Of CD137 0.000000 80 400 2000 10000 0.000000 80 400 2000 10000 0 0.64 2 3.2 16 20 80 200400 0

Selection of a Bispecific Trivalent HER2 x CD137 TRIDENT™ Format Providing Optimal Tumor-anchored Immune Co-stimulation 1560 Liqin Liu1, Chia-Ying K. Lam2, Ralph Alderson1, Vatana Long1, Yinhua Yang1, Robert Burns1, Lusiana Widjaja1, Jonathan Li2, Christina Wolff1, Valentina Ciccarone1, James Tamura1, Gundo Diedrich1, Ezio Bonvini1, Syd Johnson1, Paul A. Moore1

1 2 MacroGenics, Inc., Rockville, MD and Brisbane, CA http://ir.macrogenics.com/events.cfm

Abstract Results

Introduction: CD137 (4-1BB) signaling provides co-stimulation of CD8 or NK cells following antigen or FcγR engagement, respectively. Efforts to leverage CD137 Geometry and Relative Valency Critical for HER2 x CD137 TRIDENT Molecule: Combinatorial Activity with Redirected T-cell Killing co-stimulation via agonistic monoclonal antibodies (mAbs) have been thwarted by limited clinical efficacy or unacceptable toxicity. Bispecific targeting + + Optimal Activity A.BB7-H3 x CD3 DART Molecule Mediates . Experimental Design to C. HER2 x CD137 Enhances B7-H3 x CD3-mediated Killing of B7-H3 HER2 Tumor Cells strategies linking CD137 activation to a tumor-targeting moiety provides an approach to localize CD137 activation to the tumor microenvironment. Increased CD137 Expression on T-cells Evaluate Combinatorial Activity HER2 HER2 CD137 CD137 B7-H3 x CD3 + Control B7-H3 x CD3 + HER2 x CD137 1.4 Control TRIDENT Here we evaluate a panel of Fc-bearing HER2 x CD137 bispecific molecules incorporating different valency and geometry to define the format providing CD137 CD137 (128 pg/mL| 0.64 ng/mL| 3.2 ng/mL| 16 ng/mL| 80 ng/mL| 0.4ug/mL) (0 pg/mL| 128 pg/mL| 0.64 ng/mL| 3.2 ng/mL| 16 ng/mL| 80 ng/mL| 0.4ug/mL) + + HER2 x CD137 TRIDENT

60 CD8+/CD137 JIMT-1 (B7-H3 , HER2 ) 100 100 1.2 optimal CD137 co-stimulation in a tumor-cell anchor-dependent manner. + Primary T Cells (E:T = 5:1) 50 CD4+/CD137 1.0 HER2 HER2 Tetravalent DART + B7-H3 x CD3 (400 ng/mL) JIMT-1 + Hi

CD137 Subset Cells Bivalent HER2; Bivalent CD137 + 40 (B7-H3 ; HER2 ) 0.8 Methods: An anti-HER2 mAb specificity that does not cross compete with margetuximab, trastuzumab or pertuzumab and a proprietary anti-CD137 + 50 50 ® TM HER2 72 hrs 0.6 B7-H3 x CD3 mAb were utilized to assemble a set of HER2 x CD137 bispecific molecules in bivalent and tetravalent DART or trivalent TRIDENT configurations. 2. 3. 4. Relative EC 0.4

20 DART Molecule

or CD4 p< 0.0001, N=4

+ B7-H3 x % Viable Cells

% Viable Cells (two-tailed paired t-test) The resulting molecules were compared in binding, signaling and co-stimulation assays in the presence or absence of tumor cells expressing HER2. CD3 0.2 CD137 CD137 HER2 HER2 CD137 Upregulation T-cells 0 0 % of CD137 0.000000 80 400 2000 10000 0.000000 80 400 2000 10000 0 0.64 2 3.2 16 20 80 200400 0

