[^99mTc]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 ^99mTc-labeled cyclic pentapeptides based on the PentixaFor scaffold. Methods: Six mas₃-conjugated CPCR4 analogs with different 4-aminobenzoic acid (Abz)-D-Ala-D-Arg-aa₃ linkers (L1–L6) as well as the corresponding HYNIC- and N₄-analogs of L6-CPCR4 were synthesized via standard SPPS. Competitive binding studies (IC₅₀ and IC₅₀inv) were carried out using Jurkat T cell lymphoma cells and [¹²⁵I]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, [^99mTc]Tc-N₄-L6-CPCR4 ([^99mTc]Tc-PentixaTec) was selected for an early translation to the human setting. Five patients with hematologic malignancies underwent [^99mTc]Tc-N₄-L6-CPCR4 SPECT/planar imaging with individual dosimetry. Results: Of the six mas₃-conjugated peptides, mas₃ -L6-CPCR4 (mas₃-dap-r-a-Abz-CPCR4) showed the highest CXCR4 affinity (IC₅₀ = 5.0 ± 1.3 nM). Conjugation with N₄ (N₄-L6-CPCR4) further improved hCXCR4 affinity to 0.6 ± 0.1 nM. [^99mTc]Tc-N₄-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, [^99mTc]Tc-N₄-L6-CPCR4 (termed [^99mTc]Tc-PentixaTec) was selected for first-in-human application. [^99mTc]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 ^99mTc-labeling strategies (mas₃ vs HYNIC vs N₄) has provided [^99mTc]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: [Figure not available: see fulltext.]