Note: Descriptions are shown in the official language in which they were submitted.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98106421
76 kDa 32 kD~,, and 50 kDa HELICOBACTER POLYPEPTIDES AND
' ('ORRESPONDING PQLYNUCLEOTIDE MOLECULES
The invention relates to Helicobacter polypeptides and
corresponding polynucleotide molecules that can be used in methods to prevent
or treat Helicobacter infection in mammals, such as humans.
Background of the Invention
Helicobacter is a genus of spiral, gram-negative bacteria that
colonize the gastrointestinal tracts of mammals. Several species colonize the
stomach, most notably H. pylori, H. heilmanii, H. felis, and H. mustelae.
Although H. pylori is the species most commonly associated with human
infection, H. heilmanii and H. felis have also been isolated from humans, but
at
lower frequencies than H. pylori. Helicobacter infects over 50% of adult
populations in developed countries and nearly 100% in developing countries
and some Pacific rim countries, making it one of the most prevalent infections
worldwide.
Helicobacter is routinely recovered from gastric biopsies of humans
with histological evidence of gastritis and peptic ulceration. Indeed, H.
pylori
is now recognized as an important pathogen of humans, in that the chronic
gastritis it causes is a risk factor for the development of peptic ulcer
diseases
and gastric carcinoma. It is thus highly desirable to develop safe and
effective
vaccines for preventing and treating Helicobacter infection.
A number of Helicobacter antigens have been characterized-or
isolated. These include urease, which is composed of two structural subunits
of
approximately 30 and 67 kDa (Hu et al., Infect. Immun. 58:992, 1990; Dunn et
al., J. Biol. Chem. 265:9464, 1990; Evans et al., Microbial Pathogenesis
10:15,
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-2-
1991; Labigne et al., J. Bact., 173:1920, 1991 ); the 87 kDa vacuolar
cytotoxin
(VacA) (Cover et al., J. Biol. Chem. 267:10570, 1992; Phadnis et al., Infect.
Immun. 62:1557, 1994; WO 93/18150); a 128 kDa immunodominant antigen
associated with the cytotoxin (CagA, also called TagA; WO 93/18150; U.S.
Patent No. 5,403,924); 13 and 58 kDa heat shock proteins HspA and HspB
(Suerbaum et al., Mol. Microbiol. 14:959, 1994; WO 93/18150); a 54 kDa
catalase {Hazell et al., J. Gen. Microbiol.137:57, 1991 ); a 15 kDa histidine-
rich
protein (Hpn) (Gilbert et al., Infect. Immun. 63:2682, 1995); a 20 kDa
membrane-associated lipoprotein (Kostrcynska et al., J. Bact. 176:5938, 1994);
a 30 kDa outer membrane protein (Bolin et al., J. Clin. Microbiol. 33:381,
1995); a lactofernn receptor (FR 2,724,936); and several porins, designated
HopA, HopB, HopC, HopD, and HopE, which have molecular weights of
48-67 kDa (Exner et al., Infect. Immun. 63:1567, 1995; Doig et al., J. Bact.
177:5447, 1995). Some of these proteins have been proposed as potential
vaccine antigens. In particular, urease is believed to be a vaccine candidate
(WO 94/9823; WO 95/22987; WO 95/3824; Michetti et al., Gastroenterology
107:1002, 1994). Nevertheless, it is thought that several antigens may
ultimately be necessary in a vaccine.
Summary of the Invention
The invention provides polynucleotide molecules that encode a
family of 76 kDa Helicobacter polypeptides, designated GHPO 386, GHPO
789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO
190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa polypeptide, -- -
designated GHPO 1360, and a SO kDa polypeptide, designated GHPO 750,
which can be used, e.g., in methods to prevent, treat, or diagnose
Helicobacter
infection. The polypeptides include those having the amino acid sequences
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-3-
shown in SEQ ID NOs:2-22 (even numbers), 66, and 68. Those skilled in the
art will understand that the invention also includes polynucleotide molecules
that encode mutants and derivatives of these polypeptides, which can result
from the addition, deletion, or substitution of non-essential amino acids, as
is
described further below.
In addition to the polynucleotide molecules described above, the
invention includes the corresponding polypeptides (i.e., polypeptides encoded
by the polynucleotide molecules of the invention, or fragments thereof), and
monospecific antibodies that specifically bind to these polypeptides.
The present invention has many applications and includes expression
cassettes, vectors, and cells transformed or transfected with the
polynucleotides
of the invention. Accordingly, the present invention provides (i) methods for
producing polypeptides of the invention in recombinant host systems and
related expression cassettes, vectors, and transformed or transfected cells;
(ii)
live vaccine vectors, such as pox virus, Salmonella typhimurium, and Vibrio
cholerae vectors, that contain polynucleotides of the invention (such vaccine
vectors being useful in, e.g., methods for preventing or treating Helicobacter
infection) in combination with a diiuent or carrier, and related
pharmaceutical
compositions and associated therapeutic and/or prophylactic methods; (iii)
therapeutic and/or prophylactic methods involving administration of
polynucleotide molecules, either in a naked form or formulated with a delivery
vehicle, polypeptides or mixtures of polypeptides, or monospecific antibodies
of the invention, and related pharmaceutical compositions; (iv) methods for
detecting the presence of Helicobacter in biological samples, which can--
involve the use of polynucleotide molecules, monospecific antibodies, or
polypeptides of the invention; and (v) methods for purifying polypeptides of
the invention by antibody-based affinity chromatography. -
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-4-
Brief Description of the Drawings
Figure 1 is an alignment of the predicted amino acid sequences of
GHPO 386 (SEQ ID N0:2), GHPO 789 (SEQ ID N0:4), and GHPO 1516
(SEQ ID N0:6), as well as a consensus sequence for the 76 kDa protein family.
Figure 2 is an alignment of the predicted amino acid sequences of
GHPO 1197 (SEQ ID N0:8), GHPO 1180 (SEQ ID NO:10), GHPO 896 (SEQ
ID N0:12), GHPO 711 (SEQ ID N0:14), GHPO 190 (SEQ ID N0:16), GHPO
185 (SEQ ID N0:18), GHPO 1417 (SEQ ID N0:20), and GHPO 1414 (SEQ
ID N0:22), as well as a consensus sequence for the 76 kDa protein family.
Detailed Description
Open reading frames (ORFs) encoding a family of new, full length,
membrane-associated 76 kDa polypeptides, designated GHPO 386, GHPO 789,
GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190,
GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa polypeptide, designated
GHPO 1360, and a 50 kDa polypeptide, designated GHPO 750, have been
identified in the H. pylori genome. The amino acid sequences of the 76 kDa
polypeptides are aligned in Figures 1 and 2. The 76 kDa, 32 kDa, and 50 kDa
polypeptides can be used, for example, in vaccination methods for preventing
or treating Helicobacter infection. For example, GHPO 750, GHPO 1360,
GHPO 190, and GHPO 1516 have been shown to be protective antigens. By
"protective antigen" is meant an antigen that is capable of reducing the
infection level after challenge, relative to a positive control. Absolute
protection from infection, although included in the invention, is not
required.
The polypeptides of the invention (except GHPO 750, see below) are
secreted polypeptides that can be produced in their mature forms (i.e., as
polypeptides that have been exported through class II or class III secretion
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-5-
pathways) or as precursors that include a signal peptide, which can be removed
in the course of excretion/secretion by cleavage at the N-terminal end of the
mature form. (The cleavage site is located at the C-terminal end of the signal
peptide, adjacent to the mature form.) The cleavage site for the polypeptides
of
the invention and, thus, the first amino acid of the mature polypeptides, was
putatively determined.
According to a first aspect of the invention, there are provided
isolated polynucleotides that encode the precursor and mature forms of
Helicobacter GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180,
GHPO 896, GHPO 71 l, GHPO 190, GHPO 185, GHPO 1417, GHPO 1414,
GHPO 1360, and GHPO 750.
An isolated polynucleotide of the invention encodes:
{i) a polypeptide having an amino acid sequence that is homologous
to a Helicobacter amino acid sequence of a polypeptide associated with the
Helicobacter membrane, the Helicobacter amino acid sequence being selected
from the group consisting of the amino acid sequences shown:
-in SEQ ID N0:2, beginning with an amino acid in any one of
positions -19 to 5, preferably in position -19 or position 1, and ending with
an
amino acid in position 689 (GHPO 386);
-in SEQ ID N0:4, beginning with an amino acid in any one of
positions -20 to 5, preferably in position -20 or position 1, and ending with
an
amino acid in position 713 (GHPO 789);
-in SEQ ID N0:6, beginning with an amino acid in any one of
~ positions -20 to 5, preferably in position -20 or position 1, and ending
with an
amino acid in position 725 (GHPO 1516);
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-6-
-in SEQ ID N0:8, beginning with an amino acid in any one of
positions -20 to 5, preferably in position -20 or position l, and ending with
an
amino acid in position 691 (GHPO 1197);
-in SEQ ID NO:10, beginning with an amino acid in any one of
S positions -20 to 5, preferably in position -20 or position 1, and ending
with an
amino acid in position 652 (GHPO 1180);
-in SEQ ID N0:12, beginning with an amino acid in any one of
positions -18 to 5, preferably in position -18 or position 1, and ending with
an
amino acid in position 673 (GHPO 896);
-in SEQ ID N0:14, beginning with an amino acid in any one of
positions -21 to 5, preferably in position -21 or position 1, and ending with
an
amino acid in position 619 (GHPO 711 );
-in SEQ ID N0:16, beginning with an amino acid in any one of
positions -17 to 5, preferably in position -17 or position l, and ending with
an
amino acid in position 635 (GHPO 190);
-in SEQ ID N0:18, beginning with an amino acid in any one of
positions -19 to 5, preferably in position -19 or position l, and ending with
an
amino acid in position 626 (GHPO 185);
-in SEQ ID N0:20, beginning with an amino acid in any one of
positions -16 to 5, preferably in position -16 or position 1, and ending with
an
amino acid in position 467 (GHPO 1417);
-in SEQ ID N0:22, beginning with an amino acid in any one of
positions -18 to S, preferably in position -18 or position 1, and ending with
an
amino acid in position 673 (GHPO 1414);
-in SEQ ID N0:66, beginning with an amino acid in any one of
positions -20 to 5, preferably in position -20 or position 1, and ending with
an
amino acid in position 279 (GHPO 1360); and
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
_'7_
-in SEQ ID N0:68, beginning with an amino acid in position 1 and
ending with an amino acid in position 399 (GHPO 750); or
~ (ii) a derivative of the polypeptide.
The term "isolated polynucleotide" is defined as a polynucleotide
that is removed from the environment in which it naturally occurs. For
example, a naturally-occurnng DNA molecule present in the genome of a
living bacteria or as part of a gene bank is not isolated, but the same
molecule,
separated from the remaining part of the bacterial genome, as a result of,
e.g., a
cloning event (amplification), is "isolated." Typically, an isolated DNA
molecule is free from DNA regions (e.g., coding regions) with which it is
immediately contiguous, at the 5' or 3' ends, in the naturally occurring
genome.
Such isolated polynucleotides can be part of a vector or a composition and
still
be isolated, as such a vector or composition is not part of its natural
environment.
A polynucleotide of the invention can consist of RNA or DNA (e.g.,
cDNA, genomic DNA, or synthetic DNA), or modifications or combinations of
RNA or DNA. The polynucleotide can be double-stranded or single-stranded
and, if single-stranded, can be the coding {sense) strand or the non-coding
(anti-
sense) strand. The sequences that encode polypeptides of the invention, as
shown in SEQ ID NOs:2-22 (even numbers), 66, and 68, can be (a) the coding
sequence as shown in SEQ ID NOs: l-21 (odd numbers), 65, and 67; (b) a
ribonucleotide sequence derived by transcription of (a); or (c) a different
coding sequence that, as a result of the redundancy or degeneracy of the
genetic
code, encodes the same polypeptides as the polynucleotide molecules having
the sequences illustrated in any of SEQ ID NOs:I-21 (odd numbers), 65, and
67. The polypeptides of the invention can be ones that are naturally secreted
or
excreted by, e.g., H. felis, H. mustelae, H. heilmanii, or H. pylori.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
_g_
By "polypeptide" or "protein" is meant any chain of amino acids,
regardless of length or post-translational modification (e.g., glycosylation
or
phosphorylation). Both terms are used interchangeably in the present
application.
By "homologous amino acid sequence" is meant an amino acid
sequence that differs from an amino acid sequence shown in any of SEQ ID
NOs:2-22 (even numbers), 66, and 68, or an amino acid sequence encoded by
the nucleotide sequence of any of SEQ ID NOs:I-21 (odd numbers), 65, and
67, by one or more non-conservative amino acid substitutions, deletions, or
additions located at positions at which they do not destroy the specific
antigenicity of the polypeptide. Preferably, such a sequence is at least 75%,
more preferably at least 80%, and most preferably at least 90% identical to an
amino acid sequence shown in any of SEQ ID NOs:2-22 (even numbers), 66,
and 68.
Homologous amino acid sequences include sequences that are
identical or substantially identical to an amino acid sequence as shown in any
of SEQ ID NOs:2-22 (even numbers), 66, and 68. By "amino acid sequence
that is substantially identical" is meant a sequence that is at least 90%,
preferably at least 95%, more preferably at least 97%, and most preferably at
least 99% identical to an amino acid sequence of reference and that differs
from
the sequence of reference, if at all, by a majority of conservative amino acid
substitutions.
Conservative amino acid substitutions typically include substitutions
among amino acids of the same class. These classes include, for example,
amino acids having uncharged polar side chains, such as asparagine, glutamine,
serine, threonine, and tyrosine; amino acids having basic side chains, such as
lysine, arginine, and histidine; amino acids having acidic side chains, such
as
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-9-
aspartic acid and glutamic acid; and amino acids having nonpolar side chains,
such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan, and cysteine.
Homology can be measured using sequence analysis software (e.g.,
Sequence Analysis Software Package of the Genetics Computer Group,
University of Wisconsin Biotechnology Center, 1710 University Avenue,
Madison, WI 53705). Similar amino acid sequences are aligned to obtain the
maximum degree of homology (i.e., identity). To this end, it may be necessary
to artificially introduce gaps into the sequence. Once the optimal alignment
has
been set up, the degree of homology (i.e., identity) is established by
recording
all of the positions in which the amino acids of both sequences are identical,
relative to the total number of positions.
Homologous polynucleotide sequences are defined in a similar way.
Preferably, a homologous sequence is one that is at least 45%, more preferably
at least 60%, and most preferably at least 85% identical to a coding sequence
of
any of SEQ ID NOs:l-21 (odd numbers), 65, and 67.
Polypeptides having a sequence homologous to one of the sequences
shown in SEQ ID NOs:2-22 (even numbers), 66, and 68 include naturally-
occurring allelic variants, as well as mutants or any other non-naturally
occurring variants that are analogous in terms of antigenicity, to a
polypeptide
having a sequence as shown in SEQ ID NOs:2-22 (even numbers), 66, and 68.
As is known in the art, an allelic variant is an alternate form of a
polypeptide that is characterized as having a substitution, deletion, or
addition
of one or more amino acids that does not alter the biological function of the
polypeptide. By "biological function" is meant a function of the polypeptide
in
the cells in which it naturally occurs, even if the function is not necessary
for
the growth or survival of the cells. For example, the biological function of a
CA 02286893 1999-10-O1
WO 98!43479 PCT/US98/06421
-10-
porin is to allow the entry into cells of compounds present in the
extracellular
medium. The biological function is distinct from the antigenic function. A
polypeptide can have more than one biological function.
Allelic variants are very common in nature. For example, a bacterial
species, e.g., H. pylori, is usually represented by a variety of strains that
differ
from each other by minor allelic variations. Indeed, a polypeptide that
fulfills
the same biological function in different strains can have an amino acid
sequence that is not identical in each of the strains. Such an allelic
variation
can be equally reflected at the polynucleotide level.
Support for the use of allelic variants of polypeptide antigens comes
from, e.g., studies of the Helicobacter urease antigen. The amino acid
sequence of Helicobacter urease varies widely from species to species, yet
cross-species protection occurs, indicating that the urease molecule, when
used
as an immunogen, is highly tolerant of amino acid variations. Even among
different strains of the single species H. pylori, there are amino acid
sequence
variations.
For example, although the amino acid sequences of the UreA and
Urea subunits of H. pylori and H. fells ureases differ from one another by
26.5% and 11.8%, respectively (Ferrero et al., Molecular Microbiology
9(2):323-333, 1993), it has been shown that H. pylori urease protects mice
from
H. fells infection (Michetti et al., Gastroenterology 107:1002, 1994). In
addition, it has beer~shown that the individual structural subunits of urease,
UreA and Urea, which contain distinct amino acid sequences, are both
protective antigens against Helicobacter infection (Michetti et al., supra}:
Similarly, Cuenca et al. (Gastroenterology 110:1770, 1996) showed that
therapeutic immunization of H. mustelae-infected ferrets with H. pylori urease
was effective at eradicating H. mustelae infection. Further, several urease
CA 02286893 1999-10-O1
WO 98/43479 PCT/LJS98/06421
-11-
variants have been reported to be effective vaccine antigens, including, e.g.,
recombinant UreA + Urea apoenzyme expressed from pORV 142 (UreA and
' UreB sequences derived from H. pylori strain CPM630; Lee et al., J. Infect.
Dis.172:161, 1995); recombinant UreA + Urea apoenzyme expressed from
pORV214 (UreA and Urea sequences differ from H. pylori strain CPM630 by
one and two amino acid changes, respectively; Lee et al., supra, 1995); a
UreA-giutathione-S-transferase fusion protein (UreA sequence from H. pylori
strain ATCC 43504; Thomas et al., Acta Gastro-Enterologica Belgica 56:54,
1993); UreA + Urea holoenzyme purified from H. pylori strain NCTCl 1637
(Marchetti et al., Science 267:1655, 1995); a UreA-MBP fusion protein (UreA
from H. pylori strain 85P; Ferrero et al., Infection and Immunity 62:4981,
1994); a Urea-MBP fusion protein (Urea from H. pylori strain 85P; Ferrero et
al., supra); a UreA-MBP fusion protein (UreA from H. fells strain ATCC
49179; Ferrero et al., supra); a Urea-MBP fusion protein (Urea from H. fells
strain ATCC 49179; Ferrero et al., supra}; and a 37 kDa fragment of Urea
containing amino acids 220-569 (Dore-Davin et al., "A 37 kD fragment of
Urea is sufficient to confer protection against Helicobacter fells infection
in
mice"). Finally, Thomas et al. (supra) showed that oral immunization of mice
with crude sonicates of H. pylori protected mice from subsequent challenge
with H. fells.
Polynucieotides, e.g., DNA molecules, encoding allelic variants can
easily be obtained by polymerase chain reaction (PCR) amplification of
genomic bacterial DNA extracted by conventional methods. This involves the
use of synthetic oligonucleotide primers matching sequences that are upstream
and downstream of the 5' and 3' ends of the coding region. Suitable primers
can be designed based on the nucleotide sequence information provided in SEQ
ID NOs:l-21 (odd numbers), 65, and 67. Typically, a primer consists of 10 to
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-12-
40, preferably 15 to 25 nucleotides. It can also be advantageous to select
primers containing C and G nucleotides in proportions sufficient to ensure
efficient hybridization, e.g., an amount of C and G nucleotides of at least
40%,
preferably 50%, of the total nucleotide amount. Those skilled in the art can
readily design primers that can be used to isolate the polynucleotides of the
invention from different Helicobacter strains.
As an example, primers useful for cloning a polynucleotide molecule
encoding a polypeptide having the amino acid sequence of unprocessed GHPO
386 (SEQ ID N0:2), including a signal peptide, are shown in SEQ ID N0:23
(matching at the 5' end) and in SEQ ID N0:25 {matching at the 3' end).
Primers useful for cloning a DNA molecule encoding a polypeptide having the
amino acid sequence of mature GHPO 386 (amino acids 1-689 of SEQ ID
N0:2), lacking a signal peptide, are shown in SEQ ID N0:24 (matching at the
5' end) and in SEQ ID N0:25 (matching at the 3' end). Primers useful for
cloning a DNA molecule encoding a polypeptide having the amino acid
sequence of GHPO 1360 (SEQ ID N0:66), are shown in SEQ ID N0:78
(matching at the S' end) and in SEQ ID N0:79 (matching at the 3' end). Use of
these primers enables amplification of the entire gene encoding GHPO 1360.
Primers having sequences shown in SEQ ID N0:82 (matching at the 5' end of
the coding sequence corresponding to the mature protein) and SEQ ID N0:79
(matching at the 3' end) can be used to amplify the portion of the gene
encoding
mature GHPO 1360. Experimental conditions for carrying out PCR can readily
be determined by one skilled in the art and illustrations of carrying out PCR
are
provided in Examples 3 and 4. - --
Thus, the first aspect of the invention includes:
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-13-
(i) isolated polynucleotide molecules (e.g., DNA molecules) that can
be amplified and/or cloned using the polymerase chain reaction from a
Helicobacter, e.g., H. pylori, genome using either:
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:23, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:25 (unprocessed GHPO 386);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:26, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:28 (unprocessed GHPO 789);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:29, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:31 (unprocessed GHPO 1 S 16);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:32, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:34 (unprocessed GHPO 1197);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:35, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:37 (unprocessed GHPO 1180);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:38, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:40 (unprocessed GHPO 896);
a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:41, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
- N0:43 (unprocessed GHPO 711 );
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:44, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:46 (unprocessed GHPO 190); -
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-14-
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:47, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:49 (unprocessed GHPO 185);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO:50, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:52 (unprocessed GHPO 1417);
a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:53, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:55 (unprocessed GHPO 1414);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:78, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:79 (unprocessed GHPO 1360); or
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:80, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:81 (GHPO 750); and
(ii) isolated polynucleotide molecules (e.g., DNA molecules) that can
be amplified and/or cloned by the polymerase chain reaction from a
Helicobacter, e.g., H. pylori, genome using either:
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:24, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:25 (mature GHPO 386);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:27, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:28 (mature GHPO 789); - --
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:30, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:31 (mature GHPO 1516);
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-15-
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:33, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:34 (mature GHPO 1197);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
S N0:36, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:37 (mature GHPO 1180);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:39, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:40 (mature GHPO 896);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:42, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:43 (mature GHPO 711 );
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:45, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:46 (mature GHPO 190);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:48, and a 3' oligonucieotide primer having a sequence as shown in SEQ ID
N0:49 (mature GHPO 185);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO:51, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:52 (mature GHPO 1417);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:54, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:55 (mature GHPO 1414); or - --
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:82, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:79 (mature GHPO 1360).
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-16-
The 5' ends of the primers described above can advantageously
include a restriction endonuclease recognition site that contains, typically,
4 to
6 nucleotides. For example, the sequences 5'-GGATCC-3' (BamHI) or 5'-
CTCGAG-3' (XhoI) can be used. Restriction sites can be selected by those
skilled in the art so that the amplified DNA, when digested, if necessary, can
be
conveniently cloned into an appropriately digested vector, such as a plasmid
vector. In addition, a 5' clamp (e.g., GCC) can be included in the primers 5'
to
the restriction endonuclease recognition site.
Useful homologs that do not occur naturally can be designed using
known methods for identifying regions of an antigen that are likely to be
tolerant of amino acid sequence changes and/or deletions. For example,
sequences of the antigen from different species can be compared to identify
conserved sequences.
Polypeptide derivatives that are encoded by polynucleotides of the
1 S invention include, e.g., fragments, polypeptides having large internal
deletions
derived from full-length polypeptides, and fusion proteins. Polypeptide
fragments of the invention can be derived from a polypeptide having a
sequence homologous to the sequences of any of SEQ ID NOs:2-22 (even
numbers), 66, and 68, to the extent that the fragments retain the substantial
antigenicity of the parent polypeptide (specific antigenicity). Polypeptide
derivatives can also be constructed by large internal deletions that remove a
substantial part of the parent polypeptide, while retaining specific
antigenicity.
Generally, polypeptide derivatives should be about at least 12 amino acids in
length to maintain antigenicity. Advantageously, they can be at least -20
amino
acids, preferably at least SO amino acids, more preferably at least 75 amino
acids, and most preferably at least 100 amino acids in length.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-17-
Useful polypeptide derivatives, e.g., polypeptide fragments, can be
designed using computer-assisted analysis of amino acid sequences in order to
identify sites in protein antigens having potential as surface-exposed,
antigenic
regions (Hughes et al., Infect. Immun. 60(9):3497, 1992). For example, the
Laser Gene Program from DNA Star can be used to obtain hydrophilicity,
antigenic index, and intensity index plots for the polypeptides of the
invention.
This program can also be used to obtain information about homologies of the
polypeptides with known protein motifs. One skilled in the art can readily use
the information provided in such plots to select peptide fragments for use as
vaccine antigens. For example, fragments spanning regions of the plots in
which the antigenic index is relatively high can be selected. One can also
select fragments spanning regions in which both the antigenic index and the
intensity plots are relatively high. Fragments containing conserved sequences,
particularly hydrophilic conserved sequences, can also be selected.
Polypeptide fragments and polypeptides having large internal
deletions can be used for revealing epitopes that are otherwise masked in the
parent polypeptide and that may be of importance for inducing a protective T
cell-dependent immune response. Deletions can also remove immunodominant
regions of high variability among strains.
It is an accepted practice in the field of immunology to use fragments
and variants of protein immunogens as vaccines, as all that is required to
induce
an immune response to a protein is a small (e.g., 8 to 10 amino acids)
immunogenic region of the protein. This has been done for a number of
vaccines against pathogens other than Helicobacter. For example, short --
synthetic peptides corresponding to surface-exposed antigens of pathogens such
as murine mammary tumor virus (peptide containing 11 amino acids; Dion et
al., Virology 179:474-477, 1990), Semliki Forest virus (peptide containing 16
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-18-
amino acids; Snijders et al., J. Gen. Virol. 72:557-565, 1991), and canine
parvovirus (2 overlapping peptides, each containing 15 amino acids; Langeveld
et al., Vaccine 12(15):1473-1480, 1994) have been shown to be effective
vaccine antigens against their respective pathogens.
Polynucleotides encoding polypeptide fragments and polypeptides
having large internal deletions can be constructed using standard methods
(see,
e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley &
Sons Inc., 1994), for example, by PCR, including inverse PCR, by restriction
enzyme treatment of the cloned DNA molecules, or by the method of Kunkel et
al. (Proc. Natl. Acad. Sci. USA 82:448, 1985; biological material available at
Stratagene) .
A polypeptide derivative can also be produced as a fusion
polypeptide that contains a polypeptide or a polypeptide derivative of the
invention fused, e.g., at the - or C-terminal end, to any other polypeptide
(hereinafter referred to as a peptide tail). Such a product can be easily
obtained
by translation of a genetic fusion, i.e., a hybrid gene. Vectors for
expressing
fusion polypeptides are commercially available, and include the pMal-c2 or
pMal-p2 systems of New England Biolabs, in which the peptide tail is a
maltose binding protein, the glutathione-S-transferase system of Pharmacia, or
the His-Tag system available from Novagen. These and other expression
systems provide convenient means for further purification of polypeptides and
derivatives of the invention.
Another particular example of fusion polypeptides included in
invention includes a polypeptide or polypeptide derivative of the inverrti-on
fused to a polypeptide having adjuvant activity, such as, e.g., subunit B of
either cholera toxin or E. toll heat-labile toxin. Several possibilities can
be
CA 02286893 1999-10-O1
WO 98/43479 PCTNS98/06421
-19-
used for producing such fusion proteins. First, the polypeptide of the
invention
can be fused to the
N-terminal end or, preferably, to the C-terminal end of the polypeptide having
adjuvant activity. Second, a polypeptide fragment of the invention can be
fused
within the amino acid sequence of the polypeptide having adjuvant activity.
Spacer sequences can also be included, if desired.
As stated above, the polynucleotides of the invention encode
Helicobacter polypeptides in precursor or mature form. They can also encode
hybrid precursors containing heterologous signal peptides, which can mature
into polypeptides of the invention. By "heterologous signal peptide" is meant
a
signal peptide that is not found in the naturally-occurnng precursor of a
polypeptide of the invention.
A polynucleotide of the invention hybridizes, preferably under
stringent conditions, to a polynucleotide having a sequence as shown in any of
SEQ ID NOs: l -21 (odd numbers), 65, and 67. Hybridization procedures are,
e.g., described by Ausubel et al. (supra); Silhavy et al. (Experiments with
Gene
Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York, 1984); and Davis et al. (A Manual for Genetic Engineering: Advanced
Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York, 1980). Important parameters that can be considered for optimizing
hybridization conditions are reflected in the following formula, which
facilitates calculatian of the melting temperature (Tm), which is the
temperature above which two complementary DNA strands separate from one
another (Casey et al., Nucl. Acid Res. 4:1539, 1977): Tm = 81.5 + 0.5 x-{%
G+C) + 1.6 log (positive ion concentration) - 0.6 x (% formamide). Under
appropriate stringency conditions, hybridization temperature (Th) is
approximately 20 to 40°C, 20 to 25°C, or, preferably, 30 to
40°C below the
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-20-
calculated Tm. Those skilled in the art will understand that optimal
temperature and salt conditions can be readily determined empirically in
preliminary experiments using conventional procedures. For example,
stringent conditions can be achieved, both for pre-hybridizing and hybridizing
incubations, (i) within 4-16 hours at 42°C, in 6 x SSC containing
SO% formamide or (ii) within 4-16 hours at 65°C in an aqueous 6 x
SSC
solution (1 M NaCI, 0.1 M sodium citrate (pH 7.0)). For polynucleotides
containing 30 to 600 nucleotides, the above formula is used and then is
corrected by subtracting (600/polynucleotide size in base pairs). Stringency
conditions are defined by a Th that is 5 to 10 ° C below Tm.
Hybridization conditions with oligonucleotides shorter than 20-30
bases do not precisely follow the rules set forth above. In such cases, the
formula for calculating the Tm is as follows: Tm = 4 x (G+C) + 2 (A+T). For
example, an 18 nucleotide fragment of 50% G+C would have an approximate
Tm of 54°C.
A polynucleotide molecule of the invention, containing RNA, DNA,
or modifications or combinations thereof, can have various applications. For
example, a polynucleotide molecule can be used (i) in a process for producing
the encoded polypeptide in a recombinant host system, (ii) in the construction
of vaccine vectors such as poxviruses, which are further used in methods and
compositions for preventing and/or treating Helicobacter infection, (iii) as a
vaccine agent, in a naked form or formulated with a delivery vehicle, and (iv)
in the construction of attenuated Helicobacter strains that can over-express a
polynucleotide of the invention or express it in a non-toxic, mutated form:
According to a second aspect of the invention, there is therefore
provided (i) an expression cassette containing a polynucleotide molecule of
the
invention placed under the control of elements (e.g., a promoter) required for
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98106421
-21-
expression; {ii) an expression vector containing an expression cassette of the
invention; (iii) a procaryotic or eucaryotic cell transformed or transfected
with
an expression cassette and/or vector of the invention; as well as (iv) a
process
for producing a polypeptide or polypeptide derivative encoded by a
polynucleotide of the invention, which involves culturing a procaryotic or
eucaryotic cell transformed or transfected with an expression cassette and/or
vector of the invention, under conditions that allow expression of the
polynucleotide molecule of the invention and, recovering the encoded
polypeptide or polypeptide derivative from the cell culture.
A recombinant expression system can be selected from procaryotic
and eucaryotic hosts. Eucaryotic hosts include, for example, yeast cells
(e.g.,
Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g., COS1,
NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9)
cells), and plant cells. Preferably, a procaryotic host such as E. coli is
used.
Bacterial and eucaryotic cells are available from a number of different
sources
that are known to those skilled in the art, e.g., the American Type Culture
Collection (ATCC; Rockville, Maryland).
The choice of the expression cassette will depend on the host system
selected, as well as the features desired for the expressed polypeptide. For
example, it may be useful to produce a polypeptide of the invention in a
particular lipidated form or any other form. Typically, an expression cassette
includes a constitutive or inducible promoter that is functional in the
selected
host system; a ribosome binding site; a start codon (ATG); if necessary, a
region encoding a signal peptide, e.g., a lipidation signal peptide; a - --
polynucleotide molecule of the invention; a stop codon; and, optionally, a 3'
terminal region (translation and/or transcription terminator). The signal
peptide-encoding region is adjacent to the polynucleotide of the invention and
CA 02286893 1999-10-O1
WO 98/43479 PCT/ITS98/06421
-22-
is placed in the proper reading frame. The signal peptide-encoding region can
be homologous or heterologous to the polynucleotide molecule encoding the
mature polypeptide and it can be specific to the secretion apparatus of the
host
used for expression. The open reading frame constituted by the polynucleotide
molecule of the invention, alone or together with the signal peptide, is
placed
under the control of the promoter so that transcription and translation occur
in
the host system. Promoters and signal peptide-encoding regions are widely
known and available to those skilled in the art and include, for example, the
promoter of Salmonella typhimurium (and derivatives) that is inducible by
arabinose (promoter araB) and is functional in Gram-negative bacteria such as
E. coli (U.S. Patent No. 5,028,530; Cagnon et al., Protein Engineering
4(7):843, 1991); the promoter of the bacteriophage T7 RNA polymerase gene,
which is functional in a number of E. coli strains expressing T7 polymerase
(U.S. Patent No. 4,952,496); the OspA lipidation signal peptide; and RIpB
lipidation signal peptide (Takase et al., J. Bact. 169:5692, 1987).
The expression cassette is typically part of an expression vector,
which is selected for its ability to replicate in the chosen expression
system.
Expression vectors (e.g., plasmids or viral vectors) can be chosen from, for
example, those described in Pouwels et al. (Cloning Vectors: A Laboratory
Manual, 1985, Supp. 1987) and can purchased from various commercial
sources. Methods for transforming or transfecting host cells with expression
vectors are well krrewn in the art and will depend on the host system
selected,
as described in Ausubel et al. (supra).
Upon expression, a recombinant polypeptide of the invention (or a
polypeptide derivative) is produced and remains in the intracellular
compartment, is secreted/excreted in the extracellular medium or in the
periplasmic space, or is embedded in the cellular membrane. The polypeptide
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-23-
can then be recovered in a substantially purified form from the cell extract
or
from the supernatant after centrifugation of the cell culture. Typically, the
recombinant polypeptide can be purified by antibody-based affinity
purification
or by any other method known to a person skilled in the art, such as by
genetic
fusion to a small aff nity-binding domain. Antibody-based affinity
purification
methods are also available for purifying a polypeptide of the invention
extracted from a Helicobacter strain. Antibodies useful for immunoaffinity
purification of the polypeptides of the invention can be obtained using
methods
described below.
Polynucleotides of the invention can also be used in DNA
vaccination methods, using either a viral or bacterial host as gene delivery
vehicle (live vaccine vector) or administering the gene in a free form, e.g.,
inserted into a plasmid. Therapeutic or prophylactic efficacy of a
polynucleotide of the invention can be evaluated as is described below.
Accordingly, in a third aspect of the invention, there is provided (i) a
vaccine vector such as a poxvirus, containing a polynucleotide molecule of the
invention placed under the control of elements required for expression; (ii) a
composition of matter containing a vaccine vector of the invention, together
with a diluent or carrier; (iii) a pharmaceutical composition containing a
therapeutically or prophylactically effective amount of a vaccine vector of
the
invention; (iv) a method for inducing an immune response against Helicobacter
in a mammal (e.g., a human; alternatively, the method can be used in
veterinary
applications for treating or preventing Helicobacter infection of animals,
e.g.,
cats or birds), which involves administering to the mammal an
immunogenically effective amount of a vaccine vector of the invention to
elicit
an immune response, e.g., a protective or therapeutic immune response to
Helicobacter; and (v) a method for preventing and/or treating a Helicobacter
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-24-
(e.g., H. pylori, H. felis, H. mustelae, or H. heilmanii) infection, which
involves
administering a prophylactic or therapeutic amount of a vaccine vector of the
invention to an individual in need. Additionally, the third aspect of the
invention encompasses the use of a vaccine vector of the invention in the
preparation of a medicament for preventing and/or treating Helicobacter
infection.
A vaccine vector of the invention can express one or several
polypeptides or derivatives of the invention, as well as at least one
additional
Helicobacter antigen such as a urease apoenzyme or a subunit, fragment,
homolog, mutant, or derivative thereof. In addition, it can express a
cytokine,
such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the
immune response. Thus, a vaccine vector can include an additional
polynucleotide molecules encoding, e.g., urease subunit A, B, or both, or a
cytokine, placed under the control of elements required for expression in a
mammalian cell.
Alternatively, a composition of the invention can include several
vaccine vectors, each of which being capable of expressing a polypeptide or
derivative of the invention. A composition can also contain a vaccine vector
capable of expressing an additional Helicobacter antigen such as urease
apoenzyme, a subunit, fragment, homolog, mutant, or derivative thereof, or a
cytokine such as IL-2 or IL-12.
In vaccination methods for treating or preventing infection in a
mammal, a vaccine vector of the invention can be administered by any
conventional route in use in the vaccine field, for example, to a mucosal
~e.g.,
ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or
urinary
tract) surface or via a parenteral (e.g., subcutaneous, intradermal,
intramuscular, intravenous, or intraperitoneal) route. Preferred routes depend
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-25-
upon the choice of the vaccine vector. The administration can be achieved in a
single dose or repeated at intervals. The appropriate dosage depends on
various
parameters that are understood by those skilled in the art, such as the nature
of
the vaccine vector itself, the route of administration, and the condition of
the
mammal to be vaccinated (e.g., the weight, age, and general health of the
mammal).
Live vaccine vectors that can be used in the invention include viral
vectors, such as adenoviruses and poxviruses, as well as bacterial vectors,
e.g.,
Shigella, Salmonella, Tlibrio cholerae, Lactobacillus, Bacille bilie de
Calmette-
Guerin (BCG), and Streptococcus. An example of an adenovirus vector, as
well as a method for constructing an adenovirus vector capable of expressing a
polynucleotide molecule of the invention, is described in U.S. Patent No.
4,920,209. Poxvirus vectors that can be used in the invention include, e.g.,
vaccinia and canary pox viruses, which are described in U.S. Patent No.
4,722,848 and U.S. Patent No. 5,364,773, respectively (also see, e.g.,
Tartaglia
et al., Virology 188:217, 1992, for a description of a vaccinia virus vector,
and
Taylor et al, Vaccine 13:539, 1995, for a description of a canary poxvirus
vector). Poxvirus vectors capable of expressing a polynucleotide of the
invention can be obtained by homologous recombination, as described in Kieny
et al. (Nature 312:163, 1984) so that the polynucleotide of the invention is
inserted in the viral genome under appropriate conditions for expression in
mammalian cells. Generally, the dose of viral vector vaccine, for therapeutic
or prophylactic use, can be from about 1 x 104 to about 1 x 10" ,
advantageously
from about 1 x 10' to about 1 x 10' °, or, preferably, from about 1 x
10' to about
1x109 plaque-forming units per kilogram. Preferably, viral vectors are
administered parenterally, for example, in 3 doses that are 4 weeks apart.
Those skilled in the art will recognize that it is preferable to avoid adding
a
__ __ __ _~_.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-26-
chemical adjuvant to a composition containing a viral vector of the invention
and thereby minimizing the immune response to the viral vector itself.
Non-toxicogenic Vibrio cholerae mutant strains that can be used in
live oral vaccines are described by Mekalanos et al. (Nature 306:551, 1983)
and in U.S. Patent No. 4,882,278 (strain in which a substantial amount of the
coding sequence of each of the two ctxA alleles has been deleted so that no
functional cholerae toxin is produced); WO 92/11354 (strain in which the irgA
locus is inactivated by mutation; this mutation can be combined in a single
strain with ctxA mutations); and WO 94/1533 (deletion mutant lacking
functional ctxA and attRSl DNA sequences). These strains can be genetically
engineered to express heterologous antigens, as described in WO 94/19482.
