Note: Descriptions are shown in the official language in which they were submitted.
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1.
WO 96/11012 PCT/US95/13736
HEkl~TOPOIE:TIC CE:LI- L-8E:L~ N I.IGAND
~liI-) AND T~E:RAPEtrq!IC8 THBRE:OF
R~R~ROC12nD OF l~le l~V~'ON
TECHNICAL FIELD
The present invention involves the
development of compounds which can regulate and
control the function of adhesion molecules.
BA~K~-~OUND ART
The adhesion molecules are involved in
the fundamental control of cell-cell interaction
and cellular migration. Adhesion molecules
regulate diverse proc~s~e- in inflammation,
hematopoiesis and tumor metasta6is. (Woodruff, et
al, 1987; Springer, et al, 1987; Sharon and Li~,
1993; Scakstein, 1993) For general review6 on
adhesion molecules see Carlos and Harlan, 1994 and
Chin et al, 1991. It would be u6eful to develop
reagents which can control and regulate the
adhesion proteins, particularly within the selectin
family.
suBsrllurE SHEET (RULE 26
.
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Tle peripheral lymph node "homing
receptor", L-selectin (CD62L), is a -75 kDa
glycoprotein which mediates attachment of
lymphocytes to lymph node (LN) high endothelial
venules (HEV), an adhesive interaction which is the
fir6t step in the migration of lymphocytes from
blood into lymphoid ti6sues (Gowans and Knight,
1964; Marchesi and Gowans, 1964). This trafficking
of lymphocytes from blood into lymph nodes is
markedly nonrandom and is initiated by specific
adherence of the lymphocytes to HEV. The "lymph
node homing receptor" or L-6electin (LECAM-l) i6
the principal lymphocyte membrane glycoprotein
mediating this attachment.
The L-selectin protein is recognized by
a variety of monoclonal antibodies (mAbs) in humans
{Gatenby et al., 1982 (Leu-8); Reinherz et al.,
1982 (TQ-l); Tedder et al., 1990 (LAM)} and is a
member of the selectin family of adhesion
molecules, which includes P-selectin (CD62P) and E-
6electin (CD62E). Selectin6 6hare a common
structure consisting of an amino-terminal calcium-
dep~nAPnt lectin domain, an epidermal growth factor
domain, a variable number of repeat sequences
bearing homology to complement regulatory and
catalytic proteins b~ ~A~n~ C3b or C4b, a
transmembrane portion, and a C-terminal cytoplasmic
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tail (Bevillcqua and Nelson, 1993; Rosen, 1993).
The molec~llA~ weight varies among leukocytes due to
posttranslational glycosylation among ~ubsets of
leukocytes.(Carlos and Harlan, 1994) The lectin
domain of these proteins directs their adhesion to
carbohydrate molecules pre6ent on the cell surface.
The adhesive interaction between
lymphocytes and HEV has been extensively analyzed
u~ing an in vitro bi~in~ a~6ay (Stamper and
Woodruff, 1976). This a6say is performed under
shear at 4C, whereby binding mediated by L-
selectin i~ maximized and effects of other adhesion
molecules are minimized (Shaw et al, 1986; Spertini
et al., 1991). The interaction of L-selectin with
its corresponAi n~ ligand(s) on HEV i~ calcium-
dep~n~nt (Woodruff et al., 1977) and requires the
presence of sialic acid (Rosen et al., 1985; True
et al., 1990) and ~ulfate (Imai et al., 1993) on
the ligand(s~. L-selectin behaves a~ a lectin and
recognizes sialylated, high mannose residue~ on its
corresponding ligand which i5 identified by the
monoclonal antibody MECA-79 (Sack~tein, 1993).
MECA-79 identifie~ an L-~electin ligand on lymph
node HEV and which cross-reacts with GLYCAM-l and
CD34. In v~tro adherence of lymphocytes via L-
selectin can be inhibited by carbohydrate6 such a6
mann~-~ 6-pho~phate (man-6-P), PPME (Phosphomannan
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monoester core from Hansenula hostii, a
phosphomannosyl-rich polysaccharide), and fucoidin
(a sulfated, fucose-rich polysaccharide) (Stoolman
and Rosen, 1983).
Ligands for L-selectin have thus far been
characterized on murine endothelial cells utilizing
a murine L-selectin IgG chimera molecule as a
probe. (Watson et al., 1990) This approach has
identified two proteins, GlyCAM-l (SgpS0) (Imai et
al., 1991) and CD34 (Sgp90) (Baumhueter et al.,
1993), present on endothelial cell6. GlyCAM-1 is
a novel sialomucin, and its role as a ligand for L-
selectin is its only known function (Lasky et al.,
1992). Although present on endothelial cells in
most tissues (Beschorner et al., 1985), CD34 is
best known for its expression on the earliest
multilineage colony-forming hematopoietic stem
cells (Civin et al., 1984).
Hematopoietic progenitor cell~
characteristically express both L-selectin and CD34
(Terstappen et al., 1992), and there is growing
evidence that L-selectin plays a role in
hematopoiesis (Terstappen et ~l., 1993; Kobayashi
et al., 1994). The characterization of L-6electin
and its ligands among progenitor cells is of
considerable interest as adhesion proteins regulate
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cell-cell and cell-stromal interactions fundamental
to hematopoiesi~.
In general assays for determining the
adhesion between lymphocyte and HEV requires the
use of frozen-gections of lymph nodes.(Stamper and
Woodruff, 1976; Sackstein et al, 1988) It would be
useful to be a~le to use cells in suspension in the
a~say also. This would enable the use of cell
lines, giving ri~e to more reproducible result~ as
well as reducing the need for surgical procedures
for lymph node removal.
