Note: Descriptions are shown in the official language in which they were submitted.
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MULTIVALENT CONSTRUCTS FOR THERAPEUTIC
AND DIAGNOSTIC APPLICATIONS
Field of the Invention
The invention relates to compositions and methods for therapeutic and
diagnostic applications.
Background of the Invention
Researchers have long been attempting to exploit the ability of targeting
moieties or ligands to bind to specific cells (via receptors or otherwise) to
target
compositions such as detectable labels or therapeutic agents to particular
tissues of an
animal (especially a human). In such situations, the ability of the targeting
moiety to
bind to the target (e.g., affinity, avidity, and/or specificity) significantly
impacts the
ability to successfully target the desired tissues.
Numerous attempts have been made to use natural (e.g. polyclonal) and
monoclonal antibodies, as targeting moieties in vivo. However, use of such
antibodies
present certain disadvantages, such as unacceptable levels of antigenicity -
even for
humanized antibodies. In addition, natural antibodies are difficult to produce
in
recombinant form, due to the number of chains, disulfide bonds, and
glycosylation.
Natural antibodies also present pharmacokinetic problems. Antibodies pose
significant
problems in imaging and radiotherapeutic applications because, due to their
large size,
accumulation in extravascular target tissue and clearance from the vascular
system are
both slow. This problem is especially critical when dealing with solid tumors,
which
present additional barriers to the ingress of large blood born compounds.
Similar
problems occur with antibodies used for imaging using other modalities, such
as
magnetic resonance imaging (MRI), ultrasound and light. If the antibody is
radiolabeled with a diagnostic or therapeutic radionuclide, lower target to
background
ratios result in the images. In addition, an undesirable distribution of
radiation exposure
between the tumor and normal tissues occurs.
In attempts to solve these problems, efforts have been directed towards the
construction of smaller entities with similar binding affinities using the
essential
features of the natural antibody binding regions. The building blocks are
typically
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single-chain Fv fragments (scFv) which are monovalent. Combining fragments of
this
type so that they have the bivalent or multivalent properties of the
antibodies has been
problematic. In order to dock to a surface it is an advantage that the two
binding sites
on the antibody are connected via a flexible hinge to the constant region.
Thus, in order
to imitate the binding efficacy of antibodies, not only must the binding site
be recreated,
but so also must the bivalency (or higher valency) and the flexibility. This
flexibility is
needed because the protein backbone that makes up the nonbinding region of the
scFv is
still bulky relative to the binding site. Once an appropriate method has been
devised to
join two scFv fragments together, different scFv fragments can be joined
together as
well as more than the customary two scFv moieties present in natural
antibodies.
Certain scFv fragments, depending both on the VHNL interface and the linker
length,
can spontaneously dimerize or multimerize. These "diabodies" are smaller than
the
natural antibody and do not have the immunological properties of the Fc
portion (which
activates complement and/or binds to Fc receptors), which they lack. The two
(or more)
binding sites are rotated relative to each other, and thus the antigen must be
correctly
positioned to accommodate this presentation.,
"Miniantibodies" have properties similar to those of diabodies, but rather
than a
short 5-20 amino acid linker they have a relatively more flexible linker that
allows freer
orientation of the binding sites relative to each other, similar to in a
natural antibody.
Like diabodies, miniantibodies do not have the high molecular weight,
immunologically
active Fc dimer fragment. They can also be made by bacterial systems. Although
they
have desired advantages over natural antibodies, miniantibodies still suffer
from having
a relatively large size, which affects their pharmacokinetics, and must be
made using
biological methods. The smallest miniantibody is about 120 kDa in size.
Attempts have been made to use bispecific antibodies (e.g. antibodies that
bind
to two separate targets) to overcome one of the major deficiencies of
antibodies,
namely, that the size of the antibodies slows accumulation in the
extravascular target
tissue and clearance from the blood. The bispecific approach taken has been
referred to
as "pretargeting." This approach uses a two-step protocol. A bispecific
antibody with
at least one arm that recognizes a tumor-associated antigen and at least one
other arm
that recognizes an epitope on a diagnostic or therapy agent is given as a
first injection.
After the unbound antibody has substantially cleared non-target tissues and
has reached
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a maximum level in the tumor, the smaller, bispecific antibody-recognizable
diagnostic
or therapeutic agent is given. It is hoped that the latter agents distribute
more rapidly
throughout the body, and either bind to the bispecific antibody localized at
the tumor, or
are cleared via the kidneys.
An alternative to this approach attempts to use a mixed antibody avidin/biotin
system in a two-step procedure. For example, a targeting antibody is
conjugated with
either avidin or biotin and then is injected whereupon it localizes in the
tumor of
interest. Thereafter, either biotin or avidin (depending on which was coupled
to the
targeting antibody), bearing an imaging or radiotherapeutic radionuclide, is
injected and
becomes localized at the site of the primary antibody by binding to avidin or
biotin
respectively.
Another approach to the use of antibodies as targeting moieties for
radiopharmaceuticals or other diagnostic imagining agents has attempted to use
a
bivalent hapten to increase the avidity for the cell bound bispecific antibody
over that of
the circulating antibody. This approach relies on bidentate binding occurring
with the
cell bound antibodies, because the surface density on the cells is
sufficiently high, but
not occurring with the circulating antibodies, because the concentration is
too low. In
effect, the system makes use of the increase in avidity caused by the closer
presentation
of the antibodies/antigen on the cells.
Peptides have also been used as targeting moieties. In an attempt to improve
the
binding bi-specific peptide constructs have been prepared with two or more
peptide
based targeting agents selective for different targets. For example, a hybrid
peptide
having ligands to two targets selected from the somatostatin-, GRP-, CCK-,
Substance
P-, or VIP receptor and aIP3 integrin was reportedly made and tested for the
ability to
bind to tumor cells. The initial evaluation showed no improved tumor uptake
for the
multiple ligand systems investigated. The investigators assumed that steric
impairment
leads to a reduction of the receptor affinities of the dimeric structures.
Others have
tested an RGD-DTPA-Octreotate hybrid peptide targeted towards both the aõ(i3
integrin
and the somatostatin-2 receptor for the ability to increase the tumor uptake
over that of
a peptide selective for one or the other targets. The different binding
affinities of the
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two targeting moieties towards their targets, blood vessels and tumor cells,
respectively,
resulted in the avidity for tumors being dominated by the stronger
(somatostatin
mediated) interaction.
A variation of these approaches uses a bispecific diabody targeted to two
different epitopes on the same antigen. This approach attempts to increase the
avidity
of the construct for the target, because, although the binding is monovalent
for each
epitope, the construct as a whole is bivalent to its target, as each of the
binding epitopes
is located within the same target molecule. In the case of the single molecule
target,
scFv fragments have been found to have insufficient affinity and an increase
in avidity
was required.
Two rationales underlie the approaches described above. The first rationale
uses
two different targeting moieties to overcome some of the pharmacokinetic
problems
associated with the delivery of antibodies to solid tumors. The second
rationale uses
two different targeting moieties to increase the avidity of the construct for
a given
target, such as a single molecule or a whole tumor. However, all of the
approaches
described suffer from various drawbacks. Thus, there remains a need for
diagnostic and
therapeutic agents with increased affinity and or avidity for a target of
interest. There
also remains a need for diagnostic and therapeutic agents that, when
administered in
vivo to a mammal, have acceptable pharmacokinetic properties.
Angiogenesis, the formation of new blood vessels, occurs not only during
embryonic development and normal tissue growth and repair, but is also
involved in the
female reproductive cycle, establishment and maintenance of pregnancy, and
repair of
wounds and fractures. In addition to angiogenesis that occurs in the normal
individual,
angiogenic events are involved in a number of pathological processes, notably
tumor
growth and metastasis, and other conditions in which blood vessel
proliferation is
increased, such as diabetic retinopathy, psoriasis and arthropathies.
Angiogenesis is so
important in the transition of a tumor from hyperplastic to neoplastic growth,
that
inhibition of angiogenesis has become an active cancer therapy research field.
Tumor-induced angiogenesis is thought to depend on the production of pro-
angiogenic growth factors by the tumor cells, which overcome other forces that
tend to
keep existing vessels quiescent and stable. The best characterized of these
pro-
angiogenic agents is vascular endothelial growth factor (VEGF) (Cohen et al.,
FASEB
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J., 13: 9-22 (1999)). VEGF is produced naturally by a variety of cell types in
response
to hypoxia and some other stimuli. Many tumors also produce large amounts of
VEGF,
and/or induce nearby stromal cells to make VEGF (Fukumura et al., Cell, 94:
715-725
(1998)). VEGF, also referred to as VEGF-A, is synthesized as five different
splice
isoforms of 121, 145, 165, 189, and 206 amino acids. VEGF121 and VEGF165 are
the
main forms produced, particularly in tumors (see, Cohen et al. 1999, supra).
VEGF121
lacks a basic domain encoded by exons 6 and 7 of the VEGF gene and does not
bind to
heparin or extracellular matrix, unlike VEGF165-
VEGF family members act primarily by binding to receptor tyrosine kinases. In
general, receptor tyrosine kinases are glycoproteins having an extracellular
domain
capable of binding one or more specific growth factors, a transmembrane domain
(usually an alpha helix), a juxtamembrane domain (where the receptor may be
regulated, e.g., by phosphorylation), a tyrosine kinase domain (the catalytic
component
of the receptor), and a carboxy-terminal tail, which in many receptors is
involved in
recognition and binding of the substrates for the tyrosine kinase. There are
three
endothelial cell-specific receptor tyrosine kinases known to bind VEGF: VEGFR-
l (Flt-
1), VEGFR-2 (KDR or Flk-1), and VEGFR-3 (Flt4). Flt-1 and KDR have been
identified as the primary high affinity VEGF receptors. While Flt-1 has higher
affinity
for VEGF, KDR displays more abundant endothelial cell expression (Bikfalvi et
al., J.
Cell. Physiol., 149: 50-59 (1991)). Moreover, KDR is thought to dominate the
angiogenic response and is therefore of greater therapeutic and diagnostic
interest (see,
Cohen et al. 1999, supra). Expression of KDR is highly upregulated in
angiogenic
vessels, especially in tumors that induce a strong angiogenic response
(Veikkola et al.,
Cancer Res., 60: 203-212 (2000)). The critical role of KDR in angiogenesis is
highlighted by the complete lack of vascular development in homozygous KDR
knockout mouse embryos (Folkman et al., Cancer Medicine, 5`h Edition (B.C.
Decker
Inc.; Ontario, Canada, 2000) pp. 132-152).
KDR (kinase domain region) is made up of 1336 amino acids in its mature form.
The glycosylated form of KDR migrates on an SDS-PAGE gel with an apparent
molecular weight of about 205 kDa. KDR contains seven immunoglobulin-like
domains in its extracellular domain, of which the first three are the most
important in
VEGF binding (Cohen et al. 1999, supra). VEGF itself is a homodimer capable of
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binding to two KDR molecules simultaneously. The result is that two KDR
molecules
become dimerized upon binding and autophosphorylate, becoming much more
active.
The increased kinase activity in turn initiates a signaling pathway that
mediates the
KDR-specific biological effects of VEGF.
Thus, not only is the VEGF binding activity of KDR in vivo critical to
angiogenesis, but the ability to detect KDR upregulation on endothelial cells
or to detect
VEGF/KDR binding complexes would be extremely beneficial in detecting or
monitoring angiogenesis. Diagnostic applications, such as detecting malignant
tumor
growth, and therapeutic applications, such as targeting tumoricidal agents or
angiogenesis inhibitors to the tumor site, would be particularly beneficial.
Hepatocyte growth factor (also known as scatter factor) is a multi-functional
growth factor involved in various physiological processes such as
embryogenesis,
wound healing and angiogenesis. It has become apparent that HGF, through
interactions with its high affinity receptor (cMet), is involved in tumor
growth, invasion
and metastasis. In fact, dysregulated cMet expression (for example, the
overexpression
of cMet in neoplastic epithelium of colorectal adenomas and in other
carcinomas as
compared to normal mucosa) and/or activity, as well as hyperactivity of the
cMet
receptor through an autocrine stimulatory loop with HGF, has been demonstrated
in a
variety of tumor tissues and induces oncogenic transformation of specific cell
lines.
In general, HGF is produced by the stromal cells, which form part of many
epithelial tumors; however, it is believed that the production of HGF by tumor
cells
themselves comprises the main pathway leading to the hyperproliferation of
specific
tumors. HGF/cMet autocrine stimulatory loops have been detected in gliomas,
osteosarcomas, and mammary, prostate, breast, lung and other carcinomas.
Interrupting the HGF interaction with the cMet receptor slows tumor
progression in animal models. In addition to stimulating proliferation of
certain cancer
cells through activation of cMet, HGF also protects against DNA-damaging agent-
induced cytotoxicity in a variety of cell lines susceptible to
hyperproliferative
phenotypes (e.g., breast cancer). Therefore, preventing HGF from binding to
cMet
could predispose certain cancer cells to the cytotoxicity of certain drugs.
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In addition to hyperproliferative disorders, cMet also has been linked to
angiogenesis. For example, stimulation of cMet leads to the production of
vascular
endothelial growth factor (VEGF), which, in turn, stimulates angiogenesis.
Additionally, stimulation of cMet also has been implicated in promoting wound
healing.
In addition to identifying the cMet receptor as a therapeutic target for
hyperproliferative disorders, angiogenesis and wound healing, the large
discrepancy
between expression levels of neoplastic and corresponding normal tissues
indicates that
cMet is an attractive target for imaging applications directed to
hyperproliferative
disorders.
Summary of the Invention
The present invention features multivalent constructs which bind to a target
of
interest, as well as various methods related to the use of these constructs.
The present
invention uses small targeting moieties which bind to different binding sites
of the same
target, allowing for improved localization to the desired target, and
providing an
improved means for detecting, imaging and/or treating the target site.
Preparation and use of multivalent (e.g., dimeric or multimeric) targeting
constructs which include two or more targeting moieties, for example binding
polypeptides, specific for different binding sites of the same target are
described herein.
These targeting constructs may be linked or conjugated to a detectable label
and/or a
therapeutic agent (as defined herein) and used to deliver the detectable label
and/or
therapeutic agent to the target of interest. Thus, in addition to the
targeting constructs
themselves, the invention includes diagnostic imaging agents and therapeutic
agents
useful in diagnostic imaging and treating various disease states. Furthermore,
the
invention includes use of the targeting constructs of the invention themselves
to treat
disease.
In one aspect, the present invention features a compound having a plurality of
binding moieties, wherein at least two binding moieties have specificity for
different
binding sites on the same target. In preferred embodiments, the plurality of
binding
moieties includes a polypeptide. In other preferred embodiments, the targeting
moieties
are all binding polypeptides which bind to different sites on the desired
target. In
certain preferred emobidments, the target is a protein, a receptor, or a
receptor/ligand
complex and the binding polypeptides bind to different epitopes on the
protein, the
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receptor, or the receptor/ligand complex. In one embodiment, the target is a
receptor
involved in angiogenesis, hyperproliferative disorders or wound healing. In
another
embodiment the target includes a family of receptors, such as, for example,
protein-
tyrosine kinase receptors. In a particularly preferred embodiment, the target
is KDR or
the KDR/VEGF complex, and the binding moieties, particularly binding peptides,
bind
to different epitopes on KDR or the KDR/VEGF complex.
In another preferred embodiment, the target is the hepatocyte growth factor
(HGF) receptor (cMet) or the HGF/cMet complex, and the binding moieties
(particularly binding polypeptides) bind to different epitopes on cMet or the
HGF/cMet
complex.
In further preferred embodiments, the affinity constant of a compound of the
invention for its target is greater than the affinity constant of a
constituent polypeptide
for the target.
In another aspect, the compounds of the invention include a labelling group or
a
therapeutic agent. In certain embodiments, the compounds of the invention
include a
linker between a binding moiety and the labelling group. For example, the
linker may
include a substituted alkyl chain, an unsubstituted alkyl chain, a
polyethylene glycol
derivative, an amino acid spacer, a sugar, an aliphatic spacer, an aromatic
spacer, a lipid
molecule, or combination thereof. Preferred labelling groups include a
radionuclide, a
paramagnetic metal ion, an ultrasound contrast agent, and/or a photolabel. For
example,
preferred paramagnetic metal ions used in compounds of the invention include
Mn2+,
Cu2+ Fe2+ Coe Ni2+ G3+ Eu3+ Dy3+ Pr3+ Cr + Co3+ Fe3+ Tis+ Tb3+ Nd3+ Sm3+
Ho3+, Era+, Pao+, and Eu2+.
Radionuclides are also preferred detectable labels and therapeutic agents. The
choice of radionuclide will be determined based on the desired therapeutic or
diagnostic
application. In a preferred embodiment, where the detectable label is a
paramagnetic
metal or a radionuclide, the compounds of the invention include a chelator or
chelating
group. Preferable chelators inlcude DTPA, DOTA, DO3A, EDTA, TETA, EHPG,
HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, or MECAM. For use as a
PET agent, a peptide may be complexed with one of the various positron
emitting metal
ions, such as 51Mn, 52Fe, 60Cu, 68Ga, 72As, 94mTc, or 10In. The
heteromultimeric
constructs can also be labeled by halogenation using radionuclides, such as
"F, 1141,
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1251, 1311, 1231,77 Br.and 76Br. Preferred metal radionuclides for
scintigraphy or
radiotherapy include 99mTc, "Cr, 67Ga, 68Ga, 47Sc, "Cr, 167Tm, 141Ce, 111In,
168Yb,
175Yb 140La, 90Y, 88Y, 153Sm 16611o 165Dy 166Dy 62Cu 64Cu 67Cu 97Ru 103Ru
186Re
188Re, 203Pb, 211Bi, 212Bi, 213Bi'214 Bi, 105Rh1109Pd, 117mSn, 149Pm2 161Th,
177Lu, 198Au and
' 99Au. The choice of metal or halogen will be determined based on the desired
therapeutic or diagnostic application. For example, for diagnostic purposes
the
preferred radionuclides include 64Cu, 67Ga, 68Ga, 99mTc, and 11 'In. For
therapeutic
purposes, the preferred radionuclides include 64Cu, 9 T,105Rh, 11'In,
117mSn,'49Pm,
153Sm, 161Th, 166Dy, 166Ho, 175Yb, 177Lu, 186/188Re, and 199Au. A most
preferred chelator
used in compounds of the invention is 1-substituted 4,7,10-tricarboxymethyl
1,4,7,10
tetraazacyclododecane triacetic acid (DO3A). Preferably, a radioactive
lanthanide, such
as, for example, 177 Lu, 9 Y,153Sm,11 'In, or 166Ho is used with DOTA or DO3A
in
compounds of the invention.
Compounds of the invention include chelators having the fol~lonwring
structure:
Y~J n
X~ X
NH HN NH HN
N N YlJn ~ (N N:
1 S HO OH HO IN
where X is CH2 or 0;
Y is C,-C10 branched or unbranched alkyl, aryl, aryloxy, arylamino,
arylaminoacyl, or
aralkyl comprising C1-Clo branched or unbranched alkyl groups, C1-C,o branched
or
unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or
polyhydroxy-
polyalkoxyalkyl groups; J is C(=O)-, OC(=O)-, SO2-, NC(=O)-, NC(=S)-, N(Y),
NC(=NCH3)-, NC(=NH)-, N=N-, a homopolyamide or a heteropolyamine derived from
synthetic or naturally occurring amino acids; and n is 1-100. Most preferably,
the
compounds further include 99mTc, 186Re, or 188Re.
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In one embodiment, compounds of the the invention include a chelator having
the following structure:
OH
O
HN O
NH H NH HN
HN
CN N COON CN N
HO OH HO OH
Most preferably, the compound further includes 99mTC, t86Re, or 188Re.
In another embodiment, the chelator comprises a compound having the
following structure:
OH N NLri:: N
NHCOCH3
Most preferably, the compound further includes 99"Tc.
In other embodiments, compounds of the invention include a chelator having the
following structure:
R R
HOOC--[ n /LCOOH
CN N
N N
HOOC-f U cooH
R CO
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where R is an alkyl group, such as CH3. Most preferably, the compound further
includes 177Lu, 90Y, 153Sm, 1 "In, or 166 Ho.
In yet another embodiment, compounds of the invention include a chelator
having the following structure:
R R
HOOC1\ F--\ /LCOOH
CN N]
N N
HOOC-( U COON
R NH
where R is an alkyl group, such as CH3. Most preferably, the compound further
includes
177Lu, 90Y, 153Sm, 111In, or 166Ho.
In other embodiments, the compound of the invention includes a chelator having
the following structure:
HOOC-\ [--1 ,--000H
C N N
I
O N N
U -000H
Most preferably, the compound further includes '77Lu, 90Y,'53Sm, 111 In, or
166Ho.
Preferred ultrasound contrast agents for use in compounds of the invention
include phospholipid stabilized microbubbles or microballoons comprising a
fluorinated
gas.
One preferred embodiment of the invention includes compounds comprising at
least two binding moieties with specificity for different binding sites on a
target.
Preferably the target is a single receptor or receptor/ligand complex such as,
for
example, KDR or the KDR/VEGF complex or cMet of the cMet/VEGF complex. In
further preferred embodiments, the binding moieties bind to different epitopes
on the
receptor or receptor/ligand complex. In a particularly preferred embodiment
the
binding moieties include a polypeptide. In other preferred embodiments, a
compound
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of the invention includes a polypeptide having the amino acid sequence of SEQ
ID
NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ
ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29. The invention also
provides a compound having one or more of the foregoing amino acid sequences
that
have been modified to include one or more amino acid substitutions, amide bond
substitutions, D-amino acid substitutions, glycosylated amino acids, disulfide
mimetic
substitutions, amino acid translocations, or has been modified to include a
retroinverso
peptide, a peptoid, a retro-inverso peptoid, and/or a synthetic peptide. In
preferred
embodiments, the compound of the invention comprises SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:26, and/or
SEQ ID NO:27. In a more preferred embodiment such compounds further include a
labelling group or therapeutic agent as described herein.
In another aspect, the invention features diagnostic imaging methods using
compounds of the invention that include a labelling group. Methods of the
invention
include the steps of administering to a patient a pharmaceutical preparation
that includes
a compound of the invention having a labelling group, and imaging the compound
after
administration to the patient. In preferred embodiments, the imaging step
includes
magnetic resonance imaging, ultrasound imaging, optical imaging,
sonoluminescence
imaging, photoacoustic imaging, or nuclear imaging. In these methods, the
administering step may include inhaling, transdermal absorbing, intramuscular
injecting, subcutaneous injecting, intravenous injecting, intraperitoneally
injecting,
intraarterial injecting or parenteral administration.
In another aspect, the compounds of the invention serve as therapeutic agents
themselves and/or include a therapeutic agent. In certain embodiments, the
compounds
of the invention include a linker between a binding moiety and the therapeutic
agent.
For example, the linker may include a substituted alkyl chain, an
unsubstituted alkyl
chain, a polyethylene glycol derivative, an amino acid or peptide spacer, a
sugar, an
aliphatic spacer, an aromatic spacer, a lipid molecule, or combination
thereof. Preferred
therapeutic agents for use with compounds of the invention include a bioactive
agent, a
cytotoxic agent, a drug, a chemotherapeutic agent, or a radiotherapeutic
agent.
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In another aspect, the invention features a method of treating a disease by
administering to a patient a pharmaceutical preparation including a compound
of the
invention. In one embodiment, where one or more binding moieties of the
compound
inhibits a biological process that contributes to a disease state, the
compound may be
administered to treat that disease state. For example, the binding moieties
may inhibit
the biological process by preventing or diminishing the activity of the
receptor(e.g. by
competition with the natural ligand for the receptor, by directly inhibiting
the receptor
activity whether or not the natural ligand is bound, or by a combination of
the two).
Thus, a heteromultimeric compound of the invention, may inhibit the activity
of, for
instance KDR or cMet, and thus inhibit angiogenesis and/or hyperproliferation
and
consequently the diseases these processes contribute to. Therefore, the
invention
features a method of treating a disease by administering to a patient a
pharmaceutical
preparation including a compound of the invention alone or attached or linked
to a
separate therapeutic agent. In preferred embodiments, the invention features a
method
of treating a disease associated with angiogenesis or hyperproliferation. In a
most
preferred embodiment, the disease is neoplastic tumor growth.
The invention also features a method of screening for heteromultimeric
compounds having improved binding affinity. This method includes the steps of
preparing a labeled heteromultimeric compound comprising a plurality of
binding
moieties, wherein at least two binding moieties bind to different binding
sites of a
target; contacting the labeled heteromultimeric compound with a target;
determining a
binding strength of the labeled heteromultimeric compound (for example, by
determining the dissociation constant); and comparing the binding strength
(e.g.,
dissociation constant) of the labeled heteromultimeric compound with the
binding
strength (e.g., dissociation constant) of one or more individual binding
moieties. In
preferred embodiments of this method one of the binding moieties includes a
polypeptide. In another preferred embodiment, the target is KDR or KDR/VEGF
complex. In a preferred embodiment, one of the polypeptides used in this
method is
SEQ ID NO: I, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ IDNO:8, SEQ IDNO:9, SEQ IDNO:I0, SEQ IDNO:11, or SEQ ID
NO: 12. Preferably, the method
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includes the step of identifying a labeled heteromultimeric compound having a
binding
strength (for example, as measure by the dissociation constant) that is less
than the
binding strength of a constituent binding moiety.
In one preferred embodiment, the invention features dimeric or multimeric
targeting constructs which include two or more KDR or VEGF/KDR complex binding
polypeptides which bind to different binding sites of KDR or the VEGF/KDR
complex.
Such polypeptides are described in detail in U.S.S.N. 60/360,851 and U.S.S.N.
60/440,441, both of which are incorporated by reference herein in their
entirety, and in
copending application U.S.S.N. , entitled "KDR and VEGF/KDR binding
peptides and their use in diagnosis and therapy," filed on the same date as
the instant
application and incorporated by reference herein in its entirety. These
constructs are
referred to herein as "KDR - targeting constructs." The KDR targeting
constructs
exhibit improved binding to KDR (e.g. increased specificity and/or affinity
and/or
avidity) compared to monomeric KDR or VEGF/KDR complex binding polypeptides,
'15 and compared to dimeric or multimeric constructs of a single KDR-binding
polypeptide.
These preferred compounds may be linked or conjugated to a detectable moiety
and
used to target these compositions to KDR-expressing cells, permitting imaging
of KDR-
expressing tissue.
In another preferred embodiment, the invention features dimeric or multimeric
targeting constructs which include two or more cMet or HGF/cMet complex
binding
polypeptides which bind to different binding sites of cMet or the HGF/cMet
complex.
Such polypeptides are described in detail in copending application U.S.S.N.
entitled "Peptides that specifically bind HGF receptor (cMet) and uses
thereof," filed on
the same date as the instant application and incorporated by reference herein
in its
entirety. These constructs are referred to herein as "cMet - targeting
constructs." The
cMet targeting constructs exhibit improved binding to cMet (e.g. increased
specificity
and/or affinity and/or avidity) compared to monomeric cMet or HGF/cMet complex
binding polypeptides, and compared to dimeric or multimeric constructs of a
single
cMet-binding polypeptide.
The cMet and KDR targeting constructs of the invention may be linked or
conjugated to a therapeutic agent and used to localize the therapeutic agent
to cMet- or
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KDR-expressing tissue. Alternatively or additionally, the cMet or KDR
targeting
constructs of the invention may also be used as therapeutics themselves, as,
described
herein.
In particularly preferred embodiments, the KDR targeting constructs of the
invention include two or more of the following KDR and VEGF/KDR complex-
binding
polypeptides: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, or
SEQ ID NO:12.
In other preferred embodiments, the cMet targeting constructs of the invention
include two or more of the following binding polypeptides: SEQ ID NO:26, SEQ
ID
NO:27, SEQ ID NO:28, and/or SEQ ID NO:29.
In another embodiment, the invention provides a novel method for screening the
KDR targeting constructs for the ability to bind the target, and thus,
identify multimeric
constructs of KDR binding polypeptides with improved binding (as determined,
for
example, by dissociation constants), as compared to binding of the constituent
polypeptides. Additionally, the method of the invention allows for rapid
determination
of whether the multimeric targeting constructs will be stable in the presence
of serum in
vivo.
Constructs comprising two or more KDR or KDR/VEGF binding polypeptides
show improved ability to bind the target molecule compared to the
corresponding
monomeric binding polypeptides. For instance, as shown in Example 6 below,
tetrameric constructs of KDR binding polypeptides provided herein showed
improved
ability to bind KDR-transfected 293H cells. Combining two or more binding
polypeptides in a single molecular construct appears to improve the avidity of
the
construct over the monomeric binding polypeptides a shown by a decrease in KD.
In addition, as demonstrated herein, constructs comprising two or more binding
polypeptides specific for different epitopes of KDR and/or KDRNEGF (e.g.,
"heteromeric" constructs) were made. Constructs comprising two or more binding
polypeptide provided herein are expected to block multiple sites on KDR or
VEGF/KDR. The heteromeric constructs show superior binding ability over both
the
corresponding monomers, as well as tetrameric constructs comprising multiple
copies
of the same binding polypetide. Furthermore, heteromeric constructs comprising
two or
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more binding peptides specific for different epitopes were also able to
efficiently bind
KDR-transfected 293H cells. Thus, inclusion of two or more binding
polypeptides that
recognize different epitopes further improves the avidity of the construct for
the target
molecule, as demonstrated by a decrease in KD.
Heteromeric constructs of the binding polypeptides provided herein show
improved ability to inhibit receptor tyrosine kinase function. Based on
experiments
described herein, dimeric and other multimeric constructs of the present
invention
comprising at least two binding polypeptides specific for different epitopes
of KDR
and/or KDRNEGF are expected to inhibit the function of receptor tyrosine
kinases. In
particular, such constructs are expected to inhibit the function of VEGFR-
2/KDR,
VEGFR-1/Flt-l and VEGFR-3/Flt-4. Additionally, heteromultimeric constructs of
the
invention comprising two or more binding moieties specific for different
epitopes of
cMet and/or cMet/HGF are expected to inhibit the function of receptor tyrosine
kinases
and, in particular the function of cMet.
For the purposes of the present invention, receptor tyrosine kinase function
can
include any one of. oligomerization of the receptor, receptor phosphorylation,
kinase
activity of the receptor, recruitment of downstream signaling molecules,
induction of
genes induction of cell proliferation, induction of cell migration, or
combination
thereof. For example, heteromeric constructs of binding polypeptides provided
herein
inhibit VEGF-induced KDR receptor inactivation in human endothelial cells,
demonstrated by the inhibition of VEGF-induced phosphorylation of the KDR
receptor.
In addition, heteromeric constructs of binding peptides provided herein
inhibit VEGF-
stimulated endothelial cell migration. As shown herein, targeting two or more
distinct
epitopes on KDR with a single binding construct greatly improves the ability
of the
construct to inhibit receptor function. Even binding peptides with weak
ability to block
receptor activity can be used to generate heteromeric constructs having
improved ability
to block VEGF-induced receptor function.
Additionally, as further demonstrated herein, constructs comprising two or
more
binding polypeptides specific for different epitopes of cMet were made.
Constructs
containing two or more cMet binding polypeptide provided herein are expected
to block
multiple sites on cMet. These heteromeric cMet targeting constructs show
superior
binding ability over the corresponding monomers.
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Therefore, the present invention is drawn to constructs comprising two or more
binding polypeptides. The multimeric constructs of the present invention
comprise two
or more binding polypeptides, such that at least two of the binding
polypeptides in the
construct are specific for different epitopes of a target, for example, KDR
and/or
KDR/VEGF and cMet and/or cMet/HGF. These constructs are also referred to
herein
as "heteromeric constructs," "hetermultimers" and/or "heteromultimeric
constructs."
The constructs of the present invention can also include unrelated, or control
peptide.
The constructs can include two or more, three or more, or four or more binding
polypeptides. Based on the teachings provided herein, one of ordinary skill in
the art is
able to assemble the binding polypeptides provided herein into multimeric
constructs
and to select multimeric constructs having improved properties, such as
improved
ability to bind the target molecule, or improved ability to inhibit receptor
tyrosine
kinase function. Such multimeric constructs having improved properties are
included in
the present invention. Furthermore, the methods and teachings provided herein
have
been shown to allow for the improved binding to a variety of different targets
(e.g.,
KDR and cMet), thus demonstrating the wide applicability of the present
invention.
Brief Description of the Drawings
FIG. 1 shows the binding of fluorescent beads to KDR-transfected and mock-
transfected cells. Neutravidin-coated beads with the indicated biotinylated
ligands
attached were tested for binding to KDR-expressing and non-expressing 293H
cells.
Specific binding to KDR was detected for both P5 (with hydrophilic spacer) and
P6.
Further details are provided in Example 2.
FIG. 2 shows the percentage inhibition of125I-labeled VEGF binding by
peptides [P6, P4, P5-X-B and P 12-X-B) at two different concentrations (30 M
and 0.3
M) to KDR-transfected 293H cells, as described in Example 3. The results for
P6, P4
and P5-X-B are the average of three experiments SD, whereas the result for P
12-X-B
is based on one experiment.
FIG. 3 depicts immunoblots of KDR immunoprecipitates from unstimulated (-
V) and VEGF-stimulated (+V) HUVECs which were resolved by SDS-PAGE, blotted,
and sequentially probed with anti-phosphotyrosine ("Phospho KDR") and anti-KDR
("Total KDR") antibodies. Activated (phosphorylated) KDR was not detected in
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unstimulated (-V) HUVECs, but was abundant in immunoprecipitates from VEGF-
stimulated (+V) HUVECs. Reprobing the blot with anti-KDR demonstrated that
comparable amounts of total KDR were present in both immunoprecipitates. This
figure is representative of twelve experiments that followed the same
protocol.
FIG. 4 depicts immunoblots demonstrating inhibition of KDR phosphorylation
(activation) with a neutralizing anti-KDR antibody, as described in Example 4.
Immunoprecipitates from unstimulated (-V), VEGF-stimulated (+V), and
simultaneously VEGF/anti-KDR (lpg/mL) (+V+a-KDR)-treated HUVECs were
resolved by SDS-PAGE, blotted, and sequentially probed with anti-
phosphotyrosine
("Phospho KDR") and anti-KDR ("Total KDR") antibodies. As described in Example
4, the neutralizing antibody was able to partially block the VEGF-induced
activation of
KDR.
FIG. 5 depicts immunoblots demonstrating inhibition of KDR phosphorylation
(activation) with a KDR-binding peptide (repeat experiment).
Immunoprecipitates from
unstimulated (-V), VEGF-stimulated (+V), and a KDR-binding peptide (10 M)
(+V+P 10)-treated HUVECs were resolved by SDS-PAGE, blotted, and sequentially
probed with anti-phosphotyrosine ("Phospho KDR") and anti-KDR ("Total KDR").
As
described in Example 4, the KDR-binding peptide P10 was clearly able to
partially
block the VEGF-induced activation of KDR at 10 M.
FIG. 6 depicts binding of Tc-labeled P12-C to mock and KDR transfected 293H
cells, as described in Example 5.
FIG. 7 depicts specific binding of Tc-labeled P12-C to KDR transfected 293H
cells, as described in Example S.
FIG. 8 depicts saturation binding of peptide/Neutravidin HRP complexes, as
described in Example 6. FIG. 8A shows the results obtained using P6-XB and P5-
XB.
FIG. 8B shows the results obtained using P12-XB and P13-XB. Calculated Kd
values
were: 10.00 nM (P6-XB), 14.87 nM (P5-XB), 4.03 nM (P12-XB) and 1.81 nM (P13-
XB).
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FIG. 9 depicts binding of peptide/neutravidin HRP complexes (P1-X-B, P5-X-B,
P6-XB, P12-XB and P13-XB) to KDR-transfected and mock-transfected 293H cells
at a
single concentration (5.5 nM), as described in Example 6.
FIG. 10 depicts binding of peptide/neutravidin HRP complexes (P1-XB, Pl-B,
P5-XB, P5-B, P6-XB and P6-B) to KDR-transfected and mock-transfected 293H
cells
at a single concentration (2.78 nM), as described in Experiment B of Example
6.
FIG. 11 depicts specific binding (binding to KDR transfected cells minus
binding to mock transfected cells) of peptide/neutravidin HRP complexes (P6-
XB, P5-
XB, P12-XB and P13-XB) with and without 40% rat serum, as described in
Experiment
C of Example 6. The concentration of peptide/avidin HRP solution was 6.66 nM
for
P6-XB and P5 AB, 3.33 nM for P12-XB and 2.22 nM for P13-XB.
FIG. 12 shows the binding of peptide/avidin HRP with mock and KDR
transfected cells, plotted as absorbance at 450 nm. The proportions of control
and KDR
binding peptides used to form each tetrameric complex are indicated in the
legend, for
each tested multimer.
FIG. 13 depicts specific binding of a P5-XB/avidin-HRP complex to KDR
transfected cells (background binding to mock-transfected cells subtracted),
plotted as
absorbance at 450 nm. Increasing concentrations (as indicated by the X axis)
of
uncomplexed peptides were added to the assay as indicated in the legend. Only
free P5-
XB was able to decrease the binding of the P5-XB/avidin complex to KDR-
transfected
cells.
FIG. 14 is a graph showing the percentage inhibition of 1251-labeled VEGF
binding by peptides (P12-XB, D2, Dl, D3, and P13-D) at three different
concentrations
(10 M, 0.3 M, and 0.03 M) to KDR-transfected 293H cells. The results are
from
one experiment carried out in tripicate +/- S.D.
FIG. 15 is a photograph showing the ability of Dl to completely block the
VEGF-induced phosphorylation of KDR in HUVECs at 10 nM and the majority of
phosphorylation at I nM. Reprobing the blot for total KDR (lower panel)
demonstrated
that the effects of the tested compounds was not due to reduced sample
loading.
Homodimers composed of the two binding sequences contained in D1 did not
interfere
with the phosphorylation at up to 100 nM.
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FIG. 16 shows that D1 potently blocks the migration/invasion of endothelial
cells induced by VEGF. Migrating cells were quantitated by fluorescence
measurement
after staining the migrated cells with a fluorescent dye.
FIG. 17 is a graph showing the binding of 125I-labeled D5 to mock and KDR
transfected 293H cells in the absence and presence of 40% mouse serum.
FIG. 18 is a graph showing the specific binding (KDR-MOCK) of145I-labeled
D5 to KDR-transfected 293H cells in the absence and presence of 40% mouse
serum.
FIG. 19 is a graph of plasma clearance as percent injected dose per mL versus
time.
FIG. 20 shows SE-HPLC profiles of plasma from the Superdex peptide column.
Top panel, sample injected; followed by 0min, 30min, and 90min. The insert
within
each panel shows time point, animal number and volume injected for HPLC
analysis.
FIG. 21 is a graph showing the results of testing of KDR peptides in HWEC
proliferation assay. A, D6; B, P12-G; C, PNC- I (negative control); F, PNC-1
(negative
control).
FIG. 22 shows the kinetic analysis of D1 (heterodimer of a truncated form of
P6-D and P12-G) binding to murine KDR-Fc. All sensograms are fit to the
bivalent
analyte model.
FIG. 23 shows the kinetic analysis of D7 (heterodimer of P5-D and P6-D)
binding to murine KDR-Fc. All sensograms are fit to the bivalent analyte
model.
FIG. 24 shows kinetic analysis of fluorescein labeled P12-G binding to murine
KDR-Fc. All sensograms are fit to the 1:1 Langmuir model.
FIG. 25 is a graph showing the specific binding (binding to KDR-transfected
cells minus binding to mock-transfected cells) of 99mTc-labeled P12C in the
presence
and absence of 40% rat serum, as described in Experiment C of Example 6.
Results are
plotted as specific CPM bound +/- s.d.
FIG. 26 is a graph depicting % inhibition s.d. of specific 125I-VEGF binding
to
KDR-transfected cells by PG-1 (squares) D 1 (diamonds).
FIG. 27 is a graph depicting % maximum VEGF-stimulated migration s.d. of
HWEC cells in the presence of the indicated concentrations of PG-l (diamonds)
D1
(squares).
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FIG. 28A is a graph depicting the binding of Tc-labeled D 10 to KDR-
transfected 293H cells as described in Example 18.
FIG. 28B is a graph depicting the lack of binding of Tc-labeled D18 to KDR-
transfected 293H cells as described in example 18. Mock = mock-transfected.
Trans =
KDR-transfected. MS = mouse serum.
FIG. 29 is a graph depicting the binding of Lu-labeled D13 to KDR-transfected
293H cells as described in Example 19. Mock = mock-transfected. Trans = KDR-
transfected. MS = mouse serum.
FIG. 30 is a graph summarizing the results of a radiotherapy study with D13
conducted in nude mice implanted with PC3 tumors. Each plotted line represents
the
growth over time for an individual tumor in a treated mouse, except for the
heavy
dashed line, which represents the average tumor growth in a set of untreated
mice, as
described in Example 20.
FIG. 31 is a graph showing the total binding of complexes of control peptide
and
the test peptides (P30-XB, P31-XB, P32-XB) with 125I-streptavidin (in the
presence of
VEGF) to mock-transfected and KDR-transfected cells. Only the complex
containing
P30-XB showed specific binding (KDR-mock).
FIG. 32 is a graph showing that D26 (squares) with its glycosylation and
modified spacer is able to block VEGF-stimulated migration even more potently
than
D24 (diamonds), which lacks those chemical modifications.
FIG. 33 is a graph showing that TK-1 enhances the potency of D6 in blocking
the biological effects of VEGF in a migration assay with cultured HUVECs.
Diamonds:
D6 alone at the indicated concentrations. Squares: D6 at the indicated
concentrations
plus I00nM TK-1 (constant).
FIG. 34 is a graph showing that homodimeric D8 (squares) is less able than
heterodimeric D17 (diamonds) to block the effects of VEGF in the migration
assay as
carried out in Example 25.
FIG. 35 is a graph showing cell proliferation data for D6 as described in
Example 31 below.
FIG. 36 shows examples of (A) a C-terminus to C-terminus linked dimer, (B) an
N-terminus to C-terminus linked dimer, and (C) an N-terminus to N-terminus
linked
dimer.
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FIG. 37 is a graph showing uptake and retention of bubble contrast in the
tumor
up to 30 minutes post injection for suspensions of phospholipid stabilized
microbubbles
conjugated to a heteromultimeric construct.
Detailed Description
The present invention is based, in part, on the discovery that compounds
having
two or more binding moieties, wherein at least two of the binding moieties
bind to
different binding sites on the same target, have unexpected and significantly
improved
ability to bind the target. Preferably the target is a receptor or a
receptor/ligand
complex. The improved ability of compounds of the invention (variously
referred to as
"multivalent targeting constructs," "heterodimers," "heterotetramers,"
"heteromultimers" and/or "heteromultimeric constructs" herein) to bind a
target may be
demonstrated by comparison to the ability of an individual, constituent,
binding moiety
to bind the target. For example, the binding strength of a heteromultimer of
the
invention may be compared to the binding strength of one of its monomers.
Preferably,
a heteromultimer of the invention exhibits an increase in affinity (as
determined, for
example, by dissociation constants), compared to an individual, constituent
monomer.
Definitions
As used herein, the term "recombinant" is used to describe non-naturally
altered
or manipulated nucleic acids, host cells transfected with exogenous nucleic
acids, or
polypeptides expressed non-naturally, through manipulation of isolated DNA and
transformation of host cells. Recombinant is a term that specifically
encompasses DNA
molecules which have been constructed in vitro using genetic engineering
techniques, and
use of the term "recombinant" as an adjective to describe a molecule,
construct, vector,
transfected cell, polypeptide or polynucleotide specifically excludes
naturally occurring
such molecules, constructs, vectors, cells, polypeptides or polynucleotides.
The term "bacteriophage" is defined as a bacterial virus containing a DNA core
and a protective shell built up by the aggregation of a number of different
protein
molecules. The terms "bacteriophage" and "phage" are used herein
interchangeably.
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The term "polypeptide" is used to refer to a compound of two or more amino
acids
joined through the main chain (as opposed to side chain) by a peptide amide
bond
(-C(:O)NH ). The term "peptide" is used interchangeably herein with
"polypeptide" but
is generally used to refer to polypeptides having fewer than 40, and
preferably fewer than
25 amino acids.
The term "binding" refers to the determination by standard assays, including
those described herein, that a binding polypeptide recognizes and binds
reversibly to a
given target. Such standard assays include, but are not limited to,
equilibrium dialysis,
gel filtration, and the monitoring of spectroscopic changes that result from
binding.
The term "binding polypeptide" as used herein refers to any polypeptide
capable of
forming a binding complex with another molecule. Also included within the
definition of
"binding polypeptides" are polypeptides that are modified or optimized as
disclosed
herein. Specific examples of such modifications are discussed in detail infra,
but
include substitution of amino acids for those in the parent polypeptide
sequence to
optimize properties, obliterate an enzyme cleavage site, etc.; C- or N-
terminal amino
acid substitutions or elongations, e.g., for the purpose of linking the
binding polypeptide
to a detectable imaging label or other substrate, examples of which include,
e.g.,
addition of a polyhistidine "tail" to assist in purification; truncations;
amide bond
changes; translocations; retroinverso peptides; peptoids;
retroinversopeptoids; the use of
N-terminal or C-terminal modifications or linkers, such as polyglycine or
polylysine
segments; alterations to include functional groups, notably hydrazide (-NH-
NH2)
functionalities or the C-terminal linker -Gly-Gly-Gly-Lys, to assist in
immobilization of
binding peptides according to this invention on solid supports or for
attachment of
fluorescent dyes; modifications which effect pharmacokinetics; structural
modifications
to retain structural features; formation of salts to increase water solubility
or ease of
formulation, and the like. In addition to the detectable labels described
further herein,
the binding polypeptides may be linked or conjugated to a radiotherapeutic
agent, a
cytotoxic agent, a tumorcidal agent or enzyme, a liposome (e.g., loaded with a
therapeutic agent, an ultrasound appropriate gas, or both). In addition,
binding
polypeptides of the invention may be bound or linked to a solid support, such
as a well,
plate, bead, tube, slide, filter, or dish. Moreover, dimers or multimers of
one or more
binding polypeptides may be formed. Such constructs may, for example, exhibit
increased
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ability to bind to the target. All such modified polypeptides are also
considered "binding
polypeptides" so long as they retain the ability to bind the targets.
"Homologues" of the binding polypeptides described herein may be produced
using any of the modification or optimization techniques described herein or
known to
those skilled in the art. Such homologous polypeptides will be understood to
fall within
the scope of the present invention and the definition of "binding
polypeptides" so long
as the substitution, addition, or deletion of amino acids or other such
modification does
not eliminate its ability to bind to the target. The term "homologous," as
used herein,
refers to the degree of sequence similarity between two polymers (i.e.,
polypeptide
molecules or nucleic acid molecules). When the same nucleotide or amino acid
residue
or one with substantially similar properties (i.e. a conservative
substitution) occupies a
sequence position in the two polymers under comparison, then the polymers are
homologous at that position. For example, if the amino acid residues at 60 of
100
amino acid positions in two polypeptide sequences match or are homologous then
the
two sequences are 60% homologous. The homology percentage figures referred to
herein reflect the maximal homology possible between the two polymers, i.e.,
the
percent homology when the two polymers are so aligned as to have the greatest
number
of matched (homologous) positions. Polypeptide homologues within the scope of
the
present invention will be at least 70% and preferably greater than 80%
homologous to at
least one of the binding sequences disclosed herein.
"KDR binding polypeptide" is a binding polypeptide that forms a complex in
vitro or in vivo with vascular endothelial growth factor receptor-2 (or KDR,
Flk-1);
"VEGF/KDR complex binding polypeptide" is a binding polypeptide that forms
a complex in vitro or in vivo with a binding complex formed between vascular
endothelial growth factor (VEGF) and KDR, in particular the complex of
homodimeric
VEGF and one or two KDR molecules that is believed to form at the surface of
endothelial cells during angiogenesis. Specific examples of KDR and VEGF/KDR
binding polypeptides include but are not limited to the peptides presented
discussed herein,
and in U.S.S.N. 60/360,851 and U.S.S.N. 60/440,441, both of which are
incorporated by
reference herein in their entirety, and in copending application U.S.S.N.
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entitled "KDR and VEGF/KDR binding peptides and their use in diagnosis and
therapy," and include hybrid and chimeric polypeptides incorporating such
peptides as
well as homologues.
"cMet binding polypeptide" is a binding polypeptide that forms a complex in
vitro or in vivo with the HGF receptor, cMet;
"cMet/HGF complex binding polypeptide" is a binding polypeptide that forms a
complex in vitro or in vivo with a binding complex formed between hepatocyte
growth
factor (HGF) and cMet. Specific examples of cMet and cMet/HGF binding
polypeptides
include but are not limited to the peptides presented discussed herein, and in
U.S.S.N.
copending provisional application U.S.S.N. , entitled "Peptides that
Specifically Bind HGF Receptor (cMet) and Uses Thereof," and include hybrid
and
chimeric polypeptides incorporating such peptides as well as homologues.
A "labelling group" or "detectable label," as used herein, is a group or
moiety
capable of generating a signal for diagnostic imaging, such as magnetic
resonance
imaging, radioimaging, ultrasound imaging, x-ray imaging, light imaging, or
carrying a
moiety such as a radioactive metal or other entity that may be used in
radiotherapy or
other forms of therapy.
