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Preclinical Characterization of MGD013, a PD-1 x LAG-3 Bispecific DART® Molecule
Abstract Results
Introduction
Presented at the Society for Immunotherapy of Cancer (SITC) 32nd Annual Meeting, November 8-12, 2017, National Harbor, Maryland © 2017 MacroGenics, Inc. All rights reserved.
Ross La Motte-Mohs, Kalpana Shah, Jennifer G. Brown, Doug Smith, Sergey Gorlatov, Valentina Ciccarone, James K. Tamura, Hua Li, Jill R. Rillema, Monica Licea, Christine Shoemaker, Leilei He, Farha Vasanwala, Jessica Hill, Arin Whiddon, Massimiliano Pascuccio, Shereen Saini, Francine Z. Chen, Anushka De Costa, Ann Easton,
Peter Lung, Jonathan Li, Kurt Stahl, Jeffrey Nordstrom, Scott Koenig, Ezio Bonvini, Syd Johnson and Paul A. Moore MacroGenics, Inc., Rockville, MD and South San Francisco, CA, USA
Conclusions
http://ir.macrogenics.com/events.cfm
NCT03219268
MGD013 Binds PD-1 and LAG-3 and Blocks Ligand Interactions
Structure of MGD013
Expression of Checkpoint(s) Across All TMAs* Overlapping Expression**
Tumor MicroArray (TMA) Spot Count for Particular Indication
LAG-3* Total
PD-1+ Total
LAG-3+PD-1+ Total
LAG-3+ PD-1+
PD-1+ LAG-3+
Lung Squamous Cell Carcinoma 18/36 50%
15/36 42%
10/36 28%
10/15 67%
10/18 56%
Lung Adenocarcinoma 19/33 58%
18/33 55%
15/33 46%
15/18 83%
15/19 79%
Triple Negative Breast Cancer 16/29 55%
12/29 41%
10/29 35%
10/12 83%
10/16 63%
*Spot Counts indicate the number of TMA positive for LAG-3, PD-1, or LAG-3 + PD-1 expression divided by the total TMAs**Spot Counts indicate the number of TMA with overlapping checkpoint expression of PD-1 and LAG-3
10-3 10-2 10-1 100 101 102
2000
4000
6000
8000
10000
Binding to PD-1+ NS0 Cells
Concentration (nM, LOG)
MFI
(APC
)
10-3 10-2 10-1 100 101 1021000
2000
3000
4000Binding to LAG-3+ NS0 Cells
Concentration (nM, LOG)
MFI
(APC
)
PD-1 and LAG-3 are Expressed on TILs
10-3 10-2 10-1 100 101 1020
5000
10000
15000
20000
PD-L1 Binding Blockade
Concentration (nM, LOG)
MFI
10-4 10-3 10-2 10-1 100 101 1020
1000
2000
3000
LAG-3 Binding Blockade
Concentration (nM, LOG)
MFI
MGD013 Enhances Antigen-driven T-cell Cytokine Function In Vitro
MGD013 Co-engages PD-1 & LAG-3
� MGA012 + MG anti-LAG-3 combination comparable to benchmark antibody combination
� MGD013 enhanced IFN-γ secretion beyond that observed with antibody combinations
MGD013 Disrupts PD1- & LAG-3- Mediated T-cell Inhibitory Signaling
MGD013 demonstrates a dose dependent blockade of the PD-1/PD-L1 axis comparable to anti-PD-1 mAbs as evaluated in PD-1 reporter models obtained from DiscoverX’s PathHunter® Enzyme Fragment Complementation Assay to inhibit SHP-2 activation (A) or Promega’s PD-1/PD-L1 Blockade Biosassay to release NF-AT blockade (B). Similarly, MGD013 demonstrates a dose dependent blockade of the LAG-3/MHC-class II axis comparable to a replica of BMS’s 25F7 [anti-LAG-3 mAb] evaluated in Promega’s LAG-3/MHC-class II Blockade Bioassay to release NF-AT blockade (C).
