Molecular basis of Valine-Citrulline-PABC linker instability in site-specific ADCs & its mitigation by linker design
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1 Molecular basis of Valine-Citrulline-PABC linker instability in site-specific ADCs & its mitigation by linker design Magdalena Dorywalska, Ph.D. Rinat, ORD, Pfizer World ADC San Diego 2016
2 ADCs require internalization & processing in target cells Cell death Target tumor specific highly expressed fast internalization Antibody high specificity high affinity pharmacokinetics Toxin potent inhibitor of cell growth permeable or non-permeable AcLys-VC-PABC -Aur0101 Linker extracellular stability intracellular processing cleavable or non-cleavable
3 ADCs require internalization & processing in target cells Cleavable linkers Non-cleavable linkers Cell death Amino-PEG6-C2 -MMAD
4 ADCs require internalization & processing in target cells Cleavable linkers Non-cleavable linkers Cell death Release of toxin prior to ADC internalization could result in: loss of potency due to DAR toxicity towards non-target cells competition by naked mab (bad news if long half life)
5 Site-specific conjugation offers important advantages Random conjugation + drug conventional through Cys or Lys + drug Site-specific conjugation control of stoichiometry control of positions Advantages of site specificity homogenous & reproducible allows to analyze & optimize ADC properties potency toxicity stability kinetics distribution
6 Site-specific conjugation using Transglutaminase Transglutaminase catalyzed conjugation Transglutaminase (Streptoverticillium mobaraense, 38 kda) single step enzymatic reaction does not target endogenous glutamines specific towards glutamine tag LLQG + mtg O N H Kashiwagi et al specific acyl donor nonspecific acyl acceptor conjugate glutamine tag payload Efficient coupling to 12 sites: 1 Fv 3 Ck 3 CH1 1 hinge 4 Fc
7 Various processes contribute to premature loss of toxin unstable coupling chemistry (maleimide) VC-PABC Alley et al. Bioconj Chem 2008 Shen et al. Nat Biotech 2012 cleavage of VC-PABC linker in circulation VC-PABC degradation of payload in circulation Strop et al. Chem Biol 2013 Dorywalska et al. Bioconj Chem 2015 Dorywalska et al. Mol Cancer Ther 2016 PEG6 Dorywalska et al. PLoS One 2015
8 Payload loss observed both in vitro & in vivo Cleavable linkers Non-cleavable linkers = loss of dolaphenine
9 ADC metabolism in mouse plasma & circulation Extracellular metabolism of cleavable & non-cleavable ADCs both processes are position-dependent both processes are observed in vitro & in vivo in both cases cleavage is more efficient in vivo Stability of cleavable ADCs using C6-vc-Aur Stability of non-cleavable ADCs using Amino-PEG6-C2-MMAD % stability % stability A B C D E F G H I 0 A B C D E F G H I
10 ADC metabolism in mouse plasma & circulation Extracellular metabolism of cleavable & non-cleavable ADCs both processes are position-dependent both processes are observed in vitro & in vivo in both cases cleavage is more efficient in vivo cleavage impacts efficacy in vitro & in vivo Stability of cleavable ADCs affects efficacy in vivo Stability of non-cleavable ADCs affects efficacy in vivo