The term "specificity" refers to a binding polypeptide having a higher binding
affinity for one target over another. Binding specificity may be characterized
by a
dissociation equilibrium constant (KD) or an association equilibrium constant
(Ka) for
the two tested target materials. In a preferred embodiment, binding
polypeptides of the
invention have a dissociation constant for a desired target that is lower than
about 10
M, more preferably lower than about I M, and most preferably less than about
0.5
M or even lower. The term "KDR specificity" refers to a KDR binding moiety
having
a higher affinity for KDR than an irrelevant target. The term "VEGF/KDR
specificity"
refers to a VEGF/KDR complex binding moiety having a higher affinity for a
VEGF/KDR complex than an irrelevant target. In a preferred embodiment,
heteromultimers according to the present invention are specific for KDR or the
VEGF/KDR complex, and preferably have a dissociation constant that is lower
than
about 10 M, more preferably less than about 1 M, most preferably less than
about
0.5 M or even lower. The term "cMet specificity" refers to a cMet binding
moiety
having a higher affinity for cMet than an irrelevant target. The term
"cMet/HGF
CA 02779639 2012-05-18
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specificity" refers to a cMet/HGF complex binding moiety having a higher
affinity for a
cMet/HGF complex than an irrelevant target. In a preferred embodiment, binding
heteromultimers according to the present invention are specific for cMet or
the
cMet/HGF complex, and preferably have a dissociation constant that is lower
than
about 10 AM, more preferably less than about 1 M, most preferably less than
about
0.5 M or even lower.
The term "patient" as used herein refers to any mammal, especially humans.
The term "pharmaceutically acceptable" carrier or excipient refers to a non-
toxic
carrier or excipient that may be administered to a patient, together with a
compound of
this invention, and which does not destroy the pharmacological activity
thereof.
The term "target" or "target molecule" refers to any substance that a binding
moiety or binding polypeptide can bind to, such as proteins or polypeptides,
cells,
receptors, carbohydrates, lipids, etc. As used herein, "target" also includes
a family of
receptors, such as, for example, protein-tyrosine kinase receptors.
The terms "therapeutic agent" or "therapeutic" refer to a compound or an agent
having a beneficial, therapeutic or cytotoxic effect in vivo. Therapeutic
agents include
those compositions referred to as, for example, bioactive agents, cytotoxic
agents,
drugs, chemotherapy agents, radiotherapeutic agents, genetic material, etc.
The following common abbreviations are used throughout this specification: 9-
fluorenylmethyloxycarbonyl (finoc or Fmoc), 1-hydroxybenozotriazole (HOBt),
N,N'-
diisopropylcarbodiimide (DIC), acetic anhydride (Ac20), (4,4-dimethyl-2,6-
dioxocyclohex-l-ylidene)-3-methylbutyl (ivDde), trifluoroacetic acid (TFA),
Reagent B
(TFA:H20:phenol:triisopropylsilane, 88:5:5:2), N,N-diisopropylethylamine
(DIEA), 0-
(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HBTU),O-(7-azabenzotriazol-l-yl)-1,1,3,3-tetramethyluronium
hexafluorphosphate
(HATU), N-hydroxysuccinimide (NHS), solid phase peptide synthesis (SPPS),
dimethyl
sulfoxide (DMSO), dichloromethane (DCM), dimethylformamide (DMF), and N-
methylpyrrolidinone (NMP).
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Dimeric and Multimeric Targeting Constructs of the Invention
The targeting constructs of the invention include two or more binding moieties
which bind to different binding sites of a single target. The binding moieties
are
specific for different sites on the same target. They may be peptidic,
peptidomimetic,
etc and include binding polypeptides as ddefined herein. Additionally, binding
moieties
include small binding molecules. In a preferred embodiment the binding
moieties
comprise binding polypeptides. These targeting constructs are by definition
dimeric or
multimeric and may be referred to as "multivalent targeting constructs,"
"heterodimers," "heteromultimers," or "heteromers." These dimeric or
multimeric
constructs exhibit improved binding, as compared to a monomeric construct.
Where the
constructs comprise binding polypeptides, the polypeptide sequences may be
attached at
their N- or C- terminus or the N-epsilon nitrogen of a suitably placed lysine
moiety (or
another function bearing a selectively derivatizable group such as a pendant
oxyamino
or other nucleophilic group), or may be joined together via one or more
linkers
employing the appropriate attachment chemistry. This coupling chemistry may
include
amide, urea, thiourea, oxime, or aminoacetylamide (from chloro or bromo
acetamide
derivatives), but is not so limited.
Preferred dimers according to the invention can be constructed by connecting a
first binding peptide to a branching group to a first spacer to a linker to
second spacer
and finally to the second binding peptide. This linking scheme for the dimers
can be
represented by the following general structure:
A-B-C-D-E-F
where A and F are two different binding peptides which bind to different sites
on the
same target, B is a branch group, C and E are spacers, and D is a linker.
Suitable
spacers and linkers are known in the art and are also provided in the Examples
below.
In various embodiments, C, D and/or E may optionally be absent. A reporter
moiety or
similar group may optionally be attached to the dimer via the branch group.
The exact
arrangement of these components can vary depending, for example, on whether
the
peptides are linked from C-terminus to C-terminus, from N-terminus to C-
terminus, or
from N-terminus to N-terminus. Examples of these different attachment schemes
are
shown in FIG. 36.
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The preparation of dimeric constructs bearing two different binding peptides
(or
two molecules of a particular peptide) and a labelling group may be
accomplished as
described herein, as well as by other methods known in the art. For example,
fully
protected binding peptides can be built up on Ellman-type safety catch resin
using
automated or manual Fmoc peptide synthesis protocols. See Backes, B.J., et
al., J. Am.
Chem. Soc. (1996), 118(12), 3055-6, which is hereby incorporated by reference
in its
entirety. Separately, using standard methods known in the art of peptide
synthesis (see,
e.g., Fields, G.B. et al., "Principles and Practice of Solid Phase Synthesis"
in Synthetic
Peptides, A Users Guide, Grant, G.A. ed., W.H. Freeman Co. NY. 1992, Chap. 3
pp 77
- 183, which is hereby incorporated by reference in its entirety), a di-lysine
derivative
can be constructed on 2-chlorotrityl resin. See Barlos, K. and Gatos, D.
"Convergent
Peptide Synthesis" in Fmoc Solid Phase Peptide Synthesis, Chan, W.C. and
White, P.D.
eds, Oxford University Press, New York, 2000, Chap 9: pp 215-228, which is
hereby
incorporated by reference in its entirety. Liberation of this derivative from
the 2-
chlorotrityl resin without removal of the side-chain protecting groups,
activation of the
carboxyl group, and coupling to any amine-functionalized labelling group
provides a di-
lysine derivative whose protected pendant nitrogen atoms may be unmasked to
give two
free amino groups. The aforementioned safety-catch resin is activated and the
desired
N-deprotected labelling group-functionalized di-lysine derivative is added to
the
activated safety-catch resin. The pendant amino groups are acylated by the
carboxy-
terminus of the safety-catch resin-bound peptide which is now detached from
the resin
and an integral part of the di-lysine structure. An excess of the safety-catch
resin-bound
peptide can be employed to insure complete reaction of the amino groups of the
di-
lysine construct. Optimization of the ratio of the reacting partners in this
scheme
optimizes the yield. The protecting groups on the binding peptides are removed
employing trifluoroacetic acid based cleavage protocols.
For example, the synthesis of dimeric and multimeric constructs wherein two or
more binding peptides are present in one construct is easily accomplished.
Orthogonal
protection schemes (such as an allyloxycarbonyl group on one nitrogen and an
Fmoc
group on the other, or employing the Fmoc group in conjunction with the iV-Dde
protecting group on the other, for example) can be employed to distinguish the
pendant
nitrogen atoms of the di-lysine derivatives described above. Unmasking of one
of the
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amino groups, followed by reaction of the resulting product with an activated
safety-
catch resin-bound binding peptide as described above, provides a di-lysine
construct
having a single binding peptide attached. Removal of the second protecting
group
unmasks the remaining nitrogen. See, e.g., Mellor, S.L. et al. "Synthesis of
Modified
Peptides" in Fmoc Solid Phase Peptide Synthesis, Chan, W.C. and White, P.D.
eds,
Oxford University Press, New York, 2000, Chap 6: pp 169-176, which is hereby
incorporated by reference in its entirety. The resulting product may be
reacted with a
second safety-catch resin bearing a different binding peptide to provide a
fully-
protected heterodimeric construct, which after removal of protecting groups
with
trifluoroacetic acid, provides the desired material.
Alternatively, a binding peptide is first assembled on a Rink-amide resin by
automated or manual peptide coupling methods, usually employing Fmoc peptide
synthesis protocols. The peptide may possess a C-terminus or N-terminus
functionalized with a linker or a linker-labelling group construct that may
possess an
additional nucleophilic group such as the N-amino group of a lysine moiety,
for
example. Cleavage of the protecting groups is accomplished by employing
trifluoroacetic acid with appropriate modifiers, depending on the nature of
the peptide.
The fully deprotected peptide is then reacted with a large excess of a
bifunctional
electrophile such as glutaric acid bis-N-hydroxysuccinimide ester
(commercially
available from Tyger Scientific Inc., 324 Stokes Avenue, Ewing, NJ, 08638).
The
resulting monoamidated, mono-N-hydroxysuccinimidyl ester of glutaric acid is
then
treated with an additional equivalent of the same peptide, or an equivalent of
a different
binding peptide. Purification of the resulting material by HPLC affords the
desired
homo- or hetero-dimeric construct bearing a suitable labelling group.
In yet another approach, a modular scheme can be employed to prepare dimeric
or higher multimeric constructs bearing suitable labelling groups as defined
above. In a
simple illustration, fmoc-lysine(iV-Dde) Rink amide resin is treated with
piperidine to
remove the fmoc moiety. Then a labelling function, such as biotin, 5-
carboxyfluorescein or N,N-Dimethyl-Gly-Ser(O-t-Bu)-Cys(Acm)-Gly-OH is coupled
to
the nitrogen atom. The resin is next treated with hydrazine to remove the iV-
Dde
group. After thorough washing, the resin is treated with cyanuric chloride and
a
hindered base such as diisopropylethylamine in a suitable solvent such as DMF,
NMP
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or dichloromethane to provide a monofunctionalized dichlorotriazine bound to
the resin.
Subsequent successive displacement of the remaining chlorine atoms either by
two
equivalents of a binding peptide or one equivalent of a binding peptide,
followed by a
second binding peptide provides a resin-bound, hetero- or homo-dimeric,
labelling
group-functionalized construct. See, e.g., Falorni, M., et al., Tetrahedron
Lett. (1998),
39(41), 7607-7610; Johnson, C.R., et al., Tetrahedron (1998), 54(16), 4097-
4106;
Stankova, M. and Lebl, M., Mol. Diversity (1996), 2(1/2), 75-80.
As appropriate, the incoming peptides may be protected or unprotected as the
situation warrants. Cleavage of protecting groups is accomplished employing
trifluoroacetic acid-based deprotection reagents as described above and the
desired
materials are purified by high performance liquid chromatography.
It is understood that in each of these methods, lysine derivatives, omithine,
or
2,3-diamino propionic acid may be serially employed to increase the
multiplicity of the
multimers. The use of related, more rigid molecules bearing the requisite
number of
masked, or orthogonally protected nitrogen atoms to act as scaffolds, to vary
the
distance between the binding peptides, and to increase the rigidity of the
construct (by
constraining the motion and relative positions of the binding peptides
relative to each
other and the reporter) is entirely within the scope of the synthetic methods
described
herein.
Direct synthesis of the binding polypeptides may be accomplished using
conventional techniques, including solid-phase peptide synthesis, solution-
phase
synthesis, etc. Solid-phase synthesis is preferred. See Stewart et al., Solid-
Phase
Peptide Synthesis (1989), W. H. Freeman Co., San Francisco; Merrifield, J. Am.
Chem.
Soc., 85:2149-2154 (1963); Bodanszky and Bodanszky, The Practice of Peptide
Synthesis (Springer-Verlag, New York 1984), incorporated herein by reference.
Polypeptides of the invention may also be prepared commercially by companies
providing peptide synthesis as a service (e.g., BACHEM Bioscience, Inc., King
of
Prussia, PA; Quality Controlled Biochemicals, Inc., Hopkinton, MA). Automated
peptide synthesis machines, such as manufactured by Perkin-Elmer Applied
Biosystems, also are available.
The polypeptide compound is preferably purified once it has been isolated or
synthesized by either chemical or recombinant techniques. For purification
purposes,
CA 02779639 2012-05-18
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there are many standard methods that may be employed, including reverse-phase
high-pressure liquid chromatography (RP-HPLC) using an alkylated silica column
such
as C4-, C8- or C18-silica. A gradient mobile phase of increasing organic
content is
generally used to achieve purification, for example, acetonitrile in an
aqueous buffer,
usually containing a small amount of trifluoroacetic acid. Ion-exchange
chromatography can also be used to separate peptides based on their charge.
The
degree of purity of the polypeptide may be determined by various methods,
including
identification of a major large peak on HPLC. A polypeptide that produces a
single
peak that is at least 95% of the input material on an HPLC column is
preferred. Even
more preferable is a polypeptide that produces a single peak that is at least
97%, at least
98%, at least 99% or even 99.5% or more of the input material on an HPLC
column.
To ensure that the peptide obtained using any of the techniques described
above
is the desired peptide for use in compositions of the present invention,
analysis of the
peptide composition may be carried out. Such composition analysis may be
conducted
using high resolution mass spectrometry to determine the molecular weight of
the
peptide. Alternatively, the amino acid content of the peptide can be confirmed
by
hydrolyzing the peptide in aqueous acid, and separating, identifying and
quantifying the
components of the mixture using HPLC, or an amino acid analyzer. Protein
sequenators, which sequentially degrade the peptide and identify the amino
acids in
order, may also be used to determine the sequence of the peptide.
For example, binding polypeptides also may be produced using recombinant
DNA techniques, utilizing nucleic acids (polynucleotides) encoding the
polypeptides of
the invention, and then expressing them recombinantly, i.e., by manipulating
host cells
by introduction of exogenous nucleic acid molecules in known ways to cause
such host
cells to produce the desired binding polypeptides. Such procedures are within
the
capability of those skilled in the art (see Davis et al., Basic Methods in
Molecular
Biolo , (1986)), which is hereby incorporated by reference in its entirety.
Recombinant production of short peptides such as those described herein may
not be
practical in comparison to direct synthesis, however recombinant means of
production
may be very advantageous where a binding moiety of this invention is
incorporated in a
hybrid polypeptide or fusion protein.
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In the practice of one embodiment of the present invention, a determination of
the affinity of the heteromultimer or a constituent binding moiety for the
target relative
to another protein or target is a useful measure, and is referred to as
affinity for the
target. Standard assays for quantitating binding and determining affinity
include
equilibrium dialysis, equilibrium binding, gel filtration, or the monitoring
of numerous
spectroscopic changes (such as a change in fluorescence polarization) that may
result
from the interaction of the binding moiety and its target. These techniques or
modifications thereof measure the concentration of bound and free ligand as a
function
of ligand (or protein) concentration. The concentration of bound
heteromultimer or
polypeptide ([Bound]) is related to the concentration of free heteromultimer
or
polypeptide ([Free]) and the concentration of binding sites for the
polypeptide, i.e., on
KDR, VEGF/KDR complex, cMet, or the cMet/HGF complex (N), as described in the
following equation:
[Bound] = N x [Free]/((l/Ka)+[Free]).
A solution of the data to this equation yields the association constant, Ka, a
quantitative
measure of the binding affinity. The association constant, Ka is the
reciprocal of the
dissociation constant, KD. The KD is more frequently reported in measurements
of
affinity. In a preferred embodiment heteromultimers of the invention and
constituent
binding polypeptides bind to the target, e.g. KDR, VEGF/KDR complex, cMet or
cMet/HGF and have a KD for the target in the range of 1 nanomolar (nM) to 100
micromolar (.tM) and preferably have KD values less than 50 M, preferably
less than 1
M, more preferably less than 50 nM, and most preferably less than 10 nM.
Where heteromultimers are employed as imaging agents, other aspects of
binding affinity may become more important. For example, such imaging agents
operate in a dynamic system in that binding of the imaging agent to the target
(such as
KDR or VEGF/KDR complex, e.g., on activated endothelium) is not in a stable
equilibrium state throughout the imaging procedure. For example, when the
imaging
agent is initially injected, the concentration of imaging agent and of agent-
target
complex rapidly increases. Shortly after injection, however, the circulating
(free)
imaging agent starts to clear through the kidneys or liver, and the plasma
concentration
of imaging agent begins to drop. This drop in the concentration of free
imaging agent
in the plasma eventually causes the agent-target complex to dissociate. The
usefulness
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of an imaging agent depends on the difference in rate of agent-target
dissociation
relative to the clearing rate of the agent. Ideally, the dissociation rate
will be slow
compared to the clearing rate, resulting in a long imaging time during which
there is a
high concentration of agent-target complex and a low concentration of free
imaging
agent (background signal) in the plasma.
An advantage of heteromultimeric binding compounds, such as those of the
present invention, is that they generally possess very slow dissociation rates
relative to
their constituent monomers (see Tissot et al., J. Immunol. Methods 236(1-
2):147-165
(2000)). In addition, heteromultimeric compounds capable of binding to two
distinct
epitopes on a target molecule simultaneously can achieve multimeric binding
regardless
of the distance between target molecules on the cell surface. Homomultimeric
binding
compounds, on the other hand, depend on the presence of two or more target
molecules
being in close enough proximity such that the homomultimer can span the
distance
between them. Thus, the heteromultimeric binding compounds of the present
invention
are particularly well suited for binding to receptors and other cell surface
molecules that
are less abundant and therefore more distant from each other on the cell
surface.
Quantitative measurement of dissociation rates may be easily performed using
several methods known in the art, such as fiber optic fluorimetry (see, e.g.,
Anderson
and Miller, Clin. Chem., 34(7):1417-21 (1988)), surface plasmon resonance
(see,
Malmborg et al., J. Immunol. Methods, 198(1):51-7 (1996) and Schuck, Current
Opinion in Biotechnology, 8:498-502 (1997)), resonant mirror, and grating
coupled
planar waveguiding (see, e.g., Hutchinson, Molec. Biotechnology, 3:47-54
(1995)).
Automated biosensors are commercially available for measuring binding
kinetics:
BlAcore surface plasmon resonance sensor (Biacore AB, Uppsala SE), lAsys
resonant
mirror sensor (Fisons Applied Sensor Technology, Cambridge GB), BIOS-1 grated
coupled planar waveguiding sensor (Artificial Sensor Instruments, Zurich CH).
Modification or Optimization of Binding Polypeptides
Modification or optimization of heteromultimers is within the scope of the
present invention. In particular, modified or optimized heteromultimers are
included
within the definition of "heteromultimers". Similarly, modified or optimized
binding
polypeptides are included within the definition of "binding polypeptides" and
the
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phrase "KDR and VEGF/KDR complex binding polypeptides" includes modified or
optimized KDR and VEGF/KDR binding polypeptides, and the phrase "cMet and
cMet/HGF complex binding polypeptides" includes modified or optimized cMet and
cMet/HGF binding polypeptides. Specifically, a polypeptide sequence for use in
the
heteromultimers of the invention can be modified to optimize its potency,
pharmacokinetic behavior, stability and/or other biological, physical and
chemical
properties.
Substitution of Amino Acid Residues
Susbtitutions of amino acids within the same class (e.g., substituting one
basic
amino acid for another) are well known in the art. For example, one can make
the
following isosteric and/or conservative amino acid changes in the parent
polypeptide
sequence with the expectation that the resulting polypeptides would have a
similar or
improved profile of the properties described above:
Substitution of alkyl-substituted hydrophobic amino acids: Including alanine,
leucine,
isoleucine, valine, norleucine, S-2-aminobutyric acid, S-cyclohexylalanine or
other
simple alpha-amino acids substituted by an aliphatic side chain from 1-10
carbons
including branched, cyclic and straight chain alkyl, alkenyl or alkynyl
substitutions.
Substitution of aromatic-substituted hydrophobic amino acids: Including
phenylalanine,
tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 2-
benzothienylalanine, 3-benzothienylalanine, histidine, amino, alkylamino,
dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy
(from C,-C4)-
substituted forms of the previous listed aromatic amino acids, illustrative
examples of
which are: 2-, 3-, or 4-aminophenylalanine, 2-, 3-, or 4-chlorophenylalanine,
2-, 3-, or
4-methylphenylalanine, 2-, 3-, or 4-methoxyphenylalanine, 5-amino-, 5-chloro-,
5-
methyl- or 5-methoxytryptophan, 2'-, 3'-, or 4'-amino-, 2'-, 3'-, or 4'-chloro-
, 2, 3, or 4-
biphenylalanine, 2'-, 3'-, or 4'-methyl- 2-, 3- or 4-biphenylalanine, and 2-
or 3-
pyridylalanine.
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Substitution of amino acids containing basic functions: Including arginine,
lysine,
histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl,
or aryl-
substituted (from C,-C1o branched, linear, or cyclic) derivatives of the
previous amino
acids, whether the substituent is on the heteroatoms (such as the alpha
nitrogen, or the
distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position
for example.
Compounds that serve as illustrative examples include: N-epsilon-isopropyl-
lysine, 3-
(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine, N,N-gamma,
gamma'-
diethyl-homoarginine. Included also are compounds such as alpha methyl
arginine,
alpha methyl 2,3-diaminopropionic acid, alpha methyl histidine, alpha methyl
ornithine
where alkyl group occupies the pro-R position of the alpha carbon. Also
included are
the amides formed from alkyl, aromatic, heteroaromatic (where the
heteroaromatic
group has one or more nitrogens, oxygens or sulfur atoms singly or in
combination)
carboxylic acids or any of the many well-known activated derivatives such as
acid
chlorides, active esters, active azolides and related derivatives) and lysine,
ornithine, or
2,3- diaminopropionic acid.
Substitution of acidic amino acids: Including aspartic acid, glutamic acid,
homoglutamic acid, tyrosine, alkyl, aryl, aralkyl, and heteroaryl sulfonamides
of 2,3-
diaminopropionic acid, ornithine or lysine and tetrazole-substituted alkyl
amino acids.
Substitution of side chain amide residues: Including asparagine, glutamine,
and alkyl or
aromatic substituted derivatives of asparagine or glutamine.
Substitution of hydroxyl containing amino acids: Including serine, threonine,
homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substituted
derivatives of
serine or threonine.
It is also understood that the amino acids within each of the categories
listed
above may be substituted for another of the same group.
Substitution of Amide Bonds
Another type of modification within the scope of the invention is the
substitution
of amide bonds within the backbone of a binding polypeptide. For example, to
reduce
or eliminate undesired proteolysis, or other degradation pathways which
diminish serum
CA 02779639 2012-05-18
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stability, resulting in reduced or abolished bioactivity, or to restrict or
increase
conformational flexibility, it is common to substitute amide bonds within the
backbone
of the peptides with functionality that mimics the existing conformation or
alters the
conformation in the manner desired. Such modifications may produce increased
binding affinity or improved pharmacokinetic behavior. It is understood that
those
knowledgeable in the art of peptide synthesis can make the following amide
bond
changes for any amide bond connecting two amino acids with the expectation
that the
resulting peptides could have the same or improved activity: insertion of
alpha-N-
methylamides or peptide amide backbone thioamides, removal of the carbonyl to
produce the cognate secondary amines, replacement of one amino acid with an
aza-
aminoacid to produce semicarbazone derivatives, and use of E-olefins and
substituted
E-olefins as amide bond surrogates.
Introduction of D-Amino Acids
Another approach within the scope of the invention is the introduction of D-
alanine, or another D-amino acid, distal or proximal to a labile peptide bond.
In this
case it is also understood to those skilled in the art that such D-amino acid
substitutions
can, and at times, must be made, with D-amino acids whose side chains are not
conservative replacements for those of the L-amino acid being replaced. This
is
because of the difference in chirality and hence side-chain orientation, which
may result
in the accessing of a previously unexplored region of the binding site of the
target
which has moieties of different charge, hydrophobicity, steric requirements,
etc., than
that serviced by the side chain of the replaced L-amino acid.
Modifications To Improve Pharmacokinetic or Pharmacodynamic Properties
It is also understood that use of the heteromultimeric constructs of the
invention
in a particular application may necessitate modifications of the peptide or
formulations
of the peptide to improve pharmacokinetic and pharmacodynamic behavior. It is
expected that the properties of the peptide may be changed by attachment of
moieties
anticipated to bring about the desired physical or chemical properties. Where
the
heteromultimer includes binding polypeptides, such moieties affecting the
pharmacokinetic and pharmacodynamic behavior may be appended to the peptide
using
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acids or amines, via amide bonds or urea bonds, respectively, to the N- or C-
terminus of
the peptide, or to the pendant amino group of a suitably located lysine or
lysine
derivative, diaminopropionic acid, ornithine, or other amino acid in the
peptide that
possesses a pendant amine group or a pendant alkoxyamino or hydrazine group.
The
moieties introduced may be groups that are hydrophilic, basic, or nonpolar
alkyl or
aromatic groups depending on the peptide of interest and the extant
requirements for
modification of its properties.
Glycosylation of Amino Acid Residues
Yet another modification within the scope of the invention is to employ
glycosylated amino acid residues (e.g. serine, threonine or asparagine
residues), singly
or in combination in the either the binding or the linker moiety or both.
Glycosylation,
which may be carried out using standard conditions, may be used to enhance
solubility,
alter pharmacokinetics and pharmacodynamics or to enhance binding via a
specific or
non-specific interaction involving the glycosidic moiety. In another approach
glycosylated amino acids such as O-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-(3-
D-
glucopyranosyl) serine or the analogous threonine derivative (either the D- or
L- amino
acids) may be incorporated into the peptide during manual or automated solid
phase
peptide synthesis, or in manual or automated solution phase peptide synthesis.
Similarly D- or L-NY-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-p-D-
glucopyranosyl)-
asparagine can be employed. The use of amino acids glycosylated on a pendant
oxygen,
nitrogen or sulfur function by the agency of suitably functionalized and
activated
carbohydrate moieties that can be employed in glycosylation is anticipated.
Such
carbohydrate functions could be monosaccharides, disaccharides or even larger
assemblies of oligosaccharides (Kihlberg, Jan. (2000) Glycopeptide synthesis.
In: Fmoc
Solid Phase Peptide Synthesis - A Practical Approach (Chan, W.C. and White,
P.D.
Eds) Oxford University Press, New York, NY Chap. 8, pp 195-213).
Also anticipated is the appendage of carbohydrate functions to amino acids by
means other than glycosylation via activation of a leaving group at the
anomeric carbon.
Linkage of the amino acid to the glycoside is not limited to the formation of
a bond to
the anomeric carbon of the carbohydrate function. Instead, linkage of the
carbohydrate
moiety to the amino acid could be through any suitable, sufficiently reactive
oxygen
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atom, nitrogen atom, carbon atom or other pendant atom of the carbohydrate
function
via methods employed for formation of C-heteroatom, C-C or heteroatom-
heteroatom
(examples are S-S, O-N, N-N, P-0, P-N) bonds known in the art.
Formation of Salts
It is also within the scope of the invention to form different salts that may
increase the water solubility or the ease of formulation of these peptides.
These may
include, but are not restricted to, N-methylglucamine (meglumine), acetate,
oxalates,
ascorbates etc.
Structural Modifications which Retain Structural Features
Yet another modification within the scope of the invention is truncation of
cyclic
polypeptides. The cyclic nature of many polypeptides of the invention limits
the
conformational space available to the peptide sequence, particularly within
the cycle.
Therefore truncation of the peptide by one or more residues distal or even
proximal to
the cycle, at either the N-terminal or C-terminal region may provide truncated
peptides
with similar or improved biological activity. A unique sequence of amino
acids, even
as small as three amino acids, which is responsible for the binding activity,
may be
identified, as noted for RGD peptides. See e.g., E.F. Plow et al., Blood
(1987), 70(1),
110-5; A. Oldberg et al., Journal of Biological Chemistry (1988), 263(36),
19433-
19436; R. Taub et al., Journal of Biological Chemistry (1989 Jan. 5), 264(1),
259-65; A.
Andrieux et al., Journal of Biological Chemistry (1989 Jun. 5), 264(16), 9258-
65; and
U.S. Patent Nos. 5,773,412 and 5,759,996, each of which is incorporated herein
by
reference in its entirety.
It has also been shown in the literature that large peptide cycles can be
substantially shortened, eliminating extraneous amino acids, but substantially
including
the critical binding residues. See U.S. Patent No. 5,556,939, which is
incorporated
herein by reference in its entirety. Shortened cyclic peptides can be formed
using
disulfide bonds or amide bonds of suitably located carboxylic acid groups and
amino
groups.
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Furthermore, D-amino acids can be added to the peptide sequence to stabilize
turn features (especially in the case of glycine). In another approach alpha,
beta,
gamma or delta dipeptide or turn mimics (such as a, 0,,y, or 8 turn mimics)
some of
which are shown in structures 1, 2 and 3, below, can be employed to mimic
structural
motifs and turn features in a peptide and simultaneously provide stability
from
proteolysis and enhance other properties such as, for example, conformational
stability
and solubility (structure 1: Hart et al., J. Org. Chem., 64, 2998-2999(1999);
structure 2:
Hanessian et al., "Synthesis of a Versatile Peptidomimetic Scaffold" in
Methods in
Molecular Medicine, Vol. 23: Peptidomimetics Protocols, W.M. Kazmierski Ed.
(Humana Press Inc. Totowa N.J. 1999), Chapter 10, pp. 161-174; structure 3: WO
01/16135.
,R1 OR1
OH \
R2--~ H N
N
N N
N
O -O
O
NHBoc COOH R3 N
R2
2
3
Substitution of Disulfide Mimetics
Also included within the scope of the invention is the substitution of
disulfide
mimetics for disulfide bonds within the binding polypeptides of the invention.
When
disulfide-containing peptides are employed in generating heteromultimeric
constructs,
the disulfide bonds might need to be replaced to avoid certain difficulties
that are
sometimes posed by the presence of a disulfide bond. For example, when
generating
heteromultimeric 99MTc (or other radionuclide)-based radiopharmaceuticals or
certain
other hetermultimeric constructs the presence of the disulfide bond can be a
significant
problem. The integrity of the disulfide bond is difficult to maintain during
procedures
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WO 03/084574 PCT/US03/06656
designed to incorporate"Tc via routes that are reliant upon the reduction of
pertechnetate ion and subsequent incorporation of the reduced Tc species into
substances bearing Tc-compatible chelating groups. This is because the
disulfide bond
is rather easily reduced by the reducing agents commonly used in kits devised
for one-
step preparation of radiopharmaceuticals. Therefore, the ease with which the
disulfide
bond can be reduced during Tc chelation may require substitution with mimetics
of the
disulfide bonds. Accordingly, another modification within the scope of the
invention is
to substitute the disulfide moiety with mimetics, utilizing the methods
disclosed herein
or known to those skilled in the art, while retaining the activity and other
desired
properties of the binding polypeptides used in the invention:
1.) Oxime linker
The oxime moiety has been employed as a linker by investigators in a number of
contexts. Of the most interest is the work by Wahl, F and Mutter, M,
Tetrahedron Lett.
(1996) 37, 6861-6864). The amino acids containing an aminoalcohol function
(4), and
containing an alkoxyamino function (5), are incorporated into the peptide
chain, not
necessarily at the end of the peptide chain. After formation of the peptide,
the sidechain
protecting groups are removed. The aldehyde group is unmasked and an oxime
linkage
is formed.
HN'Trt
I
O
Boc-NH
40 Y
'f NH NH
Fmoc-NH COOH Fmoc-NHfCOOH
Fmoc-Dap(Boc-Ser(t-Bu))-OH Fmoc-Dap(TrtAoa)-OH
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2.) Lanthionine Linker
Lanthionines are cyclic sulfides, wherein the disulfide linkage (S-S) is
replaced
by a (C-S) linkage. Thus the lability to reduction is far lower and this
linkage should be
stable to stannous chloride. Lanthionines may be prepared by a number of
methods.
Preparation of Lanthionines using Bromoacetylated Peptides
Lanthionines are readily prepared using known methods. See, for example,
Robey et al. (Robey, F.A. and Fields, R.L. Anal. Biochem. (1989)177,373-377)
and
Inman, et al. (Inman, J.K.; Highet, P.F.; Kolodny, N.; and Robey, F.A.
Bioconjugate
Chem. (1991) 2, 458-463; Ploinsky, A. Cooney, M.C. Toy-Palmer, A. Osapay, G.
and
Goodman, M. J. Med. Chem. (1992) 35, 4185-4194; Mayer, J.P.; Zhang, J.; and
Liu,
C.F. in : Tam, J.P. and Kaumaya, P.T.P. (eds), "Peptides, Frontiers of Peptide
Science,"
Proceedings of the 15th American Peptide Symposium, June 14-19 Nashville,
Tenn.
Klumer Academic Pub. Boston. pp 291-292;. Wakao, Norihiro; Hino, Yoichi;
Ishikawa, Ryuichi. Jpn. Kokai Tokkyo Koho (1995), 7 pp. JP 07300452 A2
19951114 Heisei; JP 95-49692 19950309; JP 94-41458 19940311 have published in
this area. Preparation of peptides using Boc automated peptide synthesis
followed by
coupling the peptide terminus with bromoacetic acid gives bromoacetylated
peptides in
good yield. Cleavage and deprotection of the peptides is accomplished using
HF/anisole. If the peptide contains a cysteine group its reactivity can be
controlled with
low pH. If the pH of the medium is raised to 6-7, then either polymerization
or
cyclization of the peptide takes place. Polymerization is favored at high (100
mg/mL)
concentration, whereas cyclization is favored at lower concentrations (1
mg/mL), e.g.,
in Scheme 1 below, 6 cyclizes to 7.
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Scheme I - Example of Cyclization of Cysteine with a Pendant Bromoacetamide
Function
R2
R1 O
O R1 O O
~ _ 1_4
O ~'~ N NH
H -1 mg/ML O N NH
R4 NH H
NH O
N SH NH2 N 2 R4 N H N
2 H S
O H~~r ~H 0N
O O
6 7
Inman et al. demonstrated the use of N-(Boc)-N-[N-(bromoacetyl)-o-alanyl]-
L-lysine as a carrier of the bromoacetyl group that could be employed in Boc
peptide
synthesis thus allowing placement of a bromoacetyl bearing moiety anywhere in
a
sequence. In preliminary experiments they found that peptides with 4-6 amino
acids
separating the bromoacetyl-lysine derivative from a cysteine tend to cyclize,
indicating
the potential utility of this strategy.
Preparation of Lanthionines via Cysteine Thiol Addition to Acrvlamides
Several variants of this strategy may be implemented. Resin-bound serine can
be
employed to prepare the lanthionine ring on resin either using a bromination-
dehydrobromination-thiol addition sequence or by dehydration with
disuccinimidyl
carbonate followed by thiol addition. Ploinsky et al., M. J Med. Chem.,
35:4185-4194
(1992); Mayer et al., "Peptides, Frontiers of Peptide Science", in Proceedings
of the 15`h
American Peptide Symposium, Tam & Kaumaya (eds), June 14-19, 1995, Nashville,
Tenn. (Klumer Academic Pub. Boston) pp. 291-292. Conjugate addition of thiols
to
acrylamides has also been amply demonstrated and a reference to the addition
of 2-
mercaptoethanol to acrylamide is provided. Wakao et al., Jpn. Kokai Tokkyo
Koho, JP
07300452 A2 (1995).
3.) Diaryl Ether or Diarylamine Linkage
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Diary Ether Linkage From Intramolecular Cyclization of
1 Boronic Acids and Tyrosine
The reaction of arylboronic acids with phenols, amines and heterocyclic amines
in the presence of cupric acetate, in air, at ambient temperature, in
dichloromethane
using either pyridine or triethylamine as a base to provide unsymmetrical
diaryl ethers
and the related amines in good yields (as high as 98%) has been reported. See,
Evans
et al., Tetrahedron Lett., 39:2937-2940 (1998); Chan et al., Tetrahedron
Lett., 39:2933-
2936 (1998); Lam et al., Tetrahedron Lett., 39:2941-2944 (1998). In the case
of N-
protected tyrosine derivatives as the phenol component the yields were also as
high as
98%. This demonstrates that amino acid amides (peptides) are expected to be
stable to
the transformation and that yields are high. Precedent for an intramolecular
reaction
exists in view of the facile intramolecular cyclizations of peptides to
lactams,
intramolecular biaryl ether formation based on the SNAr reaction and the
generality of
intramolecular cyclization reactions under high dilution conditions or on
resin, wherein
the pseudo-dilution effect mimics high dilution conditions.
4.) Formation of Cyclic Peptides with a Lactam Linkage via Intramolecular
Native Chemical Ligation
Scheme 2 - Formation of Cyclic Peptides with a Thiazolidine Linkage via
Intramolecular Reaction of Peptide Aldehydes with Cysteine Moieties
NH, NH= I ^i000' NH,
S,S-~ Na10, SIS 000 SH ?0, o N O
N N ON N O 0 H=N~ ' !1 O H
HO
H
0
/
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Another approach that may be employed involves intramolecular cyclization of
suitably located vicinal amino mercaptan functions (usually derived from
placement of
a cysteine at a terminus of the linear sequence or tethered to the sequence
via a side-
chain nitrogen of a lysine, for example) and aldehyde functions to provide
thiazolidines
which result in the formation of a bicyclic peptide, one ring of which is that
formed by
the residues in the main chain, and the second ring being the thiazolidine
ring. Scheme
2, above, provides an example. The required aldehyde function can be generated
by
sodium metaperiodate cleavage of a suitably located vicinal aminoalcohol
function,
which can be present as an unprotected serine tethered to the chain by
appendage to a
side chain amino group of a lysine moiety. In some cases, the required
aldehyde
function is generated by unmasking of a protected aldehyde derivative at the C-
terminus
or the N-terminus of the chain. An example of this strategy is found in:
Botti, P.; Pallin,
T.D. and Tam, J.P. J. Am. Chem. Soc. 1996,118,10018-10034,
5.) Lactams Based on Intramolecular Cyclization of Pendant Amino Groups
with Carboxyl Groups on Resin
Macrocyclic peptides can be prepared by lactam formation by either head to
tail
or by pendant group cyclization. The basic strategy is to prepare a fully
protected
peptide wherein it is possible to remove selectively an amine protecting group
and a
carboxy protecting group. Orthogonal protecting schemes have been developed.
Of
those that have been developed, the allyl, trityl and Dde methods have been
employed
most. See, Mellor et al., "Synthesis of Modified Peptides," in Fmoc Solid
Phase
Synthesis: A Practical Approach, White and Chan (eds) ([Oxfoerd University
Press,,
New York, 2000]), Chapt. 6, pp. 169-178. The Dde approach is of interest
because it
utilizes similar protecting groups for both the carboxylic acid function (Dmab
ester) and
the amino group (Dde group). Both are removed with 2-10% hydrazine in DMF at
ambient temperature. Alternatively, the Dde can be used for the amino group
and the
allyl group can be used for the carboxyl.
A lactam function, available by intramolecular coupling via standard peptide
coupling reagents (such as HATU, PyBOP etc), could act as a surrogate for the
disulfide
bond. The Dde/Dmab approach is shown in Scheme 3a, below.
44
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Scheme 3a - Lactam Surrogate for the Disulfide Bond via Quasiorthogonal
Deprotection of Lys and
Asp Followed by On-Resin Cyclization and Cleavage from Resin
R RõR,,Rn
R fn q q a'a'NHBOC R n=1o q q q N-UX'NHBoc
O NN~lN,
DMF
i O
O
, 4&~
12
NATWNOAt
RõRõRn Q
M N-Z,92,NH2 -A Rlnk q _,Jl, ,q )LNt&NHBoc
WLc
14 U
Thus, a linear sequence containing, for example, the Dde-protected lysine and
Dmab
ester may be prepared on a Tentagel-based Rink amide resin at low load (-0.1-
0.2
mmol/g). Deprotection of both functions with hydrazine is then followed by on-
resin
cyclization to give the desired products.
CA 02779639 2012-05-18
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Scheme 3b - Lactam Surrogate for the Disulfide Bond via Quasiorthogonal
Deprotection of Lys and
Asp Using Allyl-based Protecting Groups Followed by On-Resin Cyclization and
Cleavage from Resin
R,,R,,Rn 11 RõRõRn
Raakn N N H N-~N~NHBoe Rac n N N' v ^ tiN~NHBOc
H H O H H
t.) Pd(PhP) fNOAC
O~~riH NMM/DMF NH O
70 i H1
2) sodlam
dlothytdlthioarbamate
DIENDMF 13
HATUIHOAt
RõRõRn
MM R,.R,,Rn
XN H ~N N~N~NN, Tom, FA ~~ ~ N-~õN-~NNBoc
HPLC H
AN
In the ally] approach, shown in Scheme 3b, the pendant carboxyl which is to
undergo cyclization is protected as an allyl ester and the pendant amino group
is
protected as an alloc group. On resin, both are selectively unmasked by
treatment with
palladium tris-triphenylphosphine in the presence of N-methylmorpholine and
acetic
acid in DMF. Residual palladium salts are removed using sodium
diethyldithiocarbamate in the presence of DIEA in DMF, followed by subsequent
washings with DMF. The lactam ring is then formed employing HATU/HOAt in the
presence of N-methylmorpholine. Other coupling agents can be employed as
described
above. The processing of the peptide is then carried out as described above to
provide
the desired peptide lactam.
Subsequently cleavage from resin and purification may also be carried out. For
functionalization of the N-terminus of the peptide, it is understood that
amino acids,
such as trans-4-(iV-Dde)methylaminocyclohexane carboxylic acid, trans-4-(iV-
Dde)methylaminobenzoic acid, or their alloc congeners could be employed. Yet
another approach is to employ the safety catch method to intramolecular lactam
formation during cleavage from the resin.
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6.) Cyclic Peptides Based on Olefin Metathesis
The Grubbs reaction (Scheme 4, below) involves the metathesis/cyclization of
olefin bonds and is illustrated as shown below. See, Schuster et al.,
Angewandte. Chem.
Int. Edn Engl., 36:2036-2056 (1997); Miller et al., J. Am. Chem. Soc.,
118:9606-9614
(1996).
Scheme 4 - Grubbs Olefin Metathesis Cyclization
CY'
I Ph 1 \ 2 Basic Grubbs reaction
R1 2 Ci%iu-/
= Optional tether
16 17
It is readily seen that, if the starting material is a diolefin (16), the
resulting
product will be cyclic compound 17. The reaction has in fact been applied to
creation
of cycles from olefin-functionalized peptides. See, e.g., Pernerstorfer et
al., Chem.
Commun., 20:1949-50 (1997); Covalent capture and stabilization of cylindrical
(3-sheet
peptide assemblies, Clark et al., Chem.Eur. J., 5(2):782-792 (1999); Highly
efficient
synthesis of covalently cross-linked peptide helices by ring-closing
metathesis,
Blackwell et al., Angew. Chem., Int. Ed., 37(23):3281-3284 (1998); Synthesis
of novel
cyclic protease inhibitors using Grubbs olefin metathesis, Ripka et al., Med.
Chem.
Lett., 8(4):357-360 (1998); Application of Ring-Closing Metathesis to the
Synthesis of
Rigidified Amino Acids and Peptides, Miller et al., J. Am. Chem. Soc.,
118(40):9606-
9614 (1996); Supramolecular Design by Covalent Capture, Design of a Peptide
Cylinder via Hydrogen-Bond- Promoted Intermolecular Olefin Metathesis, Clark
et al.,
J. Am. Chem. Soc., 117(49):12364-12365 (1995); Synthesis of Conformationally
Restricted Amino Acids and Peptides Employing Olefin Metathesis, Miller et
al., J. Am.
Chem. Soc., 117(21):5855-5856 (1995). One can prepare either C-allylated amino
acids
or possibly N-allylated amino acids and employ them in this reaction in order
to prepare
carba-bridged cyclic peptides as surrogates for disulfide bond containing
peptides.
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One may also prepare novel compounds with olefinic groups. Functionalization
of the tyrosine hydroxyl with an olefin-containing tether is one option. The
lysine s-
amino group may be another option with appendage of the olefin-containing unit
as part
of an acylating moiety, for example. If instead the lysine side chain amino
group is
alkylated with an olefin containing tether, it can still function as a point
of attachment
for a reporter as well. The use of 5-pentenoic acid as an acylating agent for
the lysine,
ornithine, or diaminopropionic side chain amino groups is another possibility.
The
length of the olefin-containing tether can also be varied in order to explore
structure
activity relationships.
Manipulation of Peptide Sequences
Other modifications within the scope of the invention include manipulations of
peptide sequences which can be expected to yield peptides with similar or
improved
biological properties. These include amino acid translocations (swapping amino
acids
in the sequence), use of retroinverso peptides in place of the original
sequence or a
modified original sequence, peptoids, retro-inverso peptoid sequences, and
synthetic
peptides. Structures wherein specific residues are peptoid instead of
peptidic, which
result in hybrid molecules, neither completely peptidic nor completely
peptoid, are
contemplated as well.
Linkers
Additionally, modifications within the invention include introduction of
linkers
or spacers between the targeting sequence of the binding moiety or binding
polypeptide
and the detectable label or therapeutic agent. For example, use of such
linkers/spacers
may improve the relevant properties of the binding peptides (e.g. increase
serum
stability, etc.). These linkers may include, but are not restricted to,
substituted or
unsubstituted alkyl chains, polyethylene glycol derivatives, amino acid
spacers, sugars,
or aliphatic or aromatic spacers common in the art.
For example, suitable linkers include homobifunctional and heterobifunctional
cross-linking molecules. The homobifunctional molecules have at least two
reactive
functional groups, which are the same. The reactive functional groups on a
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homobifunctional molecule include, for example, aldehyde groups and active
ester
groups. Homobifunctional molecules having aldehyde groups include, for
example,
glutaraldehyde and subaraldehyde.
Homobifunctional linker molecules having at least two active ester units
include
esters of dicarboxylic acids and N-hydroxysuccinimide. Some examples of such N-
succinimidyl esters include disuccinimidyl suberate and dithio-bis-
(succinimidyl
propionate), and their soluble bis-sulfonic acid and bis-sulfonate salts such
as their
sodium and potassium salts.
Heterobifunctional linker molecules have at least two different reactive
groups.
Some examples of heterobifunctional reagents containing reactive disulfide
bonds
include N-succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson et al., 1978,
Biochem
J. 173:723-737), sodium S-4-succinimidyloxycarbonyl-alpha-
methylbenzylthiosulfate,
and 4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene. N-
succinimidyl
3-(2-pyridyldithio)propionate is preferred. Some examples of
heterobifunctional
reagents comprising reactive groups having a double bond that reacts with a
thiol group
include succinimidyl 4-(N-maleimidomethyl)cyclohexahe- l -carboxylate and
succinimidyl m-maleimidobenzoate. Other heterobifunctional molecules include
succinimidyl 3-(maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-
phenyl)butyrate, sulfosuccinimidyl 4-(N-maleimidomethyl-cyclohexane)-1-
carboxylate,
maleimidobenzoyl-5N-hydroxy-succinimide ester.
Furthermore, linkers which are combinations of the molecules and/or moieties
described above, can also be employed to confer special advantage to the
properties of
the peptide. Lipid molecules with linkers may be attached to allow formulation
of
ultrasound bubbles, liposomes or other aggregation based constructs. Such
constructs
could be employed as agents for targeting and delivery of a diagnostic
reporter, a
therapeutic agent (e.g. a chemical "warhead" for therapy), or a combination of
these.
Uses of Heteromultimeric Constructs
Heteromultimeric constructs of the present invention can be used in a
multitude
of applications, including immunoassays (e.g., ELISA), as pharmaceuticals
useful for
treatments of various diseases, as well as in in vivo diagnostic and
therapeutic uses. For
example, the heteromultimeric constructs described herein will be extremely
useful for
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detection and/or imaging of target containing tissue in vitro or in vivo. For
example,
KDR or VEGF/KDR complex binding heteromultimeric constructs will be extremely
useful for detection and/or imaging of KDR or VEGF/KDR complex containing
tissue,
and particularly for detection and/or imaging of sites of angiogenesis, in
which VEGF
and KDR are intimately involved, as explained above. Any suitable method of
assaying
or imaging KDR or VEGF/KDR complex may be employed. Similarly, cMet or
HGF/cMet complex binding heteromultimeric constructs will be extremely useful
for
detection and/or imaging of cMet or HGF/cMet complex containing tissue, and
particularly for detection and/or imaging tumors or other sites of
hyperproliferation , in
which HGF and cMet are intimately involved, as explained above. Any suitable
method of assaying or imaging cMet or HGF/cMet complex may be employed.
The compounds of the invention also have utility in the treatment of a variety
of
disease states, whether used alone or in combination with another therapeutic
agent.
For example, as discussed, a compound of the invention that inhibits a
biological
process that contributes to a disease state may itself be used as a
therapeutic or
pharmaceutical composition. Alternatively (or in combination), a compound of
the
invention may include one or more additional therapeutic agents. In one
embodiment,
the invention includes heteromultimers including KDR or VEGF/KDR complex
binding
moieties which may themselves be used as therapeutics or may be used to
localize one
or more therapeutic agents (e.g. a chemotherapeutic, a radiotherapeutic,
genetic
material, etc.) to KDR expressing cells, including sites of angiogenesis, or
those
associated with a number of pathogens.. In another embodiment, the invention
includes heteromultimers including Chet or HGF/cMet complex binding moieties
which may themselves be used as therapeutics or may be used to localize one or
more
therapeutic agents (e.g. a chemotherapeutic, a radiotherapeutic, genetic
material, etc.) to
cMet expressing cells, including tumors, sites of hyperproliferation or sites
of
angiogenesis.
The heteromultimeric constructs of the present invention are particularly
useful
as therapeutic agents for treating conditions that involve endothelial cells.
