U.S. patent application number 14/814179 was filed with the patent office on 2015-11-19 for methods of treating hematological proliferative disorders by targeting epha3 expressed on aberrant vasculature in bone marrow.
The applicant listed for this patent is KaloBios Pharmaceuticals, Inc.. Invention is credited to Mark Baer, Christopher R. Bebbington, Varghese Palath, Geoffrey T. Yarranton.
Application Number | 20150329647 14/814179 |
Document ID | / |
Family ID | 47677671 |
Filed Date | 2015-11-19 |
United States Patent
Application |
20150329647 |
Kind Code |
A1 |
Yarranton; Geoffrey T. ; et
al. |
November 19, 2015 |
Methods of Treating Hematological Proliferative Disorders by
Targeting EphA3 Expressed on Aberrant Vasculature in Bone
Marrow
Abstract
The invention provides diagnostic and therapeutic methods for
the treatment of hematological proliferative disorders.
Inventors: |
Yarranton; Geoffrey T.;
(South San Francisco, CA) ; Palath; Varghese;
(South San Francisco, CA) ; Bebbington; Christopher
R.; (South San Francisco, CA) ; Baer; Mark;
(South San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KaloBios Pharmaceuticals, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
47677671 |
Appl. No.: |
14/814179 |
Filed: |
July 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13571827 |
Aug 10, 2012 |
9109032 |
|
|
14814179 |
|
|
|
|
61523104 |
Aug 12, 2011 |
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Current U.S.
Class: |
424/133.1 ;
424/158.1; 435/330; 435/7.23; 530/388.22 |
Current CPC
Class: |
G01N 2333/715 20130101;
C07K 2317/41 20130101; C07K 2317/33 20130101; G01N 33/57492
20130101; A61P 35/02 20180101; A61P 7/00 20180101; G01N 2333/912
20130101; C07K 16/2866 20130101; G01N 33/6863 20130101; C07K 16/40
20130101; A61P 35/00 20180101; G01N 2800/52 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method of treating a patient that has a hematological
proliferative disorder where the hematological proliferative
disorder cells are substantially EphA3.sup.- and aberrant
vasculature in bone marrow is EphA3.sup.+, the method comprising
administering a therapeutically effective amount of an anti-EphA3
antibody to the patient, wherein 20% or fewer of the hematological
disorder cells express EphA3.
2. The method of claim 1, wherein 10% or fewer of the hematological
proliferative disorder cells express EphA3.
3. The method of claim 1, wherein the hematological proliferative
disorder cells are myelodysplastic syndrome (MDS) cells.
4. The method of claim 1, wherein the hematological proliferative
disorder cells are myelofibrosis cells.
5. The method of claim 1, wherein the anti-EphA3 antibody comprises
a human heavy chain gamma-1 or gamma-3 constant region.
6. The method of claim 5, wherein the anti-EphA3 antibody is
provided in a hypofucosylated or afucosylated antibody
preparation.
7. The method of claim 1, wherein the anti-EphA3 antibody is a
monoclonal antibody.
8. The method of claim 1, wherein the anti-EphA3 antibody induces
ADCC.
9. The method of claim 1, wherein: (a) the myeloid proliferative
disorder cells are myelofibrosis cells or MDS cells; and (b) the
anti-EphA3 is a monoclonal antibody that (i) activates EphA3 (ii)
induces ADCC; (iii) binds to an epitope on EphA3 to which an
antibody having a V.sub.H region CDR1 having a sequence SYWIN (SEQ
ID NO:2), a V.sub.H region CDR2 having a sequence DIYPGSGNTNYDEKFKR
(SEQ ID NO:3), a V.sub.H region CDR3 having a sequence SGYYEDFDS
(SEQ ID NO:4), a V.sub.L region CDR1 having a sequence RASQEISGYLG
(SEQ ID NO:9), a V.sub.L region CDR2 having a sequence AASTLDS (SEQ
ID NO:10), and a V.sub.L region CDR3 having a sequence VQYANYPYT
(SEQ ID NO:11) binds; (iv) has framework regions from a human
immune system; (v) comprises a human heavy chain gamma-1 constant
region; and (vi) is provided in a hypofucosylated or afucosylated
antibody preparation.
10. The method of claim 1, wherein the antibody is a conjugate.
11. The method of claim 1, further comprising administering at
least one additional therapeutic agent, wherein the at least one
additional therapeutic agent is a chemotherapeutic agent.
12. A method of determining that a patient having a hematological
proliferative disorder is a candidate for treatment with an
anti-EphA3 antibody, the method comprising: providing a bone marrow
sample from the patient; and detecting expression of EphA3 on
aberrant vasculature present in the bone marrow.
13. The method of claim 12, wherein EphA3 expression is detected in
the endothelial layer of the aberrant vasculature.
14. The method of claim 12, wherein EphA3 expression is detected in
the tunica media or tunica externa of the aberrant vasculature.
15. The method of claim 12, wherein the step of detecting
expression of EphA3 comprises contacting an anti-EphA3 antibody
with the sample.
16. The method of claim 15, wherein the anti-EphA3 antibody is
produced by a hybridoma deposited with the American Type Culture
Collection (ATCC) having the Patent Deposit Designation PTA-12227;
or produced by a hybridoma deposited with the ATCC having the
Patent Deposit Designation PTA-12228.
17. The method of claim 12, wherein the patient has AML, CML, CMML,
JMML, MDS, PV, ET, IM, chronic lymphocytic leukemia, multiple
myeloma, diffuse large B-cell lymphoma, non-Hodgkin lymphoma,
mantle cell lymphoma, chronic neutrophilic leukemia, chronic
eosinophilic leukemia, or mast cell disease.
18. A hybridoma deposited with the American Type Culture Collection
having the Patent Deposit Designation PTA-12227 or having the
Patent Deposit Designation PTA-12228.
19. A monoclonal antibody produced by the hybridoma of claim
18.
20. A kit containing at least one reagent for detecting EphA3
expression, wherein the at least one reagent is a monoclonal
antibody of claim 19.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/571,827 filed Aug. 10, 2012, allowed, which claims benefit
of U.S. provisional application No. 61/523,104 filed Aug. 12, 2011,
which applications are herein incorporated by reference for all
purposes.
REFERENCE TO A "SEQUENCE LISTING" SUBMITTED AS AN ASCII TEXT
FILE
[0002] The sequence listing written in file
SEQTXT.sub.--87142.sub.--004220US.sub.--951005.txt, created on Jul.
30, 2015; 5,940 bytes, machine format IBM-PC, MS-Windows operating
system, is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] EphA3 is a receptor tyrosine kinase that is expressed on the
surface of certain solid tumors and on the vasculature of solid
tumors (see, WO 2008/112192). In addition, EphA3 has been reported
to be overexpressed on CD34.sup.+ cells in chronic myeloid leukemia
(CML) patients in the accelerated phase and blast crisis stage
(Cilloni et al., American Society of Hematology, Abstract 1092,
2008). EphA3 is also expressed on the surface of neoplastic myeloid
cells, including neoplastic myeloid stem cells, in the bone marrow
and peripheral blood samples obtained from patients that have
chronic myeloid leukemia (CML), acute myeloid leukemia (AML),
chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic
leukemia (JMML), myelodysplastic syndrome (MDS), polycythemia vera
(PV), essential thrombocythemia (ET), or idiopathic myelofibrosis
(IM) (see, WO 2010/102244).
[0004] Angiogenesis is increased in bone marrow of AML and
myelofibrosis patients (see, e.g., Padro et al., Blood 95:2637,
2000; Gianelli et al, Am. J. Clin. Path. 128:966, 2007) and high
microvessel density is a poor prognostic factor in AML (e.g., Kukzu
et al, Leukemia and Lymphoma 45:1185, 2004). Anti-angiogenic
treatment with endostatin inhibits progression of AML in a mouse
model (Schuch et al., Leukemia 19:1312, 2005). Further, new blood
vessel formation is also observed in other hematological
malignancies such as multiple myeloma, acute leukemias, and
hematological proliferative neoplasms (see, e.g., Medinger &
Mross, J. Angiogenesis Res. 2:10, 2010; and Frater et al.,
Diagnostic Pathol. 3:16, 2008).
BRIEF SUMMARY OF THE INVENTION
[0005] The invention is based, in part, on the discovery that
vasculature in bone marrow from patients that have a hematological
proliferative disorder express EphA3. Agents that target EphA3,
such as activating EphA3 antibodies, can be employed for the
treatment of hematological proliferative disorders in patients
where the neoplastic cells themselves do not expression EphA3, or
express low levels of EphA3. In some embodiments, the patient has
chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML),
acute myeloid leukemia (AML), chronic myelomonocytic leukemia
(CMML), juvenile myelomonocytic leukemia (JMML), multiple myeloma
(MM), diffuse large B-cell lymphoma, non-Hodgkin lymphoma, mantle
cell lymphoma (MCL), myelodysplastic syndrome (MDS), chronic
neutrophilic leukemia, chronic eosinophilic leukemia, mast cell
disease, polycythemia vera (PV), essential thrombocythemia (ET), or
idiopathic myelofibrosis (IM).
[0006] It is the discovery of the inventors that EphA3 is
selectively expressed on the aberrant vasculature in bone marrow of
patients that have a hematological proliferative disorder. Thus, in
one aspect, the invention provides a method of treating a patient
that has a hematological proliferative disorder, the method
comprising administering an anti EphA3 antibody to a patient that
has a hematological proliferative disorder in which EphA3 is not
expressed on the surface of the hematological proliferative
disorder cells, or is expressed at low levels on the hematological
proliferative disorder cells. In some embodiments, the patient has
AML. In some embodiments, the patient has CML, CMML, JMML, MDS, PV,
ET, or IM. In some embodiments, the patient has CLL, MM, diffuse
large B-cell lymphoma, non-Hodgkin lymphoma (NHL), MCL, chronic
neutrophilic leukemia, chronic eosinophilic leukemia, or mast cell
disease. Not to be bound by theory, a therapeutic anti-EphA3
antibody kills cells, e.g., vascular endothelial cells, that
express EphA3, but it is not necessarily anti-angiogenic in the
sense that the antibody may not prevent proliferation of
endothelial cells and angiogenesis.
[0007] In some embodiments, the therapeutic anti-EphA3 antibody
multimerizes EphA3. In some embodiments, the therapeutic anti-EphA3
antibody activates EphA3 and causes morphological changes to target
cells, e.g., rounding. In some embodiments, the therapeutic
anti-EphA3 antibody kills the target cells by inducing
antibody-dependent cell-mediated cytotoxicity (ADCC). In some
embodiments, the therapeutic anti-EphA3 antibody (i) activates
EphA3 and (ii) induces antibody-dependent cell-mediated
cytotoxicity (ADCC).
[0008] In an additional aspect, the invention provides a method of
treating a hematological proliferative disorder patient that has
aberrant bone marrow vasculature that selectively expresses EphA3,
but where the hematological proliferative disorder cells do not
express EphA3 on the surface or express EphA3 at a low level, the
method comprising administering a therapeutically effective amount
of an anti-EphA3 antibody to the patient, wherein the anti-EphA3
antibody activates EphA3 and/or induces ADCC.
