Pyc Therapeutics Limited · ASX:PYC
Pyc Therapeutics — Phylogica — Investor Presentation (January 2017)
62 slides from Pyc Therapeutics's investor presentation, Jan 11, 2017
Slide 1: Using novel cell penetrating Phylomer peptides to access intracellular targets
For personal use only Using novel cell penetra0ng Phylomer pep0des to access intracellular targets with biologics Paul Wa' Director and Chief Scien5fic Advisor BREAKTHROUGH PEPTIDE THERAPEUTICS
Slide 2: Disclaimer
Disclaimer The purpose of the presenta5on is to provide an update of the business of Phylogica Limited (ASX:PYC) For personal use only [‘Phylogica’]. These slides have been prepared as a presenta5on aid only and the informa5on they contain may require further explana5on and/or clarifica5on. According
Slide 3: Phylogica: unique capabilities for intracellular discovery and delivery of biologics
Phylogica: unique capabili0es for intracellular discovery and delivery of biologics drugs For personal use only Poten0al to expand the druggable intracellular landscape by >10-fold with Phylomer Func%onal Penetra%ng Pep%des (FPP) - Phylogica’s proprietary cell penetra0ng pep0des. s 3
Slide 4: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm § Oncology Programs - Myc, Stat5, YB1 § Collabora5on Opportuni5es and Summary 4 4
Slide 5: About Phylogica
About Phylogica § ASX Listed company (ASX:PYC) ASX: PYC 6 month share price – Based in Perth, Western Australia with 22 For personal use only Scien5sts and market cap. of ~A$50M – Founded by Telethon Kids Ins5tute of WA and Fox Chase Cancer Centre in Philadelphia § Owns unique proprietary class of p
Slide 6: Phylomers: peptides from parts of biodiverse proteins
Phylomers: pep0des from parts of biodiverse proteins § Encoded by >35 biodiverse microbial genomes (derived from volcanoes, For personal use only geysers and undersea vents) § Rich source of diverse natural secondary/ter5ary structures § Average size ~30 amino acid residues § Libraries encode hundre
Slide 7: More structures for better hits against more targets
More structures for beNer hits against more targets For personal use only Wa: 2009 Future Medicinal Chemistry. 1 (2) 257-265. § Phylomer libraries comprise thousands of unique structural families § Phylomer screens allow the target to ‘choose’ the scaffold from thousands of folds § High structural di
Slide 8: Phylomer peptides have complementary capabilities
Phylomer pep0des have complementary capabili0es Phylomers: efficiently targe0ng Protein-Protein Interac0ons For personal use only Phylomers: Discovering and valida0ng new targets Func0onal Penetra0ng Phylomers (FPPs): Efficiently delivering biologics to cells 8
Slide 9: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm – Enables endosomal escape – Compa5ble with wide range of biologics cargoes – Outperforms other intracellular delivery technologies – Func5onal in mul5ple 5ssues – Demonstrable evidence of ac5on – Eviden
Slide 10: Biologics drugs are trapped within endosomes resulting in inefficient delivery
Biologics drugs are trapped within endosomes resul0ng in inefficient delivery Drug For personal use only Drug Drug contacts cell Budding membrane endosome Cell Contact triggers Membrane forma0on of endosomes Drug target § Conven5onal CPPs Cell – mostly trapped inside endosomes Membrane – therefore nor
Slide 11: Phylomer FPP cargo sequences enter cells and escape the endosome
Phylomer FPP - cargo sequences are able to enter cells and escape the endosome Phage-displayed Phylomer libraries Screen captures very rare events For personal use only (1 in a billion) RBD Phylomer Avitag Bio0n Trapped Phylomer SA RBD Phylomer Avitag RBD Phylomer Avitag Phylom RBD Avitag er Escaped