An effective vaccine dose of a V. cholerae strain capable of expressing a
polypeptide or polypeptide derivative encoded by a polynucleotide molecule of
the invention can contain, e.g., about 1 x 1 OS to about 1 x 109, preferably
about
1x106 to about 1x108 viable bacteria in an appropriate volume for the selected
route of administration. Preferred routes of administration include all
mucosal
routes, but, most preferably, these vectors are administered intranasally or
orally.
Attenuated Salmonella typhimurium strains, genetically engineered
for recombinant expression of heterologous antigens, and their use as oral
vaccines, are described by Nakayama et al. (Bio/Technology 6:693, 1988) and
in WO 92/11361. Preferred routes of administration for these vectors include
all mucosal routes. Most preferably, the vectors are administered intranasally
or orally. _ __
Others bacterial strains useful as vaccine vectors are described by
High et al. (EMBO 11:1991, 1992) and Sizemore et al. (Science 270:299,
1995; Shigella flexneri); Medaglini et al. (Proc. Natl. Acad. Sci. USA
92:6868,
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-27-
1995; (Streptococcus gordonii); Flynn (Cell. Mol. Biol. 40 (suppl. I):31,
1194),
and in WO 88/6626, WO 90/0594, WO 91/13157, WO 92/1796, and WO
92/21376 (Bacille Calmette Guerin). In bacterial vectors, a polynucleotide of
the invention can be inserted into the bacterial genome or it can remain in a
free
state, for example, carried on a plasmid.
An adjuvant can also be added to a composition containing a
bacterial vector vaccine. A number of adjuvants that can be used are known to
those skilled in the art. For example, preferred adjuvants can be selected
from
the list provided below.
According to a fourth aspect of the invention, there is also provided
(i) a composition of matter containing a polynucleotide of the invention,
together with a diluent or Garner; (ii) a pharmaceutical composition
containing
a therapeutically or prophylactically effective amount of a polynucleotide of
the
invention; {iii) a method for inducing an immune response against
Helicobacter, in a mammal, by administering to the mammal an
immunogenically effective amount of a polynucleotide of the invention to
elicit
an immune response, e.g., a protective immune response to Helicobacter; and
(iv) a method for preventing and/or treating a Helicobacter (e.g., H. pylori,
H.
fells, H. mustelae, or H. heilmanii) infection, by administering a
prophylactic or
therapeutic amount of a polynucleotide of the invention to an individual in
need
of such treatment. Additionally, the fourth aspect of the invention
encompasses
the use of a polynucleotide of the invention in the preparation of a
medicament
for preventing and/or treating Helicobacter infection. The fourth aspect of
the
invention preferably includes the use of a polynucleotide molecule placed
under conditions for expression in a mammalian cell, e.g., in a plasmid that
is
unable to replicate in mammalian cells and to substantially integrate into a
mammalian genome. -
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-28-
Polynucleotides (for example, DNA or RNA molecules) of the
invention can also be administered as such to a mammal as a vaccine. When a
DNA molecule of the invention is used, it can be in the form of a plasmid that
is unable to replicate in a mammalian cell and unable to integrate into the
mammalian genome. Typically, a DNA molecule is placed under the control of
a promoter suitable for expression in a mammalian cell. The promoter can
function ubiquitously or tissue-specifically. Examples of non-tissue specific
promoters include the early Cytomegalovirus {CMV) promoter (U.S. Patent
No. 4,168,062) and the Rous Sarcoma Virus promoter (Norton et al., Molec.
Cell Biol. 5:281, 1985). The desmin promoter (Li et al., Gene 78:243, 1989; Li
et al., J. Biol. Chem. 266:6562, 1991; Li et al., J. Biol. Chem. 268:10403,
1993) is tissue-specific and drives expression in muscle cells. More
generally,
useful promoters and vectors are described, e.g., in WO 94/21797 and by
Hartikka et al. (Human Gene Therapy 7:1205, 1996).
For DNA/RNA vaccination, the polynucleotide of the invention can
encode a precursor or a mature form of a polypeptide of the invention. When it
encodes a precursor form, the precursor sequence can be homologous or
heterologous. In the latter case, a eucaryotic leader sequence can be used,
such
as the leader sequence of the tissue-type plasminogen factor (tPA).
A composition of the invention can contain one or several
polynucleotides of the invention. It can also contain at least one additional
polynucleotide encoding another Helicobacter antigen, such as urease subunit
A, B, or both, or a fragment, derivative, mutant, or analog thereof. A
polynucleotide encoding a cytokine, such as interleukin-2 (IL-2) or
interleukin-
12 (IL-12), can also be added to the composition so that the immune response
is enhanced. These additional polynucleotides are placed under appropriate
control for expression. Advantageously, DNA molecules of the invention
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-29-
and/or additional DNA molecules to be included in the same composition are
carried in the same plasmid.
Standard methods can be used in the preparation of therapeutic
polynucleotides of the invention. For example, a polynucleotide can be used in
a naked form, free of any delivery vehicles, such as anionic liposomes,
cationic
lipids, microparticles, e.g., gold microparticles, precipitating agents, e.g.,
calcium phosphate, or any other transfection-facilitating agent. In this case,
the
polynucleotide can be simply diluted in a physiologically acceptable solution,
such as sterile saline or sterile buffered saline, with or without a carrier.
When
present, the carrier preferably is isotonic, hypotonic, or weakly hypertonic,
and
has a relatively low ionic strength, such as provided by a sucrose solution,
e.g.,
a solution containing 20% sucrose.
Alternatively, a polynucleotide can be associated with agents that
assist in cellular uptake. It can be, e.g., (i) complemented with a chemical
agent that modifies cellular permeability, such as bupivacaine (see, e.g.,
WO 94/16737), (ii) encapsulated into liposomes, or (iii) associated with
cationic lipids or silica, gold, or tungsten microparticles.
Anionic and neutral liposomes are well-known in the art (see, e.g.,
Liposomes: A Practical Approach, RPC New Ed, IRL Press, 1990, for a
detailed description of methods for making liposomes) and are useful for
delivering a large range of products, including polynucleotides.
Cationic lipids can also be used for gene delivery. Such lipids
include, for example, LipofectinTM, which is also known as DOTMA (N-[1-
(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2-
bis(oleyloxy)-3-(trimethylammonio)propane), DDAB
(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl
spermine), and cholesterol derivatives. A description of these cationic lipids
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-30-
can be found in EP 187,702, WO 90/11092, U.S. Patent No. 5,283,185,
WO 91/15501, WO 95/26356, and U.S. Patent No. 5,527,928. Cationic lipids
for gene delivery are preferably used in association with a neutral lipid such
as
DOPE (dioleyl phosphatidylethanolamine; WO 90/11092). Other transfection-
facilitating compounds can be added to a formulation containing cationic
liposomes. A number of them are described in, e.g., WO 93/18759,
WO 93/19768, WO 94/25608, and WO 95/2397. They include, e.g., spermine
derivatives useful for facilitating the transport of DNA through the nuclear
membrane (see, for example, WO 93/18759) and membrane-permeabilizing
compounds such as GALA, Gramicidine S, and cationic bile salts (see, for
example, WO 93/19768).
Gold or tungsten microparticles can also be used for gene delivery,
as described in WO 91/359, WO 93/17706, and by Tang et al. (Nature 356:152,
1992). In this case, the microparticle-coated polynucleotides can be injected
via intradermal or intraepidermal routes using a needleless injection device
("gene gun"), such as those described in U.S. Patent No. 4,945,050, U.S.
Patent
No. 5,015,580, and WO 94/24263.
The amount of DNA to be used in a vaccine recipient depends, e.g.,
on the strength of the promoter used in the DNA construct, the immunogenicity
of the expressed gene product, the condition of the mammal intended for
administration (e.g., the weight, age, and general health of the mammal), the
mode of administration, and the type of formulation. In general, a
therapeutically or prophylactically effective dose from about 1 ~.g to about
1 mg, preferably, from about 10 ~,g to about 800 ~.g, and, more preferably,
from
about 25 ~.g to about 250 ~,g, can be administered to human adults. The
administration can be achieved in a single dose or repeated at intervals.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-31-
The route of administration can be any conventional route used in the
vaccine field. As general guidance, a polynucleotide of the invention can be
administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary,
oral,
intestinal, rectal, vaginal, or urinary tract surface, or via a parenteral
route, e.g.,
by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal,
or intramuscular route. The choice of administration route will depend on,
e.g.,
the formulation that is selected. A polynucleotide formulated in association
with bupivacaine is advantageously administered into muscle. When a neutral
or anionic liposome or a cationic lipid, such as DOTMA, is used, the
formulation can be advantageously injected via intravenous, intranasal (for
example, by aerosolization), intramuscular, intradermal, and subcutaneous
routes. A polynucleotide in a naked form can advantageously be administered
via the intramuscular, intradenmal, or subcutaneous routes. Although not
absolutely required, such a composition can also contain an adjuvant. A
systemic adjuvant that does not require concomitant administration in order to
exhibit an adjuvant effect is preferable.
The sequence information provided in the present application enables
the design of specific nucleotide probes and primers that can be used in
diagnostic methods. Accordingly, in a fifth aspect of the invention, there is
provided a nucleotide probe or primer having a sequence found in, or derived
by degeneracy of the genetic code from, a sequence shown in any of SEQ ID
NOs:l-21 (odd numbers), 65, and 67, or a complementary sequence thereof.
' The term "probe" as used in the present application refers to DNA
(preferably single stranded) or RNA molecules (or modifications or - --
combinations thereof) that hybridize under the stringent conditions, as
defined
above, to polynucleotide molecules having sequences homologous to those
shown in any of SEQ ID NOs:l-21 (odd numbers), 65, and 67, or to a -
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-32-
complementary or anti-sense sequence of any of SEQ ID NOs: l-21 (odd
numbers), 65, and 67. Generally, probes are significantly shorter than the
full-
length sequences shown in any of SEQ ID NOs: l-21 (odd numbers), 65, and
67. For example, they can contain from about 5 to about 100, preferably from
about 10 to about 80 nucleotides. In particular, probes have sequences that
are
at least 75%, preferably at least 85%, more preferably 95% homologous to a
portion of a sequence as shown in any of SEQ ID NOs: l-21 (odd numbers), 65,
and 67, or a sequence complementary to such sequences.
Probes can contain modified bases, such as inosine, methyl-5-
deoxycytidine, deoxyuridine, dimethylamino-S-deoxyuridine, or diamino-2, 6-
purine. Sugar or phosphate residues can also be modified or substituted. For
example, a deoxyribose residue can be replaced by a polyamide (Nielsen et al.,
Science 254:1497, 1991 ) and phosphate residues can be replaced by ester
groups such as diphosphate, alkyl, arylphosphonate, and phosphorothioate
esters. In addition, the 2'-hydroxyl group on ribonucleotides can be modified
by addition of, e.g., alkyl groups.
Probes of the invention can be used in diagnostic tests, or as capture
or detection probes. Such capture probes can be immobilized on solid supports,
directly or indirectly, by covalent means or by passive adsorption. A
detection
probe can be labeled by a detectable label, for example a label selected from
radioactive isotopes; enzymes, such as peroxidase and alkaline phosphatase;
enzymes that are able to hydrolyze a chromogenic, fluorogenic, or luminescent
substrate; compounds that are chromogenic, fluorogenic, or luminescent;
nucleotide base analogs; and biotin. - --
Probes of the invention can be used in any conventional
hybridization method, such as in dot blot methods (Maniatis et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,-Cold
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-33-
Spring Harbor, New York, 1982), Southern blot methods (Southern, J. Mol.
Biol. 98:503, 1975), northern blot methods {identical to Southern blot to the
exception that RNA is used as a target), or a sandwich method (Dunn et al.,
Cell 12:23, 1977). As is known in the art, the latter technique involves the
use
of a specific capture probe and a specific detection probe that have
nucleotide
sequences that are at least partially different from each other.
Primers used in the invention usually contain about 10 to
40 nucleotides and are used to initiate enzymatic polymerization of DNA in an
amplification process (e.g., PCR), an elongation process, or a reverse
transcription method. In a diagnostic method involving PCR, the primers can
be labeled.
Thus, the invention also encompasses (i) a reagent containing a
probe of the invention for detecting and/or identifying the presence of
Helicobacter in a biological material; {ii) a method for detecting andlor
identifying the presence of Helicobacter in a biological material, in which
(a) a
sample is recovered or derived from the biological material, (b) DNA or RNA
is extracted from the material and denatured, and {c) the sample is exposed to
a
probe of the invention, for example, a capture probe, a detection probe, or
both,
under stringent hybridization conditions, so that hybridization is detected;
and
(iii) a method for detecting and/or identifying the presence of Helicobacter
in a
biological material, in which (a) a sample is recovered or derived from the
biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is
contacted with at least one, or, preferably two, primers of the invention, and
amplified by the polymerase chain reaction, and (d) an amplified DNA - --
molecule is produced.
As mentioned above; polypeptides that can be produced by
expression of the polynucieotides of the invention can be used as vaccine
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-34-
antigens: Accordingly, a sixth aspect of the invention features a
substantially
purified polypeptide or polypeptide derivative having an amino acid sequence
encoded by a polynucleotide of the invention.
A "substantially purified polypeptide" is defined as a polypeptide
that is separated from the environment in which it naturally occurs and/or a
polypeptide that is free of most of the other polypeptides that are present in
the
environment in which it was synthesized. The polypeptides of the invention
can be purified from a natural source, such as a Helicobacter strain, or can
be
produced using recombinant methods.
Homologous polypeptides or polypeptide derivatives encoded by
polynucleotides of the invention can be screened for specific antigenicity by
testing cross-reactivity with an antiserum raised against a polypeptide having
an amino acid sequence as shown in any of SEQ ID NOs:2-22 (even numbers),
66, and 68. Briefly, a monospecific hyperimmune antiserum can be raised
against a purified reference polypeptide as such or as a fusion polypeptide,
for
example, an expression product of MBP, GST, or His-tag systems, or a
synthetic peptide predicted to be antigenic. The homologous polypeptide or
derivative that is screened for specific antigenicity can be produced as such
or
as a fusion polypeptide. In the latter case, and if the antiserum is also
raised
against a fusion polypeptide, two different fusion systems are employed.
Specific antigenicity can be determined using a number of methods, including
Western blot (Towl~in et al., Proc. Natl. Acad. Sci. USA 76:4350, 1979), dot
blot, and ELISA methods, as described below.
In a Western blot assay, the product to be screened, either as-a-
purified preparation or a total E. coli extract, is fractionated by SDS-PAGE,
as
described, for example, by Laemmli (Nature 227:680, 1970). After being
transferred to a filter, such as a nitrocellulose membrane, the material is
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-35-
incubated with the monospecific hyperimmune antiserum, which is diluted in a
range of dilutions from about 1:50 to about 1:5000, preferably from about
1:100 to about 1:500. Specific antigenicity is shown once a band
corresponding to the product exhibits reactivity at any of the dilutions in
the
range.
In an ELISA assay, the product to be screened can be used as the
coating antigen. A purified preparation is preferred, but a whole cell extract
can also be used. Briefly, about 100 ~,1 of a preparation of about 10 ~.g
protein/ml is distributed into wells of a 96-well ELISA plate. The plate is
incubated for about 2 hours at 37°C, then overnight at 4°C. The
plate is
washed with phosphate buffered saline (PBS) containing 0.05% Tween 20
(PBS/Tween buffer) and the wells are saturated with 250 ~,l PBS containing
1% bovine serum albumin (BSA), to prevent non-specific antibody binding.
After 1 hour of incubation at 37°C, the plate is washed with PBS/Tween
buffer.
The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA,
and 100 p,l dilutions are added to each well. The plate is incubated for
90 minutes at 37 ° C, washed, and evaluated using standard methods. For
example, a goat anti-rabbit peroxidase conjugate can be added to the wells
when the specific antibodies used were raised in rabbits. Incubation is
carried
out for about 90 minutes at 37°C and the plate is washed. The reaction
is
developed with the appropriate substrate and the reaction is measured by
colorimetry (absorbance measured spectrophotometrically). Under these
experimental conditions, a positive reaction is shown once an O.D. value of
1.0
is detected with a dilution of at least about 1:50, preferably of at least
about
1:500.
In a dot blot assay, a purified product is preferred, although a whole
cell extract can be used. Briefly, a solution of the product at a
concentration of
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-36-
about 100 p,g/ml is serially diluted two-fold with 50 mM Tris-HCl (pH 7.5).
One hundred ~.1 of each dilution is applied to a filter, such as a 0.45 ~m
nitrocellulose membrane, set in a 96-well dot blot apparatus (Biorad). The
buffer is removed by applying vacuum to the system. Wells are washed by
addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried. The
membrane is saturated in blocking buffer (50 mM Tris-HCI (pH 7.5), 0.15 M
NaCI, 10 g/L skim milk) and incubated with an antiserum diluted from about
1:50 to about 1:5000, preferably about 1:500. The reaction is detected using
standard methods. For example, a goat anti-rabbit peroxidase conjugate can be
added to the wells when rabbit antibodies are used. Incubation is carried out
for about 90 minutes at 37°C and the blot is washed. The reaction is
developed
with the appropriate substrate and stopped. The reaction is then measured
visually by the appearance of a colored spot, e.g., by colorimetry. Under
these
experimental conditions, a positive reaction is associated with detection of a
colored spot for reactions carried out with a dilution of at least about 1:50,
preferably, of at least about 1:500. Therapeutic or prophylactic efficacy of a
polypeptide or polypeptide derivative of the invention can be evaluated as is
described below.
According to a seventh aspect of the invention, there is provided (i) a
composition of matter containing a polypeptide of the invention together with
a
diluent or carrier; (ii) a pharmaceutical composition containing a
therapeutically or prophylactically effective amount of a polypeptide of the
invention; (iii) a method for inducing an immune response against Helicobacter
in a mammal by administering to the mammal an immunogenically effective
amount of a polypeptide of the invention to elicit an immune response, e.g., a
protective immune response to Helicobacter; and (iv) a method for preventing
and/or treating a Helicobacter (e.g., H. pylori, H. felis, H. mustelae, or-H.
CA 02286893 1999-10-O1
WO 981434'79 PCT/US98lU6421
-37-
heilmanii) infection, by administering a prophylactic or therapeutic amount of
a
polypeptide of the invention to an individual in need of such treatment.
Additionally, this aspect of the invention includes the use of a polypeptide
of
the invention in the preparation of a medicament for preventing and/or
treating
Helicobacter infection.
The immunogenic compositions of the invention can be administered
by any conventional route in use in the vaccine field, for example, to a
mucosal
(e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal,
vaginal, or
urinary tract) surface or via a parenteral (e.g., subcutaneous, intradermal,
intramuscular, intravenous, or intraperitoneal) route. The choice of the
administration route depends upon a number of parameters, such as the
adjuvant used. For example, if a mucosal adjuvant is used, the intranasal or
oral route will be preferred, and if a lipid formulation or an aluminum
compound is used, a parenteral route will be preferred. In the latter case,
the
subcutaneous or intramuscular route is most preferred. The choice of
administration route can also depend upon the nature of the vaccine agent. For
example, a polypeptide of the invention fused to CTB or to LTB will be best
administered to a mucosal surface.
A composition of the invention can contain one or several
polypeptides or derivatives of the invention. It can also contain at least one
additional Helicobacter antigen, such as the urease apoenzyme, or a subunit,
fragment, homolog, mutant, or derivative thereof.
For use in a composition of the invention, a polypeptide or
polypeptide derivative can be formulated into or with liposomes, such as w
neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles
(VLPs), to facilitate delivery and/or enhance the immune response. These
compounds are readily available to those skilled in the art; for example; see
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-3 8-
Liposomes: A Practical Approach (supra). Adjuvants other than liposomes can
also be used in the invention and are well known in the art (see, for example,
the list provided below).
Administration can be achieved in a single dose or repeated as
necessary at intervals that can be determined by one skilled in the art. For
example, a priming dose can be followed by three booster doses at weekly or
monthly intervals. An appropriate dose depends on various parameters,
including the nature of the recipient (e.g., whether the recipient is an adult
or an
infant), the particular vaccine antigen, the route and frequency of
administration, the presence/absence or type of adjuvant, and the desired
effect
(e.g., protection and/or treatment), and can be readily determined by one
skilled
in the art. In general, a vaccine antigen of the invention can be administered
mucosally in an amount ranging from about 10 ~,g to about 500 mg, preferably
from about 1 mg to about 200 mg. For a parenteral route of administration, the
dose usually should not exceed about 1 mg, and is, preferably, about 100 ~,g.
When used as components of a vaccine, the polynucleotides and
polypeptides of the invention can be used sequentially as part of a multi-step
Immunization process. For example, a mammal can be initially primed with a
vaccine vector of the invention, such as a pox virus, e.g., via a parenteral
route,
and then boosted twice with a polypeptide encoded by the vaccine vector, e.g.,
via the mucosal route. In another example, liposomes associated with a
polypeptide or polypeptide derivative of the invention can be used for
priming,
with boosting being carried out mucosally using a soluble polypeptide or
polypeptide derivative of the invention, in combination with a mucosal - --
adjuvant (e.g., LT).
PoIypeptides and polypeptide derivatives of the invention can also be
used as diagnostic reagents for detecting the presence of anti-Helicobacter
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-39-
antibodies, e.g., in blood samples. Such polypeptides can be about 5 to about
80, preferably, about 10 to about 50 amino acids in length and can be labeled
or
unlabeled, depending upon the diagnostic method. Diagnostic methods
involving such a reagent are described below.
Upon expression of a polynucleotide molecule of the invention, a
polypeptide or polypeptide derivative is produced and can be purified using
known methods. For example, the polypeptide or polypeptide derivative can be
produced as a fusion protein containing a fused tail that facilitates
purification.
The fusion product can be used to immunize a small mammal, e.g., a mouse or
a rabbit, in order to raise monospecific antibodies against the polypeptide or
polypeptide derivative. The eighth aspect of the invention thus provides a
monospecific antibody that binds to a polypeptide or polypeptide derivative of
the invention.
By "monospecific antibody" is meant an antibody that is capable of
reacting with a unique, naturally-occurring Helicobacter polypeptide. An
antibody of the invention can be poiyclonal or monoclonal. Monospecific
antibodies can be recombinant, e.g., chimeric (e.g., consisting of a variable
region of murine origin and a human constant region), humanized (e.g., a
human immunoglobulin constant region and a variable region of animal, e.g.,
murine, origin), and/or single chain. Both polyclonal and monospecific
antibodies can also be in the form of immunoglobulin fragments, e.g., F(ab)'2
or Fab fragments. The antibodies of the invention can be of any isotype, e.g.,
IgG or IgA, and polyclonal antibodies can be of a single isotype or can
contain
- a mixture of isotypes.
The antibodies of the invention, which can be raised to a polypeptide
or polypeptide derivative of the invention, can be produced and identified
using
standard immunologicai assays, e.g., Western blot assays, dot blot assays, or
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/064Z1
-40-
ELISA (see, e.g., Coligan et al., Current Protocols in Immunology, John Wiley
& Sons, Inc., New York, NY, 1994). The antibodies can be used in diagnostic
methods to detect the presence of Helicobacter antigens in a sample, such as a
biological sample. The antibodies can also be used in affinity chromatography
methods for purifying a polypeptide or polypeptide derivative of the
invention.
As is discussed further below, the antibodies can also be used in prophylactic
and therapeutic passive immunization methods.
Accordingly, a ninth aspect of the invention provides (i) a reagent for
detecting the presence of Helicobacter in a biological sample that contains an
antibody, polypeptide, or polypeptide derivative of the invention; and (ii) a
diagnostic method for detecting the presence of Helicobacter in a biological
sample, by contacting the biological sample with an antibody, a polypeptide,
or
a polypeptide derivative of the invention, so that an immune complex is
formed, and detecting the complex as an indication of the presence of
Helicobacter in the sample or the organism from which the sample was
derived. The immune complex is formed between a component of the sample
and the antibody, polypeptide, or polypeptide derivative, and that any unbound
material can be removed prior to detecting the complex. A polypeptide reagent
can be used for detecting the presence of anti-Helicobacter antibodies in a
sample, e.g., a blood sample, while an antibody of the invention can be used
for
screening a sample, such as a gastric extract or biopsy sample, for the
presence
of Helicobacter polypeptides.
For use in diagnostic methods, the reagent (e.g., the antibody,
polypeptide, or polypeptide derivative of the invention) can be in a free
state or
can be immobilized on a solid support, such as, for example, on the interior
surface of a tube or on the surface, or within pores, of a bead.
Immobilization
can be achieved using direct or indirect means. Direct means include passive
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98106421
-41-
adsorption (i. e., non-covalent binding) or covalent binding between the
support
and the reagent. By "indirect means" is meant that an anti-reagent compound
that interacts with the reagent is first attached to the solid support. For
example, if a polypeptide reagent is used, an antibody that binds to it can
serve
as an anti-reagent, provided that it binds to an epitope that is not involved
in
recognition of antibodies in biological samples. Indirect means can also
employ a ligand-receptor system, for example, a molecule, such as a vitamin,
can be grafted onto the polypeptide reagent and the corresponding receptor can
be immobilized on the solid phase. This concept is illustrated by the well
known biotin-streptavidin system. Alternatively, indirect means can be used,
e.g., by adding to the reagent a peptide tail, chemically or by genetic
engineering, and immobilizing the grafted or fused product by passive
adsorption or covalent linkage of the peptide tail.
According to a tenth aspect of the invention, there is provided a
process for purifying, from a biological sample, a polypeptide or polypeptide
derivative of the invention, which involves carrying out antibody-based
affinity
chromatography with the biological sample, wherein the antibody is a
monospecific antibody of the invention.
For use in a purification process of the invention, the antibody can be
polyclonal or monospecific, and preferably is of the IgG type. Purified IgGs
can be prepared from an antiserum using standard methods (see, e.g., Coligan
et al., supra). Conventional chromatography supports, as well as standard
methods for grafting antibodies, are described, for example, by Harlow et al.
(Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Cold
Spring Harbor, New York, 1988).
Briefly, a biological sample, such as an H. pylori extract, preferably
in a buffer solution, is applied to a chromatography material, which is, -
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-42-
preferably, equilibrated with the buffer used to dilute the biological sample,
so
that the polypeptide or polypeptide derivative of the invention {i.e., the
antigen)
is allowed to adsorb onto the material. The chromatography material, such as a
gel or a resin coupled to an antibody of the invention, can be in batch form
or in
a column. The unbound components are washed off and the antigen is eluted
with an appropriate elution buffer, such as a glycine buffer, a buffer
containing
a chaotropic agent, e.g., guanidine HCI, or a buffer having high salt
concentration (e.g., 3 M MgCl2). Eluted fractions are recovered and the
presence of the antigen is detected, e.g., by measuring the absorbance at 280
nm.
An antibody of the invention can be screened for therapeutic efficacy
as follows. According to an eleventh aspect of the invention, there is
provided
(i) a composition of matter containing a monospecific antibody of the
invention, together with a diluent or carrier; (ii) a pharmaceutical
composition
containing a therapeutically or prophylactically effective amount of a
monospecific antibody of the invention; and (iii) a method for treating or
preventing Helicobacter (e.g., H. pylori, H. felis, H. mustelae, or H.
heilmanii)
infection, by administering a therapeutic or prophylactic amount of a
monospecific antibody of the invention to an individual in need of such
treatment. In addition, the eleventh aspect of the invention includes the use
of a
monospecific antibody of the invention in the preparation of a medicament for
treating or preventing Helicobacter infection.
The monospecific antibody can be polyclonal or monoclonal, and is,
preferably, predominantly of the IgA isotype. In passive immunization- --
methods, the antibody is administered to a mucosal surface of a mammal, e.g.,
the gastric mucosa, e.g., orally or intragastrically, optionally, in the
presence of
a bicarbonate buffer. Alternatively, systemic administration, not requiring a
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-43-
bicarbonate buffer, can be earned out. A monospecific antibody of the
invention can be administered as a single active agent or as a mixture with at
least one additional monospecific antibody specific for a different
Helicobacter
polypeptide. The amount of antibody and the particular regimen used can be
readily determined by one skilled in the art. For example, daily
administration
of about 100 to 1,000 mg of antibody over one week, or three doses per day of
about 100 to 1,000 mg of antibody over two or three days, can be effective
regimens for most purposes. .
Therapeutic or prophylactic efficacy can be evaluated using standard
methods in the art, e.g., by measuring induction of a mucosal immune response
or induction of protective and/or therapeutic immunity, using, e.g., the H.
fells
mouse model and the procedures described by Lee et al. (Eur. J.
Gastroenterology & Hepatology 7:303, 1995) or Lee et al. (J. Infect. Dis.
172:161, 1995). Those skilled in the art will recognize that the H. fells
strain of
the model can be replaced with another Helicobacter strain. For example, the
efficacy of polynucleotide molecules and polypeptides from H. pylori is,
preferably, evaluated in a mouse model using an H. pylori strain. Protection
can be determined by comparing the degree of Helicobacter infection in the
gastric tissue assessed by, for example, urease activity, bacterial counts, or
gastritis, to that of a control group. Protection is shown when infection is
reduced by comparison to the control group. Such an evaluation can be made
for polynucleotides, vaccine vectors, polypeptides, and polypeptide
derivatives,
as well as for antibodies of the invention.
For example, various doses of an antibody of the invention can-be
administered to the gastric mucosa of mice previously challenged with an H.
pylori strain, as described, e.g., by Lee et al. (supra). Then, after an
appropriate period of time, the bacterial load of the mucosa can be estimated
by
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-44-
assessing urease activity, as compared to a control. Reduced urease activity
indicates that the antibody is therapeutically effective.
Adjuvants that can be used in any of the vaccine compositions
described above are described as follows. Adjuvants for parenteral
administration include, for example, aluminum compounds, such as aluminum
hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The
antigen can be precipitated with, or adsorbed onto, the aluminum compound
using standard methods. Other adjuvants, such as RIBI (ImmunoChem,
Hamilton, MT), can also be used in parenteral administration.
Adjuvants that can be used for mucosal administration include, for
example, bacterial toxins, e.g., the cholera toxin (CT), the E. toll heat-
labile
toxin (LT), the Clostridium docile toxin A, the pertussis toxin (PT), and
combinations, subunits, toxoids, or mutants thereof. For example, a purified
preparation of native cholera toxin subunit B (CTB) can be used. Fragments,
homologs, derivatives, and fusions to any of these toxins can also be used,
provided that they retain adjuvant activity. Preferably, a mutant having
reduced toxicity is used. Suitable mutants are described, e.g., in WO 95/17211
(Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO
95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants
that can be used in the methods and compositions of the invention include,
e.g.,
Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-1 I2-Asp mutants. Other
adjuvants, such as the bacterial monophosphoryl lipid A (MPLA) of, e.g., E.
toll, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri;
saponins, and polylactide glycolide (PLGA) microspheres, can also be used in
mucosal administration. Adjuvants useful for both mucosal and parenteral
administrations, such as polyphosphazene (WO 95/2415), can also be used.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-45-
Any pharmaceutical composition of the invention, containing a
polynucleotide, polypeptide, polypeptide derivative, or antibody of the
invention, can be manufactured using standard methods. It can be formulated
with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline
solution, such as PBS, optionally, including a bicarbonate salt, such as
sodium
bicarbonate, e.g., 0.1 to 0.5 M. Bicarbonate can advantageously be added to
compositions intended for oral or intragastric administration. In general, a
diluent or carrier can be selected on the basis of the mode and route of
administration, and standard pharmaceutical practice. Suitable pharmaceutical
carriers and diluents, as well as pharmaceutical necessities for their use in
pharmaceutical formulations, are described in Remington's Pharmaceutical
Sciences, a standard reference text in this field and in the USP/NF.
The invention also includes methods in which gastroduodenal
infections, such as Helicobacter infection, are treated by oral administration
of
a Helicobacter polypeptide of the invention and a mucosal adjuvant, in
combination with an antibiotic, an antisecretory agent, a bismuth salt, an
antacid, sucralfate, or a combination thereof. Examples of such compounds
that can be administered with the vaccine antigen and an adjuvant are
antibiotics, including, e.g., macrolides, tetracyclines, ~i-lactams,
aminoglycosides, quinolones, penicillins, and derivatives thereof (specific
examples of antibiotics that can be used in the invention include, e.g.,
amoxicillin, clarithromycin, tetracycline, metronidizole, erythromycin,
cefuroxime, and erythromycin); antisecretory agents, including, e.g., H2-
receptor antagonists (e.g., cimetidine, ranitidine, famotidine, nizatidine;
and
roxatidine), proton pump inhibitors (e.g., omeprazole, lansoprazole, and
pantoprazole), prostaglandin analogs (e.g., misoprostil and enprostil), and
anticholinergic agents (e.g., pirenzepine, telenzepine, carbenoxolone, and
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-46-
proglumide); and bismuth salts, including colloidal bismuth subcitrate,
tripotassium dicitrate bismuthate, bismuth subsalicylate, bicitropeptide, and
pepto-bismol (see, e.g., Goodwin et al., Helicobacter pylori, Biology and
Clinical Practice, CRC Press, Boca Raton, FL, pp 366-395, 1993; Physicians'
S Desk Reference, 49"' edn., Medical Economics Data Production Company,
Montvale, New Jersey, 1995). In addition, compounds containing more than
one of the above-listed components coupled together, e.g., ranitidine coupled
to
bismuth subcitrate, can be used. The invention also includes compositions for
carrying out these methods, i. e., compositions containing a Helicobacter
antigen (or antigens) of the invention, an adjuvant, and one or more of the
above-listed compounds, in a pharmaceutically acceptable carrier or diluent.
Amounts of the above-listed compounds used in the methods and
compositions of the invention can readily be determined by one skilled in the
art. In addition, one skilled in the art can readily design
treatmentlimmunization schedules. For example, the non-vaccine components
can be administered on days 1-14, and the vaccine antigen + adjuvant can be
administered on days 7, 14, 21, and 28.
Methods and pharmaceutical compositions of the invention can be
used to treat or to prevent Helicobacter infections and, accordingly,
gastroduodenal diseases associated with these infections, including acute,
chronic, and atrophic gastritis, and peptic ulcer diseases, e.g., gastric and
duodenal ulcers. a.
A 76 kDa protein band containing GHPO 386, GHPO 789, and
GHPO 1516 (hereinafter the "purified 76 kDa proteins"), GHPO 1360 and
GHPO 750 were purified from Helicobacter pylori strain ATCC number 43579
(American Type Culture Collection, Rockville, Maryland) by immunoaffinity-
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-47-
based chromatography using the methods described below in Example 1, and
were shown to be effective vaccine antigens as follows.
Groups of 10 mice each were orally immunized with l, 5, or 25 ~.g of
the purified 76 kDa proteins, purified GHPO 1360, or purified GHPO 750 in
combination with 5 ~,g of the heat-labile enterotoxin (LT) of E. coli. Twenty
five ~.g of recombinant urease, in combination with 5 ~g LT, was used as a
positive control, and 5 ~g of LT in PBS was used as a negative control. The
immunizations were carried out four times each, on days 0, 7, 14, and 21 of
the
experiment. On day 33, blood samples were collected from the mice and, on
day 34, saliva samples were collected. On day 35, all of the mice were
challenged by intragastric administration of 1 x 10' streptomycin-resistant,
mouse-adapted H. pylori. On day 49, additional saliva samples were collected
and, about two weeks after challenge, on days 52-53, the mice were sacrificed.
Stomachs were removed from the mice and were analyzed for Helicobacter
infection by measuring urease activity in the intact stomach tissue and by a
quantitative culture study (Table 1 ).
Briefly, these studies showed that the gastric urease activities in
samples from mice immunized with all three amounts of the purified 76 kDa
proteins (i.e., 1, 5, and 25 ~.g), in combination with LT, were generally
lower
than the gastric urease activities of samples from mice immunized with LT
alone or mice that were not treated prior to challenge. Levels of gastric
urease
activity generally decreased with increasing amounts of the protein
administered, with the gastric urease activity levels for the 25 ~,g doses
generally approaching those of mice immunized with 25 ~.g of recombinant
urease and LT.
The quantitative culture analyses showed that the levels of
Helicobacter detected in the stomachs of mice immunized with the purified 76
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-48-
kDa proteins, purified GHPO 1360, or purified GHPO 750, which generally
decreased with increasing dosages, were less than the levels detected in the
stomachs of control mice that were immunized with LT alone or untreated
before Helicobacter challenge (Tables 1 and 2). The percentages of mice
protected by immunization with the purified 76 kDa proteins, purified GHPO
1360, or purified GHPO 750 met or approached the percentages of mice
protected by treatment with urease (Tables 1 and 2). These results show that
the purified 76 kDa proteins, GHPO 1360, and GHPO 750 are effective vaccine
antigens for use in preventing Helicobacter infection.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-49-
Table 1
Prophylactic
Immunization
with PMsv
Antigens
as
Oral Dose
Response
Against H.
pylori Challenge
BALB/c mice Fisher's exactCFU/ml (1/4 Wilcoxon rank
test antrum) sums
# mice infectedinfection Mean t SD test
status (based
Antrum on quantitative CFU treatment
Asso group
(based on ratios, treatment v. LT only
quantitative control
Asso, 0.148 group v. LT (group 1 1
O.D. only )
Treatment cutoff) (group 11))p-value p-value
S I ug 50 kDa 60% (6/10) 0.3034 30825 f 23210 0.1736
+ LT
pg 50 kDa 40% (4/10) 0.0573 18910 t 16341 0.0588
+ LT
25 pg 50 kDa 30% (3/10) 0.0198 22710 f 32397 0.0821
+ LT
1 pg 32 kDa+ 50% (5/10) 0.1409 44225 f 87824 0.0756
LT
5 Etg 50 kDa I 0% ( 1 / 0.0011 11811 t l 15?90.0191
+ LT I 0)
1 25 pg 50 kDa 0 (0/9) 0.0001 1608 t 23917 0.01 I 4
~ + LT
25 wg rUre 0 (0/9) 0.0001 8208 t 8021 0.0179
+ LT
LT 90% (9/10) - 107340 t 127949-
90% (9/10) not determined46173 t 42325 0.2568
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-50-
Table 2
Prophylactic
Immunization
with PMsv
Antigens
as
Oral Dose
Response
Against H.
pylori Challenge
BALB/c mice Fisher's exactCFU/ml (1/4 Wilcoxon rank
test antrum) sums
# mice infectedinfection Mean t SD test
status (based
Antrum on quantitative CFU treatment
Asso group
(based on ratios, treatment v. LT only
quantitative control
Asso, 0.148 group v. LT (group 1 1
O.D. only )
Treatment cutoff) (group 11 p-value
)) p-value
1 pg 76 kDa 56% (5/9) 0.1409 39922 f 34708 0.2203
+ LT
5 pg 76 kDa 8U% (415) 1 8802 t 7788 0.0864
+ LT
25 pg 76 kDa 33% (3/9) 0.0198 9712 t 12183 0.0178
+ LT
25 pg rUre 0 (0/9) 0.0001 8208 t 8021 0.017)
+ LT
LT 90% (9/lOj - 107340 t 127949-
90% (9/10) not determined46173 f 42325 0.2568
The invention is further illustrated by the following examples.
Example 1 describes purification of GHPO 1516 (76 kDa), GHPO 1360 (32
kDa), and GHPO 750 (50 kDa) from Helicobacter cultures. Example 2
describes identification of genes, e.g., genes encoding 76 kDa proteins, such
as
GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896,
GHPO 7I l, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa
protein (GHPO 1360), and a 50 kDa protein (GHPO 750) in the Helicobacter
genome, as well as identification of signal sequences, and primer design for
amplification of genes lacking signal sequences. Example 3 describes cloning
of DNA encoding GHPO 386, GHPO 789, GHPO 1 S 16, GHPO 896, GHPO
1360, and GHPO 750 into a vector that provides a histidine tag, and production
and purification of the resulting his-tagged fusion proteins. Example 4
describes methods for cloning DNA encoding the polypeptides of the invention
so that they can be produced without His-tags, Example 5 describes methods
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-51-
for purifying recombinant polypeptides of the invention, and Example 6
describes use of the GHPO 1360 polypeptide as a serodiagnostic tool for H.
pylori infection
EXAMPLE 1: Purification and partial sequence analysis of GHPO 1516
(76 ltDa), GHPO 1360 (32 lzDa), and GHPO 750 (50 kDa) protein from
Helicobacter pylori
1.A. Culture and initial purification steps
Frozen seeds from H. pylori strain ATCC 43579 are used to seed a
75 cm2 flask containing a biphasic medium (a solid phase made of Colombia
gelose containing 6% fresh sheep blood and a liquid phase made of triptcase
soja containing 20% fetal calf serum). After 24 hours of culturing under
microaerophilic conditions, the liquid phase is used for seeding several 75
cm2
flasks containing biphasic medium lacking sheep blood. After 24 hours of
culture, the liquid phase is used to seed a 2 L biofermentor in triptcase soja
liquid phase containing 10 g/L beta-cyclodextrine. At OD 1.5-1.8, this culture
is diluted in a 10 L biofermentor containing the liquid medium. After 24
hours,
the bacteria are spun in a centrifuge at 4,000 x g for 30 minutes at
4°C. A 10 L
culture contains about 20 to 30 g (wet weight) bacteria.
