeptides which interact with HER4
expressed on the surface of certain cells thereby
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affecting their growth and/or differentiation. The
invention is also directed to anti-HER4 antibodies,
which have a variety of uses including but not limited
to their use as components of novel biological
approaches to human cancer diagnosis and therapy
provided by the invention.
The invention also relates to the identification
of HER4 ligands and methods for their purification.
The invention also relates to the discovery of an
apparent functional relationship between HER4 and
HER2, and the therapeutic aspects of the invention
include those which are based on applicants'
preliminary understanding of this relationship.
Applicants' data strongly suggests that HER4 interacts
~5 with HER2 either by heterodimer formation or receptor
crosstalk, and that such interaction appears to be one
mechanism by which the HER4 receptor mediates effects
on cell ~ehavior. The reciprocal consequence is that
HER2 activation is in some circumstances mediated
through HER4.
In this connection, it appears that although
heregulin induces phosphorylation of HER2 in cells
expressing HER2 and HER4. Heregulin does not directly
stimulate ~ER2 but acts by stimulating tyrosine
phosphorylation of HER4.
Recognition of HER4 as a primary component of the
heregulin signal transduction pathway opens a number
of novel approaches to the diagnosis and treatment of
human cancers in which the aberrant expression and/or
function of heregulin and/or HER4 are involved. The
therapeutic aspects of this invention thus include
mediating a ligand's affect on HER4 and HER2 through
antagonists, agonists or antibodies to HER4 ligands or
HER4 receptor itself.
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The invention also relates to chimeric proteins
that specifically target and kill HER4 expressing
tumor cells, polynucleotides encoding such chimeric
proteins, and methods of using both in the therapeutic
treatmènt of cancer and other human malignancies.
Applicants' data demonstrate that such recombinant
chimeric proteins specifically bind to the HER4
receptor and are cytotoxic against tumor cells that
express HER4 on their surface. The bifunctional
1() retention of both the specificity of the cell-binding
portion of the molecule and the cytotoxic potential of
the toxin portion makes for a very potent and targeted
reagent.
The invention further relates to a method
1~ allowing determination of the cytotoxic activity of
HER4 directed cytotoxic substances on cancer cells,
thereby providing a powerful diagnostic tool; this
will be of particular interest for prognosis of the
effectiveness of these substances on an individual
2() malignancy prior their therapeutic use.
4. Brief Description of the Figures
Figures l/l through l/5. Nucleotide sequence
[SEQ ID No:l~ and deduced amino acid sequence of HER4
2~ of the coding sequence from position 34 to 3961 (1308
amino acid residues) [SEQ ID No:2]. Nucleotides are
numbered on the left, and amino acids are numbered
above the sequence.
Figures 2/l through 2/4. Nucleotide sequence
3() [SEQ ID No:3] and deduced amino acid sequence ([SEQ ID
No:4] of cDNAs encoding HER4 with alternate 3' end and
without autophosphorylation domain. This sequence is
identical with that of HER4 shown in Figures l/l
through l/5 up to nucleotide 3168, where the sequence
3~ diverges and the open reading frame stops after 13
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amino acids, followed by an extended, unique 3'-
untranslated region.
Figures 3/l through 3/3. Nucleotide sequence
[SEQ ID No:5] and deduced amino acid sequence [SEQ ID
No:6] of cDNA encoding HER4 with a N-terminal
truncation. This sequence contains the 3'-portion of
the HER4 sequence where nucleotide position 156 of the
truncated sequence aligns with position 2335 of the
complete HER4 sequence shown in Figures 1/1 through
1() l/5 (just downstream from the region encoding the ATP-
binding site of the HER4 kinase). The first 155
nucleotides of the truncated sequence are unique from
HER4 and may represent the 5'-untranslated region of a
transcript derived from a cryptic promoter within an
l~ intron of the HER4 gene. (Section 6.2.2., infra).
Figures 4/1, 4/2 and 5. The deduced amino acid
sequence of two variant forms of human HER4 aligned
with the full length HER4 receptor as represented in
Figures 1/1 through 1/5. Sequences are displayed
2() using the single-letter code and are numbered on the
right with the comple~e HER4 sequence on top and the
variant sequences below. Identical residues are
indicated by a colon between the aligned residues.
Figures 4/1 and 4/2. HER4 with alternate 3'-end,
lacking an autophosphorylation domain [SEQ ID No. 4].
This sequence is identical with that of HER4, shown in
Figures 1/1 through 1/5, up to amino acid 1045, where
the sequence diverges and continues for 13 amino acids
before reaching a stop codon.
() Figure 5. HER4 with N-terminal truncation [SEQ
ID No. 6]. This sequence is identical to the 3'-
portion of the HER4 shown in Figures 1/1 through 1/5
beginning at amino acid 768. (Section 6.2.2., lnfra).
Figures 6/1 and 6/2. Deduced amino acid sequence
3~ of human HER4 and alignment with other human EGFR-
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family members (EGFR [SEQ ID No:7]; HER2 [SEQ ID
No:8]; HER3 [SEQ ID No:9]). Sequences are displayed
using the single-letter code and are numbered on the
left. Identical residues are denoted with dots, gaps
are introduced for optimal alignment, cysteine
residues are marked with an asterisk, and N-linked
glycosylation sites are denoted with a plus (+).
Potential protein kinase C phosphorylation sites are
indicated by arrows (HER4 amino acid positions 679,
685, and G99). The predicted ATP-binding site is
shown with 4 circled crosses, C-terminal tyrosines are
denoted with open triangles, and tyrosines in HER4
that are conserved with the major autophosphorylation
sites in the EGFR are indicated with black triangles.
l~ The predicted extracellular domain extends from the
boundary of the signal sequence marked by an arrow at
position 25, to the hydrophobic transmembrane domain
which is overlined from amino acid positions 650
through 675. Various subdomains are labeled on the
2() right: I, II, III, and IV = extracellular subdomains
(domains II and IV are cysteine-rich); TM =
transmembrane domain; TK = tyrosine kinase domain.
Domains I, III, TK are boxed.
Figure 7. Hydropathy profile of HER4, aligned
2~ with a comparison of protein domains for HER4 (1308
amino acids), EGFR (1210 amino acids), HER2 (1255
amino acids), and HER3 (134Z amino acids). The signal
peptide is represented by a stippled box, the
cysteine-rich extracellular subdomains are hatched,
3() the transmembrane domain is filled, and the
cytoplasmic tyrosine kinase domain is stippled. The
percent amino acid sequence identities between HER4
and other EGFR-family members are indicated. Sig,
signal peptide; I, II, III, and IV, extracellular
3~ domains; TM, transmembrane domain; JM, juxtamembrane
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domain; CaIn, calcium influx and internalization
domain; 3'UTR, 3' untranslated region.
Figures 8A and 8B. Northern blot analysis from
human tissues hybridized to HER4 probes. RNA size
markers (in kilobases) are shown on the left. Lanes l
; through 8 represent 2 ~g of poly(A)+ mRNA from
pancreas, kidney, skeletal muscle, liver, lung,
placenta, brain, and heart, respectively. Figure 8A,
Northern blot analysis of mRNA from human tissues
hybridized to HER4 probes from the 3'-
autophosphorylation domain; Figure 8B, Northern blot
analysis from human tissues hybridized to HER4 probes
from the 5'-extracellular domain (see Section 6.2.3.,
infra ) .
Figures 9A and sB. Immunoblo~ analysis of
recombinant HER4 stably expressed in CHO-KI cells,
according to procedure outlined in Section 7.l.3,
infra. Membrane preparations from CHO-KI cells
expressing recombinant HER4 were separated on 7~ SDS-
2() polyacrylamide gels and transferred to nitrocellulose.
In Figure 9A, blots were hybridized with a monoclonal
antibody to the C-terminus of HER2 (Ab3, Oncogene
Science, Uniondale, NY) that cross-reacts with HER4.
In Figure 9B, blots were hybridized with a sheep
2~ antipeptide polyclonal antibody to a common epitope of
HER2 and HER4. Lane l, parental CHO-KI cells; lanes 2
- 4, CHO-KI/HER4 cell clones 6, 21, and 3,
respectively. Note the 180 kDa HER4 protein and the
130 kDa cross-reactive species. The size in
,() kilodaltons of prestained high molecular weight
markers (BioRad, Richmond, CA) is shown on the left.
~igures lOA through lOD. Specific activation of
HER4 tyrosine kinase by a breast cancer
differentiation factor (see Section 8., infra) . Four
3~ recombinant cell lines, each of which was engineered
r to overexpress a single member of EGFR-family of
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tyrosine kinase receptors (EGFR, HER2, HER3, and
HER4), were prepared according to the methods
described in Sections 7.1.2 and 8.1., infra. Cells
from each of the four recombinant cell lines were
stimuiated with various ligand preparations and
assayed for receptor tyrosine phosphorylation using
the assay described in Section 8.2., infra . Figure
lOA, CHO/HER4 #3 cells; Figure lOB, CHO/HER2 cells;
Figure lOC, NRHER5 cells; and Figure lOD, 293/HER3
cells. Cells stimulated with: lane 1, buffer control;
lane 2, 100 ng/ml EGF; lane 3, ZO0 ng/ml amphiregulin;
lane 4, lO ml phenyl, column fraction 17 (Section 9,
infra); lane 5, 10 ~1 phenyl column fraction 14
(Section 9., infra , and see description of Figure 11,
1~ below). The size (in kilodaltons) of the prestained
molecular weight markers are labeled on the left of
each panel. The phosphorylated receptor in each
series migrates just below the 221 kDa marker. Bands
at the bottom of the gels are extraneous and are due
2() to the reaction of secondary antibodies with the
antibodies used in the immunoprecipitation.
Figures llA through llF. Biological and
biochemical properties of the MDA-MB-453-cell
differentiation activity purified from the conditioned
2~ media of HepG2 cells (Section 9., infra) . Figures llA
and llB show induction of morphologic differentiation.
Conditioned media from HepG2 cells was subjected to
ammonium sulfate fractionation, followed by dialysis
against PBS. Dilutions of this material were added to
3() MDA-MB-453 monolayer at the indicated protein
concentrations. Figure llA, control; Figure llB, 80
ng per well; Figure llC, 2.0 ~g per well; Figure
llD, Phenyl-5PW column elution profile monitored at
230 nm absorbance; Figure llE, Stimulation of MDA-MB-
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453 tyrosine autophosphorylation with the following
ligand preparations: None (control with no factor
added); TGF-a (50 ng/ml); CM (16-fold concentrated
HepG2 ~conditioned medium tested at 2 ~1 and 10 ~1 per
well~; fraction (phenyl column fractions 13 to 20, 10
~1 per well). Figure llF, Densitometry analysis of
the phosphorylation signals shown in Figure llE.
Figures 12A and 12B. NDF-induced tyrosine
phosphorylation. Figure 12A, MDA-MB-453 cells (lane
1() 1, mock transfected COS cell supernatant; lane 2, NDF
transfected COS cell supernatant); Figure lZB,
CHO/HER4 21-2 cells (lanes 1 and 2, mock transfected
COS cell supernatant; lanes 3 and 4, NDF transfected
COS cell supernatant). See Section 10., infra.
I~ Tyrosine phosphorylation was determined by the
tyrosine kinase stimulation assay described in Section
8.2., infra .
Figures 13A and 13B. Regional location of the
HER4 gene to human chromosome 2 band q33. Figure 13A,
2() Distribution of 124 sites of hybridization on human
chromosomes; Figure 13B, Distribution of
autoradiographic grains on diagram of chromosome 2.
Figure 14. Amino acid sequence of HER4-Ig fusion
protein [SEQ ID No:10] (Section 5.4., infra).
2~ Figure 15. Recombinant heregulin induces
tyrosine phosphorylation of HER4. Tyrosine
phosphorylated receptors were detected by Western
blotting with an anti-phosphotyrosine Mab. Arrows
indicate the HER2 and HER4 proteins. Monolayers of
3() MDA-MB453 or CHO/HER4 cells were incubated with media
from COS-1 cells transfected with a rat heregulin
expression plasmid (HRG), or with a cDM8 vector
control (-). The media was either applied directly
(lx) or after concentrating ZO-fold (20x, and vector
3~ control). Solubilized cells were immunoprecipitated
with anti-phosphotyrosine Mab. Monolayers of CHO/HER2
cells were incubated as above with transfected Cos-1
cell supernatants or with two stimulatory Mabs to HER2
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(Mab 28 and 29). Solubilized cells were
immunoprecipitated with anti-HER2 Mab.
Figures 16A through 16C. Expression of
recombinant HER2 and HER4 in human CEM cells.
Transfected CEM cells were selected that stably
express either HER2, HER4, or both recombinant
receptors. In Figure 16A, recombinant HER2 was
detected by immunmoprecipitation of cell lysates with
anti-HER2 Mab (Ab-2) and Western blotting with another
Il) anti-HER2 Mab (Ab-3). In Figure lGB, Recombinant HER4
was detected by immunoprecipitation of 35S-labeled cell
lysates with HER4-specific rabbit anti-peptide
antisera. In Figure 16C, Three CEM cell lines were
selected that express one or both recombinant
l~ receptors and aliquots of each were incubated with
media control (-), with two HER2-stimulatory Mabs (Mab
28 and 29), or with an isotype matched control Mab
(18.4). Solubilized cells were immunoprecipitated
with anti-HER2 Mab (Ab-2) and tyrosine phosphorylated
2(~ HER2 was detected by Western blotting with an anti-
phosphotyrosine Mab. The size in kilodaltons of
prestained high molecular weight markers (Bio-Rad) is
shown on the left and arrows indicate the HER2 and
HER4 proteins.
2~ Figures 17A through 17C. Heregulin induces
tyrosine phosphorylation in CEM cells expressing HER4.
Three CEM cell lines that express either HER2 or HER4
alone (CEM 1-3 and CEM 3-13) or together (CEM 2-9)
were incubated with 7x concentrated supernatants from
3() mock-(-) or heregulin-transfected (+) COS-1 cells.
Solubilized cells were immunoprecipitated (IP) with
anti-phosphotyrosine Mab (PY20); in Figure 17A,
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HER2-specific anti-HER2 Mab (Ab-2); in Figure 17B,
HER4-specific Mab (6-4); in Figure 17C, in each case
tyrosine phosphorylated receptors were detected by
Western blotting with anti-phosphotyrosine Mab. The
size in kilodaltons of prestained molecular weight
- markers (BioRad) is shown on the left and arrows
indica~e the HER2 and HER4 proteins. HRG, recombinant
rat heregulin.
Figure 18. Covalent cross-linking of iodinated
1() heregulin to HER4. l I-heregulin was added to
CHO/HER4 or CHO/HER2 cells for 2 h at 4 C. Washed
cells were cross-linked with Bs3, lysed, and the
proteins separated using 7% PAGE. Labeled bands were
detected on the phosphorimager. Molecular weight
l~ markers are shown on the left.
Figures l9A through l9D. Purification of p45
from HepG2 conditioned media. Column fractions were
tested for their potential to induce differentiation
of MDA-MB-453 cells. Active fractions were pooled as
2~ indicated by an horizontal bar. Figure lsA,
Concentrated HepG2 conditioned medium was subjected to
50% ammonium sulfate precipitation. Supernatant
resulting from this step was subjected to hydrophobic
interaction chromatography using phenyl-Sepharose.
2~ Pooled fractions were then loaded on a DEAE-Sepharose
column. Figure l9B, the DEAE-Sepharose column flow-
through was subjected to CM-Sepharose chromatography.
Figure l9C, Affinity Chromatography of the MDA-MB-453
differentiation factor using heparin-5PW column.
3() Fractions 35-38 eluting around 1.3M NaCl were pooled.
Figure l9D, Size Exclusion chromatography of the
differentiation factor. The molecular masses of
calibration standards are indicated in kilodaltons.
Figure 20. Aliquots (25 microliter) of the
active size exclusion column fractions (30 and 32)
were electrophoresed under reducing conditions on a
12 5~ polyacrylamide gel. The gel was silver-stained.
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Molecular masses of Bio-Rad silver stain standards are
indicated in kilodaltons.
Figures 21A through 21C. Stimulation of tyrosine
phosphorylation by p45. Figure 21A, Size exclusion
column fractions were tested on MDA-MB-453 cells for
the induction of tyrosine phosphorylation. Cell
lysates were then electrophoresed on a 4-lS%
polyacrylamide gel. After transfer to nitrocellulose,
proteins were probed with a phosphotyrosine antibody
1() and phosphoproteins detected by chemiluminescence.
The molecular mass of the predominantly phosphorylated
protein is indicated. Figure 2lB, the experiments
were performed on cells that had been transfected with
expression plasmids for either HER4 (CHO/HER4) or HER2
I~ (CHO/HER2). Cell monolayers were incubated in the
absence or the presence of p45 (size exclusion column
fraction 32, lOO ng/ml). Samples were then processed
as indicated in Figure 21A except that a 7.5%
polyacrylamide gel was used to separate the CHO/HER2
2() cell lysates. Figure 21C, CHO/HER2 cells were
incubated in the presence or the absence of N29
monoclonal antibody to the extracellular domain of
pl85 . Cell lysates were immunoprecipitated with
the Ab-3 monoclonal antibody to pl85
2~ Precipitated proteins were subjected to SDS-PAGE, and
phosphoproteins were detected as indicated under
Section 13.4., supra.
Figures 22A and 22B. Binding and cross-linking
of I-p45 to CHO-KI, CHO-HER2 and CHO/HER4 cells.
~) Figure 22A, Scatchard analysis of the binding of 1 I-
p45 to CHO/HER4 cells. Increasing concentrations of
l25I-p45 were incubated with cell monolayers for 2 h at
4 C. Nonspecific binding was subtracted from all
cell-associated radioactivity data values. A
Scatchard plot as well as a saturation curve of the
binding data are shown. Figure 22B, Covalent cross- ,
linking. I-p45 was added to the cells in the
presence or absence of an excess of unlabeled p45 for
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2 h at 4 C. After washing of the cells to remove
unbound iodinated material, the cross-linking reagent
bis-(sulfosuccinimidyl)-suberate was added to the
_ cells for 45 min. at 4 C. Cells were lysed and
proteins separated by electrophoresis on a 7.5%
polyacrylamide gel. Molecular masses of protein
standards are indicated in kilodaltons. A Molecular
Dynamics PhosphoImager was used to visualize the
radioactive species.
1~) Figures 23A and 23B. Construction of the HAR-TX
~2 expression plasmid, encoding the hydrophilic leader
sequence of amphiregulin (AR), heregulin ~2, and PE40,
under control of the IPTG inducible T7 promoter;
Figure 23A, schematic diagram of the expression
1~ plasmid pSE 8.4, encoding HAR-TX ~2; Figure 23B,
amino acid sequence of HAR ~2, the ligand portion of
HAR-TX ~2, composed of the AR leader sequence and rat
heregulin ~2 [SEQ ID No:40].
Figures 24A and 24B. cDNA sequence [SE~ ID
2() No:4l] and deduced amino acid sequence [SEQ ID No:42]
of the chimera HAR-TX ~2, comprising the amphiregulin
(AR) leader sequence and the coding sequences of rat
heregulin Pseudomonas exotoxin PE40. The linker
sequence between the two portions is indicated by a
2~ bar above the sequence, the ligand portion is located
at the 5' (N-terminal), the PE40 exotoxin portion is
located at the 3' (C-terminal) part of the sequence.
Nucleotides are numbered on the right side, and amino
acids are numbered below the sequence.
Figure 25. Purification of the chimeric HAR-TX
b2 protein: shown is a Coomassie brilliant blue
stained SDS-PAGE (4-20~) of the different purification
steps. Lanes l - 5 have been loaded under reducing
conditions. Lane l, MW standards; lane 2, refolded
3~ HAR-TX ~2, 20x concentrated; lane 3, POROS HS flow-
through, 20x concentrated; lane 4, POROS HS eluate;
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lane 5, Source 15S eluate (pure HAR-TX ~2, 2 ~g); lane
6, 2 ~g HAR-TX ~2, loaded under non-reducing
conditions.
~ igure 26. Membrane-based ELISA binding
analysis, performed to determine the binding activity
of the purified HAR-TX ~2 protein. Binding of HAR-TX
p2 (0) and PE40 (-) to membranes prepared from the
HER4 expressing human breast carcinoma cell line.
Figure 27. HAR-TX b~2 induced tyrosine
1~ phosphorylation in transfected CEM cells. CEM cells
co-expressing HER4 and HER2 (H2,4), or expressing HER4
(H4), HER2 (H2), HERl (Hl) alone, respectively, were
incubated in the presence (+) or absence (-) of HAR-TX
~2, then solubilized, and immunoblotted with the
1~ monoclonal anti-phosphotyrosine antibody PY20. The
arrow indicates the phosphorylated receptor band, the
molecular weight is indicated in kDA.
Figures 28A and 28B. Cytotoxic effect of HAR-TX
~2 on tumor cell lines. Figure 28A, following 48
2() hours incubation with HAR-TX ~2, the cell killing
effect of HAR-TX ~2 on the tumor cell lines LNCaP (-),
AU565 (0), SKBR3 (-), and SKOV3 (i-') by quantification
of fluorescent calcein cleaved from calcein-AM.
Figure 28B, Competitive cytotoxicity of HAR-TX ~2 with
2~ heregulin ~2-Ig. LNCaP cells were co-incubated with 50
ng/ml HAR-TX ~2 and increasing concentrations (2-5000
ng/ml) of either heregulin ~2-Ig (r~) or LG-Ig (-). The
data represent the mean of triplicate assays.
Figure 29. HAR-TX ~2 induced tyrosine
3() phosphorylation in tumor cells expressing HER3 (L2987)
or co-expressing HER2 and HER3 (H3396). Cells were
incubated in the presence (+) or in the absence (-) of
HAR-TX ~2, solubilized, and immunoblotted with the
monoclonal anti-phosphotyrosine antibody PY20.
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Phosphorylated receptors are indicated by an arrow,
the molecular weight is indicated in kDa.
5. Detailed Description of the Invention
The present invention is directed to
; HER4/pl80 r B ("HER4"), a closely related yet distinct
member of the Human EGF Receptor (HE~)/neu subfamily
of receptor tyrosine kinases, as well as HER4-encoding
polynucleotides (e.g., cDNAs, genomic DNAs, RNAs,
anti-sense RNAs, etc.), the production of mature and
precursor forms of HER4 from a HER4 polynucleotide
coding sequence, recombinant HER4 expression vectors,
HER4 analogues and derivatives, anti-HER4 antibodies,
HER4 ligands, and diagnostic and therapeutic uses of
1~ HER4 polynucleotides, polypeptides, ligands, and
antibodies in the field of human oncology and
neurobiology.
As discussed in section 2, supra, HER2 has been
reported to be associated with a wide variety of human
2() malignancies, thus the understanding of its activation
mechanisms as well as the identification of molecules
involved are of particular clinical interest. This
invention uncovers an apparent functional relationship
between the HER4 and HER2 receptors involving HER4-
2~ mediated phosphorylation of HER2, potentially viaintracellular receptor crosstalk or receptor
dimerization. In this connection, the invention also
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provides HER4 ligands capable of inducing cellular
differentiation in breast carcinoma cells that appears
to involve HER4-mediated phosphorylation of HER2.
Furthermore, applicants' data provide evidence that
heregulin mediates biological effects on such cells
not directly through HER2, as has been reported (Peles
et al ., 1992, Cell 69:205-216), but instead by means
of a direct interaction with HER4, and/or through an
interaction with a HER2/ HER4 complex. In cell lines
expressing both HER2 and HER4, binding of heregulin to
HER4 may stimulate HER2 either by heterodimer
formation of these two related receptors or by
intracellular receptor crosstalk.
Recently, also HER3 has been reported to bind
heregulin (see Section 2, supra). However, various
observations indicate that the heregulin-mediated
activation of HER3 varies considerably, depending on
the context of expression, suggesting that other
cellular components may be involved in the modulation
of HER3 activity (reviewed in: Carraway and Cantley,
1994, Cell 78:5-8).
Unless otherwise indicated, the practice of the
present invention utilizes standard techniques of
molecular biology and molecular cloning, micro~iology,
immunology, and recombinant DNA known in the art.
Such techniques are described and explained throughout
the literature, and can be found in a number of more
comprehensive publications such as, for example,
Sambrook et al ., Molecular Cloning; A Laboratory
Manual (Second Edition, 1989).
5.1. ~R~ Polynucleotides
One aspect of the present invention is directed
to HER4 polynucleotides, including recombinant
polynucleotides encoding the prototype HER4
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polypeptide shown in FIG. lA and lB, polynucleotides
r which are related or are complementary thereto, and
recombinant vectors and cell lines incorporating such
recombinant polynucleotides. The term "recombinant
5 polynucleotide" as used herein refers to a
polynucleotide of genomic, cDNA, synthetic or
semisynthetic origin which, by virtue of its origin or
manipulation, is not associated with any portion of
the polynucleotide with which it is associated in
10 nature, and may be linked to a polynucleotide other
than that to which it is linked in nature, and
includes single or double stranded polymers of
ribonucleotides, deoxyribonucleotides, nucleotide
analogs, or combinations thereof. The term also
15 includes various modifications known in the art,
including but not limited to radioactive and chemical
labels, methylation, caps, internucleotide
modifications such as those with charged linkages
(e.g., phosphorothothioates, phosphorodithothioates,
20 etc.) and uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidites,
carbamites, etc.), as well as those containing pendant
moeties, intercalcators, chelators, alkylators, etc.
Related polynucleotides are those having a contiguous
25 stretch of about 200 or more nucleotides and sharing
at least about 80% homology to a corresponding
sequence of nucleotides within the nucleotide sequence
disclosed in FIG. lA and lB. Several particular
embodiments o f such HER4 polynucleotides and vectors
30 are provided in example Sections 6 and 7, inf~a.
HER4 polynucleotides may be obtained using a
variety of general techniques known in the art,
including molecular cloning and chemical synthetic
methods. One method by which the molecular cloning of
35 cDNAs encoding the prototype HER4 polypeptide of the
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invention (FIG. lA and 1 B), as well as several HER4
polypeptide variants, is described by way of example
in Section 6., in f ra . Conserved regions of the
sequences of EGFR, HER2, HER3, and Xmrk are used for
selection of the degenerate oligonucleotide primers
which are then used to isolate HER4. Since many of
these sequences have extended regions of amino acid
identity, it is difficult to determine if a short PCR
fragment represents a unique molecule or merely the
species-specific counterpart of EGFR, HER2, or HER3.
Often the species differences for one protein are as
great as the differences within species for two
distinct proteins. For example, fish Xmrk has regions
of 47/55 (85~) amino acid identity to human EGFR,
suggesting it might be the fish EGFR, however
isolation of another clone that has an amino acid
sequence identical to Xmrk in this region (57/57)
shows a much higher homology to human EGFR in its
flanking sequence (92% amino acid homology) thereby
suggesting that it, and not Xmrk, is the fish EGFR
(Wittbrodt et al., 1989, Nature 342:415-421). As
described in Section 6., infra, it was necessary to
confirm that a murine HER4/erbB4 PCR fragment was
indeed a unique gene, and not the murine homolog of
EGFR, HER2, or HER3, by isolating genomic fragments
corresponding to murine EGFR, erbB2 and erbB3.
Sequence analysis of these clones confirmed that this
fragment was a novel member of the EGFR family.
Notably a region of the murine clone had a stretch of
60/64 amino acid identity to human HER2, but
comparison with the amino acid and DNA sequences of
the other EGFR homologs from the same species (mouse)
firmly established it encoded a novel transcript.
HER4 polynucleotides may be obtained from a
variety of cell sources which produce HER4-like
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activities and/or which express HER4-encoding mRNA.
In this connection, applicants have identified a
number of suitable human cell sources for HER4
; polynucleotides, including but not limited to brain,
cerebellum, pituitary, heart, skeletal muscle, and a
variety of breast carcinoma cell lines (see Section
6., infra).
For example, polynucleotides encoding HER4
polypeptides may be o~tained by cDNA cloning from RNA
lo isolated and purified from such cell sources or by
genomic cloning. Either cDNA or genomic libraries of
clones may be prepared using techniques well known in
the art and may be screened for particular HER4-
encoding DNAs with nucleotide probes which are
substantially complementary to any portion of the HER4
gene. Various PCR cloning techniques may also be used
to obtain the HER4 polynucleotides of the invention.
A number of PCR cloning protocols suitable for the
isolation of HER4 polynucleotides have been reported
in the literature (see, for example, PCR Protocols: A
Guide to Methods and APplications, Eds. Inis et al.,
Academic Press, 1990).
For the construction of expression vectors,
polynucleotides containing the entire coding region of
the desired H~R4 may be isolated as full length clones
or prepared by splicing two or more polynucleotides
together. Alternatively, HER4-encoding DNAs may be
synthesized in whole or in part by chemical synthesis
using techniques standard in the art. Due to the
inherent degeneracy of nucleotide coding sequences,
any polynucleotide encoding the desired HER4
polypeptide may be used for recombinant expression.
Thus, for example, the nucleotide sequence encoding
the prototype HER4 of the invention provided in FIG.
3s
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lA and lB may be altered by substitu~ing nucleotides
such that the same HER4 product is obtained.
The invention also provides a number of useful
applications of the HER4 polynucleotides of the
S invention, including but not limited to their use in
the preparation of HER4 expression vectors, primers
and probes to detect and/or clone HER4, and diagnostic
reagents. Diagnostics based upon HER4 polynucleotides
include various hybridization and PCR assays known in
the art, utilizing HER4 polynucleotides as primers or
probes, as appropriate. One particular aspect of the
invention relates to a PCR kit comprising a pair of
primers capable of priming cDNA synthesis in a PCR
reaction, wherein each of the primers is a HER4
polynucleotide of the invention. Such a kit may be
useful in the diagnosis of certain human cancers which
are characterized by aberrant HER4 expression. For
example, certain human carcinomas may overexpress HER4
relative to their normal cell counterparts, such as
human carcinomas of the breast. Thus, detection of
HER4 overexpression mRNA in breast tissue may be an
indication of neoplasia. In another, related
embodiment, human carcinomas characterized by
overexpression of HER2 and expression or
overexpression of HER4 may be diagnosed by a
polynucleotide-based assay kit capable of detecting
both HER2 and HER4 mRNAs, such a kit comprising, for
example, a set of PCR primer pairs derived from
divergent sequences in the HER2 and HER4 genes,
respectively.
5.2. HER4 Polypeptides
Another aspect of the invention is directed to
HER4 polypeptides, including the prototype HER4
polypeptide provided herein, as well as polypeptides
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derived from or having su~stantial homology to the
amino acid sequence of the prototype HER4 molecule.
The term "polypeptide~ in this context refers to a
r polypeptide prepared by synthetic or recom~inant
5 means, or which is isolated from na~ural sources. The
term "substantially homologous~ in this context refers
to polypeptides of about 80 or more amino acids
sharing greater than about 90% amino acid homology to
a corresponding contiguous amino acid sequence in the
lO prototype HER4 primary structure (FIG. lA and lB).
The term "prototype HER4" refers to a polypeptide
having the amino acid sequence of precursor or mature
HER4 as provided in FIG. lA and lB, which is encoded
by the consensus cDNA nucleotide sequence also
15 provided therein, or by any polynucleotide sequence
which encodes the same amino acid sequence.
HER4 polypeptides of the invention may contain
deletions, additions or substitutions of amino acid
residues relative to the sequence of the prototype
20 HE~4 depicted in FIG. lA and lB which result in silent
changes thus producing a bioactive product. Such
amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity and/or the amphipathic
25 nature of the resides involved. For example,
negatively charged amino acids include aspartic acid
and glutamic acid; positively charged amino acids
include lysine and arginine; amino acids with
uncharged polar head groups or nonpolar head groups
30 having similar hydrophilicity values include the
following: leucine, isoleucine, valine; glycine,
alanine; asparagine, glutamine; serine, threonine;
phenylalanine, tyrosine.
The HER4 polypeptide depicted in FIG. lA and lB
35 has all of the fundamental structural features
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characterizing the EGFR-family of receptor tyrosine
kinases (Hanks et al., 1988, Science 241:42-S2). The
precursor contains a single hydrophobic stretch of 26
amino acids characteristic of a transmembrane region
that bisects the protein into a 625 amino acid
extracellular ligand binding domain, and a 633 amino
acid C-terminal cytoplasmic domain. The ligand
binding domain can be further divided into 4
subdomains (I - IV), including two cysteine-rich
regions (II, residues 186-334; and IV, residues 496-
633), and two flanking domains (I, residues 29-185;
and III, residues 335-495) that may define specificity
for ligand binding (Lax et al., 1988, Mol. Cell. Biol.
8:1970-78). The extracellular domain of HER4 is most
lS similar to HER3, where domains II-IV of HER4 share 56-
67% identity to the respective domains of HER3. In
contrast, the same regions of EGFR and HER2 exhibit
43-51~ and 34-46% homology to HER4, respectively (FIG.
6A and 6B). The 4 extracellular subdomains of EGFR
and HER2 share 39-50% identity. HER4 also conserves
all 50 cysteines present in the extracellular portion
of EGFR, HER2, and HER3, except that the HER2 protein
lacks the fourth cysteine in domain IV. There are 11
potential N-linked glycosylation sites in HER4,
conserving 4 of 12 potential sites in EGFR, 3 of 8
sites in HER2, and 4 of 10 sites in HER3.
Following the transmembrane domain of HER4 is a
cytoplasmic juxtamembrane region of 37 amino acids.
This region shares the highest degree of homology with
EGFR (73% amino acid identity) and contains two
consensus protein kinase C phosphorylation sites at
amino acid residue numbers 679 (serine) and 699
tThreonine) in the FIG. lA and lB sequence, the latter
of which is present in EGFR and HER2. Notably, HER4
lacks a site analogous to Thr654 of EGFR.
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Phosphorylation of this residue in the EGFR appears to
block ligand-induced internalization and plays an
important role in its transmembrane signaling (Livneh
r et al ., 1988, Mol. Cell. Biol. 8 : 2302-08) . HER4 also
contains Thr692 analogous to Thr694 of HER2. This
threonine is absent in EGFR and HER3 and has been
proposed to impart cell-type specificity to the
mitogenic and transforming activity of the HER2 kinase
(DiFiore et al . 1992, EMBO J. 11: 3927-33) . The
juxtamembrane region of HER4 also contains a MAP
kinase consensus phosphorylation site at amino acid
number 699 (Threonine), in a position homologous to
Thr699 of EGFR which is phosphorylated by MAP kinase
in response to EGF stimulation (Takishima et 21.,
1991, Proc. Natl. Acad. Sci. U.S.A. 88:2520-25).
