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

We have devised and estimated a new strategy to prolong the residence time of radiolabeled antibodies in tumor in which an octaarginine peptide (R?) was used as an anchoring molecule to fix antibodies against CD20 (NuB2; IgG2a) on tumor cells. Conjugation of R? with antibodies was performed by maleimide-thiol chemistry using thiol groups generated by reducing the disulfide bonds of the antibody. The R?-conjugated NuB2 was then reacted with succinimidyl meta-[12?I]iodobenzoate to prepare [12?I]SIB-NuB2(I) (0.92 R?/NuB2) and [12?I]SIB-NuB2(III) (3.38 R?/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, [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2(III) exhibited 1.4 and 4.0 times higher cell-associated radioactivity than [12?I]SIB-NuB2. After the cells were washed and reincubated in a fresh medium for 3 h, [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2(III) exhibited significantly higher cell-associated radioactivity than [12?I]SIB-NuB2. In biodistribution studies in normal mice, while both [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2 exhibited similar biodistribution profiles, [12?I]SIB-NuB2(III) showed faster clearance from the blood and higher hepatic radioactivity levels than [12?I]SIB-NuB2. In SCID mice bearing CD20-positive xenografts, [131I]SIB-NuB2(I) exhibited significantly higher radioactivity in xenografts than those of [12?I]SIB-NuB2 with no significant increase being observed in other tissues. The findings indicate that appropriate R? modification of antibodies satisfies both specific targeting ability of antibody and strong cell-association property of R?, 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.