U.S. patent application number 12/974539 was filed with the patent office on 2011-09-01 for anti-p-selectin antibodies and methods of their use and identification.
Invention is credited to Richard Alvarez, Ziad S. Kawar, Rodger P. McEver, Scott Rollins, Russell Rother.
Application Number | 20110212096 12/974539 |
Document ID | / |
Family ID | 44505400 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110212096 |
Kind Code |
A1 |
Rollins; Scott ; et
al. |
September 1, 2011 |
ANTI-P-SELECTIN ANTIBODIES AND METHODS OF THEIR USE AND
IDENTIFICATION
Abstract
Antibodies are disclosed which bind specifically to P-selectin
and which block the binding of PSGL-1 to P-selectin. These
anti-P-selectin antibodies may also cause dissociation of preformed
P-selectin/PSGL-1 complexes. The disclosure identifies a heretofore
unrecognized, near N-terminal, antibody binding domain (a
conformational epitope) of P-selectin to which the
function-blocking antibodies (which may be chimeric, human or
humanized antibodies for example) bind. Antibodies are disclosed
which bind to the conformational epitope of P-selectin and which
have a dual function in blocking binding of PSGL-1 to P-selectin,
and in causing dissociation of preformed P-selectin/PSGL-1
complexes. Such single and dual function anti-P-selectin antibodies
and binding fragments thereof may be used in the treatment of a
variety of inflammatory and thrombotic disorders and conditions.
Screening methods for identifying such antibodies are also
disclosed.
Inventors: |
Rollins; Scott; (Oklahoma
City, OK) ; Alvarez; Richard; (Edmond, OK) ;
Rother; Russell; (Oklahoma City, OK) ; McEver; Rodger
P.; (OKlahoma City, OK) ; Kawar; Ziad S.;
(Oklahoma City, OK) |
Family ID: |
44505400 |
Appl. No.: |
12/974539 |
Filed: |
December 21, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12516987 |
Mar 2, 2010 |
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PCT/US07/24692 |
Nov 30, 2007 |
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12974539 |
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60872170 |
Dec 1, 2006 |
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Current U.S.
Class: |
424/139.1 ;
435/243; 435/325; 530/387.3; 530/387.9; 536/23.53 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 7/02 20180101; A61P 11/06 20180101; C07K 2317/33 20130101;
A61P 37/06 20180101; A61P 9/10 20180101; C07K 2317/76 20130101;
C07K 2317/34 20130101; A61P 35/00 20180101; C07K 16/2854 20130101;
A61P 17/06 20180101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 530/387.3; 536/23.53; 435/243; 435/325 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28; C07K 16/46 20060101
C07K016/46; C07H 21/04 20060101 C07H021/04; C12N 1/00 20060101
C12N001/00; C12N 5/00 20060101 C12N005/00; A61P 29/00 20060101
A61P029/00; A61P 17/06 20060101 A61P017/06; A61P 35/00 20060101
A61P035/00; A61P 7/02 20060101 A61P007/02; A61P 11/06 20060101
A61P011/06; A61P 9/10 20060101 A61P009/10 |
Claims
1. An isolated antibody or binding fragment thereof which
specifically binds to a conformational epitope of P-selectin,
wherein the conformational epitope is within amino acid positions
1-35 of SEQ ID NO:1.
2. The isolated antibody or binding fragment of claim 1 wherein the
conformational epitope is within amino acids 4-23 of SEQ ID
NO:1.
3. The isolated antibody or binding fragment of claim 1 wherein the
conformational epitope comprises amino acid positions 4, 14, 17,
21, and 22 of SEQ ID NO:1.
4. The isolated antibody or binding fragment of claim 3 wherein the
conformational epitope further comprises amino acid positions 20
and 23 of SEQ ID NO:1.
5. The isolated antibody or binding fragment of claim 3 wherein the
amino acids in amino acid positions 4, 14, 17, 21, and 22 are H, I,
K, N, and R, respectively.
6. The isolated antibody or binding fragment thereof of claim 3
wherein binding is abrogated when any one or more of amino acid
positions 4, 14, 17, 21, or 22 is substituted with N,N, V, R, or H,
respectively.
7. The isolated antibody or binding fragment of claim 1 comprising
the ability to block the binding of P-selectin glycoprotein
ligand-1 (PSGL-1) to P-selectin.
8. The isolated antibody or binding fragment of claim 5 further
comprising the ability to cause dissociation of a preformed
P-selectin-PSGL-1 complex.
9. The isolated antibody or binding fragment of claim 1 comprising
the ability to block the function of P-selectin by inhibiting the
binding of activated endothelial cells to leukocytes, lymphocytes,
sickled red cells, and/or platelets.
10. The isolated antibody or binding fragment of claim 1 comprising
the ability to block the function of P-selectin by inhibiting the
binding of activated platelets to leukocytes, lymphocytes, sickled
red cells, and/or platelets.
11. The isolated antibody or binding fragment of claim 1 comprising
the ability to cause dissociation of cell-cell binding between
activated endothelial cells and leukocytes, lymphocytes, sickled
red cells, and/or platelets.
12. The isolated antibody or binding fragment of claim 1 comprising
the ability to cause dissociation of cell-cell binding between
activated platelets and leukocytes, lymphocytes, sickled red cells,
and/or platelets.
13. The isolated antibody or binding fragment of claim 1 wherein
the antibody or fragment thereof is monoclonal.
14. The isolated antibody or binding fragment of claim 1 wherein
the antibody or fragment thereof is chimeric, human, or
humanized.
15. The isolated antibody or binding fragment of claim 1 comprising
an immunoglobulin selected from the class consisting of IgA, IgD,
IgE, IgG, and IgM.
16. The isolated antibody or binding fragment of claim 15 wherein
the isolated antibody or binding fragment thereof is an IgG
selected from an isotype consisting of IgG1, IgG2, IgG3, IgG4, or
an IgG2/G4 chimera.
17. The binding fragment of claim 1 comprising at least one of a
Fab, Fab', F(ab)2, or scFv fragment.
18. The isolated antibody or binding fragment of claim 1 which
binds to the conformational epitope with a K.sub.d.ltoreq.1000 nM,
a K.sub.d.ltoreq.500 nM, a K.sub.d.ltoreq.100 nM, a
K.sub.d.ltoreq.50 nM, a K.sub.d.ltoreq.25 nM, a K.sub.d<10 nM, a
K.sub.d.ltoreq.5 nM, a K.sub.d.ltoreq.1 nM, or a K.sub.d.ltoreq.0.1
nM.
19. A composition comprising the isolated antibody or binding
fragment of claim 1 disposed within a pharmaceutically-acceptable
carrier, vehicle, or diluent.
20. A nucleic acid which encodes the antibody or binding fragment
thereof of claim 1.
21. A cell line which expresses the antibody or binding fragment of
claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
12/516,987, filed May 29, 2009, which claims priority under 35
U.S.C. 371 from International Application No. PCT/US2007/024692,
filed Nov. 30, 2007, which claims benefit of U.S. Provisional
Application No. 60/872,170, filed Dec. 1, 2006. Each of the above
applications is hereby expressly incorporated herein by reference
in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
BACKGROUND
[0003] The present invention relates to antibodies and antibody
fragments which bind to specific conformational epitopes of
P-selectin, and to methods of their use and identification.
[0004] In normal hemostasis and immune surveillance, leukocytes
circulate freely in the blood and respond to injury and infection
in a sequential process of adhesion signaling mediated by cell
adhesion molecules (1-3). In inflammatory and thrombotic disease,
this process is dysregulated and can sustain pathology wherein
leukocytes attack the body's own tissue and can cause serious and
sometimes deadly complications. It is well known that leukocyte
adhesion plays a major role in the pathology of many inflammatory
and thrombotic disorders such as vasoocclusion in sickle cell
disease, reperfusion injury, thrombosis, atherosclerosis, asthma,
rheumatoid arthritis, psoriasis and tumor metastasis (4-15) deep
venous thrombosis (DVT). P-selectin is also involved in other
disease processes, such as tissue and organ damage associated with
inflammation, e.g., ischemia-reperfusion injury. P-selectin is thus
a target for intervention in human inflammatory and thrombotic
diseases.
[0005] Selectins are a family of adhesion proteins which are known
to play key roles in the recruitment of leukocytes to activated
endothelium and platelets. P-selectin is a member of the selectin
family of adhesion glycoproteins which also includes L- and
E-selectins (16). The selectins mediate the recruitment, initial
tethering and rolling, and adherence of leukocytes to sites of
inflammation (1). P-selectin is stored in Weibel-Palade bodies of
endothelial cells and alpha-granules of platelets and is rapidly
mobilized to the plasma membrane upon stimulation by vasoactive
substances such as histamine and thrombin (17).
[0006] Sickle Cell Disease
[0007] Sickle cell disease (SCD) is a rare inherited blood disorder
that causes chronic anemia and vasoocclusion, affecting primarily
people of African-American heritage in the United States. Sickle
cell disease is the most common single gene disorder in African
Americans, affecting approximately 1 in 375-600 persons of African
ancestry (18, 19). Sickle cell conditions are also common among
people of Mediterranean countries, Africa the Caribbean and parts
of South and Central America (18, 19).
[0008] SCD is an autosomal recessive disease caused by a single
missense mutation (Val6Ala) in the .beta.-globin gene that renders
the mutant hemoglobin less soluble and prone to polymerization upon
deoxygenation. The polymerization of hemoglobin causes deformation
of the erythrocyte to give the cell a sickled shape (20).
[0009] SCD has three common variants: homozygous sickle cell
disease (hemoglobin SS disease), doubly heterozygous sickle
hemoglobin C disease (hemoglobin SC disease) and the sickle
.beta.-thalassemias. The most common and severe form of the disease
occurs in individuals who inherit two copies of the HbS variant
(HbSS) and the primary hemoglobin in their red blood cells is
sickle hemoglobin. Other individuals can be affected as compound
heterozygotes with varying severity of the disease. They have one
copy of the HbS variant paired with a copy of another .beta.-globin
gene variant. HBSC results in a mild form of the disease. Hb
.beta.-thalassemia variants (resulting in the inability to produce
the normal .beta.A globin chain (.beta..sup.o) or a reduction in
its production (.beta..sup.+) result in a range of clinical
severities. HbS .beta..sup.0 is a severe form, whereas HbS
.beta..sup.+ can be moderate or mild based on the contribution of
each variant to the total hemoglobin of the patient. Other more
rare variants can result if in conjunction with the S gene, another
abnormal hemoglobin is inherited from the other parent, such as D,
G or O. The predominant form of sickle cell is present in
individuals with one copy of HbS and one copy of the normal
.beta.-globin gene (HbA). These individuals carry the sickle cell
trait (18).
[0010] SCD affects an estimated 50-100,000 people in the US
(21-24). All individuals that are homozygous or compound
heterozygous for HbS show some clinical manifestations of SCD.
Symptoms usually appear within the first 6 months of life but there
is considerable variability in SCD severity (25). Individuals with
HbSS are most severely affected, followed by individuals with HBbS
.beta..sup.o-thalassemia (22, 26). In addition to genotype,
additional factors affect disease severity such as the levels of
fetal hemoglobin and the haplotye of the .beta.-globin cluster, a
region that contains 5 genes that code for the .beta. portion of
hemoglobin. Despite the capacity to determine genetic risk factors,
the ability to predict disease course from birth is limited
(27).
[0011] In the USA, sickle cell screening at birth is mandated in
all 50 states and the District of Columbia (28) and offers an
opportunity for early intervention. Diagnostic testing methodology
usually comprises a complete blood count in conjunction with one or
more of hemoglobin electrophoresis, isoelectric focusing,
high-performance liquid chromatography and DNA testing (22).
[0012] Chronic Anemia and Hemolysis
[0013] The sickled erythrocyte has a shorter half-life than the
normal erythrocyte and results from the instability of HbS and the
effects of repeated episodes of hemoglobin
polymerization/depolymerization in the circulation. This affects
membrane ionic permeability, cellular viscosity and deformability
(20) and promotes oxidative membrane damage (29). Sickle cell
disease patients are anemic by 2 to 3 months of age and develop
symptoms and complications associated with chronic anemia and
hemolysis (22, 30) such as renal disease, ophthalmic disorders, leg
ulcers, priapism and pulmonary hypertension (26, 31-37). Hemoglobin
values for SCD patients range from 6 to 10 g/dL and the hemoglobin
S molecule has a poor affinity for oxygen. Triggers for transfusion
in patients are a hemoglobin value of 5 or less or a precipitous
drop in hemoglobin of 2 g/dL or more. Transfusions are typically
given to restore hemoglobin values to baseline levels established
for each patient as excessive hemotocrit can precipitate sickling
(38). SCD patients are more susceptible to parvovirus B19 infection
which can arrest erythropoiesis and lead to aplastic anemia crisis
(39).
[0014] Vasoocclusive Pain Crisis
[0015] Vascular occlusion is central to the clinical course of SCD
and likely involves both the micro and macro circulation. Occlusion
occurring in the microvasculature can culminate in acute painful
episodes or vasoocclusive pain crises. Vasoocclusive pain crisis is
the clinical hallmark of microvascular occlusions and accounts for
over 90% of hospital admissions of adults SCD patients. It is well
known that polymerization of hemoglobin S during deoxygenation and
cell sickling leads to blockage of the microvasculature (40).
However, it has recently become clear that hemoglobin S
polymerization is not solely responsible for vasoocclusion. It has
now been demonstrated that such events as sickled red cell lysis,
cell membrane damage and oxidative stress, repeated ischemic
damage, and microvasculature injury due to the adhesive
interactions between sickle red cells and the endothelium that
culminate in a proinflammatory environment (41-43). In this
environment of chronic vascular inflammation, the adherence of
leukocytes, platelets and sickled red cells to activated blood
vessel endothelium and to each other is believed to be a primary
cause of microvasculature blockage and vasoocclusive pain crisis
(43-47). Additional factors such as the rigidity of sickled cells,
increased blood viscosity, and local vasoconstriction have also
been identified as potentially contributing to the vasoocclusion
process.
[0016] Long-term repeated vasoocclusive events and occlusions
occurring in the macrovasculature can cause life-threatening
complications leading to organ damage and failure, stroke and death
(40). There is an approximately 20 to 30 year reduction in life
expectancy in sickle cell disease patients (48). Chronic pain in
SCD is not just a continuation of the pain of vasoocclusion: it is
usually secondary to avascular necrosis of bone at various joints
(49). Sickled red cells can become trapped in the spleen causing it
to become enlarged and precipitating splenic sequestration crisis
causing sudden and severe anemia. Functional asplenia leaves
patients susceptible to infection (18). Bone growth retardation,
renal (32), ophthalmic (33) and cerebrovascular complications
(ranging from clinically evident acute stroke to transient silent
ischemic infarct) (50) are seen as major clinical consequences of
SCD and vasoocculsive injury (22). Acute chest syndrome is another
major complication (51), and is a significant cause of morbidity
and mortality (52).
[0017] Pain episodes appear to be triggered by a number of factors
including cold, stress and physical exertion (38, 53). The
frequency, severity, location and duration of pain crises can vary
considerably, even within a specific disease subtype. Patients with
homozygous sickle cell and sickle cell .beta..sup.o-thalassemia
have a higher frequency of vasoocciusive pain crises than patients
with hemoglobin SC and sickle cell-.beta..sup.o-thalassemia
genotype (54). Disease severity is thought to depend on a complex
interaction of genetic, rheologic and hematologic factors, as well
as microvascular and endothelial factors. Crises commonly involve
pain in the back, legs, knees, arms, chest and abdomen (53). The
frequency of crisis and pain severity varies considerably among
patients and in the same patient over time. One study evaluating
pain rates in patients ranging from newborns to age 50 years
indicated that 5.2 percent of patients with sickle cell disease
have three to 10 episodes of severe pain every year (54). In an
independent study, over 30% of sickle cell patients in the US
(approximately 27,000 patients) have three or more pain crises per
year (55). Moreover, a recent study (PiSCES) evaluating health
related quality of life issues in SCD patients indicated that pain
crisis might be significantly underreported among SCD patients
(56).
[0018] Current Therapies For Vascular Occlusion
[0019] Vascular occlusion in SCD patients can manifest in multiple
ways including vasoocciusive pain crisis, acute chest syndrome,
cerebrovascular events and multiple types of organ failure.
Therefore, treatment modalities for vascular occlusion depend on
the clinical course and severity of the disease and are generally
symptomatic or palliative in nature. Patient education in the
avoidance of initiating factors that precipitate vasoocciusive pain
crisis has shown some prophylactic benefit. The two most common
symptomatic treatments are blood transfusions and analgesics. Most
SCD patients commonly have hemoglobin values between 6 and 10 g/dL
and hemoglobin values typically drop at least 1 g per dL during a
vasoocclusive pain crisis. Severe pain resulting from vasoocclusive
crisis can be treated with narcotics but their use is controversial
due to concerns of narcotic addiction and tolerance. Other
complications with narcotic use are drug-seeking behavior, sedation
and respiratory depression. Oxygen management has been utilized to
treat vasoocclusive pain crisis despite the lack of strong evidence
supporting its effectiveness. Rehydration is also sometime used
during vasoocclusive pain crises with some benefit (22, 38).
[0020] Bone marrow transplantation may be considered and can be
curative, but its use is restricted to a limited number of
patients, and carries a high risk of morbidity and mortality
(22).
[0021] Hydroxyurea (Droxia) is the only FDA approved drug for
treatment of SCD pain crises. The mechanisms by which it produces
its beneficial effects are uncertain but may involve increasing
hemoglobin F levels in RBCs thereby decreasing the level of
hemoglobin S polymerization. Hydroxyurea is cytotoxic,
myelosuppressive and teratogenic (57, 58) which implies a
carcinogenic risk to SCD patients. The long-term effects however,
on hematologic toxicities, organ damage and carcinogenicity are
currently unknown (59, 60).
[0022] In summary, most therapies for vasoocclusive pain crisis in
SCD patients provide symptomatic relief and do not address the
underlying cause of this debilitating condition. To date only one
therapy has been approved by the FDA for the treatment of pain
crisis, thus, patients with SCD represent a major unmet medical
need in a life-threatening disease with severe morbidities.
[0023] P-selectin as a Therapeutic Target for SCD
[0024] In SCD, as noted above, interactions between sickled red
cells, platelets, leukocytes and the microvasculature are
P-selectin-dependent processes and result in vasoocclusion and
painful crisis. Studies in transgenic mice engineered to express
human .beta. hemoglobin S (.beta..sup.s) have shown that
antibody-mediated inhibition of P-selectin function can prevent
and/or reduce vasoocciusion, indicating a therapeutic potential for
this target. In addition mice expressing the .beta..sup.s
hemoglobin that lack P-selectin (due to gene deletion) do not
suffer vasoocciusion, further supporting a key role for this
molecule in this morbidity.
[0025] The hyper-inflammatory state in SCD patients is
characterized by activated monocytes and vascular endothelium
(61-63). A similar pro-inflammatory phenotype was demonstrated in
resting state .beta..sup.s mice which exhibit elevated levels of
peripheral leukocytes and neutrophils, an increased number of
rolling and adherent leukocytes, and reduced blood flow volume and
red blood cell velocities (64). The .beta..sup.s mice were
hypersensitive to hypoxia/reoxygenation resulting in an
inflammatory response represented by a significant increase in the
number of adherent and emigrated leukocytes. This inflammatory
response was completely blocked by a functionally blocking
anti-mouse P-selectin antibody, but not by a functionally blocking
anti-mouse E-selectin antibody, demonstrating a critical role for
P-selectin in this process.
[0026] Inflammatory Bowel Disease
[0027] Inflammatory Bowel Disease ("IBD") is the collective term
used to describe two chronic, idiopathic inflammatory diseases of
the gastrointestinal tract: ulcerative colitis ("UC") and Crohn's
Disease ("CD"). UC and CD are considered together because of their
overlapping clinical, etiologic, and pathogenetic features. From a
therapeutic and prognostic standpoint, however, it is useful to
distinguish them.
