Note: Descriptions are shown in the official language in which they were submitted.
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W O 97/00079 PCTrUS96/10043
P--S~T-~CTIN LIGA~nDS
A~nD R~T.~n MOLEClnLES l~ND ~EI~HODS
St~tement as to Federally S~onsored Research
sThis invention wa3 made with Government ~o~L
under NIH grant DK43031, and the Government therefore has
certain rights in this invention.
Bac~Lou~ld of the Invention
This invention relates to P-selectin ligand
10 molecules, DNAs, and uses thereof.
P-selectin i8 an integral membrane C--type lectin
found within the Weibel-Palade bodies of endo~h~~
cells and the alpha granules of platelets (McEver et
al., J. Clin. Invest., 84:92-99, 1989; Bonfanti et al.,
15 Blood, 73:1109-1112, 1989; Hsu-Lin et al., J. Biol.
Chem., 259:9121-9126, 1984; St~h~rg et al., J. Cell
Biol., 101:880-886, 1985). Its translocation to the
plasma membrane can be in~llce~ by thrombin, histamine and
other -~Ators released by mast cell activation,
20 complement C5b-9 complex or C5a fragment, ~e~uxides, and
QY;~ed low-density lipoprotein (Hsu-Lin et al-, J.
Biol. Chem., 259:9121-9126, 1984; St~nhe~g et al., J.
Cell Biol., 101:880-886, 1985; Hattori et al., J. Biol.
Chem., 264:9053-9060, 1989; Kubes and Kanwar, J.
25 Immunol., 152:3570-2577, 1994; Thorlacius et al.,
Biochem. Biophys. Res. Communications, 203:1043-1049,
1994; Foreman et al., J. Clin. Invest., 94:1147-1155,
1994; Patel et al., J. Cell Biol., 112:749-759, 1991;
Lehr et al., Laboratory Invest., 71:380-386, 1994;
30 Gebuhrer et al., Biochem. J., 306:293-298, 1995). Once
displayed on the cell surface, P-selectin supports the
attachment of myelomonocytes to platelets or endothelial
cells (Larsen et al., Cell, 59:305-312, 1989; ~A hll~ger
and Mc~ver, Blood, 75:550-554 1990; Geng et al., Nature,
35 343:757-760, 1990; Gamble et al., Science, 249:414-417,
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W O 97/00079 PCTrUS96/10043
1990). In the latter setting, its appearance heralds an
underlying ~ insult and supports the initial ~tep ~n
leukocyte extravasation, the rolling of neuLro~hils along
the postcapillary venule wall (Lawrence and Springer,
5 Cell, 65:859-873, 1991). Mice which are homozygously
deficient for the P-selectin structural gene exhibit
decreased leukocyte rolling and show delayed recruitment
of granulocytes to sites of experimentally ;n~llce~
inflammation (Mayadas et al., CQ11~ 74:541-554, 1993).
10 Generally, the mediators which ;n~llc~ P-selectin
expression are involved in 8ign;-1ing trauma or wollnrlin~
One of the first reço~ni~e~ res~n~~- to ~ trauma is
mast cell activation, which is accompanied by release of
histamine, serotonin, and other diffusible mediators.
15 Other ~_ -~ events include thrombus formation at sites
of vA~ A~ lu~Lu e and complement alternative pathway
engagement by foreign bodies. P-selectin ~x~e~sion is
~n~ll~e~ by signals generated in each of these contexts.
Al~hs~yh induction of P-selectin mediated neu~.o~hil
20 rolling has been thought to be an inevitable con~ yuence
of surgical intervention, cromolyn, an agent which blocks
mast cell degranulation, has been shown to prevent such
rolling, thereby providing an elegant ~r nctration of
the role of the mast cell as the link between trauma and
25 extravasation (Kubes and Kanwar, J. Immunol., 152:3570-
3577, 1994).
Sut~ary of the Invention
In a first aspect, the invention features an
organic molecule to which there is covalently hQn~e~ a
30 sialyl-LeX dete- ;nAnt and a sulfated determinant, at
least one of these determinants being positioned at a
non-naturally occurring site on the molecule.
In a ~ecQ~A aspect, the invention features a P-
selectin ligand selected from the group consisting
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~ ~?~tially of: (a) amino acids 21-57 of Fig. 8A and (b)
amino acids 38-57 of Fig. 8A.
In a third aspect, the invention features fusion
proteins that include a P-selectin ligand joined to an
5 antibody ~t ~in (for example, one or more of the hinge,
CH2, ~nd CH3 domains).
In related aspects, the invention features
purified nucleic acid ~co~;n~ ~ protein cont~n;ng sites
for the attachment of a sialyl-LeX determinan~ and a
10 sulfated determinant, at least one of these determinants
being positioned at a non-naturally o~ul.ing site on the
protein; purified nucleic acid ~nco~ing any one of the P-
selectin ligands of the invention; purified nucleic acid
~nco~ng a P-selectin-antibody ~usion protein; and
15 vector and recombinant cells including any of these
nucleic acids. Also included in the invention is the use
of P-selectin ligands or organic molecules bearing such
ligands (if desired, in combination with other proteins
such as antibody or ~l-acid gly~u~Lein domains) in the
20 manufacture of a medicament for the treatment of any of
the conditions described below.
In another related aspect, the invention features
a method of inhibiting the b~ n~ i ng of a cell bearing a P-
selectin protein to a molecule or cell bearing a sialyl-
25 LeX determinant and a sulfated determinant. The methodinvolves contacting the P-selectin protein-bearing cell
with either an organic molecule bearing sialyl-LeX and
~ulfated determinants, at least one of these determinants
being positioned at a non-naturally occurring site on the
30 molecule; a P-selectin-antibody fusion protein; or any of
the P-selectin ligands of the invention.
In another related aspect, the invention features
a method of re~llc; ng inflammation in a mammal involving
administering to the patient a therapeutically-effective
35 amount of either an organic molecule bearing sialyl-LeX
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W O 97/00079 PCT~US96/10043
and sulfated determinants, at least one of these
determinants being positioned at a non-naturally
occurring site on the molecule; a P-selectin-antibody
fusion protein; or any one of the P-selectin ligands of
5 the invention.
In yet another related aspect, the invention
features a method of reducing or protecting a mammal
against any extravasation-~r~p~n~nt adverse reaction
(including, without limitation, extravasation-r~en~nt
10 organ damage and/or clotting associated with adult
respiratory distress ~y~.d~me, glomerular nephritis, and
ischemic myocardial injury). The ~ethod involves
administering to the mammal a therapeutically-effective
amount of either an organic molecule to which there is
15 covalently hQn~ a sialyl-LeX and a sulfated
determinant, at least one of these determinants being
positioned at a non-naturally occurring site on the
molecule; a P-selectin-antibody fusion protein; or any ~f
the P-selectin ligands of the invention.
In a final aspect, the invention features a met~od
of reducing or protecting a mammal against an adver~e
immune reaction, involving administQring to the mammal
therapeutically-effective amount of either an organic
molecule to which there is covalently hQ~ a sialyl-LeX
25 and a sulfated determinant, at least one of these
determinants being positioned at a non-naturally
occurring site on the molecule; a P-selectin-antibody
fusion protein; or any of the P-selectin ligands of the
invention. Preferably, this method involves treating the
- -1 for an adverse immune reaction ~hich is in~l~ce~ by
a microbial factor. such microbial factors include,
without limitation, gram-negative bacteria
lipopolysaccharides (LPS), peptidoglycans from gram-
positive orgAni~ , -nnA~ from fungal cell walls,
35 polysaccharides, extracellular e~:ymes (e.g.,
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W O 97/00079 PCTAUS96/10043
~L~e~,~e~inAre) and ~Qytn~: (e.g., toxic shock entero~Qyinc
of staphyloro~ci). In other preferred emho~;ments~ the
method involves treating a mammal for any adverse immune
reaction which is i~Allce~ by a host factor. Such host
5 factor~ include, without limitation, metabolites of
compl ~ent, lr; n;n, and coagulation systems, factors
relea ed from stimulated cells (e.g., cytQkin~c such as
interleukin 1 (IL-1) and tumor necrosis factor-~ (TNF)),
enzym~ and Qy~ t~ from polymorrhon-~clear leukocytes
(PMNs), vasopeptides (e.g., histamine), and products of
the metabolism of ar~h;~o~;c acid. In other preferred
embodiments, the adverse immune reaction is ;nAllce~ by
recombinant TNF-~ or is in~ll~e~ by recombinant IL-l. In
yet other preferred ~ ho~; ments, the adverse immune
15 reaction is septic shock or is septicemia.
In preferred emho~l; ments of each of the above
aspects, the organic molecule or protein also inhibits
the b~nAing of a cell bearing an E-selectin (ELAM-1)
protein to a molecule or cell bearing a sialyl-LeX
20 dete- ;n~nt and thus inhibits E-selectin-mediated
inflammation, extravasation-A~r~nAent adverse reactions,
and adverse immune reactions; the sialyl-LeX and sulfated
determinants are present on a P-selectin ligand
consisting essentially of: amino acids 21-S7 of Fig. 8A
(for example, amino acids 38-57 of Fig. 8A); the sialyl-
LeX determinant is N-linked or 0-l;nkeA; the molecule or
protein contains multiple sialyl-LeX and/or multiple
sulfated determinants; the organic molecule is a protein
(for example, an antibody (for example, IgG or IgM), ~1-
30 acid glycoprotein (AGP), or an antibody fusion protein(for example, an AGP-antibody fusion protein), the
protein is an antibody, AGP, or an antibody fusion
protein (for example, an AGP-antibody fusion protein) to
which any of the P-selectin ligands described herein is
35 appended (for example, at the pro~in's amino-terminus);
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PCI/U~ 96 ~ Oa~3
IPEA~US 1 ~ J A N 1997
the antibody or antibody fusion protein (for example, the
AGP-antibody ~usion protein) includes, as an antibody
portion, an IgG1 CH2, CH3, and/or hinge domain; the
antibody, AGP, or antibody fusion protein includes one or
5 more of the N-linked glycan addition sites of ~l-acid
glycoprotein; the antibody portion o~ the molecule bears
one or more non-naturally occurring sialyl-LeX
determinants; the sialyl-LeX determinant interferes with
the antibody's ability to fix complement or bind an Fc
10 receptor (for example, due to a sialyl-~eX determinant
attached to one or more of amino acids 274, 287, or 322
'~ of the sequence shown in Fig. lOA-E); and the organic
., "~ ..
molecule is soluble.
By a "P-selectin ligand", as used herein, is meant
15 any amlno acid sequence capable of mediating an
interaction with the P-selectin receptor and includes
those proteins referred to as P-selectin counter-
receptors. Preferable P-selectin ligands include,
without limitation, amino acids 21-57, and more
20 preferably amino acids 38-57, of Fig. 8A. P-selectin
ligands according to the invention may be used in
conjunction with additional protein domains (for example,
antibody domains) to produce fusion proteins useful in
the invention.
By ~non-naturally occurring" is meant a sialyl-LeX
or sulfated determinant that is not one which is
naturàlly bound to the molecule at that amino acid
location.
By ''inflammation" is meant a pathologic process
30 consisting of cytologic and histologic reactions that
occur in the affected blood vessels and adjacent tissues
in response to an injury or abnormal stimulation caused
by a physical, chemical, or biologic agent.
Inflammation, as used herein, includes any acute
inflammatory response (~or example, during or following
AMEI\IDED S~lEET
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W O 97/00079 PCTrUS96/10043
adult respiratory di~ ~Le~;S syndrome or ischemic
myocardial injury) as well as any chronic inflammatory
r~ron~e (for example, rheumatoid arthritis, psoriasi~,
or pemphigus vulgaris).
S By "purified nucleic acid" is meant DNA that is
free of the genes which, in the naturally-occurring
genome of the organi~m from which the DNA of the
invention is derived, flank the gene. The term therefore
include~, for example, a recombinant DNA which i8
10 incorporated into a vector; into an autonomously
replicating plasmid or virus; or into the genomic DNA of
a pr4karyote or eukaryote; or which exists as a separate
molecule (e.g., a cDNA or a genomic or cDNA fragment
prsAl~c~A by PCR or ~ LL iction enAon~lclease digestion)
15 ;nA~p~nA~nt of other seqU~nc~C. It also includes a
recombinant DNA which is part of a hybrid gene ~n~oA i n~
additional polypeptide ~equence.
By "N-linked" is meant hQ~A~A to the amide
nitrogen of an asparagine residue of a protein.
