RADIOLABLED HER2 BINDING PEPTIDES
SEQUENCE LISTING
[0001] The instant application contains a Sequence Listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by reference in
its entirety. Said ASCII copy, created on December 13, 2010, is named 2355971.txt
and is 4,957 bytes in size.
FIELD
[0002] The invention relates generally to imaging agents that bind to human
epidermal growth factor receptor type 2 (HER2) and methods for making and using
such agents.
BACKGROUND
[0003] Human epidermal growth factor receptor type 2 (HER2) is a
transmembrane protein and a member of erbB family of receptor tyrosine kinase
proteins. HER2 is a well-established tumor biomarker that is over-expressed in a wide
variety of cancers, including breast, ovarian, lung, gastric, and oral cancers.
Therefore, HER2 has great value as a molecular target and as a diagnostic or
prognostic indicator of patient survival, or a predictive marker of the response to
antineoplastic surgery.
[0004] Over the last decade, noninvasive molecular imaging of HER2 expression
using various imaging modalities has been extensively studied. These modalities
include radionuclide imaging with Positron Emission Tomography (PET) and Single
Photon Emission Tomography (SPECT). PET and SPECT imaging of HER2 (HER2-
PET and HER2-SPECT, respectively) provide high sensitivity, high spatial resolution.
PET imaging of HER2 also provides strong quantification ability. HER2-PET and
HER2-SPECT are particularly useful in real-time assays of overall tumor HER2
expression in patients, identification of HER2 expression in tumors over time,
selection of patients for HER-targeted treatment (e.g., trastuzumab-based therapy),
prediction of response to therapy, evaluation of drug efficacy, and many other
applications. However, no PET or SPECT-labeled HER2 ligands have been
developed that have a chemistry and exhibit in vivo behaviors which would be
suitable for clinical applications.
[0005] Naturally occurring Staphylococcal protein A comprises domains that form
a three-helix structure (a scaffold) that binds to the fragment, crystallizable region
(Fc) of immunoglobulin isotype G (IgG). Certain polypeptides, derived from the Zdomain
of protein A, contain a scaffold composed of three a-helices connected by
loops. Certain amino acid residues situated on two of these helices constitute the
binding site for the Fc region of IgG. Alternative binder molecules have been
prepared by substituting surface-exposed amino acid residues (13 residues) situated
on helices 1 and 2, to alter the binding ability of these molecules. One such example
is HER2 binding molecules or HER2 binders. These HER2 binders have been labeled
with PET or SPECT-active radionuclides. Such PET and SPECT-labeled binders
provide the ability to measure in vivo HER2 expression patterns in patients and would
therefore aid clinicians and researchers in diagnosing, prognosing, and treating HER2-
associated disease conditions.
[0006] HER2 binding affibody molecules, radiolabeled with the PET-active
radionucleide, F, have been evaluated as imaging agents for malignant tumors that
over express HER2. HER2 binding Affibody molecules, conjugated with mTc via
the chelators such as maGGG (mercaptoacetyltriglycyl), CGG (cysteine-diglycyl),
CGGG (SEQ ID NO: 6) (cysteine-triglycyl) or AA3, have also been used for
diagnostic imaging. The binding of these molecules to target HER2 expressing
tumors has been demonstrated in mice.
[0007] In most of the cases, the signal-generating F group is introduced to the
Affibody through a thiol-reactive maleimide group. The thiol reactive maleimide
group is prepared using a multi-step synthesis after F incorporation. However, this
chemistry only provides a low radiochemical yield. Similarly, the conjugation of
mTc with the Affibody is a multistep, low yield, process. In addition, Tc reduction
and the complex formation with chelates, require high pH (e.g., pH=ll) conditions
and long reaction times.
[0008] Though the in vivo performance of F labeled Affibody molecules was
moderately good, there is significant room for improvement. For example, in some
studies, the tumor uptake was found to be only 6.36+1.26 %ID/g 2 hours postinjection
of the imaging agent. On the other hand, mTc labeled Affibody molecules
have predominant hepatobiliary clearance, which causes a high radioactivity
accumulation in the intestine, which restricts its use for detecting HER2 tumors and
metastates in the abdominal area.
[0009] Therefore, there is a need for chemistries and methods for synthesizing
radiolabeled polypeptides in which the radioactive moiety, such as F can be
introduced at the final stage, which in turn will provide high radiochemical yields. In
addition, there is a need for a new HER2 based imaging agent for PET or SPECT
imaging with improved properties particularly related to renal clearance and toxicity
effects.
BRIEF DESCRIPTION
[0010] The compositions of the invention are a new class of imaging agents that
are capable of binding specifically to HER2 or variants thereof.
[0011] In one or more embodiments, the imaging agent composition comprises an
isolated polypeptide comprising SEQ. ID No. 1, SEQ. ID. No 2 or a conservative
variant thereof, conjugated with a mTc via a diaminedioxime chelator. The
diaminedioxime chelator may comprise Pn216, cPn216, Pn44, or derivatives thereof.
The isolated polypeptide binds specifically to HER2 or variants thereof.
[0012] In one or more embodiments, the imaging agent composition comprises an
isolated polypeptide comprising SEQ. ID No. 1, SEQ. ID. No 2 or a conservative
variant thereof, conjugated with Ga or Ga via a NOTA chelator. The isolated
polypeptide binds specifically to HER2 or variants thereof.
[0013] In one or more embodiments, the imaging agent composition comprises an
isolated polypeptide comprising SEQ. ID No. 1, SEQ. ID. No 2 or a conservative
variant thereof, conjugated with F via a linker. The linker comprises a group
derived from an aminoxy group, an azido group, or an alkyne group. The isolated
polypeptide binds specifically to HER2 or variants thereof.
[0014] An example of the methods of the invention, for preparing an imaging
agent composition, comprises (i) providing an isolated polypeptide comprising SEQ.
ID No. 1, SEQ. ID No. 2 or a conservative variant thereof; and (ii) reacting a
diaminedioxime chelator with the polypeptide to form a chelator conjugated
polypeptide. In another example, the method comprises (i) providing an isolated
polypeptide comprising SEQ. ID No. 1, SEQ. ID No. 2 or a conservative variant
thereof; (ii) reacting the polypeptide with a linker; and (iii) reacting the linker with an
F moiety to form a F conjugated polypeptide. The linker may comprise an
aminoxy group, an azido group, or an alkyne group.
FIGURES
[0015] These and other features, aspects, and advantages of the present invention
will become better understood when the following detailed description is read with
reference to the accompanying figures wherein:
[0016] FIGs. 1A and IB are graphs of the surface plasmon resonance (SPR) of the
binding affinity of two anti-HER2 polypeptides, Z477 (SEQ. ID No. 3) and (Z477)2
(SEQ. ID No. 5), respectively, at eight different concentrations, to human HER2.
[0017] FIGs. 2A and FIG. 2B are graphs of the qualitative flow cytometry of C6
(rat glioma, control) and human anti-HER2 antibody to SKOV3 (human ovarian
carcinoma) respectively. FIG. 2C shows a bar chart for Her2 receptors per cell for
SKOV3 and C6 cell lines.
[0018] FIG. 3 is a bar graph of ELISA assays for Her2 with respect to a panel of
tumor types SKOV3 2-1, SKOV3 3-1, SKOV3 3-4, with respect to SKOV3 cells, and
blank.
[0019] FIG. 4 is a reverse phase HPLC gamma chromatogram of mTc labeled
Z00477 (SEQ. ID No. 3).
[0020] FIG. 5A is a size exclusion HPLC gamma chromatogram of aggregated
mTc(CO) (His6)Z00477 (SEQ. ID. No. 4) ('His6' disclosed as SEQ ID NO: 7) at pH
9. FIG. 5B a size exclusion HPLC gamma chromatogram of non aggregated
mTc(CO) (His6)Z00477 ('His6' disclosed as SEQ ID NO: 7) labeled affibody
standard.
[0021] FIG. 6 is a graph of biodistribution profile of Z00477 (SEQ. ID No. 3) in
blood, tumor, liver, kidney and spleen samples from SKOV3 tumor bearing mice,
including the tumonblood ratio over time.
[0022] FIG. 7 is a diagram of the chemical structure for a Mal-cPN216 linker.
[0023] FIG. 8A is a graph of the electrospray ionization time of flight mass
spectrum (ESI-TOF-MS) and FIG. 8B is a graph of mass deconvolution result for the
purified Z00477 (SEQ. ID No. 3)-cPN216.
[0024] FIG. 9 is a reverse phase HPLC gamma trace chromatogram for Z02891-
cPN216 (SEQ. ID No. 2) labeled with mTc.
[0025] FIG. 10 is a graph of the biodistribution profile of Z02891 (SEQ. ID No. 2)
labeled with mTc via cPN216 ( ID, % injected dose)) in blood, liver, kidneys,
spleen, and tail samples from SKOV3 tumor bearing mice.
[0026] FIG. 11 is a graph of the biodistribution profile of Z02891 (SEQ. ID No. 2)
labeled with mTc via cPN216 ( ID, % injected dose) in tumor, blood, liver,
kidneys , bladder/urine, tail, intestine and spleen samples from SKOV3 tumor bearing
mice.
[0027] FIG. 12 is a graph of the biodistribution profile for Z02891 (SEQ. ID No.
2) in SKOV3 tumor bearing mice showing the tumor: blood ratio.
