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 immuno-PET; however, better methods for labeling sdAbs with ¹⁸F are needed. Herein, we evaluate a site-specific strategy using an ¹⁸F residualizing motif and the anti-epidermal growth factor receptor 2 (HER2) sdAb 5F7 bearing an engineered C-terminal GGC tail (5F7GGC). 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 2 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. Small-animal PET/CT imaging after administration of ¹⁸F-5F7GGC in the above mouse model was also performed. 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 coincubated 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. Small-animal PET/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 immuno-PET.

Keywords: HER2; click chemistry; immuno-PET; single-domain antibody fragment; site-specific labeling.

Graphical abstract

FIGURE 1.

Scheme for synthesis of ¹⁸F-FN-PEG₄-GK-TCO-Tz-PEG₄-Mal-5F7GGC (¹⁸F-5F7GGC).

FIGURE 2.

Sensorgrams showing dose–response curves and kinetic profiles for binding of 5F7 (A) and Tz-5F7GGC (B) to HER2-Fc extracellular domain.

FIGURE 3.

Paired-label uptake and internalization of iso-¹²⁵I-SGMIB-5F7 and ¹⁸F-5F7GGC by HER2-positive BT474M1 breast carcinoma cells. Data (mean ± SD) are percentage of initially added activity that was bound to cells (membrane + internalized) (A) and internalized (B).

FIGURE 4.

Paired-label biodistribution of ¹⁸F-5F7GGC and iso-¹²⁵I-SGMIB-5F7 sdAb conjugates in athymic mice bearing subcutaneous HER2-expressing BT474M1 breast carcinoma xenografts.

FIGURE 5.

Tumor-to-tissue ratios after injection of ¹⁸F-5F7GGC and iso-¹²⁵I-SGMIB-5F7 sdAb conjugates in athymic mice bearing subcutaneous BT474M1 xenografts.

FIGURE 6.

Maximum-intensity-projection ¹⁸F-5F7GGC immuno-PET images of representative mouse bearing subcutaneous HER2-positive BT474M1 xenograft obtained 1, 2, and 3 h after injection. Positions of tumor (T), kidney (K), and bladder (B) are indicated.