Slide 12: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm – Enables endosomal escape – Compa5ble with wide range of biologics cargoes – Outperforms other intracellular delivery technologies – Func5onal in mul5ple 5ssues – Demonstrable evidence of ac5on – Eviden
Slide 13: Phylomer FPPs are able to deliver diverse cargo classes
Phylomer FPPs are able to deliver diverse cargo classes Examples of cargoes delivered with FPP01 or its deriva5ves (FPP1.1, FPP1.2): For personal use only Cargo Class Cargo Size/Charge IC50* **MED Toxin / large protein Bouganin 28kDa, pI 7.8 20nM ND Small protein Omomyc 11kDa, pI 9.6 0.7-5µM ND scaff
Slide 14: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm – Enables endosomal escape – Compa5ble with wide range of biologics cargoes – Outperforms other intracellular delivery technologies – Func5onal in mul5ple 5ssues – Demonstrable evidence of ac5on – Eviden
Slide 15: FPP delivery of Pro-Apoptotic Peptide demonstrates superiority over TAT
FPP delivery of Pro-Apopto0c Pep0de (PAP) into cells demonstrates superiority over TAT at low concentra0ons § Pro-apopto5c pep5de PAP domain (KLAKLAK)2 induces intrinsic apoptosis1 For personal use only - Decreased prolifera5on of triple nega5ve breast cancer cell line (MDA-MB-468) PHYLOMER FPP – GA
Slide 16: Higher potency achieved by FPP-PAP vs TAT-PAP
Higher potency achieved by FPP-PAP vs TAT-PAP § Potent func5onal ac5vity of a pro-apopto5c pep5de (PAP) For personal use only – Increased apoptosis – Measured by % Annexin V staining MDA468 24h - Total Apoptosis 100 % Total Apoptotic 80 FPP_01-PAP 60 TAT-PAP 40 PAP 20 0 2.0 2.5 3.0 3.5 4.0 Log10 [nM
Slide 17: Split GFP assay shows superior FPP uptake versus other CPPs
Split GFP assay shows greatly superior FPP uptake and cytoplasmic delivery versus % complemented ( other CPPs in live cells cells HCC-827) For personal use only 100 Protein Conc. (%) 20µM Signal+ve) 80 10µM 160 fold cells (SplitGFP 5µM 37 fold 2.5µM 60 1.25µM Fluorescence 0.667µM 40 % Green 20 GFP 0
Slide 18: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm – Enables endosomal escape – Compa5ble with wide range of biologics cargoes – Outperforms other intracellular delivery technologies – Func5onal in mul5ple 5ssues – Demonstrable evidence of ac5on – Eviden
Slide 19: FPP mediated delivery of biologics into diverse cell types
FPP mediated delivery of biologics into diverse cell types Disease Cell/Tissue type Cargoes delivered For personal use only Breast cancer (in vitro and • Breast tumour epithelial (T47D, Pro-apopto5c pep5de (PAP) in vivo) MDA-MB-468, murine T11) PPI inhibitor (DPMI⍺) Protein scaffold (Omomyc) Phylomer
Slide 20: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm – Enables endosomal escape – Compa5ble with wide range of biologics cargoes – Outperforms other intracellular delivery technologies – Func5onal in mul5ple 5ssues – Demonstrable evidence of ac5on - assays
Slide 21: Proprietary Split-GFP complementation assay
Proprietary Split-GFP complementa0on assay developed to validate endosomal escape of penetra0ng Phylomer fusions For personal use only § Only biologics delivered to the cytoplasm can fluoresce in this assay FPP RBD GFP S11 + GFP 1-10 GFP 1-11 !! !! !! !! !! !! !! !! !! !! !! !! Uptake Release Complem
Slide 22: Split GFP assay shows superior FPP uptake vs other CPPs