Because an
important function of endothelial cells is angiogenesis, or the formation of
blood
vessels, the heteromultimers of the invention are particularly useful for
treating
conditions that involve angiogenesis include, for example, solid tumors, tumor
CA 02779639 2012-05-18
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metastases and benign tumors. Such tumors and related disorders are well known
in the
art and include, for example, melanoma, central nervous system tumors,
neuroendocrine
tumors, sarcoma, multiple myeloma as well as cancer of the breast, lung,
prostate,
colon, head & neck, and ovaries. Additional tumors and related disorders are
listed in
Table I of U.S. Patent No. 6,025,331, issued February 15, 2000 to Moses, et
al., the
teachings of which are incorporated herein by reference. Benign tumors
include, for
example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic
granulomas. Other relevant diseases that involve angiogenesis include for
example,
rheumatoid arthritis, psoriasis, and ocular disease, such as diabetic
retinopathy,
retinopathy of prematurity, macular degeneration, corneal graft rejection,
neovascular
glaucoma, retrolental fibroplasias, rebeosis, Oster-Webber syndrome,
myocardial
angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints,
angiofibroma and wound granulation. Other relevant diseases or conditions that
involve
blood vessel growth include intestinal adhesions, atherosclerosis,
scleroderma, and
hypertropic scars, and ulcers. Furthermore, the heteromultimers of the present
invention can be used to reduce or prevent uterine neovascularization required
for
embryo implantation, for example, as a birth control agent.
For detection of the target in solution, a heteromultimer according to the
invention can be detectably labeled, e.g., fluorescently labeled,
enzymatically labeled,
or labeled with a radionuclide or paramagnetic metal or attached to bubbles,
then
contacted with the solution, and thereafter formation of a complex between the
heteromultimer and the target can be detected. As an example, a fluorescently
labeled
KDR or VEGF/KDR complex binding heteromultimeric construct may be used for in
vitro KDR or VEGF/KDR complex detection assays, wherein the heteromultimeric
construct is added to a solution to be tested for KDR or VEGF/KDR complex
under
conditions allowing binding to occur. The complex between the fluorescently
labeled
KDR or VEGF/KDR complex binding heteromultimer and KDR or VEGF/KDR
complex target can be detected and quantified by measuring the increased
fluorescence
polarization arising from the KDR or VEGF/KDR complex-bound heteromultimer
relative to that of the free heteromultimer. Heteromultimers comprising cMet
binding
moieties may be used similarly.
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Alternatively, a sandwich-type "ELISA" assay may be used, wherein a
heteromultimeric construct is immobilized on a solid support such as a plastic
tube or
well, then the solution suspected of containing the target is contacted with
the
immobilized heteromultimeric construct, non-binding materials are washed away,
and
complexed target is detected using a suitable detection reagent, such as a
monoclonal
antibody recognizing the target. The monoclonal antibody is detectable by
conventional
means known in the art, including being detectably labeled, e.g.,
radiolabeled,
conjugated with an enzyme such as horseradish peroxidase and the like, or
fluorescently
labeled.
For example, for detection or purification of soluble target in or from a
solution,
heteromultimers of the invention can be immobilized on a solid substrate such
as a
chromatographic support or other matrix material, then the immobilized
heteromultimer
can be loaded or contacted with the solution under conditions suitable for
formation of a
heteromultimer:target complex . The non-binding portion of the solution can be
removed and the complex maybe detected, e.g., using an antibody against the
target,
such as an anti-binding polypeptide antibody (e.g., anti-KDR, anti-VEGF/KDR
complex, anti-cMet, or anti-cMet/HGF complex antibody), or the
heteromultimer:target
complex may be released from the binding moiety at appropriate elution
conditions.
The biology of angiogenesis and the roles of VEGF and KDR in initiating and
maintaining it have been investigated by many researchers and continues to be
an active
field for research and development. In furtherance of such research and
development, a
method of purifying bulk amounts of KDR or VEGF/KDR complex in pure form is
desirable, and the KDR and VEGFIKDR complex heteromultimers described herein
are especially useful for that purpose, using the general purification
methodology
described above. Similarly, the biology of tumors and other hyperproliferative
tissue
and the roles of cMet and HGF in initiating and maintaining these have been
investigated by many researchers and continues to be an active field for
research and
development. In furtherance of such research and development, a method of
purifying
bulk amounts of cMet or HGF/cMet complex in pure form is desirable, and the
cMet or
HGF/cMet complex heteromultimers described herein are especially useful for
that
purpose, using the general purification methodology described above.
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Diagnostic Imaging
Appropriately labeled heteromultimeric constructs of the present invention may
be used in in vivo diagnostic applications to image specific tissues or
cellular disorders.
A particularly preferred use for the heteromultimeric constructs according to
the present
invention is for creating visually readable images of target expressing or
containing
tissue. For this embodiment, the heteromultimers of the invention are
conjugated with
a label appropriate for diagnostic detection, optionally via a linker Suitable
linkers can
be substituted or unsubstituted alkyl chains, amino acid chains (e.g.,
polyglycine),
polyethylene glycols, polyamides, and other simple polymeric linkers known in
the art.
Preferably, a heteromultimer exhibiting much greater specificity for the
target than for
other serum proteins is conjugated or linked to a label appropriate for the
detection
methodology to be employed. For example, heteromultimers of the invention may
be
conjugated with or without a linker to a paramagnetic chelate suitable for
magnetic
resonance imaging (MRI), with a radiolabel suitable for x-ray, PET or
scintigrapic
imaging (including if necessary a chelator, such as those described herein,
for a
radioactive metal) with an ultrasound contrast agent (e.g. a stabilized
microbubble, a
microballoon, a microsphere or what has been referred to as a gas filled
"liposome")
suitable for ultrasound detection, or with an optical imaging dye.
For example, KDR or VEGF/KDR complex binding heteromultimeric
constructs of the invention or cMet or HGF complex binding heteromultimeric
constructs of the invention may be used to image neoplastic tumors, which
require
angiogenesis for survival and metastasis, or other sites of angiogenic
activity. In this
embodiment, heteromultimeric constructs including KDR and VEGF/KDR complex
binding polypeptides or cMet or HGF/cMet complex binding polypeptides are
converted to imaging reagents by conjugation with a label appropriate for
diagnostic
detection, optionally via a linker, as described herein.
In general, the technique of using a detectably labeled heteromultimeric
construct is based on the premise that the label generates a signal that is
detectable
outside the patient's body. For example, in one embodiment, when a detectably
labeled
heteromultimer of the invention is administered to the patient in which
angiogenesis,
e.g., due to a tumor, is occurring, the high affinity of the KDR or VEGF/KDR
complex
binding moieties included in the heteromultimeric constructs for KDR or
VEGF/KDR
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complex causes the heteromultimeric construct to bind to the site of
angiogenesis and
accumulate label at the site of angiogenesis. Sufficient time is allowed for
the labeled
heteromultimeric construct to localize at th site of angiogenesis. The signal
generated
by the labeled peptide is detected by a scanning device which will vary
according to the
type of label used, and the signal is then converted to an image of the site
of
angiogenesis.
In another embodiment, rather than directly labelling a heteromultimer of the
invention with a detectable label or radiotherapeutic construct,
heteromultimers of the
invention can be conjugated with for example, avidin, biotin, or an antibody
or antibody
fragment that will bind the detectable label or radiotherapeutic. For example,
in one
embodiment, heteromultimers can be conjugated to streptavidin or avidin for in
vivo
binding to target-containing or expressing cells. After the unbound
heteromultimer has
cleared from the body, a biotinylated detectable label or radiotherapeutic
construct (e.g.
a chelate molecule complexed with a radioactive metal) can be infused which
will
rapidly concentrate at the site where the targeting construct is bound. This
approach in
some situations can reduce the time required after administering the
detectable label
until imaging can take place. It can also increase signal to noise ratio in
the target site,
and decrease the dose of the detectable label or radiotherapeutic construct
required.
This is particularly useful when a radioactive label or radiotherapeutic is
used as the
dose of radiation that is delivered to normal but radiation-sensitive sites in
the body,
such as bone-marrow, kidneys, and liver is decreased. This approach, sometimes
referred to as pre-targeting or two-step, or three-step approaches was
reviewed by S.F.
Rosebrough (Q. J. Nucl. Med. 40:234-251; 1996, incorporated by reference
herein).In a
preferred embodiment, heteromultimeric constructs including KDR or VEGF/KDR
binding moieties are used. In another preferred embodiment, heteromultimeric
constructs including cMet or HGF/cMet binding moieties are used.
A. Magnetic Resonance Imaging
The heteromultimers of the present invention may advantageously be conjugated
with one or more paramagnetic metal chelates in order to form a contrast agent
for use
in MRI. Preferred paramagnetic metal ions have atomic numbers 21-29,42,44, or
57-
83. This includes ions of the transition metal or lanthanide series which have
one, and
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more preferably five or more, unpaired electrons and a magnetic moment of at
least 1.7
Bohr magneton. Preferred paramagnetic metals include, but are not limited to,
chromium (III), manganese (I1), manganese (III), iron (II), iron (III), cobalt
(II), nickel
(II), copper (II), praseodymium (III), neodymium (III), samarium (III),
gadolinium (III),
terbium (III), dysprosium (II1), holmium (III), erbium (II1), europium (III)
and
ytterbium (III). Additionally, heteromultimers of the present invention may
also be
conjugated with one or more superparamagnetic particles.
Gd(III) is particularly preferred for MRI due to its high relaxivity and low
toxicity, and the availability of only one biologically accessible oxidation
state. Gd(III)
chelates have been used for clinical and radiologic MR applications since
1988, and
approximately 30% of MR exams currently employ a gadolinium-based contrast
agent.
One skilled in the art will select a metal according to dose required to
detect
target containing tisssue and considering other factors such as toxicity of
the metal to
the subject. See, Tweedle et al., Magnetic Resonance Imaging (2nd ed.), vol.
1, Partain
et al., eds. (W.B. Saunders Co. 1988), pp. 796-7. Generally, the desired dose
for an
individual metal will be proportional to its relaxivity, modified by the
biodistribution,
pharmacokinetics and metabolism of the metal. The trivalent cation, Gd3+ is
particularly preferred for MRI contrast agents, due to its high relaxivity and
low
toxicity, with the further advantage that it exists in only one biologically
accessible
oxidation state, which minimizes undesired metabolization of the metal by a
patient.
Another useful metal is Cr3+, which is relatively inexpensive.
The paramagnetic metal chelator is a molecule having one or more polar groups
that act as a ligand for, and complex with, a paramagnetic metal. Suitable
chelators are
known in the art and include acids with methylene phosphonic acid groups,
methylene
carbohydroxamine acid groups, carboxyethylidene groups, or carboxymethylene
groups. Examples of chelators include, but are not limited to,
diethylenetriamine
pentaacetic acid (DTPA), 1,4,7,1 0-tetraazacyclotetradecane- 1,4,7, 1 0-
tetraacetic acid
(DOTA), I-substituted 1,4,7; tricarboxymethyl 1,4,7,10 teraazacyclododecane
triacetic
acid (DO3A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,11 -
tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Additional
chelating ligands
are ethylenebis-(2-hydroxy-phenylglycine) (EHPG), and derivatives thereof,
including
5-CI-EHPG, 5Br-EHPG, 5-Me-EHPG, 5t-Bu-EHPG, and 5sec-Bu-EHPG;
CA 02779639 2012-05-18
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benzodiethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof,
including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl
DTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and
derivatives
thereof; the class of macrocyclic compounds which contain at least 3 carbon
atoms,
more preferably at least 6, and at least two heteroatoms (0 and/or N), which
macrocyclic compounds can consist of one ring, or two or three rings joined
together at
the hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA,
where
NOTA is 1,4,7-triazacyclononane N,N',N"-triacetic acid, benzo-TETA,
benzo-DOTMA, where DOTMA is 1,4,7,1 0-tetraazacyclotetradecane- 1,4,7,
10-tetra(methyl tetraacetic acid), and benzo-TETMA, where TETMA is 1,4,8,11-
tetraazacyclotetradecane- 1,4,8,11 -(methyl tetraacetic acid); derivatives of
1,3-propylenediaminetetraacetic acid (PDTA) and
triethylenetetraaminehexaacetic acid
(TTHA); derivatives of 1,5,10-N,N',N"-tris(2,3-dihydroxybenzoyl)-
tricatecholate
(LICAM) and 1,3,5-N,N',N"-tris(2,3-dihydroxybenzoyl) aminomethylbenzene
(MECAM). A preferred chelator for use in the present invention is DTPA.
Examples
of representative chelators and chelating groups contemplated by the present
invention
are described in WO 98/18496, WO 86/06605, WO 91/03200, WO 95/28179, WO
96/23526, WO 97/36619, PCT/US98/01473, PCT/US98/20182, and U.S. 4,899,755,
U.S. 5,474,756, U.S. 5,846,519 and U.S. 6,143,274, each of which is hereby
incorporated by reference in its entirety. Use of the chelate DO3A is
particularly
preferred.
In one embodiment of the present invention, the chelator(s) of the MRI
contrast
agent is coupled to a heteromultimer, such as, for example one comprised of
KDR or
VEGF/KDR complex binding polypeptides or cMet or HGF/cMet complex binding
polypeptides. The positioning of the chelate(s) should be selected so as not
to interfere
with the binding affinity or specificity of the heteromultimeric construct.
Preferably,
the chelate(s) will be appended either to the N-terminus or the C-terminus,
however the
chelate(s) may also be attached anywhere within the sequence. In preferred
embodiments, a chelator having a free central carboxylic acid group (e.g.,
DTPA-
Asp(p-COOH)-OtBu) makes it easy to attach at the N-terminus of a binding
peptide by
formation of an amide bond. The chelate(s) could also be attached at the C-
terminus
with the aid of a linker. Alternatively, isothiocyanate conjugation chemistry
could be
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employed as a way of linking the appropriate isothiocyante group bearing DTPA
to a
free amino group anywhere within the peptide sequence.
For example, the heteromultimer can be bound directly or covalently to the
metal chelator(s) (or other detectable label), or it may be coupled or
conjugated to the
metal chelator(s) using a linker, which may be, without limitation, amide,
urea, acetal,
ketal, double ester, carbonyl, carbamate, thiourea, sulfone, thioester, ester,
ether,
disulfide, lactone, imine, phosphoryl, or phosphodiester linkages; substituted
or
unsubstituted saturated or unsaturated alkyl chains; linear, branched, or
cyclic amino
acid chains of a single amino acid or different amino acids (e.g., extensions
of the N- or
C- terminus of the binding moieties); derivatized or underivatized
polyethylene glycol,
polyoxyethylene, or polyvinylpyridine chains; substituted or unsubstituted
polyamide
chains; derivatized or underivatized polyamine, polyester, polyethylenimine,
polyacrylate, poly(vinyl alcohol), polyglycerol, or oligosaccharide (e.g.,
dextran)
chains; alternating block copolymers; malonic, succinic, glutaric, adipic and
pimelic
acids; caproic acid; simple diamines and dialcohols; any of the other linkers
disclosed
herein; or any other simple polymeric linkers known in the art (see, e.g., WO
98/18497,
WO 98/18496). Preferably the molecular weight of the linker can be tightly
controlled.
The molecular weights can range in size from less than 100 to greater than
1000.
Preferably the molecular weight of the linker is less than 100. In addition,
it may be
desirable to utilize a linker that is biodegradable in vivo to provide
efficient routes of
excretion for the imaging reagents of the present invention. Depending on
their location
within the linker, such biodegradable functionalities can include ester,
double ester,
amide, phosphoester, ether, acetal, and ketal functionalities.
In general, known methods can be used to couple the metal chelate and a
heteromultimer of the invention using such linkers. See, e.g., WO 95/28967, WO
98/18496, WO 98/18497 and discussion therein. For example, a heteromultimer
can be
linked through the N- or C-terminus of a component binding moiety via an amide
bond,
for example, to a metal coordinating backbone nitrogen of a metal chelate or
to an
acetate arm of the metal chelate itself. The present invention contemplates
linking of
the chelate(s) on any position, provided the metal chelate retains the ability
to bind the
metal tightly in order to minimize toxicity. Similarly, a component binding
moiety of a
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heteromultimer may be modified or elongated in order to generate a locus for
attachment to a metal chelate, provided such modification or elongation does
not
eliminate its ability to bind the target.
MRI contrast reagents prepared according to the disclosures herein may be used
in the same manner as conventional MRI contrast reagents. When imaging target
containing tissue such as, for example, a site of angiogenesis, certain MR
techniques
and pulse sequences may be preferred to enhance the contrast of the site to
the
background blood and tissues. These techniques include (but are not limited
to), for
example, black blood angiography sequences that seek to make blood dark, such
as fast
spin echo sequences (see, e.g., Alexander et al., Magnetic Resonance in
Medicine,
40(2): 298-310 (1998)) and flow-spoiled gradient echo sequences (see, e.g.,
Edelman et
al., Radiology, 177(1): 45-50 (1990)). These methods also include flow
independent
techniques that enhance the difference in contrast, such as inversion-recovery
prepared
or saturation-recovery prepared sequences that will increase the contrast
between target
containing tissue, such as an angiogenic tumor, and background tissues.
Finally,
magnetization transfer preparations may also improve contrast with these
agents (see,
e.g., Goodrich et at., Investigative Radiology, 31(6): 323-32 (1996)).
The labeled reagent is administered to the patient in the form of an
injectable
composition. The method of administering the MRI contrast agent is preferably
parenterally, meaning intravenously, intraarterially, intrathecally,
interstitially, or
intracavitarilly. For imaging active angiogenesis, intravenous or
intraarterial
administration is preferred.
For MRI, it is contemplated that the subject will receive a dosage of contrast
agent sufficient to enhance the MR signal at the target (e.g. a site of
angiogenesis) at
least 10%. After injection of the heteromultimeric construct including the MRI
reagent, , the patient is scanned in the MRI machine to determine the location
of any
sites containing the target. In therapeutic settings, upon target
localization, a cytotoxic
or therapeutic agent can be immediately administered, if necessary, and the
patient can
be subsequently scanned to visualize the therapeutic effect.
In a preferred embodiment, heteromultimers including KDR or VEGF/KDR
complex binding moieties are conjugated to one or more paramagnetic metal
chelates or
one or more superparamagnetic particles, optionally via a linker. In another
preferred
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embodiment, heteromultimeric constructs including cMet or HGF/cMet complex
binding moieties are used. Such heteromultimeric constructs are complexed with
one or
more paramagnetic metal and adminitered in a dose sufficient to enhance the MR
signal
at the site of angiogenesis at least 10%. After injection, the patient is
scanned to
determine the location of any sites of angiogenesis (e.g. angiogenic tumors,
etc.) or
hyperproliferative tissue. If necessary, upon location of an angiogenic or
hyperproliferative site, an anti-angiogenic or tumoricidal agent, such as, for
example, an
inhibitor of VEGF (or VEGF activation of KDR) may be administered. If
necessary,
the patient may be scanned again to visualize/track the tumor regression,
arrest of
angiogenesis, etc.
B. Ultrasound Imaging
When ultrasound is transmitted through a substance, the acoustic properties of
the substance will depend upon the velocity of the transmissions and the
density of the
substance. Changes in the acoustic properties will be most prominent at the
interface of
different substances (solids, liquids, gases). Ultrasound contrast agents are
intense
sound wave reflectors because of the acoustic differences between the agent
and the
surrounding tissue. Gas containing or gas generating ultrasound contrast
agents are
particularly useful because of the acoustic difference between liquid (e.g.,
blood) and
the gas-containing or gas generating ultrasound contrast agent. Because of
their size,
ultrasound contrast agents comprising microbubbles, microballoons, and the
like may
remain for a longer time in the blood stream after injection than other
detectable
moieties; thus a targeted ultrasound agent may demonsrate superior imaging of
tissue
expressing or containing the target.
In this aspect of the invention, the heteromultimeric constructs may include a
material that is useful for ultrasound imaging. For example, heteromultimers
of the
invention may be linked to materials employed to form vesicles (e.g.,
microbubbles,
microballoons, microspheres, etc.), or emulsions containing a liquid or gas
which
functions as the detectable label (e.g., an echogenic gas or material capable
of
generating an echogenic gas). Materials for the preparation of such vesicles
include
surfactants, lipids, sphingolipids, oligolipids, phospholipids, proteins,
polypeptides,
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carbohydrates, and synthetic or natural polymeric materials. See e.g. WO
98/53857,
WO 98/18498, WO 98/18495, WO 98/18497, WO 98/18496, and WO 98/18501
incorporated herein by reference in their entirety.
For contrast agents comprising suspensions of stabilized microbubbles (a
preferred embodiment), phospholipids, and particularly saturated phospholipids
are
preferred. The preferred gas-filled microbubbles can be prepared by means
known in
the art, such as, for example, by a method described in any one of the
following patents:
EP 554213, US 5,413,774, US 5,578,292, EP 744962, EP 682530, US 5,556,610, US
5,846,518, US 6,183,725, EP 474833, US 5,271,928, US 5,380,519, US 5,531,980,
US
5,567,414, US 5,658,551, US 5,643,553, US 5,911,972, US 6,110,443, US
6,136,293,
EP 619743, US 5,445,813, US 5,597,549, US 5,686,060, US 6,187,288, and US
5,908,610, each of which is incorporated by reference herein in its entirety.
In a
preferred embodiment, at least one of the phospholipid moieties has the
structure of
formula 18 or formula 19 shown below and described in U.S. Patent No. U.S.
5,686,060, which is herein incorporated by reference in its entirety.
O H
O
11
0 O/\O00")
OH
O NH
OC
18
CA 02779639 2012-05-18
WO 03/084574 PCT/US03/06656
O
O H
O
0 O' 1 " o
OH
HN
19
Examples of suitable phospholipids include esters of glycerol with one or two
molecules of fatty acids (the same or different)and phosphoric acid, wherein
the
phosphoric acid residue is in turn bonded to a hydrophilic group, such as
choline,
serine, inositol, glycerol, ethanolamine, and the like groups. Fatty acids
present in the
phospholipids are in general long chain aliphatic acids, typically containing
from 12 to
24 carbon atoms, preferably from 14 to 22, that may be saturated or may
contain one or
more unsaturations. Examples of suitable fatty acids are lauric acid, myristic
acid,
palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid,
linoleic acid, and
linolenic acid. Mono esters of phospholipid are also known in the art as the
"lyso"
forms of the phospholipids.
Further examples of phospholipids are phosphatidic acids, i.e. the diesters of
glycerol-phosphoric acid with fatty acids, sphingomyelins, i.e. those
phosphatidylcholine analogs where the residue of glycerol diester with fatty
acids is
replaced by a ceramide chain, cardiolipins, i.e. the esters of 1,3-
diphosphatidylglycerol
with a fatty acid, gangliosides, cerebrosides, etc.
As used herein, the term phospholipids includes either naturally occurring,
semisynthetic or synthetically prepared products that can be employed either
singularly
or as mixtures.
Examples of naturally occurring phospholipids are natural lecithins
(phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg
yolk
lecithins.
Examples of semisynthetic phospholipids are the partially or fully
hydrogenated
derivatives of the naturally occurring lecithins.
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Examples of synthetic phospholipids are e.g., dilauryloyl-phosphatidylcholine
("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoyl-
phosphatidylcholine
("DPPC"), diarachidoylphosphatidylcholine ("DAPC"), distearoyl-
phosphatidylcholine
("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-
mymstoylphosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatid-
ylcholine
("PSPC"), 1-stearoyl-2-palmitoyl-phosphatidylcholine ("SPPC"),
dioleoylphosphatidylycholine ("DOPC"), 1,2 Distearoyl-sn-glycero-3-
Ethylphosphocholine (Ethyl-DSPC), dilauryloyl-phosphatidylglycerol ("DLPG")
and its
alkali metal salts, diarachidoylphosphatidylglycerol ("DAPG") and its alkali
metal salts,
dimyristoylphosphatidylglycerol ("DMPG") and its alkali metal salts,
dipalmitoyl-
phosphatidylglycerol ("DPPG") and its alkali metal salts,
distearolyphosphatidylglycerol ("DSPG") and its alkali metal salts,
dioleoylphosphatidylglycerol ("DOPG") and its alkali metal salts, dimyristoyl
phosphatidic acid ("DMPA") and its alkali metal salts, dipalmitoyl
phosphatidic acid
("DPPA") and its alkali metal salts, distearoyl phosphatidic acid ("DSPA"),
diarachidoyl phosphatidic acid ("DAPA") and its alkali metal salts,
dimyristoyl
phosphatidyl-ethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine
("DPPE"), distearoyl phosphatidyl-ethanolamine ("DSPE"), dimyristoyl
phosphatidylserine ("DMPS"), diarachidoyl phosphatidylserine ("DAPS"),
dipalmitoyl
phosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS"),
dioleoylphosphatidylserine ("DOPS"), dipalmitoyl sphingomyelin ("DPSP"), and
distearoyl sphingomyelin ("DSSP").
Other preferred phospholipids include dipahnitoylphosphatidylcholine,
dipalmitoylphosphatidic acid and dipalmitoylphosphatidylserine. The
compositions
may also contain PEG-4000 and/or palmitic acid. Any of the gases disclosed
herein or
known to the skilled artisan may be employed; however, inert gases, such as
SF6, or
fluorocarbons, such as CF4, C3Fg and C4F10, are preferred.
The preferred microbubble suspensions may be prepared from phospholipids
using known processes such as a freeze-drying or spray-drying solutions of the
crude
phospholipids in a suitable solvent or using the processes set forth in EP
554213, US
5,413,774, US 5,578,292, EP 744962, EP 682530, US 5,556,610, US 5,846,518, US
6,183,725, EP 474833, US 5,271,928, US 5,380,519, US 5,531,980, US 5,567,414,
US
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5,658,551, US 5,643,553, US 5,911,972, US 6,110,443, US 6,136,293, EP 619743,
US
5,445,813, US 5,597,549, US 5,686,060, US 6,187,288, and US 5,908,610, each of
which is incorporated by reference herein in its entirety. Most preferably,
the
phospholipids are dissolved in an organic solvent and the solution is dried
without
going through a liposome formation stage. This can be done by dissolving the
phospholipids in a suitable organic solvent together with a hydrophilic
stabilizer
substance or a compound soluble both in the organic solvent and water and
freeze-
drying or spray-drying the solution. In this embodiment the criteria used for
selection
of the hydrophilic stabilizer is its solubility in the organic solvent of
choice. Examples
of hydrophilic stabilizer compounds soluble in water and the organic solvent
are e.g. a
polymer, like polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA),
polyethylene
glycol (PEG), etc., malic acid, glycolic acid, maltol and the like. Such
hydrophilic
compounds also aid in homogenizing the microbubbles size distribution and
enhance
stability under storage. Any suitable organic solvent may be used as long as
its boiling
point is sufficiently low and its melting point is sufficiently high to
facilitate subsequent
drying. Typical organic solvents include, for example, dioxane, cyclohexanol,
tertiary
butanol, tetrachlorodifluoro ethylene (C2C14F2) or 2-methyl-2-butanol however,
2-
methyl-2-butanol and C2C14F2 are preferred.
Prior to formation of the suspension of microbubbles by dispersion in an
aqueous carrier, the freeze-dried or spray-dried phospholipid powders are
contacted
with air or another gas. When contacted with the aqueous carrier the powdered
phospholipids whose structure has been disrupted will form lamellarized or
laminarized
segments that will stabilize the microbubbles of the gas dispersed therein.
This method
permits production of suspensions of microbubbles that are stable even when
stored for
prolonged periods and are obtained by simple dissolution of the dried
laminarized
phospholipids (which have been stored under a desired gas) without shaking or
any
violent agitation.
Alternatively, microbubbles can be prepared by suspending a gas into an
aqueous solution at high agitation speed, as disclosed e.g. in WO 97/29783. A
further
process for preparing microbubbles is disclosed in co-pending European patent
application no. 03002373, herein incorporated by reference, which comprises
preparing
an emulsion of an organic solvent in an aqueous medium in the presence of a
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phospholipid and subsequently lyophilizing said emulsion, after optional
washing
and/or filtration steps.
Additives known to those of ordinary skill in the art can be included in the
suspensions of stabilized microbubbles. For instance, non-film forming
surfactants,
including polyoxypropylene glycol and polyoxyethylene glycol and similar
compounds,
as well as various copolymers thereof; fatty acids such as myristic acid,
palmitic acid,
stearic acid, arachidic acid or their derivatives, ergosterol, phytosterol,
sitosterol,
lanosterol, tocopherol, propyl gallate, ascorbyl palmitate and butylated
hydroxytoluene
may be added. The amount of these non-film forming surfactants is usually up
to 50%
by weight of the total amount of surfactants but preferably between 0 and 30%
by
weight.
Other gas containing suspensions include those disclosed in, for example, US
5,798,091 and WO 97/29783, incorporated herein by reference in their entirety.
These
agents may be prepared as described in US 5,798,091 or W097/29783, each of
which is
incorporated by reference in its entirety.
Another preferred ultrasound contrast agent comprises microballoons. The term
"microballoon" refers to gas filled bodies with a material boundary or
envelope. More
on microballoon formulations and methods of preparation may be found in EP-A-0
324
938 US 4,844,882; US 5,711,933; US 5,840,275; US 5,863,520; US 6,123,922; US
6,200,548; US 4,900,540; US 5,123,414; US 5,230,882; 5,469,854; 5,585,112; US
4,718,433; US 4774,958; WO 9501187; US 5,529,766; US 5,536,490 and US
5,990,263, each of which is incorporated herein by reference in its entirety.
The preferred microballoons have an envelope including a biodegradable
physiologically compatible polymer or, a biodegradable solid lipid. The
polymers
useful for the preparation of the microballoons of the present invention can
be selected
from the biodegradable physiologically compatible polymers, such as any of
those
described in any of the following patents: EP 458745, US 5,711,933, US
5,840,275, EP
554213, US 5,413,774 and US 5,578,292, the entire contents of which are
incorporated
herein by reference. In particular, the polymer can be selected from
biodegradable
physiologically compatible polymers, such as polysaccharides of low water
solubility,
polylactides and polyglycolides and their copolymers, copolymers of lactides
and
lactones such as s-caprolactone, y-valerolactone and polypeptides. Other
suitable
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polymers include poly(ortho)esters (see e.g., US 4,093,709; US 4,131,648; US
4,138,344; US 4,180,646); polylactic and polyglycolic acid and their
copolymers, for
instance DEXON (see J. Heller, Biomaterials 1 (1980), 51; poly(DL-lactide-co-
s-
caprolactone), poly(DL-lactide-co- y -valerolactone), poly(DL-lactide-co- 'y-
butyrolactone), polyalkylcyanoacrylates; polyamides, polyhydroxybutyrate; poly-
dioxanone; poly-l3-aminoketones (A. S. Angeloni, P. Ferruti, M. Tramontini and
M.
Casolaro, The Mannich bases in polymer synthesis: 3. Reduction of poly(beta-
aminoketone)s to poly(gamma-aminoalcohol)s and their N-alkylation to
poly(gamma-
hydroxyquaternary ammonium salt)s, Polymer 23, pp 1693-1697, 1982.);
polyphosphazenes (Allcock, Harry R. Polyphosphazenes: new polymers with
inorganic backbone atoms (Science 193(4259), 1214-19 (1976)) and
polyanhydrides.
The microballoons of the present invention can also be prepared according to
the
methods of WO-A-96/15815, incorporated herein by reference, where the
microballoons are made from a biodegradable membrane comprising biodegradable
lipids, preferably selected from mono- di-, tri-glycerides, fatty acids,
sterols, waxes and
mixtures thereof. Preferred lipids are di- or tri-glycerides, e.g. di- or tri-
myristin, -
palmityn or -stearin, in particular tripalmitin or tristearin.
The microballoons may employ any of the gases disclosed herein or known to
the skilled artisan; however, inert gases such as fluorinated gases are
preferred. The
microballoons may be suspended in a pharmaceutically acceptable liquid carrier
with
optional additives known to those of ordinary skill in the art and
stabilizers.
Other gas-containing contrast agent formulations include microparticles
(especially aggregates of microparticles) having gas contained therein or
otherwise
associated therewith (for example being adsorbed on the surface thereof and/or
contained within voids, cavities or pores therein). Methods for the
preparation of these
agents are as described in EP 0122624, EP 0123235, EP 0365467, US 5,558,857,
US
5,607,661, US 5,637,289, US 5,558,856, US 5,137,928, WO 9521631 and WO
9313809, each of which is incorporated herein by reference in its entirety.
Any of these ultrasound compositions should also be, as far as possible,
isotonic
with blood. Hence, before injection, small amounts of isotonic agents may be
added to
any of above ultrasound contrast agent suspensions. The isotonic agents are
physiological solutions commonly used in medicine and they comprise aqueous
saline
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solution (0.9% NaCl), 2.6% glycerol solution, 5% dextrose solution, etc.
Additionally,
the ultrasound compositions may include standard pharmaceutically acceptable
additives, including, for example, emulsifying agents, viscosity modifiers,
cryoprotectants, lyoprotectants, bulking agents etc.
Any biocompatible gas may be used in the ultrasound contrast agents useful in
the invention. The term "gas" as used herein includes any substances
(including
mixtures) substantially in gaseous form at the normal human body temperature.
The gas
may thus include, for example, air; nitrogen; oxygen; C02; argon; xenon or
krypton,
fluorinated gases (including for example, perfluorocarbons, SF6, SeF6) a low
molecular
weight hydrocarbon (e.g. containing from 1 to 7 carbon atoms), for example, an
alkane
such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such
as
cyclopropane, cyclobutane or cyclopentene, an alkene such as ethylene,
propene,
propadiene or a butene, or an alkyne such as acetylene or propyne and/or
mixtures
thereof. However, fluorinated gases are preferred. Fluorinated gases include
materials
which contain at least one fluorine atom such as SF6, freons (organic
compounds
containing one or more carbon atoms and fluorine, i.e. CF4, C2F6,C3F8,
C4F8 C4F10,CBrF3, CCI2F2 C2CIF5, and CBrCIF2) and perfluorocarbons. The term
perfluorocarbon refers to compounds containing only carbon and fluorine atoms
and
includes, in particular, saturated, unsaturated, and cyclic perfluorocarbons.
The
saturated perfluorocarbons, which are usually preferred, have the formula
CõFõ+2, where
n is from 1 to 12, preferably from 2 to 10, most preferably from 3 to 8 and
even more
preferably from 3 to 6. Suitable perfluorocarbons include, for example, CF41
C2F6, C3F8,
C4F8, C4F10, CSF121 C6F12, C7F14, C3F18, and C9F2o. Most preferably the gas or
gas
mixture comprises SF6 or a perfluorocarbon selected from the group consisting
of C3F8
C4F8, C4F10, CSF121 C6F121 C7F14, C8F18, with C4Flo being particularly
preferred. See
also WO 97/29783, WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO
98/18497, WO 98/18501, WO 98/05364, and WO 98/17324.
In certain circumstances it may be desirable to include a precursor to a
gaseous
substance (e.g. a material that is capable of being converted to a gas in
vivo, often
referred to as a "gas precursor"). Preferably the gas precursor and the gas it
produces
are physiologically acceptable. The gas precursor may be pH-activated, photo-
activated, temperature activated, etc. For example, certain perfluorocarbons
may be
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used as temperature activated gas precursors. These perfluorocarbons, such as
perfluoropentane, have a liquid/gas phase transition temperature above room
temperature (or the temperature at which the agents are produced and/or
stored) but
below body temperature; thus, they undergo a phase shift and are converted to
a gas
within the human body.
As discussed, the gas can include a mixture of gases. The following
combinations are particularly preferred gas mixtures: a mixture of gases (A)
and (B) in
which, at least one of the gases (B), present in an amount of between 0.5 -
41% by vol.,
has a molecular weight greater than 80 daltons and is a fluorinated gas and
(A) is
selected from the group consisting of air, oxygen, nitrogen, carbon dioxide
and
mixtures thereof, the balance of the mixture being gas A.
Since ultrasound vesicles may be larger than the other detectable labels
described herein, they may be linked or conjugated to a plurality of
heteromultimeric
constructs in order to increase the targeting efficiency of the agent.
Attachment to the
ultrasound contrast agents described above (or known to those skilled in the
art) may be
via direct covalent bond between a binding polypeptide and the material used
to make
the vesicle or via a linker, as described previously. For example, see WO
98/53857
generally for a description of the attachment of a peptide to a bifunctional
PEG linker,
which is then reacted with a liposome composition. See also, Lanza et al.,
Ultrasound
in Med. & Bio., 23(6): 863-870 (1997).
A number of methods may be used to prepare suspensions of microbubbles
conjugated to heteromultimers. For example, one may prepare maleimide-
derivatized
microbubbles by incorporating 5 % (w/w) of N-MPB-PE (1, 2-dipalmitoyl-sn-
glycero-
3-phosphoethanolamine-4-(p-maleimido-phenyl butyramide), (Avanti Polar-Lipids,
Inc)
in the phospholipid formulation. Then, solutions of mercaptoacetylated
heteromultimers (10 mg/mL in DMF), which have been incubated in deacetylation
solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine HCI, pH 7.5)
are added to the maleimide-activated microbubble suspension. After incubation
in the
dark, under gentle agitation, the heteromultimer conjugated microbubbles may
be
purified by centrifugation.
Compounds that can be used for derivatization of microbubbles typically
include
the following components: (a) a hydrophobic portion, compatible with the
material
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forming the envelope of the microbubble or of the microballoon, in order to
allow an
effective incorporation of the compound in the envelope of the vesicel; said
portion is
represented typically by a lipid moiety (dipalmitin, distearoyl); and (b) a
spacer
(typically PEGs of different molecular weights), which may be optional in some
cases
(microbubbles may for instance present difficulties to be freeze dried if the
spacer is too
long e.g) or preferred in some others (e.g. peptides may be less active when
conjugated
to a microballoon with short spacers); and (c) a reactive group capable of
reacting with
a corresponding reacting moiety on the peptide to be conjugated (e.g.
maleimido with
the -SH group of cysteine).
Alternatively, heteromultimers conjugated to microbubbles may be prepared
using biotin/avidin. For example, avidin-conjugated microbubbles may be
prepared
using a maleimide-activated phospholipid microbubble suspension, prepared as
described above, which is added to mercaptoacetylated-avidin (which has been
incubated with deacetylation solution). Biotinylated heteromultimers (prepared
as
described herein), are then added to the suspension of avidin-conjugated
microbubbles,
yielding a suspension of microbubbles conjugated to the heteromultimers.
Unless it contains a hyperpolarized gas, known to require special storage
conditions, the lyophilized residue may be stored and transported without need
of
temperature control of its environment and in particular it may be supplied to
hospitals
and physicians for on site formulation into a ready-to-use administrable
suspension
without requiring such users to have special storage facilities. Preferably in
such a case
it can be supplied in the form of a two-component kit, which can include two
separate
containers or a dual-chamber container. In the former case preferably the
container is a
conventional septum-sealed vial, wherein the vial containing the lyophilized
residue of
step b) is sealed with a septum through which the carrier liquid may be
injected using an
optionally prefilled syringe. In such a case the syringe used as the container
of the
second component is also used then for injecting the contrast agent. In the
latter case,
preferably the dual-chamber container is a dual-chamber syringe and once the
lyophilizate has been reconstituted and then suitably mixed or gently shaken,
the
container can be used directly for injecting the contrast agent. In both cases
means for
directing or permitting application of sufficient bubble forming energy into
the contents
of the container are provided. However, as noted above, in the stabilised
contrast
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agents according to the invention the size of the gas microbubbles is
substantially
independent of the amount of agitation energy applied to the reconstituted
dried
product. Accordingly, no more than gentle hand shaking is generally required
to give
reproducible products with consistent microbubble size.
It can be appreciated by one ordinary skilled in the art that other two-
chamber
reconstitution systems capable of combining the dried powder with the aqueous
solution
in a sterile manner are also within the scope of the present invention. In
such systems,
it is particularly advantageous if the aqueous phase can be interposed between
the
water-insoluble gas and the environment, to increase shelf life of the
product. Where a
material necessary for forming the contrast agent is not already present in
the container
(e.g. a targeting ligand to be linked to the phospholipid during
reconstitution), it can be
packaged with the other components of the kit, preferably in a form or
container
adapted to facilitate ready combination with the other components of the kit.
No specific containers, vial or connection systems are required; the present
invention may use conventional containers, vials and adapters. The only
requirement is
a good seal between the stopper and the container. The quality of the seal,
therefore,
becomes a matter of primary concern; any degradation of seal integrity could
allow
undesirable substances to enter the vial. In addition to assuring sterility,
vacuum
retention is essential for products stoppered at ambient or reduced pressures
to assure
safe and proper reconstitution. As to the stopper, it may be a compound or
multicomponent formulation based on an elastomer, such as poly(isobutylene) or
butyl
rubber.
Ultrasound imaging techniques which may be used in accordance with the
present invention include known techniques, such as color Doppler, power
Doppler,
Doppler amplitude, stimulated acoustic imaging, and two- or three-dimensional
imaging
techniques. Imaging may be done in harmonic (resonant frequency) or
fundamental
modes, with the second harmonic preferred.
In ultrasound applications the contrast agents formed by phospholipid
stabilized
microbubbles may, for example, be administered in doses such that the amount
of
phospholipid injected is in the range 0.1 to 200 gg/kg body weight, preferably
from
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about 0.1 to 30 gg/kg. Microballoons-containing contrast agents are typically
administered in doses such that the amount of wall-forming polymer or lipid is
from
about 10 tg/kg to about 20 mg/kg of body weight.
In a preferred embodiment, the ultrasound contrast agents described herein are
conjugated to one or more heteromultimers comprised of KDR or VEGF/KDR complex
binding moieties, and target tissue expressing KDR. These targeted ultrasound
contrast
agents will localize at sites of angiogenesis and other tissue expressing KDR
and may
demonstrate superior imaging of angiogenic tissue. In another preferred
embodiment,
the ultrasound contrast agents described herein are conjugated to one or more
heteromultimers comprised of or cMet or HGF/cMet complex binding moieties, and
target tissue expressing cMet. These targeted ultrasound contrast agents will
localize at
sites of hyperproliferation or angiogenesis (including tumors) and other
tissue
expressing cMet and may demonstrate superior imaging of such tissue.
C. Optical Imaging, Sonoluminescence or Photoacoustic Imaging
In accordance with the present invention, a number of optical parameters may
be
employed to determine the location of a target, such as a KDR, VEGF/KDR
complex,
cMet or HGF/cMet complex, with in vivo light imaging after injection of the
subject
with an optically-labeled heteromultimeric construct. Optical parameters to be
detected
in the preparation of an image may include transmitted radiation, absorption,
fluorescent or phosphorescent emission, light reflection, changes in
absorbance
amplitude or maxima, and elastically scattered radiation. For example,
biological tissue
is relatively translucent to light in the near infrared (NIR) wavelength range
of 650-
1000 nm. NIR radiation can penetrate tissue up to several centimeters,
permitting the
use of heteromultimeric contructs of the invention to image target-containing
tissue in
vivo. For example, heteromultimeric constructs comprised of KDR, VEGF/KDR
complex, cMet, or HGF/cMet binding polypeptides may be used for optical
imaging of
KDR, VEGF/KDR complex, cMet, or HGF/cMet complex in vivo.
In another embodiment, the heteromultimeric constructs of the invention may be
conjugated with photolabels, such as optical dyes, including organic
chromophores or
fluorophores, having extensive delocalized ring systems and having absorption
or
emission maxima in the range of 400-1500 nm. The compounds of the invention
may
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alternatively be derivatized with a bioluminescent molecule. The preferred
range of
absorption maxima for photolabels is between 600 and 1000 nm to minimize
interference with the signal from hemoglobin. Preferably, photoabsorption
labels have
large molar absorptivities, e.g. > 105 cm 1M'1, while fluorescent optical dyes
will have
high quantum yields. Examples of optical dyes include, but are not limited to
those
described in WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO
98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO 98/47538, and references
cited therein. For example, the photolabels may be covalently linked directly
to
heteromultimers of the invention, such as, for example, heteromultimers
comprised of
KDR or VEGF/KDR complex binding peptides or linked to such a heteromultimers
via
a linker, as described previously.
After injection of the optically-labeled heteromultimeric construct, the
patient is
scanned with one or more light sources (e.g., a laser) in the wavelength range
appropriate for the photolabel employed in the agent. The light used may be
monochromatic or polychromatic and continuous or pulsed. Transmitted,
scattered, or
reflected light is detected via a photodetector tuned to one or multiple
wavelengths to
determine the location of target-containing tissue(, e.g., tissue containing
KDR,
VEGF/KDR complex, cMet, or HGF/cMet complex) in the subject. Changes in the
optical parameter may be monitored over time to detect accumulation of the
optically-
labeled reagent at the target site (e.g. the site of angiogenesis). Standard
image
processing and detecting devices may be used in conjunction with the optical
imaging
reagents of the present invention.
The optical imaging reagents described above may also be used for acousto-
optical or sonoluminescent imaging performed with optically-labeled imaging
agents
(see, U.S. 5,171,298, WO 98/57666, and references therein). In acousto-optical
imaging, ultrasound radiation is applied to the subject and affects the
optical parameters
of the transmitted, emitted, or reflected light. In sonoluminescent imaging,
the applied
ultrasound actually generates the light detected. Suitable imaging methods
using such
techniques are described in WO 98/57666.
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D. Nuclear Imaging (Radionuclide Imaging) and Radiotherapy.
Heteromultimers of the invention may be conjugated with a radionuclide
reporter appropriate for scintigraphy, SPECT or PET imaging or with a
radionuclide
appropriate for radiotherapy. Constructs in which the heteromultimers of the
invention
are conjugated with both a chelator for a radionuclide useful for diagnostic
imaging and
a chelator for a radionuclide useful for radiotherapy are within the scope of
the
invention.
For use as a PET agent, a heteromultimer may be complexed with one of the
various positron emitting metal ions, such as 51Mn, 52Fe, 60Cu, 68Ga, 72As,
94mTc, or
' in. The heteromultimeric constructs can also be labeled by halogenation
using
radionuclides, such as'8F, 1241, 1251, 1311, 123I777Br, and 76Br. Preferred
metal
radionuclides for scintigraphy or radiotherapy include 99mTc, 51Cr, 67Ga,
68Ga, 47Sc,
51Cr, 167TM, 141Ce, 111In, 168yb, 175Yb, 140La, 90Y, 88Y, 153Sm, 166110,
165Dy, 166DY, 62Cu,
64Cu, 67Ga 97Ru 103Ru 186Re 1ssRe 203Pb 211Bi 212Bi 213Bi 214Bi 1051h 109Pd
117mSn
149Pm,'61Tb,'77Lu, 198Au and 199Au. The choice of metal or halogen will be
determined based on the desired therapeutic or diagnostic application. For
example, for
diagnostic purposes the preferred radionuclides include 64Cu, 67Ga, 68Ga,
99mTc, and
11 'In. For therapeutic purposes, the preferred radionuclides include 64 Cu,
90Y,105Rh,
1111n, 117mSn, 149Pm, 153Sm, 161Tb, 1661)y, 16610, 175Yb, 177Lu, 186/188Re,
and 199Au. 99mTC
is particularly preferred for diagnostic applications because of its low cost,
availability,
imaging properties, and high specific activity. The nuclear and radioactive
properties of
Tc-99m make this isotope an ideal scintigraphic imaging agent. This isotope
has a
single photon energy of 140 keV and a radioactive half-life of about 6 hours,
and is
readily available from a 99Mo-"c generator.
The metal radionuclides may be chelated by, for example, linear, macrocyclic,
terpyridine, and N3S, N2S2, or N4 chelants (see also, U.S. 5,367,080, U.S.
5,364,613,
U.S. 5,021,556, U.S. 5,075,099, U.S. 5,886,142), and other chelators known in
the art
including, but not limited to, HYNIC, DTPA, EDTA, DOTA, TETA, and bisamino
bisthiol (BAT) chelators (see also U.S. 5,720,934). For example, N4 chelators
are
described in U.S. Patent Nos. 6,143,274; 6,093,382; 5,608,110; 5,665,329;
5,656,254;
and 5,688,487. Certain N3S chelators are described in PCT/CA94/00395,
PCT/CA94/00479, PCT/CA95/00249 and in U.S. Patent Nos. 5,662,885; 5,976,495;
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and 5,780,006. The chelator may also include derivatives of the chelating
ligand
mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N3S, and N2S2
systems such as MAMA (monoamidemonoaminedithiols), DADS (N2S
diaminedithiols), CODADS and the like. These ligand systems and a variety of
others
are described in Liu and Edwards, Chem Rev. 1999, 99, 2235-2268 and references
therein.
The chelator may also include complexes containing ligand atoms that are not
donated to the metal in a tetradentate array. These include the boronic acid
adducts of
technetium and rhenium dioximes, such as are described in U.S. Patent Nos.
5,183,653;
5,387,409; and 5,118,797, the disclosures of which are incorporated by
reference
herein, in their entirety.
In another embodiment, disulfide bonds of a binding polypeptide of the
invention are used as two ligands for chelation of a radionuclide such as 9
Tc. In this
way the peptide loop is expanded by the introduction of Tc (peptide-S-S-
peptide
changed to peptide-S-Tc-S-peptide). This has also been used in other disulfide
containing peptides in the literature (J. Q. Chen, A. Cheng, N. K. Owen, T. H.
Hoffman,
Y. Miao, S. S. Jurisson, T. P. Quinn. J. Nucl. Med. 2001, 42, 1847-1855) while
maintaining biological activity. The other chelating groups for Tc can be
supplied by
amide nitrogens of the backbone, another cystine amino acid or other
modifications of
amino acids.
Particularly preferred metal chelators include those of Formula 20, 21, and 22
(for 'In and lanthanides such as paramagnetic Gd3+ and radioactive
lanthanides, such
as, for example '77Lu, 90Y, 153Sm, and'66Ho) and those of Formula 23, 24, and
25 (for
radioactive 99mTc, 186Re, and'88Re) set forth below. These and other metal
chelating
groups are described in U.S. Patent Nos. 6,093,382 and 5,608,110, which are
incorporated by reference herein in their entirety. Additionally, the
chelating group of
formula 22 is described in, for example, U.S. Patent No. 6,143,274; the
chelating group
of formula 24 is described in, for example, U.S. Patent Nos. 5,627,286 and
6,093,382,
and the chelating group of formula 25 is described in, for example, U.S.
