U.S. patent application number 15/265625 was filed with the patent office on 2017-08-10 for treatment of leukemias and chronic myeloproliferative diseases with antibodies to epha3.
The applicant listed for this patent is KaloBios Pharmaceuticals, Inc.. Invention is credited to Christopher R. Bebbington, Varghese Palath, Geoffrey T. Yarranton.
Application Number | 20170226227 15/265625 |
Document ID | / |
Family ID | 42370943 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170226227 |
Kind Code |
A1 |
Bebbington; Christopher R. ;
et al. |
August 10, 2017 |
Treatment of Leukemias and Chronic Myeloproliferative Diseases with
Antibodies to EphA3
Abstract
The invention provides methods and compositions comprising
anti-EphA3 antibodies for the treatment of myeloproliferative
disorders.
Inventors: |
Bebbington; Christopher R.;
(Brisbane, CA) ; Yarranton; Geoffrey T.;
(Brisbane, CA) ; Palath; Varghese; (Brisbane,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KaloBios Pharmaceuticals, Inc. |
Brisbane |
CA |
US |
|
|
Family ID: |
42370943 |
Appl. No.: |
15/265625 |
Filed: |
September 14, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14473821 |
Aug 29, 2014 |
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15265625 |
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12718768 |
Mar 5, 2010 |
8834870 |
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14473821 |
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61168130 |
Apr 9, 2009 |
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61158285 |
Mar 6, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/732 20130101;
A61K 45/06 20130101; C07K 2317/21 20130101; C07K 2317/52 20130101;
C07K 2317/75 20130101; G01N 2333/705 20130101; C07K 16/40 20130101;
A61P 35/02 20180101; A61K 2039/505 20130101; C07K 2317/41 20130101;
C07K 2317/565 20130101; G01N 33/57426 20130101; A61K 39/3955
20130101; C07K 16/2866 20130101; C07K 2317/24 20130101; C07K
2317/73 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; A61K 45/06 20060101 A61K045/06; G01N 33/574 20060101
G01N033/574; A61K 39/395 20060101 A61K039/395 |
Claims
1. A method of killing myeloproliferative disorder cells that
express EphA3 on the cell surface, the method comprising contacting
the cells with an anti-EphA3 antibody, wherein the anti-EphA3
antibody (i) activates EphA3 and (ii) induces antibody-dependent
cell-mediated cytotoxicity (ADCC), wherein the myeloproliferative
disorder cells are chronic myeloid proliferative disorder cells; or
myeloid leukemia cells.
2. The method of claim 1, wherein the myeloproliferative disorder
cells are PV, ET, MDS, or IM chronic myeloproliferative disorder
cells.
3. The method of claim 2, wherein the myeloproliferative disorder
cells are IM cells.
4. The method of claim 1, wherein the myeloproliferative disorder
cells are CMML or JMML myeloid leukemia cells.
5. The method of claim 1, wherein the meyloproliferative disorder
cells are AML myeloid leukemia cells.
6. 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.
7. The method of claim 1, wherein the anti-EphA3 antibody comprises
a human heavy chain gamma-1 or gamma-3 constant region.
8. The method of claim 1, wherein the anti-EphA3 antibody is
hypofucosylated.
9. The method of claim 1, wherein the anti-EphA3 antibody competes
with an antibody that comprises 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) for binding to
EphA3.
10. The method of claim 1, wherein the anti-EphA3 antibody is a
recombinant or chimeric antibody.
11. The method of claim 1, wherein the anti-EphA3 antibody is a
human antibody.
12. The method of claim 1, wherein the anti-EphA3 antibody is a
monoclonal antibody.
13. The method of claim 1, wherein the anti-EphA3 antibody
comprises 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).
14. A method of treating a patient that has a chronic
myeloproliferative disorder or a myeloid leukemia and has
myeloproliferative disorder cells that express EphA3 on the cell
surface, the method comprising administering a therapeutically
effective amount of an anti-EphA3 antibody to the patient, wherein
the anti-EphA3 antibody (i) activates EphA3 and (ii) induces
antibody-dependent cell-mediated cytotoxicity (ADCC).
15. The method of claim 14, wherein the myeloproliferative disorder
is PV, ET, MDS, or IM.
16. The method of claim 15, wherein the myeloproliferative disorder
is IM.
17. The method of claim 14, wherein the myeloproliferative disorder
is CMML or JMML.
18. The method of claim 14, wherein the myeloproliferative disorder
is AML.
19. The method of claim 14, further comprising administering at
least one additional therapeutic agent, wherein the at least one
additional therapeutic agent is a chemotherapeutic agent.
20. The method of claim 14, wherein the anti-EphA3 antibody
comprises a human heavy chain gamma-1 or gamma-3 constant
region.
21. The method of claim 14, wherein the anti-EphA3 antibody is
hypofucosylated.
22. The method of claim 14, wherein the anti-EphA3 antibody
competes with an antibody that comprises 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) for binding to
EphA3.
23. The method of claim 14, wherein the anti-EphA3 antibody is a
recombinant or chimeric antibody.
24. The method of claim 14, wherein the anti-EphA3 antibody is a
human antibody.
25. The method of claim 14, wherein the anti-EphA3 antibody is a
monoclonal antibody.
26. The method of claim 14, wherein the anti-EphA3 antibody
comprises 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).
