Preclinical investigation of the promise and challenges of agonistic anti-gitr antibody therapy. Amy Beebe Merck Research Laboratory March 16, 2017

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Preclinical investigation of the promise and challenges of agonistic anti-gitr antibody therapy Amy Beebe Merck Research Laboratory March 16, 2017

Rationale for anti-gitr agonist in cancer GITR is a co-stimulatory member of the TNF receptor family GITR expressed constitutively on Treg; inducible by activation on Teff, NK cells GITR agonism provides a positive costimulatory signal to primed effector T cells GITR antibody leads to localized depletion of Treg in tumor (mouse) 2 Knee et. al. Eur J Cancer, 2016

Efficacy of anti-gitr (mdta-1) in MC38 tumor model associated with increased CD8:Treg ratio in tumor 3 3

Experiments in FoxP3 lineage tracing mice indicate mdta-1 does not convert intra-tumoral Treg FoxP3 lineage tracing mice Cells are GFP+ only while expressing FoxP3 Cells that have ever stably expressed FoxP3 are permanently tdtomato+ MC38 tumor model in FoxP3 lineage tracing mice. Flow plots gated on intratumoral CD4+ T cells 5d after dosing No increase in exfoxp3 observed for B16 either 4 Mahne et. al. Canc Res, 2017

MFI DTA-1 preferentially reduces highly activated intra-tumoral Tregs 1500 1000 500 0 LAG-3 ** 8000 6000 4000 2000 0 ICOS **** 6000 4000 2000 0 CD44 ** 25000 20000 15000 10000 5000 0 CD39 **** 1500 1000 500 0 TIGIT *** mdta-1 (tumor Treg) Isotype control (tumor Treg) Isotype control (dln Treg) MC38 tumor model in FoxP3 lineage tracing mice. Flow plots gated on intratumoral Treg 1 day after dosing 5 Mahne et. al. Canc Res, 2017

mdta-1 Fc mutant & FoxP3.LuciDTR mice to dissect role of GITR agonism vs Fc-mediated depletion mdta-1 IgG2a and N297A isotypes both agonize GITR; not FcγR-dependent Splenocyte cultures Intratumoral Treg cultures pnfkb p65 ( phosflow ) Mahne et. al. Canc Res, 2017 FoxP3.LuciDTR mice for flow cytometry (GFP) imaging (Luc) and diptheria toxin-mediated depletion of Treg (DTR) Suffner et. al. J Immunol, 2010 6

Minimal tumor Treg depletion or change in activation markers with Fc mutant mdta-1 migg2a mdta-1 mdta-1 N297A DT FoxP3-GDL mice with MC38 tumors Day 5 after dosing 7 Mahne et. al. Canc Res, 2017

Both GITR agonism and Treg depletion contribute to efficacy and reversal of CD8+ T cell exhaustion 8 Mahne et. al. Canc Res, 2017

dln effects ( size and IFNγ+ CD8 T cells) induced by either GITR agonism or Treg depletion alone 9 Mahne et. al. Canc Res, 2017

Counts GITR is expressed on T cells in spleen and tumor of humanized mice with SKMEL-5 tumors Spleen Tumor human Treg (blue line) non-treg CD4+ T cells (dashed line) CD8 T cells (shaded grey) GITR Hu-C34+ cord blood-engrafted NSG mice with >25% human CD45+ immune cell chimerism 12+ weeks after reconstitution Implanted with SKMEL-5 melanoma tumor cell line SC 10 Mahne et. al. Canc Res, 2017

MK-4166 slows tumor growth, increases CD8:Treg ratio in spleen, and decreases activation markers on Treg in the tumor Hu-CD34+ humanized mice dosed implanted with SKMEL-5 tumors 10mg/kg MK-4166 (antihugitr IgG1) when tumors reach 130 mm 3 11 Mahne et. al. Canc Res, 2017

Anti-GITR dosing rationale What is the best way to dose a GITR agonist for optimal effector T cell co-stimulation and intratumoral Treg depletion? Considerations: Internalization after antibody binding to GITR Mechanism-based ADA and immune complex formation 12

