HER2 BINDING PEPTIDES LABELLED WITH
A 18F- CONTAINING ORGANOSILICON COMPOUND
FIELD
[0001] 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
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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 18F incorporation. However, this
chemistry only provides a low radiochemical yield. Similarly, the conjugation of
mTc with the Affibody® is a multistep process. In addition, Tc reduction and the
complex formation with chelates, require high pH (e.g., pH=ll) conditions and long
reaction times.
[0007] 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.
[0008] Therefore, there is a need for chemistries and methods for synthesizing
radiolabeled polypeptides in which a radioactive moiety, such as, for example, 18F,
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 targeting imaging agent for PET
or SPECT imaging with improved properties particularly related to renal clearance
and toxicity effects.
SUMMARY OF THE INVENTION
[0009] The compositions of the invention are a new class of imaging agents that are
capable of binding specifically to HER2 or variants thereof.
[0010] 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.
[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 Ga or Ga via a NOTA chelator. 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 an Al18F-NOTA chelate. 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] 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 18F via an isotopic fluorine exchange chemistry. The
isolated polypeptide binds specifically to HER2 or variants thereof.
[0015] In one or more embodiments, methods of making an imaging agent
composition as described herein are provided. An example of a method 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.
[0016] 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 NOTA- chelator to form, a NOTA
chelator conjugated polypeptide and (iii) reacting the NOTAchelator conjugated
polypeptide with an Al 18F moiety to form a Al18F-NOTA chelator conjugated
polypeptide.
[0017] 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 silicon fluoride (e.g. [ F]-silicon
fluoride)-containing moiety to form a silicon fluoride conjugated polypeptide; and
(iii) reacting the silicon fluoride conjugated polypeptide with an F moiety to form an
F-silicon fluoride conjugated polypeptide.
FIGURES
[0018] 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:
[0019] 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.
[0020] 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.
[0021] 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.
[0022] FIG. 4 is a reverse phase HPLC gamma chromatogram of mTc labeled
Z00477 (SEQ. ID No. 3).
[0023] 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.
[0024] 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.
[0025] FIG. 7 is a diagram of the chemical structure for a Mal-cPN216 linker.
[0026] 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.
[0027] FIG. 9 is a reverse phase HPLC gamma trace chromatogram for Z02891-
cPN216 (SEQ. ID No. 2) labeled with mTc.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] FIG. 13A and 13B are diagrams of the chemical structures for Boc-protected
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-lH-pyrrol-lyl)
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.HCl).
[0032] 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.
[0033] 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).
[0034] 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.
[0035] FIG. 17 is a graph of the biodistribution profile of Z02891 (SEQ. ID No. 2)
polypeptide labeled with 18F ( ID, % injected dose) and the tumor: blood ratio from
SKOV3-tumored animals.
[0036] 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.
[0037] FIG. 19 is a diagram of the chemical structure of the Mal-NOTA linker.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] FIG. 23 is an analytical HPLC of formulated 2 [top: UV channel at 280 nm
showing ascorbate, 0.5 min and peptide precursor 3, 4.5 min; bottom: radioactivity
channel showing 2, 5.1 min (RCP 95%) and a decomposition product at 4.6 min.
[0042] FIG. 24 is a FASTlab M cassette layout for the preparation of 2 using tC2
SepPak purification.
[0043] FIG. 25 is an analytical HPLC of formulated 2 prepared using FASTlab™
[top: radioactivity channel, showing 2 (7.7 min), F-FBA (10.4 min) and an unknown
impurity (12.2 min); middle: UV channel at 280 nm showing p-aminobenzoic acid
formulation additive (3 min); bottom: UV channel at 350 nm showing
dimethylaminobenzaldehyde by-product (10.2 min) and an unknown impurity (3.8
min)].
[0044] FIG. 26 is a FASTlab™ cassette layout for the preparation of 2 using
Sephadex purification.
[0045] FIG. 27 is an analytical HPLC of formulated 2 prepared using FASTlab with
Sephadex purification [top: radioactivity channel, showing 2 (7.1 min), F-FBA (8.8
min) and an unknown impurity (10.2 min); middle: UV channel at 280 nm; bottom:
UV channel at 350 nm showing dimethylaminobenzaldehyde by-product (10.0 min)].
[0046] FIG. 28 is an analytical HPLC of formulated 5 [top: radioactivity channel,
showing the product (4.7 min, 92 ) and a by-product (3.9 min, 8 ); bottom: UV
channel at 280 nm].
[0047] FIG. 29 depicts a time course study of 5 showing labelling efficiencies as
measured by analytical radio HPLC.
[0048] FIG. 30 is an analytical RCY of 5 after increasing the peptide/AlCl
concentration (P: product, BP: by-product, see Fig. FIG 28).
[0049] FIG. 3 1 is an analytical HPLC profile of a labelling mixture of 5. (Top trace:
radioactivity channel, bottom trace: UV channel at 280 nm).
[0050] FIG. 32 is an analytical radioactivity channel HPLC of isolated 7 (Red:
radioactivity channel, blue: UV channel at 280 nm).
[0051] FIG. 33 depicts HER2 protein expression in tumour sections from the NCIN87
and A431 xenograft models by immunohistochemistry the HERCEPTEST by
DAKO. Pictures on the left are x2 magnification, pictures on the right are xlO of the
highlighted square.
[0052] FIG. 34 shows naive mice biodistributions of 9, 2, 5, and 7.
[0053] FIG. 35 shows biodistributions of 9, 2, 5, and 7 in the NC87/A431 tumour
bearing mice.
[0054] FIG. 36 shows biodistribution profile of 2 in the dual tumour xenograft model.
[0055] FIG. 37 shows NCI-N87 xenograft biodistribution profile of 2 using
increasing concentrations of cold precursor.
[0056] FIG. 38 shows preliminary imaging with 2 in the dual tumour xenograft model
(A) and comparison to Affibody® 9 imaging study (B).
DETAILED DESCRIPTION
[0057] The imaging agent compositions of the invention generally comprise an
isolated polypeptide of SEQ. ID No. 1, SEQ. ID No. 2 or a conservative variant
thereof, conjugated with a radioisotope such as, for example, F, mTc, Ga or Ga,
In, I, 4 I, Zr, or ^Cu; 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] The polypeptides may be about 49 residues to about 130 residues in length.
The specific polypeptide sequences are listed in Table 1.
Table 1
[0063] 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).
[0064] The polypeptides listed in Table 1 may be conjugated with F via a linker;
mTc via a diaminedioxime chelator, with Ga or Ga via a NOTA chelator, with F
via an Al18F-N0TA, with 18F via SiFA ( i.e., silicon fluoride acceptor) or silicon
fluoride exchange chemistry, with i In via DOTA chelator chemistry, with I or 4I
via fluorobenzaldehyde-like chemistry using iodobenzaldehyde, or with 4Cu via
NOTA-chelator chemistry. Table 2 provides the isoelectric point (pi), of these
polypeptides.
Table 2
[0065] 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 F.
The F may be incorporated at a C terminus, at a N-terminus, or at an internal
position of the isolated polypeptide.
[0066] 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 18F moiety to form the
F conjugated polypeptide.
[0067] 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 a
maleimido-aminoxy, maleimido-alkyne or maleimido-azide group, to form a linker
18 conjugated polypeptide; and reacting the linker with an F moiety to form the 18F
conjugated polypeptide.
[0068] 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 18F 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 the 18F
conjugated polypeptide.
[0069] Using the above-described examples, fluorine or radiofluorine atom(s), such
as F, 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, the linker 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.
[0070] A linker-conjugated polypeptide or the F-conjugated linker for use in the
preparation of an imaging agent or imaging agent composition of the invention may
be prepared by a method of the invention that is 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 an 18F-conjugated linker (e.g., 18F-
fluorobenzaldehydeX" 18F-FBA") is simpler than the procedures known in the art. 18F
conjugated-linker is prepared in one step by the direct nucleophilic incorporation of
F onto the trimethylanilinium precursor. F-linker (i.e., F-FBA) is then
conjugated to the polypeptide, such as, for example, an Affibody® and those
described herein. 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.
[0071] The fluorine-labeled imaging agent compositions are highly desired materials
in diagnostic applications. 18F labeled imaging agent compositions may be visualized
using established imaging techniques such as PET.
