Note: Descriptions are shown in the official language in which they were submitted.
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SPECIFIC HUMAN ANTIB~DIES
FIELD OF THE INVENTI~N
[Ol] The present invention relates to antibodies that bind to particular
epitopes
that are present on cells, such as cancer cells, metastatic cells, leukemia
cells, leukocytes,
and platelets, and that are important in such diverse physiological phenomena
as cell
rolling, metastasis, inflammation, and auto-immune diseases. More
particularly, the
antibodies may have anti-cancer activity, anti-metastatic activity, anti-
leukemia activity,
anti-viral activity, anti-infection activity, andlor activity against other
diseases, such as
inflammatory diseases, autoixnmune diseases, viral infection, cardiovascular
diseases such
as myocardial infarction, retinopathic diseases, and diseases caused by
sulfated tyrosine-
dependent protein-protein interactions. In addition, the antibodies of the
present invention
may be used as a targeting agent to direct a therapeutic to a specific cell or
site within the
body.
BACI~~I~~U F THE I NTIN
[02] Leukemia, lymphoma, and myeloma axe cancers that originate in the bone
marrow and lymphatic tissues and are involved in uncontrolled growth of cells.
Acute
lymphoblastic leukemia (ALL) is a heterogeneous disease that is defined by
specific
clinical and inununological characteristics. Like other forms of ALL, the
definitive cause
of most cases of B cell ALL (B-ALL) is not known; although, in many cases, the
disease
results from acquired genetic alterations in the I~NA of a single cell,
causing abnormalities
and continuous multiplication. Prognosis for patients afflicted with B-ALL is
significantly
worse than for patients with other leukemias, both in children and in adults.
Chronic
lymphocytic leukemia (CLL), one example of which is B cell CLL (B-CLL), is a
slowly
progressing form of leukemia, characterized by an increased number of
lymphocytes.
Acute myelogenous leukemia (AML) is a heterogeneous group of neoplasms having
a
progenitor cell that, under normal conditions, gives rise to terminally
differentiated cells of
the myeloid series (erythrocytes, granulocytes, monocytes, and platelets). As
in other
forms of neoplasia, AML is associated with acquired genetic alterations that
result in
replacement of normally differentiated myeloid cells with relatively
undifferentiated
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blasts, exhibiting one or more type of early myeloid differentiation. AML
generally
evolves in the bone marrow and, to a lesser degree, in the secondary
hematopoietic organs.
Primarily, AML affects adults and peaks in incidence between the ages of 15-
40, but it is
also known to affect both children and older adults. Nearly all patients with
AML require
treatment immediately after diagnosis to achieve clinical remission, in which
there is no
evidence of abnormal levels of circulating undifferentiated blast cells.
Li~and for Is~lated scFv Antibody Molecules
[03] Platelets, fibrinogen, GPIb, selectins, and PS(~L-1 (P-Selectin
Glycoprotein
Ligand-I) each play an impprtant role in several pathogenic conditions or
disease states,
such as abnormal or pathogenic inflammation, abnormal or pathogenic immune
reactions,
autoimmune reactions, metastasis, abnormal or pathogenic adhesion, thrombosis
and/or
restenosis, and abnormal or pathogenic aggregation. Thus, antibodies that bind
to or
cross-react with platelets and with these molecules would be useful in the
diagnosis and
treatment of diseases and disorders involving these and other pathogenic
conditions.
Platelets
[0~.] Platelets are well-characterised components of the blood system and play
several important roles in hemostasis, thrombosis and/or restenosis. I?amage
to blood
vessel sets in motion a process known as hemostasis, which is characterised by
a series of
sequential events. The initial reaction to damaged blood vessels is the
adhesion of
platelets to the affected region on the inner surface of the vessel. The next
step is the
aggregation of many layers of platelets onto the previously adhered platelets,
forming a
hemostatic plug and sealing the vessel wall. The hemostatic plug is further
strengthened
by the deposition of fibrin polymers. The clot or plug is degraded only when
the damage
has been repaired.
[OS] Circulating platelets are cytoplasmic particles released from the
periphery
of megakaryocytes. Platelets play an important role in hemostasis. Upon
vascular injury,
platelets adhere to damaged tissue surfaces and attach to one another
(cohesion). This
sequence of events occurs rapidly, forming a structureless mass (commonly
called a
platelet plug or thrombus) at the site of vascular injury. The cohesion
phenomenon, also
known as aggregation, may be initiated in vitro by a variety of substances, or
agonists,
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such as collagen, adenosine-diphosphate (ADP), epinephrine, serotonin, and
ristocetin.
Aggregation is one of the numerous iya vitro tests performed as a measure of
platelet
function.
Importance of Platelets in Metastasis
[06j Tumor metastasis is perhaps the most important factor limiting the
survival
of cancer patients. Accumulated data indicate that the ability of tumor cells
to interact
with host platelets represents one of the indispensable determinants of
metastasis
(~leksowicz, Tlay~~mb~sis Res. 79: 261-74 (1995)). When metastatic cancer
cells enter the
blood stream, multicellular complexes composed of platelets and leukocytes
coating the
tumor cells are formed. These complexes, which may be referred to as
microemboli, aid
the tumor cells in evading the immune system. The coating of tumor cells by
platelets
requires expression of P-selectin by the platelets.
[07] It has been demonstrated that the ability of tumor cells to aggregate
platelets correlates with the tumor cells' metastasis potential, and
inhibition of tumor-
induced platelet aggregation has been shown to correlate with the suppression
of
metastasis in rodent models. It has been demonstrated that tumor cell
interacti~n with
platelets involves membrane adhesion molecules and agonist secretion.
Expression of
immuno-related platelet glycoproteins has been identified on tumor cell lines.
It was
dmnonstrated that platelet immuno-related glycoproteins, GPIb, GPIIb/IIIa,
GPIb/I~ and
the integrin ~V subunit are expressed on the surface of breast tumor cell
lines (~leksowicz,
(1995), supra; I~amiyama et al., .l. Lab. Clip. Med. 117(3): 209-17 (1991)).
[08] Gasic et al. (PNAS 61:46-52 (1968)) showed that antibody-induced
thrombocytopenia markedly reduced the number and volume of metastases produced
by
CT26 colon adenocarcinoma, Lewis lung carcinoma, and B 16 melanoma (Karpatkin
et al.,
J. Cliri. Invest. 81(4): 1012-19 (1988); Clezardin et al., CayacerRes. 53(19):
4695-700
(1993)). Furthermore, a single polypeptide chain (60kd) was found to be
expressed on
surface membrane of HEL cells that is closely related to GPIb and corresponds
to an
incompletely or abnormally O-glycosylated GPIba subunit (Kieffer et al., J.
Biol. Chem.
261(34): 15854-62 (1986)).
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GPIb Comulex
[09] Each step in the process of hemostasis requires the presence of receptors
on
the platelet surface. ~ne receptor that is important in hemostasis is the
glycoprotein Ib-IX
complex (also known as CD42). This receptor mediates adhesion (initial
attachment) of
platelets to the blood vessel wall at sites of injury by binding von
Willebrand factor (vWF)
in the subendothelium. It also has crucial roles in two other platelet
functions important in
hemostasis: (a) aggregation of platelets induced by high shear in regions of
arterial
stenosis and (b) platelet activation induced by low concentrations of
thrombin.
[IO] The GPIb-IX complex is one of the major components of the outer surface
of the platelet plasma membrane. This complex comprises three membrane-
spanning
polypeptides - a disulfide-linked 130 kDa oc-chain and 25 kDa (3-chain of GPTb
and a
noncovalently associated GPIX (22 kDa). All of the subunits are presented in
equimolar
amounts on the platelet membrane for efficient cell-surface expression and
function of
CD42 complex, indicating that proper assembly of the three subunits into a
complex is
required for full expression on the plasma membrane. The ~,-chain of GPIb
consists of
three distinct structural domains: (1) a globular N-terminal peptide domain
containing
leucine-rich repeat sequences and Cys-bonded flanking sequences; (2) a highly
glycosylated mucin-like macroglycopeptide domain; and (3) a membrane-
associated C-
terminal region that contains the disulfide bridge to GPIbcc and transmembrane
and
cytoplasmic sequences.
[11] Several lines of evidence indicate that the vWF and thrombin-binding
domain of the GPIb-IX complex reside in a globular region encompassing
approximately
300 amino acids at the amino terminus of GPIbcc. As human platelet GPIb-IX
complex is
a key membrane receptor mediating both platelet function and reactivity,
recognition of
subendothelial-bound vWF by GPIb allows platelets to adhere to damaged blood
vessels.
Further, binding of vWF to GPIba also induces platelet activation, which may
involve the
interaction of a cytoplasmic domain of the GPIb-IX with cytoskeleton or
phospolipase A2.
Moreover, GPIba, contains a high-affinity binding site for a-thrombin, which
facilitates
platelet activation by an as-yet poorly defined mechanism.
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[12] The N-terminal globular domain of GPIboc contains a cluster of negatively
charged amino acids. Several lines of evidence indicate that in transfected
CHO cells
expressing GPIb-IX complex and in platelet GPIba, the three tyrosine residues
contained
in this domain (Tyr-276, Tyr-278, and Tyr-279) undergo sulfation.
Protein Sulfation
[13] Protein sulfation is a widespread post-translational modification that
involves enzymatic covalent attachment of sulfate, either to sugar side chains
or to the
polypeptide backbone. This modification occurs in the traps-Golgi compartment.
Sulfated proteins include secretory proteins, proteins targeted for granules,
and the '
extracellular regions of plasma membrane proteins. Tyrosine is an amino acid
residue
presently known to undergo sulfation. I~ehoe et al., Clzefn. Bi~l. 7: R57-61
(2000). ~ther
amino acids, e.g., threonine, may also undergo sulfation, particularly in
diseased cells.
[14~] A number of proteins have been found to be tyrosine-sulfated, but the
presence of three or more sulfated tyrosines in a single polypeptide, as was
found on GPTb,
is not common. GPTba (CD42), which is expressed by platelets and
megakaryocytes and
mediates platelet attachment to and rolling on subendothelium via binding with
vWF', also
contains numerous negative charges at its N-terminal domain. Such a highly
acidic and
hydrophilic environment is thought to be a prerequisite for sulfation, because
tyrosylprotein sulfotransferase specifically recognizes and sulfates tyrosines
adjacent to
acidic amino residues (Dundgaard et al., J: Bi~l. Claem. 272:21700-OS (1997)).
Full
sulfation of the acidic region of GPIba yields a region with a remarkable
negative charge
density -13 negative charges within a 19 amino acid stretch - and is a
candidate site for
electrostatic interaction with other proteins.
[15] It is also thought that sulfated N-terminal tyrosines influence the role
of
CC-chemokine receptors, such as CCRS, which serve as co-receptors with related
seven
transmembered segment (7TMS) receptor for entry of human and simian
immunodeficiency viruses (HIV-1, HIV-2, and SIV) into target cells. For
example, it is
thought that sulfated N-terminal tyrosines contribute to the binding of CCRS
to MIP-1 a,
MIP-1 (3, and HIV-1 gp120/CD4 complexes and to the ability of HIV-1 to enter
cells
expressing CCRS and CD4.. CXCR4, another important HIV-1 co-receptor, is also
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sulfated (Farzan et al., Cell 96(5): 667-76 (1999)). Tyrosine sulfation plays
a less
significant role in CXCR4-dependent HIV-1 entry than CCRS-dependent entry;
thus
demonstrating a possible role for tyrosine sulfation in the CXC-chemokine
family and
underscoring a general difference in HIV-1 utilization of CCRS and CXCR4
(Farzan et al.,
J. Biol. Chem. 277(33): 29,484-89 (2002)).
Selectins and PSGI~-1
[16] The P-, E-, and L- Selectins are members of a family of adhesion
molecules
that, among other functions, mediate rolling of leukocytes on vascular
endothelium. P-
Selectin is stored as granules in platelets and is transported to the surface
after activation
by thrombin, histamine, phorbol ester, or other stimulatory molecules. P-
Selectin is also
expressed on activated endothelial cells. E-Selectin is expressed on
endothelial cells, and
L-Selectin is expressed on neutrophils, monocytes, T cells, and ~ cells.
[17] PSGL-1 (also called C1~162) is a mucin glycoprotein ligand for P-
Selectin,
E-Selectin, and L-Selectin that shares structural similarity with GPIb (Afshar-
I~harghan et
aI. (2001), sup~cz). PSGL-1 is a disulfide-linked homodimer that has a PACE
(Paired
Easic Amino Acid Converting Enzymes) cleavage site. PSGL-1 also has three
potential
tyrosine sulfation sites followed by 10-16 decamer repeats that are high in
proline, serine,
and threonine. The extracellular portion of PSGL-1 contains three N-linked
glycosylation
sites and has numerous sialylated, fucosylated ~-linked oligosaccharide
branches (I~oore
et al., J. Bi~l. C'laena. 118: 44.5-56 (1992)). Most of the N-glycan sites and
many of the ~-
glycan sites are occupied. The structures of the ~-glycans of PSGL-1 from
human HL-60
cells have been determined. Subsets of these ~-glycans are core-2, sialylated
and
fucosylated structures that are required for binding to selectins. Tyrosine
sulfation of an
amino-terminal region of PSGL-1 is also required for binding to P-Selectin and
L-Selectin.
Further, there is an N-terminal propeptide that is probably cleaved post-
translationally.
[18] PSGL-1 has 361 residues in HL60 cells, with a 267 residue extracellular
region, 25 residue trans-membrane region, and a 69 residue intracellular
region, and forms
a disulfide-bonded homodimer or heterodimer on the cell surface (Afshar-
Kharghan et al.,
Blood 97: 3306-12 (2001)). The sequence encoding PSGL-1 is in a single exon,
so
alternative splicing should not be possible. However, PSGL-1 in HL60 cells,
and in most
cell lines, has 15 consecutive repeats of a 10 residue consensus sequences
present in the
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extracellular region, although there are 14 and 16 repeats of this sequence in
polyrnorphonuclear leukocytes, monocytes, and several other cell lines,
including most
native leukocytes.
[19] PSGL-1 is expressed on neutrophils as a dimer, with apparent molecular
weights of both 250 kDa and 160 kDa, whereas on HL60 the dimeric form is
approximately 220 kDa. When analyzed under reducing conditions, each subunit
is
reduced by half. Differences in molecular mass may be due to polymorphisms in
the
molecule caused by the presence of different numbers of decamer repeats
(Leukocyte
Typing VI. Edited by T. I~ishimoto et al. (1997)).
[20] Most blood leukocytes, such as neutrophils, monocytes, leukocytes, subset
of B cells, and all T cells express PSGL-1 (Kishimoto et al. (1997), supra).
PSGL-1
mediates rolling of leukocytes on activated endothelium, on activated
platelets, and on
other leukocytes and inflammatory sites and mediates rolling of neutrophils on
P-Selectin.
PSGL-1 may also mediate neutrophil-neutrophil interactions via binding with L-
Selectin,
thereby mediating inflammation (Snapp et al., ~lo~d 91(1): 154-64 (1998)).
[21] Leukocyte rolling is important in inflammation, and interaction between P-
Selectin (expressed by activated endothelium and on platelets, which may be
immobilized
at sites of injury) and PSGL-1 is instrumental for tethering and rolling of
leukocytes on
vessel walls (I~amachandran et al., .~I~A~° 98(1 ~): 10166-71 (2001);
Afshar-I~harghan et al.
(2001), supa~a). Cell rolling is also important in metastasis, and P- and E-
Selectin on
endothelial cells is believed to bind metastatic cells, thereby facilitating
extravasation from
the blood stream into the surrounding tissues.
[22] Thus, PSGL-1 has been found on all leukocytes: neutrophils, monocytes,
lymphocytes, activated peripheral T cells, granulocytes, eosinophils,
platelets and on some
CD34 positive stem cells and certain subsets of B cells. P-Selectin is
selectively expressed
on activated platelets and endothelial cells. Interaction between P-Selectin
and PSGL-1
promotes rolling of leukocytes on vessel walls, and abnormal accumulation of
leukocytes
at vascular sites results in various pathological inflammations. Stereo-
specific
contributions of individual tyrosine sulfates on PSGL-1 are important for the
binding of P-
Selectin to PSGL-1. Charge is also important for binding: reducing NaCl (from
150 to 50
mM) enhanced binding (Kd ~75nM). Tyrosine-sulfation on PSGL-1 enhances, but is
not
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ultimately required for PSGL-1 adhesion on P-Selectin. PSGL-1 tyrosine
sulfation
supports slower rolling adhesion at all shear rates and supports rolling
adhesion at much
higher shear rates (Rodgers et al., Bioplays. .I. 81: 2001-09 (2001)).
