Ang2 Antibodies
The present invention relates to the field of medicine. More particularly, the
present invention relates to antibodies that bind human angiopoietin-2 (Ang2), and may
be useful for treating cancer, especially tumor metastasis, and in combination with human
vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors for solid tumors
driven by VEGFR2 and Ang2, including gastric, hepatocellular carcinoma, ovarian,
colorectal, and breast cancers.
A hallmark of cancer is persistent new blood vessel formation, called
angiogenesis. Angiopoietin-1 (Angl) and Ang2 are members of a key pathway that
regulate angiogenesis; Angl and Ang2 are secreted factors that bind to the endothelial
cell-specific receptor tyrosine kinase Tie2. Angl is constitutively secreted by pericytes
and stabilizes blood vessel integrity via the Tie2 receptor. Ang2 is released from
endothelial cells only in response to stimulus (e.g. wound healing, tumor growth) and
facilitates blood vessel sprouting and inhibits pericyte-endothelial cell interaction via Tie2
signaling. Studies have suggested that inhibition of Ang2 is more important than Angl
for inhibiting angiogenesis, but in certain other studies, inhibition of Ang2 and Angl has
shown benefits (Cascone et al., J Clin One (2012) 30(4):441).
An antibody against human Ang2, when dosed in combination with the VEGF
blocker aflibercept, has been shown to inhibit tumor growth and to decrease tumor
vascularity in mouse xenograft tumor models (REGN910 in Daly et al., Cancer Res
(2013) 73(1): 108 and H1H685P in WO201 1014469). In Brown et al. (Mol Cancer Ther
(2010) 9(1):145), mAb 3.19.3, a human Ang2 antibody, is reported to bind Angl with at
least 500x less affinity than Ang2, while in Examples 4 and 9 of WO2006068953, 3.19.3
is disclosed by the applicants to have strong cross-reactivity to Angl with a KD for Ang2
of approximately 5 pM and a KD for Angl of approximately 30 pM. In a SW620
xenograft study, 3.19.3 was dosed in combination with DC101, a monoclonal antibody
that binds murine VEGFR2.
There remains a need to provide alternative antibodies that inhibit the Ang/Tie2
angiogenesis pathway by binding human Ang2 with high affinity and neutralizing human
Ang2. In particular, there remains a need to provide antibodies that bind human Ang2
with high affinity and neutralize human Ang2, but do not bind with high affinity or
neutralize human Angl.
Accordingly, an embodiment of the present invention provides an antibody,
comprising a light chain (LC) and a heavy chain (HC), wherein the light chain comprises
a light chain variable region (LCVR) and the heavy chain comprises a heavy chain
variable region (HCVR), wherein the LCVR has the amino acid sequence given in SEQ
ID NO: 3, and the HCVR has the amino acid sequence given in SEQ ID NO: 1.
Certain antibodies of the present invention bind human Ang-2 with a high affinity
that is greater than human Ang2 antibodies known in the art. Furthermore, certain
antibodies of the present invention demonstrate a positive response in tumor xenograft
models, such as EL 1997 for triple negative breast cancer, SKOV3x.l for ovarian cancer,
and PC3 for prostate cancer, indicating potential as a cancer therapeutic. Additionally,
certain antibodies of the invention selectively bind human Ang-2 over human Ang-1, and
avoid potential off-target liabilities for human Angl.
In a further embodiment, the present invention provides an antibody, wherein the
LC has the amino acid sequence given in SEQ ID NO: 4, and the HC has the amino acid
sequence given in SEQ ID NO: 2.
In a further embodiment, the present invention provides an antibody, comprising
two light chains and two heavy chains, wherein each light chain has the amino acid
sequence given in SEQ ID NO: 4, and each heavy chain has the amino acid sequence
given in SEQ ID NO: 2.
In a further embodiment, the present invention provides an antibody, wherein one
of the heavy chains forms an inter-chain disulfide bond with one of the light chains, and
the other heavy chain forms an inter-chain disulfide bond with the other light chain, and
one of the heavy chains forms two inter-chain disulfide bonds with the other heavy chain.
In a further embodiment, the present invention provides an antibody, wherein the
antibody is glycosylated.
In an embodiment, the present invention provides an antibody that binds human
Ang2 (SEQ ID NO: 7), comprising a light chain (LC) and a heavy chain (HC), wherein
the light chain comprises light chain complementarity determining regions LCDR1,
LCDR2, and LCDR3 consisting of the amino acid sequences KASQDVYIAVA (SEQ ID
NO: 8), YWASTRDT (SEQ ID NO: 9), and HQYSSYPPT (SEQ ID NO: 10),
respectively, and wherein the heavy chain comprises heavy chain complementarity
determining regions HCDR1, HCDR2, and HCDR3 consisting of the amino acid
sequences GYSFTDYNMV (SEQ ID NO: 11), YIDPYNGGTGYNQKFEG (SEQ ID
NO: 12), and ARTRDRYDVWYFDV (SEQ ID NO: 13), respectively.
In an embodiment, the present invention provides an antibody that binds human
Ang2 (SEQ ID NO: 7), comprising a light chain (LC) and a heavy chain (HC), wherein
the light chain comprises a light chain variable region (LCVR) and the heavy chain
comprises a heavy chain variable region (HCVR), wherein the LCVR has the amino acid
sequence given in SEQ ID NO: 3, and the HCVR has the amino acid sequence given in
SEQ ID NO: 1.
In a further embodiment, the present invention provides an antibody that binds
human Ang2 (SEQ ID NO: 7), wherein the LC has the amino acid sequence given in SEQ
ID NO: 4, and the HC has the amino acid sequence given in SEQ ID NO: 2.
In a further embodiment, the present invention provides an antibody that binds
human Ang2 (SEQ ID NO: 7), comprising two light chains and two heavy chains,
wherein each light chain has the amino acid sequence given in SEQ ID NO: 4, and each
heavy chain has the amino acid sequence given in SEQ ID NO: 2.
In a further embodiment, the present invention provides an antibody that binds
human Ang2 (SEQ ID NO: 7), wherein one of the heavy chains forms an inter-chain
disulfide bond with one of the light chains, and the other heavy chain forms an inter-chain
disulfide bond with the other light chain, and one of the heavy chains forms two inter
chain disulfide bonds with the other heavy chain.
In a further embodiment, the present invention provides an antibody that binds
human Ang2 (SEQ ID NO: 7), wherein the antibody is glycosylated.
In a further embodiment, the present invention provides an antibody of the present
invention that has a dissociation equilibrium constant, KD, of less than about 150 pM for
human Ang2. The antibody of the present invention is further characterized by a o rate
to human Ang2 of about 1 x 106 M^sec 1. The antibody of the present invention is further
characterized by a o rate to human Ang2 of about 0.7 x 10 4 sec 1. The KD, o , and koff
values are established by binding kinetics at 37°C as described in "Binding kinetics,
affinity, and selectivity" in the Assays section.
The antibody of the present invention binds to human Ang2 with high affinity.
For the purposes of the present disclosure, the term "high affinity" refers to a KDof less
than about 150 pM for human Ang2. The KD values are established by binding kinetics at
37°C as described in "Binding kinetics, affinity, and selectivity" in the Assays section.
