Drug – bio-affecting and body treating compositions – Radionuclide or intended radionuclide containing; adjuvant... – Attached to antibody or antibody fragment or immunoglobulin;...
Reexamination Certificate
2000-08-24
2002-03-19
Chan, Christina Y. (Department: 1644)
Drug, bio-affecting and body treating compositions
Radionuclide or intended radionuclide containing; adjuvant...
Attached to antibody or antibody fragment or immunoglobulin;...
C530S345000, C530S408000
Reexamination Certificate
active
06358489
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns methods for radiolabeling proteins and peptides with fluorine-18 (F-18). More particularly, these proteins and peptides are radiolabeled with F-18 by reacting a thiol group contained therein with an F-18-bound labeling reagent which also has a group that is reactive with thiols. The resulting F-18-labeled proteins and peptides are useful in imaging targeted tissue by clinical positron emission tomography.
2. Description of the Related Art
Positron emission tomography (PET) is a high resolution, non-invasive, imaging technique for the visualization of human disease. In PET, 511 keV gamma photons produced during positron annihilation decay are detected. In the clinical setting, fluorine-18 (F-18) is one of the most widely used positron-emitting nuclides. F-18 has a half-life (t½) of 110 minutes, and emits &bgr;+particles at an energy of 635 keV. It is 97% abundant.
The short half-life of F-18 has limited or precluded its use with longer-lived specific targeting vectors such as antibodies, antibody fragments, recombinant antibody constructs and longer-lived receptor-targeted peptides. In addition, complicated chemistry has been required to link the inorganic fluoride species to such organic targeting vectors. In typical synthesis methods, an intermediate is radiofluorinated, and the F-18-labeled intermediate is purified for coupling to protein amino groups. See, e.g., Lang et al.,
Appl. Radiat. Isol
., 45 (12): 1155-63 (1994); Vaidyanathan et al.,
Bioconj. Chem
., 5: 352-56 (1994).
These methods are tedious to perform and require the efforts of specialized professional chemists. They are not amenable to kit formulations for use in a clinical setting. Multiple purifications of intermediates are commonly required, and the final step, involving linkage to protein lysine residues, usually results in 30-60% yields, necessitating a further purification step prior to patient administration. In addition, these methods result in fluorinated targeting species which accumulate in the kidney, somewhat like radiometals.
It was recently reported that
18
F-fluoroiodomethane (
18
FCH
2
I) is a useful intermediate for the fluorination of organic intermediates. Zheng et al.,
J. Nucl. Med
., 38: 177P (Abs. 761) (1997). In this process, diiodomethane is fluorinated with the F-18 ion by a room temperature reaction in acetonitrile solvent, resulting in up to a 40% yield. The
18
FCH
2
I is then distilled into reaction vials containing various strong nucleophiles in anhydrous acetonitrile and allowed to react at 80° C. for fifteen minutes. Nucleophilic attack by carboxylates, thiolates, phenolates, and amines in particular, replaces the remaining iodine of
18
FCH
2
I, with overall yields of 10 to 35%. The reaction products can be purified by reverse-phase HPLC. Fluoromethyl diethylamine, fluoromethyl benzoate, fluoromethyl benzyl thioether and fluoromethyl 4-(2-hydroxy-3-aminopropoxy)-carbazole have been made by this method.
As discussed above, the currently available methods for labelling protein-based targeting vectors with F-18 are unsuitable. There is a need, therefore, for a simple, efficient method for incorporating the F-18 radionuclide into peptide-containing targeting vectors, such as proteins, antibodies, antibody fragments, and receptor-targeted peptides, to allow the use of such targeting vectors in routine clinical positron emission tomography.
SUMMARY OF THE INVENTION
The present invention provides methods for incorporating the F-18 radionuclide into peptide-containing targeting vectors.
