U.S. patent application number 15/621571 was filed with the patent office on 2017-10-05 for methods and compositions for improved f-18 labeling of proteins, peptides and other molecules.
The applicant listed for this patent is Immunomedics, Inc.. Invention is credited to Christopher A. D'Souza, David M. Goldenberg, William J. McBride.
Application Number | 20170283442 15/621571 |
Document ID | / |
Family ID | 51297882 |
Filed Date | 2017-10-05 |
United States Patent
Application |
20170283442 |
Kind Code |
A1 |
D'Souza; Christopher A. ; et
al. |
October 5, 2017 |
Methods and Compositions for Improved F-18 Labeling of Proteins,
Peptides and Other Molecules
Abstract
The present application discloses compositions and methods of
synthesis and use of .sup.18F- or .sup.19F-labeled molecules of use
in PET, SPECT and/or MR imaging. Preferably, the .sup.18F or
.sup.19F is conjugated to a targeting molecule by formation of a
complex with a group IIIA metal and binding of the complex to a
bifunctional chelating agent, which may then be directly or
indirectly attached to the targeting molecule. In other
embodiments, the .sup.18F or .sup.19F labeled moiety may comprise a
targetable construct used in combination with a bispecific antibody
to target a disease-associated antigen. The disclosed methods and
compositions allow the simple and reproducible labeling of
molecules at very high efficiency and specific activity in 30
minutes or less. In preferred embodiments, the bifunctional
chelating agent bound to .sup.18F- or .sup.19F-metal complex may be
conjugated to the molecule to be labeled at a reduced temperature,
e.g. room temperature.
Inventors: |
D'Souza; Christopher A.;
(Pomona, NY) ; McBride; William J.; (Boonton,
NJ) ; Goldenberg; David M.; (Mendham, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immunomedics, Inc. |
Morris Plains |
NJ |
US |
|
|
Family ID: |
51297882 |
Appl. No.: |
15/621571 |
Filed: |
June 13, 2017 |
Related U.S. Patent Documents
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 475/04 20130101;
C07B 59/002 20130101; C07D 207/452 20130101; C07D 403/12 20130101;
C07D 405/12 20130101; C07K 7/06 20130101; C07B 2200/05 20130101;
C07D 255/02 20130101; C07F 5/069 20130101 |
International
Class: |
C07F 5/06 20060101
C07F005/06; C07D 207/452 20060101 C07D207/452; C07D 405/12 20060101
C07D405/12; C07D 475/04 20060101 C07D475/04; C07D 255/02 20060101
C07D255/02; C07B 59/00 20060101 C07B059/00; C07K 7/06 20060101
C07K007/06; C07D 403/12 20060101 C07D403/12 |
Claims
1. A method of imaging comprising: a) labeling a bifunctional
chelating agent with a complex of (i) .sup.18F or .sup.19F, and
(ii) a group IIIA metal; b) attaching the .sup.18F or .sup.19F
labeled bifunctional chelating agent to a molecule to form an
.sup.18F- or .sup.19F-labeled molecule, wherein the molecule is
selected from the group consisting of an antibody, antigen-binding
antibody fragment, bispecific antibody, affibody, diabody,
minibody, scFv, aptamer, avimer, targeting peptide, somatostatin,
bombesin, octreotide, RGD peptide and folate; c) administering the
.sup.18F- or .sup.19F-labeled molecule to a subject; and d) imaging
the distribution of the .sup.18F- or .sup.19F-labeled molecule by
positron emission tomography (PET), magnetic resonance imaging (MM)
or single photon emission computer tomography (SPECT).
2. The method of claim 1, wherein the .sup.18F-metal or
.sup.19F-metal complex is attached to the bifunctional chelating
agent at a temperature between 90.degree. C. and 110.degree. C.
3. The method of claim 1, wherein the bifunctional chelating agent
is attached to the molecule at room temperature.
4. The method of claim 1, wherein the bifunctional chelating agent
is selected from the group consisting of NODA-BA, NODA-BAEM,
NODA-BM, NODA-butyne, NODA-EA, NODA-EBA, NODA-EPA, NODA-EPN,
NODA-HA, NODA-MBA, NODA-MBEM, NODA-MPAA, NODA-MPAA NHS ester,
NDOA-MPAEM, NODA-MPAPEG.sub.3M. sub.3, NODA-MPH, NODA-MPN,
NODA-2-nitroimidazole, NODA-PA, NODA-PAEM and NODA-propyl
amine.
6. The method of claim 1, wherein the group IIIA metal is
aluminum.
7. The method of claim 1, wherein a metal-.sup.18F or
metal-.sup.19F complex is attached to the bifunctional chelating
agent by heating in an aqueous medium at a temperature between
50.degree. C. and 110.degree. C.
8. The method of claim 1, wherein the efficiency of labeling with
.sup.18F is at least 35% at 50.degree. C.
9. The method of claim 1, wherein the specific activity of the
labeled molecule is at least 4,000 Ci/mmol.
10. The method of claim 1, wherein a metal-.sup.18F or
metal-.sup.19F complex is attached to the bifunctional chelating
agent in an aqueous medium comprising aluminum, trehalose,
potassium biphthalate, ethanol and ascorbic acid at a pH between
3.9 and 4.2.
11. The method of claim 1, wherein multiple copies of the
bifunctional chelating agent are attached to the molecule.
12. The method of claim 1, wherein the .sup.18F- or
.sup.19F-labeled molecule is produced in less than 30 minutes from
the start of the method.
13. The method of claim 1, wherein the bifunctional chelating agent
is attached to the molecule by a click chemistry reaction or by a
maleimide-sulfhydryl reaction.
14. The method of claim 1, wherein the yield of labeled molecule is
at least 95%.
15. The method of claim 11, further comprising: e) analyzing the
distribution of the labeled molecule to detect, diagnose or image
the presence of a disease in the subject, wherein the disease is
selected from the group consisting of cancer, a cardiovascular
disease, an infectious disease, an inflammatory disease, an
autoimmune disease, an immune dysfunction disease, graft versus
host disease, organ transplant rejection and a neurological
disease.
16. The method of claim 15, wherein the labeled molecule binds to
an antigen selected from the group consisting of carbonic anhydrase
IX, CCL19, CCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A,
CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25,
CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52,
CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80,
CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4, CXCR7,
CXCL12, HIF-1.alpha., AFP, PSMA, CEACAMS, CEACAM-6, c-met, B7, ED-B
of fibronectin, Factor H, Flt-1, Flt-3, folate receptor,
GRO-.beta., HMGB-1, hypoxia inducible factor (HIF), HM1.24,
insulin-like growth factor-1 (ILGF-1), IFN-.gamma., IFN-.alpha.,
IFN-.beta., IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R,
IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP,
MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, NCA-95,
NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le(y), RANTES,
T101, TAC, Tn antigen, Thomson-Friedenreich antigens, tumor
necrosis antigens, TNF-.alpha., TRAIL receptor R1, TRAIL receptor
R2, VEGFR, EGFR, P1GF, complement factors C3, C3a, C3b, C5a, and
C5.
17. The method of claim 15, wherein the labeled molecule is an
antibody selected from the group consisting of hR1 (anti-IGF-1R),
hPAM4 (anti-pancreatic cancer mucin), hA20 (anti-CD20), hA19
(anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2
(anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14
(anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-EGP-1) and hMN-3
(anti-CEACAM6).
18. The method of claim 1, wherein the bifunctional chelating agent
is conjugated to a molecule via an amide, ester, anhydride,
carbonate, carbamate, dithiocarbamate, ether, thioether, disulfide,
urea, thiourea, triazoyl, amine, imine, oxime or hydrazone
bond.
19. The method of claim 1, wherein the .sup.18F-metal or
.sup.19F-metal complex is attached to the bifunctional chelating
agent in the presence of an organic solvent selected from the group
consisting of ethanol, acetonitrile, MeCN, DMF and THF.
20. The method of claim 1, wherein the molecule is selected from
the group consisting of IMP449, IMP460, IMP461, IMP467, IMP469,
IMP470, IMP471, IMP479, IMP485, IMP486, IMP487, IMP488, IMP490,
IMP493, IMP495, IMP497, IMP500, IMP508 and IMP517.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/755,712, filed Jun. 30, 2015, which is a divisional of
U.S. patent application Ser. No. 14/509,679 (now U.S. Pat. No.
9,115,172), filed Oct. 8, 2014, which was a divisional of Ser. No.
14/199,625 (now U.S. Pat. No. 8,889,100), filed Mar. 6, 2014, which
was a divisional of U.S. patent application Ser. No. 13/897,849
(now U.S. Pat. No. 8,709,382), filed May 20, 2013, which was a
continuation-in-part of U.S. patent application Ser. No. 13/850,591
(now U.S. Pat. No. 8,617,518), filed Mar. 26, 2013, which was a
divisional of Ser. No. 13/474,260 (now U.S. Pat. No. 8,444,956),
filed May 17, 2012, which was a divisional of Ser. No. 12/958,889
(now U.S. Pat. No. 8,202,509), filed Dec. 2, 2010, which was a
continuation-in-part of Ser. No. 12/433,212 (now U.S. Pat. No.
8,153,100), filed Apr. 30, 2009, which was a continuation-in-part
of Ser. No. 12/343,655 (now U.S. Pat. No. 7,993,626), filed Dec.
24, 2008, which was a continuation-in-part of Ser. No. 12/112,289
(now U.S. Pat. No. 7,563,433), filed Apr. 30, 2008, which was a
continuation-in-part of Ser. No. 11/960,262 (now U.S. Pat. No.
7,597,876), filed Dec. 19, 2007, which claimed the benefit under 35
U.S.C. 119(e) of Provisional U.S. Patent Application No.
60/884,521, filed Jan. 11, 2007. This application is
continuation-in-part of U.S. patent application Ser. No. 13/752,877
(now U.S. Pat. No. 8,496,912), filed Jan. 29, 2013, which was a
divisional of Ser. No. 13/309,714 (now U.S. Pat. No. 8,398,956),
filed Dec. 2, 2011, which was a continuation-in-part of Ser. No.
12/958,889 (now U.S. Pat. No. 8,202,509), filed Dec. 2, 2010, which
was a continuation-in-part of Ser. No. 12/433,212 (now U.S. Pat.
No. 8,153,100), filed Apr. 30, 2009, which was a
continuation-in-part of Ser. No. 12/343,655 (now U.S. Pat. No.
7,993,626), filed Dec. 24, 2008, which was a continuation-in-part
of Ser. No. 12/112,289 (now U.S. Pat. No. 7,563,433), filed Apr.
30, 2008, which was a continuation-in-part of Ser. No. 11/960,262
(now U.S. Pat. No. 7,597,876), filed Dec. 19, 2007, which claimed
the benefit under 35 U.S.C. 119(e) of Provisional U.S. Patent
Application No. 60/884,521, filed Jan. 11, 2007. This application
is a continuation-in-part of U.S. patent application Ser. No.
13/323,139 (now U.S. Pat. No. 8,545,809), filed Dec. 12, 2011,
which claimed the benefit under 35 U.S.C. 119(e) of Provisional
U.S. Patent Application Nos. 61/422,258, filed Dec. 13, 2010;
61/479,660, filed Apr. 27, 2011; 61/492,613, filed Jun. 2, 2011;
61/523,668, filed Aug. 15, 2011; 61/540,248, filed Sep. 28, 2011;
61/547,434, filed Oct. 14, 2011, and also was a
continuation-in-part of Ser. No. 13/309,714 (now U.S. Pat. No.
8,398,956), filed Dec. 2, 2011, which claimed the benefit under 35
U.S.C. 119(e) of Provisional U.S. Patent Application No.
61/419,082, filed Dec. 2, 2010. U.S. Ser. No. 13/309,714 was a
continuation-in-part of Ser. No. 12/958,889 (now U.S. Pat. No.
8,202,509), filed Dec. 2, 2010, which claimed the benefit under 35
U.S.C. 119(e) of Provisional U.S. Patent Application Nos.
61/266,773, filed Dec. 4, 2009; 61/302,280, filed Feb. 8, 2010;
61/316,125, filed Mar. 22, 2010; 61/347,486, filed May 24, 2010;
61/381,720, filed Sep. 10, 2010; 61/388,268, filed Sep. 30, 2010.
U.S. Ser. No. 12/958,889 was also a continuation-in-part of Ser.
No. 12/433,212 (now U.S. Pat. No. 8,153,100), filed Apr. 30, 2009,
which was a continuation-in-part of Ser. No. 12/343,655 (now U.S.
Pat. No. 7,993,626), filed Dec. 24, 2008, which was a
continuation-in-part of Ser. No. 12/112,289 (now U.S. Pat. No.
7,563,433), filed Apr. 30, 2008, which was a continuation-in-part
of Ser. No. 11/960,262 (now U.S. Pat. No. 7,597,876), filed Dec.
19, 2007, which claimed the benefit under 35 U.S.C. 119(e) of
Provisional U.S. Patent Application No. 60/884,521, filed Jan. 11,
2007. This application claims the benefit under 35 U.S.C. 119(e) of
Provisional U.S. Patent Application No. 61/649,526, filed May 21,
2012. The text of each priority application is incorporated herein
by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 20, 2013, is named IMM310US15_SL.txt and is 28,825 bytes in
size.
FIELD
[0003] The present invention concerns methods of labeling peptides
or other molecules with .sup.18F or .sup.19F that are of use, for
example, in PET or NMR in vivo imaging. Preferably, the .sup.18F or
.sup.19F is attached as a complex with aluminum or another metal,
such as a Group IIIA metal, via a chelating moiety, which may be
covalently linked to a protein, peptide or other molecule. The
chelating moiety may be attached to a protein, peptide or other
molecule either before or after binding to the metal-.sup.18F or
metal-.sup.19F complex. Although labeling may occur at an elevated
temperature, such as 70.degree. C., 80.degree. C., 90.degree. C.,
95.degree. C., 100.degree. C., 105.degree. C., 110.degree. C., or
any temperature in between, preferably labeling of heat sensitive
molecules may occur at a lower temperature, such as room
temperature.
[0004] In certain embodiments, the labeled molecule may be used for
targeting a cell, tissue, organ or pathogen to be imaged or
detected. Exemplary targeting molecules include, but are not
limited to, an antibody, antigen-binding antibody fragment,
bispecific antibody, affibody, diabody, minibody, ScFvs, aptamer,
avimer, targeting peptide, somatostatin, bombesin, octreotide, RGD
peptide, folate, folate analog or any other molecule known to bind
to a disease-associated target.
[0005] Using the techniques described herein, .sup.18F-labeled
molecules of high specific activity may be prepared in 30 minutes
or less and are suitable for use in imaging techniques without the
need for HPLC purification of the labeled molecule. Labeling may
occur in a saline medium suitable for direct use in vivo. In
alternative embodiments an organic solvent may be added to improve
the labeling efficiency. The labeled molecules are stable under
physiological conditions, although for certain purposes, such as
kit formulations, a stabilizing agent such as ascorbic acid,
trehalose, sorbitol or mannitol may be added. In other alternative
embodiments, a chelating moiety may be preloaded with aluminum and
lyophilized for storage, prior to labeling with .sup.18F.
BACKGROUND
[0006] Positron Emission Tomography (PET) has become one of the
most prominent functional imaging modalities in diagnostic
medicine, with very high sensitivity (fmol), high resolution (4-10
mm) and tissue accretion that can be adequately quantitated (Volkow
et al., 1988, Am. J. Physiol. Imaging 3:142). Although
[.sup.18F]-deoxy-2-fluoro-D-glucose ([.sup.18F]FDG) is the most
widely used PET imaging agent in oncology (Fletcher et al., 2008,
J. Nucl. Med. 49:480), there is a keen interest in developing other
labeled compounds for functional imaging to complement and augment
anatomic imaging methods (Torigian et al., 2007, CA Cancer J. Clin.
57:206), especially with the hybrid PET/computed tomography systems
currently in use. Thus, there is a need to have facile methods of
conjugating positron emitting radionuclides to various molecules of
biological and medical interest.
[0007] Peptides or other targeting molecules can be labeled with
the positron emitters .sup.18F, .sup.64Cu, .sup.11C, .sup.66Ga,
.sup.68Ga, .sup.76Br, .sup.94mTc, .sup.86Y, and .sup.124I. A low
ejection energy for a PET isotope is desirable to minimize the
distance that the positron travels from the target site before it
generates the two 511 keV gamma rays that are imaged by the PET
camera. Many isotopes that emit positrons also have other emissions
such as gamma rays, alpha particles or beta particles in their
decay chain. It is desirable to have a PET isotope that is a pure
positron emitter so that any dosimetry problems will be minimized.
The half-life of the isotope is also important, since the half-life
must be long enough to attach the isotope to a targeting molecule,
analyze the product, inject it into the patient, and allow the
product to localize, clear from non-target tissues and then image.
.sup.18F (.beta..sup.+97%, 635 keV, t.sub.1/2 110 min) is one of
the most widely used PET emitting isotopes because of its low
positron emission energy, lack of side emissions and suitable
half-life.
[0008] Conventionally, .sup.18F is attached to compounds by binding
it to a carbon atom (Miller et al., 2008, Angew Chem Int Ed
47:8998-9033), but attachments to silicon (Shirrmacher et al.,
2007, Bioconj Chem 18:2085-89; Hohne et al., 2008, Bioconj Chem
19:1871-79) and boron (Ting et al., 2008, Fluorine Chem 129:349-58)
have also been reported. Binding to carbon usually involves
multistep syntheses, including multiple purification steps, which
is problematic for an isotope with a 110-min half-life, and
typically results in poor radiochemical yields. Current methods for
.sup.18F-labeling of peptides typically involve the labeling of a
reagent at low specific activity, HPLC purification of the reagent
and then conjugation to the peptide of interest. The conjugate is
often repurified after conjugation to obtain the desired specific
activity of labeled peptide.
[0009] An example is the labeling method of Poethko et al. (J.
Nucl. Med. 2004; 45: 892-902) in which
4-[.sup.18F]fluorobenzaldehyde is first synthesized and purified
(Wilson et al, J. Labeled Compounds and Radiopharm. 1990; XXVIII:
1189-1199) and then conjugated to the peptide. The peptide
conjugate is then purified by HPLC to remove excess peptide that
was used to drive the conjugation to completion. Other examples
include labeling with succinimidyl [.sup.18F]fluorobenzoate (SFB)
(e.g., Vaidyanathan et al., 1992, Int. J. Rad. Appl. Instrum. B
19:275), other acyl compounds (Tada et al., 1989, Labeled Compd.
Radiopharm.XXVII:1317; Wester et al., 1996, Nucl. Med. Biol.
23:365; Guhlke et al., 1994, Nucl. Med. Biol 21:819), or click
chemistry adducts (Li et al., 2007, Bioconj Chem. 18:1987). The
total synthesis and formulation time for these methods ranges
between 1-3 hours, with most of the time dedicated to the HPLC
purification of the labeled peptides to obtain the specific
activity required for in vivo targeting. With a 2 hr half-life, all
of the manipulations that are needed to attach the .sup.18F to the
peptide are a significant burden. These methods are also tedious to
perform and require the use of equipment designed specifically to
produce the labeled product and/or the efforts of specialized
professional chemists. They are also not conducive to kit
formulations that could routinely be used in a clinical
setting.
[0010] A need exists for a rapid, simple method of .sup.18F
labeling of targeting moieties, such as proteins or peptides,
preferably at high radiochemical yield, which results in targeting
constructs of suitable specific activity and in vivo stability for
detection and/or imaging, while minimizing the requirements for
specialized equipment or highly trained personnel and reducing
operator exposure to high levels of radiation. More preferably a
need exists for methods of preparing .sup.18F-labeled targeting
peptides of use in pretargeting technologies. A further need exists
for prepackaged kits that could provide compositions required for
performing such novel methods. An additional need exists for
methods of efficiently labeling temperature sensitive
molecules.
SUMMARY
[0011] In various embodiments, the present invention concerns
compositions and methods relating to .sup.18F- or .sup.19F-labeled
molecules of use for PET or NMR imaging. As discussed herein, where
the present application refers to .sup.18F the skilled artisan will
realize that either .sup.18F, .sup.19F or another radionuclide,
such as .sup.68Ga, may be utilized. In an exemplary approach, the
.sup.18F is bound to a metal and the .sup.18F-metal complex is
attached to a ligand on a peptide or other molecule. As described
below, the metals of group IIIA (aluminum, gallium, indium, and
thallium) are suitable for .sup.18F binding, although aluminum is
preferred. Lutetium may also be of use. The metal binding ligand is
preferably a chelating agent, such as NOTA, NODA, NETA, DOTA, DTPA
and other chelating groups discussed in more detail below.
Alternatively, one can attach the metal to a molecule first and
then add the .sup.18F to bind to the metal. In still other
embodiments, one may attach an .sup.18F-metal to a chelating moiety
first and then attach the labeled chelating moiety to a molecule,
such as a temperature sensitive molecule. In this way, the
.sup.18F-metal may be attached to a chelating moiety at a higher
temperature, such as between 90.degree. to 110.degree. C., more
preferably between 95.degree. to 105.degree. C., and the
.sup.18F-labeled chelating moiety may be attached to a temperature
sensitive molecule at a lower temperature, such as at room
temperature. In preferred embodiments, the labeling method uses a
biofunctional chelator that forms a physiologically stable complex
with metal-.sup.18F, which contains reactive groups that can bind
to proteins, peptides or other targeting molecules at, e.g., room
temperature. More preferably, labeling can be accomplished in 10 to
15 minutes in aqueous medium, with a total synthesis time of about
30 minutes.
[0012] The skilled artisan will realize that virtually any delivery
molecule can be attached to .sup.18F for imaging purposes, so long
as it contains derivatizable groups that may be modified without
affecting the ligand-receptor binding interaction between the
delivery molecule and the cellular or tissue target receptor.
Although the Examples below primarily concern .sup.18F-labeled
peptide moieties, many other types of delivery molecules, such as
oligonucleotides, hormones, growth factors, cytokines, chemokines,
angiogenic factors, anti-angiogenic factors, immunomodulators,
proteins, nucleic acids, antibodies, antibody fragments, drugs,
interleukins, interferons, oligosaccharides, polysaccharides,
siderophores, lipids, etc. may be .sup.18F-labeled and utilized for
imaging purposes.
[0013] Exemplary targetable construct peptides described in the
Examples below, of use for pre-targeting delivery of .sup.18F or
other agents, include but are not limited to IMP449, IMP460,
IMP461, IMP467, IMP469, IMP470, IMP471, IMP479, IMP485, IMP486,
IMP487, IMP488, IMP490, IMP493, IMP495, IMP497, IMP500, IMP508,
IMP517, comprising chelating moieties that include, but are not
limited to, DTPA, NOTA, benzyl-NOTA, alkyl or aryl derivatives of
NOTA, NODA, NODA-GA, C-NETA, succinyl-C-NETA and bis-t-butyl-NODA.
In a preferred embodiment, a chelating moiety based on NODA-propyl
amine (e.g., (tBu).sub.2NODA-propyl amine) may be derivatized to
form a reactive thiol, maleimide, azide, alkyne or aminooxy group,
which may then be conjugated to a targeting molecule at a reduced
temperature via azide-alkyne coupling, thioether, amide,
dithiocarbamate, thiocarbamate, oxime or thiourea formation.
[0014] In certain embodiments, the exemplary .sup.18F-labeled
peptides may be of use as targetable constructs in a pre-targeting
method, utilizing bispecific or multispecific antibodies or
antibody fragments. In this case, the antibody or fragment will
comprise one or more binding sites for a target associated with a
disease or condition, such as a tumor-associated or autoimmune
disease-associated antigen or an antigen produced or displayed by a
pathogenic organism, such as a virus, bacterium, fungus or other
microorganism. A second binding site will specifically bind to the
targetable construct. Methods for pre-targeting using bispecific or
multispecific antibodies are well known in the art (see, e.g., U.S.
Pat. No. 6,962,702, the Examples section of which is incorporated
herein by reference.) Similarly, antibodies or fragments thereof
that bind to targetable constructs are also well known in the art,
such as the 679 monoclonal antibody that binds to HSG (histamine
succinyl glycine) or the 734 antibody that binds to In-DTPA (see
U.S. Pat. Nos. 7,429,381; 7,563,439; 7,666,415; and 7,534,431, the
Examples section of each incorporated herein by reference).
Generally, in pretargeting methods the bispecific or multispecific
antibody is administered first and allowed to bind to cell or
tissue target antigens. After an appropriate amount of time for
unbound antibody to clear from circulation, the e.g. 18F-labeled
targetable construct is administered to the patient and binds to
the antibody localized to target cells or tissues. Then an image is
taken, for example by PET scanning.
[0015] In alternative embodiments, molecules that bind directly to
receptors, such as somatostatin, octreotide, bombesin, folate or a
folate analog, an RGD peptide or other known receptor ligands may
be labeled and used for imaging. Receptor targeting agents may
include, for example, TA138, a non-peptide antagonist for the
integrin .alpha..sub.v.beta..sub.3 receptor (Liu et al., 2003,
Bioconj. Chem. 14:1052-56). Other methods of receptor targeting
imaging using metal chelates are known in the art and may be
utilized in the practice of the claimed methods (see, e.g., Andre
et al., 2002, J. Inorg. Biochem. 88:1-6; Pearson et al., 1996, J.
Med., Chem. 39:1361-71).
[0016] The type of diseases or conditions that may be imaged is
limited only by the availability of a suitable delivery molecule
for targeting a cell or tissue associated with the disease or
condition. Many such delivery molecules are known. For example, any
protein or peptide that binds to a diseased tissue or target, such
as cancer, may be labeled with .sup.18F by the disclosed methods
and used for detection and/or imaging. In certain embodiments, such
proteins or peptides may include, but are not limited to,
antibodies or antibody fragments that bind to tumor-associated
antigens (TAAs). Any known TAA-binding antibody or fragment may be
labeled with .sup.18F by the described methods and used for imaging
and/or detection of tumors, for example by PET scanning or other
known techniques.
[0017] Certain alternative embodiments involve the use of "click"
chemistry for attachment of .sup.18F-labeled moieties to targeting
molecules. Preferably, the click chemistry involves the reaction of
a targeting molecule such as an antibody or antigen-binding
antibody fragment, comprising a functional group such as an alkyne,
nitrone or an azide group, with a .sup.18F-labeled moiety
comprising the corresponding reactive moiety such as an azide,
alkyne or nitrone. Where the targeting molecule comprises an
alkyne, the chelating moiety or carrier will comprise an azide, a
nitrone or similar reactive moiety. The click chemistry reaction
may occur in vitro to form a highly stable, .sup.18F-labeled
targeting molecule that is then administered to a subject.
[0018] In other alternative embodiments, a prosthetic group, such
as a NODA-maleimide moiety, may be labeled with .sup.18F-metal and
then conjugated to a targeting molecule, for example by a
maleimide-sulfhydryl reaction. Exemplary NODA-maleimide moieties
include, but are not limited to, NODA-MPAEM, NODA-PM, NODA-PAEM,
NODA-BAEM, NODA-BM, NODA-MPM, and NODA-MBEM.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The following Figures are included to illustrate particular
embodiments of the invention and are not meant to be limiting as to
the scope of the claimed subject matter.
[0020] FIG. 1. Biodistribution of .sup.18F-labeled agents in
tumor-bearing nude mice by microPET imaging. Coronal slices of 3
nude mice bearing a small, subcutaneous LS174T tumor on each left
flank after being injected with either (A) .sup.18F-FDG, (B)
Al.sup.18F(IMP449) pretargeted with the anti-CEA.times.anti-HSG
bsMAb, (C) Al.sup.18F(IMP449) alone (not pretargeted with the
bsMAb). Biodistribution data expressed as percent-injected dose per
gram (% ID/g) are given for the tissues removed from the animals at
the conclusion of the imaging session. Abbreviations: B, bone
marrow; H, heart; K, kidney; T, tumor.
[0021] FIG. 2. Dynamic imaging study of pretargeted
Al.sup.18F(IMP449) given to a nude mouse bearing a 35-mg LS174T
human colorectal cancer xenograft in the upper flank. The top 3
panels show coronal, sagittal, and transverse sections,
respectively, taken of a region of the body centering on the
tumor's peripheral location at 6 different 5-min intervals over the
120-min imaging session. The first image on the left in each
sectional view shows the positioning of the tumor at the
intersection of the crosshairs, which is highlighted by arrows. The
animal was partially tilted to its right side during the imaging
session. The bottom 2 panels show additional coronal and sagittal
sections that focus on a more anterior plane in the coronal section
to highlight distribution in the liver and intestines, while the
sagittal view crosses more centrally in the body. Abbreviations:
Cor, coronal; FA, forearms; H, heart; K, kidney; Lv, liver; Sag,
sagittal; Tr, transverse; UB, urinary bladder.
[0022] FIG. 3. In vivo tissue distribution with Al.sup.18F(IMP466)
bombesin analogue.
[0023] FIG. 4. Comparison of biodistribution of Al.sup.18F(IMP466)
and .sup.68Ga(IMP466) at 2 hours post-injection in AR42J
tumor-bearing mice (n=5). As a control, mice in separate groups
(n=5) received an excess of unlabeled octreotide to demonstrate
receptor specificity.
[0024] FIG. 5. Coronal slices of PET/CT scan of Al.sup.18F(IMP466)
and .sup.68Ga(IMP466) at 2 hours post-injection in mice with an
s.c. AR42J tumor in the neck. Accumulation in tumor and kidneys is
clearly visualized.
[0025] FIG. 6. Biodistribution of 6.0 nmol .sup.125I-TF2 (0.37 MBq)
and 0.25 nmol .sup.68Ga(IMP288) (5 MBq), 1 hour after i.v.
injection of .sup.68Ga(IMP288) in BALB/c nude mice with a
subcutaneous LS174T and SK-RC52 tumor. Values are given as
means.+-.standard deviation (n=5).
[0026] FIG. 7. Biodistribution of 5 MBq FDG and of 5 MBq
.sup.68Ga(IMP288) (0.25 nmol) 1 hour after i.v. injection following
pretargeting with 6.0 nmol TF2. Values are given as
means.+-.standard deviation (n=5).
[0027] FIG. 8. PET/CT images of a BALB/c nude mouse with a
subcutaneous LS174T tumor (0.1 g) on the right hind leg (light
arrow) and a inflammation in the left thigh muscle (dark arrow),
that received 5 MBq .sup.18F-FDG, and one day later 6.0 nmol TF2
and 5 MBq .sup.68Ga(IMP288) (0.25 nmol) with a 16 hour interval.
The animal was imaged one hour after the .sup.18F-FDG and
.sup.68Ga(IMP288) injection. The panel shows the 3D volume
rendering (A), transverse sections of the tumor region (B) of the
FDG-PET scan, and the 3D volume rendering (C), transverse sections
of the tumor region (D) of the pretargeted immunoPET scan.
[0028] FIG. 9. Biodistribution of 0.25 nmol Al.sup.18F(IMP449) (5
MBq) 1 hour after i.v. injection, following 6.0 nmol TF2
administered 16 hours earlier. Biodistribution of
Al.sup.18F(IMP449) without pretargeting, or biodistribution of
[Al.sup.18F]. Values are given as means.+-.standard deviation.
[0029] FIG. 10. Static PET/CT imaging study of a BALB/c nude mouse
with a subcutaneous LS174T tumor (0.1 g) on the right side (arrow),
that received 6.0 nmol TF2 and 0.25 nmol Al.sup.18F(IMP449) (5 MBq)
intravenously with a 16 hour interval. The animal was imaged one
hour after injection of Al.sup.18F(IMP449). The panel shows the 3D
volume rendering (A) posterior view, and cross sections at the
tumor region, (B) coronal, (C) sagittal.
[0030] FIG. 11. Structure of IMP479 (SEQ ID NO:54).
[0031] FIG. 12. Structure of IMP485 (SEQ ID NO:55).
[0032] FIG. 13A. Structure of IMP487 (SEQ ID NO:56).
[0033] FIG. 13B. Structure of IMP490 (SEQ ID NO:52).
[0034] FIG. 13C. Structure of IMP493 (SEQ ID NO:53).
[0035] FIG. 13D. Structure of IMP495 (SEQ ID NO: 57).
[0036] FIG. 13E. Structure of IMP496 (SEQ ID NO: 58).
[0037] FIG. 13F. Structure of IMP500.
[0038] FIG. 14. Synthesis of bis-t-butyl-NODA-MPAA.
[0039] FIG. 15. Synthesis of maleimide conjugate of NOTA.
[0040] FIG. 16. Chemical structure of exemplary NODA-based
bifunctional chelators.
[0041] FIG. 17. Chemical structures of NODA-BM derived bifunctional
chelators.
[0042] FIG. 18. Further exemplary structures of NODA-based
bifunctional chelators: (A) NODA-HA, (B) NODA-MPN, (C) NODA-EPN,
(D) NODA-MBA, (E) NODA-EPA, (F) NODA-MPAA, (G) NODA-BAEM, (H)
NODA-MPAEM, (I) NODA-BM, (J) NODA-MBEM, (K) NODA moiety with
maleimide reactive group, (L) alternative NODA moiety with
maleimide reactive group, (M) NODA-BA, (N) NODA-EA, (0) NODA-MPH,
(P) NODA-butyne, (Q) NODA-MPAPEG.sub.3N.sub.3, (R) NODA moiety with
carboxyl reactive group, (S) NODA moiety with nitrophenyl reactive
group, (T) NODA moiety with carboxyl and nitrophenyl reactive
groups, (U) another NODA moiety with carboxyl reactive group, (V)
another NODA moiety with carboxyl reactive group, (W) another NODA
moiety with carboxyl reactive group, (X) another NODA moiety with
carboxyl reactive group, (Y) another NODA moiety with carboxyl
reactive group, (Z) another NODA moiety with carboxyl reactive
group, (AA) another NODA moiety with carboxyl reactive group, (BB)
another NODA moiety with carboxyl reactive group, (CC) another NODA
moiety with carboxyl reactive group.
[0043] FIG. 19A and FIG. 19B. Radiochromatograms of the
.sup.18F-labeled functionalized TACN ligands.
[0044] FIG. 20A and FIG. 20B. Radiochromatograms of
.sup.18F-hMN14-Fab', its stability in human serum and
immunoreactivity with CEA.
[0045] FIG. 21. Schematic diagram of automated synthesis module for
.sup.18F-labeling via [Al.sup.18F]-chelation.
[0046] FIG. 22. NODA-propyl amine derived bifunctional chelating
moieties.
[0047] FIG. 23. (A) Structure of IMP 508 (SEQ ID NO: 59). (B)
Structure of IMP517 (SEQ ID NO: 60). (C) NODA-2-nitroimidazole. (D)
NOTA-DUPA-Peptide.
[0048] FIG. 24. Labeling efficiency as a function of
temperature.
DETAILED DESCRIPTION
[0049] The following definitions are provided to facilitate
understanding of the disclosure herein. Terms that are not
explicitly defined are used according to their plain and ordinary
meaning.
[0050] As used herein, "a" or "an" may mean one or more than one of
an item.
[0051] As used herein, the terms "and" and "or" may be used to mean
either the conjunctive or disjunctive. That is, both terms should
be understood as equivalent to "and/or" unless otherwise
stated.
[0052] As used herein, "about" means within plus or minus ten
percent of a number. For example, "about 100" would refer to any
number between 90 and 110.
[0053] As used herein, a "peptide" refers to any sequence of
naturally occurring or non-naturally occurring amino acids of
between 2 and 100 amino acid residues in length, more preferably
between 2 and 10, more preferably between 2 and 6 amino acids in
length. An "amino acid" may be an L-amino acid, a D-amino acid, an
amino acid analogue, an amino acid derivative or an amino acid
mimetic.
[0054] As used herein, the term "pathogen" includes, but is not
limited to fungi, viruses, parasites and bacteria, including but
not limited to human immunodeficiency virus (HIV), herpes virus,
cytomegalovirus, rabies virus, influenza virus, hepatitis B virus,
Sendai virus, feline leukemia virus, Reovirus, polio virus, human
serum parvo-like virus, simian virus 40, respiratory syncytial
virus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue
virus, rubella virus, measles virus, adenovirus, human T-cell
leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps
virus, vesicular stomatitis virus, Sindbis virus, lymphocytic
choriomeningitis virus, wart virus, blue tongue virus,
Streptococcus agalactiae, Legionella pneumophila, Streptococcus
pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria
meningitidis, Pneumococcus, Hemophilus influenzae B, Treponema
pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,
Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis
and Clostridium tetani.
[0055] As used herein, a "radiolysis protection agent" refers to
any molecule, compound or composition that may be added to an
.sup.18F-labeled complex or molecule to decrease the rate of
breakdown of the .sup.18F-labeled complex or molecule by
radiolysis. Any known radiolysis protection agent, including but
not limited to ascorbic acid, may be used.
.sup.18F Labeling Techniques
[0056] A variety of techniques for labeling molecules with .sup.18F
are known. Table 1 lists the properties of several of the more
commonly reported fluorination procedures. Peptide labeling through
carbon often involves .sup.18F-binding to a prosthetic group
through nucleophilic substitution, usually in 2- or 3-steps where
the prosthetic group is labeled and purified, attached to the
compound, and then purified again. This general method has been
used to attach prosthetic groups through amide bonds, aldehydes,
and "click" chemistry (Marik et al., 2006, Bioconj Chem 17:1017-21;
Poethko et al., 2004, J Nucl Med 45:892-902; Li et al., 2007,
Bioconj Chem 18:989-93). The most common amide bond-forming reagent
has been N-succinimidyl 4-.sup.18F-fluorobenzoate (.sup.18F-SFB),
but a number of other groups have been tested (Marik et al., 2006).
In some cases, such as when .sup.18F-labeled active ester
amide-forming groups are used, it may be necessary to protect
certain groups on a peptide during the coupling reaction, after
which they are cleaved. The synthesis of this .sup.18F-SFB reagent
and subsequent conjugation to the peptide requires many synthetic
steps and takes about 2-3 h.
[0057] A simpler, more efficient .sup.18F-peptide labeling method
was developed by Poethko et al. (2004), where a
4-.sup.18F-fluorobenzaldehyde reagent was conjugated to a peptide
through an oxime linkage in about 75-90 min, including the dry-down
step. The newer "click chemistry" method attaches .sup.18F-labeled
molecules onto peptides with an acetylene or azide in the presence
of a copper catalyst (Li et al, 2007; Glaser and Arstad, 2007,
Bioconj Chem 18:989-93). The reaction between the azide and
acetylene groups forms a triazole connection, which is quite stable
and forms very efficiently on peptides without the need for
protecting groups. Click chemistry produces the .sup.18F-labeled
peptides in good yield (.about.50%) in about 75-90 min with the
dry-down step.
TABLE-US-00001 TABLE 1 Summary of selected .sup.18F-labeling
methods. Author/Ref. Schirrmacher Hohne Li et al. Glaser &
Poethko Marik et et al. (2007) et al. (2008) (2007) Arstad (2007)
et al. (2004) al (2006) Attachment Silicon Silicon Click Click
Aldehyde/ Amide oxime Rx steps 2 1 2 2 2 many Rx time 40 115-155
110 65-80 75-90 min 110.sup.+ (min).sup.a (estimated) (estimated)
Yield.sup.b 55% 13% 54% 50% 40% 10% HPLC- 1 1 2 1 + 1 .sup. 2
purification distillation steps Specific 225-680 62 high high high
high Activity (GBq/.mu.mol) .sup.aIncluding dry-down time
.sup.bDecay corrected
[0058] A more recent method of binding .sup.18F to silicon uses
isotopic exchange to displace .sup.19F with .sup.18F (Shirrmacher
et al., 2007). Performed at room temperature in 10 min, this
reaction produces the .sup.18F-prosthetic aldehyde group with high
specific activity (225-680 GBq/.mu.mol; 6,100-18,400 Ci/mmol). The
.sup.18F-labeled aldehyde is subsequently conjugated to a peptide
and purified by HPLC, and the purified labeled peptide is obtained
within 40 min (including dry-down) with .about.55% yield. This was
modified subsequently to a single-step process by incorporating the
silicon into the peptide before the labeling reaction (Hohne et al,
2008). However, biodistribution studies in mice with an
.sup.18F-silicon-bombesin derivative showed bone uptake increasing
over time (1.35.+-.0.47% injected dose (ID)/g at 0.5 h vs.
5.14.+-.2.71% ID/g at 4.0 h), suggesting a release of .sup.18F from
the peptide, since unbound .sup.18F is known to localize in bone
(Hohne et al., 2008). HPLC analysis of urine showed a substantial
amount of .sup.18F activity in the void volume, which presumably is
due to fluoride anion (.sup.18F.sup.-) released from the peptide.
It would therefore appear that the .sup.18F-silicon labeled
molecule was not stable in serum. Substantial hepatobiliary
excretion was also reported, attributed to the lipophilic nature of
the .sup.18F-silicon-binding substrate, and requiring future
derivatives to be more hydrophilic. Methods of attaching .sup.18F
to boron also have been explored; however, the current process
produces conjugates with low specific activity (Ting et al.,
2008).
[0059] Antibodies and peptides are coupled routinely with
radiometals, typically in 15 min and in quantitative yields (Meares
et al., 1984, Acc Chem Res 17:202-209; Scheinberg et al., 1982,
Science 215:1511-13). For PET imaging, .sup.64Cu and .sup.68Ga have
been bound to peptides via a chelate, and have shown reasonably
good PET-imaging properties (Heppler et al., 2000, Current Med Chem
7:971-94). Since fluoride binds to most metals, we sought to
determine if an .sup.18F-metal complex could be bound to a chelator
on a targeting molecule (Tewson, 1989, Nucl Med Biol. 16:533-51;
Martin, 1996, Coordination Chem Rev 141:23-32). We have focused on
the binding of an Al.sup.18F complex, since aluminum-fluoride can
be relatively stable in vivo (Li, 2003, Crit Rev Oral Biol Med
14:100-114; Antonny et al., 1992, J Biol Chem 267:6710-18). Initial
studies showed the feasibility of this approach to prepare an
.sup.18F-labeled peptide for in vivo targeting of cancer with a
bispecific antibody (bsMAb) pretargeting system, a highly sensitive
and specific technique for localizing cancer, in some cases better
than [.sup.18F]FDG (fluorodeoxyglucose) (McBride et al., 2008, J
Nucl Med (suppl) 49:97P; Wagner, 2008, J Nucl Med 49:23N-24N;
Karacay et al., 2000, Bioconj Chem 11:842-54; Sharkey et al., 2008,
Cancer Res 68; 5282-90; Gold Et al., 2008, Cancer Res 68:4819-26;
Sharkey et al., 2005, Nature Med 11:1250-55; Sharkey et al., 2005,
Clin Cancer Res 11:7109s-7121s; McBride et al., 2006, J Nucl Med
47:1678-88; Sharkey et al., 2008, Radiology 246:497-508). These
studies revealed that an Al.sup.18F complex could bind stably to a
1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), but the yields
were low.
[0060] In the Examples below, new labeling conditions and several
new chelating moieties were examined that enhanced yields from
about 10% to about 80%, providing a feasible method for
.sup.18F-labeling of peptides and other molecules of use in PET
imaging.
Targetable Constructs
[0061] In certain embodiments, the moiety labeled with .sup.18F or
other diagnostic and/or therapeutic agents may comprise a peptide
or other targetable construct. Labeled peptides (or proteins), for
example RGD peptide, octreotide, bombesin or somatostatin, may be
selected to bind directly to a targeted cell, tissue, pathogenic
organism or other target for imaging, detection and/or diagnosis.
In other embodiments, labeled peptides may be selected to bind
indirectly, for example using a bispecific antibody with one or
more binding sites for a targetable construct peptide and one or
more binding sites for a target antigen associated with a disease
or condition. Bispecific antibodies may be used, for example, in a
pretargeting technique wherein the antibody may be administered
first to a subject. Sufficient time may be allowed for the
bispecific antibody to bind to a target antigen and for unbound
antibody to clear from circulation. Then a targetable construct,
such as a labeled peptide, may be administered to the subject and
allowed to bind to the bispecific antibody and localize at the
diseased cell or tissue. The distribution of .sup.18F-labeled
targetable constructs may be determined by PET scanning or other
known techniques.
[0062] Such targetable constructs can be of diverse structure and
are selected not only for the availability of an antibody or
fragment that binds with high affinity to the targetable construct,
but also for rapid in vivo clearance when used within the
pre-targeting method and bispecific antibodies (bsAb) or
multispecific antibodies. Hydrophobic agents are best at eliciting
strong immune responses, whereas hydrophilic agents are preferred
for rapid in vivo clearance. Thus, a balance between hydrophobic
and hydrophilic character is established. This may be accomplished,
in part, by using hydrophilic chelating agents to offset the
inherent hydrophobicity of many organic moieties. Also, sub-units
of the targetable construct may be chosen which have opposite
solution properties, for example, peptides, which contain amino
acids, some of which are hydrophobic and some of which are
hydrophilic. Aside from peptides, carbohydrates may also be
used.
[0063] Peptides having as few as two amino acid residues,
preferably two to ten residues, may be used and may also be coupled
to other moieties, such as chelating agents. The linker should be a
low molecular weight conjugate, preferably having a molecular
weight of less than 50,000 daltons, and advantageously less than
about 20,000 daltons, 10,000 daltons or 5,000 daltons. More
usually, the targetable construct peptide will have four or more
residues, such as the peptide
DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH.sub.2 (SEQ ID NO: 1), wherein
DOTA is 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid and
HSG is the histamine succinyl glycyl group. Alternatively, DOTA may
be replaced by NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid),
TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid),
NETA
([2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylme-
thyl-amino]acetic acid) or other known chelating moieties.
[0064] The targetable construct may also comprise unnatural amino
acids, e.g., D-amino acids, in the backbone structure to increase
the stability of the peptide in vivo. In alternative embodiments,
other backbone structures such as those constructed from
non-natural amino acids or peptoids may be used.
[0065] The peptides used as targetable constructs are conveniently
synthesized on an automated peptide synthesizer using a solid-phase
support and standard techniques of repetitive orthogonal
deprotection and coupling. Free amino groups in the peptide, that
are to be used later for conjugation of chelating moieties or other
agents, are advantageously blocked with standard protecting groups
such as a Boc group, while N-terminal residues may be acetylated to
increase serum stability. Such protecting groups are well known to
the skilled artisan. See Greene and Wuts Protective Groups in
Organic Synthesis, 1999 (John Wiley and Sons, N.Y.). When the
peptides are prepared for later use within the bispecific antibody
system, they are advantageously cleaved from the resins to generate
the corresponding C-terminal amides, in order to inhibit in vivo
carboxypeptidase activity. Exemplary methods of peptide synthesis
are disclosed in the Examples below.
[0066] Where pretargeting with bispecific antibodies is used, the
antibody will contain a first binding site for an antigen produced
by or associated with a target tissue and a second binding site for
a hapten on the targetable construct. Exemplary haptens include,
but are not limited to, HSG and In-DTPA. Antibodies raised to the
HSG hapten are known (e.g. 679 antibody) and can be easily
incorporated into the appropriate bispecific antibody (see, e.g.,
U.S. Pat. Nos. 6,962,702; 7,138,103 and 7,300,644, incorporated
herein by reference with respect to the Examples sections).
However, other haptens and antibodies that bind to them are known
in the art and may be used, such as In-DTPA and the 734 antibody
(e.g., U.S. Pat. No. 7,534,431, the Examples section incorporated
herein by reference).