In an embodiment, the present invention provides a mammalian cell, comprising a
DNA molecule comprising a polynucleotide sequence encoding a polypeptide having an
amino acid sequence of SEQ ID NO: 4 and a polynucleotide sequence encoding a
polypeptide having an amino acid sequence of SEQ ID NO: 2, wherein the cell is capable
of expressing an antibody comprising a light chain having an amino acid sequence of
SEQ ID NO: 4 and a heavy chain having an amino acid sequence of SEQ ID NO: 2.
In an embodiment, the present invention provides a process for producing an
antibody, comprising a light chain having an amino acid sequence of SEQ ID NO: 4 and a
heavy chain having an amino acid sequence of SEQ ID NO: 2, comprising cultivating a
mammalian cell of the present invention under conditions such that the antibody is
expressed, and recovering the expressed antibody.
In an embodiment, the present invention provides an antibody produced by a
process of the present invention.
In an embodiment, the present invention provides a pharmaceutical composition,
comprising an antibody of the present invention, and an acceptable carrier, diluent, or
excipient.
In an embodiment, the present invention provides a method of treating cancer,
comprising administering to a patient in need thereof, an effective amount of an antibody
of the present invention. In a further embodiment, the present invention provides a
method of treating cancer, comprising administering to a patient in need thereof, an
effective amount of an antibody of the present invention, wherein the cancer is breast
cancer, ovarian cancer, gastric cancer, colorectal cancer, or hepatocellular carcinoma. In
a further embodiment, these methods comprise the administration of an effective amount
of the antibody of the present invention in simultaneous, separate, or sequential
combination with one or more anti-tumor agents selected from the group consisting of
cisplatin, carboplatin, liposomal doxorubicin, docetaxel, cyclophosphamide and
doxorubicin, navelbine, eribulin, paclitaxel protein-bound particles for injectable
suspension, ixabepilone, capecitabine, ramucirumab, FOLFOX (leucovorin, fluorouracil,
and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), and cetuximab.
In a further embodiment, these methods comprise the administration of an
effective amount of the compound of the present invention in simultaneous, separate, or
sequential combination with one or more immuno-oncology agents selected from the
group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, and
durvalumab.
In an embodiment, the present invention provides a method of treating breast
cancer, comprising administering to a patient in need thereof, an effective amount of an
antibody of the present invention. In a further embodiment, these methods comprise the
administration of an effective amount of the antibody of the present invention in
simultaneous, separate, or sequential combination with one or more anti-tumor agents
selected from the group consisting of ramucirumab, docetaxel, cyclophosphamide and
doxorubicin, navelbine, eribulin, paclitaxel protein-bound particles for injectable
suspension, ixabepilone, and capecitabine.
In an embodiment, the present invention provides a method of treating ovarian
cancer, comprising administering to a patient in need thereof, an effective amount of an
antibody of the present invention. In a further embodiment, these methods comprise the
administration of an effective amount of the antibody of the present invention in
simultaneous, separate, or sequential combination with one or more anti-tumor agents
selected from the group consisting of ramucirumab, cisplatin, carboplatin, and liposomal
doxorubicin.
In an embodiment, the present invention provides a method of treating gastric
cancer, comprising administering to a patient in need thereof, an effective amount of an
antibody of the present invention. In a further embodiment, these methods comprise the
administration of an effective amount of the antibody of the present invention in
simultaneous, separate, or sequential combination with ramucirumab.
In an embodiment, the present invention provides a method of treating
hepatocellular carcinoma, comprising administering to a patient in need thereof, an
effective amount of an antibody of the present invention. In a further embodiment, these
methods comprise the administration of an effective amount of the antibody of the present
invention in simultaneous, separate, or sequential combination with ramucirumab.
In an embodiment, the present invention provides a method of treating colorectal
cancer, comprising administering to a patient in need thereof, an effective amount of an
antibody of the present invention. In a further embodiment, these methods comprise the
administration of an effective amount of the antibody of the present invention in
simultaneous, separate, or sequential combination with one or more anti-tumor agents
selected from the group consisting of ramucirumab, FOLFOX (leucovorin, fluorouracil,
and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), and cetuximab.
In an embodiment, the present invention provides an antibody of the present
invention, for use in therapy. In an embodiment, the present invention provides an
antibody of the present invention, for use in the treatment of cancer. In a further
embodiment, the present invention provides an antibody of the present invention, for use
in the treatment of cancer, wherein the cancer is breast cancer, ovarian cancer, gastric
cancer, colorectal cancer, or hepatocellular carcinoma. In a further embodiment, for use
in the treatment of cancer, the antibody of the present invention in simultaneous, separate,
or sequential combination with one or more anti-tumor agents selected from the group
consisting of cisplatin, carboplatin, liposomal doxorubicin, docetaxel, cyclophosphamide
and doxorubicin, navelbine, eribulin, paclitaxel protein-bound particles for injectable
suspension, ixabepilone, capecitabine, ramucirumab, FOLFOX (leucovorin, fluorouracil,
and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), and cetuximab.
In a further embodiment, for use in the treatment of cancer, the compound of the
present invention in simultaneous, separate, or sequential combination with one or more
immuno-oncology agents selected from the group consisting of nivolumab, ipilimumab,
pidilizumab, pembrolizumab, and durvalumab.
In an embodiment, the present invention provides an antibody of the present
invention, for use in the treatment of breast cancer. In a further embodiment, for use in
the treatment of breast cancer, the antibody of the present invention in simultaneous,
separate, or sequential combination with one or more anti-tumor agents selected from the
group consisting of ramucirumab, docetaxel, cyclophosphamide and doxorubicin,
navelbine, eribulin, paclitaxel protein-bound particles for injectable suspension,
ixabepilone, and capecitabine.
In an embodiment, the present invention provides an antibody of the present
invention, for use in the treatment of ovarian cancer. In a further embodiment, for use in
the treatment of ovarian cancer, the antibody of the present invention in simultaneous,
separate, or sequential combination with one or more anti-tumor agents selected from the
group consisting of ramucirumab, cisplatin, carboplatin, and liposomal doxorubicin.
In an embodiment, the present invention provides an antibody of the present
invention, for use in the treatment of gastric cancer. In a further embodiment, for use in
the treatment of gastric cancer, the antibody of the present invention in simultaneous,
separate, or sequential combination with ramucirumab.
In an embodiment, the present invention provides an antibody of the present
invention, for use in the treatment of hepatocellular carcinoma. In a further embodiment,
for use in the treatment of hepatocellular carcinoma, the antibody of the present invention
in simultaneous, separate, or sequential combination with ramucirumab.
In an embodiment, the present invention provides an antibody of the present
invention, for use in the treatment of colorectal cancer. In a further embodiment, for use
in the treatment of colorectal cancer, the antibody of the present invention in
simultaneous, separate, or sequential combination with one or more anti-tumor agents
selected from the group consisting of ramucirumab, FOLFOX (leucovorin, fluorouracil,
and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), and cetuximab.
In a further embodiment, the present invention provides the use of an antibody of
the present invention for the manufacture of a medicament for the treatment of cancer. In
a further embodiment, the present invention provides the use of an antibody of the present
invention for the manufacture of a medicament for the treatment of cancer, wherein the
cancer is breast cancer, ovarian cancer, gastric cancer, colorectal cancer, or hepatocellular
carcinoma.
