Workshop F: Linker Design: Why so complex?

Transcription

1 Genomics Institute of the Novartis Research Foundation Workshop F: Linker Design: Why so complex? Bernhard Geierstanger October 10, 2016

2 Antibody Drug Conjugates Novel targets & Biology Diverse set of antibodies Cytotoxin (cell- or non-permeable) Targeting Linker Attachment Payload n Robust manufacturing Efficient, controlled & site-specific methods New payload attachment & linker chemistries Optimal Target-Antibody-Attachment-Linker-Payload Combo 2 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

3 Targeted delivery of LMW drugs via ADC Vasalou et al., A mechanistic tumor penetration model to guide ADC design (2015) PLoS ONE Target antigen differential tumor vs. normal tissue PK and total exposure Conjugate stability Tumor penetration Non-antigen mediated cellular uptake mab affinity for target Internalization & recycling rate Rate & location of payload release Delivery to lysosome Rate & mechanism of catabolism Lysosomal transfer & permeability Metabolite potency & accumulation 3 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

4 Intrinsic need for linker Targeting With or without cleavage element Linker Attachment Payload n Antibody attachment Payload attachment Covalent attachment of drug payload Stoichiometry of drug loading Intrinsic chemical stability Designed cleavage element Covalent attachment to antibody 4 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

5 Linker Design: Why so complex? Linker properties essential for many aspects of ADC mechanism of action Linker attachment to drug Design of cleavable linker elements Linker attachment to antibody Bernhard Geierstanger, GNF Tetsuo Uno, GNF Changshou Gao, MedImmune James Patterson, Sorrento 5 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

6 Linkers affect ADC properties and MoA Manufacturing of ADC Biophysical characteristics Drug loading stoichiometry Stability in circulation Intracellular drug release mechanism and location Structure and properties of drug metabolite including clearance New linker designs for novel applications 6 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

7 Conjugation One-step vs two-step Linker n* Rx Ry Linker n* Rx Payload Linker Ry n n * Rz P ayload Linker Payload n Drug-to-Antibody Ratio DAR, n = ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

8 Manufacturing of ADC Efficiency of conjugation (enzymatic or chemical) Solubility of linker-payload or linker & payload, and conjugates critical Reactivity of end group determines required excess Maleimide-Cys chemistry highly efficient Jain et al., Current ADC linker chemistry, Pharm Res 2015, 32, ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

9 Biophysical properties of ADC Adding payload to hydrophilic antibody intrinsically increases hydrophobicity High DAR species clear more rapidly Lower MTD & TI for high DAR species Low hydrophobicity is desirable HIC PK cac10 vc-mmae ADCs ADC MTD BALB/c [mg/kg] TI (50% regress ion) E E E Hamblett et al., Clin Cancer Res 2004, 10, ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

10 Reducing hydrophobicity of homogeneous ADCs improves PK and TI Modifying linker-payload DAR 8 on reduced interchain disulfides! (a) Structures of drug linkers 1 3. (b) Plasma pharmacokinetics of h1f6 ADCs (prepared with 1 3 at a DAR of 8) and unmodified h1f6 antibody in mice (mean ± s.d.; n = 3 animals per time point per group). (c) Characterization h1f6 antibody and ADCs by hydrophobic interaction chromatography. (d) Effect of DAR on in vivo activity of h1f6-1 (dosed at 2 mg/kg) and h1f6-3 (dosed at 0.5 mg/kg) in a model of renal cell carcinoma (CD O tumor fragments implanted subcutaneously in nude mice; single dose; data are mean of n = 6 animals per group). Lyon et al., Nature Biotechnol 2015, 33, Higher DAR species are not intrinsically poorly behaved 10 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

11 Reducing hydrophobicity of homogeneous ADCs improves PK and TI Adding PEG side-chain DAR 8 on reduced interchain disulfides! (a) Structures of drug linkers 4 6 (top) and glucuronide-mmae (bottom). (b) Characterization of DAR 8 cac10 ADCs by hydrophobic interaction chromatography. PEGylation of druglinker 4 increased (5) or decreased (6) apparent ADC hydrophobicity, depending upon the configuration of the PEG. (c) Plasma pharmacokinetics in rats of cac10 ADCs prepared with compounds 4 6 at a DAR of 8 (data are mean ± s.d.; n = 3 animals per time point per group). (d) Antitumor activity of DAR 8 cac10 ADCs prepared with compounds 4 6 in the Hodgkin's lymphoma model L540cy (data are means; n = 6 animals per group) at a single dose of 1 mg/kg (cac10-4 and -6) or 2 mg/kg (cac10-5). Lyon et al., Nature Biotechnol 2015, 33, Different approaches to reducing hydrophobicity work 11 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