The remaining cytoplasmic portion of HER4
consists of a 276 amino acid tyrosine kinase domain,
an acidic helical structure of 38 amino acids that is
homologous to a domain required for ligand-induced
20 internalization of the EGFR (Chen et al., 1989, Cell
S9:33-43), and a 282 amino acid region containing 18
tyrosine residues characteristic of the
autophosphorylation domains of other EGFR-related
proteins (FIG. 6A and 6B). The 276 amino acid
tyrosine kinase domain conserves all the diagnostic
structural motifs of a tyrosine kinase, and is most
related to the catalytic domains of EGFR (79%
identity) and HER2 (77% identity), and to a lesser
degree, HER3 (63% identity). In this same region,
EGFR and HER2 share 83% identity. Examples of the
various conserved structural motifs include the
- following: the ATP-binding motif (GXGXXG) [SEQ ID
No:11] with a distal lysine residue that is predicted
to be involved in the phosphotransfer reaction (Hanks
35 et al., 198, Science 241:42-52; Hunter and Cooper, in
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The Enzymes Vol. 17 (eds. Boyer and Krebs) pp. 191-246
(Academic Press 1986)); tyrosine-kinase specific
signature sequences (DLAARN [SEQ ID No:12] and PIKWMA
[SEQ ID No:13~) and Tyr875 (FIG. 6A and 6B), a residue
that frequently serves as an autophosphorylation site
in many tyrosine kinases (Hunter and Cooper, supra);
and approximately 15 residues that are either highly
or completely conserved among all known protein
kinases (Plowman et al., 1990, Proc. Natl. Acad. Sci.
lo U.S.A. 87:4905-09; Hanks et al ., supra) . The C-
terminal 282 amino acids of HER4 has limited homology
with HER2 (27%) and EGFR (19%). However, the C-
terminal domain of each EGFR-family receptor is
proline-rich and conserves stretches of 2-7 amino
acids that are generally centered around a tyrosine
residue. These residues include the major tyrosine
autophosphorylation sites of EGFR at TyrlO68, TyrlO86,
Tyrll48, and Tyrll73 (FIG. 6A and 6B, filled
triangles; Margolis et al., 1989, J. Biol. Chem.
264:10667-71).
5.3. ~ecombinant Syntheqi~ of ~BR4 Polypeptides
The HER4 polypeptides of the invention may be
produced by the cloning and expression of DNA encoding
the desired HER4 polypeptide. Such DNA may be ligated
into a number of expression vectors well known in the
art and suitable for use in a number of acceptable
host organisms, in fused or mature form, and may
contain a signal sequence to permit secretion. Both
prokaryotic and eukaryotic host expression systems may
be employed in the production of recombinant HER4
polypeptides. For example, the prototype HER4
precursor coding sequence or its functional equivalent
may be used in a host cell capable of processing the
precursor correctly. Alternatively, the coding
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seq~ence for mature HER4 may be used to directly
express the mature HER4 molecule. Functional
equivalents of the HER4 precursor coding sequence
include any DNA sequence which, when expressed inside
the appropriate host cell, is capable of directing the
synthesis, processing and/or export of HER4.
Production of a HER4 polypeptide using
recom~inant DNA technology may be divided into a four-
step process for the purposes of description: (l)
isolation or generation of DNA encoding the desired
HER4 polypeptide; (2) construction of an expression
vector capable of directing the synthesis of the
desired HER4 polypeptide; (3) transfection or
transformation of appropriate host cells capable of
l~ replicating and expressing the HER4 coding sequence
and/or processing the initial product to produce the
desired HER4 polypeptide; and (4) identification and
purification of the desired HER4 product.
5.3.l. Isolation or Generation of HER4
Encoding DNA
HER4-encoding DNA, or functional equivalents
thereof, may be used to construct recombinant
expression vectors which will direct the expression of
the desired HER4 polypeptide product. In a specific
embodiment, DNA encoding the prototype HER4
polypeptide (FIG. lA and lB), or fragments or
functional equivalents thereof, may be used to
generate the recombinant molecules which will direct
the expression of the recombinant HER4 product in
appropriate host cells. HER4-encoding nucleotide
sequences may be obtained from a variety of cell
sources which produce HER4-like activities and/or
which express HER4-encoding m~NA. For example, HER4-
encoding cDNAs may be obtained from the breastadenocarcinoma cell line MDA-MB-4s3 (ATCC HTBl31) as
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described in Section 6., infra . In addition, a number
of human cell sources are suitable for obtaining HER4
cDNAs, including but not limited to various epidermoid
and breast carcinoma cells, and normal heart, kidney,
and brain cells (see Section 6.2.3., infra) .
The HER4 coding sequence may be obtained by
molecular cloning from RNA isolated and purified from
such cell sources or by genomic cloning. Either cDNA
or genomic libraries of clones may be prepared using
techniques well known in the art and may be screened
for particular HER4-encoding DNAs with nucleotide
probes which are substantially complementary to any
portion of the HER4 gene. Alternatively, cDNA or
genomic DNA may be used as templates for PCR cloning
with suita~le oligonucleotide primers. Full length
clones, i.e., those containing the entire coding
region of the desired HER4 may be selected for
constructing expression vectors, or overlapping cDNAs
can be ligated together to form a complete coding
sequence. Alternatively, HER4-encoding DNAs may be
synthesized in whole or in part by chemical synthesis
using techniques standard in the art.
5.3.2. Construction of ~ER4 Espression
Vectors
Various expression vector/host systems may be
utilized e~ually well by those skilled in the art for
the recombinant expression of HER4 polypeptides. Such
systems include but are not limited to microorganisms
such as bacteria transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA
expression vectors containing the desired HE~4 coding
sequence; yeast transformed with recombinant yeast
expression vectors containing the desired HER4 coding r
sequence; insect cell systems infected with
recombinant virus expression vectors (e.g.,
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baculovirus) containing the desired HER4 coding
sequence; plant cell systems infected with recombinant
virus expression vectors (e.g., cauliflower mosaic
virus CaMV; tobacco mosaic virus, TMV) or transformed
with recombinant plasmid expression vectors (e . g ., Ti
plasmid) containing the desired HER4 coding sequence;
or animal cell systems infected with recombinant virus
expression vectors (e.g., adenovirus, vaccinia virus)
including cell lines engineered to contain multiple
copies of the H~R4 DNA either stably amplified (e.g.,
CHO/dhfr, CH0/glutamine synthetase) or unstably
amplified in double-minute chromosomes (e.g., murine
cell lines).
The expression elements of these vectors vary in
their strength and specificities. Depending on the
host/vector system utilized, any one of a number of
suitable transcription and translation elements may be
used. For instance, when cloning in ~r~ lian cell
systems, promoters isolated from the genome of
mammalian cells, (e.g., mouse metallothionein
promoter) or from viruses that grow in these cells,
(e.g., vaccinia virus 7.5K promoter or Moloney murine
sarcoma virus long terminal repeat) may be used.
Promoters produced by recombinant DNA or synthetic
techniques may also be used to provide for
transcription of the inserted sequences.
Specific initiation signals are also required for
sufficient translation of inserted protein coding
sequences. These signals include the ATG initiation
codon and adjacent sequences. In cases where the
entire HER4 gene including its own initiation codon
- and adjacent sequences are inserted into the
appropriate expression vectors, no additional
translational control signals may be needed. However,
in cases where only a portion of the coding sequence
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is inserted, exogenous translational control signals,
including the ATG initiation codon must be provided.
Furthermore, the initiation codon must be in phase
with the reading frame of the H~R4 coding sequences to
ensure translation of the entire insert. These
exogenous translational control signals and initiation
codons can be of a variety of origins, both natural
and synthetic. The efficiency of expression may be
enhanced by the inclusion of transcription attenuation
sequences, enhancer elements, etc.
For example, in cases where an adenovirus is used
as a vector for driving expression in infected cells,
the desired HER4 coding sequence may be ligated to an
adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader
sequence. This chimeric gene may then be inserted in
the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of
the viral genome (e.g., region E3 or E4) will result
in a recombinant virus that is viable and capable of
expressing HER4 in infected hosts. Similarly, the
vaccinia 7.5K promoter may be used. An alternative
expression system which could be used to express HER4
is an insect system. In one such system, Autographa
californica nuclear polyhidrosis virus (AcNPV) is used
as a vector to express foreign genes. The virus grows
in Spodoptera frugiperda cells. The HER4 coding
sequence may be cloned into non-essential regions (for
example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the
polyhedrin promoter). Successful insertion of the
HER4 coding sequence will result in inactivation of
the polyhedrin gene and production of non-occluded
recombinant virus (i.e., virus lacking the
proteinaceous coat encoded by the polyhedrin gene).
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These recombinant viruses are then used to infect
Spodoptera frugiperda cells in which the inserted gene
is expressed. Yet another approach uses retroviral
vectors prepared in amphotropic packaging cell lines,
5 which permit high efficiency expression in numerous
cells types. This method allows one to assess cell-
type specific processing, regulation or function of
the inserted protein coding se~uence.
In addition, a host cell strain may be chosen
which modulates the expression of the inserted
sequences, or modifies and processes the gene product
in the specific fashion desired. Expression from
certain promoters can be elevated in the presence of
certain inducers (e.g., zinc and cadmium ions for
lS metallothionein promoters). Therefore, expression of
the recombinant HER4 polypeptide may be controlled.
This is important if the protein product of the cloned
foreign gene is lethal to host cells. Furthermore,
modifications (e.g., phosphorylation) and processing
(e.g., cleavage) of protein products are important for
the function of the protein. Different host cells
have characteristic and specific mechanisms for the
post-translational processing and modification of
protein. Appropriate cell lines or host systems can
be chosen to ensure the correct modification and
processing of the foreign protein expressed.
5.3.3. Transformants Expressing ~ER~ Gene
Products
The host cells which contain the recombinant
coding sequence and which express the desired ~ER4
polypeptide product may be identified by at least four
general approaches (a) DNA-DNA, DNA-~NA or RNA-
antisense RNA hybridization; (b) the presence or
absence of "mar~er" gene functions; (c) assessing the
level of transcription as measured by the expression
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of HER4 mRNA transcripts in the host cell; and (d)
detection of the HER4 product as measured by
immunoassay and, ultimately, by its biological
activities.
In the first approach, for example, the presence
of HER4 coding sequences inserted into expression
vectors can be detected by DNA-DNA hybridization using
hybridization probes and/or primers for PCR reactions
comprising polynucleotides that are homologous to the
HER4 coding sequence.
In the second approach, the recombinant
expression vec~or/host system can be identified and
selected based upon the presence or absence of certain
"marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics, resistance to
methotrexate (MTX), resistance to methionine
sulfoximine (MSX), transformation phenotype, occlusion
body formation in baculovirus, etc.). For example, if
the HER4 coding sequence is inserted within a marker
gene sequence of the vector, recombinants containing
that coding sequence can be identified by the absence
of the marker gene function. Alternatively, a marker
gene can be placed in tandem with the HER4 sequence
under the control of the same or different promoter
used to control the expression of the HER4 coding
sequence. Expression of the marker in response to
induction or selection indicates expression of the
HER4 coding sequence. In a particular embodiment
described by way of example herein, a HER4 expression
vector incorporating glutamine synthetase as a
selectable marker is constructed, used to transfect
CHO cells, and amplified expression of HER4 in CHO
cells is obtained by selection with increasing
concentration of MSX.
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In the third approach, transcriptional activity
for the HER4 coding region can be assessed by
hybridization assays. For example, polyadenylated RNA
can be isolated and analyzed ~y Northern blot using a
probe homologous to the HER4 coding sequence or
particular portions thereof. Alternatively, total
nucleic acids of the host cell may be extracted and
assayed for hybridization to such probes.
In the fourth approach, the expression of HER4
can be assessed immunologically, for example by
Western blots, immunoassays such as
radioimmunoprecipitation, enzyme-linked immunoasSays
and the like. Alternatively, expression of HER4 may
be assessed by detecting a biologically active
product. Where the host cell secretes the gene
product the cell free media obtained from the cultured
transfectant host cell may be assayed for HER4
activity. Where the gene product is not secreted,
cell lysates may be assayed for such activity. In
either case, assays which measure ligand binding to
HER4, HER4 phosphorylation, or other ~ioactivities of
HER4 may be used.
5.4. Anti-HER4 Antibodies
The invention is also directed to polyclonal and
monoclonal antibodies which recognize epitopes of HER4
polypeptides. Anti-HER4 antibodies are expected to
have a variety of useful applications in the field of
oncology, several of which are described generally
below. More detailed and specific descriptions of
various uses for anti-HER4 antibodies are provided in
the sections and subsections which follow. Briefly,
anti-HER4 antibodies may be used for the detection and
quantification of HER4 polypeptide expression in
cultured cells, tissue samples, and in vivo. Such
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immunological detection of HER4 may be used, for
example, to identify, monitor, and assist in the
prognosis of neoplasms characterized by aberrant or
attenuated HER4 expression and/or function.
Additionally, monoclonal antibodies recognizing
epitopes from different parts of the HER4 structure
may be used to detect and/or distinguish between
native HER4 and various subcomponent and/or mutant
forms of the molecule. Anti-HER4 antibody
preparations are also envisioned as useful
biomodulatory agents capable of effectively treating
particular human cancers. In addition to the various
diagnostic and therapeutic utilities of anti-HER4
antibodies, a number of industrial and research
applications will be obvious to those skilled in the
art, including, for example, the use of anti-HER4
antibodies as affinity reagents for the purification
of HER4 polypeptides, and as immunological probes for
elucidating the biosynthesis, metabolism and
biological functions of HER4.
Anti-HER4 antibodies may be useful for
influencing cell functions and behaviors which are
directly or indirectly mediated by HER4. As an
example, modulation of HER4 biological activity with
anti-HER4 antibodies may influence HER2 activation
and, as a consequence, modulate intracellular signals
generated by HER2. In this regard, anti-HER4
antibodies may be useful to effectively block ligand-
induced, HER4-mediated activation of HER2, thereby
affecting HER2 biological activity. Conversely, anti-
HER4 antibodies capable of acting as HER4 ligands may
be used to trigger HER4 biological activity and/or
initiate a ligand-induced, HER4-mediated effect on
HER2 biological activity, resulting in a cellular
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response such as differentiation, growth inhibition,
etc.
Additionally, anti-HER4 antibodies conjugated to
; cytotoxic compounds may be used to selectively target
such compounds to tumor cells expressing ~ER4,
resulting in tumor cell death and reduction or
eradication of the tumor. In a particular embodiment,
toxin-conjugated antibodies having the capacity to
bind to HER4 and internalize into such cells are
administered systemically for targeted cytotoxic
effect. The preparation and use of radionuclide and
toxin conjugated anti-HER4 antibodies are further
described in Section 5.5., infra.
Overexpression of HER2 is associated with several
human cancers. Applicants' data indicate that HER4 is
expressed in certain human carcinomas in which HER2
overexpression is present. Therefore, anti-HER4
antibodies may have growth and differentiation
regulatory effects on cells which overexpress HER2 in
zO com~ination with HER4 expression, including but not
limited to breast adenocarcinoma cells. Accordingly,
this invention includes antibodies capable of binding
to the HER4 receptor and modulating HER2 or HER2-HER4
functionality, thereby affecting a response in the
target cell. For the treatment of cancers involving
HER4-mediated regulation of HER2 biological activity,
agents capable of selectively and specifically
affecting the intracellular molecular interaction
between these two receptors may be conjugated to
internalizing anti-HER4 antibodies. The specificity
of such agents may result in biological effects only
in cells which co-express HER2 and HER4, such as
breast cancer cells.
Various procedures known in the art may be used
for the production of polyclonal antibodies to
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epitopes of HER4. For the production of polyclonal
antibodies, a number of host animals are acceptable
for the generation of anti-~ER4 antibodies by
immunization with one or more injections of a HER4
polypeptide preparation, including but not limited to
rabbits, mice, rats, etc. Various adjuvants may be
used to increase the immunological response in the
host animal, depending on the host species, including
but not limited to Freund~s (complete and incomplete),
mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic
polyols, polyanions, oil emulsions, keyhole lympet
hemocyanins, dinitrophenol, and potentially useful
human adjuvants such as BCG (bacille Calmette-Guerin)
and Corynebacterium parvum.
A monoclonal antibody to an epitope of HER4 may
be prepared by using any technique which provides for
the production of antibody molecules by continuous
cell lines in culture. These include but are not
limited to the hybridoma technique originally
described by Kohler and Milstein (1975, Nature 256,
495-497), and the more recent human B-cell hybridoma
technique (Kosbor et al ., 1983, ImmunoloqY TodaY 4:72)
and EBV-hybridoma technique (Cole et al., 1985,
Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96). In addition, techniques
developed for the production of ~chimeric antibodies"
by splicing the genes from a mouse antibody molecule
of appropriate antigen specificity together with genes
from a human antibody molecule of appropriate
biological activity may be used (Morrison et al.,
1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger
et al., 1984, Nature, 312:604-608; Takeda et al.,
1985, Nature, 314:452-454). Alternatively, techniques
described for the production of single chain
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antibodies (U.S. Patent ~,946,778) can be adapted to
produce HER4-specific single chain antibodies.
Recombinant human or humanized versions of anti-HER4
monoclonal antibodies are a preferred embodiment for
human therapeutic applications. Humanized antibodies
may be prepared according to procedures in the
literature (e.g., Jones et al., 1986, Nature 321:522-
25; Reichman et al., 1988, Nature 332:323-27;
Verhoeyen et al., 1988, Science 239:1534-36). The
recently described "gene conversion mutagenesis"
strategy for the production of humanized anti-HER2
monoclonal antibody may also be employed in the
production of humanized anti-HER4 antibodies (Carter
et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:4285-
89). Alternatively, techniques for generating a
recombinant phage library of random com~inations of
heavy and light regions may be used to prepare
recombinant anti-HER4 antibodies (e . g ., Huse et al .,
1989, Science 246:1275-81).
As an example, anti-HER4 monoclonal antibodies
may be generated by immunization of mice with cells
selectively overexpressing HER4 (e.g., CH0/HER4 21-2
cells as deposited with the ATCC) or with partially
purified recombinant HER4 polypeptides. In one
2S embodiment, the full length HER4 polypeptide (FIG. lA
and lB) may be expressed in Baculovirus systems, and
membrane fractions of the recombinant cells used to
immunize mice. Hybridomas are then screened on
CHOtHER4 cells (e.g., CH0 HER4 21-2 cells as deposited
30 with the ATCC) to identify monoclonal antibodies
reactive with the extracellular domain of HER4. Such
r monoclonal antibodies may be evaluated for their
ability to block NDF, or HepG2-differentiating factor,
binding to HER4; for their ability to bind and stay
35 resident on the cell surface, or to internalize into
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cells expressing HER4; and for their ability to
directly upregulate or downregulate HER4 tyrosine
autophosphorylation and/or to directly induce a HER4-
mediated signal resulting in modulation of cell growth
or differentiation. In this connection, monoclonal
antibodies N28 and N29, directed to HER2, specifically
bind HER2 with high affinity. However, monoclonal N29
binding results in receptor internalization and
downregulation, morphologic differentiation, and
inhibition of HER2 expressing tumor cells in athymic
mice. In contrast, monoclonal N28 binding to HER2
expressing cells results in stimulation of
autophosphorylation, and an acceleration of tumor cell
growth both in vitro and in vivo (Bacus et al., 1992,
Cancer Res. 52:2580-89; Stancovski et al., 1991, Proc.
Natl. Acad. Sci. U.S.A. 88:8691-95). In yet another
embodiment, a soluble recombinant HER4-Immunoglobulin
(~ER4-Ig) fusion protein is expressed and purified on
a Protein A affinity column. The amino acid sequence
of one such HER4-Ig fusion protein is provided in FIG.
14. The soluble HER4-Ig fusion protein may then be
used to screen phage libraries designed so that all
available combinations of a variable domain of the
antibody binding site are presented on the surfaces of
the phages in the library. Recombinant anti-HER4
antibodies may be propagated from phage which
specifically recognize the HER4-Ig fusion protein.
Antibody fragments which contain the idiotype of
the molecule may be generated by known techniques.
For example, such fragments include but are not
limited to: the F(ab)~E2 fragment which can be
produced by pepsin digestion of the intact antibody
molecule; the Fab' fragments which can be generated by
reducing the disulfide bridges of the F(ab')2
fragment, and the two Fab fragments which can be
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generated by treating the antibody molecule with
papain and a reducing agent. Alternatively, Fab
expression libraries may be constructed (Huse et al.,
1989, Science, 246:1275-1281) to allow rapid and easy
identification of monoclonal Fab fragments with the
desired specificity to HER4 protein.
5.5. HER4 ~igands
one aspect of the present invention is directed
to HER4 ligands. As defined herein, HE~4 ligands are
capable of binding to the 180K transmembrane protein,
~ER4/pl80'rb~4 or functional analogues thereof, and
activating tyrosine kinase activity. Functional
analogues of HER4/pl80~r~a4-ligands are capable of
lS activating HER4 tyrosine kinase activity. Activation
of the tyrosine kinase activity may stimulate
autophosphorylation and may affect a biological
activity mediated by HER4. It has been observed in
systems described in Section 12 and 13 that binding of
HER4 ligands to HER4 triggers tyrosine phosphorylation
and affects differentiation of breast cancer cells.
The HER4 ligands of the present invention include
NDF, a 44 kDa glycoprotein isolated from ras-
transformed rat fibroblasts (Wen et al., 1992, Cell
2s 69:559-572); heregulin, its human homologue, which
exists as multiple isoforms (Peles et al., 1992, Cell
69:205-218 and Holmes et al ., 1992, Science 256:1205-
1210) including p45, a 45K heparin-binding
glycoprotein that shares several features with the
heregulin-family of proteins including molecular
weight, ability to induce differentiation of breast
cancer cells, activation of tyrosine phosphorylation
in MDA-MB453 cells, and N-terminal amino acid sequence
(Section 13, infra), gp30, and p75 (LupU et al., 1990,
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Science 249:1552-1555 and Lupu et al ., 1992, Proc.
Natl. Acad. Sci. USA 89:2287-2291).
HE~4 ligands of the present invention can be
prepared by synthetic or recombinant means, or can be
isolated from natural sources. The HER4 ligand of the
present invention may contain deletions, additions or
substitutions of amino acid residues relative to the
sequence of NDF, p45 or other heregulins or any HER4
ligand known in the art as long as the ligand
lo maintains HER4 receptor binding and tyrosine kinase
activation capacity. Such amino acid substitutions
may be made on the basis of similarity in polarity,
charge, solubility, hydropho~icity, hydrophilicity
and/or the amphipathic nature of the resides involved.
For example, negatively charged amino acids include
aspartic acid and glutamic acid; positively charged
amino acids include lysine and arginine; amino acids
with uncharged polar head groups or nonpolar head
groups having similar hydrophilicity values include
the following: leucine, isoleucine, valine; glycine,
alanine; asparagine, glutamine; serine, threonine;
phenylalanine, tyrosine.
5.5.1. Recom~inant Expression of HER4
Ligan~s
The HER4 ligands of the present invention may be
produced by the cloning and expression of DNA encoding
the desired HER4 ligand. Such DNA may be ligated into
a number of expression vectors well known in the art
and suitable for use in a number of acceptable host
organisms, in fused or mature form, and may contain a
signal sequence to permit secretion. Both prokaryotic
and eukaryotic host expression systems may be employed
in the production of recombinant HER4 ligands. For
example, a HER4 ligand precursor coding sequence or
its functional equivalent may be used in a host cell
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capable of processing the precursor correctly.
Alternatively, the coding sequence for a mature HER4
ligand may be used to directly express the mature HER4
ligand molecule. Functional equivalents of the HER4
S ligand precursor coding sequence include any DNA
sequence which, when expressed inside the appropriate
host cell, is capable of directing the synthesis,
processing and/or export of the HER4 ligand.
Production of a HER4 ligand using recombinant DNA
technology may be divided into a four-step process for
the purposes of description: (1) isolation or
generation of DNA encoding the desired HER4 ligand;
(2) construction of an expression vector capable of
directing the synthesis of the desired HER4 ligand;
(3) transfection or transformation of appropriate host
cells capable of replicating and expressing the HER4
ligand coding sequence and/or processing the initial
product to produce the desired HER4 ligand; and (4)
identification and purification of the desired HER4
ligand product.
5.5.2. Isol~tion of HER4 Enco~ing DNA
HER4 ligand-encoding nucleic acid sequences may
be obtained from human hepatocellular carcinoma cell
lines, specifically the HepG2 cells available from the
ATCC, accession number HB 8065. In addition, a number
of human cell sources are suitable for obtaining HER4
ligand nucleic acids, including MDA-MB-231 cells
available from the ATCC, accession number HTB 26,
brain tissue (Falls et al., 1993, Cell 72:801-815 and
Marchionni et al., 1993 Nature 362:312-318), and any
cell source capable of producing an activity capable
of binding to the 180K transmembrane protein,
HER4/pl80'rb~4, encoded by the HER4/ERBB4 gene and
activating tyrosine kinase activity.
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Methods useful in assaying for the identification
of HER4 ligands is disclosed in Section 5.8., infra.
The techniques disclosed in Sections 5.3.2. and
5.3.3., infra apply to the construction of HER4 ligand
expression vectors and identification of recombinant
transformants expressing HER4 ligand gene products.
5.5.3. Anti-HER4 Ligand Antibodies
The present invention is also directed to
polyclonal and monoclonal antibodies which recognize
eptitopes of HER4 ligand polypeptides. Anti-HER4
ligand antibodies are expected to have a variety of
useful applications in the field of oncology.
Briefly, anti-HER4 ligand antibodies may be used for
the detection and quantification of HER4 ligand
polypeptide expression in cultured cells, tissue
samples, and in vivo. For example, monoclonal
antibodies recognizing epitopes from different parts
of the HER4 ligand structure may be used to detect
and/or distinguish binding from non-binding regions of
the ligand. Anti-HER4 ligand antibody preparations
are also envisioned as useful biomodulatory agents
capable of effectively treating particular human
cancers. An anti-HER4 ligand antibody could be used
to block signal transduction mediated through HER4,
thereby inhibiting undesirable biological responses.
In addition to the various diagnostic and therapeutic
utilities of anti-HER4 ligand antibodies, a number of
industrial and research applications will be obvious
to those skilled in the art, including, for example,
the use of anti-HER4 ligand antibodies as affinity
reagents for the purification of HER4 ligand
polypeptides, and as immunological probes for
elucidating the biosynthesis, metabolism and
biological functions of HER4 ligands.
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Anti-HER4 ligand antibodies may ~e useful for
influencing cell functions and behaviors which are
directly or indirectly mediated by HE~4. As an
example, modulation of HER4 biological activity with
s anti-HER4 ligand antibodies may influence HER2
activation and, as a consequence, modulate
intracellular signals generated by HER2. In this
regard, anti-HER4 ligand antibodies may be useful to
effectively block ligand-induced, HER4-mediated
activation of HER2, thereby affecting HER2 biological
activity. Conversely, anti-HER4 ligand antibodies
capable of acting as HER4 ligands may be used to
trigger HER4 biological activity andtor initiate a
ligand-induced, HER4-mediated ef~ect on HER2
lS biological activity, resulting in a cellular response
such as differentiation, growth inhibition, etc.
Additionally, anti-HER4 ligand antibodies
conjugated to cytotoxic compounds may be used to
selectively target such compounds to tumor cells
expressing HER4, resulting in tumor cell death and
reduction or eradication of the tumor.
Various procedures known in the art may be used
for the production of antibodies to epitopes of HER4
ligand (see Section 5.4, supra) .
5.6. Diagnostic Methods
The invention also relates to the detection of
human neoplastic conditions, particularly carcinomas
of epithelial origin, and more particularly human
breast carcinomas. In one embodiment, oligomers
corresponding to portions of the consensus HER4 cDNA
sequence provided in FIG. lA and l~ are used for the
quantitative detection of HER4 m~NA levels in a human
biological sample, such as blood, serum, or tissue
biopsy samples, using a suitable hybridization or PCR
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format assay, in order to detect cells or tissues
expressing abnormally high levels of HER4 as an
indication of neoplasia. In a related embodiment,
detection of HER4 mRNA may be combined with the
detection HER2 mRNA overexpression, using appropriate
HER2 sequences, to identify neoplasias in which a
functional relationship between HER2 and HER4 may
exist.
In another embodiment, labeled anti-HER4
antibodies or antibody derivatives are used to detect
the presence of HER4 in biological samples, using a
variety of immunoassay formats well known in the art,
and may be used for in situ diagnostic
radioimmunoimaging. Current diagnostic and staging
techniques do not routinely provide a comprehensive
scan of the body for metastatic tumors. Accordingly,
anti-HER4 antibodies labeled with, for example,
fluorescent, chemiluminescent, and radioactive
molecules may overcome this limitation. In a
preferred embodiment, a gamma-emitting diagnostic
radionuclide is attached to a monoclonal antibody
which is specific for an epitope of HER4, but not
significantly cross-reactive with other EGFR-family
members. The labeled antibody is then injected into a
patient systemically, and total body imaging for the
distribution and density of HER4 molecules is
performed using gamma cameras, followed by localized
imaging using computerized tomography or magnetic
resonance imaging to confirm and/or evaluate the
condition, if necessary. Preferred diagnostic
radionuclides include but are not limited to
technetium-9sm, indium-111, iodine-123, and iodine-
131.
Recombinant antibody-metallothionein chimeras
(Ab-MTs) may be generated as recently described (Das
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et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:9749-
53). Such Ab-MTs can be loaded with technitium-99m by
virtue of the metallothionein chelating function, and
- may offer advantages over chemically conjugated
chelators. In particular, the highly conserved
metallothionein structure may result in minimal
immunogenicity.
5.7. Assayq for the Identification of ~ER4
lo Ligands
Cell lines overexpressing a single member of the
EGFR-family can be generated by transfection of a
variety of parental cell types with an appropriate
expression vector as described in Section 7., infra.
Candidate ligands, or partially purified preparations,
may be applied to such cells and assayed for receptor
binding and/or activation. For example, a CHO-KI cell
line transfected with a HER4 expression plasmid and
lacking detectable EGFR , HER2, or HER3 may be used to
screen for HER4-specific ligands. A particular
embodiment of such a cell line is described in Section
7 ., in~ra , and has been deposited with the ATCC
(CH0/HER4 21-2). Ligands may be identified by
detection of H~R4 autophosphorylation, stimulation of
DNA synthesis, induction of morphologic
differentiation, relief from serum or growth factor
requirements in the culture media, and direct binding
of labeled purified growth factor. The invention also
relates to a bioassay for testing potential analogs of
~ER4 ligands based on a capacity to affect a
biological activity mediated by the HER4 receptor.
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5.8. Use Of The Invention in Cancer Therapy
5.8.1. T~rgeted Cancer Therapy
The invention is also directed to methods for the
treatment of human cancers involving abnormal
expression and/or function of HER4 and cancers in
which HER2 overexpression is combined with the
proximate expression of HER4, including but not
limited to human breast carcinomas and other neoplasms
overexpressing HER4 or overexpressing HER2 in
combination with expression of HER4. The cancer
therapy methods of the invention are generally based
on treatments with unconjugated, toxin- or
radionuclide- conjugated HER4 antibodies, ligands, and
derivatives or fragments thereof. In one specific
embodiment, such HER4 antibodies or ligands may be
used for systemic and targeted therapy of certain
cancers overexpressing HER2 and/or HER4, such as
metastatic breast cancer, with minimal toxicity to
normal tissues and organs. Importantly, in this
connection, an anti-HER2 monoclonal antibody has been
shown to inhibit the growth of human tumor cells
overexpressing HER2 (~acus et al., 1992, Cancer Res.
52:2580-89). In addition to conjugated antibody
therapy, modulation of heregulin signaling through
HER4 provides a means to affect the growth and
differentiation of cells overexpressing HER2, such as
certain breast cancer cells, using HER4-neutralizing
monoclonal antibodies, NDF/HER4 antagonists,
monoclonal antibodies or ligands which act as super-
agonists for HER4 activation, or agents which block
the interaction between HER2 and HER4, either by
disrupting heterodimer formation or by blocking HER-
mediated phosphorylation of the HER2 substrate.
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For targeted immunotoxin-mediated cancer therapy,
various drugs or toxins may be conjugated to anti-HER4
antibodies and fragments thereof, such as plant and
bacterial toxins. For example, ricin, a cytotoxin
from the Ricinis communis plant may be conjugated to
an anti-HER4 antibody using methods known in the art
(e.g., Blakey et al., 1988, Proq. Allerqy 45:50-so;
Marsh and Neville, 1988, J. Immunol. 140:3674-78).
Once ricin is inside the cell cytoplasm, its A chain
inhibits protein synthesis by inactivating the 60S
ribosomal subunit (May et al., 1989, EMBO J. 8:301-
08). Immunotoxins of ricin are therefore extremely
cytotoxic. However, ricin immunotoxins are not
ideally specific because the B chain can bind to
virtually all cell surface receptors, and immunotoxins
made with ricin A chain alone have increased
specificity. Recombinant or deglycosylated forms of
the ricin A chain may resuIt in improved survival
(i.e., slower clearance from circulation) of the
immunotoxins. Methods for conjugating ricin A chain
to antibodies are known (e.g., Vitella and Thorpe, in:
Seminars in Cell Bioloqy, pp 47-58; Saunders,
Philadelphia l99l). Additional toxins which may be
used in the formulation of immunotoxins include but
are not limited to daunorubicin, methotrexate,
ribosome inhibitors (e.g., trichosanthin, trichokirin,
gelonin, saporin, mormordin, and pokeweed antiviral
protein) and various bacterial toxins (e.g.,
Pseudomonas exotoxin). Immunotoxins for targeted
cancer therapy may be administered by any route which
will result in antibody interaction with the target
cancer cells, including systemic administration and
injection directly to the site of tumor. Another
therapeutic strategy may be the administration of
immunotoxins by sustained-release systems, such as
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semipermeable matrices of solid hydrophobic polymers
containing the therapeutic agent. Various of
sustained-release materials have been established and
are well known by those skilled in the art.
Sustained-release capsules may, depending on their
chemical nature, release immunotoxic molecules for a
few weeks up to over 100 days. Depending on the
chemical nature and the biological stability of the
therapeutic reagent, additional strategies for protein
stabilization may be employed.
For targeted radiotherapy using anti-HER4
antibodies, preferred radionuclides for labeling
include alpha, beta, and Auger electron emitters.
Examples of alpha emitters include astatine 211 and
bismuth 212; beta emitters include iodine 131, rhenium
188, copper 67 and yttrium 90; and iodine 12S is an
example of an Auger electron emitter.
Similarly as suggested for the use of toxin-
conjugated antibodies as therapeutic agents for
targeted cancer therapy, purified ligand molecules may
be chemically conjugated to cytotoxic substances. In
addition, recombinant chimeric polypeptides comprising
a HER4 binding (=ligand) portion fused to all or part
of a cytotoxin may be engineered by constructing
vectors comprising DNA encoding the ligand in reading
frame with DNA encoding the toxin or part thereof.