In a further embodiment , the present invention provides the use of an antibody of
the present invention in simultaneous, separate, or sequential combination with one or
more anti-tumor agents selected from the group consisting of cisplatin, carboplatin,
liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine,
eribulin, paclitaxel protein-bound particles for injectable suspension, ixabepilone,
capecitabine, ramucirumab, FOLFOX (leucovorin, fluorouracil, and oxaliplatin),
FOLFIRI (leucovorin, fluorouracil, and irinotecan), and cetuximab for the manufacture of
a medicament for the treatment of cancer.
An antibody of the present invention is an engineered, non-naturally occurring
polypeptide complex. A DNA molecule of the present invention is a non-naturally
occurring DNA molecule that comprises a polynucleotide sequence encoding a
polypeptide having the amino acid sequence of one of the polypeptides in an antibody of
the present invention.
An antibody of the present invention is designed to have engineered CDRs and
have some portions of the antibody (all or parts of the frameworks, hinge regions, and
constant regions) to be of human origin that are identical with or substantially identical
(substantially human) with frameworks and constant regions derived from human
genomic sequences. Fully human frameworks, hinge regions, and constant regions are
those human germline sequences as well as sequences with naturally-occurring somatic
mutations and those with engineered mutations. An antibody of the present invention
may comprise framework, hinge, or constant regions derived from a fully human
framework, hinge, or constant region containing one or more amino acid substitutions,
deletions, or additions therein. Further, an antibody of the present invention is preferably
substantially non-immunogenic in humans.
The antibody of the present invention is an IgG type antibody and has four amino
acid chains (two "heavy" chains and two "light" chains) that are cross-linked via intraand
inter-chain disulfide bonds. Each heavy chain is comprised of an N-terminal HCVR
and a heavy chain constant region ("HCCR"). Each light chain is comprised of a LCVR
and a light chain constant region ("LCCR"). When expressed in certain biological
systems, antibodies having native human Fc sequences are glycosylated in the Fc region.
Typically, glycosylation occurs in the Fc region of the antibody at a highly conserved Nglycosylation
site. N-glycans typically attach to asparagine. Antibodies may be
glycosylated at other positions as well.
Optionally, the antibody of the present invention contains an Fc portion which is
derived from human IgG4 Fc region because of a reduced ability to engage Fc receptormediated
inflammatory mechanisms or to activate complement resulting in reduced
effector function.
Further, certain antibodies of the present invention contain an IgG4-PAA Fc
portion. The IgG4-PAA Fc portion has a serine to proline mutation at position 229, a
phenylalanine to alanine mutation at position 235, and a leucine to alanine mutation at
position 236. The S229P mutation is a hinge mutation that prevents half-antibody
formation (phenomenon of dynamic exchange of half-molecules in IgG4 antibodies). The
F235A and L236A mutations further reduce effector function of the already low human
IgG4 isotype.
The HCVR and LCVR regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions ("CDRs"), interspersed with
regions that are more conserved, termed framework regions ("FR"). Each HCVR and
LCVR is composed of three CDRs and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and
HCDR3" and the three CDRs of the light chain are referred to as "LCDR1, LCDR2 and
LCDR3". The CDRs contain most of the residues which form specific interactions with
the antigen. There are currently three systems of CDR assignments for antibodies that are
used for sequence delineation. The Kabat CDR definition (Kabat et a , "Sequences of
Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991))
is based upon antibody sequence variability. The Chothia CDR definition (Chothia et a ,
"Canonical structures for the hypervariable regions of immunoglobulins", Journal of
Molecular Biology, 196, 901-917 (1987); Al-Lazikani et ah, "Standard conformations for
the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-
948 (1997)) is based on three-dimensional structures of antibodies and topologies of the
CDR loops. The Chothia CDR definitions are identical to the Kabat CDR definitions
with the exception of HCDR1 and HCDR2. The North CDR definition (North et al, "A
New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology,
406, 228-256 (201 1)) is based on affinity propagation clustering with a large number of
crystal structures.
For the purposes of the present invention, a combination of the three methods is
used to define CDRs. In the case of the light chain CDRs, the North CDR definitions are
used. In the case of HCDRl, a hybrid of the Kabat and Chothia CDR definitions is used.
The Kabat definition of HCDRl starts at the ninth residue after the first cysteine of the
heavy chain and is typically five to seven residues in length, whereas the Chothia
definition of HCDRl starts at the fourth residue after this cysteine and is typically seven
to nine residues in length. The HCDRl of the antibody of the present invention is defined
by the Chothia starting position and the Kabat end position. In the case of HCDR2, the
Kabat CDR definition is used. In the case of HCDR3, a hybrid of the North, Kabat and
Chothia CDR definitions is used. The Kabat definition of HCDR3 comprises residues
99-1 10 of the heavy chain (SEQ ID NO: 2 for the antibody of the present invention) and
typically starts three residues after a cysteine. The Chothia definition of HCDR3 is the
same as the Kabat definition. The North definition of HCDR3 comprises residues 97-1 10
of the heavy chain (SEQ ID NO: 2 for the antibody of the present invention) and typically
starts immediately after the cysteine residue. The HCDR3 of the antibody of the present
invention is defined by the North starting position and the Kabat/Chothia/North end
position.
An isolated DNA encoding a HCVR region can be converted to a full-length
heavy chain gene by operably linking the HCVR-encoding DNA to another DNA
molecule encoding heavy chain constant regions. The sequences of human, as well as
other mammalian, heavy chain constant region genes are known in the art. DNA
fragments encompassing these regions can be obtained e.g., by standard PCR
amplification.
An isolated DNA encoding a LCVR region may be converted to a full-length light
chain gene by operably linking the LCVR-encoding DNA to another DNA molecule
encoding a light chain constant region. The sequences of human, as well as other
mammalian, light chain constant region genes are known in the art. DNA fragments
encompassing these regions can be obtained by standard PCR amplification. The light
chain constant region can be a kappa or lambda constant region.
The polynucleotides of the present invention will be expressed in a host cell after
the sequences have been operably linked to an expression control sequence. The
expression vectors are typically replicable in the host organisms either as episomes or as
an integral part of the host chromosomal DNA. Commonly, expression vectors will
contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to
permit detection of those cells transformed with the desired DNA sequences.
The antibody of the present invention may readily be produced in mammalian
cells such as CHO, NSO, HEK293 or COS cells. The host cells are cultured using
techniques well known in the art.
The vectors containing the polynucleotide sequences of interest (e.g., the
polynucleotides encoding the polypeptides of the antibody and expression control
sequences) can be transferred into the host cell by well-known methods, which vary
depending on the type of cellular host.
Various methods of protein purification may be employed and such methods are
known in the art and described, for example, in Deutscher, Methods in Enzymology 182:
83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition,
Springer, NY (1994).
In another embodiment of the present invention, the antibody, or the nucleic acids
encoding the same, is provided in isolated form. As used herein, the term "isolated"
refers to a protein, peptide, or nucleic acid which is free or substantially free from any
other macromolecular species found in a cellular environment. "Substantially free" as
used herein means the protein, peptide, or nucleic acid of interest comprises more than
80% ( on a molar basis) of the macromolecular species present, preferably more than
90%, and more preferably more than 95%.
The antibody of the present invention, or pharmaceutical compositions comprising
the same, may be administered by parenteral routes (e.g., subcutaneous and intravenous).
