SBIR-STTR Award

Despite their inherent potential, the use of [18F]-protein radiotracers in both preclinical and clinical settings is hampered by a lack of robust radiolabeling techniques. We aim to address this by developing a fully automated radiolabeling methodology that is site-specific, occurs rapidly under mild aqueous conditions, and requires only small amounts of precursor peptide for high radiolabeling yields. The most frequent protein radiofluorination approach, conjugation of N-succinimidyl 4-[18F]-fluorobenzoate to lysine residues, is limited by a lengthy multi-step synthesis, low yields, poor control over labeling site (which can lead to reduced immunoreactivity), and the frequent requirement for large amounts of protein precursor. Other prosthetic groups have been reported, however to date none address all of these issues. The lack of an optimal, broadly applicable, radiolabeling strategy for proteins motivated us to investigate enzymes as radiolabeling catalysts. Preliminary, proof-of-concept data shows that the enzyme lipoic acid ligase can site-specifically ligate a [18F]- prosthetic to a model protein tagged with a 13-amino acid acceptor sequence (`LAP-tag'). The reaction was high yielding at neutral pH and ambient temperature, ensuring full retention of the protein's biological activity. In this proposal, we will build on these studies by developing a second generation prosthetic with a streamlined radiosynthesis and improved metabolic stability. Crucially, all radiosynthetic steps will be performed on an automated radiosynthesizer to enable reproducibility and to facilitate the immediate transfer of labeling protocols between different sites. In Specific Aim 1 we will synthesize the second generation aryl [18F]-fluoride prosthetic, confirm its ligation to a model protein-LAP construct, and purify the resulting radiotracer. In Specific Aim 2, we will transfer this protocol to UCSF where the radiolabeling will be reproduced on their radiosynthesizer. Finally, the radiotracer will be characterized for purity, specific activity, retention of biological activity, and metabolic stability. Upon completion of these studies we anticipate having a fully optimized radiofluorination protocol in hand along with proof-of-concept data for our model protein. In Phase II of this project, we will apply our methodology to several different classes of protein, including affibodies, nanobodies, diabodies, and Fab antibody fragments, with the goal of defining its full scope and benchmarking its performance against the current gold standard in the field in anticipation of producing a fully automated, commercial available, kit-based approach for protein radiofluorination.

Public Health Relevance Statement:
PROJECT NARRATIVE The translation of protein-based radiotracers from the lab to the clinic for use in cancer diagnostics is currently hampered by a lack of suitable radiolabeling methodologies. This project will use enzymes to catalyze protein radiolabeling on a commercially available automated radiosynthesizer to facilitate widespread adoption of the technique. If successful, this approach will be highly efficient, require smaller amounts of protein precursor compared to other techniques, enable site-specific labeling, and will take place under mild, aqueous conditions.

Project Terms:
Address; Adoption; Affinity; Amino Acids; Antibodies; aqueous; Automation; base; Benchmarking; Biodistribution; Biological; Biological Models; Biological Sciences; Blood Circulation; Buffers; Cancer Diagnostics; carboxylate; catalyst; Cells; Chemicals; Clinic; Clinical; Clinical Research; cohort; commercialization; companion diagnostics; Data; Development; Diagnostic; Disease; Dose; Ensure; Enzymes; Epitopes; Exhibits; Exposure to; flexibility; Fluorides; Generations; Glucose; glucose analog; Goals; Gold; Growth; Half-Life; Hand; Image; imaging probe; Immunoglobulin Fragments; Immunoglobulin G; immunoreactivity; improved; Injection of therapeutic agent; innovation; Isotopes; Kidney; Label; Lead; Life; Ligands; Ligase; Ligation; Lysine; Measures; Metabolic; Methodology; Methods; Modeling; molecular imaging; Molecular Sieve Chromatography; Molecular Weight; Mus; mutant; nanobodies; novel therapeutics; Patients; Penetrance; Peptides; Performance; Pharmacologic Substance; Phase; physical property; Positron; Positron-Emission Tomography; pre-clinical; pre-clinical research; Production; Property; Prosthesis; Protein Precursors; Proteins; Protocols documentation; Radiation; radiochemical; Radiolabeled; radiotracer; Reaction; Reporting; Reproducibility; Resistance; scaffold; Scaffolding Protein; Scanning; screening; Serum; Site; small molecule; System; targeted imaging; Techniques; Technology; Temperature; Test Result; Thioctic Acid; Time; Tissues; Translations; uptake

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.

Thesaurus Terms:
Address; Affinity; Amino Acid Sequence; Anti-Pd-L1; Aqueous; Base; Benchmarking; Biological; Biology; Characteristics; Clinic; Clinical; Clinical Translation; Clinically Relevant; Collaborations; Communities; Complex; Cyclic Gmp; Data; Development; Disease; Drug Kinetics; Efficacy Testing; Enzyme Stability; Enzymes; Equipment; Experience; Fluorides; Fluorine; Future; Generations; Gold; Grant; Half-Life; Imaging Probe; Imaging Properties; Improved; In Vitro; In Vivo Model; Individual; Industrialization; Industry; Industry Collaboration; Industry Partner; Instruction; Label; Ligands; Ligase; Ligation; Literature; Manuscripts; Measures; Metabolic; Methodology; Modeling; Mus; Mutant; Novel; Nuclear; Online Systems; Peptides; Pharmaceutical Preparations; Phase; Positron-Emission Tomography; Pre-Clinical; Process; Production; Property; Prosthesis; Protein Engineering; Protein Precursors; Protein Purification; Proteins; Protocols Documentation; Publishing; Radiochemical; Radiochemistry; Radioisotopes; Radiolabeled; Radiotracer; Reaction; Reagent; Research Personnel; Route; Running; Scale Up; Screening; Site; Sleb2 Gene; Source; Symposium; Technical Expertise; Techniques; Technology; Temperature; Test Result; Testing; Thioctic Acid; Time; Tool; Translating; Translations; Validation; Vision; Work; Xenograft Model;