U.S. patent application number 14/219218 was filed with the patent office on 2014-09-25 for reagents and methods for detecting pnh type ii white blood cells and their identification as risk factors for thrombotic disorders.
This patent application is currently assigned to ALEXION PHARMACEUTICALS, INC.. The applicant listed for this patent is ALEXION PHARMACEUTICALS, INC.. Invention is credited to Susan FAAS MCKNIGHT, Andrea ILLINGWORTH, Mayur MOVALIA, Russell P. ROTHER.
Application Number | 20140286962 14/219218 |
Document ID | / |
Family ID | 43970419 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140286962 |
Kind Code |
A1 |
MOVALIA; Mayur ; et
al. |
September 25, 2014 |
REAGENTS AND METHODS FOR DETECTING PNH TYPE II WHITE BLOOD CELLS
AND THEIR IDENTIFICATION AS RISK FACTORS FOR THROMBOTIC
DISORDERS
Abstract
The disclosure relates to methods for detecting PNH Type II cell
populations in biological samples as well as methods for
determining whether a patient is at an increased risk for
developing thrombocytopenia or thrombosis based on the percentage
of PNH Type II cells in the patient's blood. The disclosure also
features reagents and conjugates for use in the methods.
Inventors: |
MOVALIA; Mayur; (Brewer,
ME) ; ILLINGWORTH; Andrea; (Holden, ME) ; FAAS
MCKNIGHT; Susan; (Old Lyme, CT) ; ROTHER; Russell
P.; (Oklahoma City, OK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALEXION PHARMACEUTICALS, INC. |
Cheshire |
CT |
US |
|
|
Assignee: |
ALEXION PHARMACEUTICALS,
INC.
Cheshire
CT
|
Family ID: |
43970419 |
Appl. No.: |
14/219218 |
Filed: |
March 19, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13508909 |
Oct 1, 2012 |
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PCT/US2010/055997 |
Nov 9, 2010 |
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14219218 |
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61280897 |
Nov 9, 2009 |
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Current U.S.
Class: |
424/158.1 ;
424/93.72; 424/94.67; 435/7.24; 514/169; 514/457; 514/56;
514/7.8 |
Current CPC
Class: |
A61K 38/4886 20130101;
A61P 7/02 20180101; G01N 33/56972 20130101; A61P 7/00 20180101;
G01N 2800/226 20130101; G01N 33/6893 20130101; A61K 31/56 20130101;
A61K 31/352 20130101; A61K 35/19 20130101; A61K 38/196 20130101;
A61K 31/727 20130101; G01N 33/5094 20130101; C07K 16/40
20130101 |
Class at
Publication: |
424/158.1 ;
514/457; 514/56; 424/93.72; 514/169; 514/7.8; 424/94.67;
435/7.24 |
International
Class: |
G01N 33/50 20060101
G01N033/50; A61K 31/727 20060101 A61K031/727; C07K 16/40 20060101
C07K016/40; A61K 31/56 20060101 A61K031/56; A61K 38/19 20060101
A61K038/19; A61K 38/48 20060101 A61K038/48; A61K 31/352 20060101
A61K031/352; A61K 35/14 20060101 A61K035/14 |
Claims
1. A method for predicting whether a patient is at an increased
risk for developing thrombosis, the method comprising: determining
the percentage of paroxysmal nocturnal hemoglobinuria (PNH) Type II
white blood cells of the total white blood cells of the same
histological type in a biological sample from a patient; and
predicting whether the patient is at an increased risk for
developing thrombosis, wherein the patient is at an increased risk
for developing thrombosis if the percentage of PNH Type II white
blood cells is greater than or equal to 1.2%.
2. The method of claim 1, wherein the white blood cells are
granulocytes or monocytes.
3. The method of claim 1, wherein the biological sample is a whole
blood sample.
4. The method of claim 1, wherein a PNH Type II white blood cell
population that is between 1.2% to 65.3%, inclusive of 1.2% and
65.3%, indicates that the patient is at an increased risk for
thrombosis.
5. The method of claim 1, wherein a PNH Type II white blood cell
population that is greater than or equal to a) 5%, b) 10%, c) 20%,
or d) 50% indicates that the patient is at an increased risk for
thrombosis.
6. The method of claim 1, further comprising monitoring the patient
for the development of at least one symptom of thrombosis if the
patient is at an increased risk of developing thrombosis.
7. The method of claim 1, further comprising selecting an
anti-thrombotic therapy for the patient if the patient is at an
increased risk of developing thrombosis.
8. The method of claim 7, wherein the anti-thrombotic therapy is an
anticoagulant or thrombolytic agent.
9. The method of claim 8, wherein the anticoagulant is coumadin,
heparin, or derivatives thereof.
10. The method of claim 8, wherein the thrombolytic agent is a
tissue plasminogen activator, streptokinase, or a urokinase-type
plasminogen activator.
11. The method of claim 1, further comprising administering to the
patient an anti-thrombotic therapy if the patient is at an
increased risk for developing thrombosis.
12. A method for selecting a therapy for a patient, the method
comprising: selecting one or both of an anti-thrombotic therapy and
an anti-thrombocytopenic therapy for a patient determined to have a
paroxysmal nocturnal hemoglobinuria (PNH) Type II white blood cell
population of greater than or equal to 1.2%.
13. The method of claim 12, wherein the anti-thrombotic therapy is
an anticoagulant or thrombolytic agent.
14. The method claim 12, wherein the anti-thrombocytopenic therapy
is a platelet transfusion
15. The method of claim 12, wherein a non-lytic variant form of
aerolysin protein is used to determine the percentage of PNH Type
II white blood cells.
16. A method for treating a patient, the method comprising
administering to a patient in need thereof one or both of an
anti-thrombotic therapy and an anti-thrombocytopenic therapy if the
patient has a paroxysmal nocturnal hemoglobinuria (PNH) Type II
white blood cell population of greater than 1.2%.
17. The method of claim 16, wherein the anti-thrombotic therapy is
an anticoagulant or thrombolytic agent.
18. The method claim 16, wherein the anti-thrombocytopenic therapy
is a platelet transfusion.
19. The method of claim 16, wherein a non-lytic variant form of
aerolysin protein is used to determine the percentage of PNH Type
II white blood cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
Provisional Patent Application Ser. No. 61/280,897, filed Nov. 9,
2009, and entitled "Reagents and methods for detecting PNH type II
cells", the entire contents of which are incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Nov. 5,
2010, is named ALXN150WO1.txt, and is 26,000 bytes in size.
TECHNICAL FIELD
[0003] The field of the invention is medicine, immunology,
molecular biology, and protein chemistry.
BACKGROUND
[0004] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare,
debilitating disease that is characterized by, among other things,
abnormal hematopoiesis, complement-mediated intravascular
hemolysis, and a propensity for thrombosis. See, e.g., Rosse and
Nishimura (2003) Int J Hematol 77(2):121-124 and Brodsky (2008)
Blood Rev 22(2):65-74. PNH is caused by a somatic mutation in the
X-linked phosphatidylinositol glycan complementation class A (PIGA)
gene, which encodes an enzyme that is necessary for the initial
step of glycosylphosphatidylinositol (GPI) anchor biosynthesis. See
Miyata et al. (1993) Science 259:1318-1320 and Bessler et al.
(1994) EMBO J 13:110-117. GPI anchors attach a number of proteins
to the surface of hematopoietic cells. These so called GPI-anchored
proteins include, among others, complement regulatory proteins such
as CD55 (DAF) and CD59. Depending on the type of mutation that
befalls the PIGA gene, a partial or complete loss of GPI anchor
biosynthesis can result, which corresponds to a partial or complete
loss in the presence of GPI-anchored proteins (e.g., GPI-anchored
CD55 and CD59) on the cell surface. See Rosse (1997) Medicine
76:63-93. The partial or complete absence of complement regulatory
proteins on the surface of red blood cells (RBCs) results in the
heightened sensitivity of these cells for complement-mediated lysis
and associated symptoms of PNH in afflicted patients. See
Nicholson-Weller et al. (1983) Proc Natl Acad Sci USA 80:5066-5070
and Yamashina et al. (1990) N Engl J Med 323:1184-1189.
[0005] Traditionally, diagnosis of PNH and monitoring of PNH
patients involved analysis of CD55 and CD59 expression on the
surface of RBCs and granulocytes using flow cytometry. Sutherland
et al. (2009) Am J Clin Pathol 132:564-572. More recently developed
diagnostic methods for PNH have employed a recombinant, non-lytic
form of the bacterial protein aerolysin, which binds to GPI-anchors
on the surface of hematopoietic cells. See U.S. Pat. No. 6,593,095
issued to Buckley and Brodsky. Both traditional and new methods
have allowed medical practitioners to classify RBCs or white blood
cells from PNH patients into one of three groups: Type I cells
having normal or nearly normal cell-surface expression of
GPI-anchored proteins; PNH Type III cells, which have nil or
completely absent cell-surface expression of GPI-anchored proteins;
and PNH Type II cells having an intermediate level of cell-surface
expression of GPI-anchored proteins. Brodsky et al. (2000) Am J
Clin Pathol 114:459-466. The characterization of Type II cells
among white blood cell lineages has not been performed due to the
difficulty in distinguishing these cells from normal Type I white
blood cells.
SUMMARY
[0006] The disclosure is based, at least in part, on the discovery
by the inventors that patients having a PNH Type II white blood
cell population of at least 1.2% or a PNH Type II red blood cell
population of at least 0.02% are more likely to have
thrombocytopenia as compared to patients who do not have PNH Type
II cell populations or who have PNH Type II cell populations that
are smaller than 1.2% or 0.02% for white and red blood cells,
respectively. Patients with thrombocytopenia resulting from
platelet destruction are much more likely to develop thrombosis,
and among PNH patients, thrombosis is the leading cause of death.
Accordingly, the disclosure provides methods for determining
whether a patient is at an increased risk for thrombocytopenia
and/or thrombosis based on the relative population of PNH Type II
cells in the patient. Identification of the nexus between PNH Type
II cells and thrombocytopenia was aided, in part, by the
development of improved methods for detecting PNH Type II
cells.
[0007] Thus, the disclosure also provides reagents and methods
useful for detecting PNH Type II cells (e.g., Type II white blood
cells and/or Type II red blood cells) in, e.g., biological samples
from patients. The disclosure also provides methods for diagnosing
and treating patients based on the presence or amount of PNH Type
II cells in the patient. For example, the disclosure features a
method for determining risk of thrombocytopenia in a patient based
on the percentage of PNH Type II white blood cells detected in a
biological sample from a patient suspected of having PNH. The
diagnostic methods described herein have a number of advantages
over prior art methods. For example, the methods described herein
can more effectively separate PNH Type II white blood cells from
Type I cells, which allows for a more accurate and precise
measurement of the percentage of the Type II cells in a biological
sample, as well as a more accurate assessment of the total PNH
clone size, which comprises both Type II and Type III cells. In
addition, PNH diagnostic methods that rely on GPI-expression on
Type II RBCs can be unreliable because of a high turnover of red
blood cells (the inherent shorter life-span of PNH Type III RBCs
due to elevated sensitivity to complement-mediated lysis) and
frequent RBC transfusions received by PNH patients. Therefore, the
diagnostic methods described herein not only allow a practitioner
to accurately and precisely quantify the percentage of Type II
white blood cells in a biological sample, and thus the total
abnormal clone size in the sample, but the methods are also more
reliable than prior methods that relied on detecting relatively
unstable populations of PNH Type II RBCs.
[0008] In one aspect, the disclosure features a method for
predicting whether a patient is at an increased risk for
thrombosis. The method includes determining whether a patient is at
an increased risk for thrombosis based on the percentage of PNH
Type II cells of the total number of cells of the same histological
type (same lineage) in a biological sample from the patient
indicates that the patient is at an increased risk for
thrombosis.
[0009] In another aspect, the disclosure features a method for
predicting whether a patient is likely to be thrombocytopenic. The
method includes determining whether a patient is likely to be
thrombocytopenic based on the percentage of PNH Type II cells
(e.g., Type II red blood cells and/or Type II white blood cells) of
the total number of cells of the same histological type (same
lineage) in a biological sample from the patient indicates that the
patient is likely to be thrombocytopenic.
[0010] In another aspect, the disclosure features a method for
determining whether a patient is at an increased risk for
thrombosis. The method includes providing (or receiving)
information on the percentage of PNH Type II cells of the total
cells of the same histological type (same lineage) in a biological
sample from a patient; and determining whether a patient is at an
increased risk for thrombosis, wherein the percentage of PNH Type
II cells of the total number of cells of the same histological type
in the biological sample indicates that the patient is at an
increased risk for thrombosis.
[0011] In another aspect, the disclosure features a method for
predicting whether a patient is at risk for developing thrombosis,
which method includes determining the percentage of PNH Type II
cells in a biological sample from a patient; and providing a
prediction of whether the patient is at an increased risk for
thrombosis, wherein the percentage of PNH Type II cells of the
total number of cells of the same histological type in the
biological sample indicates that the patient is at an increased
risk for thrombosis.
[0012] In some embodiments, the PNH Type II cells are white blood
cells (e.g., granulocytes or monocytes). In some embodiments, the
PNH Type II cells are red blood cells.
[0013] In some embodiments of any of the methods described herein,
the combination of a percentage of PNH type II white blood cells
that is greater than or equal to 1.2% and a percentage of PNH type
II red blood cells that is greater than or equal to 0.02% is
predictive of whether the patient is at an increased risk of
developing thrombosis or is likely to be thrombocytopenic.
[0014] In some embodiments of any of the methods described herein,
the patient is at an increased risk of developing thrombosis
(and/or likely to be thrombocytopenic) when the percentage of PNH
Type II white blood cells is at least 1.2 (e.g., at least 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 65.3, or 70 or more) %. In some embodiments of any
of the methods described herein, the patient is at an increased
risk of developing thrombosis (and/or likely to be
thrombocytopenic) when the percentage of PNH Type II red blood
cells is at least 0.02 (e.g., at least 0.02, 0.03, 0.04, 0.05,
0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,
3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 71.3, or 75
or more) %.
[0015] In some embodiments of any of the methods described herein,
a PNH Type II white blood cell population that is between 1.2% to
65%, inclusive of 1.2% and 65%, indicates that the patient is at an
increased risk for thrombosis (and/or likely to be
thrombocytopenic). In some embodiments, a PNH Type II white blood
cell population that is greater than or equal to 5% indicates that
the patient is at an increased risk for thrombosis (and/or likely
to be thrombocytopenic). In some embodiments, a PNH Type II white
blood cell population that is greater than or equal to 10%, 20%, or
even 50% indicates that the patient is at an increased risk for
thrombosis (and/or likely to be thrombocytopenic).
[0016] In some embodiments, any of the methods described herein can
further include obtaining the biological sample from the patient.
The biological sample can be, e.g., a whole blood sample.
[0017] In another aspect, the disclosure features a method for
predicting whether a patient is at an increased risk for developing
thrombosis (and/or likely to be thrombocytopenic). The method
includes determining the percentage of Type II white blood cells of
the total white blood cells of the same histological type in a
biological sample from a patient; and predicting whether the
patient is at an increased risk for developing thrombosis, wherein
the patient is at an increased risk for developing thrombosis
(and/or likely to be thrombocytopenic) if the percentage of Type II
white blood cells is greater than or equal to 1.2 (e.g., greater
than or equal to 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,
3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 65.3, or 70 or
more) %.
[0018] In another aspect, the disclosure features a method for
predicting whether a patient is at an increased risk for developing
thrombosis (and/or likely to be thrombocytopenic). The method
includes determining the percentage of Type II red blood cells of
the total red blood cells of the same histological type in a
biological sample from a patient; and predicting whether the
patient is at an increased risk for developing thrombosis (and/or
likely to be thrombocytopenic), wherein the patient is at an
increased risk for developing thrombosis (and/or likely to be
thrombocytopenic) if the percentage of Type II red blood cells is
greater than or equal to 0.02 (e.g., greater than or equal to 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 13, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,
2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3,
3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71,
71.3, or 75 or more) %.
[0019] In yet another aspect, the disclosure features a method for
selecting a therapy for a patient, which method includes selecting
one or both of an anti-thrombotic therapy and an
anti-thrombocytopenic therapy for a patient determined to have a
PNH Type II white blood cell population of greater than or equal to
1.2 (e.g., greater than or equal to 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
65.3, or 70 or more) % and/or a PNH Type II red blood cell
population of greater than or equal to 0.02 (e.g., greater than or
equal to 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,
3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 71, 71.3, or 75 or more) %.