It would be useful to have ~trategies
which would allow regulation of hematopoiesis cince
it i6 regulated by cell-cell and cell-stromal
interaction~. For example, Terstappen et al (1993)
have shown that activation of L-selectin increases
the clonogenic capacitiy of stem cells.
During recovery of immune function
following bone marrow transplantation pathologic
changes have been observed following
transplantation which interfer with lymphocyte
migration and HEV integrity. Further, in addition
to changes in lymph node structure, alterations in
lymphocyte migration can occur ~econAAry to the
effect of pharmacologic agent6 used in
posttransplant therapy such as corticosteroid6
(Sackstein, 1993). It would be useful to ha~e an
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agent which can assist in reestablishing lymphocyte
trafficking and 80 immune function following bone
marrow tran~plantation.
The crucial role of adhesion molecules in
controlling and directing the inflammatory process
indicate~ that a reagent which interfere~ with the
proce~s, i.e. anti-adhesive, could have anti-
inflammatory properties.
Further, cell adhesion molecules are
involved in metastasis, therefore it would be
u~eful to develop an anti-adhesive which has anti-
metastatic properties. In particular, with the
identification of L-selectin on hematopoietic
cells, it would be useful to have an anti-adhesive
that affects L-~electin in leukemia to decrea~e the
growth and ~pread of malignant hematopoietic cell~
throughout the body.
Further, it would be useful to have
additional cell marker~; and monoclonal an~; hoA; es
directed against these cell markers to allow for
cell targeting.
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.
8~MMARY OF THE lNv~..~ON AND ADvaNTAGE8
According to the present invention, an
isolated and purified glycoprotein and functional
analogs are disclosed. The glycoproteins are
characterized by being expressed on at least
primative hematopoietic cell6, and being a ligand
for L-selectin. The binA;n~ of ligand to L-
selectin i~ not inhibited by anti-CD34 antibodies
nor by MECA-79 monoclonal antibody.
BRIEF D -~PTPTION OF T~E DRA~ING8
Other advantages of the pre~ent invention
will be readily appreciated as the same becomes
better understood by reference to the following
detailed description when considered in connection
with the accompanying drawings wherein:
FIGURE lA-B are photomi~G~L~ph6 of
cytospin preparations of RGla cells demonstrating
adherence of lymphocytes (6mall dark dots), (A)
Lymphocytes adhere to KGla in the pre6ence of CD45
or i60type control Abs, (B) Lymphocyte binding
a6say in the presence of LAN1-3 Ab (anti-L-
selectin);
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F~GURE 2 is FACS profiles of lymphocytes
used in the binding assay after incubation with
isotope-matched IgG control, LAM1-3, or anti-CD45
Abs, followed by GAM-FITC, results shown are
representative of 3 independent experiments;
FIGURE 3A-D are FACS profile6 of KGla
cells sorted by FACS prior to the binding assay
into CD34+ and.CD34- fractions using mAb HPCA-2PE,
sorted cell fractions were restained for CD34 using
mAb QBENDlO-FITC and analyzed, positive and
negative sorted fractions were >90% and <lO~
positive for CD34, respectively, results shown are
representative of 3 independent experiments;
FIGURE 4 i8 a photomicrograph showing the
lymphocyte adherence assay performed on the sorted
cells, and no differences in lymphocyte adherence
were evident among the CD34+ and CD34- populations,
adherence to the CD34 negative fraction is shown;
and
FIGURE 5A-F are FACS profiles of COS-7
cell~ were transfected with either CD34-pCDM8 (E,F)
or pCDM8 (mock~ C,D), then analyzed by FACS and
compared to KGla (A,B) for CD34 expression, Abs
used were isotype-matched IgGl ~u.lLLol and anti-
CD34 mAb QBENDlO, Lymphocytes did not adhere to
CD34-transfected COS-7 cells, despite higher levels
of CD34 expres6ion a6 compared to KGla cells.
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D~T~TT~n DE8CRIPTION OF THB PRE:FERRED E:NBODIME:NT
The present invention provides an
isolated and purified glycoprotein and functional
analogs thereof. Analog is defined as a molecule
that will be generally at lea6t 70~ homologous over
any portion that i5 functionally relevant. In more
preferred embodiments the homology will be at least
80% and can approach 95% homology to the amino acid
sequence of the protein segment of the
glycoprotçin. The homology will extend over a
region of at least 8 contiguous amino acids to 80
contiguous amino acid~. The amino acid 6equence of
an analog may differ from that of the glycoprotein
of the present in~ention when at least one residue
is deleted, inserted or substituted. The molec~lAr
weight of the glycoprotein may vary between the
analog and the present invention due to
carbohydrate differences. Differences in
glycosylation may be present between the analog and
the present invention.
The gly~oproLein has the folIowing
functional characteri6tic6. It is expres6ed on at
least primative hematopoietic cell6. The
gly~o~o~ein is a ligand for L-6electin. The
ligand binding to L-select1n i8 not inhibited by
anti-CD34 ant~hoAies and is not reco~n~zed by the
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--10--
. I
MECA-79 monoclonal antibody. The glycoprotein is
designated hereinafter as hematopoietic cell L-
selectin ligand, HLL.
Further, the glycoprotein, HLL, is a
membrane associated glycoprotein and functions as
an adhecion protein ligand. The gly~oplGLein
facilitates attachment of lymphocyte~ to
hematopoietic cells including primitive
hematopoietic cells.
The present invention also provides for
an antibody directed against the glycoprotein, HLL.
The anti hoA~ es may be either monoclonal or
polyclonal. Murine monoclonal ant~boA~es are
initially r~; fi~A against RGla cells. The
monoclonals that are generated are then screened
for the ability to- block lymphocyte binding to
KGla.
Utilizing the~e monoclonal antiho~est
the gl~o~G~ein is isolated by immunoprecipitation
of KGla membrane lysates as is stA~A~rd in the art
and used for the production of further antibodies
as needed. Such methods can be found described
Sambrook et al, Molecular Clon~n~: A L&boratory
MPn~7 ~ Cold Springs Harbor, New York, 1989, as
well as additional methods of isolation and
purification as are known in the art.