Patent Nos.
5,662,885; 5,780,006; and 5,976,495.
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R R R R
HOOC---\ /~ )_COOH
HOOC- J--000H N N
N N INN
HOOC U COON HOOC R -( U COOH
R CO NH
R
(20) (21)
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HOOC--\ n //--000H
CN N]
O N N
L -000H
(22)
OH
O
HN O
NH HN
NH HN
(N N: COON N N)
HO OH HO OH
(23a) (23b)
Y,.J )n
X X
(NH HN (NH HN
N X
N Y
HO OH n HO OH
(24a) (24b)
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OH
O O
O N N,=
N S''
NHCOCH3
(25)
In the above Formulas 20 and 21, R is alkyl, preferably methyl. In the above
Formula 24, X is either CH2 or 0, Y is either C, -CIO branched or unbranched
alkyl; Y is
aryl, aryloxy, arylamino, arylaminoacyl; Y is arylkyl - where the alkyl group
or groups
attached to the aryl group are C1-C10 branched or unbranched alkyl groups, C1-
C1o
branched or unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl
or
polyhydroxy-polyalkoxyalkyl groups, J is C(O)-, OC(=O)-, SO2-, NC(=O)-, NC(=S)-
,
N(Y), NC(=NCH3)-, NC(=NH)-, N=N-, homopolyamides or heteropolyamines derived
from synthetic or naturally occurring amino acids; all where n is 1-100. Other
variants
of these structures are described, for example, in U.S. Patent No. 6,093,382.
The
disclosures of each of the foregoing patents, applications and references are
incorporated by reference herein, in their entirety.
The chelators may be covalently linked directly to the heteromultimers or
linked
to heteromultimers via a linker, as described previously, and then directly
labeled with
the radioactive metal of choice (see, WO 98/52618, U.S. 5,879,658, and U.S.
5,849,261).
Complexes of radioactive technetium are particularly useful for diagnostic
imaging and complexes of radioactive rhenium are particularly useful for
radiotherapy.
In forming a complex of radioactive technetium with the reagents of this
invention, the
technetium complex, preferably a salt of Tc-99m pertechnetate, is reacted with
the
reagent in the presence of a reducing agent. Preferred reducing agents are
dithionite,
stannous and ferrous ions; the most preferred reducing agent is stannous
chloride.
Means for preparing such complexes are conveniently provided in a kit form
comprising a sealed vial containing a predetermined quantity of a reagent of
the
invention to be labeled and a sufficient amount of reducing agent to label the
reagent
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with Tc-99m. Alternatively, the complex may be formed by reacting a
heteromultimer
of this invention conjugated with an appropriate chelator with a pre-formed
labile
complex of technetium and another compound known as a transfer ligand. This
process
is known as ligand exchange and is well known to those skilled in the art. The
labile
complex may be formed using such transfer ligands as tartrate, citrate,
gluconate or
mannitol, for example. Among the Tc-99m pertechnetate salts useful with the
present
invention are included the alkali metal salts such as the sodium salt, or
ammonium salts
or lower alkyl ammonium salts.
Preparation of the complexes of the present invention where the metal is
radioactive
rhenium may be accomplished using rhenium starting materials in the +5 or +7
oxidation state. Examples of compounds in which rhenium is in the Re(VII)
state are
NH4ReO4 or KReO4. Re(V) is available as, for example, [ReOC14](NBu4),
[ReOCl4](AsPh4), ReOCl3(PPh3)2 and as Re02(pyridine)4+. (Ph is phenyl; Bu is n-
butyl). Other rhenium reagents capable of forming a rhenium complex may also
be
used.
Radioactively-labeled scintigraphic imaging agents provided by the present
invention are provided having a suitable amount of radioactivity. In forming
Tc-99m
radioactive complexes, it is generally preferred to form radioactive complexes
in
solutions containing radioactivity at concentrations of from about 0.01
millicurie (mCi)
to 100 mCi per mL.
Generally, the unit dose to be administered has a radioactivity of about 0.01
mCi
to about 100 mCi, preferably 1 mCi to 20 mCi. The solution to be injected at
unit
dosage is from about 0.01 mL to about 10 mL.
Typical doses of a radionuclide-labeled heteromultimeric construct imaging
agent of the invention provide 10-50 mCi. After injection of the
heteromultimeric
radionuclide imaging agent into the patient, a PET camera or a gamma camera
calibrated for the gamma ray energy of the nuclide incorporated in the imaging
agent is
used to image areas of uptake of the agent and quantify the amount of
radioactivity
present in the site. Imaging of the site in vivo can take place in a matter of
a few
minutes. However, imaging can take place, if desired, in hours or even longer,
after the
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radiolabeled peptide is injected into a patient. In most instances, a
sufficient amount of
the administered dose will accumulate in the area to be imaged within about
0.1 of an
hour to permit the taking of scintiphotos.
Proper dose schedules for the radiotherapeutic compounds of the present
invention are known to those skilled in the art. The compounds can be
administered
using many methods which include, but are not limited to, a single or multiple
IV or IP
injections, using a quantity of radioactivity that is sufficient to cause
damage or ablation
of the targeted tissue, but not so much that substantive damage is caused to
non-target
(normal tissue). The quantity and dose required is different for different
constructs,
depending on the energy and half-life of the isotope used, the degree of
uptake and
clearance of the agent from the body and the mass of the tumor. In general,
doses can
range from a single dose of about 30-50 mCi to a cumulative dose of up to
about 3
Curies.
The radiotherapeutic compositions of the invention can include physiologically
acceptable buffers, and can require radiation stabilizers to prevent
radiolytic damage to
the compound prior to injection. Radiation stabilizers are known to those
skilled in the
art, and may include, for example, para-aminobenzoic acid, ascorbic acid,
gentistic acid
and the like.
A single, or multi-vial kit that contains all of the components needed to
prepare
the radiopharmaceuticals of this invention, other than the radionuclide, is an
integral
part of this invention.
A single-vial kit preferably contains a chelating ligand (if a metal
radionuclide is
used), a source of stannous salt (if reduction is required, e.g., when using
technetium),
or other pharmaceutically acceptable reducing agent, and is appropriately
buffered with
pharmaceutically acceptable acid or base to adjust the pH to a value of about
3 to about
9. The quantity and type of reducing agent used would depend highly on the
nature of
the exchange complex to be formed. The proper conditions are well known to
those
that are skilled in the art. It is preferred that the kit contents be in
lyophilized form.
Such a single vial kit may optionally contain labile or exchange ligands such
as
glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and can
also contain
reaction modifiers such as diethylenetriamine-pentaacetic acid (DPTA),
ethylenediamine tetraacetic acid (EDTA), or a, p, or y cyclodextrin that serve
to
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improve the radiochemical purity and stability of the final product. The kit
may also
contain stabilizers, bulking agents such as mannitol, that are designed to aid
in the
freeze-drying process, and other additives known to those skilled in the art.
A multi-vial kit preferably contains the same general components but employs
more than one vial in reconstituting the radiopharmaceutical. For example, one
vial
may contain all of the ingredients that are required to form a labile Tc(V)
complex on
addition of pertechnetate (e.g. the stannous source or other reducing agent).
Pertechnetate is added to this vial, and after waiting an appropriate period
of time, the
contents of this vial are added to a second vial that contains the ligand, as
well as
buffers appropriate to adjust the pH to its optimal value. After a reaction
time of about 5
to 60 minutes, the complexes of the present invention are formed. It is
advantageous
that the contents of both vials of this multi-vial kit be lyophilized. As
above, reaction
modifiers, exchange ligands, stabilizers, bulking agents, etc. may be present
in either or
both vials.
Other Therapeutic Applications
The heteromultimeric constructs of the present invention can be used to
improve
the activity and/or efficacy of therapeutic agents by, for example, improving
their
affinity for or residence time at the target. In this embodiment
heteromultimers are
conjugated with the therapeutic agent. Alternatively, as discussed above, a
liposome or
bubble containing a therapeutic agent may be conjugated to heteromultimers of
the
invention. The therapeutic agent may be a radiotherapeutic, discussed above, a
drug,
chemotherapcutic or tumorcidai agent, genetic material, or a gene delivery
vehicle, etc.
The heteromultimer portion of the conjugate causes the therapeutic to "home"
to the
sites of target expression/localization and to improve the affinity of the
conjugate for
these sites, so that the therapeutic activity of the conjugate is more
localized and
concentrated at the target sites. For example, in one embodiment
heteromultimers
including KDR or VEGF/KDR complex binding polypeptides, can be used to improve
the activity of therapeutic agents (such as anti-angiogenic or tumorcidal
agents) against
undesired angiogenesis such as occurs in neoplastic tumors, by providing or
improving
their affinity for KDR or the VEGF/KDR complex and their residence time at a
KDR or
VEGF/KDR complex on endothelium undergoing angiogenesis. In this aspect of the
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invention, hybrid agents are provided by conjugating KDR or VEGF/KDR complex
binding heteromultimers with a therapeutic agent. Such heteromultimeric
constructs
will be useful in treating angiogenesis associated diseases, especially
neoplastic tumor
growth and metastasis, in mammals, including humans. The method of treatment
comprises administering to a mammal in need thereof an effective amount of a
heteromultimeric construct comprising KDR or VEGF/KDR complex binding
polypeptides conjugated with a therapeutic agent. The invention also provides
the use
of such conjugates in the manufacture of a medicament for the treatment of
angiogenesis associated diseases in mammals, including humans.
Heteromultimeric
constructs of the invention comprising cMet or HGF/cMet complex binding
moieties
may be used similarly to treat disease associated with hyperproliferation or
angiogenesis.
Suitable therapeutic agents for use in this aspect of the invention include,
but are
not limited to: antineoplastic agents, such as platinum compounds (e.g.,
spiroplatin,
cisplatin, and carboplatin), methotrexate, adriamycin, mitomycin, ansamitocin,
bleomycin, cytosine, arabinoside, arabinosyl adenine, mercaptopolylysine,
vincristine,
busulfan, chlorambucil, melphalan (e.g., PAM, a,L-PAM or phenylalanine
mustard),
mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin
(actinomycin D),
daunorubcin, hydrochloride, doxorubicin hydrochloride, taxol, mitomycin,
plicamycin
(mithramycin), aminoglutethimide, estramustine phosphate sodium, flutamide,
acetate,
megestrol acetate, tamoxifen citrate, testolactone, trilostane, amsacrine (m-
AMSA),
ASPARAGINASE (L-ASPARAGINASE) Erwina aparaginase, etoposide (VP-16),
interferon
cx-2a, Interferon cx-2b, teniposide (VM-26, vinblastine sulfate (VLB),
vincristine
sulfate, bleomycin sulfate, adriamycin, and arabinosyl; anti-angiogenic agents
such as;
tyrosine kinase inhibitors with activity toward signaling molecules important
in
angiogenesis and/or tumor growth such as SU5416 and SU6668 (Sugen/Pharmacia &
Upjohn), endostatin (EntreMed), angiostatin (EntreMed), Combretastatin
(oxigene),
cyclosporine, 5-fuorouracil, vinblastine, doxorubicin, paclitaxel,
daunorubicin,
immunotoxins; coagulation factors; antivirals such as acyclovir, amantadine
azidothymidine (AZT or Zidovudine), ribavirin and vidarabine monohydrate
(adenine
arahinoside, ara-A); antibiotics, antimalarials, antiprotozoans such as
chloroquine,
hydroxychloroquine, metronidazole, quinine and meglumine antimonate; anti-
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inflammatories such as diflunisal, ibuprofen, indomethacin, meclofenamate,
mefenamic
acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac,
tolmetin,
aspirin and salicylates.
Where heteromultimeric constructs target other tissue and are useful in
treating
other disease states the skilled artisan may substitute an appropriate
therapeutic agent.
The heteromultimeric constructs of the present invention may also be used to
target genetic material to specific cells. For example, the heteromultimeric
constructs
of the present invention may be used to localize genetic material to cells or
tissue
containing the desired target. Thus such constructs may be useful in gene
therapy. The
genetic material may include nucleic acids, such as RNA or DNA, of either
natural or
synthetic origin, including recombinant RNA and DNA and antisense RNA and DNA.
Types of genetic material that may be used include, for example, genes carried
on
expression vectors such as plasmids, phagemids, cosmids, yeast artificial
chromosomes
(YACs) and defective or "helper" viruses, antigene nucleic acids, both single
and
double stranded RNA and DNA and analogs thereof, such as phosphorothioate and
phosphorodithioate oligodeoxynucleotides. Additionally, the genetic material
maybe
combined, for example, with lipids, proteins or other polymers. Delivery
vehicles for
genetic material may include, for example, a virus particle, a retroviral or
other gene
therapy vector, a liposome, a complex of lipids (especially cationic lipids)
and genetic
material, a complex of dextran derivatives and genetic material, etc.
In a preferred embodiment the heteromultimeric constructs of the invention are
utilized in gene therapy for treatment of diseases associated with
angiogenesis. In this
embodiment, genetic material, or one or more delivery vehicles containing
genetic
material, e.g., useful in treating an angiogenesis-related disease, may be
conjugated to
one or more KDR or VEGF/KDR complex binding heteromultimers or cMet or
HGF/cMET complex binding heteromultimers of the invention and administered to
a
patient.
Constructs including genetic material and the KDR binding heteromultimers of
the invention may be used, in particular, to selectively introduce genes into
angiogenic
endothelial cells, which may be useful not only to treat cancer, but also
after
angioplasty, where inhibition of angiogenesis may inhibit restenosis.
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Therapeutic agents and heteromultimers of the invention can be linked or fused
in known ways, using the same type of linkers discussed herein. Preferred
linkers will
be substituted or unsubstituted alkyl chains, amino acid chains, polyethylene
glycol
chains, and other simple polymeric linkers known in the art. More preferably,
if the
therapeutic agent is itself a protein, for which the encoding DNA sequence is
known,
the therapeutic protein and a binding polypeptide of the invention may be
coexpressed
from the same synthetic gene, created using recombinant DNA techniques, as
described
above. For example, the coding sequence for a binding polypeptide may be fused
in
frame with that of the therapeutic protein, such that the peptide is expressed
at the
amino- or carboxy-terminus of the therapeutic protein, or at a place between
the termini,
if it is determined that such placement would not destroy the required
biological
function of either the therapeutic protein or the binding polypeptide. A
particular
advantage of this general approach is that concatamerization of multiple,
tandemly
arranged binding polypeptides is possible, thereby increasing the number and
concentration of binding sites associated with each therapeutic protein. In
this manner
binding peptide avidity is increased which would be expected to improve the
efficacy of
the recombinant therapeutic fusion protein.
Similar recombinant proteins containing one or more coding sequences for a
binding polypeptide may be useful in imaging or therapeutic applications. For
example,
in a variation of the pre-targeting applications discussed infra, the coding
sequence for a
KDR, VEGF/KDR complex, cMet, or HGF/cMet binding peptide may be fused in
frame to a sequence encoding an antibody (or an antibody fragment or
recombinant
DNA construct including an antibody, etc.) which, for example, binds to a
chelator for a
radionuclide (or another detectable label). The antibody expressing the KDR,
VEGF/KDR complex, cMet, or HGF/cMet binding polypeptide is then administered
to
a patient and allowed to localize and bind to KDR- or cMet-expressing tissue.
After the
non-binding antibodies have been allowed to clear, the chelator-radionuclide
complex
(or other detectable label), which the antibody recognizes is administered,
permitting
imaging of or radiotherapy to the KDR- or cMet-expressing tissues.
Additionally, the
coding sequence for a binding peptide may be fused in frame to a sequence
encoding,
for example, serum proteins or other proteins that produce biological effects
(such as
apoptosis, coagulation, internalization, differentiation, cellular stasis,
immune system
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stimulation or suppression, or combinations thereof). The resulting
recombinant
proteins are useful in imaging, radiotherapy, and therapies directed against
cancer and
other diseases that involve angiogenesis or diseases associated with the
pathogens
discussed herein.
Additionally, heteromultimers of the present invention may themselves be used
as therapeutics to treat a number of diseases. For example, where binding of a
protein
or other molecule (e.g. a growth factor, hormone etc.) is necessary for or
contributes to
a disease process and a binding moiety inhibits such binding, heteromultimers
including
such binding moieties may be useful as therapeutics. Similarly, where binding
of a
binding moiety itself inhibits a disease process, heteromultimers containing
such
binding moieties may also be useful as therapeutics.
As binding of VEGF and activation of KDR is necessary for angiogenic activity,
in one embodiment heteromultimers including KDR or VEGF/KDR complex binding
polypeptides that inhibit the binding or inhibit VEGF to KDR (or otherwise
inhibit
activation of KDR) may be used as anti-angiogenic agents. Certain
heteromultimers of
the invention that inhibit activation of KDR are discussed in the Examples. A
particularly preferred heteromultimer is the heterodimer-containing construct
D 1
(structure shown below in Example 9). Other preferred heterodimer constructs
include
D4, D5, and D6 (structures provided in Examples 9 and 15 below). These and
other
heteromultimers may be useful in the treatment of cancer or other diseases
associated
with inappropriate or excessive angiogenesis, such as, for example arthritis
and
atherosclerotic plaques, trachoma, corneal graft neovascularization,
psoriasis,
sleroderma, hemangioma and hypertrophic scarring, vascular adhesions,
angiofibroma,
and ocular diseases, such as diabetic retinopathy, retinopathy of prematurity,
macular
degeneration, corneal graft rejection, neovascular glaucoma, retrolental
fibroplasia,
rebeosis, Osler-Webber Syndrome, myocardial angiogenesis, plaque
neovascularization,
telangiectasia, hemophiliac joints, angiofibroma and wound granulation. Other
conditions that involve angiogenesis include, for example, solid tumors, tumor
metastases and benign tumors. Such tumors and related disorders are well known
in the
art and include, for example, melanoma, central nervous system tumors,
neuroendocrine
tumors, sarcoma, multiple myeloma as wells as cancer of the breast, lung,
prostate,
colon, head & neck, and ovaries. Additional tumors and related disorders are
listed in
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Table I of U.S. Patent No. 6,025,331, issued February 15, 2000 to Moses, et
al., the
teachings of which are incorporated herein by reference. Benign tumors
include, for
example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic
granulomas. Other relevant diseases or conditions that involve blood vessel
growth
include intestinal adhesions, atherosclerosis, scleroderma, and hypertropic
scars, and
ulcers. Furthermore, the heteromultimers of the present invention can be used
to reduce
or prevent uterine neovascularization required for embryo implantation, for
example, as
a birth control agent.
Heteromultimers of this invention can also be useful for treating vascular
permeability events that can result when VEGF binds KDR. In renal failure it
has been
shown that anti-VEGF antibodies can reverse damage and in a similar way the
compounds of the invention can reverse renal permeability pathogenesis in, for
example, diabetes.
As the interruption of the HGF interaction with the cMet receptor slows tumor
progression, in another embodiment, the heteromultimers include cMet or
HGF/cMet
complex binding polypeptides that inbhit the binding of cMet to HGF (or
otherwise
inhibit the activation of cMet) may be used to treat tumors and other
hyperproliferative
disorders. Particular heteromultimers that inhibit cMet are discussed in the
Examples.
A preferred heteromultimer is D28 (structure shown below in Example 9).
Furthermore, heteromultimers of the present invention may be useful in
treating
diseases associated with certain pathogens, including, for example, malaria,
HIV, SW,
Simian hemorrhagic fever virus, etc. Sequence homology searches of KDR-binding
peptides identified by phage display using the BLAST program at NCBI has
identified a
number of homologous proteins known or expected to be present on the surface
of
pathogenic organisms. Homologies were noted between KDR and VEGF/KDR
complex binding polypeptides and proteins from various malaria strains, HIV,
SW,
simian hemorrhagic fever virus, and an enterohemorrhagic E. coli strain. Some
of the
homologous proteins, such as PtEMPI and EBL-1, are hypermutable adhesion
proteins
known to play roles in virulence. These proteins possess multiple binding
sites that are
capable of binding to more than one target molecule on the host's surface.
Their high
mutation and recombination rates allow them to quickly develop new binding
sites to
promote survival and/or invasion. Similarly, proteins such as gp120 of HIV
(which also
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has homology to some of the KDR-binding peptides disclosed herein) play
critical roles
in the adhesion of pathogens to their hosts. Although not reported previously,
it is
possible that many of the pathogen proteins with homology to the KDR-binding
peptides disclosed herein also bind to KDR. Comparison of the pathogen protein
sequences with the corresponding peptide sequences may suggest changes in the
peptide sequence or other modifications that will enhance its binding
properties.
Additionally, heteromultimeric constructs including the KDR-binding peptide
sequences disclosed herein may have usefulness in blocking infection with the
pathogen
species that possesses the homology. Indeed, a strategy is being employed to
block
HIV infection by trying to prevent virus envelope proteins from binding to
their known
cellular surface targets such as CD4. Howie SE, et al., FASEB J 1998 Aug;
12(11):991-8, "Synthetic peptides representing discontinuous CD4 binding
epitopes of
HIV-1 gp 120 that induce T cell apoptosis and block cell death induced by gp
120."
Thus, KDR may represent a previously unknown target for a number of pathogens
and
the heteromultimeric constructs including KDR or VEGF/KDR complex binding
peptides may be useful in treating the diseases associated with these
pathogens.
In the above treatment methods, the compounds may be administered by any
convenient route customary for therapeutic agents, for example parenterally,
enterally
or intranasaly, and preferably by infusion or bolus injection, or by depot or
slow release
formulation. In a preferred embodiment, the composition may be formulated in
accordance with routine procedures as a pharmaceutical composition adapted for
intravenous administration to human beings. Typically, compositions for
intravenous
administration are solutions in sterile isotonic aqueous buffer. Other
pharmaceutically
acceptable carriers include, but are not limited to, sterile water, saline
solution, buffered
saline (including buffers like phosphate or acetate), alcohol, vegetable oils,
polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc,
silicic acid,
paraffin, etc. Where necessary, the composition may also include a
solubilizing agent
and a local anaesthetic such as lidocaine to ease pain at the site of the
injection,
preservatives, stabilizers, wetting agents, emulsifiers, salts, lubricants,
etc. as long as
they do not react deleteriously with the active compounds. Similarly, the
composition
may comprise conventional excipients, i.e. pharmaceutically acceptable organic
or
inorganic carrier substances suitable for parenteral, enteral or intranasal
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which do not deleteriously react with the active compounds. Generally, the
ingredients
will be supplied either separately or mixed together in unit dosage form, for
example, as
a dry lyophilized powder or water free concentrate in a hermetically sealed
container
such as an ampoule or sachette indicating the quantity of active agent in
activity units.
Where the composition is to be administered by infusion, it can be dispensed
with an
infusion bottle containing sterile pharmaceutical grade "water for injection"
or saline.
Where the composition is to be administered by injection, an ampoule of
sterile water
for injection or saline may be provided so that the ingredients may be mixed
prior to
administration.
The quantity of material administered will depend on the seriousness of the
condition.
For example, for treatment of anangiogenic condition, e.g., in the case of
neoplastic
tumor growth, the position and size of the tumor will affect the quantity of
material to
be administered. The precise dose to be employed and mode of administration
must per
force in view of the nature of the complaint be decided according to the
circumstances
by the physician supervising treatment. In general, dosages of the
heteromultimer/therapeutic agent conjugate will follow the dosages that are
routine for
the therapeutic agent alone, although the improved affinity of a
heteromultimer of the
invention for its target may allow a decrease in the standard dosage.
Such conjugate pharmaceutical compositions are preferably formulated for
parenteral administration, and most preferably for intravenous or intra-
arterial
administration. Generally, and particularly when administration is intravenous
or intra-
arterial, pharmaceutical compositions may be given as a bolus, as two or more
doses
separated in time, or as a constant or non-linear flow infusion.
The hetermultimers can be administered to an individual over a suitable time
course depending on the nature of the condition and the desired outcome. The
heteromultimeric constructs can be administered prophylactically, e.g., before
the
condition is diagnosed or to an individual predisposed to a condition.
Alternatively, the
heteromultimers of the invention can be administered while the individual
exhibits
symptoms of the condition or after the symptoms have passed or otherwise been
relieved (such as after removal of a tumor). In addition, the heteromultimers
of the
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present invention can be administered a part of a maintenance regimen, for
example to
prevent or lessen the recurrence or the symptoms or condition. As described
herein, the
heteromultimers of the present invention can be administered systemically or
locally.
As used herein the term "therapeutic" includes at least partial alleviation of
symptoms of a given condition. The heteromultimeric constructs of the present
invention do not have to produce a complete alleviation of symptoms to be
useful. For
example, treatment of an individual can result in a decrease in the size of a
tumor or
diseased area, or prevention of an increase in size of the tumor or diseased
area or
partial alleviation of other symptoms. Treatment can result in reduction in
the number
of blood vessels in an area of interest or can prevent an increase in the
number of blood
vessels in an area of interest. Treatment can also prevent or lessen the
number or size of
metastic outgrowths of the main tumor(s).
In one embodiment, symptoms that can be alleviated include physiological
characteristics such as VEGF receptor activity and migration ability of
endothelial cells.
The heteromultimers of the present invention can inhibit activity of VEGF
receptors,
including VEGF-2/KDR, VEGF-1/Flt-1 and VEGF-3/Flt-4. Such inhibition can also
be
detected, for example, by measuring the phosphorylation state of the receptor
in the
presence of or after treatment with the binding polypeptides or constructs
thereof.
Based on the teachings provided herein, one of ordinary skill in the art would
know how
and be able to administer a suitable dose of binding polypeptide or construct
thereof as
provided herein and measured before and after treatment. In another
embodiment, the
phosphorylation state of the relevant receptor, or the migration ability of
endothelial in
an area of interest can be measured in samples taken from the individual. The
VEGF
receptors or endothelial cells can be isolated from the sample and used in
assays
described herein.
The dosage of the heteromultimers may depend on the age, sex, health, and
weight of the individual, a well as the nature of the condition and overall
treatment
regimen. The biological effects of the multimers are described herein.
Therefore, based
on the biological effects of the heteromultimers provided herein, and the
desired
outcome of treatment, the referred dosage is determinable by one of ordinary
skill in the
art through route optimization procedures. Typically, the daily regiment is in
the range
of about 0.1 pg/kg to about 1 mg/kg.
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The heteromultimers provided herein can be administered as the sole active
ingredient together with a pharmaceutically acceptable excipient, or can be
administered together with other binding polypeptides and constructs thereof,
other
therapeutic agents, or combination thereof. In addition, the heteromultimers
can be
conjugated to therapeutic agents, for example, to improve specificity,
residence time in
the body, or therapeutic effect. Such other therapeutic agents include, for
example,
other antiangiogenic compounds, and tumoricidal compounds. The therapeutic
agent
can also include antibodies.
Furthermore, the heteromultimers of the present invention can be used as an
endothelial cell homing device. Therefore, the heteromultimeric constructs can
be
conjugated to nucleic acids encoding, for example, a therapeutic polypeptide,
in order to
target the nucleic acid to endothelial cells. Once exposed to the nucleic
acid, thereby
delivering the therapeutic peptide to the target cells.
In another embodiment of the invention, the therapeutic agent can be
associated
with an ultrasound contrast agent composition, said ultrasound contrast agent
including
the KDR, VEGF/KDR complex, cMet, or HGF/cMet binding peptides of the invention
linked to the material employed to form the vesicles (particularly
microbubbles or
microballoons) comprised in the contrast agent, as previously described. For
instance,
said contrast agent/therapeutic agent association can be carried out as
described in US
6,258,378, herein incorporated by reference. Thus, after administration of the
ultrasound contrast agent and the optional imaging of the contrast agent bound
to the
pathogenic site expressing the KDR, VEGF/KDR complex, cMet, or HGF/cMet
complex, the pathogenic site can be irradiated with an energy beam (preferably
ultrasonic, e.g. with a frequency of from 0.3 to 3 MHz), to cause the bursting
of
microvesicles, as disclosed for instance in the above cited U.S. Patent No.
6,258,378.
The therapeutic effect of the therapeutic agent can thus be advantageously
enhanced by
the energy released by the burst of the microvesicles, in particular causing
an effective
delivery of the therapeutic agent to the targeted pathogenic site.
As discussed above, the heteromultimers can be administered by any suitable
route. Suitable routes of administration include, but are not limited to,
topical
application, transdermal, parenteral, gastrointestinal, intravaginal, and
transalvcolar.
Compositions for the desired route of administration can be prepared by any of
the
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methods well known in the pharmaceutical arts. Details concerning dosages,
dosage
forms, modes of administration, composition and the like are further discussed
in a
standard pharmaceutical text, such as Remington's Pharmaceutical Sciences,
18th ed.,
Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990), which is
hereby
incorporated by reference.
For topical applications, the heteromultimers can be suspended, for example,
in
a cream, gel or rinse which allows the polypeptides or constructs to penetrate
the skin
and enter the blood stream, for systemic delivery, or contact the are of
interest, for
localized delivery. Compositions suitable for topical application include any
pharmaceutically acceptable base in which the polypeptides are at least
minimally
soluble.
For transdermal administration, the heteromultimers can be applied in
pharmaceutically acceptable suspension together with a suitable transdermal
device or
"patch." Examples of suitable transdermal devices for administration of the
heteromultimers of the present invention are described, for example, in U.S.
Patent No.
6,165,458, issued December 26, 2000 to Foldvari, et al., and U.S. Patent No.
6,274,166B1, issued August 4, 2001 to Sintov, et al., the teachings of which
are
incorporated herein by reference.
For parenteral administration, the heteromultimers can be suspended, for
example, in a pharmaceutically acceptable sterile isotonic solution, such as
saline and
phosphate buffered saline. The constructs of the invention can then be
injected
intravenously, intramuscularly, intraperitoneally, or subcutaneously.
For gastrointestinal and intravaginal administration, the heteromultimers can
be
incorporated into pharmaceutically acceptable powders, pills or liquids for
ingestion,
and suppositories for rectal or vaginal administration.
For transalveolar, buccal or pulmonary administration, the heteromultimers can
be suspended in a pharmaceutically acceptable excipient suitable for
aerosolization and
inhalation or as a mouthwash. Devices suitable for transalveolar
administration such as
atomizers and vaporizes are also included within the scope of the invention.
Suitable
formulations for aerosol delivery of polypeptides using buccal or pulmonary
routes can
be found, for example in U.S. Patent No. 6,312,665B1, issued November 6, 2001
to
Pankaj Modi, the teachings of which are incorporated herein by reference.
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In addition, the heteromultimers of the present invention can be administered
nasally or ocularly, where the heteromultimers are suspended in a liquid
pharmaceutically acceptable agent suitable for dropwise dosing.
The heteromultimers of the present invention can be administered such that the
polypeptide is released in the individual over an extended period of time
(sustained or
controlled release). For example, the heteromultimers can be formulated into a
composition such that a single administration provides delivery of the
constructs of the
invention for at least one week, or over the period of a year or more.
Controlled release
systems include monolithic or reservoir-type microcapsules, depot implants,
osmotic
pumps, vesicles, micelles, liposomes, transdermal patches and iontophoretic
devices. In
one embodiment, the heteromultimers of the present invention are encapsulated
or
admixed in a slow degrading, non-toxic polymer. Additional formulations
suitable for
controlled release of constructs of the invention are described in U.S. Patent
No.
4,391,797, issued July 5, 1983, to Folkman, et al., the teachings of which are
incorporated herein by reference.
Another suitable method for delivering the heteromultimers of the present
invention to an individual is via in vivo production of the polypeptides.
Genes encoding
the polypeptides can be administered to the individual such that the encoded
polypeptides are expressed. The genes can be transiently expressed. In a
particular
embodiment, the genes encoding the polypeptide are transfected into cells that
have
been obtained from the patient, a method referred to as ex vivo gene therapy.
Cells
expressing the polypeptides are then returned to the patient's body. Methods
of ex vivo
gene therapy are well known in the art, and are described, for example, in
U.S. Patent
No. 4,391,797, issued March 21, 1998 to Anderson, et al., the teachings of
which are
incorporated herein by reference.
Preparation and tests of heteromultimeric constructs in accordance with this
invention will be further illustrated in the following examples. The specific
parameters
included in the following examples are intended to illustrate the practice of
the
invention, and they are not presented to in any way limit the scope of the
invention.
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EXAMPLE 1
Peptide Synthesis and Fluorescein Labelling
Selected KDR or VEGF/KDR binding peptides corresponding to positive phage
isolates were synthesized on solid phase using 9-fluorenylmethoxycarbonyl
protocols
and purified by reverse phase chromatography. Peptide masses were confirmed by
electrospray mass spectrometry, and peptides were quantified by absorbance at
280 rim.
For synthesis, two N-terminal and two C-terminal amino acids from the phage
vector
sequence from which the peptide was excised were retained and a -Gly-Gly-Gly-
Lys-
NH2 linker was added to the C-terminus of each peptide. Peptides with selected
lysine
residues were protected with 1-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-3-
methybutyl (ivDde), which allows selective coupling to the C-terminal lysine,
is not
removed during peptide cleavage, and can be removed after coupling with 2%
hydrazine in DMF or 0.5 M hydroxylamine, pH 8, in water.
Each peptide was labeled with fluorescein on the C-terminal lysine using
Fluorescein (N-hydroxysuccinimide ester derivative) or Fluorescein
Isothiocyanate
(FITC) in DMF, 2% diisopropylethylamine (DIPEA). If the peptide contained an
ivDde
protected lysine, the reaction was quenched by the addition of 2% hydrazine,
which
reacts with all free NHS-fluorescein and removes the internal protecting
group. For all
other peptides, the reaction was quenched by the addition of an equal volume
of 0.5 M
hydroxylamine, pH 8. The quenched reactions were then diluted with water to
less than
10% DMF and then purified using C18 reverse phase chromatography. The peptides
were characterized for purity and correct mass on an LC-MS system (HP 1100
HPLC
with in-line SCIEX AP ISO single quadrapole mass spectrometer).
Fluorescence Anisotropy Measurements and BiaCore Assays
Fluorescence anisotropy measurements were performed in 384-well microplates
in a volume of 10 L in binding buffer (PBS, 0.01% Tween-20, pH 7.5) using a
Tecan
Polarion fluorescence polarization plate reader. In some cases, heparin (0.5
g/mL) or
10% human serum was added to the binding buffer. The concentration of
fluorescein
labeled peptide was held constant (20 nM) and the concentration of KDR-Fc (or
similar
target) was varied. Binding mixtures were equilibrated for 10 minutes in the
microplate
at 30 C before measurement. The observed change in anisotropy was fit to
Equation
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(1) below via nonlinear regression to obtain the apparent KD. Equation (1)
assumes that
the synthetic peptide and HSA form a reversible complex in solution with 1:1
stoichiometry:
robs = rjree + (rbound - rjree ) (K,, + KDR + P) - (K KDR + P)2 - 4 KDR P (1)~
2. P
where robs is the observed anisotropy, rfree is the anisotropy of the free
peptide, rboafla is
the anisotropy of the bound peptide, KD is the apparent dissociation constant,
KDR is
the total KDR concentration, and P is the total fluorescein-labeled peptide
concentration.
KDR-Fc (or another protein target) was cross-linked to the dextran surface of
a
CM5 sensor chip by the standard amine coupling procedure (0.5 mg/mL solutions
diluted 1:20 with 50 mM acetate, pH 6.0, RL KDR-Fc = 12859). Experiments were
performed in HBS-P buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005%
polysorbate
20 (v/v)). Peptide solutions quantitated by extinction coefficient were
diluted to 400
nM in HBS-P. Serial dilutions were performed to produce 200, 100, 50, and 25
nM
solutions. For association, peptides were injected at 20 L/min for 1 minute
using the
kinject program. Following a 1-minute dissociation, any remaining peptide was
stripped from the target surface with a quick injection of 1M NaCl for 25 sec.
at 50
gUmin. All samples were injected in duplicate. Between each peptide series a
buffer
injection and a non-target binding peptide injection served as additional
controls.
Sensorgrams were analyzed using the simultaneous ka/kd fitting program in the
BlAevaluation software 3.1.
The following common abbreviations are used throughout this specification: 9-
fluorenylmethyloxycarbonyl (fmoc or Fmoc), 1-hydroxybenozotri azole (HOBt),
N,N'-
diisopropylcarbodiimide (DIC), N-methylpyrrolidinone (NMP), acetic anhydride
(Ac20), (4,4-dimethyl-2,6-dioxocyclohex- I -ylidene)-3-methylbutyl (ivdde),
trifluoroacetic acid (TFA), Reagent B (TFA:H20:phenol:triisopropylsilane,
88:5:5:2),
diisopropylethylamine (DIEA), O-(1H-benzotriazole-1-yl)-N,N,N',N'-
tetramethyluronium hexafluorophosphate (HBTU),O-(7-azabenzotriazol-1-yl)-
1,1,3,3-
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tetramethyluronium hexafluorphosphate (HATU), N-hydroxysuccinimide (NHS),
solid
phase peptide synthesis (SPPS), dimethyl sulfoxide (DMSO), dichloromethane
(DCM),
dimethylformamide (DMF), human serum albumin (HSA), and radiochemical purity
(RCP).
Experimental Methods
The following methods were employed in the Examples.
Method I for ACT 357 MPS and ACT 496 MOS Synthesizers
The peptides were synthesized on NovaSyn TGR (Rink amide) resin (0.2
mmol/g) using the Advanced ChemTech ACT 357 or ACT 496 Synthesizers employing
Fmoc peptide synthesis protocols, specifically using HOBt/DIC as the coupling
reagents and NMP as the solvent. The Fmoc was removed by treating the Nova-Syn
TGR (Rink amide-available from NovaBiochem, San Diego, CA) resin-bound peptide
with 25% piperidine in DMF twice (4 min and 10 min). All amino acids were
dissolved
in NMP (DMF was added when the amino acid was not soluble in pure NMP). The
concentration of the amino acid was 0.25 M, and the concentration for both
HOBt and
DIC was 0.5 M.
For a 0.04 mmol scale synthesis:
A typical amino acid coupling cycle (not including wash steps) was to dispense
piperidine solution (2.4 mL) to each well and mix for 4 min, then empty all
wells. NMP
(320 L), HOBt solution (320 L, 4eq), amino acid (640 L, 4eq) and DIC (320
L,
4eq) solutions were dispensed to each well. The coupling time was 3 h; then
the resin
was washed. The cycle was repeated for each amino acid. After the last amino
acid
coupling, the resin-bound peptide was treated with 25% piperidine to remove
the Fmoc
protecting group. After washing, the resin bound peptide was capped with 1.0 M
Ac2O
(1.2 mL per well) and diisopropylethylamine in DMF, optionally including
varying
amounts of HOBt in the mixture for 30 min. The resin was washed first with
methanol
and then with dichloromethane and dried. Cleavage of the peptides from the
resin and
side-chain deprotection was accomplished using Reagent B for 4.5h. The
cleavage
solutions were collected and the resins were washing with an additional
aliquot of
Reagant B. The combined solutions were concentrated to dryness. Ether was
added to
the residue with swirling or stirring to precipitate the peptides. The ether
was decanted,
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and solid was collected. This procedure was repeated 2-3 times to remove
impurities.
The crude peptides were dissolved in DMSO and water mixture, and purified by
HPLC
(column: Water's Associates Xterra C18, 19 x 50 mm; solvents: H2O with 0.1%
TFA
and CH3CN with 0.1 % TFA; UV 220 nm; Flow rate: 50-60 mL/min). The solutions
containing the peptide were lyophilized to give the desired peptides as white
fluffy
lyophilizates (> 90% purity).
The purified linear di-cysteine containing peptides were dissolved in water,
mixtures of water-acetonitrile, or mixtures of water-DMSO at concentrations
between
0.1 mg/mL and 2.0 mg/mL. The choice of solvent was a function of the
solubility of
the crude peptide in the solvent. The pH of the solution was adjusted to 7.5-
8.5 with
aqueous ammonia, aqueous ammonium carbonate or aqueous ammonium bicarbonate.
The mixture was stirred vigorously in air for 24-48 h. In the case of non-DMSO
containing solvent systems, the pH of the solution was adjusted to 2 with
aqueous
trifluoroacetic acid. The mixture was lyophilized to provide the crude cyclic
disulfide
containing peptide. The cyclic disulfide peptide was then dissolved to a
volume of 1-2
mL in aqueous (0.1 % TFA) containing a minimum of acetonitrile (0.1 % TFA).
The
resulting solution was loaded onto a reverse phase column and the desired
compound
obtained by a gradient elution of acetonitrile into water, employing a C18, or
C8 reverse
phase semipreparative or preparative HPLC column. In the case of the DMSO-
containing solutions, the solution was diluted until the DMSO concentration
was
minimal without precipitation of the peptide. The resulting mixture was
quickly
acidified to pH 2 with dilute trifluoroacetic acid and loaded onto the reverse
phase
HPLC system and purified as described. Fractions containing the desired
materials
were pooled and the peptides isolated by lyophilization.
Method 2 for ACT 357 MPS and ACT 496 MOS Synthesizers
The peptides were synthesized as in Method 1, with the following changes.
HBTU/HOBt/DIEA were used as the coupling reagent and NMP as the solvent. A low
load (-Ø2 mmol/g) Fmoc-GGGK(Boc)-NovSyn-TGR-resin prepared from the above-
described Nova-Syn TGR resin was employed for peptides synthesis on 0.01 mmol
scale synthesis.
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For a 0.01 mmol scale synthesis:
After the Fmoc group was removed, a standard coupling procedure used a
solution of HOBt (720 L, 6 eq), amino acid (804 L, 6.6 eq), HBTU (720 L, 6
eq)
and DIEA (798 L, 13.3 eq). The mixture was agitated for 15 min, emptied and
the
resin washed. After all couplings and after cleavage and purification as
above, the
solutions containing desired linear peptides were lyophilized to give the
peptides as
white fluffy solids (> 90% purity).
The crude ether-precipitated linear di-cysteine containing peptides were
cyclized
by dissolution in water, mixtures of aqueous acetonitrile (0.1 % TFA), or
aqueous
DMSO and adjustment of the pH of the solution to 7.5 - 8.5 by addition of
aqueous
ammonia, aqueous ammonium carbonate, or aqueous ammonium bicarbonate solution.
The peptide concentration was between 0.1 and 2.0 mg/mL. The mixture was
stirred in
air for 24-48 h, acidified to a pH of 2 with aqueous trifluoroacetic acid and
then purified
by preparative reverse phase HPLC employing a gradient of acetonitrile into
water.
15, Fractions containing the desired material were pooled and the peptides
were isolated by
lyophilization.
Method 3 for the ACT 496 MOS Synthesizer
The peptides were synthesized using an Advanced ChemTech ACT 496 MOS
Synthesizer as in Method 1. The low load (-0.2 mmol/g) GGGK(Boc)-NovaSyn-TGR
resin was employed for peptide synthesis. The coupling solvent was NMP/DMSO
8:2.
The synthesis was performed at a 0.02 mmol scale using a coupling time of 3h.
The
crude linear peptides were further processed as described above for Method 1.
Method 4 for the ACT 496 MOS Synthesizer
The peptides were synthesized using method 3 on the ACT 496 with
HBTU/DIEA as the coupling reagents, and NMP as the solvent. 2,4,6-collidine as
a 1
M solution was used as the base. The low load Fmoc-GGGK(ivDde)-Novsyn-TGR
resin (-0.2 mmol/g) was used for peptide synthesis. The coupling time was 30
minutes.
The crude linear peptides were further processed as described above for Method
1.
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Method 5 for the ABI 433A Synthesizer
Synthesis of peptides was carried out on a 0.25 mmol scale using the FastMoc
protocol (Applied Biosystems Inc.) In each cycle of this protocol, 1.0 mmol of
a dry
protected amino acid in a cartridge was dissolved in a solution of 0.9 mmol of
HBTU, 2
mmol of DIEA, and 0.9 mmol of HOBt in DMF with additional NMP added. The
peptides were made using 0.1 mmol of NovaSyn TGR (Rink amide) resin (resin
substitution 0.2 mmol/g). The coupling time in this protocol was 21 min. Fmoc
deprotection was carried out with 20% piperidine in NMP. At the end of the
last cycle,
the synthesized peptide was acetylated using acetic anhydride/DIEA/HOBt/NMP.
The
peptide resin was washed and dried for further manipulations or cleaved from
the resin
(using reagent B). Generally, the cleaved peptides were cyclized, as in Method
1,
above.
Method 6: Biotinylation of Resin Bound Peptides
The peptides were prepared by Method 5. The ivDde protecting group on the C-
terminal lysine was selectively removed by treatment with 10% hydrazine in
DMF. The
resin was then treated with a solution of Biotin-N-hydroxysuccinimidyl ester
in DMF in
the presence of DIEA. After washing, the resin was dried and cleavage was
performed
using Reagent B. The resin was filtered off and the filtrate concentrated to
dryness. The
biotinylated peptide was dissolved in neat DMSO and treated with DIEA and
stirred for
4-6 h to effect disulfide cyclization. The crude mixture was purified by
preparative
HPLC.
In a typical experiment, 200 mg of the resin-bound peptide was treated with
10% hydrazine in DMF (2 x 20 mL) and washed with DMF (2 x 20 mL) and then with
dichloromethane (1 x 20 mL). The resin was resuspended in DMF (10 mL) and
treated
with a solution of Biotin-NHS ester (0.2 mmol, 5 equivalent) and DIEA (0.2
mmol) and
the resin was mixed with the reagents for 4 h. The completion of the reaction
was
checked by the ninhydrin test. The peptide was then released from the resin by
treatment with Reagent B (10 mL) for 4 h. The resin was filtered off, Reagent
B was
removed in vacuo and the peptide was precipitated by addition of anhydrous
ether. The
solid formed was collected, washed with ether and dried. The solid was
dissolved in
anhydrous DMSO and the mixture was adjusted to pH 7.5 with DIEA and stirred
for 4-6
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h to effect disulfide cyclization. The disulfide cyclization reaction was
monitored by
analytical HPLC. After completion of the cyclization, the mixture solution was
diluted
with 25% acetonitrile in water and directly purified by HPLC on reverse phase
C-18
column using a gradient of acetonitrile into water (both containing 0.1 %
TFA).
Fractions were analyzed by analytical HPLC and those containing the pure
product
were collected and lyophilized to obtain the required biotinylated peptide.
Method 7: Biotinylation of Purified Peptides
The purified peptide (10 mg, prepared by methods 1-5) containing a free amino
group was dissolved in anhydrous DMF or DMSO (I mL) and Biotin-NHS ester of (5
equivalents) and DIEA (5 equivalents) were added. The reaction was monitored
by
HPLC and after the completion of the reaction (1-2 h), the crude reaction
mixture was
directly purified by preparative HPLC. Fractions were analyzed by analytical
HPLC
and those containing the pure product were collected and lyophilized to obtain
the
required biotinylated peptide.
Method 8: Biotinylation of Resin Bound Peptides Containing Linkers
In a typical experiment, 400 mg of the resin-containing peptide (made using
the
ABI-433 A Synthesizer and bearing an ivDde-protected lysine) was treated with
10%
hydrazine in DMF (2 x 20 mL). The resin was washed with DMF (2 x 20 mL) and
DCM (1 x 20 mL). The resin was resuspended in DMF (10 mL) and treated with
Fmoc-
aminodioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC (0.4 mmol), DIEA (0.8
mmol) with mixing for 4 h. After the reaction, the resin was washed with DMF
(2 x 10
ml) and with DCM (lx 10 mL). The resin was then treated with 20% piperidine in
DMF
(2 x 15 mL) for 10 min each time. The resin was washed and the coupling with
Fmoc-
diaminodioxaoctanoic acid and removal of the Fmoc protecting group were
repeated
once more. The resulting resin, containing a peptide with a free amino group,
was
treated with a solution of Biotin-NHS ester (0.4 mmol, 5 equivalent) and DIEA
(0.4
mmol, 5 equivalents) in DMF for 2 h. The peptide-resin was washed and dried as
described previously and then treated with reagent B (20 mL) for A. The
mixture was
filtered and the filtrate concentrated to dryness. The residue was stirred
with ether to
produce a solid that was collected, washed with ether, and dried. The solid
was
dissolved in anhydrous DMSO and the pH adjusted to pH 7.5 with DIEA. The
mixture
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was stirred for 4-6 hr. to effect the disulfide cyclization reaction which was
monitored
by analytical HPLC. After the completion of the cyclization, the DMSO solution
was
diluted with 25% acetonitrile in water and applied directly to a reverse phase
C-18
column. Purification was effected using a gradient of acetonitrile into water
(both
containing 0.1 % TFA). Fractions were analyzed by analytical HPLC and those
containing the pure product were collected and lyophilized to provide the
required
biotinylated peptide.
Method 9: Formation of 5-Carboxyfluorescein Labeled Peptides
Peptide-resin obtained via from Method 5, containing an ivDde protecting group
on the epsilon nitrogen of lysine, was mixed with a solution of hydrazine in
DMF (10%
hydrazine/DMF, 2 x 10 mL, 10 min) to remove the ivDde group. The epsilon
nitrogen
of the lysine was labeled with fluorescein-5-isothiocyanate (0.12 mmol) and
diisopropylethylamine (0.12 mmol) in DMF. The mixture was agitated for 12 h
(fluorescein-containing compounds were protected from light). The resin was
then
washed with DMF (3 x 10 mL) and twice with CH2C12 (10 mL) and dried under
nitrogen for lh. The peptide was cleaved from the resin using Reagent B for 4h
and the
solution collected by filtration. The volatiles were removed under reduced
pressure
and the residue was dried under vacuum. The peptide was precipitated with
ether,
collected and the precipitate was dried under a stream of nitrogen. The
precipitate was
added to water (1mg/mL) and the pH of the mixture was adjusted to 8 with 10%
aqueous meglumine. Cyclization of the peptide was carried out for 48 h and the
solution was freeze-dried. The crude cyclic peptide was dissolved in water and
purified
by RP-HPLC on a C18 column with linear gradient of acetonitrile into water
(both
phases contained 0.1%TFA). Fractions containing the pure product were
collected and
freeze dried. The peptides were characterized by ES-MS and the purity was
determined
by RP-HPLC (linear gradient of acetonitrile into water/0.1 % TFA).
