Metabolism and Renal Clearance of ¹¹¹In-Labeled DOTA-Conjugated Antibody Fragments

Abstract

Radiometal-labeled antibody fragments are promising reagents for radioimmunotherapy due to their high tumor uptake and rapid pharmacokinetics, but their therapeutic potentials are limited by high uptake and retention in the kidney. Identification of metabolic products is a first step in designing rationale approaches to lower kidney uptake. Previous studies in rats have shown that 111In-labeled DTPA-conjugated antibody fragments (via lysine residues) were degraded to an DTPA-epsilon-amino-lysine derivative and retained in the lysosomal compartments of the liver and kidney [Rogers et al. (1995) Cancer Res. 55, 5714s-5720s]. To determine the metabolic profile of another widely used metal-chelate, [111In]DOTA conjugated to lysines in antibody fragments via active ester chemistry, we analyzed kidney homogenates from nude mice injected with an [111In]DOTA-Fab generated enzymatically from the anti-lymphoma intact antibody Rituxan. The major kidney metabolite was identified as [111In]DOTA-epsilon-amino-lysine by comparison to an authentic synthetic standard. This end product was also identified in the urine, along with relatively small amounts of [111In]DOTA-Fab. Since injection of [111In]DOTA-epsilon-amino-lysine into nude mice resulted in rapid clearance into the urine without kidney retention, it is likely that the renal retention observed was due to kidney uptake of [111In]DOTA-Fab, followed by lysosomal degradation to [111In]DOTA-epsilon-amino-lysine, which is only slowly cleared from this compartment. This observation is supported by autoradiographs of the kidney showing rapid localization of radioactivity into the distal regions of the kidney cortex. To extend this analysis to clinical trials, we have also analyzed urine taken from a patient injected with the intact antibody [111In]DOTA-cT84.66. In that example, we found that the major radioactive species was also [111In]DOTA-epsilon-amino-lysine.

111In-Labeled Galectin-3–Targeting Peptide as a SPECT Agent for Imaging Breast Tumors

“…The molecular mechanism of renal uptake of radiolabeled peptides is not completely understood. High levels of accumulation of radiolabeled polypeptides in the kidneys may be due to the long residence time of radiometabolites produced by lysosomal degradation within renal cells (29)(30)(31). Increased renal uptake could also be attributed to the presence of lysine (32,33), free sulfhydryls, or disulfide bonds in the polypeptide (34).…”

In vivo evaluation of 188Re‐labeled alpha‐melanocyte stimulating hormone peptide analogs for melanoma therapy

“…[33][34][35][36] It was reported that high renal accumulation of radiolabeled polypeptides and antibody fragments was caused by the long residence time of the radiometabolites produced by lysosomal degradation within the renal cells. 34 -36 Recently, several groups have investigated methods to lower renal radioactivity levels by chemical modification of radiolabeled antibody fragments.…”

Optimizing Radiolabeled Engineered Anti-p185HER2 Antibody Fragments forIn vivoImaging

“…Kidney lysate was prepared as described (31). Briefly, kidneys from normal mice were removed, rinsed with PBS, and homogenized in an equal volume of PBS containing a cocktail of protease inhibitors (Complete tablets, Roche/Boehringer Mannheim, Indianapolis, IN) using a Kinematica homogenizer (Brinkman Instruments, Westbury, NY).…”