Note: Descriptions are shown in the official language in which they were submitted.
W093/2~2~ PCT/US93/~43~
-~ 2134898 `:
COMPOSITIONS AND METHODS FOR VACCINATION
AGAINST CORONAVIRUSES `~
: .
Cross-Reference to,Related apPlications ~,
This is a continuation-in-part of pending U.S. patent ':'`'
application Serial No. 07/882,171, filed May 8, 1992, which
is a continuation-in-part of U.S. patent application Serial ' .
No. 07/698,927, filed May 1~, 1991, which is a :;`
continuation-in-part of U.S. patent application Serial No. ~:~
07/613,066, filed November 14, 1990.
~; '
Field of t,he Invention .
This invention relates generally to ~ompositions :.
useful for vaccination against, and treatment of, ';'~
coronavirus infections, as well as methods for l~sing these ~ -
compositions. ' ` '".
. ,.~ .;,
,Backaround _f the Invention ~`'
Feline Tnfectious Peritonitis Virus tFIPV) is a
: ~ coronavirus which causes a highly Iethal immune complexing ,~.~
di:sease in cats. The exact mechanism(s) of FIP disease ~,'`
~ pathogenesls~ is not understGod, but Iesions are found in
all major:internal organs due to the deposition of immune
complexes. Several strains of FIPV have been isolated from
: diseased cats. ~Some are highly virulent and cause disease
: ~ after a :primary infection (Type II). Gther virulent '
strains (Type I) ca:nnot initiate disease unless the cat has
been previously exposed to FIPV. ^,~
~nother feline coronavirus other than FIPV is known.
Feline enteric coronavirus (FECV) was isolated from cats
but dif~ers in pathogenesis from FIPV. For example, FECV,
' 30 in contrast to FIPV, specifically infects intestinal
, epithelial cells and causes only a mild enteritis in cats. ,~
In addition, FECV has not been reported to sensitize cats ~,-
to disease following challenge with FIPV. -'
'.
W093/2~22 2 1 3 ~1 ~ 9 8 P~T/US93/~3~
Other corona~iruses have been isolated from other
animal species, i.e., transmissible gastroenteritis virus
of swine (TGEV), porcine respiratory coronavirus (PRCV),
bovine coronavirus (BCV), avian coronavirus (ACV), murine
coronavirus (MCV) and human coronavirus (HCV). However,
these coronaviruses do not share the same pathogenesis as
FIPV. As with FECV, these other coronaviruses cause ~ither
respiratory or mild gastroenteritis illnesses.
Coronaviruses encode three structural proteins: S -
surface glycoprotein or spike; M - the envelope matrix
protein; and N - the nucleoprotein whi~h interacts with the
RNA genome to make the viral capsid. The S protein induces
an immune response including the production of neutralizing
antibodies following infection. It is also responsible for
attachment to infected cells and mediates cell fusion,
aiding spread of the virus. Thus the S protein is the
primary focus for effective coronavirus vaccine development
because it is the target of virus neutralizing antibodies
and it contains sensitizing epitopes. In particular, no
other FI~V gene products (M or N) have been found to
stimulate effective immùnity to that virus.
The sequence of the S gene of the wild-type WSU 1146
- strain of FIPV has been published [DeGroot et al, J. Gen.
Virol., 68:2639-2646 (1987)]. Type II wild-type FIPV
strains are highly virulent in cats; the S gene alone of a
virulent FIPV can sensitize cat5 to disease. A strong
humoral response to the S gene appears responsible for the
immune complexes found in diseased cats. However, little
is known about the location of antigenic/neutralizing
epitopes on the FIPV S gene.
Partial sequences of other coronavirus S genes ha~e
been identified. See, for example, the sequences of PRCV
S gene tGenbank~ database, Intell Genetics, Mountain View,
CA~; BCV strain Mebus S gene ~Abraham et al, Virol.,
W093f2~22 2 1 3 ~1 8 9 8 PC~`/VS93/043~
176:2g6-301 (1990)]; strain F15 S gene [Boireau et al, J.
Gen. Virol., 71:487-492 (lsso)]i ACV S gene ~M. Binnes et
al, J. Gen. Virol., 66:719-726 (1985)]; MCV S gene [I.
Schmidt et al, J. Gen. Virol~, 68:47-56 (1987)] and HCV S
gene ~T. Ra2be et al, J. Gen. Virol., 71:1065-1073 (1990)~.
Only on the TGEV spike protein have four major antigenic
and neutralizing sites been defined [Correa et al, J. Gen.
Virol., 71:271-279 ~l990~j and Delmas et al, J. Gen.
_irol., 71:1313-1323 (1990)].
Attempts to product a safe, effective vaccine against
FIPV have been largely unsuccessful. Administration of
sublethal doses of virulent FIPV, avirulent FIPV strains or
other antigenically related coronaviruses has not been
effective. Previous exposure to the virus typically
}5 exacerbates the disease due to the strong humoral response
to FIPV infection. More recently, a vaccine has been
produced which consists of a live virus temperature-
sensitive strain of DF2 FIPV [Primucell FIP~; SmithKline
Beecham]. When this vaccine is administered oro-nasally,
it protects, but does not sensitize, cats to wild-type DF2
FIPV. However, this vaccine does not elicit a strong
cross-strain reaction.
Recombinant vaccines ~ave been engineered which
consist of FIPV gene products produced in a heterologous
~25 expression system. Vennema et al, J. Virol., 64:1407-1409
(1990) constructed vaccinia virus recombinants expressing
the S, M, and N genes of FIPV tType II, WSU 1146 strain~.
Cats immunized with the recombinant vaccinia virus
expressing thé S gene developed FIP and died sooner than
non-vaccinated animals following FIPV challenge. The
vaccinia recombinants expressing the FIPV M or N proteins
neither conclusively protected nor sensitized cats to ~IPV.
While no protection from challenge was observed, these
results clearly illustrate that the S gene is implicated in
FIP disease development.
W093/2~22 ~ 1 3~ 1 8 9 ~: PCT/US93/043~
~ ,
There remains a need for effective compositions for
use in treating and vaccinating animals against FIPV and
serologically related infections.
Summarv o f the I nvention
In one aspect, the present invention provides chimeric
coronavirus spike (S) proteins which are useful in treating
and preventing coronavirus infections. A chimeric S
protein of this invention can induce an enhanced response
to a single coxonavirus strain or can elicit an immune
respons~ to more than one corona~irus strain and/or
species.
In another aspect, the present invention provides a
polynucleotide sequence which ~ncodes a chimeric S protèin
or S fragment of the invention.
In another aspect, the present invention provides a
recombinant polynucleotide molecule comprising a chimeric
S gene polynucleotide sequence in operative association
with regulatory se~uences capable of directing the
replication and expression thereof in a selected host cell.
In still a further aspect the present invention
provides a host cell transformed with a recombinant
polynucleotide molecule as described above.
In still another aspect, the invention provides a
pharmaceutical or vaccinal composition comprising an
~S e~fective or immunogenic amount of one or more of the
chimeric coronavirus S proteins or S protein fragments of
the invention or fragments thereof in a suitable carrier.
These chimeric coronavirus S proteins are useful in the
treatment or prophylaxis of a disease caused by the
coronavirus from which its fragments are derived and may
induce cross-strain and cross-species responses,
W093/2~22 PC~/U~g3/~3~ ~
21~1~98 ~
Thusl in another aspect, the invention provides a
method for vaccinating a naive animal against a
coronavirus, or treating an infected animal for coronavlrus
infection, both by administering an effective dosage of a
pharmaceutical composition of the invention to the animal.
In another aspect the present invention provides a
method and a diagnostic kit for use in clinical
laboratories, for distinguishing between animals vaccinated
with a chimeric S protein of the invention and an animal
which has been naturally exposed to the coronavirus or
coronaviruses which comprise the chimeric S protein.
Other aspects and advantages of the present invention
are described further in the following detailed description
of the preferred embodiments thereof~
Detailed Descrition of the_lnvention
The present invention provides novel chimeric
coronavirus S proteins useful for vaccination against, and
treatment of, coronavirus infection, as well as methods for
producing these proteins and pharmaceutical compositions
containing them. Methods for using these novel chimeric
proteins in the identification of the immune sensitizing
and protective epitopes of the coronavirus spike protein
are also disclosed.
-- I. Definitions
As defined herein, the term "chim~ric coronavirus S
proteins", "chimeric proteins of the invention",
"chimerics", and the like include proteins composed of an
aminio acid se~uence encoded by a whole or a partial S gene
sequence from a selected coronavirus fused in any order to
one or more additional amino acid sequence(s) encoded by a
W093/2~22 ~ 1 3 ~ 8 ~ ~ PCT/US93/043
whole or partial S gene sequence from a selected
coronavirus. Optionally, these chimeric proteins may also
contain one or more non-S protein or non-coronavirus
protein sequences fused to the chimeric protein, S protein
amino acid sequences or fragments thereof. ;
In one embodiment, a chimeric coronavirus S protein
according to this invention may contain a first coronavirus
S protein fragment from a selected coronavirus and at least
one additional coronavirus S protein fraqment from a
selected coronavirus, with the fragments fused in any
desired order by an optional amino acid linker, the protein ~-
fragments forming the desired chimeric protein being
isolated or derived from at least two different strains or
species of coronavirus. -
In another embodimentj a chimeric coronavirus S
protein according to this invention contains a first
corona~irus S protein fragment from a selected coronavirus
fused~ in any order to at least one discontinuous
~ coronavi~us S protein fragment (i.e., the fragments are
encoded by different regions of the S gene) from the same
selected coronavirus, the corona~irus contributing the
fragments being a Type I FIPV strain, a Type II FIPV
- strain, a FEV strain, a CCV strain, a PRCV strain, an
avian coronavirus strain, a human coronavirus strain, a
bovine coronavirus strain and a murine coronavirus strain.
; ~ Desirably, these amino acid sequences forming the
chimeric protein are fused in such a way that they are
translated in the same reading frame and form a protein,
i.e.lfused inrframe. See, for example, A. Shatzman and`M.
Rosenberg, He~atoloay, 7(1):30S-35S (1987) and G.M.
Weinstock et al, Proc. Natl. Acad. Sci., ~Q:4432-4436 (July
1983). Fusion can be direct, i.e., S protein amino acid
sequence to S protein amino acid sequence, or indirect,
i.e., through another amino acid sequence which can serve
as a spacer or linker.
WOg3/2~Z2 PCT/US93/~3~
213~898
As defined herein, an amino acid fragment as it
relates to amino acid sequences of the S prutein refers to
any amino acid sequence from at least about 8 amino acids
in length up to about the full-length S gene protein (about
1454 amino acids). A nucleotide fragment as it relates to
nucleotide sequences of a coronavirus S gene defines a
nucleotide sequence which encodes from at least about 8
amino acids in length up to about the full-length S gene
protein. Preferably, these fragments are immunogenic.
As used herein, the term "immunogenic" means any
molecule, protein, peptide, carbohydrate, virus, or region
thereof which is capable of eliciting a protPctive immune
response in a host into which it is introduced. The term
"antigenic" refers only to the ability of a molecule,
protein, peptide, carbohydrate, virus-, or region thereof to
elicit antibody formation in a host (not necessarily
protective).
As used herein, the term "region" refers to all or a
portion of a gene or protein. Such a region may contain
one or more fragments as defined above.
As used herein, the term "epitope" refers to a xegion
of a protein which is involved in its immunogenicity, and
can include regions which induce B cell and/or T cell
responses.
As used herein, the term "B cell site or T cell site"
defines a region of the protein which is a site for B cell
or T cell binding. Preferably this term refers to sites
which are involved in the immunogenicity of the protein.
~As used ~erein, a sensitizing region is defined as a
region of the coronavirus S gene which, when used as an
immunogen, results in exacerbating disease following
challenge, theoretically by causing a strong humoral
response.
W093/~22 2 1 3 ~ ~ 9 8 ~` ~
II. Source of S Protein Se~uences for Use in the Chimeric
Proteins of the Invention
Depending upon the disease type and corona~-rus
species against which protection (or treatment) is desired,
one of skill in the art can select the appropriate S
protein fragments from a desired coronavirus to construct
a chimeric protein of this invention. Particularly desired
coronaviruses for use in supplying S protein fragments for
the chimeric proteins of this invention include FECV, and
FIPV strains referred to as DF2, DF2-HP, TS-8P, TS, TN406,
UCD-2, UCD-1, UCD-3, UCD-4.
Other FIPV strains may also be useful in providing S
protein fragments. In addition, other coronavirus species,
~ including CCV, TGEV, PRCV, BCV, ACV, MCV, or HCV are
expected~to be useful in supplying S protein fragments for
; the chimeric S proteins of this invention. In view of the
teachings of the present invention, one of skill in the art
could~construct suitable chimeric coronavirus S proteins by
; using~immunogen-ic fragments analogous to those identified
~20 herein.
The S~protein sequences or partial S protein sequences
and~the corresponding nucleotide sequences of several of
the~above-~identified coronaviruses and their respective SEQ
ID-~NOS are ~provided in Table I below. To provide a
reférence point, the amino acid positions of the S protein
sequence provided for each strain are listed in column 2 of
Table~ Amino acid 1 refers~to the first amino acid of
the S protein. Those proteins having 1454 or (for canine
i. CCV,~ il452) aminQ ac`ids are full-length S proteins. The
~30 ~ remaining proteins aré partial S proteins.
, ~
~ ~
'
' ' '':
"'"
:;
::
W093/2~22 PCT/US93/043~
.. , ~
213 i~9~ ~
9 ~ `
TABLE I
S Protein/Gene
Contributing SEQ ID NOS. ~
Strains AA AA Nucleotide
S , -- _
FECV 1-14 54 12 11 :
FIPV DF2 1--14 542
FIPV DF2-HP 1-7 4 8 4 3 ` ,.
FIPV TS-BP 1-7 4 8 6 5
FIPV TS 1-1454 8 7
FIPV TN406 102-223 10 9 "~:
FIPV UCD-2 1-125 14 13
Consensus sequence 1-1454 16 15 ~
Canine CV 1-1452 18 17 .
The inventors have defined several immunogenic sites
on the DF2 FIPV S protein for use in the chimeric proteins
of this invention and have identified corresponding regions
in other related coronaviruses which are anticipated to be
usef~l in the same manner. The following information,
including the sequence listing and tables herein, provide -
predicted immunogenic regions of the coronaviruses spike
genes illustrated herein. One of skill in the art,
provided with this disclosure and the predicted regions,
could readily select suitable immunogenic regions for use
in the chimeric proteins of the invention from any
desirable coronavirus.
For ~ase in reading, the fragments based on amino acid
- position and the associated SEQ ID NOS from the S proteins
from which they are derived are set out in Table II for B
cell site and T cell site fragments.
A. B Cell Sites
Because prior exposure to FIPV exacerbates the
disease, epitopes on the S protein which induce a B cell or
humoral raspon~e are believed to be involved in virus- -
3S induced pathogenicity. The majority of conserved amino
acid residues are located in the carboxy terminal two-
thirds of the S protein of the coronaviruses discussed
W093/2~22 2 I ~ 1 S 9 ~ PCT/US93/~3~
.
specifi~ally herein, and illustrated in Table II and the
attached sequence listing, e.g. from about amino acid
position 350 to about 450, and from about position 650 to
about 1454. Heterogeneity is largely confined to the N-
terminal residues, about the first 350 amino acids of thecoronaviruses (see Table II). Another moderately
heterologous region is located between about amino acid
positions 450 to about 650. The sensitizing regions on the
coronavirus S gene for feline as well as other species
coronaviruses are predicted to lie within the regions of
antigenicity mapped between avirulent FECV and virulent
FIPV. If antibodies to such regions are found to be
protective, these regions can be useful in vaccine
compositions.
15One antigenic region is a major neutralizing site in
the area of amino acid residues ~525-650 of DF2 FIPV. This
- sequence and its analogous sequences on FECV, FIPV ~DF2-HP,
TS, and TS-BP), CCV and other coronaviruses is a desirable
S protein fragment for use in a chimeric protein of this
invention.
~- ~ Another major antigenic site was mapped to amino acids
-~#350-550 and #1170-1190 of DF2 FIPV. Again, FIPV tDF2-HP,
TS, and TS-BP3, FECV and other coronaviruses are believed
to contain an antigenic site in these regions. Thus, these
--25sequences are anothèr desirable region for an S protein ~`
fragment for use in a chimeric protein of this invention.
In addition, other suitable fragments of a feline,
canine or a related coronavirus may be used in a chimeric
molecule of the invention. Because these sites are
antigenic and were mapped by monoclonal antibodies, they
are suspected to be B cell sites.
:,
W093/2~2 2 ~ 3 4 ~ 9 ~ PCT/US93/043~
While not wishing to be bound by theory, the inventors
believe that antibodies to all or some of these sites may
contribute to the immune sensitization characteristic of
FIPV infection. By analogy with TGEV, only some of the
antigenic sites on FIPY S induce the formation of virus
neutralizing antibodies. Since virus neutralization (VN)
is often important to controlling virus replication, ;
induction of VN antibodies may he important for clearing
infecting virus and may not contribute to sensitization.
Antibodies to the non-neutralizing antigenic epitopes,
then, may be responsible for exacerbation of disease. Any
sites, neutralizing or non-neutralizing, which contribute
to sensitization are desirable regions for deletion from a
chimeric S gene of the invention. ~-
B. T Cell Sites ; ~;
Certain chimeric proteins of the present invention can
also contain epitopes which do not stimulate antibody
production but rather induce T cell immunity, i.e., T cell
sites. The majority of the T cell sites predi~ted by the
inventors and identified in Table II below lie within the
C-terminus of the surface glycoprotein. The other
coronaviruses are expected to haYe T cell sites in
approximately these regions.
One of skill in the art may select other S fragments
-25 suitable for use in chimeric S proteins of this invention
using the information provided in the present disclosure
and conventional computèr programs which predict T cell
sites on a protein using several parameters. Suitable
! ' exemplary programs include EpiPlot, (Louis Menendez-Arias,
Uni~ersity o~ Madrid), the GCG Program, and a T Cell
predictive program available from ~obert ~ew (University of
Massachusettsj.
W093/2~22 ~1^3~ 8 PCT/US93/~3~
.
.
12
As can be seen from the attached Sequence Listing with
reference to Table II, in the listed S protein T cell
sites, DF2-HP sequences are identical to DF2 insofar as
DF2-HP has been sequenced; and TS-BP sequences are
identical to TS in~ofar as TS-BP has ~een sequenced. With
respect to all the strains (with the noted exception of
TN406 below) the amino acid positions provided correlate
identically to the S protein and the sequence listing
number. With respect to TN406, tXe S protein fragment .
reported in SEQ ID N0: 10 actually spans amino acids 102-
223 of the S protein. However, in SEQ ID N0: 10 those
amiho acid positions are reported as 1-122. Therefore,
the fragment identified as amino acid 38-50 of SEQ ID N0~
corresponds to amino acid 139-151 of the TN406 S
protein. The fragment identified as amino acid 59-72 of.
SEQ ID N0: 10 corresponds to amino acid 160-173 of the
TN4Q6:S protein. ~`
~i ' '' ' ;;`
~; :' ,.:
.
''-
',~
W093/2~22 PCT/US93/043~ :
' :' 213 l898 '~:
TABLE I I ::
Virus/ Amino Acid Fraqments
Strain B Cell Sites T Cell Sites
5 SEQ ID N0: 12
- FECV 1 - 35 Q
44 -57
96-144
139-151 ;
160-173 -
344-386
350-450
350-550 .
377-386
lS 378-390 -
400-650
450-650
525-650
542-597
650-1454 ~-
1170-1190
1344-1404 ~.
1409-1418 -:
1426-1438
77-89
408 -427
482-496
9Z2-934
1133-1147
1308-1322
1368 -1391
1379-1391
SEQ ID NO: 2
FIPV/DF2 1-350
44-57
g 6-144
139-151
160-173
344-386
350 4 S 0
350-5S0
s 377 - 386
378-390 :
400-650
450-650
525~65
542-597
6S0-1454
W093~2~22 2 1 ~ 1 8 9 8 PCT/US93/~3~
i; . ~ . .
14
TAB~E II (cont ' d) .:
Virus/ Amino Acid Fraqment .
Strain B Cell Sites T Cell Sites
.
1170-1190
1344-1404
1409-1418
1426-1438
77-89 ~:
40~-427 ~:~
482-496 ;
: 922-934
1133-1147 . :
~308-1322
~ 1368-1391
1379 - 1391 - ~
',
SEQ ID NO: 4
FIPV/
DF2-HP~ 3~5:0
20~ 4~4 - 5:7
96-~144
39~- l51 ~
160 - 173 ;; :
: 344-386
;- 2-5 ~ 350 - :450 j ;~
3:5~0 - 550 ,
: 3~77-3.8:6;:
378-~3g0
0~ 450:-~650
5-6597~
408~- 427 .`
3 g~ 4;8 2 4 9 6 r
:SEQ ID N0: 8 ;~
~ FI~ITS -350
; ~- 96-144
~40 ` ~ ` ~ 139--151
344-386~; ~
350~550 i`
4S ~ ` ~ 3~77-386
3 7 8 -3 9 0
~ .
W093/2~22 ~ 13~898 PCT/US93/~3~
;.
TABLE II (cont'd)
Virus/ Amino Acid Fraqment
Strain B Cell Sites T Cell Sites
400-650 ::-
450-650
525-650
542-597
6S0-1454 -:
1170-1190
1344-1404
1409-141
1426-1438
77-89
4Q8-427
4~2-496
922-g34
1133-1147
1308-1322
1368-1391 -
1379-1391
SEQ ID NO: 6
FIPV/
TS-BP 1-350
44-57 .:
2S 139-151 ::
160-173 ~:
344-386
- ~ 350-450
: 350-550
377-386
378-390
: 400-650 .
450-650
525-650
S42-597
77-89
408-427
482-496
~ . I i
SEQ ID NO: 10
-40 FIPV/
TN406 38-50
W093/2~22 PCT/US93/~3~
2 ~ 3 1 g ~
16
TABLE II (cont'd~
Virus/Amino Acid Fraqment ~ '
Strain B Cell Sites T Cell Sites
SEQ ID NO: 14 ~
S FIPV/ . i:
UCD-2 44-57
77-89 :::
SEQ ID N0: 18 `:::
CCV/1-71 1-350
50-250
350-450 ::
375-42S
450-470 :`
450-6S0 :.
525-650 .~:
550-600 .
650-700 :
65~-1452 -
770-850 .
900-1~25 - ~-
-: 1150-1225 ..
1250-1452
_47 :~
~ 63-81
187-lgl :
241-274
335-341 :;
: 395-428
: 468-494
8460860 ;
916-952
977-~92
1068-1145 -:
1366-1391
-. .
I , ! , ~ ~
"'
,: '~` '
WO 93/23422 PC~/US93/043~4
"-` 213~9~
C. Other Immunogenlc Fragments of the Coronavirus S
Gene Protein
As discussed above, additional B and T cell sites can
be predicted based upon analogy to the sequences herein.
For example, these fragments may be selected from
previously amplified regions of feline coronaviruses. The
amplification procedure and suitable fragments are
described in more detail in co-owned, co-pending U.S.
Patent Application Ser. No. 07/698,927, and its
corresponding published International Application No.
W092/08487, published May 29, 1992 which is incorporated by
reference herein.
Additional exemplary fragments useful in making
chimeric proteins of the invention include those fragments
identified in the examples, and other fragments, all of
which are set forth in Table III below. Fragments similar
to the fragments of Table III can be identified in other
related coronaviruses.
W093/~22 P~T/US93/~3~
21~ 1898 i~
18 ~: ~
~,.
TABL~ III
Virus Strain Amino Acid Fra~ment
FECV 1-138 :
1-352 -
1-542 ~
213-555 -
352-748 . ~
352-1454 .
387-1454 '
598-1454 :-
1-311 ;
311-1454 ~
1115-1238
872-1454 ' ~
311-872 ~:
1 - 872
894-1040 ~;
1029-1454
894-1203 ~
`:
FIPV DF2 1-352
. ~ 352-399 ~:
352 -1454
: 594-1454 :~`
651-1454
: 25 ~ 311
~ : 311-1454 ':`-
:~ -- 894 - 1454
~- ~ 1115-lZ38 -;;
~ 872-1454
- 30 3~1 - 872 i;
1 - 872
894-1040 .
- 10~29 - 1454
894-1203
i .,.
FIPV DF2 - HP 1-352
213-555 ~ `.
,~ ; i j ~ 352 - 399 '```
1-311
':
.:
. ..
W093/2~22 2 1 3 ~ 8 9 8 PCT/US93/043~
; ;:;
TABLE III - (cont'd)
Virus Strain Amino Acid Fraqment
FIPV TS 1-352
352-399
352-1454
594-1454
651-1454
1-311
311-1454
1115-1238
872-1454
311-872
894-1040
1029-1454
894-1203
1-105
94-223
213-362
352-748
737-1040
894-1040
1029-1454
FIPV TS-BP 1-352
213-555
352-399
1-311
CCV 1-71 1-343
152-376
985-1108
1-1343
1405-1452
1115-1~38 .
1-35~
1344-1404
352-1452
. .
W093/2~2 213 ~ PCT/US~3/043~
;
III. Chimeric Proteins of the Invention ;
The chimeric proteins of the invention, as defined
above, are capable of eliciting an enhanced immune response
against one coronavirus strain and/or an immune response to
more than one coronavirus strain or species. These
chimeric proteins are also believed to be capable of
eliciting an immune response in more than one animal
species.
Presently, in the production of a chimeric protein of
1~ this invention, it is desirable that the chimeric protein ~;
be a full length S protein. However, the chimeric proteins
of this invention may be as small as about 16 amino acids
in length, or may be as large as at least 2 full length S
proteins. It is anticipated that useful chimeric proteins -;
will be within the above stated lower and upper amino acid
lengths. i~j
One embodiment of a chimeric protein can comprise one ;;
or more protein or peptide segments of FIPV S protein fused j;~
to one or more protein or peptide segments of FECV S ~a~
protein to form a full length, chimeric S protein. See
Tables I, II or III ~or relevant SEQ ID NOS. One specific
chimeric protein is formed by the fùsion of an S gene
protein fragment from amino acid #1-311 of a selected
strain of FIPV S protein to the carboxy terminal amino ;~
-25 acids #311-1454 of FECV S protein. An~ther such chimeric
protein is formed by the fusion of the N terminal fragment
of the ~ECV S protein (from about amino acid ~1-311) to the
carboxy terminal protein sequence (amino acid ~311-1454) of
a selected FIPV strain. In such fusions, amino acid 3li is
present only once (iOe., the total chimeric is 1454 amino
acids in length). These chimeric constructs are
illustrated in Example 3 below.
W093/2~22 PC~/US93/043~
9 8
In another alternative, the chimeric proteins of the ~;
invention consist of a fragment of the FECV S protein
inserted into the FIPV S protein to replace a homologous
fragment thereof, so that both the amino and carboxy
S terminal regions are FIPV S protein fragments. In still
another alternative, the reverse construct can be made, so
that both the amino and carboxy terminal S protein
fragments are FECV S gene protein fragments. Such chimeric
constructs are illustrated in Example 4 below.
Another currently preferred embodiment of the chimeric
protein of the invention comprises the fusion of desirable
B and T cell sites, as identified above. Particularly
suitable regions of a feline or canine coronavirus include
the B cell sites provided in Table II above. Thus, ~an
exemplary chimeric S gene may comprise FECV amino acids 1-
542 fused to a selected FIPV strain amino acids 542-597,
fused to FECV 598-1454. As above, the chimeric is 1454
amino acids in length.
Other chimerics may be constructed by inserting one or
more of the above-identified amino acid regions from one
coronavirus into the corresponding region of another
coronavirus. Other chimeric S proteins of the invention
can be made using any of the feline coronavirus strains
identified herein or by substituting any of the other
- 5 species coronaviruses for either coronavirus S protein
fragment of the constructs described above.
In still another embodiment, a chimeric S protein of
the invention may comprise a selected strain of FIPV amino
! ' acids 1-311 fused to one or more of the following T cell
amino acid regions identified in Table II. Because these
T cell regions are derived largely from the conserved C
terminus, it is expected that a strong cross-species T cell
response will be elicitedl particularly when the T cell
W093/2~22 ~ PCT/US93/~3~
,
site is fused to a B cell site. Thus, it is expected that
even if these B and T cell sites are from ~he same
coronavirus strain, the chimeric will elicit a cross~
species response.
s Other ~himerics of the invention which comprise less
than full length S proteins are those containing a deletion
corresponding to one or more B cell sites, sensitizing
regions, or other non-critical fragments of the S protein
identified herein. Alternatively, a chimeric S protein of
the invention may be an S protein which has been truncated
at the amino or carboxyl termini~ provided that the
immunogenicity of the S gene protein is not lost. For
example, it may be desirable to remove the transmembrane
domain of the S gene in order to facilitate secretion of
the protein into the extracellular medium and to facilitate
purification. Examples of these chimerics include:
FECV amino acids 1-542 fuæed to FIPV 594-1454 (e.g.,
D~2 or TS), resulting in an S protein having a deletion in
the region of amino acid residues 542-597;
FECV amino acids 1-138 fused to CCV amino acids 152-
376, fused to FECV amino acids 387-1454, resulting in an S
protein having a deletion in the regions of 139-151 and
377-386;
CCV amino acids 1-1343 fused to amino acids 1405-1452,
resulting in an S protein having a deletion in the regions
of 1344-1404; and
FIPV amino acids 352-399 fused to FIPV amino acids
651-1452, resulting in an S protein having a deletion in
~ the regions of~amino acids 1-350 and 400-550.
Currently, preferred chimeric corona~irus S proteins
are provided by the fusion of FIPV (Type I, Type II or
both) and FECV S protein fragments for use in compositions
to be administered to felines. Another composition which
is anticipated to be useful is a chimeric feline/canine
,.:
!: ~ I 3 1 8 9 8 PCT/US93/~3~
coronavirus S protein. Such a chimeric may comprise FIPV
and/or FECV proteins fused in frame to CCV protein(s).
Other chimerics which may be useful in preventing disease
in animals, and particularly in more than one animal
(cross-species protection), include fusions of S protein
fragments from FIPV/TGEV, FECV/TGEV, FECV/CCV, FIPV/FECV/
TGEV, FIPV/T~EV/PRCV, ACV/TGEV, PRCV/ACV/TGEV, or BCV/ACV/
TGEV. However, in view of the~teachings of the present
invention, one of skill in the art could construct other
suitable chimeric coronavirus S proteins for desired
therapeutic or vaccinal uses as described below.
~ In another example of the invention, the chimeric S
proteins of the invention may be fused to a fusion partner, ~r
e.g, a larger, carrier molecule. The fusion partner may be
a preferred signal sequence, a sequence which is
characterized by enhanced secretion in a selected host cell -~
system, or a sequence which enhances the stability of the
S-derived~peptide. Such~exemplary fusion partners include,
without ~limitation, ubiquitin and ~- mating factor for
yeast expression systems, and ~-galactosidase and influenza
NS-1 protein for bacterial systems. One of skill in the
art can readily select an appropriate fusion partner. The
present invention is ~ not limited: to the use of any
particular~fus~ion~;partner.~
25 ~ Any~of the~above-identified S protein fragments which
--~ can be useful~in a chimeric protein may optionally be fused
to;~each other or to a fusion partner through a conventional
linker sequence, i.e., containing about 1 to 50 amino
acids, and morre preferably, about 2 to about 20 amino acids
in length. This optional linker may provide space between
~;~ the two linked sequences. Alternatively, this linker
- sequence may encode, if desired, a polypeptide which is
~ selectively cleavable or digestible by conventional
W093/2~22 2 1 3 `1 8 9 ~ PC~/US93/~3~
. ;,
:
24
chemical or enzymatic methods. For example, the selected
cleavage site may be an enzymatic cleavage site, including
sites for cleavage by a proteolytic enzyme, such as
enterokinase, factor Xa, tr~psin, collagenase and thrombin.
s Alternatively, the cleavage site in the linker may be a
site capable of being cleaved upon exposure to a selected
chemical, e.g., cyanogen bromide or hydroxylamine. The
cleavage site, if inserted into a linker useful in the
fused sequences of this invention, does not limit this
invention. Any desired cleavage site, of which many are
known in the art, may be used for this purpose.
lV. Modîfied Amino Acid and Nucleotide Sequences
In addition to the amino acid sequences and
corresponding nucleotide sequences of the specifically-
recited embodiments of chimeric proteins of this inventionwhich are described herein and il}ustrated in the attached
sequence listing, the invention also encompasses other DNA
and amino acid sequences of the chimeric proteins of this
invention. Such other nucleic acid sequences include those
sequences capable of hybridizing to the relevant S gene
fragments disclosed herein under conditions of at least 85%
stringency, i.e. having at least 85%- homology to the
selected S gene fragment encoding a portion of the chimeric
protein, more~preferably at least 90% homology, and most
~5 preferably at least 95~ homology. For example, allelic
variations (naturally-occurring base changes in the species
population which may or may not result in an amino acid
change) of DNA sequences encoding the various S gene amino
! ' acid or DNA sequ~nces from the illustrated coronaviruses
are also included in the present invention, as well as
analogs or derivatives thereof.
W093/2~22 ~ 1 3 ~ ~ 9 8 PCT/US93/~384
Similarly, DNA sequences which code for protein
sequences of the invention but which differ in codon
sequence due to the degeneracies of the genetic code or
variations in the DNA sequence encoding these proteins
which are caused by point mutations or by induced
modifications to enhance the activity, half-life or
production of the peptide encoded thereby are also
encompassed in the invention.
Variations in the amino acid sequences of this
invention may typically include analogs that differ by only
1 to about 4 codon changes at the nucleotide level. Other
examples of analogs include polypeptides with minor amino
acid variations from the natural amino acid sequence of S -
gene proteins and/or the fusion partner; in particular,
conservative amino acid replacements. Conservative
replacements are those that take place within a family of
amino acids that are related in their side chains.
Genetically encoded amino acids are generally divided into
four families: (1) acidic = aspartate, glutamate; (2) basic
~-20 ~ ~ = lysine, arginine, histidine; (3) non-polar = alanine,
valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan; and (4) uncharged polar = glycine,
asparagine, glutamine, cysteine, serine, threonine,
tyrosine. Phenylalanine, tryptophan, and tyrosine are
sometimes clas5ified jointly as aromatic amino acids. For
~ .
example, it is reasonable to expect that an isolated
replacement of a leucine with an isoleucine or valine, an
aspartate with a glutamate, a threonine with a serine, or
a similar conser~ative replacement of an amino acid with a
structurally related amino acid will not have a significant
effect on its activity, especially if the replacement does
not involve an amino acid at an epitope of the polypeptides
of this in~ention.
W093/2~22 PCT/US93/04~
2~3 1~9~
26
V. Methods of Producing Chimeric Proteins and Fragments
The chimeric proteins or coronavirus fragments of the
invention can be prepared by chemical synthesis techniques.
Preferably, however, they are prepared by using known
recombinant DNA techniques by cloning and expressing within
a host microorganism or cell a DNA fragment carrying a
coding sequence for the selected chimeric protein. Coding
sequences for the S gene protein fragments and other viral
proteins, e.g., fusion partners, of the coronaviruses can
~e prepared synthetically or can be derived from viral RN~
by known techniques, or from available cDNA-containing
plasmids.
Systems for cloning and expressing the vaccinal
polypeptide in various viral vectors, microorganisms and
cells, are known and available from private and public
laboratories and depositories and from commercial vendors.
Currently, the most preferred expression systems include
those involving ~iral vectors, particularly poxvirus
vectors, such as vaccinia or swinepox. Also useful are
feline herpes virus vectors ~See, e.g. R. Wardley et al, ~.
Gen. Virol., 73(7):1811-1818 (1992) and J. Nunberg et al,
Vaccines, 91:191-196 ~1991)3. Another preferred expression
system includes mammalian cells, such as Chinese Hamster
ovary cells ~CHO) or COS-l cells. Another desirable
expression system includes insect cell systems, such as the
baculovirus expression system. The selection of other
suitable host cells and methods for transformation,
culture, amplification, screening and product production
and purification can be performed by one of skill in the
art by reference to known techniques. See, e.g., Gething
and Sambrook, Nature, 293:620-625(1981).
When produced by conventional recombinant means, the
chimeric proteins may be isolated either from the cellular
contents by conventional lysis techniques or from cell
medium by conventional methods, such as chromatography.
W093~2~22 PCT/~S93/043~
2131g98
27 `
See, e.g., Sambrook et al, Molecular Cloninq. A LaboratorY
Manual., 2d Edit., Cold Spring Harbor Laboratory, New York
(1989).
The resulting chimeric proteins may be screened for
efficacy as ~ vaccine or therapeutic agent in an animal
model system involving challenging animals immunized with
varcinia recombinants expressing a selected coronavirus'
gene products with the virulent FIPV strain [Vennema et al,
J. Virol., 64:1407-1409 ~199Oj].
VI. Vaccinal Compositions
The present invention provides vaccine compositions
containing an immunogenic amount of a chimeric protein
together with a carrier suitable for administration as a
composition for prophylactic treatment of coronavirus
lS infections. The protein of the invention can be employed
alone, or in a vaccine composition containing additional
antigens, 2 . g . other chimeric or recombinant coronavirus S
gene proteins of the invention, other proteins from the
~applicable coronavirus, or other proteins or peptides from
other pathogens.
Alternatively, the selected coronavirus chimeric S
gene or fragment may be incorporated into a live vector,
e.g., adeno~irus, vaccinia virus and the like. The
expression o~ ~accinal proteins in such live ~ectors are
~-25 ~ well-known to those in the art [See, e.g., U. S. Patent No. -~
4,920,209]. See, also, Examples 7 through 9 below. `
For example, a vaccine composition containing a
FIPV/FECV chimeric S protein may be formulated to further
contain the temperature sensitive FIPV`vaccine dèscribedlin
detail in co-owned, co-pending U.S. Patent Application Ser.
No. 07/428,796 filed October 30, 1989, incorporated by
reference herein. Alternati~ely, such a vaccine may also
contain, e.g. antigens directed against feline leukemia.
wo 93,?~22 ~ i3 ~ PCT/US93f~3~
28
Other suitable feline antigens include rabies, feline
calici ~irus, chlamydia, feline immunodeficiency virus,
feline parvovirus and feline rhinotracheitis. For
chimerics which will be used to vaccinate pigs, other
suitable antigens include, for example, pseudorabies,
porcine parvovirus, and bacterial antigens. For chimerics
which will be used to vaccinate dogs, other suitable
antigens include, among others, rabies, canine distemper,
Borrelia burgdorferi, canine Bordetella, canine parvovirus,
canine rotavirus, canine parainfluenza, canine adenovirus,
and Leptospira species.
The preparation of a pharmaceutically acceptable
vaccine composition, having appropriate pH isotonicity,
stability and other conventional characteristics is within
the skill of the art. Thus such ~accines may optimally
contain other conventional components, such as adjuvants
and/or carriers, e.g. aqueous suspensions of aluminum and
magnesium hydroxides, liposomes and the like.
The vaccine composition containing a coronavirus
2 0 chimeric S protein according to the invention can be
employed to vaccinate naive animals (i.e., animals not
preYiously exposed to the coronavirus) against at least the
c}inical symptoms associated with the coronaviruses from
which the chimeric protein was derived. For example, an
2S FIPV/CCV chimeri~ is useful in vaccinating against
infection with FIP or canine coronavirus. However, it is
anticipated that the chimerics of the invention will also
provide cross-species protection against corona~irus
infection. The vaccines according to the present invention
can be administered by an appropriate route, e.g., by the
oral, intranasal, subcutaneous, intraperitoneal or
intramuscular routes. The presently preferred methods of
administration are the subcutaneous and intranasal routes.
wo g3/2~22 ~ 1 3 4 ~ 9 8 PCT/US93/~3~
The amount of the chimeric coronavirus S protein of
the invention present in each vaccine dose is selected wlth
regard to consideration of the animal's age, weight, sex,
general physical condition and the like. The amount
S required to induce an immunoprotective response in the
animal without significant adverse side effects may Yary
depending upon the recombinant protein employed as
immunogen and the optional presence of an adjuvant.
Generally, it is expected that each dose will comprise
0.05-5000 micrograms of protein per mL, and preferably 0.1-
100 micrograms per mL of a sterile solution of an
immunogenic amount of a protein or peptide of this
invention. Initial doses may be optionally followed by
repeated boosts, where desirable. -
lS The vaccine compositions of the invention may be used
~to protect a naive animal against coronavirus infection.
Desirably, these vaccine compositions are expected to be -
capable of protecting ~ats against infection with one or
- more coronaviruses. Further, these vaccine compositions
are expected to be able to protect against coronavirus-
related disease in other species, particularly dogs and
pigs.
Another vaccine agent of the present invention is an
~`~ anti-sense RNA sequence generated to a sequence of a
-~Z5 ~ chimeric protein. This sequence may easily be generated by
one of skill in the art either synthetically or
recombinantly. Suitable techniques are known ~See, e.g. S.
Crooke, Biotechnoloqv, 10:882-886 (Aug. 1992)]. Under
appropriate delivery, such an anti-sense RNA sequence whe~
administered to an infected animal should be capable of
binding to the RNA of a selected coronavirus, thereby
preventing viral- replication in the cell.
- ','',.
.. .
:.
a
WO 93/234~2 PCr/US93/04384
!
VII. Pha~maceutical Compositions
The invention also provides a pharmaceutic~al
composition useful in the treatment of animals infected
with a coronavirus comprising the chimeric coronavirus S
proteins prepared according to the present invention and a
pharmaceutically effective carrier. Alternatively or
additionally, these pharmaceutical compositions may contain
a recombinant S gene protein as defined herein, or as
described in co-pending U.S. application Ser. No.
07/698,927, and its corresponding published PCT
application, WO 9208487.
Suitable pharmaceutically effective carriers for
internal administration are known to those skilled in the
art. One selected carrier is sterile saline. Other anti-
viral agents can also be employed in compositions for the
treatment of coronavirus infection. The pharmaceutical
composition can be adapted for administration by any
appropriate route, but is designed preferentially for
administration by injection or intranasal administration.
VIII. Antibodies of the Invention
The present invention provides antibodies to a
selected chimeric S protein or S protein fragment. These
antibody may be generated using conventional techniques for
production of monoclonal ~W. D. Huse et al, Science,
~25 246:1275-1281 (1989); Kohler and MiIstein] or polyclonal
antibodies.
For diagnostic purposes, the antibodies may be
associated with individual labels, and where more than one
~ antibody is employed ln a diagnostic method, the labels are
desirably interactive to produce a detectable signal. Most
desirably, the label is detectable ~isually, e.g.
colorimetrically. Detectable labels for attachment to
antibodies useful in the diagnostic assays of this
W093/2~22 2 1 3 ~ 8 ~ 8 PCT/USg3/~
invention may also be easily selected by one skilled in the
art of diagnostic assays. Labels detectable visually are
preferred for use in clinical applications due to the
rapidity of the signal and its easy readability. For
S c~lorime~ric detection, a variety of enzy~e systems have
been described in the art which will operate appropriately.
Colorimetric enzyme systems include, e.g., horseradish
peroxidase (HRP) or alkaline phosphatase (AP). Other
proximal enzyme systems are known to those of skill in the
art, including hexokinase in conjunction with glucose-6-
phosphate dehydrogenase. Also, bialuminescence or
chemiluminescence can be detected ùsing, respectively, NAD
oxidoreductase with luciferase and substrates NADH and FMN
or peroxidase with luminol and su~strate peroxide. Other
~;~;lS conventional label systems that may be employed include
fluorescent compounds, radioactive compounds or elements,
or i~munoelectrodes. These and other appropriate label
systems~ and~methods for coupling them to antibodies or
peptides are known to those of skill in the art.
Antibodies may also be used therapeutically as
~ targeting agents to deli~er ~irus-toxic or infected cell-
S~ toxic agents ~to infected cells. Rather than being
associated with labels for diagnostic uses, a therapeutic
agent can employ the antibody linked to an agent or ligand
-^ ~5 capable of disa~ling the replicating mechanism of the virus
-~ or~of destroying the virally-infected cell. The identity
- ~ of the toxic ligand~does not limit the present in~ention.
It is expected that preferred antibodies to peptides
~ encoded by the~chimeric S proteins identified herein can be
- ~30 screened for the ability to internalize into the infected
-`~ cell and deliver the ligand into the cell.
,,
'-
: ~
WO 93/23422 2 i 3 1 ~i ~ 8 PCr/US93/043 ~
32
IX. Diagnostic Rit
The present invention also pro~ides a diagnostic ~it
which enables distinction between native coronavirus
exposure and animals vaccinated with the chimeric or
recombinant moIecules of the inventicn. Such a kit may
contain a sufficient amount~ of at least one chimeric S
protein or at least one recombinant S protein of the
invention and such components as are necessary to practice
the assay. It is anticipated that of the chimeric S
proteins, those comprising less than a full length S
protein and having a deletion of one or more epitopes will
~be particularly useful in such a kit. Such assays are
conventional, and the necessary reagents and other
compohents of such a kit are well known to those of s~ill
-~15~ ~ in~the art.
Such a kit, and other diagnostic formats using a
chimeric S protein or recombinant S protein fragment of the
invent~ion,~are useful for distinguishing between animals
v w oinated~with a chimeric protein of the invention and an
`~20 ~ ~animal which~has been naturally exposed to the coronavirus
or coronaviruses which comprise the chimeric S protein.
The~following examples illustrate preferred methods
for~preparing~chimeric molecules of the invention. These
~ examples~are~ strative only and do not limit the scope
-~25~ of~the~invention.~ For instance, although these examples
uti1ize ~FECV~ another av~irulent or non-sensitizing
coronavirus, i.e. FIPV strain UCD-2, PRCV, BCV, or ACV,
could be used in its place in these chimerics.
Alternatively, coronaviruses such as CCV or TGEV can be
,,
~30 used in these chimerics. In addition, Type I FIPV
sequences (i.e., TN406) may be substituted for WT DF2 FIPV
~ (Type II)~ to expand cross-strain protection.
,~ ~
: : :
W093/2~22 PCT/US93/~3~
: ;~ 213~$9~
Exam~le 1 - PCR AmPlification
A. Source of Cells and Viruses
Norden Laboratories Feline Kidney (NLFK) [Christianson
et al, Arch. Virol., 109:185-196 (1989)] cells were grown
in Basal Medium Eagle (BME) supple~ented with 5% fetal
bovine serum (FBS) and 10 mM Hepes. For vaccinia virus
infections, human thymidine-kinase-negative (HuTK-) [ATCC
CRL 8303] and African green monkey kidney (CV-l) cell lines
~ATCC CCL 70] were used and maintained in Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal bovine
serum.
The DF2 wildtype FIPV vir-~s was originally isolated at
SBAH, Lincoln, from a cat liver explant. After several
passages of tissue homogenates in specific pathogen free
(SPF) cats, the virus was adapted to NLFK cells by
coculti~ation with infected primary spleens and later
plaque-purified. A feline enteric coronavirus, FECV (WSU-
1683), was obtained from Washington State University.
Vaccinia virus strain WR was re~eived from Dr. Bernard
,
~20 ~Moss, NIH.
B. Oligonucleotide Design and synthesis
1. Primers for Cloning AA 1-352/AA 352-1454
Chimeric Proteins
The DF2 FIPV spike gene sequence contained a PstI site
at amino~acid 352 while the FECV and CCV spike genes did
;~ not. Oligonucleotide primers were specifically designed to
allow PCR amplification of FECV and CCV spike protein amino
acid regions 1-352 and 352-1454. PCR primers were 30-40
'~ base pairs ini length and included a SmaI site in the
upstream (5') primer and a ~stI site in the downstream t3')
primer (1-352 amino acids) and a ~stI in the upstream
primer and a StuI in the downstream primer t3S2-1454 amino
- ~ acids). These sites were incorporated into the primers to
allow the reconstruction of full-length chimeric FECV/FIPV
S genes through ligation of the PstI sites.
W093/2~22 PCT/US93/~3~
2 i 3 1 ~ 9 8
34
Oligonucleotide primers were synthesized on an Applied
Biosystem Model 380B DNA Synthesizer using the phosphor-
amidite method. The primers were gel-purified prior to
use. The primers used are as follows.
The top/left strand PCR primer specific for
amplification of full length FECV S contains sequence for
amino acids 1-9 fused to the recognition site for XmaI.
This first 6 bp are a restriction site and are nan-specific
sequence. The seventh bp is a spacer to ensure that the
primer is correctly translated. The FECV specific sequence
starts at the 8th bp position of the primer, which is the
following SEQ ID N0: 19
5'-CCCGGGCATG ATTGTGCTCG TAACTTGCC TCTTGTTGTTA TGC.
Also a top/left strand PCR primer specific for FECV
contains additional stabilizing sequen~e (GTGC from the
published sequence upstream of the ATG) upstream of the
SmaI/XmaI site to help with digestion, which primer is SEQ
ID N0: 20
5'-GTGCCCCGGG CATGATTGTG CTCGTAACTT GCCTCTTGTT GTTATGC.
The following two primers are FECY Pst primers and are
- used to regenerate the PstI site in FECV and CCV whlch does
not result in an amino acid change when compared to the WT
WSU 1146 published strain. Thase primers contain the PstI
site in the middle and contain one FECV specific base pair.
The top strand primer is SEQ ID N0: 21:
5'- CAATCTTAAT TTCACTGCAG ATGTACAATC TGGTATGGGT GCT,
and is used with the StuI bottom primer. The bottom strand
primer is SEQ ID N0: 22:
1 5'- AGCACCCATA'CCAGATTGTA CATCTGCAGT GAAATTAAGA TTG,
and is used with the ~I top primer.
The following FECV Pst primers are also useful in
regenerating the PstI site in FECV and CCV which does not
result in an amino acid change when compared to the WSU
strain. The top strand primer containing the PstI site is
SEQ ID N0: 23: 5'-CTGCAGATGT ACAATCTGGT ATGGGTGCT, and is
W093/2~22 PCT/US93/~384
213 ~89~ -
3S
used with the StuI bottom primer. The bottom strand primer
containing the PstI site is SEQ ID NO: 24:
5 ' - CTGCAGT:;AA ATTAAGATTG AATCTA~TA,
and is used with the XmaI top primer.
2. Primers for Cloning AA 1-311/AA 311-872/AA
872-1454 Chimeric Proteins
Also useful is the following Table IV of primers which
were identified in the parent application (U. S. Ser. No.
07~698,927). The following Table IV primers were used to
PCR amplify the full length S gene from DF2 FIPV and FECV
~See Table I). The top strand primer for amplification of
full length DF2 FIPV S gene contained the nucleotide
sequence encoding amino acids 1-9 fused to the recognition
site for XmaI [SEQ ID N0: 25]. The top strand primer for
FECV S was SEQ ID N0: 19, identified above. The bottom
: ~ strand primer for full length F~PV S and for FECV S
contained sequence for amino acids 1450~1454 fused to the
recognition site for StuI for each S sequence ~ SEQ ID NO:
42~.
.
~ "
:
WO 93/23422 . PCl~/US93/04384
1 i `.