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Additionally, the antibodies may be
prepared against a synthetic peptide based on the
6equence, or prepared recombinantly by cloning
~er-h~ ques or the natural gene product and/or
s portions thereof may be isolated and used as the
immunogen. Such proteins or peptides can be used
to produce antibodies by stAn~rd antibody
production technology well known to those skilled
in the art as described generally in Harlow and
Lane, Ant~ho~;es: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY, 1988.
For producing polyclonal antihoAies a
host, such as a rabbit or goat, is immunized with
the protein or peptide, generally with an adjuvant
and, if neces~ary, coupled to a carrier; ant;ho~;es
to the protein are collected from the sera.
For producing monoclonal antibodies the
te~n;gue involves hyperimmunization of an
a~plo~iate donor with the protein or peptide
fragment, generally a mouse, and isolation of
splenic antibody producing cells. These cells are
fused to a cell having immortality, such as a
myeloma cell, to provide a fused cell hybrid which
has immortality and secretes the required antibody.
The cells are then cultured, in bulk, and the
monoclonal ant1hoA~e6 harvested from the culture
media for use.
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The antibody can be bound to a solid
support substrate or conjugated with a detectable
moiety or be both bound and conjugated a6 is well
known in the art. (For a general discu~sion of
conjugation of fluorescent or enzymatic moieties
see Johnstone & Thorpe, Immunochemistry ~n
Pr~ctice, Blackwell Scientiflc Publlcations,
Oxford, 1982.) The binding of an~ihoA~efi to a
~olid DU~O~ ~ substrate i6 also well known in the
art. (see for a general ~irc~ ion Harlow & Lane
Ant;ho~ies: A L~boratory Mqm~AI, Cold Spring Harbor
Laboratory Publications, New York, 1988) The
detectable moieties contemplated with the present
invention can include, but are not limited to,
fluorescent, metallic, enzymatic and radioactive
markers such as biotin, gold, ferritin, alkaline
pho~phata~e, B-galactosida~e, peroxida6e, urease,
fluore6cein, rhodamine, tritium, 14C and
iodination.
The method of targeting cells includes
the step6 of preparing an~ho~e6 directed against
the gly~o~L~e~n as described above and coupling
the ant~h~ies to the a~op~iate agent whether for
cell killing, cell selection or cell
identification. For cell k~ to~Y~nR such a6
ricin A chain, ~ omonas exotoxin A, diphtheria
toxin, other plant and bacterial toY~n~ a6 well as
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chemotheraplutic compounds can be coupled in the
present invention forming an immunotoxin. For a
general review of the antibody-toxin art see
Ramakr~h~n, 1990.
Cell targeting requires exposing a
population of cell~ to the immunotoxin. A toxin
bound antibody can be administered to the
appropriate patient and targeted cells killed ~n
v~vo. The immunotoxin is administered and dosed in
accordance with good medical practice, taking into
account the clinical condition of the individual
patient, the site and method of administration,
~r~ ng of administration, and other factors
known to medical practitioners. The "effective
amount" for purposes herein is thus determined by
such considerations as are known in the art. The
amount must be effective to achieve at least 25% of
the treated patient~ exhibit im~ovement including
but not limited -to improved survival rate, more
rapid recovery, or improvement or elimination of
6ymptoms.
Alternatively, cells can be removed from
the patient and treated ex v~vo selectively. For
example, cells expressing HLL can be removed
through complement-mediated lysis from the ex v~vo
population and the remaining cells r eL~L..ed to the
patient. Additional cell removal can be undertaken
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utilizing ce~l sorting, l~pAnning~ and magnetic bead
separation. Further, utilizing cell sorting,
~lp~nning~l magnetic bead separation and the like
cell populations can be enriched for HLL bearing
cells and this enriched cell population returned to
the patient.
The targeted cells to be removed are
cell~ expressing HLL and can be selected from the
group consisting of leukemic cells, malignant
hemopoietic progenitor cell~, or any malignant cell
expressing the marker.
The present invention also provides a
method of regulating hematopoiesis, particularly in
reconstitution of the immune system following bone
marrow transplantation. The present invention
includes the steps of selecting those cells with
high(+) or low(-) expres6ion of HLL depenAing on
the growth characteristics associated with the
marker density needed by the patient. The
selection proGed~e utilizes ex v~vo methods a6
described herein. After selection, the selected
cell type is cultured ~n v~tro, if ne~A to eYp~nA
the population using s~A~rd methods known in the
art. The patient is then infused with the
~rAnAPA, enriched HIL+ or HLL- population as
n~eA~d ~
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T~e present invention further provides a
method of regulating inflammatory re~ponse by
interrupting cellular migration into lymph nodes
and sites of both acute and chronic inflammation
including the step of administering to the patient
antibody directed agains HLL thereby disturbing
cellular migration mediated by ~-T- by blocking the
the lymphocyte attachment site and can be injected
directly at the inflammed ~ite if nee~eA. The
regulation of the inflammatory response would be
useful in autoimmune disorders, post-ischemic
tis6ue injury and sepsis (Calos and Harlan, 1994).
Administration and effective dose are as described
for immunotoxins hereinabove.
Studies by applicant on the interaction
of L-selectin and hematopoietic CD34 function as an
adhesive receptor-ligand pair, in v~tro binding
studies of lymphocytes to XGla, a primitive CD34-
positive human cell line derived from an acute
myeloid leukemia (Civin et ~1., 1994; Koeffler et
al., 1980) lead to the pre~ent invention. These
studies surprisingly revealed highly specific
adherence of lymphocytes to KGla cells mediated by
L-selectin on the lymphocyte, but uneYrectedly not
involving CD34 as the corresronA~ ligand as had
been previously Le~O~ Led (Baumhueter et al, 1993;
Oxley and Sackstein, 1994). The result6 indicated
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j
the presence of a ligand, designated HLL, for L-
selectin on the surface of this hematopoietic
progenitor cell line and provide the fir~t evidence
of L-selectin-mediated adhesion between lymphocytes
and a non-endothelial cell type.