[0028] FIG. 13A and 13B are diagrams of the chemical structures for Bocprotected
malimide-aminoxy (Mal-AO-Boc) and malimide-aminoxy (Mal-AO)
linkers. 13A is the chemical structure for tert-butyl 2-(2-(2,5-dioxo-2,5-dihydro-lHpyrrol-
l-yl)ethylamino)-2-oxoethoxycarbamate (Mal-AO-Boc) and 13B is the
chemical structure for 2-(aminooxy)-N-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)
ethyl)acetamide hydrochloride (Mal-AO. HC1).
[0029] FIG. 14A is the reverse phase HPLC chromatogram of Z00342 (SEQ. ID
No. 1) starting material and 14B is the reverse phase HPLC chromatogram of the
purified Z00342 (SEQ. ID No. l)-AO imaging agent composition, both analyzed at
280 nm.
[0030] FIG. 15 is the reverse phase HPLC gamma chromatogram for the crude
reaction mixtures and purified final products of 18F-fluorobenzyl-Z00342 (SEQ. ID
No. 1) and F-fluorobenzyl-Z02891' (SEQ. ID No. 2).
[0031] FIG. 16 is a graph of the biodistribution profile ( ID, % injected dose) of
the Z02891 (SEQ. ID No. 2) polypeptide labeled with F from SKOV3-tumored
animals.
[0032] FIG. 17 is a graph of the biodistribution profile of Z02891 (SEQ. ID No. 2)
polypeptide labeled with F ( ID, % injected dose) and the tumor: blood ratio from
SKOV3-tumored animals.
[0033] FIG. 18 is bar graph of the biodistribution profile ( ID, % injected dose)
of F labeled Z00342 (SEQ. ID No. 1) and F labeled Z02891 (SEQ. ID No. 2) in
blood, tumor, liver, kidneys, spleen and bone samples.
[0034] FIG. 19 is a diagram of the chemical structure of the Mal-NOTA linker.
[0035] FIG. 20A is a graph of the electrospray ionization time of flight mass
spectrum (ESI-TOF-MS), and 20B is a graph of the ESI-TOF-MS mass deconvolution
result for Z00477 (SEQ. ID No. 3)-NOTA.
[0036] FIG. 2 1 is a graph of the reverse phase HPLC gamma trace for the crude
reaction mixture of Ga-labeled Z00477 (SEQ. ID No. 3)-NOTA after 1 hour of
reaction.
[0037] FIG. 22 is a graph of the reverse phase HPLC gamma trace for the purified
Ga-labeled NOTA Z00477 (SEQ. ID No. 3)-NOTA polypeptide.
DETAILED DESCRIPTION
[0038] The imaging agents of the invention generally comprise an isolated
polypeptide of SEQ. ID No. 1, SEQ. ID No. 2 or a conservative variant thereof,
conjugated with F, mTc, Ga or Ga; and methods for making and using the
compositions. The isolated polypeptide binds specifically to HER2 or its variant
thereof. In one or more embodiments, the sequence of the isolated polypeptide has at
least 90% sequence similarity to any of SEQ. ID No. 1, SEQ. ID No. 2 or
conservative variant thereof.
[0039] The isolated polypeptide may comprise natural amino acids, synthetic
amino acids, or amino acid mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified, for example,
hydroxyproline, g-carboxyglutamate, O-phosphoserine, phosphothreonine, and
phosphotyrosine.
[0040] The isolated polypeptides may be prepared using standard solid phase
synthesis techniques. Alternatively, the polypeptides may be prepared using
recombinant techniques. When the polypeptides are prepared using recombinant
techniques, the DNA encoding the polypeptides or conservative variants thereof may
be isolated. The DNA encoding the polypeptides or conservative variants thereof
may be inserted into a cloning vector, introduced into a host cell (e.g., a eukaryotic
cell, a plant cell, or a prokaryotic cell), and expressed using any art recognized
expression system.
[0041] The polypeptide may be substantially comprised of a single chiral form of
amino acid residues. Thus, polypeptides of the invention may be substantially
comprised of either L-amino acids or D-amino acids; although a combination of Lamino
acids and D-amino acids may also be employed.
[0042] As the polypeptides provided herein are derived from the Z-domain of
protein A, residues on the binding interface may be non-conservatively substituted or
conservatively substituted while preserving binding activity. In some embodiments,
the substituted residues may be derived from any of the 20 naturally occurring amino
acids or any analog thereof.
[0043] The polypeptides may be about 49 residues to about 130 residues in length.
The specific polypeptide sequences are listed in Table 1.
Table 1
KFNKEMRNAYWEIALLPNLNVAQK
RAFIRSLYDDPSQSANLLAEAKKLN IDAQAPKVDC |
[0044] Additional sequences may be added to the termini to impart selected
functionality. Thus, additional sequences may be appended to one or both termini to
facilitate purification or isolation of the polypeptide, alone or coupled to a binding
target (e.g., by appending a His tag to the polypeptide).
[0045] The polypeptides listed in Table 1 may be conjugated with F via a linker;
mTc via a diaminedioxime chelator, or with Ga or Ga via a NOTA chelator.
Table 2 provides the isoelectric point (pi), of these polypeptides.
Table 2
[0046] In one or more embodiments, the isolated polypeptide, comprising SEQ. ID
No. 1, SEQ. ID No. 2 or a conservative variant thereof, may be conjugated with 18F.
The F may be incorporated at a C terminus, at a N-terminus, or at an internal
position of the isolated polypeptide.
[0047] In one or more embodiments, the F may be conjugated to the isolated
polypeptide via a linker. The linker may comprise, an aminoxy group, an azido group,
or an alkyne group. The aminoxy group of the linker may be attached with an
aldehyde, such as a fluorine-substituted aldehyde. An azide group of the linker may
be attached with a fluorine substituted alkyne. Similarly, an alkyne group of the
linker may be attached with a fluorine substituted azide. The linker may also comprise
a thiol reactive group. The linker may comprise of a maleimido-aminoxy, maleimidoalkyne
or maleimido- azide group. The F conjugated polypeptide may be prepared
by: (i) providing the isolated polypeptide comprising SEQ.ID No. 1, SEQ.ID No. 2, or
a conservative variant thereof; (ii) reacting the polypeptide with a linker, wherein the
linker comprises an aminoxy group, an azido group, or an alkyne group, to form a
linker conjugated polypeptide; and reacting the linker with an F moiety.
[0048] In another embodiment, the method may comprise: (i) providing an isolated
polypeptide comprising SEQ. ID No. 1, SEQ. ID No. 2, or a conservative variant
thereof; (ii) providing a linker; (iii) reacting the linker with the F moiety to form a
F labeled linker; and (iv) reacting the F labeled linker with the isolated polypeptide
of SEQ. ID No 1, SEQ ID no 2, or a conservative variant thereof, to form a linker
conjugated polypeptide.
[0049] Using the above-described examples, fluorine or radiofluorine atom(s),
such as 18F, may be introduced onto the polypeptides. A fluorine-substituted
polypeptide results when a fluorine-substituted aldehyde is reacted with the aminoxy
group of the linker conjugated polypeptide. Similarly, a fluorine substituted
polypeptide results, when a fluorine substituted azide or alkyne group is reacted with
the respective alkyne or azide group of the linker conjugated polypeptide. A
radiofluorine-labeled polypeptide or imaging agent composition results, when a
radiofluorine-substituted aldehyde, azide or alkyne is reacted with the respective
aminoxy, alkyne or azide group of the linker conjugated polypeptide. Further, each of
the aldehydes, azides or alkynes may have a radiofluorine (18F) substituent, to prepare
radiofluorine-labeled imaging agent compositions. The methods for introducing
fluorine onto the polypeptide may also be used to prepare a fluorinated imaging agent
composition of any length. Thus, in some embodiments the polypeptide of the
imaging agent composition may comprise, for example, 40 to 130 amino acid
residues.
[0050] The chemistry for the synthesizing linker-conjugated polypeptide of the
imaging agents is facile, and the one step reaction of the methods are more efficient
than previously known methods and result in higher yields. The methods are easier to
carry out, faster and are performed under milder, more user friendly, conditions. For
example, the method for labeling a polypeptide with F conjugated with a linker
(e.g., 18F- fluorobenzaldehyde) is simpler than the procedures known in the art. 18F
conjugated-linker is prepared in one step by the direct nucleophilic incorporation of
18F onto the trimethylanilinium precursor. 18F-linker (i.e., 18F-FBA) is then
conjugated to the polypeptide, such as an affibody. The preparation of the linker is
also easier than previously known methods in the art. Moreover, radiolabeled
aminoxy based linker-conjugated polypeptides, and the cPn family of chelator
conjugated polypeptides (e.g., affibody), show significantly better biodistribution and
better tumor uptake, as well as better clearance with less liver uptake.
[0051] The fluorine-labeled compositions are highly desired materials in
diagnostic applications. F labeled imaging agent compositions may be visualized
using established imaging techniques such as PET.
[0052] In another embodiment, the polypeptide may be conjugated with mTc via
a diamindioxime chelator of formula (1).
OH OH
wherein R , R , R , R is independently H or C1-10 alkyl, C _i 0 alkylary, C2-10
alkoxyalkyl, C O hydroxyalkyl, C O alkylamine, C O fluoroalkyl, or 2 or more R
groups, together with the atoms to which they are attached to form a carbocyclic,
heterocyclic, saturated or unsaturated ring, wherein R may be H, C O alkyl, C -10
alkylary, C2-10 alkoxyalkyl, Ci_io hydroxyalkyl, C O alkylamine, or CM O fluoroalkyl.