[0020] In another aspect, the disclosure features a method for
treating a patient. The method includes administering to a patient
in need thereof one or both of an anti-thrombotic therapy and an
anti-thrombocytopenic therapy if the patient is determined to have
a PNH Type II white blood cell population of greater than or equal
to 1.2 (e.g., greater than or equal to 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,
3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 65.3, or 70 or more) % and/or a PNH Type II red blood cell
population of greater than or equal to 0.02 (e.g., greater than or
equal to 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,
3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 71, 71.3, or 75 or more) %.
[0021] In some embodiments of any of the methods described herein,
the anti-thrombocytopenic therapy can be, e.g., platelet
transfusion.
[0022] In yet another aspect, the disclosure features a
computer-based method for determining whether a patient is at an
increased risk for developing thrombosis, which method includes
receiving data including a medical profile of a PNH patient, the
profile comprising information on the percentage of PNH Type II
white blood cells of the total white blood cells of the same
histological type (same lineage) in a biological sample from the
patient; and processing at least the portion of the data containing
the information to determine whether the patient is at an increased
risk for developing thrombosis, wherein the patient is at an
increased risk for developing thrombosis if the percentage of Type
II white blood cells is greater than or equal to 1.2 (e.g., greater
than or equal to 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,
3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 65.3, or 70 or
more) %.
[0023] In yet another aspect, the disclosure features a
computer-based method for determining whether a patient is at an
increased risk for developing thrombosis, which method includes
receiving data including a medical profile of a PNH patient, the
profile comprising information on the percentage of PNH Type II red
blood cells of the total red blood cells of the same histological
type (same lineage) in a biological sample from the patient; and
processing at least the portion of the data containing the
information to determine whether the patient is at an increased
risk for developing thrombosis, wherein the patient is at an
increased risk for developing thrombosis if the percentage of Type
II red blood cells is greater than or equal to 0.02 (e.g., greater
than or equal to 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 71, 71.3, or 75 or more) %.
[0024] In another aspect, the disclosure features a computer-based
method for determining whether a patient is at an increased risk
for developing thrombosis, which method includes providing
information on the percentage of PNH Type II white blood cells of
the total white blood cells of the same histological type (same
lineage) in a biological sample from the patient; inputting the
information into a computer; and calculating a parameter indicating
whether the patient is at an increased risk for thrombosis using
the computer and the input information, wherein the patient is at
an increased risk for developing thrombosis if the percentage of
Type II white blood cells is greater than or equal to 1.2 (e.g.,
greater than or equal to 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,
2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 65.3, or 70 or
more) %.
[0025] In another aspect, the disclosure features a computer-based
method for determining whether a patient is at an increased risk
for developing thrombosis, which method includes providing
information on the percentage of PNH Type II white blood cells of
the total white blood cells of the same histological type in a
biological sample from the patient; inputting the information into
a computer; and calculating a parameter indicating whether the
patient is at an increased risk for thrombosis using the computer
and the input information, wherein the patient is at an increased
risk for developing thrombosis if the percentage of Type II white
blood cells is greater than or equal to 0.02 (e.g., greater than or
equal to 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,
3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 71, 71.3, or 75 or more) %.
[0026] In some embodiments, any of the computer-based methods
described herein can further include storing the parameter on a
computer-readable medium and/or outputting the parameter.
[0027] In some embodiments, any of the methods described herein can
include the step of monitoring the patient for the development of
at least one symptom of thrombosis if the patient is at an
increased risk of developing thrombosis. In some embodiments, any
of the methods described herein can include selecting an
anti-thrombotic therapy for the patient if the patient is at an
increased risk of developing thrombosis. In some embodiments, any
of the methods described herein can include administering to the
patient an anti-thrombotic therapy if the patient is at an
increased risk for developing thrombosis. The anti-thrombotic
therapy can be, e.g., an anticoagulant or a thrombolytic agent. The
anticoagulant can be, e.g., coumadin, heparin, or derivatives
thereof. The thrombolytic agent can be, e.g., a tissue plasminogen
activator, streptokinase, or a urokinase-type plasminogen
activator.
[0028] In some embodiments of any of the methods described herein,
a non-lytic variant form of aerolysin protein can be used to
determine the percentage of PNH Type II white blood cells in the
biological sample.
[0029] In some embodiments, any of the methods described herein can
include recording the determined percentage of PNH Type II cells in
the biological sample. In some embodiments, any of the methods
described herein can include recording the prediction of whether
the patient is at an increased risk for developing thrombosis or
whether the patient is not at an increased risk for developing
thrombosis. The recordation can be on a computer-readable medium.
The recordation can also be, e.g., on a tangible medium (e.g., a
patient's physical record or chart).
[0030] In yet another aspect, the disclosure features a method for
classifying white blood cells. The method contacting a plurality of
white blood cells with a reagent that binds to: (i) GPI or (ii) a
GPI-anchored protein; and classifying one or more of the white
blood cells as PNH Type II cells based on the amount of reagent
bound to the cells.
[0031] In another aspect, the disclosure features a method for
classifying white blood cells, which method includes contacting a
plurality of white blood cells with a reagent that binds to: (i)
GPI or (ii) a GPI-anchored protein; interrogating at least a
portion of the white blood cells contacted with the reagent based
on the amount of reagent bound to the cells; and classifying one or
more of the interrogated cells as PNH Type II cells.
[0032] In another aspect, the disclosure features a method for
distinguishing between different white blood cell populations. The
method includes contacting a plurality of white blood cells with a
reagent that binds to: (i) GPI or (ii) a GPI-anchored protein; and
distinguishing at least a portion of the white blood cells from
other white blood cells of the plurality based on the amount of
reagent bound the cells, wherein the PNH Type II white blood cells,
if present, are sufficiently distinguished from the Type I white
blood cells and PNH Type III cells of the same histological type
(same lineage) to allow the percentage of PNH Type II white blood
cells of the total white blood cells of the same histological type
in the plurality to be determined. The method can also include
determining the percentage of PNH Type II white blood cells.
[0033] In yet another aspect, the disclosure features a method for
determining the percentage of PNH Type II white blood cells in a
sample, which method includes interrogating a plurality of white
blood cells contacted with a reagent based on the amount of reagent
bound to the cells, wherein the reagent binds to: (i) GPI or (ii) a
GPI-anchored protein, wherein the interrogating sufficiently
distinguishes the PNH Type II white blood cells, if present, from
the Type I white blood cells and PNH Type III cells of the same
histological type to allow the percentage of PNH Type II white
blood cells of the total white blood cells of the same histological
type in the plurality to be determined; and determining the
percentage of PNH Type II white blood cells.
[0034] In some embodiments of any of the methods described herein,
the plurality of white blood cells are contacted with a reagent
that binds to GPI and a reagent that binds to a GPI-linked
protein.
[0035] In some embodiments of any of the methods described herein,
the distinguishing or interrogating of white blood cells (and/or
the determination of the percentage of PNH Type II white blood
cells) includes flow cytometry.
[0036] In some embodiments of any of the methods described herein,
the plurality of white blood cells to be interrogated are obtained
from a patient having, suspected of having, or at risk of
developing PNH. In some embodiments, the patient is one for whom a
percentage of PNH Type II red blood cells has been previously
determined, but was suspect and/or inconclusive.
[0037] In some embodiments, any of the methods described herein can
further include recording the percentage of PNH Type II white blood
cells. The recordation can be on a computer-readable medium or a
tangible medium (e.g., a patient chart or record).
[0038] In some embodiments of any of the methods described herein,
the reagent can bind to a human GPI moiety. The reagent can be,
e.g., an antibody or an antigen-binding fragment thereof, or an
aerolysin protein. The aerolysin protein can be, e.g., a variant
form of aerolysin protein that is non-lytic or is substantially
non-lytic as compared to the wildtype form of the protein. The
non-lytic or substantially non-lytic aerolysin protein can comprise
the amino acid sequence depicted in SEQ ID NO:2 or 7 wherein the
threonine at position 253 is substituted with a cysteine and the
alanine at position 300 is substituted for a cysteine.
[0039] In some embodiments of any of the methods described herein,
the reagent can bind to a GPI-anchored protein. For example, the
reagent can be, e.g., an antibody or an antigen-binding fragment
thereof that binds to a GPI-anchored protein. The GPI-anchored
protein can be, e.g., alkaline phosphatase, 5' nucleotidease
acetylcholinesterase, dipeptidase, LFA-3, NCAM, PH-20, CD55, CD59,
Thy-1, Qa-2, CD14, CD33, CD16 (the Fc.sub..gamma. receptor III),
carcinoembryonic antigen (CEA), CD24, CD66b, CD87, CD48, CD52, or
any other GPI-anchored protein that is known in the art and/or set
forth herein.
[0040] In some embodiments, a patient determined to have a PNH Type
II white blood cell population of greater than or equal to 1.2% or
a PNH Type II red blood cell population of greater than or equal to
0.02% can be diagnosed as having PNH. In some embodiments, a
patient diagnosed as having PNH or a previously diagnosed PNH
patient who is determined to have a PNH Type II white blood cell
population greater than or equal to 1.2% or a PNH Type II red blood
cell population that is greater than or equal to 0.02% can be
prescribed and/or treated with a complement inhibitor such as, but
not limited to, eculizumab.
[0041] In yet another aspect, the disclosure features an antibody
or an antigen-binding fragment thereof that binds to a human GPI
moiety. The antibody or antigen-binding fragment thereof can be,
e.g., a recombinant antibody, a diabody, a chimerized or chimeric
antibody, a deimmunized human antibody, a fully human antibody, a
single chain antibody, an Fv fragment, an Fd fragment, an Fab
fragment, an Fab' fragment, and an F(ab').sub.2 fragment.
[0042] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure pertains. In
case of conflict, the present document, including definitions, will
control. Preferred methods and materials are described below,
although methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
presently disclosed methods and compositions. All publications,
patent applications, patents, and other references mentioned herein
are incorporated by reference in their entirety.
[0043] Other features and advantages of the present disclosure,
e.g., methods for determining risk of thrombocytopenia or
thrombosis in a subject, will be apparent from the following
description, the examples, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 is a two color dot plot depicting a population of
human peripheral blood granulocytes that were incubated with a
solution containing both a non-lytic variant of aerolysin
conjugated with Alexa Fluor.RTM. 488 and an antibody that binds to
CD24 conjugated to phyocoerythrin (PE). The non-lytic aerolysin
binds specifically to the GPI anchor, and therefore cells
expressing any GPI-anchored proteins are labeled with this
fluorescent protein. CD24 is a GPI-linked protein expressed on
granulocytes, so cells expressing CD24 will be bound by both the
anti-CD24 antibody and the non-lytic aerolysin. The X-axis
represents the log intensity of detectable signal produced from the
aerolysin conjugate bound to the cells and the Y-axis represents
the log intensity of the detectable signal produced from the
anti-CD24 antibody conjugate bound to the cells. Three populations
of granulocytes are revealed by this analysis: Type III cells,
which are devoid of GPI-linked proteins and thus appear unlabeled
with either the anti-CD24 antibody and the non-lytic aerolysin;
Type I granulocytes, which express high levels of GPI-linked
proteins relative to cells lacking GPI-anchors; and Type II
granulocytes, which express intermediate levels of GPI-linked
proteins and thus are labeled with both the anti-CD24 and non-lytic
aerolysin at lower levels than those seen on normal (Type I)
granulocytes.
[0045] FIG. 2 is a scatter plot depicting the absolute platelet
count versus the percentage of PNH Type II granulocytes in the
blood of patients with PNH. The Y-axis represents the platelet
count in 1 .mu.L of patient blood (.times.10.sup.-3) and the X-axis
represents the percentage of PNH Type II granulocytes within the
total granulocyte population. The left half of the plot is a
distribution of the platelet counts observed among PNH patients
(N=141) that have no detectable PNH Type II granulocyte
populations. The right half of the plot is a distribution of the
platelet counts observed among PNH patients (N=19) who have
detectable PNH Type II granulocyte populations.
DETAILED DESCRIPTION
[0046] The present disclosure features a variety of diagnostic and
therapeutic applications that are useful for, inter alia,
determining whether a patient has a PNH Type II cell population
and/or is at an increased risk for developing thrombocytopenia
and/or thrombosis. The disclosure also features reagents that can
be used in the methods. While in no way intended to be limiting,
exemplary reagents, conjugates, and methods for using any of the
foregoing are elaborated on below and are exemplified in the
working Examples.
Reagents
[0047] The disclosure features a number of reagents that are useful
in the diagnostic and therapeutic methods described herein. In some
embodiments, the reagent binds to a glycosylphosphatidylinositol
(GPI) moiety, which anchors many cell surface proteins to the cell
membrane. GPI moieties generally contain a core of
ethanolamine-HPO.sub.4-6Man.alpha.1-2Man.alpha.1-6Man.alpha.1-4GlcNH.sub.-
21-6myo-inositol-1HPO.sub.4-diacyl-glycerol (or alkylacylglycerol
or ceramide). See, e.g., Paulick and Bertozzi (2008) Biochemistry
47(27):6991-7000. However, a number of variations on this core
structure have been reported. For example, the glycan core can be
modified with side chains such as, but not limited to,
phosphoethanolamine, mannose, galactose, sialic acid, or other
sugars. Id.
[0048] In some embodiments, the reagent can be an aerolysin
protein, e.g., a non-lytic aerolysin protein. Aerolysin is a
channel-forming cytolytic protein that is expressed by virulent
Aeromonas species such as, but not limited to, Aeromonas hydrophila
and Aeromonas salmonicida. Aerolysin is secreted from the bacterial
cell as a 52 kDa precursor that is converted to the active form
(activated) by proteolytic removal of a C-terminal peptide. The
aerolysin precursor can be activated by host proteases as well as
proteases secreted by an aerolysin-expressing bacterium. Once bound
to a cell, aerolysin oligomerizes to produce channels in, and
ultimately lyse, the cell (Howard and Buckley (1985) J Bacteriol
163:336-340).
[0049] The amino acid sequences of the aerolysin polypeptide
produced by each of various members of the Aeromonas family are
highly conserved. Accordingly, an aerolysin polypeptide, as used
herein, can be from any species of Aeromonas such as, but not
limited to, A. hydrophila, A. caviae, A. veronii (biotype sobria),
A. veronii (biotype veronii), A. jandaei, A. salmonicida, and A.
schubertii.
[0050] In some embodiments, the aerolysin polypeptide is from A.
hydrophila or A. salmonicida. In some embodiments, the aerolysin
polypeptide is a proform containing a 24 amino acid signal peptide.
In some embodiments, the proform aerolysin polypeptide can have, or
consist of, a polypeptide having the amino acid sequence depicted
in SEQ ID NO:1 or SEQ ID NO:6.
[0051] In some embodiments, the aerolysin polypeptide is a form of
the protein in which the signal sequence has been removed. For
example, the aerolysin polypeptide can have, or consist of, a
polypeptide having an amino acid sequence depicted in SEQ ID NO:2
or SEQ ID NO:7.
[0052] In some embodiments, the aerolysin polypeptide is an active
form of the protein. For example, the aerolysin polypeptide can
have, or consist of, a polypeptide having an amino acid sequence
depicted in SEQ ID NO:3.
[0053] As used herein, "polypeptide," "peptide," and "protein" are
used interchangeably and mean any peptide-linked chain of amino
acids, regardless of length or post-translational modification. The
aerolysin polypeptides described herein can contain or be wildtype
proteins or can be variants of the wild-type polypeptides that have
not more than 50 (e.g., not more than one, two, three, four, five,
six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50)
conservative amino acid substitutions. Conservative substitutions
typically include substitutions within the following groups:
glycine and alanine; valine, isoleucine, and leucine; aspartic acid
and glutamic acid; asparagine, glutamine, serine and threonine;
lysine, histidine and arginine; and phenylalanine and tyrosine.
[0054] The aerolysin polypeptides described herein also include
"GPI-binding fragments" of the polypeptides, which are shorter than
the full-length, proform polypeptides, but retain at least 10%
(e.g., at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 50%, at least 55%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98%, at least 99%, at least 99.5%, or 100% or more)
of the ability of the active polypeptide to bind to a GPI moiety.