[0067] The skilled artisan will realize that although the majority
of targetable constructs disclosed in the Examples below are
peptides, other types of molecules may be used as targetable
constructs. For example, polymeric molecules, such as polyethylene
glycol (PEG) may be easily derivatized with chelating moieties to
bind Al.sup.18F. Many examples of such carrier molecules are known
in the art and may be utilized, including but not limited to
polymers, nanoparticles, microspheres, liposomes and micelles. For
use in pretargeted delivery of .sup.18F, the only requirement is
that the carrier molecule comprise one or more chelating moieties
for attachment of metal-.sup.18F and one or more hapten moieties to
bind to a bispecific or multispecific antibody or other targeting
molecule.
Chelating Moieties
[0068] In some embodiments, an .sup.18F-labeled molecule may
comprise one or more hydrophilic chelating moieties, which can bind
metal ions and also help to ensure rapid in vivo clearance.
Chelators may be selected for their particular metal-binding
properties, and may be readily interchanged.
[0069] Particularly useful metal-chelate combinations include
2-benzyl-DTPA and its monomethyl and cyclohexyl analogs.
Macrocyclic chelators such as NOTA
(1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA, TETA
(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) and NETA
are also of use with a variety of metals, that may potentially be
used as ligands for .sup.18F-labeling.
[0070] DTPA and DOTA-type chelators, where the ligand includes hard
base chelating functions such as carboxylate or amine groups, are
most effective for chelating hard acid cations, especially Group
IIa and Group IIIa metal cations. Such metal-chelate complexes can
be made very stable by tailoring the ring size to the metal of
interest. Other ring-type chelators such as macrocyclic polyethers
are of interest for stably binding nuclides. Porphyrin chelators
may be used with numerous metal complexes. More than one type of
chelator may be conjugated to a carrier to bind multiple metal
ions. Chelators such as those disclosed in U.S. Pat. No. 5,753,206,
especially thiosemicarbazonylglyoxylcysteine (Tscg-Cys) and
thiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelators are
advantageously used to bind soft acid cations of Tc, Re, Bi and
other transition metals, lanthanides and actinides that are tightly
bound to soft base ligands. It can be useful to link more than one
type of chelator to a peptide. Because antibodies to a di-DTPA
hapten are known (Barbet et al., U.S. Pat. No. 5,256,395) and are
readily coupled to a targeting antibody to form a bispecific
antibody, it is possible to use a peptide hapten with cold diDTPA
chelator and another chelator for binding an .sup.18F complex, in a
pretargeting protocol. One example of such a peptide is
Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys)-NH.sub.2 (core peptide
disclosed as SEQ ID NO:2). Other hard acid chelators such as DOTA,
TETA and the like can be substituted for the DTPA and/or Tscg-Cys
groups, and MAbs specific to them can be produced using analogous
techniques to those used to generate the anti-di-DTPA MAb.
[0071] Another useful chelator may comprise a NOTA-type moiety, for
example as disclosed in Chong et al. (J. Med. Chem., 2008,
51:118-25). Chong et al. disclose the production and use of a
bifunctional C-NETA ligand, based upon the NOTA structure, that
when complexed with .sup.177Lu or .sup.205/206Bi showed stability
in serum for up to 14 days. The chelators are not limiting and
these and other examples of chelators that are known in the art
and/or described in the following Examples may be used in the
practice of the invention.
[0072] It will be appreciated that two different hard acid or soft
acid chelators can be incorporated into the targetable construct,
e.g., with different chelate ring sizes, to bind preferentially to
two different hard acid or soft acid cations, due to the differing
sizes of the cations, the geometries of the chelate rings and the
preferred complex ion structures of the cations. This will permit
two different metals, one or both of which may be attached to
.sup.18F, to be incorporated into a targetable construct for
eventual capture by a pretargeted bispecific antibody.
Antibodies
[0073] Target Antigens
[0074] Targeting antibodies of use may be specific to or selective
for a variety of cell surface or disease-associated antigens.
Exemplary target antigens of use for imaging or treating various
diseases or conditions, such as a malignant disease, a
cardiovascular disease, an infectious disease, an inflammatory
disease, an autoimmune disease, a metabolic disease, or a
neurological (e.g., neurodegenerative) disease may include
.alpha.-fetoprotein (AFP), A3, amyloid beta, CA125, colon-specific
antigen-p (CSAp), carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1,
CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19,
CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38,
CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e,
CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138,
CD147, CD154, CXCR4, CXCR7, CXCL12, HIF-1.alpha., AFP, CEACAM5,
CEACAM6, c-met, B7, ED-B of fibronectin, EGP-1, EGP-2, Factor H,
FHL-1, fibrin, Flt-3, folate receptor, glycoprotein IIb/IIIa,
GRO-.beta., human chorionic gonadotropin (HCG), HER-2/neu, HMGB-1,
hypoxia inducible factor (HIF), HM1.24, HLA-DR, Ia, ICAM-1,
insulin-like growth factor-1 (IGF-1), IGF-1R, IFN-.gamma.,
IFN-.alpha., IFN-.beta., IL-2, IL-4R, IL-6R, IL-13R, IL-15R,
IL-17R, IL-18R, IL-1, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,
IL-25, IP-10, KS-1, Le(y), low-density lipoprotein (LDL), MAGE,
mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5a-c,
MUC16, NCA-95, NCA-90, as NF-.kappa.B, pancreatic cancer mucin,
PAM4 antigen, placental growth factor, p53, PLAGL2, Pr1, prostatic
acid phosphatase, PSA, PRAIVIE, PSMA, P1GF, tenascin, RANTES, T101,
TAC, TAG72, TF, Tn antigen, Thomson-Friedenreich antigens,
thrombin, tumor necrosis antigens, TNF-.alpha., TRAIL receptor (R1
and R2), TROP2, VEGFR, EGFR, complement factors C3, C3a, C3b, C5a,
C5, and an oncogene product.
[0075] In certain embodiments, such as imaging or treating tumors,
antibodies of use may target tumor-associated antigens. These
antigenic markers may be substances produced by a tumor or may be
substances which accumulate at a tumor site, on tumor cell surfaces
or within tumor cells. Among such tumor-associated markers are
those disclosed by Herberman, "Immunodiagnosis of Cancer", in
Fleisher ed., "The Clinical Biochemistry of Cancer", page 347
(American Association of Clinical Chemists, 1979) and in U.S. Pat.
Nos. 4,150,149; 4,361,544; and 4,444,744, the Examples section of
each of which is incorporated herein by reference. Reports on tumor
associated antigens (TAAs) include Mizukami et al., (2005, Nature
Med. 11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets
5:229-48); Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44);
and Ren et al. (2005, Ann. Surg. 242:55-63), each incorporated
herein by reference with respect to the TAAs identified.
[0076] Tumor-associated markers have been categorized by Herberman,
supra, in a number of categories including oncofetal antigens,
placental antigens, oncogenic or tumor virus associated antigens,
tissue associated antigens, organ associated antigens, ectopic
hormones and normal antigens or variants thereof. Occasionally, a
sub-unit of a tumor-associated marker is advantageously used to
raise antibodies having higher tumor-specificity, e.g., the
beta-subunit of human chorionic gonadotropin (HCG) or the gamma
region of carcinoembryonic antigen (CEA), which stimulate the
production of antibodies having a greatly reduced cross-reactivity
to non-tumor substances as disclosed in U.S. Pat. Nos. 4,361,644
and 4,444,744.
[0077] Another marker of interest is transmembrane activator and
CAML-interactor (TACI). See Yu et al. Nat. Immunol. 1:252-256
(2000). Briefly, TACI is a marker for B-cell malignancies (e.g.,
lymphoma). TACI and B-cell maturation antigen (BCMA) are bound by
the tumor necrosis factor homolog--a proliferation-inducing ligand
(APRIL). APRIL stimulates in vitro proliferation of primary B and
T-cells and increases spleen weight due to accumulation of B-cells
in vivo. APRIL also competes with TALL-I (also called BLyS or BAFF)
for receptor binding. Soluble BCMA and TACI specifically prevent
binding of APRIL and block APRIL-stimulated proliferation of
primary B-cells. BCMA-Fc also inhibits production of antibodies
against keyhole limpet hemocyanin and Pneumovax in mice, indicating
that APRIL and/or TALL-I signaling via BCMA and/or TACI are
required for generation of humoral immunity. Thus, APRIL-TALL-I and
BCMA-TACI form a two ligand-two receptor pathway involved in
stimulation of B and T-cell function.
[0078] Where the disease involves a lymphoma, leukemia or
autoimmune disorder, targeted antigens may be selected from the
group consisting of CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21,
CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54,
CD67, CD74, CD79a, CD80, CD126, CD138, CD154, B7, MUC1, Ia, Ii,
HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, an oncogene
(e.g., c-met or PLAGL2), an oncogene product, CD66a-d, necrosis
antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4) and TRAIL-R2
(DR5).
[0079] In some embodiments, target antigens may be selected from
the group consisting of (A) proinflammatory effectors of the innate
immune system, (B) coagulation factors, (C) complement factors and
complement regulatory proteins, and (D) targets specifically
associated with an inflammatory or immune-dysregulatory disorder or
with a pathologic angiogenesis or cancer, wherein the latter target
is not (A), (B), or (C). Suitable targets are described in U.S.
patent application Ser. No. 11/296,432, filed Dec. 8, 2005, the
Examples section of which is incorporated herein by reference.
[0080] The proinflammatory effector of the innate immune system may
be a proinflammatory effector cytokine, a proinflammatory effector
chemokine or a proinflammatory effector receptor. Suitable
proinflammatory effector cytokines include MIF, HMGB-1 (high
mobility group box protein 1), TNF-.alpha., IL-1, IL-4, IL-5, IL-6,
IL-8, IL-12, IL-15, and IL-18. Examples of proinflammatory effector
chemokines include CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A,
MIP-1B, ENA-78, MCP-1, IP-10, GRO-.beta., and eotaxin.
Proinflammatory effector receptors include IL-4R (interleukin-4
receptor), IL-6R (interleukin-6 receptor), IL-13R (interleukin-13
receptor), IL-15R (interleukin-15 receptor) and IL-18R
(interleukin-18 receptor).
[0081] The targeting molecule may bind to a coagulation factor,
such as tissue factor (TF) or thrombin. In other embodiments, the
targeting molecule may bind to a complement factor or complement
regulatory protein. In preferred embodiments, the complement factor
is selected from the group consisting of C3, C5, C3a, C3b, and C5a.
When the targeting molecule binds to a complement regulatory
protein, the complement regulatory protein preferably is selected
from the group consisting of CD46, CD55, CD59 and mCRP.
[0082] MIF is a pivotal cytokine of the innate immune system and
plays an important part in the control of inflammatory responses.
Originally described as a T lymphocyte-derived factor that
inhibited the random migration of macrophages, the protein known as
macrophage migration inhibitory factor (MIF) was an enigmatic
cytokine for almost 3 decades. In recent years, the discovery of
MIF as a product of the anterior pituitary gland and the cloning
and expression of bioactive, recombinant MIF protein have led to
the definition of its critical biological role in vivo. MIF has the
unique property of being released from macrophages and T
lymphocytes that have been stimulated by glucocorticoids. Once
released, MIF overcomes the inhibitory effects of glucocorticoids
on TNF-.alpha., IL-1.beta., IL-6, and IL-8 production by
LPS-stimulated monocytes in vitro and suppresses the protective
effects of steroids against lethal endotoxemia in vivo. MIF also
antagonizes glucocorticoid inhibition of T-cell proliferation in
vitro by restoring IL-2 and IFN-gamma production. MIF is the first
mediator to be identified that can counter-regulate the inhibitory
effects of glucocorticoids and thus plays a critical role in the
host control of inflammation and immunity. MIF is particularly of
use in cancer, pathological angiogenesis, and sepsis or septic
shock. More recently, CD74 has been identified as an endogenous
receptor for MIF, along with CD44, CXCR2 and CXCR4 (see, e.g.,
Baron et al., 2011, J Neuroscience Res 89:711-17). Targeting
molecules that bind to MIF, CD74, CD44, CXCR2 and/or CXCR4 may be
of use for imaging various of these conditions.
[0083] HMGB-1, a DNA binding nuclear and cytosolic protein, is a
proinflammatory cytokine released by monocytes and macrophages that
have been activated by IL-1.beta., TNF, or LPS. Via its B box
domain, it induces phenotypic maturation of DCs. It also causes
increased secretion of the proinflammatory cytokines IL-1 .alpha.,
IL-6, IL-8, IL-12, TNF-.alpha. and RANTES. HMGB-1 released by
necrotic cells may be a signal of tissue or cellular injury that,
when sensed by DCs, induces and/or enhances an immune reaction.
Palumbo et al. report that HMBG1 induces mesoangioblast migration
and proliferation (J Cell Biol, 164:441-449, 2004). Targeting
molecules that target HMBG-1 may be of use in detecting, diagnosing
or treating arthritis, particularly collagen-induced arthritis,
sepsis and/or septic shock. Yang et al., PNAS USA 101:296-301
(2004); Kokkola et al., Arthritis Rheum, 48:2052-8 (2003); Czura et
al., J Infect Dis, 187 Suppl 2:S391-6 (2003); Treutiger et al., J
Intern Med, 254:375-85 (2003).
[0084] TNF-.alpha. is an important cytokine involved in systemic
inflammation and the acute phase response. TNF-.alpha. is released
by stimulated monocytes, fibroblasts, and endothelial cells.
Macrophages, T-cells and B-lymphocytes, granulocytes, smooth muscle
cells, eosinophils, chondrocytes, osteoblasts, mast cells, glial
cells, and keratinocytes also produce TNF-.alpha. after
stimulation. Its release is stimulated by several other mediators,
such as interleukin-1 and bacterial endotoxin, in the course of
damage, e.g., by infection. It has a number of actions on various
organ systems, generally together with interleukins-1 and -6.
TNF-.alpha. is a useful target for sepsis or septic shock.
[0085] The complement system is a complex cascade involving
proteolytic cleavage of serum glycoproteins often activated by cell
receptors. The "complement cascade" is constitutive and
non-specific but it must be activated in order to function.
Complement activation results in a unidirectional sequence of
enzymatic and biochemical reactions. In this cascade, a specific
complement protein, C5, forms two highly active, inflammatory
byproducts, C5a and CSb, which jointly activate white blood cells.
This in turn evokes a number of other inflammatory byproducts,
including injurious cytokines, inflammatory enzymes, and cell
adhesion molecules. Together, these byproducts can lead to the
destruction of tissue seen in many inflammatory diseases. This
cascade ultimately results in induction of the inflammatory
response, phagocyte chemotaxis and opsonization, and cell
lysis.
[0086] The complement system can be activated via two distinct
pathways, the classical pathway and the alternate pathway. Some of
the components must be enzymatically cleaved to activate their
function; others simply combine to form complexes that are active.
Active components of the classical pathway include C1q, C1r, C1s,
C2a, C2b, C3a, C3b, C4a, and C4b. Active components of the
alternate pathway include C3a, C3b, Factor B, Factor Ba, Factor Bb,
Factor D, and Properdin. The last stage of each pathway is the
same, and involves component assembly into a membrane attack
complex. Active components of the membrane attack complex include
C5a, C5b, C6, C7, C8, and C9n.
[0087] While any of these components of the complement system can
be targeted, certain of the complement components are preferred.
C3a, C4a and C5a cause mast cells to release chemotactic factors
such as histamine and serotonin, which attract phagocytes,
antibodies and complement, etc. These form one group of preferred
targets. Another group of preferred targets includes C3b, C4b and
C5b, which enhance phagocytosis of foreign cells. Another preferred
group of targets are the predecessor components for these two
groups, i.e., C3, C4 and C5. C5b, C6, C7, C8 and C9 induce lysis of
foreign cells (membrane attack complex) and form yet another
preferred group of targets.
[0088] Coagulation factors also are preferred targets, particularly
tissue factor (TF) and thrombin. TF is also known also as tissue
thromboplastin, CD142, coagulation factor III, or factor III. TF is
an integral membrane receptor glycoprotein and a member of the
cytokine receptor superfamily. The ligand binding extracellular
domain of TF consists of two structural modules with features that
are consistent with the classification of TF as a member of type-2
cytokine receptors. TF is involved in the blood coagulation
protease cascade and initiates both the extrinsic and intrinsic
blood coagulation cascades by forming high affinity complexes
between the extracellular domain of TF and the circulating blood
coagulation factors, serine proteases factor VII or factor VIIa.
These enzymatically active complexes then activate factor IX and
factor X, leading to thrombin generation and clot formation.
[0089] TF is expressed by various cell types, including monocytes,
macrophages and vascular endothelial cells, and is induced by IL-1,
TNF-.alpha. or bacterial lipopolysaccharides. Protein kinase C is
involved in cytokine activation of endothelial cell TF expression.
Induction of TF by endotoxin and cytokines is an important
mechanism for initiation of disseminated intravascular coagulation
seen in patients with Gram-negative sepsis. TF also appears to be
involved in a variety of non-hemostatic functions including
inflammation, cancer, brain function, immune response, and
tumor-associated angiogenesis. Thus, targeting molecules that
target TF are of use in coagulopathies, sepsis, cancer, pathologic
angiogenesis, and other immune and inflammatory dysregulatory
diseases.
[0090] In other embodiments, the targeting molecule may bind to a
MEW class I, MHC class II or accessory molecule, such as CD40,
CD54, CD80 or CD86. The binding molecule also may bind to a T-cell
activation cytokine, or to a cytokine mediator, such as
NF-.kappa.B. Targets associated with sepsis and immune
dysregulation and other immune disorders include MIF, IL-1, IL-6,
IL-8, CD74, CD83, and C5aR. Antibodies and inhibitors against C5aR
have been found to improve survival in rodents with sepsis
(Huber-Lang et al., FASEB J 2002; 16:1567-1574; Riedemann et al., J
Clin Invest 2002; 110:101-108) and septic shock and adult
respiratory distress syndrome in monkeys (Hangen et al., J Surg Res
1989; 46:195-199; Stevens et al., J Clin Invest 1986;
77:1812-1816). Thus, for sepsis, preferred targets are associated
with infection, such as LPS/C5a. Other preferred targets include
HMGB-1, TF, CD14, VEGF, and IL-6, each of which is associated with
septicemia or septic shock.
[0091] In still other embodiments, a target may be associated with
graft versus host disease or transplant rejection, such as MIF (Lo
et al., Bone Marrow Transplant, 30(6):375-80 (2002)), CD74 or
HLA-DR. A target also may be associated with acute respiratory
distress syndrome, such as IL-8 (Bouros et al., PMC Pulm Med,
4(1):6 (2004), atherosclerosis or restenosis, such as MIF (Chen et
al., Arterioscler Thromb Vasc Biol, 24(4):709-14 (2004), asthma,
such as IL-18 (Hata et al., Int Immunol, Oct. 11, 2004 Epub ahead
of print), a granulomatous disease, such as TNF-.alpha. (Ulbricht
et al., Arthritis Rheum, 50(8):2717-8 (2004), a neuropathy, such as
carbamylated EPO (erythropoietin) (Leist et al., Science
305(5681):164-5 (2004), or cachexia, such as IL-6 and
TNF-.alpha..
[0092] Other targets include C5a, LPS, IFN-gamma, B7; CD2, CD4,
CD14, CD18, CD11a, CD11b, CD11c, CD14, CD18, CD27, CD29, CD38,
CD40L, CD52, CD64, CD83, CD147, CD154. Activation of mononuclear
cells by certain microbial antigens, including LPS, can be
inhibited to some extent by antibodies to CD18, CD11b, or CD11c,
which thus implicate .beta..sub.2-integrins (Cuzzola et al., J
Immunol 2000; 164:5871-5876; Medvedev et al., J Immunol 1998; 160:
4535-4542). CD83 has been found to play a role in giant cell
arteritis (GCA), which is a systemic vasculitis that affects
medium- and large-size arteries, predominately the extracranial
branches of the aortic arch and of the aorta itself, resulting in
vascular stenosis and subsequent tissue ischemia, and the severe
complications of blindness, stroke and aortic arch syndrome (Weyand
and Goronzy, N Engl J Med 2003; 349:160-169; Hunder and Valente,
In: Inflammatory Diseases of Blood Vessels. G. S. Hoffman and C. M.
Weyand, eds, Marcel Dekker, New York, 2002; 255-265). Antibodies to
CD83 were found to abrogate vasculitis in a SCID mouse model of
human GCA (Ma-Krupa et al., J Exp Med 2004; 199:173-183),
suggesting to these investigators that dendritic cells, which
express CD83 when activated, are critical antigen-processing cells
in GCA. In these studies, they used a mouse anti-CD83 MAb (IgG1
clone HB15e from Research Diagnostics). CD154, a member of the TNF
family, is expressed on the surface of CD4-positive T-lymphocytes,
and it has been reported that a humanized monoclonal antibody to
CD154 produced significant clinical benefit in patients with active
systemic lupus erythematosus (SLE) (Grammar et al., J Clin Invest
2003; 112:1506-1520). It also suggests that this antibody might be
useful in other autoimmune diseases (Kelsoe, J Clin Invest 2003;
112:1480-1482). Indeed, this antibody was also reported as
effective in patients with refractory immune thrombocytopenic
purpura (Kuwana et al., Blood 2004; 103:1229-1236).
[0093] In rheumatoid arthritis, a recombinant interleukin-1
receptor antagonist, IL-1 Ra or anakinra, has shown activity (Cohen
et al., Ann Rheum Dis 2004; 63:1062-8; Cohen, Rheum Dis Clin North
Am 2004; 30:365-80). An improvement in treatment of these patients,
which hitherto required concomitant treatment with methotrexate, is
to combine anakinra with one or more of the anti-proinflammatory
effector cytokines or anti-proinflammatory effector chemokines (as
listed above). Indeed, in a review of antibody therapy for
rheumatoid arthritis, Taylor (Curr Opin Pharmacol 2003; 3:323-328)
suggests that in addition to TNF, other antibodies to such
cytokines as IL-1, IL-6, IL-8, IL-15, IL-17 and IL-18, are
useful.
[0094] Methods for Raising Antibodies
[0095] MAbs can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A or Protein-G
Sepharose, size-exclusion chromatography, and ion-exchange
chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and
pages 2.9.1-2.9.3. Also, see Baines et al., "Purification of
Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10,
pages 79-104 (The Humana Press, Inc. 1992). After the initial
raising of antibodies to the immunogen, the antibodies can be
sequenced and subsequently prepared by recombinant techniques.
Humanization and chimerization of murine antibodies and antibody
fragments are well known to those skilled in the art, as discussed
below.
[0096] Chimeric Antibodies
[0097] A chimeric antibody is a recombinant protein in which the
variable regions of a human antibody have been replaced by the
variable regions of, for example, a mouse antibody, including the
complementarity-determining regions (CDRs) of the mouse antibody.
Chimeric antibodies exhibit decreased immunogenicity and increased
stability when administered to a subject. General techniques for
cloning murine immunoglobulin variable domains are disclosed, for
example, in Orlandi et al., Proc. Nat'l Acad. Sci. USA 6: 3833
(1989). Techniques for constructing chimeric antibodies are well
known to those of skill in the art. As an example, Leung et al.,
Hybridoma 13:469 (1994), produced an LL2 chimera by combining DNA
sequences encoding the V.sub.K and V.sub.H domains of murine LL2,
an anti-CD22 monoclonal antibody, with respective human .kappa. and
IgG.sub.1 constant region domains.
[0098] Humanized Antibodies
[0099] Techniques for producing humanized MAbs are well known in
the art (see, e.g., Jones et al., Nature 321: 522 (1986), Riechmann
et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534
(1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992),
Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J.
Immun. 150: 2844 (1993)). A chimeric or murine monoclonal antibody
may be humanized by transferring the mouse CDRs from the heavy and
light variable chains of the mouse immunoglobulin into the
corresponding variable domains of a human antibody. The mouse
framework regions (FR) in the chimeric monoclonal antibody are also
replaced with human FR sequences. As simply transferring mouse CDRs
into human FRs often results in a reduction or even loss of
antibody affinity, additional modification might be required in
order to restore the original affinity of the murine antibody. This
can be accomplished by the replacement of one or more human
residues in the FR regions with their murine counterparts to obtain
an antibody that possesses good binding affinity to its epitope.
See, for example, Tempest et al., Biotechnology 9:266 (1991) and
Verhoeyen et al., Science 239: 1534 (1988). Preferred residues for
substitution include FR residues that are located within 1, 2, or 3
Angstroms of a CDR residue side chain, that are located adjacent to
a CDR sequence, or that are predicted to interact with a CDR
residue.
[0100] Human Antibodies
[0101] Methods for producing fully human antibodies using either
combinatorial approaches or transgenic animals transformed with
human immunoglobulin loci are known in the art (e.g., Mancini et
al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005,
Comb. Chem. High Throughput Screen. 8:117-26; Brekke and Loset,
2003, Curr. Opin. Pharmacol. 3:544-50). A fully human antibody also
can be constructed by genetic or chromosomal transfection methods,
as well as phage display technology, all of which are known in the
art. See for example, McCafferty et al., Nature 348:552-553 (1990).
Such fully human antibodies are expected to exhibit even fewer side
effects than chimeric or humanized antibodies and to function in
vivo as essentially endogenous human antibodies.
[0102] In one alternative, the phage display technique may be used
to generate human antibodies (e.g., Dantas-Barbosa et al., 2005,
Genet. Mol. Res. 4:126-40). Human antibodies may be generated from
normal humans or from humans that exhibit a particular disease
state, such as cancer (Dantas-Barbosa et al., 2005). The advantage
to constructing human antibodies from a diseased individual is that
the circulating antibody repertoire may be biased towards
antibodies against disease-associated antigens.
[0103] In one non-limiting example of this methodology,
Dantas-Barbosa et al. (2005) constructed a phage display library of
human Fab antibody fragments from osteosarcoma patients. Generally,
total RNA was obtained from circulating blood lymphocytes (Id.).
Recombinant Fab were cloned from the .mu., .gamma. and .kappa.
chain antibody repertoires and inserted into a phage display
library (Id.). RNAs were converted to cDNAs and used to make Fab
cDNA libraries using specific primers against the heavy and light
chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol.
222:581-97). Library construction was performed according to
Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual,
Barbas et al. (eds), 1.sup.st edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22). The
final Fab fragments were digested with restriction endonucleases
and inserted into the bacteriophage genome to make the phage
display library. Such libraries may be screened by standard phage
display methods, as known in the art. Phage display can be
performed in a variety of formats, for their review, see e.g.
Johnson and Chiswell, Current Opinion in Structural Biology
3:5564-571 (1993).
[0104] Human antibodies may also be generated by in vitro activated
B-cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, incorporated
herein by reference in their entirety. The skilled artisan will
realize that these techniques are exemplary and any known method
for making and screening human antibodies or antibody fragments may
be utilized.
[0105] In another alternative, transgenic animals that have been
genetically engineered to produce human antibodies may be used to
generate antibodies against essentially any immunogenic target,
using standard immunization protocols. Methods for obtaining human
antibodies from transgenic mice are disclosed by Green et al.,
Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994),
and Taylor et al., Int. Immun. 6:579 (1994). A non-limiting example
of such a system is the XenoMouse.RTM. (e.g., Green et al., 1999,
J. Immunol. Methods 231:11-23, incorporated herein by reference)
from Abgenix (Fremont, Calif.). In the XenoMouse.RTM. and similar
animals, the mouse antibody genes have been inactivated and
replaced by functional human antibody genes, while the remainder of
the mouse immune system remains intact.
[0106] The XenoMouse.RTM. was transformed with germline-configured
YACs (yeast artificial chromosomes) that contained portions of the
human IgH and Igkappa loci, including the majority of the variable
region sequences, along with accessory genes and regulatory
sequences. The human variable region repertoire may be used to
generate antibody producing B-cells, which may be processed into
hybridomas by known techniques. A XenoMouse.RTM. immunized with a
target antigen will produce human antibodies by the normal immune
response, which may be harvested and/or produced by standard
techniques discussed above. A variety of strains of XenoMouse.RTM.
are available, each of which is capable of producing a different
class of antibody. Transgenically produced human antibodies have
been shown to have therapeutic potential, while retaining the
pharmacokinetic properties of normal human antibodies (Green et
al., 1999). The skilled artisan will realize that the claimed
compositions and methods are not limited to use of the
XenoMouse.RTM. system but may utilize any transgenic animal that
has been genetically engineered to produce human antibodies.
[0107] Known Antibodies
[0108] The skilled artisan will realize that the targeting
molecules of use for imaging, detection and/or diagnosis may
incorporate any antibody or fragment known in the art that has
binding specificity for a target antigen associated with a disease
state or condition. Such known antibodies include, but are not
limited to, hR1 (anti-IGF-1R, U.S. patent application Ser. No.
12/772,645, filed Mar. 12, 2010) hPAM4 (anti-pancreatic cancer
mucin, U.S. Pat. No. 7,282,567), hA20 (anti-CD20, U.S. Pat. No.
7,251,164), hA19 (anti-CD19, U.S. Pat. No. 7,109,304), hIMMU31
(anti-AFP, U.S. Pat. No. 7,300,655), hLL1 (anti-CD74, U.S. Pat. No.
7,312,318), hLL2 (anti-CD22, U.S. Pat. No. 7,074,403), hMu-9
(anti-CSAp, U.S. Pat. No. 7,387,773), hL243 (anti-HLA-DR, U.S. Pat.
No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat. No. 6,676,924),
hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,662,378, U.S. patent
application Ser. No. 12/846,062, filed Jul. 29, 2010), hRS7
(anti-TROP2), U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S.
Pat. No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No.
7,138,496) the Examples section of each cited patent or application
incorporated herein by reference.
[0109] Alternative antibodies of use include, but are not limited
to, abciximab (anti-glycoprotein IIb/IIIa), alemtuzumab
(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR),
gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20),
panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab
(anti-CD20), trastuzumab (anti-ErbB2), abagovomab (anti-CA-125),
adecatumumab (anti-EpCAM), atlizumab (anti-IL-6 receptor),
benralizumab (anti-CD125), CC49 (anti-TAG-72), AB-PG1-XG1-026
(anti-PSMA, U.S. patent application Ser. No. 11/983,372, deposited
as ATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575),
tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25),
daclizumab (anti-CD25), efalizumab (anti-CD11a), GA101 (anti-CD20;
Glycart Roche), muromonab-CD3 (anti-CD3 receptor), natalizumab
(anti-.alpha.4 integrin), omalizumab (anti-IgE); anti-TNF-.alpha.
antibodies such as CDP571 (Ofei et al., 2011, Diabetes 45:881-85),
MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (Thermo Scientific,
Rockford, Ill.), infliximab (CENTOCOR, Malvern, Pa.), certolizumab
pegol (UCB, Brussels, Belgium), anti-CD70L (UCB, Brussels,
Belgium), adalimumab (Abbott, Abbott Park, Ill.), Benlysta (Human
Genome Sciences); and antibodies against pathogens such as CR6261
(anti-influenza), exbivirumab (anti-hepatitis B), felvizumab
(anti-respiratory syncytial virus), foravirumab (anti-rabies
virus), motavizumab (anti-respiratory syncytial virus), palivizumab
(anti-respiratory syncytial virus), panobacumab (anti-Pseudomonas),
rafivirumab (anti-rabies virus), regavirumab
(anti-cytomegalovirus), sevirumab (anti-cytomegalovirus), tivirumab
(anti-hepatitis B), and urtoxazumab (anti-E. coli).
[0110] Other antibodies are known to target antigens associated
with diseased cells, tissues or organs. For example, bapineuzumab
is in clinical trials for therapy of Alzheimer's disease. Other
antibodies proposed for Alzheimer's disease include Alz 50
(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47),
gantenerumab, and solanezumab. Anti-CD3 antibodies have been
proposed for type 1 diabetes (Cernea et al., 2010, Diabetes Metab
Rev 26:602-05). Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1)
are known and in clinical trials as imaging agents for disclosing
fibrin clots and pulmonary emboli, while anti-granulocyte
antibodies, such as MN-3, MN-15, anti-NCA95, and anti-CD15
antibodies, can target myocardial infarcts and myocardial ischemia.
(See, e.g., U.S. Pat. Nos. 5,487,892; 5,632,968; 6,294,173;
7,541,440, the Examples section of each incorporated herein by
reference) Anti-macrophage, anti-low-density lipoprotein (LDL) and
anti-CD74 (e.g., hLL1) antibodies can be used to target
atherosclerotic plaques. Abciximab (anti-glycoprotein IIb/IIIa) has
been approved for adjuvant use for prevention of restenosis in
percutaneous coronary interventions and the treatment of unstable
angina (Waldmann et al., 2000, Hematol 1:394-408). Anti-CD3
antibodies have been reported to reduce development and progression
of atherosclerosis (Steffens et al., 2006, Circulation
114:1977-84). Antibodies against oxidized LDL induced a regression
of established atherosclerosis in a mouse model (Ginsberg, 2007, J
Am Coll Cardiol 52:2319-21). Anti-ICAM-1 antibody was shown to
reduce ischemic cell damage after cerebral artery occlusion in rats
(Zhang et al., 1994, Neurology 44:1747-51). Commercially available
monoclonal antibodies to leukocyte antigens are represented by: OKT
anti-T cell monoclonal antibodies (available from Ortho
Pharmaceutical Company) which bind to normal T-lymphocytes; the
monoclonal antibodies produced by the hybridomas having the ATCC
accession numbers HB44, HB55, HB12, HB78 and HB2; G7E11, W8E7,
NKP15 and GO22 (Becton Dickinson); NEN9.4 (New England Nuclear);
and FMC11 (Sera Labs). A description of antibodies against fibrin
and platelet antigens is contained in Knight, Semin. Nucl. Med.,
20:52-67 (1990).
[0111] Known antibodies of use may bind to antigens produced by or
associated with pathogens, such as HIV. Such antibodies may be used
to detect, diagnose and/or treat infectious disease. Candidate
anti-HIV antibodies include the anti-envelope antibody described by
Johansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), as well as the
anti-HIV antibodies described and sold by Polymun (Vienna,
Austria), also described in U.S. Pat. No. 5,831,034, U.S. Pat. No.
5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos
et al., Antimicrob. Agents Chemother. 2006; 50(5):1773-9, all
incorporated herein by reference.
[0112] Antibodies against malaria parasites can be directed against
the sporozoite, merozoite, schizont and gametocyte stages.
Monoclonal antibodies have been generated against sporozoites
(cirumsporozoite antigen), and have been shown to bind to
sporozoites in vitro and in rodents (N. Yoshida et al., Science
207:71-73, 1980). Several groups have developed antibodies to T.
gondii, the protozoan parasite involved in toxoplasmosis (Kasper et
al., J. Immunol. 129:1694-1699, 1982; Id., 30:2407-2412, 1983).
Antibodies have been developed against schistosomular surface
antigens and have been found to bind to schistosomulae in vivo or
in vitro (Simpson et al., Parasitology, 83:163-177, 1981; Smith et
al., Parasitology, 84:83-91, 1982: Gryzch et al., J. Immunol.,
129:2739-2743, 1982; Zodda et al., J. Immunol. 129:2326-2328, 1982;
Dissous et al., J. Immunol., 129:2232-2234, 1982)
[0113] Trypanosoma cruzi is the causative agent of Chagas' disease,
and is transmitted by blood-sucking reduviid insects. An antibody
has been generated that specifically inhibits the differentiation
of one form of the parasite to another (epimastigote to
trypomastigote stage) in vitro and which reacts with a cell-surface
glycoprotein; however, this antigen is absent from the mammalian
(bloodstream) forms of the parasite (Sher et al., Nature,
300:639-640, 1982).
[0114] Anti-fungal antibodies are known in the art, such as
anti-Sclerotinia antibody (U.S. Pat. No. 7,910,702);
antiglucuronoxylomannan antibody (Zhong and Priofski, 1998, Clin
Diag Lab Immunol 5:58-64); anti-Candida antibodies (Matthews and
Burnie, 2001, 2:472-76); and anti-glycosphingolipid antibodies
(Toledo et al., 2010, BMC Microbiol 10:47).
[0115] Where bispecific antibodies are used, the second MAb may be
selected from any anti-hapten antibody known in the art, including
but not limited to h679 (U.S. Pat. No. 7,429,381) and 734 (U.S.
Pat. Nos. 7,429,381; 7,563,439; 7,666,415; and 7,534,431), the
Examples section of each of which is incorporated herein by
reference.
[0116] Various other antibodies of use are known in the art (e.g.,
U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744;
6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702;
7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;
7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239 and U.S.
Patent Application Publ. No. 20060193865; each incorporated herein
by reference.) Such known antibodies are of use for detection
and/or imaging of a variety of disease states or conditions (e.g.,
hMN-14 or TF2 (CEA-expressing carcinomas), hA20 or TF-4 (lymphoma),
hPAM4 or TF-10 (pancreatic cancer), RS7 (lung, breast, ovarian,
prostatic cancers), hMN-15 or hMN3 (inflammation), anti-gp120
and/or anti-gp41 (HIV), anti-platelet and anti-thrombin (clot
imaging), anti-myosin (cardiac necrosis), anti-CXCR4 (cancer and
inflammatory disease)).
[0117] Antibodies of use may be commercially obtained from a wide
variety of known sources. For example, a variety of antibody
secreting hybridoma lines are available from the American Type
Culture Collection (ATCC, Manassas, Va.). A large number of
antibodies against various disease targets, including but not
limited to tumor-associated antigens, have been deposited at the
ATCC and/or have published variable region sequences and are
available for use in the claimed methods and compositions. See,
e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403;
7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803; 7,041,802;
7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598; 6,998,468;
6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018; 6,964,854;
6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244; 6,946,129;
6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533; 6,919,433;
6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625; 6,887,468;
6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568;
6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226; 6,838,282;
6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206; 6,793,924;
6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681; 6,764,679;
6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966; 6,709,653;
6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355; 6,682,737;
6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852; 6,635,482;
6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279; 6,596,852;
6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618; 6,545,130;
6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227; 6,518,404;
6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408; 6,479,247;
6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356; 6,455,044;
6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404; 6,432,402;
6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,276;
6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654; 6,372,215;
6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244; 6,346,246;
6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393; 6,254,868;
6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289; 6,077,499;
5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554; 5,776,456;
5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953, 5,525,338.
These are exemplary only and a wide variety of other antibodies and
their hybridomas are known in the art. The skilled artisan will
realize that antibody sequences or antibody-secreting hybridomas
against almost any disease-associated antigen may be obtained by a
simple search of the ATCC, NCBI and/or USPTO databases for
antibodies against a selected disease-associated target of
interest. The antigen binding domains of the cloned antibodies may
be amplified, excised, ligated into an expression vector,
transfected into an adapted host cell and used for protein
production, using standard techniques well known in the art.
Antibody Fragments
[0118] Antibody fragments which recognize specific epitopes can be
generated by known techniques. The antibody fragments are antigen
binding portions of an antibody, such as F(ab').sub.2, Fab',
F(ab).sub.2, Fab, Fv, sFv and the like. F(ab').sub.2 fragments can
be produced by pepsin digestion of the antibody molecule and Fab'
fragments can be generated by reducing disulfide bridges of the
F(ab').sub.2 fragments. Alternatively, Fab' expression libraries
can be constructed (Huse et al., 1989, Science, 246:1274-1281) to
allow rapid and easy identification of monoclonal Fab' fragments
with the desired specificity. An antibody fragment can be prepared
by proteolytic hydrolysis of the full length antibody or by
expression in E. coli or another host of the DNA coding for the
fragment. These methods are described, for example, by Goldenberg,
U.S. Pat. Nos. 4,036,945 and 4,331,647 and references contained
therein, which patents are incorporated herein in their entireties
by reference. Also, see Nisonoff et al., Arch Biochem. Biophys. 89:
230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman et al., in
METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and
Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0119] A single chain Fv molecule (scFv) comprises a V.sub.L domain
and a V.sub.H domain. The V.sub.L and V.sub.H domains associate to
form a target binding site. These two domains are further
covalently linked by a peptide linker (L). Methods for making scFv
molecules and designing suitable peptide linkers are described in
U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M.
Whitlow, "Single Chain Fvs." FASEB Vol 9:73-80 (1995) and R. E.
Bird and B. W. Walker, "Single Chain Antibody Variable Regions,"
TIBTECH, Vol 9: 132-137 (1991), incorporated herein by
reference.
[0120] An scFv library with a large repertoire can be constructed
by isolating V-genes from non-immunized human donors using PCR
primers corresponding to all known V.sub.H, V.sub.kappa and
V.sub.80 gene families. See, e.g., Vaughn et al., Nat. Biotechnol.,
14: 309-314 (1996). Following amplification, the V.sub.kappa and
V.sub.lambda pools are combined to form one pool. These fragments
are ligated into a phagemid vector. The scFv linker is then ligated
into the phagemid upstream of the V.sub.L fragment. The V.sub.H and
linker-V.sub.L fragments are amplified and assembled on the J.sub.H
region. The resulting V.sub.H-linker-V.sub.L fragments are ligated
into a phagemid vector. The phagemid library can be panned for
binding to the selected antigen.
[0121] Other antibody fragments, for example single domain antibody
fragments, are known in the art and may be used in the claimed
constructs. Single domain antibodies (VHH) may be obtained, for
example, from camels, alpacas or llamas by standard immunization
techniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001;
Yau et al., J Immunol Methods 281:161-75, 2003; Maass et al., J
Immunol Methods 324:13-25, 2007). The VHH may have potent
antigen-binding capacity and can interact with novel epitopes that
are inaccessible to conventional VH-VL pairs. (Muyldermans et al.,
2001) Alpaca serum IgG contains about 50% camelid heavy chain only
IgG antibodies (Cabs) (Maass et al., 2007). Alpacas may be
immunized with known antigens and VHHs can be isolated that bind to
and neutralize the target antigen (Maass et al., 2007). PCR primers
that amplify virtually all alpaca VHH coding sequences have been
identified and may be used to construct alpaca VHH phage display
libraries, which can be used for antibody fragment isolation by
standard biopanning techniques well known in the art (Maass et al.,
2007). These and other known antigen-binding antibody fragments may
be utilized in the claimed methods and compositions.
General Techniques for Antibody Cloning and Production
[0122] Various techniques, such as production of chimeric or
humanized antibodies, may involve procedures of antibody cloning
and construction. The antigen-binding V.kappa. (variable light
chain) and V.sub.H (variable heavy chain) sequences for an antibody
of interest may be obtained by a variety of molecular cloning
procedures, such as RT-PCR, 5'-RACE, and cDNA library screening.
The V genes of a MAb from a cell that expresses a murine MAb can be
cloned by PCR amplification and sequenced. To confirm their
authenticity, the cloned V.sub.L and V.sub.H genes can be expressed
in cell culture as a chimeric Ab as described by Orlandi et al.,
(Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V gene
sequences, a humanized MAb can then be designed and constructed as
described by Leung et al. (Mol. Immunol., 32: 1413 (1995)).
[0123] cDNA can be prepared from any known hybridoma line or
transfected cell line producing a murine MAb by general molecular
cloning techniques (Sambrook et al., Molecular Cloning, A
laboratory manual, 2.sup.nd Ed (1989)). The V.kappa. sequence for
the MAb may be amplified using the primers VK1BACK and VK1FOR
(Orlandi et al., 1989) or the extended primer set described by
Leung et al. (BioTechniques, 15: 286 (1993)). The V.sub.H sequences
can be amplified using the primer pair VH1BACK/VH1FOR (Orlandi et
al., 1989) or the primers annealing to the constant region of
murine IgG described by Leung et al. (Hybridoma, 13:469 (1994)).
Humanized V genes can be constructed by a combination of long
oligonucleotide template syntheses and PCR amplification as
described by Leung et al. (Mol. Immunol., 32: 1413 (1995)).
[0124] PCR products for V.kappa. can be subcloned into a staging
vector, such as a pBR327-based staging vector, VKpBR, that contains
an Ig promoter, a signal peptide sequence and convenient
restriction sites. PCR products for V.sub.H can be subcloned into a
similar staging vector, such as the pBluescript-based VHpBS.
Expression cassettes containing the V.kappa. and V.sub.H sequences
together with the promoter and signal peptide sequences can be
excised from VKpBR and VHpBS and ligated into appropriate
expression vectors, such as pKh and pG1g, respectively (Leung et
al., Hybridoma, 13:469 (1994)). The expression vectors can be
co-transfected into an appropriate cell and supernatant fluids
monitored for production of a chimeric, humanized or human MAb.
Alternatively, the V.kappa. and V.sub.H expression cassettes can be
excised and subcloned into a single expression vector, such as
pdHL2, as described by Gillies et al. (J. Immunol. Methods 125:191
(1989) and also shown in Losman et al., Cancer, 80:2660
(1997)).
[0125] In an alternative embodiment, expression vectors may be
transfected into host cells that have been pre-adapted for
transfection, growth and expression in serum-free medium. Exemplary
cell lines that may be used include the Sp/EEE, Sp/ESF and Sp/ESF-X
cell lines (see, e.g., U.S. Pat. Nos. 7,531,327; 7,537,930 and
7,608,425; the Examples section of each of which is incorporated
herein by reference). These exemplary cell lines are based on the
Sp2/0 myeloma cell line, transfected with a mutant Bcl-EEE gene,
exposed to methotrexate to amplify transfected gene sequences and
pre-adapted to serum-free cell line for protein expression.
Bispecific and Multispecific Antibodies
[0126] Certain embodiments concern pretargeting methods with
bispecific antibodies and hapten-bearing targetable constructs.
Numerous methods to produce bispecific or multispecific antibodies
are known, as disclosed, for example, in U.S. Pat. No. 7,405,320,
the Examples section of which is incorporated herein by reference.
Bispecific antibodies can be produced by the quadroma method, which
involves the fusion of two different hybridomas, each producing a
monoclonal antibody recognizing a different antigenic site
(Milstein and Cuello, Nature, 1983; 305:537-540).
[0127] Another method for producing bispecific antibodies uses
heterobifunctional cross-linkers to chemically tether two different
monoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631;
Perez, et al. Nature. 1985; 316:354-356). Bispecific antibodies can
also be produced by reduction of each of two parental monoclonal
antibodies to the respective half molecules, which are then mixed
and allowed to reoxidize to obtain the hybrid structure (Staerz and
Bevan. Proc Natl Acad Sci USA. 1986; 83:1453-1457). Other methods
include improving the efficiency of generating hybrid hybridomas by
gene transfer of distinct selectable markers via retrovirus-derived
shuttle vectors into respective parental hybridomas, which are
fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA. 1990,
87:2941-2945); or transfection of a hybridoma cell line with
expression plasmids containing the heavy and light chain genes of a
different antibody.
[0128] Cognate V.sub.H and V.sub.L domains can be joined with a
peptide linker of appropriate composition and length (usually
consisting of more than 12 amino acid residues) to form a
single-chain Fv (scFv), as discussed above. Reduction of the
peptide linker length to less than 12 amino acid residues prevents
pairing of V.sub.H and V.sub.L domains on the same chain and forces
pairing of V.sub.H and V.sub.L domains with complementary domains
on other chains, resulting in the formation of functional
multimers. Polypeptide chains of V.sub.H and V.sub.L domains that
are joined with linkers between 3 and 12 amino acid residues form
predominantly dimers (termed diabodies). With linkers between 0 and
2 amino acid residues, trimers (termed triabody) and tetramers
(termed tetrabody) are favored, but the exact patterns of
oligomerization appear to depend on the composition as well as the
orientation of V-domains (V.sub.H-linker-V.sub.L or
V.sub.L-linker-V.sub.H), in addition to the linker length.