213 1~
36
TABL~ IV
S' (~equence ~mc pdDriq ~ pub~hcd WSU llUi, conlAiDs Xm~ u~c)
Po~ition ~n Po~ition (AA) Scaualco
5' Xm~13' Xm~
XIN
65-69n~96(stuv 1-9 GTGCCCCCGGGTATGATTGTGCTCGTAACTTGCCTCTTO
SEQ lD ~10:25 ~-rt codon
351-3551356-380 95-104 AATACCCGGC;GC~CTG~AATOCACGTGaT~ACC
SEQ ID NO:26
705 709nl0 733 213-219 CTATTcccGGGCACGCTCAAGcAcTacTAccTGGa
SPQ D~ NO:27
1121-1125/11261153 352-360 ~aATCCCGaGOTACAATCTGaTATG~3TGCT~CAa
SEQ D) NO:28
1698-1702/170a-1730 544-554 GCTTAC~CGaOaTG53TTATaaTCAACCCATAaCCTCaAC
SEIQ ID NO:29
2277-2281/7282-2309 737-746 TGToAcccaGGcGcc~TclToAToTAAacocAc:~Aacoac
SeQ ID NO:30
2749-z753m54-m9 894-903 aCJ~ATCCCGaGGa~TGCCAOACTTOAAAACATGOAGa
SEQ n~ NO:31
3155-315M160-3185 1030-1~138 CATrAC~C1~0Wl~GaC
SEQID NO:32
3642-36W3647-3674 ll92 l201 TA~a_l~QATTCW~TTCTOTOO
3 0 SeQ ID NO:33
- ~ ~ 3' (~equcoe4 re~cnc eolopluoeot Or publi~04~ WSU 1146, Cool-ioJ 8t~ C)
Podtion ~3n Po~itian ~M) ~ enco
S' Sn~l/3' SOJ I
3 5 385.381nt0-356 97-t05 ATAATAGGCCTGGmACCAC~GCATTACCAGTGC
SEQ lD NO:34
738.73:33-710 213-223 GTATr~GK~TCCX AOGT~aC;~CrrGK TTOACK:GnX3
SE~ nD NO:35
4 0 1155.1151/1150-1126 353-362 AiL~T~L~GGCC~C~raTACXCACCY;~TAC~CAaATTGTAC
SEU2nD NO:36
1735-t731/lt301t03S4~555 TrAaT~CK}CX3TaTCQAaaCTAlX3GK71TaACC~JL~CX AC
SEU2 DD NO:37
4 5 2314-231CU231CS'2282 739L748 TJ~ACA~aC4~CTaCCXXCrTGTCK CK rTACATCACATCK3~0
'~ SEQ m NO:38
27U-2780Q779-2754196-905 ATCA~AOGCCTCCTCC~Tal m C M OOCACCC
SBQ rD NO:39
3190-31t6/31t5-3160Iml-1040 aTAT~AaaccTaccAcoocoacAccAccAAaTocAcc
5 0 S~Q nD NO:40
3679~36tS/3~t4-3647 119~1203 C~TT~ ~ cAaAATcco~ATcTcrQAaAcTaAo
SE~2 DD NCk41
4433-4429/4428~4405(91op) 1444-1452 T~L~u~T~9y~ÇSa~TTACrraGACATCK~CIlrrTTC/J~TTG~
SEU2 nD NO:42 rcp codo~
W093t2~22 2 1 3 ~1 8 ~ 8 PCT/US93/043~
C. RNA Purification
Roller bottles of confluent NLFK cells were infected
with either FIPV or FECV virus using the following
protocol. The growth medium was removed and virus (MOI =
0.1~ was adsorbed in 50 ml of BME supplemented with 2% FBS.
FIPV infections were performed in serum-free medium. The
virus was absorbed for 2 hours and then 250 ml of growth
medium added. The cultures were monitored for cytopathic
ef~ect (CPE) and ~ypically harvested at 24-36 hours post-
infection. Total cytoplasmic ~NA was prepared from the
infected monolayers by guanidine isothiocyanate extraction
[Chirgwin et al, Biochem., 18:5294 (1979)].
D. Generation of PCR amplified fragments
All PCR reagents were produced by Perkin Elmer-Cetus
(Norwalk, CT). PCR amplified fragments were generated
using the following procedure. Synthesis of cDNA from
total RNA isolated from cells infected with a specific
coronavirus was performed for each virus~
In a final reaction volume of 20 ~l of lX PCR buffer
(lOX PCR buffer; 100 mM Tris-HCl, 500 mM ~Cl, 15 mM MgCl2,
0.01% (wlv) gelatin), the following components were
assembled in RNAse free siliconized 500 ~1 microcentrifuge
tubes: 1.0 mM of each dNTP, 20 units of RNAsin (Promega
Corporation, Madison, WI), 100 picomoles of random hexamer
oligonucleotides (Pharmacia, Milwaukee, WI), 100 picomolest
~1 solution in TE buffer (10 mM Tris-HC1, 1 mM E~TA, pH
7.5), 200 units of reverse transcriptase (Moloney MuLV,
Bethesda Research Labs, Gaithersburg, MD) and 1.0 ~g of
respective RNA isolated as described above. To avoid
pipetting errors and contamination, all solutions were
aliquoted from master mixes made with diethyl pyrocarbonate
(DE~C) treated water and consisted of all of the reaction
components except the RNA which was added last. The
mixture was incubated in a programmable thermal cycler
WO93J2~22 ~ 1 3 ~ 8 9 8 PCT/US ~
(Perkin-Elmer Cetus, Norwalk, CT) at 20C for ten minutes
followed by 42C for one hour then 95C for five minutes
and finally held at 4C until PCR amplification.
Amplification of the cDNA was performed essentially
according to the method of Saiki et al, Science, 230:1350-
1354 (1985) using the Taq polymerase. Briefly, to the 20
~l CDNA reaction mix from above was added: 8.0 ~l lOX PCR
buffer, 1.0 ~l of each upstream and downstream primer
previously diluted in water to 30 picomoles per microliter
and 5.0 units of Taq polymerase (Perkin-Elmer Cetus,
Norwalk, CT). Final volume was made up to 100 ~1 of
mineral oil. As above, master mixes were prepared to avoid
contamination. The reaction was performed in the Perkin-
Elmer Cetus thermal cycler for one cycle by denaturing at
95C for 1 minute, annealing at 37C for 2' followed by an
extension at 72C for 40 minutes. This initial cycle
increased the likelihood of first strand DNA synthesis. A
standard PCR profile was then performed by a 95C-l minute
denaturation, 37C-2 minutes annealing, 72C-3 minutes
~0 extension for 40 cycles. A final extension cycle was done
by 95C-1 minute denaturation, 37C-2 minutes annealing,
72C-15 minutes extension and held at 4C until analyzed.
E. Analysis of PCR Products
PCR products were analyzed by electrophoresiny 5.0 ~l
of the reaction on a 1.2% agarose gel run 16-17 hours.
Amplified fragments wexe purified prior to use for cloning
by column chromatography with the PrimeErase Quik method
(Stratagene, LaJolla, CA). Bands were visualized by
i ethidium bromide staining the gel and fluorescence by W
irradiation at 256 nm. Photography using Polaroid type 55
film provided a negative that could be digitized for sample
distance migration and comparison against markers run on
each gel. The actual sizes of the bands were then
calculated using the Beckman Microgenie software running on
an IBM AT.
W093/2~22 2 1 3 ~ ~ 9 8 PCT/US93/~3~
39
ExamPle 2 - Cloninq of DF2 FIPV and FECV S Proteins
For use in the challenge model, a full length wild-
type (WT) DF2 FIPV S protein was employed as a positlve
control for sensitization; and a full length FECV S protein
was also tested.
Full length DF2 FIPV and FECV S genes were cloned in
vaccinia vector pSC11 [Chakrabarti et al, Mol. and Cell
Biol., 5:3403-3409 (1985)], as follows.
Pa~ pSCll
Cloning procedures were as described by Sambrook et
al, cited above (1989) and all plasmids were transformed
into the ~. coli host strain, HB101. pSCll is a vector
designed to allow expression of a cloned gene of interest
inserted into an unique SmaI site downstream of the p7.5
promoter of vaccinia virus. pSC11 contains vaccinia TK
gene~sequences for homologous recombination into the virus
and~the~ampici}lin gene for selection in E. coli . This
vector~also contains the pll vaccinia virus promoter
regulating the E. coli lacZ gene. Expression from this
promoter results in the synthesis of beta-galactosidase
protein. Recombinant viruses containing the pSC11 plasmid
will~therefore appear as blue plaques in the presence of X-
gal or Bluo-gal ~Sigma]. All clones are introduced into
the ~I site of this vector as blunt-ended fragments and
~- ~5 the resulting clones oriented with respect to the p7.5
-~ promoter.
.- ~ B . : pO TS~F3 3
Cloning procedures were as described by Sambrook et
al, 'cited above. The bacterial expression vector,
pOTSKF33, shown schematically in Figure l of the parent
application, is being maintained at SmithKline Beecham
Laboratories and is available to the public through the
,~
company.
:''
,
W093/2~2~ ~ l 3 4 8 9 ~ PCT/~S93/043~_~;
This plasmid is a derivative of pBR322 [Bethesda
Research Laboratories] and carries regulatory signals from
bacteriophage lambda. The system provides a promoter which
can be controlled ~PL), and an antitermination mechanism to
ensure efficient transcription across any gene insert, high
vector stability, antibiotic selection, and flexible sites
for insertion of any gene downstream of the regulatory
sequences. The pOTSKF33 vector also contains the coding
sequence for 52 amino acids of the enzyme galactokinase,
immediately adjacent to the ~PL promoter. The sequence of
this enzyme has been manipulated to permit insertion of
foreign genes and the construction of fusion proteins.
Linkers containing restriction sites for fusion in any
of the three reading frames, stop codons for each frame and
some additional cloning sites for fusion in any of th~
three reading frames, have been introdu~ed after the first
5Z amino acids of galactokinase.
T~anscription from the PL promoter is tightly
controlled by maintaining the plasmid in bacteria
expressing the cl+ repressor protein. Induction of foreign
protein expression is obtained by removing the repressor.
In the bacterial strains used in this study, the repressor
protein is temperature-sensitive. At the permissive
temperature, 32C, the repressor functions normally to
inhibit transcription from the PL regulatory sequences. An
increase in growth temp~rature (to 42C) results in
degradation of the repressor and expression of the fusion
polypeptide is induced.
Full-length DF2 FIPV and FECV spike protein 1-1454
amino acid inserts were isolated from established pOTSKF33
plasmid clones (as described in the above-identified parent
application, incorporated by reference herein), by SmaI/
StuI digestion of plasmid DNA and the excised gene cloned
W093/2~Z2 2 ~ 3 ~ 8 9 ~ PCT/US93/~3~
41
into pSC11/SmaI-digested, dephosphorylated vector using
conventional techniques. Insert-bearing clones were
identified by BamHI digest of mini-prep DNA. Full length
clones were oriented with respect to vector by digestion
with XbaI ( FECV) and NcoI ( FIPV) .
With respect to the description of all chimeric
proteins in the following examples, the fusions of the
indi~idual fragments are performed so that overlapping
amino acids are entered only once. For example in SEQ ID
NO: 45 of Example 3 below, the overlapping amino acid
position 311 of FIPV and FECV appears only once in the
chimeric protein, as does the amino acid position 872 of
FECV and FIPV, resulting in a chimeric pro~ein of 1454
amino acids in length.
ExamPle 3 - Cloninq of Chimeric S Genes Encodinq AA 1-
311/AA_311-1454 FIPV/FECV in ~SCll
Full length chimeric S genes were engineered to encode
(a) 1-311 amino acid of FECV fused to 311-1454 amino acid
of WT DF2 FIPV, the chimeric is identified by SEQ ID NO:
43; and (b) 1-311 amino acid of WT DF2 FIPV fused to 311-
1454 amino acid of FECV, the chimeric is identified by SEQ
- ID NO: 44. Amino acid 311 appears only once in the
chimeric so that the S gene is 1454 amino acid in total
length.
FECV and DF2 FIPV SmaI/MscI 1-311 inserts and
SmaI/MscI 311-1454 amino acid plasmid DNA fragments were
isolated from an established pSCll plasmid containing the
full-length DF2 FIPV or FECV spike gene. Digests were
loaded onto agarose gels prepared and run in Tris-Borate-
EDTA buffer. DNA fragments were isolated and eluted using
GeneClean (BiolO1 Inc., La Jolla, CA) according to the
manufacturer's instructions.
W093t2~22 2 1 3 ~ 3 PCT/US93/~3
42
The corresponding plasmid and AA 1-311 insert
fragments were ligated together overnight at 15C. HB101
host cells [ATCC] were transformed with the ligation mixes
and full length clones identified by BamHI digestion of
mini prep DNA.
The FIPV and FECV specific spike gene regions in the
chimeric clones were identified by diagnostic restriction
enzyme digestions: (1) 1-311 amino acid FIPV, NcoI;
(2) 311-1454 amino acid FIPV, XbaI; ~3) 1-311 amino acid
FECV, XbaI; and (4) 311-1454 amino acid FECV, XmnI.
Restriction enzymes were purchased from New England Biolabs
(Beverly, MA) or Bethesda Research Labs (Gaithersburg, MD)
and used according to manufacturer's specifications.
ExamPle 4 - Cloninq of Chimeric S Genes Encodinq AA 1-
i5 311!AA 311-872lAA 872-1454 FIPV/FECV/FIPV and FECV/FIPV/
FECV in pSC11
Chimerics having AA 1-311 DF2 FIPV fused to AA 311-872
: ~ FECV fused to AA 872-1454 FIPV are identified by SEQ ID N0:
45. Chimerics having AA 1-311 FECV fused to AA 311-872 DF2
FIPV, fused to AA 872-1454 FECV, are identified herein by
SEQ ID N0: 46. As in Example 3 above, the chimerics are
1454 amino acid in total length.
Nucleotide fragments encoding FIPV and FECV
MscI/BstEII AA311-872 amino acid insert fragments and
25~ MscI/BstEII AAl-311/872-1454 plasmid fragments were
isolated from established clones containing the fl~ll length
FIPV or FECV spike genes in pSC11.
The appropriate isolated fragments were then ligated
together overnight at 15C. Full length clones were
identified by BamHI dige~t of mini prep DNA. These clones
were then oriented with respect to the 7.5 K promoter in
the vector and verified for presence of swapped 311-872
amino acids by PstI digest (present in FIPV but not in FECV
W093/2~2 . PCT/US93/~3~
,~...... ';. 213~898
43
at nucleotide position 1056, AA position 352). The
specificity of FIPV and FECV insert regicns in the chimeric
clones were identified by diagnostic restriction enzyme
digestions: (1) 1-311 amino acids FIPV, NcoI; (2) 872-
1454 amino acid FIPV, XbaI; (3) 1-311 amino acid FECV,
XbaI; and (4~ 87Z-1454 amino acid FECV, XmnI.
Example 5 - Cloninq of Chimeric S Genes Encodinq AA 1-872/
872-14~4 FIPV/FECV in Pscll
DF2 FIPV ~nd FECV SmaI/ st~II 1-87~ amino acid insert
and SmaI/BstEII 872-1454 amino acid plasmid fragments were
isolated from esta~lished pSC11 clones containing the full
length FIPV and F~CV spike genes, respectively. The
appropriate isolated fragments were then ligated together
overnight at 15C. Full length clones were identified by
BamHI digest of mini prep DN~. Full length clones were
then oriented and specific insert regions identified by
PstI (DF2 FIPV AA 1-872), AccI (FECV AA 1-872), XbaI (DF2
FIPV AA 872-1454), and XmnI (FECV AA 872-1454~ restriction
enzyme digests.
Chimerics having 1-872 amino acid DF2 FXPV fused to
872-14S4 amino acid FECV are identified by SEQ ID N0: 47.
Chimerics having 1-872 amino acid FECV fused to 872-1454
amino acid DF2 FIPV are identified by SEQ ID N0: 4~. These
chimerics are 14~4 amino acid in length.
Example 6 - Cloninq of T-Cell Rich_Reqions in PSC11 and
Recombinant Ex~ression
1. Cloning of FIPV AA 894-1040, AA 894-1203 and
1~29-1454
The following example demonstrates that the amino acid
regions from 894-1040 (DF2, TS, FECV) and 894-1203 are rich
in T-cell sites, which are capable of stimulating a cell-
mediated immune response.
W093/2~22 ~ 1 3 ~ 3 ~ ~ PCT/US93/043
A FIPV SmaI/StuI insert was isolated from an
established plasmid [pOTSKF33] containing the 894-1040
amino acid region of the DF2 FIPV spike protein. The
isolated fragment was purified using GeneClean (Bio 101
Inc., LaJolla, CA) according to the manufacturer's
instructions. Purified insert was ligated with pSCll SmaI-
digested, dephosphorylated vector overnight at 15C.
Insert-bearing clones were identified by BamHI digest of
mini-prep DNA. Full-length clones were oriented with
respect to the 7.SK promoter in the vector by DraIII,
XhoI/DraIII, and BamHI/DraIII digests. PCR DNAs encoding
the DF2 S FIPV 894-1203 amino acid fragment were purified
using Prime Erase Quik Columns (Stratagene Cloning Systems,
LaJolla, CA). Purified PCR DNAs were then digested
SmaI/StuI, excised from agarose gels, and purified with
GeneClean. Purified inserts were ligated to pSC11/SmaI-
digested, dephosphorylated vector overnight at 15C. Full
length clones were identified by BamHI digest of mini-prep
DNA and oriented with respect to vector by HindIII and
XhoI/HindIII digests.
The TS FIPV 1029-1454 amino acid region, expressed as
a galK fusion protein and used to immunize mice, induced a
, ~ ,
strong CMI response when splenocytes from these animals
were sti~ulated with inactivated TS FIP~ virus. A
~25 ~ SmaI/StuI insert was isolated from an established plasmid
;~ (pOTSKF33) containing the 1029-1454 amino acid region of
the TS FIPV S gene. The fragment was purified with
GeneClean and ligated to pSCll SmaI-digested, dephos-
~ t' I phoxylated vectoxi as above. Full length clones were
-~30 identified and oriented by BamHI and HindIII digests of
mini prep DNA, respectively.
~: '
W093/2~22 PCT~US~/043~
8 g ~
2. Expression of Predicted T Cell Sites Alone
As described in the parent and grandparent of the
present application, previous experiments with an ~. coli
fusion protein containing TS FIPV 894-1040 amino acids
induced a cellular immune response in mice and protected
50~ of cats from FIPV challenge. Another region, TS FIPV
AA 1029-1454, also elicited a strong CMI response in mice
stimulated with inactivated vaccine virus (TS). Therefore,
the following recombinant viruses were made to determine
whether or not a cell mediated immune response could be
selectively stimulated by immunizing cats with one or more
of the predicted major T cell sites on the DF2 FIPV S gene
expressed in vaccinia virus recombinants.
Recombinant vaccinia viruses were engineered to
lS contain selected regions of the WT DF2 S gene encoding the
predicted FIPV T cell sites: (a) 894-1040 amino acids
(predicted to contain a T cell site at 922-934 amino acid);
~- (b) 894-1203 amino acids (the above clone was expanded to
add strong T cell:site at residues 1133-1147); and (c) TS
1029-1454 amino acids.
Example 7 - Generation of ~ecombinant Vaccinla Viruses
Standard vaccinia virus transfection procedures were
-:: used to isolate recombinant viruses expressing full length
: ~ DF2 FIPV S, FECV S, selected portions of the DF2 and TS
FIPV S snd chimeric FIPV/FECV spike genes ~D. M. Glover,
DNA Clonina A Practical A~roach, Volume 2, IRL Press,
1985)]- Briefly, plasmid DNA prepared from partial
! (Example 6), full-length (Example `2) or chimeric S
(Examples 3-5)/pSC11 clones was used to transfect CV-l
~ATCC CCL 70] monolayers infected with the WR strain of
vaccinia virus. Transfected monolayers were harvested 2
days post-infection (p.i.) and were plaque-assayed on HuTK-
W0~3/2~22 2 ~ PCT~US93/~3~
~,, ~, ,
46
cell [ATCC CRL 8303] monolayers in 60 mm dishes. At 3 days
p.i. dishes were stained with an agarose overlay containing
300 ~g/ml Bluo-gal [Sigma]. After 4-6 hours, blue BGal~
plaques were identified and picked with stexile Pasteur
pipettes into 0.5 ml DMEM + 5% fetal bo~ine serum. First-
round plaques were plaque-purified twice more on HuTK-
cells and then used to infect CV-1 and HuTK- monolayers in
T25 flasks. Infected monolayers were harvested at 2 days
p.i. and frozen at -20~C for use as ~rude virus stocks.
Exam~le 8 - Characterization of ~ecombinant Vaccinia
Viruses
The presence of DNA in 3X plaque-puri~ied virus stoc~s
was verified by DNA dot blot, using standard methods ~D. M.
Glover, DNA Clonin~. A Practical AProach, Volume 2, IRL
Press, (1985)]. Expression of recombinant S genes was
evaluated by Western blot. HuTK- or CV-1 monolayers
infected with recombinant viruses were harvested at 2 days
p.i. by scraping into the medium. Pelleted cells were
washed with PBS and resuspended in 0.6 ml RIPA buffer (0.15
M NaCl, 0.1% SDS, 1.0~ Na deoxycholate, 1.0% Triton X-lOo,
S mM EDTA, 20 mM Tris, pH 7.4). RIPA lysates were
frozen/thawed 3X and sonicated briefly. Non-reducing
sample bu~fer (2~ SDS, 80 mM Tris, pH 6.8, 10% glycerol,
0.02% bromophenol blue) was added and samples boiled 10
25~ minutes prior to electrophoresis on 10% SDS-polyacrylamide
gels as described by Laemmli. Proteins were transferred to
Immobilon-P (Millipore) at 20-25 mA for 18 hours in
Tris~Glycine/~ethanol buffer. Filters were blocked in !2%
skim milk, 1% gelatin, and TBS (20 mM Tris, pH 7.5, 500 mM
-~0 NaCl) for 1-2 hours (h) at room temperature (RT), rinsed -
with TTBS (TBS + 0.05% Tween-20) and incubated with anti-
FIPV cat serum at a 1:50 dilution in TTBS and l~ gelatin
for 1-2 hours at RT.
;.
W093/2~22 2 1 3 ~ ~ 9 ~ PCT/US93/~3~
47 ~:
Anti-FIPV serum was produced at SmithKline Beecham
Animal Health, Lincoln, Nebraska in cats that were
vaccinated two times intranasally with Primucell
(SmithKline Be~cham) vaccine containing a temperature-
sensitive DF2 FIPV at three week intervals and challengedorally with 1 mL DF2 FIPV two weeks following the third
vaccination. Animals were then bled by conventional means.
Filters were washed in TTBS 3X for 10 minutes each and
incubated with goat anti-cat alkaline-phosphatase labeled
IgG at a 1:1000 dilution for 1 hour [Kirkegaard-Perry
Laboratoriesp Inc.~. Filters were washed as before and
incubated 5-15 minutes in BCIP/NBT substrate according to
the manufacturer's instructions [Kirkegaard-Perry
La~oratories, Inc.]. Filters were then rinsed in water and
air-dried.
Example 9 - Çats I~muni~ed with Vaccinia Recombinants
Ex~ressina Chimeric FECV/~IPV_S Genes
To evaluate immunogenicity of these molecules,
recombinant vaccinia viruses expressing FIPV S (v/FIPV S),
FECV S (v/FECV S), or FIPV/FECV chimeric S genes (v/FIPV +
FECV S) are used to immunize kittens. 14-week-old specific
pathogen fxee kittens, ten per group (5 from Liberty Labs
and 5 from Sprague Dawley), are immunized subcutaneously
with 5 X 107 PFU of the recombinant ~accinia viruses. A ;
group of ten kittens receives wild-type WR vaccinia ~irus
(v/WR) as a negative control. Kittens are clinically
examined daily and rectal temperatures ta~en. A second
immuniization with the same amount of virus is given after
3 weeks. Two weeks after the second immunization kittens
are challenged orally with either 1.25 x 104 PFU (low) or 1
x 106 PFU (high) of WT DF2 FIPV and survi~al monitored.
W093/2~22 ~1 3 ~1 t~ 9 ~ PCT/US93/043~
1~
48
Virus-ne~tralization titers are determined on the day of
challenge and one and two weeks post-challenge. Serum
samples are taken on the days of first and second
vaccination, challenge, and post-challenge days 3, 7, 14,
21, and 28.
The results are predicted in Table V below.
TABLE V
Grou~ # Cats ~noculum Sensitiæation
. Challenge Dose
Hiqh Low
Group 1 10 WR V no no
Group 2 10 V/FIPV S yes yes
Group 3 10 V/FECV S yes yes
Group 410 V/FIPV AA1-311 yes no
:: Group 5 10 V/FIPV AA1-872 yes yes
Group 6 10 V/FIPV AA311-872 yes yes
Group 7 ~10 V/FIPV AA872-1454 no no
.:Exam~le 10 - Chimeric FECV/FIPV Genes
-
~-~Q~:A predicted location of the major neutralizing epitope
on~the feline coronavirus S is at about amino acids 525-650
(DF2~ DF~-HP, ~TS,~ TS-BP, FECV). To evaluate the role of
the~neutralizing epitope in immunity to FIPV, full-length
'chimeric:' S~proteins were constructed in which amino acids
~ Z5~ ~ 311-872 FIPV~DF2 are replaced by the analogous region of
: FECV or con~ersely, amino acids 311-872 of FECV are
interchànged for amino acids 311-872 of FIPV DF2.
.Full length S proteins were constructed which contain:
(a) 1-311 amino acid of FECV fused to 311-872 amino acid of
~ WT DF2 FIPV, fuséd to 872-1454 amino acid of FECV, this
;chimeric is identified by SEQ ID N0: 46; and (b) 1-311
~ ~ ,
"
,~
amino acid of WT ~F? FIPV fused to 311-872 amino acid of
FECV, fused to 872-14S4 amino acid of WT DF2 FIPV, this
chimeric is identified by SEQ ID NO: 45, were engineered.
Alternate cloning sites between amino acids 650 and
872 could be used in place of BstEII (i.e., PvuII, Hq~I) to
excise the middle portion of the S gene encompassing the
predicted VN epitope(s~.
ExamPle 11- Chimeric CCVlFIPV Genes
A full length chimeric S gene encoding A~1-352 of the
CCV S protein and 352-1454 of the DF2 FIPV spike protein
respectively, was constructed as follows. Briefly, 10 ~1
of a PCR DNA encoding the CCV S protein amino acids 1-352
was digested with XmaI/PstI and ligated overnight at 15°C
to a PstItStuI a purified insert fragment encoding
AA352-1454 isolated from a DF2 FIPV spike gene plasmid.
The DF2 FIPV S gene clone, from which a 3~2-1454 amino acid
PstI and StuI insert was isolated, was cloned by digesting
a PCR ~NA encoding DF2 FIPV S AA1-1454 with XmaI and StuI.
Ligation products were then ligated for 4 hours at room
temperature to pOTSKF33 vector ~SmithKline Beecham
Pharmaceuticals, Swedeland, PA], digested with XmaI/StuI
and dephosphorylated. Transformants in E. coll strain
AR120 ~SmithKline Beecham~ were screened by BamHI and SPhI
digests of mini prep DNAs to identify insert-bearing
clones.
Ligation products of the CCV and DF2 FIPV ligation
were ligated into pOTSK33 vectors under similar conditions
to those desicribed above. AR120 transformants were
screened by BamHI/SPhI and PstI digests of mini prep DNAs.
The presence of an unique StuI site at amino acid #128 of
the CCV S gene was confirmed by StuI digest. Clone #2324
was identified as containing a full length chimeric spike
gene encoding ~A1-352 of CCV and AA352-1454 of DF2 FIPV.
WO g3t23422 PCr/USg3~04384
~ 1 3 1 ~ 9 ~
I
Exam~le 12 - In Vitro T-Cell Proliferation AssaY
Immunizing antigens were administered as pelle~t-3
bacterial lysates of pOTSKF33 galK/TS FIPV S fusion
proteins, as described in Example 7 of co-pending, co-owned
U.S. Patent ~.pplication Ser. No. 07/698,g27. A
corresponding application was published on May 29, 1992,
; Publication No. WO 92/08487, and is incorporated by
reference herein.
The pellet 3 fractions were obtained essentially as
~ described~below.~,The inactivated~extract was~centrifuged
at 27,00~0~ x ~g for 30~min (JA20). The supernatant was
`~ discarded and the pellet resuspended by vortexin~ for lO
minutes in 200 mls of Buffer A plus 0.2% sodium deoxycholic
acid and 1% Triton X-lOO. Buffer A contains 50 mM Tris-
~15~ HCl,~pH~8~.5~, 5~mM~EDTA,~1 mM~DTT, and 5%~ glycerol. The
extract~was~centrifuged ~at 27~,000 X g~for 30 minutes and
again the~resulting~supernatant was discarded. The pellet
r ~ ended~ by~ vbrtexing lO minutes~in 200 mls of
'"~ u~r~er `A,~co~nta-i'ning~Triton~X-10~0 (l%) and 0.5 M KCl.
Fo~llowing;~cèntrifugation (27,000 x g, 30 minutes), the
pel1~et ~ was'~ resuspendod by vortexing and brief
oge~iz tion~ in~5-lO~ml~a~of ~B~S tl~l5 g dibasic sodium
' phosph ~ ,;~0.2~g monobasic~sodium phosphate and 8.5 g NaCl
per~liter~ pH~,~7.4~ This~completes the pellet-3 antigen.
The~,amount~of~àpecific~galK/TS FIPV fusion protein in each
pe}let-3;~1y&jate~was`~qyantitated by Western blot against a
galX protein'standard~
8alb/C mice tlO per group, 12 weeks of age) were
immuniz~d witX 15-40 ~g of pellet 3 antigens formulated in
O~ an~ad~uvant~ emu1sion~containing ~25~ g of Qu11 A and 5~
-, Aihydrogel ~Superfos ~ Biosectorals, Denmark]. These
antigèn~s~represented~TS~FIPV S gene regions from amino
i:G ~
. . ~ ` ~ :
' ', . " : ~ ~ '
W093/2~22 PCT/USg3/~3~
;-- 213~89~
51
acids 1-105, 94-223, 213-362, 35Z-748, 737-1040, 894-1040
and 1029-1454. Two immunizations were administered
intraperitoneally (i.p.), 0.1 mL dose on days 1 and 21.
(Mice vaccinated with AA352-748 pellet-3 antigen received
10 ~g of fusion protein per dose.)
Ten days after the second immunization (day 31),
spleens were removed aseptically from mice immunized with
the antigens identified above. Spleens from 5 mice of each
group were pooled. Splenocytes were seeded (5 x 1o6
cells/mL) into each well of 96-well polystyrené plates in
triplicate in a total of 200 ~L RPMI 1640 ~Sigma] plus 10%
fetal bovine serum, Z0 mm HEPES, Pen Strep (100 unitstmL
penicillin; 100 ~g/mL streptomycin), 0.1% ITS Growth Media
(insulin 5 ~glmL, transferrin 5 ~g/mL, selenious acid 5
~15 ~g/mL), and 2 g sodium bicarbonate/L, with or without
mitogens or specified antigens (biethylimine inactivated TS
FIPV or pellet-3 antigens). The mitogens included
concanavalin A (ConA), lipopolysaccharide (LPS), and
~ pokeweed mitogen and were used as controls.
-~ ~20 The cultures were incubated at 37C in an atmosphere
; ~ of 5~ CO2. After 72 hours (mitogens) or 96 hours (antigens)
the cultures werz labeled with tritiated thymidine (0.5
~Ci/well) [New England Nuclear (NEN), 2 ci/mmol]. Cultures
were incubated for an additional 18 to 2~ hours. Cultures
-~25~ were harvested onto glass fiber paper and counted on a ~
counter. The results were expressed as the mean of
triplicate counts per minute (cpm) for each culture and are
provided below in qualitative terms. (Background counts
obta~ned from pOTSKF33 negative control pellet-3 lysate
were subtracted~)
The following Table VI illustrates the reacti~ity of
mouse splenocytes to TS FIPV inacti~ated by BEI treatment
or pellet-3 antigens in the in vitro proliferation assay.
W093t2~22 PCT~US93/~3iX4
~13i~38
52
The immunizing antigens in column 1 of Table VI are galK
fusion proteins containing the indicated S protein
fragments of SEQ ID NO: 8, as described above. The
stimulating antigens in columns 3, 4, and 5 indicate S
protein regions identified in the pellet-3 antigens. One
plus (+) indicates 10-15,000 cpm, two pluses (++) indicates
15-20,000 cpm, +++ indicates 30,000 cpm, +++~ indicates 35-
50,000 cpm, - indicates S 10 K cpm.
TABLE VI
Reactivity of Mouse Spl:enocytes to TS FIPV by In Vitro
ProIiferation Assay
Stimulatinq Antiqen
TS-FIPV ~ Killed
Immunizing Antigen Virùs Amino Acid Positions T cell
: S Protein Reqion TS FIPV 1-105 737-1040 1029-1454 Site
AAl-105~ 77-89
AA94:-22:3~ - ND ND ND
AA--2-13-3~62 : ~+ ND ND ND
z~:AA352-748~: - ND ND ND 408-427
2.0~ 482-496
AA73~7-10`40~ ++ ~; - + + 922-934
- ~ AA894-1040~ ~ ++++ ND ~ ND ND 922-934
AA10~2~9-14~54~ +++ - : ++ +++1133-1147
~25::-: ~ .: : 1368-1391
: Splenocytes from mice that received the FIPV TS AA213-
362, AA894-1040 and AA1029-1454 fusion protein antigens
responded well to TS FIPV antigen in a T cell proliferation
~ass~ay. The latter two of these antigens represent regions
-.~3~0~ of~the` TS FIPV:spike~gene that are predicted to contain
strong T.cell epitopes. The computer programs utilized to
. ~. predict T cell epitopes are typically 80~ correct.
~,,:
W093/2~22 PCT/US93/~
',.;'""' 213~39~1
53
Vaccination with the AA213-362 fusion protein clearly
stimulates a proliferative T cell response and so must also
contain a T cell site. Serological responses of vaccinated
mice as measured by ELISA, VN and Western blot are still
being evaluated.
Numerous modifications and variations of the present
invention are included in the above-identified
specification and are expected to be obvious to one of
skill in the art. Such modifications and alterations to
the compositions and processes of the present invention are
believed to be encompassed in the scope of the claims
appended hereto.
WO 93/234~2 PCl`/US93/~43~
2 1 3 ~
54
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(~) APPLICANT: Miller, Timothy J.
Rlepfer, Sh~ron
Reed, Albert Paul
Jone~, Elalne V.
( ii) TITEE nF INVENTION- Compos1tio~ and Methoda for Vaccination
A~ain~t CoronavirusQs
(iii) NUMBER OF SEQUENCES: 48
~v) CORRESPO~DENCE ADDRESS:
~A) ~DD~ESSEE: Smith~lln~ Beecham Corporation - Corporate
Patents
(Bl STREET: 709 Swedeland Road
(C) CITY: ~Lng of Prussia
(D) STATE: PA
tE) COU~TRY: USA
(F) ZI~s 19406-2799
(v) COMPUTER READABLE FOR~:
(A) MEDIUM TYPE: Floppy d~k
(B) COMPUTER: IBM PC compatlble
~C) OPERATING SYSTEM: PC-DOS/MS~DOS
~D) SOFTWARE: Pat~t~n R~la~ao ~1.0, V~rslon ~1~25
(vi) CURREN~ APPLICA~ION DATA:
tA) APP~ICATION NUMBER: US
~B) FILING DATE:
~C) CLASSIFICATION:
~vll) PRIOR APPL~CATION ~ATA:
~A) APP~ICATION NUM8ER: US 07/882,171
~8) FILING ~ATE: 08-MAY-1992
~vii) PRIOR APPLICATION DATA:
~A) APPLICATION NUM3ER: US 07/658,927
~B) FI~ING DATE: 13-~AY-l99l
~vii) PRIOR APPL~CATION DATA:
(A) APP~ICATION NUMBE~: US 07/613,066
~B) FIL~NG DATE: 14-NOV-1990
(viil) ATTORNEY/AGENT INFORMAT~ON:
5A) NAME: Schreck, Patrlca A.
(B) REGISTRAT~ON NUMBER: 33,777
~C) REFERENCE/DOCXET NUMBER: SBC H85009-1
(lx) TELECOMMUNICATION INFORMATION:
- (A) TE~EPHONE: (215) 270-5015
~B~ TELEFAX: (215) 270-5090
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CH~RACTERISTICS:
(A) ~NGT~: 4365 ba~c pair~
~ B ) TYPE: nuclQ$c acid
( C ) STRANDEDNE:SS: doubl~
(D) TOPOLOGY: unknown
SUBSTITUTE SHEEr
WO 93/2342~ PCl`/US93/0~384
` ~ ~131~98`
(il) MOLECULE TYPE: DNA (genomic)
( ix ) FEATURE:
(A) NAME/~EY: CDS
(B) LOCATION: 1..4362
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATG ATT GTG CTC GTA ACT TGC CTC TTG TTG TTA TGT TCA TAC CAC ACA 48
Met Ile Yal Leu Val Thr Cy~ Leu Leu ~e~ Leu Cy~ S~r Tyr Hl~ Thr
1 5 10 15
GTT TTG AGT ACA ACA AAT AAT GAA TGC ATA CAA GTT AAC GTA ACA CAA 96
Val Leu Ser Thr Thr A~n Asn Glu Cy Ile Gln Val Asn Val Thr Gln
20 25 30
TTG GCT GGC AAT GAA AAC CTT ATC AGA GAT TTT CTG TTT AGT AAC TTT 144
Leu Ala Gly A~n Glu Asn Leu Ile Arg Asp Phe ~eu Phe Ser Asn Ph~
35 40 45
AAA GAA GAA GGA AGT GTA GTT GTT GGT GGT TAT TAC CCT ACA GAG GTG 192
Lys Glu 51u Gly Ser Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
50 55 6~
TGG TAC AAC TGC TCT AGA ACA GCA CAA ACT AC~ GCC TTT CAC TAT TTT 2 4 0
Trp Tyr Aon Cy~ Ser Arg Thr Ala Gln Thr Thr Ala Phe Gln Tyr Phe
65 70 ~5 80
AAT AAT ATA CAT GCC TTT TAT TTT GTT ATG G~A GCC ATG GAA AAT AGC 288
Asn A ~ Ile His Ala Phe Tyr Phe Val Met Glu Ala ~et Glu Asn Ser
~5 90 95
ACT GGT AAT GCA CGT GGT A~A CCA TTA TTA m CAT GTG CAT GGT GAG 336
Thr Gly Asn Ala Arg Gly Lys Pro Leu L~u Ph~ Hi~ Val Hi~ Gly Glu
100 105 110
CCT GTT AGT GTT ATT ATA TAT ATA TC0 GCT TAT AGG CAT GAT GTG CAA ~84
Pro Val Ser Val Ile Ile Tyr Ile Ser Ala Tyr Arg Asp Asp Val Gln
115 l~0 125
CAA AGG CCC CTT TTA AAA CAT GGG TTA GTG TGC ATA ACT AAA AAT CGC 432
Gln Arg Pro Leu Leu Lys Hi~ Gly Leu Val Cys Ile Thr Ly~ Asn Arg
130 135 140
CA~ ATT AAC TAT GAA CAA TTC ACC TCC AAC CAG TGG AAT TCC ACA TGT 480
His I1~ Asn Tyr Glu Gln Phe Thr Ser A~n Gln Trp A~n Ser Thr Cys
145 150 155 160
ACG GGT GCT GAC AGA AAA ATT CCT TTC TCT GTC ATA CCC ACG GAC AAT 528
Thr Gly Ala Asp Arg Lys Ile Pro Phe Ser Y~l Ile Pro Thr Asp Asn
165 170 175
GGA ACA AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TTT GTT ACA GCT 576
Gly Thr Lys Ile Tyr Gly Leu Glu Trp A~n Asp Asp Phe Val Thr Ala
180 185 l90
TAT ATT AGT GGT CGT TCT TAT CAC TTG AAC ATC AAT ACT AAT TGG TTT 624
Tyr Ile Ser Gly Arg Ser Tyr His Leu A~n Ile Asn Thr Asn Trp Phe
lg5 200 205
AAC AAT GTC ACA CTT TTG TAT TCA CGC TCA AGC ACT GCT ACC TGG GAA 672
Asn Asn Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
210 215 220
SUBSTITlJTE SHEET
WO 93~23422 PCT/US93/04384
2131~8 i
TAC AGT GCT GCA TAT GCT TAC CAA GGT GTT TCT AAC TTC ACT TAT TAC 720
Tyr Ser Ala Ala Tyr Ala Tyr Gln Gly Val Ser A~n Phe Thr Tyr Tyr
225 230 235 240
AAG TTA AAT AAC ACC AAT GGT CTA AA~ ACC TAT GAA TTA TGT GAA GAT 7 6 8
Ly~ Leu Asn A~n Thr A~n Gly Leu Ly~ Th~ Tyr Glu Leu cy~ Glu A~p
245 250 ~5S
TAT GAA CAT TGC ACT GGC TAT GCT ACC AAT GTA TTT GCT CCG ACA TCA 816
Tyr Glu HLs Cy~ Thr Gly Tyr Ala Thr Asn Val Phe Ala Pro Thr Ser
260 265 270
GGT GGT TAC ATA CCT GAT GGA TTT AGT TTT A~C AAT TGG TTC TTG CTT 854
Gly Gly Tyr Ile Pro A p Gly Phe Ser Phe A~n A~n Trp Phe Leu Leu
275 280 285
ACA AAT AGT TCC ACT TTT GTT AGT GGC AGG TTT GTA ACA AAT CAA CCA 912
Thr Asn Ser Ser Thr Phe Val Ser Gly Arg Pha Val Thr Asn Gln Pro
290 295 30Q
TSA TTG ATT AAT TGC ~T5 TGG CC~ GTG CCC AGT TTT GG~ GTA GCA GCA 960
Leu Leu Ile Asn Cy~ ~eu Trp Pro Val Pro Ser Ph~ Gly Val Ala Al~
305 310 315 320
CAA GAA TTT TGT TTT GAA GGT GCA CAG TTT AGC CAA TGT AAT GGT GTG 1008
Gln Glu Phe Cys Phe Glu Gly Ala Gln Phe Ser Gln Cy~ Asn Gly Val
325 330 335
TCT TTA AAT AAC ACA GTG GAT GTT ATT AGA TTC AAC CTT AAT TTC AC~ 1056
Se~ Leu Asn Asn Thr Val Asp Val Ile Arg Phe Asn L~u A~n Phe Thr
340 345 350
GCA GAT GTA CAA TCT GGT ATG GGT GCT ACA GTA TTT TCA CTG AAT ACA 1104
Ala Acp Val Gln Ser Gly Met Gly Ala Thr Val Phe Sar Leu Asn Thr
355 360 365
ACA GGT GGT GTC ATT CTT GAA ATT TCA TGT TAT AGT GAC ACA GTG AGT llS2
Thr Gly Gly Val Ile Leu Glu Ile Ser Cy~ Tyr Ser A~p Thr VA1 Ser
370 375 380
GAG TCT AGT TCT TAC AGT TAT GGT GAA ATC CCG TTC GGC ATA ACT GAC 1200
Glu Ser Ser Ser Tyr Ser Tyr Gly Glu Ile Pro PhQ Gly Ile Thr A8p
385 390 395 400
GGA CCA CGA TAC TGT TAT GTA CTT TAC AAT GGC ACA GCT CTT AAA TAT 1248
Gly Pro Arg Tyr Cy~ Tyr Val Lou Tyr A~n Gly Shr A1A L~U ~Y~ Tyr
405 410 415
TTA GGA ACA TTA CCA CCC AGT GTA AAG GAA ATT GCT ATT AGT AAG TGG 1296
Leu Gly Thr Leu Pro Pro Ser Val Lys Glu Il~ Ala Ile Ser Ly~ Trp
420 42S 430
GGC CAT TTT TAT ATT AAT GGT TAC AAT TTC TTT AGC ACA TTT CCT ATT 1344
Gly ~g Phe ~yr ~le Asn Gly Tyr A~n Phe Phe Ser Thr Phe Pro Il~
435 440 445
GGT TGT ATA TCT TTT AAT TTA ACC ACT GGT GCT AGT GGA GCT TTT TGG 1392
Gly Cy~ Ile Ser Phe Asn Leu Thr Thr Gly Ala Ser Gly Ala Phe Trp
450 455 460
ACA ATT GCT TAC ACA TCG TAT ACT GAA GCA TTA GTA C~A GTT GAA AAC 1440
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu Asn
465 470 4~S 480
SUBSTITlJTE SHEET
WO 93/23422 PCr~US93/04384
`' 213~g8
ACA GCT ATT AAA AAT GTG ACC TAT TGT AAC AGT CAC ATT AAT AAC ATT 1488
Thr Ala Ile Ly~ Asn Val Thr Tyr Cyo A~n S~r His Ile Aon Asn Ile
485 490 495
AAA TGT ~CT CAA cTr ACT GCT AAT ~TG AAT AAT GGA T~T TAT CCT GTT 15 3 6
Lys Cys Ser Cln Leu Thr Ala Asn Leu Asn Asn Gly Phe Tyr Pro Val
500 50S 510
GCT TCA AGT GAA GTA GGT TTC GTT AAT AAG ACT GTT GTG TTA TSA CCT 1584
Ala Ser Ser Glu V~l Gly Phe Val Asn Ly~ Ser Val Val Leu Leu Pro
515 520 525
ACC TTT TTC ACA CAC ACC GCT CTC AAT ATA ACC ATT GAT CTT CGT ATC 1632
Ser Phe Phe-Thr His Thr Ala Val A~n Ile Thr Ile Asp Leu Cly Met
530 535 540
AAG CTT AG$ GGT TAT GGT CAA CCC A$A GCC TCG ACA CTA AGT AAC ATC 1680
Lys Leu Ser Gly Tyr Cly Gln Pro Il~ Ala Ser Thr Leu Ser Asn Ile
545~ ~ 550 ~ 555 560
ACA CTA CCA ATG CAC GAT AAC AAT ACT GAT GTG TAC TGT ATT CGT TCT 1728
Thr Leu Pro Met Gln Asp Asn Asn Thr Asp Val Tyr Cys Ile Arg Ser
565 570 5~5
AAC CAA TTC TCA GTT TAT GTT CCT TCC AC$ TGC AAA AGT TCT TTA TGG 1776
Asn Gln Phe Ser Val Tyr Val Pro Ser Thr Cy~ Lys Ser Ser Leu Trp
580 :~ 585 590
GAC AAT ATT TTT AAT~CAA:GAC TGC-ACG GAT GT~:TTA GAG GCT ACA GCT 1824
Asp::Asn Ile Phe:Asn Cln A~p Cys Thr Asp Val ~eu Clu Ala Thr Ala
Sg5: ~ 600 ~ 605
GTT~ATA:~AAA~ACT OGT ACT TGT CCT TTC TCA TTT GAT AAA TTG AAC AAT 18~2
:Va1~Ile~Lys~Thr~Gly Thr`Cys Pro Phe Ser~ Ph~ A p Lys Leu A~n A3n
TAC TTG ACT TTT AAC AAG~TTC TGT TTG TCG TTG AGT CCT GTT GGT GCT 1920
Tyr Leu Thr Pho Asn L:y8 Phe cy8 Leu Ser L~u Ser Pro Val Gly Ala
625 630 635 640
AAT~TGC~ TTT~CAT~CTT GCT GCA CGT acA AGA ACC AAT GAG CAC GTT 1968
A~n~Cys-~y8~Phe:Arp Val Ala Ala Arg Thr Arg Thr A~n Glu Gln Val
645~ 650 655
GTT:AGA AGT~C~A:TA~GTA ATA::TAT: GAA;:GAA GGA GAC AAC ATA GTG GGT 2016
Val Arg Ser Leu Tyr Val:Ile~Tyr Glu Glu Gly A~p Asn Ile Val Gly
660~ 665 6~0
GTA CCG TCT GAT AAS AGC GGT CTG CAC GAT TTG TCT CTC CTA CAC CTA 2064
Val Pro Ser Asp A~n Ser Gly su H~s Asp ~eu Ser Val ~eu H~s r-eu
675 ~ 680 685
GAC TCC TGT ACA GAT TAC AAT ATA TAT GGT AGA ACT GGT GTT GGT ATT 2112
Asp Ser Cy8 Thr A~p Tyr A~n Ile Tyr Gly Arg Thr Cly Val Gly Ile
690 695 700
ATT AGA CGA ACT AAC AGT ACG CTA CTT AGT GGC TTA TAT TAC ACA TCA 2160
Ile Arg Arg Thr A8n Ser Thr ~eu Leu Ser Gly ~-u Tyr Tyr Thr Ser
~10 715 720
CTA TCA GGT GAT ~TG TTA GGC TTT AAA AAT GTT AGT GAT GGT GTC ATT 2208
Leu Ser Gly Asp Leu ~eu Gly Phe ~ys A8n Val Ser Asp Gly Val Ile
: ~25 730 7~5
SUBSTlTlJTE SHEET
.:
WO 93/Z3422 2 ~ 3 ~ 8 9 ~ PCI'/US93/04384
58
TAT TCT GTG ACG CCA TGT GAT GTA AGC GCA CAA GCG GCT GTT ATT GAT 2256
Tyr Ser Val Thr Pro Cys Asp Val Ser Al~ Gln Ala Ala Val Il~ A~p
740 745 750
GGT GCC ATA GTT GGA GCT ATG ACT TCC ATT AAC AGT GAA CTG TTA GGT 2304
Gly Ala Ile Val Gly Ala Met Thr Ser Ile Asn Ser Glu L~u Leu Gly
7S5 760 765
CTA ACA CAT TGG ACA ACG ACA CCT AAT TTT TAT TAC TAC TCT ATA TAT 2352
Leu Thr Hl~ Trp Thr Thr Thr Pro A~n Phe Tyr Tys Tyr SQr I1R Tyr
770 775 780
AAT TAC ACA AGT GAG AGG ACT CGT GGC ACT GQ ATT GAC AGT AAC :;AT 2400
Asn Tyr Thr Ser ~lu Arg Thr Arg Gly Thr Ala Il~ Asp Ser A~n A~p
785 790 795 800
GTT GAT TGT GAA CCT GTC ATA ACC TAT TCT AAT ATA S;GT GTT TGT AAA 2448
Val Asp Cya Glu Pro Val Ile Thr Tyr Ser Asn Ile Gly Val Cys Lys
805 810 815
AAT GGT GCT ~TG GTT TTT ATT AAC GTC ACA CAT TCT GAC GGA GAC GTG 2496
Asn Gly Ala Leu Val Phe Ile Asn Val Thr Hls Ser Asp Gly A~p Yal
820 825 B30
CAA CCA ATT AGC ACT GGT AAT GTC ACG ATA CCT ACA AAT TTT ACC ATA ~ 2544
Gln Pro Ile Ser Thr Gly A~n Val $hr Ile Pro Thr A~n Phe Thr Ile
835 8~0 845
TCT GTG CAA GTT GAA TAC ATG CAG GTS TAC ACT ACA CCA GTA TCA ATA 2 5 g 2
Ser Val Gln Val Glu Tyr Met Gln Val Tyr Thr Shr Pro Val Ser Ile
850 855 860
GAT TGT GCA AGA TAC GTT TGT AAT GGT AAC CCT AGA TGT AAC AAA TTG 2640
A~p Cys Ala Arg Tyr Val Cys Asn Gly A~n Pro Arg Cys A~n Ly~ Leu
865 870 875 880
TTA ACA CAA TAT GTG TCT GCA TGT CAA ACT ATT GAA CAA GCA CTT GCA 2688
Leu Thr Gln Tyr Val Ser Ala Cy~ Gln Thr ILe Glu Gln Al~ Leu Ala
885 890 8~5
ATG GGT GCC AGA CTT GAA AAC ATG GAG GTT GAT TCC ATG TTG TTT GTC 2 7 3 6
Met Gly Ala Arg Leu Glu Asn Met Glu Val Asp S~r Met Lau Phe Val
900 905 910
TCG GAA AAT GCC CTT AAA TTG GCA TCT GTT GAG GCG TTC AAT AGT ACA 2784
Ser Glu Asn Ala I,eu LyY Leu Ala Ser VA1 G1U Ala Phe Ann Ser Thr
915 920 925
GAA AAT TTA GAT CCT ATT TAC AAA GAA TGG CCT AGC ATA GGT GGT TCT 2832
Glu Asn Leu A~p Pro ~le ~yr ~ya Glu Trp Pro Ser Il~ Gly Gly Ser
930 935 940
TGG CTA GGA GGT CTA AAA GAT ATA CTA CCG TCC CAT AAT AGC AAA CGT 2880
Trp Leu Gly Gly Leu Lys Asp Ile Leu Pro Ser Hls As~ Ser ~ys Arg
945 950 9S5 960
AAG TAT GGT TCT GCT ATA GAA GAT TTG CTT TTT G~T AAA GTT GTA ACA 2 9 2 R
~y~ Tyr Gly Ser Ala Ile Glu Asp LQU ~u Ph~ A~p Lys Val Yal Thr
965 970 975
TCT GGT TTA GGT ACA GTT GAT GAA GAT TAT AAA CGT TGT ACT GGT GGT 29~6
Ser Gly Leu Gly Thr Val A p Glu Asp Tyr Ly~ Arg Cys Thr Gly Gly
980 985 990
SUBSTITUTE SHEET
~WO 93/23422 2 1 3 ~ 8 9 ~3 PCr/US93/04384
ss
TAC GAC ATA GCA GAC TTG-GTG TGT GCT CAA TAT TAC AAT GGC ATC ATG 3024
Tyr Asp Ile Ala AQP Leu Val Cy8 Ala Gln Tyr Tyr A~n Gly Ile Met
995 1000 1005
GTT CTA CCA GGT GTA GCT AAT GCT GAC AAG ATG ACT ATG TAC ACA GCA 3072
Val Leu Pro Gly Val Ala Asn Ala Asp Lys Met Thr Met Tyr Thr Ala
1010 1015 1020
TCA CTT GCA GGT GGT ATA ACA TTA GGT GCA CTT GGT GGT ~:;GC GCC CTG 3120
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala LRU Gly Gly Gly Ala Val
1025 1030 1035 1040
GCT ATA CCT TTT GCA GTA GCA GTA CAG GCT AGA CTT AAT TAT GTT GCT 3168
Ala Ile Pro Phe Ala Val Ala Val Gln Ala ~rg Leu Asn Tyr Val Ala
1045 lOS0 1055
CTA CAA ACT GAT GTA T~G AAT AAA AAC CAA CAC ATC CTG GCT AAT GC:T 3 216
Leu Gln Thr Asp Val Lsu Asn Lys A~n Gln Gln Ile Leu Ala Asn Ala
1060 1065 1070
TTC A~T CAA GCT ATT GGT AAC ATT ACA CAG GCT TTT GGT AAG GTT AAT 3264
Phe Asn Gln Ala Ile Gly A~n Il~ Thr Gln Ala Phe Gly Lys Val Asn
10~5 1080 1085
GAT GCT ATA CAT CAA ACA TCA CA~ GGT CTT GCC ACT GTT GCT ~ GCG ~ 33~2
A p Ala Ile H~ Gln Thr Ser Gln Gly Leu Ala Thr Val Ala Lys Ala
1090. 1095 1100
TT& GCA AAA GTG CAA GAT GTT GTC AAC ACA CAA GGG CAA GCT TTA AGT 3~60
Leu Ala Ly~ Val Gln A~p Val Val A~n Thr Gln Cly Gln Ala Leu S~r
llOS 1110 lllS 1120
CAC CTT ACA GTA CAA TTG CAA AAT AAT TTT CAA GCC ATT AGT AGT TCT 340B
~18 LQU Thr Val Gln L~u Gln A~n Asn Phe Gln Al~ Ile Ser ser Ser
1125 1130 1135
ATT AGT GAT ATT TAT AI~C AGG CTT GP.C GAA CTG AGT GCT GAT GCA CAA 3456
Ile Ser A~p Ile Tyr Asn Arg ~eu A~p Clu I,eu Ser Ala ABP Ala Gln
114~ 1145 1150
GTT GAT AGG CTG ATT ACA GGT AGA CTT ACA GCA CTT AAT GCA ~TT GTG 3504
Val Asp Arg Leu Il~ T~r Gly Arg Lau Thr Ala L~u A~n Ala Ph~ Val
1155 1160 1165
TCT CAG ACT CTA ACC AGA CAA GCA GAG GTT AGG GCT AGT AGA CAA CTT 35 S 2
Ser Gln Thr Lesl Thr Arg Gln Ala Glu Val Arg Al~ S~r Arg Gln ~eu
11?0 1175 1180
GCC AAA GAC AAG GTT AAT GAA TGT GTT AGG TCT CAG TCT CAG AGA TTC 3600
Ala ~y8 P.~p T ys Val A~n Glu Cy~ Val Arg Ser Gln Ser Gln Arg Phe
1185 ll90 1195 1200
GGA TTC TGT GGT AAT GGT AC~ CAT TTG TTT TCA CTA GCA AAT GCA GCA 3648
Gly Phe Cy~ Gly A~n Gly Thr His Leu Ph~ Ser Leu Ala Asn Ala Ala
1205 1210 1215
CCA AAT GGC ATG ATT TTC TTT CAT ACA GTA CTA T'rA CCA ACA GCT TAT 3696
Pro Aqn Gly Met Ile Phe Phe ~ Thr Val Leu ~u Pro Thr Ala Tyr
1220 1225 1230
GAA ACT GTA ACA GCT TGG TCA GGT ATT TGT GCT TCA GAT GGC GAT CGC 3744
Glu Thr Val Thr Ala Trp Ser Gly Ile Cy8 Ala Ser ABP Cly asp Arg
1235 1240 1245
SUBSTITUTE SHEE~
WO 93/23422 PCI/US93/04384~
2 ~ 9 8 !;-
ACT TTC GGA CTT GTC GTT AAA GAT GTG CAG TTG ACG TTG TTT CGT AAT 3792
Thr Phe Gly Leu Val Val Lys Asp Val Gln Leu Thr Leu Phe Arg Asn
1250 1255 1260
CTA GAT GAC AAG T~C TAT TTG ACC CCC AGA ACT ATG TAT CAG CCT AGA 3840
Leu Asp Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln Pro Arg
1265 1270 1275 1280
GTT GCA ACT AGT TCT GAT TTT GTT CAA ATT GAA GGG TGT GAT GTG TTG 3888
Val Ala Thr S~r Ser Asp PhQ Val Gln I1R C1U Cly Cy~ Asp Val Leu
1285 1290 1295
~TT GTC A~C GCG ACT GTA ATT GAT TTG CCT AGT ATT ATA CCT GAC TAT 3936
Phe Val Asn Ala Thr Val Ile Asp Leu Pro Ser Ile Ile Pro A~p Tyr
1300 1305 1310
ATT GAC ATT AAT CAA ACT GTT CAA GAC ATA TTA GAA ~AT TAC AGA CCA 3984
Ile ~8p Ile A~n Gln Thr Val Gln Asp Il~ L~u Glu Asn Tyr Arg Pro
1315 1320 1~25
AAC TGG ACT GTA CCT GAA TTT A Q CTT GAT ATT ~TC AAC GCA ACC TAT 4032
Asn Trp Thr Val Pro Glu Ph~ Thr Leu Asp Ile P~ Asn Als ~hr Tyr
1330 1335 1340
TTA AAT CTG ACT GGT GAA ATT GAT GAC TTA GAG TTT AGG TCA GAA AAG 4080
Leu Asn Leu Thr Gly Glu Ile Asp Asp Leu Glu Phe Arg S~r Glu Ly~
1345 1350 1355 1360
C~A CAT AAC ACT ACA GTA GAA CTS GCC ATT CSC ATT GAT ACC ATT AAT 4128
Leu Hls Asn Thr Thr Val Glu Leu Ala Ile Leu Ile A~p Thr Ile Asn
1365 13~0 1375
AAT ACA TTA GTC AAT CTT GAA TGG CTC AAT AGA ATT GAA AC~ TAT GTA 4176
Asn Thr Leu Val Asn Leu Glu Trp Leu Asn Arg Il~ Glu Thr Tyr Val
1380 1385 1390
AAA TGG CCT TGG ~AT GTG SGG C~A CSG ATA CG~ CSA GTA GTA GTA ~TT 4224
Lys Trp Pro ~rp Syr Yal Trp Leu Leu Ile Gly Leu Val Val Val Phe
1395 1400 1405
TGC ATA CCA TTA CTG CTA TTT TGC TGT TTT AGC ACA GGT TGT TGT GGA 4272
Cy~ Ile Pro Leu Leu Leu Phe Cys Cys Ph~ Scr Th~ Cly Cy~ Cy~ Gly
1410 1415 1420
TGC ATA GGT TGT TTA GGA AGT TGT TGT CAC ~CT A~A TGT AGS AGA AGA 4320
Cys Ile Gly Cya Leu Gly Ser Cy8 Cy~ ~is Ser Ile Cy~ S~r Arg Arg
1425 1430 1435 1440
CAA TTT GAA TAT T~T GAA CCA ATT GAA AAA GTG CAT GTC C~C 4362
Gln Phe Glu Tyr Tyr Glu Pro Ile Glu EYH Val H1~ Val 8is
1445 1450
TAA 436S
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQOENCE CXARACTE~ISTICS:
(A) LENGTH: 1454 amino acids
(B) TYPE: amino acid
(D) TOPOEOGY: linear
ii ) MOLECULE TYPE: protein
SUBSTITIJTE SHEET
. W O 93/23422 2 1 3 ~ ~ 9 8 PCT/USg3/04384
~; ~ .............................. .
