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Mechanism of DNA Alkylation by Duocarmycin SA Copyright of Femtogenix Ltd: Not for further distribution 91 Structures of Second-Generation Duocarmycin ADCs Medarex (MDX-1203) Synthon (SYD-985) Copyright of Femtogenix Ltd: Not for further distribution Phase I Non-Hodgkin's lymphoma; Renal cancer Phase I Breast cancer; Gastric cancer; Solid tumours 92 MDX-1203 (Medarex) Duocarmycin-Based ADC Drug B Peptide Linker Intracellular Activation of MDX-1203 Protecting Group Lysosomal protease Drug A The duocarmycin payload is released from the antibody through protease cleavage of the dipeptide linker, and is then converted from its pro-drug form to the cyclopropane via carboxylesterase cleavage of the carbamate. The carboxylesterase CES2 is claimed to be present in higher concentrations in cancer cells, suggesting that the active duocarmycin molecule should not be activated in normal cells. Carboxylesterase Copyright of Femtogenix Ltd: Not for further distribution 93 Duocarmycin Dimers (A-A Cross-linkers) Patent: WO2015095212 The duocarmycin dimers are extremely cytotoxic, and studies have suggested potencies in the low picomolar to femtomolar ranges in certain cell lines. For example, Genentech have shown that a CBI dimer containing a pentamethylene linker has an average cytotoxicity of 6pM across a number of cell lines (e.g., BJAB, HCC1937) Pfizer also programme dimers. One example in their patent has low picomolar potencies (i.e., 9pM in N87). have an developing active CBI Patent: WO2015110935 Copyright of Femtogenix Ltd: Not for further distribution 94 The Pyrrolobenzodiazepines (PBDs) Structures of the naturally occurring anthramycin (1), the PBD C8-Conjugates GWL-78 (2) and KMR-28-39 (3), and examples of C7/C7’-linked (4) and C8/C8’-linked (e.g., DSB-120, 5a, n = 3 and SJG-136, 6a, n = 3) PBD Dimers. Copyright of Femtogenix Ltd: Not for further distribution Evolution of the Pyrrolobenzodiazepine (PBD) Family Copyright of Femtogenix Ltd: Not for further distribution 96 Mechanism of Action of the Pyrrolobenzodiazepines (PBDs) A. The mechanism of covalent binding of a PBD to DNA once it has located in a low energy position within the minor groove; B. Molecular model of the crystal structure of anthramycin (PDB ID 274D) covalently bound to G19 (magenta) of the sequence 5’-CC(G)AACGTTGG-3’, as an example of a PBD-DNA adduct. Due to the perfect fit of anthramycin in the DNA minor groove (as a consequence of its 3-dimensional shape created by the C11a(S) chiral centre), normal base-pairing is maintained (cyan) with negligible distortion of the minor groove. 97 Copyright of Femtogenix Ltd: Not for further distribution Modes of Interaction of Pyrrolobenzodiazepine Dimers with DNA Diagram of the various possible adduct types that can be form ed between SJG-136 and DNA (i.e., interstrand and intrastrand crosslinks, and mono-adducts) (X = any base). Copyright of Femtogenix Ltd: Not for further distribution 98 Modes of DNA Cross-Linking by SJG-136 A & C, Schematic diagram and molecular models, respectively, of the interstrand cross-linked adduct formed by interaction of SJG-136 (1) with Pu-GATC-Py. B and D, Schematic diagram and molecular models, respectively, of the intrastrand adduct formed by interaction of SJG-136 (1) with Pu-GAATG-Py. Note: In models C and D, SJG-136 and the DNA strands to which it is bound are colored purple, whereas the non-covalently bonded DNA strands