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-E in tumor cells, which suppresses natural killer (NK) cell activity via ligation of the NK inhibitory receptor CD94/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 significantly more powerful than NKG2A+ NK cells against HLA-E-expressing tumors. Thus, NKG2A downregulation evades the HLA-E cancer immune-checkpoint, and increases the anti-tumor 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.

Keywords: Immunology; Immunotherapy; Innate immunity; NK cells; Oncology.

Figure 1. Expression of HLAE in tumors and its relation with KLRC1 (NKG2A) expression.

(A) Expression of HLAE in 10,375 specimens from 33 tumor types; box plots with data from every sample superimposed show log₁₀ HLAE gene transcripts per kilobase million (TPM) in adrenocortical carcinoma (ACC), BLCA, BRCA, CESC, cholangiocarcinoma (CHOL), COAD, diffuse large B cell lymphoma (DLBC), esophageal carcinoma (ESCA), GBM, HNSC, kidney chromophobe (KICH), KIRC, kidney renal papillary cell carcinoma (KIRP), AML, LGG, liver hepatocellular carcinoma (LIHC), LUAD, LUSC, mesothelioma (MESO), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), PCPG, PRAD, READ, SARC, SKCM, STAD, testicular germ cell tumors (TGCT), THCA, THYM, uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS), uveal melanoma (UVM). Box boundaries, first and third quartile range; whisker, interquartile range (first quartile to third quartile range) ×1.5. (B) Shown is log₂ normalized expression of HLAE with KLRC1, KLRD1 (CD94), or KLRC2 (NKG2C) in 9520 tumors analyzed (tumors lacking KLRC1 expression were excluded). Pearson’s correlation coefficient and linear regression line are shown. (C) Relation between log₂ normalized expression of HLAE and KLRC1. The 9520 tumor specimens were ordered by expression of HLAE or KLRC1; the corresponding expression of KLRC1 and HLAE is shown. (D) Relation between HLAE and KLRC1 expression in tumors with high Pearson’s correlation coefficient.

Figure 2. Downregulation of NKG2A expression in NK cells with anti-NKG2A PEBLs.

(A) Schematic representation of the anti-NKG2A PEBL constructs and their mechanisms of action. The PEBL constructs consist of a CD8α signal peptide and an anti-NKG2A scFv followed, at the C terminus, by the sequences listed in the box, according to each PEBL. PEBL1 binds to the KDEL receptor, which joins the COPI. PEBLs 2–4 bind directly to COPI. VL, light chain variable domain; VH, heavy chain variable domain. (B) Downregulation of NKG2A expression in NK92 cells. Flow cytometric histograms show surface expression of NKG2A, as detected by anti-NKG2A APC (Miltenyi Biotech), after transduction with a vector containing GFP only (Control) or GFP plus PEBLs 1–4. (C) NKG2A⁺ expanded human NK cells were purified by magnetic bead–positive selection and transduced with anti–NKG2A-PEBL2 or with GFP only. Shown are the percentages of NKG2A⁺ cells before and after purification and after transduction (10 experiments with NK cells from 9 donors), as measured by flow cytometry. ****P < 0.0001, t test. (D) Representative flow cytometry dot plots of 2 of the experiments shown in C. The right area in each dot plot encloses GFP⁺ (i.e., transduced) NK cells; the percentages of NKG2A⁺ and NKG2A cells among these cells are shown.

Figure 3. Phenotypic and functional effects of NKG2A downregulation by PEBL.

(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.

Figure 4. Downregulation of NKG2A increases NK cell cytotoxicity against tumor cells expressing HLA-E with HLA-G signal peptide (GpHLA-E).

(A) Four-hour cytotoxicity assays with NKG2A⁺ NK cells transduced with anti-NKG2A PEBL or GFP only (Control). Target cell lines were transduced with GpHLA-E (see Supplemental Figure 7) and luciferase. 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 cell killing using target cells cultured without NK cells as reference. NK cells from 11 donors were tested with K562, 6 with U2OS, 8 with ES8, and 5 with EW8, all in triplicate. (B) Expression of CD107a among NK cell subsets after 4-hour coculture with K562-GpHLA-E cells. Percentages are shown as mean of triplicate measurements with NK cells from 1 donor. (C) Four-hour cytotoxicity (measured as in A) of GFP-transduced NK cells against GpHLA-E–transduced target cells in the presence of the anti-NKG2A antibody Z199 compared with that of anti-NKG2A PEBL-transduced NK cells. An isotype-matched (mIgG2b) nonreactive immunoglobulin served as a control. Data are shown as mean (± SD) of triplicate measurements with NK cells from 2 donors (K562, ES8) or 1 donor (U2OS). (D) Long-term cytotoxicity of PEBL-transduced and control NK cells against GpHLA-E⁺ target cells. Experiments were performed at E/T 1:8 for K562, 1:2 for U2OS, and 1:4 for ES8. Tumor cell growth was measured with IncuCyte Zoom System. Data are shown as mean (± SD) of triplicate measurements with NK cells from 1 representative donor (out of 3 tested) compared with growth of the cell line without NK cells. (E) Spheroid tumors formed with U2OS-GpHLA-E cells transduced with mCherry were cocultured with PEBL-transduced or control NK cells at a 2:1 E/T. Images were collected with the IncuCyte Zoom System. Scale bars: 300 μm. Numerical data are shown in Supplemental Figure 9. *P < 0.05; **P < 0.01; ****P < 0.0001, t test.

