preliminary translational results from an ongoing phase 1 ... · primary aml samples were...
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Preliminary Translational Results from an Ongoing Phase 1 Study of Flotetuzumab, a CD123 x CD3 DART® Protein, in AML/MDS: Rationale for Combining Flotetuzumab and Anti-PD-1/PD-L1 Immunotherapies
Michael P Rettig, PhD1, John Godwin, MD2, Norbert Vey, MD3, Bernard Fox, PhD2, Carmen Ballesteros-Merino, PhD2, Carlo B Bifulco, MD2, Daner Li, MS4, Daniel Primo, PhD5, Joan Ballesteros, PhD5, Jichao Sun, PhD4, Helene Lelièvre, PhD, PharmD6, Jan Baughman, MPH4, Ross La Motte-Mohs, PhD4, John Muth, MS4, Paul Moore, PhD4, Ezio Bonvini, MD4, Jon Wigginton4, John F DiPersio, MD, PhD1 and Jan Davidson-Moncada, MD, PhD4
1Division of Oncology, Washington University School of Medicine, Saint Louis, MO; 2Providence Cancer Center, Portland, OR; 3Clinical Hematology, Paoli-Calmettes Institute, Marseille, France; 4MacroGenics, Inc., Rockville, MD; 5Parque Científico de Madrid, Vivia Biotech, Madrid, Spain; 6Centre de Recherche en Cancérologie de Marseille, Servier, Suresnes, France
©2017 MacroGenics, Inc. All rights reserved.Presented at the 59th American Society of Hematology Annual Meeting, December 9–12, 2017, Atlanta, GA
http://ir.macrogenics.com/events.cfm
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PD-L1 Upregulation in Residual Bone Marrow BlastsPatients on flotetuzumab with residual disease showed stable CD123 expression and increased expression of PD-L1 positive AML blasts vs. basal levels
Baseline (BL)A.
B.
Cycle 1 Day 25 (C1D25)
CD274 (PD-L1)
CD12
3
98.1 1.46 78.0 21.7
00 0.320.39
106
105
104
103
102
101
100
100 101 102 103 104 105 106100 101 102 103 104 105 106
CD274 (PD-L1)
BL C1D25 BL C1D25
CD1230
500
1000
1500
gMFI
(mea
n ±
SEM
)
Representative flow cytometry analysis of CD123 and PD-L1 in AML blast bone marrow samples pre (BL D-14) and post flotetuzumab (C1D25) (A). Geometric mean fluorescent intensity (gMFI) of PD-L1 and CD123 expression in responding patients at baseline (BL, grey symbols) or at cycle 1 day 25 (C1D25, orange squares) (B).
PD-1/PD-L1 Blockade Enhances Flotetuzumab ActivitySynergistic in vitro cytotoxicity was observed after treatment of an AML cell line with flotetuzumab in presence of CPI
PBS 0.01 ng 0.1 ng 1 ng 0
50
100
Flotetuzumab (ng/mL)
Viab
le C
ells
(%)
PBS ISOanti-PDL1*anti-PD1*
KG1A cells incubated with human T cells at E:T ratio 0.25:1 in presence of flotetuzumab ±10 μg/mL anti-checkpoint antibody or isotype control. *Statistically significant (p<0.05)
Conclusions■■ Flotetuzumab has demonstrated clinical activity in patients with relapsed/refractory AML■■ Treatment with flotetuzumab enhances T-cell infiltration and activation in the bone marrow■■ T-cell activation is associated with enhanced PD-1 expression and IFN-gamma secretion, and upregulation of PD-L1 on AML blasts ■■ Enhanced PD-L1 expression by AML blasts was associated with reduced flotetuzumab activity in vitro and in vivo■■ Residual bone marrow AML blasts show higher expression of PD-L1 positive compared to baseline■■ Synergistic in vitro T-cell mediated cytotoxicity was observed after treatment of an AML cell line with flotetuzumab in combination with PD-1/PD-L1 inhibitors ■■ These data suggest that combination with anti-PD-1/PD-L1 directed therapy could enhance the anti-leukemic effects of flotetuzumab in patients with relapsed/refractory AML
PD-L1 Expression is Associated With Decreased Flotetuzumab Anti-leukemic Activity In Vitro
80
60
40
20
100
0
Expr
essi
on o
nA
ML
Blas
ts (%
)
MGD006 (µg/mL)
110100
908070605040302010
0
Live
Cel
ls (%
)
Sample # 1 Sample # 2 Sample # 3 Sample # 4 Sample # 5 Sample # 6
Live CD25+ T cellsLive AML blasts
CD12
3CT
LA4
LAG
3PD
1PD
L1PD
L2TI
M3
GIT
R
CD12
3CT
LA4
LAG
3PD
1PD
L1PD
L2TI
M3
GIT
R
CD12
3CT
LA4
LAG
3PD
1PD
L1PD
L2TI
M3
GIT
R
CD12
3CT
LA4
LAG
3PD
1PD
L1PD
L2TI
M3
GIT
R
CD12
3CT
LA4
LAG
3PD
1PD
L1PD
L2TI
M3
GIT
R
CD12
3CT
LA4
LAG
3PD
1PD
L1PD
L2TI
M3
GIT
R
96.70 98.30
0.00 0.00 0.00 0.04 0.40 0.15 0.98 0.32 0.45 0.45 0.00 0.55 0.13 1.71
99.67
0.35 0.17 0.09 1.18 1.23 0.11
24.77
99.06
0.82 1.19 0.29 0.11
27.81
0.44
66.09
98.89
13.31
40.45
0.2542.85
59.78
0.28
83.73
96.18
0.01
18.42
0.43 0.36
82.79
0.28
84.43
A.