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ile Val Leu Val Thr Cy8 Leu Leu Leu Leu Cye Ser Tyr His Thr
1 S 10 15
Val Leu Ser Thr Thr Aen Asn Glu Cy8 Ile Gln Val Asn Val Thr Gln
~0
Leu Ala GIy Aen Glu Ren Leu Ile Arg A~p Phe Leu Ph~ Ser Aen Phe
LYQ Glu Glu Gly Ser Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
Trp Tyr A~n Cy~ Ser Arg Thr Ala Gln Thr Thr Ala Ph~ Gln Syr Phe
Asn Asn Ile H~e Ala Phe Tyr Phe Val Met Glu Ala Met Glu Asn Ser
9S
Thr Gly Aen Ala Arg Gly Ly~ Pro Leu Leu Phe His Val ~is Gly Glu
100 105 110
Pro Val Se Val Ile Ile Tyr Ile Ser Ala Tyr Arg A p A~p Val Gln
Gln Arg ~ro Leu Leu Lys Hi~ Gly Leu Val Cys Ile Thr Lys Asn Arg
130 135 140
~' 9 Ile AQn Syr Glu Gln Phe Thr Ser Asn GIn Trp Asn Ser Thr Cy8
~hr Gly Ala Aep Arg Lys Ile Pro Phe Ser Val Ile Pro Shr Asp Asn
165 1~0 175
Gly Thr Ly~ Ile Syr Gly Leu Glu Trp Asn Asp Asp Phe Val Thr Ala
. : 180 185 190
,
Tyr Ile Ser Gly Arg Ser Tyr Hle Leu Asn Ile Asn Thr Aen Trp Phe
195 200 ~ 205
A n Asn Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
210 215 220
Tyr Ser Ala Ala Tyr Ala Tyr Gln Gly Val Ser A8n Phe Thx Tyr Tyr
225 230 235 240
Ly~ Leu A~n Asn Thr A~n Gly Leu Lye Thr Tyr G1U LeU Cye Glu A~p
: ~ 245 250 255
Tyr Glu Hi8 Cyg Thr Gly Tyr Ala Thr A~n Val Phe Ala Pro Thr Ser
. i 260 ~ / 265 270
Gly Gly Tyr Il~ Pro A~p Gly Phe Ser Phe A8n Asn Trp PhQ Leu Leu
275 280 285
Thr Aen Ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr Aen Gln Pro
290 295 300
Leu Leu Ile A~n Cy8 Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
305 310 315 ~20
SUBSTITUTE SHEET
WO 93~3422 PCr/US93/04384
213~Qg8
62
Gln Glu Phe Cy~ Phe Glu Gly Ala Gln Ph Ser Gln Cy8 Asn Gly Val
Se~ Leu Asn A~n Thr Val Asp Val Ile Arg Phs A~n Leu Asn Phe Thr
340 345 350
Ala A~p Val Gln Ser Gly Met Gly Ala Thr Val Phe Ser Leu Asn Thr
355 360 365
Thr Gly Gly Val Ile Leu Glu Ile Ser Cy~ Tyr Ser Asp Thr Val Ser
Glu Ser Ser Ser Tyr Ser Tyr Gly Glu Ile Pro Pha Oly Il~ Thr Asp
Gly Pro Arg Tyr Cys Tyr Val Leu Tyr Asn Gly Thr Ala Leu Lys Tyr
40S 4}0 415
Le~ Gly Thr Leu Pro Pso Ser Val Lys Glu Ile Ala I1Q Ser Lys Trp
Gly H~ Phe Tyr Ile A~n Gly Tyr Asn Phe Phe Ser Thr Phe Pro Ile
Gly Cy9 Ile Ser Phe A~n Leu Thr Thr Gly Ala Ser Gly Ala Phe Trp
45U 455 460
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu Asn
465 470 475 480
Thr Ala Ile Ly~ Asn Val Thr Tyr Cys Asn Ser ~18 Il~ Asn Asn Ile
485 490 495
Ly~ Cys Ser Gln L~u Thr Ala A~n Leu Aan Asn Cly Ph~ Tyr Pro Val
500 505 S10
Ala.Ser Ser Glu Val Gly Phe Val Asn Lys Ser Val Yal Leu Leu Pro
515 520 525
Ser Phe Phe Thr His Thr Ala Val A8n Ile Thr Ile Aap Leu Gly Met
530 535 540
Ly~ Leu Ser Gly ~yr Gly Gln Pro Ile Ala S~r Thr Leu Ser A~n Ile
545 550 555 560
Thr Leu Pro Met &1 A~p A-n A~n Thr A;p Val Tyr Cys Ile Arg S0r
A~n Gln Pha Ser Val Tyr Val Pro 5er Thr Cy8 Lys Ser Ser Leu Trp
580 585 590
Asp Asn Ile Phe A9n Gln A8p Cys Thr Asp Val Leu Glu Ala Thr Ala
595 600 605
Val Ile Lys Thr Gly Thr Cy5 Pro Phe Ser Phe Aqp Lys Leu Asn A~n
610 515 620
Tyr Leu Thr Phe A~n Ly8 Phe Cy8 Leu Ser LQU Ser Pro Val Gly Ala
625 630 635 640
A3n Cy8 Ly~ Phe A~p Val Ala Ala Arg Thr Arg Thr Asn Glu Gln VB1
645 6S0 655
SUBSTITUTE SHEET
WO 93/23422 . 2 1 3 ~ ~ 9 8 PCT/US93/04384
63
Val Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly A~p Asn Ile Val Gly
660 665 670
Val Pro Ser A~p A~n Ser Gly Leu Hi~ Aqp Leu Ser Val Leu Hl~ Leu
67~ 680 685
A~p Ser Cy~ Thr Aap Tyr A~n Ile Tyr Gly Arg Thr Gly V~l Gly Ile
690 695 700
Ile Arg Arg Thr Asn Ser Thr Leu Leu Ser Gly Leu Tyr ~yr Th. Ser
70s 710 715 720
Leu Ser Gly Aqp Leu Leu Gly Phe Ly~ Asn Val Ser Asp Gly Val Ile
~2s 730 735
yr Ser Val Thr Pro Cya A~p Val Ser Ala Gln Ala Al~ Val Ile A~p
740 745 750
Gly Ala Ile Val Gly Ala Met Thr Ser Ile A~n Ser Glu L8U Leu Gly
7ss 760 765
L~u Thr ~i~ Trp Thr Thr Thr Pro A~n Phe Tyr Tyr Tyr S~r I1Q Tyr
770 775 780
Asn Tyr Thr Ser Glu Arg Thr Arg Gly Thr Ala Ile A~p Ser A~n Asp
~85 790 795 800
al Asp Cy~ Glu Pro Val Ile Thr Tyr Ser As~ Ile Gly Val Cy~ Lys
805 810 815
~n Gly A}2 Leu Val Phe Ile Asn Val Thr H~ 8 Ser A~p Gly Aap Val
825 830
Gln Pro Ile Ser Thr Gly A~n Val Thr Ile Pro Thr Asn Phe Thr Ile
835 840 845
Ser Val Gln Val Glu Tyr Met Gln Val Tyr Thr Thr Pro Val Ser Ile
8s0 85s 860
Asp Cys Ala Arg Tyr Val Cys Asn Gly Asn Pro Arg Cys A~n Lys Leu
865 870 87S 8~0
eu Thr Gln Tyr Val ser Ala Cys Gln Thr Ile Glu Gln Ala Leu Ala
ass 890 895
et Gly Ala Arg Lau Glu ~8n Met Glu Val A6p sQr M~t L~u PhQ Val
900 90S 910
Ser Glu A~n Ala Leu Lys Leu Ala Ser Val Glu Ala Phe A~n Ser Thr
915 920 925
Glu Asn Leu Asp Pro Ile Tyr Lys Glu Trp Pro S-r Ile Gly Cly Ser
930 935 940
rp Leu Gly Gly Leu LYB Asp Ile Leu Pro Ser ~i8 A~n Ser LYB Arg
950 9SS 960
ys Tyr Gly ser Ala Ile Glu A~p Leu Leu Phe Asp Lys Val Val Thr
965 970 975
er Gly Leu Gly Thr Val A8p Glu Asp Tyr Ly~ Ars Cys Thr Gly Gly
980 985 990
SUBSTITUl-l~ SHEET
WO 93/2 3422 P~/US93/0438~`
~., .. ~
2l3 i~83~
64
Tyr A~p I1Q Ala Asp Leu Val Cy Ala ~ln Tyr Tyr Asn Gly Ile Met
Val Leu Pro Gly Val Ala A n ~la Asp Lys Met hr Met ~y~ Thr Ala
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
Ala Ile Pro Phe Ala Yal Ala Val Gln Ala Arg Leu A~n Tyr Val Ala
1045 1050 1055
Leu Gln Thr Asp Val Leu A~n Lys Asn Gln Gln Ile Leu Ala Asn Ala
1060 1065 1070
Phe Asn Glln Ala Ile Gly Asn Ile Thr Gln Ala Phe Gly Ly~ Val Asn
Asp Ala Ile Hi~ Gln Thr Ser Gln Gly Leu Ala Thr VA1 Ala LYR Ala
Leu Ala Lys Val Gln Asp Val Val Asn ~hr Gln Gly Gln Ala Leu ser
1105 1110 ~ 1115 1120
Hi~ Leu Thr Val Gln Leu Gln A~n Asn Phe Gln Ala Ile Ser Ser Ser
1125 1130 1135
Ile Ser Asp Ile Tyr A~n Arg Leu A3p Glu ~eu Ser Ala A;p Ala Gln
Val Asp Arg Leu Ile ~hr Gly Arg Leu Thr Ala Leu Asn Ala Phe Val
1155 1160 1165
Ser Gln Thr Lau Thr Arg Gln Ala Glu Val Arg Ala S~r Arg Gln Leu
1170 1175 1180
Ala Lys Asp Lys Val A3n Glu Cy~ Val Arg Ser Gln Ser Gln Arg Phe
1190 1195 1200
Gly Phe Cy3 Gly A~n Gly ~hr Hi~ Leu Phe Ser Leu Ala Asn Ala Ala
1205 1~10 1215
Pro Asn Gly Met Ile Phe Phe His Thr Val Leu Leu Pro Thr Ala Tyr
1220 1225 1230
Glu Thr Val Thr Ala ~rp Ser Gly Il~ Cy~ Ala Sar A~p Gly A8p Arg
1235 1240 1245
Thr Ph~ Gly Leu Val Val LYR Asp Val Gln Leu Thr Leu PhQ Arg Asn
1250 1255 1260
Leu A~p A~p LYB Ph~ Tyr ~eu Thr Pro Arg Thr Met Tyr Gln Pro Arg
1265 ` 12~0 1275 1280
Val Ala Thr Ser Ser Asp Phe Val Gln Ile Glu Gly Cys Asp Val ~eu
1285 1290 1295
Phe Val Asn Ala Thr Val Ile Aep Leu Pro Ser Ile Ile Pro A~p Tyr
1300 1305 1310
Ile Asp Ile Asn Gln Thr Val Gln Asp Ile Le~ Glu A~n Tyr Arg Pro
1315 1320 1325
SUBSTITUTE SHEET
~YO 93/23422 PCT/USg3/04384
.~,, 2~3l~g8
A~n Trp Thr Val Pro Glu Phe Thr Leu A~p Ile Phe A~n Ala Thr Tyr
1330 1335 1340
Leu Asn Leu Thr Gly Glu Ile Asp A~p Leu Glu Phe Arg Ser Glu LYB
1345 1~50 1355 1360
~eu His Aan Thr Thr Val Glu Leu Ala Ile Leu Ile A~p Thr Ile A~n
1~65 1370 13~5
A~n Thr Leu Val Asn Leu Glu Trp Leu A~n Arg Ile Glu Thr Tyr Val
1380 13~5 1390
Lys Trp Pro Trp Tyr Val Trp Leu Leu Ile Gly L~u V~l Val Val Phe
1395 1400 1405
Cys Ile Pro Leu Leu Leu Phe Cy~ Cys Phe Ser Thr Cly Cys Cya Gly
1410 14IS 1420
Cy~ Ile Gly Cy~ Leu Gly Ser Cy~ Cya His Ser Ile Cy~ Ser Arg Arg
1425 1430 1435 1440
Gln Phe Glu Tyr Tyr Glu Pro Ile Glu Lys Val Hi~ Val Hi~
1445 1450
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGS~: 2246 base pair~
~ (B) T~PEs nucleic acid
:: : (C) STRANDEDNESS: doubls
(D) TOPOLOGY: unknown
(li) MOLECU~E TYPE: DNA ~ genomic)
( ix ) FEATUPE:
(A) NAME/XEYs CDS
(B) LOCATION: 1..2244
(x~) SEQUENCE DESCRIPTION: SEQ ID NO:3:
-:ATG ATT GTG C$C GTA ACT $GC CTC T~G TTG rTA TGT TCA ~TAC CAC ACA 48
Met I1Q Val Leu Yal Thr Cys Leu ~eu Leu Leu Cys Ser ~yr H~& Thr
- 1 5 ~ ~ 10 15
GTT TTG AGT ACA A~A AAT AAT GAA TGC ~TA QA GTT AAC GT~ ACA CAA 96
Val ~eu S~r Thr Thr A8n A~n:Glu Cy~ Ile Gln Val A~n Val Thr Gln
20 : 25 30
TTG GCT GGC AAT GAA AAC CTT ATC AGA GAT TTT CTG TTT AGT AAC TTT 144
! I ' Leu Ala GIy A~n Glu- Asn Leu Ile Arg A~p Phe Leu Phe Ser A~n Phe
35 40 45
~:- . AAA GAA GAA GGA A~T GTA GTT G$T GGT GG$ TAT TAC CCT ACA GAG G$G 192
: : Ly~ Glu Glu Gly Ser Val Val Val Gly Gly $yr Tyr Pro $hr Glu Val
: 5~ 55 60
TGG TAC AAC TGC TCT AGA ACA GCT CGA ~CT ACT GCC TTT C~G TAT TTT 240
Trp Tyr A~n Cy~ Ser Arg Thr Ala Arg Thr $hr Ala Phe Gln Tyr Phe
65 70 75 80
~'
SUBSTITUTE SHEET
WO~3/23422 PCr/US93/0~384 ~
2~3~898
AAT AAT ATA GAT GCC TTT TAT TTT GTT ATG GAA GCC ATG GAA AAT AGC 288
Asn Aqn Ile Hi~ Ala Phe Tyr Phe Val Met Glu Ala Met Clu Asn Ser
85 90 95
ACT GGT AAT GCA GGT GGT AAA CCA TTA TTA TTT CAT GTG CAT GGT GAG 336
Thr Gly Asn Ala Arg Gly LYB Pro LeU Leu Phe H~s Val His Gly Glu
100 105 110
CCT GTT AGT GTT ATT ATA TAT A~A TCG GCT TAT AGC GA~ GAT GTG CAA 384
Pro Val Ser Val Ile Il~ Tyr Ile Ser Ala Tyr As~ Asp A~p Val Gln
115 120 125
CAA AGG CCC CTT TTA GAA CAT GGG TTA G~G TGC ATA ACT ~AA AAT CGC 432
Gln Arg Pro Leu Leu Glu H1~ Gly Leu Val Cy~ Ile Thr Lys Asn Arg
130 135 140
CAT ATT AAC TAT GAA QA TTC ACC TCC AAC CAG TGG AAT TCC ACA ~GT 480
~L~ A~n Tyr Glu Gln Ph~ Thr SQr A~n Gln Trp Asn SQr $hr Cy~
145 lS0 155 160
ACG GGT GCT GAC AGA AAA ATT CCT TTC TCT GTC ATA CCC ACG GAC AAT 52 8
Thr Gly Ala Asp Arg Lys Ile Pro Phe Ser Val Il~ Pro Thr Asp Asn
165 170 175
GGA ACA AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TT~ GTT ACA GCT 576
Gly Thr Ly~ Ile Tyr Gly Lau Glu Trp Asn Asp Asp Ph~ Val Thr Ala
180 18~ 190
TAT ATT AGT GGT CGT TCT TAT CAC TTG AAC ATC AAT ACT AAT TGG TTT 624
Tyr Ile Ser Gly Arg Ser Tyr Hls Leu Asn Il~ Asn Thr Asn Trp Phe
195 2~0 205
AAC AAT GTC ACA CTT TTG TAT TCA CGC TC~ AGC ACT GCT ACC TGC GAA 672
A6n Asn Val Thr Lau Leu ~yr Sffr Arg S~r S~r Thr Ala Thr Trp Clu
210 215 220
TAC AGT GCT GCA TAT GCT TAC CAA GGT GTT TCS AAC TTC ACT TA~ TAC 720
Tyr Ser Ala Ala $yr Ala Tyr Gln Gly Val ser Asn Phe Thr Tyr Tyr
22; 230 235 240
AAG TTA AAT AAC ACC AAT GGT CTA AAA ACC TAT GAA TTA TGT GAA GAT 768
Lys ~eu A~n Asn Thr Asn Gly Leu Lys Thr ~yr Clu Leu Cy~ Glu Aap
i~ 245 250 255
: - TAT GAA CAT $GC ACT GGC TAT GCT ACC AAT GTA TTS GCT CCG ACA ~CA 816
Tyr qlu HLs Cy~ Thr Gly Tyr Ala Thr A~n Val Phe A1B Pro Thr Ser
: 260 - 265 270
GGT GGT TAC ATA CCT GAT GGA T$T AGT TTT AAT AAT TGC TTC TTC CTT 864
Gly Gly Tyr Ile Pro Asp Gly Ph~ S~r Pha A~n A~n Trp Ph~ ~u ~ ~u
275 280 285
ACA AAT AGT $CC ACT TTT GTT AGT GGC AGG TTT GTA ACA ~AT CAA CCA 912
Thr A~n Ser Ser Thr Phe Val Ser Gly Arg PhQ Val Thr Asn Gln Pro
290 295 300
TTA TTG ATT AAT TGC TTG TGG CCA GTG CCC AGT TTT GGT GTA GCA GCA 960
L~u Leu Ile A~n Cy~ Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
305 310 315 320
CAA GAA TTT TGT TTT GA~ GGT GCA CAG TTT AGC CAA TGT AAT GGT GTG 1008
Gln Glu Phe Cy9 Phe Glu Gly Ala Gln Phe Ser Gln Cy8 Asn Gly Val
325 330 335
SUBSTITUTE SHEET
W O g3/23422 PCT/~S93/04384
``` 213489~
67
TCT TTA AAT AAC ACA GTG GAT GTT ATT AGA TTC AAC CTT AAT TTC ACT 10 5 6
Ser Leu A~n Asn Thr Val Asp Val Ile Arg Phe A~n Leu A~n Phe Thr
340 345 î50
GCA GAT GTA CAA TCT GGT ATG GGT GCC ACA GTA TTT TCA CTG AAT ACA 1104
Ala Asp Val Gln Ser Gly Met Gly Ala Thr Val Phe Ser L~u A~n Thr
355 360 365
ACA GGT GGT GTC ATT CTT GAA ATT TCA TGT TAT AGT GAC ACA GTG AGT 11 S 2
Thr Gly Gly Val Ile Leu Glu Ile Ser Cys Tyr Ser A~p Thr Val Ser
370 375 380
GAG TCT AGT TCT TAC AGT TAT GGT GAA ATC CCG TTC GGC ATA ACT GAC 1200
Glu Ser SQr Sar Tyr Ser Tyr Gly Glu Ile Pro Phe Gly Il~ Thr A~p
385 390 395 400
GGA CCA CGA TAC TGT TAT GTA CTT TAC AAT GGC ACA GCT CTT AAA TA~ 12 4 8
Gly Pro Arg Tyr Cyo Tyr Val Leu Tyr A~n Gly Thr Ala Leu Lys Tyr
405 410 415
TTA GGA ACA TTA CCA CCC AGT GTA AAG GAA ATT GCT ATT ACT AAG TGC 12 9 6
Leu Gly Shr Leu Pro Pro Ser Val Ly~ Glu Ile Ala Ile Ser Ly~ Trp
420 425 430
GGC CAT TTT TAT ATT AAT GGT TAC AAT TTC TTT AGC ACA TTT CCT ATT ; 13 4 4
Gly His Phe Tyr Ile A~n Gly Tyr A~n Phe Phe Ser Thr Phe Pro Ile
435 440 445
GGT TGT ATA SCT TTT AAT TTA ACC ACT GGT GTT AGT GGA GCT TTT TGG 13 9 2
Gly Gys: Ile S~r Pho Asn Leu Thr Thr Gly Val Ser Cly Ala PhQ Trp
450 ~ ~ 455 460
ACA ATT GCT TAC ACA TCG TAT ACT GAA GCA $TA GSA C~A GTT GAA AAC 14 4 0
Thr Ile Ala ~yr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu A~n
465~ 470 475 480
ACA GCT ATT AAA AAT GTC ACG TAT TGT AAC ACT CAC ATT AAT AAC ATT 1488
Thr Ala Ile Ly~ A~n Val Thr Tyr Cya A-n Ser H~s Ile A~n Asn Ile
485 490 495
AAA TGT TCT CAA C$T ~ ACT GCT AAT TTG AA$ AAT G¢A TTT TAT CCT GTT 15 3 6
Ly Cys Ser Gln Leu Thr Ala Asn Leu A~n A~n Gly Phe $yr Pro Val
500 ~ ~ 50S . S10
GCS TCA AGT GAA ~GTA GGT TTC G$T AAT AAG A¢$ GTT GTG TTA TTA CCT 1584
Ala Sar S~r Clu Val Cly Pho Val A~n Ly~ Ser V~l Val Lou I,~u Pro
515 ~ 520 525
AGC T$T TTC ACA TAC ACC GCT GTC AAT ATA ACC AT$ GAT C$$ GGT A$G 16 3 2
S er Phe Phe 'rhr $yr Thr Ala Val Asn Ile $hr Ile Asp Leu Gly Met
530 535 540
AAG CTT ACT GGT TA$ GGT CAA CCC ATA GCC TCG ACA CTA ACT AAC ATC 1680
Ly~ Leu Ser Gly Tyr Gly Gln Pro rlQ Ala S~r Thr L~u Ser A~n r1Q
545 550 555 560
ACA: CTA CC~ ATG CAG GAT AAC AAT ACT GAT GTG Tac TGT ATT CGT TCT 1728
Thr Leu Pro Met Gln A8p Asn Asn Thr A8p V~l Tyr Cy~ Ile Arg Ser
565 570 575
AAC CAA TTC $CA GT~ TAT GTT CAT TCC ACT TGC AAA AGT TCT TTA TGG 17 7 6
A~n Gln Pho Ser Val Tyr V~l H~EI Ser Thr Cys Lys Ser Ser I.eu Trp
580 585 S90
.
:; SUBSTITUTE SHEET
WO 93/23422 PCr/US93/0~3~4 -
2 1 3 1 ~ 9 8 d . r ~ .
68
GAC AAT ATC TTT AAT CAA GAC TGC ACG GAT ~TT TTA GAG GCT ACA GCT 1824
A8p A~ Ile Pht~t Asn Gln Asp Cys Thr Asp Val Leu Glu Ala Thr Ala
595 600 605
GTT ATA AAA AC~ GGT ACT TGT CCT TTC TCA TTT GAT AAA TTG AAC AAT 1872
Val Ile Lys Thr Gly Thr Cy~ Pro Phe Ser Phe Asp Lys Leu A3n Asn
61Q 615 620
TAC TTG ACT TTT AAC AAG TTC TGT TTC TCG TTG A5T CCT GTT GGT GCT 1 g 2 O
Tyr Leu Thr Phe Asn Lys Phe Cy~ Leu Ser Leu Ser Pro Val Gly Ala
625 630 635 640
AAT TGC AAG TTT GAT GTT GCT GCA CGT ACA AGA ACC AAT GAG CAG GTT 1968
Asn Cyt3 Lys Phe AsFtt Val Ala Ala Arg Thr Arg Thr Asn Glu Gln Val
645 650 655
GTT AGA AGT CTA TAT GTA ATA TAT GAA GAA GGA GAC AAC ATA GTG GGT 2016
Val Arg Ser Ltatu Tyr Val l1Q Tyr Glu Glu Gly Aap Asn Ile Val Gly
660 665 . 670
GTA CCG TCT GAT GAT AGC GGT CTG CAC GAT TTG TCT GTG CTA CAC CTA 2064
Val Pro Ser A~p Asp Ser Gly Leu Hl8 Asp Leu Ser Val Leu His Leu
675 680 685
GAC TCC TGT ACA GAT TAC AAT ATA TAT GGT AGA ACT GGT GTT CGT ATT 2112
A~p Ser Cy~ Thr Aap ~yr A n Ile Tyr Gly Arg Thr Gly Val Cly Ilta
ATT:AGA CGA ACT AAC AGT ACG CTA CTT AGT GGC TTA TAT TAC ACA TCA 2160
Ile~Arg Arg Thr A~n Ser Thr Leu L-u S-r Gly Leu Tyr Tyr Thr S r
CTA TC~ GGT aAT TTG TTA GCC TST AAA AAT CTT ACT GAT GGT tvTC A$T 2208
Leu Ser Gly A~p ~eu Leu Cly Pho ~-y~ A~n Val ser Atap Gly Val Ile
725 730 735
TAT TCT GTG ACG CCA TGT GAT GTA AGC GCA CAA GCG GC 2 2 4 6
Tyr Ser VaL Thr Pro Cy~t Asp Val Ser Ala Gln Ala
740 745
(2J INFORMATION FOR SEQ ID NO:4:
. ( L ) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 748 amtino atcids
~ ~ ~ (8) TYP~: amino ac~d
D) TOPO~OtGY: linear
(ii) MOLECULE TYPE: Ftrotein
t~ xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ile Val ~eu Val Thr Cy8 _eu _eu Leu _eu Cy8 Ser Tyr Hi~ ~hr
Val _eu Ser Thr Thr A~n A~n Glu Cy8 Ile Gln Val A~n Val Thr Gln
- Leu Ala Gly Agn Glu A~n Leu Ile Arg A~p Phe Leu Phe Ser A~n Phe
SUBSTITUTE SHEET
-WO 93/23422 PCr/US93J043~4
21 3 1898
69
Lys Glu Glu Gly Ser Val Val Val Gly Gly Tyr Tyr Pxo Thr Glu Val
Trp Tyr Asn CY8 Ser Arg Thr Ala Arg Thr Thr Ala Phe Gln Tyr Phe
Asn Asn Ile Hi~ Ala Phe Tyr Phe Val Met G1U Ala Mat Glu Asn S~r
~5 90 9S
Thr Gly Asn Ala Arg Gly Ly~ Pro Leu Leu Phe H1~ Val H~ Gly Glu
~00 105 110
Pro Val Ser Val I1Q I1e Tyr I1Q Ser Ala Tyr Arg A~p A~p Val Gln
llS 120 125
Gln Arg Pro Leu Leu Glu Hl~ Gly Leu Val Cyg Ile Thr Ly~ Asn Arg
130 135 140
His Ile Asn Tyr Glu Gln Phe Thr Ser Asn Gln Trp Asn Ser Thr Cy9
145 150 155 160
Thr Gly Ala A~p Arg Ly~ Ile Pro Phe Se~ Val Ile Pro Th~ A~p Asn
~65 170 17S
G1Y Thr Lys Ile Tyr Gly Le~ Glu Trp Asn Asp Asp Phe Val Thr Ala
180 185 190
Tyr Ile Ser Gly Arg Ser Tyr ~1~ Leu Asn Ile Asn Thr Asn Trp Phe
l9S 200 20~
Asn A~n Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp G1U
: -~ 21~ ~15 220
Tyr Ser Ala Ala Tyr Ala Tyr Gln Gly Val Ser A~n Phe Thr Tyr Tyr
Lys Leu Asn Asn Thr Asn Gly Leu Lys Thr Tyr Glu L~u Cy~ Glu ~sp
245 250 255
,, ~
Tyr Glu ~ 8: Cy~ Thr Gly Tyr Ala Thr A~n Val Phe Ala Pro Thr Ser
: ~ 250 265 270
,:
Gly Gly Tyr Ile Pro Asp Gly Phe Ser Phe A~n A~n Trp Phe Leu Leu
275 280 2~5
Thr Aan ser Ser Thr Phe Val Ser Gly Arg Phe Val ~hr A~n Gln Pro
290 295 300
:
: Leu ~eu Ile Asn Cy~ Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
305 310 315 320
l 1~GIn alu Phe Cy8 Phe Glu Gly Ala Gln Phe 5er Gln Cys Asn Gly Val
; 325 ` 330 335
Ser Leu Asn Asn Thr Val Asp Val Ile Arg Phe Asn Leu A~n Phe Thr
340 345 350
:Ala Asp Val Gln Ser Gly Met Gly Ala Thr Val Phe Ser Leu Asn Thr
;:~ 355 360 365
Thr Gly Gly Val Ile Lau Glu Ila S~r Cy~ Tyr Ser A~p Thr Val Ser
370 375 380
.
SUBSTITIJTE SHEET
WO 93/23422 ~ ~ ~ r~ PCr~US93/0438
Glu Ser Ser Ser Tyr Ser Tyr Gly Glu Ile Pro Phe Gly Ile Thr Asp
Gly Pro Arg Tyr Cy8 Tyr Val Leu Tyr A~n Gly Thr Ala Leu Lys Tyr~
405 410 415
Leu Gly Thr Lsu Pro Pro Ser Val Lys Glu Ile Ala Ile Ser Lys Trp
420 425 430
Gly His Phe Tyr Ile A~n Gly Tyr A~n Phe Phe Ser T~r Phe Pro Ile
4~5 440 445
Gly Cys Ile Ser Phe A~n Leu Thr Thr Gly Val Ser Gly Ala Phe Trp
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu Aqn
465 470 475 480
Thr Ala Ile Ly~ A~ Val Thr Tyr Cy~ Asn Ser ~i~ Ile Asn Asn Ile
485 490 495
Lys Cys Ser Gln Leu Thr Ala Asn Leu Asn Asn Gly Phe Tyr Pro Val
500 505 510
Ala ser Ser Glu Yal Gly Phe Val Asn Ly~ Ser Val Val L~u L~u Pro
515 520 525
Ser Phe Phe Thr ~yr Thr Ala Val A~n ~le Thr Ile ABP Leu Gly Met
Lys Leu Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu Ser A~n Ile
545 550 555 560
Thr L~u Pro Met Gln Asp Asn Asn Thr A p Val Tyr Cy~ Ile Arg Ser
Asn Gln Phe Ser Val Tyr Val Hi~ Ser Thr Cy8 Lys Ser Ser Leu Trp
580 585 590
Asp A~n Ile Phe Asn Gln Asp Cy3 Thr Asp Val Leu 51u Ala Thr Ala
595 600 605
Val Ile Ly~ Thr Gly Thr Cy~ Pro Phe Ser Phe A~p Lya Leu Asn Asn
610 615 620
Tyr Leu Thr Phe Asn Lys Phe Cy9 Leu Ser Leu Ser Pro Val Gly Ala
625 630 635 640
Asn Cy8 Lys Pha A~p Val Ala Ala Arg Thr Arg Thr A~n Glu Gln Val
645 650 655
. Val Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly A~p Asn Ile Val Gly
660 665 670
Val Pro Ser A9p Agp Ser Gly Leu Hi8 Asp Leu Ser Val Leu His Leu
675 680 685
Asp Ser Cys Thr Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Val Gly Ile
690 695 700
Ile Arg Arg Thr A~n Ser Thr Leu Leu Ser Gly Leu Tyr Tyr Thr Ser
705 710 715 720
SUBSTITUTE SHEET
W O 93/2342~ 2 1 3 -1 8 9 8 PCT/US93/04384
Leu Ser Gly Asp Leu Leu Gly Phe Ly~ A~n Val Ser A~ip Gly Val Ile
725 730 735
Tyr Ser Val Thr Pro Cy~ Aap Val Ser Ala Gln Ala
740 745
(2) INFO~ATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 2246 ~ase pair~
(B) TYPE: nucl~lc acld
(C) STRANDEDNESSs double
~D) TOPOLOGYs unknown
(ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(~) NAME/XEY: CDS
(8) LOCATION: 1..2244
~xi) SEQUENCE DESCRIP~ION: SEQ ID NO:5:
ATG ATT GTG CTC GTA ACT TGC CTC TTG TTG TTA TGT TCA TAC CAC ACA 4 8
Met I le Val Leu Val Thr Cyi9 Leu Leu Leu Leu Cy~ Ser Tyr H~B Thr
5 10 15
GTT TTG AGT ACA ACA AAT AAT GAA TGC ATA C~ CTT AAC GTA ACI~ CAA 9 6
Val Leu Ser Tbr Thr A~n Asn Glu Cy~ Gln Val Asn Val Thr Gln
20 25 30
TTG GCT GGC AAT GAA AAC CTT ATC AGA GAT TTT CTG TTT AGT AAC TTT 144
Leu Ala Gly Asn S;lu A~n Leu Ile Arg Asp Phe Lau Phe Ser A~n Phe
35 40 4S
AA~ GAA GAA Gt;:A AGT GTA GTT GTT GGT GGT TAT TAC CCT ACA GAG GTG 19 2
Ly~ Glu Glu Gly Ser Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Yal
50 55 60
TGG TAC AAC TGC TCT AGA ACA GCT CGA ACT AC'r GCC TTT CAG TAT TTT 240
Trp Tyr Ai~n Cyi~ Ser Arg ~hr Ala Arg Irhr Thr Al2 Phe Gln ~yr Phe
65 70 ~5 R0
AAT A.AT ATA CAT GCC TTT TAT TT2 GTT ATG GAA GCC ATG GAA AAT AGC 288
A~n Asn Ile His Ala Phe Tyr Ph~ Val 21et Glu Al~ M~t Glu A~n S~r
85 90 95
ACT GGT AAT GCA CGT GGT AAA CCA TTA T~A TTT CAT GTG CAT GGT GAG 3 3 6
Thr Gly Asn Ala Arg Gly Lys Pro Leu Leu Ph~ HiE Val H~ ~ Gly Gls~
100 105 110
CCT GTT AGT GTT P~TT ATA TAT ATA TCG GCT TAT AGG GAT GAT CTG CAA 384
Pro Val Ser Val Ile Ile Tyr Ile Ser Ala Tyr Arg A~p Asp Val Gln
115 120 125
CAA AGG CCC CTT TTA AAA CAT GGG TTA GTG TGC~ ATA ACT AAA AAT CGC . 432
Gln Arg Pro L~u Leu Ly0 Hi~ Gly L~u Val Cys Il~ Thr Lys Asn Arg
130 135 140
CAT ATT A~C TAT GAA CI~A TTC ACC TCC AAC C~G TGG AAT TCC ACA TGT 480
Hi Ile Asn Tyr Glu Gln Phe Thr Ser Asn Gln Trp Asn Ser Thr Cys
145 150 155 160
SU~STITUTE SHEt~T
W Q 93/23422 2 ~ 3 ~ 8 9 ~ PCT/US93/~43~_
ACG GGT GCT GAC AGA AAA ATT CCT TTC TCT GTC ATA CCC ACG GAC ~AT 528
Thr Gly Ala Asp Arg ~y8 Il~ Pro Phe Ser Val Ile Pro Thr ABP As~
165 170 175
GGA ACA AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TTT GTT ACA GCT 576
Gly Thr Lys Ile Tyr Gly Leu Glu Trp Asn Asp A~p Phe Val Thr Ala
180 185 190
TAT ATT AGT GGT CGT TCT TAT CAC TTG AAC ATC AAT ACT AAT TGG TTT 624
Tyr Ile Ser Gly Arg Ser Tyr H1~ L~u A~n Ila A~n Thr Asn Trp Phe
195 200 205
AAC AAT GTC ACA CTT TTG TAT TCA CGC TCA AGC ACT GCT ACC TGG GAA 672
AYn Asn Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
215 220
TAC AGT GCT GCA TAT GCT TAC CAA GGT GTT TCT A~C TTC ACT TAT TAC 720
Ty~ Ser Al~ Ala Tyr Ala Tyr Gln Gly Val S~r Aan Phe Thr Tyr Tyr
230 235 240
AAG TTA AAT AAC ACC AAT GGT CTA AAA ACC TAT GAA TTA TGT GAA GAT 768
Ly~ Leu Asn A~n Thr A~n Gly Leu Ly~ Thr Tyr Glu Leu Cys Glu A~p
245 250 255
TAT GAA CAT TGC ACT GGC TAT GCT ACC A~T GTA TTT GCT CCG ACA TCA 816
Tyr Glu His Cys Thr Gly Tyr Ala Thr Asn V~l Phe Ala Pro Thr Ser
260 265 2~0
GGT GGT TAC ATA CCT GAT GGA TTT AGT TTT AAT AAT TGG TTC TTG CTT 864
Gly Gly Tyr Ile Pro AQP Gly Phe 5er Phe A~n A3n Trp Phe ~eu Leu
275 280 285
ACA AAT AGT TCC ACT TTS GTT AGT GGC AGG TTT GTA ACA AAT CAA CCA 912
Thr Asn Ser Ser Thr Phe V~l Ser Gly Arg Phe V~l Thr A~n Gln Pro
295 300
TTA TTG ATT AAT TGC TTC TGG CCA GTG CCC AGT TTT GGT GTA GCA GCA 960
Leu Leu Ile A-n Cy~ Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
CAA G~A TTT TGT TTT G~A GGT GCA CAG TTT AGC CAA TGT AAT GGT GTG 1008
Gln Glu Pha Cy~ Phe Glu Gly Ala Gln Phe S~r Gln Cy~ Asn Gly Val
325 330 335
TCT TTA AAT AAC ACA GTG GAT GTT ATT AGA TTC A~C CTT AAT TTC ACT 1056
Ser Leu A~n A~n Thr Val A~p Val Ile Arg Pha A~n Leu A~n Phe Thr
340 345 350
GCA GAT GTA CAA TCT GGT ATG GGT GCT ACA GTA TTT TCA CTG AAT ACA 1104
Ala Asp Val Gln S2r Gly Met Gly Ala Thr Val Ph~ S~r Leu A~n Thr
355 360 365
; !: ACA GGT GGT GTC ATT CTT GAA ATT TCA TGT TAT AGT GAC ACA GTG AGT 1152
Thr Gly Gly Val 118 L~u Glu ~1Q Ser Cy~ Tyr Ser A~p Thr Val Ser
370 375 380
GAG TCT AGT TCT TAC AGT TAT GGT GAA ATC CCG TTC GGC ATA ACT GAC 1200
Glu Ses ser Sor Tyx Ser Tyr Gly Glu Ile Pro Ph~ Gly Ile Thr A~p
385 390 395 400
GGA CCA CGA TAC TGT TAT GTA CTT TAC AAT GGC ACA GCT CTT AAA TAT 1248
Gly Pro Arg Tyr Cys Tyr Val Leu Tyr Asn Gly Thr Ala Leu Lys Tyr
405 410 41S
SUBSTITlJTE SHEET
WO 93/23422 2 1 3 ~ 8 9 8 PCr/US93/04384
73
TTA GGA ACA TTA CCA CCC AGT GTA AAG GAA ATT GCT ATT AGT AAG TGG 1296Leu Gly Thr Leu Pro Pro Ser Val Ly~ Glu Ile Ala Ile Ser Lys Trp
. 420 425 430
GGC CAT TTT TAT ATT AAT GGT TAC AAT TTC TTT AGC ACA TTT CCT ATT~ 1344
Gly Hi~ Phe Tyr Ile Asn Gly Tyr A~n Phe Phe Ser Thr Phe Pro Ile
435 440 445
GAT TGT ATA TCT TTT AAT TTA ACC ACT GGT GTT AGT GGA GCT TTT TGG 1392
AqP CY9 Ile Ser Phe Asn Leu Thr Thr Gly Val Ser Gly Ala Phe Trp
450 455 460
ACA ATT GCT TAC ACA TCG TAT ACT GAA GCA TTA GTA CAA GTT GAA AAC 1440
Thr Ile Rl~ Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln V~l Glu Asn
465 470 475 480
ACA GCT ATT AAA AAT GTG ACG TAT TGT AAC AGT CAC ATT AAT AAC ATT 1488
Thr Ala Ile Ly3 Asn Val Thr Tyr Cy~ Asn Ser Hi~ Ile Asn A~n Ile
485 490 495
AAA TGT TCT CAA CTT ACT GCT AAT TTG AAT AAT GGA TTT TAT CCT GTT 1536
Lys Cy~ Ser Gln Leu Thr Ala A~n Leu A~n ~sn Gly Phe ~yr Pro Val
500 505 510
GCT TCA AGT GAA GTA GGT TTC GTT AAT AAG AGT GTT GTG TTA TTA CCT : 1584
Ala Ser Ser Glu Val Gly Phe Val Asn Ly~ S~r Val Val L~u Leu Pro
515 520 52~
AGC TTT TTC ACA TAC ACC GCT GTC AAT ATA ACC ATT GAT CTT GGT ATG 1632
Ser Phe Phe Thr Tyr Thr Ala Val A~n Il~ Thr Il~ A~p LQU Gly M~t
530 535 540
AAG CTT AGT GGT TAT GGT CAA CCC ATA GCC TCG ACA CTA AGT AAC ATC 1680
Lys Le~ Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu Ser Asn Ile
545 550 555 560
ACA CTA CCA ATG CAG GAT AAC AAT ACT GAT GTG TAC TGT ATT CGT TCT 1728
Thr Leu Pro MQt Gln Asp Asn A~n Thr Asp V~l Tyr Cy~ Ile Arg S~r
5~5 570 575
AAC CAA TTC TCA GTT TAT GTT CAT TCC ACT TGC AAA AGT TCT TTA TGG 17 7 6Asn Gln Phe Ser Va1 TYr Va1 Hi~ Ser Thr Cy~ Lya Ser ser Leu Trp
S80 5~5 590
GAC AAT ATT TTT AAT CAA GAC TCC ACG GAT GTT TTA GAG GCT ACA GCT 1824
Asp Asn Ile Phe A~n Gln ARP CY8 Thr Asp V~l Leu Glu Al~ Thr Ala
595 600 605
GTT ATA AAA ACT GGT ACT TGT CCT TTC TCA TTT GAT ~AA TTG AAC AAT 1872
Val Ile Ly~ Thr Gly Thr Cy8 Pro Phe Ser Phe Asp Ly~ Leu Asn Asn
610 615 620
TAC TTG ACT TTT AAC AAG TTC TGT TTG TCC TTG AGT CCT GTT GGT GCT 1920
Tyr Leu Thr Phe As~ Lys Phe Cys Leu Ser LRU S~r Pro Val Gly Ala
625 630 635 640
AAT TGC AAG TTT GAT GTT GCT GCA CGT ACA AGA ACC AAT GAG CAG GTT 1968
Asn Cys Lys Phe A~p Val Ala Ala Arg Thr Arg Thr A8n Glu Gln Val
645 650 655
GTT AGA AGT CTA TAT GTA aTa TAT GAA GAA GGA CAC AAC ATA GTG aGT 2016
Val Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly A8p Asn Il~ V~l Gly
660 665 570
SUBSTITUTE SHEET
. . . . ~ .
