541 An Unbiased Screen Identifies a CD137×PD-L1 Bispecific IgG1 Antibody With Unique T-Cell Activation and Binding Properties Presented at the Cecile Geuijen,1 Paul Tacken,1 Rinse Klooster,1 Horacio Nastri,2 Shaun Stewart,2 Jing Zhou,2 Steve Wang,2 Cheng-Yen Huang,2 Arjen Kramer,1 Linda Kaldenberg-Hendriks,1 John de Kruif,1 AACR Annual Meeting 2019 Renate den Blanken-Smit,1 Vanessa Zondag-van der Zande,1 Abdul Basmeleh,1 Willem Bartelink,1 Patrick Mayes,2 Gregory Hollis,2 Reid Huber,2 Mark Throsby1 Atlanta, GA, USA • March 29-April 3, 2019 1Merus NV, Utrecht, The Netherlands 2Incyte Corporation, Wilmington, DE Abstract Antibody Generation Unbiased Screening CD137×PD-L1 Biclonics® Library MCLA-145 Blocks Ligand Binding CD137 (4-1BB) is a transmembrane costimulatory receptor on T and ● A library of ~200 CD137xPD-L1 Biclonics® was produced and purified ● Binding of CD137L to CD137L Blocking PD-1 Blocking NK cells that enhances adaptive immune responses and is a critical ● CD137xPD-L1 Biclonics® were screened in reporter and/or T cell transactivation assay in the absence and presence of PD-L1 expressing cells CD137 is blocked by mediator of antitumor immunity. CD137 signaling requires receptor MCLA-145 MCLA-145 ® MCLA-145 in a FACS- 9 0 0 0 clustering normally facilitated by the trimeric CD137 ligand ● CD137xPD-L1 Biclonics potently activate T cells in the presence of PD-L1 9000 M CUrelumabL A -1 4 5 analog 1.51 .5 Atezolizumab analog M C L A -1 4 5 based ligand binding U re lu m a b a n a lo g A te z o liz u m a b a n a lo g (CD137L). Alternatively, CD137 signaling can be triggered either U toUtomilumabm ilu m a b a n analoga lo g Negative control Ab assay 7 5 0 0 y y t ExampleExample ofof TT cellcell activationactivation assayassay screenscreen onon PDPD--L1xCD137L1xCD137 panelpanel (IL(IL--22 readout)readout) 7500t i directly by agonistic monoclonal antibodies (mAbs) or indirectly via Example of T-Cell Activation Assay Screen on CD137×PD-L1 Panel (IL-2 readout) i N eNegativeg C trl A b control Ab N e g C trl A b s IgGIgG s n n e ● MCLA-145 blocks PD-1 e t crosslinking of CD137 binding mAbs by Fcγ receptors on 12,00012000 t n (ng/mL)(ng/mL) n 6 0 0 0 1 .0 I I 6000 1.0 e neighboring cells. The development of CD137 targeted agents for binding to PD-L1 in an e nm c c 10,00010000 1000010,000 m n n n L1 L1 e e - 0 - ELISA-based assay c cancer therapy has been hampered by on-target off-tumor toxicity in c 4 5 0 0 5 s 5000 4500s 4 e 80008000 e r ILIL--22 r the case of agonist, monospecific, bivalent mAbs or limited antitumor D o 2500 ● Analogs are bivalent o O u ++ 2500 u l l OD450, F activity in the case of crosslinking mAbs. To address the issues of 6000 F 3 0 0 0 0 .5 Counts 6000 antibodies 2 counts 2 2 counts 2 3000 0.5 n 1250 n - - 2 a a - Il Il e toxicity and efficacy, a highly selective and potent CD137xPD-L1 e CHO PD CHO CHO PD CHO IL 4000 M 4000 625 M bispecific antibody (bAb) was identified by applying an unbiased 15001 5 0 0 20002000 313 functional screening approach. Collections of common light chain MeanIntensity Fluorescence Fabs recognizing CD137 and PD-L1 were produced based on 0 0 .0 0 0 0 .0 1 0 .1 1 1 0 1 0 0 0.0 ® 0 .1 1 1 0 antibody panels from immunized MeMo mice. A large and diverse PB14618p05 PB16814p01 PB14676p03 PB14585p05 PB14675p04 PB14584p05 PB16817p01 PB16841p01 PG6619p07 PG1337p218 0.01 0.1 Ig G (1g /m L ) 10 100 0.1 1 10 Ig G ( g /m L ) panel of CD137xPD-L1 bAbs was then produced by combining IgG IgG, µg/mL IgG, µg/mL different CD137 and PD-L1 Fabs based on epitope and sequence 12,00012000 (ng/mL) diversity in the IgG1 Biclonics® format. The bAbs were screened for 10,00010000 10000 activity in reporter cell lines expressing the receptors. This unbiased 5000 combinatorial screening identified a CD137xPD-L1 bAb (MCLA-145) 80008000 MCLA-145 Epitope Binding to CD137 2500 for which CD137-mediated activation is dependent on the presence 6000 Counts 6000 CHO CHO 2 counts 2 VH, heavy chain variable domain ILIL--22 counts 2 1250 - - 2 - Il of PD-L1 on a neighboring cell and, as such, the antibody acts in Il ++ IL 40004000 625 ● Binding epitopes were identified using CD137 alanine shotgun mutagenesis libraries and ‘trans’. Flow cytometry experiments demonstrated that MCLA-145 is ● The MeMo® and Biclonics® technoloies were used to generate MCLA-145 313 Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) fully cross-reactive to cynomolgus monkey CD137 and PD-L1. The – MeMo® is a transgenic mouse that generates human common light chain (cLC) antibodies 20002000 ● MCLA-145 binds the ligand binding domain, CRDII/2, of CD137 CD137 Fab arm blocks the interaction of CD137 with CD137L as – Biclonics® are bispecific human IgG1 molecules comprising 2 different cLC and heavy chain 00 demonstrated in a