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.

Arano and co-workers (Arano et al. (1999) Cancer Res. 59, 128-134) have synthesized peptides with an N-terminal radioiodinated hippuric acid and a C-terminal lysine linked to antibody fragments via the epsilon-amino group of lysine that show reduced kidney uptake compared to antibody fragments directly radioiodinated. This approach takes advantage of the lysine specific carboxypeptidase activity of the kidney brush border enzymes that cleave off the radiolabeled peptide linker from the antibody fragment prior to uptake by proximal tubule cells. On the basis of their approach, we have synthesized a tetrapeptide with an N-terminal DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and a C-terminal (N(epsilon)-maleoyl)lysine that was site-specifically conjugated to an anti-CEA diabody (Yazaki et al. (2001) Bioconjugate Chem. 12, 220-228) that was engineered to contain a C-terminal cysteine (Cys-diabody). Biodistributions of the In-111-radiolabeled conjugate in nude mice show significantly reduced kidney uptake (a maximum of 82%ID/g at 6 h) compared to In-111 radiolabeled DOTA-diabody (184%ID/g at 6 h) in which DOTA was conjugated to endogenous lysine residues using DOTA-active ester chemistry. To further reduce kidney uptake, a homologous compound with a C-terminal (N(epsilon)-amino-1,6-hexane-bis-vinyl sulfone)lysine was synthesized and site-specifically conjugated to the Cys-diabody. Biodistributions of this In-111-labeled conjugate reduced kidney uptake to 54%ID/g at 6 h. To explore the effect of the relative positions of the chelate vs the cys-diabody on kidney uptake, we also synthesized a tetrapeptide with an N-terminal bromoacetate for conjugation to Cys-diabody and a C-terminal (N(epsilon)-amidino-propyl-3-thio-vinylsulfonyl-DO3A)lysine. This peptide essentially reverses the positions of the chelate and Cys-diabody attachment points on the peptide, while retaining the linker length on the epsilon-amino group of the lysine. In this case, biodistributions of the In-111-radiolabeled conjugate in nude mice showed high kidney uptake (189%ID/g at 6 h), comparable to that obtained with the In-111-radiolabeled active ester conjugated DOTA-diabody (184%ID/g at 6 h). We conclude that the peptide linker strategy of Arano and co-workers to reduce kidney uptake can be successfully applied to chelate/radiometal complexes and requires that the chelate/radiometal be located at the N-terminus of the peptide and the antibody fragment attachment site on the epsilon-amino group of the lysine. Furthermore, we demonstrated a role for the attachment chemistry to the epsilon-amino group of the lysine on the magnitude of kidney uptake.