11 Extracellular ADC cleavage is enzyme-catalyzed Inhibition of C6-VC-PABC-Aur0101 cleavage in mouse plasma Inhibitor Inhibitor specificity C6-VC-PABC- Aur0101 stability in mouse plasma (%) preheat plasma at 65 C for 1 h - 99 E64, 30 um Cys proteases, trypsin 3 Leupeptin, 100 um Cys & Ser proteases 4 Antipain, 100 um Cys & Ser proteases 4 Chymostatin, 100 um Cys & Ser proteases 6 N-ethylmaleimide, 1 mm Cys proteases 17 Aprotinin, 5 um Ser proteases 3 Pefabloc, 1 mm Ser proteases 96 v Benzamidine, 4 mm Ser proteases 6 3,4-Dichloroisocoumarin, 1 mm Ser proteases 28 Pepstatin A, 1 um Asp proteases 4 Bestatin, 135 um Metalloproteases 3 EDTA, 5 mm Metalloproteases 5 Carboxypeptidase inhibitor, 10 um Carboxypeptidases 4 BNPP, 100 um Ser esterases 96 α2-macroglobulin, 0.3 um Broad spectrum 5 Inhibition of Amino-PEG6-C2-MMAD cleavage in mouse plasma Inhibitor Inhibitor specificity PEG6-C2-MMAD cleavage in mouse plasma - - yes preheat plasma at 65 C for 1 h - no E64, 30 um Cys proteases, trypsin yes Leupeptin, 100 um Cys & Ser proteases yes Antipain, 100 um Cys & Ser proteases yes Chymostatin, 100 um Cys & Ser proteases yes N-ethylmaleimide, 1 mm Cys proteases partial Aprotinin, 5 um Ser proteases yes Pefabloc, 1 mm Ser proteases no v Benzamidine, 4 mm Ser proteases yes 3,4-Dichloroisocoumarin, 1 mm Ser proteases partial Pepstatin A, 1 um Asp proteases yes Bestatin, 135 um Metalloproteases yes EDTA, 5 mm Metalloproteases yes Carboxypeptidase inhibitor, 10 um Carboxypeptidases yes BNPP, 100 um Ser esterases no
12 Extracellular VC-PABC cleavage is not catalyzed by Cathepsin B Lysosomal Cathepsin B and mouse plasma hydrolase: show different ph dependence Mouse Cathepsin B stability (%) show different inhibitor sensitivity Human Cathepsin B stability (%) Mouse plasma stability (%) Human plasma stability (%) ph Inhibitor Mouse Cathepsin B stability (%) Human Cathepsin B stability (%) Mouse plasma stability (%) Human plasma stability (%) ph Inhibitor E64, 10 um Leupeptin, 10 um Pefabloc, 1 mm show different activity among species cleavage of VC-PABC in lysosomes & in plasma is by different enzymes
13 Mouse serum fractionation to identify VC-PABC hydrolase mouse serum > 4000 proteins total protein ~ 70 mg/ml albumin ~ 40 mg/ml globulins ~ 25 mg/ml fibrinogen ~ 4 mg/ml other < 1 mg/ml IgG depletion albumin depletion affinity removal of most abundant contaminants Top3 column cation exchange HiTrap SP NaOAc ph 5.2 elution with NaCl size exclusion Superdex 200 Tris HCl ph 8.5 (NH 4 ) 2 SO 4 fractionation 2 M remove ppt 3 M collect ppt anion exchange HiTrap Q Tris HCl ph 8.5 elution with NaCl 2.E+06 serum fractionation & Aur0101 release assay 2.E+06 1.E+06 1.E+06 1.E+06 8.E+05 free Aur0101 release assayed by LC/MS inhibition assays & proteomics 6.E+05 4.E+05 2.E+05 0.E+00 S200 fxn A9 S200 fxn A10 S200 fxn A11 S200 fxn A12 S200 fxn B12 S200 fxn B11 S200 fxn B10 S200 fxn B9 S200 fxn B8 S200 fxn B7 S200 fxn B6 S200 fxn B5 S200 fxn B4 S200 fxn B3 S200 fxn B2 S200 fxn B1 S200 fxn C1 S200 fxn C2 S200 fxn C3 S200 fxn C4 S200 fxn C5 S200 fxn C6 S200 fxn C7 S200 fxn C8 S200 fxn C9 S200 fxn C10 S200 fxn C11 S200 fxn C12 S200 fxn D12