W O 93/23422 PCT/US93/~4384
8 9 8
74
GTA CCG TCT GAT GAT AGC GGT CTG CP.C GAT TTG TCT GTG CTA CAC CTA 2064
Val Pro Ser A~p Asp Ser Gly Leu ~18 A~p Leu Ser V~l L~u Hi~ Leu
675 680 685
GAC TCC TGT ACA GAT TAC AAT ATA TAT GGT AGA ACT GGT GTT GGT ATT 2 l 12
A~p Ser Cy8 Thr Asp Tyr A n Ile Tyr Gly Arg Thr Gly Val Gly Ile
ATT AGA CGA ACT AAC AGT ACG CTA CTT AGT GGC TTA TAT TAC ACA TCA 2160
Ile Arg Arg Thr A~ Ser Thr Leu Leu Ser Gly Leu Tyr Tyr Thr Ser
CTA TCA GGT GAT TTG TTA GGC TTT AAA A~T GTT AGT GAT GGT GTC ATT 2208
Leu Ser Gly Asp Leu Leu Gly Phe Lys Asn Val Sar Asp Gly Val Ile
TAT T T GTG ACC CCA TGT GAT GTA AGC GCA CAA GCG GC 2246
~yr Ser Val Thr Pro Cys Asp Val Ser Ala Gln Al~
(2) INFO~MATION FOR SEQ ID No:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 748 amino acide
(B) TYP~: am~no ~cid
(D) TOPOLOGY: llnear
(ii) MOLECULE TYPE: protein
(xi) SEQUE~CE DESCRIPTION: SEQ ID NO:6:
Met Il~ Val Leu Val $hr Cy~ Leu ~eu Leu Leu Cy~ Ser Tyr H~ $hr
Val Leu Ser Thr Thr A~n Asn G1U Cys Ile Gln Val A8n Val Thr Gln
20 2S 30
Leu Ala Gly Asn Glu Asn Leu ~le Arg Asp Phe Leu Phe Ser Asn Ph~
35 40 45
Lys Glu Glu Gly Ser Val Val Val Gly Gly $yr ~yr Pro Thr Glu Val
50 S5 60
Trp Tyr Asn Cy8 Ser Arg Thr Ala Arg Thr Thr Ala PheiGln Tyr Phe
Asn A n Ile Hi~ Ala Phe Tyr Phe Val Met Glu Ala Mat Glu Asn Ser
85 90 95
Thr Gly Asn Ala Arg Gly Lys Pro Leu Leu Phe Hls Val H1~ Gly Glu
100 105 110
Pro Val Ser Val Ile Ile Tyr Ile Ser Ala Tyr Arg Asp Asp Val Gln
115 120 125
Gln Arg Pro Leu Leu Ly~ His Gly Leu Val Cy~ Ila Thx Lys As~ Arg
130 135 140
Hi~ Ile Asn Tyr Glu Gln Phe Thr Ser Asn Gln Trp Asn Ser Thr Cy~ -
145 150 155 160
SUBST17UTE SHEET
W O 93/234~2 2 1 3 1 ~ 9 8 P~T/U~g3/04384
Thr Gly Ala Asp Arg Ly~ Ile Pro Phe Ser Val Ile Pro Thr A~p A~n
165 170 175
ly Thr Lys Ile Tyr Gly Leu Glu Trp Asn A~p Asp Phe Val Thr Ala
180 185 l90
Tyr Ile Ser Gly Arg Ser ~yr ~iB Leu Asn Ile A~n Thr Asn Trp Phe
195 200 205
A~n Asn Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
210 215 220
Tyr Ser Ala Ala Tyr Ala Tyr Gln Gly Val Ser Asn Ph~ Thr Tyr Tyr
225 230 235 240
y~ Leu A3n Asn Thr Asn Gly Leu Lys Thr Tyr Glu Leu Cy~ Glu Asp
245 250 255
yr Glu Hi~ Cy~ Thr Gly Tyr Ala Thr Asn Val Phe Ala Pro Thr Ser
260 265 ~70
Gly Gly Tyr Ile Pro A8p Gly Phe Ser Phe Asn A~n Trp Phe Leu Leu
275 280 285
Thr A~n Ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr Asn Gln Pro
290 295 300
Leu Leu Ile A~n Cy8 L~u Trp Pro Val Pro Ser Phe Gly Val Ala Ala
305 310 315 320
ln Glu Phe Cys Phe Glu Gly Ala Gln Phe Ser Gln CYB Asn Gly Val
325 330 ~35
er Leu Asn Asn Thr Val Asp Yal Ile Arg Phe Asn Leu Asn Phe Thr
340 345 350
Ala A3p Val Gln Ser Gly Met Gly Ala Thr Val Ph~ Ser Leu Ann Thr
355 360 ~65
Thr Gly Gly Val Ile Leu Glu Ile Ser Cy8 Tyr Ser Asp Thr Val Ser
3~0 375 ~80
Glu Ser Ser Ser Tyr Ser Tyr Gly Glu Ile Pro Phe Gly I1Q Thr Asp
385 390 395 400
ly Pro A~g Tyr Cy~ Tyr V~l Leu $yr A~n Gly Thr Al~ Leu LYB Tyr
405 41~ 415
eu Gly Thr Leu Pro Pro Ser Val Lys Glu Ile Ala Ile ser Lys Trp
420 425 430
ly Hi~ Phe Tyr Il~ Aan Gly Tyr A~n Phe Phe Ser Thr Pho Pro Ile
435 440 445
Asp Cys Ile Ser Phe Asn Leu Thr Thr Gly Val Ser Gly Ala Phe Trp
450 455 460
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu A3n
465 470 475 480
hr Ala Ile Lys A~n Val ~hr Tyr Cy8 Asn Ser Hl~ Asn Aon Ile
485 490 495
SUBSTITUTE SHEET
WO 93/23422 2 i 3 4 ~ 9 8 PCT/US93tO4384
76
Lys Cya Ser Gln Leu Thr Ala Asn Leu A~n A~n Gly Phe yr Pro Val
Ala Ser Ser Glu Val Gly Phe VaI A~n Lys Ser Val Val Leu Leu Pro
Ser Phe Phe Thr Tyr Thr Ala Val Asn Ile Thr Il A8p Leu Gly Met
Lys Leu Ser Gly ~yr GIy Gln Pro Ile Ala Ser Thr L~u Ses A~n Ile
Thr Leu Pro Met Gln Asp Asn A~n Thr Asp Val Tyr cys Ile Arg Ser
A~n Gln Phe Sez Val Tyr Val Hi~ Ser Thr Cy8 Lys Ser Ser Leu TFp
Asp A~n Ile Phe Asn GLn A3p Cys Thr A~p Val Leu Glu Ala Thr Ala
595 60g 605
Val Ile Lys Thr Gly Thr Cy Pro Phe Ses Phe Asp Lys Lau Asn Asn
Tyr Leu Thr Phe Asn Lys Phe Cy~ Leu Ser Leu Ser Pro Val Gly Ala
A~n Cys Lys Phe Asp Val Ala Ala Arg ~hr Arg Thr Asn Glu Gln Val
645 650 65S
Val Arg Ser L u Tyr Val Il~ Tyr Glu alu Gly A-p Asn Ile Val Gly
Val Pro Ser A-p A~p Ser Gly Leu HiD Asp Leu Ser Val L~u Hi~ Leu
Asp Ser Cy~ Thr Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Val Gly Ile
I le Arg Arg Thr Asn S r Thr LQU Leu Ser G}y Leu Tyr Tyr Thr Ser
Leu Ser Gly Asp Leu Leu Gly Phe Lys Asn Val Ser Asp Gly Val Ile
. 725 730 ~35
Tyr Ser Val Thr Pro Cy~ A~p Val Ser Ala Gln Ala
740 ~45
: : :
~2) INFORMATION FOR SEQ ID NO:7:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 4365 ba~e palr~
(8) TYPE: nucleic acid
( C ) S~}~ANDEDNESS s doubl~
( D ) TOPOLOGY: unknown
( i i ) MOLECULE TYPE: cDNA
SUBSTITUTE SHEET
WO 93/23422 . . . PCI'/US93/04384
~.:.', 213~98
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCA~ION: 1..4362
(xi) SEQUENCE DESC~IPTION: SEQ ID NO:7:
ATG ATT GTG CTC GTA ACT TGC CTC TTG TTG TTA TGT TCA TAC CAC ACA 48
Met Ile Val Leu Val Thr Cy~ Leu Leu Leu Leu CYB Ser Tyr His Thr
5 10 15
GTT TTG AGT ACA ACA AAT AAT GAA TGC ATA CAA GTT AAC GTT ACA CAA 96
Val Leu Sar Thr Thr Asn A~n Glu Cy~ Ila Gln Val A~n Val Thr Cln
20 25 30
TTG GCT GGC AAT GAA A~C CTT ATC AGA GAT TTT CTG TTT AGT AAC TTT 14 4
Leu Ala Gly ~sn Glu A~n Leu Ile Arg Asp Phe Leu Phe Ser A~n Phe
35 40 45
AAA GAA GAA GGA AGT GTA GTT GTT GGT GGT TAT TAC CCT ACA GAG GTG 192
Lys Glu Glu Gly Ser Val Val Val Gly Cly Tyr Tyr Pro Thr Clu Val
50 55 60
TGG TAC AAC TGC TCT AGA ACA GCT CGA ACT ACT GCC TTT CAG TAT TTT 240
Trp Tyr A~n Cy~ Ser Arg Thr Ala Arg Thr Thr Ala Phe Gln Tyr Phe
65 70 75 80
A~T AAT ATA CAT GCC TTT TAT TTT GTT ATG GAA CCC ATG GAA AAT AGC 288
A~n A~n Ile Hia Ala Pho Tyr Phe Val Met Clu Ala M~t Glu Asn SQr
85 90 95
ACT GGT AAT GCA CGT GGT AAA CCA TTA TTA TTT C~T GTG CAT GGT GAG 336
Thr Gly Asn Ala Arg Gly Lys Pro Leu Leu Phe Hl~ Val ~18 Gly Glu
100 105 110
CCT GTT AGT GTT ATT ATA TAT ATA TCG GCT TAT AGG GAT GAT GTG CAA 384
Pro Val Ser Val Ile Ile Tyr Ile Ser Ala Tyr Arg Asp ~8p Val Gln
115 120 125
CAA AGG CCC CTT TTA AAA CAT GGG TTA GT5 TGC ATA ACT AAA AAT CGC 432
Gln Arg Pro Leu Leu Lys His Gly Leu Val Cy5 Ile Thr Lys Asn Arg
130 13S 140
CAT ATT AAC TAT GAA CAA TTC ACC TCC AAC CAG TGG A~T TCC ACA TGT 480
His I le Asn Tyr Glu Gln Phe Thr Ser Asn Gln Trp Aan Ser Thr CYB
145 150 155 160
ACG GGT GCT GAC AGA AAA ATT CCT TTC TCT GTC ATA CCC ACG GAC AAT 528
Thr Gly Ala Asp Arg Ly~ Ile Pro Phe Ser Val Ile Pro Thr Asp Asn
GGA ACA AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TTT GTT A Q GCT 576
Gly Thr Lys Ile Tyr Gly Leu Glu Trp Asn A~p Asp Phe Val Tbr Ala
1 180 185 190
.AT ATT AGT GGT CG~ TCT TAT CAC TTG AAC ATC AAT ACT AAT TGG TTT 624
Tyr Ile Ser Gly Arg S2r Tyr His Leu Asn Il~ Asn Thr Asn Trp Phe
195 200 205
AAC AAT GTC ACA CTT TTG TAT TCA CGC TCA AGC ACT GCT ACC TGG GAA 672
Asn Asn Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
210 215 220
SUBSTITUTE SHEET
W 0 93/23422 2 ~ 3 ~ ~ 9 8 PCT/US93/04384
TAC AGT GCT GCA TAT GCT TAC CAA GGT GTT TCT AAC TTC ACT TAT TAC 720
Tyr Ser Ala Ala Tyr Ala Tyr Gln Gly Val Ser A~n Phe Thr Tyr Tyr
AAG TTA AAT AAC ACC AAT GGT CTA AAA ACC TAT GAA TTA TGT GAA GAT 768
Ly~ Leu Asn A~n Thr Asn Gly Leu LYB Thr Tyr Glu Leu Cys Glu Asp
TAT GAA CAT TGC ACT GCC TAT GCT ACC AAT GTA TTT GCT CCG ACA TCA 816
Tyr Glu Hl~ Cyu Thr Gly ~yr Ala Thr Asn V~l Phe Ala Pro Thr Ser
GGT GGT TAC ATA CCT GAT GGA TTT AGT TTT AAT AAT TGG TTC TTG CTT 864
Gly Gly Tyr Ile Pro Asp Gly Phe Ser Phe Aon Asn Trp Phe Leu Leu
280 285
ACA AAT AGT TCC ACT TTT GTS AGT GGC AGG TTT GTA ACA AAT CAA CCA 912
Thr Asn Ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr A~n Gln Pro
290 295 . 300
TTA TTG ATT AAT TGC TTG TGG CCA GTG CCC AGT TTT GGT GTA GTA GCA 960
Leu Leu Ile Asn Cy8 Leu Trp Pro Val Pro Ser Phe Gly Val Val Ala
CAA GAA TTT TGT TTT GAA GGT GCA CAG TTT AGC CAA TGT AAT GGT GTG 1008
Gln Glu Phe Cys Phe Glu Gly Ala Gln Phe Ser Gln Cyfl A~n Gly Val
330 335
TCT TTA AAT AAC ACA GTG GAT GTT ATT AGA TTC AAC CTT AAT TTC ACT 1056
Ser Leu Asn Aon Thr Val A~p Val Ile Arg Phe Asn Leu Aon Phe Thr
; 340 345 350
GCA GAT GTA CAA TCT GGT ATG GGT GCT ACA GTA TTT TCA CTG AAT ACA 1104
Ala Asp Val Gln Ser Gly Met Gly AIa Thr Va~ Phe Ser Leu Asn Thr
355 360 36S
ACA GGT GGT GTC ATT CTT GAA ATT ~CA TGT TAT AGT GAC ACA GTG AGT llS2
Thr GIy Gly Val Ile Leu Glu Ile Ser Cy~ Tyr Ser Aop Thr Val Ser
aAG TCT AGT TCT TAC AGT TAT GOT GAA ATC CCG TTC GGC AT~ ACT GAC 1200
15~Ser S-F~s-r Tyr 3Sgo Tyr Oly Glu Ile Pro Phe Gly Ile Thr Asp
GGA CCA CGA TAC TG~ TAT GTA CTT TAC AAT GGC ACA GCT CTT AAA TAT 1248
Gly Pro Arg Tyr 3 :Tyr Val Leu Tyr AAn Gly Thr Ala Leu Ly~ Tyr
410 415
TTA GGA ACA TTA CCA CCC AGT GTA AAG GAA ATT GCT ATT AGT AAG TGG 1296
~eu Gly Thr ~eu Pro Pro Ser Val LYB Glu Il~ Ala Ile Ser LY8 Trp
420 425 430
GGC CAT TTT TAT ATT AAT GGT TAC AAT TTC TTT AGC ACA TTT CCT ATT 1344
Gly His Phe Tyr Ile Asn Gly Tyr Asn Phe Phe Ser Thr Phe Pro Ile
: 435 440
GAT TGT ATA TCT TTT AAT TTA ACC ACT GGT GTT AGT GGA GCT TTT TGG 1392
Asp Cyo Ile Ser Pho Aon Leu Thr Thr Gly Val Ser Gly Ala Phe Trp
450 455 460
ACA ATT GCT ~AC ACA TCG TAT ACT GAA GCA TTA GTA CAA GTT GAA AAC 1440
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu Asn
46~ 470 475 480
; -SUBSTITUTE SHEET
WO 93/234i22 2 1 3 ~ ~ 9 8 PCI /US93/04i3X4
ACA GCT ATT AAA AAT GTG ACG TAT TGT AAC AGT CAC ATT AAT A~C ATT 1488
Thr Ala Ile Ly~ Aen Val Thr Tyr Cy~ A~n Ser Hl~ Asn Asn Il
485 490 495
AAA TGT TCT CAA CTT ACT GCT AAT TTG AAT AAT GGA TTT TAT CCT GTT 1536
Ly~ Cys Ser Gln Leu Thr Ala Asn Leu A~n A~n Gly Ph~ Tyr Pro Val
500 505 510
GCT TCA AGT GAA GTA GGT TTC GTT AAT AAG AGT GTT GTG TTA TTA CCT 1584
Ala Ser Ser Glu Val Gly Phe Val Asn Lys Ser Val Val Leu Leu Pro
515 520 525
AGC TTT TTC ACA TAC ACC GCT GTC AAT ATA ACC ATT GAT CTT GGT ATG 1632
Ser Phe Phe Thr Tyr Thr Ala Val A~n Ile Thr Ile A~p L~u Gly Met
530 535 540
AAG CTT AGT GGT TAT GGT CAA CCC ATA GCC TCG ACA CTA AGT AAC ATC 1680
Lys Leu Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu Ser Asn Ile
545 550 555 560
ACA CTA CCA ATG CAG GAT AAC AAT ACT GAT GTG TAC TGT ATT CGT TCT 1728
Thr Leu Pro Met Gln Asp A~n Asn Thr Asp Val Tyr Cys Ile Arg Ser
565 570 575
AAC CAA TTC TCA GTT TAT GTT CAT TCC ACT TGC AAA AGT TCT TTA TGG 1776
Asn Gln Phe Ser Val Tyr Val Hi~ Ser Thr Cy~ Lye Ser Ser Leu Trp
580 585 590
GAC AAT ATT TTT AAT CAA GAC TGC ACG GAT GTT TTA GAG GCT ACA GCT 1824
A~p Asn Ile Phe Asn Gln Asp Cy~ Thr Aep Val Leu Glu Ala ~hr Ala
59S 600 605
~ ~ ,
GTT ATA AAA ACT GGT ACT TGT CCT TTC TCA TTT GAT AAA TTG AAC AAT 1872
Val Ile Lys Thr Gly Thr Cye Pro Phe Ser Phe Asp Lys LQU Asn Asn
610 615 620
TAC TTG ACT TTT AAC ACG TTC TGT TTG TCG TTG AGT CCT GTT GGT GCT 1920
Tyr Leu Thr Phe Asn Thr Phe Cys Leu Ser Leu Ser Pro Val Gly Ala
62S 630 635 640
AAT TGC AAG TTT GAT GTT GCT GCA CGT ACA AGA ACC AAT GAG CAG GTT 1968
: Aen Cys Lys Phe Aep Val Ala Ala Arg Thr Arg Thr Asn Glu Gln Val
~: 645 650 655
GTT AGA AGT CTA TAT ATA ATA TAT GAA GAA GGA aAc AAC ATA CTG GGT 2016
Val Arg S:er ~eu Tyr Ile IIe Tyr Glu Glu Gly Aep Asn Ile Val Gly
660 665 670
GTA CCG T~T GAT GAT AGC GGT CTG CAC GAT TTG TCT GTG CTA CAC CTA 2064
. Val Pro Ser Asip Asp Ser Gly Leu ~i8 Aep Leu Ser V~l Leu His Leu
675 680 685
. GAC' rcc TGT ACA GAT TAC AAT ATA TAT GGT AGA ACT GGT GTT GGT ATT 2112
Asp ser Cy~ Thr A~p Tyr Aen Ile Tyr Gly Arg Thr Gly Val Gly Ile
; 690 695 700
. .,
~: ATT AGA CGA ACT AAC AGT ACG CTA CTT AGT GGC TTA TAT TAC ACA TCA 2160
~- Ile Arg Arg Thr A~n Ser Thr Leu Leu Ser Gly Leu Tyr Tyr Thr Ser
705 710 715 720
CTA TCA GGT GAT TTG TTA GGC TTT AAA AAT GTT AGT GAT GGT GTC ATT 2208
Leu Ser Gly Aep leu I,eu Gly Phe Lys Aen Val ser Aup Gly Val Ile
725 7~0 735
SUBSTITUTE SHEET
WO 93/23422 PCT/US93/04384- .
2 13 ~
TAT TCT GTG ACG CCA TGT GAT GTA AGC GCA CAA GCG GCT GTT ATT GAT 2256
Tyr Ser Val Thr Pro Cya A~p Val Ser Ala Gln Ala Ala Val Ile ARP
740 745 750
GGT GCC ATA GTT GGA GCT ATG ACT TCC ATT AAC AGT GAA CTG TTA GGT 2304
Gly Ala Ile Val Gly Ala Met Thr Ser Ile Asn Ser Glu Leu Leu Gly
755 760 765
CTA ATA CAT TGG ACA ACG ACA CCT AAT T~T SAT TAC TA~ TC~ ATA TAT 2352
Leu Ile His Trp Thr Thr Thr Pro A~n Phe Tyr Tyr ~yr Ser Tle Tyr
770 775 780
AAT TAC ACA AGT GAG AGG ACT CGT GGC ACT GC~ ATT GAC AGT AAC GAT . 2400
Asn Tyr Thr Ser Glu Arg Thr Arg Gly Thr Ala Ile Asp Ser A3n A3p
785 790 795 800
GTT GAT TGT GAA CCT GTC ATA ACC TAT TCT AAT ATA GGT GTT TGT AAA 244&
Val Asp cys Glu Pro Val Ile Thr Tyr Ser Asn Ils Gly Val Cys Lys
805 810 815
AAT GGT GCT TTG GTS T~T ATT AAC GTC ACA CAT TCT GAC GGA GAC GTG 2496
Asn Gly Ala Leu Val Phe Ile Aan Val Thr ~is S~r Asp Gly Asp Yal
820 825 . 830
CAA CCA ATT AGC ACT GGT AAT GTC ACG A2A CCT ACA AAT TTT ~CC ATA 2544
Gln ~ro Ile Ser Thr Gly A~n Val Thr Ile Pro Thr Asn Phe Thr Il~
835 840 845
TCT GTG CAA GTT GAA SAC ATG CAG GTT TAC ACT ACA CCA GTA TCA ATA 2592
Ser Val Gln YaL Glu Tyr Met Cln Val Tyr Thr Thr Pro Yal Ser Ils
8~50 855 860 -
GAT TGT GCA AGA TAC G~T TGT AAT GGT AAC CCT AGA TGT AAC AAA ~TG 2640
A~p Cy~ Al~:Arg ~yr Val CYH A~n Gly A~n Pro ~rg Cy~ Asn Ly~ Leu
865 870 875 880
TTA ACA CAA TAT GTG TCT GCA TGT CAA ACT ATT GAA CRA GCA CTT GCA 2688
Leu Thr Gln Tyr Val Ser Ala Cys Gln Thr Ile Glu Gln Ala Leu Ala
885 890 895
ATG GGT GCC AGA CTT GAA AAC ATG GAG G~T GAT TCC ATG TTG TTT GTC 2736
Met Gly Ala Arg Leu GLu A~n Met Glu V~l A~p Ser Met Leu Phe VaL
900 905 910
TCG GAA AAT GCC CTT AAA TTG GCA TCT GTT GAG GCG TTC AAT AGT ACA 2784
Ser Glu Asn Ala Leu Lys Leu Ala Ser Val Glu Ala Phe Asn Ser Thr
915 ~ 920 925
GAA AAT TTA GAT CCT ATT TAC AAA GAA TGG CCT AGC ATA GGT GGT TCT ~832
Glu Asn Leu Asp Pro Ile Tyr Ly~ Glu Trp Pro Ser ~le Gly ~ly Ser
930 . 935 94Q
TGG CTA GGA GGT CTA AAA GAT ATA CTA CCG TCC CAT AAT AGC AAA CGT 2880
Trp Leu Gly Gly Leu Lys Asp Ile Leu Pro Ser Hls A8n Ser Lys Arg
945 950 . 955 960
A~G TAT GGT TCT GCT ATA GAA GAT TTG CTT TTT GAT AAA GTT GTA ACA 2928
Ly~ Tyr Gly Ser Ala Ile Glu A8p Leu Leu Phe A~p Ly8 Val Val Thr
965 970 97S
TCT GGT TTA GGT ACA GTT GAT GAA GAT TAT AAA CGT TGT ACT GGT GGT 2976
Ser Gly Leu Gly Thr Val A8p Glu A8p Tyr Ly8 Arg Cy8 Thr Gly Gly
980 985 990
SUBSTITUTE SHEET
WO 93/23422 PCI`/I~S93/04384
,: ..
2131~98
TAC G~C ATA GCA GAC TTG GTG T~T GCT CAA TAT TAC AAT GGC ATC ATG 3024
Tyr Asp Ile Ala Aqp Leu Val Cy9 Ala Gln Tyr Tyr Asn Gly Ile Met
995 1000 1005
GTT CTA CCA GGT GTA GCT AAT GCT GAC AAG ATG ACT ATG TAC ACA GCA ~ 3072
Val Leu Pro Gly Val Ala A~n Al~ Asp Ly~ Met Thr Met Tys Thr Ala
1010 1015 1020
TCA CTT GCA GGT GGT ATA ACA TTA GGT GCA CTT GGT GGT GGC GCC GTG 3120
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
102~ 1030 1035 1040
GCT ATA CCT TTT GCA GTA GCA GTA CAG GCT AGA CTT AAT TAT GTT GCT 3168
Ala Ile Pro Phe Ala Val Ala Val Gln Al~ Arg Leu A~n Tyr Val Ala
1045 lOS0 1055
CTA CAA ACT GAT GTA ~TG AAT AAA AAC C~A CAG ATC CTG GCT AAT GCT 3216
Leu Gln Thr A~p Val Leu Aan Lys Asn Gln Gln Ile Leu Ala Asn Ala
1060 1065 1070
TSC AAT CAA GCT A~T GGT AAC ATT ACA CAG GCT TTT GGT AAG GTT AAT 3264
Phe Asn Gln Ala Ils Gly A~n Ile Thr Gln Al~ Ph~ Gly Lys Val A~
1075 108~ 1085
GAT GCT ATA CAT CAA ACA TCA CAA GGT CTT GCC ACT GTT GCT AAA GCG 3312
Asp Ala Ile Hi9 Gln Thr Ser Gln Gly Leu Ala Thr Val Ala Lys Ala
1090 1095 1100
TTG GCA AAA GTG CAA GAT GTT GTC AAC ACA CAA GGG CAA GCT ~TA AG~ 3360
Leu Ala Lya Val Gln Asp Val Val A~n Thr Gln Gly Gl~ Ala Leu Ser
1105 1110 lllS 1120
CAC CTT ACA GTA CA~ TTG CAA AAT AAT TTT CAA GCC ATT AGT AGT TCT 3408
HLs Leu Thr Val Gln ~eu Gln A~n Asn Phe Gln Ala Ile S~r Ser SQr
1125 1130 1135
ATT AGT GAT ATT TAT AAC AGG CTT GAC GAA CTG AGT GCT GAT GCA CAA 3456
Ile Ser Asp Ile Tyr Asn Arg Leu A~p Glu Leu Ser Ala A~p Ala Gln
1140 1145 llS0
GTT GAT AGG CT5 A~T ACA GGT AGA CTT ACA GCA CTT AAT GCA TTT GTG 3504
Yal Asp Arg ~eu Ile Thr Gly Arg Leu Thr Ala Leu Asn Ala Phe Val
llSS li60 1165
TCT CAG ACT CTA ACC AGA CAA GCA GAG GTT AGG GCT AGT AGA CAA CTT 35S2
Ser Gln Thr ~eu T~r Arg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu
-. 1170 1175 11~0
GCC AAA GAC AAG GTT AAT GA~ TGT GTT AGG TC~ CAG TCT CAG AGA TTC 3600
Ala Ly~ A~p Lys Yal Asn Glu Cys Val Arg Ser Gln Ser Gln Arg Phe
1185 1190 1195 1200
GGA TTC TGT GGT AAT GGT ACA CAT TTG TTT TCA CTA GCA AAT GCA GCA 3648
Gly Phe Cy~ Gly A~n Gly Thr His Leu Phe Ser Leu Ala Asn Ala Ala
1205 1210 1215
CCA AAT GGC ATG ATT TTC TTT CAT ACA GTA CTA TTA CCA ACA GCT TAT 3696
Pro Asn Gly Met Ile Phe Phe Hi~ Thr Yal Leu Le~ Pro Thr Ala Tyr
1220 1225 1230
GAA ACT GTA ACA GCT TGG TCA GGT ATT TGT GCT TCA GAT GGC GAT CGC 3744
Glu Thr Val Thr Ala Trp Ser Gly Ile Cy~ Ala Ser A~p Gly Asp Arg
1235 1240 1245
SUBSTITUTE SHEE~
W O 93~23422 2 1 3 4 8 ~ 8 PCT/US93/04384,
82
ACT TTC GGA CTT GTC GTT AAA GAT GTG CAG TTG ACG TTG TTT CGT AAT 3792
Thr Phe Gly Leu Val Val Ly~ A~p Val Gln Leu Thr Leu Phe Arg A~n
1250 1255 1260
CTA GAT GAC AAG TTC TAT TTG ACC CCC AGA ACT ATG TAT CAG CCT AGA 3840
Leu A~p Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln Pro Arg
1265 1270 1275 1280
GTT GCA ACT AGT TCT GAT TTT GTT CAA ATT GAA GGG TGT GAT GTG TTG 3888
Val Ala Shr Ser Ser ABP Phe Val Gln Ile Glu Gly CY8 Asp Val Leu
1285 1290 1295
TTT GTC AAC GCG ACT GTA ATT GAT TTG CCT AGT ATT ATA CCT GAC TAT 3936
Phe Val Asn Ala Thr Val Ile Asp LQU Pro Ser Il~ Ile Pro Asp Tyr
1300 1305 1310
ATT GAC ATT AAT CAA ACT GTT CAA GAC ATA TTA GAA AAT TAC AGA CCA 3984
Ile ABP Ile Asn Gln Thr Val Gln Asp Ile Leu Glu Asn Tyr Arg Pro
1315 1320 1325
AAC TGG ACT GTA CCT GAA TTS ACA CTT GAT ATT TTC AAC ACA ACC TAT 4032
Asn lT33p0Thr Val Pro Glu Phe Thr Leu A~p Ile Phe Asn Thr Thr Tyr
TSA AAT C~G ACT GGT GAA ATT GAT GAC TTA GAG TST AGG TCG GAA AAG 4080
1345 Thr Gly lG3150Ile A~p Asp Leu Glu Phe Arg Ser Glu Lys
CTA CAT AAC ACT ACA GTA GAA CTT GCC ATT CTC ATT GAT AAC ATT AAT 4128
L~u Hls A~n Thr Thr Val Glu L~u Ala ~ u Il~ A~p A~n I1Q ~n
1365 1370 1375
AA~ ACA TTA G~C AAT CTT GAA TGG CTC AAT AGA ATT GAA ACT TAT GTA 4176
Asn~Thr Lsu Val Asn Leu Glu Trp Eeu A~n Arg I1~ Glu Thr Tyr Val
1380 1385 1390
AAA TGG CCT TGG TAT GTG TGG CTA CTG ATA GGT TTA GTA GTA GTA TTT 4224
Ly~ Trp Pro Trp Tyr Val Trp Leu Leu Ile Gly Leu Val Val Val PhQ
1395 1400 1405
TGC ATA CCA TTA CTG CTA TTT TGC TGT TTT AGC ACA GGT TGT TGT GGA 4272
Cy9 Ile Pro Leu Leu Leu Phe Cys Cy~ Phe Ser Thr Gly Cy~ Cys Gly
1410 1415 1420
TGC ATA GGT TGT TTA GGA AGT TGT TG~ CAC TCT ATA TCT AGT AGA AGA 4320
Cy~ Ile Gly Cys Leu Gly Ser Cy~ Cys Hl~ Ser Ile Cys Ser Ar~ Arg
1425 1430 1435 1440
CAA TTT GAA AAT TAS GAA CCA ATT GAA AAA GTG CAT GTC CAC 4-~62
Gln Phe Glu A~n Tyr Glu Pro Ile G1U ~ys Val Hl~ Val Hls
1445 1450
TAAi I i 4365
t2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHaRACTERISTICS:
(A) ~ENGTH: 1454 amlno acLds
(B) TYPE: amino acid
(D) TOPOLOGY: linear
~i~) MO~ECUEE TYPE: prot~in
SUBSTITUTE SHEET
:- .
WO ~3~23422 '~ S 9 ~ PCI/US93/04384
. .,
..... .
!
83
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Ile ~al L~u Val Thr Cya L2u Leu Leu Leu CYB Ser Tyr H~ 8 Thr
Val Leu Ser Thr Thr A~n Asn Glu Cy9 Ile Gln Val A~n Val Thr Gln
Leu Ala Gly Asn Glu A~n Leu Ile Arg A~p Phe Leu Phe Ser Asn Phe
Lys 51u Glu Gly Ser Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
Trp Tyr Asn Cy~ Ser Arg Thr Ala Arg ~hr Thr Ala Phe Gln Tyr Phe
Asn A~n Ile Hi~ Ala Phe Tyr Phe Val Met Glu Ala Met Glu A~n Ser
Thr Gly Asn Ala Arg Gly Lys Pro Leu Leu Phe ~1~ Val His Gly Glu
100 105 110
Pro Val Ser Val Ile Ile Tyr Ile Ser Ala Tyr Arg Asp A~p Val G1~ i
Gln Arg Pro Leu Leu Lys His Gly L~u Val Cy~ Ile ~hr LYB A~n Arg
130 135 140
~is Ile A~n Tyr Glu Gln Phe Thr Ser Asn Gln Trp A~n Ser Thr Cy~
145 150 lS5 160
Thr Gly Ala Asp Arg Lys Ile Pro Phe Ser Val Ile Pro Thr A~p Asn
165 170 1~5
Gly Thr Lya Ile ~yr Gly Leu Glu Trp A~n Asp Asp Ph~ Val Thr Ala
180 185 190
Tyr Ile Ser Gly Arg Ser Tyr His Leu Asn Ile A~n Thr A~n Trp Phe
195 200 205
Asn Asn Val Thr Leu Leu ~yr Ser Arg Ser Ser Thr Ala Thr Trp Glu
210 215 220
Tyr Ser Ala Ala Tyr Ala Tyr Gln Gly Val Ser A~n Phe Thr ~yr Tyr
225 230 ~ 235 240
Lys Leu A~n Asn ~hr A3n Gly Leu Lyu Thr Tyr Glu Lau Cy~ Glu A~p
245 250 25S
Tyr Glu His Cys Thr Gly ~yr Ala Thr Asn Val Phe Ala Pro Thr Ser
260 265 270
Gly Gly Tyr Ile Pro Asp Gly Phe Ser Phe Asn Asn Trp Phe Leu ~eu
2~5 280 285
Thr Asn Ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr Asn Gln Pro
290 295 300
Leu Leu Ile Asn Cy~ Leu Trp Pro Val Pro Ser Phe Gly Val Val Ala
305 310 315 320
SUBSTlTlJTE SHEET
WO 93/23422 2 1 3 ~1 ~ 9 ~ PCr/US93/04384
, .,
84
Gln Glu Phe Cy~ Phe Glu Gly Ala Gln Phe Ser Gln Cy~ Asn Gly Val
Ser Leu Asn A~n Thr Val Asp Val Ile Arg Phe Asn Leu A~n Phe Thr
340 345 350
Ala Asp Val Gln Ser Gly Met Gly Ala Thr Val Ph~ Se Leu Asn Thr
Thr Gly Gly Val Ile Leu Glu Ile Ser Cys ~yr S~r A~p Thr Val Ser
370 375 380
Glu Ser Ser Ser Tyr Ser Tyr Gly Glu Ile Pro Phe Gly Ile Thr A.sp
Gly Pro Arg Tyr Cys Tyr Val Leu Tyr Asn Gly Thr Ala L~u Ly Tyr
Leu Gly Thr Leu Pro Pro Ser Val Lys Glu Ile Ala Ile S~er Lys Trp
Gly His Phe Tyr Ile Asn Gly Tyr Asn Phe Phe Ser Th Phe Pro Ile
Asp Cy ~le Ser Phe A n Leu Thr Thr Gly Val Ser Gly Ala Phe Trp
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala L u Val Gln Val Glu Asn
Thr Ala Ile Lys Asn Val Thr Tyr Cy8 A~n Ser EIi8 ~le Aon Asn Il~
- ~ - 485 490 495
Ly- Cys Ser Gln: Leu Shr Ala Asn Leu A~n Asn Gly Phe yr Pro Val
Ala Ser Ser C1u Val Gly Phe Val Asn Lys S~r Val Val Lau Leu Pro
Ser Phe Phe Thr Tyr Thr Ala Val A~n I le Thr Ile A~p Leu Gly Me~
Lys Leu Ser Gly Tyr ti;ly Gln Pro Ile Ala Ser Thr L-u Ser A~n Ile
545 5~0 555 S60
Thr Leu Pro M-t Gln AYP A-n Asn Thr Asp Val Tyr Cys Ile Arg Ser
Asn Gln Phe Ser Val Tyr Val Hi~ Ser Thr Cy~ Ly~ Ser Ser Leu Trp
580 585 590
A~p A~n Ile Phe Asn Gln Asp Cys Thr Asp Val Leu Glu Ala Thr Ala
595 600 605
Val Ile Lys ~hr Gly Thr Cys Pro Phe Ser Phe A~p Ly~ Leu A~n Asn
610 615 620
Tyr Leu Thr Phe Asn Thr Phe Cys Leu Ser Leu S~r Pro Val Gly Ala
625 ~ 630 635 640
Asn Cys Ly~ Phe Asp Val Ala Ala Arg Thr Arg Thr A~n Glu Gln Val
645 650 655
SUBSTITUTE SHEET
~ WO 93~23422 PCr/US93/04384
` -` i 2 1 3 1 $ 98
Val Arg Ser Leu Tyr Ile Ile Tyr Glu Glu Gly A~p A~n Ile Val Gly
660 665 670
Val Pro Ser Asp A~p Ser Gly Leu Hi~ A~p Leu Ser Val Leu His Leu
675 680 685
Asp Ser Cys Thr Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Val Gly Ile
690 695 70~
Ile Arg Arg Thr A~n S~r Thr Leu Leu Ser Gly Leu ~yr Tyr Thr Ser
~OS 710 715 720
Leu Ser Gly Asp Leu L~u Gly Phe Ly~ A~n Val Ser A~p Gly Val Il~
725 730 735
Tyr Ser Val Thr Pro Cy~ Asp Val Ser Ala Gln Ala Ala Val Ile Asp
740 745 750
Gly Ala Ile Val Gly Ala Met Thr Ser Il~ A~n S~r G1U L~U Leu Gly
755 760 765
Leu Ile His Trp Thr Shr Thr Pro Asn Phe Tyr Tyr Tyr Ser Ile ~yr
770 775 780
Asn Tyr Thr Ser Glu Arg Thr Arg Gly Thr Ala Ile Asp ser A~n Asp
785 790 795 800
Val Asp Cy~ Glu Pro Val Ile Thr Tyr Ser A~n I1Q Gly Val Cy8 Lya
805 ~lO 815
Asn Gly Ala Leu Val Phe Ile Asn Val Thr Hls Ser Asp Gly Asp Val
820 825 830
Gln Pro Ile Ser Thr Gly A~n Val Thr Ile Pro Thr A n Phe Thr Ile
~: 835 ~40 845
Ser Val Gln Val Glu Tyr Met Gln Val Tyr ~hr Thr Pro Val Ser Ile
850 855 860
Asp Cys Ala Arg Tyr Val Cys A~n Gly Asn Pro Arg Cy9 Asn Lys Leu
B65 870 8~5 880
Leu Thr Gln Tyr Val Ser Ala Cy~ Gln Thr Ile Glu Gln Ala Leu Ala
885 890 89S
Met Gly Ala Arg Leu Glu Aan ~et Glu V~l Asp Ser Met Leu Ph~ Val
900 905 910
Ser Glu Asn Ala Leu Lya L~u Ala Ser Val Glu Ala Phe Asn Ser Thr
91S 9~0 925
Glu;Asn Leu ABP Pro Ile Tyr Lya Glu Trp Pro Ser Ile Gly Gly Ser
930 935 9~0
Trp Leu Gly Gly Leu Ly~ Asp Ile Leu Pro Ser Hi~ A~n Ser Ly~ Arg
945 9S0 955 g60
LYB Tyr Gly Ser Ala I1Q Glu Asp Leu Leu Ph~ Asp Lys Val Val Thr
965 970 975
Ser Gly Leu Gly Thr Val A~p Glu Asp Tyr Lys Arg Cy~ Thr Gly Gly
980 985 990
SUBSTITUTE SHEET
WO 93/23422 2 ~ 3 1 ~ ~ 8 PCr/US93/04384~
86
~yr Rsp Ile Ala A~p ~eu Val Cy~ Ala Gln Tyr Tyr Asn Gly Ile ~et
995 1000 lOOS
Val Leu Pro Gly Val Ala Asn Ala A~p Ly~ Met Thr Met Tyr Thr Ala
1010 lOlS 1020
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
1~25 1~30 103S 1040
Ala Ile Pro Phe Ala Val Ala Val Gln Ala Arg Leu Asn Tyr Val Ala
1045 1050 1055
Leu Gln Thr Asp Val Leu Asn Lys A~n Gln Gln Ile Leu Ala A~n Ala
1060 1065 1070
Phe Asn Gln Ala Ile Gly A~n Ile Thr Gln Ala Phe Gly Ly~ Val Asn
1075 1080 1085
Asp Ala Ile His Gln Thr Ser Gln Gly Leu Ala Thr Val Ala Lys Ala
1090 1095 1100
Leu Ala Lye Val Gln A~p Val Val A~n Thr Gln Gly Gln Ala L~u Ser
1105 1110 1115 1120
His Leu Thr Val Gln Leu Gln Asn A~n Phe Gln Ala Ile Ser Ser Ser
1125 . 1130 1135
Ile Ser ABP Ile Tyr A8n Arg Leu A8p Glu Leu Ser Ala A8p Ala Gln
1140 1145 llS0
Val ABP Arg ~eu Ile Thr Gly Arg Leu Thr Ala LRU Asn Ala PhQ Val
llSS 1160 1165
ser Gln Thr Leu Thr Ar~. Gln Ala Glu Val Ar~ Ala Ser Arg Gln Leu
0 1175 1180
Ala Ly~ Asp ~y~ Val A8n Glu Cy~ Val Arg Ser Gln Sar Gln Arg Phe
1185 : 1190 1195 1200
Gly Phe Cy~ Gly Asn Gly Thr Hi9 Leu Phe Ser Leu Ala A~n Ala Ala
I205 1210 1215
Pro Asn Gly Met Ile Phe Phe ~i8 Thr Val: ~eu Leu Pro Thr A1a Tyr
1220 1225 1230
Glu Thr Val ~hr Ala Trp Ser Gly Ile Cy~ Ala Ser A~p Gly A~p Arg
~ 1235 :1240 124S
: Thr Phe Gly ~eu Val Val Ly~ A~p Yal Gln Leu Thr Leu Phe Arg Asn 1250 1255 1260
Leu A0p!Asp Ly8 Phe Tyr Leu Thr Pro Arg Thr Met TYr Gln Pro Arg
1265 1270 1275 1280
Val Ala Thr Ses Ser A~p Phe Val Gln Ile Glu Gly CY9 A~p Val Lau
128S 12gO 1295
Phe Val A~n Ala Thr Val Ile A5p Leu Pro Ser Ile Ile Pro AQP Tyr
1300 1305 1310
Ile A~p Ile A~n Gln Thr V~l Gln A~p Ile ~u Glu Asn Tyr Arg Pro
1315 1320 1325
SUBSTITUTE SHEET
WO 93/23422 PCI`/USg3/04384
213~I898
87
Asn Trp Thr Val Pro Glu Phe Thr Leu Asp Il~ Phe Asn Thr ~hr Tyr
1330 1335 1340
Leu Asn Leu Thr Gly Glu Ile Asp Asp Leu Glu Phe Axg Ser Glu Ly~
1345 1350 135~ 1360
Leu His A~n Thr Thr Val Glu Leu Ala Ile Leu Ile A~p A~n Ile A~n
1365 1370 1375
Asn Thr Leu Val Asn Leu Glu Trp Leu A~n Arg Ile Clu Thr Tyr Val
1380 1385 1~90
Ly~ Trp Pro Trp Tyr Val Trp Leu Leu Ile Gly Leu Val Val Val Phe
1395 1400 1405
Cys Ile Pro Leu Le-~ Leu Phe Cys Cy~ Phe Ser Thr Gly Cy~ Cy8 Gly
1~10 1415 1420
Cys Ile Gly Cy~ Leu Gly Ser Cy~ CYB Hi~ Ser Ile Cy~ Ser Arg Arg
1425 1430 1435 1440
Gln Phe Glu A3n Tyr Glu Pro Ile Glu Ly~ Val Hls Val H~ g
~ 1445 1450
12) INFO~MATION FOR SEQ ID NO:9:
i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 370 ba0e p~lr~
(B) TYPE: nucle~c acid
C ) ST~ DEDNESS: double
D ) TOPOLOGY: unknown
~ ) MOLECULE TYPE: cDNA
: ~:: ( ix ) FEATURE :
~: ~A) NAME/lCEY: CDS
~B) LOCATION: 3..368
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GT GGT AAA CCA TTA TTA $TT CAT GTC CAT CGT GA¢ CCT GTT AGT GTT 47
- Gl-y Ly~ Pro L~u Leu Phe His V~l EI1~ Gly Glu Pro V~l Ser V~l
5 10 15
ATT ATA TAT: ATA TCG GCT TAT AGG GAT GAT GTG CAA C~A AGG CCC CTT 95
Ile Ile Tyr Ile Ser Ala Tyr Arq ABP Asp Val Gln Gln Arg Pro Leu
~ 20: 25 ~0
- TTA ~A CAT GGG TTA OTG TGC ATA ACT AAA AAT CCC CAT ATT AAC TAT ~:43
Leu Ly~ Gly Leu Val Cy9 Ile Thr Lys Asn Ary E~is Ile A~n Tyr
~ i 35- ~ 40 45
GAA CAA TTC ACC TCC AAC CAG TGG AAT TCC ACA TGT ACG GGT GCT GAC l 91
~: G~u Gln Phe Thr Ser Asn Gln Trp A8n Ser Thr Cy8 Thr Gly Ala Asp
50 55 60
~':
AGA AAA ATT CCT TTC TCT GTC ATA CCC ACG GAC AAT Gt:A ACA AAA ATC 2 3 9
Arg Ly~ Ile Pro Phe Ser Val Ile Pro Thr Asp Asn Gly Thr Lys Ile
~: ~ 65 7~ 75
,
SUBSTITIJTE SHEE~
.
W O 93/~3422 P~T/US93/04384
2 3 3~8
88
TA~ GGT CTT GAG TGG AAT GAT GAC TTT G~T ACA GCT TAT ATT AGT GGT 2 87
Tyr Gly Leu Glu Trp A n A~p A~p Phe Val Thr Ala Tyr Ile Ser Gly
CGT TCT TAT CAC TTG AAC ATC AAT ACT AAT TGG TTT AAC AAT GTC ACA ~ ~S
Arg ser Tyr Hi~ Leu A~n Il~ A~n Thr Asn Trp Ph~ ~8~ ~n Val Thr
100 105 110
CTT TTG TAT TCA CGC TCA AGC ATT GCT ACC TGG GA 370
L~u Leu Tyr Ser Arg Ser Sar Ile Ala Thr Trp
115 120
(2) INFORMATION FOR SEQ ID NO:10~
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGT~: 122 ~mlno acids
(B3 TYPE: amlno acld
(D) TOPOLOGY. lLnear
~ii) MOLECULE TYPE: protei~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Gly Lys Pro Leu Leu Phe His Val His Gly Glu Pro Val Ser Val Ile
1 5 10 15
Ile Tyr Ile Ser Ala Tyr Arg A~p Asp Val &ln Gln Arg Pro Leu Leu
-~ Lys His Gly Leu Val Cys Ile Thr Lys Asn Arg Hls Ile Asn Tyr Glu
:~: 35 40 45
~ ~ .