Combination studies were performed in vitro and in immune deficient mice reconstituted with human PBMCs. 1. in CD8 B7-H3 x CD3 (ng/mL) B7-H3 x CD3 (ng/mL) Concentration (ng/mL) 10-1 100 101 102 103 104 105 HER2 HER2 CD137 Results: TRIDENT molecules bearing bivalent CD137 and monovalent HER2 binding achieve optimal HER2-dependent tumor-cell anchored CD137 B7-H3 x CD3 (ng/mL) Bivalent DART HER2 D. HER2 x CD137 Enhances IFN-γ and TNF-α Release Monovalent CD137; CD137 CD137 Trivalent TRIDENT 250 HER2 x CD137 TRIDENT 300 HER2 x CD137 TRIDENT immune cell co-stimulation. CD137 co-stimulation increases proportionally with the level of HER2 expression as observed with HER2 1+ (MCF7 breast), Monovalent HER2 104 104 Re-expose T Cells and HER2 x CD137 +2000 (ng/mL) +2000 (ng/mL) + + Bivalent HER2; Monovalent CD137 JIMT-1/GF Cells (E:T = 3:1) to 200 250 Monovalent HER2; Bivalent CD137 B7-H3 x +200 (ng/mL) +200 (ng/mL) HER2 2+ (JIMT1 breast) and 3+ (N87 gastric) tumor cells and was paralleled with increased HER2 /CD137 cell association. No CD137 activation is observed CD137 HER2 HER2 103 103 B7-H3 x CD3 ± HER2 x CD137 200 CD137+ CD3 150 +2 (ng/mL) +2 (ng/mL) in the absence of HER2-expressing tumor cells. HER2 x CD137 bispecifics enhance NK-cell proliferation and IFN-γ release induced by margetuximab, +0.2 (ng/mL) 150 +0.2 (ng/mL) 2 2 10 10 100 +0.000 (ng/mL) +0.000 (ng/mL) 5. 6. 7. 48 hrs 100 an Fc-optimized anti-HER2 mAb that up-regulates CD137 expression on NK-cells concomitant with enhanced ADCC against HER2-positive cells. CD137 CD137 CD137 T cells 50 101 101 50 IFN- γ (pg/mL)

JIMT-1 TNF- α (pg/mL) Similarly, HER2 x CD137 bispecific molecules enhanced the in vitro activity of MGD009, a B7-H3 x CD3 bispecific DART molecule that up-regulates DART/TRIDENT proteins (1–7) with varying valency of CD137 and/or HER2 binding were expressed in CHO cells and purified (B7-H3+; HER2Hi) 0 0 100 100 Evaluate Viability and 10-2 100 102 104 10-2 100 102 104 to homogeneity. Binding of each molecule to soluble HER2 and CD137 was confirmed by ELISA and to natively expressed cell 100 101 102 103 104 100 101 102 103 104 + + Cytokine Levels CD137 during T-cell redirected killing. Finally, in vivo mouse studies demonstrate the ability of HER2 x CD137 molecules to expand tumor-associated surface antigen by FACS analyses of JIMT-1 (HER2 ) tumor cells or activated CD8 T cells (CD137 ). Co-stimulatory activity on CD8 CD8 B7-H3 x CD3 (ng/mL) B7-H3 x CD3 (ng/mL) primary T cells was evaluated by co-incubating each test article with primary T cells sub-optimally primed with anti-CD3 in CD8 cells when co-administered with a tumor targeted CD3 bispecific molecule and support enhance anti-tumor activity. presence of HER2+ N87 Gastric cancer cell line. A. Top panel: CD137 expression on T cells was induced by B7-H3 x CD3 DART molecule incubated in presence of JIMT-1 B7-H3+, C. HER2 x CD137 TRIDENT molecule enhances the potency of T-cells primed with B7-H3 x CD3 DART molecule to sustain redirected HER2+ tumor cells (E:T = 2:1; 24hr). Bottom panel: Representative flow-cytometry plots demonstrating CD137 expression no T-cell killing B7-H3+/HER2+ JIMT-1 breast cancer cells. D. HER2 x CD137 TRIDENT molecule enhances secretion of IFNg and TNFa Conclusions: An optimal HER2 x CD137 bispecific format providing maximal CD137 activation in a HER2- dependent manner was identified as a trivalent CD8 T-cells population before (left) and after treatment (right). mediated by B7-H3 x CD3 DART molecule. Tumor TRIDENT molecule bearing bivalent CD137 and monovalent HER2 binding. Combinatorial activity with HER2 x CD137 bispecifics was observed with both HER2 CD137 Format/ HER2 x CD137 Anchored a HER2-directed therapeutic mAb and a CD3-engaging tumor-targeted bispecific molecule. HER2 x CD137 TRIDENT molecules have therapeutic potential Binding Binding Valency Orientation Costimulatory (JIMT-1) (activated CD8) and provide a structural template for incorporating alternate tumor- and/or co-stimulatory-targeting arms. Activity HER2 x CD137 TRIDENT Molecule Enhances Anti-Tumor HER2 x CD137 Combinatorial Activity With Activity of CD3 Bispecific (5T4 x CD3) Margetuximab, an anti-HER2 Fc-engaging mAb DART/ 1. HER2 x CD137 ü ü + Bivalent NSG/Class I-/- Mice 800 Vehicle A. Margetuximab Mediates Increased B. HER2 x CD137 TRIDENT Combines with Background 5T4 x CD3 PBMC + SKOV3 (HER2+, 5T4+) CD137 Expression on NK Cells Margetuximab to Increase NK Cell 700 HER2 x CD137 Proliferation and IFN-γ Production 2. (HER2/CD137) 2x ü ü ++ 5T4 x CD3 + HER2 x CD137