[0028] IBD occurs world-wide and is reported to afflict as many as
two million people. Onset has been documented at all ages; however,
IBD predominately begins in young adulthood. The three most common
presenting symptoms of IBD are diarrhea, abdominal pain, and fever.
The diarrhea may range from mild to severe and is often accompanied
by urgency and frequency. In UC, the diarrhea is usually bloody and
may contain mucus and purulent matter as well. Anemia and weight
loss are additional common signs of IBD. Reports of an increasing
occurrence of psychological problems, including anxiety and
depression, are perhaps not surprising secondary effects of what is
often a debilitating disease that occurs in people in the prime of
life.
[0029] A battery of laboratory, radiological, and endoscopic
evaluations are combined to derive a diagnosis of IBD and to assess
the extent and severity of the disease. Nevertheless,
differentiating UC from CD, as well as other types of inflammatory
conditions of the intestines, such as irritable bowel syndrome,
infectious diarrhea, rectal bleeding, radiation colitis, and the
like, is difficult, because the mucosa of the small and large
intestines reacts in a similar way to a large number of different
insults. Once other types of bowel disorders have been ruled out,
the final diagnosis is often made on the basis of the progression
of the disease. In many patients, though, the colitis must still be
regarded as indeterminate because of the overlapping features of UC
and CD, particularly with CD of the colon.
[0030] The leading early symptoms of UC and CD are chronic
recurrent diarrhea, bloody diarrhea, recurrent abdominal pain,
nausea, weight loss general evidence of inflammation without any
obvious explanation (fever, raised ESR, leucocytosis,
thrombocytosis and dysproteinenemia or anemia). Among these
symptoms, diarrhea and anemia are more characteristic of UC while
pain and weight loss and marked evidence of inflammation are more
common in CD. While the history and physical examination of a
patient can help, the final confirmation of the diagnosis has
traditionally been made endoscopically, histologically and, in
relation to the small intestine, radiologically as well.
[0031] The SAMP-1/Yit mouse model of spontaneous iletis closely
resembles human Crohn's Disease (65, 66). Therapeutic inhibition of
PSGL-1 binding uniquely ameliorates ileitis in this model whereas
blockade of individual selectins does not (67). Inhibition of TNF
in this model does reduce the severity of ileitis in a manner
similar to anti-PSGL-1 binding although the therapeutic effect does
not appear to be as potent as anti-PSGL-1. Thus, the SAMP-1 model
appears to closely mirror human Crohn's Disease not only in its
pathophysiology but also in its response to therapeutic
intervention. This evidence points to the conclusion that
therapeutic substances which inhibit P-selectin-PSGL-1 binding
activity in humans (and other primates) would also be effective as
treatments of Crohn's Disease.
[0032] P-selectin plays its central role in the recruitment of
leukocytes to inflammatory and thrombotic sites by binding to its
counter-receptor, P-selectin glycoprotein ligand-1 (PSGL-1) (or a
PSGL-1-like receptor on sickled red blood cells), which is a
mucin-like glycoprotein constitutively expressed on leukocytes
including neutrophils, monocytes, platelets, and on some
endothelial cells (68). The ultimate physiologic function of the
selectins is to promote extravasation of leukocytes into inflamed
or damaged tissues. The initial binding of P-selectin on the
endothelium to PSGL-1 on the leukocytes is essential and central to
this process. The predominant mechanism for rolling and tethering
of leukocytes to activated endothelium and platelets is the binding
of leukocyte PSGL-1 to the P-selectin on these cells (68, 69).
PSGL-1 binds to P-, L- and E-selectin (70). P-selectin and SGP-3, a
glycosulfopeptide modeled from the N-terminus of PSGL-1, have been
co-crystallized and the contact residues for lectin-ligand binding
have been identified (71).
[0033] The selectins share common structural motifs including a
lectin domain (or carbohydrate recognition domain), an epidermal
growth factor-like domain (EGF), a varying series of consensus
repeats, a transmembrane domain and a cytoplasmic tail (70). As
noted, the initial tethering and rolling of leukocytes is mediated
by the interaction of P-selectin and PSGL-1. Thus the blocking of
P-selectin function by using (1) antibodies to P-selectin, (2)
antibodies to PSGL-1, (3) fragments of PSGL-1 or recombinant forms
of PSGL-1, (4) small molecules that mimic the binding domain of
PSGL-1, and (5) other molecules that disrupt the binding of
P-selectin to PSGL-1, can block leukocyte rolling and tethering and
thus prevent firm adhesion to endothelial cells or platelets. Mice
deficient in P-selectin or PSGL-1 also fail to support leukocyte
tethering and rolling on activated endothelial cells (72, 74).
L-selectin plays a dual role in that it is constitutively expressed
on circulating leukocytes and can initiate "secondary binding" by
interaction with PSGL-1 on other leukocytes (75). This process
leads to further recruitment of new leukocytes to the inflamed
area. L-selectin binding to PSGL-1 also plays a role in homing of
lymphocytes to the high endothelial vasculature (HEV) venules in
the secondary lymphatic system (76). E-selectin is
transcriptionally regulated and is expressed on activated
endothelial cells hours after P-selectin mediated events.
E-selectin can bind PSGL-1 with low affinity but can also bind
other ligands. Single transgenic knockout mice for each selectin
have shown that these molecules possess compensatory selectin
mechanisms for leukocyte homing and rolling (77).
[0034] In view of the above, there is a well-established need for
new treatments, such as antibodies, that target P-selectin as a
means of treating inflammatory and thrombotic diseases by
disrupting the binding of P-selectin and PSGL-1. It is therefore a
preferred goal of the present invention to block P-selectin binding
to PSGL-1 to block the adherence of blood cells that contribute to
vasoocclusion in SCD and other thrombotic disorders.
SUMMARY OF THE DISCLOSURE
[0035] The presently disclosed and claimed inventive concepts, in
one embodiment, are directed to "function-blocking" antibodies
which bind specifically to P-selectin and which block the binding
of PSGL-1 to P-selectin. These anti-P-selectin antibodies may also
cause dissociation of preformed P-selectin/PSGL-1 complexes. The
present disclosure describes a heretofore unrecognized antibody
binding domain (a conformational epitope) within the lectin domain
(e.g., carbohydrate recognition domain, CRD) of P-selectin to which
the function-blocking antibodies (which may be chimeric, human or
humanized antibodies or fragments thereof for example) bind. The
presently disclosed and claimed inventive concepts, in one
embodiment, are also directed to anti-P-selectin antibodies which
bind to the conformational epitope described herein and which have
a dual function in (1) blocking binding of PSGL-1 to P-selectin,
and (2) causing dissociation of preformed P-selectin/PSGL-1
complexes. The presently disclosed and claimed inventive concepts
in particular are directed to using such single and dual function
anti-P-selectin antibodies or antibody fragments as described
herein in treatments for inflammatory, thrombotic or other
conditions or disorders in primates (including humans) which
involve platelet, sickled red cell, leukocyte, lymphocyte, and/or
endothelial cell adhesion, wherein the condition or disorder
comprises or is associated with (but not limited to) at least one
of sickle cell vasoocclusive pain crisis, inflammatory bowel
disease (e.g., Crohn's Disease, ulcerative colitis, enteritis),
arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic
arthritis), graft rejection, graft versus host disease, asthma,
chronic obstructive pulmonary disease, psoriasis, dermatitis,
sepsis, nephritis, lupus erythematosis, scleroderma, rhinitis,
anaphylaxis, diabetes, multiple sclerosis, atherosclerosis,
thrombosis, tumor metastasis, allergic reactions, thyroiditis,
ischemic reperfusion injury (e.g., due to myocardial infarction,
stroke, or organ transplantation), and conditions associated with
extensive trauma, or chronic inflammation, such as for example,
type IV delayed hypersensitivity, associated for example with
infection by Tubercle bacilli, or systematic inflammatory response
syndrome, or multiple organ failure. Other embodiments of the
inventive concepts disclosed and claimed herein will be apparent in
the Detailed Description below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 shows a homology comparison at the amino acid level
of human and mouse P-selectin indicating the location of lectin,
EGF, CR1 and CR2 domains (transition between domains is indicated
by .dwnarw.). Nonlinear conformational domains A, B, C1, D, E1, C2,
E2, C3, and F are indicated by dashed boxes. Amino acid differences
are indicated in boldface.
[0037] FIG. 2 shows a representative sensogram showing the ability
of the G1 test antibody to bind various P-selectin chimeras: SEQ ID
NO:1 (human P-selectin), SEQ ID NO:2 (mouse P-selectin), and SEQ ID
NO:4 (human cluster A (aa 1-35) replaces mouse as 1-35 in mouse
P-selectin). The G1 antibody binds to human P-selectin (SEQ ID
NO:1), does not bind to mouse P-selectin (SEQ ID NO:2), and binds
mouse P-selectin when human amino acids from Cluster A have been
inserted into the mouse sequence in residues 3-23 (SEQ ID
NO:4).
[0038] FIG. 3 shows representative two-step BIACORE P-selectin
chimera binding data for G1, G3, G4 and G5 anti-P-selectin
antibodies binding to SEQ ID NO:1-4, 7-10, 18 and 19. G4 was an
uncharacterized anti-P-selectin antibody and demonstrated
P-selectin binding properties similar to G1.
[0039] FIG. 4 shows BIACORE sensograms demonstrating dissociation
of the preformed P-selectin/PSGL-1 complex upon exposure to dual
function anti-P-selectin antibodies (G1, G4, hSEL001). PSGL-1 is
represented by GSP-6 peptide, a PSGL-1 mimetic. Initial RU increase
shows binding of P-selectin to biotin-GSP-6 coupled to a
streptavidin coated BIACORE chip. Once steady state binding of the
P-selectin/GSP-6 complex was reached (i.e., after normal
dissociation of the complex had reached near-equilibrium), test
antibodies G1, G3, and G5 were injected and assessed for
dissociation properties. G1 caused dissociation of the preformed
P-selectin/GSP-6 complex. G5 bound to the preformed complex, but
did not cause its dissociation. G3 did not bind or dissociate the
preformed P-selectin/PSGL-1 complex. A new anti-P-selectin
antibody, G4, and a humanized anti-P-selectin antibody named
hSEL001, also both bound and caused dissociation of the preformed
P-selectin/PSGL-1 complex, indicating dual function
capabilities.
[0040] FIG. 5 shows a 3-D representation of a human P-selectin
molecule with GSP-6 binding thereto. Lectin and EGF domains are
demarcated by a dashed line. Binding region 1 indentifies a Cluster
A conformational epitope that is distal to the lectin/ligand
binding domain. Test antibody G1 bound to region 1, Cluster A. G4,
and a humanized anti-P-selectin antibody, hSEL001, also bound
region 1, Cluster A.
[0041] FIG. 6 shows graphs of results of cell-based in vitro
rolling assays under flow of human neutrophils on low (A) and high
(B) density P-selectin. Results demonstrate blocking and/or
dissociation of the preformed P-selectin/PSGL-1 complex and
subsequent release of neutrophils upon exposure to antibodies G1,
G3 and hSel001. Antibodies were introduced at equivalent
concentrations of 20 .mu.g/ml for the duration of the study. There
is a lag time of about 1 minute before the antibody reaches the
chamber due to the dead volume of the system. At 1-minute intervals
thereafter, cells remaining bound were counted and expressed as %
cells bound. Panel (A) shows neutrophils rolling at average
velocity of 5 .mu.m/s on low density (50 sites/.mu.m.sup.2)
membrane P-selectin. Panel (B) shows neutrophils rolling at an
average velocity of 1 .mu.m/s on high density P-selectin (380
sites/.mu.m.sup.2).
DETAILED DESCRIPTION
[0042] As indicated above, the presently disclosed and claimed
inventive concepts, in one embodiment, are directed to antibodies
which bind specifically to P-selectin and which block the binding
of PSGL-1 to P-selectin (and are referred to herein as
"function-blocking" antibodies). In some embodiments, these
anti-P-selectin antibodies may also cause dissociation of preformed
P-selectin/PSGL-1 complexes. The disclosure describes a heretofore
unrecognized antibody binding domain (a conformational epitope)
within the lectin domain (e.g., carbohydrate recognition domain,
CRD) of P-selectin to which the function-blocking antibodies (which
may be chimeric, human or humanized antibodies, or fragments
thereof for example) bind. The presently disclosed and claimed
inventive concepts also directed to anti-P-selectin antibodies
which bind to the conformational epitope described herein and which
have a dual function in (1) blocking binding of PSGL-1 to
P-selectin and (2) causing dissociation of preformed
P-selectin/PSGL-1 complexes. The presently disclosed and claimed
inventive concepts in particular are directed to using such single
and dual function anti-P-selectin antibodies or antibody fragments
as described herein in treatments for inflammatory, thrombotic or
other conditions or disorders in primates (including humans) which
involve platelet, sickled red cell, leukocyte, lymphocyte, and/or
endothelial cell adhesion, wherein the condition or disorder
comprises or is associated with (but not limited to) at least one
of sickle cell vasoocclusive pain crisis, inflammatory bowel
disease (e.g., Crohn's Disease, ulcerative colitis, enteritis),
arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic
arthritis), graft rejection, graft versus host disease, asthma,
chronic obstructive pulmonary disease, psoriasis, dermatitis,
sepsis, nephritis, lupus erythematosis, scleroderma, rhinitis,
anaphylaxis, diabetes, multiple sclerosis, atherosclerosis,
thrombosis, tumor metastasis, allergic reactions, thyroiditis,
ischemic reperfusion injury (e.g., due to myocardial infarction,
stroke, or organ transplantation), and conditions associated with
extensive trauma, or chronic inflammation, such as for example,
type IV delayed hypersensitivity, associated for example with
infection by Tubercle bacilli, or systematic inflammatory response
syndrome, or multiple organ failure. The presently disclosed and
claimed inventive concepts are also directed to a screening assay
for the detection of anti-P-selectin antibodies which bind to the
conformational epitope of P-selectin described here, and, in one
embodiment, also block binding of PSGL-1 to P-selectin, and which,
in another embodiment, cause reversal of the binding of PSGL-1 to
P-selectin (i.e., dissociation of the preformed complex).
[0043] It is noted that as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural reference
unless the context clearly dictates otherwise. Thus, for example,
reference to "a cell" includes a plurality of such cells and
equivalents thereof known to those skilled in the art, and so
forth. As well, the terms "a" (or "an"), "one or more" and "at
least one" can be used interchangeably herein. It is also to be
noted that the terms "comprising", "including", "characterized by"
and "having" can be used interchangeably.
[0044] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the presently
disclosed and claimed inventive concepts, the preferred methods and
materials are now described.
[0045] "Leukocyte rolling," as used herein, includes weak adhesion
of leukocytes to endothelial cells of blood vessels and rolling of
leukocytes along endothelial cells of blood vessels prior to firm
adhesion and transmigration of leukocytes into endothelial tissue.
Following leukocyte rolling, these adherent leukocytes can migrate
through the endothelium and destroy ischemic tissue during
reperfusion. Accordingly, reduction of leukocyte rolling results in
a reduction of damage to tissues and organs caused by acute
inflammatory responses.
[0046] As used herein, a "P-selectin antagonist" includes any agent
which is capable of antagonizing P-selectin, e.g., by inhibiting
interaction between P-selectin and a P-selectin glycoprotein
ligand-1, e.g., by inhibiting interactions of P-selectin expressing
endothelial cells and activated platelets with PSGL-1 expressing
leukocytes.
[0047] As noted herein, the presently disclosed and claimed
inventive concepts are directed to purified antibodies (including
but not limited to chimeric, human, or humanized antibodies and
fragments thereof), which recognize (i.e., bind to) P-selectin (SEQ
ID NO:1) and which block binding of P-selectin to PSGL-1 (or
PSGL-1-like receptors), and to methods for screening for such
antibodies and binding fragments thereof, and to therapeutic
methods of use thereof.
[0048] More particularly, the presently disclosed and claimed
inventive concepts are directed to purified antibodies (or
fragments thereof), against P-selectin, host cells that produce
such anti-P-selectin antibodies (or fragments thereof), screening
assays to identify anti-P-selectin antibodies (or fragments
thereof) which block leukocytes, sickled erythrocytes, lymphocyte,
platelet and endothelial cell P-selectin-mediated adhesion and
optionally further cause dissociation of preformed adhesions or
cell complexes that were mediated through PSGL-1/P-selectin
interactions, and therapeutic methods using such antibodies (or
binding fragments thereof). The presently disclosed and claimed
inventive concepts include novel antibodies against primate
(including human) P-selectin and binding fragments thereof,
particularly including, in one embodiment, hSel001 antibody.
Preferred antibodies of the disclosure are capable of specifically
binding primate (particularly human) P-selectin, and inhibiting one
or more P-selectin activities in vitro and/or in vivo. Where used
herein, the term "PSGL-1" is also intended to include "PSGL-1-like
receptor" on sickled red cells (erythrocytes).
Antibodies
[0049] Antibody molecules belong to a family of plasma proteins
called immunoglobulins, whose basic building block, the
immunoglobulin fold or domain, is used in various forms in many
molecules of the immune system and other biological recognition
systems. A typical immunoglobulin has four polypeptide chains,
containing an antigen binding region known as a variable region and
a non-varying region known as the constant region.
[0050] Native antibodies and immunoglobulins are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable
domain (VH) followed by a number of constant domains. Each light
chain has a variable domain at one end (VL) and a constant domain
at its other end. The constant domain of the light chain is aligned
with the first constant domain of the heavy chain, and the light
chain variable domain is aligned with the variable domain of the
heavy chain.
[0051] Depending on the amino acid sequences of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are at least five major classes of immunoglobulins:
IgA, IgD, IgE, IgG and IgM, and several of these may be further
divided into subclasses (isotypes), e.g. IgG.sub.1, IgG.sub.2,
IgG.sub.3 and IgG.sub.4 and IgA.sub.1 and IgA.sub.2. The constant
domains of the heavy chains that correspond to the different
classes of immunoglobulins are called alpha (.alpha.), delta
(.delta.), epsilon (.epsilon.), gamma (.gamma.) and mu (.mu.),
respectively. The light chains of antibodies can be assigned to one
of two clearly distinct types, called kappa (.kappa.) and lambda
(.lamda.), based on the amino sequences of their constant domain.
The subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0052] The term "variable" in the context of variable domain of
antibodies, refers to the fact that certain portions of the
variable domains differ extensively in sequence among antibodies.
The variable domains are for binding and determine the specificity
of each particular antibody for its particular antigen. However,
the variability is not evenly distributed through the variable
domains of antibodies. It is concentrated in three segments per
chain called complementarity determining regions (CDRs), also known
as hypervariable regions, both in the light chain and the heavy
chain variable domains.
[0053] The more highly conserved portions of variable domains are
called the framework (FR). The variable domains of native heavy and
light chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each light and heavy chain are
held together in close proximity by the FR regions and contribute
to the formation of the antigen-binding site of the antibody. The
constant domains are not involved directly in binding an antibody
to an antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
[0054] An antibody of the presently disclosed and claimed inventive
concepts thus can be in any of a variety of forms, including a
whole immunoglobulin, an antibody fragment such as Fv, Fab, and
similar fragments, a single chain antibody which includes the
variable domain complementarity determining regions (CDRs), and the
like forms, all of which fall under the broad term "antibody", as
used herein. In preferred embodiments, in the context of both the
therapeutic and screening methods described below, an antibody or
fragment thereof is used that is immuno-specific for an antigen or
epitope of the presently disclosed and claimed inventive concepts
as described herein.
[0055] The term "antibody fragment" as used herein refers to a
portion of a full-length antibody, generally the antigen binding or
variable region. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2 and Fv fragments. Papain digestion of antibodies
produces two identical antigen binding fragments, called the Fab
fragment, each with a single antigen binding site, and a residual
"Fc" fragment, so-called for its ability to crystallize readily.
