Streptavidin in Antibody Pretargeting. 5. Chemical Modification of Recombinant Streptavidin for Labeling with the α-Particle-Emitting Radionuclides <sup>213</sup>Bi and <sup>211</sup>At (2007)

We are investigating the use of recombinant streptavidin (rSAv) as a carrier molecule for the short-lived alpha-particle-emitting radionuclides 213Bi ( t 1/2 = 45.6 min) and 211At ( t 1/2 = 7.21 h) in cancer therapy. To utilize rSAv as a carrier, it must be modified in a manner that permits rapid chelation or bonding with these short-lived radionuclides and also modified in a manner that diminishes its natural propensity for localization in the kidney. Modification for labeling with (213)Bi was accomplished by conjugation of rSAv with the DTPA derivative p-isothiocyanato-benzyl-CHX-A'' (CHX-A''), 3a. Modification for direct labeling with 211At was accomplished by conjugation of rSAv with an isothiocyanatophenyl derivative of a nido-carborane (nCB), 3b, or an isothiocyanatophenyl-dPEG/decaborate(2-) derivative, 3c. After conjugation of the chelating or bonding moiety, rSAv was further modified by reaction with an excess (50-100 equivalents) of succinic anhydride. Succinylation of the lysine amines has previously been shown to greatly diminish kidney localization. rSAv modified by conjugation with 3a and succinylated rapidly radiolabeled with 213Bi (<5 min), providing a 72% isolated yield. 211At labeling of modified rSAv was accomplished in aqueous solution using chloramine-T as the oxidant. Astatination of rSAv conjugated with 3b and succinylated occurred very rapidly (<1 min), providing a 50% isolated radiochemical yield. Astatination of rSAv conjugated with 3c and succinylated was also very rapid (<1 min) providing 66-71% isolated radiochemical yields. Astatination of succinylated rSAv, 2a, which did not have conjugated borane cage moieties, resulted in a much lower radiolabeling yield (18%). The 213Bi or 211At-labeled modified rSAv preparations were mixed with the corresponding 125 I-labeled rSAv, and dual-label in vivo distributions were obtained in athymic mice. The in vivo data show that 213Bi-labeled succinylated rSAv [ 213Bi] 6a has tissue concentrations similar to those of 125 I-labeled modified rSAv [ 125 I] 6b, suggesting that (213)Bi is quite stable toward release from the chelate in vivo. In vivo data also indicate that the (211)At-labeled rSAv conjugated with 3b or 3c and succinylated are stable to in vivo deastatination, whereas succinylated rSAv lacking a boron cage moiety is subject to some deastatination. The modified rSAv conjugated with nido-carborane derivative 3b has a higher retention in many tissues than rSAv without the carborane conjugated. Interestingly, the rSAv conjugated with 3c, which also contains an m-dPEG 12 moiety, has significantly decreased concentrations in blood and other tissues when compared with those of direct-labeled rSAv, suggesting that it may be a good candidate for further study. In conclusion, rSAv that has been modified with CHX-A'' and succinylated (i.e., 5a) may be useful as a carrier of 213Bi. The encouraging results obtained with the PEGylated decaborate(2-) derivative 3c and succinylated (i.e., 5c) suggests that its further study as a carrier of 211At in pretargeting protocols is warranted.