Poster - Stemline Therapeutics, Inc.

Preclinical Studies of SL-401, a Targeted Therapy Directed To The Interleukin-3 Receptor (IL-3R), in Blastic Plasmacytoid Dendritic
Cell Neoplasm (BPDCN): Potent Activity in BPDCN Cell Lines, Primary Tumor, and in an In Vivo Model
Fanny Angelot-Delettre1, Arthur E. Frankel2, Estelle Seilles1, Sabeha Biichle1,
Eric Deconinck1,4, Francis Bonnefoy1, Baptiste Lamarthee1, Christopher Brooks3, Eric Rowinsky3, Philippe Saas1, and Francine Garnache Ottou1
1INSERM
UMR1098, Besancon, France; 2University of Texas Southwestern Medical Center, Dallas, TX USA;
3Stemline Therapeutics, Inc., New York, NY USA; 4Hematology, Hôpital Jean Minjoz, Besancon, France.
B.
A.
DAUDI (negative control) CAL-1
SL-401 (365pM)
SL-401 (365pM)
34%
**
Percent survival
Cells alone
C.
100
(BPDCN cell line)
**
86%
NSG survival rate
A.
C.
10%
Patient # 1
SL-401 (365 pM)
6%
80
60
40
20
7AAD
0
0
20
0. 0
08
fM
0.
7
fM
0.
00
6
pM
0.
06
pM
0.
49
pM
4.
5
p
40 M
.6
pM
36
5
pM
0
40
20
0
ce
lls
SL-401 concentrations
60
VI
N
ID
A
40
Drugs
Figure depicts the viability of primary blasts from BPDCN
(AV-/7AAD- cells) patients (#1-9, #11 and #12) as a
function of SL-401 concentration. Drug treatment was for
18 h. Untreated cells were considered 100% viable. Each
gray line represents blasts from a single BPDCN patient,
whereas the black line represents the mean
concentration values ± SEM.
30
40
50
60
days
B.
no SL-401
SL-401
BDCA-4
m CD45
h CD45
CD123
CD4
CD34
SSC
FSC
CD3
YT
D
EX
60
20
80
C
80
*
100
nl
SL y
-4
01
ER
W
K
ID
M
TX
C
YC
Cell culture and patient sample processing: Peripheral blood
or bone marrow aspirates were obtained for diagnostic purposes
from 12 BPDCN patients (#1-12) who were diagnosed using
clinical, morphological, and immunophenotypic criteria previously
established (Swerdlow et al. 2008, Feuillard et al. (2002),
Garnache-Ottou et al. (2009), Angelot-Delettre et al. (2012). Two
BPDCN cell lines (GEN2.2 and CAL-1), as well as positive (TF-1H-Ras) and negative (DAUDI) controls, were cultured in
RPMI1640 media supplemented with 10% fetal bovine serum.
Drugs and culture: SL-401 (Stemline Therapeutics, New York,
NY, USA) was tested at concentrations from 365 pM to 0.08 fM.
Cells were incubated at 3x105 cells/mL with or without SL-401 or
the indicated drugs for 18 h at 37°C and 5% CO2.
Flow cytometric (FC) analysis: Cell viability was determined by
flow cytometry using Annexin V (AV) and 7-aminoactinomycin D
(7-AAD) staining according to a standard protocol on a Canto II™
flow cytometer (BD Biosciences).
Mice: Six-week-old NOD-SCID IL2Rγnull (NSG) mice (n=16) were
irradiated (2 Grays) and innoculated IV 24 h later with 1x106
GEN2.2 cells. Mice were then treated IP 8 days later with 5 daily
injections of SL-401 (2 µg/mouse/injection) or PBS (control).
Animals were monitored weekly for circulating BPDCN cells by
flow cytometry.
Statistical analysis: Statistical analysis was performed using
StatEL software 2.6 (Adscience, Paris, France). Data were
analyzed using the Mann-Whitney or Student t-tests. A p
value < 0.05 was considered statistically significant.
% viability
MATERIALS AND METHODS
100
#1
#2
#3
#4
#5
#6
#7
#8
#9
#11
#12
mean
SL-401 is more effective against
BPCDN than most
chemotherapeutic agents tested
O
SL-401 is cytotoxic against BPDCN
primary cells in a dose-dependent
manner
10
CD4
(A) Dot plots showing Annexin V (AV) and 7-AAD staining of SL-401 treated cells. The top panels show staining of
DAUDI (negative control; left panel) or the CAL-1 BPDCN cell line (right panel) after treatment with SL-401 for 18 h.
