Simplified 211At-labeling of internalizing proteins by preconjugation with the tin precursor of a residualizing prosthetic agent: Application to an anti-HER2 single domain antibody fragment

1143 Objectives: Labeling proteins with the α-particle emitting radiohalogen 211At generally is accomplished with pre-labeled prosthetic agents such as N-succinimidyl 3-[211At]astatobenzoate (SAB) via a two-step procedure. However, given the 7.2 h half-life of 211At, the total synthesis time is not ideal nor is its adaptability to kit formulation. To overcome this, a method for preconjugating a protein with tin precursor for SAB has been reported (J Nucl Med 2008; 49:1537). In the current study, we have evaluated the possibility of extending this preconjugation strategy labeling with the tin precursor of N-succinimidyl 5-guanidinomethyl-3-[211At]astatobenzoate (iso-[211At]SAGMB), a promising residualizing prosthetic agent for labeling internalizing proteins with 211At. The anti-HER2 single domain antibody (sdAb) fragment 2Rs15d was used as the model internalizing protein for this study. Methods: N-succinimidyl 5-guanidinomethyl-3-(tri-n-methyl)stannyl benzoate (iso-SGMTB), synthesized in multiple steps, was conjugated to 2Rs15d at pH 8.5 and the conjugate evaluated by MALDI-TOF. Iso-SGMTB-2Rs15d was labeled with 211At at pH 5.5 using N-iodosuccinimide and purified by gel filtration. The radiochemical purity (RCP) of iso-[211At]SAGMB-2Rs15d was determined by ITLC, methanol precipitation, and SDS-PAGE/phosphor imaging. To evaluate the site(s) of labeling on the protein (prosthetic moiety-lysine vs. constituent tyrosines), iso-[211At]SAGMB-2Rs15d was proteolyzed using pronase and the proteolysate was analyzed by HPLC and TLC. For comparison, 2Rs15d was also labeled by reaction with iso-[131I]SGMIB. HER2-specific binding affinity and paired-label cell uptake/internalization of iso-[211At]SAGMB-2Rs15d and iso-[131I]SGMIB-2Rs15d were compared on HER2-positive SKOV-3 ovarian cancer cells. Immunoreactive fraction (IRF) was determined for binding to HER2 extracellular domain affixed to magnetic beads and paired-label biodistribution was carried out in mice bearing SKOV-3 xenografts at 1, 4, and 24 h post-injection. Results: MALDI-TOF indicated that an average of 1 iso-SGMTB has been conjugated per 2Rs15d molecule. The conjugate was labeled with 211At with radiochemical yield of 55.6±0.1% in a total preparation time of 20 min vs. ~3 h for the two-step procedure with RCP of 95-96% and IRF of 71±8%. Analysis of proteolysate indicated that >93% of 211At activity corresponded to the lysine-conjugate. HER2 binding affinity was 7.4±0.8 nM for iso-[211At]SAGMB-2Rs15d with internalized activity at 6 h (38.1±8.3%) higher than that for iso-[131I]SGMIB-2Rs15d (25.9±1.4%). SKOV-3 xenograft uptake for iso-[211At]SAGMB-2Rs15d was 9.2±1.2, 13.1±5.7, and 3.9±0.8% ID/g at 1, 4, and 24 h, respectively, and similar to that for iso-[131I]SGMIB-2Rs15d (8.6±1.6, 12.2±4.5, and 6.1±0.7% ID/g); however, lower tumor-to-normal tissue ratios were observed for kidneys, lungs, liver, and stomach with iso-[211At]SAGMB-2Rs15d. Conclusions: Preconjugation of iso-SGMTB to this sdAb reduced 211At-labeling time considerably without compromising radiochemical yield, HER2 binding, or intracellular trapping. Moreover, the iso-[211At]SAGMB-2Rs15d conjugate demonstrated excellent tumor targeting. The simplicity of this procedure should be readily adaptable to labeling with the high activity levels that will be required for clinical evaluation of iso-[211At]SAGMB-sdAb conjugates in patients with HER2-expressing carcinomas.