12 Site-specific conjugation enables conjugates with hydrophobic payloads Cys Engineered ADC & PBD DNA damaging payload Table 2 h1f6- PBD(4) h1f6- PBD(2) h1f6 239C -PBD 0.1 mg/kg (tolerated at 2.5 mg/kg) % Aggreg. % Unconj. mab use of endogenous interchain disulfides for conjugation of this PDB linker was not a viable approach Jeffrey et al., Bioconj. Chem. 2013, 24, 1256 Sutherland et al., (SGN-CD33A for AML), Blood 2013, 122, ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

13 TEINE CONJUGATION. (E,F) PHARMACOKINETICS OF THE UNCONJUGATED ANTI-M1S1 ANTIBODY (BLACK), SITE-SPECIFIC ADC WITH DAR6 AND DAR8_1 (RED) AND CONVENTIONAL DAR8 (BLUE) IN MOUSE (E) AND RAT (F). ERROR BARS ± S.E.M. Site-specific conjugation improves TI of ADC with high drug loading DAR 8 with transglutaminase conjugation to LLQG tags! (a) Structure of the linker-payload used in the study. (b,c) In vitro comparison of cytotoxicity of increasingly higher-loaded, site-specifically and conventionally conjugated antibody to tumor-associated calcium signal transducer 2 (anti-m1s1) ADCs in cells with high (M1S1+++) (b) and low (M1S1+) (c) target expression. (d) In vivo comparison of higher-loaded sitespecifically (DAR6 and DAR 8_1) and conventionally (DAR8) conjugated anti-m1s1 ADCs in the low target expressing Colo205 xenograft model. Error bars, mean ± s.e.m. SS, site specific; Cys, conventional cysteine conjugation. (e,f) Pharmacokinetics of the unconjugated anti-m1s1 antibody (black), sitespecific ADC with DAR6 and DAR8_1 (red) and conventional DAR8 (blue) in mouse (e) and rat (f). Error bars ± s.e.m. Strop et al., Nat Biotechnol 2015 Jul;33(7): ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

14 Y COMBINING DAR2 (HC C-TERMINAL) AND DAR6) CONJUGATES RELATIVE TO THE UNCONJUGATED MAB (WT). (F) PK COMPARISON OF DAR8_1, DAR8_2 AND CONVENTIONAL DAR8 CONJUGATES RELATIVE TO THE UNCONJUGATED MAB (WT). Clearance depends on site of attachment Worst single site dominates (a) Site-specific DAR8_1. The spheres indicate positions of the conjugated payloads for different ADCs. The numbers indicate position numbers and sequence information, defined in Supplementary Figure 1. (b) Site-specific DAR8_2. (c) Conventional DAR8 conjugates. (d) PK of site-specific DAR2 (HC), DAR6 and DAR8_1 conjugates relative to the unconjugated mab (WT). (e) PK of site-specific DAR2 (HC C-terminal), DAR6 and DAR8_2 (created by combining DAR2 (HC C-terminal) and DAR6) conjugates relative to the unconjugated mab (WT). (f) PK comparison of DAR8_1, DAR8_2 and conventional DAR8 conjugates relative to the unconjugated mab (WT). Strop et al., Nat Biotechnol Jul;33(7): ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

15 Increased drug loading stoichiometry Polymeric linkers for DAR >20 for higher potency Synthesis of trastuzumab PHF vinca ADCs (Mersana) Yurkovetskiy et al. Cancer Res 2015;75: ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

16 Targeted delivery of LMW drugs via ADC Target antigen differential tumor vs. normal tissue PK and total exposure Conjugate stability Tumor penetration Non-antigen mediated cellular uptake Vasalou et al., (2015) PLoS ONE mab affinity for target Internalization & recycling rate Rate & location of payload release Delivery to lysosome Rate & mechanism of catabolism Lysosomal transfer & permeability Metabolite potency & accumulation 16 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