[0009] In some embodiments, the therapeutic anti-EphA3 antibody for
use in the methods of the invention is a recombinant or chimeric
antibody. In some embodiments, the therapeutic anti-EphA3 antibody
is a human antibody. The therapeutic anti-EphA3 antibody may be a
polyclonal antibody or a monoclonal antibody. In some embodiments,
the therapeutic anti-EphA3 antibody is a multivalent antibody that
comprises a Fab, a Fab', or an Fv. In some embodiments, the
antibody is a F(ab').sub.2. In some embodiments, the therapeutic
anti-EphA3 antibody competes for EphA3 binding with mAb IIIA4. In
some embodiments, the therapeutic antibody binds to the same
epitope as mAb IIIA4. In typical embodiments, the therapeutic
antibody does not block ephrin ligand binding, e.g., ephrinA5
binding, to EphA3. In some embodiments the therapeutic anti-EphA3
antibody comprises the V.sub.H and V.sub.L regions of mAb IIIA4. In
some embodiments, the therapeutic anti EphA3 antibody comprises the
V.sub.H and V.sub.L region CDR1, CDR2 and CDR3 of mAb IIIA4. In
some embodiments, the therapeutic antibody comprises the V.sub.H
region CDR3 and V.sub.L region CDR3 of mAb IIIA4. In some
embodiments, the therapeutic antibody induces ADCC. Thus, in some
embodiments the therapeutic antibody has an active isotype, e.g.,
the antibody has a human heavy chain constant region that is a
gamma-1 or gamma-3 region. In some embodiments, the therapeutic
antibody does not induce ADCC, e.g., the antibody has a human heavy
chain constant region that is a gamma-2 or gamma-4 region, or is a
multimeric fragment.
[0010] In the context of this invention, "an anti-EphA3 antibody
that activates EphA3 or induces ADCC" refers to an antibody that
(i) activates EphA3 (ii) induces ADCC, or (iii) activates and
induces ADCC.
[0011] In some embodiments of the invention, a hematological
proliferative disorder patient is treated with an anti-EphA3
antibody as described herein and also receives treatment with
another therapeutic agent for the disease. Thus, in some
embodiments, the method comprises administering one or more
additional therapeutic agents. For example, when the hematological
proliferative disorder is CML, additional therapeutic agents may
include imatinib mesylate, nilotinib, dasatinib, or another
chemotherapeutic agent. When the hematological proliferative
disorder is AML, the additional therapeutic agents may be, e.g.,
cytosine arabinoside alone or in combination with daunorubicin.
[0012] In additional aspects, the invention provides a method of
identifying a hematological proliferative disorder patient having a
hematological proliferative disorder that is a candidate for
treatment with an anti-EphA3 antibody, wherein the method comprises
detecting EphA3 expression on the aberrant bone marrow vasculature
in the hematological proliferative disorder patient In some
embodiments, the patient has AML. In some embodiments, the patient
has CML, CMML, JMML, MDS, PV, ET, or IM. In some embodiments, the
patient has CLL, MM, diffuse large B-cell lymphoma, NHL, MCL,
chronic neutrophilic leukemia, chronic eosinophilic leukemia, or
mast cell disease.
[0013] In some embodiments, the invention provides a method of
monitoring the efficacy of treatment of a hematological
proliferative disorder patient that has aberrant bone marrow
vasculature that expresses EphA3, the method comprising: obtaining
a bone marrow sample from a patient that has been treated with a
therapeutic agent for the hematological proliferative disorder; and
detecting expression of EphA3 on the aberrant bone marrow
vasculature.
[0014] EphA3 expression can be detected using commonly known
techniques. Thus, in some embodiments detecting expression of EphA3
comprises detecting protein expression on the vasculature, e.g.,
using immunohistochemistry. In some embodiments, an antibody used
to detect EphA3 expression on aberrant vasculature binds to an
epitope different from the epitope that is bound by the therapeutic
antibody. In some embodiments, the antibody is the monoclonal
antibody SL-2, which is produced by a hybridoma deposited with the
American Type Culture Collection (ATCC), 10801 University Blvd.,
Manassas, Va. 20110, USA on Nov. 8, 2011 under the conditions of
the Budapest Treaty and assigned patent deposit number PTA-12227.
In some embodiments, the antibody is the monoclonal antibody SL-7,
which is produced by a hybridoma deposited with ATCC on Nov. 8,
2011 under the conditions of the Budapest Treaty and assigned
patent deposit number PTA-12228.
[0015] In a further aspect, the invention provides monoclonal
antibodies, e.g., for detection of EphA3 expression on aberrant
vasculature. In one embodiment, the monoclonal antibody SL-2, which
is produced by a hybridoma deposited under ATCC deposit number
PTA-12227. In one embodiment, the monoclonal antibody is SL-7,
which is produced by a hybridoma deposited under ATCC deposit
number PTA-12228. The invention additional provides kits that
include a monoclonal antibody SL-2 and/or SL-7. Such kits can,
e.g., also contain additional reagents, such as reagents for
immunohistochemical analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 provides illustrative data from an ELISA
demonstrating antibody binding to EphA3. Supernatants from six
hybridomas were diluted in PBS to 5 .mu.g/ml and applied to EphA3
coated plates. Binding was detected with a goat anti mouse HRP
conjugate. Data were analyzed with Prizm 5.0 software.
[0017] FIG. 2 provides illustrative data that shows binding of
purified SL antibodies to LK63 cells analyzed by FACS. Binding of
antibodies is shown (open histogram) in comparison with an
irrelevant isotype control antibody (filled histogram) (A) 25 nM
mouse mAb IIIA4, (B) 25 nM SL-2, (C) 25 nM SL-5, and D) 25 nM SL-7
antibodies to LK63 cells is shown. Data for each sample represents
10,000 events excluding dead cells.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0018] The term "hematological proliferative disorders" as used
herein refers to certain diseases that involve abnormal
proliferation of hematological cells, including leukemias,
lymphomas, and various myeloid neoplasias and dysplasias. In the
current invention, hematological proliferative diseases thus
include chronic hematological proliferative disorders (CMPDs);
acute myeloid leukemia (AML); chronic myelomonocytic leukemia
(CMML); juvenile myelomonocytic leukemia (JMML), chronic
lymphocytic leukemia (CLL), chronic myeloid leukemia (CML),
multiple myeloma (MM), diffuse large B-cell lymphoma, non-Hodgkin
lymphoma (NHL), mantle cell lymphoma (MCL), myelodysplastic
syndrome (MDS), chronic neutrophilic leukemia, chronic eosinophilic
leukemia, mast cell disease, polycythemia vera (PV), essential
thrombocythemia (ET), and idiopathic myelofibrosis (IM). The term
"JMML" encompasses all diagnoses referred to as Juvenile Chronic
Myeloid Leukemia (JCML), Chronic Myelomonocytic Leukemia of
Infancy, and Infantile Monosomy 7 Syndrome. Hematological
proliferative disorders can be diagnosed using known criteria,
e.g., the World Health Organization (WHO) criteria, the
French-American-British (FAB) classification system, the
International Prognostic Scoring System (IPSS), and the like. In
the 2008 WHO classification, CMPDs are referred to as hematological
proliferative neoplasms (MPNs). Hematological proliferative
disorders are often characterized by the presence of particular
mutations. For example, CML is characterized by the presence of
BCR-ABL. PV, ET, and IM are "non-BCR-ABL" (also referred to herein
as "BCR-ABL minus" or "BCR-ABL negative") CMPDs, as these disorders
do not have BCR-ABL. However, BCR-ABL negative disorders are often
characterized by the presence of JAK2 mutations, which are rare in
CML. As used herein, the term "CML" includes both Philadelphia
chromosome positive and Philadelphia chromosome negative CML.
[0019] The term "hematological proliferative disorder cells" as
used in the context of this invention refers to neoplastic
hematological cells that are characteristic of a hematological
proliferative disorder. The term "neoplastic" encompasses cells
that may not yet be considered to be malignant, e.g., such as the
myeloid cells that are characteristic of myelodysplastic syndrome,
as well as malignant cells, such as malignant acute leukemia cells.
"Hematological proliferative disorder cells" encompasses both blast
cells and stem cells, but does not include aberrant bone marrow
vasculature associated with the aberrant angiogenesis that occurs
in the bone marrow of patients that have a hematological
proliferative disorder, e.g., AML patients.
[0020] As used in the context of this application, "aberrant bone
marrow angiogenesis" refers to increased, and structurally
abnormal, angiogenesis relative to the level of angiogenesis in
normal bone marrow and the structure of the vasculature in normal
bone marrow. As used herein "aberrant vasculature" refers to
anomalies of microvascular architecture including: enhanced
irregularity of shape and tortuosity, increased fibers, increased
microvessel density, and complex branching of irregular shaped
sinuses (see, e.g., Kvansnicka & Thlele, Histol. Pathol.
19:1245-1260, 2004).
[0021] The term "selectively expressed" as applied to EphA3
expression in the context of expression or "selective expression of
EphA3" on aberrant vasculature refers to EphA3 expression on
abnormal vasculature, but not normal vasculature. EphA3 can be
expressed in the endothelium of the abnormal vasculature or in
other parts of vessels, e.g., the tunica media or tunica
externa.
[0022] In the context of this invention, "low level" or "minimal"
expression of EphA3 on hematological proliferative disorder cells
refers to expression in a population of cells such that less than
50%, or in some embodiments, less than 40%, 30%, 20%, 10%, or less
than 5%, of the hematological proliferative disorder cells express
EphA3 as assessed by flow cytometry or other means known in the
art. In some embodiments, hematological proliferative disorder
cells in CML, PV, ET, IM, AML, MDS, CMML, or JMML exhibit low level
expression of EphA3, where EphA3 is expressed at a level of 5% or
less of the hematological proliferative disorder cell population as
assessed by flow cytometry.
[0023] The term "EphA3.sup.+" when used in the context of
expression on aberrant vasculature refers to EphA3 expression that
is above background. The term "substantially Epha3.sup.-" in the
context of expression of EphA3 on neoplastic hematological disorder
cells refers to no expression, i.e., where expression is not
observed on any of the cells or where less than 50%, typically less
than 40%, less than 30%, less than 20%, less than 10% or less than
5% of the neoplastic cells express EphA3.
[0024] The terms "cancer cell" and "tumor cell" are used
interchangeably to refer to a neoplastic cell. The term includes
cells that are benign as well as malignant. Neoplastic
transformation is associated with phenotypic changes of the tumor
cell relative to the cell type from which it is derived. The
changes can include morphological changes, and aberrant growth.
(see, Freshney, Culture of Animal Cells a Manual of Basic Technique
(3.sup.rd edition, 1994).
[0025] "Inhibiting growth of hematological proliferative disorder
cells" in the context of the invention refers to slowing growth
and/or reducing the cancer cell burden of a patient that has a
hematological proliferative disorder. "Inhibiting growth of a
cancer", e.g., AML, thus includes killing cancer cells.
[0026] As used herein "EphA3" refers to the Eph receptor A3. This
receptor has also been referred to as "Human embryo kinase", "hek",
"eph-like tyrosine kinase 1", "etk1" or "tyro4". EphA3 belongs to
the ephrin receptor subfamily of the protein-tyrosine kinase
family. EPH and EPH-related receptors have been implicated in
mediating developmental events. Receptors in the EPH subfamily
typically have a single kinase domain and an extracellular region
containing a Cys-rich domain and 2 fibronectin type III repeats.
The ephrin receptors are divided into 2 groups based on the
similarity of their extracellular domain sequences and their
affinities for binding ephrin-A and ephrin-B ligands. EphA3 binds
ephrin-A ligands. EphA3 nucleic acid and protein sequences are
known. An example of a human EphA3 amino acid sequence is available
under accession number EAW68857.
[0027] In the context of this invention, "an anti-EphA3 antibody
that activates EphA3 or induces ADCC" refers to an antibody that
(i) activates EphA3 (ii) induces ADCC, or (iii) activates and
induces ADCC.