Such recombinant ligand-toxins may be used to
specifically target HER4 expressing cancer cells. A
particular embodiment of such a ligand-toxin is
disclosed herein and described in more detail in
Sections 5.8.2., inf~a , and Section 15, infra .
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5.8.2. The Generation Of A Heregulin-toxin
8pecifically Targeting ~ER4 Expressing
- Tumor Cells
Another aspect of the invention relates to the
deyelopment of a strategy to selectively target and
kill HER4 expressing tumor cells. More particularly,
HER4 expressing tumor cells may be specifically
targeted and killed by contacting such tumor cells
with a fusion protein comprising a cytotoxic
polypeptide covalently linked to a polypeptide which
is capable of activating HER4 expressed on such cells.
In a specific embodiment described by way of
example in Section 15, infra, a fusion protein
comprising a chimeric heregulin ~2 ligand and the
cytotoxic substance PE40 is generated by expression of
the corresponding chimeric coding sequence. PE40 is a
derivative of the Pseudomonas exotoxin PE, a potent
cell killing agent made by Pseudomonas aeruginosa
(Fitzgerald et al., 1980, Cell 21:867-873). The
wildtype protein PE contains three domains whose
functions are cell recognition, membrane
translocation, and ADP ribosylation of elongation
factor 2. It kills cells by binding to a cell surface
receptor, entering the cell via an endocytotic vesicle
and catalyzing ADP-ribosylation of elongation factor
2. The derivative PE40 lacks the cell ~inding
function of the wildtype protein, but still exhibits
strong cytotoxic activity. Generation of PE40 fusion
proteins with specific cell targeting molecules have
been described (Kondo et al., 1988, J. Biol. Chem.
263:9470-9475 (PE40 fusions with different monoclonal
antibodies); Friedman et al., 1993, Cancer Res.
53:334-339 (BR96/PE40 fusions); U.S. Pat. No. 5206353
- (CD4/PE40 fusions); U.S. Pa~. No. 5082927 (IL-4/PE40
fusions) and U.S. Pat. No. 4892827 (TGF-~/PE40 and IL-
2/PE40 fusions)).
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The chimeric heregulin-toxin protein HAR-TX ~2
described in Section 15, infra, contains the
amphiregulin (AR) leader sequence thereby facilitating
the purification of the recombinant protein. As
confirmed by applicants' data, the AR leader has no
influence on the binding specificity of the
recombinant heregulin-toxin. Related embodiments
include, for example, PE40 linked to other members of
the heregulin family, like heregulin-~1 and heregulin-
~, and other molecules capable of activating HER4.
In a cytotoxicity assay with cultured tumor celllines, the applicants demonstrate specificity of the
cytotoxic effect of the chimeric heregulin-PE40
protein to HER4 expressing cancer cells; they include
but are not limited to prostate carcinoma, bladder
carcinoma, and a considerable number of different
breast cancer types, including breast carcinoma cells
with amplified HER2 expression. The bifunctional
retention of both the specificity of the cell binding
portion of the molecule and the cytotoxic potential of
PE40 provides a very potent and targeted reagent.
An effective therapeutic amount of heregulin-
toxin will depend upon the therapeutic objectives, the
route of administration, and the condition of the
patient. Accordingly, dosages should be titrated and
the route of administration modified as required to
obtain the optimal therapeutic effect. A typical
daily dosage may be in the range of 0.1 mg/kg - l
mg/kg, preferably between 0.1 and 0.5 mg/kg, with
intravenous administration. For regression of solid
tumors, it may take 3-5 doses, with schedules such as
3 doses, each four days apart. Also the use of
sustained-release preparations (see Section 5.8.1.,
supra) may be considered for administration of the
reagent. The therapeutic efficacy of heregulin-toxin
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may ~e between 2 and l0, which means that a tumor
regression effect would be expected between 2- and lO-
fold below the toxic dose (see Section 15, infra).
- Desirably, the heregulin-toxin will be administered at
a dose and frequency that achieves the desired
therapeutic effect, which can be monitored using
conventional assays.
Cancer therapy with heregulin-toxins of the
invention may be combined with chemotherapy, surgery,
and radiation therapy, depending on the type of tumor.
One advantage of using a low molecular weight toxin
drug is that they are capable of targeting metastatic
lesions that cannot be located and removed by surgery.
Heregulin-toxins may also be particularly useful on
patients that are MDR (Multi Drug Resistance) positive
since their mechanism of action is not inhibited by
the p-glycoprotein pump of MDR positive cells as are
many standard cancer therapeutic drugs.
5.9. Other ~herapeutic ~se of HER4 Ligands
Additional therapeutic uses of HER4 ligands may
include other diseases ca~sed by deficient H~R4
receptor tyrosine kinase activation rather than by
hyperactivation. In this regard, type II diabetes
mellitus is the consequence of deficient insulin-
mediated signal transduction, caused by mutations in
the insulin-receptor, including mutations in the
ligand-binding domain (Taira et al., 1889, Science
245:63-66; Odawara et al ., 1989, Science 245:66-68;
Obermeier-Kusser et al., 1989, J. Biol. Chem.
264:9497-9504). Such diseases might be treated by
administration of modified ligands or ligand-analogues
which re-establish a functional ligand-receptor
interaction.
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5.10. HER4 Analogues
The production and use of derivatives, analogues
and peptides related to HER4 are also envisioned and
are within the scope of the invention. Such
derivatives, analogues and peptides may be used to
compete with native HER4 for binding of HER4 specific
ligand, thereby inhibiting HER4 signal transduction
and function. The inhibition of HER4 function may be
utilized in several applications, including but not
lo limited to the treatment of cancers in which HER4
biological activity is involved.
In a specific embodiment, a series of deletion
mutants in the HER4 nucleotide coding sequence
depicted in FIG. lA and lB may be constructed and
analyzed to determine the minimum amino acid sequence
requirements for binding of a HER4 ligand. Deletion
mutants of the HER4 coding sequence may be constructed
using methods known in the art which include but are
not limited to use of nucleases and/or restriction
enzymes; site-directed mutagenesis techniques, PCR,
etc. The mutated polypeptides expressed may be
assayed for their ability to bind HER4 ligand.
The DNA sequence encoding the desired HER4
analogue may then be cloned into an appropriate
expression vector for overexpression in either
bacteria or eukaryotic cells. Peptides may be
purified from cell extracts in a number of ways
including but not limited to ion-exchange
chromatography or affinity chromatography using HER4
ligand or antibody. Alternatively, polypeptides may
be synthesized by solid phase techniques followed by
cleavage from resin and purification by high
performance liquid chromatography.
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6. Example: Isolation of cDNAs Encoding HER4
EGFR and the related proteins, H~R2, HER3, and
Xmrk exhibit extensive amino acid homology in their
- tyrosine kinase domains (Kaplan et al., 1991, Nature
350:15~-160; Wen et al., 1992, Cell 69:559-72i Holmes
et al., 1992, Science 256:1205-10; Hirai et al.,
Science 1987 238:1717-20). In addition, there is
strict conservation of the exon-intron boundaries
within the genomic regions that encode these catalytic
domains (Wen et al., supra; Lindberg and Hunter, 1990,
Mol. Cell. Biol. 10:6316-24; and unpublished
observations). Degenerate oligonucleotide primers
were designed based on conserved amino acids encoded
by a single exon or adjacent exons from the kinase
domains of these four proteins. These primers were
used in a polymerase chain reaction (PCR) to isolate
genomic fragments corresponding to murine EGFR, erbB2
and erbB3. In addition, a highly related DNA fragment
(designated MER4) was identified as distinct from
these other genes A similar strategy was used to
obtain a cDNA clone corresponding to the human
homologue of MER4 from the breast cancer cell line,
MDA-MB-453. Using this fragment as a probe, several
breast cancer cell lines and human heart were found to
be an abundant source of the EGFR-related transcript.
cDNA libraries were constructed using RNA from human
heart and MDA-MB-453 cells, and overlapping clones
were isolated spanning the complete open reading frame
of HER4/erbB4.
6.1. Materials and Methods
6.1.1. ~olecular Cloning
Several pools of degenerate oligonucleotides were
synthesized based on conserved sequences from EGFR-
family members (Table I) (5'-ACNGTNTGGGARYTNAYHAC-3'
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[SEQ ID No:14]; 5'-CAYGTNAARATHACNGAYTTYGG-3' [SEQ ID
No:16]; 5'-GACGAATTCCNATHAARTGGATGGC-3' [SEQ ID
No:17]; 5'-AANGTCATNARYTCCCA-3' tSEQ ID No:18]; 5'-
TCCAGNGCGATCCAYTTDATNGG-3' [SEQ ID No:19]; 5'-
S GG~TCDATCATCCARCCT-3' tSEQ ID No:20]; 5'-
CTGCTGTCAGCATCGATCAT-3' ~SEQ ID No:21~; TVWELMT [SEQ
ID No:22]; HVKITDFG [SEQ ID No:23]; PIKWMA [SEQ ID
No:13]; VYMIILK [SEQ ID No:24]; WELMTF [SEQ ID No:25];
PIKWMALE [SEQ ID No:26]; CWMIDP tSEQ ID No:27]. Total
genomic DNA was isolated from subconfluent murine
K1735 melanoma cells and used as a template with these
oligonucleotide primers in a 40 cycle PCR
amplification. PCR products were resolved on agarose
gels and hybridized to 32P-labeled probes from the
kinase domain of human EGFR and HER2. Distinct DNA
bands were isolated and subcloned for se~uence
analysis. Using the degenerate oligonucleotides
H4VWELM and H4VYMIIL as primers in a PCR amplification
(Plowman et al., 1990, Proc. Natl. Acad. Sci. U.S.A.
87:4905-09), one clone (MER4-8s) was identified that
contained a 144 nucleotide insert corresponding to
murine erbB4. This 32P-labeled insert was used to
isolate a 17-kilo~ase fragment from a murine T-cell
genomic library (Stratagene, La Jolla, CA) that was
found to contain two exons of the murine erbB4 gene.
A specific oligonucleotide (4M3070) was synthesized
based on the DNA sequence of an erbB4 exon, and used
in a PCR protocol with a degenerate 5'-oligonucleotide
(H4PIKWMA) on a template of single stranded MDA-MB-453
cDNA. This reaction generated a 260 nucleotide
fragment (pMDAPIK) corresponding to human HER4. cDNA
libraries were constructed in lambda ZAP II
(Stratagene) from oligo(dT)- and specific-primed MDA-
MB453 and human heart RNA (Plowman et al,, supra ;
Plowman et al., 1990, Mol. Cell. Biol. 10:1969-81).
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HER4-specific clones were isolated by pro~ing the
libraries with the 32P-labeled insert from pMDAPIK. To
complete the cloning of the 5'-portion of HER4, we
used a PCR strategy to allow for rapid amplification
of cDNA ends (Plowman et al ., supra ; Frohman et al .,
1988, Proc. Natl. Acad. Sci. U.S.A. 85:8998-9002).
All cDNA clones and several PCR generated clones were
sequenced on both strands using T7 polymerase with
oligonucleotide primers (Tabor and ~ichardson, 1987,
Proc. Natl. Acad. Sci. U.S.A. 84:4767-71).
TAB~E I
O~IGON~CLEOTIDE PREPAR~TIONs FOR C~ONING ~ER4
Nucleoeide Encoded
3esi~nacion Sequen~e' ~ a~ Sequen~e Orien~a~ior. S-~.ID No.
VWELM S'-A~ln~ARYTNA~AC-3~ 256-fold TVWELMT 3en~e 14
~4~ITDFG S'-CAYGTNAARATYAC~GAYTTYGG-3' 768-~old HVX~TDFG sense lS
H4PIKWMA S'-GACGAATTCCNATHAARTGGATGGC 4a-fold P~WMA gcn~e lS
U4VYMIIL S~-ACAYTTNARDATDATCATRT~NAC-3~ ;76-~old VYMIILK an~i~ense 17
H4WSLMTF 5'-AANGTCAT~ARYTCCCA-3' 32-fold WELMTF an~en3e 18
~4PI~WMA S'-TCCAGNGCGATCC~YTTDATNGG-3' 96-~old p~WMALE an~isense l9
H4CWMIDP S'- oe RT0 ATCAICCARCCT-3' 12-~old CWM}DP aneisense 20
2 O 4M3070 5~ ~AGCATCG~TC~T-3' ~ero er~R4 exon an~isense 21
:Ceg-~eraee r.ucleotlde re3idue de~lgna~ons:
D - A. G, or T;
H . A, C, or T:
A, C, G. or T:
y . c or T.
6.l.2. Northern Blot An~ly~is
3'- and 5'-HER4 specific [~32P]UTP-labeled
antisense RNA probes were synthesized from the
linearized plasmids pHtlBl.6 (containing an 800 bp
~ER4 fragment beginning at nucleotide 3098) and
p5'H4E7 (containing a l kb fragment from the 5'-end of
the HER4 sequence), respectively. For tissue
distribution analysis (Section 6.2.3., infra ), the
Northern blot (Clontech, Palo Alto, CA) contained 2 Mg
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poly(A)+ mRNA per lane from 8 human tissue samples
immobilized on a nylon membrane. The filter was
prehybridized at 60 C for several hours in RNA
hybridization mixture (50% formamide, 5X SSC, 0.5%
SDS, lOx Denhardt's solution, 100 /~g/ml denatured
herring sperm DNA, 100 ~g/ml tRNA, and 10 ~g/ml
polyadenosine) and hybridized in the same buffer at
60 C, overnight with 1-1.5 x 106 cpm/ml of 32P-
labeled antisense RNA probe. The filters were washed
in O.lXSSC/0.1% SDS, 65 C, and exposed overnight on a
PhosphoImager (Molecular Dynamics, Sunnyvale, CA).
6.1.3. Semi-Quantitative PCR Detection of
HER4
RNA was isolated from a variety of human cell
lines, fresh frozen tissues, and primary tumors.
Single stranded cDNA was synthesized from 10 ~g of
each RNA by priming with an oligonucleotide containing
a T17 track on its 3'-end
(XSCT17:5'GACTCGAGTCGACATCGA~ lllllllllllll-3')
[SEQ ID No:28].
1% or 5% of each single strand template preparation
~as then used in a 35 cycle PCR reaction with two
HER4-specific oligonucleotides:
4H2674: 5'-GAAGAAAGACGACTCGTTCATCGG-3'
[SEQ ID No:29],
and
4H2965: 5'-GACCATGACCATGTAAACGTCAATA-3'
[SEQ ID No:30].
Reaction products were electrophoresed on 2% agarose
gels, stained with ethidium bromide and photographed
on a W light box. The relative intensity of the 291-
bp HER4-specific bands were estimated for each sample
as shown in Table II.
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6.2. Re~ults
6.2.1. Sequence Analysis of cDNA Clones
Encodi~g ~ER4
cDNA clones encoding parts of the HER4 coding and
non-coding nucleotide sequences were isolated by PCR
cloning according to the method outlined in Section
6.1.1., supra. The complete HER4 nucleotide sequence
assembled from these cDNAs is shown in FIG. lA and lB
and contains a single open reading frame encoding a
lo polypeptide of 1308 amino acids. The HER4 coding
region is flanked by a 33 nucleotide 5'-untranslated
region and a 1517 nucleotide 3'-untranslated region
ending with a poly(A) tail. A 25 amino acid
hydrophobic signal sequence follows a consensus
initiating methionine at position number 1 in the
amino acid sequence depicted in FIG. lA and lB. In
relation to this signal sequence, the mature HER4
polypeptide would be predicted to begin at amino acid
residue number 26 in the sequence depicted in FIG. lA
and lB (Gln), followed by the next 1283 amino acids in
the sequence. Thus the prototype mature HER4 of the
invention is a polypeptide of 1284 amino acids, having
a calculated Mr of 144,260 daltons and an amino acid
sequence corresponding to residues 26 through 1309 in
FIG. lA and lB.
Comparison of the HER4 nucleotide and deduced
amino acid sequences (FIG. lA and lB) with the
available DNA and protein sequence databases indicated
that the HE~4 nucleotide sequence is unique, and
revealed a 60/64 amino acid identity with HER2 and a
54/54 amino acid identity to a fragment of a rat EGFR
homolog, tyro-2.
6.2.2. ~equence Analysis of Related cDNAs
Several cDNAs encoding polypeptides related to
the prototype HER4 polypeptide (FIG. lA and lB) were
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also isolated from the MDA-MB-453 cDNA library and
comprised two forms.
The first alternative type of cDNA was identical
to the consensus HER4 nucleotide sequence up to
nucleotide 3168 (encoding Arg at amino acid position
1045 in the FIG. lA and lB) and then abruptly diverges
into an apparently unrelated sequence (FIG. 2A and 2B,
FIG. 4). Downstream from this residue the open
reading frame continues for another 13 amino acids
before reaching a stop codon followed by a 2 kb 3'-
untranslated sequence and poly(A) tail. This cDNA
would be predicted to result in a HER4 variant having
the C-terminal autophosphorylation domain of the
prototype HER4 deleted.
A second type of cDNA was isolated as 4
independent clones each with a 3'-sequence identical
to the HER4 consensus, but then diverging on the 5'-
side of nucleotide 2335 (encoding Glu at amino acid
position 768 in the FIG. lA and lB), continuing
upstream for only another 114-154 nucleotides (FIG. 3,
FIG. 5). Nucleotide 2335 is the precise location of
an intron-exon junction in the HER2 gene (Coussens et
al., 1985, Science 230:1132-39; Semba et al., 1985,
Proc. Natl. Acad. Sci. U.S.A. 82:6497-6501),
suggesting these cDNAs could be derived from mRNAs
that have initiated from a cryptic promoter within the
flanking intron. These 5'-truncated transcripts
contain an open reading frame identical to that of the
HER4 cDNA sequence of FIG. lA and lB, beginning with
the codon for Met at amino acid position 772 in FIG.
lA and lB. These cDNAs would be predicted to encode a
cytoplasmic HER4 variant polypeptide that initiates
just downstream from the ATP-binding domain of the
HER4 kinase.
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6.2.3. Human Tissue Distribution of HER4
Expression
`Northern blots of poly(A)l mRNA from human tissue
samples were hybridized with antisense RNA probes to
the 3'-end of HER4, encoding the autophosphorylation
domain, as described in Section 6.l. 2 ., supra. A HER4
mRNA transcript of approximately 6kb was identified,
and was found to be most abundant in the heart and
skeletal muscle (FIG. 8, Panel l). An mRNA of greater
than approximately 15 kb was detected in the brain,
with lower levels also detected in heart, skeletal
muscle, kidney, and pancreas tissue samples.
The same blot was stripped and rehybridized with
a probe from the 5'-end of HER4, within the
extracellular domain coding region, using identical
procedures. This hybridization confirmed the
distribution of the 15 kb HER4 mRNA spe~ies, and
detected a 6.5 kb mRNA species in heart, skeletal
muscle, kidney, and pancreas tissue samples (FIG. 8,
Panel 2) with weaker signals in lung, liver, and
placenta. In addition, minor transcripts of l.7-2.6
kb were also detected in pancreas, lung, brain, and
skeletal muscle tissue samples. The significance of
the different sized RNA transcripts is not known.
Various human tissues were also examined for the
presence of HER4 mRNA using the semi-quantitative PCR
assay described in Section 6.l.3., supra. The results
are shown in Table II, together with results of the
assay on primary tumor samples and neoplastic cell
lines tSection 6.2.4., immediately below). These
results correlate well with the Northern and solution
hybridization analysis results on the selected RNA
samples. The highest levels of HER4 transcript
expression were found in heart, kidney, and brain
tissue samples. In addition, high levels of HER4 mRNA
expression were found in parathyroid, cerebellum,
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pituitary, spleen, testis, and breast tissue samples.
Lower expression levels were found in thymus, lung,
salivary gland, and pancreas tissue samples, Finally,
low or negative expression was observed in liver,
prostate, ovary, adrenal, colon, duodenum, epidermis,
and bone marrow samples.
6.2.4. HE~4 mRNA ExpresYion i~ Prim~ry
Tumor~ and Variou~ Cell Line~ of
Neoplastic Origin
HER4 mRNA expression profiles in several primary
tumors and a number of cell lines of diverse
neoplastic origin were determined with the semi-
quantitative PCR assay (Section 6.1.3, supra) using
primers from sequences in the HER4 kinase domain. The
results are included in Table II. This analysis
detected the highest expression of H~R4 ~NA in 4 human
mammary adenocarcinoma cell lines (T-47D, MDA-M3-453,
BT-474, and H3396), and in neuroblastoma (SK-N-MC),
and pancreatic carcinoma (Hs766T) cell lines.
Intermediate expression was detected in 3 additional
m~m~ry carcinoma cell lines (MCF-7, MDA-MB-330, MDA-
MB-361). Low or undetectable expression was found in
other cell lines derived from carcinomas of the breast
(MDB-MB-231, MDA-MB-157, MDA-MB-468, SK-BR-3), kidney
(Caki-1, Caki-2, G-401), liver (SK-HEP-1, HepG2),
pancreas (PANC-l, AsPC-1, Capan-l), colon (HT-29),
cervix (CaSki), vulva (A-41), ovary (PA-1, Caov-3),
melanoma (SK-MEL-28), or in a variety of leukemic cell
lines. Finally, high level expression was observed in
Wilms (kidney) and breast carcinoma primary tumor
samples.
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TABLE II
; HER4 EXPRES8ION BY PRC ANALYSI8
VERY STRONG STRONG MEDIUM
5 T47D (breast) MDA-MB-453 (breast) MCF-7 (breaQt)
BT-474 (brea~t) MDA-MB-330 (breast)
H3396 (brea~t) MDA-MB-157 (breast)
H~766T (pancreatic) JEG-3
(choriocarcinoma)
SK-N-MC (neural) HEPM (palate)
Wilms Tumor (~idney) 458(medullablastoma)
BreaQt Carcinoma
10 Xidney Brain Skeletal Muscle
Heart Cerebellum Thymus
Parathyroid Pituitary Pancreas
Breast Lung
Testi~ Salivary Gland
Spleen
~EAR NEGATIVE
MDB-MB-231 (breast) MDA-MB-468 (breast)
MDA-M3-157 (breast) G-401 (kidney)
S~-BR-3 (breast) HepG2 (liver)
A-431 (vulva) PANC-l (pancreas)
Caki-1 (kidney) AsPC-l(pancrea~)
Caki-2 ~kidney) Capan-l (pancreas)
SR-HEP-1 (liver) HT-29 (~olon)
THP-1 (macrophage) CaSki (cer~ix)
PA-1 (ovary)
Prostate Caov-3 (ovary)
Adrenal SK-MEL-28 (melanoma)
ovary HUF (fibroblast)
Colon H2981 (lung)
Placenta Ovarian tumor
GEO (colon)
ALL bone marrow
AML bone marrow
Duodenum
Epidermi~
Liver
Bone marrow stroma
7. Example: Recombinant Expression of HER4
30 7.l. Materials and ~ethods
7.l.l. C~O-RI Cells and Culture
Conditions
CHO-KI cells were obtained from the ATCC
(Accession Number CCL 61). These cells lack any
detectable EGFR, HER2, or HER3 by immunoblot, tyrosine
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phosphorylation, and 35S-labeled immunoprecipitation
analysis. Transfected cell colonies expressing HER4
were selected in glutamine-free Glasgow modified
Eagle's medium (GMEM-S, Gibco) supplemented with 10%
dialyzed fetal bovine serum an increasing
concentrations of methionine sulfoximine (Bebbington,
1991, in Methods: A Companion to Methods in EnzymolooY
2:136-14S Academic Press).
7.1.2. Expression Vector Construction and
Transfections
The complete 4 kilobase coding sequence of
prototype HER4 was reconstructed and inserted into a
glutamine synthetase expression vector, pEE14, under
the control o f the cytomegalovirus immediate-early
promoter (Bebbington, supra) to generate the HER4
expression vector pEEHER4. This construct (pEEHER4)
was linearized with MluI and transfected into CHO-KI
cells by calcium phosphate precipitation using
standard techniques. Cells were placed on selective
media consisting of GMEM-S supplemented with 10%
dialyzed fetal bovine serum and methionine sulfoximine
at an initial concentration of 25 ~M (L-MSX) as
described in Bebbington, su pra, for the selection of
initial resistant colonies. After 2 weeks, isolated
colonies were transferred to 48-well plates and
expanded for HER4 expression immunoassays as described
immediately below. Subsequent rounds of selection
using higher concentrations of MSX were used to
isolate cell colonies tolerating the highest
concentrations of MSX. A number of CHO/HER4 clones
selected at various concentrations of MSX were
isolated in this manner.
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7.l.3. HER4 ~xpression Im~unoassay
Confluent cell monolayers were scraped into
hypotonic lysis buffer (lO mM Tris pH7.4, 1 mM XCl, 2
mM MgCl2) at 4 C, dounce homogenized with 30 strokes,
and the cell debris was removed by centri~ugation at
3500 x g, 5 min. Membrane fractions were collected by
centrifugation at lOO,OOO x g, 20 min, and the pellet
was resuspended in hot Laemmli sample buffer with 2-
mercaptoethanol. Expression of the HER4 polypeptide
was detected by immunoblot analysis on solubilized
cells or membrane preparations using HE~2
immunoreagents generated to either a l9 amino acid
region of the HER2 kinase domain, which coincidentally
is identical to the HER4 sequence (residues 927-945),
or to the C-terminal 14 residues of HER2, which share
a stretch of 7 consecutive residues with a region near
the C-terminus of HER4. on further amplification,
HER4 was detected from solubilized cell extracts by
immunoblot analysis with PY20 anti-phosphotyrosine
antibody (ICN Biochemicals), presumably reflecting
autoactivation and autophosphorylation of HER4 due to
receptor aggregation resulting from abberantly high
receptor density. More specifically, expression was
detected by immunobloting with a primary murine
monoclonal antibody to HER2 (Neu-Ab3, Oncogene
Science) diluted 1:50 in blotto (2.5% dry milk, 0.2%
NP40 in PBS) using 12sI-goat anti-mouse Ig F(ab')2
(Amersham, UK) diluted l:500 in blotto as a second
antibody. Alternatively, a sheep polyclonal
antipeptide antibody against HER2 residues 929-947
(Cambridge Research Biochemicals, Valleystream, NY)
was used as a primary immunoreagent diluted l:lOO in
blotto with ~2sI-Protein G (Amersham) diluted l:200 in
blotto as a second antibody. Filters were washed with
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blotto and exposed overnight on a phosphoImager
(Molecular Dynamics).
7.2. Results
CHO-KI cells transfected with a vector encoding
the complete human prototype HER4 polypeptide were
selected for amplified expression in media containing
increasing concentrations of methionine sulfoximine as
outlined in Section 7.1., et seq., supra. Expression
of HER4 was evaluated using the immunoassay described
in Section 7.1. 3 ., supra . Several transfected CHO-KI
cell clones stably expressing HER4 were isolated. One
particular clone, CHO/HER4 21-2, was selected in media
supplemented with 250 ~M MSX, and expresses high
levels of HER4. CHO/HER4 21-2 cells have been
deposited with the ATCC.
Recombinant HER4 expressed in CHO/HER4 cells
migrated with an apparent Mr of 180,000, slightly less
than HER2, whereas the parental CHO cells showed no
cross-reactive bands (FIG. 9). In addition, a 130 kDa
band was also detected in the CHO/HER4 cells, and
presumably represents a degradation product of the 180
kDa mature protein. CHO/HER4 cells were used to
identify ligand specific binding and
autophosphorylation of the HER4 tyrosine kinase (see
Section 9., et seq., infra ) .
8. Example: Assay for Detecting EGFR-Family Ligands
8.1. Cell Lines
A panel of four recombinant cell lines, each
expressing a single member of the human EGFR-family,
were generated for use in the tyrosine kinase
stimulatory assay described in Section 8.2., below.
The cell line CHO/HE~4 3 was generated as described in
Section 7.1. 2, supra.
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CHO/HER2 cells (clone 1-2500) were selected to
express high levels of recombinant human pl85'rb~2 by
dihydrofolate reductase-induced gene amplification in
dhfr-deficient CHO cells. The HER2 expression
plasmid, cDNeu, was generated by insertion of a full
length HER2 coding sequence into a modified pCDM8
(Invitrogen, san Diego, CA) expression vector (Seed
and Aruffo, 19~7, Proc. Natl. Adad. Sci. U.S.A.
84:3365-69) in which an expression cassette from
pSV2DHFR (containing the murine dhfr cDNA driven by
the SV40 early promoter) has ~een inserted at the
pCDM8 vëctor's unique BamHI site. This construct
drives HER2 expression from the CMV immediate-early
promoter.
NRHER5 cells (Velu et al., 1987, Science 1408-lo)
were obtained from Dr. Hsing-Jien Kung (Case Western
Reserve University, Cleveland, OH). This murine cell
line was clonally isolated from NR6 cells infected
with a retrovirus stock carrying the human EGFR, and
was found to have approximately 1o6 human EGFRs per
cell.
The cell line 293/HER3 was selected for high
level expression of pl60'rb33. The parental cell line,
293 human embryonic kidney cells, constitutively
expresses adenovirus Ela and have low levels of EGFR
expression. This line was established by
cotransfection of linearized cHER3 (Plowman et al.,
1990, Proc. Natl. Acad. Sci. U.S.A. 87:4905-09) and
pMClneoPolyA (neomycin selectable marker with an
Herpes simplex thymidine kinase promoter, Stratagene),
with selection in DMEM/F12 media containing 500~g/ml
G418.
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8.2. Tyrosine Kinase Stimulation ASSAY
Cells were plated in 6-well tissue culture plates
(Falcon), and allowed to attach at 37 C for 18-24 hr.
Prior to the assay, the cells were changed to serum-
free media for at least 1 hour. Cell monolayers were
then incubated with the amounts of ligand preparations
indicated in Section 7.3., below for 5 min at 37 C.
Cells were then washed with PBS and solubilized on ice
with 0.5 ml PBSTDS containing phosphatase inhibitors
(10 mM NaHPO4, 7.25, 150 mM NaCl, 1% Triton X-100,
0.5% deoxycholate, 0.1% SDS, 0.2% sodium azide, 1 mM
NaF, 1 mM EGTA, 4 mM sodium orthovanadate, 1%
aprotinin, 5 mg/ml leupeptin). Cell debris was
removed by centrifugation (12000 x g, 15 min, 4 C)
and the cleared supernatant reacted with 1 mg murine
monoclonal antibody to phosphotyrosine (PY20, ICN
Biochemicals, Cleveland, Ohio) for CHO/HER4 and
293/HER3 cells, or 1 mg murine monoclonal antibody to
HER2 (Neu-Ab3, Oncogene Sciences) for CHO/HER2 cells,
or 1 mg murine monoclonal antibody EGFR-1 to human
EGFR (Amersham) for NRHER5 cells. Following a 1 hr
incubation at 4 C, 30 ~1 of a 1:1 slurry (in PBSTDS)
of anti-mouse IgG-agarose (for PY20 and Neu-Ab3
antibodies) or protein A-sepharose (for EGFR-R1
antibody) was added and the incu~ation was allowed to
continue an additional 30 minutes. The beads were
washed 3 times in PBSTDS and the complexes resolved by
electrophoresis on reducing 7% SDS-polyacrylamide
gels. The gels were transferred to nitrocellulose and
blocked in TNET (10 mM Tris pH7.4, 75 mM NaCl, 0.1
Tween-20, 1 mM EDTA). PY20 antiphosphotyrosine
antibody diluted 1:1000 in TNET was used as the
primary antibody followed by l2'I-goat anti-mouse Ig
F(ab')2 diluted 1:500 in TNET. Blots were washed
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with TNET and exposed on a phosphorimager (Molecular
Dynamics).
8.3. Results
Several EGF-family member polypeptide and ligand
preparations were tested for their ability to
stimulate tyrosine phosphorylation of each of four
EGFR-family receptors expressed in recombinant CHO
cells using the tyrosine phosphorylation stimulation
assay described in Section 8.2., above. The
particular preparations tested for each of the four
recombinant cell lines and the results obtained in the
assay are tabulated below, and autoradiographs of some
of these results are shown in FIG. 10.
TAB~E III
8TIMULATION OF q~YR PEO8P~IORYLATION
OF EGFR--FAMILY RE~ ~ .L O~S
PREPARATION RECOMBINANT CELLS
CHO/HER4#3 CHO/HER2 NRHER5 2293/HER3
EGF - - +
AMPHIREGULIN - - +
TGF-~ - - +
25 HB-EGF - _ +
FRACTION 17* + - _ -
FRACTION 14*
* The identification of the HER4 tryrosine kinase
stimulatory activity within the conditioned media
of HepG2 cells and the isolation of these
preparations is described in Section 9, infra.
;
The results indicate that EGF, AR, TGF-~, and HB-
EGF, four related ligands which mediate their growth
regulatory signals in part through interaction with
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EGFR, were able to stimulate tyrosine phosphorylation
of EGFR expressed in recombinant NIH3T3 cells (for
EGF, see FIG. lO, Panel 3, lane 2), but not HER4,
HER2, or HER3 expressed in recombinant CHO or 293 ~'
cells (FIG. lO, Panel l, 2, 4, lanes 2 and 3).
Additionally, as discussed in more detail below, the
assay identified a HepG2-derived preparation (fraction
17) as a HER4 ligand capable of specifically
stimulating tyrosine phoshorylation of HER4 expressed
in CHO/HER4 cells alone.
9. Example: Icolation of a ~ER4 Ligand
9.l. Material~ A~d Methods
9.l.l. Cell Differentiation Aqsay
For the identification of ligands specific for
HER2, HER3 or HER4, the receptor expression profile of
MDA-MB-453 cells offers an excellent indicator for
morphologic differentiation inducing activity. This
cell line is known to express HER2 and HER3, but
contains no detectable EGFR. The results of the semi-
quantitative PCR assays (Table III) indicated high
level expression of HER4 in MDA-MB-453 cells. In
addition, cDNA encoding the prototype HER4 polypeptide
of the invention was first isolated from this cell
line (Section 6., supra).
MDA-MB-453 cells (7500/well) were grown in 50 ml
DMEM supplemented with 5% FBS and lx essential amino
acids. Cells were allowed to adhere to 96-well plates
for 24 hr. Samples were diluted in the above medium,
added to the cell monolayer in 50 ml final volume, and
the incubation continued for an additional 3 days.
Cells were then examined by inverted light microscopy
for morphologic changes.
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9 .1. 2 . 8Ource Cells
Serum free media from a panel of cultures of
human cancer cells were screened for growth regulatory
activity on MDA-MB-453 cells. A human hepatocarcinoma
cell line, HepG2, was identified as a source of a
factor which induced dramatic morphologic
differentiation of the MDA-MB-453 cells.
9.1.3. Purification of ~ER4 Ligand
lo The cell differentiation assay described in
Section 10.1.1., supra, was used throughout the
purification procedure to monitor the column fractions
that induce morphological changes in MDA-MB-453 cells.