The bottom panel shows staining of blasts from Patient #1 untreated (left panel) or treated with SL-401 (right panel)
for 18 h. The value shown in each dot plot represents the percentage of viable cells (AV-/7-AAD- cells). (B and C)
Percent viability of (B) BPDCN cell lines CAL-1 (n=3) and GEN 2.2 (n=5) and (C) primary BPDCN cells (n = 9
independent experiments) (patients #1 to #11) after culture with or without SL-401, ** p < 0.001.
Viability (%)
Since BPDCN cells express high levels of IL-3R, the antitumor
effect of SL-401, a novel targeted therapy directed to IL-3R, is
being evaluated in clinical and preclinical studies. SL-401, which is
a biologic comprised of human IL-3 fused to a truncated diphtheria
toxin payload, potently inhibits protein synthesis following IL-3Rmediated binding and internalization. The aim of this study was to
further our evaluation of the activity of SL-401 against BPDCN
cells, not only in vitro on primary BPDCN cells and established
BPDCN cells lines, but also in vivo, in a mouse model of human
BPDCN. These results further support the later stage clinical trials
of SL-401 in this indication.
SL-401 is active against BPDCN cell lines and BPDCN patient samples
AV FITC
Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), an
aggressive malignancy derived from plasmacytoid dendritic cells
(pDCs) that typically presents as cutaneous lesions and can also
involve bone marrow, lymph nodes and the spleen, invariably
terminates in an acute leukemic phase. Its diagnosis is based on
its clinical presentation as well as the unique pathological and
immuno-phenotypic features of a cutaneous PDC neoplasm, with
high expression of CD123 (interleukin-3 receptor [IL-3R]), CD304,
and/or CD303 (Garnache-Ottou F et al., 2009); CD56 and CD4
are also commonly expressed. There is no accepted standard of
care to treat BPDCN and therapeutic strategies have never been
prospectively evaluated. Treatment approaches include:
symptomatic therapy, intensive multi-agent chemotherapy, and
allogenic stem cell transplantation. Although chemotherapy-naive
patients often respond to chemotherapy, most subjects experience
recurrent disease shortly thereafter; median overall survival
estimates
range
from
9
to
13
months.
SL-401 significantly increases overall survival
of mice bearing a human BPDCN xenograft
after a single cycle of treatment
RESULTS
INTRODUCTION
ASH2013
Poster #3942
Mean percentage of viable cells (AV-/7-AAD- cells)
from BPDCN patients #7-9 and #11 untreated (cells
alone) or treated with SL-401 (365 pM), erwinase
(ERW, 10 UI/ml), kidrolase (KID, 10 UI/ml),
methotrexate (MTX, 4.5 µg/ml), cyclophosphamide
(CYC, 100 µM), cytarabine (CYT, 80 µg/ml),
dexamethasone (DEX, 250 µg/ml), vincristine (VIN,
20 ng/ml), or idarubicin (IDA, 0.078 µg/ml).
Untreated cells were considered as 100% viable.
Histograms represent the mean ± SEM of 3
independent experiments
(*p < 0.05 between SL-401 and all other drugs
pairwise except IDA).
CD56
h CD45
(A) SL-401 (IP; 2 µg/mouse/injection) was administered daily for 5 days starting
8 days post-implantation (pink area). Overall Survival (OS): The solid line
corresponds to saline-treated controls (no SL-401, n=3) while the dotted line
corresponds to SL-401-treated animals (SL-401, n=4). Data from one
representative experiment out of 3 are shown, p < 0.001.
(B) The
immunophenotype of peripheral blood from a BPDCN mouse on day 53 postSL-401 treatment showing mouse (blue) and human (red) cells. BPDCN
infiltration (52% blastic cells) was observed. h: human; m: murine (C) Implanted
GEN 2.2 cells detected in a nodal tumor (standard MGG; magnification x1000).
CONCLUSIONS
This is an extension of our previous study to evaluate the in vitro and in vivo
sensitivity of BPDCN cells to SL-401, a novel targeted therapy directed to the
IL-3R. We have demonstrated that SL-401 has potent cytotoxicity against both
cell lines and primary BPDCN blasts sampled from patients; IC50 values are in
the low femtomolar range. In addition, SL-401 significantly increased the overall
survival of mice bearing a human BPDCN xenograft after a single cycle of
treatment. These results indicate that SL-401 may provide a clinical benefit for
patients with BPDCN, an unmet medical need. A Phase 2 multi-cycle trial in this
orphan indication is currently being planned in North America and Europe.
Disclosures: Brooks: Stemline: Employment and equity ownership. Rowinsky: Stemline: Employment and equity
ownership. Frankel: Stemline: Patents and Royalties and Research Funding.