Poster Session I: Acute lymphoblastic leukemia - Biology & translational research
Targeting CD19 antigen by BiTE molecule blinatumomab or CAR-T cells has shown great efficacy and survival benefits in patients with relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, a significant number of patients will relapse and up to 60% of relapses are CD19 negative. CD123 is highly expressed in BCP-ALL and as shown by our group (EHA 2018) can be targeted with flotetuzumab, a bispecific DART molecule that is currently being clinically evaluated for treatment of acute myeloid leukemia. For both blinatumomab and flotetuzumab, cytokine release syndrome has been observed, which is dose dependent for both bispecific constructs.
1) To verify if blinatumomab and flotetuzumab act synergistically in primary BCP-ALL, and
2) to decipher if the release of cytokines can be reduced by using a suboptimal dose of both constructs while maintaining optimal activity.
Bone marrow cells were isolated from BCP-ALL patients and incubated either with flotetuzumab alone (0.01 to 1 ng/ml) or blinatumomab alone (0.1 to 10 ng/ml), or with their combination. T-cell activation and cytokine production were measured after 24 h. The number of T cells and blasts was determined on days 4 and 6. Statistical significance was determined by unpaired Student's t-test.
Flotetuzumab, blinatumomab and their combination induced dose-dependent T-cell activation, proliferation, cytokine production and cytotoxicity. After 6 days, maximal killing of primary BCP-ALL blasts was induced either with 0.1 ng/ml flotetuzumab or 1 ng/ml blinatumomab (median 97.01% (93.88% - 98.75%) and 98.39% (98.26% - 99.34%) killed blasts respectively, vs 45.44% (39.58% - 60.43%) in untreated control group, n = 3, Figure 1A). Although both 0.01 ng/ml flotetuzumab and 0.1 ng/ml blinatumomab resulted in suboptimal cytotoxicity, their combination induced the significant killing of BCP-ALL blasts (median 85.78% (67.35% - 90.68%) killed blasts, vs 45.44% in untreated control group, p = 0.025, n = 3, Figure 1A). In addition, we compared the cytokine release by T cells in primary BCP-ALL samples treated with optimal concentrations of flotetuzumab or blinatumomab as single agents or blinatumomab + flotetuzumab combinations that resulted in significant blast killing (Figure 1B). Compared to the samples treated with 0.1 ng/ml flotetuzumab, secretion of cytokines was significantly lower in samples treated with the combination of 0.01 ng/ml flotetuzumab and 0.1 ng/ml blinatumomab (median 388.65 pg/ml vs 4.33 pg/ml IL-2, p = 0.042; median 40.75 pg/ml vs 7.73 pg/ml IL10, p = 0.043; and median 769.5 pg/ml vs 24.37 pg/ml TNFα, p = 0.0074; n = 3). Furthermore, compared to optimal concentration of 0.1 ng/ml flotetuzumab TNFα production (median 769.5 pg/ml) was significantly lower in samples treated with combination of higher flotetuzumab and blinatumomab doses (0.025 ng/ml flotetuzumab + 0.1 ng/ml blinatumomab: median 40.31 pg/ml, p = 0.0088, n = 3; and 0.5 ng/ml flotetuzumab + 0.1 ng/ml blinatumomab: median 96.98 pg/ml p = 0.014; n = 3). Finally, TNFα concentration was significantly lower in the samples treated with 0.01 ng/ml flotetuzumab + 0.1 ng/ml blinatumomab combination than in those treated with 1 ng/ml blinatumomab (median 24.37 pg/ml vs 214.93 pg/ml, p = 0.039, n = 3).
Our results indicate that dual targeting of CD123 and CD19 with combination of low doses of flotetuzumab and blinatumomab is an attractive approach to prevent antigen escape of BCP-ALL, resulting in significant reduction of cytokine production by effector cells.