GPI-binding fragments of an aerolysin polypeptide include terminal
as well internal deletion variants of the protein. Deletion
variants can lack one, two, three, four, five, six, seven, eight,
nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid
segments (of two or more amino acids) or non-contiguous single
amino acids. GPI-binding fragments can be at least 40 (e.g., at
least 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
200, 250, 300, or 325 or more) amino acid residues in length (e.g.,
at least 40 contiguous amino acid residues of SEQ ID Nos:1-3). In
some embodiments, the GPI-binding fragment of an aerolysin
polypeptide is less than 400 (e.g., less than 350, 325, 300, 275,
250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,
95, 90, 85, 80, 75, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, or
40) amino acid residues in length (e.g., less than 400 contiguous
amino acid residues of SEQ ID NOs:1-3). In some embodiments, the
GPI-binding fragment of an aerolysin polypeptide is at least 40,
but less than 400, amino acid residues in length.
[0055] In some embodiments, the GPI-binding fragment of an
aerolysin polypeptide can include, or consist of, a polypeptide
having the following amino acid sequence: L
DPDSFKHGDVTQSDRQLVKTVVGWAVNDSDTPQSGYD
VTLRYDTATNWSKTNTYGLSEKVTTKNKFKWPLVGET
ELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKIP VKIELYKADISYPY (SEQ ID
NO:4).
[0056] In some embodiments, the GPI-binding fragment of an
aerolysin polypeptide can include, or consist of, a polypeptide
having the following amino acid sequence: L
DPDSFKHGDVTQSDRQLVKTVVGWAVNDSDTPQSGYD
VTLRYDTATNWSKTNTYGLSEKVTTKNKFKWPLVGEC
ELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKIP VKIELYKCDISYPY (SEQ ID
NO:5).
[0057] In some embodiments, the aerolysin polypeptide can have an
amino acid sequence that is, or is greater than, 70 (e.g., 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) % identical to the
aerolysin sequence having the amino acid sequence depicted in any
one of SEQ ID NOs:1-3, 6, or 7 (see below).
[0058] Percent (%) amino acid sequence identity is defined as the
percentage of amino acids in a candidate sequence that are
identical to the amino acids in a reference sequence, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Alignment for
purposes of determining percent sequence identity can be achieved
in various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full-length of the sequences
being compared can be determined by known methods.
[0059] Depending on the intended application, in some embodiments
it may be preferable to use a variant aerolysin polypeptide that
lacks the ability to lyse cells. Such variant forms of the
aerolysin polypeptide are known in the art and described in, e.g.,
Brodsky et al. (2000) Am J Clin Pathol 114:459-466. In some
embodiments, the non-lytic, variant form of aerolysin contains, or
consists of, the amino acid sequence depicted in SEQ ID NO:2 or SEQ
ID NO:7 wherein one or more of the histidine at position 132 is
substituted for an asparagine (His132Asn); the glycine at position
202 is a cysteine; the threonine at position 253 is a cysteine and
the alanine at position 300 is a cysteine; and the threonine at
position 225 is a glycine. One exemplary non-lytic variant of
aerolysin comprises the amino acid sequence depicted in SEQ ID NOs:
2 or 7, wherein the threonine at position 253 is a cysteine and the
alanine at position 300 is a cysteine. As described above, the
variant forms will retain the ability to bind to GPI moieties.
[0060] In some embodiments, the variant aerolysin polypeptide has
less than 10 (e.g., less than 9, 8, 7, 6, 5, 4, 3, 2, or 1) % of
the ability of the non-variant counterpart aerolysin polypeptide to
lyse target cells. In some embodiments, the variant aerolysin
polypeptide has no detectable cytolytic activity.
[0061] Methods for determining whether a variant aerolysin
polypeptide binds to a GPI moiety are known in the art and
exemplified in the working examples. For example, cell-based
methods for detecting the binding between a variant aerolysin
polypeptide and a GPI moiety on a cell surface can be determined
using flow cytometry techniques and a dectectably-labeled (e.g., a
fluorophore-labeled) variant aerolysin polypeptide. See, e.g., Hong
et al. (2002) EMBO J 21(19):5047-5056.
[0062] Likewise, methods for detecting and/or quantitating the
cytolytic activity of an aerolysin polypeptide or variant thereof
are also known in the art. For example, the hemolytic activity of a
variant Aerolysin polypeptide can be determined by contacting the
variant polypeptide to normal human erythrocytes and measuring the
amount of hemoglobin released from the erythrocytes. See, e.g.,
Howard and Buckley (1982) Biochemistry 21(7):1662-1667; Avigad and
Bernheimer (1976) Infection and Immunity 13(5):1378-1381; Garland
and Buckley (1988) Infection and Immunity 56(5):1249-1253; and
Bernheimer and Avigard (1974) Infection and Immunity 9:1016-1021. A
decreased amount, or the absence of, cytolytic activity by the
variant, as compared to the amount of cytolytic activity possessed
by the non-variant counterpart polypeptide, is an indication that
the variant polypeptide has reduced or absent cytolytic
activity.
[0063] Methods for obtaining an aerolysin polypeptide, or producing
a variant of the polypeptide as described herein, are known in the
art of molecular biology and exemplified in the working Examples.
See, e.g., Sambrook et al. (1989) "Molecular Cloning: A Laboratory
Manual, 2.sup.nd Edition," Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. and Ausubel et al. (1992) "Current
Protocols in Molecular Biology," Greene Publishing Associates.
Template DNA encoding an aerolysin polypeptide can be obtained from
any of the Aeromonas species described herein using standard
techniques (see, e.g., Sambrook et al. (1989), supra). For example,
Howard et al. describes the isolation and characterization of a
nucleic acid sequence encoding an aerolysin polypeptide from
Aeromonas hydrophila (Howard et al. (1987) J Bacteriol
169(6):2869-2871). An aerolysin polypeptide isolated from Aeromonas
salmonicida is described in Buckley (1990) Biochem. Cell Biol.
68:221-224 and Wong et al. (1989) J Bacteriol. 171:2523-2527.
[0064] In some embodiments, an aerolysin polypeptide can contain
internal or terminal (carboxy or amino-terminal) irrelevant or
heterologous amino acid sequences (e.g., sequences derived from
other proteins or synthetic sequences not corresponding to any
naturally occurring protein). The sequences can be, for example, an
antigenic tag (e.g., FLAG, polyhistidine, hemagglutinin (HA),
glutathione-S-transferase (GST), or maltose-binding protein (MBP)).
Heterologous sequences can also include proteins useful as
diagnostic or detectable markers, for example, luciferase, green
fluorescent protein (GFP), or chloramphenicol acetyl transferase
(CAT).
[0065] Exemplary aerolysin polypeptides as well as methods for
preparing and purifying the polypeptides are described in U.S.
provisional patent application Ser. No. 61/200,655, the disclosure
of which is incorporated herein by reference in its entirety.
[0066] In some embodiments, the reagent can be an antibody that
binds to a GPI moiety. Antibodies that bind to non-human GPI
moieties have been identified and isolated. See, e.g., Naik et al.
(2006) Infection and Immunity 74(2):1412 (isolation of a
naturally-occurring antibody that binds to the GPI moieties of
Plasmodium falciparum.). As described in detail herein, it is well
within the capability of an ordinarily skilled artisan to generate
an antibody that binds to a human GPI moiety.
[0067] As used herein, the term "antibody" refers to a whole or
intact antibody molecule (e.g., IgM, IgG (including IgG1, IgG2,
IgG3, and IgG4), IgA, IgD, or IgE) or any antigen-binding fragment
thereof. The term antibody includes, e.g., a chimerized or chimeric
antibody, a humanized antibody, a deimmunized antibody, and a fully
human antibody. Antigen-binding fragments of an antibody include,
e.g., a single chain antibody, a single chain Fv fragment (scFv),
an Fd fragment, an Fab fragment, an Fab' fragment, or an
F(ab).sub.2 fragment. An scFv fragment is a single polypeptide
chain that includes both the heavy and light chain variable regions
of the antibody from which the scFv is derived. In addition,
intrabodies, minibodies, triabodies, and diabodies (see, e.g.,
Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and
Kortt (1999) J Immunol Methods 231(1):177-189; Poljak (1994)
Structure 2(12):1121-1123; Rondon and Marasco (1997) Annual Review
of Microbiology 51:257-283, the disclosures of each of which are
incorporated herein by reference in their entirety) are also
included in the definition of antibody and are compatible for use
in the methods described herein. Bispecific antibodies are also
embraced by the term "antibody." Bispecific antibodies are
monoclonal, preferably human or humanized, antibodies that have
binding specificities for at least two different antigens. Methods
for generating an antibody or a fragment thereof are discussed
herein.
[0068] In some embodiments, the reagent can bind to a GPI-anchored
protein. For example, the reagent can be an antibody that binds to
a GPI-anchored protein. In some embodiments, the reagent can be a
ligand for a GPI-anchored protein. GPI-anchored proteins are myriad
and include, without limitation, alkaline phosphatase, 5'
nucleotidease acetylcholinesterase, dipeptidase, LFA-3, NCAM,
PH-20, CD55, CD59, Thy-1, Qa-2, CD14, CD33, CD16 (the
Fc.sub..gamma. receptor III), carcinoembryonic antigen (CEA), and
CD52. Antibodies that bind to GPI-anchored proteins are well known
in the art and are described in, e.g., Hall and Rosse (1996) supra,
Richards et al. (2008) Cytometry B Clin Cytom 76B(1):47-55;
Richards and Barnett (2007) Clin Lab Med 27(3):577-590; Luzzatto et
al. (2006) Int J Hematol 84(2):104-112; and Thomason et al. (2004)
Am J Clin Pathol 122(1):128-134. Such antibodies are also
commercially available from, e.g., Santa Cruz Biotechology, Inc.
(Santa Cruz, Calif.), Novus Biologicals (Littleton, Colo.), and
R& D Systems (Minneapolis, Minn.).
[0069] Suitable methods for generating an antibody that binds to a
GPI-anchored protein or a GPI moiety for use in the diagnostic
and/or therapeutic methods described are well known in the art and
described in the following section.
Methods for Generating an Antibody
[0070] Suitable methods for producing an antibody (e.g., an
antibody that binds to a GPI moiety or a GPI-anchored protein), or
antigen-binding fragments thereof, in accordance with the
disclosure are known in the art and described herein. For example,
monoclonal anti-CD55 antibodies may be generated using human
CD55-expressing cells, a CD55 polypeptide, or an antigenic fragment
of CD55 polypeptide, as an immunogen, thus raising an immune
response in animals from which antibody-producing cells and in turn
monoclonal antibodies may be isolated. The sequence of such
antibodies may be determined and the antibodies or variants thereof
produced by recombinant techniques. Recombinant techniques may be
used to produce chimeric, CDR-grafted, humanized and fully human
antibodies based on the sequence of the monoclonal antibodies as
well as polypeptides capable of binding to a GPI-anchored protein
or a GPI moiety.
[0071] Moreover, antibodies derived from recombinant libraries
("phage antibodies") may be selected using, e.g., cells expressing
a GPI moiety or a GPI-anchored protein, recombinant GPI-linked
proteins, or free GPI moieties as bait to isolate the antibodies or
polypeptides on the basis of target specificity. The production and
isolation of non-human and chimeric antibodies are well within the
purview of the skilled artisan.
[0072] Recombinant DNA technology can be used to modify one or more
characteristics of the antibodies produced in non-human cells.
Thus, chimeric antibodies can be constructed in order to decrease
the immunogenicity thereof in diagnostic or therapeutic
applications. Moreover, immunogenicity can be minimized by
humanizing the antibodies by CDR grafting and, optionally,
framework modification. See, U.S. Pat. Nos. 5,225,539 and
7,393,648, the contents of each of which are incorporated herein by
reference.
[0073] Antibodies can be obtained from animal serum or, in the case
of monoclonal antibodies or fragments thereof, produced in cell
culture. Recombinant DNA technology can be used to produce the
antibodies according to established procedure, including procedures
in bacterial or preferably mammalian cell culture. The selected
cell culture system preferably secretes the antibody product.
[0074] In another embodiment, a process for the production of an
antibody disclosed herein includes culturing a host, e.g. E. coli
or a mammalian cell, which has been transformed with a hybrid
vector. The vector includes one or more expression cassettes
containing a promoter operably linked to a first DNA sequence
encoding a signal peptide linked in the proper reading frame to a
second DNA sequence encoding the antibody protein. The antibody
protein is then collected and isolated. Optionally, the expression
cassette may include a promoter operably linked to polycistronic
(e.g., bicistronic) DNA sequences encoding antibody proteins each
individually operably linked to a signal peptide in the proper
reading frame.
[0075] Multiplication of hybridoma cells or mammalian host cells in
vitro is carried out in suitable culture media, which include the
customary standard culture media (such as, for example Dulbecco's
Modified Eagle Medium (DMEM) or RPMI 1640 medium), optionally
replenished by a mammalian serum (e.g. fetal calf serum), or trace
elements and growth sustaining supplements (e.g. feeder cells such
as normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages, 2-aminoethanol, insulin, transferrin, low density
lipoprotein, oleic acid, or the like). Multiplication of host cells
which are bacterial cells or yeast cells is likewise carried out in
suitable culture media known in the art. For example, for bacteria
suitable culture media include medium LE, NZCYM, NZYM, NZM,
Terrific Broth, SOB, SOC, 2xYT, or M9 Minimal Medium. For yeast,
suitable culture media include medium YPD, YEPD, Minimal Medium, or
Complete Minimal Dropout Medium.
[0076] In vitro production provides relatively pure antibody
preparations and allows scale-up production to give large amounts
of the desired antibodies. Techniques for bacterial cell, yeast,
plant, or mammalian cell cultivation are known in the art and
include homogeneous suspension culture (e.g. in an airlift reactor
or in a continuous stirrer reactor), and immobilized or entrapped
cell culture (e.g. in hollow fibers, microcapsules, on agarose
microbeads or ceramic cartridges).
[0077] Large quantities of the desired antibodies can also be
obtained by multiplying mammalian cells in vivo. For this purpose,
hybridoma cells producing the desired antibodies are injected into
histocompatible mammals to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially mineral oils such as pristane (tetramethyl-pentadecane),
prior to the injection. After one to three weeks, the antibodies
are isolated from the body fluids of those mammals. For example,
hybridoma cells obtained by fusion of suitable myeloma cells with
antibody-producing spleen cells from Balb/c mice, or transfected
cells derived from hybridoma cell line Sp2/0 that produce the
desired antibodies are injected intraperitoneally into Balb/c mice
optionally pre-treated with pristane. After one to two weeks,
ascitic fluid is taken from the animals.
[0078] The foregoing, and other, techniques are discussed in, for
example, Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat.
No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual,
(1988) Cold Spring Harbor, the disclosures of which are all
incorporated herein by reference. Techniques for the preparation of
recombinant antibody molecules are described in the above
references and also in, e.g.: WO97/08320; U.S. Pat. No. 5,427,908;
U.S. Pat. No. 5,508,717; Smith (1985) Science 225:1315-1317;
Parmley and Smith (1988) Gene 73:305-318; De La Cruz et al. (1988)
Journal of Biological Chemistry 263:4318-4322; U.S. Pat. No.
5,403,484; U.S. Pat. No. 5,223,409; WO88/06630; WO92/15679; U.S.
Pat. No. 5,780,279; U.S. Pat. No. 5,571,698; U.S. Pat. No.
6,040,136; Davis et al. (1999) Cancer Metastasis Rev. 18(4):421-5;
and Taylor et al. (1992) Nucleic Acids Research 20: 6287-6295;
Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97(2): 722-727,
the contents of each of which are incorporated herein by reference
in their entirety.
[0079] The cell culture supernatants are screened for the desired
antibodies, preferentially by immunofluorescent staining of GPI or
GPI-anchored protein-expressing cells, by immunoblotting, by an
enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a
radioimmunoassay.
[0080] For isolation of the antibodies, the immunoglobulins in the
culture supernatants or in the ascitic fluid may be concentrated,
e.g. by precipitation with ammonium sulfate, dialysis against
hygroscopic material such as polyethylene glycol, filtration
through selective membranes, or the like. If necessary and/or
desired, the antibodies are purified by the customary
chromatography methods, for example gel filtration, ion-exchange
chromatography, chromatography over DEAE-cellulose and/or (immuno-)
affinity chromatography, e.g. affinity chromatography with a
GPI-moiety, a cell expressing GPI-anchors at its surface, a
GPI-anchored polypeptide, a cell expressing a GPI-anchored protein
at its surface, or with Protein-A or -G.