Moreover, it has been
suggested that PSGL-1 expression on platelets is 25-100 fold lower than that
of leukocytes
(Frenette et al., J. Exp. Med. 191(8): 1413-22 (2000)).
[23] A commercially available monoclonal antibody to human PSGL-1, KPLl,
has been shown to inhibit the interactions between PSGL-1 and P-selectin and
between
PSGL-1 and L-selectin. The KPLl epitope was mapped to the tyrosine sulfation
region of
PSGL-1 (YEI~LD~'D) (SEQ ID N~:l) (Snapp et al., Bl~od 91(1):154-64 (1998)).
[24] Pretreatment of tumor cells with ~-sialoglycoprotease, which removes
sialylated, fucosylated mucin ligands, also inhibited tumor cell- platelet
complex
formation. Ira viv~ experiments indicate that either of these treatments
results in greater
monocyte association with circulating tumor cells, suggesting that reducing
platelet
binding increases access by immune cells to circulating tumor cells (Varki and
Varki,
B~az. ,I. Bi~l. IZes. 34(6): 711-17 (2001)).
Fibran~~en
[2~] There are two forms of normal human f brinogen - normal (y) and y prime,
each of which is found in normal individuals. Normal fibrinogen, which is the
more
abundant form (approximately 90% of the total fibrinogen found in the body),
is
composed of two identical 55 kDa cc chains, two identical 95 kDa ~i chains,
and two
identical 49.5 kDa y chains. Normal variant fibrinogen, which is the less
abundant form
(approximately 10% of the fibrinogen found in the body), is composed of two
identical 55
kDa a, chains, two identical 95 kDa (3 chains, one 49.5 kDa y chain, and one
variant 50.5
kDa y prime chain. The gamma and gamma prime chains are both coded for by the
same
gene, with alternative splicing occurring at the 3' end. Normal gamma chain is
composed
of amino acids 1-411 and normal variant gamma prime chain is composed of 427
amino
acids, of which amino acids 1-407 are the same as those in the normal gamma
chain and
amino acids 408-427 are VRPEHPAETEYDSLYPEDDL (SEQ m N~:2). This region is
normally occupied with thrombin molecules.
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[26] Fibrinogen is converted into fibrin by the action of thrombin in the
presence of ionized calcium to produce coagulation of the blood. Fibrin is
also a
component of thrombi, and acute inflammatory exudates.
[27] Therefore, an object of the invention is to provide various antibodies or
polypeptides that bind sulfated PSGL-1 and methods of use thereof.
[2~] Specifically, an object of the invention is to provide methods of
activating
ADCC or stimulating natural killer (NIA) or T cells by administering the
antibodies of the
present invention.
[29] Another specific object of the invention is to provide a method of
inducing
cell death.
[30] Yet another specific object of the invention is to provide a method of
preventing infection by a virus, such as HIV, comprising administering to a
patient in need
thereof an antibody as herein.
[31] Another specific object of the invention is to provide a method of
introducing an agent into a cell that expresses sulfated PSGL-1 having the
following steps:
coupling or complexing the agent to an antibody as described herein and
administering the
antibody-agent couple or complex to the cell is provided
[32] Finally, it is a specific objective of the present invention to provide
methods of diagnosis, prognosis, and staging using the present antibodies.
SUMMARY ~F THE INVENTI~N
[33] The present invention provides antibodies or polypeptides that bind an
epitope of PSGL-1 comprising the motif D-X-Y-D (SEQ ID N0:3), wherein X
represents
any amino acid or the covalent linkage between D and Y, and Y is sulfated,
which
antibody can be coupled to or complexed with multiple copies of an agent
selected from
the group consisting of anti-cancer, anti-leukemic, anti-metastasis, anti-
neoplastic, anti-
disease, anti-adhesion, anti-thrombosis, anti-restenosis, anti-autoimmune,
anti-
aggregation, anti-bacterial, anti-viral, and anti-inflammatory agents.
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[34] The antibodies of the invention can be used in a method of inducing
antibody-dependent cell cytotoxicity and/or stimulating natural killer (NK)
cells or T cells.
Tn addition, by administering these antibodies to a patient in need thereof, a
method of
inducing cell death is provided. A method of preventing infection by a virus
(e.g., HIV)
by administering to a patient in need thereof an antibody of the present
invention is also
provided. The present invention also provides a method of introducing an agent
into a cell
that expresses sulfated PSGL-1 by coupling or complexing an agent to an
antibody of the
present invention and administering the antibody-agent couple or complex to
the cell. The
present invention further provides a method of identifying, isolating and
purifying tumor
cell markers. Finally, the present invention provides methods of diagnosis,
prognosis and
staging using the present antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
[35] The invention is herein described in more detail, by way of example only,
and not by way of limitation, with reference to the accompanying drawings
described
below, wherein:
[~6] Figure 1 shows a Western blot of partially purified AML-I~1 cell lysate
before and after passage through Y1-IgG affinity column.
[37] Figure 2 shows that, of three tyrosines in the purified protein's
sulfated-
tyrosine motif, tyrosines 2 and 3 are sulfated.
[38] Figure 3 shows Y1-IgG (20 ~,g/ml) mediated A:DCC (percent cytotoxicity)
in primary B-CLL samples.
[39] Figure 4 shows Yl-IgG-mediated ADCC (percent cytotoxicity) by PBMC
against AML cells.
[40] Figure S shows increaseed ADCC (percent cytotoxicity) in ML-2 cells as a
function of Y1-IgG concentration.
[41] Figure 6 shows ADCC (percent cytotoxicity) by PBMC against ML-2 as a
function of competition between Y1-IgG and KPL-1.
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11
[42] Figure 7A shows analysis of Y1-IgG-mediated ADCC (percent
cytotoxicity) by natural killer cells from normal donors and B-CLL patients
against ML2
cells. Figure 7S shows the involvement of CD14+ cells (monocytes) in ADCC
against
M12 targets.
[43] Figure ~ shows expression of CD69 (an early activation marker) on NK
cells mediated by Yl .
[44] Figure 9 shows apoptotic effect of Yl-IgG on mononuclaer cells (CD19+,
CDS+) from B-CLL patients by FAGS analysis.
[45] Figure 10 shows analysis of ADCC activity (percent cytotoxicity) against
mononuclear cells from human B-CLL patients, i.e., primary human B-CLL cells
(KBC115 and KBC116 cells) mediated by Yl-IgG and Rituximab.
[46] Figure 11 shows analysis of CDC activity (percent lysis) against
mononuclear cells from human B-CLL patients (KBC156, KBC159, KBC160, KBC166,
and RAJI sells) mediated by Y1-IgG, Rituximab, and Campath~ in the presence
and
absence ~f patient plasma.
[47] Figure 12 shows reaction scheme for preparation of antibody linked to
morpholino-doxoru~bicin.
[4~] Figure 13 shows cytotoxicity of an antibody-agent complex, namelyYl-
morpholinodaunorubicin (Y1-M-DNR) and Yl-morpholinodoxorubicin (Y1-M-Dox)
complexes in cord blood and AML cells.
[49] Figure 14 shows cytotoxicity of the antibody-agent complex Y1 M I~NR
against 2 patient AML samples (M4 and MS stage) and against CD34+ cells.
[50] Figure 15 shows cytotoxicity of various Y1 complexes as a percent of
control in B-ALL cells.
[51] Figure 16A shows binding of Yl scFv to KU812 cells and Figure 16B
shows the surface expression of GPIb on sulfate starved KU812 cells.
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12
[52] Figure 17A shows inhibitory effects of sulfated peptides DLYDYYPE on
the binding of Yl-scFv to platelets. Figure 17B shows the effects of
substitution mutant
peptides in Yl-scFv platelet binding assay.
[53) Figure 18A shows effects of mutant peptides in the inhibition of Yl-scFv
binding to purified glycocalicin. Figure 18B shows the binding of YlscFv to
peptides
covalently coupled to CovaLinkTM Plates by ELISA.
[54] Figure 19 shows binding of Y1 to immobilized, sulfated peptides derived
from PSGL-1.
[55] Figure 20 shows percent activity of Yl binding to non-sulfated PSGL-1
and to PSGL-1 sulfated in the first, second, and third positions.
~ [56] Figure 21 some potential Yl binding motifs that are highly acidic and
have
sulfated tyrosines.
[57] Figure 22 shows recognition of small cell lung carcinoma (SCLC) lysate
by Y1.
[58] Figure 23 shows endocytosis of Yl into primary AML cells.
[59] lFigure 24 shows endocytosis of Yl into primary AML cells.
[60] Figure 25 shows analysis of Yl binding to healthy CD34+ stem cells.
[61] Figure 26 shows analysis of Yl binding to healthy CD34+ stem cells.
[62] Figure 27 shows internalization of Y1 into primary AML cells at
37° C.
[63] Figure 28 shows visualization of Yl staining in primary AML cells after
stripping membrane-bound protein by acid treatment.
[64] Figure 29 shows visualization of Yl staining in primary AML cells after
stripping membrane-bound protein by pronase treatment.
[65] Figure 30 shows visualization of Yl staining in primary AML cells after
acid treatment or sucrose pre-incubation at 4° C (Fig. 30A) and at
37° C (Fig. 30B).
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13
[66] Figure 31 shows that Y1-scFv effectively inhibits binding of activated
human platelets to ML2 cells.
[67] Figure 32 shows the effect of Y1-scFv (10 ~.g/ml) on ML2 cell rolling on
immobilised rh-P-Selectin at low density (0.2 ~,g/ml).
[68] Figure 33 shows the effect of Y1-scFv (10 ~,g/ml) on ML2 cell rolling on
immobilized rh-P-Selectin at high density (1.0 ~,g/ml).
[69] Figure 34 shows the effect of Yl-IgG (1 ~,g/ml) on ML2 cell rolling on
immobilized rh-P-Selectin (1.0 ~.glml) at various shear stress forces.
[70] Figure 35 shows the effect of increasing concentrations of Yl-scFv on
human neutrophil rolling on immobilized rh-P-Selectin at high density (1.0
~g/ml).
[71] Figure 36 shows the effect of Y1-IgG on human neutrophil rolling on
immobilized rh-P-Selectin at lugh density (1.0 ~,g/ml).
I~E7°A1LEI~ I~F~C F°F1~l~T ~F TT~IF 1 L~TI~N
[72] Antibodies (Abs), or immunoglobulins (Igs), are protein molecules that
bind to antigen. Each f~xn.ctional binding unit of naturally occurring
antibodies is
composed of units of four polypeptide chains (2 heavy and 2 light) linked
together by
disulfide bonds. Each of the chains has a constant and variable region.
Naturally
occurring antibodies can be divided into several classes including IgG, IgM,
IgA, IgD, and
IgE, based on their heavy chain component. The IgG class encompasses several
sub-
classes including, but not restricted to, IgGI, IgG~, IgG3, and IgG4.
Immunoglobulins are
produced in viv~ by B lymphocytes, and each such molecule recognizes a
particular
foreign antigenic determinant and facilitates clearing of that antigen.
[73] Antibodies may be produced and used in many forms, including antibody
complexes. As used herein, the term "antibody complex" or "antibody complexes"
is used
to mean a complex of one or more antibodies with another antibody or with an
antibody
fragment or fragments, or a complex of two or more antibody fragments.
Examples of
antibody fragments include Fv, Fab, F(ab')2, Fc, and Fd fragments. Therefore,
an antibody
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according to the present invention encompasses a complex of an antibody or
fragment
thereof.
[74] As used herein in the specification and in the claims, an Fv is defined
as a
molecule that is made up of a variable region of a heavy chain of a human
antibody and a
variable region of a light chain of a human antibody, which may be the same or
different,
and in which the variable region of the heavy chain is connected, linked,
fused, or
covalently attached to, or associated with, the variable region of the Light
chain. The Fv
can be a single chain Fv (scFv) or a disulfide stabilized Fv (dsFv). An scFv
is comprised
of the variable domains of each of the heavy and light chains of an antibody,
linked by a
flexible amino-acid polypeptide spacer, or linker. The linker may be branched
or
unbranched. Preferably, the linker is 0-15 amino acid residues, and most
preferably the
linker is (Gly4Ser)3.
[75] The Fv molecule, itself, is comprised of a first chain and a second
chain,
each chain having a first, second and third hyper~ariable region. The
hypeavariable loops
within the variable domains of the light and heavy chains are termed
Complementary
Determining Legions (CDLs). There are CDLl, CDL2, and CDL3 regions in each of
the
heavy and light chains. These regions are believed to fornl the antigen
binding site and
can be specifically modified to yield enhanced binding activity. The most
variable of
these regions in nature is the CDL3 region of the heavy chain. The CDL3 region
is
understood to be the most exposed region of the Ig molecule and, as shown and
provided
herein, is the site primarily responsible for the selective and/or specific
binding
characteristics observed.
[76] A fragment of an Fv molecule is defined as any molecule smaller than the
original Fv that still retains the selective and/or specific binding
characteristics of the
original Fv. Examples of such fragments include but are limited to (1) a
minibody, which
comprises a fragment of the heavy chain only of the Fv, (2) a microbody, which
comprises
a small fractional unit of antibody heavy chain variable region (International
Application
No. PCT/IL99/00581), (3) similar bodies having a fragment of the light chain,
and
(4) similar bodies having a functional unit of a light chain variable region.
It should be
appreciated that a fragment of an Fv molecule can be a substantially circular
or looped
polypeptide.
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[77] As used herein the term "Fab fragment" is a monovalent antigen-binding
fragment of an immunoglobulin. A Fab fragment is composed of the light chain
and part
of the heavy chain.
[78] An F(ab')2 fragment is a bivalent antigen binding fragment of an
immunoglobulin obtained by pepsin digestion. It contains both light chains and
part of
both heavy chains.
[79] An Fc fragment is a non-antigen-binding portion of an immunoglobulin. It
contains the carboxy-terminal portion of heavy chains and the binding sites
for the Fc
receptor.
[80] An Fd fragment is the variable region and first constant region of the
heavy
chain of an immunoglobulin.
[81] Polyclonal antibodies are the product of an immune response and axe
formed by a number of different ~ lymphocytes. l~Ionoclonal antibodies are
derived from
one clonal 13 cell.
[82] A cassette, as applied to polypeptides and as defined iil the present
invention, refers to a given sequence of consecutive amino acids that serves
as a
framework and is considered a single unit and is manipulated as such. Amino
acids can be
replaced, inserted into, removed, or attached at one or both ends. Likewise,
stretches of
amino acids can be replaced, inserted into, removed, or attached at one or
bath ends.
[83] The term "epitope" is used herein to mean the antigenic determinant or
recognition site or antigen site that interacts with an antibody, antibody
fragment, antibody
complex or a complex having a binding fragment thereof or T cell receptor. The
term
epitope is used interchangeably herein with the terms ligand, domain, and
binding region.
[84] Selectivity is herein defined as the ability of a targeting molecule to
choose
and bind one entity or cell state from a mixture of entities or entity states,
all entities or
entity states of which may be specific for the targeting molecule.
[85] The term "affinity" as used herein is a measure of the binding strength
(association constant) between a binding molecule (e.g., one binding site on
an antibody)
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and a ligand (e.g., antigenic determinant). The strength of the sum total of
noncovalent
interactions between a single antigen-binding site on an antibody and a single
epitope is
the affinity of the antibody for that epitope. Low affinity antibodies bind
antigen weakly
and tend to dissociate readily, whereas high-affinity antibodies bind antigen
more tightly
and remain bound longer. The term "avidity" differs from affinity, because the
former
reflects the valence of the antigen-antibody interaction.
[86] Specificity of antibody-antigen interaction: Although the antigen-
antibody
reaction is specific, in some cases antibodies elicited by one antigen can
cross-react with
another unrelated antigen. Such cross-reactions occur if two different
antigens share a
homologous or similar structure, epitope, or an anchor region thereof, or if
antibodies
specific for one epitope bind to an unrelated epitope possessing similar
structure
I
conformation or chemical properties. .