For these 6tudies, the lymphocyte-HEV
adherence assay was used which is an in vitro
approximation of physiologic adhe6ion mediated by
L-~electin was used. It ha6 been a fundamental
tool in 6tudying the function of L-6electin in its
native state on the surface of lymphocytes. This
binding assay was novelly adapted to examine
lymphocyte-hematopoietic cell adhesion, and the
results provide the unexpected results of L-
selectin-dependent adhesive interactions between
lymphocytes and non-endothelial cell6.
The adaptation of the assay allowed for
the first time the u~e of cell lines in a
lymphocyte-HEV adhe~e,.ce assay. In this assay,
slides were prepared of KGla cell suspensions which
were used in place of slides of frozen lymph node
sections a8 taught by the prior art. The KGla
cells were placed on the slides by using cyto-spin
centrifugation as further described herP~nhPlow.
Several ~n~Pp~n~ent lines of evidence
indicate that lymphocyte b~n~1ng to RGla is
mediated primarily, if not 6clely, by L-~electin.
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Fir6t, an anti-L-6electin mAb (LAM1-3) previously
shown to block L-selectin-mediated adherence to LN
HEV tspertini et al., 1991), completely inhibited
PBL from binding to KGla or LN HEV, wherea6 anti-
CD45 and i60type control Abs did not blocklymphocyte binding. Second, L-selectin-mediated
binding is a calcium-dependent event, and
lymphocytes were unable to bind to KGla in the
presence of the calcium chelator EDTA. Third,
carbohydrates 6uch as man-6-P, PPME, and fucoidin
inhibited lymphocyte adherence to RGla. These
compounds are all known to bind to L-6electin and
to inhibit lymphocyte binding to HEV in the in
vitro a6say (Stoolman and Rosen, 1983; Stoolman et
al., 1984). Lastly, it is known that PMA treatment
of lymphocytes causes shedding of membrane L-
selectin via a protein kinase C activation pathway,
and corre6ponds to the loss of lymphocyte binding
to LN HEV in the ~n v~ tro as6ay (Tedder et al.,
1990). In these studies, PNA-treated PBL were no
longer able to bind to XGla.
The nature of the ligand wa6 investigated
by determining the effect6 of various enzyme
treatment6 of KGla on the binding capacity.
Previou6 6tudies have shown that ligand expres~ion
of sialic acid is es6ential for L-selectin-mediated
b~ n~ ~ ng of lymphocytes to LN HEV (Rosen et al.,
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1985). In the pre6ent example6, neuraminida6e-
treated KGla showed a complete 1058 of lymphocyte
binding, indicating that 6ialic acid re6idue6 are
al60 a neces6ary component on the RGla L-6electin
ligand; a6 ~uch, lymphocyte adherence to KGla
involves carbohydrate motif6 and i6 not ba6ed
6trictly on protein-protein interactions. This
fin~;ng, combined with the re6ults of protease
experiment~, indicate6 that the KGla ligand i8 a
10 glycoI~GLein.
To examine whether 0-linked
glyco6ylations on the ligand play a central role in
the adhesive interaction, KGla were digested with
the enzyme 0-fiialoglycu~LoLein endopeptidase which
6pecifically cleave6 protein6 at 6ite6 of o-linked
sialo-glycosylation (Abdullah et al., 1992) and
which has been shown to differentially cleave
epitope6 of the CD34 molecule (Sutherland et al.,
1992). The data reveal that treatment of KGla in
suspension with the enzyme actively de~LLoyed CD34
epitopes, yet had no effect on lymphocyte
adherence. The6e re6ult6 sugge6t that ligand
sialic acid residues critical to b~t~Ai nq are
pre6ent on N-linked rather than on 0-linked
glycosylations.
CD34 has been Le~oLLed to be a ligand for
L-selectin hAr~ on the f1n~ng that a murine L-
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--19--
selectin-IgG chimera molecule precipitated CD34
from a murine lymph node lysate (Baumhueter et al.,
1993). The result6 a~ set forth in the examples
indicate that CD34 as expressed on KGla is not a
functional ligand for lymphocyte L-selectin, as no
difference in lymphocyte binding to sorted CD34-
and CD34+ KGla cells was observed.(F-igures 3 and 4)
Titration studies using varying proportions of KGla
and HL60 have demonstrated that the amount of
lymphocyte adherence is directly proportional to
the percentage of input KGla cells, indicating that
differences in lymphocyte binding to the positive
and negative sorted fractions would have been
evident if CD34 were the ligand. It is unlikely
lS that a particular bi nAi nq epitope of CD34 as
selected, as this experiment was done using two
different anti-CD34 mAbs to sort the XGla. Two
forms of CD34 on XGla have been reported
(ntruncated" and "full lengthn) (Xrause et al.,
1993); however, these differences do not account
for the data here as sorting was also performed
u~ing QBENDlO, which recoqn1~es both forms.
In addition to sorting experiments,
evidence that CD34 i8 not the L-selectin ligand on
KGla is derived from mAb bloçk~ng studies and
adherence assays using other CD34 positive cell6.