By ~O-link~" is mQant honA~A to the hydroxyl-
group oxygen of a serine, threQn;n~, or hyd~ox~lysine
re~idue of a protein.
By an "extravasation-A~r~nA~nt adverse reaction"
is meant any reaction which is detrimental to the host
25 and which results directly or indirectly from the
ina~L OPL iate attachment of neutrophils to endothelium at
or proximate to a site of inflammation, ti~ ~ damage, or
thrombus formation and results in migration of those
neuL.ophils into the at~A~h~ blood vessel or organ.
30 Organs which may be affected by such damage include,
without limitation, the heart, lungs, and kidneys.
By an "adverse immune reaction" is meant any
reaction mediated by an immune cell (i.e., any B cell, T
cell, monocyte/macrophage, natural killer cell, mast
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- 8 -
cell, basophil, or granulocyte) and which is detrimental
to the host.
ne~ Descr;~tion
The drawings will first be briefly described.
Fig. lA is a schematic representation of the
structure of the PSGL-1 deletion mutantD. Systematic
deletion of the ectodomain of PSGL-1 was accomplished
with ~v~l~ional PCR methods. A representative
10-residue repeat (stippled; SEQ ID N0:1) and the
10 transmembrane domain (hatched) are illustrated. Fig. lB
i8 histogram which represents P-selectin b; n~ i n~ activity
of transfected COS cells expressing the deletions shown
in Fig. lA. 51Cr-labeled cells were allowed to adhere to
soluble P-Delectin adsorbed to microtiter wells. The
15 cells were washed, the bound cells were then lysed, and
51Cr levels were counted. Deletion constructs were
illL.~ ce~ into cells either in the Ah~n~e (bar 2) or
presen~e (remaining 7 bars) of the human FTVII
fucosyltransferase.
Fig. 2A is a schematic representation of chimeras
of PSGL-1 and CD43. The membrane proximal extracellular
domain, transmembrane, and intracellular domains of
PSGL-1 were replaced with the cognate se~l~n~e~ of CD43.
The resulting molecule lacks cyst~n~c and thus cannot
25 form a ~ fide linke~ dimer. Fig. 2B is a histogram
~e~eDenting P-selectin b;n~;n~ activity of transfected
CoS cells expressing the chimeras shown in Fig. 2A.
n, cotransfection with the human FTVII
fucosyltransferase.
Fig. 3A is a schematic representation of chimeric
mucins bearing the PSGL-l apical domain appended to
intact or truncated mucin C-termini. The PSGL-1
N-terminus (stippled; SEQ ID N0:1) and the tr~n~ ~mbrane
(TM) domains (hatched) are illustrated. The sequence of
35 PSGL-l-NH2/CD43 "repeats" are represented by SEQ ID N0:2.
_
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PSGL-l was fused to the N-terminus of the predicted
mature CD34 and GlyCAM-1 molecules, and to the N-terminus
of the repeat region of CD43. Fig. 3B is a hi~togram
enting P-selectin bi nA i ng activity of transfected
5 CoS cells expressing the constructs shown in Fig. 3A.
h, human FTVII fucosyltransferase.
Fig. 4A is a schematic 1~ e~entation of PSGL
deletion mutants. The amino terminal domain was ~pre
to PSG~ molecules having varying numbers of the repeated
10 element. Fig. 4B is a histogram r e~r ~~ ~nting P-selectin
bi nA i n~ activity of transfected COS cells expressing the
chimeras illustrated in Fig. 4A.
Fig. 5 is a photograph of an autoradiogram of
mucin:immunogloh~ n fusion proteins labeled with
15 35S ~ fate and electrophoresed on an 8% denaturing
polyacrylamide gel under reducing conditions. Lane A,
supernatant of CDM8 transfected cells; Lane B,
cupernatant of cells transfected with Ig expression
vector (no mucin insert); Lane C, supernatant of cells
20 expressing PSGL-l:Ig; Lane D, supernatant of cells
expres~ing CD43:Ig; Lane E, supernatant of cells
expre~sing CD34:Ig; and Lane F, supernatant o~ cells
expre~sing GlyCAM-l:Ig.
Fig. 6A and Fig. 6B are histograms representing
25 bi n~ ~ ng to immobilized P- and E-selectin of COS cells
expressing PSGL-l with or without fucosyltransferase and
in the ~-en~e or Ah~~n~e of 10 mM NaC103. Fig. 6A is a
histogram representing binding of cells to P-selectin.
Fig. 6B is a ~to~am reprasenting bi n~; n~ of cells to
30 E-selectin.
Fig. 7 is a photograph of an autoradiogram of
PSGL-l:immunoglobulin fusion proteins labeled with
35S-sulfate in the pr~Fen~e or Ah-~n~e of 10 mM NaC103 and
electrophoresed on an 8% denaturing polyacrylamide gel
35 under reducing conditions. The photograph indicates that
, CA 0222462~ 1997-12-12
P~TIUS 96 / 1 ~ a 4 3
IP~AIU~ 1 4 J A N ~997
- 10 -
chlorate inhibits incorporation of 35S-sulfate into
soluble mucin chimeras. ~ane A, supernatant of CDM8
transfected cells in the absence of chlorate; Lane B,
supernatant of cells expressing PSGL-l:Ig in the absence
5 of chlorate; Lane C, supernatant of CDM8 in the presence
of chlorate; and Lane D, supernatant of cells expressing
PSGL-l:Ig in the presence of chlorate.
Fig. 8A is a listing of the sequence endpoints of
various PSGL-l deletion mutants (indicated by the arrows)
10 The uppermost sequence is SEQ ID NO:3; the middle
sequence is SEQ ID NO:13; the lowermost sequence i8 SEQ
ID NO:14. Fig. 8B is a histogram representing P-selectin
binding activity of transfected COS cells expressing the
deletion mutants having the endpoints shown in Fig. 8A.
Fig. 9A is a schematic diagram of the constructs
employed to measure the effect of appending wildtype and
mutant variants of PSGL-1 residues 38-57 to deleted
PSGL-1 or CD43. The inserted sequences are shown at
bottom left. Fig. 9B is a histogram representing
20 P-selectin binding activity of transfected COS cells
expressing the chimeras illustrated in Fig. 9A.
Fig. lOA-E are a listing of the nucleotide
- sequence (SEQ ID NO:8) encoding IgG1 (SEQ ID NO:9) and
mutations designed to create N-linked glycan addition
25 sites (SEQ ID NO:12).
Fig. llA-B illustrate the nucleotide sequence (SEQ
ID NO:10) and Fig. llC is the amino acid sequence (SEQ ID
NO:11) of an AGP-IgG1 fusion protein.
Fig. 12A is a schematic diagram of immunoglobulin
fusion proteins consisting of either intact PSGL-1 tSEQ
ID NO:4) or 20 residue peptides joined to the hinge, CH2,
and CH3 domains of human IgG1. Construct Y/F-hIgG bears
SEQ ID NO 5; construct T/AhIgG bears SEQ ID NO:6;
construct Y/F-T/A-hIgG bears SEQ ID NO:7. Fig. 12B is a
35 photograph of an 8~ polyacrylamide gel used to assess
A~ENDED SHEET
CA 02224625 1997-12-12
W O 97/00079 PCTAUS96/10043
in~o-~ ation of [35S]cysteine and methionine by the
fusion proteins shown in Fig. 12A following transfection
into COS cell~. Lane A, supernatant of cells transfected
with CDM8 control. Lane B, supernatant of cell~
5 transfected with PSGh-l-immunoglohlll in fusion protein.
Lane C, ~u~e~ ~-atant of cells transfected with WT-hIgG.
Lane D, supernatant of cells transfected with Y/F-hIgG.
~ane E, supernatant of cells transfected with T/A-hIgG.
Lane F, ~u~e~l~atant of cells transfected with Y/F-T/A-
10 hIgG. Fig. 12C is a photograph of an 8% polyacrylamidegel used to AC-eC~ inc~ ation of [35S] sulfate by the
fusion proteins shown in Fig. 12A following transfection
of COS cells. In addition, a ~vllL~ol fusion protein
bearing no amino-terminal addition was included (Lane B).
15 Lanes C through G correspond to Lanes B through F in Fig.
12B.
Fig. 13 is a bar graph of interacting HL-60 cells
per video-ca~L~Led field. The cells were infused into a
parallel plate flow chamber precoated with either P-
~electin-immunoglQh~ n chimera or a CD4-immunglQhlll~n
chimera col.L ~1. The cells were subjected to a shear
stress of 0. 75 dynes/cm2. Each bar represents the
average number of cells (tSEM) per field from eight
frames taken at 15 ~~con~ intervals. Cells rolling or
flowing appear a~ streaks on the video image. The bars
represent, from left to right: HL-60 cells rolling or
flowing over P-selectin-immunoglobin chimera, HL-60 cells
pretreated in a sulfate-free medium with 10 mM sodium
chlorate, and HL-60 cells flowing over CD4-immunoglobulin
30 chimera.
Sialyl-Lewis X (S3 alyl-LeX) and culfated
determinants were shown to interact with P-selectin and
facilitate bin~ing by the following experiments. These
examples are presented to illustrate, not limit, the
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- 12 -
invention. The methods used in the following experiments
will first be described.
Pr~uc~-~on of Solllhle P-Select~n
P-selectin and E-selectin Ig chimeras were
5 prepared by transient expression in COS cells of an
expression plasmid encoAin~ the lectin, EGF-related, an~
first two short con~~ncus repeat related domains of
P-selectin joined to the hinge, CH2, and CH3 domains of
human IgGl (Aruffo et al., EMB0 J., 6:3313-3316, 1991;
10 Walz et al., Science, 250:1132-1135, 1990). The PSGL-l
cDNA qo~i n~ sequence was ob~i n~ by PCR amplification of
an HL-60 cDNA library, and the sequence confirmed by DN~
sequencing. The co~i ng segment for the mature
extracellular, transmembrane, and intracellular domain
15 was inserted into an expression vector ba~ed on CDM8
which lacks the polyoma virus origin of replication and
contains the leader sequence for the CD5 antigen
positioned just ~-LLeam of the co~; n~ region for an
influenza hemagglutinin (flu) peptide (Field et al., Mol.
20 Cell. Biol. 8:2159-2165, 1988) epitope tag.
Co~ tion of PSG-T-1 Delet;ons
Amino terminal PSGL-1 deletion constructs were
prepared by PCR amplification using primers en~oAi n~ tha
desired endpoint of the deletion ~utant located
25 downstream of an XbaI site in frame two (~nco~ Leu
Asp). The resulting se~l~nce~ enco~e~ a polypeptide in
which the residues listed below i ~ tely followed the
aspartic acid (D) of the Xba site: A118, A128, A138,
A148, A158, A168, G178, A188, Al98, A208, A218, A228,
30 A238, A248, A258, and T268 of the PSGL-1 precursor. The
PCR fragments were then inserted in the CD5 leader flu
tag expression vector used for expression of the intact
PSGL-l. The flu tag terminates in an XbaI site in the
frame described above. Sequences at the flu tag junction
35 were verified, and expression was confirmed in CoS cells
CA 02224625 l997-l2-l2
W 097/00079 PCTnUS96/10043
- 13 -
by indirQct immunofluore~c~n~ microscopy and flow
cytometry. A series of internal deletions with an EcoRI
~ite at the ~ite of the daletion in frame one (oncoA~
glutamic acid phenylAlAnin~) was also prepared by first
5 creating deletion variants with amino termini (rQsidues
immediately following phenylAlAninQ tF] of the EcoRI site
aV~ ol~l;n~ to A118, A128, A138, A148, A158, A168,
G178, A188, A198, A208, A218, A228, A238, A248, and A258
of the peptide sequence of the precursor. To each of
10 these deleted variants was appen~ a flu-tagged amino-
terminal PSGL-1 domain en~in~ with an EcoRI site in the
glutamic acid phenyl~l ~ni n~ frame immediately do~..~L~eam
of PSGL--1 ~e~ ~or A117. The resulting constructs
contained deletions between A117 and the various
15 Qndpoints above.
Muc; n Dc ~in Tnterchanges
CD34, CD43, and GlyCAM-1 mucins were prepared for
addition of the PSGL-l amino-terminal domain by Apre~ i n~
an EcoRI site to either the mature amino terminus (CD34
20 or GlyCAM-l), or to the beginning of a region of
threonine/proline-rich repeats (CD43). As above, the
EcoRI site was in the frame glutamic acid pheny~ n~ne
(frame 1). The CD34 sequence began at residue F30 of the
precursor, the Gly-CAM-l at precursor Ll9, and the CD43
25 at precursor I135. To each of these was app~n~ the
flu-tagged PSG~-l domain terminating in EcoRI as above.
