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CD16+NK-92 and anti-CD123 antibody therapy for AML
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Figure 2. NK-92 cytotoxicity against sorted leukemic stem cells and clonogenic leukemic cells relative to bulk leukemia cells. (A) Primary acute myeloid leukemia (AML) samples were sorted into CD34+CD38– and CD34+CD38+ fractions for subsequent testing in a chromium release assay with NK-92 at a 25:1 E:T ratio. Data are presented as the mean percent (%) lysis of triplicate samples (+/-Standard Deviation) representative of two separate experiments. (B) Four primary AML samples were incubated with or without NK-92 at a 25:1 E:T ratio for 4 hours in 96-well U bottom plates and utilized in either a chromium release assay (CRA) or a methyl- cellulose cytotoxicity assay (MCA) conducted on the same day. The % lysis values and % colony inhibition values are plotted together (B). An example of the methyl- cellulose cytotoxicity assay (C) shows a representative assay for one sample (080179) with a control (AML only) (i), low density control (AML + NK-92 infused into methylcellulose only) (ii), and treatment group (AML + NK-92 co-incubated together in a 96-well plate well and then infused in methylcellulose) (iii).
Irradiated NK-92 reduces leukemic stem cell fraction in the secondary transplantation assay
To assess the cytotoxic effect of irradiated NK-92 (iNK-92) on LSCs in the in vivo setting, secondary trans- plantation experiments were conducted to evaluate total engraftment and fraction of LSCs in secondary recipients. Primary AML cells (3x106) were injected by tail vein into two cohorts of 4 mice and treated with or without iNK-92 from day 2 (15x106 iNK-92 cells) twice weekly to a total dose of 75x106 iNK-92 cells. At six weeks, mice were sac- rificed and BM (1x106 cells) from each of the 4 primary recipients (donor mice) in control or treatment groups transplanted into 4 secondary recipient NSG mice (Figure 3A). Evaluation of BM from secondary recipients inocu- lated with BM from AML-infused mice untreated by iNK- 92 revealed a high proportion of human CD45+ cells (80.8, 93.3, 80.4, 96.4 Av=87.7%), while one mouse from AML infused iNK-92 treatment group was leukemia-free with engraftment at background levels of non-injected mice (96.4, 94.7, 1.8, 95.7 Av=72.2%). There was no significant difference between secondary engraftment of AML between groups receiving BM from AML-infused mice in the control and iNK-92 therapy groups (Figure 3B). However, the proportion of CD34+CD38–CD123+ cells in secondary transplanted mice for the AML control group was 7.85% (8.01, 9.48, 8.66, 5.25) and for the AML + iNK- 92 group was 3.66% (7.13, 3.46, 0.03, 4.00), which was significantly lower (P=0.05) (Figure 3C).
NK-92 prolongs survival in a primary AML xenograft model
We next sought to assess the impact of NK-92 on sur- vival of mice inoculated with primary human AML. NSG mice inoculated with 3x106 primary AML cells received 10x106 non-irradiated NK-92 weekly for three doses (Figure 4A). This treatment increased median survival from 57 to 72 days (log rank test P<0.01), although most ultimately succumbed to disease (Figure 4B). Autopsy revealed enlarged spleens and pale fragile bones compared with controls. Flow cytometry of BM from NSG mice inoculated with AML only (Online Supplementary Figure S4A), or AML + NK-92 treatments that became sympto- matic (Online Supplementary Figure S4B), had 99% engraft- ment by human leukemia in the bone marrow, while the mouse that survived long term (~9 months) was healthy at sacrifice and did not have evidence of leukemic infiltration in the marrow (Online Supplementary Figure S4C) or splenomegaly. To determine the impact of irradiation on the in vivo activity of NK-92, the cells were irradiated with 1000 cGy prior to infusion into NSG mice inoculated ten days before with 3x106 primary AML cells. NSG mice were administered 20x106 iNK-92 [intraperitoneal injec- tion (i.p.)] weekly x 5 doses and monitored for signs of leukemia (Figure 5A). Survival was improved in the treat- ment group (26-48 days) to near statistical significance (P=0.0566), but all mice ultimately succumbed to disease (Figure 5B).
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