[0129] These techniques for producing multispecific or bispecific
antibodies exhibit various difficulties in terms of low yield,
necessity for purification, low stability or the
labor-intensiveness of the technique. More recently, a technique
known as "dock and lock" (DNL), discussed in more detail below, has
been utilized to produce combinations of virtually any desired
antibodies, antibody fragments and other effector molecules (see,
e.g., U.S. Patent Application Publ. Nos. 20060228357; 20060228300;
20070086942; 20070140966 and 20070264265, the Examples section of
each incorporated herein by reference). The DNL technique allows
the assembly of monospecific, bispecific or multispecific
antibodies, either as naked antibody moieties or in combination
with a wide range of other effector molecules such as
immunomodulators, enzymes, chemotherapeutic agents, chemokines,
cytokines, diagnostic agents, therapeutic agents, radionuclides,
imaging agents, anti-angiogenic agents, growth factors,
oligonucleotides, siderophores, hormones, peptides, toxins,
pro-apoptotic agents, or a combination thereof. Any of the
techniques known in the art for making bispecific or multispecific
antibodies may be utilized in the practice of the presently claimed
methods.
Dock-And-Lock (DNL)
[0130] In preferred embodiments, bispecific or multispecific
antibodies or other constructs may be produced using the
dock-and-lock technology (see, e.g., U.S. Pat. Nos. 7,550,143;
7,521,056; 7,534,866; 7,527,787 and 7,666,400, the Examples section
of each incorporated herein by reference). The DNL method exploits
specific protein/protein interactions that occur between the
regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and
the anchoring domain (AD) of A-kinase anchoring proteins (AKAPs)
(Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,
Nat. Rev. Mol. Cell Biol. 2004; 5: 959). PKA, which plays a central
role in one of the best studied signal transduction pathways
triggered by the binding of the second messenger cAMP to the R
subunits, was first isolated from rabbit skeletal muscle in 1968
(Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure of the
holoenzyme consists of two catalytic subunits held in an inactive
form by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443).
Isozymes of PKA are found with two types of R subunits (RI and MI),
and each type has .alpha. and .beta. isoforms (Scott, Pharmacol.
Ther. 1991; 50:123). The R subunits have been isolated only as
stable dimers and the dimerization domain has been shown to consist
of the first 44 amino-terminal residues (Newlon et al., Nat.
Struct. Biol. 1999; 6:222). Binding of cAMP to the R subunits leads
to the release of active catalytic subunits for a broad spectrum of
serine/threonine kinase activities, which are oriented toward
selected substrates through the compartmentalization of PKA via its
docking with AKAPs (Scott et al., J. Biol. Chem. 1990; 265;
21561)
[0131] Since the first AKAP, microtubule-associated protein-2, was
characterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA.
1984; 81:6723), more than 50 AKAPs that localize to various
sub-cellular sites, including plasma membrane, actin cytoskeleton,
nucleus, mitochondria, and endoplasmic reticulum, have been
identified with diverse structures in species ranging from yeast to
humans (Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The
AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (Carr
et al., J. Biol. Chem. 1991; 266:14188). The amino acid sequences
of the AD are quite varied among individual AKAPs, with the binding
affinities reported for RII dimers ranging from 2 to 90 nM (Alto et
al., Proc. Natl. Acad. Sci. USA. 2003; 100:4445). AKAPs will only
bind to dimeric R subunits. For human RII.alpha., the AD binds to a
hydrophobic surface formed by the 23 amino-terminal residues
(Colledge and Scott, Trends Cell Biol. 1999; 6:216). Thus, the
dimerization domain and AKAP binding domain of human RII.alpha. are
both located within the same N-terminal 44 amino acid sequence
(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO
J. 2001; 20:1651), which is termed the DDD herein.
[0132] We have developed a platform technology to utilize the DDD
of human RII.alpha. and the AD of AKAP as an excellent pair of
linker modules for docking any two entities, referred to hereafter
as A and B, into a noncovalent complex, which could be further
locked into a binding molecule through the introduction of cysteine
residues into both the DDD and AD at strategic positions to
facilitate the formation of disulfide bonds. The general
methodology of the "dock-and-lock" approach is as follows. Entity A
is constructed by linking a DDD sequence to a precursor of A,
resulting in a first component hereafter referred to as a. Because
the DDD sequence would effect the spontaneous formation of a dimer,
A would thus be composed of a.sub.2. Entity B is constructed by
linking an AD sequence to a precursor of B, resulting in a second
component hereafter referred to as b. The dimeric motif of DDD
contained in a.sub.2 will create a docking site for binding to the
AD sequence contained in b, thus facilitating a ready association
of a.sub.2 and b to form a binary, trimeric complex composed of
a.sub.2b. This binding event is made irreversible with a subsequent
reaction to covalently secure the two entities via disulfide
bridges, which occurs very efficiently based on the principle of
effective local concentration because the initial binding
interactions should bring the reactive thiol groups placed onto
both the DDD and AD into proximity (Chmura et al., Proc. Natl.
Acad. Sci. USA. 2001; 98:8480) to ligate site-specifically. Using
various combinations of linkers, adaptor modules and precursors, a
wide variety of DNL constructs of different stoichiometry may be
produced and used, including but not limited to dimeric, trimeric,
tetrameric, pentameric and hexameric DNL constructs (see, e.g.,
U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and
7,666,400.)
[0133] By attaching the DDD and AD away from the functional groups
of the two precursors, such site-specific ligations are also
expected to preserve the original activities of the two precursors.
This approach is modular in nature and potentially can be applied
to link, site-specifically and covalently, a wide range of
substances, including peptides, proteins, antibodies, antibody
fragments, and other effector moieties with a wide range of
activities. Utilizing the fusion protein method of constructing AD
and DDD conjugated effectors described in the Examples below,
virtually any protein or peptide may be incorporated into a DNL
construct. However, the technique is not limiting and other methods
of conjugation may be utilized.
[0134] A variety of methods are known for making fusion proteins,
including nucleic acid synthesis, hybridization and/or
amplification to produce a synthetic double-stranded nucleic acid
encoding a fusion protein of interest. Such double-stranded nucleic
acids may be inserted into expression vectors for fusion protein
production by standard molecular biology techniques (see, e.g.
Sambrook et al., Molecular Cloning, A laboratory manual, 2.sup.nd
Ed, 1989). In such preferred embodiments, the AD and/or DDD moiety
may be attached to either the N-terminal or C-terminal end of an
effector protein or peptide. However, the skilled artisan will
realize that the site of attachment of an AD or DDD moiety to an
effector moiety may vary, depending on the chemical nature of the
effector moiety and the part(s) of the effector moiety involved in
its physiological activity. Site-specific attachment of a variety
of effector moieties may be performed using techniques known in the
art, such as the use of bivalent cross-linking reagents and/or
other chemical conjugation techniques.
Pre-Targeting
[0135] Bispecific or multispecific antibodies may be utilized in
pre-targeting techniques. Pre-targeting is a multistep process
originally developed to resolve the slow blood clearance of
directly targeting antibodies, which contributes to undesirable
toxicity to normal tissues such as bone marrow. With pre-targeting,
a radionuclide or other diagnostic or therapeutic agent is attached
to a small delivery molecule (targetable construct) that is cleared
within minutes from the blood. A pre-targeting bispecific or
multispecific antibody, which has binding sites for the targetable
construct as well as a target antigen, is administered first, free
antibody is allowed to clear from circulation and then the
targetable construct is administered.
[0136] Pre-targeting methods are disclosed, for example, in Goodwin
et al., U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med.
29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr
et al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.
29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989;
Kalofonos et al., J. Nucl. Med. 31:1791, 1990; Schechter et al.,
Int. J. Cancer 48:167, 1991; Paganelli et al., Cancer Res. 51:5960,
1991; Paganelli et al., Nucl. Med. Commun. 12:211, 1991; U.S. Pat.
No. 5,256,395; Stickney et al., Cancer Res. 51:6650, 1991; Yuan et
al., Cancer Res. 51:3119, 1991; U.S. Pat. Nos. 6,077,499;
7,011,812; 7,300,644; 7,074,405; 6,962,702; 7,387,772; 7,052,872;
7,138,103; 6,090,381; 6,472,511; 6,962,702; and 6,962,702, each
incorporated herein by reference.
[0137] A pre-targeting method of treating or diagnosing a disease
or disorder in a subject may be provided by: (1) administering to
the subject a bispecific antibody or antibody fragment; (2)
optionally administering to the subject a clearing composition, and
allowing the composition to clear the antibody from circulation;
and (3) administering to the subject the targetable construct,
containing one or more chelated or chemically bound therapeutic or
diagnostic agents.
Immunoconjugates
[0138] Any of the antibodies, antibody fragments or antibody fusion
proteins described herein may be conjugated to a chelating moiety
or other carrier molecule to form an immunoconjugate. Methods for
covalent conjugation of chelating moieties and other functional
groups are known in the art and any such known method may be
utilized.
[0139] For example, a chelating moiety or carrier can be attached
at the hinge region of a reduced antibody component via disulfide
bond formation. Alternatively, such agents can be attached using a
heterobifunctional cross-linker, such as N-succinyl
3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56:
244 (1994). General techniques for such conjugation are well-known
in the art. See, for example, Wong, CHEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al.,
"Modification of Antibodies by Chemical Methods," in MONOCLONAL
ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages
187-230 (Wiley-Liss, Inc. 1995); Price, "Production and
Characterization of Synthetic Peptide-Derived Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge
University Press 1995).
[0140] Alternatively, the chelating moiety or carrier can be
conjugated via a carbohydrate moiety in the Fc region of the
antibody. Methods for conjugating peptides to antibody components
via an antibody carbohydrate moiety are well-known to those of
skill in the art. See, for example, Shih et al., Int. J. Cancer 41:
832 (1988); Shih et al., Int. J. Cancer 46: 1101 (1990); and Shih
et al., U.S. Pat. No. 5,057,313, the Examples section of which is
incorporated herein by reference. The general method involves
reacting an antibody component having an oxidized carbohydrate
portion with a carrier polymer that has at least one free amine
function. This reaction results in an initial Schiff base (imine)
linkage, which can be stabilized by reduction to a secondary amine
to form the final conjugate.
[0141] The Fc region may be absent if the antibody used as the
antibody component of the immunoconjugate is an antibody fragment.
However, it is possible to introduce a carbohydrate moiety into the
light chain variable region of a full length antibody or antibody
fragment. See, for example, Leung et al., J. Immunol. 154: 5919
(1995); U.S. Pat. Nos. 5,443,953 and 6,254,868, the Examples
section of which is incorporated herein by reference. The
engineered carbohydrate moiety is used to attach the functional
group to the antibody fragment.
[0142] Other methods of conjugation of chelating agents to proteins
are well known in the art (see, e.g., U.S. Pat. No. 7,563,433, the
Examples section of which is incorporated herein by reference).
Chelates may be directly linked to antibodies or peptides, for
example as disclosed in U.S. Pat. No. 4,824,659, incorporated
herein in its entirety by reference.
Click Chemistry
[0143] In various embodiments, immunoconjugates may be prepared
using the click chemistry technology. The click chemistry approach
was originally conceived as a method to rapidly generate complex
substances by joining small subunits together in a modular fashion.
(See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31; Evans,
2007, Aust J Chem 60:384-95.) Various forms of click chemistry
reaction are known in the art, such as the Huisgen 1,3-dipolar
cycloaddition copper catalyzed reaction (Tornoe et al., 2002, J
Organic Chem 67:3057-64), which is often referred to as the "click
reaction." Other alternatives include cycloaddition reactions such
as the Diels-Alder, nucleophilic substitution reactions (especially
to small strained rings like epoxy and aziridine compounds),
carbonyl chemistry formation of urea compounds and reactions
involving carbon-carbon double bonds, such as alkynes in thiol-yne
reactions.
[0144] The azide alkyne Huisgen cycloaddition reaction uses a
copper catalyst in the presence of a reducing agent to catalyze the
reaction of a terminal alkyne group attached to a first molecule.
In the presence of a second molecule comprising an azide moiety,
the azide reacts with the activated alkyne to form a
1,4-disubstituted 1,2,3-triazole. The copper catalyzed reaction
occurs at room temperature and is sufficiently specific that
purification of the reaction product is often not required.
(Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe et al.,
2002, J Org Chem 67:3057.) The azide and alkyne functional groups
are largely inert towards biomolecules in aqueous medium, allowing
the reaction to occur in complex solutions. The triazole formed is
chemically stable and is not subject to enzymatic cleavage, making
the click chemistry product highly stable in biological systems.
However, the copper catalyst is toxic to living cells, precluding
biological applications.
[0145] A copper-free click reaction has been proposed for covalent
modification of biomolecules in living systems. (See, e.g., Agard
et al., 2004, J Am Chem Soc 126:15046-47.) The copper-free reaction
uses ring strain in place of the copper catalyst to promote a [3+2]
azide-alkyne cycloaddition reaction (Id.) For example, cyclooctyne
is a 8-carbon ring structure comprising an internal alkyne bond.
The closed ring structure induces a substantial bond angle
deformation of the acetylene, which is highly reactive with azide
groups to form a triazole. Thus, cyclooctyne derivatives may be
used for copper-free click reactions, without the toxic copper
catalyst (Id.)
[0146] Another type of copper-free click reaction was reported by
Ning et al. (2010, Angew Chem Int Ed 49:3065-68), involving
strain-promoted alkyne-nitrone cycloaddition. To address the slow
rate of the original cyclooctyne reaction, electron-withdrawing
groups are attached adjacent to the triple bond (Id.) Examples of
such substituted cyclooctynes include difluorinated cyclooctynes,
4-dibenzocyclooctynol and azacyclooctyne (Id.) An alternative
copper-free reaction involved strain-promoted alkyne-nitrone
cycloaddition to give N-alkylated isoxazolines (Id.) The reaction
was reported to have exceptionally fast reaction kinetics and was
used in a one-pot three-step protocol for site-specific
modification of peptides and proteins (Id.) Nitrones were prepared
by the condensation of appropriate aldehydes with
N-methylhydroxylamine and the cycloaddition reaction took place in
a mixture of acetonitrile and water (Id.) However, an attempt to
use the reaction with nitrone-labeled monosaccharide derivatives
and metabolic labeling in Jurkat cells was unsuccessful (Id.)
[0147] In some cases, activated groups for click chemistry
reactions may be incorporated into biomolecules using the
endogenous synthetic pathways of cells. For example, Agard et al.
(2004, J Am Chem Soc 126:15046-47) demonstrated that a recombinant
glycoprotein expressed in CHO cells in the presence of
peracetylated N-azidoacetylmannosamine resulted in the
incorporation of the corresponding N-azidoacetyl sialic acid in the
carbohydrates of the glycoprotein. The azido-derivatized
glycoprotein reacted specifically with a biotinylated cyclooctyne
to form a biotinylated glycoprotein, while control glycoprotein
without the azido moiety remained unlabeled (Id.) Laughlin et al.
(2008, Science 320:664-667) used a similar technique to
metabolically label cell-surface glycans in zebrafish embryos
incubated with peracetylated N-azidoacetylgalactosamine. The
azido-derivatized glycans reacted with difluorinated cyclooctyne
(DIFO) reagents to allow visualization of glycans in vivo.
[0148] The Diels-Alder reaction has also been used for in vivo
labeling of molecules. Rossin et al. (2010, Angew Chem Int Ed
49:3375-78) reported a 52% yield in vivo between a tumor-localized
anti-TAG72 (CC49) antibody carrying a trans-cyclooctene (TCO)
reactive moiety and an .sup.111In-labeled tetrazine DOTA
derivative. The TCO-labeled CC49 antibody was administered to mice
bearing colon cancer xenografts, followed 1 day later by injection
of .sup.111In-labeled tetrazine probe (Id.) The reaction of
radiolabeled probe with tumor localized antibody resulted in
pronounced radioactivity localized in the tumor, as demonstrated by
SPECT imaging of live mice three hours after injection of
radiolabeled probe, with a tumor-to-muscle ratio of 13:1 (Id.) The
results confirmed the in vivo chemical reaction of the TCO and
tetrazine-labeled molecules.
[0149] Antibody labeling techniques using biological incorporation
of labeling moieties are further disclosed in U.S. Pat. No.
6,953,675 (the Examples section of which is incorporated herein by
reference). Such "landscaped" antibodies were prepared to have
reactive ketone groups on glycosylated sites. The method involved
expressing cells transfected with an expression vector encoding an
antibody with one or more N-glycosylation sites in the CH1 or
V.kappa. domain in culture medium comprising a ketone derivative of
a saccharide or saccharide precursor. Ketone-derivatized
saccharides or precursors included N-levulinoyl mannosamine and
N-levulinoyl fucose. The landscaped antibodies were subsequently
reacted with agents comprising a ketone-reactive moiety, such as
hydrazide, hydrazine, hydroxylamino or thiosemicarbazide groups, to
form a labeled targeting molecule. Exemplary agents attached to the
landscaped antibodies included chelating agents like DTPA, large
drug molecules such as doxorubicin-dextran, and acyl-hydrazide
containing peptides. However, the landscaping technique is not
limited to producing antibodies comprising ketone moieties, but may
be used instead to introduce a click chemistry reactive group, such
as a nitrone, an azide or a cyclooctyne, onto an antibody or other
biological molecule.
[0150] Modifications of click chemistry reactions are suitable for
use in vitro or in vivo. Reactive targeting molecule may be formed
either by either chemical conjugation or by biological
incorporation. The targeting molecule, such as an antibody or
antibody fragment, may be activated with an azido moiety, a
substituted cyclooctyne or alkyne group, or a nitrone moiety. Where
the targeting molecule comprises an azido or nitrone group, the
corresponding targetable construct will comprise a substituted
cyclooctyne or alkyne group, and vice versa. Such activated
molecules may be made by metabolic incorporation in living cells,
as discussed above. Alternatively, methods of chemical conjugation
of such moieties to biomolecules are well known in the art, and any
such known method may be utilized. The disclosed techniques may be
used in combination with the .sup.18F or .sup.19F labeling methods
described below for PET or NMR imaging, or alternatively may be
utilized for delivery of any therapeutic and/or diagnostic agent
that may be conjugated to a suitable activated targetable construct
and/or targeting molecule.
Affibodies
[0151] Affibodies are small proteins that function as antibody
mimetics and are of use in binding target molecules. Affibodies
were developed by combinatorial engineering on an alpha helical
protein scaffold (Nord et al., 1995, Protein Eng 8:601-8; Nord et
al., 1997, Nat Biotechnol 15:772-77). The affibody design is based
on a three helix bundle structure comprising the IgG binding domain
of protein A (Nord et al., 1995; 1997). Affibodies with a wide
range of binding affinities may be produced by randomization of
thirteen amino acids involved in the Fc binding activity of the
bacterial protein A (Nord et al., 1995; 1997). After randomization,
the PCR amplified library was cloned into a phagemid vector for
screening by phage display of the mutant proteins.
[0152] A .sup.177Lu-labeled affibody specific for HER2/neu has been
demonstrated to target HER2-expressing xenografts in vivo
(Tolmachev et al., 2007, Cancer Res 67:2773-82). Although renal
toxicity due to accumulation of the low molecular weight
radiolabeled compound was initially a problem, reversible binding
to albumin reduced renal accumulation, enabling radionuclide-based
therapy with labeled affibody (Id.)
[0153] The feasibility of using radiolabeled affibodies for in vivo
tumor imaging has been recently demonstrated (Tolmachev et al.,
2011, Bioconjugate Chem 22:894-902). A maleimide-derivatized NOTA
was conjugated to the anti-HER2 affibody and radiolabeled with
.sup.111In (Id.) Administration to mice bearing the HER2-expressing
DU-145 xenograft, followed by gamma camera imaging, allowed
visualization of the xenograft (Id.)
[0154] The skilled artisan will realize that affibodies may be used
as targeting molecules in the practice of the claimed methods and
compositions. Labeling with metal-conjugated .sup.18F may be
performed as described in the Examples below. Affibodies are
commercially available from Affibody AB (Solna, Sweden).
Phage Display Peptides
[0155] In some alternative embodiments, binding peptides may be
produced by phage display methods that are well known in the art.
For example, peptides that bind to any of a variety of
disease-associated antigens may be identified by phage display
panning against an appropriate target antigen, cell, tissue or
pathogen and selecting for phage with high binding affinity.
[0156] Various methods of phage display and techniques for
producing diverse populations of peptides are well known in the
art. For example, U.S. Pat. Nos. 5,223,409; 5,622,699 and
6,068,829, each of which is incorporated herein by reference,
disclose methods for preparing a phage library. The phage display
technique involves genetically manipulating bacteriophage so that
small peptides can be expressed on their surface (Smith and Scott,
1985, Science 228:1315-1317; Smith and Scott, 1993, Meth. Enzymol.
21:228-257).
[0157] The past decade has seen considerable progress in the
construction of phage-displayed peptide libraries and in the
development of screening methods in which the libraries are used to
isolate peptide ligands. For example, the use of peptide libraries
has made it possible to characterize interacting sites and
receptor-ligand binding motifs within many proteins, such as
antibodies involved in inflammatory reactions or integrins that
mediate cellular adherence. This method has also been used to
identify novel peptide ligands that may serve as leads to the
development of peptidomimetic drugs or imaging agents (Arap et al.,
1998a, Science 279:377-380). In addition to peptides, larger
protein domains such as single-chain antibodies may also be
displayed on the surface of phage particles (Arap et al.,
1998a).
[0158] Targeting amino acid sequences selective for a given target
molecule may be isolated by panning (Pasqualini and Ruoslahti,
1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J. Nucl.
Med. 43:159-162). In brief, a library of phage containing putative
targeting peptides is administered to target molecules and samples
containing bound phage are collected. Target molecules may, for
example, be attached to the bottom of microtiter wells in a 96-well
plate. Phage that bind to a target may be eluted and then amplified
by growing them in host bacteria.
[0159] In certain embodiments, the phage may be propagated in host
bacteria between rounds of panning. Rather than being lysed by the
phage, the bacteria may instead secrete multiple copies of phage
that display a particular insert. If desired, the amplified phage
may be exposed to the target molecule again and collected for
additional rounds of panning. Multiple rounds of panning may be
performed until a population of selective or specific binders is
obtained. The amino acid sequence of the peptides may be determined
by sequencing the DNA corresponding to the targeting peptide insert
in the phage genome. The identified targeting peptide may then be
produced as a synthetic peptide by standard protein chemistry
techniques (Arap et al., 1998a, Smith et al., 1985).
Aptamers
[0160] In certain embodiments, a targeting molecule may comprise an
aptamer. Methods of constructing and determining the binding
characteristics of aptamers are well known in the art. For example,
such techniques are described in U.S. Pat. Nos. 5,582,981,
5,595,877 and 5,637,459, each incorporated herein by reference.
[0161] Aptamers may be prepared by any known method, including
synthetic, recombinant, and purification methods, and may be used
alone or in combination with other ligands specific for the same
target. In general, a minimum of approximately 3 nucleotides,
preferably at least 5 nucleotides, are necessary to effect specific
binding. Aptamers of sequences shorter than 10 bases may be
feasible, although aptamers of 10, 20, 30 or 40 nucleotides may be
preferred.
[0162] Aptamers need to contain the sequence that confers binding
specificity, but may be extended with flanking regions and
otherwise derivatized. In preferred embodiments, the binding
sequences of aptamers may be flanked by primer-binding sequences,
facilitating the amplification of the aptamers by PCR or other
amplification techniques. In a further embodiment, the flanking
sequence may comprise a specific sequence that preferentially
recognizes or binds a moiety to enhance the immobilization of the
aptamer to a substrate.
[0163] Aptamers may be isolated, sequenced, and/or amplified or
synthesized as conventional DNA or RNA molecules. Alternatively,
aptamers of interest may comprise modified oligomers. Any of the
hydroxyl groups ordinarily present in aptamers may be replaced by
phosphonate groups, phosphate groups, protected by a standard
protecting group, or activated to prepare additional linkages to
other nucleotides, or may be conjugated to solid supports. One or
more phosphodiester linkages may be replaced by alternative linking
groups, such as P(O)O replaced by P(O)S, P(O)NR.sub.2, P(O)R,
P(O)OR', CO, or CNR.sub.2, wherein R is H or alkyl (1-20C) and R'
is alkyl (1-20C); in addition, this group may be attached to
adjacent nucleotides through O or S. Not all linkages in an
oligomer need to be identical.
[0164] Methods for preparation and screening of aptamers that bind
to particular targets of interest are well known, for example U.S.
Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163, each incorporated
by reference. The technique generally involves selection from a
mixture of candidate aptamers and step-wise iterations of binding,
separation of bound from unbound aptamers and amplification.
Because only a small number of sequences (possibly only one
molecule of aptamer) corresponding to the highest affinity aptamers
exist in the mixture, it is generally desirable to set the
partitioning criteria so that a significant amount of aptamers in
the mixture (approximately 5-50%) is retained during separation.
Each cycle results in an enrichment of aptamers with high affinity
for the target. Repetition for between three to six selection and
amplification cycles may be used to generate aptamers that bind
with high affinity and specificity to the target.
Avimers
[0165] In certain embodiments, the targeting molecules may comprise
one or more avimer sequences. Avimers are a class of binding
proteins somewhat similar to antibodies in their affinities and
specificities for various target molecules. They were developed
from human extracellular receptor domains by in vitro exon
shuffling and phage display. (Silverman et al., 2005, Nat.
Biotechnol. 23:1493-94; Silverman et al., 2006, Nat. Biotechnol.
24:220.) The resulting multidomain proteins may comprise multiple
independent binding domains, that may exhibit improved affinity (in
some cases sub-nanomolar) and specificity compared with
single-epitope binding proteins. (Id.) Additional details
concerning methods of construction and use of avimers are
disclosed, for example, in U.S. Patent Application Publication Nos.
20040175756, 20050048512, 20050053973, 20050089932 and 20050221384,
the Examples section of each of which is incorporated herein by
reference.
Methods of Administration
[0166] In various embodiments, bispecific antibodies and targetable
constructs may be used for imaging normal or diseased tissue and
organs (see, e.g. U.S. Pat. Nos. 6,126,916; 6,077,499; 6,010,680;
5,776,095; 5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996;
5,697,902; 5,328,679; 5,128,119; 5,101,827; and 4,735,210, each
incorporated herein by reference in its Examples section).
[0167] The administration of a bispecific antibody (bsAb) and an
.sup.18F-labeled targetable construct may be conducted by
administering the bsAb antibody at some time prior to
administration of the targetable construct. The doses and timing of
the reagents can be readily devised by a skilled artisan, and are
dependent on the specific nature of the reagents employed. If a
bsAb-F(ab').sub.2 derivative is given first, then a waiting time of
24-72 hr (alternatively 48-96 hours) before administration of the
targetable construct would be appropriate. If an IgG-Fab' bsAb
conjugate is the primary targeting vector, then a longer waiting
period before administration of the targetable construct would be
indicated, in the range of 3-10 days. After sufficient time has
passed for the bsAb to target to the diseased tissue, the
.sup.18F-labeled targetable construct is administered. Subsequent
to administration of the targetable construct, imaging can be
performed.
[0168] Certain embodiments concern the use of multivalent target
binding proteins which have at least three different target binding
sites as described in patent application Ser. No. 60/220,782.
Multivalent target binding proteins have been made by cross-linking
several Fab-like fragments via chemical linkers. See U.S. Pat. Nos.
5,262,524; 5,091,542 and Landsdorp et al. Euro. J. Immunol. 16:
679-83 (1986). Multivalent target binding proteins also have been
made by covalently linking several single chain Fv molecules (scFv)
to form a single polypeptide. See U.S. Pat. No. 5,892,020. A
multivalent target binding protein which is basically an aggregate
of scFv molecules has been disclosed in U.S. Pat. Nos. 6,025,165
and 5,837,242. A trivalent target binding protein comprising three
scFv molecules has been described in Krott et al. Protein
Engineering 10(4): 423-433 (1997).
[0169] Alternatively, a technique known as "dock-and-lock" (DNL),
described in more detail below, has been demonstrated for the
simple and reproducible construction of a variety of multivalent
complexes, including complexes comprising two or more different
antibodies or antibody fragments. (See, e.g., U.S. Pat. Nos.
7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400, the
Examples section of each of which is incorporated herein by
reference.) Such constructs are also of use for the practice of the
claimed methods and compositions described herein.
[0170] A clearing agent may be used which is given between doses of
the bispecific antibody (bsAb) and the targetable construct. A
clearing agent of novel mechanistic action may be used, namely a
glycosylated anti-idiotypic Fab' fragment targeted against the
disease targeting arm(s) of the bsAb. In one example, anti-CEA
(MN-14 Ab).times.anti-peptide bsAb is given and allowed to accrete
in disease targets to its maximum extent. To clear residual bsAb
from circulation, an anti-idiotypic Ab to MN-14, termed WI2, is
given, preferably as a glycosylated Fab' fragment. The clearing
agent binds to the bsAb in a monovalent manner, while its appended
glycosyl residues direct the entire complex to the liver, where
rapid metabolism takes place. Then the .sup.18F-labeled targetable
construct is given to the subject. The WI2 Ab to the MN-14 arm of
the bsAb has a high affinity and the clearance mechanism differs
from other disclosed mechanisms (see Goodwin et al., ibid), as it
does not involve cross-linking, because the WI2-Fab' is a
monovalent moiety. However, alternative methods and compositions
for clearing agents are known and any such known clearing agents
may be used.
[0171] Formulation and Administration
[0172] The .sup.18F-labeled molecules may be formulated to obtain
compositions that include one or more pharmaceutically suitable
excipients, one or more additional ingredients, or some combination
of these. These can be accomplished by known methods to prepare
pharmaceutically useful dosages, whereby the active ingredients
(i.e., the .sup.18F-labeled molecules) are combined in a mixture
with one or more pharmaceutically suitable excipients. Sterile
phosphate-buffered saline is one example of a pharmaceutically
suitable excipient. Other suitable excipients are well known to
those in the art. See, e.g., Ansel et al., PHARMACEUTICAL DOSAGE
FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger
1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th
Edition (Mack Publishing Company 1990), and revised editions
thereof.
[0173] The preferred route for administration of the compositions
described herein is parenteral injection. Injection may be
intravenous, intraarterial, intralymphatic, intrathecal,
subcutaneous or intracavitary (i.e., parenterally). In parenteral
administration, the compositions will be formulated in a unit
dosage injectable form such as a solution, suspension or emulsion,
in association with a pharmaceutically acceptable excipient. Such
excipients are inherently nontoxic and nontherapeutic. Examples of
such excipients are saline, Ringer's solution, dextrose solution
and Hank's solution. Nonaqueous excipients such as fixed oils and
ethyl oleate may also be used. A preferred excipient is 5% dextrose
in saline. The excipient may contain minor amounts of additives
such as substances that enhance isotonicity and chemical stability,
including buffers and preservatives. Other methods of
administration, including oral administration, are also
contemplated.
[0174] Formulated compositions comprising .sup.18F-labeled
molecules can be used for intravenous administration via, for
example, bolus injection or continuous infusion. Compositions for
injection can be presented in unit dosage form, e.g., in ampoules
or in multi-dose containers, with an added preservative.
Compositions can also take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and can contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the compositions can be in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use.
[0175] The compositions may be administered in solution. The pH of
the solution should be in the range of pH 5 to 9.5, preferably pH
6.5 to 7.5. The formulation thereof should be in a solution having
a suitable pharmaceutically acceptable buffer such as phosphate,
TRIS (hydroxymethyl) aminomethane-HCl or citrate and the like. In
certain preferred embodiments, the buffer is potassium biphthalate
(KHP), which may act as a transfer ligand to facilitate
.sup.18F-labeling. Buffer concentrations should be in the range of
1 to 100 mM. The formulated solution may also contain a salt, such
as sodium chloride or potassium chloride in a concentration of 50
to 150 mM. An effective amount of a stabilizing agent such as
glycerol, albumin, a globulin, a detergent, a gelatin, a protamine
or a salt of protamine may also be included. The compositions may
be administered to a mammal subcutaneously, intravenously,
intramuscularly or by other parenteral routes. Moreover, the
administration may be by continuous infusion or by single or
multiple boluses.
[0176] Where bispecific antibodies are administered, for example in
a pretargeting technique, the dosage of an administered antibody
for humans will vary depending upon such factors as the patient's
age, weight, height, sex, general medical condition and previous
medical history. Typically, for imaging purposes it is desirable to
provide the recipient with a dosage of bispecific antibody that is
in the range of from about 1 mg to 200 mg as a single intravenous
infusion, although a lower or higher dosage also may be
administered as circumstances dictate. Typically, it is desirable
to provide the recipient with a dosage that is in the range of from
about 10 mg per square meter of body surface area or 17 to 18 mg of
the antibody for the typical adult, although a lower or higher
dosage also may be administered as circumstances dictate. Examples
of dosages of bispecific antibodies that may be administered to a
human subject for imaging purposes are 1 to 200 mg, more preferably
1 to 70 mg, most preferably 1 to 20 mg, although higher or lower
doses may be used.
[0177] In general, the dosage of .sup.18F label to administer will
vary depending upon such factors as the patient's age, weight,
height, sex, general medical condition and previous medical
history. Preferably, a saturating dose of the .sup.18F-labeled
molecules is administered to a patient. For administration of
.sup.18F-labeled molecules, the dosage may be measured by
millicuries. A typical range for .sup.18F imaging studies would be
five to 10 mCi.
[0178] Administration of Peptides
[0179] Various embodiments of the claimed methods and/or
compositions may concern one or more .sup.18F-labeled peptides to
be administered to a subject. Administration may occur by any route
known in the art, including but not limited to oral, nasal, buccal,
inhalational, rectal, vaginal, topical, orthotopic, intradermal,
subcutaneous, intramuscular, intraperitoneal, intraarterial,
intrathecal or intravenous injection. Where, for example,
.sup.18F-labeled peptides are administered in a pretargeting
protocol, the peptides would preferably be administered i.v.
[0180] In certain embodiments, the standard peptide bond linkage
may be replaced by one or more alternative linking groups, such as
CH.sub.2--NH, CH.sub.2--S, CH.sub.2--CH.sub.2, CH.dbd.CH,
CO--CH.sub.2, CHOH--CH.sub.2 and the like. Methods for preparing
peptide mimetics are well known (for example, Hruby, 1982, Life Sci
31:189-99; Holladay et al., 1983, Tetrahedron Lett. 24:4401-04;
Jennings-White et al., 1982, Tetrahedron Lett. 23:2533; Almquiest
et al., 1980, J. Med. Chem. 23:1392-98; Hudson et al., 1979, Int.
J. Pept. Res. 14:177-185; Spatola et al., 1986, Life Sci
38:1243-49; U.S. Pat. Nos. 5,169,862; 5,539,085; 5,576,423,
5,051,448, 5,559,103.) Peptide mimetics may exhibit enhanced
stability and/or absorption in vivo compared to their peptide
analogs.
[0181] Peptide stabilization may also occur by substitution of
D-amino acids for naturally occurring L-amino acids, particularly
at locations where endopeptidases are known to act. Endopeptidase
binding and cleavage sequences are known in the art and methods for
making and using peptides incorporating D-amino acids have been
described (e.g., U.S. Patent Application Publication No.
20050025709, McBride et al., filed Jun. 14, 2004, the Examples
section of which is incorporated herein by reference).
Imaging Using Labeled Molecules
[0182] Methods of imaging using labeled molecules are well known in
the art, and any such known methods may be used with the
.sup.18F-labeled molecules disclosed herein. See, e.g., U.S. Pat.
Nos. 6,241,964; 6,358,489; 6,953,567 and published U.S. Patent
Application Publ. Nos. 20050003403; 20040018557; 20060140936, the
Examples section of each incorporated herein by reference. See
also, Page et al., Nuclear Medicine And Biology, 21:911-919, 1994;
Choi et al., Cancer Research 55:5323-5329, 1995; Zalutsky et al.,
J. Nuclear Med., 33:575-582, 1992; Woessner et. al. Magn. Reson.
Med. 2005, 53: 790-99.
[0183] In certain embodiments, .sup.18F-labeled molecules may be of
use in imaging normal or diseased tissue and organs, for example
using the methods described in U.S. Pat. Nos. 6,126,916; 6,077,499;
6,010,680; 5,776,095; 5,776,094; 5,776,093; 5,772,981; 5,753,206;
5,746,996; 5,697,902; 5,328,679; 5,128,119; 5,101,827; and
4,735,210, each incorporated herein by reference. Such imaging can
be conducted by direct .sup.18F labeling of the appropriate
targeting molecules, or by a pretargeted imaging method, as
described in Goldenberg et al. (2007, Update Cancer Ther. 2:19-31);
Sharkey et al. (2008, Radiology 246:497-507); Goldenberg et al.
(2008, J. Nucl. Med. 49:158-63); Sharkey et al. (2007, Clin. Cancer
Res. 13:5777s-5585s); McBride et al. (2006, J. Nucl. Med.
47:1678-88); Goldenberg et al. (2006, J. Clin. Oncol. 24:823-85),
see also U.S. Patent Publication Nos. 20050002945, 20040018557,
20030148409 and 20050014207, each incorporated herein by
reference.
[0184] Methods of diagnostic imaging with labeled peptides or MAbs
are well-known. For example, in the technique of
immunoscintigraphy, ligands or antibodies are labeled with a
gamma-emitting radioisotope and introduced into a patient. A gamma
camera is used to detect the location and distribution of
gamma-emitting radioisotopes. See, for example, Srivastava (ed.),
RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum
Press 1988), Chase, "Medical Applications of Radioisotopes," in
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al.
(eds.), pp. 624-652 (Mack Publishing Co., 1990), and Brown,
"Clinical Use of Monoclonal Antibodies," in BIOTECHNOLOGY AND
PHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993).
Also preferred is the use of positron-emitting radionuclides (PET
isotopes), such as with an energy of 511 keV, such as .sup.18F,
.sup.68Ga, .sup.64Cu, and .sup.124I. Such radionuclides may be
imaged by well-known PET scanning techniques.
[0185] In preferred embodiments, the .sup.18F-labeled peptides,
proteins and/or antibodies are of use for imaging of cancer.
Examples of cancers include, but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
More particular examples of such cancers are noted below and
include: squamous cell cancer (e.g. epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, rectal cancer, colorectal cancer, endometrial cancer or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, anal carcinoma, penile carcinoma, as well as head and
neck cancer. The term "cancer" includes primary malignant cells or
tumors (e.g., those whose cells have not migrated to sites in the
subject's body other than the site of the original malignancy or
tumor) and secondary malignant cells or tumors (e.g., those arising
from metastasis, the migration of malignant cells or tumor cells to
secondary sites that are different from the site of the original
tumor).
[0186] Other examples of cancers or malignancies include, but are
not limited to: Acute Childhood Lymphoblastic Leukemia, Acute
Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid
Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular
Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease,
Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult
Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft
Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies,
Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone
Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of
the Renal Pelvis and Ureter, Central Nervous System (Primary)
Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma,
Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)
Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood
Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia,
Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma,
Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell
Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma,
Childhood Hypothalamic and Visual Pathway Glioma, Childhood
Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood
Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial
Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer,
Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,
Childhood Visual Pathway and Hypothalamic Glioma, Chronic
Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer,
Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma,
Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal
Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic
Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor,
Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer,
Gaucher's Disease, Gallbladder Cancer, Gastric Cancer,
Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ
Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia,
Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease,
Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer,
Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma,
Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer,
Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung
Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male
Breast Cancer, Malignant Mesothelioma, Malignant Thymoma,
Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary
Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,
Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple
Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous
Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal
Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer,
Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma
Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic
Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant
Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian
Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant
Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura,
Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary
Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central
Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer,
Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,
Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck
Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal
and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic
Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer,
Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[0187] The methods and compositions described and claimed herein
may be used to detect or diagnose malignant or premalignant
conditions. Such uses are indicated in conditions known or
suspected of preceding progression to neoplasia or cancer, in
particular, where non-neoplastic cell growth consisting of
hyperplasia, metaplasia, or most particularly, dysplasia has
occurred (for review of such abnormal growth conditions, see
Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co.,
Philadelphia, pp. 68-79 (1976)).
[0188] Dysplasia is frequently a forerunner of cancer, and is found
mainly in the epithelia. It is the most disorderly form of
non-neoplastic cell growth, involving a loss in individual cell
uniformity and in the architectural orientation of cells. Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation. Dysplastic disorders which can be detected include,
but are not limited to, anhidrotic ectodermal dysplasia,
anterofacial dysplasia, asphyxiating thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral
dysplasia, cervical dysplasia, chondroectodermal dysplasia,
cleidocranial dysplasia, congenital ectodermal dysplasia,
craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial
dysplasia, ectodermal dysplasia, enamel dysplasia,
encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,
epithelial dysplasia, faciodigitogenital dysplasia, familial
fibrous dysplasia of jaws, familial white folded dysplasia,
fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal
dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic
dysplasia, mammary dysplasia, mandibulofacial dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous
dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia,
oculoauriculovertebral dysplasia, oculodentodigital dysplasia,
oculovertebral dysplasia, odontogenic dysplasia,
opthalmomandibulomelic dysplasia, periapical cemental dysplasia,
polyostotic fibrous dysplasia, pseudoachondroplastic
spondyloepiphysial dysplasia, retinal dysplasia, septo-optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial
dysplasia.
[0189] Additional pre-neoplastic disorders which can be detected
include, but are not limited to, benign dysproliferative disorders
(e.g., benign tumors, fibrocystic conditions, tissue hypertrophy,
intestinal polyps, colon polyps, and esophageal dysplasia),
leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar
cheilitis, and solar keratosis.
[0190] Additional hyperproliferative diseases, disorders, and/or
conditions include, but are not limited to, progression, and/or
metastases of malignancies and related disorders such as leukemia
(including acute leukemias (e.g., acute lymphocytic leukemia, acute
myelocytic leukemia (including myeloblastic, promyelocytic,
myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid
tumors including, but not limited to, sarcomas and carcinomas such
as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, emangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0191] In a preferred embodiment, diseases that may be diagnosed,
detected or imaged using the claimed compositions and methods
include cardiovascular diseases, such as fibrin clots,
atherosclerosis, myocardial ischemia and infarction. Antibodies to
fibrin (e.g., scFv(59D8); T2G1s; MH1) are known and in clinical
trials as imaging agents for disclosing said clots and pulmonary
emboli, while anti-granulocyte antibodies, such as MN-3, MN-15,
anti-NCA95, and anti-CD15 antibodies, can target myocardial
infarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos.
5,487,892; 5,632,968; 6,294,173; 7,541,440, the Examples section of
each incorporated herein by reference) Anti-macrophage,
anti-low-density lipoprotein (LDL) and anti-CD74 (e.g., hLL1)
antibodies can be used to target atherosclerotic plaques. Abciximab
(anti-glycoprotein IIb/IIIa) has been approved for adjuvant use for
prevention of restenosis in percutaneous coronary interventions and
the treatment of unstable angina (Waldmann et al., 2000, Hematol
1:394-408). Anti-CD3 antibodies have been reported to reduce
development and progression of atherosclerosis (Steffens et al.,
2006, Circulation 114:1977-84). Treatment with blocking MIF
antibody has been reported to induce regression of established
atherosclerotic lesions (Sanchez-Madrid and Sessa, 2010, Cardiovasc
Res 86:171-73). Antibodies against oxidized LDL also induced a
regression of established atherosclerosis in a mouse model
(Ginsberg, 2007, J Am Coll Cardiol 52:2319-21). Anti-ICAM-1
antibody was shown to reduce ischemic cell damage after cerebral
artery occlusion in rats (Zhang et al., 1994, Neurology
44:1747-51). Commercially available monoclonal antibodies to
leukocyte antigens are represented by: OKT anti-T-cell monoclonal
antibodies (available from Ortho Pharmaceutical Company) which bind
to normal T-lymphocytes; the monoclonal antibodies produced by the
hybridomas having the ATCC accession numbers HB44, HB55, HB12, HB78
and HB2; G7E11, W8E7, NKP15 and GO22 (Becton Dickinson); NEN9.4
(New England Nuclear); and FMC11 (Sera Labs). A description of
antibodies against fibrin and platelet antigens is contained in
Knight, Semin. Nucl. Med., 20:52-67 (1990).
[0192] In one embodiment, a pharmaceutical composition may be used
to diagnose a subject having a metabolic disease, such amyloidosis,
or a neurodegenerative disease, such as Alzheimer's disease,
amyotrophic lateral sclerosis (ALS), Parkinson's disease,
Huntington's disease, olivopontocerebellar atrophy, multiple system
atrophy, progressive supranuclear palsy, corticodentatonigral
degeneration, progressive familial myoclonic epilepsy, strionigral
degeneration, torsion dystonia, familial tremor, Gilles de la
Tourette syndrome or Hallervorden-Spatz disease. Bapineuzumab is in
clinical trials for Alzheimer's disease therapy. Other antibodies
proposed for Alzheimer's disease include Alz 50 (Ksiezak-Reding et
al., 1987, J Biol Chem 263:7943-47), gantenerumab, and solanezumab.
Infliximab, an anti-TNF-.alpha. antibody, has been reported to
reduce amyloid plaques and improve cognition. Antibodies against
mutant SOD1, produced by hybridoma cell lines deposited with the
International Depositary Authority of Canada (accession Nos.
ADI-290806-01, ADI-290806-02, ADI-290806-03) have been proposed for
therapy of ALS, Parkinson's disease and Alzheimer's disease (see
U.S. Patent Appl. Publ. No. 20090068194). Anti-CD3 antibodies have
been proposed for therapy of type 1 diabetes (Cernea et al., 2010,
Diabetes Metab Rev 26:602-05). In addition, a pharmaceutical
composition of the present invention may be used on a subject
having an immune-dysregulatory disorder, such as graft-versus-host
disease or organ transplant rejection.
[0193] The exemplary conditions listed above that may be detected,
diagnosed and/or imaged are not limiting. The skilled artisan will
be aware that antibodies, antibody fragments or targeting peptides
are known for a wide variety of conditions, such as autoimmune
disease, cardiovascular disease, neurodegenerative disease,
metabolic disease, cancer, infectious disease and
hyperproliferative disease. Any such condition for which an
.sup.18F-labeled molecule, such as a protein or peptide, may be
prepared and utilized by the methods described herein, may be
imaged, diagnosed and/or detected as described herein.
Kits
[0194] Various embodiments may concern kits containing components
suitable for imaging, diagnosing and/or detecting diseased tissue
in a patient using labeled compounds. Exemplary kits may contain an
antibody, fragment or fusion protein, such as a bispecific antibody
of use in pretargeting methods as described herein. Other
components may include a targetable construct for use with such
bispecific antibodies. In preferred embodiments, the targetable
construct is pre-conjugated to a chelating group that may be used
to attach an Al.sup.18F complex or a complex of .sup.18F with a
different metal. However, in alternative embodiments it is
contemplated that a targetable construct may be attached to one or
more different diagnostic agents, such as .sup.68Ga.
[0195] A device capable of delivering the kit components may be
included. One type of device, for applications such as parenteral
delivery, is a syringe that is used to inject the composition into
the body of a subject. Inhalation devices may also be used for
certain applications.
