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Preclinical Activity and Safety of MGD006/S80880, a CD123 x CD3 Bispecific
DART® Molecule for the Treatment of Hematological Malignancies
EORTC 2014
Abstract #: 138
Poster #: 132
P. Moore1, G.R. Chichili1, L. Huang1, H. Li1, S. Burke1, F. Chen2, L. He1, Q. Tang1, L. Jin1, S. Gorlatov1, V. Ciccarone1, S. Koenig1, M. Shannon1, R. Alderson1, S. Johnson1, E. Bonvini1 | 1MacroGenics, Rockville MD, USA, 2MacroGenics, South San Francisco CA, USA
Abstract
Figure 5: Non-Human Primate Functional Cross Reactivity
T-cell directed killing of tumor cells using bispecific molecules may hold promise for cancer treatment. MGD006 (also encoded as S80880 and known as CD123 x CD3 DART) is a bispecific
diabody-based molecule consisting of two polypeptides, each composed of the VH of one antibody in tandem with the VL of the second antibody and covalently linked at the COOH termini via
a disulfide bridge. MGD006 simultaneously binds T lymphocytes and cells expressing CD123 (IL3RA), an antigen up-regulated in several hematological malignancies and differentially expressed
in AML blasts compared to normal hematopoietic stem and progenitor cells. MGD006 mediates dose-dependent T-cell redirected killing of CD123-positive cell lines as well as primary AML
blasts. Furthermore, MGD006 demonstrates potent activity in tumor-bearing mice engrafted with human peripheral blood mononuclear cells or when tumors were co-inoculated with human
T lymphocytes. MGD006 binds to both human and cynomolgus monkey antigens and can redirect T cells from either species to kill CD123-expressing cells. Preclinical safety and pharmacology
was therefore assessed in monkeys that were infused with escalating doses of MGD006 on continuous or intermittent schedules over a period of 4 weeks. Monkeys infused with MGD006
showed depletion of circulating CD123-positive cells, an activity biomarker, as early as 72h after treatment initiation and persisting throughout the 4-week treatment period. Furthermore, T
cells from treated monkeys exhibited efficient ex vivo redirected target cell lysis, indicating no functional exhaustion. Transient release of cytokines, particularly IL-6, was observed following
the first MGD006 infusion, but not after subsequent administrations even when the dose was escalated. A reversible, decrease in red cell mass with concomitant reduction in CD123-positive
progenitors in the bone marrow was also observed. No significant changes in circulating platelets or neutrophil levels were observed. The preclinical safety and efficacy profile of MGD006
supports advancement to clinical evaluation. A phase 1 safety study of MGD006 in relapse/refractory AML is currently recruiting patients. (http://clinicaltrials.gov/ct2/show/NCT02152956).
Autologous Depletion of
Peripheral CD123+ cells
Redirected T cell killing of
CD123+ AML Cells
Figure 7: Prolonged MGD006 Exposure In Vivo
has no Negative Impact on T-Cell Effector Function Ex Vivo
Figure 6C: T-Cell Kinetics and Activation Profile
A
T-cell Activation
MGD006 Exposure
Ex Vivo CTL Activity
B
(Left) MGD006 mediates dose-dependent depletion of CD14-/CD123+ population in cynomolgus monkey PBMC.
Cynomolgus PBMC also support MGD006-mediated T cell cytotoxicity (middle) and activation (right) in the presence
of CD123+ leukemic cell line Kasumi-3 (E:T = 15:1). Activity is consistent with that observed with human PBMC.
Note: Studies involving animal subjects were performed following institutional IACUC approval.
