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Characteristics of products for NK-cell infusion prepared by CD3 depletion followed by CD56 enrichment: requirements for rare event analysis

      NK cell immunotherapy delivered early after HLA-haploidentical BMT can prevent relapse. Such products require processing that rigorously depletes T cells to reduce GVHD risk while sparing NK cells. Here we describe characteristics of 18 products processed by two labs supporting a multi-center clinical trial. Marrow donors underwent a single non-mobilized apheresis day 6 post-BMT with next day processing by a 2-step method of T cell depletion/NK cell enrichment using the Miltenyi CD3/CD56 CliniMACS system. Starting products and all fractions were characterized by flow for T(CD3+CD56-), NK-T(CD3+CD56+), NK(CD3-CD56+), & B cells(CD19+) & monocytes (CD14+). T, B, & NK cells were assessed after gating on a low side scatter population negative for dead cells (7-AAD), monocytes and myeloid cells (CD33) to reduce background staining. Starting products did not differ between sites however, trial products did have a higher % of T cells compared to DLI products (N=17, p=0.03). CD3-depletion alone preserved NK cells (81.2±11.0% recovery) but removed only 3.1±0.3 logs of T cells and 2.0±0.5 logs of NK-T cells. Subsequent CD56-enrichment reduced T cells to 5.6±0.7 logs and NK-T cells to 2.4±0.3 logs. Final NK cell recovery was 52.4±8.1% (39.5% -66.7%) with B cells constituting only 0.2±0.2% of the final product. Both labs produced final products with >5.0 logs of TCD and with similar NK cell purity (91% and 84.1%) and recovery (52.9% and 52.7%). Monocytes constituted 6.9±7.1%(range 1.2-28.2%) of the final product that did not differ by lab. We conclude that using the Miltenyi system with a 2-step T cell depletion/NK cell enrichment labs can consistently achieve good recovery of NK cells with extremely low T & B cell content allowing NK infusions up to 5.0E6/kg. A consensus flow cytometry analysis method was critical to detect rare events and ensure safe levels of T cells for infusion across major HLA barriers.
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