The amino terminus and repeat elements of PSGL-l were
app~n~e~ to the membrane proximal, transmembrane, and
intracellular domains of CD43 through an EcoRI site in
30 the glutamic acid phenylal ~n; ne frame positioned
immediately ~eam of the se~nc~ S225 of the CD43
precursor. The complementary fragment from PSGL-l
correspon~ to the amino-terminal residues of the
precur~or up to T267.
35 Fine Structllre Ma~ing of ~he ~ino-Ter~in~l Dom~Ln
CA 02224625 1997-12-12
W O 97/00079 PCT~US96/10043
~ 14 ~
A similar strategy was employed for the
construction of deletions in the amino-terminal domain,
in which PCR generated deletions were formed using
primers bearing an XbaI site in the leucine aspartic acid
5 frame (frame 2). Immediately downstream of the residues
~n~o~ ~ ng aspartic acid were the PSGL-1 se~l~ncefi
~oLLe~ ng to precursor R38, E58, P78, and A98. For
the definition of the amino-terminal domain, duplex
oligonucleotides were synthesized corre~pon~;n~ to the
10 residues between 38 and 57 with the indicated sequence
changes to mutate threon;ne or tyrosine residues to
~lAn;~ or phenylAlAnlne. All constructs were confirmed
by dideoxy sequencing.
Cell ~hesion AssaYs
Transfected cells were de~Ach~ from culture
h~C with 0.5 m~ EDTA in phosphate buffered ~ A1 in~
(PBS) 48 to 60 hours after transfection. The cells were
then loaded with 100 ~1 51CrO4 (1 mCi/ml; DuPont, Boston,
MA) in 0.9% NaCl plus 100 ml medium by inGllh~ting them ~t
20 37~C for 1 hour. TOAt1~1 cells were washed twice in PBS
and r~ cr~n~e~ in 0.2% BSA, 0.15 M NaCl, 3 mM CaC12.
Variation in labeling rate (counts incorporated per cell)
between cells prepared in parallel with the same batch of
labeled chromate was typically minimal. The labeled
25 cells were incllhAted in wells of 96-well microculture
plates which had been coated with affinity purified goat
anti-human IgG antibody (100 ~1 of 20 ~g/ml anti-human
IgG Fc (heavy chain specific) in PBS) for 2 hours in a
humid ch~mher at room temperature. After the plate was
30 washed twice with PBS, additional protein-b; n~; ng sitQs
were blocked by an overnight ;n~lhAtion with 200 ~1 3%
BSA in PBS. The plate was washed with PBS four times and
in~lhAted with 200 ~1 of fusion protein supernatants for
2 hours. Following three PBS washes and one additional
35 wash (in 0.2~ BSA, 0.15 M NaCl, 3 mM CaC12), 2 x 105
CA 02224625 1997-12-12
W O 97/00079 PCTrUS96/10043
cells/well (in 200 ~1 0.2% BSA, 0.15 M NaCl, 3 mM CaC12)
were ~e~ and allowed to bind for lS minutes at room
temperature while the plate rotated on a rotary platform
(80 rpm). The plate was washed three times by f~ n~
5 the wells with 200 ~1 0.15 M NaCl/3 mM CaC12 and then
inverting the plate. Adherent cells were lysed by the
addition of 200 ~1 2% SDS, and labeled chromate was
counted with a gamma ray spectrometer.
n~-f 1 llnrescence An;~ 1 VS i ~
Cells were prepared for cytometry by ;n~nh~tion
with the primary monoclonal antibody (a 1:200 dilution of
ascites or 5 ~g/ml of purified antibody is suitable) in
PBS con~ining 3% BSA for 30 to 45 minutes. The cells
were w~ twice with PBS and incllh~ted with 2 ~g/ml
15 FITC~conjugated affinity purified antibody tc eithQr
mouse IgG (12CA5) or 0~ IgM (CSLEX-l) for 30 to 45
minuteC in PBS/3% BSA. ~he cells were then wA~s~ twice
with PBS and resusr~nA~ in 1 ml of 1% freshly
depolymerized paraformaldehyde in PBS prior to analysis.
20 For immunofluore~~en~e microscopy, transfected cells were
fixed with 4% freshly depolymerized paraformaldehyde,
WZ~ ~A~ ~!YpQ~~~ to BSA al: 3% in PBS for 30 minutes, and
then incllh~ted with primary antibody (ascites, 1:250) for
30-45 minutes. The cellc were then washed twice with PBS
25 and ~ncl~h~ted for 30-45 minute3 with FITC-conjugated
affinity-purified antibody to mouse IgG (Cappell; 2 ~g/ml
in PBS cont~;n;n~ 3% BSA). Finaly, the cells were washed
twice with PBS and analyzed.
Metabolic T~helinq with 35so~
COS cells transfected with expression plasmids
enso~ ng mucin:immunoglobulin chimeras were trypsinized
one day after transfection and transferred to new plates
in complete medium (DMEM with 10% calf serum). Prior to
labeling, the medium was removed, the cells were washed
35 once with PBS, and the medium was replaced w~th either
CA 02224625 1997-12-12
W O 97/00079 PCTrUS96/10043
- 16 -
cysteine and methionine-free medium for labeling with
[35S]cysteine and methionine (TrAn~TAhel, ICN) or with
sulfate-free CRCN-30 medium (Sigma Chemical Co.) ~or
lAheli~ with 35S04. Serum was not added, and
5 radionuclide was typically present at a co~centration of
200 ~LCi/ml. After a 1 ~hel i ng interval of 12 to 16 hour
the supernatants were harvested, and the fusion proteins
were collected by adsorption to goat anti-human IgG
agarose (Cappel). Adsorbed proteins were ~ubjected to
10 denaturing electrophoresis on 8% polyacrylamide gels
under reducing conditions.
Chlorate Inhibition of Adhesion
COS cells were transfected with D~F dextran and
~n~-lh~ted immediately in DMEM con~;n; ng 10% calf serum
15 and 10 mM sodium chlorate. One day after transfection
the cells were trypsinized and ~nc~lhAted in fresh ~fih_~
in the ~ame medium for 6 hours. The medium was then
removed, the cells were washed with PBS, and then
inc~lh~ted for 18 additional hours in a custom prepared
20 DMEM medium (Life Technologie~) lacking sulfate and
con~1 n i n~ 2% of the conventional levels of cysteine and
methionine with 10% dialyzed fetal bovine serum in the
pr6-Ance of 10 mM sodium chlorate (Baeuerle and Huttner,
Biochem. Biophys. Res. Comm., 141:870-877, 1986). Cell~
25 were then harvested for use in the adhesion and
immunofluore~Ac~rce assays. CollL~ol cells were treated
similarly but were ~ncllh~ted in DMEM con~ning
n~ yzed serum.
~-6Q Cell Rolling
Video images of HL-60 cells rolling through a
parallel plate rectangular flow chamber (FCS2, Bioptechs,
Incorporated, Butler, PA) with a temperature controlled
stage set at 37~C were acquired with an AIMS Technology
(Bronx, NY) CCD camera mounted on a Zeiss ICM 405
35 inverted microscope equipped with a 2.5x objective. The
CA 02224625 1997-12-12
W O 97/00079 PCTrUS96/10043
chamber height was 250 ~m. Cells were withdrawn through
the chamber at a defined flow rate with the aid of a
Harvard Apparatu~ (South Natick, MA) model I/W 22 syringe
pump. Images were analyzed using NIH Image. To inhibt
5 ~ulfation, HL-60 cells were washed once with PBS and
grown for 18 hours in sulfate free medium con~Ai~in~ 2%
of the normal levels of cysteine and methionine, 10 mN
codium chlorate, and dialyzed serum as deccribed above.
For each experiment, 106 cells were sus~n~ in 1 ml of
10 0.15 M NaCl, 3 mM CaC12 and drawn through the chamber.
Gla s coverslips were coated with affinity-purified goat
anti-human IgG antibody at a ~o~Dntration of 10 ~g/ml in
50 mM Tri~-HCl (pH 9.0) for 2 hours, washed twice with
PBS, and blocked overnight with 0.2% BSA in PBS. The
15 treated coverslips were then immersed in ~upernatants of
COS cells transfected with the a~ iate i~munogloh~
chimers expression plasmids, washed twice with PBS, and
assembled in the flow chamber.
The Ami no T~rminus of PS~T~ NecessarY for P-Selecti n
20 hin~l~n~
Deletions of the amino terminus of the PSGL-l
mucin were created with PCR t~hn i ques, and the reeulting
truncated cDNAs were inserted downstream of a secretory
peptide ~equence which had been fused to a short
25 oligopeptide tag derived from influenza hemagglutinin
(HA). Expression plasmids ~n~o~i n~ the truncated
molecules (Fig. lA) were transfected into COS cells in
the pre-~nc~ of a specific myeloid fucosyltransferase,
designated FTVII, which directs the expression of ~LeX
30 determinants exclusively (S~ki et al., J. Biol. Chem.,
269:14730-14737, 1994; Natsuka et al., [published erratum
appears in J. Biol. Chem, 269:20806, 1994], J. Biol.
Chem., 269:16789-16794, 1994). Expression of the
deletion mutants at the cell surface was confirmed by
35 indirect immunofluorer-~n~e using anti-HA monoclonal
CA 02224625 1997-12-12
W O 97/00079 PCTrUS96/10043
- 18 -
ant~hoAies. The pr~~nc~ of sLeX on the cell surface wa~
similarly confirmed using the monoclonal antibody
CSLEX-l. The ability of radiolabeled transfected cell~
to bind to plastic wells precoated with
5 P-selectin:immunoglohlll in fusion protein was determined.
These experiments revealed that deletion of the amino
terminal 100 residues (referred to herein as the apical
domain) of PSGL-l was sufficient to abolish bi n~ i ng of
the transfectants to immobilized P-~electin (Fig. lB).
10 ~hese experiments also demonstrate that sLeX mediates P-
selectin b;nA;n~, as expression of FTVII was required for
P-se~ectin b;n~;n~ (Fig. lB; compare bar 2 with bar 3).
Ex~le~sion of the deletion variants at the cell surface
was confirmed by indirect immunofluorer~~nce using anti-
15 HA monoclonal ant;h~Aies, and the prer~n~e of ~LeX on thecell surface was confirmed using the monoclonal antibody
CSLEX-l. Table 1 shows the mean f 1UOL ~ -~e~Ce intensity
(MFI) of COS cells that were cotransfected with human
~lvllh and the deletion constructs (shown in Fig. lA),
20 and ~ubjected to indirect immunofluore~c~nc~ with
antibody against the amino terminal flu peptide or sLeX.
Table 1
CQnstruct Ex~ression (MFI)
Flu Slex
PSGL-1-flu 5.0 37
Xbal 17.0 26
Xba3 20.0 28
Xba6 21.0 28
Xba9 12.0 26
Xbal2 6.0 30
Xbal6 6.0 25
CA 02224625 1997-12-12
W O 97/00079 PCT~US96/10043
-- 19 --
Tn the Context Ql~ T.Arge, Sulfated Mucins~ the AminQ
Terminus of PS~T-l is Sufficient for P-Selec~-;n B;n~in~
To determine whether PSGL-1 seql~ncec other than
those found in the first 100 N--ter ;nAl amino acid~
(i.e., the apical domain) of PSGL-l were required for
bi nA; ng to P-selectin, the transmembrane and cytoplasmic
regions of PSGL - 1 were replaced with those of the CD43
antigen (Pallant et al., Proc. Natl. Acad. Sci., 86:1328-
1332, 1989; Shelley et al., Proc. Natl. Acad. Sci.,
10 86:2819-2823, 1989). The refiulting molecule, which did
not contain cysteine residues, holln~ P-selectin with the
same efficiency as PSGL-l did (Fig. 2A and Fig. 2B).
Thus, neither disulfide bond formation nor a specific
~embrane An~oring segment is required for P-selectin
15 b; n~ ~ ~ activity.
The predicted first lOo amino acids o~ PSGL-l were
then genetically grafted onto the amino termini of
mucin-like repeat elementc of several unrelated mucins to
determine whether or not ~he PSGL-l apical domain is
20 sufficient for P-selectin ligand (i.e., counterreceptor)
activity (Fig. 3A). Certain of these chimeric mucins
were able to ~u~o-L P-selectin bin~in~ in this setting.