[0072] 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, C3-10
alkylary, C2-10 alkoxyalkyl, C Ohydroxyalkyl, CM 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.
[0073] 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 (II).
[0074] 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.
[0075] 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-chelator 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 NOTAchelator
conjugated polypeptide. The NOTA chelator may be further conjugated with
67^Ga or 6 Ga.
[0076] 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 (III).
[0077] In one or more embodiments, the polypeptide may be conjugated with Al F
via NOTA (l,4,7-triazacyclononane-N,N',N"-triacetic acid) chelator. The
NOTAchelator 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
chelator conjugated polypeptide. The NOTA- chelator conjugated polypeptide may
18 then be further conjugated with Al F to form the Al 18F-NOTA-chelator conjugated
polypeptide.
[0078] In one or more embodiments, the polypeptide may be conjugated with F via
NOTA- chelator. The NOTA- chelator 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 a source of F (e.g.,
A1 F) to form an F-NOTA- chelator; and (iii) reacting the F-NOTA- chelator with
the isolated polypeptide to form the 18F-NOTA-chelator conjugated polypeptide.
[0079] In one or more embodiments, a chelator may comprise a chelate moiety (e.g.
NOTA, DOTA) alone or a chelate moiety and a linker, each as described herein. By
way of example, a NOTA-chelator can represent a NOTA chelate moiety alone or a
NOTA chelate moiety attached to a linker as described herein.
[0080] In one or more embodiments, the polypeptide may be conjugated with F via
SiFA chemistry. The 18F-SiFA 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 a silicon fluoride acceptor (SiFA) group, to form a SiFA conjugated
polypeptide; and (iii) reacting the SiFA conjugated polypeptide with an 18F moiety or
a source of F. The F moiety or source of F can any such moiety or source
capable of reacting with a SiFA group and undergo isotopic fluorine exchange
chemistry. Scheme I below illustrates radiolabelling of Z0289 1 (SEQ. ID No. 2) using
[1 F]SiF coupling:
Scheme I
[0081] In one or more embodiments, the methods of making a radiolabeled imaging
agent or imaging agent composition of the invention as described herein, are
automated. For example, a radiolabeled imaging agent or imaging agent composition
of the invention may be conveniently prepared in an automated fashion by means of
an automated radiosynthesis apparatus. There are several commercially-available
examples of such platform apparatus, including TRACERlab™ (e.g., TRACERlab™
MX) and FASTlab™ (both from GE Healthcare Ltd.). Such apparatus commonly
comprises a "cassette", often disposable, in which the radiochemistry is performed,
which is fitted to the apparatus in order to perform a radiosynthesis. The cassette
normally includes fluid pathways, a reaction vessel, and ports for receiving reagent
vials as well as any solid-phase extraction cartridges used in post-radiosynthetic clean
up steps. Optionally, in a further embodiment of the invention, the automated
radiosynthesis apparatus can be linked to a high performance liquid chromatograph
(HPLC).
[0082] The present invention therefore provides a cassette for the automated synthesis
of a radiolabeled imaging agent or imaging agent composition of the invention, each
as defined herein.
[0083] The invention also comprises methods of imaging at least a portion of a
subject. In one embodiment, the method comprises administering a radiolabeled
imaging agent or an imaging agent composition of the invention to a subject and
imaging the subject. The subject may be imaged, for example, with a diagnostic
device.
[0084] In one or more embodiments, a method of imaging may further comprise the
steps of monitoring the delivery of the agent or 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, single
photon emission computed tomography (SPECT), positron emission tomography
(PET), or combinations thereof.
[0085] A radiolabeled imaging agent or an imaging agent composition of the
invention may be administered to humans and other animals parenterally as a
pharmaceutical composition. A pharmaceutical composition of the invention
comprises a radiolabeled imaging agent or an imaging agent composition, as
described herein, and a pharmaceutically acceptable carrier, excipient, solvent or
diluent.
[0086] For example, a pharmaceutical composition 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.
[0087] A pharmaceutical composition of the invention may also contain an 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.
[0088] A radiolabeled imaging agent or an imaging agent composition of the
invention 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.
[0089] An imaging agent composition or a pharmaceutical composition of the
invention 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.
[0090] An imaging agent composition or a pharmaceutical composition of the
invention 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.
[0091] 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
[0092] The following examples are provided for illustration only and should not be
construed as limiting the invention.
[0093] MATERIALS
[0094] 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
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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).
[0099] 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
[00100] 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.
[00101] 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.
[00102] 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.
[00103] 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).
[00104] 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.
[00105] 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.
[00106] 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.
[00107] 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.
[00108] 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
[00109] 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 WmTc(CO) (His6)-Polypeptides ('His6' disclosed as SEQ
ID NO: 7)
[00110] 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
[00111] 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.
[00112] 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.
[00113] 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
[00114] 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.
[00115] 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.
[00116] 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.
[00117] 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 CN), HPLC-grade trifluoroacetic acid (TFA), and Millipore 18 ihW water were
used for HPLC purifications.
[00118] Example 1.
[00119] 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 Mal-cPN216 was obtained as a white powder (230 mg, 80% yield). IH-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).
[00120] The polypeptide Z00477 (SEQ ID No. 3)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).
[00121] 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.
[00122] 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%).
[00123] 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).
[00124] Example 2.
[00125] 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.
[00126] Example 3.
[00127] 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
[00128] 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.
[00129] 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/ddH20 (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.
[00130] 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.
[0131] 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.
[0132] 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 L· 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).
[0133] 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.
[0134] 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
g) were obtained in 3 to 4 weeks in greater than 80% of animals injected.
[0135] Mice were given tail-vein injections of ~ 1 ug of mTc-labeled polypeptides
(-10 m I 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).
[0136] 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.
[0137] 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- mTc
Table 7. Z02891 (SEQ. ID No. 2) cPN216 uptake (%ID/g) in SKOV3 tumor bearing
mice
[0138] Example 4.
[0139] 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).
[0140] Example 5.
[0141] To incorporate F into the Polypeptide molecules, a bifunctional linker
Mal-aminooxy 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.
[0142] General
[0143] 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.
[0144] Example 6.
[0145] 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, CDCI 3) : 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).
[0146] Example 7.
[0147] 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) )
[0148] Example 8.
[0149] The polypeptide (Z00477(SEQ ID No. 4), Z00342 (SEQ ID No. 1)
Z02891 (SEQ ID. No. 2)) was dissolved with freshly degassed PBS buffer (lx,
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
Mai-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).
[0150] 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.
[0151] 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)-ONH2, Z00342 (SEQ.
ID No. l)-ONH 2, and Z02891 (SEQ. ID No. 2) -ONH 2, respectively.
[0152] Example 9. Preparation of 18FBA.
[0153] 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 K2C0 (EMD Science) were purchased and used as received.
Optima™-grade acetonitrile was used as both HPLC and reaction solvents.
[0154] 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 [ F~] 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 (ddH20 )
containing Kryptofix K222 (376 g.mol 1, 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 K F K222, K2CO 3 . The reaction mixture
was heated to 90°C for 15 min and immediately cooled and quenched with 3 mL of
ddH20 . This mixture was subsequently passed through a cation exchange cartridge
(Waters SepPak Light Accell Plus CM), diluted to 10 mL with ddH20 , and loaded
onto a reverse phase C18 SepPak (Waters SepPak Plus CI 8). The SepPak was flushed
with 10 mL of ddH20 then purged with 30 mL of air. [18F]4-fluorobenzaldehyde
( FBA), was eluted in 1.0 mL of methanol.
[0155] Example 10.
[0156] 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
FBA in methanol (see Example 9) 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 of ddH20 with 0.1% TFA was used to dilute the solution to approx.
500 mL· 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 L· 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
m of a 0.1 Ci/ L· 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 FBA 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 F-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
[0157] Analytical HPLC conditions used are as follows: Analysis performed on an
HP Agilent 1100 with a G1311A QuatPump, G1313A autoinjector with IOOmE
syringe and 2.0mL seat capillary, Phenomenex Gemini CI 8 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 1) : 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 mLmin 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.
[0158] 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.
[0159] Example 11
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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) F-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.