competition assay by flow cytometry. The PD-L1 variable region binding domains, and a Fc region that preferentially forms the bispecific PB14618p05 PB16814p01 PB14676p03 PB14585p05 PB14675p04 PB14584p05 PB16817p01 PB16841p01 PG6619p07 PG1337p218 Fab arm blocks the interaction between PD-1 and PD-L1 as CandidateCandidate PDCD137PD--L1L1 x ×x PD CD137CD137-L1 Biclonics Biclonics®Biclonics®® UrelumabUrelumab NegNegativeNeg CtrlCtrl heterodimer via charge engineering analoganalogAnalog Control Ab demonstrated in ELISA. Binding epitopes were mapped by shotgun ® IL-2, interleukin 2 CD137 Mutations Affecting MCLA-145 Binding mutagenesis using a flow-based screen. In addition, hydrogen- ● To discover MCLA-145, a large diverse library of Biclonics was generated ® deuterium exchange experiments were performed to map the – MeMo mice were immunized with protein or DNA encoding CD137 or PD-L1 binding domain on CD137. Data show that MCLA-145 binds the – High-affinity, target-specific human Fab fragments were selected via phage display technology CD137 binding reactivity (%WT) ligand binding domain of CD137 domain (CRDII). The PD-L1 Fab based on the repertoire generated in MeMo® MCLA-145–Mediated CD137 MCLA-145 Target Binding arm binds PD-L1 in the PD-1 binding N-terminal V domain. Both – Selected cLC Fabs were reformatted as Biclonics® to generate libraries Activation Depends on Characteristics Mutation MCLA-145 Control Ab epitope mapping data sets are consistent with the CD137 and PD-L1 – The CH2 region of these Biclonics® was engineered to block Fcγ receptor interaction ligand blocking activity of MCLA-145. Monovalent binding affinities Activation in ‘trans’ R66A 4.54 73.4 were measured by surface plasmon resonance (SPR) and ● The strength of MCLA-145 for CD137 and PD-L1 was assessed using radioactive iodine labeling and demonstrated affinities in the low nM radiolabeling and cell-based assays as well as surface plasmon G70A -1.7 70.6 (CD137) and subnanomolar (PD-L1) range. SPR experiments also Screening CD137 and PD-L1 Panels ● MCLA-145 induces CD137 signaling in CD137 Jurkat NF-kB/luc reporter resonance (SPR) confirmed that MCLA-145 was able to bind simultaneously to both cells in the presence of PD-L1 expressing CHO-PD-L1 cells, but not control V71A 13.2 93.0 CD137 and PD-L1 recombinant proteins. The unique binding ● Large cLC mAb panels were generated for CD137 and PD-L1, and Fab clones were selected for cells without PD-L1 (CHO-K1) Antibody Human (SPR) Cynomolgus (SPR) Human 125I on Cells properties of MCLA-145 may result in an increased therapeutic screening based on V sequence diversity F72A -2.7 74.4 H ● Positive control reference bivalent anti-CD137 urelumab analog induces window by specifically activating CD137 expressing cells in the K CD137 1.9 nM 0.6 nM 2.02 nM ● Panels were screened for affinity, cross-reactivity with cynomolgus monkey and mouse CD137 signaling regardless of PD-L1 expression D WT, wild type tumor niche where PD-L1 is expressed while simultaneously orthologues, PD-1 and CD137 ligand (CD137L) blocking, domain-binding specificity, and ability to K PD-L1 0.51 nM 0.32 nM 0.49 nM blocking inhibitory input from the PD-1/PD-L1 axis. induce CD137 receptor activation ● Bivalent monospecific mAb carrying 2 MCLA-145 CD137 Fab arms fails to D induce CD137 signaling KD, dissociation constant ● No CD137 signaling with control Abs carrying the MCLA-145 CD137 or CD137 Signaling PD-1/PD-L1 Blocking Activity ● MCLA-145 binds human PD-L1 while already in complex with CD137 as PD-L1 Fab arm combined with mock arms (CD137×Mock and Mock×PD-L1) well as CD137 while already in complex with PD-L1 Introduction 1201 2 0 Ab1 A b 1120 RU PB17311 PD-L1 CD137 Dual Binding 66 1101 1 0 Ab2 A b 2 11 ×1100 MCLA-145 Conclusions A b 3 5050 ● CD137 (4-1BB) is a transmembrane costimulatory receptor 1001 0 0 Ab3 100 CD137×CD137 Ab4 A b 4 on T and NK cells that enhances adaptive immune 909 0 A b 5 CD137×Mock ® Ab5 80 ) 4040 CD137 + PD-L1 binding ● MCLA-145 is an Fc-silenced Biclonics that engages human CD137 and PD-L1 and blocks ligand binding to both receptors A b 6 88 ×1100 55 responses and is a critical mediator of antitumor immunity 8 0 U n 80 Ab6 A b 7 Mock×PD-L1 o L ● MCLA-145 induces CD137 signaling provided PD-L1 is present in its environment i ● The development of CD137-targeted agents for cancer t 7 0 Ab7 A b 8 60 c 70 R Urelumab analog M C L A -1 4 5 ( u 3030 ● The unique binding properties of MCLA-145 may result in an increased therapeutic window by specifically activating d 6 0 Ab8 therapy has been hampered by on-target off-tumor toxicity 60 Blocking % PD-L1 + CD137 binding (RLU) n y 5 I 40 5 Negative control Ab C D 1 3 7 x C D 1 3 7 PD-L1 binding (RU) Negative t 66 ×1100 CD137-expressing cells in the tumor niche where PD-L1 is