CD34 and CD43, two relatively large mucins found
predominantly on human hematopoietic cells, were both
25 able to ~u~LL bin~in~ In ~o,.L ast, an artificially
~n~h~red variant of GlyCAM-1, a mucin expressed on high
endothelial venules that has L-selectin ligand activity
(Lasky et al., Science, 258:964-969, 1992), was inactive
in this assay (Fig. 3B). The GlyCAM-1 ~ucin domain in
30 these experiments was tethered to the cell surface via
the extracellular stalk, transmembrane ~ i n ~ and
cytoplasmic Anchoring segments of CD7 (Aruffo et al.,
EMBO J., 6:3313-3316, 1987). Cell surface expression of
the different mucins and mucin chimeras was confirmed by
35 indirect immunofluor~rc~ using an~ihoAies agains flu
CA 02224625 1997-12-12
W O 97/00079 PCTAUS9~10043
- 20 -
tag, sLe~, or the ~e_~e-tive mucins. Table 2 shows mean
fluoreccence intensity (MFI) measurements of expression
of flu tag or sLeX by COS cells transfected with the
constructs analyzed in Fig. 3B. CD34 and CD43 construc~s
5 were positive for expression by indirect
immunofluorescence using cognate anti-CD ant~ho~te
T~hle ~
Construct ~xl~e-s~on (J~ T)
10 ~ --- 52
PSGL-1-flu 9.8 43
CD43 --- 60
PSGL-l-NH2/CD43 rep. 12 67
CD34 --_ 50
PSGL--l--NH2/CD34--COOH 8.0 33
Glycam-flu 12 32
PSGL--l--NH2/Glycam--COOH 8.0 28
The apparent mol~clllAr masses of CD43 and CD34
expressed in COS cells are Le~G~Led to be 100-130 kD
(Shelley et al., Proc. Natl. Acad. Sci., 86:2819-2823,
1989) and 100 kD (Simmons et al., J. Immunol., 148:267-
271, 1992), respectively; the PSGL-1 monomer exhibits an
effective mol~c~ ~ mass of 110 kD (Sako et al., Cell,
75:1179-1186, 1993). GlyCAM-1, in its native (untethered)
2S state comigrates with 50 kD proteins, suggesting that it
i8 substantially ~;maller (Lasky et al., Science, 258:964-
969, 1992). In our studies, the larger mucins were able
to ~u~po~L P-selectin b; n~; ng when the apical domain of
PSGL-1 was appen~ to the amino terminus of the mucins.
CA 02224625 1997-12-12
W O 97/00079 PCTAUS96/10043
- 21 -
Sequential deletion of the internal repeat elements of
PSGL-1 allowed us to shorten the molecule in a systematic
manner without compromising potential global tertiary
a~sociations (Fig. 4A). As the~e repeat elements were
5 delet~d, the bin~;ng activity of PSGL-1 declined,
consi~tent with the conclusion that distance from the
plasma membrane is an important determinant of P-selectin
bin~in~ activity (Fig. 4B).
Our data also indicate that sulfation i8 one
10 determinant of the ability of ~ucins to ~ o-~ apical
domain-directed bi nA;ng. We AÇ:r~ the ability of
vari~us ~ucins to undergo sulfation in COS cells.
PSGL-l~ CD34, CD43, and GlyCAM-1 soluble mucin chimeras
readily inco~ ated sodium 35S-sulfate when ex~ in
15 COS cells (Fig. 5).
Tn~h;tion of Sulfation Blocks PSGL-1 Bjndin~ to P-
Select~ n
We have found that inhibition of sulfation blocks
PSGL-l bin~ing to P-selectin. COS cells were
20 cotransfected with PSGL-l and FTVII, or transfected with
PSGL-l and FTVII separately. During the time period in
which maximum synthesis of PSGL-l was expected, the cells
were inr~hAted in a modified DMEM medium lacking sulfate
and cont~i n ~ ng 10 mM sodium chlorate, a relativelly
25 selective inhibitor of sulfation (NaC103). We observed a
significant decrease in the ability of chlorate-treated
cotransfected cells to bind to immobilized P-selectin
(Fig. 6A), whereas the same cells showed little or no
decrement in b~r~A~ ng to immobilized
30 E-selectin (Fig. 6B). Cell surface expression of either
the sLeX antigen and the PSGL-l amino terminal tag
sequence was not inhibited by NaC103 treatment. In fact,
as shown in Table 3, an increase in the mean fluorec~nce
intensity of the transfected cells, representing both
35 anti-sLeX and anti-flu tag, was observed following
CA 0222462~ 1997-12-12
W O 97/00079 PCTrUS96/10043
- 22 -
chlorate treatment, suggesting that chlorate may affect
inter~Al~7~tion or cell surface export.
Table 3
Expression (MFI)
w/o N~C103 W/10 m~ N~C103
sLex Flu sLex Flu
~ ln 23 --- 30 --
PSGL-l-flu --- 9 --- 22
PSGL-lflu + ~L~llh15 10 35 34
A soluble PSGL-1 im oglobulin chimera
synthesized under comparable conditions showed
10 essentially complete inhibition of 35s-sulfate
in~r~ation (Fig. 7), under conditions in which prote~n
synthesis as measured by [35S]cysteine and methionine
inco~Lation was not inhibited. These data demG.lDL~ate
that sulfation of the P-selectin ligand is required for
15 P-selectin b1 n~ i ng activity.
Fin~ Structure Deletion Analvsis o~ the A~ical Domain of
PSt'-T~--l
To loç~li7e the elements within the 100 amino acid
apical domain which contribute to P-selectin ligand
20 activity, we prepared a collection of deletion mutants in
which various regions of the apical domain were deleted
(Fig. 8A). Each amino terminal deletion mutant was then
placed downstream of the CD5 leader/flu tag element to
monitor cell surface expression. The fine structure
25 deletion mutants showed little variability in their
ability to express the epitope tag, as ~ by
indirect immunofluorescence. Removal of the first 20
amino acids of the N-terminus of the mature PSGL-l did
not affect P-selectin bin~;nq activity. In ooIlL~ast,
30 removal of the first 40 amino acids of the N-terminus
abrogated bi nA i ng (Fig. 8B). Further deletions of PSGL
did not affect P-selectin b;n~ing activity. Accordingly,
CA 02224625 1997-12-12
W O 97/00079 PCT~US96/10043
- 23 -
amino acid residues 20 to 40 of PSGL (i.e., residues 38
to 57 of the predicted precursor having the signal
seguence) are required for P-selectin b;n~;nq.
To demonstrate that residues 38 to 57 are
5 sufficient for PSGL-l apical domain-directed activity, we
Arp~nA~ this segment to the amino termini of PSGL-1 and
CD43 mucin cores from which the apical domains had been
deleted (Fig. 9A). In both cases, addition of amino acids
38-57 of PSGL-l peptide element conferred P-selectin
10 bi n~ i n~ activity upon the mucin core. In both cases, the
level of P-selectin bin~i ng activity was equivalent to
that Pf native PSGL-l (Fig. 9B).
Specific Residues With;n the Amino Termin~l Pe~ de Are
Re~li~ed for P-selectin Bin~;ng Activ;ty
The 20 amino acid region which i8 n~ce~ ~y for
P-~electin binAin~ contains three potential tyrosinQ
~ulfation sitQs and two thr~on;n~ residues for 0-l;nk~
gly~ylation. To A ~-~ C the importance of these
rQsidues, the tyrosines were converted to phenylAlAninQ
(Fig. 9A). In a ~ecQ~A peptide, the thr~on;n-- werQ
converted to Al An; n~fi~ In addition, a third peptide,
contA; n; ng a quintuple mutation, was prepared such that
both conver~ions were made in a single peptide. Each
mutated peptide was then positioned, separately,
25 downstream of the flu tag and u~L.eam of either (1) the
truncated PSGL-l lA~k;ng the apical ~ ~;n~ or (2) the
CD43 repeat elements and transmembrane domain. Cells
expressing the resulting chimeras were tested for their
ability to bind to immobilized P-selectin (Fig. 9A).
30 Conversion of the tyrosines to phenylAlAn;r~- resulted in
a loss of binding activity to P-selectin. Replacement of
the threonine residues with Al~ n i n~ dimi n; ~he~ b; n~; ~g ~
but did not abolish it entirely. Expression of the flu
tag or sLeX epitope was not affected in these cells.
35 Binding -~;Ated by the apical 20 esidues was, like that
CA 02224625 1997-12-12
W O 97/00079 PCT~US96/10043
- a4 -
of native PSGL-l, ~p~n~nt on the pr~en~e of calcium.
These data indicate that sulfation of tyrosines at
positions 46, 48, and 51 is required for P-selectin
hin~inq activity. E-selectin bin~ing was unaff~cted
S under the same condition. In addition, these data
indicate that the thr~Qrl;n~- at position~3 44 and 57 are
required. These thr~onin? residues can serve as sites
for o-l ink~ glycan addition. These experiments, in
con~unction with our experiments showing that FTVII
10 e~Le~sion is n?ce~A~y for P-selectin bin~inq~ provide
cv;~nce that P-selectin b; n~;nq requires sLeX at
thr~?o~in~3 44 and 57. In sum, the above--described
experiments demonstrate that amino acids 38-57,
contA; ni~q three residues for sulfation and two residues
15 for sLeX addition, are sufficient to confer P-selectin
bi n~nq activity.
Residues Within the Amino-Terminal 20 Amino Acids ~e
Slllfated on TYrosine
To determine whether the amino-terminal segment
20 was capable of being sulfated in vivo, we created fusion
proteins consisting of the native or mutant peptide
5e~l~n~e~ ~oined to human immunoglobulin Gl (IgG1) (Fig.
12A). The resulting fusion proteins were e~ in
COS cells, and their ability to assimilate inorganic
25 sulfate was A~cecrr~ (Fig. 12B). Immunogloh~ n chimeras
bearing the native peptide se~l~nc~c were capable of
incorporating sulfate, whereas those bearing
phenyl~lAnine substituted for tyrosine were not (Fig.
12C). Replacement of threonine with AlAnin~ had no
30 effect on ulfate incorporation (Fig. 12C).
Tnh;hitors of Sl~lfation Block ~T.--I;0Rollina on P--
Select;n-T~unoqlobulin Chimeras
To explore whether inhibition of sulfation would
compromise a physiologically relevant adhesion, we
35 subjected HL-60 cells to growth in medium contAining
CA 02224625 1997-12-12
W O 97/00079 PCTAJS96/10043
- 25 -
chlorate and examined the ability of the reculting cells
to attach and roll on coverslips coated with P-selectin-
i unogloh - l i n chimeras under conditions of defined fluid
shear ~L~ (Lawrence et al., Blood, 75:227-237, 1990).
5 HL-60 cells were capable of attaching to and rolling upon
coverslips precoated with P-selectin-immunoglQhllin
chimeras, whereas no such interaction was observed with
lips coated with a cD4-immunoglQh~ n chimera (Fig.
13). Growth of HL-60 cells in chlorate dramatically
10 r~llce~ the frequency of cell interaction with the
substrate (Fig. 13).
An~ih-~dies ;-ntl Ant;hody Fusion Proteins Beari~c7 Sia
T,~X Antl Sl~lfAt~rl Determinilnts
In one emhoA;ment, the invention features an
15 antibody bearing sialyl-LeX and sulfated determinants.
Such an anti~ody may be created by ill~r~ducing sulfation
sit2s ti.e.~ a tyrosine in an acidic context) into an
existing antibody molecule in the vicinity of an
il~ ollc~A or existing sialyl-LeX addition site (for
20 example, by s~AnAA~d site-directed mutagenesis).
Alternatively, a~o~iate sialyl-LeX and/or sulfation
site~ may be added by Arr~A i n~ any P-selectin ligand
~equence (for example, any P-selQctin domain dQscribQd
herein) to a naturally-occurring antibody sequence (for
25 example, IgG or IgM) by stAnAA~d recombinant DNA
~chn i que8 to produce a P-selectin-antibody fusion
prot~in. Preferably, the P-selQctin ligand ssquQnce is
Arr~A~A to the amino-terminus of the antibody molecule.
Such an~ihoAies are useful for disrupting undesirable
30 interactions between cells or proteins, or, generally,
for disrupting any interaction between two molecules, one
of which bears a determinant carried by the antibody.