are shown in green. Copyright of Femtogenix Ltd: Not for further distribution 99 Examples of C8/C2'- and C2/C2’-Linked PBD Dimers Examples of C8/C2'- and C2/C2’-linked PBD dimers synthesized by the Thurston, Kamal and Lown groups. Dimer C differs from the others in that it possesses only one electrophilic N10C11 imine moiety, and so cannot cross-link DNA. Copyright of Femtogenix Ltd: Not for further distribution 100 Structure-Activity Relationships (SARs) for the Pyrrolobenzodiazepines (PBDs) and Indolinobenzodiazepines (IGNs) Summary of the Structure Activity Relationships (SARs) for the PBD monomers (A), the PBD dimers (B), and the indolinobenzodiazepine analogues (C). Modifications which enhance activity are shown in blue, and those which reduce activity are in red. Copyright of Femtogenix Ltd: Not for further distribution 101 DNA Cross-Linking Ability of SJG-136 (a) Autoradiograph of a neutral agarose gel showing DNA interstrand cross-linking of linear 32P-end-labeled pBR322 DNA (0.2 nM) by SJG-136 following a 2 h incubation at 37 °C. The lanes are: C, double-stranded DNA control; 0, single-stranded DNA control; and 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0 μM SJG-136. DS and SS are double- and single-stranded DNA, respectively; (b) Quantification of the gel in (a) to showing the concentration dependence of DNA cross-linking for SJG-136 in linear DNA. 32P-end-labeled pBR322 "Reprinted with permission from Gregson, S. J., et al, (2001) Design, synthesis, and evaluation of a novel pyrrolobenzodiazepine DNA-interactive agent with highly efficient crosslinking ability and potent cytotoxicity, Journal of Medicinal Chemistry, 44, 737-748 (2001). Copyright © 2001 American Chemical Society." Copyright of Femtogenix Ltd: Not for further distribution 102 Footprinting and In Vitro Stop Assay Results for SJG-136 A, Footprinting gels showing the interaction of SJG-136 with the MS2 DNA sequence (10 nM) at concentrations of 0.1, 1, 3, 10, 30, and 100 μM (left panel = top strand [MS2-F]; right panel = bottom strand [MS2-R]). The labels to the right of each gel correspond to the potential cross-linking sites; B, T-Stop assay showing the effect of incubation time on the ability of SJG-136 (at 1.0 μM) to inhibit transcription. The right-hand side of the panel shows that most stop sites are already visible after only 15 mins incubation of SJG-136 and the DNA duplex together prior to the addition of transcription buffer, and that there is little significant change up to 60 mins incubation. The left-hand side of the panel shows that, once transcription had started (t = 0), there was little effect upon adding SJG-136 at either 30 or 60 min time points (labeled as −30 and −60 min, respectively). Martin, C., Ellis, T., McGurk, C.J., Jenkins, T.C., Hartley, J.A., Waring, M.J. and Thurston, D.E., “Sequence-Selective Interaction of the Minor-Groove Interstrand Cross-Linking Agent SJG-136 with Naked and Cellular DNA: Footprinting and Enzyme Inhibition Studies”, Biochemistry, 44, 4135-4147 (2005). Copyright of Femtogenix Ltd: Not for further distribution 103 HPLC/MS Studies of the Interaction of SJG-136 With Oligonucleotides A. Structures of the double-stranded oligonucleotides sequences used to study the interaction with of SJG-136 in initial using HPLC/MS studies methodology; B. Graph of % cross-linking versus time for duplexes Seq-1 to Seq-4 for a molar ratio of 4:1 (SJG-136/DS-DNA); C. Same data as in Panel B but plotted