The pellet obtained using the method described above is washed with
500 ml PBS (7.650 g NaCI, 0.724 g disodium phosphate, and 0.210 g
monopotassium phosphate for one liter (pH 7.2)) for a one liter culture. The
- bacteria are then spun in a centrifuge again under the same conditions. - -
The pellet (C 1 } is suspended in 1 % N-octyl-D-glucopyranoside
(NOG; 30 ml/L; Sigma). The bacterial suspension is incubated for 1 hour at
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-52-
room temperature while stirring, spun in a centrifuge at 17,600 x g for
30 minutes at 4°C, and the pellet (C2) is recovered.
The supernatant (S2) is dialyzed against PBS overnight at 4°C
while
stirring. The precipitate is recovered by centrifugation at 2,600 x g for
30 minutes at 4°C. The supernatant (S2d) is discarded and the pellet
(Cs2d) is
recovered and stored at -20°C.
The pellet (C2) is resuspended in 20 mM Tris-HCl buffer (pH 7.5)
and
100 ~.M Pefabloc (Buffer A), and is homogenized with an ultra-turrax (3821,
Janke and Kungel). Lysozyme and EDTA are added at 0.1 mg/ml and 1 mM,
respectively.
The homogenate is sonicated three times for 2 minutes each at 4°C,
and then is spun in an ultracentrifuge at 210,000 x g for 30 minutes at
4°C. The
supernatant (S3), which contains the cytoplasmic and periplasmic proteins, is
eliminated, while the pellet is recovered, washed with buffer A, and spun in
an
ultracentrifuge at 210,000 x g for 30 minutes at 4°C. The supernatant
(S4) is
eliminated and the pellet (C4) is stored at -20°C. This pellet (C4)
contains
membrane proteins.
The pellet (C4) is washed in 50 mM NaCO, (pH 9.5) and 100 ~M
Pefabloc (buffer B). The suspension is spun in an ultracentrifuge at
210,000 x g for 30 minutes at 4°C. The supernatant (SS) is eliminated,
and the
pellet (CS) is then washed and spun in an ultracentrifuge as is described
above.
The supernatant (S6) is eliminated and the pellet (C6) is stored at -
20°C.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-53-
1.B. Purification of the proteins of membrane fraction C4 by preparative
SDS-PAGE
SDS-PAGE is carried out according to the method of Laemmli
(supra), using a biphasic gel consisting of a 5% polyacrylamide concentrating
gel and a 10% polyacrylamide separating gel. The membrane fraction C4 is
resuspended in buffer A, diluted in an equal volume of 2x sample buffer, and
heated for 5 minutes at 95°C. About 19 mg of protein is applied to the
gel
( 16 x 12 cm; 5 mm thick). Pre-migration is carried out for 2 hours at 50 V,
and
is followed by migration overnight at 65 V. After Coomassie blue staining,
five major bands are revealed that have apparent molecular weights of 87, 76,
54, 50, and 32 kDa. Bands at 50 and 32 kDa appear to be slightly contaminated
with bands at 47 and 35 kDa, respectively.
A band corresponding to the purified 76 kDa proteins, 32 kDa
protein (GHPO 1360), or 50 kDa protein (GHPO 750) is cut out from the gel
and is pounded with an ultra-turrax in 10-20 ml extraction buffer (25 mM Tris-
HCI (pH 8.8), 8 M urea, 10% SDS, 100 ~,M phenyl methyl sulfonyl fluoride
(PMSF), and 10 wM Pefabloc (buffer C)).
Each homogenate is filtered through a Millipore AP20 filter under
7 bars at room temperature, washed with 5-10 ml buffer C, and then filtered
again. Each filtrate is precipitated with three volumes of a 50/50 mixture of
75% methanol and 75% isopropanol, and then is spun in a centrifuge at
240,000 x g for 16 hours at 10°C.
Each pellet is resuspended in 2 ml of 10 mM NaPO, (pH 7.0)
containing 1 M NaCI, 0.1 % Sarkosyl, 100 ~.M PMSF, and 6 M urea (buffer D).
The solubilized sample is dialyzed, in order, against 100 ml buffer D
containing
4 M urea, 100 ml buffer D containing 2 M urea and 0.5% Sarkosyl, and twice
against 100 ml buffer D that does not contain urea or Sarkosyl. The dialyses
CA 02286893 1999-10-O1
WO 98143479 PCT/US98/06421
-54-
are carried out for 1 hour each while stirring at room temperature. The last
dialysate is incubated for 30 minutes in an ice bath, and then is spun in a
centrifuge at low speed for 10 minutes at 4°C. The supernatant is
recovered,
filtered through a Millipore filter (0.45 ~,m), and stored at -20°C.
1.C. Purification of the 76 ltDa, 32 kDa, or 50 kDa protein by
immunoaffinity-based chromatography
1.C.1. Antiserum preparation
Specific polyclonal serum against the purified 76 kDa proteins, the
32 kDa protein (GHPO 1360), or the .50 lcDa protein (GHPO 750), which are
purified by preparative SDS-PAGE, is prepared by hyperimmunizing rabbits as
follows. On day 0, a preparation containing 50 ~g of the protein mixed with
complete Freund's adjuvant is administered subcutaneously to the rabbits at
multiple sites. The rabbits are boosted at days 21 and 42 with 25 ~,g of the
protein in incomplete Freund's adjuvant, and are sacrificed at day 60.
Complement is removed from the serum by heating for 30 minutes at
56°C.
The hyperimmune serum is then sterilized by filtration through a Millipore
membrane (0.22 ~tm).
1.C.2. IgG purification
The hyperimmune serum prepared as described above is applied to a
Protein A Sepharose Fast Flow column (Pharmacia) that is equilibrated with
100 mM Tris-HCI (pH 8.0). The column is washed with 10 column volumes of
100 mM Tris-HCl (pH 8.0), and then with 10 column volumes of 10 mM Tris-
HCl (pH 8.0). IgGs are eluted in 0.1 M glycine buffer (pH 3.0), and are --
collected as 5 ml fractions, to each of which 0.25 ml of Tris-HCl (pH 8.0) is
added. The optical density of each fraction is measured at 280 nm, the IgG-
containing fractions are pooled together and, if necessary, frozen at -
70°C.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-55-
1.C.3. Preparation of the column
An appropriate amount of CNBr-activated Sepharose 4B gel
- (Pharmacia; reference: I 7-0430-O 1 ) is suspended in I mM NaCI buffer ( 1 g
dry
gel provides for 3.5 ml hydrated gel; 5 to 10 mg IgGs can be retained per ml
of
hydrated gel). The gel is then washed using a buchner by adding small
quantities of 1 mM HCI. The total volume of 1 mM HCl that is used amounts
to 200 ml/g of gel.
Purified IgGs are dialyzed for 4 hours at room temperature against
50 volumes of 500 mM sodium phosphate buffer (pH 7.5). The IgGs are then
diluted to 3 mg/ml with the same buffer. IgGs are incubated with the gel
overnight at 5~3°C while stirring. The gel is packed in a
chromatography
column and is washed with 2 column volumes of 500 mM phosphate buffer
{pH 7.5). The gel is then transferred to a tube and is incubated with 100 mM
ethanolamine (pH 7.5), and then it is washed with 2 column volumes of PBS.
The gel can be stored in PBS/merthiolate, 1/10,000.
1.C.4. Adsorption and elution
The 7G kDa protein is adsorbed and eluted as follows. The
membrane fraction Cs2d is suspended in 50 mM Tris-HCl (pH 8.0), 2 mM
EDTA, and then is filtered through a 0.45 ~m membrane. The supernatant is
applied to the column, which is equilibrated with 50 mM Tris-HCl (pH 8.0),
2 mM EDTA, at a flow rate of about 10 ml/hour. The column is washed with
20 column volumes of 50 mM Tris-HCI (pH 8.0), 2 mM EDTA, and then with
2 to 6 volumes 10 mM phosphate buffer (pH 6.8).
The antigen is eluted with 100 mM glycine buffer (pH 2.5). The
eluate is collected in 3 ml fractions, to each of which is added 150 ~,1 1 M
phosphate buffer (pH 8.0). The optical density of each fraction is measured at
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-56-
280 nm, fractions containing the 76 kDa protein are pooled, and stored at -
70°C.
Analysis by 10% SDS-PAGE reveals a single band at 76 kDa. N-
terminal sequence was carried out on this purified 76 kDa preparation, and the
sequence obtained is as follows: EDDGFYTSVGYQIGEAAQMV (SEQ ID
N0:58).
The 32 kDa protein (GHPO 1360) or the 50 kDa protein (GHPO
750) is purified by immunoaffinity-based chromatography as follows. In order
to separate the 32 or 50 kDa protein from the contaminating proteins (the 47
and 35 kDa proteins, respectively), membrane fraction C4 is solubilized in 50
mM NaC03 (pH 9.5) for 30 minutes at room temperature under stirring and the
preparation is centrifuged for 30 minutes at 200,000 x g at 4°C. The 47
and 35
kDa proteins are insoluble in the NaC03 buffer and are eliminated in the
pellet.
The supernatant is dialyzed against 50 mM Tris-HCL (pH 8.0), 2
mM EDTA, and then is filtered through a 0.45 ~m membrane. The filtered
supernatant is applied to the column, which is equilibrated with 50 mM Tris-
HCL (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour. The column is
washed with 20 column volumes of 50 mM Tris-HCL (pH 8.0), 2 mM EDTA,
and then with 2 to 6 volumes of 10 mM phosphate buffer (pH 6.8).
The antigen is eluted with 100 mM glycine buffer (pH 2.5). The
eluate is collected in 3 ml fractions, to each of which is added 150 ~1 1 M
phosphate buffer (pH 8.0). The optical density of each fraction is measured at
280 nm, and fractions containing the SO or 32 kDa protein are pooled and
stored at -70°C.
Analysis of the purified protein by 10% SDS-PAGE reveals-single
bands at 50 and 32 kDa. N-terminal sequencing is carried out with the purified
50 kDa protein preparation. The sequence found is as follows:
MKEKFNRTKPHVNIGTIGHVDH (SEQ ID N0:73). Similarly, N-terminal
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-5 7-
and internal sequencing is carried out with the purified 32 ltDa preparation.
The sequences found are as follows: AHNANNATHNTKK (SEQ ID N0:74)
and KPAHNA (SEQ ID N0:75) (N-terminal), and IDKQPKAKK (SEQ ID
N0:76) and FWAKKQAE (SEQ ID N0:77) (internal).
1.D. Purification of the 76 IzDa protein from membrane fraction Cs2d and
purification of the 32 kDa and 50 kDa proteins from membrane fraction
C4
The 76 kDa protein can also be purified as follows. A 40 ml Q-
Sepharose column (diameter: 2.5 cm; height: 8 cm) is prepared according to the
. 10 manufacturer's instructions (Pharmacia). The column is washed and
equilibrated with buffer B, containing 50 mM NaCO, (pH 9.5), 100 ~,M
Pefabloc, and 0.1 % Zwittergent 3-14. The chromatography is monitored by
measuring absorbance at 280 nm at the column exit.
One hundred and forty mg of protein from the membrane fraction
Cs2d resuspended in buffer B are applied to the column. The column is
washed with 0.1 M NaCI in buffer B, and then a 0.1-0.5 M NaCI gradient is
applied to the column. The fraction eluted between 0.35 and 0.45 M NaCI is
further purified on a 10 ml S-Sepharose column (diameter: 1.5 cm; height:
5 cm; up to 10 mg protein/ml of gel), which is prepared according to the
manufacturer's instructions (Pharmacia). The fraction obtained is dialyzed
against 50 mM acetate (pH 5.0) containing 100 ~,M Pefabloc and
0.1% Zwittergent 3-14, and then is applied to the column, which is
equilibrated
. with the acetate buffer.
The column is washed with the acetate buffer until the absorbance at
280 nm is stabilized (about 3 column volumes are required). Proteins are
CA 02286893 1999-10-O1
WO 98/43479 PCT/LJS98/06421
-58-
eluted with a 0-0.5 M NaCI gradient in acetate buffer. The fraction eluted at
0.15 M NaCl is enriched with the 76 kDa protein.
The 32 kDa protein (GHPO 1360) can also be purified as follows.
Membrane fraction C4 is solubilized in 50 mM NaCO, buffer (pH 9.5) at room
temperature for 30 minutes under stirring. The suspension is then centrifuged
at 200,000 x g for 30 minutes at 4°C. This allows the 32 and 35 kDa
proteins
to be separated, since the 35 kDa protein is insoluble in the NaC03 buffer.
The
supernatant is dialyzed against 50 mM NaP04 buffer (pH 7.0), and then is
applied to an SP-Sepharose column, which is equilibrated with the NaP04
buffer. The column is washed with the NaPO, buffer, and then an 0-0.5 M
NaCI gradient is applied to the column. The fraction eluted between 0.26 and
0.31 M contains the 32 kDa protein.
The 50 kDa protein can also be purified as follows. Membrane fraction
C4 is solubilized in 50 mM NaCO, buffer (pH 9.5) at room temperature for
30 minutes while stirring. The suspension is then centrifuged at 200,000 x g
for 30 minutes at 4°C. This allows the 50 and 47 kDa proteins to be
separated,
since the 47 kDa protein is insoluble in the NaC03 buffer. The supernatant is
dialyzed against 50 mM NaPO, buffer (pH 7.0).
A 40 ml Q-Sepharose column (diameter: 2.5 cm; height: 8 cm) is
prepared according to the manufacturer's instructions (Pharmacia), washed, and
equilibrated with buffer B (pH 9.5) (50 mM NaCO" 100 p,M Pefabloc, and
0.1% Zwittergent 3-14).
The chromatography is monitored by UV detection at 280 nm at the
column exit. One hundred and forty mg of protein solubilized as is described
above are applied to the column, which is then washed with buffer B until the
absorbance at 280 nm is stabilized. The proteins are eluted with a 0.1-
0.5 M NaCl gradient in buffer B ( 10 fold VT), which is followed by washing in
CA 02286893 1999-10-O1
WO 98/43479 PCTlUS98/06421
-59-
buffer B containing 0.5, and then 1, M NaCI (2 fold V,.). The fractions are
recovered, analyzed by SDS-PAGE, and pooled according to their
electrophoretic profiles.
Fraction 9, which corresponds to the beginning of the washing at 1 M
NaCI and contains acidic proteins, is further purified as follows. A 10 ml
DEAF Sepharose column (diameter: 1.5 cm, height: 5 cm) is prepared
according to the manufacturer's instructions (Pharmacia) {up to 10 mg
proteinlml of gel). The column is washed and equilibrated with buffer B.
Chromatography is monitored as is described above.
Fraction 9 is dialyzed against buffer B and contains about 10 mg protein.
Fraction 9 is applied to the DEAE-Sepharose column. The column is washed
with buffer B until the absorbance at 280 nm is stabilized. The proteins are
eluted with a 0-0.5 M NaCI gradient in buffer B ( 10 fold VT), followed by
washing in buffer B, containing 1 M NaCI (2 fold VT). Fractions are recovered
and analyzed by SDS-PAGE. The 50 kDa protein is found in the fractions
eluted at 0.3-0.4 M NaCI.
EXAMPLE 2: Identification of genes in the H. pylori genome, such as
genes encoding the 76 kDa proteins, the 32 kDa protein (GHPO 1360), and
the 50 kDa protein (GHPO 750) identification of signal sequences, and
primer design for amplification of genes lacking signal sequences
2.A. Creating H. pylori genomic databases
The H. pylori genome was provided as a text file containing a single
contiguous string of nucleotides that had been determined to be 1.76
Megabases in length. The complete genome was split into 17 separate files
using the program SPLIT (Creativity in Action), giving rise to 16 contigs,
each
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-60-
containing 100,000 nucleotides, and a 17t" contig containing the remaining
76,000 nucleotides. A header was added to each of the 17 f les using the
format: >hpg0.txt (representing contig 1), .hpgl.txt (representing contig 2),
etc.
The resulting 17 files, named hpg0 through hpgl G, were then copied together
to
S form one file that represented the plus strand of the complete H. pylori
genome.
The constructed database was given the designation "H." A negative strand
database of the H. pylori genome was created similarly by first creating a
reverse complement of the positive strand using the program SeqPup (D.G.
Gilbert, Indiana University Biology Department) and then performing the same
procedure as described above for the plus strand. This database was given the
designation "N."
The regions predicted to encode open reading frames (ORFs) were
defined for the complete H. pylori genome using the program GENEMARKTM
(Borodovsky et al., Comp. Chem. 17:123, 1993}. A database was created from
a text file containing an annotated version of all ORFs predicted to be
encoded
by the H. pylori genome for both the plus and minus strands, and was given the
designation "O." Each ORF was assigned a number indicating its location on
the genome and its position relative to other genes. No manipulation of the
text
file was required.
2.B. Searching the H. pylori databases
The databases:,constructed as is described above were searched using the
program FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444-2448,
1988). FASTA was used for searching either a DNA sequence against either of
the gene databases ("H" and/or "N"), or a peptide sequence against the ORF
library ("O"). TFASTX was used to search a peptide sequence against all
possible reading frames of a DNA database ("H" and/or "N" libraries). -
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-61-
Potential frameshifts also being resolved, FASTx was used for searching the
translated reading frames of a DNA sequence against either a DNA database, or
a peptide sequence against the protein database.
2.C. Isolation of DNA sequences from the H. pylori genome
The FASTA searches against the constructed DNA databases identified
exact nucleotide coordinates on one or more of the isolated contigs, and
therefore the location of the target DNA. Once the exact location of the
target
sequence was known, the contig identified to carry the gene was exported into
the software package MapDraw (DNAStar, Inc.) and the gene was isolated.
Gene sequences with flanking DNA was then excised and copied into the
EditSeq. Software package (DNAStar, Inc.) for further analysis.
2.D. Identification of signal sequences
The deduced protein encoded by a target gene sequence is analyzed
using the PROTEAN software package (DNAStar, Inc.). This analysis predicts
I 5 those areas of the protein that are hydrophobic by using the Kyte-
Doolittle
algorithm, and identifies any potential polar residues preceding the
hydrophobic core region, which is typical for many signal sequences. For
confirmation, the target protein is then searched against a PROSITE database
(DNAStar, Inc.) consisting of motifs and signatures. Characteristic of many
signal sequences and hydrophobic regions in general, is the identification of
predicted prokaryotic lipid attachment sites. Where confirmation between the
two approaches is apparent at the N-terminus of any protein, putative cleavage
sites are sought. Specifically, this includes the presence of either an
Alanine
(A), Serine (S), or Glycine (G) residue immediately after the core hydrophobic
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-62-
region. In the case of lipoproteins, a Cysteine (C) residue would be
identified
as the +1 residue, post-cleavage.
2.E. Rational design of PCR primers based on the identification of signal
sequences
In order to clone gene sequences as N-terminus translational fusions for
the generation of recombinant proteins with N-terminal Histidine tags, the
gene
sequence that specifies the signal sequence is omitted. The 5'-end of the gene-
specific portion of the N-terminal primer is designed to start at the first
codon
beyond the cleavage site. In the case of lipoproteins, the 5'-end of the N-
terminal primer begins at the second codon, immediately after the modifiable
residue at position +1 post-cleavage. The omission of the signal sequence from
the recombinant allows for one-step purification, and potential problems
associated with insertion of signal sequences in the membrane of the host
strain
carrying the hybrid construct are avoided.
EXAMPLE 3: Preparation of isolated DNA encoding GHPO 386, GHPO
789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO 750, and
production of these proteins as a histidine-tagged fusion proteins
3.A. Preparation of genomic DNA from Helicobacter pylori
Helicobacter pylori strain ORV2001, stored in LB medium containing
50% glycerol at -70°C, is grown on Colombia agar containing 7% sheep
blood
for 48 hours under microaerophilic conditions (8-10% C02, 5-7% 02,~and 85-
87% Nz). Cells are harvested, washed with PBS (pH 7.2), and DNA is then
extracted from the cells using the Rapid Prep Genomic DNA Isolation kit
(Pharmacia Biotech). -
CA 02286893 1999-10-O1
WO 98!43479 PCTJUS98/06421
-63-
3.B. PCR amplification
DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896,
GHPO 1360, and GHPO odd numbers), 65, and 67 is amplified from genomic
DNA, as can be prepared as is described above, by the Polymerase Chain
Reaction (PCR) using the following primers:
GHPO 386:
N-terminal primer:
5'-CTGAATTCGATTTCAAGGAGAAAACATGAAA-3' (SEQ ID N0:59);
and
C-terminal primer:
5'-CCGCTCGAGTTAGTAAGCGAACACATAATT-3' (SEQ ID N0:60).
GHPO 789:
N-terminal primer:
5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID N0:61 );
and
C-terminal primer:
5'-CCGCTCGAGTTAGTAAGCGAACACATAGTTCAA-3' (SEQ ID N0:62).
GHPO 1516:
N-terminal primer:
5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID N0:56);
and
C-terminal primer:
5'-CCGCTCGAGTTAAGTAAGCGAACACATATTCAA-3' (SEQ ID N0:57).
GHPO 896:
N-terminal primer:
' S'-CGCGGATCCGAAGTTTCTTTGTATCAAAG-3' (SEQ ID N0:63); and
C-terminal primer: -
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-64-
5'-CCGCTCGAGTTAGTAAGCAAACACATAATTGTG-3' (SEQ ID N0:64).
GHPO 1360:
N-terminal primer:
5'-CGCGGATCCGAATGAAAAAAAATATCTTAAAT-3' (SEQ ID N0:69);
and
C-terminal primer:
5'-CCGCTCGAGTTACTTGTTGATAACAATTTT-3' (SEQ ID N0:70).
GHPO 750:
N-terminal primer:
S'-CGCGGATCCGAATGGCAAAAGAAAAGTTTAAC-3' (SEQ ID N0:71);
and
C-terminal primer:
5'-CCGCTCGAGTTATTCAATAATATTGCTCAC-3' (SEQ ID N0:72).
GHPO 711:
N-terminal primer:
5'-GGGAATTCAAAAAAACGAAAAAAACG-3' (SEQ ID N0:83); and
C-terminal primer:
5'-CCCCTCGAGTTAATAGGCAAACAC-3' (SEQ ID N0:84).
The N-terminal and C-terminal primers for each clone both include a 5'
clamp and a restriction enzyme recognition sequence for cloning purposes
(BamHI (GGATCC) and XhoI (CTCGAG) recognition sequences).
Amplification of gene-specific DNA is carried out using a heat-stable
DNA Polymerase (e.g., Thermalase DNA Polymerase (Amresco)) according to
the manufacturer's instructions. The reaction mixture, which is brought to a
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-65-
final volume of 100 ~1 with distilled water, is as follows:
dNTPs mix 200 ~M
lOx ThermoPol buffer 10 ~1
primers 300 nM each
DNA template 50 ng
DNA polymerise 2 units
Appropriate amplification reaction conditions can readily be determined
by one skilled in the art. In the present case, the following conditions were
used. For GHPO 386 and GHPO 789, in a reaction containing Taq DNA
polymerise (Appligene), a denaturing step was carried out at 95°C for
30
seconds, followed by an annealing step at 50°C for one minute, and an
extension step at 72°C for 2 minutes and 30 seconds. Twenty five cycles
were
carried out. For GHPO 896, in a reaction containing Taq DNA polymerise, a
denaturing step was carried out at 97 ° C for 30 seconds, followed by
an
annealing step at 50°C for one minute, and an extension step at
72°C for 2
minutes and 30 seconds. Twenty five cycles were carried out. The same
reaction conditions were used for GHPO 1516 as GHPO 896, except that Vent
DNA polymerise was used for clone GHPO 1516, instead of Taq DNA
polymerise, and the annealing temperature was 55 °C. For GHPO 1360 and
GHPO 750, Thermaiase DNA polymerise was used. A denaturing step was
carried out at 95°C for 30 seconds, followed by an annealing step at
55°C for
one minute, and an extension step at 72 ° C for 2 minutes. Thirty
cycles were
carried out. For GHPO 711, Vent DNA polymerise was used. A denaturing
step was carried out at 94°C for 30 seconds, followed by an annealing
step at
50°C for 30 seconds, and an extension step at 72°C for 1 minute.
Twenty five
cycles were carried out.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-66-
3.C. Transformation and selection of transformants
A single PCR product is thus amplified and is then digested at
37°C for
2 hours with BamHI and XhoI concurrently in a 20 ~.l reaction volume. The
digested product is ligated to similarly cleaved pET28a (Novagen) that is
dephosphorylated prior to the ligation by treatment with Calf Intestinal
Alkaline Phosphatase {CIP). The gene fusion constructed in this manner allows
one-step affinity purification of the resulting fusion protein because of the
presence of histidine residues at the N-terminus of the fusion protein, which
are
encoded by the vector.
The ligation reaction (20 ~,l) is carried out at i4°C overnight and
then is
used to transform 100 ~,l fresh E. toll XL1-blue competent cells (Novagen).
The cells are incubated on ice for 2 hours, then heat-shocked at 42
° C for
30 seconds, and returned to ice for 90 seconds. The samples are then added to
I ml LB broth in the absence of selection and grown at 37°C for 2
hours. The
cells are then plated out on LB agar containing kanamycin (50 ~g/ml) at a IOx
and neat dilution and incubated overnight at 37°C. The following day,
50
colonies are picked onto secondary plates and incubated at 37°C
overnight.
Five colonies are picked into 3 ml LB broth supplemented with
kanamycin (100 ~g/ml) and are grown overnight at 37°C. Plasmid DNA is
extracted using the Quiagen mini-prep. method and is quantitated by agarose
gel electrophoresis.
PCR is performed with the gene-specific primers under the conditions
stated above and transformant DNA is confirmed to contain the desired insert.
If PCR-positive, one of the five plasmid DNA samples (500 ng) extracted from
the E. toll XLl-blue cells is used to transform competent BL21 (~,DE3) E. toll
competent cells (Novagen; as described previously). Transformants ( 10) are
CA 02286893 1999-10-O1
WO 98/43479 PCTNS98106421
-67-
picked onto selective kanamycin (50 ~,g/ml) containing LB agar plates and
stored as a research stock in LB containing 50% glycerol.
3.D. Purification of recombinant proteins
One ml of frozen glycerol stock prepared as described in 3.C. is used to
inoculate 50 ml of LB medium containing 25 ~,g/ml of kanamycin in a 250 ml
Erlenmeyer flask. The flask is incubated at 37°C for 2 hours or
until the
absorbance at 600 nm (OD~oo) reaches 0.4-1Ø The culture is stopped from
growing by placing the flask at 4°C overnight. The following day, 10 ml
of the
overnight culture are used to inoculate 240 ml LB medium containing
kanamycin (25 lzg/ml), with the initial OD~oo about 0.02-0.04. Four flasks are
inoculated for each ORF.
The cells are grown to an ODboo of 1.0 (about 2 hours at 37°C), a 1
mi
sample is harvested by centrifugation, and the sample is analyzed by SDS-
PAGE to detect any leaky expression. The remaining culture is induced with 1
mM IPTG and the induced cultures are grown for an additional 2 hours at
37°C.
The final OD6oo is taken and the cells are harvested by centrifugation at
5,000 x g for 15 minutes at 4°C. The supernatant is discarded and the
pellets
are resuspended in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. Two hundred and
fifty ml of buffer are used for a 1 L culture and the cells are recovered by
centrifugation at 12,000 x g for 20 minutes. The supernatant is discarded and
the pellets are stored at -45°C.
3. E. Protein purification
Pellets obtained from 3.D. are thawed and resuspended in 95 ml of SO
mM Tris-HCl (pH 8.0). Pefabloc and lysozyme are added to final
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-68-
concentrations of 100 ~,M and 100 ~g/ml, respectively. The mixture is
homogenized with magnetic stirring at 5°C for 30 minutes. Benzonase
(Merck)
is added at a 1 U/ml final concentration, in the presence of 10 mM MgCl2, to
ensure total digestion of the DNA. The suspension is sonicated (Branson
Sonifier 450) for 3 cycles of 2 minutes each at maximum output. The
homogenate is spun in a centrifuge at 19,000 x g for 15 minutes and both the
supernatant and the pellet are analyzed by SDS-PAGE to detect the cellular
location of the target protein in the soluble or insoluble fractions, as is
described further below.
3.E.1. Soluble fraction
If the target protein is produced in a soluble form (i.e., in the supernatant
obtained in 3.E.) NaCI and imidazole are added to the supernatant to final
concentrations of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCI, and 10 mM
imidazole (buffer A). The mixture is filtered through a 0.45 ~.m membrane and
loaded onto an IMAC column (Pharmacia HiTrap chelating Sepharose; 1 ml)
that has been charged with nickel ions according to the manufacturer's
recommendations. After loading, the column is washed with 50 column
volumes of buffer A and the recombinant target protein is eluted with 5 ml of
buffer B (50 mM Tris-HCl (pH 8.0), 0.5 M NaCI, 500 mM imidazole).
The elution profile is monitored by measuring the absorbance of the
fractions at 280 nm. Fractions corresponding to the protein peak are pooled,
dialyzed against PBS containing 0.5 M arginine, filtered through a 0.22 ~.m
membrane, and stored at -45°C.
3.E.2. Insoluble fraction - --
If the target protein is expressed in the insoluble fraction (pellets
obtained from 3.E.), purification is conducted under denaturing conditions.
NaCI, imidazole, and urea are added to the resuspended pellet to final -
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-69-
concentrations of 50 mM Tris-HCl {pH 8.0), 0.5 M NaCI, 10 mM imidazole,
and 6 M urea (buffer C). After complete solubilization, the mixture is
filtered
through a 0.45 ~m membrane and loaded onto an IMAC column.
The purification procedures on the IMAC column are the same as
described in 3.E.1., except that 6 M urea is included in all buffers used and
10
column volumes of buffer C are used to wash the column after protein loading,
instead of 50 column volumes.
The protein fractions eluted from the IMAC column with buffer D
(buffer C containing 500 mM imidazole) are pooled. Arginine is added to the
solution to final concentration of 0.5 M and the mixture is dialyzed against
PBS
containing 0.5 M arginine and various concentrations of urea (4 M, 3 M, 2 M, 1
M, and 0.5 M) to progressively decrease the concentration of urea. The final
dialysate is filtered through a 0.22 ~.m membrane and stored at -45°C.
Alternatively, when the above purification process is not as efficient as it
should be, two other processes may be used as follows. A first alternative
involves the use of a mild denaturant, N-octyl glucoside (NOG). Briefly, a
pellet obtained in 3.E. is homogenized in 5 mM imidazole, 500 mM sodium
chloride, 20 mM Tris-HCl (pH 7.9) by microfluidization at a pressure of 15,000
psi and is clarified by centrifugation at 4,000-5,000 x g. The pellet is
recovered, resuspended in 50 mM NaP04 (pH 7.5) containing 1-2% weight
/volume NOG, and homogenized. The NOG-soluble impurities are removed by
centrifugation. The~ellet is extracted once more by repeating the preceding
extraction step. The pellet is dissolved in 8 M urea, 50 mM Tris (pH 8.0). The
urea-solubilized protein is diluted with an equal volume of 2 M arginine, -50
mM Tris (pH 8.0), and is dialyzed against 1 M arginine for 24-48 hours to
remove the urea. The final dialysate is filtered through a 0.22 ~,m membrane
and stored at -45°C. -
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-70-
A second alternative involves the use of a strong denaturant, such as
guanidine hydrochloride. Briefly, a pellet obtained in 3.E. is homogenized in
5
mM imidazole, 500 mM sodium chloride, 20 mM Tris-HCl (pH 7.9) by
microfluidization at a pressure of 15,000 psi and clarified by centrifugation
at
S 4,000-5,000 x g. The pellet is recovered, resuspended in 6 M guanidine
hydrochloride, and passed through an IMAC column charged with Ni~. The
bound antigen is eluted with 8 M urea (pH 8.5). Beta-mercaptoethanol is added
to the eluted protein to a final concentration of 1 mM, then the eluted
protein is
passed through a Sephadex G-25 column equilibrated in 0.1 M acetic acid.
Protein eluted from the column is slowly added to 4 volumes of 50 mM
phosphate buffer (pH 7.0). The protein remains in solution.
3.F. Evaluation of the protective activity of the purified protein
A protection test is described above that was carried out for testing the
protective activity of the purified, native proteins. This test can also be
used for
testing the protective efficacy of recombinant proteins. Alternatively, the
following test can be used.
Groups of 10 OF1 mice (IFFA Credo) are immunized rectally with 25
p.g of the purified recombinant protein, admixed with 1 ~,g of cholera toxin
(Berna) in physiological buffer. Mice are immunized on days 0, 7, 14, and 21.
Fourteen days after the last immunization, the mice are challenged with H.
pylori strain ORV2001 grown in liquid media (the cells are grown on agar
plates, as described in 1.A., and, after harvest, the cells are resuspended in
Brucella broth; the flasks are then incubated overnight at 37 ° C).
Fourteen days
after challenge, the mice are sacrificed and their stomachs are removed. The
amount of H. pylori is determined by measuring the urease activity in the
stomach and by culture. -
CA 02286893 1999-10-O1
WO 98/43479 PCT/IJS98/06421
-71-
3.G. Production of monospecific polyclonal antibodies
3.G.1. Hyperimmune rabbit antiserum
' New Zealand rabbits are injected both subcutaneously and
intramuscularly with 100 ~,g of a purified fusion polypeptide, as obtained in
3.E.1. or 3.E.2., in the presence of Freund's complete adjuvant and in a total
volume of approximately 2 ml. Twenty one and 42 days after the initial
injection, booster doses, which are identical to priming doses, except that
Freund's incomplete adjuvant is used, are administered in the same way.
Fifteen days after the last injection, animal serum is recovered,
decomplemented, and filtered through a 0.45 ~m membrane.
3.G.2. Mouse hyperimmune ascites fluid
Ten mice are injected subcutaneously with 10-50 p.g of a purified fusion
polypeptide, as obtained in 3.E.1. or 3.E.2., in the presence of Freund's
complete adjuvant and in a volume of approximately 200 ~.1. Seven and 14
days after the initial injection, booster doses, which are identical to the
priming
doses, except that Freund's incomplete adjuvant is used, are administered in
the
same way. Twenty one and 28 days after the initial infection, mice receive SO
~.g of the antigen alone intraperitoneally. On day 21, mice are also injected
intraperitoneally with sarcoma 180/TG cells CM26684 (Lennette et al.,
Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections, 5th
Ed., Washington DC, American Public Health Association, 1979). Ascites
fluid is collected 10-13 days after the last injection.
. EXAMPLE 4: Methods for producing transcriptional fusions lacking His-
tags
Methods for amplification and cloning of DNA encoding the
polypeptides of the invention as transcriptional fusions lacking His-tags are
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-72-
described as follows. Two PCR primers for each clone are designed based
upon the sequences of the polynucleotides that encode them (SEQ ID NOs: l-21
(odd numbers), 65, and 67). These primers can be used to amplify DNA
encoding the polypeptides of the invention from any Helicobacter pylori
strain,
including, for example, ORV2001 and the H. pylori strain deposited with the
American Type Culture Collection (ATCC, Rockville, Maryland) as ATCC
number 43579, as well as from other Helicobacter species.
The N-terminal primers are designed to include the ribosome binding
site of the target gene, the ATG start site, the signal sequence (if any), and
the
cleavage site. The N-terminal primers can include a 5' clamp and restriction
endonuclease recognition site, such as that for BamHI (GGATCC), which
facilitates subsequent cloning. Similarly, the C-terminal primers can include
a
restriction endonuclease recognition site, such as that for XhoI (CTCGAG),
which can be used in subsequent cloning, and a TAA stop codon. Specific
primers that can be used are listed above.
Amplification of a genes encoding the polypeptides of the invention can
be carried out using Vent DNA polymerase (New England Biolabs) or Taq
DNA polymerase (Appligene) under the conditions described above in
Example 3. Alternatively, Thermalase DNA polymerase or Pwo DNA
polymerase (Boehringer Mannheim) can be used, according to instructions
provided by the manufacturers.
A single PCRaproduct for each clone is amplified and can be cloned into
BafnHI XhoI cleaved pET24, resulting in construction of transcriptional
fusions
that permit expression of the proteins without His-tags. The expressed
products
can be purified as denatured proteins that are refolded by dialysis into 1 M
arginine.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-73-
Cloning into pET 24 allows transcription of genes from the T7 promoter,
which is supplied by the vector, but relies upon binding of the RNA-specific
' DNA polymerase to the intrinsic ribosome binding site of the genes, and
thereby expression of the complete ORF. The amplification, digestion, and
cloning protocols are as described above for constructing translational
fusions.
EXAMPLE 5: Purification of the polypeptides of the invention by
immunoaffinity
S.A. Purification of specific IgGs
An immune serum, as prepared as is described in section 3.G., is applied
to a protein A Sepharose Fast Flow column (Pharmacia) equilibrated in 100
mM Tris-HCl (pH 8.0). The resin is washed by applying 10 column volumes
of 100 mM Tris-HCl and 10 volumes of 10 mM Tris-HCl (pH 8.0) to the
column. IgG antibodies are eluted with 0.1 M glycine buffer (pH 3.0) and are
collected in S ml fractions to each of which is added 0.25 ml 1 M Tris-HCl
(pH 8.0). The optical density of the eluate is measured at 280 nm and the
fractions containing the IgG antibodies are pooled, dialyzed against 50 mM
Tris-HCl (pH 8.0), and, if necessary, stored frozen at -70°C.
S.B. Preparation of the column
An appropriate amount of CNBr-activated Sepharose 4B gel ( 1 g of
dried gel provides for approximately 3.5 ml of hydrated gel; gel capacity is
from 5 to 10 mg coupled IgG/ml of gel) manufactured by Pharmacia (17-0430-
O 1 ) is suspended in 1 mM HCl buffer and washed using a buchner by adding
small quantities of 1 mM HCl buffer. The total volume of buffer is 200 ml per
gram of gel.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98J06421
-74-
Purified IgG antibodies are dialyzed for 4 hours at 205 °C against
50 volumes of 500 mM sodium phosphate buffer (pH 7.5). The antibodies are
then diluted in 500 mM phosphate buffer (pH 7.5) to a final concentration of 3
mg/ml.
IgG antibodies are mixed with the gel overnight at St3 °C. The gel
is
packed into a chromatography column and is washed with 2 column volumes of
500 mM phosphate buffer (pH 7.5), and 1 column volume of 50 mM sodium
phosphate buffer, containing S00 mM NaCI (pH 7.5). The gel is then
transferred to a tube, mixed with 100 mM ethanolamine (pH 7.5) for 4 hours at
room temperature, and washed twice with 2 column volumes of PBS. The gel
is then stored in 1/10,000 PBS/merthiolate. The amount of IgG antibodies
coupled to the gel is determined by measuring the optical density (OD) at 280
nm of the IgG solution and the direct eluate, plus washings.
S.C. Adsorption and elution of the antigen
An antigen solution in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, for
example, the supernatant obtained in 3.E.1. or the solubilized pellet obtained
in
3.E.2., after centrifugation and filtration through a 0.45 p.m membrane, is
applied to a column equilibrated with 50 mM Tris-HCl (pH 8.0), 2 mM EDTA,
at a flow rate of about 10 ml/hour. The column is then washed with
20 volumes of SO mM Tris-HCl (pH 8.0), 2 mM EDTA. Alternatively,
adsorption can be achieved by mixing overnight at 5~3 °C.