27. A method of monitoring the efficacy of treatment of a patient
having a myeloproliferative disorder with EphA3.sup.+
myeloproliferative cells, wherein the myeloproliferative disorder
is AML or MDS, the method comprising: obtaining a sample comprising
myeloproliferative disorder stem cells and/or blast cells from the
patient following a therapeutic treatment for the
myeloproliferative disorder; and detecting expression of EphA3 on
the myeloproliferative disorder stem cells and/or blast cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/473,821, filed Aug. 29, 2014, which is a continuation of
U.S. application Ser. No. 12/718,768, filed Mar. 5, 2010, which
claims benefit of U.S. provisional application No. 61/158,285,
filed Mar. 6, 2009 and U.S. provisional application No. 61/168,130
filed Apr. 9, 2009. Each application is herein incorporated by
reference.
REFERENCE TO A "SEQUENCE LISTING" SUBMITTED AS AN ASCII TEXT
FILE
[0002] This application includes a Sequence Listing as a text file
named "SEQTXT 918065.txt" created Aug. 29, 2014, and containing
5,252 bytes. The material contained in this text file is
incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0003] Eph receptor tyrosine kinases (Ephs) belong to a large group
of receptor tyrosine kinases (RTKs), kinases that phosphorylate
proteins on tyrosine residues. Ephs and their membrane bound ephrin
ligands (ephrins) control cell positioning and tissue organization
(Poliakov, et al., Dev Cell 7:465-80, 2004). In contrast to other
receptor tyrosine kinases, Eph receptor activation does not only
require ligand binding and dimerization but also involves preformed
ligand oligomers. Thus, tyrosine phosphorylation of Eph receptors
requires presentation of ephrin ligands in their clustered or
membrane-attached forms (Davis et al., Science 266:816-819, 1994).
Functional and biochemical Eph responses occur at higher ligand
oligomerization states (Stein et al., Genes Dev 12:667-678,
1998).
[0004] Among other patterning functions, various Ephs and ephrins
have been shown to play a role in vascular development. The
de-regulated re-emergence of some ephrins and their receptors in
adults also has been observed to contribute to tumor invasion,
metastasis and neo-angiogenesis. For example, dominant-negative,
soluble EphA2 or A3 proteins exhibit effects on ephrin-induced
endothelial cell function in vitro, and tumor angiogenesis and
progression in vivo (Nakamoto,. et al., Microsc Res Tech 59:58-67,
2002; Brantley-Sieders, et al., Curr Pharm Des 10:3431-42, 2004;
Brantley, et al. Oncogene 21:7011-26, 2002; Cheng, et al. Neoplasia
5:445-56, 2003; and Dobrzanski, et al. Cancer Res 64:910-9, 2004).
Furthermore, Eph family members have been found to be
over-expressed on tumor cells from a wide variety of human solid
tumors (Brantley-Sieders, et al., Curr Pharm Des 10:3431-42, 2004;
Marme, Ann Hematol 81 Suppl 2:S66, 2002; and Booth, et al., Nat Med
8:1360-1, 2002).
[0005] EphA3 has also been reported to be activated and
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
(available online Nov. 14, 2008)). Cilloni et al. reported that
when primary CML cells or EphA3-transfected normal cells were
incubated with a specific monoclonal antibody that they referred to
as a blocking antibody, the antibody induced a significant
reduction of proliferation in primary cells and transfected cells,
reduced colony growth and induced changes to the adhesion
properties. The antibody did not induce any significant changes in
normal control cells or cells from CML patient in the chronic
stage.
[0006] There have been no reports that EphA3 is a therapeutic
target in other myeloproliferative disorders.
BRIEF SUMMARY OF THE INVENTION
[0007] The invention is based, in part, on the discovery that
neoplastic myeloid cells, including neoplastic myeloid stem cells,
in the bone marrow and peripheral blood samples obtained from a
patient that has 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), express EphA3 protein on the cell
surface and that such cells can be killed using an activating
anti-EphA3 antibody or an antibody that induces ADCC.
[0008] In one aspect, the invention provides a method of killing
AML cells, MDS cells, CMML cells, JMML cells, CML cells, PV cells,
ET cells, or IM cells, the method comprising contacting the cells
with an anti-EphA3 antibody. In one aspect, the invention provides
a method of treating a patient that has AML, CCML, JMML, MDS, CML,
PV, ET or IM, the method comprising administering an anti-EphA
antibody to the patient. In some embodiments, the anti-EphA3
antibody dimerizes EphA3. In some embodiments, the anti-EphA3
antibody activates EphA3 and kills the target cells by apoptosis.
In some embodiments, the anti-EphA3 antibody kills the target cells
by inducing antibody-dependent cell-mediated cytotoxicity (ADCC).
In some embodiments, the invention provides a method of killing
myeloproliferative disorder cells that express EphA3 on the
surface, the method comprising contacting the cells with an
anti-EphA3 antibody, wherein the anti-EphA3 antibody (i) activates
EphA3 and (ii) induces antibody-dependent cell-mediated
cytotoxicity (ADCC). In some embodiments, the invention provides a
method of treating a patient that has a myeloproliferative disorder
and has myeloproliferative disorder cells the express EphA3 on the
cell surface, the method comprising administering a therapeutically
effective amount of an anti-EphA3 antibody to the patient, wherein
the anti-EphA3 antibody (i) activates EphA3 and (ii) induces ADCC.