against log time to provide rate data from the gradients (Units = % cross-linking, log h−1). Note: For both Panels (B and C), 0 h corresponds to approximately 5 mins after initial mixing of the duplex DNA and SJG-136. All data points are the means of triplicate measurements from independent experiments, with error bars showing ± standard errors. Narayanaswamy M., Griffiths, W.J., Howard, P.W., Thurston D.E., “An assay combining high-performance liquid chromatography and mass spectrometry to measure DNA interstrand cross-linking efficiency in oligonucleotides of varying sequences”, Analytical Biochemistry, 374, 173-181 (2008). Copyright of Femtogenix Ltd: Not for further distribution 104 Comparison of the reaction rates of SJG-136 with 12-mer duplex oligonucleotides containing Pu-GATC-Py, Pu-GATG-Py, Pu-GAATC-Py and Pu-GAATG-Py sequences. Reactions were monitored by HPLC in separate experiments with a 4:1 molar ratio of SJG136/DNA, and with adduct molecular weights and stoichiometries confirmed by MS. Copyright of Femtogenix Ltd: Not for further distribution 105 New Mechanism of Action: PBDs Bonding to Terminal Guanine Residues Mantaj J, Jackson PJM, Karu K, Rahman KM, Thurston DE. Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments. PLoS One. 2016;11(4):e0152303. Copyright of Femtogenix Ltd: Not for further distribution 106 Examples of Methods of Attachment of PBD Dimers A. N10-attachment (e.g., Stemcentrx and Genentech): B. N10-attachment with second imine deactivated (ADC Therapeutics): C. C2-attachment (e.g., Seattle Genetics, ADC Therapeutics) with or without PEG: Copyright of Femtogenix Ltd: Not for further distribution 107 Examples of Linker Technologies Copyright of Femtogenix Ltd: Not for further distribution 108 PBD Dimers Connected to C-ring via a PEG Unit Copyright of Femtogenix Ltd: Not for further distribution 109 Intra-tumour Catabolites Used for Determining Efficacy of Antibody Drug Conjugates ZHANG, D., YU, S.-F., MA, Y., XU, K., DRAGOVICH, P. S., PILLOW, T. H., LIU, L., DEL ROSARIO, G., HE, J., PEI, Z., SADOWSKY, J. D., ERICKSON, H. K., HOP, C. E. C. A. & KHOJASTEH, S. C. 2016. Chemical Structure and Concentration of Intratumor Catabolites Determine Efficacy of Antibody Drug Conjugates. Drug Metabolism and Disposition, 44, 1517-1523. Copyright of Femtogenix Ltd: Not for further distribution 110 Copyright of Femtogenix Ltd: Not for further distribution Other New Analytical Method: DM1 Evaluation in Human Serum On-line solid phase extraction (SPE)—liquid chromatography–tandem mass spectrometry (LC–MS/MS) used for the quantitation of maytansinoid (DM1) in human serum DM1 contains a free thiol moiety, likely to readily dimerize or react with other thiol-containing molecules in serum Samples pre-treated with a reducing agent [tris (2-carboxyethyl) phosphine] (TCEP) and further blocked with N-ethylmaleimide (NEM). DM1-NEM exhibited sufficiently stability under all relevant analytical conditions and no DM1 losses from the ADC were observed. The assay was used for DM1 determination in human serum concentration after the intravenous administration of an investigational antibody drug conjugate (ADC) containing DM1 as payload. HEUDI, O., BARTEAU, S., PICARD, F. & KRETZ, O. 2016. Quantitative analysis of maytansinoid (DM1) in human serum by on-line solid phase extraction coupled with liquid chromatography tandem mass spectrometry Method validation and its application to clinical samples. J Pharm