In one embodiment, n may vary from 0-5. Examples of methods for preparing
diaminedioxime chelators are described in PCT Application, International Publication
No.WO2004080492(Al) entitled "Methods of radio fluorination of biologically active
vector", and PCT Application, International Publication No.WO2006067376(A2)
entitled "Radio labelled conjugates of RGD-containing peptides and methods for their
preparation via click-chemistry", which are incorporated herein by references.
[0053] The mTc may be conjugated to the isolated polypeptide via the
diamindioxime at the N-terminus of the isolated polypeptide. The chelator may be a
bifunctional compound. In one embodiment, the bifunctional compound may be MalcPN216.
The Mal-cPN216 comprises a thiol-reactive maleimide group for
conjugation to a terminal cysteine of the polypeptide of SEQ ID No. 1 or SEQ ID No
2 and a bis-amineoxime group (diamindioxime chelator) for chelating with mTc.
The Mal-cPN216 may have a formula (2).
[0054] The diamindioxime chelator conjugated peptide may be prepared by (i)
providing an isolated polypeptide comprising SEQ.ID No. 1, SEQ. ID No. 2 or a
conservative variant thereof, (ii) reacting a diamindioxime chelator with the
polypeptide to form the diamindioxime conjugated polypeptide. The diamindioxime
chelator may be further conjugated with mTc.
[0055] In one or more embodiments, the polypeptide may be conjugated with Ga,
or Ga via NOTA (l,4,7-triazacyclononane-N,N',N"-triacetic acid) chelator. The
NOTA conjugated polypeptide may be prepared by (i) providing an isolated
polypeptide comprising SEQ.ID No. 1, SEQ. ID No. 2 or a conservative variant
thereof, (ii) reacting a NOTA chelator with the polypeptide to form the NOTA
conjugated polypeptide. The NOTA chelator may be further conjugated with Ga or
Ga.
[0056] In one embodiment, the Ga, specifically Ga, may be conjugated to the
isolated polypeptide via NOTA chelator. The NOTA chelator may be functionalized
with a maleimido group, as described in formula (3).
[0057] The invention also comprises methods of imaging at least a portion of a
subject. In one embodiment, the method comprises administering the imaging agent
composition to the subject and imaging the subject. The subject may be imaged, for
example, with a diagnostic device.
[0058] The method may further comprise the steps of monitoring the delivery of
the composition to the subject and diagnosing the subject with a HER2-associated
disease condition (e.g., breast cancer). In one embodiment, the subject may be a
mammal, for example, a human. In another embodiment, the subject may comprise
cells or tissues. The tissues may be used in biopsy. The diagnostic device may
employ an imaging method chosen from magnetic resonance imaging, optical
imaging, optical coherence tomography, X-ray, computed tomography, positron
emission tomography, or combinations thereof.
[0059] The imaging agent compositions may be administered to humans and other
animals parenterally. Pharmaceutical compositions of this invention for parenteral
injection comprise pharmaceutically-acceptable sterile aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions as well as sterile powders for
reconstitution into sterile injectable solutions or dispersions just prior to use.
Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles
include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil),
and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained,
for example, by using coating materials such as lecithin, by adjusting the particle size
in dispersions, and by using surfactants.
[0060] These imaging agent compositions may also contain adjuvant such as
preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention
of the action of microorganisms may be ensured by the inclusion of various
antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol
sorbic acid, and the like. It may also be desirable to include isotonic agents such as
sugars, sodium chloride, and the like. Prolonged absorption of the injectable
pharmaceutical form may be brought about by the inclusion of agents, which delay
absorption such as aluminum monostearate and gelatin.
[0061] The imaging agent compositions may be dispersed in physiologically
acceptable carrier to minimize potential toxicity. Thus, the imaging agents may be
dispersed in a biocompatible solution with a pH of about 6 to about 8. In some
embodiments, the agent is dispersed in a biocompatible solution with a pH of about 7
to about 7.4. In other embodiments, the agent is dispersed in a biocompatible solution
with a pH of about 7.4.
[0062] The imaging agent compositions may be combined with other additives that
are commonly used in the pharmaceutical industry to suspend or dissolve the
compounds in an aqueous medium, and then the suspension or solution may be
sterilized by techniques known in the art. The imaging agent composition may be
administered in a variety of forms and adapted to the chosen route of administration.
For example, the agents may be administered topically (i.e., via tissue or mucus
membranes), intravenously, intramuscularly, intradermally, or subcutaneously. Forms
suitable for injection include sterile aqueous solutions or dispersions and sterile
powders for the preparation of sterile injectable solutions, dispersions, liposomal, or
emulsion formulations. Forms suitable for inhalation use include agents such as those
dispersed in an aerosol. Forms suitable for topical administration include creams,
lotions, ointments, and the like.
[0063] The imaging agent compositions may be concentrated to conveniently
deliver a preferred amount of the agents to a subject and packaged in a container in
the desired form. The agent may be dispensed in a container in which it is dispersed
in a physiologically acceptable solution that conveniently facilitates administering the
agent in concentrations between 0.1 mg and 50 mg of the agent per kg body weight of
the subject.
[0064] In one or more embodiments, the target tissue may be imaged about four
hours after administering the agents. In alternative embodiments, the target tissue
may be imaged about 24 hours after administering the agents to the subject.
Examples
[0065] The following examples are provided for illustration only and should not be
construed as limiting the invention.
[0066] A panel of tumorigenic cell lines with a reasonable probability of
expressing HER2 was selected based on available literature (Bruskin, et. al. Nucl.
Med. Biol. 2004: 31: 205; Tran, et. al. Imaging agent composition Chem. 2007: 18:
1956), as described in Table3.
Table 3
Cell line Species Type Purpose
SKOV3 Human Ovarian carcinoma Candidate
SKBR3 Human Breast carcinoma Candidate
C6 Rat Glioma control
[0067] All cell lines were obtained from the American Type Culture Collection
(ATCC) and cultured as recommended. Cells were cultured to > 90% confluence
prior to use. Flow cytometry (Beckman Coulter Cytomics FC500 MPL) was carried
out on the cell lines listed in table 4 using anti-Her2 primary antibodies (R&D
Systems, PN MAB1129) and the Dako QIFIKIT (PN K0078) for quantitative analysis
of indirect immunofluorescence staining. Calibration beads with 5 different
populations bearing different numbers of Mab molecules were used in conjunction
with the cell lines to determine number of surface receptors per cell. In all cases,
appropriate isotype controls were obtained from the corresponding vendors.
[0068] Adherent cells were released from their flasks using cell dissociation buffer
(PBS + 10 mM EDTA) rather than trypsin to avoid proteolysis of cell surface
receptors. Cells were washed twice in PBS and resuspended in ice-cold FC buffer
(PBS + 0.5 % BSA w/v) to a concentration of 5-10 x 106 cells/ml. 100 ΐ aliquots of
cells were mixed with 5 g of primary antibody and incubated, on ice, for 45 minutes.
Cells were then washed with 1 ml of ice cold flow cytometry (FC) buffer (PBS with
2% bovine serum albumin), centrifuged at 300 x g for 5 min, and resuspended in 0.5
m of FC buffer. 100 m of 1:50 dilution with PBS of the secondary antibody
fragment (F(ab)2 FITC-conjugated goat anti-mouse Immunoglobulins) was added and
incubated, on ice and in the dark, for 45 minutes. Cells were then washed twice with
1 mL of ice cold FC buffer, centrifuged at 300 x g for 5min, and resuspended in 500
m of FC buffer. All stained cells were passed through a 100-micron filter prior to
flow cytometry to prevent clogs of the flow cell.
[0069] Flow cytometry was carried out on a Beckman Coulter Cytomics FC500
MPL. A minimum of 5 x 104 events was collected for each tube. All analyses were
single color, with detection of FITC in FL1. Forward scatter (FS) and side scatter
(SS) data demonstrated that all cell populations were tightly grouped.
[0070] Flow cytometry was used to evaluate the cells for their HER2 expression in
vitro (Figs.2A, 2B, and 2C) with SKOV3 cells showing the highest level of HER2
expression (Fig. 3). The results in Fig. 3 were reproducible (n=3).
[0071] The highest expressing cell line was SKOV3. These cells were injected
into 6-12 week old immuno-compromised mice and allowed to grow tumors. Tumor
growth curves and success rates were dependent on the number of cells inoculated.
Optimal tumor growth was obtained with three to four million cells/mouse
[0072] In vivo studies were carried out with female CD-I nude mice (Charles
River Labs, Hopkinton, MA) with an age range between 6 and 15 weeks. Mice were
housed in a ventilated rack with food and water ad libitum and a standard 12 hour
day-night lighting cycle. For xenografts, animals were injected with 100 mΐ of cells in
PBS. Cells were implanted subcutaneously in the right hindquarter. Implantation was
performed under isoflurane anesthesia. For SKOV3, between 3 x 106 to 4 x 106 cells
were implanted in each mouse. Under these conditions, useable tumors (100 to 300
g) were obtained in 3 to 4 weeks in greater than 80% of animals injected.
[0073] Tumors were collected from mice by dissection, and whole tumors were
stored at -20°C until processing. Tumors were ground on ice in 1 ml of RIPA buffer
supplemented with a protease inhibitor cocktail (Santa Cruz Biotech, Santa Cruz, CA
#24948) in a Dounce homogenizer. Homogenates were then incubated on ice for 30
minutes, then centrifuged at 10,000 x G for 10 minutes in a refrigerated centrifuge.
Supernatants were collected and stored on ice or at 4°C until further processing.