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Method 10: Preparation of Peptidic Chelate for Binding to Tc
By Coupling of Single Amino Acids
Peptides were synthesized starting with 0.1 mmol of NovaSyn -TGR resin (0.2
mmol/g substitution). Deprotected (ivDde) resin was then treated according to
the
protocol A for the incorporation of Fmoc (Gly)-OH, Fmoc-Cys(Acm)-OH and, Fmoc-
Ser(tBu)-OH.
Protocol A for manual coupling of single amino acid:
1. Treat with 4 equivalents of corresponding Fmoc-amino acid and 4.1
equivalents of
hydroxy benzotriazole and 4.1 equivalents of HOB and 4.1 equivalents of DIC
for 5h.
2. Wash with DMF (3 X 10 mL)
3. Treat with 20% piperidine in DMF (2 X10 mL, 10 min)
4. Wash with DMF (3 X 10 mL)
The Fmoc-protected peptide loaded resin was then treated with 20% piperidine
in DMF (2 X 10 mL, 10 min) and washed with DMF (3 X 10 mL). A solution of N,N-
dimethylglycine (0.11 mmol), HATU (1 mmol), and DIEA (0.11 mmol) in DMF (10
mL) was then added to the peptide loaded resin and the manual coupling was
continued
for 5 h. After the reaction the resin was washed with DMF (3 x 10 mL) and
CH2Cl2 (3 x
10 mL) and dried under vacuum.
Method 11: Formation of Mercapto-acetylated Peptides Using S-
Acetylthioglycolic
Acid N-Hydroxysuccinimide Ester
To a solution of a peptide (0.005 mmol, obtained from Methods 1-5 with a free
amine) in DMF (0.25 mL) was added S-acetylthioglycolic acid N-
hydroxysuccinimide
ester (SATA) (0.0055mmol) and the reaction mixture was stirred at ambient
temperature for 6 h. The volatile were removed under vacuum and the residue
was
purified by preparative HPLC using acetonitri le-water containing 0.1 %TFA.
Fractions
containing the pure product were collected and freeze-dried to yield the
mercaptoacetylated peptide. The mercaptoacetylated peptide was characterized
by ESI-
MS and the purity was determined by reverse phase PHLC analysis employing a
linear
gradient of acetonitrile into water (both containing 0.1% TFA).
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Method 12: Formation of Mercaptoacetylated Peptide Using S-Acetylthioglycolic
Acid
Purified peptides from Method 5, after disulfide cyclization, were coupled
with
S-acetylthioglycolic acid (1.5-10 eq.)/HOBt (1.5-10 eq.)/DIC (1.5-10 eq.) in
NMP for
2-16h at room temperature. The mixture was then purified by preparative HPLC
and the
fractions containing pure peptide combined and lyophilized. In the case of
compounds
with another lysine protected by an ivDde group, the deprotection reaction
employed
2% hydrazine in DMSO for 3h at room temperature. Purification of the reaction
mixture afforded pure peptide.
In the case of preparing a compound with S-acetylthioglycolic acid coupled to
two aminodioxaoctanoic acid groups and the peptide, the purified peptide from
Method
5 (having a free amino group, was coupled to AcSCH2-CO-(NH-CH2-CH2-O-CH2-CH2-
O-CH2-CO) 2-OH (30 eq.)/HOBt (30 eq.)/DIC (30 eq.) in NMP for 40 h at room
temperature. The mixture was purified and the ivDde group was removed. A
second
purification gave the final product as a white lyophilizate.
Alternatively Fmoc aminodioxaoctanoic acid was coupled twice successively to
the peptide (produced by method 5) followed by Fmoc removal and coupling to S-
acetylthioglycolic acid.
Method 13: Preparation of Homodimers and Heterodimers
The required purified peptides were prepared by SPPS using Method 5. To
prepare homodimers, half of the peptide needed to prepare the dimer was
dissolved in
DMF and treated with 10 equivalents of glutaric acid bis N-hydoxysuccinimidyl
ester
The progress of the reaction was monitored by HPLC analysis and mass
spectroscopy.
At completion of the reaction, the volatiles were removed in vacuo and the
residue was
washed with ethyl acetate to remove the unreacted bis-NHS ester. The residue
was
dried, re-dissolved in anhydrous DMF and treated with another half portion of
the
peptide in the presence of 2 equivalents of DIEA. The reaction was allowed to
proceed
for 24 hr. This mixture was applied directly to a Waters Associates C-18
XTerra RP-
HPLC column and purified by elution with a linear gradient of acetonitrile
into water
(both containing 0.1% TFA).
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In the case of heterodimers, one of the monomers was reacted with the bis NHS
ester of glutaric acid and after washing off the excess of bis NHS ester, the
second
amine was added in the presence of DIEA. After the reaction, the mixture was
purified
by preparative I-IPLC.
Preparation of KDR and VEGF/KDR Complex Binding Polypeptides
Utilizing the methods described above, the KDR and VEGF/KDR complex
binding polypeptides in Table 1 were prepared. As used in Table 1, the letter
"J" in the
peptide sequences refers to the spacer or linker group, 8-amino-3,6-
dioxaoctanoyl. Also
as used in Table 1, the designation "C*" refers to a cysteine residue that
contributes to a
disulfide bond. The ability of the biotinylated polypeptides to bind to KDR
was
assessed using the assay set described below.
The following biotinylated peptides bound well to the KDR-expressing cells:
P 13-XB (Kd 1.81 nM +/- 0.27), P5-XB (Kd 14.87 +/- 5.07 nM, four experiment
average), P6-XB (Kd 10.00 +/- 2.36 nM, four experiment average), P12-XB (Kd
4.031
+/- 0.86 nM, three experiment average), P6-F-XB (Kd 6.94 +/- 1.94 nM, one
experiment), and P12-F-XB (Kd 3.02 +/- 0.75 nM, one experiment).
Table 1. Sequence or Structure of Peptides and Peptide Derivatives
Ref. Structure or Sequence SEQ.
Number ID NO
PI Contol Peptide
P1-B Biotinylated Control Peptide
Pl-XB Biotinylated Control Peptide with Spacer
P2 AGWIECYHPDGICYHFGT 1
P2-D Ac-AGWIEC*YHPDGIC*YNFGTGGGK-NH2
P3 AGWLECYAEFGHCYNFGT 2
P3-D Ac-AGWLEC*YAEFGHC*YNFGTGGGK-NH2
P4 AGDSWCSTEYTYCEMIGT 3
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P4-D Ac-AGDSWC*STEYTYC*EMIGT-GGGK-NH2
P5 AGPKWCEEDWYYCMITGT 4
P5-D Ac-AGPKWC*EEDWYYC*MITGT-GGGK-NH2
P5-E Ac-AGPK(ivDde)WC*EEDWYYC*MITGTGGGK-NH2
P5-B Ac-AGPKWC*EEDWYYC*MITGT-GGGK-(Biotin)-NH2
P5-XB Ac-AGPKWC*EEDWYYC*MITGTGGGK-(Biotin-JJ-)-NH2
P6 GDSRVCWEDSWGGEVCFRYDP 5
P6-D Ac-GDSRVC*WEDSWGGEVC*FRYDPGGGK-NH2
P6-B Ac-GDSRVC*WEDSWGGEVC*FRYDP-GGGK-(Biotin)-NH2
P6-XB Ac-GDSRVC*WEDSWGGEVC*FRYDPGGGK-(Biotin-JJ-)-NH2
P6-F-XB Ac-VC*WEDSWGGEVC*FRYDPGGGK-(Biotin-JJ-)-NH2
P7 GDWWECKREEYRNTTWCAWADP 6
P7-D Ac-GDWWEC*KREEYRNTTWC*AWADPGGGK-NH2
P7-E Ac-GDWWEC*K(ivDde)REEYRNTTWC*AWADPGGGK-NH2
P8 GDPDTCTMWGDSGRWYCFPADP 7
P8-D Ac-GDPDTC*TMWGDSGRWYC*FPADPGGGK-NH2
P9 AQEPEGYAYWEVITLYHEEDGDGG 8
P9-D Ac-AQEPEGYAYWEVITLYHEEDGDGGK-NH2
PIO AQAFPRFGGDDYWIQQYLRYTDGG 9
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P10-D Ac-AQAFPRFGGDDYWIQQYLRYTDGGK-NH2
Pi l AQGDYVYWEIIELTGATDHTPPGG 10
P11-D Ac-AQGDYVYWEIIELTGATDHTPPGGGK-NH2
P12 AGPTWCEDDWYYCWLFGT 11
P12-D Ac-AGPTWC*EDDWYYC*WLFGT-NH2
P12-XB Ac-AGPTWC*EDDWYYC*WLFGT-GGGK-(Biotin-JJ-)-NH2
P12-F-XB Ac-AGPTWCEDDWYYCWLFGTJK-(Biotin-JJ-)-NH2
P12-C Ac-AGPTWC*EDDWYYC*WLFGTGGGKJJGC(Acm)-
,N-dimeth 1-GSC Acm)-NH2
P13 AQDWYYDEILSMADQLRHAFLSGG 12
P13-D Ac-AQDWYYDEILSMADQLRHAFLSGG-NH2
P13-XB Ac-AQDWYYDEILSMADQLRHAFLSGG-GGGK-(Biotin-JJ-)-
NH2
P14 GSDHHCYLHNGQWICYPFAPGGGK 13
P14-D Ac-GSDHHC*YLHNGQWIC*YPFAPGGGK-NH2
P15 GDYPWCHELSDSVTRFCVPWDPGGGK 14
P15-D Ac-GDYPWC*HELSDSVTRFC*VPWDPGGGK-NH2
P16 GDDHMCRSPDYQDHVFCMYWDPGGGK 15
P16-D Ac-GDDHMC*RSPDYQDHVFC*MYWDPGGGK-NH2
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P17 GDPPLCYFVGTQEWHHCNPFDPGGGK 16
P17-D Ac-GDPPLC*YFVGTQEWHHC*NPFDPGGGK-NH2
P18 GDGSWCEMRQDVGKWNCFSDDPGGGK 17
P18-E Ac-GDGSWC*EMRQDVGK(-ivDde-)WNC*FSDDPGGGK-NH2
P19 AQRGDYQEQYWHQQLVEQLKLLGGGK 18
P19-E Ac-AQRGDYQEQYWHQQLVEQLK(-ivDde-)LLGGGK-NH2
P20 GDNWECGWSNMFQKEFCARPDPGGGK 19
P20-E Ac-GDNWEC*GWSNMFQK(-ivDde-)EFC*ARPDPGGGK-NH2
P21 AGPGPCK -ivDde- GYMPHQCWYMGTGGGK 20
P21-E Ac-AGPGPC*K(-ivDde-)GYMPHQC*WYMGTGGGK-NH2
P22 AGYGPCAEMSPWLCWYPGTGGGK 21
P22-D Ac-AGYGPC*AEMSPWLC*WYPGTGGGK-NH2
EXAMPLE 2
Bead-binding assay to confirm ability of peptides identified by nhaae display
to bind
KDR-expressing cells
The following experiments were performed to assess the ability of KDR-binding
peptides to bind to KDR-expressing cells. In this experiment, KDR-binding
peptides
P5-B and P5-XB and P6-B and P6-XB were conjugated to fluorescent beads and
their
ability to bind to KDR-expressing 293H cells was assessed. The experiments
show that
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both peptide sequences can be used to bind particles such as beads to KDR-
expressing
sites. In general, the P6 peptides exhibited better binding to the KDR
expressing cells
than P5. However, the binding of both peptides improved with the addition of a
spacer.
Biotinylation of an anti-KDR antibody
Anti-KDR from Sigma (V-9134), as ascites fluid, was biotinylated using a kit
from Molecular Probes (F-6347) according to the manufacturer's instructions.
Preparation of peptide-conjugated fluorescent beads
0.1 mL of a 0.2 mM stock solution of each biotinylated peptide (prepared as
set
forth above, in 50% DMSO) was incubated with 0.1 mL of Neutravidin-coated red
fluorescent microspheres (2 micron diameter, custom-ordered from Molecular
Probes)
and 0.2 mL of 50 mM MES (Sigma M-8250) buffer, pH 6.0 for 1 hour at room
temperature on a rotator. As a positive control, biotinylated anti-KDR
antibody was
incubated with the Neutravidin-coated beads as above, except that 0.03 mg of
the
biotinylated antibody preparation in PBS (Gibco 14190-136) was used instead of
peptide solution. Beads can be stored at 4 C until needed for up to 1 week.
Transfection of 293H cells
293H cells were transfected using the protocol described in Example 6.
Transfection was done in black/clear 96-well plates (Becton Dickinson, cat. #
354640).
The cells in one half of the plate (48 wells) were mock-transfected (with no
DNA) and
those in the other half of the plate were transfected with KDR cDNA. The cells
were
80-90% confluent at the time of transfection and completely confluent the next
day, at
the time of the assay; otherwise the assay was aborted.
Binding assay
From the above bead preparations, 0.12 mL was spun for 10 minutes at 2000
rpm in a microcentrifuge at room temperature. The supernatant was removed and
0.06
mL of MES pH 6.0 was added. Each bead solution was then vortexed and sonicated
in
a water bath 15 min. To 1.47 mL of DMEM, high glucose (GIBCO 11965-084) with
lx
MEM Non-Essential Amino Acids Solution (NEAA) (GIBCO 11140-050) and 40%
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FBS (Hyclone SH30070.02) 0.03 mL of the sonicated bead preparations was added.
96-well plates seeded with 293H cells which have been mock-transfected in
columns 1
to 6, and KDR-transfected in columns 7 to 12 (as described above), were
drained and
washed once with DMEM, high glucose with lx NEAA and 40% FBS. To each well
was added 0.1 mL of bead solution, six wells per bead preparation. After
incubating at
room temperature for 30 minutes, the wells were drained by inverting the
plates and
washed four times with 0.1 mL PBS with Ca++Mg++ (GIBCO 14040-117) with shaking
at room temperature for 5 minutes each wash. After draining, 0.1 mL of PBS was
added per well. The plates were then read on a Packard FluoroCount fluorometer
at
excitation 550nm/emission 620nm. Unconjugated Neutravidin beads were used as a
negative control while beads conjugated with a biotinylated anti-KDR antibody
were
used as the positive control for the assay.
To calculate the number of beads bound per well, a standard curve with
increasing numbers of the same fluorescent beads was included in each assay
plate. The
standard curve was used to calculate the number of beads bound per well based
on the
fluorescence intensity of each well.
As shown in FIG. 1, the positive control beads with anti-KDR attached clearly
bound preferentially to the KDR-expressing cells while avidin beads with
nothing
attached did not bind to either cell type. Biotinylated P5 beads did not bind
to the
KDR-transfected cells significantly more than to mock-transfected cells, but
adding a
hydrophilic spacer between the peptide moiety and the biotin group enhanced
binding to
KDR cells without increasing the binding to mock-transfected cells.
Biotinylated P6
beads showed greater binding to KDR-transfected cells. As was the case for P5,
adding
a hydrophilic spacer between the peptide portion and the biotin of the
molecule
significantly improved the specific binding to KDR in the transfected cells.
Thus the
peptide sequences of both P5 and P6 can be used to bind particles such as
beads to KDR
expressing sites.
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EXAMPLE 3
Competition of KDR binding peptides and '251-labeled VEGF for binding to KDR-
transfected 293H cells
The following experiment assesses the ability of KDR-binding peptides to
compete with 1251-labeled VEGF for binding to KDR expressed by transfected
293H
cells. While KDR-binding polypeptide P4 did not compete significantly with
125I-
labeled VEGF, P5-XB, P6 and P12-XB competed very well with '2'1-labeled VEGF,
inhibiting 96.29 2.97% and 104.48+2.07% of125I-labeled VEGF binding.
Transfection of 293H cells
293H cells were transfected using the protocol described in Example 6.
Transfection was done in black/clear 96-well plates (Becton Dickinson, cat. #
354640).
The cells in one half of the plate (48 wells) were mock-transfected (with no
DNA) and
those in the other half of the plate were transfected with KDR cDNA. The cells
were
80-90% confluent at the time of transfection and completely confluent the next
day, at
the time of the assay; otherwise the assay was aborted.
Preparation of M199 media
To prepare M199 medium for the assay, one M199 medium packet (GIBCO,
cat. # 31100-035), 20 mL of 1 mM HEPES (GIBCO, cat. #15630-080), and 2 g of
DIFCO Gelatin (DIFCO, cat. # 0143-15-1) were added to 950 mL of double
distilled
(dd) H2O and the pH of the solution was adjusted to 7.4 by adding
approximately 4 mL
of IN NaOH. After pH adjustment, the M199 medium was warmed to 37 C in a
water
bath for 2 h to dissolve the gelatin, then filter sterilized using 0.2 m
filters (Coming,
cat. # 43109), and stored at 4 C to be used later in the assay.
Preparation of peptide solutions
3 mM stock solutions of peptides P6, P4, P5-XB , and PI 2-XB , (prepared as
described above) in 50% DMSO were prepared.
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Preparation of125I-labeled VEGF solution for the assay
25 Ci of lyophilized 125I-labeled VEGF (Amersham, cat. # IM274) were
reconstituted with 250 gL of ddH2O to create a stock solution, which was
stored at -80
C for later use. For each assay, a 300 pM solution of 125I-labeled VEGF was
made
fresh by diluting the above stock solution in M199 medium. The concentration
of 125I-
labeled VEGF was calculated daily based on the specific activity of the
material on that
day.
Preparation of 30 uM and 0.3 M peptide solution in 300 pM 1251-labeled VEGF
For each 96 well plate, 10 mL of 300 pM1251-labeled VEGF in M199 medium
was prepared at 4 C. Each peptide solution (3 mM, prepared as described
above) was
diluted 1:100 and 1:10000 in 300 L of M199 media with 300 pM 125I-labeled
VEGF to
prepare 30 M and 0.3 gM peptide solutions containing 300 pM of t25l-labeled
VEGF.
Once prepared, the solutions were kept on ice until ready to use. The dilution
of
peptides in M199 media containing 300 pM125I-labeled VEGF was done freshly for
each experiment.
Assay to detect competition with 125I-labeled VEGF in 293H cells
Cells were used 24 h after transfection, and to prepare the cells for the
assay,
they were washed 3 times with room temperature M199 medium and placed in the
refrigerator. After 15 minutes, the M 199 medium was removed from the plate
and
replaced with 75 L of 300 pM 1251-labeled VEGF in M199 medium (prepared as
above). Each dilution was added to three separate wells of mock and KDR
transfected
cells. After incubating at 4 C for 2 h, the plates were washed 5 times with
cold binding
buffer, gently blotted dry and checked under a microscope for cell loss. 100
L of
solubilizing solution (2% Triton X-100, 10% Glycerol, 0.1% BSA) was added to
each
well and the plates were incubated at room temperature for 30 minutes. The
solubilizing
solution in each well was mixed by pipeting up and down, and transferred to
1.2 mL
tubes. Each well was washed twice with 100 L of solubilizing solution and the
washes
were added to the corresponding 1.2 mL tube. Each 1.2 mL tube was then
transferred to
a 15.7 mm X 10 cm tube to be counted in an LKB Gamma Counter (1251 window for
1
minute).
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Competition of peptides with1251-labeled VEGF in 293H cells
The ability of KDR-binding peptides P6, P4, P5-XB, and P 1 2-X13, to
specifically block125I-labeled VEGF binding to KDR was assessed in mock-
transfected
and KDR-transfected cells. P4 was used in the assay as a negative control. It
was
selected because it exhibits only poor binding to KDR in FP assays, and thus
would not
be expected to displace or compete with VEGF. To calculate the specific
binding to
KDR, the binding of 1251-labeled VEGF to mock-transfected cells was subtracted
from
KDR-transfected cells. Therefore, the binding of 125I.. labeled VEGF to sites
other than
KDR (which may or may not be present in 293H cells) is not included when
calculating
the inhibition of 125I-labeled VEGF binding to 293H cells by KDR-binding
peptides.
FIG. 2 shows the percentage inhibition of 125I-labeled VEGF binding by
peptides (P6 P4 , P5-XB , and P12-XB) at two different concentrations (30 M
and 0.3
AM) to KDR-transfected 293H cells. Percentage inhibition was calculated using
formula [(Y1-Y2)xI00/Y1], where Y1 is specific binding to KDR-transfected 293H
cells in the absence of peptides, and Y2 is specific binding to KDR-
transfected 293H
cells in the presence of peptides or DMSO (vehicle). Specific binding to KDR-
transfected 293H cells was calculated by subtracting binding to mock-
transfected 293H
cells from binding to KDR-transfected 293H cells. Results for P6, P4 and P5-XB
are
the average of three experiments SD, whereas the result for P12-XB is from
one
experiment.
As shown in FIG. 2, P4, which, due to its relatively high Kd (>2 AM, measured
by FP against KDR-Fc), was used as a negative control, did not compete
significantly
with 125I-labeled VEGF,12.69+7.18% at 30 M and -5.45 9.37% at 0.3 M (FIG.
2).
At the same time, P6, and P12-XB competed very well with 125I-labeled VEGF,
inhibiting 96.29+2.97% and 104.48 2.07% of 125I-labeled VEGF binding at 30 M
and
52.27+3.78% and 80.96+3.8% at 0.3 AM, respectively. The percentage inhibition
with
P5-X-B was 47.95 5.09% of12SI-labeled VEGF binding at 30 M and 24.41+8.43% at
0.3 M (FIG. 2). Thus, as one would expect, a peptide that only binds KDR
poorly did
not block VEGF binding, while three other KDR-binding peptides did compete
with
VEGF, and their potency increased with their binding affinity. This assay
should also be
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useful for identifying peptides that bind tightly to KDR but do not compete
with VEGF,
a feature that may be useful for imaging KDR in tumors, where there is
frequently a
high local concentration of VEGF that would otherwise block the binding of KDR-
targeting molecules.
EXAMPLE 4
Inhibition of VEGF-induced KDR receptor activation by tides identified by ha e
display
The ability of KDR-binding peptides identified by phage display to inhibit
VEGF induced activation (phosphorylation) of KDR was assessed using the
following
assay. A number of peptides of the invention were shown to inhibit activation
of KDR
in monomeric and/or tetrameric constructs, including P5-D, P6-D, P10-D and P11-
D.
As discussed above, peptides that inhibit activation of KDR may be useful as
anti-
angiogenic agents.
Human umbilical vein endothelial cells (HUVECs) (Biowhittaker Cat No. CC-
2519) were obtained frozen on dry ice and stored in liquid nitrogen until
thawing. These
cells were thawed, passaged, and maintained as described by the manufacturer
in EGM-
MV medium (Biowhittaker Cat No. CC-3125). Cells seeded into 100 mm dishes were
allowed to become confluent, then cultured overnight in basal EBM medium
lacking
serum (Biowhittaker Cat No. CC-3121). The next morning, the medium in the
dishes
was replaced with 10 mL fresh EBM medium at 37 C containing either no
additive
(negative control), 5 ng/mL VEGF (Calbiochem Cat No. 676472 or Peprotech Cat
No.
100-20) (positive control), or 5 ng/mL VEGF plus the indicated concentration
of the
KDR-binding peptide (prepared as described above). In some cases, a
neutralizing anti-
KDR antibody (Cat No. AF357, R&D Systems) was used as a positive control
inhibitor
of activation. In such cases, the antibody was pre-incubated with the test
cells for 30
min at 37 C prior to the addition of fresh medium containing both VEGF and
the
antibody. After incubating the dishes 5 min in a 37 C tissue culture
incubator they were
washed three times with ice-cold Dulbecco's phosphate buffered saline (D-PBS)
containing calcium and magnesium and placed on ice without removing the last
10 mL
of D-PBS. The first dish of a set was drained and 0.5 mL of Triton lysis
buffer was
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added (20 mM Tris base pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2
mM
EDTA (ethylenediaminetetraacetic acid), 1 mM PMSF
(phenylmethylsulfonylfluoride),
1 mM sodium orthovanadate, 100 mM NaF, 50 mM sodium pyrophosphate, 10 .tg/mL
leupeptin, 10 gg/mL aprotinin). The cells were quickly scraped into the lysis
buffer
using a cell scraper (Falcon, Cat No. 353087), dispersed by pipeting up and
down
briefly, and the resulting lysate was transferred to the second drained dish
of the pair.
Another 0.5 mL of lysis buffer was used to rinse out the first dish then
transferred to the
second dish, which was then also scraped and dispersed. The pooled lysate from
the two
dishes was transferred to a 1.5 mL Eppindorf tube. The above procedure was
repeated
for each of the controls and test samples (KDR-binding peptides), one at a
time. The
lysates were stored on ice until all the samples had been processed. At this
point
samples were either stored at -70 C or processed to the end of the assay
without
interruption.
The lysates, either freshly prepared or frozen and thawed, were precleared by
adding 20 L of protein A-sepharose beads (Sigma 3391, preswollen in D-PBS),
washed three times with a large excess of D-PBS, reconstituted with 6 mL D-PBS
to
generate a 50% slurry) and rocked at 4 C for 30 min. The beads were pelleted
by
centrifugation for 2 min in a Picofuge (Stratgene, Cat No. 400550) at 2000xg
and the
supernatants transferred to new 1.5 mL tubes. 20 g of anti-Flk-1 antibody
(Santa Cruz
Biotechnology, Cat No. sc-504) was added to each tube, and the tubes were
incubated
overnight (16-18 h) at 4 C on a rotator to immunoprecipitate KDR. The next day
40 L
of protein A-sepharose beads were added to the tubes, which were then
incubated at
4 C for I h on a rotator. The beads in each tube were subsequently washed
three times
by centrifuging for 2 min in a Picofuge, discarding the supernatant, and
dispersing the
beads in 1 mL freshly added TBST buffer (20 mM Tris base pH 7.5, 137 mM NaCl,
and
0.1 % Tween 20). After centrifuging and removing the liquid from the last
wash, 40 L
of Laemmli SDS-PAGE sample buffer (Bio-Rad, Cat No. 161-0737) was added to
each
tube and the tubes were capped and boiled for 5 min. After cooling, the beads
in each
tube were pelleted by centrifuging and the supernatants containing the
immunoprecipitated KDR were transferred to new tubes and used immediately or
frozen and stored at -70 C for later analysis.
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Detection of phosphorylated KDR as well as total KDR in the
immunoprecipitates was carried out by immunoblot analysis. Half (20 L) of
each
immunoprecipitate was resolved on a 7.5% precast Ready Gel (Bio-Rad, Cat No.
161-
1154) by SDS-PAGE according to the method of Laemmli (U: K. Laemmli "Cleavage
of structural proteins during assembly of the head of bacteriophage T4."
Nature (1970);
227, 680-685).
Using a Bio-Rad mini-Protean 3 apparatus (Cat No. 165-3302). The resolved
proteins in each gel were electroblotted to a PVDF membrane (Bio-Rad, Cat. No.
162-
0174) in a Bio-Rad mini Trans-Blot cell (Cat No. 170-3930) in CAPS buffer (10
mM
CAPS, Sigma Cat No. C-6070, 1% ACS grade methanol, pH 11.0) for 2 h at 140 mA
according to the method of Matsudaira (P. Matsudaira. "Sequence from picomole
quantities of proteins electroblotted onto polyvinylidine diflouride
membranes." J. Biol.
Chem. (1987); 262, 10035-10038). Blots were blocked at room temperature in 5%
Blotto-TBS (Pierce Cat No. 37530) pre-warmed to 37 C for 2 h. The blots were
first
probed with an anti-phosphotyrosine antibody (Transduction Labs, Cat No.
P11120),
diluted 1:200 in 5% Blotto-TBS with 0.1 % Tween 20 added for 2 h at room temp.
The
unbound antibody was removed by washing the blots four times with D-PBS
containing
0.1 % Tween 20 (D-PBST), 5 min per wash. Subsequently, blots were probed with
an
HRP-conjugated sheep anti-mouse antibody (Amersham Biosciences Cat No. NA93 1)
diluted 1:25,000 in 5% Blotto-TBS with 0.1% Tween 20 added for 1 h at room
temperature, and washed four times with D-PBST. Finally, the blots were
incubated
with 2 mL of a chemiluminescent substrate (ECL Plus, Amersham Cat No. RPN2132)
spread on top for 2 min, drip-drained well, placed in plastic sheet protector
(C-Line
Products, Cat No. 62038), and exposed to X-ray film (Kodak BioMax ML, Cat No.
1139435) for varying lengths of time to achieve optimal contrast.
To confirm that similar amounts of KDR were compared in the assay, the blots
were stripped by incubating for 30 min at 37 C in TBST with its pH adjusted
to 2.4
with HCI, blocked for 1 h at room temp with 5% Blotto-TBS with 0.1% Tween 20
(Blotto-TBST), and reprobed with an anti-Flk-1 polyclonal antibody (Cat No. sc-
315
from Santa Cruz Biotech), 1:200 in 5% Blotto-TBST with 1% normal goat serum
(Life
Tech Cat No. 16210064) for 2 h at room temp. The unbound antibody was removed
by
washing the blots four times with D-PBST, 5 min per wash. Subsequently, the
blots
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were probed with an HRP-conjugated donkey anti-rabbit secondary antibody
(Amersham Biosciences Cat No. NA934) diluted 1:10,000 in 5% Blotto-TBST for I
h
at room temperature, and washed four times with D-PBST. Finally, the blots
were
incubated with 2 mL of chemiluminescent substrate and exposed to X-ray film as
described above.
Immunoblots of KDR immunoprecipitates prepared from HUVECs with and
without prior VEGF stimulation, shown in FIG. 3, demonstrated that activated
(phosphorylated) KDR could be detected when the HUVECs were stimulated with
VEGF. An anti-phosphotyrosine antibody (PY-20) detected no phosphorylated
proteins
close to the migration position of KDR from unstimulated HUVECs on the blots,
but
after five minutes of VEGF stimulation, an intense band was consistently
observed at
the expected location (FIG. 3, upper panel). When the blots were stripped of
bound
antibodies by incubation in acidic solution then reprobed with an anti-KDR
antibody
(sc-315), the identity of the phosphorylated protein band was confirmed to be
KDR.
Moreover, it was observed that immunopreciptates from unstimulated HUVECs
contained about as much total KDR as immunoprecipitates from VEGF-stimulation
HUVECs (FIG. 3, lower panel).
It is reasonable to conclude that the phosphorylated KDR detected was formed
from pre-existing KDR through autophosphorylation of KDR dimers resulting from
VEGF binding as five minutes is not enough time to synthesize and process a
large
glycosylated cell-surface receptor such as KDR.
The ability of the assay to detect agents capable of blocking the VEGF
activation of KDR was assessed by adding a series of compounds to HUVECs in
combination with VEGF and measuring KDR phosphorylation with the immunoblot
assay described above. As negative and positive controls, immunoprecipitates
from
unstimulated HUVECs and from HUVECs stimulated with VEGF in the absence of any
test compounds were also tested in every assay. When a neutralizing anti-KDR
antibody (Cat No. AF-357 from R&D Systems) was combined with the VEGF, the
extent of KDR phosphorylation was greatly reduced (FIG. 4, upper panel),
indicating
that the antibody was able to interfere with the ability of VEGF to bind to
and activate
KDR. This result was expected because the ability of the antibody to block
VEGF-
induced DNA synthesis is part of the manufacturer's quality control testing of
the
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antibody. Re-probing the blot with an anti-KDR antibody (FIG. 4, lower panel)
indicated that slightly less total KDR was present in the VEGF+antibody-
treated lane
(+V+a-KDR) relative to the VEGF-only-treated lane (+V), but the difference was
not
great enough to account for the much lower abundance of phosphorylated KDR in
the
antibody-treated lane.
To assess the potency of a KDR-binding peptide (Pl0-D) identified by phage
display, the experiment was repeated with P10-D in the presence of VEGF. P10-D
was
able to largely inhibit the VEGF-induced phosphorylation of KDR. Re-probing
the blot
for total KDR showed that there is even more total KDR in the VEGF+PI O-D-
treated
cells (+V+P 10-D) than in the VEGF only-treated cells (+V) (FIG. 5, lower
panel).
Thus, it is clear that the decreased phosphorylation of KDR in the presence of
P 10-D is
not due to differential sample loading, but rather the ability of the compound
to inhibit
VEGF-activation of KDR.
Using the methods of this Example, the following peptides demonstrated at
least
a 50% inhibition of VEGF-induced KDR phosphorylation at 10 M:
P2-D, P3-D, P6-D, P7-E, P8-D, P9-D, P10-D, P11-D.
P2 and P6 were the most potent compounds in the assay, producing at least a
50% inhibition of VEGF-induced KDR phosphorylation at 1 M.
The following peptides were tested in the assay and did not produce
significant
inhibition of KDR activation at 10 M:
P5-E, P14-D, P15-D, P16-D, P17-D, P18-E, P19-E, P20-E, P21-E, P23-D
In addition, tetrameric complexes of biotinylated derivatives P6-XB or P12-XB
(prepared as described above and discussed in Example 6, infra) produced at
least a
50% inhibition of VEGF-induced KDR phosphorylation at 10 nM.
EXAMPLE 5
Binding of Tc-labeled polypeptide to KDR-transfected 293H cells
In this Example, the ability of Tc-labeled P12-C to bind KDR was assessed
using KDR-transfected 293H cells. The results show that Tc-labeled P12-C bound
significantly better to KDR transfected 293H cells than to mock transfected
293H cells,
and binding increased with concentration of the Tc-labeled polypeptide in a
linear
manner.
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Preparation of peptidic chelate (P12-C) for binding to Tc by SPPS
tBuO
0
O P
HO NH N
NJJ"~H H
O
T N
Trt Mel Me
To a 250 ml of SPPS reaction vessel was added 6.64 mmol of H-Gly-2-Cl-trityl
resin (0.84 mmol/g, Novabiochem). It was swelled in 80 mL of DMF for lh. For
each
coupling cycle the resin was added 26.6 mmol of DIEA, 26.6 mmol of a Fmoc-
amino
acid in DMF (EM Science), 26.6 mmol of HOBT (Novabiochem) in DMF, and 26.6
mmol of DIC. The total volume of DMF was 80 mL. The reaction mixture was
shaken
for 4h. The resin then was filtered and washed with DMF (3 x 80 mL). A
solution of
20% piperidine in DMF (80 mL) was added to the resin and it was shaken for 10
min.
The resin was filtered and this piperidine treatment was repeated. The resin
finally was
washed with DMF (3 x 80 mL) and ready for next coupling cycle. At the last
coupling
cycle, N,N-dimethyl glycine (Aldrich) was coupled using HATU/DIEA activation.
Thus, to a suspension of N,N-dimethyl glycine (26.6 mmol) in DMF was added a
solution of 26.6 mmol of HATU (Perseptive Biosystems) in DMF and 53.1 mmol of
DIEA. The clear solution was added to the resin and shaken for 16 h. Following
the
synthesis, the resin was filtered and washed with DMF (3 x 80 mL), CH2C12 (3 x
80 ml)
and dried. The resin was mixed with 80 mL of AcOH/CF3CH2OH/DCM (1/1/8, v/v/v)
and shaken for 45 min. The resin was filtered and the filtrate was evaporated
to a paste.
Purification of the crude material by silica gel chromatography using 25%
MeOH/DCM
afforded 2.0 g of the final product.
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Coupling of the peptidic chelate (P12-C to the peptide (Fragment coupling)
To a mixture of purified Me2N-Gly-Cys-(Trt)-Ser(tBu)-Gly-OH-and
hydroxybenzotriazole (0.0055mmol) in DMF (0.25 mL), diisopropylcarbodiimide
(0.0055 mmol) was added and the mixture was stirred at RT for 6 h. The peptide
(0.005
mmol) in DMF (0.25 mL) was then added to the reaction mixture and stirring was
continued for an additional 6 h. DMF was removed under vacuum and the residue
was
treated with reagent B and stirred for 3h. TFA was removed under reduced
pressure and
the residue was purified by preparative HPLC using acetonitrile-water
containing 0.1%
TFA. Fractions containing the pure product were collected and freeze dried to
yield the
peptide. The peptide was characterized by ES-MS and the purity was determined
by
RP-HPLC (acetonitrile-water/0.1% TFA) gradient.
Synthesis of 99mTc-labeled peptide
A stannous gluconate solution was prepared by adding 2 mL of a 20 g/mL
SnCl2'2H20 solution in nitrogen-purged IN HCl to 1.0 mL of nitrogen-purged
water
containing 13 mg of sodium glucoheptonate. To a 4 mL autosampler vial was
added 20-
40 pL (20 - 40 g) ofP12-C ligand dissolved in 50/50 ethanol/H2O, 6-12 mCi of
99mTCO4in saline and 100 L of stannous glucoheptonate solution. The mixture
was
heated at 100 C for 22 min. The resulting radiochemical purity (RCP) was 10-
47%
when analyzed using a Vydac C 18 Peptide and Protein column that was eluted at
a flow
rate of 1 mL/min with 66% H2O (0.1 % TFA)/34% ACN(0.085% TFA). The reaction
mixture was purified by HPLC on a Vydac C 18 column (4.6 mm x 250 mm) at a
flow
rate of 1 mL/min, using 0.1% TFA in water as aqueous phase and 0.085% TFA in
acetonitrile as the organic phase. The following gradient was used; 29.5% org.
for 35
min., ramp to 85% org. over 5 min, hold for 10 min. The fraction containing
99MTc-
P12-C (which no longer contained the ACM protecting group) was collected into
500
L of a stabilizing buffer containing 5 mg/mL ascorbic acid and 16 mg/mL
hydroxypropyl-y-cyclodextrin in 50 mM phosphate buffer. The mixture was
concentrated using a speed vacuum apparatus to remove acetonitrile, and 200 L
of
0.1% HSA in 50 mM pH 5 citrate buffer was added. The resulting product had an
RCP
of 100%. Prior to injection into animals, the compound was diluted to the
desired
radioconcentration with normal saline.
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Transfection of 293H cells
293H cells were transfected using the protocol described in avidin HRP
example. Transfection was done in black/clear 96-well plates (Becton
Dickinson, cat. #
354640). The cells in one half of the plates (48 wells) were mock-transfected
(with no
DNA) and the cells in the other half of the plate were transfected with KDR
cDNA. The
cells were 80-90% confluent at the time of transfection and completely
confluent the
next day, at the time of the assay; otherwise the assay was aborted.
Preparation of opti-MEMI media with 0.1 % HSA
Opti-MEMI was obtained from Invitrogen (cat. # 11058-021) and human serum
albumin (HSA) was obtained from Sigma (cat. # A-3782). To prepare opti-MEMI
media with 0.1 % HSA, 0.1 % w/v HSA was added to opti-MEMI, stirred at room
temperature for 20 minutes, and then filter sterilized using 0.2 M filter.
Preparation of Tc-labeled peptide dilutions for the assay
Stock solution of Tc-labeled P12-C (117 Ci/ml) was diluted 1:100, 1:50, 1:25
and 1:10 in opti-MEMI with 0.1 % HSA to provide solutions with final
concentration of
1. 17, 2.34, 4.68 and 11.7 ttCi/mL of Tc-labeled P12-C
Assay to detect the binding of Tc-labeled peptide
Cells were used 24 h after transfection, and to prepare the cells for the
assay,
they were washed 1 X with 100 L of room temperature opti-MEMI with 0.1 % HSA.
After washing, the opti-MEMI with 0.1 % HSA was removed from the plate and
replaced with 70 L of 1. 17, 2.34, 4.68 and 11.7 Ci/mL of Tc-labeled P12-C
(prepared
as above). Each dilution was added to three separate wells of mock and KDR
transfected cells. After incubating at room temperature for 1 h, the plates
were
transferred to 4 C for 15 minutes and washed 5 times with 100 L of cold
binding
buffer (opti-MEMI with 0.1 % HSA), gently blotted dry and checked under a
microscope for cell loss. 100 L of solubilizing solution (2% Triton X-100,
10%
Glycerol, 0.1% BSA) was added to each well and the plates were incubated at 37
C for
10 minutes. The solubilizing solution in each well was mixed by pipeting up
and down,
and transferred to 1.2 mL tubes. Each well was washed once with 100 L of
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solubilizing solution and the washes were added to the corresponding 1.2 mL
tube.
Each 1.2 mL tube was then transferred to a 15.7 mm X 100 cm tube to be counted
in an
LKB Gamma Counter (Tc-window for 20 sec).
Binding of Tc-labeled peptide to KDR transfected cells
The ability of Tc-labeled P 12-C to bind specifically to KDR was demonstrated
using transiently transfected 293H cells. As shown in FIG. 6, Tc-labeled P12-C
bound
significantly better to KDR transfected 293H cells, as compared to mock
transfected
293H cells. To calculate specific binding to KDR, the binding of Tc-labeled
P12-C to
mock transfected cells was subtracted from the binding to KDR transfected
cells. As
shown in FIG. 7, a linear increase in the specific binding of Tc-labeled P12-C
to KDR
was observed with increasing concentration of Tc-labeled P12-C. Linear binding
was
expected because the concentration of Tc-labeled P12-C was only 100 pM (even
at the
highest concentration, 11.7 gCi/mL, tested in the assay), which is far below
the KD
value of -3-4 nM of P12 (as calculated using the avidin HRP assay), so no
saturation of
binding would be expected.
EXAMPLE 6
Binding of KDR binding peptide/avidin HRP complex to KDR transfected 293H
cells
To determine the binding of peptides identified by phage display to KDR
expressed in transiently-transfected 293H cells, a novel assay that measures
the binding
of biotinylated peptides complexed with neutravidin HRP to KDR on the surface
of the
transfected cells was developed. This assay was used to screen the
biotinylated peptides
described above. Neutravidin HRP was used instead of streptavidin or avidin
because it
has lower non-specific binding to molecules other than biotin, due to the
absence of
lectin binding carbohydrate moieties and also due to the absence of the cell
adhesion
receptor-binding RYD domain in neutravidin.
In the experiments described herein, tetrameric complexes of KDR-binding
peptides P6-XB, P5-XB, P12-XB , and P13-XB and a control peptide, P1-XB were
prepared and tested for their ability to bind 293H cells that were transiently-
transfected
with KDR. All four tetrameric complexes of KDR-binding peptides bound to the
KDR-
expressing cells; however, P13-XB exhibited the best Kd (1.81 nM). The
tetrameric
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complexes of KDR-binding peptides P6-XB and P5-XB exhibited improved binding
over monomers of the same peptides. Moreover, inclusion of a spacer (between
the
KDR-binding peptide and the biotin) in these constructs was shown to improve
binding
in Experiment B.
Experiment C, demonstrates the use of this assay to assess the effect of serum
on
binding of peptides of the invention to KDR and VEGF/KDR complex. The binding
of
P5-XB, P6-XB and P13-XB was not significantly affected by the presence of
serum,
while the binding of P 12-XB was reduced more than 50% in the presence of
serum.
Experiment D demonstrates that this assay is useful in evaluating distinct
combinations of KDR and VEGF/KDR complex binding polypeptides for use in
multimeric targeting constructs which contain more than one KDR and VEGF/KDR
complex binding polypeptide. Moreover, Experiments D and E establish that
heteromeric constructs, which have two or more KDR binding peptides that bind
to
different binding sites, exhibited superior binding to "homotetrameric"
constructs of the
targeting peptides alone.
Experiment A
Preparation of m-RNA and 5' RACE ready cDNA library
HUVEC cells were grown to almost 80% confluence in 175 cm2 tissue culture
flasks (Becton Dickinson, Biocoat, cat # 6478) and then 10 ng/mL of bFGF
(Oncogene,
cat # PF003) was added for 24 h to induce expression of KDR. MRNA was isolated
using the micro-fast track 2.0 kit from Invitrogen (cat. # K1520-02). 12 g of
mRNA
(measured by absorbance at 260 nM) was obtained from two flasks (about 30
million
cells) following the kit instructions. Reverse transcription to generate cDNA
was
performed with 2 .tg of mRNA, oligo dT primer (5'-(T)25GC-3') and/or smart II
oligo
(5'AAGCAGTGGTAACAACGCAGAGTA CGCGGG-3') using Moloney Murine
Leukemia Virus (MMLV) reverse transcriptase. The reaction was performed in a
total
volume of 20 L and the reaction mix contained 2 p.L of RNA, 1 tL smart II
oligo, 1 L
of oligo dT primer, 4 L of 5x first-strand buffer (250 mM Tris HCl pH 8.3,
375 mM
KCI, 30 mM MgCl2) 1 L DTT (20 mM, also supplied with reverse transcriptase),
1 L
dNTP mix (10 mM each of dATP, dCTP, dGTP, and dTTP in ddH2O, Stratagene, cat.
#
200415), 9 L ddH2O and I gL MMLV reverse transcriptase (Clonetech, cat #8460-
1).
The reverse transcription reaction was performed for 90 minutes at 42 C, and
the
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reaction was stopped by adding 250 L of tricine-EDTA buffer (10 mM tricine,
1.0 mM
EDTA). The reverse transcription product, a 5' RACE ready cDNA library, can be
stored for 3 months at -20 C. All water used for DNA and RNA applications was
DNAse and RNAse free from USB (cat. # 70783).
Cloning of s-KDR into TOPOII Vector
To clone s-KDR, a 5' oligo (G ATG GAG AGC AAG GTG CTG CTG G) and a
3' oligo (C CAA GTT CGT CTT TTC CTG GGC A) were used. These were designed
to amplify the complete extracellular domain of KDR (-2.2 kbps) from the 5'
RACE
ready cDNA library (prepared above) using polymerase chain reaction (PCR) with
pfu
polymerase (Stratagene, cat. # 600135). The PCR reaction was done in total
volume of
50 L and the reaction mix contained 2 L 5' RACE ready cDNA library, I L 5'
oligo
(10 M), 1 L 3' oligo (10 M), 5 .tL lOX PCR buffer [PCR buffer (200 mM Tris-
HCI
pH 8.8, 20 mM MgSO4, 100 mM KCI, 100 mM (NH4)2SO4) supplied with pfu enzyme
plus 1% DMSO and 8% glycerol], 1 L dNTP mix (10 mM) and 40 L ddH2O. The
PCR reaction was performed by using a program set for 40 cycles of 1 minute at
94 C,
1 minute at 68 C and 4 minutes at 72 C. The PCR product was purified by
extraction
with 1 volume of phenol, followed by extraction with 1 volume of chloroform
and
precipitated using 3 volume of ethanol and 1/10 volume of 3M sodium acetate.
The
PCR product was resuspended in 17 L of ddH2O, the 2 L of l OX Taq polymerase
buffer (100 mM Tris-HCI pH 8.8, 500 mM KCI, 15 mM MgCl2, 0.01% gelatin) and 1
L of Taq polymerase (Stratagene, cat. # 600131) was added to generate an A
overhang
to each end of the product. After incubating for 1 hour at 72 C the modified
product
was cloned directly into a TOPOII vector from invitrogen (cat. # K4600-01)
following
the manufacturer's protocol to give TOPO-sKDR. The TOPO vector allows easy
cloning of PCR products because of the A-overhang in Taq (PCR enzyme)-treated
PCR
products.
Cloning the transmembrane and cytoplasmic domains of KDR into TOPO II Vector
To clone the transmembrane and cytoplasmic domains of KDR, a 5' oligo (TCC
CCC GGG ATC ATT ATT CTA GTA GGC ACG GCG GTG) and a 3' oligo (C AGG
AGG AGA GCT CAG TGT GGT C) were used. These were designed to amplify the
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complete transmembrane and cytoplasmic domains of KDR (-1.8 kbps) from the 5'
RACE ready cDNA library (described above) using polymerase chain reaction
(PCR)
with pfu polymerase. PCR reaction conditions and the program were exactly the
same
as described above for s-KDR. Just as with the s-KDR sequence, the PCR product
was
purified using phenol chloroform extraction, treated with Taq polymerase and
cloned
into TOPOII vector from invitrogen to give TOPO-CYTO.
Cloning of full-length KDR into pcDNA6 Vector
To create the full-length receptor, the extra-cellular domain and the
cytoplasmic
domain (with trans-membrane domain) were amplified by PCR separately from TOPO-
sKDR and TOPO-CYTO respectively and ligated later to create the full-length
receptor.
An oligo with a NotI site at the 5' end of the extracellular domain (A TAA GAA
TGC
GGC CGC AGG ATG GAG AGC AAG GTG CTG CTG G) and an oligo
complimentary to the 3' end of the extracellular domain (TTC CAA GTT CGT CTT
TTC CTG GGC ACC) were used to amplify by PCR the extracellular domain from
TOPO-sKDR. Similarly, the 5' oligo (ATC ATT ATT CTA GTA GGC ACG GCG
GTG) and the 3' oligo, with a Notl site (A TAA GAA TGC GGC CGC AAC AGG
AGG AGA GCT CAG TGT GGT C), were used to amplify by PCR the cytoplasmic
domain of KDR (with transmembrane domain) from TOPO-CYTO. Both PCR products
were digested with Notl and ligated together to create the full-length
receptor. The
cDNA encoding the full-length receptor was purified on an agarose gel and
ligated into
the Notl site of the pcDNA6N5-HisC vector. Purification of DNA and ligation
was
done as described earlier for psKDR. The ligation reaction was used to
transform a
culture of DH5a bacteria and a number of individual clones were analyzed for
the
presence and orientation of insert by restriction analysis of purified plasmid
from each
clone with EcoRI enzyme.
Cell Culture
293H cells were obtained from Invitrogen (cat. # 11631) and grown as
monolayer cultures in their recommended media plus 1mL/L pen/strep
(Invitrogen, cat.