None of the anti-CD34 mAh8 tested, or any
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combination~thereof, was able to block lymphocyte
binding to KGla. Furthermore, lymphocytes did not
adhere to another primitive CD34+ cell line, RPNI
8402, and tran6fection of CD34 into COS-7 cells did
not confer lymphocyte binding capacity. While
potential glycosylation differences of the CD34
molecule expressed by the~e cell types could affect
their ability to support lymphocyte adherence, thi~
explanation i6 ~ kely in light of equivalent
adherence observed among the sorted CD34+ and CD34-
KGla cell6. Taken together, the data presented
here indicate that the CD34 glycoform present on
hematopoietic cell6 is not a ligand for L-selectin.
Moreover, flow cytometric analy~is of the various
lS cell lines utilized in the bi n~ i ng assay provides
evidence that membrane structures such as LFA-l,
VLA-4, CD44, Sialyl LeX and CD43 do not play a
primary role in lymphocyte adherence to KGla since
each of these molecules were also present on at
least one other cell line te~ted that did not
demonstrate lymphocyte b; nAi n~.
HLL is not recognized by MECA-79
monoclonal antibody which identifies L-6electin
ligands on lymph node HEV. ImmunofluorP~cence
analysi~ of KGla using NECA-79 shows no evi~nce of
the protein identified by NECA-79. HLL is shown to
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be unique from L-6electin ligands thus far
identified.
In the present study, direct cell-cell
interactions were utilized to detect the presence
of an L-selectin ligand on a hematopoietic cell.
Other studies directed at identifying L-selectin
ligands have relied on moler~lAr approaches
utilizing a murine L-selectin-IgG chimera molecule,
synthesized in a human embryonal kidney cell line,
as a probe (Watson et al., 1990). Of note, studies
utilizing this chimera have failed to demonstrate
binding of the molecule to KGla cells (Majdic et
al., 1994). In general, tissue- and species-
specific patterns of glycosylations are well
described, (Yamashita et al., 1983; Cullen et al.,
1981; Yamashita et al., 1985) and such differences
can affect the biological activity of proteins
expressed in different cells (Cowing 1983; Huff et
al., 1983). A6 it i6 known that glycosylation of
L-selectin varies among different cell6 expressing
the protein (Lewinsohn et al., 1987, Ord et al.,
1990; Griffin et al., 1990), such differences may
a~o~lL for the observation here that native L-
~-lectin, expressed on lymphoc-;e membranes,
selectively binds to a corr~pQnA~r~ ligand on KGla
cell6 while the chimera apparently does not.
Similarly, differences in glycosylation of CD34
SU~STlTUTE SHEET (RULE 26)
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among endot1elial cells and hematopoietic cell6 may
account for the differential capacity of this
protein to participate in L-selectin interactions
among these cell type~.
L-6electin ligands have been recognized
heretofore only on endothelia-l cells. The
detection of an L-selectin ligand on a non-
enaothelial cell eYp~nA~ the physiologic
implicatiQns of L-selectin function beyond it~
well-characterized role in regulating leukocyte
trafficking.
The above discussion provides a factual
basis for the characterization and use of }~-T-. The
methodc used with and the utility of the present
invention can be shown by the following examples.
~ raXPLE8
GENER~T METHODS:
Cell L~ne~. Cell lines u~ed in these
studies were obtained from the following sources:
KGla and Nalm 16, gift of Dr. William E. Janssen;
HL60, K562, and Ra~i, gift of Dr. Lynn Moscin~ki;
COS-7, gift of Dr. Ke~th Zukerman (all from H.
Lee Moffitt CAncer Center, Tampa, FL); RPMI 8402,
gift of Dr. Daniel G. Tenen ~Harvard Medical
School~ Boston, MA). All cell6 were cultured in
RPMI 1640 (Gibco-BRL, Gaither~burg, MD)
SllBSnTU~E SHEET (RULE 26)
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.
6upplemente with 10% heat-inactivated fetal bovine
serum (FBS) in a humidified chamber at 37C with 5%
C2 in air.
Preparat~on of Lymphocytes. Human
S peripheral blood lymphocytes (PBL) were i60lated by
Ficoll density gradient from blood drawn in sodium
citrate. To obtain rat thoracic duct lymphocytes
(TDL), thoracic duct~ sf rats were cannulated as
described by Bollman et al.(1948). Lymph was
collected in phosphate buffered saline (PBS) with
O.l~ penicillin/streptomycin and 5 U/ml heparin.
PBL or TDL were washed three times in RPMI 1640
medium without bicarbonate (Gibco-BRL), pH 7.4, and
suspended at l x 107 cells/ml in above medium with
s% FBS and kept on ice until u6e in the adherence
assay.
Lymphocyte Adherence As6~y. The
procedu~e for the ~n vitro binding of human or rat
lymphocytes to KGla was adapted from the rat
lymphocyte-lymph note b~nA1ng a6say which has been
described by Stamper and Woodruff (1976) and
Sackstein et al.(1988). Cytospin preparations of
KGla or other cell lines were made on a Cytospin 3
Cytocentrifuge (chAnAo~ Lipshaw, Pittsburgh, PA).
Frozen rat LN sections 8~m thick were mounted on
6lides, and lymphocyte b~lA1 ng to LN HEV 6erved as
a po6itive ~ ol in all experiment6. Slide6 were
SUBSTITUTE SHEET (RULE 26)
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air dried, ~ixed in 3~ glutaraldehyde (Electron
Microscopy Sciences, Fort Wa6hington, PA) in PBS,
rin~ed with PBS, incubated in 0.2M L-ly6ine (Sigma
Chemical Company, St. Louis, M0) to block unreacted
glutaraldehyde, then rin6ed and held in RPMI 1640
with 1% FBS at 4C until u6e in experiment6.
Lymphocyte su6pensions (200~l) were
overlaid onto cytospin or LN 6ection6 in duplicate
and placed on a rotating platform (80rpm) at 4C
for 30 minutes. Slides were then rinsed in cold
PBS to remove non-adherent lymphocytes, fixed in 3%
glutaraldehyde, and stAi n~A with methyl green-
thionin. Slides were examined under the light
microscope for adherence of lymphocytes to KGla or
LN HEV.