Figure 6: Safety and PD Modeling in Non-Human Primates
B
Chain 1
H2N-­‐
AntiCD123
Chain 2
AntiCD3
E
3
A
96 h 144 h B
96 h 144 h M G D 006
C D 3x4420
VL1
VH2
Anti-­‐CD123
H2N-­‐
Anti-­‐CD3
VL2
VH1
Anti-­‐CD3
-­‐COOH
Cys
Anti-­‐CD123
O D450nM
A
Bi-specific Binding
Expansion of Residual T cells -­‐COOH
Cys
4420xC D 123
2
Untreated 86.6% 90.3% 1
0
0 .0 0 0 1
0 .0 0 1
0 .0 1
0 .1
1
D A R T ( mg /m L )
C
D
Reduced F
Non-­‐reduced MGD006
98 kDa 98 kDa 5 .0 ´1 0
62 kDa 62 kDa 5
59500
Human
T Cells
Cynomolgus Monkey
T Cells
of two separate chains (B) yielding a homogenous product with anticipated MW as shown by MassSpectrometry (C) and SDS-PAGE (D).
• ELISA-based bispecific binding as demonstrated by CD123 capture and CD3 detection (E). Flow cytometry
binding to CD123+ MOLM13 cells (upper panel) and both CD3+ human T-cells (lower left) and CD3+
cynomolgus monkey T-cells (lower right) (F). Binding with CD3 x 4420 control DART was restricted to T-cells.
Figure 2: MGD006 Mediated Redirected T-cell Killing:
Dependence on CD123 Expression
A: Targeted Lysis of CD123+ve AML Cell line
10 ng/mL
10 4
200
100
200
500
0
1
2
3
10 10 10 10
Anti-CD123 PE
200
# Microspheres
Med.low
Blank
100
100
100 101 102 103 104
Anti-CD123 PE
10
0
10
1
10
Vehicle Vehicle Vehicle 100 100 100 300 300 300 300 600 600 300 600 1000 300 600 1000 Recovery
36-­‐65 -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ 2
Anti-CD123 PE
10
3
10
FL1-H: FL1-Height
FL1-H: FL1-Height
1
2
10
10
3
FL4-H: FL4-Height
10
4
10
100
10
3
10
3
10
2
10
2
10
1
10
1
10
0
10
0
0
10
1
10
2
3
10
FL4-H: FL4-Height
4
102
103
FL4-H: FL4-Height
104
0
10
1
2
10
3
10
FL4-H: FL4-Height
4
10
10 0
101
102
FL4-H: FL4-Height
103
10 4
AML patient PBMCs were incubated in the presence of MGD006 or a control DART (CD3 x 4420) or left untreated.
(A) The CD123+ leukemic blast population (defined as CD45+/CD33+) is eliminated in presence of MGD006
(B) A concomitant increase in the level of CD4+ and CD8+ T cells observed in the presence of MGD006 but not
with the controls.
10
1
10
0
0
10
1
10
2
10
3
10
FL4-H: FL4-Height
10
3
10
2
10
1
10
0
10
0
10
1
10
2
FL4-H: FL4-Height
10
3
E
100
102
103
FL4-H: FL4-Height
104
10 0
101
102
FL4-H: FL4-Height
103
10 4
Cytokines
4
Circulating levels of CD14-/CD123+ (top) or CD303+ (pDC) leukocytes (bottom) shown by treatment group.
Treatment intervals are indicated by the filled grey bars. All MGD006 treated animals displayed rapid depletion of
CD123+ circulating cells during the treatment phase. Subsequent to treatment, the levels of circulating CD123+ve
cells returned to baseline levels – consistent with sparing of the CD123-ve hematopoietic stem cell (HSC).
Figure 6B: Depletion of CD123+ Cells in Bone Marrow,
But No Impact on HSC Population
C
Vehicle
MGD006
RCC cells (lower). Experiments performed with human T-cells (E:T = 10:1) for 18h.
• (B) MGD006 mediated autologous depletion of CD123+ cell populations in human PBMC – both CD14-/
CD123+ (predominantly pDCs) and CD14+/CD123+ cells (monocytes) in time (upper panel) and dosedependent manners (lower panel). No depletion of CD123-ve cell populations observed.
Tumor volumes (mean ± SEM) measured in human
PBMC reconstituted mice (8/group) treated with
indicated doses of MGD006. Filled grey area indicates
the continuous delivery of MGD006 or control DART.
