N-(Maleimidoethyl)-3-(guanidinomethyl)-5-[131I]iodobenzmide ([131I]MEGMIB): A Residualizing Prosthetic Agent for Site-Specific Radioiodination of Internalizing Single Domain Antibody Fragments.

581 Objectives: Anti-HER2 single domain antibody fragments (sdAbs; a.k.a. nanobodies, VHH molecules) are an attractive platform for delivering radionuclides for imaging and targeted radionuclide therapy (TRT). Earlier, we demonstrated excellent tumor uptake and retention as well as low normal tissue uptake when the anti-HER2 sdAb 5F7 was labeled using the residualizing agent N-succinimidyl 3-guanidinomethyl-5-[*I]iodobenzoate (iso-[*I]SGMIB). Because site-specific labeling can have advantages over random methods such as iso-[*I]SGMIB, we developed a maleimide moiety-containing analogue of iso-[*I]SGMIB, N-(maleimidoethyl)-3-(guanidinomethyl)-5-[131I]iodobenzmide ([131I]MEGMIB) and employed it for labeling a 5F7 variant having a cysteine tail (GGC) at its C-terminus (5F7GGC). Methods: The residualizing prosthetic agent MEGMIB and its protected tin precursor Boc2-MEGMTB were synthesized and characterized. Unlabeled MEGMIB was conjugated to reduced 5F7GGC and the binding affinity of the MEGMIB-5F7GGC conjugate to HER2 was measured by surface plasmon resonance. The tin precursor Boc2-MEGMTB was labeled with 131I using NCS as the oxidant and the Boc groups were removed by TFA treatment. The resultant [131I]MEGMIB was conjugated to 5F7GGC at pH of 6.3. Radiochemical purity (RCP) of the labeled conjugate was determined by SDS-PAGE and size-exclusion chromatography. Immunoreactive fraction (IRF) was determined by the Lindmo method and binding affinity (Kd) using the HER2-positive SKOV-3 ovarian cancer cell line. Paired-label internalization of [131I]MEGMIB-5F7GGC and iso-[125I]SGMIB-5F7 was determined on SKOV-3 cells. Paired-label biodistribution of [131I]MEGMIB-5F7GGC and iso-[125I]SGMIB-5F7 was assessed in nude mice bearing subcutaneous SKOV-3 xenografts. Results: [131I]MEGMIB was synthesized in 87 ± 6% (n=11) yield, comparable to yields for iso-[131I]SGMIB. Conjugation of [131I]MEGMIB to 5F7GGC was achieved in an yield of 45% ± 7% (n=10) with a RCP, Kd and IRF of [131I]MEGMIB-5F7GGC of higher than 99%, 0.94 ± 0.27 nM and 81%, respectively. Specific uptake by SKOV-3 cells after a 1 h incubation was 10.6 ± 0.6% and 8.4 ± 0.3% for 131I and 125I, respectively with the difference statistically significant. The amount of initially bound activity that was internalized at 1 h was 50.30 ± 3.36% for 131I similar to that for 125I (55.95 ± 3.27% Fig. 2). The uptake (%ID/g) of [131I]MEGMIB-5F7GGC in SKOV-3 xenografts was 8.43 ± 2.84, 6.11 ± 1.63, and 3.60 ± 1.75% ID/g at 1, 4 and 24 h, respectively; compared to 8.35 ± 2.66, 6.11 ± 1.56 and 3.13 ± 1.52% ID/g for co-injected iso-[125I]SGMIB-5F7. The kidney activity for [131I]MEGMIB-5F7GGC at 1 h (20.39 ± 5.34 %ID/g) was significantly lower (P=0.01) than that for iso-[125I]SGMIB-5F7 (41.82 ± 13.33 %ID/g). The difference in kidney levels for the two tracers also was significant at 4 h (P=0.02). Tumor-to-kidney ratios for [131I]MEGMIB-5F7GGC also were significantly higher (0.41 ± 0.09 vs 0.21 ± 0.09 at 1h; P=0.008). Conclusion: This novel residualizing prosthetic agent allows site-specific labeling of 5F7GGC, a HER2-specific sdAb with a free cysteine at the C-terminus, in good yield with excellent retention of affinity, immunoreactivity, intracellular retention and tumor localizing capacity. Importantly, tumor uptake of [131I]MEGMIB-5F7GGC was comparable to that for the previous lead agent iso-[125I]SGMIB-5F7, but with up to two fold lower uptake in the kidneys, the dose limiting tissue for radiolabeled scFv fragments. Acknowledgements: This work was supported in part by NIH Grant CA42324.