In some embodiments, the invention provides a method of killing
myeloproliferative disorder cells that express EphA3 on the
surface, the method comprising contacting the cells with an
anti-EphA3 antibody that activates EphA3 or induces ADCC, wherein
the myeloproliferative disorder cells are acute myeloid leukemia
(AML) cells or myelodysplastic syndrome (MDS) cells. In some
embodiments, the invention provides a method of treating a patient
that has a myeloproliferative disorder and has myeloproliferative
disorder cells the express EphA3 on the cell surface, the method
comprising administering a therapeutically effective amount of an
anti-EphA3 antibody to the patient, wherein the anti-EphA3 antibody
activates EphA3 or induces ADCC, wherein the myeloproliferative
disorder is AML or MDS.
[0009] In some embodiments, the anti-EphA3 antibody for use in the
methods of the invention is a recombinant or chimeric antibody. In
some embodiments, the anti-EphA3 antibody is a human antibody. The
anti-EphA3 antibody may be a polyclonal antibody or a monoclonal
antibody. In some embodiments, the 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 anti-EphA3 antibody competes for EphA3 binding
with mAb IIIA4. In some embodiments, the antibody binds to the same
epitope as mAB IIIA4. In typical embodiments, the antibody does not
block ephrin ligand binding, e.g., ephrinA5 binding, to EphA3. In
some embodiments the anti-EphA3 antibody comprises the V.sub.H and
V.sub.L regions of mAb IIIA4. In some, embodiments, the anti EphA3
antibody comprises the V.sub.H and V.sub.L region CDR1, CDR2 and
CDR3 of mAb IIIA4. In some embodiments, the antibody comprises the
V.sub.H region CDR3 and V.sub.L region CDR3 of mAb IIIA4. In some
embodiments, the antibody induces ADCC. Thus, in some embodiments
the 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 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.
[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 myeloproliferative
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 myeloproliferative disorder is CML,
additional therapeutic agents include imatinib mesylate, nilotinib,
dasatinib, or another chemotherapeutic agent. When the
myeloproliferative disorder is AML, the additional therapeutic
agents may be cytosine arabinoside alone or in combination with
daunorubicin.
[0012] Normal myeloid blast cells and stem cells do not express
EphA3 on the cell surface. Thus, in additional aspects, the
invention provides a method of identifying a patient having a
myeloproliferative disorder that is a candidate for treatment with
an anti-EphA3 antibody, wherein the method comprises detecting
EphA3 expression by myeloid blast cells and/or stem cells from the
patient.
[0013] In some embodiments, the invention provides a method of
determining that an AML patient or MDS patient is a candidate for
treatment with an anti-EphA3 antibody, the method comprising:
providing a sample from the patient, where the sample comprises
myeloproliferative disorder cells; and detecting expression of
EphA3 on the myeloproliferative disorder cells. In some
embodiments, the invention provides a method of determining that a
CMPD patient is a candidate for treatment with an anti-EphA3
antibody, the method comprising: providing a sample comprising
neoplastic stem cells from the patient; and detecting expression of
EphA3 by the neoplastic stem cells. In some embodiments, the
invention provides a method of monitoring the efficacy of treatment
of a patient having a myeloproliferative disorder with EphA3+
myeloproliferative cells, wherein the myeloproliferative disorder
is AML or MDS, the method comprising: obtaining a sample comprising
myeloproliferative disorder stem cells and/or blast cells from the
patient following a therapeutic treatment for the
myeloproliferative disorder; and detecting expression of EphA3 on
the myeloproliferative disorder stem cells and/or blast cells. In
some embodiments, the invention provides a method of monitoring the
efficacy of treatment of a CMPD patient that has neoplastic
myeloproliferative disorder stem cells that express EphA3, the
method comprising: obtaining a sample comprising the neoplastic
stem cells from the patient following a therapeutic treatment for
the CMPD; and detecting expression of EphA3 on the stem cells.
[0014] EphA3 expression can be detected using commonly known
techniques. Thus, in some embodiments detecting expression of EphA3
comprises detecting protein expression on the cell surface, e.g.,
using flow cytometry. In some embodiments, the step of detecting
expression of EphA3 comprises detecting EphA3 RNA levels, e.g.,
using an amplification reaction such as RT-PCR.
[0015] The invention further provides a pharmaceutical composition
comprising an anti-EphA3 antibody as described herein for use in
treating a patient that has a myeloproliferative disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A-1D provides data showing binding of an engineered
human anti-EphA3 antibody to leukemic stem cells. AML primary bone
marrow cells were stained with: engineered human anti-EphA3
antibody or IgG1 control and FITC-conjugated anti-human IgG;
PE-conjugated anti-CD34; PEcy5-conjugated anti-CD38; and
APC-conjugated anti-CD123 antibodies for flow cytometry analysis
(50, 000 events per sample). (FIG. 1A) isotype control gating for
CD34 analysis. (FIG. 1B) Sample stained with anti-EphA3 and
anti-CD34. (FIG. 1C) Sample stained for CD34 and CD38 (R2
represents CD34+ CD38- cells). (FIG. 1D) Identification of EphA3
and CD123 expression on CD34+ CD38- cells (R2 gate).
[0017] FIG. 2 provides data showing induction of CD16-mediated ADCC
activity by an engineered human anti-EphA3 antibody. Peripheral
blood mononuclear cells from a patient suffering from Essential
Thrombocythemia were used as the target. PBMC effector cells from a
normal individual were added at an effector: target ratio of 200:1
in the presence of anti-EphA3 antibody at the concentrations shown.
ADCC activity was analyzed in the presence of anti-CD16 antibody to
inhibit Fc-mediated effector function (circles) or in the absence
of CD16-blocking antibody (triangles) by measuring LDH release
after 16 hours.