In accordance with one embodiment of the invention there is provided a method for radiolabeling thiol-containing peptides with fluorine-18 (F-18), comprising reacting a peptide comprising a free thiol group with a labelling reagent having the general formula
18
F—(CH
2
)
m
—CR
1
R
2
—(CH
2
)
n
—X, wherein:
n is 0, 1 or 2;
m is 0, 1 or 2;
and n+m is 0, 1, or 2;
X is selected from the group consisting of iodide, bromide, chloride, azide, tosylate, mesylate, nosylate, triflate, unsubstituted maleimide, maleimide substituted with one or two alkyl groups, and maleimide substituted with a sulfonate group; and
R
1
and R
2
are the same or different and are selected from the group consisting of iodide, bromide, chloride, azide, tosylate, mesylate, nosylate, triflate, hydrogen, —CONH
2
, carboxyl, hydroxyl, sulfonic acid, tertiary amine, quaternary ammoniumun, unsubstituted alkyl, substituted alkyl, —COOR′, —CONR′
2
, or COR′, wherein the substituents of the substituted alkyl groups are selected from the group consisting of —CONH
2
, carboxyl, hydroxyl, sulfonic acid, tertiary amine and quaternary ammonium and wherein R′ is a C
1
-C
6
alkyl or phenyl.
In accordance with another embodiment, there is provided a method for radiolabeling thiol-containing peptides with F-18, comprising reacting a peptide comprising a free thiol group with a F-18 fluorinated alkene, wherein at least one of the two double-bonded carbon atoms bears at least one leaving group selected from the group consisting of iodide, bromide, chloride, azide, tosylate, mesylate, nosylate and triflate.
In accordance with another embodiment of the invention, a peptide that has been radiolabeled with F-18 as described above is delivered to a targeted tissue using a bispecific antibody (bsMAb) or a bispecific antibody fragment (bsFab) containing at least one arm that is specific to the targeted tissue and at least one other arm that is specific to the F-18-labeled peptide or a low molecular weight hapten conjugated to the F-18-labeled peptide.
In this methodology, the bsMAb or the bsFab is administered to a patient and allowed to localize to the targeted tissue. Some time later (after the unbound bsMAb or the unbound bsFab is allowed to clear), the F-18-labeled peptide or the hapten conjugate thereof is administered to the patient. Since at least one of the arms of the bsMAb or the bsFab is specific to the F-18-labeled peptide or the hapten conjugated to the F-18-labeled peptide, the F-18-labeled peptide is also localized to the target. After the unbound F-18-labeled peptide or the unbound hapten conjugate thereof is allowed to clear, the target is then visualized by routine clinical positron emission tomography.
The bsMAb or bsFab is ideally monoclonal and humanized. Preferably, the F-18-labeled peptide contains a thiol group. Examples of suitable peptides are X-Gly-D-Tyr-D-Trp-Gly-D-Lys(X)-Gly-D-Tyr-D-Trp-OH wherein X represents a free or protected amino acid group, Ac-Cys(Y)-D-Tyr-D-Trp-Gly-D—Cys(Y)-Gly-D-Tyr-D-Trp-OH wherein Y represents a free or protected thiol group, and Ac-Gly-D-iodo-Tyr-D-Trp-Gly-D-Lys(Ac)-Gly-D-iodo-Tyr-D-Trp-OH. The hapten can be a metal chelate complex comprising, for example, manganese, iron, or gadolinium which are useful in magnetic resonance imaging (MRI).
The bsMAb, bsFab, and associated methodologies described above are disclosed in U.S. Provisional Application Serial No. 60/090,142 (entitled “Production and use of novel peptide-based agents for use with bispecific antibodies” and filed Jun. 22, 1998), the entire contents of which are herein incorporated by reference.
These and other objects and aspects of the invention will become apparent to the skilled artisan in view of the teachings contained herein.
REFERENCES:
patent: 98/16254 (1998-04-01), None
Page et al., “Preclinical Evaluation And Pet Imaging Of 18F-Labeled Mel-14 F(ab')2 Fragment In Normal Dogs”,Nuclear Medicine And Biology:, vol. 21(7):911-919, (1994).
Wilbur, “Radiohalogenation of Protein: An Overview of Radionuclides, Labeling Methods, And Reagents For Conjugate Labeling”,Bioconjugate Chemistry, vol. 3(6):433-470, (1992).
Shiue et al., “Synthesis Of 18F-Labelled N-(P-'18Fluorophenyl) Maleimide And Its Derivatives For Labelling Monoclonal Antibody With 18F”,Journal of Labelled Compounds And Radiopharmaceuticals, vol. 26:287-289, (1989).
Kilbourn et al., “Fluorine-18 Labeling of Proteins”,Journal Of Nuclear Medici
Chan Christina Y.
Huynh Phuong N.
Immunomedics Inc.
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