Gln Phe Thr S-r A~n Gln Trp Asn Ser Thr Cy~ Shr Gly Ala Asp Arg
Lys Ile Pro Phe Ser Val Ile Pro Thr Asp Asn Gly Thr Lys I1Q Tyr
Gly Leu Glu Trp As Asp Asp Phe Val Thr Al~ Tyr Ile Ser Gly Arg
Ser Tyr His Leu Asn Ile Asn Thr A~n Trp Phe A~n Asn Val Thr Leu
100 105 110
Leu Tyr Ser Ar~ Sar S~r Ila Ala Thr Trp
115 120
(2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 4365 base palrs
(B) TYPg: nucleic ac~d
~C) STRANDEDNESS: double
(D) ~OPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/XEY: CDS
~B) LOCA~ION: 1..4362
S~JBSTITlJTE SHEET
PCT/VS~3/04384
W O 93/23422 2 ~ 3 ~ ~ 9 8
8g
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ATG ATT GTG CTC GTA ACT TGC CTC TTG TTG TTA TGC TCA TAC CAC ACT 48
Met I1e Va1 LeU Va1 Thr CY~ LeU L~U LeU L~U CYB S~r TYr H18 Thr
1 5 10 15
GTT TCG AGT ACG TCA AAC AAT GAT TGT AGA CAA GTT AAC GTA ACA CAA 96
Va1 Ser Ser Thr Ser A3n Asn A3p Cy~ Arg Gln Val Asn Val Thr Gln
20 25 30
TTA GCT GGC A~T GAA AAC CTT ATT AGA GAC T~r TTG TTT CAA AGT TTT 14 4
LeU A1a G1Y A~n G1U Asn LQU I1e Arg A8P Ph~ ~eu Phe Gln Ser Phe
35 40 45
AAA GAA GAA GGA ATT GTA GTT GTT GGT GGT TAT TAC CCT ACA GAG GTG 192
LYS G1U G1U Gly I1e Val Val Val G1Y G1Y TYr Tyr PrO Tbr G1U Val
50 55 60
TGG TAC AAC TGC TCT AGA ACA GCA ACT ACC AC~ GCC TAT GAG TAT TTT 240
Trp Tyr Asn CY8 Ser Arg Thr Ala ~hr Thr Thr Ala Tyr Glu ryr Phe
65 70 ~5 80
AAT AAT ATA CAT GCC TTT T~T TTT GAT ATG GAA GCT ATG GAA AAT AGC 288
Asn Asn I le His Ala Phe Tyr Phe Asp Met Glu Ala M~t Glu Asn Ser
85 90 g5
ACT GGT AAT GCA CGT GGT AAA CCT CTA TTA ~TT CAT GTT CAT GGT GAA 336
Thr Gly A~n Al~ Arg Gly Ly~ Pro L~u Leu Pho HL~ V~l H1~ Gly Glu
100 105 110
CCT GTT AGT ATC ATC ATA TAT ASA TCA GCT TAT GGG GAT GAT GTG CAA 384
Pro Val Ser Il~ Ile Ile Tyr Ile ser Al~ Tyr Gly A~p Asp Val Gln
115 120 125
CAA AGG CCA CTT TTA GAA CAT GGG TTA TTG TGC ATT A~T AAA AAT CGC 432
Gln Arg Pro L~u Leu Glu Hls Gly L~u Leu Cys Ile Thr Ly~ Asn Arg
130 135 140
AAT ATT GAC TAT AAC ACC TTC ACC AGC AAC CAG TGG GAT TCC ATA TGT 480
A~n Ile A P Tyr Asn Thr Phe Thr Ser Asn Gln Trp A~p Ser Ile Cys
145 150 155 160
ACG GGT AAT GAC AGA AAA ATT CCT TTC TCT GTC ATA CCC AGG GAT AAT 528
Thr Gly Asn Asp Arg Lys Il~ Pro Phe Ser V~ Pro Arg Asp Asn
165 170 175
GGA ACA AAA ATC TAT GGG CTT GAG TGG AAT GAT GAA TTT GTT ACA GCG 576
Gly Thr Lys Ile Tyr Gly L~u Glu Trp Asn Asp Glu Ph~ Val Thr Ala
180 185 190
TAT ATT AGT GGT CGT TCT TAT AAT TGG AAC ATC AAT AAT AAC TGG TTT 624
Tyr Ile Ser Gly Arg Ser Tyr A~n Trp Asn Ile Asn Asn Asn Trp Phe
1 195 200 205
AAC AAT GTC ACA CTT TTG TAT TCA CGC TCA AGC ACT GCT ACC TGG GAA 672
Asn A~n Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
210 215 220
TAC AGT GCT GCA TAT ~-TT TAC CAA GGT GTT TCT AAC TTC ACT TAT TAC 720
Tyr Ser Ala Ala Tyr Val Tyr Gln Gly Yal Ser A~n Phe Thr Tyr Tyr
225 230 235 240
SUBSTITUTE SHEET
W O 93/2342~ PCT/US93/04384
2 ~ 9 0 '`` -~:
AAG TTA AAT AAC ACC AAT G :;T TTA AAA ACC TAT GAA TTT TGT GAG GAT 768
Lys Leu A~n Asn Thr Asn Gly Leu Lys Thr Tyr Glu Phe Cy~ Glu Aqp
245 250 255
TAT GAA TAT TGC ACT GGC TAC GCC ACT AAT GTC TTI' GC~ CC A ACT GTG 816
Tyr Glu Tyr CY8 Thr Gly Tyr Ala Thr A~n Val PhQ ~la Pro Thr Val
260 265 270
GGA GGT TAC ATA CCT GAT GGA TTT AGT TTT AAC AAT TGG TTT I'TG CTT 864
Gly Gly Tyr Ile Pro Asp Gly Ph~ Ser Phel A~n A~n Trp Phe Leu Leu
275 280 285
ACA AAT AGC TCC ACT TTT GTT AGT GGC AGA TTT GTA ACA AAC CAA CCA 912
Thx Asn 5er Ser Thr Phe Val S~2r Gly Arg Ph~ Val ~hr Asn Gln Pro
290 295 30û
CTA TTA GTT AAC TGC TTA TGG CCA GTG CCC AGT TTT GGT GTA GCA GCA 960
Leu ~eu Val A~n Cys Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
310 315 320
CAA GAA TTT TGT TTT GAA GGT GCG CAG TTT AGT CAG TGT AGT GGT GTA 1008
Cln Glu Ph~ Cya PhOE Glu Cly Ala Gln Phe S~r Gln Cys S~r Cly Val
325 330 335
TCT TTA AAT AAC ACA GTA GAT GTT ATI' AGA TTC AAT CTT AAT TTC ACC 1056
5er Leu Asn Asn Thr Val Asp Val Ile Arg Phe Asn Leu A~n Phe Thr
340 345 350
GCA GAT GTA CAA TCT GGT ATG GGT GCT ACA GTG TTT TCG ~TG AAT ACA 1106
Ala Asp Val Gln Ser Gly Met Gly Ala Thr Val Phe S~r Leu A~n Thr
355 360 365
ACG GGT GGT GTC ATT CTT GAA GTT TCA TGT TAT AAT GAC ACA GTG AGT 1152
Thr Gly Gly Val Ile Leu Glu Val Ser Cy~ Tyr Asn A~p Thr Yal Ser
3~0 375 380
GAG TCT AGT TTT TAC ACT TAT CCT eAA ATT CCC TTC CCC ATA ACT CAT 1200
15 Ser S-r Ph- Tyr S r Tyr Gly Glu Ilo Pro Ph- Gly Il~ Thr Asp
GGA CCA CGG TAC TGT TAT GTA CTT TAC AAT GGC ACA GCT CTT AAG TAT 1248
Gly Pro Arg Tyr Cys Tyr Val ~eu Tyr Asn Gly Thr Ala Leu Lys Tyr
405 410 415
TTA GGA ACA TTA CCA CCT AGT GTA AAG GAA ATT GCT ATT AGT AAG TGG 1296
Lou Gly Thr Lou Pro Pro Sor Val Lys Glu Il~ Ala Ile Sor Lys Trp
420 ~ 425 430
GGC CAT TTT TAT ATT AAT GGT TAC AAT TTC TTT AGC ACA TTT CCT ATT 1344
Gly His Phe Tyr Ile Asn Gly Tyr Asn Phe Phe Ser Thr Phe Pro Ile
435 440 445
GAT TGT! ATA TCT TTT A~C TTA ACC ACT GGT GAT AGT aGA GCT TTT TGG 1392
Asp Cys Ile Sor Ph~ Aon Leu Thr Thr Cly Aop Sor Cly Ala PhQ Trp
450 455 460
ACA ATT GCT TAC ACA TCt: TAC ACT GAG GCA TTA GTA CAA GTT GAA A~C 1440
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu A~n
465 4~0 475 480
ACA GCT AST AAA AAC CTC ACC TAT TCT AAC AGT CAC ATT AAT AAC ATT 1488
Thr Ala Il~ Lys Lys Val Thr Tyr Cys A~n SQr Hi~ Asn A-n I1Q
48S 490 495
SUBSTITUTE SHEET
W O 93/23422 PCT/US93/04384
2~3~898
91
AAG TGT TCT CAA CTT ACT GCT AAT TTG AAT AAT GGA TTT TAT CCT GTT 1536
Lys Cy~ Ser Gln Leu Thr Ala A~n Leu Asn Asn Gly Phe Tyr Pro Val
500 SOS S10
GCT TCA AGT GAG GTT GGT CTT GTG AAT AAG AGT GTT GTG TTA TTA CCT 1584
Ala Ser Ser Glu Val Gly Leu Val Asn Lys Ser Val Val Leu Leu Pro
515 S20 525
ATC TTT TTC GCA CAT ACC GCT ATC AAT ATA ACC ATT GAT CTT GGT ATG 1632
Ile PhQ Phe Ala His Thr ALa I1Q Asn Ile Thr Ile Asp Leu Gly Met
530 535 540
AAG CGT AGC GGT TAT GGT CAA CCC ATA GCA TCA ACA TTA AGT AAC ATT 1680
Lys Arg ser Gly Tyr Gly Gln Pro Ile Ala ser Thr Leu Ser Asn Ile
545 550 , 555 . 560
ACA CTA CCA ATG CAG CAT AAT AAC ACA GAT GTG TAC TGT ATT CGT TCT 1728
. Thr Leu Pro Met Gln Asp Asn Asn Thr A~p VaI Tyr Cys ILe Arg Ser
: : 565~ 570 575
AAC~CAG TTT TCA GTT TAT GTT CAS TCT ATT SGT AAG AGT TCT TTA SGG 1776
Asn Gln Phe Ser Val Tyr Val His Ser Ile Cys Lys Ser ser Leu Trp
580 , 585 590
GAC AAT ASS TTS AAT CAA GAA TGC~ACG GAT GTT TTA GAT GCC ACA GCT 1824
A~p A-n II Ph- A~n Cln Clu Cys Thr A-p;V~l L-u A p Ala Thr Ala
GTT ATA~:AAG ACT~GGT ACT TGT CCT TTC TCA STT GAT AAA TSG AAC AAT 1872
V~ Ile~Lys;Thr.~Gly Thr Cy Pro Ph- s-r Ph- ABP Lys Leu Asn A~n
:T~C~TTA~ACT~STT`:AAC~AAG~TT:C TGT:TTG TCG TSG AGS ~CS GTT GGC GCT 1920
.Le~:~Shr~Phe~A-n~Ly ~ Ph-~ Cyu L-u S-r Leu Ser Pro Val al y Ala
~" ~
AAC TGC AAG STT~GAT GTS GCC GCA CGS ACA AGA~ACC AAT GAG CAA GTT 1968
~'. Asn Cys Lye Phe A~p Val Ala Ala Arg Thr Arg Thr A~n Glu Gln Val
: 64~5 ~ 650~ 655
GTT~AGA:,AGT~CTA~:TAT~GTA;~ATA~TM GAA GAA~CGA GAC AAC ATA GTT GGT 2016
;,Val Arg~Srr,L ~ yr~ Var~ Tyr~Glu~Glu Cly A-p A~n Ile Val Oly
GSA CCG SCS~:GAT~:AAT AGC GGT CTG CAC GAT TTG TCT GTG CTA CAC CTA 2064
Va~l~Pro~Ser~Asp~A~n Ser~Gly~eù His Asp Leu Ser Val Leu Hie ~eu
675 ~ 680: ~: 685
GAC:~TCC TGT~ACA~CAG:TAT AAT~ATA TAT GGT AGA ACT GGT GTT GGS ATS 2112
A-p~Ser Cy8 Thr Glu Tyr A~n Ile Tyr Gly Arg Thr Gly Val Gly Ile
' 690 695 700
' ATT AGA CAA ACT AAC AGT ACG CTA CTT AGC GGC TTA TAT TAC ACA TCA 2160
~ Ile Arg Gln Thr A~n Ser Thr ~eu Leu Ser Gly Leu Tyr Tyr Thr Ser
,,-,, ~ 705 710 715 720
CTA TCA GGT~GAT TTG TTA GGC TTT AAA AAT GTT ACT GAT GGT GTC ATC 2208
: Leu Ser Cly:X~p ~-u Leu Gly Phe Ly- A~n.Val S-r A-p Cly Val Ile
?25 730 735
: TAT TCT GTC ACC CCA TGT GAT GTA AGC GCA CAA GCG GCT GTT ATT GAT 2256
: Tyr Ser Val Thr Pro Cy~ A~p Val Ser~Ala Gln Ala Ala Val Ile Asp
40 745 7S0
SUBSTITUTE SHEET
., ~
WO 93~23422 PCT/US93/04384
GGT G~C ATA GTT GGA GCT ATG ACT TCC ATT AAC AGT GAA CTG TTA GGT 2304
Gly A1a I1Q Va1 G1Y A1a Met Thr Ser Ile A~n Ser G1U LQU LQU Gly
755 760 765
CTA AAA CAC TGG ACA ACA ACA CCT AAT TTT TAT TAC TAC TCT ATA TAT ~ ~ 52Leu Ly~ Trp Thr Thr ~hr Pro A~n Phe Tyr ~yr Tyr Sor Il~ Tyr
770 775 780
AAT TAT ACA AAT GAG AGG ACT CGT GGC ACT GCA ATT GAC AGT AAC GAT 2400
ARn Tyr Thr A~n Glu Arg Thr Arg Gly Thr Ala Ilo Asp Ser Asn A~p
785 790 795 800
GTT GAT TGT GAA CCT ATC ATA ACC TAT TCT AAC ATA GGT GTT TGT AAA 2448
Val A9P CY~ Glu Pro Ile Il~ Thr Tyr S~r A~n Il~ Cly Val Cya Lya
805 81~ 81S
AAT GGT GCT TTG GTT TTT ATT AAC GTC ACA CAT TCT GAT GGA GAC GTG 2496
Aqn Gly Ala Leu Val Phe Ilo A8n Val Thr Hl~ Ser Aap Gly A8p Val
820 825 83~
CAA CCA ATT AGC ACT GGT ACT GTC ACG ATA CCT ACA AAC TTT ACC ATA .2544
Gln Pro Ila Ser Thr Gly Thr Val Thr Ile Pro Thr A~n Phe Thr Ile
835 840 845
TCT GTG CAA GTC GAA TAC ATT CAG GTT TAC ACC ACA CCA GTA TCA ATA 2592
Ser Val Gln Yal Glu Tyr Ile Gln Val Tyr Thr Thr Pro Val Ser Ile
850 8S5 860
GAT TGT GCA AGA TAC GTT TGC AAT GGT AAC CCT AGA TCT AAC AAA TTC 2640
Asp Cy~ Ala Arg Tyr Val Cy~ Asn Gly A3n Pro Arg Cy8 Asn Ly~ Leu
865 870 8~5 880
TTA ACA CAA TAT GTT TCT GCA TGT CAA ACT ATT GAG C~A GCA CTT GCA 2688
Leu Thr Gl~ Tyr Val Ser Ala Cy~ Gln Thr Ile Glu Gl~ Ala Leu Ala
885 890 ~95
ATG GGT GCC AGA CTT GAA AAC ATG GAG GTT CAT TCC ATG TTG TTC GTT 2736
~et Gly Ala Arg Leu Glu Aan Met Glu Val Asp Ser M~t Leu Phe Val
900 , 90S 910
TCT GAA AAT GCC CTT AAA TTG GCA TCT GTT GAG GCG TTC AAT AGT ACA 2784
Ser Glu AQn Ala Leu Ly~ Leu Ala Ser Val Glu Ala Phe Asn Ser Thr
915 920 925
GAA AAT TTA GAC CCT ATT TAC AAA CAA TGC CCT AAC ATA GCT GCT TCT 2832
Glu A~n Lou Asp Pro Ile Tyr Lys Glu Trp Pro A~n I1Q Gly Gly Ser
930 935 940
TGG TTA GGA GGS TTA AAA GAC ATA CTG CCG TCC CAT AAT AGC A~A CGT 2880
Trp ~eu Gly Gly Leu Ly~ Asp Ile Leu Pro Ser ~I D AB~ Ser LYB Arg
945 950 955 960
AAG TAT CGT TCT GCT ATA GAA GAC TTG CTT TTT GAT AAG GTT GTA ACT 2928
LY9 TYr Arg Ser Ala Ile Glu ABP LQU LOU Phu Asp LYB Va~ VA1 Thr
96S g~o 9~5
TCT GGT TTA GGT ACA GTT GAT GAA GAT TAT AAA CGT TGT ACA GGT GGT 2~g76
Ser Gly Leu Gly Thr Val A8p Glu A8p Tyr Ly~ Arg Cy~ Thr Gly Gly
980 985 990
TAT GAC ATA CCC CAC T$A ¢TC TGT GCT CAA TAT TAC AAT CCC ATC ATC 3024
Tyr A~p Il~ Al~ A~p LOU V~l Cy~ Ala Gln Tyr Tyr A~n Gly Ilo Met
995 1000 100S
SUBSTITUTE SHEET
WO 93/2342~ PCI/US93104384
~ 213~9~
93
GTG TTA CCT GG$ GTA GCT AAT GAT GAC AAG ATC AC~ ATC TAC ACA CCA 3072
Val Leu Pro Gly Val Ala Ann A~p A~p Ly~ Met Thr ~t Tyr Thr Ala
1~10 lOlS 1020
TCT CTT GCA GGT GGT ATA ACA CTA GGT GCA CTT GCT GGT GGC GCC GTT ~ 3120
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
102S 1030 1035 1040
.
GCT ATA CCT TTT CCA GTA GCA GTT CAA GCT AGA CTT AAT TAT GTT GCT 3168
Ala Ile Pro Phe Ala Val Ala Val Gln Al~ Arg Leu Asn ~yr Val Ala
1045 1050 lOSS
CTA CAA ACT GAT GTA TTG AAT AAA AAC CAG QG ATC CTG GCT AAT GCT 3216
Leu Gln Thr Asp Val Leu Asn Lys A~n Gln Gln Ile Leu Ala Asn Ala
1060 1065 1070
TTC-AAT CAA GCT ATT GGT AAC ATT ACA CAC GCA TTT GCC AAG GTT AAT 3264
Phe A~n Gln Ala Ile Gly A~n Ile Thr Gln Ala Phe Gly Ly~ Val Asn
1075 1080 1085
GAT GCT ATA CAT CAA ACA TCA AAA GGT CTT GCA ACT GT$ GCT AAA GCA 3312
Asp Ala Ile Hi~ Gln Thr Ser LYB Gly L~u Ala Thr Val Ala Lys Ala
1090 1095 1100
TTG GCA AAA GTG CAA ¢AT GTT GTC AAC ACA CAA GGC CAA GCT TTA AGC 3360
Leu Ala rya Val Gln Asp Val Val A~n ~hr Gln Gly Gln Ala L~ Ser
1105 1110 1115 1120
CAC CTA ACA GTA CAA TTG CAA AAT AAT $TT CAA GCC ATT AGT AGC TCT 3408
Hls Leu Thr Val Gln Leu Gln A~n Asn Phe Gln Ala Ile Ser Ser Ser
. 1125 1130 1135
A~T AG~ GAT:AST TAT AAC ACG CTT CAC GAA CTC AaT GC~ GAT GCA CAA 3456
Ile S~r A~p I}~ Tyr A~n Arg LQU A~p Glu L~u Ser Ala Asp Ala Gln
1140 1145 1150
GTT GAT AGG CTG ATT ACA GGA AGA CTT ACA GCA CTT AAT CCA TTT GTG 3504
Val A~p Arg Leu Ile Thr Gly Arg ~eiu Thr Ala ~eu Asn Ala Phe Val
llS5 1160 1165
TCT CAG:ACT CTA ACC AG~ CAA GCC GAC GTT ACG GCT ACT AGA CAA CTT 3552
Ser Gln Thr L~u Thr Arg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu
1170 1175 1180
GCC AAG GAC AAG GTT AAT GAA TGT GTT AGA TCC C~A TCT CAG AGA TTT 3600
Ala Lys A~p Ly~ Val A~n Glu CYB Val Arg S~r Gln Ser Gln Arg Phe
I185 1190 1195 1200
GGA TTC TGT GGT AAT GGT ACA CAC TTG TTT TCA CTT CCA ~T GCA GC~ 3648
Cly Ph~ Cy- Oly A~n Gly Thr 8~- Leu Phe Ser T eu Ala A~n Ala Ala
1205 1210 1215
CCAI AAT GGC ATG ATT TTC TTT CAT ACA GTG CTA TTA CCA ACG GCT TAT 3696
Pro Asn Gly Met Ile Phe Phe Hi~ Thr Val Leu Leu Pro Thr Ala Tyr
1220 1225 1230
GAA ACT GTA ACA GCT TGG CCA GGT ATT TGT GCT TCA GAT GGC GAT CGC 3744
Glu Thr Val Thr Ala Trp Pro Gly Ile Cys Ala Ser A~p Gly A~p Arg
. 1235 1240 1245
ACT TTT GGA CTT GTC GTT AAA GAT GTA CAG TTG ACG TTG TTT CGT AAC 3792
Thr Phe Gly Leu Val Val Ly8 Agp Val Gln Leu Thr Leu Phe Asg Asn
1250 1255 1260
SUBSTITUTE SHEET
WO 93/23422 2 ~ PCT/US93/04384
94
CTA GAT GAC AAG TTC TAT T~G ACT CCC AGA ACT ATG TAT CAG CCT AGA 3 840
Leu Asp Asp Ly~ Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln Pro Arg
1270 1275 1280
GCT GCA ACT AGT TCT GAT TTT GTT CAA ATT GA~ GGG TGC GAT GTG TTC 3888
Ala Ala Thr Ser S~r A8P Phe Val Gln Ile Glu Gly Cy~ Asp Val L~u
12a5 1290 1295
TTT GTC AAT GCA ACT GTA ATT GAC TTG CCT AGT ATT ATA CCT GAC TAT 3936
Phe Val A~n Ala Thr Val Iie Asp Leu Pro Ser rle Ile Pro Asp Tyr
1300 1305 1310
ATT GAC ATC AAT CAC ACT GT$ CAA GAT ATA T$A CAA AAT TAC AGA CCA 3984
Ile A~p Ile A~n Cln Thr Val Gln A~p Ile Leu Glu Aan Tyr Arg Pro
1315 1320 1325
AAC TGG ACT:GTA CCT GAA TTG ACA CTT GAT ATT TTT AAC GCA ACC TAT 4032
Asn Trp Thr VA1 Pro Glu Leu Th~ Leu Asp Ile Phe A~n Ala Thr Tyr
1330 1335 1340
TTA AAT CTG ACT GGT GAA ATT GAT GAC TTA CAA TTT MG TCA CAA AAG 4080
Leu A~n Leu Thr Gly Glu Ile A~p A~p L~u Glu Ph~ Arg Ser Clu LYB
1345 1350 1355 1360
CTA CAC AAT ACC ACT GTA GAA CTT GCC ATT CTC ATT CAC AAC ATT AAC 4128
Leu His Asn Thr Thr Val Glu Leu Ala Ile Leu ~le A~p A~n Ile Asn
1365 13~0 13~5
~AAC ACA TTA GTC AAT CTT CAA TGG CTC AAT AGA ATT GAA ACT TAT GTA 4176
A-n Thr L-u Val A-n L-u Gla Trp L u A-n Arg Ile Glu Thr Tyr Val
AAA TGG CCT TCG~TAT GTC TGG CTA CTA ATA GGC TTA GTA GTA ATA TTT 4224
Ly~ Trp Pro Trp Tyr Val Trp LQU Leu Ile Gly Leu ~al ~al ~le Phe
1395 : 1400 1405
TGC ATA CCA TTA TTG CTA TTT TCC TGT TGT ACT ACA GGT TCT TCT CGA 4272
CYB Il-`Pro Leu Leu Leu PhQ Cy~ Cy~ Cyc Ser Thr Cly Cy~ Cy~ Gly
1410 1415 1420
TGC ASA GGT TGC TTA GGA AGT TGT TGT CAC TCT ASG TGT ~GT AGA AGA 4320
y25Ile Gly Cy~ Leu:~ll3y05-r Cy~ Cy~ Hi8 S;r M~t Cy~ Ser Asg Arg
CAA ~TT~ GAA AAT TAT GAA CCA ATT GAA AAA CTC CAT GTC CAC 4362
Gln Ph~ Clu:A-n Tyr Glu Pro ~le Glu Lyc Val Hl~ V~l Hi~
1~445 1450
TAA~ 4365
-
' ! (2) ~NFO~MATlON FOR SEQ ID NO:12: ' ! I
(1) SEQUENCE CHARACTERIS~CS:
~A) LENGTH: 1454 amlno acld~ ~
~-~- (B) TYPE: amlno acid
: (D) ~OPOLOGY: linear
~; ( ii ) MOLECUEE TYPE : protein
SUBSl ITUTE SHEET
W O 93/23422 PCT/U~93/04384
- - 2131898
(xl) SEQUENCE DESC~IPTION: SE~ ID NO:12s
~et Ile Val Leu Val Thr Cy~ Leu Leu Leu Leu Cy~ Ser Tyr Hls Thr
1 5 10 15
Val Ser Ser Thr Ser A~n Asn ~sp Cy~ Arg Gln Val Aan Val Thr Gln
Leu Ala Gly Asn Glu AQn Leu Ile Arg A~p Phe Leu Phe Gln Ser Phe
Ly~ Glu Glu Gly Ile Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
Trp Tyr AQn Cys Ser Arg Thr Ala Thr Thr Thr Ala Tyr Glu Tyr Phe
A~n A~n Ile Hi~ Ala Phe Tyr Ph~ A~p Met Glu Ala Met Glu Asn Ser
Thr Gly Asn Ala Arg Gly LYB Pro Leu Leu Phe ~ 8 Val ~is Gly Glu
100 105 110
Pro Val Ser Ile Ile Ile Tyr Ile Ser Ala Tyr Gly Asp Asp Val Gln
115 120 125
Gln Arg Pro Leu Leu Glu H~ Gly Le~ L~u Cy~ Ile Shr Ly~ Aan Arg
130 135 140
Asn Ile A3p Tyr Asn Thr Phe Thr Ser A~n Gln Trp A~p Ser Ile Cys
145 150 155 160
Thr Gly A~n Aap Arg Ly~ Pro Phe Ser Val Ile Pro Arg Asp A~n
- 165 170 175
Gly Thr Lys Ile Tyr Gly Leu Glu Trp Asn A~p Glu Phe Val Thr Ala
180 lR5 190
Tyr Ile Ser Gly Arg Ser Tyr A~ Trp Asn Ile Asn Aan A~n ~rp Phe
195 ~ 200 205
A~n Asn Val Thr ~u Leu Tyr Ser Ary Ser Ser ~hr Al a Thr Trp Glu
210 215 220
- $yr Ser Ala Ala Tyr Val Tyr Gln Gly Val Ser Asn Phe Thr Tyr Tyr
225 ~30 235 240
Lys Leu Asn Asn Thr A~n Gly Leu Ly~ Thr Tyr Glu Phe Cy~ Clu A~p
245 250 255
Tyr Glu Tyr Cy~ Thr Gly Tyr Ala Thr Asn V~l Phe Al~ Pro Thr V~l
, 260 265 270 :
Gly Gly Tyr Ile Pro Asp Gly Phe Ser Phe Asn Asn Trp Phe Leu Leu
275 280 285
Thr Asn S~r S~r Thr Pha Val Ser Gly Arg PhQ Val Thr ~n Gln Pro
290 295 300
Leu Leu Val Ain Cy9 Leu Trp Pro Val Pro Ser Ph~ Gly Val Ala Ala
305 310 315 320
SUBSTITUTE SHEET
.
WO 93~23422 PCT/US93/0~384
2 1 3 1 8 9 ~ ;
96
Gln Glu Phe Cy~ Phe Glu Gly Ala Gln Phe Ser Gln Cy8 Ser Gly Val
325 330 335
Ser Leu Asn Aqr~ Thr Val A~p Val Ile Arg Phe A~n Leu Asn Phe Thr
t40 345 ~50
Ala Asp v~l Gln ser Gly Met Gly Ali~ Thr Val Phe Ser Leu A~n Thr
355 360 365
Thr Gly Gly Val Ile Leu Glu Val Ser Cy8 Tyr Asn ABP Thr Val Ser
370 3~5 380
Glu Ser Ser Phe Tyr Ser Tyr Gly Glu Ile Pro Phe Gly Ile Thr A~p
385 390 395 400
Gly Pro Arg ~yr Cy9 Tyr Val Leu Tyr Asn Gly Thr Ala Leu Lys Tyr
Leu Gly Thr Leu Pro Pro Ser Val Lys Glu Ile Ala Ile Ser Lys Trp
Gly Hiq Phe Tyr Ile Acin Gly Tyr Asn Phe Phe Ser Thr Phe Pro Ile
435 440 445
Asp Cys Ile Ser Phe Asn Leu Thr Thr Gly Asp Ser Gly Ala Phe Trp
450 455 460
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu Asn
4~5 470 475 480
Thr Ala Ile Ly8 Ly~ Val Thr Tyr Cy~ A~n Ser H~ a Ile Asn Asn I1Q
485 490 495
Lys Cys Ser Gln Leu Thr Ala Asn Leu Asn Asn Gly Phe Tyr Pro Val
500 505 510
Ala Ser Ser Glu Val Gly Leu Val Asn Lys Ser Val Val Leu Leu Pro
515 520 525
Ile Phe Phe Ala His ~hr Ala Ile Asn Ile Thr Ile A~p Leu Gly Miet
530 535 540
Lys Arg Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu Ser Asn Ile
545 550 555 560
Thr Leu Pro Met Gln Asp A~n A~n Thr Asp Val Tyr Cy8 Ile Arg Ser
565 570 575
A~n G! n Phe Ser Val Tyr Val His Ser Ile Cy8 Lys Ser Ser Leu Trp
580 585 590
A~p Asn Ile Phe Asn Gln Glu Cy~ Thr Asy Val Leu Asp Ala Thr Ala
i 595 600 ` 605
Val Ile Lys Thr Gly Thr Cy~ Pro Phe Ser Phe Asp Lys Leu Asn Asn
610 615 620
Tyr Leu Thr Phe Asn Lys Phe Cys Leu Ser Leu Ser Pro Val Gly Ala
625 630 635 640
Asn Cy~ Lys Phe A~p Val Ala Ala Arg Thr Arg Thr Asn Glu Gln Val
645 650 655
SUBSTITUTE SHEEl~
WO 93/23422 PCT/US93/043~4
213Ig98
Val Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly A~p Asn Ile Val Gly
660 665 570
Val Pro Ser Asp Aqn Ser Gly Leu Hi~ Asp Leu Ser Val Leu His Leu
675 680 685
Asp Ser Cys Thr Glu Tyr Asn Ile Tyr Gly Arg Thr Gly Val Gly Ile
~gO 695 700
Ile Arg Gln Thr A~n Ser Thr Leu L~u Ser Gly Leu Tyr Tyr Thr Ser
705 710 715 720
Leu Ser Gly Asp Leu Leu Gly Phe Ly~ Asn Val Ser A~p Gly Val Ile
725 730 735
Tyr Ser Val Thr Pro Cy~ A~p Val S~r Ala Gln Ala Ala Val Ile A~p
740 745 750
GIy Ala Ile Val Gly Ala Met Thr Ser Il~ Asn Ser Glu Leu Leu Gly
755 760 765
Leu Lys His Trp Thr Thr Thr Pro Asn Phe ~yr Tyr Tyr Ser Ile Tyr
770 775 780
Asn Tyr Thr A~n Glu Arg ~hr Arg Gly Thr Ala ~le A~p Ser A~n Asp
785 7gO 795 800
Val A~p Cys Glu Pro Ile Ile Thr Tyr Ser Asn Ile Gly Val Cy~ Ly~
805 81~ 815
Asn Gly Ala Leu Val Phe I1Q Aan Val Thr Hia Ser A~p Gly A~p Val
820 825 830
Gln Pro Ile Ser Thr GIy Thr Val Thr Ile Pro Thr Asn Phe Thr Ile
835 840 845
Ser Val Gln Val Glu Tyr Ile Gln Val Tyr Thr Thr Pro Val Ser Ile
8~0 855 860
Asp Cys Ala Arg Tyr Val Cys A~n Gly Asn Pro Arg CYB Aan Lys Leu
865 870 875 880
: Leu Thr Gln Tyr Val Ser Ala Cy- Gln Thr lle Glu Gln Ala ~eu Ala
as 890 89S
Met Gly A}a Arg ~eu Glu Asn Met Glu Val Asp Ser ~et Leu Phe Val
905 910
Ser Glu Asn Ala Leu Lys Leu Ala Ser Val Glu Ala Phe Asn Ser Thr
~ 915 920 925
- - Glu A8n Leu Asp Pro Ile Tyr Ly~ Glu Trp Pro Asn Ile Gly Gly Ser
; , 9!30 1 935 940
: Trp Leu Gly Gly Leu Ly~ Asp Ile Leu Pro Ser His A~n Ser Lys Arg
945 950 955 960
Lys Tyr Arg Ser Ala Ile Glu Asp Leu Leu Phe Asp Ly~ Val VA1 Thr
965 . 970 g75
Ser Gly Leu Gly Thr Val Asp Glu Asp Tyr Lys Arg Cy~ Thr Gly Gly
980 985 990
~' :
SUBSTITUT~ SHEET
:'~
WO 93~23422 2 1 3 ~ 8 9 ~ PC'r/US93/04384
98
Tyr Asp gle Ala Asp Leu Val Cys Ala Gln Tyr Tyr A n Gly Ile Met
Val LPU Pro Gly Val Ala A~n ARP A~p Ly~ ~t Thr M~t ~yr Thr Ala
1010 1015 1020
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly A1B Val
la Ile Pro Phe Ala Val Ala Val Gln Ala Arg Leu As~ Tyr Val Ala
1045 1050 1055
eu Gln Thr A~p Val Leu Asn Lye Asn Gln Gln Il~ Leu Al~ ARn Ala
1060 1065 107~
he A3n Gln Ala Ile Gly A~n Ile Thr Gln Ala Phe Gly Ly~ Val Asn
sp Ala Ile His Gln ~hr er Lys Gly Leu Ala Thr Val Ala Ly~ Ala
;u Ala Ly~ Val Gln A p Val Val Asn Thr Gln Gly Gln Ala Leu S;r
is Leu Thr Val Gln Leu Gln Asn Asn Phe Gln Ala Ile Ser Ser Ser
112S 1130 1135
le Ser A~p Ile Tyr Asn Arg Leu Asp Glu Leu Ser Ala Asp Ala Gln
1140 1145 1150
Val Asp Arg Leu Ile Thr Gly Arg ~eu Thr Ala Leu A~n Ala Phe Val
1155 1160 1165
Ser Gln Thr Leu Thr Arg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu
Ala Lys Aqp Ly~ Val A n Glu Cy~ Val Arg Ser Gln Ser Gln Arg Phe
ly Phe Cy9 Gly A~n Gly Thr Hi~ Leu Phe Ser Leu Ala Asn Ala Ala
1205 1210 ~215
ro Asn Gly Met Ile Phe Phe His Thr Val Leu Leu Pro Thr Ala Tyr
1220 1225 1230
lu Thr 1235Thr Ala Trp Pro Gly Ile Cys Ala Ser Asp Gly A~p Arg
hr Phe Gly Leu Val Val Lys Asp Val Gln Leu hr Leu Ph~ Arg Asn
Leu A~p A~p Ly~ Phe Tyr Leu Thr Pro Arg ~hr ~et Tyr Cln Pro Arg265 1270 1275 1280
la Ala ~hr Ser Ser A~p Phe Val Gln Ile Glu Gly Cy~ Asp Val Leu
1285 1290 1295
he Val Asn Ala Thr Yal Ile Asp Leu Pro Ser Ile Ile Pro AQP Tyr
1300 130~ 1310
Ile Asp Ile Asn Gln Thr Val Gln A~p Ile Leu Glu A~n Tyr Arg Pro
1315 - 1320 13~5
SUBSTITUTE SHEET
WO 93/23422 PCI /lJS93/0438~
:`` 2134~`98
gg
A~n Trp Thr Val Pro Glu Leu Thr Leu A~p Ile Phn Asn Ala Thr Tyr
1330 1335 1340
Leu A~n Leu Thr Gly Glu Ile Asp Aap Leu Glu Phe Arg Ser Glu Ly3
1345 1350 1355 1360
Leu His A~n Thr Thr Val Glu Leu Ala Ile Leu Ile A~p Asn Ile A~n
136S 1370 1375
A~n Thr L~u Val A~ Leu Glu Trp Leu A~ Asg Ile Glu Thr Tyr Val
1380 1385 1390
Lys Trp Pro Trp Tyr Val Trp Leu Leu Ile Gly Leu Val Val Ile Phe
1395 1400 1405
Cys Ile Pro L~u Leu L~u Phe Cy~ Cy~ Cy~ Sar Thr Gly Cys Cy~ Gly
1410 1415 1420
Cy~ Ile Gly Cy~ Leu Gly Ser Cys CYB H~s Ser ~et Cy~ Ser Arg Arg
1425 1430 1435 1~40
Gln Phe Glu A~n Tyr Glu Pro Ile Glu Lys Val HLs Val ~18
1445 1450
(2) INFORMAT~ON FOR SEQ ID NO:13:
(i) SEQUENCE C~ARACTERISTICS: :
(A1 ~ENGTH: 377 ba~e pairs
: (B) TYPE: nucleic acid
~C) ST~ANDE~NESS: double
: ~D) TOPO~OGY: unknown
~ii) MOLECULE TYPE: cDNA
ix) FEATURE:
(A) NAME/KEY: CDS
B) LOCATION: 1..375
xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
~:~AAT GCT CGT GGT AAA CCA TTA TTA TTT CA~ GTG QT GGT GAG CCT GTT 48
:~ ~ Asn Ala Arg Gly Lyg Pro Leu Leu Phe ~i8 Val ~i8 Gly Glu Pro Val
5 10 ~ 15
AGT GT~ ATT ATA TAT ATA TCG GCT TAT Aac GA~ GAT GTC CAA CAA AGG 96
Ser Val Ile Ile Tyr Ile Ser Ala Tyr Ar~ A~p A~p Val Gln Gln Axg
20 ~ 25 30
CCC CTT TTA AAA CAT GGG TTA GTG TGC ATA ACT AAA AAT CGC CAT ATT 144
Pro Leu Leu Lys H1s~Gly~Leu Val Cy8 Ile Thr Ly~ A n Arg H~B Il~
AAC TAT GAA CAA TTC ACC TCC AAC CAG TGG AAT TCC ACA TGT ACG GGT 192
Aan Tyr Glu Gln Phe Ths S~r A~n Gln Tr~ Aon Sar Thr Cy~ ~hr Gly
:~50 5S 60
GCT GAC AGA AAA ATT CCT TTC TCT GTC ATA CCC ACG GAC AAT GGA ACA 240
Ala A~p Arg LYB Ile Pro Phe Ser Val Ile Pro Thr A~p ABn Gly Thr
65 70 75 80
::
- SUBSTITUTE SHEET
WO 93/23422 PCI /US93/04384
2 i 3 ~ (~ 9 ~r
100
AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TTT GTT ACA GCT TAT ATT 288
Ly~ Ile Tyr Gly Leu Glu Trp Aqn A~p Asp Phe Val Thr Ala Tyr Ile
85 90 95
AGT GGT CGT TCT TAT CAC TTG AAC ATC AAT ACT AAT TGG TTT AAC AAT ^ 336
Ser Gly Arg Ser Tyr His Leu Asn Ile A~n Thr Asn Trp Phe A~n A~n
100 105 110
GTC ACA CTT TTG TAT TCA CGC TCA AGC ACT GCT ACC TGG GA 377
Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp
115 120 125
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) L~GTH: 125 amino acids
~8) TYPE: amlno acid
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
A3n Ala Arg Gly Ly~ Pro Leu Leu Phe Hi3 Val ~is Gly Glu Pro Val
1 S 10 15
Ser Val Ile Ile Tyr Ile Ser Ala Tyr Arg A~p Asp Val Gln Gln Arg
Pro Leu Leu Lys His Gly Leu Val Cy~ Ile Thr Lys Asn Arg H~s Ile
Asn Tyr Glu Gln Phe Thr Ser A~n Gln Trp A~n Ser Thr Cys Thr Gly
5~ 60
Ala Asp Arg Ly~ Ile Pro Phe ser Val Ile Pro Thr A~p A~n Gly Thr
Lys Ile Tyr Gly Leu Glu Trp Asn Asp Acp Phe Val Thr Ala Tyr Ile
Ser Gly Arg Ser Tyr ~i~ Leu Asn Ile Asn Thr A~n Trp Phe Asn Asn
100 105 110
Val Thr Leu Leu Tyr S~r Ar~ Ser Ser Thr Ala Thr Trp
115 120 125
(2) INFORMATION FOR SEQ ID NO:15:
ti) SEQUENCE CHAAAC~ERISTICS:
(A) LENGTH: 4365 b~c pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
~D) TOPOLOGY: unknown
(ii) MO~ECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
~B) LOCATION: 1..4362
SUBSTITUTE SHEE r
WO 93/23422 PCr/~lS93/04384
``` 213~98
101
.
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:15:
ATG ATT GTG CTC GTA ACT TGC CTC TTG TTG TTA TGT TCA TAC CAC ACA 4 8
Met Ile Val Leu Val Thr Cys Leu Leu Leu Leu Cys Ser Tyr Hi~ Thr
1 5 lO 15
GTT TTC AGT ACA ACA AAT AAT GAA TGC ATA CAA GTT AAC GTA ACA CAA 9 6
Val Leu Ser Thr Thr Asn Asn Glu Cya Il~ Gln Val A~n Val Thr Gln
20 25 30
TTG GCT GGC AAT GAA AAC CTT ATC AGA GAT TTT CTG TTI' AGT AAC TTT 144
Leu Ala Gly Asn Glu Asn Leu Ile Arg Asp Phe Leu Phe Ser Asn Phe
35 40 45
AAA GAA GAA GGA AGT GTA GTT GTT GGT GGT TAT TAC CCT ACA GAG GTG 19 2
Lys Glu Glu Gly Ser Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
50 55 60
TGG TAC AAC TGC TCT AGA ACA GCT CGA ACT ACT GCC TTT CAG TAT TTT 2 4 0
Trp Tyr Asn Cy~ Ser Arg Thr Ala A-g Thr Thr Ala Phe Gln Tyr Phe
65 70 75 80
AAT AAT ATA CAT GCC TTT TAT TTT GTT ATG GAA GCC ATG GAA ~AT AGC 288
Asn Asn Ile His Ala Phe Tyr Ph~ Val M~at Glu Ala Met Glu Asn Ser
85 90 95
ACT GGT AAT GCA CGT Gt:;T AAA CCA TTA TTA TTT CAT GTt; CAT GGT GAG 336
Thr Gly Asn Ala Arg Gly Ly~ Pro Leu Leu Phe Hi~ Val HL~ Gly Glu
100 105 110
CCT GTT AGT GTT ATT ATA TAT ATA TCG GCT TAT A~G GAT GAT GTG CAA 3 84
Pro Val ser Val Il~ l1Q Tyr I1Q Ser Ala Tyr Arg ABP A~p Val Gln
115 120 125
CAA AGG CCC CTT TTA AAA CAT GGG TTA GTG TGC ASA ACT AA~ AAI~ CGC 432
Gln Arg Pro Leu Leu Lys His Gly Leu Val Cy Ile Thr Ly~ A~n Arg
: ~ 130 1~5 140
CAT AT~ AAC TAT GAA CAA TSC ACC TCC AAC CAC TGG ~ T SCC ACA TGT 480
Hi~ Ile A~n Tyr Glu Gln Phe Thr Ses Asn Cln Trp Asn Ser ~hr Cy8
- 14S 150 155 160
ACG GGT GCT GAC AGA AAA ATT CCT TTC TCT GTC ATA CCC AcG GAC AAT 528
Thr Gly Ala A p Arg Lys Ile Pro Phe Ser Val Ile Pro Thr A~p Aon
165 170 175
- GGA ACA AAA ATC TAT GGT CTT GAG TGG AAT GAT GAC TTT GTT ACA GCT 5 7 6
Gly Thr Ly~ Ile Tyr Gly Leu Glu Trp A~n Asp A~p Phe Val Thr Ala
~ 180 las lso
TAT ATT AGT GGT CGT TCT TAT CAC TTG AAC A'rC AAT ACT AAT TGG TTT 6 2 4
Tyr Ile Ser Gly Arg Ser Tyr Hl~ Leu A~n Ile Asn Thr Asn Trp Phe
- 195 ~ 200 205
AAC AAT GTC ACA CTT TTG TAT TCA CGC TCA AGC ACT GCT ACC TGG GAA 6 7 2
A~n Aan Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
~10 21S 220
TAC AGT GCT GCA TAT GCT TAC CAA GGT GTT TCT AAC TTC ACT TAT TAC 720
Tyr Ser AIa Ala Tyr Ala Tyr Gln Gly Val Ser Asn Phe Thr Tyr Tyr
225 230 ~35 240
SUBSTITUTE SHEE~
WO 93/23422 PCI`/US93/04384
~ 1 3 L¦ ~3 9 8 `;
102
AAG TTA AAT AAC ACC AAT GGT CTA AAA ACC TAT GAA TTA TGT GAA GAT 768
Lys Leu A~n A n Thr A~n Gly Leu Lys Thr Tyr Glu Leu Cy~ Gl~ Asp
245 250 255
TAT GAA CAT TGC ACT GGC TAT GCT ACC AAT GTA TTT GCT CCG ACA TCA~ 816
Tyr Glu ~18 cys Thr Gly Tyr Ala Thr Asn Val Phe Ala Pro Thr Ser
260 265 270
GGT GGT TAC ATA CCT GAT GGA TTT AGT TTT AAT AAT TGG TTC TTG CTT 864
Gly Gly Tyr Ile Pro Asp Gly Phe Ser Phe ABn Asn Trp Ph~ Leu Leu
275 280 285
ACA AAT AGT TCC ACT TTT GTT AGT GGC AGG TTT GTA ACA AAT CAA CCA 912
Thr Asn Ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr A~n Gln Pro
290 295 300
.TA TTG ATT AAT TGC TTG TGG CCA GTG CCC AGT TTT GGT GTA GCA GCA 960
Leu Leu Ile Asn Cys Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
30; 310 315 320
CAA GAA TTT TGT TTS GAA GGT GCA CAG TTT AGC CAA TGT AAT GGT GTG 1008
Gln Glu Phe Cys Phe Glu Gly Ala Gln Phe Ser Gln Cy8 A~n Gly Val
325 330 335
TCT TTA AAT AAC ACA GTG GAT GTT ATT AGA TTC AAC CTT AAT TTC ACT 1056
Ser Leu A~n Asn Thr Val Asp Val Ile Arg Phe Aan Leu Asn Phe Thr
340 34S 350
GCA~GAT GTA CAA~TCT GGT ATG GGT GCT ACA GTA TTT TCA CTG AAT ACA 1104
Ala Asp Val Gln ser Gly Met Gly Ala Thr Val Phe Ser Leu Asn Thr
355 360 365
~ACA GGT GGT G$C ATT CTT GAA ATT TCA TGT TAT AGT GAC ACA GTG AGT 1152
Thr Gly Gly Val Ile Leu Glu Ile Ser Cys Tyr Ser~Asp Thr Val Ser
3~0 3~5 380
GAG TCT AGT TCT TAC AGT TAT GGT GAA ATC CCG TTC GGC ATA ACT GAC 1200
Glu Ser Ser ser Tyr Ser Tyr Gly Glu Ile Pro Phe Gly Ile Thr Asp
385 390 395 . 4~0
GGA CCA CGA TAC TCT TAT GTA CTT TAC AAT GGC ACA GCT CTT AAA TAT 1248
:Gly Pro Arg Tyr Cys Tyr Val Leu Tyr A8n Gly Thr Ala Leu Lys Tyr
TTA GGA ACA T~A CCA CCC AGT GTA AAG GAA ATT GCT ATT AGT AAG TGG 1296
Leu Gly Thr Leu Pro Pro Ser Val Lys Glu Ile Ala Ile Ser Lys Trp
420 425 430
~: ~
GGC CAT T~T TAT ATT AAT GGT TAC AAT TTC TTT AGC ACA TTT CCT ATT 1344
Gly His Phe Tyr Ile Asn Gly Tyr Asn Phe Phe Ser Thr Phe Pro Ile
435 440 445
GAT TGT ATA TCT TTT; AAT TTA ACC ACT GGT GTT ACT GGA GCT TTT TGG 1392
Asp Cyq I le Ser Phe Asn Leu Thr Thr Gly Val Ser Gly Ala Phe Trp
450 455 460
ACA ATT GCT TAC ACA TCG TAT ACT GAA GCA TTA GTA CAA GTT GAA AAC 1440
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val G~u A~n
465 470 475 480
ACA GCT AT~ AAA AAT GTG ACG TAT TGT AAC AGT CAC ATT AAT AAC ATT 1488
Thr Ala Ile Ly~ A8n Val Thr Tyr Cy~ A~n Ser Rls Ile A8n Asn Ile
485 490 49S
SUBSTITIJTE SHEET
WO 93/23422 - PCrJUS93/043~4
- 21~8.98
103
AAA TGT TCT CAA CTT ACT GCT AAT TTG A~T AAT GGA TTT TAT CCT GTT 1536
Ly-q Cys Ser Gln Le~ Thr Ala A~n Leu Aun Asn Gly Ph~ Tyr Pro Val
500 505 510
GCT TCA AGT GAA GTA GGT TTC GTT AAT AAG AGT GTT GTG TTA TTA CCT ~ 1584
Ala Ser Ser Glu Val Gly Phe Val Asn Ly~ Ser Val Val Leu Leu Pro
515 5~0 52S
AGC TTT TTC ACA TAC ACC GCT GTC AAT ATA ACC ATT GAT CTT GGT ATG 1632
Ser Phe Phe Thr Tyr Thr Ala Val Asn Ile Thr Ile A~p Leu Gly Met
530 5~5 S40
AAG CTT AGT GGT TAT GGT CAA CCC ATA GCC TCG ACA CTA AGT AAC ATC 1680
Lyq Leu Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr L~u S~r A~n Ile
S45 550 555 560
ACA CTA CCA ATG CAG GAT AAC AAT ACT GAT GTG TAC TGT ATT CGT TCT 1728
Thr Leu Pro Met Gln ABP Asn Asn Thr Asp Val Tyr Cy~ Ile Arg Ser
565 570 S75
AAC CAA TTC TCA GTT TAT GTT CAT TCC ACT TGC AAA AGT TCT TTA TGG 1776
Asn Gln Phe Ser Val Tyr Val Hig Ser Thr Cy~ Lys Ser Ser Leu Trp
580 585 590
GAC AAT ATT TTT AAT CAA GAC TGC ACG GAT GTT TTA GAG GCT ACA GCT 1824
Asp Asn Ile Phe Asn Gln A~p Cy~ Thr A~p Val Leu Glu Ala Thr Ala
595 600 60~
GTT ATA AAA ACT GGT ACT TGT CCT TTC TCA TST GAT AAA TTG AAC AAT 1872
Val Ile Lys Thr Gly Thr Cy~ Pro Phe Ser Ph2 A~p Lys Leu Asn A~n
610 615 620
TAC TTG ACT T~T AAC AAG TTC TGT TTG TCG TTG AGT CCT GTT GGT GCT 1920
Tyr Leu Thr Phe Asn Lys Phe Cys Leu Ser Leu Ser Pro Val Gly Ala
6~25 630 635 640
AAT TGC AAG TTT GAT GTT GCT GCA CGT ACA AGA ACC AA$ GAG CAG GTT 1968
Asn Cys Lys Phe Asp Val Ala Ala Arg Thr Arq Thr Asn Glu Gln Val
645 650 655
GTT AGA AGT CTA TAT GTA ATA TAT GAA GAA GGA GAC AAC ATA GTG GGT 2016
Val Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly Asp Asn Ile Val Gly
660 665 6~0
GTA CCG TCT GAT GAT AGC GGT CTG CAC GAT TTG TCT GTG CTA CAC CTA 2064
Val Pro Ser A~p Asp Ser Gly Leu ~i~ AEP Leu Ser V~l Leu H~ 8 Leu
675 680 6B5
GAC TCC TGT ACA GAT TAC AAT ATA TAT GGT AGA ACT GGT GTT GGT ATT 2112
Asp Ser Cy~ Thr Asp Tyr A~n Ile Tyr Gly Arg Thr Gly Val Gly Ile
690 695 ~00
ATT ~GA CGA ACT AAC AGT ACG CTA CTT AGT GGC TTA TAT TAC ACA TCA ~ 2160
Ile Arg Arg Thr Asn Ser Thr Leu Leu Ser Gly Leu Tyr Tyr Thr Ser
705 710 715 720
CTA TCA GGT OAT TTG T$A GGC TTT AAA AAT GTT ACT CAT GGT CTC ATT 2208
Leu Ser Gly Asp Leu Leu Gly Phe Lys Asn Val S~r Asp Gly Val Ile
725 730 735
TAT TCT GTG ACG CCA TGT GAT GTA AGC GCA CAA GCG GCT GTT ATT GAT 2256
Tyr Ser Val Thr Pro Cy~ A~p Val Ser Ala Gln Ala Ala Val Ile Asp
740 745 750
SU8STITUTE S~EEr
WO 93/234~2 2 1 3 1 8 9 8 PCI`/US93/043~
104
GGT GCC ATA GTT GGA GCT ATG ACT TCC ATT AAC AGT GAA CTG TTA GGT 2304
Gly Ala Ile Val Gly Ala Met Thr Ser Ile A~n Ser Glu Leu Leu Gly
CTA AAA CAT TGG ACA ACG ACA CCT AAT TTT TAT TAC TAC TCT ATA TAT 2352
Leu Lys His Trp Thr Thr Thr Pro ARn Phe Tyr Tyr Tyr Ser Ile Tyr
?70 775 780
AAT TAC ACA AGT GAG AGG ACT CGT GGC ACT GCA ATT GAC AGT AAC GAT 2400
Asn Tyr Thr Ser Glu Arg Thr Arg Gly Thr Ala Ile Asp Ser Asn Asp
785 790 795 800
GTT GAT TGT GAA CCT 5TC ATA ACC TAT TCT AAT ATA GGT GTT TGT AAA 2448
Val Asp Cy~ Glu Pro Val Il~ Thr Tyr Ser Asn Ile Gly Val Cy~ Lys
805 810 815
AAT GGT GCS TTG GTT TTT ATT AAC GTC ACA CAT TCT GAC GGA GAC GTG 2496
A~n Gly Ala L~u Val Phe Ile Asn Val Thr ~ 8 Ser Asp Gly A~p Val
. 820 825 830
CAA CCA ATT AGC ACT GGT ATT GTC ACG ATA CCT ACA AAT TTT ACC ATA 2544
Gln Pro Ile Ser Thr Gly Ile Val Thr Ile Pro Thr Asn Ph~ Shr Ile
835 840 845
TCT GTG CAA GTT GAA TAC ATG CAG GTT TAC ACT ACA CCA GTA TCA ATA 2S92
Ser Val Gln Val Glu Tyr Met Gln Val Tyr Thr Thr Pro Val Ser Ile
850 855 : 860
GAT TGT GCA AGA TAC CTT TGT AAT GCT AAC CCT ACA TGT AAC AAA TTG 2640
A p Cy~ Ala~Arq Tyr Val Cy~ A~n Gly Asn Pro Arg Cy~ A~n Lya Leu
TTA ACA CAA TAT GTG TCT :GCA TGT CAA ACT ATT GAA CAA GCA CTT GCA 2688
Leu Thr Cln Tyr Val ser Ala CYQ Gln Thr Il~ Clu Cln Ala Leu Ala
: 885 890 895
ATG GGT GCC ACA CT~ CAA AAC ATG CAG CTT CAT TCC ATG TTG TST GTC 2736
Met Gly Ala Arg Leu Glu A~n Met Glu Val ABP Ser Met ~eu Phe Val
900 905
TCG:GAA AAT GCC CTT AAA TTG GCA TCT GTT GAG GCG TTC AAT AGT ACA 2784
Ser GLu Agn~Ala Leu Lys Leu Ala Ser Val Glu Ala Phe ARn Sex Thr
GAA AAT TTA~GAT CCT ATT TAC AAA:GAA ~GG CCS ACC ASA GGT CGT TCT 2832
Glu Asn Leu Asp~Pro Ile Tyr Ly~ Glu Trp Pro Ser Ile Gly Gly Ser
930 ~ 935: 940
TGO:CTA GGA GCT CTA AAA~GAT ATA CTA CCG TCC CAT AAT AGC AAA CGT 2880
Trp Leu Gly Gly Leu Lys Asp Ile Leu Pro Ser H~ A~n Ser Lys Arg
945 950 955 960
AAG TAT GGT TCT GCT ATA GAA GAT TTG CTS TTT GAT AAA GTT GTA ACT 292B
Lys Tyr Gly Ser Ala Ile Glu Asp Leu Leu Phe Asp Lys Val Val Thr
965 970 975
: '
TCT GGT TTA GGT ACA GTT GAT GAA GAT TAT AAA CGT TGT ACT GGT GGT 29~6
~ Ser Gly Leu Gly Thr Val Agp Glu Agp Tyr Lyg Arg Cys Thr Gly Gly
J ~ 980 985 990
~ TAC GAC ATA GCA GAC TT~ GTG TGT GCT CAA TA~ TAC AAT GGC ATC ATG 3024
: Tyr AQP Ile Ala A~p Phe Val CY5 Ala Gln Tyr Tyr A~n Gly Ile Met
~ 995 1000 1005
SUBSTITUTE SHEET
:' .
