Isolation of CD56⁺ Natural Killer Cells Using the CliniMACS® MARRS Tubing Set, a Newly Developed Tubing Set Including a Cross-Flow Filtration Module for Removal of Unbound Reagent.

Use of Natural Killer (NK) cells in cellular immunotherapy requires processing of large numbers of NK cells in a closed system consisting of clinical grade components. We have previously developed a two step NK cell isolation process in which the CliniMACS® ^plus Instrument and CliniMACS® CD3 and CD56 Reagents are used for CD3⁺ cell depletion and subsequent CD56⁺ cell enrichment, respectively. We have recently optimized the CD3 depletion step for very large scale cell processing by development of the CliniMACS® Depletion Tubing Set (“DTS”) and appropriate cell separation software, reducing the processing time of the CD3 depletion step by 50%. We now have developed the CliniMACS® MARRS Tubing Set (MARRS = MAgnetic Reagent Removal System), a tubing set which includes a cross-flow filtration module for removal of unbound magnetic reagent after magnetic labelling. The cross-flow filtration module allows to directly connect a magnetically labelled cell product to the tubing set without centrifugational washing steps, reducing the processing time by 45 minutes. We isolated CD56⁺ cells from buffy coats as well as apheresis harvests using the CliniMACS® MARRS Tubing Set as compared to the standard CliniMACS® Tubing Set. Both tubing sets (MARRS vs. standard) yielded CD56⁺ cells of comparable purity (94.1% vs. 92.3%), comparable depletion of CD56⁻CD3⁺ T cells (3.2 log vs. 3.0 log) and comparable recovery (72.9% vs. 76.7%) with a trend towards better performance of the CliniMACS® MARRS Tubing Set. To assess the functionality of CliniMACS® separated CD56⁺ cells we performed flow cytometric cytotoxicity tests against the MHC class I negative target cell line K562. Specific killing could be increased from <2% (unseparated sample) to 47% (MARRS) and 53% (standard) at an effector to target ratio of 50:1. Thus, K562 killing cells are efficiently enriched and cytotoxicity is similar for both CD56⁺ cells isolated using the standard CliniMACS® Tubing Set and the CliniMACS® MARRS Tubing Set. We additionally assessed whether the killing efficiency of CD56⁺ cells correlates with NK receptor expression (NCR, KIR, NKG2D, costimulatory molecules) on different NK and NKT subsets. Results of these analyses will be presented. We conclude that the CliniMACS® MARRS Tubing Set can be used to efficiently isolate functional CD56⁺ cells from buffy coats or medium scale apheresis harvests (e.g. non-mobilized donors), clearly reducing total processing time. For larger scale NK cell selection using apheresis harvests (e.g. mobilized donors) the combination of Depletion Tubing Set for CD3⁺ cell depletion and the standard CliniMACS® Tubing Set for CD56⁺ cell enrichment is suitable.