[0028] For the purposes of the present invention, "activation" of
EphA3 causes phosphorylation of EphA3. An antibody that activates
EphA3 or "an activating antibody" causes phosphorylation of EphA3
and shape change, e.g., rounding, of endothelial cells and is
therefore considered to be an agonist in the context of this
invention. EphA3 can be activated by dimerization, which leads to
shape change, e.g., rounding, in endothelial cells and may lead to
apoptosis. In some embodiments, an antibody that activates EphA3
competes with mAb IIIA4 for binding to EphA3. Typically, an
"activating" antibody binds to the ligand binding domain (amino
acids 29-202 of EphA3) wherein amino acid residues 131, 132, and
136 are important for binding. In some embodiments, the activating
antibody binds to a site encompassing the residues 131, 132, and
136 within the ligand binding domain of human EphA3 protein.
[0029] In the present application, the terms "EphA3 antibody" or
"anti-EphA3 antibody" are used interchangeably to refer to an
antibody that specifically binds to EphA3. In some embodiments, the
antibody can dimerize EphA3. The term encompasses antibodies that
bind to EphA3 in the presence of ephrin ligand (e.g., ephrin-A5)
binding, as well as antibodies that bind to the ligand binding
site.
[0030] An "EphA3 antibody that binds to EphA3 in the presence of
binding of an ephrin ligand" refers to an antibody that does not
significantly prevent binding of an ephrin ligand, such as
ephrin-A5, to EphA3. The presence of such an antibody in a binding
reaction comprising EphA3 and an ephrin ligand, e.g., ephrin-A5,
reduces ephrin ligand binding to EphA3 by less than about 30%,
typically less than 20% or 10%.
[0031] The term "mAb IIIA4" refers to monoclonal antibody IIIA4
that was originally raised against LK63 human acute pre-B leukemia
cells to affinity isolate EphA3 (Boyd, et al. J Biol Chem
267:3262-3267, 1992). mAb IIIA4 binds to the native EphA3 globular
ephrin-binding domain (e.g., Smith, et al., J. Biol. Chem
279:9522-9531, 2004). It is deposited in the European Collection of
Animal Cell Cultures under accession no. 91061920 (see, e.g., EP
patent no. EP0590030).
[0032] An "antibody having an active isotype" as used herein refers
to an antibody that has a human Fc region that binds to an Fc
receptor present on immune effector cells. "Active isotypes"
include IgG1, IgG3, IgM, IgA, and IgE. The term encompasses
antibodies that have a human Fc region that comprises
modifications, such as mutations or changes to the sugar
composition and/or level of glycosylation, that modulate Fc
effector function.
[0033] An "Fc region" refers to the constant region of an antibody
excluding the first constant region immunoglobulin domain. Thus, Fc
refers to the last two constant region immunoglobulin domains of
IgA, IgD, and IgG, and the last three constant region
immunoglobulin domains of IgE and IgM, and the flexible hinge
N-terminal to these domains. For IgA and IgM Fc may include the J
chain. For IgG, Fc comprises immunoglobulin domains C.gamma.2 and
C.gamma.3 and the hinge between C.gamma.1 and C.gamma.. It is
understood in the art that the boundaries of the Fc region may
vary, however, the human IgG heavy chain Fc region is usually
defined to comprise residues C226 or P230 to its carboxyl-terminus,
using the numbering is according to the EU index as in Kabat et al.
(1991, NIH Publication 91-3242, National Technical Information
Service, Springfield, Va.). The term "Fc region" may refer to this
region in isolation or this region in the context of an antibody or
antibody fragment. "Fc region" includes naturally occurring allelic
variants of the Fc region as well as modifications that modulate
effector function. Fc regions also include variants that don't
result in alterations to biological function. For example, one or
more amino acids can be deleted from the N-terminus or C-terminus
of the Fc region of an immunoglobulin without substantial loss of
biological function. Such variants can be selected according to
general rules known in the art so as to have minimal effect on
activity (see, e.g., Bowie, et al., Science 247:306-1310,
1990).
[0034] As used herein, an "antibody" refers to a protein
functionally defined as a binding protein and structurally defined
as comprising an amino acid sequence that is recognized by one of
skill as being derived from the framework region of an
immunoglobulin encoding gene of an animal producing antibodies. An
antibody can consist of one or more polypeptides substantially
encoded by immunoglobulin genes or fragments of immunoglobulin
genes. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0035] A typical immunoglobulin (antibody) structural unit is known
to comprise a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0036] The term "antibody" as used herein includes antibody
fragments that retain binding specificity. For example, there are a
number of well characterized antibody fragments. Thus, for example,
pepsin digests an antibody C-terminal to the disulfide linkages in
the hinge region to produce F(ab').sub.2, a dimer of Fab which
itself is a light chain joined to VH--CH1 by a disulfide bond. The
F(ab').sub.2 may be reduced under mild conditions to break the
disulfide linkage in the hinge region thereby converting the
(Fab').sub.2 dimer into an Fab' monomer. The Fab' monomer is
essentially an Fab with part of the hinge region (see, Fundamental
Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more
detailed description of other antibody fragments). While various
antibody fragments are defined in terms of the digestion of an
intact antibody, one of skill will appreciate that fragments can be
synthesized de novo either chemically or by utilizing recombinant
DNA methodology. Thus, the term antibody, as used herein also
includes antibody fragments either produced by the modification of
whole antibodies or synthesized using recombinant DNA
methodologies.
[0037] Antibodies include V.sub.H-V.sub.L dimers, including single
chain antibodies (antibodies that exist as a single polypeptide
chain), such as single chain Fv antibodies (sFv or scFv) in which a
variable heavy and a variable light region are joined together
(directly or through a peptide linker) to form a continuous
polypeptide. The single chain Fv antibody is a covalently linked
V.sub.H-V.sub.L which may be expressed from a nucleic acid
including V.sub.H- and V.sub.L-encoding sequences either joined
directly or joined by a peptide-encoding linker (e.g., Huston, et
al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). While the
V.sub.H and V.sub.L are connected to each as a single polypeptide
chain, the V.sub.H and V.sub.L domains associate non-covalently.
Alternatively, the antibody can be another fragment. Other
fragments can also be generated, e.g., using recombinant
techniques, as soluble proteins or as fragments obtained from
display methods. Antibodies can also include diantibodies and
miniantibodies. Antibodies of the invention also include heavy
chain dimers, such as antibodies from camelids. For the purposes of
this inventor, antibodies are employed in a form that can activate
EphA3 present on the surface of hematological proliferative cells
or that can kill hematological proliferative cells by ADCC. Thus,
in some embodiments an antibody is dimeric. In other embodiments,
the antibody may be in a monomeric form that has an active isotype.
In some embodiments the antibody is in a multivalent form, e.g., a
trivalent or tetravalent form, that can cross-link EphA3.
[0038] As used herein, "V-region" refers to an antibody variable
region domain comprising the segments of Framework 1, CDR1,
Framework 2, CDR2, and Framework3, including CDR3 and Framework 4,
which segments are added to the V-segment as a consequence of
rearrangement of the heavy chain and light chain V-region genes
during B-cell differentiation.
[0039] As used herein, "complementarity-determining region (CDR)"
refers to the three hypervariable regions in each chain that
interrupt the four "framework" regions established by the light and
heavy chain variable regions. The CDRs are primarily responsible
for binding to an epitope of an antigen. The CDRs of each chain are
typically referred to as CDR1, CDR2, and CDR3, numbered
sequentially starting from the N-terminus, and are also typically
identified by the chain in which the particular CDR is located.
Thus, a V.sub.H CDR3 is located in the variable domain of the heavy
chain of the antibody in which it is found, whereas a V.sub.L CDR1
is the CDR1 from the variable domain of the light chain of the
antibody in which it is found.
[0040] The sequences of the framework regions of different light or
heavy chains are relatively conserved within a species. The
framework region of an antibody, that is the combined framework
regions of the constituent light and heavy chains, serves to
position and align the CDRs in three dimensional space.
[0041] The amino acid sequences of the CDRs and framework regions
can be determined using various well known definitions in the art,
e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT),
and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk,
1987, Canonical structures for the hypervariable regions of
immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al.,
1989, Conformations of immunoglobulin hypervariable regions. Nature
342, 877-883; Chothia C. et al., 1992, structural repertoire of the
human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al.,
J. Mol. Biol 1997, 273(4)). Definitions of antigen combining sites
are also described in the following: Ruiz et al., IMGT, the
international ImMunoGeneTics database. Nucleic Acids Res., 28,
219-221 (2000); and Lefranc, M.-P. IMGT, the international
ImMunoGeneTics database. Nucleic Acids Res. January 1; 29(1):207-9
(2001); MacCallum et al, Antibody-antigen interactions: Contact
analysis and binding site topography, J. Mol. Biol., 262 (5),
732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86,
9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153,
(1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et
al, In Sternberg M. J. E. (ed.), Protein Structure Prediction.
Oxford University Press, Oxford, 141-172 1996).
[0042] "Epitope" or "antigenic determinant" refers to a site on an
antigen to which an antibody binds. Epitopes can be formed both
from contiguous amino acids or noncontiguous amino acids juxtaposed
by tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
e.g., Epitope Mapping Protocols in Methods in Molecular Biology,
Vol. 66, Glenn E. Morris, Ed (1996).
[0043] As used herein, "chimeric antibody" refers to an
immunoglobulin molecule in which (a) the constant region, or a
portion thereof, is altered, replaced or exchanged so that the
antigen binding site (variable region) is linked to a constant
region of a different or altered class, effector function and/or
species, or an entirely different molecule which confers new
properties to the chimeric antibody, e.g., an enzyme, toxin,
hormone, growth factor, drug, etc.; or (b) the variable region, or
a portion thereof, is altered, replaced or exchanged with a
variable region, or portion thereof, having a different or altered
antigen specificity; or with corresponding sequences from another
species or from another antibody class or subclass.
[0044] As used herein, "humanized antibody" refers to an
immunoglobulin molecule in CDRs from a donor antibody are grafted
onto human framework sequences. Humanized antibodies may also
comprise residues of donor origin in the framework sequences. The
humanized antibody can also comprise at least a portion of a human
immunoglobulin constant region. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. Humanization can be
performed using methods known in the art (e.g., Jones et al.,
Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327,
1988; Verhoeyen et al., Science 239:1534-1536, 1988); Presta, Curr.
Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No. 4,816,567),
including techniques such as "superhumanizing" antibodies (Tan et
al., J. Immunol. 169: 1119, 2002) and "resurfacing" (e.g., Staelens
et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc.
Natl. Acad. Sci USA 91: 969, 1994).
[0045] A "HUMANEERED.TM." antibody in the context of this invention
refers to an engineered human antibody having a binding specificity
of a reference antibody. An engineered human antibody for use in
this invention has an immunoglobulin molecule that contains minimal
sequence derived from a donor immunoglobulin. In some embodiments,
the engineered human antibody may retain only the minimal essential
binding specificity determinant from the CDR3 regions of a
reference antibody. Typically, an engineered human antibody is
engineered by joining a DNA sequence encoding a binding specificity
determinant (BSD) from the CDR3 region of the heavy chain of the
reference antibody to human V.sub.H segment sequence and a light
chain CDR3 BSD from the reference antibody to a human V.sub.L
segment sequence. A "BSD" refers to a CDR3-FR4 region, or a portion
of this region that mediates binding specificity. A binding
specificity determinant therefore can be a CDR3-FR4, a CDR3, a
minimal essential binding specificity determinant of a CDR3 (which
refers to any region smaller than the CDR3 that confers binding
specificity when present in the V region of an antibody), the D
segment (with regard to a heavy chain region), or other regions of
CDR3-FR4 that confer the binding specificity of a reference
antibody. Methods for engineering human antibodies are provided in
U.S. Pat. No. 7,981,843 and US patent application publication no.