WO 93/23422 P~/US93/04384
``` - 21~ ~9~
!
105
GTT CTA CCA GGT GTA GCT AAT GCT GAC A~G ATG ACT ATG TAC ACA GCA 3072
Val Leu Pro Gly Val Ala Asn P.la A~p Ly~ Met Thr Met Tyr Thr Ala
1010 lOlS 1020
TCA CTT GCA GGT GGT ATA ACA TTA GGT GCA CTT GGT GGT GGC GCC GTG 3120
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
1025 103û 1035 1040
GCT ATA CCT TTT GCA GTF- GCP~ GTT CAA GCT AGA CTT AAT TAT GTT GCT 3168
Ala Ile Pro Phe Ala Val Ala Val Gln Ala Arg Leu A~n Tyr Val Ala
1045 1050 1055
CTA C~A ACT GAT GTA TTG AAT AAA AAC CAA CAG ATC CTG GCT AAT GCT 3216
Leu Gln Thr A~p Val Leu Asn Lys A~n Gln Cln Ile Leu Ala A~n Ala
1060 1065 1070
TTC AAT CAA GCT ATT GGT AAC ATT ACA CAG GCT TTT GGT A~G GTT AAT 3264
Phe A~n Gln Ala Ile Gly Asn Ile Thr Gln Ala Phe Gly Lyo Val A~n
1075 1080 1085
GAT GCT ATA CAT CAA ACA TCA CAA GGT CTT GCC ACT GTT GCT AA~ GCG 3312
Asp Ala Ile His Gln Thr Ser Gln Gly L~u Ala Thr Val Ala Ly~ Ala
1090 lOgS 1100
TTG GCA AAA GTG CAA GAT GTT GTC AAC ACA CAA GGG CA~ GCT TTA AGT 3360
Leu Ala Ly~ Val Gln A~p Val Val Asn Thr Gln Gly Gln Ala Leu Ser
llOS 1110 1115 1120
CAC CTT ACA GTA CAA TTG CAA AAT AAT TTT CAA GCC ATT AGT AGT TCT 3408
His Leu Thr Val Gln LRU Gln A~n A~n Phe Gln Ala Ile Ser Ser Ser
1125 1130 ll~S
ATT AGT GAT ATT TAT AAC AGG CTT GAC GA~ CTG AGT GCT GAT GCA CAA 3456
r~ Ile~Ser Asp IIe Tyr Asn Arg Leu Asp Glu L~au ser Ala A~p Ala Gln
~ 1140 1145 llS0
~.~
GTT GAT AGG CTG ATT ACA GGT AGA CTT ACA GCA CTT AAT GC~ TTT GTG 3504
Val A~p Arg Leu Ile Thr Gly Ars~ Leu Thr Ala Leu Asn Ala Phe Val
1155 1160 1165
TCT CAG ACT CTA ACC AGA CAA GCA GAG GTT AGG GCT AGT AGA CAA CTT 3552
Ser Gln Thr Leu Thr Arg Gln Ala Glu Val Arg P.la Ser Arg Gln Leu
1170 1175 1180
-- GCC AAA GAC AAG GTT AAT GAA TGT GTT AGG TC$ CAG TCT CAG AGA TTC 3600Ala Ly~ A p Ly~ Val Asn Glu Cy~ Val Arg Ser Gln Ser Gln Arg Phe
1185 llgO 1195 1200
GGA TTC TGT GGT AAT GGT ACA CAT TTG TTT TCA CTC GCA AAT GCA GCA 364R
Gly Phe Cy~ Gly A~n Gly Thr ~i9 Leu Phe Ser Leu Ala Asn Ala Ala
1205 1210 1215
CCA AAT GGC ATG ATT TTC TTT CAT ACA GTA CTA TTA CCA ACA GCT TAT 3696
Pro A~n Gly Met Ile Phe Phe Hi~ Thr Val Leu LBU Pro Thr Ala Tyr
1220 1225 1230
GAA ACT GTA ACA GC~ TGG TCA GGT ATT TGT GCS TCA GAT GGC GAT CGC 3744
Glu Thr Val Thr Al~ Trp Sér Gly Ile Cy~ Ala Sor A~p Gly Aup Arg
1235 1240 1245
ACT TTC GGA CTT GTC GTT AAA GAT GTG CAG TTG ACG TTG TTT CGT AAT 3792
Thr Phe Gly Leu Val Val Ly~ A~p Val Gln Leu Ths Leu Phe Arg Asn
12~0 1255 1260
SUBSTITUTE SHEET
WO 93/23422 PCI`/US93/04384~
2 ~ ~ ~ gg;8` `'''''~'
106
CTA GAT GAC AAG TTC TAT TTG ACC CCC AGA ACT ATG TAT CAG CCT AGA 3840
Leu Asp Asp Ly~ Phe Tyr L~u Thr Pro Arq Thr Met Tyr Gln Pro Arg
1265 1270 1275 12a0
GTT GCA AC~ AGT TCT GAT TTT GTT CAA ATT GAA GGG TGT GAT GTC TTG 3888
Val Ala Thr Ser S~r A~p Phe Val Gln Ile Glu Gly Cy~ A~p Val Leu
1285 1290 1295
TTT GTC AAT GCA ACT GTA ATT GAT TTG CCT AGT ATT ATA CCT GAC TAT 3936
Phe Val Asn Ala Thr Val Ile Asp Leu Pro Ser Ilo Ile Pro Asp Tyr
ATT GAC ATT AAT CAA ACT GTT CAA GAC ATA TTA GAA AAT TAC AGA CCA 3984
Ile A~p Ile Asn Gln Thr Val Gln Asp Ile Leu Glu Asn Tyr Arq Pro
1315 1320 1325
AAC TGG ACT GTA CCT GAA TTT ACA CTT GAT ATT TTC AAC GCA ACC TAT 4032
Asn Trp Thr Val Pro Glu:Phe Thr Leu A~p Ile Phe Asn Ala Thr Tyr
1335 1340
STA AAT CTG ACT GGT GAA ATT GAT GAC TTA GAG TTT AGG TCG GAA AAG 4080
Leu Asn Leu Thr Gly Glu~Ile Asp Asp Leu Glu PhQ Arg~;Ser Glu Lys
1345 1350 1355 1360
CTA CAT AAC ACT ACA GTA GAA CTT GCC ATT CTC ATT GAT AAC ATT AAT 4128
Leu ~is Asn Thr Thr Val Glu Leu Ala Ile Leu Ile Asp Asn }le Aan
1365 1370 1375
AAT~ACA TSA GTC AAT CTT GAA TGG CTC AAT AGA ATT GAA AC2 TAT GTA 4176
A~n~Thr~Leu~Val Asn Leu Glu Trp Leu Asn Arg Ile Clu Thr Tyr Val
1380~ ~ 1385 1390
AAA~TGO CCT~2CC~TAT~GTC TGC CTA CTA ATA GGT TTA GSA GTA GTA TTT 4224
~ys Trp Pro Trp Tyr Val ~rp Leu Leu lle Gly L~u Val Val Val Phe
1395 ~1400 1405
TCC A2A CCA~TTA CTG CTA TTT TCC TGT STT AGC ACA GCT TGT SGT GGA . 4272
Cy~ Ile~Pro~Leu~Leu Leu ~he Cys Cys Phe Ser Thr Gly Cys Cys Gly
410~ 141S 1420
TGC:~2A GGT`~TGT TTA~GGA AGT TGT TGT CAC TCT ATA TGT AGT AGA ACA 4320
:Cys~le Gly Cys:~ou:Gly~Ser Cys Cy8 H~ Sor II~ Cy8 Sar Arg Arg
~ 1425 ::~ 1430 ~ ~ ~ 1435 1440
i~; CAA TTT GAA~AAT TAT:~GAA CCA ATT GAA AAA GTG CAT GTC CAC 4362
;:; : Gln~:Phe Glu Asn ~Tyr Glu Pro Ile~Glu Lys Val Hls Val Hl8
1445 : ~ 1450
: TAA
: 4365
: ~ (2) ~NF~RMATION FOR SEQ ~D NO:16:
- (1) SEQUENCE CHARACTERISTICS:
~ - (A1 LENGTH: 1454 ~mino aclds
Ci~ : (B) TYPE: amino acLd
D) TOPO~OGY: lLnear
(Ll) MOLECU~E TYPE: protein
,
~' .
SUBSTITUTE SHEET
WO 93/23422 PCI /US93/04384
213 1~9~
107
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Met Ile Val Leu Val Thr Cys Leu Leu Leu Leu Cys Ser Tyr Hls Thr
1 5 10 15
Val Leu Ser Thr Thr A~n Asn Glu Cy~ Ile Gln Val Asn Val Thr Cln
Leu Ala Gly Asn Glu Asn Leu Ile Arg A~p Phe Leu Ph~ ser AQn Phe
Lys Glu Glu Gly Ser Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
Trp Tyr Asn Cys Ser Arg Thr Ala Arg Thr Thr Ala Ph~ Gln Tyr Phe
A~n Asn Ile HLs Ala Phe Tyr Phe Val Met Glu Ala Met Glu Asn Ser
~5 90 95
Thr Gly Asn Ala Arg Gly Lys Pro Leu Leu Phe Hi3 Val Hi~ Gly Glu
1~0 105 110
Pro Val Ser Val Ile Ile Tyr Ile Ser Ala Tyr Arg Asp Asp Val Gln
115 120 125
Gln Arg Pro Leu Leu Lys H~s Gly Leu Val Cy8 Ile Thr Ly~ A~n Arg
130 135 140
His Ile Asn Tyr Glu Gln Phe Thr Ser Asn Gln Trp A~n Ser Thr Cys
- 145 150 155 160
Thr Gly Ala A~p Arg ~y8 Ile Pro Phe Ser Val Ile Pro Thr Aqp Asn
165 170 175
Gly Thr Lys Ile Tyr Gly Leu Glu Trp A~n A~p A~p Phe Val Thr Ala
180 18S 190
Tyr Ile Ser Gly Arg Ser Tyr Hi~ Leu Asn Ile A~n Thr A~n Trp Phe
195 200 205
A3n Asn Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
210 215 220
Tyr Ser Ala Ala Tyr Ala Tyr Gln Gly Val Ser Asn Phe Thr Tyr Tyr
225 230 2~5 240
Lys Leu Asn Asn Thr A~n Gly Leu Ly~ Thr Tyr Clu Leu Cys Glu Asp
245 250 2S5
Tyr Glu H~s Cys Thr Gly Tyr Ala Thr Asn Val Phe Ala Pro Thr Ser
- 260 265 270
Gly Gly Tyr ~le Pro Asp Gly Phe Ser Phe Asn A~n Trp Phe Leu Leu
275 2~0 285
Thr A~n Ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr A~n Gln Pro
290 295 300
Leu Leu Ile Asn Cys Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
305 310 ~15 320
SUBSTIT~JTE SHEET
W O 9~/23422 PCT/U~93/04384
2i3 189~
108
Gln Glu Phe Cys Phe Glu Gly Ala Gln Phe Ser Gln Cy~ ARn Gly Val
3 4 0 3 4 5 3 5 0
Ala Asp Val Gln Ser Gly Met Gly Ala Thr Val Phe Ser Leu Asn Thr
Thr Gly Gly Val Ile Leu Glu Ile S~r cy~ Tyr Ser A~p Thr Val S~r
370 375 380
Glu Ser Ser Ser Tyr Ser Tyr Gly Glu Ile Pro Phe Gly Ile Thr Asp
Gly Pro Arg Tyr Cy9 Tyr Val Leu Tyr Asn Gly Thr Ala Leu Ly~ Tyr
405 410 415
Leu Gly Thr Leu Pro Pro Ser Val Lys Glu Ile Ala Ile Ser Lys Trp
Gly His Phe Tyr Ile Asn Gly Tyr A~n Phe Phe Ser Thr Ph~ Pro Ile
435- 440 445
Asp Cys Ile Ser Phe Asn Leu Thr Thr Gly Val Ser Gly Ala Phe Trp
Thr Ile Ala Tyr Thr S~r Tyr Thr Glu Ala Leu V~l Gln Val Glu A~n
465 470 475 480
Thr Ala Ile Lys Aon Val Thr Tyr Cy9 A~n Ser H~ 8 Ile Asn A~n Ile
485 490 495
Ly~ Cys Ser ~Gln LQU Thr Ala Asn Leu A~n Asn Gly Pha Tyr Pro Val
Ala Ssr Ser Glu Val Gly Phe Val A~n Lys Ser Val Val Leu L~u Pro
515 520 525
Ser Phe Phe Thr Tyr Thr Ala Val Asn I le Thr I 1 Asp Leu Gly Met
Lys Leu Ser Gly ~yr Gly Gln Pro Ile Ala Ser Thr Leu Ser Asr~ Ile
545 550 555 560
Thr Leu Pro Met Gln A~p A8n Asn Thr Asp Val ~yr Cy~ Ile Arg Ser
Asn Gln Phe Ser Val Tyr Val His Ser Thr Cys Ly~ Ser Ser Leu Trp
Asp Asn lle Phe Asn Gln Asp Cy8 Thr Asp Y~l L~u Glu Ala Thr Ala
! ' 595 600 605
Val I le Lys Thr Gly ~hr Cys Pro Phe Ser Phe Asp Lys Leu Aqn Asn
610 615 620
Tyr Leu Thr Phe ~sn Lys Phe Cy8 Leu Ser Leu Ser Pro Val Gly Ala
625 630 63; 640
A~n Cys Lys Phe Asp Val Ala Ala Arg Thr Arg Thr Asn Glu Gln Val
645 650 655
SUBSTITUTE SHEEl
WO 93/23422 PCI /US93/04384
213 ~98
109
al Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly Asp Asn Ile Val Gly
660 665 670
Val Pro Ser Asp A~p Ser Gly Leu HLs A~p Leu Ser Val Leu Hl~ Leu
675 680 685
Asp Ser Cys Thr Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Val Gly Ile
690 695 700
Ile Arg Arg Thr Asn Ser Thr Leu Leu Ser Gly Leu Tyr Tyr ~hr Ser
705 710 715 720
Leu Ser Gly Asp Leu Leu Gly Phe Lys A~n Val Ser Asp Gly Val Ile
~25 730 735
Tyr Ser Val Thr Pro Cys Asp Val Ser Ala Gln Ala Ala Val Ile Aep
740 745 750
Gly Ala Ile Val Gly Ala Met Thr Ser Ile Asn Ser Glu Leu Leu Gly
755 760 765
Leu Lys His Trp Thr Thr Thr Pro A~n Phe Tyr Tyr Tyr Ser Ile Tyr
770 775 780
Aqn Tyr Thr Ser Glu Arg Thr Arg Gly Thr Ala Ile Asp Ser Asn Asp
785 790 ~95 890
Val Asp Cy~ Glu Pro Val Ile Thr Tyr Ser Asn Ile Gly Val Cys Lys
805 810 815
Asn: Gly Al:a Leu Val Phe Ile Asn Val Thr H~ Ser ABP Gly A~p Val
820 825 830
Gln Pro Ile Ser Shr Gly Ile Val Thr Ile Pro Thr Asn Phe Thr Ile
835 840 ~45
Ser Val Gln Val Glu Tyr Met Gln Val Tyr Thr Thr Pro Val Ser Ile
8S0 855 860
A3p Cys Ala Arg :Tyr Val Cy~ A~n Gly A~n Pro Arg Cy~ Asn LYD Leu
865 8~0 875 880
Leu Thr Gln Tyr Val Ser Ala Cys Gln Thr Ile Glu Gln Ala Leu Ala
885 890 8~5
Met Gly Ala Arg Lc~u Glu A~n Met Glu Val A~p Ser Mot Lau Phe Val
900 905 910
Ser Glu Asn Ala Leu Lys Leu Ala Ser Val Glu Ala Phe Asn Ser Thr
915 920 925
Glu Asln Leu Asp Pro Ile Tyr Lys Glu Trp Pro Ser Ile Gly Gly Ser
930 935 940
Trp Leu Gly Gly Leu Lys Asp Ile Leu Pro Ser H$~ Asn Ser Lys Arg
945 950 955 960
Ly3 Tyr Gly Ser Ala Ile Glu A~p Leu Leu Phe A~p Lys Val Val Thr
965 970 975
Ser Gly Leu Gly Thr Val Asp Glu ~0p Tyr Lys Arg Cys Thr Gly Gly
g80 985 990
SUBSTITUTE SHEE~
WO g3/23422 P~/US~3/0~3~4
2131~
110
Tyr Asp Ile Ala Aqp Phe Val Cy~ Ala Gln Tyr Tyr Asn Gly Ile Met
995 1000 1005
Val Leu Pro Gly Val Ala A~n Ala A~p Ly~ Met Thr Met Tyr Thr Ala
1010 1015 1020
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
1025 1030 1035 1040
Ala Ile Pro Pha Ala Val Ala Val Gln Ala Arg Leu A~n Tyr Val Ala
1045 . 1050 1055
Le~ Gln Thr A~p Val Leu A~n Ly~ A~n Gln Gln Ile Leu Ala A~n Ala
1060 1065 1070
Phe Asn Gln Ala Ile Gly Asn Ile Thr Gln Ala Phe Gly Ly~ Val Aan
1075 1080 1085
Asp Ala Ile H~s Gln Thr Ser Gln Gly Leu Al~ Thr V~l Al~ Lys Ala
1090 . 1095 1100
Leu Ala Lys Val G1~ Asp Val Val A~n ~hr Gln Gly Gln Ala Leu Ser
1105 1110 1115 1120
Hi~ Leu Thr Val Gln Leu Gln A~n Asn Phe Gln Ala Ile Ser Ser Ser
1125 1130 11~5
~le Ser A~p Ile Tyr A~n Arg Leu A~p Glu Leu Ser Ala Asp Ala Gln
1140 1145 llS0
Val Asp Arg Le~ Ile Thr Gly Arg Leu Thr Ala Leu Asn Ala Phe Val
1155 1160 1165
~:~ Ser Gln Thr Leu Thr Arg Gln Ala Clu Val Arg Ala Ser Arg Gln Leu
1170 1175 1180
Ala Lya Asp Lys Val A~n Glu Cy~ Val Arg Ser Gln Sor Gln Arg Phe
1185 1190 1195 1200
Gly Phe Cy~ Gly Asn Gly Thr Hi9 Leu Phe Ser Leu Ala Asn Ala Ala
1205 1210 1215
Pro Asn Gly Met Ile Phe Phe ~is Thr Val Leu Leu Pro Thr Ala Tyr
: 1~20 122S 1230
Glu Thr Val Thr Ala Trp Ser Gly Ile Cys Ala Ser Asp Gly Asp Arg
1235 1240 1245
Thr Phe Gly Leu Val Val Ly~ A8p Val Gln Leu Thr Leu Phe Arg Asn
1250 1255 1260
Leu A~p Asp Lys Phe Tyr Leu Thr Pro Arg ~hr Met Tyr Gln Pro Arg
1265 1270 1275 1280
Val Ala Thr Ser Ser A~p Phe Val Gln Ile Glu Gly Cy~ A9p Val Leu
1285 1290 1295
Phe Val Acn Ala Thr Val Ile A~p Leu Pro S~r Ile ~le Pro A~p Tyr
1300 1305 1310
Ile A~p Ile A~n Gln Thr Val Gln Aap Ile Leu Glu A8n Tyr Arg Pro
1315 1320 1325
SllBSTlTll~E SHEET
W O 93/23422 PfCT/~S93/W384
... ,,,., 213~g98
111
AQn Trp Thr Val Pro Glu Phe Thr Leu Affffp Ile Phe Aff~ffn Ala Thr Tyr
1330 1335 1340
Leu Asn Leu Thr Gly Glu Ile ABP Asp Leu Glu Phe Arg Ser Glu Ly
1345 1350 1~55 1360
Leu His Acfffn Thr Thr Val Glu Leu Ala Ile Leu Ile A~p Asn Ile Asn
1365 1370 1375
A3n Thr Leu Val Asn Leu Glu Trp Leu A~n Arg Ile Glu Thr Tyr Val
1380 1385 1390
Ly3 Trp Pro Trp Tyr Val Trp LfafU LQU Ilff2f Gly Leu Val Val Val Phe
1395 1400 1405
Cys I le Pro Leu Leu Leu Phe Cyf~ Cyffff Phe Ser Thr Gly Cys Cys Gly
1410 1415 1420
Cyffff Ile Gly Cy8 Leu Gly Ser Cyf Cyffsf Hl~f Ser Ile Cy~ Ser Arg Arg
1425 1430 1435 1440
Gln Phe Glu Aff3n Tyr Glu Pro Ilfreff Glu Ly~ Val ~f~f Val HifJ
1445 1450
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4359 base pairfa
(B~ TYPE: nucle~c acid `
~C) ST~ANDEDNESS: doublf3
(D) TOPOLOGY: unknown
: (ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY.: CDS
(B) LOCATION: 1..4356
(xi) SEQUENCE DESf~RlPTION: SEQ ID NO:17:
ATG ATT GTG CTC GTA ACT TGC CTf~ TTG TTT TCG ~AC AAT AGT GTG ATT 48
Met Ile Val Leu Val Thr Cys Leu Leu Pbe Ser ~yr Aan Ser Val Ile
~ 1 5 10 15
TGT ACA TCA AAC AAT GAC TGT GTA CAA GTT AAT GTG ACfA CAA TTG CCT 96
Cy~f Thr Sf~fr Asn Asn Aafp Cys Val Gln Yal Af~ Val Thr f~ln Leu Pro
20 25 30
GGC AAT GAA AAC ATT ATT AAA GAT TTT CTA TTT CAC ACC TTC AAA GAA 144
f fGly A3ln Glu Asn I~le Ile Lys A~fp Phe Leu Phe Hls Thr Phe Lys Glu
35 40 4S
G~A GGA AGT GTA GTT GTT GGT GGT TAT TAC CCT Af~A GAG GTG TCG frAT 192
Glu Gly Ser Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val Trp Tyr
S0 55 60
AAC SGC SCC ACA AGC GCA ACA ACC ACC GCT TAC AAG ~AT TTT AGT AAT 240
. Asn CYB S~r Arg Ser Ala Thr Thr Thr Ala ~yr LYB A~p Phe Ser Asn
65 70 75 80
SUBSTITUTE SHEET
WO 93/23422 PCr/US93/04384
21318~8
112
ATA CAT GCA TTC TAT TTT GAT ATG GAA GCC ATG GAG AAT AGT ACT GGC 288
Ile HiR Ala Phe Tyr Phe Asp Met Glu Ala Met Glu Asn Ser Thr Gly
90 ~S
AAT GCA CGA GG~ AAA cC~ TTA C~A GTA CAT GTT CAT GGT GAT CCT GTT ~ 336
Asn Ala Arg Gly Ly~ Pro Leu Leu Val Hls Val His Gly Asp Pro Val
100 105 110
AGT ATC ATC ATA TAT ATA TCG GCT TAT AGA CAT GAT GTG CAA GGA AGG 384
Ser Ile Ile Ile Tyr Ile Ser Ala Tyr Arg Asp Asp Val Gln Gly Arg
120 12S
CCT CTT TTA AAA CAT GGT TTG TTG TGT ATA ACT AAA AAT AAA ATC ATT 432
Pro Leu Leu Lys His Gly Leu Leu Cys Ile Thr Lys A~n Lys Ile Ile
130. 135 140
GAC TAT AAC ACG TTT ACC AGC GCA CAG TGG ACT GCC ATA TGT TTG GGT 480
Asp Tyr A~n Thr Phe Thr S-r Ala Gln Trp Ser Ala Ile Cys Leu Gly
150 155 160 '
: GAT GAC AGA AAA ATA CCA TTC TCT GTC ATA CCC ACA GGT AAT GGT ACA 528
Asp Asp Arg Lys Ile Pro Phe Ser Val Ile Pro Thr Gly Asn Gly Thr
170 175
AAA ATA TTT GGT CTT GAG TGG AAT GAT GAC TAT GTT ACA GCC TAT ATT , 576
Ly~ Ile Phe Gly LQU G1U Trp Asn Asp A~p Tyr Val Thr Ala ~yr Ile
le0 185 - 190
AGT GAT CGT TCT CAC CAT TTG AAC ATC AAT AAT AAT TGG TTT AAC AAT 624
;~ ~ Ser Asp Arg S-r Y.i- Hls Leu Asn Ile A~n Asn Asn Trp Phe Asn A~n
GTG~ACA~ATC~C$A SAC TC$ CGA SCA AGC ACT GCT A~G $GG CAG AAG AGT 672
; 2~10~ Le L-u;:Tyr~Ser~Arg S-r S-r Thr Al Th
GCT:,GCA TAT GTT $AT CAA GGT GTT TCA AAT $$T ACT TAT TAC AAG T$A 720
Ala~Ala Tyr Val Tyr Gln Gly Val Ser Asn Phe Shr Tyr Tyr Ly~ ~eu
2ZS: :, 2~30 ~: 235 240
AAT AAC ACC AAT~GGC TTC AAA AGC $AS GAA TTG TGT GAA GAT TAT GAA 768
,,,;~ , 245Y~ Leu Ly- S-r Tyr Glu Leu Cy~ Clu A~p Ty Cl
TGC;TGC ACT:~GGC~TAT~:GC~ ACC AAC G~A T$T GCC CCO ACA GTG GGC GGT 816
Cys Cy3:Thr Gly~Tyr Ala:Thr A~n Val Phe Ala Pro Thr Val Gly Gly
260;~ 265 ~ 270
TAT ATA CCT~GAT::GC:TTC~AGT TTT AAC AAT TGG TTT ATG CTT ACA AAC 864
Tyr Ile Pro'A~p Gly Phe Ser Phe~A~n A~n Trp Phe Met ~eu Thr A~n
275 280 285
S~, AGT ,SCÇ ACG TTS GT T AOT GGC AGA~TST GTA ACA AAT CAA CCA TTA TTG ` 912
Ser Ser Thr Phe Val Ser Gly Arg Phe V~l Thr A~n Gln Pro Leu ~eu
~: ~ 290 295 300
:: GTT AAT TGT TTG TGC CCA CTC CCC ACT CTT CCT CTC GCA CCA CAA GAA 960
Val Asn Cy8 Leu'Trp Pro Val Pro Sor ~-u Cly Val Ala Ala Gln Clu
305 :: '310 : 315 320
S:; TGT TTT GAA GGT GCO CAG T$T AGC CAA TGS AAT OGT GTG TCT SSA 1008
~',,,:: Phe Cy~Phe Glu Gly Ala Gln Ph- Ser Gln Cy~ A~n Gly Val Ser ~eu
: 325 ' 330 335
, "
~ ~ ` SUBSTITUTE SHEET
~:
:
WO 93/23422 PCr/US93/04384
:'-'; : '
2~ 3489~
113
AAC AAT ACA GTG GAT GTC ATT AGA TTC AAC CTT AAT TTT ACC ACA GAT 1056
Asn Asn Thr Val Asp Val Ile Arg Phe A~n Leu A~n Phe Thr Thr Asp
340 345 350
GTA CAA TCT GGT ATG GGT GCT ACA GTA TTT TCA CTG AAT ACA ACA 5GT ~ 1104
Val Gln ser Gly ~et Cly Ala Thr Val Phe Ser Leu A~n Thr Thr Gly
355 360 365
GGT GTC ATT CT~ GAG ATT TCT TGT TAT AAT GAT ACA GTG AGT GAG TCA 1152
Gly Val Ile Leu Glu Il~ Ser Cy~ Tyr A~n A~p Thr Val Ser Glu Ser
370 375 80
AGT TTC TAC AGT TAT GGT GAA ATT TCA TTC GGC GTA ACT GAT GGA CCG 1200
Ser Phe Tyr Ser Tyr Gly Glu Ile Ser Phe Gly Val Thr Asp Gly Pro
385 390 395 4~0
CGT TAC TGT TAC GCA CTC TAT AAT GGC ACG GCT CTT AAG TAT TTA GGA 1248
Arg Tyr Cy Tyr Ala Leu Tyr Asn Gly Thr Ala Leu LYB Tyr Leu Gly
40S 410 415
ACA TTA CCA CCT AGT GTC AAG GAA ATT GCT ATT AGT AAG TGG GGC CAT 1296
Thr Leu Pro Pro Ser Val Lys Glu Il~ Ala Ile Ser Lys Trp Gly Hi~
420 425 430
TTT TAT ATT AAT GGT TAC AAT TTC TTT AGC ACT TTT CCT ATT GAT TGT ~ 13 44
Phe Tyr Ile Asn Gly Tyr A~n Phe Phe Ser Thr Phe Pro Ile Asp Cys
435 440 445
ATA TCT TTT AAT TTA ACC ACT GGT GAT AGT GGA GCA TTT TGG ACA ATT 1392
Ile Ser Phe Asn Leu Thr Thr Gly Asp Ser Gly Ala Ph~ Trp Thr Ile
450 455 460
GCT TAC ACA TCG TAC ACT GA~ GCA TTA GTA CAA GTT GAA AAC ACA GCT 1440
Ala Tyr Thr Ser Tyr Thr A~p Ala Leu Val Gln Val Glu Asn Thr Ala
465 470 47S 480
ATT AAA AAG GTG ACG TAT TGT AAC.ACT CAC ATT A~T AAC ATT AAA TGT 1488
Ile Lys Ly~ Val Thr Tyr Cy~ A~n Ser H~ 8 Il~ Asn A~n Ile Lys Cys
485 490 495
TCT CAA CTT ACT GCT AAT TTG CAA AAT GGA TTT TAT CCT GTT GCT TCA 1536
Ser Gln Leu Shr Ala A~n Leu Gln Asn Gly Phe Tyr Pro VA1 Ala Ser
500 5~S 510
AGT GAA GTT GGT CTT GTC AAT AAG AGT GTT GTG TTA CTA CCT AGT TTC 1584
Ser Glu VaL Gly Leu Val A~n Lys Ser Val Val Leu Leu Pro Ser PhQ
515 520 525
TAT TCA CAT ACC AGT GTT AAT ATA ACT ATT GAT CTT GaT ATG AAG CGT 1632
Tyr Ser ~ hr Ser Val Asn Ile Thr Ile ABP Leu Gly Met Ly~ Arg
530 535 540
AGT GGT TAT GGT CAA CCC ATA GCC TCA ACA TTA AGT AAC ATC ACA CTA 1680
Ser Gly Tyr Gly Gln Pro Il~ Ala Ses Thr Leu Ser A~n Ile Thr Leu
545 550 555 560
CCA ATG CAG GAT AAT AAC ACC GAT GTG TAC TGC ATT CGT TCT AAC CAA 1728
Pro Met Gln Asp A~n Asn Thr A~p Val Tyr Cys Ile Arg Ser Asn Gln
565 570 5~5
TTT TCA GTT $AC GTT CAT TCC ACT TGT AAA ACT TCT TTA TCG CAC CAT 1776
Phe Ser Val $yr Val H1B Ser Thr Cy8 Ly~ SQr Sor L~u Trp A~p A~p
580 SB5 590
SUBSTITUTE SHEE~
W093/23422 213'~8g~ pCT/US93/04384
114
GTG TTT AAT TCC GAC TGC ACA GAT GTT TTA TAT GCT ACA GCT GTT ATA 1824
Val Phe Asn Ser Asp Cy8 Thr Aap Val Leu Tyr Ala Thr Ala Val Il~
S95 600 605
AAA ACT GGT ACT TG~ CCT TTC TCG TT~ GAT AAA TTG AAC AAT TAC TTA 1872
Lyq Thr Gly ~hr Cy8 Pro Phe Ser Phe A~p Lye Leu Asn Asn Tyr Leu
5 620
ACT TTT AAC AAG TTC TGT TTG TCA TTG AAT CCT GTT GGT GCC AAC TGC 1920
Thr Phe Asn Lye PhQ Cys Leu Ser LQU Asn Pro Val Gly Ala Asn Cys
635 640
AAG TTT GAT GTT GCC GCT CGT ACA AGA ACC AAT GAG CAG GTT GTT AGA 1968
Lyq Phe Asp Val Ala Ala Arg Thr Arg Thr Asn Glu Gln Val Val Arg
645 650 6S5
AGT TTA TAT GTA ATA TAT GAA GAA GGA GAC AAC ATA GTG GGT GTG CCG 2016
Ser Leu Tyr Val Ile Tyr Glu Glu Gly Asp Asn Il~ Yal Gly Val Pro
660 6~5 670
TCT GAC AAT AGT GGT CTT CAC GAC TTG TCA GTG CTA CAC TTA GAC TCC 2064
Ser Asp Asn Ser Gly Leu His Asp Leu Ser Val Leu His Leu Asp Ser
675 680 685
TGT ACA GAT TAT AAT ATA TAT GGT AGA ACT GGT GTT GGT ATT ATT AGA 2112
Cys Thr Aup Tyr A~n Il~ Tyr Gly Arg Thr Gly Val Cly Il~ ~le Arg
CAA ACT~AAC AGT ACG CTA CTT AGT GGC TTA TAT TAC ACA TCA CTA TCA 2160
~- Gln Thr Asn Ser Thr Leu LQU Ser GIy Leu Tyr Tyr Thr Ser Leu Ser
710 . 715 720
: GGT GAC TTG STA G~G TTT AAA AAT GTT AGT GAT GGT GTC ATC TAT TCT 2208
Gly Asp Leu Leu Gly Phe Lys Asn Val Ser A~p Cly Val Ile Tyr Ser
:~ ~ :725 73~ 735
GTC:ACG:CCA TGT GAT GTA AGC:GCA CAA GCT GCT GTT ATT GAT GGC GCC 2256
Val~hr Pro 7CyO~A-p Val ~seF Ala Gln Ala Ala V~l Ile Asp Gly Ala
- ATA GTT GGA GCT ATG ACT ~CC ATT AAT AGT GAA ATG TTA GCT CTA ACA 2304
Ilo.Val Cly Ala Met Thr Ser I1Q A~n Ser Glu Me~ L~u Cly Leu Thr
55 760 765
CAT TGG ACA ACA ACA CCT AAT TTT TAT ~AT TAT TCT ATA TAT AAT TAT 2352
Hls Trp Thr Thr Thr Pro Asn Phe Tyr Tyr Tyr Ser Ile Tyr Asn Tyr
770 775 : 780
: ACC AAT GA~AGG~ACT CGT GGC ACA GCA ATT GAT ACS AAC GAT GTT GAT 2400
~- Thr A~n Glu Arq Thr Arg Cly Thr Ala Ile Asp S~r Asn Asp Val A~p
785 790 795 800
TGT GAA CCT ATC ATA ACC TAT TCT AAT ATA GGT GTT TGT AAA AAT GGA 2448
: Cy~ Glu Pro Ile Ile Thr Tyr Ser A~n Ile Gly Val Cys Lys A~n Gly
805 810 815
~-~ GCT TTG GT~ TTT ATT AAC GTC ACA CAT TCT GAT GGA GAC GTT CAA CCA 2496
Ala Leu Val Phe Ile Asn Val Thr HiS Ser Asp Cly Asp Val aln Pro.
.820~ B25 8~0
ATT AGC ACC GGT AAT GTC ACG ATA CCT ACA AAT TTT ACC ATA TCT GTG 2544
Ile Ser Thr Gly Asn Val Thr Ile Pro Thr Asn Phe Thr Ile Ser Val
835 8~0 845
: .
~ SUBSTITUTE SHEET
PCI /US93/043B4
WO 93/23422
'' ~'' 2~ 3D~S9~
115
CAA GTT GAG TAC ATT CAG GTT TAC ACT ACA CCG GTG TCA ATA GAT T~T 2 5 92
Gln Val Glu Tyr Ile Gln Val Tyr Thr Thr Pro Val Ser Ile Asp Cy~
850 855 860
TCA AGG TAC GTT TGC AAT GGT AAC CCT AGA TGC AAT A~A TTG TTA ACG ~ 2640
Ser Arg Tyr Val CY8 A~ Gly Asn Pro Arg Cy~ Asn Ly3 Leu Leu Thr
865 870 875 880
CAA TAC GTT TCT GCA TGT CAA ACT ATT GAG CAA GCA CTT GCA ATG GGT 2688
Gln Tyr Val Ser Ala Cya Gln Thr Ile Glu Gln Ala Leu Ala Met Gly
885 890 895
GCC AGA CTT GAA AAC ATC GA~ ATT GAT TCC ATC TT~ TTT GTT TCC GAA 2736
Ala Ar~ Leu Glu Aqn Met Glu Il~ A~p Ser Met Leu Phe Val Ser Glu
900 905 9lO
AAT GCC CTT AAA TTG GCA TCT GTT GAA GCA TTC AAT AGT ACG GAA ACT 2184
Asn Ala Leu Ly~ Leu Ala Ser Val Glu Ala Phe A~n Ser Thr Glu Thr
9lS 920 925
TTA GAT CCT ATT TAC AAA GAA TGG CCT AAC ATT GGT GGT TCT TGG CTA 2832
Leu Asp Pro Ile Tyr Ly~ Glu Trp Pro Aan Il~ Gly Gly Ser ~p Leu
930 935 940
GGA GGT TTA AAA GAC ATA TTG CCA TCT CAC AAC AGC AAA CGT AAG TAC 2880
Gly Gly Leu Ly~ A~p Ile Leu Pro Ser Hi~ A~n Ser Lya Arg Ly~ Tyr
9qS 9S0 955 960
CGG TCG GCT ATA GAA GAT TTG CTT TTT GAT AAC GTT GTA ACA TCT GGC 2928
Arg Ser Al~ Glu Asp ~u LHU Phe Aup Ly~ Val V~l Thr Ser Gly
965 970 g75
TTA GGT ACA GTT GAT GAA GAT TAT AAA CGT TGT ACA GGT GGT ~AT GAC 2976
Leu Gly Thr Val A~p Glu A~p Tyr ~y~ Arg Cy~ Thr Gly Gly Tyr A3p
980 985 990
ATA GCT GAC TTA GTG TGT GCA CAA TAT TAC AAT GCC ATC ATG CTG CTA- 3024
Ile Ala A8P Leu Val Cy~ Ala Gln Tyr Tyr A~n Gly I1Q ~t Val Leu
995 lO00 lOOS
CCT GGT GTA GCT AAT GAT GAC AAG ATG GCT ATG TAC ACT GCA TCT CTT 3072
Pro Gly Val Ala Asn A8p A8p Lys Met Ala Met Tyr Thr Ala Ser Leu
lOlO l~lS 1020
GCA GGT GGT ATA ACA TTA GGT GCA CTT GGT GGT CCC GCA GTG TCT ATA 3120
Ala Gly Gly Ile Thr Le~ Gly Ala Leu Gly Gly Gly Ala Val Ser Ile
1025 1030 1035 1040
CCT TTT GCA ATA GCA GTT CAA GCC AGA CTT AAT TAT GTT GCT CTA CAA 3l68
Pro Phe Ala Ile Ala Val Gln Ala Arg Leu A~n ~yr Val Ala Leu Gln
1045 lOS0 1055
ACT GAT GSA TTG AGC AAG AAC CAG CAG ATC CTG GCT AAT GCT TTC AAT 32l6
Thr A~p Val Leu Se~ Lys A~n Gln Gln Ile L~u Ala Aon Ala Ph~ A~n
1060 1065 1070
CAA GCT ATT GGT AAC ATT ACA CAG GC~ TTT GGT AA~ GTT AAT GAT GCT 3264
Gln Ala Ile Gly Asn Ile Thr Gln Ala Phe Gly Ly~ Val Asn Asp Ala
1075 1080 1085
ATA CAT C~A ACC TCA CAA GCT CTT GCT ACT GTT GCT AAA CCA TTG GCA 33l2
Ile His Gln Thr Ser Gln Gly Leu Ala Thr Val Ala ~y~ Ala L~u Ala
lO90 1095 llO0
S~JBSTIT~iTE SHEE~
W 0 93/23422 ~ ~ 3 i ~ ~ ~ PCT/U~93/04384
116
AAA GTG CAA GAT GTT GTT AAC ACA CAA GGG CAA GCT TTA AGC C~C CTA 3360
1105 Gln Asp Val Val Asn Thr Gln Gly Gln Ala Leu Ser His L
n Leu Gln Asn A~n Phe GClA GlCC ATT AGT AGT TCC ATT AG ~ 3408
1125 1130 1135
GAC ATT TAT AAC AGG CTT GAT GAA TTG-AGT GCT GAT GCA CAA GTT GAC 3456
Asp Ile Tyr Asn Arg Leu Asp Glu L u Sor Ala Asp Ala Gln Val Asp
AGG CTG ATT ACA GGA AGA CTT ACA GCA CTT AAT GCA m GTG TCT CAG 3504
Arg Leu lle Thr Gly Arg Leu Thr Ala Leu A~n Ala Phe Val Ser Gln
1l7UOThr Arg Gln Ala Glu Val AGG AlCT AGC ACA AG C~T GCT AAA 3552
a p Ly~ V~l A~n Glu Cy~ Val Arg Ser Gln Ser Gln Arg Phe Gly Phe 3600
y ly A3n Cly Thr Nis Leu Phe Ser Leu Al~ Asn Ala Ala pCCA AAT 3648
1205 1210 1215
y t Il~12he Ph- 8~g Thr Val LTuA LTTA CCA ACA GCT AT GAA ACC 3696
Val ThCrC AlC TGG~TCA CCT ATT TCT CCA TCA CAT CCC GAT CGT ACT TTT
1240 1245
;Gly l250Val Val Ly- A~p~V 1 Gln Leu Thr Leu Pho ACgc AsAT LTA GAT 3792
G~C;~AAA TTC TAT TTG ACT CCC AOA ACT ATC TAT CAC CCT AGA GTT CCA 3840
; A p Ly~ Ph- Tyr L-u Thr Pro Arg Thr M-t Tyr GIn Pro Arg Val Ala
ACT AGT TCT GAT TTT GTT CAA ATT CAA GGA TGT GAT GTG TTG TTT GTT 3888
Thr Ser S-r~A~p Ph V-l Gln Il- Glu Cly Cy- ABP V~1 Leu l295Val
Thr l300Ilo A~p Leu proT SAGT AT~ ATA CCT GAC TAT ATT 3936
, ~ ATT AATiCAA ACT GTT CAG GAC ATA TTA GAA AAT TTC AGA CCA AAT TGG 3984
1320 1325 Trp
- ACT GTA CCT CAG TTG CCA CTT GAC ATT TTC AAT GCA ACC TAC TTA AAC 4032
Thr Val Pro Glu Leu Pro Leu A~p Ile Pho A~n Ala Thr Tyr Leu Asn
-1330 1335 1340
~ ~ .
CTG AC? GGT GAA ATT AAT GAC TTA GAA TTT AGG TCA GAA AAG TTA CAT 4080
L34u Thr Gly Glu Ile Asn Asp Leu Glu Phe Arg Ser Glu Lys Leu Hls
.; .
