Despite their inherent potential, the use of 18F-labeled protein PET probes is hampered by a lack of robust radiolabeling techniques. This proposal seeks to address this by developing a commercially available kit for the fully automated production of 18F-labeled proteins via Enzymatically Catalyzed Radiolabeling (ECR) on the ELIXYS FLEX/CHEM automated radiosynthesizer platform. ECR uses lipoic acid ligases to ligate a 18F-labeled prosthetic to a protein tagged with a specific 13-amino acid sequence (?LAP-tag?). Ligation is site-specific, rapid, and high yielding in mild, aqueous conditions (neutral pH, near ambient temperature). Only minimal amounts of protein are required (10 nmol), making the production of high specific activity 18F-labeled proteins achievable. Phase I developed a second-generation aryl fluoride prosthetic, [18F]FPOA, which was shown to i) be compatible with ECR, and ii) have improved physiochemical properties and metabolic stability. Moreover, [18F]FPOA synthesis, subsequent ligation to a model protein and purification of the resulting 18F-labeled protein was automated on ELIXYS. In this Phase II proposal, the ECR process will be rigorously optimized, then translated into a commercial kit. In SA1, various synthetic routes to [18F]FPOA will be screened for radiochemical yields and ease of purification. The optimal route will be fully automated on ELIXYS and its individual components converted into a partial ECR kit. In SA2, lipoic acid ligase production will be scaled-up and the stability of the enzyme characterized under various conditions. The Ligation Reagent, containing the enzyme and other necessary co-factors, will then be developed. In addition, the ELIXYS purification protocol for 18F-labeled proteins will be refined. A complete ECR kit, containing components for [18F]FPOA synthesis and purification, the Ligation Reagent, and 18F-labeled protein purification cartridges, will be finalized and tested. Finally, the protocol for introducing a LAP-tag into a protein will be optimized, creating straightforward instructions for researchers and/or commercial CROs to follow. In SA3, we will apply ECR to a clinically relevant anti-PD-L1 model. An anti-PD-L1 biologic will be radiolabeled using ECR, and the resulting radiotracer rigorously tested using established in vitro and murine xenograft models. Data will be benchmarked against literature precedent and radiolabeling with the current gold standard, [18F]SFB, to fully understand the capabilities of ECR. Finally, proof-of-concept clinical production runs will be performed. For future Phase III endeavors, we will encourage the use of the ECR platform technology by publishing manuscripts and conference abstracts and providing ECR synthesis data via the SOFIE Network, an online portal enabling PET probe synthesis sharing and discussion among the radiochemistry community; likewise, we?ll work with our academic and industry partners to help define ECR for specific applications of novel biologic-based PET probes. We will also partner with regulatory consultants to establish cGMP sources for all required materials and subsequently submit Drug Manufacturing Files on the ECR methodology to the FDA.
The translation of protein-based radiotracers from the lab to the clinic is currently hampered by a lack of suitable radiolabeling methodologies. This project will fully develop the use of an enzyme kit to catalyze protein radiolabeling on a commercially available automated synthesizer. If successful, this approach will be highly efficient, requiring smaller amounts of protein precursor compared to other techniques, enable site-specific labeling, and will take place under mild, aqueous conditions; the resultant improvement in biologic-based PET probe synthesis quality and availability will facilitate their ultimate use in the lab and the clinic to better understand the biology of disease.