20060134098.
[0046] The term "human antibody" as used herein refers to an
antibody that is substantially human, i.e., has FR regions, and
often CDR regions, from a human immune system. Accordingly, the
term includes humanized and HUMANEERED.TM. antibodies as well as
antibodies isolated from mice reconstituted with a human immune
system and antibodies isolated from display libraries.
[0047] A "hypofucosylated" antibody preparation refers to an
antibody preparation in which the average content of
.alpha.1,6-fucose is less than 50% of that found in naturally
occurring IgG antibody preparations. As understood in the art,
"hypofucosylated" is used in reference to a population of
antibodies.
[0048] An "afucosylated" antibody lacks .alpha.1,6-fucose attached
to the CH2 domain of the IgG heavy chain.
[0049] A "therapeutic" antibody as used herein refers to a human
and/or chimeric antibody that is administered to a patient that has
a hematological proliferative disorder.
[0050] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not normally found in the same
relationship to each other in nature. For instance, the nucleic
acid is typically recombinantly produced, having two or more
sequences, e.g., from unrelated genes arranged to make a new
functional nucleic acid. Similarly, a heterologous protein will
often refer to two or more subsequences that are not found in the
same relationship to each other in nature.
[0051] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, e.g., recombinant cells
express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all. By the term "recombinant nucleic acid" herein is meant nucleic
acid, originally formed in vitro, in general, by the manipulation
of nucleic acid, e.g., using polymerases and endonucleases, in a
form not normally found in nature. In this manner, operably linkage
of different sequences is achieved. Thus an isolated nucleic acid,
in a linear form, or an expression vector formed in vitro by
ligating DNA molecules that are not normally joined, are both
considered recombinant for the purposes of this invention. It is
understood that once a recombinant nucleic acid is made and
reintroduced into a host cell or organism, it will replicate
non-recombinantly, i.e., using the in vivo cellular machinery of
the host cell rather than in vitro manipulations; however, such
nucleic acids, once produced recombinantly, although subsequently
replicated non-recombinantly, are still considered recombinant for
the purposes of the invention. Similarly, a "recombinant protein"
is a protein made using recombinant techniques, i.e., through the
expression of a recombinant nucleic acid as depicted above.
[0052] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction where the antibody binds to the protein of interest. In
the context of this invention, the antibody typically binds to
EphA3 with an affinity that is at least 100-fold better than its
affinity for other antigens.
[0053] The term "equilibrium dissociation constant (K.sub.D) refers
to the dissociation rate constant (k.sub.d, time.sup.-1) divided by
the association rate constant (k.sub.a, time.sup.-1, M.sup.-1).
Equilibrium dissociation constants can be measured using any known
method in the art. The antibodies of the present invention are high
affinity antibodies. Such antibodies have an affinity better than
500 nM, and often better than 50 nM or 10 nM. Thus, in some
embodiments, the antibodies of the invention have an affinity in
the range of 500 nM to 100 pM, or in the range of 25 nM to 100 pM,
or in the range of 25 nM to 50 pM, or in the range of 25 nM to 1
pM.
[0054] As used herein, "hematological proliferative disorder
therapeutic agent" refers to an agent that when administered to a
patient suffering from a hematological proliferative disorder,
e.g., AML, in a therapeutically effective dose, will cure, or at
least partially arrest the symptoms of the disease and
complications associated with the disease. In some embodiments, the
therapeutic agent may be a chemotherapeutic agent.
[0055] The terms "identical" or percent "identity," in the context
of two or more polypeptide (or nucleic acid) sequences, refer to
two or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues (or nucleotides) that
are the same (i.e., about 60% identity, preferably 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region, when compared and aligned for
maximum correspondence over a comparison window or designated
region) as measured using a BLAST or BLAST 2.0 sequence comparison
algorithms with default parameters described below, or by manual
alignment and visual inspection (see, e.g., NCBI web site). Such
sequences are also referred to herein as being "substantially
identical." "Substantially identical" sequences also includes
sequences that have deletions and/or additions, as well as those
that have substitutions, as well as naturally occurring, e.g.,
polymorphic or allelic variants, and man-made variants. As
described below, the preferred algorithms can account for gaps and
the like. Preferably, protein sequence identity exists over a
region that is at least about 25 amino acids in length, or more
preferably over a region that is 50-100 amino acids=in length, or
over the length of a protein.
[0056] A "comparison window", as used herein, includes reference to
a segment of one of the number of contiguous positions selected
from the group consisting typically of from 20 to 600, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned. Methods of alignment of sequences for comparison
are well-known in the art. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm
of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
manual alignment and visual inspection (see, e.g., Current
Protocols in Molecular Biology (Ausubel et al., eds. 1995
supplement)).
[0057] Preferred examples of algorithms that are suitable for
determining percent sequence identity and sequence similarity
include the BLAST and BLAST 2.0 algorithms, which are described in
Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul
et al., J. Mol. Biol. 215:403-410 (1990). BLAST and BLAST 2.0 are
used, with the parameters described herein, to determine percent
sequence identity for the nucleic acids and proteins of the
invention. The BLASTN program (for nucleotide sequences) uses as
defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,
N=-4 and a comparison of both strands. For amino acid sequences,
the BLASTP program uses as defaults a wordlength of 3, and
expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands.
[0058] An indication that two polypeptides are substantially
identical is that the first polypeptide is immunologically cross
reactive with the antibodies raised against the second polypeptide.
Thus, a polypeptide is typically substantially identical to a
second polypeptide, e.g., where the two peptides differ only by
conservative substitutions.
[0059] The terms "isolated," "purified," or "biologically pure"
refer to material that is substantially or essentially free from
components that normally accompany it as found in its native state.
Purity and homogeneity are typically determined using analytical
chemistry techniques such as polyacrylamide gel electrophoresis or
high performance liquid chromatography. A protein that is the
predominant species present in a preparation is substantially
purified. The term "purified" in some embodiments denotes that a
protein gives rise to essentially one band in an electrophoretic
gel. Preferably, it means that the protein is at least 85% pure,
more preferably at least 95% pure, and most preferably at least 99%
pure.
[0060] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers, those containing modified
residues, and non-naturally occurring amino acid polymer.
[0061] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, e.g., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs may have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions
similarly to a naturally occurring amino acid.
[0062] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0063] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical or associated, e.g.,
naturally contiguous, sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode most proteins. For instance, the codons GCA, GCC, GCG
and GCU all encode the amino acid alanine Thus, at every position
where an alanine is specified by a codon, the codon can be altered
to another of the corresponding codons described without altering
the encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
One of skill will recognize that in certain contexts each codon in
a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, silent variations of a nucleic acid that
encode a polypeptide is implicit in a described sequence with
respect to the expression product, but not with respect to actual
probe sequences.
[0064] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention. Typically conservative substitutions for
one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)
Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
[0065] The term "a" or "an" is generally intended to mean "one or
more" unless otherwise indicated.
Introduction
[0066] The invention is based, in part, on the discovery that bone
marrow vasculature that arises from aberrant angiogenesis in
patients that have a hematological proliferative disorder, e.g.,
AML, CML, CMML, JMML, MDS, PV, ET, IM, CLL, MM, diffuse large
B-cell lymphoma, NHL, MCL, chronic neutrophilic leukemia, chronic
eosinophilic leukemia, or mast cell disease, selectively express
EphA3. EphA3 expression in the aberrant vasculature is often in the
endothelial layer of the neovasculature, but may also be observed
in the extraendothelial layer of larger vessels, e.g., in the
tunica media and/or tunica externa. Thus, patients that have a
hematological proliferative disorder in which the hematological
proliferative disorder cells do not express EphA3, or express EphA3
at a low level, can be treated by administering an anti-EphA3
activating antibody and/or an anti-EphA3 antibody that induces
ADCC. Accordingly, in one aspect, the invention provides methods of
treating a CML, PV, ET, IM, AML, MDS, CMML, or JMML patient where
the patient does not express EphA3 on hematological proliferative
disorder cells or expresses EphA3 at a level of 5% or less,
comprising administering an activating anti-EphA3 antibody to the
patient. In some embodiments, the patient has AML. In some
embodiments, the patient has CML, CMML, JMML, MDS, PV, ET, or IM.
In some embodiments, the patient has CLL, MM, diffuse large B-cell
lymphoma, NHL, MCL, chronic neutrophilic leukemia, chronic
eosinophilic leukemia, or mast cell disease. In some embodiments,
the methods of the invention comprise administering an anti-EphA3
antibody that induces ADCC to a CML, PV, ET, IM, AML, MDS, CMML, or
JMML patient where the patient does not express EphA3 on
hematological proliferative disorder cells. In some embodiments, an
anti-EphA3 antibody that is administered to a CML, PV, ET, IM, AML,
MDS, CMML, or JMML patient that does not express EphA3 on
hematological proliferative disorder cells (i) is an activating
anti-EphA3 antibody and (ii) induces ADCC.
[0067] In some embodiments, an anti-EphA3 antibody for use in this
invention does not block binding of EphA3 to ephrin, e.g.,
ephrin-A5. In some embodiments, the antibody dimerizes EphA3. In
some embodiments, the antibody cross-links EphA3. In some
embodiments, the antibody competes with mAb IIIA4 for binding to
EphA3, e.g., such an antibody may bind to the same epitope as mAb
IIIA4. In some embodiments, the antibody has an active isotype
where the heavy chain constant domain can bind to Fc receptor
present on immune effector cells, leading to ADCC.
Therapeutic Anti EphA3 Antibodies
[0068] The anti-EphA3 antibodies for use in the methods of the
invention can be raised against EphA3 proteins, or fragments, or
produced recombinantly. Any number of techniques can be used to
determine antibody binding specificity. See, e.g., Harlow &
Lane, Antibodies, A Laboratory Manual (1988) for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity of an antibody
[0069] In some embodiments, the anti-EphA3 antibody is a polyclonal
antibody. Methods of preparing polyclonal antibodies are known to
the skilled artisan (e.g., Harlow & Lane, Antibodies, A
Laboratory manual (1988); Methods in Immunology). Polyclonal
antibodies can be raised in a mammal by one or more injections of
an immunizing agent and, if desired, an adjuvant. The immunizing
agent includes a EphA3 receptor protein, or fragment thereof.
[0070] In some embodiments, the anti-EphA3 antibody is a monoclonal
antibody. Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler & Milstein, Nature
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in
vitro.
[0071] Human monoclonal antibodies can be produced using various
techniques known in the art, including phage display libraries
(Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991); Marks et
al., J. Mol. Biol. 222:581 (1991)). The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, p. 77 (1985) and Boerner et al., J. Immunol.
147(1):86-95 (1991)). Similarly, human antibodies can be made by
introducing of human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, e.g., in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016, and in the following scientific publications: Marks et
al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev.
Immunol. 13:65-93 (1995).
[0072] In some embodiments the anti-EphA3 antibodies are chimeric
or humanized monoclonal antibodies. As noted supra, humanized forms
of antibodies are chimeric immunoglobulins in which a CDR of a
human antibody is replaced by a CDR of a non-human species such as
mouse, rat or rabbit having the desired specificity, affinity and
capacity.
