A Polar Substituent-Containing Acylation Agent for the Radioiodination of Internalizing Monoclonal Antibodies: N-Succinimidyl 4-Guanidinomethyl-3-[¹³¹I]iodobenzoate ([¹³¹I]SGMIB)

Abstract

The objective of this study was to develop an acylation agent for the radioiodination of monoclonal antibodies that would maximize retention of the label in tumor cells following receptor- or antigen-mediated internalization. The strategy taken was to add a polar substituent to the labeled aromatic ring to impede transport of labeled catabolites across lysosomal and cell membranes after antibody degradation. Preparation of unlabeled N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) was achieved in six steps from 3-iodo-4-methylbenzoic acid. Preparation of 4-guanidinomethyl-3-[131I]iodobenzoic acid from the silicon precursor, 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylsilylbenzoic acid proceeded in less than 5% radiochemical yield. A more successful approach was to prepare [131I]SGMIB directly from the tin precursor, N-succinimidyl 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylstannylbenzoate, which was achieved in 60-65% radiochemical yield. A rapidly internalizing anti-epidermal growth factor receptor variant III antibody L8A4 was labeled using [131I]SGMIB in 65% conjugation efficiency and with preservation of immunoreactivity. Paired-label in vitro internalization assays demonstrated that the amount of radioactivity retained in cells after internalization for L8A4 labeled with [131I]SGMIB was 3-4-fold higher than that for L8A4 labeled with 125I using either Iodogen or [125I]SIPC. Catabolite assays documented that the increased retention of radioiodine in tumor cells for antibody labeled using [131I]SGMIB was due to positively charged, low molecular weight species. These results suggest that [131I]SGMIB warrants further evaluation as a reagent for labeling internalizing antibodies.

Synthesis of N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate: a radio-iodination agent for labeling internalizing proteins and peptides

“…Neither Boc-SGMTB nor the corresponding iodo derivative Boc-SGMIB is commercially available, but they can be synthesized following literature protocols 19 . For the most part, 0.2 mg Boc-SGMTB is used for a radio-iodination reaction.…”

“…Boc-SGMIB, 2 (synthesize according to the literature protocol 19 ) . Boc-SGMTB, 3 (synthesize according to the literature protocol 19 ) !…”

“…Boc-SGMIB, 2 (synthesize according to the literature protocol 19 ) . Boc-SGMTB, 3 (synthesize according to the literature protocol 19 ) ! CAUTION As an active ester, SGMTB can undergo slow hydrolysis; however, when it is stored under argon at À20 1C, no detectable decomposition has been observed for months, if not years.…”

“…Guanidine has a pK a of approximately 13 and is therefore expected to remain exclusively in the protonated form within the lysosome. We have exploited this property to develop a conjugation labeling agent useful in the radio-iodination of internalizing mAbs 19 . [*I]SGMIB was derived simply by attaching a guanidinomethyl function to SIB (Fig.…”

“…1). An unlabeled standard of SGMIB (4), its Boc-protected derivative (Boc-SGMIB, 2) and the tin precursor N-succinimidyl 4-[N 1 , N 2 -bis(tert-butyloxycarbonyl)guanidinomethyl]-3-(trimethylstannyl)benzoate (Boc-SGMTB, 3) were obtained from 3-iodo-4-methylbenzoic acid (1) in multiple steps 19 . An SGMIB analog labeled with an a-particleemitting heavy halogen, 211 (20 1C).…”