The adsorbed gel is washed with 2 to 6 volumes of 10 mM sodium
phosphate buffer (pH 6.8) and the antigen is eluted with 100 mM glycine-buffer
(pH 2.5). The eluate is recovered in 3 ml fractions, to each of which is added
1 SO ~,l of 1 M sodium phosphate buffer (pH 8.0). Absorption is measured at
CA 02286893 1999-10-O1
WO 98/43479 PCTNS98106421
-75-
280 nm for each fraction; those fractions containing the antigen are pooled
and
stored at
-20°C.
EXAMPLE 6: The GHPO 1360 polypeptide is useful as a serodiagnostic
tool for H. pylori infection
The reactivity of patient sera against H. pylori proteins was analyzed by
immunoblot technique. Briefly, total lysate of H. pylori strain ORV2001 was
subjected to SDS-PAGE electrophoresis (BioRad protean II system) on a
12.5% gel. Proteins were electrotransferred onto a nitrocellulose paper for
immunoblot assay. After blocking, the nitrocellulose paper was incubated with
patient sera ( 1:500 diluted in blocking buffer) for one hour at room
temperature, washed, and further incubated with peroxidase-conjugated goat
anti-human IgG. The positive bands were revealed by incubation with the
appropriate substrates. The results showed that the H. pylori-positive ulcer
patient sera react specifically with proteins having molecular weights between
50 and 60 kDa and about 30 to 35 kDa. To identify the nature of these
proteins, the reactivities of the patient sera were analyzed by immunoblot
assay
against purified proteins with similar molecular weights: urease (67 kDa and
30
kDa), catalase (54 kDa), heat-shock protein B (60 kDa), and the GHPO 1360
polypeptide (32 kDa) expressed and purified as described in Example 5. All
patient sera showed strong reactivity against the GHPO 1360 polypeptide, but
the reactivities against other purified proteins were quite variable. These
results show that the GHPO 1360 polypeptide is a useful antigen for use tn
diagnosis of H. pylori infection.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/0642I
-76-
Other embodiments are within the following claims.
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: MERIEUX ORAVAX SOCIETE EN NOM COLLECTIF
PASTEUR MERIEUX SERUMS ET VACCINS S.A., ET
AL.
(ii) TITLE OF THE INVENTION: 76 kDa, 30 kDa, and 50 kDa
Helicobacter Polypeptides and
Corresponding Polynucleotide Molecules
(iii) NUMBER OF SEQUENCES: 84
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Clark & Elbing LLP
(B) STREET: 176 Federal Street
(C) CITY: Boston
(D) STATE: MA
(E) COUNTRY: USA
(F) ZIP: 02110
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US98/----
(B) FILING DATE: 31-MAR-98
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/834,666
(B) FILING DATE: O1-APR-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/831,310
(B) FILING DATE: O1-APR-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Clark, Paul T.
(B) REGISTRATION NUMBER: 30,162
(C) REFERENCE/DOCKET NUMBER: 06132/037W01
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617-428-0200
(B) TELEFAX: 617-428-7045
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 _ PCT/US98/06421
~g_
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 2798 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 328...2451
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 328...385
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
TGGTCCTGGCATTCCGAGGTTCGAATCCTTGCACCCCAGC CATTTTTCCT TATTTTTTGG60
CGCGGAGTAGAGCAGTCCGGTAGCTCGTTGGGCTCATAAC CCAAAGGTCA GTGGTTCAAA120
TCCATTCTCCGCAACCAATCCTTTAAACCACACCACCACC AAACGAACCA AACGAAACAA180
AAAGCATCAAAATCAAAAAAATGACAAAATTTTTAAGAAA ATGACAAAAA F~AAAAAAAAC240
GATTTTATGCTATATTAACGAAATCTTGTGATAAGATCTT ATTCTTTTAA AAGACTTATC300
TAACCATTTTAATTTCAAGGAGAAAAC AAA AAA ACC CTT TTA CTC TCT 354
ATG CTC
Met Lys Lys Thr Leu Leu Leu Ser
Leu
-15
TCT CTC TCT CTC TCG TTT TTG CTC CAC GCT GAA GAC GAC GGC TTT TAC 402
Ser Leu Ser Leu Ser Phe Leu Leu His Ala Glu Asp Asp Gly Phe Tyr
-10 -5 1 5
ACA AGC GTG GGC TAT CAA ATC GGT GAA GCC GCT CAA ATG GTG AAA AAC 450
Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val Lys Asn
15 20
ACC AAA GGC ATT CAA GAG CTT TCA GAC AAT TAT GAA AAG CTG AAC AAT 498
Thr Lys Gly Ile Gln Glu Leu Ser Asp Asn Tyr Glu Lys Leu Asn Asn
25 30 35
CTT TTG AAT AAT TAC AGC ACC CTA AAC ACC CTT ATC AAA TTG TCC GCT 546
Leu Leu Asn Asn Tyr Ser Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala
40 45 50
GAT CCG AGC GCG ATT AAC GAC GCA AGG GAT AAT CTA GGC TCA AGC TCT 594
Asp Pro Ser Ala Ile Asn Asp Ala Arg Asp Asn Leu Gly Ser Ser Ser
55 60 65 70
AGG AAT TTG CTT GAT GTC AAA ACC AAT TCC CCC GCG TAT CAA GCC GTG 642
Arg Asn Leu Leu Asp Val Lys Thr Asn Ser Pro Ala Tyr Gln Ala Val
75 80 85
CTT TTA GCA CTC AAT GCT GCA GTG GGG TTG TGG CAA GTT ACA AGC TAC 690
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
- 79- _
Leu Leu Ala Leu Asn Ala Ala Val Gly Leu Trp Gln Val Thr Ser Tyr
90 95 100
GCT TTT ACT GCT TGT GGT CCT GGC AGT AAC GAG AAT GCG AAT GGA GGG 738
Ala Phe Thr Ala Cys Gly Pro Gly Ser Asn Glu Asn Ala Asn Gly Gly
105 110 115
ATC CAA ACT TTT AAT AAT GTG CCA GGA CAA GAT ACG ACG ACC ATC ACT 786
Ile Gln Thr Phe Asn Asn Val Pro Gly Gln Asp Thr Thr Thr Ile Thr
120 125 130
TGC AAT TCG TAT TAT GAG CCA GGA CAT GGT GGG CCT ATA TCC ACT GCA 834
Cys Asn Ser Tyr Tyr Glu Pro Gly His Gly Gly Pro Ile Ser Thr Ala
135 140 145 150
AAT TAT GCG AAA ATC AAT CAA GCC TAT CAA ATC ATC CAA AAG GCT TTG 882
Asn Tyr Ala Lys Ile Asn Gln Ala Tyr Gln Ile Ile Gln Lys Ala Leu
155 160 165
ACA GCC AAT GGA GCT AAT GGA GAT GGG GTC CCC GTT TTA AGC AAC ACC 930
Thr Ala Asn Gly Ala Asn Gly Asp Gly Val Pro Val Leu Ser Asn Thr
170 175 180
ACT ACA AAA CTT GAT TTC ACT ATC AAT GGA GAC AAA AGA ACG GGG GGC 978
Thr Thr Lys Leu Asp Phe Thr Ile Asn Gly Asp Lys Arg Thr Gly Gly
185 190 195
AAA CCA AAT ACA CCT GAA AAG TTC CCA TGG AGT GAT GGG AAA TAT ATT 1026
Lys Pro Asn Thr Pro Glu Lys Phe Pro Trp Ser Asp Gly Lys Tyr Ile
200 205 210
CAC ACC CAA TGG ATT AAC ACA ATA GTA ACA CCA ACA GAA ACA AAT ATC 1074
His Thr Gln Trp Ile Asn Thr Ile Val Thr Pro Thr Glu Thr Asn Ile
215 220 225 230
AAC ACA GAA AAT AAC GCT CAA GAG CTT TTA AAA CAA GCG AGC ATC ATT 1122
Asn Thr Glu Asn Asn Ala Gln Glu Leu Leu Lys Gln Ala Ser Ile Ile
235 240 245
ATC ACT ACC CTA AAT GAG GCA TGC CCA AAC TTC CAA AAT GGT GGT AGA 1170
Ile Thr Thr Leu Asn Glu Ala Cys Pro Asn Phe Gln Asn Gly Gly Arg
250 255 260
AGT TAT TGG CAA GGG ATA AGC GGC AAT GGG ACA ATG TGC GGG ATG TTT 1218
Ser Tyr Trp Gln Gly Ile Ser Gly Asn Gly Thr Met Cys Gly Met Phe
265 270 275
AAG AAT GAA ATC AGC GCG ATC CAA GGC ATG ATC GCT AAC GCT CAA GAA 1266
Lys Asn Glu Ile Ser Ala Ile Gln Gly Met Ile Ala Asn Ala Gln Glu.___
280 285 290
GCT GTC GCG CAA AGC AAA ATC GTT AGT GAA AAC GCG CAA AAT CAA AAC 1314
Ala Val Ala Gln Ser Lys Ile Val Ser Glu Asn Ala Gln Asn Gln Asn
295 300 305 310
AAC TTG GAT ACT GGA AAA CCA TTC AAC CCT TAC ACG GAC GCC AGC TTT 1362
Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala Ser Phe
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 _gp_ PCT/US98/06421
315 320 325
GCG CAA AGC ATG CTC AAA AAC GCT CAA GCG CAA GCA GAG ATT TTA AAC 1410
Ala Gln Ser Met Leu Lys Asn Ala Gln Ala Gln Ala Glu Ile Leu Asn
330 335 340
CAA GCC GAA CAA GTA GTA AAA AAC TTT GAA AAA ATC CCT ACA GCC TTT 1458
Gln Ala Glu Gln Val Val Lys Asn Phe Glu Lys Ile Pro Thr Ala Phe
345 350 355
GTA TCAGACTCT TTAGGG GTGTGTTAT GAAGTGCAA GGT GAGCGT 1506
GGG
Val SerAspSer LeuGly ValCysTyr GluValGln GlyGly GluArg
360 365 370
AGG GGCACCAAT CCAGGT CAGGTAACT TCTAACACT TGGGGA GCCGGT 1554
Arg GlyThrAsn ProGly GlnValThr SerAsnThr TrpGly AlaGly
375 380 385 390
TGC GCGTATGTG AAACAA ACCATAACG AATTTAGAC AACAGC ATCGCT 1602
Cys AlaTyrVal LysGln ThrIleThr AsnLeuAsp AsnSer IleAla
395 400 405
CAC TTTGGCACT CAAGAG CAGCAGATA CAGCAAGCC GAAAAC ATCGCT 1650
His PheGlyThr GlnGlu GlnGlnIle GlnGlnAla GluAsn IleAla
410 415 420
GAC ACTCTAGTG AATTTC AAATCTAGA TACAGCGAA TTAGGC AACACC 1698
Asp ThrLeuVal AsnPhe LysSerArg TyrSerGlu LeuGly AsnThr
425 430 435
TAT AACAGCATC ACCACC GCGCTCTCC AAAGTCCCT AACGCG CAAAGC 1746
Tyr AsnSerIle ThrThr AlaLeuSer LysValPro AsnAla GlnSer
440 445 450
TTG CAAAACGTG GTGAGC AAAAAGAAT AACCCCTAT AGCCCT CAAGGC 1794
Leu GlnAsnVal ValSer LysLysAsn AsnProTyr SerPro GlnGly
455 460 465 470
ATAGAG AAT TACTACCTC AATCAA AATTCTTAC AACCAAATC CAA 1842
ACC
IleGlu ThrAsn TyrTyrLeu AsnGln AsnSerTyr AsnGlnIle Gln
475 480 485
ACCATC AACCAA GAACTAGGG CGTAAC CCCTTTAGG AAAGTGGGC ATC 1890
ThrIle AsnGln GluLeuGly ArgAsn ProPheArg LysValGly Ile
490 495 500
GTCAAT TCTCAA ACCAACAAT GGTGCC ATGAATGGG ATCGGTATT CAG 1938
ValAsn SerGln ThrAsnAsn GlyAla MetAsnGly IleGlyIle Gln
505 510 515
GTGGGC TATAAG CAATTCTTT GGCCAA AAAAGAAAA TGGGGCGCT AGG 1986
ValGly TyrLys GlnPhePhe GlyGln LysArgLys TrpGlyAla Arg
520 525 530
TATTAC GGCTTT TTTGACTAC AACCAT GCGTTCATT AAATCCAGC TTC 2034
-
TyrTyr GlyPhe PheAspTyr AsnHis AlaPheIle LysSerSer Phe
535 540 545 550
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98143479 PCT/US98/06421
_gl_ _
TTC AAC TCG GCT TCT GAT GTG TGG ACT TAT GGT TTT GGA GCG GAC GCT 2082
Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala
555 560 565
CTT TAT AAC TTC ATC AAC GAT AAA GCC ACC AAT TTC TTA GGC AAA AAC 2130
Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn
570 575 580
AAC AAG CTT TCC GTG GGG CTT TTT GGA GGG ATT GCG TTA GCG GGC ACT 2178
Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr
585 590 595
TCA TGG CTT AAT TCT GAG TAT GTG AAT TTA GCC ACC GTG AAT AAC GTC 2226
Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val
600 605 610
TAT AAC GCT AAA ATG AAT GTG GCG AAT TTC CAA TTC TTA TTC AAT ATG 2274
Tyr Asn Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe Asn Met
615 620 625 630
GGA GTG AGG ATG AAT TTA GCC AGA TCC AAG AAA AAA GGC AGC GAT CAT 2322
Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His
635 640 645
GCG GCT CAG CAT GGG ATT GAA CTA GGG CTT AAA ATC CCC ACC ATC AAC 2370
Ala Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn
650 655 660
ACG AAC TAT TAT TCT TTC ATG GGG GCT GAA CTC AAA TAC AGA AGG CTT 2418
Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu
665 670 675
TAT AGC GTG TAT TTG AAT TAT GTG TTC GCT TAC TAAGCTTTTT GTGAAACTCC 2471
Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr
680 685
CTTTTTAAGGGGTTTTTTTTTGAACTCTCTTTTTAAATTCTCTTTTTAAA 2531
GAGATTTCTT
TTTTTTAAGCTTTTTTTTGAATTCTTTTTTTTGAATTCTTTGTTTTTAAGCTTTTTTTAA2591
ACCCTTTCGTTTTTAAACTCCCTTTTTTAAGGGATTTCTTTTTTTAAACTCTTTTTTTTT2651
AAACTCTTTTTTTTAAACCCTCTTTTTTTAAGGGATTTCTTTTTAAAGCTTTTTTGAAGT2711
CTTTTTTTAAATTCTTTTTTTGGGGGTTTGATCTTTCTTTTTGCCAATCCCCACTACTTT2771
CGCTTTTTAATCTTTAGGTTTTATTTT 2798
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENC~CHARACTERISTICS:
(A) LENGTH: 708 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...19
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Phe Leu
-15 -10 -5
Leu His Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile
1 5 10
Gly Glu Ala Ala Gln Met Val Lys Asn Thr Lys Gly Ile Gln Glu Leu
15 20 25
Ser Asp Asn Tyr Glu Lys Leu Asn Asn Leu Leu Asn Asn Tyr Ser Thr
30 35 40 45
Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn Asp
50 55 60
Ala Arg Asp Asn Leu Gly Ser Ser Ser Arg Asn Leu Leu Asp Val Lys
65 70 75
Thr Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Leu Asn Ala Ala
80 85 90
Val Gly Leu Trp Gln Val Thr Ser Tyr Ala Phe Thr Ala Cys Gly Pro
95 100 105
Gly Ser Asn Glu Asn Ala Asn Gly Gly Ile Gln Thr Phe Asn Asn Val
110 115 120 I25
Pro Gly Gln Asp Thr Thr Thr Ile Thr Cys Asn Ser Tyr Tyr Glu Pro
130 135 140
Gly His Gly Gly Pro Ile Ser Thr Ala Asn Tyr Ala Lys Ile Asn Gln
145 150 155
Ala Tyr Gln Ile Ile Gln Lys Ala Leu Thr Ala Asn Gly Ala Asn Gly
160 165 170
Asp Gly Val Pro Val Leu Ser Asn Thr Thr Thr Lys Leu Asp Phe Thr
175 180 185
Ile Asn Gly Asp Lys Arg Thr Gly Gly Lys Pro Asn Thr Pro Glu Lys
190 195 200 205
Phe Pro Trp Ser Asp Gly Lys Tyr Ile His Thr Gln Trp Ile Asn Thr
210 215 220
Ile Val Thr Pro Thr Glu Thr Asn Ile Asn Thr Glu Asn Asn Ala Gln
225 230 235
Glu Leu Leu Lys Gln Ala Ser Ile Ile Ile Thr Thr Leu Asn Glu Ala
240 245 250
Cys Pro Asn Phe Gln Asn Gly Gly Arg Ser Tyr Trp Gln Gly Ile Ser
255 260 265
Gly Asn Gly Thr Met Cys Gly Met Phe Lys Asn Glu Ile Ser Ala Ile
270 275 280 285
Gln Gly Met Ile Ala Asn Ala Gln Glu Ala Val Ala Gln Ser Lys Ile
290 295 300
Val Ser Glu Asn Ala Gln Asn Gln Asn Asn Leu Asp Thr Gly Lys Pro
305 310 315
Phe Asn Pro Tyr Thr Asp Ala Ser Phe Ala Gln Ser Met Leu Lys Asn
320 325 330
Ala Gln Ala Gln Ala Glu Ile Leu Asn Gln Ala Glu Gln Val Val Lys
335 340 345
Asn Phe Glu Lys Ile Pro Thr Ala Phe Val Ser Asp Ser Leu Gly Val
350 355 360 365
Cys Tyr Glu Val Gln Gly Gly Glu Arg Arg Gly Thr Asn Pro Gly Gln_
370 375 380
Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Lys Gln Thr
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-83
385 390 395
Ile Thr Asn Leu Asp Asn Ser Ile Ala His Phe Gly Thr Gln Glu Gln
400 405 410
Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu Val Asn Phe Lys
415 420 425
5er Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala
430 435 440 445
Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln Asn Val Val Ser Lys
450 455 460
Lys Asn Asri Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu
465 470 475
Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu Gly
480 485 490
Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn Ser Gln Thr Asn Asn
495 500 505
Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe
510 515 520 525
Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr
530 535 540
Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val
545 550 555
Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp
560 565 570
Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val G1y Leu
575 580 585
Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr
590 595 600 605
Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Val
610 615 620
Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala
625 630 635
Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln His Gly Ile Glu
640 645 650
Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met
655 660 665
Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr
670 675 680 685
Val Phe Ala Tyr
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2699 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 199..,2397
(D) OTHER INFORMATION:
SUBSTITUTE SHEET (RULE 2B)
CA 02286893 1999-10-O1
WO 98/43479 PCT/US98/06421
-84
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 199...259
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
TAAAATCCAATTAAAAGCGT TCAAAGGTAA CGCAAAAAAACAAAAPATGACGCAATTTTT60
TCAAAATGACF~AAAAP,AAAC GCTTTATGCT ATAATACCCCAAATACATTCTAATAGCAAA120
TGCGTTCTAATGCAAATGCA TTCCAATGTA TGAAATCCCTAATACTAAATCCAATTTAAT180
CCAAAAAGGAGAAAAAAC ATG AAA AAA CAC TCA TTA TTA GGC 231
ATC CTT GCT
Met Lys Lys His Ile Leu Ser Leu Leu Gly
Ala
-20 -15 -10
TCG CTT TTA GTT TCC ACT TTG AGC GCT GAA GAC GAC GGC TTT TAC ACA 279
Ser Leu Leu Val Ser Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr
-5 1 5
AGC GTA GGC TAT CAG ATC GGT GAA GCC GCT CAA ATG GTA ACA AAC ACC 327
Ser Val Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr
15 20
AAA GGC ATC CAA CAG CTT TCA GAC AAT TAT GAA AAT TTG AAC AAC CTT 375
Lys Gly Ile Gln Gln Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu
25 30 35
TTA ACG AGA TAC AGC ACC CTA AAC ACC CTT ATC AAA TTG TCC GCT GAT 423
Leu Thr Arg Tyr Ser Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp
40 45 50 55
CCG AGC GCA ATT AAT GCG GTG CGG GAA AAT CTG GGC GCG AGC GCG AAG 471
Pro Ser Ala Ile Asn Ala Val Arg Glu Asn Leu Gly Ala Ser Ala Lys
60 65 70
AAT TTG ATC GGC GAT AAA GCC AAC TCC CCC GCC TAT CAA GCC GTG CTT 519
Asn Leu Ile Gly Asp Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Leu
75 BO 85
TTA GCG ATC AAC GCG GCG GTA GGG TTT TGG AAT GTC GTG GGC TAT GTG 567
Leu Ala Ile Asn Ala Ala Val Gly Phe Trp Asn Val Val Gly Tyr Val
90 95 100
ACG CAA TGT GGG GGT AAC GCC AAT GGT CAA GAA AGC ACC TCT TCA ACC 615
Thr Gln Cys Gly Gly Asn Ala Asn Gly Gln Glu Ser Thr Ser Ser Thr
105 ~"" 110 115
ACC ATC TTC AAC AAC GAG CCA GGG TAT CGA TCC ACT TCC ATC ACT TGT 663
Thr Ile Phe Asn Asn Glu Pro Gly Tyr Arg Ser Thr Ser Ile Thr Cys
120 125 130 135
TCT TTG AAC GGG CAT AAG CCT GGA TAC TAT GGC CCT ATG AGC ATT GAG 711
Ser Leu Asn Gly His Lys Pro Gly Tyr Tyr Gly Pro Met Ser Ile Glu
140 145 150
AAT TTT AAA AAG CTT AAC GAA GCC TAT CAG ATC CTC CAA ACG GCT TTA 759
Asn Phe Lys Lys Leu Asn Glu Ala Tyr Gln Ile Leu Gln Thr Ala Leu
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98143479 _85_ PCT/(JS98/06421
155 160 165
AAA AAC GGC TTA CCC GCG CTC AAA GAA AAC AAC GGG AAG GTC AGT GTA 807
Lys Asn Gly Leu Pro Ala Leu Lys Glu Asn Asn Gly Lys Val Ser Val
170 175 180
ACC TAT ACC TAC ACA TGC TCA GGG CAA GGG AAT AAT AAC TGC TCG CCA 855
Thr Tyr Thr Tyr Thr Cys Ser Gly Gln Gly Asn Asn Asn Cys Ser Pro
185 190 195
AGT GTC AAC GGA ACC AAA ACC ACA ACC CAA ACC ATA GAC GGC AAA AGC 903
Ser Val Asn Gly Thr Lys Thr Thr Thr Gln Thr Ile Asp Gly Lys Ser
200 205 210 215
GTA ACC ACC ACG ATC AGT TCA AAA GTG GTT GGT AGC ATC GCT AGT GGC 951
Val Thr Thr Thr Ile Ser Ser Lys Val Val Gly Ser Ile Ala Ser Gly
220 225 230
AAC ACA TCA CAT GTC ATC ACC AAC AAA TTA GAC GGT GTG CCT GAT AGC 999
Asn Thr Ser His Val Ile Thr Asn Lys Leu Asp Gly Val Pro Asp Ser
235 240 245
GCT CAA GCG CTC TTA GCG CAA GCG AGC ACG CTC ATC AAC ACC ATC AAC 1047
Ala Gln Ala Leu Leu Ala Gln Ala Ser Thr Leu Ile Asn Thr Ile Asn
250 255 260
GAA GCA TGC CCG TAT TTC CAT GCT ACT AAT AGT AGT GAG GCT AAC GCC 1095
Glu Ala Cys Pro Tyr Phe His Ala Thr Asn Ser Ser Glu Ala Asn Ala
265 270 275
CCA AAA TTC TCT ACT ACT ACT GGG AAA ATA TGC GGC GCT TTT TCA GAA 1143
Pro Lys Phe Ser Thr Thr Thr Giy Lys Ile Cys Gly Ala Phe Ser Glu
280 285 290 295
GAA ATC AGC GCG ATC CAA AAG ATG ATC ACG GAC GCG CAA GAG CTA GTT 1191
Glu Ile Ser Ala Ile Gln Lys Met Ile Thr Asp Ala Gln Glu Leu Val
300 305 310
AAT CAA ACG AGC GTC ATT AAC AGC AAC GAA CAA TCA ACT CCG GTA GGC 1239
Asn Gln Thr Ser Val Ile Asn Ser Asn Glu Gln Ser Thr Pro Val Gly
315 320 325
AAT AAT AAT GGC AAG CCT TTC AAC CCT TTC ACG GAC GCA AGT TTT GCG 1287
Asn Asn Asn Gly Lys Pro Phe Asn Pro Phe Thr Asp Ala Ser Phe Ala
330 335 340
CAA GGC ATG CTC GCT AAC GCT AGC GCG CAA GCT AAA ATG CTC AAT TTA 1335
Gln Gly Met Leu Ala Asn Ala Ser Ala Gln Ala Lys Met Leu Asn Leu
345 350 355
GCC CAT CAG GTG GGG CAA GCC ATT AAC CCA GAG AAT CTT AGC GAG AAT 13E3
Ala His Gln Val Gly Gln Ala Ile Asn Pro Glu Asn Leu Ser Glu Asn
360 365 370 375
TTT AAA AAT TTT GTT ACA GGC TTT TTA GCC ACA TGC AAT AAC AAA TCA_ 1431
Phe Lys Asn Phe Val Thr Gly Phe Leu Ala Thr Cys Asn Asn Lys Ser
380 385 390
sues~TUTE sHesr ~RU~ 2s~
CA 02286893 1999-10-O1
WO 98/43479 _86_ PCT/US98106421
ACA GCT GGC ACT GGT GGC ACA CAA GGT TCA GCT CCA GGC ACA GTG ACC 1479
Thr Ala Gly Thr Gly Gly Thr Gln Gly Ser Ala Pro Gly Thr Val Thr
395 400 405
ACT CAA ACT TTC GCT TCT GGT TGC GCG TAT GTG GAG CAA ACC CTA ACG 1527
Thr Gln Thr Phe Ala Ser Gly Cys Ala Tyr Val Glu Gln Thr Leu Thr
410 415 420
AAC TTA GGC AAC AGC ATC GCT CAC TTT GGC ACT CAA GAG CAG CAG ATA 1575
Asn Leu Gly Asn Ser Ile Ala His Phe Gly Thr Gln Glu Gln Gln Ile
425 430 435
CAG CAA GCC GAA AAC ATC GCT GAC ACT CTA GTG AAT TTC AAA TCT AGA 1623
Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu Val Asn Phe Lys Ser Arg
440 445 450 455
TAC AGC GAA TTA GGC AAC ACC TAT AAC AGC ATC ACC ACC GCG CTC TCC 1671
Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu Ser
460 465 470
AAA GTC CCT AAC GCG CAA AGC TTG CAA AAC GTG GTG AGC AAA AAG AAT 1719
Lys Val Pro Asn Ala Gln Ser Leu Gln Asn Val Val Ser Lys Lys Asn
475 480 485
AAC CCC TAT AGC CCT CAA GGC ATA GAG ACC AAT TAC TAC CTC AAT CAA 1767
Asn Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu Asn Gln
490 495 500
AAT TCT TAC AAC CAA ATC CAA ACC ATC AAC CAA GAA CTA GGG CGT AAC 1815
Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu Gly Arg Asn
505 510 515
CCC TTT AGG AAA GTG GGC ATC GTC AAT TCT CAA ACC AAC AAT GGT GCC 1863
Pro Phe Arg Lys Val Gly Ile Val Asn Ser Gln Thr Asn Asn Gly Ala
520 525 530 535
ATG AAT GGG ATC GGT ATT CAG GTG GGC TAT AAG CAA TTC TTT GGC CAA 1911
Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe Gly Gln
540 545 550
AAA AGA AAA TGG GGC GCT AGG TAT TAC GGC TTT TTT GAT TAC AAC CAT 1959
Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His
555 560 565
GCG TTC ATC AAA TCC AGC TTT TTC AAC TCG GCT TCT GAC GTG TGG ACT 2007
Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr
570 575 580
TAT GGT TTT GGA GCG GAC GCG CTT TAT AAC TTC ATC AAC GAT AAA GCC 2055
Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Ala
585 590 595
ACC AAT TTC TTA GGC AAA AAC AAC AAG CTT TCT TTG GGG CTT TTT GGC 2103
Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Leu Gly Leu Phe Gly
600 605 610 615
GGG ATT GCG TTA GCG GGC ACT TCA TGG CTC AAT TCT GAG TAC GTG AAT 2151
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 _g~_ PCTNS98/06421
Gly Ile Ala Leu Ala Giy Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn
620 625 630
TTA GCC ACC GTG AAT AAC GTC TAT AAC GCT AAA ATG AAT GTG GCG AAT 2199
Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Val Ala Asn
635 640 645
TTC CAA TTC TTA TTC AAT ATG GGA GTG AGG ATG AAT TTA GCC AGA TCC 2247
Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser
650 655 660
AAG AAA AAA GGC AGC GAT CAT GCA GCT CAG CAT GGG ATT GAG TTA GGG 2295
Lys Lys Lys Gly Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly
665 670 675
CTT AAA ATC CCC ACC ATC AAC ACG AAC TAT TAT TCC TTT ATG GGG GCT 2343
Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala
680 685 690 695
GAA CTC AAA TAC AGA AGG CTC TAT AGC GTG TAT TTG AAC TAT GTG TTC 2391
Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe
700 705 710
GCT TAC TAATGTTTGG CTCTTTGTGA AACTCCCTTT TTAAGGGGTT TTTTTTTGAA CT 2449
Ala Tyr
CTCTTTTTAA ATTCTCTTTT TAAAGAGATTTCTTTTTTT'TAAGCTTTTTT TTGAATTCTT2509
TTTTTTTGAA TTCTTTGTTT TTAAGCTTTTTTTAAACCCTTTCGTTTTTA AACTCCCTTT2569
TTTAAGGGAT TTCTTTTTTT GAACTCCCTTTTTTGAACCCTTTTTTTTAA ACCCTCTTTT2629
TTTAAGGGGT TTCTTTTTAA AGCTTTTTTGAAGTCTTTTTTTAAATTCTT TTTTTGGGGG2689
TTTGATCTTT 2699
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 733 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
ix) FEATC7RE
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Lys Lys His Ile Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser
-20 -15 -10 -5
Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln
1 5 10
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 _8S_ PCT/US98/06421
Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Gln
15 20 25
Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser
30 35 40
Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn
45 50 55 60
Ala Val Arg Glu Asn Leu Gly Ala Ser Ala Lys Asn Leu Ile Gly Asp
65 70 75
Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Ile Asn Ala
80 85 90
Ala Val Gly Phe Trp Asn Val Val Gly Tyr Val Thr Gln Cys Gly Gly
95 100 105
Asn Ala Asn Gly Gln Glu Ser Thr Ser Ser Thr Thr Ile Phe Asn Asn
110 115 120
Glu Pro Gly Tyr Arg Ser Thr Ser Ile Thr Cys Ser Leu Asn Gly His
125 130 135 140
Lys Pro Gly Tyr Tyr Gly Pro Met Ser Ile Glu Asn Phe Lys Lys Leu
145 150 155
Asn Glu Ala Tyr Gln Ile Leu Gln Thr Ala Leu Lys Asn Gly Leu Pro
160 165 170
Ala Leu Lys Glu Asn Asn Gly Lys Val Ser Val Thr Tyr Thr Tyr Thr
175 180 185
Cys Ser Gly Gln Gly Asn Asn Asn Cys Ser Pro Ser Val Asn Gly Thr
190 195 200
Lys Thr Thr Thr Gln Thr Ile Asp Gly Lys Ser Val Thr Thr Thr Ile
205 210 215 220
Ser Ser Lys Val Val Gly Ser Ile~Ala Ser Gly Asn Thr Ser His Val
225 230 235
Ile Thr Asn Lys Leu Asp Gly Val Pro Asp Ser Ala Gln Ala Leu Leu
240 245 250
Ala Gln Ala Ser Thr Leu Ile Asn Thr Ile Asn Glu Ala Cys Pro Tyr
255 260 265
Phe His Ala Thr Asn Ser Ser Glu Ala Asn Ala Pro Lys Phe Ser Thr
270 275 280
Thr Thr Gly Lys Ile Cys Gly Ala Phe Ser Glu Glu Ile Ser Ala Ile
285 290 295 300
Gln Lys Met Ile Thr Asp Ala Gln Glu Leu Val Asn Gln Thr Ser Val
305 310 315
Ile Asn Ser Asn Glu Gln Ser Thr Pro Val Gly Asn Asn Asn Gly Lys
320 325 330
Pro Phe Asn Pro Phe Thr Asp Ala Ser Phe Ala Gln Gly Met Leu Ala
335 340 345
Asn Ala Ser Ala Gln Ala Lys Met Leu Asn Leu Ala His Gln Val Gly
350 355 360
Gln Ala Ile Asn Pro Glu Asn Leu Ser Glu Asn Phe Lys Asn Phe Val
365 370 375 380
Thr Gly Phe Leu Ala Thr Cys Asn Asn Lys Ser Thr Ala Gly Thr Gly
385 390 395
Gly Thr Gln Gly Ser Ala Pro Gly Thr Val Thr Thr Gln Thr Phe Ala
400 405 410
Ser Gly Cys Ala Tyr Val Glu Gln Thr Leu Thr Asn Leu Gly Asn Ser
415 420 425
Ile Ala His Phe Gly Thr Gln Glu Gln Gln Ile Gln Gln Ala Glu Asn
430 435 440
Ile Ala Asp Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly
445 450 455 460
Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 $g PCT/US98/06421
465 470 475
Gln Ser Leu Gln Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro
480 485 490
Gln Gly Ile Glu Thr Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln
495 500 505
Ile Gln Thr Ile Asn Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val
5I0 515 520
Gly Ile Val Asn Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly
525 530 535 540
Ile Gln Val Gly Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp Gly
545 550 555
~ Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser
560 565 570
Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala
575 580 585
Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly
590 595 600
Lys Asn Asn Lys Leu Ser Leu Gly Leu Phe Gly Gly Ile Ala Leu Ala
605 610 615 620
Gly Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn
625 630 635
Asn Val Tyr Asn Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe
640 645 650
Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser
655 660 665
Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr
670 675 680
Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg
685 690 695 700
Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr
705 710
(2) INFORMATION FOR 5EQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2915 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 365...2597
(D) OTHER INFORMATION:
{A) NAME/KEY: Signal Sequence ---
{B) LOCATION: 365...425
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 90 PCT/US98/06421
TTTTAGGCGA CAAAATCGCT TATGTTGGGG ATAAAGGCAA CCCGCACAAT TTCGCTCACA 60
AGAAATAAAC CGCTCATAAG GGGCAAACGC CCCAP.AAAAG CGATTTTTAA AGAGGTTACG 120
GCAAAATCAA GCTCTTTAGT ATTTAATCTT AAAAAATGCT AAAAGCCTTT TTATGGGCTA 180
ACACCACACA AAAAGCATCA AAATCAAAAA AATGACAAAA TTTTTAAGAA AATGACAAAA 240
AAAAACGCTT TATGCTATAA TACCCCAAAT ACATTCTAAT AGCAAATGCG TTCTAATGCA 300
AATGCATTCC AATGTATGAA ATCCCTAATA CTAAATCCAA TTTAATCCAA AAAGGAGAAA 360
AAAC ATG AAA AAA CAC ATC CTT TCA TTA GCT TTA GGC TCG CTT TTA GTT 409
Met Lys Lys His Ile Leu Ser Leu Ala Leu Gly Ser Leu Leu Val
-20 -15 -10
TCC ACT TTG AGC GCT GAA GAC GAC GGC TTT TAC ACA AGC GTA GGC TAT 457
Ser Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr
-5 1 5 10
CAG ATC GGT GAA GCC GCT CAA ATG GTA ACA AAC ACC AAA GGC ATC CAA 505
Gln Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln
15 20 25
CAG CTT TCA GAC AAT TAT GAA AAT TTG AAC AAC CTT TTA ACG AGA TAC 553
Gln Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr
30 35 40
AGC ACC CTA AAC ACC CTT ATC AAA TTG TCC GCT GAT CCG AGC GCA ATT 601
Ser Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile
45 50 55
AAT GCG GTG CGG GAA AAT CTG GGC GCG AGC ACG AAG AAT TTG ATC GGC 649
Asn Ala Val Arg Glu Asn Leu Gly Ala Ser Thr Lys Asn Leu Ile Gly
60 65 70 75
GAT AAA GCC AAC TCC CCG GCG TAT CAA GCC GTG TTT TTA GCG ATC AAC 697