Protein concentrations in lysates were determined using a BCA protein assay kit
(Pierce Biotechnology 23225). Lysates were diluted to a standard concentration to
yield 20 g of protein/well in the microtiter plate. ELISA's were run with a
commercially available human HER2 kit (R&D Systems, DYC1129) according to the
manufacturer's instructions. Each sample was run in triplicate, and data are reported
as pg HER2^g total protein, errors are reported as standard deviations.
[0074] Target expression in vivo was measured by ELISA. Excised tumors were
homogenized and analyzed for HER2 using a commercially available matched pair kit
(R&D systems, DYC1129, Minneapolis, MN). The results, in FIG. 3, show that the
SKOV3 cell line grows a high-expressing tumor. ELISA controls were cell-culture
lysates of the negative control lines used for flow cytometry. These results indicate
that tumor xenografts of SKOV3 are appropriate for the in vivo study of molecules
targeting human HER2.
[0075] All polypeptides were received from Affibody AB in Sweden. The
polypeptides are referred to by their numeric internal development codes, which are
prefixed with "Z". Table 1 details the polypeptides described herein. The
polypeptides include polypeptide Z00342 (SEQ. ID No. 1); polypeptide Z02891
(SEQ. ID No. 2); polypeptide Z00477 (SEQ. ID No. 3 and 4), and dimer of Z00477,
i.e., (Z00477)2 (SEQ. ID No. 5).
[0076] Binding interactions between the polypeptids and the HER2/neu antigen
were measured in vitro using surface plasmon resonance (SPR) detection on a
Biacore™ 3000 instrument (GE Healthcare, Piscataway, NJ). The extracellular
domain of the Her2/neu antigen was obtained as a conjugate with the Fc region of
human IgG (Fc-Her2) from R&D Systems (Minneapolis, MN) and covalently
attached to a CM-5 dextran-functionalized sensor chip (GE Healthcare, Piscataway,
NJ) pre-equilibrated with HBS-EP buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM
EDTA, 0.005% v/v surfactant P20) at 10 mE/min and subsequently activated with
EDC and NHS. The Fc-HER2 (5 mg/ml) in 10 mM sodium acetate (pH 5.5) was
injected onto the activated sensor chip until the desired immobilization level (-3000
Resonance Units) was achieved (2 min). Residual activated groups on the sensor chip
were blocked by injection of ethanolamine ( 1 M, pH 8.5). Any non-covalently bound
conjugate was removed by repeated (5x) washing with 2.5 M NaCl, 50 mM NaOH. A
second flow cell on the same sensor chip was treated identically, except with no Fc-
HER2 immobilization, in order to serve as a control surface for refractive index
changes and non-specific binding interactions with the sensor chip. Prior to the
kinetic study, binding of the target analyte was tested on both surfaces and a surface
stability experiment was performed to ensure adequate removal of the bound analyte
and regeneration of the sensor chip following treatment with 2.5 M NaCl, 50 mM
NaOH. SPR sensorgrams were analyzed using the BIAevaluation software (GE
Healthcare, Piscataway, NJ). The robustness of the kinetic model was determined by
evaluation of the residuals and standard error for each of the calculated kinetic
parameters, the "goodness of the fit" (c2 < 10), and a direct comparison of the
modeled sensorgrams to the experimental data. SPR measurements were collected at
eight analyte concentrations (0-100 nM protein) and the resulting sensorgrams were
fitted to a 1:1 Langmuir binding model.
[0077] FIG. 1 shows example surface plasmon resonance (SPR) data obtained for
Z00477 (SEQ. ID No. 3) and (Z00477) 2 (SEQ. ID No. 5) when run on human HER2-
functionalized surfaces. This relationship holds true for all polypeptides for which the
affinities are known (Table 2), in which the values for the dimer Z(477)2 (SEQ. ID
No. 5) are estimates based on avidity affect.
[0078] Labeling of His6 (SEQ ID NO: 7)-tagged Polypeptides with the fac-
[ mTc(CO)3]+ core was accomplished using modifications to a previously published
procedure (Waibel, R.; et al., A. Nat. Biotechnol. 1999, 17, 897.). Briefly,
Na[ mTc0 4] in saline (4 mCi, 2 mL) was added to an Isolink® boranocarbonate kit
(Alberto, R. et al, J . Am. Chem. Soc. 2001, 123, 3135.). The resulting solution was
heated to 95°C for 15-20 minutes, to give fac-[ mTc(CO) (H20)3] + . A portion (2
mCi, 1 mL) of the solution was removed and neutralized to pH ~7 with 1 N HC1. A
325 m aliquot was removed and added to a solution of the His6-Polypeptide (SEQ ID
NO: 7) (40 mg). The resulting solution was heated in a water bath at 35-37°C for 40
minutes. Typical radiochemical yields ranged from 80-95% (determined by ITLCSG,
Biodex, 0.9% NaCl). The crude reaction products were chromatographed on a
NAP-5 column (GE Healthcare, 10 mM PBS) to give products of >99%
radiochemical purity. Typical specific activities obtained were 3-4 mCi m . The
resulting solution was then diluted with 10 mM PBS to give the proper concentration
for subsequent biodistribution studies.
[0079] HPLC was carried out on an Agilent 1100 series HPLC equipped with a
Grace- Vydac Peptide/Protein C4 (4.6 x 250 mm) column and a Raytest GABI
radioactivity detector. Solvent A was 95:5 watenMeCN with 0.1% TFA, and solvent
B was 5:95 watenMeCN with 0.1% TFA. The gradient was as follows (all changes
linear; time/%B): 0/0, 4/20, 16/60, 20/100, 25/100, 26/0, 31/0.
[0080] Each polypeptide was labeled with the tricarbonyltechnetium core in high
yield (>90%) before purification. Purification by NAP-5 chromatography gave
samples of mTc-labeled Polypeptides in >99% radiochemical purity (Table 4)
Table 4
[0081] Representative HPLC chromatograms of NAP-5 purified radiolabeled
polypeptides are shown in FIG. 4. The retention time of the radiolabeled species was
virtually unchanged from the corresponding unlabeled polypeptide's retention time in
a 220 nm UV chromatogram (except for the time difference due to the physical
separation of the UV and gamma detectors; data not shown).
Animal Models used to study Tc(CO) (His6)-Polypeptides ('His6' disclosed as SEQ
ID NO: 7)
[0082] In vivo studies were carried out with female CD-I nude mice (Charles
River Labs, Hopkinton, MA) with an age range between 6 and 15 weeks. Mice were
housed in a ventilated rack with food and water ad libitum and a standard 12 hour
day-night lighting cycle. For xenografts, animals were injected with 100 mΐ of cells in
PBS. Cells were implanted subcutaneously in the right hindquarter. Implantation was
performed under isoflurane anesthesia. For SKOV3, between 3 x 106 to 4 x 106 cells
were implanted in each mouse. Under these conditions, useable tumors (100 to 300
g) were obtained in 3 to 4 weeks in greater than 80% of animals injected.
Biodistribution
[0083] Mice were given tail-vein injections of ~ 1 mg of mTc-labeled polypeptides
(~3 m l mg). Mice were placed in filter-paper lined cages until euthanasia. Three
mice were euthanized at each timepoint and tissues of interest dissected and counted
on a Perkin Elmer Wallac Wizard 1480 Gamma Counter. Data were collected for
blood, kidney, liver, spleen, and injection site (tail). Urine from cages was pooled
with the bladder and also counted. The remaining tissues were counted and the sum
of all tissues plus urine for each animal was summed to provide the total injected
dose. The % injected dose for each organ was determined based on this total, and
organs were weighed for determination of the % injected dose per gram, (%ID/g).
Data is reported as mean value for all three mice in the timepoint with error bars
representing the standard deviation of the group.
[0084] The mTc labeled Z00477 (SEQ. ID No. 4) polypeptide was injected into
SKOV3 mice. FIG. 6 shows the tumor and blood curves for these experiments. The
Z00477 (SEQ. ID No. 4) polypeptide shows good tumor uptake in target-expressing
SKOV3 tumors, with a maximal value of approximately 3% of the injected dose per
gram of tissue at 30 minutes post-injection (PI), and a peak tumor: blood ratio of more
than 8 at 240 minutes PI.
[0085] Polypeptides exhibit a monoexponential clearance from the blood with
half-lives of less than two minutes. This clearance is primarily mediated by the liver
and kidneys. Polypeptide uptake in the spleen was moderate, and moderate to high
uptake in the liver is observed, as described in Table 5.
Table 5. Z00477 (SEQ. ID No. 3) His6 (SEQ ID NO: 7)tagged uptake (%ID/g) in
SKOV3 tumor bearing mice
[0086] Bivalent polypeptides exhibit higher affinity than the corresponding
monomers, presumably due to the avidity effect. Their larger size, however, may
hinder tumor penetration. For the HER2 polypeptides, bivalent forms of each the four
high affinity polypeptides were available. The Z00477 (SEQ. ID No. 3) dimer,
(Z00477)2 (SEQ. ID No. 5), was radiolabeled and used for a four-hour biodistribution
experiment in SKOV3-tumored mice.
[0087] The monovalent and bivalent polypeptides otherwise exhibit similar
biodistribution characteristics, and blood half-lives are observed for both in the one to
two minute range. The results clearly indicate that both monomeric and divalent
polypeptides can be targeted to HER2 in vivo.
[0088] To introduce the mTc chelator cPN216 (FIG. 7), a bifunctional compound
Mal-cPN216 was synthesized comprising of a thiol-reactive maleimide group for
conjugation to a terminal cysteine of a polypeptide and an amine oxime group for
chelating mTc.