MGD006
Figure 9: Overview of Ongoing Phase 1 Study
D
A
(A) Reversible, modest reductions were observed in RBCs. Neutrophils (B) and platelets (C) while fluctuating prior,
during and post-treatment, remained within the normal reference range (horizontal dotted lines indicate the
reference range in cynomolgus monkeys).
CD3 x 4420
MGD006
Treatment of AML patient primary PBMCs with MGD006 for 144h leads to: (A) decrease in absolute counts of
leukemic blasts, (B) concomitant increase in T cells, accompanied by (C) enhanced CD25, (D) granzyme B expression
on CD4/CD8 T-cells and (E) cytokine secretion (predominantly IFNγ and IL-6).
Flow cytometry analysis performed on bone marrow aspirates collected pre-dose, post-final dose (4 animals per
group) and following a 4-week recovery period (remaining 4 animals per group) revealed (A) decreased frequency
of CD123+ cells for all MGD006 treatment groups, that returns to baseline following recovery. (B) No significant
difference in the frequency of the HSC population (defined as CD34+/CD38-/CD45RA-/CD90+ cells) between vehicle
and MGD006 treated groups.
MGD006-mediated IL-6 release is primarily associated with the first dose with its magnitude decreasing with each
successive dose even when the doses were escalated (i.e., Groups 3-6)
Expansion Cohort
(n=12)
Establish safety, determine MTD, characterize PK and describe early evidence of
anti-leukemic activity
Patient Population:
Relapsed or refractory acute myeloid leukemia (AML)
Inclusion Criteria:
Confirmed diagnosis of primary or secondary AML
Unlikely to benefit from cytotoxic chemotherapy
Eighteen (18) years of age or older
Dosing Regimen:
4 Weekly cycles of IV infusion
Principal Investigator:
John DiPersio, MD (Washington University, St. Louis, MO)
MGD006/S80880 – Conclusions
Potent in vitro and in vivo depletion of CD123+ve populations
– AML patient samples
– CD123+ve Xenograft in Humanized Murine Model
• Well tolerated in primate toxicology at doses up to 1000ng/kg/day
– On target depletion of CD123+ve populations in periphery and bone marrow
– Minimal bone marrow toxicity
– No evidence of significant neutropenia/thrombocytopenia
– No evidence of off-target effects
• Suitable dosing strategy to address Cmax-associated cytokine release
• Phase 1 studies:
– Relapse/refractory AML (ongoing)
– John DiPersio, MD (Washington University, St. Louis, MO)
– http://clinicaltrials.gov/ct2/show/NCT02152956
•
B
Dose Escalation
5 Escalating Dose Levels
“3+3” design
Objectives:
Figure 6E: Cytokine Response “Desensitization”
18 h
• (A) Potent cytotoxicity by MGD006 against CD123+ Kasumi-3 AML cells (upper) but not against CD123- A498
NSG/β2m k/o mice were subcutaneously implanted
with the KG-1a (AML M0) cells on Day 0 and injected
intraperitoneally with human PBMCs on Day 1. MGD006
was administered via continuous infusion (IP-implanted
osmotic pumps) on Day 16 - 22.
Figure 6D: Absence of Neutropenia or Thrombocytopenia
C
4
101
101
(A) The absolute number of circulating CD4 and CD8 T cells fluctuates during intermittent MGD006 dosing
(middle panel) and displays a sustained decrease following continuous dosing (right panel) with levels rebounding
following cessation of treatment. Relative values (mean percent ± SEM) of CD25+, CD69+, PD-1+ and Tim-3+ of
CD4 (B) or CD8 T cells (C) reveal increased expression of PD1 on both CD4 and CD8 T-cells, which trends back to
baseline subsequent to dosing. Treatment intervals are indicated by the filled grey bars.