[0081] Another embodiment provides a process for the preparation of
a bacterial cell line secreting antibodies directed against a GPI
moiety or a GPI-anchored protein in a suitable mammal. For example,
a rabbit is immunized with a GPI-moiety, a cell expressing
GPI-anchors at its surface, a GPI-anchored polypeptide, a cell
expressing a GPI-anchored protein at its surface, or fragments
thereof. A phage display library produced from the immunized rabbit
is constructed and panned for the desired antibodies in accordance
with methods well known in the art (such as, e.g., the methods
disclosed in the various references incorporated herein by
reference).
[0082] Hybridoma cells secreting the monoclonal antibodies are also
disclosed. The preferred hybridoma cells are genetically stable,
secrete monoclonal antibodies described herein of the desired
specificity, and can be expanded from deep-frozen cultures by
thawing and propagation in vitro or as ascites in vivo.
[0083] In another embodiment, a process is provided for the
preparation of a hybridoma cell line secreting monoclonal
antibodies against a GPI moiety or a GPI-anchored protein. In that
process, a suitable mammal, for example a Balb/c mouse, is
immunized with one or more polypeptides or antigenic fragments of,
e.g., CD55 or CD14 or with one or more polypeptides or antigenic
fragments derived from a CD55-expressing cell, the CD55-expressing
cell itself, or an antigenic carrier containing a purified
polypeptide as described. Similarly, the mammal can be immunized
with a human GPI moiety, a fragment thereof, or cells that express
the human GPI moiety, perhaps at a high amount. Antibody-producing
cells of the immunized mammal are grown briefly in culture or fused
with cells of a suitable myeloma cell line. The hybrid cells
obtained in the fusion are cloned, and cell clones secreting the
desired antibodies are selected. For example, spleen cells of
Balb/c mice immunized with, e.g., a GPI-anchored protein or a GPI
moiety are fused with cells of the myeloma cell line PAI or the
myeloma cell line Sp2/0-Ag 14. The obtained hybrid cells are then
screened for secretion of the desired antibodies and positive
hybridoma cells are cloned.
[0084] Methods for preparing a hybridoma cell line include
immunizing Balb/c mice by injecting subcutaneously and/or
intraperitoneally an antigen of interest several times, e.g., four
to six times, over several months, e.g., between two and four
months. Spleen cells from the immunized mice are taken two to four
days after the last injection and fused with cells of the myeloma
cell line PAI in the presence of a fusion promoter, preferably
polyethylene glycol. Preferably, the myeloma cells are fused with a
three- to twenty-fold excess of spleen cells from the immunized
mice in a solution containing about 30% to about 50% polyethylene
glycol of a molecular weight around 4000. After the fusion, the
cells are expanded in suitable culture media as described supra,
supplemented with a selection medium, for example HAT medium, at
regular intervals in order to prevent normal myeloma cells from
overgrowing the desired hybridoma cells.
[0085] The antibodies and fragments thereof can be "chimeric."
Chimeric antibodies and antigen-binding fragments thereof comprise
portions from two or more different species (e.g., mouse and
human). Chimeric antibodies can be produced with mouse variable
regions of desired specificity spliced into human constant domain
gene segments (for example, U.S. Pat. No. 4,816,567). In this
manner, non-human antibodies can be modified to make them more
suitable for human clinical application (e.g., methods for
detecting Type II PNH cells).
[0086] The monoclonal antibodies of the present disclosure include
"humanized" forms of the non-human (e.g., mouse) antibodies.
Humanized or CDR-grafted mAbs are particularly useful as
therapeutic agents for humans because they are not cleared from the
circulation as rapidly as mouse antibodies and do not typically
provoke an adverse immune reaction. Generally, a humanized antibody
has one or more amino acid residues introduced into it from a
non-human source. These non-human amino acid residues are often
referred to as "import" residues, which are typically taken from an
"import" variable domain. Methods of preparing humanized antibodies
are generally well known in the art. For example, humanization can
be essentially performed following the method of Winter and
co-workers (see, e.g., Jones et al. (1986) Nature 321:522-525;
Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al.
(1988) Science 239:1534-1536), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody. Also
see, e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In some
embodiments, humanized forms of non-human (e.g., mouse) antibodies
are human antibodies (recipient antibody) in which hypervariable
(CDR) region residues of the recipient antibody are replaced by
hypervariable region residues from a non-human species (donor
antibody) such as a mouse, rat, rabbit, or non-human primate having
the desired specificity, affinity, and binding capacity. In some
instances, framework region residues of the human immunoglobulin
are also replaced by corresponding non-human residues (so called
"back mutations"). In addition, phage display libraries can be used
to vary amino acids at chosen positions within the antibody
sequence. The properties of a humanized antibody are also affected
by the choice of the human framework. Furthermore, humanized and
chimerized antibodies can be modified to comprise residues that are
not found in the recipient antibody or in the donor antibody in
order to further improve antibody properties, such as, for example,
affinity or effector function.
[0087] Fully human antibodies are also provided in the disclosure.
The term "human antibody" includes antibodies having variable and
constant regions (if present) derived from human gel mline
immunoglobulin sequences. Human antibodies can include amino acid
residues not encoded by human germline immunoglobulin sequences
(e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in vivo). However, the term "human
antibody" does not include antibodies in which CDR sequences
derived from the germline of another mammalian species, such as a
mouse, have been grafted onto human framework sequences (i.e.,
humanized antibodies). Fully human or human antibodies may be
derived from transgenic mice carrying human antibody genes
(carrying the variable (V), diversity (D), joining (J), and
constant (C) exons) or from human cells. For example, it is now
possible to produce transgenic animals (e.g., mice) that are
capable, upon immunization, of producing a full repertoire of human
antibodies in the absence of endogenous immunoglobulin production.
(See, e.g., Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA
90:2551; Jakobovits et al. (1993) Nature 362:255-258; Bruggemann et
al. (1993) Year in Immunol. 7:33; and Duchosal et al. (1992) Nature
355:258.) Transgenic mice strains can be engineered to contain gene
sequences from unrearranged human immunoglobulin genes. The human
sequences may code for both the heavy and light chains of human
antibodies and would function correctly in the mice, undergoing
rearrangement to provide a wide antibody repertoire similar to that
in humans. The transgenic mice can be immunized with the target
protein (e.g., a GPI moiety or a GPI-anchored protein such as CD55
or CD14) to create a diverse array of specific antibodies and their
encoding RNA. Nucleic acids encoding the antibody chain components
of such antibodies may then be cloned from the animal into a
display vector. Typically, separate populations of nucleic acids
encoding heavy and light chain sequences are cloned, and the
separate populations then recombined on insertion into the vector,
such that any given copy of the vector receives a random
combination of a heavy and a light chain. The vector is designed to
express antibody chains so that they can be assembled and displayed
on the outer surface of a display package containing the vector.
For example, antibody chains can be expressed as fusion proteins
with a phage coat protein from the outer surface of the phage.
Thereafter, display packages can be screened for display of
antibodies binding to a target.
[0088] In addition, human antibodies can be derived from
phage-display libraries (Hoogenboom et al. (1991) J. Mol. Biol.
227:381; Marks et al. (1991) J. Mol. Biol., 222:581-597; and
Vaughan et al. (1996) Nature Biotech 14:309 (1996)). Synthetic
phage libraries can be created which use randomized combinations of
synthetic human antibody V-regions. By selection on antigen fully
human antibodies can be made in which the V-regions are very
human-like in nature. See, e.g., U.S. Pat. Nos. 6,794,132,
6,680,209, 4,634,666, and Ostberg et al. (1983), Hybridoma
2:361-367, the contents of each of which are incorporated herein by
reference in their entirety.
[0089] For the generation of human antibodies, also see Mendez et
al. (1998) Nature Genetics 15:146-156, Green and Jakobovits (1998)
J. Exp. Med. 188:483-495, the disclosures of which are hereby
incorporated by reference in their entirety. Human antibodies are
further discussed and delineated in U.S. Pat. Nos. 5,939,598;
6,673,986; 6,114,598; 6,075,181; 6,162,963; 6,150,584; 6,713,610;
and 6,657,103 as well as U.S. Patent Publication Nos. 20030229905
A1, 20040010810 A1, US 20040093622 A1, 20060040363 A1, 20050054055
A1, 20050076395 A1, 20050287630 A1. See also International
Publication Nos. WO 94/02602, WO 96/34096, and WO 98/24893, and
European Patent No. EP 0 463 151 B1. The disclosures of each of the
above-cited patents, applications, and references are hereby
incorporated by reference in their entirety.
[0090] In an alternative approach, others, including GenPharm
International, Inc., have utilized a "minilocus" approach. In the
minilocus approach, an exogenous Ig locus is mimicked through the
inclusion of pieces (individual genes) from the Ig locus. Thus, one
or more V.sub.H genes, one or more D.sub.H genes, one or more
J.sub.H genes, a mu constant region, and a second constant region
(preferably a gamma constant region) are formed into a construct
for insertion into an animal. This approach is described in, e.g.,
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825; 5,625,126;
5,633,425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318;
5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825;
5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, the
disclosures of which are hereby incorporated by reference. See also
European Patent No. 0 546 073 B 1, International Patent Publication
Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO
93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and
WO 98/24884, the disclosures of each of which are hereby
incorporated by reference in their entirety. See further Taylor et
al. (1992) Nucleic Acids Res. 20: 6287; Chen et al. (1993) Int.
Immunol. 5: 647; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA
90: 3720-4; Choi et al. (1993) Nature Genetics 4: 117; Lonberg et
al. (1994) Nature 368: 856-859; Taylor et al. (1994) International
Immunology 6: 579-591; Tuaillon et al. (1995) J. Immunol. 154:
6453-65; Fishwild et al. (1996) Nature Biotechnology 14: 845; and
Tuaillon et al. (2000) Eur. J. Immunol. 10: 2998-3005, the
disclosures of each of which are hereby incorporated by reference
in their entirety.
[0091] In certain embodiments, de-immunized antibodies (e.g.,
antibodies that bind to a human GPI moiety or to a GPI-anchored
protein) or antigen-binding fragments thereof are provided.
De-immunized antibodies or antigen-binding fragments thereof may be
modified so as to render the antibody or antigen-binding fragment
thereof non-immunogenic, or less immunogenic, to a given species.
De-immunization can be achieved by modifying the antibody or
antigen-binding fragment thereof utilizing any of a variety of
techniques known to those skilled in the art (see, e.g., PCT
Publication Nos. WO 04/108158 and WO 00/34317). For example, an
antibody or antigen-binding fragment thereof may be de-immunized by
identifying potential T cell epitopes and/or B cell epitopes within
the amino acid sequence of the antibody or antigen-binding fragment
thereof and removing one or more of the potential T cell epitopes
and/or B cell epitopes from the antibody or antigen-binding
fragment thereof, for example, using recombinant techniques. The
modified antibody or antigen-binding fragment thereof may then
optionally be produced and tested to identify antibodies or
antigen-binding fragments thereof that have retained one or more
desired biological activities, such as, for example, binding
affinity, but have reduced immunogenicity. Methods for identifying
potential T cell epitopes and/or B cell epitopes may be carried out
using techniques known in the art, such as, for example,
computational methods (see e.g., PCT Publication No. WO 02/069232),
in vitro or in silico techniques, and biological assays or physical
methods (such as, for example, determination of the binding of
peptides to MHC molecules, determination of the binding of
peptide:MHC complexes to the T cell receptors from the species to
receive the antibody or antigen-binding fragment thereof, testing
of the protein or peptide parts thereof using transgenic animals
with the MHC molecules of the species to receive the antibody or
antigen-binding fragment thereof, or testing with transgenic
animals reconstituted with immune system cells from the species to
receive the antibody or antigen-binding fragment thereof, etc.). In
various embodiments, the de-immunized antibodies described herein
include de-immunized antigen-binding fragments, Fab, Fv, scFv, Fab'
and F(ab').sub.2, monoclonal antibodies, murine antibodies,
engineered antibodies (such as, for example, chimeric, single
chain, CDR-grafted, humanized, fully human antibodies, and
artificially selected antibodies), synthetic antibodies and
semi-synthetic antibodies.
[0092] In some embodiments, a recombinant DNA comprising an insert
coding for a heavy chain variable domain and/or for a light chain
variable domain of an antibody-expressing cell line is produced.
The term DNA includes coding single stranded DNAs, double stranded
DNAs consisting of said coding DNAs and of complementary DNAs
thereto, or these complementary (single stranded) DNAs
themselves.
[0093] Furthermore, a DNA encoding a heavy chain variable domain
and/or a light chain variable domain of an antibody (e.g., an
anti-GPI antibody or an anti-GPI-anchored protein antibody) can be
enzymatically or chemically synthesized to contain the authentic
DNA sequence coding for a heavy chain variable domain and/or for
the light chain variable domain, or a mutant thereof. A mutant of
the authentic DNA is a DNA encoding a heavy chain variable domain
and/or a light chain variable domain of the above-mentioned
antibodies in which one or more amino acids are deleted, inserted,
or exchanged with one or more other amino acids. Preferably said
modification(s) are outside the CDRs of the heavy chain variable
domain and/or of the light chain variable domain of the antibody in
humanization and expression optimization applications. The term
mutant DNA also embraces silent mutants wherein one or more
nucleotides are replaced by other nucleotides with the new codons
coding for the same amino acid(s). The term mutant sequence also
includes a degenerate sequence. Degenerate sequences are degenerate
within the meaning of the genetic code in that an unlimited number
of nucleotides are replaced by other nucleotides without resulting
in a change of the amino acid sequence originally encoded. Such
degenerate sequences may be useful due to their different
restriction sites and/or frequency of particular codons which are
preferred by the specific host, particularly E. coli, to obtain an
optimal expression of the heavy chain murine variable domain and/or
a light chain murine variable domain.
[0094] The term mutant is intended to include a DNA mutant obtained
by in vitro mutagenesis of the authentic DNA according to methods
known in the art.
[0095] For the assembly of complete tetrameric immunoglobulin
molecules and the expression of chimeric antibodies, the
recombinant DNA inserts coding for heavy and light chain variable
domains are fused with the corresponding DNAs coding for heavy and
light chain constant domains, then transferred into appropriate
host cells, for example after incorporation into hybrid
vectors.
[0096] Recombinant DNAs including an insert coding for a heavy
chain murine variable domain of an antibody of interest fused to a
human constant domain IgG, for example .gamma.1, .gamma.2, .gamma.3
or .gamma.4, in particular embodiments .gamma.1 or .gamma.4, may be
used. Recombinant DNAs including an insert coding for a light chain
murine variable domain of an antibody fused to a human constant
domain .kappa. or .lamda., preferably .kappa., are also
provided.
[0097] Another embodiment pertains to recombinant DNAs coding for a
recombinant polypeptide wherein the heavy chain variable domain and
the light chain variable domain are linked by way of a spacer
group, optionally comprising a signal sequence facilitating the
processing of the antibody in the host cell and/or a DNA sequence
encoding a peptide facilitating the purification of the antibody
and/or a cleavage site and/or a peptide spacer and/or an agent. The
DNA coding for an agent is intended to be a DNA coding for the
agent useful in diagnostic or therapeutic applications. Thus, agent
molecules which are toxins or enzymes, especially enzymes capable
of catalyzing the activation of prodrugs, are particularly
indicated. The DNA encoding such an agent has the sequence of a
naturally occurring enzyme or toxin encoding DNA, or a mutant
thereof, and can be prepared by methods well known in the art.
[0098] Accordingly, the monoclonal antibodies or antigen-binding
fragments of the disclosure can be naked antibodies or
antigen-binding fragments that are not conjugated to other agents,
for example, a therapeutic agent or detectable label.
Alternatively, the monoclonal antibody or antigen-binding fragment
can be conjugated to an agent such as, for example, a cytotoxic
agent, a small molecule, a hormone, an enzyme, a growth factor, a
cytokine, a ribozyme, a peptidomimetic, a chemical, a prodrug, a
nucleic acid molecule including coding sequences (such as
antisense, RNAi, gene-targeting constructs, etc.), or a detectable
label (e.g., an NMR or X-ray contrasting agent, fluorescent
molecule, etc.). In certain embodiments, an antibody or an
antigen-binding fragment thereof (e.g., Fab, Fv, single-chain scFv,
Fab', and F(ab).sub.2) is linked to a molecule that increases the
half-life of the antibody or antigen-binding fragment (see the
section entitled "Conjugates").