B.
Primary AML samples were co-incubated with flotetuzumab at different concentrations for 144 hours in vitro. ■■Figure A: Immunophenotyping of AML samples expressed as percent of total cells. Boxes highlight expression on PD-L1 in flotetuzumab sensitive samples (green) vs resistant samples (red).■■Figure B: Flotetuzumab concurrently induced T-cell activation (as measured by increased cell counts of CD25+ T cells (green line)) and depleted AML blasts cells (red line) in concentration-dependent manner.
PD-L1 Expression is Associated With Decreased Flotetuzumab Activity In Vivo
Early Progressors Responders0
50
100
PD-L
1+ AM
L Bl
asts
at
BL (%
)
*p=0.0205
Patients that progressed early (<15 days) on flotetuzumab treatment had higher baseline levels of PD-L1 on AML cells than patients that had evidence of anti-leukemic activity (SD, OB, PR, CR). AML samples collected during screening were analyzed for PD-L1 expression by flow cytometry. Data is expressed as mean ± distribution.
PD-L1 Expression Is Upregulated in Bone Marrow Blasts
0
500
1000
Baseline Cycle 1 Baseline Cycle 1
1500
2000
0
1000
2000
3000
4000CD3+ PD-L1+
Baseline Cycle 1N
umbe
r of
Cel
ls/m
m2
PD-L1CD3CD8FoxP3DAPI
Multiplex immunohistochemical staining of bone marrow from AML patient treated with flotetuzumab. FFPE bone marrow sections were immunolabeled and quantifies for flotetuzumab as well as PD-L1, CD3, CD8, FoxP3 and DAPI. Analysis showed a statistically-significant increase in the cell density of CD8 (p=0.0025) and PD-L1 (p<0.001) after one cycle of treatment.
Background■■ Immunotherapy has transformed cancer treatment■■Although checkpoint inhibitors (CPI) have demonstrated striking clinical activity and are approved for the treatment of patients with various solid tumors and Hodgkin’s disease, their role in the treatment of acute myeloid leukemia (AML) remains unclear■■Activated T-cells can upregulate PD-1 expression, and become functionally exhausted when PD-1 is engaged by ligands including PD-L1 and PD-L2■■As part of our ongoing Phase I study of flotetuzumab, a novel CD123 x CD3 DART protein, in patients with AML and myelodysplastic syndrome (MDS), we have completed translational studies that support a rationale for combining flotetuzumab with PD-1/PD-L1 inhibition
Phase 1 Flotetuzumab Study Design (CP-MGD006-01)
Dose EscalationSingle patient
dose escalation(3, 10, 30, 100 ng/kg/day)
Expansion CohortRelapsed/Refractory AML
Expansion CohortHypomethylation Failure
MDS
Dose Escalation3 + 3 Multi-patient
dose escalation4-week cycles
■■Patient Population: Patients with relapsed/refractory AML and hypomethylation failure Int-2/High Risk MDS■■Dosing Regimen: Continuous intravenous infusion with lead-in dosing■■Anti-leukemic activity in patients with relapsed/refractory AML has been reported previously (ESMO 2017; ASH 2017 Oral Presentation #637)
Methods
Flotetuzumab Mode of ActionFlotetuzumab redirects T-cells to lyse AML cells in conjunction with induction of T-cell activation, cytokine release and proliferation
T cell
Tumor celllysis
Tumor Cell
FlotetuzumabEngagement
T cellTumor Cell
T-cell Activation,Expansion, andDifferentiation
Activation,CytokineRelease
T cell
Tumor Cell
T cell
Tumor cellDeath
Target CellKilling
Activation,Cytokine andGranulesRelease
Flotetuzumb(CD123 x CD3)Perforin/GranzymeGranule(s) ReleaseCytokine(s) Release
Flotetuzumab expands CD8 lytic cells in bone marrow in vivoA.