[0073] An antibody that is employed in the invention can be in
numerous formats. In some embodiments, the antibody can include an
Fc region, e.g., a human Fc region. For example, such antibodies
include IgG antibodies that bind EphA3 and that have an active
isotype. In some embodiments, the antibody can be an active
fragment (e.g., it can dimerize EphA3) or can comprise a derivative
of an antibody such as an Fab, Fab', F(ab').sub.2, Fv, scFv, or a
single domain antibody ("dAb"). For example, in some embodiments,
the antibody may be a F(ab').sub.2. Other exemplary embodiments of
antibodies that can be employed in the invention include activating
nanobodies or activating camellid antibodies. Such antibodies may
additionally be recombinantly engineered by methods well known to
persons of skill in the art. As noted above, such antibodies can be
produced using known techniques. As appreciated by one of skill in
the art, in some embodiments when an antibody is in a format that
can be monovalent, e.g., an Fv or Fab format, the antibody may be
employed as a multivalent antibody, such as a trivalent or
tetravalent antibody. Methods of generating multivalent antibodies
are known (see, e.g., King et al., Cancer Res. 54:6176-6185,
1994).
[0074] In many embodiments, an antibody for use in the invention
has an Fc constant region that has an effector function, e.g.,
binds to an Fc receptor present on immune effector cells. Exemplary
"effector functions" include C1q binding; complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor), and the like. Such effector
functions generally require the Fc region to be combined with a
binding domain (e.g. an antibody variable domain) and can be
assessed using known assays (see, e.g., the references cited
hereinbelow.)
[0075] Anti-EphA3 antibodies that have an active isotype and are
bound to Fc-receptors on effector cells, such as macrophages,
monocytes, neutrophils and NK cells, can induce cell death by
ADCC.
[0076] The Fc region can be from a naturally occurring IgG1, or
other active isotypes, including IgG3, IgM, IgA, and IgE. "Active
isotypes" include antibodies where the Fc region comprises
modifications to increase binding to the Fc receptor or otherwise
improve the potency of the antibody. Such an Fc constant region may
comprise modifications, such as mutations, changes to the level of
glycosylation and the like, that increase binding to the Fc
receptor. There are many methods of modifying Fc regions that are
known in the art. For example, U.S. Patent Application Publication
No. 20060039904 describes variants of Fc receptors that have
enhanced effector function, including modified binding affinity to
one or more Fc ligands (e.g., Fc.gamma.R, C1q). Additionally, such
Fc variants have altered antibody-dependent cell-mediated
cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC)
activity. Other Fc variants include those disclosed by Ghetie et
al., Nat Biotech. 15:637-40, 1997; Duncan et al, Nature
332:563-564, 1988; Lund et al., J. Immunol 147:2657-2662, 1991;
Lund et al, Mol Immunol 29:53-59, 1992; Alegre et al,
Transplantation 57:1537-1543, 1994; Hutchins et al., Proc Natl.
Acad Sci USA 92:11980-11984, 1995; Jefferis et al, Immunol Lett.
44:111-117, 1995; Lund et al., FASEB J 9:115-119, 1995; Jefferis et
al, Immunol Lett 54:101-104, 1996; Lund et al, J Immunol
157:4963-4969, 1996; Armour et al., Eur J Immunol 29:2613-2624,
1999; Idusogie et al, J Immunol 164:4178-4184, 200; Reddy et al, J
Immunol 164:1925-1933, 2000; Xu et al., Cell Immunol 200:16-26,
2000; Idusogie et al, J Immunol 166:2571-2575, 2001; Shields et
al., J Biol Chem 276:6591-6604, 2001; Jefferis et al, Immunol Lett
82:57-65. 2002; Presta et al., Biochem Soc Trans 30:487-490, 2002;
Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005-4010, 2006; U.S.
Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375;
5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,194,551; 6,737,056;
6,821,505; 6,277,375; 7,335,742; and 7,317,091; and PCT
Publications WO 94/2935; WO 99/58572; WO 00/42072; WO 02/060919,
and WO 04/029207.
[0077] In some embodiments, the natural glycosylation of Fc regions
may be modified. For example, a modification may be aglycosylation,
for example, by removing one or more sites of glycosylation within
the antibody sequence. Such an approach is described in further
detail in U.S. Pat. Nos. 5,714,350 and 6,350,861. An Fc region can
also be made that has an altered type of glycosylation, such as a
hypofucosylated Fc variant having reduced amounts of fucosyl
residues or an Fc variant having increased bisecting GlcNAc
structures. Such carbohydrate modifications can be accomplished by,
for example, expressing the antibody in a host cell with altered
glycosylation machinery. Cells with altered glycosylation pathways,
including mammalian cells, yeast and plants, have been described in
the art and can be used as host cells in which to express
recombinant antibodies of the invention to thereby produce an
antibody with altered glycosylation. Techniques for modifying
glycosylation include those disclosed e.g., in Umana et al, Nat.
Biotechnol 17:176-180, 1999; Davies, et al., Biotechnol. Bioeng.
74:288-294, 2001; Shields et al, J Biol Chem 277:26733-26740, 2002;
Shinkawa et al., J Biol Chem 278:3466-3473, 2003; Niwa et al.
Clinc. Cancer Res. 1-:6248-6255, 2004; Presta et al., Biochem Soc
Trans 30:487-490, 2002; Kanda et al, Glycobiology 17:104-118, 2006;
U.S. Pat. Nos. 6,602,684; 6,946,292; and 7,214,775; U.S. Patent
Application Publication Nos. 20070248600; 20070178551; 20080060092;
20060253928; PCT publications WO 00/61739; WO 01/292246; WO
02/311140; and WO 02/30954; and Potelligent.TM. technology (Biowa,
Inc. Princeton, N.J.); and GlycoMAb.TM. glycosylation engineering
technology (GLYCART biotechnology AG, Zurich, Switzerland).
Non-glycosylated antibodies may also be produced using ProBioGen
technology (von Horsten et al., Glycobiology 20(12): 1607-1618,
2010). In a hypofucosylated antibody preparation, typically at
least 50 to 70% of the antibody molecule, often at least 80% of the
molecules, or at least 90% of the molecules, lack fucose.
[0078] In some embodiments of the invention, the antibody is
additionally engineered to reduce immunogenicity, e.g., so that the
antibody is suitable for repeat administration. Methods for
generating antibodies with reduced immunogenicity include
humanization and humaneering procedures and modification techniques
such as de-immunization, in which an antibody is further
engineered, e.g., in one or more framework regions, to remove T
cell epitopes.
[0079] In some embodiments, the antibody is a HUMANEERED.TM.
antibody. A HUMANEERED.TM. antibody is an engineered human antibody
having a binding specificity of a reference antibody, obtained by
joining a DNA sequence encoding a binding specificity determinant
(BSD) from a CDR, typically a CDR3 of the heavy chain of the
reference antibody to human V.sub.H segment sequences, and a BSD
from a CDR, typically a CDR3, of the light chain of the reference
antibody to human V.sub.L segment sequences. Methods for generating
such antibodies are provided in U.S. Pat. No. 7,981,843 and U.S.
Patent Application Publication No. 20060134098.
[0080] An antibody can further be de-immunized to remove one or
more predicted T-cell epitopes from the V-region of an antibody.
Such procedures are described, for example, in WO 00/34317.
[0081] In some embodiments, the variable region is comprised of
human V-gene sequences.
[0082] For example, a variable region sequence can have at least
80% identity, or at least 85% or at least 90% identity, or higher,
to human germ-line V-gene sequences.
[0083] An antibody used in the invention can include a human
constant region. The constant region of the light chain may be a
human kappa or lambda constant region. The heavy chain constant
region is often a gamma chain constant region, for example, a
gamma-1 or gamma-3 constant region.
[0084] In some embodiments, e.g., where the antibody is a fragment,
the antibody can be conjugated to another molecule, e.g., to
provide an extended half-life in vivo such as a polyethylene glycol
(pegylation) or serum albumin. Examples of PEGylation of antibody
fragments are provided in Knight et al., Platelets 15:409, 2004
(for abciximab); Pedley et al., Br. J. Cancer 70:1126, 1994 (for an
anti-CEA antibody); and Chapman et al., Nature Biotech. 17:780,
1999.
Antibody Specificity
[0085] An antibody for use in the methods of the invention
activates EphA3 and/or kills EphA3.sup.+ cells by ADCC. An example
of an antibody suitable for use with the present invention is an
antibody that has the binding specificity of mAb IIIA4. The
monoclonal antibody mAb IIIA4 binds to the native EphA3 globular
ephrin-binding domain (Smith et al., J. Biol. Chem. 279:9522-9531,
2004; and Vearing et al., Cancer Res. 65:6745-6754, 2005). High
affinity mAb IIIA4 binding to the EphA3 surface has little effect
on the overall affinity of ephrin-A5 interactions with EphA3.
[0086] In some embodiments, a monoclonal antibody that competes
with mAb IIIA4 for binding to EphA3, or that binds the same epitope
as mAb IIIA4, is used. Any of a number of competitive binding
assays can be used to measure competition between two antibodies
for binding to the same antigen. For example, a sandwich ELISA
assay can be used for this purpose. In an exemplary assay, ELISA is
carried out by using a capture antibody to coat the surface of a
well. A subsaturating concentration of tagged-antigen is then added
to the capture surface. This protein will be bound to the antibody
through a specific antibody:antigen interaction. After washing, a
second antibody that is linked to a detectable moiety is added to
the ELISA. If this antibody binds to the same site on the antigen
as the capture antibody, or interferes with binding to that site,
it will be unable to bind to the target protein as that site will
no longer be available for binding. If however this second antibody
recognizes a different site on the antigen it will be able to bind.
Binding can be detected by quantifying the amount of detectable
label that is bound. The background is defined by using a single
antibody as both capture and detection antibody, whereas the
maximal signal can be established by capturing with an antigen
specific antibody and detecting with an antibody to the tag on the
antigen. By using the background and maximal signals as references,
antibodies can be assessed in a pair-wise manner to determine
specificity. The ability of a particular antibody to recognize the
same epitope as another antibody is typically determined by such
competition assays.
[0087] A first antibody is considered to competitively inhibit
binding of a second antibody, if binding of the second antibody to
the antigen is reduced by at least 30%, usually at least about 40%,
50%, 60% or 75%, and often by at least about 90%, in the presence
of the first antibody using any of the assays described above.
[0088] In some embodiments, the antibody binds to the same epitope
as mAb IIIA4. The epitope for mAb IIIA4 and human engineered
derivatives resides in the N-terminal globular ligand binding
domain of EphA3 (amino acids 29-202 in the partial human EphA3
sequence below):
TABLE-US-00001 (SEQ ID NO: 1) 1 MDCQLSILLL LSCSVLDSFG ELIPQPSNEV
NLLDSKTIQG ELGWISYPSH GWEEISGVDE 61 HYTPIRTYQV CNVMDHSQNN
WLRTNWVPRN SAQKIYVELK FTLRDCNSIP LVLGTCKETF 121 NLYYMESDDD
HGVKFREHQF TKIDTIAADE SFTQMDLGDR ILKLNTEIRE VGPVNKKGFY 181
LAFQDVGACV ALVSVRVYFK KC
[0089] The mAb IIIA4 antibody binds adjacent to but does not
interfere substantially with binding of EphrinA5 to the receptor.