An antibody of the present invention may be administered to a patient alone with
pharmaceutically acceptable carriers, diluents, or excipients in single or multiple doses.
Pharmaceutical compositions of the present invention can be prepared by methods well
known in the art (e.g., Remington: The Science and Practice of Pharmacy, 19th ed.
(1995), A. Gennaro et al., Mack Publishing Co.) and comprise an antibody, as disclosed
herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
The term "treating" (or "treat" or "treatment") refers to slowing, interrupting,
arresting, alleviating, stopping, reducing, or reversing the progression or severity of an
existing symptom, disorder, condition, or disease.
"Binds" as used herein in reference to the affinity of an antibody for human Ang2
is intended to mean, unless indicated otherwise, a KD of less than about 1 x 10 8 M,
preferably, less than about 1 x 10 9 M as determined by common methods known in the
art, including by use of a surface plasmon resonance (SPR) biosensor at 25°C or 37°C
essentially as described herein. The term "selective" or "selectivity" used herein in
reference to a compound of the present invention refers to a compound that binds a target,
such as human Ang2, with a KD about 1000-, 500-, 200-, 100-, 50-, or about 10-fold
lower than the compound binds other proteins, including member of the target family
such as human Angl, as measured by surface plasmon resonance at 25°C or 37°C.
Additionally, or alternatively, an Ang2 selective antibody of the present invention binds
human Ang2 but does not bind or only minimally binds human Angl when assayed by
the methods described in the Example herein below.
"Effective amount" means the amount of an antibody of the present invention or
pharmaceutical composition comprising an antibody of the present invention that will
elicit the biological or medical response of or desired therapeutic effect on a tissue,
system, animal, mammal or human that is being sought by the researcher, medical doctor,
or other clinician. An effective amount of the antibody may vary according to factors
such as the disease state, age, sex, and weight of the individual, and the ability of the
antibody to elicit a desired response in the individual. An effective amount is also one in
which any toxic or detrimental effect of the antibody is outweighed by the therapeutically
beneficial effects.
This invention is further illustrated by the following non-limiting example.
Example 1: Antibody expression and purification
The polypeptides of the variable regions of the heavy chain and light chain, the
complete heavy chain and light chain amino acid sequences of Antibody A, and the
nucleotide sequences encoding the same, are listed below in the section entitled "Amino
Acid and Nucleotide Sequences." In addition, the SEQ ID NOs for the light chain, heavy
chain, light chain variable region, and heavy chain variable region of Antibody A are
shown in Table 1.
The antibodies of the present invention, including, but not limited to, Antibody A
can be made and purified essentially as follows. An appropriate host cell, such as HEK
293 or CHO, can be either transiently or stably transfected with an expression system for
secreting antibodies using an optimal predetermined HC:LC vector ratio (such as 1:3 or
1:2) or a single vector system encoding both HC and LC. Clarified media, into which the
antibody has been secreted, may be purified using any of many commonly-used
techniques. For example, the medium may be conveniently applied to a MabSelect
column (GE Healthcare), or KappaSelect column (GE Healthcare) for Fab fragment, that
has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH
7.4). The column may be washed to remove nonspecific binding components. The
bound antibody may be eluted, for example, by pH gradient (such as 20 mM Tris buffer
pH 7 to 10 mM sodium citrate buffer pH 3.0, or phosphate buffered saline pH 7.4 to 100
mM glycine buffer pH 3.0). Antibody fractions may be detected, such as by SDS-PAGE,
and then may be pooled. Further purification is optional, depending on the intended use.
The antibody may be concentrated and/or sterile filtered using common techniques.
Soluble aggregate and multimers may be effectively removed by common techniques,
including size exclusion, hydrophobic interaction, ion exchange, multimodal, or
hydroxyapatite chromatography. The purity of the antibody after these chromatography
steps is greater than 95%. The product may be immediately frozen at -70°C or may be
lyophilized.
Table 1: SEQ ID NOs
Assays
Binding kinetics, affinity, and selectivity
The binding kinetics, affinity, and selectivity for multiple species of soluble Ang2
such as human, cynomolgous, mouse, rabbit and dog, may be determined for antibodies
of the present invention at 25°C or 37°C by use of a surface plasmon resonance (SPR)
biosensor such as a BIAcore® 2000, BIAcore® 3000, or a BIAcore® T100 (GE
HealthCare) according to methods known in the art.
Human Ang2 may be purchased from R&D Systems. Protein A surface for
capture of antibodies may be prepared using the following methods. Immobilization of
soluble Protein A (Calbiochem, Cat: 539202) on a CM4 (Biacore #BR-1005-34) or CM5
(Biacore #BR-1000-99) may be prepared using EDC/NHS amine coupling method
(Biacore # BR-1000-50). Briefly, the surfaces of all four flow cells may be activated by
injecting a 1:1 mixture of EDC/NHS for seven minutes at 5 mΐ/min or 10 m /h h. After
which, soluble protein A may be diluted in 10 mM acetate buffer, pH 4.5, and
immobilized for seven minutes onto flow cell (Fc) 2, 3 or 4 at a flow rate of 5 m /h h or
10 m /h h. Un-reacted sites still remaining on the chip surface may be blocked with a
seven minute injection of ethanolamine at 5 m n i h or 10 m n i h. Running buffer may
be HBS-EP+ (Biacore # BR-1006-69). Antibody samples may be prepared at 1 mg/mL or
2 mg/mL by dilution into running buffer. Discrete concentrations of Ang2 ligands
ranging from 200 nM to 3.1 nM may be prepared using a two-fold serial dilution into
running buffer. Each analysis cycle may consist of a series of five separate steps: (1)
capture of antibody onto separate flow cells (Fc2, Fc3, and Fc4), (2) injection (using
kinject) of 250 mI_, (300-second surface contact time) of discrete concentrations of Ang2
over all Fc at 50 m / h h, (3) return to buffer flow for 20 minutes to monitor dissociation
phase, (4) regeneration of chip surfaces with a 10 mI_, (30-second contact time) injection
of 10 mM glycine, pH 1.5, (5) equilibration of chip surface with a 15 mI_, (45-second
contact time) injection of HBS-EP+ running buffer. Resultant data may be processed
using standard double-referencing and fit to a 1:1 binding model using Biacore 2000
Evaluation software, version 4.1, to determine the association rate (kon, M_1s_1 units),
dissociation rate (koff, s 1 units). Calculation of the equilibrium dissociation constant (KD)
may be calculated from the following relationship, KD = o on and is presented in molar
units.
H1H685P-293HEK is a human IgGl Ang2 antibody expressed in 293 HEK cells
that utilizes an HCVR sequence (SEQ ID NO: 18 from WO201 1014469) and a LCVR
sequence (SEQ ID NO: 20 from WO201 1014469). 3.19.3-293HEK is a human IgGl
antibody expressed in 293 HEK cells that binds Ang2 and utilizes the 3.19.3 HCVR
sequence (SEQ ID NO: 2 from WO2009097325) and the MEDIl LCVR sequence (SEQ
ID NO: 3 fromWO2009097325).
In experiments performed essentially as described in this assay, Antibody A binds
to human Ang2 with a KDapproximately 4x and 8x lower than H1H685P-293HEK and
3.19.3-293HEK, respectively (Table 2).