[0018] FIG. 3 provides data showing enhanced ADCC activity shown by
an engineered human anti-EphA3 antibody (IgG1k) deficient in
.alpha.1,6 fucose. LK63 target cells were incubated with
fucosylated anti-EphA3 antibody (hatched bars) or antibody
deficient in .alpha.1,6 fucose produced from kifunensine-treated
cells (solid bars) at the concentrations shown. PBMC effector cells
were added at an effector: target ratio of 100:1 for 16 hours and
ADCC activity was determined by measuring LDH release.
[0019] FIG. 4 provides data showing apoptosis activity of a human
engineered antibody. Bone marrow cells (98% EphA3.sup.+ by flow
cytometry) from a CML patient were incubated in 96-well microtiter
wells (2.times.10.sup.5 cells per well) with human engineered
anti-EphA3 antibody or IgG1 control antibody at the concentrations
shown for 24 hours. Cells were then stained with Annexin V-FITC and
propidium iodide and analyzed by flow cytometry. Percent cells
undergoing apoptosis (Annexin V-positive) are shown.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0020] The term "myeloproliferative disorders" as used herein
refers to certain chronic myeloproliferative diseases classified as
chronic myeloid proliferative disorders (CMPDs); acute myeloid
leukemia (AML); myeloid dysplastic syndrome (MDS); chronic
myelomonocytic leukemia (CMML); and juvenile myelomonocytic
leukemia (JMML). In the context of this invention, a
"myeloproliferative disorder" thus refers to chronic myeloid
leukemia (CML); polycythemia vera (PV); essential thrombocythemia
(ET); idiopathic myelofibrosis (IM), which is also referred to as
primary myelofibrosis; AML; MDS; CMML; and JMML, The term "JMML"
encompasses all diagnoses referred to as Juvenile Chronic Myeloid
Leukemia (JCML), Chronic Myelomonocytic Leukemia of Infancy, and
Infantile Monosomy 7 Syndrome. Myeloproliferative 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
myeloproliferative neoplasms (MPNs). Myeloproliferative disorders
are often characterized by the presence of particular mutations.
For example, CIVIL 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.
[0021] The term "myeloid stem cells" or "stem cells" as used herein
are hematopoietic stem cells that are characterized as CD34.sup.-,
CD123.sup.+, and CD38.sup.-.
[0022] The term "myeloproliferative disorder cells" refers to
neoplastic myeloid cells that are characteristic of a
myeloproliferative disorder. The term encompasses myeloid 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.
The term encompasses both blast cells and stem cells.
[0023] The terms "cancer cell" or "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 loss of contact inhibition, morphological
changes, and aberrant growth. (see, Freshney, Culture of Animal
Cells a Manual of Basic Technique (3.sup.rd edition, 1994).
[0024] "Inhibiting growth of a cancer" in the context of the
invention refers to slowing growth and/or reducing the cancer cell
burden of a patient that has a myeloproliferative disorder.
"Inhibiting growth of a cancer" thus includes killing cancer
cells.
[0025] 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 exemplary human EphA3 amino acid sequence is available
under accession number (EAW68857).
[0026] For the purposes of the present invention, "activation" of
EphA3 causes phosphorylation of EphA3 and apoptosis. An antibody
that activates EphA3 or "an activating antibody" causes
phosphorylation of EphA3 and apoptosis and is therefore considered
to be an agonist in the context of this invention. EphA3 can be
activated by dimerization, which leads 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.
[0027] In the present invention, "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.
[0028] 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%.
[0029] 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).
[0030] 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.
[0031] 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).
[0032] 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.
[0033] 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.
[0034] 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)'2, a dimer of Fab which itself is
a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may
be reduced under mild conditions to break the disulfide linkage in
the hinge region thereby converting the (Fab')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.
[0035] 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 myeloproliferative cells or that
can kill myeloproliferative 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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).
[0040] "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).
[0041] 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.
[0042] 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).
[0043] 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
US patent application publication no 20050255552 and US patent
application publication no. 20060134098.
[0044] 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.
[0045] 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.
[0046] An "afucosylated" antibody lacks .alpha.1,6-fucose attached
to the CH2 domain of the IgG heavy chain.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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 50 or 25 nM to
100 pM, or in the range of 50 or 25 nM to 50 pM, or in the range of
50 nM or 25 nM to 1 pM.
[0051] As used herein, "cancer therapeutic agent" refers to an
agent that when administered to a patient suffering from cancer, in
a therapeutically effective dose, will cure, or at least partially
arrest the symptoms of the disease and complications associated
with the disease.
[0052] 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 then said to be "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.
[0053] 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)).
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] "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, often silent variations of a nucleic acid
which encodes a polypeptide is implicit in a described sequence
with respect to the expression product, but not with respect to
actual probe sequences.
[0061] 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)).
[0062] The term "a" or "an" is generally intended to mean "one or
more" unless otherwise indicated.
Introduction
[0063] The invention is based, in part, on the discovery that
EphA3-expressing neoplastic blast and/or neoplastic stem cells in
patients that have a myeloproliferative disorder can be killed by
contacting the EphA3-expressing myeloproliferative disorder cells
with an activating antibody and/or an 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,
comprising administering an activating anti-EphA3 antibody to the
patient. 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. In some embodiments,
an anti-EphA3 antibody that is administered to a CML, PV, ET, IM,
AML, MDS, CMML, or JMML patient (i) is an activating anti-EphA3
antibody and (ii) induces ADCC.
[0064] 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.