Asp Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Phe Leu Ala Ile Asn
80 85 90
GCG GCG GTA GGG TTG TGG AAT ACC ATC GGC TAT GCG GTC ATG TGC GGG 745
Ala Ala Val Gly Leu Trp Asn Thr Ile Gly Tyr Ala Val Met Cys Gly
95 100 105
AAC GGG AAC GGC ACA GAG AGT GGG CCT GGC AGC GTG ATC TTT AAT GAC 793
Asn Gly Asn Gly Thr Glu Ser Gly Pro Gly Ser Val Ile Phe Asn Asp
110 115 120
CAA CCA GGA CAG GAT TCC ACG CAA ATT ACT TGC AAC CGC TTT GAA TCA 841
Gln Pro Gly Gln Asp Ser Thr Gln Ile Thr Cys Asn Arg Phe Glu Ser
125 130 135
ACT GGG CCT GGT AAA AGC ATG TCT ATT GAT GAA TTC AAA AAA CTC AAT 889
Thr Gly Pro Gly Lys Ser Met Ser Ile Asp Glu Phe Lys Lys Leu Asn
140 145 150 155
GAA GCC TAT CAA ATC ATC CAG CAA GCT TTA AAA AAT CAA AGT GGG TTT 937
Glu Ala Tyr Gln Ile Ile Gln Gln Ala Leu Lys Asn Gln Ser Gly Phe
160 165 170
CCT GAA TTA GGC GGG AAC GGC ACA AAA GTG AGT GTT AAT TAC AAT TAC 985
Pro Glu Leu Gly Gly Asn Gly Thr Lys Val Ser Val Asn Tyr Asn Tyr
175 180 185
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 91 PCT/US98/06421
GAA TGC AGA CAA ACT GCT GAT ATC AAC GGC GGT GTG TAT CAG TTC TGC 1033
Glu Cys Arg Gln Thr Ala Asp Ile Asn Gly Gly Val Tyr Gln Phe Cys
190 195 200
AAG GCT AAA AAT GGT AGT AGT AGC AGT AGT AAT GGC GGT AAT GGC AGT 1081
Lys Ala Lys Asn Gly Ser Ser Ser Ser Ser Asn Gly Gly Asn Gly Ser
205 210 215
AGC ACG CAA ACA ACC GCG ACA ACC ACG CAA GAC GGC GTA ACG ATC ACC 1129
Ser Thr Gln Thr Thr Ala Thr Thr Thr Gln Asp Gly Val Thr Ile Thr
220 225 230 235
ACT ACC TAT AAT AAT AAC AAA GCC ACC GTC AAA TTT GAC ATC ACC AAT 1177
Thr Thr Tyr Asn Asn Asn Lys Ala Thr Val Lys Phe Asp Ile Thr Asn
240 245 250
AAC GCT GAA CAG CTG TTA AAT CAA GCG GCA AAC ATC ATG CAA GTC CTT 1225
Asn Ala Glu Gln Leu Leu Asn Gln Ala Ala Asn Ile Met Gln Val Leu
255 260 265
AAT ACG CAA TGC CCT TTA GTG CGT TCC ACG AAT AAC GAA AAC ACT CCA 1273
Asn Thr Gln Cys Pro Leu Val Arg Ser Thr Asn Asn Glu Asn Thr Pro
270 275 280
GGG GGT GGT CAA CCA TGG GGT TTA AGC ACA TCC GGG AAT GCG TGC AGC 1321
Gly Gly Gly Gln Pro Trp Gly Leu Ser Thr Ser Gly Asn Ala Cys Ser
285 290 295
ATC TTC CAA CAA GAA TTT AGC CAG GTT ACT AGC ATG ATC AAA AAC GCC 1369
Ile Phe Gln Gln Glu Phe Ser Gln Val Thr Ser Met Ile Lys Asn Ala
300 305 310 315
CAA GAA ATA ATC GCG CAA AGC AAA ATC GTT AGT GAA AAC GCG CAA AAT 1417
Gln Glu Ile Ile Ala Gln Ser Lys Ile Val Ser Glu Asn Ala Gln Asn
320 325 330
CAA AAC AAC TTG GAT ACT GGA AAA CCA TTC AAC CCT TAC ACG GAC GCC 1465
Gln Asn Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala
335 340 345
AGC TTT GCG CAA AGC ATG CTC AAA AAC GCT CAA GCG CAA GCA GAG ATG 1513
Ser Phe Ala Gln Ser Met Leu Lys Asn Ala Gln Ala Gln Ala Glu Met
350 355 360
TTC AAT TTG AGC GAA CAA GTG AAA AAG AAC TTG GAA GTC ATG AAA AAC 1561
Phe Asn Leu Ser Glu Gln Val Lys Lys Asn Leu Glu Val Met Lys Asn
365 370 375
AAC AAT AAT GTT AAC GAG AAA TTA GCA GGA TTT GGG AAA GAA GAA GTA 1609
, Asn Asn Asn Val Asn Glu Lys Leu Ala Gly Phe Gly Lys Glu Glu Val--
380 385 390 395
ATG ACC AAT TTT GTT AGC GCC TTT TTG GCA AGC TGC AAe~ GAT GGT GGC 1657
Met Thr Asn Phe Val Ser Ala Phe Leu Ala Ser Cys Lys Asp Gly Gly
400 405 410
ACA TTG CCT AAT GCA GGG GTT ACT TCT AAC ACT TGG GGG GCG GGT TGC 1705
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 92 PCT/US98/06421
Thr Leu Pro Asn Ala Gly Val Thr Ser Asn Thr Trp Gly Ala Gly Cys
415 420 425
GCG TAT GTG GGA GAG ACG ATA AGC GCC CTA ACC AAC AGC ATC GCT CAC 1753
Ala Tyr Val Gly Glu Thr Ile Ser Ala Leu Thr Asn Ser Ile Ala His
430 435 440
TTT GGC ACT CAA GAG CAG CAG ATA CAG CAA GCC GAA AAC ATC GCT GAC 1801
Phe Gly Thr Gln Glu Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp
445 450 455
ACT CTA GTG AAT TTC AAA TCT AGA TAC AGC GAA TTA GGC AAC ACC TAT 1849
Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr
460 465 470 475
AAC AGC ATC ACC ACC GCG CTC TCC AAA GTC CCT AAC GCG CAA AGC TTG 1897
Asn Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu
480 485 490
CAA AAC GTG GTG AGC AAA AAG AAT AAC CCC TAT AGC CCT CAA GGC ATA 1945
Gln Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile
495 500 505
GAG ACC AAT TAC TAC CTC AAT CAA AAT TCT TAC AAC CAA ATC CAA ACC 1993
Glu Thr Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr
5I0 515 520
ATC AAC CAA GAA CTA GGG CGT AAC CCC TTT AGG AAA GTG GGC ATC GTC 2041
Ile Asn Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val
525 530 535
AAT TCT CAA ACC AAC AAT GGT GCC ATG AAT GGG ATC GGC ATT CAG GTG 2089
Asn Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Ile Gln Val
540 545 550 555
GGC TAT AAG CAA TTC TTT GGC CAA AAA AGA AAA TGG GGC GCT AGG TAT 2137
Gly Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr
560 565 570
TAC GGC TTT TTT GAT TAC AAC CAT GCG TTC ATC AAA TCC AGC TTT TTC 2185
Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe
575 580 585
AAC TCG GCT TCT GAC GTG TGG ACT TAT GGT TTT GGA GCG GAC GCG CTT 2233
Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu
590 595 600
TAT AAC TTC ATC AAC GAT AAA GCC ACC AAT TTC TTA GGC AAA AAC AAC 2281
Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn
605 610 615 - -- -
AAG CTT TCT TTG GGG CTT TTT GGC GGG ATT GCG TTA GCG GGC ACT TCA 2329
Lys Leu Ser Leu Gly Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser
620 625 630 635
TGG CTC AAT TCT GAG TAC GTG AAT TTA GCC ACC GTG AAT AAC GTC TAT 2377
Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 93 PCT/US98/06421
640 645 650
AAC GCT AAA ATG AAT GTG GCG AAT TTC CAA TTC TTA TTC AAT ATG GGA 2425
Asn Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe Asn Met Gly
655 660 665
GTG AGG ATG AAT TTA GCC AGA TCC AAG AAA AAA GGC AGC GAT CAT GCA 2473
Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala
670 675 6B0
GCT CAG CAT GGG ATT GAG TTA GGG CTT AAA ATC CCC ACC ATC AAC ACG 2521
Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr
685 690 695
AAC TAT TAT TCC TTT ATG GGG GCT GAA CTC AAA TAC AGA AGG CTC TAT 2569
Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr
700 705 710 715
AGC GTG TAT TTG AAT NAT GTG TTC GCT TAC TAAGCTTTTT GTGAAACTCC 2619
Ser Val Tyr Leu Asn Xaa Val Phe Ala Tyr
720 725
CTTTTTAAGG GGTTTTTTTT TGAACTCTCT TTTAAATTCT CTTTTTAAAG AGATTTCTTT 2679
TTTTAAGCTT TTTTTTGAAC TTTTTTTTGA ATTCTTTGTT TTTAAGCTTT TTTTAAACCC 2739
TTTCGTTTTT AAACTCCCTT TTTTAAGGGA TTTCTTTTTT TGAACTCCCT TTTTTGAACC 2799
CTTTTTTTTA AACCCTCTTT TTTTAAGGGG TTTCTTTTTA AAGCTTTTTT GAAGTCTTTT 2859
TTTAAATTCT TTTTTTGGGG GTTTGATCTT TCTTTTTGCC AATCCCCACT ACTTTC 2915
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 745 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Lys Lys His Ile Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser -
-20 -15 -10 -5
Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln
1 5 10
Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Gln_
15 20 25
Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 94 PCT/IJS98/06421
30 35 40
Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn
45 50 55 60
Ala Val Arg Glu Asn Leu Gly Ala Ser Thr Lys Asn Leu Ile Gly Asp
65 70 75
Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Phe Leu Ala Ile Asn Ala
80 85 90
Ala Val Gly Leu Trp Asn Thr Ile Gly Tyr Ala Val Met Cys Gly Asn
95 100 105
Gly Asn Gly Thr Glu Ser Gly Pro Gly Ser Val Ile Phe Asn Asp Gln
110 115 120
Pro Gly Gln Asp Ser Thr Gln Ile Thr Cys Asn Arg Phe Glu Ser Thr
125 130 135 140
Gly Pro Gly Lys Ser Met Ser Ile Asp Glu Phe Lys Lys Leu Asn Glu
145 150 155
Ala Tyr Gln Ile Ile Gln Gln Ala Leu Lys Asn Gln Ser Gly Phe Pro
160 165 170
Glu Leu Gly Gly Asn Gly Thr Lys Val Ser Val Asn Tyr Asn Tyr Glu
175 180 185
Cys Arg Gln Thr Ala Asp Ile Asn Gly Gly Val Tyr Gln Phe Cys Lys
190 195 200
Ala Lys Asn Gly Ser Ser Ser Ser Ser Asn Gly Gly Asn Gly Ser Ser
205 210 215 220
Thr Gln Thr Thr Ala Thr Thr Thr Gln Asp Gly Val Thr Ile Thr Thr
225 230 235
Thr Tyr Asn Asn Asn Lys Ala Thr Val Lys Phe Asp Ile Thr Asn Asn
240 245 250
Ala Glu Gln Leu Leu Asn Gln Ala Ala Asn Ile Met Gln Val Leu Asn
255 260 265
Thr Gln Cys Pro Leu Val Arg Ser Thr Asn Asn Glu Asn Thr Pro Gly
270 275 280
Gly Gly Gln Pro Trp Gly Leu Ser Thr Ser Gly Asn Ala Cys Ser Ile
285 290 295 300
Phe Gln Gln Glu Phe Ser Gln Val Thr Ser Met Ile Lys Asn Ala Gln
305 310 315
Glu Ile Ile Ala Gln Ser Lys Ile Val Ser Glu Asn Ala Gln Asn Gln
320 325 330
Asn Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp AIa Ser
335 340 345
Phe Ala Gln Ser Met Leu Lys Asn Ala Gln Ala Gln Ala Glu Met Phe
350 355 360
Asn Leu Ser Glu Gln Val Lys Lys Asn Leu GIu Val Met Lys Asn Asn
365 370 375 380
Asn Asn Val Asn Glu Lys Leu Ala Gly Phe Gly Lys Glu Glu Val Met
385 390 395
Thr Asn Phe Val Ser Ala Phe Leu Ala Ser Cys Lys Asp Gly Gly Thr
400 405 410
Leu Pro Asn Ala Gly Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala
4I5 420 425
Tyr Val Gly Glu Thr Ile Ser Ala Leu Thr Asn Ser Ile Ala His Phe-
430 435 440
Gly Thr Gln Glu Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr
445 450 455 460
Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn
465 470 475
Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln
480 485 490
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 95 PCT/US98106421
Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile Glu
495 500 505
Thr Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile
510 515 520
Asn Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn
525 530 535 540
Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly
545 550 555
Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr
560 565 570
Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn
575 580 585
Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr
590 595 600
Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys
605 610 615 620
Leu Ser Leu Gly Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp
625 630 635
Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn
640 645 650
Ala Lys Met Asn Va1 Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val
655 660 665
Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala
670 675 680
Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn
685 690 695 700
Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser
705 710 715
Val Tyr Leu Asn Xaa Val Phe Ala Tyr
720 725
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2603 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 210...2342
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 210...270
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
ATGACCTTTA TTGGTTTAAT ATTTGTTTAG AAATAACACA AAAACCTTTT TTTTTTTTTT 60
SU9STITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 96 PCT/US98/06421
TGAAAGGGCA AAAACGCCTA ATTAATATCA AAATCCCATG AATTTATACT ATATTAACGA 120
AAGCTTGCGG TATGGTTTCA CCTAAAGACA CACTTCCGCA AGATTTACTA ACAATTTCAA 180
TCTTATTTCA AGTAATAAAA GGAGAAAAC ATG AAG AAA AAA TTT CTG TCA TTA 233
Met Lys Lys Lys Phe Leu Ser Leu
-20 -15
ACC TTA GGT TCG CTT TTA GTT TCC GCT TTA AGC GCT GAA GAC AAC GGC 281
Thr Leu Gly Ser Leu Leu Val Ser Ala Leu Ser Ala Glu Asp Asn Gly
-10 -5 1
TTT TTT GTG AGT GCG GGC TAT CAA ATC GGT GAA TCC GCT CAA ATG GTG 329
Phe Phe Val Ser Ala Gly Tyr Gln Ile Gly Glu Ser Ala Gln Met Val
10 15 20
AAA AAC ACT AAA GGC ATT CAA GAT CTT TCA GAT AGC TAT GAA AGA CTG 377
Lys Asn Thr Lys Gly Ile Gln Asp Leu Ser Asp Ser Tyr Glu Arg Leu
25 30 35
AAC AAT CTT TTA ACG AGT TAT AGT GCC CTA AAC ACT CTT ATT AGG CAG 425
Asn Asn Leu Leu Thr Ser Tyr Ser Aia Leu Asn Thr Leu Ile Arg Gln
40 45 50
TCC GCC GAC CCC AAC GCT ATC AAT AAC GCA AGG GGC AAT TTG AAC GCT 473
5er Ala Asp Pro Asn Ala Ile Asn Asn Ala Arg Gly Asn Leu Asn Ala
55 60 65
AGT GCG AAG AAT TTG ATC AAT GAT AAA AAG AAT TCC CCG GCG TAT CAA 521
Ser Ala Lys Asn Leu Ile Asn Asp Lys Lys Asn Ser Pro Ala Tyr Gln
70 75 80
GCG GTG CTT TTA GCC TTG AAT GCG GCA GCG GGG TTG TGG CAA GTC ATG 569
Ala Val Leu Leu Ala Leu Asn Ala Ala Ala Gly Leu Trp Gln Val Met
85 90 95 100
AGC TAT TCG ATC AGC GTT TGT GGC CCT GGC TCT GAC AAA AAT AAA AAT 617
Ser Tyr Ser Ile Ser Val Cys Giy Pro Gly Ser Asp Lys Asn Lys Asn
105 110 115
GGG GGC GTC CAA ACC TTT GAA AAT GTG CCG TCA AAT GGG GGG ACT ACC 665
Gly Gly Val Gln Thr Phe Glu Asn Val Pro Ser Asn Gly Gly Thr Thr
120 125 130
ATT GCT TGC GAT TCA TTT TAT GAA CCA GGA AAG TGG AGC GGT ATA TCC 713
Ile Ala Cys Asp Ser Phe Tyr Glu Pro Gly Lys Trp Ser Gly Ile Ser
135 140 145
ACT GAA AAT TAC GCA AAA ATC AAT AAA GCC TAT CAA ATC ATC CAA AAG 761
Thr Glu Asn Tyr Ala Lys Ile Asn Lys Ala Tyr Gln Ile Ile Gln Lys
150 155 160
GCT TTT GGA GCA AGC GGG CAA GAT ATT CCT GCC TTA AGC GAC ACC AAA 809
Ala Phe Gly Ala Ser Gly Gln Asp Ile Pro Ala Leu Ser Asp Thr Lys
165 170 175 180
GAA CTT AAT TTT GAA ATT AAA GGG AAA AAA AAT GAT AGC GTC CAG CCA 857
Glu Leu Asn Phe Glu Ile Lys Gly Lys Lys Asn Asp Ser Val Gln Pro
185 190 195
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 97 PCT/US98106421
GGA GAA AGA TGG AAA TTC CCA TGG ACT AAT GGA AAA TTT GTT TCA GTC 905
Gly Glu Arg Trp Lys Phe Pro Trp Thr Asn Gly Lys Phe Val Ser Val
200 205 210
AAG TGG GTG AAT GGG AAG TAT GAA GAA ATT AAA GAA GAC ATC AAA GTG 953
Lys Trp Val Asn Gly Lys Tyr Glu Glu Ile Lys Glu Asp Ile Lys Val
215 220 225
TCA AAT AAC GCT CAA GAG CTT TTA AAA CAG GCT AGC ACT ATT TTA ACC 1001
Ser Asn Asn Ala Gln Glu Leu Leu Lys Gln Ala Ser Thr Ile Leu Thr
230 235 240
ACT CTT AAT GAA GCA TGC CCA TGG TTG AGT AAT GGT GGT GCA GGC AAT 1049
Thr Leu Asn Glu Ala Cys Pro Trp Leu Ser Asn Gly Gly Ala Gly Asn
245 250 255 260
GTG GCC GGT GGC AAT AGT TTA TGG GCC GGA ATA GAT AAA GGC GAC GGG 1097
Val Ala Gly Gly Asn Ser Leu Trp Ala Gly Ile Asp Lys Gly Asp Gly
265 270 275
AGC GCA TGC GGG ATT TTT AAA AAT GAA ATC AGC GCG ATT CAA GAC ATG 1145
Ser Ala Cys Gly Ile Phe Lys Asn Glu Ile Ser Ala Ile Gln Asp Met
280 285 290
ATC AAA AAC GCT GAA ATA GCC GTA GAG CAA TCC AAA ATC GTT ACC GCC 1193
Ile Lys Asn Ala Glu Ile Ala Val Glu Gln Ser Lys Ile Val Thr Ala
295 300 305
AAC GCG CAA AAC CAG CAC AAC CTA GAC ACT GGG AAA GCA TTC AAC CCC 1241
Asn Ala Gln Asn Gln His Asn Leu Asp Thr Gly Lys Ala Phe Asn Pro
310 315 320
TAT AAA GAC GCC AAC TTC GCC CAA AGC ATG TTC GCT AAC GCT AGA GCG 1289
Tyr Lys Asp Ala Asn Phe Ala Gln Ser Met Phe Ala Asn Ala Arg Ala
325 330 335 340
CAA GCG GAG ATT TTA AAC CGC GCT CAA GCA GTG GTG AAG GAC TTT GAA 1337
Gln Ala Glu Ile Leu Asn Arg Ala Gln Ala Val Val Lys Asp Phe Glu
345 350 355
AGA ATC CCT GCA GCG TTC GTG AAA GAC TCT TTA GGA GTA TGC CAT GAA 1385
Arg Ile Pro Ala Ala Phe Val Lys Asp Ser Leu Gly Val Cys His Glu
360 365 370
AAG GGT AGC GAC GGC AAT CTC CGT GGC ACG CCA TCT GGC ACG GTT ACT 1433
Lys Gly Ser Asp Gly Asn Leu Arg Gly Thr Pro Ser Gly Thr Val Thr
375 380 385
TCT AAC ACT TGG GGA GCC GGC TGC GCG TAT GTG GGA GAA ACC GTA ACG 1481
' Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Gly Glu Thr Val Thr
390 395 400
- AAT CTA AAA AAC AGC ATC GCT CAT TTT GGC GAC CAA GCG GAG CGA ATC 1529
Asn Leu Lys Asn Ser Ile Ala His Phe Gly Asp Gln Ala Glu Arg Ile
405 410 415 420
CAT AAT GCG CGA AAT CTC GCC TAC ACT TTA GCG AAT TTC AGC GGC CAG 1577
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 98 PCT/US98/06421
His Asn Ala Arg Asn Leu Ala Tyr Thr Leu Ala Asn~Phe Ser Gly Gln
425 430 435
TAC AAA AAG CTA GGC GAA CAC TAT GAC AGC ATC ACA GCG GCG CTC TCT 1625
Tyr Lys Lys Leu Gly Glu His Tyr Asp Ser Ile Thr Ala Ala Leu Ser
440 445 450
AGC TTG CCT GAT GCG CAA TCT TTA CAA AAT GTG GTG AGC AAA AAG ACT 1673
Ser Leu Pro Asp Ala Gln Ser Leu Gln Asn Val Val Ser Lys Lys Thr
455 460 465
AAC CCT AAC AGC CCG CAA GGC ATA CAG GAT AAT TAC TAC ATT GAC TCC 1721
Asn Pro Asn Ser Pro Gln Gly Ile Gln Asp Asn Tyr Tyr Ile Asp Ser
470 475 480
AAC ATC CAT TCT CAA GTG CAA TCT AGG AGT CAA GAA CTC GGC AGT AAC 1769
Asn Ile His Ser Gln Val Gln Ser Arg Ser Gln Glu Leu Gly Ser Asn
485 490 495 500
CCT TTC AGA CGC GCC GGG CTA ATC GCC GCT TCT ACC ACC AAT AAC GGC 1817
Pro Phe Arg Arg Ala Gly Leu Ile Ala Ala Ser Thr Thr Asn Asn Gly
505 510 515
GCG ATG AAT GGG ATT GGC TTT CAA GTG GGC TAT AAG CAA TTC TTT GGG 1865
Ala Met Asn Gly Ile Gly Phe Gln Val Gly Tyr Lys Gln Phe Phe Gly
520 525 530
AAA AAC AAA CGA TGG GGC GCG AGA TAC TAC GGC TTT GTG GAT TAC AAC 1913
Lys Asn Lys Arg Trp Gly Ala Arg Tyr Tyr Gly Phe Val Asp Tyr Asn
535 540 545
CAC ACC TAT AAC AAG TCC CAA TTT TTC AAC TCC GAT TCT GAT GTT TGG 1961
His Thr Tyr Asn Lys Ser Gln Phe Phe Asn Ser Asp Ser Asp Val Trp
550 555 560
ACT TAT GGC GTG GGG AGC GAT TTG TTA GTG AAT TTC ATC AAC GAT AAA 2009
Thr Tyr Gly Val Gly Ser Asp Leu Leu Val Asn Phe Ile Asn Asp Lys
565 570 575 580
GCC ACT AAA CAC AAT AAA ATT TCT TTT GGC GCG TTT GGC GGT ATC CAA 2057
Ala Thr Lys His Asn Lys Ile Ser Phe Gly Ala Phe Gly Gly Ile Gln
585 590 595
CTA GCC GGG ACT TCA TGG CTT AAT TCT CAG TAT GTG AAT TTA GCG AAT 2105
Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Tyr Val Asn Leu Ala Asn
600 605 610
GTG AAC AAT TAT TAT AAA GCT AAA ATC AAC ACC TCT AAC TTC CAA TTC 2153
Val Asn Asn Tyr Tyr Lys Ala Lys Ile Asn Thr Ser Asn Phe Gln Phe
615 620 625
TTA TTC AAT CTG GGC TTA AGG ACC AAT CTC GCC AGA AAT AAA AGA ATA 2201
Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Arg Asn Lys Arg Ile
630 635 640
GGC GCT GAT CAT AGC GCG CAA CAT GGC ATG GAA TTA GGC GTG AAG ATC 2249
Gly Ala Asp His Ser Ala Gln His Gly Met Glu Leu Gly Val Lys Ile
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 99 PCT/US98106421
645 650 655 660
CCC ACG ATC AAC ACA AAT TAC TAT TCT TTG CTA GGC ACT ACC TTG CAA 2297
Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Thr Leu Gln
665 670 675
TAC AGA AGG CTT TAT AGC GTG TAT CTC AAC TAT GTG TTT GCT TAC TAAAA 2347
Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr
680 6B5 690
GCTTAAACTC CTTTTTAAACTCCCTTTTTA GGGGGTTTAATCTTTTTAACTGACTTTTCT2407
TTTAGCTTTT TTTAATTTTTTCCACCAAAC AAAGTTTTTTGACTTCAAGCGTTAATCACA2467
AAAAATACTC AAAGGCGTTTTTTGCAATCT AAATAAAAAATTAGCGTTATTCAAGCGATC2527
ATTTTAAACC ACCCAAGCAAGAAACCCCAA ACATCTTTAGCGTTCGCGCGCTCCACTAAC258'7
CAAAAAACGC CCCAAA 2603
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 711 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
Met Lys Lys Lys Phe Leu Ser Leu Thr Leu Gly Ser Leu Leu Val Ser
-20 -15 -10 -5
Ala Leu Ser Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln
1 5 10
Ile Gly Glu Ser Ala Gln Met Val Lys Asn Thr Lys Gly Ile Gln Asp
15 20 25
Leu Ser Asp Ser Tyr Glu Arg Leu Asn Asn Leu Leu Thr Ser Tyr Ser
30 35 40
Ala Leu Asn Thr Leu Ile Arg Gln Ser Ala Asp Pro Asn Ala Ile Asn
45 50 55 60
Asn Ala Arg Gly Asn Leu Asn Ala Ser Ala Lys Asn Leu Ile Asn Asp
65 70 75
Lys Lys Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Leu Asn Ala
' 80 85 90
Ala Ala Gly Leu Trp Gln Val Met Ser Tyr Ser Ile Ser Val Cys Gly
95 100 105
- Pro Gly Ser Asp Lys Asn Lys Asn Gly Gly Val Gln Thr Phe Glu Asn
110 115 120
Val Pro Ser Asn Gly Gly Thr Thr Ile Ala Cys Asp Ser Phe Tyr Glu
125 130 135 140
Pro Gly Lys Trp Ser Gly Ile Ser Thr Glu Asn Tyr Ala Lys Ile Asn
SU85TtTUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 100 PCT/US98/06421
145 150 155
Lys Ala Tyr Gln Ile Ile Gln Lys Ala Phe Gly Ala Ser Gly Gln Asp
160 165 170
Ile Pro Ala Leu Ser Asp Thr Lys Glu Leu Asn Phe Glu Ile Lys Gly
175 180 185
Lys Lys Asn Asp Ser Vai Gln Pro Gly Glu Arg Trp Lys Phe Pro Trp
190 195 200
Thr Asn Gly Lys Phe Val Ser Val Lys Trp Val Asn Gly Lys Tyr Glu
205 210 215 220
Glu Ile Lys Glu Asp Ile Lys Val Ser Asn Asn Ala Gln Glu Leu Leu
225 230 235 _
Lys Gln Ala Ser Thr Ile Leu Thr Thr Leu Asn Glu Ala Cys Pro Trp
240 245 250
Leu Ser Asn Gly Gly Ala Gly Asn Val Ala Gly Gly Asn Ser Leu Trp
255 260 265
Ala Gly Ile Asp Lys Gly Asp Gly Ser Ala Cys Gly Ile Phe Lys Asn
270 275 280
Glu Ile Ser Ala Ile Gln Asp Met Ile Lys Asn Ala Glu Ile Ala Val
285 290 295 300
Glu Gln Ser Lys Ile Val Thr Ala Asn Ala Gln Asn Gln His Asn Leu
305 310 315
Asp Thr Gly Lys Ala Phe Asn Pro Tyr Lys Asp Ala Asn Phe Ala Gln
320 325 330
Ser Met Phe Ala Asn Ala Arg Ala Gln Ala Glu Ile Leu Asn Arg Ala
335 340 345
Gln Ala Val Val Lys Asp Phe Glu Arg Ile Pro Ala Ala Phe Val Lys
350 355 360
Asp Ser Leu Gly Val Cys His Glu Lys Gly Ser Asp Gly Asn Leu Arg
365 370 375 380
Gly Thr Pro Ser Gly Thr Val Thr Ser Asn Thr Trp Gly Ala Gly Cys
385 390 395
Ala Tyr Val Gly Glu Thr Val Thr Asn Leu Lys Asn Ser Ile Ala His
400 405 410
Phe Gly Asp Gln Ala GIu Arg Ile His Asn Ala Arg Asn Leu Ala Tyr
415 420 425
Thr Leu Ala Asn Phe Ser Gly Gln Tyr Lys Lys Leu Gly Glu His Tyr
430 435 440
Asp Ser Ile Thr Ala Ala Leu Ser Ser Leu Pro Asp Ala Gln Ser Leu
445 450 455 460
Gln Asn Val Val Ser Lys Lys Thr Asn Pro Asn Ser Pro Gln Gly Ile
465 470 475
Gln Asp Asn Tyr Tyr Ile Asp Ser Asn Ile His Ser Gln Val Gln Ser
480 485 490
Arg Ser Gln Glu Leu Gly Ser Asn Pro Phe Arg Arg Ala Gly Leu Ile
495 500 505
Ala Ala Ser Thr Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Phe Gln
510 515 520
Val Gly Tyr Lys Gln Phe Phe Gly Lys Asn Lys Arg Trp Gly Ala Arg
525 530 535 540
Tyr Tyr Gly Phe Val Asp Tyr Asn His Thr Tyr Asn Lys Ser Gln Phe
545 550 555
Phe Asn Ser Asp Ser Asp Val Trp Thr Tyr Gly Val Gly Ser Asp Leu
560 565 570
Leu Val Asn Phe Ile Asn Asp Lys Ala Thr Lys His Asn Lys Ile Ser
575 580 585
Phe Gly Ala Phe Gly Gly Ile Gln Leu Ala Gly Thr Ser Trp Leu Asn
590 595 600
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 101 PCT/US98/06421
Ser Gln Tyr Val Asn Leu Ala Asn Val Asn Asn Tyr Tyr Lys Ala Lys
605 610 615 620
Ile Asn Thr Ser Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Thr
625 630 635
Asn Leu Ala Arg Asn Lys Arg Ile Gly A1a Asp His Ser Ala Gln His
640 645 650
Gly Met Glu Leu Gly Val Lys Ile Pro Thr I1e Asn Thr Asn Tyr Tyr
655 660 665
Ser Leu Leu Gly Thr Thr Leu Gln Tyr Arg Arg Leu Tyr Ser Val Tyr
670 675 6B0
Leu Asn Tyr Val Phe Ala Tyr
6B5 690
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2427 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 232...2247
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 232...292
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
AAAACGCGCAGCAAAAAATC TCTGTTAAGCTTTTATCATT AGCGTTCCAT TGAAACAAAA60
TCTAAAAACCCTTTCCAATA CCACCCAAACAAACGCGCAA AAAATGCAAA AATTCTAAAT120
TTTCTCCAAATGACAAi~AAA AAAAAAAACGATTTTATGCT ACAATGCTTT TAATACATTC1B0
TTACTTAATGTATAAAATCT CAATCACTCAATTTAATTTC AAAGGATATT T ATG 237
AAA
Met Lys
-20
AAA ACC CTT TTA CTC TCT CTC TCT CTC TCT CTC TCG TCA TCG CTT TTA 285
Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Ser Leu Leu
-15 -10 -5
AAC GCT GAA GAC AAC GGC TTT TTT ATC AGC GCG GGC TAT CAA ATC GGT 333
Asn Ala Glu Asp Asn Gly Phe Phe Ile Ser Ala Gly Tyr Gln Ile Gly
1 5 10
GAA GCC GCT CAA ATG GTG AAA AAC ACC GGC GAA TTG AAA AAA CTT TCA 381
Glu Ala Ala Gln Met Val Lys Asn Thr Gly Glu Leu Lys Lys Leu Ser
15 20 25 30
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 102 PCT/US98/06421
GAC ACT TAT GAG AAT TTG AGC AAC CTT TTA ACC AAT TTT AAC AAC CTC 429
Asp Thr Tyr Glu Asn Leu Ser Asn Leu Leu Thr Asn Phe Asn Asn Leu
35 40 45
AAT CAA GCG GTA ACG AAC GCG AGC AGC CCT TCA GAA ATC AAT GCC ACG 477
Asn Gln Ala Val Thr Asn Ala Ser Ser Pro Ser Glu Ile Asn Ala Thr
50 55 60
ATC GAT AAT TTA AAA GCA AAC ACG CAA GGG CTG ATT GGC GAA AAA ACC 525
Ile Asp Asn Leu Lys Ala Asn Thr Gln Gly Leu Ile Gly Glu Lys Thr
65 70 75
AAT TCC CCG GCG TAT CAA GCG GTG TAT TTG GCG CTC AAT GCG GCG GTG 573
Asn 5er Pro Ala Tyr Gln Ala Val Tyr Leu Ala Leu Asn Ala Ala Val
80 85 90
GGG CTG TGG AAT GTG ATA GCC TAT AAT GTC CAA TGC GGT CCT GGT AAG 621
Gly Leu Trp Asn Val Ile Ala Tyr Asn Val Gln Cys Gly Pro Gly Lys
95 100 105 110
AGT GGG GAT CAA AGC GTA ATT TTT GAT GGC CAA CCA GGA CAT GAT TCA 669
Ser Gly Asp Gln Ser Val Ile Phe Asp Gly Gln Pro Gly His Asp Ser
115 120 125
AGA TCC ATT AAT TGC AAT TTA ACC GGT TAT AAC AAC GGG GTT AGC GGC 717
Arg Ser Ile Asn Cys Asn Leu Thr Gly Tyr Asn Asn Gly Val Ser Gly
130 135 140
CCT TTA TCC ATT GAC AAT TTT AAA ACG CTT AAT CAA GCT TAT CAA ACT 765
Pro Leu Ser Ile Asp Asn Phe Lys Thr Leu Asn Gln Ala Tyr Gln Thr
145 150 155
ATC CAA CAA GCT TTA AAA CAA GAT AGC GGA TTT CCT GTT TTG GAT AGT 813
Ile Gln Gln Ala Leu Lys Gln Asp Ser Gly Phe Pro Val Leu Asp Ser
160 165 170
AAA GGA AAA CAA GTA ACT ATA AAA ATA ACA ACA CAA ACT AAT GGA GCT 861
Lys Gly Lys Gln Val Thr Ile Lys Ile Thr Thr Gln Thr Asn Gly Ala
175 180 185 190
AAT AAA AGT GAA ACT ACT ACT ACT ACT ACT ACT ACT AAT GAC GCT CAA 909
Asn Lys Ser Glu Thr Thr Thr Thr Thr Thr Thr Thr Asn Asp Ala Gln
195 200 205
ACC CTT TTG CAA GAA GCC AGT AAA ATG ATA AGC GTC CTC ACT ACA AAC 957
Thr Leu Leu Gln Glu Ala Ser Lys Met Ile Ser Val Leu Thr Thr Asn
210 215 220
TGC CCA TGG GTA AAT ACC GCT CAT AAC TCA AAC GGG GGT GCA CCG TGG __ 1005
Cys Pro Trp Val Asn Thr Ala His Asn Ser Asn Gly Gly Ala Pro Trp
225 230 235
AAT TTA AAT ACG ACA GGG AAT GTG TGT CAG GTT TTT GCC ACG GAG TTT 1053
Asn Leu Asn Thr Thr Gly Asn Val Cys Gln Val Phe Ala Thr Glu Phe
240 245 250
AGC GCC GTT ACT AGC ATG ATC AAA AAC GCG CAA GAA ATC GTA ACG CAA 1101
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 103 . PCT/US98/06421
Ser Ala Val Thr Ser Met Ile Lys Asn Ala Gln Glu Ile Val 'Thr Gln
255 260 265 270
GCT CAA AGC CTT AAC AAC CCG CAA AGC AAT CAA AAC GCG CCG AAA GAT 1149
Ala Gln Ser Leu Asn Asn Pro Gln Ser Asn Gln Asn Ala Pro Lys Asp
275 280 285
TTC AAT CCT TAC ACC TCT GCT GAT AGG GCT TTC GCT CAA AAC ATG CTC 1197
Phe Asn Pro Tyr Thr Ser Ala Asp Arg Ala Phe Ala Gln Asn Met Leu
290 295 300
AAT CAC GCG CAA GCG CAA GCC AAG ATG CTT GAA CTA GCC GAT CAA ATG 1245
Asn His Ala Gln Ala Gln Ala Lys Met Leu Glu Leu Ala Asp Gln Met
305 310 315
AAA AAA GAC CTT AAC ACT ATC CCA AAA CAA TTT ATC ACA AAC TAC TTG 1293
Lys Lys Asp Leu Asn Thr Ile Pro Lys Gln Phe Ile Thr Asn Tyr Leu
320 325 330
GCA GCT TGC CGC AAT GGG GGT GGG ACA TTA CCT GAT GCA GGG GTT ACT 1341
Ala Ala Cys Arg Asn Gly Gly Gly Thr Leu Pro Asp Ala Gly Val Thr
335 340 345 350
TCT AAC ACT TGG GGG GCC GGT TGC GCC TAT GTG GAA GAG ACG ATA ACC 1389
Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Glu Glu Thr Ile Thr
355 360 365
GCC CTA AAT AAC AGC CTT GCG CAT TTT GGC ACT CAA GCC GAT CAA ATC 1437
Ala Leu Asn Asn Ser Leu Ala His Phe Gly Thr Gln Ala Asp Gln Ile
370 375 380
AAG CAA TCT GAG TTG TTG GCG CGC ACG ATA CTT GAT TTT AGA GGC AGC 1485
Lys Gln Ser Glu Leu Leu Ala Arg Thr Ile Leu Asp Phe Arg Gly Ser
385 390 395
CTT AAG GAT TTA AAC AAC ACT TAT AAC AGC ATC ACC ACG ACC GCT TCA 1533
Leu Lys Asp Leu Asn Asn Thr Tyr Asn Ser Ile Thr Thr Thr Ala Ser
400 405 410
AAC ACG CCC AAT TCC CCA TTC CTT AAA AAT TTG ATA AGC CAA TCC ACT 1581
Asn Thr Pro Asn Ser Pro Phe Leu Lys Asn Leu Ile Ser Gln Ser Thr
415 420 425 430
AAC CCT AAT AAC CCC GGG GGC TTA CAG GCC GTT TAT CAA GTC AAC CAA 1629
Asn Pro Asn Asn Pro Gly Gly Leu Gln Ala Val Tyr Gln Val Asn Gln
435 440 445
AGC GCT TAT TCG CAA TTA TTA AGC GCC ACG CAA GAA TTA GGG CAT AAC 1677
Ser Ala Tyr Ser Gln Leu Leu Ser Ala Thr Gln Glu Leu Gly His Asn
450 455 460
CCT TTC AGA CGC GTT GGC TTA ATC AGC TCT CAA ACC AAC AAC GGT GCG 1725
Pro Phe Arg Arg Val Gly Leu Ile Ser Ser Gln Thr Asn Asn Gly Ala
465 470 475
ATG AAT GGG ATC GGC GTG CAA ATA GGG TAT AAA CAA TTT TTT GGT GAA 1773
Met Asn Gly Ile Gly Val Gln Ile Gly Tyr Lys Gln Phe Phe Gly Glu
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 104 PCT/LTS98/06421
480 485 490
AAA AGA AGA TGG GGG TTA AGG TAT TAT GGT TTT TTT GAT TAC AAC CAT 1821
Lys Arg Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His
495 500 505 510
GCT TAT ATC AAA TCC AGC TTT TTC AAC TCC GCC TCT GAT GTG TTC ACT 1869
Ala Tyr Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Phe Thr
515 520 525
TAT GGG GTA GGA ACA GAT GTC CTC TAT AAC TTT ATC AAC GAT AAA GCC 1917
Tyr Gly Val Gly Thr Asp Val Leu Tyr Asn Phe Ile Asn Asp Lys Ala
530 535 540
ACC AAA AAC AAT AAG ATT TCT TTT GGG GTG TTT GGG GGG ATT GCG TTA 1965
Thr Lys Asn Asn Lys Ile Ser Phe Gly Val Phe Gly Gly Ile Ala Leu
545 550 555