[0099] Several possible vector systems are available for the
expression of cloned heavy chain and light chain genes in mammalian
cells. One class of vectors relies upon the integration of the
desired gene sequences into the host cell genome. Cells which have
stably integrated DNA can be selected by simultaneously introducing
drug resistance genes such as E. coli gpt (Mulligan and Berg (1981)
Proc. Natl. Acad. Sci. USA, 78:2072) or Tn5 neo (Southern and Berg
(1982) Mol. Appl. Genet. 1:327). The selectable marker gene can be
either linked to the DNA gene sequences to be expressed, or
introduced into the same cell by co-transfection (Wigler et al.
(1979) Cell 16:77). A second class of vectors utilizes DNA elements
which confer autonomously replicating capabilities to an
extrachromosomal plasmid. These vectors can be derived from animal
viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc.
Natl. Acad. Sci. USA, 79:7147), polyoma virus (Deans et al. (1984)
Proc. Natl. Acad. Sci. USA 81:1292), or SV40 virus (Lusky and
Botchan (1981) Nature 293:79).
[0100] Since an immunoglobulin cDNA is comprised only of sequences
representing the mature mRNA encoding an antibody protein,
additional gene expression elements regulating transcription of the
gene and processing of the RNA are required for the synthesis of
immunoglobulin mRNA. These elements may include splice signals,
transcription promoters, including inducible promoters, enhancers,
and termination signals. cDNA expression vectors incorporating such
elements include those described by Okayama and Berg (1983) Mol.
Cell. Biol. 3:280; Cepko et al. (1984) Cell 37:1053; and Kaufman
(1985) Proc. Natl. Acad. Sci. USA 82:689.
[0101] As is evident from the disclosure, the anti-GPI moiety
antibodies or anti-GPI-anchored protein antibodies can be used in
methods for diagnosing disease (e.g., diagnosing PNH or an
increased risk of developing thrombocytopenia), monitoring of
disease progression, and the selection of appropriate therapies,
including combination therapies, for treating PNH,
thrombocytopenia, or thrombosis in a subject.
[0102] In the diagnostic embodiments of the present disclosure,
bispecific antibodies are contemplated. Bispecific antibodies are
monoclonal, preferably human or humanized, antibodies that have
binding specificities for at least two different antigens. In the
present case, one of the binding specificities is for a GPI moiety
or a GPI-anchored protein on a cell (such as, e.g., a white blood
cell or a red blood cell), the other one is for any other antigen,
and preferably for a cell-surface protein or receptor or receptor
subunit.
[0103] Methods for making bispecific antibodies are within the
purview of those skilled in the art. Traditionally, the recombinant
production of bispecific antibodies is based on the co-expression
of two immunoglobulin heavy-chain/light-chain pairs, where the two
heavy chains have different specificities (Milstein and Cuello
(1983) Nature 305:537-539). Antibody variable domains with the
desired binding specificities (antibody-antigen combining sites)
can be fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy-chain constant
domain, including at least part of the hinge, C.sub.H2, and
C.sub.H3 regions. DNAs encoding the immunoglobulin heavy-chain
fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are co-transfected
into a suitable host organism. For further details of illustrative
currently known methods for generating bispecific antibodies see,
e.g., Suresh et al. (1986) Methods in Enzymology 121:210; PCT
Publication No. WO 96/27011; Brennan et al. (1985) Science 229:81;
Shalaby et al., J. Exp. Med. (1992) 175:217-225; Kostelny et al.
(1992) J. Immunol. 148(5):1547-1553; Hollinger et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448; Gruber et al. (1994) J. Immunol.
152:5368; and Tutt et al. (1991) J. Immunol. 147:60. Bispecific
antibodies also include cross-linked or heteroconjugate antibodies.
Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0104] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. (See, e.g., Kostelny et al. (1992)
J. Immunol. 148(5):1547-1553). The leucine zipper peptides from the
Fos and Jun proteins may be linked to the Fab' portions of two
different antibodies by gene fusion. The antibody homodimers may be
reduced at the hinge region to form monomers and then re-oxidized
to form the antibody heterodimers. This method can also be utilized
for the production of antibody homodimers. The "diabody" technology
described by Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA
90:6444-6448 has provided an alternative mechanism for making
bispecific antibody fragments. The fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) by a linker which is too short to allow pairing between the
two domains on the same chain. Accordingly, the VH and VL domains
of one fragment are forced to pair with the complementary VL and VH
domains of another fragment, thereby forming two antigen-binding
sites. Another strategy for making bispecific antibody fragments by
the use of single-chain Fv (scFv) dimers has also been reported.
(See, e.g., Gruber et al. (1994) J. Immunol. 152:5368.)
Alternatively, the antibodies can be "linear antibodies" as
described in, e.g., Zapata et al. (1995) Protein Eng.
8(10):1057-1062. Briefly, these antibodies comprise a pair of
tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which form a
pair of antigen binding regions. Linear antibodies can be
bispecific or monospecific.
Conjugates
[0105] In some embodiments, a reagent described herein (e.g., a
non-lytic aerolysin polypeptide or an antibody that binds to a GPI
moiety or a GPI-anchored protein) can be conjugated to a
heterologous moiety. The heterologous moiety can be, e.g., a
heterologous protein (see above), a therapeutic agent (e.g., a
toxin or a drug), or a detectable label such as, but not limited
to, a radioactive label, an enzymatic label, a fluorescent label,
or a luminescent label. Suitable radioactive labels include, e.g.,
.sup.32P, .sup.33P, .sup.14C, .sup.125I, .sup.131I, .sup.35S, and
.sup.3H. Suitable fluorescent labels include, without limitation,
fluorescein, fluorescein isothiocyanate (FITC), Alexa Fluor.RTM.
488, Alexa Fluor.RTM. 647, GFP, DyLight 488, phycoerythrin (PE),
propidium iodide (PI), PerCP, PE-Alexa Fluor.RTM. 700, Cy5,
allophycocyanin, Cy7, and PE-Alexa Fluor.RTM. 750. Luminescent
labels include, e.g., any of a variety of luminescent lanthanide
(e.g., europium or terbium) chelates. For example, suitable
europium chelates include the europium chelate of diethylene
triamine pentaacetic acid (DTPA). Enzymatic labels include, e.g.,
alkaline phosphatase, CAT, luciferase, and horseradish
peroxidase.
[0106] Suitable methods for conjugating a heterologous moiety to
the reagent are well-known in the art of protein chemistry. For
example, two proteins can be cross-linked using any of a number of
known chemical cross linkers. Examples of such cross linkers are
those which link two amino acid residues via a linkage that
includes a "hindered" disulfide bond. In these linkages, a
disulfide bond within the cross-linking unit is protected (by
hindering groups on either side of the disulfide bond) from
reduction by the action, for example, of reduced glutathione or the
enzyme disulfide reductase. One suitable reagent,
4-succinimidyloxycarbonyl-.alpha.-methyl-.alpha.(2-pyridyldithio)toluene
(SMPT), forms such a linkage between two proteins utilizing a
terminal lysine on one of the proteins and a terminal cysteine on
the other. Heterobifunctional reagents that cross-link by a
different coupling moiety on each protein can also be used. Other
useful cross-linkers include, without limitation, reagents which
link two amino groups (e.g.,
N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups
(e.g., 1,4-bis-maleimidobutane) an amino group and a sulfhydryl
group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an
amino group and a carboxyl group (e.g.,
4-[p-azidosalicylamido]butylamine), and an amino group and a
guanidinium group that is present in the side chain of arginine
(e.g., p-azidophenyl glyoxal monohydrate).
[0107] Radioactive labels can be conjugated to the reagent by
covalent or non-covalent (e.g., ionic or hydrophobic) bonds. They
can be bound to any part of the protein provided that the
conjugation does not interfere with the ability of the reagent to
bind to a GPI moiety or to a GPI-anchored protein. In some
embodiments, where the reagent is a protein, the radioactive label
can be directly conjugated to the amino acid backbone of the
reagent. Alternatively, the radioactive label can be included as
part of a larger molecule (e.g., .sup.125I in
meta-[.sup.125I]iodophenyl-N-hydroxysuccinimide ([.sup.125I]mIPNHS)
which binds to free amino groups to form meta-iodophenyl (mIP)
derivatives of relevant proteins (see, e.g., Rogers et al. (1997)
J. Nucl. Med. 38:1221-1229) or chelate (e.g., to DOTA or DTPA)
which is in turn bound to the protein backbone. Methods of
conjugating the radioactive labels or larger molecules/chelates
containing them to the reagents described herein are also known in
the art. Such methods involve incubating the reagent with the
radioactive label under conditions (e.g., pH, salt concentration,
and/or temperature) that facilitate binding of the radioactive
label or chelate to the reagent (see, e.g. U.S. Pat. No. 6,001,329,
the disclosure of which is incorporated herein by reference in its
entirety).
[0108] Methods for conjugating a fluorescent label (sometimes
referred to as a "fluorophore") to a reagent (e.g., a non-lytic
aerolysin protein or an antibody) are known in the art of protein
chemistry. For example, fluorophores can be conjugated to free
amino groups (e.g., of lysines) or sulfhydryl groups (e.g.,
cysteines) of proteins using succinimidyl (NHS) ester or TFP ester
moieties attached to the fluorophores. In some embodiments, the
fluorophores can be conjugated to a heterobifunctional cross-linker
moiety such as sulfo-SMCC. Suitable conjugation methods involve
incubating the reagent with the fluorophore under conditions that
facilitate binding of the fluorophore to the reagent. See, e.g.,
Welch and Redvanly (2003) "Handbook of Radiophaimaceuticals:
Radiochemistry and Applications," John Wiley and Sons (ISBN:
0471495603). A variety of kits are commercially available for use
in conjugating a fluorophore to a protein, e.g., the Alexa
Fluor.RTM. 488 Protein Labeling Kit and the Alexa Fluor.RTM. 647
Protein Labeling Kit (Molecular Probes, Invitrogen.TM.) In some
embodiments, the fluorophore can be conjugated to a reagent at 1-2
mol dye per mol of protein.
[0109] In some embodiments, the reagents (e.g., an aerolysin
protein or an antibody that binds to a GPI moiety or a GPI-anchored
protein) can be modified, e.g., with a moiety that improves the
stabilization and/or retention of the antibodies themselves in
circulation, e.g., in blood, serum, or other tissues. For example,
a reagent described herein can be PEGylated as described in, e.g.,
Lee et al. (1999) Bioconjug. Chem 10(6): 973-8; Kinstler et al.
(2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et
al. (2002) Advanced Drug Delivery Reviews 54:459-476. The
stabilization moiety can improve the stability, or retention of,
the reagent in a subject's body (e.g., blood or tissue) by at least
1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more)
fold.
Biological Samples and Sample Collection
[0110] Suitable biological samples for use in the methods described
herein include any biological fluid, population of cells, or tissue
or fraction thereof, which includes one or more white blood cells
and/or one or more red blood cells. A biological sample can be, for
example, a specimen obtained from a subject (e.g., a mammal such as
a human) or can be derived from such a subject. For example, a
sample can be a tissue section obtained by biopsy, or cells that
are placed in or adapted to tissue culture. A biological sample can
also be a biological fluid such as urine, whole blood or a fraction
thereof (e.g., plasma), saliva, semen, sputum, cerebral spinal
fluid, tears, or mucus. A biological sample can be further
fractionated, if desired, to a fraction containing particular cell
types. For example, a whole blood sample can be fractionated into
serum or into fractions containing particular types of blood cells
such as red blood cells or white blood cells (leukocytes). If
desired, a biological sample can be a combination of different
biological samples from a subject such as a combination of a tissue
and fluid sample.
[0111] The biological samples can be obtained from a subject, e.g.,
a subject having, suspected of having, or at risk of developing,
paroxysmal nocturnal hemoglobinuria (PNH). Any suitable methods for
obtaining the biological samples can be employed, although
exemplary methods include, e.g., phlebotomy, swab (e.g., buccal
swab), lavage, or fine needle aspirate biopsy procedure.
Non-limiting examples of tissues susceptible to fine needle
aspiration include lymph node, lung, thyroid, breast, and liver.
Biological samples can also be obtained from bone marrow. Samples
can also be collected, e.g., by microdissection (e.g., laser
capture microdissection (LCM) or laser microdissection (LMD)),
bladder wash, smear (PAP smear), or ductal lavage.
[0112] Methods for obtaining and/or storing samples that preserve
the activity or integrity of cells in the biological sample are
well known to those skilled in the art. For example, a biological
sample can be further contacted with one or more additional agents
such as appropriate buffers and/or inhibitors, including protease
inhibitors, the agents meant to preserve or minimize changes in the
cells (e.g., changes in osmolarity or pH) or denaturation of cell
surface proteins (e.g., GPI-linked proteins) or GPI moieties on the
surface of the cells. Such inhibitors include, for example,
chelators such as ethylenediamine tetraacetic acid (EDTA), ethylene
glycol tetraacetic acid (EGTA), protease inhibitors such as
phenylmethylsulfonyl fluoride (PMSF), aprotinin, and leupeptin.
Appropriate buffers and conditions for storing or otherwise
manipulating whole cells are described in, e.g., Pollard and Walker
(1997), "Basic Cell Culture Protocols," volume 75 of Methods in
Molecular Biology, Humana Press; Masters (2000) "Animal cell
culture: a practical approach," volume 232 of Practical approach
series, Oxford University Press; and Jones (1996) "Human cell
culture protocols," volume 2 of Methods in molecular medicine,
Humana Press."
[0113] A sample also can be processed to eliminate or minimize the
presence of interfering substances. For example, a biological
sample can be fractionated or purified to remove one or more
materials (e.g., cells) that are not of interest. Methods of
fractionating or purifying a biological sample include, but are not
limited to, flow cytometry, fluorescence activated cell sorting,
and sedimentation.
Diagnostic and Therapeutic Methods
[0114] As noted above and elaborated on in the working examples,
the inventors have discovered a clinical relationship between the
presence or amount of PNH Type II hematopoietic cells (e.g., Type
II white blood cells and/or Type II red blood cells) and
thrombocytopenia in a patient. For example, the inventors have
determined that a patient with a PNH Type II white blood cell
population of at least 1.2 (e.g., at least 1.2, 1.5, 2, 3, 5, 7, 9,
10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47, 50,
52, 55, 57, 60, 62, 65, or 65.3 or more) % as compared to the total
white blood cells of the same histological type (the same lineage)
in the biological sample tested is much more likely to be
thrombocytopenic than a patient who does not have a detectable PNH
Type II white blood cell population or a patient with a PNH Type II
white blood cell population lower than 1.2%. Patient samples with
PNH Type II granulocyte populations had similar peripheral white
blood cell counts, peripheral red blood cell counts, absolute
neutrophil counts, and hemoglobin (Hgb) levels, compared to patient
samples without detectable Type II granulocyte populations,
indicating that differences in platelet counts are likely not due
to differences in underlying bone marrow production. In other
words, the decreased platelet counts in patients with detectable
PNH Type II granulocyte clones may be due to increased terminal
complement-mediated platelet consumption or destruction, which can
be associated with thrombosis, the leading cause of death among PNH
patients. See, e.g., Franchini (2006) Hematology 11(3):139-146.
Accordingly, the present disclosure features methods for using
information related to the percentage of PNH Type II white blood
cells in a patient sample for determining whether the patient is at
an increased risk of developing thrombocytopenia and/or thrombosis.
Similarly, the inventors have determined that a patient with a PNH
Type II RBC population of at least 0.02 (e.g., at least 0.02, 0.03,
0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4,
3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71,
71.3, or 75 or more) % is much more likely to be thrombocytopenic
than a patient who does not have a detectable PNH Type II RBC
population or a patient with a PNH Type II RBC population lower
than 0.02%. Thus, the present disclosure also features methods for
using information related to the PNH Type II RBC clone size in a
patient for determining whether the patient is at risk of
developing thrombocytopenia and/or thrombosis.