Because thQse determinants normally act to facilitate
interactions involving E seleatin and P-selectin (e.g.,
35 interactions between neutrophils and endothelial cells
. CA 0222462~ 1997-12-12
PCT/US 96 /1 ~ 043
IP~A/US ~ 4 J A N 1997
-
_ - 26 -
lining the blood vessel walls), the abllity to disrupt
such interactions provides many therapeutic applications,
for example, in minimizing inflammation and decreasing
extravasation-dependent organ damage and/or clotting.
In addition, if desired, one or more sialyl-LeX
moieties which mask the CH2 portion of the immunoglobulin
molecule and thus inhibit complement fixation and Fc
receptor binding may also be incorporated into the
antibody sequence. Because the carbohydrate moieties
10 block the immunoglobulin domain which triggers complement
fixation and Fc receptor binding, such antibodies do not
'~, elicit the undesirable side effects (i.e., those
resulting from complement ~ixation and Fc receptor
binding) frequently associated with antibody-based
15 therapies. Preferably, the carbohydrate groups serve not
only to inhibit undesirable complement fixation and Fc
receptor binding, but also perform the function of
competitively inhibiting an E-selectin and/or P-selectin
mediated intracellular interaction.
To inhibit complement fixa~ion and Fc receptor
binding, sialyl-LeX determinants may be added to the
antibody molecule at any appropriate site. N-linked
glycan addition sites are well known to be: N X S/T
(where N is asparagine, S is serine, T is threonine, and
25 X is any amino acid except proline). Accordingly, an
exemplary molecule may be designed that includes several
such sites for attachment of sialyl-LeX side chains.
Inspection of the IgG1 sequence (Fig. lOA-E) reveals at
least five sites at which N-linked glycan addition sites
30 may be introduced into the molecule in advantageous
locations, where complement ~ixing and Fc receptor binding
ability will be impaired by the process. These sites
include amino acid residues 274, 287, 295, 322, and 335.
Although these are preferred sites of N-linked glycan
35 addition, they are not the only candidates; other
AMENDED S~lEET
CA 02224625 1997-12-12
W O 97/00079 PCTAUS96/10043
- 27 -
u3eful sites may be identified and in~-~v~ated into the
IgGl sequence using, a~ ~l;~Ance~ the following criteria:
(1) the sites are, preferably, located in the CH2 region
of the immunoglohlll ;n molecule, i.e., in the portion of
5 the molecule responsible for complement fixation and Fc
receptor b; nA; n~; (2) the sites are located in regions of
the sequence, predicted by their hydrophilic nature, to
be pre~ent on the outside of the immunoglobulin molecule
and therefore A~ce~cible to the enzymes r~cpQ~ible for
10 attachment of carbohydrate side rhA;n~; (3) the sites are
located in a region which is minimally disruptive to the
prim~ry Amino acid sequence and, thus, the predicted
~e~-onAAry amino acid structure. For example, a
naturally-occurring site which differs from an N-l~nkeA
15 glycan addition site by a single amino acid would be
preferable to a site requiring two alterations in the
amino acid sequence. Moreover, it is preferable to
create an N-linked glycan addition site by substituting
amino acids of similar charge or polarity (e.g.,
20 substitution of one l~n~hArged amino acid for another).
One or more N-li nk~A glycan addition site substitutions
may be engineered into a particular IgGl-~ncoA i ng
seguence; such sequences (i.e., those which ~nroA~ an
antibody molecule to which sialyl-LeX moieties are
25 attA~h~A) are termed IgG1-sialyl-LeX or IgGl-LeX.
The il-L~duction of additional glycosylation sites
at amino acids ~274, ~287, and #322 within the C~2 domain
created a molecule that was unr~cogni~ed by Fc receptor
or complement using assays that are st~n~Ard in the art;
30 exemplary co~plement fixation assays include Weir et al.,
~nAhook of Experimental Immunology, Blackwell, Oxford;
and Coligan et al. Current Protocols In Immunology, Wiley
Interscience, 1995.
A particular IgGl molecule bearing sialyl-LeX
35 moieties ic proA~lre~ as follows. The IgGl gene is
. CA 0222462~ 1997-12-12
PCTi1J~ ~6 /1 0 043
IP~ ; 1 4 J A N 1997
- 28 -
publically available, and its sequence is shown in Fig.
10. The gene is mutagenlzed by s~andard methods of in
vitro site-dlrected mutagenesis in order to introduce one
or more N-linked glycan addition sites (e.g., those
5 described above and shown above the naturally-occurrlng
sequence in Fig. lOA-E). The gene is then inserted into
a vector designed to express the protein in a eukaryotic
cell (see, e.g., those vectors described ln Glllles et
al., U.S. Patent No. 4,663,281, hereby incorporated by
10 reference). The eukaryotic host cell is preferably a
m~mm~l lan cell (e.g., a CHO or lecll cell), and the
expression vector containing the mutated IgGl-LeX-encoding
sequence is introduced into the hoqt cell by transient or
stable transfection using standard techniques. Such host
15 cells are also transfected (translently or stably) with a
vector capable of expressing an ~(1,3)fucosyltransferase
capable of attaching the slalyl-LeX groups to the antibody
molecule at the glycosylation sites. The
~(1,3)fucosyltransferase gene may be expressed from a
20 vector dlstinct ~rom that encoding IgGl-LeX, or both genes
may be carried on, and expressed from, a common vector.
~mm~l ian cells are particularly useful hosts for the
synthesis of IgGl-LeX because they provide all required
precursors for slalyl-LeX production.
To produce the sialyl-LeX-modified and sulfated
antibodies of the invention, the gene encoding the
antibody sequence is preferably expressed in a cell which
also expresses an ~(1,3)fucosyltransferase that
exclusively catalyzes ~(1,3)fucose linkages; such an
30 enzyme is described in Walz et al., Science 250:1132-1135
(1990) and in Seed, U.S.S.N. 08/483,151, entitled
"Fucosyltransferase Genes and Uses Thereof," filed June
7, 1995 (hereby incorporated by reference). Less
preferab y, the ~(1,3)fucosyltransferase cDNA described
AMEND~D SHEET
CA 02224625 1997-12-12
W O 97/00079 PCTrUS96/10043
- 29 -
in Lowe et al. (Cell ~:475, 1990) may be utilized. This
fucosyltransferase re~o~n;7es a sialylated precursor
molecule and adds either an ~(1,3)- or an ~(1,4)-link~A
fucose moiety to N-acetylgll~o~mine side ~h~ i n~ . The
5 sialyl-Le~ determinant is characterized by an ~(1,3)-
l~n~A~Q, and, as such, the ~(1,3)fucosyltrans~erase
enzyme of Lowe (~u~r~) proA-lr~ both the desired sialyl-
LeX-modified molecules and products bearing ~gl,4)-l;n~e~
fucose which, al~ho~yh not active in bin~ to P-
10 selectin and E-selectin, do not interfere with the action
of the sialyl-Le~-modified molecules nor produce other
unde~irable side effects.
Host cells expres~ing ~(1,3)fucosyltransferase and
the antibody to be modified are grown by 8t~n~d
15 methodfi, and the antibody i8 purified from a cell lysate
hAr~~ on its affinity for a Protein A column or any other
8~n~d te~hn;que of antibody isolation and
purification.
~l-Acid Glyco~-o~ein-Antibody Fusion Proteins Bearing
20 Sialyl-LeX and Sulfated Determinants
As ~c~ herein, antibody fusion proteins
modifi~d by sulfation and sialyl-LeX addition have
importAnt therapeutic and diagnostic uses. Previous work
has demo~.~L~ ated that large amounts of antibody fusion
25 proteins may be generated and secreted transiently from
tran~fected mammalian cells (for example, COS cells). In
general, to produce an AGP antibody fusion protein
accor~ing to the invention, DNA ~nco~; ng an AGP and a P-
selectin ligand domain are fused in-frame to human IgG
30 domains (for example, constant domains) by st~n~d
techn;ques, and the fusion protein is expressed, also by
s~A~d ~echn;ques. The antibody portion of the
molecule facilitates fusion protein purification and also
prolongs the plasma half-life of otherwise short-lived
35 polypeptides or polypeptide domains. Preferably,
. CA 0222462~ 1997-12-12
PCTIU~96/~04~
IPEA/IIs 1 4 J A N 1997
_ - 30 -
antibody fusion proteins are expressed according to the
methods disclosed in Seed et al., U.S.S.N. 08/483,151
entitled "Fucosyltransferase Genes and Uses Thereof,"
filed June 7, 1995 (which is hereby incorporated by
5 reference), e.g., using IgG or IgM antibodies or portions
thereof (see also Zettlemeisl et al., DNA Cell Biol.
9:347 (1990) for IgM fusion proteins).
Recombinant plasmids expressing particular AGP-
antibody fusion proteins (e.g., AGP-Hinge-CH2-CH3 and
10 AGP-CH2-CH3 proteins) have been constructed as follows.
A cDNA encoding the acute phase ~l-AGP gene was cloned
from a human liver cDNA library by polymerase chain
reaction (PCR) using oligonucleotide primers
corresponding to the 5' and 3' coding regions of ~1-AGP
(Board et al., Gene 44:127, 1986) according to standard
techniques. The 5' AGP primer was designed to contain a
HindIII restriction site and the 3' primer was designed
to contain a BamHI restriction site rather than the AGP
stop codon. The PCR-amplified product was digested with
20 HindIII/BamHI and cloned into a HindIII/BamHI-cut plasmid
expression cassette (see Aruffo et al., Cell, 61:1303,
1990) containing constant domains of human IgG1 (i.e.,
Hinge-CH2-CH3 or CH2-CH3). A nucleotide sequence and
amino acid sequence of this AGP-IgG fusion protein are
25 shown in Figs. llA-B and Fig. llC, respectively.
To create a molecule that blocks P-selectin-
mediated interactions, sites for sulfation and, if
necessary, sialyl-LeX addition are introduced into the
antibody fusion protein sequence (for example, the
30 antibody fusion proteins described above). Such sites
may be incorporated into an existing fusion molecule, for
example, by introducing one or more sulfation sites
(i.e., a tyrosine in an acidic context) in the vicinity
of an introduced or existing sialyl-LeX addition site (for
35 example, by standard techniques of site-directed
AMENDED SHEET
CA 02224625 1997-12-12
W O 97/00079 PCTAUS96/10043
mutagenesis), or a P-selectin ligand sequence (for
example, any of the P-seleetin ligand se~l~ne~c deseribed
herein) may be Arren~ to the antibody fusion protein
sequence using s~n~A~d ~ec~n~ques of reeombinant DNA
5 te~n~logy.
The P-selectin-AGP-antibody fusion genes are then
il.L~ e~ into expression plasmids, and the plasmids are
transfeeted into any a~ iate fucosyltransferase-
~x~ ing cell for the production of soluble antibody
10 fu8ion protein~.
To prepare an antibody fusion protein eapable of
~nh~h~ting eomplement fixation and Fc reeeptor bin~ng~
additi~nAl 5ialyl-LeX rQn~nCllc gly~ylation sites (N-X-
T/S) may be i.~L~ol.l~eA into the CH2 domain of human IgGl
15 a8 described above.
RAf-~'1 on this eonE;truction strategy, any number of
reeombinant P-selQctin-AGP-antibody fusion protQins may
be designed having long plasma half-lives and the ability
to inhibit undesirable eell-eell interaetions (for
20 Qxample, the interactions between leukocytes and
selectin-bearing cells). To generate molecules with
heightened inhibitory potency, c~n~ te molec~ are
de~igned and screened using the assays described above.
In one particular example, molecules may be screened for
25 their ability to incorporate sialyl-LeX and ~ulfated
aeterminants and block the b;n~;ng of ne~Llo~hils to
activated endothelial cells; such molecules find use in
the inhibition of ~electin-~ren~nt inflammatory
reactions and tissue injury inflicted by invading
30 leukocytes.
Molecl~les Ca~able of Interferinq with P-Selectin-Mediated
An~ E-Selectin-Mediated Interactions
Because both P-selectin- and E-selectin-mediated
intracellular interactions are involved in inflammation
35 and heCs~ ? the crucial determinants involved in those
CA 0222462~ 1997-12-12
W O 97/00079 PCTrUS96/10043
- 32 -
interactions have now been idQntified, it i~ possible to
de~ign a single molecule capable of interfering with both
typQs of deleterious interactions. In particular,
molQculQs (for example, proteins) may be c~l.~L ~cted that
5 include both a P-selectin ligand domain (i.e., a domain
bearing sialyl-LeX and sulfated moieties) and an E-
~electin ligand ~ ~ ; n ( i . ~ ., a domain bearing a sialyl-
LeX moie~y). Such a molecule may be constructed by
combining domains, for example, by A~r~n~ing a P-select~n
10 ligand domain to a sialylated molecule (for example, a
sialylated antibody or antibody fusion protein described
hereLn). Alternatively, a~Lop~iate sialyl-LeX and/or
~ulfation sites may be illL.~llce~ into an existing
sequence, for example, by site directed mutagenesis.
