Poster Presentation at the EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics 2024 titled, “Discovery and pre-clinical development of AKY-1189, a potent and selective Nectin-4 miniprotein binder optimized for use as a targeted radiopharmaceutical.”

EV 225Ac-AKY-1189 Vehicle 225Ac-AKY-1189 [2/uni03BCCi] Enfortumab vedotin [3mg/kg, QWx3] 0 3 6 9 12 15 18 21 24 27 30 33 36 39 Days Tumor volume (mm3) 1000 800 600 1600 140 120 400 200 0 Anti-tumor efficacy HT-1376-[L] 100 10 1 [EV], Plasma (µg/mL) 0 20 45 60 80 100 120 AUCd0-28 (g*day/mL) Cmax (µg/mL) T1/2 (days) 94 (modeled) AUCd0-7 (*day/mL) 31 23.2 1.9 Time (hr) Enfortumab-vedotin exposure (3mg/kg dose) Tolerability HT-1376-[L] 0 3 6 9 12 15 18 21 Body weight (% of initial weight) 120 100 80 60 40 20 0 Days 24 27 30 33 36 39 EV 225Ac-AKY-1189 Lorem ipsum HT-1376 -[L] Nucleus (DAPI) Actin (Phalloidin-AF488) Biotin-AKY-1 189 (Streptavidin-PE) HT-1376 -KO Figure 3. On-cell in vitro target selectivity of biotin-AKY-1189. NOTE: Specificity of biotin-AKY-1189 binding to Nectin-4 was additionally verified across 6200+ human cell surface and secreted proteins using Retrogenix Cell Microarray Technology with >450-fold selectivity over demonstrated Ki. Dose N T1/2 AUCINF CL Vss (mg/kg) (min) (min*ng/mL) (mL/min/kg) (mL/kg) 0.03 3 20.9 (1.8) 2850 (229) 10.6 (0.9) 202 (24) 0.1 6 26.4 (3.5) 15009 (1263) 6.9 (0.5) 182 (8.3) 0.3 3 26.5 (0.9) 41017 (2015) 7. 3 (0. 4) 199 (24) 62.4* 12 26.6 (17) 8.79 e 6 (6.1e 5) 7 .4 (0.4) 222 (9.9) Discovery and pre-clinical development of AKY-1189, a potent and selective Nectin-4 miniprotein binder optimized for use as a targeted radiopharmaceutical James Way1, William Blackwell1, Andrew Clay1, Marci Copeland1, Michael Doligalski1, Isaiah Gober1, Hyun Joo Kil1, Tatsiana Kosciuk1, Daša Lipovšek2, Wai Lau2, Mehran Makvandi1, Iris Paulus1, Trevor Price1, Howard Sauls1, Srividhya Subramanian2, Erin Swiger1, Mark Woodward1, Jeffrey Kovacs1, Paul Feldman1 Aktis Oncology, Discovery and Preclinical Translation, Durham NC, USA1 and Boston MA, USA2. INTRODUCTION • Miniproteins are an ideal targeting chemotype for radiopharmaceuticals due to their ability to fold into diverse three-dimensional structures, their potent and selective binding to a variety of targets, and their small size that facilitates rapid clearance and deep tumor penetration. • The “fast-in, fast-out” pharmacokinetics and selectivity of miniproteins, combined with the extremely potent but short-range energy emissions of a-particles, help minimize side effects and drive robust efficacy. • Nectin-4 is a promising target for precision radiopharmaceutical development given its restricted normal tissue expression profile in adults and its overexpression in 55% to 90% of urothelial, breast, lung, head/neck, and cervical tumors.1-4 AKY-1189 Selectively Binds Nectin-4 on Cancer Cells • Nectin-4 CRISPR/Cas9-mediated knockout (KO) in HT-1376 cells was confirmed via flow cytometry (Figure 3A). • Biotin-AKY-1189 demonstrates on-target binding to cell surface Nectin-4 in vitro (Figure 3B, C). • Biotin-AKY-1189 does not bind Nectin-4 CRISPR/Cas9 KO cells (Figure 3B, C). 