In experiments performed essentially as described in this assay, Antibody A binds
human, cynomolgus, mouse, rabbit and dog Ang2 with affinities of 80, 107, 109, 210 and
140 pM, respectively (Table 3).
Table 2
Table 3
Antibody A Biacore SPR Parameters for Ang2 Binding (37°C, n=3)
kon (1/Ms) koff (l /s) ¾ (pM)
human Ang2 1.0 (± 0.4) x l 06 0.7 (± 0.2) x 10 4 80 ± 50
cyno Ang2 2.7 (± 0.2) x l 06 2.9 (± 0.1) x 10~4 107 ± 6
mouse Ang2 1.0 (± 0.2) x l 06 1.1 (± 0.1) x 10~4 109 ± 9
rabbit Ang2 7.3 (± 1.9) x 105 1.5 (± 0.1) x 10 4 210 ± 40
dog Ang2 7.9 (± 3.2) x l 05 1.1 (± 0.4) x 10~4 140 ± 10
rat Ang2 1.2 (± 0.04) x l O6 2.0 (± 0.2) x 10 4 180 ± 20
Affinity measurement with Kinexa
A KinExA 3200 instrument (Sapidyne Inst. Inc.) may be used to measure binding
kinetics. Briefly, human Ang2 may be covalently coupled to sepharose beads and the
binding of free Mab to the beads may be detected on the KinExA 3200. To measure KD,
individual tubes containing Mab ( 1 pM, 2 pM, or 5 pM) with decreasing serially diluted
human Ang2 may be incubated for a few days at 25°C in PBS containing lmg/ml BSA.
After the incubation, free Mab in each equilibrated sample may be determined. values
may be determined using N-curve analysis with KinExA software.
In experiments performed essentially as described in this assay, Antibody A binds
to human Ang2 with a KDabout 8x lower than H1H685P-293HEK (Table 4).
Table 4
Antibody A inhibits interaction of human Ang2 to human Tie2 via solid phase
ELISA assay
The blocking of human Ang2 binding to its receptor human Tie2 by an antibody
of the present invention may be measured in a solid phase in vitro ELISA assay.
For the assay, high binding 96-well ELISA plates (Costar #2592) may be coated
with 4 mg/ml (in 100 mΐ ) recombinant human Tie2-Fc (R&D Systems #313-TI), overnight
at room temperature. The plates may be washed 3X with TBST (Tris buffered saline
containing 0.05% Tween 20) and then may be blocked with 300 mΐ/well of blocking
buffer (0.5% BSA/D-PBS) (BSA: Jackson ImmunoResearch #001-000-162; IgG-free,
protease-free) for 1-2 hours at room temperature on an orbital shaker. During the
blocking step, in separate polypropylene mutiwell plates, 75 mΐ of 2X test antibodies
(serially diluted 1:3 in blocking buffer) may be added with 75 mΐ of 2X biotinylated
human Ang2 (R&D Systems #BT623) (also diluted in blocking buffer). The
antibody/biotinylated Ang2 mixtures may then be incubated for 1 hour at 37°C (final
biotinylated Ang2 concentration was 100 ng/ml). The blocking solution may be removed
from the Tie2-Fc coated ELISA plates, after which 50 mΐ per well of the
antibody/biotinylated Ang2 mixtures may be added (in duplicate wells). The plates may
then be incubated for 2 hours at room temperature, covered with plate sealers, on an
orbital shaker. Plates may then be washed 3X, after which 100 mΐ per well of
streptavidin-HRP (R&D Systems #DY998, may be diluted 1:200 in blocking buffer) may
be added. Plates may then be incubated for 45 minutes at room temperature, covered
with plate sealers, on an orbital shaker. Plates may then be washed again 3X.
Plates may then be developed by adding 100 mΐ per well of One Component TMB
substrate (may be warmed to room temperature) (Surmodics/BioFX Labs #TMBW-1000-
01). Development may be allowed to progress for 10 minutes at room temperature (plates
may be covered with aluminum foil). Development may be stopped with 100 mΐ per well
of acid stop solution (TMB stop solution, Surmodics/BioFX Labs #LSTP-1000-01).
Plates may be mixed on an orbital shaker after which they may be read at 450 nm on an
ELISA reader (Molecular Devices SpectraMax 190), using SOFTmax PRO 5.4.1
software (Molecular Devices Corp.). The A450 values reflect the amount of biotinylated
Ang2 that remained bound to Tie-2-Fc. Reduction of A450 values reflected blocking of
biotinylated Ang2 binding to Tie-2-Fc.
IC50 values for inhibition of Ang2 binding to Tie-2 may be calculated with
GraphPad Prism 6, using Log-transformed X values. Nonlinear regression (curve fit)
analysis (sigmoidal dose response, variable slope) may be performed on the logtransformed
data to obtain IC50 values. If an experiment is performed more than once,
the geometric mean IC50 value (and 95% confidence interval) between experiments may
be calculated.
In experiments performed essentially as described in this assay, Antibody A
specifically blocked binding of Ang2 to Tie-2 in a dose-dependent manner, resulting in
geometric mean IC50 value (n=2) of 0.027 nM (95% confidence interval 0.00089 -
0.8029 nM ). Antibody A and H1H685P-293HEK had comparable neutralization IC50
values.
Neutralization of Ang2 Induced phosphorylation of Tie-2 but not Angl mediated
phosphorylation.
The in vitro cell-based inhibition of human Tie-2 by an antibody of the present
invention may be measured in a cell-based assay where Angl and Ang2 bind to and
induce human Tie-2 phosphorylation in a dose-dependent manner. The in vitro cell-based
assay may be used to evaluate the ability of Antibody A to selectively neutralize Ang2
and not Angl mediated phosphorylation of the Tie-2 receptor in a dose-dependent
manner. An Ang2 antibody, an Angl/2 cross-reactive antibody, and a control human
IgG4 PAA isotype antibody may be included as positive and negative controls
respectively.
The CHO-Tie-2 cell line may be generated by stable transfection of a full-length
human Tie-2 receptor (with a 3X FLAG tag at the C-terminus). CHO-Tie-2 cells may be
maintained in complete medium of Hams F-12 (CellGro/Mediatech #10-080-CV), 10%
heat inactivated FBS (Life Technologies/Invitrogen #10082-147), IX antibioticantimycotic
(Life Technologies/Invitrogen #15240-062), 1.25 mg/ml G418 (Corning
Cellgro #30-234-CI), 10 iΐ puromycin (Calbiochem #540411), and 0.078% sodium
bicarbonate (Thermo Hyclone #SH30033.01).