Gly~o_ylation or sulfation of an engineered
molecule may be tested, for example, as described herein
and in Walz et al., Science 250:1132-1135 (1990). The
ability of a ~ialyl-LeX-modified and/or sulfated molecule
to interfere with intracellular interactions may also be
20 tested as de~cribed in Walz et al., supra, or by any
5~n~d techn;~ue, for example, by assaying the ability
of increasing concentrations of the determinant-bearing
molecule to i~hibit adherence of T lymphocytes or ~yelo~d
cells to immobilized P-selectin and/or E-selectin.
25 Use
For administering a protein or organic molecule of
the invention to a patient, the pharmaceutically-pure
protein or molecule is suspe~P~ in an acceptable
carrier, e.g., physiological ~l in~, and is delivered to
30 the patient by any appropriate route (for example,
intravenously) in a single dose or in multiple doses.
Optimally, a sufficient guantity of the therapeutic is
provided to saturate all P-selectin and, for a dual
function molecule, all E-selectin b;n~;ng sites on an
35 endothelial cell. Typically, thi~ may be achieved with
CA 02224625 1997-12-12
W O 97/00079 PCT~US96/10043
~ 33 ~
doses of 0.1 mg/kg or greater. The preferred dosage is
in the range of 0.1-2.0 mg/kg.
The sialyl-LeX-modified and sulfated molecules and
proteins of the invention (for example, the modified
5 an~iho~ies and antibody fusion proteins described herein)
may be used, in one example, for the treatment of
extravasation-A~r~n~ent organ damage and/or clotting. In
particular, heC~l~re r --1 ectin mediates the attachment of
n~LL-J~ overlying ~ites of inflammation or ~ 7~
10 damage or proximate to thrombus formation, the molecules
and protein~ of the invention provide useful therapeutics
for bl~ n~ such interactions. For example, P-selectin
likely mediates the migration of neutrophils into the
lung following adult respiratory distress syndrome and
15 into the heart following ~ h~mic myocardial injury
(i.e., infarction), and may play a role in glomerular
damage to the k;~ln~y8 under certain condition~.
Accordingly, a sialyl-LeX modified and sulfated mole
or protein of the invention may be administered to a
20 patient suffering from such a ~ir~e or condition. Such
treatment attenuates extravasation-A~ren~nt damage by
competitively inhibiting the interaction between the
invading ne~ro~hils and the endothelial cell of the
blood vessel or organ. The comrolln~c of the invention,
25 particularly, P-selectin ligand-AGP fusion proteins and
P-~eleatin ligand-AGP-antibody fusion proteinc may also
be used, as described above, for the treatment of septic
shock or septicemia.
In addition, an~iho~ies or antibody fusion
30 proteins according to the invention may be used in
conventional ~erhn iques of antibody-based therapies or in
v vo diagnostics, ~Aking advantage of the antibody's
specificity to target therapeutic or diagnostic sites.
In one particular example, the P-selectin ligand domain
35 of an antibody fusion protein acc~-ding to the invention
CA 0222462~ 1997-12-12
W O 97tO0079 PCT~US96/10043
targets that protein to a site of inflammation and
provides both a therapeutic (useful for blor~i n~
deleterious P-selectin-mediated intracellular
interactions) and a ~;Agn~ctic (useful for tagging the
5 site of inflammation). Agstin, attA~he~ ~ialyl-LeX
determinants may be used to mask the CH2 domain of the
antibody and block the undesirable effects of complemen~
fixation and Fc receptor b;n~in~.
Other ~odiments
Other QmbodimQnts are within the claims. For
~xample, for the yu~o~e of blocking interactions between
cQlls or proteins, any other ap~lop~iate carrier molecule
to which a sialyl-LeX and a ~ulfated determinant may be
attachad may be utilized in the invention. Generally,
15 proteins are preferred h~CAl~e of their relatively long
half-lives in serum. One class of carrier proteins are
serum proteins such as albumin (e.g., bovine serum
albumin or human serum albumin), transferrin, or ~-2
macroglobulin. The carrier proteins may contain
20 endogenous sulfation and glycan addition sitQs in
addition to which site~ are i"Llollc~ into the DNA
seguence of the carrier protein (as described above) byp
for example, site-directed mutagenesis. The carrier
molecule, less preferably, may be a lipid. In one
25 example, the lipid, with one or more attA~h~ sialyl-LeX
and sulfated determinants is delivered as a liposome to a
target cell wall (e.g., an endothelial cell wall). The
l~rQ~ome may block a cell or protein interaction or may
be used to deliver a drug to its a~ iate ~ite of
30 action.
Production of carrier molecules bearing sialyl-LeX
and sulfated determinants may be carried out in a cell,
preferably, a eukaryotic cell other than yeast.
Nammalian cells, e.g., mammalian cell lines, provide
CA 02224625 1997-12-12
W O 97/00079 PCTrUS96/10043
- 35 -
particularly suitable hosts. These cellc generally
synthe~ize the n~aeFCAry precursor molecules and produce
or can be engineered to produce the enzymes ~~Gl.sible
for sulfation and carbohydrate attachment. For the
5 attachment of sialyl-~e~ determinants, mammalian cell
lines ~uch as CH0 and lecll are particularly ~uitable.
Alternatively, either or both of the sialyl-LeX and
sulfated determinants may be attA~hPA to a carrier
molecule in vitro, i.e., extracellularly. In one
10 example, ~(1,3)fucosyltransferase would be bound to a
solid ~u~p~LL (e.g., a column) and a sulfated carrier
molec~le pA~~~ over the ho~n~ fucosyltransferase enzyme,
under conditions which facilitate attachment of sialyl-
LeX y-O~y~ to their a~Liate site(s) on the carrier
15 molecule.
The invention also encompAsses the use of sulfated
and sialyl-Le~-modified AGP-antibody fusion proteins for
protecting against, inhibiting, or treating a shock-
inducing event, the clinical manifestations of '-~9C!l', or
20 both which are caused by microbial factors (e.g.,
opolycAc~hArides (LPS)), microbial toyinc (e.g., toxic
~oc~ enterotoYinc)~ host mediators (e.g., cytQk~n ), or
anti-tumor therapies (e.g., administration of tumor
necrosis factor (TNF) or interleukin-l (IL-l)), or any
25 combination thereof. For example, such an antibody
fusion protein can be administered to a human patient to
alleviate the effects of septic shock inAllceA by
microbial LPS. The ability of an antibody fusion protein
to protect against, treat, or inhibit the effects of
30 ~hock (e.g., septicemia or toxic shock syndrome) is
evaluated according to st~nA~rd methods known in the art
(e.g., those described in Libert et al. (1994) ~. Exp.
M~d. 180: 1571-1575).
All publications, patents, and patent applications
35 mentioned in thi_ specification are herein in~L~o~ated
CA 02224625 1997-12-12
W O 97/00079 PCT~US96/10043
- 36 -
by reference to the same extent as if each individual
publication, patent, and patent application was
specifically and indiv~ Ally indicated to be in~GLated
by reference.
CA 02224625 1997-12-12
W O 97/00079 PCTrUS96/10043
SEQUEN OE LISTING
(1) GENERaL INFORMATIONs
(i) APPLICANT: The General Hospital C~.~G.aLion
(ii) TITLE OF lhv~-lON: P-SELECTIN LIGANDS AND ~TAT~n ~T~C~T~S
AND ~T~O~S
(iii) NUMBER OF SEQU~N OE S: 14
(~v) ~ rONDEN OE An~SSs
~A~I AnD~ Y~s Fish & Richardson P C
,BI STREET: 225 Franklin Street
C, CITYs Boston
D STATB: MA
E~ ~UUh ~: USA
~F, ZIP: 02210-2804
(v) OOMPUTBR ~n~RT~ FORM:
IAJ M~DIUM TYPE: Floppy disk
B CX~u.~n: IBM PC c~ ,-';hle
,C OPERATING SYSTEM: PC-DOS/NS-DOS
~DJ SOFTWARE: P~ten~Tn n~le~e ~1.0, Version ~1 30
(vii) PRIOR APPLI Q TION DATAs
(A) APPLI Q TION Nur~R~s US 60/000,Z13
~B) FILING DATEs 14-JUN-1995
(C) CLASSIFICATIONs
(viii) A~.~nN~/AGENT lN~Ok~ATIONs
(A) NAMEs Lech, Raren F.