Anti EphA3 Antibodies
[0065] The anti-EphA3 antibodies 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
[0066] 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.
[0067] 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.
[0068] 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).
[0069] 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.
[0070] 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).
[0071] 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.)
[0072] 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.
[0073] 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 J9:115-119, 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,
[0074] In some embodiments, the glycosylation of Fc regions may be
modified. For example, a modification may be aglycosylation, for
example, by altering 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
machinery, including 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 Potillegent.TM. technology (Biowa,
Inc. Princeton, N.J.); and GlycoMAb.TM.. glycosylation engineering
technology (GLYCART biotechnology AG, Zurich, Switzerland). 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.
[0075] 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.
[0076] 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 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. Methods for generating such antibodies are provided in US
patent application publication no. 20050255552 and US patent
application publication no.
[0077] 20060134098.
[0078] 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.
[0079] In some embodiments, the variable region is comprised of
human V-gene sequences. 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.
[0080] 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.
[0081] 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
[0082] An antibody for use in 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.
[0083] 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.
[0084] 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.
[0085] In some embodiments, the antibody binds to the same epitope
as mAb IIIA4. The epitope for 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
[0086] The IIIA4 antibody binds adjacent to but does not interfere
substantially with binding of EphrinA5 to the receptor. The epitope
for antibody 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 IIIA4. Mutation of Valine 133 to
Glutamic acid (V133E) reduces binding of EphA3 to 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
IIIA4 or a human engineered derivative of IIIA4. Thus, G132, V133
and R136 (bolded and underlined in the sequence above) are
important amino acids within the IIIA4 epitope.
Binding Affinity
[0087] 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)).
[0088] The anti-EphA3 antibody can bind to any region of EphA3. In
some embodiments, the anti-EphA3 antibody activates EphA3. Often,
the antibody 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 myeloproliferative 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.
[0089] 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.
[0090] 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 CDRH1 (SEQ ID CDRH2 CDRH3 AFFINITY antibody
NO:) (SEQ ID NO:) (SEQ ID NO:) (nM) IIIA4 SYWIN (2)
DIYPGSGNTNYDEKFKR (3) SGYYEDFDS (4) 2.5 FA3A4- TYWIS (5)
DIYPGSGNTNYDEKFQG (6) SGYYEEFDS (7) 3.2 H12A K3D TYWIS (5)
DIYPGSGNTNYDEKFEG (8) SGYYEEFDS (7) 25 CDRL1 CDRL2 CDRL3 AFFINITY
antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (nM) IIIA4
RASQEISGYLG (9) AASTLDS (10) VQYANYPYT (11) 2.5 FA3A4- RASQGIISYLA
(12) AASSLQS (13) VQYANYPYT (11) 3.2 H12A K3D RASQGIISYLA (12)
AASSLQS (13) VQYMNYPYT (14) 25
[0091] 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 n apoptosis assay as
described in the examples), the ability to induce ADCC (e.g., using
an ADCC assay as described in the examples), and for binding
specificity and affinity using assays described above.
Non-Antibody EphA3 Binding Agents
[0092] Other proteins that bind to EphA3 and dimerize or activate
EphA3 receptor may also be administered to a patient that has a
leukemia or CMPD. Such proteins include a soluble Ephrin A5-Fc
protein.
[0093] 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 dimerizes EphA3. For example, the antibody
mimetic may be used in a dimeric or multivalent format.
[0094] 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.
[0095] U.S. Pat. Nos. 6,818,418 and 7,115,396 disclose an antibody
mimic 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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
disulphide 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.
[0100] 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.
[0101] 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
[0102] The invention also provides methods of determining whether a
patient having a myeloproliferative disorder is a candidate for
treatment with an anti-EphA3 antibody. The methods comprise
detecting the expression of EphA3 on myeloproliferative disorder
cells from the patient. In some embodiments, expression of EphA3 is
detected on blast cells. In some embodiments, EphA3 expression is
detected on stem cells. In some embodiments, EphA3 expression is
detected on both blast and stem cells.
[0103] In some embodiments, a blood sample, e.g., a serum or plasma
sample, from a myeloproliferative disorder patient can be evaluated
for elevated levels (e.g., in comparison to a normal patient that
does not have a myeloproliferative disorder) of soluble EphA3 to
determine if the patient is a candidate for treatment with an
anti-EphA3 antibody. In some embodiments, levels of soluble EphA3
can be determined in a patient to monitor the efficacy of treatment
with an anti-EphA3 antibody. Soluble EphA3 can be detected using
known immunoassays, e.g., an ELISA.
[0104] EphA3 expression can be detected using methods well known in
the art. Often, an immunological assay can be used to detect levels
of EphA3 protein. Immunological assays include ELISA,
fluorescent-activated cell sorting, and the like. Alternatively
EphA3 expression can be detected by detecting the level of mRNA
encoding EphA3. Often, a nucleic acid amplification method, e.g.,
an RT-PCR is employed to quantify the amount of RNA.
[0105] A sample comprising myeloproliferative disorder cells is
obtained from the patient for evaluating EphA3 expression. The
sample is often a peripheral blood sample, but other suitable
samples, e.g., a bone marrow sample, may also be analyzed.
[0106] A patient is considered to be a candidate for treatment with
an anti-EphA3 antibody if blast cells, stem cells, or both that are
present in the sample comprising myeloproliferative disorder cells
express EphA3. Accordingly, "an EphA3.sup.+ patient" as used here
is a patient that shows EphA3 expression on myeloproliferative
disorder cells relative to cells from normal controls, e.g.,
patients who do not have a hematopoietic disorder.