[0196] The kit components may be packaged together or separated
into two or more containers. In some embodiments, the containers
may be vials that contain sterile, lyophilized formulations of a
composition that are suitable for reconstitution. A kit may also
contain one or more buffers suitable for reconstitution and/or
dilution of other reagents. Other containers that may be used
include, but are not limited to, a pouch, tray, box, tube, or the
like. Kit components may be packaged and maintained sterilely
within the containers. Another component that can be included is
instructions to a person using a kit for its use.
EXAMPLES
Example 1. .sup.18F-Labeling of Peptide IMP272
[0197] The first peptide that was prepared and .sup.18F-labeled was
IMP272:
[0198] DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH.sub.2 (SEQ ID
NO:3)
[0199] Acetate buffer solution--Acetic acid, 1.509 g was diluted in
.about.160 mL water and the pH was adjusted by the addition of 1 M
NaOH then diluted to 250 mL to make a 0.1 M solution at pH
4.03.
[0200] Aluminum acetate buffer solution--A solution of aluminum was
prepared by dissolving 0.1028 g of AlCl.sub.3 hexahydrate in 42.6
mL DI water. A 4 mL aliquot of the aluminum solution was mixed with
16 mL of a 0.1 M NaOAc solution at pH 4 to provide a 2 mM Al stock
solution.
[0201] IMP272 acetate buffer solution--Peptide, 0.0011 g,
7.28.times.10.sup.-7 mol IMP272 was dissolved in 364 .mu.L of the
0.1 M pH 4 acetate buffer solution to obtain a 2 mM stock solution
of the peptide.
[0202] .sup.18F-Labeling of IMP272--A 3 .mu.L aliquot of the
aluminum stock solution was placed in a REACTI-VIAL.TM. and mixed
with 50 .mu.L .sup.18F (as received) and 3 .mu.L of the IMP272
solution. The solution was heated in a heating block at 110.degree.
C. for 15 min and analyzed by reverse phase HPLC. The HPLC trace
(not shown) showed 93% free .sup.18F and 7% bound to the peptide.
An additional 10 .mu.L of the IMP272 solution was added to the
reaction and it was heated again and analyzed by reverse phase HPLC
(not shown). The HPLC trace showed 8% .sup.18F at the void volume
and 92% of the activity attached to the peptide. The remainder of
the peptide solution was incubated at room temperature with 150
.mu.L PBS for .about.1 hr and then examined by reverse phase HPLC.
The HPLC (not shown) showed 58% .sup.18F unbound and 42% still
attached to the peptide. The data indicate that Al.sup.18F(DTPA)
complex may be unstable when mixed with phosphate.
Example 2. IMP272 .sup.18F-Labeling with Other Metals
[0203] A .about.3 .mu.L aliquot of the metal stock solution
(6.times.10.sup.-9 mol) was placed in a polypropylene cone vial and
mixed with 75 .mu.L .sup.18F (as received), incubated at room
temperature for .about.2 min and then mixed with 20 .mu.L of a 2 mM
(4.times.10.sup.-8 mol) IMP272 solution in 0.1 M NaOAc pH 4 buffer.
The solution was heated in a heating block at 100.degree. C. for 15
min and analyzed by reverse phase HPLC. IMP272 was labeled with
indium (24%), gallium (36%), zirconium (15%), lutetium (37%) and
yttrium (2%) (not shown). These results demonstrate that the
.sup.18F metal labeling technique is not limited to an aluminum
ligand, but can also utilize other metals as well. With different
metal ligands, different chelating moieties may be utilized to
optimize binding of an .sup.18F-metal conjugate.
Example 3. Production and Use of a Serum-Stable .sup.18F-Labeled
Peptide IMP449
[0204] NOTA-benzyl-ITC-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH.sub.2
(SEQ ID NO:4)
[0205] The peptide, IMP448
D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH.sub.2 (SEQ ID NO:5) was made
on Sieber Amide resin by adding the following amino acids to the
resin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc
was cleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH,
the Aloc was cleaved, Fmoc-D-Ala-OH with final Fmoc cleavage to
make the desired peptide. The peptide was then cleaved from the
resin and purified by HPLC to produce IMP448, which was then
coupled to ITC-benzyl NOTA.
[0206] IMP448 (0.0757 g, 7.5.times.10.sup.-5 mol) was mixed with
0.0509 g (9.09.times.10.sup.-5 mol) ITC benzyl NOTA and dissolved
in 1 mL water. Potassium carbonate anhydrous (0.2171 g) was then
slowly added to the stirred peptide/NOTA solution. The reaction
solution was pH 10.6 after the addition of all the carbonate. The
reaction was allowed to stir at room temperature overnight. The
reaction was carefully quenched with 1 M HCl after 14 hr and
purified by HPLC to obtain 48 mg of IMP449.
[0207] .sup.18F-Labeling of IMP449
[0208] IMP449 (0.002 g, 1.37.times.10.sup.-6 mol) was dissolved in
686 .mu.I, (2 mM peptide solution) 0.1 M NaOAc pH 4.02. Three
microliters of a 2 mM solution of Al in a pH 4 acetate buffer was
mixed with 15 .mu.L, 1.3 mCi of .sup.18F. The solution was then
mixed with 20 .mu.I, of the 2 mM IMP449 solution and heated at
105.degree. C. for 15 min. Reverse Phase HPLC analysis showed 35%
(t.sub.R.about.10 min) of the activity was attached to the peptide
and 65% of the activity was eluted at the void volume of the column
(3.1 min, not shown) indicating that the majority of activity was
not associated with the peptide. The crude labeled mixture (5
.mu.L) was mixed with pooled human serum and incubated at
37.degree. C. An aliquot was removed after 15 min and analyzed by
HPLC. The HPLC showed 9.8% of the activity was still attached to
the peptide (down from 35%). Another aliquot was removed after 1 hr
and analyzed by HPLC. The HPLC showed 7.6% of the activity was
still attached to the peptide (down from 35%), which was
essentially the same as the 15 min trace (data not shown).
[0209] High Dose .sup.18F-Labeling of IMP449
[0210] Further studies with purified IMP449 demonstrated that the
.sup.18F-labeled peptide was highly stable (91%, not shown) in
human serum at 37.degree. C. for at least one hour and was
partially stable (76%, not shown) in human serum at 37.degree. C.
for at least four hours. Additional studies were performed in which
the IMP449 was prepared in the presence of ascorbic acid as a
stabilizing agent. In those studies (not shown), the
.sup.18F-metal-peptide complex showed no detectable decomposition
in serum after 4 hr at 37.degree. C. The mouse urine 30 min after
injection of .sup.18F-labeled peptide was found to contain .sup.18F
bound to the peptide (not shown). These results demonstrate that
the .sup.18F-labeled peptides disclosed herein exhibit sufficient
stability under approximated in vivo conditions to be used for
.sup.18F imaging studies.
[0211] Since IMP449 peptide contains a thiourea linkage, which is
sensitive to radiolysis, several products are observed by RP-HPLC.
However, when ascorbic acid is added to the reaction mixture, the
side products generated are markedly reduced.
Example 4. Preparation of DNL Constructs for .sup.18F Imaging by
Pretargeting
[0212] The DNL technique may be used to make dimers, trimers,
tetramers, hexamers, etc. comprising virtually any antibodies or
fragments thereof or other effector moieties. For certain preferred
embodiments, IgG antibodies, Fab fragments or other proteins or
peptides may be produced as fusion proteins containing either a DDD
(dimerization and docking domain) or AD (anchoring domain)
sequence. Bispecific antibodies may be formed by combining a
Fab-DDD fusion protein of a first antibody with a Fab-AD fusion
protein of a second antibody. Alternatively, constructs may be made
that combine IgG-AD fusion proteins with Fab-DDD fusion proteins.
For purposes of .sup.18F detection, an antibody or fragment
containing a binding site for an antigen associated with a target
tissue to be imaged, such as a tumor, may be combined with a second
antibody or fragment that binds a hapten on a targetable construct,
such as IMP 449, to which a metal-.sup.18F can be attached. The
bispecific antibody (DNL construct) is administered to a subject,
circulating antibody is allowed to clear from the blood and
localize to target tissue, and the .sup.18F-labeled targetable
construct is added and binds to the localized antibody for
imaging.
[0213] Independent transgenic cell lines may be developed for each
Fab or IgG fusion protein. Once produced, the modules can be
purified if desired or maintained in the cell culture supernatant
fluid. Following production, any DDD.sub.2-fusion protein module
can be combined with any corresponding AD-fusion protein module to
generate a bispecific DNL construct. For different types of
constructs, different AD or DDD sequences may be utilized. The
following DDD sequences are based on the DDD moiety of PKA
RII.alpha., while the AD sequences are based on the AD moiety of
the optimized synthetic AKAP-IS sequence (Alto et al., Proc. Natl.
Acad. Sci. USA. 2003; 100:4445).
TABLE-US-00002 DDD1: (SEQ ID NO: 6)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2: (SEQ ID NO: 7)
CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1: (SEQ ID NO: 8)
QIEYLAKQIVDNAIQQA AD2: (SEQ ID NO: 9) CGQIEYLAKQIVDNAIQQAGC
[0214] The plasmid vector pdHL2 has been used to produce a number
of antibodies and antibody-based constructs. See Gillies et al., J
Immunol Methods (1989), 125:191-202; Losman et al., Cancer (Phila)
(1997), 80:2660-6. The di-cistronic mammalian expression vector
directs the synthesis of the heavy and light chains of IgG. The
vector sequences are mostly identical for many different IgG-pdHL2
constructs, with the only differences existing in the variable
domain (VH and VL) sequences. Using molecular biology tools known
to those skilled in the art, these IgG expression vectors can be
converted into Fab-DDD or Fab-AD expression vectors. To generate
Fab-DDD expression vectors, the coding sequences for the hinge, CH2
and CH3 domains of the heavy chain are replaced with a sequence
encoding the first 4 residues of the hinge, a 14 residue Gly-Ser
linker and the first 44 residues of human RII.alpha. (referred to
as DDD1). To generate Fab-AD expression vectors, the sequences for
the hinge, CH2 and CH3 domains of IgG are replaced with a sequence
encoding the first 4 residues of the hinge, a 15 residue Gly-Ser
linker and a 17 residue synthetic AD called AKAP-IS (referred to as
AD1), which was generated using bioinformatics and peptide array
technology and shown to bind RIIa dimers with a very high affinity
(0.4 nM). See Alto, et al. Proc. Natl. Acad. Sci., U.S.A. (2003),
100:4445-50.
[0215] Two shuttle vectors were designed to facilitate the
conversion of IgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1
expression vectors, as described below.
[0216] Preparation of CH1
[0217] The CH1 domain was amplified by PCR using the pdHL2 plasmid
vector as a template. The left PCR primer consisted of the upstream
(5') end of the CH1 domain and a SacII restriction endonuclease
site, which is 5' of the CH1 coding sequence. The right primer
consisted of the sequence coding for the first 4 residues of the
hinge followed by four glycines and a serine, with the final two
codons (GS) comprising a Bam HI restriction site. The 410 bp PCR
amplimer was cloned into the pGemT PCR cloning vector (Promega,
Inc.) and clones were screened for inserts in the T7 (5')
orientation.
[0218] A duplex oligonucleotide was synthesized by to code for the
amino acid sequence of DDD1 preceded by 11 residues of a linker
peptide, with the first two codons comprising a BamHI restriction
site. A stop codon and an EagI restriction site are appended to the
3' end. The encoded polypeptide sequence is shown below, with the
DDD1 sequence underlined.
TABLE-US-00003 (SEQ ID NO: 10)
GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA
[0219] Two oligonucleotides, designated RIIA1-44 top and RIIA1-44
bottom, that overlap by 30 base pairs on their 3' ends, were
synthesized (Sigma Genosys) and combined to comprise the central
154 base pairs of the 174 bp DDD1 sequence. The oligonucleotides
were annealed and subjected to a primer extension reaction with Taq
polymerase. Following primer extension, the duplex was amplified by
PCR. The amplimer was cloned into pGemT and screened for inserts in
the T7 (5') orientation.
[0220] A duplex oligonucleotide was synthesized to code for the
amino acid sequence of AD1 preceded by 11 residues of the linker
peptide with the first two codons comprising a BamHI restriction
site. A stop codon and an EagI restriction site are appended to the
3'end. The encoded polypeptide sequence is shown below, with the
sequence of AD1 underlined.
TABLE-US-00004 (SEQ ID NO: 11) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA
[0221] Two complimentary overlapping oligonucleotides encoding the
above peptide sequence, designated AKAP-IS Top and AKAP-IS Bottom,
were synthesized and annealed. The duplex was amplified by PCR. The
amplimer was cloned into the pGemT vector and screened for inserts
in the T7 (5') orientation.
[0222] Ligating DDD1 with CH1
[0223] A 190 bp fragment encoding the DDD1 sequence was excised
from pGemT with BamHI and NotI restriction enzymes and then ligated
into the same sites in CH1-pGemT to generate the shuttle vector
CH1-DDD1-pGemT.
[0224] Ligating AD1 with CH1
[0225] A 110 bp fragment containing the AD1 sequence was excised
from pGemT with BamHI and NotI and then ligated into the same sites
in CH1-pGemT to generate the shuttle vector CH1-AD1-pGemT.
[0226] Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors
[0227] With this modular design either CH1-DDD1 or CH1-AD1 can be
incorporated into any IgG construct in the pdHL2 vector. The entire
heavy chain constant domain is replaced with one of the above
constructs by removing the SacII/EagI restriction fragment
(CH1-CH3) from pdHL2 and replacing it with the SacII/EagI fragment
of CH1-DDD1 or CH1-AD1, which is excised from the respective pGemT
shuttle vector.
[0228] Construction of h679-Fd-AD1-pdHL2
[0229] h679-Fd-AD1-pdHL2 is an expression vector for production of
h679 Fab with AD1 coupled to the carboxyl terminal end of the CH1
domain of the Fd via a flexible Gly/Ser peptide spacer composed of
14 amino acid residues. A pdHL2-based vector containing the
variable domains of h679 was converted to h679-Fd-AD1-pdHL2 by
replacement of the SacII/EagI fragment with the CH1-AD1 fragment,
which was excised from the CH1-AD1-SV3 shuttle vector with SacII
and EagI.
[0230] Construction of C-DDD1-Fd-hMN-14-pdHL2
[0231] C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for
production of a stable dimer that comprises two copies of a fusion
protein C-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at
the carboxyl terminus of CH1 via a flexible peptide spacer. The
plasmid vector hMN14(I)-pdHL2, which has been used to produce
hMN-14 IgG, was converted to C-DDD1-Fd-hMN-14-pdHL2 by digestion
with SacII and EagI restriction endonucleases to remove the CH1-CH3
domains and insertion of the CH1-DDD1 fragment, which was excised
from the CH1-DDD1-SV3 shuttle vector with SacII and EagI.
[0232] The same technique has been utilized to produce plasmids for
Fab expression of a wide variety of known antibodies, such as hLL1,
hLL2, hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others.
Generally, the antibody variable region coding sequences were
present in a pdHL2 expression vector and the expression vector was
converted for production of an AD- or DDD-fusion protein as
described above. The AD- and DDD-fusion proteins comprising a Fab
fragment of any of such antibodies may be combined, in an
approximate ratio of two DDD-fusion proteins per one AD-fusion
protein, to generate a trimeric DNL construct comprising two Fab
fragments of a first antibody and one Fab fragment of a second
antibody.
[0233] C-DDD2-Fd-hMN-14-pdHL2
[0234] C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for
production of C-DDD2-Fab-hMN-14, which possesses a dimerization and
docking domain sequence of DDD2 appended to the carboxyl terminus
of the Fd of hMN-14 via a 14 amino acid residue Gly/Ser peptide
linker. The fusion protein secreted is composed of two identical
copies of hMN-14 Fab held together by non-covalent interaction of
the DDD2 domains.
[0235] Two overlapping, complimentary oligonucleotides, which
comprise the coding sequence for part of the linker peptide and
residues 1-13 of DDD2, were made synthetically. The
oligonucleotides were annealed and phosphorylated with T4 PNK,
resulting in overhangs on the 5' and 3' ends that are compatible
for ligation with DNA digested with the restriction endonucleases
BamHI and PstI, respectively.
[0236] The duplex DNA was ligated with the shuttle vector
CH1-DDD1-pGemT, which was prepared by digestion with BamHI and
PstI, to generate the shuttle vector CH1-DDD2-pGemT. A 507 bp
fragment was excised from CH1-DDD2-pGemT with SacII and EagI and
ligated with the IgG expression vector hMN14(I)-pdHL2, which was
prepared by digestion with SacII and EagI. The final expression
construct was designated C-DDD2-Fd-hMN-14-pdHL2. Similar techniques
have been utilized to generated DDD2-fusion proteins of the Fab
fragments of a number of different humanized antibodies.
[0237] H679-Fd-AD2-pdHL2
[0238] h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14
as A. h679-Fd-AD2-pdHL2 is an expression vector for the production
of h679-Fab-AD2, which possesses an anchor domain sequence of AD2
appended to the carboxyl terminal end of the CH1 domain via a 14
amino acid residue Gly/Ser peptide linker. AD2 has one cysteine
residue preceding and another one following the anchor domain
sequence of AD1.
[0239] The expression vector was engineered as follows. Two
overlapping, complimentary oligonucleotides (AD2 Top and AD2
Bottom), which comprise the coding sequence for AD2 and part of the
linker sequence, were made synthetically. The oligonucleotides were
annealed and phosphorylated with T4 PNK, resulting in overhangs on
the 5' and 3' ends that are compatible for ligation with DNA
digested with the restriction endonucleases BamHI and SpeI,
respectively.
[0240] The duplex DNA was ligated into the shuttle vector
CH1-AD1-pGemT, which was prepared by digestion with BamHI and SpeI,
to generate the shuttle vector CH1-AD2-pGemT. A 429 base pair
fragment containing CH1 and AD2 coding sequences was excised from
the shuttle vector with SacII and EagI restriction enzymes and
ligated into h679-pdHL2 vector that prepared by digestion with
those same enzymes. The final expression vector is
h679-Fd-AD2-pdHL2.
Example 5. Generation of TF2 DNL Construct
[0241] A trimeric DNL construct designated TF2 was obtained by
reacting C-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2
was generated with >90% yield as follows. Protein L-purified
C-DDD2-Fab-hMN-14 (200 mg) was mixed with h679-Fab-AD2 (60 mg) at a
1.4:1 molar ratio. The total protein concentration was 1.5 mg/ml in
PBS containing 1 mM EDTA. Subsequent steps involved TCEP reduction,
HIC chromatography, DMSO oxidation, and IMP 291 affinity
chromatography. Before the addition of TCEP, SE-HPLC did not show
any evidence of a.sub.2b formation. Addition of 5 mM TCEP rapidly
resulted in the formation of a.sub.2b complex consistent with a 157
kDa protein expected for the binary structure. TF2 was purified to
near homogeneity by IMP 291 affinity chromatography (not shown).
IMP 291 is a synthetic peptide containing the HSG hapten to which
the 679 Fab binds (Rossi et al., 2005, Clin Cancer Res
11:7122s-29s). SE-HPLC analysis of the IMP 291 unbound fraction
demonstrated the removal of a.sub.4, a.sub.2 and free kappa chains
from the product (not shown).
[0242] Non-reducing SDS-PAGE analysis demonstrated that the
majority of TF2 exists as a large, covalent structure with a
relative mobility near that of IgG (not shown). The additional
bands suggest that disulfide formation is incomplete under the
experimental conditions (not shown). Reducing SDS-PAGE shows that
any additional bands apparent in the non-reducing gel are
product-related (not shown), as only bands representing the
constituent polypeptides of TF2 are evident. MALDI-TOF mass
spectrometry (not shown) revealed a single peak of 156,434 Da,
which is within 99.5% of the calculated mass (157,319 Da) of
TF2.
[0243] The functionality of TF2 was determined by BIACORE assay.
TF2, C-DDD1-hMN-14+h679-AD1 (used as a control sample of
noncovalent a.sub.2b complex), or C-DDD2-hMN-14+h679-AD2 (used as a
control sample of unreduced a.sub.2 and b components) were diluted
to 1 .mu.g/ml (total protein) and passed over a sensorchip
immobilized with HSG. The response for TF2 was approximately
two-fold that of the two control samples, indicating that only the
h679-Fab-AD component in the control samples would bind to and
remain on the sensorchip. Subsequent injections of WI2 IgG, an
anti-idiotype antibody for hMN-14, demonstrated that only TF2 had a
DDD-Fab-hMN-14 component that was tightly associated with
h679-Fab-AD as indicated by an additional signal response. The
additional increase of response units resulting from the binding of
WI2 to TF2 immobilized on the sensorchip corresponded to two fully
functional binding sites, each contributed by one subunit of
C-DDD2-Fab-hMN-14. This was confirmed by the ability of TF2 to bind
two Fab fragments of WI2 (not shown).
Example 6. Production of TF10 DNL Construct
[0244] A similar protocol was used to generate a trimeric TF10 DNL
construct, comprising two copies of a C-DDD2-Fab-hPAM4 and one copy
of C-AD2-Fab-679. The TF10 bispecific ([hPAM4].sub.2.times.h679)
antibody was produced using the method disclosed for production of
the (anti CEA).sub.2.times.anti HSG bsAb TF2, as described above.
The TF10 construct bears two humanized PAM4 Fabs and one humanized
679 Fab.
[0245] The two fusion proteins (hPAM4-DDD2 and h679-AD2) were
expressed independently in stably transfected myeloma cells. The
tissue culture supernatant fluids were combined, resulting in a
two-fold molar excess of hPAM4-DDD2. The reaction mixture was
incubated at room temperature for 24 hours under mild reducing
conditions using 1 mM reduced glutathione. Following reduction, the
DNL reaction was completed by mild oxidation using 2 mM oxidized
glutathione. TF10 was isolated by affinity chromatography using IMP
291-affigel resin, which binds with high specificity to the h679
Fab.
Example 7. Sequence Variants for DNL
[0246] In certain preferred embodiments, the AD and DDD sequences
incorporated into the cytokine-MAb DNL complex comprise the amino
acid sequences of AD1 or AD2 and DDD1 or DDD2, as discussed above.
However, in alternative embodiments sequence variants of AD and/or
DDD moieties may be utilized in construction of the DNL complexes.
For example, there are only four variants of human PKA DDD
sequences, corresponding to the DDD moieties of PKA RI.alpha.,
RII.alpha., RI.beta. and RII.beta.. The RII.alpha. DDD sequence is
the basis of DDD1 and DDD2 disclosed above. The four human PKA DDD
sequences are shown below. The DDD sequence represents residues
1-44 of RII.alpha., 1-44 of RII.beta., 12-61 of RI.alpha. and 13-66
of RI.beta.. (Note that the sequence of DDD1 is modified slightly
from the human PKA RII.alpha. DDD moiety.)
TABLE-US-00005 PKA RI.alpha. (SEQ ID NO: 12)
SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEE AK PKA RI.beta.
(SEQ ID NO: 13) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEEN
RQILA PKA RII.alpha. (SEQ ID NO: 14)
SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RII.beta. (SEQ ID
NO: 15) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER
[0247] The structure-function relationships of the AD and DDD
domains have been the subject of investigation. (See, e.g.,
Burns-Hamuro et al., 2005, Protein Sci 14:2982-92; Carr et al.,
2001, J Biol Chem 276:17332-38; Alto et al., 2003, Proc Natl Acad
Sci USA 100:4445-50; Hundsrucker et al., 2006, Biochem J
396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Gold et
al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell
24:397-408, the entire text of each of which is incorporated herein
by reference.)
[0248] For example, Kinderman et al. (2006, Mol Cell 24:397-408)
examined the crystal structure of the AD-DDD binding interaction
and concluded that the human DDD sequence contained a number of
conserved amino acid residues that were important in either dimer
formation or AKAP binding, underlined in SEQ ID NO:6 below. (See
FIG. 1 of Kinderman et al., 2006, incorporated herein by
reference.) The skilled artisan will realize that in designing
sequence variants of the DDD sequence, one would desirably avoid
changing any of the underlined residues, while conservative amino
acid substitutions might be made for residues that are less
critical for dimerization and AKAP binding.
TABLE-US-00006 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
[0249] As known in the art, conservative amino acid substitutions
have been characterized for each of the twenty common L-amino
acids. Thus, based on the data of Kinderman (2006) and conservative
amino acid substitutions, potential alternative DDD sequences based
on SEQ ID NO:6 are shown in Table 2. In devising Table 2, only
highly conservative amino acid substitutions were considered. For
example, charged residues were only substituted for residues of the
same charge, residues with small side chains were substituted with
residues of similar size, hydroxyl side chains were only
substituted with other hydroxyls, etc. Because of the unique effect
of proline on amino acid secondary structure, no other residues
were substituted for proline. Even with such conservative
substitutions, there are over twenty million possible alternative
sequences for the 44 residue peptide
(2.times.3.times.2.times.2.times.2.times.2.times.2.times.2.times.2.times.-
2.times.2.times.2.times.2.times.2.times.2.times.4.times.2.times.2.times.2.-
times.2.times.2.times.4.times.2.times.4). The skilled artisan will
realize that an almost unlimited number of alternative species
within the genus of DDD moieties can be constructed by standard
techniques, for example using a commercial peptide synthesizer or
well known site-directed mutagenesis techniques. The effect of the
amino acid substitutions on AD moiety binding may also be readily
determined by standard binding assays, for example as disclosed in
Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50).
TABLE-US-00007 TABLE 2 Conservative Amino Acid Substitutions in
DDD1 (SEQ ID NO: 6). Consensus sequence disclosed as SEQ ID NO: 16.
S H I Q I P P G L T E L L Q G Y T V E V L R T K N A S D N A S D K R
Q Q P P D L V E F A V E Y F T R L R E A R A N N E D L D S K K D L K
L I I I V V V
[0250] Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50)
performed a bioinformatic analysis of the AD sequence of various
AKAP proteins to design an RII selective AD sequence called AKAP-IS
(SEQ ID NO:8), with a binding constant for DDD of 0.4 nM. The
AKAP-IS sequence was designed as a peptide antagonist of AKAP
binding to PKA. Residues in the AKAP-IS sequence where
substitutions tended to decrease binding to DDD are underlined in
SEQ ID NO:8 below. The skilled artisan will realize that in
designing sequence variants of the AD sequence, one would desirably
avoid changing any of the underlined residues, while conservative
amino acid substitutions might be made for residues that are less
critical for DDD binding. Table 3 shows potential conservative
amino acid substitutions in the sequence of AKAP-IS (AD1, SEQ ID
NO:8), similar to that shown for DDD1 (SEQ ID NO:6) in Table 2
above.
[0251] Even with such conservative substitutions, there are over
thirty-five thousand possible alternative sequences for the 17
residue AD1 (SEQ ID NO:8) peptide sequence
(2.times.3.times.2.times.4.times.3.times.2.times.2.times.2.times.2.times.-
2.times.2.times.4). Again, a very large number of species within
the genus of possible AD moiety sequences could be made, tested and
used by the skilled artisan, based on the data of Alto et al.
(2003). It is noted that FIG. 2 of Alto (2003) shows an even large
number of potential amino acid substitutions that may be made,
while retaining binding activity to DDD moieties, based on actual
binding experiments.
TABLE-US-00008 AKAP-IS (SEQ ID NO: 8) QIEYLAKQIVDNAIQQA
TABLE-US-00009 TABLE 3 Conservative Amino Acid Substitutions in AD1
(SEQ ID NO: 8). Consensus sequence disclosed as SEQ ID NO: 17. Q I
E Y L A K Q I V D N A I Q Q A N L D F I R N E Q N N L V T V I S
V
[0252] Gold (2006, Mol Cell 24:383-95) utilized crystallography and
peptide screening to develop a SuperAKAP-IS sequence (SEQ ID
NO:18), exhibiting a five order of magnitude higher selectivity for
the RII isoform of PKA compared with the RI isoform. Underlined
residues indicate the positions of amino acid substitutions,
relative to the AKAP-IS sequence, which increased binding to the
DDD moiety of RII.alpha.. In this sequence, the N-terminal Q
residue is numbered as residue number 4 and the C-terminal A
residue is residue number 20. Residues where substitutions could be
made to affect the affinity for RII.alpha. were residues 8, 11, 15,
16, 18, 19 and 20 (Gold et al., 2006). It is contemplated that in
certain alternative embodiments, the SuperAKAP-IS sequence may be
substituted for the AKAP-IS AD moiety sequence to prepare DNL
constructs. Other alternative sequences that might be substituted
for the AKAP-IS AD sequence are shown in SEQ ID NO:19-21.
Substitutions relative to the AKAP-IS sequence are underlined. It
is anticipated that, as with the AD2 sequence shown in SEQ ID NO:9,
the AD moiety may also include the additional N-terminal residues
cysteine and glycine and C-terminal residues glycine and
cysteine.
TABLE-US-00010 SuperAKAP-IS (SEQ ID NO: 18) QIEYVAKQIVDYAIHQA
Alternative AKAP sequences (SEQ ID NO: 19) QIEYKAKQIVDHAIHQA (SEQ
ID NO: 20) QIEYHAKQIVDHAIHQA (SEQ ID NO: 21) QIEYVAKQIVDHAIHQA
[0253] FIG. 2 of Gold et al. disclosed additional DDD-binding
sequences from a variety of AKAP proteins, any of which could be
utilized to design a DNL construct.
[0254] Stokka et al. (2006, Biochem J 400:493-99) also developed
peptide competitors of AKAP binding to PKA, shown in SEQ ID
NO:22-24. The peptide antagonists were designated as Ht31 (SEQ ID
NO:22), RIAD (SEQ ID NO:23) and PV-38 (SEQ ID NO:24). The Ht-31
peptide exhibited a greater affinity for the RII isoform of PKA,
while the RIAD and PV-38 showed higher affinity for RI.
TABLE-US-00011 Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD
(SEQ ID NO: 65) LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66)
FEELAWKIAKMIWSDVFQQC
[0255] Hundsrucker et al. (2006, Biochem J 396:297-306) developed
still other peptide competitors for AKAP binding to PKA, with a
binding constant as low as 0.4 nM to the DDD of the RII form of
PKA. The sequences of various AKAP antagonistic peptides are
provided in Table 1 of Hundsrucker et al., reproduced in Table 4
below. AKAPIS represents a synthetic RII subunit-binding peptide.
All other peptides are derived from the RII-binding domains of the
indicated AKAPs.
TABLE-US-00012 TABLE 4 AKAP Peptide sequences Peptide Sequence
AKAPIS QIEYLAKQIVDNAIQQA (SEQ ID NO: 8) AKAPIS-P QIEYLAKQIPDNAIQQA
(SEQ ID NO: 25) Ht31 KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 26)
Ht31-P KGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 27)
AKAP7.delta.-wt-pep PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 28)
AKAP7.delta.-L304T-pep PEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 29)
AKAP7.delta.-L308D-pep PEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 30)
AKAP7.delta.-P-pep PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 31)
AKAP7.delta.-PP-pep PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 32)
AKAP7.delta.-L314E-pep PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 33)
AKAP1-pep EEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 34) AKAP2-pep
LVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 35) AKAP5-pep
QYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 36) AKAP9-pep
LEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 37) AKAP10-pep
NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 38) AKAP11-pep
VNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 39) AKAP12-pep
NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 40) AKAP14-pep
TQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 41) Rab32-pep
ETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 12)
[0256] Residues that were highly conserved among the AD domains of
different AKAP proteins are indicated below by underlining with
reference to the AKAP IS sequence (SEQ ID NO:8). The residues are
the same as observed by Alto et al. (2003), with the addition of
the C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al.
(2006), incorporated herein by reference.) The sequences of peptide
antagonists with particularly high affinities for the RII DDD
sequence were those of AKAP-IS, AKAP7.delta.-wt-pep,
AKAP7.delta.-L304T-pep and AKAP7.delta.-L308D-pep.
TABLE-US-00013 AKAP-IS (SEQ ID NO: 8) QIEYLAKQIVDNAIQQA
[0257] Carr et al. (2001, J Biol Chem 276:17332-38) examined the
degree of sequence homology between different AKAP-binding DDD
sequences from human and non-human proteins and identified residues
in the DDD sequences that appeared to be the most highly conserved
among different DDD moieties. These are indicated below by
underlining with reference to the human PKA RIIa DDD sequence of
SEQ ID NO:6. Residues that were particularly conserved are further
indicated by italics. The residues overlap with, but are not
identical to those suggested by Kinderman et al. (2006) to be
important for binding to AKAP proteins. The skilled artisan will
realize that in designing sequence variants of DDD, it would be
most preferred to avoid changing the most conserved residues
(italicized), and it would be preferred to also avoid changing the
conserved residues (underlined), while conservative amino acid
substitutions may be considered for residues that are neither
underlined nor italicized.
TABLE-US-00014 (SEQ ID NO: 6)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
[0258] A modified set of conservative amino acid substitutions for
the DDD1 (SEQ ID NO:6) sequence, based on the data of Carr et al.
(2001) is shown in Table 5. Even with this reduced set of
substituted sequences, there are over 65,000 possible alternative
DDD moiety sequences that may be produced, tested and used by the
skilled artisan without undue experimentation. The skilled artisan
could readily derive such alternative DDD amino acid sequences as
disclosed above for Table 2 and Table 3.
TABLE-US-00015 TABLE 5 Conservative Amino Acid Substitutions in
DDD1 (SEQ ID NO: 6). Consensus sequence disclosed as SEQ ID NO: 43.
S H I Q I P P G L T E L L Q G Y T V E V L R T N S I L A Q Q P P D L
V E F A V E Y F T R L R E A R A N I D S K K L L L I I A V V
[0259] The skilled artisan will realize that these and other amino
acid substitutions in the DDD or AD amino acid sequences may be
utilized to produce alternative species within the genus of AD or
DDD moieties, using techniques that are standard in the field and
only routine experimentation.
Example 8. In Vivo Imaging Using .sup.18F-Labeled Peptides and
Comparison with .sup.18F-FDG
[0260] In vivo imaging techniques using pretargeting with
bispecific antibodies and labeled targeting peptides were used to
successfully detect tumors of relatively small size. The .sup.18F
was purified on a WATERS.RTM. ACCELL' Plus QMA Light cartridge. The
.sup.18F eluted with 0.4 M KHCO.sub.3 was mixed with 3 .mu.L 2 mM
Al.sup.3+ in a pH 4 acetate buffer. The Al.sup.18F solution was
then injected into the ascorbic acid IMP449 labeling vial and
heated to 105.degree. C. for 15 min. The reaction solution was
cooled and mixed with 0.8 mL DI water. The reaction contents were
loaded on a WATERS.RTM. OASIS.RTM. 1 cc HLB Column and eluted with
2.times.200 .mu.L 1:1 EtOH/H.sub.2O. TF2 was prepared as described
above. TF2 binds divalently to carcinoembryonic antigen (CEA) and
monovalently to the synthetic hapten, HSG
(histamine-succinyl-glycine).
[0261] Biodistribution and microPET Imaging.
[0262] Six-week-old NCr nu-m female nude mice were implanted s.c.
with the human colonic cancer cell line, LS174T (ATCC, Manassas,
Va.). When tumors were visibly established, pretargeted animals
were injected intravenously with 162 .mu.g (.about.1 nmole/0.1 mL)
TF2 or TF10 (control non-targeting tri-Fab bsMAb), and then 16-18 h
later, .about.0.1 nmol of Al.sup.18F(IMP449) (84 .mu.Ci, 3.11
MBq/0.1 mL) was injected intravenously. Other non-pretargeted
control animals received .sup.18F alone (150 .mu.Ci, 5.5 MBq),
Al.sup.18F complex alone (150 .mu.Ci, 5.55 MBq), the
Al.sup.18F(IMP449) peptide alone (84 .mu.Ci, 3.11 MBq), or
.sup.18F-FDG (150 .mu.Ci, 5.55 MBq). .sup.18F and .sup.18F-FDG were
obtained on the day of use from IBA Molecular (Somerset, N.J.).
Animals receiving .sup.18F-FDG were fasted overnight, but water was
given ad libitum.
[0263] At 1.5 h after the radiotracer injection, animals were
anesthetized, bled intracardially, and necropsied. Tissues were
weighed and counted together with a standard dilution prepared from
each of the respective products. Due to the short physical
half-life of .sup.18F, standards were interjected between each
group of tissues from each animal. Uptake in the tissues is
expressed as the counts per gram divided by the total injected
activity to derive the percent-injected dose per gram (% ID/g).
[0264] Two types of imaging studies were performed. In one set, 3
nude mice bearing small LS174T subcutaneous tumors received either
the pretargeted Al.sup.18F(IMP449), Al.sup.18F(IMP449) alone (not
pretargeted), both at 135 .mu.Ci (5 MBq; 0.1 nmol), or .sup.18F-FDG
(135 .mu.Ci, 5 MBq). At 2 h after the intravenous radiotracer
injection, the animals were anesthetized with a mixture of
O.sub.2/N.sub.2O and isoflurane (2%) and kept warm during the scan,
performed on an INVEON.RTM. animal PET scanner (Siemens Preclinical
Solutions, Knoxville, Tenn.).
[0265] Representative coronal cross-sections (0.8 mm thick) in a
plane located approximately in the center of the tumor were
displayed, with intensities adjusted until pixel saturation
occurred in any region of the body (excluding the bladder) and
without background adjustment.
[0266] In a separate dynamic imaging study, a single LS174T bearing
nude mouse that was given the TF2 bsMAb 16 h earlier was
anesthetized with a mixture of O.sub.2/N.sub.2O and isoflurane
(2%), placed supine on the camera bed, and then injected
intravenously with 219 .mu.Ci (8.1 MBq) Al.sup.18F(IMP449) (0.16
nmol). Data acquisition was immediately initiated over a period of
120 minutes. The scans were reconstructed using OSEM3D/MAP. For
presentation, time-frames ending at 5, 15, 30, 60, 90, and 120 min
were displayed for each cross-section (coronal, sagittal, and
transverse). For sections containing tumor, at each interval the
image intensity was adjusted until pixel saturation first occurred
in the tumor. Image intensity was increased as required over time
to maintain pixel saturation within the tumor. Coronal and sagittal
cross-sections without tumor taken at the same interval were
adjusted to the same intensity as the transverse section containing
the tumor. Background activity was not adjusted.
[0267] Results
[0268] While .sup.18F alone and [Al.sup.18F] complexes had similar
uptake in all tissues, considerable differences were found when the
complex was chelated to IMP449 (Table 6). The most striking
differences were found in the uptake in the bone, where the
non-chelated .sup.18F was 60- to nearly 100-fold higher in the
scapula and .about.200-fold higher in the spine. This distribution
is expected since .sup.18F, or even a metal-fluoride complex, is
known to accrete in bone (Franke et al. 1972, Radiobiol. Radiother.
(Berlin) 13:533). Higher uptake was also observed in the tumor and
intestines as well as in muscle and blood. The chelated
Al.sup.18F(IMP449) had significantly lower uptake in all the
tissues except the kidneys, illustrating the ability of the
chelate-complex to be removed efficiently from the body by urinary
excretion.
[0269] Pretargeting the Al.sup.18F(IMP449) using the TF2 anti-CEA
bsMAb shifted uptake to the tumor, increasing it from 0.20.+-.0.05
to 6.01.+-.1.72% injected dose per gram at 1.5 h, while uptake in
the normal tissues was similar to the Al.sup.18F(IMP449) alone.
Tumor/nontumor ratios were 146.+-.63, 59.+-.24, 38.+-.15, and
2.0.+-.1.0 for the blood, liver, lung, and kidneys, respectively,
with other tumor/tissue ratios>100:1 at this time. Although both
.sup.18F alone and [Al.sup.18F] alone had higher uptake in the
tumor than the chelated Al.sup.18F(IMP449), yielding tumor/blood
ratios of 6.7.+-.2.7 and 11.0.+-.4.6 vs. 5.1.+-.1.5, respectively,
tumor uptake and tumor/blood ratios were significantly increased
with pretargeting (all P values <0.001).
[0270] Biodistribution was also compared to the most commonly used
tumor imaging agent, [.sup.18F]FDG, which targets tissues with high
glucose consumption and metabolic activity (Table 6). Its uptake
was appreciably higher than the Al.sup.18F(IMP449) in all normal
tissues, except the kidney. Tumor uptake was similar for both the
pretargeted Al.sup.18F(IMP449) and .sup.18F-FDG, but because of the
higher accretion of [.sup.18F]FDG in most normal tissues,
tumor/nontumor ratios with .sup.18F-FDG were significantly lower
than those in the pretargeted animals (all P values <0.001).
TABLE-US-00016 TABLE 6 Biodistribution of TF2-pretargeted
Al.sup.18F(IMP449) and other control .sup.18F-labeled agents in
nude mice bearing LS174T human colonic xenografts. For
pretargeting, animals were given TF2 16 h before the injection of
the Al.sup.18F(IMP449). All injections were administered
intravenously. Percent Injected Dose Per Gram (Mean .+-. SD) at 1.5
hr Post-Injection Al.sup.18F(IMP449) TF2-pretargeted .sup.18F alone
[Al.sup.18F] alone alone Al.sup.18F(IMP449) .sup.18F-FDG Tumor 1.02
.+-. 0.45 1.38 .+-. 0.39 0.20 .+-. 0.05 6.01 .+-. 1.72 7.25 .+-.
2.54 Liver 0.11 .+-. 0.02 0.12 .+-. 0.02 0.08 .+-. 0.03 0.11 .+-.
0.03 1.34 .+-. 0.36 Spleen 0.13 .+-. 0.06 0.10 .+-. 0.03 0.08 .+-.
0.02 0.08 .+-. 0.02 2.62 .+-. 0.73 Kidney 0.29 .+-. 0.07 0.25 .+-.
0.07 3.51 .+-. 0.56 3.44 .+-. 0.99 1.50 .+-. 0.61 Lung 0.26 .+-.
0.08 0.38 .+-. 0.19 0.11 .+-. 0.03 0.17 .+-. 0.04 3.72 .+-. 1.48
Blood 0.15 .+-. 0.03 0.13 .+-. 0.03 0.04 .+-. 0.01 0.04 .+-. 0.02
0.66 .+-. 0.19 Stomach 0.21 .+-. 0.13 0.15 .+-. 0.05 0.20 .+-. 0.32
0.12 .+-. 0.18 2.11 .+-. 1.04 Small Int. 1.53 .+-. 0.33 1.39 .+-.
0.34 0.36 .+-. 0.23 0.27 .+-. 0.10 1.77 .+-. 0.61 Large Int. 1.21
.+-. 0.13 1.78 .+-. 0.70 0.05 .+-. 0.04 0.03 .+-. 0.01 2.90 .+-.
0.79 Scapula 6.13 .+-. 1.33 9.83 .+-. 2.31 0.08 .+-. 0.06 0.04 .+-.
0.02 10.63 .+-. 5.88 Spine 19.88 .+-. 2.12 19.03 .+-. 2.70 0.13
.+-. 0.14 0.08 .+-. 0.03 4.21 .+-. 1.79 Muscle 0.16 .+-. 0.05 0.58
.+-. 0.36 0.06 .+-. 0.05 0.10 .+-. 0.20 4.35 .+-. 3.01 Brain 0.15
.+-. 0.06 0.13 .+-. 0.03 0.01 .+-. 0.01 0.01 .+-. 0.00 10.71 .+-.
4.53 Tumor wt (g) 0.29 .+-. 0.07 0.27 .+-. 0.10 0.27 .+-. 0.08 0.33
.+-. 0.11 0.25 .+-. 0.21 N 6 7 8 7 5
[0271] Several animals were imaged to further analyze the
biodistribution of Al.sup.18F(IMP449) alone or Al.sup.18F(IMP449)
pretargeted with TF2, as well [.sup.18F]FDG. Static images
initiated at 2.0 h after the radioactivity was injected
corroborated the previous tissue distribution data showing uptake
almost exclusively in the kidneys (FIG. 1). A 21-mg tumor was
easily visualized in the pretargeted animal, while the animal given
the Al.sup.18F(IMP449) alone failed to localize the tumor, having
only renal uptake. No evidence of bone accretion was observed,
suggesting that the Al.sup.18F was bound firmly to IMP 449. This
was confirmed in another pretargeted animal that underwent a
dynamic imaging study that monitored the distribution of the
Al.sup.18F(IMP449) in 5-min intervals over 120 minutes (FIG. 2).
Coronal and sagittal slices showed primarily cardiac, renal, and
some hepatic uptake over the first 5 min, but heart and liver
activity decreased substantially over the next 10 min, while the
kidneys remained prominent throughout the study. There was no
evidence of activity in the intestines or bone over the full
120-min scan. Uptake in a 35-mg LS174T tumor was first observed at
15 min, and by 30 min, the signal was very clearly delineated from
background, with intense tumor activity being prominent during the
entire 120-min scanning.
[0272] In comparison, static images from an animal given
.sup.18F-FDG showed the expected pattern of radioactivity in the
bone, heart muscle, and brain observed previously (McBride et al.,
2006, J. Nucl. Med. 47:1678; Sharkey et al., 2008, Radiology
246:497), with considerably more background activity in the body
(FIG. 1). Tissue uptake measured in the 3 animals necropsied at the
conclusion of the static imaging study confirmed much higher tissue
.sup.18F radioactivity in all tissues (not shown). While tumor
uptake with .sup.18F-FDG was higher in this animal than in the
pretargeted one, tumor/blood ratios were more favorable for
pretargeting; and with much less residual activity in the body,
tumor visualization was enhanced by pretargeting.
[0273] These studies demonstrate that a hapten-peptide used in
pretargeted imaging can be rapidly labeled (60 min total
preparation time) with .sup.18F by simply forming an
aluminum-fluoride complex that can then be bound by a suitable
chelate and incorporated into the hapten-peptide. This can be made
more general by simply coupling the [Al.sup.18F]-chelate to any
molecule that can be attached to the chelating moiety and be
subsequently purified.
[0274] This report describes a direct, facile, and rapid method of
binding .sup.18F to various compounds via an aluminum conjugate.
The [Al.sup.18F] peptide was stable in vitro and in vivo when bound
by a NOTA-based chelate. Yields were within the range found with
conventional .sup.18F labeling procedures. These results further
demonstrate the feasibility of PET imaging using metal.sup.18F
chelated to a wide variety of targeting molecules.
Example 9. Preparation and Labeling of IMP460 with Al-.sup.18F
[0275] IMP460 NODA-Ga-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH.sub.2
(SEQ ID NO:44) was chemically synthesized. The NODA-Ga ligand was
purchased from CHEMATECH.RTM. and attached on the peptide
synthesizer like the other amino acids. The peptide was synthesized
on Sieber amide resin with the amino acids and other agents added
in the following order Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc
removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc
removal, Fmoc-D-Ala-OH, and NODA-GA(tBu).sub.3. The peptide was
then cleaved and purified by HPLC to afford the product.
[0276] Radiolabeling of IMP460
[0277] IMP 460 (0.0020 g) was dissolved in 732 .mu.L, pH 4, 0.1 M
NaOAc. The .sup.18F was purified as described above, neutralized
with glacial acetic acid and mixed with the Al solution. The
peptide solution, 20 .mu.L was then added and the solution was
heated at 99.degree. C. for 25 min. The crude product was then
purified on a WATERS.RTM. HLB column. The [Al.sup.18F] labeled
peptide was in the 1:1 EtOH/H.sub.2O column eluent. The reverse
phase HPLC trace in 0.1% TFA buffers showed a clean single HPLC
peak at the expected location for the labeled peptide (not
shown).
Example 10. Synthesis and Labeling of IMP461 and IMP462
NOTA-Conjugated Peptides
[0278] The simplest possible NOTA ligand (protected for peptide
synthesis) was prepared and incorporated into two peptides for
pretargeting--IMP461 and IMP462.
