A Comparison of Cleavable and Noncleavable Hydrazinopyridine Linkers for the^99mTc Labeling of Fab‘ Monoclonal Antibody Fragments

Abstract

The design and synthesis of hydrazinopyridine bifunctional chelating agents (BCA's) featuring amide, ester, and disulfide groups are described. The BCA's site-specifically react with the free thiol groups of the tumor-specific monoclonal antibody fragment C46.3 using a one-pot in situ reduction and conjugation procedure from the F(ab')2 to give Fab'-linker conjugates. Molar substitution ratios (MSR's) of the hydrazinopyridine conjugates were comparable to the theoretical (maximum) number of thiols per fragment determined by free hydrazine and residual thiol assays. The series of C46.3 Fab'-linker conjugates were 99mTc-labeled in greater than 95% radiochemical purity by incubation with 99mTc-tricine for 1 h at room temperature. In order to evaluate the conjugates for radiopharmaceutical applications, the tumor localization and biodistribution properties of the radiolabeled Fab'-linker conjugates, compared to the direct labeled fragment, were tested in nude mice bearing LS174T xenografts. Depending upon the structure of the linker connecting the radiolabeled hydrazinopyridine group to the antibody fragment, we observed a variation in kidney uptake and whole-body clearance. Diester- and monoester-linked conjugates exhibited lower kidney uptake and faster whole-body clearance than the corresponding linker containing amide groups. This result may be interpreted as evidence for rapid metabolism of ester compared to amide groups in the kidney following uptake. At 24-h postinjection, the monoester-linked conjugate 99mTc-C46.3 Fab'-BA displayed the highest tumor: blood ratio (16.2) compared to the directly labeled conjugate (6.6) and is therefore a potential clinical candidate for imaging breast and ovarian cancer.

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4′-dithiodipyridine

“…1). Given the conflicting numbers on 4-thiopyridone absorption (between 19,800 M -1 cm -1 and 24,000 M -1 cm -1 were reported for ε 324 [13,21,22,25]), the first task of this study was calibration of the spectrophotometric response of DTDP reagent to a thiol.…”

“…The first task was to resolve the confusion about the molar absorptivity (ε 324 ) of 4-TP [13,21,22,25] and the molar absorptivity change (∆ε 324 ) which accompanies the reaction of a mercaptan with DTDP (see Fig. 2), taking into account the pH dependence [20,21] of 4-TP absorbance (see Fig.…”

6-Hydrazinonicotinamide Derivatives as Bifunctional Coupling Agents for 99mTc-Labeling of Small Biomolecules

“…These complexes can be readily prepared by reacting XV120 directly with 99m TcO 4 -in the presence of excess tricine, watersoluble phosphine coligand, and stannous chloride. The radiolabeling yield for RP444 is usually 90% or greater using 5 µg XV120 and 100 mCi 99m TcO 4 -. The XV120-to-technetium ratio is about 7:1 under these conditions.…”

“…Since then, the HYNIC technology has successfully been used for the 99m Tc-labeling of antibodies [3][4][5][6][7][8] and small biomolecules (BMs), including chemotactic peptides [9][10][11][12][13][14][15][16][17][18], somatostatin analogs [19][20][21][22][23][24][25],"stealth" liposomes [26,27], antisense oligonucleotides [28][29][30][31], a folate receptor ligand [32], and polypeptides [32][33][34]. A ternary ligand system (HYNIC, tricine, and trisodium triphenylphosphine-3,3¢,3≤-trisulfonate, TPPTS) has been used for the 99m Tc-labeling of chemotactic peptides [35] and leukotriene B 4 (LTB 4 ) receptor antagonists [36][37][38][39] for imaging infection and inflammation, integrin a v b 3 receptor antagonists for tumor imaging [40], and a glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonist for imaging thrombosis [41][42][43][44][45][46]…”

Technetium-binding in labelled HYNIC-peptide conjugates: Role of coordinating amino acids

“…1) as a bifunctional linker for labelling biomolecules with 99m Tc [1][2][3][4][5], it has proven to be exceptionally efficient and has become widely used, with many applications and refinements being reported [6][7][8][9][10][11][12][13][14][15][16][17][18][19]. HYNIC is typically [7,[20][21][22][23] used in the form of an activated ester, and conjugated to a peptide by reaction with the N-terminal amine group.…”