SUMMARY/ABSTRACT In 2016, over 33,000 organ transplants were performed in the United States, an increase of 20% over the past 5 years. Organ transplantation requires lifelong immunosuppression to prevent rejection of the transplant. Tacrolimus, a macrolide antibiotic, is one of the most effective immunosuppressants, with >90% of solid organ transplant recipients receiving it as part of their maintenance immunosuppression. Tacrolimus has a narrow therapeutic window, with sub-therapeutic levels putting patients at risk for rejection and supra-therapeutic levels leading to toxicity. In addition to significant pharmacokinetic variability, it is also estimated that >25% of patients are non-adherent to their immunosuppressant regimen, significantly increasing graft rejection and failure rates and increasing the financial burden on the US healthcare system. Because transplant recipients require a lifetime of immunosuppression, any methods that facilitate improved dosing and rapid detection of non-adherence are of great clinical utility. Because tacrolimus levels cannot be ascertained at the point of care, detection of non-therapeutic levels in the outpatient setting is challenging. Patients must either attend a separate lab appointment ahead of their routine follow-up visits or they have levels drawn on the day of their visit. In the former, an extra burden is placed on the patient to attend an additional appointment while the latter precludes analysis of levels prior to the patient’s appointment. Delays in medication management or identification of patient non-compliance place the patient at risk for graft rejection (sub-therapeutic levels) or drug-associated toxicities (supra-therapeutic levels). To address this technical hurdle, Affinergy plans to develop a point-of-care lateral flow assay that will enable frequent, accurate and affordable monitoring of tacrolimus levels. We have already generated a proprietary capture reagent and identified several detection reagent candidates that bind with nanomolar affinity to tacrolimus. At the conclusion of Phase I, we will have a prototype lateral flow assay with established limits of quantitation. In Phase II, we will scale up production of our assay, optimize performance characteristics, establish storage conditions, determine stability and evaluate our assay using transplant recipient blood specimens. Successful completion of this project will lead to improved long-term maintenance of patients receiving tacrolimus for immunosuppression and ease the burden on both patients and providers.

PROJECT NARRATIVE Tacrolimus concentration must be closely monitored in transplant recipients as a part of their lifelong immunosuppression. However, patients receiving tacrolimus in the outpatient setting must have blood levels shipped out or drawn the day of their visit, leading to unacceptable delays in dosage adjustment. In this Fast- Track application, we propose to develop a novel peptide-based sandwich assay to facilitate rapid, affordable, point-of-care measurement of tacrolimus concentration in biological fluids.