17 ADC stability in circulation Prevent premature release of payload Stability of drug attachment Reduction of disulfide linkers Acid-labile hydrazone linkage Stability of linker Enzymatic cleavage of VC-PABC linker in mouse but not cyno plasma (Dorywalska et al., Bioconjug Chem 2015, 26, 650-9; Mol Cancer Ther 2016, 15, ) Stability of antibody attachment Many stable chemistries; maleimide chemistry discussed next Chemical stability of drug Enzymatic degradation of MMAD in mouse but not cyno plasma (Dorywalska et al., PLoS One. 2015, 10(7):e More in next presentations 17 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

18 Maleimide deconjugation results in DAR loss Retro-Michael mechanism (Alley et al., Bioconjug Chem 2008, 19, ; Tumey et al., Bioconjug Chem 2014, 25, 1871) Mitigation by: Choice of attachment site (Shen et al., Nat Biotechnol 2012, 30, ) Linker modification (Lyon et al., Nat Biotechnol 2014, 32, ; Burke et al., Mol Cancer Ther 2016, 15, ; Fontaine, Santi et al., Bioconjug Chem 2015, 26, ) Modified maleimides (Christie et al., J. Control Release 2015, 220, ) Using other thiol-reactive groups (Alley et al., Bioconjug Chem 2008, 19, ) More in next presentations 18 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016 Wei et al., Anal. Chem. 2016, 88,

19 Majority of deconjugated maleimide payload transferred to albumin Near quantitative recovery of payload from incubated samples after enzymatic cleavage of the ADC-depleted plasma (little recovery from protein depleted samples). Deconvoluted mass spectrum of payload-adduct from 5 mg/kg ADC postdosed and ADC-depleted cyno serum at 168 h. Albumin-vc-MMAE adduct Wei et al., Anal. Chem. 2016, 88, Verifies earlier suggestions (Alley et al., Bioconjug Chem 2008, 19, ) 19 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

20 Instability of ADC drug in rodent plasma MMAD In vitro 13% cleaved In vivo 80% cleaved Transglutaminase conjugation Aur3377 Position-dependent degradation of MMAD C-terminus but not of Aur3377 Likely by serine-based hydrolase in mouse plasma Less pronounced in rat, not observed in cyno and human plasma Dorywalska et al. (2015), PLoS ONE 10(7): e doi: /journal.pone ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

21 Intracellular delivery of LMW drugs via ADC Vasalou et al., (2015) PLoS ONE mab affinity for target Internalization & recycling rate Rate & location of payload release Delivery to lysosome Rate & mechanism of catabolism Lysosomal transfer & permeability Metabolite potency & accumulation 21 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

22 ADC drug release mechanism & location Cleavable linker Protease cleavage, ph, disulfide reduction Endosome & lysosome Vasalou et al., (2015) PLoS ONE Non-cleavable linker Antibody degradation in lysosome Amino acid adduct generated Jain et al., Pharm Res 2015, 32, Different linker = different drug metabolite structures = different properties 22 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

23 Lys-MCC-DM1 & other non-cleavable ADC drug metabolites are generated in lysosome Inhibitor of lysosmal proteases cells 10 nm (DM1 equivalents) anti-cd70 ADC for 24 hours. Only Lys-MCC-DM1 detected; not detected in media; low cell permeability Brooke M. Rock et al. Drug Metab Dispos 2015;43: ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

24 Transporter protein facilitates lysosome to cytoplasm ADC drug metabolite transfer Solute carrier family 46 member A3 SLC46A3 shrna protects against anti-cd70-mcc-dm1 ADC SLC46A3 sirna inhibits potency of MCC-DM1 but not mcmmaf ADCs Kevin J. Hamblett et al. Cancer Res 2015;75: ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

25 SLC46A3 sirna is specific for maytansines & blocks metabolite escape from lysosome cells Functional effect of SLC46A3 on lysine-mcc-dm1 concentration in cells. Solute carrier family 46 member A3 SLC46A3 sirna protects against different maytansine linker-payloads & increases lysine-mcc-dm1 metabolite concentration in lysosome Kevin J. Hamblett et al. Cancer Res 2015;75: ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

26 SLC46A3 protein required to transport drug metabolite of MCC-DM1 ADC from lysosome Kevin J. Hamblett et al. Cancer Res 2015;75: ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

