Page 161 - 2018_10-Haematologica-web

CD16+NK-92 and anti-CD123 antibody therapy for AML
Figure 6. CD16+NK-92 in vitro ADCC assay against primary acute myeloid leukemia (AML) and OCI/AML5. OCI/AML5 cells were labeled with 100 mCi of Na251CrO4 for 2 hours +/- 10 mg/mL of 7G3 (anti-CD123 mAb) prior to treatment with CD16+NK-92 in 96-well plates in a standard chromium release assay. Data are presented as the mean percent lysis of triplicate samples (+/- Standard Deviation) from a representative experiment carried out twice.
expressing a tumor-associated antigen.18 CD16+NK-92 cytotoxicity against OCI/AML5 was enhanced via ADCC when target cells were coated with a murine IgG2a anti- human CD123 mAb (7G3), indicating the ability to redi- rect CD16+NK-92 against a LSC-associated antigen. Cross-species interaction between murine Fc gamma receptors and human immune effectors cells has been reported, with murine IgG2a being the most effective iso- type subclass in facilitating ADCC.27,28 However, the enhancement in CD16+NK-92 cytotoxicity seen here with 7G3 is likely an underestimate of the full potential of these cells to mediate ADCC, as the antibodies were murine rather than human in origin.
We then sought to combine iCD16+NK-92 and 7G3 therapy in our AML xenograft model and built upon prior work which demonstrated that systemic treatment with 7G3 alone in an AML NOD/SCID xenograft model reduced BM engraftment of human leukemia.29
We initially used a very small quantity of 7G3 in a single dose, preceded by blocking with a non-specific isotype- matched antibody to block Fc receptors, and improve cir- culation and binding of 7G3 to CD123, as established by Leyton et al.30 This approach worked in increasing the effi- cacy of the iCD16+NK-92 cells and improved survival. In a follow-up experiment, we did not use an Fc blocking pre-dose strategy, but gave 100 mg of 7G3 or isotype con- trol antibody BM4 for five doses with or without the iCD16+NK-92 cells. In this experiment, iCD16+NK-92 alone prolonged survival over control. The BM4 antibody had no therapeutic effect and did not enhance iCD16+NK- 92, while 7G3 alone had a modest survival benefit above control, which was not statistically significant above BM4 isotype control group. Of note, the best outcome was in the iCD16+NK-92 + 7G3 treated group which had a 10- day improvement in median survival compared with the iCD16+NK-92 + BM4 treatment group. Our data, there- fore, demonstrate that the combination of iCD16+NK-92 and 7G3 can improve survival by antibody-dependent cell-mediated cytotoxicity. Furthermore, this represents the first demonstration of in vivo efficacy of the CD16+NK-92 cell line alone and in combination with anti- body, which has only previously been tested in vitro.18 The
Fc optimized anti-CD123 humanized monoclonal anti- body CSL362 (derived from 7G3) can facilitate ADCC from peripheral blood-derived allogeneic NK cells against primary AML and CD123-expressing cell-line targets31 and is currently being tested in several clinical trials for AML. A recent study demonstrated potent CSL362-mediated NK cell ADCC against primary AML blasts and LSCs with comparable results seen with NK cells from AML patients and healthy donors.32 CD16+NK-92 cells which express the high affinity CD16 receptors could be combined with CSL362 as a potentially potent treatment of AML.
Our strategy of targeting LSCs by anti-CD123-facilitat- ed ADCC in vivo had a comparable improvement in medi- an survival to CD123 CAR T-cell therapy in an AML NSG xenograft model.33 The latter, however, used the KG1a line and not primary AML cells, as in our case. Using a differ- ent CAR vector, another group tested CD123 CAR T cells in a primary AML model with no improvement in median survival, but had approximately 40% long-term survivors at day 100 (approx. 70 days after control median survival), which was statistically significant.34 One other study of CD123 CAR T cells demonstrated efficacy against LSCs using primary and secondary engraftment models, but did not have a survival end point.35 Another group used CD123 CAR-transduced cytokine-induced killer (CIK) cells, and showed in vitro efficacy against a CD123+ cell line and primary AML targets.36 A limitation of CD123 CAR T-cell therapy is that it can target HSCs with low CD123 expression, making it a potentially myeloablative therapy, suitable only in conjunction with stem cell trans- plantation.34 In contrast, a clinical study of antibody based targeting of CD123 using CSL360 did not exhibit mye- loablation, despite having the potential for NK-mediated ADCC against LSCs with low expression of CD123.37 Therefore, we anticipate that our approach utilizing irra- diated CD16+NK-92 in combination with anti-CD123 antibodies may have a reduced risk of causing life-threat- ening myelosuppression, but this remains to be shown in clinical studies of both these approaches.
In summary, we have shown that NK-92 preferentially targets LSCs over bulk leukemia blasts in vitro and irradiat- ed NK-92 can improve survival in an AML xenograft
haematologica | 2018; 103(10)
1727