# 15140-148). All the cells were grown in presence of antibiotic for everyday
culture
but were split into antibiotic free media for 16-20 hour prior to
transfection.
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Preparation of DNA for Transfection
E. coll. bacteria DH5u containing pf-KDR was streaked onto LB with 50 g/mL
ampicillin (LB agar from US biologicals, cat. # 75851 and ampicillin from
Sigma, cat.
#A2804) plates from a glycerol stock and plates were left in a 37 C incubator
to grow
overnight. Next morning, a single colony was picked from the plate and grown
in 3 mL
of LB/ampicillin media (LB from US biologicals, cat. # US75852) at 37 T. After
8
hours, 100 pL of bacterial culture from the 3 mL tube was transferred to 250
mL of
LB/ampicillin media for overnight incubation at 37 T. Bacteria were grown up
with
circular agitation in a 500 mL bottle (Beckman, cat. # 355605) at 220 rpm in a
Lab-Line
incubator shaker. The next day, the bacterial culture was processed using maxi-
prep kit
(QIAGEN, cat. # 12163). Generally, about 1 mg of plasmid DNA (as quantitated
by
absorbance at 260 nm) was obtained from 250 mL of bacterial culture.
Transfection of 293H cells in 96 well plate
Transfection was done as recommended in the lipofectamine 2000 protocol
(Invitrogen, cat# 11668-019) using a poly-D-lysine-coated 96 well plate. 320
ng of
KDR DNA (pc-DNA6-fKDR)/per well in 0.1 mL was used for 96 well plate
transfections. Transfection was done in serum-containing media, the
transfection
reagent mix was removed from cells after 6-8 hours and replaced with regular
serum-
containing medium. Transfection was done in black/clear 96-well plates (Becton
Dickinson, cat. # 354640). The cell in one half of the plate (48 wells) were
mock-
transfected (with no DNA) and the cells in the other half of the plate were
transfected
with KDR cDNA. The cells were 80-90% confluent at the time of transfection and
completely confluent next day, at the time of the assay, otherwise the assay
was
aborted.
Preparation of M199 media
M199 media was prepared as described above
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Preparation of SoftLink soft release avidin-sepharose
SoftLink soft release avidin-sepharose was prepared by centrifuging the
sepharose obtained from Promega (cat. # V2011) at 12,000 rpm for 2 minutes,
washing
twice with ice cold water (centrifuging in-between the washes) and
resuspending the
pellet in ice cold water to make a 50% slurry in ddH2O. A fresh 50% slurry of
avidin-
sepharose was prepared for each experiment.
Preparation of peptide/neutravidin HRP solution
Biotinylated peptides P6-XB, P5-XB, P12-XB, P 13-XB and the biotinylated
control peptide, P1-XB, (prepared as described above) were used to prepare 250
M
stock solutions in 50% DMSO and a 33 M stock solution of Neutravidin HRP was
prepared by dissolving 2 mg of Neutravidin HRP (Pierce, cat. # 31001) in 1 mL
of
ddH2O. Peptide stock solutions were stored at -20 C, whereas the Neutravidin
HRP
stock solution was stored at -80 C. The structures of the biotinylated
peptides are
shown in Table 1. To prepare peptide/neutravidin HRP complexes, 10 pL of 250
M
biotinylated peptide stock solution and 10 gL of 33 p.M Neutravidin HRP were
added to
I ml of M199 medium. This mixture was incubated on a rotator at 4 C for 60
minutes,
followed by addition of 50 L of soft release avidin-sepharose (50% slurry in
ddH2O) to
remove excess peptides and another incubation for 30 minutes on a rotator at 4
C.
Finally, the soft release avidin-sepharose was pelleted by centrifuging at
12,000 rpm for
5 minutes at room temperature, and the resulting supernatant was used for the
assays.
Fresh peptide/neutravidin HRP complexes were prepared for each experiment.
Preparation of peptide/neutravidin HRP dilutions for the assay
For saturation binding experiments, 120 L, 60 L, 20 L, 10 L, 8 L, 6 L, 4
L and I pL of peptide/neutravidin HRP complex were added to 1.2 ml aliquots of
M199 medium to create dilutions with final concentrations of 33.33 nM, 16.65
nM, 5.55
nM, 2.78 nM, 1.67 nM, 1.11 nM and 0.28 nM complex, respectively.
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Preparation of blocking solution for transfected 293H cells
Blocking solution was prepared by adding 20 mL of M199 medium to 10 mg of
lyophilized unlabeled neutravidin (Pierce, cat. # 31000). Fresh blocking
solution was
used for each experiment.
Assay to detect the binding of peptide/neutravidin HRP
24 h after transfection, each well of the 293H cells was washed 1 X with 100
L
of M199 medium and incubated with 80 gL of blocking solution at 37 C. After
one
hour, cells were washed 2X with 100 gL of M 199 media and incubated with 70 L
of
peptide/neutravidin HRP dilutions of P1-XB, P6-XB, P5-XB, P12-XB and P13-XB
for
two and half hours at room temperature. Each dilution was added to three
separate wells
of mock as well as KDR-transfected 293H cells (two plates were used for each
saturation binding experiment). After incubation at room temperature, plates
were
transferred to 4 C for another half-hour incubation. Subsequently, cells were
washed
five times with ice-cold M199 media and IX with ice-cold PBS (in that order).
After the
final wash, 100 gL of ice cold TMB solution (KPL, cat. # 50-76-00) was added
to each
well and each plate was incubated for 30 minutes at 37 C in an air incubator.
Finally,
the HRP enzyme reaction was stopped by adding 50 L of IN phosphoric acid to
each
well, and binding was quantitated by measuring absorbance at 450 nm using a
microplate reader (BioRad Model 3550).
Binding of peptide/ neutravidin HRP to KDR-transfected cells
In this assay, complexes of P6-XB, P5-XB, P12-XB, P13-XB peptides, and the
control peptide, P1-XB, with neutravidin HRP were prepared as described above
and
tested for their ability to bind 293H cells that were transiently-transfected
with KDR.
During the peptide/neutravidin complex preparation, a 7.5 fold excess of
biotinylated
peptides over neutravidin HRP was used to ensure that all four biotin binding
sites on
neutravidin were occupied. After complex formation, the excess of free
biotinylated
peptides was removed using soft release avidin-sepharose to avoid any
competition
between free biotinylated peptides and neutravidin HRP-complexed biotinylated
peptides.
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The experiment was performed at several different concentrations of
peptide/neutravidin HRP, from 0.28 nM to 33.33 nM, to generate saturation
binding
curves for P5-XB and P6-XB (FIG. 8A) and 0.28 to 5.55 nM to generate
saturation
binding curve for P12-XB and P13-XB (FIG. 8B). To draw the saturation binding
curve, the background binding to mock-transfected cells was subtracted from
the
binding to KDR-transfected cells for each distinct peptide/neutravidin HRP
complex at
each concentration tested. Therefore, absorbance on the Y-axis of FIG. 8 is
differential
absorbance (KDR minus mock) and not the absolute absorbance. Analysis of the
saturation binding data in FIG. 8 using Graph Pad Prism software (version 3.0)
yielded
a Kd of 10.00 nM (+/-2.36) for the tetrameric P6-XB, 14.87 nM (+/- 5.07) for
the
tetrameric P5-XB, 4.03 nM (+/- 0.86) for the tetrameric P12-XB and 1.81 nM (+/-
0.27)
for the tetrameric P13-XB peptide complexes. These binding constants are, as
expected, lower than those measured by FP against the KDRFc construct for the
related
monodentate peptides P6 (69 nM) and P5 (280 nM) (fluoresceinated) but similar
for the
monodentate peptide P12 (3 nM). As expected, no saturation of binding for the
control
P1-X-B peptide/neutravidin HRP-complex was observed. As shown in FIG. 9, the
binding of peptide/neutravidin HRP complexes at a single concentration (5.55
nM) was
plotted to demonstrate that a single concentration experiment can be used to
differentiate between a KDR binding peptide (P6-XB, P5-XB and P12-XB ) from a
non-binding peptide (P1-XB).
Experiment B
Experiment B was designed to look at the effect of a spacer (X) between the
KDR binding sequence (P6 and P5) and biotin. In this experiment, biotinylated
P6 and
P5 (with and without spacer X, prepared as set forth above) were tested, and P
1 (with
and without spacer, prepared as set forth above) was used as a negative
control.
This experiment was performed as set forth in Experiment A described above,
except that it was only done at single concentration of 2.78 nM. It is evident
from the
results, shown in FIG. 10, that a spacer (X) is required for effective binding
of P6 and
P5. The spacer (X) between the binding sequence and biotin can be helpful in
enhancing binding to target molecule by multiple mechanisms. First, it may
help
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reduce the steric hindrance between four biotinylated peptides after their
binding to a
single avidin molecule. Second, it may provide extra length necessary to reach
multiple
binding sites available on a single cell.
Experiment C
Experiment C examined the serum effect on the binding of P6-XB, P5-XB, P12-
XB, and P 13-XB. In this experiment, biotinylated peptide/avidin HRP complexes
of
P6-XB, P5-XB, P12-XB, and P13-XB were tested in M199 media (as described above
in Experiment A) with and without 40% rat serum. This experiment was performed
as
described for Experiment A, except that it was only done at single
concentration of 6.66
nM for P6-XB and P5-XB, 3.33 nM for P12-XB and 2.22 nM for Pl3XB.
The results, shown in FIG. 11, indicate that binding of P6-XB, P5-XB and P13-
XB was not significantly affected by 40% rat serum whereas binding of P12-XB
dropped more than 50% in presence of 40% rat serum. More than an 80% drop in
the
binding of Tc-labeled P12-C (P12 with Tc-chelate), prepared by the method
described
in Example 5 above, was observed in the presence of 40% rat serum (data shown
in
FIG. 25). Because the serum effect on the binding of Tc-labeled P12-C is
mimicked in
the avidin HRP assay disclosed herein, this assay may be used to rapidly
evaluate the
serum effect on the binding of peptide(s) to KDR.
Experiment D
Experiment D was designed to evaluate the binding of tetrameric complexes of
KDR and VEGF/KDR complex-binding polypeptides P6-XB and P5-XB, particularly
where the constructs included at least two KDR binding polypeptides. The KDR
binding peptides and control binding peptide (P1-XB) were prepared as
described
above. This experiment was performed using the protocol set forth for
Experiment A,
except the procedures set forth below were unique to this experiment.
Preparation of peptide/neutravidin HRP solutions
250 gM stock solutions of biotinylated peptides P1-X-B, P6-XB, and P5-XB
were prepared in 50% DMSO and a 33 M stock solution of Neutravidin HRP was
prepared by dissolving 2 mg of Neutravidin HRP (Pierce, cat. # 31001) in 1 mL
of
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ddH2O. Peptide stock solutions were stored at -20 C, whereas the Neutravidin
HRP
stock solution was stored at -80 C. To prepare peptide/neutravidin HRP
complexes, a
total 5.36 gL of 250 gM biotinylated peptide stock solution (or a mixture of
peptide
solutions, to give peptide molecules four times the number of avidin HRP
molecules)
and 10 pL of 33 gM Neutravidin HRP were added to 1 mL of M199 medium. This
mixture was incubated on a rotator at 4 C for 60 minutes, followed by
addition of 50
1iL of soft release avidin-sepharose (50% slurry in ddH20) to remove excess
peptides
and another incubation for 30 minutes on a rotator at 4 C. Finally, the soft
release
avidin-sepharose was pelleted by centrifuging at 12,000 rpm for 5 minutes at
room
temperature, and the resulting supernatant was used for the assays. Fresh
peptide/neutravidin HRP complexes were prepared for each experiment.
Assay to detect the binding of peptide/neutravidin HRP
The procedure described above was used to detect binding of the
peptide/neutravidin HRP. The results of this experiment establish that P6-XB
and P5-
XB bind to KDR in multimeric fashion, and cooperate with each other for
binding to
KDR in 293H transfected cells.
P1-XB is a biotinylated derivative of P1, a control peptide that does not bind
to
KDR. As expected, a tetrameric complex of P1-XB with avidin-HRP did not show
enhanced binding to KDR-transfected cells. As shown in FIG. 12, tetrameric
complexes of P6-XB or P5-XB bound to KDR-transfected cells significantly
better than
to mock-transfected cells. P6-XB tetramers however, bound much better than P5-
X
tetramers. When P1-XB was added to the peptide mixture used to form the
tetrameric
complexes, the binding to the KDR-transfected cells was decreased. The ratios
of
specific binding of tetramer to monomer, dimer and trimer were calculated by
dividing
the specific binding (obtained by subtracting the binding to mock transfected
cells from
KDR transfected cells) of tetramer, trimer & dimer with that of monomer.
Results
suggest that there is co-operative effect of multimerization of P5-XB, P6-XB
and P 13-
XB on the binding to KDR transfected cells.
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Table 2. Enhanced binding of homomultimeric constructs over monomers
Ref. Number Tetramer Trimer Dimer
P5-XB 45.4 5 4.3
P6-XB* 38.6 7.1 2.7
P12-XB 1 1.1 1.1
P13-XB 16 5.7 2.3
*Monomeric Peptide binding at 3.33 nM was zero and therefore the ratios were
calculated using binding at 5.55 nM.
A mixture of 25% P1-XB with 75% P5-XB did not bind significantly over
background to KDR-transfected cells, indicating that multivalent binding is
critical for
the P5-XB/avidn-HRP complex to remain bound to KDR throughout the assay. This
phenomenon also held true for P6-XB, where substituting 50% of the peptide
with P1-
XB in the tetrameric complex abolished almost all binding to KDR on the
transfected
cells.
A peptide mixture composed of 50% P1-XB with 25% P6-XB and 25% P5-XB
bound quite well to KDR-transfected cells relative to mock-transfected cells,
indicating
that there is a great advantage to targeting two binding sites on the same
target
molecule. Furthermore, it was noted that tetrameric complexes containing
different
ratios of P6-XB and P5-XB (3:1, 2:2, and 1:3) all bound much better to KDR-
transfected cells than pure tetramers of either peptide, in agreement with the
idea that
targeting two distinct sites on a single target molecule is superior to
multimeric binding
to a single site. This may be because multimeric binding to a single target
requires that
the multimeric binding entity span two or more separate target molecules which
are
close enough together for it to bind them simultaneously, whereas a multimeric
binder
which can bind two or more distinct sites on a single target molecule does not
depend
on finding another target molecule within its reach to achieve multimeric
binding. The
ratios of specific binding of heterotetramer, heterotrimer and heterodimer to
monomer
were calculated by dividing the specific binding (obtained by subtracting the
binding to
mock transfected cells from KDR transfected cells) of tetramer, trimer and
dimer with
that of monomer. Monomer, which was used to calculate the ratios, for each set
of
heteromers is recorded at the end of each heteromer listing in the table and
given the
ratio of 1.
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Table 3. Enhanced binding of heteromultimeric constructs over monomers
Peptide Mix Heteromer/Monomer Conc.
Where (1X,2X,3X) is the occupancy of the Ratio (nM)
four avidin sites
P6-XB(IX)+P5-XB(3X) 529 3.33
P6-XB(2X)+ P5-XB (2X) 777 3.33
P6-XB (3X)+ P5-XB (1X) 633 3.33
P1-XB(1X)+ P6-XB (1X)+ P5-XB (2X) 213 3.33
P1-XB (1X)+ P6-XB (2X)+ P5-XB (1X) 242 3.33
P 1-XB (2X)+ P6-XB (I X)+ P5-XB (IX) 109 3.33
P5-XB (1X)+ P1-XB (3X) 1 3.33
P6-XB (1X)+P12-XB(3X) 46 2.78
P6-XB (2X)+ P1 2-XB (2X) 42 2.78
P6-XB (3X)+ P12-XB (1X) 43 2.78
P1-XB (IX)+ P6-XB (IX)+ P12-XB (2X) 47 2.78
P1-XB (1X)+ P6-XB (2X)+ P12-XB (1X) 52 2.78
P1-XB (2X)+ P6-XB (1X)+ P12-XB (1X) 40 2.78
P1-XB (3X)+ P6-XB 1 X* 1 5.55
P1-XB (1X)+P13-XB(lX)+ P12-XB (2X) 5 2.78
P1-XB (1X)+ P13-XB (2X)+ P12-XB (IX) 7 2.78
P1-XB (2X)+ P13-XB (1X)+ P12-XB (1X) 2 2.78
P13-XB 1X + P1-XB (3X) 1 2.78
P1-XB (IX)+ P6-XB (1X)+ P13-XB (2X) 83 2.78
P1-XB (IX)+ P6-XB (2X)+ P13-XB (1X) 31 2.78
P1-XB (2X)+ P6-XB (IX)+ P13-XB (1X) 31 2.78
P 1-XB (3X)+ P6-XB 1 X* 1 5.55
The enhanced binding ratios of the homodimers range from about 1-4 fold as
seen in table 2 whereas the binding of the heterodimers ranges from 2-110
fold,
demonstrating the synergistic effect on binding strength of complementary
sequences
(Table 3).
Experiment E
Experiment E was designed to confirm that P6-XB and P5-XB bind to distinct
binding sites on KDR. If the peptides bind to the same site on KDR, they would
likely
compete with each other for binding to KDR, whereas if the peptides bind to
different
sites, there should be no competition between the peptides for binding to KDR.
This
experiment was performed using a single concentration of P5-XB/avidin HRP
(3.33
nM) solution in each well and adding a varying concentration (0-2.5 JIM) of P
I -XB, P5-
XB and P6-XB, none of which were complexed with avidin.
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It is evident from the results, shown in FIG. 13, that P5-XB does compete with
P5-XB/avidin HRP solution for binding to KDR transfected cells whereas PI-XB
and
P6-XB do not compete with P5-XB/avidin HRP solution for binding to KDR
transfected cells. Thus, P5-XB and P6-XB bind to distinct and complementary
sites on
KDR.
EXAMPLE 7
Preparation of Heterodimeric Constructs
To obtain a higher affinity peptide binder to the KDR receptor, two linear
peptides (P9, P10) were linked together to form a heterodimer. As determined
by
VEGF competition assays, these two peptides bind different sites on KDR. It is
possible, therefore, that both peptides in the heterodimer could bind a single
protein
molecule at the same time and as result, bind with a higher overall affinity
for the
receptor. Two forms of the heterodimer were synthesized in an effort to
determine the
optimal orientation for this bidentate binding event. The peptides were either
linked
together in a tail-to-tail orientation via their C-terminal lysine residues or
in a head-to-
head orientation via their N-terminal amino groups.
The peptides were synthesized using standard Fmoc solid-phase peptide
synthesis protocols. To add spacing between the two peptides in the dimer,
each
individual peptide monomer was modified at either the C-terminal lysine (to
make the
tail-to-tail dimer) or N-terminal amino (to make the head-to-head dimer) with
a
monodispersed PEG-based amino acid linker (Fmoc-NH-PEG4-CO2H). After
deprotection of the Fmoc group of each PEG linker, the P9 peptide was labeled
with
levulinic acid (CH3(C=O)(CH2)2CO2H) and the P10 peptide was labeled with Boc-
amino-oxyacetic acid. After deprotection, cleavage and purification, the two
peptides
were ligated together in a 1:1 ratio in denaturing buffer (8M Urea, 0.1M
sodium acetate,
pH 4.6) to form an oxime linkage
(-CH=N-O-) between the two different peptides. Using the two different sets of
monomers, the tail-to-tail and head-to-head heterodimers were formed in
solution and
purified to homogeneity by standard reverse phase protocols. A more detailed
description of this linkage chemistry is found in K. Rose, et al. JACS, 1999,
121: 7034-
7038, which is hereby incorporated by reference in its entirety.
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Assay for Binding Affinity of Heterodimeric Constructs
To assay for improved binding affinity relative to either monomeric peptide,
each heterodimer was assayed for binding using a surface plasmon resonance
instrument (Biacore 3000). Soluble KDR receptor was cross-linked to the
dextran
surface of a CM5 sensor chip by the standard amine coupling procedure. A
0.5mg/mL
solution was diluted 1:40 with 50mM acetate, pH 6.0 to immobilize a total RL
of 12721.
Experiments were performed in PBST buffer (5.5mM phosphate, pH 7.65, 0.15M
NaCI,
0.1 % Tween-20 (v/v)). Peptide solutions quantified by extinction coefficient
were
diluted to produce 1000, 500, 250, 125, 62.5 and 31.3nM solutions. For
association,
peptides were injected at 20 .tL/min for 2 minutes using the kinject program.
Following
a 3 minute dissociation, any remaining peptide was stripped from the KDR
surface with
a quickinject of 50mM NaOH, 1M NaCI for 15s at 75 gUmin. Monomeric P9 and P10
were run as standards. Sensorgrams were analyzed by global analysis using
BIAevaluation software 3.1.
The peptide dimers investigated in this study by BlAcore analysis bind KDR
with significantly higher affinity than either of the constituent monomers. By
design,
the interaction of a dimeric peptide with KDR is expected to proceed through
two
kinetic steps. With more detailed analysis it may be possible to accurately
dissect the
individual rate constants for these steps. However, an apparent KD was
calculated for
the dimer interaction using the rate describing the initial encounter (ke,l)
and the
predominant off-rate (kd,2). From this analysis, the apparent KD of the head-
to-head
dimer was 2.2 nM and that of the tail-to-tail dimer was 11 nM (Table 4). These
estimates represent an increase in affinity over the individual monomers of
greater than
60-fold for the comparison of the P9 to T-T dimer KD (732 nM /11.1 nM) and
greater
than 560-fold for the P10 to H-H dimer KD (1260 nM/2.24 nM).
Table 4. Summary of Kinetic Parameters
Peptide ka 1/Ms kd 1 1/s Ku 1 n Chi2*
P9 4.53 x 10 3.32 x 10" 732 0.67
P10 3.60x10 4.5x10" 1260 1.2
Head-to-head dimer 1.11 x 10 2.49 x 10 2.24 1.25
Tail-to-tail dimer 1.15 x 10 1.28 x 10" 11.1 2.33
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EXAMPLE 8
The following methods were used for the preparation of individual peptides and
dimeric peptide constructs described in Examples (8-12).
Automated Peptide Synthesis
Peptide synthesis was carried out on an ABI-433A Synthesizer (Applied
Biosystems Inc., Foster City, CA) on a 0.25 mmol scale using the FastMoc
protocol. In
each cycle of this protocol preactivation was accomplished by dissolution of
1.0 mmol
of the requisite dry N-Fmoc side-chain protected amino acid in a cartridge
with a
solution of 0.9 mmol of HBTU, 2 mmol of DIEA, and 0.9 mmol of HOBt in a DMF-
NMP mixture. The peptides were assembled on NovaSyn TGR (Rink amide) resin
(substitution level 0.2 mmol/g). Coupling was conducted for 21 min. Fmoc
deprotection was carried out with 20% piperidine in NMP. At the end of the
last cycle,
the N-terminal Fmoc group was removed and the fully protected resin-bound
peptide
was acetylated using acetic anhydride / DIEA / HOBt / NMP.
Cleavage, Side-chain Deprotection and Isolation of Crude Peptides
Cleavage of the peptides from the resin and side-chain deprotection was
accomplished using Reagent B for 4.5h at ambient temperature. The cleavage
solutions
were collected and the resins were washed with an additional aliquot of
Reagant B. The
combined solutions were concentrated to dryness. Diethyl ether was added to
the
residue with swirling or stirring to precipitate the peptides. The liquid
phase was
decanted, and solid was collected. This procedure was repeated 2-3 times to
remove
impurities and residual cleavage cocktail components.
Cyclization of Di-cysteine Peptides
The crude ether-precipitated linear di-cysteine containing peptides were
cyclized
by dissolution in water, mixtures of aqueous acetonitrile (0.1% TFA), aqueous
DMSO
or 100% DMSO and adjustment of the pH of the solution to 7.5 - 8.5 by addition
of
aqueous ammonia, aqueous ammonium carbonate, aqueous ammonium bicarbonate
solution or DIEA. The mixture was stirred in air for 16-48 h, acidified to pH
2 with
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aqueous trifluoroacetic acid and then purified by preparative reverse phase
HPLC
employing a gradient of acetonitrile into water. Fractions containing the
desired
material were pooled and the purified peptides were isolated by
lyophilization.
Preparation of Peptides Containing Linkers
In a typical experiment, 400 mg of the resin-bound peptide bearing an ivDde-
protected lysine) was treated with 10% hydrazine in DMF (2 x 20 mL). The resin
was
washed with DMF (2 x 20 mL) and DCM (1 x 20 mL). The resin was resuspended in
DMF (10 mL) and treated with Fmoc-8-amino-3,6-dioxaoctanoic acid (0.4 mmol),
HOBt (0.4 mmol), DIC (0.4 mmol) and DIEA (0.8 mmol) with mixing for 4 h. After
the reaction, the resin was washed with DMF (2 x 10 mL) and with DCM (1 x 10
mL).
The resin was then treated with 20% piperidine in DMF (2 x 15 mL) for 10 min
each
time. The resin was washed and the coupling with Fmoc-8-amino-3,6-
dioxaoctanoic
acid and Fmoc protecting group removal were repeated once more.
The resulting resin-bound peptide with a free amino group was washed and
dried and then treated with reagent B (20 mL) for 4 h. The mixture was
filtered and the
filtrate concentrated to dryness. The residue was stirred with ether to
produce a solid,
which was washed with ether and dried. The solid was dissolved in anhydrous
DMSO
and the pH adjusted to 7.5 with DIEA. The mixture was stirred for 16h to
effect the
disulfide cyclization and the reaction was monitored by analytical HPLC. After
completion of the cyclization, the reaction mixture was diluted with 25%
acetonitrile in
water and applied directly to a reverse phase C-18 column. Purification was
effected
using a gradient of acetonitrile into water (both containing 0.1% TFA).
Fractions were
analyzed by HPLC and those containing the pure product were combined and
lyophilized to provide the required peptide.
Preparation of Biotinylated Peptides Containing Linkers
In a typical experiment, 400 mg of the resin-bound peptide bearing an ivDde-
protected lysine, was treated with 10% hydrazine in DMF (2 x 20 mL). The resin
was
washed with DMF (2 x 20 mL) and DCM (1 x 20 mL). The resin was resuspended in
DMF (10 mL) and treated with Fmoc-8-amino-3,6-dioxaoctanoic acid (0.4 mmol),
HOBt (0.4 mmol), DIC (0.4 mmol) and DIEA (0.8 mmol) with mixing for 4 h. After
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the reaction, the resin was washed with DMF (2 x 10 mL) and with DCM (lx 10
mL).
The resin was then treated with 20% piperidine in DMF (2 x 15 mL) for 10 min
each
time. The resin was washed and the coupling with Fmoc-8-amino-3,6-
dioxaoctanoic
acid and removal of the Fmoc protecting group were repeated once more.
The resulting resin-bound peptide with a free amino group was treated with a
solution of Biotin-NHS ester (0.4 mmol, 5 equiv.) and DIEA (0.4 mmol, 5
equiv.) in
DMF for 2 h. The resin was washed and dried as described previously and then
treated
with Reagent B (20 mL) for 4 h. The mixture was filtered and the filtrate
concentrated
to dryness. The residue was stirred with ether to produce a solid that was
collected,
washed with ether, and dried. The solid was dissolved in anhydrous DMSO and
the pH
adjusted to 7.5 with DIEA. The mixture was stirred for 4-6 h to effect the
disulfide
cyclization which was monitored by HPLC. Upon completion of the cyclization,
the
reaction mixture was diluted with 25% acetonitrile in water and applied
directly to a
reverse phase C-18 column. Purification was effected using a gradient of
acetonitrile
into water (both containing 0.1 % TFA). Fractions were analyzed by HPLC and
those
containing the pure product were collected and lyophilized to provide the
required
biotinylated peptide.
Preparation of DOTA-Conjugated Peptides for Labeling with Selected Gadolinium
or
Indium Isotopes
In a typical experiment, 400 mg of the resin-bound peptide bearing an Ne-ivDde-
protected lysine moiety was treated with 10% hydrazine in DMF (2 x 20 mL). The
resin was washed with DMF (2 x 20 mL) and DCM (1 x 20 mL). The resin was
resuspended in DMF (10 mL) and treated with Fmoc-8-amino-3,6-dioxaoctanoic
acid
(0.4 mmol), HOBt (0.4 mmol), DIC (0.4 mmol), DIEA (0.8 mmol) with mixing for 4
h.
After the reaction, the resin was washed with DMF (2 x 10 mL) and with DCM (lx
10
mL). The resin was then treated with 20% piperidine in DMF (2 x 15 mL) for 10
min
each time. The resin was washed and the coupling with Fmoc-8-amino-3,6-
dioxaoctanoic acid and removal of the Fmoc protecting group were repeated
once. The
resulting resin-bound peptide with a free amino group was resuspended in DMF
(10
mL) and treated with a solution of 1,4,7, 1 0-tetraazacyclododecane- 1,4,7,1 0-
tetraacetic
acid,-1,4,7-tris-t-butyl ester (DOTA-tris-t-butyl ester, 0.4 mmol, 5 equiv.),
HOBt (0.4
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mmol), DIC (0.4 mmol) and DIEA (0.8 mmol) in DMF (10 mL) with mixing for 4 h.
Upon completion of the reaction, the resin was washed with DMF (2 x 10 mL) and
with
DCM (1 x 10 mL) and treated with Reagent B (20 mL) for 4 h. The mixture was
filtered and the filtrate concentrated to dryness. The residue was stirred in
ether to
produce a solid that was collected, washed with ether, and dried. The solid
was
dissolved in anhydrous DMSO and the pH adjusted to 7.5 with DIEA. The mixture
was
stirred for 16 h to effect the disulfide cyclization, which was monitored by
HPLC. Upon
completion of the cyclization, the mixture was diluted with 25% acetonitrile
in water
and applied directly to a reverse phase C-18 HPLC column. Purification was
effected
using a gradient of acetonitrile into water (both containing 0.1 % TFA).
Fractions were
analyzed by HPLC and those containing the pure product were combined and
lyophilized to provide the required biotinylated peptide.
The following monomeric peptides of Table 5 were prepared by the above
methods.
Table 5. Sequence or Structure of Monomeric Peptides and Peptide Derivatives
Ref. Number Structure or Sequence Seq
Id.
No:
P12-XB-K Ac-AGPTWC*EDDWYYC*WLFGTGGGK(BiotinJJ-K -NH2 --
P12-XDT-K Ac-AGPTWC*EDDWYYC*WLFGTJK DOTAJJ-K -NH2 --
P12-X Ac-AGPTWC*EDDWYYC*WLFGTJK JJ -NH2 --
P12-E Ac-AGPTWC*EDDWYYC*WLFGTGGGK K ivDde) -NH2 --
P6-F-XB-K Ac-VC*WEDSWGGEVC*FRYDPGGGK iotin-JJK -NH2 --
P6-F-X Ac-VC*WEDSWGGEVC*FRYDPGGGK(JJ)-NH2 --
P13-EB-K Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(ivDde)K(Biotin- _
JJ)-NH2
P13-X Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(J)-NH2
P13-K-E Ac-AQDWYYDEILSMADQLRHAFLSGGGGGKK(ivDde) --
P6-X Ac-GDSRVC*WEDSWGGEVC*FRYDPGGGK(JJ)-NH2 --
P12-A Ac-AGPTWC*EDDWYYC*WLFGTGGGK[(PnAO6- --
C =O CH2 3C =O -K -NH2
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P12-XDT-K-E Ac-AGPTWC*EDDWYYC*WLFGTGGGK[KBTA-JJK(iV-Dde)]- _
z
P6-F Ac-VC*WEDSWGGEVC*FRYDPGGGK-NHz
P12-0 Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(BOA)]-NHz --
P13-A-E Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK[PnAO6- --
C(=0 CH2 3C =O)-K iV-Dde -NH2
P23 AQDWYYEILJGRGGRGGRGGK 22
P23-K-E Ac-AQDWYYEILJGRGGRGGRGGK[K(ivDde)]-NHz
P24 APGTWCDYDWEYCWLGTFGGGK 23
P24-A Ac-APGTWC*DYDWEYC*WLGTFGGGK[(6PnAO-
C(=0 (CH2)3C =O)-K -NHz
P25 GVDFRCEWSDWGEVGCRSPDYGGGK 24
P25-X Ac-GVDFRC*EWSDWGEVGC*RSPDYGGGK(JJ)-NHz
P12-BK Ac-AGPTWC*EDDWYYC*WLFGTGGGK(Biotin-K)-NHz --
P12-JE JJAGPTWC*EDDWYYC*WLFGTGGGK(iV-Dde)-NHz --
P6-J-F JJVC*WEDSWGGEVC*FRYDPGGG-NHz
P12-JA [-JJAGPTWCEDDWYYCWLFGTGGGGK(PnA06-Glut)-NH2]- --
P12-S Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(SATA)]-NHz --
P12-SX-K Ac-AGPTWC*EDDWYYC*WLFGTGGGK[SATA-JJ-K]-NHz --
--
P12-NE H2N-AGPTWC*EDDWYYC*WLFGTGGGK[K(iV-Dde)]-NH2
P12-Q Ac-AGPTWC*EDDWYYC*WLFGTGGGK{Biotin-JJK[NH2- --
Se GaINAc-al ha-D -Gl -Ser GaINAc3-al ha-D -NHz
P6-F-Q Ac-VC*WEDSWGGEVC*FRYDPGGGK(NH2-Ser(GaINAc-alpha- --
D -Gly-Ser Ga1NAc-al ha-D -NHz
P26 GSPEMCMMFPFLYPCNHHAPGGGK 25
P26-A Ac-GSPEMC*MMFPFLYPC*NHHAPGGGK[(PnA06)-
C(=0 (CHz 3C(=O)-K] -NHz
P27 GSFFPCWRIDRFGYCHANAPGGGK 26
P27-X Ac-GSFFPC*WRIDRFGYC*HANAPGGGK(JJ)-NH2
P27-A Ac-GSFFPC*WRIDRFGYC*HANAPGGGK[(PnAO6)-
C =0 CHZ 3C =0 -K -NHz
P28 AQEWEREYFVDGFWGSWFGIPHGGGK 27
P28-X Ac-AQEWEREYFVDGFWGSWFGIPHGGGK(JJ)-NH2-
P29 GDYSECFFEPDSFEVKCYDRDPGGGK 28
P29-X Ac-GDYSEC*FFEPDSFEVKC*YDRDPGGGK(JJ)-NH2
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As used in Table 5 above and elsewhere herein, the designation "C*" refers to
a
cysteine residue that contributes to a disulfide bond. In general, the
monomeric
peptides described herein are prepared as cyclic disulfide peptides and then
linked
together to form dimers. Consequently, even if a cysteine residue lacks the
"C*"
designation, the presence of a disulfide bond to the nearest cysteine in the
monomer can
generally be assumed. The monomer components of the dimers will also generally
contain such disulfide bonds, regardless of whether the cysteine residues
contain the
"C*" designation or not. However, one skilled in the art will appreciate that
the dimers
and other heteromultimers of the present invention could alternatively be
prepared by
performing the cyclization of Di-cysteine peptides after the monomers are
linked to
form dimers, and the present invention is not intended to be limiting with
respect to the
presence or absence of such disulfide bonds.
EXAMPLE 9
The purified peptide monomers mentioned above in Example 8 were used in the
preparation of various homodimeric and heterodimeric constructs.
Preparation of Homodimer-Containing Constructs
To prepare homodimeric compounds, half of the peptide needed to prepare the
dimer was dissolved in DMF and treated with 10 equivalents of glutaric acid
bis-N-
hydoxysuccinimidyl ester. The progress of the reaction was monitored by HPLC
analysis and mass spectroscopy. At completion of the reaction, the volatiles
were
removed in vacuo and the residue was washed with ethyl acetate to remove the
unreacted bis-NHS ester. The residue was dried, re-dissolved in anhydrous DMF
and
treated with another half portion of the peptide in the presence of 2
equivalents of
DIEA. The reaction was allowed to proceed for 24 h. This mixture was applied
directly to a YMC reverse phase HPLC column and purified by elution with a
linear
gradient of acetonitrile into water (both containing 0.1% TFA).
Preparation of Heterodimer-Containing Constructs
In the case of heterodimers, one of the monomers ("A") was reacted with the
bis-NHS ester of glutaric acid and after washing off the excess of bis-NHS
ester (as
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described for the homodimeric compounds), the second monomer ("B") was added
in
the presence of DIEA. After the reaction the mixture was purified by
preparative
HPLC. Typically, to a solution of glutaric acid bis N-hydoxysuccinimidyl ester
(0.02
mmol, 10 eqivalents) in DMF (0.3 mL) was added a solution of peptide A and
DIEA (2
equiv) in DMF (0.5mL) and the mixture was stirred for 2 h. The progress of the
reaction was monitored by HPLC analysis and mass spectroscopy. At completion
of the
reaction, the volatiles were removed in vacuo and the residue was washed with
ethyl
acetate (3 x 1.0 mL) to remove the unreacted bis-NHS ester. The residue was
dried, re-
dissolved in anhydrous DMF (0.5 mL) and treated with a solution of peptide B
and
DIEA (2 equiv) in DMF (0.5 mL) for 24 h. The mixture was diluted with water
(1:1,v/v) and applied directly to a YMC C-18 reverse phase HPLC column and
purified
by elution with a linear gradient of acetonitrile into water (both containing
0.1 % TFA).
Fractions were analyzed by analytical HPLC and those containing the pure
product
were combined and lyophilized to obtain the required dimer. The following
dimers
were prepared by this method (structure, name, compound reference number):
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ONH~ M
AC-AGPTWCEDDWYYCWLFGTGGG-0 H N` ^p^~O~/~N1,0,^0i.,.,NHBIOTIN
HN 0
0
NH
I
HN '0
H 1~0
Ac=VCWEDSWGGEVCFRYDPGGG-N NH
ONH.
Ac-AGPTWCEDDWYYC WLFGTGGGK[(Biotin-JJK-(O=)C(CH2)3C(=O)-JJ-NH(CH2)4-
(S)-CH((Ac-VCWEDSWGGEVCFRYDPGGG)-NH)CONH2 ]-NH2 : Dl
~O~,O^,NHBIOTIN
AcJ-GPTWCEDDWYYCWLFGTGGG H ON M ~p/~O"-NQ$
H
HN 0
O
NH
I
HN '0
O
Ac-AGPTWCEDDWYYCWLFGTJ_H/ .NH
CTONH_,
Ac-AGPTWCEDDWYYCWLFGTGGGK[(Biotin-JJK-(O=)C(CH2)3C(=O)-JJ-NH(CH2)4-
(S)-CH((Ac-AGPTWCEDDWYYCWLFGTJ)-NH)CONH2 ]-NH2 : D2
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ONH= H
Ac-VCWEDSWGGEVCFRYDPGGG-H 01-0,/~.O~ ,NHSIOTIN
O
HN 0
0
NH
I1
HN1O
HH 0
AaVCWEDSWGGEVCFRYDPGGG--N NH
ONH3
Ac-V C WEDS W GGEV CFRYDPGGGK[(Biotin-JJK-(O=)C(CH2)3 C(=O)-JJ-NH(CH2)4-(S)-
CH((Ac-VCWEDSWGGEVCFRYDPGGG)-NH)CONH2 ]-NH2 D3
N
O O
ONH~ H 0 ~N)/OOH
ACAGPTWCEDOWYYCWLFGTJ-0
O
HN 0
NH
I
HN)*O
F1 O
Ac=VCWEDSWGGEVCFRYDPGGG-N NH
ON
Ac-AGPT W CEDD W YYC W LFGTJK[DOTA-JJK-(O=)C(CH2)3 C(=O)-JJ-NH(CH2)4-(S)-
CH((Ac-VCWEDS WGGEVCFRYDPGGG)-NH)CONH2 ]-NH2 : D4
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RLL _ _ .,
AGAGPTYVCEDDWYYCWLFGTGGO-H ON H'
H HNI O
O
NH
HM-O
H I-r
Ac=VCWEDSWGGEVCPRYDPGGG-N NH
NH=
Ac-VCWEDS WGGEVCFRYDPGGGK(JJ-C(=O)(CH2)3C(=O)-K-NH(CH2)4-(S)-CH((Ac-
AGPTWCEDDWYYCWLFGTGGG)-NH)CONH2)-NH2 : D5
NH2
H" O~~O~~NH-Biotin
Ac-AQDWYYDEILSMADOLRHAFLSGGGGG-NH
H
H
O
O
Ni
O
S
O
O
O Nom, H, NH
Ac-AQD W YYDEILSMADQLRHAFLSGGGGG-N H
Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK {Ac-
AQDWYYDEILSMADQLRHAFLSGGGGGK(J-Glut-)-NH2}K(Biotin-JJ)-NH2 : D8
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O 0 NH2
Ac-AQDWYYDEILSMADQLRHAFLSGGGGG-NH N '~~./~~NH2
H
NH
O
O
NH
cO
O
O=~
7
cO
O
0 NHO~
2^ JNH
Ac-GDSRVCWEDSWGGEVCFRYDPGGG-NH "''~'
Ac-AQD W YYDEILSMADQLRHAFLSGGGGGK ( [Ac-
GDSRVCWEDSWGGEVCFRYDPGGGK(JJ-Glut-)]-NH2}K-NH2: D9
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0
Ac-AGPTWCEDDWYYCWLFGTGGG-NH.NH2 O
O N
--<'
NH
C-N O
N
NH HN
NH (N
O N
OH '
OH
O
NH
CO
O
O=>
(
NH
cO
O
O~
0 NHH22 NH
Ac-GDSRVCWEDSWGGEVCFRYDPGGG-NH ~~"
Ac-AGPTW CEDD WYYC W LFGTGGGK { [Ac-
GDSRVC WEDSWGGEVCFRYDPGGGK(JJ-Glut-NH(CH2)4-(S)-CH(PnAO6-Glut-
NH)(C=O-)]-NH2}-NH2 : D10
143
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ONHZ
H H
Ac-AGPTWCEDDWYYCWLFGTGGG-H ^H NrO~O^~N0
O
H O
0
HN
O O
NH
HOOC'N NN
<\,NJ COOH
HOOC
O~
HN O
CO
O
H
Ac-VCW EDS W GG EVCF RYDPGGG-NNH
CONH2
Ac-AGPTWCEDDWYYCWLFGTGGGK (Ac-
VCWEDSWEDS WGGEVCFRYDPGGGK[JJ-Glut-NH(CH2)4-(S)-CH(DOTA-JJ-NH-
)(C=O)-]-NH2}-NH2 : D11
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O
Ac-AGPTWCEDDYYCWLFGTGGG-NH NH2
O
O
N NH NH
H
NH HN
NH
O~ CN N/
O H OH
O
C
O
O
0 NH2 HN
Ac-VCWEDSWGGEVCFRYDPGGG-NH
Ac-AGPT W CEDD W YYC W LFGTGGGK { [PnAO6-Glut-K(Ac-
VCWEDSWGGEVCFRYDPGGGK(-C(=O)CH2(OCH2CH2)2OCH2C(=O)-)-NH2] }-NH2
D12
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O
AAGPTWCEDDWYYCWLFGTGCG-N ONH N)L _ N
H H H
0 OH
N~O
HO O N-.,,*,%
O f
N Oyi OH
(NH HO OH
O~
HN 0
CO
O
Ac-VCWEDSWGGEVCFRYDPGGG-N`^ ^'NH
CONH2 _
Ac-AGPT W CEDD W YYC WLFGTGGGK { Ac-V C WEDS W GGE V CFRYDPGGGK[JJ-
Glut-K(BOA)]-NH2}-NH2 : D13
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Ac-AQDWYYDEILSMADQLRHAFLSGGGGG-NH,NHZ O
0 H
N "O~
NH
(-N 0
H
NH HN
H "N N:
0 OH OH
O
H
c0
O
NH
CO
O
0 NH O~
H
Ac-GDSRVCWEDSWGGEVCFRYDPGGG-NH ~"'
Ac-AQD WYYDEILSMADQLRHAFLSGGGGGK {PnAO6-Glut-K[Ac-
GSDRVCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH2]}-NH2 : D14
147
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Ac-AGPTW CEDDW YYC W LFGTG GG-NH
0 NH2 O NH O O
HN ).000,
NH
0 H ri
NH HN
O
N N
NH OH OH
cO
O=(
H
cO
0
0 NH0~
H
Ac-GDSRVCWEDSWGGEVCFRYDPGGG-NH ""
Ac-AGPTWCEDDWYYCWLFGTGGGK {[[Ac-
GDSRVCWEDSWGGEVCFRYDPGGGKJJ-Glut]-NH2]-K(PnAO6-Glut)}-NH2: D15
148
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O
Ac-AGPTWCEDDWYYCWLFGTGGG-NH` x
NHZ O
H
O
N
O NH
CN
H
H HN
NH CNN
p~ OH OH
O~
^ /O
NH
O
H O
N
O O O O
O
NH
0 H2
Ac-GDSRVCWEDSWGGEVCFRYDPGGG-NH rN Ac-AGPTWCEDDWYYCWLFGTGGGK {PnAO6-Glut-K[Ac-
GDSRV CWEDS WGGEVCFRYDPGGGK[-
C(=O)CH2O(CH2CH2O)2CH2C(=O)NH(CH2)30(CH2CH2O)2(CH2)3NH
C(=O)CH2O(CH2CH2O)2CH2C(=O)-]-NH2]) -NH2 : D16
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O
Ac-AQDWYYDEIWGRGGRGGRGG-NH NHZ
O NHZ
N
H
H
O
O
NH
O
O
O=
NH
O
O
O~
O NH2 NH
Ac-VCWEDSWGGEVCFRYDPGGG-NH ''0'~
Ac-AQDWYYDEILJGRGGRGGRGGK {K[Ac-
VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NHZ]}-NH2 : D17
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0
Ac-APGTWCDYDWEYCWLGTFGGG-NH
NHZ O
CO N MH
O
N r
H
NH HN
H ~N N)
O OH OH
O
NH
cO
O
O:>
(
NH
cO
O
O~
0 NHZ NH
Ac-GVDFRCEWSDWGEVGCRSPDYGGG-NH ""
Ac-APGTWCDYDWEYCWLGTFGGGK {PnAO6-Glut-K[Ac-
GVDFRCEWSDWGEVGCRSPDYGGGK(JJ-Glut)-NH2]}-NHZ : D18
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~ONHZ 0 ~j
Ac-AC
PTWCEDDWYYCWLFGTGGG-N "N NS
H H H HH
HNyNH
O
HN
O
O
(NH
O
O
HN 0
CO
O
I-f O
H Ac-VC W E DS W G G EVC F RYD PG G G-NNH
CONH2
Ac-AGPTWCEDDWYYCWLFGTGGGK {Biotin-K[Ac-
VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH2])-NH2 : D19
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O
O
HN- NH
y^"".-~
O
O 0
O~ 1
NH HN O
O (0
O
0
0 0
VCW EDSWGGEVCFRYDPGGG-NH2
AG PTWCEDDWYYCWLFGTGGGK-NH2
(-JJAGPTWCEDDWYYCWLFGTGGGK-NH2)-Glut-
VCWEDSWGGEVCFRYDPGGG-NH2 : D20
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0 00
rH
O
0 00 NH HN 1 O
o~ 0
0 o
NH-VCW EDSWGGEVCFRYDPGGG-NH2
N-AG P T W CE DDWYYCW L FGTGGG-N
0 O ~.=~ NH2
NH HN
Hi
NH HN
N~
N
OH OH
[-JJAGPTWCEDDWYYCWLFGTGGGK(PnAO6-Glut)-NH2]-Glut-
JJVCWEDSWGGEVCFRYDPGGG-NH2 : D21
0
O
~-N-G D S R V C W E D S W G G E V C F R Y D P G G G-N\ H
H NHz
HN 0 0 O"""YH/~ IOt H
0
O
NH
H
N-A G P T W C E D D W Y V C W L F T G G G K-NH2
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Ac-GDSRVCWEDS WGGEVCFRYDPGGGK {JJ-Glut-JJ-
AGPTWCEDDWYYCWLFGTGGGK-NH2}-NH2 : D22
ONHZ
Ac-AGPTWCEDDWYYCWIFGTGGG-NH y
HN H
S
O
O
NH
O
HN O
O
Ac-VCWEDSWGGEVCFRYDPGGG-NH NH
CONHZ
Ac-AGPTWCEDDWYYCWLFGTGGGK {Ac-VCWEDSWGGEVCFRYDPGGGK[JJ-
Glut-K(SATA)]-NH2}-NH2 : D23 (D5 functionalized with the SATA (S-
Acetylthioacetyl) group)
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ONHZ O O
H
PTWCEDDWYYCWLFGTGGG-NH N N
Ac-AG
H
O
HN
O
HN p
O
(NH
O
O
NH
O
S p
HN O
O
O
Ac-VCW EDSWGGEVCFRYDPGGG-NH NH
CONH2
Ac-AGPTWCEDDWYYCWLFGTGGGK {SATA-JJK[Ac-
VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH2])-NH2 : D24
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O
Ac-AGPTWCEDDWYYCWLFGTGGG-NHNHZ
CO NHZ
N
H
H
O
O
NH
0
0
NH
cO
O
O NH0~
z NH
Ac-GDSRVCWEDSWGGEVCFRYDPGGG-NH 'r"'
Ac-AGPTWCEDDWYYCWLFGTGGGK (Ac-
GDSRVCWEDSWGGEVCFRYDPGGGK[JJ-Glut-NH(CH2)4-(S)-CH(NH2)C(=O)-]-
NH2}-NHZ : D25
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HZN-A G P T W C E D D W Y Y C W L F G T G G G G-N 0 NH2
NH2
H H
0
O H
0 0
N-V C W E D S W G G E V C F R Y D P G G G-NH2
H
Ac-AGPTWCEDDWYYCWLFGTGGGK {(-Glut-JJ-VCWEDSWGGEVCFRYDPGGG-
NH2)-K}-NH2: D26
Ac-AG PTWCEDDWYYCWLFGTGGG-NH`
y `NHZ H
~
O
~-N N
H 0
HO HO H
H O H OH H OH O
OH 0 O
OH NH-Ac
0 NH-Ac O HO Ac-NH H Ac-NH O
H H
' v v 'N N JHV 0 O H"~`H H
NH 0 H 0 O
H 1 H
Ac-VCWEDSWGGEVCPRYDPGGG-NH NHZ H H HNl
0 S
Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[S(Ga1NAc-
alpha-D)-G-S(Ga1NAc-alpha-D)-Glut-S(Ga1NAc-alpha-D)-G-S(Ga1NAc-alpha-D)-
NH(CH2)4-
(S)-CH(Biotin-JJNH-)C(=O)-]-NH2}-NH2: D27
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CONH2 0 0 0
H
AC-GSPEMCMMFPFLYPCNHHAPGGG-NH N N NH
H
NH HN
HN
O N N~
I I
OH OH
0
NH
0
O
HN
I-fo
AC-CSFFPCWRIDRFGYCHANAPGGG-NH NH
CONH2
Ac-GSPEMCMMFPFLYPCNHHAPGGGK {PnAO6-Glut-K[Ac-
GSFFPCWRIDRFGYCHANAPGGGKJJ-Glut]-NH2}-NH2 : D28 (A heterodimeric c-
Met binder)
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0 O 0
H
Ac-CSFFPCWRIDRFGYCHANAPCGG-NH O N N~
H NH
NH HN
O >(
N N)
On OH
0
NH
0
HN 0
0
I-flo
Ac-AQEWEREYFVDGFWGSWFGIPHGGG44H NH
CONH2
Ac-GSFFPC WRIDRFGYCHANAPGGGK {PnAO6-Glut-K[Ac-
AQEWEREYFVDGFWGSWFGIPHGGGK(JJ-Glut)-NHZ]}-NH2 : D29 (A
heterodimeric c-Met binder)
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ONHZ O O O
H
Ac-GSPEMCMMFPFLYPCNHHAPGGG-NH H N NH
NH HN
HN
C ~
OH OH
O
NH
0
HN O
O
IYO
Ac-GDYSECFFEPDS FEVKCYDRDPGGG-NH NH
CONH2
Ac-GSPEMCMMFPFLYPCNHHAPGGGK {PnAO6-Glut-K[Ac-
GDYSECFFEPDSFEVKCYDRDPGGGK(JJ-Glut)-NH2]}-NH2 : D30 (A heterodimeric
C-Met binder)
For the preparation of the dimer D5, after the coupling reaction of the
individual
peptides, 50 d of hydrazine was added to the reaction mixture (to expose the
lysine N'-
amino group) and the solution was stirred for 2 min. The reaction mixture was
diluted
with water (1.0 mL) and the pH was adjusted to 2 with TFA. This was then
purified by
the method described above.
