EphA2/CD137 Bicycle® tumor-targeted immune cell agonists (TICAs™) induce tumor ABSTRACT# 700 regressions, immunogenic memory, and reprogramming of the tumor immune microenvironment Kristen Hurov, Johanna Lahdenranta, Gemma Mudd, Punit Upadhyaya, Elizabeth Repash, Jun Ma, Julia Kristensson, Marianna Kleyman, Jessica Kublin, Liuhong Chen, Eric Haines, Sailaja Battula, Kevin McDonnell, Philip E. Brandish, and Nicholas Keen

ABSTRACT RESULTS RESULTS

• CD137/4-1BB is a member of the TNF receptor superfamily involved in the stimulation of several Mouse CD8 IHC immune cell types, including T and NK cells. CD137 reporter/tumor cell A. A. B. Primary PBMC/A549 co-culture • Despite compelling preclinical data, agonistic anti-CD137 antibodies have been hampered by failure to co-culture assay delineate hepatoxicity from efficacy in clinical studies [1,2]. assay • A new generation of both systemic and targeted CD137 agonists that are now entering clinical BCY12491 BCY12491 120 PC-3 (#56000) Cell line 16000 600 development rely on biologic agents with suboptimal properties for CD137 agonism due to their A549 (#23000) (EphA2 molecules/cell) BCY12759 NB BCY12759 NB

relatively large sizes and long circulating half-lives [3-5]. These properties may limit their tissue 100

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• BCY12491 is a tumor-targeted immune cell agonist (TICA™) that exemplifies a new class of fully n

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o I and a co-stimulatory molecule. We developed this new class of synthetic molecules with antibody-like F 4000 20 affinities and target selectivity to circumvent the before mentioned barriers to optimal targeted CD137 agonistic therapeutics. 0 0 0 • BCY12491, an EphA2/CD137 TICA, is designed to deliver a highly potent CD137 agonist to EphA2 -11 -10 -9 -8 -12 -11 -10 -9 -8 -7 -6 -12 -11 -10 -9 -8 -7 -6 Vehicle BCY12491 BCY13626 aCD137 overexpressing tumors, including pancreatic, gastric, and colorectal, among others. Log concentration (M) Log Concentration (M) Log Concentration (M) Specific gene set (left) and gene • BCY12491 is a potent EphA2-dependent CD137 agonist with optimal binding, pharmacologic, and Figure 3: BCY12491 led to potent EphA2-dependent activity in CD137 reporter and primary immune cell co- B. Gene expression changes in tumor (NanoString) C. (right) expression changes in tumor pharmacokinetic properties that enable anti-tumor TME remodeling and complete responses in vivo culture assays. A) NF-ĸB-Luc2/CD137 Jurkat reporter cells were co-cultured with EphA2 expressing cancer cells and the (NanoString) without the need for continuous exposure. downstream CD137 mediated NF-κB activation was measured by luminescence after treatment with BCY12491. Fold • This work unleashes a new and tractable avenue to testing a novel class of therapeutic CD137 agonists induction by BCY12491 is dependent on the levels of EphA2 expression in the co-culture cell line (mean ± SD). B) T Cell Score Pdcd1 (PD1) in humans for the treatment of cancer. BCY12491 promoted cytokine secretion in PBMC / cancer cell co-culture experiments. PBMCs from healthy donors were 1000 *** * *** * co-cultured with EphA2 expressing A549 cells in the presence of anti-CD3 and test molecules. Supernatants were analyzed 800 1000 INTRODUCTION for cytokines by Luminex, figures are representative data using PBMCs from one donor (from a total of n=5). The non- 600 750 binder (NB) control showed minimal activity (mean ± SD). 400 500 250 Simulated multi-dose plasma 200 0 0 Loop 1 Loop 2 1:1 Efficacy PK profile of BCY12491 MC38 re-challenge

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A. B. following BIW dosing in mice C. - CD137 Lead 1:2 800 Naive CTR mice *** * *** *** Bicycle 1:2 1000 3000 + ) BIW Dosing Vehicle 10000 3 ) BCY12491 CRs 750

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Phage screening identified CD137 binders with nM potency. The lead peptide was chemically optimized to 1 o 200