Treatment of Myeloproliferative Disorders
[0107] 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 AML, CML, PV, ET, IM, MDS, CMML, or
JMML and has neoplastic myeloproliferative disorder cells that
express EphA3 on the cell surface. In some embodiments, an
anti-EphA3 agent, such as an antibody, is administered to a patient
that neoplastic myeloid stem cells (characterized as CD34.sup.+,
CD123.sup.- and CD38.sup.-) that express EphA3 A patient, such as
an AML patient, that is treated with the anti-EphA3 agent, e.g., an
anti-EphA3, in accordance with the invention may therefore have
both hematopoietic stem cells and blast cells that express EphA3.
Other patients that are treated using methods and compositions
described herein may express EphA3 only on blast cells. Still other
patients may express EphA3 only on stem cells. In some embodiments,
a patient treated with the anti-EphA3 antibody is an AML or MDS
patient having myeloproliferative disorder blast cells that
expresses EphA3 on the surface.
[0108] Leukemic and myeloproliferative disorder stem 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.
[0109] 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).
[0110] 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).
[0111] 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.
[0112] 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.
[0113] The anti-EphA3 composition 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).
[0114] 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.
[0115] The pharmaceutical compositions of the invention are
administered to a patient that has a myeloproliferative disorder 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.
[0116] 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 25 mg/kg body weight or in the
range about 1 mg to about 2 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 0.1mg/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.
[0117] 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
myeloproliferative disorder.
[0118] An anti-EphA3 antibody or anti-EphA3 agonist binding agent,
e.g., that induces dimerization or activates EphA3, can be used in
combination with one or more additional therapeutic agents to treat
the myeloproliferative 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.
[0119] In some embodiments, an anti-EphA3 activating antibody an 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.).
[0120] 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.
[0121] 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.
[0122] Patients can receive one or more of these additional
therapeutic agents as concomitant therapy. Alternatively, patients
may be treated sequentially with additional therapeutic agents.
[0123] In some embodiments, an anti-EphA3 activating antibody is
administered to a patient that has undergone a bone marrow
transplant.
[0124] In some embodiments, an anti-EphA3 antibody, or other
activating EphA3 binding agent, is administered by injection or
infusion through any suitable route including but not limited to
intravenous, subcutaneous, intramuscular, intranasal, or
intraperitoneal 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.
[0125] 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 CMPDs and Leukemias that Express EphA3 on the
Surface
[0126] Flow cytometry was used to evaluate the expression of EphA3
on the surface of tumor cells from patients diagnosed with a
myeloproliferative disorder. Cells isolated from peripheral blood
(buffy coat cell preparations; peripheral blood mononuclear cells
(PBMC)) or bone marrow aspirates were suspended at 1.times.10.sup.6
cells/0.1 ml in flow cytometry buffer (PBS, 2 mM EDTA, 2% fetal
bovine serum, 0.05% sodium azide) with 1 .mu.g normal IgG to block
Fc-receptor binding (rat IgG; US Biological or anti-FcR
antibodies). Anti-EphA3 antibody or negative control human IgG1 was
added at 5 .mu.g/ml and incubated on ice for 20 min. Cells were
washed by dilution in flow cytometry buffer and centrifugation at
1000 rpm for 5 min. The cell pellet was resuspended in
FITC-conjugated goat F(ab)'.sub.2 anti-human IgG antibody (Caltag)
diluted in flow cytometry buffer (1:20) and incubated on ice for 20
min. Cells were washed once by centrifugation and resuspended in
flow cytometry buffer containing propidium iodide (Sigma) diluted
1:1000. Viable cells which exclude propidium iodide were analyzed
by flow cytometry to identify EphA3-expressing cells in comparison
with cells stained with negative control antibody.
[0127] Table 2 shows that EphA3 is detectable on the cell surface
in a proportion of acute and chronic myeloid leukemias and in
myeloproliferative disorders including idiopathic myelofibrosis and
essential thrombocythemia peripheral blood mononuclear cells.
TABLE-US-00003 TABLE 2 Summary of Flow Cytometry screen of bone
marrow and peripheral blood (PBMC) samples for surface EphA3
detected by flow cytometry Number of EphA3 Samples Tumor samples
positive positive for type tested samples* EphA3 (%) AML 41 26 63
CML 10 5 50 MDS 16 7 44 IM 1 1 (PBMC) ET 1 1 (PBMC) PV 2 1 (PBMC)
*Sample defined as positive if at least 5% of cells show higher
immunofluorescence than the fluorescence intensity in samples
stained with isotype control antibody
[0128] Leukemic stem cells in AML were also evaluated for surface
EphA3 expression. Bone marrow-derived cells from an AML patient
were stained with antibodies to CD34, CD38 and CD123 to identify
the leukemic stem cell population (characterized as CD34-positive,
CD123-positive and CD38-negative). PE-conjugated anti-CD34;
PEcy5-conjugated anti-CD38; and APC-conjugated anti-CD123
antibodies were used for flow cytometry analysis (50, 000 events
per sample). Binding of human engineered antibody specific for
EphA3 to CD34, CD38-gated cells is shown in FIG. 1. All of the
CD123-positive (CD34-positive and CD38-negative) leukemic stem
cells were positive for EphA3 expression.
[0129] EphA3 was not detectable on normal hematopoietic
CD34-positive stem cells (data not shown). Further, antibody to
EphA3 did not interfere with normal hematopoiesis in in vitro
colony formation assays.