10-4 10-3 10-2 10-1 100 1010
20
40
60
80
100
Concentration (nM, LOG)
%N
oTr
eatm
entC
trl
MGA012Negative Control
MGD013
PD-L1-induced SHP-2 Activation
10-3 10-2 10-1 100 101 1020
10000
20000
30000
40000
PD-1 Jurkat + PD-L1 CHO
Concentration (nM, LOG)
RU
LLu
min
esce
nce
25F7*Nivolumab*MGD013
10-2 100 102100000
120000
140000
160000
180000LAG-3 Jurkat + Raji (SED Stimulated)
Concentration (nM, LOG)
RU
LLu
min
esce
nce MGA012
25F7*
MGD013
A Inhibition of SHP-2 Activation PD-1 Signaling LAG-3 SignalingB C“Brakes on” “Brakes released”
SED (50ng/ml)
Raji
CD4lo TCR
HLA-DR
Raji
JurkatReporter Line
NFAT luc2NFAT luc2
CD4lo TCR
HLA-DR
LAG-3
MGD013or
αLAG-3mAb
MGD013 Enhances Immune Responses in TME Models Compares favorably to PD-1 + LAG-3 mAb combination
MGD013, MGA012 (PD-1 mAb), MG’s LAG-3 mAb were evaluated and compared against the combination of nivolumab* + 25F7* for their ability to capture immune activation under the mimicry of the tumor microenvironment (TME). HT-29 colorectal cells were cultured with fibroblasts and PBMCs to recapitulate a stromal microenviroment (left panel) or with endothelial cells and PBMCs to recapitulate a vascular microenvironment (right panel). Immune profiling of checkpoint targets including adhesion molecules, cytotoxic granules, and cytokines were measured (DiscoverX).
0 50 100 150 200 250 300 350 400 450
Control IgG
25F7*
MG anti-LAG-3
Nivolumab*
MGA012
Nivolumab* + 25F7*
MGA012 + MG anti-LAG-3
MGD013
Relative IFN-γ Induction(% of 25 nM MGA012, mean± SEM)
0.006 nM0.024 nM0.09 nM0.39 nM1.56 nM6.25 nM25 nM
***
IFN-γ release by 25 nM MGA012 = 3276 ± 744 pg/mL
Ratio-paired t-test(25 nM group):
* p < 0.0262** p < 0.0022
NS = not significant
NS+PD-1 LAG-3
orPD-1
LAG-3
MGA012 MG anti-LAG-3 MGD013 Nivo+25F7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Log
Rati
o
CD10
6/VC
AM1
CD87
/uPA
R
CEAC
AM5/
CD66
e
Colla
gen
l
Colla
gen
lll
CXCL
10/I
P10
Kera
tin
20
MM
P9 PAll
PBM
C Cy
toto
xici
ty
Gra
nzym
e B
IFN
γ
IL10
IL17
A
IL2
IL6
SRB
TNFα
sVEG
F
TIM
P2 tPA
uPA
CCL2
/MCP
1
CD10
6/VC
AM1
CD40
CD69
uPAR
CEAC
AM5/
CD66
e
Colla
gen
I V
CXCL
10/I
P10
CXCL
9/M
IG
Kera
tin
20
PBM
C Cy
toto
xici
ty
Gra
nzym
e B
IFN
γ
IL10
IL17
A
IL2
IL6
SRB
TNFα
Granzyme BIFN IL2 IL6IL17A
sVEGF
tPA Granzyme B
IFN
IL2IL6
IL10 IL17ATNF
StroHT29 VascHT29
Nivolumab* (anti-PD-1)MGD013
25F7 (anti-LAG-3)*
Maximal Binding of antigenBackground
* Replicas of nivolumab and 25F7 mAbs produced at MacroGenics based on published sequences
� MGD013 was engineered as a tetravalent bispecific DART molecule in a human hinge-stabilized IgG4 backbone.
� MGD013 is capable of simultaneously binding PD-1 and LAG-3.
� MGD013 blocks PD-1/PD-L1/PD-L2 and LAG-3/MHC-Class II interactions and resultant inhibitory signal with potency comparable to MGA012 (anti-PD-1), and replicas of nivolumab or 25F7 (anti-LAG-3).
� MGD013 enhances T-cell responses compared to individual mAbs or combination mAb blockade.
� MGD013 was well-tolerated and demonstrates favorable pharmacokinetics in cynomolgus monkeys.
Clinical testing of MGD013 in several cancer indications is ongoing [NCT03219268].
� PD-1 and LAG-3 are two coinhibitory molecules that deliver negative signals upon interaction with ligands expressed on tumor cells and/or antigen presenting cells (PD-L1, PD-L2, or MHC-II).
Background: Monoclonal antibodies (mAbs) that target the immune checkpoints, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1), have shown enhanced clinical antitumor activity when given in combination, triggering interest in determining whether additional checkpoint inhibitor combinations may afford enhanced clinical benefit. Lymphocyte-activation gene 3 (LAG-3) is another immune checkpoint expressed on activated T cells and tumor infiltrating lymphocytes (TILs). Recognizing the therapeutic potential of dual checkpoint blockade, we have engineered MGD013, a IgG4κ bispecific DART molecule, to bind PD-1 and LAG-3 concomitantly or independently and disrupt these nonredundant inhibitory pathways to further restore exhausted T-cell function.