Pepsin treatment yields an F(ab').sub.2 fragment that has two
antigen binding fragments that are capable of cross-linking
antigen, and a residual other fragment (which is termed pFc').
Additional fragments can include diabodies, linear antibodies,
single-chain antibody molecules, and multispecific antibodies
formed from anti-body fragments. As used herein, "functional
fragment" with respect to antibodies, refers to Fv, F(ab) and
F(ab').sub.2 fragments.
[0056] Antibody fragments may be as small as about 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30, 35, 40, 45 or 50 or 75 or 100 (inclusive) or
more amino acids, for example. In general, an antibody fragment of
the presently disclosed and claimed inventive concepts can have any
upper size limit so long as it is has similar or immunological
properties relative to antibody that binds with specificity to the
P-selectin-binding described herein and which blocks binding of
PSGL-1 to P-selectin. Where used herein the term "inclusive" is
intended to refer to all integers between any two numbers listed
herein.
[0057] As noted elsewhere herein, antibody fragments contemplated
herein retain the ability to selectively bind to all of or a
portion of the P-selectin binding epitope described herein.
Preferably, an antibody or binding fragment of an antibody of the
present invention is capable of binding to an epitope comprising
one or more of amino acid residues 1-35, or, more particularly,
4-23, of the sequence set forth in SEQ ID NO:1. Some types of
antibody fragments are defined as follows:
[0058] Fab is the fragment that contains a monovalent
antigen-binding fragment of an antibody molecule. A Fab fragment
can be produced by digestion of whole antibody with the enzyme
papain to yield an intact light chain and a portion of one heavy
chain.
[0059] Fab' is the fragment of an antibody molecule can be obtained
by treating whole antibody with pepsin, followed by reduction, to
yield an intact light chain and a portion of the heavy chain. Two
Fab' fragments are obtained per antibody molecule.
[0060] Fab' fragments differ from Fab fragments by the addition of
a few residues at the carboxyl terminus of the heavy chain CH1
domain including one or more cysteines from the antibody hinge
region.
[0061] (Fab').sub.2 is the fragment of an antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction. F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds.
[0062] Fv is the minimum antibody fragment that contains a complete
antigen recognition and binding site. This region consists of a
dimer of one heavy and one light chain variable domain in a tight,
non-covalent association (VH-VL dimer). It is in this configuration
that the three CDRs of each variable domain interact to define an
antigen binding site on the surface of the VH-VL dimer.
Collectively, the six CDRs confer antigen binding specificity to
the antibody. However, even a single variable domain (or half of an
Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0063] A single chain antibody (SCA) is defined herein as a
genetically engineered molecule containing the variable region of
the light chain, the variable region of the heavy chain, linked by
a suitable polypeptide linker as a genetically fused single chain
molecule. Such single chain antibodies are also referred to as
"single-chain Fv" or "sFv" or "scFv" antibody fragments. Generally,
the Fv polypeptide further comprises a polypeptide linker between
the VH and VL domains that enables the sFv to form the desired
structure for antigen binding.
[0064] As noted above, the antibodies or antibody fragments of the
presently disclosed and claimed inventive concepts in preferred
embodiments comprise immunoglobulins of the isotypes IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4 or IgG.sub.2/G.sub.4 chimeras,
preferably binds to P-selectin with a high affinity (for example
wherein the K.sub.d is .ltoreq.1000 nM) and preferably comprises a
human constant region, and preferably inhibits binding of
P-selectin to PSGL-1 and more preferably also caused reversal of
binding of P-selectin to PSGL-1 in a preformed complex. Further,
the anti-P-selectin antibody or binding fragment thereof preferably
does not activate complement via the classical pathway by
interacting with Clq and preferably does not bind Fc receptors,
collectively called antibody effector function. The presently
disclosed and claimed inventive concepts in particular are directed
to using such single and dual function anti-P-selectin antibodies
or antibody fragments as described and identified herein in
treatments for inflammatory, thrombotic or other conditions or
disorders in primates (including humans) which involve platelet,
sickled red cell, leukocyte, lymphocyte, and/or endothelial cell
adhesion, wherein the condition or disorder comprises or is
associated with (but not limited to) at least one of sickle cell
vasoocclusive pain crisis, inflammatory bowel disease (e.g.,
Crohn's Disease, ulcerative colitis, enteritis), arthritis (e.g.,
rheumatoid arthritis, osteoarthritis, psoriatic arthritis), graft
rejection, graft versus host disease, asthma, chronic obstructive
pulmonary disease, psoriasis, dermatitis, sepsis, nephritis, lupus
erythematosis, scleroderma, rhinitis, anaphylaxis, diabetes,
multiple sclerosis, atherosclerosis, thrombosis, tumor metastasis,
allergic reactions, thyroiditis, ischemic reperfusion injury (e.g.,
due to myocardial infarction, stroke, or organ transplantation),
and conditions associated with extensive trauma, or chronic
inflammation, such as for example, type IV delayed
hypersensitivity, associated for example with infection by Tubercle
bacilli, or systematic inflammatory response syndrome, or multiple
organ failure.
[0065] As noted elsewhere herein, P-selectin plays a central role
in recruitment of leukocytes and lymphocytes to inflammatory and
thrombotic sites by binding to a surface ligand (PSGL-1) on these
cells and in the binding of sickled red cells to endothelia having
activated endothelial cells. PSGL-1 is constitutively expressed on
leukocytes, including neutrophils and monocytes, and on some
endothelial cells. A PSGL-1-like receptor is expressed on sickled
red cells and enables these cells to bind P-selectin on activated
endothelial cells.
[0066] Without wanting to be bound by theory, it is believed that
the treatment of vasoocclusive sickle cell pain crisis, for example
by the anti-P-selectin antibody of the present invention, is
effective by inhibiting any one or more of the following
interactions: (1) PSGL-1 on leukocytes binding to P-selectin on
activated endothelium; (2) a PSGL-1-like ligand on sickled red
cells binding to P-selectin on activated endothelium; (3)
P-selectin on the surface of activated platelets binding PSGL-1 on
endothelial cells; (4) sickled red cells binding leukocytes through
an uncharacterized ligand-receptor interaction; (5) P-selectin on
activated platelets binding the PSGL-1 like receptor on sickled red
cells, and/or; (6) P-selectin on the surface of activated platelets
binding PSGL-1 on leukocytes. It is expected that the
function-blocking anti-P-selectin antibody blocks the initiation
and propagation of vasoocclusion at multiple levels of cell-cell
interactions in the microvasculature.
[0067] The presently disclosed and claimed inventive concepts in
one embodiment are directed to antibodies that specifically bind to
human P-selectin. CDRs in such antibodies are not limited to the
specific sequences of VH and VL shown herein or in the documents
incorporated by reference herein and may include variants of these
sequences that retain the ability to block P-selectin binding to
PSGL-1. Such variants may be produced by a skilled artisan using
techniques well known in the art. For example, amino acid
substitutions, deletions, or additions, can be made in the FRs
and/or in the CDRs as described elsewhere herein. While changes in
the FRs are usually designed to improve stability and decrease
immunogenicity of the antibody, changes in the CDRs are typically
designed to increase affinity of the antibody for its target.
Variants of FRs also include naturally occurring immunoglobulin
allotypes. Such affinity-increasing changes may be determined
empirically by routine techniques that involve altering the CDR and
testing the affinity antibody for its target.
[0068] For example, conservative amino acid substitutions can be
made within any one of the disclosed CDRs. Various alterations can
be made according to methods well known to those skilled in the art
(78). These include but are not limited to nucleotide sequences
that are altered by the substitution of different codons that
encode an identical or a functionally equivalent amino acid residue
("conservative substitutions") within the sequence, thus producing
a "silent" change. For example, the nonpolar amino acids which may
be conservatively substituted include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan, and methionine. The
polar neutral amino acids which may be substituted conservatively
include glycine, serine, threonine, cysteine, tyrosine, asparagine,
and glutamine. The positively charged (basic) amino acids which may
be conservatively substituted include arginine, lysine, and
histidine. The negatively charged (acidic) amino acids which may be
conservatively substituted include aspartic acid and glutamic acid.
Substitutes for an amino acid within the sequence may also be
selected from other members of the class to which the amino acid
belongs.
[0069] Derivatives and analogs of antibodies of the invention can
be produced by various techniques well known in the art, including
recombinant and synthetic methods (79, 80). Antibodies in which CDR
sequences differ only insubstantially from those of the variable
regions of anti-P-selectin antibodies such as hSel001, discussed in
further detail below, are also encompassed within the scope of this
invention. As noted above, typically, an amino acid is substituted
by a related amino acid having similar charge, hydrophobic, or
stereochemical characteristics. Such substitutions would be within
the ordinary skills of an artisan. Further, a skilled artisan would
appreciate that changes can be made in FRs without adversely
affecting the binding properties of an antibody. Changes to FRs
include, but are not limited to, humanizing a non-human derived or
engineering certain framework residues that are important for
antigen contact or for stabilizing the binding site, e.g., changing
the class or subclass of the constant region, changing specific
amino acid residues which might alter the effector function such as
Fc receptor binding.
[0070] As used herein, the "affinity" of the antibody for
P-selectin or the conformational epitope thereof is characterized
by its K.sub.d, or disassociation constant. A stronger affinity is
represented by a lower K.sub.d while a weaker affinity is
represented by a higher K.sub.d. As such, an antibody of the
present invention preferably has an affinity for a P-selectin
conformational epitope represented by a K.sub.d.ltoreq.1000 nM, or
.ltoreq.500 nM, or .ltoreq.100 nM, or .ltoreq.50 nM, or more
preferably by a K.sub.d.ltoreq.25 nM, and still more preferably by
a K.sub.d.ltoreq.10 nM, and even more preferably by a
K.sub.d.ltoreq.5 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM.
[0071] An antibody or antibody fragment "homolog," as used herein,
means that a relevant amino acid sequence (preferably for example
in the CDRs and/or variable domains VH and/or VL) of a protein or a
peptide is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, 99.5%, or 100% identical to a given sequence. By way
of example, such sequences may be variants derived from various
species, or the homologous sequence may be recombinantly produced.
The sequence may be derived from the given sequence by truncation,
deletion, amino acid substitution, or addition. Percent identity
between two amino acid sequences is determined by standard
alignment algorithms such as, for example, Basic Local Alignment
Tool (BLAST) and other alignment algorithms and methods of the art
(81-84).
[0072] The term "isolated" or "purified" refers to a molecule that
is substantially free of its natural environment. For instance, an
isolated protein is substantially free of cellular material or
other proteins from the cell or tissue source from which it was
derived. The term also refers to preparations where the isolated
protein is at least 70-80% (w/w) pure; or at least 80-90% (w/w)
pure; or at least 90-95% pure; or at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% (w/w) pure. In some embodiments, the
isolated molecule is sufficiently pure for pharmaceutical
compositions.
[0073] Inhibitory activity refers to a reduction in an activity of
P-selectin by a P-selectin inhibitor (such as an antibody or
fragment thereof), relative to the activity of P-selectin in the
absence of the same inhibitor. A neutralizing antibody may reduce
one or more P-selectin activities. For example, the reduction in
activity (e.g., P-selectin binding to PSGL-1) is preferably at
least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, or higher. In another example, the dissociative activity of a
dual function antibody or fragment (i.e., the percentage of
preformed P-selectin/PSGL-1 complex which may be caused to
dissociate) may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,
85%, 90%, 95%, or higher.
[0074] The term "P-selectin inhibitor" when used herein includes
any agent, such as, e.g., a neutralizing antibody, capable of
inhibiting activity, expression, processing, binding, or cell
surface localization of P-selectin. Such inhibitors are said to
"inhibit," "neutralize," or "reduce" the biological activity of
P-selectin.
[0075] The preparation of monoclonal antibodies is conventional and
well known to persons of ordinary skill in the art. Monoclonal
antibodies can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
[0076] Methods of in vitro and in vivo manipulation of monoclonal
antibodies are well known to those skilled in the art. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler and Milstein (85), or may be made by recombinant methods,
e.g., as described in U.S. Pat. No. 4,816,567, for example.
[0077] Another method involves humanizing a monoclonal antibody by
recombinant means to generate antibodies containing, for example,
human or primate specific and recognizable sequences.
[0078] Methods of making antibodies of the presently disclosed and
claimed inventive concepts which bind with high affinity to human
P-selectin or to the conformational epitopes thereof as described
herein may comprise transfecting a cell with a DNA construct, the
construct comprising a DNA sequence encoding at least a portion of
the neutralizing P-selectin specific antibodies of the invention,
culturing the cell under conditions such that the antibody protein
is expressed by the cell, and isolating the antibody protein.
[0079] Preferably, the constant region has been modified to
modulate (i.e. reduce or enhance) effector function as noted
elsewhere as compared to the effector function of a wild-type
immunoglobulin heavy chain Fc region. In various embodiments, the
IgG constant region has reduced effector function, or alternatively
it has increased effector function, for example. Fc effector
function includes, for example, antibody-dependent cellular
cytotoxicity (ADCC), phagocytosis, complement-dependent
cytotoxicity, and half-life or clearance rate function. The IgG
amino acid sequence of the Fc domain can be altered to affect
binding to Fc gamma receptors (and thus ADCC or phagocytosis
functions), or to alter interaction with the complement system
(complement-dependent cytotoxicity function).
[0080] In one embodiment, the antibody comprises a constant region
or Fc portion that has low or no affinity for at least one Fc
receptor. In an alternative embodiment, the second polypeptide has
low or no affinity for complement protein Clq. In general, an
effector function of an antibody can be altered by altering the
affinity of the antibody for an effector molecule such as an Fc
receptor. Binding affinity will generally be varied by modifying
the effector molecule binding site. Disclosure of IgG modifications
that alter interaction with effector molecules such as Fc receptors
can be found for example in U.S. Pat. Nos. 5,624,821 and
5,648,260
[0081] Antibody proteins of the presently disclosed and claimed
inventive concepts can be produced using techniques well known in
the art. For example, the antibody proteins can be produced
recombinantly in cells (79, 86).
[0082] For recombinant production, a polynucleotide sequence
encoding the antibody protein is inserted into an appropriate
expression vehicle, such as a vector which contains the necessary
elements for the transcription and translation of the inserted
coding sequence. The expression vehicle is then transfected into a
suitable target cell which will express the peptide. Transfection
techniques known in the art include, but are not limited to,
calcium phosphate precipitation (87) and electroporation (88). A
variety of host-expression vector systems may be utilized to
express the antibody proteins described herein preferably including
eukaryotic cells.
[0083] The presently disclosed and claimed inventive concepts
further provide isolated nucleic acids encoding the antibodies
disclosed or otherwise enabled herein. The nucleic acids may
comprise DNA or RNA and may be wholly or partially synthetic or
recombinant. Reference to a nucleotide sequence as set out herein
encompasses a DNA molecule with the specified sequence, and
encompasses a RNA molecule with the specified sequence in which U
is substituted for T, unless context requires otherwise.
[0084] In another embodiment, the nucleic acid molecules which
encode the antibodies of the presently disclosed and claimed
inventive concepts also comprise nucleotide sequences that are, for
example, at least 50% identical to the sequences disclosed herein.
Also contemplated are embodiments in which a sequence is at least
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a sequence
disclosed herein and/or which hybridize to a sequence of the
presently disclosed and claimed inventive concepts under conditions
of high or moderate stringency. The percent identity may be
determined by visual inspection and mathematical calculation.
[0085] Stringency, including "high stringency," as used herein,
includes conditions readily determined by the skilled artisan based
on, for example, the length of the DNA. Generally, such conditions
are defined as hybridization conditions of 50% formamide,
6.times.SSC at 42.degree. C. (or other similar hybridization
solution, such as, e.g., Stark's solution, in 50% formamide at
42.degree. C.), and with washing at approximately 68.degree. C.,
0.2.times.SSC, 0.1% SDS. The skilled artisan will recognize that
the temperature and wash solution salt concentration can be
adjusted as necessary according to factors such as the length of
the probe.
[0086] "Moderate stringency," as used herein, includes conditions
that can be readily determined by those having ordinary skill in
the art based on, for example, the length of the DNA. The basic
conditions are set forth by Sambrook et al. (79) and include use of
a prewashing solution for the nitrocellulose filters 5.times.SSC,
0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50%
formamide, 6.times.SSC at 42.degree. C. (or other similar
hybridization solution, such as Stark's solution, in 50% formamide
at 42.degree. C.), and washing conditions of 60.degree. C.,
0.5.times.SSC, 0.1% SDS.
[0087] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, generally being directed against a
single epitopic site. Furthermore, in contrast to conventional
polyclonal antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant. In
addition to their specificity, the monoclonal antibodies are
advantageous in that, in one embodiment, they are synthesized by
the hybridoma culture, uncontaminated by other immunoglobulins. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method.
[0088] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567).
[0089] Methods of making antibody fragments are also known in the
art (89) (incorporated herein by reference). Antibody fragments of
the present invention can be prepared by proteolytic hydrolysis of
the antibody or by expression in E. coli of DNA encoding the
fragment. Antibody fragments, as noted above, can be obtained by
pepsin or papain digestion of whole antibodies conventional
methods. For example, antibody fragments can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 5S
fragment denoted F(ab').sub.2. This fragment can be further cleaved
using a thiol reducing agent, and optionally a blocking group for
the sulfhydryl groups resulting from cleavage of disulfide
linkages, to produce 3.5S Fab' monovalent fragments. Alternatively,
an enzymatic cleavage using pepsin produces two monovalent Fab'
fragments and an Fc fragment directly. These methods are described,
for example, in U.S. Pat. No. 4,036,945 and U.S. Pat. No.
4,331,647, and references contained therein, which are hereby
expressly incorporated in their entireties by reference.
[0090] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the conformational epitope that is recognized by the intact
antibody. For example, Fv fragments comprise an association of VH
and VL chains. This association may be noncovalent or the variable
chains can be linked by an intermolecular disulfide bond or
cross-linked by chemicals such as glutaraldehyde. Preferably, the
Fv fragments comprise VH and VL chains connected by a peptide
linker. These single-chain antigen binding proteins (sFv) are
prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Another form of an
antibody fragment is a peptide coding for a single CDR. CDR
peptides ("minimal recognition units") are often involved in
antigen recognition and binding. CDR peptides can be obtained by
cloning or constructing genes encoding the CDR of an antibody of
interest. Such genes are prepared, for example, by using the
polymerase chain reaction to synthesize the variable region from
RNA of antibody-producing cells.
[0091] The presently disclosed and claimed inventive concepts
comprise engineered antibodies including fully human and humanized
forms of non-human (e.g., primate or murine) antibodies. Such
humanized antibodies are chimeric immunoglobulins, immunoglobulin
chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or
other antigen-binding subsequences of antibodies) that contain
minimal sequences derived from non-human immunoglobulin. For the
most part, humanized antibodies are human immunoglobulins in which
residues from a CDR of the recipient are replaced by residues from
a CDR of a nonhuman species such as mouse, rat or rabbit having the
desired specificity, affinity and capacity.
[0092] In making an engineered antibody, a DNA sequence encoding an
antibody molecule of the presently disclosed and claimed inventive
concepts is prepared synthetically by established standard methods.
For example, according to the phosphoamidine method,
oligonucleotides are synthesized, e.g. in an automatic DNA
synthesizer, purified, annealed, ligated and cloned in suitable
vectors.