27 Simulations suggest endosomal release may be desirable Simulations: Endosomal release resulted in much higher payload concentrations in cytosol than lysosomal degradation Vasalou et al., (2015) PLoS ONE 27 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

28 ADC activity driven by released payload independent of target antigen & antibody Intracellular MMAE correlates with ADC potency in L-82 cells in vitro. Intratumoral MMAE concentration correlates with ADC antitumor activity in L-82 xenograft model. Fu Li et al. Cancer Res 2016;76: ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

29 Antitumor activity correlates with payload accumulation Intratumoral MMAE correlates with cac10-vcmmae specific antitumor activity in Karpas 299 xenograft model. 2 mp/kg, single dose Fu Li et al. Cancer Res 2016;76: ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

30 Better in vivo bystander killing for VC-MMAE vs. VC-MMAF ADC b/c low MMAF lipophilicity Admixed tumor model with heterogeneous CD30 expression. Karpas 299 cells Single dose 3 mg/kg Fu Li et al. Cancer Res 2016;76: ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

31 Different cleavable linkers, similar efficacy Different payloads, different efficacy Role of linker and payload in bystander killing. Single dose Single dose 3 mg/kg (DAR 4) 3 mg/kg (DAR 4) 0.1 mg/kg (DAR 2) Fu Li et al. Cancer Res 2016;76: ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

32 Bystander killing depends on payload kinetics in and out of cells (payload accumulation) Vasalou et al., (2015) PLoS ONE Vasalou C, et al., (2015) PLoS ONE Simulations *Release of payload upon cell death and lysis into interstitial space not considered See Dhaval K.Shah et al. for other ADC simulations Structure of drug metabolite will determine cell permeability, accumulation and systemic clearance 32 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

33 Novel applications may require new linkers Antibiotic delivery Anti-S. aureus antibiotic ADC cleaved in phagolysome inside host cell (Lehar et al. Nature 2015, 527, ) sirna delivery Polyanionic payload; low eficiency; endosomal and lysosomeal escape limiting? (Cuellar et al., Nucleic Acid Res 2015, 43, ) Extracellular ADCs Inhibiting membrane proteins on cell surface (Marshall et al., Mol Therapy advance online publication 19 July 2016) 33 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

34 Potency of extracellular ADCs vary with linker length Targeting Na+/K+-ATPase plasma membrane ion pump in the proximity of cancer-related targets (CD20, CD38 etc.) (a) A549 cells treated with anti-dysadherin antibody linked to CG1 (b) A549 cells treated with CG1 (black open circle) and 4 the different Linker-CG1s PEG polymers Marshall et al., Extracellular ADC exploiting the proximity of two proteins, Mol Therapy advance online publication 19 July 2016; doi: /mt ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

35 Linkers affect ADC properties and MoA Manufacturing of ADC Biophysical characteristics Drug loading stoichiometry Stability in circulation Intracellular drug release mechanism and location Structure and properties of drug catabolites including clearance New linker designs for novel applications 35 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

36 Intrinsic need for linker Targeting With or without cleavage element Linker Attachment Payload n Antibody attachment Payload attachment Covalent attachment of drug payload Stoichiometry of drug loading Intrinsic chemical stability Designed cleavage element Covalent attachment to antibody 36 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

37 Linker Design: Why so complex? Linker properties essential for many aspects of ADC mechanism of action Linker attachment to drug Design of cleavable linker elements Linker attachment to antibody Bernhard Geierstanger, GNF Tetsuo Uno, GNF Changshou Gao, MedImmune James Patterson, Sorrento 37 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016

38 October 10, 13:00-16:00 Workshop F: Linker Design: Why so complex? Workshop Leader: Bernhard Geierstanger, Director, GNF The majority of ADCs currently in clinical development use only a limited number of chemical linkers. One of the major challenge in the development of safe and effective antibody-drug conjugates has been the generation of suitable chemical linkers between the cytotoxic drug and the monoclonal antibody. While the synthesis of linker chemistry is quite complex and several aspects must be critically balanced to guarantee efficacy, ultimately, the nature of the chemical linker being used shapes the release profile of the cytotoxin. This workshop will cover: Lessons learned from linker design Important considerations for designing the optimum linker Why linkers have failed to realize their potential? Assess cleavable vs. non-cleavable Review new linker technologies 38 ADC World Summit San Diego Bernhard Geierstanger October 10, 2016