Enhanced target-specific accumulation of radiolabeled antibodies by conjugating arginine-rich peptides as anchoring molecules.

ABSTRACT

We have devised and estimated a new strategy to prolong the residence time of radiolabeled antibodies in tumor in which an octaarginine peptide (R8) was used as an anchoring molecule to fix antibodies against CD20 (NuB2; IgG2a) on tumor cells. Conjugation of R8 with antibodies was performed by maleimide-thiol chemistry using thiol groups generated by reducing the disulfide bonds of the antibody. The R8-conjugated NuB2 was then reacted with succinimidyl meta-[¹²5I]iodobenzoate to prepare [¹²5I]SIB-NuB2(I) (0.92 R8/NuB2) and [¹²5I]SIB-NuB2(III) (3.38 R8/NuB2). Both SIB-NuB2(I) and SIB-NuB2(III) exhibited size-exclusion HPLC elution profiles and immunoreactivity to CD20-positive cells similar to those of NuB2. NuB2(I) also possessed isoelectric focusing (IEF) profile similar to NuB2. However, NuB2(III) registered a broad IEF band toward higher pI. When incubated with CD20-positive cells, [¹²5I]SIB-NuB2(I) and [¹²5I]SIB-NuB2(III) exhibited 1.4 and 4.0 times higher cell-associated radioactivity than [¹²5I]SIB-NuB2. After the cells were washed and reincubated in a fresh medium for 3 h, [¹²5I]SIB-NuB2(I) and [¹²5I]SIB-NuB2(III) exhibited significantly higher cell-associated radioactivity than [¹²5I]SIB-NuB2. In biodistribution studies in normal mice, while both [¹²5I]SIB-NuB2(I) and [¹²5I]SIB-NuB2 exhibited similar biodistribution profiles, [¹²5I]SIB-NuB2(III) showed faster clearance from the blood and higher hepatic radioactivity levels than [¹²5I]SIB-NuB2. In SCID mice bearing CD20-positive xenografts, [¹³¹I]SIB-NuB2(I) exhibited significantly higher radioactivity in xenografts than those of [¹²5I]SIB-NuB2 with no significant increase being observed in other tissues. The findings indicate that appropriate R8 modification of antibodies satisfies both specific targeting ability of antibody and strong cell-association property of R8, which was reflected in the increased radioactivity levels in tumor. These findings supported the applicability of this approach to enhance target-specific accumulation of radiolabeled antibodies.