[0279] Synthesis of Bis-t-Butyl-NOTA
[0280] NO2AtBu (0.501 g 1.4.times.10.sup.-3 mol) was dissolved in 5
mL anhydrous acetonitrile. Benzyl-2-bromoacetate (0.222 mL,
1.4.times.10.sup.-3 mol) was added to the solution followed by
0.387 g of anhydrous K.sub.2CO.sub.3. The reaction was allowed to
stir at room temperature overnight. The reaction mixture was
filtered and concentrated to obtain 0.605 g (86% yield) of the
benzyl ester conjugate. The crude product was then dissolved in 50
mL of isopropanol, mixed with 0.2 g of 10% Pd/C (under Ar) and
placed under 50 psi H.sub.2 for 3 days. The product was then
filtered and concentrated under vacuum to obtain 0.462 g of the
desired product ESMS [M-H].sup.- 415.
[0281] Synthesis of IMP461
[0282] The peptide was synthesized on Sieber amide resin with the
amino acids and other agents added in the following order
Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D-Tyr(But)-OH,
Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D-Ala-OH, and
Bis-t-butylNOTA. The peptide was then cleaved and purified by HPLC
to afford the product IMP461 ESMS MH.sup.+ 1294
NOTA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH.sub.2; SEQ ID NO:45).
[0283] Synthesis of IMP 462
[0284] The peptide was synthesized on Sieber amide resin with the
amino acids and other agents added in the following order
Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D-Tyr(But)-OH,
Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D-Asp(But)-OH,
and Bis-t-butyl NOTA. The peptide was then cleaved and purified by
HPLC to afford the product IMP462 ESMS MH.sup.+ 1338
(NOTA-D-Asp-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH.sub.2; SEQ ID
NO:46).
[0285] .sup.18F Labeling of IMP461 & IMP462
[0286] The peptides were dissolved in pH 4.13, 0.5 M NaOAc to make
a 0.05 M peptide solution, which was stored in the freezer until
needed. The F-18 was received in 2 mL of water and trapped on a
SEP-PAK.RTM. Light, WATERS.RTM. ACCELL.TM. Plus QMA Cartridge. The
.sup.18F was eluted from the column with 200 .mu.L aliquots of 0.4
M KHCO.sub.3. The bicarbonate was neutralized to .about.pH 4 by the
addition of 10 .mu.L of glacial acetic acid to the vials before the
addition of the activity. A 100 .mu.L aliquot of the purified
.sup.18F solution was removed and mixed with 3 .mu.L, 2 mM Al in pH
4, 0.1 M NaOAc. The peptide, 10 .mu.L (0.05 M) was added and the
solution was heated at .about.100.degree. C. for 15 min. The crude
reaction mixture was diluted with 700 .mu.L DI water and placed on
an HLB column and after washing the .sup.18F was eluted with
2.times.100 .mu.L of 1:1 EtOH/H.sub.2O to obtain the purified
.sup.18F-labeled peptide.
Example 11. Preparation and .sup.18F-Labeling of IMP467
[0287] IMP467 C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH.sub.2
(SEQ ID NO:47)
[0288] Tetra tert-butyl C-NETA-succinyl was produced. The
tert-Butyl
{4-[2-(Bis-(tert-butyoxycarbonyl)methyl-3-(4-nitrophenyl)propyl]-7-tert-b-
utyoxycarbonyl[1,4,7]triazanonan-1-yl} was prepared as described in
Chong et al. (J. Med. Chem. 2008, 51:118-125).
[0289] The peptide, IMP467
C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH.sub.2 (SEQ ID NO:47)
was made on Sieber Amide resin by adding the following amino acids
to the resin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH,
the Aloc was cleaved Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH,
Trt-HSG-OH, the Aloc was cleaved,
tert-Butyl{4-[Bis-(tert-butoxycarbonylmethyl)amino)-3-(4-succiny-
lamidophenyl)propyl]-7-tert-butoxycarbonylmethyl[1,4,7]triazanonan-1-yl}ac-
etate. The peptide was then cleaved from the resin and purified by
RP-HPLC to yield 6.3 mg of IMP467. The crude peptide was purified
by high performance liquid chromatography (HPLC) using a C18
column.
[0290] Radiolabeling
[0291] A 2 mM solution of IMP467 was prepared in pH 4, 0.1 M NaOAc.
The .sup.18F.sup.-, 139 mCi, was eluted through a WATERS.RTM.
ACCELL.TM. Plus SEP-PAK.RTM. Light QMA cartridge and the
.sup.18F.sup.- was eluted with 1 mL of 0.4 M KHCO.sub.3. The
labeled IMP467 was purified by HLB RP-HPLC. The RP-HPLC showed two
peaks eluting (not shown), which are believed to be diastereomers
of Al.sup.18F(IMP467). Supporting this hypothesis, there appeared
to be some interconversion between the two HLB peaks when IMP467
was incubated at 37.degree. C. (not shown). In pretargeting
techniques as discussed below, since the [Al.sup.18F]-chelator
complex is not part of the hapten site for antibody binding, the
presence of diastereomers does not appear to affect targeting of
the .sup.18F-labeled peptide to diseased tissues.
[0292] Comparison of Yield of Radiolabeled Peptides
[0293] In an attempt to improve labeling yields while maintaining
in vivo stability, 3 NOTA derivatives of pretargeting peptide were
synthesized (IMP460, IMP461 and IMP467). Of these, IMP467 nearly
doubled the labeling yields of the other peptides (Table 7). All of
the labeling studies in Table 7 were performed with the same number
of moles of peptide and aluminum. The results shown in Table 7
represent an exemplary labeling experiment with each peptide.
[0294] The .sup.18F-labeling yield of IMP467 was .about.70% when
only 40 nmol (.about.13-fold less than IMP449) was used with 1.3
GBq (35 mCi) of .sup.18F, indicating this ligand has improved
binding properties for the Al.sup.18F complex. By enhancing the
kinetics of ligand binding, yields were substantially improved
(average 65-75% yield), while using fewer moles of IMP467 (40
nmol), relative to IMP449 (520 nmol, 44% yield).
TABLE-US-00017 TABLE 7 Comparison of yields of different NOTA
containing peptides Peptide Yield IMP449 44% IMP460 5.8% IMP461 31%
IMP467 87%
Example 12. Factors Affecting Yield and Stability of IMP467
Labeling Peptide Concentration
[0295] To examine the effect of varying peptide concentration on
yield, the amount of binding of Al.sup.18F to peptide was
determined in a constant volume (63 .mu.L) with a constant amount
of Al.sup.3+ (6 nmol) and .sup.18F, but varying the amount of
peptide added. The yield of labeled peptide IMP467 decreased with a
decreasing concentration of peptide as follows: 40 nmol peptide
(82% yield); 30 nmol (79% yield); 20 nmol (75% yield); 10 nmol (49%
yield). Thus, varying the amount of peptide between 20 and 40 nmol
had little effect on yield with IMP467. However, a decreased yield
was observed starting at 10 nmol of peptide in the labeling
mix.
[0296] Aluminum Concentration
[0297] When IMP467 was labeled in the presence of increasing
amounts of Al.sup.3+ (0, 5, 10, 15, 20 .mu.L of 2 mM Al in pH 4
acetate buffer and keeping the total volume constant), yields of
3.5%, 80%, 77%, 78% and 74%, respectively, were achieved. These
results indicated that (a) non-specific binding of .sup.18F to this
peptide in the absence of Al.sup.3+ is minimal, (b) 10 nmol of
Al.sup.3+ was sufficient to allow for maximum .sup.18F-binding, and
(c) higher amounts of Al.sup.3+ did not reduce binding
substantially, indicating that there was sufficient chelation
capacity at this peptide concentration.
[0298] Kinetics of Al.sup.18F(IMP467) Radiolabeling
[0299] Kinetic studies showed that binding was complete within 5
min at 107.degree. C. (5 min, 68%; 10 min, 61%; 15 min, 71%; and 30
min, 75%) with only moderate increases in isolated yield with
reaction times as long as 30 min. A radiolabeling reaction of
IMP467 performed at 50.degree. C. showed that no binding was
achieved at the lower temperature. Additional experiments,
disclosed in the Examples below, show that under some conditions a
limited amount of labeling can occur at reduced temperatures.
[0300] Effect of pH
[0301] The optimal pH for labeling was between 4.3 and 5.5. Yield
ranged from 54% at pH 2.88; 70-77% at pH 3.99; 70% at pH 5; 41% at
pH 6 to 3% at pH 7.3. The process could be expedited by eluting the
.sup.18F.sup.- from the anion exchange column with nitrate or
chloride ion instead of carbonate ion, which eliminates the need
for adjusting the eluent to pH 4 with glacial acetic acid before
mixing with the AlCl.sub.3.
[0302] High-Dose Radiolabeling of IMP467
[0303] Five microliters of 2 mM Al.sup.3+ stock solution were mixed
with 50 .mu.L of .sup.18F 1.3 GBq (35 mCi) followed by the addition
of 20 .mu.L of 2 mM IMP467 in 0.1 mM, pH 4.1 acetate buffer. The
reaction solution was heated to 104.degree. C. for 15 min and then
purified on an HLB column (.about.10 min) as described above,
isolating 0.68 GBq (18.4 mCi) of the purified peptide in 69%
radiochemical yield with a specific activity of 17 GBq/.mu.mol (460
Ci/mmol). The reaction time was 15 min and the purification time
was 12 min. The reaction was started 10 min after the 1.3 GBq (35
mCi).sup.18F.sup.- was purified, so the total time from the
isolation of the .sup.18F.sup.- to the purified final product was
37 min with a 52% yield without correcting for decay.
[0304] Human Serum Stability Test
[0305] An aliquot of the HLB purified peptide (.about.30 .mu.L) was
diluted with 200 .mu.L human serum (previously frozen) and placed
in the 37.degree. C. HPLC sample chamber. Aliquots were removed at
various time points and analyzed by HPLC. The HPLC analysis showed
very high stability of the .sup.18F-labeled peptides in serum at
37.degree. C. for at least five hours (not shown). There was no
detectable breakdown of the .sup.18F-labeled peptide after a five
hour incubation in serum (not shown).
[0306] The IMP461 and IMP462 ligands have two carboxyl groups
available to bind the aluminum whereas the NOTA ligand in IMP467
had four carboxyl groups. The serum stability study showed that the
complexes with IMP467 were stable in serum under conditions
replicating in vivo use. In vivo biodistribution studies with
labeled IMP467 show that the Al.sup.18F-labeled peptide is stable
under actual in vivo conditions (not shown).
[0307] Peptides can be labeled with .sup.18F rapidly (30 min) and
in high yield by forming Al.sup.18F complexes that can be bound to
a NOTA ligand on a peptide and at a specific activity of at least
17 GBq/.mu.mol, without requiring HPLC purification. The
Al.sup.18F(NOTA)-peptides are stable in serum and in vivo.
Modifications of the NOTA ligand can lead to improvements in yield
and specific activity, while still maintaining the desired in vivo
stability of the Al.sup.18F(NOTA) complex, and being attached to a
hydrophilic linker aids in the renal clearance of the peptide.
Further, this method avoids the dry-down step commonly used to
label peptides with .sup.18F. As shown in the following Examples,
this new .sup.18F-labeling method is applicable to labeling of a
broad spectrum of targeting peptides.
[0308] Optimized Labeling of Al.sup.18F(IMP467)
[0309] Optimized conditions for .sup.18F-labeling of IMP467 were
identified. These consisted of eluting .sup.18F.sup.- with
commercial sterile saline (pH 5-7), mixing with 20 nmol of
AlCl.sub.3 and 40 nmol IMP467 in pH 4 acetate buffer in a total
volume of 100 .mu.L, heating to 102.degree. C. for 15 min, and
performing SPE separation. High-yield (85%) and high specific
activity (115 GBq/.mu.mol) were obtained with IMP467 in a single
step, 30-min procedure after a simple solid-phase extraction (SPE)
separation without the need for HPLC purification.
Al.sup.18F(IMP467) was stable in PBS or human serum, with 2% loss
of .sup.18F after incubation in either medium for 6 h at 37.degree.
C.
[0310] Concentration and Purification of .sup.18F.sup.-
[0311] Radiochemical-grade .sup.18F.sup.- needs to be purified and
concentrated before use. We examined 4 different SPE purification
procedures to process the .sup.18F.sup.- prior to its use. Most of
the radiolabeling procedures were performed using .sup.18F.sup.-
prepared by a conventional process. The .sup.18F.sup.- in 2 mL of
water was loaded onto a SEP-PAK.RTM. Light, Waters Accell.TM. QMA
Plus Cartridge that was pre-washed with 10 mL of 0.4M KHCO.sub.3,
followed by 10 mL water. After loading the .sup.18F onto the
cartridge, it was washed with 5 mL water to remove any dissolved
metal and radiometal impurities. The isotope was then eluted with
.about.1 mL of 0.4M KHCO.sub.3 in several fractions to isolate the
fraction with the highest concentration of activity. The eluted
fractions were neutralized with 5 .mu.L of glacial acetic acid per
100 .mu.L of solution to adjust the eluent to pH 4-5.
[0312] In the second process, the QMA cartridge was washed with 10
mL pH 8.4, 0.5 M NaOAc followed by 10 mL DI H.sub.2O.
.sup.18F.sup.- was loaded onto the column as described above and
eluted with 1 mL, pH 6, 0.05 M KNO.sub.3 in 200-.mu.L fractions
with 60-70% of the activity in one of the fractions. No pH
adjustment of this solution was needed.
[0313] In the third process, the QMA cartridge was washed with 10
mL pH 8.4, 0.5 M NaOAc followed by 10 mL DI H.sub.2O. The
.sup.18F.sup.- was loaded onto the column as described above and
eluted with 1 mL, pH 5-7, 0.154 M commercial normal saline in
200-.mu.L fractions with 80% of the activity in one of the
fractions. No pH adjustment of this solution was needed.
[0314] Finally, we devised a method to prepare a more concentrated
and high-activity .sup.18F.sup.- solution, using tandem ion
exchange. Briefly, Tygon tubing (1.27 cm long, 0.64 cm OD) was
inserted into a TRICORN.TM. 5/20 column and filled with .about.200
.mu.L of AG 1-X8 resin, 100-200 mesh. The resin was washed with 6
mL 0.4 M K.sub.2CO.sub.3 followed by 6 mL H.sub.2O. A SEP-PAK.RTM.
light Waters ACCELL.TM. Plus CM cartridge was washed with DI
H.sub.2O. Using a syringe pump, the crude .sup.18F.sup.- that was
received in 5-mL syringe in 2 mL DI H.sub.2O flowed slowly through
the CM cartridge and the TRICORN.TM. column over .about.5 min
followed by a 6 mL wash with DI H.sub.2O through both ion-binding
columns. Finally, 0.4 M K.sub.2CO.sub.3 was pushed through only the
TRICORN.TM. column in 50-4, fractions. Typically, 40 to 60% of the
eluted activity was in one 50-.mu.L fraction. The fractions were
collected in 2.0 mL free-standing screw-cap microcentrifuge tubes
containing 5 .mu.L glacial acetic acid to neutralize the carbonate
solution. The elution vial with the most activity was then used as
the reaction vial.
Example 13. Labeling by Addition of .sup.18F.sup.- to a Peptide
Complexed with Aluminum
[0315] An HSG containing peptide (IMP 465,
Al(NOTA)-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH.sub.2) (SEQ ID NO:48)
linked to macrocyclic NOTA complexed with aluminum, was
successfully labeled with F-18. .sup.18F incorporation using 40
nmol of IMP 465 was 13.20%. An intermediate peptide, IMP 461, was
made as described above. Then 25.7 mg of IMP461 was dissolved in 2
mL DI water to which was added 10.2 mg AlCl.sub.3.3H.sub.2O and the
resultant solution heated to 100.degree. C. for 1 h. The crude
reaction mixture was purified by RP-HPLC to yield 19.6 mg of
IMP465.
[0316] For .sup.18F-labeling, 50 .mu.L .sup.18F solution [0.702 mCi
of .sup.18F.sup.-] and 20 .mu.L (40 nmol) 2 mM IMP465 solution (0.1
M NaOAc, pH 4.18) was heated to 101.degree. C. for 17 minutes.
Reverse Phase HPLC analysis showed 15.38% (RT about 8.60 min) of
the activity was attached to the peptide and 84.62% of the activity
eluted at the void volume of the column (2.60 min).
[0317] In a separate experiment, the percent yield of
.sup.18F-labeled peptide could be improved by varying the amount of
peptide added. The percent yield observed for IMP465 was 0.27% at
10 nmol peptide, 1.8% at 20 nmol of peptide and 49% at 40 nmol of
peptide.
[0318] IMP467 showed higher yield than IMP461 when peptide was
pre-incubated with aluminum before exposure to .sup.18F. IMP467 was
incubated with aluminum at room temperature and then frozen and
lyophilized. The amount of aluminum added for the pre-incubation
was varied.
TABLE-US-00018 TABLE 8 Labeling of IMP467 Pre-Incubated with
Aluminum Before .sup.18F.sup.- is Added IMP467 + Al Premixed,
Isolated Frozen and Lyophilized Labeling Yield 40 nmol IMP467 + 10
nmol Al Premix 82% 40 nmol IMP467 + 20 nmol Al Premix 64% 40 nmol
IMP467 + 30 nmol Al Premix 74% 40 nmol IMP467 + 6 nmol Al Normal
77% Labeling (Mix Al + .sup.18F first)
[0319] The yields were comparable to those obtained when IMP467 is
labeled by addition of an Al.sup.18F complex. Thus, .sup.18F
labeling by addition of .sup.18F to a peptide with aluminum already
bound to the chelating moiety is a feasible alternative approach to
pre-incubating the metal with .sup.18F.sup.- prior to addition to
the chelating moiety.
Example 14. Synthesis and Labeling of IMP468 Bombesin Peptide
[0320] The .sup.18F labeled targeting moieties are not limited to
antibodies or antibody fragments, but rather can include any
molecule that binds specifically or selectively to a cellular
target that is associated with or diagnostic of a disease state or
other condition that may be imaged by .sup.18F PET. Bombesin is a
14 amino acid peptide that is homologous to neuromedin B and
gastrin releasing peptide, as well as a tumor marker for cancers
such as lung and gastric cancer and neuroblastoma. IMP468
(NOTA-NH--(CH.sub.2).sub.7CO-Gln-Trp-Val-Trp-Ala-Val-Gly-His-Leu-Met-NH.s-
ub.2; SEQ ID NO:49) was synthesized as a bombesin analogue and
labeled with .sup.18F to target the gastrin-releasing peptide
receptor.
[0321] The peptide was synthesized by Fmoc based solid phase
peptide synthesis on Sieber amide resin, using a variation of a
synthetic scheme reported in the literature (Prasanphanich et al.,
2007, PNAS USA 104:12463-467). The synthesis was different in that
a bis-t-butyl NOTA ligand was add to the peptide during peptide
synthesis on the resin.
[0322] IMP468 (0.0139 g, 1.02.times.10.sup.-5 mol) was dissolved in
203 .mu.L of 0.5 M pH 4.13 NaOAc buffer. The peptide dissolved but
formed a gel on standing so the peptide gel was diluted with 609
.mu.L of 0.5 M pH 4.13 NaOAc buffer and 406 .mu.L of ethanol to
produce an 8.35.times.10.sup.-3M solution of the peptide. The
.sup.18F was purified on a QMA cartridge and eluted with 0.4 M
KHCO.sub.3 in 200 .mu.L fractions, neutralized with 10 .mu.L of
glacial acetic acid. The purified .sup.18F, 40 .mu.L, 1.13 mCi was
mixed with 3 .mu.L of 2 mM AlCl.sub.3 in pH 4, 0.1 M NaOAc buffer.
IMP468 (59.2 .mu.L, 4.94.times.10.sup.-7 mol) was added to the
Al.sup.18F solution and placed in a 108.degree. C. heating block
for 15 min. The crude product was purified on an HLB column, eluted
with 2.times.200 .mu.L of 1:1 EtOH/H.sub.2O to obtain the purified
.sup.18F-labeled peptide in 34% yield.
Example 15. Imaging of Tumors Using .sup.18F Labeled Bombesin
[0323] A NOTA-conjugated bombesin derivative (IMP468) was prepared
as described above. We began testing its ability to block
radiolabeled bombesin from binding to PC-3 cells as was done by
Prasanphanich et al. (PNAS 104:12462-12467, 2007). Our initial
experiment was to determine if IMP468 could specifically block
bombesin from binding to PC-3 cells. We used IMP333 as a
non-specific control. In this experiment, 3.times.10.sup.6 PC-3
cells were exposed to a constant amount (.about.50,000 cpms) of
.sup.125I-Bombesin (Perkin-Elmer) to which increasing amounts of
either IMP468 or IMP333 was added. A range of 56 to 0.44 nM was
used as our inhibitory concentrations.
[0324] The results showed that we could block the binding of
.sup.125I-BBN with IMP468 but not with the control peptide (IMP333)
(not shown), thus demonstrating the specificity of IMP468.
Prasanphanich indicated an IC.sub.50 for their peptide at 3.2 nM,
which is approximately 7-fold lower than what we found with IMP468
(21.5 nM).
[0325] This experiment was repeated using a commercially available
BBN peptide. We increased the amount of inhibitory peptide from 250
to 2 nM to block the .sup.125I-BBN from binding to PC-3 cells. We
observed very similar IC.sub.50-values for IMP468 and the BBN
positive control with an IC.sub.50-value higher (35.9 nM) than what
was reported previously (3.2 nM) but close to what the BBN control
achieved (24.4 nM).
[0326] To examine in vivo targeting, the distribution of
Al.sup.18F(IMP468) was examined in scPC3 prostate cancer xenograft
bearing nude male mice; alone vs. blocked with bombesin. For
radiolabeling, aluminum chloride (10 .mu.L, 2 mM), 51.9 mCi of
.sup.18F (from QMA cartridge), acetic acid, and 60 .mu.L of IMP468
(8.45 mM in ethanol/NaOAc) were heated at 100.degree. C. for 15
min. The reaction mixture was purified on reverse phase HPLC.
Fractions 40 and 41 (3.56, 1.91 mCi) were pooled and applied to HLB
column for solvent exchange. The product was eluted in 800 .mu.L
(3.98 mCi) and 910 .mu.Ci remained on the column. iTLC developed in
saturated NaCl showed 0.1% unbound activity.
[0327] A group of six tumor-bearing mice were injected with
Al.sup.18F(IMP468) (167 .mu.Ci, .about.9.times.10.sup.-1.degree.
mol) and necropsied 1.5 h later. Another group of six mice were
injected iv with 100 .mu.g (6.2.times.10.sup.-8 mol) of bombesin 18
min before administering Al.sup.18F(IMP468). The second group was
also necropsied 1.5 h post injection. The data shows specific
targeting of the tumor with [Al.sup.18F] IMP 468 (FIG. 3). Tumor
uptake of the peptide is reduced when bombesin was given 18 min
before the Al.sup.18F(IMP468) (FIG. 3). Biodistribution data
indicates in vivo stability of Al.sup.18F(IMP468) for at least 1.5
h (not shown).
[0328] Larger tumors showed higher uptake of Al.sup.18F(IMP468),
possibly due to higher receptor expression in larger tumors (not
shown). The biodistribution data showed Al.sup.18F(IMP468) tumor
targeting that was in the same range as reported for the same
peptide labeled with .sup.68Ga by Prasanphanich et al. (not shown).
The results demonstrate that the .sup.18F peptide labeling method
can be used in vivo to target receptors that are upregulated in
tumors, using targeting molecules besides antibodies. In this case,
the IMP468 targeting took advantage of a naturally occurring
ligand-receptor interaction. The tumor targeting was significant
with a P value of P=0.0013. Many such ligand-receptor pairs are
known and any such targeting interaction may form the basis for
.sup.18F imaging, using the methods described herein.
Example 16. Synthesis and Labeling of Somatostatin Analog
IMP466
[0329] Somatostatin is another non-antibody targeting peptide that
is of use for imaging the distribution of somatostatin receptor
protein. .sup.123I-labeled octreotide, a somatostatin analog, has
been used for imaging of somatostatin receptor expressing tumors
(e.g., Kvols et al., 1993, Radiology 187:129-33; Leitha et al.,
1993, J Nucl Med 34:1397-1402). However, .sup.123I has not been of
extensive use for imaging because of its expense, short physical
half-life and the difficulty of preparing the radiolabeled
compounds. The .sup.18F-labeling methods described herein are
preferred for imaging of somatostatin receptor expressing
tumors.
TABLE-US-00019 (SEQ ID NO: 50) IMP466
NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl
[0330] A NOTA-conjugated derivative of the somatostatin analog
octreotide (IMP466) was made by standard Fmoc based solid phase
peptide synthesis to produce a linear peptide. The C-terminal Throl
residue is threoninol. The peptide was cyclized by treatment with
DMSO overnight. The peptide, 0.0073 g, 5.59.times.10.sup.-6 mol was
dissolved in 111.9 .mu.L of 0.5 M pH 4 NaOAc buffer to make a 0.05
M solution of IMP466. The solution formed a gel over time so it was
diluted to 0.0125 M by the addition of more 0.5 M NaOAc buffer.
[0331] .sup.18F was purified and concentrated with a QMA cartridge
to provide 200 .mu.L of .sup.18F in 0.4 M KHCO.sub.3. The
bicarbonate solution was neutralized with 10 .mu.L of glacial
acetic acid. A 40 .mu.L aliquot of the neutralized .sup.18F eluent
was mixed with 3 .mu.L of 2 mM AlCl.sub.3, followed by the addition
of 40 .mu.L of 0.0125 M IMP466 solution. The mixture was heated at
105.degree. C. for 17 min. The reaction was then purified on a
Waters 1 cc (30 mg) HLB column by loading the reaction solution
onto the column and washing the unbound .sup.18F away with water (3
mL) and then eluting the radiolabeled peptide with 2.times.200
.mu.L 1:1 EtOH water. The yield of the radiolabeled peptide after
HLB purification was 34.6%.
[0332] Effect of Ionic Strength
[0333] To lower the ionic strength of the reaction mixture
escalating amounts of acetonitrile were added to the labeling
mixture (final concentration: 0-80%). The yield of radiolabeled
IMP466 increased with increasing concentration of acetonitrile in
the medium. The optimal radiolabeling yield (98%) was obtained in a
final concentration of 80% acetonitrile, despite the increased
volume (500 .mu.L in 80% vs. 200 .mu.L in 0% acetonitrile). In 0%
acetonitrile the radiolabeling yield ranged from 36% to 55% in
three experiments.
Example 17. Imaging of Neuroendocrine Tumors with an .sup.18F- and
.sup.68Ga-Labeled IMP466
[0334] Studies were performed to compare the PET images obtained
using an .sup.18F versus .sup.68Ga-labeled somatostatin analogue
peptide and direct targeting to somatostatin receptor expressing
tumors.
[0335] Methods
[0336] .sup.18F Labeling--
[0337] IMP466 was synthesized and .sup.18F-labeled by a variation
of the method described in the Example above. A QMA SEPPAK.RTM.
light cartridge (Waters, Milford, Mass.) with 2-6 GBq .sup.18F (BV
Cyclotron VU, Amsterdam, The Netherlands) was washed with 3 mL
metal-free water. .sup.18F.sup.- was eluted from the cartridge with
0.4 M KHCO.sub.3 and fractions of 200 .mu.L were collected. The pH
of the fractions was adjusted to pH 4, with 10 .mu.L metal-free
glacial acid. Three .mu.L of 2 mM AlCl.sub.3 in 0.1 M sodium
acetate buffer, pH 4 were added. Then, 10-50 .mu.L IMP 466 (10
mg/mL) were added in 0.5 M sodium acetate, pH 4.1. The reaction
mixture was incubated at 100.degree. C. for 15 min unless stated
otherwise. The radiolabeled peptide was purified on RP-HPLC. The
Al.sup.18F(IMP466) containing fractions were collected and diluted
two-fold with H.sub.2O and purified on a 1-cc Oasis HLB cartridge
(Waters, Milford, Mass.) to remove acetonitrile and TFA. In brief,
the fraction was applied on the cartridge and the cartridge was
washed with 3 mL H.sub.2O. The radiolabeled peptide was then eluted
with 2.times.200 .mu.L 50% ethanol. For injection in mice, the
peptide was diluted with 0.9% NaCl. A maximum specific activity of
45,000 GBq/mmol was obtained.
[0338] .sup.68Ga Labeling--
[0339] IMP466 was labeled with .sup.68GaCl.sub.3 eluted from a
TiO.sub.2-based 1,110 MBq .sup.68Ge/.sup.68Ga generator (Cyclotron
Co. Ltd., Obninsk, Russia) using 0.1 M ultrapure HCl (J. T. Baker,
Deventer, The Netherlands). IMP466 was dissolved in 1.0 M HEPES
buffer, pH 7.0. Four volumes of .sup.68Ga eluate (120-240 MBq) were
added and the mixture was heated at 95.degree. C. for 20 min. Then
50 mM EDTA was added to a final concentration of 5 mM to complex
the non-incorporated .sup.68Ga.sup.3+. The .sup.68Ga-labeled IMP466
was purified on an Oasis HLB cartridge and eluted with 50%
ethanol.
[0340] Octanol-Water Partition Coefficient (Log
P.sub.oct/water)--
[0341] To determine the lipophilicity of the radiolabeled peptides,
approximately 50,000 dpm of the radiolabeled peptide was diluted in
0.5 mL phosphate-buffered saline (PBS). An equal volume of octanol
was added to obtain a binary phase system. After vortexing the
system for 2 min, the two layers were separated by centrifugation
(100.times.g, 5 min). Three 100 .mu.L samples were taken from each
layer and radioactivity was measured in a well-type gamma counter
(Wallac Wizard 3'', Perkin-Elmer, Waltham, Mass.).
[0342] Stability--
[0343] Ten .mu.L of the .sup.18F-labeled IMP466 was incubated in
500 .mu.L of freshly collected human serum and incubated for 4 h at
37.degree. C. Acetonitrile was added and the mixture was vortexed
followed by centrifugation at 1000.times.g for 5 min to precipitate
serum proteins. The supernatant was analyzed on RP-HPLC as
described above.
[0344] Cell Culture--
[0345] The AR42J rat pancreatic tumor cell line was cultured in
Dulbecco's Modified Eagle's Medium (DMEM) medium (Gibco Life
Technologies, Gaithersburg, Md., USA) supplemented with 4500 mg/L
D-glucose, 10% (v/v) fetal calf serum, 2 mmol/L glutamine, 100 U/mL
penicillin and 100 .mu.g/mL streptomycin. Cells were cultured at
37.degree. C. in a humidified atmosphere with 5% CO.sub.2.
[0346] IC.sub.50 Determination--
[0347] The apparent 50% inhibitory concentration (IC.sub.50) for
binding the somatostatin receptors on AR42J cells was determined in
a competitive binding assay using Al.sup.19F(IMP466),
.sup.69Ga(IMP466) or .sup.115In(DTPA-octreotide) to compete for the
binding of .sup.111In(DTPA-octreotide).
[0348] Al.sup.19F(IMP466) was formed by mixing an aluminium
fluoride (Al.sup.19F) solution (0.02 M AlCl.sub.3 in 0.5 M NaAc, pH
4, with 0.1 M NaF in 0.5 M NaAc, pH 4) with IMP466 and heating at
100.degree. C. for 15 min. The reaction mixture was purified by
RP-HPLC on a C-18 column as described above.
[0349] .sup.69Ga(IMP466) was prepared by dissolving gallium nitrate
(2.3.times.10.sup.-8 mol) in 30 .mu.L mixed with 20 .mu.L IMP466 (1
mg/mL) in 10 mM NaAc, pH 5.5, and heated at 90.degree. C. for 15
min. Samples of the mixture were used without further
purification.
[0350] .sup.115In(DTPA-octreotide) was made by mixing indium
chloride (1.times.10.sup.-5 mol) with 10 .mu.L DTPA-octreotide (1
mg/mL) in 50 mM NaAc, pH 5.5, and incubated at room temperature
(RT) for 15 min. This sample was used without further purification.
.sup.111In(DTPA-octreotide) (OCTREOSCAN.RTM.) was radiolabeled
according to the manufacturer's protocol.
[0351] AR42J cells were grown to confluency in 12-well plates and
washed twice with binding buffer (DMEM with 0.5% bovine serum
albumin). After 10 min incubation at RT in binding buffer,
Al.sup.19F(IMP466), .sup.69Ga(IMP466) or
.sup.115In(DTPA-octreotide) was added at a final concentration
ranging from 0.1-1000 nM, together with a trace amount (10,000 cpm)
of .sup.111In(DTPA-octreotide) (radiochemical purity>95%). After
incubation at RT for 3 h, the cells were washed twice with ice-cold
PBS. Cells were scraped and cell-associated radioactivity was
determined. Under these conditions, a limited extent of
internalization may occur. We therefore describe the results of
this competitive binding assay as "apparent IC.sub.50" values
rather than IC.sub.50. The apparent IC.sub.50 was defined as the
peptide concentration at which 50% of binding without competitor
was reached.
[0352] Biodistribution Studies--
[0353] Male nude BALB/c mice (6-8 weeks) were injected
subcutaneously in the right flank with 0.2 mL AR42J cell suspension
of 10.sup.7 cells/mL. Approximately two weeks after tumor cell
inoculation when tumors were 5-8 mm in diameter, 370 kBq .sup.18F
or .sup.68Ga-labeled IMP466 was administered intravenously (n=5).
Separate groups (n=5) were injected with a 1,000-fold molar excess
of unlabeled IMP466. One group of three mice was injected with
unchelated [Al.sup.18F]. All mice were killed by CO.sub.2/O.sub.2
asphyxiation 2 h post-injection (p.i.). Organs of interest were
collected, weighed and counted in a gamma counter. The percentage
of the injected dose per gram tissue (% ID/g) was calculated for
each tissue. The animal experiments were approved by the local
animal welfare committee and performed according to national
regulations.
[0354] PET/CT Imaging--
[0355] Mice with s.c. AR42J tumors were injected intravenously with
10 MBq Al.sup.18F(IMP466) or .sup.68Ga(IMP466). One and two hours
after the injection of peptide, mice were scanned on an Inveon
animal PET/CT scanner (Siemens Preclinical Solutions, Knoxville,
Tenn.) with an intrinsic spatial resolution of 1.5 mm (Visser et
al, JNM, 2009). The animals were placed in a supine position in the
scanner. PET emission scans were acquired over 15 minutes, followed
by a CT scan for anatomical reference (spatial resolution 113
.mu.m, 80 kV, 500 .mu.A). Scans were reconstructed using Inveon
Acquisition Workplace software version 1.2 (Siemens Preclinical
Solutions, Knoxville, Tenn.) using an ordered set expectation
maximization-3D/maximum a posteriori (OSEM3D/MAP) algorithm with
the following parameters: matrix 256.times.256.times.159, pixel
size 0.43.times.0.43.times.0.8 mm.sup.3 and MAP prior of 0.5
mm.
[0356] Results
[0357] Effect of Buffer--
[0358] The effect of the buffer on the labeling efficiency of
IMP466 was investigated. IMP466 was dissolved in sodium citrate
buffer, sodium acetate buffer, 2-(N-morpholino)ethanesulfonic acid
(MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
buffer at 10 mg/mL (7.7 mM). The molarity of all buffers was 1 M
and the pH was 4.1. To 200 .mu.g (153 nmol) of IMP466 was added 100
.mu.L [Al.sup.18F] (pH 4) and incubated at 100.degree. C. for 15
min. Radiolabeling yield and specific activity was determined with
RP-HPLC. When using sodium acetate, MES or HEPES, radiolabeling
yield was 49%, 44% and 46%, respectively. In the presence of sodium
citrate, no labeling was observed (<1%). When the labeling
reaction was carried out in sodium acetate buffer, the specific
activity of the preparations was 10,000 GBq/mmol, whereas in MES
and HEPES buffer a specific activity of 20,500 and 16,500 GBq/mmol
was obtained, respectively.
[0359] Effect of AlCl.sub.3 Concentration--
[0360] Three stock solutions of AlCl.sub.3 in sodium acetate, pH
4.1 were prepared: 0.2, 2.0 and 20 mM. From these solutions, 3
.mu.L was added to 200 .mu.L of .sup.18F.sup.- to form
[Al.sup.18F]. To these samples, 153 nmol of peptide was added and
incubated for 15 min at 100.degree. C. Radiolabeling yield was 49%
after incubation at a final concentration of 6 nmol AlCl.sub.3.
Incubation with 0.6 nmol AlCl.sub.3 and 60 nmol AlCl.sub.3 resulted
in a strong reduction of the radiolabeling yield: 10% and 6%,
respectively.
[0361] Effect of Amount of Peptide--
[0362] The effect of the amount of peptide on the labeling
efficiency was investigated. IMP466 was dissolved in sodium acetate
buffer, pH 4.1 at a concentration of 7.7 mM (10 mg/mL) and 38, 153
or 363 nmol of IMP466 was added to 200 .mu.L [Al.sup.18F] (581-603
MBq). The radiolabeling yield increased with increasing amounts of
peptide. At 38 nmol, radiolabeling yield ranged from 4-8%, at 153
nmol, the yield had increased to 42-49% and at the highest
concentration the radiolabeling yield was 48-52%.
[0363] Octanol-Water Partition Coefficient--
[0364] To determine the lipophilicity of the .sup.18F and
.sup.68Ga-labeled IMP466, the octanol-water partition coefficients
were determined. The log P.sub.octanol/water value for the
Al.sup.18F(IMP466) was -2.44.+-.0.12 and that of .sup.68Ga(IMP466)
was -3.79.+-.0.07, indicating that the .sup.18F-labeled IMP 466 was
slightly less hydrophilic.
[0365] Stability--
[0366] The .sup.18F-labeled IMP466 did not show release of .sup.BF
after incubation in human serum at 37.degree. C. for 4 h,
indicating excellent stability of the Al[.sup.18F]NOTA complex.
[0367] IC.sub.50 Determination--
[0368] The apparent IC.sub.50 of Al.sup.19F(IMP466) was 3.6.+-.0.6
nM, whereas that for .sup.69Ga(IMP466) was 13.+-.3 nM. The apparent
IC.sub.50 of the reference peptide, .sup.115In(DTPA-octeotride)
(OCTREOSCAN.RTM.), was 6.3.+-.0.9 nM.
[0369] Biodistribution Studies--
[0370] The biodistribution of both Al.sup.18F(IMP466) and
.sup.68Ga(IMP466) was studied in nude BALB/c mice with s.c. AR42J
tumors at 2 h p.i. (FIG. 4). Al.sup.18F was included as a control.
Tumor targeting of the Al.sup.18F(IMP466) was high with
28.3.+-.5.7% ID/g accumulated at 2 h p.i. Uptake in the presence of
an excess of unlabeled IMP466 was 8.6.+-.0.7% ID/g, indicating that
tumor uptake was receptor-mediated. Blood levels were very low
(0.10.+-.0.07% ID/g, 2 h pi), resulting in a tumor-to-blood ratio
of 299.+-.88. Uptake in the organs was low, with specific uptake in
receptor expressing organs such as adrenal glands, pancreas and
stomach. Bone uptake of Al.sup.18F(IMP466) was negligible as
compared to uptake of non-chelated Al.sup.18F (0.33.+-.0.07 vs.
36.9.+-.5.0% ID/g at 2 h p.i., respectively), indicating good in
vivo stability of the .sup.18F-labeled NOTA-peptide.
[0371] The biodistribution of Al.sup.18F(IMP466) was compared to
that of .sup.68Ga(IMP466) (FIG. 4). Tumor uptake of
.sup.68Ga(IMP466) (29.2.+-.0.5% ID/g, 2 h pi) was similar to that
of Al.sup.18F-IMP 466 (p<0.001). Lung uptake of
.sup.68Ga(IMP466) was two-fold higher than that of
Al.sup.18F(IMP466) (4.0.+-.0.9% ID/g vs. 1.9.+-.0.4% ID/g,
respectively). In addition, kidney retention of .sup.68Ga(IMP466)
was slightly higher than that of Al.sup.18F(IMP466) (16.2.+-.2.86%
ID/g vs. 9.96.+-.1.27% ID/g, respectively.
[0372] Fused PET and CT scans are shown in FIG. 5. PET scans
corroborated the biodistribution data. Both Al.sup.18F(IMP466) and
.sup.68Ga(IMP466) showed high uptake in the tumor and retention in
the kidneys. The activity in the kidneys was mainly localized in
the renal cortex. Again, the [Al.sup.18F] proved to be stably
chelated by the NOTA chelator, since no bone uptake was
observed.
[0373] FIG. 5 clearly shows that the distribution of an
.sup.18F-labeled analog of somatostatin (octreotide) mimics that of
a .sup.68Ga-labeled somatostatin analog. These results are
significant, since .sup.68Ga-labeled octreotide PET imaging in
human subjects with neuroendocrine tumors has been shown to have a
significantly higher detection rate compared with conventional
somatostatin receptor scintigraphy and diagnostic CT, with a
sensitivity of 97%, a specificity of 92% and an accuracy of 96%
(e.g., Gabriel et al., 2007, J Nucl Med 48:508-18). PET imaging
with .sup.68Ga-labeled octreotide is reported to be superior to
SPECT analysis with .sup.111In-labeled octreotide and to be highly
sensitive for detection of even small meningiomas (Henze et al.,
2001, J Nucl Med 42:1053-56). Because of the higher energy of
.sup.68Ga compared with .sup.18F, it is expected that .sup.18F
based PET imaging would show even better spatial resolution than
.sup.68Ga based PET imaging. This is illustrated in FIG. 5 by
comparing the kidney images obtained with .sup.18F-labeled IMP466
(FIG. 5, left) vs. .sup.68Ga-labeled IMP466 (FIG. 5, right). The
PET images obtained with .sup.68Ga show more diffuse margins and
lower resolution than the images obtained with .sup.18F. These
results demonstrate the superior images obtained with
.sup.18F-labeled targeting moieties prepared using the methods and
compositions described herein and confirm the utility of the
described .sup.18F-labeling techniques for non-antibody targeting
peptides.
Example 18. Comparison of .sup.68Ga and .sup.18F PET Imaging Using
Pretargeting
[0374] We compared PET images obtained using .sup.68Ga- or
.sup.18F-labeled peptides that were pretargeted with the bispecific
TF2 antibody, prepared as described above. The half-lives of
.sup.68Ga (t.sub.1/2=68 minutes) and .sup.18F (t.sub.1/2=110
minutes) match with the pharmacokinetics of the radiolabeled
peptide, since its maximum accretion in the tumor is reached within
hours. Moreover, .sup.68Ga is readily available from
.sup.68Ge/.sup.68Ga generators, whereas .sup.18F is the most
commonly used and widely available radionuclide in PET.
[0375] Methods
[0376] Mice with s.c. CEA-expressing LS174T tumors received TF2
(6.0 nmol; 0.94 mg) and 5 MBq .sup.68Ga(IMP288) (0.25 nmol) or
Al.sup.18F(IMP449) (0.25 nmol) intravenously, with an interval of
16 hours between the injection of the bispecific antibody and the
radiolabeled peptide. One or two hours after the injection of the
radiolabeled peptide, PET/CT images were acquired and the
biodistribution of the radiolabeled peptide was determined. Uptake
in the LS174T tumor was compared with that in an s.c. CEA-negative
SK-RC 52 tumor. Pretargeted immunoPET imaging was compared with
.sup.18F-FDG PET imaging in mice with an s.c. LS174T tumor and
contralaterally an inflamed thigh muscle.
TABLE-US-00020 (SEQ ID NO: 51) IMP288
DOTA-D-Tyr-D-Lys(HSG)-D-G1u-D-Lys(HSG)-NH2
[0377] Pretargeting--
[0378] The bispecific TF2 antibody was made by the DNL method, as
described above. TF2 is a trivalent bispecific antibody comprising
an HSG-binding Fab fragment from the h679 antibody and two
CEA-binding Fab fragments from the hMN-14 antibody. The
DOTA-conjugated, HSG-containing peptide IMP288 was synthesized by
peptide synthesis as described above. The IMP449 peptide,
synthesized as described above, contains a
1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelating
moiety to facilitate labeling with .sup.18F. As a tracer for the
antibody component, TF2 was labeled with .sup.125I (Perkin Elmer,
Waltham, Mass.) by the iodogen method (Fraker and Speck, 1978,
Biochem Biophys Res Comm 80:849-57), to a specific activity of 58
MBq/nmol.
[0379] Labeling of IMP288--
[0380] IMP288 was labeled with .sup.111In (Covidien, Petten, The
Netherlands) for biodistribution studies at a specific activity of
32 MBq/nmol under strict metal-free conditions. IMP288 was labeled
with .sup.68Ga eluted from a TiO-based 1,110 MBq.sup.68Ge/.sup.68Ga
generator (Cyclotron Co. Ltd., Obninsk Russia) using 0.1 M
ultrapure HCl. Five 1 ml fractions were collected and the second
fraction was used for labeling the peptide. One volume of 1.0 M
HEPES buffer, pH 7.0 was added to 3.4 nmol IMP 288. Four volumes of
.sup.68Ga eluate (380 MBq) were added and the mixture was heated to
95.degree. C. for 20 min. Then 50 mM EDTA was added to a final
concentration of 5 mM to complex the non-chelated .sup.68Ga.sup.3+.
The .sup.68Ga(IMP288) peptide was purified on a 1-mL Oasis
HLB-cartridge (Waters, Milford, Mass.). After washing the cartridge
with water, the peptide was eluted with 25% ethanol. The procedure
to label IMP288 with .sup.68Ga was performed within 45 minutes,
with the preparations being ready for in vivo use.
[0381] Labeling of IMP449--
[0382] IMP449 was labeled with .sup.18F as described above. 555-740
MBq .sup.18F (B.V. Cyclotron VU, Amsterdam, The Netherlands) was
eluted from a QMA cartridge with 0.4 M KHCO.sub.3. The Al.sup.18F
activity was added to a vial containing the peptide (230 .mu.g) and
ascorbic acid (10 mg). The mixture was incubated at 100.degree. C.
for 15 min. The reaction mixture was purified by RP-HPLC. After
adding one volume of water, the peptide was purified on a 1-mL
Oasis HLB cartridge. After washing with water, the radiolabeled
peptide was eluted with 50% ethanol. Al.sup.18F(IMP449) was
prepared within 60 minutes, with the preparations being ready for
in vivo use.
[0383] Radiochemical purity of .sup.125I-TF2, .sup.111In(IMP288)
and .sup.68Ga(IMP288) and Al.sup.18F(IMP449) preparations used in
the studies always exceeded 95%.
[0384] Animal Experiments--
[0385] Experiments were performed in male nude BALB/c mice (6-8
weeks old), weighing 20-25 grams. Mice received a subcutaneous
injection with 0.2 mL of a suspension of 1.times.10.sup.6
LS174T-cells, a CEA-expressing human colon carcinoma cell line
(American Type Culture Collection, Rockville, Md., USA). Studies
were initiated when the tumors reached a size of about 0.1-0.3 g
(10-14 days after tumor inoculation).