[0164] 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) F-fluorobenzyl oxime uptake (%ID/g) in SKOV-
3 tumor bearing mice
Table 10. Z00342 (SEQ. ID No. 1) sF-fluorobenzyl oxime uptake (%ID/g) in
SKOV-3 tumor bearing mice
Kidney 78.93 ± 2.93 (n=3) 30.94 ± 4.93 (n=3) 10.75 ± 2.17 (n=3) 4.91 ± 0.63 (n=3)
Spleen 3.85 ± 0.51 (n=3) 1.77 ± 0.34 (n=3) 0.47 ± 0.08 0.23 ± 0.05 (n=3)
(n=3)
[0165] General.
[0166] 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.
[0167] Example 12.
[0168] [ I]4-iodobenzaldehyde ( 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 L· of
ddH20 and adding 8 mL· of trifluoroacetic acid followed by the addition of 123IIBA 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 123IB-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.
[0169] Example 13. Preparation of 67Ga-NOTA-Z00477 (SEP ID No. 3)
[0170] 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)
[0171] 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.
[0172] 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)
[0173] 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.
[0174] 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)
[0175] 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).
[0176] 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.
[0177] 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.
[0178] General
[0179] Recombinant HER2 Z28921-Cys was purchased from Affibody AB, Sweden,
Eei-aminooxyacetic acid succinic ester from IRIS Biotech, and di-tertbutyldifluorosilane
was purchased from Fluorochem. Reagents and solvents were
purchased from IRIS Biotech, Merck, Romil and Fluka.
[0180] Analytical LC-MS spectra were recorded on a Thermo Finnigan MSQ
instrument by electrospray ionisation (ESI) operated in positive mode coupled to a
Thermo Finnigan Surveyor PDA chromatography system using the following
conditions: Solvent A = H2O/0.1% TFA and solvent B = ACN/0.1% TFA if not
otherwise stated, flow rate: 0.6 mL/min, column: Phenomenex Luna 3 mih C18 (2) 20
x 2 mm, detection: UV 214/254 nm.
[0181] Semi-preparative reversed-phase HPLC runs were performed on a Beckman
System Gold chromatography system using the following conditions: Solvent A =
H2O/0.1% TFA and solvent B = ACN/0.1% TFA if not otherwise stated, flow rate: 10
mL/min, column: Phenomenex Luna 5 mih C18 (2) 250 x 21.2 mm, detection: UV 214
nm.
[0182] Preparative reversed-phase HPLC runs were performed on a Waters Prep 4000
system using the following conditions: Solvent A = H2O/0.1% TFA and solvent B =
ACN/0.1% TFA if not otherwise stated, flow rate: 50 mL/min, column: Phenomenex
Luna 10m C18 (2) 250 x 50 mm, detection: UV 214/254 nm.
[0183] Abbreviations:
[0184] Ala (A): Alanine
[0185] Arg (R): Arginine
[0186] Asn (N): Asparagine
[0187] Asp (D): Aspartic acid
[0188] ACN: Acetonitrile
[0189] Boc: tert-Butyloxycarbonyl
[0190] Cys (C): Cysteine
[0191] DIPEA: Diisopropylethylamine
[0192] DMF: N,N-Dimethylformamide
[0193] DMAB: 4-dimethylamino-benzaldehyde
[0194] DOTA: l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid
[0195] EDT: 1,2-Ethanedithiol
[0196] EMS: Ethyl methyl sulphide
[0197] ESI: Electrospray ionisation
[0198] eq: Equivalent
[0199] FBA: 4-Fluorobenzaldehyde
[0200] Gin (Q): Glutamine
[0201] Glu (E): Glutamic acid
[0202] hr(s): Hour(s)
[0203] HER2: Human Epidermal growth factor receptor
[0204] HOAt: 1-Hydroxy-7-azabenzotriazole
[0205] HPLC: High perfomiance liquid cliromatography
[0206] lie (I): Isoleucine
[0207] LC-MS: Liquid chromatography - mass spectroscopy
[0208] Leu (L): Leucine
[0209] Lys (K): Lysine
[0210] Met (M): Methionine
[0211] min: Minutes
[0212] mih: Micrometre
[0213] nm: Nanometre
[0214] NMP: 1-Methyl-2-pyrrolidinone
[0215] NOTA: 1,4,7-Triazacyclononane- 1,4,7-triacetic Acid
[0216] PDA: Photodiode array
[0217] PET: Positron emission tomography
[0218] Phe (F): Phenylalanine
[0219] Pro (P): Proline
[0220] PyAOP: (7-Azabenzotriazol- 1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate
[0221] Ser (S): Serine
[0222] SiFA: 4-(Di-tert-butylfluorosilyl)benzaldehyde
[0223] TFA: Trifluoroacetic acid
[0224] Thr (T): Threonine
[0225] TIS: Triisopropylsilane
[0226] Trp (W): Tryptophan
[0227] Tyr (Y): Tyrosine
[0228] Val (V): Valine
[0229] Example 14. Semi-automated radiosynthesis of Compound 2
[ F]-FBA 2
[0230] A FASTlabTM platform (GE Healthcare) was used to prepare
[18F]Fluorobenzaldehyde ("[18FJFBA") yielding typically 7 GBq of [18FJFBA in
ethanol (1.5 mL, non-decay corrected yields 12-54%). A small fraction (92 ) of this
[18FJFBA solution was then manually conjugated to the aminoxy precursor 3 (0.4
mg, 55 nmol) in the presence of aniline hydrochloride (3.2 mg, 25 mihoΐ ) in water
(138 ) in a silanised P6 vial. The mixture was heated at 70°C for 20 minutes using
a Peltier heater. 2 was isolated via size exclusion chromatography (NAP5 cartridge,
GE Healthcare). An initial elution with 0.25 mL saline/0.1 % sodium ascorbate was
discarded. A subsequent 0.75 mL saline/0.1 % sodium ascorbate elution containing 2
was collected and formulated with the same elution mixture at pH 5-5.5 to give the
desired radioactive concentration. Non-decay corrected yields of the isolated 2 from
the conjugation step were 17-38%, and the radiochemical purity (RCP) values for the
manually prepared 2 were > 95%. (TLC system: Perkin Elmer Instant Imager using
C18 reversed-phase sheets with water/30 % acetonitrile (v/v) as mobile phase. The
labelled peptide remained at the origin.). The product was further analysed by HPLC
using a Gilson 322 pump with a Gilson UV/ViS 156 detector, a Bioscan Flow-Count
radioactivity detector, and a Luna C18 Phenomenex column (50 x 4.6 mm, 3 mih) or a
Luna C18 Phenomenex column (150 x 4.6 mm, 5 mih) . The mobile phase comprised
of solvents A (0.1 M ammonium acetate) and B (acetonitrile) running at 1 mL/min
with a linear gradient (5-95 % B in 15 min). The UV absorbance was measured at 280
and 350 nm. FIG. 23 shows a representative example of an analytical HPLC trace of
the formulation of 2.
