Pre-clinical characterization of the novel FAP targeting ligand PNT6555 for the imaging and therapy of cancer

Introduction: Background: Fibroblast Activation Protein-α (FAP) is a transmembrane glycoprotein highly expressed on activated fibroblasts. It is a constitutively active 170 kDa serine protease and a member of the dipeptide peptidase (DPP) family, sharing approximately 50% homology with DPPIV. FAP is expressed only at very low levels in normal adult tissues but is highly overexpressed in many epithelial cancers through upregulation on cancer-associated fibroblasts present in the stroma of various types of tumor. POINT Biopharma is developing PNT6555, which comprises a FAP-targeting moiety linked to a DOTA chelator and can be labeled with gallium-68 for PET imaging and lutetium-177 or actinium-225 for therapeutic applications.

Methods: Methods: PNT6555 and its radiometal chelates were evaluated for potency of FAP inhibition, target selectivity, biodistribution and efficacy using biochemical and cellular assays as well as imaging, biodistribution and efficacy studies in tumor bearing mice. Human dosimetry estimates for ⁶⁸Ga-PNT6555 and ¹⁷⁷Lu-PNT6555 were performed based on biodistribution data in mice.

Results: Results: PNT6555 and its gallium (^natGa-PNT6555) and lutetium (^natLu-PNT6555) chelates showed potent activity in FAP inhibition assays using human, mouse, and rat sources of FAP. PNT6555, ^natLu-PNT6555 and ^natGa-PNT6555 also showed significantly reduced potency when tested against PREP and DPPIV, two closely related homologous proteins. In vivo time-course biodistribution studies (by PET imaging) with ⁶⁸Ga-PNT6555 showed rapid clearance of ⁶⁸Ga-PNT6555 from blood through the kidneys and urinary tract, with rising ⁶⁸Ga-PNT6555 activity observed in the tumor through 60 minutes. At 60 minutes, the tumor was the only site of significant retained activity. In vivo biodistribution studies (by SPECT imaging and direct organ assay) with ¹⁷⁷Lu-PNT6555 showed rapid renal clearance into the bladder. After 24 hours, the tumor was the only tissue with significant activity retention. Direct organ assay showed little ¹⁷⁷Lu-PNT6555 accumulation and retention in normal tissues with a high level of tumor retention observed out to 168h. Dosimetry estimates predicted that the tissues receiving the largest dose equivalents were tumor and the urinary bladder wall. The dose-limiting organ was predicted to be red marrow. Therapeutic studies using a single dose of ¹⁷⁷Lu-PNT6555 or ²²⁵Ac-PNT6555 were completed in pre-clinical mouse models of cancer. In the HEK-mFAP model, significant dose responsive efficacy was observed in mice treated with either ¹⁷⁷Lu-PNT6555 or ²²⁵Ac-PNT6555, with no apparent weight loss observed at all tested dose levels. Several mice experienced long-term survival >100 days at several of the doses tested.

Conclusions: Conclusions: PNT6555, and its radiometal chelates, are potent and specific inhibitors of FAP. ⁶⁸Ga/¹⁷⁷Lu-PNT6555 showed rapid and prolonged uptake into FAP expressing tumors with limited uptake or retention observed in normal tissues. ¹⁷⁷Lu/²²⁵Ac-PNT6555 showed compelling efficacy in pre-clinical tumor models that expressed FAP. Clinical studies with imaging and therapeutic chelates of PNT6555 are warranted.