Biomed Anal, 120, 322-32. Copyright of Femtogenix Ltd: Not for further distribution 112 PBD-Based ADCs at the Clinical Stage Company Product Name Seattle Genetics Indication Target Payload Linkage Phase SGN-CD33A (vadastuximab talirine) AML CD33 SG3211 C2 Val-Ala I->III Seattle Genetics SGN-CD33A (vadastuximab talirine) AML (+SoC) CD33 SG3211 C2 Val-Ala I Seattle Genetics SGN-CD33A (vadastuximab talirine) AML (ASCT) CD33 SG3211 C2 Val-Ala I/II Seattle Genetics SGN-CD33A (vadastuximab talirine) AML (+HMA) CD33 SG3211 C2 Val-Ala III Seattle Genetics SGN-CD33A (vadastuximab talirine) MDS (+HMA) CD33 SG3211 C2 Val-Ala I->II Seattle Genetics Seattle Genetics Seattle Genetics SGN-CD70A SGN-CD123A SGN-CD19B NHL/RCC AML NHL/RCC CD70 CD123 CD19 SG3211 SG3211 SG3211 C2 Val-Ala C2 Val-Ala C2 Val-Ala I I I Stemcentrx (recently acquired by AbbVie) Rovalpituzumab teserine (Rova-T; SC16LD6.5) SCLC DLL3 SG3249 N10 Val-Ala I->III Stemcentrx (recently acquired by AbbVie) Rovalpituzumab teserine (Rova-T; SC16LD6.5) CisR Ovarian DLL3 SG3249 N10 Val-Ala I ADC Therapeutics ADC Therapeutics ADC Therapeutics ADCT-301 ADCT-301 ADCT-402 CD25 CD25 CD19 SG3249 SG3249 SG3249 N10 Val-Ala N10 Val-Ala N10 Val-Ala I I I ADC Therapeutics ADCT-402 Lymphoma AML CD19-positive hematological tumors CD19-positive hematological tumors CD19 SG3249 N10 Val-Ala I *Source: World ADC Berlin 2016 (ADC Therapeutics Presentation) Copyright of Femtogenix Ltd: Not for further distribution 113 Examples PBD-Based ADCs at Pre-Clinical Stage • At least 5 pre-clinical studies involving PBD-based ADCs are currently ongoing: Company Product Name Indication Target Payload Linkage Phase Seattle Genetics SGN-CD352A Multiple myeloma CD352 SG3211 C2 Val-Ala Pre-clinical Genentech Trastuzumab linked ADC Breast cancer HER2 Undisclosed Cleavable Pre-clinical Genentech hu7C2-linked ADC Breast cancer HER2 Undisclosed Cleavable Pre-clinical Pierre Fabre TBA Axl-expressing cancers Axl Cleavable Pre-clinical • All PBD-based ADCs listed above contain maleimide Ab-attachment moieties. *Source: World ADC Berlin 2016 (ADC Therapeutics Presentation) Copyright of Femtogenix Ltd: Not for further distribution 114 Interest in PBD-Based Payloads Enhanced Due to AbbVie Acquiring StemCentrx in 2016 Copyright of Femtogenix Ltd: Not for further distribution 115 BMS Works with AbbVie to Explore PBD-Based Payloads Bristol-Myers Squibb will team up with AbbVie to run an early-stage lung cancer study combining BMS’ marketed checkpoint inhibitors with AbbVie’s experimental antibody drug conjugate Rova-T (rovalpituuzumab tesirine). The Phase I/II study, scheduled to start later this year, will involve combination therapy of BMS’ YervoyTM (ipilimumab) and OpdivoTM (nivolumab) (which have licenses for melanoma and non-small lung cancer among others) with Rova-T for relapsed extensive-stage small cell lung cancer (SCLC). Copyright of Femtogenix Ltd: Not for further distribution 116 PBD-CBI (G-A Cross-Linking) Dimers as ADC Payloads Adenine Guanine CBI-PBD heterodimer with dual mode of alkylation and the University of Auckland Copyright of Femtogenix Ltd: Not for further distribution 117 PBD-CPI Dimers: G-A Cross Linking Mechanism Example of a PBD-CPI (Bizelesin) dimer: Snapshot of an MD simulation of the Hurley hybrid PBD-CPI dimer (blue) with its PBD and CPI units covalently bound to the G18 (magenta) and A8 (yellow) bases of the sequence 5’GCC(G)AATTAGC-3’, with excellent accommodation in the minor groove