[0231] Example 14a. Preparation of Compound 3
[0232] (i Preparation of Eei-aminooxyacetyl-maleimide
[0233]
[0234] N-(2-Aminoethyl)maleimide TFA salt (51 mg, 0.20 mmol) and Eei-AOAc-
OSu (77 mg, 0.30 mmol) were dissolved in NMP (2 mL). Sym.-collidine (80 , 0.6
mmol) was added and the reaction mixture stirred for 70 min. The reaction mixture
was diluted with water (7 mL) and the product, eei-aminooxyacetyl-maleimide,
purified by semi-preparative HPLC. Purification using semi-preparative HPLC
(gradient: 15-30% B over 40 min where A = water/0.1% acetic acid and B = ACN)
affording 43 mg (75%) pure Eei-aminooxyacetyl-maleimide. The purified material,
eei-aminooxyacetyl-maleimide, was characterised by LC-MS (gradient: 10-40% B
over 5): tR: 1.93 min, found m/z: 284.1, expected MH+: 284.1
[0235] (ii) Preparation of Compound 3
[0236] Recombinant Z02891-Cys (144 mg, 0.205 mmol)(purchased from Affibody
AB, Sweden) and eei-aminooxyacetyl-maleimide (17 mg, 0.60 mmol) were dissolved
in water (3 mL). The solution was adjusted to pH 6 by addition of ammonium acetate
and the reaction mixture shaken for 90 min. The reaction mixture was diluted with
water (7 mL) and the product purified by semi-prep HPLC affording 126 mg
lyophilised Eei-protected product. The eei-protected product was treated with 2.5%
TFA/water (16 mL) under a blanket of argon for 20 min. The solution was diluted
with water (144 mL), frozen using isopropanol/dry-ice bath under a blanket of argon
and lyophilised affording 149 mg (100%) Z02891-Cys-maleimide-aminooxyacetyl
(3). Lyophilised Z02891-Cys-maleimide-aminooxyacetyl (3) was analysed by
analytical LC-MS (gradient: 10-40% B over 5 min, tR: 3.28 min, found m/z: 1811.8,
expected MH4
4+ : 1811.4
[0237] Example 15. Automated radiosynthesis of Compound 2 using tC2 SepPak
purification
[0238] A FASTlabTM cassette was assembled containing a first vial (8.25 mg/21.9
mihoΐ Kryptofix, 1.16 mg/8.4 mmol K2CO , 165 m water, 660 m acetonitrile), a
second vial (1.5 mg/4.8 mmol triflate 1, 1.5 mL anhydrous DMSO), a third vial (5.5
mg/0.76 mmol 3, 8.2 mg/63 mmol aniline hydrochloride, 0.7 mL ammonium acetate
buffer pH 4.5/0.25 M), a fourth vial (4 mL, 4 % w/v aqueous ammonia), external vials
of ethanol (25 mL) and phosphoric acid ( 1 % w/w, 25 mL), a pre-conditioned QMA
light SepPak cartridge, an OASIS MCX SepPak cartridge, and two C2 SepPak
cartridges. The product vial contained an aqueous solution of p-aminobenzoic acid
(0.08 % w/w, 19 mL). The cassette layout is shown in FIG. 24.
[0239] The required programme sequence was uploaded from the PC control to the
synthesizer module and the assembled cassette mounted onto the machine. A water
bag and a product vial were attached. A vial containing [18FJwater (300 MBq, 1 mL)
was attached to the FASTlab™ module and the radiosynthesis commenced. The
process included an azeotropic drying step of the [18F]-Kryptofix/potassium carbonate
complex as eluted from the QMA cartridge, the radiosynthesis of [18F]FBA, the
purification of [18F]FBA using the MCX cartridge, ammonia solution and elution with
ethanol, the conjugation step to produce 2, and the purification and formulation step
using phosphoric acid/ethanol on the C2 cartridges. The total process took one hour
and generated 2 in 33 % non-decay corrected radiochemical yield with 94 %
radiochemical purity.
[0240] Example 16. Automated radiosynthesis of Compound 2 using Sephadex
purification
[0241] A FASTlab™ cassette was assembled containing a first vial (8.25 mg/21.9
mihoΐ Kryptofix, 1.16 mg/8.4 mihoΐ K2CO , 165 m water, 660 m acetonitrile), a
second vial (1.5 mg/4.8 mihoΐ triflate 1, 1.5 mL anhydrous DMSO), a third vial (5.0
mg/0.69 mihoΐ 3, 8.2 mg/63 mihoΐ aniline hydrochloride, 0.7 mL ammonium acetate
buffer pH 4.5/0.25 M), a fourth vial (4 mL, 4 % w/v aqueous ammonia), external vials
of ethanol (25 mL) and saline (Polyfusor, 0.9 % w/v, 25 mL), a pre-conditioned QMA
light SepPak cartridge, an OASIS MCX SepPak cartridge, and a custom packed size
exclusion cartridge (2 mL, Supelco, Cat. #57608-U) containing dry Sephadex G10
(500 mg, Sigma-Aldrich, Cat. #G10120). The cassette layout is shown in FIG. 26.
The radiosynthesis of 2 was performed as described in Example 15. After priming the
Sephadex cartridge with saline (5 mL), the crude reaction mixture was pumped
through the Sephadex cartridge and pure 2 collected in the product vial. The synthesis
time was 40 minutes and the non-decay corrected radiochemical yield was 10 %. The
radiochemical purity of the product was 95 % and the level of DMAB was 0.8 mg/mL.
FIG. 27 shows the HPLC analysis of the final product.
[0242] Example 17. Radiosvnthesis of ri8F1AlF-NOTA(COOH)2-Z0289HSEO
ID No. 2X5)
[0243]
[0244] A solution of NOTA(COOH)2-Z02891 (4) (746 g, 100 nmol) in sodium
acetate buffer (50 m , pH 4.0, 0.5 M) was mixed with a solution of AICI 3 (3 , 3.33
mg, 25 nmol in sodium acetate buffer, pH 4.0, 0.5 M) in a conical polypropylene
centrifuge vial (1.5 mL). This mixture was added to a small volume of [18FJfluoride
(50 m ) in a capped P6 vial. This vial was heated for 15 min at 100 °C. After diluting
with saline (100 m ), the reaction solution was transferred to a NAP5 size exclusion
cartridge (GE Healthcare). The final product was eluted into a P6 vial using saline
(750 m ) . The labelled peptide 5 was obtained with 11 % non-decay corrected
radiochemical yield. FIG. 28 shows the analytical HPLC of the formulated product.
Table 11 summarises the data of individual runs.
Table 11. Summary of [ SF]A1F-NOTA(COOH)2-Z02891(SEQ ID No. 2)(5)
preparations using NAP5 purification.
a End of Synthesis radiochemical yield, non-decay corrected
[0245] Example 17a. Preparation of Compound 4
[0246] i Preparation of NOTA(bis-tBu)
[0247] (a) Synthesis of Tetratosyl-N,N -bis(2-hvdroxyethyl)ethylene diamine
[0248] N,N"-bis(2-hydroxyethyl)-ethylenediamine (Aldrich, 14.8 g, 100 mmol) and
pyridine (Fluka, 200mL) was stirred at 0°C under nitrogen while a solution of
toluene-4-sulfonyl chloride (Fluka, 77 g, 400 mmol) dissolved in pyridine (Fluka,
lOOmL) was dropped into the solution over a period of 75 minutes. The temperature
was slowly raised to room temperature and continued stirred for 4 hours. Solution was
poured into a mixture of ice (250 mL) and hydrochloric acid (concentrated, 250 mL)
while stirring to afford a dark sticky oil. Solvents were removed by decantation,
product crude washed with water, decanted and re-dissolved in methanol (250mL).
The resulting slurry was isolated by filtration and the crude product was re-dissolved
in hot methanol (60°C, 600 mL) and cooled down. Solid product was filtered off and
dried in vacuo. Yield 36.36 g (47.5%). Product was verified by NMR.
[0249] (b) Synthesis of l-Benzyl-4-7-ditosyl-l A7-triazonane
[0250] Tetratosyl-N,N -bis(2-hydroxyethyl)ethylene diamine (See Example 17a(i)(a);
2.0 g, 2.6 mmol), benzyl amine (500m1, 4.6 mmol), potassium carbonate (Fluka, 792
mg, 5.7 mmol) and acetonitrile (Merck, 25 mL) was heated to 100°C and stirred
overnight. Solvents were removed from solid product by filtration. The solid was
washed with acetonitrile (2x 10 mL) and solvents were evaporated off. Solids was
dissolved in hot ethanol (15 mL) and left for three days in room temperature. Crystals
were collected by filtration and dried in vacuum overnight. Product confirmed by LCMS
(Phenomenex Luna CI8(2) 2.0x50 mm, 3 mih, solvents: A = water/0.1%
trifluoroacetic acid and B = acetonitrile/0. 1% trifluoroacetic acid; gradient 10-80% B
over 5 min; flow rate 0.6 mL/min, UV detection at 214 and 254 nm, ESI-MS) ¾ =
3.66 min. Yield 1 g (72%).
[0251] (c) Synthesis of (4-Benzyl-7-tert-butoxycarbonylmethyl-[l,4,71triazonan-lvD-
acetic acid tert-butyl ester
[0252] Sulphuric acid (Sigma, concentrated, 25 mL) was added to l-Benzyl-4-7-
ditosyl-l,4,7-triazonane (See Example 17a(i)(b); 2.5 g, 4.7 mmol) while stirring and
heated to 100 °C and left for 20 hours. The reaction mixture was cooled to room
temperature and added drop wise into diethyl ether (VWR, 500 mL). Product (white
precipitate) was filtered off and washed with acetonitrile, chloroform and
dichloromethane. Solvents were removed in vacuo. The product crude (986.3 mg, 4.5
mmol) were mixed with triethylamine (Fluka, 1.4 mL, 10 mmol) in acetonitrile (50
mL). Tert-buthyl bromoacetate (Fluka, 1.47 mL, 10 mmol) was dissolved in
acetonitrile (25 mL) and added dropwise. The reaction mixture was stirred in room
temperature overnight. pH was controlled and triethylamine added if necessary.