[0386] The interval between TF2 and IMP288 injection was 16 hours,
as this period was sufficient to clear unbound TF2 from the
circulation. In some studies .sup.125I-TF2, (0.4 MBq) was
co-injected with unlabeled TF2. IMP288 was labeled with either
.sup.111In or .sup.68Ga. IMP449 was labeled with .sup.18F. Mice
received TF2 and IMP288 intravenously (0.2 mL). One hour after the
injection of .sup.68Ga-labeled peptide, and two hours after
injection of Al.sup.18F(IMP449), mice were euthanized by
CO.sub.2/O.sub.2, and blood was obtained by cardiac puncture and
tissues were dissected.
[0387] PET images were acquired with an Inveon animal PET/CT
scanner (Siemens Preclinical Solutions, Knoxville, Tenn.). PET
emission scans were acquired for 15 minutes, preceded by CT scans
for anatomical reference (spatial resolution 113 .mu.m, 80 kV, 500
.mu.A, exposure time 300 msec).
[0388] After imaging, tumor and organs of interest were dissected,
weighed and counted in a gamma counter with appropriate energy
windows for .sup.125I, .sup.111In, .sup.68Ga or .sup.18F. The
percentage-injected dose per gram tissue (% ID/g) was
calculated.
[0389] Results
[0390] Within 1 hour, pretargeted immunoPET resulted in high and
specific targeting of .sup.68Ga-IMP288 in the tumor (10.7.+-.3.6%
ID/g), with very low uptake in the normal tissues (e.g.,
tumor/blood 69.9.+-.32.3), in a CEA-negative tumor (0.35.+-.0.35%
ID/g), and inflamed muscle (0.72.+-.0.20% ID/g). Tumors that were
not pretargeted with TF2 also had low .sup.68Ga(IMP288) uptake
(0.20.+-.0.03% ID/g). [.sup.18F]FDG accreted efficiently in the
tumor (7.42.+-.0.20% ID/g), but also in the inflamed muscle
(4.07.+-.1.13% ID/g) and a number of normal tissues, and thus
pretargeted .sup.68Ga-IMP 288 provided better specificity and
sensitivity. The corresponding PET/CT images of mice that received
.sup.68Ga(IMP288) or Al.sup.18F(IMP449) following pretargeting with
TF2 clearly showed the efficient targeting of the radiolabeled
peptide in the subcutaneous LS174T tumor, while the inflamed muscle
was not visualized. In contrast, with .sup.18F-FDG the tumor as
well as the inflammation was clearly delineated.
[0391] Dose Optimization--
[0392] The effect of the TF2 bsMAb dose on tumor targeting of a
fixed 0.01 nmol (15 ng) dose of IMP288 was determined. Groups of
five mice were injected intravenously with 0.10, 0.25, 0.50 or 1.0
nmol TF2 (16, 40, 80 or 160 .mu.g respectively), labeled with a
trace amount of .sup.125I (0.4 MBq). One hour after injection of
.sup.111In(IMP288) (0.01 nmol, 0.4 MBq), the biodistribution of the
radiolabels was determined.
[0393] TF2 cleared rapidly from the blood and the normal tissues.
Eighteen hours after injection the blood concentration was less
than 0.45% ID/g at all TF2 doses tested. Targeting of TF2 in the
tumor was 3.5% ID/g at 2 h p.i. and independent of TF2 dose (data
not shown). At all TF2 doses .sup.111In(IMP288) accumulated
effectively in the tumor (not shown). At higher TF2 doses enhanced
uptake of .sup.111In(IMP288) in the tumor was observed: at 1.0 nmol
TF2 dose maximum targeting of IMP288 was reached (26.2.+-.3.8%
ID/g). Thus at the 0.01 nmol peptide dose highest tumor targeting
and tumor-to-blood ratios were reached at the highest TF2 dose of
1.0 nmol (TF2:IMP288 molar ratio=100:1). Among the normal tissues,
the kidneys had the highest uptake of .sup.111In(IMP288)
(1.75.+-.0.27% ID/g) and uptake in the kidneys was not affected by
the TF2 dose (not shown). All other normal tissues had very low
uptake, resulting in extremely high tumor-to-nontumor ratios,
exceeding 50:1 at all TF2 doses tested (not shown).
[0394] For PET imaging using .sup.68Ga-labeled IMP288, a higher
peptide dose is required, because a minimum activity of 5-10
MBq.sup.68Ga needs to be injected per mouse if PET imaging is
performed 1 h after injection. The specific activity of the
.sup.68Ga(IMP288) preparations was 50-125 MBq/nmol at the time of
injection. Therefore, for PET imaging at least 0.1-0.25 nmol of
IMP288 had to be administered. The same TF2:IMP288 molar ratios
were tested at 0.1 nmol IMP288 dose. LS174T tumors were pretargeted
by injecting 1.0, 2.5, 5.0 or 10.0 nmol TF2 (160, 400, 800 or 1600
.mu.g). In contrast to the results at the lower peptide dose,
.sup.111In(IMP288) uptake in the tumor was not affected by the TF2
doses (15% ID/g at all doses tested, data not shown). TF2 targeting
in the tumor in terms of % ID/g decreased at higher doses
(3.21.+-.0.61% ID/g versus 1.16.+-.0.27% ID/g at an injected dose
of 1.0 nmol and 10.0 nmol, respectively) (data not shown). Kidney
uptake was also independent of the bsMAb dose (2% ID/g). Based on
these data we selected a bsMAb dose of 6.0 nmol for targeting
0.1-0.25 nmol of IMP288 to the tumor.
[0395] Pet Imaging--
[0396] To demonstrate the effectiveness of pretargeted immunoPET
imaging with TF2 and .sup.68Ga(IMP288) to image CEA-expressing
tumors, subcutaneous tumors were induced in five mice. In the right
flank an s.c. LS174T tumor was induced, while at the same time in
the same mice 1.times.10.sup.6 SK-RC 52 cells were inoculated in
the left flank to induce a CEA-negative tumor. Fourteen days later,
when tumors had a size of 0.1-0.2 g, the mice were pretargeted with
6.0 nmol .sup.125I-TF2 intravenously. After 16 hours the mice
received 5 MBq .sup.68Ga(IMP288) (0.25 nmol, specific activity of
20 MBq/nmol). A separate group of three mice received the same
amount of .sup.68Ga-IMP 288 alone, without pretargeting with TF2.
PET/CT scans of the mice were acquired 1 h after injection of the
.sup.68Ga(IMP288).
[0397] The biodistribution of .sup.125I-TF2 and [.sup.68Ga]IMP288
in the mice are shown in FIG. 6. Again high uptake of the bsMAb
(2.17.+-.0.50% ID/g) and peptide (10.7.+-.3.6% ID/g) in the tumor
was observed, with very low uptake in the normal tissues
(tumor-to-blood ratio: 64.+-.22). Targeting of .sup.68Ga(IMP288) in
the CEA-negative tumor SK-RC 52 was very low (0.35.+-.0.35% ID/g).
Likewise, tumors that were not pretargeted with TF2 had low uptake
of .sup.68Ga(IMP288) (0.20.+-.0.03% ID/g), indicating the specific
accumulation of IMP288 in the CEA-expressing LS174T tumor.
[0398] The specific uptake of .sup.68Ga(IMP288) in the
CEA-expressing tumor pretargeted with TF2 was clearly visualized in
a PET image acquired 1 h after injection of the .sup.68Ga-labeled
peptide (not shown). Uptake in the tumor was evaluated
quantitatively by drawing regions of interest (ROI), using a 50%
threshold of maximum intensity. A region in the abdomen was used as
background region. The tumor-to-background ratio in the image of
the mouse that received TF2 and .sup.68Ga(IMP288) was 38.2.
[0399] We then examined pretargeted immunoPET with .sup.18F-FDG. In
two groups of five mice a s.c. LS174T tumor was induced on the
right hind leg and an inflammatory focus in the left thigh muscle
was induced by intramuscular injection of 50 .mu.L turpentine (18).
Three days after injection of the turpentine, one group of mice
received 6.0 nmol TF2, followed 16 h later by 5 MBq
.sup.68Ga(IMP288) (0.25 nmol). The other group received
.sup.18F-FDG (5 MBq). Mice were fasted during 10 hours prior to the
injection and anaesthetized and kept warm at 37.degree. C. until
euthanasia, 1 h postinjection.
[0400] Uptake of .sup.68Ga(IMP288) in the inflamed muscle was very
low, while uptake in the tumor in the same animal was high
(0.72.+-.0.20% ID/g versus 8.73.+-.1.60% ID/g, p<0.05, FIG. 7).
Uptake in the inflamed muscle was in the same range as uptake in
the lungs, liver and spleen (0.50.+-.0.14% ID/g, 0.72.+-.0.07%
ID/g, 0.44.+-.0.10% ID/g, respectively). Tumor-to-blood ratio of
.sup.68Ga(IMP288) in these mice was 69.9.+-.32.3; inflamed
muscle-to-blood ratio was 5.9.+-.2.9; tumor-to-inflamed muscle
ratio was 12.5.+-.2.1. In the other group of mice .sup.18F-FDG
accreted efficiently in the tumor (7.42.+-.0.20% ID/g,
tumor-to-blood ratio 6.24.+-.1.5, FIG. 4). .sup.18F-FDG also
substantially accumulated in the inflamed muscle (4.07.+-.1.13%
ID/g), with inflamed muscle-to-blood ratio of 3.4.+-.0.5, and
tumor-to-inflamed muscle ratio of 1.97.+-.0.71.
[0401] The corresponding PET/CT image of a mouse that received
.sup.68Ga(IMP288), following pretargeting with TF2, clearly showed
the efficient accretion of the radiolabeled peptide in the tumor,
while the inflamed muscle was not visualized (FIG. 8). In contrast,
on the images of the mice that received .sup.18F-FDG, the tumor as
well as the inflammation was visible (FIG. 8). In the mice that
received .sup.68Ga(IMP288), the tumor-to-inflamed tissue ratio was
5.4; tumor-to-background ratio was 48; inflamed
muscle-to-background ratio was 8.9. .sup.18F-FDG uptake had a
tumor-to-inflamed muscle ratio of 0.83; tumor-to-background ratio
was 2.4; inflamed muscle-to-background ratio was 2.9.
[0402] The pretargeted immunoPET imaging method was tested using
the Al.sup.18F(IMP449). Five mice received 6.0 nmol TF2, followed
16 h later by 5 MBq Al[.sup.18F]IMP449 (0.25 nmol). Three
additional mice received 5 MBq Al.sup.18F(IMP449) without prior
administration of TF2, while two control mice were injected with
[Al.sup.18F] (3 MBq). The results of this experiment are summarized
in FIG. 9. Uptake of Al.sup.18F(IMP449) in tumors pretargeted with
TF2 was high (10.6.+-.1.7% ID/g), whereas it was very low in the
non-pretargeted mice (0.45.+-.0.38% ID/g). [Al.sup.18F] accumulated
in the bone (50.9.+-.11.4% ID/g), while uptake of the radiolabeled
IMP449 peptide in the bone was very low (0.54.+-.0.2% ID/g),
indicating that the Al.sup.18F(IMP449) was stable in vivo. The
biodistribution of Al.sup.18F(IMP449) in the TF2 pretargeted mice
with s.c. LS174T tumors were highly similar to that of
.sup.68Ga(IMP288).
[0403] The PET-images of pretargeted immunoPET with
Al.sup.18F(IMP449) show the same intensity in the tumor as those
with .sup.68Ga(IMP288), but the resolution of the .sup.18F PET
images were superior to those of the .sup.68Ga. (FIG. 10). The
tumor-to-background ratio of the Al.sup.18F(IMP449) signal was
66.
[0404] Conclusions
[0405] The present study showed that pretargeted immunoPET with the
anti-CEA.times.anti-HSG bispecific antibody TF2 in combination with
a .sup.68Ga- or .sup.18F-labeled di-HSG-DOTA-peptide is a rapid and
specific technique for PET imaging of CEA-expressing tumors.
[0406] Pretargeted immunoPET with TF2 in combination with
.sup.68Ga(IMP288) or Al.sup.18F(IMP449) involves two intravenous
administrations. An interval between the infusion of the bsMAb and
the radiolabeled peptide of 16 h was used. After 16 h most of the
TF2 had cleared from the blood (blood concentration <1% ID/g),
preventing complexation of TF2 and IMP288 in the circulation.
[0407] For these studies the procedure to label IMP288 with
.sup.68Ga was optimized, resulting in a one-step labeling
technique. We found that purification on a C18/HLB cartridge was
needed to remove the .sup.68Ga colloid that is formed when the
peptide was labeled at specific activities exceeding 150 GBq/nmol
at 95.degree. C. If a preparation contains a small percentage of
colloid and is administered intravenously, the .sup.68Ga colloid
accumulates in tissues of the mononuclear phagocyte system (liver,
spleen, and bone marrow), deteriorating image quality. The
.sup.68Ga-labeled peptide could be rapidly purified on a
C18-cartridge. Radiolabeling and purification for administration
could be accomplished within 45 minutes.
[0408] The half-life of .sup.68Ga matches with the kinetics of the
IMP288 peptide in the pretargeting system: maximum accretion in the
tumor is reached within 1 h. .sup.68Ga can be eluted twice a day
form a .sup.68Ge/.sup.68Ga generator, avoiding the need for an
on-site cyclotron. However, the high energy of the positrons
emitted by .sup.68Ga (1.9 MeV) limits the spatial resolution of the
acquired images to 3 mm, while the intrinsic resolution of the
microPET system is as low as 1.5 mm.
[0409] .sup.18F, the most widely used radionuclide in PET, has an
even more favorable half-life for pretargeted PET imaging
(t.sub.1/2=110 min). The NOTA-conjugated peptide IMP449 was labeled
with .sup.18F, as described above. Like labeling with .sup.68Ga, it
is a one-step procedure. Labeling yields as high as 50% were
obtained. The biodistribution of Al.sup.18F(IMP449) was highly
similar to that of .sup.68Ga-labeled IMP288, suggesting that with
this labeling method .sup.18F is a residualizing radionuclide.
[0410] In contrast with FDG-PET, pretargeted radioimmunodetection
is a tumor specific imaging modality. Although a high sensitivity
and specificity for FDG-PET in detecting recurrent colorectal
cancer lesions has been reported in patients (Huebner et al., 2000,
J Nucl Med 41:11277-89), FDG-PET images could lead to diagnostic
dilemmas in discriminating malignant from benign lesions, as
indicated by the high level of labeling observed with inflammation.
In contrast, the high tumor-to-background ratio and clear
visualization of CEA-positive tumors using pretargeted immunoPET
with TF2 .sup.68Ga- or .sup.18F-labeled peptides supports the use
of the described methods for clinical imaging of cancer and other
conditions. Apart from detecting metastases and discriminating
CEA-positive tumors from other lesions, pretargeted immunoPET could
also be used to estimate radiation dose delivery to tumor and
normal tissues prior to pretargeted radioimmunotherapy. As TF2 is a
humanized antibody, it has a low immunogenicity, opening the way
for multiple imaging or treatment cycles.
Example 19. Synthesis of Folic Acid NOTA conjugate
[0411] Folic acid is activated as described (Wang et. al.
Bioconjugate Chem. 1996, 7, 56-62.) and conjugated to
Boc-NH--CH.sub.2--CH.sub.2--NH.sub.2. The conjugate is purified by
chromatography. The Boc group is then removed by treatment with
TFA. The amino folate derivative is then mixed with p-SCN-Bn-NOTA
(Macrocyclics) in a carbonate buffer. The product is then purified
by HPLC. The folate-NOTA derivative is labeled with Al.sup.18F as
described in the preceding Examples and then HPLC purified. The
.sup.18F-labeled folate is injected i.v. into a subject and
successfully used to image the distribution of folate receptors,
for example in cancer or inflammatory diseases (see, e.g., Ke et
al., Advanced Drug Delivery Reviews, 56:1143-60, 2004).
Example 20. Pretargeted PET Imaging in Humans
[0412] A patient (1.7 m.sup.2 body surface area) with a suspected
recurrent tumor is injected with 17 mg of bispecific monoclonal
antibody (bsMab). The bsMab is allowed to localize to the target
and clear from the blood. The .sup.18F-labeled peptide (5-10 mCi on
5.7.times.10.sup.-9 mol) is injected when 99% of the bsMab has
cleared from the blood. PET imaging shows the presence of
micrometastatic tumors.
Example 21. Imaging of Angiogenesis Receptors by
.sup.18F-Labeling
[0413] Labeled Arg-Gly-Asp (RGD) peptides have been used for
imaging of angiogenesis, for example in ischemic tissues, where
.alpha..sub.v.beta..sub.3 integrin is involved. (Jeong et al., J.
Nucl. Med. 2008, Apr. 15 epub). RGD is conjugated to SCN-Bn-NOTA
according to Jeong et al. (2008). [Al.sup.18F] is attached to the
NOTA-derivatized RGD peptide as described above, by mixing aluminum
stock solution with .sup.18F and the derivatized RGD peptide and
heating at 110.degree. C. for 15 min, using an excess of peptide to
drive the labeling reaction towards completion. The .sup.18F
labeled RGD peptide is used for in vivo biodistribution and PET
imaging as disclosed in Jeong et al. (2008). The [Al.sup.18F]
conjugate of RGD-NOTA is taken up into ischemic tissues and
provides PET imaging of angiogenesis.
Example 22. Carbohydrate Labeling
[0414] A NOTA thiosemicarbazide derivative is prepared by reacting
the p-SCN-Bn-NOTA with hydrazine and then purifying the ligand by
HPLC. [Al.sup.18F] is prepared as described in the preceding
Examples and the [Al.sup.18F] is added to the NOTA
thiosemicarbazide and heated for 15 min. Optionally the
Al.sup.18F(NOTA thiosemicarbazide) complex is purified by HPLC. The
Al.sup.18F(NOTA thiosemicarbazide) is conjugated to oxidized
carbohydrates by known methods. The .sup.18F-labeled carbohydrate
is successfully used for imaging studies using PET scanning.
Example 23. Effect of Organic Solvents on F-18 Labeling
[0415] The affinity of chelating moieties such as NETA and NOTA for
aluminum is much higher than the affinity of aluminum for .sup.18F.
The affinity of Al for .sup.18F is affected by factors such as the
ionic strength of the solution, since the presence of other
counter-ions tends to shield the positively charged aluminum and
negatively charged fluoride ions from each other and therefore to
decrease the strength of ionic binding. Therefore low ionic
strength medium should increase the effective binding of Al and
.sup.18F.
[0416] An initial study adding ethanol to the .sup.18F reaction was
found to increase the yield of radiolabeled peptide. IMP461 was
prepared as described above.
TABLE-US-00021 TABLE 9 .sup.18F-labeling of IMP461 in ethanol # 2
mM AlCl.sub.3 .sup.18F.sup.- 2 mM IMP 461 Solvent Yield* 1 10 .mu.L
741 .mu.Ci 20 .mu.L EtOH 60 .mu.L 64.9% 2 10 .mu.L 739 .mu.Ci 20
.mu.L H.sub.2O 60 .mu.L 21.4% 3 10 .mu.L 747 .mu.Ci 20 .mu.L EtOH
60 .mu.L 46.7% 4 5 .mu.L 947 .mu.Ci 10 .mu.L EtOH 60 .mu.L 43.2%
*Yield after HLB column purification, Rxn # 1, 2 and 4 were heated
to 101.degree. C. for 5 minutes, Rxn # 3 was heated for 1 minute in
a microwave oven.
[0417] Preliminary results showed that addition of ethanol to the
reaction mixture more than doubled the yield of .sup.18F-labeled
peptide. Table 9 also shows that microwave irradiation can be used
in place of heating to promote incorporation of [Al.sup.18F] into
the chelating moiety of IMP461. Sixty seconds of microwave
radiation (#3) appeared to be slightly less (18%) effective than
heating to 101.degree. C. for 5 minutes (#1).
[0418] The effect of additional solvents on Al.sup.19F complexation
of peptides was examined. In each case, the concentration of
reactants was the same and only the solvent varied. Reaction
conditions included mixing 25 .mu.L Na.sup.19F+20 .mu.L
AlCl.sub.3+20 .mu.L IMP461+60 .mu.L solvent, followed by heating at
101.degree. C. for 5 min. Table 10 shows that the presence of a
solvent does improve the yields of Al.sup.19F(IMP461) (i.e.,
IMP473) considerably.
TABLE-US-00022 TABLE 10 Complexation of IMP 461 with Al.sup.19F in
various solvents Solvent H.sub.2O MeOH EtOH CH.sub.3CN Al-IMP461
2.97 3.03 2.13 1.54 IMP465 52.46 34.19 31.58 24.58 IMP473 14.99
30.96 33.00 37.48 IMP473 15.96 31.81 33.29 36.40 IMP461 13.63 -- --
-- Solvent IPA Acetone THF Dioxane Al-IMP461 2.02 2.05 2.20 16.67
IMP465 32.11 28.47 34.76 10.35 IMP473 27.31 34.35 29.38 27.09
IMP473 27.97 35.13 29.28 11.62 IMP461 10.58 -- 4.37 34.27 Solvent
DMF DMSO t.sub.R (min) Al-IMP461 -- -- 9.739 IMP465 19.97 37.03
10.138 IMP473 27.77 31.67 11.729 IMP473 27.34 31.29 11.952 IMP461
-- -- 12.535 Al[.sup.19F]IMP461 = IMP473
Example 24. Elution of "F" with Bicarbonate
[0419] .sup.18F, 10.43 mCi, was received in 2 mL in a syringe. The
solution was passed through a SEP-PAK.RTM. Light, WATERS.RTM.
ACCELL.TM. Plus QMA Cartridge. The column was then washed with 5 mL
of DI water. The .sup.18F was eluted with 0.4 M KHCO.sub.3 in
fractions as shown in Table 11 below.
TABLE-US-00023 TABLE 11 Elution of QMA Cartridge with KHCO.sub.3
Vol. Acetic Vol. 0.4M Activity Vial acid .mu.L KHCO.sub.3 .mu.L mCi
1 7.5 150 0.0208 2 10 200 7.06 3 5 100 1.653 4 25 500 0.548
[0420] The effects of the amount of additional solvent (CH.sub.3CN)
on .sup.18F-labeling of IMP461 was examined. In each case, the
concentration of reactants was the same and only the amount of
solvent varied. Reaction conditions included mixing 10 .mu.L
AlCl.sub.3+20 .mu.L .sup.18F+20 .mu.L IMP461+CH.sub.3CN followed by
heating at 101.degree. C. for 5 min. Table 12 shows that following
an initial improvement the labeling efficiency decreases in the
presence of excess solvent.
TABLE-US-00024 TABLE 12 .sup.18F-labeling of IMP461 using varying
amounts of CH.sub.3CN RCY CH.sub.3CN (.mu.L) .sup.18F.sup.- mCi
t.sub.R 2.70 min (%) t.sub.R 8.70 min (%) % (HLB) 0 0.642 13.48
86.52 50.7 100 0.645 1.55 98.45 81.8* 200 0.642 2.85 97.15 80.8 400
0.645 14.51 85.49 57.8 *Aqueous wash contains labeled peptide. RCY
= radiochemical yield after HLB purification
Example 25. High Dose Radiolabeling of IMP461
[0421] .sup.18F, 163 mCi, was received in 2 mL in a syringe. The
solution was passed through a SEP-PAK.RTM. Light, WATERS.RTM.
ACCELL.TM. Plus QMA Cartridge. The column was then washed with 5 mL
of DI water. The .sup.18F.sup.- was eluted with 0.4 M
K.sub.2CO.sub.3 in fractions as shown in Table 13.
TABLE-US-00025 TABLE 13 High Dose Labeling Vial Vol. Acetic acid
.mu.L Vol. 0.4M K.sub.2CO.sub.3 .mu.L Activity mCi 1 18.5 185 5.59
2 5 50 35.8 3 5 50 59.9 4 5 50 20.5 5 5 50 5.58 6 50 500 4.21
[0422] An aluminum chloride solution (10 .mu.L, 2 mM in pH 4, 2 mM
NaOAc) was added to vial number 3 from Table 13. The peptide (20
.mu.L, 2 mM in pH 4, 2 mM NaOAc) was added to the vial followed by
the addition of 170 .mu.I, of CH.sub.3CN. The solution was heated
for 10 min at 103.degree. C. the diluted with 6 mL of water. The
solution was pulled into a 10 mL syringe and injected onto two
WATERS.RTM. HLB Plus Cartridges arranged in tandem. The cartridges
were washed with 8 mL water. The radiolabeled peptide
Al.sup.18F(IMP461) was then eluted with 10 mL 1:1 EtOH/H.sub.2O,
30.3 mCi, 63.5% yield, specific activity 750 Ci/mmol. The labeled
peptide was free of unbound .sup.18F by HPLC. The total reaction
and purification time was 20 min.
Example 26. Preparation of Al.sup.19F Peptides
[0423] Products containing .sup.27Al and/or .sup.19F are useful for
certain applications like MR imaging. An improved method for
preparing [Al.sup.19F] compounds was developed. IMP461 was prepared
as described above and labeled with .sup.19F. Reacting IMP461 with
AlCl.sub.3+NaF resulted in the formation of three products (not
shown). However, by reacting IMP461 with AlF.sub.3.3H.sub.2O we
obtained a higher yield of Al.sup.19F(IMP461).
[0424] Synthesis of IMP 473:
[0425] [Al.sup.19F(IMP461)] To (14.1 mg, 10.90 .mu.mol) IMP461 in 2
mL NaOAc (2 mM, pH 4.18) solution added (4.51 mg, 32.68 .mu.mol)
AlF.sub.3.3H.sub.2O and 500 .mu.L ethanol. The pH of the solution
to adjusted to 4.46 using 3 .mu.L 1 N NaOH and heated in a boiling
water bath for 30 minutes. The crude reaction mixture was purified
by preparative RP-HPLC to yield 4.8 mg (32.9%) of IMP 473. HRMS
(ESI-TOF) MH.sup.+ expected 1337.6341; found 1337.6332
[0426] These results demonstrate that .sup.19F labeled molecules
may be prepared by forming metal-.sup.19F complexes and binding the
metal-.sup.19F to a chelating moiety, as discussed above for
.sup.18F labeling. The instant Example shows that a targeting
peptide of use for pretargeting detection, diagnosis and/or imaging
may be prepared using the instant methods.
Example 27. Synthesis and Labeling of IMP479, IMP485 and IMP487
[0427] The structures of additional peptides (IMP479, IMP485, and
IMP487) designed for .sup.18F-labeling are shown in FIG. 11 to FIG.
13. IMP485 is shown in FIG. 12. IMP485 was made on Sieber Amide
resin by adding the following amino acids to the resin in the order
shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved,
Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was
cleaved, (tert-Butyl).sub.2NODA-MPAA (methyl phenyl acetic acid).
The peptide was then cleaved from the resin and purified by RP-HPLC
to yield 44.8 mg of IMP485.
[0428] Synthesis of Bis-t-butyl-NODA-MPAA: (tBu).sub.2NODA-MPAA for
IMP485 Synthesis
[0429] To a solution of 4-(bromomethyl) phenyl acetic acid (Aldrich
310417) (0.5925 g, 2.59 mmol) in CH.sub.3CN (anhydrous) (50 mL) at
0.degree. C. was added dropwise over 1 h a solution of NO2AtBu
(1.0087 g, 2.82 mmol) in CH.sub.3CN (50 mL). After 4 h anhydrous
K.sub.2CO.sub.3 (0.1008 g, 0.729 mmol) was added to the reaction
mixture and allowed to stir at room temperature overnight. Solvent
was evaporated and the crude mixture was purified by preparative
RP-HPLC to yield a white solid (0.7132 g, 54.5%).
##STR00001##
[0430] Although this is a one step synthesis, yields were low due
to esterification of the product by 4-(bromomethyl)phenylacetic
acid. Alkylation of NO2AtBu using methyl(4-bromomethyl)
phenylacetate was employed to prevent esterification (FIG. 14).
[0431] .sup.18F-Labeling
[0432] For .sup.18F labeling studies in water, to 40 nmol of
IMP479/485/487 (formulated using trehalose+ascorbic
acid+AlCl.sub.3) was added 250 .mu.L .sup.18F.sup.- solution
[.about.919-1112 .mu.Ci of .sup.18F.sup.-] and heated to
101.degree. C. for 15 minutes. In ethanol, to 40 nmol of
IMP479/485/487 (formulated using trehalose+ascorbic
acid+AlCl.sub.3) was added 250 .mu.L .sup.18F.sup.- solution
[1.248-1.693 mCi of .sup.18F.sup.-], 100 .mu.L EtOH and heated to
101.degree. C. for 15 minutes. An exemplary experiment showing
labeling of different peptides is shown in Table 14. With minimal
optimization, radiolabeling of IMP485 has been observed with up to
an 88% yield and a specific activity of 2,500 Ci/mmol. At this
specific activity, HPLC purification of the radiolabeled peptide is
not required for in vivo PET imaging using the radiolabeled
peptide.
TABLE-US-00026 TABLE 14 Labeling of IMP479, IMP485 and IMP487
Isolated yields after HLB purification IMP # H.sub.2O EtOH IMP479
44.0% 57.5% IMP485 74.4% 79.7% IMP487 63.6% 81.6%
[0433] Stability in Serum
[0434] A kit containing 40 nmol of IMP485 or IMP487, 20 nmol
AlCl.sub.3, 0.1 mg ascorbic acid and 0.1 g trehalose adjusted to pH
3.9 was reconstituted with purified .sup.18F.sup.- in 200 .mu.L
saline and heated 106.degree. C. for 15 min. The reaction mixture
was then diluted with 800 .mu.L water and placed on an HLB column.
After washing, the column was eluted with 2.times.200 .mu.L 1:1
EtOH/H.sub.2O to obtain the purified Al.sup.18F(IMP485) in 64.6%
isolated yield. The radiolabeled peptide in 50 .mu.L was mixed with
250 .mu.L of fresh human serum in a vial and incubated at
37.degree. C.
[0435] Both radiolabeled peptides were stable at 37.degree. C. in
fresh human serum over the four hours tested (not shown).
[0436] Effect of Bulking Agents on Yield of Lyophilized Peptide
[0437] An experiment was performed to compare yield using IMP485
kits (40 nmol) with different bulking agents labeled with 2 mCi of
F-18 (from the same batch of F-18) in 200 microliters of saline.
The bulking agents were introduced at a concentration of 5% by
weight in water with a dose of 200 microliters/vial. We tested
sorbitol, trehalose, sucrose, mannitol and glycine as bulking
agents. Results are shown in Table 15
TABLE-US-00027 TABLE 15 Effects of Bulking Agents on Radiolabeling
Yield Bulking Agent Activity mCi Yield % Sorbitol 2.17 82.9 Glycine
2.17 41.5 Mannitol 2.11 81.8 Sucrose 2.11 66.1 Trehalose 2.10
81.3
[0438] Sorbitol, mannitol and trehalose all gave radiolabeled
product in the same yield. The mannitol kit and the trehalose kit
both formed nice cakes. The sucrose kit and the glycine kit both
had significantly lower yields. We also recently tested
2-hydroxypropyl-beta-cyclodextrin as a bulking agent and obtained a
58% yield for the 40 nmol kit. We have found that radiolabeling is
very pH sensitive and needs to be tuned to the ligand and possibly
even to the peptide+the ligand. In the case of IMP485 the optimal
pH is pH 4.0.+-.0.2 whereas the optimal pH for IMP467 was pH
4.5.+-.0.5. In both cases the yields drop off rapidly outside the
ideal pH zone.
[0439] Time Course of Labeling
[0440] The time course for labeling of IMP485 was examined. To 40
nmol of IMP485 (formulated using trehalose+AlCl.sub.3 (20
nmol)+ascorbic acid) was added .about.200-250 .mu.L .sup.18F.sup.-
solution (0.9% saline) and heated to 104.degree. C. for 5 to 15
minutes. The results for labeling yield were: 5 min (28.9%), 10 min
(57.9%), 15 min (83.7%) and 30 min (88.9%). Thus, the time course
for labeling was approximately 15 minutes.
[0441] Biodistribution of IMP485 Alone
[0442] The biodistribution of IMP485 in the absence of any
pretargeting antibody was examined in female Taconic nude mice (10
week old) bearing small or no BXPC3 pancreatic cancer xenografts.
The mice were injected i.v. with Al.sup.18F(IMP485), (340
2.29.times.10.sup.-9 mol, 100 .mu.L in saline). The mice, 6 per
time point, were necropsied at 30 min and 90 min post injection. In
the absence of pretargeting antibody a low level of accumulation
was seen in tumor and most normal tissues. The substantial majority
of radiolabel was found in the bladder and to a lesser extent in
kidney. Most of the activity was cleared before the 90 min time
point.
[0443] Pretargeting of IMP485 with TF2 DNL Targeting Molecule
[0444] IMP485 Radiolabeling--
[0445] (218 mCi) was purified to isolate 145.9 mCi. The purified
.sup.18F.sup.- (135 mCi) was added to a lyophilized vial containing
40 nmol of pre-complexed Al(IMP485). The reaction vial was heated
at 110.degree. C. for 17 min. Water (0.8 mL) was added to the
reaction mixture before HLB purification. The product (22 mCi) was
eluted with 0.6 mL of water:ethanol (1:1) mixture into a vial
containing lyophilized ascorbic acid. The product was diluted with
saline. The Al.sup.18F(IMP485) specific activity used for injection
was 550 Ci/mmol.
[0446] Biodistribution of Al.sup.18F(IMP485) Alone--
[0447] Mice bearing sc LS174T xenografts were injected with
Al.sup.18F(IMP485) (28 5.2.times.10.sup.-11 mol, 100 .mu.L. Mice
were necropsied at 1 and 3 h post injection, 6 mice per time
point.
[0448] Biodistribution of TF2+Al.sup.18F(IMP485) with Pretargeting
at 20:1 bsMAb to Peptide Ratio--
[0449] Mice bearing sc LS174T xenografts were injected with TF2
(163.2 .mu.g, 1.03.times.10.sup.-9 mol, iv) and allowed 16.3 h for
clearance before injecting Al.sup.18F(IMP485) (28 .mu.Ci,
5.2.times.10.sup.-11 mol, 100 .mu.L iv). Mice were necropsied at 1
and 3 h post injection, 7 mice per time point.
[0450] Urine Stability--
[0451] Ten mice bearing s.c. Capan-1 xenografts were injected with
Al.sup.18F(IMP485) (400 .mu.Ci, in saline, 100 .mu.L). Urine was
collected from 3 mice at 55 min post injection. The urine samples
were analyzed by reverse phase and SE-HPLC. Stability of the
radiolabeled IMP485 in urine was observed.
TABLE-US-00028 TABLE 16 Al.sup.18F(IMP485) Alone at 1 h post
injection: Tissue n Weight STD WT % ID/g STD % ID/g % ID/org STD %
ID/org T/NT STD T/NT Tumor 6 0.235 0.147 0.316 0.114 0.081 0.063
1.0 0.0 Liver 6 1.251 0.139 0.176 0.032 0.220 0.043 1.8 0.4 Spleen
6 0.085 0.019 0.210 0.181 0.018 0.017 1.9 0.9 Kidney 6 0.149 0.013
3.328 0.556 0.499 0.119 0.1 0.0 Lung 6 0.141 0.039 0.238 0.048
0.033 0.010 1.3 0.3 Blood 6 0.222 0.006 0.165 0.062 0.268 0.101 2.0
0.4 Stomach 6 0.478 0.083 0.126 0.110 0.057 0.045 3.5 1.6 Sm Int. 6
0.896 0.098 0.396 0.128 0.353 0.110 0.8 0.3 Lg Int. 6 0.504 0.056
0.081 0.019 0.041 0.010 3.9 0.9 Muscle 6 0.103 0.029 0.114 0.079
0.011 0.008 4.1 2.5 Scapula 6 0.057 0.015 0.107 0.019 0.006 0.001
2.9 0.7
TABLE-US-00029 TABLE 17 Al.sup.18F(IMP485) Alone at 3 h post
injection: Tissue n Weight STD WT % ID/g STD % ID/g % ID/org STD %
ID/org T/NT STD T/NT Tumor 6 0.265 0.126 0.088 0.020 0.022 0.011
1.0 0.0 Liver 6 1.219 0.091 0.095 0.047 0.114 0.056 13.6 31.4
Spleen 6 0.091 0.015 0.065 0.009 0.006 0.001 1.4 0.2 Kidney 6 0.154
0.013 2.265 0.287 0.345 0.028 0.0 0.0 Lung 6 0.142 0.008 0.073
0.019 0.010 0.003 1.3 0.6 Blood 6 0.236 0.019 0.008 0.005 0.013
0.007 21.0 27.9 Stomach 6 0.379 0.054 0.041 0.017 0.016 0.008 2.5
1.0 Sm. Int. 6 0.870 0.042 0.137 0.031 0.119 0.029 0.7 0.3 Lg. Int.
6 0.557 0.101 0.713 0.215 0.408 0.194 0.1 0.0 Muscle 6 0.134 0.038
0.013 0.007 0.002 0.001 203.9 486.6 Scapula 6 0.074 0.009 0.079
0.026 0.006 0.002 1.2 0.6
TABLE-US-00030 TABLE 18 TF2 + Al.sup.18F(IMP485), at 1 h post
peptide injection: Tissue n Weight STD WT % ID/g STD % ID/g %
ID/org STD % ID/org T/NT STD T/NT Tumor 7 0.291 0.134 28.089 4.545
8.025 3.357 1 0 Liver 7 1.261 0.169 0.237 0.037 0.295 0.033 123 38
Spleen 7 0.081 0.013 0.254 0.108 0.020 0.008 139 87 Kidney 7 0.140
0.018 3.193 0.730 0.444 0.098 9 4 Lung 7 0.143 0.014 0.535 0.147
0.075 0.018 57 22 Blood 7 0.205 0.029 0.278 0.071 0.456 0.129 110
43 Stomach 7 0.473 0.106 0.534 1.175 0.265 0.598 381 318 Sm. Int. 7
0.877 0.094 0.686 0.876 0.586 0.725 75 39 Lg. Int. 7 0.531 0.068
0.104 0.028 0.055 0.015 291 121 Muscle 7 0.090 0.014 0.136 0.102
0.012 0.009 348 274 Scapula 6 0.189 0.029 0.500 0.445 0.095 0.092
120 108
TABLE-US-00031 TABLE 19 TF2 + Al.sup.18F(IMP485), at 3 h post
peptide injection: Tissue n Weight STD WT % ID/g STD % ID/g %
ID/org STD ID/org T/NT STD T/NT Tumor 7 0.320 0.249 26.518 5.971
8.127 5.181 1 0 Liver 7 1.261 0.048 0.142 0.019 0.178 0.025 189 43
Spleen 7 0.079 0.012 0.138 0.031 0.011 0.002 195 41 Kidney 7 0.144
0.012 2.223 0.221 0.319 0.043 12 3 Lung 7 0.145 0.014 0.244 0.056
0.035 0.005 111 24 Blood 7 0.229 0.014 0.023 0.008 0.037 0.012 1240
490 Stomach 7 0.430 0.069 0.025 0.017 0.010 0.005 1389 850 Sm. Int.
7 0.818 0.094 0.071 0.029 0.059 0.028 438 207 Lg. Int. 7 0.586
0.101 0.353 0.160 0.206 0.103 86 33 Muscle 7 0.094 0.014 0.025
0.006 0.002 0.001 1129 451 Scapula 7 0.140 0.030 0.058 0.018 0.008
0.002 502 193
[0452] Conclusions
[0453] The IMP485 labels as well as or better than IMP467, with
equivalent stability in serum. However, IMP485 is much easier to
synthesize than IMP467. Preliminary studies have shown that
.sup.18F-labeling of lyophilized IMP485 works as well as
non-lyophilized peptide (data not shown). The presence of alkyl or
aryl groups in the linker joining the chelating moiety to the rest
of the peptide was examined. The presence of aryl groups in the
linker appears to increase the radiolabeling yield relative to the
presence of alkyl groups in the linker.
[0454] Biodistribution of IMP485 in the presence or absence of
pretargeting antibody resembles that observed with IMP467. In the
absence of pretargeting antibody, distribution of radiolabeled
peptide in tumor and most normal tissues is low and the peptide is
removed from circulation by kidney excretion. In the presence of
the TF2 antibody, radiolabeled IMP485 is found primarily in the
tumor, with little distribution to normal tissues. Kidney
radiolabeling is substantially decreased in the presence of the
pretargeting antibody. We conclude that IMP485 and other peptides
with aryl groups in the linker are highly suitable for PET imaging
with .sup.18F-labeling.
Example 28. Kit Formulation of IMP485 for Imaging
[0455] We report a simple, general kit formulation for labeling
peptides with .sup.18F. A ligand that contains
1,4,7-triazacyclononane-1,4-diacetate (NODA) attached to a methyl
phenylacetic acid (MPAA) group was used to form a single stable
complex with (AlF).sup.2+. The lyophilized kit contained IMP485, a
di-HSG hapten-peptide used for pretargeting. The kit was
reconstituted with an aqueous solution of .sup.18F.sup.-, heated at
100-110.degree. C. for 15 min, followed by a rapid purification by
solid-phase extraction (SPE). In vitro and in vivo stability and
tumor targeting of the Al.sup.18F(IMP485) were examined in nude
mice bearing human colon cancer xenografts pretargeted with an
anti-CEACAM5 bispecific antibody. .sup.18F-labeling of
MPAA-bombesin and somatostatin peptides also was evaluated.
[0456] The HSG peptide was labeled with .sup.18F.sup.- as a single
isomer complex, in high yield (50-90%) and high specific activity
(up to 153 GBq/.mu.mol), within 30 min. It was stable in human
serum at 37.degree. C. for 4 h, and in vivo showed low uptake
(0.06%.+-.0.02 ID/g) in bone. At 3 h, pretargeted animals had high
Al.sup.18F(IMP485) tumor uptake (26.5%.+-.6.0 ID/g), with ratios of
12.+-.3, 189.+-.43, 1240.+-.490 and 502.+-.193 for kidney, liver,
blood and bone, respectively. Bombesin and octreotide analogs were
labeled with comparable yields. In conclusion, .sup.18F-labeled
peptides can be produced as a stable, single [Al.sup.18F] complex
with good radiochemical yields and high specific activity in a
simple one-step kit.
[0457] Reagents List
[0458] Reagents were obtained from the following sources: Acetic
acid (JT Baker 6903-05 or 9522-02), Sodium hydroxide (Aldrich
semiconductor grade 99.99% 30,657-6), .alpha.,.alpha.-Trehalose (JT
Baker 4226-04), Aluminum chloride hexahydrate (Aldrich 99% 237078),
Ascorbic acid (Aldrich 25,556-4).
[0459] Acetate Buffer 2 mM--
[0460] Acetic acid, 22.9 .mu.L (4.0.times.10.sup.-4 mol) was
diluted with 200 mL water and neutralized with 6 N NaOH (.about.15
.mu.L) to adjust the solution to pH 4.22.
[0461] Aluminum Solution 2 mM--
[0462] Aluminum hexahydrate, 0.0225 g (9.32.times.10.sup.-5 mol)
was dissolved in 47 mL DI water.
[0463] .alpha.,.alpha.-Trehalose Solution--
[0464] .alpha.,.alpha.-Trehalose, 4.004 g was dissolved in 40 mL DI
water to make a 10% solution.
[0465] Peptide Solution, IMP485 2 mM--
[0466] The peptide IMP485 (0.0020 g, 1.52 .mu.mol) was dissolved in
762 .mu.L of 2 mM acetate buffer. The pH was 2.48 (the peptide was
lyophilized as the TFA salt). The pH of the peptide solution was
adjusted to pH 4.56 by the addition of 4.1 .mu.L of 1 M NaOH.
[0467] Ascorbic Acid Solution 5 mg/mL--
[0468] Ascorbic acid, 0.0262 g (1.49.times.10.sup.4 mol) was
dissolved in 5.24 mL DI water.
[0469] Formulation of Peptide Kit
[0470] The peptide, 20 .mu.L (40 nmol) was mixed with 12 .mu.L (24
nmol) of Al, 100 .mu.L of trehalose, 20 .mu.L (0.1 mg) ascorbic
acid and 900 .mu.L of DI water in a 3 mL lyophilization vial. The
final pH of the solution was about pH 4.0. The vial was frozen,
lyophilized and sealed under vacuum. Ten and 20 nmol kits have also
been made. These kits are made the same as the 40 nmol kits keeping
the peptide to Al.sup.3+ ratio of 1 peptide to 0.6 Al.sup.3+ but
formulated in 2 mL vials with a total fill of 0.5 mL.
[0471] Purification of .sup.18F.sup.-
[0472] The crude .sup.18F.sup.- was received in 2 mL of DI water in
a syringe. The syringe was placed on a syringe pump and the liquid
pushed through a Waters CM cartridge followed by a QMA cartridge.
Both cartridges were washed with 10 mL DI water. A sterile
disposable three way valve between the two cartridges was switched
and 1 mL commercial sterile saline was pushed through the QMA
cartridge in 200 .mu.L fractions. The second fraction usually
contains .about.80% of the .sup.18F.sup.- regardless of the amount
of .sup.18F.sup.- applied (10-300 mCi loads were tested).
[0473] We alternatively use commercial .sup.18F.sup.- in saline,
which has been purified on a QMA cartridge. This is a concentrated
version of the commercial bone imaging agent so it is readily
available and used in humans. The activity is supplied in 200 .mu.L
in a 0.5 mL tuberculin syringe.
[0474] Radiolabeling
[0475] The peptide was radiolabeled by adding .sup.18F.sup.- in 200
.mu.L saline to the lyophilized peptide in a crimp sealed vial and
then heating the solution to 90-110.degree. C. for 15 min. The
peptide was purified by adding 800 mL of DI water in a 1 mL syringe
to the reaction vial, removing the liquid with the 1 mL syringe and
applying the liquid to a Waters HLB column (1 cc, 30 mg). The HLB
column was placed on a crimp sealed 5 mL vial and the liquid was
drawn into the vial under vacuum supplied by a remote (using a
sterile disposable line) 10 mL syringe. The reaction vial was
washed with two one mL aliquots of DI water, which were also drawn
through the column. The column was then washed with 1 mL more of DI
water. The column was then moved to a vial containing buffered
lyophilized ascorbic acid (.about.pH 5.5, 15 mg). The radiolabeled
product was eluted with three 200 .mu.L portions of 1:1 EtOH/DI
water. The yield was determined by measuring the activity on the
HLB cartridge, in the reaction vial, in the water wash and in the
product vial to get the percent yield.
[0476] Adding ethanol to the radiolabeling reaction can increase
the labeling yield. A 20 nmol kit can be reconstituted with a
mixture of 200 .mu.L .sup.18F.sup.- in saline and 200 .mu.L
ethanol. The solution is then heated to 110.degree. C. in the crimp
sealed vial for 16 min. After heating, 0.8 mL of water was added to
the reaction vial and the activity was removed with a syringe and
placed in a dilution vial containing 2 mL of DI water. The reaction
vial was washed with 2.times.1 mL DI water and each wash was added
to the dilution vial. The solution in the dilution vial was applied
to the HLB column in 1-mL aliquots. The column and the dilution
vial were then washed with 2.times.1-mL water. The radiolabeled
peptide was then eluted from the column with 3.times.200 .mu.L of
1:1 ethanol/water in fractions. The peptide can be labeled in good
yield and up to 4,100 Ci/mmol specific activity using this
method.
[0477] The yield for this kit and label as described was 80-90%
when labeled with 1.0 mCi of .sup.18F.sup.-. When higher doses of
.sup.18F.sup.- (.about.100 mCi) were used the yield dropped.