The epitope for mAb IIIA4 has been further characterized by Smith
et al., J. Biol. Chem. 279: 9522, 2004 using site-directed
mutagenesis. In this analysis, mutation of Glycine at position 132
to Glutamic acid (G132E) abolishes binding to mAb IIIA4. Mutation
of Valine 133 to Glutamic acid (V133E) reduces binding of EphA3 to
mAb IIIA4 antibody approximately 100-fold. It has subsequently been
observed by the inventors that Arginine 136 is also part of the
epitope. This residue is changed to Leucine in the sequence of the
highly conserved EphA3 protein in the rat (R136L). Rat EphA3 does
not bind mAb IIIA4 or a human engineered derivative of mAb IIIA4.
Thus, G132, V133 and R136 (bolded and underlined in the sequence
above) are important amino acids within the mab IIIA4 epitope.
Binding Affinity
[0090] In some embodiments, the antibodies suitable for use with
the present invention have a high affinity binding for human EphA3.
For the purposes of this invention, high affinity binding between
an antibody and an antigen exists if the dissociation constant
(K.sub.D) of the antibody is <about 10 nM, for example, about 5
nM, or about 2 nM, or about 1 nM, or less. A variety of methods can
be used to determine the binding affinity of an antibody for its
target antigen such as surface plasmon resonance assays, saturation
assays, or immunoassays such as ELISA or RIA, as are well known to
persons of skill in the art. An exemplary method for determining
binding affinity is by surface plasmon resonance analysis on a
BIAcore.TM. 2000 instrument (Biacore AB, Freiburg, Germany) using
CM5 sensor chips, as described by Krinner et al., (2007) Mol.
Immunol. February; 44(5):916-25. (Epub 2006 May 11)).
[0091] The therapeutic anti-EphA3 antibody can bind to any
extracellular region of EphA3. In some embodiments, the anti-EphA3
antibody activates EphA3. Often, the antibody multimerizes, e.g.,
dimerizes, EphA3. In some embodiments, the antibody clusters EphA3.
In some embodiments, an anti-EphA3 antibody can also be employed
that has an active isotype, such as an IgG1, IgG3, IgM, IgA, or
IgE, and is cytotoxic to hematological proliferative disorder cells
via ADCC. Antibodies for use in the invention can also be
multivalent including forms of monomers that are cross-linked or
otherwise multimerized to form multivalent antibodies.
[0092] In some embodiments, an antibody employed in the invention
does not compete with an EphA3 ligand for binding to EphA3, whereas
in other embodiments an EphA3 antibody for use in the invention can
compete for binding of an EphA3 ligand such as an ephrin, e.g.,
ephrin-A5, to EphA3. Antibodies that compete with a ligand for
binding to EphA3, can be identified using techniques as described
above, where an ephrin ligand such as ephrin-A5, is used instead of
another antibody for a competition analysis.
[0093] In exemplary embodiments, the anti-EphA3 antibody comprises
the V.sub.L and V.sub.H regions of mAb IIIA4. In other embodiments,
the anti-EphA3 antibody comprises CDRs 1, 2 and 3 of mAb IIIA4. In
some embodiments, the anti-EphA3 antibody comprises CDR3 of mAb
IIIA4. Table 1 provides CDR sequences (defined according to Kabat
numbering) of antibodies that bind to the same epitope as mAb
IIIA4. Affinity for EphA3 antigen was determined by ELISA. An
antibody of the invention may thus also have heavy chain and/or
lights chain CDRs set forth in Table 1.
TABLE-US-00002 TABLE 1 AFFINITY antibody CDRH1 CDRH2 CDRH3 (nM)
IIIA4 SYWIN DIYPGSGNT SGYYEDFDS 2.5 (SEQ ID NYDEKFKR (SEQ ID NO: 2)
(SEQ ID NO: 4) NO: 3) FA3AM- TYWIS DIYPGSGN SGYYEEFDS 3.2 H12A (SEQ
ID TNYDEKFQG (SEQ ID NO: 5) (SEQ ID NO: 7) NO: 6) K3D TYWIS
DIYPGSGN SGYYEEFDS 25 (SEQ ID TNYDEKFEG (SEQ ID NO: 5) (SEQ ID NO:
7) NO: 8) AFFINITY antibody CDRL1 CDRL2 CDRL3 (nM) IIIA4
RASQEISGYLG AASTLDS VQYANYPYT 2.5 (SEQ ID (SEQ ID (SEQ ID NO: 9) NO
10) NO 11) FA3AM- RASQGIISYLA AASSLQS VQYANYPYT 3.2 H12A (SEQ ID
(SEQ ID (SEQ ID NO 12) NO 13) NO 11) K3D RASQGIISYLA AASSLQS
VQYMNYPYT 25 (SEQ ID (SEQ ID (SEQ ID NO 12) NO 13) NO 14)
[0094] Examples of therapeutic antibodies for use in the invention
are described, e.g., in WO2011/053465, which is incorporate by
reference.
[0095] Antibodies as described herein for use in the invention can
be identified using known assays for the characteristic of
interest. Thus, antibodies can be identified by screening for the
ability to activate EphA3 (e.g., using a phosphorylation assay
and/or screening for changes in cell morphology, such as rounding),
the ability to induce ADCC, and for binding specificity and
affinity. Such assays are well known in the art.
Diagnostic Antibodies
[0096] Therapeutic antibodies may also be used for diagnosis.
Accordingly, any of the antibodies described above may be employed
for identifying EphA3 expression on aberrant vasculature.
[0097] Anti-EphA3 antibodies that bind to a region other than the
extracellular domain may also be used for detecting EphA3
expression. For example, 5E11F2 (commercially available from
Invitrogen) binds to the C-terminal peptide amino acids 955-983 and
can be used diagnostically. For the purposes of detecting EphA3
expression, antibodies are employed in a format that can bind to
EphA3 present on the vasculature.
[0098] In typical embodiments the antibody is used for
immunohistochemical analysis of EphA3 expression on bone marrow
vasculature. Antibodies suitable for use in immunohistochemistry
(IHC) bind to EphA3 subjected to formaldehyde fixation, paraffin
embedding and xylene and ethanol extraction. Antibodies for use in
IHC are selective for EphA3 and can bind to any region of the EphA3
protein including the extracellular domain or the intracellular
domain. Polyclonal and or monoclonal antibodies are suitable for
use in this aspect of the invention. In some embodiments the
antibody is a monoclonal antibody such as a mouse monoclonal
anti-EphA3 antibody. Preferably, the antibody chosen for
immunohistochemistry binds to a different epitope from that
recognized by a therapeutic antibody. In some embodiments, the
antibody for detection of EphA3 expression binds to a different
epitope from that recognized by monoclonal antibody IIIA4 and does
not compete with mAb IIIA4 for binding to EphA3.
[0099] In some embodiments, the antibody for detecting Epha3
expression is SL-2, which is produced by a hybridoma deposited with
the ATCC having the accession number PTA-12227; or SL-7, which is
produced by a hybridoma deposited with the ATCC having the
accession number PTA-12228.
[0100] The invention also provides a monoclonal antibody, e.g.,
SL-2 or SL-7, that can be used to detect EphA3 expression on
aberrant vasculature; and kits that contain the antibody. A kit of
the invention includes an SL-2 or SL-7 antibody, or both, and may
include other reagents for analyzing expression, e.g., a secondary
antibody, buffers, and other reagents.
Non-Antibody EphA3 Binding Agents for Therapy
[0101] Other proteins that bind to EphA3 and dimerize or activate
EphA3 receptor may also be administered to a patient that has a
hematological proliferative disorder where the hematological
proliferative disorder cells do not express or minimally express
EphA3. Such proteins include a soluble Ephrin A5-Fc protein.
[0102] Other EphA3 binding agents include scaffolded proteins that
bind EphA3. Thus, the EphA3 binding agent can be an "antibody
mimetic" that targets and binds to the antigen in a manner similar
to antibodies. When an antibody mimetic is used, the form of the
mimetic is such that it multimerizes EphA3. For example, the
antibody mimetic may be used in a dimeric or multivalent
format.
[0103] Certain antibody mimetics use non-immunoglobulin protein
scaffolds as alternative protein frameworks for the variable
regions of antibodies. For example, Ku et al. (Proc. Natl. Acad.
Sci. U.S.A. 92:6552-6556, 1995) discloses an alternative to
antibodies based on cytochrome b562 in which two of the loops of
cytochrome b562 were randomized and selected for binding against
bovine serum albumin. The individual mutants were found to bind
selectively with BSA similarly with anti-BSA antibodies.
[0104] U.S. Pat. Nos. 6,818,418 and 7,115,396 disclose an antibody
mimetic featuring a fibronectin or fibronectin-like protein
scaffold and at least one variable loop. Known as Adnectins, these
fibronectin-based antibody mimics exhibit many of the same
characteristics of natural or engineered antibodies, including high
affinity and specificity for any targeted ligand. The structure of
these fibronectin-based antibody mimics is similar to the structure
of the variable region of the IgG heavy chain. Therefore, these
mimics display antigen binding properties similar in nature and
affinity to those of native antibodies. Further, these
fibronectin-based antibody mimics exhibit certain benefits over
antibodies and antibody fragments. For example, these antibody
mimics do not rely on disulfide bonds for native fold stability,
and are, therefore, stable under conditions which would normally
break down antibodies. In addition, since the structure of these
fibronectin-based antibody mimics is similar to that of the IgG
heavy chain, the process for loop randomization and shuffling may
be employed in vitro that is similar to the process of affinity
maturation of antibodies in vivo.
[0105] Beste et al. (Proc. Natl. Acad. Sci. U.S.A. 96:1898-1903,
1999) disclose an antibody mimic based on a lipocalin scaffold
(Anticalin.RTM.). Lipocalins are composed of a .beta.-barrel with
four hypervariable loops at the terminus of the protein. The loops
were subjected to random mutagenesis and selected for binding with,
for example, fluorescein. Three variants exhibited specific binding
with fluorescein, with one variant showing binding similar to that
of an anti-fluorescein antibody. Further analysis revealed that all
of the randomized positions are variable, indicating that
Anticalin.RTM. would be suitable to be used as an alternative to
antibodies. Thus, Anticalins.RTM. are small, single chain peptides,
typically between 160 and 180 residues, which provides several
advantages over antibodies, including decreased cost of production,
increased stability in storage and decreased immunological
reaction.
[0106] U.S. Pat. No. 5,770,380 discloses a synthetic antibody
mimetic using the rigid, non-peptide organic scaffold of
calixarene, attached with multiple variable peptide loops used as
binding sites. The peptide loops all project from the same side
geometrically from the calixarene, with respect to each other.
Because of this geometric confirmation, all of the loops are
available for binding, increasing the binding affinity to a ligand.
However, in comparison to other antibody mimics, the
calixarene-based antibody mimic does not consist exclusively of a
peptide, and therefore it is less vulnerable to attack by protease
enzymes. Neither does the scaffold consist purely of a peptide, DNA
or RNA, meaning this antibody mimic is relatively stable in extreme
environmental conditions and has a long life span. Further, since
the calixarene-based antibody mimic is relatively small, it is less
likely to produce an immunogenic response.
[0107] Murali et al. (Cell Mol Biol 49:209-216, 2003) describe a
methodology for reducing antibodies into smaller peptidomimetics,
they term "antibody like binding peptidomimetics" (ABiP) which may
also be useful as an alternative to antibodies.
[0108] WO 00/60070 discloses a polypeptide chain having CTL4A-like
.beta.-sandwich architecture. The peptide scaffold has from 6 to 9
.beta.-strands, wherein two or more of the polypeptide .beta.-loops
constitute binding domains for other molecules, such as antigen
binding fragments. The basic design of the scaffold is of human
origin, thus reducing the risk of inducing an immune response. The
.beta.-sandwich scaffold may have improved stability and
pharmacokinetic properties in vivo when compared to standard
antibodies as the molecule contains a second, non-immunoglobulin
disulfide bridge. As antigen binding domains can be located at
opposite ends of a single peptide chain, the .beta.-sandwich also
facilitates design of bispecific monomeric molecules.