Solvents were removed in vacuo and crude material dissolved in dichloromethane
(150 mL) and washed with water (2x 25 mL), 0.1 M hydrochloric acid (lx 25mL) and
water (lx 25mL). The organic phase was filtered and solvent evaporated off. Crude
material was dissolved in acetonitrile /water (1:1) and purified by preparative HPLC
(Phenomenex Luna C18 (2) 5mih 250x 21.2 mm, solvents: A = water/0.1%
trifluoroacetic acid and B = acetonitrile/0. 1% trifluoroacetic acid; gradient 10-80% B
over 60 min) and lyophilized. LC-MS (Phenomenex Luna CI 8(2) 2.0x50 mm, 3 mih,
solvents: A = water/0.1% trifluoroacetic acid and B = acetonitrile/0. 1% trifluoroacetic
acid; gradient 10-80% B over 5 min; flow rate 0.6 mL/min, UV detection at 214 and
254 nm, ESI-MS) R= 3.99 min, (Ml) 447.4. Product verified by NMR.
[0253] Product was mixed with Pd/C (10%, 235 mg) and methanol (25 mL) and
stirred under argon. Argon was then removed by vacuo and hydrogen gas was started
to be supplied. Reaction mixture was left for three hours with stirring and
continuously supply of hydrogen gas. Catalyst was removed by centrifugation and
solvents evaporated off. Crude product was purified with preparative HPLC
(Phenomenex Luna C18 (2) 5mih 250x 21.2 mm, solvents: A = water/0.1%
trifluoroacetic acid and B = acetonitrile/0. 1% trifluoroacetic acid; gradient 2-80% B
over 60 min). LC-MS (Phenomenex Luna CI 8(2) 2.0x50 mm, 3 mih, solvents: A =
water/0. 1% trifluoroacetic acid and B = acetonitrile/0. 1% trifluoroacetic acid; gradient
10-80% B over 5 min; flow rate 0.6 mL/min, UV detection at 214 and 254 nm, ESIMS)
tR = 2.55 min, (Ml) 357.9.Yield 150 mg. Product confirmed by NMR.
[0254] (d) Synthesis of (4,7-Bis-tert-butoxycarbonylmethyl-[l,4,71triazonan-lylVacetic
acid rNOTA(bis-tBu)]
[0255] (4-tert-Butoxycarbonylmethyl-[l,4,7]triazonane-l-yl)-acetic acid tert-butyl
ester (See Example 17a(i)(d); 280 mihoΐ , 100 mg) and bromoacetic acid (Fluka,
lmmol, 138.21 mg) were dissolved in methanol ( 1 mL). Potassium carbonate
dissolved in water ( 1 mL) was added with stirring. Reaction mixture was stirred at
room temperature overnight and concentrated in vacuo. The residue was dissolved in
water (2.5 mL), and pH was adjusted to 4 with hydrochloric acid ( 1 M). The crude
product was purified by preparative HPLC (Phenomenex Luna C18 (2) 5 ih 250x
21.2 mm, solvents: A = water/0.1% trifluoroacetic acid and B = acetonitrile/0. 1%
trifluoroacetic acid; gradient 10-80% B over 60 min). LC-MS (Phenomenex Luna
CI 8(2) 2.0x50 mm, 3 mih, solvents: A = water/0.1% trifluoroacetic acid and B =
acetonitrile/0. 1% trifluoroacetic acid; gradient 10-80% B over 5 min; flow rate 0.6
mL/min, UV detection at 214 and 254 nm, ESI-MS) tR = 2.40 min. Yield 117.7 mg.
Product confirmed by NMR.
NOTA(bis-tBu) was purified by preparative HPLC (gradient: 20-40% B over 40 min)
affording 72 mg pure NOTA(bis-tBu). The purified material was characterised by
LC-MS (gradient: 10-40% B over 5): R: 3.75 min, found m z 416.2, expected MH+:
416.3.
[0256] (ii) Preparation of NOTA(bis-tBu)-maleimide
[0257] N-(2-Aminoethyl)maleimide trifluoroacetic acid salt (23 mg, 0.090 mmol),
NOTA(bis-tBu) (30 mg, 0.072 mmol) and PyAOP (51 mg, 0.10 mmol) were
dissolved in N,N-dimethylformamide (DMF) (2 mL). Sym.-collidine (29 m , 0.40
mmol) was added and the reaction mixture shaken for 1 r. The mixture was diluted
with water/0. 1% trifluoroacetic acid (TFA) (6 mL) and the product purified by semipreparative
HPLC. Purification using semi-preparative HPLC (gradient: 20-50% B
over 60 min) afforded 33 mg (87%) pure NOTA(bis-tBu)-maleimide. The purified
material was characterised by LC-MS (gradient: 10-40% B over 5, ¾: 4.09 min, found
m/z: 538.2, expected MH+: 538.3
[0258] (iii) Preparation of NOTA(bis-acid)-maleimide
[0259] NOTA(bis-tBu)-maleimide (33 mg, 6 1 mihoΐ ) was treated with a solution of
2.5% triisopropylsilane (TIS) and 2.5% water in TFA (10 mL) for 4 hrs 30 min. TFA
was evaporated in vacuo, the residue dissolved in water/0. 1% TFA (8 mL) and the
product purified by semi-preparative HPLC. Purification using semi-preparative
HPLC (gradient: 0-20% B over 40 min) afforded 15 mg (58%) pure NOTA(bis-acid)-
maleimide. The purified material was characterised by LC-MS (gradient: 0-30% B
over 5): R: 1.34 min, found m/z: 426.0, expected MH+: 426.2
[0260] (iv) Preparation of 4
Recombinant Z02891-Cys (40 mg, 5.7 mihoΐ ) (purchased from Affibody AB, Sweden)
and NOTA(bis-acid)-maleimide (6.1 mg, 14 mihoΐ) were dissolved in water (1.5 mL).
The solution was adjusted to pH 6 by adding ammonium acetate and the mixture
shaken for 1 r. The reaction mixture was diluted with water/0. 1% TFA (6 mL) and
the product purified using semi-preparative HPLC. Purification using semipreparative
HPLC (gradient: 20-30% B over 40 min) afforded 38 mg (90%) pure
compound 4. Purified 4 was analysed by analytical LC-MS (gradient: 10-40% B over
5 min): ¾: 3.31 min, found m/ z 1864.5, expected MH4
4+: 1864.5
[0261] Example 18
[0262] Time course study for the radiosynthesis of r F]AlF-NOTA(COOH)v
Z0289KSEO ID No. 2 5a
[0263] Fluorine- 18 was purified using a QMA cartridge and eluted with saline as
described by W. J . McBride et al. (Bioconj. Chem. 2010, 21, 1331). A solution of Fwater
(25 , 12 MBq) was mixed with AICI 3 (1.667 mg, 12.5 nmol) in sodium
acetate buffer (1.5 , pH 4.0, 0.5 M) and compound 6 (380 mg, 50 nmol):
dissolved in sodium acetate buffer (25 m , pH 4.0, 0.5 M). The mixture was heated at
100 °C and aliquots analysed by HPLC. The analytical data are given in FIG. 29.
[0264] Example 18a. Preparation of Compound 6
[0265] (i Preparation of NOTA(tris-tBu)
(a) Synthesis of a-bromoglutaric acid 5-benzyl ester
To a solution of L-glutamic acid-5-benzylester (Fluka, 3.0 g, 0.013 mol) and sodium
bromide (Fisher, 4.6 g, 0.044 mol) in aqueous hydrobromic acid (Fluka, 1M, 22.5
mL) cooled to 0°C was added portion wise sodium nitrite (Fluka, 1.6 g, 0.023 mol).