Baseline Cycle 1 Cycle 2 Cycle 3
Num
ber
of C
ells
/mm
2
CD8+
B.
0
2500
2000
1500
1000
500
0
100
80
60
40
0
1500
1000
500
20
C. FoxP3+CD3+
* **
D.
PD-L1CD3CD8Fox P3DAPI
Baselin
e
Cycle 1
Cycle 2
Cycle 3
Baselin
e
Cycle 1
Cycle 2
Cycle 3
Baselin
e
Cycle 1
Cycle 2
Cycle 3
1500
1000
500
0Num
ber
Cells
/mm
2
CD3–
CD8+CD3+
CD8+CD3+
FOXP3+CD3+
FOXP3+
BM 16-413 BaselineBM 16-470 +1MBM 16-536 +2MBM 16-579 +3MBM 16-37 +4M
Flotetuzumab
Multiplex IHC staining of bone marrow from AML patient treated with flotetuzumab. FFPE bone marrow sections were immunolabeled and quantifies for MGD006 (A) as well as PD-L1, CD3, CD8, FoxP3 and DAPI (B). Analysis showed a statistically significant increase in the cell density of CD3+, FoxP3+ and CD8+ (1.58-fold increase, p=0.0013) cells on treatment (*p<0.05) compared to baseline (C). CD8+CD3+ T cells and CD8+CD3- cells also demonstrated an increase in cell density post flotetuzmab treatment (D).
Results
IFNγ Upregulates PD-L1 in AML Blasts
FMO control
Untreated
10 ng/mL IFNγ
CD274 (PD-L1)
88.5
0 104
0
40
80
0
40
80
22.3
0 104
Perc
ent
of M
ax
UPN250167 UPN600734
Cryopreserved cells from two AML patients were cultured on HS27 stromal cells for 48 hours. Samples were left untreated or treated with IFNγ (10 ng/mL) for an additional 48 hours. PD-L1 expression on the AML blasts was evaluated by flow cytometry.
Flotetuzumab Upregulates PD-1, PD-L1 Expression and IFNγ Secretion Ex Vivo
CD4-gatedCD3-gated CD8-gated
PD1
Basal TimeAfter 120h DART Exposure
Mean Flourescence Intensity
PD-L1
PD-L2
B7-H3
CD80
CD86
CD45
IFN
γ (p
g/m
L)
5 ng/mL
50 ng/mL
500 ng/mL
BL
0 103 104 105 0 103 104 105 0 103 104 105
3000
2500
2000
1500300
200
100
0
Baselin
e (BL)
500 (pg/m
L)
50 (pg/m
L)
5 (pg/m
L)
Basal TimeAfter 120h DART ExposurePD-L1
PD-L2
B7-H3
CD80
CD86
CD45
1 1E1 1E2
A. B. C.
Flow cytometric analysis of PD-1 expression on T cell subsets (A) and IFNγ secretion in supernatants (B) from an AML-patient donor PBMC (24% CD123-positive cells and 14.2% CD3-positive T cells). PBMC were cultured for 48 hours in the absence (grey histograms, baseline (BL)) or presence of 5 (green histograms), 50 (orange histograms), and 500 (blue histograms) pg/mL of flotetuzumab.Flow cytometric analysis of checkpoint (PD-L1, PD-L2, and B7-H3) and costimulatory (CD80, CD86, and CD45) molecules at base line (blue histograms) or 120 hours post-incubation of 50 ng/mL of flotetuzumab (green histograms) in AML blasts (C).
Flotetuzumab Upregulates PD-1 In VivoPD-1 up-regulation observed on both CD4 and CD8 T cells
Study Day
PD-1
+ (Pe
rcen
t of
Tot
al C
D4+ T
-cel
ls)
Study Day
PD-1
+ (Pe
rcen
t of
Tot
al C
8+ T-c
ells
)
1 8 15 22 25
10
20
20
30
40
60
0 01 8 15 22 25
PD-1 expression on CD4 and CD8 T cells from patients treated on CP-MGD006-01 study. Graphs show median PD-1 expression of all patients treated on study.
Induction of IFNγ and PD-L1 in Flotetuzumab-treated Patients
0
20
40
60
IFN
γ (p
g/m
L, m
ean
± SE
M)
Cohort 0 300 500 7000
1
2
3
Cohort (dose ng/kg/day)
Cohort 0 300 500 700
Cohort (dose ng/kg/day)
PD-L
1+ A
ML
Blas
ts (%
)
Patient samples from CP-MGD006-01 were evaluated for circulating IFNγ levels (left panel) and PD-L1 expression on circulating AML blasts across four dose levels in Cohort 0 (each ≤100 ng/kg/day), 300 ng/kg/day, 500 ng/kg/day and 700 ng/kg/day.
NCT02152956