[0093] The DNA sequence may then be inserted into a recombinant
expression vector, which may be any vector, which may conveniently
be subjected to recombinant DNA procedures. The choice of vector
will often depend on the host cell into which it is to be
introduced. Thus, the vector may be an autonomously replicating
vector, i.e., a vector that exists as an extrachromosomal entity,
the replication of which is independent of chromosomal replication,
e.g., a plasmid. Alternatively, the vector may be one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0094] In the vector, the DNA sequence encoding the protein should
be operably connected to a suitable promoter sequence. The promoter
may be any DNA sequence, which shows transcriptional activity in
the host cell of choice and may be derived from genes encoding
proteins either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of
the coding DNA sequence in mammalian cells include, but are not
limited to, the LTR promoter, SV 40 promoter, the MT-1
(metallothionein gene) promoter or the adenovirus 2 major late
promoter. A suitable promoter for use in insect cells is the
polyhedrin promoter. Suitable promoters for use in yeast host cells
include promoters from yeast glycolytic genes or alcohol
dehydrogenase genes or the TPI1 or ADH2-4c promoters. Suitable
promoters for use in filamentous fungus host cells are, for
instance, the ADH3 promoter or the tpiA promoter.
[0095] The DNA coding sequence may also be operably connected to a
suitable terminator, such as the human growth hormone terminator or
(for fungal hosts) the TPI1 or ADH3 promoters. The vector may
further comprise elements such as polyadenylation signals (e.g.
from SV 40 or title adenovirus 5 E1b region), transcriptional
enhancer sequences (e.g. the SV 40 enhancer) and translational
enhancer sequences (e.g., ones encoding adenovirus VA RNAs).
[0096] The recombinant expression vector may further comprise a DNA
sequence enabling the vector to replicate in the host cell in
question. An example of such a sequence (when the host cell is a
mammalian cell) is the SV 40 origin of replication. The vector may
also comprise a selectable marker, e.g. a gene the product of which
complements a defect in the host cell, such as the gene coding for
dihydrofolate reductase (DHFR) or one which confers resistance to a
drug, e.g. neomycin, hydromycin or methotrexate.
[0097] The procedures used to ligate the DNA sequences coding the
proteins, the promoter and the terminator, respectively, and to
insert them into suitable vectors containing the information
necessary for replication, are well known to persons skilled in the
art.
[0098] To obtain recombinant proteins of the presently disclosed
and claimed inventive concepts the coding DNA sequences may be
usefully fused with a second peptide coding sequence and a protease
cleavage site coding sequence, giving a DNA construct encoding the
fusion protein, wherein the protease cleavage site coding sequence
positioned between the HBP fragment and second peptide coding DNA,
inserted into a recombinant expression vector, and expressed in
recombinant host cells. In one embodiment, said second peptide
selected from, but not limited by the group comprising
glutathion-S-reductase, calf thymosin, bacterial thioredoxin or
human ubiquitin natural or synthetic variants, or peptides thereof.
In another embodiment, a peptide sequence comprising a protease
cleavage site may be the Factor Xa, with the amino acid sequence
IEGR, enterokinase, with the amino acid sequence DDDDK, thrombin,
with the amino acid sequence LVPR/GS, or Acharombacter lyticus,
with the amino acid sequence XKX, cleavage site.
[0099] The host cell into which the expression vector is introduced
may be any cell which is capable of expression of the peptides or
full-length proteins, and is preferably a eukaryotic cell, such as
invertebrate (insect) cells or vertebrate cells, e.g. Xenopus
laevis oocytes or mammalian cells, in particular insect and
mammalian cells. Examples of suitable mammalian cell lines include,
but are not limited to, the HEk293 (ATCC CRL-1573), COS (ATCC
CRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL-61)
cell lines. Methods of transfecting mammalian cells and expressing
DNA sequences introduced in the cells are well known in the
art.
[0100] Alternatively, fungal cells (including yeast cells) may be
used as host cells. Examples of suitable yeast cells include cells
of Saccharomyces spp. or Schizosaccharomyces spp., in particular
strains of Saccharomyces cerevisiae. Examples of other fungal cells
are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora
spp., in particular strains of Aspergillus oryzae or Aspergillus
niger. The use of Aspergillus spp. for the expression of proteins
is described in, e.g., EP 238 023.
[0101] The medium used to culture the cells may be any conventional
medium suitable for growing mammalian cells, such as a
serum-containing or serum-free medium containing appropriate
supplements, or a suitable medium for growing insect, yeast or
fungal cells. Suitable media are available from commercial
suppliers or may be prepared according to published recipes.
[0102] The proteins recombinantly produced by the cells may then be
recovered from the culture medium by conventional procedures
including separating the host cells from the medium by
centrifugation or filtration, precipitating the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g.
ammonium sulphate, purification by a variety of chromatographic
procedures, e.g. HPLC, ion exchange chromatography, affinity
chromatography, or the like.
[0103] The antibodies of the present invention preferably include
one or more modifications which inactivate complement. The term
"complement activity" broadly encompasses the biochemical and
physiological activities associated with activation of the
complement system, individual complement pathway associated
molecules, as well as genes encoding these molecules. Therefore,
complement activities include, e.g., structure and expression of a
gene encoding a complement pathway molecule, biochemical activity
(e.g., enzymatic or regulatory) of a complement pathway molecule,
cellular activities that initiate or result from activation of the
complement system, and presence of serum autoantibodies against
complement pathway molecules. In the hSel001 antibody the preferred
modification to inactivate complement is a replacement of a lysine
residue with alanine at position 342 in the heavy chain constant
region CH2. Other substitutions at the same position may include
for example any of gly, leu, trp, tyr, pro, thr, ser, met, asp,
asn, glu, gin, phe, ile, val, thr, and cys with the proviso that
the substitution is also effective in eliminating the ability of
the constant region to activate complement.
[0104] The terms "complement pathway associated molecules,"
"complement pathway molecules," and "complement pathway associated
proteins" are used interchangeably and refer to the various
molecules that play a role in complement activation and the
downstream cellular activities mediated by, responsive to, or
triggered by the activated complement system. They include
initiators of complement pathways (i.e., molecules that directly or
indirectly triggers the activation of complement system), molecules
that are produced or play a role during complement activation
(e.g., complement proteins/enzymes such as C3, C5, C5b-9, Factor B,
MASP-1, and MASP-2), complement receptors or inhibitors (e.g.,
clusterin, vitronectin, CR1, or CD59), and molecules regulated or
triggered by the activated complement system (e.g., membrane attack
complex-inhibitory factor, MACIF). Thus, in addition to complement
proteins noted above, complement pathway associated molecules also
include, e.g., C3/C5 convertase regulators (RCA) such as complement
receptor type 1 (also termed CR1 or CD35), complement receptor type
2 (also termed CR2 or CD21), membrane cofactor protein (MCP or
CD46), and C4bBP; MAC regulators such as vitronectin, clusterin
(also termed "SP40,40"), CRP, CD59, and homologous restriction
factor (HRF); immunoglobulin chains such as Ig kappa, Ig lambda, or
Ig gamma; C1 inhibitor; and other proteins such as CR3, CR4
(CD11b/18), and DAF (CD55).
[0105] In an alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-P-selectin antibody can be identified
and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with the conformational epitopes described herein respectively to
thereby isolate immunoglobulin library members that bind P-selectin
in accordance with the present invention. Kits for generating and
screening phage display libraries are commercially available (e.g.,
the Pharmacia Recombinant Phage Antibody System, Catalog No.
27-9400-01; and the Stratagene SurJZAP.TM.. Phage Display Kit,
Catalog No. 240612).
[0106] P-selectin mediates interaction of activated platelets or
endothelial cells with blood cells including certain red blood
cells (i.e. sickled red cells) and leukocytes including monocytes,
neutrophils, eosinophils, CD4.sup.+T cells, CD8.sup.+T cells, B
cells and natural killer (NK) cells. As noted herein, it is known
that P-selectin is involved in a number of cellular responses to
inflammation resulting from injury, infection, or physicochemical
assaults. Atherosclerosis, characterized by atherosclerotic lesions
on the inner surfaces of blood vessels, is one example of a
condition involving the binding of certain leukocytes to
P-selectin-bearing endothelial cells on the inner lining of blood
vessel walls.
[0107] As indicated above, therapies directed to blocking
P-selectin function, for example, using antibodies to P-selectin to
prevent the tethering and rolling of leukocytes and adherence of
red blood cells (i.e. sickled red cells), could have a profound
effect on numerous types of inflammatory and thrombotic diseases.
Given its pivotal roll in the initiation of rolling and tethering
of leukocytes to the endothelium and platelets, P-selectin is a
primary target for therapeutic development to treat inflammatory
and thrombotic disorders. For example, the transient nature of the
acute phase of sickle cell anemia coupled with the recurrent
chronic effects of organ damage and associated complications and
morbidity suggests that a therapeutic intervention that exhibits
both blocking initial adhesion due to binding of P-selectin and
PSGL-1, and inducing dissociation of prior, ongoing or
preestablished adhesion, would have novel application to this and
other inflammatory and thrombotic diseases. The present invention
thus encompasses, in one embodiment, a method of using a
conformational antibody binding epitope of P-selectin to screen for
and identify "single function"-blocking antibodies to P-selectin
which block P-selectin/PSGL-1 binding, and "dual function"
antibodies to P-selectin which not only block P-selectin-PSGL-1
binding, but which also cause dissociation of preformed
P-selectin/PSGL-1 complex (and thus cell-cell complex), and the use
of such antibodies for therapeutic treatment of diseases, such as,
but not limited to, inflammatory and thrombotic diseases.
[0108] As noted above, novel conformational binding epitopes of
P-selectin have been discovered. These binding epitopes have
enabled the identification herein of antibodies to human P-selectin
which block the binding of PSGL-1 to P-selectin and thus block the
function of P-selectin (thus the antibodies may be referred to
herein as "function-blocking" antibodies). The discovery of these
conformational epitopes have further led to the discovery of dual
function anti-P-selectin antibodies which bind with high
specificity to the conformational epitope and which not only block
the binding of P-selectin and PSGL-1, but which also induce the
dissociation of preformed P-selectin/PSGL-1 complexes (i.e., the
reversal of P-selectin-PSGL-1 binding) thereby causing the
dissociation of cell complexes such as leukocyte/endothelial cell,
leukocyte/platelet, lymphocyte/endothelial cell,
lymphocyte/platelet, sickled red cell/endothelial cell or sickled
red cell/platelet complexes.
[0109] The binding regions for some antibodies to P-selectin have
been previously mapped using constructs of large functional domains
encompassing the lectin, epidermal growth factor (EGF) and
consensus repeat (CR) regions of the native protein P-selectin in
mouse and human (90, 70, 91-95). These results indicated the
primary binding areas for some function-blocking antibodies to
P-selectin were in the lectin binding domain, a region that spans
amino acid residues 1-120 of the native P-selectin protein, or in
the EGF domain spanning amino acids 121-154.
[0110] The present disclosure describes the discovery of novel
nonlinear (e.g., conformational) epitopes by using mouse/human
chimeras containing the lectin, EGF, CR1 and CR2 domains of
P-selectin that were probed with function-blocking test antibodies
to P-selectin. Previous work has shown that, at a minimum,
expression of the lectin and EGF domains are required for proper
folding and conformation of P-selectin constructs (91). A
comparison of the amino acid sequences of human and mouse
P-selectin indicated that there is homology in the lectin domain
with a specific number of amino acid residue differences between
human and mouse. Herein, three-dimensional (3-D) homology was used
to compare the human and mouse lectin, EGF, CR1 and CR2 domains of
P-selectin (FIG. 1) to identify amino acid differences between
human and mouse P-selectins (sequences and numbering according to
the mature proteins).
[0111] Where used herein the term "mouse amino acid" refers to an
amino acid residue which is found in mouse P-selectin but is not
found in the corresponding position in human P-selectin. Where used
herein the term "human amino acid" refers to an amino acid residue
which is found in human P-selectin but is not found in the
corresponding position in mouse P-selectin.
[0112] The method used 3-D modeling of P-selectin to compare the
positions of amino acid differences between human and mouse
P-selectin on the exposed surface of the protein and to identify
clusters of amino acid differences between human and mouse in the
lectin and EGF domains which are located on the surface of the
folded protein. This 3-D method represents clusters of amino acid
differences which result from juxtaposition of discontinuous amino
acids brought into proximity to one another by folding of the
protein. For example, some amino acids will form conformational
epitopes by virtue of being on the same surface, e.g. face, of
helical structures. Homology comparison of such clusters allowed
for selection of amino acid substitutions to test the effect of
such changes on the binding of function-blocking (PSGL-1 blocking)
antibodies to human P-selectin.
[0113] The method further involved mapping of conserved restriction
sites in the open reading frames of the cDNA to identify a strategy
for constructing chimeric proteins that span the lectin, EGF, CR1
and CR2 domains and would enable substitution of single or multiple
amino acids at specific sites in the human or mouse P-selectin to
identify those amino acids which optimize antibody binding to human
P-selectin. Chimeras were constructed with known molecular cloning
techniques, using human and mouse P-selectin N-terminal regions
spanning the ATG through CR2 domain with a suitable vector such as
pBluescript (pBS-hPsel and pBS-mPsel). The chimeras were inserted
into another suitable vector such as pIG1 (pIG-hPsel and pIG-mPsel)
where the constructs were fused to the Fc region of human IgG1
containing the hinge, CH2 and CH3 region. These constructs
preserved structures that are consistent with the native
conformation of P-selectin. Thus domains that were exposed on the
surface of the native protein were also present on the chimera
constructs and thus served as putative epitopes for binding of test
antibodies. Such constructs could be transfected and transiently
expressed using molecular and cell expression techniques known to
persons having ordinary skill in the art.
[0114] Using this method, test antibodies, can be evaluated for
binding to the human/mouse chimeras using fluorescence-activated
cell sorting (FACS) and surface plasmon resonance (BIACORE) methods
known to persons having ordinary skill in the art. The effects of
changes in amino acids in various positions in the chimera
constructs by substitution of mouse amino acids, for example, into
the human sequence, conversely, or human amino acids into the mouse
sequence, for example, that abrogated or enabled binding of
antibodies directed to human P-selectin were evaluated and thus
enabled identification of particular amino acids for optimal
binding.
[0115] Characterization of Chimera Constructs
[0116] Amino acids of mouse P-selectin which have been substituted
into the human P-selectin sequence are indicated in boldface in the
chimeras described below. Amino acids of human P-selectin which
have been substituted into the mouse sequence are indicated in
italicized boldface. Substitution of glutamine for histidine is
indicated as underlined boldface.
Native Protein Constructs
TABLE-US-00001 [0117] Human P-selectin lectin, EGF, CR1, CR2
domains SEQ ID NO: 1 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Mouse P-selectin lectin,
EGF, CR1, CR2 domains-Amino acid differences from human in
boldface. SEQ ID NO: 2 42
WTYNYSTKAYSWNNSRVFCRRHFTDLVAIQNKNEIAHLNDVIPFFNSYYWIGIRKINNKW 102
TWVGTNKTLTEEAENWADNEPNNKKNNQDCVEIYIKSNSAPGKWNDEPCFKRKRALCYTA 162
SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 222
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 282
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Human/Mouse Chimera
Constructs Chimera-1 (mouse substitutions in human cluster
A-N.sub.4N.sub.14V.sub.17F.sub.18R.sub.20R.sub.21H.sub.22F.sub.23)
SEQ ID NO: 3 1
WTYNYSTKAYSWNNSRVFCRRHFTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-2 (human cluster
A to I.sub.35-mouse thereafter) SEQ ID NO: 4 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIAHLNDVIPFFNSYYWIGIRKINNKW 61
TWVGTNKTLTEEAENWADNEPNNKKNNQDCVEIYIKSNSAPGKWNDEPCFKRKRALCYTA 121
SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Chimera-3 (substitutions
in human cluster A-to mouse N.sub.4N.sub.14V.sub.17F.sub.18) SEQ ID
NO: 5 1
WTYNYSTKAYSWNNSRVFCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-4 (substitutions
in human cluster A-to mouse R.sub.20R.sub.21H.sub.22F.sub.23) SEQ
ID NO: 6 1
WTYHYSTKAYSWNISRKYCRRHFTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-5 (single amino
acid change-human H.sub.4 to mouse N.sub.4) SEQ ID NO: 7 1
WTYNYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-5Q (substitution
of Q for H.sub.4 in cluster A-removes putative glycosylation site)
SEQ ID NO: 8 1
WTYQYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-6 (human
sequence to EGF-S.sub.121-mouse EGF, CR1 and CR2) SEQ ID NO: 9 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Chimera-7 (human
sequence to G.sub.177-mouse thereafter) SEQ ID NO: 10 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Chimera-7B (human
sequence to end of EGF-V.sub.156-mouse thereafter) SEQ ID NO: 11 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Chimera-8 single amino
acid change-human I.sub.14 to mouse N.sub.14) SEQ ID NO: 12 1
WTYNYSTKAYSWNNSRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-9 (single amino
acid change-human K.sub.17 to mouse V.sub.17) SEQ ID NO: 13 1
WTYHYSTKAYSWNISRVYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-10 (single amino
acid change-human Y.sub.18 to mouse F.sub.18) SEQ ID NO: 14 1
WTYHYSTKAYSWNISRKFCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-11 (single amino
acid change-human Q.sub.20 to mouse R.sub.20) SEQ ID NO: 15 1
WTYHYSTKAYSWNISRKYCRNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-12 (single amino
acid change-human N.sub.21 to mouse R.sub.21) SEQ ID NO: 16 1
WTYHYSTKAYSWNISRKYCQRRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-13 (single amino
acid change-human R.sub.22 to mouse H.sub.22) SEQ ID NO: 17 1
WTYHYSTKAYSWNISRKYCQNHYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-14 (single amino
acid change-human Y.sub.23 to mouse F.sub.23) SEQ ID NO: 18 1
WTYHYSTKAYSWNISRKYCQNRFTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-15 (human
sequence to cluster C2-S.sub.97-mouse thereafter) SEQ ID NO: 19 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSNSAPGKWNDEPCFKRKRALCYTA 121
SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA
Chimera-16 (substitution of human .sub.4 .sub.14 .sub.17 .sub.21
.sub.22 into mouse Cluster A) SEQ ID NO: 20 1 WTY YSTKAYSWN SR FCR
FTDLVAIQNKNEIAHLNDVIPFFNSYYWIGIRKINNKW 61
TWVGTNKTLTEEAENWADNEPNNKKNNQDCVEIYIKSNSAPGKWNDEPCFKRKRALCYTA 121
SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Chimera-17 (human
sequence to Cluster B to I.sub.35-mouse Cluster B to I.sub.42-human
to CR1 to E.sub.154-mouse CR1, CR2) SEQ ID NO: 21 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIAHLNDVIPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Chimera-17B (human
sequence to Cluster B to I.sub.35-mouse Cluster B to I.sub.42-human
thereafter) SEQ ID NO: 22 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIAHLNDVIPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-18 (human
sequence with mouse cluster C (Cl, C2, C3) and mouse CR1, CR2) SEQ
ID NO: 23 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPFFNSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSNSAPGKWNDEHCLKKKRALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242
FAYDSSKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Chimera-18B (human
sequence with mouse cluster C (Cl, C2, C3) SEQ ID NO: 24 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPFFNSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSNSAPGKWNDEHCLKKKRALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-19 (human
sequence with mouse Cluster D and mouse CR1, CR2) SEQ ID NO: 25 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKINNKW 61
TWVGTNKTLTEEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Chimera-19B (human
sequence with mouse Cluster D) SEQ ID NO: 26 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKINNKW 61
TWVGTNKTLTEEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-20 (human
sequence with mouse Cluster E and mouse CR1, CR2) SEQ ID NO: 27 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEPCFKRKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181
FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242
FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA Chimera-20B (human
sequence with mouse Cluster E) SEQ ID NO: 28 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEPCFKRKHALCYTA 121
SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK--- Chimera-21 (human
sequence with mouse Cluster F) SEQ ID NO: 29 1
WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW 61
TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121
SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181
FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242
FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK---
[0118] FACS Analysis of Anti-P-Selectin Antibodies to Human/Mouse
Chimeras
[0119] Antibody binding to human/mouse chimeras of P-selectin was
analyzed using FACS analysis on a system such as a BD BIOSCIENCES
FACS ARIA CELL SORTER to measure binding of anti-P-selectin
antibodies to human/mouse chimeras which were coupled to beads
coated with a goat anti-human Fc antibody. Such beads coated with
chimeras were incubated with test anti-P-selectin antibodies that
were then detected with anti-mouse Fc or isotype specific
antibodies labeled with reporters, such as FITC, suitable for
detection by the FACS system.