After stirring for 2h at 0°C, concentrated sulphuric acid (Merck, 1.2 mL) was added
followed by diethyl ether (Eternell). The water phase was extracted three times with
diethyl ether. The combined organic phases was washed four times with brine, dried
over sodium sulphate and evaporated under reduced pressure. The crude product was
purified using normal phase chromatography (Silica column (40 g), solvents: A=
hexane, B= ethyl acetate, gradient: 10 -35% B over 20 min, flow rate 40mL/min, UV
detection at 214 and 254 nm) affording 1.81 g of the pure product. Yield 46 %.
Structure verified by NMR.
(b) Synthesis of a-bromo2lutaric acid 5-benzyl ester 1-tert-butyl ester
{Bioorg. Med. Chem. Lett. 2000 10, 2133-2135)
To a solution of a-bromoglutaric acid-5-benzylester (See Example 18a(i)(a); 1.2 g,
4.0 mmol) in chloroform (Merck, 5 mL) a solution of tert-butyl 2,2,2-
trichloroacetimidate (Fluka, 1.57 mL, 8.52 mmol) in cyclohexane (Merck, 5 mL) was
added dropwise over 5 minutes. N,N-Dimethylacetamide (Fluka, 0.88 mL) was added
followed by boron trifluoride ethyl etherate (Aldrich, 80 ) as catalyst. The reaction
mixture was stirred for 5 days at room temperature. Hexane was added and the
organic phase washed with brine three times, dried over sodium sulphate and
evaporated under reduced pressure. The crude product was purified using normal
phase chromatography (Silica column (40 g), solvents: A= hexane, B= ethyl acetate,
gradient: 10 to 35 % B over 15 min, flow rate 40mL/min, UV detection at 214 and
254 nm) affording 1.13 g (79%) of the pure product. Structure was verified by NMR
(c) Synthesis of 2-[l,4,71triazonan-l-yl-pentanedioic acid 5-benzyl ester 1-tertbut
l ester
A solution of a-bromoglutaric acid-5-benzylester 1-tert-butyl ester (See Example
18a(i)(a); 513 mg, 1.44 mmol) in chloroform (Merck, 20 rriL) was added over a
period of 3 hours to a solution of 1,4,7 triazacyclononane (Fluka, 557 mg, 4.31 mmol)
in chloroform (Merck, 20 mL). The mixture was stirred for 3 days at room
temperature and concentrated in vacuo to a light yellow oil. The crude product was
purified using normal phase chromatography (Silica column (40 g), solvents: A=
ethanol: ammonia (aq) 95:5, B= chloroform: ethanol: ammonia (aq) 385:175:20,
gradient: 0% B over 6 min, 100% B over 12 min, flow rate 40mL/min, UV detection
at 214 and 254 nm) affording the semi-pure product (289 mg). Yield 49 %. Product
confirmed by LC-MS (column Phenomenex Luna C18(2) 2.0x50 mm, 3 mih, solvents:
A = water/0. 1% trifluoroacetic acid and B = acetonitrile/0. 1% trifluoroacetic acid;
gradient 10-50% B over 5 min; flow rate 0.6 mL/min, UV detection at 214 and 254
nm, ESI-MS) R = 2.5 min, m z (MH+), 406.3.
(d) Synthesis of 2-(4, 7-bis-tert-butoxycarbonylmethyl-[l,4,71triazonan-l-ylpentanedioic
acid 5-benzyl ester 1-tert-butyl ester
2-[l,4,7]Triazonan-l-yl-pentanedioic acid 5-benzyl ester 1-tert-butyl ester (See
Example 18a(i)(b); 600 mg, 1.48 mmol) in dry acetonitrile (40 mL) was cooled to
zero degrees before tert-butyl bromoacetate (Fluka, 548 mg, 414 m , 2.81 mmol) in
dry acetonitrile (10 mL) was added drop wise over a period of 15 minutes. The
reaction mixture was stirred for additional 15 minutes before dry potassium carbonate
(Fluka, 1.13 g, 814 mmol) was added and the reaction mixture warmed slowly to
room temperature over 4 hours. The mixture was filtered over Celite and evaporated
to dryness to afford the crude product. Product was confirmed by LC-MS (column
Phenomenex Luna C18(2) 2.0x50 mm, 3 mih , solvents: A = water/0. 1%
trifluoroacetic acid and B = acetonitrile/0. 1% trifluoroacetic acid; gradient 10-80% B
over 5 min; flow rate 0.6 mL/min, UV detection at 214 and 254 nm, ESI-MS) R= 3.9
min, m/z (MH+), 634.4.
(e Synthesis of 2-(4, 7-bis-tert-butoxycarbonylmethyl -ri,4,71triazonan-l-ylpentanedioic
acid 1-tert-butyl ester [NOTA(tris-tBu)1
2-(4, 7-Bis-tert-butoxycarbonylmethyl-[l ,4,7]triazonan- l-yl-pentanedioic acid 5-
benzyl ester 1-tert-butyl ester (See Example 18a(i)(c); 938 mg, 1.48 mmol) was
dissolved in 2-propanol (Arcus, 115 mL) and 10% Pd/C (Koch-Light, 315 mg)
suspended in water (3 mL) was added. The mixture was treated with hydrogen (4 atm)
for 3 hours, filtered over Celite and evaporated to dryness. The residue was
chromatographed on silica gel (Silica column (4g), solvents: 2-propanol:ammonia
95:5, flow rate 40mL/min, UV detection at 214 and 254 nm) affording a semi-pure
product (225 mg). Product was confirmed by LCMS (Phenomenex Luna C18 (2),
2.0x50mm, 3mih ; solvents: A = water/0. 1% trifluoroacetic acid and B =
acetonitrile/0. 1% trifluoroacetic acid, gradient 10-80% B over 5 min, flow rate 0.6
mL/min, UV detection at 214 and 254 nm, ESI-MS) tR 2.4min, MH+ 544.5.
Purified NOTA(tris-tBu) was characterised by LC-MS (gradient: 10-80% B over 5):
R: 2.4 min, found m . 544.5, expected MH+: 544.4.
[0266] (ii) Preparation of NOTA(tris-tBu)-NH-CH2CH2-NH2
[0267] PyAOP (96 mg, 0.18 mmol) dissolved in NMP ( 1 mL) was added to a solution
of NOTA(tris-tBu) (100 mg, 0.18 mmol) and ethylenediamine (1.2 mL, 18 mmol) in
NMP ( 1 mL). The reaction mixture was shaken for 1 hr and then a second aliquot of
PyAOP (38 mg, 0.073 mmol) was added. Shaking was continued for 30 min. 20%
ACN/water (5 mL) was added and the product purified by semi-preparative HPLC.
Purification using semi-preparative HPLC (gradient: 20-50% B over 40 min) afforded
123 mg (98%) pure NOTA(tris-tBu)-NH-CH 2CH2-NH2. The purified material was
characterised by LC-MS (gradient: 20-50% B over 5): R: 1.95 min, found mJr. 586.4,
expected MH+: 586.4
[0268] (in) NOTA(tris-tBu)-NH-CH2CH2-NH-maleimide
[0269] NOTA(tris-tBu)-NH-CH 2CH2-NH2 (123 mg, 0.176 mmol), 3-maleimidopropionic
aid NHS ester (70 mg, 0.26 mmol) and sym.-collidine (346 m , 2.60 mmol)
were dissolved in NMP (2 mL). The reaction mixture was stirred for 6 hr. Water (6
mL) was added and the product purified by semi-preparative HPLC. Purification
using semi-preparative HPLC (gradient: 20-50% B over 40 min) afforded 115 mg
(87%) pure NOTA(tris-tBu)-NH-CH 2CH2-NH-maleimide. The purified material was
characterised by LC-MS (gradient: 10-60% B over 5): ¾: 3.36 min, found m/z: 737.4,
expected MH+: 737.4
[0270] iv Preparation of NOTA(tris-acid)-NH-CH2CH2-NH-maleimide
[0271] NOTA(tris-tBu)-NH-CH 2CH2-NH-maleimide (115 mg, 0.150 mmol) was
treated with a solution of 2.5% TIS and 2.5% water in TFA (10 mL) for 4 hrs. The
solvents were evaporated in vacuo, the residue re-dissolved in water (8 mL) and the
product purified by semi-preparative HPLC. Purification using semi-preparative
HPLC (gradient: 0-20% B over 40 min) afforded 80 mg (90%) pure NOTA(tris-acid)-
NH-CH2CH2-NH-maleimide. The purified material was characterised by LC-MS
(gradient: 0-30% B over 5): tR: 2.1 min, found m/z: 569.5, expected MH+: 569.2
[0272] (v) Preparation of Compound 6
[0273] (a) Preparation of Synthetic Z02891-Cvs
[0274] Sequence:
EAKYAKEMRNAYWEIALLPNLTNQQKRAFIRKLYDDPSQSSELLSEAKKLND
SQAPKVDC was assembled on a CEM Liberty microwave peptide synthesiser using
Fmoc chemistry starting with 0.05 mmol NovaPEG Rink Amide resin. 0.5 mmol
amino acid was applied in each coupling step (5 min at 75 °C) using 0.45 mmol
HBTU/0.45 mmol HOAt/ 1.0 mmol DIPEA for in situ activation. Fmoc was removed
by 5% piperazine in DMF. Double coupling of both Arg was applied. Asp-Ser and
Leu-Ser pseudoproline dipeptides (0.5 mmol) were incorporated into the sequence.