) 1200 Margetuximab 60 3 600 0.1 µg/mL HER2 x CD137 CD137 (4-1BB)-Conditional Agonist: Rationale Properties of anti-CD137 Arm DART/ CTRL mAb 0.1 µg/mL HER2 x Control 0.1 µg/mL Control x CD137 3. (HER2)2 x (CD137)2 ü ü +++ 500 900 ■■ Tetravalent Cells 40 CD137 (4-1BB), a TNFR superfamily member expressed on immune cells, The CD137 antibody (MG-24) mAb was selected for incorporation into HER2 x + Day 0 600 in Gate d interacts with CD137L leading to CD137 pathway activation CD137 bispecifics based on conditional agonistic activity. This is in contrast to 4. (CD137)2 x (HER2)2 ü ü ++ 400 + CD5 6 urelumab that can directly agonize independent of secondary cross-linking. - 20 ––↑ T, NK-cell effector function, proliferation and survival 300 300 CD3 Endothelial adhesion molecules and chemokines resulting in CD8 homing to 5. HER2 x CD137 x CD137 ü ü ++++ % Ki67 ––↑ A. Reporter Assay B. Primary T-cell Costimulation CD4 Depletion (OKT4) + Tumor Volume (mm 200 0 0

tumor site MFI of CD137 on NK Cells CD137 Signaling via NF-κB TRIDENT/ 5T4 x CD3 DART molecule -6 -5 -4 -3 -2 -1 0 1 2 3 -4 -2 0 2 4 6. HER2 x CD137 x HER2 ü ü + 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 ––↑ DC maturation and cross-priming of antigen-specific T cells Without Crosslinking Without Crosslinking Trivalent (10 µg/kg; sub-efficacious 100 Margetuximab (ng/mL) Margetuximab (ng/mL) 25000 MG-24 10000 mono-therapy dose) 180 Survival 0.1 µg/mL HER2 x CD137 Survival Proliferation 20000 Urelumab* 8000 HER2 x CD137 TRIDENT molecule (5 mg/kg) 0 7. HER2 x HER2 x CD137 ü ü - 105 105 0.1 µg/mL HER2 x Control TNF, IL-6, IL-8 Proliferation Cytolytic Activity Activation Negative Control Ab 0510 15 20 25 30 35 40 45 15000 6000 Study Day 0.1 µg/mL Control x HER2 M-CSF, ROS Migration 120 104 104 Differentiation RL U 10000 4000 The (HER2)2 x (CD137)2 DART molecule (3), a 2 x 2 DART and the HER2 x CD137 x CD137 TRIDENT molecule (5), a 1 x 3 TRIDENT supported greatest conditional CD137 co-stimulation and were selected for further analyses. Correlation with Increased CD8 Infiltrate and T-cell Activation IFN-γ (pg/mL) IFN- γ (pg/mL) 3 3

