Fig 2: eNK cells armed with Pin-CD20 exhibit long-term binding in vitro and in vivo. (A) Representative dot plot showing UCB-derived NK cells purity before (left) and after 14 days of expansion (right). NK cell frequency is indicated within the dot plot. (B) eNK cytotoxicity against K562 cell lines at effector to target ratio 1:1 and 3:1 at 4 hours. n=7 individuals eNK donors represented, Wilcoxon matched-pairs test. (C) eNK phenotypic analysis of indicated markers. The bar graph shows individual eNK donors (n=6) and mean±SEM is shown. (D) Pin-mAbs eNK cells manufacturing scheme. Mutated Fc region (Pin-Fc): red; FcγR CD16a: blue. UCB-derived eNK cells are incubated with Pin-mAbs, allowing its arming onto CD16a expressing eNK cells. Created with BioRender.com. (E and F) Comparison of arming efficiency in rituximab-armed eNK (RTX eNK) and Pin-CD20-armed eNK (Pin-CD20 eNK) over a 72 hours kinetic, eNK cells represent the baseline. Representative histogram plot for one eNK donor (F) and bar graphs representing the frequency of armed cells (left) and the arming geometric mean of fluorescence intensity (gMFI, right) detected by an anti-rituximab idiotype antibody (E) are shown. n=9 eNK donors; two-way analysis of variance with Tukey’s test, difference between RTX eNK and Pin-CD20 eNK groups is shown; mean±SEM is shown. (G) Arming stability in vivo. Pin-CD20 eNK cells arming efficiency was assessed 24 hours after intraperitoneal injection in SCID mice. eNK, n=2; Pin-CD20 eNK, n=2. mean±SEM is shown. eNK, expanded NK; mAb, monoclonal antibodies; NK, natural killer; UCB, umbilical cord blood.

Fig 3: Comparisons of percentage cell proportions in asymptomatic and symptomatic individualsShown are CD4+/CD8+ ratios, CD4+ T cells, CD8+ T cells, B cells (CD19+), and antibody-secreting plasma cells (CD27+CD38+). The scatterplots and bar graphs show the proportion of classical memory B cells (CD21+CD27+), naive B cells (CD21+CD27−), atypical memory B cells (CD21−CD27−), activated memory B cells (CD21−CD27+), classical monocytes (CD14+), and non-classical monocytes (CD16+) between symptomatic and asymptomatic individuals. Cell proportions were determined from PBMCs of patients with symptomatic (n = 155) and asymptomatic (n = 62) COVID-19. A horizontal line across the scatterplots shows the median baseline samples per participant, while the lower and upper dotted lines represent the first and third percentiles, respectively. The bar plots show the mean with standard error bars. Statistical significance between symptomatic and asymptomatic patients was determined by a two-tailed Mann-Whitney U test. Multiple comparisons between different groups were tested using Kruskal-Wallis with Dunn’s post hoc test (non-significant [ns], p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001).

Fig 4: Long-term binding of Pin-EGFR on eNK cells. (A) Schematic illustration of the Pin-EGFR monoclonal antibody and the preparation of Pin-EGFR-armed eNK cells. The two light chains of the mAb are in light blue, each with one variable (VL) and one constant (CL) domain. The two heavy chains are in dark blue, each with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3 domains). Fab: fragment, antigen binding; Fc: fragment, crystallizable region. Pin-EGFR mutations in the CH2 domain of the Fc part are represented by yellow stars. (B–D) eNK cells were incubated with Fc-engineered (solid blue line, Pin-EGFR) or wild-type cetuximab (dotted blue line, WT cetuximab) at 10 μg/mL for 1 h at 37 °C. Cells were then washed twice and incubated at 37 °C for 24 h, 48 h or 72 h. At the indicated hours, flow cytometry was used to analyze CD16 expression frequency and mAb binding on eNK cells using an anti-human-Fab detection antibody. The frequency of viable CD45+/CD56+/CD16+ eNK cells (right y axis, brown) and CD45+/CD56+/CD16+ armed eNK cells (left y axis, blue) are shown in (B). The fold-change in the geometric mean of fluorescence intensity (gMFI) detected by anti-Fab in CD45+/CD56+/CD16+ eNK cells normalized to unarmed eNK cells is shown in (C). Data are representative of 13 individual donors for CD16 and Pin-EGFR (frequency and gMFI) and 6 individual donors for wild-type cetuximab. Welch’s unequal variances t-test was applied to analyze differences between Pin-EGFR-armed eNK and WT cetuximab-armed eNK groups; mean ± SEM is shown; **** p < 0.0001, *** p < 0.001, ** p value < 0.01, * p value < 0.05. A representative histogram plot for one eNK donor is shown in (D).