14 Proteomics analysis identifies Carboxylesterases 1C & 1G Intensity Intensity -186 Da Carboxylesterases large family of hydrolases cleave ester & amide bonds expressed in many tissues involved in lipid metabolism expression can vary Intensity -875 Da -873 Da Intensity
15 Ces1C knockout mice show no VC-PABC cleavage
16 Linker chemistry affects VC-PABC cleavage across many sites C6-VC-PABC-Aur0101 AcLys-VC-PABC-Aur0101 % stability Stability in mouse plasma A B C D E F G H I C6 AcLys
17 Linker chemistry affects VC-PABC cleavage across many sites Linker 1 R group R group VC-PABC-Aur0101 Linker 2 Linker 3 Site Payload Mouse plasma stability (%) Rat plasma stability (%) Cyno plasma stability (%) A Linker 1-VC-PABC-Aur A Linker 2-VC-PABC-Aur A Linker 4-VC-PABC-Aur A Linker 5-VC-PABC-Aur0101 (C6) A Linker 6-VC-PABC-Aur A Linker 7-VC-PABC-Aur A Linker 8-VC-PABC-Aur A Linker 9-VC-PABC-Aur A Linker 10-VC-PABC-Aur F Linker 1-VC-PABC-Aur F Linker 2-VC-PABC-Aur F Linker 3-VC-PABC-Aur F Linker 4-VC-PABC-Aur F Linker 5-VC-PABC-Aur0101 (C6) F Linker 6-VC-PABC-Aur F Linker 7-VC-PABC-Aur F Linker 8-VC-PABC-Aur F Linker 9-VC-PABC-Aur Linker 4 Linker 5 Linker 6 Linker 7 Linker 8 Linker 9 Linker 10 = C6 = AcLys
18 Linker chemistry doesn t affect lysosomal VC-PABC processing Linker 1 R group R group VC-PABC-Aur0101 Linker 2 Linker 3 In vitro cytotoxicity BxPC3 cells (M1S1+++) In vitro cytotoxicity SW620 cells (M1S1-) Linker 4 % RLU of control ADC conc [nm] Site F-Linker 1-VC-PABC-Aur0101 (IC nm) Site F-Linker 2-VC-PABC-Aur0101 (IC nm) Site F-Linker 3-VC-PABC-Aur0101 (IC nm) Site F-Linker 4-VC-PABC-Aur0101 (IC nm) Site F-Linker 5-VC-PABC-Aur0101 (IC nm) Site F-Linker 6-VC-PABC-Aur0101 (IC nm) Site F-Linker 7-VC-PABC-Aur0101 (IC nm) Site F-Linker 8-VC-PABC-Aur0101 (IC nm) Site F-Linker 9-VC-PABC-Aur0101 (IC nm) NCC Site F-Linker 5-VC-PABC-Aur0101 % RLU of control ADC conc [nm] Site F-Linker 1-VC-PABC-Aur0101 Site F-Linker 2-VC-PABC-Aur0101 Site F-Linker 3-VC-PABC-Aur0101 Site F-Linker 4-VC-PABC-Aur0101 Site F-Linker 5-VC-PABC-Aur0101 Site F-Linker 6-VC-PABC-Aur0101 Site F-Linker 7-VC-PABC-Aur0101 Site F-Linker 8-VC-PABC-Aur0101 Site F-Linker 9-VC-PABC-Aur0101 NCC Site F-Linker 5-VC-PABC-Aur0101 Linker 5 Linker 6 Linker 7 Linker 8 Linker 9 Linker 10 = C6 = AcLys
19 Intracellular CatB & extracellular Ces1C have different substrate specificities Site A VC-PABC linker intact (%) VC-PABC linker intact (%) Linker 1 Linker 5 Linker 10 Linker cleavage in mouse plasma Linker cleavage by Ces1C Linker cleavage by CatB Site F VC-PABC linker intact (%) time (h) VC-PABC linker intact (%) time (h) VC-PABC linker intact (%) time (h) Linker 1 Linker 3 Linker 5 Linker 7 Linker 10 Site F-Linker 1-VC-PABC-Aur0101 Site F-Linker 3-VC-PABC-Aur0101 Site F-Linker 5-VC-PABC-Aur0101 Site F-Linker 7-VC-PABC-Aur0101 Site F-Linker 10-VC-PABC-Aur0101 Site F-Linker 1-VC-PABC-Aur0101 Site F-Linker 3-VC-PABC-Aur0101 Site F-Linker 5-VC-PABC-Aur0101 Site F-Linker 7-VC-PABC-Aur0101 Site F-Linker 10-VC-PABC-Aur0101 Site F-Linker 1-VC-PABC-Aur0101 Site F-Linker 3-VC-PABC-Aur0101 Site F-Linker 5-VC-PABC-Aur0101 Site F-Linker 7-VC-PABC-Aur0101 Site F-Linker 10-VC-PABC-Aur0101