For the assay, CHO-Tie-2 cells may be resuspended to 10,000 cells per well (in
100 mΐ growth medium), into the inner 60 wells of poly-lysine coated 96-well plates (BD
Biocoat #356640). 200 mΐ of D-PBS may be placed into the edge wells to reduce
evaporation. Cells may be incubated overnight at 37°C, 95% RH, 5% C0 2. The next
day, cells may be washed once and medium may be replaced with 100 mΐ serum-free
growth medium containing 0.1% BSA (Sigma #A7979, low endotoxin). Cells may then
be starved for 7.5 to 24 hours in serum-free medium at 37°C, 95% RH, 5% C0 2. During
the starvation period, antibodies (at 6X the final concentrations) may be serially diluted
1:2 in polypropylene plates in serum-free growth medium containing 0.1% BSA. Human
Ang2 (R&D Systems #623-AN, reconstituted in D-PBS/0.1% BSA) and human Angl
(R&D Systems #923-AN, reconstituted in D-PBS/0.1% BSA) may also be diluted to 6X
the final concentration in serum-free growth medium containing 0.1% BSA. Antibodies
and the Ang2 or Angl ligand may then be mixed at a 1:1 ratio in polypropylene plates
and may be incubated for 1 hour at 37°C. The antibody/ligand mixtures may then be
added at 50 mΐ per well to the cells (in triplicate wells per treatment) and may be
incubated for 13 minutes to 2 1 hours at 37°C, 95% RH, 5% C0 2. The final concentration
range of antibody may be 0.0625 - 283 nM, and the final concentration of human Ang2
and Angl may be 0.3 mg/ml (approx. 6 nM) and 0.5 mg/ml (approx. 8.9 nM), respectively.
After the incubation time, medium may be quickly and fully removed from the cells, and
cells may be lysed in 60 mΐ per well of cold IX Tris Lysis Buffer (Meso Scale Discovery
#R60TX; 150 mM NaCl, 20 mM Tris pH 7.5, 1mM EDTA, 1mM EGTA, 1% Triton X-
100) which may contain freshly added protease and phosphatase inhibitors (IX protease
inhibitor cocktail, Sigma #P8340; IX phosphatase inhibitor cocktail 2, Sigma #P5726; IX
phosphatase inhibitor cocktail 3, Sigma #P0044; 1mM final activated sodium
orthovanadate (EMD Chemicals #567540)). Plates may then be placed on ice for 10
minutes, after which they may be placed on an orbital shaker at low speed for 25 minutes
at 4°C. The plates may then be sealed and frozen at -80°C.
The day before analysis for phospho-Tie-2 (with a human phospho-Tie-2 DuoSet
ELISA kit from R&D Systems, #DYC2720), high binding ELISA plates (Greiner
BioOne, #655081) may be coated overnight at 4°C with 4 mg/ml mouse anti-human total
Tie-2 capture antibody in IX ELISA coating buffer (Surmodics/BioFX Labs #COAT-
1000-01).
The day of phospho-Tie-2 measurement, plates containing lysates may be thawed
on ice. The coated ELISA plates may be washed with wash buffer (IX TBST containing
0.05% Tween 20) and blocked with 300 mΐ per well of blocking buffer (1% BSA (Jackson
ImmunoResearch #001-000-162; IgG-free, protease-free), 0.01% sodium azide) for a
minimum of 1 hour at room temperature on an orbital shaker (while covered with plate
sealers). During blocking, lysates may be diluted 1:5 or 1:10 in polypropylene plates in
cold lysis buffer containing protease and phosphatase inhibitors. ELISA plates may be
blocked and washed 4X, and 100 mΐ per well of diluted lysates (or phospho-Tie-2 ELISA
standards) may be added and incubated for 2 hours at room temperature, covered with
sealers, on an orbital shaker. Plates may be washed 4X and 100 mΐ per well of HRP
conjugated mouse anti-phospho tyrosine (diluted as recommended on the vial, in
TBST/0.1% BSA) may be added. Plates may then be covered with sealers, and incubated
for 2 hours at room temperature on an orbital shaker. Plates may then be washed 6X and
removal of liquid from the wells may be ensured. Plates may then be developed by
adding 100 mΐ per well of One Component TMB substrate (Surmodics/BioFX Labs
#TMBW- 1000-01). Plates may be allowed to develop for 20 or 30 minutes at room
temperature covered with aluminum foil. Development may be stopped with 100 mΐ per
well of acid stop solution (TMB stop solution, Surmodics/BioFX Labs #LSTP-1000-01).
Plates may then be mixed on an orbital shaker. The ELISA plates may be read at 450 nm
on an ELISA reader (Molecular Devices SpectraMax 190), using SOFTmax PRO 5.4.1
software (Molecular Devices Corp.). Phospho-Tie-2 values for the samples may be
obtained from the standard curve (4-parameter logistic fit), and multiplied by the dilution
factor of 5 or 10. Percent inhibition may be calculated by the following formula: (pTie2
value of treatment-mean pTie2 value of Ang2 alone treatment) / (mean medium alone
pTie2 value- mean pTie2 value of Ang2 alone treatment)* 100.
IC50 values for inhibition of Ang2 induced phospho-Tie-2 may be calculated with
GraphPad Prism 4, using Log-transformed X values. Nonlinear regression (curve fit)
analysis (sigmoidal dose response, variable slope) may be performed on the logtransformed
data to obtain IC50 values. If an experiment was performed more than once,
the geometric mean IC50 value (and 95% confidence interval) between experiments may
be calculated.
In experiments performed essentially as described in this assay, Antibody A dosedependently
neutralized human Ang2 induced phospho-Tie-2 in CHO-Tie-2 cells with an
IC50 of 0.773 nM (95% confidence interval 0.412 - 1.45 nM) (n=3). Furthermore,
Antibody A did not neutralize human Angl induced phospho-Tie-2 in CHO-Tie-2 cells
when compared to the anti-Angl/2 cross-reactive antibody 3.19.3-293HEK. Antibody A
and H1H685P-293HEK had comparable Ang2 neutralization IC50 values.
Repression of Ang2 Induced Blood Vessel Development
The in vivo repression of physiological angiogenesis by an Ang2 antibody may be
measured in a model of blood vessel development in the mouse retina. The
aforementioned assay may be used to study the ability of antibodies of the present
invention to repress physiological angiogenesis in the mouse retina.
For this assay, the day of mouse pup delivery by the pregnant females may be
marked PO (postnatal day 0). Following delivery, at days two and four (P2 and P4) pups
may be injected with vehicle control (PBS) or 10 mg/kg of Antibody A. At P5 mice may
be sacrificed and eyes may be harvested and may be fixed in formalin for 5 hours and
may be washed with PBS.
Retinas may then be dissected, and may be stained with anti-CD31 diluted at
1:200 (BD Pharmingen; clone MEC 13.3; Catalog 553370), and anti-SMA-FTIC diluted
at 1:200 (Sigma; Clonel A4 Catalog F3777). For the anti-CD3 1 treated retinas an anti-
Rat Alexa-647 diluted at 1:400 (Jackson Immuno Research; Catalog 712-606-153) may
be used as a secondary antibody. Acquisition of the retinas may be done by using Nikon
Ti, and quantifications of vascular progression, number of sprouting tip cells, and
vascular density of remodeling plexus may be performed by using FIJI software. High
magnification images may be acquired using a confocal Nikon Al .
In experiments performed essentially as described in this assay, Antibody A
repressed vascular progression, reduced both the number of endothelial tip cells and
vascular density, as well as increased pericyte coverage similarly at 3 mg/kg, 10 mg/kg
and 30 mg/kg when compared to the PBS control group. This data was statistically
significant with p< 0.0001 for all treatment groups when compared to the PBS control
group (Table 5).
Table 5
Vehicle Antibody A Antibody A Antibody A
Parameters
(PBS) (30mg/kg) (lOmg/kg) (3mg/kg)
Vascular progression
Mean (%) 100 59.91 59.76 67.73
Std. Error of Mean 5.046 3.848 2.524 2.463
P value (Vehicle vs.