Split GFP assay shows greatly superior FPP uptake and cytoplasmic delivery vs other CPPs in live cells % complemented cells (HCC-827) For personal use only 100 Protein Conc. (%) 20µM Signal+ve) 80 10µM 160 fold Green cells (SplitGFP 5µM 37 fold 2.5µM 60 1.25µM CHO-K1 cells – 40x GFP%Fluorescence 0.6
Slide 23: Split GFP microscopy confirms endosomal escape
Split GFP microscopy confirms endosomal escape RBD GFP FPP RBD GFP FPP RBD GFP Receptoronly No FPP Phylomer With FPP Phylomer With FPP Phylomer personal Lowuse GFP GFP (100X) GFP (Phylomer + Cargo) DAPI (Nucleus) ForHigh Receptor GFP GFP (Phylomer + Cargo) DAPI (Nucleus) GFP – Endosomes are much larg
Slide 24: FPP allows rapid cell uptake and endosomal release
FPP allows rapid cell uptake and endosomal release § Use pH sensi5ve Naphthofluorescein (NF) dye which only fluoresces a}er For personal use only endosomal escape (at pH above 6.5)1 1 Qian et al., Chem.Commun. 51 (2015) 2162 § Most delivery occurs within 1hr and is temperature (energy) dependent 5µM N
Slide 25: Biochemical assay confirms efficient endosomal escape of FPP-cargo
A biochemical assay using BirA expressing cells confirms efficient endosomal escape of FPP-cargo For personal use only Double tagged construct 60kDa FPP Avitag Cargo V5 50kDa 26% 46% 40kDa FPP+Avi+Cargo+V5 Constructs +/- Bio5nyla5on added to HEK293-BirA 30kDa cells @ 15µM Time)(min) 30 60 Time (min) Av
Slide 26: Negligible endosomal release for TAT fusion compared to FPP fusion
Negligible endosomal release detected for TAT fusion compared to 58% release for FPP fusion For personal use only Gel 1: SA Capture, Anti-V5 IB 16µM 4µM 60 58% 50 40 30 20 15 10 3.5kDa FPPavi113 + - - - - + - - - bioC27_0113 - + - - - - + - - Tat_Avi_0113 - - + - - - - + - bioTat_0113 - - - + - - -
Slide 27: FPP permits far greater endosomal release than TAT
FPP permits far greater endosomal release than TAT § Protein conjugates applied to HEK293 BirA cells for 60min For personal use only § Cell lysates assessed for dual-tagged conjugate that has escaped the endosome (sensi5ve Streptavidin/V5 AlphaLISA proximity assay) iMyc&0113&Endosomal&Release:&FPP_0
Slide 28: FPP can enhance potency of cargo delivered via receptor mediated endocytosis
FPP can enhance potency of cargo delivered via receptor medicated endocytosis For personal use only Toxin RBD FPP FPP-cargo size 50KDa cargo HEK-EGFR Viability - 48h HEK-EGFR Apoptosis - 72h 150 30 Boug-RBD-FPP02 Boug-RBD Boug % Annexin V +ve cells RBD 100 20 % Viability IC50 787.1 nM Bouganin-RBD-F
Slide 29: Using Omomyc as a model cargo for FPP delivery
Using the validated MYC inhibitor Omomyc as a model cargo for FPP delivery § MYC: Amplified/overexpressed in most human cancers For personal use only Prolifera5on Cell Growth MYC Metabolism Apoptosis § MYC: non redundant role in cancer – associated with addic5on § OmoMyc: 92 aa pep5de derived from bH
Slide 30: Delivering Omomyc using Phylogica's FPP
Delivering Omomyc using Phylogica’s FPP § Linked a Phylomer cell penetra5ng pep5de (FPP PYJ02) to Omomyc, the For personal use only most effec5ve known Myc inhibitor when expressed in cells Func%onal Penetra%ng Phylomer PYJ02 Omomyc § Omomyc protein has very low potency due to very poor cell penetra5
Slide 31: FPP delivery of Omomyc reduces viability of resistant breast cancer cells
FPP delivery of Omomyc reduces viability of resistant breast cancer cells § Delivery of FPP-Omomyc (PYJ02-Omyc) into aggressive mul5-drug resistant For personal use only mouse basal cancer cell line in-vitro, used in a syngeneic mouse model T11 mouse basal cancer cell line – 24h 120 Omyc 100 PYJ02_O