GCT GGC ACT TCG TGG CTT AAT TCT CAA TAC GTG AAT TTA GCG ACA TTC 2013
Ala Gly Thr Ser Trp Leu Asn Ser Gln Tyr Val Asn Leu Ala Thr Phe
560 565 570
AAT AAT TTT TAC AGC GCT AAA ATG AAT GTG GCG AAT TTC CAA TTC TTA 2061
Asn Asn Phe Tyr Ser Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu
575 580 585 590
TTC AAC TTG GGC TTG AGA ATG AAT CTC GCT AAA AAC AAA AAG AAA GCG 2109
Phe Asn Leu Gly Leu Arg Met Asn Leu Ala Lys Asn Lys Lys Lys Ala
595 600 605
AGC GAT CAT GTA GCT CAG CAT GGC GTG GAA CTA GGC GTG AAG ATC CCT 2157
Ser Asp His Val Ala Gln His Gly Val Glu Leu Gly Val Lys Ile Pro
610 6I5 620
ACG ATC AAC ACG AAT TAC TAT TCT TTG CTA GGC ACT CAA CTC CAA TAC 2205
Thr Ile Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Gln Leu Gln Tyr
625 630 635
CGC AGG CTT TAT AGC GTG TAT TTG AAT TAT GTG TTT GCT TAC TAATATCTG 2256
Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr
640 645 650
TCTTTTTGTG AAACTCCCTT TTTAAGGGAT TTTTTTTGAA GCCTTTCTTT TTTTAAACCC 2316
TCTTTTTTGG GGGTCAAGCG TAAAATTCAC CCCTATCCCT TTAAGAAAAT AAAATAAAAG 2376
AAAATGCGTT TTATAACAAA ATAAGATCTA AAACAATAAA ACAAAAACCC A 2427
(2) INFORMATION FOR SEQ ID NO:10:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 672 amino acids
(B) TYPE: amino acid -
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein _
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 105 PCT/I1S98/06421
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Ser
-20 -15 -10 -5
Leu Leu Asn Ala Glu Asp Asn Gly Phe Phe Ile Ser Ala Gly Tyr Gln
1 5 10
Ile Gly Glu Ala Ala Gln Met Val Lys Asn Thr Gly Glu Leu Lys Lys
15 20 25
Leu Ser Asp Thr Tyr Glu Asn Leu Ser Asn Leu Leu Thr Asn Phe Asn
30 35 40
Asn Leu Asn Gln Ala Val Thr Asn Ala Ser Ser Pro Ser Glu Ile Asn
45 50 55 60
Ala Thr Ile Asp Asn Leu Lys Ala Asn Thr Gln Gly Leu Ile Gly Glu
65 70 75
Lys Thr Asn Ser Pro Ala Tyr Gln Ala Val Tyr Leu Ala Leu Asn Ala
80 85 90
Ala Val Gly Leu Trp Asn Val Ile Ala Tyr Asn Val Gln Cys Gly Pro
95 100 105
Gly Lys Ser Gly Asp Gln Ser Val Ile Phe Asp Gly Gln Pro Gly His
110 115 120
Asp Ser Arg Ser Ile Asn Cys Asn Leu Thr Gly Tyr Asn Asn Gly Val
125 130 135 140
Ser Gly Pro Leu Ser Ile Asp Asn Phe Lys Thr Leu Asn Gln Ala Tyr
145 150 155
Gln Thr Ile Gln Gln Ala Leu Lys Gln Asp Ser Gly Phe Pro Val Leu
160 165 170
Asp Ser Lys Gly Lys Gln Val Thr Ile Lys Ile Thr Thr Gln Thr Asn
175 180 185
Gly Ala Asn Lys Ser Glu Thr Thr Thr Thr Thr Thr Thr Thr Asn Asp
190 195 200
Ala Gln Thr Leu Leu Gln Glu Ala Ser Lys Met Ile Ser Val Leu Thr
205 210 215 220
Thr Asn Cys Pro Trp Val Asn Thr Ala His Asn Ser Asn Gly Gly Ala
225 230 235
Pro Trp Asn Leu Asn Thr Thr Gly Asn Val Cys Gln Val Phe Ala Thr
240 245 250
Glu Phe Ser Ala Val Thr Ser Met Ile Lys Asn Ala Gln Glu Ile Val
255 260 265
Thr Gln Ala Gln Ser Leu Asn Asn Pro Gln Ser Asn Gln Asn Ala Pro
270 275 280
Lys Asp Phe Asn Pro Tyr Thr Ser Ala Asp Arg Ala Phe Ala Gln Asn
285 290 295 300
Met Leu Asn His Ala Gln Ala Gln Ala Lys Met Leu Glu Leu Ala Asp
- 305 310 315
Gln Met Lys Lys Asp Leu Asn Thr Ile Pro Lys Gln Phe Ile Thr Asn
320 325 330
- Tyr Leu Ala Ala Cys Arg Asn Gly Gly Gly Thr Leu Pro Asp Ala Gly
335 340 345
Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Glu Glu Thr
350 355 360
Ile Thr Ala Leu Asn Asn Ser Leu Ala His Phe Gly Thr Gln Ala Asp
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 106 PCT/US98/06421
365 370 375 380
Gln Ile Lys Gln Ser Glu Leu Leu Ala Arg Thr Ile Leu Asp Phe Arg
385 390 395
Gly Ser Leu Lys Asp Leu Asn Asn Thr Tyr Asn Ser Ile Thr Thr Thr
400 405 410
Ala Ser Asn Thr Pro Asn Ser Pro Phe Leu Lys Asn Leu Ile Ser Gln
415 420 425
Ser Thr Asn Pro Asn Asn Pro Gly Gly Leu Gln Ala Val Tyr Gln Val
430 435 440
Asn Gln Ser Ala Tyr Ser Gln Leu Leu Ser Ala Thr Gln Glu Leu Gly
445 450 455 460
His Asn Pro Phe Arg Arg Val Gly Leu Ile Ser Ser Gln Thr Asn Asn
465 470 475
Gly Ala Met Asn Gly Ile Gly Val Gln Ile Gly Tyr Lys Gln Phe Phe
480 485 490
Gly Glu Lys Arg Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr
495 500 505
Asn His Ala Tyr Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val
510 515 520
Phe Thr Tyr Gly Val Gly Thr Asp Val Leu Tyr Asn Phe Ile Asn Asp
525 530 535 540
Lys Ala Thr Lys Asn Asn Lys Ile Ser Phe Gly Val Phe Gly Gly Ile
545 550 555
Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Tyr Val Asn Leu Ala
560 565 570
Thr Phe Asn Asn Phe Tyr Ser Ala Lys Met Asn VaI Ala Asn Phe Gln
575 580 585
Phe Leu Phe Asn Leu Gly Leu Arg Met Asn Leu Ala Lys Asn Lys Lys
590 595 600
Lys Ala Ser Asp His Val Ala Gln His Gly Val Glu Leu Gly Val Lys
605 610 615 620
Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Gln Leu
625 630 635
Gln Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr
640 645 650
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2429 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 205...2277 ___
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 205...259 -
(D) OTHER INFORMATION:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 107 PCT/US98/06421
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
TGAAAGAAGA CTGATTAGTC TTTCTTTTAG GGGCGATTCA AGCCTTAAAAGCCGGGTCAA60
AATCCCCATT TTTCCCAATT TTTACAAAAA AAAAAAAAAC AAAATCTCTAAAATTTAGAG120
CTAAAATTAG CCATAAAATT CCATTTATTG CTTATAATAT GAAGTTTCTTTGTATCAAAG180
AAAAATCTAT TAAAAGGAGA AAAC ATG AAA AAA TCC CTC TCT CTT 231
TTA CTC
Met Lys Lys Ser Leu Leu Leu Ser Leu
-15 -10
TCT CTC ATC GCT TCC TTA TCA AGA GCT GAA GAT GAC GGA TTT TAT ACG 279
Ser Leu Ile Ala Ser Leu Ser Arg Ala Glu Asp Asp Gly Phe Tyr Thr
-5 1 5
AGT GTG GGC TAT CAG ATC GGT GAA GCG GTC CAA CAA GTG AAA AAC ACA 327
Ser Val Gly Tyr Gln Ile Gly Glu Ala Val Gln Gln Val Lys Asn Thr
15 20
GGA GCA TTG CAA AAT CTT GCA GAC AGA TAC GAT AAC TTA AAC AAC CTT 375
Gly Ala Leu Gln Asn Leu Ala Asp Arg Tyr Asp Asn Leu Asn Asn Leu
25 30 35
TTA AAC CAA TAC AAT TAT TTA AAT TCC TTA GTC AAT TTA GCC AGC ACG 423
Leu Asn Gln Tyr Asn Tyr Leu Asn Ser Leu Vai Asn Leu Ala Ser Thr
40 45 50 55
CCG AGC GCG ATC ACC GGT GCG ATT GAT AAT TTA AGC TCA AGC GCG ATT 471
Pro Ser Ala Ile Thr Gly Ala Ile Asp Asn Leu Ser Ser Ser Ala Ile
60 65 70
AAC CTC ACT AGC GCC ACC ACC ACT TCC CCC GCC TAT CAA GCT GTG GCT 519
Asn Leu Thr Ser Ala Thr Thr Thr Ser Pro Ala Tyr Gln Ala Val Ala
75 80 85
TTA GCG CTC AAT GCC GCT GTG GGC ATG TGG CAA GTC ATA GCC CTT TTT 567
Leu Ala Leu Asn Ala Ala Val Gly Met Trp Gln Val Ile Ala Leu Phe
90 95 100
ATT GGC TGT GGC CCT GGC CCT ACC AAT AAT CAA AGC TAT CAA TCG TTT 615
Ile Gly Cys Gly Pro Gly Pro Thr Asn Asn Gln Ser Tyr Gln Ser Phe
105 110 115
GGT AAC ACA CCA GCC CTT AAT GGG ACC ACC ACC ACT TGC AAT CAA GCA 663
Gly Asn Thr Pro Ala Leu Asn Gly Thr Thr Thr Thr Cys Asn Gln Ala
120 1 125 130 135
TAT GGG ACA GGC CCT AAT GGC ATC CTA TCT ATT GAT GAA TAC CAA AAA 711
Tyr Gly Thr Gly Pro Asn Gly Ile Leu Ser Ile Asp Glu Tyr Gln Lys
140 145 150
CTC AAC CAA GCT TAT CAG ATC ATC CAA ACC GCT TTA AAC CAA AAT CAA 759
Leu Asn Gln Ala Tyr Gln Ile Ile Gln Thr Ala Leu Asn Gln Asn Gln
155 160 165
GGG GGT GGG ATG CCT GCC TTG AAT GAC ACC ACC AAA ACA GGG GTA GTC 807
Gly Gly Gly Met Pro Ala Leu Asn Asp Thr Thr Lys Thr Gly Val Val
SU9STITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 1 ~g PCT/OS98/06421
170 175 180
AAC ATA CAA CAA ACC AAT TAT AGG ACC ACC ACA CAA AAC AAT ATC ATA 855
Asn Ile Gln Gln Thr Asn Tyr Arg Thr Thr Thr Gln Asn Asn Ile Ile
185 190 195
GAG CAT TAT TAT ACA GAG AAT GGG AAA GAG ATC CCA GTC TCT TAT TCA 903
Glu His Tyr Tyr Thr Glu Asn Gly Lys Glu Ile Pro Val Ser Tyr Ser
200 205 210 215
GGC GGA TCA TCA TTC TCG CCT ACA ATA CAA TTG ACA TAC CAT AAT AAC 951
Gly Gly Ser Ser Phe Ser Pro Thr Ile Gln Leu Thr Tyr His Asn Asn
220 225 230
GCT GAA AAC CTT TTG CAA CAA GCC GCC ACT ATC ATG CAA GTC CTT ATT 999
Ala Glu Asn Leu Leu Gln Gln Ala Ala Thr Ile Met Gln Val Leu Ile
235 240 245
ACT CAA AAG CCG CAT GTG CAA ACG AGC AAT GGC GGT AAA GCG TGG GGG 1047
Thr Gln Lys Pro His Val Gln Thr Ser Asn Gly Gly Lys Ala Trp Gly
250 255 260
TTG AGT TCT ACG CCT GGG AAT GTG ATG GAT ATT TTT GGT CCT TCT TTT 1095
Leu Ser Ser Thr Pro Gly Asn Val Met Asp Ile Phe Gly Pro Ser Phe
265 270 275
AAC GCT ATT AAT GAG ATG ATT AAA AAC GCT CAA ACA GCC CTA GCA AAA 1143
Asn Ala Ile Asn Glu Met Ile Lys Asn Ala Gln Thr Ala Leu Ala Lys
280 285 290 295
ACC CAA CAG CTT AAC GCT AAT GAA AAC GCC CAA ATC ACG CAA CCC AAC 1191
Thr Gln Gln Leu Asn Ala Asn Glu Asn Ala Gln Ile Thr Gln Pro Asn
300 305 310
AAT TTC AAC CCC TAC ACC TCT AAA GAC AAA GGG TTC GCT CAA GAA ATG 1239
Asn Phe Asn Pro Tyr Thr Ser Lys Asp Lys Gly Phe Ala Gln Glu Met
315 320 325
CTC AAT AGA GCT GAA GCT CAA GCA GAG ATT TTA AAT TTA GCT AAG CAA 1287
Leu Asn Arg Ala Glu Ala Gln Ala Glu Ile Leu Asn Leu Ala Lys Gln
330 335 340
GTA GCG AAC AAT TTC CAC AGC ATT CAA GGG CCT ATT CAA GGG GAT TTA 1335
Val Ala Asn Asn Phe His Ser Ile Gln Gly Pro Ile Gln Gly Asp Leu
345 350 355
GAA GAA TGT AAA GCA GGA TCG GCT GGC GTG ATC ACT AAT AAC ACT TGG 1383
G1u Glu Cys Lys Ala Gly Ser Ala Gly Val Ile Thr Asn Asn Thr Trp
360 365 370 375
GGT TCA GGT TGC GCG TTT GTG AAA GAA ACT TTA AAC TCT TTA GAG CAA 14-31
Gly Ser Gly Cys Ala Phe Val Lys Glu Thr Leu Asn Ser Leu Glu Gln
380 385 390
CAC ACC GCT TAT TAC GGC AAC CAG GTC AAT CAG GAT AGG GCT TTG GCT 1479
His Thr Ala Tyr Tyr Gly Asn Gln Val Asn Gln Asp Arg Ala Leu Ala
395 400 405
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 109 PCT/US98/06421
CAA ACC ATT TTG AAT TTT AAA GAA GCC CTT AAC ACC CTG AAT AAA GAC 1527
Gln Thr Ile Leu Asn Phe Lys Glu Ala Leu Asn Thr Leu Asn Lys Asp
410 415 420
TCA AAA GCG ATC AAT AGC GGT ATC TCC AAC TTG CCT AAC GCT AAA TCT 1575
Ser Lys Ala Ile Asn Ser Gly Ile Ser Asn Leu Pro Asn Ala Lys Ser
425 430 435
CTT CAA AAC ATG ACG CAT GCC ACT CAA AAC CCT AAT TCC CCA GAA GGT 1623
Leu Gln Asn Met Thr His Ala Thr Gln Asn Pro Asn Ser Pro Glu Gly
440 445 450 455
CTG CTC ACT TAT TCT TTG GAT TCA AGC AAA TAC AAC CAG CTC CAA ACC 1671
Leu Leu Thr Tyr Ser Leu Asp Ser Ser Lys Tyr Asn Gln Leu Gln Thr
460 465 470
ATC GCG CAA GAA TTG GGC AAA AAC CCT TTC AGG CGC TTT GGC GTG ATT 1719
Ile Ala Gln Glu Leu Gly Lys Asn Pro Phe Arg Arg Phe Gly Val Ile
475 480 485
GAC TTT CAA AAC AAC AAC GGC GCA ATG AAC GGG ATC GGC GTG CAA GTG 1767
Asp Phe Gln Asn Asn Asn Gly Ala Met Asn Gly Ile Gly Val Gln Val
490 495 500
GGT TAT AAA CAA TTC TTT GGT AAA AAA AGG AAT TGG GGG TTA AGG TAT 1815
Gly Tyr Lys Gln Phe Phe Gly Lys Lys Arg Asn Trp Gly Leu Arg Tyr
505 510 515
TAT GGT TTC TTT GAT TAT AAC CAT GCT TAT ATC AAA TCT AAT TTT TTC 1863
Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Asn Phe Phe
520 525 530 535
AAC TCC GCT TCT GAT GTG TGG ACT TAT GGG GTG GGT ATG GAC GCT CTC 1911
Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Met Asp Ala Leu
540 545 550
TAT AAC TTC ATC AAC GAT AAA AAC ACC AAC TTT TTA GGC AAG AAC AAC 1959
Tyr Asn Phe Ile Asn Asp Lys Asn Thr Asn Phe Leu Gly Lys Asn Asn
555 560 565
AAG CTT TCA GTA GGG CTT TTT GGA GGC TTT GCG TTA GCC GGG ACT TCG 2007
Lys Leu Ser Val Gly Leu Phe Gly Gly Phe Ala Leu Ala Gly Thr Ser
570 575 580
TGG CTT AAT TCC CAA CAA GTG AAT TTG ACC ATG ATG AAT GGC ATT TAT 2055
Trp Leu Asn Ser Gln Gln Val Asn Leu Thr Met Met Asn Gly Ile Tyr
585 590 595
AAC GCT AAT GTC AGC ACT TCT AAC TTC CAA TTT TTG TTT GAT TTA GGC 2103
Asn Ala Asn Val Ser Thr Ser Asn Phe Gln Phe Leu Phe Asp Leu Gly
600 505 610 615
- TTG AGA ATG AAC CTC GCT AGG CCT AAG AAA AAA GAC AGC GAT CAT GCC 2151
Leu Arg Met Asn Leu Ala Arg Pro Lys Lys Lys Asp Ser Asp His Ala
620 625 630
GCT CAG CAT GGC ATT GAA CTA GGT TTT AAG ATC CCC ACG ATC AAC ACC 2199
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 110 PCT/US98/06421
Ala Gln His Gly Ile Glu Leu Gly Phe Lys Ile Pro Thr Ile Asn Thr
635 640 645
AAC TAT TAT TCT TTC ATG GGC GCT AAA CTA GAA TAC AGA AGG ATG TAT 2247
Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Glu Tyr Arg Arg Met Tyr
650 655 660
AGC CTT TTT CTC AAT TAT GTG TTT GCT TAC TAAAAATTCT TTTTGAACCC CTC 2300
Ser Leu Phe Leu Asn Tyr Val Phe Ala Tyr
665 670
TTTTTTTGGG GGAGTGTTGC AAAAATGCCC CCCTATTTGC TTGTGAGTTT TGGTTAAAAT 2360
TTTAGTTACC CACGCTTAAA AAGCGCCAAG CCTTTTACAC ACAACTCCTT TAATTTTGTT 2420
TTTAAGAAA 2429
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 691 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
{D) TOPOLOGY: linear
{ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
{A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...18
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Lys Lys Ser Leu Leu Leu Ser Leu Ser Leu Ile Ala Ser Leu Ser
-15 -10 -5
Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly
1 5 10
Glu Ala Val Gln Gln Val Lys Asn Thr Gly Ala Leu Gln Asn Leu Ala
15 20 25 30
Asp Arg Tyr Asp Asn Leu Asn Asn Leu Leu Asn Gln Tyr Asn Tyr Leu
35 40 45
Asn Ser Leu Val Asn Leu Ala Ser Thr Pro Ser Ala Ile Thr Gly Ala
50 55 60
Ile Asp Asn Leu Ser Ser Ser Ala Ile Asn Leu Thr Ser Ala Thr Thr
65 70 75
Thr Ser Pro Ala Tyr Gln Ala Val Ala Leu Ala Leu Asn Ala Ala Val
80 85 90
Gly Met Trp Gln Val Ile Ala Leu Phe Ile Gly Cys Gly Pro Gly Pro
95 100 105 110
Thr Asn Asn Gln Ser Tyr Gln Ser Phe Gly Asn Thr Pro Ala Leu Asn
115 120 125
Gly Thr Thr Thr Thr Cys Asn Gln Ala Tyr Gly Thr Gly Pro Asn Gly
130 135 140 _
Ile Leu Ser Ile Asp Glu Tyr Gln Lys Leu Asn Gln Ala Tyr Gln Ile
145 150 155
SUBSTITUTE SHEET (RULE 26)
r
CA 02286893 1999-10-O1
WO 98/43479 1 I I PCT/US98/06421
Ile Gln Thr Ala Leu Asn Gln Asn Gln Gly Gly Gly Met Pro Ala Leu
160 165 170
Asn Asp Thr Thr Lys Thr Gly Val Val Asn Ile Gln Gln Thr Asn Tyr
175 180 185 190
Arg Thr Thr Thr Gln Asn Asn Ile Ile Glu His Tyr Tyr Thr Glu Asn
195 200 205
Gly Lys Glu Ile Pro Val Ser Tyr Ser Gly Gly Ser Ser Phe Ser Pro
210 215 220
Thr Ile Gln Leu Thr Tyr His Asn Asn Ala Glu Asn Leu Leu Gln Gln
225 230 235
Ala Ala Thr Ile Met Gln Val Leu Ile Thr Gln Lys Pro His Val Gln
240 245 250
Thr Ser Asn Gly Gly Lys Ala Trp Gly Leu Ser Ser Thr Pro Gly Asn
255 260 265 270
Val Met Asp Ile Phe Gly Pro Ser Phe Asn Ala Ile Asn Glu Met Ile
275 280 285
Lys Asn Ala Gln Thr Ala Leu Ala Lys Thr Gln Gln Leu Asn Ala Asn
290 295 300
Glu Asn Ala Gln Ile Thr Gln Pro Asn Asn Phe Asn Pro Tyr Thr Ser
305 310 315
Lys Asp Lys Gly Phe Ala Gln Glu Met Leu Asn Arg Ala Glu Ala Gln
320 325 330
Ala Glu Ile Leu Asn Leu Ala Lys Gln Val Ala Asn Asn Phe His Ser
335 340 345 350
Ile Gln Gly Pro Ile Gln Gly Asp Leu Glu Glu Cys Lys Ala Gly Ser
355 360 365
Ala Gly Val Ile Thr Asn Asn Thr Trp Gly Ser Gly Cys Ala Phe Val
370 375 380
Lys Glu Thr Leu Asn 5er Leu Glu Gln His Thr Ala Tyr Tyr Gly Asn
385 390 395
Gln Val Asn Gln Asp Arg Ala Leu Ala Gln Thr Ile Leu Asn Phe Lys
400 405 410
Glu Ala Leu Asn Thr Leu Asn Lys Asp Ser Lys Ala Ile Asn Ser Gly
415 420 425 430
Ile Ser Asn Leu Pro Asn Ala Lys Ser Leu Gln Asn Met Thr His Ala
435 440 445
Thr Gln Asn Pro Asn Ser Pro Glu Gly Leu Leu Thr Tyr Ser Leu Asp
450 455 460
Ser Ser Lys Tyr Asn Gln Leu Gln Thr Ile Ala Gln Glu Leu Gly Lys
465 470 475
Asn Pro Phe Arg Arg Phe Gly Val Ile Asp Phe Gln Asn Asn Asn Gly
480 485 490
Ala Met Asn Gly Ile Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly
495 500 505 510
Lys Lys Arg Asn Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn
515 520 525
His Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp
530 535 540
Thr Tyr Gly Val Gly Met Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys
545 550 555
Asn Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe
560 565 570
~ Gly Gly Phe Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Gln Val
575 580 585 590
Asn Leu Thr Met Met Asn Gly Ile Tyr Asn Ala Asn Val Ser Thr Ser_
595 600 605
Asn Phe Gln Phe Leu Phe Asp Leu Gly Leu Arg Met Asn Leu Ala Arg
SU8ST1TUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 112 PCT/US98/06421
610 615 620
Pro Lys Lys Lys Asp Ser Asp His Ala Ala Gln His Gly Ile Glu Leu
625 630 635
Gly Phe Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly
640 645 650
Ala Lys Leu Glu Tyr Arg Arg Met Tyr Ser Leu Phe Leu Asn Tyr Val
655 660 665 670
Phe Ala Tyr
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2270 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 130...2049
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 130...193
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
ATTGAGCGCA TCAAAACACC CTAAAACTTT TTTGAAATCC AATAAATTTA TGTTATAATT 60
AAACGCATTG TAAATAAATT CTCATTTTGA TACATTTTTA CAATAAAACA TTACTTTAAG 120
GAACATCTT ATG AAA AAA ACG AAA AAA ACG ATT CTG CTT TCT CTA ACT CTC 171
Met Lys Lys Thr Lys Lys Thr Ile Leu Leu Ser Leu Thr Leu
-20 -15 -10
GCG GCG TCA TTG CTC CAT GCT GAA GAC AAC GGC GTT TTT TTA AGC GTG 219
Ala Ala Ser Leu Leu His Ala Glu Asp Asn Gly Val Phe Leu Ser Val
-5 1 5
GGT TAT CAA ATC GGT GAA GCG GTT CAA AAA GTG AAA AAC GCC GAC AAG 267
Gly Tyr Gln Ile Gly Glu Ala Val Gln Lys Val Lys Asn Ala Asp Lys
15 20 25
GTG CAA AAA CTT TCA GAC ACT TAT GAA CAA TTA AGC CGG CTT TTA ACC 315
Val Gln Lys Leu Ser Asp Thr Tyr Glu Gln Leu Ser Arg Leu Leu Thr
30 35 40
AAC GAT AAT GGC ACA AAC TCA AAG ACA AGC GCG CAA ATC AAC CAA GCG 363
Asn Asp Asn Gly Thr Asn Ser Lys Thr Ser Ala Gln Ile Asn Gln Ala_
45 50 55
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 113 PCT/US98/06421
GTT AATAAT TTGAAC GAACGCGCA AAA TTA GCCGGT GGGACAACC 411
ACT
Val AsnAsn LeuAsn GluArgAla LysThrLeu AlaGly GlyThrThr
60 65 70
AAT TCCCCT GCCTAT CAAGCCACG CTTTTAGCG TTGAGA TCGGTGTTA 459
Asn SerPro AlaTyr GlnAlaThr LeuLeuAla LeuArg SerValLeu
75 80 85
GGG CTATGG AATAGC ATGGGTTAT GCGGTCATA TGCGGA GGTTATACC 507
Gly LeuTrp AsnSer MetGlyTyr AlaValIle CysGly GlyTyrThr
90 95 100 105
AAA AGTCCA GGCGAA AACAATCAA AAAGATTTC CACTAC ACCGATGAG 555
Lys SerPro GlyGlu AsnAsnGln LysAspPhe HisTyr ThrAspGlu
110 115 120
AAT GGCAAT GGCACT ACAATCAAT TGCGGTGGG AGCACA AATAGTAAT 603
Asn GlyAsn GlyThr ThrIleAsn CysGlyGly SerThr AsnSerAsn
125 130 135
GGC ACTCAT AGTTCT AGTGGCACA AATACATTA AAAGCA GACAAAAAT 651
Gly ThrHis SerSer SerGlyThr AsnThrLeu LysAla AspLysAsn
140 145 150
GTT TCTCTA TCTATT GAGCAATAT GAAAAAATC CATGAA GCTTATCAG 699
Val SerLeu SerIle GluGlnTyr GluLysIle HisGlu AlaTyrGln
155 160 165
ATT CTTTCA AAAGCT TTAAAACAA GCCGGGCTT GCTCCT TTAAATAGC 747
Ile LeuSer LysAla LeuLysGln AlaGlyLeu AlaPro LeuAsnSer
170 175 180 185
AAA GGGGAA AAGTTA GAAGCGCAT GTAACCACA TCAAAA CCAGAAAAT 795
Lys GlyGlu LysLeu GluAlaHis ValThrThr SerLys ProGluAsn
190 195 200
AAT AGTCAA ACTAAA ACGACAACT TCTGTTATT GATACG ACTAATGAT 843
Asn SerGln ThrLys ThrThrThr SerValIle AspThr ThrAsnAsp
205 210 215
GCG CAAAAT CTTTTG ACTCAAGCG CAAACGATT GTCAAT ACCCTTAAA 891
Ala GlnAsn LeuLeu ThrGlnAla GlnThrIle ValAsn ThrLeuLys
220 225 230
GAT TATTGC CCCATG TTGATAGCG AAATCTAGT AGTGAA AGTAGTGGC 939
Asp TyrCys ProMet LeuIleAla LysSerSer SerGlu SerSerGly
235 240 245
GCA GCTACT ACAAAC GCCCCTTCA TGGCAAACA GCCGGT GGCGGCAAA 987
Ala AlaThr ThrAsn AlaProSer TrpGlnThr AlaGly GlyGly_
Lys
250 255 260 265
AAT TCATGT GCGACT TTTGGTGCG GAGTTTAGT GCCGCT TCAGACATG 1035
Asn SerCys AlaThr PheGlyAla GluPheSer AlaAla SerAspMet
270 275 280
ATT AATAAT GCGCAA AAAATCGTT CAAGAAACC CAACAA CTCAGCGCC 1083
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 114 PCTNS98/06421
Ile Asn Asn Ala Gln Lys Ile Val Gln Glu Thr Gln Gln Leu Ser Ala
285 290 295
AAC CAA CCA AAA AAT ATC ACA CAA CCC CAT AAT CTC AAC CTT AAC ACC 1131
Asn Gln Pro Lys Asn Ile Thr Gln Pro His Asn Leu Asn Leu Asn Thr
300 305 310
CCT AGC AGT CTT ACG GCT TTA GCT CAA AAA ATG CTC AAA AAT GCG CAA 1179
Pro Ser Ser Leu Thr Ala Leu Ala Gln Lys Met Leu Lys Asn Ala Gln
315 320 325
TCT CAA GCA GAA ATT TTA AAA CTA GCC AAT CAA GTG GAG AGC GAT TTT 1227
Ser Gln Ala Glu Ile Leu Lys Leu Ala Asn Gln Val Glu Ser Asp Phe
330 335 340 345
AAC AAA CTT TCT TCA GGC CAT CTT AAA GAC TAC ATA GGG AAA TGC GAT 1275
Asn Lys Leu Ser Ser Gly His Leu Lys Asp Tyr Ile Gly Lys Cys Asp
350 355 360
GCG AGC GCT ATA AGC AGT GCG AAT ATG ACA ATG CAA AAT CAA AAG AAC 1323
Ala Ser Ala Ile Ser Ser Ala Asn Met Thr Met Gln Asn Gln Lys Asn
365 370 375
AAT TGG GGG AAC GGG TGT GCT GGC GTG GAA GAA ACT CTG TCT TCA TTA 1371
Asn Trp Gly Asn Gly Cys Ala Gly Val Glu Glu Thr Leu Ser Ser Leu
380 385 390
AAA ACA AGT GCC GCT GAT TTT AAC AAC CAA ACG CCA CAA ATC AAT CAA 1419
Lys Thr Ser Ala Ala Asp Phe Asn Asn Gln Thr Pro Gln Ile Asn Gln
395 400 405
GCG CAA AAC CTA GCC AAC ACC CTT ATT CAA GAA CTT GGC AAC AAC CCT 1467
Ala Gln Asn Leu Ala Asn Thr Leu Ile Gln Glu Leu Gly Asn Asn Pro
410 415 420 425
TTT AGG AAT ATG GGC ATG ATC GCT TCT TCA ACC ACG AAT AAC GGC GCC 1515
Phe Arg Asn Met Gly Met Ile Ala Ser Ser Thr Thr Asn Asn Gly Ala
430 435 440
TTG AAT GGC CTT GGG GTG CAA GTG GGT TAT AAG CAA TTT TTT GGG GAA 1563
Leu Asn Gly Leu Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu
445 450 455
AAG AAA AGA TGG GGG TTA AGG TAT TAT GGT TTC TTT GAT TAC AAC CAC 1611
Lys Lys Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His
460 465 470
GCC TAT ATC AAA TCC AAT TTC TTT AAC TCG GCT TCT GAT GTG TGG ACT 1659
Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr
475 480 485
TAT GGG GTG GGC AGC GAT TTA TTG TTT AAT TTC ATC AAT GAT AAA AAC 1707
Tyr Gly Val Gly Ser Asp Leu Leu Phe Asn Phe Ile Asn Asp Lys Asn
490 495 500 505
ACC AAC TTT TTA GGC AAG AAT AAC AAG ATT TCA GTG GGA TTT TTT GGA 1755
Thr Asn Phe Leu Gly Lys Asn Asn Lys Ile Ser Val Gly Phe Phe Gly
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 115 PCT/US98/06421
510 515 520
GGT ATC GCC TTA GCA GGG ACT TCA TGG CTT AAT TCT CAA TTC GTG AAT 1803
Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Phe Val Asn
525 530 535
TTA AAA ACC ATC AGC AAT GTT TAT AGC GCT AAA GTG AAT ACG GCT AAC 1851
Leu Lys Thr Ile Ser Asn Val Tyr Ser Ala Lys Val Asn Thr Ala Asn
540 545 550
TTC CAATTT TTATTCAAT TTGGGC TTGAGAACC AATCTCGCT AGACCT 1899
Phe GlnPhe LeuPheAsn LeuGly LeuArgThr AsnLeuAla ArgPro
555 560 565
AAG AAAAAA GATAGTCAT CATGCG GCTCAACAT GGCATGGAA TTGGGC 1947
Lys LysLys AspSerHis HisAla AlaGlnHis GlyMetGlu LeuGly
570 575 580 585
GTG AAAATC CCTACCATT AACACG AATTATTAT TCTTTTCTA GACACT 1995
Val LysIle ProThrIle AsnThr AsnTyrTyr SerPheLeu AspThr
590 595 600
AAA CTAGAA TATCGAAGG CTTTAT AGCGTGTAT CTCAATTAT GTGTTT 2043
Lys LeuGlu TyrArgArg LeuTyr SerValTyr LeuAsnTyr ValPhe
605 610 615
GCC TATTAAAAACCCT TTTAAAAAAC CTCTAAAGAT
2101
CTTTTTAAAA AA
AAGGGGGGGC
Ala Tyr
AAATTTTCAA AAAACAATCA TTAAACCCTA AAAAAGAAAT TTTAAGGTAT AATGCTTTCG 2161
CCATTTTTAA TTTTCCATGG CAAACTCCTT TTTAGAATTT ATCCCCATAA TCGCTCTTAT 2221
GGGGCGTTTG TTTTGCAACA ATCTTTTCGA AACTATCCAA CAAGCTTTA 2270
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 640 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...21
(D) OTHER INFORMATTON:
' (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Lys Lys Thr Lys Lys Thr Ile Leu Leu Ser Leu Thr Leu Ala Ala
-20 -15 -10
Ser Leu Leu His Ala Glu Asp Asn Gly Val Phe Leu Ser Val Gly Tyr
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 116 PCT/US98/064Z1
-5 1 5 10
Gln Ile Gly Glu Ala Val Gln Lys Val Lys Asn Ala Asp Lys Val Gln
15 20 25
Lys Leu Ser Asp Thr Tyr Glu Gln Leu Ser Arg Leu Leu Thr Asn Asp
30 35 40
Asn Gly Thr Asn Ser Lys Thr Ser Ala Gln Ile Asn Gln Ala Val Asn
45 50 55
Asn Leu Asn Glu Arg Ala Lys Thr Leu Ala Gly Gly Thr Thr Asn Ser
60 65 70 75
Pro Ala Tyr Gln Ala Thr Leu Leu Ala Leu Arg Ser Val Leu Gly Leu
80 B5 90
Trp Asn Ser Met Gly Tyr Ala Val Ile Cys Gly Gly Tyr Thr Lys Ser
95 100 105
Pro Gly Glu Asn Asn Gln Lys Asp Phe His Tyr Thr Asp Glu Asn Gly
110 115 120
Asn Gly Thr Thr Ile Asn Cys Gly Gly Ser Thr Asn Ser Asn Gly Thr
125 130 135
His Ser Ser Ser Gly Thr Asn Thr Leu Lys Ala Asp Lys Asn Val Ser
140 145 150 155
Leu Ser Ile Glu Gln Tyr Glu Lys Ile His Glu Ala Tyr Gln Ile Leu
160 165 170
Ser Lys Ala Leu Lys Gln Ala Gly Leu Ala Pro Leu Asn Ser Lys Gly
175 180 185
Glu Lys Leu Glu Ala His Val Thr Thr Ser Lys Pro Glu Asn Asn Ser
190 195 200
Gln Thr Lys Thr Thr Thr Ser Val Ile Asp Thr Thr Asn Asp Ala Gln
205 210 215
Asn Leu Leu Thr Gln Ala Gln Thr Ile Val Asn Thr Leu Lys Asp Tyr
220 225 230 235
Cys Pro Met Leu Ile Ala Lys Ser Ser Ser Glu Ser Ser Gly Ala Ala
240 245 250
Thr Thr Asn Ala Pro Ser Trp Gln Thr Ala Gly Gly Gly Lys Asn Ser
255 260 265
Cys Ala Thr Phe Gly Ala Glu Phe Ser Ala Ala Ser Asp Met Ile Asn
270 275 280
Asn Ala Gln Lys Ile Val Gln Glu Thr Gln Gln Leu Ser Ala Asn Gln
285 290 295
Pro Lys Asn Ile Thr Gln Pro His Asn Leu Asn Leu Asn Thr Pro Ser
300 305 310 315
Ser Leu Thr Ala Leu Ala Gln Lys Met Leu Lys Asn Ala Gln Ser Gln
320 325 330
Ala Glu Ile Leu Lys Leu Ala Asn Gln Val Glu Ser Asp Phe Asn Lys
335 340 345
Leu Ser Ser Gly His Leu Lys Asp Tyr Ile Gly Lys Cys Asp Ala Ser
350 355 360
Ala Ile Ser Ser Ala Asn Met Thr Met Gln Asn Gln Lys Asn Asn Trp
365 370 375
Gly Asn Gly Cys Ala Gly Val Glu Glu Thr Leu Ser Ser Leu Lys Thr
380 385 390 395
Ser Ala Ala Asp Phe Asn Asn Gln Thr Pro Gln Ile Asn Gln Ala Gln
400 405 410
Asn Leu Ala Asn Thr Leu Ile Gln Glu Leu Gly Asn Asn Pro Phe Arg
415 420 425
Asn Met Gly Met Ile Ala Ser Ser Thr Thr Asn Asn Gly Ala Leu Asn
430 435 440
Gly Leu Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu Lys Lys
445 450 455
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 j j ~ PCT/US98/06421
Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr
460 465 470
475
Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly
480 485 490
Val Gly Ser Asp Leu Leu Phe Asn Phe Ile Asn Asp Lys Asn Thr Asn
495 500 505
Phe Leu Giy Lys Asn Asn Lys Ile Ser Val Gly Phe Phe Gly Gly Ile
510 515 520
Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Phe Val Asn Leu Lys
525 530 535
Thr Ile Ser Asn Val Tyr Ser Ala Lys Val Asn Thr Ala Asn Phe Gln
540 545 550 555
Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Arg Pro Lys Lys
560 565 570
Lys Asp Ser His His Ala Ala Gln His Gly Met Glu Leu Gly Val Lys
575 580 585
Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Leu Asp Thr Lys Leu
590 595 600
Glu Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr
605 610 615
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2248 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 173...2128
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 173...224
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
TGGTTTTATC GTTACAAAAT TCAACATTTC AAAGATAAAT AAGTTAAAAT ACCCCAAAAT 60
CTTTTTTTTT TTTTTGAAAT CCAATCAATT TATAGTAAAA TTAGGTTCAT TGTAAATATA 120
TTATCACTTC ATGATATTCT TACAACAAAA ACATTACTTT AAGGAACATT TT ATG AAA 178
Met Lys
AAG ACA ATT CTG CTC TCT CTC TCT GCT TCA TCG CTC TTG CAC GCT GAA 226
Lys Thr Ile Leu Leu Ser Leu Ser Ala Ser Ser Leu Leu His Ala Glu
-15 -10 -5 1 _
GAC AAC GGC TTT TTT GTG AGC GCC GGC TAT CAA ATC GGC GAA GCG GTG 274
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 118 PCT/US98/06421
Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln Ile Gly Glu Ala Val
S 10 15
CAA ATG GTC AAA AAC ACC GGT GAA TTG AAA AAC TTG AAC GAA AAA TAC 322
Gln Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu Asn Glu Lys Tyr
20 25 30
GAG CAA TTA AGC CAG TAT TTA AAT CAA GTG GCT TCG TTG AAG CAA AGC 370
Glu Gln Leu Ser Gln Tyr Leu Asn Gln Val Ala Ser Leu Lys GIn Ser
35 40 45
ATT CAA AAC GCC AAC AAC ATT GAG CTG GTC AAT AGC TCT TTA AAC TAT 418
Ile Gln Asn Ala Asn Asn Ile Glu Leu Val Asn Ser Ser Leu Asn Tyr
50 55 60 65
TTA AAA AGC TTT ACC AAC AAC AAC TAT AAC AGC ACC ACC CAA TCG CCC 466
Leu Lys Ser Phe Thr Asn Asn Asn Tyr Asn Ser Thr Thr Gln Ser Pro
70 75 80
ATC TTT AAT GCC GTG CAA GCC GTT ATC ACT TCG GTA TTG GGT TTT TGG 514
Ile Phe Asn Ala Val Gln Ala Val Ile Thr Ser Val Leu Gly Phe Trp
85 90 95
AGT CTT TAT GCG GGG AAT TAC TTC ACT TTT TTT GTG GGT AAA AAG GTG 562
Ser Leu Tyr Ala Gly Asn Tyr Phe Thr Phe Phe Val Gly Lys Lys Val
100 105 110
GGT GAT AGT GGG CAA CCC GCT AGT GTC CAG GGT AAC CCT CCT TTT AAA 610
Gly Asp Ser Giy Gln Pro Ala Ser Val Gln Gly Asn Pro Pro Phe Lys
