Fig 2: Monalizumab Unleash NK Cell Function In Vitro, Related to Figure 5(A) FACS profiles showing HLA-E expression at the cell surface in SCCHN cell lines. White histograms: isotype control; gray histograms: anti-HLA-E mAb. NK cells were co-cultured with the cell line indicated, in the presence or absence of monalizumab. The frequencies of CD107- and CD137-producing NKG2A+ NK cells are shown. Each donor is represented by a single dot. Wilcoxon matched-pairs signed-rank test, * p < 0.05, ** p < 0.01.(B) FACS profiles showing HLA-E expression at the cell surface in ovarian cancer cell lines. White histograms: isotype control; gray histograms: anti-HLA-E mAb. NK cells were co-cultured with the cell line indicated (after overnight stimulation of the cell line with IFN-?) in the presence or absence of monalizumab. The frequencies of CD107- and CD137-producing NKG2A+ NK cells are shown. Each donor is represented by a single dot. Wilcoxon matched-pairs signed-rank test, * p < 0.05, ** p < 0.01.

Fig 3: HLA-E and PD-L1 Expression in Solid Cancers, Related to Figure 4Formalin-fixed paraffin-embedded (FFPE) tissue microarrays were stained by immunohistochemistry with anti-HLA-E (clone MEM-E/02) or anti-PD-L1 (clone E1L3N) antibodies. For each indication, scatterplots represent the percentage of the area stained for HLA-E or PD-L1 for each spot. Quantification was performed with Halo (Indicalabs). Colored bars indicate the mean (±SD). Lung (n = 45), pancreas (n = 79), stomach (n = 76), colon (n = 109), H&N (n = 70) and liver tumors (n = 106) were studied.

Fig 4: Monalizumab and Durvalumab Unleash NK and CD8+ T Cell Function In Vitro(A) NK cells were co-cultured with K562 or K562 cells expressing HLA-E in the presence or absence of monalizumab. The frequencies of CD107-positive NK cells are shown. Box and whiskers plot, with the means indicated by crosses. N = 8. The whiskers are drawn down to the 25th percentile minus 1.5 times IQR (interquartile range) and up to the 75th percentile plus 1.5 times IQR. Friedman analysis followed by Dunn’s multiple comparisons test. **p = 0.006; ***p = 0.0001.(B) Purified 7 days IL-2-activated NK cells were co-cultured or K562 cells expressing HLA-E in the presence or absence of monalizumab. The frequencies of CD107 and IFN-?-positive NK cells are shown. Non-parametric Wilcoxon matched-pairs rank test. N = 6; *p = 0.0313.(C) NK cells were stimulated in vitro with IL-15 for 9 days. The data shown are the frequencies of NK cells expressing NKG2A or PD-1 before (day 0) and after (day 9) culture.(D) The NK cells generated in (C) were co-cultured with K562 cells expressing HLA-E or co-expressing HLA-E and PD-L1 without (control) or with monalizumab (mona), durvalumab (durva), or both these antibodies (combo). The data shown are the frequencies of CD107-expressing NKG2A+ PD-1+or PD-1- NK cells. Box and whiskers plot, with the means indicated by crosses like in Figure 5A. N = 13 donors. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.(E) CD8+ T cells were co-cultured in vitro with monocytes in the presence of IL-15 and Flu peptide for 9 days. Top panel: one representative dot plot showing the frequency of Flu tetramer positive (TMr+) CD8+ T cells after culture (n = 14). Bottom panel: frequencies of NKG2A+ and/or PD-1+ cells after gating on TMr+ CD8+ T cells (n = 14).(F) The CD8+ T cells generated in (E) were co-cultured with Flu peptide-pulsed K562 cells expressing PD-L1, HLA-E and HLA-A2 without (control) or with monalizumab (mona), durvalumab (durva), or both antibodies (combo). The data shown are the frequencies of CD107-expressing (upper panels) and IFN-?-secreting (lower panels) NKG2A+ or NKG2A- CD8+ T cells (n = 17). The whiskers are drawn like in Figure 5A. *p = 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.(G) The CD8+ T cells generated in (E) were co-cultured with Flu peptide-pulsed K562 cells expressing PD-L1, HLA-E, and HLA-A2 loaded with Cr51 without (control) or with monalizumab (mona), durvalumab (durva), or both antibodies (combo). The data shown are the frequencies of K562 target cell lysis. N = 14 donors. The whiskers are drawn like in Figure 5A. *p < 0.05; **p < 0.01; ***p < 0.001.See also Figures S5 and S6.

Fig 5: Characterization of Monalizumab, Related to Figures 5 and 6(A) Titration of monalizumab on Ba/F3 cells expressing human NKG2A or NKG2C. Monalizumab was detected with a secondary antibody conjugated to PE.(B) Titration of monalizumab on NKG2A+ NK and CD8+ T cells from the PBMCs of healthy donors.(C) Monalizumab inhibits the binding of HLA-E tetramers to CD94/NKG2A receptors on human peripheral NK cells. Human PBMCs (n = 4 with NKG2C+ NK cells < 3.5%) were incubated with various concentrations of monalizumab (ranging from 0 to 10 µg/mL), washed and then incubated with PE-conjugated HLA-E tetramers. NK cells were defined as CD56+ CD3- lymphocytes. The percentage inhibition was calculated as follows: inhibition (%) = 100 – 100*[(MFI PE (at X µg/mL of monalizumab) – MFI PE FMO)/(MFI PE (at 0 µg/mL of monalizumab) - MFI FMO)]. MFI: mean fluorescence intensity; FMO: fluorescence minus one.