Compounds) < 0.0001 < 0.0001 < 0.0001
(Dunnett's test)
P value (30 vs. 10 and
3mg/kg) > 0.9999 0.2457
(Dunnett's test)
P value (10 vs. 3mg/kg)
0.2892
(Dunnett's test)
Number of tip cells
Mean (%) 100 41.82 47.79 52.25
Std. Error of Mean 6.181 3.25 4.647 3.474
P value (Vehicle vs.
Compounds) < 0.0001 < 0.0001 < 0.0001
(Dunnett's test)
P value (10 and 3mg/kg)
0.6685 0.1899
(Dunnett's test)
P value (10 vs. 3mg/kg)
0.8290
(Dunnett's test)
Vascular density
Mean (%) 100 58.51 55.97 60.06
Std. Error of Mean 4.48 2.685 3.569 3.056
P value (Vehicle vs.
Compounds) < 0.0001 < 0.0001 < 0.0001
(Dunnett's test)
P value (10 and 3mg/kg)
0.9105 0.9735
(Dunnett's test)
P value (10 vs. 3mg/kg)
0.7396
(Dunnett's test)
In Vivo anti-angiogenic effects of Antibody A in PC3 prostate cancer xenograft
model
The anti-angiogenic effect of Ang2 antibodies and Ang2 antibodies in
combination with VEGFR2 antibodies may be evaluated in a PC3 prostate cancer
xenograft model.
Mice bearing PC3 xenograft tumors at approximately 250 mm3 volume,
randomized at 10 mice/group, may be treated with control IgGl, DC101 (an anti-
VEGFR2 murine antibody), Antibody A, DC101 + Antibody A for 6 days (2QW; 20
mg/kg). After six days, tumors may be collected, fixed and sectioned. The sectioned
tumors may be stained for CD105 and the total vessel area may be determined.
In experiments performed essentially as described, when compared to the control
IgGl group, the total vessel area was reduced by 65% in the DC101 treatment group,
69% in the Antibody A treatment group, and 84% in the combination treatment group.
When compared to the IgGl control group, these results were statistically significant with
a p<0.0001 for all the treatment groups.
Antibody A and anti-VEGFR2 antibody inhibits in vivo tumor growth
The efficacy of the antibodies of the present invention may be measured via in
vivo xenograft models. The antitumor efficacy of DClOl (an anti-VEGFR2 murine
antibody), Antibody A and its combination may be assessed in the subcutaneous triple
negative patient derived breast cancer model (EL1997) and the subcutaneous ovarian
xenograft model (SKOV3x.l). Mice bearing tumors may be treated with antibodies
diluted in PBS, on a twice weekly basis via intra-peritoneal injection. Tumor growth may
be determined by three dimensional caliper measurements of tumor volumes twice
weekly during the course of treatment.
EL1997 triple negative breast patient derived xenografts: Immuno-deficient
mice bearing ELI 997 triple negative breast patient derived xenografts (TNBC PDX) at
approximately 350mm3volume randomized at n=7 mice/group may be treated with
vehicle control, DClOl monotherapy at 20 mg/kg, Antibody A monotherapy at 20 mg/kg,
Cyclophosphamide and Doxorubicin combination at 100 mg/kg and 10 mg/kg
respectively, DClOl and Antibody A combination or Cyclophosphamide, Doxorubicin,
DClOl and Antibody A combination dosed at the respective monotherapy concentrations.
Treatments may be administered twice a week for 4 consecutive weeks.
In experiments performed essentially as described, DClOl and Antibody A
monotherapy treatment groups exhibited a %T/C (change in tumor volume) of 35.2% and
56.7%, respectively. The combination of the two antibodies, DClOl and Antibody A
resulted in a %T/C of 19.9%. Furthermore, the combination of Cyclophosphamide,
Doxorubicin, DClOl and Antibody A resulted in a %T/C of 0.4%, with a statistically
significant (p=0.0019) reduction in tumor volume when compared to the combination of
DClOl and Antibody A. The combination treatment of the chemotherapeutic agents
alone, Cyclophosphamide and Doxorubicin resulted in a %TC of 32%. These results
indicate that the monotherapy treatment with DClOl or Antibody A is efficacious, and
has potential for greater efficacy in combination; which is further enhanced with the
addition of chemotherapeutic agents.
SKOV3x.l ovarian xenografts: Immuno-deficient mice bearing SKOV3x.l
ovarian xenografts at approximately 350mm3 volume, randomized at n=10 mice/group,
may be treated with vehicle control, Antibody A monotherapy at 3, 10 or 30 mg/kg,
Paclitaxel monotherapy at 25 mg/kg or the combination of Paclitaxel and Antibody A at
25 mg/kg and lOmg/kg respectively. Treatments may be administered twice a week for 4
weeks.
In experiments performed essentially as described, the results showed a %T/C
values of 39.1, 32.5 and 27.3 for 3, 10 and 30 mg/kg doses of Antibody A respectively
and %T/C of 20.3 for 25 mg/kg paclitaxel monotherapy with p values of <0.001
compared with the control. The combination of 25 mg/kg Paclitaxel and 10 mg/kg
Antibody A enhanced the efficacy of the two monotherapies, showing a %T/C of 7.1%
when compared to the vehicle control group.
SKOV3x.l ovarian xenografts: Immuno-deficient mice bearing SKOV3x.l
ovarian xenografts at approximately 250mm3 volume, randomized at n=10 mice/group,
may be treated with vehicle control, DClOl monotherapy at 20 mg/kg, Paclitaxel
monotherapy at 25 mg/kg, DClOl and Antibody A combination at 25 mg/kg each, or
Paclitaxel, DClOl and Antibody A combination dosed at the respective monotherapy
concentrations. Treatments may be administered twice a week for 4 weeks.
In experiments performed essentially as described, monotherapy treatment of
DClOl resulted in a %T/C of 9.6%, whereas the combination of the two antibodies,
DClOl and Antibody A, resulted in an improved %T/C of 0.1% when compared to the
vehicle control group. The combination of Paclitaxel, DClOl and Antibody A resulted in
tumor regressions of about -33% compared to a %T/C of 17.8% for the Paclitaxel
monotherapy. These results indicate in the xenograft model that VEGFR2 inhibition with
DClOl has potential for greater efficacy in combination with Antibody A. The benefit of
this combination is further enhanced with the addition of a chemotherapeutic agent
leading to statistically significant regressions in tumor volumes.