Example 2
Evaluation of the Ability of an anti-EphA3 Antibody to Induce
Apoptosis of Myeloproliferative Disorder Cells
[0130] This example demonstrates that an anti-EphA3 antibody
induced apoptosis in myeloproliferative disorder cells.
[0131] An engineered human activating antibody that binds to EphA3
was evaluated for the ability to induce apoptosis in vitro in
primary cells isolated from patients or individuals suffering from
myeloproliferative disorders. Cells were seeded at
2.5.times.10.sup.5 cells/ well in 96-well "U"-bottom plates in 0.1
ml culture medium (RPMI 1640 with 10% fetal bovine serum).
Anti-EphA3 antibody or human IgG1 isotype control antibody was
added to final concentrations between 10 .mu.g/ml and 1 ng/ml and
the plates were incubated at 37.degree. C. and 5% carbon dioxide in
a tissue-culture incubator for 24 hours. As a positive control for
apoptosis induction, separate cell samples were incubated with
camptothecin (10 .mu.M; Calbiochem). At the end of the incubation,
cells were harvested and washed by centrifugation at 1000 rpm for 5
min followed by incubation in 0.1 ml of 1.times. Annexin V binding
buffer (BD Pharmingen , Cat # 556547, component no.51-66121E)
containing 5.mu.l FITC-conjugated Annexin V (BD Pharmingen,
component no. 51-65874X) and 5 .mu.l Propidium Iodide (component
no.51-66211E) for 15 minutes at room temperature in the dark. Four
hundred .mu.l 1X binding buffer was added to each tube and annexin
V-staining apoptotic cells were identified by flow cytometry. FIG.
4 provides data showing apoptosis activity of a human engineered
antibody.
[0132] The results shown in Table 3 demonstrate that the antibody
induced apoptosis in several samples at levels comparable to
camptothecin. In samples in which only a small proportion of the
cells express EphA3, the anti-EphA3 antibody induced apoptosis in a
similar small proportion of the cells, indicating that the
induction of apoptosis is specific for EphA3-positive cells.
TABLE-US-00004 TABLE 3 Induction of apoptosis by an engineered
human activating antibody that binds to EphA3. (PB, peripheral
blood; BM, bone marrow). Anti-EphA3- Camptothecin- mediated
mediated EphA3.sup.+ apoptosis apoptosis Sample Disease cells (%)
(% cell death) (% cell death) PB-1 ET 27 64 78 PB-2 PV 6 1.8 73.2
BM, 06 AML 65 85.5 59.8 BM, 07 AML 80 46.7 47.8
Example 3
Evaluation of the Ability of an anti-EphA3 Antibody to Induce ADCC
in Myeloproliferative Disorder Cells
[0133] Preparation of anti EphA3 Antibody deficient in a
1,6-fucose
[0134] CHO cells expressing a recombinant engineered human
anti-EphA3 antibody (IgG1k) were cultured in CHO-SFM II medium
(Invitrogen) containing 2 .mu.l kifunensine to generate antibody
with a modified glycosylation pattern defective in
.alpha.1,6-fucose as described (Zhou et al., Biotechnol. Bioeng.
99:652-665, 2008). Antibody purified by Protein A affinity
chromatography showed significant reduction in the level of
.alpha.1,6-fucose determined by binding of Lens culinaris Lectin
(Sigma) on protein blots with less than 10% antibody molecules
containing this sugar moiety. ADCC assay
[0135] Human PBMC effector cells were isolated from buffy coat
samples by Ficoll-hypaque density separation according to standard
techniques. Primary mononuclear cells from bone marrow or
peripheral blood from patients with leukemia or myeloproliferative
disorders were used as target cells in ADCC assays. Tumor target
cells were incubated for 16 hours with human effector cells at an
effector: target ratio of 100:1 or 200:1 for PBMC. Lactate
dehydrogenase (LDH) released from dead cells was determined by
CytoTox 96 assay (Promega). In this assay, incubation of target
cells with antibody in the absence of effector cells showed no
detectable cytotoxicity.
[0136] Results of a representative ADCC assay in which killing of
human essential thrombocythemia cells was induced by an anti-EphA3
antibody (IgG1k) in the presence of PBMC effector cells are shown
in FIG. 2. The antibody showed potent ADCC activity in this assay.
Inclusion of an antibody to CD16 abrogates the cytotoxic activity
of the anti-EphA3 antibody, indicating that ADCC is mediated by the
CD16 receptor (FcRIII). Anti-CD16 antibody (BD Pharmingen) was
added at a concentration of 5 .mu.g/ml.
[0137] The antibody preparation deficient in .alpha.1,6 fucose was
evaluated in comparison with fucosylated antibody in ADCC assays.
In the assay shown in FIG. 3, a pre-B cell leukemia derived cell
line LK63 was used as the target. The antibody deficient in
.alpha.1,6 fucose was significantly more potent than the
fucosylated antibody in this assay. ADCC activity was detected with
low levels of defucosylated antibody (0.1 ng/ml), a concentration
at which fucosylated antibody showed no detectable ADCC
activity.
[0138] The engineered human anti-EphA3 antibody also shows potent
ADCC activity against primary human tumor cells from bone marrow
samples from AML patients and shows ADCC activity against
EphA3-positive cells in the peripheral blood of polycythemia vera
patients as shown in Table 4.
