Page 53 - Haematologica August 2018

Activity of SL-401 in AML and MDS
Patients with MDS who are refractory to hypomethylat- ing agents also have very limited therapeutic options.7
CD33 is a widely expressed myeloid marker present on the majority of AML cells, and CD33-targeted immunotherapies have shown promising results.8,9 However, LSC of immature AML lack CD33 expression and are not eliminated by CD33 targeted agents.8 CD123 is the a chain of the interleukin-3-receptor (IL-3R) het- erodimer that has affinity and specificity for interleukin-3 (IL-3).10 LSC are known to express CD12311,12 and several efforts to target CD123 to eradicate these cells have emerged.13-18 To date, however, each of these agents exhib- ited shortcomings that limited their development. While a prototype monoclonal antibody against CD123 showed no promising response in AML, new clones of antibod- ies/single chain fragment variable (ScFv) are currently under investigation for antibody-drug conjugates (ADC) and antibody-dependent cellular cytotoxicity (ADCC) functions.13-15 CD123 targeted chimeric antigen receptor T cells (CART cells) are now being evaluated in early phase clinical trials and fatalities from cytokine release syn- drome in patients receiving CD123-CART therapy were reported as of this writing. Recently, preclinical effects of a dual affinity retargeting (DART) molecule, generated from CD3 and CD123 antibodies, were described in AML.18
SL-401 is a recombinant fusion protein consisting of human IL-3 and truncated diphtheria toxin, previously reported to be active in CD123+ neoplasms.19,20 The IL-3 domain dictates the specificity for CD123 expressing cells, and the catalytic unit of diphtheria toxin upon internaliza- tion inhibits the translational machinery to initiate cell death. Although, most chemotherapy regimens are effec- tive in eradicating tumor bulk in the periphery, progenitor tumor cells in the marrow are less exposed to treatments and receive protective support from the niche. Bone mar- row stromal cells are a part of the bone marrow microen- vironment and play an important role in the proliferation of AML by offering various forms of protection mediated through soluble factors and contact-dependent survival signals to leukemic cells.21-24 Co-culturing of AML cells on human bone marrow stromal cell line HS-5 monolayers increases proliferation, viability, and colony formation of the AML cells, while diminishing chemotherapy-induced apoptosis.25 Furthermore, bone marrow-derived mes- enchymal stromal cells (MSC) from AML patients exhibit overlapping as well as distinct cytogenetic abnormalities versus AML blasts and provide a conducive cytokine and growth milieu for leukemia cells.24,26 Therefore, it is critical to assess potential AML therapies in the context of the bone marrow microenvironment with autologous MSCs, which might be unique for each patient. In the present study, we report the potent activity of SL-401 in CD123 expressing primary AML blasts and MDS samples and its relation to CD123 levels. We also show in vitro studies of SL-401 when AML cells are co-cultured with MSCs and in vivo studies using patient derived xenograft (PDX) mouse models.
Whether CD123 is sufficiently specific for leukemic stem cells is controversial. We show here definitively that CD123 targeted SL-401 is cytotoxic to both normal cord blood-derived hematopoietic stem cells and CD123+ blasts in AML and MDS. These findings suggest that CD123 tar- geting may cause pancytopenia as a consequence of on- target off-tumor effects and have translational relevance
for use of CD123 targeting as a “bridge to transplant” in AML and MDS. Whether MDS may be less likely to develop on-target and off-tumor side effects is being explored in combination studies of SL-401 and hypomethylating agents in early phase clinical trials (clini- caltrials.gov identifier 03113643).
Methods
Cells
AML cell lines, MV4-11 and MOLM-13 were obtained from DSMZ in 2015, confirmed to have FLT3-ITD mutation by DNA sequencing, and routinely tested for mycoplasma using MycoAlertTM Mycoplasma Detection Kit (Lonza). Primary AML and MDS cells were obtained from patient apheresis products or bone marrow aspirates following written informed consent under an institutional review board (IRB) approved protocol, according to the Declaration of Helsinki. Umbilical cord blood samples were received through the Leukemia tissue bank and collected under an IRB-approved protocol. AML primary cells were cultured in RPMI 1640 (Life Technologies, Grand Island, NY) with 10-20% fetal bovine serum (FBS) (Sigma-Aldrich, St Louis, MO); 2mM L-gluta- mine; penicillin (100 U/ml); streptomycin (100 μg/ml) (Life Technologies) and supplemented with 10ng/ml GM-CSF and SCF (R&D Systems) overnight before treatment with SL-401. MDS samples were cultured similarly, with additional IL-3 (10ng/ml; R&D Systems). AML subtype and mutational characteristics of AML patient samples used in this study are listed in Online Supplementary Table S1.
Bone marrow-derived mesenchymal stromal cell expansion
For AML-stromal cell co-culture experiments, AML cells were cultured in RPMI 1640 base medium with 10% FBS without any growth factors on the monolayer of the HS-5 human fibroblast cell line expressing green fluorescent protein (GFP) or with autol- ogous mesenchymal stromal cells (MSC) derived from bone mar- row of AML patients. To derive MSC for each AML, bone marrow cells were incubated for 24-36 hours in DMEM (Life Technologies) with 10% FBS and antibiotics. The plastic-adherent, elongated spindle- to stellate- shaped cells were expanded following aspira- tion of non-adherent cells and weekly exchange of media and pas- saging when confluent. MSC in passage 3 or 4 were trypsinized and stained for CD45, CD73, CD90 and CD105 with a viability stain and assessed via flow cytometry for purity.
Chemicals and reagents
SL-401 was provided by Stemline Therapeutics Inc. and is now in clinical trials20 (clinicaltrials.gov identifier 02270463). Additional information in Online Supplementary Methods.
Cell viability and staining
Apoptosis was measured by annexin-V-FITC and propidium iodide as described previously.27 For co-culture experiments, stro- mal cells were gated out based on GFP+ for HS-5 and CFSE+ for MSC, and the viability of leukemic cells was determined by annexin-V PE and 7-AAD staining. Cell counts were measured using a Tali cytometer (Invitrogen) according to the manufacturer’s instruction. LIVE/DEAD stain (Invitrogen) was used in combina- tion with surface markers for gating live cells in flow cytometric experiments. CD123 Molecules of Equivalent Soluble Fluorochrome (MESF) on AML cells was calculated using QuantumTM MESF microsphere kit (Bangs Laboratories, Fishers, IN) and CD123 antibody (Clone: SSDCLY107D2; Beckman
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