The Promise of DARPins for Site-Specific Drug Conjugation & Pharmacokinetic Optimization

Department of Biochemistry The Promise of DARPins for Site-Specific Drug Conjugation & Pharmacokinetic Optimization Fabian Brandl, Plückthun Group, University of Zurich / University of Bern World ADC Berlin, 8 th -10 th February 2016

1. Non-IgG Binding Proteins Monoclonal Antibodies Alternative non-igg Binding Proteins Specific binding proteins Well validated Various approved human therapeutics Very successful BUT a range of restrictions Large in Size Complex multidomain structure Stability / aggregation propensity High production costs Homogeneous and site-specifically conjugated ADCs difficult to obtain Very challenging Specific binding proteins Antibody-like non-ig class proteins >50 different scaffolds proposed Numerous clinical studies ongoing Favorable properties High stability Low aggregation propensity Simple structure Low production costs (E. coli) Homogeneous and site-specific conjugates easy to obtain High freedom of engineering Facile incorporation of functional groups Compatible with non-natural amino acids Merten, H., et al. (2015). Bioconjug Chem 26(11):2176-85 Plückthun, A. (2015). Annu Rev Pharmacol Toxicol 55:489-511 February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 2

2. DARPins Next Generation non-igg Binding proteins Designed Ankyrin Repeat Proteins - DARPins (one continuous protein) Very robust non-igg protein scaffold High affinity - Efficient selection of binders by Phage and Ribosome Display Immunogenicity Systemic administration found to be safe in clinical trials Methionine and Cysteine-free unique engineering possibilities for site-specific conjugation Binz et al., J Mol Bio, 2003; Binz et al., Nat Biotechnol, 2004 February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 3

3. Facile and Defined Bioconjugation Site-Specific Conjugation Chemistries Maleimide-Thiol Coupling Introduced codon: cysteine ph 6.5 7.5 Very fast Click Chemistry Introduced codon: methionine Methionine Azidohomoalanine (Aha) Physiological ph slow Rate Optimized DBCO (100x faster) Aha easily incorporated by E. coli during expression Kiick, K. L., et al. (2002). Proc Natl Acad Sci USA 99(1): 19-24; Lim, R. K. and Q. Lin (2010). Chem Commun (Camb) 46(10): 1589-1600. February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 4

3. Facile and Defined Bioconjugation Click Chemistry Any position Thiol Chemistry Dye DARPin Polymer Half-life module Toxin Any position Engineering flexibility allows generation of bifunctional binders with defined stoichiometry Bifunctional binders for various applications Simon, M., et al. (2012). Bioconjug Chem 23(2): 279-286 February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 5

Intermediate Results DARPins are promising candidates for biomedical applications High stability, aggregation and protease resistance, ease of production, etc. Very versatile scaffold with high freedom of engineering Site-Specific and stoichiometrically defined conjugation Aim Translate advantageous properties of DARPins to therapeutic proteins Exploit site-specific conjugation for tumor targeting Drug conjugation Pharmacokinetic optimization February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 6

An Attractive Tumor Target - Epithelial Cell Adhesion Molecule (EpCAM) Tumor-associated antigen K D = 68 pm Low Levels healthy epithelia Basolateral cell surface High Levels epithelial tumors Homogeneously distributed Antibody binding has not demonstrated significant anti-tumor effects Several ADCs and Fusion-Toxins under preclinical and clinical investigation Efficient internalization on ligand binding Well suited for intracellular drug delivery Maetzel, D., et al. (2009). Nat Cell Biol 11, 162-171 Simon, M. et al. (2013). Expert Opin Drug Deliv 10(4):451-468 February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 7

Toxins add an effector function to EpCAM-targeting DARPins Monomethyl Auristatin F - MMAF Derivative of dolasatin-10 Synthetic antimitotic pentapeptide Microtubule destabilizing Highly potent small molecule drug IC 50 : 2.3 6.3 nm on HT29 and SKBR3 Dolabella Auricularia Wedge Sea Hare Targeted MMAF is 2-3 orders of magnitude more potent February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 8

Orthogonal Assembly of a DARPin-Auristatin Bioconjugate Click Chemistry Maleimide-Thiol Chemistry Cys34 DARPin: Targeting Auristatin (MMAF): Cytotoxicity Serum Albumin: Serum Half-Life Extension Serum Albumin DBCO-PEG4-Maleimide February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 9

In vitro Performance of DARPin-Auristatin Bioconjugates for Tumor Targeting Binding Kinetics (SPR) Cytotoxicity against Various Tumor Cell Lines (XTT) Simon, M., et al. (2013). Bioconjug Chem 24(11): 1955-1966 February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 10

DARPin-Auristatin Bioconjugates for Tumor Targeting In vivo half-life In vivo efficacy AzEc1-MMAF SA-Ec1-MMAF HT29 tumor 150 mg/kg 0,2,5,8 day DARPin: 10.6 min SA-DARPin: 17.4 h Simon, M., et al. (2013). Bioconjug Chem 24(11): 1955-1966 February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 11