[0120] One-Step Surface Plasmon Resonance (BIACORE)
[0121] In one aspect of the presently disclosed and claimed
inventive concepts, BIACORE chips were used to capture a test
anti-P-selectin antibody. Human-mouse P-selectin chimeras described
herein were disposed onto the chip and test antibodies were added
to the prebound chip. Binding of the chimeras to test antibodies
was measured by resonance response units.
[0122] Two-Step Surface Plasmon Resonance (BIACORE) Analysis
[0123] In another aspect of the presently disclosed and claimed
inventive concepts, a capture chip, such as a BIACORE chip was
provided with a goat anti-human IgG Fc polyclonal antibody
covalently attached to its surface. P-selectin chimeric human/mouse
constructs of the lectin, EGF, CR1 and CR2 domain on a human IgG Fc
were injected onto the chip and captured at concentrations that
achieve a standardized level of surface coating as measured by the
resonance response. The resonance response level achieved after
loading each P-selectin chimera construct was designated as a new
"secondary baseline" level. Test anti-P-selectin antibodies (e.g.,
mouse monoclonal anti-P-selectin antibodies G1, G3 and G5) were
then injected onto the BIACORE chip and incubated for binding to
the P-selectin chimera construct already captured on the surface.
The method could be modified to test humanized antibodies by
creating P-selectin constructs on mouse IgG Fc and capturing with a
goat anti-mouse IgG Fc polyclonal antibody and then probing with
test humanized anti-P-selectin antibodies. Antibodies which bind to
the P-selectin constructs cause an increase of the resonance
response level from the secondary baseline. The resulting increase
in resonance response may be measured as "added resonance units
(RUs)" and thus indicate the level of binding to the P-selectin
construct pre-coated onto the capture chip of the test antibody.
Using these methods, optimal requirements for the binding of
anti-P-selectin antibodies to P-selectin chimeras were precisely
mapped to particular conformational epitopes.
[0124] Identification of Dual Function Anti-P-Selectin Antibodies
(Antibodies that Both Block and Dissociate Binding of P-Selectin to
PSGL-1)
[0125] BIACORE analysis was also used to discover dual
functionality of specific anti-P-selectin antibodies, i.e., as
discussed above, they can both block and dissociate (reverse)
binding interactions between P-selectin and PSGL-1. In this method,
PSGL-1, or small molecule mimetics of the binding epitope of PSGL-1
such as a biotinylated glycosulfopeptide mimetic (e.g., GSP-6), or
chimeric proteins containing the N-terminus of PSGL-1, are captured
on a BIACORE chip, such as a streptavidin chip, using methods known
to persons having ordinary skill in the art (GSP-6 is a
glycosylated, sulfated 18 amino acid peptide mimetic of amino acids
42-60 of the exposed N-terminus of PSGL-1 described in detail in
U.S. Pat. No. 6,593,459, for example). First, to demonstrate
"function-blocking" ability, in one embodiment, an anti-P-selectin
antibody is pre-mixed with soluble P-selectin and incubated for a
period to allow formation of the P-selectin/antibody complex. The
resulting anti-P-selectin antibody/P-selectin complex is introduced
onto the chip bearing the PSGL-1 (or PSGL-1 mimetic) and binding to
the PSGL-1 or its mimetic is measured. Anti-P-selectin antibodies,
which prevent binding of P-selectin to the PSGL-1 or PSGL-1 mimetic
on the chip, are designated as function-blocking antibodies.
[0126] Second, anti-P-selectin antibodies which have been shown (by
the above-method or another similar method) to be function-blocking
antibodies (i.e., which block PSGL-1 binding to P-selectin), can be
tested for an additional function, that is, having the ability to
dissociate (reverse) binding between preformed P-selectin PSGL-1
complex. Such antibodies can be tested for "dual function"
properties using the method of BIACORE analysis discussed herein.
In one embodiment, to demonstrate the dual function property,
PSGL-1, or a mimetic thereof such as GSP-6, is coupled to a BIACORE
chip. P-selectin is then disposed on the chip and allowed to bind
to the PSGL-1, or the mimetic. After equilibrium binding of
P-selectin to PSGL-1, or the mimetic, is indicated by the
sensogram, function-blocking anti-P-selectin antibodies are
introduced and the dissociation of P-selectin binding to PSGL-1, or
the mimetic is measured by any appropriate method. Such antibodies
that are shown to dissociate (i.e., reverse), P-selectin/PSGL-1
binding are designated as "dissociating antibodies" and are
characterized as dual function antibodies, i.e., they possess both
function-blocking and dissociating properties in disrupting binding
of P-selectin to PSGL-1. Such dual function antibodies are a
particularly preferred embodiment of the invention as they are
especially suitable for therapeutic application as treatments of
acute and chronic inflammatory and thrombotic diseases such as are
described elsewhere herein.
[0127] Discovery of Conformational Epitopes
[0128] The three-dimensional (3-D) structure of the mature human
and mouse P-selectin proteins were analyzed and compared as to
amino acid differences in the lectin and EGF domains. Six clusters
of conformational amino acid differences were identified on exposed
surfaces of the proteins. These were designated as clusters A, B,
C, D, E and F (FIG. 1). The N-termini of human and mouse
P-selectins spanning residues 1-35 contain 8 amino acid
differences. Cluster A was arbitrarily defined by the boundary of
the first amino acid difference (H.sub.4) and the last amino acid
difference (Y.sub.23). Cluster A contains 20 amino acids and forms
a rigid alpha helix with a cysteine bond near the N-terminus of the
protein (see region "1" in FIG. 5). Cluster B (FIG. 1) is a
conformational epitope spanning amino acid residues 36-42 and
contains 4 amino acid differences between human and mouse
P-selectin. Where used herein, the term "conformational epitope" is
intended to refer to an epitope which is not recognized under
reducing conditions. Clusters C and E (FIG. 1) are conformational
and discontinuous and are brought into proximity by folding of the
native P-selectin protein. Cluster C has three conformational
regions (C1, C2, C3) containing 5 amino acid differences between
human and mouse P-selectin. Cluster C1 is separated from C2 by 51
amino acids and cluster C2 is separated from C3 by 15 amino acids.
Likewise cluster E has two conformational epitopes (E1, E2)
containing five amino acid differences between human and mouse
P-selectin with cluster E1 being separated from E2 by 19 amino
acids. Clusters A, B, C, D and E lie within the consensus lectin
domain of P-selectin (FIG. 1). Cluster F resides in the EGF domain
and has 3 amino acid differences out of 11 amino acids. Clusters
C1, E1, C2, E2 and C3 encompass key contact residues which have
previously been identified for interaction of P-selectin with its
physiological ligand PSGL-1 (Somers et al). Clusters A and B are
distal to (upstream of) these contact residues.
[0129] The open reading frames of cDNAs for human and mouse
P-selectin were analyzed to identify common restriction sites that
could be used to assemble chimeras spanning the clusters. PCR and
chemical DNA synthesis was used to generate cDNAs coding for
specific protein or chimera constructs such as SEQ ID NO: 1-29
(described above, and in the Sequence Listing). Restriction cloning
was used to construct plasmids coding for the human/mouse chimeras.
The chimeras were transiently expressed in COS-7 cells and utilized
for FACs and BIACORE analysis. P-selectin chimeras were tested for
binding function using BIACORE by analyzing their binding to GSP-6
bound to a BIACORE chip. As noted above, GSP-6 is a small molecule
that mimics the N-terminus of human PSGL-1 (96). All tested
chimeras bound to the GSP-6 on the chip, though to varying degrees,
as mouse P-selectin has a lower binding affinity to human PSGL-1
than does human P-selectin. This indicated that chimeras had
maintained function after expression and purification.
[0130] FACS Analysis
[0131] The results of FACs analysis of anti-P-selectin antibodies,
using the constructs or chimeras corresponding to SEQ ID NOs.:1-29
are summarized in Table 1 (below). Three anti-P-selectin test
antibodies (G1, G3 and G5) were isolated from hybridomas generated
by immunization of mice with a human recombinant P-selectin
containing the lectin and EGF domains (90). Previous studies had
shown that these antibodies were specific to human P-selectin and
that G1 and G3 are function-blocking antibodies and G5 is
non-blocking (90, and unpublished data). G1 and G3 were determined
herein to bind to human P-selectin (SEQ ID NO:1) but did not bind
to mouse P-selectin (SEQ ID NO:2). When the corresponding eight
mouse amino acids were substituted in cluster A of the human
sequence (Chimera 1, SEQ ID NO:3), binding by G1 antibody was
abolished, indicating that at least one or more of the
corresponding eight amino acid positions in cluster A was essential
for binding of the G1 antibody to P-selectin and that the
substitution with the "mouse" amino acids in those one or more
positions abolished the binding. To further evaluate the binding
specificity of the G1 test antibody, the eight different human
amino acids in positions 1-23 were substituted in cluster A of the
mouse sequence (SEQ ID NO:4, chimera-2) and G1 binding was
achieved. Chimeras containing the human P-selectin lectin domain
with mouse amino acid substitutions in the EGF, CR1 and CR2 domains
(SEQ ID NO:9, chimera-6), and human sequence through the EGF domain
with mouse CR1 and CR2 domains (SEQ ID NO:11, chimera-7B), were
bound by G1 and G3 indicating that the primary binding epitopes
remained intact after substitution of these mouse amino acids and
that the conformation of the antibody binding epitopes were not
adversely affected.
[0132] Using these methods, the test antibody G3 was shown to bind
human P-selectin, did not bind mouse P-selectin and in contrast to
G1, bound to SEQ ID NO:3 (chimera-1), indicating that G3 binds to
an epitope distinct from the epitope bound by G1. Specifics of the
G3 epitope mapping are outlined in Table 1 below.
[0133] The test antibody G5, previously shown to be non-blocking,
was also analyzed using this method. G5 was shown to be specific
for human P-selectin, did not bind mouse P-selectin, and was
confirmed as a non-blocking antibody. G5 bound to SEQ ID NO:1
(human P-selectin), SEQ ID NO:3, and SEQ ID NO:10 that spans the
EGF domain and includes the first part of CR1 to N.sub.178, but G5
did not bind to SEQ ID NO:2 (mouse P-selectin) or to SEQ ID NO:9
that spans to S.sub.128, or to SEQ ID NO:11 that spans to the start
of CR1 at V155. These results indicate that antibody G5 binds to
the first part of CR1 and requires at least amino acids R.sub.157
through N.sub.178.
TABLE-US-00002 TABLE 1 Results of binding of various antibodies
(G1, G3, G4, G5, hSel001) to human and mouse P-selectin and chimera
constructs thereof. Human/Mouse Chimeras Mouse Domains Inserted in
Human SeqID P-selectin Human Mouse G1 G3 G5 G4 hSel001 1 None 1-279
0 +.sup.1,2,3 +.sup.1,2,3 +.sup.1,3 +.sup.3 +.sup.2 2 all 0 1-282
-.sup.1,2,3 -.sup.1,2,3 -.sup.1,3 -.sup.3 -.sup.2 3
A.sub.4,14,17,18,20,21,22,23 1-3, 24-279 4-23 -.sup.1,2,3 +.sup.1,3
+.sup.1 -.sup.3 4 B, C, D, E, F 1-35 36-282 +.sup.1,2,3 -.sup.3
+.sup.3 5 A.sub.4,14,17,18 19-279 1-18 -.sup.2 6 A.sub.20,23 1-19,
24-279 20-23 -.sup.2 7 A.sub.4 1-3, 5-279 4 +.sup.2w,3w +.sup.3
+.sup.3w 8 none 1-3, 5-279 0 +.sup.2,3 +.sup.3 +.sup.3 9 F, CR1,
1-128 129-282 +.sup.1,2,3 +.sup.1,3 -.sup.1,3 +.sup.3 CR2 10 CR1,
CR2 1-177 178-282 +.sup.1,2,3 +.sup.1,3 +.sup.1,3 +.sup.3 11 CR1,
CR2 1-156 157-282 +.sup.2 +.sup.2 -.sup.2 12 A.sub.14 1-13, 15-279
14 -.sup.2 13 A.sub.17 1-16, 18-279 17 -.sup.2 14 A.sub.18 1-17,
19-279 18 +.sup.2 15 A.sub.20 1-19, 21-279 20 +.sup.2 16 A.sub.21
1-20, 22-279 21 +.sup.2w 17 A.sub.22 1-21, 23-279 22 -.sup.2 18
A.sub.23 1-22, 24-270 23 +.sup.2 19 C2, E2, C3, 1-97 98-282
+.sup.2,3 -.sup.3 +.sup.3 F, CR1, CR2 20 B, C, D, E, F 1-35 H/M
hybrid 36-282 +.sup.2,3 -.sup.2,3 +.sup.3 +.sup.2 21 B, CR1, CR2
1-35, 43-156 36-42, 157-282 +.sup.3 +.sup.3 -.sup.3 22 B 1-35,
43-279 36-42 +.sup.3 +.sup.3 23 C1, C2, C3, CR1, 1-43, 47-97,
44-46, 98, 114, +.sup.3 -.sup.3 -.sup.3 CR2 99-113, 115-156 157-282
24 C1, C2, C3 1-43, 47-97, 44-46, 98, 114 -.sup.3 +.sup.3 99-113,
115-279 25 D, CR1, CR2 1-55, 72-156 56-71, 157-282 +.sup.3 +.sup.3
26 D 1-55, 72-279 56-71 +.sup.3 +.sup.3 27 E1, E2, CR1, 1-84,
89-107, 85-88, 108-112, +.sup.3 +.sup.3 CR2 113-156 157-282 28 E1,
E2 1-84, 89-107, 85-88, 108-112 +.sup.3 +.sup.3 113-279 29 F 1-128,
140-279 129-139 +.sup.3 +.sup.3 Critical Amino A C First A A Acid
Positions: (4, 14, 17, part of (4, 14, 17, (4, 14, 17, 21, 22) CR1
7, 21, 22) 21, 22) (157-164) .sup.1By FACS, .sup.2By BIACORE
1-step, .sup.3By BIACORE 2-step, .sup.wWeak binding
[0134] One-Step Surface Plasmon Resonance (BIACORE)
[0135] To further investigate the importance of the cluster A
domain (amino acids 4-23) to the binding of G1 antibody to
P-selectin, several chimeric constructs were made in which single
or multiple mouse amino acids were inserted into the human
P-selectin sequence and binding to G1 was analyzed using the
surface plasmon resonance ("one-step" BIACORE) methods disclosed
herein. The one-step BIACORE binding results are presented in Table
1. The G1 test antibody was captured on a BIACORE chip and the
binding of various chimeras was measured as response units. A
representative sensogram showing binding of G1 to constructs of
human P-selectin (SEQ ID NO:1), mouse P-selectin with human cluster
A (SEQ ID NO:4) and mouse P-selectin (SEQ ID NO:2) is shown in FIG.
2. Using this method, it was shown that G1 bound to human
P-selectin and to SEQ ID NO:4 (where human amino acids were
substituted in cluster A of mouse P-selectin), but did not bind to
mouse P-selectin (SEQ ID NO:2), which showed this method to be
consistent with previous results.
[0136] Mouse P-selectin (SEQ ID NO:2) has a putative glycosylation
site (N) at position 4 whereas human P-selectin (SEQ ID NO:1) does
not. To test the importance of this difference, SEQ ID NO:1 was
substituted at position 4 with N (forming SEQ ID NO:7) and Q
(forming SEQ ID NO:8) were made in the human chimera and its effect
on G1 antibody binding measured. Inserting N into human P-selectin
at position 4 diminished G1 binding suggesting that glycosylation
at this site in human P-selectin would interfere with antibody
binding. Substitution of glutamine (Q) at this position did not
prevent G1 binding.
[0137] To further identify amino positions in cluster A that are
optimal or essential for G1 antibody binding, single amino acid
substitutions of mouse sequence amino acids into the human
P-selectin (SEQ ID NO:1) were made, and binding of the resulting
chimeras to G1 was measured using the one-step BIACORE method
disclosed herein. The chimeras tested (and substitution) were: SEQ
ID NO:7 (H.sub.4N); SEQ ID NO:12 (I.sub.14N); SEQ ID NO:13
(K.sub.IN); SEQ ID NO:14 (Y.sub.189; SEQ ID NO:15 (Q.sub.20R); SEQ
ID NO:16 (N.sub.21R); SEQ ID NO:17 (R.sub.22H); and SEQ ID NO:18
(Y.sub.23F). The G1 antibody bound to SEQ ID NO:14, 15 and 18, but
did not bind SEQ ID NO:12, 13, and 17 and only weakly to SEQ ID
NO:7 and SEQ ID NO:16. This result confirmed the results, shown
with SEQ ID NO:20, that amino acid positions 4, 14, 17, 21, and 22
are each individually required positions for G1 binding. In a
preferred embodiment, these amino acids are H.sub.4, I.sub.14,
K.sub.12, N.sub.21 and R.sub.22, respectively.
[0138] Two-Step Surface Plasmon Resonance (BIACORE)
[0139] To assess G1 binding to additional chimeras and the binding
of other anti-P-selectin test antibodies including G3 and G5
(non-blocking), the two-step surface plasmon resonance ("two-step"
BIACORE) assay described herein was used. The results of the
"two-step" assays for test antibodies G1, G3 and G5 are presented
in Table 1, and in FIG. 3. Using this method, none of the test
antibodies investigated bound to mouse P-selectin and all bound to
human P-selectin demonstrating their specificity to human
P-selectin. G1, G3 and G5 test antibodies all bound to SEQ ID NO:11
indicating they all bind to a region spanning the N-terminus
through the lectin and EGF domains of human P-selectin. The G5
non-blocking antibody did not bind to SEQ ID NO:9, but did bind SEQ
ID NO:10 confirming that G5 binds to the CR1 domain. G5 binding
properties were not further evaluated.
[0140] Further analysis using this method showed that G3 did not
bind SEQ ID NO:23, which has mouse amino acids inserted in cluster
C1, C2, C3, CR1, and CR2, nor did it bind SEQ ID NO:24 which has
mouse amino acids in C1, C2, and C3. G3 also did not bind to other
chimeras that had mouse sequence in cluster C, that is SEQ ID NO:19
and SEQ ID NO:20. These results indicate that the blocking test
antibody G3 requires cluster C for binding and demonstrates the
novel finding that conformational clusters of amino acids brought
into proximity by protein folding can serve as binding domains
(conformational epitopes) for anti-P-selectin antibodies. The
method also confirmed that G3 can block binding of PSGL-1 and
P-selectin and thus has function-blocking properties (FIG. 4).
However, the method also showed that G3 did not bind to or
dissociate (reverse) the binding of P-selectin/PSGL-1 complex (FIG.
4) and thus does not have the dual function properties of the
preferred antibodies of the presently disclosed and claimed
inventive concepts.
[0141] Amino acid positions which optimize binding of G1 were
identified by generating a human/mouse hybrid in cluster A. The
hybrid cluster A chimera (SEQ ID NO:20) contains human P-selectin
amino acids at positions 4, 14, 17, 21 and 22 (H, I, K, N, and R,
respectively) and mouse P-selectin amino acids at positions 18, 20,
and 23 (Y, Q, and Y, respectively). As indicated in Table 1, G1
bound SEQ ID NO:20. This result when taken with the previous data
indicates that amino acid positions 4, 14, 17, 21, and 22 comprise
positions which are each required for optimal binding to
P-selectin. These results comprise a novel finding that G1 binds an
epitope in the helix structure of cluster A that is distal to the
lectin-ligand binding domain contact residues previously identified
for P-selectin (71). In a preferred embodiment the amino acids at
positions 4, 14, 17, 21, and 22 are H, I, K, N, and R,
respectively. 3-D analysis of this epitope revealed a rigid helical
structure with the required amino acids occupying sites on the same
face of the helix; thus cluster A is designated as comprising a
conformational epitope (FIG. 5). BIACORE analysis shown in FIG. 4
confirmed that G1 can block and also dissociate the binding of
P-selectin and PSGL-1 and thus this antibody has the dual function
properties of the preferred embodiments of the presently disclosed
and claimed inventive concepts.