115 120 125
ACG ATT ATA GAG AAC TGC TCA GGA ATT GAA AAC TGC GCT ATG GAT CAA 658
Thr Ile Ile Glu Asn Cys Ser Gly Ile Glu Asn Cys Ala Met Asp Gln
130 135 140 145
ACC ACT TAT GAT AAG ATG AAA AAA CTC GCT GAA GAC CTC CAA GCG GCT 706
Thr Thr Tyr Asp Lys Met Lys Lys Leu Ala Glu Asp Leu Gln Ala Ala
150 155 160
CAA ACA AAC TCT GCC ACT AAA GGC AAC AAT CTT TGC GCT TTA TCC GGG 754
Gln Thr Asn Ser Ala Thr Lys GIy Asn Asn Leu Cys Ala Leu Ser Gly
165 170 175
TGT GCT GCA ACA GAC TCA ACA TCA AAC CCA CCA AAC TCA ACC GTG AGC 802
Cys Ala Ala Thr Asp Ser Thr Ser Asn Pro Pro Asn Ser Thr Val Ser
180 185 190
AAC GCT CTT AAT TTG GCG CAA CAG CTT ATG GAT TTA ATC GCA AAC ACT 850
Asn Ala Leu Asn Leu Ala Gln Gln Leu Met Asp Leu Ile Ala Asn Thr
195 200 205
AAA ACG GCT ATG ATG TGG AAA AAT ATC GTC ATC AGT GGC GTT TCA AAC 898
Lys Thr Ala Met Met Trp Lys Asn Ile Val Ile Ser Gly Val Ser Asn
210 215 220 225
ACA TCC GGT GCT ATC ACA TCC ACT AAT TAC CCA ACG CAA TAC GCG GTG 946
Thr Ser Gly Ala Ile Thr Ser Thr Asn Tyr Pro Thr Gln Tyr Ala Val
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 119 PCT/US98/06421
230 235 240
TTT AAC AAC ATT AAG GCG ATG ATA CCC ATT TTG CAA CAA GCG GTT ACG 994
Phe Asn Asn Ile Lys Ala Met Ile Pro Ile Leu Gln Gln Ala Val Thr
245 250 255
CTT TCT CAA AGC AAC CAC ACC CTA TCT GCT AGC TTG CAA GCT CAA GCC 1042
Leu Ser Gln Ser Asn His Thr Leu Ser Ala Ser Leu Gln Ala Gln Ala
260 265 270
ACA GGA TCT CAA ACA AAC CCT AAA TTC GCT AAA GAC ATC TAC ACT TTC 1090
Thr Gly Ser Gln Thr Asn Pro Lys Phe Ala Lys Asp Ile Tyr Thr Phe
275 280 285
GCT CAA AAC CAA AAG CAA GTC ATC TCT TAC GCT CAA GAC ATT TTC AAC 1138
Ala Gln Asn Gln Lys Gln Val Ile Ser Tyr Ala Gln Asp Ile Phe Asn
290 295 300 305
CTC TTT AAT TCT ATC CCT GCA GAG CAG TAT AAG TAT CTA GAG AAA GCT 1186
Leu Phe Asn Ser Ile Pro Ala Glu Gln Tyr Lys Tyr Leu Glu Lys Aia
310 315 320
TAC TTG AAA ATA CCC AAT GCG GGT TCA ACG CCT ACT AAC CCT TAC AGA 1234
Tyr Leu Lys Ile Pro Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg
325 330 335
CAAGTG GTGAAT TTAAACCAA GAAGTTCAG ACGATT AAT GTG 1282
AAA
AAC
GlnVal ValAsn LeuAsnGln GluValGln ThrIleLys AsnAsn Val
340 345 350
AGTTAT TATGGT AACCGGGTG GATGCGGCT TTAAGCGTG GCTAGA GAT 1330
SerTyr TyrGly AsnArgVal AspAlaAla LeuSerVal AlaArg Asp
355 360 365
GTTTAT AACCTA AAATCCAAT CAAGCAGAA ATCGTAACC GCCTAT AAC 1378
ValTyr AsnLeu Lys5erAsn GlnAlaGlu IleValThr AlaTyr Asn
370 375 380 385
GACGCT AAGACT TTGAGCGAA GAGATTTCT AAACTCCCG CACAAT CAA 1426
AspAla LysThr LeuSerGlu GluIleSer LysLeuPro HisAsn Gln
390 395 400
GTCAAT ACAAAA GACATTGTT ACACTACCT TACGATAAA AACGCC CCA 1474
ValAsn ThrLys AspIleVal ThrLeuPro TyrAspLys AsnAla Pro
405 410 415
w
GCAGCA GGCCAA TCCAACTAC CAAATCAAC CCAGAGCAG CAATCC AAT 1522
AlaAla GlyGln SerAsnTyr GlnIleAsn ProGluGln GlnSer Asn
420 425 430
CTT AAC CAA GCT TTA GCA GCG ATG AGC AAT AAC CCC TTT AAA AAA GTG 1570
Leu Asn Gln Ala Leu Ala Ala Met Ser Asn Asn Pro Phe Lys Lys Val
435 440 445
GGC ATG ATC AGC TCT CAA AAC AAT AAC GGC GCT TTG AAC GGG CTT GGC 1618
Gly Met Ile Ser Ser Gln Asn Asn Asn Gly Ala Leu Asn Gly Leu Gly
450 455 460 465
SUBSTITUTE SHEET (RULE 26~
CA 02286893 1999-10-O1
WO 98/43479 120 PCT/I1S98/06421
GTG CAA GTG GGT TAT AAG CAA TTC TTT GGC GAA AGC AAA AGA TGG GGG 1666
val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu Ser Lys Arg Trp Gly
470 475 480
TTA AGG TAT TAC GGA TTC TTT GAT TAC AAC CAC GGC TAC ATC AAA TCC 1714
Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Gly Tyr Ile Lys Ser
485 490 495
AGC TTC TTT AAC TCT TCT TCT GAT ATA TGG ACT TAT GGC GGT GGG AGC 1762
Ser Phe Phe Asn Ser Ser Ser Asp Ile Trp Thr Tyr Gly Gly Gly Ser
500 505 510
GAT TTG TTA GTG AAT ATT ATC AAC GAT AGC ATC ACA AGA AAG AAC AAC 1810
Asp Leu Leu Val Asn Ile Ile Asn Asp Ser Ile Thr Arg Lys Asn Asn
515 520 525
AAG CTC TCC GTG GGT CTT TTT GGA GGC ATC CAA CTA GCA GGG ACT ACA 1858
Lys Leu Ser Val Gly Leu Phe Gly Gly Ile Gln Leu Ala Gly Thr Thr
530 535 540 545
TGG CTT AAT TCT CAA TAC GTG AAT TTA ACC GCG TTC AAT AAC CCT TAC 1906
Trp Leu Asn Ser Gln Tyr Val Asn Leu Thr Ala Phe Asn Asn Pro Tyr
550 555 560
AGC GCG AAA GTC AAT GCT ACC AAT TTC CAA TTC TTG TTC AAT CTC GGC 1954
Ser Ala Lys Val Asn Ala Thr Asn Phe Gln Phe Leu Phe Asn Leu Gly
565 570 575
TTG AGG ACG AAT CTC GCT ACA GCT AGG AAA AAA GAC AGC GAA CAT TCC 2002
Leu Arg Thr Asn Leu Ala Thr Ala Arg Lys Lys Asp Ser Glu His Ser
580 585 590
GCG CAA CAT GGC ATT GAA TTG GGT ATT AAA ATC CCC ACC ATT ACC ACG 2050
Ala Gln His Gly Ile Glu Leu Gly Ile Lys Ile Pro Thr Ile Thr Thr
595 600 605
AAT TAC TAT TCT TTT CTA GGC ACT CAA TTG CAA TAC AGA AGG CTC TAT 2098
Asn Tyr Tyr Ser Phe Leu Gly Thr Gln Leu Gln Tyr Arg Arg Leu Tyr
610 615 620 625
AGC GTG TAT CTC AAT TAT GTG TTC GCT TAC TGAGTGATTC AAGCTCTCTT CTT 2151
Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr
630 635
TAAGGGGGTT TAGAAAAATC GCAACGCCAA GCTTTTTATC GTTGGTGATA AAATCTACAA 2211
AACTAACGGC GCGACAACAA ACCCTAACGC TACGCTC 2248
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 652 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 121 PCT/US98/06421
(ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...17
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Lys Lys Thr Ile Leu Leu Ser Leu Ser Ala Ser Ser Leu Leu His
-15 -10 -5
Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln Ile Gly Glu
1 5 10 15
Ala Val Gln Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu Asn Glu
20 25 30
Lys Tyr Glu Gln Leu Ser Gln Tyr Leu Asn Gln Val Ala Ser Leu Lys
35 40 45
Gln Ser Ile Gln Asn Ala Asn Asn Ile Glu Leu Val Asn Ser Ser Leu
50 55 60
Asn Tyr Leu Lys Ser Phe Thr Asn Asn Asn Tyr Asn Ser Thr Thr Gln
65 70 75
Ser Pro Ile Phe Asn Ala Val Gln Ala Val Ile Thr Ser Val Leu Gly
80 85 90 95
Phe Trp Ser Leu Tyr Ala Gly Asn Tyr Phe Thr Phe Phe Val Gly Lys
100 105 110
Lys Val Gly Asp Ser Gly Gln Pro Ala Ser Val Gln Gly Asn Pro Pro
115 120 125
Phe Lys Thr Ile Ile Glu Asn Cys Ser Gly Ile Glu Asn Cys Ala Met
130 135 140
Asp Gln Thr Thr Tyr Asp Lys Met Lys Lys Leu Ala Glu Asp Leu Gln
145 150 155
Ala Ala Gln Thr Asn Ser Ala Thr Lys Gly Asn Asn Leu Cys Ala Leu
160 165 170 175
Ser Gly Cys Ala Ala Thr Asp Ser Thr Ser Asn Pro Pro Asn Ser Thr
180 185 190
Val Ser Asn Ala Leu Asn Leu Ala Gln Gln Leu Met Asp Leu Ile Ala
195 200 205
Asn Thr Lys Thr Ala Met Met Trp Lys Asn Ile Val Ile Ser Gly Val
210 215 220
Ser Asn Thr Ser Gly Ala Ile Thr Ser Thr Asn Tyr Pro Thr Gln Tyr
225 230 235
Ala Val Phe Asn Asn Ile Lys Ala Met Ile Pro Ile Leu Gln Gln Ala
240 245 250 255
Val Thr Leu Ser Gln Ser Asn His Thr Leu Ser Ala Ser Leu Gln Ala
260 265 27p
Gln Ala Thr Gly Ser Gln Thr Asn Pro Lys Phe Ala Lys Asp Ile Tyr
275 280 285
Thr Phe Ala Gln Asn Gln Lys Gln Val Ile Ser Tyr Ala Gln Asp Ile
' 290 295 300
Phe Asn Leu Phe Asn Ser Ile Pro Ala Glu Gln Tyr Lys Tyr Leu Glu
305 310 315
Lys Ala Tyr Leu Lys Ile Pro Asn Ala Gly Ser Thr Pro Thr Asn Pro
320 325 330 335
Tyr Arg Gln Val Val Asn Leu Asn Gln Glu Val Gln Thr Ile Lys As~1
340 345 350
Asn Val Ser Tyr Tyr Gly Asn Arg Val Asp Ala Ala Leu Ser Val Ala
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 122 PCT/US98/06421
355 360 365
Arg Asp Val Tyr Asn Leu Lys Ser Asn Gln Ala Glu Ile Val Thr Ala
370 375 380
Tyr Asn Asp Ala Lys Thr Leu Ser Glu Glu Ile Ser Lys Leu Pro His
385 390 395
Asn Gln Val Asn Thr Lys Asp Ile Val Thr Leu Pro Tyr Asp Lys Asn
400 405 410 415
Ala Pro Ala Ala Gly Gln Ser Asn Tyr Gln Ile Asn Pro Glu Gln Gln
420 425 430
Ser Asn Leu Asn Gln Ala Leu Ala Ala Met Ser Asn Asn Pro Phe Lys
435 440 445
Lys Val Gly Met Ile Ser Ser Gln Asn Asn Asn Gly Ala Leu Asn Gly
450 455 460
Leu Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu Ser Lys Arg
465 470 475
Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Gly Tyr Ile
480 485 490 495
Lys Ser Ser Phe Phe Asn Ser Ser Ser Asp Ile Trp Thr Tyr Gly Gly
500 505 510
Gly Ser Asp Leu Leu Val Asn Ile Ile Asn Asp Ser Ile Thr Arg Lys
515 520 525
Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Ile Gln Leu Ala Gly
530 535 540
Thr Thr Trp Leu Asn Ser Gln Tyr Val Asn Leu Thr Ala Phe Asn Asn
545 550 555
Pro Tyr Ser Ala Lys Val Asn Ala Thr Asn Phe Gln Phe Leu Phe Asn
560 565 570 575
Leu Gly Leu Arg Thr Asn Leu Ala Thr Ala Arg Lys Lys Asp Ser Glu
580 585 590
His Ser Ala Gln His Gly Ile Glu Leu Gly Ile Lys Ile Pro Thr Ile
595 600 605
Thr Thr Asn Tyr Tyr Ser Phe Leu Gly Thr Gln Leu Gln Tyr Arg Arg
610 615 620
Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr
625 630 635
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2161 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
( ix ) FEATURE ~'
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 122...2056
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 122...179 _
(D) OTHER INFORMATION:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 123 PCT/US98/06421
(xi) SEQUENCE DESCRIPTION:
SEQ
ID
N0:17:
CAAAAATCTT TTTTTTTTTT C ATGGTAAAGT TAAACATATT
60
TTTTGAAAT CAATAAATTT
GTAAATAAAT TTTAATTTCT AATTACTTTA AGGAACATTT
120
ATTCATGTTT
ACAATAAAAA
T TG TC CT TC CA CG 169
A AAA T CTC TCG T CTC
AAG TCT CTT
ACA C T
ATT
CTA
C
M et ys ys le Leu eu er eu Ser eu er eu er er
L L Thr L S L L S L S S Leu
I
- 15 -10 - 5
TTGCACGCT GAA AAC TTT TTTGTG GCCGGCTAT CAAATC 217
GAC GGC AGC
LeuHisAla GluAspAsn Phe PheVal AlaGlyTyr GlnIle
Gly Ser
1 5 10
GGCGAACGG GTGCAAATG AAA AACACC GAATTGAAA AACTTG 265
GTC GGC
GlyGluArg ValGlnMet Lys AsnThr GluLeuLys AsnLeu
Val Gly
15 20 25
AACGAAAAA TACGAGCAA AGC CAATCT GCCCAACTG GCTTCG
TTA TTA
313
AsnGluLys TyrGluGln Ser GlnSer AlaGlnLeu AlaSer
Leu Leu
30 35 40 45
TTAAAAAAA AGCATTCAA GCG AACAAC CAGGCTGTC AACAAT 361
ACG ATT
LeuLysLys SerIleGln Ala AsnAsn GlnAlaVal AsnAsn
Thr Ile
50 55 60
GCTTTAAGC GATTTAAAA TTT GCGAGT AACCACACA AACAAA 409
AGC AAC
AlaLeuSer AspLeuLys Phe AlaSer AsnHisThr AsnLys
Ser Asn
65 70 75
GAAACATCG CCCATCTAC ACC GCGCAA GTTATCACT TCAGTA 457
AAC GCT
GluThrSer ProIleTyr Thr AlaGln ValIleThr SerVal
Asn Ala
80 85 90
TTGGCTTTT TGGAGTCTT GCA GGGAAC ACCAGTTTT CATGTG 505
TAT GCT
LeuAlaPhe TrpSerLeu Ala GlyAsn ThrSerPhe HisVal
Tyr Ala
95 100 105
ACCGGTTTG AATGATGGA AAT GCTCCT GGAAGAATC CATCAA 553
TCT CTT
ThrGlyLeu AsnAspGly Asn AlaPro GlyArgIle HisGln
Ser Leu
110 115 120 125
GATGGGAAC TGCACAGGA CAA CAATGT ATGAATAAA GAAACT 601
TTA TTT
AspGlyAsn CysThrGly Gln GlnCys MetAsnLys GluThr
Leu Phe
130 135 140
TATGATAAA ATGAAAGCG GCC GAAAAT CAAAAAGCT CAAGGC 649
CTT CTC
TyrAspLys MetLysAla Ala GluAsn GlnLysAla GlnGly
Leu Leu
145 150 155
AAT CTC TGT GCC TTA TCA GAA TGC CCT AGC GAT CAA TTA AAT GGA AAC 697
Asn Leu Cys Ala Leu Ser Glu Cys Pro Ser Asp Gln Leu Asn Gly Asn
160 165 170
AAT GGA AAC AAA ACT TCC ATG ACT AAA GCT CTT GAA ACC GCG CAA CAG_ 745
Asn Gly Asn Lys Thr Ser Met Thr Lys Ala Leu Glu Thr Ala Gln Gln
175 180 185
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 124 PCT/US98/06421
CTT ATG GAT TTA ATC GCA AAC ACT AAA ACG GCT ATG ATG TGG AAA AAT 793
Leu Met Asp Leu Ile Ala Asn Thr Lys Thr Ala Met Met Trp Lys Asn
190 195 200 205
ATC GTC ATC GCA GGT GTT ACA AAC AGA CCC GGT GGT GCT GGC GCT ATC 841
Ile Val Ile Ala Gly Val Thr Asn Arg Pro Gly Gly Ala Gly Ala Ile
210 215 220
ACA TCC ACT GGT CCT GTA ACC GAC TAT GCG GTG TTT AAC AAC ATT AAG 889
Thr Ser Thr Gly Pro Val Thr Asp Tyr Ala Val Phe Asn Asn Ile Lys
225 230 235
GCG ATG ATA CCC ATT TTG CAA CAA GCG GTT ACG CTT TCT CAA AGC AAC 937
Ala Met Ile Pro Ile Leu Gln Gln Ala Val Thr Leu Ser Gln Ser Asn
240 245 250
CAC ACC CTA TCT GCT AGC TTG CAA GCT CAA GCC ACA GGA TCT CAA ACA 985
His Thr Leu Ser Ala Ser Leu Gln Ala Gln Ala Thr Gly Ser Gln Thr
255 260 265
AAC CCT AAA TTC GCT AAA GAC ATC TAC ACT TTC GCT CAA AAC CAA AAG 1033
Asn Pro Lys Phe Ala Lys Asp Ile Tyr Thr Phe Ala Gln Asn Gln Lys
270 275 280 285
CAA GTC ATC TCT TAC GCT CAA GAC ATT TTC AAC CTC TTT AAT TCT ATC 1081
Gln Val Ile Ser Tyr Ala Gln Asp Ile Phe Asn Leu Phe Asn Ser Ile
290 295 300
CCT GCA GAG CAG TAT AAG TAT CTA GAG AAA GCT TAC TTG AAA ATA CCC 1129
Pro Ala Glu Gln Tyr Lys Tyr Leu Glu Lys Ala Tyr Leu Lys Ile Pro
305 310 315
AAT GCG GGT TCA ACG CCT ACT AAC CCT TAC AGA CAA GTG GTG AAT TTA 1177
Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg Gln Val Val Asn Leu
320 325 330
AAC CAA GAA GTT CAG ACG ATT AAA AAC AAT GTG AGT TAT TAT GGT AAC 1225
Asn Gln Glu Val Gln Thr Ile Lys Asn Asn Val Ser Tyr Tyr Gly Asn
335 340 345
CGG GTG GAT GCG GCT TTA AGC GTG GCT AGA GAT GTT TAT AAC CTA AAA 1273
Arg Val Asp Ala Ala Leu Ser Val Ala Arg Asp Val Tyr Asn Leu Lys
350 355 360 365
TCC AAT CAA GCA GAA ATC GTA ACC GCC TAT AAC GAC GCT AAG ACT TTG 1321
Ser Asn Gln Ala Glu Ile Val Thr Ala Tyr Asn Asp Ala Lys Thr Leu
370 375 380
AGC GAA GAG ATT TCT AAA CTC CCG CAC AAT CAA GTC AAT ACA AAA GAC 1369
Ser Glu Glu Ile Ser Lys Leu Pro His Asn Gln Val Asn Thr Lys Asp
385 390 395
ATT GTT ACA CTA CCT TAC GAT AAA AAC GCC CCA GCA GCA GGC CAA TCC 1417
Ile Val Thr Leu Pro Tyr Asp Lys Asn Ala Pro Ala Ala Gly Gln Ser
400 405 410 _
AAC TAC CAA ATC AAC CCA GAG CAG CAA TCC AAT CTT AAC CAA GCT TTA 1465
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 125 PCT/US98/0642I
Asn Tyr Gln Ile Asn Pro Glu Gln Gln Ser Asn Leu Asn Gln Ala Leu
415 420 425
GCA GCG ATGAGC AATAACCCC TTTAAA AAAGTGGGC ATGATCAGC TCT 1513
Ala Ala MetSer AsnAsnPro PheLys LysValGly MetIleSer Ser
430 435 440 445
CAA AAC AATAAC GGCGCTTTG AACGGG CTTGGCGTG CAAGTGGGT TAT 1561
Gln Asn AsnAsn GlyAlaLeu AsnGly LeuGlyVal GlnValGly Tyr
450 455 460
AAG CAA TTCTTT GGCGAAAGC AAAAGA TGGGGGTTA AGGTATTAC GGA 1609
Lys Gln PhePhe GlyGluSer LysArg TrpGlyLeu ArgTyrTyr Gly
465 470 475
TTC TTT GATTAC AACCACGGC TACATC AAATCCAGC TTCTTTAAC TCT 1657
Phe Phe AspTyr AsnHisGly TyrIle LysSerSer PhePheAsn Ser
4B0 485 490
TCT TCT GATATA TGGACTTAT GGCGGT GGGAGCGAT TTGTTAGTG AAT 1705
Ser Ser AspIle TrpThrTyr GlyGly GlySerAsp LeuLeuVal Asn
495 500 505
ATT ATC AACGAT AGCATCACA AGAAAG AACAACAAG CTCTCCGTG GGT 1753
Ile Ile AsnAsp SerIleThr ArgLys AsnAsnLys LeuSerVal Gly
510 515 520 525
CTT TTT GGAGGC ATCCAACTA GCAGGG ACTACATGG CTTAATTCT CAA 1801
Leu Phe GlyGly IleGlnLeu AlaGly ThrThrTrp LeuAsnSer Gln
530 535 540
TAC GTG AATTTA ACCGCGTTC AATAAC CCTTACAGC GCGAAAGTC AAT 1849
Tyr Val AsnLeu ThrAlaPhe AsnAsn ProTyrSer AlaLysVal Asn
545 550 555
GCT ACC AATTTC CAATTCTTG TTCAAT CTCGGCTTG AGGACGAAT CTC 1897
Ala Thr AsnPhe GlnPheLeu PheAsn LeuGlyLeu ArgThrAsn Leu
560 565 570
GCT ACA GCTAGG AAAAAAGAC AGCGAA CATTCCGCG CAACATGGC ATT 1945
Ala Thr AlaArg LysLysAsp SerGlu HisSerAla GlnHisGly Ile
575 580 585
GAA TTG GGTATT AAAATCCCC ACCATT ACCACGAAT TACTATTCT TTT 1993
Glu Leu GlyIle LysIlePro ThrIle ThrThrAsn TyrTyrSer Phe
590 595 600 605
CTA GGC ACTCAA TTGCAATAC AGAAGG CTCTATAGC GTGTATCTC AAT 2041
Leu Gly ThrGln LeuGlnTyr ArgArg LeuTyrSer ValTyrLeu Asn
___
610 615 620
TAT GTG TTCGCT TATTAAAAAATCT CTTCATCAAA
2097
TCTTTTTAAA T
ATAGGGGGAG
Tyr Val PheAla Tyr
625
CTATTTTGAT TCAAAAACAAAAT 2157
AGTTATCAAT AAACCAAATC
ATTTGATGAA
AATAAAG
ACCC 2161
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 126 PCT/US98/06421
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 645 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...19
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Lys Lys Thr Ile Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Leu
-15 -10 -5
Leu His Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln Ile
1 5 10
Gly Glu Arg Val Gln Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu
15 20 25
Asn Glu Lys Tyr Glu Gln Leu Ser Gln Ser Leu Ala Gln Leu Ala Ser
30 35 40 45
Leu Lys Lys Ser Ile Gln Thr Ala Asn Asn Ile Gln Ala Val Asn Asn
50 55 60
Ala Leu Ser Asp Leu Lys Ser Phe Ala Ser Asn Asn His Thr Asn Lys
65 70 75
Glu Thr Ser Pro Ile Tyr Asn Thr Ala Gln Ala Val Ile Thr Ser Val
80 85 90
Leu Ala Phe Trp Ser Leu Tyr Ala Gly Asn Ala Thr Ser Phe His Val
95 100 105
Thr Gly Leu Asn Asp Gly Ser Asn Ala Pro Leu Gly Arg Ile His Gln
110 115 120 125
Asp Gly Asn Cys Thr Gly Leu Gln Gln Cys Phe Met Asn Lys Glu Thr
130 135 140
Tyr Asp Lys Met Lys Ala Leu Ala Glu Asn Leu Gln Lys Ala Gln Gly
145 150 155
Asn Leu Cys Ala Leu Ser Glu Cys Pro Ser Asp Gln Leu Asn Gly Asn
160 165 170
Asn Gly Asn Lys Thr Ser Met Thr Lys Ala Leu Glu Thr Ala Gln Gln
175 180 185
Leu Met Asp Leu Ile Ala Asn Thr Lys Thr Ala Met Met Trp Lys Asn
190 195 200 205
Ile Val Ile Ala Gly Val Thr Asn Arg Pro Gly Gly Ala Gly Ala Ile__
210 215 220
Thr Ser Thr Gly Pro Val Thr Asp Tyr Ala Val Phe Asn Asn Ile Lys
225 230 235
Ala Met Ile Pro Ile Leu Gln Gln Ala Val Thr Leu Ser Gln Ser Asn
240 245 250
His Thr Leu Ser Ala Ser Leu Gln Ala Gln Ala Thr Gly Ser Gln Thr
255 260 265
Asn Pro Lys Phe Ala Lys Asp Ile Tyr Thr Phe Ala Gln Asn Gln Lys
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 127 PCT/US98/06421
270 275 280 ~ 285
Gln Val Ile Ser Tyr Ala Gln Asp Ile Phe Asn Leu Phe Asn Ser Ile
290 295 3D0
Pro Ala Glu Gln Tyr Lys Tyr Leu Glu Lys Ala Tyr Leu Lys Ile Pro
305 310 315
Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg Gln Val Val Asn Leu
320 325 330
Asn Gln Glu Val Gln Thr Ile Lys Asn Asn Val Ser Tyr Tyr Gly Asn
335 340 345
Arg Val Asp Ala Ala Leu Ser Val Ala Arg Asp Val Tyr Asn Leu Lys
350 355 360 365
Ser Asn Gln Ala Glu Ile Val Thr Ala Tyr Asn Asp Ala Lys Thr Leu
370 375 380
Ser Glu Glu Ile Ser Lys Leu Pro His Asn Gln Val Asn Thr Lys Asp
385 390 395
Ile Val Thr Leu Pro Tyr Asp Lys Asn Ala Pro Ala Ala Gly Gln Ser
400 405 410
Asn Tyr Gln Ile Asn Pro Glu Gln Gln Ser Asn Leu Asn Gln Ala Leu
415 420 425
Ala Ala Met Ser Asn Asn Pro Phe Lys Lys Val Gly Met Ile Ser Ser
430 435 440 445
Gln Asn Asn Asn Gly Ala Leu Asn Gly Leu Gly Val Gln Val Gly Tyr
450 455 460
Lys Gln Phe Phe Gly Glu Ser Lys Arg Trp Gly Leu Arg Tyr Tyr Gly
465 470 475
Phe Phe Asp Tyr Asn His Gly Tyr Ile Lys Ser Ser Phe Phe Asn Ser
480 485 490
Ser Ser Asp Ile Trp Thr Tyr Gly Gly Gly Ser Asp Leu Leu Val Asn
495 500 505
Ile Ile Asn Asp Ser Ile Thr Arg Lys Asn Asn Lys Leu Ser Val Gly
510 515 520 525
Leu Phe Gly Gly Ile Gln Leu Ala Gly Thr Thr Trp Leu Asn Ser Gln
530 535 540
Tyr Val Asn Leu Thr Ala Phe Asn Asn Pro Tyr Ser Ala Lys Val Asn
545 550 555
Ala Thr Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu
560 565 570
Ala Thr Ala Arg Lys Lys Asp Ser Glu His Ser Ala Gln His Gly Ile
575 580 5B5
Glu Leu Gly Ile Lys Ile Pro Thr Ile Thr Thr Asn Tyr Tyr Ser Phe
590 595 600 605
Leu Gly Thr Gln Leu Gln Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn
610 615 620
Tyr Val Phe Ala Tyr
625
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1799 base pairs
(B) TYPE: nucleic acid -
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA _
(ix) FEATURE:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 128 PCT/US98/06421
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 185...1633
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 185...233
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
TACTCAAAAC ATTTTTCACTATCAAAAACCTTTTTTTTAA ATCCAAAAAA AAAGCAAAAT60
TTCTTAATTT TTGCTCAATTTTATTAAAAATTCAATAAAT TTATGGCACA ATTTAAACTT120
ATTGTAAATA AAGTTTCAATTTGATACGATTTTACAAACA AAACATTACT TTAAGGAACA180
TTTT ATG AAA AAA TCT CTT ATG GTT TCA TCG CTC 229
ACG ATT TTA CTT CTC
Met Lys Lys Thr Ile Leu Ser Leu Met Val Ser Ser Leu
Leu Leu
-15 -10 -5
GCT GAA AAT GAC GGC GTT TTT ATG AGC GTG GGC TAT CAA ATC GGC GAA 277
Ala Glu Asn Asp Gly Val Phe Met Ser Val Gly Tyr Gln Ile Gly Glu
1 5 10 15
GCG GTT CAA CAA GTG AAA AAC ACC GGC GAA ATC CAA AAA GTC TCC AAC 325
Ala Val Gln Gln Val Lys Asn Thr Gly Glu Ile Gln Lys Val Ser Asn
20 25 30
GCT TAC GAA AAT TTG AAC AAT CTT TTA ACC CGC TAT AAC GAA CTC AAA 373
Ala Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Asn Glu Leu Lys
35 40 45
CAA ACG GCC TCT AAC ACC AAT TCA AGT ACC GCT CAA GCG ATT GAT AAT 421
Gln Thr Ala Sex Asn Thr Asn Ser Ser Thr Ala Gln Ala Ile Asp Asn
50 55 60
CTA AAA GAG AGC GCT AGC CGA TTG AAA ACG ACC CCC AAT AGC GCT AAT 469
Leu Lys Glu Ser Ala Ser Arg Leu Lys Thr Thr Pro Asn Ser Ala Asn
65 70 75
CAA GCC GTG TCT TCA GCG CTC AGC TCT GCG GTA GCC ATG TGG CAA GTA 517
Gln Ala Val Ser Ser Ala Leu Ser Ser Ala Val Ala Met Trp Gln Val
80 85 90 95
ATA GTC TCT AAT TTA GCC AAT AAC TCG CTA CCC ACT AGT GAA TAC AAC 565
Ile Val Ser Asn Leu Ala Asn Asn Ser Leu Pro Thr Ser Glu Tyr Asn
100 105 110
AAA ATC AAT GCG ATT TCT CAA TCG CTC CAA AAC ACC CTA GAA AAT AAA 613
Lys Ile Asn Ala Ile Ser Gln Ser Leu Gln Asn Thr Leu Glu Asn Lys
115 120 125
AAC AAT GAT CTT AAA ATT GAA AAT GAC TAC GAC CAT CTT TTA ACT CAA 661
Asn Asn Asp Leu Lys Ile Glu Asn Asp Tyr Asp His Leu Leu Thr Gln_
130 135 140
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 129 PCT/US98/06421
GCT AGCACC ATTATTAAT ACCCTTCAA CAA TGCCCAGGC GAC 709
AGC ATA
Ala SerThr IleIleAsn ThrLeuGln Gln CysProGly Asp
Ser Ile
145 150 155
GGA GGCAAT GGCAAACCA TGGGGCATT GCA AGCGGGAAC GCATGC 757
AAT
Gly GlyAsn GlyLysPro TrpGlyIle AsnAla SerGlyAsn AlaCys
160 165 170 175
AAT ATTTTT GGCAACACC TTTAACGCC ATCACT AGCATGATA GATAGC 805
Asn IlePhe GlyAsnThr PheAsnAla IleThr SerMetIle AspSer
180 185 190
GCT AAAAAA GCCGCCGCA GATGCCCGA AGAACT GCCCCAGAA AGTCCA 853
Ala LysLys AlaAlaAla AspAlaArg ArgThr AlaProGlu SerPro
195 200 205
AAC CAACCA AGTGCGTTT AACAACGCT GATTTC AATAAAAAC CTTAAT 901
Asn GlnPro SerAlaPhe AsnAsnAla AspPhe AsnLysAsn LeuAsn
210 215 220
CAA GTCTCA AGCGTTATT AATGACACG ATCTCT TACCTCAAA GGGGAC 949
Gln ValSer SerValIle AsnAspThr IleSer TyrLeuLys GlyAsp
225 230 235
AAT TTAGCA ACCATCTAC AACACCCTT CAAAAA ACGCCCGAT TCTAAA 997
Asn LeuAla ThrIleTyr AsnThrLeu GlnLys ThrProAsp SerLys
240 245 250 255
GGG TTTCAA AGTTTGGTG AGCCGATCT AGCTAT AGTTATTCC CTCAAC 1045
Gly PheGln SerLeuVal SerArgSer SerTyr SerTyrSer LeuAsn
260 265 270
GAA ACCCAA TATTCTGAA TTCCAAACT ACCACC AAAGAGTTT GGCCAT 1093
Glu ThrGln TyrSerGlu PheGlnThr ThrThr LysGluPhe GlyHis
275 280 285
AAC CCTTTT AGAAGCGTG GGTTTAATC AACTCT CAAAGCAAT AACGGA 1141
Asn ProPhe ArgSerVal GlyLeuIle AsnSer GlnSerAsn AsnGly
290 295 300
GCG ATGAAT GGCGTGGGC GTGCAATTA GGCTAT AAGCAATTC TTTGGG 1189
Ala MetAsn GlyValGly ValGlnLeu GlyTyr LysGlnPhe PheGly
305 310 315
AAA AATAAA TTTTTTGGG ATCCGTTAT TATGCC TTTTTTGAT TACAAC 1237
Lys AsnLys PhePheGly IleArgTyr TyrAla PhePheAsp TyrAsn
320 325 330 335
CAT GCCTAT ATCAAATCC AACTTTTTC AACTCC GCTTCCAAT GTTTTC 1285
His AlaTyr IleLysSer AsnPhePhe AsnSer AlaSerAsn ValPhe
340 345 350
ACT TATGGC GCAGGCAGT GATCTTTTA TTGAAT TTCATCAAT GGCGGA 1333
Thr TyrGly AlaGlySer AspLeuLeu LeuAsn PheIleAsn GlyGly
355 360 365
TCC GATAAA CGCAAA GTCTCTTTT GGCATT TTTGGAGGC ATCGCT 1381
AAC
SU9STtTUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 130 PCT/US98/06421
Ser Asp Lys Asn Arg Lys Val Ser Phe Gly Ile Phe Gly Gly Ile Ala
370 375 380
CTA GCA GGC ACG ACA TGG CTT AAT TCC CAA TTT ATG AAT TTA AAA ACC 1429
Leu Ala Gly Thr Thr Trp Leu Asn Ser Gln Phe Met Asn Leu Lys Thr
385 390 395
ACC AAT AGC GCC TAC AGC GCT AAG ATC AAC AAC ACC AAT TTC CAA TTC 1477
Thr Asn Ser Ala Tyr Ser Ala Lys Ile Asn Asn Thr Asn Phe Gln Phe
400 405 410 415
TTA TTC AAT ACT GGT TTA AGG CTT CAA GGG ATT CAC CAT GGC GTT GAA 1525
Leu Phe Asn Thr Gly Leu Arg Leu Gln Gly Ile His His Gly Val Glu
420 425 430
TTAGGC GTG AAA ATC CCC ACC ATC AAC TAC TAT TCT TTC 1573
ACG AAT ATG
LeuGly Val Lys Ile Pro Thr Ile Asn Tyr Tyr Ser Phe
Thr Asn Met
435 440 445
GGCGCT AAA TTA GCA TAC CGA AGA CTT GTG TAT TTC AAT 1621
TAT AGC TAT
GlyAla Lys Leu Ala Tyr Arg Arg Leu Val Tyr Phe Asn
Tyr Ser Tyr
450 455 460
GTTTTG GCC TAT TGATATTGAA TCGGTTCTCA 1678
TTACTAATGA GGACAAAGCC AAACT
ValLeu Ala Tyr
465
TTTTGGCTCT TTTTACAAAA AACACACTAA1738
CAATGAATAA
CGGCATCATT
TTACTTGACT
AATTTCTTTT AGCGGTAGAG TAGGCGGCTG1798
TCTTTTTTGA
GCGAAATTCC
AGATTAGCTC
T 1799
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...16
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION:SEQID N0:20:
MetLys Thr IleLeu Leu LeuMetVal Ser Leu Leu
Lys Ser Ser Ala
-15 -10 -5
GluAsn Gly ValPhe Met ValGlyTyr Ile Gly Glu
Asp Ser Gln Ala
1 5 10 15 -
ValGln Val LysAsn Thr GluIleGln Val Ser Asn
Gln Gly Lys Ala
20 25 30
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 131 PCT/US98/06421
Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Asn Glu Leu Lys Gln
35 40 45
Thr Ala Ser Asn Thr Asn Ser Ser Thr Ala Gln Ala Ile Asp Asn Leu
50 55 60
Lys Glu Ser Ala Ser Arg Leu Lys Thr Thr Pro Asn Ser Ala Asn Gln
65 70 75 80
Ala Val Ser Ser Ala Leu Ser Ser Ala Val Aia Met Trp Gln Val Ile
. 85 90 95
Val Ser Asn Leu Ala Asn Asn Ser Leu Pro Thr Ser Glu Tyr Asn Lys
100 105 110
Ile Asn Ala Ile Ser Gln Ser Leu Gln Asn Thr Leu Glu Asn Lys Asn
115 120 125
Asn Asp Leu Lys Ile Glu Asn Asp Tyr Asp His Leu Leu Thr Gln Ala
130 135 140
Ser Thr Ile Ile Asn Thr Leu Gln Ser Gln Cys Pro Gly Ile Asp Gly
145 150 155 160
Gly Asn Gly Lys Pro Trp Gly Ile Asn Ala Ser Gly Asn Ala Cys Asn
165 170 175
Ile Phe Gly Asn Thr Phe Asn Ala Ile Thr Ser Met Ile Asp Ser Ala
180 185 190
Lys Lys Ala Ala Ala Asp Ala Arg Arg Thr Ala Pro Glu Ser Pro Asn
195 200 205
Gln Pro Ser Ala Phe Asn Asn Ala Asp Phe Asn Lys Asn Leu Asn Gln
210 215 220
Val Ser Ser Val Ile Asn Asp Thr Ile Ser Tyr Leu Lys Gly Asp Asn
225 230 235 240
Leu Ala Thr Ile Tyr Asn Thr Leu Gln Lys Thr Pro Asp Ser Lys Gly
245 250 255
Phe Gln Ser Leu Val Ser Arg Ser Ser Tyr Ser Tyr Ser Leu Asn Glu
260 265 270
Thr Gln Tyr Ser Glu Phe Gln Thr Thr Thr Lys Glu Phe Gly His Asn
275 280 285
Pro Phe Arg Ser Val Gly Leu Ile Asn Ser Gln Ser Asn Asn Gly Ala
290 295 300
Met Asn Gly Val Gly Val Gln Leu Gly Tyr Lys Gln Phe Phe Gly Lys
305 310 315 320
Asn Lys Phe Phe Gly Ile Arg Tyr Tyr Ala Phe Phe Asp Tyr Asn His
325 330 335
Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asn Val Phe Thr
340 345 350
Tyr Gly Ala Gly Ser Asp Leu Leu Leu Asn Phe Ile Asn Gly Gly Ser
355 360 365
Asp Lys Asn Arg Lys Val Ser Phe Gly Ile Phe Gly Gly Ile Ala Leu
370 375 380
Ala Gly Thr Thr Trp Leu Asn Ser Gln Phe Met Asn Leu Lys Thr Thr
385 X390 395 400
Asn Ser Ala Tyr Ser Ala Lys Ile Asn Asn Thr Asn Phe Gln Phe Leu
405 410 415
Phe Asn Thr Gly Leu Arg Leu Gln Gly Ile His His Gly Val Glu Leu
' 420 425 430
Gly Val Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly
435 440 445
Ala Lys Leu Ala Tyr Arg Arg Leu Tyr Ser Val Tyr Phe Asn Tyr Val
450 455 460
Leu Ala Tyr
465
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 132 PCTNS98/06421
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2338 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 146...2218
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 146...200
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
ACTTAAAATT GTTTTTTTTT TTTTTCAAAA TATAAATTTT AAGCCAAAAA TAAGCATTTT 60
ATGGTAAAAT GGCGAACTTT CATAAACATG ACTATTATGG GAATGTCATG GGAATGTGAA 120
GAAAAATCTA TTAAAA GGA GAA AAC ATG AAA AAA TCC CTC TTA CTC TCT CTT 172
Met Lys Lys Ser Leu Leu Leu Ser Leu
-18 -15 -10
TCT CTC ATC GCT TCC TTA TCA AGA GCT GAA GAT GAC GGA TTT TAT ACG 220
Ser Leu Ile Ala Ser Leu Ser Arg Ala Glu Asp Asp Gly Phe Tyr Thr
-5 1 5
AGT GTG GGC TAT CAG ATC GGT GAA GCG GTC CAA CAA GTG AAA AAC ACA 268
Ser Val Gly Tyr Gln Ile Gly Glu Ala Val Gln Gln Val Lys Asn Thr
15 20
GGA GCA TTG CAA AAT CTT GCA GAC AGA TAC GAT AAC TTA AAC AAC CTT 316
Gly Ala Leu Gln Asn Leu Ala Asp Arg Tyr Asp Asn Leu Asn Asn Leu
25 30 35
TTA AAC CAA TAC AAT TAT TTA AAT TCC TTA GTC AAT TTA GCC AGC ACG 364
Leu Asn Gln Tyr Asn Tyr Leu Asn Ser Leu Val Asn Leu Ala Ser Thr
40 45 50 55
CCG AGC GCG ATC ACC GGT GCG ATT GAT AAT TTA AGC TCA AGC GCG ATT 412
Pro Ser Ala Ile Thr Gly Ala Ile Asp Asn Leu Ser Ser Ser Ala Ile
60 65 70
AAC CTC ACT AGC GCC ACC ACC ACT TCC CCC GCC TAT CAA GCT GTG GCT 460
Asn Leu Thr Ser Ala Thr Thr Thr Ser Pro Ala Tyr Gln Ala Val Ala
75 BO 85
TTA GCG CTC AAT GCC GCT GTG GGC ATG TGG CAA GTC ATA GCC CTT TTT 508
Leu Ala Leu Asn Ala Ala Val Gly Met Trp Gln Val Ile Ala Leu Phe
