CD33 Delineates Two Functionally Distinct NK Cell Populations Divergent in Cytokine Production and Antibody-Mediated Cellular Cytotoxicity

Abstract

The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33− subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33− NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33− NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.

Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies

“…To define optimal conditions required for the generation of CAR-expressing primary human NK cells and their expansion under Good Manufacturing Practice (GMP)-compliant conditions recently published in cooperation with Miltenyi Biotec ( 26 ), we systematically tested a number of variables that are known to influence the cytotoxicity of genetically modified NK cells.…”

“…Primary human NK cells were isolated and expanded as previously described ( 26 ) and then transduced with serially diluted (1:10 to 1:10,000) non-concentrated supernatants of freshly produced lentiviral vector particles on Retronectin-coated 96-well plates. Three to four days after transduction, NK cells were analyzed by flow cytometry for EGFP expression.…”

“…This cytotoxicity is often mediated by the expression of antigens on the leukemic blasts that are ligands for activating receptors on NK cells ( 42 ). As in vitro expanded NK cells are highly activated ( 26 ), they often recognize and thus kill leukemia cells independently of CAR antigen binding ( 42 ). In order to eliminate this “background” killing for follow-up assays, we planned to systematically block activating receptors of the NK cells in cytotoxic experimental settings.…”

“…In our studies, the use of the transduction enhancer Retronectin was associated with an 8-fold increase in gene transfer efficiency over the control wells without any enhancer and thus superior to Vectofusin, which mediated a 7-fold increase. For research purposes, these values are quite comparable; however, for clinical applications, Miltenyi has adapted the use of their Vectofusin reagent for the automated processes in the closed tube system on the CliniMACS Prodigy ® ( 26 ). Thus, using Vectofusin will thus be the easiest way to achieve high transduction efficiencies for clinical CAR NK cell products.…”

“…In this study, we intended to establish the transduction of primary human NK cells derived from peripheral blood in a feeder-cell-free and CliniMACS Prodigy™-compatible system ( 26 ) using the NK MACS medium from Miltenyi Biotec and also addressed several issues in genetic engineering of NK cells, including optimized transgene expression, alternative lentiviral pseudotypes, and transduction enhancers. We also established a methodology for the enrichment of human CAR NK cells and developed an assay system to functionally test the CAR-specific cytotoxic activity against leukemic cell lines and primary AML blasts in vitro.…”