Slide 32: FPP delivery of Omomyc results in apoptosis of cancer cells
FPP delivery of Omomyc results in apoptosis of cancer cells For personal use only § Caspase assay shows increased apoptosis in FPP01-Omyc treated cells only § FPP-01 itself had no apopto5c ac5vity 32
Slide 33: FPP-Omomyc shows potent activity in multiple Myc-driven cancer cell lines
FPP-Omomyc shows potent ac0vity in mul0ple Myc-driven cancer cell lines FPP_01-Omyc Cell line Disease Cell line characteris0cs (IC50 (µM)) For personal use only T47D Breast cancer p53 mut(hetero), Myc+++ 4.5 MDA-MB-468 Breast cancer p53 mut(hetero), triple -ve 1.7 SUM159 * Breast cancer Basal, Tripl
Slide 34: Global gene expression profiling
Global gene expression profiling Color Key Pearson's correlation Mul0-dimensional scaling Multi−Dimensional Scaling Pearson’s correla0on For personal use only ● ● 1.0 ● 0.9 0.94 0.98 Value X1746_3h_rep1 X1746_3h_rep2 Omyc_3h_rep1 PBS_3h_rep1 0.5 ● Omyc_3h_rep1 ●● ● ●● X1746_3h_rep1 ● Dimension 2 ● PB
Slide 35: Gene set enrichment analysis: Myc pathway
Gene set enrichment analysis: Myc pathway § Differen5ally regulated genes enriched for a priori defined set of ac5vated For personal use only and repressed Myc pathway genes § Zeller et. al.,(2003) Genome Biology 4: R69. § Savino et al., (2011) PLoS ONE, 6(7), e22284 Myc repressed genes – ac0vated by
Slide 36: Modulation of Myc-pathway target genes
Modula0on of Myc-pathway target genes Tfap4 mRNA expression levels by ddPCR p21 mRNA expression levels by ddPCR For personal use only (normalised to Eef1a1) (normalised to Eef1a1) copies per 8000 copies per 8000 ** p=0.002 106 Eef1a1 106 Eef1a1 8-fold ** p=0.009 6000 * p=0.01 6000 8-fold 4000 4000 2
Slide 37: FPP-Omomyc doesn't significantly affect other pathways (Notch and Wnt)
Gene set enrichment analysis: FPP-Omomyc doesn’t significantly affect other pathways (Notch and Wnt) § No evidence for off-target regula5on of other pathways unrelated/upstream For personal use only of Myc WNT Pathway (no correla0on) GSEA of WNT and Notch from KEGG ( http://www.genome.jp/kegg/) pathway
Slide 38: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm – Enables endosomal escape – Compa5ble with wide range of biologics cargoes – Outperforms other intracellular delivery technologies – Func5onal in mul5ple 5ssues – Demonstrable evidence of ac5on – Eviden
Slide 39: Protocol for syngeneic allograft breast cancer model
Protocol for syngeneic allograr breast cancer model: intratumoral delivery For personal use only Intratumoural injections (40nanomoles every second day) 2.5x105 T11 cells/mouse (BALB/c) D0 D4 D6 D8 D10 D16 D17 D22 Weigh prior to start of exp Weigh (body weight) Tumour volume (mm3) Tumour samples tak
Slide 40: Intratumoral FPP-Omomyc inhibits growth in basal breast cancer cells
Intratumoral FPP-Omomyc inhibits growth in basal breast cancer cells (T11) in immunocompetent mice T11 Syngeneic triple negative breast cancer graft For personal use only 1200 1000 FPP01_Omyc Tumour volume (mm3) Omyc 800 Treatment Period FPP01 PBS (Vehicle) 600 Intratumoural injections 400 200 0 0 1
Slide 41: FPP-Omomyc inhibition of T11 tumour engraftment: intravenous delivery
FPP-Omomyc inhibi0on of T11 tumour engrarment: intravenous delivery Intravenous injec5ons (40 nmoles) For personal use only Intravenous&injection 120 PBS FPP01.10Omyc 100 Tumour&&Volume&(mm3) 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 Day&post3inoculation&of&cells 41
Slide 42: Tumour Immunohistochemistry: FPP-Omomyc induces apoptosis and blocks proliferation
