VEGFR2/Ang2 Compounds
The present invention relates to the field of medicine. More particularly, the
present invention relates to compounds that bind to human vascular endothelial growth
factor receptor-2 (VEGFR2) and human angiopoietin-2 (Ang2), and may be useful for
treating cancer, especially solid tumors driven by VEGFR2 and Ang2, including gastric,
hepatocellular carcinoma, ovarian, colorectal, non-small cell lung, biliary tract, and breast
cancers.
A hallmark of cancer is persistent new blood vessel formation, called
angiogenesis. The vascular endothelial growth factor (VEGF) pathway is an important
signaling cascade in the regulation of angiogenesis; human VEGFR2 is a key receptor in
the VEGF pathway. Ramucirumab (IMC-1 121B) is an IgGl antibody that targets human
VEGFR2, and has been shown to have an antitumor effect in certain clinical studies (see,
for example, Zhu et al., Clin Cancer Res (2013) 19:6614).
Angiopoietin- 1 (Angl) and Ang2 are members of another 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. 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 (Daly et al., Cancer Res (2013) 73(1): 108).
Multiple investigational Ang2 antibodies are currently in clinical trials.
Inhibition of both the VEGF and Ang/Tie2 pathways of angiogenesis has been
proposed for the potential to improve the outcome against cancer (see, for example, Daly
et al., Cancer Res (2013) 73:108). Currently, co-administration of a VEGFR2 antibody
and Ang-2 antibody would require either injections or infusions of two separate products
or administration of a co-formulation of an antibody mixture. Separate administration
would permit flexibility of dose amount and timing, but would be a potential issue for
patient compliance and inconvenience due to increased infusion time. A co-formulation
might provide some flexibility of dosage amounts, but can be challenging to find
formulation conditions that permit chemical and physical stability of both antibodies due
to different molecular characteristics of the two different antibodies. Furthermore, co
administration or co-formulation involves the additive costs of two drug therapies.
WO20 12/009705 disclosed complexes containing one or more modular
recognition domains (MRDs) attached to scaffolds that include antibodies. Ang2 was
listed as contemplated for the MRD portion of the complex, and a VEGFR2 antibody was
specified as an antibody which MRDs could be attached. A MRD against Ang2 attached
to a VEGFR2 antibody was not exemplified. Brown et al. (Mol Cancer Ther (2010)
9(1): 145) disclosed a human monoclonal Ang2 antibody, 3.19.3. In a SW620 xenograft
study, 3.19.3 was dosed in combination with DC101, a monoclonal antibody that binds
murine VEGFR2.
When generating compounds (Compound A and B) that contain a VEGFR2
antibody portion containing the light chain variable region (LCVR) and heavy chain
variable region (HCVR) of the IMC- 1121B antibody in an IgGl or IgG4 backbone, fused
to a single chain Fv (scFv) portion binding human Ang2, expression and stability
problems were observed by Applicant as part of the present invention. Specifically,
chemical instability and unacceptable product quality in recombinantly expressed
materials due to free light chain mis-pairing were surprisingly observed. As observed by
Applicant as part of the present invention, compound engineering to improve chemical
instability and product quality in the materials obtained from cell culture unexpectedly led
to certain compounds with increased product heterogeneity due to Mab-diabody
formation.
There remains a need to provide compounds that inhibit two angiogenesis
pathways by binding and neutralizing both human VEGFR2 and human Ang2. In
particular, there remains a need to provide compounds that inhibit two angiogenesis
pathways by binding and neutralizing both human VEGFR2 and human Ang2, and
without compromising significant Ang2 in vitro binding activity due to the use of an
Ang2 scFv, and without compromising significant in vitro cell-based assay activity due to
the combination of the VEGFR2 antibody and Ang2 scFv into one compound. There also
remains a need to provide compounds that inhibit two angiogenesis pathways by binding
and neutralizing both human VEGFR2 and human Ang2, and avoid at least one of the
above listed stability and product heterogeneity problems.
Accordingly, an embodiment of the present invention provides a compound,
comprising an antibody fused by two linkers to two single chain fragment variable (scFv)
polypeptides, wherein:
a) the antibody comprises two identical heavy chains (HCs) and two identical
light chains (LCs), wherein each HC comprises a heavy chain variable region
(HCVR) whose amino acid sequence is given in SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, or SEQ ID NO: 4, and wherein each LC comprises a light chain
variable region (LVCR) whose amino acid sequence is given in SEQ ID NO: 13,
SEQ ID NO: 14, or SEQ ID NO: 15,
b) the two scFv polypeptides are identical and each comprise an HCVR
operably linked to an LCVR, wherein each HCVR has the amino acid sequence
given in SEQ ID NO: 21, or SEQ ID NO: 22, and wherein each LCVR has the
amino acid sequence given in SEQ ID NO: 23, or SEQ ID NO: 24, and
c) the two linkers are identical glycine-rich linkers that each operably link the
carboxy-terminus of one HC of the antibody to the amino-terminus of one of the
scFv polypeptides.
In a further embodiment, the present invention provides a compound comprising
an antibody fused by two linkers to two scFv polypeptides, wherein the two scFv
polypeptides each comprise the carboxy-terminus of the LCVR of one scFv polypeptide
operably linked to the amino-terminus of the HCVR of one scFv polypeptide.
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein each HCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 1, each LCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 13, the HCVR of each of the
scFv polypeptides has the amino acid sequence given in SEQ ID NO: 21, and the LCVR
of each of the scFv polypeptides has the amino acid sequence given in SEQ ID NO: 23
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein each HCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 2, each LCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 14, the HCVR of each of the
scFv polypeptides has the amino acid sequence given in SEQ ID NO: 21, and the LCVR
of each of the scFv polypeptides has the amino acid sequence given in SEQ ID NO: 23.
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein each HCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 3, each LCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 15, the HCVR of each of the
scFv polypeptides has the amino acid sequence given in SEQ ID NO: 21, and the LCVR
of each of the scFv polypeptides has the amino acid sequence given in SEQ ID NO: 23.
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein each HCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 4, each LCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 13, the HCVR of each of the
scFv polypeptides has the amino acid sequence given in SEQ ID NO: 22, and the LCVR
of each of the scFv polypeptides has the amino acid sequence given in SEQ ID NO: 24.
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein the antibody comprises
two heavy chains (HCs) and two light chains (LCs), wherein each HC has the amino acid
sequence given in one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:
8, and each LC has the amino acid sequence given in one of SEQ ID NO: 16, SEQ ID
NO: 17, or SEQ ID NO: 18.
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein each HC of the antibody
has the amino acid sequence given in SEQ ID NO: 5, and each LC of the antibody has the
amino acid sequence given in SEQ ID NO: 16.
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein each HC of the antibody
has the amino acid sequence given in SEQ ID NO: 6, and each LC of the antibody has the
amino acid sequence given in SEQ ID NO: 17.
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein each HC of the antibody
has the amino acid sequence given in SEQ ID NO: 7, and each LC of the antibody has the
amino acid sequence given in SEQ ID NO: 18.
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein each HC of the antibody
has the amino acid sequence given in SEQ ID NO: 8, and each LC of the antibody has the
amino acid sequence given in SEQ ID NO: 16.
In an embodiment, the present invention provides a compound comprising an
antibody fused by two linkers to two scFv polypeptides, wherein each scFv polypeptide
has the identical amino acid sequence given in one of SEQ ID NO: 19 or SEQ ID NO: 20.
In an embodiment, the present invention provides a compound comprising an antibody
fused by two linkers to two scFv polypeptides, wherein each scFv polypeptide has the
amino acid sequence given in SEQ ID NO: 19. In an embodiment, the present invention
provides a compound comprising an antibody fused by two linkers to two scFv
polypeptides, wherein each scFv polypeptide has the amino acid sequence given in SEQ
ID NO: 20.
In an embodiment, the present invention provides a compound comprising two
first polypeptides and two second polypeptides wherein each of the first polypeptides has
the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID
NO: 12, and each of the second polypeptides has the amino acid sequence of SEQ ID NO:
16, SEQ ID NO: 17, or SEQ ID NO: 18. As shown in Table 1, the two first polypeptides
comprise the HC of the antibody, the linker, and the scFv polypeptide; the two second
polypeptides comprise the LC of the antibody.
In an embodiment, the present invention provides a compound comprising two
first polypeptides and two second polypeptides wherein each of the first polypeptides has
the amino acid sequence of SEQ ID NO: 9, and each of the second polypeptides has the
amino acid sequence of SEQ ID NO: 16. In an embodiment, the present invention
provides a compound comprising two first polypeptides and two second polypeptides
wherein each of the first polypeptides has the amino acid sequence of SEQ ID NO: 10,
and each of the second polypeptides has the amino acid sequence of SEQ ID NO: 17. In
an embodiment, the present invention provides a compound comprising two first
polypeptides and two second polypeptides wherein each of the first polypeptides has the
amino acid sequence of SEQ ID NO: 11, and each of the second polypeptides has the
amino acid sequence of SEQ ID NO: 18. In an embodiment, the present invention
provides a compound comprising two first polypeptides and two second polypeptides
wherein each of the first polypeptides has the amino acid sequence of SEQ ID NO: 12,
and each of the second polypeptides has the amino acid sequence of SEQ ID NO: 16.
In an embodiment, the present invention further provides a compound comprising
two first polypeptides and two second polypeptides wherein each of the first polypeptides
forms an inter-chain disulfide bond with each of the second polypeptides, and the first
polypeptide forms two inter-chain disulfide bonds with the other first polypeptide, and
each of the first polypeptides forms an intra-chain disulfide bond.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein:
a) the antibody comprises two identical heavy chains (HCs) and two identical
light chains (LCs), wherein each HC comprises a heavy chain variable region
(HCVR) whose amino acid sequence is given in SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, or SEQ ID NO: 4, and wherein each LC comprises a light chain
variable region (LVCR) whose amino acid sequence is given in SEQ ID NO: 13,
SEQ ID NO: 14, or SEQ ID NO: 15,
b) the two scFv polypeptides are identical and each comprise an HCVR
operably linked to an LCVR, wherein each HCVR has the amino acid sequence
given in SEQ ID NO: 21, or SEQ ID NO: 22, and wherein each LCVR has the
amino acid sequence given in SEQ ID NO: 23, or SEQ ID NO: 24, and
c) the two linkers are identical glycine-rich linkers that each operably link the
carboxy-terminus of one HC of the antibody to the amino-terminus of one of the
scFv polypeptides.