(B) REGISTRATION NUMBBRs
(C) n~Y~N OE /DOCKET r~rUR~s 00786/28WOl
(iX) T~T~ rlcATIoN lN~ OndATION:
(A) TELEPHONE: 617/542-5070
(B) TELEFAXs 617/542-8906
(C) TELEYs 200154
(2) lN~O~ATION FOR SEQ ID NOsls
(i) SEQU~N OE CHARACTERISTICSs
A LENGTH: 10 ~mLno acid~
~B TYPE: amino acid
C, sT~Nn~n~Ss not relevant
~DJ TOPOLOGY: lin~ar
(ii) Y~r~CnT~ TYPEs prot~n
(xi) SEQUENCE D~CC~TPTION: SEQ ID NO:l:
Ala Thr Glu Ala Gln Thr Thr Pro Pro Ala
1 5 10
(2) lh~O_~ATION FOR SEQ ID NOs2s
CA 02224625 l997-l2-l2
W O 97/00079 PCT~US96/10043
- 38 -
~i) 8EQU~N OE ~TPRTSTICSs
IAJ LENGTHs 18 amino acids
rBJ TYPES amino acid
rC, STR~N~-F.~r-~-CSS not rel~vant
~DJ TOPOLOGYs linear
(ii) MnT-~C~T-T~ TYPEs protein
(xi) SEQUEN OE P~RTPTIONs SEQ ID NOs2s
Met Ala Thr Asn Ser Leu Glu Thr Ser Thr Gly Thr Ser Gly Pro Pro
1 5 10 15
Val Thr
(2) INFOPMATION FOR SEQ TD NOs3s
(i) SEQUEN OE ~AR~rT~RT~TICSs
(A'l LENGTHs 42 amino ncid~
Bl TYPEs amino acid
.,C, STR~N~ cs: not rele~ant
~D~ TOPOLOGY: linear
(ii) M~T~CnT~ TYPEs prot~in
(xi) SEQUEN OE D~-qCRTPTION: SEQ ID NOs3s
Gln Leu Trp A~p Thr Trp Ala A~p Clu Ala Glu Ly~ Ala LRU Gly Pro
1 5 10 15
Leu LQU Ala Arg A~p Arg Arg Gln Ala Thr Glu Tyr Glu Tyr L~u Assp
20 25 30
Tyr A~p Phe Leu Pro Glu Thr Glu Pro Pro
(2) ~rORiLATION FOR SEQ ID NOs4s
(i) SEQUBNCE ~R~T~RTCTICSs
rA~ L~NGTHs 20 amino acid~
B TYPBs amino acid
C, S~R~N~ CSs not rel~vant
~DJ TOPOLOGYs linHar
( ii ) M~T-~C~T ~ TYPBs prot~in
(xi) SEQUEN OE p~C~RTPTIONs SEQ ID NOs4s
Arg Anp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr LQU Asp Tyr Asp Phe
1 5 10 15
LQU Pro Glu Thr
(2) lNr~K~ATION FOR SEQ ID NOs5s
(i) SEQUENOE ~R~T~PTSTICSs
CA 02224625 l997-l2-l2
W O 97/00079 P ~ fiUS96~0~43
- 39 -
~Aj T- _~S 20 amino acid~
~B TYPE: amino acid
'C STRP~ Ss not ralevant
~D~ TODOLOGYs linear
(ii) ~T~Cn~-R TYPEs protoin
(xi) SEQUENCE ~CC~TPTION: SEQ ID NO:5:
Arg A~p Arq Ar~ Gln Ala Thr Glu Ph~ Glu PhQ Leu A~p PhQ A~p Ph~
1 5 10 15
~ou Pro Glu Thr
(2) lN ~RMATION FOR SEQ ID NO:6:
(i) SEQUEN OE r~R~DT~TICSs
~A~l LENGTHs 20 amino acid~
BI TYPBs amino acid
C, S~RP~ ASs not r~levant
DJ TOPOLOGY: linear
( ii ) ~nT~CnT-~ TYPE: protQin
(xi) SEQUEN OE ~ACRTPTION SEQ ID NOs6:
Arg Asp Arg Arg Gln Ala Ala Glu Tyr G u Tyr Leu Asp Tyr A p Phe
L~u Pro Glu Ala
(2) INFOR~TION FOR SEQ ID NOs7s
(i) SBQUEN OE r~R~rT~RTRTICSs
~AJ LENGTHs 20 amino acid~
IB TYPEs iino acid
,C, STD~r~ -CSs not r~lovant
~DJ TOPOLOGYs linear
ii ) ~r-~C~n-~ TYPE: protein
(xi) SEQUENCE D~-~RTPTION: SEQ ID NOs7:
Arg A~p Arg Arg Gln Ala Ala Glu Phe Glu Phe Leu A~p Phe A~p Phe
1 5 10 15
Leu Pro Glu Ala
(2) lN~OR~ATION FOR SEQ ID NOs8:
CA 02224625 1997-12-12
W O 97/00079 PCTrUS96/10043
- 40 -
(i) ~~QuENcB ~RaRa~T~T.CTICS
~A, LENGTH~ 2287 ba~Q pair~
BI TYPEs ~l~i~ acid
~C ST~ ASS ~ingl~
,DJ TOPOLOGY: lin~r
(ii) MnTRC~TR TYPB: DNA (~
(xi) SlSQUll:NOE l:!~CCPTPTIONs SE:Q ID NO~8s
AAGCTTACCA CCATGGACTG GACCTGGAGG ~~C~ .,v.Gv.GGC ~~~r~CT~A~ 60
Gv.v.C~AGT CCCAGGTGCA G~vv~v~AG -,~,GGGv~,G AGGTr-aar-aa GC~-G~v.~C 120
~Gv~vAAGG -~ ~AA GG~,,~-,vvA GC~~~CTTCA Gc~cTaTGc TATCAGCTCG 180
GTGC~~~ CC~-~GvACA AGGG~..vAG TGGATGGGAG GGAT Q TCCC TA-, vv- 240
rA~A~~T rA~ar~ GTTcr~y~Gc AGAGT Q CGA T~ArCGCGr-a CGAATC QCG 300
Ar~ ccT A QTGGAGCT G~ -CCTG AvATCTGAGG Ara~GC~. GTATTACTGT 360
açr~raTa ATGGAGCGTA TTGTAGTGGT GGTAGCTGCT A~,~G~,G GTTcr-acccc 420
~GrJG~ -G G~C~,vv, Q CC-v~ ~-. T Q GGTGAGT ACTGAATTCT Av~ GG 480
G~~a~~Cr~~ CCCTr-AACTT GG~.. GGGG rarGr-A~GC~ GCT~ r~TGA GGAa~GTGaC 540
Gcr~A~a~GT Ccarar,crAA TGCCQTvAG ccr~--AA~AT Gr-l~CCCTGAA C~ ,~ A~ 600
AGTTAAraA- Ct~aAGGGc~ GCI;~vG Gccra-GcTcT GTCC~ ararC GCGGTQQT 660
GrJrarraAC7T ~ v ;ACC cTcra~raa- GG---CrAq'CGG ~ 'C'CC~;. G~ra'CCTCC 720
T~r,raa~~--~a C~ vvGGG ra~a~CGCCC ;~vGG~-vCC TGGTQLGGA CTA ;--CCCC 780
G ~ vA Cvv-v~,v~v GAACTQGGC GCC~-~vACCA GC~G~;A QC~,.. ~CG 840
~ v~ AC Av~ ;AGG ACTCTACTCC CTrar-~~~C'G TGGTGACCGT GCC;~,-.C~AGC 900
AG~.. ~;A cC-ra~~~c~a CATCTCCAAC CTCAATQQ a~Ccra~~aa ra~rar~¢TG 960
c-A~a-~ ~aa-- -.. v.vAGAG GCrar---a~a~- G----A~Cr'~ G ~v- v~,- vG aa~~ra~GCTC 1020
Avw~;~.v CX TG---A~C~C--a - ~CC~-~ A'r C~~Acccra~ Tcrar,G,--~raA- ra~ r~~~- 1080
CC~v.~,~vCC TCTTQCCCG GAGC~;. vC' C;~-CC~ T QTCCTQGG C-A~''~Gr,TCT 1140
TCTGGCl~ T~r,r~ra~GCTC Tr,-,~Aa~Gra ra~Gc~Tar~GT Cc~cc~ Aar~r rP~cc~c~,vc 1200
a~a~aaa~GC GCaG~.v~.v GGCTQGACC Tr~---Ca-Arl'A~C' QTATCCGGC a~~---A',CCTGC 1260
CccTr-arcTA r--C~--r~AC7CC AaArCcr,AAA ~ c~;Ar-Tc CCTCAGCTCv GACACCTTCT 1320
I;~C~CC ;AG ATTCQGTAA CTCCQATCT ~ ;A aAGccraAA~ v~vAQA 1380
AACTQQCA TGC-rrarrGT GCC~rar,GTAA Gcr-ar~ccr-AA~ GC ;-CGCC~;- CQGCTQAG 1440
GOOGrA~---a---a2- ,C:CC -.r~~~ TAG~;;.v~;AT cra~=--r ~ar Gcccr7~r~A-cG GGTGCTCAQ 1500
C~TC Q CCTC Q ~v~ 'C Tr-ar-rACCTG AA~ -~;~-GGG r~---r-aCCGTQ ~v~C~ - 1560
~CC'CC~'-AAA ACC~AAr~G~-r~ ACCCT QTGA ~ 'c'~r~ hC CCCTGAGGTC A Q'~v~-v~vG 1620
CA 02224625 1997-12-12
W O 97/00079 PCTrUS96/10043
~ G~ACGT ~r~rar~a GACCCTGAGG TCAAGTTCAA CTGvTACGTG r-a~GO~GG 1680
AVVTC~ATaA ~C~P--~~A AA~GGG ~--~--~~r,~A ~Ar~rl~G TA~'C~X~,~G 1740
TCAGC~-C~- CACC~.C~.C ~aCr~ ~T GGCTGAATGG r~ ,TAC AAGTC~a-~ 1800
TCTCr~ r~C~C~A GCC'CC~CG ~ -r~T CTCr~~C~ AAAC~v~ 1860
aGG~ cc~ r~ CATGr~-ara G~C~lOG cccr~-ccTc ,~CC~.~AGA 1920
GTG~~CG~TG Tp~~r-ApccTc ~V~c~Ar-~G GGr~CCCCG AnAAr,rarAr GTGTAr~~CC 1980
,GCCCC~A~C CCC~_~TGAG cT~raAr-A ACCAGGT Q G CCTGACCTGC ~.vv.~fAAG 2040
G~-~ ATCC r~~Cr~~~TC vC~AGT CC~ T~G~c-A~CCr~ G~ ~aa~T 2100
~-~-A-~r~G G~-'~v~G CTGGACTCCG AOGG~ .. ~..O~- --AC P~A~_CTCA 2160
A rar~rAr~GTGG r~ rJcGrA AC~-~ ~~C A~ C~-G ATGCATGAGG 2220
~,~.A~-A~ CrarTarA~C r-A~Ar-A~C~ ~ C ~coGG~AaA TGA~TGCGAC 2280
a~ccc~c 2287
(2) INFORMATION FOR SEQ ID NOs9s
~i) SEQUENCF CHARACTERISTICS:
lAj L~NGTHs 442 amino acid~
~B~l TYPEs amino acid
~C, ST~ -CSs not r~.dr.L
~DJ TOPOLOGY: linear
(ii) M~n~CnT-~ TYPEs prot~in
(xi) 8EQUEN OE ~~DTPTIONs 8EQ ID NOs9s
Ly~ Leu Thr Thr Met Asp Trp Thr Trp Arg Phe Leu Phe Phe Val Val
1 5 10 15
Ala Ala Ala Thr Gly Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly
Ala &lu Val Lyn Lys Pro Gly Ser Ser Val Ly- Val 8er Cy~ Ly- Ala
8er Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly LQU Glu Trp ~Qt Gly Gly I1~ I1Q Pro Ile Phe Gly
Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala
Asp Glu Ser Thr Ala Arg Asp Ann Gly Ala Tyr Cyo 8er Gly Gly Ser
100 105 110
CA 02224625 1997-12-12
W O 97/00079 PCTAJS96/10043
- 42 ~
Cyu Tyr 8Qr Gly Trp PhQ Asp Pro Trp Gly Gln Gly Thr Leu Val Thr
115 120 125
Val 8er 80r Ala 8er Thr Ly-~ Gly Pro 8er Val Phe Pro LQU Ala Pro
130 135 140
8er Ser Lys Ser Thr Ser Gly 61y Thr Ala Ala Leu Gly Cyl~ Leu Val
145 150 155 160
~ys Asp Tyr Phe Pro Glu Pro Val Thr Val 8er Trp A~n Ser Gly Ala
165 170 175
~eu Thr 8er Gly Val Hi~ Thr Phe Pro Ala Val Leu Gln 8er 8er Gly
180 185 190
L~u Tyr 8er Leu 8er Ser Val Val Thr Val Pro Ser 8er 8er ABP Ly~
195 200 205
Lys Val Glu Pro Lys SQr Cys Asp Lys Thr Hi~ Thr Cy~ Pro Pro Cy~
210 215 220
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe LQU Phe Pro Pro
2Z5 230 235 240
~y~ Pro Ly~ Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
~al Val Val A~p Val 8er Hi-~ Glu ABP Pro Glu Val Lyl~ Phe A~n Trp
260 265 270
Tyr Val A~p Gly Val Glu Val Hi~ A~n Ala Ly~ Thr Ly~ Pro Arg Glu
275 280 285
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val LQU
290 295 300
Hi~ Gln Aep Trp Leu A~n Gly Ly~ Glu Tyr Lys Cy~ Ly~ VA 1 8~r Affn
305 310 315 320
~y~ Ala Leu Pro Ala Pro IlQ Glu Lys Thr Ile Ser Ly~ Ala Lyff Gly
325 330 335
~ln Pro Arg Glu Pro Gln Val Tyr Thr L~u Pro Pro SQr Arg A~P G1U
340 345 350
LQU Thr Ly~ A~n Gln Val Ser Leu Thr Cy~ Leu Val Ly~ Gly Pho Tyr
355 360 365
Pro Ser A~p Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
A~n Tyr Lyll Thr Thr Pro Pro Val LQU A~p Ser Asp Gly Ser Phe Phe
385 390 395 ~00
~eu Tyr Ser Ly~ Leu Thr Val A~p Ly~ Ser Arg Trp Gln Gln Gly A~n
405 410 415
~al Phe Ser Cys Ser Val Met HLs Glu Ala Leu Hi~ Asn HL~ Tyr Thr
420 425 430
Gln Lyl~ 8er LQU Ser Leu Ser Pro Gly Ly
435 440