2000 γ 5000 5000 Vehicle 2500 Vehicle 10 10 CD137 CD137 60 0 0 Day 7–22 5T4 x CD3 5T4 x CD3 -2 -1 0 1 HER2 x CD137 HER2 x CD137 2 2 IFN- APC 10 10 10 10 MG-24 Urelumab* Negative Control 10 10 mAb (µg/mL) 4000 5T4 x CD3 + HER2 x CD137 2000 5T4 x CD3 + HER2 x CD137 101 102 103 104 105 101 102 103 104 105 0 With Crosslinking With Crosslinking CD137L 10000 CD156 CD56 10-4 10-2 100 102 104 25000 MG-24 HER2 x CD137 (1x2) TRIDENT Molecule Supports 3000 1500 Activation Margetuximab (ng/mL) Costimulation 20000 Urelumab* 8000 Tumor Excision & Flow Cytometry Negative Control Ab Optimal Conditional CD137 Agonism 2000 1000 CD137 expression on NK cells was increased %Ki67 CD3-CD56+ cells upon incubation of purified CD137 15000 6000 Top panel: Top panel: Activated by margetuximab but not control mAb in the presence of NK cells with JIMT-1 HER2++ tumor cells (E:T = 2:1; 16hr) in the (Count/1 mg Tumor) (Count/1 mg Tumor) + RL U 10000 4000 T-cell Co-stimulatory +++ ++ + + ++ T or NK Cell A. N87 (HER2 ) JIMT-1 (HER2 )MCF-7 (HER2 ) 1000 500 JIMT-1 HER2 tumor cells (E:T = 2:1; 16hr). Bottom panel: presence of margetuximab and HER2 x CD137 TRIDENT molecule Activity 1 ng/mL|10 ng/mL|0.1 µg/mL|1 µg/mL representative flow cytometry plots demonstrating CD137 or control TRIDENT molecules. Bottom panel: IFN-g levels in