For large-scale production of conditioned medium,
HepG2 cells were cultured in DMEM containing 10~ fetal
bovine serum using Nunc cell factories. At about 70
confluence, cells were washed then incubated with
serum-free DMEM. Conditioned medium (HepG2-C~) was
collected 3 days later, and fresh serum-free medium
added to the cells. Two additional harvests of HepG2-
CM were collected per cell factory. The medium was
centrifuged and stored at -20 C in the presence of
500 mM PMSF.
Ten litres of HepG2-CM were concentrated 16-fold
using an Amicon ultrafiltration unit (10,000 molecular
weight cutoff membrane), and subjected to sequential
precipitation with 20% and 60% ammonium sulfate.
After centrifugation at 15,000 x g, the supernatant
was extensively dialyzed against PBS and passed
through a DEAE-sepharose (Pharmacia) column pre-
e~uilibrated with PBS. The flow-through fraction was
then applied onto a 4 ml heparin-acrylic (Bio-Rad)
column equilibrated with PBS. Differentiation
inducing activity eluted from the heparin column
between 0.4 and 0.8 M NaCl. Active heparin fractions
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were pooled, brought to 2.0 M ammonium sulfate,
centrifuged at 12,000 x g for 5 min, and the resulting
supernatant was loaded onto a phenyl-5PW column (8 x
75 mm, Waters). Bound proteins were eluted with a
decreasing gradient from 2.0 M ammonium sulfate in 0.1
M Na2HPO4, pH 7.4 to 0.1 M Na2HPO4. Dialyzed fractions
were assayed for tyrosine phosphorylation of MDA-MB-
453 cells, essentially as described (Wen et a7., 1992,
Cell 69:559-72), except PY20 was used as the primary
antibody and horseradish peroxidase-conjugated goat
F(ab')2 anti-mouse Ig (Cappell) and chemiluminescence
were used for detection. Phosphorylation signals were
analyzed using the Molecular Dynamics personal
densitometer.
9.2. Results
Semi-purified HepG2-derived factor demonstrated a
capacity to induce differentiation in MDA-MB-453 cells
(FIG. 11, Panel 1-3). With reference to the
micrographs shown in FIG. 11, Panel 1-3, untreated
MDA-MB-453 cells are moderately adherent and show a
rounded morphology (FIG. 11, Panel 1). In contrast,
the addition of semi-purified HepG2-derived factor
induces these cells to display a noticeably flattened
morphology with larger nuclei and increased cytoplasm
(FIG. 11, Panel 2 and 3). This HepG2-derived factor
preparation also binds to heparin, a property which
was utilized for purifying the activity.
on further purification, the HepG2-derived factor
was found to elute from a phenyl hydrophobic
interaction column at l.OM ammonium sulfate
(fractions 16 to 18). FIG. 11, Panel 4, shows the
phenyl column elution profile. Tyrosine
phosphorylation assays of the phenyl column fractions
revealed that the same fractions found to induce
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differentiation of the human breast carcinoma cells
are also able to stimulate tyrosine phosphorylation of
a 185 kDa protein in MDA-MB-453 cells (FIG. 11, Panel
5). In particular, fraction 16 induced a 4.5-fold
increase in the phosphorylation signal compared to the
baseline signal observed in unstimulated cells, as
determined by densitometry analysis (FIG. 11, Panel
6).
The phenyl fractions were also tested against the
panel of cell lines which each overexpress a single
member of the EGFR-family (Section 9.1., supra).
Fraction 17 induced a significant and specific
activation of the HER4 kinase ( FIG. 10, Panel 1, lane
4) without directly affecting the phosphorylation of
HER2, EGFR, or HER3 (FIG. 10, Panel 1-4, lane 4).
Adjacent fraction 14 was used as a control and had no
effect on the phosphorylation of any of the EGFR-
family receptors ~FIG. lO, Panel 1-4, lane 5).
Further purification and analysis of the factor
present in fraction 17 indicates that it is a
glycoprotein of 40 to 45 kDa, approximately the same
size as NDF and HRG. The HepG2-derived factor also
has functional properties similar to NDF and HRG,
inasmuch as it stimulates tyrosine phosphorylation of
HER2/pl~5 in MDA-M~-453 cells, but not EGFR in NR5
cells, and induces morphologic differentiation of HER2
overexpressing human breast cancer cells.
Recently, several groups have reported the
identification of specific ligands for HER2 (see
Section 2., supra ., including NDF and HRG-~. In
contrast to these molecules, the HepG2-derived factor
described herein failed to stimulate phosphorylation
of HER2 in CH0/HER2 cells, but did stimulate
phosphorylation of HER4 in CH0/HER4 cells. These
findings are intriguing in view of the ability of the
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HepG2-derived factor to stimulate phosphorylation of
MDA-MD-453 cells, a cell line known to overexpress
HER2 and HER3 and the source from which HER4 was
cloned. Since EGFR and HER2 have been shown to act
S synergistically, it is conceivable that HER4 may also
interact with other EGFR-family members. In this
connection, these results suggest that NDF may bind to
HER4 in MDA-MB-453 cells resulting in the activation
of HER2. The results described in Section 10.,
immediately below, provide evidence that NDF interacts
directly with HER4, resulting in activation of HER2.
10. Example: Reconbinant NDF-Induced, HER4 Mediated
Phosphoryl~tion of ~ER2
Recombinant NDF was expressed in COS cells and
tested for its activity on HER4 in an assay system
essentially devoid of other known members of the EGFR-
family, notably EGFR and HER2.
A full length rat NDF cDNA was isolated from
normal rat kidney RNA and inserted into a cDM8-based
expression vector to generate cNDFl.6. This construct
was transiently expressed in COS cells, and
conditioned cell supernatants were tested for NDF
activity using the tyrosine kinase stimulation assay
described in Section 8.2., supra . Supernatants from
cNDF1.6 transfected cells upregulated tyrosine
phosphorylation in MDA-MB-453 cells relative to mock
transfected COS media FIG. 12, Panel 1.
Phosphorylation peaked 10-lS minutes after addition on
NDF.
The crude NDF supernatants were also tested for
the ability to phosphorylate EGFR (NRS cells), HER2
(CHO/HER2 1-2500 cells), and HER4 (CH0/HER4 21-2
cells). The NDF preparation had no effect on
phosphorylation of EGFR, or HER2 containing cells, but
induced a 2.4 to 4 fold increase in tyrosine
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phosphorylation of HER4 after 15 minutes incubation
(see FIG. 12, Panel 2). These findings provide
preliminary evidence that NDF/HRG-~ mediate their
effects not through direct binding to HER2, but
instead by means of a direct interaction with HER4.
In cell lines expressing both HER2 and HER4, such as
MDA-MB-453 cells and other breast carcinoma cells,
binding of NDF to HER4 may stimulate HER2 either by
heterodimer formation of these two related
transmembrane receptors, or by intracellular
crosstalk. Formal proof of the direct interaction
between NDF and HER4 will require crosslinking of l2sI-
NDF to CHO/HER4 cells and a detailed analysis of its
binding characteristics.
. ~rle: Chromosomal ~apping of the ~E~4 Gene
A HE~4 cDNA probe corresponding to the 5' portion
of the gene (nucleotide positions 34-1303) was used
for in situ hybridization mapping of the HER4 gene.
In situ hybridization to metaphase chromosomes from
lymphocytes of two normal male donors was conducted
using the HER4 probe labeled with 3H to a specific
activity of 2.6 x 10' cpm/~g as described (~arth et
al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:7400-04).
The final probe concentration was o.oS ~g/~l of
hybridization mixture. Slides were exposed for one
month. Chromosomes were identified by Q banding.
11.1. Results
A total of 58 metaphase cells with
autoradiographic grains were examined. Of the 124
hybridization sites scored, 38 (31%) were located on
the distal portion of the long arm of chromosome 2
(FIG. 13). The greatest number of grains (21 grains)
was located at band q33, with significant numbers of
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grains on bands q34 (10 grains) and q35 (7 grains).
No significant hybridization on other human
chromosomes was detected.
12. ~xample: Activation of the ~ER4 Receptor i5
Involved in ~ignal Transduction by ~eregulin
12.1. RecombinAnt ~eregulin Induction of
Tyrosine Phosphorylation of HER4
lZ.l.l ~ateriAls an~ Metho~s
CHO cells expressing recombinant HER4 or HER2
were generated as previously described in Section 8.
Cells (1 x 105 of CHO/HER2 and CHO/HER4, and 5 x 105 of
MDA-MB453) were seeded in 24 well plates and cultured
24 h. Cells were starved in serum free media for 1-6
h prior to addition of conditioned media from
transfected COS cells, or 25 ~g/ml HER2-stimulatory
Mab (N28 and N29) (Stancovski et al ., l991, Proc.
Natl. Acad. Sci. U.S.A. 88:8691-8695). Following 10
min treatment at room temperature, cells were
solubilized (Section 13, infra) and immunoprecipitated
with 2 ~g anti-phosphotyrosine Mab (PY20, ICN
Biochemicals) or anti-HER2 Mab (c-neu Ab-2, Oncogene
Sciences) and anti-mouse IgG-agarose (Sigma). Western
blots were performed using PY20 as described supra,
and bands were detected on a Molecular Dynamics
phosphorimager-
Recombinant rat heregulin was produced as
follows. A 1.6 kb fragment encoding the entire open
reading frame of rat heregulin (and 324 bp of S'-
untranslated sequence) was obtained by PCR using
normal rat kidney RNA as a template. This fragment
was inserted into a CDM8-based expression vector
(Invitrogen) to generate cND~1.6. The expression
plasmid was introduced into COS-1 cells using the
DEAE-dextranchloroquine method (Seed et al., Proc.
Natl. Acad. Sci. U.S.A. 1987, 84:3365-3369). After
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two days of growth in Dulbecco's Modified Eagle Medium
(DMEM?/10% FBS, the medium was replaced with DM~M and
the incubation continued for an additional 48 h.
Clarified conditioned medium was either used directly
or was dialyzed against 0.1 M acetic acid for 2 days,
dried, and resuspended as a 20-fold concentrate in
DMEM.
12.1.2. ~E~ Tyrosine Phosphorylation
As shown in FIG. 15, recombinant heregulin
induces tyrosine phosphorylation of HER4. Tyrosine
phosphorylated receptors were detected by Western
blotting with an anti-phosphotyrosine Mab a,
Monolayers of MDA-MB453 or CH0/HER4 cells were
incubated with media from COS-1 cells transfected with
a rat heregulin expression plasmid (HRG), or with a
cDM8 vector control (-). The media was either applied
directly (lx) or after concentrating 20-fold (20x, and
vector control). Solubilized cells were
immunoprecipitated with anti-phosphotyrosine Mab. b,
Monolayers of CH0/HER2 cells were incubated as above
with transfected Cos-1 cell supernatants or with two
stimulatory Mabs to HER2 (Mab 28 and 29). Solubilized
cells were immunoprecipitated with anti-HER2 Mab.
Arrows indicate the HER2 and HER4 proteins.
12 .1. 3 . ~e8ult5
In order to determine if HER4 is involved in
signaling by heregulin, the ability of recombinant rat
heregulin to stimulate tyrosine phosphorylation in a
panel of Chinese hamster ovary (CHo) cells that
ectopically express human HER2 or HER4 was examined.
The activity of recombinant heregulin was first
confirmed by its ability to stimulate differentiation
of human breast cancer cells (data not shown) and to
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induce tyrosine phosphorylation of a high molecular
weight protein in MDA-MB453 cells (FIG. 15, Panel l).
Heregulin had no effect on CHO cells expressing only
HER2 (FIG. 15, Panel 3), yet these cells were shown to
have a functional receptor since their tyrosine kinase
activity could be stimulated by either of two
antibodies specific to the extracellular domain of
HER2 (FIG. 15, Panel 3). However, heregulin was able
to induce tyrosine phosphorylation of a 180K protein
in CHO cells expressing HER4 (FIG. 15, Panel 2).
Species differences in ligand-receptor
interactions have been reported for EGF receptor (Lax
et al., 1988, Mol. Cell. Biol. 8:1970-1978) . It is
unlikely that such differences are responsible for our
failure to detect a direct interaction between rat
heregulin and human HER2, since previous studies have
shown that rat heregulin does not directly interact
with rat HER2/neu (Peles et al., supra). In addition,
rat, rabbit, and human heregulin share high sequence
homology and have been shown to induce tyrosine
phosphorylation in their target cells of human origin
(Wen D. et al., supra; Holmes et al., supra; and Falls
et al., supra).
12.2. Expr~ssion o~ ~ecombin~nt ~ER2 ~nd ~ER4
in ~uman CEM Cells
12.2.1. ~aterials and MethodQ
cNHER2 and cNHER4 expression plasmids were
generated by insertion of the complete coding
sequences of human HER2 and HER4 into cNE0, an
expression vector that contains an SV2-NE0 expression
unit inserted at a unique BamHI site of CDM8. These
constructs were linearized and transfected into CEM
cells by electroporation with a Bio-Rad Gene Pulser
apparatus essentially as previously described (Wen et
al., supra). Stable clones were selected in RPMI/10%
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FBS supplemented with 500 ~g/ml active Geneticin.
HER2 immunoprecipitations were as described in FIG.
15, using 5 x lo6 cells per reaction, and the HER2
_ Western blots were performed with a second anti-HER2
Ma~ (c-neu Ab-3, Oncogence Sciences). For metabolic
labeling of HER4, 5 x l06 cells were incubated for 4-6
h in methionine and cysteine-free Minimal Essential
Medium (MEM) supplemented with 2% FBS and 250 ~Ci/ml
[3sS]Express protein labeling mix (New England
Nuclear). Cells were washed twice in RPMI and
solubilized as above. Lysates were then incubated for
6 h, 4- C with 3 ~l each of two rabbit antisera raised
against synthetic peptides corresponding to two
regions of the cytoplasmic domain of human HER4
(66qLARLLEGDEKEYNADGGe8 [SEQ ID No:3l] and
1010EEDLEDMMDAEEY1022 [SEQ ID No:32]). Immune complexes
were precipitated with 5 ~g goat anti-rabbit Ig
(Cappel) and Protein G Sepharose (Pharmacia).
Proteins were resolved on 7~ SDS-polyacrylamide gels
and exposed on the phosphorimager. For Mab-
stimulation assays, 5 x lo6 cells were resuspended in
l00 ~l RPMI and 25 /~g/ml Mab was added for 15 min at
room temperature. Control Mab 18.4 is a murine IgG1
specific to human amphiregulin (Plowman et al., l990,
Mol. Cell. Biol. l0:1969-1981). Following Mab-
treatment, cells were washed in PBS, solubilized
(Section 13, infra), and immunoprecipitated with anti-
HER2 Mab (Ab-2). Tyrosine phosphorylated HER2 was
detected by PY20 Western blot as in FIG. 15.
12.2.2. ExprQssion of HER2 and HER4 in
~uman CEM Cell~
Expression of recombinant HER2 and HER4 in human
CEM cells is shown in FIG. 16. Transfected CEM cells
were selected that stably express either HER2, HER4,
or both recombinant receptors. In FIG. 16, Panel l,
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recombinant HER2 was detected by immunmoprecipitation
of cell lysates with anti-HER2 Mab (Ab-2) and Western
blotting with another anti-HER2 Mab (Ab-3). In FIG.
16, Panel 2, recombinant HER4 was detected by
immunoprecipitation of 35S-labeled cell lysates with
HER4-specific rabbit anti-peptide antisera. In FIG.
16, Panel 3, three CEM cell lines were selected that
express one or both recombinant receptors and aliquots
of each were incubated with media control (-), with
two HER2-stimulatory Mabs (Mab 28 and 29), or with an
isotype matched control Mab (18.4). Solubilized cells
were immunoprecipitated with anti-HER2 Mab (Ab-2) and
tyrosine phosphorylated HER2 was detected by Western
blotting with an anti-phosphotyrosine Mab. The size
in kilodaltons of prestained high molecular weight
markers (Bio-Rad) is shown on the left and arrows
indicate the HER2 and HER4 proteins.
12.2.3. Results
These findings of Example 12 support the earlier
observation that HER2 alone is not sufficient to
transduce the heregulin signal. To further address
this possibility, a panel of human CEM cells that
express the recombinant receptors either alone or in
combination was established. The desired model system
was of human origin, since many of the reagents
against erbB family members are specific to the human
homologues. CEM cells are a human T lymphoblastoid
cell line and were found to lack expression of EGF
receptor, HER2, HER3, or HER4, by a variety of
immunologic, biologic, and genetic analyses (data not
shown). FIG. 16 demonstrates the selection of three
CEM cell lines that express only HER2 (CEM 1-3), only
HER4 (CEM 3-13), or both HER2 and HER4 (CEM 2-9). The
presence of a functionally and structurally intact
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HER2 in the appropriate cells was confirmed by the
induction of HER2 tyrosine phosphorylation by each of
the two antibodies specific to the extracellular
- domain of HER2, but not by an isotype matched control
antibody (FIG. 16, Panel 3).
12.3. Heregulin In~uction of Tyrosine
Phosphorylation in CEM Cells Expressing
HER4
12.3.1. Materials and Methods
~ecombinant rat heregulin was prepared as in FIG.
15, and diluted to 7x in RPMI. The HER4-specific Mab
was prepared by immunization of mice with recombinant
HER4 (manuscript in preparation). CEM cells (5 x 106)
were treated with the concentrated supernatants for 10
min, room temperature and precipitated with PY20 or
anti-HER2 Mab (Ab-2) as described in FIG. 15.
Immunoprecipitation with anti-~ER4 Mab was performed
by incubation of cells lysates with a 1:5 dilution of
hy~ridoma supernatent for several hours followed by 2
~g rabbit anti-mouse Ig (cappel) and Protein A
Sepharose CL-4B (Pharmacia). PY20 Westerns as
described in FIG. 15.
12.3.2. ~eregulin Induction of Tyrosine
Phosphorylation in CEM Cells
Expre 9 sing ~ER4
As shown in FIG. 17, heregulin induces tyrosine
phosphorylation in CEM cells expressing HER4. Three
CEM cell lines that express either HER2 or HER4 alone
30 (CEM 1-3 and CEM 3-13) or together (CEM 2-9) were
incubated with 7x concentrated supernatants from mock-
(-) or heregulin-transfected (+) COS-1 cells.
Solubilized cells were immunoprecipitated (IP) with
anti-phosphotyrosine Mab (PY20) (FIG. 17, Panel 1);
35 HER2-specific anti-HER2 Mab (Ab-2) (FIG. 17, Panel 2);
or HER4-specific Mab (6-4) (FIG. 17, Panel 3). In
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each case, tyrosine phosphorylated receptors were
detected by Western blotting with anti-phosphotyrosine
Mab. The size in kilodaltons of prestained molecular
weight markers (BioRad) is shown on the left and
arrows indicate the HER2 and HER4 proteins.
12.3.3 Result~
The panel of cEM cells were then analyzed by
phosphotyrosine Western blots of cells lysates
following treatment with heregulin and
immunoprecipitation with three different monoclonal
antibodies (Mabs). Precipitation with an anti-
phosphotyrosine antibody (PY20) again demonstrates
that heregulin is able to stimulate tyrosine
phosphorylation in cells expressing HER4, but not in
cells expressing only HER2 (FIG. 17, Panel 1).
However, precipitation with an antibody specific to
the extracellular domain of HER2 demonstrates that
HER2 is tyrosine phosphorylated in response to
heregulin in cells that co-express HER4 (FIG. 17,
Panel 2). Furthermore, precipitation with a HER4-
specific Mab confirms that heregulin induces tyrosine
phosphorylation of HER4 irrespective of HER2
expression (FIG. 17, Panel 3). Due to co-expression
of HER2 and HER4 in many breast carcinomas, these
findings suggest that earlier studies of heregulin-
HER2 interactions may require reevaluation.
12.4. Covalent Cross-linking of Iodinated
Heregulin to HER4
12.4.1. Materi~ls ~nd Methods
To facilitate purification, recombinant heregulin
was produced as an epitope-tagged fusion with
amphiregulin. The 63 amino acid EGF-structural motif
of rat heregulin (Wen et al., supra) from serine 177
to tyrosine 239 was fused to the N-terminal 141 amino
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acids of the human amphiregulin precursor (Plowman et
al., supra). This truncated portion of heregulin has
previously been shown to be active when expressed in
E. coli (Holmes et al ., supra ), and the N-terminal
residues of amphiregulin provide an epitope for
immunologic detection and purification of the
recombinant protein. This cDNA fragment was spliced
into a cDM8 based expression vector for transient
expression in COS-1 cells. Recombinant heregulin was
lo purified by anion exchange and reverse phase
chromatography as shown to be active based on the
specific stimulation of HER4 tyrosine phosphorylation.
Purified heregulin was iodinated with 250 ~Ci of l2sI-
labeled Bolton-Hunter reagent (NEN). CHO/HER4 or
CHO/HER2 cells were incubated with l2sI-heregulin (105-
cpm) for 2 h at 4 C. Monolayers were washed in PBS
and 3 mM Bis(sulfosuccinimidyl) suberate (BS3, Pierce)
was added for 30 min on ice. The cells were washed in
tris-buffered saline, dissolved in SDS sample buffer,
run on a 7% polyacrylamide gel, and visualized on the
phosphorimager.
12.4.2. Results
As shown in FIG. 18, previous binding and
covalent cross-linking studies have demonstrated that
p45 binds specifically to HER4 and displays a single
high-affinity site with a Kd of 5 nM on CH0/HE~4 cells
(Section 13, infra). Preliminary cross-linking
studies have been performed on these cells with
recombinant heregulin revealing a high molecular
weight species that corresponds to the heregulin-HER4
receptor complex.
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12.5 Resultg
As the data demonstrate heregulin induces
tyrosine phosphorylation of HER4 in the absence of
HER2. In contrast, heregulin does not directly
- 5 stimulate HER2. However, in the presence of HER4,
heregulin induces phosphorylation of HER2, presumably
either by transphosphorylation or through receptor
heterodimerization. Together, these experiments
suggest that HER4 is the receptor for heregulin.
Most breast cancer cells that overexpress HER2
have been shown to be responsive to heregulin, whereas
HER2-positive ovarian and fibroblast lines do not
respond to the ligand. This observation could be
explained by the fact that HER4 is co-expressed with
HER2 in most or all of the breast cancer cell lines
studied, but not in the ovarian carcinomas.
Furthermore, overexpression of HER2 in heregulin-
responsive breast cancer cells leads to increased
binding, whereas expression of HER2 in heregulin-
unresponsive ovarian or fibroblast cells has no effect(Peles et al., supra).
Northern and in situ hybridization analyses
localizes HER4 to the white matter and glial cells of
the central and peripheral nervous system, as well as
to cardiac, skeletal, and smooth muscle. This
distribution is consistent with HER4 being involved in
signaling by the neurotropic factors, GGF, and ARIA.
Recognition of HER4 as a primary component of the
heregulin signal transduction pathway will assist in
deciphering the molecular mechanisms that results in
its diverse biologic effects.
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13. Example: Purification of the ~ER4 ligand, p45
13.1 Material~ an~ ~ethods
13.1.1. Cell Culture and Reagents
MDA-MB 453 cells were obtained from the American
S Type Culture Collection (Rockville, MD) and cultured
in Dulbecco's modified Eagle's medium tDMEM)
supplemented with 10% fetal bovine serum and amino
acids (Life Technologies, Inc.). HepG2 cells were
obtained from Dr. S. Radka and cultured in 10~ fetal
bovine serum containing DMEM. For large scale
production of serum-free conditioned medium, HepG2
cells were propagated in Nunc cell factories. Chinese
hamster ovary cells (CHO-KI) expressing high levels of
either recombinant human pl85'rb~2 (CHO/HER2) or
recombinant human pl80'rb34 (CHO/HER4) were generated
and cultured as described in Section 8. N29 monoclonal
antibody to the extracellular portion of the human
HER2 receptor was a gift from Dr. Y. Yarden. Ab-3 c-
neu monoclonal antibody that reacts with the human
pl85'rb32 was from Oncogene Science Inc.
13 .1. 2. Human Breast Cancer Cell
Differentiation Assay
MDA-MB-453 human breast cancer cells overexpress
pl85'rba2 but do not express the EGFR at their surface
(Kraus, 1987, EMBO J. 6:605-610). A cell
differentiation assay was used to monitor the
chromatography fractions for their ability to induce
phenotypic differentiation in MDA-MB-453 cells.
13.1.3. Purification of p45
; Medium conditioned by HepG2 cells (HepG2-CM, 60
liters) was concentrated 26-fold using an Amicon
ultrafiltration unit (10,000 molecular weight cutoff
membranes) and then subjected to 50~ ammonium sulfate
((NH4)2SO44) precipitation. After centrifugation at
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25,000 x g for 1 h, the supernatant was loaded, as
five separate runs, on a phenyl-Sepharose column ~2.5
x 24.5 cm, Pharmacia LKB Biotechnology Inc.)
equilibrated with 1.9 M (NH4)2S04 in 0.1 M Na2HP04, pH
7.4. Bound proteins were eluted with a 240 ml linear
decreasing gradient from 1.9 M to 0 M (NH4)2S04 in 0.1
M phosphate buffer, pH 7.4. The flow rate was 70
ml/h, and 5.8-ml fractions were collected. Active
fractions were pooled, concentrated, dialyzed against
PBS, and then applied (three separate runs) to a DEAE-
Sepharose column (2.5 x 25 cm, Pharmacia) equilibrated
with PBS, pH 7.3. The flow rate was 1 ml/min. The
column flow-through was then loaded (two separate
runs) on a CM-Sepharose Fast Flow column (2.5 x 13.5
cm, Pharmacia) pre-equilibrated with PBS, pH 7.3.
Proteins were eluted at 1 ml/min. with a 330-ml
gradient from PBS to 1 M NaCl in PBS. Fractions of 5
ml were collected. The active material was loaded on
a TSKgel heparin-5PW HPLC column (7.5 x 75 mm,
TosoHaas) equilibrated with PBS. The flow rate was
0.5 ml/min. A 50-ml linear NaC1 gradient (PBS to 2 M
in PBS) followed by an isocratic elution with 2 M NaCl
was used to elute the bound proteins. Fractions of 1
ml were collected. Active fractions corresponding to
the 1.3 M NaC1 peak of protein were pooled and
concentrated. A Protein Pak SW-200 size exclusion
chromatography column (8 x 300 mm, Waters)
equilibrated with lO0 mM Na2HP04, pH7.4, 0.01% Tween 20
was used as a final step of purification. The flow
rate was 0.5 ml/min., and 250-~l fractions were
collected. Column fractions were then analyzed by
SDS-PAGE (12.5% gel) under reducing conditions and
proteins detected by silver staining.
~5
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13.1.4. Detection of ~yrosine-
Phosphorylated Proteins by Western
Blotting
Aliquots of PBS-dialyzed column fractions were
diluted to 200 ~l in PBS, then added to individual
wells of 4 8-well plated containin~ either 5 x 105 MDA-
MB-453 cells, 2 x 104 CHOJHER2 cells or s x 104
CHO/HER2 cells. Following a 10-min. incubation at 37
C, cells were washed and then lysed in 100 ~1 of
boiling electrophoresis sample buffer. Lysates were
heated at 100 C for 5 min., cleared by centrifugation,
and then subjected to SDS-PAGE. After
electrophoresis, proteins were transferred to
nitrocellulose. The membrane was blocked for 2 h at
room temperature with 6% hovine serum albumin in 10 mM
15 Tria-HC1, pH 8.0, 150 mM NaC1, 0.05% Tween 20. PY20
monoclonal anti-phosphotyrosine antibody (ICN, 2 h at
22 C) and horseradish peroxidase-conjugated goat anti-
mouse IgG F~ab')2 (Cappel, lh at 22 C) were used as
primary and secondary probing reagents, respectively.
Proteins phosphorylated on tyrosine residues were
detected with a chemiluminescence reagent (Amersham
Corp.).
13 .1. 5. C~O/HER2 Stimulation As~ay
CHO/HERZ cells were seeded in 24-well plates at 1
X 105 cells/well and cultured 24 h. Monoclonal
antibody N29 specific to the extracellular domain of
pl85'rb32 (Stancovski et al., l991, PNAS 88:8691-8695)
was added at 25 ~g/ml. Following a 20-min. incubation
3 at room temperature, media were removed and cells were
solubilized for 10 min. on ice in PBS-TDS (10 mM
sodium phosphate, pH 7.25, 150 mM NaC1, 1% Triton,
o.5% sodium deoxycholate, 0.1% SDS, 0.2% NaN3, 1 mM
r NaF, 1 m M phenylmethylsulfonyl fluoride, 20 ~g/ml
aprotinin~ with occasional vortexing. Clarified
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extracts were incubated for 2 h at 4 C with an antip-
185'rb~32 antibody (Ab-3 c-neu, Oncogene Science Inc.).
Rabbit anti-mouse IgG (Cappel) and protein A-Sepharose
were then added, and samples were incubated an
additional 30 min. Immune complexes were washed 3
times with PBS-TDS, resolved on a 7% polyacrylamide
gel, and electrophoretically transferred to
nitrocellulose. Phosphorylation of the receptor was
assessed by Western blot using a 1:1000 dilution of
PY20 phosphotyrosine primary antibody (ICN
Biochemicals) and a 1:500 dilution of 125I-sheep anti-
mouse F(ab') 2 (Amersham Corp.).
13.1.6. Covalent Cross-linking of
Iodinated p45
HPLC-purified p45 (1.5 ~g) was iodinated with 250
~Ci of l24I-labeled Bolton-Hunter reagent obtained from
Du Pont-New England Nuclear. l25I-p4s was purified by
filtration through a Pharmacia PD-10 column. The
specific activity was 104 cpm/ng. 12sI-p45 retained its
biological activity as confirmed in a differentiation
assay as well as a kinase stimulation assay (data not
shown). Binding of radiolabeled p45 was performed on
2 x 105 CHO/HER4 cells and 4 x 105 CHO-KI or CHO/HER2
cells in 12-well plates. Cell monolayers were washed
twice with 1 ml of ice-cold binding buffer (DMEM
supplemented with 44 mM sodium bicarbonate, 50 mM BES
[N-, N-Bis (2-hydroxyethyl) -2-aminoethan-sulfonic
acid], pH 7.0, 0.1~ bovine serum albumin) and then
incubated on ice for 2 h with 50 ng/ml 125I-p45 in the
absence or the presence of 250 ng/ml unlabeled p45.
The monolayers were washed twice with PBS and then
incubated in the presence of 1 mM
bis (sulfosuccinimidyl)suberate (BS3, Pierce) in PBS for
45 min. on ice. Supernatants were discarded, and the
reaction was quenched by adding 0.2 M glycine in PBS.
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Cells were washed and then lysed by adding 150 ~l of
boiling electrophoresis sample buffer containing o.l M
dithiothreitol. Samples were boiled for 5 min. and 50
. ~l of each sample was loaded on 7.5% polyacrylamide
- 5 gels. Dried gels were analyzed using a Molecular
Dynamics PhosphorImager and then exposed to Kodak X-
Omat AR films.
13.l.7. Binding Analysis of Iodinated p45
CHO/~ER4 cells, CHO-KI cells (lO; cells/well), and
CHo/HER2 cells (2 x lOs cells/well) were seeded in 24-
well plates. After 48 h, cells were washed with
binding buffer and then incubated with increasing
concentrations of 12~I-p45. Nonspecific binding was
determined in the presence of excess unla~eled p45.
After a 2-h incubation at 4 c, the cells were washed
three times with binding buffer and then lysed in 500
~l of 0.5M NaOH, O.l~ SDS. Cell-associated
radioactivity was determined by using a ~-counter.
Scatchard analysis was performed using the
computerized LIGAND program (Munson and Rodbard, 1980,
Anal. Biochem 107:220-239).
13.l.8. N-terminal Amino Acid ~equence
The N-terminal sequence analysis of p45 (z5 pmol)
was performed as previously described (Shoyab et al.,
l990, Proc. Natl. Acad. Sci. 87:7912-7916).
13.2. Purification of the ~ER4 ligand, p45
Sixty liters of medium conditioned by HEPG2 cells
was used as a starting material, and throughout the
purification procedure, bioactivity was assessed by a
cell differentiation assay described in Section
lO.1.1., supra. After concentration (1540 mg of
protein) and ammonium sulfate precipitation, the
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active material (1010 mg of protein~ was loaded on a
phenyl-Sepharose column (FIG. l9, Panel 1). Column
fractions 40-85 (348 mg of protein eluting between lM
ammonium sulfate and OM ammonium sulfate) were found ,~
to induce morphological changes in MDA-MB-453 cells.
The biologically active column flow-through (174 mg of
protein) was subjected to a cation-exchange
chromotography (FIG. 19, Panel 2) with activity
eluting between 0.35 and 0.48 M NaCl. The active
fractions were pooled (1.5 mg of protein) and applied
to an analytical heparin column (FIG. 19, Panel 3).
The differentiation activity eluted from the heparin
column between 0.97 and 1.45 M NaCl (fractions 27-38).
Size exclusion chromatography of the heparin column
fractions 35-38 achieved a homogeneous preparation of
the human breast cancer cell differentiation factor.
A major protein peak eluted with a molecular weight
greater than 70,000 (FIG. 19, Panel 4). Fractions 30
and 32 assayed at 30 ng/ml confirmed the bioactivity
of this protein with phenotypic changes being apparent
after 24 hours. SDS-PAGE analysis of these column
fractions followed by silver staining of the gel
showed that the biologically active peak contained a
single protein migrating around 45 kDA (FIG. 20). The
faint 67 kDa band corresponds to a staining artifact,
as evidenced by the left lane of the gel, which
contained no sample. The amount of pure protein
recovered in fractions 30-33 was estimated to be 6
micrograms. The difference in the molecular weight
estimated by size exclusion chromatography and SDS-
PAGE indicates that this protein may form dimers or
oligomers under non-denaturing conditions.
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13.3. N-terminal Amino ACid Sequence of p45
Twenty-five pmol of p45 was subjected to direct
amino acid sequencing, identifying the sequence Ser-
Gly-X-Lys-Pro-X-X-Ala-Ala [SEQ ID No:33]. An X
denotes a sequenator cycle in which a precise amino
acid could not be assigned. comparison of this
partial sequence with two protein data bases (GenBank
release 73, EMBL release 32) revealed a perfect
homology between the identified residues and a region
of the amino terminus of heregulin (Holmes et al.,
supra) The N-terminal serine residue of p45
corresponds to residue 20 of the deduced amino acid
sequence of heregulins.
13.~. p45 Stimulateq Protein Phosphorylation
FIG. 21, Panel 1 shows the stimulatory effect of
sequential fractions from the size exclusion
chromatography column on tyrosine phosphorylation in
MDA-MB-453 cells. Densitometric analysis of the
20 autoradiogram revealed that ~ractions 30-34 were
essentially equipotent. Homogeneously purified p45
specifically stimulated tyrosine phosphorylation of
pl8 O~rbB4 ( FIG. 21, Panel 2). p45 was not able to
stimulate phosphorylation in CH0/HER2 cells, and the
25 cell were found to express functional p1ss~rbB2 receptor
as evidenced by immunoreactivity with 5 monoclonal
antibodies specific to different regions of pl85'rbB2.
p45 has an N-terminal amino acid sequence similar to
the recently isolated pl85'rbB2 ligand.