B
4
10
Efficacy of MGD006 Against Established CD123 +ve KG1a Xenografts in Immune Deficient
Mice Adoptively Transferred with Human PBMC
T im -3 +
Establish Starting Dose
4 Dose Levels
“1+3” design
102
100
P D -1 +
B
103
1
100
2
104
102
101
10
10
103
102
3
10
104
3
Primary AML Patient Sample
E:T = 1:300
A
C D 69+
CD4 10 4
10
10
101
MGD006 0.1ng/ml CD123 APC
50
0
10
104
100
Med.High
Low
0
104
10
18 h
High
150
0
104
0 0
10 10 1 10 2 10 3 10 4
Anti-CD123 PE
4
10
10
QSC Micropheres
200
200
0
1
10
FL1-H: FL1-Height
300
250
300
0
10
U937
400
10
100
100
0
10
1
102
FL1-H: FL1-Height
300
0 0
10 101 102 103 104
Anti-CD123 PE
6h
400
# Cells
300
RS4-11
500
400
# Cells
400
2
103
10
0 0
10 10 1 10 2 10 3 10 4
Anti-CD123 PE
Molm-16
500
15 22 29 A
0.1 ng/mL
10 4
104
FL1-H: FL1-Height
TF1
10
FL1-H: FL1-Height
100 101 102 103 104
Anti-CD123 PE
3 4 5 C D 25+
MGD006
FL1-H: FL1-Height
100
0 0
10 101 102 103 104
Anti-CD123 PE
3
FL1-H: FL1-Height
200
200
100
500
# Cells
300
300
10
FL1-H: FL1-Height
100
3h
# Cells
# Cells
# Cells
200
# Cells
400
400
300
THP-1
500
CD14 FITC
400
0
Molm-13
1 8 Figure 4: Mechanism of Action in AML Patient is Associated with T-cell
Activation, Expansion, Cytokine Release and Upregulation of Granzyme B
B: Autologous Depletion of CD123+ve Cells in Human PBMC
No Treatment CD3 x 4420
500
Group 6 (4M/4F) Vehicle 100 Figure 8: Potent Anti-Tumor Activity in
Human PBMC Reconstituted Xenograft Model
Figure 6A: Depletion of Circulating CD123+ Cells During
MGD006 Treatment Phase
CD33 • MGD006 is a DART-based diabody comprising bi-specificity for CD3 and CD123 (A) derived from co-expression
Kasumi-3
5.61% 47.5% M a s s (D a )
500
Group 2 Group 3 Group 4 Group 5 (4M/4F) (4M/4F) (4M/4F) (4M/4F) Vehicle Vehicle Vehicle Vehicle 100 100 100 100 MGD006 safety and
pharmacodynamic
properties were
evaluated in
cynomolgus
monkeys following
either intermittent
(Groups 2-5) or
continuous (Group 6)
dosing compared to
vehicle (Group 1)
over a 4-week
treatment period.
28 kDa 28 kDa 59000
MGD006 (7-­‐day-­‐on) ng/kg/day 1 2 38 kDa 38 kDa 58500
Molm-13
(CD123 +)
49 kDa 49 kDa 0
No DART
CD3x4420 0.1ng/ml Vehicle MGD006 (4-­‐day-­‐on/3-­‐day-­‐off) ng/kg/day Group 1 (4M/4F) Vehicle Vehicle CD8 1 .0 ´1 0
85.6% 87.4% CD3x4420
6
CD45 In t e n s it y ( C o u n t s )
5 8 8 9 8 .8
C
Study Design
Study Day Design and Structural Features
Figure 3: Effective Blast Elimination in AML Patient Ex Vivo
Infusion No. Figure 1: MGD006 – A Bispecific CD123 x CD3
Dual-Affinity Re-Targeting (DART) Molecule
(A) End-of-infusion serum concentrations of MGD006 in cynomolgus monkeys after receiving 7-day continuous
infusions at dose levels of 100, 300, 600 or 1000ng/kg/day (Group 6) confirming increased Cmax with dose.
(B) Comparable MGD006-mediated cytotoxicity against CD123+ Kasumi-3 cells with PBMCs from either naïve
monkeys or those treated with weekly escalating doses of MGD006 up to 1000 ng/kg/d for 4 weeks.
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