12000 12000 12000 Her2 x Control CD8 IFN- γ (pg/mL) 2000 PD-1 5000 + Control x CD137 expression on CD56 population before (left) and after supernatant of NK/tumor cell co-cultures following treatment HER2 0 0 4000 Urelumab* Day 29 0 0 treatment (right). with margetuximab and HER2 x CD137 or single arm controls. ■■ 10-2 10-1 100 101 MG-24 Urelumab* Negative Control Cell Lines 8000 8000 2 x 2 DART CD137 expression is up-regulated on T cells following TCR engagement with mAb (µg/mL) 72h 1 x 2 TRIDENT IFN-γ cognate MHC:peptide complex Evaluation of CD137 mAb MG-24 to activate (A) Jurkat CD137 NF-kB-luc reporter line or (B) support primary T cell co-stimulation Release 4000 4000 1 IF N γ (pg/mL) ––Expressed on tumor-reactive T-cell lineages in the absence (upper panels) or presence (lower panels) of 4 x ahFC cross-linking reagent. For panel (B) 0.1 µg/mL and 1 µg/mL of Sub-optimally each test article were evaluated. *A replica of Urelumab was employed as control for non-cluster dependent activation. Stimulated 000 ––Synergy of CD137 agonism with PD-1 checkpoint blockade2 T Cells 1 ng/mL|10 ng/mL|0.1 µg/mL|1 µg/mL1 ng/mL|10 ng/mL|0.1 µg/mL|1 µg/mL1 ng/mL|10 ng/mL|0.1 µg/mL|1 µg/mL ––CD3 based bispecifics induce CD137 expression on T cells Conclusions ■■ Monoclonal antibodies can further induce CD137 expression on NK cells upon B. CD137-mediated 8x106 8x106 8x106 1 x 2 TRIDENT ■■ NF-κB Activation 2 x 2 DART Proof-of-concept studies confirm HER2+ tumor-cell conditional activation of the CD137 pathway by various HER2 x CD137 DART and Urelumab replica ligation of FcgRIIIA Properties of anti-HER2 Arm 6x106 4x106 4x106 HER2 x Control 3 4 5 +,++,+++ HER2 3x106 3x106 Control x CD137 TRIDENT proteins ––Synergy of CD137 agonism with rituximab , trastuzumab , cetuximab 6 The HER2-1 mAb selected for incorporation into HER2 x CD137 bispecifics Cell Lines 4x10 6 6 ■■ 6h 2x10 2x10 ■■ Agonistic CD137 mAbs exhibit potent antitumor activity in preclinical models 2x106 HER2 x CD137 (1x2) TRIDENT molecule bearing monovalent HER2 binding and bivalent CD137 engagement provides optimal activity does not compete with margetuximab (same epitope as trastuzumab) or Luciferase 1x106 1x106 but display unwanted toxicity or insufficient activity in the clinic1 + pertuzumab, facilitating potential combination with existing mAb therapy. Luminescence (RLU ) Jurkat 10-2 10-1 100 101 102 103 104 10-2 10-1 100 101 102 103 104 10-2 10-1 100 101 102 103 104 ––CD137 pathway activation and T-cell co-stimulation dependent on HER2 expression level on co-engaged tumor cells ■ NF-κB-luciferase ■ + Concentration (ng/mL) Concentration (ng/mL) Concentration (ng/mL) + + + Goal: Develop optimal bi-specific conditional CD137 pathway activation of Cold mAb/ αHER2-1 margetuximab pertuzumab (CD137 ) 1000 1000 1000 ––Increased activity consistent with increased loading of HER2 cells and level of HER2 cell:CD137 cell association tumor immune infiltrate Labeled mAb 900 900 900 800 800 800 ■■ 700 700 700 80000 HER2 x CD137 TRIDENT molecule enhances anti-tumor activity of CD3-based bispecific DART molecules 80000 80000 1 x 2 TRIDENT 600 600 600 2 x 2 DART αHER2-1 500 500 500 60000 MF I MF I MF I 60000 Control x CD137 400 400 400 ––Sustains CTL capacity of T cells primed by CD3-based DART molecule and increases secreted levels of IFN-g and TNF-a a Fluor 488 a Fluor 488 a Fluor 488 12000 300 300 300 10000 40000 40000 +

200 200 200 MFI Al ex Al ex Al ex C. HER2 Cell-surface 8000 2500 ––Enhances anti-tumor activity in xenograft model of HER2 ovarian cancer HER2 x CD137 Bi-specific: Desired Molecule Profile 100 100 100 2000 α hFc AP C 6000 Binding 1500 0 0 0 20000 4000 10-7 10-6 10-7 10-5 10-4 10-3 10-2 10-1 100 102 10-7 10-6 10-7 10-5 10-4 10-3 10-2 10-1 100 102 10-7 10-6 10-7 10-5 10-4 10-3 10-2 10-1 100 102 1000 ■■ 2000 500 HER2 x CD137 TRIDENT molecule enhances activity mediated by an anti-HER2 antibody (margetuximab) αHER2 x αCD137 Properties Purpose Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL) 0 0 0 10-5 10-4 10-3 10-2 10-1 100 101 10-5 10-4 10-3 10-2 10-1 100 101 10-5 10-4 10-3 10-2 10-1 100 101 1000 1000 1000 Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL) ––Binds an epitope distinct from trastuzumab (margetuximab) or pertuzumab Bispecific format providing optimal CD137 Co-stimulation of TILs to enhance anti-tumor 900 900 900 co-stimulation contingent on tumor HER2 immunity in HER2+ tumors (breast, gastric, 800 800 800 ––Increases NK cell proliferation and IFN-g secretion 700 700 700 D. Cell-cell Conjugation 600 600 600 expression level ovarian, CRC) margetuximab 14 14 14 1 x 2 TRIDENT + 500 500 500 +,++,+++ ■■ MF I MF I MF I HER2 400 400 400 (%) 12 12 12 2 x 2 DART + Data support further evaluation of HER2 x CD137 to enhance anti-tumor immunity in HER2 cancers alone or in combination with

xa Fluor 488 xa Fluor 488 xa Fluor 488 Cell Lines Control x CD137 CD137 mediated agonistic activity 300 300 300 10 10 10 Co-stimulatory activity restricted to tumor sites 200 200 200 (CFSE) Ale Ale Ale 8 8 8 existing anti-HER2 therapy, checkpoint inhibitors or with CD3-directed bispecific molecules dependent on cross-linking to HER2 100 100 100

