Development of a potency assay for CD34+ cell-based therapy for post-acute myocardial infarction

Abstract

Abstract Background: Acute myocardial infarction (AMI) results from obstruction of a coronary artery and affects around 126 million individuals globally. We have shown in a small pilot study that intracardiac delivery of autologous CD34+ cells after myocardial infarction is safe and leads to long term improvement. The improvement was such that three patients initially recommended for early heart transplantation in this study, no longer required it years after the cell injection. After the successful results of this pilot study, we started a multicenter, randomized, controlled Phase I/IIb study in post-AMI to investigate the safety and efficacy of intramyocardial injection of expanded autologous CD34+ cells (ProtheraCytes®) (NCT02669810). While patient recruitment is ongoing, we have developed a potency assay for the batch release of ProtheraCytes®. Methods: The proposed mechanisms of action by which ProtheraCytes® promote cardiac regeneration and revascularisation of damaged myocardial tissue is via the secretion of angiogenic factors and endothelial differentiation. We conducted a series of in vitro studies characterizing the growth factor secretion, exosome secretion, gene expression, cell surface markers, differentiation potential, and angiogenic potential of ProtheraCytes® clinical batches to develop a potency assay. Results: Here we show that ProtheraCytes® secrete vascular endothelial growth factor (VEGF) and its concentration is significantly correlated with the number of CD34+ cells obtained after expansion (Pearson r = 0.7484; p-value = 0.0009). ProtheraCytes® also secrete exosomes containing proangiogenic miRNAs (126, 130a, 378, 26a), antiapoptotic miRNAs (21 and 146a), antifibrotic miRNAs (133a, 24, 29b, 132), and miRNAs promoting myocardial regeneration (199a and 590). We also show that ProtheraCytes® have in vitro angiogenic activity, express surface markers of endothelial and cardiomyocyte progenitor cells, and can differentiate in vitro into endothelial cells. Conclusions: The potency assay should represent the product's mechanism of action, quantitatively measure the relevant biological product attribute, and have lot to lot consistency. Developing a potency assay is a required step before commencing the pivotal Phase 3 clinical studies. After the in vitro characterization of multiple ProtheraCytes® clinical batches, we established that measuring the concentration of VEGF provided the most practical, reliable, and consistent potency assay.