TABLE-US-00005 TABLE 4 ADCC activity of an engineered human
anti-EphA3 antibody against cells from patients with leukemia or
myeloproliferative disease. (PB, peripheral blood; BM, bone
marrow). Anti-EphA3- EphA3.sup.+ mediated ADCC (% Sample Disease
cells (%) cytotoxicity at 16 h) PB-1 ET 27 70 PB-2 PV 6 8 BM, 06
AML 65 85.5 BM, 07 AML 80 46.7 BM, 157260 AML 65 70
[0139] Table 5 summarizes data on the cell phenotype of
EphA3-expressing cells from a larger panel of primary samples from
bone marrow aspirates from AML and myelodysplastic syndrome
patients and shows the proportion of cells in each sample killed by
anti-EphA3 antibody either by direct induction of apoptosis or by
effector-cell mediated ADCC activity. In these samples, in each
case in which CD123.sup.+ CD34.sup.+ CD38.sup.- leukemic stem cells
(LSC) could be identified, 100% of these LSC were also positive for
EphA3 expression. In the majority of samples, there is good
correlation between the percent of cells killed either by ADCC or
apoptosis mediated by an engineered human anti-EphA3 antibody and
the proportion of cells detected as positive for EphA3 by flow
cytometry, indicating specificity of the antibody for
EphA3-expressing cells.
TABLE-US-00006 TABLE 5 Summary of expression of EphA3 on malignant
blast and leukemic stem cells: A human engineered antibody kills
EphA3+ cells by two independent mechanisms. Flow Cytometry Analysis
on Bone Marrow Samples Leukemic Stem Cells Anti-EphA3 (CD34+
activity CD34+ Bone CD38- % Total marrow CD123+) % Total Cells
EphA3+ CD34+ EphA3+ LSC Cells Killed (% of (% of (% of (% of EphA3+
Killed by Patient total total CD34+ total (% of by Apop- Sample
cells) cells) cells) cells) LSC) ADCC tosis AML1 0 0 0 0 N/A 0 0
AML2 51 59 98 0 N/A 86 86 AML3 83 81 100 N/D N/A 47 50 AML4 88 40
100 25 100 95 79 AML5 55 90 64 0 N/A 72 79 AML6 21 20 100 12 99 20
22 AML7 16 77 12 10 100 20 15 AML8 24 0 0 0 N/A 22 20 AML9 31 16 36
0 N/A 40 45 AML10 41 43 92 0 N/A 50 48 AML11 55 56 99 0 N/A 65 75
AML12 14 27 22 0 N/A 20 15 MDS 1 15 17 22 1 100 25 20 MDS 2 9 28 35
3 100 20 19
[0140] 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 human Eph receptor A3 (EphA3,
Eph receptor tyrosine kinase A3, human embryo kinase, hek, eph-like
tyrosine kinase 1, etk1, tyro4) partial sequence 1Met Asp Cys Gln
Leu Ser Ile Leu Leu Leu Leu Ser Cys Ser Val Leu 1 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 Asn 65 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 Arg 145 150 155 160
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 antibody IIIA4 CDRH1
2Ser Tyr Trp Ile Asn 1 5 317PRTArtificial Sequencesynthetic
anti-EphA3 antibody IIIA4 CDRH2 3Asp Ile Tyr Pro Gly Ser Gly Asn
Thr Asn Tyr Asp Glu Lys Phe Lys 1 5 10 15 Arg 49PRTArtificial
Sequencesynthetic anti-EphA3 antibody IIIA4 CDRH3 4Ser Gly Tyr Tyr
Glu Asp Phe Asp Ser 1 5 55PRTArtificial Sequencesynthetic
anti-EphA3 antibody FA3AM-H12A and K3D CDRH1 5Thr Tyr Trp Ile Ser 1
5 617PRTArtificial Sequencesynthetic anti-EphA3 antibody FA3AM-H12A
CDRH2 6Asp Ile Tyr Pro Gly Ser Gly Asn Thr Asn Tyr Asp Glu Lys Phe
Gln 1 5 10 15 Gly 79PRTArtificial Sequencesynthetic anti-EphA3
antibody FA3AM-H12A and K3D CDRH3 7Ser Gly Tyr Tyr Glu Glu Phe Asp
Ser 1 5 817PRTArtificial Sequencesynthetic anti-EphA3 antibody K3D
CDRH1 8Asp Ile Tyr Pro Gly Ser Gly Asn Thr Asn Tyr Asp Glu Lys Phe
Glu 1 5 10 15 Gly 911PRTArtificial Sequencesynthetic anti-EphA3
antibody IIIA4 CDRL1 9Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Gly 1
5 10 107PRTArtificial Sequencesynthetic anti-EphA3 antibody IIIA4
CDRL2 10Ala Ala Ser Thr Leu Asp Ser 1 5 119PRTArtificial
Sequencesynthetic anti-EphA3 antibody IIIA4 CDRL3 11Val Gln Tyr Ala
Asn Tyr Pro Tyr Thr 1 5 1211PRTArtificial Sequencesynthetic
anti-EphA3 antibody FA3AM-H12A CDRL1 12Arg Ala Ser Gln Gly Ile Ile
Ser Tyr Leu Ala 1 5 10 137PRTArtificial Sequencesynthetic
anti-EphA3 antibody FA3AM-H12A CDRL2 13Ala Ala Ser Ser Leu Gln Ser
1 5 149PRTArtificial Sequencesynthetic anti-EphA3 antibody K3D
CDRL3 14Val Gln Tyr Met Asn Tyr Pro Tyr Thr 1 5
* * * * *