[87] A platelet is a disc-like cytoplasmic fragment of a megakaryocyte that is
shed in the marrow sinus and subsequently circulates in the peripheral blood
stream.
Platelets have several physiological functions including a major role in
clotting. A platelet
contains centrally located granules and peripheral clear protoplasm, but has
no definite
nucleus.
[88] Agglutination as used herein means the process by which suspended
bacteria, cells, discs, or other particles of similar size are caused to
adhere and form into
clumps. The process is similar to precipitation but the particles are larger
and are izz
suspension rather than being in solution.
[89] The term aggregation means a clumping of platelets induced in vitro, and
thrombin and collagen, as part of a sequential mechanism leading to the
formation of a
thrombus or hemostatic plug.
[90] Conservative amino acid substitution is defined as a change in the amino
acid composition by way of changing one or two amino acids of a peptide,
polypeptide or
protein, or fragment thereof. The substitution is of amino acids with
generally similar
properties (e.g., acidic, basic, aromatic, size, positively or negatively
charged, polarity,
non-polarity) such that the substitutions do not substantially alter peptide,
polypeptide or
protein characteristics (e.g., charge, isoelectric point, affinity, avidity,
conformation,
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solubility) or activity. Typical substitutions that may be performed for such
conservative
amino acid substitution may be among the groups of amino acids as follows:
glycine (G), alanine (A), valine (V), Ieucine (L) and isoleucine (1)
aspartic acid (D) and glutamic acid (E)
alanine (A), serine (S) and threonine (T)
,,
histidine (H), lysine (K) and arginine (R)
asparagine (N) and glutamine (Q)
phenylalanine (F), tyrosine (I') and tryptophan (Vt~
[91] Conservative amino acid substitutions can be made in, e.g., regions
flanking the hypervariable regions primarily responsible for the selective
and/or specific
binding characteristics of the molecule, as well as other parts of the
molecule, e.g.,
variable heavy chain cassette. Additionally or alternatively, modification can
be
accomplished by reconstructing the molecules to form full-sire antibodies,
diabodies
(dimers), triabodies (timers), and/or tetrabodies (tetramers) or to form
minbodies or
microbodies.
[~2] A phagemid is defined as a phage particle that carries plasmid I~NA.
Phagemids are plasmid vectors designed to contain an origin of replication
from a
filamentous phage, such as m13 of fd. Since it carnes plasmid DNA, the
phagemid
particle does not have sufficient space to contain the full complement of the
phage
genome. The component that is missing from the phage genome is information
essential
for packaging the phage particle. In order to propagate the phage, therefore,
it is necessary
to culture the desired phage particles together with a helper phage strain
that complements
the missing packaging information.
[93] A promoter is a region on DNA at which RNA polymerase binds and
initiates transcription.
[94] A phage display Library (also termed phage peptide/antibody library,
phage
library, or peptide/antibody library) comprises a Large population of phages
(108 or larger),
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each phage particle displaying a different peptide or polypeptide sequence.
These peptide
or polypeptide fragments may constructed to be of variable length. The
displayed peptide
or polypeptide can be derived from, but need not be limited to, human antibody
heavy or
light chains.
[95] A pharmaceutical composition refers to a formulation which comprises an
antibody or peptide or polypeptide of the invention and a pharmaceutically
acceptable
carrier, excipient or diluent thereof, or an antibody-pharmaceutical agent
(antibody-agent)
complex and a pharmaceutically acceptable carrier, excipient or diluent
thereof.
[96] An agent in the context of the present invention is useful in the
treatment of
active disease, prophylactic treatment, or diagnosis of a mammal including,
but not
restricted to, a human, bovine, equine, porcine, marine; canine, feline, or
any other warm-
blooded animal. The agent is selected from the group of radioisotope, toxin,
oligonucleotide, recombinant protein, antibody fragment, anti-cancer agents,
anti-
leukemic, anti-metastasis, anti-neoplastic, anti-disease, anti-adhesion, anti-
thrombosis,
anti-restenosis, anti-autoimmune, anti-aggregation, anti-bacterial, anti-
viral, and anti-
inflammatory agents. ~ther examples of such agents include, but are not
limited to anti-
viral agents including acyclovir, ganciclovir, and zidovudine; anti-
thrombosis/restenosis
agents including cilostazol, dalteparin sodium, reviparin sodium, and aspirin;
anti-
inflammatory agents including zaltoprofen, pranoprofen, droxicam, acetyl
salicylic 17,
diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin,
celecoxib,
indomethaciii, rofecoxib, and nimesulid; anti-autoimmune agents including
leflunomide,
denileukin iliftitox, subreum, WinRho SDF, defibrotide, and cyclophosphamide;
and anti-
adhesion/anti-aggregation agents including limaprost, clorcromene, and
hyaluronic acid.
[97] An anti-leukemia agent is an agent with anti-leukemia activity. For
example, anti-leukemia agents include agents that inhibit or halt the growth
of leukemic or
immature pre-leukemic cells, agents that kill leukemic or pre-leukemic cells,
agents that
increase the susceptibility of leukemic or pre-leukemic cells to other anti-
leukemia agents,
and agents that inhibit metastasis of leukemic cells. In the present
invention, an anti-
leukemia agent may also be an agent with anti-angiogenic activity that
prevents, inhibits,
retards or halts vascularization of tumors.
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[98] The expression pattern of a gene can be studied by analyzing the amount
of
gene product produced under various conditions, at specific times, in various
tissues, etc.
A gene is considered to be "over-expressed" when the amount of gene product is
higher
than that found in a normal control, e.g., non-diseased control.
[99] A given cell may express on its surface a protein having a binding site
(or
epitope) for a given antibody, but that binding site may exist in a cryptic
form (e.g~., be
sterically hindered or blocked, or lack features needed for binding by the
antibody) in the
cell in a state, which may be called a first stage (stage I). Stage I may be,
e.g., a normal,
healthy, non-diseased status. When the epitope exists in cryptic form, it is
not recognized
by the given antibody, i.e., there is no binding of the antibody to this
epitope or to the
given cell at stage I. However, the epitope may be exposed by, e.g.,
undergoing
modifications itself, or being unblocked because nearby or associated
molecules are
modified or because a region undergoes a conformational change. Examples of
modifications include changes in folding, changes in post-translational
modifications,
changes in phospholipidation, changes in sulfation, changes in glycosylation,
and the like.
Such modifications may occur when the cell enters a different state, which may
be called a
second stage (stage Il). Examples of second states, or stages, include
activation,
proliferation, transformation, or in a malignant status. Upon being modified,
the epitope
may then be exposed, and the antibody may bind.
[100] Peptido-mimetics (peptide mimetics) are molecules (e.g., antibodies)
that
no longer contain any peptide bonds, i.e., amide bonds, between amino acids;
however, in
the context of the present invention, the term peptide mimetic is intended to
include
molecules that are no longer completely peptidic in nature, such as pseudo-
peptides, semi-
peptides and peptoids. Whether completely or partially non-peptide,
peptidomimetics
according to this invention provide a spatial arrangement of reactive chemical
moieties
that closely resembles the three-dimensional arrangement of active groups in
the peptide
on which the peptidomimetic is based. These molecules include small molecules,
lipids,
polysaccharides, or conjugates thereof.
[101] Phagemids are plasmid vectors designed to contain an origin of
replication
from a filamentous phage, such as M13 or fd.
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[102] A wide spectrum of diseases exists that involves diseased, altered, or
otherwise modified cells that express cell-specific and/or disease-specific
ligands on their
surfaces. These ligands can be utilized to effect recognition, selection,
diagnosis and
treatment of specific diseases through recognition, selection, diagnosis and
treatment of
each individual cell. The subject invention provides for peptides or
polypeptides that
comprise an Fv molecule, a construct thereof, a fragment thereof, a construct
of a fragment
thereof, or a fragment of a construct, all of which have enhanced binding
characteristics.
These binding characteristics allow the peptide or polypeptide molecule to
bind selectively
and/or specifically to a target cell in favor of other cells, the binding
specificity and/or
selectivity being primarily determined by a first hypervariable region. The Fv
can be a
scFv or a dsFv.
[103] The Fv molecule described above can be used to target a diseased cell.
The
diseased cell can be, for example, a cancer cell. Examples of types of cancer
that are
amenable to diagnosis and/or treatment by specific targeting include, but are
not limited
to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma,
seminoma,
and melanoma. Leukemia, lymphoma, and myeloma are cancers that originate in
the bone
marrow and lymphatic tissues and are involved in uncontrolled growth of cells.
[104] Antibodies that bind to PSC~L-1 and/or GPIb were identified using a
phage
display library and disclosed in LT.S. Application Nos. 10/032,423;
10/032,037;
10/029,9~~; 10/029,926; 091751,11; 10,19,032; and 60/25~,94~~ and
International
Application Nos. PCT/USO1/49442 and PCT/CTS01/49440. Specific examples of
antibodies disclosed in these applications include the Yl, Y17, and L32
antibodies. These
antibodies were isolated from the germ line (DP32) and were discovered to
specifically
bind to an epitope, found on proteins of the hematopoetic cells, which is
sulfated at an N-
terminal tyrosine and is thought to be involved in cell migration, e.g. tumor
metastasis.
[105] The sulfated epitopes binding to Yl/Y17/L32 axe characterized by the
presence of sulfated moieties, such as sulfated tyrosine residues or sulfated
carbohydrate
or lipid moieties, preferably within a cluster of two or more acidic amino
acids, which are
found on ligands and receptors that play important roles in such diverse
processes as
inflammation, immune reactions, infection, autoimmune reactions, metastasis,
adhesion,
thrombosis and/or restenosis, cell rolling, and aggregation. Such epitopes are
also found
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on diseased cells, such as T-ALL cells, B-leukemia cells, B-CLL cells, AML
cells,
multiple myeloma cells, and metastatic cells. These epitopes are useful
targets for the
therapeutic mediation of these processes (as well as targeting agents) and for
diagnostic
procedures.
[106] Moreover, it was found for these antibodies of the present invention
that
binding is dependent on the stage of development of the cell (AML subtype is
classified
based on the French-American-British system using the morphology observed
under
routine processing and cytochemical staining): the antibodies bind to AML
cells that are of
subtype M3 or above, but not MO or M1 subtype cells. In addition, the
antibodies may or
may not bind M2 subtype cells. Accordingly, the antibodies of the present
invention do
not bind normal, healthy bone marrow (e.g., CI~34+ cells). It is thought that
such
differences are based on alterations in PSGL-1 expression and/or sulfation, as
well as
possible conformational changes in PSGL-1 that expose a slightly different
epitope.
[107] In particular, it has been found that KLT812 cells, a human chronic
myeloid
leukemia cell line that expresses low levels of CaPIb, binds the Yl antibody.
Following
growth of I~U812 cells in sodium chlorate, which inl>ibits sulfation but not
expression of
the (aPTb protein, binding of IjI to the cells was reduced by 50~10. In
addition, it has been
found that tyrosine-sulfated peptides based on amino acids 273 to 2~5 of
C"aPTb
competitively inhibit binding of the Y1 antibody to platelets, while non-
sulfated peptides
do not inhibit binding of the Y1 antibody to platelets.
[108] The invention comprises or employs an antibody or fragment thereof that
recognizes and binds to an epitope comprising a sulfated tyrosine motif. Such
a motif
comprises a peptide sequence that is rich in acidic residues (aspartate and
glutamate) and
contains at least one tyrosine. Recognition and binding depend at least in
part on at least
one of the tyrosines being sulfated. One such antibody is Yl or a fragment
thereof.
Although Yl is the antibody referred to in the embodiments described herein,
this should
not be understood as limiting the invention to embodiments that employ Yl. The
invention includes embodiments that use other antibodies that bind to an
epitope
comprising a sulfated tyrosine motif, including but not limited to antibodies
related to Y1
and fragments thereof that retain binding specificity.
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[109] According to the present invention, provided is an antibody or fragment
thereof that binds to an epitope comprising a sulfated tyrosine motif, wherein
the binding
is dependent on at least one tyrosine of the motif being sulfated. In one
embodiment, the
antibody mediates antibody-dependent cell cytotoxicity. In a preferred
embodiment, the
antibody is Y1 or a related antibody, or a fragment thereof.
[110] The invention further provides an agent complexed with (e.g.,
associated,
combined, fused, or linked to) such an antibody or fragment thereof. between 1
and 16
agent molecules, or more, can be bound to each antibody. The antibody has four
disulfide
bonds at the lunge region that can be selectively reduced to eight thiol
groups. ~y using a
linker that can covalently bond to thiol functions and which carries one agent
molecule, up
to eiglit agent molecules can be attached to the antibody. By using a linker
that similarly
reacts with thiol functions but carries n agent molecules, up to 8n agent
molecules can be
attached to the antibody. In one embodiment only the heavy chains are
complexed with
the agent or only the light chains are complexed with the agent, while in a
more prefered
embodiment, each heavy chain is complexed with about 2 copies of the agent and
each
light chain is complexed with about 2 copies of the agent.
[111] To those of ordinary skill in the art it is known that an even greater
number
of agent molecules can be linked to the antibody by using intermediate drug
carriers such
as natural (e.g. dextran) and synthetic (e.g. I~'l~~A) polymers as well as
liposomes (e.g.,
antibody-linker-carrier-agent). Agents can also be linked directly or
indirectly to free
amino groups of the antibody. For example, agents can be linked to free ~- or
o~-amino
groups via a linker. Typically an agent is joined to a linker directly, or
first to a carrier,
which is then joined to a linker. The linker-agent or linker-Garner-agent
complex is then
joined to the antibody. The antibody-agent complex can be internalized by a
tumor cell,
wherein the agent brings about the cell's death. In one embodiment, the
antibody-agent
linkage can be broken inside the cell by, for example, acid cleavage or enyzme
cleavage.
In a preferred embodiment, the antibody is Y1 or a related antibody, or a
fragment thereof.
(112] The invention further provides a composition for treating a disease
comprising Yl or a Y1-agent complex.
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[113] In one embodiment, the present invention provides methods of inducing or
activating ADCC by administering the antibodies of the present invention.
Accordingly,
these antibodies may activate ADCC and/or stimulate natural killer (NK) cells
(e.g.
CD56+), y~ T-cells, and/or monocytes, which may result in cell lysis.
Generally,
following administration of an antibody comprising an Fc region or portion of
the
antibody, said antibody binds to an Fc receptor (FcR) on effector cells, for
example, NK
cells, triggering the release of perforin and granzyme B and/or induction of
Fas B
expression, which then leads to apoptosis. Binding of Fast expressed on
effector cells to
the Fas receptor on the target cell surface may induce target cell apoptosis
via activation of
the Fas receptor signal transduction pathway. In one embodiment, the antibody
of the
invention induces Fast expression on effector cells. Various factors can
affect ADCC,
including the type of effector cells involved, cytokines (IL-2 and G-CSF, for
example),
incubation time, the number of receptors present on the surface of the cells,
and antibody
affinity.
[114] In yet another embodiment, a method of inducing cell death by
administering to a patient in need thereof an antibody of the present
invention coupled or
complexed to an agent, wherein the antibody-agent couple or complex enters the
cell by
internalization and the antibody-agent conjugate or complex is cleaved,
releasing the agent
is provided. Internalization can take place by any suitable means, for
example, by
endocytosis or by phagocytosis. The invention thus provides a means of
treating a disease
(e.g., treating can include ameliorating the effects of a disease, preventing
a disease, or
inhibiting the progress of a disease) in a patient.
[115] Specifically, an antibody is used to introduce an agent into a cell. The
antibody binds to proteins preferentially expressed on the surface of diseased
cells, such as
proteins with sulfated tyrosine residues. In a preferred embodiment, the agent
is a toxin
such as doxorubicin, morpholino-doxorubicin, or morpholino-daunorubicin. In a
more
preferred embodiment, the toxin is linked to the antibody via an adipic acid
linker or an
[N-s-Maleimidocaproic acid hydrazide linker. The adipic acid linker has been
used to
bind to the a amino groups, whereas the N-[maleimidocaproic acid] hydrazide
linker has
been used to bind to both the a amino groups and also to the SH groups of the
reduced
disulphide linkages (via the maleimido group to form a C-S bond). In addition,
a
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hydrazone bond is formed between the drug and the N-[maleimidocaproic acid]
hydrazide
linker.