However if ethanol is added to the labeling mixture the yield goes
up. If the peptides are diluted too much in saline the yields will
drop. The labeling is also very sensitive to pH. For our peptide
with this ligand we have found that the optimal pH for the final
formulation was pH 4.0.+-.0.2.
[0478] The purified radiolabeled peptide in 50 .mu.L 1:1
EtOH/H.sub.2O was mixed with 150 .mu.L of human serum and placed in
the HPLC autosampler heated to 37.degree. C. and analyzed by
RP-HPLC. No detectable .sup.18F above background at the void volume
was observed even after 4 h.
TABLE-US-00032 TABLE 20 IMP 485 Labeling Activity nmol of iso-
Specific of Volume Volume Activity lated Activity Vial #/ Pep-
Saline EtOH at start product % Ci/ Peptide tide .mu.L .mu.L mCi mCi
Yield mmol 1. IMP485 10 100 0 20.0 7.10 62 2. IMP485 10 50 50 19.4
9.43 78 3. IMP485 10 200 0 19.07 5.05 38 4. IMP485 20 100 100 37.3
22.3 80 5. IMP485 10 100 0 45.7 16.2 42 6. IMP485 20 200 200 175.6
82.7 58 4135
[0479] Synthesis and Radiolabeling of IMP486: Al-OH(IMP485)
[0480] IMP485 (21.5 mg, 0.016 mmol) was dissolved in 1 mL of 2 mM
NaOAc, pH 4.4 and treated with AlCl.sub.3.6H.sub.2O (13.2 mg, 0.055
mmol). The pH was adjusted to 4.5-5.0 and the reaction mixture was
refluxed for 15 minutes. The crude mixture was purified by
preparative RP-HPLC to yield a white solid (11.8 mg).
[0481] The pre-filled Al(NODA) complex (IMP486) was also
radiolabeled in excellent yield after formulating into lyophilized
kits. The labeling yields with IMP486 (Table 21) were as good as or
better than IMP485 kits (Table 20) when labeled in saline. This
high efficiency of radiolabeling with chelator preloaded with
aluminum was not observed with any of the other Al(NOTA) complexes
tested (data not shown). The equivalency of labeling in saline and
in 1:1 ethanol/water the labeling yields was also not observed with
other chelating moieties (not shown).
TABLE-US-00033 TABLE 21 IMP486 Labeling Activity of Specific
Peptide Volume Volume Activity at isolated % Activity 20 nmol
Saline EtOH start mCi product mCi Yield Ci/mmol IMP486 100 .mu.L 0
46 28 76 2800 IMP485 100 .mu.L 100 .mu.L 41 25 83 IMP486 100 .mu.L
100 .mu.L 43 22 81 IMP486 200 .mu.L 0 42 18 73
[0482] Effect of Bulking Agents
[0483] An experiment was performed to compare yield using IMP485
kits (40 nmol) with different bulking agents. The peptide was
labeled with 2 mCi of .sup.18F.sup.- from the same batch of
.sup.18F.sup.- in 200 microliters saline. The bulking agents were
introduced in water at a concentration of 5% by weight, with a dose
of 200 microliters/vial. We tested sorbitol, trehalose, sucrose,
mannitol and glycine as bulking agents. Results are shown in Table
22.
TABLE-US-00034 TABLE 22 Effects of Bulking Agents on Radiolabeling
Yield (Refer table 15) Bulking Agent Activity mCi Yield % Sorbitol
2.17 82.9 Glycine 2.17 41.5 Mannitol 2.11 81.8 Sucrose 2.11 66.1
Trehalose 2.10 81.3
[0484] Sorbitol, mannitol and trehalose all gave radiolabeled
product in the same yield. The sucrose kit and the glycine kit both
had significantly lower yields. Trehalose was formulated into
IMP485 kits at concentrations ranging from 2.5% to 50% by weight
when reconstituted in 200 .mu.L. The same radiolabeling yield,
.about.83%, was obtained for all concentrations, indicating that
the .sup.18F-radiolabeling of IMP485 was not sensitive to the
concentration of the trehalose bulking agent. IMP 485 kits were
formulated and stored at 2-8.degree. C. under nitrogen for up to
three days before lyophilization to assess the impact of
lyophilization delays on the radiolabeling. The radiolabeling
experiments indicated that yields were all .about.80% at time zero,
and with 1, 2, and 3 days of delay before lyophilization.
[0485] Ascorbic or gentisic acid often are added to
radiopharmaceuticals during preparation to minimize radiolysis.
When IMP485 (20 nmol) was formulated with 0.1, 0.5 and 1.0 mg of
ascorbic acid at pH 4.1-4.2 and labeled with .sup.18F.sup.- in 200
.mu.L saline, final yields were 51, 31 and 13% isolated yields,
respectively, suggesting 0.1 mg of ascorbic acid was the maximum
amount that could be included in the formulation without reducing
yields. Formulations containing gentisic acid did not label well.
Ascorbic acid was also included in vials used to isolate the HLB
purified product as an additional means of ensuring stability
post-labeling. The IMP485 to Al.sup.3+ ratio appeared to be optimal
at 1:0.6, but good yields were obtained from 1:0.5 of up to a ratio
of 1:1. The radiolabeling reaction was also sensitive to peptide
concentration, with good yields obtained at concentrations of
1.times.10.sup.-4 M and higher.
[0486] Effect of pH on Radiolabeling
[0487] The effect of pH on radiolabeling of IMP485 is shown in
Table 23. The efficiency of labeling was pH sensitive and decreased
at either higher or lower pH relative to the optimal pH of about
4.0.
TABLE-US-00035 TABLE 23 Effect of pH on IMP485 Radiolabeling
Efficiency. pH Yield % 3.27 33 3.53 61 3.84 85 3.99 88 4.21 89 4.49
80 5.07 14
[0488] Collectively, these studies led to a final formulated kit
that contained 0.5 mL of a sterile solution with 20 nmol IMP485, 12
nmoles Al.sup.3+, 0.1 mg ascorbic acid, and 10 mg trehalose
adjusted to 4.0.+-.0.2, which was then lyophilized
[0489] Biodistribution
[0490] Biodistribution studies were performed in Taconic nude mice
bearing subcutaneous LS174T tumor xenografts.
[0491] Al.sup.18F(IMP485) Alone:
[0492] Mice bearing sc LS174T xenografts were injected with
Al.sup.18F(IMP485) (28 .mu.Ci, 5.2.times.10.sup.-11 mol, 100 .mu.L,
iv). Mice were necropsied at 1 and 3 h post injection, 6 mice per
time point.
[0493] TF2+Al.sup.18F(IMP485) Pretargeting at 20:1 bsMab to Peptide
Ratio:
[0494] Mice bearing sc LS174T xenografts were injected with TF2
(163.2 1.03.times.10.sup.-9 mol, iv) and allowed 16.3 h for
clearance before injecting Al.sup.18F(IMP485) (28 .mu.Ci,
5.2.times.10.sup.-11 mol, 100 .mu.L, iv). Mice were necropsied at 1
and 3 h post injection, 7 mice per time point.
TABLE-US-00036 TABLE 24 Biodistribution of TF2 pretargeted
Al.sup.18F(IMP485) or Al.sup.18F(IMP485) alone at 1 and 3 h after
peptide injection in nude mice bearing LS174T human colonic cancer
xenografts. Percent-injected dose per gram tissue (mean .+-. SD; N
= 7) TF2 pregargeted Al.sup.18F(IMP485) Al.sup.18F(IMP485) alone
Tissue 1 h 3 h 1 h 3 h Tumor 28.09 .+-. 4.55 26.52 .+-. 5.97 0.32
.+-. 0.11 0.09 .+-. 0.02 Liver 0.24 .+-. 0.04 0.14 .+-. 0.02 0.18
.+-. 0.03 0.10 .+-. 0.05 Spleen 0.25 .+-. 0.11 0.25 .+-. 0.11 0.21
.+-. 0.18 0.07 .+-. 0.01 Kidney 3.19 .+-. 0.73 2.22 .+-. 0.22 3.33
.+-. 0.56 2.27 .+-. 0.29 Lung 0.54 .+-. 0.15 0.24 .+-. 0.06 0.24
.+-. 0.05 0.07 .+-. 0.02 Blood 0.28 .+-. 0.07 0.02 .+-. 0.01 0.17
.+-. 0.06 0.09 .+-. 0.01 Stomach 0.53 .+-. 1.18 0.03 .+-. 0.02 0.13
.+-. 0.11 0.04 .+-. 0.02 Sm. Int. 0.69 .+-. 0.88 0.07 .+-. 0.03
0.40 .+-. 0.13 0.14 .+-. 0.03 Lg. Int. 0.10 .+-. 0.03 0.35 .+-.
0.16 0.08 .+-. 0.02 0.71 .+-. 0.22 Muscle 0.14 .+-. 0.10 0.03 .+-.
0.01 0.11 .+-. 0.08 0.01 .+-. 0.01 Scapula 0.5 .+-. 0.45 0.06 .+-.
0.02 0.11 .+-. 0.02 0.03 .+-. 0.01
[0495] Synthesis of IMP492 or Al.sup.19F(IMP485)
[0496] IMP485 (16.5 mg, 0.013 mmol) was dissolved in 1 mL of 2 mM
NaOAc, pH 4.43, 0.5 mL ethanol and treated with AlF.sub.3.3H.sub.2O
(2.5 mg, 0.018 mmol). The pH was adjusted to 4.5-5.0 and the
reaction mixture was refluxed for 15 minutes. On cooling the pH was
once again raised to 4.5-5.0 and the reaction mixture refluxed for
15 minutes. The crude was purified by preparative RP-HPLC to yield
a white solid (10.3 mg).
[0497] Synthesis of IMP490
TABLE-US-00037 (SEQ ID NO: 52)
NODA-MPAA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl
[0498] The peptide was synthesized on threoninol resin with the
amino acids added in the following order: Fmoc-Cys(Trt)-OH,
Fmoc-Thr(But)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH,
Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH and
(tBu).sub.2NODA-MPAA. The peptide was then cleaved and purified by
preparative RP-HPLC. The peptide was cyclized by treatment of the
bis-thiol peptide with DMSO.
[0499] Synthesis of IMP491 or Al.sup.19F(IMP490)
[0500] The Al.sup.19F(IMP490) was prepared as described above
(IMP492) to produce the desired peptide after HPLC
purification.
[0501] Synthesis of IMP493
TABLE-US-00038 (SEQ ID NO: 53)
NODA-MPAA-(PEG).sub.3-Gln-Trp-Ala-Val-Gly-His-Leu- Met-NH.sub.2
[0502] The peptide was synthesized on Sieber amide resin with the
amino acids added in the following order: Fmoc-Met-OH, Fmoc-Leu-OH,
Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH,
Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-(PEG).sub.3-COOH and
(tBu).sub.2NODA-MPAA. The peptide was then cleaved and purified by
preparative RP-HPLC.
[0503] The affinity of the Al.sup.19F complex of IMP493 was
EC.sub.50=183 nm versus EC.sub.50=59 nm for .sup.125I-bombesin. The
IMP493 kit radiolabeled with .about.100 MBq of .sup.18F.sup.- had a
70% yield. Radiolabeling IMP490 with 100 MBq of .sup.18F.sup.-
resulted in 80% yield, which was reduced to 65% when 2.11 GBq
.sup.18F.sup.- was used. The peptide is eluted as a single
radiolabeled peak at 15.4 min using HPLC (not shown).
[0504] Synthesis of IMP494 or Al.sup.19F(IMP493)
[0505] The Al.sup.19F(IMP493) was prepared as described above
(IMP492) to produce the desired peptide after HPLC
purification.
Example 29. Other Prosthetic Group Labeling Methods Using
Al.sup.18F
[0506] In certain embodiments, the aluminum fluoride labeling
method may be performed using prosthetic group labeling methods for
molecules that are sensitive to heat. Prosthetic group conjugation
may be carried out at lower temperatures for heat-sensitive
molecules.
[0507] The prosthetic group NOTA is labeled with .sup.18F as
described above and then it is attached to the targeting molecule.
In one non-limiting example, this is performed with an aldehyde
NOTA that is then attached to an amino-oxy compound on a targeting
molecule. Alternatively an amino-oxy maleimide is reacted with the
aldehyde and then the maleimide is attached to a cysteine on a
targeting molecule (Toyokuni et al., 2003, Bioconj Chem
14:1253).
[0508] In another alternative, the AlF-chelator complexes are
attached to targeting molecules through click chemistry. The
ligands are first labeled with Al.sup.18F as discussed above. The
Al.sup.18F-chelate is then conjugated to a targeting molecule
through a click chemistry reaction. For example, an alkyne NOTA is
labeled according to Marik and Stucliffe (2006, Tetrahedron Lett
47:6681) and conjugated to an azide containing targeting agent.
[0509] Radiolabeling of Kits with .sup.18F.sup.- in Saline
[0510] The .sup.18F.sup.- (0.01 mCi or higher) is received in 200
.mu.L of saline in a 0.5 mL syringe and the solution is mixed with
200 .mu.L of ethanol and injected into a lyophilized kit as
described above. The solution is heated in the crimp sealed
container at 100-110.degree. C. for 15 min. The solution is diluted
with 3 mL water and eluted through an HLB cartridge. The reaction
vial and the cartridge are washed with 2.times.1 mL portions of
water and then the product is eluted into a vial containing
buffered ascorbic acid using 1:1 ethanol water in 0.5 mL fractions.
Some of the ethanol may be evaporated off under a stream of inert
gas. The solution is then diluted in saline and passed through a
0.2 .mu.m sterile filter prior to injection.
Example 30. Maleimide Conjugates of NOTA for .sup.18F-Labeling
[0511] The Examples above describe a novel method of
.sup.18F-labeling, which captures a ([.sup.18F]AlF).sup.2+ complex,
using a NOTA-derived ligand bound on a peptide. These labeled
peptides were stable in vivo and retained their binding abilities
(McBride et al., 2009, J. Nucl. Med. 50, 991-998; McBride et al.,
2010, Bioconjug. Chem. 21, 1331-1340; Laverman et al., 2010, J.
Nucl. Med. 51, 454-461; McBride et al. 2011, J. Nucl. Med. 52
(Suppl. 1), 313-314P (abstract 1489)). Although this procedure
allows peptides to be radiofluorinated in one simple step within 30
min, it requires agents to be heated to .about.100.degree. C.,
which is unsuitable for most proteins and some peptides. We and
others have found that an aromatic group attached to one of the
nitrogen atoms of the triazacylcononane ring of NODA can enhance
the yield for the ([.sup.18F]AlF).sup.2+ complexation compared to
some alkyl and carboxyl substituents (D'Souza et al., 2011,
Bioconjug. Chem. 1793-1803; McBride et al., 2010, Bioconjug. Chem.
21, 1331-1340; Shetty et al. 2011, Chem. Comm. DOI:
10.1039/c1cc13151f). In the present Example, we explored the
potential for labeling heat-labile compounds with
([.sup.18F]AlF).sup.2+, using a new ([.sup.18F]AlF).sup.2+-binding
ligand that contains 1,4,7-triazacyclononane-1,4-diacetate (NODA)
attached to a methyl phenylacetic acid group (MPAA). This was
conjugated to N-(2-aminoethyl)maleimide (N-AEM) to form NODA-MPAEM.
(Details of the synthesis are shown in FIG. 15.) The NODA-MPAEM was
labeled with ([.sup.18F]AlF).sup.2+ at 105.degree. C. in 49-82%
yield and conjugated at room temperature to an antibody Fab'
fragment in 69-80% yield (total time .about.50 min) and with
retention of immunoreactivity. These data indicate that the rapid
and simple [Al.sup.18F]-labeling method can be easily adapted for
preparing heat-sensitive compounds with .sup.18F quickly and in
high yields.
[0512] Synthesis of Bis-t-Butyl-NODA-MPAA NHS Ester:
(tBu).sub.2NODA-MPAA NHS Ester
[0513] To a solution of (tBu).sub.2NODA-MPAA (175.7 mg, 0.347 mmol)
in CH.sub.2Cl.sub.2 (5 mL) was added 347 .mu.L (0.347 mmol) DCC (1
M in CH.sub.2Cl.sub.2), 42.5 mg (0.392 mmol) N-hydroxysuccinimide
(NETS), and 20 .mu.L N,N-diisopropylethylamine (DIEA). After 3 h
DCU was filtered off and solvent evaporated. The crude mixture was
purified by flash chromatography on (230-400 mesh) silica gel
(CH.sub.2Cl.sub.2:MeOH, 100:0 to 80:20) to yield (128.3 mg, 61.3%)
of the NHS ester. The FIRMS (ESI) calculated for
C.sub.31H.sub.46N.sub.4O.sub.8 (M+H).sup.+ was 603.3388, observed
was 603.3395.
[0514] Synthesis of NODA-MPAEM: (MPAEM=Methyl Phenyl Acetamido
Ethyl Maleimide)
[0515] To a solution of (tBu).sub.2NODA-MPAA NHS ester (128.3 mg,
0.213 mmol) in CH.sub.2Cl.sub.2 (5 mL) was added a solution of 52.6
mg (0.207 mmol) N-(2-aminoethyl) maleimide trifluoroacetate salt in
250 .mu.L DMF and 20 .mu.L DIEA. After 3 h the solvent was
evaporated and the concentrate was treated with 2 mL TFA. The crude
product was diluted with water and purified by preparative RP-HPLC
to yield (49.4 mg, 45%) of the desired product. HRMS (ESI)
calculated for C.sub.25H.sub.33N.sub.5O.sub.7 (M+H).sup.+ was
516.2453, observed was 516.2452.
[0516] .sup.18F-Labeling of NODA-MPAEM
[0517] The NODA-MPAEM ligand (20 nmol; 10 .mu.L), dissolved in 2 mM
sodium acetate (pH 4), was mixed with AlCl.sub.3 (5 .mu.L of 2 mM
solution in 2 mM acetate buffer, 200 .mu.L of .sup.18F (0.73 and
1.56 GBq) in saline, and 200 .mu.L of acetonitrile. After heating
at 105-109.degree. C. for 15-20 min, 800 .mu.L of deionized (DI)
water was added to the reaction solution, and the entire contents
removed to a vial (dilution vial) containing 1 mL of deionized (DI)
water. The reaction vial was washed with 2.times.1 mL DI water and
added to the dilution vial. The crude product was then passed
through a 1-mL HLB column, which was washed with 2.times.1 mL
fractions of DI water. The labeled product was eluted from the
column using 3.times.200 .mu.L of 1:1 EtOH/water.
[0518] Conjugation of Al.sup.18F(NODA-MPAEM) to hMN-14 Fab'
[0519] Fab' fragments of humanized MN-14 anti-CEACAMS IgG
(labetuzumab) were prepared by pepsin digestion, followed by TCEP
(Tris(2-carboxyethyl)phosphine) reduction, and then formulated into
a lyophilized kit containing 1 mg (20 nmol) of the Fab' (2.4
thiols/Fab') in 5% trehalose and 0.025 M sodium acetate, pH 6.72.
The kit was reconstituted with 0.1 mL PBS, pH 7.01, and mixed with
the Al.sup.18F(NODA-MPAEM) (600 .mu.L 1:1 EtOH/H.sub.2O). After
incubating for 10 min at room temperature, the product was purified
on a 3-mL SEPHADEX G50-80 spin column in a 0.1 M, pH 6.5 sodium
acetate buffer (5 min). The isolated yield was calculated by
dividing the amount of activity in the eluent by the total activity
in the eluent and the activity on the column.
[0520] Immunoreactivity of the purified product was analyzed by
adding an excess of CEA and separating on SE-HPLC, comparing to a
profile of the product alone. The product was also analyzed by
RP-HPLC to assess percent-unbound Al.sup.18F(NODA-MPAEM).
[0521] .sup.99mTc-CEA-Scan.RTM.
[0522] A CEA-SCAN.RTM. kit containing 1.2 mg of IMMU-4, a murine
anti-CEACAM5 Fab' (anti-CEA, 2.4.times.10.sup.-2 .mu.mol), was
labeled with 453 MBq .sup.99mTcO.sub.4.sup.-Na.sup.+ in 1 mL saline
according to manufacturer's instructions and used without further
purification.
[0523] Animal Study
[0524] Nude mice were inoculated subcutaneously with CaPan-1 human
pancreatic adenocarcinoma (ATCC Accession No. HTB-79, Manassas,
Va.). When tumors were visible, the animals were injected
intravenously with 100 .mu.L of the radiolabeled Fab'. The
Al.sup.18F(NODA-MPAES)-hMN-14 Fab' was diluted in saline to 3.7
MBq/100 .mu.L containing .about.2.8 .mu.g of Fab'. A
.sup.99mTc-IMMU-4 Fab' aliquot (16.9 MBq) was removed and diluted
with saline (0.85 MBq/100 .mu.L containing .about.2.8 .mu.g of
Fab'). The animals were necropsied at 3 h post injection, tissues
and tumors removed, weighed, and counted by gamma scintillation,
together with standards prepared from the injected products. The
data are expressed as percent injected dose per gram.
[0525] Results
[0526] Synthesis and Reagent Preparation
[0527] The NODA-MPAEM was produced as shown in FIG. 15, where the
(tBu).sub.2NODA-MPAA was coupled to 2-aminoethyl-maleimide and then
deprotected to form the desired product. The crude product was
diluted with water and purified by preparative RP-HPLC to yield
(49.4 mg, 45%) of the desired product [HRMS (ESI) calculated for
C.sub.25H.sub.33N.sub.5O.sub.7 (M+H).sup.+ 516.2453, found
516.2452].
[0528] Radiolabeling
[0529] The NODA-MPAEM (20 nmol) was mixed with 10 nmol of Al.sup.3+
and labeled with 0.73 GBq and 1.56 GBq of .sup.18F.sup.- in saline.
After SPE purification, the isolated yields of
Al.sup.18F(NODA-MPAEM) were 82% and 49%, respectively, with a
synthesis time of about 30 min. The Al.sup.18F(NODA-MPAES)-hMN-14
Fab' conjugate was isolated in 74% and 80% yields after spin-column
purification for the low and high dose protein labeling,
respectively. The total process was completed within 50 min. The
specific activity for the purified Al.sup.18F(NODA-MPAES)-hMN-14
Fab' was 19.5 GBq/.mu.mol for the high-dose label and 10.9
GBq/.mu.mol for the low dose label.
[0530] SE-HPLC analysis of the labeled protein for the 0.74-GBq run
showed the .sup.18F-labeled Fab' as a single peak and all of the
activity shifted when excess CEA was added (not shown). RP-HPLC
analysis on a C4 column showed the labeled maleimide standard
eluting at 7.5 min, while the purified .sup.18F-protein eluted at
16.6 min (not shown). There was no unbound Al.sup.18F(NODA-MPAEM)
in the spin-column purified product.
[0531] Serum Stability
[0532] The Al.sup.18F(NODA-MPAES)-hMN-14 Fab' was mixed with fresh
human serum and incubated at 37.degree. C. SE-HPLC analysis over a
3-h period, with and without CEA showed that the product was stable
and retained binding to CEA (not shown).
[0533] Biodistribution
[0534] The biodistribution of the Al.sup.18F(NODA-MPAES)-hMN-14
Fab' and the .sup.99mTc-IMMU-4 murine Fab' was assessed in nude
mice bearing Capan-1 pancreatic cancer xenografts. At 3 h
post-injection, both agents showed an expected elevated uptake in
the kidneys, since Fab' is renally filtered from the blood (Table
25). The [Al.sup.18F]-Fab' concentration in the blood was
significantly (P<0.0001) lower than the .sup.99mTc-Fab', with a
correspondingly elevated uptake in the liver and spleen. The faster
blood clearance of the [Al.sup.18F]-Fab' likely contributed to the
lower tumor uptake as compared to the .sup.99mTc-Fab' (2.8.+-.0.3
vs. 6.8.+-.0.7, respectively), but it also resulted in a more
favorable tumor/blood ratio for the fluorinated Fab' (5.9.+-.1.3
vs. 0.9.+-.0.1, respectively). Bone uptake for both products was
similar, suggesting the Al.sup.18F(NODA) was tightly held by the
Fab'.
TABLE-US-00039 TABLE 25 Biodistribution of
Al.sup.18F(NODA-MPAES)-hMN-14 Fab' and .sup.99mTc-IMMU-4 Fab' at 3
h after injection with 0.37 MBq (~3 .mu.g) of each conjugate in
nude mice bearing Capan-1 human pancreatic cancer xenografts (N =
6). Al.sup.18F(NODA-MPAES)- .sup.99mTc CEA Scan hMN-14 Fab' IMMU-4
Fab' Tissue % ID/g T/NT % ID/g T/NT Capan-1 2.8 .+-. 0.3 -- 6.8
.+-. 0.7 -- (weight .+-. SD) (0.22 .+-. 0.08 g) (0.16 .+-. 0.05 g)
Liver 17.5 .+-. 3.8 0.2 .+-. 0.04 4.6 .+-. 0.4 1.5 .+-. 0.1 Spleen
11.3 .+-. 1.6 0.3 .+-. 0.04 3.5 .+-. 0.5 2.0 .+-. 0.3 Kidney 216
.+-. 30.9 0.0 .+-. 0.0 183 .+-. 22.5 0.04 .+-. 0.01 Lung 4.2 .+-.
1.6 0.8 .+-. 0.5 4.4 .+-. 0.8 1.6 .+-. 0.3 Blood 0.5 .+-. 0.1 5.9
.+-. 1.3 7.6 .+-. 0.9 0.9 .+-. 0.1 Stomach 0.6 .+-. 0.1 4.7 .+-.
1.2 2.4 .+-. 0.3 2.9 .+-. 0.4 Sm. Int. 2.2 .+-. 0.2 1.3 .+-. 0.1
3.4 .+-. 0.4 2.0 .+-. 0.2 Lg. Int. 1.1 .+-. 0.4 2.8 .+-. 0.7 5.2
.+-. 1.0 1.3 .+-. 0.3 Muscle 0.4 .+-. 0.1 6.7 .+-. 1.3 1.1 .+-. 0.2
6.1 .+-. 1.0 Scapula 1.6 .+-. 0.4 1.8 .+-. 0.4 2.0 .+-. 0.2 3.4
.+-. 0.5
DISCUSSION
[0535] We prepared a simple NODA-MPAEM ligand for attachment to
thiols on temperature-sensitive proteins or other molecules bearing
a sulfhydryl group. To avoid exposing the heat-labile compound to
high temperatures, the NODA-MPAEM was first mixed with Al.sup.3+
and .sup.18F.sup.- in saline and heated at 100-115.degree. C. for
15 min to form the Al.sup.18F(NODA-MPAEM) intermediate. This
intermediate was rapidly purified by SPE in 49-82% isolated yield
(67.7.+-.13.0%, n=5), depending on the amount of activity added to
a fixed amount (20 nmol) of the NODA-MPAEM. The
Al.sup.18F(NODA-MPAEM) was then efficiently (69-80% isolated yield,
74.3.+-.5.5, n=3) coupled to a reduced Fab' in 10-15 min, using a
spin column gel filtration procedure to isolate the radiolabeled
protein, in this case an antibody Fab' fragment. The entire
two-step process was completed in .about.50 min, and the labeled
product retained its molecular integrity and immunoreactivity.
Thus, the feasibility of extending the simplicity of the
[Al.sup.18F]-labeling procedure to heat-sensitive compounds was
established.
[0536] The [Al.sup.18F]-ligand complex has been shown to be very
stable in serum in vitro, and in animal testing, minimal bone
uptake is seen (McBride et al., 2009, J. Nucl. Med. 50, 991-998;
D'Souza et al., 2011a, J. Nucl. Med. 52 (Suppl. 1), 171P (abstract
577)). In this series of studies, .sup.18F associated with the
NODA-MPAEM compound conjugated to a Fab' was stable in serum in
vitro, and the conjugate retained binding to CEA. When injected
into nude mice, there was selective localization in the tumor,
providing a .about.6:1 tumor/blood ratio. Bone uptake was similar
for the Al.sup.18F(NODA-MPAES)-hMN-14 Fab' and the
.sup.99mTc-IMMU-4 murine Fab', again reflecting in vivo stability
of the .sup.18F or Al.sup.18F complex. However, [Al.sup.18F]-Fab'
hepatic and splenic uptake was higher as compared to the
.sup.99mTc-IMMU-4. The specific NODA derivative can be modified in
different ways to accommodate conjugation to other reactive sites
on peptides or proteins. However, use of this particular derivative
showed that the Al.sup.18F-labeling procedure can be adapted for
use with heat-labile compounds.
[0537] Conclusions
[0538] NODA-MPAEM was labeled rapidly with .sup.18F.sup.- in saline
and then conjugated to the immunoglobulin Fab' protein in high
yield. The labeling method uses only inexpensive disposable
purification columns, and while not requiring an automated device
to perform the labeling and purification, it can be easily adapted
to such systems. Thus, the NODA-MPAEM derivative established that
this or other NODA-containing derivatives can extend the capability
of facile ([.sup.18F]AlF).sup.2+ fluorination to heat-labile
compounds.
Example 31. Improved .sup.18F-Labeling of NOTA-Octreotide
[0539] The aim of this study was to further improve the rapid
one-step method for .sup.18F-labeling of NOTA-conjugated
octreotide. Octreotide was conjugated with a NOTA ligand and was
labeled with .sup.18F in a single-step, one-pot method. Aluminum
(Al.sup.3+) was added to .sup.18F.sup.- and the AlF.sup.2+ was
incorporated into NOTA-octreotide, as described in the Examples
above. The labeling procedure was optimized with regard to
aluminum:NOTA ratio, ionic strength and temperature. Radiochemical
yield and specific activity were determined.
[0540] Under optimized conditions, NOTA-octreotide was labeled with
Al.sup.18F in a single step with 98% yield. The radiolabeling,
including purification, was performed in 45 min. Optimal labeling
yield was observed with Al:NOTA ratios around 1:20. Lower ratios
led to decreased labeling efficiency. Labeling efficiencies in the
presence of 0%, 25%, 50%, 67% and 80% acetonitrile in Na-acetate pH
4.1 were 36%, 43%, 49%, 70% and 98%, respectively, indicating that
increasing concentrations of the organic solvent considerably
improved labeling efficiency. Similar results were obtained in the
presence of ethanol, DMF and THF. Labeling in the presence of DMSO
failed. Labeling efficiencies in 80% MeCN at 40.degree. C.,
50.degree. C. and 60.degree. C. were 34%, 65%, 83%, respectively.
Labeling efficiency was >98% at 80.degree. C. and 100.degree. C.
Specific activity of the .sup.18F-labeled peptide was higher than
45,000 GBq/mmol.
[0541] Optimal .sup.18F-labeling of NOTA-octreotide with Al.sup.18F
was performed at 80-100.degree. C. in Na-acetate buffer with 80%
(v/v) acetonitrile and a Al:NOTA ratio between 1:20 and 1:50.
Labeling efficiency was typically >98%. Since labeling
efficiency at 60.degree. C. was 83%, this method may also allow
.sup.18F-labeling of temperature-sensitive biomolecules such as
proteins and antibody fragments. These conditions allow routine
.sup.18F-labeling of peptides without the need for purification
prior to administration and PET imaging.
Example 32. Functionalized Triazacyclononane Ligands for Molecular
Imaging
[0542] The present Example relates to synthesis and use of a new
class of triazacyclonane derived ligands and their complexes useful
for molecular imaging. Exemplary structures are shown in FIG. 16 to
FIG. 18. The ligands may be functionalized with a .sup.19F moiety
selected from the group consisting of fluorinated alkyls,
fluorinated acetates, fluoroalkyl phosphonates, fluoroanilines,
trifluoromethyl anilines, and trifluoromethoxy anilines in an
amount effective to provide a detectable .sup.19F NMR signal. The
complexation of these ligands with radioisotopic or paramagnetic
cations renders them useful as diagnostic agents in nuclear
medicine and magnetic resonance imaging (MRI). Preferably, the
Al.sup.18F and .sup.68Ga complexes of these ligands are useful for
PET imaging, while the .sup.111In complexes can be used in SPECT
imaging. Methods for conjugating these radiolabeled ligands to a
targeting molecule like antibody, protein or peptide are also
disclosed.
[0543] The disclosed bifunctional chelators (BFCs) can be
radiolabeled with .sup.111In, .sup.68Ga, .sup.64Cu, .sup.177Lu,
Al.sup.18F, .sup.99mTc or .sup.86Y or complexed with a paramagnetic
metal like manganese, iron, chromium or gadolinium, and
subsequently attached to a targeting molecule (biomolecule). The
labeled biomolecules can be used to image the hematological system,
lymphatic reticuloendothelial system, nervous system, endocrine and
exocrine system, skeletomuscular system, skin, pulmonary system,
gastrointestinal system, reproductive system, immune system,
cardiovascular system, urinary system, auditory or olfactory system
or to image affected cells or tissues in various medical
conditions.
Synthesis of Bifunctional Chelators
2-{4-(carboxymethyl)-7-[2-(4-nitrophenyl)ethyl]-1,4,7-triazacyclononan-1-y-
l)acetic acid. NODA-EPN
[0544] To a solution of 4-nitrophenethyl bromide (104.5 mg, 0.45
mmol) in anhydrous CH.sub.3CN at 0.degree. C. was added dropwise
over 20 min a solution of (tBu).sub.2NODA (167.9 mg, 0.47 mmol) in
CH.sub.3CN (10 mL). After 1 h, anhydrous K.sub.2CO.sub.3 (238.9 mg,
1.73 mmol) was added to the reaction mixture and allowed to stir at
room temperature overnight. Solvent was evaporated and the
concentrate was acidified with 4 mL TFA. After 5 h, the reaction
mixture was diluted with water and purified by preparative RP-HPLC
to yield a pale yellow solid (60.8 mg, 32.8%). HRMS (ESI)
calculated for C.sub.18H.sub.26N.sub.4O.sub.6 (M+H).sup.+ 395.1925;
found 395.1925.
2-{4-(carboxymethyl)-7-[2-(4-nitrophenyl)methyl]-1,4,7-triazacyclononan-1--
yl)acetic acid. NODA-MPN
[0545] To a solution of 4-nitrobenzyl bromide (61.2 mg, 0.28 mmol)
in anhydrous CH.sub.3CN at 0.degree. C. was added dropwise over 20
min a solution of (tBu).sub.2NODA (103.6 mg, 0.29 mmol) in
CH.sub.3CN (10 mL). After 1 h, anhydrous K.sub.2CO.sub.3 (57.4 mg,
0.413 mmol) was added to the reaction mixture and allowed to stir
at room temperature overnight. Solvent was evaporated and the
concentrate was acidified with 3 mL TFA. After 5 h, the reaction
mixture was diluted with water and purified by preparative RP-HPLC
to yield a pale yellow solid (19.2 mg, 17.4%). HRMS (ESI)
calculated for C.sub.17H.sub.24N.sub.4O.sub.6 (M+H).sup.+ 381.1769;
found 381.1774.
6-(4,7-bis{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyclononan-1-yl)he-
xanoic acid. (tBu).sub.2NODA-HA
[0546] To a solution of (tBu).sub.2NODA (208.3 mg, 0.58 mmol) in 10
mL CH.sub.3CN was added 6-bromohexanoic acid (147.3 mg, 0.755 mmol)
and K.sub.2CO.sub.3 (144.5 mg, 1.05 mmol). The reaction flask was
placed in a warm water-bath for 48 h. Solvent was evaporated and
the concentrate was diluted with water and purified by preparative
RP-HPLC to yield a white solid (138.5 mg, 50.1%). ESMS calculated
for C.sub.24H.sub.45N.sub.3O.sub.6 (M+H).sup.+ 472.3381; found
472.27.
4-[(4,7-bis{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyclononan-1-yl)
methyl]benzoic acid. (tBu).sub.2NODA-MBA
[0547] To a solution of .alpha.-bromo-p-toluic acid (126.2 mg, 0.59
mmol) in anhydrous CH.sub.3CN was added dropwise over 20 min a
solution of (tBu).sub.2NODA (208 mg, 0.58 mmol) in CH.sub.3CN (10
mL) and allowed to stir at room temperature for 48 h. Solvent was
evaporated and the concentrate was diluted with water/DMF and
purified by preparative RP-HPLC to yield a white solid (74.6 mg).
HRMS (ESI) calculated for C.sub.26H.sub.41N.sub.3O.sub.6
(M+H).sup.+492.3068; found 492.3071.
4-[(4,7-bis{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyclononan-1-yl)e-
thyl]benzoic acid. (tBu).sub.2NODA-EBA
[0548] To a solution of 4-(2-bromoethyl)benzoic acid (310.9 mg,
1.36 mmol) in anhydrous CH.sub.3CN was added dropwise over 20 min a
solution of (tBu).sub.2NODA (432.3 mg, 1.21 mmol) in CH.sub.3CN (10
mL) and K.sub.2CO.sub.3 (122.4 mg, 0.89 mmol). The reaction was
stirred at room temperature for 72 h. Solvent was evaporated and
the concentrate was diluted with water/DMF and purified by
preparative RP-HPLC to yield a white solid (35.1 mg). HRMS (ESI)
calculated for C.sub.27H.sub.43N.sub.3O.sub.6 (M+H).sup.+ 506.3225;
found 506.3234.
2-[7-but-3-ynyl-4-(carboxymethyl]-1,4,7-triazacyclononan-1-yl)acetic
acid. NODA-Butyne
[0549] To a solution of (tBu).sub.2NODA (165.8 mg, 0.46 mmol) in 5
mL CH.sub.3CN was added 4-bromo-1-butyne (44 .mu.L, 62.3 mg, 0.47
mmol) and reaction mixture was stirred at room temperature for 72
h. Solvent was evaporated and the concentrate was purified by
preparative RP-HPLC to yield an oil. HRMS (ESI) calculated for
C.sub.22H.sub.39N.sub.3O.sub.4 (M+H).sup.+ 410.3013; found
410.3013. The purified product was acidified with 2 mL TFA and
after 5 h diluted with water, frozen and lyophilized. HRMS (ESI)
calculated for C.sub.14H.sub.23N.sub.3O.sub.6 (M+H).sup.+298.1761;
found 298.1757.
tert-butyl-2-(7-(4-aminobutyl)-4-{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-t-
riazacyclononan-1-yl)acetic acid. (tBu).sub.2NODA-BA
[0550] To a solution of (tBu).sub.2NODA (165.2 mg, 0.46 mmol) in 5
mL CH.sub.3CN was added 4-(Boc-amino)butyl bromide (124.7 mg, 0.49
mmol), a pinch of K.sub.2CO.sub.3 and reaction mixture was stirred
at room temperature for 72 h. Solvent was evaporated and the
concentrate was treated with 1 mL CH.sub.2Cl.sub.2 and 0.5 mL TFA.
After 5 min the solvents were evaporated and the crude oil was
diluted with water/DMF and purified by preparative RP-HPLC to yield
a white solid (137.2 mg, 69.3%). HRMS (ESI) calculated for
C.sub.22H.sub.44N.sub.4O.sub.4 (M+H).sup.+429.3435; found
429.3443.
[0551] NODA-BAEM: (BAEM=Butyl Amido Ethyl Maleimide)
[0552] To a solution of (tBu).sub.2NODA-BM (29.3 mg, 0.068 mmol) in
CH.sub.2Cl.sub.2 (3 mL) was added a 13-maleimido propionic acid NHS
ester (16.7 mg, 0.063 mmol), 20 .mu.L DIEA and stirred at room
temperature overnight. Solvent was evaporated and the concentrate
was acidified with 1 mL TFA. After 3 h, the reaction mixture was
diluted with water and purified by preparative RP-HPLC to yield a
white solid. HRMS (ESI) calculated for
C.sub.21H.sub.33N.sub.5O.sub.7 (M+H).sup.+ 468.2453; found
468.2441.
2-{4-[(4,7-bis-tert-butoxycarbonylmethyl)-[1,4,7]-triazacyclononan-1-yl)me-
thyl]phenyl}acetic acid. (tBu).sub.2NODA-MPAA
[0553] To a solution of 4-(bromomethyl)phenylacetic acid (593 mg,
2.59 mmol) in anhydrous CH.sub.3CN (50 mL) at 0.degree. C. were
added dropwise over 1 h a solution of (tBu).sub.2NODA (1008 mg,
2.82 mmol) in CH.sub.3CN (50 mL). After 4 h, anhydrous
K.sub.2CO.sub.3 (100.8 mg, 0.729 mmol) was added to the reaction
mixture and allowed to stir at room temperature overnight. Solvent
was evaporated and the crude was purified by preparative RP-HPLC
(Method 5) to yield a white solid (713 mg, 54.5%). .sup.1NMR (500
MHz, CDCl.sub.3, 25.degree. C., TMS) .delta. 1.45 (s, 18H),
2.64-3.13 (m, 16H), 3.67 (s, 2H), 4.38 (s, 2H), 7.31 (d, 2H), 7.46
(d, 2H); .sup.13C (125.7 MHz, CDCl.sub.3) .delta. 28.1, 41.0, 48.4,
50.9, 51.5, 57.0, 59.6, 82.3, 129.0, 130.4, 130.9, 136.8, 170.1,
173.3. HRMS (ESI) calculated for C.sub.27H.sub.43N.sub.3O.sub.6
(M+H).sup.+ 506.3225; found 506.3210.
2-(4-(carboxymethyl)-7-{[4-(carboxymethyl)phenyl]methyl}-1,4,7-triazacyclo-
nonan-1-yl)acetic acid. NODA-MPAA
[0554] To a solution of 4-(bromomethyl)phenylacetic acid (15.7 mg,
0.068 mmol) in anhydrous CH.sub.3CN at 0.degree. C. was added
dropwise over 20 min a solution of (tBu).sub.2NODA (26 mg, 0.073
mmol) in CH.sub.3CN (5 mL). After 2 h, anhydrous K.sub.2CO.sub.3 (5
mg) was added to the reaction mixture and allowed to stir at room
temperature overnight. Solvent was evaporated and the concentrate
was acidified with 2 mL TFA. After 3 h, the reaction mixture was
diluted with water and purified by preparative RP-HPLC to yield a
white solid (11.8 mg, 43.7%). .sup.1H NMR (500 MHz, DMSO-d.sub.6,
25.degree. C.) .delta. 2.65-3.13 (m, 12H), 3.32 (d, 2H), 3.47 (d,
2H), 3.61 (s, 2H), 4.32 (s, 2H), 7.33 (d, 2H), 7.46 (d, 2H);
.sup.13C (125.7 MHz, DMSO-d.sub.6) 40.8, 47.2, 49.6, 50.7, 55.2,
58.1, 130.4, 130.5, 130.9, 136.6, 158.4, 158.7, 172.8, 172.9. HRMS
(ESI) calculated for C.sub.19H.sub.27N.sub.3O.sub.6 (M+H).sup.+
394.1973; found 394.1979.
[0555] (tBu).sub.2NODA-MPAA NHS Ester.
[0556] To a solution of (tBu).sub.2NODA-MPAA (175.7 mg, 0.347 mmol)
in CH.sub.2Cl.sub.2 (5 mL) was added (1 M in CH.sub.2Cl.sub.2) DCC
(347 .mu.L, 0.347 mmol), N-hydroxysuccinimide (NHS) (42.5 mg, 0.392
mmol), and 20 .mu.L N,N-diisopropylethylamine (DIEA). After 3 h,
dicyclohexylurea (DCU) was filtered off and solvent evaporated. The
crude product was purified by flash chromatography on (230-400
mesh) silica gel (CH.sub.2Cl.sub.2:MeOH (100:0 to 80:20) to yield
the NHS ester (128.3 mg, 61.3%). HRMS (ESI) calculated for
C.sub.31H.sub.46N.sub.4O.sub.8 (M+H).sup.+ 603.3388; found
603.3395.
[0557] NODA-MPAEM: (MPAEM=Methyl Phenyl Acetamido Ethyl
Maleimide)
[0558] To a solution of (tBu).sub.2NODA-MPAA NHS ester (128.3 mg,
0.213 mmol) in CH.sub.2Cl.sub.2 (5 mL) was added a solution of
N-(2-aminoethyl) maleimide trifluoroacetate salt (52.6 mg, 0.207
mmol) in 250 .mu.L DMF and 20 .mu.L DIEA. After 3 h, the solvent
was evaporated and the concentrate treated with 2 mL TFA. The crude
product was diluted with water and purified by preparative RP-HPLC
to yield a white solid (49.4 mg, 45%). HRMS (ESI) calculated for
C.sub.25H.sub.33N.sub.5O.sub.7 (M+H).sup.+ 516.2453; found
516.2452.
tert-butyl-2-(7-(4-aminopropyl)-4-{[(tert-butyl)oxycarbonyl]methyl}-1,4,7--
triazacyclononan-1-yl)acetic acid. (tBu).sub.2NODA-PA
[0559] To a solution of (tBu).sub.2NODA (391.3 mg, 1.09 mmol) in 5
mL CH.sub.3CN was added Benzyl-3-bromo propyl carbamate (160 .mu.L)
and reaction mixture was stirred at room temperature for 28 h.
Solvent was evaporated and the concentrate was dissolved in 40 mL
2-propanol, mixed with 128.7 mg of 10% Pd-C and placed under 43 psi
H.sub.2 overnight. The product was then filtered and the filtrate
concentrated. The crude product was diluted with water/DMF and
purified by preparative RP-HPLC to yield a white solid (353 mg).
HRMS (ESI) calculated for C.sub.21H.sub.42N.sub.4O.sub.4
(M+H).sup.+ 415.3291; found 415.3279.
[0560] NODA-PAEM: (PAEM=Propyl Amido Ethyl Maleimide)
[0561] To a solution of (tBu).sub.2NODA-PM (109.2 mg, 0.263 mmol)
in CH.sub.2Cl.sub.2 mL) was added a .beta.-maleimido propionic acid
NHS ester (63.6 mg, 0.239 mmol), 20 .mu.L DIEA and stirred at room
temperature overnight. Solvent was evaporated and the concentrate
was acidified with 1 mL TFA. After 3 h, the reaction mixture was
diluted with water and purified by preparative RP-HPLC to yield a
white solid (79 mg). HRMS (ESI) calculated for
C.sub.20H.sub.31N.sub.5O.sub.7 (M+H).sup.+ 454.2319; found
454.2296.
[0562] .sup.18F-Labeling of Functionalized Triazacyclononane
Ligands
[0563] The functionalized triazacyclononane ligand (20 nmol; 10
.mu.L), dissolved in 2 mM sodium acetate (pH 4), was mixed with
AlCl.sub.3 (5 .mu.L of 2 mM solution in 2 mM acetate buffer, 25-200
.mu.L of .sup.18F.sup.- in saline, and 25-200 .mu.L of ethanol.
After heating at 90-105.degree. C. for 15-20 min, 800 .mu.L of
deionized (DI) water was added to the reaction solution, and the
entire contents removed to a vial (dilution vial) containing 1 mL
of deionized (DI) water. The reaction vial was washed with
2.times.1 mL DI water and added to the dilution vial. The crude
product was then passed through a 1-mL HLB column, which was washed
with 2.times.1 mL fractions of DI water. The labeled product was
eluted from the column using 3.times.200 .mu.L of 1:1 EtOH/water.
Radiochromatograms of the .sup.18F-labeling of functionalized TACN
ligands are shown in FIG. 19.