Number of lymphocytes adherent to
confluent area of KGla were counted by light
microscopy using an oc~llAr grid under 250X
magnifi~ation. Quantitation wa6 performed by
examining two fields per ~lide, minimum of two
~lides per experiment, three ~eparate experiment6.
Re6ult~ are pre6ented aG % b~ nA ~ ~g compared to
corre6ponA~q untreated control 6ection6.
Treatment of Lymphocytes w~ th Potent ~ al
Inhibltor6. Lymphocyte6 in RPMI 1640 medium with
5~ FBS were pre-1nc~h~ted (30 min on ice) and the
a66ay performed in the prer~.nce of the following
SUBSJITUTE SHEET (RULE 26)
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inhibitor6: lmN EDTA (no pre-incubation period); lO
mM D-mannose-6-phosphate (Sigma); lO ~g/ml PPNB
(kindly provided by Dr. M.E. Slodki, USDA, Peoria,
IL); and 5 ~g/ml fucoidin (Sigma).
S An tibody Bl ock ~ ng Experimen ts .
Lymphocytes (l x 107 cells/ml) were pre-incubated
on ice for 20 minute6 with mAbs at l.O ~g/ml and
used in the binding assay without further washing.
The following ~Abs were used: LAM1-3 (anti-L-
selectin) (kind gift of Dr. ~homas Tedder, Duke
University, Durham, N.C., and also obtained from
Coulter Corp., Hialeah, FL~; anti-CD45 (leukocyte
Common Antigen) (Becton Dickinson, San Jose, CA);
and IgGl (i60type control) (Coulter). In 60me
lS experiments, prepared KGla filides were incubated
with 0.2 ~g of anti-CD34 Abs {HCPAa-l (clone MylO)
and HPCA-2 (clone 8Gl2) (Becton Dickinson), QBENDlO
(AMAC) and 12.8 (kindly provided by Dr. Pat Roth,
Coulter cor`p.)~ in RPNI 1640 with 5% FBS for 30
minutes prior to the b~n~ng as~ay.
PMA ~rreatment of Lymphocyte~.
Lymphocytes were suspended at l X 107 cells/ml in
cell culture medium and ~n~llh~ted l hour at 37C
with or without lO ng/ml PMA (Gibco-BRL). Cell6
were then washed twice in PBS and used in either
the lymphocyte b~nA~g a6say or analyzed for
~urface antigens by flow cytometry (see below).
SUB~IJUTE SHEET (RULE 26)
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Enzyme Treatment of ~Gla or LN. cytospin
preps KGla or LN frozen sections were
glutaraldehyde-fixed, then treated with various
enzymes prior to the bi~A~ng assay. For treatment
with neuraminidase (sialidase), 61ides were rin6ed
twice with enzyme buffer (50 mM NaAc, 154 mM NaCl,
9 mM CaC12, pH 5.5), then inc~lh~ted 30 min. at 37C
with 50 ~1 of buffer (oonL.ol) or undiluted
neuramin1AA~ (1.2 U/ml, Boehringer MAnn~e;m~
Tn~iAn-Apolis, IN). In protease studies, slides
were incubated with RPMI 1640 alone or RPMI 1640
contAinin~ enzymes: 100 U/ml chymotrypsin (Sigma)
(115 min. at 37C), or 0.1% bromelA;n (Sigma) (30
min. at 37C); to assess specificity, the protease
inhibitors PMSF (1.0 mg/ml, Sigma) and chymostatin
(900 ~g/ml, Boehringer MAnnhplm) were coinr~h~ted
with chymotrypsin (100 U/ml) for 15 min. at 37C.
Following enzyme treatments, slides were washed X3
with RPMI 1640 and placed in RPNI 1640 with 1% FBS
untll use in the binA;n~ assay.
XGla cell~ in su6pension (4 x 107
cells/ml) were incllhAted with 0-sialogly~yLG~ein
endopeptidase (Accurate Chemical and ScientifiC
Corp., Westbury, NY) (0.24 mg/ml, 37 C, 30 min.),
washed X3 with 2~ FBS in PBS, and cytospin
preparations were made for use in the b~nA~n~
a6say. To verify the activity of the enzyme, cells
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were tested for the cleavage of CD34 by flow
cytometry using QBEND10 mAb.
Antigen Express~on by Flow Cytometry.
Flow cytometric analysi~ was performed using the
following commercially-available mAbs together with
isotype-matched control~: TQ1 (anti-L-selectin),
LAM1-3 (Anti-L-selectin), 4B4 -(Anti-VLA-4) (all -
from Coulter Corp.); QBEND10 (anti-CD34) (AMAC,
Westbrook, NE); anti-CD44, LFA-1-~ (anti-CD18),
LFA-l-~ (anti-CDlla), HPCAS-2 (anti-CD34), anti-
CD45, Leukosialin (anti-CD43), anti-Sialyl-LeX (all
from Becton Dickinson). Cells (1 X 106) in 100 ~l
of PBS with 2% FBS were incubated on ice for 25
minute~ with Ab as per manufacturer's
recommendations, wA~h~A X3 and analyzed on a
FACStarP~Us (Becton Dickin~on).
Fluorescence Act~vated Cell Sort~ng of
~Gl~ cells. XGla cells were st~n~A with anti-CD34
mAbs ~QBE~10-FITC in two experiments, HPCA-2-PE in
one experiment) and positive and negative
expre~sing cell6 were sorted on a FAC~tarPLU8 flow
cytometer equipped with an argon laser tuned at 488
nm (Becton Di~nr~n). Sorted cell population6
were rest~n^~ with anti-CD34 Ab directed at
epitopes not used for ~orting and were analyzed to
determine the efficiency of the sort. Cytospin
preparat1ons were made of the positive and negative
SUBSllME SHEET (R(~LE 26)
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sorted fraction6 and were used in the lymphocyte
binding assay.