[0115] The following methods can be employed to detect the
presence, or to determine the percentage, of PNH Type II
hematopoietic cells (e.g., Type II PNH white blood cells) as
compared to the total amount of cells of the same histological type
(same lineage) in a biological sample from the patient. In
embodiments where a plurality of white blood cells are
interrogated, the methods are useful in allowing a practitioner to
distinguish between populations of PNH Type I, Type II, and Type
III white blood cells of the same histological type (same lineage)
in order to accurately determine the size of the total abnormal PNH
population (i.e., Type II plus Type III cells) as compared to the
total number of white blood cells of the same histological type in
the plurality (and/or allow the practitioner to determine the
percentage of PNH Type II white blood cells in the plurality).
First, a population of cells (e.g., white blood cells, red blood
cells, or a combination of white and red blood cells) is contacted
with a reagent that binds to: (i) a GPI moiety or (ii) a GPI-linked
protein for a period of time and under conditions that allow for
the binding of the reagent to the GPI moiety or GPI-linked protein
if present on the surface of cells present in the sample. The
population of cells can be present in a biological sample (e.g., a
whole blood sample; see the section entitled "Biological Samples
and Sample Collection"), e.g., a biological sample obtained from a
patient. For example, cells present in a whole blood sample from a
patient can be contacted with an aerolysin protein (e.g., a
non-lytic form of aerolysin) or an antibody that binds to a GPI
moiety or a GPI-anchored protein such as CD59. See, e.g., Hall and
Rosse (1996) Blood 87(12):5332-5340 and U.S. Pat. No. 6,593,095. At
least a portion of the cells (e.g., white blood cells or RBCs)
contacted with the reagent can be distinguished based on the amount
of reagent bound to the surface of the cells. For example, where a
detectably-labeled reagent was used, the amount of reagent bound to
the surface can be determined as a function of the total amount of
signal produced from detectably labeled reagent bound to the
surface of the cell. As described above, the amount of binding of
the reagent to the cell reflects the amount of expression of GPI
moieties and/or GPI-anchored proteins, which are indicative of
whether cells are PNH Type III cells (little or no expression of
GPI and GPI-anchored proteins), normal cells (Type I cells; having
a relatively high level of expression of GPI and GPI-anchored
proteins as compared to the Type III cells), and PNH Type II cells
(having an intermediate level of expression of GPI and GPI-anchored
proteins as compared to Type I cells and Type III cells). The
distinguishing or interrogating process can involve, e.g., flow
cytometry.
[0116] In some embodiments, the methods are used to detect the
amount of binding of a reagent to RBCs from a patient sample. In
some embodiments, the methods can be used to detect the amount of
binding of a reagent to white blood cells from a patient sample.
White blood cells that are particularly amenable to evaluation in
the diagnostic methods described herein include, e.g., granulocytes
and monocytes (e.g., macrophages).
[0117] The samples can be from patients who have, are suspected of
having, or at risk for developing paroxysmal nocturnal
hemoglobinuria (PNH). In some embodiments, the patients have one or
more symptoms including, e.g., Coombs negative intravascular
hemolysis, elevated LDH levels, recurrent iron deficiency anemia,
thrombosis in unusual sites, episodic dysphagia, or abdominal
pain.
[0118] In some embodiments, to aid in the identification of normal
cells or PNH cells, a set of control cell populations can also be
subjected to the detection method. The control populations can be
evaluated before, concurrently, or after the evaluation of the cell
population of interest. As discussed in more detail below, a
practitioner can choose to subject a control population of cells
known to be PNH Type II cells, a control population of cells known
to be PNH Type III cells, and/or a control population of cells
known to be normal or Type I cells to the methods to deteii tine
the typical amount, or average amount, of binding of the reagent
used to a particular type of cell. This control information can be
used to classify or identify cells (e.g., white blood cells or red
blood cells) of interest as normal cells, PNH Type II cells, and/or
PNH Type III cells.
[0119] Depending on the particular composition of the cell
population within the biological sample, at least some cells of the
population can be distinguished from other cells based on a high
amount of bound reagent, a low amount of bound reagent, or an
intermediate level of bound reagent. In some cases, only cells with
a high amount of bound reagent will be present (for example, cells
from a healthy patient or a patient who does not have PNH). In some
cases, a larger percentage of cells will have little, or no,
reagent bound to their surface (for example, white blood cells or
RBCs from a PNH patient having a high percentage of PNH Type III
cells). In some embodiments, a population of cells contacted with
the reagent can be classified into high, low, and intermediate
categories based on the amount of reagent bound to the cells.
[0120] In some embodiments, one or more cells (e.g., one or more
distinguished or interrogated cells) can be classified based on the
amount of reagent bound to their cell surface. As exemplified in
the working examples and depicted in FIG. 1, individual cells in a
population can be readily classified as highly reagent bound, low
or poorly reagent bound, and intermediately reagent bound using
flow cytometry methods. For example, white blood cells obtained
from a patient with PNH are contacted with two different reagents:
a first reagent that binds to a GPI moiety (e.g., a
detectably-labeled, non-lytic aerolysin protein) and a second
reagent that binds to a GPI-anchored protein (e.g., a
detectably-labeled antibody that binds to human CD24). The first
reagent and second reagent are labeled with different detectable
labels. The contacted cells are then subjected to flow cytometry.
An artisan skilled in the art of flow cytometry would be readily
able to use the methods to distinguish between cells based on the
amount of binding of each reagent to the cells. See, e.g., Macey
(2007) "Flow Cytometry: principles and applications," Humana Press
(ISBN: 1588296911) and Brodsky et al. (2000) Am J Clin Pathol
114:459-466. As shown in FIG. 1, the flow cytometry methods can
readily be used to classify granulocytes obtained from a PNH
patient as having a high amount of binding of each of the reagents
(cell population at upper right; normal or Type I cells), a low
amount of binding of each reagent (cell population at lower left;
Type III cells), and an intermediate amount of binding of each
reagent (cell population at bottom center; Type II cells).
[0121] The classification of a cell can be performed by comparing
the amount of the reagent bound to the cell to a control amount
(e.g., a control amount of binding of the reagent to PNH Type I
cells, PNH Type II cells, and/or PNH Type III cells). The control
amount of binding of the reagent to PNH Type I cells can be based
on, e.g., the average amount of observed binding of the reagent to
cells of the same histological type obtained from one or more
(e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20,
25, 30, 35, or 40 or more) healthy individuals. The control amount
of binding of the reagent to PNH Type III cells can be based on,
e.g., the average amount of binding observed to cells of the same
type obtained from one or more (e.g., two, three, four, five, six,
seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 or more) patients
with PNH. The control amount of binding of the reagent to PNH Type
II cells can be based on, e.g., the average amount of binding
observed to cells of the same type obtained from one or more (e.g.,
two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25,
30, 35, or 40 or more) patients with PNH and having a detectable
population of PNH Type II cells of the same histological type (same
lineage). For example, to classify a white blood cell of interest
based on the amount of reagent bound to the surface of the cell, a
practitioner can compare the amount of reagent bound to the cell
with the typical amount, or average amount, of reagent bound to a
white blood cell known to be a PNH Type I white blood, a white
blood cell known to be a PNH Type II white blood cell, and/or a
white blood cell known to be a PNH Type III white blood cell.
[0122] In some embodiments, the distinguishing or classifying of a
cell (e.g., a white blood cell or RBC) of interest can be performed
by determining whether the amount of a reagent bound to the cell
falls within a predetermined range indicative of PNH Type I cells,
PNH Type II cells, or PNH Type III cells of the same histological
type. In some embodiments, the distinguishing or classifying of a
hematopoietic cell of interest can include determining if the
amount of reagent bound to the surface of the cell falls above or
below a predetermined cut-off value. A cut-off value is typically
the amount of reagent bound to the surface of a cell (or the amount
of signal detected from a cell) above or below which is considered
indicative of a certain class of cells, namely PNH Type I cells,
PNH Type II cells, or PNH Type III cells.
[0123] Some cut-off values are not absolute in that diagnostic
correlations (e.g., an amount of reagent bound to the surface of
the cell and likelihood that the cell is a PNH Type II cell) can
still remain significant over a range of values on either side of
the cutoff. It is understood that refinements in optimal cut-off
values could be determined depending on the quality of reagents
used, the sophistication of statistical methods and detection
device (e.g., flow cytometry) used, and on the number and source of
samples interrogated. Therefore, cut-off values can be adjusted up
or down, on the basis of periodic re-evaluations or changes in
methodology or sample distribution.
[0124] As used herein, "thrombocytopenia" refers to a condition in
which a patient has a platelet count of less than 200,000 (e.g.,
less than 150,000; less than 140,000; less than 130,000; less than
120,000; less than 110,000; less than 100,000; or less than 90,000)
platelets per .mu.L of blood. In some embodiments, a patient with
thrombocytopenia has a platelet count of less than 100,000
platelets per .mu.L of blood.
[0125] As described above, information related to the percentage of
PNH Type II cells can be used in methods for determining whether a
patient is at increased risk for developing thrombosis. The
information related to the percentage of PNH Type II cells (e.g.,
Type II white blood cells and/or Type II red blood cells) in a
biological sample from a patient can be communicated (e.g.,
electronic or printed form) to a medical practitioner to be used by
the practitioner for selecting an appropriate therapeutic regimen
for the patient. Based on a PNH Type II white blood cell population
of at least 1.2 (e.g., at least 12, 1.5, 2, 3, 5, 7, 9, 10, 12, 15,
17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47, 50, 52, 55, 57,
60, 62, 65, or 65.3 or more) %, as compared to the total number of
white blood cells of the same histological type in the biological
sample tested, the practitioner may determine that the patient is
at risk of developing thrombocytopenia, or may likely be
thrombocytopenic. Likewise, a patient with a PNH Type II RBC
population of at least 0.02 (e.g., at least 0.02, 0.03, 0.04, 0.05,
0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,
3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 71.3, or 75
or more) % of the total RBC in the biological sample tested is much
more likely to be thrombocytopenic than a patient who does not have
a detectable PNH Type II RBC population or a patient with a PNH
Type II RBC population lower than 0.02%. A patient with a PNH Type
II white blood cell population of at least 1.2% or a PNH Type II
red blood cell population of at least 0.02% can be, e.g., at least
1.5 (e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,
9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 20, 30, or even 40 or
more) times as likely to develop a thrombus than a normal
individual or a patient that does not have that percentage of PNH
Type II cells.
[0126] The medical practitioner may request additional tests to
determine the platelet counts in the patient. Methods for
determining platelet counts in a blood-derived sample from a
subject are well known in the art of medicine and described in,
e.g., Sallah et al. (1998) Postgraduate Medicine 103:209-210;
Kottke-Marchant (1994) Hematol Oncol Clin North Am. 8:809-853;
Redei et al. (1995) J Crit. Illn 10:133-137; Butkiewicz et al.
(2006) Thrombosis Research 118(2):199-204; Tomita et al. (2000) Am
J Hematol 63(3):131-135; and Schrezenmeier et al. (1998) Br J
Haematol 100(3):571-576.
[0127] If the patient is determined by the medical practitioner to
be thrombocytopenic or to likely be thrombocytopenic, the
practitioner may select, prescribe, or administer to the patient an
anti-thrombocytopenic therapy. The anti-thrombocytopenic therapy
can be, e.g., a corticosteroid, platelet transfusion, a
splenectomy, or a platelet production-stimulating agent. The
platelet production-stimulating agent can be, e.g., thrombopoietin
(TPO) or a thrombopoietin mimetic. See, e.g., Kuter and Begley
(2002) Blood 100:3457-3469; Li et al. (2001) Blood 98:3241-3248;
and Vadhan-Raj et al. (2000) Ann Intern Med 132:364-368. A TPO
mimetic peptide can have the amino acid sequence depicted in FIG. 5
of U.S. Patent Application Publication No. 20030049683, the
disclosure of which (particularly FIG. 5) is incorporated by
reference in its entirety.
[0128] If the percentage of PNH Type II white blood cells in a
biological sample from a patient is about 1.2%, the medical
practitioner may also determine that the patient is at an increased
risk for developing thrombosis. The medical practitioner may then
select for the patient an appropriate anti-thrombotic therapy. For
example, the practitioner may select, prescribe, or administer to
the patient an anticoagulant or thrombolytic agent. The
anticoagulant can be, e.g., coumadin, heparin, or derivatives
thereof. The thrombolytic agent can be, e.g., a tissue plasminogen
activator (e.g., Retavase.TM., Rapilysin.TM.), streptokinase, or a
urokinase-type plasminogen activator.
[0129] In some embodiments, a patient determined to have a PNH Type
II white blood cell population of greater than or equal to 1.2% or
a PNH Type II red blood cell population of greater than or equal to
0.02% can be diagnosed as having PNH. In some embodiments, a
patient diagnosed with having PNH or a previously diagnosed PNH
patient who is determined to have a PNH Type II white blood cell
population greater than or equal to 1.2% or a PNH Type II red blood
cell population that is greater than or equal to 0.02% can be
prescribed and/or treated with a complement inhibitor.
[0130] Any compounds which bind to or otherwise block the
generation and/or activity of any of the human complement
components may be utilized in accordance with the present
disclosure. For example, an inhibitor of complement can be, e.g., a
small molecule, a nucleic acid or nucleic acid analog, a
peptidomimetic, or a macromolecule that is not a nucleic acid or a
protein. These agents include, but are not limited to, small
organic molecules, RNA aptamers, L-RNA aptamers, Spiegelmers,
antisense compounds, double stranded RNA, small interfering RNA,
locked nucleic acid inhibitors, and peptide nucleic acid
inhibitors. In some embodiments, a complement inhibitor may be a
protein or protein fragment.
[0131] In some embodiments, antibodies specific to a human
complement component are useful herein. Some compounds include
antibodies directed against complement components C1, C2, C3, C4,
C5 (or a fragment thereof; see below), C6, C7, C8, C9, Factor D,
Factor B, Factor P, MBL, MASP-1, and MASP-2, thus preventing the
generation of the anaphylatoxic activity associated with C5a and/or
preventing the assembly of the membrane attack complex associated
with C5b.
[0132] Also useful in the present methods are naturally occurring
or soluble forms of complement inhibitory compounds such as CR1,
LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175,
complestatin, and K76 COOH. Other compounds which may be utilized
to bind to or otherwise block the generation and/or activity of any
of the human complement components include, but are not limited to,
proteins, protein fragments, peptides, small molecules, RNA
aptamers including ARC 187 (which is commercially available from
Archemix Corporation, Cambridge, Mass.), L-RNA aptamers,
spiegelmers, antisense compounds, serine protease inhibitors,
molecules which may be utilized in RNA interference (RNAi) such as
double stranded RNA including small interfering RNA (siRNA), locked
nucleic acid (LNA) inhibitors, peptide nucleic acid (PNA)
inhibitors, etc.
[0133] In some embodiments, the complement inhibitor inhibits the
activation of complement. For example, the complement inhibitor can
bind to and inhibit the complement activation activity of C1 (e.g.,
C1q, C1r, or C1s) or the complement inhibitor can bind to and
inhibit (e.g., inhibit cleavage of) C2, C3, or C4. In some
embodiments, the inhibitor inhibits formation or assembly of the C3
convertase and/or C5 convertase of the alternative and/or classical
pathways of complement. In some embodiments, the complement
inhibitor inhibits terminal complement formation, e.g., formation
of the C5b-9 membrane attack complex. For example, an antibody
complement inhibitor may include an anti-C5 antibody. Such anti-C5
antibodies may directly interact with C5 and/or C5b, so as to
inhibit the formation of and/or physiologic function of C5b.
Exemplary anti-C5 antibodies include, e.g., eculizumab
(Soliris.RTM.; Alexion Pharmaceuticals, Inc., Cheshire, Conn.; see,
e.g., Kaplan (2002) Curr Opin Investig Drugs 3(7):1017-23; Hill
(2005) Clin Adv Hematol Oncol 3(11):849-50; and Rother et al.
(2007) Nature Biotechnology 25(11):1256-1488) and pexelizumab
(Alexion Pharmaceuticals, Inc., Cheshire, Conn.; see, e.g., Whiss
(2002) Curr Opin Investig Drugs 3(6):870-7; Patel et al. (2005)
Drugs Today (Bare) 41(3):165-70; and Thomas et al. (1996) Mol
Immunol. 33(17-18):1389-401).
[0134] Methods for administering an appropriate anti-thrombotic
therapy and/or an anti-thrombocytopenic therapy to a patient in
need thereof are well known in the art of medicine.