Figure 5. Downregulation of NKG2A increases ADCC activity.

(A) Expression of HLA-E in the SK-BR-3 cell line transduced with HLA-E plus HLA-G signal peptide (GpHLA-E) or nontransduced (WT). Cells were labeled with APC-conjugated anti–HLA-E APC (blue) or isotype-matched nonreactive antibody (gray). (B) Four-hour cytotoxicity with NK cells transduced with anti-NKG2A PEBL or GFP alone (Control) against SK-BR-3-GpHLA-E cells expressing luciferase. BrightGlo was added after 4 hours of coculture, and luminescence was measured using a Flx 800 plate reader. Box (25th–75th percentile, median) and whiskers (minimum-maximum) graphs indicate the collective results of triplicate measurements obtained with NK cells from 2 donors, at a 1:1 or 1:2 E/T. Trastuzumab was added at 10 μg/ml. Horizontal bars correspond to median value. ***P = 0.0001; *P = 0.018, t test.

Figure 6. Cytotoxicity of NKG2A^null NK cells against tumor cells with endogenous HLA-E expression.

(A) Four-hour cytotoxicity of NK cells transduced with anti-NKG2A PEBL or GFP only (Control) against cell lines expressing endogenous HLA-E (see Supplemental Figure 10). EW8 and PLC/PRF/5 were transduced with luciferase. BrightGlo was added after 4 hours of coculture, and luminescence was measured using a Flx 800 plate reader. Cytotoxicity of U937 and OP-1 was measured by flow cytometry. Box (25th–75th percentile, median) and whiskers (minimum-maximum) plots from 3 experiments with cells from 3 donors (EW8, PLC/PRF/5), and 6 experiments with cells from 2 donors (U937, OP-1) in triplicate, at 2:1, 1:1, or 1:2 E/T. (B) Spheroid tumors of U2OS-mCherry were cocultured with NK cells at 1:2 E/T in triplicate and analyzed with IncuCyte Zoom System. Data are shown as mean (± SD) red calibrated unit (RCU)/μM². Representative images at end of culture are shown. Scale bars: 300 μm. (C) Four-hour cytotoxicity against cell lines exposed to IFN-γ (300 ng/ml; 12 hours). Plots are from 3 experiments with NK cells from 3 donors (EW8, PLC/PRF/5), and 6 with NK cells from 2 donors (U937) in triplicate at 2:1, 1:1, or 1:2 E/T. (D) Similar experiments targeting cells exposed for 12 hours to conditioned medium (C.M.) from 24-hour cocultures of NK cells with the respective cell lines. Four-hour cytotoxicity was compared with that against cells not exposed to conditioned medium. (E) Four-hour cytotoxicity against primary AML cells from 4 patients, exposed to IFN-γ (300 ng/ml; 12 hours). Data are from 4 experiments with NK cells from 2 donors in triplicate at 2:1 and 1:1 E/T. (F) NKG2A-negative NK cells from 3 donors were stimulated with K562-mb15-41BBL for 7 days, transduced with anti-NKG2A PEBL or GFP alone, and then exposed to IL-12 (20 ng/ml) for 5 days. Percentage of NKG2A⁺ cells at each stage is shown. (G) PEBL-transduced and control NK cells were exposed to IL-12 and tested in 4-hour cytotoxicity assays against K562-GpHLA-E cells. Data are shown as mean (± SD) of triplicate measurements at each E/T. **P < 0.01; ***P < 0.001; ****P < 0.0001, t test.

Figure 7. Antitumor capacity of anti-NKG2A PEBL-transduced NK cells in immunodeficient mice.

(A) ES8 cells (2 × 10⁵) transduced with GpHLA-E and luciferase were injected i.p. in 23 NOD/SCID IL2RG^null mice. One day later, mice were treated with 1 × 10⁷ expanded NKG2A⁺ NK cells transduced with either GFP alone (Control) or with anti-NKG2A PEBL (n = 7 for each group); another group of mice received tissue culture medium instead (no NK; n = 9). One additional injection of NK cells or medium was given 4 days later. All mice received i.p. injections of IL-2 (20,000 IU each) 3 times per week. Bioluminescence was measured with a Xenogen IVIS Spectrum System, with imaging beginning 5 minutes after i.p. injection of d-luciferin (150 μg/g body weight) and analyzed with Living Image 3.0 software. Collated ventral images of mice in 2 independent experiments are shown, 1 with 9 mice (3 in each group) and the other with 14 mice (6 injected with tumor alone, 4 treated with control NK cells, and 4 with anti-NKG2A PEBL NK cells). (B) Luminescence measurements of tumor cell growth. Each symbol corresponds to the sum of bioluminescence measurements by ventral and dorsal imaging in each mouse. (C) Kaplan-Meier curves and log-rank test for overall survival. Mice were euthanized when the sum of ventral and dorsal bioluminescence signal reached 5 × 10¹⁰ photons per second. **P = 0.0034; ***P < 0.001.