Preclinical validation of AMPDA-HB derivatives integrating Hsp70-specific TPP motif and anti-CD2 single domain antibody radiolabeled by Al18F approach for PET tumor imaging

Background Al(III) complexes are gaining increasing interest for their potential use in Positron Emission Tomography (PET) imaging by formation of ternary complexes with the radioisotope fluorine-18 ( 18 F). The non-macrocyclic semi-rigid pentadentate chelator AMPDA-HB has been recently proposed for the radiolabeling of peptides and temperature sensitive biomolecules with [Al 18 F] 2+ under mild conditions. In this work, we aimed to validate the [ 18 F][AlF-AMPDA-HB complex by preparing two bifunctional derivatives of AMPDA-HB chelator and conjugating them with the Heat shock protein 70 (Hsp70) specific peptide TPP and a CD2 reactive single domain antibody (sdAb), followed by in vivo PET imaging of correlated tumor models. Methods Bifunctional derivatives of the AMPDA-HB chelator bearing NHS-activated ester (AMPDA-HB-NHS) and maleimide (AMPDA-HB-mal) groups were synthesized and conjugated to an anti-CD2 sdAb and to a membrane Hsp70 targeting tumor cell-penetrating specific peptide (TPP), respectively, and radiolabeled according to the [ 18 F][AlF] 2+ approach. The purity of the tracers was confirmed via radio-HPLC and radio-TLC, whereas their stabilities were measured in vitro (up to 4h in PBS, Serum and EDTA solutions) and in vivo in mice. Tracer biodistribution and tumor uptake were investigated in mice subcutaneously injected with mouse 4T1 breast cancer cells and human Jurkat cells expressing Hsp70 and CD2 on the tumor cell membrane, respectively. Results The labeling with [ 18 F][AlF] 2+ of the bioconjugates AMPDA-HB-antiCD2 sdAb and AMPDA-HB-mal-TPP resulted in a radiochemical yield of 30-50% (radio-TLC) and radiochemical purity > 98%. An in vitro stability >95% was confirmed in human serum and PBS solution. In vivo PET/MR imaging carried out at 1h post injection confirmed targeting ability in mice bearing 4T1 and Jurkat tumors, and good stability as shown by biodistribution. Possible limitations of this work are the single time point (1 h) in vivo imaging assessment and modest tumor uptake of the [ 18 F]AlF-(AMPDA-HB-TPP) tracer. Attaching PEG chains or ABD domains might potentially help prolonging bloodstream circulation, increasing tumor uptake and consequently the PET signal of AMPDA-HB conjugates. Conclusion The development of the AMPDA-HB-based pentadentate bifunctional chelators and their conjugation to selected biomolecules brings important innovation in the field of [ 18 F][AlF] 2+ radiolabeling providing a new tool for the application of this approach to oncological or ImmunoPET imaging.