[0135] In some embodiments, methods for determining whether a
patient is at an increased risk for developing thromobocytopenia or
thrombosis can be aided by computer. For example, the methods can
include receiving data including a medical profile of a PNH
patient, the profile comprising information on the percentage of
PNH Type II white blood cells of the total white blood cells of the
same histological type (same lineage) in a biological sample from
the patient; and processing at least the portion of the data
containing the information to determine whether the patient is at
an increased risk for developing thrombosis. In another example,
the methods can include providing information on the percentage of
PNH Type II white blood cells of the total white blood cells of the
same histological type in a biological sample from the patient;
inputting the information into a computer; and calculating a
parameter indicating whether the patient is at an increased risk
for thrombosis using the computer and the input information. The
relative risk of the patient for developing thrombocytopenia or
thrombosis can be output by the computer in print and/or can be
stored on a computer-readable medium.
Kits
[0136] Also featured herein are kits for use in: determining
whether a biological sample from a patient contains a PNH Type II
white blood cell population and/or determining if a patient is at
increased risk for developing thrombocytopenia or thrombosis. The
kits can include, e.g., one or more of detectably-labeled
conjugates selected from: an aerolysin conjugate (e.g., a non-lytic
variant aerolysin protein conjugate) or conjugates of antibodies
that bind to GPI-anchored proteins. The kits can also include a
control sample containing a GPI expressing cell or GPI bound
particle; and optionally, instructions for detecting the presence
of a GPI expressing cell. The kits can also include one or more
means for obtaining a biological sample (e.g., a blood sample) from
a human and/or any of the kit components described above.
[0137] The following examples are intended to illustrate, not
limit, the invention.
EXAMPLES
Example 1
Materials and Methods
[0138] A total of 2,921 patient peripheral blood samples were
obtained to test for the presence of PNH Type II cells and PNH Type
III cells. The blood samples were drawn into sterile vials
containing EDTA.
[0139] To determine the percentage of normal white blood cells
(Type I cells), PNH Type II, and PNH Type III white blood cells
present in each of the patient samples, the peripheral blood was
mixed and stained with one or more of the following conjugates: a
non-lytic aerolysin variant protein conjugated to AlexaFluor.RTM.
488 (Protox Biotech FL2-S), an anti-CD24 antibody conjugated to
phycoerythrin (PE) (Beckman Coulter Clone ALB9), an anti-CD15
antibody conjugated to PC5 (Clone 80H5), and an anti-CD45 antibody
conjugated to PC7 (Clone J.33). After incubation of the blood with
one or more of the above reagents for 15-30 minutes at room
temperature, the blood was lysed with Immunoprep.TM. (Beckman
Coulter) and washed twice with PBA buffer (phosphate-buffered
saline, 1% bovine serum albumin, and 10 mM NaN.sub.3). Cells are
then re-suspended in PBA buffer and analyzed using the FC 500 Flow
Cytometer (Beckman Coulter). If a PNH Type II or Type III
granulocyte population was identified, the monocytes were also
interrogated for Type II or Type III population using patient blood
contacted with one or more of the following conjugates: a non-lytic
aerolysin variant protein conjugated to AlexaFluor.RTM. 488 (Protox
Biotech FL2-S), an antibody that binds to CD33 conjugated to
phycoerythrin (PE) (Clone D3HL60.251), an antibody that binds to
CD14 conjugated to ECD (Clone RMO52), an antibody that binds to
CD64 conjugated to PC5 (Clone 22), and an antibody that binds to
CD45 conjugated to PC7 (Clone J.33), which allowed for
lineage-specific gating on monocytes.
[0140] To determine the percentage of normal red blood cells (Type
I cells), PNH Type II, and PNH Type III red blood cells 20 ml of
peripheral blood in EDTA was placed in 3 mL of phosphate buffered
saline (PBS) and mixed thoroughly. 50 .mu.l of diluted patient
blood was contacted with one or more of the following conjugates:
an anti-CD235a antibody conjugated to FITC (Beckman Coulter clone
11E4B-7-6/KC16) and an anti-CD59 antibody conjugated to PE
(Invitrogen Clone MEM-43). The blood and conjugates were incubated
at room temperature in the dark for one hour while vortexing every
15 minutes. After the one hour incubation, the blood was washed
twice with PBS, resuspended in PBS, and analyzed on an FC 500 Flow
Cytometer (Beckman Coulter).
Example 2
[0141] Whole blood samples from 2,921 patients, which samples were
submitted for diagnostic testing for PNH, were analyzed using a
high-sensitivity flow cytometry-based assay to detect the
expression level of GPI and GPI-anchored proteins on white blood
cells (particularly granulocytes) to thereby determine the Type I,
PNH Type II, and PNH Type III white blood cell (granulocyte)
populations in each of the samples. The assay was also used to
detect the Type I, PNH Type II, and PNH Type III red blood cell
populations in each of the patient samples. The methods employed a
fluorescently-labeled non-lytic aerolysin protein variant along
with antibodies to specific GPI-anchored lineage-specific protein
antigens. An exemplary flow-cytometry analysis of one patient
sample is depicted in FIG. 1. Cells from a whole blood sample were
contacted with the fluorescently-labeled aerolysin reagent (Alexa
Fluor) and a phycoeyrthrin (PE)-labeled antibody that binds to the
GPI-anchored protein CD24. The cells of the whole blood sample were
subjected to flow cytometry analysis and the granulocytes therein
displayed based on the amount of signal detected from each reagent
bound to the surface of the granulocytes. As shown in FIG. 1,
granulocytes with the highest amount of signal detected from the
AlexaFluor and PE labels (upper right; Type I cells) were separated
from populations of granulocytes having a very low or absent signal
(lower left; Type III granulocytes) and granulocytes producing an
intermediate amount of signal (middle population; Type II
granulocytes).
[0142] The PNH red blood cell populations were interrogated using
two reagents: a detectably-labeled antibody that binds to CD235 and
a detectably-labeled reagent that binds to CD59. The PNH white
blood cell populations were interrogated using the
detectably-labeled aerolysin protein and several antibodies to
GPI-anchored lineage-specific cell surface proteins including CD24,
CD14, CD16, CD66b, and CD55.
[0143] 216 of the patient samples had a detectable PNH Type III
granulocyte population that was >0.01% of the total number of
granulocytes in the sample and an absolute count of at least 50 PNH
Type III granulocytes. Clinical information related to several
parameters (e.g., hemoglobin levels, LDH levels, and platelet
counts) was available for 162 of these patients (see Table 1).
TABLE-US-00001 TABLE 1 Clinical Features of Patients with a
Detectable PNH Type III or II granulocyte population (where
clinical data were available.) Cases Cases without with Type Type
II II granulocytes granulocytes P-value (N = 19) (N = 143) Wilcoxin
Median (range) Total PNH 87.20 11.40 <0.01 granulocyte
population (9.2-99.5) (0.01-99.9) (%) Median (range) PNH Type 7.10
n/a n/a II granulocyte population (1.2-65.3) (%) Median (range) PNH
Type 76.0 11.40 0.02 III granulocyte population (4.5-96.4)
(0.01-99.9) (%) Median (range) PNH Type 3.30 0.20 <0.01 II RBC
population (%) (0.02-71.3) (0-76.20) Median (range) PNH Type 16.10
2.90 0.01 III RBC population (%) (0.03-86.70) (0-92.9) Median white
blood cell 3.80 4.20 0.44 (.times.10.sup.9/L) Median absolute 2.07
2.15 0.70 neutrophil count (cells/.mu.L) Median RBC
(.times.10.sup.12/L) 3.08 3.14 0.80 Median hemoglobin (g/dL) 10.6
10.4 0.87 Median LDH (IU/L) 336 315 0.88 Median platelets
(.times.10.sup.9/L) 54 116 0.01 Platelets <100 .times.10.sup.9/L
(%) 68.4 44.0 0.05* (13/19) (62/141) *Fisher's exact test.
Of the samples from patients in which clinical information was
available, 19 (8.8%) patient samples contained distinct Type II
granulocyte populations, ranging from 1.2-65.3% of the total
granulocyte population, with a median clone size of approximately
7%. In 4 of the 19 patient samples, the Type II granulocyte
population represented >50% of the total abnormal population
(e.g., PNH Type H and Type III cells). In 10 of the 19 patient
samples, a PNH Type II monocyte population was also detected. An
evaluation of the ability of various antibodies, specific for
individual GPI-linked proteins found on granulocytes, to detect PNH
Type II granulocytes indicated that the Type II granulocyte
population was detectable in all cases using the detectably-labeled
aerolysin reagent, but in decreasing percentages using antibodies
specific for CD66b (88%), CD55 (50%), CD24 (47%), and CD16 (0%)
(see Table 2). These results indicate that the aerolysin-based
conjugate is particularly useful to accurately detect PNH Type II
granulocyte populations in patient samples.
TABLE-US-00002 TABLE 2 Detection of Type II granulocytes using
aerolysin or other anti-GPI anchored-protein antibodies. Aerolysin
CD24 CD66b CD55 CD16 19/19 (100%) 9/19 (47%) 8/9 (88%) 4/8 (50%)
0/9 (0%)
Patient samples containing PNH Type II granulocyte populations had
a significantly larger median total combined PNH Type II and PNH
Type III granulocyte population than those without Type II
granulocytes (87% versus 11%; p=0.0003), as well as larger median
Type II and Type III RBC populations, which reflects an increased
ability of the method to detect PNH Type II white blood cell
populations in patient samples with overall larger PNH cell
populations.
[0144] After comparison with the clinical data it was discovered
that patient samples with PNH Type II granulocyte populations also
had lower median platelet (plt) counts (54.times.10.sup.9/L;
p<0.01). See FIG. 2. Patient samples with PNH Type II
granulocyte populations had similar peripheral white blood cell
counts, peripheral red blood cell counts, absolute neutrophil
counts, and hemoglobin (Hgb) levels, compared to patient samples
without detectable Type II granulocyte populations (Table 1),
indicating that differences in platelet counts are likely not due
to differences in underlying bone marrow production. In other
words, while the disclosure is in no way limited by any particular
theory or mechanism of action, as PNH patients have dysregulated
complement control due to the lack of the GPI-linked complement
regulatory proteins CD55 and CD59, the decreased platelet counts
observed in patients with detectable PNH Type II granulocyte clones
may be due to increased terminal complement-mediated platelet
consumption or destruction, which may in turn be associated with
thrombosis, the leading cause of death among PNH patients.
[0145] While the present disclosure has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the disclosure. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present disclosure. All such
modifications are intended to be within the scope of the
disclosure.
Sequence CWU 1
1
71492PRTAeromonas hydrophila 1Gln Lys Ile Lys Leu Thr Gly Leu Ser
Leu Ile Ile Ser Gly Leu Leu 1 5 10 15 Met Ala Gln Ala Gln Ala Ala
Glu Pro Val Tyr Pro Asp Gln Leu Arg 20 25 30 Leu Phe Ser Leu Gly
Gln Gly Val Cys Gly Asp Lys Tyr Arg Pro Val 35 40 45 Asn Arg Glu
Glu Ala Gln Ser Val Lys Ser Asn Ile Val Gly Met Met 50 55 60 Gly
Gln Trp Gln Ile Ser Gly Leu Ala Asn Gly Trp Val Ile Met Gly 65 70
75 80 Pro Gly Tyr Asn Gly Glu Ile Lys Pro Gly Thr Ala Ser Asn Thr
Trp 85 90 95 Cys Tyr Pro Thr Asn Pro Val Thr Gly Glu Ile Pro Thr
Leu Ser Ala 100 105 110 Leu Asp Ile Pro Asp Gly Asp Glu Val Asp Val
Gln Trp Arg Leu Val 115 120 125 His Asp Ser Ala Asn Phe Ile Lys Pro
Thr Ser Tyr Leu Ala His Tyr 130 135 140 Leu Gly Tyr Ala Trp Val Gly