[116J After the antibody-agent complex binds to the cell surface protein, the
cell
internalizes the complex. Enzymes within the cell then cleave the antibody-
toxin linkage,
and the toxin acts on the cell to bring about its death. In another
embodiment, the
invention provides a composition for treating a disease comprising such an
antibody-toxin
conjugate.
[117] Another embodiment provides an analogous method for introducing a non-
toxic agent into a cell. The non-toxic agent can be used to change the
behavior or activity
of the cell, for example by directly or indirectly activating or repressing
the activity of a
specific gene.
[11~] In one other embodiment, the present invention provides a method of
preventing infection by a virus comprising administering to a patient in need
thereof an
antibody as herein. Thus, a means of treating a disease is accomplished by
administering
an antibody that blocks infection. The cell expresses on its surface a protein
containing a
sulfated-tyrosine motif containing epitope that is recognized by the antibody
and that is
also necessary for infection by the infectious agent. The antibody binds to
the protein,
thereby blocking infection. Proteins that the preferred antibody is lmovsm to
bind via a
sulfated tyrosine motif containing epitope include fibrinogen y chain, GPlb-c~
chain,
complement C4, and PSGL-1. Proteins that the preferred antibody is believed to
bind via
a sulfated tyrosine motif -containing epitope include CCRS and CXCR4. Either
of CCRS
and CXCR4 can function as a coreceptor necessary for HIV infection. In a
preferred
embodiment, the antibody could be used to block infection by an HIV strain.
Preferably
the antibody is ~'1.
[119] Finally, a method is provided for introducing an agent into a cell that
expresses sulfated PSGL-1 having the following steps: coupling or complexing
the agent
to an antibody as described herein and administering the antibody-agent couple
or
complex to the cell.
[120] The antibody of the present invention binds to sulfated PSGL-1. White
cells involved in inflammation, such as monocytes, neutrophils, and
lymphocytes, axe
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primarily recruited by the four adhesion molecules, PSGL-I, P-selectin, VLA-4,
and
VCAM-1 in the inflammatory processes of diseases such as atherosclerosis (Huo
and Ley,
Acta Playsiol. Scaad., 173: 35-43 (2001); Libby, Sci. Arn. May: 48-55 (2002);
Wang et al.,
.I. Am. Coll. Car~diol. 38: 577-582 (2001)). The antibody's interference with
any of these
central molecules may suggest a potential role for the antibody in abrogating
related
diseases. Specifically, P-selectin controls cell attachment and rolling.
Additionally, P-
selectin - PSGL-1 interactions activate a number of other molecules on cells
which are
integrally connected with tumorigenesis (when concerned with malignant cells)
and
inflammatory responses (when concerned with white blood cells) (Shebuski and
Kilgore,
,I. Pha~fraacol. ExP. They. 300: 729-735 (2002)). used on this understanding
of P-
selectin's ability to regulate cellular processes, it is apparent that the
antibody's enhanced
scF°v selectivity for sulfated PSGL-1 may make it a superior molecule
for treating a variety
of malignant and inflammatory diseases. Moreover, models of malignant disease
have
shown that P-selectin binding to malignant cells requires sulfation of PSGL-1
(Ma and
Geng, ~: Immunol. 168: 1690-1696 (2002)). This requirement is similar to that
for binding
of the antibody. Thus, one can expect that the antibody could abrogate P-
selectin
facilitation of progressing malignant disease.
[121] Preferably, the antibody of the present invention binds to an epitope
present
on at least one cell type involved in inflammation or tumorigenesis, including
T-ALL
cells, AML cells, Pre-~-ALL cells, ~-Leukemia cells, ~-CLL cells, multiple
myeloma
cells, and metastatic cells. Further preferably, the antibody of the present
invention may
bind to epitopes on a lipid, carbohydrate, peptide, glycolipid, glycoprotein,
lipoprotein,
and/or lipopolysaccharide molecule. Such epitopes preferably have at least one
sulfated
moiety. Alternatively, but also preferably, the antibody of the present
invention
crossreacts with two or more epitopes, each epitope having one or more
sulfated tyrosine
residues, and at least one cluster of two or more acidic amino acids, an
example of which
is PSGL-1.
[122] These antibodies, antigen-binding fragment or complex thereof, of the
present invention may be internalized into a cell following binding to PSGL-1
on the
surface of the cell. Such internalization may occur via endocytosis as an
active process,
which is manner, time and temperature dependent. For example, Y1 is
specifically
internalized into cells from AML patients via PSGL-1.
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[123] The antibody of the present invention binds to proteins having tyrosine
sulfation sites. Such proteins include PSGL-1, GPlb, a-2antiplasmin;
aminopeptidase B;
CC chemokine receptors such as CCR2, CCRS, CCR3, CXCR3, CXCR4, CCRB, CCR2b,
and CXCI; seven-transmembrane-segment (7TMS) receptors; coagulation factors
such as
factor V, VIII, and IX; fibrinogen gamma chain; heparin cofactor II;
secretogranins such
as secretogranin I and II; vitronectin, amyloid precursor, a-2-antiplasmin;
cholecystokinin;
a-choriogonadotropin; complement C4; dermatan sufaieproteiglycan; fibrinectin;
and
castrin. In a preferred embodiment, the antibody of the present invention
binds to sulfated
CC chemokine receptors such as CCRS, CXCR4, CXCI, and CCR2b. As mentioned
previously, sulfated tyrosines may contribute to the binding of CCRS to MIP-1
a, MIP[3,
and HIV-1 gp120/CD4 and to the ability of HIV-1 to enter cells expressing CCRS
and
CD4.
[124] Antibodies, peptides, polypeptides, proteins, and fragments and
constructs
thereof can be produced in either prokaryotic or eukaryotic expression
systems. Methods
for producing antibodies and fragments in prokaryotic and eukaryotic systems
are well-
known in the art.
[125] A eukaryotic cell system, as defined in the present invention and as
discussed, refers to an expression system for producing peptides or
polypeptides by
genetic engineering methods, wherein the host cell is a eukaryote. A
eukaryotic
expression system may be a mammalian system, and the peptide or polypeptide
produced
in the mammalian expression system, after purification, is preferably
substantially free of
mammalian contaminants. ~ther examples of a useful eukaryotic expression
system
include yeast expression systems.
[126] A preferred prokaryotic system for production of the peptide or
polypeptide
of the invention uses E. coli as the host for the expression vector. The
peptide or
polypeptide produced in the E. coli system, after purification, is
substantially free of E.
coli contaminating proteins. Use of a prokaryotic expression system may result
in the
addition of a methionine residue to the N-terminus of some or all of the
sequences
provided for in the present invention. Removal of the N-terminal methionine
residue, after
peptide or polypeptide production to allow for full expression of the peptide
or
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polypeptide, can be performed as is known in the art, one example being with
the use of
Aeromonas aminopeptidase under suitable conditions (LT.S. Fatent No.
5,763,215).
[127] The antibodies and polypeptides of the subject invention can be
complexed
with e.g. associated with, combined, fused, or linked to various
pharmaceutical agents,
such as drugs, toxins, and radioactive isotopes and optionally, with a
pharmaceutically
effective carrier, to form peptide-drug compositions comprising an
antibody/polypeptide
and a pharmaceutical agent having anti-disease and/or anti-cancer activity.
Such
compositions may also be used for diagnostic purposes.
[128] For example,, conjugation or complexing of anthracyclines to antibodies
is
generally known in the art (Dubowchik ~ Walker, Phaa~yyaacol. ~ Tl2eYa. 83: 67-
123
(1999); Trail et al., C'arzeef~ Irramuuol. Immurlother. 52: 328-337 (2003)).
Such conjugation
can be by direct conjugation or via linkers, such as acid cleavable linkers or
enzyme
cleavable linkers and may involve the use of intermediate carriers such as
dextran and
synthetic polymers. ~nthracyclines have been complexed to the antibodies of
the present
invention via (1) o, amino groups (about pH 8) to produce a drug:antibody
ratio of 4:1 (in
which case two drug molecules are attached to the heavy chain and two to the
light chain);
and (2) disulfide linkages to produce a drug antibody ratio of between 4:1 and
S:1
depending or the method used. As is known in the art, the drug antibody ratio
can, for
example, be doubled, tripled or quadrupled, etc, by using a two, three, four,
etc., branched
linker. ~ne skilled in the art may make chemical modifications to the
antibody, linker,
carrier and/or drug in order to make reactions more convenient for the
purposes of
preparing a conjugate.
(129] In one embodiment of the present invention, the two disulfide linkages
in
the Fc region were reduced with mercaptoethylamine and was then reacted with
the drug
linker at about pH 7, which leads to a drug antibody ratio of 4:1 (in which
case all the four
drugs are attached in the heavy chains). In another embodiment, the four
disulfide bonds
in the hinge region were reduced with DTT (about pH 7) and was then reacted
with the
drug linker, which leads to a drug antibody ratio of about 7:1 to 8:1 (in
which case 5 or 6
of the drug molecules are attached to the heavy chains and one or two of the
drug
molecules are attached to the light chains).
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[130] Examples of carriers useful in the invention include dextran, HPMA (a
hydrophilic polymer), ox any other polymer, such as a hydrophilic polymer, as
well as
derivatives, combinations and modifications thereof. Alternatively, decorated
liposomes,
also known as immunoliposomes, can be used, such as liposomes decorated with
scFv Yl
molecules, such as Doxil, a commercially available liposome containing large
amounts of
doxorubicin. Such liposomes can be prepared to contain one or more desired
agents and
be admixed with the antibodies of the present invention to provide a high drug
to antibody
ratio.
[131] Alternatively, the link between the antibody or polypeptide and the
agent
may be a direct link. A direct link between two or more neighboring molecules
may be
produced via a chenucal bond between elements or groups of elements in the
molecules.
The chemical bond can be, for example, an ionic bond, a covalent bond, a
hydrophobic
bond, a hydrophilic bond, an electrostatic bond, or a hydrogen bond. The bonds
can be,
for example, amide, carbon-sulfide, peptide, and/or disulfide bonds. In order
to attach the
the antibody to the agent or linker, amine, carboxy, hydroxyl, thiol and ester
functional
groups may be used, as is known in the art to form covalent bonds.
[132] The link between the peptide and the agent or between the peptide and
carrier, or between the carrier and agent may be via a linker compoiuld. As
used herein, a
linker compound is defined as a compound that joins two or more moieties. 'The
linker
can be straight-chained or branched. A branched linker compound may be
composed of a
double-branch, triple branch, or quadruple or more branched compound. Linker
compounds useful in the present invention include those selected from the
group having
dicarboxylic acids, malemido hydrazides, PDPH, carboxylic acid hydrazides; and
small
peptides.
[133] More specific examples of linker compounds useful, according to the
present invention, include: (a) dicarboxylic acids such as succinic acid,
glutaric acid, and
adipic acid; (b) maleimido hydrazides such as N-[maleimidocaproic acid]
hydrazide, 4-[N-
rnaleimidomethyl]cyclohexan-1-carboxylhydrazide, and N-[maleimidoundecanoic
acid]
hydrazide; (c) (3-[2-pyridyldithio]propionyl hydrazide) derivatives,
combinations,
modifications and analogs thereof; and (d) carboxylic acid hydrazides selected
from 2-5
carbon atoms.
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[134] Linking via direct coupling using small peptide linkers is also useful.
For
example, direct coupling between the free sugar of, for example, the anti-
cancer drug
doxorubicin and a scFv may be accomplished using small peptides. Examples of
small
peptides include AUl, AUS, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH,
IRS, KT3, Protein C, S-TAG~, T7, V5, VSV-G, and K.AI~.
[135] Antibodies and polypeptides of the present invention may be bound to,
conjugated to, complexed with, or otherwise associated with imaging agents
(also called
indicative markers), such as radioisotopes, and these conjugates can be used
for diagnostic
and imaging purposes. Fits having such radioisotope-antibody (or fragment)
complexes
are provided.
[136] Examples of radioisotopes useful for diagnostics include lllindium,
113indium, 99mrhenium, losrhenium, lolrhenium, 99mtechnetium, lalmtellurium,
lzzmtellurium,
lasmtelluriunm lssthulium, 167thulium 168thulium la3iodine, la6iodine,
131iodine, 1331odme,
8lmtOn, 33xen~n, 90yttriunl, 213biSmuth, 77bromlne, lBfluOrine, 951lltherilum,
97T11t11emu1Tl,
103'~'~t~hle°lllunl, los~the~~,~, 107merCUry, 203merCUTy, 67gallium,
and 68gallium. Preferred
radioactive isotopes, are opaque to X-rays or any suitable paramagnetic ions.
[137] The indicative marker molecule may also be a fluorescent marker
molecule.
Examples of fluorescent marker molecules include fluorescein, phycoerythrin,
or
rhodamine, or modifications or conjugates thereof.
[13~] Antibodies and polypeptides conjugated to indicative markers may be used
to diagnose, prognose, or monitor disease states. Generally, such methods
include
providing a sample of at least one cell from a patient and determining whether
the
antibody or fragment thereof of the present invention binds to the cell of the
patient,
thereby indicating that the patient is at risk for or has the disease. Such
monitoring may be
carned out ira viva, ita vitro, or ex vivo. Where the monitoring or diagnosis
is carried out in
vivo or ex viv~, the imaging agent is preferably physiologically acceptable in
that it does
not harm the patient to an unacceptable level. Acceptable levels of harm may
be
determined by clinicians using such criteria as the severity of the disease
and the
availability of other options.
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[139] With respect to cancer, staging a disease in a patient generally
involves
determining the classification of the disease based on the size, type,
location, and
invasiveness of the tumor. One classification system to classify cancer by
tumor
characteristics is the "TNM Classification of Malignant Tumours" (6th Edition)
(L.H.
Sobin, Ed.), which is incorporated by reference herein and which classifies
stages of
cancer into T, N, and M categories with T describing the primary tumor
according to its
size and location, N describing the regional lymph nodes, and M describing
distant
metastases. In addition, the numbers I, II, III and IV are used to denote the
stages and
each number refers to a possible combination of TNM factors. For example, a
Stage I
breast cancer is defined by the TMN group: T1, N0, MO which mean:Tl - Tumor is
2 cm
or less in diameter, NO - No regional lymph node metastasis, MO - No distant
metastasis.
Another system is used to stage AML, with subtypes of classified based on the
French-
American-Eritish system using the morphology observed under routine processing
and
cytochemical staining.
[Ol] In addition, a recently proposed World Health ~rganization (WH~) staging
or classification of neoplastic diseases of the hematopoietic and lymphoid
tissues includes
(specifically for AMLs) traditional FAE-type categories of disease, as well as
additional
disease types that correlate with specific cytogenetic findings and AML
associated with
myelodysplasia. ~thers have also proposed pathologic classifications. For
example, one
proposal specific for AML includes disease types that correlate with specific
cytogenetic
translocations and can be recognized reliably by morphologic evaluation and
immunophenotyping and that incorporate the importance of associated
myelodysplastic
changes. This system would be supported by cytogenetic or molecular genetic
studies and
could be expanded as new recognizable clinicopathologic entities are described
(Artier,
Arn. .I. Clin. Pathol. 115(4): 552-60 (2001)).
[140] The present invention provides for a diagnostic kit for in vitro
analysis of
treatment efficacy before, during, or after treatment, having an imaging agent
having a
peptide of the invention linked to an indicative marker molecule, or imaging
agent. The
invention further provides for a method of using the imaging agent for
diagnostic
localization and imaging of a cancer, more specifically a tumor, having the
following
steps: (a) contacting the cells with the composition; (b) measuring the
radioactivity bound
to the cells; and hence (c) visualizing the tumor.
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[141] Examples of suitable imaging agents include fluorescent dyes, such as
FITC, PE, and the like, and fluorescent proteins, such as green fluorescent
proteins. Other
examples include radioactive molecules and enzymes that react with a substrate
to produce
a recognizable change, such as a color change.