[0089] cPN216-amine was obtained from GE Healthcare. N-b-
maleimidopropionic acid was purchased from Pierce Technologies (Rockford, IL). Nmethylmorpholine,
(benzotriazol- 1-yloxy) tripyrrolidinophosphonium
hexafluorophosphate (PyBoP), dithiothreitol (DTT), ammonium bicarbonate, and
anhydrous DMF were purchased from Aldrich (Milwaukee, WI). PBS buffer (lx, pH
7.4) was obtained from Invitrogen (Carlsbad, CA). HPLC-grade acetonitrile
(CH 3CN), HPLC-grade trifluoroacetic acid (TFA), and Millipore 18 ihW water were
used for HPLC purifications.
[0090] To an ice-cooled solution of N-b-maleimidopropionic acid (108 mg, 0.64
mmol), cPN216-amine (200 mg, 0.58 mmol), and PyBoP (333 mg, 0.64 mmol) in
anhydrous DMF at 0 °C was added 0.4 M of N-methylmorpholine in DMF (128 ,
1.16 mmol). The ice bath was removed after 2 hrs, and the mixture was stirred at
room temperature overnight before being subjected to HPLC purification. The
product was obtained as a white powder (230 mg, 80% yield). 1H-NMR (400MHz,
DMSO-d6): d 1.35 (m, 2 H), 1.43 (s, 12 H), 1.56 (m, 5 H), 1.85 (s, 6 H), 2.33 (dd, J l
= 8 Hz, J2 = 4 Hz, 2 H), 2.78 (m, 4 H), 3.04 (m, 2 H), 3.61 (dd, J l = 8 Hz, J2 = 4 Hz,
2 H), 7.02 (s, 2 H), 8.02 (s, 1 H), 8.68 (s, 4 H), 11.26 (s, 2 H); m/z = 495.2 for
[M+H]+ (C24H43N605, Calculated MW = 495.3).
[0091] The polypeptide was dissolved with freshly degassed PBS buffer (lx, pH
7.4) at a concentration of approximately 1 mg/mL. The disulfide linkage in the
polypeptide was reduced by the addition of DTT solution in freshly degassed PBS
buffer (lx, pH 7.4). The final concentration of DTT was 20 mM. The reaction
mixture was vortexed for 2 hours and passed through a Zeba desalt spin column
(Pierce Technologies) pre-equilibrated with degassed PBS buffer (lx, pH 7.4) to
remove excess of DTT reagent. The eluted reduced polypeptide molecule was
collected, and the bifunctional compound Mal-cPN216 was added (20 equivalents per
equivalent of the polypeptide) as a solution in DMSO, and the mixture was vortexed
at room temperature for 3 hours and frozen with liquid-nitrogen. The reaction mixture
was stored overnight before being subject to Reverse phase HPLC purification (FIGs.
8A and 8B).
[0092] The HPLC purification was performed on a MiCHROM Magic C18AQ 5 m
200A column (MiChrom Bioresources, Auburn, CA). Solvent A: H20 (with 0.1%
formic acid), Solvent B: CH CN (with 0.1% formic acid). Gradient: 5-100% B over
30 mins.
[0093] The fractions containing desired product were combined and neutralized
with 100 mM ammonium bicarbonate solution, and the solvents were removed by
lyophilization to give the desired imaging agent composition as a white solid (yield
41%).
[0094] LC-MS analysis of the purified product confirmed the presence of the
desired product, and the MW suggested that only one cPN216 label was added to
polypeptide constructs (Z00477 (SEQ. ID No. 3)-cPN216: calculated MW: 7429 Da,
found: 7429 Da; Z02891 (SEQ. ID No. 2) -cPN216 calculated MW: 7524 Da, found:
7524 Da).
[0095] To a 20 mL vial was added 10.00 mL of distilled, deionized water.
Nitrogen gas was bubbled through this solution for approximately 30 minutes prior to
addition of the NaHC0 (450 mg, 5.36xl0 3 mol), Na2C0 (60 mg, 5.66xl0 4 mol)
and sodium para-aminobenzoate (20 mg, 1.26xl0 4 mol). All reagents were weighed
independently and added to the vial containing water. Tin chloride (1.6 mg, 7.09xl0 6
mol) and MDP (2.5 mg, 1.42xl0 5 mol) were weighed together into a 1 dram vial and
subsequently transferred (with 1 subsequent wash) by rapid suspension in
approximately 1 mL of the carbonate buffer mixture. 10 aliquots were removed
and transferred under a stream of nitrogen to silanized vials, immediately frozen and
maintained in a liquid nitrogen bath until lyophilization. Each vial was partially
capped with rubber septa and placed in a tray lyophilizer overnight. Vials were sealed
under vacuum, removed from the lyophilizer, crimp-sealed with aluminum caps, repressurized
with anhydrous nitrogen and stored in a freezer until future use.
[0096] Synthesis of the radiolabeled polypeptide was performed using a one-step
kit formulation produced in house (Chelakit A+) containing a lyophilized mixture of
stannous chloride as a reducing agent for technetium, methylene diphosphonic acid,
p-aminobenzoate as a free -radical scavenger and sodium bicarbonate/sodium
carbonate (pH 9.2) as buffer. In rapid succession, 20 of a 2 g/ L solution of
polypeptide in saline was added to the Chelakit, followed immediately by Na mTcC>4
(0.8 mCi, 29.6 MBq) in 0.080 mL of saline (0.1 5M NaCl) obtained from Cardinal
Health (Albany, NY). The mixture was agitated once and allowed to sit at ambient
temperature for 20 min. Upon completion, the crude radiochemical yield was
determined by ITLC (Table 6 below according to ITLC-SG, Biodex, 0.9% NaCl).
Table 6
[0097] The reaction volume was increased to 0.45 mL with 0.35 mL of 150 mM
sterile NaCl, and the final product purified by size exclusion chromatography (NAP5,
GE Healthcare, charged with 10 mM PBS). The crude reaction mixture was loaded
onto the NAP5 column, allowed to enter the gel bed and the final purified product
isolated after elution with 0.8 mL of 10 mL PBS. Final activity was assayed in a
standard dose calibrator (CRC-15R, Capintec, Ramsey, NJ). Radiochemical yield
(Table6) and purity were determined by ITLC (>98.5%), C4 RP-HPLC (FIG. 9) and
SEC-HPLC analysis. The final product (10-15 m /m , 0.2 - 0.5 m m
(0.37MBq^g, 7.4MBq/mL)) was used immediately for biodistribution studies.
[0098] The HPLC conditions used for this experiment were as follows: C4 RPHPLC
method 1: Solvent A: 95/5 H20/CH CN (with 0.05% TFA), Solvent B: 95/5
CH CN/ddH 20 (distilled, deionized water) with 0.05% TFA. Gradient elution: 0 min.
0%B, 4 min. 20%B, 16min. 60%B, 20 min. 100%B, 25 min. 100%B, 26 min. 0%B,
3 1 min. 0%B.
[0099] C4 RP-HPLC method 2 : Solvent A: 0.06% NH in water, Solvent B:
CH CN. Gradient elution: 0 min. 0%B, 4 min. 20%B, 16min. 60%B, 20 min. 100%B,
25 min. 100%B, 26 min. 0%B, 3 1 min. 0%B.
[0100] RP-HPLC analysis performed on an HP Agilent 1100 with a G1311A
QuatPump, G1313A autoinjector with IOOm syringe and 2.0mL seat capillary, Grace
Vydac - protein C4 column (S/N E050929-2-1, 4.6 mmxl50 mm), G1316A column
heater, G1315A DAD and Ramon Star - GABI gamma-detector.
[0101] SEC HPLC: Solvent: l x (10 mM) PBS (Gibco, Invitrogen, pH 7.4
containing CaCl2 and MgCl2) . Isocratic elution for 30 min. Analysis performed on a :
Perkin Elmer SEC-4 Solvent Environmental control, Series 410 LC pump, ISS 200
Advanced LC sample processor and Series 200 Diode Array Detector. A Raytest
GABI with Socket 8103 0111 pinhole (0.7 mm inner diameter with 250 E volume)
flow cell gamma detector was interfaced through a Perkin Elmer NCI 900 Network
Chromatography Interface. The column used was a Superdex 75 10/300 GL High
Performance SEC column (GE Healthcare code: 17-5174-01, ID no. 0639059).
[0102] The operating pH of the Chelakits used to incorporate mTc into the
cPN216 chelate (pH = 9.2) nearly matched the calculated pi of the Z00477 (SEQ. ID
No. 3) polypeptide. Labeling under these conditions were determined to cause
aggregation in the final product (FIGs. 5A and 5B). Aggregation was confirmed by
size exclusion HPLC and through the increased blood residence time and liver uptake
observed in the biodistribution studies. By altering the isoelectric point of the
polypeptide, mTc was successfully incorporated onto the Z02891 (SEQ. ID No. 2)
construct. Size exclusion HPLC confirmed the presence of a species with the
appropriate molecular weight and biodistribution studies showed uptake of the tracer
into the tumor xenografts.
[0103] In vivo studies were carried out with female CD-I nude mice (Charles
River Labs, Hopkinton, MA) with an age range between 6 and 15 weeks. Mice were
housed in a ventilated rack with food and water ad libitum and a standard 12 hours
day-night lighting cycle. For xenografts, animals were injected with 100 mΐ of cells in
PBS. Cells were implanted subcutaneously in the right hindquarter. Implantation was
performed under isoflurane anesthesia. For SKOV3, between 3 x 106 to 4 x 106 cells
were implanted in each mouse. Under these conditions, useable tumors (100 to 300
) were obtained in 3 to 4 weeks in greater than 80% of animals injected.