In a further embodiment, the present invention provides a compound that binds
human VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2
(SEQ ID NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2
(SEQ ID NO: 33), wherein the carboxy-terminus of the LCVR of each scFv polypeptide
is operably linked to the amino-terminus of the HCVR.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein each HCVR of the antibody has the amino acid sequence given in SEQ
ID NO: 1, each LCVR of the antibody has the amino acid sequence given in SEQ ID NO:
13, the HCVR of each of the scFv polypeptides has the amino acid sequence given in
SEQ ID NO: 21, and the LCVR of each of the scFv polypeptides has the amino acid
sequence given in SEQ ID NO: 23
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein each HCVR of the antibody has the amino acid sequence given in SEQ
ID NO: 2, each LCVR of the antibody has the amino acid sequence given in SEQ ID NO:
14, the HCVR of each of the scFv polypeptides has the amino acid sequence given in
SEQ ID NO: 21, and the LCVR of each of the scFv polypeptides has the amino acid
sequence given in SEQ ID NO: 23.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein each HCVR of the antibody has the amino acid sequence given in SEQ
ID NO: 3, each LCVR of the antibody has the amino acid sequence given in SEQ ID NO:
15, the HCVR of each of the scFv polypeptides has the amino acid sequence given in
SEQ ID NO: 21, and the LCVR of each of the scFv polypeptides has the amino acid
sequence given in SEQ ID NO: 23.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein each HCVR of the antibody has the amino acid sequence given in SEQ
ID NO: 4, each LCVR of the antibody has the amino acid sequence given in SEQ ID NO:
13, the HCVR of each of the scFv polypeptides has the amino acid sequence given in
SEQ ID NO: 22, and the LCVR of each of the scFv polypeptides has the amino acid
sequence given in SEQ ID NO: 24.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein the antibody comprises two heavy chains (HCs) and two light chains
(LCs), wherein each HC has the amino acid sequence given in one of SEQ ID NO: 5,
SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and each LC has the amino acid
sequence given in one of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein each HC of the antibody has the amino acid sequence given in SEQ ID
NO: 5, and each LC of the antibody has the amino acid sequence given in SEQ ID NO:
16.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein each HC of the antibody has the amino acid sequence given in SEQ ID
NO: 6, and each LC of the antibody has the amino acid sequence given in SEQ ID NO:
17.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein each HC of the antibody has the amino acid sequence given in SEQ ID
NO: 7, and each LC of the antibody has the amino acid sequence given in SEQ ID NO:
18.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein each HC of the antibody has the amino acid sequence given in SEQ ID
NO: 8, and each LC of the antibody has the amino acid sequence given in SEQ ID NO:
16.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 and human Ang2 comprising an antibody that binds human VEGFR2 (SEQ ID
NO: 32) fused by two linkers to two scFv polypeptides that bind human Ang2 (SEQ ID
NO: 33), wherein each scFv polypeptide has the identical amino acid sequence given in
one of SEQ ID NO: 19 or SEQ ID NO: 20. In an embodiment, the present invention
provides a compound that binds human VEGFR2 and human Ang2 comprising an
antibody that binds human VEGFR2 (SEQ ID NO: 32) fused by two linkers to two scFv
polypeptides that bind human Ang2 (SEQ ID NO: 33), wherein each scFv polypeptide
has the amino acid sequence given in SEQ ID NO: 19. In an embodiment, the present
invention provides a compound that binds human VEGFR2 and human Ang2 comprising
an antibody that binds human VEGFR2 (SEQ ID NO: 32) fused by two linkers to two
scFv polypeptides that bind human Ang2 (SEQ ID NO: 33), wherein each scFv
polypeptide has the amino acid sequence given in SEQ ID NO: 20.
In an embodiment, the present invention provides a compound that binds human
VEGFR2 (SEQ ID NO: 32) and human Ang2 (SEQ ID NO: 33) comprising two first
polypeptides and two second polypeptides wherein each of the first polypeptides has the
amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID
NO: 12, and each of the second polypeptides has the amino acid sequence of SEQ ID NO:
16, SEQ ID NO: 17, or SEQ ID NO: 18. In an embodiment, the present invention
provides a compound that binds human VEGFR2 (SEQ ID NO: 32) and human Ang2
(SEQ ID NO: 33) comprising two first polypeptides and two second polypeptides wherein
each of the first polypeptides has the amino acid sequence of SEQ ID NO: 9, and each of
the second polypeptides has the amino acid sequence of SEQ ID NO: 16. In an
embodiment, the present invention provides a compound that binds human VEGFR2
(SEQ ID NO: 32) and human Ang2 (SEQ ID NO: 33) comprising two first polypeptides
and two second polypeptides wherein each of the first polypeptides has the amino acid
sequence of SEQ ID NO: 10, and each of the second polypeptides has the amino acid
sequence of SEQ ID NO: 17. In an embodiment, the present invention provides a
compound that binds human VEGFR2 (SEQ ID NO: 32) and human Ang2 (SEQ ID NO:
33) comprising two first polypeptides and two second polypeptides wherein each of the
first polypeptides has the amino acid sequence of SEQ ID NO: 11, and each of the second
polypeptides has the amino acid sequence of SEQ ID NO: 18. In an embodiment, the
present invention provides a compound that binds human VEGFR2 (SEQ ID NO: 32) and
human Ang2 (SEQ ID NO: 33) comprising two first polypeptides and two second
polypeptides wherein each of the first polypeptides has the amino acid sequence of SEQ
ID NO: 12, and each of the second polypeptides has the amino acid sequence of SEQ ID
NO: 16.
In an embodiment, the present invention further provides a compound that binds
human VEGFR2 (SEQ ID NO: 32) and human Ang2 (SEQ ID NO: 33) comprising two
first polypeptides and two second polypeptides wherein each of the first polypeptides
forms an inter-chain disulfide bond with each of the second polypeptides, and the first
polypeptide forms two inter-chain disulfide bonds with the other first polypeptide, and
each of the first polypeptides forms an intra-chain disulfide bond.
In a further embodiment, the present invention provides a compound of the
present invention that has a dissociation equilibrium constant, K , of about 300 pM to
about 1400 pM for human VEGFR2 and a KD of about 300 pM to about 800 pM for
human Ang2. The compound of the present invention is further characterized by a kon
rate to human VEGFR2 of about 0.1 x 105M to about 0.5 x 105 M^sec and a kon
rate to human Ang2 of about 1 x 105 M^sec to about 3 x 105 M^sec 1 . The compound of
the present invention is further characterized by a k ff rate to human VEGFR2 of about
0.1 x 10 4 sec 1 to about 0.5 x 10 4 sec and a k0 rate to human Ang2 of about 0.8 x 10
sec 1 to about 1.2 x 10 4 sec 1 . The KD, kon, and k values are established by binding
kinetics at 25°C as described in "Binding kinetics, affinity, and selectivity" in the Assays
section.
The compound of the present invention binds to human VEGFR2 and human
Ang2 with high affinity. For the purposes of the present disclosure, the term "high
affinity" refers to a K of less than about 1500 pM for human VEGFR2 and of less than
about 1000 pM for human Ang2. The K values are established by binding kinetics at
25°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 a encoding a polypeptide selected
from the group consisting of a polypeptide consisting of SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11 and SEQ ID NO: 12 and a polynucleotide sequence encoding a
polypeptide selected from the group consisting of a polypeptide consisting of SEQ ID
NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, wherein the cell is capable of expressing a
compound comprising a first polypeptide having an amino acid sequence selected from
the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID
NO: 12 and a second polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
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: 9 and a polynucleotide sequence encoding a
polypeptide having an amino acid sequence of SEQ ID NO: 16, wherein the cell is
capable of expressing a compound comprising a first polypeptide having an amino acid
sequence of SEQ ID NO: 9 and a second polypeptide having an amino acid sequence of
SEQ ID NO: 16.
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: 10 and a polynucleotide sequence encoding a
polypeptide having an amino acid sequence of SEQ ID NO: 17, wherein the cell is
capable of expressing a compound comprising a first polypeptide having an amino acid
sequence of SEQ ID NO: 10 and a second polypeptide having an amino acid sequence of
SEQ ID NO: 17.
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: 11 and a polynucleotide sequence encoding a
polypeptide having an amino acid sequence of SEQ ID NO: 18, wherein the cell is
capable of expressing a compound comprising a first polypeptide having an amino acid
sequence of SEQ ID NO: 11 and a second polypeptide having an amino acid sequence of
SEQ ID NO: 18.
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: 12 and a polynucleotide sequence encoding a
polypeptide having an amino acid sequence of SEQ ID NO: 16, wherein the cell is
capable of expressing a compound comprising a first polypeptide having an amino acid
sequence of SEQ ID NO: 12 and a second polypeptide having an amino acid sequence of
SEQ ID NO: 16.
In an embodiment, the present invention provides a process for producing a
compound comprising two first polypeptides selected from the group consisting of SEQ
ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and two second
polypeptides selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and
SEQ ID NO: 18, comprising cultivating the mammalian cell of the present invention
under conditions such that the compound is expressed, and recovering the expressed
compound.
In an embodiment, the present invention provides a process for producing a
compound comprising two first polypeptides of SEQ ID NO: 9 and two second
polypeptides of SEQ ID NO: 16, comprising cultivating the mammalian cell of the
present invention under conditions such that the compound is expressed, and recovering
the expressed compound.
In an embodiment, the present invention provides a process for producing a
compound comprising two first polypeptides of SEQ ID NO: 10 and two second
polypeptides of SEQ ID NO: 17, comprising cultivating the mammalian cell of the
present invention under conditions such that the compound is expressed, and recovering
the expressed compound.
In an embodiment, the present invention provides a process for producing a
compound comprising two first polypeptides of SEQ ID NO: 11 and two second
polypeptides of SEQ ID NO: 18, comprising cultivating the mammalian cell of the
present invention under conditions such that the compound is expressed, and recovering
the expressed compound.
In an embodiment, the present invention provides a process for producing a
compound comprising two first polypeptides of SEQ ID NO: 12 and two second
polypeptides of SEQ ID NO: 16, comprising cultivating the mammalian cell of the
present invention under conditions such that the compound is expressed, and recovering
the expressed compound.
In an embodiment of the above-described processes, the two polynucleotide
sequences in the mammalian cell of the present invention are part of the same nucleic
acid molecule.
In an embodiment, the present invention provides a compound obtainable by one
of the aforementioned processes.
In an embodiment, the present invention provides a pharmaceutical composition,
comprising a compound 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 a compound
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 a compound of the present invention, wherein the cancer is breast
cancer, ovarian cancer, gastric cancer, colorectal cancer, or hepatocellular carcinoma. 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 a compound
of the present invention, wherein the cancer is breast cancer, ovarian cancer, gastric
cancer, colorectal cancer, non-small cell lung cancer, biliary tract cancer, or
hepatocellular carcinoma. 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 anti-tumor agents
selected from the group consisting of cisplatin, carboplatin, liposomal doxorubicin,
docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel proteinbound
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 a further embodiment, the present invention provides a method of treating
cancer, comprising administering to a patient in need thereof, an effective amount of a
compound of the present invention in simultaneous, separate, or sequential combination
with one or more immuno-oncology agents, wherein the cancer is bladder cancer, kidney
cancer, prostate cancer, or testicular cancer, and wherein the immuno-oncology agents are
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 a
compound of the present invention. In a further embodiment, these methods of treating
breast cancer comprise the administration of an effective amount of the compound of the
present invention in simultaneous, separate, or sequential combination with one or more
anti-tumor agents selected from the group consisting of 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 a
compound of the present invention. In a further embodiment, these methods of treating
ovarian cancer comprise the administration of an effective amount of the compound 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, 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 a
compound of the present invention. In a further embodiment, these methods of treating
gastric cancer comprise the administration of an effective amount of the compound 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 a compound of the present invention. In a further embodiment, these
methods of treating hepatocellular carcinoma comprise the administration of an effective
amount of the compound 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 a
compound of the present invention. In a further embodiment, these methods of treating
colorectal cancer comprise the administration of an effective amount of the compound of
the present invention in simultaneous, separate, or sequential combination with one or
more anti-tumor agents selected from the group consisting of FOLFOX (leucovorin,
fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), and
cetuximab.