[142] In one example, the imaging agent of the kit is a fluorescent dye, such
as
FITC, and the kit provides for analysis of treatment efficacy of cancers, more
specifically
blood-related cancers, e.g., leukemia, lymphoma, and myeloma. FAGS analysis is
used to
determine the percentage of cells stained by the imaging agent and the
intensity of staining
at each stage of the disease, e.g., upon diagnosis, during treatment, during
remission and
during relapse.
[143] Antibodies and polypeptides of the present invention may be bound to,
conjugated to, or otherwise associated with anti-cancer agents, anti-
neoplastic agents, anti-
viral agents, anti-metastatic agents, anti-inflammatory agents, anti-
thrombosis agents, anti-
restenosis agents, anti-aggregation agents, anti-autoimmune agents, anti-
adhesion agents,
anti-cardiovascular disease agents, pharmaceutical agents, or other anti-
disease. An agent
refers to an agent that is useful in the prophylactic treatment or diagnosis
of a mammal
including, but not restricted to, a human, bovine, equine, porcine, marine,
canine, feline,
or any other warm-blooded animal.
[144] Examples of such agents include, but are not limited to, anti-viral
agents
including acyclovir, ganciclovir and zidovudine; anti-thrombosislrestenosis
agents
including cilostazol, dalteparin sodium, reviparin sodium, and aspirin; anti-
inflammatory
agents including zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17,
diclofenac,
ibuprofen, dexibuprofen, sulindac, naproxen, anltOlmetm, celecoxib,
indomethacin,
rofecoxib, and nimesulid; anti-autoimmune agents including leflunomide,
denileukin
diftitox, subreum, WinRho SI~F, defibrotide, and cyclophosphamide; and anti-
adhesion/anti-aggregation agents including limaprost, clorcromene, and
hyaluronic acid.
[145] Exemplary pharmaceutical agents include anthracyclines such as
doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, caxminomycin,
epirubicin, esorubicin, morpholinodoxorubicin, morpholinodaunorubicin,
methoxymorpholinyldoxorubicin, methoxymorpholinodaunorubicin and
methoxymorpholinyldoxorubicin and substituted derivatives, combinations and
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modifications thereof. Further exemplary pharmaceutical agents include cis-
platinum,
taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide,
prednisone,
fludarabine, idarubicin, chlorambucil, interferon alpha, hydroxyurea,
temozolomide,
thalidomide and bleomycin, and derivatives, combinations and modifications
thereof.
[146] An anti-cancer agent is an agent with anti-cancer activity. For example,
anti-cancer agents include agents that inhibit or halt the growth of cancerous
or immature
pre-cancerous cells, agents that kill cancerous or pre-cancerous cells, agents
that increase
the susceptibility of cancerous or pre-cancerous cells to other anti-cancer
agents, and
agents that inhibit metastasis of cancerous cells. In the present invention,
an anti-cancer
agent may also be an agent with anti-angiogenic activity that prevents,
inhibits, retards, or
halts vascularization of tumors.
[147] Inhibition of growth of a cancer cell includes, for example, the (i)
prevention of cancerous or metastatic growth, (ii) slowing down of the
cancerous or
metastatic growth, (iii) the total prevention of the growth process of the
cancer cell or the
metastatic process, while leaving the cell intact and alive, (iv) interfering
contact of cancer
cells with the microenvironment, or (v) killing the cancer cell. For example,
an antibody
could effect the killing of a cancer cell by binding to the cancer cell and
thereby
stimulating T cells or natural killer cells to kill the bound cell by antibody-
dependent cell
cytotoxicity.
[14~~] ~n anti-Leukemia agent is an agent with anti-leukemia activity. For
example, anti-leukemia agents include agents that inhibit or halt the growth
of leukemic or
immature pre-leukemic cells, agents that kill leukemic or pre-leukemic, agents
that
increase the susceptibility of leukemic or pre-leukemic cells to other anti-
leukemia agents,
and agents that inhibit metastasis of leukemic cells. Tn the present
invention, an anti-
leukemia agent may also be agent with anti-angiogenic activity that prevents,
inhibits,
retards or halts vascularization of tumors.
[149] Inhibition of growth of a leukemia cell includes, for example, the (i)
prevention of leukemic or metastatic growth, (ii) slowing down of the Ieukemic
or
metastatic growth, (iii) the total prevention of the growth process of the
leukemia cell or
the metastatic process, while leaving the cell intact and alive, (iv)
interfering contact of
cancer cells with the microenvironment, or (v) killing the leukemia cell.
»~.~.~ ~ ~~~
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[150] Examples of anti-disease, anti-cancer, and anti-leukemic agents to which
antibodies and fragments of the present invention may usefully be linked
include toxins,
radioisotopes, and pharmaceuticals.
[151J Examples of toxins include gelonin, Pseudomonas exotoxin (PE), PE40,
PE3 ~, diphtheria toxin, ricin, or derivatives, combinations and modifications
thereof.
[152] Examples of radioisotopes include gamma-emitters, positron-emitters, and
x-ray emitters that may be used for localization and/or therapy, and beta-
emitters and
alpha-emitters that may be used for therapy. The radioisotopes described
previously as
useful for diagnostics are also useful for therapeutics.
[153] Non-limiting examples of anti-cancer or anti-leukemia agents include
anthracyclines such as doxorubicin (adriamycin), daunorubicin, idarubicin,
detorubicin,
carminomycin, epirubicin, esorubicin, morpholinodoxorubicin,
morpholinodaunorubicin,methoxymorpholinyldoxorubicin,methoxymorpholinodaunorubi
c
in and methoxymorpholinyldoxorubicin and substituted derivatives, combinations
and
modifications thereof. Exemplary pharmaceutical agents include cis-platinum,
taxol,
calicheamicin, vincristii~e, cytarabine (Ara-C), cyclophosphamide, prednisone,
daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha,
hydroxyurea,
temozolomide, thalidonude, and bleomycin, and derivatives, combinations and
modifications thereof.
[154] In one embodiment, the pharmaceutical compositions of the present
invention have an antibody or polypeptide of the present invention and a
pharmaceutically
acceptable Garner. The antibody or polypeptide can be present in an amount
effective to
inhibit cell rolling, inflammation, auto-immune disease, metastasis, growth
and/or
replication of tumor cells or leukemia cells, or increase in number of tumor
cells in a
patient having a tumor or leukemia cells in a patient having leukemia.
Alternatively, the
antibody or polypeptide can be present in an amount effective to increase
mortality of
tumor cells or leukemia cells. Also alternatively, the antibody or polypeptide
can be
present in an amount effective to alter the susceptibility of diseased cells
to damage by
anti-disease agents, tumor cells to damage by anti-cancer agents, or leukemia
cells to
damage by anti-leukemia agents. Further alternatively, the antibody or
polypeptide can be
present in an amount effective to decrease number of tumor cells in a patient
having a
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tumor or leukemia cells in a patient having leukemia. Yet further
alternatively, the
antibody or polypeptide can be present in an amount effective to inhibit
restenosis. The
antibody, or polypeptide can also be present in an amount effective to inhibit
HIV entry.
Alternatively, the antibody or polypeptide, can be used as a targeting agent
to direct a
therapeutic to a specific cell or site.
[155] Antibodies and polypeptides of the present invention may be administered
to patients in need thereof via any suitable method. Exemplary methods include
intravenous, intramuscular, subcutaneous, topical, intratracheal, intrathecal,
intraperitoneal, intralymphatic, nasal, sublingual, oral, rectal, vaginal,
respiratory, buccal,
intradermal, transdermal, or intrapleural administration.
[156] For intravenous administration, the formulation preferably will be
prepared
so that the amount administered to the patient will be an effective amount
from about 0.1
mg to about 1000 mg of the desired composition. More preferably, the amount
administered will be in the range of about 1 mg to about 500 mg of the desired
composition. The compositions of the invention are effective over a wide
dosage range
and depend on factors such as the particulars of the disease to be treated,
the half life of
the peptide, or polypeptide-based pharmaceutical composition in the body of
the patient,
physical and chemical characteristics of any agent complexed to antibody or
fragment
thereof and of the pharmaceutical composition, mode of administration of the
pharmaceutical composition, particulars of the patient to be treated or
diagnosed, as well
as other parameters deemed important by the treating physician.
[157] Pharmaceutical composition for oral administration may be in any
suitable
form. Examples include tablets, liquids, emulsions, suspensions, syrups,
pills, caplets, and
capsules. Methods of making pharmaceutical compositions are well known in the
art (See,
e.g., Remington, The Science and Practice of Pharmacy, Alfonso R. Gennaro
(Ed.)
Lippincott, Williams & Wilkins (pub)).
[158] The pharmaceutical composition may also be formulated so as to
facilitate
timed, sustained, pulsed, or continuous release. The pharmaceutical
composition may also
be administered in a device, such as a timed, sustained, pulsed, or continuous
release
device.
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[159] The pharmaceutical composition for topical administration can be in any
suitable form, such as creams, ointments, lotions, patches, solutions,
suspensions,
lyophilizates, and gels.
[160] Compositions having antibodies, constructs, conjugates, and fragments of
the subject invention may comprise conventional pharmaceutically acceptable
diluents,
excipients, carriers, and the like. Tablets, pills, caplets, and capsules may
include
conventional excipients such as lactose, starch, and magnesium stearate.
Suppositories
may include excipients such as waxes and glycerol. Injectable solutions
comprise sterile
pyrogen-free media such as saline, and may include buffering agents,
stabilising agents or
preservatives. Conventional enteric coatings may also be used.
[161] The antibodies and polypeptides of the present invention and
pharmaceutical compositions thereof, can be used in methods of ameliorating
the effects
of a disease, preventing a disease, treating a disease, or inhibiting the
progress of a disease
in patients in need thereof. Such methods include inhibiting cell rolling,
inflammation,
autoimmune disease, metastasis, growth and/or replication of tumor cells or
leukemia
cells, or increase in number of tumor cells in a patient having a tumor or
leukemia cells in
a patient having leukemia. In addition, such methods include increasing the
mortality rate
of tumor cells or leukemia cells, alter the susceptibility of diseased cells
to damage by
anti-disease agents, tumor cells to damage by anti-cancer agents, or leukemia
cells to
damage by anti-cancer agents. Such methods also include decreasing number of
tumor
cells in a patient having tumor or leukemia cells in a patient having
leukemia. Such
methods also include inhibiting or decreasing HIV entry in cells. Such methods
further
include preventing or inhibiting cardiovascular diseases such as restenosis.
[162] The present invention moreover provides a method of manufacturing a
medicament for the treatment of various disease states such as, e.g., AML, T-
ALL, B-
leukemia, B-CLL, Pre-B-ALL, multiple myeloma, metastasis, HIV infection,
cardiovascular diseases, or other diseases in which such cellular functions or
actions as
cell rolling, inflammation, immune reactions, infection, autoimmune reactions,
metastasis,
play a significant role. Such medicament comprises the antibodies and the
polypeptides of
the present invention.
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[163] In one embodiment, the invention provides a method of diagnosing cancer
in a person by assaying the ability of Yl to bind specifically to a tissue
sample and
comparing Y1 binding to binding by a control antibody such as I~FL-1. In one
embodiment, the method comprises isolating cell samples from blood or solid
tissue from
the person, incubating the cells with an antibody or a fragment thereof that
recognizes a
sulfated tyrosine motif containing epitope ("the experimental antibody"),
washing away
the non-specifically bound antibody, and comparing the results to those of a
corresponding
staining procedure performed with a reference standard such as a control
antibody with
known binding activity. A control antibody is one that recognizes an epitope
containing
the unsulfated fornl of the tyrosine motif or antigens that contain such. The
presence of
tumor cells is indicated when the experimental antibody binding is
substantially greater
than binding by the control, as determined by the strength of the staining.
The staining
procedure can be performed by standard methods. For example, the first
antibody call be
visualized by using secondary antibodies that recognize the first antibody and
that are
conjugated to an enzyme substrate which produces a color reaction when acted
on by the
enzyme. Alternatively, the presence of tumor cells is indicated when both the
experimental antibody and the control antibody bind to the cells, but the
cells internalize
Y1 and do not internalize the control antibody. In one embodiment, the cancer
is a solid
tumor. In another embodiment, the cancer is a blood-borne tumor. In a
preferred
embodiment, the experimental antibody is Yl or a fragment thereof, or a
related antibody
or a fragment thereof. Iu another preferred embodiment, the control antibody
is I~Ll.
[164] In another embodiment, the invention provides a method of diagnosing a
cancer comprising screening cell samples from blood or solid tissue for the
presence of
tumor cells. Western blots are performed on cell sample lysates using Yl or a
fragment
thereof, or a related antibody or a fragment thereof. Yl binding can be
observed by
tagging Yl itself with a detectable label, or by using standard methods that
employ a
detectable anti-human antibody. The presence of tumor cells is indicated when
Y1
binding is substantially greater than binding by the control, where the
control is defined as
above. The presence of tumor cells is indicated when Y1 binding is
substantially greater
than binding by the control.
[165] In another embodiment, the invention provides a method of identifying
protein markers of blood-borne or solid tumors by preparing a cell lysate and
purifying the
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lysate by passing it through an affinity column. The affinity column
incorporates Y1 or a
fragment thereof, or a related antibody or fragment thereof. In one
embodiment, the cell
lysate is derived from a primary tissue sample collected from a human being.
In another
embodiment, the cell lysate is derived from a tumor cell line. In a further
embodiment, the
tumor cell line can be an immortalized cell line.
[166] In another embodiment, the invention provides a method of monitoring the
stage of a blood-borne cancer comprising isolating white blood cells from a
patient with a
blood-borne cancer, incubating the cells with Y1, determining the extent of Yl
binding
relative to reference standard.
[167] As an alternative approach for therapeutic targeting of sulfated
tyrosine
epitopes present on proteins, such as GPIb and PSGL-1, a small inorganic
chemical entity
may be identified by screening of an appropriate combinatorial library. Such a
chemical
entity may have a number of advantages over a scFv or IgG-based therapeutic
agent. For
example, an inorganic chemical entity may be administered orally and have an
enhanced
biosafety profile, including reduced immuno-crossreactivity. It may provide
enhaslced
selectivity towards the target, particularly following rational drug design to
optimize an
initially selected lead compound. Gther advantages include lower production
costs, longer
shelf life and a less complicated regulatory approval process.
[I~~] Since a number of embodiments of the epitope of the invention have been
identified, e.g. on GPIb and PSGL-1, a ligand-driven approach may be taken to
identify
inorganic chemical entities, which have very narrow specificity, or
alternately, target more
than one sulfated tyrosine epitope for disease states such as re-perfusion
injury which
involves more than one distinct target each bearing such an epitope. The
ligand-driven
approach significantly shortens the screening process for identifying targets
for therapeutic
intervention, and enables simultaneous target validation with lead
optimization, which
may be carried out with a series of focused libraries.
[169] A library of inorganic chemical entities specialized for targeting
sulfated
tyrosine epitopes rnay be designed and developed first by analyzing the three
dimensional
interaction between an antibody such as Y1 and its known taxgets such as
residues sulfated
Tyr-276 and Asp-277 of GPIb. Chemical libraries composed of entities that
mimic the Y1
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38
binding site and which provide increased affinity to the target may be
developed by
computer assisted combinatorial library design.
[170] Throughout this application, reference has been made to various
publications, patents, and patent applications. The teachings and disclosures
of these
publications, patents, and patent applications in their entireties are hereby
incorporated by
reference into this application to more fully describe the state of the art to
which the
present invention pertains.
EXAMPLES
[171] The following examples are set forth to aid in understanding the
invention
but are not intended and should not be construed, to limit its scope in any
way. Although
specific reagents and reaction conditions are described, modifications can be
made that are
meant to be encompassed by the scope of the invention. The following examples,
therefore, axe provided to further illustrate the invention.
E PLE 1: Identification of Yl ligand from p~nxnarg~ L cells (1a1191~-3)
[172] 1.1 Primary AML cells (stage M4) were collected from a patient and
lysed. The lysate was subjected to purification comprising affinity
chromatography on a
Y1-IgG column (see Fig.1). The isolated protein was digested with
endoproteinase Asp-
~T, and the resulting peptide sequence was determined using mass spectrometry.
The
sequence was identical to the published human PSGL-11!T-terminal amino acid
sequence.