[0104] Mice were given tail-vein injections of ~ 1 ug of mTc-labeled polypeptides
(-10 m( ί 1 mg). Mice were placed in filter-paper lined cages until euthanasia. Three
mice were euthanized at each timepoint and tissues of interest dissected and counted
on a Perkin Elmer Wallac Wizard 1480 Gamma Counter. Data were collected for
blood, kidney, liver, spleen, and injection site (tail). Urine from cages was pooled
with the bladder and also counted. The remaining tissues were counted and the sum
of all tissues plus urine for each animal was summed to provide the total injected
dose. The % injected dose for each organ was determined based on this total, and
organs were weighed for determination of the % injected dose per gram, (%ID/g).
Data is reported as mean value for all four to five mice in the time point with error
bars representing the standard deviation of the group. Four time points were taken
over four hours (5, 30,120, and 240 minutes post-injection).
[0105] The Z02891 (SEQ. ID No. 2) -cPN216- mTc polypeptide shows strong
tumor uptake in target-expressing SKOV3 tumors, with a value of 7.1 1 ± 1.69% (n=5)
of the injected dose per gram of tissue at 30 minutes post-injection (PI), which
remains fairly constant over the time-course of the study up to 240 min PI. Tumor:
blood ratios were 2, 5, and 5 at 30, 120, and 240 min post injection, respectively.
FIG. 10, 11 and 12 show the tumor, blood and tumor: blood curves for these
experiments.
[0106] The Polypeptides exhibit a monoexponential clearance from the blood with
half-lives of less than two minutes. This clearance is primarily mediated by the
kidneys, with 10.58 ± 2.96 (n=5) ID/organ at 240 min post-injection PI. Activity is
secreted primarily in the urine. Polypeptide uptake in the spleen was moderate to
high due to possible aggregation, and moderate uptake in the liver is observed, e.g.,
12 %ID/organ (equivalent in value in mice to %ID/g) over the course of the study.
Biodistribution results for Z02891 (SEP. ID No. 2)-cPN216- 99mTc
Table 7. Z02891 (SEQ. ID No. 2) cPN216 uptake (%ID/g) in SKOV3 tumor bearing
mice
5 Minutes 30 Minutes 120 Minutes 240 Minutes
Blood 8.69 ± 0.99 (n=5) 3.32 ± 0.48 (n=5) 1.33 ± 0.05 (n=5) 1.05 ± 0.09 (n=5)
Tumor 3.19 ± 1.78 (n=4) 7.11 ± 1.69 (n=5) 7.18 ± 3.33 (n=5) 5.07 ± 3.47 (n=5)
Liver 9.87 ± 0.81 (n=5) 11.07±1.06 (n=5) 8.33 ± 0.50 (n=5) 9.38 ± 0.69 (n=5)
Kidney 67.61±9.24 (n=5) 74.15±4.17 (n=5) 37.14±3.48 (n=5) 29.67±10.87 (n=5)
Spleen 7.07 ± 1.84 (n=5) 4.51 ± 1.25 (n=5) 3.91 ± 0.44 (n=5) 2.85 ± 0.62 (n=5)
[0107] Z00477 (SEQ. ID. NO. 4), Z00342 (SEQ. ID No. 1) and Z02891 (SEQ. ID
No. 2)-cysteine polypeptides were functionalized with an aminoxy group via an
engineered C-terminal cysteine. The purity of the polypeptide molecules provided
was determined to be >95 by High Performance Liquid Chromatography (HPLC).
[0108] To incorporate F into the Polypeptide molecules, a bifunctional linker
Mai-AO was synthesized comprising of two orthogonal groups: a thiol-reactive
maleimide group for conjugation to the engineered cysteine and an aldehyde-reactive
aminoxy group (FIGs. 13A and 13B). This linker was prepared by reacting N-(2-
aminoethyl) malemide with 2-(tert-butoxycarbonylaminooxy) acetic acid using 1-
ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) -mediated coupling conditions
yielding the Boc-protected form of the linker. The Boc protecting group was then deprotected
by acid cleavage to give the final Mai-AO product in quantitative yield. The
final product was used directly without further purification.
[0109] Dichloromethane, 2-(tert-butoxycarbonylaminooxy) acetic acid,
triethylamine, N-(2-aminoethyl)maleimide trifluoroacetic acid (TFA) salt, Nhydroxybenzotriazole
hydrate (HOBT), l-ethyl-3-[3-
dimethylaminopropyljcarbodiimide (EDC), dithiothriotol (DTT), and all other
standard synthesis reagents were purchased from Sigma-Aldrich Chemical Co. (St.
Louis, MO). All chemicals were used without further purification. PBS buffer (lx, pH
7.4) was obtained from Invitrogen (Carlsbad, CA). HPLC-grade ethyl acetate,
hexanes, acetonitrile (CH 3CN), trifluoroacetic acid (TFA), and Millipore 18 ihW
water were used for purifications.
[0110] To a solution of 2-(tert-butoxycarbonylaminooxy)acetic acid (382 mg, 2
mmol) in anhydrous dichloromethane (20 mL) was added sequentially triethylamine
(307 m , 2.2 mmol), N-(2-aminoethyl)maleimide-TFA salt (508 mg, 2 mmol),
HOBT(306 mg, 2 mmol), and EDC (420 mg, 2.2 mmol). After being stirred for 24
hrs at room temperature, the reaction mixture was diluted with ethyl acetate (50 mL)
and washed with saturated sodium bicarbonate solution (3 x 30 mL), water (30 mL),
and brine (30 mL). The organic layer was dried over anhydrous magnesium sulfate
and filtered. The filtrate was concentrated to a pale yellow solid, which was purified
by column chromatography (70% ethyl acetate in hexanes) to give the product as a
white powder (500 mg, 80% yield). *H-NMR (400MHZ, CDCI3) : d 1.50 (s, 9 H), 3.55
(tt, Jl= 6.0 Hz, J2= 6.5 Hz, 2 H), 3.77 (dd, J= 7.6 Hz, 2 H), 4.30 (s, 2 H), 6.3 (s, 2 H).
[0111] A solution of 9.3 mg of Mal-AO-Boc in 1 mL of 3M HC1 in methanol was
stirred at room temperature for 18 hours. Solvents were removed under vacuum to
yield Mal-AO as a light yellow solid. (80% yield). *H-NMR (400MHZ , DMSO-d6) :
d 3.27 CH2 (t, J= 4.0 Hz, 2H), 3.49 CH2 (t, J= 4.0 Hz, 2H), 4.39 CH20 (s, 2H), 7.00
CH=CH (s, 2H); m/z = 214.07 for [M+H]+ (C H12N 0 4, Calculated MW = 214.11) )
[0112] The polypeptide was dissolved with freshly degassed PBS buffer (lx, pH
7.4) at a concentration of approximately 1 mg/mL. The disulfide linkage in the
polypeptide was reduced by the addition of dithiothreitol (DTT) solution in freshly
degassed PBS buffer (lx, pH 7.4). The final concentration of DTT is 20 mM. The
reaction mixture was vortexed for 2 hours and eluted through a Zeba desalt spin
column (Pierce Technologies) pre-equilibrated with degassed PBS buffer to remove
excess of DTT reagent. The reduced polypeptide was collected, and the bifunctional
Mal-AO compound was added (15 equivalents per equivalent of the polypeptide) as a
solution in DMSO. After being vortexed at room temperature overnight, the reaction
mixture was purified with High Performance Liquid Chromatography (HPLC) (FIGs.
14A and 14B).
[0113] The HPLC purification was performed on a MiCHROM Magic C18AQ 5m
200A column (MiChrom Bioresources, Auburn, CA). Solvent A: H20 (with 0.1%
formic acid), Solvent B: CH CN (with 0.1% formic acid). Gradient: 5-100% B over
30 mins. The fractions containing desired product was combined and neutralized with
100 mM ammonium bicarbonate solution, and the solvents were removed by
lyophilization to give the aminoxy-modified polypeptide as a white solid.
[0114] ESI-TOF-MS analysis confirmed the presence of target product with the
expected molecular weights (calculated MW: 6964 Da, 8531 Da, and 7243 Da, found:
6963 Da, 8532 Da, and 7244 Da for Z00477 (SEQ. ID No. 4)-ONH 2, Z00342 (SEQ.
ID No. l)-ONH 2, and Z02891 (SEQ. ID No. 2) -ONH 2, respectively.
[0115] Methods: All reactions were performed either under a nitrogen atmosphere
or in a crimp-top sealed vial purged with nitrogen prior to use. Kryptofix 222
(Aldrich) and K2CC>3 (EMD Science) were purchased and used as received.
Optima™-grade acetonitrile was used as both HPLC and reaction solvents.
