This study demonstrated for the first time that human peripheral blood mononuclear cells (PBMCs) from chronic lymphocytic leukemia (CLL) patients can be converted into NK cells, CD8+ T cells, NKT cells, and TCRγδ T cells (SUPLEXA cells) despite low numbers of normal immune cells at baseline and the known immunologic impairment present in CLL patients. Importantly, SUPLEXA cells derived from CLL patients acquire potent tumor killing activity that is indistinguishable from SUPLEXA cells prepared from NHVs. Taken together, these findings support the feasibility of converting PBMCs from CLL patients with low percentages of NK and T cells into an autologous cellular therapy for cancer.

Engineered Leukocyte Immuno Stimulatory (ENLIST) cell lines were engineered by expressing curated immunomodulatory proteins in the SK-MEL-2 melanoma cell line. Two million PBMCs from patients with CLL or controls were incubated with 0.4 M freeze/thaw killed ENLIST cells for 5 days in XVIVO-15 medium with 2% heat-inactivated human AB serum (XAB2) and then split 1:15 in XAB2 containing IL-7 and IL-15 to expand. After 9 days, SUPLEXA cells were harvested and cryopreserved. Original PBMCs and matched SUPLEXA cells from each donor were thawed and characterized by mass cytometry (CyTOF). CyTOF staining results of PBMCs from CLL patients demonstrated approximately 95% leukemia cells and few T cells, NK cells, B cells, and monocytes. CyTOF staining of SUPLEXA cells from all CLL patients showed expansion of NK cells, CD8 T cells, and CD4 T cells that were similar in phenotype to SUPLEXA cells from controls showing high expression of granzymes and perforin that are indicative of potent tumor cell killing activity. Cancer cells in the original CLL PBMC samples were reduced to 0.78%. However, a population of non-T/non-B cells was detected in SUPLEXA cells from all CLL patients that require further characterization. Next, SUPLEXA cells from CLL and controls patients were comparatively tested for tumor cell killing activity at 2:1, 1:1, and 1:2 effector to target cell ratios. Percent killing of tumor cells by SUPLEXA cells prepared from CLL patients and controls were nearly identical at all effectors to target ratios.

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