13.5. Binding and Covalent Cro~-linking of
P45 to p18o~rb8~
Binding and cross-linking studies were performed
f in order to confirm that p45 was able to bind to
pl80'rb94. Binding studies revealed that while no
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specific binding of 125I-p45 to CH0-KI and CHO/HER2
cells could be measured, CH0/HER4 cells displayed a
single high affinity site (Kd about 5nM) with 7 x 104
receptors/cell (FIG. 22, Panel 1). The results of
iodinated p45 cross-linking to CH0-KI, CHO/HER2, or
CHO/HER4 cells are presented in FIG. 22, Panel 2.
Whereas no cross-linked species was observed in either
CHO-KI or CHO/HER2 cells, four distinct bands were
observed in CHO/HER4 cells, migrating as 45-, 100-,
and 210-kDa species, and a very high molecular weight
species. In the presence of unlabeled p45, ~25I-p45
binding was greatly reduced. The 45 kDa band
represents uncross-linked yet p180'rb~4 associated 125I-
p45. The 210 kDa band corresponds to the p45-pl80'rb~4
complex (assuming an equimolar stoichiometry of ligand
and receptor), whereas the high molecular weight band
is presumed to be a dimerized form of the receptor-
ligand complex. The 100 kDa band could represent a
truncated portion of the extracellular domain of the
pl80'rb~4 receptor complexed to 125I-p45 or a covalently
associated p45 dimer. The c-kit ligand provides
precedence for cross-linked dimers (Williams et al.,
1990, Cell 63:167-174).
13.6. Re~ults
The HER4 ligand, p45, purified from medium
conditioned by HepG2, induces differentiation of
breast cancer cells and activates tyrosine
phosphorylation of a 185 kDa protein in MDA-MB-453
cells. p45 is not capable of directly binding to
pl85'rbB2 but shows specificity to HER4/pl80erb~4.
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1~. Example: Targeted Cytotoxicity Mediated By A
Chimeric Heregulin-Toxin Protein
14.1. Materials an~ Methods
14.1.1. Reagents and Cell ~ines
Heregulin ~2-Ig and the mouse monoclonal antibody
directed against the Pseudomonas exotoxin (PE) was
supplied by Dr. J.-M. Colusco and by Dr. Tony Siadek,
respectively (Bristol-Myers-squibb, Seattle, WA). The
cell lines BT474, MDA-MB-453, T47D, SKBR-3, and MCF-7
(all breast carcinoma), LNCaP (prostate carcinoma),
CEM (T-cell leukemia) and SXOV3 (ovarian carcinoma)
were obtained from ATCC (Rockville, MD). The H3396
breast carcinoma cell line and the L2987 lung
carcinoma cell line were established at Bristol-Myers-
Squibb ~Seattle, WA). The AU565 breast carcinoma cell
line was purchased from the Cell Culture laboratory,
Naval Biosciences Laboratory (Naval Supply Center,
Oakland, CA). All cell lines were of human origin.
BT474 and T47D cells were cultured in IMDM
supplemented with 10~ fetal bovine serum (FBS) and 10
~g/ml insulin~ MCF-7, H3396, LNCaP and L2987 were
cultured in IMDM supplemented with 10~ FBS. SKBR3 and
SKOV3 cells were grown in McCoys media supplemented
with 10% FBS and 0.5% non-essential amino acids.
AU565 cells were cultured in RPMI 1640 media
supplemented with 15% FBS and CEM transfectants (see
section 15.1.5., infra) were cultured in RPMI 1640
supplemented with 10% FBS and 500 ~g/ml G418.
14.1.2. Construction of HA~-T2 ~2 Expression
Plasmid
Rat heregulin cDNA (Wen et al., 1994, Mol. Cell.
; Biol. 14:1909-1919) was isolated by RT-PCR using mRNA
from rat kidney cells as template. The cDNA was
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prepared in chimeric form with the AR leader sequence
by a two-step PCR insertional cloning protocol using
cARP (Plowman et al., 1990, Mol. Cell. Biol. 10:1969-
1981) as template to amplify the 5' end of the
chimeric ligand using the oligonucleotide primers
CARP5:
(5'-CGGAAGCTTCTAGAGATCCCTCGAC-3') [SEQ ID No:34]
and
ANSHLIK2:
(3'CCGCACACTTTATGTGTTGGCTTGTGTTTCTTCTA~ llCCA
G- 5') [SEQ ID No:35].
The EGF-like domain PCR was amplified from
cNDF1.6 (Plowman et al., 1993, Nature 366:473-475)
using the oligonucleotide primers
ANSHLIK1:
(5'-CAAAAATGGAAAAAATAGAAGAAACAGAAGCCATCTCATAA
AGTGTGCGG-3') t SEQ ID No:36]
and
XNDF1053:
(3'-GTCTCTAGATTAGTAGAGTTCCTCCG~~ CTTG-5') [ SEQ ID
No:37].
The products were com~ined and reamplified using
the oligonucleotide primers CARP5 and XNDF1053. The
HAR (heregulin-amphiregulin) construct (cNANSHLIK) was
PCR amplified in order to insert an Nde I restriction
site on the 5' end and a ~ind III restriction site on
the 3' end with the oligonucleotide primers
NARP1:
(5'-GTCAGAGTTCATATGGTAGTTAAGCCCCCCCAAAAC-3') [ SEQ ID
No:38]
and
NARP4:
(3'-GGCAGTTCTATGAACACGTTCACGGGCTTGCTTAAATGACCGCTGGCA
ACGGTCTTGATACAATACCGTAGAAAAATGTTTAGCCTCCTTGAGATGTTCGAA
TCTCCTAGAAAC-5') [SEQ ID No:39].
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The resulting 287 bp DNA fragment was digested
with Nde I and Hind III, followed by ligation into the
compatibly digested expression plasmid pBW 7.0 which
contained, in frame at the 5~ fusion site, the
nucleotide sequence encoding for of PE40 (Friedman et
al., 1993, Cancer Res. 53:334-339). The resulting
expression plasmid pSE 8.4 then contained the gene
fusion encoding the chimeric heregulin-toxin protein,
under the control of a IPTG-inducible T7 promoter.
14.l.3. Expre~ion and Isolation of Recombinant
HAR-T~ ~2 Protein
The plasmid pSE 8.4 encoding the chimeric protein
HAR-TX ~2 was transformed into the E. coli strain BL21
(~DE3). Cells were grown by fermentation in T broth
containing lO0 ~g/ml ampicillin at 37C to a optical
density of A6;0 = 4.8, followed by induction of protein
expression with l mM isopropyl-l-thio-~-D-
galactopyranoside (IPTG). After 90 minutes the cells
were harvested by centrifugation. The cell pellet was
frozen at -70C, then thawed and resuspended at 4C in
solubilization buffer (50 mM Tris-HCl (pH 8.0), lO mM
EDTA, l ug/ml leupeptin, 2 ug/ml aprotinin, l ug/ml
pepstatin-A, 0.5 mM PMSF) containing l~ tergitol by
homogenization and sonication. The insoluble material
of the suspension, containing inclusion bodies with
the HAR-TX ~z protein, was pelleted by centrifugation
and washed three times with solubilization buffer
containing 0.5% tergitol (first wash), l M NaCl
(second wash), and buffer alone (third wash).
The resulting pellet containing pre-purified
inclusion bodies was dissolved in 6.5 M guanidine-HCl,
O.l M Tris-HCl (pH 8.0), 5 mM EDTA; sonicated; and
refolded by rapid dilution (lO0-fold) into O.l M Tris-
HCl (pH 8.0), 1.3 M urea, 5 mM EDTA, 1 mM glutathione,
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and 0.1 mM oxidized glutathione at 4C. The addition of
the denaturating agent urea at low concentration was
utilized to allow slow refolding and avoid the
formation of aggregates. The refolded HAR-TX ~2 ~'
protein was diluted 2-fold with 50 mM sodium phosphate
(pH 7.0) and applied to a cation-exchange resin (POROS
50 HS, PerSeptive Biosystems, Cambridge, MA), pre-
equilibrated in the same buffer. The HAR-TX ~2
protein was eluted with a 450 nM NaCl step gradient in
50 mM sodium phosphate (pH 7.0) and fractions were
analyzed using SDS-PAGE and Coomassie blue staining.
Final purification of pooled fractions was performed
by chromatography using Source 15S cation-exchange
media (Pharmacia, Uppsala, Sweden) equilibrated with
50 mM sodium phosphate (pH 6.0). Chimeric HAR-TX ~2
protein was eluted with a gradient of 0-1 M NaCl in
the same buffer and analyzed by SDS-PAGE.
14.1.4. ELI~A Test for Determination of Binding
Activity
Membranes from 5 x 10' MDA-MB-453 cells were
prepared and coated to 96 well plates as previously
described for H3396 human breast carcinoma cells
(Siegall et al., 1994, J. Immunol. 152:2377-2384).
Subsequently, the membranes were incubated with
titrations of either HAR-TX ~2 or PE40 ranging from
0.3 - 300 ug/ml and the mouse monoclonal anti-PE
antibody EXA2-lH8 as the secondary reagent (Siegall et
al ., supra ) . The isolate of the toxin portion PE40
alone was used to determine unspecific binding
activity to ~he membrane preparations, in comparison
with the specific binding activity of HAR-TX ~2.
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14.l.5. Phosphotyrosine Analysis of tr~n~fected
CEM cell lines
`CEM cells expressing various receptors of the
EGF - R family (1-5 x lo6 cells) were stimulated with 500
q 5 nglml HAR-TX ~2 for 5 minutes at room temperature.
The cells were pelleted and resuspended in O.l ml
lysis buffer (50 mM Tris-HCl, pH 7.4, l50 mM NaCl, 5
mM MgCl2, 1~ NP40, 0.5% deoxycholate, 0.1% sodium
dodecylsulfate, 1 mM sodium orthovanadate) at 4c.
lO Insoluble material was pelleted by centrifugation at
lO,000 x g for 30 seconds, and samples were analyzed
by SDS-PAGE and subsequent Western blot analysis using
the anti-phosphotyrosine antibodies 4GlO (ICN, Irvine,
CA) and PY20 (Upstate Biotechnology, Lake Placid, New
15 York).
14.l.6. Cytotoxicity Assays
For cytotoxicity assays, tumor cells (lOs
cells/ml) in growth medium were added to 96-well flat
20 bottom tissue culture plates (o.l ml/well) and
incubated at 37c for 16 h. Cells were incubated with
HAR-TX ~2 for 48 h at 37C, washed twice with phosphate
buffered saline (PBS), followed by addition of 200
~l/well of l.5 ~M calcein-AM (Molecular Probes Inc.,
25 Eugene, OR). The plates were incubated for 40 minutes
at room temperature (RT), and the fluorescence
measured using a Fluorescence Concentration Analyzer
(Baxter Heathcare Corp., Mundelein, IL) at
excitation/emission wavelengths of 485t530 nm.
30 Calcein-AM is mem~rane permeable and virtually non-
fluorescent. When it is hydrolyzed by intracellular
esterases, an intensely fluorescent product, calcein
is formed. The % cytotoxicity was calculated as
previously described (Siegall et al., supra). To
35 determine the specificity of the cytotoxic effect of
HAR-TX ~2 competitive assays were performed on LNCaP
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and on MDA-MB-453 cells. Treated essentially as
described above, plates were incubated with increasing
concentrations of HAR-TX ~2 in presence heregulin ~2-
Ig (0.002-5.0 ~g/ml) or with HAR-TX ~2 (50 ng/ml).
Isotype matched L6-Ig (Hellstrom et al., 1986, Cancer
Res. 46:3917-3923) was used as negative control for
the competition assay.
14.1.7. Generation of Monoclonal Antibodies to
HE~4
HER4, expressed in baculovirus, was used as the
immunogen for subcutaneous injection into 4-6 week old
female BALB/c mice. Immunization was performed 4
times (approximately 1 month apart) with 20 ~g of HER4
protein given each time. Spleen cells from immunized
mice were removed four days after the final
immunization and fused with the mouse myeloma line
P2x63-Ag8.653 as previously described (Siegall et al.,
supra ) . Positive hybridoma supernatants were selected
by ELISA screening on plates coated with HER4
transfected CH0 cells (Plowman et al., 1993, Nature
366:473-475) and selected against parental CHO cells
and human fibroblasts. Secondary screening was
performed by ELISA on plates coated with
baculovirus/HER4 membranes. Positive hybridomas were
rescreened by two additional rounds of ELISA using
CHO/HER4 and HER4 negative cells, and identified false
positive were removed. Positive hybridomas were
cloned in soft agar and tested for reactivity with the
HER4 positive MDA-MB-453 human breast carcinoma cell
line and CEM cells co-transfected with HER4 and HER2.
Anti-HER4 hybridoma line 6-4-11 (IgGl) was cloned in
soft agar and screened for reactivity to native and
denatured HER4. A second antibody (7-142, IgG2a) was
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also selected and found to bind to the cytoplasmic
domain of HER4.
The characteristics for both antibodies are
summarized in Table VI (see section 15.2.8., infra)
14.1.8. Quantitation of ~ER2, ~R3, and HER4
Protein in tumor cell line~
Cell-surface expression of HER2, HER3, and HER4
protein was determined by quantification of specific
antibody binding, detected by the CAS Red Chromagen
system (Becton Dickson Cellular Imaging System,
Elmhurst, IL). HER2 staining was performed by using
mouse anti-HER2 mAb 24.7 (Stancovski et al., l991,
Proc. Natl. Acad. Sci. USA 88:8691-8695) as primary,
and biotinylated goat anti-mouse IgG (Jackson Labs,
West Grove, PA) as secondary antibody as previously
described (Bacus et al., 1993, Cancer Res. 53:5251-
5261). For detection of HER3 and HER4 the primary
antibodies used were, respectively, mouse anti-HER3
mAb RTJ2 (Santa Cruz Biotech, Santa Cruz, CA) at 2.5
~g/ml concentration or mouse anti-HER4 mAb 6-4-11 at
15 ~g/ml concentration followed by incubation with
biotinylated rabbit anti-mouse IgG (Zymed Labs, South
San Francisco, CA).
The staining procedure was performed at RT as
follows: cells were fixed in 10% neutral buffered
formalin for 60 minutes, washed with H2O and rinsed
with Tris buffered saline (TBS; 0.05 M Tris, 0.15 M
NaCl, pH 7.6). Unspecific binding sites were blocked
by incubation with 10~ goat serum (for HER2) or rabbit
serum (for HER3 and HER4) in 0.1~ bovine serum
albumin/TBS for 15 minutes. Subsequently, cells were
incubated with primary and secondary antibodies for 30
and 20 minutes, respectively, followed by incubation
with alkaline phosphatase conjugated streptavidin
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(Jackson Labs) for 15 minutes, with TBS washing
between the steps. Detection of antibody binding was
achièved using CAS Red Chromagen (Becton Dickinson
Cellular Imaging System, supra) for 4 minutes (HER2),
~-lO minutes (HER3), and 10-12 minutes (HER4). Cells
were counterstained as described in the CAS DNA stain
protocol (Becton Dickinson Cellular Imaging System).
14.1.9. Image Analy~is
Image analysis was performed as previously
described (Bacus et al., 1993, supra ; Bacus et al .,
1992, Cancer Res. 52:2580-2589; Peles et al., 1992,
Cell 69:205-216). In the quantitation of HER2, both
solid state imaging channels of the CAS 200 Image
Analyzer (Becton Dickinson Cellular Imaging System), a
microscope-based, two-color system were used. The two
imaging channels were specifically matched to the two
components of the stains used. one channel was used
for quantitating the total DNA of the cells in the
field following Feulgen staining as described (Bacus
et al., 1990, Mol. Carcinoq. 3:350-362), and the other
for quantitating the level of HER2, HER3, and HER4
proteins following immunostaining. ~hen the total DNA
amount per cell was known, the average total HER2,
HER3, and HER4 per cell were computed. sparsely
growing AU565 cells were used for calibrating the HER2
protein. Their level of staining was defined as 100%
of HER2 protein content (1.0 relative amounts - 10,000
sum of optical density); all other measurementS of
HER2, HER3, and HE~4 protein were related to this
value.
14.1.10. Determination of the LD50 of HAR-TX ~2
For toxicity studies, HAR-TX ~2 at different
concentrations was administered intravenous in 0.2 ml
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PBS. Per group each two mice and two rats were
injected.
14.2. RE~TS
514.2.1. Con~tructiOn, ~xpr~ssion, and
Purification of HAR-TX ~2
The HAR-TX ~2 expression plasmid, encoding the
hydrophilic leader sequence from amphiregulin (AR),
heregulin ~2, and PE40, under control of the IPTG
inducible T7 promoter, was constructed as described in
Section 15.1.2., supra , and is diagrammatically shown
in FIG. 23, Panel 1. The AR leader se~uence was added
to the N-terminus of heregulin to facilitate the
purification procedure (FIG. 23, Panel 2). FIG. 24A
and 24B show the nucleotide sequence and the deduced
amino acid sequence of the cDNA encoding HAR-TX ~2
Chimeric HAR-TX ~2 protein was expressed in E.
col i of inclusion bodies. Recombinant protein was
denatured and refolded as described in Section
15.1.2., supra, and applied to cation-exchange
chromatography on a POROS HS column. Semi-purified
HAR-TX ~2 protein was detected by PAGE and Coomassie
blue staining as major band migrating at 51 kDa (FIG.
25, lane 2). The column flow-through from POROS HS
contained only small amounts of HA~-TX ~2 (FIG. 25,
lane 3). POROS HS chromatography resulted in ~50%
purity of HAR-TX ~2 (FIG. 25, lane 4). Further
purification, to >95~ purity, was done by
chromatography using Source 15S cation-exchange resin
(FIG. 25, lane 5). The monomeric nature of purified
-HAR-TX ~2 was determined by non-reducing SDS-PAGE
(FIG. 25, lane 6) which exhibited the same migration
pattern as under reducing conditions (FIG. 25, lane
5).
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14.2.2. ~inding of EAR-~X ~2 to MDA-MB-453
Cell Membrane~
To determine the specific binding activity of
HAR-TX ~2, an ELISA assay was performed using
membranes of the HER4 positive human breast carcinoma
cell line MDA-MB-4s3 as the target for binding. HAR-
TX ~2 was found to bind to the immobilized cell
membranes in a dose-dependent fashion up to 300 ~g/ml
(FIG. 26). PE40, the toxin component of HAR-TX ~2
used as negative control, was una~le to bind to MDA-
MB-453 membranes.
14.2.3. Tyrosine Phosphorylation of HER
Forms on Transfected CEM Cells
To test the biological activity of HAR-TX ~2 a
HER4 receptor phosphorylation assay was performed as
previously described for heregulin (Carraway et al.,
1994, J. Biol. Chem. 269:14303-14306). CEM cells
expressing different HER family mem~ers were exposed
to HAR-TX ~2 and stimulation of tyrosine
phosphorylation was analyzed by phosphotyrosine
immunoblot analysis (Section 4, supra; Section
15.1.5., supra) . As shown in FIG. 27, HAR-TX ~2
induced tyrosine phosphorylation in CEM cells
expressing HER4 either alone or together with HER2,
but not in cells expressing only HER2 or HERl. This
result demonstrates that HER4 is sufficient and
necessary for induction of tyrosine phosphorylation in
response to HAR-TX ~2, which is not true for HERl and
for HER2. The fact that HAR-TX ~2 does not induce
tyrosine phosphorylation in CEM cells transfected with
HERl confirms that the hydrophilic leader sequence of
amphiregulin does not affect the specificity of the
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heregulin moiety in its selective interaction between
receptor family members.
14.2.4. Cytotoxicity of HAR-TX ~2 Against
Tumor Cells
The cell killing activity of HAR-TX ~2 was
determined against a variety of human cancer cell
lines. AU56~ and SKBR3 breast carcinomas and LNCaP
prostate carcinoma were sensitive to HAR-TX ~2 with
1 ECso values of 25, 20, 4.5 ng/ml, respectively, while
SKOV3 ovarian carcinoma cells were insensitive to HAR-
TX ~2 (EC50 ~2000 ng/ml) (FIG. 28, Panel 1). Addition
of heregulin ~2-Ig to LNCaP cells reduced the
cytotoxic activity of HAR-TX ~2 (FIG. 28, Panel 2).
In contrast, L6-Ig, a chimeric mouse-human antibody
with a non-related specificity but matching human FC
domains (Hellstrom et al., supra), did not inhibit the
HAR-TX ~2 cytotoxic activity (FIG. 28, Panel 2).
Thus, the cytotoxic effect of HAR-TX ~2 was due to
specific heregulin-mediated binding. Similar data
were obtained using MDA-MB-453 cells (not shown).
14.2.5. E~2, ~ER3, and HER4 Receptor
Density on Human Tumor Cells:
2~ Correlation with ~AR-TX ~2-
Hediated cytotoxicity
To understand why cell lines differed in their
sensitivity to HAR-TX ~2, their levels of HER2, HER3,
and HER4 were quantitated by image analysis (see
Section 15.1.8. and 15.1.9., supra) using receptor
specific monoclonal antibodies (Table IV). The data
strongly indicate that HER4 expression is required for
heregulin directed cytotoxic activity. All seven of
the tumor cell lines which expressed detectable levels
of HER4 were found to be sensitive to HAR-TX ~2-
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mediated killing with ECso values ranging from 1-125
ng/ml. Moreover, the sensitivity of the différent
cell lines correlates directly with the expressiOn
level of HER4: MCF-7 cells displaying the lowest
detectable levels of HER4 were found to be the least
sensitive (ECso = 125 ng/ml) of the cells which did
respond. All four cell lines which were found to be
devoid of any detectable HER4 expression on their
surface were found to be resistant to HAR-TX ~2.
Three of them, SKOV3, L2987 and H3396, displayed both
HER2 and HER3 in the absence of HER4.
TABLE IV
Comparative ~ER2, HER3, and ~ER4 cell ~urface receptor
density and cytotoxicity of ~AR-TX ~2 on human tumor
cell lines
RELATIVE AMOUNTS
EC50 ~
Cell Line Type HER2 HER3 ~ER4 (nq/ml)
BT474 Breast 1.6 0.32 0.3
MDA-MD-453 Breast 1.2 1.08 0.3 2
25 LNCaP Prostate 0.7 2.6 0.85 4.5
T47D Breast 0.04 0.1 0.1 9.5
SKBR3 Breast 4.6 2.5 0.56 20
AU565 Breast 4.6 0.73 0.18 25
MCF-7 Breast 0.04 1.8 0.05 125
H3396 Breast 0.6 2.5 -- >2000
SKOV3 Ovarian 0.64 1.3 -- >2000
L2987 Lung 0.16 1.4 -- >2000
30 CEM T leukemia -- -- -- >2000
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14 . 2 . 6 . ~AR~ 2 Induces Tyrosi~e
Phosphorylation in Tumor Cells
That Do Not Express ~ER4
In contrast to reports that heregulin directly
binds to both HER3 and HER2/HER3 in a heterodimer
configuration (carraway et al., 1994, J. Biol. Chem.
269:14303-14306; Sliwkowski et al., 1994, J. Biol.
Chem. 269:14661-15665), tumor cells that express HER3
alone (L2987) or co-express HER2 and HER3 (H3396 and
SKOV3 ) were insensitive to HAR-TX ~2. Direct
interaction of H3396 and L2s87 cells with the chimeric
protein was determined by phosphotyrosine immunoblots
following HAR-TX ~2 induction. HAR-TX ~2 was found to
1~ induce tyrosine phosphorylation in both tumor cell
types (FIG. 29) similar to that previously seen in
COS-7 cells transfected with HER2 and HER3 (Sliwkowski
et al., supra). SROV3 cells were found to exhibit the
same tyrosine phosphorylation pattern in the presence
or absence of heregulin and thus direct interaction
between receptors and heregulin could not be
established (data not shown). However, previous
studies indicate that heregulin does not bind to these
cells (Peles et al., supra).
14.2.7. Toxicity of HAT-TX ~2
For the toxicity studies, HAR-TX ~2 was
administered as described in section 15.1.10. In
mice, 2/2 animals died at 2 mg/kg, 2/2 died at 1
mg/~g, 1/2 died at 0.75 mg/kg, and 0/2 died at 0.~
mg/kg, thus the LDso is about 0.75 mg/kg (Ta~le V). In
rats the determined LDs was slightly higher, as 50% of
the animals died at 1 mg/kg (Table V).
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TABLE V
Toxicity of HAR-TX ~2
8peciesdose[mg/ng] Lethality [%]
mouse 0.5 o
0.75 50
100
2 lOo
rat 1 50
2 loo
14.2.8. Characteristics of HER4 ~pecific
Monoclonal Antibodies
lS The characteristics of the HER4 specific
monoclonal antibodies disclosed herein are summarized
in Table VI.
TABLE VI
Characteristics of HER4 Antibodies
Abbreviations: Cyto, cytoplasmic domain;
ECD, extracellular domain; FACS, fluorescence-activated cell
sorter analysis; fibro, fibroblasts; ICC, immunocytochemistry;
RIP, receptor immunoprecipitation;
E~yorldo~I~oeyp~ R~P~--t-r~ ACS EI~R4Ig ~CC TCC
f ~ ~ro C~O/E14
EIAR2 I g
6-4-11 IgGl ~ - ECD
7-142 IgG2a - ~ Cyto - - - ~
15. Microorganism and Cell De~osit~
The following microorganisms and cell lines have been
deposited with the American Type Culture Collection,
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and have been assigned the following accession
numbers:
Microorqanism Plasmid Accession Number
- E.coli SCS-1 pBSHER4Y 69131
(con~aining the complete human HER4 coding sequence)
Cell Line Accession Number
CHO/HER4 21-2 C~L11205
Hybridoma Cell line 6-4-11 H~11715
Hybridoma Cell line 7-142 HB11716
The present invention is not ~o be limited in
scope by the microorganisms and cell lines deposited
or the embodiments disclosed herein, which are
intended as single illustrations of one aspect of the
invention, and any which are functionally equivalent
are within the scope of the invention. Indeed,
various modifications of the invention, in addition to
those shown and described herein, will become apparent
to those skilled in the art from the foregoing
description. Such modifications are intended to fall
within the scope of the appended claims. All base
pair and amino acid residue numbers and sizes given
for polynucleotides and polypeptides are approximate
and used for the purpose of description.
All publications and patent applications
mentioned in this specification are indicative of the
level of skill of those skilled in the art to which
the invention pertains. All publications and patent
applications are herein incorporated by reference to
the same extent as if each individual publication or
patent application was specifically and individually
indicated to be incorporated by reference.
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SEQUENCE LISTING
(1) GENERAL~INFORMATION:
(i) APPLICANTS: Plowman, Gregory D.
Culouscou, Jean-Michel
Shoyab, Mohammed
Siegall, Clay B.
Hellstrom, Ingegerd
Hellstrom, Karl E.
(ii) TITLE OF INVENTION: HER4 HUMAN RECEPTOR TYROS ~iE KINASE
(iii) NUMBER OF SEQUENCES: 42
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Pennie & Edmonds
(B) STREET: 1155 Avenue of the Americas
(C) CITY: New York
(D) STATE: New York
(E) COUNTRY: U.S.A.
(F) ZIP: 10036-2711
(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: To be assigned.
(B) FILING DATE: Concurrently herewith.
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/150,704
(B) FILING DATE: 10-NOV-1993
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Misrock, S. Leslie
(B) REGISTRATION NUMBER: 18,872
(C) REFERENCE/DOCKET NUMBER: 5624-230
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 790-9090
(B) TELEFAX: (212) 869-a864/974
(C) TELEX: 66141 PENNIE
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5501 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 34..3961
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(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AATTGTCAGC ACGGGATCTG AGACTTCCAA AAA ATG AAG CCG GCG ACA GGA CTT 54
-Met Lys Pro Ala Thr Gly Leu
-TGG GTC TGG GTG AGC CTT CTC GTG GCG GCG GGG ACC GTC CAG CCC AGC 102
~Trp Val Trp Val Ser Leu Leu Val Ala Ala Gly Thr Val Gln Pro Ser
= 10 15 20
GAT TCT CAG TCA GTG TGT GCA GGA ACG GAG AAT AAA CTG AGC TCT CTC 150
Asp Ser Gln Ser Val Cys Ala Gly Thr Glu Asn Lys Leu Ser Ser Leu
25 30 35
TCT GAC CTG GAA CAG CAG TAC CGA GCC TTG CGC AAG TAC TAT GAA AAC 198
Ser Asp Leu Glu Gln Gln Tyr Arg Ala Leu Arg Lys Tyr Tyr Glu Asn
40 45 50 55
TGT GAG GTT GTC ATG GGC AAC CTG GAG ATA ACC AGC ATT GAG CAC AAC 246
Cys Glu Val Val Met Gly Asn Leu Glu Ile Thr Ser Ile Glu His Asn
60 65 70
CGG GAC CTC TCC TTC CTG CGG TCT GTT CGA GAA GTC ACA GGC TAC GTG 294
Arg Asp Leu Ser Phe Leu Arg Ser Val Arg Glu Val Thr Gly Tyr Val
75 80 85
TTA GTG GCT CTT AAT CAG TTT CGT TAC CTG CCT CTG GAG AAT TTA CGC 342
Leu Val Ala Leu Asn Gln Phe Arg Tyr Leu Pro Leu Glu Asn Leu Arg
90 95 lOO
ATT ATT CGT GGG ACA AAA CTT TAT GAG GAT CGA TAT GCC TTG GCA ATA 390
Ile Ile Arg Gly Thr Lys Leu Tyr Glu Asp Arg Tyr Ala Leu Ala Ile
105 110 115
TTT TTA AAC TAC AGA AAA GAT GGA AAC TTT GGA CTT CAA GAA CTT GGA 438
Phe Leu Asn Tyr Arg Lys Asp Gly Asn Phe Gly Leu Gln Glu Leu Gly
120 125 130 135
TTA AAG AAC TTG ACA GAA ATC CTA AAT GGT GGA GTC TAT GTA GAC CAG 486
Leu Lys Asn Leu Thr Glu Ile Leu Asn Gly Gly Val Tyr Val Asp Gln
140 145 150
AAC AAA TTC CTT TGT TAT GCA GAC ACC ATT CAT TGG CAA GAT ATT GTT 534
Asn Lys Phe Leu Cys Tyr Ala Asp Thr Ile His Trp Gln Asp Ile Val
155 160 165
CGG AAC CCA TGG CCT TCC AAC TTG ACT CTT GTG TCA ACA AAT GGT AGT 582
Arg Asn Pro Trp Pro Ser Asn Leu Thr Leu Val Ser Thr Asn Gly Ser
170 175 180 185
TCA GGA TGT GGA CGT TGC CAT AAG TCC TGT ACT GGC CGT TGC TGG GGA 630
Ser Gly Cys Gly Arg Cys His Lys Ser Cys Thr Gly Arg Cys Trp Gly
l90 195
CCC ACA GAA AAT CAT TGC CAG ACT TTG ACA AGG ACG GTG TGT GCA GAA 678
Pro Thr Glu Asn His Cys Gln Thr Leu Thr Arg Thr Val Cys Ala Glu
200 . 205 210 215
CAA TGT GAC GGC AGA TGC TAC GGA CCT TAC GTC AGT GAC TGC TGC CAT 726
Gln Cys Asp Gly Arg Cys Tyr Gly Pro Tyr Val Ser Asp Cys Cys His
220 225 230
CGA GAA TGT GCT GGA GGC TGC TCA GGA CCT AAG GAC ACA GAC TGC TTT 774
Arg-Glu Cys Ala Gly Gly Cys Ser Gly Pro Lys Asp Thr Asp Cys Phe
235 240 245
SUBSTITUTE SHEET (RUl_E 26~
CA 02202~33 1997-04-11
W O96/12019 PCTrUS95/1352
- 112 -
GCC TGC ATG AAT TTC AAT GAC AGT GGA GCA TGT GTT ACT CAG TGT CCC 822
Ala Cys Met Asn Phe Asn Asp Ser Gly Ala Cys Val Thr Gln Cys Pro
250 255 260
CAA ACC TTT ~GTC TAC AAT CCA ACC ACC TTT CAA CTG GAG CAC AAT TTC 870
Gln Thr Phe Val Tyr Asn Pro Thr Thr Phe Gln Leu Glu His Asn Phe
265 270 275
AAT GCA AAG TAC ACA TAT GGA GCA TTC TGT GTC AAG AAA TGT CCA CAT 918
Asn Ala Lys Tyr Thr Tyr Gly Ala Phe Cys Val Lys Lys Cys Pro His
280 285 290 295
AAC TTT GTG GTA GAT TCC AGT TCT TGT GTG CGT GCC TGC CCT AGT TCC 966
Asn Phe Val Val Asp Ser Ser Ser Cys Val Arg Ala Cys Pro Ser Ser
300 305 310
AAG ATG GAA GTA GAA GAA AAT GGG ATT AAA ATG TGT AAA CCT TGC ACT 1014
Lys Met Glu Val Glu Glu Asn Gly Ile Lys Met Cys Lys Pro Cys Thr
315 320 325
GAC ATT TGC CCA AAA GCT TGT GAT GGC ATT GGC ACA GGA TCA TTG ATG 1062
Asp Ile Cys Pro Lys Ala Cys Asp Gly Ile Gly Thr Gly Ser Leu Met
330 335 340
TCA GCT CAG ACT GTG GAT TCC AGT AAC ATT GAC AAA TTC ATA AAC TGT 1110
Ser Ala Gln Thr Val Asp Ser Ser Asn Ile Asp Lys Phe Ile Asn Cys
345 350 355
ACC AAG ATC AAT GGG AAT TTG ATC TTT CTA GTC ACT GGT ATT CAT GGG 1158
Thr Lys Ile Asn Gly Asn Leu Ile Phe Leu Val Thr Gly Ile His Gly
365 370 375
GAC CCT TAC AAT GCA ATT GAA GCC ATA GAC CCA GAG AAA CTG AAC GTC 1206
Asp Pro Tyr Asn Ala Ile Glu Ala Ile Asp Pro Glu Lys Leu Asn Val
380 385 390
TTT CGG ACA GTC AGA GAG ATA ACA GGT TTC CTG AAC ATA CAG TCA TGG 1254
Phe Arg Thr Val Arg Glu Ile Thr Gly Phe Leu Asn Ile Gln Ser Trp
395 400 405
CCA CCA AAC ATG ACT GAC TTC AGT GTT TTT TCT AAC CTG GTG ACC ATT 1302
Pro Pro Asn Met Thr Asp Phe Ser Val Phe Ser Asn Leu Val Thr Ile
410 415 420
GGT GGA AGA GTA CTC TAT AGT GGC CTG TCC TTG CTT ATC CTC AAG CAA 1350
Gly Gly Arg Val Leu Tyr Ser Gly Leu Ser Leu Leu Ile Leu Lys Gln
425 430 435
CAG GGC ATC ACC TCT CTA CAG TTC CAG TCC CTG AAG GAA ATC AGC GCA 1398
Gln Gly Ile Thr Ser Leu Gln Phe Gln Ser Leu Lys Glu Ile Ser Ala
440 445 450 455
GGA AAC ATC TAT ATT ACT GAC AAC AGC AAC CTG TGT TAT TAT CAT ACC 1446
Gly Asn Ile Tyr Ile Thr Asp Asn Ser Asn Leu Cys Tyr Tyr His Thr
460 465 470
ATT AAC TGG ACA ACA CTC TTC AGC ACA ATC AAC CAG AGA ATA GTA ATC 1494
Ile Asn Trp Thr Thr Leu Phe Ser Thr Ile Asn Gln Arg Ile Val Ile
475 480 485
CGG GAC AAC AGA AAA GCT GAA AAT TGT ACT GCT GAA GGA ATG GTG TGC 1542
Arg Asp Asn Arg Lys Ala Glu Asn Cys Thr Ala Glu Gly Met Val Cys
495 500
AAC CAT CTG TGT TCC AGT GAT GGC TGT TGG GGA CCT GGG CCA GAC CAA 1590
Asn His Leu Cys Ser Ser Asp Gly Cys Trp Gly Pro Gly Pro Asp Gln
505 510 515
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
W O 9~12019
PCT/US95/13~2.1
- 113 -
TGT CTG TCG TGT CGC CGC TTC AGT AGA GGA AGG ATC TGC ATA GAG TCT 1638
Cys Leu Ser Cys Arg Arg Phe Ser Arg Gly Arg Ile Cys Ile Glu Ser
520 525 530 535
TGT AAC CTC TAT GAT GGT GAA TTT CGG GAG TTT GAG AAT GGC TCC ATC 1686
- Cys Asn Leu Tyr Asp Gly Glu Phe Arg Glu Phe Glu Asn Gly Ser Ile
540 S45 550
TGT GTG GAG TGT GAC CCC CAG TGT GAG AAG ATG GAA GAT GGC CTC CTC 1734
Cys Val Glu Cys Asp Pro Gln Cys Glu Lys Met Glu Asp Gly Leu Leu
555 560 565
ACA TGC CAT GGA CCG GGT CCT GAC AAC TGT ACA AAG TGC TCT CAT TTT 1782
Thr Cys His Gly Pro Gly Pro Asp Asn Cys Thr Lys Cys Ser His Phe
570 575 580
AAA GAT GGC CCA AAC TGT GTG GAA AAA TGT CCA GAT GGC TTA CAG GGG 1830
Lys Asp Gly Pro Asn Cys Val Glu Lys Cys Pro Asp Gly Leu Gln Gly
585 590 595
GCA AAC AGT TTC ATT TTC AAG TAT GCT GAT CCA GAT CGG GAG TGC CAC 1878
Ala Asn Ser Phe Ile Phe Lys Tyr Ala Asp Pro Asp Arg Glu Cys Xis
600 605 610 615
CCA TGC CAT CCA AAC TGC ACC CAA GGG TGT AAC GGT CCC ACT AGT CAT 1926
Pro Cys His Pro Asn Cys Thr Gln Gly Cys Asn Gly Pro Thr Ser ~is
620 625 630
GAC TGC ATT TAC TAC CCA TGG ACG GGC CAT TCC ACT TTA CCA CAA CAT 1974
Asp Cys Ile Tyr Tyr Pro Trp Thr Gly His Ser Thr Leu Pro Gln His
635 640 645
GCT AGA ACT CCC CTG ATT GCA GCT GGA GTA ATT GGT GGG CTC TTC ATT 2022
Ala Arg Thr Pro Leu Ile Ala Ala Gly Val Ile Gly Gly Leu Phe Ile
650 655 660
CTG GTC ATT GTG GGT CTG ACA TTT GCT GTT TAT GTT AGA AGG AAG AGC 2070
Leu Val Ile Val Gly Leu Thr Phe Ala Val Tyr Val Arg Arg Lys Ser
665 670 675
ATC AAA AAG AAA AGA GCC TTG AGA AGA TTC TTG GAA ACA GAG TTG GTG 2118
Ile Lys Lys Lys Arg Ala Leu Arg Arg Phe Leu Glu Thr Glu Leu Val
630 685 690 695
GAA CCA TTA ACT CCC AGT GGC ACA GCA CCC AAT CAA GCT CAA CTT CGT 2166
Glu Pro Leu Thr Pro Ser Gly Thr Ala Pro Asn Gln Ala Gln Leu Arg
700 705 710
ATT TTG AAA GAA ACT GAG CTG AAG AGG GTA AAA GTC CTT GGC TCA GGT 2214
Ile Leu Lys Glu Thr Glu Leu Lys Arg Val Lys Val Leu Gly Ser Gly
715 720 725
GCT TTT GGA ACG GTT TAT AAA GGT ATT TGG GTA CCT GAA GGA GAA ACT 2262
Ala Phe Gly Thr Val Tyr Lys Gly Ile Trp Val Pro Glu Gly Glu Thr
730 735 740
GTG AAG ATT CCT GTG GCT ATT AAG ATT CTT AAT GAG ACA ACT GGT CCC 2310
Val Lys Ile Pro Val Ala Ile Lys Ile Leu Asn Glu Thr Thr Gly Pro
; 745 750 755
AAG GCA AAT GTG GAG TTC ATG GAT GAA GCT CTG ATC ATG GCA AGT ATG 23S8
Lys Ala Asn Val Glu Phe Met Asp Glu Ala Leu Ile Met Ala Ser Met
765 770 775
GAT CAT CCA CAC CTA GTC CGG TTG CTG GGT GTG TGT CTG AGC CCA ACC 2406
Asp His Pro His Leu Val Arg Leu Leu Gly Val Cys Leu Ser Pro Thr
780 785 790
SUBSTITUTE SHEET (RULE 26~
_ . . _ . .
