A COMPARISON OF CLEAVABLE AND NONCLEAVABLE HYDRAZINOPYRIDINE LINKERS FOR THE TC-99M LABELING OF FAB' MONOCLONAL-ANTIBODY FRAGMENTS

The design and synthesis of hydrazinopyridine bifunctional chelating a gents (BCA's) featuring amide, ester, and disulfide groups are describ ed. The BCA's site-specifically react with the free thiol groups of th e tumor-specific monoclonal antibody fragment C46.3 using a one-pot in situ reduction and conjugation procedure from the F(ab')(2) to give F ab'-linker conjugates. Molar substitution ratios (MSR's) of the hydraz inopyridine conjugates were comparable to the theoretical (maximum) nu mber of thiols per fragment determined by free hydrazine and residual thiol assays. The series of C46.3 Fab'-linker conjugates were Tc-99m-l abeled in greater than 95% radiochemical purity by incubation with Tc- 99m-tricine for 1 h at room temperature. In order to evaluate the conj ugates for radiopharmaceutical applications, the tumor localization an d biodistribution properties of the radiolabeled Fab'-linker conjugate s, compared to the direct labeled fragment, were tested in nude mice b earing LS174T xenografts. Depending upon the structure of the Linker c onnecting the radiolabeled hydrazinopyridine group to the antibody fra gment, we observed a variation in kidney uptake and whole-body clearan ce. Diester- and monoester-linked conjugates exhibited lower kidney up take and faster whole-body clearance than the corresponding linker con taining amide groups. This result may be interpreted as evidence for r apid metabolism of ester compared to amide groups in the kidney follow ing uptake. At 24-h postinjection, the monoester-linked conjugate Tc-9 9m-C46.3 Fab'-BA displayed the highest tumor: blood ratio (16.2) compa red to the directly labeled conjugate (6.6) and is therefore a potenti al clinical candidate for imaging breast and ovarian cancer.