Amino Acid and Nucleotide Sequences
SEQ ID NO: 1 (HCVR of Antibody A)
QVQLVQSGAEVKXPGASVKVSCKASGYSFTDYNMVWVRQAPGQGLEWMGYID
PYNGGTGYNQKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARTRDRYDV
WYFDVWGQGTLVTVSS
SEQ ID NO: 2 (HC of Antibody A)
QVQLVQSGAEVKXPGASVKVSCKASGYSFTDYNMVWVRQAPGQGLEWMGYID
PYNGGTGYNQKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARTRDRYDV
WYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTKTYTCNVDHKPSNTKV
DKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQED
PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLG
SEQ ID NO: 3 (LCVR of Antibody A)
DIQMTQSPSSVSASVGDRVTITCKASQDVYIAVAWYQQKPGKAPKLLIYWASTR
DTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPPTFGGGTKVEIK
SEQ ID NO: 4 (LC of Antibody A)
DIQMTQSPSSVSASVGDRVTITCKASQDVYIAVAWYQQKPGKAPKLLIYWASTR
DTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPPTFGGGTKVEIKRTV
AAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 5 (DNA of LC of Antibody A)
GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAG
AGTCACCATCACTTGTAAGGCCAGTCAGGATGTGTATATTGCTGTAGCCTGGT
ATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTGGGCATCCAC
CCGGGACACTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAT
TTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTG
TCACCAATATAGCAGCTATCCTCCTACGTTCGGCGGAGGGACCAAGGTGGAG
ATCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC
CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA
CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC
AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC
GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA
ACAGGGGAGAGTGC
SEQ ID NO: 6 (DNA of HC of Antibody A)
CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAG
TGAAGGTCTCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATGGTG
TGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATATATTGATC
CTTACAATGGTGGTACTGGCTACAACCAGAAGTTCGAGGGCAGAGTCACCAT
GACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGA
TCTGACGACACGGCCGTGTATTACTGTGCGAGAACGAGGGATAGGTACGACG
TCTGGTACTTCGATGTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCC
TCCACCAAGGGCCCATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTC
CGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG
GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGC
CCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATG
CCCACCCTGCCCAGCACCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCC
CCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTG
CGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC
GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCC
TCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGG
TGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAA
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT
CCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTG
GCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT
SEQ ID NO: 7 (human Ang2)
YNNFRKSMDSIGKKQYQVQHGSCSYTFLLPEMDNCRSSSSPYVSNAVQRDAPLE
YDDSVQRLQVLENIMENNTQWLMKXENYIQDNMKKEMVEIQQNAVQNQTAVM
IEIGTNLLNQTAEQTRKLTDVEAQVLNQTTRLELQLLEHSLSTNKLEKQILDQTSEI
NKXQDK^SFLEKKVLAMEDKHIIQLQSIKEEKDQLQVLVSKQNSIIEELEKKIVTA
TVNNSVLQKQQHDLMETVNNLLTMMSTSNSAKDPTVAKEEQISFRDCAEVFKSG
HTTNGIYTLTFPNSTEEIKAYCDMEAGGGGWTIIQRREDGSVDFQRTWKEYKVGF
GNPSGEYWLGNEFVSQLTNQQRYVLKIHLKDWEGNEAYSLYEHFYLSSEELNYR
IHLKGLTGTAGKISSISQPGNDFSTKDGDNDKCICKCSQMLTGGWWFDACGPSNL
NGMYYPQRQNTNKFNGIKWYYWKGSGYSLKATTMMIRPADF
SEQ ID NO: 8 (LCDR1 of Antibody A)
KASQDVYIAVA
SEQ ID NO: 9 (LCDR2 of Antibody A)
YWASTRDT
SEQ ID NO: 10 (LCDR3 of Antibody A)
HQYSSYPPT
SEQ ID NO: 11 (HCDR1 of Antibody A)
GYSFTDYNMV
SEQ ID NO: 1 (HCDR2 of Antibody A)
YIDPYNGGTGYNQKFEG
SEQ ID NO: 13 (HCDR3 of Antibody A)
ARTRDRYDVWYFDV

CLAIM
An antibody that binds human Ang2 (SEQ ID NO: 7), comprising a light
chain (LC) and a heavy chain (HC), wherein the light chain comprises
light chain complementarity determining regions LCDR1, LCDR2, and
LCDR3 consisting of the amino acid sequences KASQDVYIAVA (SEQ
ID NO: 8), YWASTRDT (SEQ ID NO: 9), and HQYSSYPPT (SEQ ID
NO: 10), respectively, and wherein the heavy chain comprises heavy chain
complementarity determining regions HCDR1, HCDR2, and HCDR3
consisting of the amino acid sequences GYSFTDYNMV (SEQ ID NO:
11), YIDPYNGGTGYNQKFEG (SEQ ID NO: 12), and
ARTRDRYDVWYFDV (SEQ ID NO: 13), respectively.
An antibody, comprising a light chain (LC) and a heavy chain (HC),
wherein the light chain comprises a light chain variable region (LCVR)
and the heavy chain comprises a heavy chain variable region (HCVR),
wherein the LCVR has the amino acid sequence given in SEQ ID NO: 3,
and the HCVR has the amino acid sequence given in SEQ ID NO: 1.
The antibody of any one of Claims 1 or 2, wherein the LC has the amino
acid sequence given in SEQ ID NO: 4, and the HC has the amino acid
sequence given in SEQ ID NO: 2.
The antibody of any one of Claims 1-3, comprising two light chains and
two heavy chains, wherein each light chain has the amino acid sequence
given in SEQ ID NO: 4, and each heavy chain has the amino acid
sequence given in SEQ ID NO: 2.
The antibody of any one of Claims 1-4, wherein one of the heavy chains
forms an inter-chain disulfide bond with one of the light chains, and the
other heavy chain forms an inter-chain disulfide bond with the other light
chain, and one of the heavy chains forms two inter-chain disulfide bonds
with the other heavy chain.
6. The antibody of any one of Claims 1-5, wherein the antibody is
glycosylated.
7. A mammalian cell comprising a DNA molecule comprising a
polynucleotide sequence encoding a polypeptide having an amino acid
sequence of SEQ ID NO: 4 and a polynucleotide sequence encoding a
polypeptide having an amino acid sequence of SEQ ID NO: 2, wherein the
cell is capable of expressing an antibody comprising a light chain having
an amino acid sequence of SEQ ID NO: 4 and a heavy chain having an
amino acid sequence of SEQ ID NO: 2.
8. A process for producing an antibody comprising a light chain having an
amino acid sequence of SEQ ID NO: 4 and a heavy chain having an amino
acid sequence of SEQ ID NO: 2, comprising cultivating the mammalian
cell of Claim 7 under conditions such that the antibody is expressed, and
recovering the expressed antibody.
9. An antibody produced by the process of Claim 8.
10. A pharmaceutical composition, comprising the antibody of any one of
Claims 1-6 or 9, and an acceptable carrier, diluent, or excipient.
11. A method of treating cancer, comprising administering to a patient in need
thereof, an effective amount of the antibody of any one of Claims 1-6 or 9.
12. The method of Claim 11, wherein the cancer is breast cancer, ovarian
cancer, gastric cancer, colorectal cancer, or hepatocellular carcinoma.
13. The method of Claim 11 or 12, further comprising administering
simultaneously, separately, or sequentially an effective amount of
ramucirumab.
14. The antibody of any one of Claims 1-6 or 9, for use in therapy.
15. The antibody of any one of Claims 1-6 or 9, for use in the treatment of
cancer.
16. The antibody for use of Claim 15, wherein the cancer is breast cancer,
ovarian cancer, gastric cancer, colorectal cancer, or hepatocellular
carcinoma.
17. The antibody of any one of Claims 1-6 or 9 in simultaneous, separate, or
sequential combination with ramucirumab, for use in the treatment of
cancer.
18. The combination for use of Claim 17, wherein the cancer is breast cancer,
ovarian cancer, gastric cancer, colorectal cancer, or hepatocellular
carcinoma.