20 Structural differences contribute to substrate selectivity of Ces1C & CatB Human Ces1 Bencharit et al Human CatB Musil et al. 1991
21 VC-PABC linker stability affects ADC efficacy in vivo Linker stability PK in mouse conc (ng/ml) Linker 1 Linker time (h) Linker 3 Linker 4 Linker 5 Linker 6 conc (ng/ml) Linker 7 Linker 8 Linker 9 Site F-Linker 3-VC-PABC-Aur0101 (mab) Site F-Linker 3-VC-PABC-Aur0101 (ADC) Site F-Linker 4-VC-PABC-Aur0101 (mab) Site F-Linker 4-VC-PABC-Aur0101 (ADC) Site F-Linker 5-VC-PABC-Aur0101 (mab) Site F-Linker 5-VC-PABC-Aur0101 (ADC) Site F-Linker 6-VC-PABC-Aur0101 (mab) Site F-Linker 6-VC-PABC-Aur0101 (ADC) In vivo efficacy in BxPC3 model Stability in mouse plasma 800 %stability Linker 5 Linker 7 0 Site D Linker 5 Site D Linker 7 VC-PABC-MMAD VC-PABC-Aur Days post tumor implantation NCC Site D-Linker 5-VC-PABC-MMAD, 3 mg/kg C16 Site D-Linker 5-VC-PABC-MMAD, 3 mg/kg C16 Site D-Linker 7-VC-PABC-MMAD, 3 mg/kg
22 Ces1C cleaves VC-PABC linkers in maleimide-linked ADCs Plasma incubation results in maleimide decoupling in the presence of thiols i.e. albumin
23 Ces1C cleaves VC-PABC linkers in maleimide-linked ADCs Incubate in the absence of plasma mab Ces1C mab -875 Da -871 Da
24 Some lessons learned Site-specific conjugation allows analysis & optimization of individual ADC properties ADC stability is dependent on site of conjugation & linker chemistry => Linker stbilisation expands the repertoire of available sites ADC efficacy is strictly defined by linker stability prior to internalization into target cells VC-PABC linkers are substrates to extracellular & intracellular proteases Different substrate specificities of plasma Ces1C & lysosomal CatB allow the design of ADCs stable in circulation & efficiently processed in the lysosome Maleimide-VC-PABC-auristatins are substrates of mouse Ces1C Interspecies differences in enzyme activities affect the relevance of preclinical in vivo models for both site-specific and conventional ADCs => Design of VC-PABC linkers stable in mouse & rat is essential to carry out clinically relevant in vivo studies
25 Acknowledgements Pavel Strop Santiago Farias Victor Lui Meri Galindo Casas Mathias Rickert Kathy Delaria Shu-Hui Liu Thomas Tran Adela Hasa-Moreno Jody Melton-Witt Davide Foletti Mike Chin Colleen Brown Ishita Barman Yasmina Abdiche Kevin Lindquist Dan McDonough Janette Sutton Carole Loo Gary Bolton Teresa Radcliffe German Vergara Russell Dushin Ludivine Moine Dahui Zhou Chris O Donnell Jeremy Myers Anna Wang Frank Loganzo Hans-Peter Gerber Bob Abraham Arvind Rajpal Dave Shelton Jaume Pons Strop & Liu et al. Chem Biol (2013) 20: Farias & Strop. et al. Bioconj Chem (2014) 25: Dorywalska & Strop et al. BioconjChem (2015) 26: Strop & Delaria et al. Nat Biotech (2015) 33: Dorywalska & Strop et al. PLOS One (2015) 10: e Dorywalska et al. Mol Cancer Ther (2016) 15:
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