[0142] These results indicate that antibodies that bind to a
conformational epitope located within amino acids 1-35, and more
particularly within amino acids 4-23, of SEQ ID NO:1 which is
distal to the lectin-ligand binding domain in human P-selectin,
will have unique dual function properties. Without wanting to be
bound by theory, it is contemplated that the antibodies which bind
to this epitope act by contributing allosteric forces that exert on
the lectin-ligand binding interface to induce a conformational
change that dissociates P-selectin binding to PSGL-1. Thus G1 is
able to bind the distal epitope at cluster A and block and
dissociate the complex by binding and disrupting the molecular
interactions at the lectin-ligand binding site on P-selectin. In
contrast, antibodies such as G3, that bind to an epitope in the
lectin-ligand binding domain of P-selectin can block P-selectin
binding to PSGL-1 by allosteric hindrance, but cannot cause
dissociation of the P-selectin/PSGL-1 complex since the antibody
cannot bind to the conformational epitope of Cluster C when it is
occupied by the ligand. The test antibody G5 bound to the cluster
of P-selectin CR1 and was shown to be non-blocking (FIG. 4).
[0143] In summary, antibodies which bind to P-selectin have been
characterized as having three possible activities in regard to the
interaction of P-selectin, and its ligand, PSGL-1. First,
P-selectin antibodies can bind to P-selectin but not interfere with
the binding of PSGL-1 to P-selectin ("non-blocking"). For example,
as shown herein, the non-blocking antibody G5 binds amino acids
157-164 and requires R.sub.157/E.sub.161, L.sub.162, E.sub.163 and
L.sub.164 of CR1 for binding. Second, P-selectin antibodies can
bind to P-selectin and interfere with the binding of PSGL-1 to
P-selectin ("function-blocking"), but not interfere with a
preformed P-selectin/PSGL-1 complex. For example, the results
described herein also showed that antibody G3 binds to
conformational clusters in a different part of P-selectin that span
the lectin-ligand binding domain. G3 binds cluster C and requires
C1 amino acids Y.sub.44, Y.sub.45, S.sub.46 and C2 amino acid
P.sub.98 and C3 amino acid H.sub.114 and thus requires a
conformational epitope. Third, P-selectin antibodies can bind to
P-selectin and block the binding of PSGL-1 to P-selectin
(function-blocking antibody) and furthermore can cause reversal of
preformed P-selectin/PSGL-1 binding (dissociative binding). Such
antibodies are referred to herein as "dual function" antibodies.
The results disclosed herein demonstrate, for example, that the
test antibody G1 binds a conformational epitope in cluster A, and
that G1 binding had an absolute requirement for a conformational
epitope comprising amino acid positions 4, 14, 17, 21, and 22,
preferably wherein those amino acids are H, I, K, N, and R,
respectively. As discussed elsewhere herein, substitution of the
"human" amino acid (H, I, K, N, and R) at any one of these
positions, respectively, with the corresponding "mouse" amino acid
(N, N, V, R, and H) will result in the abrogation of binding by the
dual function antibodies described and claimed herein.
[0144] In another embodiment of the presently disclosed and claimed
inventive concepts, a previously uncharacterized mouse monoclonal
anti-P-selectin antibody clone designated G4, generated using
standard hybridoma methods, was tested for binding to the
conformational epitope of cluster A, and was tested for dual
function capabilities using the methods described herein. G4 was
tested for binding to human/mouse chimeras SEQ ID NOs.:1-4, 7-10,
19 and 20. G4 was shown to bind SEQ ID NO:20 and had similar
binding specificity as described for G1 (see Table 1 and FIG. 3).
G4 was then shown to block binding of P-selectin to PSGL-1 (FIG. 4)
and also to cause dissociation of preformed P-selectin/PSGL-1
complex, thus characterizing G4 as a dual function P-selectin
antibody which binds an epitope (in cluster A) which is distal to
the lectin-ligand binding domain of P-selectin and blocks and
dissociates binding of P-selectin to PSGL-1. The G1 and G4
antibodies thus both bound to an epitope in cluster A and both
demonstrated dual function properties. This result demonstrates the
use of cluster A or specific binding positions or amino acids
thereof as an epitope able to be used to screen anti-P-selectin
antibodies for dual function capabilities (as well as for
function-blocking activity alone). Such dual function antibodies
possess unique properties for therapeutic applications where
initiation of P-selectin-mediated adhesion and/or ongoing
P-selectin-mediated adhesion in acute or chronic settings may be
treated. Using the methods described herein, other antibodies
having dual function properties (as well as for function-blocking
activity alone) can be identified using the method of screening
using the cluster A epitope or specific positions or amino acids
thereof.
[0145] In another embodiment of the presently disclosed and claimed
inventive concepts, a humanized IgG2 anti-P-selectin antibody
lacking effector function called hSel001 (a humanized P-selectin
binding antibody comprising CDRs of mouse antibody G1 grafted into
human framework regions and previously characterized in U.S. patent
application Ser. No. 12/516,987) was also screened using the
screening method described herein. A summary of the data (Table 1)
shows that hSel001 antibody bound to the same chimeras (SEQ ID
NO:4, 7, 8, 9, 10, 19 and 20) as antibody G1. hSel001 binding was
specific to the conformational epitope described herein located
within cluster A. Results showed that antibody hSel001 possesses
dual function properties enabling it to both block binding of
P-selectin to PSGL-1 and dissociate preformed P-selectin/PSGL-1
complexes (FIG. 4). Thus hSel001 is another antibody encompassed by
the presently disclosed and claimed inventive concepts and can be
used as a therapeutic treatment for inflammatory and thrombotic
diseases as described herein, and wherein P-selectin binding to
PSGL-1 is blocked, and dissociation of preformed P-selectin/PSGL-1
complex is promoted.
[0146] Cell-Based In Vitro Rolling Assays Under Flow with Human
Neutrophils
[0147] To further evaluate the blocking and dissociative properties
of antibodies G1, G3 and hSel001, cell-based in vitro rolling
assays were performed with freshly isolated human neutrophils that
were introduced under a flow of 1.0 dyn/cm.sup.2 in a flow chamber
coated with low and high levels of membrane P-selectin. The low
density P-selectin was coated at 0.25 ug/ml and the high density
P-selectin was coated at 2 ug/ml. Site densities were determined
using I.sup.125-labeled G1 mAb to be 50 sites/mm.sup.2 (low) and
380 sites/mm.sup.2 (high). On low density P-selectin, neutrophils
rolled at an average velocity of 5 .mu.m/s. On high density
P-selectin, neutrophils rolled at an average velocity of 1 .mu.m/s.
Neutrophils are introduced in buffer under flow and allowed to
begin rolling and tethering. Once equilibrated, test antibodies
(G1, G3, hSel001) were introduced in cell-free buffer under flow.
There is a dead volume of about 1 minute interval before the
antibody reaches the chamber. At 1 minute intervals thereafter,
cells remaining bound are counted and expressed as % cells bound.
Results were recorded on video microscopy for approximately 0-20
minutes and the data analyzed post run.
[0148] Results in FIG. 6 panel (A) showed that neutrophils rolled
at a higher velocity on low density P-selectin. Thus as the
P-selectin/PSGL-1 complex released due to normal on/off kinetics of
the lectin/ligand binding, neutrophils traveled greater distances
at higher velocity to the next P-selectin binding site. As the
complex releases, the previously occupied P-selectin becomes
available for binding by anti-P-selectin antibodies. Thus all three
antibodies, G1, G3, and hSel01, showed equivalent blocking
functionality over the course of 1-4 minutes.
[0149] Results in FIG. 6 panel (B) on high density P-selectin
showed that neutrophils roll much slower (1 .mu.m/s) as a greater
number of P-selectin binding sites are available. Many neutrophils
come to a rolling stop on P-selectin at this density. Under these
conditions the murine antibody G1 and the humanized antibody
hSel001 were able to release rolling and tethering neutrophils by
dissociating the P-selectin/PSGL-1 complex immediately and over the
course of 1-8 minutes. In contrast the G3 anti-P-selectin antibody
required up to 20 minutes to block rolling neutrophils. This
indicated that the G3 antibody was only able to bind unoccupied
P-selectin sites and thus block P-selectin/PSGL-1 complexes, but
was not able to bind and dissociate the pre-formed complex. These
cell-binding assays under flow confirm the BIACORE results reported
previously herein which demonstrate that murine antibody G1 and
humanized antibody hSel001 both have dual function properties
causing both blockage and dissociation of preformed
P-selectin/PSGL-1 complexes. Thus the hSel001 antibody has the dual
function anti-P-selectin properties of the preferred embodiments of
the presently disclosed and claimed inventive concepts.
[0150] The isolated dual function antibody claimed as an embodiment
of the presently disclosed and claimed inventive concepts does not
comprise the murine antibody G1.
[0151] Antibodies of the presently disclosed and claimed inventive
concepts provided by any of the above described methods are
preferably used in the manufacture of a pharmaceutical composition
for the therapeutic treatment of a pathological condition, wherein
such treatment comprises mitigating, reversion, or inhibiting an
inflammatory response or thrombosis.
[0152] It is an important objective of the presently disclosed and
claimed inventive concepts to use the antibodies, or functionally
active fragments or variants of said antibodies for the manufacture
of a pharmaceutical composition for prevention and/or treatment of
inflammatory responses or diseases or thrombosis such as described
herein.
[0153] The presently disclosed and claimed inventive concepts in
particular are directed to using such single and dual function
anti-P-selectin antibodies or antibody fragments as described and
identified herein in treatments for inflammatory, thrombotic or
other conditions or disorders in primates (including humans) which
involve platelet, sickled red cell, leukocyte, lymphocyte, and/or
endothelial cell adhesion, wherein the condition or disorder
comprises or is associated with (but not limited to) at least one
of sickle cell vasoocclusive pain crisis, inflammatory bowel
disease (e.g., Crohn's Disease, ulcerative colitis, enteritis),
arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic
arthritis), graft rejection, graft versus host disease, asthma,
chronic obstructive pulmonary disease, psoriasis, dermatitis,
sepsis, nephritis, lupus erythematosis, scleroderma, rhinitis,
anaphylaxis, diabetes, multiple sclerosis, atherosclerosis,
thrombosis, tumor metastasis, allergic reactions, thyroiditis,
ischemic reperfusion injury (e.g., due to myocardial infarction,
stroke, or organ transplantation), and conditions associated with
extensive trauma, or chronic inflammation, such as for example,
type IV delayed hypersensitivity, associated for example with
infection by Tubercle bacilli, or systematic inflammatory response
syndrome, or multiple organ failure. The term "primate" as used
herein refers to humans, monkeys, including baboons and cynomolgus
monkeys, and apes, the latter including chimpanzees, gorillas,
gibbons and orangutans, for example.
[0154] In the pharmaceutical composition of a medicament according
to the presently disclosed and claimed inventive concepts, the
antibodies may be formulated by any of the established methods of
formulating pharmaceutical compositions, e.g. as described in the
latest edition of Remington's Pharmaceutical Sciences or described
elsewhere herein. The composition may typically be in a form suited
for local or systemic injection or infusion and may, as such, be
formulated with sterile water or an isotonic saline or glucose
solution. The compositions may be sterilized by conventional
sterilization techniques, which are well known in the art. The
resulting aqueous solutions may be packaged for use or filtered
under aseptic conditions and lyophilized, the lyophilized
preparation being combined with the sterile aqueous solution prior
to administration. The composition may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions, such as buffering agents, tonicity
adjusting agents and the like, for instance sodium acetate, sodium
lactate, sodium chloride, potassium chloride, calcium chloride,
etc. The concentration of proteins may vary widely, for example,
from less than about 0.01% to as much as 15-20% or more by weight.
A unit dosage of the composition may contain for example from about
1 .mu.g to about 1000 mg of an antibody or antibody fragment.
[0155] The antibodies or antibody fragments of the presently
disclosed and claimed inventive concepts may be administered
topically or by injection. Dosages will be prescribed by the
physician according to the particular condition and the particular
individual to be treated. Dosages and frequency is carefully
adapted and adjusted according to parameters determined by the
physician in charge. Preferred administration routes may be oral,
via inhalation, subcutaneous, intravenous, intramuscular,
intratracheal, intravesical, or intraperitoneal injections and may
be given per 24 to 48 hours, or per week, every 14 days, every 4
weeks for example in the range of from 0.01-1000 mg, especially 0.1
mg to 100 mg, in particular 1-10 mg per kg body weight. The dose
may be administered continuously through a catheter or in
individual boluses. The antibody of the invention may be
administered in an efficacious quantity such as, but not limited
to, the ranges between 1 ng/kg to 1 .mu.g/kg, 0.01 .mu.g/kg to 50
.mu.g/kg, 0.1 .mu.g/kg to 1 .mu.g/kg, 1 .mu.g/kg to 5 .mu.g/kg, 5
.mu.g/kg to 10 .mu.g/kg, 10 .mu.g/kg to 50 .mu.g/kg, 50 .mu.g/kg to
100 .mu.g/kg, 100 mg/kg to 1 mg/kg, 1 mg/kg to 10 mg/kg, or 10
mg/kg to 100 mg/kg body weight.
[0156] Pharmaceutical compositions used in the presently disclosed
and claimed inventive concepts comprising antibodies described
herein may additionally be supplemented by other therapeutic
compounds which are routinely prescribed by the physician according
to the particular condition and the particular individual to be
treated such as an anti-inflammatory drug, wherein said drugs are
prescribed by the physician according to the particular condition
and the particular individual to be treated.
[0157] As noted elsewhere herein, the phenomenon of
P-selectin/PSGL-1 binding has functional importance in sickled red
cell, endothelial cell leukocyte and platelet interactions, and/or
microvesicle adhesion, leukocyte rolling, recruitment, aggregation;
leukocyte secretion of cytokines; promotion of coagulation; and
other aspects of inflammation, thrombosis, coagulation, immune
response, and signal transduction including, but not limited to,
sickle cell vasoocclusive pain crisis, inflammatory bowel disease
(e.g., Crohn's Disease, ulcerative colitis, enteritis), arthritis
(e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis),
graft rejection, graft versus host disease, asthma, chronic
obstructive pulmonary disease, psoriasis, dermatitis, sepsis,
nephritis, lupus erythematosis, scleroderma, rhinitis, anaphylaxis,
diabetes, multiple sclerosis, atherosclerosis, thrombosis, tumor
metastasis, allergic reactions, thyroiditis, ischemic reperfusion
injury (e.g., due to myocardial infarction, stroke, or organ
transplantation), and conditions associated with extensive trauma,
or chronic inflammation, such as for example, type IV delayed
hypersensitivity, associated for example with infection by Tubercle
bacilli, or systematic inflammatory response syndrome, or multiple
organ failure. A neutralizing antibody to P-selectin as described
herein will inhibit one or more of these activities in a patient as
mediated through P-selectin/PSGL-1 receptor binding (or in the case
of sickled red cells, P-selectin/PSGL-1 like receptor binding), in
vivo or in vitro, for example. Thus, the inhibition of
P-selectin/PSGL-1 binding with a neutralizing antibody described
herein is useful in the treatment in a patient of various
conditions and disorders including but not limited to, those
described herein.
[0158] The P-selectin specific antibodies or binding fragments
described herein can be linked to another molecule. For example,
antibodies may be linked to another peptide or protein, toxin,
radioisotope, cytotoxic or cytostatic agents. The antibodies can be
linked covalently by chemical cross-linking or by recombinant
methods. The antibodies may also be linked to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or
4,179,337. The antibodies can be chemically modified by covalent
conjugation to a polymer, for example, to increase their stability
or half-life. Exemplary polymers and methods to attach them are
also shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and
4,609,546.
[0159] The antibodies may also be tagged with a detectable label. A
detectable label is a molecule which, by its chemical nature,
provides an analytically identifiable signal which allows the
detection of a molecular interaction. A protein, including an
antibody, has a detectable label if it is covalently or
non-covalently bound to a molecule that can be detected directly
(e.g., by means of a chromophore, fluorophore, or radioisotope) or
indirectly (e.g., by means of catalyzing a reaction producing a
colored, luminescent, or fluorescent product). Detectable labels
include a radiolabel such as 131I or .sup.99Tc, a heavy metal, or a
fluorescent substrate, such as Europium, for example, which may
also be attached to antibodies using conventional chemistry.
Detectable labels also include enzyme labels such as horseradish
peroxidase or alkaline phosphatase. Detectable labels further
include chemical moieties such as biotin, which may be detected via
binding to a specific cognate detectable moiety, e.g., labeled
avidin.
[0160] The presently disclosed and claimed inventive concepts are
also directed to methods of screening for anti-P-selectin
antibodies and binding fragments thereof which block both
P-selectin/PSGL-1 binding and/or which cause dissociation of
preformed P-selectin/PSGL-1 complexes.
[0161] As noted above, the presently disclosed and claimed
inventive concepts are directed to antibodies against P-selectin,
host cells that produce such anti-P-selectin antibodies, vectors
that contain DNA which encode such anti-P-selectin antibody
expression and screening methods to identify anti-P-selectin
antibodies which block P-selectin/PSGL-1 binding and in a further
embodiment has a "dual function" in also causing dissociation of
preformed P-selectin/PSGL-1 complex. Thus, in one embodiment the
presently disclosed and claimed inventive concepts is directed to
methods of identifying anti-P-selectin antibodies that specifically
bind to a conformational epitope in amino acids 1-35, and more
preferably in amino acids 4-23, of human P-selectin (SEQ ID NO:1)
(such as the conformational epitope described herein) and which
block PSGL-1, or mimetics thereof, from binding to P-selectin, and
which can reverse such binding thereto, thus exhibiting a dual
function in blocking selectin-mediated adhesion due to
P-selectin/PSGL-1 binding and in causing dissociation of preformed
P-selectin/PSGL-1 complexes.
[0162] The screening method in a preferred embodiment comprises in
vitro assays that can be used to identify anti-P-selectin
antibodies that abolish P-selectin/PSGL-1 binding and preferably
cause dissociation of preformed P-selectin/PSGL-1 complexes. Test
anti-P-selectin antibodies can be screened for dual function
capability with a series of assays such as, but not limited to,
those described herein which will identify those antibodies that
bind to a conformational epitope within amino acids 1-35, and more
particularly within amino acids 4-23, of P-selectin, and that block
the binding of the PSGL-1 ligand to P-selectin, and which
preferably cause dissociation of preformed P-selectin/PSGL-1
complexes. No anti-P-selectin antibodies have heretofore been shown
to have the ability to both block PSGL-1 binding to P-selectin and
to cause dissociation of preformed P-selectin/PSGL-1 complexes.
[0163] In a first step of the screening method, for example, test
antibodies generated against P-selectin are assayed for the ability
to block binding of PSGL-1 to P-selectin. Test antibodies which
block binding of PSGL-1 to P-selectin are screened to determine
their ability to cause dissociation of preformed P-selectin/PSGL-1
complexes. Test antibodies identified as having dual function of
blocking both PSGL-1 binding to P-selectin, and causing
dissociation of P-selectin/PSGL-1 complex comprise particularly
preferred embodiments of the presently disclosed and claimed
inventive concepts and can be used in the methods of the presently
disclosed and claimed inventive concepts.