In an embodiment, the present invention provides a compound of the present
invention, for use in therapy. In an embodiment, the present invention provides a
compound of the present invention, for use in the treatment of cancer. In a further
embodiment, the present invention provides a compound 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, the
present invention provides a compound of the present invention, for use in the treatment
of cancer, wherein the cancer is breast cancer, ovarian cancer, gastric cancer, colorectal
cancer, non-small cell lung cancer, biliary tract cancer, or hepatocellular carcinoma. 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 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 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, wherein the cancer is bladder cancer, kidney cancer, prostate
cancer, or testicular cancer, and wherein the immuno-oncology agents are selected from
the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, and
durvalumab.
In an embodiment, the present invention provides a compound 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 compound of the present invention in simultaneous,
separate, or sequential combination with one or more anti-tumor agents selected from the
group consisting of docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin,
paclitaxel protein-bound particles for injectable suspension, ixabepilone, and
capecitabine.
In an embodiment, the present invention provides a compound 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 compound 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, and liposomal doxorubicin.
In an embodiment, the present invention provides a compound 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 compound of the present invention in simultaneous,
separate, or sequential combination with ramucirumab.
In an embodiment, the present invention provides a compound 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 compound of the present
invention in simultaneous, separate, or sequential combination with one or more anti
tumor agents selected from the group ramucirumab.
In an embodiment, the present invention provides a compound 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 compound of the present invention in
simultaneous, separate, or sequential combination with one or more anti-tumor agents
selected from the group consisting of FOLFOX (leucovorin, fluorouracil, and
oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), and cetuximab.
In an embodiment, the present invention provides the use of a compound 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 a compound 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 a
compound 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, non-small cell lung cancer, biliary tract cancer, or hepatocellular carcinoma.
In a further embodiment , the present invention provides the use of a compound 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.
A compound 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 a compound of
the present invention.
The antibody portion of the compound 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 naturallyoccurring
somatic mutations and those with engineered mutations. The antibody portion
of the compound 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, the antibody
portion of the compound of the present invention is preferably substantially nonimmunogenic
in humans.
The antibody portion of the compound of the present invention is an IgG type
antibody and has four amino acid chains (two "heavy" chains and two "light" chains) that
are covalently stabilized via intra- and 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 N-glycosylation site. N-glycans typically attach to
asparagine. Antibodies may be glycosylated at other positions as well.
Optionally, the antibody portion of the compound of the present invention
contains an Fc portion which is derived from human IgG4 Fc region because of a reduced
ability to engage Fc receptor-mediated inflammatory mechanisms or to activate
complement resulting in reduced effector function.
Further, the antibody portion of certain compounds of the present invention
contains an IgG4-PAA Fc portion. The IgG4-PAA Fc portion has a serine to proline
mutation at position 224, a phenylalanine to alanine mutation at position 230, and a
leucine to alanine mutation at position 231. The S224P mutation is a hinge mutation that
prevents half-antibody formation (phenomenon of dynamic exchange of half-molecules in
IgG4 antibodies). The F230A and L231A mutations further reduce effector function of
the already low human IgG4 isotype.
An isolated DNA molecule encoding a HCVR region can be converted to a fulllength
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.
A "single chain fragment variable" or "scFv" or "scFv polypeptide" refers to an
engineered, non-naturally occurring single folded polypeptide comprising the LCVR and
the HCVR of an antibody linked through a scFv linker molecule. The scFv polypeptide
portion of the compound of the present invention is an engineered, non-naturally
occurring scFv that has been designed to have engineered CDRs and have some portions
of the scFv (all or parts of the frameworks) to be of human origin that are identical with
or substantially identical (substantially human) with frameworks derived from human
genomic sequences. Fully human frameworks are those human germline sequences as
well as sequences with naturally-occurring somatic mutations and those with engineered
mutations. The scFv polypeptide portion of the compound of the present invention may
comprise framework derived from a fully human framework containing one or more
amino acid substitutions, deletions, or additions therein. Further, the scFv polypeptide
portion of the compound of the present invention is preferably substantially nonimmunogenic
in humans. Optionally, the scFv polypeptide portion of the compound can
have 44-100 disulfides from cysteine 44 in HCVR and cysteine 100 in LCVR (Cys44 and
CyslOO numbering corresponds with a numbering that starts with the first amino acid of
the scFv polypeptide). In such a scFv polypeptide, the HCVR and LCVR domains can be
either in the HCVR - scFv linker - LCVR or LCVR - scFv linker - HCVR order. The
scFv linker can be a flexible glycine-rich peptide linker which enables the HCVR and
LCVR chains to be folded as a functional monomeric unit for recognizing an antigen.
Optionally, the scFv linker is a glycine-rich linker such as a 2x G4S linker, a 3x G4S
linker, a 4x G4S linker, or a 5x G4S linker.
Fusion of a scFv to an antibody can allow for multiple structures to form during
expression and secretion. Firstly, scFv elements fused to an antibody can fold
independently via an intramolecular interaction by which individual HCVR and LCVR
elements located within the same polypeptide fold to form two separate and autonomous
units, referred here to as Mab-scFv. Secondly, via an alternative folding pathway, scFv
elements fused to an antibody can fold via intermolecular interactions by which a HCVR
present in one polypeptide will fold with a LCVR present in the adjacent polypeptide to
form a single co-folded species, here referred to as Mab-Diabody.
The term "linker" and "scFv linker" both refer to glycine-rich peptide linkers.
The "linkers" are utilized in certain embodiments of the invention to link the antibody to
the scFv, and the "scFv linkers" are utilized in certain embodiments of the invention to
link the LCVR of the scFv to the HCVR of the scFv. Preferably, the peptide linkers are
glycine-rich peptides with at least 5 amino acids, preferably of at least 10 amino acids,
more preferably between 10 and 50 amino acids. In some embodiments of the present
invention, said glycine-rich peptide linker is (GxS) with G=glycine, S=serine, (x=3 and
n=3, 4, 5 or 6) or (x=4 and n=2, 3, 4 or 5). For example, in some embodiments of the
present invention, said glycine-rich peptide linker is (GxS) with G=glycine, S=serine,
x=4 and n=2, 3, 4 or 5 (i.e., GGGGSGGGGS (SEQ ID NO: 34),
GGGGSGGGGSGGGGS (SEQ ID NO: 35), GGGGSGGGGSGGGGSGGGGS (SEQ ID
NO: 36), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 37), respectively.
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 compound 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 compound 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 compound, 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 compound of the present invention, or pharmaceutical compositions
comprising the same, may be administered by parenteral routes (e.g., subcutaneous and
intravenous). A compound 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 a compound, 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. A patient refers to a mammal,
preferably a human with a disease, disorder, or condition that would benefit from
inhibition of VEGFR2 and/or Ang2.
"Binds" as used herein in reference to the affinity of a compound, antibody, or
scFv polypeptide for human VEGFR2 or human Ang2 is intended to mean, unless
indicated otherwise, a K of less than about 1 x 10~8 M , preferably, less than about 1 x 10
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 K D 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 compound 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 a compound of the present invention or
pharmaceutical composition comprising a compound 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 compound may vary according to factors
such as the disease state, age, sex, and weight of the individual, and the ability of the
compound to elicit a desired response in the individual. An effective amount is also one
in which any toxic or detrimental effect of the compound is outweighed by the
therapeutically beneficial effects.
This invention is further illustrated by the following non-limiting examples.
Example 1: Compound expression and purification
The polypeptides of the antibody portions, the scFv portions, and the antibodylinker-
scFv of Compound C, Compound D, Compound E, and Compound F, 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 antibody portions,
the scFv portions, and the antibody-linker-scFv of Compound C, Compound D,
Compound E, and Compound F are shown in Table 1.
The compounds of the present invention, including, but not limited to Compound
C, Compound D, Compound E, and Compound F, 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 compounds using an optimal
predetermined HC-linker-scFv:LC vector ratio (such as 1:3 or 1:2) or a single vector
system encoding both HC-linker-scFv and LC. Clarified media, into which the
compound 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 compound 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). Compound fractions may be detected, such as by SDSPAGE,
and then may be pooled. Further purification is optional, depending on the
intended use. The compound 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 compound 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
Compound C Compound D Compound E Compound F
HCVR of antibody 1 2 3 4
HC of antibody 5 6 7 8
HC of antibody + 9 10 11 12
linker + scFv
polypeptide
LCVR of antibody 13 14 15 13
LC of antibody 16 17 18 16
scFv polypeptide 19 19 19 20
HCVR of scFv 2 1 2 1 2 1 22
polypeptide
LCVR of scFv 23 23 23 24
polypeptide
Assays
Binding kinetics, affinity, and selectivity
The binding kinetics, affinity, and selectivity to human Ang2 and to human
VEGFR2, for compounds of the present invention, may be determined 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.
The kinetics and equilibrium dissociation constant (¾) for multiple species of
soluble Ang2 (human, cyno, mouse, rabbit and dog) and VEGFR2 extracellular domain
(ECD) (human, cyno, rabbit and dog) may be determined for compounds of the present
invention at 25°C or 37°C using Biacore surface plasmon resonance methods. Human
Ang2 and VEGFR2-ECD may be purchased from R&D Systems and Sino Biological,
respectively. 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 10 mE/min. After which, soluble protein A may be diluted to 50-100 g/mL 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 10 mE/min. Un-reacted sites still remaining on the chip surface may
be blocked with a seven minute injection of ethanolamine at 10 mE/min. Running buffer
may be HBS-EP+ (Biacore # BR- 1006-69). Compound samples may be prepared at 1
g/mL by dilution into running buffer. Discrete concentrations of Ang2 ligands ranging
from 50 nM to 1.56 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
compound onto separate flow cells (Fc2, Fc3, and Fc4), (2) injection (using kinject) of
250 E (300-second surface contact time) of discrete concentrations of Ang2 or
VEGFR2-ECD over all Fc at 50 mE/min, (3) return to buffer flow for 20 minutes to
monitor dissociation phase, (4) regeneration of chip surfaces with a 10 E (30-second
contact time) injection of 10 mM glycine, pH1.5, (5) equilibration of chip surface with a
15 E (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
V1units), dissociation rate (k ff, s units). Calculation of the equilibrium dissociation
constant (KD) may be calculated from the following relationship, K = k0ff/kon, and is
presented in molar units.
In experiments performed essentially as described in this assay, Compound C,
Compound D, Compound E, and Compound F bind human Ang2 at 25°C with a K in the
range of 407 pM to 673 pM (Table 2). Compound C, Compound D, Compound E, and
Compound F bind human VEGFR2-ECD at 25°C with a KD in the range of 528 pM to
1110 pM (Table 3). These compounds of the present invention demonstrate high affinity
for both human Ang2 and human VEGFR2.
In experiments performed essentially as described in this assay, Compound F
exhibited comparable binding kinetics and affinities for human Ang2 relative an Ang2
antibody with the same HCDRs and LCDRs as the scFv polypeptide portion of
Compound F (Tables 4 and 5). This demonstrates that the potent binding kinetics of the
Ang2 antibody with the same HCDRs and LCDRs as the scFv polypeptide portion of
Compound F is retained in Compound F.
In experiments performed essentially as described in this assay, Compound F
binds human, cynomolgus monkey, mouse rabbit and dog Ang2 with comparable high
affinities, and Compound F binds human, cynomolgus monkey, rabbit and dog VEGFR2
with comparable high affinities.