Tumour Immunohistochemistry: FPP-Omomyc induces apoptosis and blocks prolifera0on For personal use only Vehicle Control FPP_Omomyc Omomyc TUNEL Apoptosis Ki-67 Prolifera0on +++ +/- ++ 42
Slide 43: Tumour Immunohistochemistry: FPP-Omomyc decreases PDL1
Tumour Immunohistochemistry: FPP-Omomyc decreases PDL1 (Myc target gene) For personal use only FPP- PBS Omomyc FPP01.1 Omomyc H&E Ki-67 PDL1 § Increased cell death, decreased prolifera5on & PDL1 expression in tumours treated with FPP-Omomyc versus FPP, Omomyc or vehicle controls § Poten5al immuno-on
Slide 44: Efficacy following intravenous delivery in EµMyc lymphoma model
Evidence of efficacy following intravenous delivery in EµMyc lymphoma model Mice injected IV daily with Phylomer FPP-Omomyc fusion (36nmol, 52mg/kg) For personal use only EuMyc cells/mouse (in 100uL PBS) At each time point: D0 D3 D4 D5 D6 D7 D8 D9 - 2 mice per group are culled and weighed - Spleens (a
Slide 45: Enhanced delivery of antisense morpholino oligonucleotides using FPPs: DMD Model
Enhanced delivery in vitro of an0sense morpholino oligonucleo0des using FPPs: DMD Model For personal use only § Morphilino an5sense oligo (AO) targe5ng dystrophin gene (mutated in Duchenne muscular dystrophy; DMD) § DMD oligo is designed to induce exon skipping (Δ23) in muscle cells to produce a sho
Slide 46: FPP enhances dystrophin protein levels in muscles
FPP enhances dystrophin protein levels in muscles Mouse DMD animal model For personal use only Tibialis anterior Muscle Diaphragm Muscle Heart Muscle PMO FPP-PMO § ~4 day old mdx mice treated with five IP injec5ons of 4 nmoles of FPP-PMO § Muscle 5ssue sec5ons stained for Dystrophin protein 2 weeks a
Slide 47: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm – Enables endosomal escape – Compa5ble with wide range of biologics cargoes – Outperforms other intracellular delivery technologies – Func5onal in mul5ple 5ssues – Demonstrable evidence of ac5on – Eviden
Slide 48: FPP peptide not cytotoxic (membrane integrity assay)
FPP pep0de not cytotoxic (membrane integrity assay) For personal use only § Phylomer FPPs are non-toxic – even at 100µM § Posi5ve control: Pep5de with known human cytotoxicity (haemolysis) – Brevinin an AMP from the skin of the frog, Rana brevipoda porsa Membrane permeability staining of HEK293T cel
Slide 49: FPP peptide not cytotoxic (reduction potential assay)
FPP pep0de not cytotoxic (reduc0on poten0al assay) For personal use only 120 IC50 [uM] FPPs 100 FPP1.2-PAP 6.1 FPP1.2 (P_T)-PAP 6.0 80 % viability FPP1.2 (KT)-PAP 7.3 60 FPP1.2 (KTQE)-PAP 3.9 FPP1.2 (P_T) >30 40 FPP1.2 (KT) >30 20 FPPs + PAP-toxin cargo 0 3.0 3.5 4.0 4.5 Protein Log [nM] § CHO-K1 ce
Slide 50: Additional promising safety signals
Addi0onal promising safety signals § No evidence of in vivo toxicity For personal use only – We rou5nely inject 200µM of FPP-fusion proteins or pep5des i.v., i.p., and intratumorally into mice with no toxic side effects observed – Mice have received up to 6 doses (4 x i.v. + 2 x i.p. over 6 days) § N
Slide 51: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm – Enables endosomal escape – Compa5ble with wide range of biologics cargoes – Outperforms other intracellular delivery technologies – Func5onal in mul5ple 5ssues – Demonstrable evidence of ac5on – Eviden
Slide 52: Phylogica's Intracellular Oncology Pipeline
Phylogica’s Intracellular Oncology Pipeline § Phylomer screens against validated and clinically relevant oncology targets For personal use only – cMyc, N-Myc, Stat5 and YB1 § Validated hits already exceed potency of gold standard inhibitors § Stat5 and YB 1 collabora5ons with Dana Farber Ins5tute Fu