[0116] K F (40mCi mL 1 (1480 MBq mL ) in purified water) was obtained from
IBA Molecular (Albany, NY) and PETNET Solutions (Albany, NY) and were used as
received. The [18F ] fluoride was first immobilized on a Chromafix 30-PS-HCO3
anion exchange cartridge (ABX, Radeberg, Germany), then eluted into a drydown
vessel with a 1 mL, 4:1 mixture of acetonitrile: distilled, deionized H20 (ddH 20 )
containing Kryptofix K222 (376 g.mol , 8 mg, 2.13xl0 5 mol) and potassium
carbonate (138.2 g.mol 1, 2.1 mg, 1.52xl0 5 mol). The solvent was removed under
partial vacuum and a flow of nitrogen with gentle heating (~ 45°C) (-15 min). The
source vial and anion exchange cartridge were then washed with 0.5mL of acetonitrile
containing K222 (8 mg) and the reaction mixture again brought to dryness under
partial vacuum and gentle heating ( 10 min). The reaction vessel was repressurized
with nitrogen and the azeotropic drydown repeated once with an additional 0.5mL of
acetonitrile. 4-formyl-N,N,N-trimethylanilinium triflate (313.30 g mol , 3.1 mg,
9.89X10 6 mol) was dissolved in 0.35 mL of anhydrous DMSO (Acros) and added
directly to the reaction vessel containing the K18F K222, K2CC>3. The reaction mixture
was heated to 90°C for 15 min and immediately cooled and quenched with 3 mL of
ddH 20 . This mixture was subsequently passed through a cation exchange cartridge
(Waters SepPak Light Accell Plus CM), diluted to 10 mL with ddH 20 , and loaded
onto a reverse phase C18 SepPak (Waters SepPak Plus CI 8). The SepPak was flushed
with 10 mL of ddH 20 then purged with 30 mL of air. [ F]4-fluorobenzaldehyde
( FBA), was eluted in 1.0 mL of methanol.
[0117] Separately, a high recovery vial (2mL, National Scientific) was charged
with either the Z00477-(SEQ. ID No. 3)-ONH2 (0.35-0.5mg), Z00342-(SEQ. ID
No.l)-ONH 2 (0.35-0.5mg) or Z02891-(SEQ. ID No. 2)-ONH2 (0.35-0.5mg). The
solid was suspended in 25 L· of ddH20 and 8 L· of trifluoroacetic acid. 25 L· of
18FBA in methanol (see above) was transferred to the reaction vial. The vessel was
capped, crimped, placed in a heating block and maintained at 60°C for 15 minutes; at
which point a small aliquot (<5 ) was removed for analytical HPLC analysis . 450
m of ddH20 with 0.1% TFA was used to dilute the solution to approx. 500 L· in
preparation for semi-preparative HPLC purification. FB-Polypeptide was isolated
and purified by semi-preparative HPLC . The HPLC fraction containing the product
(0.113 mCi/4.18MBq) was diluted 5:1 with ddH20 and subsequently immobilized on
a tC18 Plus Sep Pak (Waters). The SepPak was flushed first with 5 mL of ddH20 then
30 mL of air. 18FB-Polypeptide was isolated in a minimal amount of ethanol by first
eluting the void volume (approx. 0.5mL) followed by collecting 250 to 300 of
eluent in a separate flask. RP-HPLC analysis was performed on the isolated product in
order to establish radiochemical and chemical purity. Typically, 10 L· of a 0.1
solution was injected for post formulation analysis. Isolated radiochemical
yields are indicated in Table 9 and are decay corrected from the addition of
polypeptide to 18FBA and radiochemical purity of >99%. Alternatively, 18F-labeled
polypeptides were isolated by NAP5 size exclusion chromatography by diluting the
reaction mixture to approximately 0.5mL with lOmM PBS and loading onto the gel.
The 18F-labled polypeptides were isolated by eluting the column with 0.8 mL of
lOmM PBS and used without further modification. These results are illustrated in
Table 8, and FIG. 15.
Table 8
[0118] Analytical HPLC conditions used are as follows: Analysis performed on an
HP Agilent 1100 with a G1311A QuatPump, G1313A autoinjector with IOOm
syringe and 2.0mL seat capillary, Phenomenex Gemini CI8 column
(4.6mmxl50mm), 5m, 100A (S/N 420477-10), G1316A column heater, G1315A
DAD and Ramon Star - GABI gamma-detector. 95:5 ddH20:CH CN with 0.05%
TFA, Solvent B: CH CN with 0.05% TFA. Gradient elution (1.0 mLmin ) : 0 min.
0%B, 1 min. 15%B, 21min. 50%B, 22 min. 100%B, 26 min. 100%B, 27 min. 0%B,
32 min. 0%B. or gradient elution (1.2 mL min 1) : 0 min. 0%B, 1 min. 15%B, lOmin.
31%B, 10.5 min. 100%B, 13.5 min. 100%B, 14 min. 0%B, 17 min. 0%B.
[0119] Semipreparative HPLC conditions used are as follows: Purification was
performed on a Jasco LC with a DG-2080-54 4-line Degasser, an MX-2080-32
Dynamic Mixer and two PU-2086 Plus Prep pumps, an AS-2055 Plus Intelligent
autoinjector with large volume injection kit installed, a Phenomenex 5 Luna CI 8(2)
100A, 250 x 10 mm, 5 m column with guard (S/N 295860-1, P/N 00G-4252-N0), an
MD-2055 PDA and a Carroll & Ramsey Associates Model 105S Analogue Ratemeter
attached to a solid-state SiPIN photodiode gamma detector. Gradient elution: 0 min.
5%B, 32 min. 20%B, 43 min. 95%B, 46 min. 95%B, 49 min. 5%B, Solvent A:
ddH20:CH CN with 0.05% TFA, Solvent B: CH CN with 0.05% TFA.
[0120] In vivo studies were carried out with female CD-I nude mice (Charles
River Labs, Hopkinton, MA) with an age range between 6 and 15 weeks. Mice were
housed in a ventilated rack with food and water ad libitum and a standard 12 hour
day-night lighting cycle. For xenografts, animals were injected with 100 mΐ of cells in
PBS. Cells were implanted subcutaneously in the right hindquarter. Implantation was
performed under isoflurane anesthesia. For SKOV3, between 3 x 106 to 4 x 106 cells
were implanted in each mouse. Under these conditions, useable tumors (100 to 300
mg) were obtained in 3 to 4 weeks in greater than 80% of animals injected.
[0121] Mice were given tail-vein injections of ~ 1 ug of F-labeled polypeptide
(~4 uCi/1 g). Mice were placed in filter-paper lined cages until euthanasia. Three
mice were euthanized at each timepoint and tissues of interest dissected and counted
on a Perkin Elmer Wallac Wizard 1480 Gamma Counter. Data were collected for
blood, kidney, liver, spleen, bone and injection site (tail). Urine from cages was
pooled with the bladder and also counted. The remaining tissues were counted and
the sum of all tissues plus urine for each animal was summed to provide the total
injected dose. The percent injected dose for each organ was determined based on this
total, and organs were weighed for determination of the percent injected dose per
gram, (%ID/g). Data is reported as mean value for all three mice in the timepoint
with error bars representing the standard deviation of the group.
[0122] The polypeptides underwent biodistribution studies in SKOV3 cell
xenograft models. Four time points were taken over four hours (5, 30, 120, and 240
minutes post-injection). Complete biodistribution data are included in table 12
(%ID/g Z02891 (SEQ. ID No. 2) - F-fluorobenzyl oxime in SKOV3 Tumor Bearing
Mice) and table 13 (%ID/g Z00342 (SEQ. ID No. 1) F-fluorobenzyl oxime in
SKOV3 Tumor Bearing Mice). FIGs. 16, 17 and 18 show the tumor, blood, tumor:
blood, and clearance curves for these tests.
[0123] The Z02891 (SEQ. ID No. 2) F-fluorobenzyl oxime polypeptide shows
strong tumor uptake in target-expressing SKOV3 tumors, with a value of 17.47 ± 2.89
(n=3) of the injected dose per gram of tissue at 240 minutes post-injection (PI).
Tumor: blood ratios were approximately 3, 34, and 128 at 30, 120, and 240 min post
injection, respectively. The Z00342 (SEQ. ID No. 1) 18F-fluorobenzyl oxime
polypeptide shows strong tumor uptake in target-expressing SKOV3 tumors, with a
value of 12.45 ± 2.52 (n=3) of the injected dose per gram of tissue at 240 minutes PI.
Tumor: blood ratios were approximately 3, 32 and 53 at 30, 120 and 240 min post
injection, respectively.
[0124] The polypeptides exhibit a monoexponential clearance from the blood with
half-lives of less than two minutes. This clearance of Z02891 (SEQ. ID No. 2) is
primarily mediated by the kidneys, with 0.95 ± 0.07 (n=3) ID/organ at 240 min PI.
Activity is secreted primarily in the urine. Polypeptide uptake in the spleen was
minimal, and low uptake in the liver is observed, ca. 1.8 %ID/organ (equivalent in
value in mice to %ID/g) over the course of the study (four hours post injection).
Table 9. Z02891 (SEQ. ID No. 2) sF-fluorobenzyl oxime uptake (%ID/g) in SKOV-
3 tumor bearing mice
Tumor 2.39 ± 1.13 (n=3) 8.91 ± 2.09(n=3) 13.47 ± 3.61 17.47 ± 2.89 (n=3)
(n=3)
Liver 4.68 ± 0.45 (n=3) 3.85 ± 0.95 1.57 ± 0.42 (n=3) 1.59 ± 0.83 (n=3)
(n=3)
Kidney 72.42 ± 35.02 ± 5.22 ± 0.65 (n=3) 2.49 ± 0.17 (n=3)
15.61(n=3) 5.76(n=3)
Spleen 3.04 ± 1.15 (n=3) 1.46 ± 0.05 0.37 ± 0.01 (n=3) 0.26 ± 0.04 (n=3)
(n=3)
Table 10.Z00342 (SEQ. ID No. 1) sF-fluorobenzyl oxime uptake (%ID/g) in SKOV-
3 tumor bearing mice
[0125] All reactions are performed either under a nitrogen atmosphere or in a
crimp-top sealed vial purged with nitrogen. Optima™-grade acetonitrile is used as
both HPLC and reaction solvents.