and little distortion of the central AT base-pairing (cyan). Copyright of Femtogenix Ltd: Not for further distribution 118 Publications on PBD-CBI Dimers The use of molecular dynamics simulations to evaluate the DNA sequence-selectivity of G-A cross-linking PBDduocarmycin dimers Jackson Paul, Rahman Khondaker Miraz and Thurston David E. Bioorganic Medicinal Chemistry Letters (In Press) Copyright of Femtogenix Ltd: Not for further distribution 119 PBD-CBI Dimers as ADC Payloads: Poster at AACR 2016 (New Orleans) Copyright of Femtogenix Ltd: Not for further distribution 120 Indolinobenzodiazepines (IGNs) Distinguished from PBD dimer ADC constructs by four unique structural features: Two additional D-rings attached to the C-rings of the PBD skeletons. Conversion of one N10-C11 imine to a non-electrophilic secondary amine to create a payload that mono-alkylates rather than cross-links DNA. The central aniline ring within the C8/C8’-linker to which the antibody is attached, a concept originally developed by Sanofi. Joining of the DGN462 payload to the antibody via a cleavable disulfide linker. Copyright of Femtogenix Ltd: Not for further distribution 121 Tetrahydroisoquinolinebenzodiazepine (THIQBD) Dimers Patent: WO2016115191 Contains a 6-7-6-6 (A-B-C-D) scaffold with G-G cross-linking ability. A variety of analogues have been produced, and when conjugated to an antibody, cytoxicities in the low picomolar to femtomolar ranges have been obtained. For example, when conjugated to the anti-fucosyl GM1 antibody, the ADC outlined below was shown to have a cytotoxicity of 30pM against N87 cells. Copyright of Femtogenix Ltd: Not for further distribution 122 Pro-PBDs and Related Payload Technologies pro-PBDs (latent DNA cross-linkers) pro-DRH-417- containing SMDC pro-IBD (indolinobenzodiazepines) pro-PBD-Hoechst dye pro-PBD-seco-CBI hybrids Distamycin hybrids Copyright of Femtogenix Ltd: Not for further distribution 123 Endocyte’s Concept of Pro-PBD Conjugates Slide provided by: Iontcho R. Vlahov, VP Discovery Chemistry, Endocyte, Inc. Copyright of Femtogenix Ltd: Not for further distribution 124 Calicheamicins (DNA-Cleaving Agent) Structure of calicheamicin Caliche clay found in central Texas (USA) of the type that the calicheamicin-producing microorganism (Micromonospora echinospora ssp calichensis) was first isolated from. Calicheamycin is a natural product isolated from the bacterium Micromonospora echinospora sp. Calichensis. It was discovered in caliche clay (a type of soil found in Texas) by Wyeth-Ayerst researchers (now Pfizer). It works by cleaving DNA Copyright of Femtogenix Ltd: Not for further distribution 125 Calicheamicins (DNA-Cleaving Agent) Structure of Calicheamicin γ1 Molecular model of Calicheamicin γ1 interacting with DNA Copyright of Femtogenix Ltd: Not for further distribution 126 Mechanism of Biological Action of Dynamicin A Copyright of Femtogenix Ltd: Not for further distribution 127 Synthesis of ADCs Containing Calicheamicin Copyright of Femtogenix Ltd: Not for further distribution 128 Example: MylotargTM (Gentuzumab ozogamicin) Copyright of Femtogenix Ltd: Not for further distribution 129 129 MylotargTM Loses Licence in 2010 Copyright of Femtogenix Ltd: Not for further distribution 130 Novel DNA-Interactive Agents • 1,5-Diaryl-3-oxo-1,4-pentadienyl agents (University of Saskatchewan) • Major-groove binding agents (Femtogenix Ltd) • PNU-159682 (bioactive metabolite of nemorubicin; 3,000-fold more