These results indicate that primary AML cells at stage 4 express FSGL-1 that
can be
bound by Yl-IgG. It was further determined that the purified protein was
sulfated at
tyrosines 2 and 3 of the Y1 recognition motif (see Fig. 2). Internal controls
were used to
verify the specificity of the immunomodulatory effects of Yl e.g, no induction
of mouse
interleukin-6 secretion was detected.
EXAMPLE 2: Antibody-dependent cell cytotoxicity
[173] 2.1 Effect of Yl-IgG:
[174] Studies to determine whether Y1-IgG is capable of mediating antibody
dependent
cell cytotoxicity (A17CC) have shown this antibody mediates effector cell
cytotoxicity of
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39
various target cells, including ML2 (an AML-derived cell line which served as
a target in
our model system) and B-CLL cells from patient clinical samples. Yl-IgG binds
these
cell types via CD162 (PSGL-1), a molecule which is substantially absent on
healthy B-
cells and early stage AML.
[175] The effector cell populations that are involved in Yl-IgG ADCC have been
defined. For Yl-IgG to mediate ADCC, natural killer (NK) cells (CD56+), y8T
cells, and
monocytes (CD14+) are required, but T-helper cells (CD4+) and cytotoxic T
cells (CDR+)
are not required. This was confirmed with donor cells from bath healthy
subjects and B-
CLL patients.
[176] Furthermore, even in the absence of target cells, Y1-IgG mediates
activation of
different types of effector cells, as measured by the appearance of an early
activation
marker (CD69+), secretion of cytokines, such as TNFcc and IFNy and induction
of Fast.
Hyper cross-linking (XL) of Y1-IgG with secondary anti-human Fc antibodies
demonstrated that an apoptotic mechanism also contributes to cell killing.
[177] ~1-IgG activity towards primary B-CLL cells iya vit~~ was compared to
that of two
commercially available hmnanized antibodies currently used extensively for
treatment of
various lymphoid malignancies: Rituximab (which binds CD20) and Campath (which
binds CD52). i~lhile the mechanism of action of Rituximab against B-CLL is not
clear, its
cytotoxic effects against CD20-positive malignant B cells may involve one or
more of
complement-dependent cytotoxicity (CDC), ADCC and induction of apoptosis. The
cytotoxic effects of Campath against CD52-positive malignant B cells, as well
as normal
B and T cells, involves CDC, ADCC and induction of apoptosis. Campath
administration
is associated with complete ablation of all mature normal B and T cells,
leading to severe
hematological toxicity.
[178] For ADCC experiments, mononuclear effector and target cells were
separated on
FICOLL~. Target cells were then labeled with PKH26, which stably incorporates
a
fluorescent dye within the lipid regions of the cell membranes. Cells were
then washed
and incubated with effector cells at various Effector:Taxget (E:T) ratios, in
the absence or
presence of different concentrations of Y1-IgG or control antibodies for 24
hours. Dead
cells were stained by TOPRO~(Molecular Probes, Inc., Eugene , OR): and
analyzed by
FRCS on gated target cells.
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[179] For CDC experiments, mononuclear cells from B-CLL patients were
separated on
FICOLL~. Cells were incubated with or without Y1-IgG or control antibodies for
24
hours in the presence or absence of the patient's plasma. Apoptotic cells were
then stained
with Annexin-PI and analyzed by FACE.
[1~0] To assess effector cell activation, mononuclear cells from healthy
donors were
separated on FICOLL~. Cells were incubated with or without Y1-IgG or control
antibodies for 24 hours. FAGS analysis with a,CD69 (an early activation
marker) antibody
was performed for different types of effector cells. Secretion of cytokines
such as TNFoc
and IFNy were measured by ELISA.
[l~l] For apoptosis experiments, mononuclear cells from B-CLL patients were
separated
on FICOLL~. Cells were incubated in the presence or absence of Yl-IgG or
control
antibodies for 10 minutes at 37°C. Anti-human Fc antibodies were then
added and
incubated for 4-24 hours at 37°C. Diseased cells (CD19+, CDS+) were
then stained for
apoptotic markers, Annexin-TOPI~O~ and analyzed by FAGS.
[1~2] Comparative studies with Y1-IgG and Rituximab indicate that Yl-IgG is
superior
to lZituximab with respect to mediation of ADCC and induction of apoptosis
against B-
CLL cells. In contrast to Campath~, Yl-IgG was found to be incapable of
mediating CDC
against primary B-CLL cells. These i~a vit~~ results indicate that Yl-IgG may
be useful as
a therapeutic agent for treatment of B-CLL based on its ADCC activity.
[1R3] 2.1.1 CC in primarg~ B-cltr~nic lymph~cg~tic le~~emia (B-CLL):
To determine if Yl-,IgG mediates ADCC in primary B-CLL, B-CLL cells from
different
patients were co-incubated for 24 hours with PBMC effector cells at different
effector/target cell ratios. Analysis of thirteen different B-CLL clinical
samples indicated
that Y1-IgG mediated effector cell cytotoxicity in all cases (Fig. 3) with the
average extent
of cell lysis about 21.4%. Four of thirteen samples (30%) exhibited more than
30% lysis,
while only two of thirteen samples (15%) exhibited less than 10% lysis. In
some cases, a
high degree of lysis was seen even at a low E:T ratio, e.g. KBC171, which
exhibited about
62% lysis, while in other cases, a low degree of lysis was seen even at a high
E:T ratio,
e.g. KBC 104, which exhibited only about 7% lysis. Variation may be attributed
to the
effector cell samples obtained from different healthy donors, as well as to
differences
among the B-CLL samples.
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[184] 2.1.2 Y1-IgG-mediated ADCC by PBMC against acute myeloid
leukemia cells: PBMC also effected ADCC against primary AML cells from a
patient
using varying ratios of PBMC:AML (10, 20, or 40: in the presence of 10 or 20
~,g/ml Y1-
IgG. For example, at a cell ratio of 10:1, 7.9% of AML cells died in the
absence of
antibody and 6% died in the presence of human IgG. In the presence of 10 and
20 ~.g/ml
Yl-IgG 14.2% and 17.6% of the AML cells died, respectively (Fig. 4). The
degree of
cytotoxicity increased with increasing Y1-IgG concentration (10 or 20 ~.g/ml).
Human
IgG did not induce ADCC. A similar result was obtained for one additional
primary AML
sample (data not shown).
[185] ML-2 cells provide a good model for ADCC since Y1-IgG binds without
undergoing detectable internalization.
[186] a. ML2 ADCC increases with Yl-IgG concentration: After 24 hours of
incubation, cytotoxicity was higher in the presence of Yl-IgG than in its
absence at four
different effector (PBMC) to target ratios (5:1, 10:1, 20:1, 40:1) (Fig. 5).
This effect was
diminished or absent when mouse anti-PSGL-1 antibody I~FL1 was substituted for
Y1-
IgG and diminished even further when human TgG (which binds to the Fc receptor
on
effector cells) was used instead of Y1-IgG. A Yl-IgG concentration as low as 5
p,g/ml
could induce ADCC when the effectoraarget ratio was 40:1.
[187] b. competition between ~Il-TgG and I'LL-1: Y1-IgG (20 and 50 ~,g/ml)
induced ADCC against ML2 cells at effectoraarget ratios of 20:1 and 40: l .
The mouse
anti-PSGL-1 antibody KPL-1 alone did not induce AI~CC, and could therefore be
used as
a competitor for Yl-binding induced ADCC. KPL-1 partially inhibited Y1-IgG-
induced
ADCC (Fig. 6), and for example, while 74.1% cytotoxicity was observed after 48
hours
incubation in the presence of Yl-IgG (20 ~,g/ml; effector/target ratio 20:1),
the additional
presence of KPLl (20 pg/ml) resulted in only 58.8% cytotoxicity. Thus, Y1-IgG-
induced
ADCC of ML2 involves binding of PSGL-1 by Yl-IgG.
[188] c. Involvement of Natural Killer, y8T cells and Monocytes in Y1-IgG
Mediated ADCC: Positively selected effector cells were analyzed for their
capability of
effecting Y1-IgG mediated ADCC of ML2 or B-CLL target cells. Natural killer
(NK)
cells (CD56+), Y6T cells and cytotoxic T-cells (CD8+) from normal donors and B-
CLL
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42
patients were isolated using commercially available magnetic beads. As shown
in Fig.
7A, NK cells from both normal donors and from B-CLL patients (KCS samples in
Fig.
7A) are capable of effecting ADCC on ML2 and B-CLL targets (KCS samples in
Fig.
7A), resulting in 13 to 68% lysis over control. Also, y~T cells were shown to
mediate
ADCC of ML2 cells. In contrast, cytotoxic T-cells do not appear to be involved
in Y1-
IgG mediated cytotoxicity.
[189] Negative selection of specific cell populations from PBMC showed that
CD14+
cells (monocytes) are also involved in ADCC against ML2 targets, in addition
to NIA cells
(CD56+) and y~+ T cells (Fig. 713). All of the effector cells which are
involved in Yl-IgG
mediated toxicity express the Fc receptor, CD16.
[190] d. Activati~n ~f NK -cells by Y1: Y1 mediates ADCC by natural killer
cells, as measured by expression of CD69. Effector cells from six healthy
donors were
incubated for 24 hours at 37° C in the presence of Y1-IgG or human IgG
or a marine anti-
CD62 antibody (I~PLl, PLl or PL2) or in the absence of any antibody (contTOl).
FAGS
analysis was then performed and expression of the early activation marker CD69
on
natural killer (NIA) cells (CD56+) was determined. As shown in Fig. 8,
activation of NIA
cells by '~1-IgG was mediated with all six donor cells. In contrast, no effect
could be
detected by either human IgG or by anti-CD162 mouse antibodies I~PLl, PL1 or
PL2.
Preliminary studies have also shown induction of Fast expression on effector
cells
following incubation with ~1-IgG (data not shown).
[191] e. 5~1-IgG ineluced Ap~pt~sis
[192] Mononuclear cells (CD19+, CDS+) from B-CLL patients incubated in the
presence of Yl-IgG exhibited about 5% apoptosis within 24 hours, as assessed
by FAGS
analysis (Fig. 9). Addition of secondary antibodies that cross-link the Yl-IgG
elicited an
additional 50% of apoptosis within 24 hours (Fig. 9).
[193] These results suggest that cross-linking of an antibody directed to a
sulfated
epitope on PSGL-1 triggers signals for apoptosis of primary B-CLL cells. This
implies
that PSGL-1 can be a target for inducing apoptosis in B-CLL patients irz vivo,
wherein the
cross-linking effect may be mediated by Fc receptor bearing cells, e.g.
monocytes, CD56+
NK cells and y8f T cells.
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43
[194] The apoptotic and cross-linking effects described above may be inhibited
using the anti-PSGL-1 antibody KPL1 (data not shown). This antibody on its own
does
not induce apoptosis. This provides confirmation that the apoptotic signal is
mediated via
an epitope on PSGL-1.
[195] f. The ADCC effect of Yl-IgG on B-CLL relative to Rituximab
[196] The percentage of cell death induced by the Y1-IgG antibody in two
primary human B-CLL patient samples was significantly higher than that
obtained by
P.ituximab. Figure 10 shows that Y1 induced 25% to 35% cytotoxicity over
control
compared to only 10°/~ to 13°1° induced by Rituximab.
Saturation of receptor molecules on
the target cells was achieved at 10~,g/ml of Yl-IgG antibody but not by the
same
concentration of hituximab.
[197] Taken together, the results suggest that Y1-IgG is a promising candidate
as a
therapeutic agent in the treatment of B-CLL, as it cytotoxic and apoptotic
effects appear to
be mediated via specific recognition of a PSGL-1 sulfated epitope expressed on
these
diseased cells.
[19~] g. ~aalysis of the CDC cffect of Yl-IgG~ °tuximab and ~ampath on
B-ALL
[199] l~lononuclear cells from B-CLL patients were incubated with Yl-IgG,
I~ituximab or Caanpath~ in the presence and absence of 25% of the patients'
plasma. As
shown in Figure 11, only Campath~ mediated cytotoxicity of primary B-CLL cells
via
CDC. Neither Rituximab nor Y1-IgG induced cytotoxicity via complement
fixation.
[200] These in vitro results indicate that Y1-IgG may be useful as a
therapeutic agent for
treatment of B-CLL based on its ADCC activity.
E~~AMPLE 3: Yl-IgG-M-Daunorubicin Derivative
[201] 3.1 Preparation of Yl-IgG-M-Daunorubicin Derivative: Antibody-
toxin conjugates such as morpholino-doxorubicin-Y1-IgG (Fig.13) and antibody-M-
daunorubicin conjugates (see below) were prepared. Daunorubicin was modified,
joined
to one of two different linkers, and then joined to the antibody via the
antibody's free
"""
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44
amino groups or via the antibody's reduced disulfide bonds. The term M-DNR-
L1NI~ER
refers to both (6-Maleimidocaproyl)hydrazone of Morphlinyldaunorubicin acetate
and to
M-DNR-AES.
[202] a. Preparation of 3'-Deamino-3'-(4-morpholinyl) daunorubicin
acetate (M-DNR-Ac)
[203] Dry triethylamine was added to a solution of daunorubicin hydrochloride
in dry
dimethylformamide, under argon, followed by bis(2-iodoethyl) ether. The
reaction
mixture was protected from light and stirred for 36 hours at room temperature.
[204] The resulting aqueous nuxture was extracted with methylene chloride. The
organic phase was dried over anhydrous sodium sulfate, filtered through celite
and
evaporated to dryness. The crude product was purified by silica gel column
chromatography, and the relevant fractions pooled together and evaporated to
yield the 1VI-
DNR free base as a red oil, which was found to be 98°/~ pure (by HPLC).
The yield was
55%.
[209] After reaction with acetic acid, the resulting free base was isolated as
its solid
acetate salt followed by lyophilization. M-DNR-Ac is stable for at least 12
months under
argon at -20° C.
[206] b. Preparation of (6-Malegmndo~apr~yl)lgg~draa~one of
l~~rphlinyldaunorubicin acetate
[207] 6-maleimidocaproylhydrazide was added to a solution of M-DNR-Ac in dry
methanol, under argon, followed by trifluoroacetic acid. The clear solution
was protected
from light and stirred for 24 hours at room temperature.
[208] The methanolic solution was evaporated to dryness under reduced pressure
at 25°
C, resulting in a red oily residue, which was dissolved in dry methanol. To
this solution,
dry ether was added and the precipitated red solid isolated by centrifugation.
The pure
crystalline product, which had a purity of 98% and a yield of 88%, was
obtained after
three triturations with dry ether, dried under high vacuum, and kept under
argon at -20°C.
(6-Maleimidocaproyl)hydrazone of Morphlinyldaunorubicin acetate is stable for
at least 4
months under argon at -20° C.
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[209] c. Preparation of N-hydroxysuccimimide ester of Adipic acid
monohydrazone of Morpholinodaunorubicin (M-DNR-AES)
[210] 1. Preparation of Adipic acid monohydrazone of
morpholinodaunorubicin
[211] The following were combined and stirred at room temperature under Ar
while
being protected from light for 1 hour: morpholinodaunorubicin acetate salt,
dry Me~H,
freshly prepared methanolic solution (hydrazidoadipic acid hydrochloride and
Et3N and
TFA stock solutions).
[212] The solvent was removed under reduced pressure and the resulting residue
dissolved in NH4~Ac:AN and injected into a semiprep HPLC column. The mixture
was
washed and concentrated under isocratic conditions. The desired product was
collected
after about 4.5 min and concentrated by C-18 Sep Pak cartridge. The product
was eluted,
lyophilized, and stored at 20°C under Ar. The product was obtained as a
red solid with
an 80% yield and 95% purity.
[213] 2. Preparation of N-hydroxysuccimimide ester of Adipic
acid monohydrazone of Morpholinodaunorubicin
[214] Hydroxysuccinimide in dry THF and DCC in dry THF were added to the
adipic
acid monohydra~one of morpholinodaunorubicin in dry THF. The clear, red
solution was
stirred for 24h at room temperature under I~r. The end of the reaction was
determined by
analytical HPLC, and the solvent was removed. The glacial solid was then
dissolved in
buffer solution (N-methylinorpholinium acetate/AI~ and filtered through
cotton.
[215] The product was isolated by RP-HPLC, diluted with two volumes of n-
Methylinorpholinium acetate solution and loaded on a Sep-Pak (900mg). The
product was
eluted and lyophilized to obtain a powder with 73% yield and 97.4% purity.