[0126] [123I]4-iodobenzaldehyde (123 I BA) is added to a high recovery vial (2 mL,
National Scientific) containing the polypeptide-ONH 2 (Z02891, SEQ. ID No. 2),
0.35-0.5mg). The reaction commences by dissolving the polypeptide in 25 mL· of
ddH20 and adding 8 mL· of trifluoroacetic acid followed by the addition of IIBA in
methanol. The vessel is capped, crimped, placed in a heating block and maintained at
60°C for 15 minutes; removing a small aliquot (<5 ) for analytical HPLC analysis
is done to assess the status of the reaction. The reaction mixture is diluted to a
minimum 1:1 mixture of ddH20 : Acetonitrile mixture containing 0.1% TFA in
preparation for semi-preparative HPLC purification. 123IB-Polypeptide is isolated and
purified by semi-preparative HPLC or NAP5 size exclusion chromatography. The
HPLC fraction containing the product is further diluted (5:1) with ddH20 and
subsequently immobilized on a tC18 Plus Sep Pak (Waters). Flushing the SepPak
first with 5 mL of ddH20 then 30 mL of air gives the IB-Polypeptide in a minimal
amount of ethanol by first eluting the void volume (approx. 0.5mL) followed by
collecting 250 to 300 L· of eluent in a separate flask. RP-HPLC analysis is performed
on the isolated product to establish radiochemical and chemical purity.
[0127] Polypeptide Z00477 (SEQ. ID 3) was labeled with Ga, specifically Ga,
after a NOTA (l,4,7-triazacyclononane-N,N',N"-triacetic acid) chelator was
conjugated to the polypeptide. (Fig. 19)
[0128] Bioconjugation of Mal-NOTA to polypeptide molecules was accomplished
as follows. The polypeptide was dissolved with freshly degassed PBS buffer (lx, pH
7.4) at a concentration of approximately 1 mg/mL. The disulfide linkage in the
polypeptide was reduced by the addition of DTT solution in freshly degassed PBS
buffer (lx, pH 7.4). The final concentration of DTT was 20 mM. The reaction
mixture was vortexed for 2 hours and passed through a Zeba desalt spin column
(Pierce Technologies) pre-equilibrated with degassed PBS buffer (lx, pH 7.4) to
remove excess of DTT reagent. The eluted reduced polypeptide molecule was
collected, and the bifunctional compound mal-NOTA was added (15 equivalents per
equivalent of the polypeptide) as a solution in DMSO, and the mixture was vortexed
at room temperature. The reaction was allowed to proceed overnight to ensure the
complete conversion of the polypeptide molecules.
[0129] The HPLC purification was performed on a MiCHROM Magic C18AQ 5m
200A column (MiChrom Bioresources, Auburn, CA). Solvent A: H20 (with 0.1%
formic acid), Solvent B: CH CN (with 0.1% formic acid). Gradient: 5-100% B over
30 mins. (Fig. 20A)
[0130] The fractions containing desired product were combined and neutralized
with 100 mM ammonium bicarbonate solution, and the solvents were removed by
lyophilization to give the conjugated polypeptide as a white solid.
[0131] LC-MS analysis of the purified product confirmed the presence of the
desired product, and the MW suggested that only one NOTA chelator was added to
the polypeptide construct (calculated MW: 7504 Da, found: 7506 Da for Z00477
(SEQ. ID No. 3)-NOTA). (Fig. 20B)
[0132] Radiolabeling was subsequently accomplished as follows: 25m1 HEPES
solution (63mM) was initially added to a screw top vial followed by IOmI GaCl
(GE Healthcare) in 40.5 MBq of 0.04M HC1. 30 g (MW = 7506, 4.0xl0 9 mol) of
the NOTA Z00477 (SEQ. ID No. 3) in 30 mΐ H20 was then added to the reaction
mixture to give a final NOTA Z00477 (SEQ. ID No. 3) concentration of 6 1 mM with a
pH of 3.5-4.0. The reaction vial was sealed and the reaction maintained at ambient
temperature. Reverse phase HPLC analysis of the crude reaction mixture determined
the radiochemical purity of the Ga-NOTA Z00477 (SEQ. ID No. 3) was determined
to be 95% by HPLC after 2 hours at room temperature. (Fig. 21) The Ga-NOTA
Z00477 (SEQ. ID No. 3) was purified by HPLC after a reaction time of 1 day. 22MBq
of Ga-NOTA Z00477 (SEQ. ID No. 3) was injected onto the HPLC for the
purification. 15MBq of the Ga labeled product was obtained from the purification
(radiochemical yield = 68%). HPLC solvents were removed under vacuum to give a
solution with an approximate volume of 0.5 mL. Approximately 1.45 mL of
Dulbecco's phosphate buffered saline was then added to give a final solution at pH 6-
6.5 with a radioactivity concentration of 7.7 MBq/mL. Purified, formulated Ga-
NOTA Z00477 (SEQ. ID No. 3) was found to be stable for at least 2 hr at room
temperature. (RCP = 96% by HPLC) (Fig. 22).
[0133] Analytical HPLC conditions used are as follows: A Grace Vydac C4 protein
5 micron, 300A, 4.6 x 250 mm HPLC column. Solvent A = 95/5 H20 / MeCN in
0.05% trifluoroacetic acid (TFA) Solvent B = 95/5 CH CN / H20 in 0.05% TFA.
HPLC gradient (Min/%B): 0/0, 4/20, 16/60, 20/100, 25/100, 26/0.
[0134] Semi-preparative HPLC conditions used are as follows: Column: Grace Vydac
C4 protein 5 micron, 300A, 4.6 x 250 mm. Solvent A = 95/5 H20 / MeCN in 0.05%
trifluoroacetic acid (TFA) Solvent B = 95/5 CH CN / H20 in 0.05% TFA. HPLC
gradient (Min/%B): 0/0, 4/20, 16/60, 20/100, 25/100, 26/0.
[0135] While only certain features of the invention have been illustrated and
described herein, many modifications and changes will occur to those skilled in the
art. It is, therefore, to be understood that the appended claims are intended to cover
all such modifications and changes as fall within the true spirit of the invention.

CLAIMS:
1. An imaging agent composition comprising :
an isolated polypeptide comprising SEQ. ID No 1, SEQ. ID No 2, or a
conservative variant thereof, conjugated with a mTc via a diaminedioxime
chelator; wherein the isolated polypeptide binds specifically to HER2, or a variant
thereof.
2. An imaging agent composition comprising :
an isolated polypeptide comprising SEQ. ID No 1, SEQ. ID No 2, or a
conservative variant thereof, conjugated with Ga or Ga via a NOTA chelator;
wherein the isolated polypeptide binds specifically to HER2, or a variant thereof.
3. An imaging agent composition comprising :
an isolated polypeptide comprising SEQ. ID No 1, SEQ. ID No 2, or a
conservative variant thereof, conjugated with F via a linker; wherein the linker
comprises a group derived from an aminoxy group, an azido group, or an alkyne
group; and wherein the isolated polypeptide binds specifically to HER2, or a
variant thereof.
4. The composition of claim 1, wherein the diaminedioxime chelator comprises
Pn216, cPn216, Pn44, or derivatives thereof.
5. The composition of claim 3, wherein the 18F is attached to the isolated
polypeptide via the aminoxy linker at (the) N-terminus of the isolated polypeptide.
6. The composition of claim 4, wherein the mTc is conjugated to the isolated
polypeptide via the cPn216 chelator at (the) N-terminus of the isolated
polypeptide.
7. A method of imaging at least a portion of a subject comprising:
administering the composition of claim 1 to the subject, and
imaging the subject with a diagnostic device.
8. The method of claim 7, further comprising the steps of:
monitoring delivery of the composition of claim 1 to the subject; and
diagnosing the subject with a HER2-associated disease condition.
9. A method of imaging at least a portion of a subject comprising:
administering the composition of claim 2 to the subject, and
imaging the subject with a diagnostic device.
10. The method of claim 9, further comprising the steps of:
monitoring delivery of the composition of claim 2 to the subject; and
diagnosing the subject with a HER2-associated disease condition.
11. A method of imaging at least a portion of a subject comprising:
administering the composition of claim 3 to the subject, and
imaging the subject with a diagnostic device.
12. The method of claim 11, further comprising the steps of:
monitoring delivery of the composition of claim 2 to the subject; and
diagnosing the subject with a HER2-associated disease condition.
13. The method of claim 12, wherein the HER2-associated disease condition is
breast cancer.
14. The method of claim 11, wherein the diagnostic device employs an imaging
method selected from the group consisting of MRI, PET, SPECT, radioimaging,
and combinations thereof.
15. A method for preparing a chelator conjugated polypeptide composition
comprising:
(i) providing an isolated polypeptide comprising SEQ. ID No 1 or SEQ. ID No
2, or a conservative variant thereof;
(ii) reacting a diaminedioxime chelator with the polypeptide to form the
chelator conjugated polypeptide.
16. The method of claim 15, wherein the diaminedioxime chelator is conjugated
with Tc.
17. A method for preparing a 18F conjugated polypeptide composition
comprising:
(i) providing an isolated polypeptide comprising SEQ. ID No. 1 or SEQ. ID
No. 2 or a conservative variant thereof;
(ii) reacting the polypeptide with a linker wherein the linker comprises a group
derived from an aminoxy group, an azido group, or an alkyne group; and
(iii) reacting the linker with a F moiety to form the F conjugated
polypeptide.