225Ac-AKY-1189 Demonstrates Improved Anti-Tumor Activity Compared to Enfortumab Vedotin in a Model of Urothelial Cancer • A single administration of 225Ac-AKY-1189 drives greater anti-tumor effects than 3 administrations of enfortumab vedotin (EV) in HT-1376-[L] xenografts (Figure 9A) at well-tolerated dosing regimens (Figure 9B). • Exposures of EV were optimized to align with plasma exposures achieved in patients at the approved clinical doses of EV (Figure 9C and table inset). CONCLUSIONS • AKY-1189’s discovery exemplifies the synergy of biological (YSD) and medicinal chemistry optimization to a radiopharmaceutical clinical candidate. • AKY-1189 has been engineered for high-affinity and specificity for Nectin-4 as well as a favorable pharmacokinetic profile and reduced renal retention. • 225Ac-AKY-1189 drives robust anti-tumor responses in translationally relevant models of metastatic urothelial carcinoma after a single dose while displaying improved anti-tumor effects compared to three doses of enfortumab vedotin. • AKY-1189 labeled with either 68Ga (for PET/CT) or 177Lu (for SPECT/CT) has been evaluated in a first in human imaging assessment. For details, please attend the presentation of Abstract #10 on Friday, October 25, 2024. • 225Ac-AKY-1189 is a first-in-class Nectin-4 miniprotein-based radiopharmaceutical discovered at Aktis Oncology, which is progressing towards IND filing. REFERENCES 1. Fabre-Lafay S, Monville F , Garrido-Urbani S, et al. Nectin-4 is a new histological and serological tumor associated marker for breast cancer. BMC Cancer. 2007;7(73). 2. Takano A, Ishikawa N, Nishino R, et al. Identification of nectin-4 oncoprotein as a diagnostic and therapeutic target for lung cancer. Cancer Res. 2009;69(16):6694-703. 3. Rosenberg J, Sridhar SS, Zhang J, et al. EV-101: a phase I study of single-agent enfortumab vedotin in patients with nectin-4-positive solid tumors, including metastatic urothelial carcinoma. J Clin Oncol. 2020;28(10):1041-1049. 4. Sanders C, Lau J-F , Dietrich D, et al. Nectin-4 is widely expressed in head and neck squamous cell carcinoma. Oncotarget. 2022;13:116 6 -1173. 5. Pill J, Kraenzlin B, Jander J, et al. Fluorescein-labeled sinistrin as marker of glomerular filtration rate. Eur J Med Chem. 2005;40(10):1056-61. 6. Powles TB, Valderrama BP , Gupta S, et al. Enfortumab vedotin and pembrolizumab in untreated advanced urothelial cancer. N Engl J Med. 2024;390:875-888. 7. www.accessdata.fda.gov/drugsatfda_docs/nda/2019/761137Orig1s000MultiDiscliplineR.pdf Figure 2. Biotin-AKY-1189 binding to Nectin-4. A. SPR binding of biotin-AKY-1189 to recombinant human Nectin-4 protein. B. Dose-response curve of biotin-AKY-1189. binding to Nectin-4 endogenously expressed on HT-1376 human urothelial cancer cells. HT-1376 Low [L] Expression HT-1376 Representative [R] Expression Representative Patient Biopsy Model Treatment Median survival (Days) Significance (Gehan-Breslow-Wilcoxon test) HT-1376-[L] Vehicle 28.5 p=0.0006225Ac- AK Y-1189 49.0 HT-1376-[R] Vehicle 26.5 p=0.0017225Ac- AK Y-1189 Not Reached Biotin-AKY-1189 (log M) Fluorescence Unit (615 nm) 20000 15000 10000 5000 0 -11 -10 -9 -8 -7 -6 -5 Translational Relevance of Cell Line-Derived Xenograft (CDX) Models • HT-1376 CDX models endogenously or exogenously expressing Nectin-4 were evaluated via immunohistochemistry and categorized as Low (H-score = 175) and Representative (H-score = 291) (Figure 6A, B) given that the median H-score for patients with metastatic urothelial carcinoma is 2806 (patient biopsy shown; H-score = 286) (Figure 6C). A. B. Identification of High-Affinity Miniprotein Binders to Nectin-4 • Affinity maturation coupled with subsequent rounds of medicinal chemistry optimization and characterization (affinity, selectivity, physicochemical properties) led to the discovery of candidate molecules with improved properties. Stage KD (nM) Off Rate (sec–1) Original Hit 47 ,000 0.13 0 Affinity matured miniprotein 1 90 0.016 Affinity matured miniprotein 2 9.7 0.025 Lead1 5.8 0.020 Optimized exemplar miniprotein1,2 3.0 0.005 AK Y-11891 ,2 0.22 0.001 Table 1. Affinities of synthesized miniproteins. 