The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.

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.

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