SUBSTITUTE SHEEl
,
WO 93/2342~ PCr/US93/04384
; 213489~
117
AAC ACC ACA GTA GAA CTT GCT ATT C~C ATT GAT AAT ATT A~T AAC ACA 412 8
Asn Thr Thr Val Glu Leu Ala Ile L~au Ila Asp Asn Ile A~n A~n I~hr
1365 1370 1375
TTA GTC AAT CT~ GAA ~GC CTC AAT AGA ATT G~A ACT TAT CTA AAA TCC 417 6
Leu Val A~n L~u Glu Trp Leu ADn Arg Ile Glu Thr Tyr V~ll Ly~ Trp
1380 1385 ~390
CCT TGG TAT GTG TGG CTA CTA ATT GGA TTA GTA CTA ATA TTC TGC ATA 4 2 2 4
Pro ~rp Tyr Val Trp Leu Leu Ile Gly Leu Yal Val Ile Phe Cy~ Ile
1395 1400 1405
ccc ATA TTG CTA T~T TGT TGT TGT ACC ACT GGT ~GT TGT GCA TGT Al'T 4272
Pro Ile Leu Leu Ph~ Cy~ Cye Cy8 Ser Thr Cly cy~ Cy~ Gly cy~ Ile
1410 1415 1420
GGG TGT TTA GGA AGC TGT TGT CAT TCC ATA TG$ AGT AGA AGG CGA TTT 4~20
Gly Cy~ Leu Gly Ser Cy8 CYE~ Hi~ Ser Ile Cys Ser Arg Arg Arg Phe
1425 1430 1435 1440
GAA AGT TAT GAA CCA ATT GAA A~ GT :: CAT GTC CAC TAA 4 3 5 9
Glu Ser Tyr Glu Pro Ile Glu Lys Val H~ 8 Val H~s
1445 1450
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENOE CHARACTE~ISTICS:
(A) LEt~GTH: 1452 am~no acid~
(B) TYPE~ amlno ~cid
(D) TOPOLOGY: llnear
ii) MOLECULB TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Met Ile Val Leu Val Thr Cy~ Leu Leu PhE Ser Tyr A~n Ser Val Ile
- 1 5 10 15
CYR Thr Ser Asn Asn Asp Cya Val Gln Val Asn Val Thr Gln Leu Pro
Gly Asn Glu Asn Ile Ile Lys Asp Phe Leu Phe H~s Thr Phe Lys Glu
Glu Gly Ser Yal Val Val Gly Gly Tyr Tyr Pro Thr Gl~ Val Trp Tyr
Asn Cy5 Ser Arg Ser Ala ~hr Thr Thr Ala Tyr Ly~ Asp Phe Ser Aen
6 5 7 0 7 5 8 0
I le His Ala Phe Tyr Phe Asp Met Glu Ala Met Glu Asn Ser Thr Gly
B5 90 95
A~n Ala Arg Gly Lys Pro Leu Leu Yal Hls Val H~ 8 Gly Asp Pro Val
100 105 110
Ser Ile Ile Ile Tyr Ile Ser Ala Tyr Arg Asp Asp Val Gln Gly Arg
115 120 125
Pro Leu Leu Ly~ ~is Gly LQU Leu Cys Ile Thr Lyo A~n Lys Ile Ile
130 135 140
SlJ8STITUTE SHEET
WO 93/2342~ PCr/US93~04384
2 ~ 3 ~
118
A p Tyr A~n Thr Ph~ Thr Ser Ala Gln Trp Ser Ala Ile Cy~ Leu GIy
Asp Asp Arg Lys I Ie Pro Phe Ser Val Ile Pro Thr Gly A~n Gly Thr
Lyq Ile Phe Gly Leu Glu Trp Asn Asp A~p Tyr Val Thr Ala Tyr Ile
Ser A~p Arg Ser Hi~ Hla Leu Asn Ile Asn Asn Asn Trp Ph~ Asn Asn
Val Thr Ile Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Gin Lys Ser
Ala Ala Tyr Val Tyr Gln Gly Val Ser Asn Phe Thr Tyr Tyr Lys Leu
Asn Asn ~hr Asn ly Leu Ly Ser Tyr Glu Leu Cy~ Glu A~p yr Glu
Cys Cys Thr Gly Tyr Ala Thr Asn Val Phe Ala Pro Thr Val Gly Gly
~yr Ile Pro Asp Gly Phe Ser Phe Asn Asn Trp Ph~ Met Leu Thr Asn
ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr Aan Gln Pro Leu Leu
Val A~n Cys Leu Trp Pro Val Pro Ser Leu Gly Val Ala Alà Gln Glu
Phe Cy~ Phe Glu Gly Ala Gln Phe Ser Gln Cy~ A~n Gly Val Ser Leu
A~n Asn Thr Val A~p Val Ile Arg Phe A~n Leu Aan Phe Thr Thr Asp
Val Gln Ser Gly Met Gly Ala Thr Val Phe Ser Leu Asn Thr Thr Gly
355 360 3~5
Gly Val Il- Leu G1u Il- Ser cy8 Tyr Aan Asp Thr Val Ser Glu Ser
Ser Phe Tyr Ser Tyr Gly Glu Ile Ser Phe Gly Val Thr A-p Gly Pro
Arg Tyr Cys Tyr Ala Leu Tyr Asn Gly Thr Ala Leu Ly~ Tyr Leu Gly
Thr Leu Pro Pro Ser Val Ly~Glu Ile Ala Ile Ser Lys Trp Gly His
Phe Tyr Ile A~n Gly. Tyr A~n Phe Phe Ser Thr Phe Pro Ile A~p Cy~
Ils Ser Phe A~n Leu Thr Thr Gly Aap Ser Gly Al~ Phe Trp Thr lle
450 455 460
Ala Tyr Thr Ser Tyr Thr Asp Ala Leu Val Gln Val Glu Asn Thr Ala
465 470 475 480
SUBSTITUTE SHEET
WO 93/23422 PCr/US93~04384
~:- 213~98
llg
Ile Lys Lys Val Thr Tyr Cy~ Aqn Ser Hi~ Ile A~n Asn Ile Lys Cys
485 490 495
Ser Gln Leu Thr Ala As~ Leu Cln Asn Gly Phe Tyr Pro Val Ala Ser
50~ 505 510
Ser Glu Val Cly L~u Val Asn Ly~ Ser Val Val Leu L~u Pro Ser Phe
515 520 525
Tyr Ser ~9 Thr Ser Val Asn Ile Thr Ile Asp Leu ~ly ~et Lys Arg
530 535 540
Ser Gly Tyr Gly Gln Pro ~1~ Ala Ser Thr Leu S~r Asn ~1~ Thr Leu
545 550 555 560
Pro Met Gln Asp Asn A~n ~hr Asp Val Tyr CYB Ile Arg ser Asn Gln
565 5~0 575
Phe Ser Val Tyr Val HLs Ser Thr Cy~ Ly~ Ser Ser Leu Trp Asp Asp
580 585 590
Val Phe Asn Ser Asp Cya Thr Asp Val Leu Tyr Ala Thr Ala Val Ile
595 600 605
~y~ Thr Gly Thr Cy8 Pro Phe Ser Phe Asp Lys Leu Asn Asn Tyr Leu
610 615 620
Thr Phe A~n Ly8 Phe Cy8 L~u Ser Leu A~n Pro Val Gly Ala Aen Cy~
625 630 535 640
Ly~ Phe Asp Val Ala Ala Arg Thr Arg Thr A~n Glu Gln Val Val ~ry
645 650 6S5
Ser Leu Tyr Val Ile Tyr Glu Glu Gly A3p Asn Ile Val Gly Val Pro
660 665 670
Ser Asp Asn Ser Gly Leu His Asp Leu Ser Val Leu His L~u Asp Ser
675 680 685
Cys Thr ASP TYr Aqn Ile Tyr Gly Arg Thr Gly Val Gly Ile Ile Arg
690 695 700
Gln Thr Asn Ser Thr Leu Leu Ser Gly L~u TYr TYr Thr Ser Leu Ser
705 ~10 715 ~20
Gly Asp Leu Leu Gly Phe Lys A~n Val Ser A~p Gly Val Ile Tyr Ser
725 ~30 735
ValjThr Pro Cy~ Asp Val Ser Ala Gln Ala Ala Val Ile Asp Gly Ala
740 t45 ~50
Ile Val Gly Ala M~t Thr Ser Ile A~n Ser Glu Met L~u Gly LQU Thr
7S5 760 765
His Trp $hr Thr Thr Pro Asn Phe Tyr Tyr Tyr Ser Ile Tyr Asn Tyr
770 775 780
Thr Asn ~lu Arg Thr Arg Gly Thr Rla Ile A~p Ser Asn Asp Yal Asp
~85 790 7g5 800
Cy~ Glu Pro Il~ Ile Thr Tyr Ser A~n Ile Gly Val Cy~ Ly~ A~n Gly
805 310 815
SUE~S~ITUTE SHEET
WO 93/23422 ~ 3 ~ PCI`/US93/04384
120
Ala Leu Val Phe Ile Asn Val Thr ~ S~r A~p ~:ly Asp Val Gln Pro
82 0 82 5 8 ~ 0
Ile Ser Thr Gly A~n Val Thr Ile Pro Thr A~n Phe Thr Ile Ser Val
835 A40 845
Glr~ Val Glu Tyr Ile C:ln al Tyr Thr Thr Pro Val Ser I1Q Asp Cy~
Ser Arg ~yr Val Cy~ A~n Gly Asn Pro Arg Cy~ A~n Lys Leu Leu Thr
86~ 870 875 880
Gln Tyr Val Ser Ala Cy6 Gln Thr Ile ~lu Gln Ala Leu Ala Met Gly
885 8gO 895
Ala Arg Leu Glu A~n Met Glu Ile A3p Ser Met ~eu Phe Val Ser Glu
, goo 9~5 910
A~n Ala Leu Lys Leu Ala Se~ Val Glu Ala Phe A~n Ser Thr Glu Thr
91S 920 925
Leu Asp Pro Ile Tyr I,y~ Glu Trp Pro A3n I1Q Gly Gly Ser Trp Leu
Gly Gly Leu Ly~ Asp Ile Leu Pro Ser Hi- Asn Ser Lys Arg Ly~ Tyr
~ - ,
~Arg- Ser Ala Ile Glu Asp Leu Leu Phe A8p Lya Val Val Thr Ser Gly
: ~ 965 970 975
Leu G}y Thr Val Asp Glu A3p Tyr Ly~ Arg Cys Thr Gly Gly Tyr Asp
~ sao 985 99o
Ile Ala A~p Leu Val Cys Al Gln Tyr Tyr A~n Gly Ile Met V~l Leu
99S 1000 1005
Pro Gly Val Ala Asn A~p Asp Lys ~et Ala Met Tyr Thr Ala Ser Leu
1010 1015 1020
Ala Gly Gly Ile Thr Leu Cly Ala L~u Gly Gly Gly Ala Val Ser Ile
1025 : 1030 1035 1040
Pro Phe Ala lle Ala Val Gln Ala Arg ~eu A~n Tyr Val Ala Leu Gln
104~ 1050 1055
Thr Asp Val Leu Ser Ly~ Asn Gln Gln Il~ L~u Ala Agn Ala Phe A~n
1060 1065 1070
Gln Ala Ile Gly A~n Il~ Thr Gln Ala Pho Gly Ly~ Val Asn Asp Ala
1075 108Q 1085
Ile ~19 Gln Thr Ser Gln Gly Leu Ala Thr Val Ala Lys Ala Leu Ala
1090 1095 1100
Lys Val Gln Asp Val Val A~n Thr Gln Gly Gln Ala Leu Ser His Leu
1105 1110 1115 1120
Thr Val Gln Le~ Gln Asn Asn Phe Gln Ala Ile Ser Ser Ser Ile Ser
11~5 1130 1135
Asp Ile Tyr A~n Arg Leu Asp Glu Leu Ser Ala A~p Ala Gln Val Asp
1140 1145 11~0
SUBSTITIJTE SHEET
W O 93/23422 PCT/USg3~04384
.~; .
9~8
121
Arg Leu Ile Thr Gly Arg Leu Thr Ala Leu Asn Ala Phe Val Ser Gln
1155 1160 1165
Thr Leu Thr Arg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Ly~
1170 1175 1180
A~p Ly~ Val A~n Glu Cy8 Val Arg Ser Gln Ser Gln Arg Phe Gly Phe
1185 1190 1195 1200
CYQ Gly Aen Gly Thr ~i8 Leu Phe Ser Leu Ala Asn Ala Ala Pro Asn
1205 1210 1215
Gly Met Ile Phe Phe Hia Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr
1220 1225 123~
Val Thr Ala ~rp Ser Gly Ile Cy8 Ala Ser Asp Gly ABP Arg Thr Phe
1235 1240 1245
Gly Leu Val Val Ly~ Asp Val Gln Lau Thr LQU Phe Arg Asn LQU A~p
1250 1255 1260
Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln Pro Arg Val Ala
1265 1270 1275 1280
Thr Ser Ser A~p Phe Val Gln Ile Glu Gly Cys A~p Val Leu Phe Val
1285 1290 1295
; Asn Ala Thr Val ~le Asp Leu Pro Ser Ile Ile Pro Asp Tyr Ile Asp
1300 1305 1310
Ile A~n Gln Thr Val Gln Asp Ile ~eu Glu A~n Phe Arg Pro A~n Trp
~;~ 1315 1320 1325
Thr Val Pro Glu Leu Pro Leu A~p Ile Phe A~n Ala ~hr Tyr Leu Asn
13;30` 1335 1340
Leu Thr Gly Glu Ile A~n A~p Lou Clu Phe Arg Ser Clu Lys LRU H1~
1345~ . 1350 1355 1360
A~n Thr Thr Val Glu Leu Ala Ile Leu Ile A8p Asn Ile Asn Asn Thr
: 1365 1370~ 137S
Leu:Val AQn ~eu Glu Trp Leu A3n Arg Ile Glù Thr ~yr Val Lys Trp
1380 ~ 1385 1390
: Pro Trp Tyr Val~Trp Leu Leu Ile Gly Leu Val Val Ile Phe Cy_ Ile
1395 ~ : 1400 1405
Pro Ile Lau Leu Phe CYB CY~ CY~ Ser ~hr Gly Cy3 Cy~ Gly Cy~ Ile
l: . 1415 1420
Gly Cy~ Leu Gly ser Cy~ Cy~ Hi~ Ser I1Q CY~ Ser Arg Arg Arg Phe
1425 1430 1435 1440
; Glu Ser Tyr Glu Pro Ile Glu LYJ Val Hl~ Val Hl~
: 1445 1450
, ~ ,
SUBSTITUTE SHEEr
WO 93/23422 PCr/VS93/04384
21 3~ $98
122
( 2 ) INFORMATION FOR SEQ ID NO: 19: -.
1 ) SEQUENCE CEIIW~CTE~I STI CS:
(A) LENGTH: 43 ba~e pairs
(8) TYPE: nucletc ac~d
(C) STRANDEDNESS: ~lngle
(D) TOPOLOGYs unknown
: (ii) MOLECULE TYPE: DNA ~genom~c)
(xi) SEQUENCE DESCRIPTION: SEQ SD NOs19:
CCCGGGCATG ATTGTGCTCG TAACTTGCCT CTTGTTGTTA TGC 43
(2) INFORMATION FO~ SEQ ID NO: 20:
(1) SEQUENCE CHARAC~ERISTSCS:
~A) LENGTH: 47 b~e patr~
(8) TYPE: nucle~c acid
(C) STRANDEDNESS: ~lngle
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (g~nomlc)
~ ~ :
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GTGCCCCGGG~CATGATSGTG CTCGTAACTT GCCTCTTGTT GTTATGC 47
(2) SNFORMATION EOR SEQ ID NO:21:
~1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 b~e palr~
(B) TYPE: nucleLc acld
(C) STRANDEDNESS: ~ingle
:~ (D) TOPO~OGY: unknown
(Li) MOEECUEE~ ~YPE: DNA ~g-nomic)
(xl) SEQUENCE DESCRIP~ION: SEQ ID NO:21:
CAATCTTAAT TTCACTGCAG ATGTACAATC TGGTATGGGT GCT 43
(2) INFORMATION FOR,SEQ ID NO:22:
~1) SEQUENCE C~ARACTERISTICS:
(A) ~ENGTH: 43 ba~e pair~
~B) TYPE: nucleLc acid
~:C (C) STRANDEDNESS: ~in~le
: ~D) TOPOTOGY: unknown
~ (ii) MOTECU~E TYPE: DNA (genomlc)
: (xl) SEQUENCE DESCRIPTION: SEQ I3 NO:22:
AGCACCCATA CCAGATTGTA C~TCTGCAGT GaAATTAAGA TTG 4 3
SUBSTITUTE SHEET
WO 93/23422 PCI'/US93/W384
` : ` 2 i 3~ 8 9 ~
123
(2) INFORMATION FOR SEQ ID NO:23:
(~) SEQUENCE CHARACTERISTICS:
(A) LENGTHs 29 ba~e palr~
(8) TYPE: nucl~ic ac~d
(C) ST~ANDEDNESS: sln~le
(i) TOPOLOGY: unknown
(ii) MOLECUEE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CTGCAGATGT ACAATCTGG~ A~GGGTGCT 29
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGT~: 29 ba~ pa~rs
(B) TYPE: nucloic ac~d
(C) STRANDEDNESS: single
(D) TOPOEOGY: unknown
(~1) MOLECU~E TYPE: DNA ig~nomlc)
(x~ SQUENCE DESCRIPSrON: SQ ID NO:24:
: C~GCAGTGAA AT~AAGATTG AATC~AATA 29
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CH~RACTER~ST~CS:
(A) ~ENGTHs 39 ba-- paLrs
~B) TYPE: nucl~c acld
-~ (C1 STRANDEDNESSs~ ~lngl-
D J TOPOLOGY: unknown
ii) MOLECULE::TYP: DNA (genomic)
:- ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
...
~ GTGCCCCCGG GTATGATTGT GCTCOTAACS TGC C~G 39
:,~':-
(2) INFORMATION FOR SEQ ID NO:26:
(~) SEQUENCE C~ARACTERISTICS:
~A) ~ENGTH: 35 ba-e pair~
8 ) TYPE: nuclelc ~c~d
(C) STRANDEDNESS: ~nglo
~S ~D) TOPO~OGY: unknown
,
) MOLECU$E TYPE: DNA tgenoml~)
(X~) SEQUENCE DESCRIPT~ON: SEQ ID NO:26:
AATACCCGGG GCACT¢GTAA TGCACGTGGT AAACC 35
SUBSTITUTE SHEET
WO 93/23422 PC~/US93/0438~
2-~3~g8
~.
124
(2) INFORMATION FOR SEQ ID NO:27: ~ ~`
(i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: 35 ba~e pair~
(8) TYPE: AUClelC acld
(C) STRANDEDNESS: single .;
(~) TOPOLOGY: unknown
~ii) MOLECULE TYPE: DNA (genomic) .
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GTATTCCCGG GCACGCTC~A GCACTGCTAC CTGGG 35
(2) INFORMATION FOR SEQ ID NO:28:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 36 b~se palrs
~B3 SYPE: nucle~c acld
(c) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(i$) MOLECULE TYPE: DNA (genom$c)
(x13 ~SEQ~ENOE DESCR PT~ON: SEQ ID NO:28:
- CAGATCCCGC GOTACAATCT GGTATGGOTG CTACAG 36
.
(2) INFORMATION FOR SEQ ID NO:29:
(l3 SEQUENCE C~ARACTERISTICS:
~A) LENGTH: 39 base pairs
(B3 TYPE: nucleic acld
(C) STRANDEDNESS: si~gle
~D) TOPO~OGY: unknown
(ii) MO~ECULE TYPE: DNA ~g-nomic)
- ~ ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
~- OCTTACCCGG GGTGGTTATG GTCAACCCAT AGCCTCGAC 39
, ', ' ' .
~2) INFORMATION FOR SEQ ID NO:30:
! '
(i3 SEQUENCE CaA~ACTERISTICS:
~A) ~ENGTH: 39 b~se palrs
: ~B) TYPE: nucleic ~cid
~C) STRANDEDNESSs ~ingle
~D) TOPOLOGY: unknown
~ii) MO~ECULE TYPE: DNA ~g-nomic)
~xl) SEQUENCE DESCRIPTION: SEQ ID NO:30:
TGTGACCCGG GCGCCATGTG ATGTAAGCGC ACAAGCGGC 39
SlJBSTlTlJTE SHEET
WO 93/23422 2 1 3 4 ~ 9 ~ PCI~US93/W384
125
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGT~: 37 ba~ pa~rs
(B) TYPE: nuclei~ acid
~C) 5TRANDEDNESS: ~lngle
~D) ~OPOLOGY: unknown
(i1) MOLECULE TYPE: DNA tgenomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
GCAATCCCGG GGGGTGCCAG ACTTGAAAAC ATGGAGG 37
(~) INFO~MATION FOR SEQ ID NO:32:
(i) SEQUENCE CEARACTERISTICS:
(A) LENGT~: 37 ~a~e pair~ :
(B) TYPE: nucle~c acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: un~nown
(ii) MOLECULE TYPE: DNA ~genomic)
(xi) SE9UE~CE DESCRIPTION: SEQ ID NO:32:
CATTACCCGG GGGTGCACTT GGTGGTGGCG CCGSGGC 37
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGT~: 39 b~s~ palr~
(B) TYPE: nucle~c acid
(C) ST~ANDEDNESS: ~Ingle
( D ) TOPOLOGY: unknown
(ii) ~OT.ECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3~:
TAGGTCCCGG GCTCAGTCTC AGAGATTCGG ATTCTGTGG 39
~2~ INFO~MATION FOR SEQ ID NO:34:
(i) SEQUENC~ CHARACTERISTICS:
(A) LENGTH: 36 basQ pa~r~
~B) TYPE: nucleic ac~d
~C) STRANDEDNESS: slngle
(D) TOPOLOGY: unknown
~ii) MO~ECULE TYPE: DNA ~9enomic)
(xL) SEQUSNCE DESCRIPTION: SEQ ID NO:34:
ATAATAGGCC TGGTT$ACCA CGTGCATTAC CAGTaC 36
SUBSTITUTE SHEET
WO g3/23422 PCr/US93/0438~ ~ ~
j .; .,; 3
1 3 i ~ 9 8
~ .
126
~2) INFORHATION FOR SEQ ID NO:35:
~i) SEQUENCE CHARAC~ERISTICS:
(A) LENGTH: 35 baeQ paLrs
(B) TYPE: nucleLc acld
~C) STRANDEDNESS: single ~-
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE- DNA (genomic)
(xL) SEQUENCE DESCRIPTION: SEQ ID NO:35:
GTATTAGGCC TCCCAGGTAG CAGTGCTTGA GCGTG 3s .
~`,.
(2) INFORMAT}ON FOR SEQ ID NO:36: ;-~
(L) SEQUENOE CHARACTERISTICS:
(A) ~ENGTH: 36 bacQ pairs
(8) TYPE: nucleLc acld ~;.-
(C) STRANDEDNESS: sLngle
(D) TOPOLOGY: unknown ~`.
(i~) HOEECULE TYPE: DNA (genom~c) ~`
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:36:
AAATAAGGCC~TCTGTAGCAC CCATACCAGA TTGTAC 36
(2) ~NFORMA~ION FOR SEQ ID NO:37: .
(i) SEQUENCE CaARACTERISTICS: ';;.
- : (A) LENGTH: 39 ba~e paLr~ :
: (B) TYPE: nucleLc acid
(C:)~S:~RANDEDNESS: single
(D~)~ SOPOLOCY: unknown
(Ll) MO-ECUEE TYPE. DNA (~g~nomic)
(xL) SEQUENCE::DESCRIP~ION: SEQ ID NO:37:
.. . .
TTAGTAGGCC~TGTCGAGGCT ATGGQTTCAC CASAACCAC 39
(2l INFORMATION FOR SEQ ID NO:38:
(l) SEQUENCEiCHARACTERISTICS:
~A) ~ENGTH: 39 ba-e palr~
(8) TYPE: nuclclc acLd
-; (C) STRANDEDNESS: ~lngl~
~D) TOPO~OGY: unknown
(lL) M~EECUTE TYPE: DNA (genomLc)
(xL) SEQUENCE DESCRIPTION: SEQ ID NO:38:
TAACAAGGCC TGCCGCTTGT GCGCTTACAT CACATGGCG 3g
SUBSTITUTE SHEET
WO 93/23422 ~ 1 ~ A 8 9 ~ PCT/US93/04384
. . .
127
~2) INFORMATION FOR SEQ ID NO:39:
~i) SEQUENCE CHARACTERISTICS:
(A) ~NGT~s 37 baoR pa~ro
(B) TYPE: nucl~Lc acid
~C) STRANDEDNESSs ~ingl~ -
~D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA ~genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:-~9:
ATCAAAGGCC TCCTCCATGT ~T~CAAGTCT GGCACCC 37
(2) INFORMA~ION FOR SEQ ID NO:40:
(i) SEQUENCE CaARACTE~STICS:
~A) LENGTH: 37.ba~ pair~
(B) SYPE: nucleic acid
(C) STRANDEDNESS: 8 ing le
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(x$) SEQUENCE DESCRIPTION: SEQ ID NO:40:
GTATAAGGCC TGCCACGGOG CCACCACC~A GTGCACC 37
~2) INFO~MATION FOR S~Q ID NO:41:
(i) SEQUENCE CHARACTERIST~CS:
(A) LENGTH: 39 b~Q palr~
~B) TYPE: nucl~ic a~ld
~C1 ST~ANDEDNESS: ~ingI~
- ~ ~D) TOPOLOGY: unknown
($l) MOE~CU~E TYPE: DNA ~g~nomlc)
(xi) SEQUE~CE DESCRIPTION: SEQ ID NO:41:
CATTAAGGCC TCCACAGAAT CCG~ATC~CT GAGACTGAG39
(2) INFORMATION FOR~SEQ ID NO:42:
~i) SEQUENCE CHARACTERISTICS:
(A) LE~GTH: 38 ba~e pair~
(B) TYPE: nucl~lc ~cid
(C) STRANDEDNESSs ~ingle
(~) TOPOrOGY: unknowm
(ii) MOLECU~E 2YPE: DNA (g~nomtc)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
TAAATAGGCC TTTAG~GGAC ATGCACTTTT TCAATTGG38
SUBSTIT~.JTE SHEET
WO 93/23422 2 ~ 3 ~ PCI/US93/04384
128 `~
(2) INFORMATION ~OR SEQ ID NO:43:
~i) SEQUENCE C~ARACTERISTICS~
(A) LENGTH: 1454 amino acidq ;::
( B ~ TYPE: amino acid :'
~D) TOPOLOGY: unknown
(ii) ~OLECULE TYPE: protein ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: -
Met Ile Val Leu Val Thr Cys Leu Leu Leu Leu Cys Ser Tyr His Thr
5 10 15 , ",
Val Ser Ser Thr Ser A~n A~n Asp Cy~ Arg Gln Val Asn Val Thr Gln
. 20 25 30
Leu Ala Gly Asn Glu Asn Leu Ile Arg Asp Phe Leu Phe Gln Ser Phe
35 40 45 ~'
Lys Glu Glu Gly Ile Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
Trp Tyr Asn Cy3 Ser Arg Thr Ala Thr Thr Thr Ala Tyr Glu Tyr Phe
Asn Asn Ile Hi3 hla Phe Tyr Phe Asp Met Glu Ala Met Glu Asn Ser .
85 90 95 ;-
Thr Gly Asn ~la Arg Gly Lys Pro Leu Leu Phe ~is Val His Gly Glu
100 105 110 .
Pro Val Ser Ile Ile Ile Tyr Ile Ser Ala Tyr Gly Asp A~p Val Gln
115 120 125
Gln Arg Pro Leu Leu Glu His fily Leu Leu Cys Ile Thr Lys Asn Arg
130 135 140
Asn Ile Asp Tyr A3n Thr Phe Thr Ser Asn Gln Trp Asp Ser Ile Cy9
145 150 155 160 :.
Thr Gly A~n Asp Arg Lys Ile Pxo Phe Ser Val Ile Pro Arg Asp Asn
165 170 1~5
Gly Thr Lys Ile Tyr Gly Leu Glu Trp Asn Asp Glu Phe Val Thr Ala
180 185 190
Tyr Ile Ser Gly Arg Ser Tyr Aqn Trp A~n Ile Asn Asn AYn Trp Phe
195 200 205
Asn Asn Val Thr Leu Leu Tyr ser Arg Ser Ser Thr Ala Thr Trp Glu
210 215 220
Tyr Ser Ala Ala Tyr Val Tyr Gln Gly Val Ser A~n Phe Thr Tyr Tyr
225 230 235 240
Lys Leu Asn Asn Thr Asn Gly Leu Ly~ Thr Tyr Glu Phe Cy~ Glu Asp
245 250 255
Tyr Glu Tyr Cys Thr Gly Tyr Ala Thr Asn Val Phe Ala Pro Thr Val
260 26~ 270
W O 93/23422 PCT/~S93/043~4
~ ~13~98
129
Gly Gly Tyr Ile Pro Asp Gly Phe Ser Phe Asn Asn Trp Phe Leu Leu
275 280 285
Thr Asn Ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr A~n Gln Pro .
290 295 300
Leu Leu Val A~n Cy~ Leu Trp Pru Val Pro Ser Phe Gly Val Ala Ala
305 310 315 320
ln Glu Phe Cy8 Phe Glu Gly Ala Gln Phe Ser Gln Cys Asn Gly Val
32S 330 335
Ser Leu Asn A~n Thr Val A~p Val Ile Arg Phe A~n Leu Asn Phe Thr
340 345 3S0 ~:
Ala Asp Val Gln Ser Gly Met Gly Ala Thr Val Phe Ser Leu Asn Thr
35S 360 365
Thr Gly Gly Val Ile Leu Glu Ile Ser Cye Tyr Ser Asp Thr Val Ser
370 37S 380
Glu Ser Ser Ser Tyr Ser Tyr Gly Glu ~le Pro Phe Gly Ile Thr Asp
385 390 395 400
Gly Pro Arg Tyr Cys Tyr Val Leu Tyr Asn Gly Thr Ala Leu Lys Tyr
405 410 415
I.eu Gly Thr Leu Pro Pro Ser Val Lys Glu Ile Ala Ile Ser Lys Trp
420 42S 430
Gly His Phe Tyr Ile Asn Gly Tyr Asn Phe Phe Ser Thr Phe Pro Ile
43S 440 445
Gly Cy~ Ile Ser Phe Asn ~eu Thr Thr Gly Ala Ser Gly Ala Phe Trp
450 455 460 :
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu A~n
46S 470 475 480
. Thr Ala Ile Ly~ Asn Val Thr Tyr Cys Asn Ser ~i~ Ile Asn Asn Ile
4B5 490 495
Ly~ Cys Ser Gln Leu Thr Ala Asn Leu Aan Asn Gly Phe Tyr Pro Val
~ 500 505 510 ~:
Ala Ser Ser Glu ~al Gly Phe Val A~n Ly~ Ser Val Val Leu Leu Pro :~
515 520 525 .
Ser Phe Phe Thr ~i~ Thr Ala Yal Asn Ile Thr Ile A~p Leu Gly Met
530 535 540
Ly~ Le~ Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu Ser A~n Ile
545 550 555 560
Thr Leu Pro Met Gln A~p ~sn Asn Thr Asp Val Tyr Cys Ile Arg Ser
565 570 575
A~n Gln Phe Ser Val Tyr Val Pro Ser Thr Cy~ Lys Ser Ser Leu Trp
5B0 585 590
A~p Asn Ile Phe A~n Gln Asp Cys Thr Asp Val Leu Glu Ala Thr Ala
595 600 605
WO 93~2~422 PCl`tUS93/0~384
~ i 3 ~
130 ,,
Val Ile Lys Thr Gly Thr Cys Pro Phe Ser Phe Asp Lys Leu Asn Asn
610 615 620
Tyr Leu Thr Phe Asn Ly~ Phe Cys Leu Ser Leu Ser Pro Val Gly Ala
625 630 635 640
Asn Cya Ly~ Phe A~p Val Ala Ala Arg Thr Axg Thr Asn Glu Gln Val
645 6~0 65S
Va1 Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly ARP A~n Ile Val Gly
660 665 670
Va1 Pro Ser A8p Asn Ser Gly Leu His Asp Leu Ser Val Leu His Leu
675 680 685
Asp Ser Cy~ Thr A~p ~yr Asn Ile Tyr Gly Arg Thr Gly Val Gly Ile
690 695 700
Ile Arg Arg Thr A~n Ser Thr Leu Leu Ser Gly Leu Tyr Tyr Thr Ser
705 710 715 720 :`~
Leu Ser Gly Asp Leu Leu Gly Phe Ly~ Asn Val Ser Asp Gly Val Ile
125 730 735
Tyr Ser Val Thr Pro ~y~ Asp Va1 Ser Ala Gln Ala Ala Val Ile Asp
740 745 750
Gly Ala Ile Val Gly Ala Met Thr Ser Ile Asn Ser Glu Leu Leu Gly
7S5 760 765 :
Leu Thr Hia Trp Thr Thr Thr Pro A~n Phe Tyr Tyr Tyr Ser Ile Tyr
770 775 780
A~n Tyr Thr Ser Glu Arg Thr Arg Gly Thr Ala Ile A~p Ser Asn ~5p
78$ 790 795 800
Va1 A~p Cy~ Glu Pro Val Ile Thr Tyr Ser A3n Ile Gly Val Cy~ Lys
805 810 815
ARn Gly Ala Leu Va1 Phe Ile A~n Val Thr Hi~ Ser A~p Gly A3p Val
820 825 830
Gln Pro Ile Ser Thr Gly A~n Val Thr Ile Pro Thr Asn Phe Thr Ile
835 840 845
Ser Val Gln Val Glu Tyr Met Gln Val Tyr Thr Thr Pro Val Ser Ile
85~ 85~ 860
A p Cy~ Al~ Arg Tyr Val Cy~ Aon Gly A~n Pro Arg Cy~ A~n Ly~ Leu
865 870 87S 880
Leu Thr Gln Tyr Val Ser Ala Cy~ Gln Thr Ile Glu Gln Ala Leu Ala
885 890 895
Met Gly Ala Arg Leu Glu A~n Met Glu Val A~p Ser Met Leu Phe Val
900 905 9lO
Ser Glu Asn Ala Leu Ly~ Leu Ala Ser Val Glu Ala Phe Asn ser Thr
915 ~20 925
Blu A~n Leu ABP Pro Ile Tyr Ly~ Glu Trp Pro Ser Ile Gly Gly Ser
930 935 940
W O 93/23422 PCT/US93/04384
. l.
213 4~ 98
131
Trp Leu Gly Gly Leu Ly~ Asp Ile Leu Pro Ser His Asn Ser Lys Arg
945 950 955 960
Lys Tyr Gly Ser Ala Ile Glu Asp Leu Leu Phe A~p Lyq Val Val Thr
965 970 975
Ser Gly Leu Gly Thr Val Asp Glu A~p Tyr Ly~ Arg Cys Thr Gly Gly
980 985 990
Tyr Asp Ile Ala Asp Leu V~l Cy~ Ala Gln Tyr Tyr A~n Gly Ile Met
995 1000 1005
Val Leu Pro Gly Val Ala Asn Ala Asp Ly~ Met Thr ~et Tyr Thr Ala
1010 1015 1020
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
1025 1030 1035 1040
Ala Ile Pro Phe Ala Val Ala Val Gln Ala Arg Leu A~n Tyr Val Ala
104S 1050 1055
Leu Gln Thr Asp Val Leu A~n Ly~ Asn Gln Gln Ile Leu Ala Asn Ala
1060 1065 1070 . :~
Phe Asn Gln Ala Ile Gly AQn Ile Thr Gln Ala Phe Gly Lys Val Asn
1075 1080 1085
Asp Ala Ile Hi3 Gln Thr Ser Gln Gly Leu Ala Thr Val Ala Ly3 Ala
1090 1095 1100 :
Leu Ala Lys Val Gln~Asp Val Val Asn Thr Gln Gly Gln Ala Leu Ser
1105 1110 1115 . 11~0
Hi~ Leu Thr Ual Gln Leu Gln Asn Asn Phe Gln Ala Ile Ser Ser Ser
1125 1130 1135
~Ile Ser A3p Ile Tyr Asn Arg Leu Anp GlU Leu Ser Ala Asp Ala Gln :~.
1140 1145 1150
Val Asp Arg Leu Ile Thr Gly Arg Leu Thr A}a Leu Asn Ala Phe Val
: 1155 11~0 1165 `
s:er Gln Thr Leu Thr Axg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu i;
-- 1170 1175 1180 .
Ala Lys Asp Ly~ Val A~n Glu Cys Val Arg Ser Gln Ser Gln Arg Phe
1185 ~1190 ~ 1195 1200
Gly Phe Cys Gly A205Gly Thr ~g Leu P12hel0Ser 1215
Pro A~n Gly Met Ile Phe Phe Hi~ Thr Val Leu Leu Pro Thr Ala Tyr
1220 1225 1230 .
- Glu Thr Val Thr Ala Trp Ser Gly Ile Cy~ Ala Ser Asp Gly Asp Arg
1235 1240 1245 :
Thr Phe Gly Leu Val Val Lys Asp Val Gln Leu Thr Leu Phe Arg A~n
1250 1255 1260
Leu A~p A~p Ly~ Phe Ty2r Leu Thr Pro Arg T2h~ ~et Tyr Gln Pro Arg
'
WO 93/23422 PCI`/US93/04384 . . -
2~ 3.~ 8~8 ;
132
Val Ala Thr Ser Ser ~p Phe Val ~ln Ile Glu Gly Cy~ A~p Val Leu
1285 1290 1295
Phe Val A~n Ala Thr Val Ile A~p Leu Pro Ser Ile Ile Pro Asp Tyr
1300 1305 1310
Ile Asp Ile A~n Gln Thr Val Gln Asp Ile Leù Glu A~n Tyr Arg Pro
1315 1320 l~Z5
A~n Trp Thr Val Pro Glu Phe Thr Leu Asp Ile Phe Asn Ala Thr Tyr
1~30 13~5 1340
Leu A3n Leu Thr Gly Glu Ile Asp A~p Leu Glu Phe Arg Ser Glu Lys
1345 1350 1355 1360
Leu His Asn Thr Thr Val Glu Leu Ala Ile Leu Ile Aqp Thr Ile Asn
1365 1370 1375
A3n Thr Leu Val A~n Leu Glu Trp Leu Asn Arg Ile Glu Thr Tyr Val
1380 1385 1390
Lys Trp Pro Trp Tyr Val Trp Leu Leu Ile Gly Leu Val Val Val Phe
1395 1400 . 1405
Cy~ Ile Pro Leu Leu Leu Phe Cys Cy~ Phe Ser Thr Gly Cys Cys Gly
1410 1415 1420
Cys Ile Gly Cya Leu Gly Ser Cy5 Cy~ His Ser Ile Cy3 Ser Arg Arg
1425 1430 1435 1440
Gln Phe Glu Tyr Tyr Glu Pro Ile Glu Lys Val ~i~ Val Hi~
144S 1450
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CH~RACTERISTICS:
: ~ (A) L~NGT~: 14:54 amino acids
(B) TYPE: amino acid
(D) SOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
:~
: - ~ (xi) SEQUENC:E DESCRIPTION: SEQ ID NO:44:
Met Ile Val Leu Val Thr Cy~ Leu Leu Leu Leu Cy9 Ser Tyr ~i~ Thr
1 5 10 15
Val ~eu Ser Thr Thr Asn Asn Glu Cy~ Ile Gln Val Asn Val Thr Gln
Leu Ala Gly Asn Glu A;sn Leu Ile Arg Asp Phe Leu Phe Ser Asn Phe
Lys Glu Glu Gly Ser Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
5S 60
Trp Tyr Asn Cys Ser Arg Thr Ala Gln Thr Thr Ala Phe Gln Tyr Phe
W 0 93~23422 PCT/US93/043842 1 3 1 ~ 9 8
:
133
Asn Asn Ile His Ala Phe Tyr Phe Yal Met Glu Ala Met Glu Asn Ser
~0 9S
Thr Gly Asn Ala Arg Gly Ly~ Pro Leu Leu Phe Hi~ Val His Gly Glu
100 105 110
Pro Val Ser Val Ile Ile Tyr Ile Ser Ala Tyr Arg Asp Asp Val Gln
115 120 125
Gln Arg Pro Leu Leu Lys His Gly Leu Val Cys Ile Thr Ly~ Asn Arg
130 135 ~ 140
His Ile~Asn Tyr~GIu Gln Phe Thr Ser Asn~Gln Trp Asn Ser Thr Cys
145~ ~ ~ 150 155 160
Thr Gly Ala Asp~Ag Lys Ile Pro Phe Ser Val Ile Pro Thr Asp Asn
165~ ~ 170 175
Gly~T~hr Lys~Ile Tyr Gly Leu Glu~Trp~Asn A~p A~p Phe Val Thr Ala
lq0~ 185 ~ 190
Tyr Ile Ser Gly Arg Ser Tyr His Leu Asn Ile Asn Thr Aqn Trp Phe
195 ~ ~ - 200 ~ 205
;Asn Asn Val Th~Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
Ala~Syr Ala Tyr~Oln cly~V~ Ser Asn Phe Thr Syr Tyr
Lys~Leu~Asn~A~n`Thr A-n~Gly~Leu Lys Thr ~yr Gl Leu Cy3 Glu Asp
Tyr~Glu Hl~-Cy~ Thr~Oly~Tyr A1a~Thr Asn Val Phe Ala Pro Thr Ser
260~ 26~5; ~ 270
~Lc ~ro~A-p Gly Phe~S-r~Phe A~n~Asn Trp;Phe Leu Leu
~;` ' Th~ A ~ ;Serl Sér~Thr~Ph- V 1 Ser~Cly~Arg~Phe~VaI Thr Asn Gln Pro
Léu ILe~ n Cy~ T-rp~Pro Val ~ro S r Ph- Gly Va ~Ala Ala
n~Glù~Phe~Gy~Phè~Glù~-Gly~Ala~Gln~Phe~Ser GIn Cys Ser Gly Val
- 3`25~ 330~ 335
Ser Leu A~n Asn~hr Val Agp Val lle Arg Phe A~n Leu Asn Phe Thr -~
~; ; 3~0 ~ 345 350
~-9 ~1~ Ala~A~p Val Gln Ser Gly Met Gly Ala Thr Val Phe Ser Leu Asn Thr
355 ~ 360~ 365
Thr Gly Gly~ Leu~Glu~V-l 9-r Cys Tyr Asn Asp Thr Val Ser ¦`
$~S-r~8-r Pb-~Tyr~Ser Tyr~Gly~Clu~Ile Pro Phe GIy Ile Thr Asp ¦r
Gly~ro~Arg~Tyr~-Cy Tyr Val Leu Tyr~A n Gly Thr Ala Leu Lys Tyr
W093/23422 ~ 3 ~ PCr~US93/M38
134
Leu Gly Thr Leu Pro Pro 5er Val Lys ~lu Ile Ala Ile Ser Lys Trp
420 425 430
Gly Hiq Phe Tyr Ile Asn Gly Tyr Aqn Phe Phe Ser Thr Phe Pro Ile
435 440 445 -
Asp Cy~ Ile Ser Phe Asn Leu Thr Thr Gly A~p Ser Gly Ala Phe Trp
450 455 460
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu ~sn
465 470 475 480
Thr Ala Ile Lys Lys Val Thr Tyr Cys Asn Ser His Ile Asn Asn Ile
485 4~0 495
Lys Cy~ Ser Gln Leu Thr Ala Asn Leu AQn Asn Gly Phe Tyr Pro Val
500 ~05 510
Ala Ser Ser Glu Val Gly Leu Val Asn Lys Ser Val Val Leu Leu Pro
515 520 525
Ile Phe Phe Ala Hia Thr Ala Ile A3n Ile Thr Ile Asp Leu Gly Met
530 535 540
Lys Arg Ser Gly Tyr Gly ~ln Pro Ile Ala Ser Thr Leu Ser Aqn Ile
54S 550 555 560 ;
Thr Leu Pro Met Gln Asp Asn Asn ~hr Asp Val Tyr Cy9 Ile Arg Ser
565 S70 S75
Aon Gln Phe Ser Val Tyr Val Hi~ Ser Ile Cys ~ys Ser Ser Leu Trp
Asp A~n Ile Phe A~n Gln Glu Cy~ Thr A~p VaI Leu A~p Ala Thr Ala
595 600 60S
Val Ile Lys Thr Gly Thr Cy9 Pro Phe Ser Phe Asp Lys Leu Asn Asn
610 615 620
Tyr Leu ~hr Phe A~n Ly~ Phe Cy~ Leu Ser Leu Ser Pro Val Gly Ala
625 ~ 630 635 640
Asn Cy~ Ly Phe Asp Val Ala Ala Arg Thr Arg Thr Acn Glu Gln Val
645 650 6S5
Val Arg Ser Leu ~yr Val Ile Tyr Glu Glu Gly Asp Asn Ile Val Gly
660 665 6~0
Val Pro Ser Asp Asn Ser Gly Leu His Asp Leu Ser Val Leu ~i8 Leu
675 680 68S
Asp Ser Cy~ Thr Glu Tyr Asn Ile Tyr Gly Arg Thr Gly Val Gly Ile
690 695 700
Ile Arg Gln Thr Asn Ser Thr Leu Leu Ser Gly Leu Tyr Tyr Thr Ser
705 71~ 715 720
Leu Ser Gly AQP Leu Leu Gly Phe Lys Asn Val Ser Asp Gly Val Ile
725 730 735
Tyr Ser Val Thr Pro Cys A~p Val Ser Ala Gln Ala Ala Val Ile Asp
740 745 750
WO 93/23422 PCl`/US93fO4384
2 1 3 4 ~ 9 8 ~ ~
135 ~:
Gly Ala Ile Val Gly Ala Met Thr Ser Ile Asn Ser Glu Leu Leu Gly
755 7~0 765
Leu Lys Hi~ Trp Thr Thr ~hr Pro A~n Phe ~yr Tyr Tyr Ser I le Tyr
770 775 780
A~n Tyr Thr Asn Glu Arg Thr Arg Gly Thr Ala Ile Asp Ser Asn Asp ;:
785 790 795 800 '
Val A~p Cy~ Glu Pro Ile Ile Thr Tyr Ser Asn Ile Gly Val Cys Lys
. : 805 810 815
Asn:Gly Ala Leu Val Phe Ile Asn Val Thr His Ser Asp Gly Asp Val
820 825 830
Gln Pro Ile Ser Thr Gly ~:Thr Val $hr Ile ~Pro Thr Asn Phe Thr Ile
: 835 840 845 -:
, ~ :
~Ser Va1 Gln ~Val Glu Tyr Ile Gln Val Tyr Thr 'Thr Pre Val Ser Ile '.
850 ~ 855 - 860
Asp Cys Ala Arg Tyr Val Cys Asn Gly Asn Pro Arg Cy~ Asn Lys Leu ,~
865 870 : 875 880 -~
Leu Thr Gln Tyr Val Ser Ala Cys Gln Thr Ile Glu Gln Ala Leu Ala
8:85 ~ 890~ : 895 -.,
Met:Gly~Ala~Arg Leu~Glu A~n~Met Glu~Val A~p ~Ser Met Leu ~Phe Val
900~ : 905 ~ 910
Ser~Glu ~Asn:~Ala Leu Lys Leu Ala ~Ser Val Glu Ala Phe Asn Ser Thr
:91~ : :: 920 : : 925
7.-p~Pro ~ yr~ Ly-~ Glu rrp Pro A Il- Gly Gly Ser
Trp Leu~ Gly~;:Gly~Leu~ ys A-p:Ile'Leù Pro Ser 8l~ Asn Ser Lys Arg ,`
Tyr~ Arg~ Sër '~A~a Il- ~C~lu A-p~Lou Leu~ Phe Asp Lys Val Val Thr
9 6 5 ~ 9 7 0 9 7 5
Ser:~Gly:~éu-,~Gly~Thr:~Val~:~A~p~Glu~Asp Tyr ~y~ Arg Cy9 Thr Gly Gly .
980:~ 985~ 990 '~
,Asp ~lle~Ala~Asp`~eu~Val~Cys~Ala~Gln Tyr Tyr Asn Gly Ile Met '~Y
9~9S ,~ l000~ 1005 i~-
Val Leu Pro: Gly~' Val Ala Asn Asp~Asp Lys Met Thr Met Tyr Thr Ala '~
1010 , 1015: ~ 1020
ser Leu: Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
025 ~ :1030 : : 1035 1040
Al~a~Ile~Pro ~Ph-~:Alà ~Val Ala~Val Gln Ala Arg Leu Asn Tyr Val Ala 5
1045 ~, 1050 1055
::Leu~Gln~$hr Asp Val ~eu Asn Lys Asn Oln Gln:Ile Leu Ala A6n Ala
::1060 ~ ~ : 1065 1070
Phe::As`n GIn Ala Ile Gly Asn I}e Thr Gln Ala Phe Gly Ly3 Val Asn ?i`'
1075 1080 i 1085 t!'~
?
`'., ': ~ :: '
~: :
WO 93/23422 P~/US93/0438a,.
213~9~
136
Asp Ala Ile Hi~ Gln T~r Ser Lya Gly Leu Ala Thr Val Ala Lys Ala
1090 1095 11~0
Leu Ala Lys Val Gln Asp Val Val Asn Thr Gln Gly Gln Ala L~u Ser
1105 1110 1115 1120 .
His Leu Thr Val Gln Leu Gln A~n Asn Phe Gln Ala Ile Ser Ser Ser
' 1125 1130 1135
Ile Ser Asp Ile Tyr Asn Arg Leu Asp Glu Leu Ser Ala Asp Ala Gln
1140 114~ 1150
Val Asp Arg Leu Ile Thr Gly Arg Leu Thr Ala Leu A~n Ala Phe Val
ll5S 1160 1165
Ser Gln Thr Leu Thr Arg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu
1170 1175 1180
Ala Lys Asp Ly~ Val Asn Glu Cy~ Val Arg Ser Gln Ser ~ln Arq Phe
1}85 1190 1195 1200
Gly Phe Cy~ Gly A~n Gly Thr Hi~ Leu Phe Ser Leu Ala A~n Ala Ala
1205 1210 1215
Pro Asn Gly Met Ile Phe Phe His Thr Val Leu Leu Pro Thr Ala Tyr
1220 1225 1230
Glu Thr Val Thr Ala Trp Pro Gly Ile Cys Ala Ser Asp Gly Asp Arg
; ~ 1235 1240 1245
Thr Phe Gly Leu Val Val Lys Asp Val Gln Leu Thr Leu Phe Arg Aqn
1250 I255 1260
Leu Asp Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln Pro Arg
1265 1270 12~5 1280 -~
Ala Ala Thr Ser Ser Asp Phe Val Gln Ile Glu Gly Cys Asp Val Leu
1285 1290 1295
~he Val Asn Ala Thr Va} Ile Asp Leu Pro Ser Ile Ile Pro A~p Tyr
1300 13~5 1310
Ile Asp Ile A n Gln ~hr Val Gln Asp Ile Leu Glu A~n Tyr Arg Pro
1315 . 1320 1325
Aan Trp Thr Val Pro Glu LQU Thr Lou A~p I1~ Phe Asn Ala Thr Tyr
1330 1335 1340
Leu A3n Leu Thr Gly Glu Ile A~p Asp Leu Glu Phe Arg Ser Glu Ly~
1345 1350 135S 1360
Leu His Asn Thr Thr Val Glu Leu Ala Ile Leu Ile Asp Asn Ile Asn
1365 1370 1375
Asn Thr Leu Val A~n Leu Glu Trp Leu A~n Arg Ile Glu Thr Tyr Val
1380 1385 1390
Lys Trp Pro Trp Tyr Val Trp Leu Leu Ile Gly Leu Val Val Ile Phe
1395 1400 1405
Cy~ Ile Pro Leu Leu Leu Phe y8 Cys Cy~ Ser Thr Gly Cy~ Cy~ Gly
1410 1415 1420
WO 93/23422 2 1 ~ I 8 9 8 PCl'~US93/04384 `:
. .
137 ~:
Cys Ile Gly CY8 Leu Gly Ser Cys Cy~ Hi~ Ser Met CYQ Ser Arg Arg -:
1425 1430 1435 1440 :~
Gln Phe Glu A~n Tyr Glu Pro Ile Glu Ly~ Val His Val 1~1~
1445 1450 ~`
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CaARACTERISTICS:
(A) LENGTH: 1454 amine acids
(B3 TYPE: amino acid
(D) TOPOLOGY: unknown
( ii ) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Met Ile Val Leu Val Thr cys Leu Leu Leu Leu Cy~ Ser Tyr His Thr
1~ 15
Val Leu Ser Thr Thr A~n Asn Glu Cy~ Ile Gln Val ~sn Val Thr Gln
20 25 30 ~:
Leu Ala Gly Asn Glu A~n Leu Ile Arg A~p Phe Leu Phe Ser AQn Phe
35 40 45 -:
Lys: Glu Glu Gly Ser Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
~ ~ ~ 50 55 60
- - ~ Trp Tyr Asn Cys Ser Arg Thr Ala Gln $hr Thr Ala Phe Gln Tyr Phe
A~n A~n Ile Hi~ Ala Phe Tyr Phe Val Met Glu Ala Met Glu Asn Ser
Thr Gly Agn Ala Arg Gly Lyc Pro Leu Leu Phe ~i~ Val His Gly Glu ~`
100 105 110
Pro Val Ser Val Ile Ile Tyr Ile Ser Ala Tyr Arg Asp Asp Val Gln
115 120 125
Gln Arg Pro Leu Lau Lyo ~is Gly ~eu Val Cys Ile Thr Ly~ Asn Arg ;~
130 135 140 ~.