/PKH26 6 6 6 0 0 0 CD137 + αHER2 binding not competing with Enable combination with existing HER2 10-7 10-6 10-7 10-5 10-4 10-3 10-2 10-1 100 102 10-7 10-6 10-7 10-5 10-4 10-3 10-2 10-1 100 102 10-7 10-6 10-7 10-5 10-4 10-3 10-2 10-1 100 102 4 4 4 ■■The TRIDENT format, bearing monovalent tumor targeting and bivalent CD137 binding, provides a framework for other tumor antigen-

Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL) CHO/CD137 CFSE CD137 2 2 2 trastuzumab/margetuximab/pertuzumab mAb-based therapies Cells 6 1000 1000 1000 (PKH26) 10-3 10-2 10-1 100 101 10-3 10-2 10-1 100 101 10-3 10-2 10-1 100 101 anchored CD137 bispecifics 900 900 900 Avoid any FcgR interactions and cross-linking of Concentration (ng/mL) Concentration (ng/mL) Concentration (ng/mL) Effector function “null” Fc 800 800 800 CD137; retain FcRn binding for prolonged PK 700 700 700 600 600 600 ■■ pertuzumab 500 500 500 T-cell costimulation (A) and NF-kB pathway activation (B) strictly dependent on Cyomolgus monkey αHER2 and αCD137 MF I 400 MF I 400 MF I 400 Enable NHP PK/Tox study xa Fluor 488 300 xa Fluor 488 300 xa Fluor 488 300 HER2 tumor cell surface expression; no activity observed in presence of low cross-reactivity 200 200 200 References Ale Ale Ale 100 100 100 expressing HER2 (MCF-7) or absent HER2 expression (data not shown) Combinatorial activity with mAbs 0 0 0 10-7 10-6 10-7 10-5 10-4 10-3 10-2 10-1 100 102 10-7 10-6 10-7 10-5 10-4 10-3 10-2 10-1 100 102 10-7 10-6 10-7 10-5 10-4 10-3 10-2 10-1 100 102 1. Chester et al., (2018) Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. BLOOD 131 (1): 49-57. 2. Chen et al (2015) Combination of 4-1BB agonist and PD-1 antagonist promotes antitumor effector/memory CD8 ■ (including Fc-optimized), checkpoint Combination therapy with other IO agents Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL) ■ Enhanced conditional CD137 activation correlates with increase HER2 cell T cells in a poorly immunogenic tumor model. Cancer Immunol. Res 3 (2): 149-160. 3. Kohrt et al., (2011) CD137 stimulates anti-lymphoma activity of anti-CD20 antibodies. BLOOD 117(8): 2423-2432. 4. Masu et al., (2018) Anti-CD137 monoclonal antibody inhibitors and CD3-directed bispecifics AlexaFluor 488-labeled mAbs binding to N87 cell line in the presence of 10 μg/mL unlabeled mAbs as competitor. surface binding (C) and consequential ability to support cell:cell association (D) enhances trastuzumab-induced, natural killer cell-mediated cytotoxicity against pancreatic cancer cell lines with low human epidermal growth factor-like receptor 2 expression. PLoS One, Dec 31;13(12):e0200664. 5. Kohrt et al., (2014) Targeting CD137 enhances the efficacy of cetuximab.J.Clin. Invest. 124: 2668-2682. 6. Liu et al., (2019) Tumor-antigen 5T4-dependent Activation of the CD137 Costimulatory Pathway by Bispecific 5T4 x CD137 x CD137 TRIDENT™ Molecules. AACR 2019 Abstract 3476.