[216] d. Preparation of M-DNR-Yl-IgG Conjugate
[217] M-DNR-LINKER in dry DMF was added to the MAb solution at a molar ratio M-
DNR-LINKER/Yl-IgG of 23. The mixture was gently shaken overnight at room
temperature under argon, then centrifuged. The supernatant was filtered
through SPIN-X
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46
tubes (Costar) and shaken with Bio-Beads SM-2 (Biorad) for 1 hour at room
temperature.
The mixture was allowed to stand for 10 minutes. The supernatant was passed
through
PD-10 columns (Pharmacia) that had been equilibrated with PBS. The conjugate
was
eluted with PBS and the protein-containing fractions combined. The purified
conjugate
was sterilized by SPIN-X filtration. The conjugate solutions were frozen and
stored at -
70°C. The product conjugate was obtained in 45-50% yield and had the
following
characteristics: 5°/~ aggregates; 2°/~-5% absorbed, non-
covalently linked M-DNI~
derivatives; average molecular ratio of drug to antibody is 4.
[218] e. Preparati~n ~f 1VI-I?NR-YlIgG c~njugate (via reduction ~f the
S-S b~nds in the Fc region)
[219] Y1-IgG in a buffer composed of NaCI, MES and EDTA was added to
cysteamine
hydrochloride solution (Merck) in the same buffer. The mixture was incubated
at 37° C
for 1.5 hours under argon. The reaction mixture was loaded onto a PD-10 column
(Sephadex G-25, Pharmacia) that had been equilibrated with PBS/EDTA. The
reduced
protein was eluted with PBS/EDTA. The fractions with the highest protein
concentrations
were combined and stored at 4° C. The molecular ratio of free
sulfhydryl groups to
antibody was at least 3.5. (6-Maleimidocaproyl)hydrazone of
morphlinyldaunorubicine
acetate was diluted in DMF and added to the solution of reduced protein and
incubated for
30 minutes at 4° C. The reaction mixture was passed through a PD-10
column
preequilibrated with PBS, then eluted with PBS. The protein fractions were
combined,
then sterilized by SPIN-~ filtration (Costar). The purified conjugate was
aliquoted and
stored at -70°C. The product conjugate was obtained in ~50% yield and
had the following
characteristics: less than 5% aggregates; 1-2% free M-DNR derivatives; average
molecular ratio of drug to antibody of 4.
[220] f. Preparation of IgG-Yl-M-I)NR conjugate (via reduction of
the S-S bonds)
[221] IgG-Yl was reduced by passing it through a PD-10 column (Sephadex -25M,
Pharmacia) in EDTA and eluting in PBS/EDTA. The protein-containing fractions
were
pooled. The molecular ratio of free sulthydryl groups to antibody was at least
6. (6-
Maleimidocaproyl)hydrazone of morphlinyldaunorubicin acetate in dry
dimethylformamide was diluted in DMF and added to the reduced protein. The
solution
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was incubated for 30 minutes at 4°C. The protein conjugate was purified
on a PD-10
column in PBS and the protein fractions were sterilized by SPIN-X filtration
(Corning Life
Sciences). The conjugate was frozen and stored at -70°C. Yield was
about 50% relative
to original antibody. The final product contained less than 5% aggregates,
free M-DNR
was between 0-2%, and the average molecular ratio of drug to antibody was 7.
[222] 3.2 Cytotoxicity of Yl-IgG-M-DNI~ Derivative (Fig.12-14): The
specific cytotoxic effect of Yl-drug conjugates was assessed in semisolid
(1VIETHOCULT~; StemCell Technologies Inc., Vancouver BG, Canada) clonogenic
assays. Cells (CD34+ stem cells from cord blood or primary AML patient
samples) were
incubated with free or conjugated drugs at concentrations of up to 10-6M for 1
hour at
37°C. The cells were washed, seeded in METH~CULT~ and grown for 10-12
days, after
which colonies were counted. Bovine (b)-IgG-M-DNR was used as a non-specific
conjugate control.
[223] The results (Fig.13) showed limited effect of the conjugate Y1-IgG-M-
DIVI~ on the cord blood samples. That is, non-target cells (healthy CD34+ stem
cells)
incubated in the presence of 1 ~,M Yl-IgG-M-D~ survived at least as well as
control
cells and cells incubated in the presence of 1 ~.M b-IgG-M-DID or 0.1 ~.M M-
DIVI~ (i.e. a
non-lethal dose of free drug). Thus, no significant effect was seen in all the
cord blood
saanples.
[224] In contrast, in an AML sample (M7 megakaryocytic leukemia), Y1-IgG M-
DNR was three times more inhibitory in inhibiting colony growth, relative to
the non-
specific b-IgG-M-DNR conjugate at the same concentration (Fig.13). That is, 1
~M Yl-
IgG-M-DNR reduced viability of primary AML cells to 40% relative to the
control (target
cells incubated alone), while 0.1 ~M M-DNl~ and 1 ~.M b-IgG-M-DN12 each
reduced
viability of primary AML cells to 80% relative to the control.
[225] The specificity of the Y1-IgG-M-DNR conjugate was further demonstrated
in a similar experiment showing that following incubation with 1 ~,M Y1-IgG-M-
DNR,
colony formation from primary AML-M4 and AML-MS cells (obtained from patients)
was
inhibited to 60% and 35% respectively, relative to control (Fig.14). In
contrast, all AML-
M4 cells and 78% of AML-MS cells gave rise to colonies following incubation
with 1 ~,M
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48
h-IgG-M-DNR. Cells incubated with 1 ~,M M-DNR did not give rise to colonies,
confirming that the cells were sensitive to the drug.
[226] B-ALL cells, which do not express a Y1 epitope (as assessed by failure
to
bind Yl), are not sensitive to Yl-IgG-M-DNR and thus gave rise to colonies
following
incubation with 1 ~,M Yl-IgG-M-DNR at the same rate as the control (Fig.15).
EXAMPLE 4: Yl recognition motif and sulfation
[227] To evaluate the potential effect of tyrosine sulfation on the binding of
Yl-
scFv to GPTb, the human chronic myeloid leukemia cell line KU812 expressing
GPIb was
grown in sulfate-free medium in the presence of 100 mM sodium chlorate to
inhibit
sulfation. Under the conditions employed, tyrosine sulfation is inhibited by
up to 95°/~
without affecting protein synthesis or other post-translational~modifications.
As show in
Figure 16A, binding of Y1-scFv to KU812 cells was reduced by 50% following
growth
with sodium chlorate, as compared to control cells grown in complete medium
lacking
sodium chlorate. Under the same conditions, surface expression of GPIb on
sulfate-
starved cells was unchanged, as indicated by FACS analysis using the mouse
anti-GPIb
monoclonal antibody AI~2-FITC (Fig.16B).
[22~] To further evaluate whether tyrosine sulfate modification plays a role
in
Y1-scFv binding to GPTb, various synthetic peptides based on residues 273-285
of GPTb
were assessed for the ability to inhibit binding of Y1-scFv to platelets.
[229] Briefly, peptides were synthesized by solid phase methodology using an
ABIMED AMS-422 multiple peptide synthesizer, and as required, tyrosine sulfate
was
incorporated using FMOC-Try sodium salt. Synthetic peptides were purified
using a
Lichrosorb RP-18 column. For the Yl-scFv platelet binding assay, a mixture of
synthetic
peptide (2.5, 25 or 200 p,M) and Y1-scFv (10 p,g) was incubated with washed.
Following
washing, platelets were incubated with R-phycoerythrin labeled-anti scFv,
washed and
analyzed by FACS.
[230] As shown in Figure 17A, peptide DLYSDYsYSPE (SEQ ID N0:4)
(comprising 3 sulfated tyrosines) at 25 ~,M effectively inhibited binding Y1-
scFv to
platelets, while the corresponding non-sulfated control DLYDYYPE (SEQ ID
NO:S), had
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49
no effect even at 200 ~M. Furthermore, each of peptides DLYSDYYPE (SEQ ID
NO:6),
DLYSDYsYPE (SEQ ID NO:7) and DLYSDYYsPE (SEQ ~ NO:B) (sulfated at the first,
the first and second, and the first and third tyrosines, respectively) at 25
wM effectively
inhibited Yl-scFv binding. Peptides DLYDYSYSPE (SEQ ID NO:9) and DLYDYYSPE
(SEQ ID NO:10) (sulfated at the second and third, and third tyrosines,
respectively) had
no effect on Yl-scFv binding even at 200 ~,M (Fig. I7A). These results clearly
demonstrate that sulfation at Tyr-276 in GPIb, i.e. the "first" tyrosine
position, is important
for significant competition and therefore for Y1-scFv binding to GPIb.
[231] To assess whether additional amino acids within the region of residues
273-
2~5 of GPIb contribute to Y1-scFv binding, substitution mutant peptides based
on the
peptide DLYsDYYPE were tested in the Yl-scFv platelet binding assay (Fig.
I7)3).
When sulfated Tyr-276 was replaced with a negatively charged Glu residue, the
mutant
peptide DLEDYYPE (SEQ ID NO:11) exerted no substantial inhibition on Yl-scFv
binding to platelets, in contrast to DLYsDYYPE. Similarly, mutant peptides
DLYSEYYPE (SEQ ~ NO:12), DLYsN~YPE (SEQ ID NO:13) and DLYsAYYPE(SEQ
ID NO:14), having Asp-277 replaced by Glu, Asn, and Ala respectively were
substantially
incapable of inhibiting Y1-scFv binding to platelets. On the other hand,
replacement of
Leu-275 by Ala (DAYsDYYPE) (SEQ ~ NO:15)and various replacements of amino
acids 278 to 280 [DLYsDFYPE (SEQ ~ NO:16), DLYsDAYPE (SEQ ~ NO:17),
DLYsDYYAE (SEQ ID 1~T0:18) and DLYsDYYPA (SEQ ID NO:19)] yielded mutant
peptides which all inhibited Yl-scF°v binding to platelets, in a manner
substantially
identical to that of DLYsDYYPE (Fig.17i~).
[232] To validate the platelet binding inhibition assay, the mutant peptides
were
also tested for inhibition of Yl-scFv binding to purified glycocalicin
(Fig.18A). Briefly,
glycocalicin immobilized on microtiter plates was incubated with Yl-scFv (5
~,g/ml) and
peptide (25 pM). Following washing, bound Y1-scFv was detected using
polyclonal anti-
scFv (generated by immunization of a rabbit with an scFv mixture) and anti-
rabbit IgG
antibody conjugated to horseradish peroxidase, and reading the absorbance at
450 nm in
an ELISA reader.
[233] The results obtained in the glycocalicin binding inhibition assay
(Fig.18A)
indicated that both sulfated Tyr-276 and the adjacent residue Asp-277 of GPIb
are
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important for Y1-scFv binding, thus confirming the results obtained in the
platelet binding
inhibition assay (Fig.17A and B). Additional confirmation was obtained by
evaluating
by ELISA the direct binding of Yl-scFv to peptides covalently coupled to
CovaLink
Plates (Fig.1~S). This study indicated that GPIb-derived mutant peptides
having
replacements of sulfated Tyr-276 or of Asp-277 were substantially incapable of
direct
binding by Y1-scFv, in contrast to mutant peptides having replacements at
positions 275,
27~, 279 or 2~0, or having non-sulfated Tyr-27S or Tyr-279, all of which were
substantially capable of direct binding by Yl-scFv.
[234] Analogous direct binding experiments using synthetic peptides based on
the
residues 42-5~ of PSGL-1 confirmed that PSGL-1 tyrosine sulfation is important
for Y1-
scrv binding (see Fig. I9). Specifically, sulfation of the third tyrosine
position in the
PSGL-1 sequence QATEYEYLDYDFLPETE (SEQ ~ N~:20) results in approximately
100% Yl-scFv binding activity relative to the corresponding unsulfated control
peptide
(see Fig. 20). In contrast, sulfation of the same linear peptide at the second
tyrosine
position confers only about 40% binding relative to the control, and sulfation
at the first
tyrosine position confers binding activity substantially indistinguishable
from the control.
[235] Taken together, the results obtained using synthetic peptides based on
GPIb
and PSGL-1 indicate that the sequence YsD, which is found within the motif
D~YSD
(SEQ ~ N~:21), wherein X represents any amino acid and Ys represents sulfated
tyrosine, is important for Y1 binding to its epitope.
[236] Several proteins are known to contain the sequence YSD found within the
motif DXYSD and/or within a highly acidic environment (Fig. 21). It is
believed that such
an acidic environment is significant for tyrosine sulfation in vivo. It is
predicted that Yl is
capable of binding such proteins at this sulfated sequence, as has been shown
for GPIb and
PSGL-1.
[237] 4.2 Yl binds to solid tumor antigens while KPL-1 does not:
Western blotting of a small cell lung carcinoma (SCLC) cell line lysate was
performed to
compare binding of Y1 and the commercially available mouse anti-PSGL-1
antibody
KPL1 (Fig. 22). A single broad band was observed in the Yl blot, whereas no
band was
observed in the KPLl blot. This indicates that PSGL-1 may serve as a target
for
immunotherapy in SCLC patients using Yl.
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51
EXAMPLE 5: Y1-IgG-mediated endocytosis via PSGL-1
[238] PSGL-1 is highly expressed on .AML patients' blood cells. The results
below indicate that Yl specifically recognizes and is internalized by tumor
cells
expressing PSGL-1. Commercially available I~PL-1 (anti-PSGL-1) binds to tumor
cells
but is not internalized. This indicates that PSGL-1 may serve as a target for
immunotherapy in AML patients using Yl.
[239] 5.1 Confocal Microscopy Studies: Patients' white blood cells were
isolated on FIC~LL~ gradient. Cells were incubated for different time periods,
at 37°C, in
the presence of fluorescent anti-PSGL-1 antibodies (Yl or commercial
antibodies KPLl
and PL1). Fluorescent antibody localization was determined in live cells by
visualizing
the cells by confocal microscopy.
[240] Figures 23 and 24 show live AML patient's cells visualized by confocal
microscopy following incubation of the cells at 37°C for 2 hours with
Yl-PE (left), I~PL1-
PE (middle) and Yl-IgG-FITC (right). Cells were scanned in three-dimensional
manner
(X, Y and ~ plans) and the pictures presented here were taken from the center
of the
sphere in respect to the Z plan. As shown, following incubation Yl-IgG was
present in
the interior part of the cells (but not in the nucleus), while I~PLl was
present on the cell
membranes and did not internalize.
[241] ~.1 Fluorescence I~ncroscopy Stendne~: Patients' white blood cells
were isolated on FIC~LL gradient. Cells were incubated for different time
periods, at
37°C, in the presence of Yl-IgG. For detection of the Y1-IgG, cells
were fixed,
permeabilized and then stained with rhodamine-labeled anti-human (Fc)
antibodies. The
cells were visualized by fluorescence microscopy.
[242] Figures 25 and 26 show live CD34+ cells (from healthy bone marrow and
from healthy cord blood, respectively) visualized by Confocal Microscopy
following
incubation of the cells at 37°C for 2 hours with Y1-IgG-FITC (left) and
I~l'L1-PE (right)
and with anti-CI~34-PE or FITC. As shown, Y1-IgG did not bind normal CD34+
stem
cells. In contrast, KPL-1-PE labeled normal cells including CD34+ cells, as
evidenced by
double staining of some cells in the lower right panel.
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52
[243] 5.3 Monitoring Endocytosis: Cell surface binding of Y1-IgG was
detected after incubation of the cells in the presence of the antibody, at
4°C with 0.1
NaN3 (which inhibits active process of internalization). Incubation at
37°C for lOmin-2hrs
(without NaN3) resulted in capping and patching and in internalized staining.
Similar
pictures were obtained with different AML samples either by means of confocal
microscopy or by means of fluorescence microscopy.
[244] Figure 27 shows four individual cells from an AML patient sample
incubated with non-labeled Yl-IgG for different time periods, at 37°C
or at 4°C. As
shown, cell surface binding of Y1-IgG was detected after incubation of the
cells in the
presence of the antibody, at 4°C with 0.1% NaN3. Incubation of Y1-IgG
at 37°C for 10
minutes - 2hours (without NaN3) resulted in capping and patching and in
internalized
staining. Internalization increased with time. No internalization was observed
in cells
leept at 4°C. Yl-IgG was detected with rhodamine-labeled anti-human
(Fc) antibodies.