active than doxorubicin) • Rebeccamycin (inhibitor of topoisomerase, intercalates into DNA). • Sandramycin (DNA topoisomerase I inhibitor) • Sanguinarine (induces DNA damage) • Sinefungin (blocks methylation of bases in DNA and RNA) • Telomestatin (telomerase inhibitor) Rebeccamycin Copyright of Femtogenix Ltd: Not for further distribution Sandramycin 131 Examples of Mechanistic Studies Gel-Based Studies DNA Footprinting N7 Guanine Major Groove: Hot aqueous piperidine → Cleavage. N3 Adenine Minor Groove: Thermal cleavage of adducts. Competition experiments with minor and major groove binding agents. A. 1000 1000 Seq-1, RT 26.0 mins 500 500 0 mAU mAU 0 0 10 20 30 40 Minutes 1000 B. 2/Seq-1 adduct, RT 27.0 mins Seq-1, RT 26.0 mins 500 Free 2, RT 38.5 mins 0 Determine whether a molecule intercalates DNA 0 0 10 20 40 2/Seq-1 adduct, RT 27.0 mins C. mAU HPLC-MS Assays and Biophysical Studies 30 Minutes 1000 500 Seq-1, RT 26.0 mins 500 Free 2, RT 38.5 mins 0 1000 500 0 0 10 20 30 40 Minutes Designed duplex and hairpin oligonucleotides Inosine replacement studies FRET-based DNA melting studies High-field NMR and/or X-Ray Studies Copyright of Femtogenix Ltd: Not for further distribution mAU 1000 132 132 mAU Viscosity studies mAU 6. Small-Molecule Payloads Currently at the Discovery or Development Stages RNA Polymerase II and III Inhibitors • α-Amanitin, β-Amanitin, Epsilon-Amanitin, gamma • Amatoxin – Heidelberg Pharma • Tubulysins (e.,g. A) • Pironetin • Podophyllotoxin Tubulin Polymerisation Inducer • Epothilone A and B • Ixabepilone • Protein toxins – PE18, SarcinDI, Diptheria toxin, Bouganin toxin Cytokines, chemokines (Philogen) Novel Tubulin Inhibitors Copyright of Femtogenix Ltd: Not for further distribution • Bafilomycin A (V-ATPase Inhibitor) Rapamycin Kinase Inhibitors AZD8055 Chaetosin (Specific inhibitor of lysine-specific methyltransferase SU) Ansamitocin P-3 (Oskar) Apoptolidin (F0F1-ATPase Inhibitor) Methyltransferase Inhibitors • • Spliceostatins Cryptophycins (Genentech) Major-groove binding agents (Femtogenix Ltd) ATPase Inhibitors • • 1,5-Diaryl-3-oxo-1,4-pentadienyl agents (University of Saskatchewan) Tubulin Inhibitors • Novel DNA-Interacting Agents Actin Cytoskeleton Polymerisation Inhibitors • Cucurbitacin E HDAC Inhibitor • Tubastatin A eIF4A Inhibitors (e.g., Flavaglines) Oxysterol Binding Protein (OSBP) Interactors (e.g., OSW-1) Leptomycin 133 Spliceostatin-Based RNA Splicing Inhibitors Under development by Pfizer Pre-mRNA splicing is catalyzed by the large ribonucleoprotein spliceosome. Spliceosome assembly is a highly dynamic process in which the complex transitions through a number of intermediates. The potent antitumour compound Spliceostatin A (SSA) has been shown to inhibit splicing and to interact with an essential component of the spliceosome, SF3b. Potent anti-proliferative agents capable of targeting both actively dividing and quiescent cells. Copyright of Femtogenix Ltd: Not for further distribution Thailanstatin A: A Novel Spliceostatin Analog Antibody-thailanstatin conjugates have been shown to disrupt RNA splicing in tumour cells. They have potent cytotoxicity in a variety of cell lines including multi-drug resistant lines, and are inactive in non-target expressing cells. Spliceostatin The patent literature suggests conjugates prepared at Pfizer have a superior safety profile to the approved ADC T-DM1. Thailanstatin A Copyright of Femtogenix Ltd: Not for further distribution 135