Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells
Takahiro Kamiya, … , Murray Robinson, Dario Campana
Takahiro Kamiya, … , Murray Robinson, Dario Campana
Published March 12, 2019
Citation Information: J Clin Invest. 2019;129(5):2094-2106. https://doi.org/10.1172/JCI123955.
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Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells

Abstract

A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA class I molecule (HLA-E) in tumor cells, which suppresses NK cell activity via ligation of the NK inhibitory receptor CD94/NK group 2 member A (NKG2A). Gene expression data from approximately 10,000 tumor samples showed widespread HLAE expression, with levels correlating with those of KLRC1 (NKG2A) and KLRD1 (CD94). To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum–retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A protein expression blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E–expressing tumor cells. Transduction of anti-NKG2A PEBL produced more potent cytotoxicity than interference with an anti-NKG2A antibody and prevented de novo NKG2A expression without affecting NK cell proliferation. In immunodeficient mice, NKG2Anull NK cells were substantially more powerful than NKG2A+ NK cells against HLA-E–expressing tumors. Thus, NKG2A downregulation evades the HLA-E cancer immune checkpoint and increases the antitumor activity of NK cell infusions. Because this strategy is easily adaptable to current protocols for clinical-grade immune cell processing, its clinical testing is feasible and warranted.

Authors

Takahiro Kamiya, See Voon Seow, Desmond Wong, Murray Robinson, Dario Campana

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Figure 3

Phenotypic and functional effects of NKG2A downregulation by PEBL.

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Phenotypic and functional effects of NKG2A downregulation by PEBL. (A) D...
(A) Downregulation of NKG2A is accompanied by decrease of surface CD94 expression. Flow cytometry dot plots show expression of NKG2A (CD159a PE) and of CD94 (anti-CD94 APC) in a representative sample of NK cells expanded by coculture with K562-mb15-41BBL and transduced with either anti-NKG2A PEBL-2 or GFP alone (Control). (B) Summary of CD94 versus NKG2A expression results obtained with NK cells from 7 donors. (C) NKG2C (CD159c APC) versus NKG2A expression obtained with NK cells from 8 donors. (D) Expression of CD25 in PEBL-transduced and control NK cells. Flow cytometry histograms show expression of CD25 (red, detected with anti–CD25 PE-Cy7); staining with isotype-matched nonreactive antibody (mouse IgG1 PE-Cy7) is shown in gray. (E) Survival and expansion of PEBL-transduced and control NK cells from 6 donors cultured with IL-2 (400 IU/ml). Data are shown as mean (± SD) of triplicate measurements at each time point. (F) Results of 4-hour cytotoxicity assays performed against luciferase-labeled K562 cells. BrightGlo was added after 4 hours of coculture, and luminescence was measured using a Flx 800 plate reader. Data are shown as mean (± SD) of triplicate measurements with NK cells from 8 donors. (G) Long-term cytotoxicity of PEBL-transduced and control NK cells against mCherry-transduced K562 cells at 1:8 E/T. K526 cell growth was measured with IncuCyte Zoom System. Data are shown as mean (± SD) of triplicate measurements with NK cells from 1 donor and of cultures without NK cells.