Fig 2: hEGFRvIII:CD3 bi-scFv binds only human CD3-expressing cells. (A) hEGFRvIII:CD3 bi-scFv binding to PBMCs from human, cynomolgus, beagle, mouse, pig, rabbit, and rat were tested using flow cytometry. Binding studies using only the secondary detection PEPvIII multimer served as control. (B) hEGFRvIII:CD3 bi-scFv binding to PBMCs from three human donors. Dashed line shows EC20. EC20, 20% of the maximal effective concentration; EGFRvIII, epidermal growth factor receptor variant III; PBMCs, peripheral blood mononuclear cells.

Fig 3: hEGFRvIII:CD3 bi-scFv extends survival in vivo. Immunodeficient NSG mice were coimplanted with U87-MG-EGFRvIII and human PBMCs. Subsequently, mice in the treatment group received 50 µg hEGFRvIII:CD3 bi-scFv daily for 5 days via intravenous tail injection. Bar indicates time of hEGFRvIII:CD3 bi-scFv treatment. EGFRvIII, epidermal growth factor receptor variant III; NSG, NOD-scid gamma; PBMCs, peripheral blood mononuclear cells.

Fig 4: Cryopreserved NK cells exert antitumor effect in an in vivo PDX mouse model. Ascites were collected from an NSG mouse that had PDX tumor injected intraperitoneally. Spheroids were obtained from the ascites, and 20,000 spheroids were injected in suspension back to the intraperitoneal (i.p.) cavity of female NSG mice and allowed to seed for 3 days. Mice were then treated with 2 doses of cryopreserved NK cells (1 × 107/dose, days 0 and 5) from two donors, which were thawed and directly injected i.p. along with IL-2 (25,000 U, 3×/week). Health was monitored periodically, and abdominal wash was collected from one mouse treated with frozen NK cells euthanized 64 days posttreatment. A schematic of the experiment is shown (A). Mice injected with frozen NK cells from donor 1 (blue dashed line) and donor 2 (red dotted line) had a significant increase in survival compared to untreated mice (black solid line) (B). Flow cytometry dot plots are shown for gating on CD45+ cells in the abdominal wash of a treated mouse euthanized on day 64 posttreatment (C). The percentages of hNKT (CD3+, CD56+), hNK (CD3-,CD56+), and hT cells (CD3+) were determined in the hCD45+ population. Statistical significance was determined by comparing survival curves using the Gehan–Breslow–Wilcoxon test.

Fig 5: NK cells are viable after cryopreservation. NK cells were expanded with PM21 particles (PM21-NK cells) from T-cell-depleted PBMCs obtained from multiple donors (N = 19). Cells were cryopreserved and stored in liquid nitrogen until use. Frozen PM21-NK cells were thawed and counted for number of viable NK cells, and no loss of NK cells was seen. Representative flow cytometry dot plots gating for viable (Draq7-) NK (CD3-, CD56+) cells immediately post-thaw are shown (A). Thawed NK cells were placed in media at 1 × 107 of viable cells and rested overnight at 37°C. After 16 h, the total number of viable NK cells was counted. Percent recovery at 16 h post-thaw was calculated by dividing the total number of viable cells, remaining after 16 h rest, by 1 × 107. Mean recovery was 73% ± 22% (B). Data are represented as a scatter plot with an error bar representing standard deviation.