[0109] In addition to non-immunoglobulin protein frameworks,
antibody properties have also been mimicked in compounds comprising
RNA molecules and unnatural oligomers (e.g., protease inhibitors,
benzodiazepines, purine derivatives and beta-turn mimics).
Accordingly, non-antibody EphA3 binding agents can also include
such compounds.
[0110] In some embodiments, the EphA3 binding agents employed in
the invention competed with mAb IIIA4 for binding to EphA3. Such
agents can be identified using known assays, such as the exemplary
competition assays described herein.
Identification of Patients Who are Candidate for Treatment with
Anti-EphA3
[0111] The invention also provides methods of determining whether a
patient having a hematological proliferative disorder is a
candidate for treatment with an anti-EphA3 antibody. The methods
comprise detecting the expression of EphA3 on aberrant bone marrow
vasculature in the patient.
[0112] EphA3 expression can be detected using methods well known in
the art. Often, an immunological assay employing
immunohistochemistry is used to detect EphA3 expression on aberrant
bone marrow vasculature. Alternatively EphA3 expression can be
detected by detecting mRNA encoding EphA3 in the aberrant bone
marrow vasculature. For example, a nucleic acid amplification
method that comprises RT-PCR may be employed.
[0113] In evaluating a patient that is a candidate for treatment, a
bone marrow sample comprising hematological proliferative disorder
cells is obtained from the patient for evaluating EphA3 expression.
A patient is considered to be a candidate for treatment with an
anti-EphA3 antibody if the aberrant vasculature expresses EphA3.
Accordingly, "an EphA3.sup.+ patient" as used here is a
hematological proliferative disorder patient that shows EphA3
expression aberrant bone marrow vasculature.
Treatment of Hematological Proliferative Disorders
[0114] In one aspect, the methods of the present invention comprise
administering an anti-EphA3 agent, typically an anti-EphA3
antibody, to a patient that has a hematological proliferative
disorder that has aberrant bone marrow vasculature that expresses
EphA 3, but has EphA3- hematological proliferative disorder cell
expression. In some embodiments, an anti-EphA3 agent, such as an
antibody, is administered to the patient. In some embodiments, the
patient has AML. In some embodiments, the patient has CML, CMML,
JMML, MDS, PV, ET, or IM. In some embodiments, the patient has CLL,
MM, diffuse large B-cell lymphoma, NHL, MCL, chronic neutrophilic
leukemia, chronic eosinophilic leukemia, or mast cell disease.
[0115] Various methods, including immunological methods such as
flow cytometry and PCR methods can be employed to identify whether
hematological proliferative disorder cells express EphA3. Leukemic
and other hematological proliferative disorder cells can be
identified by commonly used techniques such as immunophenotyping
using flow cytometry, or by in vitro cell culture techniques or in
vivo transplantation experiments.
[0116] Stem cells are multipotent progenitor cells that may be
further defined functionally as cells with self-renewing capacity
(see, e.g., Reya et al., Nature 414:105-111, 2001, and references
cited therein). This may be demonstrated, for example, in long-term
culture initiating cell (LTC-IC) assays in which cells are cultured
on irradiated bone-marrow stromal feeder cells. In this assay, the
presence of stem cells is revealed by the ability to serially
transfer colonies for extended periods (e.g., at least 5 weeks e.g.
Guan and Hogge (2000) Leukemia 14: 2135). Serial transfer assays
may also be carried out by culturing stem cell-derived colonies in
methyl cellulose in the presence of growth factors, such as a
combination of stem cell factor (SCF), interleukin-3 (IL3),
granulocyte macrophage colony stimulating factor (GM-CSF) and
erythropoietin (EPO).
[0117] In vivo transplantation to identify stem cells is carried
out by passaging by serial transfer in mice with defective immune
systems (SCID/NOD mice; van Rhenen et al., Clin. Cancer. Res. 11:
6520-6527, 2005).
[0118] In flow cytometry analysis, leukemic or chronic
myeloproliferative disorder (CMPD) stem cells are typically present
in the CD34-positive, CD38-negative cell compartment (although
approximately 10% of AML cases are CD34-negative). Leukemic or CMPD
stem cells can be identified in the CD38-negative cell compartment
as CD123-positive cells (Jordan et al., Leukemia 14: 1777-1784,
2000) although other markers may also be used to identify stem
cells including the presence of CD117, CD45RA or CD133.
[0119] Blast cells are unipotent cells that are able to participate
in granulopoiesis. Blast cells are larger cells than normal human
mononuclear and polymorphonuclear blood cells and can be identified
by microscopy from blood smears or by flow cytometry analysis on
the basis of high forward scatter (FSC) and side scatter (SSC)
compared with monocytes and granulocytes.
[0120] The anti-EphA3 composition for administering to the patient
can be formulated for use in a variety of drug delivery systems.
One or more physiologically acceptable excipients or carriers can
also be included in the compositions for proper formulation.
Suitable formulations for use in the present invention are found in
Remington: The Science and Practice of Pharmacy, 21st Edition,
Philadelphia, Pa. Lippincott Williams & Wilkins, 2005. For a
brief review of methods for drug delivery, see, Langer, Science
s249: 1527-1533 (1990).
[0121] The anti-EphA3 antibody for use in the methods of the
invention is provided in a solution suitable for injection into the
patient such as a sterile isotonic aqueous solution for injection.
The anti-EphA3 antibody is dissolved or suspended at a suitable
concentration in an acceptable carrier. In some embodiments the
carrier is aqueous, e.g., water, saline, phosphate buffered saline,
and the like. The compositions may contain auxiliary pharmaceutical
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents, and the like.
[0122] The pharmaceutical compositions of the invention are
administered to a patient that has a hematological proliferative
disorder in which aberrant bone marrow vasculature expresses EphA3,
but the hematological proliferative disorder cells do not, in an
amount sufficient to at least partially arrest the disease or
symptoms of the disease and its complications. An amount adequate
to accomplish this is defined as a "therapeutically effective
dose." A therapeutically effective dose is determined by monitoring
a patient's response to therapy. Typical benchmarks indicative of a
therapeutically effective dose are known in the art, depending on
the disease. For example, therapeutic efficacy may be indicated by
the decrease of the number of abnormal myeloid cells that are
characteristic of the particular myeloid proliferation disorder in
the blood or bone marrow.
[0123] The dose of the anti-EphA3 antibody is chosen in order to
provide effective therapy for the patient and is in the range of
about 0.1 mg/kg body weight to about 10 mg/kg body weight or in the
range about 1 mg to about 1 g per patient. The dose is often in the
range of about 0.5 mg/kg or about 1 mg/kg to about 10 mg/kg, or
approximately about 50 mg to about 1000 mg/patient. In some
embodiments, the antibody is administered in an amount less than
about 0.1 mg/kg body weight, e.g., in an amount of about 20
mg/patient or less. The dose may be repeated at an appropriate
frequency which may be in the range once per day to once every
three months, depending on the pharmacokinetics of the antibody
(e.g. half-life of the antibody in the circulation) and the
pharmacodynamic response (e.g. the duration of the therapeutic
effect of the antibody). In some embodiments where the antibody or
modified antibody fragment has an in vivo half-life of between
about 7 and about 25 days and antibody dosing is repeated between
once per week and once every 3 months. In other embodiments, the
antibody is administered approximately once per month.
[0124] Amounts that are administered that are effective will depend
upon the severity of the disease and the general state of the
patient's health, including other factors such as age, weight,
gender, administration route, etc. Single or multiple
administrations of the anti EphA3 antibody may be administered
depending on the dosage and frequency as required and tolerated by
the patient. In any event, the methods provide a sufficient
quantity of the anti EphA3 antibody to effectively treat the
hematological proliferative disorder.
[0125] An anti-EphA3 antibody or anti-EphA3 agonist binding agent,
e.g., that induces multimerization or activates EphA3, can be used
in combination with one or more additional therapeutic agents to
treat the hematological proliferative disorder. Therapeutic agents
that can be administered in conjunction with anti-EphA3 binding
agents include compounds such as MYLOTARG.RTM. (gemtuzumab
ozogamicin for injection); a tyrosine kinase inhibitor such as
imatinib mesylate (GLEEVEC.RTM.), nilotinib (TASIGNA.RTM.), and
dasatinib (SPRYCEL.RTM.); interferon-.alpha., and various
chemotherapeutic agents.
[0126] In some embodiments, an anti-EphA3 activating antibody can
be used in combination with one or more additional therapeutic
agents to treat a patient that has chronic myeloid leukemia where
leukemic stem cells from the patient express EphA3. Such
therapeutic agents include various chemotherapeutic agents and
imatinib mesylate (GLEEVEC.RTM.).
[0127] In some embodiments, an anti-EphA3 antibody, e.g., an
activating antibody, can be used in combination with one or more
additional agents to treat acute myeloid leukemia. Such agents
include cytosine arabinoside alone and in combination with
daunorubicin.
[0128] In some embodiments, an anti-EphA3 activating antibody can
be used in combination with one or more additional therapeutic
agents to treat a patient that has a BCR-ABL negative CMPD. Such
inhibitors include JAK2 inhibitors, which are known in the art and
undergoing clinical evaluation.
[0129] Patients can receive one or more of these additional
therapeutic agents as concomitant therapy. Alternatively, patients
may be treated sequentially with additional therapeutic agents.
[0130] In some embodiments, an anti-EphA3 activating antibody is
administered to a patient that has undergone a bone marrow
transplant, where the patient has hematological proliferative
disorder cells that are negative for EphA3 expression.
[0131] In some embodiments, an anti-EphA3 antibody, or other
activating Epha3 binding agent, is administered by injection or
infusion through any suitable route, typically intravenous routes.
In some embodiments, the anti-EphA3 antibody is diluted in a
physiological saline solution for injection prior to administration
to the patient. The antibody is administered, for example, by
intravenous infusion over a period of between 15 minutes and 2
hours.
[0132] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill in the art will
readily recognize a variety of non-critical parameters that could
be changed or modified to yield essentially similar results.
EXAMPLES
Example 1
Identification of an Anti-EphA3 Monoclonal Antibody for Use in
Immunohistochemistry
[0133] A panel of monoclonal antibodies that bind the extracellular
domain of EphA3 were generated by immunization of mice with human
EphA3-Fc fusion protein. Spleen cells from immunized mice were
fused with the SP2/0 hybridoma. Supernatants from seven hybridomas
(named SL-1-SL-7), each secreting a murine IgG1 isotype monoclonal
antibody, were assayed for EphA3 binding by ELISA (FIG. 1). ELISA
plates (Costar, cat #3590) were coated with 50 ng/well of EphA3-Fc
for 1 hr at 37.degree. C. Wells were washed once with
phosphate-buffered saline with 0.1% Tween 20 (PBST) and blocked
with 5% Skimmed Milk (Marvel) in PBST for 1 hr at 37.degree. C.
Following one wash with PBST, a two-fold dilution series of
antibody supernatant in PBS (50 .mu.l/well) was added across the
plates (10 dilutions). The plates were incubated for 1 hr at RT.
Plates were washed 3.times. with PBST and 100 .mu.l of a 1:2000
dilution of goat anti-mouse HRP (DAKO, cat # P0447) was added to
each well. Plates were incubated for 45 minutes at RT. Plates were
washed 3.times. in PBST, 1.times. in PBS and developed with 100
.mu.l/well of TMB reagent (Sigma, T0440-1L). Reactions were stopped
with 100 .mu.l of 2M H2SO4 and absorbance measured at 450 nm
(Spectramax Plus, Molecular Devices). Antibody concentrations in
the supernatants were quantified by biolayer interferometry using a
ForteBio Octet biosensor.