Visualizing the cells was performed by fluorescence microscopy.
[245] 5.4 Acid Stripping: 'Treatment of the cells with SOn~IVI Glycine (pH
2.5) resulted in removing of cell surface binding of Y1-IgG and enabled
detection of
internalized Yl-IgG.
[246] Figure 28 shows AI~II, patient's cells that were incubated with non-
labeled
Y1-IgG for 1 hour, at 37°C. Cells shown in the lower row were then
incubated at room
temperature for 5 minutes with SOmM glycine, pH 2.5 to remove surface bound Y1-
IgG.
As shown, the upper panel represents cell surface capping and patching and
internalized
Y1-IgG. In the lower panel, only internalized Y1-IgG was detected.
[247] S.S Pronase: Removing of cell surface proteins by the proteolytic
enzyme, pronase, also resulted in removing of cell surface binding of Yl-IgG
and enabled
detection of internalized Y1-IgG.
[248] Figure 29 shows AML patient's cells that were incubated with non-labeled
Yl-IgG for 1 hour at 37°C. Cells shown in the lower row were then
incubated at room
temperature for 60 minutes with lmg/ml pronase to remove surface bound Yl-IgG.
As
shown, the upper panel represents cell surface capping and patching and
internalized Yl-
IgG. In the lower panel, only internalized Y1-IgG is detected. Note that the
uropods were
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53
removed by pronase, which implies that the uropods formed by Y1-IgG cross-
linking of
CD162 are formed on the outer surface of the cell surface.
[249] 5.6 Coated-pits mediated endocytosis: Receptor mediated
endocytosis can occur via coated-pits. Coated-pits-mediated endocytosis can be
blocked
by incubation of the cells with 0.45M sucrose for 15 minutes at 37°C
prior to incubation
with Yl-IgG. This method inhibited endocytosis of Yl-IgG without affecting
binding of
Yl-IgG to the cell surface.
[250] Figure 30 shows AML patient's cells that were incubated with non-labeled
Yl-IgG for lhr, at 4° C (Fig. 30A) or at 37° C (Fig. 30B). Cells
shown in the middle row
were then incubated at room temperature for 5 minutes with 50 mM Glycine pH
2.5 to
remove surface bound Y1-IgG. As shown (Fig. 30A), the upper panel represents
cell
surface staining of Yl-IgG. The surface bound Yl-IgG was removed by the acid
wash
(middle panel). At 37° C (Fig. 30B) capping and patching and
internalization of YI-IgG
was observed (upper panel). Acid wash removed cell surface bound Yl-IgG and
only the
internalized antibody could be detected (middle panel).
[251] Blocking of coated-pits mediated endocytosis (bottom rows) was obtained
by incubation of the cells with 0.451VI Sucrose for 15 minutes at 37°C
prior to incubation
with Y1-IgG. As shown in the lower panels, treating the cell with 0.451'
sucrose did not
affect binding of Y1-IgG to the cells at 4~°C (Fig. 30A) but inhibited
internalization at
37°C (Fig. 30B).
[252] E~~AMFLE 6: Yl-IgG-Inhibition of Leukocyte-Flatelet
Interactions
[253] Adherence of leukocytes to vascular surfaces results in organ injury in
various
disorders, including reperfusion injury, stroke, mesentaric and peripheral
vascular disease;
organ transplantation and circulatory shock. Reperfusion injury is associated
with
adherence of leukocytes to vascular endothelium in the ischemic zone,
presumably in part
due to activation of platelets and endothelium by thrombin and cytokines,
which renders
their surfaces adhesive for leukocytes. The main initiator of reperfusion
injury is the
interaction between von Willbrand factor (vWF) and platelet GPIb receptor.
Cardiac
patients who are treated with thrombolytic agents such as tissue plasminogen
activator and
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54
streptokinase to relieve coronary artery obstruction, may still suffer
myocardial necrosis
due to reperfusion injury. Thus, there is a need for drugs which are capable
of reducing
leukocyte adherence to vascular surfaces and which may be administered in
conjunction
with thrombolytic agents to improve outcome of cardiovascular disorders.
[254] Since Y1 (both scFv and full IgG) binds to distinct sulfated molecules
on platelets
(i.e. GPIb) and leukocytes (i.e. PSGL-1), this antibody has potential as a
therapeutic agent
for inhibiting various cell-cell interactions.
[255] Figure 31 shows that Y1-scFv effectively inhibits the binding of
activated human
platelets to MLZ cells (a human AML derived cell line expressing PSGL-I).
~ptimal
inhibition was obtained when the antibody was incubated simultaneously with
both
platelets and ML2 cells, while partial inhibition was obtained when the
antibody was
initially incubated with either platelets or ML2 cells, followed by removal of
non-bound
antibody and subsequent addition of the remaining cell type (Fig. 3I).
[256] Figure 31 also shows that the marine antibody KPLI (directed against
human
PSGL-1 ~-terminal d~main, but not tyrosine sulfation dependent) was also
effective in
inhibiting binding of activated platelets to ML2, but the inhibition was less
than that
exerted by YI-scFv. This might be due to the fact that I~PLl does not
recognize an
epitope present on both cell types, as does YI-scFv. 1~1o inhibition was
observed with the
marine antibody, PL2 antibody which is also directed against human PSCIL-1
(not shown).
[257] E PLE 7: Yl-IgG-Inliibiti~n ~f Cell ~lling ~n Ipnrn~bilized
F-Selectin under flow conditi~ns
[258] For preparation of a ligand-coated substrate, recombinant human (rh)-P-
Selectin (R&D Systems, Minneapolis, MN) was diluted to 0.2-1.0 ~g/ml in
coating
medium (PBS supplemented with 20 mM bicarbonate, pH 8.5) and immediately
adsorbed
onto a polystyrene plate overnight at 4° C, followed by washing with
PBS containing 2
~,g/ml human serum albumin (Calbiochem) at 4° C for 1 hr.
[259] For laminar flow assays a polystyrene plate on which purified ligand was
immobilized was assembled in a parallel plate laminar flow chamber as
described
previously (Lawrence & Springer, Cell 65, 859-873 (I991)). Human neutrophils
(isolated
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from anti-coagulated blood by dextran sedimentation and density separation
over
FICOLL) or ML-2 cells were washed in H/H medium (Hanks' balanced salt
solution, 10
mM HEPES , resuspended in cell binding medium (H/H medium supplemented with 2
mM CaCl2) at 2 ~ 106 cells/ml, and perfused at room temperature through the
flow
chamber at a rate generating wall shear stress at the desired flow rate,
generated with an
automated syringe pump (Harvard Apparatus, Natick, MA). Upon reaching the
upstream
side of the test adhesive substrate, the flow rate was elevated to generate a
shear stress of 1
dyn/cma, and all cellular interactions were visualized at two different fields
of view (each
0.17 mm2 in area) using a 10~ objective of an inverted phase contrast
microscope (Diaphot
300, Nikon Inc., Tokyo, Japan). An imaging system was used for analysis of
instantaneous velocities of leukocytes, WSCAN-Array-3 (Galai, Migdal-Ha'emek,
Israel)
as described previously (Dwir et al., J: Bi~l. Chew. 275, 18682-18691 (2000)).
[260] Accumulation of rolling leukocytes on the test fields was determined by
computerized cell motion tracking. The frequency of rolling cells was defined
as the
number of cells out of the cell flux that initiated persistent rolling on the
adhesive substrate
lasting at least 3 s after initial tethering. Cells were incubated with
antibodies at different
concentrations and perfused to the flow chamber with binding medium containing
the
same concentration of antibody. Cell rolling was analyzed either following
washing of the
reagent ("washed") or in the presence of the reagent.
[261] For image analysis, an imaging system was developed for quantitative
analysis of instantaneous velocities of cell rolling on different adhesive
substrates. Video
frame images consisting of 768 ~ 574 pixels (with a pixel size of 1.15 ~,m
using a l Ox
objective), were digitized using a Matrox Pulsar frame grabber (Matrox
Graphics Inc.,
Dorval, Quebec, Canada), and images were scanned and processed by the WSCAN-
Array-
3 imaging software (Galai, Migdal-Ha'emek, Israel), running on an Atlas
pentium MMX-
200 work station. Cell motions were identified from images tracked at 0.02-s
intervals.
The program output provided the co-ordinates of the center point of each cell
in successive
interlaced fields at 0.02 s apart.
[262] A computer program for cell motion analysis was developed in
collaboration with the laboratory of Professor David Malah (Electric
Engineering Faculty,
Technion, Haifa, Israel). The software runs under Matlab 5.2 and compares
instantaneous
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56
positions of individual cells at successive video images over a period of up
to 5 seconds.
Tethers of individual cells rolling persistently on the ligand-coated field or
moving
through it in a jerky motion were determined according to changes in
instantaneous cell
velocities in the flow direction. A rolling pause was defined as an
instantaneous velocity
drop to below 29 ~,m/s at shear stresses of 1-1.75 dyn/cm2. This threshold
velocity value
gave optimal correlation between pause analysis performed on representative
cells by the
computerized system and manually, directly from the video monitor. The step
distances
between successive pauses of an individual rolling cell were averaged to yield
the mean
step distance of a given rolling cell.
[263] Figure 32 shows the effect of Yl-scFv (10~.g/ml) on ML2 cell rolling on
immobilized rh- P-Selectin at low density (0.2 ~,glml). The analysis showed
that at shear
force of 1 dyn/cma, the number of rolling cells per field was totally
eradicated in the
presence of Yl-scFv. No such effect was obtained when equal amounts of the
scFv-N06
(negative control) was used.
[264] Figure 33 shows the effect of Yl-scFv (10 ~,g/ml) on ML2 cell rolling on
immobilized rh-P-Selectin at high density (1.0 ~,ghnl) at various shear
forces. The
analysis showed that at shear forces of 1, 5 and 10 dyn/cm~, the number of
rolling cells per
field was inhibited by 83°/~, 98% and 100%, respectively in the
presence of Yl-scFv. No
such effect was obtained with the negative control, N06, or when cells were
washed
following incubation with Yl-scFv and then tested (Y1 wash).
[265] Figure 34 shows the effect of Y1-IgG (1 ~,g/ml) on ML2 cell rolling on
immobilized rh-P-Selectin (1 ~.g/ml) at various shear stress forces. The
analysis showed
that at shear force of ldyn/cm2, cell rolling was inhibited by 89%, and that
at shear forces
of 5 and 10 dyn/crna cell rolling was inhibited by 100%. When cells were
washed
following incubation with Y1-IgG and then tested (Yl-IgG wash), cell rolling
was
inhibited by 46%, 48% and 54% at shear forces 1, 5 and 10 dyn/cma,
respectively. The
marine anti-PSGL-1 antibody KPL1 was also capable of 100% inhibition of cell
rolling at
all shear forces.
[266] Figure 35 shows the effect of increasing concentrations of Y1-scFv on
human neutrophil rolling on immobilized rh-P-Selectin at high density (1.0
~.g/ml). The
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57
analysis showed that at a shear force ldyn/cm2, Y1-scFv at 1, 5 and 10 ~g/ml
inhibited the
number of rolling neutrophils by 20%, ~ 1 % and 100%, respectively.
[267j Figure 36 shows the effect of Yl-IgG on human neutrophil rolling on
immobilized rh-P-Selectin at high density (1.0 p.g/ml). The analysis showed
that at a shear
force ldyn/cma, the number of rolling neutrophils per field was totally
eradicated (100%
inhibition) in the presence of Yl-IgG (1 ~,g/ml). Similar results were
obtained with KPL-
1.
[26~] Ea~ample ~: Screening of Inorganic Compound Library
[269] Synthetic sulfated peptide (sulfated on a given specific tyrosine
residue
within the known amino acid sequence of the peptide) derived from a specific
receptor
(protein) can be prepared with a biotin tag (biotinylated) coupled to the
synthetic peptide
via a short linker such as caproic acid. Control peptides using the same
synthetic peptide
can be prepared without sulfation and without the biotin tag ("B"). In
addition, synthetic
sulfated peptides derived from other, non-related proteins can be prepared
without having
the biotin tag ("C") as additional controls.
[270] The biotinylated peptide above ("A") cam be coupled to strepavidin-
coated
magnetic beads and excess unbound biotinylated peptide then washed away. The
biotin-
stretavidin peptide conjugate ("I~") can be screened against a small chemical
entity library
in the presence of large excess of non-sulfated control peptide ("E") under
physiological
conditions (37~ C, pIi 7.0-7.4, salts concentration, conductivity etc.) for
molecules that
bind to "A". The coupled-magnetic beads are then washed twice with buffer,
each time
centrifuged to remove excess unbound molecules. Molecules bound to the
magnetic beads
("E") can be eluted, chemically identified and prepared in larger quantity for
further
screening.
[271] Confirmation of binding to biotinylated sulfated peptides by the
selected
chemical compounds ("E") can be carried out by a further screening process.
This process
includes either competition with unrelated sulfated peptides that are
biotinylated (process
1) or competition with an antibody or fragments thereof (e.g. scFv) that bind
specifically
to the biotinylated peptide, "A" (process 2).
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[272] 8.1 Re-screening by competition with unrelated biotinylated sulfated
peptides (Process 1)
[273] In order to ensure that the compounds bind specifically to "A", a second
round of screeing can be carried out. Biotin-streptavidin peptide conjugate
("I~") can be
re-screened with the selected compounds "E" in the presence of large excess of
unrelated
biotinylated sulfated peptides, "C". The tube is then centrifuged, the biotin-
stretavidin
peptide conjugate coupled magnetic beads washed twice with buffer and
centrifuged each
time to remove excess unbound molecules. Compounds that bound to the magnetic
beads
can be eluted for chemical identification. Larger quantities of the chemical
compound can
be prepared for further studies, such as validation of selective binding to
"A", and efficacy
testing in vitr~ and irZ vivo.
[274] 8.2 Re-screening by competition with specific scFv anti-sulfated
antibody (Process 2)
[275] Compounds with preferred binding affinity to "A" can be re-screened by
competing the binding of biotin-streptavidin peptide conjugate ("I~") to each
of the
selected compounds "E" in the presence of large excess of specific schv
antibody that
specifically recognises and binds to "A". Chemical compounds that are
specifically
inlubited from binding to "A" by the ecru antibody can be prepared for further
studies,
such as validation of selective binding to "A", and eff cacy testing i~a
vita~~ and i~a ~~iv~.
[276] The invention has been described with reference to specific examples,
materials and data. As one skilled in the art will appreciate, alternate means
for using or
preparing the various aspects of the invention may be available. Such
alternate means are
to be construed as included within the intent and spirit of the present
invention as defined
by the following 'claims:
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SEQ ID NO: 1
YEYLDYD
SEQ ID NO: 2
VRPEHI'AETEYDSLYPEDDL
SEQ ID NO: 3
DXYD
SEQ ID NO: 4
DLYSDYSYSFE
SEQ ID NO: 5
DLYDYYPE
SEQ ID NO: 6
DLYSDYYPE
SE(~ ID NO: 7
DLYSDYSYPE
SEQ ID NO: ~
DLYSDYYSFE
SEQ LiD NO: 9
DLYDYSYSPE
SEQ ID N~: 10
DLYDYS~SPE
SEQ ID 1~0: 11
DLEDYYPE
SEQ TD NO: 12
DLYSEYYPE
SEQ ID NO: 13
DLYSNYYPE
SE(~ ID NO: 14
DLYSAYYPE
SEQ ID NO: 15
DAYSDYYPE
SEQ ID NO: 16
DLYSDFYPE
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SEQ ID NO: 17
DLYSDAYPE
SEQ ID NO: 18
DLYSDYYAE
SE(~ ID NO: 19
DLYSDYYFA
SEQ ID NO: 20
QATEYEYLDYDFLFETE
SE(~ ID NO: 21
DXYSD
SEQ ID NO: 22
DEGDTDLYDYYPEEDTEGD
SE(~ ID NO: 23
EHPAETEYDSLYPED
SEA ID NO: 24
I~EA1~TEDYEDYEYDELPAI~
SEA ID N~: 2~
C~YYDYDFPL