His Ile A~n Tyr Glu Gln Phe Thr Ser Asn Gln Trp Asn Ser Thr Cys `~
14~ 150 155 160
Thr Gly Ala Asp Arg Ly~ Ile Pro Phe Ser Val Ile Pro Thr Asp Asn
16S 110 175
Gly Thr Lys Ile Tyr Gly Leu Glu Trp Asn Asp Asp Phe Val Thr Ala
180 185 190
Tyr Ile Ser Gly Arg Ser Tyr His Leu A~n Ile Asn Thr AQn Trp Phe
195 200 205
Asn A~n Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr ~la Thr Trp Glu
210 215 220
Tyr Ser Ala Ala Tyr Ala Tyr Gln Gly Val Ser Asn Phe Thr Tyr Tyr
225 230 23~ 240 :
WO 93/234~2 PCI/US93/0438d
2 1 ~ 8
138
Lys Leu A~n Asn Thr Asn Gly Leu Lya Thr Tyr Glu Leu Cys Glu Asp
245 250 255
Tyr Glu His Cy~ Thr Gly Tyr Ala Thr Asn Val Phe Ala Pro Thr Ser
2~0 265 270
Gly Gly Tyr Ile Pro Asp Gly Phe Ser Phe A~n A3n Trp Phe Leu Leu
~7S 280 2F~i
Thr Asn Ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr Asn Gln Pro
290 295 300
Leu Leu Ile Asn Cy8 Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
305 310 315 3~0
Gln Glu Phe CYB Phe Glu Gly Ala Gln Phe Ser Gln Cys Ser Gly Val
325 330 335
Ser Leu Asn.Asn Thr Val Asp Val Ile Arg Phe Asn Leu Asn Phe Thr
340 345 350
Ala Asp Val Gln Ser Gly Met Gly Ala Thr Val Phe Ser Leu A3n Thr
355 360 365
Thr Gly Gly Val Ile Leu Glu Val Ser Cy~ Tyr Asn A~p Thr Val Ser
370 375 380
Glu Ser Ser Phe Tyr Ser Tyr Gly Glu Ile Pro Phe Gly Ile Thr Asp
385 390 395 400
Gly Pro Arg Tyr Cys Tyr Val Leu Tyr Asn Gly Thr Ala Leu Ly3 Tyr
~05 410 415
Leu Gly Thr Leu Pro Pro Ser Val Ly8 Glu Ile Ala Ile Ser Ly~ Trp
420 425 430
Gly Hi3 Phe Tyr Ile Asn Gly Tyr A~n Phe Phe Ser Thr Phe Pro Ile
- 435 440 44S
A~p Cys Ile Ser Phe Asn Leu Thr Thr Gly Asp Ser Gly Ala Phe Trp
450 455 460
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu Asn
465 470 475 480
Thr Ala Ile Ly~ Lys Val Thr Tyr Cy8 A~n Ser Hi~ Ile Aan A3n Ile
485 490 495
Lys Cys Ser Gln Leu Thr Ala A~n Leu A8n A n Gly Phe Tyr Pro Val
500 505 510
Ala Ser Ser Glu Val Gly Leu Val A~n Ly~ Ser Val Val Leu Leu Pro
515 520 525
Ile Ph~ Phe Ala Hi3 Thr Ala Ile A~n Il~ Thr Ile Asp Leu Gly Met
530 53S 540
Lys Arg Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu Ser Asn Ile
545 550 555 ~ 560
Thr Leu Pro Met Gln Asp A5n A~n Thr Asp Val Tyr Cy8 Ile Arg Ser
565 570 5~5
W O 93/23422 P ~ /VSg3/043&4
213~98 '~
139 `;:
Asn Gln Phe Ser Val Tyr Val Hi~ Ser Ile Cy~ Ly~ Ser Ser Leu Trp
580 585 S90
A~p A-qn Ile Phe A~n Gln Glu Cy~ Thr Asp Val Leu Asp Ala Thr Ala
595 600 605
Val Ile Ly~ Thr Gly Thr Cys Pro Phe Ser Phe Asp Lyq Leu Asn Asn ;.
610 615 620 ~:~
Tyr Leu Thr Phe A~n Ly~ ~he Cy~ Leu Ser Leu Ser Pro Val Gly Ala
625 630 635 640
A~n Cy8 Lya Phe Asp Val Ala Ala Arg Thr Arg Thr A~n Glu Gln Val ~-
645 650 655
Val Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly Asp Asn Ile Val Gly
660 665 670 .
Val Pro Ser Asp A3n Ser Gly Leu Hiq Asp Leu Ser Val Leu Hi~ Leu
67S 680 685
Asp Ser Cys Thr Glu Tyr Asn Ile Tyr Gly Arg Thr Gly Val Gly Ile
690 695 700 .
Ile Arg Gln Thr Asn Ser Thr Leu Leu Ser Gly Leu Tyr Tyr Thr Ser ~`
70S 710 715 720
Leu Ser Gly Asp Leu Leu Gly Phe Ly~ A~n Val Ser Asp Gly Val Ile
725 730 735
Tyr Ser Val Thr Pro Cy~ A3p Val Ser Ala ~ln Ala Ala Val Ile Asp `
740 745 750 ~`
Gly Ala Ile Val Gly Ala Met Thr Ser Ile A~n Ser Glu Leu Leu Gly
755 760 765
Leu Lys His Trp Thr Thr Thr Pro A3n Phe Tyr Tyr Tyr Ser Ile Tyr
770 775 ~80 `~
Asn Tyr Thr Asn Glu Arg Thr Arg Gly Thr Ala Ile Asp Ser Asn Asp
785 790 795 800 -
Val As~ Cys Glu Pro Ile Ile Thr Tyr Ser A n Ile Gly Val Cys Lys
.. 805 810 815
~.
A~n Gly Ala Leu V~l Phe Ile Asn Val Thr His Ser A p Gly A p Val .:
820 825 830
Gln Pro Ile Ser Thr Gly Thr Val Thr Ile Pro Thr A~n Phe Thr Ile
835 840 845
ser Val Gln Val Glu Tyr Ile Gln Val Tyr Thr Thr Pro Val Ser Ile
850 855 860 ~
Asp Cys Ala Ar~ Tyr Val Cys A~n Gly Asn Pro Arg Cy9 Asn Lys Leu -.
865 870 875 880 ~
Leu Thr Gln Tyr Val Ser Ala Cy8 Gln Thr Ile Glu Gln Ala Leu Ala ,~;;
. 885 890 895 `~
Met Gly Ala Ar~ Leu Glu A~n Met Glu Val Asp Ser Met Leu Phe Val
900 905 910
'
WO 93/23422 P~/US93/04384
,........... .
2 1 3 4
140
Ser Glu A~n Ala Leu Lys Leu Ala Ser Val Glu Ala Phe Asn Ser Thr
915 920 925
Glu Aqn Leu A8p Pro Ile Tyr Ly~ Glu Trp Pro Ser Ile Gly Gly Ser
930 935 94~
Trp Leu Gly Gly Leu Lys Asp Ile Leu Pro Ser Hi~ Asn Ser Ly~ Arg
945 950 9s5 960
Lys Tyr Gly Ser Ala Ile Glu A3p Leu Leu Phe Asp Ly~ Val Val Thr
965 970 975
Ser Gly Leu Gly ~hr Val A~p Glu A~p Tyr Lya Arg Cy8 Thr Gly Gly
9~0 985 99Q
Tyr Asp Ile Ala Asp Leu ~al Cy~ Ala Gln Tyr Tyr Asn Gly Ile Met
995 1000 1005
Val Leu Pro Gly Val Ala A~n Ala Asp Lys Met Thr Met Tyr Thr Ala
1010 1015 1020
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
1025 10~0 1035 1040 :
Ala Ile Pro Phe Ala Val Ala Val Gln Ala Arg Leu Asn Tyr Val Ala
1045 1050 1055
Leu Gln Thr Asp Val Leu Asn Lys A~n Gln Gln Ile Leu Ala Asn Ala
1060 1065 1070
Phe A~n Gln Ala Ile Gly A~n Ile ~hr Gln Ala Phe Gly Ly~ Val Asn
107~ 10~0 1085
Asp Ala Ile His Gln Thr Ser Gln Gly Leu Ala Thr Vai Ala Lys Ala
1090 1095 1100
Leu Ala Ly~ Val Gln A~p Val Val A~n Thr Gln Gly Gln Ala Leu Ser
1105 1110 1115 1120
HL9 Leu Thr Val Gln Leu Gln Asn Asn Phe Gln Ala Ile Ser Ser Ser
1~125 1130 1135
Ile Ser A~p Ile Tyr Asn Arg Leu Asp Glu Leu Ser Ala Asp Ala Gln
.. 1140. 1145 1150
Val Asp Arg Leu lle ~hr Gly Arg Leu Thr Ala Leu A~n Ala Phe Val
115~ 1160 1165
Ser Gln Thr Leu Thr Arg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu
1170 1175 1180
Ala Ly~ ABP Lys Val Asn Glu Cy8 Val Arg Ser Gln Ser Gln Arg Phe
1185 1190 1195 1200
Gly Phe Cy8 Gly A~n Gly Thr Hi~ Leu Phe Ser Leu Ala Asn Ala Ala
1205 1210 1215
Pro Asn Gly Met Ile Phe Phe His Thr Val Leu Leu Pro Thr Ala Tyr
12ZO 1225 1230
Glu Thr Val Thr Ala Trp Ser Gly Ile Cy8 Ala Ser A~p Gly A~p Arg
1235 . 1240 1245
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. 13~89'~
141
Thr Phe Gly Leu Val Val Ly~ Asp Val Gln Leu Thr Leu Phe Arg Asn
1250 1255 1260
Leu Asp Asp Ly~ Phe Tyr Leu Thr Pro ~rg Thr Met Tyr Gln Pro Arg ~-
1265 1270 1275 1280
Val Ala Thr Ser Ser Asp Phe Val Gln Ile Glu Gly Cys Asp Val Leu
1285 1290 . 12g5
Phe Val Aon Ala Thr Val Ile A~p Leu Pro Ser Ile Ile Pro Asp Tyr
1300 13~5 1310
Ile Asp Ile A3n Gln Thr Val Gln A~p Ile Leu Glu Asn Tyr Arg Pro
1315 1320 1325
Asn Trp Thr Val Pro Glu Phe Thr Leu Asp Ile Phe Asn Ala Thr Tyr
1330 1335 1340 .
Leu Asn Leu Thr Gly Glu Ile Asp A~p Leu Glu Phe Arg Ser Glu Lys
1345 1350 1355 1360
Leu His A n Thr Thr Val Glu Leu Ala Ile Leu Ile Asp Thr Ile AQn
1365 1370 1375
A~n Thr Leu Val A~n Leu Glu Trp Leu Asn Arg Ile Glu Thr Tyr Val
1380 1385 1390
Lys Trp Pro Trp Tyr Val Trp Leu Leu Ile Gly Leu Val Val Val Phe
1395 1400 1405 .
Cys Ile Pro Leu Leu Leu Phe Cy~ Cy~ Phe Ser Thr Gly Cy Cys Gly:~
1410 1415. 1420 :
Cy9 Ile Gly Cys LQU Gly 5er Cy~ Cys Hi~ Ser Ile Cya Ser Arg Arg ~:
1425 lg30 1435 1440
Gln Phe Glu Tyr Tyr Glu Pro Ile Glu Lyq Val Hia Val Hls i`
1445 1450 ~'
~ .
(2) INFORMATION FOR SEQ ID No:46:
(i) SEQUENCE CHARACTE~ISTICS: ;~
(A) LENGTH: 14S4 amino acida
(B) TYP~: amino acid .
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQU~NCE DESCRIPTION: SEQ ID NO:46:
Met Ile Val Leu Val Thr Cys Leu Leu Leu Leu Cy8 Ser Tyr Hls Thr
1 5 10 15
Val Ser Ser Thr Ser Asn Asn A~p Cys Arg Gln Val Asn Val Thr Gln
Leu Ala Gly A~n Glu Asn Leu Ile Arg Asp Phe Leu Phe Gln Ser Phe
3S 40 45
Lys Glu GlU Gly Ile Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Yal
WO 93/~ 2 2 :~ 3 4 ~ 9 8 PCI/US93/04384
142
Trp Tyr Asn Cys Ser Arg Thr Ala Thr ~hr Thr Ala Tyr Glu Tyr Phe
Asn A6n Ile His Ala Phe Tyr Phe A~p Met Glu Ala Met Glu Asn Ser
so 95
Thr Gly Asn Ala Arg Gly Lys Pro Leu Leu Phe ~is Val His Gly Glu
100 105 110
Pro Val Ser Ile Ile Ile Tyr Ile Ser Ala Tyr Gly Asp Asp Val Gln
115 120 125
Gln Arg Pro Leu Leu Glu His Gly Leu Leu Cy8 Ile Thr Lys Asn Arg
130 135 140 :
~sn Ile Asp Tyr Asn Thr Phe Thr Ser Asn Gln Trp Asp Ser Ile Cys
lg5 : ~ 150 155 ~ 160
Thr Gly Asn Asp Arg Lys Ile Pro Phe Ser Val Ile Pro Arg Asp Asn -~
165 ~ 170 175
Gly Thr Lys Ile Tyr Gly Leu Glu Trp Asn Asp Glu Phe Val Thr Ala
180 185 190
Tyr Ile Ser Gly Arg Ser Tyr Asn Trp Asn Ile Asn Asn Asn Trp Phe
lgS ~ 200 205
A~n Asn Val: Thr Leu Leu Tyr Ser Arg Ser Ser ~ Thr Ala Thr Trp Glu
210 ~ 215 220
Tyr ~Ser~Al-: Ala~Ty:r Val~ Tyr Gln Gly Val Ser Asn Phe Thr Tyr Tyr
225~ : 2:30 235 240
Leu -;Asn~ABn ~Thr A-n: ~Gly Leu Lys Thr Tyr Glu Phe Cys Glu Asp
Z45 250 255
5~ ~ Tyr~ Glu Tyr Cy~ Thr Gly Tyr Ala Thr A~n Val Phe Ala Pro Thr Val
: ~ ~260 265 270
Gly; Gly Tyr Ile Pro A~p Gly Phe Ser Phe Asn Asn Trp Phe Leu Leu
27~5~ ZB0 ~ 285
hr~-hsn: S:e~r~ Ser Thr Phe Val Ser Gly Arg Phe Val Thr A~n Gln Pro
290 ~ 295: ~ 300
- Leu I.eu Val As n Cy8 Leu~Trp~ Pro Val Pro Ser Phe Gly Val Ala Ala
305~ 310~ : 315 320
Gln ~Glu Phe Cyg Pho ~Glu Gly Ala Gln Phe Ser aln Cy9 Asn Gly Val
325 330 335
r ~i ' S-r ~ eu AJn Asn Thr Vai Asp Val Ile Arg Phe Asn Leu Asn Phe Thr
340 345 350
Ala Asp Val Gln Ser Gly Met Gly Ala Thr Val Phe Ser Leu Asn Thr
355 360 365
:Thr Gly Gly Val Ile Leu Glu Ile Ser Cys Tyr Ser Asp Thr Val Ser
370 - 375 380
~: : : Glu~ Ser Ser Ser Tyr S-r Tyr Gly Glu Ile Pro Phe Gly Ile Thr A~p
385 ~ 39C 395 400
' :
. . ,
.
.
W 0 93/23422 PCT/USg3/043~4
213~9~ ,
.:
143
Gly Pro Arg Tyr Cy~ Tyr Val Leu Tyr A~n Gly Thr Ala Leu Lys Tyr
405 410 41S ;~-
Leu Gly Thr Leu Pro Pro Ser Val Lys Glu Ile Ala Ile Ser Lys Trp
420 425 ~30
Gly His Phe Tyr Ile Asn Gly Tyr A~n Phe Phe Ser Thr Phe Pro Ile
43~ 440 445 :~
Gly Cys Ile Ser Phe Asn Leu Thr Thr Gly Ala Ser Gly Ala Phe Trp
450 455 460
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu Asn
465 47~ 475 480
Thr Ala Ile Lys Asn Val Thr Tyr Cy~ Asn Ser Hi~ Ile Asn Asn Ile
485 490 495
Lys Cys Ser Gln Leu Thr Ala Asn Leu Asn A~n Gly Phe Tyr Pro Val
500 5QS S10
Ala Ser Ser Glu Val Gly Phe Val Asn Lys Ser Val Val Leu Leu Pro
515 520 ~25 ~`
Ser Phe Phe Thr His Thr Ala Val Asn Ile Thr Ile Asp Leu Gly Met
530 535 540
Lys Leu Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu ser Asn Ile ,-
545 550 555 560
Thr Leu Pro Met Gln A~p Asn Asn Thr Asp Val Tyr Cy~ Ile Arg Ser
- 565 570 575
Asn Gln Phe Ser Val Tyr Val Pro Ser ~hr Cy~ Lys Ser Ser Leu Trp
580 585 590
A3p Asn Ile Phe A~n Gln Asp Cys Thr Asp Val Leu Glu Ala Thr Ala
595 600 605
Val Ile Lys Thr Gly Thr Cy9 Pro Phe Ser Phe Asp Ly3 Leu Asn Asn
610 61S 620
Tyr Leu Thr Phe A~n Lys Phe Cy8 Leu Ser Leu Ser Pro Val Gly Ala
625 630 635 640
A~n Cy5 Ly3 Phe Asp Val Ala Ala Arg Thr Arg Thr Asn Glu Gln Val
645 650 655
Val Arg Ser Leu Tyr Val Ile Tyr.Glu Glu Gly Asp A~n Ile Val Gly
6~0 665 670
., ,
Val Pro Ser A~p A~n Ser Gly Leu ~i~ Asp Leu Ser Val Leu Hi~ Leu
675 680 685
Asp Ser Cy~ Thr A~p Tyr A~n Ile Tyr Gly Arg Thr Gly Val Gly Ile
690 695 700
Ile Arg Arg Thr A~n Ser Thr Leu Leu Ser Gly Leu Tyr Tyr Thr Ser
705 710 715 720
Leu Ser Gly Asp Leu Leu Gly Phe Lys Asn Val Ser Asp Gly Val Ile
725 730 735
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144
Tyr Ser Val Thr Pro Cy~i Asp Val Ser Ala Gln Ala Ala Val Ile Asip
740 745 750
Gly Ala Ile Val Gly Ala Met Thr Ser Ile Asn Ser Glu Leu Leu Gly
755 760 765
Leu Thr His Trp ~hr Thr Thr Pro Ai3n Phe Tyr Tyx Tyr Ser Ile Tyr
770 77S 780
A~n Tyr Thr Ser Glu Arg Thr Arg Gly Thr Ala Ile Asp Ser A~in Asip
785 790 79S 800
Val Asp Cy8 Glu Pro Val Ile Thr Tyr Ser AEin Ile Gly Val Cy~i Ly~
805 810 815
Asn Gly Ala Leu Val Phe Ile Asn Val Thr His Ser A~p ~ly Asip Val
820 825 . 830
Gln Pro Ile Ser Thr Gly Asn Val Thr Ile Pro Thr Asn Phe Thr Ile
835 840 845
Ser Val Gln Val Glu Tyr Met Gln Val Tyr Thr Thr Pro Val Ser Ile
850 855 860
Asp Cys Ala Arg Tyr Val Cys Asn Gly A~n Pro Arg Cy~ Asn Lys Leu
865 ~70 87~ 880
Leu Thr Gln Tyr Val Ser Ala Gys Gln Thr Ile Glu Gln Ala Leu Ala
885 890 8g5
Met Gly Ala Arg Leu Glu Asn Met Glu Val A~p Ser Met Leu Phe Val
goo . 905 910
Ser Glu Asn Ala Leu Lya Leu Ala Ser Val Glu Ala Phe Asn ser Thr
915 920 925
Glu A~in Leu Asp Pro Ile Tyr LyEi Glu Trp Pro Asn Ile Gly Gly Ser
930 935 940
Trp Leu Gly Gly Leu Lys Asp Ile Leu Pro Ser His Asin Ser Lys Arg
945 950 gS5 960
Lys Tyr Arg Ser Ala Ile Glu A~p Leu Leu Phe Asp Lys Val Val Thr
965 970 975
Ser Gly Leu Gly Thr Val AEiP Glu A~p Tyr Lya Arg CYEi Thr Gly Gly
980 985 990
Tyr Asip Ile Ala Asp Leu Val Cys Ala Gln Tyr Tyr Asn Gly Ile ~et
995 1000 1oo5
Val Leu Pro Gly Val Ala Asn Asp Asp Lya Met Thr Met Tyr Thr Ala
1010 1015 1020
S~r Leu Ala Gly GLy Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
1025 1030 1035 1040
Ala Ile Pro Phe Ala Val Ala Val Gln Ala Arg Leu Asn Tyr Val Ala ;:
1045 1050 1055 :
Leu Gln Thr A~ip Val Leu Ai~in LYB A~in Gln Gln Ile Leu Ala Ai3n Ala
1060 1065 1070
W~ 93/23422 2 1 3 1 8 9 ~ PCr/US93/04384
14~
Phe Asn Gln Ala Ile Gly Asn Ile Thr Gln Ala Phe Gly Lys Val Aqn
10~5 1080 108S .
Asp Ala Ile His Gln Thr Ser Ly~ Gly Leu Ala Thr Val Ala Lys Ala
1090 1095 1100
Leu Ala Ly~ Val Gln Asp Val Val A~n Thr Gln Gly Gln Ala Leu Ser
1105 1110 . 1115 1120 :
is Leu Thr Val Gln Leu Gln A~n Asn Phe Gln Ala Ile Ser Ser Ser
1125 1130 1135
le Ser Asp Ile Tyr Asn Arg Leu Asp Glu Leu Ser Ala Asp Ala Gln
1140 114S 1150 .
al Asp Arg Leu Ile Thr Gly ~rg Leu Thr Ala Leu Asn Ala Phe Val
1155 1160 1165
Ser Gln Thr Leu Thr Arg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu
1110 -1175 118
Ala Ly3 Asp Ly~ Val Asn G~u Cys Val Arg Ser Gln S~r Gln Arg Phe
1185 1190 1195 1200
ly Phe Cy8 Gly Asn Gly Thr Hi9 Leu Phe Ser LQU Ala A~n ALa Ala
1205 1210 1215
ro A~n Gly Met Ile Phe Phe Hi~ Thr Val Leu Leu Pro Thr Ala Tyr
1220 1225 1230
lu Thr Val Thr Ala Trp Pro Gly Ile Cy~ Ala Ser A~p Gly Asp Arg
1235 ~240 1245
Thr Phe Gly Leu Val Val Lys Asp Yal Gln Leu Thr Leu Phe Arg Asn
1250 1255 1260
Leu Asp Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln Pro Arg
1265 .1270 1275 1280
la Ala Thr Ser Ser A~p Phe Val Gln Ile Clu Gly Cys A3p Val Leu
12a5 1290 1295
he Val Asn Ala Thr Val Ile A~p Leu P~o Ser Ile Ile Pro Asp Tyr
1300 1305 1310
le Asp Ile A~n Gln Thr Val Gln A p Ile Leu Glu Asn Tyr Arg Pro
1315 1320 1325 ~.
A~n Trp Thr Val Pro Glu Leu Thr Leu Asp Ile Phe Asn Ala Thr Tyr
1330 1335 1340
Leu Asn Leu Thr Gly Glu Ile Asp Asp Leu Glu Phe Arg Ser Glu Lys
1345 1350 1355 1360
eu ~ A~n Thr Thr Val Glu Leu Ala Ile Leu Ile A~p Asn Ile Asn
1365 1370 1375
~n Thr Leu Val Asn Leu Glu Trp Leu A~n Arg Ile Glu Thr Tyr Val
1380 1385 1390
Lys ~rp Pro Trp Tyr Val Trp Leu Leu Ile Gly Leu Val Val Ile Phe
13gS 1400 1405
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2~31~
146
Cys Ile Pro Leu Leu Leu Phe Cys Cys Cys Ser Thr Gly Cys Cys Gly
1410 1415 1420
Cys Ile Gly Cy~ Leu Gly Ser Cyn Cya Hi~ Ser Met Cy~ Ser Arg Arg
1425 1430 1435 1440
Gln Phe Glu Asn Tyr Glu Pro Ile Glu Lys Val ~i8 Val His
1445 1450 ~-
, .
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE~C~ARACTERISTICS:
(A) ~ENGTH: 1454 amino acids
(8) TYPE: amino acid
(~D) TOPOLOGY: unknown
~ii) MOLECULE TYPE: protein
(xi~j SEQUENCE DESC~IPTION: SEQ ID NO:47:
Met Ile Val Leu Val Thr Cys Leu Leu Leu Leu Cys Ser Tyr ~is Thr
1 S ~ 10 15
Val Leu Ser Thr Thr Asn Aqn Glu CYB Ile Gln Val Asn Val Thr Gln
~ 20 25 : 30
Leu Ala Gly Asn Glu:Asn Leu Ile Arg A~p Phe Leu Phe Ser Asn Phe
3S~ : 40 4S :.
Ly- GLu clu~Cly~s~r~V~l~Val:VaL Gly~Gly Tyr~yr Pro Thr Glu Val
Trp~Tyr~Asn Cys~S-r:Arg Thr Ala~Gln Thr Thr Ala Phe Gln Tyr Phe
A~n'A n Ile~:H~q Ala Phe Tyr Phe Val Met;Glu Ala Met Glu Aqn Ser
:85:: ~ : : 90 9i ~'
Thr Gly Asn Ala~Arg Gly Lys~Pro Leu Leu Phe His Val His Gly Glu ".
10~0~ 105 110 ,~:
: Pro Val Ser Val~ Ile Tyr~Ile:~S~er~ A~l- Tyr Arg Asp Asp ~al Gln '~
Gln~Arg~Pro Leu~Leu:'Ly~His Gly T-eu Val Cys Ile Thr Lys Asn Arg
30~ 3;~ 140 .
~' His Ile Asn Tyr Glu Gln Phe Thr Ser A~n Gln Trp Asn Ser Thr Cys :'
145 150 155 160 .~
~: Thr Gly Ala A~p Arg ~y~ Ile Pro Phe Ser Val Ile Pro Thr Asp Asn ::.
165 ' 1~0 l~S '.
'Gly Thr Lys Ile Tyr Gly Leu Glu Trp Asn Asp A~p Phe Val Thr Ala :~
180 185 190
,~ Tyr Ile Ser ¢ly Arg Ser Tyr H1~ Leu A~n Ile A~n Thr Asn Trp Phe .
195~ 200~ 205 : ',,'
; A~sn Asn Val Thr Leu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu `,.
' :210~ ~ 215 . 220
,, - ~ ~:
::
W O 93/23~22 PCT/US93/04384
2 13~9~
147
Tyr Ser Ala Ala Tyr Ala Tyr Gln Gly Val Ser Asn Phe Thr Tyr Tyr
225 230 23S 240
yq Leu Aqn Asn Thr Asn Gly Leu Lys Thr Tyr Glu Leu Cys Glu Asp
24S 250 2S5
Tyr Glu Hi~ Cys Thr Gly Tyr Ala Thr Asn Val Phe P,la Pro Thr Ser
260 265 270 ~.
Gly Gly Tyr Ile Pro Asp Gly Phe Ser Phe Asn Asn Trp Phe Leu Leu
275 280 285
Thr Asn Ser Ser Thr Phe Val Ser Gly Arg Phe Val Thr AYn Gln Pro
290 295 300
Leu Leu Ile Aqn Cy~ Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
305 310 315 320
Gln Gl~ Phe cys Phe Glu Gly Ala Gln Phe Ser Gln Cys Asn Gly Val
325 330 3~5
Ser I eu Asn Asn Thr Val Asp Val Ile Arg Fhe Asn Leu Asn Phe Thr
340 34~ ~50 -
Ala Asp Val ~ln Ser Gly ~et Gly Ala Thr Val Phe Ser Leu Asn Thr
355 360 365
Thr Gly Gly Val Ile Leu Glu Ile Ser Cys Tyr Ser Asp Thr Val Ser
370 375 380
Glu Ser Ser Ser Tyr Sar Tyr Gly Glu Ile Pro Phe Gly Ile Thr Asp
385 390 395 400 ~-
Gly Pro Arg Tyr Cy5 Tyr Val Leu Tyr Asll Gly Thr Ala Leu Lys Tyr
405 410 415
Leu Gly Thr Leu Pro Pro Ser Val Lys Glu Ile Alil Ile Ser Lys Trp
420 425 430
Gly His Phe Tyr ILe Asn Gly Tyr Asn Phe Phe Ser Thr Phe Pro Ile
435 440 445
Gly Cys Ile Ser Phe Asn Leu Thr Thr Gly Ala Ser Gly Ala Phe Trp
450 455 460
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu Asn
465 470 475 480
Thr Ala Ile Ly~ Asn Val ~hr Tyr Cy3 Asn Ser His Ile Asn Asn Ile
485 490 495
Lys Cyq Ser Gln Leu Thr Ala Asn Leu A~n A~n Gly Phe Tyr Pro Val
500 505 510
Ala Ser Ser Glu Val Gly Phe Val Asn Lys Ser Val Val Leu Leu Pro 515 520 52S
Ser Phe ~he Thr His Thr Ala Val A~n Ile Thr Ile Asp Leu Gly Met
530 535 540
LyY Leu Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu Ser A~n Ile
545 550 555 560
WO 93/23422 PCT'/US93/04384
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148
Thr Leu Pro Met Gln Aqp Asn Asn Thr Asp Val ~yr Cys Ile Arg Ser
565 570 575
Asn Gln Phe Ser Val Tyr Val Pro Ser Thr Cys LyE~ Ser Ser Leu Trp
580 ' 585 S90
A~p Asn Ile Phe Asn Gln Asp Cys Thr Asp Val Leu Glu Ala Thr Ala
595 600 605
Val Ile Ly~ Thr Gly Thr Cys Pro Phe Ser Phe Asp Lys leu Asn Asn
610 615 620
Tyr Leu Thr Phe A~n Lye Phe Cy~ Leu Ser Leu Ser Pro Val Gly Ala
625 ~ 630 635 640
Aan Cys Ly8 Phe Asp Val Ala Ala Arg Thr Ary ~hr Asn Glu Gln Val ~;
645 650 655
Val Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly A~;p A~n Ile Val Gly
660 ~ ~665 670
Val Pro Ses A~p A~n Ser Gly Leu Hi~ A~p Leu Ser Val Leu His Leu
675 680 685
A_p Ser Cy8 Thr A8p Tyr Asn Ilè Tyr Gly Arg Thr Gly Val Gly I le
~690 695 700
Ile Arg Arg Thr A8n Ser Th Leu~Leu Ser Gly Leu Tyr Tyr Thr Ser
705~ 7~10 ~ ~ ~715 720
5` ~ Leu~ S~r~ Gly~Asp Leu Leu Gly Phe Ly~ Aan Val Ser A3p Gly Val Ile
7Z5~- ~ 730 735
Tyr~ 5er~Val Thr~ Pro~Cy8 Asp Val Ser Ala Gln Ala Ala Val Ile Asp
740 ~ 7 q 5 750
Gly;Ala~ILe Val~Cly Ala M-t~ Thr S-r Il- A-n~ S-r Glu Leu ~Leu Gly
L-u~Thr HL~ Trp Thr~Thr Thr~Pro A~n Phe~ Tyr Tyr Tyr Ser Ile Tyr
A~n ~TYr Thr~Ser~ Glu~Arg~ Thr ~Arg Cly Thr~Ala~ sp Ser A8n Asp
7~8~5~ 790 ~ ?ss ~ 800
Y~l~ABp~cy~ Gl- P~o V~ Thr Tyr S r~A--n~ Gly Val Cy Ly8
A~sl Gly Ala Leu Val Phe~Ile A~n Val Thr Hi8 Ser Alap Gly Asp Val ~i
820 ~ 825 B30 :~
Gln Pro Ile Scr Thr Gly A~n Val Thr Ile Pro Thr Asn Phe Thr Ile
835 840 845
Ser Val Gln Val GlU Tyr Met aln Val Tyr Thr Thr Pro Val Ser Ile
8:SO ~ 855 860
Asp~ Cys~ ~Ala l~g Tyr Val~ cys~AEln Gly As~n Pro Arg Cy8 Asn Lys Leu
:865 ~ 870 ~ 875 880
u Thr Cln~Tyr Val S-r Ala Cya Gln Thr Tl- Clu Gln Ala Leu Ala
88S 890 895
WO 93/23422 2 1 3 1 ~ 9 8 PCI/US93/04384
149
Met Gly Ala Arg Leu Glu Asn Met Glu Val A~p Ser Met Leu Phe Val
900 9~5 910
Ser Glu Asn Ala Leu Lya Leu Ala Ser Val Glu Ala Phe A~n Ser Thr
915 920 925
. Glu A~n Leu A~p Pro Ile Tyr Lya Glu Trp Pro Asn Ile Gly Gly Ser
930 935 940
Trp Leu Gly Gly Leu Lys Asp Ile Leu Pro 5er His Asn Ser Lys Arg
945 g~o 955 960
Lys Tyr Arg Ser Ala Ile Glu hsp Leu Leu Phe Asp Lys Val Val Thr
965 970 975 -
Ser Gly Leu Gly Thr Val ABP Glu Asp Tyr Ly~ Arg Cy~ Thr Gly Gly
980 985 99o
Tyr Asp Ile Ala A~p Leu Yal Cys Ala Gln Tyr Tyr Asn Gly Ile Met
ggs looo 1005
Val Leu Pro Gly Val Ala Asn A3p Asp Lys Met Thr Met Tyr Thr Ala
1010 ~ 1015 1020
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
1025 1030 1035 1040
Ala Ile Pro Phe Ala Val Ala Val Gln Ala Arg Leu Asn Tyr Val Ala
1045 1050 1055
Leu Gln Thr A~p Val Leu A~n Ly~ Asn Gln Gln Ile Leu Ala A~n Ala
1060 1065 1070 .
Phe A~n Gln Ala Ile Gly Asn Ile Thr Gln Ala Phe Gly Ly~ Val Asn
: 1075 1080 1085
Asp Ala Ile ~i~ Gln Thr Ser Ly~ Gly Leu Ala Thr Val Ala Ly~ Ala
logo 1095 lloo
Leu Ala Ly~ Val G}n Asp Val Val A~n Thr Gln Gly Gln Ala Leu Ser
~1105 1110 1115 1120
~His Leu Thr Val Gln Leu Gln Asn A3n Phe Gln Ala Ile Ser Ser Ser
1125 1130 1135
Ile Ser Asp Ile ~yr Asn Arg Leu A3p Glu Leu Ser Ala A3p Ala Gln
1140 1145 1150
Val A~p Arg Leu Ile Thr Gly Arg Leu Thr Ala Leu Asn Ala Phe Val
1155 1160 1165
Ser Gln Thr Leu Thr Arg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu
1170 1175 1180
Ala LYB ABP Ly~ Val A~n Glu CYE Val Arg 5er Gln Sor Gln Arg Phe
1185 llgO 1195 1200
Gly Phe Cy~ Gly A~n Gly Thr H1~ Leu Phe Ser Leu Ala Asn Ala Ala
1205 1210 1215
Pro A~n Gly Met Ile Phe Phe Hi~ Thr Val Leu Leu Pro Thr Ala Tyr
1220 1~25 1230
WO 93/23422 PCl'/US93/04384 .
~ L 3 ~ 8 9
i~
I
150 ' '
Glu Thr Val Thr Ala Trp Pro Gly Ile Cy_ Ala Ser Asp Gly Asp Arg
1235 1240 1245
Thr Phe Gly Leu Val Val Ly~ A~p Val Gln Leu Thr Leu Phe Arg Aqn ;:
1250 1255 1260
Leu A~p Asp Lys Phe ~yr Leu Thr Pro Arg Thr Met Tyr Gln Pro Arg
1265 1270 1275 1280
Ala Ala Thr Ser Ser Asp Phe Val Cln Ile Glu Gly Cy~ A3p Val Leu
: 1285 : 1290 1295
Phe Val Aun Ala Thr Val Ile Asp Leu Pro Ser Ile Ile Pro Aqp Tyr
1300 1305 1310 ..
Ile~Asp Ile~Asn~G~ln Thr Val Gln Asp }Le Leu GIu Aqn Tyr Arg Pro
- ~ ~ 1315 ~ 1320 1325
~ ~ .
Asn Trp~Thr Val Pro Glu Leu Thr Leu Asp Ile Phe Asn Ala Thr Tyr
1330 ~ 1335 ~ ~ : 1340
Leu Asn Leu Thr Gly Glu Ile Asp A~p Leu Glu Phe Arg Ser Glu Lys
1345 : ~ 1350 : 1355 i 1360 , -~
~L-u~Hi~ A~n~Thr~Thr V-l Glu ~eu Ala~Ile Leu~ A-p Asn Ile Asn
Asn Thr~Leu~Val A8n~Leu~Glu~Trp~eu A~n Arg Ile Glu Thr: ~yr Val
3~80~ 1385 : ~ ~ 1390 l'.
Eys Trp~Pro~Trp-Tyr~Val~rp ~Leu Leiu Ile Gly Leu Val Val Ile Phe !:''
1395-~ : : : 1400~ : 1405
Ile~:Pro-~u ~eu~L0 ~Ph-~Cys:~Cy~ Cy- Ser ~Thr Gly Cy8 Cy8 Gly
4 ~ l435 l440
61n~Ph-~Glu~A-n~Tyri~ lu Pro~ Clu~Ly Val Hl-~V~l HL8 ~ ,:
J~ IN 0 ~ TTON~ ~ ~S ~ ID NO-48
no ~-acid~
~S ~ :8) ~: TYPB: amino: ~acid
D:) q~OPO~OGY~s ~unknown:
ll) MOI.ECUI.E TYPÉI~ prot-Ln ~
l) SEQVENCE DE8CRIPTION:~8EQ:~ID NO:48s
Met ~ Val L~u~ Val Thr~Cy~Leu Leu Leu Leu ~ Ser Tyr H$3 Thr ,~"
: Vai Ser~ ~S~r.~Thr~: Ser: A~n: A~n A~p :Cys: Arg Gln: Val Asn Val Thr Gln E
y;~ 20~ 25 :~ : 30: .
L-u Ala Gly~ Glu A-n~L ~le Arg A-p Pb- L-u Phe Gln S-r Phe
W O 93/23422 2 1 ~ ~ ~ 9 8 PCT/US93/04384
151
Ly~ Glu Glu Gly Ile Val Val Val Gly Gly Tyr Tyr Pro Thr Glu Val
Trp Tyr Asn Cys Ser Arg Thr Ala Thr Thr Thr Ala Tyr Glu Tyr Phe
6~ 70 75 80
sn Asn Ile Hi~ Ala Phe Tyr Phe A~p Met Glu Ala Met Glu Asn Sex
g5
hr Gly A~n Ala Arg Gly Ly~ Pro Leu Leu Phe Hi~ Val Hi~ Gly Gl~
100 105 110
Pro Val Ser Ile Ile Ile Tyr Ile Ser Ala Tyr Gly Asp Asp Val Gln
Gln Arg Pro L~u Leu Glu His Gly Leu Leu Cy5 IIe Thr Lys Asn Arg
Asn Ile ABP Tyr Asn Thr Phe Thr Ser Asn Gln Trp A~p Ser Ile Cys
145 150 155 160
hr Gly A3n A~p Arg Lys Ile Pro Phe Ser Val Ile Pro Arg Asp Asn
165 170 175
ly Thr ~ys Ile Tyr Gly Leu Glu Trp Asn Asp Glu Phe Val Thr Ala
180 185 190
Tyr Ile Ser Gly Arg Ser Tyr Asn Trp Asn Ile A~n Asn Asn Trp Phe
195 200 205
A~n Asn Val Thr ~eu Leu Tyr Ser Arg Ser Ser Thr Ala Thr Trp Glu
210 215 220
Tyr Ser Ala A}a Tyr Val Tyr Gln Gly Val Ser Asn Phe Thr Tyr Tyr
225 230 235 240
ys Leu Asn Asn Thr Asn Gly Leu Lys Thr Tyr Glu Phe Cy3 Glu Asp
245 250 2S5
yr Glu Tyr Cys Thr Gly Tyr Ala Thr A~n Val Phe Ala Pro Thr Val
260 265 270
Gly Gly Tyr Ile Pro Asp Gly Phe Ser Phe Asn Asn Trp Phe Leu Leu
275 280 285
Thr Asn Ser Ser Thr Phe Val Ser Gly Asg Phe Val Thr Asn Gln Pro
290 295 300
Leu Leu Val Asn Cy~ Leu Trp Pro Val Pro Ser Phe Gly Val Ala Ala
305 310 315 320
Gln Glu Phe Cys Phe Glu Gly Ala Gln Phe Ser Gln Cys Ser Gly Val
325 330 335
er Leu A~n Agn Thr Val Anp Yal Ile Arg Phe A~n Leu Asn Phe Thr
340 345 350
Ala Asp Val Gln Ser Gly Met Gly Ala Thr Yal Phe Ser Leu Asn Thr
355 360 365
Thr Gly Gly Val Ile ~eu Glu Yal Ser Cyg Tyr Asn Asp Thr Val Ser
370 375 380
WO 93~23422 PCr/US93/Q4384
152 -
Glu Ser Ser Phe Tyr Ser Tyr Gly Glu Ile Pro Phe Gly Ile Thr Asp
385 3~0 395 400
Gly Pro Arg Tyr Cy~ Tyr Val Leu Tyr A~n Gly Thr Ala Leu Ly~ Tyr
4~5 410 415
Leu Gly Thr Leu Pro Pro Ser Val Ly~ Glu Ile Ala Ile Ser Lys Trp
420 425 430
Gly His Phe Tyr Ile A~n Gly Tyr Asn Phe ~he Ser Thr Phe Pro Ile
435 440 445 :
Asp Cys Ile Ser Phe Asn Leu Thr Thr Gly A~p Ser Gly Ala Phe Trp
450 455 4~0
Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val ~lu Asn
465 470 475 480
Thr Ala Ile Lya Lya Val Thr Tyr Cya Asn Ser Hi~ Ile Asn Asn Ile
485 490 495
Ly~ Cy~ Ser Gln Leu Thr Ala A~n Leu Asn Aan Gly Phe Tyr Pro Val
500 505 510 `~:
A}a Ser Ser Glu Val Gly Leu Val Asn Ly~ Ser Val Val Leu Leu Pro
515 520 525
Ile Phe Phe Ala Hi~ Thr Ala Ile Asn Ile Thr Ile Asp Leu Gly Met ~:.
530 : 535 540
Ly~ Arg Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu Ser A~n Ile
545~ 55~ 555 560
Thr Leu Pro Met Gln A~p Asn A~n Thr A p Val Tyr Cys Ile Arg Ser
- : Asn:Gln Phe Ser: Val Tyr Val His Ser Ile Cys Ly~ Ser Ser Leu ~rp
5 80 5 8 5 5 9 0
Asp~A5n Ile Phe A~n Gln Glu Cy8 Thr Asp Val Leu Asp Ala Thr Ala
~ 595 ~ 600 605
:~ ~ Val Ile Lys Thr Gly Thr Cys Pro Phe Ser Phe Asp Lys Leu Asn Asn ~-
~ 615 620
Tyr Leu Thr Phe A~n Lys Phe Cy~ Leu Ser Leu Ser Pro Val Gly Ala ..
625 ~ 630 ~ 635 640
Asn Cy~ Ly~ Phe A8p Val Ala Ala Arg Thr Arg Thr A~n Glu Gln Val `~
645 650 655 ~ :
1, , ~ , .
Val Arg Ser Leu Tyr Val Ile Tyr Glu Glu Gly A~p Asn Ile Val Gly ~.
660 665 670
Val Pro Ser Asp Asn Ser Gly Leu Hi~ A5p Leu Ser Val Leu His Leu i~^~
675 680 685 ~;
Asp Ser Cy~ Thr Glu Tyr Asn Ile Tyr Gly Arg Thr Gly Val Gly Ile l:~
690 69S 700
I le Arg Gln Thr Asn Ser Thr Leu Leu S2r Gly Leu Tyr Tyr Thr Ser
705 710 715 720
WO 93/2342~ PCI/US93/04384
- 213~89~
153
Leu Ser Gly Asp Leu Leu Gly Phe Ly~ A~n Val Ser ~sp Gly Val Ile
725 730 735
Tyr Ser Val Thr Pro Cy~ Asp Val Ser Ala ~-ln Ala Ala Val Ile Asp
740 74S 750
Gly Ala Ile Val Gly Ala Met Thr Ser Ile Asn Ser Glu Leu Leu Gly
755 ?60 765
Leu Ly~ Hia Trp Thr Thr Thr Pro Asn Phe Tyr Tyr Tyr Ser Ile Tyr
770 775 780
Asn Tyr Thr Aan Glu Arg Thr Arg Gly Thr Ala Ile A9p Ser Asn A3p
785 790 795 800
Val Asp Cys Glu Pro Ile Ile Thr Tyr Ser Asn Ile Gly Val Cys Lys
805 810 815
Asn Gly Ala Leu Val Phe Ile Asn Val Thr His Ser Asp Gly Asp Val
820 - a25 830
Gln Pro Ile Ser Thr Gly Thr Val Thr Ile Pro Thr Asn Phe Thr Ile
8-~5 840 ~45
Ser Val Gln Val Glu Tyr Ile Gln Val Tyr Thr Thr Pro Val Ser Ile
850 85S 860
Asp Cys Ala Arg Tyr Val Cy~ Asn Gly Asn Pro Arg Cy9 Ao~n Lys Leu
865 ~ 870 875 880
Leu Thr ~ln Tyr Val Ser Ala Cy~ Gln Thr Ile Glu Gln Ala Leu Ala
885 890 895
Met Gly Ala Arg Leu Glu Asn Met Glu Val A~p Ser Met Leu Phe Val
900 905 910
Ser Glu Asn Ala Leu Ly~ Leu Ala Ser Val Glu Ala Phe Asn Ser Thr
g15 920 925
Glu A~n Leu A3p Pro Ile Tyr Ly~ Glu Trp Pro Ser Ile Gly Gly Ser
930 935 940
Trp Leu Gly Gly keu Ly~ A~p Ile Leu Pro Ser Hi~ Asn Ser Lys Arg
94S 950 g55 960
Lys Tyx Gly Ser Ala Ile Glu A~p Leu ~eu Phe Asp Lys Val Val Thr
97 975
Ser Gly Leu Gly Thr Val ABP Glu Asp Tyr Lya Arg Cy8 Thr Gly Gly
980 985 990
Tyr Aop Ile Ala A3p Leu Val Cys Ala Gln Tyr Tyr A~n Gly Ile Met
995 1000 1005
Val Leu Pro Gly Val Ala A~n Ala Asp Lys Met Thr Met Tyr Thr Ala
lO10 1015 1020
Ser Leu Ala Gly Gly Ile Thr Leu Gly Ala Leu Gly Gly Gly Ala Val
1025 1030 103S 1040
Ala Ile Pro Phe Ala Val Ala Val Gln Ala Arg Leu A3n Tyr Val Ala
1045 lOS0 1055
WO 93/23422 PCI'/US93/W384
?, ~ 3 ~
154
Leu Gln Thr Asp Val Leu Asn Lys Asn Gln Gln Ile Leu Ala Aqn Ala
1060 1065 1070
Phe A3n Gln Ala Ile Gly Asn Ile Thr Gln Ala Phe Gly Ly~ Val Asn
107S 1080 1085
Asp Ala Ile ~is Gln Thr Ser Gln Gly Leu Ala Thr Val Ala Ly~ Ala
1090 1095 1100
Leu Ala Ly~ Val Gln Asp Val Val Asn Thr Gln Gly Gln Ala Leu Ser
1105 1110 1115 1120
i~ Leu Thr Val Gln Leu Gln A~n Asn Phe Gln Ala Ile Ser Ser Ser
1125 1130 1135
le Ser A~p Ile Tyr A~n Arg Leu Asp Glu Leu Ser Ala Asp Ala Gln
1140 1145 1150
Val A~p Arg Leu Ile Thr Gly Arg Leu Thr Ala Leu A3n Ala Phe Val
1155 1160 1165
Ser Gln Thr Leu Thr Arg Gln Ala Glu Val Arg Ala Ser Arg Gln Leu
1170 1175 1180
Ala Ly~ Asp Lys Val Asn Glu Cy~ Val Ar~ Ser Gln Ser Gln Arg Phe
1185 1190 1195 1200
ly Phe Cys Gly A~n Gly Thr Hiq Leu Phe Ser Leu Ala Asn Ala Ala
1205 1210 1215
ro Asn Gly Met Ile Phe Phe His Thr Val Leu Leu Pro Thr Ala Tyr
1220 1225 1230
Glu Thr Val Thr Ala Trp Ser Gly Ile Cy8 Ala Ser Asp Gly Asp Arg
1235 1240 1245
Thr Phe Gly Leu Val Val LYQ Asp Val Gln Leu Thr Leu Phe Arg Asn
1250 1255 1260
Leu Aap Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln Pro Arg
1265 1270 1275 1280
al Ala Thr Ser Ser Asp Phe Val Gln Ile Glu Gly Cy8 Asp Val Leu
1285 1290 1295
he Yal A3n Ala Thr Val Ile Asp Leu Pro Ser Ile Ile Pro A-~p Tyr
130~ 1305 ~310
Ile Asp Ile A~n Gln Thr Yal Gln Asp Ile Leu Glu Asn Tyr Arg Pro
1315 1320 1325
A~n Trp Thr Val Pro Glu Phe Thr Leu Aap Ile Phe A~n Ala Thr Tyr
1330 133~ 1340
Leu A~n Leu Thr Gly Glu Ile Asp Asp Leu Glu Phe Arg Ser Glu Lys
1345 1350 13SS 1360
eu Hi~ Asn Thr Thr Val Glu Leu Ala Ile Leu Ile Asp Thr Ile Asn
1365 1370 1375
sn Thr Leu Val A~n Leu Glu Trp Leu A~n Arg Ile Glu Thr Tyr Val
1380 1385 1390
WO 93/23422 PC~/US93~04384
155
Lys Trp Pro Trp Tyr Val Trp Leu Leu Ile Gly Leu Val Yal Val Phe
13g5 1400 1405
Cys Ile Pro Leu Leu Leu Phe Cys Cy8 Phe Ser Thr Gly Cys Cys Gly
1410 1415 1420
Cys Ile Gly Cys Leu Gly Ser Cys Cy~ His Ser Ile Cy~ Ser Arg Arg
1425 1430 ~435 1440
Gln Phe Glu Tyr Tyr Glu Pro Ile Glu Ly~ Val His Val His
1445 1450
