Site-Specific and Residualizing Linker for ¹⁸F-Labeling with Enhanced Renal Clearance: Application to an Anti-HER2 Single Domain Antibody Fragment

Abstract

Single domain antibody fragments (sdAbs) are promising vectors for immunoPET; however, better methods for labeling sdAbs with ¹⁸F are needed. Herein, we evaluate a site-specific strategy utilizing an ¹⁸F residualizing motif and the anti-HER2 sdAb 5F7 bearing an engineered C-terminal GGC tail. Methods: 5F7GGC was site-specifically attached with a tetrazine-bearing agent via thiol-maleimide reaction. The resultant conjugate was labeled with ¹⁸F by inverse electron demand Diels-Alder cycloaddition with a trans-cyclooctene attached to 6-¹⁸F-fluoronicotinoyl moiety via a renal brush border enzyme-cleavable linker and a PEG4 chain (¹⁸F-5F7GGC). For comparisons, 5F7 sdAb was labeled using the prototypical residualizing agent, N-succinimidyl 3-(guanidinomethyl)-5-¹²⁵I-iodobenzoate (iso-¹²⁵I-SGMIB). The two labeled sdAbs were compared in paired-label studies performed in the HER2-expressing BT474M1 breast carcinoma cell line and athymic mice bearing BT474M1 subcutaneous xenografts. MicroPET/CT imaging after administration of ¹⁸F-5F7GGC in the above mouse model was also carried out. Results: ¹⁸F-5F7GGC was synthesized in an overall radiochemical yield of 8.9 ± 3.2% with retention of HER2 binding affinity and immunoreactivity. The total cell-associated and intracellular activity for ¹⁸F-5F7GGC was similar to that for co-incubated iso-^125I-SGMIB-5F7. Likewise, the uptake of ¹⁸F-5F7GGC in BT474M1 xenografts in mice was similar to that for iso-¹²⁵I-SGMIB-5F7; however, ¹⁸F-5F7GGC exhibited significantly more rapid clearance from the kidney. MicroPET/CT imaging confirmed high uptake and retention in the tumor with very little background activity at 3 h except in the bladder. Conclusion: This site-specific and residualizing ¹⁸F labeling strategy could facilitate clinical translation of 5F7 anti-HER2 sdAb as well as other sdAbs for immunoPET.