Transfection of C05-7 w~ th CD34 cDNA .
COS-7 cells were transiently transfected with human
full-length CD34 cDNA in pCDM8 plasmid (a gift from
Dr. Daniel Tenen, Boston, MA) using a DEAE Dextran
transfection method (Selden, 1992). Briefly, COS-7
cells were incubated for 4 hours at 37C with lO ml
of transfection solution contAining 20-40 ~g of
plasmid DNA, 10% Nu Serum (Collaborative Biomedical
Products, Bedford, MA), 400 ~g/ml DEAE Dextran
(Sigma), and lO0 ~ chloroquine (Sigma) in
D~llheccols Modified Eagles Medium (Gibcon-8RL).
Cells were then rinsed and treated with lO~ DMS0
lS (Sigma) in PBS for two minutes at room temperature,
rinsed in PBS, and incubated in tissue culture
media for 3 day6. In-one set of experiments,
trypsinization was avoided by growing transfected
cells directly on glass slides for subsequent use
in the binding assay or for analysis of CD34
expres~ion by fluorescence microscopy. In other
experiments, COS-7 cells grown on lO cm plates were
removed with trypsin/EDTA (0.25%/lmM, Gibco-BRL),
then analyzed for CD34 expression by flow
cytometry. These trypsinl~ed cells were then
placed on slides by cytospin for use in the
lymphocyte b~n~ n~ a8~ay.
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B~AMPL~ 1
Lymphocytes Bind to RGla. Lymphocyte~
(both PBL and TDL) adhered specifically and
reproducibly to KGla, ~ut not to RPMI 8402, HL60,
Nalm 16, K562, or Raji cell lines in the in vitro
binding assay (Table 1). All experiment6 were
performed in parallel with LN frozen sections ar
positive controls. Lymphocyte binding to RGla was
observed under conditions identical to those
whereby L-selectin mediates binding of lymphocytes
to LN HEV.
Lymphocyte pin~ing to RGla is Med~ated by
L-selectin. To directly examine whether lymphocyte
attachment was mediated by L-selectin, PBL were
pre-incubated with the anti-L-~electin mAb LAM1-3,
anti-CD45, or IgG1 isotype cGnLlol Abs. The LAM1-3
Ab completely inhibited lymphocyte binding to KGla
and LN ~on~.ol, while CD45 and isotype control mAbs
did not affect binding (Fig. lA & lB). In order to
quantify the relative amounts of Ab attachment to
lymphocytes, Ab-treated lymphocytes were incllh~ted
with goat-anti-mouse FITC-conjugated ~econAAry Ab
and analyzed by flow cytometry. Although the
amount of anti-CD45 Ab on lymphocytes was
significantly greater than that of LAM1-3 a~
indicated by mean ahAnnel fluorerc~nae (Fig. 2),
SUBSrmJTE SHEE~ (RULE 26J
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j
LAMl-3 alone blocked lymphocyte adherence to KGla
and LN HEV, indicating that this ef~ect was
specific and not secondary to charge or 6teric
alterations of the lymphocyte membrane.
The Effect of Enzyme Treatment of ~Gla on
Lymphocyte B~ndin~. Pretreatment of both KGla and
LN col.LLol sections with neuraminida6e (60 mU),
chymotrypsin (lO0 U/ml) or bromelain (O.l~) prior
to the binding a~ay abrogated binding of
lymphocytes, while treatment with buffer or medium
alone did not alter binding capacity. In addition,
the effects of chymG~Ly~in were confirmed by
coincubation with the protease inhibitors
lS chymostatin and PMSF, which prevented chymotrypsin
effectc on lymphocyte binA;ng. However,
pretreatment of KGla with 0-6ialogly~0~LGLein
endopeptidase had no effect on lymphocyte binA~ng
despite complete enzymatic removal of the CD34
epitope recognized by QBENDlO mAb. (Table 2)
Lymphocyte B~nd~ng to RGla ~ C~lcium
Dependent. Lymphocyte b;nA~n~ to KGla and to LN
control ~ections was completely inhibited by the
presence of EDTA, indicating a calcium requirement
for lymphocyte-KGla b;n~ng.
SUBSTITIJTE SHEET (RULE 26)
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M~nnose-6-Phosph~te, PPME, and Fucoidin
Inhib~t Lymphocyte Binding to RGla. The
specificity of lymphocyte-RGla binding wa~
investigated by treating PBL or TDL with
carbohydrate inhibitors of L-6electin-HEV
interactions prior to the adherence assay. Man-6-P
(10 mM), PPME (10 ~g/ml), and fucoidin (5 ~g/ml)
all inhibited lymphocyte binding to both KGla and
LN control section6. (Table 2)
PMA Treatment of Lymphocytes Results ~n
the Loss of Binding to ~Gla. PBL were incubated
for 1 hour at 37C with 10 ng/ml PNA, then used in
the lymphocyte binding assay. PMA-treated PBL were
unable to bind to either KGla or LN HEV, while
control PBL demonstrated high amounts of b;n~in~.
(Table 2)
Loss of surface L-selectin was a~sessed
by flow cytometric analysis of TQl levels in
~oll~Lol and PMA-treated PBL. PNA-treated
lymphocytes ~howed a dramatic decrease in TQl mean
c~n~l fluorP~cence (to levels less than 10% of
that of untreated cells) in three separate
experiments. PMA-treated PBL were also analyzed
for expression of CD44, LFA-l (both ~ and B
ChA~lF)., and VI,A-4, and expression of these
SUBSrlTUI~ SHEEt (RULE 26)
CA 02202472 1997-04-11
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.
adhesion mo~lecules following PMA exposure was
identical to expre6sion on control PBL.
