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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 10 0 10 1 10 2 2000 4000 6000 8000 10000 Binding to PD-1 + NS0 Cells Concentration (nM, LOG) MFI (APC) 10 -3 10 -2 10 -1 10 0 10 1 10 2 1000 2000 3000 4000 Binding 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 10 0 10 1 10 2 0 5000 10000 15000 20000 PD-L1 Binding Blockade Concentration (nM, LOG) MFI 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 0 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 10 0 10 1 0 20 40 60 80 100 Concentration (nM, LOG) % No Treatment Ctrl MGA012 Negative Control MGD013 PD-L1-induced SHP-2 Activation 10 -3 10 -2 10 -1 10 0 10 1 10 2 0 10000 20000 30000 40000 PD-1 Jurkat + PD-L1 CHO Concentration (nM, LOG) RUL Luminescence 25F7* Nivolumab* MGD013 10 -2 10 0 10 2 100000 120000 140000 160000 180000 LAG-3 Jurkat + Raji (SED Stimulated) Concentration (nM, LOG) RUL Luminescence MGA012 25F7* MGD013 A Inhibition of SHP-2 Activation PD-1 Signaling LAG-3 Signaling B C “Brakes on” “Brakes released” SED (50ng/ml) Raji CD4 lo TCR HLA -DR Raji Jurkat Reporter Line NFAT luc2 NFAT luc2 CD4 lo TCR HLA -DR LAG-3 MGD013 or α LAG-3 mAb 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 nM 0.024 nM 0.09 nM 0.39 nM 1.56 nM 6.25 nM 25 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 or PD-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 Ratio CD106/VCAM1 CD87/uPAR CEACAM5/CD66e Collagen l Collagen lll CXCL10/IP10 Keratin 20 MMP9 PAll PBMC Cytotoxicity Granzyme B IFNγ IL10 IL17A IL2 IL6 SRB TNFα sVEGF TIMP2 tPA uPA CCL2/MCP1 CD106/VCAM1 CD40 CD69 uPAR CEACAM5/CD66e Collagen IV CXCL10/IP10 CXCL9/MIG Keratin 20 PBMC Cytotoxicity Granzyme B IFNγ IL10 IL17A IL2 IL6 SRB TNFα Granzyme B IFN IL2 IL6 IL17A sVEGF tPA Granzyme B IFN IL2 IL6 IL10 IL17A TNF StroHT29 VascHT29 Nivolumab* (anti-PD-1) MGD013 25F7 (anti-LAG-3)* Maximal Binding of antigen Background * 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 Structure mAb1 VL mAb1 VH mAb2 VH mAb2 VL mAb1 VH mAb2 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) Triple Negative Breast Cancer H&E LAG-3 PD-1 T-cell Clonal Expansion Cytokine Secretion Effector 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 10 0 10 2 0.0 1.0 2.0 3.0 4.0 5.0 Concentration (nM, LOG) Relative Dimerization Activity Nivolumab* 25F7* MGD013 Nivolumab* + 25F7* Light MGD013 (PD1 x LAG3) hinge stabilized IgG4 tetravalent, bispecific DART molecule
