Fully automated enzymatic radiolabeling of biomolecules

PROJECT SUMMARY ABSTRACT Despite their inherent potentialthe use ofF labeled protein PET probes is hampered by a lack of robust radiolabeling techniquesThis proposal seeks to address this by developing a commercially available kit for the fully automated production ofF labeled proteins via Enzymatically Catalyzed RadiolabelingECRon the ELIXYS FLEX CHEM automated radiosynthesizer platformECR uses lipoic acid ligases to ligate aF labeled prosthetic to a protein tagged with a specificamino acid sequenceLAP tagLigation is site specificrapidand high yielding in mildaqueous conditionsneutral pHnear ambient temperatureOnly minimal amounts of protein are requirednmolmaking the production of high specific activityF labeled proteins achievablePhase I developed a second generation aryl fluoride prostheticF FPOAwhich was shown to ibe compatible with ECRand iihave improved physiochemical properties and metabolic stabilityMoreoverF FPOA synthesissubsequent ligation to a model protein and purification of the resultingF labeled protein was automated on ELIXYSIn this Phase II proposalthe ECR process will be rigorously optimizedthen translated into a commercial kitIn SAvarious synthetic routes toF FPOA will be screened for radiochemical yields and ease of purificationThe optimal route will be fully automated on ELIXYS and its individual components converted into a partial ECR kitIn SAlipoic acid ligase production will be scaled up and the stability of the enzyme characterized under various conditionsThe Ligation Reagentcontaining the enzyme and other necessary co factorswill then be developedIn additionthe ELIXYS purification protocol forF labeled proteins will be refinedA complete ECR kitcontaining components forF FPOA synthesis and purificationthe Ligation ReagentandF labeled protein purification cartridgeswill be finalized and testedFinallythe protocol for introducing a LAP tag into a protein will be optimizedcreating straightforward instructions for researchers and or commercial CROs to followIn SAwe will apply ECR to a clinically relevant anti PD LmodelAn anti PD Lbiologic will be radiolabeled using ECRand the resulting radiotracer rigorously tested using established in vitro and murine xenograft modelsData will be benchmarked against literature precedent and radiolabeling with the current gold standardF SFBto fully understand the capabilities of ECRFinallyproof of concept clinical production runs will be performedFor future Phase III endeavorswe will encourage the use of the ECR platform technology by publishing manuscripts and conference abstracts and providing ECR synthesis data via the SOFIE Networkan online portal enabling PET probe synthesis sharing and discussion among the radiochemistry communitylikewisewe ll work with our academic and industry partners to help define ECR for specific applications of novel biologic based PET probesWe 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 PROJECT NARRATIVE The translation of protein based radiotracers from the lab to the clinic is currently hampered by a lack of suitable radiolabeling methodologiesThis project will fully develop the use of an enzyme kit to catalyze protein radiolabeling on a commercially available automated synthesizerIf successfulthis approach will be highly efficientrequiring smaller amounts of protein precursor compared to other techniquesenable site specific labelingand will take place under mildaqueous conditionsthe 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