Discovery of a Transferrin Receptor 1-Binding Aptamer and Its Application in Cancer Cell Depletion for Adoptive T-Cell Therapy Manufacturing.

The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across companies, current methods do not actively eliminate the cancer cells in the patient's apheresis product from the healthy immune cells. Alarmingly, it has been found that transduction of a single leukemic B cell with the CAR gene can confer resistance to CAR T-cell therapy and lead to treatment failure. In this study, we report the identification of a novel high-affinity DNA aptamer, termed tJBA8.1, that binds transferrin receptor 1 (TfR1), a receptor broadly upregulated by cancer cells. Using competition assays, high resolution cryo-EM, and de novo model building of the aptamer into the resulting electron density, we reveal that tJBA8.1 shares a binding site on TfR1 with holo-transferrin, the natural ligand of TfR1. We use tJBA8.1 to effectively deplete B lymphoma cells spiked into peripheral blood mononuclear cells with minimal impact on the healthy immune cell composition. Lastly, we present opportunities for affinity improvement of tJBA8.1. As TfR1 expression is broadly upregulated in many cancers, including difficult-to-treat T-cell leukemias and lymphomas, our work provides a facile, universal, and inexpensive approach for comprehensively removing cancerous cells from patient apheresis products for safe manufacturing of adoptive T-cell therapies.

Using A Spin-Coater to Capture Adhesive Species during Polydopamine Thin-Film Fabrication.

Spin-coating was evaluated as a technique to study events that occur during polydopamine (PDA) thin-film formation. The reaction variants studied included type of oxidant, dopamine (DA) concentration, pH, adhesion time prior to spin, substrate chemistry, and notably, DA solution aging time. A strong oxidant, sodium periodate (SP), and a weak oxidant, atmospheric oxygen were chosen. It was found that reactions in solution were much faster and produced much thicker PDA films with SP than with oxygen. PDA thickness correlated positively with DA concentration, SP solution pH, and adhesion time. DA oxidation and aggregation is a dynamic process, which is reflected in the DA aging-time parameter. PDA film thickness reached a maximum value as DA solution aged. Color photography, UV-vis spectroscopy, and dynamic light scattering indicated that the optimal DA aging time for PDA adhesion is the result of the evolution of PDA particle size and chemistry over time. The capture of the optimal aging-time window was identified as the critical parameter for preparing PDA films with continuity and appreciable thickness. When these conditions were applied in a modified dip-coating method, comparable PDA films were fabricated as those obtained from spin-coating. Native silicon wafers (SiO2) as well as wafers that were modified with polydimethylsiloxane (PDMS) and amine-containing polydimethylsiloxane (PADMS) were chosen to represent a wide range of substrates with different substrate-PDA interactions. The main effect of substrate structural difference was on PDA film morphology. "Island" morphologies were obtained on PDMS where only hydrophobic interactions are responsible for PDA adhesion, while "speck" morphologies were observed on SiO2 and PADMS. The stabilities of the fabricated PDA films were tested in 0.1 M HCl and DMSO. The SP-derived PDA films exhibited very little mass loss compared to those fabricated using either the conventional dip-coating method or oxygen as an oxidant. Choosing a strong oxidant, understanding the DA reaction dynamics, and taking advantage of the optimal DA aging time are important in the fabrication of stable PDA films on a variety of substrates.