Table 2
Biacore SPR hAng2 at 25°C
kon (10 1/Ms) koff (10 4 l/s) KD (pM)
Compound A 1.83 <0.1 <100pM
Compound B 2.85 <0.1 <100pM
Compound C 2.26 1.05 462
Compound D 1.75 0.86 489
Compound E 2.39 0.97 407
Compound F 1.56 1.05 673
Table 3
Biacore SPR hVEGFR2-ECD at 25°C
kon (10 1/Ms) koff (10 4 l/s) KD (pM)
Compound A 0.68 0.48 711
Compound B 0.81 0.41 513
Compound C 0.38 0.20 528
Compound D 0.34 0.29 860
Compound E 0.32 0.35 1110
Compound F 0.25 0.18 721
Table 4
Table 5
Compound F Biacore SPR Ang2 Binding at 37°C (n=3)
kon (1/Ms) k0ff(l/s) KD (pM)
human Ang2 8.2 (+ 2.4) x 105 0.7 (+ 0.3) x 10 4 100 + 70
cyno Ang2 9.6 (+ 1.3) x 105 2.8 (+ 0.1) x 10 4 300 + 30
mouse Ang2 3.6 (+ 0.9) x 105 0.8 (+ 0.1) x 10 4 230 + 20
rabbit Ang2 2.7 (+ 0.7) x 105 1.1 (+ 0.1) x 10 4 420 + 60
dog Ang2 3.6 (+ 1.6) x 105 0.9 (+ 0.3) x 10 4 240 + 30
Table 6
Compound F Biacore SPR VEGFR2-ECD Binding at 37°C (n=3)
kon (1/Ms) koff (l/s) KD (nM)
human VEGFR2 2.5 (+ 0.1) x 104 1.2 (+ 0.2) x 10 4 4.6 + 0.7
cyno VEGFR2 3.4 (+ 0.2) x 104 1.1 (+ 0.1) x 10 4 3.3 + 0.4
rabbit VEGFR2 3.8 (+ 0.5) x 104 2.3 (+ 0.1) x 10 4 6.1 + 1.0
dog VEGFR2 4.1 (+ 0.3) x 104 1.5 (+ 0.7) x 10 3 37 + 1
Inhibition of human Ang2 to human Tie2 interaction
The blocking of human Ang2 binding to its receptor human Tie 2 by a compound
of the present invention may be measured in a solid phase in vitro ELISA assay. The
aforementioned in vitro cell-based assay may also be used to establish comparable
blocking activity of a compound of the present invention to an Ang2 antibody with the
same HCDRs and LCDRs sequences as the scFv polypeptide portion of the compound.
For the assay, high binding 96-well ELISA plates (Costar #2592) may be coated
with 4 g/ml (in 100 ul) 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 per 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, Compound F and
an Ang2 antibody with the same HCDRs and LCDRs as the scFv polypeptide portion of
Compound F result in geometric mean IC50 values (n=2) of 0.034 nM and 0.027 nM
respectively. Compound F dose dependently blocks human Ang2 binding to human Tie-
2 comparably to the Ang2 antibody with the same HCDRs and LCDRs as the scFv
polypeptide portion of Compound F. This data indicates that the Ang2 scFv polypeptide
portion of the compound has maintained potency in this assay that is comparable to that
of the Ang2 antibody.
Neutralization of Ang2 induced phosphorylation of Tie2, but not Angl mediated
phosphorylation.
The in vitro cell-based inhibition of human Ang2 by a compound of the present
invention may be measured in a cell-based assay where Angl and Ang2 bind to and
induce human Tie2 phosphorylation in a dose-dependent manner. The in vitro cell-based
assay may be used to evaluate the ability of compounds of the present invention to
selectively neutralize Ang2 and not Angl mediated phosphorylation of the Tie-2 receptor
in a dose-dependent manner. An Ang2 antibody, an Angl antibody, and a control human
IgG4 PAA isotype antibody may be included as positive and negative controls
respectively.
The CHO-Tie2 cell line may be generated by stable transfection of a full-length
human Tie2 receptor (with a 3X FLAG tag at the C-terminus). CHO-Tie2 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 antibiotic -
antimycotic (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-Tie2 cells may be resuspended to 10,000 cells per well (in
lOOul 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, compounds (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. Compounds
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 compound/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 compounds may be 0.0625 - 283 nM, and the final concentration of human
Ang2 and Angl may be 0.3 g/ml (approx. 6 nM) and 0.5 g/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; 1 mM
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-Tie2 (with a human phospho-Tie2 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
Tie2 capture antibody in IX ELISA coating buffer (Surmodics/BioFX Labs #COAT-
1000-01).
The day of phospho-Tie2 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-Tie2 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-Tie2 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-Tie2 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, Compound F
dose-dependently neutralizes human Ang2 induced phospho-Tie2 in CHO-Tie2 cells with
an IC50 of 0.587 nM (n=3) while an Ang2 antibody with the same HCDRs and LCDRs as
the scFv polypeptide portion of Compound F has an IC50 of 0.773 nM. The results
indicate that Compound F neutralizes Ang2 induced phospho-Tie2, but does not
neutralize human Angl induced phospho-Tie2 in CHO-Tie-2 cells when compared to the
positive control Angl antibody. Moreover, this data indicates that the Ang2 scFv
polypeptide portion of Compound F has maintained potency in this assay that is
comparable to that of the Ang2 antibody with the same HCDRs and LCDRs as the scFv
polypeptide portion of Compound F.
Neutralization of VEGF165 induced phosphorylation of VEGFR2
The in vitro cell-based inhibition of human VEGFR2 may be measured in a cellbased
assay where binding of VEGF165 to the VEGFR2 on a VEGFR2 expressing cell
line, induces VEGFR2 phosphorylation in a dose-dependent manner. The
aforementioned assay may be used to evaluate the ability of a compound of the present
invention to selectively neutralize VEGF165 mediated phosphorylation of the VEGFR2
receptor in a dose-dependent manner. A VEGFR2 antibody and an irrelevant antibody
human IgG4 PAA isotype may be included as a positive and negative control,
respectively.
For the assay, VEGFR2 expressing human ECFC (endothelial colony forming
cells, derived from umbilical cord blood endothelial progenitors) (Endgenitor
Technologies, Lot 100506- 14-P4, passages 10-12) may be seeded at 14,000 cells per well
(in 100 mΐ growth medium), into the inner 60 wells of collagen I coated 96-well plates
(BD Biocoat #35-4407) in growth medium: EGM-2MV BulletKit (Lonza #CC-4147).
Components of the included EGM-2MV Singlequot bag may be added to 500 ml of
EBM-2 basal medium, adjusted to 10% final FBS concentration (Life
Technologies/Invitrogen #10082-147, heat inactivated). 250 mΐ of growth medium may
be placed into the edge wells to reduce evaporation. Cells may be incubated ON at 37°C,
95% RH, 5% C0 2. The next day, medium may be removed and replaced with 100 mΐ
serum-free EBM-2 basal medium containing 0.1% BSA (Sigma #A7979, low endotoxin).
Cells may be starved for 6 ½ hours at 37°C, 95% RH, 5% C0 2. During the starvation
period, compounds (at 6X the final concentrations) may be serially diluted 1:4 in
polypropylene plates in EBM-2/0.1% BSA. Human VEGF165 may be diluted to 6X the
final concentration in EBM-2/ 0.1% BSA. Compounds (or EBM-2/0. 1% BSA medium
alone) may be added in triplicate at 25 mΐ to the cells, and cells may be incubated for 45
minutes at 37°C, 95% RH, 5% C0 2. Cells may be treated with 25 mΐ of 6X VEGF165 for
5 minutes at 37°C, 95% RH, 5% C0 2. (The final concentration range of compounds may
be 0.018 - 300 nM, and the final concentration of human VEGF165 may be 0.16 nM).
Medium may be 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, 1 mM EGTA, 1% Triton X-100) containing freshly added IX protease
and phosphatase inhibitors (included with the phospho-VEGFR2 assay kit). Plates may
be placed on ice for 10 minutes, then on an orbital shaker at low speed for 20 minutes at
4°C. Plates may then be sealed and frozen at -80°C.
The day of phospho-VEGFR2 measurement, plates containing lysates may be
thawed on ice. Phospho-VEGFR2 levels may be measured using a phospho-VEGFR2
(Tyrl054) whole cell lysate kit, (Meso Scale Discovery #K151DJD). Meso Scale assay
plates, pre-coated with an antibody against phospho-VEGFR2, may be blocked with 150
mΐ per well of blocking buffer (3% blocker A in TBST) for a minimum of 1 hour at room
temperature on an orbital shaker (while covered with plate sealers). The plates may be
washed 3X with IX Meso Scale wash buffer, and 50 mΐ of lysates may be added per well
(may be incubated for 1 hour at room temperature, covered with sealers, on an orbital
shaker). The plates may be washed, and 3X, 25 mΐ per well of IX MSD SULFO-TAG™
conjugated anti-total VEGFR2 (diluted in the manufacturer's recommended antibody
diluent) may be added, and incubated for 1 hour at room temperature, covered with
sealers, on an orbital shaker. Plates may be washed 3X and removal of liquid from the
wells may be ensured. 150 ul per well of IX Read Buffer T may be added to the plates,
and they may be read immediately on a Meso Scale Discovery SECTOR Imager
MA6000. Percent inhibition may be calculated by the following formula: (signal value of
treatment-mean signal value of VEGF+huIgG treatment) / (mean starve medium alone
signal value-mean signal value of VEGF+huIgG treatment)* 100.
IC50 values for inhibition of VEGF165 induced phospho-VEGFR2 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 log-transformed 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, Compound F dose
dependently neutralizes human VEGF165-induced phospho-VEGFR2 in ECFC with an
IC50 mean of 0.83 nM (n=3), while IMC-1121B has an IC50 mean of 0.52 nM (n=3).
This indicates that the VEGFR2 antibody portion of the compound has maintained
potency in Compound F that is comparable to that of IMC- 112 IB in this cell based assay.
Neutralization of VEGF165 induced cell proliferation.
The in vitro cell-based inhibition of human VEGFR2 by a compound of the
present invention may be measured in a cell-based assay where VEGF165 induces human
VEGFR2 proliferation in a dose-dependent manner. The ability of a compound of the
present invention to neutralize human VEGF165 induced proliferation via VEGFR2 may
be measured in human HMVEC-d (dermal microvascular endothelial cells). VEGFR2,
Ang2, and irrelevant human IgG4 PAA antibodies may be included as positive and
negative controls.
Human dermal microvascular endothelial cells may be isolated from newborn
male foreskin, and may be confirmed for CD31, VEGFR2, and acetylated LDL
expression. HMVEC-d may be maintained in complete growth medium MCDB13 1
(Mediatech #15-100-CV), 2 mM L-glutamine (Thermo Scientific #SH30034.01), IX
penicillin-streptomycin (Life Technologies/Invitrogen #15140), MGVS supplement (Life
Technologies/Invitrogen #S-005-25) as directed for 500 ml medium; which may be
supplemented to contain 4.9% FBS, 1 g/ml hydrocortisone, 3ng/ml human FGF, 10
g/ml heparin, 1 ng/ml human EGF, and 0.08 mM dibutyryl cyclic AMP.