Slide 53: Multiple Phylomer Hits (iMyc) Targeting MYC
Mul0ple Phylomer Hits (iMyc) Targe0ng MYC Myc reporter gene assay In silico modelling For personal use only (p< 0.0001) 150 Modelled&structures&of&hits **** **** % Relative response ratio (normalised to empty vector) NMy_0136 Omomyc/cMyc dimer 100 50 NMy_0148 0 yc or 36 30 48 81 47 ct M 01 01 01 00
Slide 54: High affinity primary 'iMyc' Phylomers against Myc
High affinity primary ‘iMyc’ Phylomers against Myc § Primary hits before any sequence matura5on can exhibit target binding For personal use only affini5es comparable to natural ligand (Max) and Omomyc protein – Binding affini5es determined by Bio-Layer Interferometry NMy_0113 NMy_0114 cMB_0177 166 ± 16.3
Slide 55: FPP-iMyc conjugates are stable over hours in serum
FPP-iMyc conjugates are stable over hours in serum 0-3 hrs incubation in serum (100% FCS, 37°C) 0-24 hrs incubation in serum (100% FCS, 37°C) For personal use only 200000 200000 FPP-iMyc_0114 FPP-iMyc_0114 Stability (AL units) FPP-iMyc_0178 Stability (AL units) FPP-iMyc_0178 150000 FPP-iMyc_0355 150
Slide 56: Synergistic targeting of Myc and prosurvival proteins
Synergis0c targe0ng of Myc and prosurvival proteins For personal use only Cancer Death Dual Treatment More n era0o Cell D Prolif eath More Less P h rolife s Ce ll Deat ra0o n Les BCL-2 FPP-Bim BCL-2 BCL-x MYC Small molecule Bcl BCL-x + MYC FPP-iMyc MCL-1 Inhibitors MCL-1 § Strategic opportunity for
Slide 57: Addition of FPP-Bim synergistically achieves nanomolar potencies
Addi0on of FPP-Bim synergis0cally achieves nanomolar potencies with FPP01-spyC-Omomyc Eu-myc cells For personal use only 120 Omomyc + 0µM Bim Eu-myc (BurkiN’s Lymphoma model) cells 100 Omomyc + 5µM Bim 80 Omomyc + 2.5µM Bim [FPP-Bim ] 0 5uM 2.5uM 1.25uM 625nM % Viability Omomyc + 1.25µM Bim IC50 2.5
Slide 58: Potent activity of single compound FPP-Bim-Omomyc-FPP bispecific
Potent ac0vity of a single compound FPP-Bim-Omomyc- FPP bispecific in EµMyc lymphoma cells For personal use only FPP Bim -S-S-cells (24h) Omomyc/Bim bispecific in Eu-myc Omomyc FPP Reducible Eµ-myc cells Disulphide Bond 100 IC50 = 2.2 µM % Viability FPP-omomyc FPP-Bim 50 FPP-omomyc/FPP-Bim bispecific
Slide 59: Table of Contents
Table of Contents For personal use only § Company and Technology Overview § FPP Delivery PlaYorm – Enables endosomal escape – Compa5ble with wide range of biologics cargoes – Outperforms other intracellular delivery technologies – Func5onal in mul5ple 5ssues – Demonstrable evidence of ac5on – Eviden
Slide 60: Phylogica is open to alliances with Pharma
Phylogica is open to alliances with Pharma § Phylogica’s business model is open to alliances at any stage: For personal use only – Discovery alliances – Alliances around our internal programs § Open to mul5ple licensing structures: – License exis5ng FPPs for par5cular cargoes or targets – Develop cu
Slide 61: Expanding the intracellular landscape for novel biotherapeutics with Phylomer FPP technology
Expanding the intracellular landscape for novel biotherapeu0cs with Phylomer FPP technology For personal use only Novel endosomal escape trap for detec5on of efficient CPPs Streptavidin Valida5on of hits as best-in-class for cytoplasmic delivery, using an avitag Western blot and the Split GFP assay !!
Slide 62: Contact Details
Contact Details Dr. Paul WaN Director and Chief Scien0fic Advisor Tel: +61 8 9489 7777 For personal use only Fax: +61 8 9489 7700 Mobile: +61 421 550 213 paulw@phylogica.com www.phylogica.com 62