[0564] .sup.18F-Labeling of NODA-MPAEM
[0565] To 10 .mu.L (20 nmol) 2 mM NODA-MPAEM solution was added 5
.mu.L 2 mM AlCl.sub.3, 200 .mu.L .sup.18F.sup.- solution [15.94
mCi, Na.sup.18F, PETNET] followed by 200 .mu.L CH.sub.3CN and
heated to 110.degree. C. for 15 minutes. The crude reaction mixture
was purified by transferring the resultant solution into a
Oasis.RTM. HLB 1 cc (30 mg) cartridge (P#186001879, L#099A30222A)
and eluting with DI H.sub.2O to remove unbound .sup.18F.sup.-
followed by 1:1 EtOH/H.sub.20 to elute the .sup.18F-labeled
peptide. The crude reaction solution was pulled through the HLB
cartridge into a 10 mL vial and the cartridge washed with 6.times.1
mL fractions of DI H.sub.2O (4.34 mCi). The HLB cartridge was then
placed on a new 3 mL vial and eluted with 4.times.150 .mu.L 1:1
EtOH/H.sub.2O to collect the labeled peptide (7.53 mCi). The
reaction vessel retained 165.1 .mu.Ci, while the cartridge retained
270 .mu.Ci of activity. 7.53 mCi61.2% of Al
[.sup.18F]NODA-MPAEM.
[0566] .sup.18F-Labeling of NODA-MPAEM:
[0567] To 10 .mu.L (20 nmol) 2 mM NODA-MPAEM solution was added 5
.mu.L 2 mM AlCl.sub.3, 200 .mu.L .sup.18F.sup.- solution [15.94
mCi, Na.sup.18F, PETNET] followed by 200 .mu.L CH.sub.3CN and
heated to 110.degree. C. for 15 minutes. The crude reaction mixture
was purified by transferring the resultant solution into a
Oasis.RTM. HLB 1 cc (30 mg) cartridge (P#186001879, L#099A30222A)
and eluting with DI H.sub.2O to remove unbound .sup.18F.sup.-
followed by 1:1 EtOH/H.sub.20 to elute the .sup.18F-labeled
peptide. The crude reaction solution was pulled through the HLB
cartridge into a 10 mL vial and the cartridge washed with 6.times.1
mL fractions of DI H.sub.2O (4.34 mCi). The HLB cartridge was then
placed on a new 3 mL vial and eluted with 4.times.150 .mu.L 1:1
EtOH/H.sub.2O to collect the labeled peptide (7.53 mCi). The
reaction vessel retained 165.1 .mu.Ci, while the cartridge retained
270 .mu.Ci of activity. 7.53 mCi61.2% of Al
[.sup.18F]NODA-MPAEM.
TABLE-US-00040 TABLE 26 .sup.18F-labeling of 20 nmol NODA-MPAEM +
10 nmol Al.sup.3+ Activity.sup.a Na.sup.18F Aqueous CH.sub.3CN
Isolated activity.sup.b RCY.sup.c (mCi) (.mu.L) (.mu.L) (mCi) (%)
2.02 80 -- 1.09 64.1 1.86 40 40 1.43 91.0 1.96 50 50 1.371 90.8
3.26 200 200 2.05 76.0 15.08 200 200 8.24 70.3 15.94 200 200 7.53
61.2 .sup.a10 .mu.L NODA-MPAEM, 5 .mu.L Al.sup.3+, 105-110.degree.
C., 15 min. .sup.bIsolated activity in (1:1) EtOH/H.sub.2O after
HLB column purification (SPE). .sup.cdecay corrected RCY - based on
synthesis time of 27-42 minutes.
[0568] Conjugation of hMN14-Fab'-SH with
Al.sup.18F(NODA-MPAEM):
[0569] To the vial containing hMN14-Fab'-SH (1 mg, .about.20
nmoles) L #112310 was added 200 .mu.L PBS, pH 7.38 and 600 .mu.L of
the HLB purified Al.sup.18F(NODA-MPAEM) (EtOH:H.sub.2O::1:1). The
crude reaction mixture was passed through a sephadex (G-50/80, 0.1
M NaOAc, pH 6.5) 3 mL spin column. The activity in the eluate was
4.27 mCi, while 1.676 mCi was retained on the spin column and 0.178
mCi in the empty reaction vial. 4.27 mCi68.6% of
[Al.sup.18F]-hMN14-Fab.
To 800 .mu.L of PBS was added 1 .mu.L of eluateinjected 40 .mu.L
(SEC-HPLC) at 2.08 p.m. Major product at 10.312 min.
[0570] Serum Stability:
[0571] In an autosampler vial 200 .mu.L of fresh human serum+50
.mu.L of eluate149.6 .mu.Ci at 2.45 p.m. Incubated at 37.degree.
C.[Al.sup.18F]-hMN14-Fab in serum.
To 4 .mu.L of [Al.sup.18F]-hMN14-Fab in serum added 280 .mu.L of
buffer B (PBS) injected 40 .mu.L (SEC-HPLC) at 3.51 p.m. Major
product at 10.331 min.
[0572] Immunoreactivity of .sup.18F-hMN14-Fab:
[0573] To 100 .mu.g carcinoembryonic antigen (CEA) [L #2371505,
Scripp's Labs] was added 200 .mu.L 1% HSA in PBS, pH 7.38+100 .mu.L
PBS, pH 7.38CEA in PBS. Added 4 .mu.L of [Al.sup.18F]-hMN14-Fab in
serum at 37.degree. C. to 150 .mu.L CEA in PBSinjected 40 .mu.L
(SEC-HPLC) at 4.33 p.m. Major product at 7.208 min.
Radiochromatograms of spin column purified [Al.sup.18F]-hMN14-Fab,
stability of [Al.sup.18F]-hMN14-Fab in human serum and its
immunoreactivity with CEA are shown in FIG. 20.
[0574] Exemplary synthetic schemes for the bifunctional chelators
are shown below.
##STR00002##
##STR00003##
##STR00004##
##STR00005##
##STR00006##
##STR00007##
##STR00008##
##STR00009##
##STR00010##
##STR00011##
##STR00012##
##STR00013##
##STR00014##
##STR00015##
##STR00016##
##STR00017##
##STR00018##
##STR00019##
[0575] Exemplary structures of .sup.18F-labeled probes are shown
below.
##STR00020## ##STR00021## ##STR00022##
[0576] Conclusions
[0577] We have found that a novel class of triazacyclononane (TACN)
derived BFCs, possessing a functionality that provides for an easy
linkage onto biomolecules via solid phase or in solution, form
stable complexes with a variety of metals. These BFCs also form
remarkably stable Al.sup.18F chelates. Most .sup.18F-labeling
methods are tedious to perform, require the efforts of a
specialized chemist, involve multiple purifications of the
intermediates, anhydrous conditions, and generally end up with low
RCYs. An advantage of this new class of BFCs is that they can be
radiofluorinated rapidly in one step with high specific activity in
an aqueous medium.
Example 33. Further Optimization of Kit Formulation
[0578] The effect of varying buffer composition on labeling
efficiency was determined. Kits were formulated with 20 nmol IMP485
and 10 nmol AlCl.sub.3.6H.sub.2O in 5% .alpha.,.alpha.-trehalose.
The buffers and ascorbic acid were varied in the different
formulations. The peptide and trehalose were dissolved in DI water
and the AlCl.sub.3.6H.sub.2O was dissolved in the buffer
tested.
[0579] MES Buffer--
[0580] 4-morpholineethanesulfonic acid (YMS, Sigma M8250), 0.3901 g
(0.002 mol) was dissolved in 250 mL of DI H.sub.2O and adjusted to
pH 4.06 with acetic acid (8 mM buffer).
[0581] KHP Buffer--
[0582] Potassium biphthalate (KHP, Baker 2958-1), 0.4087 g (0.002
mol) was dissolved in 250 mL DI H.sub.2O pH 4.11 (8 mM buffer).
[0583] HEPES Buffer--
[0584] N-2 hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES,
Calbiochem 391338) 0.4785 g (0.002 mol) dissolved in 250 mL DI
H.sub.2O and adjusted to pH 4.13 with AcOH (8 mM buffer).
[0585] HOAc Buffer--
[0586] Acetic acid (HOAc, Baker 9522-02), 0.0305 g (0.0005 mol) was
dissolved in 250 mL DI H.sub.2O and adjusted to pH 4.03 with NaOH
(2 mM buffer).
[0587] The AlCl.sub.3.6H.sub.2O (Aldrich 23078) was dissolved in
the buffers to obtain a 2 mM solution of Al.sup.3+ in 2 mM buffer.
IMP 485 0.0011 g (MW 1311.67, 8.39.times.10.sup.-7 mol) was
dissolved in 419 .mu.L DI H.sub.2O._Ascorbic acid, 0.1007 g
(Aldrich 25,556-4, 5.72.times.10.sup.-4 mol) was dissolved in 20 mL
DI H.sub.2O.
[0588] A variety of kits (summarized in Table 27) were prepared and
adjusted to the proper pH by the addition of NaOH or HOAc as
needed. The solution was then dispensed in 1 mL aliquots into 4, 3
mL lyophilization vials, frozen on dry ice and lyophilized. The
initial shelf temperature for the lyophilization was -10.degree. C.
The samples were placed under vacuum and the shelf temperature was
increased to 0.degree. C. The samples were lyophilized for 15 hr
and the shelf temperature was increased to 20.degree. C. for 1 h
before the vials were sealed under vacuum and removed from the
lyophilizer. The kits were prepared with different buffers, at
different pH values, with or without ascorbic acid and with or
without acetate. After lyophilization, the kits were dissolved in
400 .mu.L of saline and the pH was measured with a calibrated pH
meter with a micro pH probe.
[0589] Radiolabeling--
[0590] The kits were all labeled with .sup.18F.sup.- in saline (200
.mu.L, PETNET) with ethanol (200 .mu.L) and heated to
.about.105.degree. C. for 15 min. The labeled peptides were diluted
with 0.6 mL DI H.sub.2O and then added to a dilution vial
containing 2 mL DI H.sub.2O. The reaction vial was washed with
2.times.1 mL portions of DI H.sub.2O, which were added to the
dilution vial. The diluted solution was filtered through a 1 mL (30
mg) HLB cartridge (1 mL at a time) and washed with 2 mL DI
H.sub.2O. The cartridge was moved to an empty vial and eluted with
3.times.200 .mu.L 1:1 EtOH/DI H.sub.2O. The Al[.sup.18F]MP485 was
in the 1:1 EtOH/DI H.sub.2O fractions. The isolated yield was
determined by counting the activity in the reaction vial, the
dilution vial, the HLB cartridge, the DI H.sub.2O column wash and
the 1:1 EtOH/DI H.sub.2O wash adding up the total and then dividing
the amount in the 1:1 EtOH/DI H.sub.2O fraction by the total and
multiplying by 100.
[0591] Results
[0592] The results of the studies are shown in Table 27. All the
labeling in the presence of 0.1 mg of ascorbic acid went well. The
ascorbic acid appears to serve as a significant non-volatile buffer
that keeps the pH the same before and after lyophilization (kits
1-4). When ascorbic acid is not used (kits 5-8) the pH can change
significantly along with the radiolabeling yield. The KHP buffer,
kit 8, was the best kit in the second batch. Higher levels of
ascorbate might also stabilize the Al.sup.18F complex in solution
and act as a transfer ligand for Al.sup.18F. The KHP buffer might
also act as a transfer ligand for Al.sup.18F so the amount of KHP
was increased from 5.times.10.sup.-7 mol/kit for kit 8 to
6.times.10.sup.-6 mol/kit for kit 11. The increase in KHP
stabilized the pH better than kit 8 and gave a much better labeling
yield. The kits with KHP+ascorbate (kit 12) and KHP+MES (kit 13)
had slightly higher labeling yields. It may be that the higher
levels of KHP and ascorbate act both as buffers and as transfer
ligands to increase the labeling yields with those excipients.
Citric acid is not a good buffer for [Al.sup.18F]-labeling (kit
14), it gives low labeling yields even when only 50 .mu.L of 2 mM
citrate was used in the presence 0.1 mg of ascorbate. Increasing
amounts of KHP, 0.1 M and above (kits 16-18) lead to lower labeling
yields with more activity found in the aqueous wash from the HLB
column.
TABLE-US-00041 TABLE 27 Results of labeling and pH studies pH
before pH after Isolated Kit/lot Buffer lyophilization
lyophilization % yield 1. HEPES + ascorbic 4.07 3.83 82.6 2. MES +
ascorbic 4.08 3.99 83.5 3. NaOAc + ascorbic 4.10 4.10 83.5 4. KHP +
ascorbic 4.12 4.10 86.1 5. HEPES No ascorbic + 4.10 4.24 45.4 HOAc
6. MES No ascorbic + HOAc 4.07 4.25 66.4 7. HOAc No ascorbic + 4.11
4.50 35.2 HOAc 8. KHP No ascorbic + HOAc 4.01 4.09 71 9. HEPES No
ascorbic 4.07 4.44 69 or NaOAc 10. MES No ascorbic 4.06 4.62 75 or
NaOAc 11. BM 20-57 KHP 4.07 4.05 83.9 alone 0.015M 12. BM 20-57 KHP
4.03 3.96 85.8 0.015M + ascorbic 13. BM 20-57 KHP 4.10 4.09 87.0
0.015M + MES 0.015M 14. Citric acid 4.03 3.93 31.2 15. Ascorbic
acid 4.13 4.04 80.0 16. KHP 0.1M 4.09 3.82 80.7 17. KHP 0.2M 4.05
3.79 78.1 18. KHP 0.4M 4.02 3.79 69.0
[0593] It appears from these results that potassium biphthalate is
an optimal buffer for labeling. The peptide labeling kits were
therefore reformulated to utilize KHP in the labeling buffer. The
reformulated kits gave very high isolated labeling yields of about
97% when 100 nmol of peptide was labeled in 1:1 ethanol/saline. The
labeling and purification time was also simplified and reduced to
20 min. In addition to using the new buffering agent, potassium
biphthalate (KHP), we also added more moles of buffer, which may
help stabilize the pH during labeling. The peptide is purified
through an Alumina N cartridge by adding more saline to the
reaction after heating and pushing crude product through the
cartridge. The unbound .sup.18F.sup.- and Al.sup.18F stick to the
alumina and the labeled peptide is eluted very efficiently from the
cartridge with saline. The formulation shown below is for a 20 nmol
peptide kit but the same formulation is used for a 100 nmol peptide
kit by adding more peptide and more Al.sup.3+ (60 nmol Al.sup.3+
for the 100 nmol peptide kit).
[0594] 2 mM Al.sup.3+ in 2 mM KHP--
[0595] Aluminum chloride hexahydrate, 0.0196 g
(8.12.times.10.sup.-5 mol, Aldrich 23078, MW 241.43) was dissolved
in 40.6 mL of 2 mM potassium biphthalate (KHP, JT Baker 2958-1, MW
204.23). This can be stored at room temperature for months.
[0596] Ascorbic Acid--
[0597] Ascorbic acid, 0.100 g was dissolved in 20 mL DI H.sub.2O.
This is made fresh on the day of use.
[0598] 5% Trehalose--
[0599] .alpha.,.alpha.-Trehalose dihydrate, 2.001 g (JT Baker,
4226-04, MW 378.33) was dissolved in 20 mL DI H.sub.2O. This can be
stored at room temperature for weeks.
[0600] KHP Kit Buffer--
[0601] KHP, 0.2253 g was dissolved in 18 mL DI H.sub.2O (0.06 M).
This solution can be kept for months at room temperature.
[0602] IMP485 Solution--
[0603] IMP485, 0.0049 g (3.74.times.10.sup.-6 mol, MW 1311.67) was
dissolved in 1.494 mL DI H.sub.2O (2.5.times.10.sup.-3M). This
solution can be stored for months at -20.degree. C.
[0604] 1M KOH--
[0605] Potassium hydroxide (99.99% semiconductor grade, MW 56.11,
Aldrich 306568) was dissolved in DI H.sub.2O to make a 1 M
solution.
[0606] Kit Formulation (20 nmol kit, 40 kits)
[0607] The peptide, IMP485 (320 .mu.L, 8.times.10.sup.-7 mol) was
placed in a 50 mL sterile polypropylene centrifuge tube (metal
free) and mixed with 240 .mu.L of the 2 mM Al.sup.3+ solution
(4.8.times.10.sup.-7 mol) 800 .mu.L of the ascorbic acid solution,
1600 .mu.L of the 0.06 M KHP solution, 8 mL of the 5% trehalose
solution and the mixture was diluted to 40 mL with DI H.sub.2O. The
solution was adjusted to pH 3.99-4.03 with a few microliters of 1 M
KOH. The peptide solution was dispensed 1 mL/vial with a 1 mL
pipette into 3 mL glass lyophilization vials (unwashed).
[0608] Lyophilization--
[0609] The vials were frozen on dry ice, fitted with lyophilization
stoppers and placed on a -20.degree. C. shelf in the lyophilizer.
The vacuum pump was turned on and the shelf temperature was raised
to 0.degree. C. after the vacuum was below 100 mtorr. The next
morning the shelf temperature was raised to 20.degree. C. for 4 hr
before the samples were closed under vacuum and crimp sealed.
[0610] Radiolabeling--
[0611] The .sup.18F.sup.- in saline was received from PETNET in 200
.mu.L saline in a 0.5 mL tuberculin syringe. Ethanol, 200 .mu.L,
was pulled into the .sup.18F.sup.- solution and then the mixture
was injected into a lyophilized kit containing the peptide. The
solution was then heated in a 105.degree. C. heating block for 15
min. Sterile saline, 0.6 mL was then added to the reaction vial and
the solution was removed from the vial and pushed through an
alumina N cartridge (SEP-PAK light, WAT023561, previously washed
with 5 mL sterile saline) into a collection vial. The reaction vial
was washed with 2.times.1 mL saline and the washes were pushed
through the alumina column. The total labeling and purification
time was about 20 min.
Example 34. Labeling at Reduced Temperature
[0612] The effect of varying the chelator structure on efficiency
of labeling at reduced temperature was examined. A comparison of
low temperature labeling of IMP466 (NOTA-Octreotide) with IMP485
showed that the simple NOTA ligand labels much better at low
temperature than the NODA-MPAA ligand.
[0613] In one embodiment, a temperature sensitive molecule, such as
a protein, may be conjugated to multiple copies of a simple NOTA
ligand. The protein can then be purified and formulated for
Al.sup.18F-labeling (e.g., lyophilized). The protein kit was
reconstituted with .sup.18F in saline, heated for the appropriate
length of time and purified by gel filtration or an alumina column.
Tables 27 and 28 show the temperature effects of labeling IMP466
vs. IMP485.
TABLE-US-00042 TABLE 28 Temperature-dependent labeling for
Al.sup.18F(IMP466) Temp % Yield % Yield % Yield % Yield % Yield
.degree. C. 25 .mu.M 50 .mu.M 100 .mu.M 250 .mu.M 500 .mu.M 50 3.3
8.6 14.5 20.5 37.1 70 21.3 47.4 58.0 82.8 93.6 90 29.0 50.7 70.3
83.0 93.5 100 34.3 55.8 77.4 84.0 94.5 110 34.9 60.3 78.1 87.9
90.5
TABLE-US-00043 TABLE 29 Temperature-dependent labeling for
Al.sup.18F(IMP485) Temp % Yield % Yield % Yield % Yield % Yield
.degree. C. 25 .mu.M 50 .mu.M 100 .mu.M 250 .mu.M 500 .mu.M 50 1.31
3.29 3.18 6.10 12.99 70 7.01 12.8 22.90 36.8 39.8 90 22.2 38.3 82.3
85.4 85.3 100 48.6 76.1 91.8 94.6 96.6 110 61.6 74.4 96.4 94.0
96.8
[0614] The data show that by switching to a different chelating
moiety, the efficiency of low temperature labeling with Al.sup.18F
may be tripled at 50.degree. C. Further modification of the
chelating moiety may provide additional improvement of low
temperature labeling. However, the 37% efficiency observed with
IMP466 is sufficient to enable .sup.18F PET imaging with
temperature sensitive molecules if a sufficient number of chelating
moieties are attached to the molecule.
[0615] We have also examined the effect of peptide concentration on
low temperature labeling of IMP485. Kits were made with 10, 20, 40,
100 and 200 nmol of peptide and 0.6 equivalents of Al.sup.3+
respectively. The rest of the formulation was the same for all of
the kits. The kits were labeled with 400 .mu.L saline/EtOH and
heated at 50-110.degree. C. for 15 min and then purified through
the Alumina N cartridge. The labeling results are reported as
isolated yields in Table 30. At any temperature, increasing the
concentration of peptide increased the efficiency of labeling. The
results indicate that if the reaction volume can be decreased with
the use of a microfluidics device then we can greatly reduce the
amount of peptide and .sup.18F.sup.- needed to prepare a single
dose of labeled peptide for PET imaging.
TABLE-US-00044 TABLE 30 Effect of peptide concentration on
efficiency of labeling as a function of temperature. Temp % Yield %
Yield % Yield % Yield % Yield .degree. C. 25 .mu.M 50 .mu.M 100
.mu.M 250 .mu.M 500 .mu.M 50 1.31 3.29 3.18 6.10 12.99 70 7.01 12.8
22.90 36.8 39.8 90 22.2 38.3 82.3 85.4 85.3 100 48.6 76.1 91.8 94.6
96.6 110 61.6 74.4 96.4 94.0 96.8
Example 35. Automated Synthesis of .sup.18F-Labeled Molecules
[0616] This Example compared the automated synthesis of
.sup.18F-FBEM published by Kiesewetter et al., (2011, Appl Radiat
Isot 69:410-4) to that of Al.sup.18F(NODA-MPAEM). The automated
synthesis of .sup.18F-FBEM was accomplished using a sophisticated
synthesis module (see below), with a RCY of 17% in 95 min. Our
synthesis module (FIG. 21) would include a heating device and a HLB
cartridge or HPLC column. With NODA-MPAEM we were able to get
67-79% RCY (decay corrected) in 40 min in one single step.
##STR00023##
[0617] In both .sup.18F-FBEM and .sup.18F-FDG-MHO, the .sup.18F is
introduced first followed by a maleimide (Scheme 20 and 21). While
NODA-MPAEM--a maleimide containing BFC--is .sup.18F-labeled in one
final step (Scheme 19).
##STR00024##
##STR00025##
TABLE-US-00045 Prosthetic Synthesis Synthesis group module time
Specific activity RCY (%) .sup.18F-SFB TRACERlab .TM. 98 min 44.3
.+-. 2.5 250-350 8-12 GBq MX.sub.FDG GBq/.mu.mol (216-324 (6.8-9.5
Ci/.mu.mol) mCi) RCY* (%) .sup.18F-FBEM Eckert & 95 min 17.3
.+-. 7.1 181-351 Ziegler GBq/.mu.mol (4.9-9.5 Ci/.mu.mol) With 8.2
GBq (222 mCi) .sup.18F.sup.- provide 1.87 GBq (50.6 mCi) of
.sup.18F-FBEM in 96 min (22.8% uncorrected; 41.7% corrected for
decay). *not decay corrected.
Example 34. Room Temperature Labeling of Targeting Molecules Using
Bifunctional Chelator (BFC) Moieties
[0618] The objective of this Example was to perform
.sup.18F-labeling of temperature sensitive molecules at reduced
temperatures, such as room temperature, with high radiochemical
yield and high specific activity of the labeled molecule.
Preferably, the labeling reaction is accomplished in 10 to 15
minutes in aqueous medium, with a total synthesis time of 30
minutes or less. More preferably, the labeling technique involves
the initial reaction of a metal-.sup.18F or metal-.sup.19F with a
bifunctional chelating (BFC) moiety at elevated temperature (e.g.,
90 to 105.degree. C.), followed by site-specific attachment of the
BFC to the targeting molecule at a reduced temperature (e.g., room
temperature). In certain embodiments, the BFC may be derived from
the structure of NODA-propyl amine (FIG. 22).
[0619] IMP508 (FIG. 23A) and IMP517 (FIG. 23B) were synthesized as
disclosed below.
[0620] The NODA chelating moiety formed according to schemes 22 and
23 was attached to a bis-HSG peptide (IMP508), formulated into 20
nmol peptide kits and labeled with .sup.18F.
##STR00026##
##STR00027##
[0621] The methyl ester was synthesized as follows. The
NO.sub.2AtBu, 1.0033 g (2.807.times.10.sup.-3 mol) was mixed with
0.4638 g (2.810.times.10.sup.-3 mol) of the methyl
6-formylnicotinate and dissolved in 10 mL THF.
Triacetoxyborohydride, 0.6248 g (2.948.times.10.sup.-3 mol) was
added and the reaction was stirred at room temperature for two days
and an additional 0.3044 g of the borohydride was added. The
reaction was quenched with H.sub.2O after stirring 6.5 hr more at
room temp. The product was extracted with dichloromethane, dried
over Na.sub.2SO.sub.4, filtered and concentrated under reduced
pressure to obtain the crude brown product. The product was
purified by flash chromatography eluting with hexanes, 25%
EtOAc/hexanes, 50% EtOAc/hexanes, 75% EtOAc/hexanes, 100% EtOAc,
dichloromethane, 5% MeOH/94% dichloromethane/1% triethylamine and
10% MeOH/89% dichloromethane/1% triethylamine. The product, was
isolated as a brown tar 0.455 g and was in the
MeOH/dichloromethane/triethylamine fractions.
[0622] To synthesize the acid, the methyl ester (0.411 g,
8.12.times.10.sup.-4 mol) was dissolved in 5 mL dioxane and stirred
with 0.8 mL of 1 M NaOH. The reaction was stirred for 18 hr at room
temperature and another 1.3 mL of NaOH was added in portions as the
reaction stirred at room temperature for another 8 hr. The reaction
was quenched with 1 M citric acid and adjusted to pH 4.91 with 1 M
NaOH. The product was extracted with dichloromethane. Some
saturated NaCl solution was added to the aqueous layer and the
solution was again extracted with dichloromethane. The organic
layers were combined, dried over Na.sub.2SO.sub.4, and concentrated
to obtain 0.3421 g of the product (85% yield).
[0623] IMP517 was produced as disclosed in Scheme 24. The methyl
ester triazole precursor was hydrolyzed and conjugated to the
bis-HSG peptide to obtain IMP517 (FIG. 23B).
##STR00028##
[0624] IMP517 was test labeled with different concentrations of
peptide in 400 .mu.L of saline.
[0625] IMP485 was also labeled in 400 .mu.L of saline for
comparison.
TABLE-US-00046 Peptide/nmol labeled in 400 .mu.L saline 110.degree.
C., 15 min Isolated % Yield IMP517 2.5 nmol 5.54 IMP517 5 nmol 31.1
IMP517 10 nmol 66.9 IMP517 20 nmol 85.8 IMP485 20 nmol LSNE Kit
78.3
[0626] IMP517 was labeled with F-18 in 400 .mu.L of 1:1 EtOH/saline
at different temperatures for 15 min.
TABLE-US-00047 IMP517 (20 nmol) Labeling temp. .degree. C. Isolated
% Yield 50 5.8 60 19.3 70 31.6 90 72.4 100 86.9 110 93.1
[0627] FIG. 24 compares the labeling of IMP517 20 nmol kits in 400
.mu.L of 1:1 EtOH/saline heated for 15 min. IMP517 gave the highest
labeling yields of the ligands tested so far and also gave high
yields in saline alone. New NODA derivatives with different
functional groups in the vicinity of the 1,4,7-triazacyclononane
ring were prepared and attached to a standard test peptide. The
peptides were radiolabeled over a range of temperatures from 50 to
110.degree. C. with and without a co-solvent. Two of these
derivatives containing a pyridyl or a triazole group showed
improved labeling yields at lower temperatures as well as labeling
equal or better than the benzyl-NODA standard at higher
temperatures. Adding ethanol to the triazole derivative did not
increase yields as much as the other derivatives, indicating that
it may be possible to improve the radiolabeling yield at lower
temperatures and reduce or eliminate the need for a co-solvent.
[0628] Alterations to the NODA/NOTA ligand on a peptide can have a
positive effect on the radiolabeling yield of the peptide, and may
lead to ligands that can be used for direct one-step .sup.18F
labeling of some temperature-sensitive molecules.
Example 35. Non-Peptide, Small Molecule-Imaging Agents
[0629] A NODA-2-nitroimidazole derivative (50 nmol, I mL) (FIG.
23C) used for hypoxia imaging was labeled in 0.1 M, pH 4, NaOAc
buffer by mixing with 22.5 .mu.L of 2 mM AlCl.sub.3.6H.sub.2O (45
nmol) in 0.1 M pH 4 NaOAc, and 50 .mu.L of .sup.18F.sup.- in
saline, then heating at 110.degree. C. for 10 min to obtain the
labeled complex in 85% yield. In vivo studies with the
Al.sup.18F-NODA-2-nitroimidazole showed the expected
biodistribution and tumor targeting, with no evidence of product
instability. The NOTA-DUPA-Pep molecule (FIG. 23D) was made for
targeting the prostate-specific membrane antigen (PSMA). The
.sup.18F-labeled molecule was synthesized in 79% yield after HPLC
purification to remove the unlabeled targeting agent.
Example 36. Large Peptide and Protein Labeling
[0630] NOTA-N-ethylmaleimide was attached to a cysteine side chain
of the 40 amino acid exendin-4 peptide, which targets the
glucagon-like peptide type-1 receptor (GLP-1 receptor) (Kiesewetter
et al., 2012, Tharanostics 2:999-1009). The peptide was labeled
with .sup.18F.sup.-, using unpurified cyclotron target water to
obtain the labeled peptide in 23.6.+-.2.4% uncorrected yield in 35
min. The Al.sup.18F-labeled peptide had 15.7.+-.1.4% ID/g in the
tumor and 79.25.+-.6.20% ID/g in the kidneys at 30 min, with low
uptake in all other tissues.
[0631] The NOTA-affibody Z.sub.HER2:2395 (58-amino acid, 7 kDa) was
labeled at 90.degree. C. for 15 min with Al.sup.18F, the affibody,
and acetonitrile (Heskamp et al., 2012, J Nucl Med 53:146-53). The
labeling and purification process took about 30 min and the yield
was 21.+-.5.7%. Again, biodistribution studies supported the
stability of the product with negligible bone uptake.
[0632] We also examined a two-step labeling method for
temperature-sensitive molecules. The NODA-MPAA ligand was attached
to N-ethylmaleimide to make NODA-MPAEM. The NODA-MPAEM (20 nmol in
10 .mu.L 2 mM, pH 4, NaOAc) was mixed with 5 .mu.L 2 mM AlCl.sub.3
in 2 mM, pH 4, NaOAc followed by 200 .mu.L .sup.18F.sup.- in saline
and 200 .mu.L of acetonitrile. The solution was heated at
105-109.degree. C. for 15 min and purified by SPE to produce the
Al.sup.18F-NODA-MPAEM in 80% yield. This product was then coupled
to a pre-reduced antibody Fab' fragment (20 nmol) by mixing the
purified Al.sup.18F-NODA-MPAEM at room temperature for 10 min,
followed by isolation of the labeled Fab' by gel filtration. The
labeled protein was obtained in an 80% yield. The total synthesis
time for both steps combined was about 50 min, with an overall
decay-corrected yield of about 50-60%.
Example 37. Residualization and In Vivo Clearance of Al.sup.18F
Complexes
[0633] Lang et al. compared the biodistribution of .sup.18F on
carbon, Al.sup.18F and .sup.68Ga attached to the same NOTA-PRGD2
peptide in the U-87 MG human glioblastoma model (Lang et al., 2011,
Bioconjugate Chem 22:2415-22). They found that tumor uptake of the
.sup.18F-PPRGD2 peptide was 3.65.+-.0.51% ID/g at 30 min PI
compared to 1.85.+-.0.30% ID/g at 2 h, indicating that the .sup.18F
activity was slowly clearing from the tumor between 30 min and 2 h
(51% retention). The metal-complexed RGD peptides had higher tumor
retention [4.20.+-.0.23% ID/g (30 min), 3.53.+-.0.45% ID/g (2 h) or
84% retention for Al.sup.18F-NOTA-PRGD2, and 3.25.+-.0.62% ID/g (30
min), 2.66.+-.0.32% ID/g (2 h), or 82% retention
.sup.68Ga-NOTA-PRGD2] over the same period. These data show that
the chelated AlF complex may be retained better in the tumor than
the radiofluorinated compound with .sup.18F bound to a carbon atom.
The retention of activity also was seen with the exendin peptide
and the affibody, where the activity cleared from the kidneys when
the .sup.18F was attached to a carbon atom (Kiesewetter et al.,
2012, Eur J Nucl Med Mol Imaging 39:463-73; Kramer et al., 2008,
Eur J Nucl Med Mol Imaging 35:1008-18), but was retained with the
Al.sup.18F complex (Kiesewetter et al., 2012, Theranostics
2:999-1009; Heskamp et al., 2012, J Nucl Med 53:146-53). Retention
of the radionuclide in a tissue could provide a targeting
advantage, particularly in rapidly metabolizing tissues, such as
damaged heart tissue.
Sequence CWU 1
1
6014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term
DOTAMOD_RES(2)..(2)Lys(HSG)MOD_RES(4)..(4)Lys(HSG)C-term
amidatedsee specification as filed for detailed description of
substitutions and preferred embodiments 1Phe Lys Tyr Lys 1
24PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term
acetylatedMOD_RES(1)..(1)Lys(DTPA)MOD_RES(3)..(3)Lys(DTPA)MOD_RES(4)..(4)-
Lys(Tscg-Cys); branched sequence with C-term Lys which has a
(Tscg-Cys) side-chain; Cys is not the C-term residueC-term amidated
2Lys Tyr Lys Lys 1 35PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term
DTPAMOD_RES(3)..(3)Lys(HSG)MOD_RES(4)..(4)D-TyrMOD_RES(5)..(5)Lys(HSG)C-t-
erm amidated 3Gln Ala Lys Tyr Lys 1 5 44PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
NOTA-ITC
benzyl-ITCMOD_RES(1)..(1)D-AlaMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3-
)..(3)D-TyrMOD_RES(4)..(4)D-Lys(HSG)C-term amidated 4Ala Lys Tyr
Lys 1 54PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(1)..(1)D-AlaMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3)..(3)D-TyrM-
OD_RES(4)..(4)D-Lys(HSG)C-term amidated 5Ala Lys Tyr Lys 1
644PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro
Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 745PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 7Cys Gly His Ile Gln Ile
Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly 1 5 10 15 Tyr Thr Val Glu
Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe 20 25 30 Ala Val
Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 45
817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 921PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 9Cys Gly Gln Ile Glu Tyr
Leu Ala Lys Gln Ile Val Asp Asn Ala Ile 1 5 10 15 Gln Gln Ala Gly
Cys 20 1055PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser His Ile Gln Ile 1 5 10 15 Pro Pro Gly Leu Thr Glu Leu Leu Gln
Gly Tyr Thr Val Glu Val Leu 20 25 30 Arg Gln Gln Pro Pro Asp Leu
Val Glu Phe Ala Val Glu Tyr Phe Thr 35 40 45 Arg Leu Arg Glu Ala
Arg Ala 50 55 1129PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 11Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gln Ile Glu Tyr 1 5 10 15 Leu Ala Lys Gln Ile Val Asp
Asn Ala Ile Gln Gln Ala 20 25 1251PRTHomo sapiens 12Ser Leu Arg Glu
Cys Glu Leu Tyr Val Gln Lys His Asn Ile Gln Ala 1 5 10 15 Leu Leu
Lys Asp Val Ser Ile Val Gln Leu Cys Thr Ala Arg Pro Glu 20 25 30
Arg Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Lys Leu Glu Lys Glu 35
40 45 Glu Ala Lys 50 1354PRTHomo sapiens 13Ser Leu Lys Gly Cys Glu
Leu Tyr Val Gln Leu His Gly Ile Gln Gln 1 5 10 15 Val Leu Lys Asp
Cys Ile Val His Leu Cys Ile Ser Lys Pro Glu Arg 20 25 30 Pro Met
Lys Phe Leu Arg Glu His Phe Glu Lys Leu Glu Lys Glu Glu 35 40 45
Asn Arg Gln Ile Leu Ala 50 1444PRTHomo sapiens 14Ser His Ile Gln
Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val
Glu Val Gly Gln Gln Pro Pro Asp Leu Val Asp Phe Ala Val 20 25 30
Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Arg Gln 35 40 1544PRTHomo
sapiens 15Ser Ile Glu Ile Pro Ala Gly Leu Thr Glu Leu Leu Gln Gly
Phe Thr 1 5 10 15 Val Glu Val Leu Arg His Gln Pro Ala Asp Leu Leu
Glu Phe Ala Leu 20 25 30 Gln His Phe Thr Arg Leu Gln Gln Glu Asn
Glu Arg 35 40 1644PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMOD_RES(1)..(1)Ser or
ThrMOD_RES(2)..(2)His, Lys or ArgMOD_RES(4)..(4)Gln or
AsnMOD_RES(8)..(8)Gly or AlaMOD_RES(10)..(10)Thr or
SerMOD_RES(11)..(11)Glu or AspMOD_RES(14)..(14)Gln or
AsnMOD_RES(15)..(15)Gly or AlaMOD_RES(17)..(17)Thr or
SerMOD_RES(19)..(19)Glu or AspMOD_RES(22)..(22)Arg or
LysMOD_RES(23)..(24)Gln or AsnMOD_RES(27)..(27)Asp or
GluMOD_RES(30)..(30)Glu or AspMOD_RES(32)..(32)Ala, Leu, Ile or
ValMOD_RES(34)..(34)Glu or AspMOD_RES(37)..(37)Thr or
SerMOD_RES(38)..(38)Arg or LysMOD_RES(40)..(40)Arg or
LysMOD_RES(41)..(41)Glu or AspMOD_RES(42)..(42)Ala, Leu, Ile or
ValMOD_RES(43)..(43)Arg or LysMOD_RES(44)..(44)Ala, Leu, Ile or Val
16Xaa Xaa Ile Xaa Ile Pro Pro Xaa Leu Xaa Xaa Leu Leu Xaa Xaa Tyr 1
5 10 15 Xaa Val Xaa Val Leu Xaa Xaa Xaa Pro Pro Xaa Leu Val Xaa Phe
Xaa 20 25 30 Val Xaa Tyr Phe Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 35 40
1717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Gln or AsnMOD_RES(2)..(2)Ile, Leu
or ValMOD_RES(3)..(3)Glu or AspMOD_RES(4)..(4)Tyr, Phe, Thr or
SerMOD_RES(5)..(5)Leu, Ile or ValMOD_RES(7)..(7)Lys or
ArgMOD_RES(8)..(8)Gln or AsnMOD_RES(11)..(11)Asp or
GluMOD_RES(12)..(12)Asn or GlnMOD_RES(15)..(16)Gln or
AsnMOD_RES(17)..(17)Ala, Leu, Ile or Val 17Xaa Xaa Xaa Xaa Xaa Ala
Xaa Xaa Ile Val Xaa Xaa Ala Ile Xaa Xaa 1 5 10 15 Xaa
1817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Gln Ile Glu Tyr Val Ala Lys Gln Ile Val Asp Tyr
Ala Ile His Gln 1 5 10 15 Ala 1917PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 19Gln Ile Glu Tyr Lys Ala
Lys Gln Ile Val Asp His Ala Ile His Gln 1 5 10 15 Ala
2017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Gln Ile Glu Tyr His Ala Lys Gln Ile Val Asp His
Ala Ile His Gln 1 5 10 15 Ala 2117PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 21Gln Ile Glu Tyr Val Ala
Lys Gln Ile Val Asp His Ala Ile His Gln 1 5 10 15 Ala
2224PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp
Ala Val Ile Glu 1 5 10 15 Gln Val Lys Ala Ala Gly Ala Tyr 20
2318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Leu Glu Gln Tyr Ala Asn Gln Leu Ala Asp Gln Ile
Ile Lys Glu Ala 1 5 10 15 Thr Glu 2420PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Phe
Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser Asp Val 1 5 10
15 Phe Gln Gln Cys 20 2517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 25Gln Ile Glu Tyr Leu Ala Lys
Gln Ile Pro Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala 2625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Lys
Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala 1 5 10
15 Val Ile Glu Gln Val Lys Ala Ala Gly 20 25 2725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Lys
Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Pro Asp Ala 1 5 10
15 Pro Ile Glu Gln Val Lys Ala Ala Gly 20 25 2825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 2925PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Pro
Glu Asp Ala Glu Leu Val Arg Thr Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 3025PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Asp Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 3125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 31Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 3225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn 1 5 10
15 Ala Pro Leu Lys Ala Val Gln Gln Tyr 20 25 3325PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Glu Lys Ala Val Gln Gln Tyr 20 25 3425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Glu
Glu Gly Leu Asp Arg Asn Glu Glu Ile Lys Arg Ala Ala Phe Gln 1 5 10
15 Ile Ile Ser Gln Val Ile Ser Glu Ala 20 25 3525PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Leu
Val Asp Asp Pro Leu Glu Tyr Gln Ala Gly Leu Leu Val Gln Asn 1 5 10
15 Ala Ile Gln Gln Ala Ile Ala Glu Gln 20 25 3625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 36Gln
Tyr Glu Thr Leu Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn 1 5 10
15 Ala Ile Gln Leu Ser Ile Glu Gln Leu 20 25 3725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Leu
Glu Lys Gln Tyr Gln Glu Gln Leu Glu Glu Glu Val Ala Lys Val 1 5 10
15 Ile Val Ser Met Ser Ile Ala Phe Ala 20 25 3825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Asn
Thr Asp Glu Ala Gln Glu Glu Leu Ala Trp Lys Ile Ala Lys Met 1 5 10
15 Ile Val Ser Asp Ile Met Gln Gln Ala 20 25 3925PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 39Val
Asn Leu Asp Lys Lys Ala Val Leu Ala Glu Lys Ile Val Ala Glu 1 5 10
15 Ala Ile Glu Lys Ala Glu Arg Glu Leu 20 25 4025PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Asn
Gly Ile Leu Glu Leu Glu Thr Lys Ser Ser Lys Leu Val Gln Asn 1 5 10
15 Ile Ile Gln Thr Ala Val Asp Gln Phe 20 25 4125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Thr
Gln Asp Lys Asn Tyr Glu Asp Glu Leu Thr Gln Val Ala Leu Ala 1 5 10
15 Leu Val Glu Asp Val Ile Asn Tyr Ala 20 25 4225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 42Glu
Thr Ser Ala Lys Asp Asn Ile Asn Ile Glu Glu Ala Ala Arg Phe 1 5 10
15 Leu Val Glu Lys Ile Leu Val Asn His 20 25 4344PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMOD_RES(1)..(1)Ser or ThrMOD_RES(4)..(4)Gln or
AsnMOD_RES(10)..(10)Thr or SerMOD_RES(18)..(18)Val, Ile, Leu or
AlaMOD_RES(23)..(23)Gln or AsnMOD_RES(33)..(33)Val, Ile, Leu or
AlaMOD_RES(34)..(34)Glu or AspMOD_RES(37)..(37)Thr or
SerMOD_RES(38)..(38)Arg or LysMOD_RES(40)..(40)Arg or
LysMOD_RES(42)..(42)Ala, Leu, Ile or ValMOD_RES(44)..(44)Ala, Leu,
Ile or Val 43Xaa His Ile Xaa Ile Pro Pro Gly Leu Xaa Glu Leu Leu
Gln Gly Tyr 1 5 10 15 Thr Xaa Glu Val Leu Arg Xaa Gln Pro Pro Asp
Leu Val Glu Phe Ala 20 25 30 Xaa Xaa Tyr Phe Xaa Xaa Leu Xaa Glu
Xaa Arg Xaa 35 40 444PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term
NODA-GaMOD_RES(1)..(1)D-AlaMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3)..(3)D-TyrM-
OD_RES(4)..(4)D-Lys(HSG)C-term amidated 44Ala Lys Tyr Lys 1
454PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term
NOTAMOD_RES(1)..(1)D-AlaMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3)..(3)D-TyrMOD_-
RES(4)..(4)D-Lys(HSG)C-term amidated 45Ala Lys Tyr Lys 1
464PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term
NOTAMOD_RES(1)..(1)D-AspMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3)..(3)D-TyrMOD_-
RES(4)..(4)D-Lys(HSG)C-term amidated 46Asp Lys Tyr Lys 1
473PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term
C-NETA-succinylMOD_RES(1)..(1)D-Lys(HSG)MOD_RES(2)..(2)D-TyrMOD_RES(3)..(-
3)D-Lys(HSG)C-term amidated 47Lys Tyr Lys 1 484PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
NOTAMOD_RES(1)..(1)D-AlaMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3)..(3)D-TyrMOD_-
RES(4)..(4)D-Lys(HSG)C-term amidated 48Ala Lys Tyr Lys 1
4910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term NOTA-NH-(CH2)7COC-term amidated 49Gln Trp
Val Trp Ala Val Gly His Leu Met 1 5 10 508PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
NOTAMOD_RES(1)..(1)D-PheMOD_RES(4)..(4)D-TrpMOD_RES(8)..(8)Threoninol
50Phe Cys Phe Trp Lys Thr Cys Thr1 5 514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
DOTAMOD_RES(1)..(1)D-TyrMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3)..(3)D-GluMOD_-
RES(4)..(4)D-Lys(HSG)C-term amidated 51Tyr Lys Glu Lys 1
528PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term
NODA-MPAAMOD_RES(1)..(1)D-Phemisc_feature(2)..(7)CyclicMOD_RES(4)..(4)D-T-
rpMOD_RES(8)..(8)Threoninol 52Phe Cys Phe Trp Lys Thr Cys Thr1 5
538PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term NODA-MPAA-(PEG)3C-term amidated 53Gln Trp
Ala Val Gly His Leu Met 1 5 544PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term
NODA-HAMOD_RES(1)..(1)D-AlaMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3)..(3)D-TyrM-
OD_RES(4)..(4)D-Lys(HSG)C-term amidated 54Ala Lys Tyr Lys 1
553PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term
NODA-MPAAMOD_RES(1)..(1)D-Lys(HSG)MOD_RES(2)..(2)D-TyrMOD_RES(3)..(3)D-Ly-
s(HSG)C-term amidated 55Lys Tyr Lys 1 563PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
NODA-EPA-succinylMOD_RES(1)..(1)D-Lys(HSG)MOD_RES(2)..(2)D-TyrMOD_RES(3).-
.(3)D-Lys(HSG)C-term amidated 56Lys Tyr Lys 1 574PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideN-term
NODA-EBAMOD_RES(1)..(1)D-GluMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3)..(3)D-Tyr-
MOD_RES(4)..(4)D-Lys(HSG)C-term amidated 57Glu Lys Tyr Lys1
584PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term
NODA-MBAMOD_RES(1)..(1)D-GluMOD_RES(2)..(2)D-Lys(HSG)MOD_RES(3)..(3)D-Tyr-
MOD_RES(4)..(4)D-Lys(HSG)C-term amidated 58Glu Lys Tyr Lys1
593PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term NODA
derivativeMOD_RES(1)..(1)D-Lys(HSG)MOD_RES(2)..(2)D-TyrMOD_RES(3)..(3)D-L-
ys(HSG)C-term amidatedsee specification as filed for detailed
description of substitutions and preferred embodiments 59Lys Tyr
Lys 1 603PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term NODA
derivativeMOD_RES(1)..(1)D-Lys(HSG)MOD_RES(2)..(2)D-TyrMOD_RES(3)..(3)D-L-
ys(HSG)C-term amidatedsee specification as filed for detailed
description of substitutions and preferred embodiments 60Lys Tyr
Lys 1
* * * * *