Aptamer-Based Chromatographic Methods for Efficient and Economical Separation of Leukocyte Populations

Abstract

The manufacturing process of chimeric antigen receptor T cell therapies includes isolation systems that provide pure T cells. Current magnetic-activated cell sorting and immunoaffinity chromatography methods produce desired cells with high purity and yield but require expensive equipment and reagents and involve time-consuming incubation steps. Here, we demonstrate that aptamers can be employed in a continuous-flow resin platform for both depletion of monocytes and selection of CD8⁺ T cells from peripheral blood mononuclear cells at low cost with high purity and throughput. Aptamer-mediated cell selection could potentially enable fully synthetic, traceless isolations of leukocyte subsets from a single isolation system.

Keywords: CAR T cell manufacturing; aptamers; cell isolation; chromatography.

Figure 1.

Monocyte aptamer (Mono.A2) depletion studies. A) Monocyte depletion scheme. Thawed PBMCs are run first through uncoupled resin, yielding pre-cleared PBMCs (PC PBMCS) in the flowthrough that are subsequently loaded into a Mono-A2-conjugated resin. B) Optimization of Mono.A2 aptamer concentration for resin coupling was performed with a single PBMC donor using concentrations ranging from 3 to 25 nmol/g resin, where a concentration of 6.25 nmol/g resin depleted the monocytes the most. C) Representative flow cytometry histograms of CD14 expression in the initial PC PBMCs compared to the monocyte-depleted Mono.A2 flowthrough fractions. D) Final CD14⁺ monocyte purities in pre-sort PBMCs, PC PBMCs, and flowthrough PBMCs. The circles, squares, and triangles represent different donors from separate isolation experiments. Horizontal lines and error bars represent the mean ± s.d.; ns = not significant; ***p<0.001 (one-way ANOVA test)

Figure 2.

CD8⁺ T cell aptamer (CD8.A3t) selection optimization studies. A) PBMC cell loading protocol for pre-clearing step compared to control for CD8⁺ T cell selection on CD8.A3t-conjugated resin. Thawed PBMCs were run through unfunctionalized resin first in the pre-clearing procedure. The resulting uncoupled flowthrough was run through CD8.A3t-coupled resin. This was compared to the initial procedure of thawed PBMCs run through one round of CD8.A3t-coupled resin alone. B) Flow cytometry histograms of CD8 expression comparing isolation purities from elution fractions with pre-clearing procedure and without pre-clearing procedure. C) Purity and yield analysis comparing PBMCs isolated through CD8.A3t- and 12dT-CD8.A3t-coupled resin, performed on the same donor. D) Optimization of 12dT-CD8.A3t aptamer concentration for resin coupling performed across two PBMC donors using concentrations ranging from 0.625 and 25 nmol/g resin, where the highest purity and yield occurred at 0.625 nmol/g resin. E) Optimization of 12dT-CD8.A3t-coupled resin volumes after pre-clearing using 0.3, 0.4, and 0.5 mL volumes.

Figure 3.

Final 12dT-CD8.A3t-mediated resin chromatography results for selection of CD8⁺ T cells. A) CD8⁺ selection scheme where thawed PBMCs are first run on unfunctionalized resin, resulting in pre-cleared PBMCs (PC PBMCs) in the flowthrough that are subsequently run in CD8.A3t-coupled resin in the first round (R1). The eluted flush from this round is finally run through a second round (R2) of CD8.A3t-coupled resin. B) Representative flow cytometry histograms of CD8⁺ expression in each round of isolation. C) Final CD8⁺ purity and yield data in samples from cell isolation experiments. The circles, squares, and triangles represent different donors from three separate isolation experiments. Horizontal lines and error bars represent the mean ± s.d.; **p<0.01, ***p<0.001, ****p<0.0001, ns not significant (one-way ANOVA test).

Scheme 1.

Conjugation of thiol-labelled aptamers to epoxidized polystyrene resin for chromatographic cell separation. Epoxidized polystyrene resin particles are rehydrated, washed, pre-blocked with salmon sperm DNA, and conjugated with TCEP-reduced, thiol-labelled aptamers in modified DPBS, pH 8 for 2hr. After conjugation, aptamer-coupled resin is washed and blocked with 2% BSA, 1% Tween-20 in modified DPBS solution.