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Synthesis of D27
Scheme 1- Synthesis of Compound 2 (P12-Q)
Ac-AGPTWCEDDWYYCWLFGTGGGK(iV-Dde)-NH Resin (Pal-PEG-PS)
1
1. 10% hydrazine in DMF (2 x 10 min)
2. Fmoc-Lys(iV-Dde)-OH/HOBt/DIC/DMF
3. 20% piperidine in DMF (2 x 10 min)
4. Fmoc-NH-JJ-Biotin/HOBt/DIC/DMF
5. NH2NH2/DMF (10%, 2 x 10 min)
6. Fmoc-Ser(GaINAc(Ac)3-ar.-D)-OH/HATU/DIEA/DMF
7. 20% piperidine in DMF (2 x 10 min)
8. Fmoc-Gly-OH/HOBt/DIC/DMF
9. 20% piperidine/DMF (2 x 10 min)
10. Fmoc-Ser(GaINAc(Ac)3 (x-D)-OH/HATU/DIEA/DMF
11. 20% piperidine in DMF (2 x 10 min)
12. Reagent B
13. DMSO/aq. N-Methylglucamine/pH 8/air/2 days
ONH2
O H
N-JJ-Biotin
Ac-AGPTWCEDDWYYCWLFGTGGG H""..
N
H
HNC
2 Ser(GaINAc(Ac)3-alpha-D)
GIy.
Ser(GaINAc(Ac)3-alpha-D)
H2N
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Scheme 2 - Synthesis of Compound 4 (P6-F-Q)
Ac-VCWEDSWGGEVCFRYDPGGGK(iV-Dde)-NH Resin (Pal-PEG-PS)
3
1. 10% hydrazine in DMF (2 x 10 min)
2. Fmoc-Ser(GaINAc(Ac)3-a-D)-OH/HATU/DIEA/DMI
3. 20% piperidine in DMF (2 x 10 min)
4. Fmoc-Gly-OH/HOBt/DIC/DMF
5. 20% piperidine in DMF (2 x 10 min)
6. Fmoc-Ser(GaINAc(Ac)3 a-D)-OH/HATU/DIEA/DMI
7. 20% piperidine in DMF (2 x 10 min)
8. reagent B
9. DMSO/N-methylglucamine/pH 8/air/2 days
Ac-VCWEDSWGGEVCFRYDPGGG-N\ ,CONH2
4
HNC
)Ser(GaINAc(Ac)3alpha-D)
GIy
H N',Ser(GaINAc(Ac)3alpha-D)
z
Synthesis of 1 and 3
Synthesis of the monomers were carried out as described in Method 5 on a 0.25
mmol scale employing as the starting resin Fmoc-GGGK(iV-Dde)NH-PAL-PEG-PS
resin. The peptide resin was washed and dried before cleavage or further
derivatization
by automated or manual methods.
Procedure for Synthesis of 2 and 4
Appendage of Biotin-JJ, Lysyl, Glycyl and Serinyl(Ga1NAc(Ac)3-a-D moieties
onto 1 and 3 was done by manual SPPS such as described in Method 6 and Method
8.
The coupling of amino acids was carried out in DMF using HOBt/DIC activation
(except for Ser(Ga1NAc(Ac)3-a-D). Fmoc removal was carried out with 20%
piperidine
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in DMF. All couplings were 5-16 hours duration. After each coupling, the
completion
was confirmed by the Kaiser test. In the case of Ser(Ga1NAc(Ac)3-a-D, the
coupling
was performed in DMF employing HATU/DIEA as the coupling agent. In cases where
the Kaiser test indicated unreacted amino groups the coupling was repeated.
Removal of
the N-terminal Fmoc group and cleavage from resin was performed. The crude
peptide
was precipitated in ether and washed twice by ether and dried under vacuum.
The linear
crude peptide was directly cyclized by dissolving the peptide in DMSO (40
mg/mL).
The pH of the solution was adjusted to 8 by addition of aqueous N-
methylglucamine
and the solution was was stirred in air for 48h at room temperature. The
peptides were
then purified employing gradient HPLC as described in Method 1 employing a
Waters-
YMC C-18 ODS preparative column (250 mm x 4.6 mm i.d.). The pure product-
containing fractions were combined and lyophilized to provide the needed
peptides.
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Scheme 3 - Synthesis of D27 (6)
0 0
DMFIDIEA
4 + v ~I i{
O 0 0 0
Ac-VCWEDSWGGEVCFRYDPGGG--N ICONH2
GI compounds/DII:A/DMF
)Ser(GaINAc(Ac);alpha-D)
HN Ser(GaINAc(Ac);alpha-D) NH2NH2AWH (15% v/4)
j
O
p O
7
0 N.
J ONH2
Ac AGPTWCEDDWYYCWLFJJ-Biotin
GTGGG H
N
Ac-VCWEDSWGGEVCFRYDPGGG-lj CONH2 H
(GaINAc-alpha-D)Ser`NH Gly \
:i:)ser(Ga1NAc1Pha)
(GaINAc-alpha-D)Ser<HN N ~ -Ser(GaINAc-alpha-D)
H ""'~Y
O 0
6
Procedure: Synthesis of D27 - Compound 6
To a solution of glutaric acid bis-NHS ester (0.122 mmol, Pierce Scientific
Co.)
in anhydrous DMF was added dropwise a solution of 4 in DMF (40 mg, 0.0122
mmol).
DIEA was added to neutralize the trifluoroacetic acid bound to the peptide and
N-
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hydroxysuccinimide formed during the reaction. This 0.7 mL solution was
stirred for
4 h. The reaction was monitored by HPLC and mass spectroscopy. DMF was removed
under vacuum. The excess diester was removed by addition of ethyl acetate
which
precipitated the peptide-monoester 5 while dissolving glutaric acid bis-NHS
ester. The
mixture was centrifuged and the liquid portion decanted. This was repeated
twice. The
residue was kept under vacuum for 10 min. The residue was dissolved in DMF and
mixed with a solution of 2 (37 mg, 0.009 mmol) in DMF (pH 7). It was stirred
at
ambient temperature for 16 h. The volatiles were removed under high vacuum and
the
acetate functions were removed by treatment of the residue with I mL of
hydrazine/MeOH (15/85, v/v) solution with stirring for 2.5 h at ambient
temperature.
Acetone was added to quench the excess of hydrazine and the volatiles were
removed
under vacuum. The resulting residue was dissolved in DMSO and purified by
preparative HPLC as described above to provide 9 mg of the pure material.
Analytical Data
The HPLC analysis data and mass spectral data for the dimeric peptides D1-5
and D8-30 identified above, as well as that for peptide components of dimer
D27, are
given in Table 6 below.
Table 6. Analytical Data for Homodimeric and Heterodimeric Peptide Constructs
Compound # Retention Mass Spectral data (API-ES, - ion)
Time
(System)
D1 8.98 min. (A) 1987.7 (M-3H)/3, 1490.6 (M-4H)/4, 1192.3 (M-5H)/5
D2 16.17 min (B) 2035.3 (M-3H)/3, 1526.1 (M-4H)/4, 1220.7 (M-5H)/5
D3 8.74 min (C) 1933.6 (M-3H)/3, 1449.9 (M-4H)/4, 1159.4 (M-5H)/5
D4 10.96 min (D) 2032.8 (M-3H)/3
D5 6.57 min (E) 1816.2 (M-3H)/3, 1361.8 (M-4H)/4, 1089.4 (M-5H)/5,
907.7 (M-6H)/6
D8 4.96 min; (F) 2379.3 [M-3H]/3
D9 5.49 min; (G) 2146.4 [M-3H]/3
D10 5.44 min; (H) 2082.7 [M-3H]/3,1561.7 [M-4H]/4, 1249.1 [M-5H]/5,
1040.7 [M-6H ]/6
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D11 7.23 min; (E) 2041.8 [M-3H]/3, 1531.1 [M-4H]/4, 1224.6 [M-5H]/5
D12 5.84 min; (H) 1877.1 [M-3H]/3, 1407.6 [M-4H]/4, 1125.9 [M-5H]/5,
938.1 [M-6H ]/6.
D13 5.367 min; (E) 1965.3 [M-3H]/3, 1473.8 [M-4H]/4, 1178.8 [M-5H]/5,
982.2 [M-6H ]/6
D14 4.78 min; (I) 2275.0 [M-3H]/3, 1362.8 [M-5H]/5
D15 5.41 min; (H) 1561.3 [M-4H]/4, 1249.1 [M-5H]/5, 1040.8 [M-6H]/6,
891.8 [M-7H ]/7.
D16 5.44 min; (J) 2150.8 [M-3H]/3, 1613.1 [M-4H]/4, 1289.9 [M-5H]/5,
1074.8 M-6H /6, 920.9 (M-7H]/7.
D17 4.78 min; (K) 1789.4 [M-3H]/3, 1347.7 [M-4H]/4.
D18 4.74 min; (L) 2083.1 [M-3H]/3, 1562.7 [M-4H]/4, 1249.5 [M-5H]/5.
D19 7.13 min; (0) 1891.9 [M-3H]/3,1418.4 [M-4H]/4,1134.8 [M-5H]/5,
945.5 (M-6H]/6.
D20 9.7 min; (P) 2700.4 [M-2H]/2,1799.3[M-3H]/3
D21 6.1 min; (P) 2891.3 [M-2H]/2, 1927.2[M-3H]/3, 1445.1 [M-4H]/4,
1155.8 [M-5H ]/5.
D22 6.23 min; (Q) 1994.4 [M-3H]/3, 1495.7 [M-4H]/4, 1196.3 [M-5H]/5
D23 7.58 min; (J) 1854.4 [M-3H]/3, 1390.8 [M-4H]/4, 1112.7 [M-5H]/5,
927 [M-6H]/6
D24 8.913 min; (R) 1952.1 [M-3H]/3, 1463.4 [M-4H]/4, 1171.1 [M-5H]/5,
975.3 [M-6H ]/6
D25 5.95 min; (E) 1954.9 [M-3H]/3, 1466.1 [M-4H]/4, 1172.4 [M-5H]/5,
976.8 M-6 /6.
D26 6.957 min; (S) 1759.1 [M-3H]/3, 1319.6 (M-4H]/4,1055.1 [M-5H]/5
D27 5.50 min; (M) 2317.6 [M-3H]/3, 1737.2[M-4H]/4, 1389.3[M-5H]/5,
1157.7 [M-6H ]/6.
D28 4.89 min; (N) 6229 [M+H]
D29 5.01 min; (N) 2258.1 [M-3H + TFA]/3
D30 4.35 min; (N) 2176.0 [M-3H]/3, 1631.5 [M-4H]/4, 1302.6 [M-
5H /5, 1087.7 -6H /6, 932.1 [m-min
P 12-Q 7.4 min (T) 2041.3 [M - 2H]/2
P6-F-Q 8.0 min (T) 1636.3 [M - 2H]/2
HPLC Analysis Systems
System A: Column: YMC C-4 (4.6 x 250 mm); Eluents: A: Water (0.1% TFA),
B: Acetonitrile (0.1 % TFA); Elution: Initial condition, 25% B, Linear
Gradient 25-60%
B in 10 min; Flow rate: 2.0 ml/ min; Detection: UV @ 220 nm.
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System B: Column: YMC C-4 (4.6 x 250 mm); Eluents: A: Water (0.1% TFA),
B: Acetonitrile (0.1 % TFA); Elution: Initial condition, 25 % B, Linear
Gradient 25-60%
B in 20 min; Flow rate: 2.0 mL/min; Detection: UV @ 220 nm.
System C: Column: YMC C-4 (4.6 x 250 mm); Eluents: A: Water (0.1% TFA),
B: Acetonitrile (0.1% TFA); Elution: Initial condition, 30% B, Linear Gradient
30-60%
B in 10 min; Flow rate: 2. 0 mU min; Detection: UV @ 220 nm.
System D: Column: YMC C-4 (4.6 x 250 mm); Eluents: A: Water (0.1% TFA),
B: Acetonitrile (0.1% TFA); Elution: Initial condition, 20% B, Linear Gradient
20-60%
B in 10 min; Flow rate: 2. 0 mU min; Detection: UV @ 220 nm.
System E: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water
(0.1 %TFA), B: Acetonitrile (0.1 %TFA) : Elution: Initial condition, 10 % B,
Linear
Gradient 10-60 % B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 rim.
System F: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water
(0.1%TFA), B: Acetonitrile (0.1%TFA) : Elution: Initial condition, 30 % B,
Linear
Gradient 30-70 % B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 rim.
System G: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water
(0.1%TFA), B:Acetonitrile (0.1%TFA) : Elution: Initial condition, 30 % B,
Linear
Gradient 30-75 % B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 nm.
System H: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water
(0.1 %TFA), B: Acetonitrile (0.1 %TFA) : Elution: Initial condition, 20 % B,
Linear
Gradient 20-52 % B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 Mn.
System I: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water (0.1%TFA),
B: Acetonitrile (0.1%TFA) : Elution: Initial condition, 10 % B, Linear
Gradient 10-65
% B in 10 min; Flow rate: 3.0 mUmin; Detection: UV @ 220 run.
System J: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water (0.1%TFA),
B: Acetonitrile (0.1%TFA) : Elution: Initial condition, 20 % B, Linear
Gradient 20-60
% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 nm.
System K: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water
(0.1 %TFA), B: Acetonitrile (0.1 %TFA) : Elution: Initial condition, 5 % B,
Linear
Gradient 5-60 % B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 nm.
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System L: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water
(0.1 %TFA), B: Acetonitrile (0.1 %TFA) : Elution: Initial condition, 5 % B,
Linear
Gradient 5-65 % B in 10 min; Flow rate: 3.0 mUmin; Detection: UV @ 220 nm.
System M: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water
(0.1 %TFA), B: Acetonitrile (0.1 %TFA) : Elution: Initial condition, 15 % B,
Linear
Gradient 15-50 % B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 nm.
System N: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water
(0.1%TFA), B: Acetonitri le (0.1%TFA) : Elution: Initial condition, 10 % B,
Linear
Gradient 20-80 % B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 nm.
System 0: Column: YMC-C18, 4.6 x 250 mm; Eluents:A: Water (0.1%TFA),
B: Acetonitrile (0.1%TFA) : Elution: Initial condition, 30 % B, Linear
Gradient 30-60
% B in 10 min; Flow rate: 2.0 mUmin; Detection: UV @ 220 nm.
System P: Column: YMC-C18, 4.6 x 250 mm; Eluents:A: Water (0.1%TFA),
B: Acetonitrile (0.1 %TFA) : Elution: Initial condition, 20 % B, Linear
Gradient 20-80
% B in 20 min; Flow rate: 2.0 mL/min; Detection: UV @ 220 nm.
System 0: Column: YMC-C18, 4.6 x 250 mm; Eluents:A: Water (0.1%TFA),
B: Acetonitrile (0.1%TFA) : Elution: Initial condition, 20 % B, Linear
Gradient 20-60
% B in 6 min; Flow rate: 2.0 mL/min; Detection: UV @ 220 nm.
System R: Column: YMC-C18, 4.6 x 250 mm; Eluents:A: Water (0.1%TFA),
B: Acetonitrile (0.1 %TFA) : Elution: Initial condition, 25 % B, Linear
Gradient 25-60
% B in 10 min; Flow rate: 2.0 mL/min; Detection: UV @ 220 nm.
System S: Column: YMC-C18, 4.6 x 100 mm;; Eluents:A: Water (0.1%TFA),
B: Acetonitrile (0.1%TFA) : Elution: Initial condition, 10 % B, Linear
Gradient 10-60
% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 nm.
System T: Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: Water
(0.1%TFA), B: Acetonitrile (0.1%TFA) : Elution: Initial condition, 15 % B,
Linear
Gradient 15-50 % B in 8 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 nm.
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EXAMPLE 10
Competition with 125I-VEGF for binding to KDR on HUVECs and KDR-transfected
cells
The following experiment assessed the ability of KDR-binding polypeptides,
homodimers and heterodimers of the invention to compete with125I-labeled VEGF
for
binding to KDR expressed by transfected 293H cells.
Protocol:
293H cells were transfected with the KDR cDNA or mock-transfected by
standard techniques described herein. The cells were incubated with 125I-VEGF
in the
presence or absence of competing compounds (at 10 M, 0.3 M, and 0.03 M).
After
washing the cells, the bound radioactivity was quantitated on a gamma counter.
The
percentage inhibition of VEGF binding was calculated using the formula [(Y1-
Y2)x100/Y1], where Y1 is specific binding to KDR-transfected 293H cells in the
absence peptides, and Y2 is specific binding to KDR-transfected 293H cells in
the
presence of peptide competitors. Specific binding to KDR-transfected 293H
cells was
calculated by subtracting the binding to mock-transfected 293H cells from the
binding
to KDR-transfected 293H cells.
Results:
As shown in FIG. 14, all of the KDR-binding compounds assayed were able to
compete with 125I-VEGF for binding to KDR-transfected cells. The heterodimer
(D1)
was clearly the most effective at competing with 125I-VEGF, even over the two
homodimers (D2 and D3), confirming the superior binding of Dl.
EXAMPLE 11
Receptor Activation Assam
The ability of KDR-binding multimeric constructs, including heteromultimers of
the invention, to inhibit VEGF induced activation (phosphorylation) of KDR was
assessed using the following assay (see also Example 4 above).
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Protocol:
Dishes of nearly confluent HUVECs were placed in basal medium lacking
serum or growth factors overnight. The next day, the dishes in group (c) below
were
pretreated for 15 min in basal medium with a KDR-binding peptide, then the
cells in the
dishes in groups (a), (b), and (c) were placed in fresh basal medium
containing:
(a) no additives (negative control),
(b) 5 ng/mL VEGF (positive control), or
(c) 5 ng/mL VEGF plus the putative competing/inhibiting peptide.
After 5 min of treatment, lysates were prepared from the dishes.
Immunoprecipitated
KDR from the lysates was analyzed sequentially by inununoblotting for
phosphorylation with an anti-phosphotyrosine antibody, and for total KDR with
an anti-
KDR antibody (to control for sample loading).
Results:
As shown in FIG. 15, D I was able to completely block the VEGF-induced
phosphorylation of KDR in HUVECs at 10 nM. More than half of the
phosphorylation
was inhibited by the compound at 1 nM. Homodimers D2 and D3, made up of the
two
individual binding moieties that are contained in D1, had no effect on
phosphorylation
at up to 100 nM, demonstrating the benefit achievable by using an appropriate
heterodimer to block a receptor-ligand interaction. In multiple experiments,
the ICSO for
D1 in this assay varied between 0.5 and 1 nM. A different heterodimer
containing
unrelated binding sequences, D31 (structure shown below), had no effect on
phosphorylation at 100 nM in spite of it's high binding affinity (11 nM for
KDR by
Biacore), suggesting that the choice of KDR-binding moieties is important when
constructing a multimer to compete with VEGF for binding to KDR. Even though
the
affinity of D 1 for KDR is 10-fold higher than that of D2 (by SPR analysis),
it's IC50 in
the activation assay is at least 100-fold lower, suggesting that targeting two
distinct
epitopes on KDR with a single binding molecule can generate greater steric
hindrance
than a molecule with similar affinity that only binds to a single epitope on
KDR.
Similarly, it should be pointed out that the two KDR-binding moieties within D
1 when
tested as monomeric free peptides (P12-XB and P6-D) in the receptor activation
assay
had IC50s of 0.1 and 1 micromolar respectively, which is 100 to 1000-fold
higher than
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the IC50 for Dl in the assay and 14 to 30-fold higher than the KDS for the
fluoresceinated derivatives of the monomeric peptides. Thus, creating a dimer
containing two peptides with weak VEGF-blocking activity has resulted in a
molecule
with very potent VEGF-blocking activity that goes well beyond the increased
binding
affinity of D1.
Ac--A Q E P E G Y A Y W E V I T L Y H E E D G D G G-N 0H
~~\/N,
HN JR~
IN
NH,
AC--A Q A F P R F G G D D Y W I Q Q Y L R Y T D G G-
D31: Ac-AQEPEGYAYWEVTTLYHEEDGDGGK(Ac-
AQAFPRFGGDDYWIQQYLRYTDGGK(-
(O=)C(CH2CH2O)4CH2CH2NHC(=O)CH2CH2C(CH3)=NOCH2C(=O)NH(CH2CH2O)4
CH2CH2C(=O)-)NH2)NH2
EXAMPLE 12
Migration Assay
The following experiment assessed the ability of D1, a heteromultimer of the
invention, to block the VEGF-induced migration of HUVECs in culture.
Protocol:
Serum-starved HUVECs were placed, 100,000 cells per well, into the upper
chambers of BD Matrigel-coated FluoroBlok 24-well insert plates (#354141).
Basal
medium, containing either nothing or different attractants such as VEGF
(10ng/mL) or
serum (5% FBS) in the presence or absence of potential VEGF-
blocking/inhibiting
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compounds, was added to the lower chamber of the wells. After 22 hours,
quantitation
of cell migration/invasion was achieved by post-labeling cells in the insert
plates with a
fluorescent dye and measuring the fluorescence of the invading/migrating cells
in a
fluorescent plate reader. The VEGF-induced migration was calculated by
subtracting
the migration that occurred when only basal medium was placed in the lower
chamber
of the wells.
Results:
VEGF induced a large increase in endothelial cell migration in the assay,
which
was potently blocked by Dl. At 5 nM D1, the VEGF-stimulated endothelial cell
migration was 84% blocked (see FIG. 16). At 25 nM D1, this migration was
almost
completely blocked. In other experiments, a known KDR inhibitor, SU-1498 ((E)-
3-
(3,5-Diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-
propyl)aminocarbonyl]acrylonitrile]was tested in the assay. SU1498 at 3
micromolar
did not block the VEGF-induced migration as well as Dl (47% blocked at 3
micromolar). D7 (structure provided below), at 50 nM, also produced
essentially
complete inhibition of the migration stimulated by VEGF. Serum was a very
powerful
attractant in the assay when used in place of VEGF, but its effect was not
significantly
diminished by Dl, indicating that D1 specifically inhibits endothelial
migration induced
by VEGF.
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Ac-A G P K W C E E D W Y Y C M I T G T G G G-N_ JL--NH2
N
0 ?r-H
0
O
NH
AC-0 D S R V C W E 0 S W G G E V C F R Y D P G 6 G-H NH2
0
D7: Ac-AGPKWC*EEDWYYC*MITGTGGGK(Ac-
GDSRVC* WEDSWGGEVC*FRYDPGGGK(-
(0=)C(CH2CH2O)4CH2CH2NHC(=O)(CH2)3C(=O)NHCH2CH2(OCH2CH2)4C(=O)-
)NH2)NH2
EXAMPLE 13
The following experiments describe methods used to prepare Tc, In, Lu, and I-
labelled compounds.
Preparation of 99mTc-P12-P
SnCI2'2H20 (20 mg) was dissolved in 1 mL of 1 N HCI, and 10 L of this
solution was added to 1 mL of a DTPA solution that was prepared by dissolving
10 mg
of Ca Nat DTPA*2.5 H2O (Fluka) in 1 mL of water. The pH of the stannous DTPA
solution was adjusted to pH 6-8 using IN NaOH. 50 g of P12-P (Ac-
AGPTWC*EDDWYYC*WLFGTGGGK(PnAO6-NH-(O=)C(CH2)3C(=O)-JJ)-NH2) in
50 L of 10% DMF was mixed with 20 gL of 99riTcO4 (2.4 to 4 mCi, Syncor),
followed
by 100 L of the stannous Sn-DTPA solution. After 30 minutes at RT, the
radiochemical purity (RCP) was 93%. The product was purified on a Supelco
Discovery C16 amide column (4 x 250 mm, 5 um pore size) eluted at a flow rate
of 0.5
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mL'min using an aqueous/organic gradient of lg/L ammonium acetate in water (A)
and
acetonitrile (B). The following gradient was used: 30.5% B to 35% B in 30
minutes,
ramp up to 70% B in 10 min. The compound, which eluted at a retention time of
21.2
minutes was collected into 500 L of 50 mM citrate buffer (pH 5.2) containing
1%
ascorbic acid and 0.1% HSA, and acetonitrile was removed using a Speed Vacuum
(Savant). After purification, the compound had an RCP of >98%.
Preparation of "' 1n-P 12-XDT
50 pg of P12-XDT (Ac-AGPTWCEDDWYYCWLFGTJK(JJ-DOTA)-NH2) in
50 .iL of 10% DMF was mixed with' 111nC13 (50 .LL, 400 pCi, Mallinckrodt) and
100
L of 0.2M ammonium acetate or citrate buffer at a pH of 5.3. After being
heated at
85 C for 45 minutes, the radiochemical purity (RCP) ranged from 44% to 52.2%
as
determined using HPLC. The "'In-labeled compound was separated from unlabeled
ligand using a Vydac C18 column (4.6 x 25 cm, 5 micron pore size) under the
following
conditions: aqueous phase, I g/L ammonium acetate (pH 6.8); organic phase,
acetonitrile. Gradient: 23% org. to 25% org. in 30 minutes, up to 30% org. in
2
minutes, hold for 10 minutes. The compound, which eluted at a retention time
of 20.8
min, was collected into 200 pL of 50 mM citrate buffer (pH 5.2) containing 1%
ascorbic
acid and 0.1 % HSA, and the acetonitrile was removed using a Speed Vacuum
(Savant).
After purification the compound had an RCP of >93%.
Preparation of111 In-D4
A histidine buffer was prepared by adjusting a 0.1M solution of histidine
(Sigma) to pH 6.25 with concentrated ammonium hydroxide. Ammonium acetate
buffer was prepared by adjusting a 0.2 M solution of ammonium acetate (99.99%,
Aldrich) to pH 5.5 using concentrated HCl (J. T. Baker, Ultra Pure). High
purity
111InC13 (100 p.L, 1.2 mCi, Mallinckrodt) was added to D4 (200 Pg in 200 of
50% DMF,
10% DMSO, 20% acetonitrile and 20% water), followed by addition of 300 .tL of
histidine buffer. The final pH was 5.5. After incubation of the reaction
mixture at 85 C
for 45 minutes, the RCP was 20%.
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Alternatively, I1'InC13 provided with a commercially available OctreoScanTM
Kit
(134 L, 0.6 mCi, Mallinkrodt) was added to D4 (135 g) in 162 gL of 0.2M
ammonium acetate buffer. The final pH was 5.5. After incubation of the
reaction
mixture at 85 C for 45 min. the RCP was 20%.
Preparation of 125I- D5
D5 (200 g), in 30 L of DMF that had been previously adjusted to pH 8.5-9.0
using diisopropyl amine, was added to 1 mCi of mono-iodinated 125I Bolton-
Hunter
Reagent (NEX-120, Perkin-Elmer) that had been evaporated to dryness. The vial
was
shaken and then incubated on ice for 30 minutes with occasional shaking. After
this
time, the RCP was 23%. 125I-D5 (shown below) was purified by HPLC at a flow
rate of
1 mL/min using a Vydac C18 column (4.6 x 250 mm, 5 micron pore size) under the
following conditions. Aqueous phase: 0.1% TFA in water; organic phase: 0.085%
TFA
in acetonitrile. Gradient: 30% org. to 36% org. in 30 minutes, up to 60% org.
in 5
minutes, hold for 5 minutes. The compound was collected into 200 L of 50 mM
citrate buffer (pH 5.2) containing I% ascorbic acid and 0.1 % HSA.
Acetonitrile was
removed using a Speed Vacuum (Savant). The resulting compound had an RCP of
97%.
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ONH2 51
Ac---AGPTWCEDDWYYCWLFGTGGG /~^N `H
H H HN O OH
O
NH
ry
O
HNO
AC- VCWEDSWGGEVCFRYDPGGG-NNH
CONH2
125 I-D5
Preparation of 77Lu-Dl 1
D 11 (5 iiL of a -1 gg/.tL solution in 0.05N NH4OH/10% EtOH) was added to a
glass insert microvial containing 80 tL of 0.2M NaOAc buffer, pH 5.6. Enough
'77 Lu
was added to bring the ligand:Lu ratio to 2:1 (1-5 mCi). The vial was crimp-
sealed
and heated at 100 C for 15-20 minutes, cooled for 5 minutes, and treated with
3 L of
1% Na2EDTA.2H20 in H20. The entire reaction mixture was injected onto a
Supelco
Discovery RP Amide C16 column (4 mm x 250 mm x 5 m). The following HPLC
conditions were used: Column temperature= 50 C, Solvent A = H2O w/ 0.1% TFA,
Solvent B = ACN w/ 0.085% TFA, gradient 0.6/0.25 mL/min A/B at t = 0 minutes
to
0.5/0.4 mL/min A/B at t = 60 minutes. The retention time for Dl l was -40
minutes;
that of 171Lu-D11 1334 was -42 minutes. The radioactive peak was collected
into 0.7
ml of 0.05M citrate buffer, pH 5.3 containing 0.1 % Human Serum Albumin
Fraction V
and 1.0% Ascorbic Acid, and the mixture was spun down in a Savant Speed Vac to
remove organic solvents. Radiochemical purities of greater than 80% were
obtained.
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Preparation of 99mTc-D 12
SnC122H20 (20 mg) was dissolved in 1 mL of I N HCI, and 10 L of this
solution was added to I mL of a DTPA solution that was prepared by dissolving
10 mg
of Ca Nat DTPA*2.5 H2O (Fluka) in 1 mL of water. D12 (100 g in 100 L of 50%
DMF) was mixed with 75 gL of 0.1 M, pH 9 phosphate buffer and 60 L of
99niTcO4
(2.4 to 4 mCi, Syncor), followed by 100 L of the stannous Sn-DTPA solution.
After 10
min at 40 C, the radiochemical purity (RCP) was 16%. The product was purified
on a
Supelco Discovery C16 amide column (4 x 250 mm, 5 urn pore size) eluted at a
flow
rate of 0.7 mL/min using an aqueous/organic gradient of 0.1 % TFA in water (A)
and
0.085% TFA in acetonitrile (B). The following gradient was used: 30% B to 42%
B in
36 min, ramp up to 70% B in 10 min. The compound, which eluted at a retention
time
of 37.1 min. was collected into 500 L of 50 mM citrate buffer (pH 5.2)
containing
0.2% HSA, and acetonitrile was removed using a Speed Vacuum (Savant). After
purification, the compound had an RCP of >90%.
Preparation of 99rtiTc-D14
SnC12'2H20 (20 mg) was dissolved in 1 mL of 1 N HCI, and 10 gL of this
solution was added to I mL of a DTPA solution that was prepared by dissolving
10 mg
of Ca Nat DTPA'2.5 H2O (Fluka) in I mL of water. D14 (100 g in 100 L of 50%
DMF) was mixed with 50 L of 99niTcO4 (6 mCi, Syncor) and 125 gL of 0.1 M
phosphate buffer, pH 9 followed by 100 L of the stannous Sn-DTPA solution.
After 15
min at 40 , the radiochemical purity (RCP) was 21%. The product was purified
on a
Vydac peptide C18 column (4.6 x 250 mm) eluted at a flow rate of 1 mL/min
using an
aqueous/organic gradient of 0.1% TFA in water (A) and 0.085% TFA in
acetonitrile
(B). The following gradient was used: 30% B to 45% B in 40 min. The compound,
which eluted at a retention time of 34.9 min., was collected into 500 L of 50
mM
citrate buffer (pH 5.3) containing 0.2% HSA, and acetonitrile was removed
using a
Speed Vacuum (Savant). After purification, the compound had an RCP of 92.5 %.
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EXAMPLE 14
Binding of'25I-labeled heteromultimers of the Invention to KDR-Transfected
Cells
An experiment was performed to test the ability of 125I-labeled D5 to bind to
KDR-transfected 293H cells. In this experiment, different amounts of 125I-
labeled D5
(1-4 Ci/ml, labeled with 125I-Bolton-Hunter reagent and HPLC-purified) were
incubated with mock & KDR-transfected 293H cells in 96-well plates for I hr at
room
temperature. Binding was performed with and without 40% mouse serum to
evaluate
the serum effect on binding to KDR-transfected cells. After washing away the
unbound
compound, the cells in each well were lysed with 0.5 N NaOH and the lysates
were
counted with a gamma counter.
The results of this experiment are summarized in FIG. 17 and FIG. 18. It is
clearly evident from these results that 125I-labeled D5 is able to
specifically bind to
KDR-transfected cells and its binding is not affected by the presence of 40%
mouse
serum. Somewhat more binding to KDR-transfected cells was observed in the
absence
of serum as compared to binding in the presence of 40% mouse serum. However,
the
binding of 125I-D5 to mock-transfected cells was also increased by about the
same
extent when serum was omitted during the assay, indicating that the increased
binding
in the absence of serum was non-specific (FIG. 17). Specific binding to KDR-
transfected cells (after subtracting binding to mock-transfected cells) looked
almost
identical with or without mouse serum (as shown in FIG. 18). In this
experiment, 10-
14% of the total CPM added were specifically bound to KDR-transfected cells
(data not
shown).
EXAMPLE 15
A peptide heterodimer (D6, shown below) was prepared as previously described
in Example 9 using glutaric acid bis N-hydoxysuccinimidyl ester. The
heterodimer was
tested for binding to KDR-Fc using Biacore and an affinity contant was
determined as
follows.
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O NH2
Ac-GDSRVCWEDSWGGEVCFRYDPGGG-N- "-' NH
H (-1-O
0
O
HN~
r_1__O
O1
O
D
HN
O
O
HD
H
OD
O
1-f-O
HD
OJ
O
AC-AGPTWCEDDWYYCWLFGTGGG-N NH
O N
Molecular Weight =6030.58
Exact Mass =6024
Molecular Formula =C H3mN66086S4
Molecular Composition =C 53.58% H 6.15% N 15.33% 0 22.82% S 2.13%
Peptide Heterodimer: D6
Three densities of KDR-Fc were cross-linked to the dextran surface of a CM5
sensor chip by the standard amine coupling procedure (0.5 mg/mL solution
diluted
1:100 or 1:50 with 50 mM acetate, pH 6.0). Flow cell 1 was activated and then
blocked
to serve as a reference subtraction. Final immobilization levels achieved:
RL Fe 2 KDR-Fc = 1607
RL Fc 3 KDR-Fc = 3001
RL Fc 4 KDR-Fc = 6319
Experiments were performed in PBS buffer (5.5 mM phosphate, pH 7.65, 0.15 M
NaCI)
+ 0.005% P-20 (v/v)). D6 was diluted to 250nM in PBS and serial dilutions were
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performed to produce 125, 62.5, 31.3 15.6, 7.8, and 3.9nM solutions. All
samples were
injected in duplicate. For association, peptides were injected at 20 ILL/min
for 12.5
minutes using the kinject program. Following a 10 minute dissociation, any
remaining
peptide was stripped from the KDR surface with a quickinject of 50mM NaOH + IM
NaCl for 12s at 75 ILL/min. Sensorgrams were analyzed using BlAevaluation
software
3.1 and a hyperbolic double rectangular regression equation in SigmaPlot 6Ø
Heterodimer steady state binding affinities (KDAV) were determined at all
three KDR
immobilization densities (Table 7).
Table 7. Summary of Parameters
KD1(nM) RMax1 KDAV (nM) RMaXAV R2*
vs. 1600RU 46 13.1 1.5 12.6 0.995
6
vs. 3000RU 25.5 21.2 0.665 22.7 0.991
vs. 6000R17 61.3 0.662 62.2 0.993
From this data, it appears that at the higher immobilization densities, the
heterodimer
binds KDR with a sub-nanomolar affinity (-P0.6 nM).
To assess the in-vivo clearance of this peptide heterodimer, a small amount of
material was iodinated using iodogen and Na125I according to standard
protocols
(Pierce). Radio iodination was done in the Radiation Safety lab, within the
designated
hood. One tube coated with the iodogen reagent was pre-wet with 1 mL of 25 mM
Tris,
0.4M NaCI, pH 7.5. This was discarded and 100 l of the same buffer added.
Using a
Hamilton syringe 11 L of the 125I-Nal was transferred to the reaction tube.
Based on
original estimates of the Na125I concentration of 143.555 mCi/ml, the 11 L
should
contain about 1.5 mCi. No dose calibrator was in the room. After addition, the
sample
was swirled and set in a lead pig to incubate for 6min with a swirl every 30
sec. After 6
min, the entire sample was transferred to the peptide that was in an Eppendorf
tube.
The sample was swirled and set to incubate for 8 min, with a swirl every 30
sec. After
8 min the reaction was quenched (terminated) with tyrosine (IOmg/mL, a
saturated
solution), allowed to sit for 5 min, and then 2 L was removed for a standard.
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For purification a 10 mL column of the D-salt polyacrylamide 1800 was used to
separate the labeled peptide from labeled tyrosine. The column was first
washed with
mL saline, then 5 mL of 25 mM Tris, 0.4M NaCl, pH 7.5 containing 2.5% HSA to
block non-specific sites. After the HSA buffer wash, the column was eluted
with 60mL
5 of the 25 mM Tris, 0.4 M NaCl buffer, and the column was stored overnight at
4 C.
The labeled sample contained 1.355 mCi, as determined by the dose calibrator.
The 2
l sample that was removed as a standard contained 8.8 Ci. The peptide sample
was
applied to the D-salt 1800 column and eluted with the Tris/ NaCl buffer, pH
7.5. The
flow was controlled by applying single 0.5ml aliquots for each fraction, #1-
14, and then
10 1.0 mL for fractions 25-43. The peak of activity in fractions # 9, 10, and
11, was
assumed to be the peptide. The radioactivity in 24 through -40 is likely the
labeled
tyrosine. From this purification, fractions #9-12 were pooled together and
used for the
subsequent clearance study (concentration of 125I-D6 in pool is 7.023 g/mL;
100 gL =
0.702 g with 8.6 G).
A total of 15 mice were injected with 100 gL 1251-D6 and mice (in sets of 3)
were sacrificed at the following time points: 0, 7, 15, 30, 90 minutes. Actual
activity
injected was about 6 Ci. With 6 Ci injected the corresponding peptide
administered
was - 0.5 g per animal. Once sacrificed, the counts were determined in a 50
L
plasma sample from each animal . For each set of three animals at each time
point, the
counts were averaged, converted to % injected dose/ml plasma (ID%/mL), and
then
plotted to assess the rate of clearance (FIG. 19). Then this data was fit to
either a 4 or 5
parameter equation to determine the biphasic half life of this molecule. The 4
parameter fit resulted in a T1,2 of 2.55 minutes and a T1/2p of 64.66 minutes.
The 5
parameter fit resulted in a Tln of 2.13 minutes and a T1,2p of 23.26 minutes.
Besides taking counts from the plasma samples, larger volumes of plasma were
taken from mice sacrificed at the 0, 30, and 90 minute time points. These
samples were
injected onto a Superdex peptide column (Pharmacia) coupled to a radioactivity
detector to assess the association of the peptide with serum proteins (FIG.
20). As
shown, the labeled peptide does associate with higher MW proteins, which could
explain its biphasic half life clearance behavior.
To help assess the potency of the peptide as an anti-angiogenesis inhibitor,
D6
was tested in an endothelial cell proliferation assay using HUVECs and BrdU
detection.
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Briefly, freshly isolated HUVECs (between p3 - 6) were cultured in RPMI + 10%
FCS
+1% antibiotics + 1% 1-glutamin + 0.4% BBE (bovine brain extract) and seeded
per
well, 5000-10000/well in100 L. The cells were allowed to recover for 24 h
prior to
use. Then the cells were washed with PBS twice and treated for 48 h with anti-
VEGF
antibody (positive control) or peptides A, B and C (0.1 and 10 ug/mL) in RPMI
+ 0.1 %
BSA + I% 1-glutamin. The following 6 variables were tested in 2 series (n=4):
Series I: w/o VEGF
Series II: w/ VEGF (30 ng/mL)
1. Standard medium: RPMI + 10% FCS +l % antibiotics + I% 1-glutamin +
0.4% BBE
2. Negative control 1: RPMI (true starvation)
3. Negative control 2: RPM! + 0.1 % BSA + 1 % 1-glutamin
4. Positive control: anti-VEGF 10 g/ml in RPMI + 0.1 % BSA + 1 % 1-
glutamin
5. 0.1 g/ml KDR peptides in RPMI + 0.1% BSA + 1% 1-glutamin
6. 10 pg/ml KDR peptides in RPMI + 0.1 % BSA + 1 % 1-glutamin
Protocol:
1) cells are incubated for 48 hours under various conditions
2) 10 L BrdU dilution (1:100 in EBM) is added to each well at 24 hours
3) incubate for another 24 hours (total 48 hrs)
4) aspirate the culture medium
5) add 100 L FixDenat to each well, incubate at room temperature for 30 min.
6) Discard FixDenat solution
7) 100 L antibody-solution (PBS I% BSA and anti-BrdU PO) added to each well.
8) incubate at RT for 90 minutes.
9) wash 3 times with PBS, 200pL/well, 5 min.
10) add substrate solution (TMB), incubate for 10-30 minutes
11) transfer all to a flexibel plate
12) stop the reaction by adding 2M H2S04, 25 L/well
13) read absorbance at 450 nm within 5 minutes after stopping the reaction.
Note: Background binding was determined by omitting the anti-BrdU antibody in
4
wells with control cells (cultured in complete medium; EBM + BulletKit) and by
complete labeling of cells that was not exposed to BrdU.
Of the two KDR binding constructs tested (D6 and P 12-G (Ac-
AGPTWC*EDDWYYC*WLFGT-GGGK-NH2)) as shown in Figure 21, D6 completely
inhibits HUVEC proliferation at 10 g/mL in the presence of VEGF, similar to
an anti-
VEGF antibody (positive control). PNC-1 (Ac-
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AEGTGDLHCYFPWVCSLDPGPEGGGK-OH) (SEQ ID NO: 29) was used as
negative control. On the other hand, P12-G (one of the peptides that make up
the
heterodimer) does not inhibit proliferation this assay at the highest
concentration tested
(10 gg/mL). As a result, the heterodimer clearly shows an enhanced ability to
compete
with VEGF in comparison with P12-G alone.
EXAMPLE 16
BlAcore Analysis-murine KDR-Fc Binding of Peptide Dimers Dl and D7
Using BlAcore, determine the binding constants of peptide dimers D1 (a
heterodimer of P 12-G and a truncated form of P6-D) and D7 (a heterodimer of
P5-D
and P6-D) for murine KDR-Fc.
Procedure
Three densities of recombinant murine KDR-Fc were cross-linked to the dextran
surface of a CM5 sensor chip by the standard amine coupling procedure (0.5
mg/mL
solution diluted 1:100 or 1:40 with 50 mM acetate, pH 6.0). Flow cell 1 was
activated
and then blocked to serve as a reference subtraction. Final immobilization
levels
achieved:
RL Fc 2 KDR-Fc = 2770
R,, Fc 3 KDR-Fc = 5085
R,, Fc 4 KDR-Fc = 9265
Experiments were performed in PBS buffer (5.5 mM phosphate, pH 7.65, 0.15
M NaCl) + 0.005% P-20 (v/v)). P12-G, run as a control, was diluted to 125nM in
PBS.
Serial dilutions were performed to produce 62.5, 31.3, 15.6, 7.8, and 3.9 nM
solutions.
D1 and D7 were diluted to 5OnM in PBS and serial dilutions were performed to
produce
25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39nM solutions. All samples were
injected in
duplicate. For association, peptides were injected at 30 gUmin for 3 minutes
using the
kinject program. Following a 10 minute dissociation, any remaining peptide was
stripped from the rmKDR-Fc surface with a quickinject of 50mM NaOH + 1M NaCl
for
12s at 75 gL/min.
Sensorgrams were analyzed using the simultaneous ka/kd fitting program in the
BlAevaluation software 3.1. The Results are shown in Table 8 and FIGs 22-24.
The
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fact that about the same KD2 constant was achieved for both heterodimers even
when
the density of receptor on the sensor chip was reduced by half is consistent
with
multimeric binding of the heterodimers to individual receptors rather than
cross-link-
type binding between receptors.
Table 8. Summary of Kinetic Parameters.
kal ka2 KD1# KD2
1/Ms kd1 1/s 1/RUs kd2 1/s nM Chit'
Ivs. 27007.94E+05 0.0139 3.31E-04 5.96E-04 17.5 0.751 0.077
D1 vs. 5000R 5.54E+05 8.88E-03 1.17E-04 4.57E-04 16.0 0.825 0.323
Ivs.2700R 7.59E+05 0.011 3.36E-04 6.44E-04 14.5 0.848 0.082
D7 vs. 50008 5.21E+05 7.39E-03 1.17E-04 I.68E-04 14.2 0.898 0.278
Fluorescein vs. 2700RU 1.02E+06 0.037 - - 36.4 - 0.073
labeled P12-G
vs. 5000R5.18E+05 0.0174 - - 33.6 - 0.167
# KDi is a calculated KD based on kd1/ka1
' KD2 is a calculated KD based on kd2/ka1 (i.e. avidity factor)
The chi2 value is a standard statistical measure of the closeness of the fit.
For good
fitting to ideal data, chi2 is of the same order of magnitude as the
instrument noise in
RU (typically < 2).
EXAMPLE 17
Demonstration of the distinction between binding affinity and biological
potency
through in vitro assays
The following experiments showed that heteromultimers can display much
greater biological potency than a monomeric peptide with similar binding
affinity to the
same target.
Protocol experiment 1:
293H cells were transfected with the KDR cDNA or mock-transfected by
standard techniques described in Example 6. The cells were incubated with 125I-
VEGF
in the presence or absence of PG-1 (Ac-ERVTTCWPGEYGGVECYSVAY-NH2) (SEQ
ID NO: 30) or D1 (at 300, 30, 3, and 0.3 nM). After washing the cells, the
bound
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radioactivity was quantitated on a gamma counter. The percentage inhibition of
VEGF
binding was calculated using the formula [(Y1-Y2)xI00/Y1], where Y1 is
specific
binding to KDR-transfected 293H cells in the absence peptides, and Y2 is
specific
binding to KDR-transfected 293H cells in the presence of peptide competitors.
Specific
binding to KDR-transfected 293H cells was calculated by subtracting the
binding to
mock-transfected 293H cells from the binding to KDR-transfected 293H cells.
Protocol experiment 2:
Serum-starved HWECs were placed, 100,000 cells per well, into the upper
chambers of BD fibronectin-coated FluoroBlok 24-well insert plates. Basal
medium,
with or without VEGF (10 ng/mL) in the presence or absence of increasing
concentrations of PG-I or D1, was added to the lower chamber of the wells.
After 22
hours, quantitation of cell migration/invasion was achieved by post-labeling
cells in the
insert plates with a fluorescent dye and measuring the fluorescence of the
invading/migrating cells in a fluorescent plate reader. VEGF-stimulated
migration was
derived by subtracting the basal migration measured in the absence of VEGF.
Results experiment 1:
As shown in FIG. 26, PG-1 and D1 competed about equally well with 125I-
VEGF for binding to KDR-transfected cells, indicating that they possess
comparable
binding affinities as well as a comparable ability to inhibit VEGF from
binding to KDR.