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(2) ~ ~TION FOR 8BQ ID NOslOs
(i) SEQUBN OE rUARArT~TSTICSs
~A'l LENGTHs 1894 ba~e pair~
BI TY~Es n~ls~c ~cid
C I STRP~ css singlQ
~DJ TOPOLOGYs linear
( ii ) ~ICIT.~T ~r TYPl!: S DNA ( j i C~ )
(xi) SEQU~N OE n~-~r~TPTION: SEQ ID NOslOs
A.GvCv~- v~ C~.vvv.. ~-. TACAGTCCTG AvC~. ~.AC ~.~.v~.G~A PGCCr~~~TC 60
C Q .,v.v~G ~~CT~~T ACCvv.vCcC ATr-~Cr~A~G CC~f~C-~ 7A CCAGAT Q CT 120
vGCAAGTGGT TTTATATCGC A-.CGo~ ~r-AAA~rAr7G AGTArAATAA v.C4v..~AG 180
CAGATCCAAG Q AC~ .. TTACTTCACC ccrAAr~ rr~ r GA. ... C 240
r~~~~~TACC r~~~cr-~~A Gr-Acr-~-TGc ATCTA~AArA Cr~~CTP~cT GAATGTCCAG 300
C~C~~'TG Gr-~rJ~TCTC ~T~rGT~v G~-~GGCr~- AGCA...C~vC~ TCA~..v~.v 360
ATCCTCAGGG p~~~r~ CTACATGCTT G~-~ GACv Tr-AArr~TGA ~ ~T¢~v 420
CG6~v~ 7 TCTATC~TvA r~~Cr~~~~ ~r~~r~C P~CTGGv AGAGTTCTAC 480
GAAC~---~a A~v~-~vCYv QTTCCQAG TCAGATGTCG TGTAr~ TTGr~ ~ 540
~'~T~r--TVT~7 ~r~~TGVA ~r--~r~ r--~ G~r'~~~r~G~ G~~CGGr~ 600
TCGGATCCCG AGGGTGAGTA CTAAr~CTTQ GCG~.~.GC CTGr-Ar~~AT CC~G~A~G 660
r~C~r~nT ~r~ AAr~ r-acc CCv- vCCT cTTr-Arc~GG AGC~ vCC 720
CGC~Cr~rTC ATGCT Q GGG ~ ~h.~ Gv~ CCr~~GCTCT G~C~~CC'~ 780
~'CCT7~'GTG CCC~TAArCC ~_GCC~;A r~ AAr,GGG Qv~ aG GCTr~ CT 840
GCr~P~~CC ATA.C~GGvA Gr-Ar,CCTGCC CCTC~~CTAA GCCr~rCCCA APGGCC~ 900
TCTCCACTCC CTCAGCTCGv ACAC~ C~;~ 'C-;AGA TTCCAGTAAC TCCCAATCTT 960
~, GCA~ P~CC~AAATC TT~vTC~AA ACTr~r~r~T G~CrArCGTG CCCAGGTAAG 1020
C~CCr-~-G C~-,~CC~,' QGCTQAGG CGGr-~r~r-GT GCC~T~r-~r-T AGC~ATC 1080
r~~Gr~~~~~, CCCCAr-CCGG ~.~.~A Q C GTC Q CCTCC A~ ~-, r~rr~rCTGA 1140
A~.C~.GGGG GGACCGT QG ~..C~. .. CCCCC'~AAA ccrAAr,rA~A CCCT Q TGAT 1200
~.CCCG'-~~C CCTGAGGTCA Q-.GC~.~. GGTGGACGTG AGCCAr~-AAC ACCCTGAGGT 1260
CAAGTT QAC TGGTACGTGG A~GGC~.GCA GCTGrATAAT GCrAAr-ArAA ~GccGc~cr-~ 1320
G~PGC~C,TAC AArAr,rArGT ACC~G~.~. CAGC~-~.C ACC~.C~.GC ACr-~Gr-Ar,TG 1380
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GCTGAATGGC aAr-r-~r-TACA AGTGCAAGGT CTC~Ar~AA G~C~.CC~AG CCCC~CGA 1440
~ --r~C TCr~''-CC~ AAG~-~G~AC CO~.~4G~.G Cr-~rAGGC~r ATGr-~r~r-~r- 1500
GC~C~h-,OCG C~C'~CCTCT GCC~.~AGAG TC~-OCCTGT ~ -CTCT GTCCT~ 1560
G~r-'Y'CO~ r'-'-c TGT~-'-CCT GK'CC'C~CC CGGGATGAGC T-'-~''-~' 1620
CCAGGTCAGC CTGACCTGCC TGGTCAAAGG CTTCTATCCC ~-C~'-A~C~ CC~.~AGTG 1680
a--~--~----~T occ--~--ccc~ Ar~ArTA r~Ar~r~ ~--C~--GC TGGACTCOGA 1740
C~GL.C~.-C ..C~.~ACA G~CTCAC CGTGr-~r~Ar- AGCAGGTGGC ~--'-GrJr~' 1800
. ~A ~ 3C~-~A TGCATGAGGC TCTC-~-''- CAC~GC ~r~ ~c~ 1860
~-.C~.~.~-- CC~.AAAT GAGTGCr-~rG GCCG 1894
(2) lh~ ~ATIoN FOR SEQ ID NOslls
~i) 8EQUEN OE ~U~T~TCTICSs
~AJ LENGTHs 437 amino ~cLd~
rB TYPEs ~mino ~cid
,C~ S~Nn~nNESSs not r~l~_~L
~DJ TOPOLOGYs linear
( Li ) MOT-~C~T-~ TYPBs prot~in
(xi) SEQUBNCL D~CrPTPTIONs 8EQ ID NOslls
Met Ala LeU 8er Trp Val Leu Thr Val L~U 8~r LOU L~U Pro LeU L U
1 5 10 15
Glu Ala Gln Ile Pro Leu Cy# Ala Asn Leu Val Pro Val Pro Ile Thr
2S 30
A~n Ala Thr Leu Asp Cln I 1Q Thr Gly Ly~ Trp PhQ Tyr Il- Ala 8er
~5
Ala Phe Arg A0n Glu Clu Tyr A~n Lys Ser Val Gln Glu Ile Gln Ala
Thr Phe Phe Tyr Phe Thr Pro Asn Lys Thr Glu Asp Thr Ile Phe Leu
Arg Clu Tyr Gln Thr Arg Gln Asp Gln Cy~ Il~ Tyr Asn Thr Thr Tyr
Leu Asn Val Gln Arg Glu A~n Gly Thr Ile Ser Arg Tyr Vnl Gly Gly
100 105 110
Gln Glu HL~ Phe Ala Hi~ Leu Leu Ile Leu Arg Asp Thr Ly~ Thr Tyr
115 120 125
MQt L~U Ala Phe Asp Val Asn Asp Glu Ly~ A~n Trp Gly Leu 8er Val
130 135 140
Tyr Ala Asp Lys Pro Glu Thr Thr Lys Glu Gln LQU Gly Glu Phe Tyr
145 150 155 160
Glu Ala Leu Asp Cys Leu Arg Ile Pro Lys Ser Asp Val Val Tyr Thr
165 170 175
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Asp Trp Lys Lys Asp Lys Cys Glu Pro LQU Glu Lys Gln Hi~ Glu Ly~
180 185 190
alu Arg Ly~ Gln Glu Glu Gly Glu 8~r A~p Pro Glu Gly Glu Pro Ly~
195 200 205
S~r Cys Asp Lys Thr E~is Thr CYB Pro Pro Cy8 Pro Aln Pro Glu Leu
210 215 Z20
LQu Gly ¢ly Pro 8Rr Val Ph~ IA~U PhQ Pro Pro Lys Pro I.y~ Asp Thr
225 230 235 240
~Qu M~t I 1~ 8er Arg Thr Pro Glu Val Thr Cy~ Val Val ~al Asp Val
245 250 255
~~r Hi- Glu Asp Pro Glu Val Lys Ph~ Asn Trp Tyr Val Asp Gly Val
260 265 270
Clu Val EIis Asn Ala Lys Thr Lys Pro Arg Glu Glu ¢ln Tyr A~n Sor
275 280 285
Thr Tyr Arg Val Val S~r Val L~u Thr Val Lou His Gln Asp Trp L~u
290 295 300
Asn Gly Lyi~ Glu Tyr Lys Cy8 Lys Val S~r Asn Lys Ala L~u Pro Ala
305 310 315 320
~ro Ilo Glu Ly~ Thr Ilo 8er Lyt~ Ala Lys Gly Gln Pro Arg Glu Pro
325 330 335
~ln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Ly~ A~n Gln
340 345 350
Val SQr LQU Thr Cy~ Lau Val Lya Gly ~PhQ Tyr Pro S~r A~p I 1Q Ala
355 360 365
Val Glu Trp Glu S--r Asn Gly Gln Pro Glu Asn A-~n Tyr Lyff Thr Thr
370 375 380
Pro Pro Val L~u Asp Ser Asp Gly S~r Ph~ Ph~ L~u Tyr Slar Lys L~u
385 390 395 400
~hr Val A~p Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 8~r Cy~ S~r
405 410 415
~al N~t His Glu Ala L~u HLs Asn EIi8 Tyr Thr Gln Lys 8~r ~au Scr
420 425 430
L~u S~r Pro Gly Lys
435
(2) lN~.~ATION FOR SEQ ID NOsl2s
i ) SEQUENOE CHARACTkRISTICS s
lA' LENGTH: 442 amino acids
B, TYPE : amino acid
~C, sT~ n~ lzcSs not rolHvant
~D~ TOPOLOGYs lin~ar
TYPE s prot~in
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(xi) 8EQU~N OE ~-C~TPTIONs SEQ ID NOsl2s
Ly~ L u Thr Thr ~et Aop Trp Thr Trp Arg Phe Leu Phe Ph~ Val Val
1 5 10 15
~la Ala Ala Thr Gly Vnl Gln S~r Gln Val Gln Leu Val Gln 8Qr Gly
Aln Glu Val Ly~ Lys Pro Gly Ser S~r Val Lys Val Ser Cy8 Lys Ala
Ser Gly Gly Thr Phe 8er Ser Tyr Ala Il- Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Lou Glu Trp ~et Cly Gly Ilo Ile Pro Ile Pho Gly
~hr Ala Asn Tyr Aln Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala
~~p Glu Ser Thr Ala Arg Asp Asn Gly Ala Tyr Cy~ Ser Gly Gly Ser
100 105 110
Cys Tyr 8Or Gly Trp PhQ Asp Pro Trp Gly Gln Gly Thr Lou Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Ly~ Ser Thr Ser Gly Gly Thr Ala Ala Lou Gly Cyo L~u Val
145 150 155 160
~yo Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn SQr Gly Ala
165 170 175
~~u Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Sor Val Val Thr Val Pro Ser Ser S~r A~p Lyu
195 200 205
Ly- Val Glu Pro Lys S~r Cy8 Asp Lys Thr Uis Thr Cy~ Pro Pro Cy-
210 215 220
Pro Ala Pro Glu Leu L~u Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
~y~ Pro Ly- A~p Thr Lou Nbt Ile Sor Arg Thr Pro Clu Val Thr Cy~
245 250 255
~al Val Val Asp Val Ser His Glu Asp Pro Glu Val Asn Phs Ser Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Asn Lys Thr Lys Pro Arg Glu
275 280 285
Glu A~n Tyr Ssr Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
Hio Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lya Cys Asn Val Ser Asn
305 310 315 320
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Ly0 Ala LQu Pro Ala Pro Il~ Glu Lys Asn Il~ 8~r ~y~ Ala Lys Gly
325 330 335
ln Pro Arg alu Pro aln Val Tyr Thr L~u Pro Pro S~r Arg Asp alu
340 345 350
Lou Thr Lys Asn Gln Val 8~r Leu Thr Cy~ LQu Val Ly~ Gly Ph~ Tyr
355 360 365
Pro S~r A~p Il~ Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu A~n
370 375 380
A~n Tyr Ly~ Thr Thr Pro Pro V~l L u Asp 8~r Asp ¢ly 8Or Pho Pho
385 390 395 400
Lou Tyr 8er Lys Leu Thr V 1 Asp Ly~ Ser Arg Trp Gln Gln Gly A~n
405 410 415
Val Phe Scr Cys sQr Val ~t ~is Glu Ala Leu His Asn His Tyr Thr
, 420 425 430
Gln Lys 8er Lou Ser Leu Ser Pro Gly Lys
435 ~40
(2) INFORMATION FOR SEQ ID NO t 13s
(i) SEQUEN OE rUA~CT~RTCTICSs
~A' LENGTHs 42 amino acid~
IBI TYPBs ino acid
,CJ ST~ ASS not ~al~.~n~
~DJ TOPOLOGYs linQar
~ii) M~T~C~T-~ TYPEs prot~in
(xi) SEQUBN OE ~C~RTPTIONs SEQ ID NOsl3s
Pro alu Mct L u Arg Asn S~r Thr Asp Thr Thr Pro L~u ~hr aly
1 5 10 15
Pro Gly Thr Pro Glu S~r Thr Thr Val Glu Pro Ala Ala Arg Arg sQr
20 25 30
Thr aly L~u A~p Ala Cly Gly Ala Val Thr Glu
~2) l~ndATION FOR SEQ ID NOs14s
(i) SEQUEN OE C~ARACT~RISTICSs
~AJ LENGTHs 16 am~no acids
IBI TYPEs amino acid
,C, S~R~Nn~n'~CSs not rQlQvant
~DJ TOPOLOGYs linoar
(ii) ~nT.~.~ TYPEs prot~in
(~i) SEQUEN OE D~-C~-RTPTIONs SEQ ID NOsl4s
LQu Thr Thr Glu L~u Ala A~n NQt Gly Asn L~u S~r Thr Asp S~r Ala
1 5 10 15