Internalization of GITR on T cells after dosing: Receptor availability assay for target engagement 13

PK/PD analyses for determination of doses for saturating GITR in the blood and total body Receptor Availability in Blood mdta-1 PK (6 days) 4.2 ng/ml mdta-1 serum concentration gives 50% saturation of GITR on T cells in blood Based on receptor availability assay data >1 μg/ml mdta-1 serum concentration predicted to give 50% saturation of total body GITR Based on analysis of observed non-linear PK in the dose range tested 14

A higher DTA-1 dose is required for saturation of GITR on T cells in tumor compared to blood 15

Efficacy does not improve when doses exceed that needed to saturate GITR in the tumor 16

Efficacy does not improve with repeat 5 mg/kg doses 17

mdta-1 concentrations decrease rapidly in mice after re-dosing with mdta-1 PK after 1 st dose mdta-1 PK after 3 rd dose mdta-1 All mice have detectable ADA by d9 after 1 st dose and rapid drop in mdta-1 levels by d10 96hr serum mdta-1 concentrations Naïve mice: 28 μg/ml (5 mg/kg) and 161 μg/ml (30 mg/kg) Pre-dosed mice: 0.1 μg/ml (5 mg/kg) and 1.5 μg/ml (30 mg/kg) Based on this, expect that re-dosing at 5 mg/kg would be ineffective 18 Brunn et al. J Pharmacol Exp Ther, 2016

Pre-existing ADA increases the dose required for GITR saturation and single-dose anti-tumor efficacy 19 19 Brunn et al. J Pharmacol Exp Ther, 2016

mdta-1 forms high molecular weight complexes in plasma from mdta-1 pre-dosed (ADA+) mice with In vivo dosed mdta-1 Ex vivo spiked mdta-1 HP-SEC analysis of plasma after dosing 30 mg/kg DyLight 650-mDTA-1 in mice with pre-existing ADA HP-SEC analysis of plasma 15 min after spiking DyLight 650-mDTA-1 into plasma from mice with preexisting ADA 20 Brunn et al. J Pharmacol Exp Ther, 2016

Third dose First dose Hepatic uptake and catabolism of mdta-1 after 3 rd dose consistent with clearance of anti-drug:drug IC Tissue/blood ratio Liver Kidney Spleen Ileum Heart Concentration Liver Kidney Spleen Ileum Heart Blood Tissue/blood ratio increases dramatically after 3 rd dose Liver lysates show antibody-derived catabolites after 3 rd dose Immunofluorescence shows mdta-1 uptake in liver keratinocytes 24hrs after 3 rd dose F4/80+ mdta-1 merged 1 st dose 3rd dose 21 Brunn et al. J Pharmacol Exp Ther, 2016

Anti-drug antibody response is mechanism-driven: No ADA in GITR -/- mice 22

Summary: Preclinical investigation of the promise and challenges of agonistic anti-gitr antibody therapy Potential benefits of anti-gitr agonist in cancer immunotherapy Reduced regulatory T cells in the tumor Direct co-stimulation of activated T cells to further tip the balance Indications / patients that may benefit Challenges Cancers with high GITR and FoxP3 co-expression in tumor Patients with tumors that have immune infiltrate but are not fully responsive to PD-1 inhibitors Optimal dose regimen determination for an agonist Duration of saturation required, criteria for re-dosing Compatibility with optimal dose regimen for targeted killing? Potential impact of ADA on achieving optimal exposure 23

Acknowledgements Smita Mauze Danling Gu Roanna Ueda Renu Jain Ashley Mahne Barbara Joyce-Shaikh Daniel Cua Elaine Pinheiro Yanhong Ma Mohammad Tabrizifard Meric Ovacek Leo Tseng Enrique Escandon Nicholas Brunn Douglas Hodges Shuli Zhang Derek Wiswell Eddie Bowman Jane Xia 24

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