For the assay, HMVEC-d cells at passage 5 may be washed once in pre-warmed
growth medium, and may be resuspended to 2,000 cells per well (in 100 mΐ growth
medium), into the inner 60 wells of white-walled, clear bottom 96-well plates (BD #35-
3377). 250 mΐ of supplement-free medium may be added to the edge wells to reduce
evaporation. Compounds and human VEGF165 (Lot ALY-BE01241-033) at 4X the final
concentrations may be serially diluted 1:4 in polypropylene plates in supplement-free
medium. Compounds (or supplement-free medium alone) may be added to the cells in
triplicates at 50 mΐ per well, which may be followed by 50 mΐ per well of the 4X
VEGF165. The plates may then be incubated for 5 days at 37°C, 95% RH, 5% C02. The
final concentration range of compounds may be 0.012 - 800 nM, and the final
concentration of human VEGF165 may be 0.5 nM.
After the incubation period, the plates and CellTiter Glo substrate (Promega
#G7571) may be equilibrated to room temperature for 30 minutes. 100 mΐ per well of
CellTiter Glo reagent may be added, and plates may be placed on an orbital shaker for 2
minutes at room temperature. Plates may be incubated for an additional 10 minutes, then
luminescence may be recorded ( 1 second integration time) on a Perkin Elmer Wallac
Victor 3 Model 1420 reader.
IC50 values for inhibition of VEGF165 induced proliferation may be calculated
with GraphPad Prism 6, using Log-transformed X values. Medium alone values may be
included as the highest point of curves; the X-value (concentration) for medium alone
may be set to 100X higher than the highest X value. Also, the VEGF alone values may
be included as the lowest point of the curves; the concentration for VEGF alone may be
set to 100X lower than the lowest X value. Nonlinear regression (curve fit) analysis
(sigmoidal dose response, variable slope) may be performed on the log-transformed data
to obtain IC50 values.
In experiments performed essentially as described in this assay, Compound F dose
dependently neutralizes human VEGF165-induced proliferation of HMVEC-d similarly
to IMC-1121B with IC50 means of 19.24 nM and 31.36 nM, respectively (n=2). This
indicates that the VEGFR2 antibody portion of the compound has maintained potency in
Compound F that is comparable to IMC-1121B in this cell based assay.
Inhibition of VEGF165 Induced Cord Formation
The in vitro inhibition of VEGF induced cord formation may be measured in an in
vitro co-culture system. The aforementioned assay may be used to measure inhibition of
VEGF induced cord formation by a compound of the present invention. A VEGFR2
antibody may be included as a positive control.
For this assay, adipose derived stem cells (ADSC; Lonza # PT5006, lot#OF4505-
01) may be cultured on Corning culture flasks (Corning #431082) in EGM-2MV medium
(Lonza # CC3202). Endothelial colony forming cells (ECFC; Lonza, lot# EGTECFC100506r)
may be cultured on Collagen I coated flasks (BD Biosciences #356486) in
EGM-2MV medium supplemented with 5% heat inactivated FBS ( Gibco # 10082-147).
ADSC at passages 4-6 may be harvested from culture flasks which may be rinsed with
DPBS (Hyclone #SH30028.03) followed by TrypLE Express (Gibco #12605-010).
ADSC cells may be suspended in Basal Medium (MCDC-131 (Gibco #10372-019)
supplemented with 10 g/ml insulin, 1 mM dexamethasone, 30 g/ml ascorbic acid, 10
g/ml human transferrin and 50 mg/ml tobramycin). Viable cell count may be determined
and cells may be seeded onto black, clear bottomed 96-well plates (BD Falcon #353219)
at 4xl0 4 cells per well in 100 mΐ Basal Medium. Cells may be incubated at 37°C in 5%
C0 2 overnight to allow attachment. Next day, ECFC at passages 7-10 may be harvested
in Basal Medium as above and viable cell count may be adjusted to 4xl0 4 cells per ml.
Medium may be removed from ADSC cells and 100 mΐ ECFC cell suspension may be
added to each well. Plates may be incubated at 37°C in 5% C0 2 for 2-3 hours to allow
cells to settle on top of the ADSC monolayer. IMC-1 121B and compounds of the present
invention may be diluted to 80 mg/ml in Basal Medium, and then may be serially diluted
1:3 with Basal Medium to produce a nine point dose response series. 50 mΐ of each
dilution of compound may be added to the co-culture. 50 mΐ of an 80 ng/ml solution of
rhVEGF (R&D #293-VE/CF, 50 mg/ml in DPBS) prepared in Basal Medium may be
added to the co-culture + compound combination. Final concentrations for compounds
and rhVEGF may be 20 mg/ml and 20 ng/ml respectively. Positive control for the assay
may include 20 ng/ml rhVEGF in the absence of compound. Negative control for the
assay may include Basal Medium without rhVEGF. Plates may then be incubated at 37°C
in 5% CO2 for 3 days to allow cords to form.
At the end of the incubation period, medium may be aspirated from each well and
lOOul room temperature 80% ethanol may be carefully added. Plates may be incubated at
room temperature for 20 minutes. Ethanol solution may be aspirated and wells may be
washed twice with 150ul DPBS. Anti-huCD31 (R&D #AF806 Affinity purified sheep
IgG, 200ug/ml) and MAB Anti-Actin, alpha-Smooth Muscle-Cy3 (Sigma #C6198) may
be each diluted 1:250 in 2.5% FBS/DPBS. 100 mΐ antibody mix may be added to wells
and plates may be incubated at 37°C in 5% C0 2 for 2 hours. Plates may then be aspirated
and wells may be washed twice with 150ul DPBS. Alexa Fluor 488 donkey anti-sheep
IgG (H+L) (Life Technologies #A11015) may be diluted 1:400 and Hoescht 33342 (Life
Technologies #H3570) may be diluted 1:1000 in 2.5% FBS/DPBS and 100 mΐ per well
may be added to plates. Plates may be incubated at room temperature protected from
light for 30 minutes. Wells may then be washed twice with 150 mΐ DPBS. 150 mΐ DPBS
may be added to each well and plates may be sealed with black adhesive seals
(PerkinElmer #6050173).
Plates may be read on the ArrayScan VTI HCS Reader (Cellomics-Thermo
Fisher) using the Tube Formation Bio-application. Total Tube Area data may be plotted
against compound concentrations in nM in GraphPad Prism 6. Compound concentrations
may be transformed into log data and IC50 values for inhibition may be calculated by
nonlinear regression (sigmoidal dose response, variable slope). Each experiment may
represent the mean of triplicates and triplicate experiments may be expressed as the
geometric means and 95% confidence intervals may be calculated.
In experiments performed essentially as described in this assay, Compound F dose
dependently inhibits human VEGF-induced cord formation in the ADSC/ECFC coculture
system, comparably to IMC-1121B with mean IC50 of 6.04 nM and 5.6 nM,
respectively (n=3). This indicates that the VEGFR2 antibody portion of the compound
has maintained potency in Compound F that is comparable to IMC-1 121B in this cell
based assay.
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 compounds 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 P0 (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 Ang2 antibody or 13.5 mg/kg
of the compound to maintain comparable molar amounts of the molecules. 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; ClonelA4 Catalog F3777). For the anti-CD31 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, Compound F and
an Ang-2 Ab with the same HCDRs and LCDRs as the scFv polypeptide portion of
Compound F comparably repress vascular progression, reduce both the number of
endothelial tip cells and vascular density, as well as increase pericyte coverage (Table 7).
These results from this in vivo model indicate that the Ang2 scFv polypeptide portion of
Compound F has maintained function and potency that is comparable to an Ang2
antibody with the same HCDRs and LCDRs as the scFv polypeptide portion of
Compound F.
Table 7
Parameters Vehicle Ang2 mAb Compound F
Vascular progression
Mean ( ) 100 57.29 49.01
Std. Error of Mean 2.956 4.296 3.679
P value (Vehicle vs.
Compounds) < 0.0001 < 0.0001
(Dunnett' s test)
P value (Ang2 mAb vs.
Compound F) 0.2543
(Dunnett' s test)
Number oi' tip cells
Mean ( ) 100 61.77 47.26
Std. Error of Mean 6.749 6.658 4.719
P value (Vehicle vs.
Compounds) < 0.0001 < 0.0001
(Dunnett' s test)
P value (Ang2 mAb vs.
Compound F) 0.1354
(Dunnett' s test)
Vascular density
Mean ( ) 100 60.52 62.97
Std. Error of Mean 5.728 2.178 2.935
P value (Vehicle vs.
Compounds) < 0.0001 < 0.0001
(Dunnett' s test)
P value (Ang2 mAb vs.
Compound F) 0.9476
(Dunnett' s test)
Physical stability, chemical stability, and product quality
Product qualities, including aggregate level, homogeneity during expression,
physical stability, and chemical stability, are evaluated to identify any issues and ensure
suitability for therapeutic uses.
Free light chain mis-pairing
During the purification and analysis of Compound A (two first polypeptides of
SEQ ID NO: 38 and two second polypeptides of SEQ ID NO: 39), and Compound B (two
first polypeptides of SEQ ID NO: 40 and two second polypeptides of SEQ ID NO: 39),
the presence of non-covalent light chains from the antibody portion of the compound is
detected. Purification to remove the mis-folded species results in poor final yield of the
desired compound. The instability of the interface between VH and VL of the antibody
portion of the compound, and the folding of the domains of the scFv polypeptide are both
found to contribute to the problem. Mutations in the framework of the antibody portion
of the compound are used to eliminate this light chain mis-pairing problem, however, the
problem was only reduced and not resolved. A number of germline frameworks in the
scFv polypeptide are tried, but the expression profiles are not improved. Engineering of
the CDRs and frameworks of the antibody portion of the compound in combination with
scFv polypeptide engineering is required to produce the desired result of acceptable levels
of light chain mis-pairing.
Mass-spec analysis confirms the existence of free LC associated with Compound
A, either as non-covalently linked association or as covalently linked species. To further
quantify the percentage of LC associations, a HIC-HPLC (TSKgel butyl-NPR 4.6mm ID
x 10cm, 2.5um; TOSOH cat# 42168) method is employed. In this analysis, a protein
sample is injected onto a Butyl-NPR column and eluted according to their
hydrophobicity. Through weak hydrophobic interaction, monomer, aggregates and
various states of LC associated species are resolved sequentially during elution. Method
development such that test articles are prepared in 1 mg/ml solution in buffer 50 mM
Potassium Phosphate, 1M ammonium sulphate, pH 6.7 and 50 g of sample is injected
onto a TSKgel butyl-NPR column at flow rate of 1 ml/min on Agilent LC 1260 system.
With a salt gradient from 1M ammonium sulphate to zero in 50 mM Potassium
Phosphate buffer, pH 6.7, three protein peaks are resolved and fractionated. Each
fractionated peak is analyzed by LCMS and identified as monomer Mab-scFv, bicysteinylated
Mab-scFv with one extra cysteinylated LC, and bi-cysteinylated Mab-scFv
with two extra cysteinylated LC. With peak assignments confirmed by LCMS, the final
chromatograph (A214 detection) is integrated to calculate the total percentage of LC
association.
In experiments performed as described above, the compound with an M111L
mutation of Compound B to fix oxidation was found to exhibit 55% LC association.
Through engineering of the variable domain of VR2 and the frameworks, the final
molecules Compound C, Compound D, Compound E, and Compound F were found to
exhibit no detectable LC association as detected by HIC-HPLC analysis (Table 8).