CA 02202~33 1997-04-11
W O96/12019 PCTrUS95/13~2
- 114 -
ATC CAG CTG GTT ACT CAA CTT ATG CCC CAT GGC TGC CTG TTG GAG TAT 2454
Ile Gln Leu Val Thr Gln Leu Met Pro His Gly Cys Leu Leu Glu Tyr
795 800 805
GTC CAC GAG~CAC AAG GAT AAC ATT GGA TCA CAA CTG CTG CTT AAC TGG 2502
Val His Glu His Lys Asp Asn Ile Gly Ser Gln Leu Leu Leu Asn Trp
810 815 820
TGT GTC CAG ATA GCT AAG GGA ATG ATG TAC CTG GAA GAA AGA CGA CTC 2550
Cys Val Gln Ile Ala Lys Gly Met Met Tyr Leu Glu Glu Arg Arg Leu
825 830 835
GTT CAT CGG GAT TTG GCA GCC CGT AAT GTC TTA GTG AAA TCT CCA AAC 2598
Val ~is Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn
840 845 850 855
CAT GTG AAA ATC ACA GAT TTT GGG CTA GCC AGA CTC TTG GAA GGA GAT 2646
His Val Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu Glu Gly Asp
860 865 870
G~A AAA GAG TAC AAT GCT GAT GGA GGA AAG ATG CCA ATT AAA TGG ATG 2694
Glu Lys Glu Tyr Asn Ala Asp Gly Gly Lys Met Pro Ile Lys Trp Met
875 880 885
GCT CTG GAG TGT ATA CAT TAC AGG AAA TTC ACC CAT CAG AGT GAC GTT 2742
Ala Leu Glu Cys Ile His Tyr Arg Lys Phe Thr His Gln Ser Asp Val
890 895 9oO
TGG AGC TAT GGA GTT ACT ATA TGG GAA CTG ATG ACC TTT GGA GGA AAA 2790
Trp Ser Tyr Gly Val Thr Ile Trp Glu Leu Met Thr Phe Gly Gly Lys
905 910 915
CCC TAT GAT GGA ATT CCA ACG CGA GAA ATC CCT GAT TTA TTA GAG AAA 2838
Pro Tyr Asp Gly Ile Pro Thr Arg Glu Ile Pro Asp Leu Leu Glu Lys
920 925 930 935
GGA GAA CGT TTG CCT CAG CCT CCC ATC TGC ACT ATT GAC GTT TAC ATG 2886
Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met
940 945 950
GTC ATG GTC AAA TGT TGG ATG ATT GAT GCT GAC AGT AGA CCT AAA TTT 2934
Val Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys Phe
955 960 965
AAG GAA CTG GCT GCT GAG TTT TCA AGG ATG GCT CGA GAC CCT CAA AGA 2982
Lys Glu Leu Ala Ala Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg
970 975 980
TAC CTA GTT ATT CAG GGT GAT GAT CGT ATG AAG CTT CCC AGT CCA AAT 3030
Tyr Leu Val Ile Gln Gly Asp Asp Arg Met Lys Leu Pro Ser Pro Asn
985 990 995
GAC AGC AAG TTC TTT CAG AAT CTC TTG GAT GAA GAG GAT TTG GAA GAT 3078
Asp Ser Lys Phe Phe Gln Asn Leu Leu Asp Glu Glu Asp Leu Glu Asp
1000 1005 1010 1015
ATG ATG GAT GCT GAG GAG TAC TTG GTC CCT CAG GCT TTC AAC ATC CCA 3126
Met Met Asp Ala Glu Glu Tyr Leu Val Pro Gln Ala Phe Asn Ile Pro
1020 1025 1030
CCT CCC ATC TAT ACT TCC AGA GCA AGA ATT GAC TCG AAT AGG AGT GAA 3174
Pro Pro Ile Tyr Thr Ser Ary Ala Arg Ile Asp Ser Asn Arg Ser Glu
1035 1040 1045
ATT GGA CAC AGC CCT CCT CCT GCC TAC ACC CCC ATG TCA GGA AAC CAG 3222
Ile Gly His Ser Pro Pro Pro Ala Tyr Thr Pro Met Ser Gly Asn Gln
1055 1060
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
WO 96/12019 PCTNS95l13~2
- 115 -
TTT GTA TAC CGA GAT GGA GGT TTT GCT GCT GAA CAA GGA GTG TCT GTG 3270
Phe Val Tyr Arg Asp Gly Gly Phe Ala Ala GlU Gln Gly Val Ser Val
1070 1075
CCC TAC AGA~GCC CCA ACT AGC ACA ATT CCA GAA GCT CCT GTG GCA CAG 3318
Pro Tyr Arg Ala Pro Thr Ser Thr Ile Pro Glu Ala Pro Val Ala Gln
1080 1085 1090 1095
GGT GCT ACT GCT GAG ATT TTT GAT GAC TCC TGC TGT AAT GGC ACC CTA 3366
Gly Ala Thr Ala Glu Ile Phe Asp Asp Ser Cys Cys Asn Gly Thr Leu
1100 1105 1110
CGC AAG CCA GTG GCA CCC CAT GTC CAA GAG GAC AGT AGC ACC CAG AGG 3414
Arg Lys Pro Val Ala Pro His Val Gln Glu Asp Ser Ser Thr Gln Arg
1115 1120 1125
TAC AGT GCT GAC CCC ACC GTG TTT GCC CCA GAA CGG AGC CCA CGA GGA 3462
Tyr Ser Ala Asp Pro Thr Val Phe Ala Pro Glu Arg Ser Pro Arg Gly
1135 1140
GAG CTG GAT GAG GAA GGT TAC ATG ACT CCT ATG CGA GAC AAA CCC AAA 3510
Glu Leu Asp Glu Glu Gly Tyr Met Thr Pro Met Arg Asp Lys Pro Lys
1150 1155
CAA GAA TAC CTG AAT CCA GTG GAG GAG AAC CCT TTT GTT TCT CGG AGA 3558
Gln Glu Tyr Leu Asn Pro Val Glu Glu Asn Pro Phe Val Ser Arg Arg
1165 1170 1175
AAA AAT GGA GAC CTT CAA GCA TTG GAT AAT CCC GAA TAT CAC AAT GCA 3606
Lys Asn Gly Asp Leu Gln Ala Leu Asp Asn Pro Glu Tyr His Asn Ala
1180 1185 1190
TCC AAT GGT CCA CCC AAG GCC GAG GAT GAG TAT GTG AAT GAG CCA CTG 3654
Ser Asn Gly Pro Pro Lys Ala Glu Asp Glu Tyr Val Asn Glu Pro Leu
1195 1200 1205
TAC CTC AAC ACC TTT GCC AAC ACC TTG GGA AAA GCT GAG TAC CTG AAG 3702
Tyr Leu Asn Thr Phe Ala Asn Thr Leu Gly Lys Ala Glu Tyr Leu Lys
1215 1220
AAC AAC ATA CTG TCA ATG CCA GAG AAG GCC AAG AAA GCG TTT GAC AAC 3750
Asn Asn Ile Leu Ser Met Pro Glu Lys Ala Lys Lys Ala Phe Asp Asn
123 0 123 5
CCT GAC TAC TGG AAC CAC AGC CTG CCA CCT CGG AGC ACC CTT CAG CAC 3798
Pro Asp Tyr Trp Asn His Ser Leu Pro Pro Arg Ser Thr Leu Gln His
1245 1250 1255
CCA GAC TAC CTG CAG GAG TAC AGC ACA AAA TAT TTT TAT AAA CAG AAT 3846
Pro Asp Tyr Leu Gln Glu Tyr Ser Thr Lys Tyr Phe Tyr Lys Gln Asn
1260 1265 1270
GGG CGG ATC CGG CCT ATT GTG GCA GAG AAT CCT GAA TAC CTC TCT GAG 38 94
Gly Arg Ile Arg Pro Ile Val Ala Glu Asn Pro Glu Tyr Leu Ser Glu
1275 1280 1285
TTC TCC CTG AAG CCA GGC ACT GTG CTG CCG CCT CCA CCT TAC AGA CAC 3942
Phe Ser Leu Lys Pro Gly Thr Val Leu Pro Pro Pro Pro Tyr Arg His
1295 1300
- CGG AAT ACT GTG GTG TAAGCTCAGT TGTGGTTTTT TAGGTGGAGA GACACACCTG 3 9 9 7
Arg Asn Thr Val Val
CTCCAATTTC CCCACCCCCC T~ lllC--~C TGGTGGTCTT CCTTCTACCC CAAGGCCAGT 4 0 5 7
A~llllGACA CTTCCCAGTG GAAGATACAG AGATGCAATG ATAGTTATGT GCTTACCTAA 4117
SUBSTITUTE SHEET (RULE 26~
.. . . . . .. . . ..
CA 02202~33 1997-04-11
WO96/12019 PCT~US95/1352
- 116 -
CTTGAACATT AGAGGGAAAG ACTGAAAGAG AAAGATAGGA GGAACCACAA lvlll~llCA 4177
TTTCTCTGCA TGGvllv~lC AGGAGAATGA AACAGCTAGA GAAGGACCAG AAAATGTAAG 4237
GCAATGCTGC CTACTATCAA ACTAGCTGTC A~lllllllC 11111~1111 TCTTTCTTTG 4297
lllClllCll Ccl-ll-lll 1lllllllll TTTTAAAGCA GATGGTTGAA ACACCCATGC 4357
TAl~lv L' 1 CC TATCTGCAGG AACTGATGTG TGCATATTTA GCATCCCTGG AAATCATAAT 4417
AAAGTTTCCA TTAGAACAAA AGAATAACAT TTTCTATAAC ATATGATAGT GTCTGAAAT. 4477
GAGAATCCAG TTTCTTTCCC CAGCAGTTTC TGTCCTAGCA AGTAAGAATG GCCAACTCAA 4537
CTTTCATAAT TTAAAAATCT CCATTAAAGT TATAACTAGT AATTATGTTT TCAACACTTT 4597
TTGGillllll TCAllllvll TTGCTCTGAC CGATTCCTTT ATATTTGCTC CCCTATTTTT 4657
GGCTTTAATT TCTAATTGCA AAGATGTTTA CATCAAAGCT TCTTCACAGA ATTTAAGCAA 4717
GAAATATTTT AATATAGTGA AATGGCCACT ACTTTAAGTA TACAATCTTT AAAATAAGAA 4777
AGGGAGGCTA ATATTTTTCA TGCTATCAAA TTATCTTCAC CCTCATCCTT TACATTTTTC 4837
AACATTTTTT TTTCTCCATA AATGACACTA CTTGATAGGC CGTTGGTTGT CTGAAGAGTA 4897
GAAGGGAAAC TAAGAGACAG TTCTCTGTGG TTCAGGAAAA CTACTGATAC TTTCAGGGGT 4957
GGCCCAATGA GGGAATCCAT TGAACTGGAA GAAACACACT GGATTGGGTA TGTCTACCTG 5017
GCAGATACTC AGAAATGTAG TTTGCACTTA AGCTGTAATT TTAlllvllC lllll~lGAA 5077
CTCCATTTTG GATTTTGAAT CAAGCAATAT GGAAGCAACC AGCAAATTAA CTAATTTAAG 5137
TACATTTTTA AAAAAAGAGC TAAGATAAAG ACTGTGGAAA TGCCAAACCA AGCAAATTAG 5197
GAACCTTGCA ACGGTATCCA GGGACTATGA TGAGAGGCCA GCACATTATC TTCATATGTC 5257
ACCTTTGCTA CGCAAGGAAA TTTGTTCAGT TCGTATACTT CGTAAGAAGG AATGCGAGTA 5317
AGGATTGGCT TGAATTCCAT GGAATTTCTA GTATGAGACT ATTTATATGA AGTAGAAGGT 5377
AACTCTTTGC ACATAAATTG GTATAATAAA AAGAAAAACA CAAACATTCA AAGCTTAGGG 5437
ATAGGTCCTT GGGTCAAAAG TTGTAAATAA ATGTGAAACA TCTTCTCAAA AAAAAAAAAA 5497
AAAA 5501
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 130~3 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) ~OLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Lys Pro Ala Thr Gly Leu Trp Val Trp Val Ser Leu Leu Val Ala
1 5 10 15
Ala Gly Thr Val Gln Pro Ser Asp Ser Gln Ser Val Cys Ala Gly Thr
Glu Asn Lys Leu Ser Ser Leu Ser Asp Leu Glu Gln Gln Tyr Arg Ala
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
WO g6/1201g ~ uS95l~352
- 117 -
Leu Arg Lys Tyr Tyr Glu Asn Cys Glu Val Val Met Gly Asn Leu Glu
,, 50 sS 60
Ile Thr Ser Ile Glu His Asn Arg Asp Leu Ser Phe Leu Arg Ser Val
Arg Glu Val Thr Gly Tyr Val Leu Val Ala Leu Asn Gln Phe Arg Tyr
85 gO 95
Leu Pro Leu Glu Asn Leu Arg Ile Ile Ary Gly Thr Lys Leu Tyr Glu
100 105 110
Asp Arg Tyr Ala Leu Ala Ile Phe Leu Asn Tyr Arg Lys Asp Gly Asn
115 120 125
Phe Gly Leu Gln Glu Leu Gly Leu Lys Asn Leu Thr Glu Ile Leu Asn
130 135 140
Gly Gly Val Tyr Val Asp Gln Asn Lys Phe Leu Cys Tyr Ala Asp Thr
145 150 155 160
Ile His Trp Gln Asp Ile Val Arg Asn Pro Tr~ Pro Ser Asn Leu Thr
165 170 175
Leu Val Ser Thr Asn Gly Ser Ser Gly Cys Gly Arg Cys His Lys Ser
180 185 190
Cys Thr Gly Arg Cys Trp Gly Pro Thr Glu Asn His Cys Gln Thr Leu
195 200 205
Thr Arg Thr Val Cys Ala Glu Gln Cys Asp Gly Arg Cys Tyr Gly Pro
210 215 220
Tyr Val Ser Asp Cys Cys His Arg Glu Cys Ala Gly Gly Cys Ser Gly
225 230 235 240
Pro Lys Asp Thr Asp Cys Phe Ala Cys Met Asn Phe Asn Asp Ser Gly
245 250 255
Ala Cys Val Thr Gln Cys Pro Gln Thr Dhe Val Tyr Asn Pro Thr Thr
260 265 270
Phe Gln Leu Glu His Asn Phe Asn Ala Lys Tyr Thr Tyr Gly Ala Phe
Z75 280 285
Cys Val Lys Lys Cys Pro His Asn Phe Val Val Asp Ser Ser Ser Cys
290 295 300
Val Arg Ala Cys Pro Ser Ser Lys Met Glu Val Glu Glu Asn Gly Ile
305 310 315 320
Lys Met Cys Lys Pro Cys Thr Asp Ile Cys Pro Lys Ala Cys Asp Gly
325 330 335
Ile Gly Thr Gly Ser Leu Met Ser Ala Gln Thr Val Asp Ser Ser Asn
340 345 350
Ile Asp Lys Phe Ile Asn Cys Thr Lys Ile Asn Gly Asn Leu Ile Phe
355 360 365
Leu Val Thr Gly Ile His Gly Asp Pro Tyr Asn Ala Ile Glu Ala Ile
370 375 380
Asp Pro Glu Lys Leu Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly
385 390 395 400
SUSSTlTlJTE SHEET (RULE ~6~
CA 02202~33 1997-04-11
WO 96/12019 PCI~/US9~/1352-1
- 118 -
Phe Leu Asn Ile Gln Ser Trp Pro Pro Asn Met Thr Asp Phe Ser Val
405 410 ~15
he Ser Asn Leu Val Thr Ile Gly Gly Arg Val Leu Tyr Ser Gly Leu
420 425 430
Ser Leu Leu Ile Leu Lys Gln Gln Gly Ile Thr Ser Leu Gln Phe Gln
435 440 445
Ser Leu Lys Glu Ile Ser Ala Gly Asn Ile Tyr Ile Thr Asp Asn Ser
450 455 460
Asn Leu Cys Tyr Tyr His Thr Ile Asn Trp Thr Thr Leu Phe Ser Thr
465 470 475 480
le Asn Gln Arg Ile Val Ile Arg Asp Asn Arg Lys Ala Glu Asn Cys
485 490 495
hr Ala Glu Gly Met Val Cys Asn His Leu Cys Ser Ser Asp Gly Cys
500 505 510
Trp Gly Pro Gly Pro Asp Gln Cys Leu Ser Cys Arg Arg Phe Ser Arg
515 520 525
Gly Arg Ile Cys Ile Glu Ser Cys Asn Leu Tyr Asp Gly Glu Phe Arg
530 535 540
Glu Phe Glu Asn Gly Ser Ile Cys Val Glu Cys Asp Pro Gln Cys Glu
545 550 555 560
vs Met Glu Asp Gly Leu Leu Thr Cys His Gly Pro Gly Pro Asp Asn
565 570 575
ys Thr Lys Cys Ser His Phe Lys Asp Gly Pro Asn Cys Val Glu Lys
580 585 590
Cys Pro Asp Gly Leu Gln Gly Ala Asn Ser Phe Ile Phe Lys Tyr Ala
595 600 605
Asp Pro Asp Arg Glu Cys His Pro Cys His Pro Asn Cys Thr Gln Gly
610 615 620
Cys Asn Gly Pro Thr Ser His Asp Cys Ile Tyr Tyr Pro Trp Thr Gly
625 630 635 640
is Ser Thr Leu Pro Gln His Ala Ary Thr Pro Leu Ile Ala Ala Gly
645 650 655
al Ile Gly Gly Leu Phe Ile Leu Val Ile Val Gly Leu Thr Phe Ala
660 665 670
Val Tyr Val Arg Arg Lys Ser Ile Lys Lys Lys Arg Ala Leu Arg Arg
675 680 685
Phe Leu Glu Thr Glu Leu Val Glu Pro Leu Thr Pro ser Gly Thr Ala
690 695 700
Pro Asn Gln Ala Gln Leu Arg Ile Leu Lys Glu Thr G1U Leu Lys Arg
705 710 715 720
al Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Ile
725 730 735
rp Val Pro Glu Gly Glu Thr Val Lys Ile Pro Val Ala Ile Lys Ile
740 745 750
eu Asn Glu Thr Thr Gly Pro Lys Ala Asn Val Glu Phe Met Asp Glu
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
W O 96/12019 PCTnUS9Sl1352l
- 119 -
755 760 765
Ala Leu Ile Met Ala Ser Met Asp His Pro His Leu Val Arg Leu Leu
0 775 780
Gly Val Cys Leu Ser Pro Thr Ile Gln Leu Val Thr Gln Leu Met Pro
7~5 790 795 800
is Gly Cys Leu Leu Glu Tyr Val His Glu His Lys Asp Asn Ile Gly
805 810 815
er Gln Leu Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Met
820 825 830
Tyr Leu Glu Glu Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn
a35 840 845
Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu
850 855 860
Ala Arg Leu Leu Glu Gly Asp Glu Lys Glu Tyr Asn Ala Asp Gly Gly
865 870 875 880
ys Met Pro Ile Lys Trp Met Ala Leu Glu Cys Ile His Tyr Arg Lys
885 890 895
he Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Ile Trp Glu
900 905 910
Leu Met Thr Phe Gly Gly Lys Pro Tyr Asp Gly Ile Pro Thr Arg Glu
915 920 925
Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile
930 935 940
Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys Trp Met Ile Asp
945 950 955 960
la Asp Ser Arg Pro Lys Phe Lys Glu Leu Ala Ala Glu Phe Ser Arg
965 970 975
et Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Asp Arg
980 985 990
Met Lys Leu Pro Ser Pro Asn Asp Ser Lys Phe Phe Gln Asn Leu Leu
995 1000 1005
Asp Glu Glu Asp Leu Glu Asp Met Met Asp Ala Glu Glu Tyr Leu Val
1010 1015 1020
Pro Gln Ala Phe Asn Ile Pro Pro Pro Ile Tyr Thr Ser Arg Ala Arg
1025 1030 1035 1040
le Asp Ser Asn Arg Ser Glu Ile Gly His Ser Pro Pro Pro Ala Tyr
1045 1050 1055
hr Pro Met Ser Gly Asn Gln Phe Val Tyr Arg Asp Gly Gly Phe Ala
1060 1065 1070
Ala Glu Gln Gly Val Ser Val Pro Tyr Arg Ala Pro Thr Ser Thr Ile
1075 1080 1085
Pro Glu Ala Pro Val Ala Gln Gly Ala Thr Ala Glu Ile Phe Asp Asp
1090 1095 1100
Ser Cys Cys Asn Gly Thr Leu Arg Lys Pro Val Ala Pro His Val Gln
1105 1110 1115 1120
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 l997-04-ll
WO 96/12019 PCTrUS9S/135Z~
- 120 -
Glu Asp Ser Ser Thr Gln Arg Tyr Ser Ala Asp Pro Thr Val Phe Ala
1125 1130 1135
ro Glu Arg Ser Pro Arg Gly Glu Leu Asp Glu Glu Gly Tyr Met Thr
1140 1145 1150
Pro Met Arg Asp ~ys Pro Lys Gln Glu Tyr Leu Asn Pro Val Glu Glu
1155 1160 1165
Asn Pro Phe Val Ser Arg Arg Lys Asn Gly Asp Leu Gln Ala Leu Asp
1170 1175 1180
Asn Pro Glu Tyr His Asn Ala Ser Asn Gly Pro Pro Lys Ala Glu Asp
1185 1190 1195 1200
lu Tyr Val Asn Glu Pro Leu Tyr Leu Asn Thr Phe Ala Asn Thr Leu
1205 1210 1215
ly Lys Ala Glu Tyr Leu Lys Asn Asn Ile Leu Ser Met Pro Glu Lys
1220 1225 1230
Aia Lys Lys Ala Phe Asp Asn Pro Asp Tyr Trp Asn His Ser Leu Pro
1235 1240 1245
Pro Arg Ser Thr Leu Gln His Pro Asp Tyr Leu Gln Glu Tyr Ser Thr
1250 1255 1260
Lys Tyr Phe Tyr Lys Gln Asn Gly Arg Ile Arg Pro Ile Val Ala Glu
1265 1270 1275 1280
Asn Pro Glu Tyr Leu Ser Glu Phe Ser Leu Lys Pro Gly Thr Val Leu
1285 1290 1295
Pro Pro Pro Pro Tyr Arg His Arg Asn Thr Val Val
1300 1305
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5555 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: un~nown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(}3) LOCATION: 34..3210
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AATTGTCAGC ACGGGATCTG AGACTTCCAA AAA ATG AAG CCG GCG ACA GGA CTT 5
Met Lys Pro Ala Thr Gly Leu
1 5
TGG GTC TGG GTG AGC CTT CTC GTG GCG GCG GGG ACC GTC CAG CCC AGC 102
Trp Val Trp Val Ser Leu Leu Val Ala Ala Gly Thr Val Gln Pro Ser
15 20
GAT TCT CAG TCA GTG TGT GCA GGA ACG GAG AAT AAA CTG AGC TCT CTC 150
Asp Ser Gln Ser Val Cys Ala Gly Thr Glu Asn Lys Leu Ser Ser Leu
30 35
SUBSTITllTE SHEET (RULE 26~
CA 02202~33 1997-04-11
O 96/12019 PCT~US9511352
- 121 -
TCT GAC CTG GAA CAG CAG TAC CGA GCC TTG CGC AAG TAC TAT GAA AAC 198
Ser Asp Leu Glu Gln Gln Tyr Arg Ala Leu Arg Lys Tyr Tyr Glu Asn
45 S0 55
TGT GAG GTT~ GTC ATG GGC AAC CTG GAG ATA ACC AGC ATT GAG CAC AAC 246
Cys Glu Val Val Met Gly Asn Leu Glu Ile Thr Ser Ile Glu His Asn
60 65 70
CGG GAC CTC TCC TTC CTG CGG TCT GTT CGA GAA GTC ACA GGC TAC GTG 294
Arg Asp Leu Ser Phe Leu Arg Ser Val Arg Glu Val Thr Gly Tyr Va
75 80 85
TTA GTG GCT CTT AAT CAG TTT CGT TAC CTG CCT CTG GAG AAT TTA CGC 342
Leu Val Ala Leu Asn Gln Phe Arg Tyr Leu Pro Leu Glu Asn Leu Arg
95 100
ATT ATT CGT GGG ACA AAA CTT TAT GAG GAT CGA TAT GCC TTG GCA ATA 390
Ile Ile Arg Gly Thr Lys Leu Tyr Glu Asp Arg Tyr Ala Leu Ala Ile
110 115
TTT TTA AAC TAC AGA AAA GAT GGA AAC TTT GGA CTT CAA GAA CTT GGA 438
Phe Leu Asn Tyr Arg Lys Asp Gly Asn Phe Gly Leu Gln Glu Leu Gly
125 130 135
TTA AAG AAC TTG ACA GAA ATC CTA AAT GGT GGA GTC TAT GTA GAC CAG 486
Leu Lys Asn Leu Thr Glu Ile Leu Asn Gly Gly Val Tyr Val Asp Gln
140 145 150
AAC AAA TTC CTT TGT TAT GCA GAC ACC ATT CAT TGG CAA GAT ATT GTT 534
Asn Lys Phe Leu Cys Tyr Ala Asp Thr Ile His Trp Gln Asp Ile Val
155 160 165
CGG AAC CCA TGG CCT TCC AAC TTG ACT CTT GTG TCA ACA AAT GGT AGT 582
Arg Asn Pro Trp Pro Ser Asn Leu Thr Leu Val Ser Thr Asn Gly Ser
175 180
TCA GGA TGT GGA CGT TGC CAT AAG TCC TGT ACT GGC CGT TGC TGG GGA 630
Ser Gly Cys Gly Arg Cys His Lys Ser Cys Thr Gly Arg Cys Trp Gly
190 195
CCC ACA GAA AAT CAT TGC CAG ACT TTG ACA AGG ACG GTG TGT GCA GAA 678
Pro Thr Glu Asn His Cys Gln Thr Leu Thr Arg Thr Val Cys Ala Glu
20S 210 215
CAA TGT GAC GGC AGA TGC TAC GGA CCT TAC GTC AGT GAC TGC TGC CAT 726
Gln Cys Asp Gly Arg Cys Tyr Gly Pro Tyr Val Ser Asp Cys Cys His
220 225 230
CGA GAA TGT GCT GGA GGC TGC TCA GGA CCT AAG GAC ACA GAC TGC TTT 774
Arg Glu Cys Ala Gly Gly Cys Ser Gly Pro Lys Asp Thr Asp Cys Phe
235 240 245
GCC TGC ATG AAT TTC AAT GAC AGT GGA GCA TGT GTT ACT CAG TGT CCC 822
Ala Cys Met Asn Phe Asn Asp Ser Gly Ala Cys Val Thr Gln Cys Pro
255 260
CAA ACC TTT GTC TAC AAT CCA ACC ACC TTT CAA CTG GAG CAC AAT TTC 870
Gln Thr Phe Val Tyr Asn Pro Thr Thr Phe Gln Leu Glu Hls Asn Phe
270 275
AAT GCA AAG TAC ACA TAT GGA GCA TTC TGT GTC AAG AAA TGT CCA CAT 918
Asn Ala Lys Tyr Thr Tyr Gly Ala Phe Cys Val Lys ~ys Cys Pro His
285 290 295
AAC TTT GTG GTA GAT TCC AGT TCT TGT GTG CGT GCC TGC CCT AGT TCC 966
Asn Phe Val Val Asp Ser Ser Ser Cys Val Arg Ala Cys Pro Ser Ser
300 305 310
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-ll
WO96/12019 PCTrUS95/1352
- 122 -
AAG ATG GAA GTA GAA GAA AAT GGG ATT AAA ATG TGT AAA CCT TGC ACT 1014
Lys Met Glu Val Glu Glu Asn Gly Ile Lys Met Cys Lys Pro Cys Thr
315 320 325
GAC ATT TGC CCA AAA GCT TGT GAT GGC ATT GGC ACA GGA TCA TTG ATG 1062
Asp Ile Cys Pro Lys Ala Cys Asp Gly Ile Gly Thr Gly Ser Leu Met
335 340
TCA GCT CAG ACT GTG GAT TCC AGT AAC ATT GAC AAA TTC ATA AAC TGT 1110
Ser Ala Gln Thr Val Asp Ser Ser Asn Ile Asp Lys Phe Ile Asn Cys
350 355
ACC AAG ATC AAT GGG AAT TTG ATC TTT CTA GTC ACT GGT ATT CAT GGG 1158
Thr Lys Ile Asn Gly Asn Leu Ile Phe Leu Val Thr Gly Ile His Gly
365 370 375
GAC CCT TAC AAT GCA ATT GAA GCC ATA GAC CCA GAG AAA CTG AAC GTC 1206
Asp Pro Tyr Asn Ala Ile Glu Ala Ile Asp Pro Glu Lys Leu Asn Val
380 385 390
TTT CGG ACA GTC AGA GAG ATA ACA GGT TTC CTG AAC ATA CAG TCA TGG 1254
Phe Arg Thr Val Arg Glu Ile Thr Gly Phe Leu Asn Ile Gln Ser Trp
395 400 405
CCA CCA AAC ATG ACT GAC TTC AGT GTT TTT TCT AAC CTG GTG ACC ATT 1302
Pro Pro Asn Met Thr Asp Phe Ser Val Phe Ser Asn Leu Val Thr Ile
415 420
GGT GGA AGA GTA CTC TAT AGT GGC CTG TCC TTG CTT ATC CTC AAG CAA 1350
Gly Gly Arg Val Leu Tyr Ser Gly Leu Ser Leu Leu Ile Leu Lys Gln
430 435
CAG GGC ATC ACC TCT CTA CAG TTC CAG TCC CTG AAG GAA ATC AGC GCA 1398
Gln Gly Ile Thr Ser Leu Gln Phe Gln Ser Leu Lys Glu Ile Ser Ala
445 450 455
GGA AAC ATC TAT ATT ACT GAC AAC AGC AAC CTG TGT TAT TAT CAT ACC 1446
Gly Asn Ile Tyr Ile Thr Asp Asn Ser Asn Leu Cys Tyr Tyr His Thr
460 465 470
ATT AAC TGG ACA ACA CTC TTC AGC ACA ATC AAC CAG AGA ATA GTA ATC 1494
Ile Asn Trp Thr Thr Leu Phe Ser Thr Ile Asn Gln Arg Ile Val Ile
475 480 485
CGG GAC AAC AGA AAA GCT GAA AAT TGT ACT GCT GAA GGA ATG GTG TGC 1542
Arg Asp Asn Ary Lys Ala Glu Asn Cys Thr Ala Glu Gly Met Val Cys
495 500
AAC CAT CTG TGT TCC AGT GAT GGC TGT TGG GGA CCT GGG CCA GAC CAA 1590
Asn His Leu Cys Ser Ser Asp Gly Cys Trp Gly Pro Gly Pro Asp Gln
510 515
TGT CTG TCG TGT CGC CGC TTC AGT AGA GGA AGG ATC TGC ATA GAG TCT 1638
Cys Leu Ser Cys Arg Arg Phe Ser Arg Gly Arg Ile Cys Ile Glu Ser
525 530 535
TGT AAC CTC TAT GAT GGT GAA TTT CGG GAG TTT GAG AAT GGC TCC ATC 1686
Cys Asn Leu Tyr Asp Gly Glu Phe Arg Glu Phe Glu Asn Gly Ser Ile
540 545 550
TGT GTG GAG TGT GAC CCC CAG TGT GAG AAG ATG GAA GAT GGC CTC CTC 1734
Cys Val Glu Cys Asp Pro Gln Cys Glu Lys Met Glu Asp Gly Leu Leu
555 560 565
ACA TGC CAT GGA CCG GGT CCT GAC AAC TGT ACA AAG TGC TCT CAT TTT 1782
Thr Cys His Gly Pro Gly Pro Asp Asn Cys Thr Lvs Cys Ser His Phe
575 580
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
W O~6/12019 PCTnUS9~113~2
- 123 -
AAA GAT GGC CCA AAC TGT GTG GAA AAA TGT CCA GAT GGC TTA CAG GGG 1830
Lys Asp Gly Pro Asn Cys Val Glu Lys Cys Pro Asp Gly Leu Gln Gly
590 S9S
GCA AAC AGT~TTC ATT TTC AAG TAT GCT GAT CCA GAT CGG GAG TGC CAC 1878
Ala Asn Ser Phe Ile Phe Lys Tyr Ala Asp Pro Asp Arg Glu Cys His
605 610 615
CCA TGC CAT CCA AAC TGC ACC CAA GGG TGT AAC GGT CCC ACT AGT CAT 1926
Pro Cys His Pro Asn Cys Thr Gln Gly Cys Asn Gly Pro Thr Ser His
620 625 630
GAC TGC ATT TAC TAC CCA TGG ACG GGC CAT TCC ACT TTA CCA CAA CAT 1974
Asp Cys Ile Tyr Tyr Pro Trp Thr Gly His Ser Thr Leu Pro Gln His
635 640 645
GCT AGA ACT CCC CTG ATT GCA GCT GGA GTA ATT GGT GGG CTC TTC ATT 2022
Ala Arg Thr Pro Leu Ile Ala Ala Gly Val Ile Gly Gly Leu Phe Ile
655 660
CTG GTC ATT GTG GGT CTG ACA TTT GCT GTT TAT GTT AGA AGG AAG AGC 2070
Leu Val Ile Val Gly Leu Thr Phe Ala Val Tyr Val Arg Arg Lys Ser
670 675
ATC AAA AAG AAA AGA GCC TTG AGA AGA TTC TTG GAA ACA GAG TTG GTG 2118
Ile Lys Lys Lys Arg Ala Leu Arg Arg Phe Leu Glu Thr Glu Leu Val
685 690 695
GAA CCA TTA ACT CCC AGT GGC ACA GCA CCC AAT CAA GCT CAA CTT CGT 2166
Glu Pro Leu Thr Pro Ser Gly Thr Ala Pro Asn Gln Ala Gln Leu Arg
700 705 710
ATT TTG AAA GAA ACT GAG CTG AAG AGG GTA AAA GTC CTT GGC TCA GGT 2214
Ile Leu Lys Glu Thr Glu Leu Lys Arg Val Lys Val Leu Gly Ser Gly
715 720 725
GCT TTT GGA ACG GTT TAT AAA GGT ATT TGG GTA CCT GAA GGA GAA ACT 2262
Ala Phe Gly Thr Val Tyr Lys Gly Ile Trp Val Pro Glu Gly Glu Thr
735 740
GTG AAG ATT CCT GTG GCT ATT AAG ATT CTT AAT GAG ACA ACT GGT CCC 2310
Val Lys Ile Pro Val Ala Ile Lys Ile Leu Asn Glu Thr Thr Gly Pro
750 7S5
AAG GCA AAT GTG GAG TTC ATG GAT GAA GCT CTG ATC ATG GCA AGT ATG 23s8
Lys Ala Asn Val Glu Phe Met Asp Glu Ala Leu Ile Met Ala Ser Met
765 770 775
GAT CAT CCA CAC CTA GTC CGG TTG CTG GGT GTG TGT CTG AGC CCA ACC 2406
Asp His Pro His Leu Val Arg Leu Leu Gly Val Cys Leu Ser Pro Thr
780 785 790
ATC CAG CTG GTT ACT CAA CTT ATG CCC CAT GGC TGC CTG TTG GAG TAT 2454
Ile Gln Leu Val Thr Gln Leu Met Pro His Gly Cys Leu Leu Glu Tyr
795 800 805
GTC CAC GAG CAC AAG GAT AAC ATT GGA TCA CAA CTG CTG CTT AAC TGG 2502
Val His Glu His Lys Asp Asn Ile Gly Ser Gln Leu Leu Leu Asn Trp
815 820
TGT GTC CAG ATA GCT AAG GGA ATG ATG TAC CTG GAA GAA AGA CGA CTC 2550
Cys Val Gln Ile Ala Lys Gly Met Met Tyr Leu Glu Glu Arg Arg Leu
830 835
GTT CAT CGG GAT TTG GCA GCC CGT AAT GTC TTA GTG AAA TCT CCA AAC 2598
Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn
845 850 855
SUBST TUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
W O96/12019 PCTrUS95/1352
- 124 -
CAT GTG AAA ATC ACA GAT TTT GGG CTA GCC AGA CTC TTG GAA GGA GAT 2646
~is Val Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu Glu Gly Asp
865 870
GAA AAA GAG TAC AAT GCT GAT GGA GGA AAG ATG CCA ATT AAA TGG ATG 2694
Glu Lys Glu Tyr Asn Ala Asp Gly Gly Lys Met Pro Ile Lys Trp Met
875 880 885
GCT CTG GAG TGT ATA CAT TAC AGG AAA TTC ACC CAT CAG AGT GAC GTT 2742
Ala Leu Glu Cys Ile His Tyr Arg Lys Phe Thr His Gln Ser Asp Val
895 900
TGG AGC TAT GGA GTT ACT ATA TGG GAA CTG ATG ACC TTT GGA GGA AAA 2790
Trp Ser Tyr Gly Val Thr Ile Trp Glu Leu Met Thr Phe Gly Gly Lys
910 915
CCC TAT GAT GGA ATT CCA ACG CGA GAA ATC CCT GAT TTA TTA GAG AAA 2838
Pro Tyr Asp Gly Ile Pro Thr Arg Glu Ile Pro Asp Leu Leu Glu Lys
925 930 935
GGA GAA CGT TTG CCT CAG CCT CCC ATC TGC ACT ATT GAC GTT TAC ATG 2886
Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met
940 945 950
GTC ATG GTC AAA TGT TGG ATG ATT GAT GCT GAC AGT AGA CCT AAA TTT 2934
Val Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys Phe
955 960 965
AAG GAA CTG GCT GCT GAG TTT TCA AGG ATG GCT CGA GAC CCT CAA AGA 2982
Lys Glu Leu Ala Ala Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg
975 980
TAC CTA GTT ATT CAG GGT GAT GAT CGT ATG AAG CTT CCC AGT CCA AAT 3030
Tyr Leu Val Ile Gln Gly Asp Asp Arg Met Lys Leu Pro Ser Pro Asn
990 995
GAC AGC AAG TTC TTT CAG AAT CTC TTG GAT GAA GAG GAT TTG GAA GAT 3078
Asp Ser Lys Phe Phe Gln Asn Leu Leu Asp Glu Glu Asp Leu Glu Asp
1005 1010 1015
ATG ATG GAT GCT GAG GAG TAC TTG GTC CCT CAG GCT TTC AAC ATC CCA 3126
Met Met Asp Ala Glu Glu Tyr Leu Val Pro Gln Ala Phe Asn Ile Pro
1020 1025 1030
CCT CCC ATC TAT ACT TCC AGA GCA AGA ATT GAC TCG AAT AGG AGT GTA 3174
Pro Pro Ile Tyr Thr Ser Arg Ala Arg Ile Asp Ser Asn Arg Ser Val
1035 1040 1045
AGA AAT AAT TAT ATA CAC ATA TCA TAT TCT TTC TGAGATATAA AATCATGTAA 3227
Arg Asn Asn Tyr Ile ~is Ile Ser Tyr Ser Phe
1055
TAGTTCATAA GCACTAACAT TTCAAAATAA TTATATAGCT CAAATCAATG TGATGCCTAG 3287
ATTAAAAATA TACCATACCC ACAAAAGATG TGCCAATCTT GCTATATGTA GTTAATTTTG 3347
GAAGACAAGC ATGGACAATA CAACATGTAC TCTGAAATAC CTTCAAGATT TCAGAAGCAA 3407
AACATTTTCC TCATCTTAAT TTATTTAAAA CAAATCTTAA CTTTAAAAAA CAATTCCAAC 3467
TAATAAAACC ATTATGTGTA TATAAATAAA TGAAAATTCC TACCAAGTAG GCTTTCTACT 3527
lrl~lllCTT AAAAAGATAT TATGATATAT TAGTCAAGAA GTAATACAAG TATAAATCTC 3587
TTTCACTTAT TTAAGAAAAA TTAAATATTT TCTGTCAAGT TGAAGTAGAA ACACAGAAAA 3647
CCGTGCAGTC CTTTGAACCT AATCACATCG AAAAGGcTGc TGAGAAGTAG Alllll~lll 3707
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
Og6/1201g PCTfUS9~/13~2
- 125 -
TTAAGAAGTA GATTTAAGTT TTGAAGGAAG TTTCTGAAAA CACTTTACAT TTTAAATGTT 3767
AAACCTACTC TATATGAATT CCAlr~lllC TTTGAAAGCT GTCAAATCCA TGCATTTATT 3827
TTTATAAATT CATTCCTCAT ACATTCAACA TATATTGAG~ ACCACTGTAT GTGAAGCATT 3887
AGTATACATT TAAGACTCAA AGAATTTTGA TACAACTTCT GCTTTCAAGA AGTGAAAACC 3947
TTAATCAAAG AATCATACAG ATAGAGGGAC TGCATAGTAA GTGCTGTAAT CCAGTATTCA 4007
CTGACCAGTA CGGAGCATGA AGAAGTAGTA AAlll~l~lC TGTAATCAGT TTCTTCCATT 4067
GATAAGATAT AAACATGATG CTTAATTTTT TCTAGAAGAT AAll~ C TCTTAATCTA 4127
AGAACATTAT CATAGCTAGT AGAACCGACA GCATCCGATT l~l~llGACC ATAGCCATAA 4187
GAATATCTTC AACTTGCTGC TCATTATCTA ACA~ACATAA TTTTCTTTAT TTCATATTGA 4247
TTGTAATAAG TAATATCCCC CTGGAAGTTT ACTATTCAAC ACATATATGT TAACCTCCTT 4307
AATTCCTTAA ACAAACTTCA TGAGGTTCTA TTATTATCAT CCCC~l~lll CAAAGGAAGA 4367
AACTTGCCAC AGAGAAGTCA GGTGATATGA CTGGTGTCAC ACAGCTAGTC AGTGGAAGAG 4427
AGGAATAAGT AATCTAGATA TCTGCCTACT ACACTGTAGG TTTGCTTCAA AGTTACTGAA 4487
GYCATGTTAT TTCCATGATG TGATTAGAGT CTGGGACTTG l~ll~lllGG GAAATTTCCC 4547
AGGTGGTTTT CTTATAAAAT GCATCTCAAA TCTGCTCTAC ACCTTTTACT CATCTACCTC 4607
CATTTAGAAG ATCTGATATG GAAAGAGACA AAGATGGAGA CCTCAATTAT 1llll~llll 4667
CTGTTAAAAA TATTATAGTA CAACTGAAAC TTATCACATG CCAATGGGGA ATAGATAACT 4727
AAAAGTTTAA AATTAGATCA ATGGATAGGT AAATGAATAA l~Nrl~llll GCTTGTGAGA 4787
GGGGAAGGAA AAGCGGTTAA GGTGGTATAA AGGAGGCTCC TCTGTACACT TGCAAAATGA 4847
