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CD16+NK-92 and anti-CD123 antibody therapy for AML
A
B
Figure 4. NK-92 therapy of primary acute
myeloid leukemia (AML)
NOD/SCID gammanull (NSG) mice. 3x106 pri- mary AML cells were injected intravenously (i.v.) via tail vein into irradiated NOD/SCID gamma null mice to establish disease in control (n=5) and therapy mice (n=10). 10x106 NK-92 were infused via tail vein weekly for three weeks starting on the day of AML inoculation in treat- ment group (A). Mice were monitored for signs of leukemia and sacrificed at humane end points. Kaplan-Meier survival curves were gen- erated to compare survival in control and treat- ment groups (P<0.01) (B).
xenografted
over control with both groups having a median survival of 32 days (P=0.619), but 7G3 significantly improved median survival to 35 days compared with the AML only control (P<0.001), but not the BM4 isotype control (P=0.1509). A combination of 7G3 and iCD16+NK-92 produced the best survival outcome, with a median survival of 42 days, which was significantly enhanced over mice infused with AML only and received no therapy (+10 days; P<0.001), 7G3 (+ 7 days; P<0.0025) and iCD16+NK-92 + BM4 (+ 10 days; P<0.0025) (Figure 7B).
Discussion
The overall long-term survival for patients with AML is approximately 40%,2 demonstrating a need for novel treat- ment strategies, particularly for patients in remission with detectable residual disease. We recently published a phase I clinical trial of NK-92 in relapsed and refractory hemato- logic malignancies (lymphoma and multiple myeloma) with minimal toxicities despite cumulative doses as high as 150 billion cells, and showed clinical responses.14 Prior stud- ies implicating NK cells as therapeutically relevant in hap- lotype transplantation for AML20 prompted us to further investigate NK-92 as therapy for AML.
We have studied the mechanism of NK-92 cytotoxicity against primary AML samples and its efficacy in a primary
human AML xenograft model. We confirmed initial reports that NK-92 mediates cytotoxicity in vitro against primary AML11 and demonstrated that killing was due pri- marily to granule exocytosis rather than ligand-mediated cytotoxicity (e.g. via Fas ligand) as shown by inhibiting cytotoxicity with the calcium chelator, EGTA.
We noted that classically defined, sorted CD34+CD38– LSCs4 were more sensitive to NK-92 killing than leukemia blast cells at low E:T ratios in a standard chromium release assay. Given the conflicting reports in the literature of the definitive immunophenotype of the LSC in AML,4,5,21,22 we opted to use a clonogenic assay to assess the effect of immune effector cells against LSCs in a larger set of sam- ples. Primary AML grows well in methylcellulose and leads to the generation of visually detectable single cell-derived colonies that identifies the frequency of individual leukemic stem and progenitor cells. Specifically, we used a methylcellulose cytotoxicity assay (MCA) established pre- viously by our lab19 that enables a comparison of the degree of killing of bulk leukemia blast cells versus colony inhibi- tion in a 4-hour period. This approach provides another means to assay differential cytotoxicity against bulk and LSC populations. The MCA demonstrated a 2-3-fold higher % colony inhibition than the % lysis meas- ured by the CRA. These results support our initial finding using cell sorted LSCs, which showed that NK-92 can pref- erentially recognize and kill LSCs over bulk leukemia.
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