B~AMPLE 2
Pretreatment of ~Gla w~th Anti-CD34
Antibodies Did Not Inhi~t Adherence of
Lymphocytes. Cyto~pin preps of KGla were
preincubated with anti-CD34 Abs and the b~n~;n~
assay was performed in the presence of the Abs.
Monoclonal ABS to four different CD34 epitopes were
used alone or in combination, including the clones
MylO, QBENDlO, 8gl2, and 12.8, in amounts ranging
from 0.2 to 17 ~g/slide. Anti-CD45 (irrelevant
control) and IgGl (isotype control) Abs were also
lS tested. None of the anti-CD34 Abs inhibited
lymphocyte binding to KGla, despite
immunohistochemical evidence of extensive Ab
binding to the glutaraldehyde-fixed KGla sections.
Other Surface Antlgens on ~Gla do not
Appe~r to Med~ate Bind~ng. The surface expression
of several antigens on KGla, RPNI 8402, HL60, Nalm
16, K562, and Raji was analyzed by flow cytometry
(Table l). LFA-l, FLA-4, CD44, Sialyl LeX, and
CD43 were all expressed by KGla and at lea6t one
other cell line that did not ~ rort lymphocyte
adherence. Of note, although RPMI 8402 cells
SUBST~TUI E SHEET (RULE 26)
CA 02202472 1997-04-11
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.
express CD34 at levels comparable to KGla, there
was no adherence of lymphocytes to these cells in
the binding assay.
CD34 Posttlve and Negative ~Gla Cells
Supported Equivalent Amount~ of Lymphocyte Bind~ng.
CD34+ and CD34- KGla cell6 were separated by
fluorescence activated cell sorting and cytospin
preparations of each population were made. The ~n
vitro adherence of lymphocytes was identical in the
CD34~ and CD34- populations despite an enrichment
of >90% and <10% CD34+ cell8 in the respective
populations (Fig. 3 and 4; Table 2).
E~AMPLB 3
CD34-Transfected aos-7 Cells Did Not
Support Lymphocyte Adherence. COS-7 cells were
transfected with CD34 and tested in the ~n v~tro
~nA~ng assay, and both trypsinized and intact
transfected COS-7 cell6 failed to ~ o~L
lymphocyte adherence. By flow cytometric analy6i6,
transfected cells were a~oximately 60% po6itive
for CD34 expre66ion, and the mean ch~nn
fluorPFcPnce wa6 greater than that of KGla ~ul.LLol
cells (Fig. 5). Intact, ~lLLy~sinized COS-7 cells
transfected with CD34 also strongly expressed CD34
SUBSTI~UJE SHE~T (RVLE 26)
CA 02202472 1997-04-11
W O96/11012 PCT~US95/13736
,
(~90% positive a6 estimated by fluorescence
microscopy)~
Throughout this application variou~
publications are referenced by citation or number.
Full citations for the publications referenced by
number are listed below. The disclosures of these
publications in their entireties are hereby
incorporated by reference into this application in
order to more fully de6cribe the ~tate of the art
to which thi6 invention pertains.
The invention has been described in an
illustrative manner, and it is to be understood
that the terminology which has been used is
intended to be in the nature of words of
description rather than of limitation.
Obviously, many modifications and
variations of the present invention are possible in
light of the above teachings. It is, therefore, to
be under~tood that within the scope of the app~AP~
claims, the invention may be practiced otherwi6e
than a~ ~pecifically described.
SUBSrlTUT~ SHEET (RUIE 26J
CA 02202472 1997-04-11
WO 96/11012 PCTtUS95tl3736
--35--
.
+ + + +
+ + + + +
C: + + + + + +
+ + + + +
z
t~ K
E~ a~
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+ I I + +
+ + +
cn
++ +
+ + + I I I _~
r~ a Z a + +
O 0 V ~ + I + + + h
+ + + + +
+ + + + , + ~
0 0 ~, + + + + O
O ~ ~ + 3
0 - ~ + + O
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+ + ~a
+ +
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SUBSrlTlJTE SHEET (RULE 26~
CA 02202472 1997-04-11
wos6/11oi2 PCT~S95/13736
-36-
j
T~ble 2. Lymphocyte A~herence to ~Gla
LYMPHOCYTE TREAq~ENrNean (SEM) o~ Bindlng
EDTA 0-3 (0 3)
Mannose-6-P 5.7 (1-0)
Fucoidin 1.4 (0-4 ?
PPME 5 4 (o 5)
LAM1-3 mAb 1.9 (0.4)
Anti--CD45 mAb 98.7 (6.3)
IgGl Control mAb 115.1 (9.0)
PMA 1.1 (0.3)
XGlA TREATMENT:
Anti CD34 mABst 116.2 (7.7)
Anti CD45 mAb 98.0 (3.6)
IgGl Control mAb 101.8 (8.5)
CD34-Positive Sort 102.8 (3.5)
CD34-Negative Sort 104.1 (4.2)
Neuraminidase 3.1 (0-7)
Neuraminidase Buffer Co.l~ol100.5 (6.7)
O-Sialogly~o~ro~ein Endopeptidase98.4 (2.3)
Bromelain 3.8 (0.4)
ChymG~ in 6.7 (0.7)
ChymG~ in, PMSF, Chymo6tatin94.0 (3-8)
tCombination o~ HPCA-l, HPCA-2, 12.8 and QBEND10 mAbs.
SU~SrlTU~E SHEET (RULE 26)
CA 02202472 1997-04-11
WO96/11012 PCT~S95/13736
-37-
j
~F~R~
Abdullah KM, Udoh EA, Shewen PE, Mellors A: A
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_38-
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SUBSrl~E SHEFr (RULE 26~
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SUB~TITUTE SHEET (RULE 26)
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SU~SrIME SHEE~ (RULE 26i~
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