Pseudomonas aeruginosa exotoxin A (ETA, PE) A highly potent protein toxin NH 2 DARPin ΔIa Ia II Ib III REDLK KDEL ADPribosyltransferase Targeting EpCAM- Targeting Translocation ER localization Adapted from: Weldon, J. E. and I. Pastan (2011). FEBS J 278(23): 4683-4700. ADP-ribosylation of the eukaryotic elongation factor 2 (eef2) Shutdown of protein synthesis Complex domain structure Targeting domain can be substituted to other targeting domains Highly potent on various cell lines (IC 50 = 5 pm) 1000x more potent than MMAF Immunogenic potential and high target-independent toxicity which limits the therapeutic window Deletion of B- and T-cell epitopes Conjugation with PEG veiling the active site February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 12

DARPin-Fusion Toxins A Very Potent Anti-Tumor Fusion Protein In vivo half-life DARPin ETA In vitro potency (IC 50 ) t ½ = 11.2 min Cell Lines Ec4-ETA Off7-ETA EpCAM + 5 fm 0.7 pm >1 nm GS-Linker EpCAM - >10 nm NA Highly potent on various EpCAM + + - Maintain potency In vivo efficacy Pronounced anti-tumor effect with complete regression Short half-life of of 11.2 min Increase half-life Dose-limiting toxicity due to to off-target toxicity Reduce unspecific toxicity Martin-Killias, P., et al. (2011). Clin Cancer Res 17(1): 100-110 February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 13

A Protease-Sensitive Prodrug-Like Fusion Toxin Site specific conjugation of half-life extension / veiling module Block of active site or intracellular transport of toxin Tumor protease cleavable linker Prodrug DARPin ETA DARPin ETA Reactivation in the tumor environment What to conjugate? Where to conjugate? How to conjugate? Maintain potency Decrease unspecific toxicity Increase half-life Reduce immunogenicity Insure proper reactivation February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 14

A Protease-Sensitive Prodrug-Like Fusion Toxin 3C protease DARPin-ETA" Aha486/AhaKDEL -3C-PEG 20kDa Click kda Di-PEGylated Aha486 Click 170 130 100 70 55 3C Protease digest reactivation Free Fusion Toxin PEG 20 kda 40 35 Methionine-free fusion protein Aha incorporation without loss of potency Site-specific PEG-conjugation veiling of the fusion toxin Reversible toxin inhibition Half-life extension Stefan, N., et al. (2014). Bioconjug Chem 25(12): 2144-2156 25 February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 15

V ia b ility (% o f u n tre a te d ) 4. Site-Specific Conjugation and Pharmacokinetic Optimization A Protease-Sensitive Prodrug-Like Fusion Toxin In vitro comparison of di-pegylated (inactivated), 3C digested (reactivated) and non-pegylated (active) fusion toxin DARPin-ETA" E486Aha+AhaKDEL -3C-PEG 20kDa Therapeutic Window Targeted 1 0 0 5 0 +PEG -PEG +PEG -PEG Non-targeted 0 1 0-1 5 1 0-1 4 1 0-1 3 1 0-1 2 1 0-1 1 1 0-1 0 1 0-9 1 0-8 1 0-7 1 0-6 C o n c e n tra tio n (M ) Toxicity Pronounced prodrug effect with model protease 3C 1000-fold toxin inhibition Highly widened therapeutic window 6 orders of magnitude difference btw. specific and unspecific toxicity February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 16

A Protease-Sensitive Prodrug-Like Fusion Toxin Half-life increased from 7 to 80 min Active toxin Deactivated toxin Improved tolerability in vivo with increased AUC Stefan, N., et al. (2014). Bioconjug Chem 25(12): 2144-2156 February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 17

5. Conclusions DARPins Promising non-igg binding proteins Stable and aggregation-resistant High yielding production in E. coli High freedom of engineering Facile incorporation of functional groups for site-specific and defined conjugation Site-specific and stiochiometrically defined bioconjugation allows for: Payloading of EpCAM-targeted DARPins with small molecule drugs Pharmacokinetic optimization of DARPin-Auristatin conjugates Generation of Prodrug-Like DARPin-ETA Fusion Toxins 10x improved half-life and highly improved tolerability of DARPin-ETA-PEG2 fusion toxins February 9, 2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 18

Thank you very much for your attention! Fabian Brandl PhD student Plückthun Lab UZH f.brandl@bioc.uzh.ch The Plückies Prof. Andreas Plückthun Prof. Uwe Zangemeister-Wittke 2/15/2016 Fabian Brandl - University of Zurich - World ADC Berlin 2016 19

Department of Biochemistry The Promise of DARPins for Site-Specific Drug Conjugation & Pharmacokinetic Optimization Fabian Brandl, Plückthun Group, University of Zurich / University of Bern World ADC Berlin, 8 th -10 th February 2016