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T 3000 attachment points, affinities, physicochemical properties. TICA’s of varying valency (1:1, 1:2 and 1:3) 0 0 0.01 2000 were constructed using different linkers. TICA’s were optimized to obtain the desired PK and 100 0 7 14 21 28 0 1 2 3 4 5 6 7 8 0 7 14 21 28 1000 pharmaceutical properties. Day Day 0 0 Time after re-challenge (day) 1 6 1 6 e 9 2 7 e 9 2 7 l 4 6 3 l 4 6 3 ic 2 3 1 ic 2 3 1 h 1 1 D h 1 1 D Efficacy Survival e Y Y C e Y Y C D. E. V C C V C C CD B B B B Activated *p<0.05,**p<0.01, ***p<0.001 one-way ANOVA with Dunnett's T cell BCY12491 100 TM 3 *** 2500 Dosing (BIW)

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CRD4 , 2000 microenvironment. MC38 tumor bearing mice (huCD137-C57Gl/6) were treated with 15 mg/kg q3d i.v. of BCY12491 or

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CD137L T BCY12491 scores; blue indicates low pathway scores. Scores are displayed on the same scale via a Z-transformation. C) Increase in T cell 0 Dosing (BIW) score, cytotoxic cell score, macrophage cell score (left column of plots), and immune checkpoint gene expression (right column Antigen 0 presenting of plots) in tumor tissue upon BCY12491 treatment. cell 0 7 14 21 28 0 7 14 21 28 *p<0.05,**p<0.01,***p<0.001, Day after treatment initiation Mantel-Cox Log-rank test Day afer treatment initiation CONCLUSIONS Figure 4: Intermittent dosing of BCY12491 led to significant anti-tumor activity and immunogenic memory in the syngeneic MC38 mouse model. CD137 Bicycle is human-specific thus humanized CD137 (huCD137) C57BL/6 mice • BCY12491 is an EphA2/CD137 Bicycle TICA that exhibits highly potent, EphA2-dependent activity in in vitro primary immune were used. BCY12491 (5 mg/kg) was administered BIW over 17 days. A) BCY12491 treatment led to anti-tumor activity cell assays (n=6 per group) whereas the non-binding control BCY13626 was inactive. B) PK parameters obtained from single IV • BCY12491 causes tumor regression, complete responses, immunogenic memory, and significant modulation of the tumor Figure 2: The concept of a Bicycle tumor targeted immune bolus dose of BCY12491 were used to simulate plasma concentrations following repeat doses of 5 mg/kg IV BIW. The TM immune microenvironment in preclinical syngeneic mouse models without continuous drug exposure in the periphery cell agonist (TICA ). CD137 is a member of the TNFR EC50 for target coverage was based on the mean EC50 for INFγ secretion (determined in vitro from 2 donors using the superfamily and requires trimerization for its activation. CD137 is PBMC/MC38 co-culture assay). Between doses, trough plasma concentrations of BCY12491 were below the corresponding • Bicycle Therapeutics is advancing an EphA2/CD137 TICA clinical candidate depicted here bound by its ligand, CD137L. An alternative in vitro EC50 for substantial periods, demonstrating intermittent plasma exposure of BCY12491 produces robust anti- approach to achieve CD137 clustering: linking a CD137 Bicycle to References: [1] Segal et al, Clin Cancer Res 23(8): 1929-36 (2017); [2] Chester et al, Blood 131(1): 49-57 (2018); tumor activity. C) Unlike in matched naïve control (CTR) mice (100% tumor growth), no tumor growth was observed in [3] Hinner et al, Clin Cancer Res 25(19): 5878-89 (2019); [4] Claus et al, Sci Transl Med 11(496): a Bicycle targeting a highly expressed tumor antigen, i.e. EphA2. complete responder mice implying a development of immunogenic memory. D) BCY12491-driven anti-tumor activity is EphA2 IHC, indicating high expression in eaav5989 (2019); [5] Eskiocak et al, JCI Insight 5(5): e133647 Binding of these molecules would result in a multivalent array of dependent on CD8+ T cells, but not NK cells. MC38 tumor-bearing mice depleted of CD8+ cells and/or NK cells or treated pancreatic cancer CD137 engaging Bicycles, enabling the clustering of the CD137 with vehicle or isotype-control antibodies received 4 doses of 15 mg/kg BCY12491 or vehicle BIW. E) Survival data receptors in a tumor antigen-dependent manner. corresponding to panel (A). (Tumor volumes are mean ± SD) Bicycle Therapeutics 4 Hartwell Place, Lexington, MA 02421 Questions? Contact [email protected] www.bicycletherapeutics.com