Page 59 - Haematologica August 2018

Activity of SL-401 in AML and MDS
limited to a sub-population of normal stem cells, few lym- phoid progenitors, basophils and AML LSCs.35-37 Moreover, blasts in about 10% of AML cases express CD123 without concurrent CD33 expression, and overall about 80% of AML patients express CD123.28 Furthermore, high numbers of CD34+CD38low/-CD123+ blasts predict poor outcome in AML patients and CD34+CD38–CD123+ population is increased in AML patients at relapse.37 While favorable and intermediate risk AML subtypes have comparable levels of CD123 expres- sion, high-risk AML sub-groups harboring a FLT3-ITD mutation tend to have higher CD123 expression.38 It was recently shown that CD123 targeted chimeric antigen receptor (CAR) T-cell therapy leads to myeloablation in primary AML xenografts.16 Therefore, CD123 serves as an important target for treating AML.
Here, we have shown that AML cells express varying levels of cell surface CD123. Importantly, we show that SL-401 is potent in killing AML cells that express even low levels of CD123 and activity of SL-401 is not dependent on surface density of CD123. Earlier reports have used IL- 3 or variant IL-3-fused toxins to investigate the CD123 tar- geted killing of leukemic cells.29-34 These studies did not include a detailed analysis on AML cell survival or activity
using in vivo PDX survival models. Previous studies involv- ing IL-3-protein-toxin have used fluorescence intensities or transcript levels to compare the CD123 expression on different cell types and colony forming ability of AML as a prime read out. To control for variability between exper- iments, we used microspheres with a standardized fluo- rochrome to derive the CD123-MESF, thus minimizing variations due to instrument or time points. In our study, we saw no correlation between CD123-MESF and sensi- tivity to SL-401 cytotoxicity. It is important to note that the previous studies utilized a different clone of antibody, assay for receptor subunits, AML culture methods and cytotoxicity assays and end points. The use of high serum containing medium to culture AML in our studies may have affected CD123 expression less likely (Online Supplementary Figure S10) and possible induction of LSC differentiation.
The fact that AMLs expressing less than 10,000 CD123- MESF are sensitive to SL-401 underscores the potency of this agent. This is corroborated by a clinical trial with SL- 401 that showed no correlation between robust responses and SL-401 pharmacokinetics.19,20 Interestingly, SL-401 mediated cytotoxicity in the presence of exogenous IL-3 (10ng/ml) in in vitro AML/MDS cultures. We think this
AB
C
P=0.0069
Figure 5. In vivo activity of SL-401 in AML PDX models. (A) Survival curves of treatment groups from busulfan preconditioned NRGS mice engrafted with primary AML (AML 28 and AML 29). AML 28 was used to engraft three animal in each group and AML 29 was used to engraft one animal in each group. Total mice used are four per group. Ten days after engraftment, mice were randomized and treated with vehicle or SL-401 (50 μg/kg administered intraperi- toneally, 3 doses: M/W/F per week for 5 weeks). (B) AML burden in bone marrow of NRGS mice engrafted with AML and treated with vehicle or SL-401 during week 3-6. Mice were treated blindly and sacrificed on week 7. Bone marrow was harvested, counted and immunophenotyped by multicolor flow cytometry. P=0.004 for Vehicle vs. SL-401 treatment. (C) Representative flow cytometric dot plots of bone marrow showing tumor burden of Vehicle and SL-401 treated PDX mice, engrafted with AML 28, at week 7.
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