TCAAATTATA TACCCTTGTA TTTATAATTT TAAGTGACAA ATTCATTACT TCTGGTTACA 4907
ACAGTGAAAT TTAAAAAAAA ATAGTTTTTC TTTCTTAGCT TGCAATGCTA TAAATCTTTT 4967
TCTTTTTATA AGAATTCTTA CATTTCAGCT lrll~llCAT TTTAATTTAT AATTCTCAGT 5027
GCAAGAAATT CTTAATAAAG GTTTGAGCTA GCTAGATGGA ATTATTGAGA CAAAGTCTAA 5087
ATCACCCGTG GACTTATTTG ACCTTTAGCC ATCATTTCTT ATTCCACATT ATAAAACAAT S147
GTTACCTGTA GAlll~llll TA~lllllCA GTCCTTGGAA AAGAAATGGT GATTAAATAT 5207
CATTATATCA TTTTATGTTC AGGCATTTAA AAAGCTTTAT TTGTCATCTA TATTGTCCTA 5267
ATAGTTTTCA GTCTGGCTTT ACGTAACTTT TACGGAAAT~ TCTAACATGT AcAAATGccA 5327
TGTTCCTCCT rl~lllCCTA CATGGCTGAA TTAGAAAACA AATTACTTCC ATTTTAAGTT 5387
TGGCTAAATT AGAAAACAAA TTACTACCAT TTTAAGTTTG GTGGCTAAAT AACGTGCTAA 5447
GGGAACATCT TAAAAAGTGA ATTTTGATCA AATATTTCTT AAGCATATGT GATAGACTTT 5507
GAAACCAAAA AA~Uu~AAAA AAAAAAAAA~ AAAAAAAAAA AAAAAAAA 5555
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTTCS:
(A) LENGTH: 1058 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 l997-04-ll
WO 96112019 PCI~/US9511352"
- 126 -
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
et Lys Pro~Ala Thr Gly Leu Trp Val Trp Val Ser Leu Leu Val Ala
la Gly Thr Val Gln Pro Ser Asp Ser Gln Ser Val Cys Ala Gly Thr
lu Asn Lys Leu Ser Ser Leu Ser Asp Leu Glu Gln Gln Tyr Arg Ala
Leu Arg Lys Tyr Tyr Glu Asn Cys Glu Val Val Met Gly Asn Leu Glu
Ile Thr Ser Ile Glu His Asn Arg Asp Leu Ser Phe Leu Arg Ser Val
rg Glu Val Thr Gly Tyr Val Leu Val Ala Leu Asn Gln Phe Arg Tyr
eu Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly Thr Lys Leu Tyr Glu
100 105 110
Asp Arg Tyr Ala Leu Ala Ile Phe Leu Asn Tyr Arg Lys Asp Gly Asn
115 120 125
Phe Gly Leu Gln Glu Leu Gly Leu Lys Asn Leu Thr Glu Ile Leu Asn
130 135 140
Gly Gly Val Tyr Val Asp Gln Asn Lys Phe Leu Cys Tyr Ala Asp Thr
145 150 155 160
le His Trp Gln Asp Ile Val Arg Asn Pro Trp Pro Ser Asn Leu Thr
165 170 175
eu Val Ser Thr Asn Gly Ser Ser Gly Cys Gly Arg Cys His Lys Ser
180 185 190
Cys Thr Gly Arg Cys Trp Gly Pro Thr Glu Asn His Cys Gln Thr Leu
195 200 205
Thr Arg Thr Val Cys Ala Glu Gln Cys Asp Gly Arg Cys Tyr Gly Pro
210 215 220
Tyr Val Ser Asp Cys Cys His Arg Glu Cys Ala Gly Gly Cys Ser Gly
225 230 235 240
ro Lys Asp Thr Asp Cys Phe Ala Cys Met Asn Phe Asn Asp Ser Gly
245 250 255
la Cys Val Thr Gln Cys Pro Gln Thr Phe Val Tyr Asn Pro Thr Thr
260 265 270
Phe Gln Leu Glu His Asn Phe Asn Ala Lys Tyr Thr Tyr Gly Ala Phe
275 280 285
Cys Val Lys Lys Cys Pro His Asn Phe Val Val Asp Ser Ser Ser Cys
290 295 300
Val Arg Ala Cys Pro Ser Ser Lys Met Glu Val Glu Glu Asn Gly Ile
305 310 315 320
ys Met Cys Lys Pro Cys Thr Asp Ile Cys Pro Lys Ala Cys Asp Gly
325 330 335
SUBSTITUTE SHEET (RULE 26~
-
CA 02202~33 1997-04-ll
WO 96/12019 PCIIUS95/1352
- 127 -
Ile Gly Thr Gly Ser Leu Met Ser Ala Gln Thr ~ral Asp Ser Ser Asn
340 345 350
? Ile Asp Lys Phe Ile Asn Cys Thr Lys Ile Asn Gly Asn Leu Ile Phe
355 ~ 360 365
Leu Val Thr Gly Ile His Gly Asp Pro Tyr Asn Ala Ile Glu Ala Ile
- 370 375 380
Asp Pro Glu Lys Leu Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly
385 390 395 400
Phe Leu Asn Ile Gln Ser Trp Pro Pro Asn Met Thr Asp Phe Ser Val
405 410 415
Phe Ser Asn Leu Val Thr Ile Gly Gly Arg Val Leu Tyr Ser Gly Leu
420 42=5 430
Ser Leu Leu Ile Leu Lys Gln Gln Gly Ile Thr Ser Leu Gln Phe Gln
435 440 445
Ser Leu Lys Glu Ile Ser Ala Gly Asn Ile Tyr Ile Thr Asp Asn Ser
450 455 460
Asn Leu Cys Tyr Tyr His Thr Ile Asn Trp Thr Thr Leu Phe Ser Thr
465 470 475 480
Ile Asn Gln Arg Ile Val Ile Arg Asp Asn Arg Lys Ala Glu Asn Cys
485 490 495
Thr Ala Glu Gly Met Val Cys Asn His Leu Cys Ser Ser Asp Gly Cys
500 505 510
Trp Gly Pro Gly Pro Asp Gln Cys Leu Ser Cys Arg Arg Phe Ser Arg
515 520 525
Gly Arg Ile Cys Ile Glu Ser Cys Asn Leu Tyr Asp Gly Glu Phe Arg
530 535 540
Glu Phe Glu Asn Gly Ser Ile Cys Val Glu Cys Asp Pro Gln Cys Glu
545 550 555 560
Lys Met Glu Asp Gly Leu Leu Thr Cys His Gly Pro Gly Pro Asp Asn
565 570 575
Cys Thr Lys Cys Ser His Phe Lys Asp Gly Pro Asn Cys Val Glu Lys
580 585 590
Cys Pro Asp Gly Leu Gln Gly Ala Asn Ser Phe Ile Phe Lys Tyr Ala
595 600 605
Asp Pro Asp Arg Glu Cys His Pro Cys His Pro Asn Cys Thr Gln Gly
610 615 620
Cys Asn Gly Pro Thr Ser His Asp Cys Ile Tyr Tyr Pro Trp Thr Gly
625 630 635 640
~is Ser Thr Leu Pro Gln His Ala Arg Thr Pro Leu Ile Ala Ala Gly
645 650 655
Val Ile Gly Gly Leu Phe Ile Leu Val Ile Val Gly Leu Thr Phe Ala
660 665 670
Val Tyr Val Arg Arg Lys Ser Ile Lys Lys Lys Arg Ala Leu Arg Arg
675 680 685
Phe Leu Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Thr Ala
SUBSTITUTE SHEET (RULE 26~ _ __ __
CA 02202~33 l997-04-ll
W O96/12019 PCTrUS95/1352
- 128 -
690 695 700
Pro Asn Gln Ala Gln Leu Arg Ile Leu Lys Glu Thr Glu Leu Lys Arg
705 710 715 720
Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Ile
725 730 735
Trp Val Pro Glu Gly Glu Thr Val Lys Ile Pro Val Ala Ile Lys Ile
740 745 750
Leu Asn Glu Thr Thr Gly Pro Lys Ala Asn Val Glu Phe Met Asp Glu
755 760 765
Ala Leu Ile Met Ala Ser Met Asp His Pro His Leu Val Arg Leu Leu
770 775 780
Gly Val Cys Leu Ser Pro Thr Ile Gln Leu Val Thr Gln Leu Met Pro
785 790 795 800
His Gly Cys Leu Leu Glu Tyr Val His Glu His Lys Asp Asn Ile Gly
805 810 815
Ser Gln Leu Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Met
820 825 830
Tyr Leu Glu Glu Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn
835 840 845
Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu
850 855 860
Ala Arg Leu Leu Glu Gly Asp Glu Lys Glu Tyr Asn Ala Asp Gly Gly
865 870 87s 880
Lys Met Pro Ile Lys Trp Met Ala Leu Glu Cys Ile His Tyr Arg Lys
885 890 895
Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Ile Trp Glu
900 905 910
Leu Met Thr Phe Gly Gly L-~s Pro Tyr Asp Gly Ile Pro Thr Arg Glu
915 920 925
Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile
930 935 940
Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys Trp Met Ile Asp
945 950 955 960
Ala Asp Ser Arg Pro Lys Phe Lys Glu Leu Ala Ala Glu Phe Ser Arg
965 970 975
Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Asp Arg
980 985 990
Met Lys Leu Pro Ser Pro Asn Asp Ser Lys Phe Phe Gln Asn Leu Leu
995 1000 1005
Asp Glu Glu Asp Leu Glu Asp Met Met Asp Ala Glu Glu Tyr Leu Val
1010 1015 1020
Pro Gln Ala Phe Asn Ile Pro Pro Pro Ile Tyr Thr Ser Arg Ala Arg
1025 1030 1035 1040
Ile Asp Ser Asn Arg Ser Val Arg Asn Asn Tyr Ile His Ile Ser Tyr
1045 1050 1055
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
WO 96112019 PCTrUS95/1352
- 129 -
Ser Phe
(2) INFORMATION FOR SEQ ID NO:S:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3321 base pairs
- (B) TYPL: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 156..1782
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:5:
CATTAGCTGC AATTGATCAA GTGACTGAGA GAAGGGCAAC ATTCCATGCA ACAGTATAGT 60
GGTATGGAAA GCCCTGGATG TTGAAATCTA GCTTCAAAAA GC~lG~ G AAATGTAGTT 120
AATTGGATGA AGTGAGAAGA GATAAAACCA GAGAG GAA GCT CTG ATC ATG GCA 173
Glu Ala Leu Ile Met Ala
1 5
AGT ATG GAT CAT CCA CAC CTA GTC CGG TTG CTG GGT GTG TGT CTG AGC 221
Ser Met Asp His Pro His Leu Val Arg Leu Leu Gly Val Cys Leu Ser
10 15 20
CCA ACC ATC CAG CTG GTT ACT CAA CTT ATG CCC CAT GGC TGC CTG TTG 269
ro Thr Ile Gln Leu Val Thr Gln Leu Met Pro His Gly Cys Leu Leu
25 30 35
GAG TAT GTC CAC GAG CAC AAG GAT AAC ATT GGA TCA CAA CTG CTG CTT 317
Glu Tyr Val His Glu His Lys Asp Asn Ile Gly Ser Gln Leu Leu Leu
~S 50
AAC TGG TGT GTC CAG ATA GCT AAG GGA ATG ATG TAC CTG GAA GAA AGA 365
Asn Trp Cys Val Gln Ile Ala Lys Gly Met Met Tyr Leu Glu Glu Arg
60 65 70
CGA CTC GTT CAT CGG GAT TTG GCA GCC CGT AAT GTC TTA GTG AAA TCT 413
Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Ser
75 80 85
CCA AAC CAT GTG AAA ATC ACA GAT TTT GGG CTA GCC AGA CTC TTG GAA 461
Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu Glu
90 95 100
GGA GAT GAA AAA GAG TAC AAT GCT GAT GGA GGA AAG ATG CCA ATT AAA 509
Gly Asp Glu Lys Glu Tyr Asn Ala Asp Gly Gly Lys Met Pro Ile Lys
110 115
TGG ATG GCT CTG GAG TGT ATA CAT TAC AGG AAA TTC ACC CAT CAG AGT 557
Trp Met Ala Leu Glu Cys Ile Hls Tyr Ary Lys Phe Thr His Gln Ser
125 130
GAC GTT TGG AGC TAT GGA GTT ACT ATA TGG GAA CTG ATG ACC TTT GGA 605
Asp Val Trp Ser Tyr Gly Val Thr Ile Trp Glu Leu Met Thr Phe Gly
140 145 150
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 l997-04-ll
W O96/12019 PCTrUS9511352
- 130 -
GGA AAA CCC TAT GAT GGA ATT CCA ACG CGA GAA ATC CCT GAT TTA TTA 653GlfLys Pro Tyr Asp Gly Ile Pro Thr Arg Glu Ile Pro Asp Leu Leu
160 165
GAG AAA GGA~GAA CGT TTG CCT CAG CCT CCC ATC TGC ACT ATT GAC GTT 701
Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val
170 175 180
TAC ATG GTC ATG GTC AAA TGT TGG ATG ATT GAT GCT GAC AGT AGA CCT 749
Tyr Met Val Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro
190 195
AAA TTT AAG GAA CTG GCT GCT GAG TTT TCA AGG ATG GCT CGA GAC CCT 797
Lys Phe Lys Glu Leu Ala Ala Glu Phe Ser Arg Met Ala Arg Asp Pro
205 210
CAA AGA TAC CTA GTT ATT CAG GGT GAT GAT CGT ATG AAG CTT CCC AGT 845
Gln Arg Tyr Leu Val Ile Gln Gly Asp Asp Arg Met Lys Leu Pro Ser
220 225 230
CCA AAT GAC AGC AAG TTC TTT CAG AAT CTC TTG GAT GAA GAG GAT TTG 893
Pro Asn Asp Ser Lys Phe Phe Gln Asn Leu Leu Asp Glu Glu Asp Leu
235 240 245
GAA GAT ATG ATG GAT GCT GAG GAG TAC TTG GTC CCT CAG GCT TTC AAC 941
Glu Asp Met Met Asp Ala Glu Glu Tyr Leu Val Pro Gln Ala Phe Asn
250 255 260
ATC CCA CCT CCC ATC TAT ACT TCC AGA GCA AGA ATT GAC TCG AAT AGG 989
Ile Pro Pro Pro Ile Tyr Thr Ser Arg Ala Arg Ile Asp Ser Asn Arg
270 275
AGT GAA ATT GGA CAC AGC CCT CCT CCT GCC TAC ACC CCC ATG TCA GGA 1037
Ser Glu Ile Gly His Ser Pro Pro Pro Ala Tyr Thr Pro Met Ser Gly
285 290
AAC CAG TTT GTA TAC CGA GAT GGA GGT TTT GCT GCT GAA CAA GGA GTG 1085
Asn Gln Phe Val Tyr Arg Asp Gly Gly Phe Ala Ala Glu Gln Gly Val
300 305 310
TCT GTG CCC TAC AGA GCC CCA ACT AGC ACA ATT CCA GAA GCT CCT GTG 1133
Ser Val Pro Tyr Arg Ala Pro Thr Ser Thr Ile.Pro Glu Ala Pro Val
315 320 325
GCA CAG GGT GCT ACT GCT GAG ATT TTT GAT GAC TCC TGC TGT AAT GGC 1181
Ala Gln Gly Ala Thr Ala Glu Ile Phe Asp Asp Ser Cys Cys Asn Gly
330 335 340
ACC CTA CGC AAG CCA GTG GCA CCC CAT GTC CAA GAG GAC AGT AGC ACC 1229
Thr Leu Arg Lys Pro Val Ala Pro His Val Gln Glu Asp Ser Ser Thr
350 355
CAG AGG TAC AGT GCT GAC CCC ACC GTG TTT GCC CCA GAA CGG AGC CCA 1277
Gln Arg Tyr Ser Ala Asp Pro Thr Val Phe Ala Pro Glu Arg Ser Pro
365 370
CGA GGA GAG CTG GAT GAG GAA GGT TAC ATG ACT CCT ATG CGA GAC AAA 1325
Arg Gly Glu Leu Asp Glu Glu Gly Tyr Met Thr Pro Met Arg Asp Lys
380 385 390
CCC AAA CAA GAA TAC CTG AAT CCA GTG GAG GAG AAC CCT TTT GTT TCT 1373
Pro Lys Gln Glu Tyr Leu Asn Pro Val Glu Glu Asn Pro Phe Val Ser
395 400 405
CGG AGA AAA AAT GGA GAC CTT CAA GCA TTG GAT AAT CCC GAA TAT CAC
1421
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 l997-04-ll
W O 96tl2019 PCTnUS95J13~2
- 131 -
Arg Arg Lys Asn Gly Asp Leu Gln Ala Leu Asp Asn Pro Glu Tyr His
410 415 420
AAT GCA TCC AAT GGT CCA CCC AAG GCC GAG GAT GAG TAT GTG AAT GAG 1~69
Asn Ala Se~ Asn Gly Pro Pro Lys Ala Glu Asp Glu Tyr Val Asn Glu
430 435
- CCA CTG TAC CTC AAC ACC TTT GCC AAC ACC TTG GGA AAA GCT GAG TAC 1517Pro Leu Tyr Leu Asn Thr Phe Ala Asn Thr Leu Gly Lys Ala Glu Tyr
' 445 450
CTG AAG AAC AAC ATA CTG TCA ATG CCA GAG AAG GCC AAG AAA GCG TTT 1565
Leu Lys Asn Asn Ile Leu Ser Me~ Pro Glu Lys Ala Lys Lys Ala Phe
460 465 470
GAC AAC CCT GAC TAC TGG AAC CAC AGC CTG CCA CCT CGG AGC ACC CTT 1613
Asp Asn Pro Asp Tyr Trp Asn His Ser ~eu Pro Pro Arg Ser Thr Leu
475 480 485
CAG CAC CCA GAC TAC CTG CAG GAG TAC AGC ACA AAA TAT TTT TAT AAA 1661
Gln His Pro Asp Tyr Leu Gln Glu Tyr Ser Thr Lys Tyr Phe Tyr Lys
490 495 500
CAG AAT GGG CGG ATC CGG CCT ATT GTG GCA GAG AAT CCT GAA TAC CTC 1709
Gln Asn Gly Arg Ile Arg Pro Ile Val Ala Glu Asn Pro Glu Tyr Leu
510 515
TCT GAG TTC TCC CTG AAG CCA GGC ACT GTG CTG CCG CCT CCA CCT TAC 1757
Ser Glu Phe Ser Leu Lys Pro Gly Thr Val Leu Pro Pro Pro Pro Tyr
525 530
AGA CAC CGG AAT ACT GTG GTG TAAGCTCAGT l~lG~lllll TAGGTGGAGA 1808
Val
535 540
GACACACCTG CTCCAATTTC CCCACCCCCC T~~ l~lC TGGTGGTCTT CCTTCTACCC
CCAGT AGTTTTGACA CTTCCCAGTG GAAGATACAG AGATGCAATG ATAGTTATGT 1928
GCTTACCTAA CTTGAACATT AGAGGGAAAG ACTGAAAGAG AAAGATAGGA GGAACCACAA 1988
TGTTTCTTCA lll~l~lGCA TGGGTTGGTC AGGAGAATGA AACAGCTAGA GAAGGACCAG 2048
AAAATGTAAG GCAATGCTGC CTACTATCAA ACTAGCTGTC A~lllllllC llll''l'~'ll'll' 2108
l~lll~lll~ llr~lll~ll C~l~ll~lll llllllllll TTTTAAAGCA GATGGTTGAA 2168
ACACCCATGC TAl~l~llCC TATCTGCAGG AACTGATGTG TGCATATTTA GCATCCCTGG 2228
AAATCATAAT AAAGTTTCCA TTAGAACAAA AGAATAACAT TTTCTATAAC ATATGATAGT 2288
GTCTGAAATT GAGAATCCAG 'l'll'~''l''l''l'CCC CAGCAGTTTC TGTCCTAGCA AGTAAGAATG 2348
GCCAACTCAA CTTTCATAAT TTAAAAATCT CCATTAAAGT TATAACTAGT AATTATGTTT 2408
TCAACACTTT TTG~llllll TCAllll~ll TTGCTCTGAC CGAllC~lll ATATTTGCTC 2468
CCCTATTTTT GGCTTTAATT TCTAATTGCA AAGATGTTTA CATCAAAGCT TCTTCACAGA 2528
ATTTAAGCAA GAAATATTTT AATATAGTGA AATGGCCACT ACTTTAAGTA TACAATCTTT 2588
AAAATAAGAA AGGGAGGCTA ATATTTTTCA TGCTATCAAA TTATCTTCAC CCTCATCCTT 2648
TACATTTTTC AACAllllll TTTCTCCATA AATGACACTA CTTGATAGGC C~L 1G~11~1 2708
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
W O96112019 PCTrUS95/1352
- 132 -
CTGAAGAGTA GAAGGGAAAC TAAGAGACAG llCl~'~lGG TTCAGGAAAA CTACTGATAC 2768
TTTCAGGGGT GGCCCAATGA GGGAATCCAT TGAACTGGAA GAAACACACT GGATTGGGTA 2828
TGTCTACCTG GCAGATACTC AGAAATGTAG TTTGCACTTA AGCTGTAATT TTAlrl~llC 2888
lrlll-lGAA CTCCATTTTG GATTTTGAAT CAAGcAATAT GGAAGCAACC AGCAAATTAA 2948
CTAATTTAAG TACATTTTTA AAAAAAGAGC TAAGATAAAG ACTGTGGAAA TGccAAAccA 3008
AGCAAATTAG GAACCTTGCA ACGGTATCCA GGGACTATGA TGAGAGGCCA GCACATTATC 3068
TTCATATGTC ACCTTTGCTA CGCAAGGAAA TTTGTTcAGT TCGTATACTT CGTAAGAAGG 3128
AATGCGAGTA AGGATTGGCT TGAATTCCAT GGAATTTCTA GTATGAGACT ATTTATATGA 3188
AGTAGAAGGT AA~r~lllGC ACATAAATTG GTATAATAAA AAGAAAAACA CAAACATTCA 3248
AAGCTTAGGG ATAGGTCCTT GGGTCAAAAG TTGTAAATAA ATGTGAAACA l~lr~lCAAA 3308
AA~U~AAAA AAA 3321
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 541 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Glu Ala Leu Ile Met Ala Ser Met Asp His Pro His Leu Val Arg Leu
1 5 10 15
Leu Gly Val Cys Leu Ser Pro Thr Ile Gln Leu Val Thr Gln Leu Met
2S 30
Pro His Gly Cys Leu Leu Glu Tyr Val His Glu Hls Lys Asp Asn Ile
Gly Ser Gln Leu Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met
Met Tyr Leu Glu Glu Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg
Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly
Leu Ala Arg Leu Leu Glu Gly Asp Glu Lys Glu Tyr Asn Ala Asp Gly
100 105 110
Gly Lys Met Pro Ile Lys Trp Met Ala Leu Glu Cys Ile His Tyr Arg
115 120 125
Lys Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Ile Trp
130 135 140
Glu Leu Met Thr Phe Gly Gly Lys Pro Tyr Asp Gly Ile Pro Thr Arg
145 150 155 160
Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro
165 170 175
Ile Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys Trp Met Ile
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
WO 96112019 PCT~S95113S2d
- 133 -
180 185 lgo
Asp Ala Asp Ser Arg Pro Lys Phe Lys Glu Leu Ala Ala Glu Phe Ser
195 200 205
Arg Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Asp
210 215 220
Arg Met Lys Leu Pro Ser Pro Asn Asp Ser Lys Phe Phe Gln Asn Leu
225 230 235 240
Leu Asp Glu Glu Asp Leu Glu Asp Met Met Asp Ala GlU Glu Tyr Leu
24s 250 255
al Pro Gln Ala Phe Asn Ile Pro Pro Pro Ile Tyr Thr Ser Arg Ala
260 265 270
Arg Ile Asp Ser Asn Arg Ser Glu Ile Gly His Ser Pro Pro Pro Ala
275 280 285
Tyr Thr Pro Met Ser Gly Asn Gln Phe Val Tyr Arg Asp Gly Gly Phe
290 29s 300
Ala Ala Glu Gln Gly Val Ser Val Pro Tyr Arg Ala Pro Thr Ser Thr
30s 310 315 320
Ile Pro Glu Ala Pro Val Ala Gln Gly Ala Thr Ala GlU Ile Phe Asp
32s 330 335
sp Ser Cys Cys Asn Gly Thr Leu Arg Lys Pro Val Ala Pro His Val
340 345 350
Gln Glu Asp Ser Ser Thr Gln Arg Tyr Ser Ala Asp Pro Thr Val Phe
355 360 365
Ala Pro Glu Arg Ser Pro Arg Gly Glu Leu Asp Glu Glu Gly Tyr Met
370 37s 380
Thr Pro Met Arg Asp Lys Pro Lys Gln Glu Tyr Leu Asn Pro Val Glu
385 390 395 400
Glu Asn Pro Phe Val Ser Arg Arg Lys Asn Gly Asp Leu Gln Ala Leu
405 410 415
sp Asn Pro Glu Tyr His Asn Ala Ser Asn Gly Pro Pro Lys Ala Glu
420 425 430
Asp Glu Tyr Val Asn Glu Pro Leu Tyr Leu Asn Thr Phe Ala Asn Thr
435 440 445
Leu Gly Lys Ala Glu Tyr Leu Lys Asn Asn Ile Leu Ser Met Pro Glu
450 45s 460
Lys Ala Lys Lys Ala Phe Asp Asn Pro Asp Tyr Trp Asn His Ser Leu
465 470 47s 480
Pro Pro Arg Ser Thr Leu Gln His Pro Asp Tyr Leu Gln Glu Tyr Ser
485 490 495
hr Lys Tyr Phe Tyr Lys Gln Asn Gly Arg Ile Arg Pro Ile Val Ala
500 505 510
Glu Asn Pro Glu Tyr Leu Ser GlU Phe Ser Leu Lys Pro Gly Thr Val
515 520 525
Leu Pro Pro Pro Pro Tyr Arg His Arg Asn Thr Val Val
530 535 540
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
WO 96112019 PCI'/US95/1352~1
- 134 -
(2) INFO~MATION ~OR SEQ ID NO: 7:
( i ) SEQUENCE: CHARACTERISTICS:
(A) LENGTH: 1210 amino acids
(B) TYPE: amino acid
(C) STR~NDEDNESS: unknown
( D ) TOPOLOGY: unknown
( ii ) MOLECUL~: TYPE: protein
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
et Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
S 10 15
la Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
ly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
lu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
llS 120 125
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 lSS 160
er Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
er Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
l9S 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
hr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
lu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
hr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
WO 96112019 PCT~S95~13~2
- 135 -
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 ` 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 33s
ys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
35s 360 365
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
le Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
eu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
he Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
sn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
lu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg
515 520 525
Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Lys Leu Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
s45 550 555 560
lu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
sp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
ys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
SUBSTITUTE SHEET (RULL 26
CA 02202~33 1997-04-11
WO 96/12019 PCI`IUS95/13S2~1
- 136 -
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
625 630 635 640
ro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
645 650 655
eu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His
660 665 670
le Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu
675 680 685
Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu
690 695 700
Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser
705 710 715 720
ly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu
725 730 735
ys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser
740 745 750
ro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser
755 760 765
Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser
770 775 780
Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp
785 790 795 800
yr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn
805 810 815
rp Cys Val Gln Ile Ala Lys Gly Met Met Tyr Leu Glu Asp Arg Arg
820 825 830
eu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro
835 840 845
Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala
850 855 860
Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp t
865 870 875 880
Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser
885 890 895
Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly
900 905 910
ys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu
915 920 925
SUBSTITUTE SHEET (RULE 26~
. CA 02202~33 1997-04-11
Wo 96/12019 PCTllUS9511352
- 137
~ Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr
- 930 935 940
Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys
950 955 960
Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln
965 970 g75
Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro
980 985 990
Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp
995 1000 1005
Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe
lolo 1015 1020
Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala
1025 1030 1035 1040
Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln
1045 1050 1055
Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp
1060 1065 1070
Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro
1075 1080 1085
Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser
1090 1095 lloo
Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser
1105 lllo 1115 1120
Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro
1125 1130 1135
Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp
1140 1145 1150
Ser Pro Ala Hls Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp
1155 1160 1165
Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn
1170 1175 1180
Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val
1185 llgo 1195 1200
Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala
1205 1210
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1255 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
SUBSTIT~TE SHEET (RU~E 26~
CA 02202~33 1997-04-11
WO 96/12019 PCr/US95/13St~
- 138 -
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu
1 5 10 15
ro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys
2S 30
eu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His
Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr
Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val
ln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu
ln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr
100 105 110
la Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro
115 120 125
Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser
130 135 140
Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln
145 150 155 160
eu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn
165 170 175
sn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys
180 185 190
is Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser
195 200 205
Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys
210 215 220
Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys
225 230 235 240
la Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu
245 250 255
is Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val
260 265 270
hr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg
275 280 285
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
WO 9~/12019 PCI'IUS9~1352
- 139 -
Tyr Thr Phe Gly Ala Ser Cy5 Val Thr Ala CyS Pro Tyr Asn Tyr Leu
290 295 300
Ser Thr Asp Val Gly Ser Cyg Thr Leu Val Cys Pro Leu His Asn Gln
30!; 310 315 320
lu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys
325 330 335
ro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu
340 345 350
al Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys
355 360 365
Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly ASp
370 375 380
Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe
385 390 395 400
lu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro
405 410 415
sp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg
420 425 430
ly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu
435 440 445
Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly
450 455 460
Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val
465 470 475 480
ro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr
485 490 495
la Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His
500 505 510
ln Leu Cys Ala Arg Arg Ala Leu Leu Gly Ser Gly Pro Thr Gln Cys
515 520 525
Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys
530 535 540
Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys
545 550 555 560
eu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys
565 570 575
he Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp
580 585 590
ro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu
595 600 605
Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln
610 615 620
Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys
SUBSTITUTE SHEET (RUL~ 26~
CA 02202~33 l997-04-ll
WO96112019 PCTrUS95/1352
- 140 -
625 630 635 640
ly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Val Ser
~ 645 650 655
la Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly
660 665 670
le Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg
675 680 685
Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly
690 695 700
Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu
705 710 715 720
Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys
725 730 735
ly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile
740 745 750
ys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu
755 760 765
Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg
770 775 780
Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu
785 790 795 800
Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg
805 . 810 815
eu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly
820 825 830
et Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala
835 840 845
Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe
850 855 860
Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp
865 870 875 880
Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg
885 890 895
rg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val
900 905 910
rp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala
915 920 925
Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro
930 935 940
Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met
945 950 955 960
Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe
965 970 975
Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu
SUBSTITUTE SHEET (RULE 26~
.