Results experiment 2:
In spite of the fact that both PG-1 and D1 potently block 1251-VEGF binding to
KDR-expressing cells to the same degree (FIG. 26), the heterodimeric D 1 was
significantly more potent in blocking the biological effects of VEGF as
demonstrated in
an endothelial cell migration assay (FIG. 27) than the monomeric PG-1. At up
to 62.5
nM, PG-I had no effect on VEGF-stimulated migration whereas D1 completely
blocked
VEGF-stimulated migration at 50 nM. These data suggest that heteromultimeric
binding can more effectively block the biological activity of a ligand than a
monomer,
even when the monomer has a comparable ability to inhibit ligand binding to
its
receptor.
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EXAMPLE 18
Binding of Tc-labeled heterodimers of the Invention KDR-transfected 293H cells
In this Example, the ability of Tc-labeled D10 to bind KDR was assessed using
KDR-transfected 293H cells. The results show that Tc-labeled D10 bound
significantly
better to KDR transfected 293H cells than to mock transfected 293H cells, and
good
binding was maintained in the presence of 40% mouse serum. In addition, a
derivative
of Tc-labeled D10 with its amino acid sequence scrambled, D18, was shown to
possess
no affinity for KDR-expressing cells, confirming the specificity of the D10
binding to
those cells.
Synthesis of 99mTc-labeled peptides
Preparation of 99i'Tc-D 10:
SnC12'2H20 (20 mg) was dissolved in 1 mL of 1 N HCI, and 10.tL of this
solution was added to 1 mL of a DTPA solution that was prepared by dissolving
10 mg
of Ca Nat DTPA*2.5 H2O (Fluka) in 1 mL of water. Dl0 (100 g in l00 L of 50%
DMF) was mixed with 75 L of 0.1 M, pH 9 phosphate buffer and 50 L of 99mTcO4
(2.4 to 5 mCi, Syncor), followed by 100 L of the stannous Sn-DTPA solution.
After
15 min at RT, the radiochemical purity (RCP) was 72%. The product was purified
on a
Supelco Discovery C16 amide column (4 x 250 mm, 5 um pore size) eluted at a
flow
rate of 0.7 mUmin using an aqueous/organic gradient of 0.1% TFA in water (A)
and
0.085% TFA in acetonitrile (B). The following gradient was used: 30% B to 42%
B in
36 min, ramp up to 70% B in 10 min. The compound, which eluted at a retention
time
of 32 min. was collected into 500 L of 50 mM citrate buffer (pH 5.2)
containing 0.2%
HSA, and acetonitrile was removed using a Speed Vacuum (Savant). After
purification,
the compound had an RCP of >90%.
Preparation of 99mTc-D 18:
SnC12.2H20 (20 mg) was dissolved in 1 mL of 1 N HCI, and 10 L of this
solution was added to I mL of a DTPA solution that was prepared by dissolving
10 mg
of Ca Nat DTPA.2.5 H2O (Fluka) in I mL of water. D18 (100 gg in 100 L of 50%
DMF) was mixed with 50 L of 0.1 M, pH 9 phosphate buffer and 90 L of
99rtiTcO4
(14 mCi, Syncor), followed by 100 L of the stannous Sn-DTPA solution. The
reaction
was warmed for 20 minutes at 37 C. The entire reaction was injected on a Vydac
218TP54 C18 column (4.6 x 250 mm, 5 um silica) and eluted at a flow rate of
1.5
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mL/min using an aqueous/organic gradient of 0.1 % TFA in water (A) and 0.085%
TFA
in acetonitrile (B). The following gradient was used: 32% to 39% B in 30
minutes,
ramp up to 80% B in 2 min. The free ligand eluted at a retention time of 19
minutes.
The complex, which eluted at 24 minutes, was collected into 500 L of 50 mM
citrate
buffer (pH 5.3) containing 0.1 % HSA and I% Ascorbic Acid. Acetonitrile &
excess
TFA were removed using a Speed Vacuum (Savant) for 40 minutes. After
purification,
the compound had an RCP of 93%.
Transfection of 293H cells
293H cells were transfected using the protocol described in Example 6.
Transfection was done in black/clear 96-well plates (Becton Dickinson, cat. #
354640).
The cells in one half of the plate (48 wells) were mock-transfected (with no
DNA) and
the cells in the other half of the plate were transfected with KDR cDNA. The
cells were
80-90% confluent at the time of transfection and completely confluent the next
day, at
the time of the assay; otherwise the assay was aborted.
Preparation of opti-MEMI media with 0.1 % HSA
Opti-MEMI was obtained from Invitrogen (cat. # 11058-021) and human serum
albumin (HSA) was obtained from Sigma (cat. # A-3782). To prepare opti-MEMI
media with 0.1% HSA, 0.1% w/v HSA was added to opti-MEMI, stirred at room
temperature for 20 minutes, and then filter sterilized using 0.2 M filter.
Preparation of Tc-labeled peptide dilutions for the assay
Stock solutions of Tc-labeled D10 and D18 were diluted in opti-MEMI with
0.1 % HSA to provide solutions with final concentrations of 1.25, 2.5, 5.0,
and 10
ICi/mL of each Tc-labeled heterodimer. A second set of dilutions was also
prepared
using a mixture of 40% mouse serum/60% opti-MEMI with 0.1 % HSA as the
diluent.
Assay to detect the binding of the Tc-labeled heterodimers
Cells were used 24 h after transfection, and to prepare the cells for the
assay,
they were washed 1 X with 100 L of room temperature opti-MEMI with 0.1 % HSA.
After washing, the opti-MEMI with 0.1 % HSA was removed from the plate and
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replaced with 70 L of 1.25, 2.5, 5.0, and 10 jCi/mL of Tc-labeled D10 or D18
(prepared as above with both diluent solutions). Each dilution was added to
three
separate wells of mock and KDR-transfected cells. After incubating at room
temperature for 1 h, the plates were washed 5 times with 100 L of cold
binding buffer
(opti-MEMI with 0.1 % HSA). 100 gL of solubilizing solution (0.5 N NaOH) was
added
to each well and the plates were incubated at 37 C for 10 minutes. The
solubilizing
solution in each well was mixed by pipeting up and down, and transferred to
1.2 mL
tubes. Each well was washed once with 100 L of solubilizing solution and the
washes
were added to the corresponding 1.2 mL tube. Each 1.2 mL tube was then
transferred to
a 15.7 mm X 100 cm tube to be counted in an LKB Gamma Counter.
Binding of Tc-labeled heterodimers to KDR transfected cells
The ability of Tc-labeled D10 and D18 to bind specifically to KDR was
demonstrated using transiently transfected 293H cells. As shown in FIG. 28A,
Tc-
labeled D10 bound significantly better to KDR transfected 293H cells, as
compared to
mock-transfected 293H cells in both the presence and absence of 40% mouse
serum,
although there was some inhibition in the presence of serum. The total
specific binding
of this Tc-labeled heterodimer to KDR-expressing cells was much greater than
that
observed previously with a Tc-labeled monomeric peptide (Example 5). Tc-
labeled
D18, on the other hand, displayed no affinity for either mock-transfected or
KDR-
transfected 293H cells, confirming the specificity of D 10 binding.
EXAMPLE 19
Binding of a Lu-labeled heterodimers to KDR-transfected 293H cells
In this Example, the ability of Lu-labeled D13 to bind KDR was assessed using
KDR-transfected 293H cells. The results show that Lu-labeled D13 bound
significantly
better to KDR transfected 293H cells than to mock transfected 293H cells, and
good
binding was maintained in the presence of 40% mouse serum.
Synthesis of'77Lu-labeled peptide
Preparation of 177Lu-D 13:
D13 (306 g) was added to a 2-mL autosampler vial with a -450 L conical
insert and dissolved in 0.01N NH4OH (50 L). To this was added 300 L of 0.5M
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Ammonium Acetate containing stabilizers. A 6.8 L aliquot of 77LuC13 in 0.05N
HCl
(39.3 mCi) was added, the vial was crimp-sealed, warmed for 15 min at 37 C,
cooled
for -' 5 minutes, and 10 L of I% Na2EDTA.2H20 in H2O was added. A 350 L
aliquot
of the reaction mixture was injected onto a Supelco Discovery RP Amide C16
column
(4 mm x 250 mm x 5 pan). The following HPLC conditions were used: Column
temperature = 37 C, Solvent A = H2O containing 2 g/L NH4OAc buffer, pH 7.0,
Solvent B = 80% ACN/20% H2O, gradient 0.56/0.24 mljmin AB at t = 0 minutes to
0.47/0.33 mL/min A/B at t = 30 minutes. The retention time for D13 was -28
minutes;
the retention time for 177Lu-D 13 was -29 minutes. The radioactive peak was
collected
into 1 mL of a buffer containing stabilizers, final pH = 7.6 adjusted with
Sodium
Hydroxide. It was then spun down -r40 minutes using a Speed Vacuum (Savant) to
remove ACN. The RCP of the isolated product was 86%.
Transfection of 293H cells
293H cells were transfected using the protocol described in Example 6.
Transfection was done in black/clear 96-well plates (Becton Dickinson, cat. #
354640).
The cells in one half of the plate (48 wells) were mock-transfected (with no
DNA) and
the cells in the other half of the plate were transfected with KDR cDNA. The
cells were
80-90% confluent at the time of transfection and completely confluent the next
day, at
the time of the assay; otherwise the assay was aborted.
Preparation of opti-MEMI media with 0.1 % HSA
Opti-MEMI media with 0.1 % HAS was prepared as in Example 18.
Preparation of Lu-labeled peptide dilutions for the assay
A stock solution of Lu-labeled D13 was diluted in opti-MEMI with 0.1% HSA
to provide solutions with final concentrations of 1.25, 2.5, 5.0, and 10
Ci/mL of
labeled heterodimer. A second set of dilutions was also prepared using a
mixture of
40% mouse serum/60% opti-MEMI with 0.1 % HSA as the diluent.
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Assay to detect the binding of the Lu-labeled heterodimers
This was carried out as detailed in Example 18 except that Lu-labeled D13 was
used in place of the Tc-labeled heterodimers.
Binding of Lu-labeled heterodimer to KDR transfected cells
The ability of Lu-labeled D13 to bind specifically to KDR was demonstrated
using transiently-transfected 293H cells. As shown in FIG. 29, Lu-labeled D13
bound
significantly better to KDR transfected 293H cells, as compared to mock-
transfected
293H cells in both the presence and absence of 40% mouse serum, although there
was
some binding inhibition in the presence of serum.
EXAMPLE 20
Radiotherapy with a Lu-labeled heterodimers in tumor-bearing mice.
In this Example, the ability of Lu-labeled D13 to inhibit the growth of PC3
cell
tumors implanted in nude mice is demonstrated.
Synthesis of'77Lu-labeled D13
177Lu-labeled D13 was prepared as described in Example 19.
Animal model
PC3 cells from ATCC, grown as recommended by the supplier, were injected
subcutaneously between the shoulder blades of nude mice. When their tumors
reached
100-400 mm3, twelve mice were injected i.v. with 500 microcuries of Lu-labeled
D13
and their growth monitored for an additional 18 days. Mice were sacrificed if
they lost
20% or more of their body weight or their tumors exceeded 2000 mm3. Tumor
growth
in the treated mice was compared with the average tumor growth in 37 untreated
nude
mice implanted with PC3 tumors.
Results
In 6 of the 12 treated mice in the study, the tumors experienced a significant
or
complete growth delay (FIG. 30) relative to untreated tumor mice, indicating
that D13
was effective in slowing PC3 tumor growth under the conditions employed
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EXAMPLE 21
Cell based assay for binding of KDRNEGF complex binders
In this experiment the ability of a KDRNEGF complex-binding peptide to
selectively bind to the KDRNEGF complex is demonstrated.
Reagent preparation
The reagents for this assay were prepared as described in Example 5 except
where noted.
Preparation of peptide-125l-streptavidin complex solution
Biotinylated peptides P30-XB, P31-XB, P32-XB and biotinylated non-binding
control peptide were used to prepare 1.25 M stock solutions in 50% DMSO. A
33.33
nM stock solution of 125I-streptavidin was purchased from Amersham. A stock
solution
of 13.33 nM 1-125 streptavidin/100 nM VEGF was prepared by mixing 850 ml of 1-
125
streptavidin with 22 pl of 10 M VEGF and 1275 pl of M199 media. Another stock
solution was prepared in the same manner, but lacking VEGF. To prepare 13.33
nM
peptide- 125I-streptavidin complex solution + VEGF, 500 l of the 1251-
streptavidin
(with & without VEGF) stock solutions (prepared in last step) were mixed with
24 pl
of 1.25 pM peptide solution of P30-XB, P31-XB, P32-XB, or control peptide. The
mixtures were incubated on a rotator at 4 C for 60 minutes, followed by
addition of 50
l of soft release avidin-sepharose (50% slurry in ddH2O) to remove excess
peptides and
another incubation for 30 minutes on a rotator at 4 C. Finally, the soft
release avidin-
sepharose was pelleted by centrifuging at 12,000 rpm for 5 minutes at room
temperature, and the resulting supernatants were used for the assays.
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Table 9. Biotinylated Peptides
Reference Structure or Sequence SEQ
Number ID
NO:
P30 AGPGPCKGYMPHQCWYMGTGGGK 31
P30-XB Ac-AGPGPCKGYMPHQCWYMGTGGGK(Biotin-JJ)-
NH2
P31 AGMPWCVEKDHWDCWWWGTGGGK 32
P31-XB Ac-AGMPWCVEKDHWDCWWWGTGGGK(Biotin-JJ)-
NH2
P32 AGYGPCKNMPPWMCWHEGTGGGK 33
P32-XB Ac-AGYGPCKNMPPWMCWHEGTGGGK(Biotin-JJ)-
N112
Binding ofpeptide/neutravidin HRP to KDR-transfected cells
In this assay, complexes of control peptide and the test peptides (P30-XB, P31-
XB, P32-XB) with125I-streptavidin in the presence or absence of VEGF (prepared
as
above) were tested for their ability to bind 293H cells that were transiently-
transfected
with KDR. The complex of P30-XB with 125I-streptavidin specifically bound to
KDR-
transfected 293H cells as compared to mock transfected cells in the presence
of VEGF
(FIG. 3IA), but not when VEGF was omitted (FIG. 31B). P30-XB was also the best
KDRNEGF complex binder among the peptides tested using fluorescence
polarization
and SPR (BiaCore) assays. See Table 9, U.S.S.N. 60/360,851, U.S.S.N.
60/440,441,
and copending U.S.S.N. entitled "KDR and VEGF/KDR Binding Peptides and
Their Use in Diagnosis and Therapy," filed on the same date as the instant
application
and incorporated by reference herein in its entirety. This example shows that
peptide
(P30-XB) can specifically bind to The KDR/VEGF complex present on the cell
surface.
Therefore, it may possibly be used in targeting the KDR/VEGF complex in vitro
and in
vivo for diagnostic or therapeutic purposes. Since the KDRNEGF binding peptide
only
detects the functional and active KDR receptor and not all the KDR present on
cell
surface, it may be useful in detecting and/or treating active angiogenesis in
tumors,
metastasis, diabetic retinopathy, psoriasis, and arthropathies. Furthermore,
these
peptides, as well as other peptides which bind KDRNEGF complex may
advantageously be included in hetermultimers of the invention.
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EXAMPLE 22
The following experiment assessed the ability of heterodimers D24 and D26 to
block the VEGF-induced migration of HUVECs in culture and demonstrated that
the
added glycosylation and/or distinct spacer structure used in D26 enhanced its
potency.
Protocol:
Serum-starved HUVECs were placed, 100,000 cells per well, into the upper
chambers of BD fibronectin-coated FluoroBlok 24-well insert plates. Basal
medium,
with or without VEGF (10 ng/mL) in the presence or absence of D24 or D26, was
added to the lower chamber of the wells. After 22 hours, quantitation of cell
migration/invasion was achieved by post-labeling cells in the insert plates
with a
fluorescent dye and measuring the fluorescence of the invading/migrating cells
in a
fluorescent plate reader. The VEGF-induced migration was calculated for each
experimental condition by subtracting the amount of migration that occurred
when only
basal medium was added to the lower chamber of the wells.
Results:
VEGF induced a large increase in endothelial cell migration in the assay,
which
was potently blocked by both D24 and D26 (FIG. 32). D26 was ten-fold more
potent
than D24 (IC50 0.5 nM and 5 nM respectively), indicating that the
glycosylation of D26
and/or its distinct spacer properties has enhanced its ability to bind KDR and
block the
effects of VEGF.
EXAMPLE 23
The following experiment assessed the ability of TK-1 (structure provided
below), a multimeric construct of the peptide TKPPR (which binds to NP-1, a
VEGF
receptor which enhances the effects of VEGF mediated by KDR), to enhance the
inhibition of the VEGF-induced migration of HUVECs in culture produced by D6.
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HO
F
0 O
0
F OH
0k-NH
0 N
NHH O
O J-N O
0 N N O H -11CO O
T K P P R-OH 0 01
HN` HN~
0 O 0
O T K P P R-OH
O
0 0 T K P P R-OH
O T K P P R-OH
5CF-Gly-N { [ CH2CH2C(=O)-Gly-N(CH2CH2C(=O)-Adoa-Thr-Lys-Pro-Pro-Arg-OH]2 } 2
where Adoa = 3,6-dioxa-8-aminooctanoyl, 5CF = 5-carboxyfluoresceinyl
TK-1
Protocol:
Serum-starved HUVECs were placed, 100,000 cells per well, into the upper
chambers of BD fibronectin-coated FluoroBlok 24-well insert plates. Basal
medium,
with or without VEGF (10 ng/mL) in the presence or absence of varying
concentrations
of D6, or varying concentrations of D6 in combination with a constant 100 nM
TK-1
(synthesized as described in WO 01/91805 A2) was added to the lower chamber of
the
wells. After 22 hours, quantitation of cell migration/invasion was achieved by
post-
labeling cells in the insert plates with a fluorescent dye and measuring the
fluorescence
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of the invading/migrating cells in a fluorescent plate reader. VEGF-induced
migration
was calculated for each experimental conditions by subtracting the amount of
migration
observed in the absence of VEGF.
Results:
VEGF induced a large increase in endothelial cell migration in the assay,
which
was potently blocked by D6 (IC50 about 12.5 nM), but not by 100 nM TK- I alone
(FIG.
33). Surprisingly however, TK-1 was able to enhance the potency of D6 by about
ten-
fold when used in the assay simultaneously with D6 (IC5o about 2.5 nM). This
indicates
that compounds containing the TKPPR sequence (or analogs) found in TK-1 can be
used to enhance the potency of certain compounds such as D6 which compete with
VEGF for binding to KDR. In addition, a heteromultimer containing the peptide
sequences found in D6 (or similar) as well as the TKPPR sequence (or analogs),
in one
or more repetitions, would likely possess enhanced activity in this assay.
(See U.S.S.N.
09/871,974 for details on the preparation of TKPPR constructs, which is herein
incorporated by reference).
EXAMPLE 24
Identification of fragments of P13-XB with KDR binding activity
The following experiment showed that fragments of P13-XB can maintain
significant KDR binding activity.
Protocol:
293H cells were transfected with the KDR cDNA or mock-transfected by
standard techniques described in Example 6. Streptavidin-HRP complexes
containing
P12-XB were prepared as in Example 6. Binding of the streptavidin-HRP
complexes to
the cells was carried out as in Example 6 with a complex concentration of 5.5
nM in the
presence of 0 to 250 nM or 0 to 1000 nM of the following competing peptides:
P13-XB,
Fl, F2, and F3. After determining the specific binding under each experimental
condition, the IC50 for each peptide was determined (where possible).
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Results:
As shown in Table 9, F1, composed of just the Asp-Trp-Tyr-Tyr binding motif
that is also shared with P12-XB along with the non-targeted Gly-Gly-Gly-Lys
sequence
that was added to most monomeric peptides synthesized based on phage display
data,
was the smallest fragment able to block P12-XB streptavidin-HRP complex
binding
with an IC50 below one micromolar. Surprisingly, a larger fragment derived
from P 13-
XB, F2, failed to significantly inhibit complex binding at one micromolar.
However,
when a solubilising motif, (Gly-Arg-Gly)3 was added to the latter peptide to
make F3, it
was able to compete with the complex for binding with an IC50 of 175 nM,
confirming
that certain fragments of P 13-XB containing the Asp-Tip-Tyr-Tyr motif retain
KDR-
binding activity. These fragments (or other fragments of the binding
polypeptides
disclosed herein), which retain the ability to bind the target, may be
utilized instead of
the full-length peptide in heteromultimers of the invention.
Table 9. Fragments of P 13-XB in a displacement assay competing with a complex
composed of P12-XB and streptavidin-HRP for binding to KDR-expressing cells.
Ref Sequence/Structure IC50, SEQ
Number nM ID
NO:
P13-XB Ac-AQDWYYDEILSMADQLRHAFLSGG-GGGK- 93 --
(Biotin-JJ-)-NH2
Fl Ac-DWYYGGGK-NH2 850 31
F2 Ac-AQDWYYDEIL-NH2 >1000 32
F3 Ac-AQDWYYDEILJGRGRGGRGG-NH2 175 33
EXAMPLE 25
Heterodimers targetin tgwo epitopes on a single target molecule results in
superior
binding to a homodimers that binds one of the two epitopes on the target
molecule.
The following experiment provides further evidence that heterodimeric
constructs are superior to homodimers in their ability to block the biological
effects of a
peptide growth factor or cytokine.
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Protocol:
Serum-starved HUVECs were placed, 100,000 cells per well, into the upper
chambers of BD fibronectin-coated FluoroBlok 24-well insert plates. Basal
medium,
containing either nothing or VEGF in the presence or absence of increasing
concentrations of homodimeric D8 or heterodimeric D17, was added to the lower
chamber of the wells. After 22 hours, quantitation of cell migration/invasion
was
achieved by post-labeling cells in the insert plates with a fluorescent dye
and measuring
the fluorescence of the invading/migrating cells in a fluorescent plate
reader.
Results:
VEGF induced a large increase in endothelial cell migration in the assay,
which
was potently blocked by D17 but not D8 (FIG. 34). D17 blocked VEGF-induced
migration with an IC50 of about 250 nM while D8 had no significant effect on
migration
even at 800 nM. This is in spite of the fact that D8 used the full targeting
sequence
found inpI3-XB while D17 contained a truncated version of the P13-XB sequence
(as
seen in F3) with a lower affinity for KDR (as demonstrated in Example 24).
Thus a
heterodimer with the capability of binding to two separate epitopes on the
same target
molecule can be more effective at blocking ligand binding to the target
molecule than a
homodimer containing the same or even more potent targeting sequences.
EXAMPLE 26
Preparation of cyclic peptides in which the disulfide bond is replaced by an
amide bond
Disulfide bond substitution analogs of P12-G (P12 with non-target GGGK
sequence) where the Cys residues at position 6 and 13 are replaced by a pair
of amino
acids, one with a carboxy-bearing side-chain (either Glu Or Asp) and the other
with an
amino-bearing side chain [(Lys or Dpr (2,3-diaminopropanoic acid)] were
prepared.
The cycle, encompassing the same sequence positions as those included in p12-G
(made
by formation of the disulfide bond) was made by condensation of the side-chain
amino
and side-chain acid moieties, resulting in a lactam ring which bridges the
residues 6-13
as does the disulfide bond of P12-G.
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Table 10. Examples of the substitutions made for Cys6 and Cys13 of P12-G in
lactam
analogs.
Lactam Analogs of P12
Ref Sequence or Structure SE Positio Positio Differenc
No. Q n6 n13 ein Ring
ID Size vs
NO P12
P12-G AGPTWCEDDWYYCWLFGTGGGK 29 Cys Cys -
(parent
seq)
--
P33 AGPTWEEDDWYYKWLFGTGGGK 34 Glu L YS
P33-L Ac- Glu Lys 4
AGPTWEEDDWYYKWLFGTGGGK-
NH2 (6-13 lacta
P34 AGPTWKEDDWYYEWLFGTGGGK 35 Lys Glu --
P34-L Ac- Lys Glu 4
AGPTWKEDDWYYEWLFGTGGGK-
NH2 (6-13 lactam)
P35 AGPTW-Dpr- 36 Dpr Asp --
EDD W YYD W LFGTGGGK-NH2
P35-L Ac-AGPTW-Dpr- Dpr Asp 0
EDDWYYDWLFGTGGGK-NH2 (6-13
lactam)
P36 AGPTWDEDDWYY-Dpr- 37 Asp Dpr --
WLFGTGGGK
P36-L Ac-AGPTWDEDDWYY-Dpr- Asp Dpr 0
WLFGTGGGK-NH2 (6-13 lactam)
P37 AGPTWDEDDWYYKWLFGTGGGK 38 Asp L YS --
P37-L Ac- Asp Lys 3
AGPTWDEDDWYYKWLFGTGGGK-
NH2 (6-13 lacta
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Representative Synthesis of Cyclic Lactam Peptides - P33-L
FmocNH Resin (PAL-PEG-PS)
l SPPS (Method 5)
Ac-
AGPT(tBu)W(Boc)E(OAII)E(OtBu)D(OtBu)D(OtBu)W(Boc)Y(tBu)Y(tBu)K(Aloc)W(Boc)LFGT(
tBu)GGGK(Boc) Resin
1
Pd(PPh3)4
NMM/HOAcIDMF/Argon
Ac,AGPT(tBu)W(Boc)EE(OtBu)D(OtBu)D(OtBu)W(Boc)Y(tBu)Y(tBu)KW(Boc)LFGT(tBu)GGGK(
Boc Resin
2
HATUINMM/DMF
O H
Ac
AGPT(tBu)W(Boc)EE(OtBu)D(OtBu)D(OtBu)W(Boc)Y(tBu)Y(tBu)KW(Boc)LFGT(tBu)GGGK(Boc
) Resin
3
reagent B
O
I
Ac-AG PTWEEDDWYYKW LFGTGGGK-NH2
4
Synthesis of Resin bound peptide 1
Synthesis of 1 was carried out using Method 5 on a 0.25 mmol scale. The
peptide resin 1 was washed and dried for further derivatization manually.
Synthesis of 4 P33-L
To 1 (240 mg, 0.06 mmol) was added NMM (N-methyl
morpholine)/HOAc/DMF 1/2/10 (v/v/v) (65 mL). Palladium tris-triphenylphosphine
[Pd(PPh3)4, 554.4 mg, 0.48 mmol] was added and the resin was shaken for 20h
shielded
from light. The resin was filtered and washed with a solution of sodium
diethyldithiocarbamate (0.5 g)/DIEA (0.5 ml)/DMF (100 mL), and finally with
DMF (3
x 70 mL). This treatment served to expose only the carboxy and amino groups of
G1u6
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and Lys13 which are required for the lactam forming reaction. The on-resin
cyclization
of 2 was carried out using HATU (114 mg, 0.3 mmol), NMM (66 L, 0.6 mmol) and
DMF (10 mL) for 3 h. The completion of the cyclization was monitored by Kaiser
test.
The peptide was cleaved from the peptide resin 3 using reagent B for 4 h. The
resin was
filtered and the filtrate was evaporated to a paste. The crude peptide was
precipitated in
ether and washed twice with ether. The cyclic peptide was purified by
preparative
reverse phase linear gradient HPLC using a Waters-YMC C-18 column (250 mm x 30
mm i.d.) with CH3CN into H2O (both with 0.1% TFA) as the eluent.
Lyophilization of
the product-containing fractions afforded 8 mg of (P33-L). P34-L, P35-L, P36-L
and
P37-L were prepared similarly.
Replacement of the disulfide bridge of P12-G while retaining KDR-binding
activity
The following experiment demonstrated that the lactam P34-L, which replaced
the chemically reactive disulfide bridge of P 12-G maintained significant KDR
binding
activity.
Protocol:
293H cells were transfected with the KDR cDNA or mock-transfected by
standard techniques described in Example 6. Streptavidin-HRP complexes
containing
P12-XB were prepared as in Example 6. Binding of the streptavidin-HRP
complexes to
the cells was carried out as in Example 6 with a complex concentration of 5.5
nM in the
presence of 0 to 250 nM P12-G, or P34-L. After determining the specific
binding under
each experimental condition, the IC50 for each peptide was determined.
Results:
As shown in Table 11, P34-L, containing a lactam disulfide bridge replacement,
was still able to compete with P12-XB-streptavidin-HRP complexes for binding
to
KDR although some affinity was lost (IC50 108 nM versus 13 nM for P12-G).
These
lactam peptides (or similarly prepared lactam analogs of binding polypeptides
disclosed
herein) may be utilized instead of the disulfide bridge-containing peptides in
heteromultimers of the invention.
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Table 11. P12-G and P34-L (disulfide bridge replacement analog) in a
displacement
assay competing with a complex composed of P 12-XB and streptavidin-HRP for
binding to KDR-expressing cells.
Fragment (Ref Number) IC50, nM
P12-G 13
P34-L 108
EXAMPLE 27
Measurement of binding of peptide dimers to cMet
Using a BlAcore machine, the binding constant was determined for the dimer
D28 binding to immobilized cMet-Fc.
Procedure
Three densities of cMet-Fc (R&D Systems) were cross- linked to the dextran
surface of a CM5 sensor chip by the standard amine coupling procedure (3 M
solution
diluted 1:100, 1:50, or 1:20 with 50 mM acetate, pH 5.5). Flow cell 1 was
activated and
then blocked to serve as a reference subtraction.
Final immobilization levels achieved:
RL Fe 2 cMet-Fc = 2582
RL Fc 3 cMet-Fc = 5048
RL Fc 4 cMet-Fc = 9721
Experiments were performed in PBST buffer (5.5 mM phosphate, pH 7.65, 0.15
M NaCl) + 0.05% (v/v) Tween-20). Peptide dimers were dissolved in deionized
H2O to
1 mg/mL solutions. Dimers were diluted to 5OnM in PBS. Serial dilutions were
performed to produce 25, 12.5, 6.25, and 3.125n\4 solutions. All samples were
injected
in duplicate. For association, dimers were injected at 30 L/minute for 3
minutes using
the kinject program. Following a 10-minute dissociation, any remaining peptide
was
stripped from the cMet surface with two quickinjects of 4M MgC12 for 2 minutes
at
50jL/minute. Sensorgrams were analyzed using BlAevaluation software 3.1.
Kd value of 0.79 nM was obtained for D28 (heterodimer of P26-A and P27-X),
which was significantly better than KD value of either heterodimer alone (see
SEQ ID
NO:369 (880 nM) and SEQ ID NO:370 (220 nM) as shown in the Table 8 of U.S.S.N.
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entitled "Peptides that specifically bind HGF receptor (cMet) and uses
thereof," filed on the same date as the instant application and incorporated
by reference
herein in its entirety.
EXAMPLE 28
In vitro cell proliferation assay
Microvascular endothelial cells (MVECs, Cascade Biologics, Portland, OR)
were used to assess the in vitro efficacy of D6 and related analogues for
their ability to
inhibit VEGF-stimulated proliferation. MVECs (passage 2) were grown to 90%
confluency, trypsinized and plated in gelatin-coated 96-well microtiter plates
at a
density of 4-8 x 103 cells/well. Sixteen to 24 hours after plating, the cells
were washed
one time (200 L/welt) with media devoid of fetal bovine serum but containing
0.1 %
bovine serum albumin (BSA). Fresh BSA-containing media was added to each well
and the cells were incubated for an additional 24 hours. After this 24 hour
period of
starvation, fresh BSA-containing media with or without D6 or other test
substances was
added and the cells were incubated for an additional 48 hours at 37 C. The
media was
removed and fresh BSA-containing media was added with or without BrdU and the
cells were incubated for an additional 24 hours prior to determining the level
of
incorporation exactly as described by the manufacturer (Oncogene Cat# QIA58).
Results are shown in FIG. 35.
EXAMPLE 29
In Vivo inhibition of tumor growth.
Conditions are described providing methods for determining efficacy of three
(3) concentrations for a test compound (dimer D6) suspected of having anti-
angiogenic
activity on SW-480 human colon carcinoma cells using an in vivo xenograft
tumor
model.
Athymic nude mice are acceptable hosts for the growth of allogenic and
heterogenic cells. Nude mice are required in Points to Consider in the
Characterization
of Cell Lines used to Produce Biologicals (Points to Consider in the
Characterization of
Cell Lines used to Produce Biologicals, FDA 1993).
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D6 is a synthetic heterodimeric peptide suspected of having anti-angiogenic
activity. This peptide binds to the human VEGF receptor 2 (KDR) with high
affinity
and competes with VEGF binding. The following experiments confirms its anti-
angiogenic activity.
SW-480 Human Carcinoma Cells
Colon carcinoma, SW-480, cells (ATCC) were cultured in Dulbecco's Modified
Eagles Medium (DMEM) supplemented with 4 mM L-glutamine, 0.1 mM non-essential
amino acids, 50 mg/mL Gentamicin, 250 mg/mL Fungizone and 10% heat inactivated
fetal bovine serum at 37 C in 95% air and 5% C02.
Exponentially growing cells were harvested, washed twice in phosphate
buffered saline (PBS) to remove any traces of trypsin or serum. Cells were
suspended
in Hanks Balanced Salt Solution (HBSS) for injections.
Sterile phosphate buffered saline (BioWhittaker) was manufactured in
accordance with cGMP regulations and was cell culture tested to assure
compatibility;
having a pH of 7.3-7.7 and an osmolarity of 271-287 mOsm/kg. PBS was the
vehicle
used to reconstitute Test Articles and for vehicle control injections.
Cisplatin (American Pharmaceutical Partners, Inc.; Los Angeles, CA) was
prepared according to manufacture's specifications. Cisplatin was prepared in
an aseptic
fashion using a BL2 BioChem guard hood.
Test System
Species/Strain: Mus musculus, Crl:NU/NU-nuBR mice (nude mice)
Sex: Female
Age: 6-8 weeks at initiation of treatment
Weight Range: No weight requirement
Source: Animals were received from the Gnottobiotic Department at Charles
River
Laboratories, Wilmington, MA.
Number: A total of 115 animals were received and injected for this study, with
90 mice
used on study.
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Method of Identification:
Mice were uniquely numbered using an ear tag system. Additionally, cages
were marked with cage cards minimally identifying group number, animal number,
study number and IACUC protocol number.
Randomization:
Animals were randomly assigned to treatment groups using Microsoft Excel
97 SR-1 program.
Animal Care
Mice were fed gamma-irradiated rodent chow ad libitum. Tap water was
sterilized and supplied via bottle and sipper tube ad libitum.
Animal Environment:
Animals were housed by groups in semi-rigid isolators. Mice were housed in
flat bottom caging containing five to ten animals. Cages contained gamma-
irradiated
contact bedding. The number of mice in each cage may have been altered due to
the
behavior of the mice, changes were noted in the isolator inventory. The
housing
conforms to the recommendations set forth in the Guide for the Care and Use of
Laboratory Animals, National Academy Press, Washington, D.C., 1996 and all
subsequent revisions.
Environmental controls were set to maintain a temperature of 16-26 C (70 f
8 F) with a relative humidity of 30-70. A 12:12 hour light: dark cycle was
maintained.
Acclimation:
Once animals were received, they were allowed to acclimate to the laboratory
environment for 24-hours prior to the study start. Mice were observed for
signs of
disease, unusual food and/or water consumption or other general signs of poor
condition. At the time of animal receipt, animals were clinically observed and
appeared
to be healthy.
Experimental Design:
Female athymic nude mice (Crl:NU/NU-nuBR) at 6-8 weeks of age were used
in this study. A total of 115 mice were injected subcutaneously into the right
lateral
thorax with 5 x 106 SW-480, human colon carcinoma cells. When tumors reached a
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target window size of approximately 150 75 mg, 90 tumor-bearing mice were
randomly selected and distributed into one of nine groups. Test compound and
vehicle
were administered intraperitoneally (IP), Cisplatin was administered
intravenously (N).
Tumor measurements were recorded twice weekly using hand-held calipers. Mice
were
monitored daily for signs of toxicity and morbidity. At study termination,
animals were
euthanized by carbon dioxide overdose and necropsied for tissue collection.
A total of nine (9) groups were used in this study. Each group contained ten
(10) tumor-bearing mice. Groups 1 and 2 contained untreated and vehicle
treated
negative control mice, respectively. Groups 3, 4, and 5 contained mice that
received
one of three different concentrations of the D6 heterodimer. Groups 6, 7, and
8
contained mice that received one of three different concentrations of a
different anti-
angiogenic peptide. Group 9 contained mice that received cisplatin, a standard
chemotherapeutic compound as a positive control.
Dose levels for each group are provided in Table 12. Dosing began the same
day that animals were randomly sorted into groups (Study Day 7). Each dose was
removed from the dose vial using aseptic technique for each animal and the
injection
site was wiped with an alcohol swab prior to dose administration. Doses were
administered with a 1.0 mL syringe and a 27-gauge x %z" needle for each mouse.
Table 12. Study Treatment Groups
Group Test Compound Concentration Number of Animals
mg/kg
1 Untreated - 10
2 Vehicle 0 10
3 D6 0.05 10
4 D6 0.5 10
5 D6 5.0 10
9 Cisplatin 6.0 10
The Test compound- and vehicle-treated mice received daily intraperitoneal
(IP)
injections for 15 days. Cisplatin was administered every other workday for a
total of
five (5) doses via an intravenous route.
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Clinical Observations of each animal were performed and recorded at least once
daily for toxicity, morbidity and mortality. Morbidity included signs of
illness such as,
but not limited to, emaciation, dehydration, lethargy, hunched posture,
unkempt
appearance, dyspnea and urine or fecal staining.
Tumor Measurements:
In accordance with the protocol, tumor measurements were taken twice weekly
throughout the study by measuring the length and width of tumors with
calibrated
calipers. Measurements occurred a minimum of 3-4 days apart, except when
animals
were euthanized and measurements were taken; this sometimes resulted in an
interval of
less than 3 days. Tumor weights were calculated using the following formula:
mg = (L
x W2)/2. Animals were euthanized either when mean tumor weight was > 1000 mg
per
group over two (2) consecutive measurements, or if tumors became ulcerated,
impaired
the animal's ability to ambulate or obtain food and water.
Unscheduled Euthanasia and Unexpected Deaths:
1. Unscheduled Euthanasia:
None of the animals required unscheduled euthanasia while on study.
2. Unexpected Deaths:
None of the animals died while on study.
Necropsy:
1. Euthanasia and Necropsy Order:
All mice in groups 1, 2, 3, 4, and 5 (50 total) were submitted for necropsy
when
tumors reached a group mean target size of> 1000 mg over two (2) consecutive
measurements within a group. Animals were submitted for necropsy to the
Charles
River Laboratories Health Monitoring Laboratory (HM), Wilmington, MA. All
animals
were euthanized on Study Day 22, short of received the full 28 day treatment
regiment
with Test Articles because mean tumor size was > 1000 mg in Test Article
Treated
Groups 3-8. All animals were humanely euthanized by carbon dioxide (CO2)
inhalation.
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Tissue Collection:
Tumors were dissected free of surrounding tissue and overlying skin.
Additionally the kidneys were collected. Any abnormalities noted on the renal
surfaces
were noted.
Frozen blocks were made of tumors and kidneys for each animal. A
representative section of the tissue (tumor, kidneys) was taken. Kidney
sections
included the cortex and medulla. Tissue sections were placed in the bottom of
a labeled
plastic-freezing mold. Tissue was embedded with OCT medium. Blocks were
submerged into isopentane chilled with dry ice until frozen. Blocks were
briefly
examined for quality, and stored on dry ice.
Blocks were labeled with the animal number and a letter code corresponding to
tissue (A = left kidney; B = right kidney; C = mass). Blocks from one animal
were
placed into a labeled bag.
Results:
A. In-Life Measurements and Observations:
1. Clinical Observations, Morbidity and Mortality Summary Statement:
All animals appeared healthy and were within normal limits throughout the
study and the Test Compound (D6) did not show any signs of toxicity at the
doses used
in this study.
Animals were euthanized on Study Day 22. All mice, except Group 9 mice,
were euthanized prior to completing Test compound administration, because mean
tumor size was > 1000 mg in Groups 1-8. Group 9, Cisplatin-treated animals
were
euthanized on Study Day 22 when mean tumor weight was 995 mg. No animals died
while on study.
Mass Palpation Summary
Throughout the study palpable masses were detected in all mice, with tumors
progressively growing for the duration of the study. As expected tumors in
untreated
and vehicle treated negative control mice (Groups l and 2) grew the fastest,
reaching a
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mean tumor size of 1000 mg on or before Study Day 20. In addition, animals
treated
with Cisplatin (Group 9) developed tumors that grew the slowest reaching a
mean
tumor size of 995 mg at study termination (Day 22).
In general, except for Group 3 mice, all animals treated with Test compounds
resulted in slower tumor growth. Animals in Group 3, which were treated with
the low
dose of D6 (0.05 mg/kg) had tumors that grew at approximately the same rate as
the
tumors in untreated and vehicle treated animals in Groups 1 and 2. Animals
treated
with higher doses of D6 (Groups 4 and 5) had tumors that grew slower; reaching
a mean
tumor size of 1000 mg on Study Day 21. When compared to control Groups I and 2
mice, Test compound treatment resulted in a delay of tumor growth of
approximately I
day.
Conclusions
Data from this study validate the model used because tumor-bearing mice in
negative control Groups I and 2 and positive control Group 9 responded as
expected.
Throughout the study palpable masses were observed in all groups. In addition,
all animals were healthy and within normal limits throughout the study.
Furthermore,
the Test compound (D6) did not appear to adversely affect the animals.
Therefore,
these data would suggest that animals treated with D6 had tumors that grew
slowly
(approximately 1 day slower over the 22 day test period than controls). Also,
since the
Test compound did not show any significant toxic effects, higher
concentrations of Test
compound could also be used with potentially better tumor regression.
EXAMPLE 30
The following example describes the preparation of an ultrasound contrast
agent
conjugated to a KDR-binding heterodimer of the invention and the ability of
the
heterdimer conjugated contrast agent to localize to KDR-expressing cells in
vitro and
angiogenic tissue in vivo.
Preparation of derivatized microbubbles for peptide conjugation.
200 mg of DSPC (distearoylphosphatidylcholine), 275 mg of DPPG.Na
(distearoylphosphatidylglycerol sodium salt) and 25 mg of N-MPB-PE were
solubilized
at 60 C in 50 ml of Hexan/isopropanol (42/8). The solvent was evaporated under
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vacuum, and then PEG-4000 (35.046 g) was added to the lipids and the mixture
was
solubilized in 106.92 g of t-butyl alcohol at 60 C, in a water bath. The
solution was
filled in vials with 1.5 ml of solution. The samples were rapidly frozen at -
45 C and
lyophilized. The air in the headspace was replaced with a mixture of C4F1o/Air
(50/50)
and vials capped and crimped. The lyophilized samples were reconstituted with
10 ml
saline solution (0.9%-NaCI) per vial, yielding a suspension of phospholipids
stabilized
microbubbles.
Peptide conjugation
D23 (a dimeric construct of P6- and P12-derived sequences) was conjugated
with a preparation of microbubbles as above described, according to the
following
methodology. The thioacetylated peptide (200 g) was dissolved in 20 1 DMSO and
then diluted in 1 ml of Phosphate Buffer Saline (PBS). This solution was mixed
to the
N-MPB-functionalized microbubbles dispersed in 18 ml of PBS-EDTA 10 mM, pH 7.5
and 2 ml of deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M
hydroxylamine.HCI, pH 7.5) was added. The headspace was filled with C4F1o/Air
(50/50) and the mixture was incubated for 2.5 hours at room temperature under
gentle
agitation (rotating wheel), in the dark. Conjugated bubbles were washed by
centrifugation. Similarly, the monomer peptides making up D23 were separately
conjugated to two different microbubble preparations according to the
methodology
described above.
In vitro assay on transfected cells
The ability of phospholipid stabilized microbubbles conjugated to
heteromultimeric constructs of the invention to bind to KDR-expressing cells
was
assessed using 293H cells transfected to expresss KDR.
Transfection of 293H cells on Thermanox coverslips:
293H cells were transfected with KDR DNA as set forth in Example 6. The
transfected cells were incubated with a suspension of peptide-conjugated
microbubbles
prepared as described above. For the incubation with the transfected cells a
small plastic
cap is filled with a suspension containing I to 3.108 peptide-conjugated
microbubbles
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and the cap covered with an inverted Thermanox coverslip is placed so that
the
transfected cells are in contact with the conjugated microbubbles. After about
20 min at
RT, the coverslip is lifted with tweezers, rinsed three times in PBS and
examined under
a microscope to assess binding of the conjugated microbubbles.
Determination of the % of surface covered by microvesicles
Images were acquired with a digital camera DC300F (Leica) and the percent of
surface covered by bound microbubbles in the imaged area was determined using
the
software QWin (Leica Microsystem AG, Basel, Switzerland). Table 13 shows the
results of the binding affinity (expressed as coverage % of the imaged
surface) of
targeted microvesicles of the invention to KDR transfected cells, as compared
to the
binding of the same targeted microvesicles to Mock-transfected cells.
Table 13.
Conjugated microbubbles
of covered surface
prepared as described above
Peptide code Batch Id KDR Mock
P6 Derivative BG1979T02 3.5% 0.9%
P12 Derivative BG1980T02 16.8% 1.0%
D23 (dieter) BG2002T02 22.9% 3.3%
D6 Deriv./
BG1958T02 12.9% 0.8%
P 12 Deriv.
When the P-6 derived sequence and the P12-derived sequence are separately
attached to phospholipid stabilized microbubbles as monomers the resulting
preparations achieve binding of the bubbles to KDR transfected cells in vitro
to a
different extent (3.5% & 16.8%). When a preparation of phospholipid stabilized
microbubbles resulting from the addition of equal quantities of each of these
peptide
monomers (but the same total peptide load) is tested in the same system 12.9%
binding
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is achieved. Binding is a little more than the average of the two but as it is
achieved
with two sequences that bind to different sites on the target will be more
resistant to
competition at one or other of the sites on the target. However, for D23, the
dimer,
binding is increased to 22.9% (with the same peptide load). These results
indicate that
hetromultimers of the invention permit increased binding and increased
resistance to
competition.
In Vivo animal models
A known model of angiogenic tissue (the rat Mat B III model) was used to
examine the ability of phospholipid stabilized microbubbles conjugated to a
heteromultimer of the invention to localize to and provide images of
angiogenic tissue.
Animals: Female Fisher 344 rat (Charles River Laboratories, France) weighing
120 to
160g were used for the MATBIII tumor implantation. Male OFA rats (Charles
River
Laboratories, France) weighing 100 to 150g were used for Matrigel injection.
Anesthesia
Rats were anesthetized with an intramuscular injection (1 ml/kg) of Ketaminol
/xylazine (Veterinaria AG/Sigma) (50/1 Omg/ml) mixture before implantation of
Matrigel or MatBIII cells. For imaging experiments, animals were anesthetized
with the
same mixture, plus subcutaneous injection of 50% urethane (lg/kg).
Rat MATBIII tumor model
A rat mammary adenocarcinoma, designated 13762 Mat B III, was obtained
from ATCC (CRL-1666) and grown in McCoy's 5a medium + 10% FCS. 1% glutamine
and 1% pen/strep (Invitrogen cat# 15290-018). Cells in suspension were
collected and
washed in growth medium, counted, centrifuged and resuspended in PBS or growth
medium at 1.107 cells per ml. For tumor induction: 1x106 cells in 0.1 ml were
injected
into the mammary fat pad of anesthetized female Fisher 344 rat. Tumors usually
grow
to a diameter of 5-8 mm within 8 days.
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In vivo ultrasound imaging
Tumor imaging was performed using an ultrasound imaging system ATL HDI
5000 apparatus equipped with a L7-4 linear probe. B-mode pulse inversion at
low
acoustic power (MI=0.05) was used to follow accumulation of peptide conjugated-
microbubbles on the KDR receptor expressed on the endothelium of neovessels.
For the
control experiments, an intravenous bolus of unconjugated microbubbles or
microbubbles conjugated to non-specific peptide was injected. The linear probe
was
fixed on the skin directly on line with the implanted tumors and accumulation
of
targeted bubbles was followed during thirty minutes.
A perfusion of SonoVue was administrated before injecting the test bubble
suspension. This allows to evaluate the vascularization status and the video
intensity
obtained after SonoVue injection is taken as an internal reference.
A baseline frame was recorded and then insonation was stopped during the
injection of the microbubbles. At various time points after injection (1, 2,
5, 10, 15, 20,
25, 30 minutes) insonation was reactivated and 2 frames of one second were
recorded
on a videotape.
Video frames from tumor imaging experiments were captured and analysed with
the video-capture and Image-Pro Plus 2.0 software respectively. The same
rectangular
Area of Interest (AOI) including the whole sectional area of the tumor was
selected on
images at different time points (1, 2, 5, 10, 15, 20, 25, 30 minutes). At each
time point,
the sum of the video pixel inside the AOI was calculated after the subtraction
of the
AOI baseline. Results are expressed as the percentage of the signal obtained
with
SonoVue , which is taken as 100%. Similarly, a second AOI situated outside the
tumor, and representing the freely circulating contrast agent, is also
analyzed.
FIG. 37 shows uptake and retention of bubble contrast in the tumor up to 30
minutes post injection for suspensions of phospholipid stabilized microbubbles
conjugated to a heteromultimeric construct of the invention prepared as
described above
(D23). In contrast, the same bubbles showed only transient (no more than 10
minutes)
visualization/bubble contrast in the AOI situated outside the tumor site.
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Other Embodiments
Although the present invention has been described with reference to preferred
embodiments, one skilled in the art can easily ascertain its essential
characteristics and
without departing from the spirit and scope thereof, can make various changes
and
modifications of the invention to adapt it to various usages and conditions.
Those
skilled in the art will recognize or be able to ascertain using no more than
routine
experimentation, many equivalents to the specific embodiments of the invention
described herein. Such equivalents are encompassed in the scope of the present
invention.
All publications and patents mentioned in this specification are herein
incorporated by reference.
We claim:
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