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 133 PCT/US98/06421
90 95 100
ATT GGC TGTGGC CCT CCT ACCAAT AAT AGC TATCAA TTT 556
GGC CAA TCG
Ile Gly CysGly ProGlyPro ThrAsn AsnGlnSer TyrGlnSer Phe
105 110 115
GGT AAC ACACCA GCCCTTAAT GGGACC ACCACCACT TGCAATCAA GCA
604
- Gly Asn ThrPro AlaLeuAsn GlyThr ThrThrThr CysAsnGln Ala
120 125 130 135
TAT GGG ACAGGC CCTAATGGC ATCCTA TCTATTGAT GAATACCAA AAA 652
Tyr Gly ThrGly ProAsnGly IleLeu SerIleAsp GluTyrGln Lys
140 145 150
CTC AAC CAAGCT TATCAGATC ATCCAA ACCGCTTTA AACCAAAAT CAA 700
Leu Asn GlnAla TyrGlnIle IleGln ThrAlaLeu AsnGlnAsn Gln
155 160 165
GGG GGT GGGATG CCTGCCTTG AATGAC ACCACCAAA ACAGGGGTA GTC 748
Gly Gly GlyMet ProAlaLeu AsnAsp ThrThrLys ThrGlyVal Val
170 175 180
AAC ATA CAACAA ACCAATTAT AGGACC ACCACACAA AACAATATC ATA 796
Asn Ile GlnGln ThrAsnTyr ArgThr ThrThrGln AsnAsnIle Ile
185 190 195
GAG CAT TATTAT ACAGAGAAT GGGAAA GAGATCCCA GTCTCTTAT TCA 844
Glu His TyrTyr ThrGluAsn GlyLys GluIlePro ValSerTyr Ser
200 205 210 215
GGC GGA TCATCA TTCTCGCCT ACAATA CAATTGACA TACCATAAT AAC 892
Gly Gly SerSer PheSerPro ThrIle GlnLeuThr TyrHisAsn Asn
220 225 230
GCT GAA AACCTT TTGCAACAA GCCGCC ACTATCATG CAAGTCCTT ATT 940
Ala Glu AsnLeu LeuGlnGln AlaAla ThrIleMet GlnValLeu Ile
235 240 245
ACT CAA AAGCCG CATGTGCAA ACGAGC AATGGCGGT AAAGCGTGG GGG 988
Thr Gln LysPro HisValGln ThrSer AsnGlyGly LysAlaTrp Gly
250 255 260
TTG AGT TCTACG CCTGGGAAT GTGATG GATATTTTT GGTCCTTCT TTT 1036
Leu Ser SerThr ProGlyAsn ValMet AspIlePhe GlyProSer Phe
265 270 275
AAC GCT ATTAAT GAGATGATT AAAAAC GCTCAAACA GCCCTAGCA AAA 1084
Asn Ala IleAsn GluMetIle LysAsn AlaGlnThr AlaLeuAla Lys
280 285 290 2g5
ACC CAA CAGCTT AACGCTAAT GAAAAC GCCCAAATC ACGCAACCC AAC 1132
Thr Gln GlnLeu AsnAlaAsn GluAsn AlaGlnIle ThrGlnPro Asn
300 305 310
AAT TTC AACCCC TACACCTCT AAAGAC AAAGGGTTC GCTCAAGAA ATG_1180
Asn Phe AsnPro TyrThrSer LysAsp LysGlyPhe AlaGlnGlu Met
315 320 325
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 134 PCTNS98/06421
CTC AAT AGA GCT GAA GCT CAA GCA GAG ATT TTA AAT TTA GCT AAG CAA 1228
Leu Asn Arg Ala Glu Ala Gln Ala Glu Ile Leu Asn Leu Ala Lys Gln
330 335 340
GTA GCG AAC AAT TTC CAC AGC ATT CAA GGG CCT ATT CAA GGG GAT TTA 1276
Val Ala Asn Asn Phe His Ser Ile Gln Gly Pro Ile Gln Gly Asp Leu
345 350 355
GAA GAA TGT AAA GCA GGA TCG GCT GGC GTG ATC ACT AAT AAC ACT TGG 1324
Glu Glu Cys Lys Ala Gly Ser Ala Gly Val Ile Thr Asn Asn Thr Trp
360 365 370 375
GGT TCA GGT TGC GCG TTT GTG AAA GAA ACT TTA AAC TCT TTA GAG CAA 1372
Gly Ser Gly Cys Ala Phe Val Lys Glu Thr Leu Asn Ser Leu Glu Gln
380 385 390
CAC ACC GCT TAT TAC GGC AAC CAG GTC AAT CAG GAT AGG GCT TTG GCT 1420
His Thr Ala Tyr Tyr Gly Asn Gln Val Asn Gln Asp Arg Ala Leu Ala
395 400 405
CAA ACC ATT TTG AAT TTT AAA GAA GCC CTT AAC ACC CTG AAT AAA GAC 1468
Gln Thr Ile Leu Asn Phe Lys Glu Ala Leu Asn Thr Leu Asn Lys Asp
410 415 420
TCA AAA GCG ATC AAT AGC GGT ATC TCC AAC TTG CCT AAC GCT AAA TCT 1516
Ser Lys Ala Ile Asn Ser Gly Ile Ser Asn Leu Pro Asn Ala Lys Ser
425 430 435
CTT CAA AAC ATG ACG CAT GCC ACT CAA AAC CCT AAT TCC CCA GAA GGT 1564
Leu Gln Asn Met Thr His Ala Thr Gln Asn Pro Asn Ser Pro Glu Gly
440 445 450 455
CTG CTC ACT TAT TCT TTG GAT TCA AGC AAA TAC AAC CAG CTC CAA ACC 1612
Leu Leu Thr Tyr Ser Leu Asp Ser Ser Lys Tyr Asn Gln Leu Gln Thr
460 465 470
ATC GCG CAA GAA TTG GGC AAA AAC CCT TTC AGG CGC TTT GGC GTG ATT 1660
Ile Ala Gln Glu Leu Gly Lys Asn Pro Phe Arg Arg Phe Gly Val Ile
475 480 485
GAC TTT CAA AAC AAC AAC GGC GCA ATG AAC GGG ATC GGC GTG CAA GTG 1708
Asp Phe Gln Asn Asn Asn Gly Ala Met Asn Gly Ile Gly Val Gln Va1
490 495 500
GGT TAT AAA CAA TTC TTT GGT AAA AAA AGG AAT TGG GGG TTA AGG TAT 1756
Gly Tyr Lys Gln Phe Phe Gly Lys Lys Arg Asn Trp Gly Leu Arg Tyr
505 510 515
TAT GGT TTC TTT GAT TAT AAC CAT GCT TAT ATC AAA TCT AAT TTT TTC _ 1804
Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Asn Phe Phe
520 525 530 535
AAC TCC GCT TCT GAT GTG TGG ACT TAT GGG GTG GGT ATG GAC GCT CTC 1852
Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Met Asp Ala Leu
540 545 550 _
TAT AAC TTC ATC AAC GAT AAA AAC ACC AAC TTT TTA GGC AAG AAC AAC 1900
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98!43479 135 PCT/US98/06421
Tyr Asn Phe Ile Asn Asp Lys Asn Thr Asn Phe Leu Gly Lys Asn Asn
555 560 565
AAG CTT TCA GTA GGG CTT TTT GGA GGC TTT GCG TTA GCC GGG ACT TCG 1948
Lys Leu Ser Val Gly Leu Phe Gly Gly Phe Ala Leu Ala Gly Thr Ser
570 575 580
TGG CTT AAT TCC CAA CAA GTG AAT TTG ACC ATG ATG AAT GGC ATT TAT 1996
Trp Leu Asn Ser Gln Gln Val Asn Leu Thr Met Met Asn Gly Ile Tyr
585 590 595
AAC GCT AAT GTC AGC ACT TCT AAC TTC CAA TTT TTG TTT GAT TTA GGC 2044
Asn Ala Asn Val Ser Thr Ser Asn Phe Gln Phe Leu Phe Asp Leu Gly
600 605 610 615
TTG AGA ATG AAC CTC GCT AGG CCT AAG AAA AAA GAC AGC GAT CAT GCC 2092
Leu Arg Met Asn Leu Ala Arg Pro Lys Lys Lys Asp Ser Asp His Ala
620 625 630
GCT CAG CAT GGC ATT GAA CTA GGT TTT AAG ATC CCC ACG ATC AAC ACC 2140
Ala Gln His Gly Ile Glu Leu Gly Phe Lys Ile Pro Thr Ile Asn Thr
635 640 645
AAC TAT TAT TCT TTC ATG GGC GCT AAA CTA GAA TAC AGA AGG ATG TAT 2188
Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Glu Tyr Arg Arg Met Tyr
650 655 660
AGC CTT TTT CTC AAT TAT GTG TTT GCT TAC TAAAAACTCT CTTTAAAAAA GGG 2241
Ser Leu Phe Leu Asn Tyr Val Phe Ala Tyr
665 670
GTTTGTTTAA AAACGCTTAA AAGCATTTTT AAAATTAAGC AGTAAAGAGC CTAGATAATC 2301
TCTTGCAACC GCTCTCAAGC GATAAAATTA AAGTGAT 2338
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 691 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...18
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Met Lys Lys Ser Leu Leu Leu Ser Leu Ser Leu Ile Ala Ser Leu Ser_
-18 -15 -10 -5
Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 136 PCT/US98/06421
1 5 10
Glu Ala Val Gln Gln Val Lys Asn Thr Gly Ala Leu Gln Asn Leu Ala
15 20 25 30
Asp Arg Tyr Asp Asn Leu Asn Asn Leu Leu Asn Gln Tyr Asn Tyr Leu
35 40 45
Asn Ser Leu Val Asn Leu Ala Ser Thr Pro Ser Ala Ile Thr Gly Ala
50 55 60
Ile Asp Asn Leu Ser Ser Ser Ala Ile Asn Leu Thr Ser Ala Thr Thr
65 70 75
Thr Ser Pro Ala Tyr Gln Ala Val Ala Leu Ala Leu Asn Ala Ala Val
80 85 90
Gly Met Trp Gln Val Ile Ala Leu Phe Ile Gly Cys Gly Pro Gly Pro
95 100 105 110
Thr Asn Asn Gln Ser Tyr Gln Ser Phe Gly Asn Thr Pro Ala Leu Asn
115 120 125
Gly Thr Thr Thr Thr Cys Asn Gln Ala Tyr Gly Thr Gly Pro Asn Gly
130 135 140
Ile Leu Ser Ile Asp Glu Tyr Gln Lys Leu Asn Gln Ala Tyr Gln Ile
145 150 155
Ile Gln Thr Ala Leu Asn Gln Asn Gln Gly Gly Gly Met Pro Ala Leu
160 165 170
Asn Asp Thr Thr Lys Thr Gly Val Val Asn Ile Gln Gln Thr Asn Tyr
175 180 185 190
Arg Thr Thr Thr Gln Asn Asn Ile Ile Glu His Tyr Tyr Thr Glu Asn
195 200 205
Gly Lys Glu Ile Pro Val Ser Tyr Ser Gly Gly Ser Ser Phe Ser Pro
210 215 220
Thr Ile Gln Leu Thr Tyr His Asn Asn Ala Glu Asn Leu Leu Gln Gln
225 230 235
Ala Ala Thr Ile Met Gln Val Leu Ile Thr Gln Lys Pro His Val Gln
240 245 250
Thr Ser Asn Gly Gly Lys Ala Trp Gly Leu Ser Ser Thr Pro Gly Asn
255 260 265 270
Val Met Asp Ile Phe Gly Pro Ser Phe Asn Ala Ile Asn Glu Met Ile
275 280 285
Lys Asn Ala Gln Thr Ala Leu Ala Lys Thr Gln Gln Leu Asn Ala Asn
290 295 300
Glu Asn Ala Gln Ile Thr Gln Pro Asn Asn Phe Asn Pro Tyr Thr Ser
305 310 315
Lys Asp Lys Gly Phe Ala Gln Glu Met Leu Asn Arg Ala Glu Ala Gln
320 325 330
Ala Glu Ile Leu Asn Leu Ala Lys Gln Val Ala Asn Asn Phe His Ser
335 340 345 350
Ile Gln Gly Pro Ile Gln Gly Asp Leu Glu Glu Cys Lys Ala Gly Ser
355 360 365
Ala Gly Val Ile Thr Asn Asn Thr Trp Gly Ser Gly Cys Ala Phe Val
370 375 380
Lys Glu Thr Leu Asn Ser Leu Glu Gln His Thr Ala Tyr Tyr Gly Asn
385 390 395
Gln Val Asn Gln Asp Arg Ala Leu Ala Gln Thr Ile Leu Asn Phe Lys '
400 405 410
Glu Ala Leu Asn Thr Leu Asn Lys Asp Ser Lys Ala Ile Asn Ser Gly
415 420 425 430
Ile Ser Asn Leu Pro Asn Ala Lys Ser Leu Gln Asn Met Thr His Ala_
435 440 445
Thr Gln Asn Pro Asn Ser Pro Glu Gly Leu Leu Thr Tyr Ser Leu Asp
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 137 PCT/US98/06421
450 455 460
Ser Ser Lys Tyr Asn Gln Leu Gln Thr Ile Ala Gln Glu Leu Gly Lys
465 470 475
Asn Pro Phe Arg Arg Phe Gly Val Ile Asp Phe Gln Asn Asn Asn Gly
480 485 490
Ala Met Asn Gly Ile Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly
495 500 505 510
Lys Lys Arg Asn Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn
515 520 525
His Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp
530 535 540
Thr Tyr Gly Val Gly Met Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys
545 550 555
Asn Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe
560 565 570
Gly Gly Phe Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Gln Val
575 580 585 590
Asn Leu Thr Met Met Asn Gly Ile Tyr Asn Ala Asn Val Ser Thr Ser
595 600 605
Asn Phe Gln Phe Leu Phe Asp Leu Gly Leu Arg Met Asn Leu Ala Arg
610 615 620
Pro Lys Lys Lys Asp Ser Asp His Ala Ala Gln His Gly Ile Glu Leu
625 630 635
Gly Phe Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly
640 645 650
Ala Lys Leu Glu Tyr Arg Arg Met Tyr Ser Leu Phe Leu Asn Tyr Val
655 660 665 670
Phe Ala Tyr
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
TCAAGGAGAA AACATGAAAA AAACCC 26
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
GAAGACGACG GCTTTTACAC AAGCGT 26
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
W O 98/43479 13 8 PCT/US98/06421
(2} INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTIC S:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
AAAGCTTAGT AAGCGAACAC ATAA 24
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTIC S:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
AAGGAGAAAA AACATGAAAA AACACATCC 29
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTIC S:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
GAAGACGACG GCTTTTACAC AAGCG 25
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTIC S:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
AACATTAGTA AGCGAACACA TAGTTC _ 26
(2) INFORMATION FOR SEQ ID N0:29:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 139 PCT/US98/06421
(i) SEQUENCE CHARACTERISTICS:
(Ay LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
AAGGAGAAAA AACATGAAAA AACACATCC
29.
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GAAGACGACG GCTTTTACAC AAGCGT 26
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
AAAAGCTTAG TAAGCGAACA CAT 23
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
AAGGAGAAAA CATGAAGAAA AAATTT 26
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 140 PCT/US98/06421
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
GAAGACAACG GCTTTTTTGT GAGTG 25
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
AGCTTTTAGT AAGCAAACAC ATAGT 25
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
AAGGATATTT ATGAAAAAAA CCCTT 25
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
GAAGACAACG GCTTTTTTAT CAGCG 25
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 141 PCT/US98/06421
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
GATATTAGTA AGCAAACACA TAATTC 26
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
AAGGAGAAAA CATGAAAAAA TCCCTCT 27
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
GAAGATGACG GATTTTATAC GAGTGT 26
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
TTTTAGTAAG CAAACACATA ATTGAG 26
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS: _
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 142 PCT/US98/06421
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
AAGGAACATC TTATGAAAAA AACG 24
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
GAAGACAACG GCGTTTTTTT AAGCG 25
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
GGTTTTTAAT AGGCAAACAC ATAAT 25
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
AAGGAACATT TTATGAAAAA GACAAT 26
(2) INFORMATION FOR SEQ ID N0:45:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 143 PCT/US98/06421
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
GAAGACAACG GCTTTTTTGT GAGCG 25
(2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
TCACTCAGTA AGCGAACACA TAA 23
(2) INFORMATION FOR SEQ ID N0:47:
' (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
AAGGAACATT TTATGAAAAA GACAA 25
(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
GAAGACAACG GCTTTTTTGT GAGCG 25
(2) INFORMATION FOR SEQ ID N0:49: '
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid _
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 144 PCT/US98/06421
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
TTTTAATAAG CGAACACATA AAAGAG 26
(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
AAGGAACATT TTATGAAAAA AACGAT 26
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
GAAAATGACG GCGTTTTTAT GAGCG 25
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
ATATCAATAG GCCAAAACAT AATTGA 26
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 145 PCT/US98/06421
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
AAGGAGAAAA CATGAAAAAA TCCCTC 26
(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
GAAGATGACG GATTTTATAC GAGTGT 26
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
TTTTAGTAAG CAAACACATA ATTGAG 26
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
CGCGGATCCG AATCCAATTT AATCCAAAAA GG 32
(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs __
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 146 PCT/US98/06421
CCGCTCGAGT TAAGTAAGCG AACACATATT CAA 33
(2) INFORMATION FOR SEQ ID N0:58
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58
Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala
1 5 10 15
Ala Gln Met Val
(2) INFORMATION FOR 5EQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
CTGAATTCGA TTTCAAGGAG AAAACATGAA A 31
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUEN E DESCRIPTION: SEQ ID N0:60:
CCGCTCGAGT TAGTAAGCGA ACACATAATT 30
(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
SUBSTITUTE SHEET (RULE 25)
CA 02286893 1999-10-O1
WO 98/43479 147 PCT/US98/06421
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
CGCGGATCCG AATCCAATTT AATCCAAAAA GG 32
(2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
CCGCTCGAGT TAGTAAGCGA ACACATAGTT CAA 33
(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
CGCGGATCCG AAGTTTCTTT GTATCAAAG 29
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
CCGCTCGAGT TAGTAAGCAA ACACATAATT GTG 33
' (2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1149 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single _
(D) TOPOLOGY: linear
SUBSTCTUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 148 PCT/US98/06421
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 106...1002
(D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 106...166
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:65:
TTACTCTTTA TTTGTTTTAA 60
ATGTGAGTTT ATTTGCTATA
TCTGTGTCAT
GATAGCTGAT
ATGTGAATTT AGTGGAGAAC ACACA 117
AATGATGAAA ATG
ATTAGTTTAG AAA
AAA
AAT
Met
Lys
Lys
Asn
-20
ATCTTAAAT TTAGCG TTAGTGGGT GCGTTGAGC ACGTCG TTTTTGATG 165
IleLeuAsn LeuAla LeuValGly AlaLeuSer ThrSer PheLeuMet
-15 -10 -5
GCTAAGCCG GCTCAT AACGCAAAT AACGCTACG CATAAC ACGAAAAAA 213
AlaLysPro AlaHis AsnAlaAsn AsnAlaThr HisAsn ThrLysLys
1 5 10 15
ACGACTGAT TCTTCA GCAGGCGTG TTAGCGACA GTGGAT GGCAGACCT 261
ThrThrAsp SerSer AlaGlyVal LeuAlaThr ValAsp GlyArgPro
20 25 30
ATCACTAAA AGCGAT TTTGACATG ATTAAGCAA CGAAAT CCTAATTTT 309
IleThrLys SerAsp PheAspMet IleLysGln ArgAsn ProAsnPhe
35 40 45
GATTTTGAC AAGCTT AAAGAGAAA GAAAAAGAA GCCTTG ATTGATCAA 357
AspPheAsp LysLeu LysGluLys GluLysGlu AlaLeu IleAspGln
50 55 60
GCTATTCGC ACCGCC CTTGTAGAA AATGAAGCT AAAACC GAGAAATTG 405
AlaIleArg ThrAla LeuValGlu AsnGluAla LysThr GluLysLeu
65 70 75 80
GACAGCACT CCAGAA TTTAAAGCG ATGATGGAA GCGGTT AAAAAACAG 453
AspSerThr ProGlu PheLysAla MetMetGlu AlaVal LysLysGln
85 90 95
GCTTTAGTG GAATTT TGGGCTAAA AAACAGGCT GAAGAA GTGAAAAAA 501
AlaLeuVal GluPhe TrpAlaLys LysGlnAla GluGlu ValLysLys
100 105 110
GTCCAAATC CCAGAA AAAGAAATG CAAGATTTT TACAAC GCTAACAAA 549
ValGlnIle ProGlu LysGluMet GlnAspPhe TyrAsn AlaAsnLys
115 120 125
GATCAGCTT TTTGTC AAGCAAGAA GCCCATGCT AGGCAT ATTTTAGTG 597
AspGlnLeu PheVal LysGlnGlu AlaHisAla ArgHis IleLeuVal
130 135 140
AAAACCGAA GATGAG GCTAAACGG ATTATTTCT GAGATT GACAAACAG 645
LysThrGlu AspGlu AlaLysArg IleIleSer GluIle AspLysGln
145 150 155 160
CCAAAGGCT AAAAAA GAAGCTAAA TTCATTGAG TTAGCC AATCGGGAT 693
ProLysAla LysLys GluAlaLys PheIleGlu LeuAla AsnArgAsp
165 170 175
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 149 PCTNS98/06421
ACG ATTGAT CCTAAC AGCAAGAAC GCGCAA AATGGCGGT GATTTGGGG 741
Thr IleAsp ProAsn SerLysAsn AlaGln AsnGlyGly AspLeuGly
180 185 190
AAA TTCCAA AAGAAC CAAATGGCT CCGGAT TTTTCTAAA GCCGCTTTC 789
Lys PheGln LysAsn GlnMetAla ProAsp PheSerLys AlaAlaPhe
195 200 205
GCT TTAACT CCTGGG GATTACACT AAAACC CCTGTTAAA ACAGAGTTT 837
Ala LeuThr ProGly AspTyrThr LysThr ProValLys ThrGluPhe
210 215 220
GGT TATCAT ATTATC TATTTGATT TCTAAA GATAGCCCT GTAACTTAT 885
Gly TyrHis IleIle TyrLeuIle SerLys AspSerPro ValThrTyr
225 230 235 240
ACT TATGAA CAGGCT AAACCTACC ATTAAG GGGATGTTA CAAGAAAAG 933
Thr TyrGlu GlnAla LysProThr IleLys GlyMetLeu GlnGluLys
245 250 255
CTT TTCCAA GAACGC ATGAATCAA CGCATT GAGGAACTA AGAAAGCAC 981
Leu PheGln GluArg MetAsnGln ArgIle GluGluLeu ArgLysHis
260 265 270
GCT AAAATT GTTATC AACAAGTAATTGATGA 1036
GGTGTTATCA
TGTTAGTTAA
AGGC
Ala LysIle ValIle AsnLys
275
AATGAAATTT TGGGGGCGTT 1096
TATTGAAAGC TAATTTCGTG
CCATAAAGAA
GGTTATGGGG
AATTTTGAAA AGGAAAATTC 1149
TGCTAAACGC CCC
TATTTTTGAA
GCAGGAAATG
(2) INFORMATION FOR SEQ ID N0:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 299 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:66:
Met Lys Lys Asn Ile Leu Asn Leu Ala Leu Val Gly Ala Leu Ser Thr
-20 -15 -10 -5
Ser Phe Leu Met Ala Lys Pro Ala His Asn Ala Asn Asn Ala Thr His
1 5 10
Asn Thr Lys Lys Thr Thr Asp Ser Ser Ala Gly Val Leu Ala Thr Val
15 20 25
Asp Gly Arg Pro Ile Thr Lys Ser Asp Phe Asp Met Ile Lys Gln Arg
30 35 40
Asn Pro Asn Phe Asp Phe Asp Lys Leu Lys Glu Lys Glu Lys Glu Ala
45 50 55 60
Leu Ile Asp Gln Ala Ile Arg Thr Ala Leu Val Glu Asn Glu Ala Lys_
65 70 75
Thr Glu Lys Leu Asp Ser Thr Pro Glu Phe Lys Ala Met Met Glu Ala
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 150 PCT/US98/06421
80 85 90
Val Lys Lys Gln Ala Leu Val Glu Phe Trp Ala Lys Lys Gln Ala Glu
95 100 105
Glu Val Lys Lys Val Gln Ile Pro Glu Lys Glu Met Gln Asp Phe Tyr
110 115 120
Asn Ala Asn Lys Asp Gln Leu Phe Val Lys Gln Glu Ala His Ala Arg
125 130 135 140
His IIe Leu Val Lys Thr Glu Asp Glu Ala Lys Arg Ile Ile Ser Glu
145 150 155
Ile Asp Lys Gln Pro Lys Ala Lys Lys Glu Ala Lys Phe Ile Glu Leu
160 165 170
Ala Asn Arg Asp Thr Ile Asp Pro Asn Ser Lys Asn Ala Gln Asn Gly
175 180 185
Gly Asp Leu Gly Lys Phe Gln Lys Asn Gln Met Ala Pro Asp Phe Ser
190 195 200
Lys Ala Ala Phe Ala Leu Thr Pro Gly Asp Tyr Thr Lys Thr Pro Val
205 210 215 220
Lys Thr Glu Phe Gly Tyr His Ile Ile Tyr Leu Ile Ser Lys Asp Ser
225 230 235
Pro Val Thr Tyr Thr Tyr Glu Gln Ala Lys Pro Thr Ile Lys Gly Met
240 245 250
Leu Gln Glu Lys Leu Phe Gln Glu Arg Met Asn Gln Arg Ile Glu Glu
255 260 265
Leu Arg Lys His Ala Lys Ile Val Ile Asn Lys
270 275
(2) INFORMATION FOR SEQ ID N0:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1448 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 118...1314
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
CTCTTGAATG GGTAGCATTT 60
GCGATAAGAC AGGAATACTT
AAAAATGTCT
TAAATTTTGT
AGGATTTTGT ATTTCAAAAA ATTAAGGAGA 120
TTAGTATAAT AATACAA
TCTAAAATCC ATG
Met
__1
GCA GAA TTTAACAGA ACTAAGCCG GTT ATTGGA ACC 168
AAA AAG CAT AAT
Ala Glu PheAsnArg ThrLysPro Val IleGly Thr
Lys Lys His Asn
5 10 15
ATT CAT GACCATGGT AAAACGACT AGT GCGATT TCA 216
GGG GTA TTG GCA
Ile His AspHisGly LysThrThr Ser AlaIle Ser
Gly Val Leu Ala
20 25 30 -
GCG CTT TTGAAAGGT CTTGCAGAA AAA TATGAT AAT 264
GTG TCT ATG GAC
Ala Leu LeuLysGly LeuAlaGlu Lys TyrAsp Asn
Val Ser Met Asp
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 151 PCT/US98/06421
35 40 45
ATT GATAAC GCCCCTGAA GAAAAA GAAAGAGGG ATCACTATC GCTACT 312
Ile AspAsn AlaProGlu GluLys GluArgGly IleThrIle AlaThr
50 55 60 65
TCT CACATT GAATATGAG ACTGAA AACAGACAC TATGCGCAT GTGGAT 360
Ser HisIle GluTyrGlu ThrGlu AsnArgHis TyrAlaHis ValAsp
70 75 80
TGC CCAGGA CACGCTGAC TATGTA AAAAACATG ATCACCGGT GCGGCG 408
Cys ProGly HisAlaAsp TyrVal LysAsnMet IleThrGly AlaAla
85 90 95
CAA ATGGAC GGAGCGATT TTGGTT GTTTCTGCA GCTGATGGC CCTATG 456
Gln MetAsp GlyAlaIle LeuVal ValSerAla AlaAspGly ProMet
100 105 110
CCT CAAACT AGGGAGCAT ATCTTA TTGTCTCGT CAAGTAGGC GTGCCT 504
Pro GlnThr ArgGluHis IleLeu LeuSerArg GlnValGly ValPro
115 120 125
CAC ATCGTT GTTTTCTTA AACAAA CAAGACATG GTAGATGAC CAAGAA 552
His IleVal ValPheLeu AsnLys GlnAspMet ValAspAsp GlnGlu
130 135 140 145
TTG TTAGAA CTTGTAGAA ATGGAA GTGCGCGAA TTGTTGAGC GCGTAT 600
Leu LeuGlu LeuValGlu MetGlu VaiArgGlu LeuLeuSer AlaTyr
150 155 160
GAA TTTCCT GGCGATGAC ACTCCT ATCGTAGCG GGTTCAGCT TTAAGA 648
Glu PhePro GlyAspAsp ThrPro IleValAla GlySerAla LeuArg
165 170 175
GCT TTAGAA GAAGCAAAG GCTGGT AATGTGGGT GAATGGGGT GAAAAA 696
Ala LeuGlu GluAlaLys AlaGly AsnValGly GluTrpGly GluLys
180 185 190
GTG CTTAAA CTTATGGCT GAAGTG GATGCCTAT ATCCCTACT CCAGAA 744
Val LeuLys LeuMetAla GluVal AspAlaTyr IleProThr ProGlu
195 200 205
AGA GACACT GAAAAAACT TTCTTG ATGCCGGTT GAAGATGTG TTCTCT 792
Arg AspThr GluLysThr PheLeu MetProVal GluAspVal PheSer
210 215 220 225
ATT GCGGGT AGAGGGACT GTGGTT ACAGGTAGG ATTGAAAGA GGCGTG 840
Ile AlaGly ArgGlyThr ValVal ThrGlyArg IleGluArg GlyVal
230 235 240
GTG AAAGTA GGCGATGAA GTGGAA ATCGTTGGT ATCAGACCT ACACAA 888
Val LysVal GlyAspGlu ValGlu IleValGly IleArgPro ThrGln
245 250 255
AAA ACGACT GTAACCGGT GTAGAA ATGTTTAGG AAAGAGTTG GAAAAA 936
Lys ThrThr ValThrGly ValGlu MetPheArg LysGluLeu GluLys
260 265 270
GGT GAAGCC GGCGATAAT GTGGGC GTGCTTTTG AGAGGAACT AAAAAA 984
Gly GluAla GlyAspAsn ValGly ValLeuLeu ArgGlyThr LysLys
275 280 285
GAA GAAGTG GAACGCGGT ATGGTT CTATGCAAA CCAGGTTCT ATCACT 1032
Glu GluVal GluArgGly MetVal LeuCysLys ProGlySer IleThr
290 295 300 305
__
CCG CACAAG AAATTTGAG GGAGAA ATTTATGTC CTTTCTAAA GAAGAA 1080
Pro HisLys LysPheGlu GlyGlu IleTyrVal LeuSerLys GluGlu
310 315 320
GGC GGGAGA CACACTCCA TTCTTC ACCAATTAC CGCCCGCAA TTCTAT 1128
Gly GlyArg HisThrPro PhePhe ThrAsnTyr ArgProGln PheTyr
325 330 335 _
GTG CGCACA ACTGATGTG ACTGGC TCTATCACC CTTCCTGAA GGCGTA 1176
Val ArgThr ThrAspVal ThrGly SerIleThr LeuProGlu GlyVal
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 152 PCT/US98/06421
340 345 350
GAA ATG GTT ATG CCT GGC GAT AAT GTG AAA ATC ACT GTA GAG TTG ATT 1224
Glu Met Val Met Pro Gly Asp Asn Val Lys Ile Thr Val Glu Leu Ile
355 360 365
AGC CCT GTT GCG TTA,GAG TTG GGA ACT AAA TTT GCG ATT CGT GAA GGC 1272
Ser Pro Val Ala Leu Glu Leu Gly Thr Lys Phe Ala Ile Arg Glu Gly
370 375 380 385
GGT AGG ACC GTT GGT GCT GGT GTT GTG AGC AAT ATT ATT GAA TAATATTAG 1323
Gly Arg Thr Val Gly Ala Gly Val Val Ser Asn Ile Ile Glu
390 395
CAAAAAGAGA GTTACCATAA AGGGTCATTA TGAAAGTTAA AATAGGGTTG AAGTGTTCTG 1383 _
ATTGTGAAGA TATCAATTAC AGCACAACCA AGAACGCTAA AACTAACACT GAAAAACTGG 1443
AGCTT
1448
(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 399 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
Met Ala Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn Ile Gly
1 5 10 15
Thr Ile Gly His Val Asp His Giy Lys Thr Thr Leu Ser Ala Ala Ile
20 25 30
Ser Ala Val Leu Ser Leu Lys Gly Leu Ala Glu Met Lys Asp Tyr Asp
35 40 45
Asn Ile Asp Asn Ala Pro Glu Glu Lys Glu Arg Gly Ile Thr Ile Ala
50 55 60
Thr Ser His Ile Glu Tyr Glu Thr Glu Asn Arg His Tyr Ala His Val
65 70 75 80
Asp Cys Pro Gly His Ala Asp Tyr Val Lys Asn Met Ile Thr Gly Ala
85 90 95
Ala Gln Met Asp Gly Ala Ile Leu Val Val Ser Ala Ala Asp Gly Pro
100 105 110
Met Pro Gln Thr Arg Glu His Ile Leu Leu Ser Arg Gln Val Gly Val
115 120 125
Pro His Ile Val Val Phe Leu Asn Lys Gln Asp Met Val Asp Asp Gln
130 135 140
Glu Leu Leu Glu Leu Val Glu Met Glu Val Arg Glu Leu Leu Ser Ala
145 150 155 160
Tyr Glu Phe Pro Gly Asp Asp Thr Pro Ile Val Ala Gly Ser Ala Leu
165 170 175
Arg Ala Leu Glu Glu Ala Lys Ala Gly Asn Val Gly Glu Trp Gly Glu
180 185 190
Lys Val Leu Lys Leu Met Ala Glu Val Asp Ala Tyr Ile Pro Thr Pro
195 200 205
Glu Arg Asp Thr Glu Lys Thr Phe Leu Met Pro Val Glu Asp Val Phe
210 215 220
Ser Ile Ala Gly Arg Gly Thr Val Val Thr Gly Arg Ile Glu Arg Gly
225 230 235 240
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 153 PCT/US98/06421
Val Val Lys Val Gly Asp Glu Val Glu Ile Val Gly Ile Arg Pro Thr
245 250 255
Gln Lys Thr Thr Val Thr Gly Val Glu Met Phe Arg Lys Glu Leu Glu
260 265 2 70
Lys Gly Glu Ala Gly Asp Asn Val Gly Val Leu Leu Arg Gly Thr Lys
275 280 285
Lys Glu Glu Val Glu Arg Gly Met Val Leu Cys Lys Pro Gly Ser Ile
290 295 300
Thr Pro His Lys Lys Phe Glu Gly Glu Ile Tyr Val Leu Ser Lys Glu
305 310 315 320
Glu Gly Gly Arg His Thr Pro Phe Phe Thr Asn Tyr Arg Pro Gln Phe
325 330 335
Tyr Val Arg Thr Thr Asp Val Thr Gly Ser Ile Thr Leu Pro Glu Gly
340 345 350
Val Glu Met Val Met Pro Gly Asp Asn Val Lys Ile Thr Val Glu Leu
355 360 365
Ile Ser Pro Val Ala Leu Glu Leu Gly Thr Lys Phe Ala Ile Arg Glu
370 375 380
Gly Gly Arg Thr Val Gly Ala Gly Val Val Ser Asn Ile Ile Glu
385 390 395
(2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
CGCGGATCCG AATGAAAAAA AATATCTTAA AT 32
(2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
CCGCTCGAGT TACTTGTTGA TAACAATTTT 30
(2) INFORMATION FOR SEQ ID N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
~ (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 154 PCT/US98/06421
CGCGGATCCG AATGGCAAAA GAAAAGTTTA AC 32
(2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single _
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
CCGCTCGAGT TATTCAATAA TATTGCTCAC 30
(2) INFORMATION FOR SEQ ID N0:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:73:
Met Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn Ile Gly Thr
1 5 10 15
Ile Gly His Val Asp His
(2) INFORMATION FOR SEQ ID N0:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:74:
Ala His Asn Ala Asn Asn Ala Thr His Asn Thr Lys Lys
1 5 10
(2) INFORMATION FOR SEQ ID N0:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 155 PCT/US98/06421
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:75:
Lys Pro Ala His Asn Ala
1 5
(2) INFORMATION FOR SEQ ID N0:76:
_ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:76:
Ile Asp Lys Gln Pro Lys Ala Lys Lys
1 5
(2) INFORMATION FOR SEQ ID N0:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:77:
Phe Trp Ala Lys Lys Gln Ala Glu
1 5
(2) INFORMATION FOR SEQ ID N0:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:78:
GTGGAGAACA CACAATGAAA AAAAATATC 29
(2) INFORMATION FOR SEQ ID N0:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:79:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 156 PCT/LJS98/06421
GCTAATATTA TTCAATAATA TTGCTCACAA C 31
(2) INFORMATION FOR SEQ ID N0:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:80:
GGAGAAATAC AAATGGCAAA AGAAAAG 27
(2) INFORMATION FOR SEQ ID N0:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:81:
GCTAATATTA TTCAATAATA TTGCTCACAA C 31
(2) INFORMATION FOR SEQ ID N0:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:82:
CATAACGCAA ATAACGCTAC GCAT 24
(2) INFORMATION FOR SEQ ID N0:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:83:
GGGAATTCAA AAAAACGAAA AAAACG 26
(2) INFORMATION FOR SEQ ID N0:84: _
(i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26)
CA 02286893 1999-10-O1
WO 98/43479 157 PCT/US98/06421
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:84:
CCCCTCGAGT TAATAGGCAA ACAC 24
SUBSTITUTE SHEET (RULE 2B)