The simultaneous removal of the side-chain protecting groups and cleavage of the
peptide from the resin was carried out in TFA (40 mL) containing 2.5% TIS, 2.5%
EDT, 2.5% EMS and 2.5% water for 1 hr. The resin was removed by filtration,
washed with TFA and the combined filtrates were evaporated in vacuo. Diethyl ether
was added to the residue, the formed precipitate washed with diethyl ether and dried.
The cleavage procedure was repeated once more. The dried precipitates were
dissolved in 20% ACN/water and left over night in order to remove remaining Trp
protecting groups. The solution was lyophilised affording 148 mg (42%) crude
Z02891-Cys. 148 mg crude Z02891-Cys was purified by semi-preparative HPLC (4
runs, gradient: 25-30% B over 40 min) affording 33 mg (9%) pure Z02891-Cys. The
purified material was characterised by LC-MS (gradient: 10-40% B over 5): ¾: 3.40
min, found m z 1758.3, expected MH4
4+: 1758.4.
Synthetic Z02891-Cys (13.7 mg, 1.95 mihoΐ) and NOTA(tris-acid)-NH-CH2CH2-NHmaleimide
(11 mg, 19.3 mihoΐ) were dissolved in water ( 1 mL). The solution was
adjusted to pH 6 by adding ammonium acetate and the mixture shaken for 3 hrs. The
reaction mixture was diluted with water/0.1% TFA (6.5 mL) and the product was
purified using semi-preparative HPLC. Purification using semi-preparative HPLC
(gradient: 15-35% B over 40 min) afforded 8.4 mg (57%) pure 6. Compound 6 was
analysed by analytical LC-MS (gradient: 10-40% B over 5 min): tR: 3.31 min, found
m/z: 1900.7, expected MH44+: 1900.2
[0275] Example 19. Impact of the AlC13/peptide ratio radiochemical yields of
ri8FlAlF-NOTA(COOH)2-Z02891(SEO ID No. 2X5)
[0276] Three solutions of 4 (149 g, 20 nmol) in sodium acetate buffer (10 , pH
4.0, 0.5 M) were mixed with solutions of A1C1 (0.33 g, 2.49 nmol; 0.66 mg, 4.98
nmol; and 1.33 mg, 9.96 nmol, respectively) in sodium acetate buffer ( 1 m , pH 4.0,
0.5 M) in conical polypropylene centrifuge vials (1.5 mL). To these vials a small
volume of [ F]fluoride (10 m ) was added. The vials were heated for 15 min at 100
°C and subsequently analyzed by HPLC. The incorporation yields are given in Table
12.
Table 12. Impact of AlCl /peptide ratio on analytical RCY of [ F]A1FNOTA(
COOH) 2-Z02891(SEQ ID No. 2)(5).
2 1/4 29 % 2 %
3 1/2 28 % 3 %
[0277] Example 20. Impact of the reagent dilution on radiochemical yields of
ri8FlAlF-NOTA(COOH)2-Z02891(SEO ID No. 2X5)
[0278] A solution of 4 (373 , 50 nmol) in sodium acetate buffer (25 , H 4.0, 0.5
M) was mixed with a solution of AICI 3 (1.66 , 12.5 nmol) in sodium acetate buffer
(1.5 , pH 4.0, 0.5 M) in a conical polypropylene centrifuge vial (1.5 mL). A small
volume of [18 F]fluoride (10 , 80 MBq) was added. Two serial dilutions of this
mixture (50 % and 25 % v/v) with sodium acetate buffer (1.5 L·, pH 4.0, 0.5 M) were
prepared. The three vials were then heated at 100 °C for 15 minutes and subsequently
analyzed by HPLC. The data are shown in Table 13.
Table 13. Impact of reagent concentration on analytical RCY of [ F]A1FNOTA(
COOH) 2-Z02891(SEQ ID No. 2)(5). The ratio of reagents was kept constant.
[0279] Example 21. Impact of the peptide/AlC13 concentration on radiochemical
yields of ri8FlAlF-NOTA(COOH)2-Z02891(SEO ID No. 2X5)
[0280] Three vials containing [ F]fluoride (25 m , 23-25 MBq), A1C1 (¼ eq. of
peptide 4 in 1.5 m sodium acetate buffer, pH 4.0, 0.5 M), and 4 (50, 100, 150 nmol)
in sodium acetate buffer (25 m , pH 4.0, 0.5 M) were heated at 100 °C for 30 minutes.
FIG. 30 shows the incorporation data after 15 and 30 minutes.
[0281] Example 22. Impact of microwave heating on radiochemical yields of
r FlAlF-NOTA(COOH),-Z02891(SEO ID No. 2X5)
[0282] A Wheaton vial (3 mL) containing [ F]fluoride (25 m , 29 MBq), A1C1 (1.66
mg, 12.5 nmol) in 1.5 m sodium acetate buffer, pH 4.0, 0.5 M), and 4 (373 mg, 50
nmol) in sodium acetate buffer (25 m , pH 4.0, 0.5 M) was heated using a microwave
device (Resonance Instruments Model 521, set temperature 80 °C, 50 W) for 5, 10,
and 15 s. Table 14 gives the summary of the HPLC analyses after these time points.
[0283] Table 14. Analytical RCY from preparation of [ sF]AlF-NOTA(COOH) 2-
Z0289KSEO ID No. 2)(5) using microwave heating.
[0284] Example 23. Preparation of ri8F]AlF-NOTA(COOH)3-Z02891(SEQ ID
No. 2)(5a)
[0286] A PP centrifuge vial (1.5 mL) containing [ F]fluoride (25 m , 29 MBq),
A1C1 (1.66 mg, 12.5 nmol) in 1.5 sodium acetate buffer, pH 4.0, 0.5 M), and 6
(380 mg, 50 nmol) in sodium acetate buffer (25 m , pH 4.0, 0.5 M) was heated at 100
°C for 15 minutes. The analytical RCY of 5a was 15-20 %. FIG. 3 1 shows the HPLC
profile of the reaction mixture.
[0287] Example 24. Preparation of ri8F]SiFA-Z02891(SEO ID No. 2X7)
[0289] A solution of peptide precursor 8 (750 g, 100 nmol) in sodium acetate buffer
(50 , pH 4.0, 0.5 M) was added to a solution of [ FJfluoride in water (50 ) in a
polypropylene centrifuge vial (1.5 mL) and heated for 15 minutes at 95 °C. After
adding saline (100 m , 0.9 % w/v), the mixture was purified using a saline
conditioned NAP5 column (GE Healthcare). The product 7 was obtained withl8 %
non-decay corrected radiochemical yield and 87 % radiochemical purity after 26
minutes. FIG. 32 shows the HPLC analysis of the final product.
