Abstract 1119: Novel fibroblast activation protein targeting radioligand for imaging and radiotherapy of solid tumors
Authors: Mukkamala Ramesh , Spencer Lindemann D. , Daniel Carlson J. , Tudi Pooja , Kaine Miller Nicholas , Horner Autumn , Madduri Srinivasarao , Low Philip S. 
Published: March 2024

Fibroblast activation protein has emerged as an attractive biomarker for the imaging and therapyof ~90% of human epithelial cancers due to its expression on cancer associated fibroblasts (CAFs), but not on fibroblasts in healthy tissues. Not surprisingly, several small molecules and peptide-based radioligands that target FAP have been introduced into human clinical trials, but preclinical and clinical data suggest that these radioligand therapies suffer from inadequate tumor uptake, short tumor residence times, or unwanted uptake in the healthy tissues. Hence, there remains an unmet need for development of new FAP-targeted radioligands with improved therapeutic properties. For this purpose, we have designed and synthesized a higher affinity and specificity FAP-targetingligand (FAP8) and linked this ligand via a PEG spacer to both a well-known metal chelator (DOTA)and a 4(-iodo)-phenyl butyric acid pharmacokinetic extender to yield the trifunctional conjugate, FAP8-IP-DOTA. Binding affinity and FAP specificity studies reveal that FAP8-IP-DOTA exhibits high affinity and selectivity for FAP (IC50 = 1.6 nM) relative to its ubiquitous homologs’ prolyloligopeptidase (IC50 = 16.5 nM) and dipeptidyl peptidase-IV (IC50 = 865.0 nM). Moreover, 177Luand 111In radiolabeled forms of the conjugate were found to bind a FAP-transduced cell line (HEK-FAP) with a Kd = 3.0 ± 2 nM, and this binding was completely suppressed upon co-incubation with unlabeled conjugate, i.e., further confirming FAP’s specificity. SPECT/CT imaging studies following intravenous injection of 111In labelled FAP8-IP-DOTA into mice expressing HT29, MDA-MB-231, KB or 4T1 tumors then demonstrated high tumor uptake and remarkably prolonged tumor retention of &gt;8 days. Biodistribution studies of the 177Lu labelledFAP8-IP-DOTA conjugate in mice implanted with either FAP-negative 4T1 tumor cells or FAP-transduced HEK tumor cells at different time points further revealed declining tumor radioactivity from 12.0 to 2.0 % ID/g and 21.0 to 12.0 %ID/g between 1h and 168h post injection, respectively. Importantly, uptake of the same conjugate in the healthy tissues was minimal over this time course, with most radioactivity clearing rapidly. Comparative dosimetry analyses showed tumor: healthy tissues ratios of &gt;5-fold for all major organs. Finally, analysis of tumor inhibiting activity demonstrated that 177Lu-FAP8-IP-DOTA suppressed tumor growth between 60% and 100% in all xenograft models tested (HT29, MDA-MB-231, KB and 4T1), with remarkably improved overall survival rates compared to untreated groups. Based on these promising results, we suggest that FAP8-IP-DOTA warrants further evaluation for possible translation into human clinical trials.

Citation Format: Ramesh Mukkamala, Lindemann D. Spencer, Carlson J. Daniel, Pooja Tudi, Miller Nicholas Kaine, Autumn Horner, Srinivasarao Madduri, Philip S. Low. Novel fibroblast activation protein targeting radioligand for imaging and radiotherapy of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1119.