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B.A. Williams et al.
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Figure 3. Effect of iNK-92 on secondary bone marrow (BM) engraftment of acute myeloid leukemia (AML) cells and leukemic stem cells (LSCs). (A) 3x106 AML cells were also infused intravenously (i.v.) into two cohorts of 4 mice and treated with and without iNK-92 from day 2 and given 15x106 cells twice weekly to a total dose of 75x106 (A). BM (1x106 cells) from each of 4 primary recipients in control and treatment was serially transplanted 1:1 into 4 new NOD/SCID gammanull (NSG) mice. These mice were sacrificed at six weeks and BM assayed for overall leukemic engraftment as determined by presence of % human CD45+ cells (B) and LSC engraft- ment as determined by % human CD34+CD38–CD123+ cells (C) (*P=0.05).
CD16+NK-92 mediates ADCC in vitro and in vivo and prolongs survival in an AML xenograft model by targeting leukemic stem cells
To develop a strategy to enhance killing of LSCs, we uti- lized a gene-modified CD16+NK-92 transduced with the high affinity CD16 receptor (NK-92.176V GFP), which is capable of mediating ADCC against antibody-coated tar- gets. The proportion of cells expressing CD16 was 2.3% for parental NK-92 and 27.9% for CD16+ NK-92 (Online Supplementary Figure S5). The chromium release assay was modified to measure ADCC by coating target cells with antibodies prior to the 2-hour chromium incubation. The CD123+ leukemia cell line OCI/AML5 was pretreated with anti-CD123 (7G3) antibody at a dose of 10 μg/mL prior to use in a chromium release assay. CD16+NK-92 showed cytotoxicity against OCI/AML5 cells at all E:T ratios, and was significantly enhanced (2-6x) when targets were coat- ed with anti-CD123 mAb (Figure 6), demonstrating effec- tive ADCC.
To enhance the approach of irradiated NK-92 against human AML in the xenograft NSG model, we treated the mice with a CD16+NK-92 cell line, in combination with an
anti-CD123 mAb (7G3), given on the same days to facili- tate targeting of leukemic stem cells by antibody-depen- dent cell-mediated cytotoxicity. A blocking dose of iso- type control antibody BM4 (200 mg) was given prior to the administration of 7G3 at a low dose (8 mg), due to the known sequestration of 7G3 in the spleen (Online Supplementary Figure S6A). In this pilot experiment, we demonstrated that 7G3 (8 mg) could enhance the therapeu- tic efficacy of iCD16+ NK-92, as determined by an improvement of median survival by 13 days (P=0.0173) (Online Supplementary Figure S6B).
We conducted a more rigorously controlled experiment of CD16+NK-92 and anti-CD123 mAb therapy utilizing an isotype control antibody (BM4) in the control arms (Figure 7A). The cohorts included: no therapy, 7G3, BM4, iCD16+NK-92, iCD16+NK-92 + 7G3, iCD16+NK-92 + BM4. The dosing schedule was iCD16+NK-92, with or without 7G3 or BM4, given on days 3, 5, 7, 10, and 12 after AML inoculation (day 0). NSG mice without therapy had a median survival of 32 days. iCD16+NK-92 alone sig- nificantly improved survival to a median of 37 days (P<0.001). Treatment with BM4 did not enhance survival
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haematologica | 2018; 103(10)
% Engraftment
% Leukemic stem cells