Methods: Proprietary PD-1 and LAG-3 mAbs were generated and selected based on binding characteristics, biophysical properties, the ability to block their respective receptor/ligand axes and to synergize in T-cell stimulation assays. Humanized sequences were incorporated into a tetravalent bispecific DART format and benchmarked against combinations of replicas of the approved PD-1 mAb (nivolumab) and BMS-986016 anti-LAG-3 mAb (25F7), which is currently under clinical evaluation. MGD013 biological activity was evaluated in various primary cell-based immune assays. Safety was assessed in cynomolgus monkey toxicology studies performed at MPI (Mattawan, MI) under Institutional Animal Care and Use Committee-approved protocols.
Results: MGD013 bound with high affinity to human and cynomolgus monkey PD-1- and LAG-3-expressing cells and blocked PD-1/PD-L1, PD-1/PD-L2 and LAG-3/HLA (MHC-II) interactions, with resultant signaling blockade. Functional characterization revealed enhanced cytokine secretion in response to antigen stimulation that was greater than that of the combination of individual equimolar amounts of PD-1 and LAG-3 mAbs. MGD013 was well tolerated in a repeated-dose (Q1Wx4) cynomolgus monkey toxicology study. Except for the occurrence of watery feces in a few animals, no MGD013-related adverse findings were noted, including hematological or clinical chemistry changes, serum cytokine levels or gross and microscopic histological findings, establishing a no-observed-adverse-effect level (NOAEL) of 100 mg/kg.
Conclusion: MGD013 is a bispecific DART molecule capable of simultaneously blocking the PD-1 and LAG-3 pathways, resulting in enhanced T-cell activation compared to single or combination mAb blockade. MGD013 has demonstrated a favorable preclinical safety and toxicological profile and is currently initiating clinical testing [NCT03219268].
Rationale
General StructuremAb1 VL
mAb1 VH
mAb2 VH
mAb2 VL
mAb1 VHmAb2 VL
mAb1 VL mAb2 VH
Fc
Fc
� High affinity binding to PD-1 and LAG-3 that compares favorably to nivolumab* (anti-PD-1) or 25F7* (anti-LAG-3)
Trip
le N
egat
ive
Brea
st C
ance
r
H&E LAG-3PD-1
T-cell Clonal ExpansionCytokine SecretionEffector Function
Tumor-directed Migration
Enhancement of T-cell response following SEB stimulation
* Indicates replicas of nivolumab and/or 25F7 (anti-LAG-3)
Well Tolerated in Cynomolgus Monkeys
Pilot Toxicology Study Design
�1-hour IV infusion at 100 (1 male) or 150mg/kg (2 males) �QW x 2
GLP Toxicology Study Design
�1-hour IV infusion at 10, 40 or 100 mg/kg (5 animals/sex/group) �QW x 4; 10-week recovery
Observations
�Well tolerated. Drug-related changes were limted to watery feces at ≥ 40 mg/kg with no impact to body weight. �Nonadverse increase in incidence of mononuclear cell infiltrates. �No cytokine release
Conclusion �NOAEL = 100 mg/kg; MTD > 150 mg/kg
� Combination mAb blockade of PD-1 and LAG-3 in animal models resulted in enhanced antitumor immunity than either mAb alone and is actively being tested clinically.
� MGD013 is a checkpoint inhibitor DART molecule currently under clinical evaluation that has been designed to restore T-cell effector function and enhance antitumor activity by simultaneously targeting PD-1 andLAG-3.
A
Binding of MGD013 to NS0-PD-1+ (A) and NS0-LAG-3+ (B) engineered cells was assessed by FACS analysis. Inhibition of soluble PD-L1 binding to NS0-PD-1+ cells (C) or soluble LAG-3 binding to class II+ Daudi cells (D), respectively, was assessed by FACS analysis. Similar data were obtained for soluble PD-L2 binding to NS0 -PD-1+ cells (data not shown).
B
C D
Indicated molecules were evaluated using enzyme fragment complementation assay employing PathHunter® U2OS PD-1/LAG-3 dimerization cell line (DiscoverX).
10-4 10-2 100 1020.0
1.0
2.0
3.0
4.0
5.0
Concentration (nM, LOG)
Rel
ativ
eD
imer
izat
ion
Act
ivity
Nivolumab*25F7*
MGD013Nivolumab* + 25F7*
Light
MGD013 (PD1 x LAG3) hinge stabilized IgG4 tetravalent, bispecific DART molecule