1 Specificity was validated using Retrogenix Cell Microarray Technology. 2 Molecule was optimized using medicinal chemistry. 100 80 60 40 20 0 Normalized to mode Biotin-AKY-1189 / Step PE [100nM] HT-1376-[L] HT-1376- Nectin-4-KO 0 10 100 3 4 MFI [398] MFI [5063] Biotin-AKY-1189 (nM) 5000 4000 3000 2000 1000 0 0.0001 0.001 0.01 0.1 1 10 100 1000 10000 HT-1376-[L] HT-1376-KO Delta MFI (Strep-PE) (AKY-1189 MFI-Vehicle MFI) 10 10 10 10 10 100 1 2 3 4 5 10 10 10 10 10 100 1 2 3 4 5 100 80 60 40 20 0 HT-1376-[L] HT-1376-[L]-Nectin-4-KO Anti-Nec4-AF488+ 82.8 Anti-Nec4-AF488+ 4.28 Normalized to mode Anti-Nectin-4-FITC Untreated Isotype-FITC Anti-Nectin-4-FITC 100 80 60 40 20 0 Normalized to mode A. B. C. Figure 6. Immunohistochemical analysis of expression of cell surface Nectin-4. Exemplar low expression (A) and representative expression (B) in HT-1376 CDX sections. C. Representative patient biopsy. Figure 7. A. SPECT/CT imaging of 111In-AKY-807 , 111In-AKY-1162, and 111In-AK Y-1189 in HT-1376 -[R] tumor-bearing mice 22 hours after dosing. B. Quantitative assessment of kidney uptake (% injected dose per gram of tissue). NOTE: please visit Abstract #308, presented on Thursday, October 24, 2024, for allometrically scaled dosimetry values. [225Ac]Ac-AKY-1189 anti-tumor efficacy PDX-Uro-ST5420B 0 3 6 9 12 15 18 21 Tumor volume (% increase over D.-1) 1000 800 600 400 200 Vehicle 225Ac-AKY-1189 Enfortumab vedotin & # & # & # # Animal removal & - Vehicle # - EV [3mg/kg QWx3] 111In-AKY-807 111In-AKY-1162 111In-AKY-1189 Kidney uptake comparison %ID/g (Kidney) 60 50 40 30 20 10 0 0 3 6 9 12 15 18 21 24 Hours 111In-AKY-807 111In-AKY-1162 111In-AKY-1189 A. B. B. C. 120 100 80 60 40 20 0 HT-1376-[L] - Vehicle HT-1376-[L] - 225Ac-AKY-1189 [1/uni03BCCi] HT-1376-[R] - Vehicle HT-1376-[R] - 225Ac-AKY-1189 [1/uni03BCCi] 100 75 50 25 0 100 20 30 Days Survival proportionsTumor volumes 40 50 60 % Survival 2000 1500 1000 500 0 50 10 15 Days 20 25 30 Tumor volume (mm3) Body weights 50 10 15 Days 20 25 30 % Body weight (normalized to D-1) Treatment Figure 8. Preclinical efficacy of 225Ac-AKY-1189 in mouse models of metastatic urothelial carcinoma. Tumor volumes (A) and body weights (B) measured bi-weekly. C. Survival proportions over 55 days. Animals were treated with a single administration of 225Ac-AKY-1189 at 1μCi of activity or vehicle. B.A. C. Figure 9. Animals received 225Ac-AKY-1189 [2μCi] at Day 0 as a single bolus intravenous (IV) administration or enfortumab vedotin (EV) as an IV injection (3 mg/kg, QWx3) . Tumor volumes (A) and body weights (B) were measured bi-weekly for 4 weeks. C. Plasma exposures of EV were measured in mice via LC/MS/MS and parameters calculated; clinical exposure (AUCd0-28) of the approved dose of EV is 111 ± 38 μg*day/mL7. + AKY-1189AK Y-1189 Miniprotein Yeast cells are transformed with plasmids encoding individual miniproteins that are expressed on the cell surface. Yeast surface display allows parallel screening for binding of tumor targets to up to 108 unique miniproteins. Solid-phase synthesis of affinity matured miniproteins accelerates lead optimization and enables site-specific modifications. 