[0134] Each of the antibodies bound to recombinant EphA3. SL-2
antibody had the highest affinity (EC50=0.24 nM).
[0135] Four antibodies with the highest affinity, SL-2, SL-5, SL-6
and SL-7 were tested for cross reactivity with other Ephs. ELISA
plates were coated with 6 different Ephs (EphA1, EphA2, EphA3,
EphA5, EphB4 and EphB6) and probed with SL-2, SL-5, SL-6 and SL-7.
All four antibodies bound EphA3 specifically and showed no
detectable binding to other Eph proteins or Fc-fusion proteins.
[0136] Antibodies were purified from hybridoma supernatant using
Protein A affinity chromatography. Cell supernatant in the range
0.7-4.2 liters was loaded onto a 25 mL rProtein A FF column (GE
Healthcare) at 3.7 mL/min at 4.degree. C. with the pump P-50 (GE
Healthcare). After loading the cell supernatant, the column was
transferred to an AKTA Prime Purification System (GE Healthcare),
washed with 10 column volumes of Buffer A (PBS pH7.0) at a
flow-rate of 4 mL/min. Antibody was eluted from the column with
Buffer B (0.3M Arg, 0.15M NaCl pH 2.8) in 9 ml, fractions. Each
fraction was neutralized with 1 mL 2M Tris pH 8.0. Fractions
containing antibody were identified by SDS-PAGE (4-20% Tris-Glycine
Gel; Invitrogen; Cat#EC60252). Selected fractions were pooled and
buffer exchanged into 1.times.PBS pH 7.0 overnight at 4.degree. C.
using a Slide-A-Lyze dialysis cassette (ThermoFisher;
Cat#17-5080-01). Following the buffer exchange, the sample was
removed from the dialysis cassette and filtered through a 0.22 um
filter.
[0137] Binding of purified monoclonal antibodies to native EphA3
expressed on the cell surface was confirmed by flow cytometry using
EphA3-positive cell line LK63. For flow cytometry analysis, LK63
cells were harvested by centrifugation at 200 g for 5 minutes,
washed once in dPBS (Invitrogen, cat #14190) and blocked with 2%
bovine serum albumin (BSA)+10 .mu.g/ml rat IgG in dPBS for 25
minutes on ice. Cells were incubated with 25 nM purified anti-EphA3
antibodies for 30 minutes on ice, washed once in cold dPBS+0.5%
BSA, and incubated in a 1:100 dilution of FITC-conjugated
anti-mouse secondary antibody (Jackson Immunoresearch 115-096-062)
for 20 minutes on ice. Cells were washed once in cold dPBS+0.5% BSA
before being re-suspended in 0.5 ml cold dPBS for FACS analysis.
Propidium iodide (1 .mu.l/sample, Sigma cat # P4864) was used to
exclude non-viable cells from FITC channel readings and flow
cytometry was carried out using a FACSCaliber instrument (Beckton
Dickinson).
[0138] As shown in FIG. 2, purified SL-2, SL-5, and SL-7 antibodies
bound to LK63 cells at levels equivalent to the mouse IIIA4
anti-EphA3 positive control antibody.
Example 2
Expression of EphA3 on AML Tumor Vasculature
[0139] Standard bone-marrow biopsy formaldehyde-fixed paraffin
embedded (FFPE) sections were used for analysis of binding of
anti-EphA3 antibodies to cells in the bone marrow of AML patients
by immunohistochemistry. Sections were treated in two changes of
xylene (5 minutes each) to remove paraffin, hydrated through an
ethanol series and rinsed in distilled water. Antigen retrieval was
performed using Proteinase K (IHC world, Cat# IW-1101) for 10
minutes at 37.degree. C. in humidified chamber followed by rinsing
in Tris-buffered saline pH7.4 with 0.1% Tween 20 (TBST) for 4
minutes. Slides were blocked using universal blocking solution
(Vector Laboratories Inc., Cat # SP-2001) for 10 minutes followed
by Avidin solution for 15 minutes and biotin solution for 15
minutes. Fresh antibody blocking solution (0.07 g skimmed milk,
+210 ul goat serum, +14 ml antibody diluent) was added to the
slides for 20 minutes at room temperature and slides were rinsed
with TBST. Primary antibodies (SL-2, SL-5 or SL-7) or isotype
control antibody were diluted to 25 .mu.g/ml in antibody diluent
(IHC World, Cat # IW-1000) and added to slides (0.5 ml per slide)
for 1 hour at room temperature. Slides were then rinsed twice in
TBST. Biotin-SP-conjugated Goat Anti-Mouse IgG: (Jackson
ImmunoResearch Laboratories, Cat#115-065-166) secondary antibody
was diluted 1:500 in antibody diluent and added to the slides.
Slides were incubated for 30 minutes at room temperature and rinsed
twice in TBST. Two to three drops of Phosphatase-Labeled
Streptavidin (KPL, Cat#: 71-00-45) was also added and the slides
were incubated for 30 minutes at room temperature. After two
rinses, New Fuchsin Substrate System (Dako, Cat# K0698) was used
according to the manufacturer's instructions to develop the
slides.
[0140] SL-2 antibody detected EphA3-positive cells in
immunohistochemistry in bone marrow from human AML patients. In AML
bone marrow (Table 2), EphA3 was detected on the endothelium in
tumor neovasculature. In larger blood vessels such as arterioles,
EphA3 was expressed in vessel tissues adjacent to the endothelial
layer. In contrast, EphA3 was not detected in normal bone
marrow.
TABLE-US-00003 TABLE 2 Detection of EphA3 in biopsy tissue sections
from normal bone marrow or bone marrow from AML patients by
immunohistochemistry using SL-2 monoclonal antibody. Patient
disease sub-type is scored using the Fench American British (FAB)
classification scheme. n/a denotes not identified. FAB sub-type,
Sample Stage Hematopoietic cells Blood vessels I-6414 normal - -
I-1360 normal - - 7709 M6 + + 7703 M5a - - 7706 M1 - - I-8930 M1
(CD34-ve) + + I-8931 M5a + + 7704 n/a, relapse - + 7710 M1 - - 7707
n/a, relapse + + 7708 M3 - + 7712 M2 - +
[0141] All publications, patent applications, accession numbers,
and other references cited in this specification are herein
incorporated by reference as if each individual publication or
patent application were specifically and individually indicated to
be incorporated by reference.
Sequence CWU 1
1
141202PRTArtificial Sequencesynthetic N-terminal globular ligand
binding domain of human Eph receptor A3 (EphA3), human embryo
kinase (hek), eph-like tyrosine kinase 1 (etk1), tyro4, partial
sequence including mAb IIIA4 epitope, amino acids 29-202 1Met Asp
Cys Gln Leu Ser Ile Leu Leu Leu Leu Ser Cys Ser Val Leu1 5 10 15
Asp Ser Phe Gly Glu Leu Ile Pro Gln Pro Ser Asn Glu Val Asn Leu 20
25 30 Leu Asp Ser Lys Thr Ile Gln Gly Glu Leu Gly Trp Ile Ser Tyr
Pro 35 40 45 Ser His Gly Trp Glu Glu Ile Ser Gly Val Asp Glu His
Tyr Thr Pro 50 55 60 Ile Arg Thr Tyr Gln Val Cys Asn Val Met Asp
His Ser Gln Asn Asn65 70 75 80 Trp Leu Arg Thr Asn Trp Val Pro Arg
Asn Ser Ala Gln Lys Ile Tyr 85 90 95 Val Glu Leu Lys Phe Thr Leu
Arg Asp Cys Asn Ser Ile Pro Leu Val 100 105 110 Leu Gly Thr Cys Lys
Glu Thr Phe Asn Leu Tyr Tyr Met Glu Ser Asp 115 120 125 Asp Asp His
Gly Val Lys Phe Arg Glu His Gln Phe Thr Lys Ile Asp 130 135 140 Thr
Ile Ala Ala Asp Glu Ser Phe Thr Gln Met Asp Leu Gly Asp Arg145 150
155 160 Ile Leu Lys Leu Asn Thr Glu Ile Arg Glu Val Gly Pro Val Asn
Lys 165 170 175 Lys Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys
Val Ala Leu 180 185 190 Val Ser Val Arg Val Tyr Phe Lys Lys Cys 195
200 25PRTArtificial Sequencesynthetic anti-EphA3 monoclonal
antibody IIIA4 V-H heavy chain region CDRH1 2Ser Tyr Trp Ile Asn1 5
317PRTArtificial Sequencesynthetic anti-EphA3 monoclonal antibody
IIIA4 V-H heavy chain region CDRH2 3Asp Ile Tyr Pro Gly Ser Gly Asn
Thr Asn Tyr Asp Glu Lys Phe Lys1 5 10 15 Arg49PRTArtificial
Sequencesynthetic anti-EphA3 monoclonal antibody IIIA4 V-H heavy
chain region CDRH3 4Ser Gly Tyr Tyr Glu Asp Phe Asp Ser1 5
55PRTArtificial Sequencesynthetic anti-EphA3 monoclonal antibody
FA3AM-H12A and K3D V-H heavy chain region CDRH1 5Thr Tyr Trp Ile
Ser1 5 617PRTArtificial Sequencesynthetic anti-EphA3 monoclonal
antibody FA3AM-H12A V-H heavy chain region CDRH2 6Asp Ile Tyr Pro
Gly Ser Gly Asn Thr Asn Tyr Asp Glu Lys Phe Gln1 5 10 15
Gly79PRTArtificial Sequencesynthetic anti-EphA3 monoclonal antibody
FA3AM-H12A and K3D V-H heavy chain region CDRH3 7Ser Gly Tyr Tyr
Glu Glu Phe Asp Ser1 5 817PRTArtificial Sequencesynthetic
anti-EphA3 monoclonal antibody K3D V-H heavy chain region CDRH2
8Asp Ile Tyr Pro Gly Ser Gly Asn Thr Asn Tyr Asp Glu Lys Phe Glu1 5
10 15 Gly911PRTArtificial Sequencesynthetic anti-EphA3 monoclonal
antibody IIIA4 V-L light chain region CDRL1 9Arg Ala Ser Gln Glu
Ile Ser Gly Tyr Leu Gly1 5 10 107PRTArtificial Sequencesynthetic
anti-EphA3 monoclonal antibody IIIA4 V-L light chain region CDRL2
10Ala Ala Ser Thr Leu Asp Ser1 5 119PRTArtificial Sequencesynthetic
anti-EphA3 monoclonal antibody IIIA4 and FA3AM-H12A V-L light chain
region CDRL3 11Val Gln Tyr Ala Asn Tyr Pro Tyr Thr1 5
1211PRTArtificial Sequencesynthetic anti-EphA3 monoclonal antibody
FA3AM-H12A and K3D V-L light chain region CDRL1 12Arg Ala Ser Gln
Gly Ile Ile Ser Tyr Leu Ala1 5 10 137PRTArtificial
Sequencesynthetic anti-EphA3 monoclonal antibody FA3AM-H12A and K3D
V-L light chain region CDRL2 13Ala Ala Ser Ser Leu Gln Ser1 5
149PRTArtificial Sequencesynthetic anti-EphA3 monoclonal antibody
K3D V-L light chain region CDRL3 14Val Gln Tyr Met Asn Tyr Pro Tyr
Thr1 5
* * * * *