Table 8
To facilitate measurement of Mab-Diabody/Mab-scFv ratios in compounds of the
present invention, the following treatments are performed. Typically 20 g of the
compound of the present invention is buffer exchanged into 50 mM sodium phosphate
containing 150 mM sodium chloride, pH 6.6 using an Amicon Ultra-0.5mL centrifugal
filter device. Samples are concentrated to approximately 30 mΐ and then mixed with 1 mΐ
of freshly prepared FABricator enzyme (Genovis, Cat: AO-FRl-020; 2000 U dissolved in
30 mΐ of ddH20 ) and incubated at 37°C overnight. Digested samples (1 mΐ) are subjected
to LC/UV/MS analysis using the Waters Acquity UPLC coupled to a Waters Xevo G2-S
mass spectrometer. The samples are loaded onto a PLRP-S 50x1.0mm, 1000 A, 5um
reverse phase column (Proxeon, Cat: PL1312-1502) at a flow rate of 0.3 ml/min and
column temperature of 80°C. Samples are eluted from the column using a gradient of
TFA in Acetonitrile. The eluate is analyzed first by UV at 214 nm with the flow then
directed to the mass spectrometer for analysis using sensitivity mode, positive polarity
with an acquisition range of 400-4000 m/z.
In experiments performed essentially as described in this assay, Compound B
exhibits less than 1% Mab-Diabody. Compound C, Compound D, and Compound E,
incorporating all the modifications made to Compound B to reduce oxidation and light
chain mis-pairing problems, surprisingly had approximately 5-6% Mab-Diabody. The
sequence changes incorporated into Compound F had a Mab-Diabody percentage of less
than 1% Mab-Diabody which is comparable to Compound B.
Oxidation
Chemical stability of compounds of the present invention may be evaluated by
producing varied formulation conditions which may be subjected to temperature hold
stress. Changes in chemical stability may be monitored by established LCMS peptide
mapping techniques. Briefly, test articles may be obtained and diluted to a final
concentration of 1 mg/ml in the following formulation buffers: lOmM Citrate, pH 5.0,
lOmM Citrate, pH 6.5, IX PBS, pH 7.4 and lOmM Tris, pH 8.0 and then subjected to
exhaustive dialysis at 4°C in each respective buffer to ensure complete buffer exchange.
Buffer exchanged samples may then be subjected to incubation at either 4°C or 40°C for
four weeks, after which samples may be analyzed by LCMS as follows. Stressed material
may be buffer exchanged into 8M guanidine to denature prior to reduction with DTT
followed by subsequent alkylation by iodoacetamide. Reduced and alkylated protein may
then be buffer exchanged into Tris, pH 7.5 and digested with trypsin at a 20: 1 molar ratio
at 37°C for four hours. Digestion may be quenched with the addition of 1 m glacial
acetic acid. Separation of digested peptide fragments may be achieved by capture onto a
Zorbax 1.8mih C18 2.1mm x 50mm pre-equilibrated 0.2% Formic Acid in water and
subsequent elution using a 0.2% Formic acid in acetonitrile gradient operated at 0.3
ml/min. Eluted peptides may be immediately analyzed using a Agilent ESI-QTOF set to
scan in positive ion mode from 300m/z to 2000m/z at 1 scan/second. The ESI source
may be set at 4000 V and the temperature at 350°C, nebulizer gas at 40 psi and cone gas
at 12 psi. Agilent Mass Hunter Bio-confirm software may be used to align the tryptic
peptide mass spectrum to the protein sequence.
In experiments performed essentially as described in this assay, Compound B
exhibits 14.2% oxidation at position Ml 11 on the heavy chain of the VEGFR2 antibody
portion of the compound when subjected to temperature stress at 40°C at pH 8.0. To
circumvent issues related to oxidation at this position, a single point mutation that
changes methionine 111 to leucine in Compounds C, D, E, and F is made to eliminate the
possibility of chemical modification at this position.
Plasma pharmacokinetics (PK) and pharmacodynamics (PD) following a single
intravenous dose to male Cynomolgus monkeys
The PK and PD of compounds of the present invention may be measured in
Cynomolgus Monkeys after a single intravenous dose.
Male Cynomolgus Monkeys (n=2/group) may be administered a single
intravenous dose of a compound of the present invention. Blood may be sampled
between 2-672 hours post-dose and plasma isolated for quantifying compound plasma
levels using three ELISA methods.
The total human IgG method may utilize an ELISA format to measure the
concentration of the compound of the present invention (total human IgG). Standards,
controls, and test samples may be incubated with goat anti-human F(ab')2 which has been
immobilized on a microtiter plate. After incubation, a mouse anti-human IgG4-HRP
(horseradish peroxidase) may be added to the wells. Once unbound enzyme is washed
away, SureBlue® TMB (tetramethylbenzidine) substrate solution may be added to the
wells. The color development may be stopped by the addition of an acidic solution and
the optical density measured at 450 nm with wavelength correction set to 630 nm. The
assay range may be 30-700 ng/ml.
The VEGFR2 antigen capture method may utilize an ELISA format to measure
the concentration of the compounds of the present invention (VEGFR2 antigen capture).
Standards, controls and test samples may be incubated on a microtiter plate coated with
human VEGFR2. After incubation, a mouse anti-human IgG4-HRP (horseradish
peroxidase) may be added to the wells. Once unbound enzyme is washed away,
SureBlue® TMB (tetramethylbenzidine) substrate solution may be added to the wells.
The color development may be stopped by the addition of an acidic solution and the
optical density measured at 450 nm with wavelength correction set to 630 nm. The assay
range may be 80-2000 ng/ml.
The Ang2 antigen capture method utilizes an ELISA format to measure the
concentration of the compounds of the present invention (Ang2 antigen capture).
Standards, controls and test samples may be incubated on a microtiter plate coated with
Ang2. After incubation, a mouse anti-human IgG4-HRP (horseradish peroxidase) is
added to the wells. Once unbound enzyme is washed away, SureBlue® TMB
(tetramethylbenzidine) substrate solution may be added to the wells. The color
development may be stopped by the addition of an acidic solution and the optical density
measured at 450 nm with wavelength correction set to 630 nm. The assay range may be
30-700 ng/ml.
Noncompartmental analysis may be performed using Phoenix WinNonlin 6.3.
Plots may be generated using SigmaPlot vl 1, and data processing may be performed
using Microsoft Excel 2010.
In experiments performed essentially as described in this assay, the PK was
measured for Compound E and Compound F after one dose at 1, 10, and 25 mg/kg. For
Compound E, terminal half-life, as measured in all three assays, was within the range of
8.96-37.9 h depending on the dose group (Table 9), while the terminal half-life for
Compound F was within the range of 18.6-79.5 h depending on the dose group (Table
10). These results from three different binding assays, that each measure different parts
of the compound, each demonstrate a higher terminal half-life for Compound F compared
to Compound E.
Table 9
Assay Dose Mean t1/2 (n=2)
(mg/kg) (h)
1 8.96
Ang2 Ag Capt 10 24.4
25 35.9
Compound E 1 35.8
Total Human IgG 10 30.2
25 36.7
1 15.6
VEGFR2 Ag
10 23.2
Capture
25 37.9
Table 10
Assay Dose Mean t1/2 (n=2)
(mg/kg) (h)
1 20.1
Ang2 Ag Capt 10 54.3
25 75.2
Compound F 1 18.6
Total Human IgG 10 52.6
25 79.5
1 27.5
VEGFR2 Ag
10 54.3
Capture
25 71.2
Amino Acid and Nucleotide Sequences
SEQ ID NO: 1 (HCVR of antibody - Compound C)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISSS
SSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTEAFDIWGQ
GTLVTVSS
SEQ ID NO: 2 (HCVR of antibody - Compound D)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMLWVRQAPGKGLEWVSAISSSS
SYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQ
GTLVTVSS
SEQ ID NO: 3 (HCVR of antibody - Compound E)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISSS
SSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQ
GTLVTVSS
SEQ ID NO: 4 (HCVR of antibody - Compound F)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMLWVRQAPGKGLEWVSAISSSS
SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQG
TLVTVSS
SEQ ID NO: 5 (HC of antibody - Compound C)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISSS
SSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTEAFDIWGQ
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL
SLSLG
SEQ ID NO: 6 (HC of antibody - Compound D)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMLWVRQAPGKGLEWVSAISSSS
SYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQ
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL
SLSLG
SEQ ID NO: 7 (HC of antibody - Compound E)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISSS
SSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQ
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL
SLSLG
SEQ ID NO: 8 (HC of antibody - Compound F)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMLWVRQAPGKGLEWVSAISSSS
SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQG
TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP
PCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV
DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE
KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS
LSLG
SEQ ID NO: 9 (HC of antibody/linker/scFv polypeptide - Compound C)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISSS
SSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTEAFDIWGQ
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL
SLSLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNM
VWVRQAPGQCLEWMGYIDPYNGGTGYNQKFEGRVTMTTDTSTSTAYMELRSLR
SDDTAVYYCARTRDRYDVWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGG
GSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQDVYIAVAWYQQKPGKAPKLLI
YWASTRDTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPPTFGCGTKV
EIK
SEQ ID NO: 10 (HC of antibody/linker/scFv polypeptide - Compound D)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMLWVRQAPGKGLEWVSAISSSS
SYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQ
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL
SLSLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNM
VWVRQAPGQCLEWMGYIDPYNGGTGYNQKFEGRVTMTTDTSTSTAYMELRSLR
SDDTAVYYCARTRDRYDVWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGG
GSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQDVYIAVAWYQQKPGKAPKLLI
YWASTRDTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPPTFGCGTKV
EIK
SEQ ID NO: 11 (HC of antibody/linker/scFv polypeptide - Compound E)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISSS
SSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQ
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL
SLSLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNM
VWVRQAPGQCLEWMGYIDPYNGGTGYNQKFEGRVTMTTDTSTSTAYMELRSLR
SDDTAVYYCARTRDRYDVWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGG
GSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQDVYIAVAWYQQKPGKAPKLLI
YWASTRDTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPPTFGCGTKV
EIK
SEQ ID NO: 12 (HC of antibody/linker/scFv polypeptide - Compound F)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMLWVRQAPGKGLEWVSAISSSS
SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQG
TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP
PCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV
DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE
KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS
LSLGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKASQDVYIAVAWY
QQKPGQPPKLLIYWASTRDTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQY
SSYPPTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSV
KVSCKASGYSFTDYNMVWVRQAPGQCLEWMGYIDPYNGGTGYNQKFEGRVTIT
ADESTSTAYMELSSLRSEDTAVYYCARTRDRYDVWYFDVWGQGTLVTVSS
SEQ ID NO: 13 (LCVR of antibody - Compound C and Compound F)
DIQMTQSPSSVSASVGDRVTITCRASRGIDNWLTWYQQKPGKAPKLLIYEASSLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKAFPPTFGGGTKVDIK
SEQ ID NO: 14 (LCVR of antibody - Compound D)
DIQMTQSPSSVSASVGDRVTITCRASQGIDNWLTWYQQKPGKAPKLLIVEASSLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKRFPPTFGGGTKVDIK
SEQ ID NO: 15 (LCVR of antibody - Compound E)
DIQMTQSPSSVSASVGDRVTITCRASQGIDNWLTWYQQKPGKAPKLLIVEASSLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKAFPPTFGGGTKVDIK
SEQ ID NO: 16 (LC of antibody - Compound C and Compound F)
DIQMTQSPSSVSASVGDRVTITCRASRGIDNWLTWYQQKPGKAPKLLIYEASSLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKAFPPTFGGGTKVDIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 17 (LC of antibody - Compound D)
DIQMTQSPSSVSASVGDRVTITCRASQGIDNWLTWYQQKPGKAPKLLIVEASSLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKRFPPTFGGGTKVDIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 18 (LC of antibody - Compound E)
DIQMTQSPSSVSASVGDRVTITCRASQGIDNWLTWYQQKPGKAPKLLIVEASSLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKAFPPTFGGGTKVDIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 19 (scFv polypeptide - Compound C, Compound D, Compound E)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNMVWVRQAPGQCLEWMGYID
PYNGGTGYNQKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARTRDRYDV
WYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVS
ASVGDRVTITCKASQDVYIAVAWYQQKPGKAPKLLIYWASTRDTGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCHQYSSYPPTFGCGTKVEIK
SEQ ID NO: 20 (scFv polypeptide - Compound F)
DIVMTQSPDSLAVSLGERATINCKASQDVYIAVAWYQQKPGQPPKLLIYWASTR
DTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYSSYPPTFGCGTKVEIKGG
GGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYNMV
WVRQAPGQCLEWMGYIDPYNGGTGYNQKFEGRVTITADESTSTAYMELSSLRSE
DTAVYYCARTRDRYDVWYFDVWGQGTLVTVSS
SEQ ID NO: 2 1 (HCVR of scFv polypeptide - Compound C, Compound D,
Compound E)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNMVWVRQAPGQCLEWMGYID
PYNGGTGYNQKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARTRDRYDV
WYFDVWGQGTLVTVSS
SEQ ID NO: 22 (HCVR of scFv polypeptide - Compound F)
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYNMVWVRQAPGQCLEWMGYID
PYNGGTGYNQKFEGRVTITADESTSTAYMELSSLRSEDTAVYYCARTRDRYDVW
YFDVWGQGTLVTVSS
SEQ ID NO: 23 (LCVR of scFv polypeptide - Compound C, Compound D,
Compound E)
DIQMTQSPSSVSASVGDRVTITCKASQDVYIAVAWYQQKPGKAPKLLIYWASTR
DTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPPTFGCGTKVEIK
SEQ ID NO: 24 (LCVR of scFv polypeptide - Compound F)
DIVMTQSPDSLAVSLGERATINCKASQDVYIAVAWYQQKPGQPPKLLIYWASTR
DTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYSSYPPTFGCGTKVEIK
SEQ ID NO: 25 (DNA of HC of antibody/linker/scFv polypeptide - Compound C)
GAGGTCCAGCTGGTGCAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAAC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCCATTAGTA
GTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCAT
CTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGA
GCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTCACAGAGGCTTTTGATA
TCTGGGGCCAAGGGACACTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC
ATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCC
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTG
GAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGT
CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG
GGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGG
TGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGC
ACCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAG
GACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGT
GAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACC
GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCC
CATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAAAGCAATGGGCAGCCG
GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTC
TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGA
GCCTCTCCCTGTCTCTGGGTGGCGGAGGCTCCGGGGGAGGGGGTAGCGGAGG
AGGGGGATCCCAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCT
GGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACTCATTCACTGACTA
CAACATGGTGTGGGTGCGACAGGCCCCTGGACAATGCCTTGAGTGGATGGGA
TATATTGATCCTTACAATGGTGGTACTGGCTACAACCAGAAGTTCGAGGGCA
GAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAG
GAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAACGAGGGAT
AGATACGACGTCTGGTACTTCGATGTCTGGGGCCAGGGAACCCTGGTCACCG
TCTCCTCAGGAGGCGGAGGTTCCGGGGGAGGGGGCAGCGGAGGAGGCGGAT
CGGGCGGAGGAGGAAGTGGAGGCGGAGGCAGCGACATCCAGATGACCCAGT
CTCCATCTTCCGTGTCTGCATCTGTTGGCGACAGAGTCACCATCACTTGTAAG
GCCAGTCAGGATGTGTATATTGCTGTAGCCTGGTATCAGCAGAAACCAGGGA
AAGCCCCTAAGCTCCTGATCTATTGGGCATCCACCCGGGACACTGGGGTCCCA
TCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA
GCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCACCAATATAGCAGCTAT
CCTCCTACGTTCGGCTGCGGGACCAAGGTGGAGATCAAA
SEQ ID NO: 26 (DNA of LC of antibody- Compound C and Compound F)
GACATCCAGATGACCCAGTCTCCATCTTCTGTGTCTGCATCTGTAGGAGACAG
AGTCACCATCACTTGTCGGGCGAGTCGTGGTATTGACAACTGGTTAACGTGGT
ATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATTTACGAAGCATCCAG
TTTGCAATCAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAT
TTCACTCTCACTATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTG
TCAACAAGCTAAGGCTTTTCCTCCCACTTTCGGCGGAGGGACCAAGGTGGAC
ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC
CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA
CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC
AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC
GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA
ACAGGGGAGAGTGC
SEQ ID NO: 27 (DNA of HC of antibody/linker/scFv polypeptide - Compound D)
GAGGTCCAGCTGGTGCAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGCTT
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCCATTAGTA
GTAGTAGTAGTTACACCTACTACGCAGACTCAGTGAAGGGCCGATTCACCAT
CTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGA
GCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTCACAGATGCTTTTGATAT
CTGGGGCCAAGGGACACTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCA
TCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGC
CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG
AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTC
CTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG
GCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGT
GGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCA
CCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGG
ACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTG
AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGG
TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCC
ATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAAAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC
CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCT
TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAG
CCTCTCCCTGTCTCTGGGTGGCGGAGGCTCCGGGGGAGGGGGTAGCGGAGGA
GGGGGATCCCAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTG
GGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACTCATTCACTGACTAC
AACATGGTGTGGGTGCGACAGGCCCCTGGACAATGCCTTGAGTGGATGGGAT
ATATTGATCCTTACAATGGTGGTACTGGCTACAACCAGAAGTTCGAGGGCAG
AGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGG
AGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAACGAGGGATA
GATACGACGTCTGGTACTTCGATGTCTGGGGCCAGGGAACCCTGGTCACCGTC
TCCTCAGGAGGCGGAGGTTCCGGGGGAGGGGGCAGCGGAGGAGGCGGATCG
GGCGGAGGAGGAAGTGGAGGCGGAGGCAGCGACATCCAGATGACCCAGTCT
CCATCTTCCGTGTCTGCATCTGTTGGCGACAGAGTCACCATCACTTGTAAGGC
CAGTCAGGATGTGTATATTGCTGTAGCCTGGTATCAGCAGAAACCAGGGAAA
GCCCCTAAGCTCCTGATCTATTGGGCATCCACCCGGGACACTGGGGTCCCATC
AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGC
CTGCAGCCTGAAGATTTTGCAACTTACTATTGTCACCAATATAGCAGCTATCC
TCCTACGTTCGGCTGCGGGACCAAGGTGGAGATCAAA
SEQ ID NO: 28 (DNA of LC of antibody- Compound D)
GACATCCAGATGACCCAGTCTCCATCTTCTGTGTCTGCATCTGTAGGAGACAG
AGTCACCATCACTTGTCGGGCGAGTCAGGGTATTGACAACTGGTTAACGTGGT
ATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCGTCGAGGCATCCAG
TTTGCAATCAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAT
TTCACTCTCACTATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTG
TCAACAGGCTAAGAGGTTTCCTCCCACTTTCGGCGGAGGGACCAAGGTGGAC
ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC
CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA
CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC
AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC
GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA
ACAGGGGAGAGTGC
SEQ ID NO: 29 (DNA of HC of antibody/linker/scFv polypeptide - Compound E)
GAGGTCCAGCTGGTGCAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAAC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCGATTAGTA
GTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCAT
CTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGA
GCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTCACAGATGCTTTTGATAT
CTGGGGCCAAGGGACACTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCA
TCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGC
CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG
AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTC
CTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG
GCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGT
GGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCA
CCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGG
ACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTG
AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGG
TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCC
ATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAAAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC
CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCT
TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAG
CCTCTCCCTGTCTCTGGGTGGCGGAGGCTCCGGGGGAGGGGGTAGCGGAGGA
GGGGGATCCCAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTG
GGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACTCATTCACTGACTAC
AACATGGTGTGGGTGCGACAGGCCCCTGGACAATGCCTTGAGTGGATGGGAT
ATATTGATCCTTACAATGGTGGTACTGGCTACAACCAGAAGTTCGAGGGCAG
AGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGG
AGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAACGAGGGATA
GATACGACGTCTGGTACTTCGATGTCTGGGGCCAGGGAACCCTGGTCACCGTC
TCCTCAGGAGGCGGAGGTTCCGGGGGAGGGGGCAGCGGAGGAGGCGGATCG
GGCGGAGGAGGAAGTGGAGGCGGAGGCAGCGACATCCAGATGACCCAGTCT
CCATCTTCCGTGTCTGCATCTGTTGGCGACAGAGTCACCATCACTTGTAAGGC
CAGTCAGGATGTGTATATTGCTGTAGCCTGGTATCAGCAGAAACCAGGGAAA
GCCCCTAAGCTCCTGATCTATTGGGCATCCACCCGGGACACTGGGGTCCCATC
AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGC
CTGCAGCCTGAAGATTTTGCAACTTACTATTGTCACCAATATAGCAGCTATCC
TCCTACGTTCGGCTGCGGGACCAAGGTGGAGATCAAA
SEQ ID NO: 30 (DNA of LC of antibody- Compound E)
GACATCCAGATGACCCAGTCTCCATCTTCTGTGTCTGCATCTGTAGGAGACAG
AGTCACCATCACTTGTCGGGCGAGTCAGGGTATTGACAACTGGTTAACGTGGT
ATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCGTCGAGGCATCCAG
TTTGCAATCAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAT
TTCACTCTCACTATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTG
TCAACAGGCTAAGGCTTTTCCTCCCACTTTCGGCGGAGGGACCAAGGTGGAC
ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC
CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA
CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC
AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC
GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA
ACAGGGGAGAGTGC
SEQ ID NO: 31 (DNA of HC of antibody/linker/scFv polypeptide - Compound F)
GAGGTCCAGCTGGTGCAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGCTT
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCCATTAGTA
GTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCAT
CTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGA
GCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTCACAGATGCTTTTGATAT
CTGGGGCCAAGGGACACTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCA
TCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGC
CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG
AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTC
CTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG
GCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGT
GGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCA
CCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGG
ACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTG
AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGG
TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCC
ATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAAAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC
CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCT
TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAG
CCTCTCCCTGTCTCTGGGTGGCGGAGGCTCCGGGGGAGGGGGTAGCGGAGGA
GGGGGATCCGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCT
GGGCGAGAGGGCCACCATCAACTGCAAGGCCAGTCAGGATGTGTATATTGCT
GTAGCCTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACT
GGGCATCCACCCGGGACACTGGGGTCCCTGACCGATTCAGTGGCAGCGGGTC
TGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCA
GTTTATTACTGTCACCAATATAGCAGCTATCCTCCTACGTTCGGCTGCGGGAC
CAAGGTGGAGATCAAAGGTGGCGGAGGATCTGGTGGAGGTGGCTCAGGAGG
TGGCGGAAGCGGCGGAGGTGGAAGTCAGGTGCAGCTGGTGCAGTCTGGGGCT
GAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGTT
ACTCATTCACTGACTACAACATGGTGTGGGTGCGACAGGCCCCTGGACAATG
CCTTGAGTGGATGGGATATATTGATCCTTACAATGGTGGTACTGGCTACAACC
AGAAGTTCGAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGC
CTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT
GCGAGAACGAGGGATAGGTACGACGTCTGGTACTTCGATGTCTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCA
SEQ ID NO: 32 (human VEGFR2 ECD)
ASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVE
VTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSD
QHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTI
PSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLV
LNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGV
TRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRIP
AKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKE
KQSHVVSLVVYVPPQIGEKSLISPVDSYQYGTTQTLTCTVYAIPPPHHIHWYWQL
EEECANEPSQAVSVTNPYPCEEWRSVEDFQGGNKIEVNKNQFALIEGKNKTVSTL
VIQAANVSALYKCEAVNKVGRGERVISFHVTRGPEITLQPDMQPTEQESVSLWCT
ADRSTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSNSTNDILIM
ELKNASLQDQGDYVCLAQDRKTKKRHCVVRQLTVLERVAPTITGNLENQTTSIG
ESIEVSCTASGNPPPQIMWFKDNETLVEDSGIVLKDGNRNLTIRRVRKEDEGLYTC
QACSVLGCAKVEAFFIIEGAQEKTNLE
SEQ ID NO: 33 (human Ang2)
YNNFRKSMDSIGKKQYQVQHGSCSYTFLLPEMDNCRSSSSPYVSNAVQRDAPLE
YDDSVQRLQVLENIMENNTQWLMKLENYIQDNMKKEMVEIQQNAVQNQTAVM
IEIGTNLLNQTAEQTRKLTDVEAQVLNQTTRLELQLLEHSLSTNKLEKQILDQTSEI
NKLQDKNSFLEKKVLAMEDKHIIQLQSIKEEKDQLQVLVSKQNSIIEELEKKIVTA
TVNNSVLQKQQHDLMETVNNLLTMMSTSNSAKDPTVAKEEQISFRDCAEVFKSG
HTTNGIYTLTFPNSTEEIKAYCDMEAGGGGWTIIQRREDGSVDFQRTWKEYKVGF
GNPSGEYWLGNEFVSQLTNQQRYVLKIHLKDWEGNEAYSLYEHFYLSSEELNYR
IHLKGLTGTAGKISSISQPGNDFSTKDGDNDKCICKCSQMLTGGWWFDACGPSNL
NGMYYPQRQNTNKFNGIKWYYWKGSGYSLKATTMMIRPADF
SEQ ID NO: 34
GGGGSGGGGS
SEQ ID NO: 35
GGGGSGGGGSGGGGS
SEQ ID NO: 36
GGGGSGGGGSGGGGSGGGGS
SEQ ID NO: 37
GGGGSGGGGSGGGGSGGGGSGGGGS
SEQ ID NO: 38 (HC of antibody/linker/scFv polypeptide - Compound A)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSS
SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQG
TMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTD
YNMVWVRQAPGQCLEWMGYIDPYNGGTGYNQKFEGRVTMTTDTSTSTAYMEL
RSLRSDDTAVYYCARTRDRYDVWYFDVWGQGTLVTVSSGGGGSGGGGSGGGG
SGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQDVYIAVAWYQQKPGKA
PKLLIYWASTRDTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPPTFGC
GTKVEIK
SEQ ID NO: 39 (LC of antibody - Compound A and Compound B)
DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLD
TGVPSRFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 40 (HC of antibody/linker/scFv polypeptide - Compound B)
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSS
SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQG
TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL
SLSLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNM
VWVRQAPGQCLEWMGYIDPYNGGTGYNQKFEGRVTMTTDTSTSTAYMELRSLR
SDDTAVYYCARTRDRYDVWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGG
GSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQDVYIAVAWYQQKPGKAPKLLI
YWASTRDTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPPTFGCGTKV
EIK

WE CLAIM:
1. A compound, comprising an antibody fused by two linkers to two single
chain fragment variable (scFv) polypeptides, wherein:
a) the antibody comprises two identical heavy chains (HCs)
and two identical light chains (LCs), wherein each HC comprises a
heavy chain variable region (HCVR) whose amino acid sequence is
given in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID
NO: 4, and wherein each LC comprises a light chain variable
region (LVCR) whose amino acid sequence is given in SEQ ID
NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15,
b) the two scFv polypeptides are identical and each comprise
an HCVR operably linked to an LCVR, wherein each HCVR has
the amino acid sequence given in SEQ ID NO: 21, or SEQ ID NO:
22, and wherein each LCVR has the amino acid sequence given in
SEQ ID NO: 23, or SEQ ID NO: 24, and
c) the two linkers are identical glycine-rich linkers that each
operably link the carboxy-terminus of one HC of the antibody to
the amino-terminus of one of the scFv polypeptides.
2. The compound of Claim 1, wherein the two scFv polypeptides each
comprise the carboxy-terminus of the LCVR of one scFv polypeptide
operably linked to the amino-terminus of the HCVR of one scFv
polypeptide.
3. The compound of Claims 1 or 2, wherein each HCVR of the antibody has
the amino acid sequence given in SEQ ID NO: 1, each LCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 13, the HCVR
of each of the scFv polypeptides has the amino acid sequence given in
SEQ ID NO: 21, and the LCVR of each of the scFv polypeptides has the
amino acid sequence given in SEQ ID NO: 23.
4. The compound of Claims 1 or 2, wherein each HCVR of the antibody has
the amino acid sequence given in SEQ ID NO: 2, each LCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 14, the HCVR
of each of the scFv polypeptides has the amino acid sequence given in
SEQ ID NO: 21, and the LCVR of each of the scFv polypeptides has the
amino acid sequence given in SEQ ID NO: 23.
5. The compound of Claims 1 or 2, wherein each HCVR of the antibody has
the amino acid sequence given in SEQ ID NO: 3, each LCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 15, the HCVR
of each of the scFv polypeptides has the amino acid sequence given in
SEQ ID NO: 21, and the LCVR of each of the scFv polypeptides has the
amino acid sequence given in SEQ ID NO: 23.
6. The compound of Claims 1 or 2, wherein each HCVR of the antibody has
the amino acid sequence given in SEQ ID NO: 4, each LCVR of the
antibody has the amino acid sequence given in SEQ ID NO: 13, the HCVR
of each of the scFv polypeptides has the amino acid sequence given in
SEQ ID NO: 22, and the LCVR of each of the scFv polypeptides has the
amino acid sequence given in SEQ ID NO: 24.
7. The compound of Claims 1 or 2, wherein the antibody comprises two
heavy chains (HCs) and two light chains (LCs), wherein each HC has the
amino acid sequence given in one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, or SEQ ID NO: 8, and each LC has the amino acid sequence
given in one of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.
8. The compound of Claim 7, wherein each HC of the antibody has the amino
acid sequence given in SEQ ID NO: 5, and each LC of the antibody has
the amino acid sequence given in SEQ ID NO: 16.
9. The compound of Claim 7, wherein each HC of the antibody has the amino
acid sequence given in SEQ ID NO: 6, and each LC of the antibody has
the amino acid sequence given in SEQ ID NO: 17.
10. The compound of Claim 7, wherein each HC of the antibody has the amino
acid sequence given in SEQ ID NO: 7, and each LC of the antibody has
the amino acid sequence given in SEQ ID NO: 18.
11. The compound of Claim 7, wherein each HC of the antibody has the amino
acid sequence given in SEQ ID NO: 8, and each LC of the antibody has
the amino acid sequence given in SEQ ID NO: 16.
12. The compound of Claim 1 or 7, wherein each scFv polypeptide has the
identical amino acid sequence given in one of SEQ ID NO: 19 or SEQ ID
NO: 20.
13. The compound of Claim 12, wherein each scFv polypeptide has the amino
acid sequence given in SEQ ID NO: 19.
14. The compound of Claim 12, wherein each scFv polypeptide has the amino
acid sequence given in SEQ ID NO: 20.
15. A compound comprising two first polypeptides and two second
polypeptides wherein each of the first polypeptides has the amino acid
sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID
NO: 12, and each of the second polypeptides has the amino acid sequence
of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.
16. The compound of Claim 15, wherein each of the first polypeptides has the
amino acid sequence of SEQ ID NO: 9, and each of the second
polypeptides has the amino acid sequence of SEQ ID NO: 16.
17. The compound of Claim 15, wherein each of the first polypeptides has the
amino acid sequence of SEQ ID NO: 10, and each of the second
polypeptides has the amino acid sequence of SEQ ID NO: 17.
18. The compound of Claim 15, wherein each of the first polypeptides has the
amino acid sequence of SEQ ID NO: 11, and each of the second
polypeptides has the amino acid sequence of SEQ ID NO: 18.
19. The compound of Claim 15, wherein each of the first polypeptides has the
amino acid sequence of SEQ ID NO: 12, and each of the second
polypeptides has the amino acid sequence of SEQ ID NO: 16.
20. The compound of any one of Claims 15-19, wherein each of the first
polypeptides forms an inter-chain disulfide bond with each of the second
polypeptides, and the first polypeptide forms two inter-chain disulfide
bonds with the other first polypeptide, and each of the first polypeptides
forms an intra-chain disulfide bond.
21. A mammalian cell comprising a DNA molecule comprising a
polynucleotide sequence a encoding a polypeptide selected from the group
consisting of a polypeptide consisting of SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11 and SEQ ID NO: 12 and a polynucleotide sequence
encoding a polypeptide selected from the group consisting of a polypeptide
consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18,
wherein the cell is capable of expressing a compound comprising a first
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ
ID NO: 12 and a second polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17
and SEQ ID NO: 18.
22. A process for producing a compound comprising two first polypeptides
selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11 and SEQ ID NO: 12 and two second polypeptides selected
from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID
NO: 18, comprising cultivating the mammalian cell of Claim 2 1 under
conditions such that the compound is expressed, and recovering the
expressed compound.
23. A compound obtainable by the process of Claim 22.
24. A pharmaceutical composition, comprising the compound of any one of
Claims 1-20, and an acceptable carrier, diluent, or excipient.
25. A method of treating cancer, comprising administering to a patient in need
thereof, an effective amount of the compound of any one of Claims 1-20.
26. The method of Claim 25, wherein the cancer is breast cancer, ovarian
cancer, gastric cancer, colorectal cancer, non-small cell lung cancer,
biliary tract cancer, or hepatocellular carcinoma.
27. The method of Claim 25, wherein the cancer is breast cancer, ovarian
cancer, gastric cancer, colorectal cancer, or hepatocellular carcinoma.
28. The compound of any one of Claims 1-20, for use in therapy.
29. The compound of any one of Claims 1-20, for use in the treatment of
cancer.
30. The compound for use of Claim 29, wherein the cancer is breast cancer,
ovarian cancer, gastric cancer, colorectal cancer, non-small cell lung
cancer, biliary tract cancer, or hepatocellular carcinoma.
31. The compound for use of Claim 29, wherein the cancer is breast cancer,
ovarian cancer, gastric cancer, colorectal cancer, or hepatocellular
carcinoma.