[ 99m Tc]Tc-PentixaTec: development, extensive pre-clinical evaluation, and first human experience

Purpose The clinical success non-invasive imaging of CXCR4 expression using [ 68 Ga]Ga-PentixaFor-PET warrants an expansion of the targeting concept towards conventional scintigraphy/SPECT with their lower cost and general availability. To this aim, we developed and comparatively evaluated a series of 99m Tc-labeled cyclic pentapeptides based on the PentixaFor scaffold. Methods Six mas 3 -conjugated CPCR4 analogs with different 4-aminobenzoic acid (Abz)-D-Ala-D-Arg-aa 3 linkers ( L1–L6 ) as well as the corresponding HYNIC- and N 4 -analogs of L6- CPCR4 were synthesized via standard SPPS. Competitive binding studies (IC 50 and IC 50 inv) were carried out using Jurkat T cell lymphoma cells and [ 125 I]FC-131 as radioligand. Internalization kinetics were investigated using hCXCR4-overexpressing Chem-1 cells. Biodistribution studies and small animal SPECT/CT imaging (1 h p.i.) were carried out using Jurkat xenograft bearing CB17/SCID mice. Based on the preclinical results, [ 99m Tc]Tc-N 4 - L6- CPCR4 ([ 99m Tc]Tc-PentixaTec) was selected for an early translation to the human setting. Five patients with hematologic malignancies underwent [ 99m Tc]Tc-N 4 - L6 -CPCR4 SPECT/planar imaging with individual dosimetry. Results Of the six mas 3 -conjugated peptides, mas 3 -L6 -CPCR4 (mas 3 -dap-r-a-Abz-CPCR4) showed the highest CXCR4 affinity (IC 50 = 5.0 ± 1.3 nM). Conjugation with N 4 (N 4 - L6 -CPCR4) further improved hCXCR4 affinity to 0.6 ± 0.1 nM. [ 99m Tc]Tc-N 4 - L6- CPCR4 also showed the most efficient internalization (97% of total cellular activity at 2 h) and the highest tumor accumulation (8.6 ± 1.3% iD/g, 1 h p.i.) of the compounds investigated. Therefore, [ 99m Tc]Tc-N 4 - L6 -CPCR4 (termed [ 99m Tc]Tc-PentixaTec) was selected for first-in-human application. [ 99m Tc]Tc-PentixaTec was well tolerated, exhibits a favorable biodistribution and dosimetry profile (2.1–3.4 mSv per 500 MBq) and excellent tumor/background ratios in SPECT and planar imaging. Conclusion The successive optimization of the amino acid composition of the linker structure and the N-terminal 99m Tc-labeling strategies (mas 3 vs HYNIC vs N 4 ) has provided [ 99m Tc]Tc-PentixaTec as a novel, highly promising CXCR4-targeted SPECT agent for clinical application. With its excellent CXCR4 affinity, efficient internalization, high uptake in CXCR4-expressing tissues, suitable clearance/biodistribution characteristics, and favorable human dosimetry, it holds great potential for further clinical use. Graphical abstract