[0290] Example 24a. Preparation of Compound 8
[0291] (i Synthesis of SiFa
n-Butyllithium in hexane (2.5 M, 3.2 mL, 7.9 mmol) was added dropwise under argon
to a cooled (-78 °C) solution of 2-(4-bromophenyl)-l,3-dioxolane (1.8 g, 7.9 mmol) in
dry tetrahydrofurna (THF) (6 mL). After stirring for 2 hrs at -78 °C, the resulting
yellow suspension was taken up in a syringe and added dropwise over a period of 20
min to a cooled solution (-70 °C) of di-ieri-butyldifluorosilane (1.5 mL, 8.33 mmol) in
THF (15 mL). The reaction mixture was stirred for 1 hr at -70 °C and then allowed to
warm to ambient temperature. A sample (3 mL) was withdrawn from the reaction
mixture after 2 hrs 30 min and quenched with water/0. 1% TFA resulting in removal
of the dioxolane protecting group. The deprotected product was purified by
preparative HPLC. Purification using preparative HPLC (gradient: 40-95% B over 60
min) afforded pure SiFA. The purified material was characterised by LC-MS
(gradient: 50-95% B over 5): t 2.05 min, found m/ z not detected, expected MH+:
267.2
[0292] (ii) Preparation of SiFA-aminooxyacetyl-maleimide
[0293]
[0294] Eei-aminooxyacetyl-maleimide (20 mg, 7 1 mihoΐ ) was added to SiFA in
water/ACN/0.1% TFA (from HPLC prep fractions). 1M HC1 ( 1 mL) was added and
the reaction mixture stirred over night. The product was purified by semi-preparative
HPLC. Purification using semi-preparative HPLC (gradient: 40-80% B over 40 min)
afforded 15 mg (45%) pure SiFA-aminooxyacetyl-maleimide. The purified material
was characterised by LC-MS (gradient: 40-70% B over 5): t 3.00 min, found m/ z
462 , expected MH+: 462.2
[0295] (iii) Preparation of Compound 8
[0296] Recombinant Z02891-Cys Affibody (24 mg, 3.4 mihoΐ ) and SiFAaminooxyacetyl-
maleimide (4.7 mg, 10 mihoΐ ) were dissolved in 50% ACN/water ( 1
mL). The solution was adjusted to pH 6 by adding ammonium acetate and the mixture
shaken for 1 hr. The reaction mixture was diluted with 10% ACN/water/0. 1% TFA (8
mL) and the product purified using semi-preparative HPLC. Purification using semipreparative
HPLC (gradient: 20-40% B over 40 min) afforded 26 mg (100%) pure
Z02891-Cys-maleimide-aminooxyacetyl-SiFA (8). Purified Z02891-Cys-maleimideaminooxyacetyl-
SiFA (8) was analysed by analytical LC-MS (gradient: 10-40% B
over 5 min): R: 3.87 min, found m/z: 1873.6, expected MH4
4+ : 1873.5
[0297] Example 25. Tumour model validation
[0298] The A431 and NCI-N87 xenograft models were validated for tumour growth
and HER2 expression. The animal model setup involved inoculation of 2xl0 6 NCIN87
or 10 A431 cells per animal (in IOOmI of 50%PBS/50%Matrigel)
subcutaneously into the right flank followed by an inoculation period of 30 days.
HER2 expression in these tumours was assessed by immunohistochemistry, using the
FDA-validated HercepTest (Dako, K5204).
[0299] FIG. 33 depicts that with the recommended intensity scale (0 - +3), NCI-N87
tumours stain strongly (+3), while A431 cells show a considerably weaker staining
intensity (+1). These data suggest that the tumour models have significantly different
HER2 expression and are therefore suitable for comparing the uptake of the HER2
targeted Affibody molecules. Based on the adequate separation of IHC scores, no
further quantitative assessment was considered necessary.
[0300] Example 26. Biodistribution of Compounds 2, 5, and 7 in normal mice
[0301] The saline formulated tracers compounds 2, 5, and 7 have been evaluated
using naive CD1 mice. Following intravenous injection of 3 MBq of activity (2.5
MBq for the 2 min time point), animals were sacrificed at 2, 90, 120 and 180min post
injection and retention of radioactivity was assessed in key organs. In the
biodistribution measurements, 5 showed significant kidney retention (70.3 % ID at 90
minutes p.i.) which was not observed for the 2 or 7 (4.8 % ID and 10 % ID,
respectively at 90 min p.i.). Defluorination of 7 was observed (bone uptake 5.3 %
ID/g at 90 min p.i.). FIG. 34 compares the biodistribution data with the corresponding
[ In]DOTA-Z02891(SEQ ID No. 2)(9) compound:
[0302] Example 27. Tumour uptake of Compounds 2, 5, and 7
[0303] In a tumour mouse model with high and low HER2 level expressing tumor
cells (NC87 and A431, respectively) a differential uptake of compounds 2, 5, and 7
was observed as expected. FIG. 35, Tables 15 and 16 compare the biodistribution data
with corresponding [ In]DOTA-Z02891(SEQ ID No. 2)(9) compound.
[0304] Table 15. Key ratios from the NCI-N87 xenograft biodistribution of
Compounds 9, 2, 5 and 7.
[0305] Example 28. Imaging of 2 in dual tumour xeno2raft model
[0306] Dual tumour xenograft mice were generated by implantation of A431 and
NCI-N87 in each of the two flanks. These mice were used to assess the biodistribution
of 2, enabling a same-animal assessment of uptake in both low and high HER2
expressing tumours. Timepoints included 30 and 60 p.i.
[0307] FIG. 36 shows that 2 performance was comparable to that observed in the
single tumour animal studies, with good separation in binder uptake between the
A431 and NCI-N87 tumours, starting from as early as 30min p.i. As far as
background tissue clearance (see Table 7 for key tissue ratios), blood levels at 60min
p.i. have reduced significantly, providing a NCI-N87 tumour: blood ratio of 4.52,
while at 30min, partial blood clearance gives a 2.39 ratio, accompanied by a positive
tumounliver ratio of 1.39. These properties suggest that the pharmacokinetics of 2 is
sufficient for imaging human subjects within a suitable imaging window.
0308] Table 17. Key ratios from the dual tumour xenograft biodistribution of 2.
[0309] Example 29. Compound 2 add-back studies in NCI-N87 tumoured mice
[0310] For the add-back studies performed in the NCI-N87 tumour model to assess
the effect of excess cold ligand in binder efficacy, the following four different
preparations were assessed at 90min p.i.:
1. Standard compound 2 preparation
2. Standard preparation plus 10( g kg per mouse cold precursor
3. Standard preparation plus 50( g kg per mouse cold precursor
4. Standard preparation plus 100(Vg/kg per mouse cold precursor
The concentration of cold precursor in the standard preparation was 12C^g/kg per
mouse, therefore this study examined the effects of cold precursor at 10 x the original
concentration used (in the mouse). FIG. 37 shows that the effect on tumour uptake
was not significant and clearance from other tissues was not significantly affected
either.
[0311] Example 30. Compound 2 in vivo imaging studies in dual flank
A431/NCI-N87 tumoured mice
[0312] The dual-tumour mouse model described in Example 28 was used to perform a
preliminary imaging study. 10 MBq of 2 were injected i.v. per animal and the mice
were imaged for 30 minutes starting at 120 min p.i. The image in FIG 38 shows that
clearance was through the kidneys and bladder, as previously demonstrated through
the biodistribution studies. The transverse imaging shows uptake in the 2 tumours,
with the NCI-N87 tumour showing considerably higher signal intensity than the A43 1
tumour, in agreement with the dual tumour biodistribution studies in Example 28.
[0313] Comparison of the current 2 imaging study with the Affibody® 9 imaging
study (FIG. 38) shows a similar difference in uptake between high and low HER2
expressing tumours. However, 2 has a considerably improved background from the
kidneys due to minimal kidney retention also seen in the biodistributions.
[0314] All patents, journal articles, publications and other documents discussed
and/or cited above are hereby incorporated by reference.
What is claimed is:
1. An imaging agent composition comprises an isolated polypeptide comprising
SEQ. ID No. 1, SEQ. ID. No 2 or a conservative variant thereof, conjugated with 18F
via an isotopic fluorine exchange chemistry wherein the isolated polypeptide binds
specifically to HER2 or variants thereof.
2. A method of making an imaging agent composition according to claim 1
comprising (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 silicon
fluoride-containing moiety to form a silicon fluoride conjugated polypeptide; and (iii)
reacting the silicon fluoride conjugated polypeptide with an 18F moiety or a source of
18F to form an 18F-silicon fluoride conjugated polypeptide.
3. A method of making an imaging agent composition according to claim 1
comprising (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 a SiFA group, to form a SiFA conjugated polypeptide;
and (iii) reacting the SiFA conjugated polypeptide with an F moiety or a source of
F.
4. A pharmaceutical composition comprising an imaging agent composition
according to claim 1 and a pharmaceutically acceptable carrier.