[0164] In one embodiment of the method, test antibodies which block
binding of PSGL-1 to P-selectin are first identified using a first
screening assay. For example, in a preferred embodiment of the
first screening assay, PSGL-1 or a synthetic PSGL-1 mimetic such as
GSP-6, or a terminal epitope portion of PSGL-1 able to bind to
P-selectin, is bound to a substrate, such as a BIACORE chip, in a
method known to persons having ordinary skill in the art. The
substrate having the PSGL-1 (or the PSGL-1 mimetic) bound thereto
is then exposed to an anti-P-selectin test antibody/P-selectin
complex. The binding of the complex to the PSGL-1-substrate is then
evaluated. If the test antibody/P-selectin complex does not bind to
the PSGL-1 on the substrate, the test antibody is identified as a
"function-blocking" antibody.
[0165] In an alternative embodiment of the first screening assay,
P-selectin, or a portion thereof which maintains the integrity of
the conformational epitope, is bound to the substrate. For example,
the minimum portion of P-selectin which maintains the
conformational epitope comprises the lectin and EGF binding domains
of P-selectin. In this embodiment, the P-selectin-substrate is
exposed to the test antibody, which binds to form the
P-selectin-antibody complex. Then PSGL-1, or a high molecular
weight mimetic thereof such as a GSP-6/biotin/avidin complex, is
exposed to the P-selectin/test antibody complex and the binding
thereto of PSGL-1 (or the mimetic) is evaluated. An antibody which
prevents or inhibits the binding of PSGL-1 to P-selectin is
identified as a "function-blocking" antibody. When the GSP-6 or
PSGL-1 mimetic is bound to the substrate, in a preferred
embodiment, it is bound to biotin, and the mimetic-biotin complex
is bound to a streptavidin coating on the substrate.
[0166] In a preferred embodiment of the second screening assay,
either PSGL-1 or a mimetic thereof is bound, as described above, to
the substrate (such as a BIACORE chip). P-selectin is then applied
to the PSGL-1/substrate to form the P-selectin/PSGL-1 (or mimetic)
complex. The test antibody is then applied and dissociation of the
complex is measured as a decrease in mass or as Response Units (RU)
since P-selectin is being dissociated away. A function-blocking
anti-P-selectin antibody, which induces dissociation of the
P-selectin/PSGL-1 complex, is designated as a dual function
anti-P-selectin antibody. In yet another embodiment of the second
assay, the anti-P-selectin antibody itself is bound to the
substrate, and a P-selectin/PSGL-1 complex is exposed to it, and
dissociation thereof is measured.
[0167] In an alternate embodiment of the second screening assay,
P-selectin may be bound to a substrate rather than the PSGL-1.
PSGL-1 or a high molecular weight mimetic thereof such as a
GSP-6/biotin/avidin complex is then exposed to the P-selectin on
the substrate and allowed to form a PSGL-1/P-selectin complex. The
PSGL-1/P-selectin complex is then exposed to a function-blocking
anti-P-selectin antibody and dissociation of the complex is
evaluated, for example using a BIACORE method as described
elsewhere herein.
[0168] Although the presently disclosed and claimed inventive
concepts and its advantages have been described in detail, it
should be understood that various changes, substitutions and
alterations can be made herein without departing from the spirit
and scope of the presently disclosed and claimed inventive concepts
as defined by the appended claims. Moreover, the scope of the
present application is not intended to be limited to the particular
embodiments of the process, machine, items of manufacture,
compositions of matter, means, methods and steps described in the
specification. As one having ordinary skill in the art will readily
appreciate from the present disclosure, processes, machines, items
of manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the presently disclosed and claimed inventive
concepts. Accordingly, the appended claims are intended to include
within their scope such processes, machines, items of manufacture,
compositions of matter, means, methods, or steps.
[0169] Each of the references, patents or publications cited herein
is expressly incorporated herein by reference in its entirety.
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Sequence CWU 1
1
291279PRTHomo sapiens 1Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser
Trp Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu
Val Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val
Leu Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn
Asn Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn
Glu Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg
Asn Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala
Pro Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His
Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120
125Lys Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys
130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu
Cys Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn
Cys Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys
Ser Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser
Lys Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro
Pro Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro
Glu Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230 235
240Phe Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe Ala
245 250 255Leu Val Gly Pro Glu Val Val Gln Cys Thr Ala Ser Gly Val
Trp Thr 260 265 270Ala Pro Ala Pro Val Cys Lys 2752282PRTMus
musculus 2Trp Thr Tyr Asn Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Asn
Ser Arg1 5 10 15Val Phe Cys Arg Arg His Phe Thr Asp Leu Val Ala Ile
Gln Asn Lys 20 25 30Asn Glu Ile Ala His Leu Asn Asp Val Ile Pro Phe
Phe Asn Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Ile Asn Asn Lys
Trp Thr Trp Val Gly 50 55 60Thr Asn Lys Thr Leu Thr Glu Glu Ala Glu
Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Lys Asn Asn Gln
Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Asn Ser Ala Pro Gly Lys
Trp Asn Asp Glu Pro Cys Phe Lys Arg 100 105 110Lys Arg Ala Leu Cys
Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Asn Gln Gly
Glu Cys Ile Glu Thr Ile Gly Ser Tyr Thr Cys Ser Cys 130 135 140Tyr
Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Lys Glu Cys Gly145 150
155 160Lys Val Asn Ile Pro Gln His Val Leu Met Asn Cys Ser His Pro
Leu 165 170 175Gly Glu Phe Ser Phe Asn Ser Gln Cys Thr Phe Ser Cys
Ala Glu Gly 180 185 190Tyr Glu Leu Asp Gly Pro Gly Glu Leu Gln Cys
Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Asn Pro Pro Lys Cys Asp
Ala Val Gln Cys Gln Ser Leu 210 215 220Glu Ala Pro Pro His Gly Thr
Met Ala Cys Met His Pro Ile Ala Ala225 230 235 240Phe Ala Tyr Asp
Ser Ser Cys Lys Phe Glu Cys Gln Pro Gly Tyr Arg 245 250 255Ala Arg
Gly Ser Asn Thr Leu His Cys Thr Gly Ser Gly Gln Trp Ser 260 265
270Glu Pro Leu Pro Thr Cys Glu Ala Ile Ala 275 2803279PRTArtificial
SequenceChimera-1 (mouse P-selecin amino acids substituted into
human P-selectin at positions 4, 14, 17, 18, 20, 21, 22, 23) 3Trp
Thr Tyr Asn Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Asn Ser Arg1 5 10
15Val Phe Cys Arg Arg His Phe Thr Asp Leu Val Ala Ile Gln Asn Lys
20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser
Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp
Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala
Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val
Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro Gly Lys Trp Asn Asp
Glu His Cys Leu Lys Lys 100 105 110Lys His Ala Leu Cys Tyr Thr Ala
Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu
Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe
Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys Gly145 150 155 160Glu
Leu Glu Leu Pro Gln His Val Leu Met Asn Cys Ser His Pro Leu 165 170
175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser Phe His Cys Thr Asp Gly
180 185 190Tyr Gln Val Asn Gly Pro Ser Lys Leu Glu Cys Leu Ala Ser
Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro Gln Cys Leu Ala Ala Gln
Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu Arg Gly Asn Met Thr Cys
Leu His Ser Ala Lys Ala225 230 235 240Phe Gln His Gln Ser Ser Cys
Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250 255Leu Val Gly Pro Glu
Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr 260 265 270Ala Pro Ala
Pro Val Cys Lys 2754282PRTArtificial SequenceChimera-2 (human
P-selectin positions 1-35; mouse P-selectin thereafter) 4Trp Thr
Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys
Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys 20 25
30Asn Glu Ile Ala His Leu Asn Asp Val Ile Pro Phe Phe Asn Ser Tyr
35 40 45Tyr Trp Ile Gly Ile Arg Lys Ile Asn Asn Lys Trp Thr Trp Val
Gly 50 55 60Thr Asn Lys Thr Leu Thr Glu Glu Ala Glu Asn Trp Ala Asp
Asn Glu65 70 75 80Pro Asn Asn Lys Lys Asn Asn Gln Asp Cys Val Glu
Ile Tyr Ile Lys 85 90 95Ser Asn Ser Ala Pro Gly Lys Trp Asn Asp Glu
Pro Cys Phe Lys Arg 100 105 110Lys Arg Ala Leu Cys Tyr Thr Ala Ser
Cys Gln Asp Met Ser Cys Ser 115 120 125Asn Gln Gly Glu Cys Ile Glu
Thr Ile Gly Ser Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe Tyr
Gly Pro Glu Cys Glu Tyr Val Lys Glu Cys Gly145 150 155 160Lys Val
Asn Ile Pro Gln His Val Leu Met Asn Cys Ser His Pro Leu 165 170
175Gly Glu Phe Ser Phe Asn Ser Gln Cys Thr Phe Ser Cys Ala Glu Gly
180 185 190Tyr Glu Leu Asp Gly Pro Gly Glu Leu Gln Cys Leu Ala Ser
Gly Ile 195 200 205Trp Thr Asn Asn Pro Pro Lys Cys Asp Ala Val Gln
Cys Gln Ser Leu 210 215 220Glu Ala Pro Pro His Gly Thr Met Ala Cys
Met His Pro Ile Ala Ala225 230 235 240Phe Ala Tyr Asp Ser Ser Cys
Lys Phe Glu Cys Gln Pro Gly Tyr Arg 245 250 255Ala Arg Gly Ser Asn
Thr Leu His Cys Thr Gly Ser Gly Gln Trp Ser 260 265 270Glu Pro Leu
Pro Thr Cys Glu Ala Ile Ala 275 2805279PRTArtificial
SequenceChimera-3 (mouse P-selecin amino acids substituted into
human P-selectin at positions 4, 14, 17, 18) 5Trp Thr Tyr Asn Tyr
Ser Thr Lys Ala Tyr Ser Trp Asn Asn Ser Arg1 5 10 15Val Phe Cys Gln
Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile
Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp
Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp Val Gly 50 55 60Thr
Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala Asp Asn Glu65 70 75
80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys
85 90 95Ser Pro Ser Ala Pro Gly Lys Trp Asn Asp Glu His Cys Leu Lys
Lys 100 105 110Lys His Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met
Ser Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn
Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys
Glu Tyr Val Arg Glu Cys Gly145 150 155 160Glu Leu Glu Leu Pro Gln
His Val Leu Met Asn Cys Ser His Pro Leu 165 170 175Gly Asn Phe Ser
Phe Asn Ser Gln Cys Ser Phe His Cys Thr Asp Gly 180 185 190Tyr Gln
Val Asn Gly Pro Ser Lys Leu Glu Cys Leu Ala Ser Gly Ile 195 200
205Trp Thr Asn Lys Pro Pro Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu
210 215 220Lys Ile Pro Glu Arg Gly Asn Met Thr Cys Leu His Ser Ala
Lys Ala225 230 235 240Phe Gln His Gln Ser Ser Cys Ser Phe Ser Cys
Glu Glu Gly Phe Ala 245 250 255Leu Val Gly Pro Glu Val Val Gln Cys
Thr Ala Ser Gly Val Trp Thr 260 265 270Ala Pro Ala Pro Val Cys Lys
2756279PRTArtificial SequenceChimera-4 (mouse P-selecin amino acids
substituted into human P-selectin at positions 20, 21, 22, 23) 6Trp
Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10
15Lys Tyr Cys Arg Arg His Phe Thr Asp Leu Val Ala Ile Gln Asn Lys
20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser
Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp
Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala
Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val
Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro Gly Lys Trp Asn Asp
Glu His Cys Leu Lys Lys 100 105 110Lys His Ala Leu Cys Tyr Thr Ala
Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu
Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe
Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys Gly145 150 155 160Glu
Leu Glu Leu Pro Gln His Val Leu Met Asn Cys Ser His Pro Leu 165 170
175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser Phe His Cys Thr Asp Gly
180 185 190Tyr Gln Val Asn Gly Pro Ser Lys Leu Glu Cys Leu Ala Ser
Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro Gln Cys Leu Ala Ala Gln
Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu Arg Gly Asn Met Thr Cys
Leu His Ser Ala Lys Ala225 230 235 240Phe Gln His Gln Ser Ser Cys
Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250 255Leu Val Gly Pro Glu
Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr 260 265 270Ala Pro Ala
Pro Val Cys Lys 2757279PRTArtificial SequenceChimera-5 (single
amino acid change in human P-selectin - human H4 to mouse N4) 7Trp
Thr Tyr Asn Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10
15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys
20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser
Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp
Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala
Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val
Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro Gly Lys Trp Asn Asp
Glu His Cys Leu Lys Lys 100 105 110Lys His Ala Leu Cys Tyr Thr Ala
Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu
Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe
Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys Gly145 150 155 160Glu
Leu Glu Leu Pro Gln His Val Leu Met Asn Cys Ser His Pro Leu 165 170
175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser Phe His Cys Thr Asp Gly
180 185 190Tyr Gln Val Asn Gly Pro Ser Lys Leu Glu Cys Leu Ala Ser
Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro Gln Cys Leu Ala Ala Gln
Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu Arg Gly Asn Met Thr Cys
Leu His Ser Ala Lys Ala225 230 235 240Phe Gln His Gln Ser Ser Cys
Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250 255Leu Val Gly Pro Glu
Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr 260 265 270Ala Pro Ala
Pro Val Cys Lys 2758279PRTArtificial SequenceChimera-5Q
(substitution of Q for H4 in human P-selectin - removes putative
glycosylation site) 8Trp Thr Tyr Gln Tyr Ser Thr Lys Ala Tyr Ser
Trp Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu
Val Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val
Leu Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn
Asn Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn
Glu Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg
Asn Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala
Pro Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His
Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120
125Lys Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys
130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu
Cys Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn
Cys Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys
Ser Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser
Lys Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro
Pro Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro
Glu Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230 235
240Phe Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe Ala
245 250 255Leu Val Gly Pro Glu Val Val Gln Cys Thr Ala Ser Gly Val
Trp Thr 260 265 270Ala Pro Ala Pro Val Cys Lys 2759282PRTArtificial
SequenceChimera-6 (human P-selectin positions 1-121; thereafter
mouse P-selectin) 9Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp
Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val
Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu
Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn
Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu
Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn
Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro
Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala
Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115
120 125Asn Gln Gly Glu Cys Ile Glu Thr Ile Gly Ser Tyr Thr Cys Ser
Cys 130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Lys
Glu Cys Gly145 150 155 160Lys Val Asn Ile Pro Gln His Val Leu Met
Asn Cys Ser His Pro Leu 165 170 175Gly Glu Phe Ser Phe Asn Ser Gln
Cys Thr Phe Ser Cys Ala Glu Gly 180 185 190Tyr Glu Leu Asp Gly Pro
Gly Glu Leu Gln Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Asn
Pro Pro Lys Cys Asp Ala Val Gln Cys Gln Ser Leu 210 215 220Glu Ala
Pro Pro His Gly Thr Met Ala Cys Met His Pro Ile Ala Ala225 230 235
240Phe Ala Tyr Asp Ser Ser Cys Lys Phe Glu Cys Gln Pro Gly Tyr Arg
245 250 255Ala Arg Gly Ser Asn Thr Leu His Cys Thr Gly Ser Gly Gln
Trp Ser 260 265 270Glu Pro Leu Pro Thr Cys Glu Ala Ile Ala 275
28010282PRTArtificial SequenceChimera-7 (human P-selectin positions
1-177; thereafter mouse P-selectin) 10Trp Thr Tyr His Tyr Ser Thr
Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln Asn Arg
Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr
Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly
Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys
Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro
Asn Asn Lys Arg Asn Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90
95Ser Pro Ser Ala Pro Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys
100 105 110Lys His Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser
Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr
Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu
Tyr Val Arg Glu Cys Gly145 150 155 160Glu Leu Glu Leu Pro Gln His
Val Leu Met Asn Cys Ser His Pro Leu 165 170 175Gly Glu Phe Ser Phe
Asn Ser Gln Cys Thr Phe Ser Cys Ala Glu Gly 180 185 190Tyr Glu Leu
Asp Gly Pro Gly Glu Leu Gln Cys Leu Ala Ser Gly Ile 195 200 205Trp
Thr Asn Asn Pro Pro Lys Cys Asp Ala Val Gln Cys Gln Ser Leu 210 215
220Glu Ala Pro Pro His Gly Thr Met Ala Cys Met His Pro Ile Ala
Ala225 230 235 240Phe Ala Tyr Asp Ser Ser Cys Lys Phe Glu Cys Gln
Pro Gly Tyr Arg 245 250 255Ala Arg Gly Ser Asn Thr Leu His Cys Thr
Gly Ser Gly Gln Trp Ser 260 265 270Glu Pro Leu Pro Thr Cys Glu Ala
Ile Ala 275 28011282PRTArtificial SequenceChimera 7B (human
P-selectin positions 1-156; thereafter mouse P-selectin) 11Trp Thr
Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys
Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys 20 25
30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser Tyr
35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp Val
Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala Asp
Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val Glu
Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro Gly Lys Trp Asn Asp Glu
His Cys Leu Lys Lys 100 105 110Lys His Ala Leu Cys Tyr Thr Ala Ser
Cys Gln Asp Met Ser Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu Glu
Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe Tyr
Gly Pro Glu Cys Glu Tyr Val Lys Glu Cys Gly145 150 155 160Lys Val
Asn Ile Pro Gln His Val Leu Met Asn Cys Ser His Pro Leu 165 170
175Gly Glu Phe Ser Phe Asn Ser Gln Cys Thr Phe Ser Cys Ala Glu Gly
180 185 190Tyr Glu Leu Asp Gly Pro Gly Glu Leu Gln Cys Leu Ala Ser
Gly Ile 195 200 205Trp Thr Asn Asn Pro Pro Lys Cys Asp Ala Val Gln
Cys Gln Ser Leu 210 215 220Glu Ala Pro Pro His Gly Thr Met Ala Cys
Met His Pro Ile Ala Ala225 230 235 240Phe Ala Tyr Asp Ser Ser Cys
Lys Phe Glu Cys Gln Pro Gly Tyr Arg 245 250 255Ala Arg Gly Ser Asn
Thr Leu His Cys Thr Gly Ser Gly Gln Trp Ser 260 265 270Glu Pro Leu
Pro Thr Cys Glu Ala Ile Ala 275 28012279PRTArtificial
SequenceChimera-8 (single substitution in human P-selectin - human
I14 tomouse N14) 12Trp Thr Tyr Asn Tyr Ser Thr Lys Ala Tyr Ser Trp
Asn Asn Ser Arg1 5 10 15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val
Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu
Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn
Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu
Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn
Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro
Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala
Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys
Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135
140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys
Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn Cys
Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser
Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser Lys
Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro
Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu
Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230 235 240Phe
Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250
255Leu Val Gly Pro Glu Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr
260 265 270Ala Pro Ala Pro Val Cys Lys 27513279PRTArtificial
SequenceChimera-9 (single substitution in human P-selectin - human
K17 tomouse V17) 13Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp
Asn Ile Ser Arg1 5 10 15Val Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val
Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu
Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn
Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu
Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn
Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro
Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala
Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys
Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135
140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys
Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn Cys
Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser
Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser Lys
Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro
Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu
Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230 235 240Phe
Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250
255Leu Val Gly Pro Glu Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr
260 265 270Ala Pro Ala Pro Val Cys Lys 27514279PRTArtificial
SequenceChimera-10 (single substitution in human P-selectin - human
Y18 to mouse F18) 14Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp
Asn Ile Ser Arg1 5 10 15Lys Phe Cys Gln Asn Arg Tyr Thr Asp Leu Val
Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu
Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn
Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu
Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn
Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro
Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala
Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys
Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135
140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys
Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn Cys
Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser
Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser Lys
Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro
Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu
Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230 235 240Phe
Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250
255Leu Val Gly Pro Glu Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr
260 265 270Ala Pro Ala Pro Val Cys Lys 27515279PRTArtificial
SequenceChimera-11 (single substitution in human P-selectin - human
Q20 to mouse R20) 15Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp
Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Arg Asn Arg Tyr Thr Asp Leu Val
Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu
Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn
Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu
Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn
Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro
Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala
Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys
Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135
140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys
Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn Cys
Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser
Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser Lys
Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro
Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu
Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230 235 240Phe
Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250
255Leu Val Gly Pro Glu Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr
260 265 270Ala Pro Ala Pro Val Cys Lys 27516279PRTArtificial
SequenceChimera-12 (single substitution in human P-selectin - human
N21 to mouse R21) 16Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp
Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln Arg Arg Tyr Thr Asp Leu Val
Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu
Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn
Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu
Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn
Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro
Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala
Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys
Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135
140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys
Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn Cys
Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser
Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser Lys
Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro
Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu
Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230 235 240Phe
Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250
255Leu Val Gly Pro Glu Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr
260 265 270Ala Pro Ala Pro Val Cys Lys 27517279PRTArtificial
SequenceChimera-13 (single substitution in human P-selectin - human
R22 to mouse H22) 17Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp
Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln Asn His Tyr Thr Asp Leu Val
Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu
Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn
Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu
Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn
Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro
Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala
Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys
Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135
140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys
Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn Cys
Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser
Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser Lys
Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro
Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu
Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230 235 240Phe
Gln His Gln Ser Ser Cys Ser Phe Ser
Cys Glu Glu Gly Phe Ala 245 250 255Leu Val Gly Pro Glu Val Val Gln
Cys Thr Ala Ser Gly Val Trp Thr 260 265 270Ala Pro Ala Pro Val Cys
Lys 27518279PRTArtificial SequenceChimera-14 (single substitution
in human P-selectin - human Y23 to mouse F23) 18Trp Thr Tyr His Tyr
Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln
Asn Arg Phe Thr Asp Leu Val Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile
Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp
Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp Val Gly 50 55 60Thr
Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala Asp Asn Glu65 70 75
80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys
85 90 95Ser Pro Ser Ala Pro Gly Lys Trp Asn Asp Glu His Cys Leu Lys
Lys 100 105 110Lys His Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met
Ser Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn
Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys
Glu Tyr Val Arg Glu Cys Gly145 150 155 160Glu Leu Glu Leu Pro Gln
His Val Leu Met Asn Cys Ser His Pro Leu 165 170 175Gly Asn Phe Ser
Phe Asn Ser Gln Cys Ser Phe His Cys Thr Asp Gly 180 185 190Tyr Gln
Val Asn Gly Pro Ser Lys Leu Glu Cys Leu Ala Ser Gly Ile 195 200
205Trp Thr Asn Lys Pro Pro Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu
210 215 220Lys Ile Pro Glu Arg Gly Asn Met Thr Cys Leu His Ser Ala
Lys Ala225 230 235 240Phe Gln His Gln Ser Ser Cys Ser Phe Ser Cys
Glu Glu Gly Phe Ala 245 250 255Leu Val Gly Pro Glu Val Val Gln Cys
Thr Ala Ser Gly Val Trp Thr 260 265 270Ala Pro Ala Pro Val Cys Lys
27519282PRTArtificial SequenceChimera-15 (human P-selectin
positions 1-97; thereafter mouse P-selectin) 19Trp Thr Tyr His Tyr
Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln
Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile
Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp
Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp Val Gly 50 55 60Thr
Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala Asp Asn Glu65 70 75
80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys
85 90 95Ser Asn Ser Ala Pro Gly Lys Trp Asn Asp Glu Pro Cys Phe Lys
Arg 100 105 110Lys Arg Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met
Ser Cys Ser 115 120 125Asn Gln Gly Glu Cys Ile Glu Thr Ile Gly Ser
Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys
Glu Tyr Val Lys Glu Cys Gly145 150 155 160Lys Val Asn Ile Pro Gln
His Val Leu Met Asn Cys Ser His Pro Leu 165 170 175Gly Glu Phe Ser
Phe Asn Ser Gln Cys Thr Phe Ser Cys Ala Glu Gly 180 185 190Tyr Glu
Leu Asp Gly Pro Gly Glu Leu Gln Cys Leu Ala Ser Gly Ile 195 200
205Trp Thr Asn Asn Pro Pro Lys Cys Asp Ala Val Gln Cys Gln Ser Leu
210 215 220Glu Ala Pro Pro His Gly Thr Met Ala Cys Met His Pro Ile
Ala Ala225 230 235 240Phe Ala Tyr Asp Ser Ser Cys Lys Phe Glu Cys
Gln Pro Gly Tyr Arg 245 250 255Ala Arg Gly Ser Asn Thr Leu His Cys
Thr Gly Ser Gly Gln Trp Ser 260 265 270Glu Pro Leu Pro Thr Cys Glu
Ala Ile Ala 275 28020282PRTArtificial SequenceChimera-16 (human
P-selectin amino acids substituted into mouse P-selectin at
positions 4, 14, 17, 21, 22) 20Trp Thr Tyr His Tyr Ser Thr Lys Ala
Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys Phe Cys Arg Asn Arg Phe Thr
Asp Leu Val Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Ala His Leu Asn
Asp Val Ile Pro Phe Phe Asn Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg
Lys Ile Asn Asn Lys Trp Thr Trp Val Gly 50 55 60Thr Asn Lys Thr Leu
Thr Glu Glu Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn
Lys Lys Asn Asn Gln Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Asn
Ser Ala Pro Gly Lys Trp Asn Asp Glu Pro Cys Phe Lys Arg 100 105
110Lys Arg Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser
115 120 125Asn Gln Gly Glu Cys Ile Glu Thr Ile Gly Ser Tyr Thr Cys
Ser Cys 130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val
Lys Glu Cys Gly145 150 155 160Lys Val Asn Ile Pro Gln His Val Leu
Met Asn Cys Ser His Pro Leu 165 170 175Gly Glu Phe Ser Phe Asn Ser
Gln Cys Thr Phe Ser Cys Ala Glu Gly 180 185 190Tyr Glu Leu Asp Gly
Pro Gly Glu Leu Gln Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn
Asn Pro Pro Lys Cys Asp Ala Val Gln Cys Gln Ser Leu 210 215 220Glu
Ala Pro Pro His Gly Thr Met Ala Cys Met His Pro Ile Ala Ala225 230
235 240Phe Ala Tyr Asp Ser Ser Cys Lys Phe Glu Cys Gln Pro Gly Tyr
Arg 245 250 255Ala Arg Gly Ser Asn Thr Leu His Cys Thr Gly Ser Gly
Gln Trp Ser 260 265 270Glu Pro Leu Pro Thr Cys Glu Ala Ile Ala 275
28021282PRTArtificial SequenceChimera-17 (human P-selectin
positions 1-35, and 43-154; mouseP-selectin positions 36-42, and
155-282) 21Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile
Ser Arg1 5 10 15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile
Gln Asn Lys 20 25 30Asn Glu Ile Ala His Leu Asn Asp Val Ile Pro Tyr
Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn Lys Thr
Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu Ala Glu
Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn Asn Glu
Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro Gly Lys
Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala Leu Cys
Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys Gln Gly
Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135 140Tyr
Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Lys Glu Cys Gly145 150
155 160Lys Val Asn Ile Pro Gln His Val Leu Met Asn Cys Ser His Pro
Leu 165 170 175Gly Glu Phe Ser Phe Asn Ser Gln Cys Thr Phe Ser Cys
Ala Glu Gly 180 185 190Tyr Glu Leu Asp Gly Pro Gly Glu Leu Gln Cys
Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Asn Pro Pro Lys Cys Asp
Ala Val Gln Cys Gln Ser Leu 210 215 220Glu Ala Pro Pro His Gly Thr
Met Ala Cys Met His Pro Ile Ala Ala225 230 235 240Phe Ala Tyr Asp
Ser Ser Cys Lys Phe Glu Cys Gln Pro Gly Tyr Arg 245 250 255Ala Arg
Gly Ser Asn Thr Leu His Cys Thr Gly Ser Gly Gln Trp Ser 260 265
270Glu Pro Leu Pro Thr Cys Glu Ala Ile Ala 275
28022279PRTArtificial SequenceChimera-17B (human P-selectin
positions 1-35 and 43-279; mouseP-selectin positions 36-42) 22Trp
Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10
15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys
20 25 30Asn Glu Ile Ala His Leu Asn Asp Val Ile Pro Tyr Tyr Ser Ser
Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp
Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala
Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val
Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro Gly Lys Trp Asn Asp
Glu His Cys Leu Lys Lys 100 105 110Lys His Ala Leu Cys Tyr Thr Ala
Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu
Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe
Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys Gly145 150 155 160Glu
Leu Glu Leu Pro Gln His Val Leu Met Asn Cys Ser His Pro Leu 165 170
175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser Phe His Cys Thr Asp Gly
180 185 190Tyr Gln Val Asn Gly Pro Ser Lys Leu Glu Cys Leu Ala Ser
Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro Gln Cys Leu Ala Ala Gln
Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu Arg Gly Asn Met Thr Cys
Leu His Ser Ala Lys Ala225 230 235 240Phe Gln His Gln Ser Ser Cys
Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250 255Leu Val Gly Pro Glu
Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr 260 265 270Ala Pro Ala
Pro Val Cys Lys 27523282PRTArtificial SequenceChimera-18 (human
P-selectin positions C (C1, C2, C3) and mouseCR1, CR2 substituted
with mouse P-selectin C (C1, C2, C3), CR1and CR2) 23Trp Thr Tyr His
Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys Tyr Cys
Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys 20 25 30Asn Glu
Ile Asp Tyr Leu Asn Lys Val Leu Pro Phe Phe Asn Ser Tyr 35 40 45Tyr
Trp Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp Val Gly 50 55
60Thr Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala Asp Asn Glu65
70 75 80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val Glu Ile Tyr Ile
Lys 85 90 95Ser Asn Ser Ala Pro Gly Lys Trp Asn Asp Glu His Cys Leu
Lys Lys 100 105 110Lys Arg Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp
Met Ser Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu Glu Thr Ile Gly
Asn Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu
Cys Glu Tyr Val Lys Glu Cys Gly145 150 155 160Lys Val Asn Ile Pro
Gln His Val Leu Met Asn Cys Ser His Pro Leu 165 170 175Gly Glu Phe
Ser Phe Asn Ser Gln Cys Thr Phe Ser Cys Ala Glu Gly 180 185 190Tyr
Glu Leu Asp Gly Pro Gly Glu Leu Gln Cys Leu Ala Ser Gly Ile 195 200
205Trp Thr Asn Asn Pro Pro Lys Cys Asp Ala Val Gln Cys Gln Ser Leu
210 215 220Glu Ala Pro Pro His Gly Thr Met Ala Cys Met His Pro Ile
Ala Ala225 230 235 240Phe Ala Tyr Asp Ser Ser Cys Lys Phe Glu Cys
Gln Pro Gly Tyr Arg 245 250 255Ala Arg Gly Ser Asn Thr Leu His Cys
Thr Gly Ser Gly Gln Trp Ser 260 265 270Glu Pro Leu Pro Thr Cys Glu
Ala Ile Ala 275 28024279PRTArtificial SequenceChimera-18B (human
P-selectin positions C (C1, C2, C3 substituted with mouse
P-selectin positions C (C1, C2, C3)) 24Trp Thr Tyr His Tyr Ser Thr
Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln Asn Arg
Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr
Leu Asn Lys Val Leu Pro Phe Phe Asn Ser Tyr 35 40 45Tyr Trp Ile Gly
Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys
Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro
Asn Asn Lys Arg Asn Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90
95Ser Asn Ser Ala Pro Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys
100 105 110Lys Arg Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser
Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr
Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu
Tyr Val Arg Glu Cys Gly145 150 155 160Glu Leu Glu Leu Pro Gln His
Val Leu Met Asn Cys Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe
Asn Ser Gln Cys Ser Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val
Asn Gly Pro Ser Lys Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp
Thr Asn Lys Pro Pro Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215
220Lys Ile Pro Glu Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys
Ala225 230 235 240Phe Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu
Glu Gly Phe Ala 245 250 255Leu Val Gly Pro Glu Val Val Gln Cys Thr
Ala Ser Gly Val Trp Thr 260 265 270Ala Pro Ala Pro Val Cys Lys
27525282PRTArtificial SequenceChimera-19 (human P-selectin
positions Cluster D, CR1, CR2 substituted with mouse P-selectin D,
CR1 and CR2) 25Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp Asn
Ile Ser Arg1 5 10 15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala
Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu Pro
Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Ile Asn Asn
Lys Trp Thr Trp Val Gly 50 55 60Thr Asn Lys Thr Leu Thr Glu Glu Ala
Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn Asn
Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro Gly
Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala Leu
Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys Gln
Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135
140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Lys Glu Cys
Gly145 150 155 160Lys Val Asn Ile Pro Gln His Val Leu Met Asn Cys
Ser His Pro Leu 165 170 175Gly Glu Phe Ser Phe Asn Ser Gln Cys Thr
Phe Ser Cys Ala Glu Gly 180 185 190Tyr Glu Leu Asp Gly Pro Gly Glu
Leu Gln Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Asn Pro Pro
Lys Cys Asp Ala Val Gln Cys Gln Ser Leu 210 215 220Glu Ala Pro Pro
His Gly Thr Met Ala Cys Met His Pro Ile Ala Ala225 230 235 240Phe
Ala Tyr Asp Ser Ser Cys Lys Phe Glu Cys Gln Pro Gly Tyr Arg 245 250
255Ala Arg Gly Ser Asn Thr Leu His Cys Thr Gly Ser Gly Gln Trp Ser
260 265 270Glu Pro Leu Pro Thr Cys Glu Ala Ile Ala 275
28026279PRTArtificial SequenceChimera-19B (human P-selectin
positions Cluster D substitutedwith mouse P-selectin Cluster D)
26Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1
5 10 15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn
Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu
Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Ile Asn
Asn Lys Trp Thr Trp Val Gly 50 55 60Thr Asn Lys Thr Leu Thr Glu Glu
Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn
Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro
Gly Lys Trp Asn Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala
Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Lys
Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135
140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys
Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn Cys
Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser
Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser Lys
Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro
Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu
Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230 235 240Phe
Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250
255Leu Val Gly Pro Glu Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr
260 265 270Ala Pro Ala Pro Val Cys Lys 27527282PRTArtificial
SequenceChimera-20 (human P-selectin positions E, CR1 and CR2
substituted with mouse P-selectin Cluster E, CR1 and CR2) 27Trp Thr
Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys
Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys 20 25
30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser Tyr
35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp Val
Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp Ala Asp
Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys Val Glu
Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro Gly Lys Trp Asn Asp Glu
Pro Cys Phe Lys Arg 100 105 110Lys His Ala Leu Cys Tyr Thr Ala Ser
Cys Gln Asp Met Ser Cys Ser 115 120 125Lys Gln Gly Glu Cys Leu Glu
Thr Ile Gly Asn Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly Phe Tyr
Gly Pro Glu Cys Glu Tyr Val Lys Glu Cys Gly145 150 155 160Lys Val
Asn Ile Pro Gln His Val Leu Met Asn Cys Ser His Pro Leu 165 170
175Gly Glu Phe Ser Phe Asn Ser Gln Cys Thr Phe Ser Cys Ala Glu Gly
180 185 190Tyr Glu Leu Asp Gly Pro Gly Glu Leu Gln Cys Leu Ala Ser
Gly Ile 195 200 205Trp Thr Asn Asn Pro Pro Lys Cys Asp Ala Val Gln
Cys Gln Ser Leu 210 215 220Glu Ala Pro Pro His Gly Thr Met Ala Cys
Met His Pro Ile Ala Ala225 230 235 240Phe Ala Tyr Asp Ser Ser Cys
Lys Phe Glu Cys Gln Pro Gly Tyr Arg 245 250 255Ala Arg Gly Ser Asn
Thr Leu His Cys Thr Gly Ser Gly Gln Trp Ser 260 265 270Glu Pro Leu
Pro Thr Cys Glu Ala Ile Ala 275 28028279PRTArtificial
SequenceChimera-20B (human P-selectin positions E substituted with
mouse P-selectin Cluster E) 28Trp Thr Tyr His Tyr Ser Thr Lys Ala
Tyr Ser Trp Asn Ile Ser Arg1 5 10 15Lys Tyr Cys Gln Asn Arg Tyr Thr
Asp Leu Val Ala Ile Gln Asn Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn
Lys Val Leu Pro Tyr Tyr Ser Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg
Lys Asn Asn Lys Thr Trp Thr Trp Val Gly 50 55 60Thr Lys Lys Ala Leu
Thr Asn Glu Ala Glu Asn Trp Ala Asp Asn Glu65 70 75 80Pro Asn Asn
Lys Arg Asn Asn Glu Asp Cys Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro
Ser Ala Pro Gly Lys Trp Asn Asp Glu Pro Cys Phe Lys Arg 100 105
110Lys His Ala Leu Cys Tyr Thr Ala Ser Cys Gln Asp Met Ser Cys Ser
115 120 125Lys Gln Gly Glu Cys Leu Glu Thr Ile Gly Asn Tyr Thr Cys
Ser Cys 130 135 140Tyr Pro Gly Phe Tyr Gly Pro Glu Cys Glu Tyr Val
Arg Glu Cys Gly145 150 155 160Glu Leu Glu Leu Pro Gln His Val Leu
Met Asn Cys Ser His Pro Leu 165 170 175Gly Asn Phe Ser Phe Asn Ser
Gln Cys Ser Phe His Cys Thr Asp Gly 180 185 190Tyr Gln Val Asn Gly
Pro Ser Lys Leu Glu Cys Leu Ala Ser Gly Ile 195 200 205Trp Thr Asn
Lys Pro Pro Gln Cys Leu Ala Ala Gln Cys Pro Pro Leu 210 215 220Lys
Ile Pro Glu Arg Gly Asn Met Thr Cys Leu His Ser Ala Lys Ala225 230
235 240Phe Gln His Gln Ser Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe
Ala 245 250 255Leu Val Gly Pro Glu Val Val Gln Cys Thr Ala Ser Gly
Val Trp Thr 260 265 270Ala Pro Ala Pro Val Cys Lys
27529279PRTArtificial SequenceChimera-21 (human P-selectin
positions Cluster F substituted with mouse P-selectin Cluster F)
29Trp Thr Tyr His Tyr Ser Thr Lys Ala Tyr Ser Trp Asn Ile Ser Arg1
5 10 15Lys Tyr Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn
Lys 20 25 30Asn Glu Ile Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser
Ser Tyr 35 40 45Tyr Trp Ile Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr
Trp Val Gly 50 55 60Thr Lys Lys Ala Leu Thr Asn Glu Ala Glu Asn Trp
Ala Asp Asn Glu65 70 75 80Pro Asn Asn Lys Arg Asn Asn Glu Asp Cys
Val Glu Ile Tyr Ile Lys 85 90 95Ser Pro Ser Ala Pro Gly Lys Trp Asn
Asp Glu His Cys Leu Lys Lys 100 105 110Lys His Ala Leu Cys Tyr Thr
Ala Ser Cys Gln Asp Met Ser Cys Ser 115 120 125Asn Gln Gly Glu Cys
Ile Glu Thr Ile Gly Ser Tyr Thr Cys Ser Cys 130 135 140Tyr Pro Gly
Phe Tyr Gly Pro Glu Cys Glu Tyr Val Arg Glu Cys Gly145 150 155
160Glu Leu Glu Leu Pro Gln His Val Leu Met Asn Cys Ser His Pro Leu
165 170 175Gly Asn Phe Ser Phe Asn Ser Gln Cys Ser Phe His Cys Thr
Asp Gly 180 185 190Tyr Gln Val Asn Gly Pro Ser Lys Leu Glu Cys Leu
Ala Ser Gly Ile 195 200 205Trp Thr Asn Lys Pro Pro Gln Cys Leu Ala
Ala Gln Cys Pro Pro Leu 210 215 220Lys Ile Pro Glu Arg Gly Asn Met
Thr Cys Leu His Ser Ala Lys Ala225 230 235 240Phe Gln His Gln Ser
Ser Cys Ser Phe Ser Cys Glu Glu Gly Phe Ala 245 250 255Leu Val Gly
Pro Glu Val Val Gln Cys Thr Ala Ser Gly Val Trp Thr 260 265 270Ala
Pro Ala Pro Val Cys Lys 275
* * * * *
References