0 20 40 60 80 100 120 1000 100 10 1 1000 100 10 1 In-AKY-1189, 0.3 mg/kg (n=3) In-AKY-1189, 0.1 mg/kg (n=6) In-AKY-1189, 0.03 mg/kg (n=3) FITC-sinistrin, 62.4 mg/kg (n=12) [In-AKY-1189], rat plasma (ng/mL) [FITC-sinistrin], rat plasma (µg/mL) Time (min) DISCOVERY Figure 1. Yeast-surface-display (YSD) platform and solid phase miniprotein synthesis supports lead optimization. • Biotin-AKY-1189 binds recombinant human Nectin-4 with an observed KD of 0.22 nM when assessed by surface plasmon resonance (SPR) (Figure 2A). • On-cell binding of biotin-AKY-1189 to Nectin-4 was demonstrated on HT-1376 human urothelial carcinoma cells with an observed Ki of 0.82 nM as assessed by a competitive binding assay (DELFIA) (Figure 2B). Figure 4. Immunofluorescent images of biotin-AKY-1189 imaged with a Streptavidin-PE conjugate in HT-1376-[L] wild type (A) and Nectin-4 KO (B) cells. AKY-1189 Is Rapidly Internalized After Binding to Cell Surface Nectin-4 • AKY-1189 potently and selectively binds endogenous Nectin-4 on the surface of HT-1376 cells and is rapidly internalized after 30 minutes (Figure 4A, white arrows) but is not bound or internalized in Nectin-4 KO cells (Figure 4B). CHARACTERIZATION IN VIVO BIODISTRIBUTION AND EFFICACY Figure 5. Pharmacokinetic analysis of In-AKY-1189 in Sprague-Dawley rats after single-dose intravenous administration. FITC-sinistrin was co-formulated with In-AKY-1189 as a control molecule that is cleared at GFR.5 AKY-1189 Is Rapidly Cleared from the Plasma In Vivo • Pharmacokinetic (PK) profile of In-AKY-1189 demonstrates “fast-in, fast- out” kinetics with near dose proportionality and plasma clearance at glomerular filtration rate (GFR) (Figure 5) in rats after a single dose. Response (Relative units, RU) 16 11 6 1 -4 -100 -50 0 50 100 Time(s) -150 200 250 300 350 AKY-1189 Binds Nectin-4 With High Affinity In Vitro and On Cells Table 3. Plasma pharmacokinetic profile of In-AKY-1189 (top three rows). Noncompartmental analysis reported values ± standard error. *Bottom row shows analysis for co-administered FITC-sinistrin. Table 4. Survival benefit analysis for 225Ac-AKY-1189-treated animals. 225Ac-AKY-1189 Demonstrates Target-Dependent Efficacy and Survival Benefit After a Single Administration • 225Ac-AKY-1189 drives robust anti-tumor effects in vivo after a single administration of drug in both the HT-1376-[L] and HT-1376-[R] models (Figure 8A) with no impacts on animal weights (Figure 8B), suggesting a well-tolerated therapeutic dose. • A prolonged survival benefit was observed (Figure 8C, Table 4) in both models, suggesting a spectrum of Nectin-4-expressing patients who may benefit. Miniprotein Sequence Modifications Improve Biodistribution Profiles • 111I n - A K Y- 8 07, 111In-AKY-1162, and 111In-AKY-1189 sequences were designed for and exhibit robust tumor uptake with improved renal clearance (Figure 7). A. H-score: 175 H-score: 291 H-score: 286 B.A. C. ACKNOWLEDGEMENTS We thank Ved Srivastava (Perpetual Medicines) and Chris Bahl and James Bowman (AI Proteins and formerly from Institute for Protein Innovation) for their contributions to this work. B.A.

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