CA 02202~33 1997-04-11
WO 96112019 PCTnUS9~/1352
- 141 -
980 985 990
Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu
995 1000 1005
eu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu
1010 1015 1020
al Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly
1025 1030 1035 1040
Gly Met Val His His Arg ~is Arg Ser Ser Ser Thr Arg Ser Gly Gly
1045 1050 1055
ly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg
1060 1065 1070
er Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly
1075 1080 1085
sp Leu Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His
1090 1095 1100
sp Pro Ser Pro Leu Gln Arg Tyr Ser GlU Asp Pro Thr Val Pro Leu
1105 1110 1115 1120
Pro Ser Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln
1125 1130 1135
ro Glu Tyr Val Asn Gln Pro ASp Val Arg Pro Gln Pro Pro Ser Pro
1140 1145 1150
rg Glu Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu
1155 1160 1165
rg Ala Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val
1170 1175 1180
he Ala Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln
1185 1190 1195 1200
Gly Gly Ala Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala
1205 1210 1215
he Asp Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala
1220 1225 1230
ro Pro Ser Thr Phe Lys Gly Thr Pro Thr Val Ala Glu Asn Pro Glu
1235 1240 1245
yr Gly Leu Asp Val Pro Val
1250 1255
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1342 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
SuBsTlTuTE-sHEET (RUEE 26~
CA 02202~33 1997-04-11
WO 96/12019 PCT/US9511352.1
- 142 -
Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser ~eu
a Ary Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr
eu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr
Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu
Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile
Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr
eu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp
100 105 110
ly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser
115 120 125
His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser
130 135 140
Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr
145 150 155 160
Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val
165 170 175
ys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly
180 185 190
rg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr
195 200 205
Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn
210 215 220
Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp
225 230 235 240
Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val
245 250 255
ro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu
260 265 270
lu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala
2~5 280 285
Ser Cys Pro Hls Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala
290 295 300
Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys
305 310 315 320
Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser
325 330 335
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
WO 961121)l9 PCTJUS95/1352
- 143 -
Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val
340 345 350
sn Cys Thr ~ Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu
35S 360 365
Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu
370 375 380
Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln
385 390 395 400
er Trp Pro Pro His Met ~lis Asn Phe Ser Val Phe Ser Asn Leu Thr
405 410 415
hr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile
420 425 430
et Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu
435 440 4~5
Ile Ser Ala Gly Ary Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr
450 455 460
His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu
465 470 475 480
rg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu
485 490 495
ly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro
500 5~5 510
ly Pro Gly Gln cys Leu Ser Cys Ary Asn Tyr Ser Arg Gly Gly Val
515 520 525
Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala
530 535 540
His Glu Ala Glu Cys Phe Ser Cys His Pro Glu Cvs Gln Pro Met Gly
545 550 555 560
ly Thr Ala Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys
565 570 575
la His Phe Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly
580 585 590
al Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn
595 600 605
Glu Cys Arg Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro
610 615 620
Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr
625 630 635 640
is Leu Thr Me~ Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile Phe
645 650 655
et Met Leu Gly Gly Thr Phe Leu Tyr Trp Arg Gly Arg Arg Ile Gln
660 665 670
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 l997-04-ll
WO 96/12019 PCI'IUS9511352~1
- 144 -
Asn Lys Arg Ala Met Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu
675 680 685
Pro Leu Asp Pro Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe
690~ 695 700
Lys Glu Thr Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe
705 710 715 720
Gly Thr Val His Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys
725 730 735
e Pro Val Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser
740 745 750
he Gln Ala Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His
755 760 765
Ala His Ile Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln
770 775 780
Leu Val Thr Gln Tyr Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg
785 790 795 800
Gln His Arg Gly Ala Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val
805 810 815
ln Ile Ala Lys Gly Met Tyr Tyr Leu Glu Glu His Gly Met Val His
820 825 830
rg Asn Leu Ala Ala Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val
835 840 845
Gln Val Ala Asp Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys
850 855 860
Gln Leu Leu Tyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu
865 870 875 880
Glu Ser Ile His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser
885 890 895
yr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr
900 905 910
la Gly Leu Arg Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu
915 920 925
Arg Leu Ala Gln Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met
930 935 940
Val Lys Cys Trp Met Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu
945 950 955 960
Leu Ala Asn Glu Phe Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu
965 970 975
al Ile Lys Arg Glu Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro
980 985 990
is Gly Leu Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu
995 1000 1005
Leu Asp Leu Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu Ala Thr
lO10 1015 1020
SUBSTITUTE SHEET (RULE 26)
CA 02202~33 1997-04-11
W O 96/12019 PCTrUS95~1352
- 145 -
Thr ~hr Leu Gly Ser Ala Leu Ser Leu Pro Val Gly Thr Leu Asn Arg
1025 1030 1035 1040
Pro Arg Gly Ser Gln Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met Pro
1045 1050 1055
et Asn Gln Gly Asn Leu Gly Gly Ser Cys Gln Glu Ser Ala Val Ser
1060 1065 1070
ly Ser Ser Glu Arg Cys Pro Arg Pro Val Ser Leu His Pro Met Pro
1075 ~080 1085
Arg Gly Cys Leu Ala Ser Glu Ser Ser Glu Gly His Val Thr Gly Ser
1090 1095 1100
G1U Ala Glu Leu Gln Glu Lys Val Ser Met Cys Arg Ser Arg Ser Arg
1105 1110 1115 1120
Ser Arg Ser Pro Arg Pro Arg Gly Asp Ser Ala Tyr His Ser Gln Arg
1125 1130 1135
is Ser Leu Leu Thr Pro Val Thr Pro Leu Ser Pro Pro Gly Leu Glu
1140 1145 1150
lu G1U Asp Val Asn Gly Tyr Val Met Pro Asp Thr His Leu Lys Gly
1155 1160 1165
Thr Pro Ser Ser Arg Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Ser
1170 1175 1180
Val Leu Gly Thr Glu Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met
1185 1190 1195 1200
Asn Arg Arg Arg Arg His Ser Pro Pro His Pro Pro Arg Pro Ser Ser
1205 1210 1215
eu Glu Glu Leu Gly Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser
1220 1225 1230
la Ser Leu Gly Ser Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile
1235 1240 1245
Met Pro Thr Ala Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr Met Asn
1250 1255 1260
rg Gl.n Arg Asp Gly Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met Gly
1265 1270 1275 1280
Ala Cys Pro Ala Ser Glu Gln Gly Tyr Glu Glu Met Arg Ala Phe Gln
1285 1290 1295
ly Pro Gly His Gln Ala Pro His Val His Tyr Ala Ary Leu Lys Thr
1300 1305 1310
eu Arg Ser Leu Glu Ala Thr Asp Ser Ala Phe Asp Asn Pro Asp Tyr
1315 1320 1325
Trp His Ser Arg Leu Phe Pro Lys Ala Asn Ala Gln Arg Thr 1330
1335 1340
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 911 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 l997-04-ll
WO 96112019 PCI/US95/1352
- 146 -
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) S~QUENCE DESCRIPTION: SEQ ID NO:10:
et Lys Pro Ala Thr Gly Leu Trp Val Trp Val Ser Leu Leu Val Ala
la Gly Thr Val Gln Pro Ser Asp Ser Gln Ser Val Cys Ala Gly Thr
lu Asn Lys Leu Ser Ser Leu Ser Asp Leu Glu Gln Gln Tyr Arg Ala
Leu Arg Lys Tyr Tyr Glu Asn Cys Glu Val Val Met Gly Asn Leu Glu
Ile Thr Ser Ile Glu His Asn Arg Asp Leu Ser Phe Leu Arg Ser Val
rg Glu Val Thr Gly Tyr Val Leu Val Ala Leu Asn Gln Phe Arg Tyr
go 95
eu Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly Thr Lys Leu Tyr Glu
100 105 110
sp Arg Tyr Ala Leu Ala Ile Phe Leu Asn Tyr Arg Lys Asp Gly Asn
115 120 125
Phe Gly Leu Gln Glu Leu Gly Leu Lys Asn Leu Thr Glu Ile Leu Asn
130 135 140
Gly Gly Val Tyr Val Asp Gln Asn Lys Phe Leu Cys Tyr Ala Asp Thr
145 150 lSS 160
le His Trp Gln Asp Ile Val Arg Asn Pro Trp Pro Ser Asn Leu Thr
165 170 175
eu Val Ser Thr Asn Gly Ser Ser Gly Cys Gly Arg Cys His Lys Ser
180 185 190
ys Thr Gly Arg Cys Trp Gly Pro Thr Glu Asn His Cys Gln Thr Leu
l9S 200 205
Thr Arg Thr Val Cys Ala Glu Gln Cys Asp Gly Arg Cys Tyr Gly Pro
210 215 220
T'yrr Val Ser Asp Cys Cys His Arg Glu Cys Ala Gly Gly Cys Ser Gly
225 230 235 240
ro Lys Asp Thr Asp Cys Phe Ala Cys Met Asn Phe Asn Asp Ser Gly
245 250 255
la Cys Val Thr Gln Cys Pro Gln Thr Phe Val Tyr Asn Pro Thr Thr
260 265 270
he Gln Leu Glu His Asn Phe Asn Ala Lys Tyr Thr Tyr Gly Ala Phe
275 280 285
Cys Val Lys Lys Cys Pro His Asn Phe Val Val Asp Ser Ser Ser Cys
290 295 300
Val Arg Ala Cys Pro Ser Ser Lys Met Glu Val Glu Glu Asn Gly Ile
SUBSTITUTE SHEET (RULE 26~
-
CA 02202~33 1997-04-11
WO 96/12019 PCIIUS9511352
- 147 -
305 310 315 320
Lys Met Cys Lys Pro Cys Thr Asp Ile Cys Pro Lys Ala Cys Asp Gly
325 330 335
le Gly Thr Gly Ser Leu Met Ser Ala Gln Thr Val Asp Ser Ser Asn
.~ 340 345 350
Ile Asp Lys Phe Ile Asn Cys Thr Lys Ile Asn Gly Asn Leu Ile Phe
355 360 365
Leu Val Thr Gly Ile His Gly Asp Pro Tyr Asn Ala Ile Glu Ala Ile
370 375 380
Asp Pro Glu Lys Leu Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly
385 390 395 400
Phe Leu Asn Ile Gln Ser Trp Pro Pro Asn Met Thr Asp Phe Ser Val
405 410 415
Phe Ser Asn Leu Val Thr Ile Gly Gly Arg Val Leu Tyr Ser Gly Leu
420 425 430
Ser Leu Leu Ile Leu Lys Gln Gln Gly Ile Thr Ser Leu Gln Phe Gln
435 440 445
Ser Leu Lys Glu Ile Ser Ala Gly Asn Ile Tyr Ile Thr Asp Asn Ser
450 455 460
Asn Leu Cys Tyr Tyr His Thr Ile Asn Trp Thr Thr Leu Phe Ser Thr
465 470 475 480
Ile Asn Gln Arg Ile Val Ile Arg Asp Asn Arg Lys Ala Glu Asn Cys
485 490 495
Thr Ala Glu Gly Met Val Cys Asn His Leu Cys Ser Ser Asp Gly Cys
500 505 510
Trp Gly Pro Gly Pro Asp Gln Cys Leu Ser Cys Arg Arg Phe Ser Arg
515 520 525
Gly Arg Ile Cys Ile Glu Ser Cys Asn Leu Tyr Asp Gly Glu Phe Arg
530 535 540
Glu Phe Glu Asn Gly Ser Ile Cys Val Glu Cys Asp Pro Gln Cys Glu
545 550 555 560
Lys Met Glu Asp Gly Leu Leu Thr Cys His Gly Pro Gly Pro Asp Asn
565 570 575
Cys Thr Lys Cys Ser His Phe Lys Asp Gly Pro Asn Cys Val Glu Lys
5B0 585 590
Cys Pro Asp Gly Leu Gln Gly Ala Asn Ser Phe Ile Phe Lys Tyr Ala
595 600 605
Asp Pro Asp Arg Glu Cys His Pro Cys His Pro Asn Cys Thr Gln Gly
610 615 620
Cys Asn Gly Pro Thr Ser His Asp Cys Ile Tyr Tyr Pro Trp Thr Gly
625 630 635 640
~Iis Ser Thr Leu Pro Gln Asp Pro Val Lys Val Lys Ala Leu Glu Gly
645 650 655
SUBSTITUTE SHEET (RUI_E 26)
CA 02202~33 1997-04-11
W O96/12019 PCTrUS95/13~2
- 148 -
Phe Pro Arg Leu Val Gly Pro Asp Phe Phe Gly Cys Ala Glu Pro Ala
660 665 670
sn Thr Phe Leu Asp Pro Glu Glu Pro Lys Ser Cys Asp Lys Thr His
675 680 685
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
690 695 700
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
705 710 715 720
ro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
725 730 735
al Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Val Ala Lys
740 745 750
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
, 755 760 765
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
770 775 780
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
785 790 795 800
er Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
805 810 815
ro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
820 825 830
al Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
835 840 845
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
850 855 860
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
865 870 875 880
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
885 890 895
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
900 905 910
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 a~ino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Gly Xaa Gly Xaa Xaa Gly
1 5
(2) INFO~MATION FOR SEQ ID NO:12:
SUBSTITUTE SHEET (RULE 26)
. CA 02202533 1997-04-11
wo 96/12019 PCrnJS9511352
- 149 -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Asp Leu Ala Ala Arg Asn
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Pro Ile Dys Trp Met Ala
1 5
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: sin~le
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ACNGTNTGGG ARYTNAYHAC 20
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 ~ase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CAYGTNAARA THACNGAYTT YGG 23
(2~ INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26)
CA 02202~33 1997-04-11
W O9611Z019 PCTrUS95/13~2
- 150 -
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GACGAATTCC NATHAARTGG ATGGC 25
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
ACAYTTNARD ATDATCATRT ANAC 24
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
AANGTCATNA RYTCCCA 17
(2) INFORMATION FOR SEQ ID NO:lg:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(8) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
TCCAGNGCGA TCCAYTTDAT NGG 23
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
SUBSTITUTE SHEET (RULE 26~
CA 02202533 1997-04-11
W O96/12019 PCTnUS95~13524
- 151 -
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GGRTCDATCA TCCARCCT l8
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CTGCTGTCAG CATCGATCAT 20
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Thr Val Trp Glu Leu Met Thr
(2) INFORMATION FOR SEQ ID NO:23:
(i) S~:OU~N~' CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
His Val Lys Ile Thr Asp Phe Gly
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
W O96/12019 PCTrUS95/1352
- 152 -
(ii) MOLECULE TYPE: peptide
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Val Tyr Met Ile Ile Leu Lys
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Trp Glu Leu Met Thr Phe
l 5
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Pro Ile Lys Trp Met Ala Leu Glu
(2) INFORMATION FOR SEQ ID NO:27:
(i) S~:Q~N~: CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Cys Trp Met Ile Asp Pro
l 5
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
SUBSTITUTE SHEET (RULE 26~
CA 02202533 1997-04-11
W O9~/12019 PCTnUS9Sl13524
- 153 -
(ii) MOLECULE TYPE: DNA (genomic~
(xi) SEQUENCE DESCRIPTIoN: SEQ ID NO:28:
GACTCGAGTC GACATCGATT ~111111111 TTTTT 35
(~) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CXARACTERISTICS:
(A) LENGT~: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
~ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GAAGAAAGAC GACTCGTTCA TCGG 24
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPL: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
GACCATGACC ATGTAAACGT CAATA 25
(2) INFORMATION FOR SEQ ID NO:3l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3l:
Leu Ala Arg Leu Leu Glu Gly Asp Glu Lys Glu Tyr Asn Ala Asp Gly
l 5 l0 15
Gly
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
SUBSTITUTE SHEET (RULE 26)
CA 02202~33 1997-04-11
W O96/12019 PCTrUS9511352
- 154 -
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Glu Glu Asp Leu Glu Asp Met Met Asp Ala Glu Glu Tyr
l 5 lO
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Xaa
(B) LOCATION: 3
(D) OTHER INFORMATION: "Xaa = Any amino acid~
(ix) FEATURE:
(A) NAME/KEY: Xaa
(B) LOCATION: 6
(D) OTHER INFORMATION: "Xaa = Any amino acid~
(ix) FEATURE:
(A) NAME/KEY: Xaa
(B) LOCATION: 7
(D) OTHER INFORMATION: "Xaa = Any amino acid~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Ser Gly Xaa Lys Pro Xaa Xaa Ala Ala
l 5
(2) INFORMATION FOR SEQ ID NO:3s:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3~:
CGGAAGCTTC TAGAGATCCC TCGAC 25
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
~C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic~
SUBSTITUTE SHEET (RULE 26~
CA 02202533 1997-04-11
W O96/12019 ~CTnUS95l13524
- 155 -
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
; ~1LL1-1ACCT TTTTTATCTT ~1-11-~1~11C G~1--1-~1~1AT TTCACACGCC 50
(2~ INFORMATION FOR SEQ ID NO:36:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 baSe PairS
(B) TYPE: nUC1eiC aCid
(C) STRANDEDNESS: UnknOWn
(D) TOPOLOGY: UnknOWn
(ii) MOLECULE TYPE: DNA (genOmiC)
(Xi) S~:UU~N~ DESCRIPTION: SEQ ID NO 36
CAAAAATGGA AAAAATAGAA GAAACAGAAG CCATCTCATAA AGTGTGCGG 5O
(2) INFORMATION EOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 baSe pairs
(B) TYPE: nUC1eiC aCid
(C) STRANDEDNESS: UnknOWn
(D) TOPOLOGY: UnknOWn
(ii) MOLECULE TYPE: DNA (genOmiC)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
-1-1--1-11-C GCCTCCTTGA GATGATTAGA TCTCTG 36
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 baSe Pair5
(B) TYPE: nUC1eiC aCid
(C) STRANDEDNESS: UnknOWn
(D) TOPOLOGY: UnknOWn
(ii) MOLECULE TYPE: DNA (genOmiC)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
GTCAGAGTTC ATATGGTAGT TAAGCCCCCC CAAAAC 36
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 baSe Pair5
(B) TYPE: nUC1eiC aCid
(C) STRANDEDNESS: UnknOWn
(D~ TOPOLOGY: UnknOWn
r (ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
W O96112019 PCTrUS95/13~2
- 156 -
CAAAGATCCT CTAAGCTTGT AGAGTTCCTC CGATTTGTAA AAAGATGCCA TAACATAGTT 60
CTGGCAACGG TCGCCAGTAA ATTCGTTCGG GCACTTGCAC AAGTATCTTG ACGG 94
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Met Val Val Lys Pro Pro Gln Asn Lys Thr Glu Ser Glu Asn Thr Ser
l 5 l0 15
sp Lys Pro Lys Arg Lys Lys Lys Gly Gly Lys Asn Gly Lys Asn Arg
rg Asn Arg Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe
Cys Val Asn Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro
Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys
65 70 75 80
Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr
85 90 95
INFORMATION FOR SEQ ID NO:4l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1389 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: l..l386
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
ATG GTA GTT AAG CCC CCC CAA AAC AAG ACG GAA AGT GAA AAT ACT TCA 48
Met Val Val Lys Pro Pro Gln Asn Lys Thr Glu Ser Glu Asn Thr Ser
l s l0 15
GAT AAA CCC AAA AGA AAG AAA AAG GGA GGC AAA AAT GGA AAA AAT AGA 96
Asp Lys Pro Lys Arg Lys Lys Lys Gly Gly Lys Asn Gly Lys Asn Ary
20 25 30
AGA AAC AGA AGC CAT CTC ATA AAG TGT GCG GAG AAG GAG AAA ACT TTC 144
Arg Asn Arg Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe
35 40 45
TGT GTG AAT GGG GGC GAG TGC TTC ACG GTG AAG GAC CTG TCA AAC CCG l92
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
W ~96/12019 PCTnUS9~J1352
- 157 -
Cys Val A5n Gly Gly Glu Cys Ph~ Thr Val Lys Asp Leu Ser Asn Pro
_ TCA AGA TAC TTG TGC AAG TGC CCG AAC GAA TTT ACT GGC GAC CGT TGC 240
Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys
65 70 75 80
CA~ AAC TAT GTT ATG GCA TCT TTT TAC AAA GCG GAG GAA CTC TAC AAG 288
Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Lys
85 90 95
CTT ATG GCC GAG GAA GGC GGC AGC CTG GCC GCG CTG ACC GCG CAC CAG 336
Leu Met Ala Glu Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln
100 105 110
GCT TGC CAC CTG CCG CTG GAG ACT TTC ACC CGT CAT CGC CAG CCG CGC 384
Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg ~is Ary Gln Pro Arg
115 120 125
GGC TGG GAA CAA CTG GAG CAG TGC GGC TAT CCG GTG CAG CGG CTG GTC 432
Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val
130 135 140
GCC CTC TAC CTG GCG GCG CGG CTG TCG TGG AAC CAG GTC GAC CAG GTG 480
Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
145 150 155 160
ATC CGC AAC GCC CTG GCC AGC CCC GGC AGC GGC GGC GAC CTG GGC GAA 528
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu
165 170 175
GCG ATC CGC GAG CAG CCG GAG CAG GCC CGT CTG GCC CTG ACC CTG GCC 576
Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala
1~30 185 190
GCC GCC GAG AGC GAG CGC TTC GTC CGG CAG GGC ACC GGC AAC GAC GAG 624
Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu
195 200 205
GCC GGC GCG GCC AAC GCC GAC GTG GTG AGC CTG ACC TGC CCG GTC GCC 672
Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala
210 215 220
GCC GGT GAA TGC GCG GGC CCG GCG GAC AGC GGC GAC GCC CTG CTG GAG 720
Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu
225 230 235 240
CGC AAC TAT CCC ACT GGC GCG GAG TTC CTC GGC GAC GGC GGC GAC GTC 768
Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val
245 250 255
AGC TTC AGC ACC CGC GGC ACG CAG AAC TGG ACG GTG GAG CGG CTG CTC 816
Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu
260 265 270
CAG GCG CAC CGC CAA CTG GAG GAG CGC GGC TAT GTG TTC GTC GGC TAC 864
Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr
275 280 285
CAC GGC ACC TTC CTC GAA GCG GCG CAA AGC ATC GTC TTC GGC GGG GTG 912
His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val
290 295 300
CGC GCG CGC AGC CAG GAC CTC GAC GCG ATC TGG CGC GGT TTC TAT ATC 960
Ars Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile
305 310 315 320
SUBSTITUTE SHEET (RULE 26~
CA 02202~33 1997-04-11
WO96/12019 PCTrUS95/1352
- 158 -
GCC GGC GAT CCG GCG CTG GCC TAC GGC TAC GCC CAG GAC CAG GAA CCC 1008
Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro
325 330 335
GAC GCA CGC GGC CGG ATC CGC AAC GGT GCC CTG CTG CGG GTC TAT GTG 1056
Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val
340 345 350
CCG CGC TCG AGC CTG CCG GGC TTC TAC CGC ACC AGC CTG ACC CTG GCC 1104
Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala
355 360 365
GGC GGC GAG GCG GCG GGC GAG GTC GAA CGG CTG ATC GGC CAT CCG CTG 1152
Gly Gly Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu
370 375 380
CCG CTG CGC CTG GAC GCC ATC ACC GGC CCC GAG GAG GAA GGC GGG CGC 1200
Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg
3~5 390 395 400
CTG GAG ACC ATT CTC GGC TGG CCG CTG GCC GAG CGC ACC GTG GTG ATT 1248
Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile
405 410 415
CCC TCG GCG ATC CCC ACC GAC CCG CGC AAC GTC GGC GGC GAC CTC GAC 1296
Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp
420 425 430
CCG TCC AGC ATC CCC GAC AAG GAA CAG GCG ATC AGC GCC CTG CCG GAC 1344
Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp
435 440 445
TAC GCC AGC CAG CCC GGC AAA CCG CCG CGC GAG GAC CTG AAG 1386
Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
450 455 460
TAA
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Met Val Val Lys Pro Pro Gln Asn Lys Thr Glu Ser Glu Asn Thr Ser
1 5 10 15
sp Lys Pro Lys Arg Lys Lys Lys Gly Gly Lys Asn Gly Lys Asn Arg
Arg Asn Arg Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe
Cys Val Asn Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro
Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys
ln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Lys
eu Met Ala Glu Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln
SUBSTITUTE SHEET (RULE 26)
CA 02202~33 1997-04-11
W O96112019 PCTnUS9~1352
- 159 -
100 105 110
Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg
115 120 125
Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val
130 135 140
- Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
14S 150 155 160
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu
165 170 175
Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg heu Ala Leu Thr Leu Ala
180 185 190
Ala Ala GlU Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu
195 200 205
Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala
210 215 220
Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu
225 230 235 240
Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val
245 250 255
Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu
Z60 265 270
Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr
275 280 285
~is Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val
290 295 300
Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile
305 310 315 320
Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro
325 330 335
Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val
340 345 350
Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala
355 360 365
Gly Gly Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu
370 375 380
Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg
385 390 395 400
Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile
405 410 415
Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp
420 425 430
Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp
435 440 445
Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
4S0 45S 460
SUBSTITUTE SHEET (RULE 26~