Gly Asn His Ser Gln Tyr Val Gly Glu 145 150 155 160 Asp Met Asp Val
Thr Arg Asp Gly Asp Gly Trp Val Ile Arg Gly Asn 165 170 175 Asn Asp
Gly Gly Cys Asp Gly Tyr Arg Cys Gly Asp Lys Thr Ala Ile 180 185 190
Lys Val Ser Asn Phe Ala Tyr Asn Leu Asp Pro Asp Ser Phe Lys His 195
200 205 Gly Asp Val Thr Gln Ser Asp Arg Gln Leu Val Lys Thr Val Val
Gly 210 215 220 Trp Ala Val Asn Asp Ser Asp Thr Pro Gln Ser Gly Tyr
Asp Val Thr 225 230 235 240 Leu Arg Tyr Asp Thr Ala Thr Asn Trp Ser
Lys Thr Asn Thr Tyr Gly 245 250 255 Leu Ser Glu Lys Val Thr Thr Lys
Asn Lys Phe Lys Trp Pro Leu Val 260 265 270 Gly Glu Thr Glu Leu Ser
Ile Glu Ile Ala Ala Asn Gln Ser Trp Ala 275 280 285 Ser Gln Asn Gly
Gly Ser Thr Thr Thr Ser Leu Ser Gln Ser Val Arg 290 295 300 Pro Thr
Val Pro Ala Arg Ser Lys Ile Pro Val Lys Ile Glu Leu Tyr 305 310 315
320 Lys Ala Asp Ile Ser Tyr Pro Tyr Glu Phe Lys Ala Asp Val Ser Tyr
325 330 335 Asp Leu Thr Leu Ser Gly Phe Leu Arg Trp Gly Gly Asn Ala
Trp Tyr 340 345 350 Thr His Pro Asp Asn Arg Pro Asn Trp Asn His Thr
Phe Val Ile Gly 355 360 365 Pro Tyr Lys Asp Lys Ala Ser Ser Ile Arg
Tyr Gln Trp Asp Lys Arg 370 375 380 Tyr Ile Pro Gly Glu Val Lys Trp
Trp Asp Trp Asn Trp Thr Ile Gln 385 390 395 400 Gln Asn Gly Leu Ser
Thr Met Gln Asn Asn Leu Ala Arg Val Leu Arg 405 410 415 Pro Val Arg
Ala Gly Ile Thr Gly Asp Phe Ser Ala Glu Ser Gln Phe 420 425 430 Ala
Gly Asn Ile Glu Ile Gly Ala Pro Val Pro Leu Ala Ala Asp Ser 435 440
445 His Ser Ser Lys Leu Gln Ser Val Asp Gly Ala Gly Gln Gly Leu Arg
450 455 460 Leu Glu Ile Pro Leu Asp Ala Gln Glu Leu Ser Gly Leu Gly
Phe Asn 465 470 475 480 Asn Val Ser Leu Ser Val Thr Pro Ala Ala Asn
Gln 485 490 2470PRTAeromonas hydrophila 2Ala Glu Pro Val Tyr Pro
Asp Gln Leu Arg Leu Phe Ser Leu Gly Gln 1 5 10 15 Gly Val Cys Gly
Asp Lys Tyr Arg Pro Val Asn Arg Glu Glu Ala Gln 20 25 30 Ser Val
Lys Ser Asn Ile Val Gly Met Met Gly Gln Trp Gln Ile Ser 35 40 45
Gly Leu Ala Asn Gly Trp Val Ile Met Gly Pro Gly Tyr Asn Gly Glu 50
55 60 Ile Lys Pro Gly Thr Ala Ser Asn Thr Trp Cys Tyr Pro Thr Asn
Pro 65 70 75 80 Val Thr Gly Glu Ile Pro Thr Leu Ser Ala Leu Asp Ile
Pro Asp Gly 85 90 95 Asp Glu Val Asp Val Gln Trp Arg Leu Val His
Asp Ser Ala Asn Phe 100 105 110 Ile Lys Pro Thr Ser Tyr Leu Ala His
Tyr Leu Gly Tyr Ala Trp Val 115 120 125 Gly Gly Asn His Ser Gln Tyr
Val Gly Glu Asp Met Asp Val Thr Arg 130 135 140 Asp Gly Asp Gly Trp
Val Ile Arg Gly Asn Asn Asp Gly Gly Cys Asp 145 150 155 160 Gly Tyr
Arg Cys Gly Asp Lys Thr Ala Ile Lys Val Ser Asn Phe Ala 165 170 175
Tyr Asn Leu Asp Pro Asp Ser Phe Lys His Gly Asp Val Thr Gln Ser 180
185 190 Asp Arg Gln Leu Val Lys Thr Val Val Gly Trp Ala Val Asn Asp
Ser 195 200 205 Asp Thr Pro Gln Ser Gly Tyr Asp Val Thr Leu Arg Tyr
Asp Thr Ala 210 215 220 Thr Asn Trp Ser Lys Thr Asn Thr Tyr Gly Leu
Ser Glu Lys Val Thr 225 230 235 240 Thr Lys Asn Lys Phe Lys Trp Pro
Leu Val Gly Glu Thr Glu Leu Ser 245 250 255 Ile Glu Ile Ala Ala Asn
Gln Ser Trp Ala Ser Gln Asn Gly Gly Ser 260 265 270 Thr Thr Thr Ser
Leu Ser Gln Ser Val Arg Pro Thr Val Pro Ala Arg 275 280 285 Ser Lys
Ile Pro Val Lys Ile Glu Leu Tyr Lys Ala Asp Ile Ser Tyr 290 295 300
Pro Tyr Glu Phe Lys Ala Asp Val Ser Tyr Asp Leu Thr Leu Ser Gly 305
310 315 320 Phe Leu Arg Trp Gly Gly Asn Ala Trp Tyr Thr His Pro Asp
Asn Arg 325 330 335 Pro Asn Trp Asn His Thr Phe Val Ile Gly Pro Tyr
Lys Asp Lys Ala 340 345 350 Ser Ser Ile Arg Tyr Gln Trp Asp Lys Arg
Tyr Ile Pro Gly Glu Val 355 360 365 Lys Trp Trp Asp Trp Asn Trp Thr
Ile Gln Gln Asn Gly Leu Ser Thr 370 375 380 Met Gln Asn Asn Leu Ala
Arg Val Leu Arg Pro Val Arg Ala Gly Ile 385 390 395 400 Thr Gly Asp
Phe Ser Ala Glu Ser Gln Phe Ala Gly Asn Ile Glu Ile 405 410 415 Gly
Ala Pro Val Pro Leu Ala Ala Asp Ser His Ser Ser Lys Leu Gln 420 425
430 Ser Val Asp Gly Ala Gly Gln Gly Leu Arg Leu Glu Ile Pro Leu Asp
435 440 445 Ala Gln Glu Leu Ser Gly Leu Gly Phe Asn Asn Val Ser Leu
Ser Val 450 455 460 Thr Pro Ala Ala Asn Gln 465 470
3439PRTAeromonas hydrophila 3Ala Glu Pro Val Tyr Pro Asp Gln Leu
Arg Leu Phe Ser Leu Gly Gln 1 5 10 15 Gly Val Cys Gly Asp Lys Tyr
Arg Pro Val Asn Arg Glu Glu Ala Gln 20 25 30 Ser Val Lys Ser Asn
Ile Val Gly Met Met Gly Gln Trp Gln Ile Ser 35 40 45 Gly Leu Ala
Asn Gly Trp Val Ile Met Gly Pro Gly Tyr Asn Gly Glu 50 55 60 Ile
Lys Pro Gly Thr Ala Ser Asn Thr Trp Cys Tyr Pro Thr Asn Pro 65 70
75 80 Val Thr Gly Glu Ile Pro Thr Leu Ser Ala Leu Asp Ile Pro Asp
Gly 85 90 95 Asp Glu Val Asp Val Gln Trp Arg Leu Val His Asp Ser
Ala Asn Phe 100 105 110 Ile Lys Pro Thr Ser Tyr Leu Ala His Tyr Leu
Gly Tyr Ala Trp Val 115 120 125 Gly Gly Asn His Ser Gln Tyr Val Gly
Glu Asp Met Asp Val Thr Arg 130 135 140 Asp Gly Asp Gly Trp Val Ile
Arg Gly Asn Asn Asp Gly Gly Cys Asp 145 150 155 160 Gly Tyr Arg Cys
Gly Asp Lys Thr Ala Ile Lys Val Ser Asn Phe Ala 165 170 175 Tyr Asn
Leu Asp Pro Asp Ser Phe Lys His Gly Asp Val Thr Gln Ser 180 185 190
Asp Arg Gln Leu Val Lys Thr Val Val Gly Trp Ala Val Asn Asp Ser 195
200 205 Asp Thr Pro Gln Ser Gly Tyr Asp Val Thr Leu Arg Tyr Asp Thr
Ala 210 215 220 Thr Asn Trp Ser Lys Thr Asn Thr Tyr Gly Leu Ser Glu
Lys Val Thr 225 230 235 240 Thr Lys Asn Lys Phe Lys Trp Pro Leu Val
Gly Glu Thr Glu Leu Ser 245 250 255 Ile Glu Ile Ala Ala Asn Gln Ser
Trp Ala Ser Gln Asn Gly Gly Ser 260 265 270 Thr Thr Thr Ser Leu Ser
Gln Ser Val Arg Pro Thr Val Pro Ala Arg 275 280 285 Ser Lys Ile Pro
Val Lys Ile Glu Leu Tyr Lys Ala Asp Ile Ser Tyr 290 295 300 Pro Tyr
Glu Phe Lys Ala Asp Val Ser Tyr Asp Leu Thr Leu Ser Gly 305 310 315
320 Phe Leu Arg Trp Gly Gly Asn Ala Trp Tyr Thr His Pro Asp Asn Arg
325 330 335 Pro Asn Trp Asn His Thr Phe Val Ile Gly Pro Tyr Lys Asp
Lys Ala 340 345 350 Ser Ser Ile Arg Tyr Gln Trp Asp Lys Arg Tyr Ile
Pro Gly Glu Val 355 360 365 Lys Trp Trp Asp Trp Asn Trp Thr Ile Gln
Gln Asn Gly Leu Ser Thr 370 375 380 Met Gln Asn Asn Leu Ala Arg Val
Leu Arg Pro Val Arg Ala Gly Ile 385 390 395 400 Thr Gly Asp Phe Ser
Ala Glu Ser Gln Phe Ala Gly Asn Ile Glu Ile 405 410 415 Gly Ala Pro
Val Pro Leu Ala Ala Asp Ser His Ser Ser Lys Leu Gln 420 425 430 Ser
Val Asp Gly Ala Gly Gln 435 4128PRTAeromonas hydrophila 4Leu Asp
Pro Asp Ser Phe Lys His Gly Asp Val Thr Gln Ser Asp Arg 1 5 10 15
Gln Leu Val Lys Thr Val Val Gly Trp Ala Val Asn Asp Ser Asp Thr 20
25 30 Pro Gln Ser Gly Tyr Asp Val Thr Leu Arg Tyr Asp Thr Ala Thr
Asn 35 40 45 Trp Ser Lys Thr Asn Thr Tyr Gly Leu Ser Glu Lys Val
Thr Thr Lys 50 55 60 Asn Lys Phe Lys Trp Pro Leu Val Gly Glu Thr
Glu Leu Ser Ile Glu 65 70 75 80 Ile Ala Ala Asn Gln Ser Trp Ala Ser
Gln Asn Gly Gly Ser Thr Thr 85 90 95 Thr Ser Leu Ser Gln Ser Val
Arg Pro Thr Val Pro Ala Arg Ser Lys 100 105 110 Ile Pro Val Lys Ile
Glu Leu Tyr Lys Ala Asp Ile Ser Tyr Pro Tyr 115 120 125
5128PRTAeromonas hydrophila 5Leu Asp Pro Asp Ser Phe Lys His Gly
Asp Val Thr Gln Ser Asp Arg 1 5 10 15 Gln Leu Val Lys Thr Val Val
Gly Trp Ala Val Asn Asp Ser Asp Thr 20 25 30 Pro Gln Ser Gly Tyr
Asp Val Thr Leu Arg Tyr Asp Thr Ala Thr Asn 35 40 45 Trp Ser Lys
Thr Asn Thr Tyr Gly Leu Ser Glu Lys Val Thr Thr Lys 50 55 60 Asn
Lys Phe Lys Trp Pro Leu Val Gly Glu Cys Glu Leu Ser Ile Glu 65 70
75 80 Ile Ala Ala Asn Gln Ser Trp Ala Ser Gln Asn Gly Gly Ser Thr
Thr 85 90 95 Thr Ser Leu Ser Gln Ser Val Arg Pro Thr Val Pro Ala
Arg Ser Lys 100 105 110 Ile Pro Val Lys Ile Glu Leu Tyr Lys Cys Asp
Ile Ser Tyr Pro Tyr 115 120 125 6492PRTAeromonas salmonicida 6Met
Lys Lys Leu Lys Ile Thr Gly Leu Ser Leu Ile Ile Ser Gly Leu 1 5 10
15 Leu Met Ala Gln Ala Gln Ala Ala Glu Pro Val Tyr Pro Asp Gln Leu
20 25 30 Arg Leu Phe Ser Leu Gly Gln Glu Val Cys Gly Asp Lys Tyr
Arg Pro 35 40 45 Val Asn Arg Glu Glu Ala Gln Ser Val Lys Ser Asn
Ile Val Gly Met 50 55 60 Met Gly Gln Trp Gln Ile Ser Gly Leu Ala
Asn Gly Trp Val Ile Met 65 70 75 80 Gly Pro Gly Tyr Asn Gly Glu Ile
Lys Pro Gly Ser Ala Ser Ser Thr 85 90 95 Trp Cys Tyr Pro Thr Asn
Pro Ala Thr Gly Glu Ile Pro Thr Leu Ser 100 105 110 Ala Leu Asp Ile
Pro Asp Gly Asp Glu Val Asp Val Gln Trp Arg Leu 115 120 125 Val His
Asp Ser Ala Asn Phe Ile Lys Pro Thr Ser Tyr Leu Ala His 130 135 140
Tyr Leu Gly Tyr Ala Trp Val Gly Gly Asn His Ser Gln Tyr Val Gly 145
150 155 160 Glu Asp Met Asp Val Thr Arg Asp Gly Asp Gly Trp Val Ile
Arg Gly 165 170 175 Asn Asn Asp Gly Gly Cys Asp Gly Tyr Arg Cys Gly
Asp Lys Thr Ser 180 185 190 Ile Lys Val Ser Asn Phe Ala Tyr Asn Leu
Asp Pro Asp Ser Phe Lys 195 200 205 His Gly Asp Val Thr Gln Ser Asp
Arg Gln Leu Val Lys Thr Val Val 210 215 220 Gly Trp Ala Ile Asn Asp
Ser Asp Thr Pro Gln Ser Gly Tyr Asp Val 225 230 235 240 Thr Leu Arg
Tyr Asp Thr Ala Thr Asn Trp Ser Lys Thr Asn Thr Tyr 245 250 255 Gly
Leu Ser Glu Lys Val Thr Thr Lys Asn Lys Phe Lys Trp Pro Leu 260 265
270 Val Gly Glu Thr Glu Leu Ser Ile Glu Ile Ala Ala Asn Gln Ser Trp
275 280 285 Ala Ser Gln Asn Gly Gly Ser Thr Thr Thr Ser Leu Ser Gln
Ser Val 290 295 300 Arg Pro Thr Val Pro Ala His Ser Lys Ile Pro Val
Lys Ile Glu Leu 305 310 315 320 Tyr Lys Ala Asp Ile Ser Tyr Pro Tyr
Glu Phe Lys Ala Asp Val Ser 325 330 335 Tyr Asp Leu Thr Leu Ser Gly
Phe Leu Arg Trp Gly Gly Asn Ala Trp 340 345 350 Tyr Thr His Pro Asp
Asn Arg Pro Asn Trp Asn His Thr Phe Val Ile 355 360 365 Gly Pro Tyr
Lys Asp Lys Ala Ser Ser Ile Arg Tyr Gln Trp Asp Lys 370 375 380 Arg
Tyr Ile Pro Gly Glu Val Lys Trp Ser Asp Trp Asn Trp Thr Ile 385 390
395 400 Gln Gln Asn Gly Leu Ser Thr Met Gln Asn Asn Leu Ala Arg Val
Leu 405 410 415 Arg Pro Val Arg Ala Gly Ile Thr Gly Asp Phe Ser Ala
Glu Ser Gln 420 425 430 Phe Ala Gly Asn Ile Glu Ile Gly Ala Pro Val
Pro Val Ala Ala Ala 435 440 445 Ser Gln Ser Ser Arg Ala Arg Asn Leu
Ser Ala Gly Gln Gly Leu Arg 450 455 460 Leu Glu Ile Pro Leu Asp Ala
Gln Glu Leu Ser Gly Leu Gly Phe Asn 465 470 475 480 Asn Val Ser Leu
Ser Val Thr Pro Ala Ala Asn Gln 485 490 7469PRTAeromonas
salmonicida 7Ala Glu Pro Val Tyr Pro Asp Gln Leu Arg Leu Phe Ser
Leu Gly Gln 1 5 10 15 Glu Val Cys Gly Asp Lys Tyr Arg Pro Val Asn
Arg Glu Glu Ala Gln 20 25 30 Ser Val Lys Ser Asn Ile Val Gly Met
Met Gly Gln Trp Gln Ile Ser 35 40 45 Gly Leu Ala Asn Gly Trp Val
Ile Met Gly Pro Gly Tyr Asn Gly Glu 50 55 60 Ile Lys Pro Gly Ser
Ala Ser Ser Thr Trp Cys Tyr Pro Thr Asn Pro 65 70 75 80 Ala Thr Gly
Glu Ile Pro Thr Leu Ser Ala Leu Asp Ile Pro Asp Gly 85 90 95 Asp
Glu Val Asp Val Gln Trp Arg Leu Val His Asp Ser Ala Asn Phe 100 105
110 Ile Lys Pro Thr Ser Tyr Leu Ala His Tyr Leu Gly Tyr Ala Trp Val
115 120
125 Gly Gly Asn His Ser Gln Tyr Val Gly Glu Asp Met Asp Val Thr Arg
130 135 140 Asp Gly Asp Gly Trp Val Ile Arg Gly Asn Asn Asp Gly Gly
Cys Asp 145 150 155 160 Gly Tyr Arg Cys Gly Asp Lys Thr Ser Ile Lys
Val Ser Asn Phe Ala 165 170 175 Tyr Asn Leu Asp Pro Asp Ser Phe Lys
His Gly Asp Val Thr Gln Ser 180 185 190 Asp Arg Gln Leu Val Lys Thr
Val Val Gly Trp Ala Ile Asn Asp Ser 195 200 205 Asp Thr Pro Gln Ser
Gly Tyr Asp Val Thr Leu Arg Tyr Asp Thr Ala 210 215 220 Thr Asn Trp
Ser Lys Thr Asn Thr Tyr Gly Leu Ser Glu Lys Val Thr 225 230 235 240
Thr Lys Asn Lys Phe Lys Trp Pro Leu Val Gly Glu Thr Glu Leu Ser 245
250 255 Ile Glu Ile Ala Ala Asn Gln Ser Trp Ala Ser Gln Asn Gly Gly
Ser 260 265 270 Thr Thr Thr Ser Leu Ser Gln Ser Val Arg Pro Thr Val
Pro Ala His 275 280 285 Ser Lys Ile Pro Val Lys Ile Glu Leu Tyr Lys
Ala Asp Ile Ser Tyr 290 295 300 Pro Tyr Glu Phe Lys Ala Asp Val Ser
Tyr Asp Leu Thr Leu Ser Gly 305 310 315 320 Phe Leu Arg Trp Gly Gly
Asn Ala Trp Tyr Thr His Pro Asp Asn Arg 325 330 335 Pro Asn Trp Asn
His Thr Phe Val Ile Gly Pro Tyr Lys Asp Lys Ala 340 345 350 Ser Ser
Ile Arg Tyr Gln Trp Asp Lys Arg Tyr Ile Pro Gly Glu Val 355 360 365
Lys Trp Ser Asp Trp Asn Trp Thr Ile Gln Gln Asn Gly Leu Ser Thr 370
375 380 Met Gln Asn Asn Leu Ala Arg Val Leu Arg Pro Val Arg Ala Gly
Ile 385 390 395 400 Thr Gly Asp Phe Ser Ala Glu Ser Gln Phe Ala Gly
Asn Ile Glu Ile 405 410 415 Gly Ala Pro Val Pro Val Ala Ala Ala Ser
Gln Ser Ser Arg Ala Arg 420 425 430 Asn Leu Ser Ala Gly Gln Gly Leu
Arg Leu Glu Ile Pro Leu Asp Ala 435 440 445 Gln Glu Leu Ser Gly Leu
Gly Phe Asn Asn Val Ser Leu Ser Val Thr 450 455 460 Pro Ala Ala Asn
Gln 465
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