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Humanized Animal Models Quasi-Humanized Models Humanized Models Transgenic, knockout, or otherwise suppressed systems that mimic human processes, but do not contain primary human cells Transgenic, knockout, or otherwise suppressed systems that allow for primary human cell xenografts CXR/Taconic transADMETTM Model YecurisTM FRGTM KO Model

A Brief History of Humanized Mice Quasi-Humanized Humanized • Alb/uPA Transgenic - Heckel 1990 • Controllable FAH-/- KO - Grompe • Repop uPA - Rhim • Repop FAH-/- - Grompe 1995 2000 • Hu uPA/SCID / HCV - Mercer • 2D6(+) + HNF4α(-) - Corchero • Intestinal 3A4 - Granvil • Hu uPA/SCID / Metabolism - Tateno • Liver 3A4 - van Herwaarden 2005 • Hu uPA/SCID / Validation - Katoh • 3a(-), 3A4(+) - van Herwaarden • Hu FRGTM / Metabolism - Azuma • 3a(-), 3A4(+) - van Waterschoot • CAR/PXR - Sheer • Hu FRGTM / HCV - Bissig 2010 • 3a(-)/3a13(+)/3A4/3A7 and CAR/PXR/3A4/3A7 DDI - Hasegawa • Hu uPA/SCID / 3A4 DDI - Hasegawa 2012 • 2d6(-), 2D6 Variants - Sheer • Hu FRGNTM / L/B Malaria - Vaughan

Why Chimeric Systems? “If you do what you’ve always done, you’ll get what you’ve always gotten.” - Tony Robbins • Drugs are failing in new and increasingly creative ways Failure by Type, Phase III • Drugs are failing late in the process • Traditional tools are useful, but gaps are forming • New tools must be more physiologically relevant, be more predictive, and on net reduce costs

Chimeric Tools in Preclinical Development Early Tox Screening Clearance Induction / Inhibition Efflux Drug-Drug Interactions Clinic Metabolite ID • Toxicology • Genotox • Immunotox • Reprotox Uni-Dimensional Systems Biology • Primary Cell Bioreactors • Rate Determination for Low Clearance Drugs • Data Continuity • Identification of Human Specific Metabolism • Increasingly Translational • Species Specific Toxicology • ADME/Tox in Disease Model Context

Yecuris Corporation Founded in 2007 by Markus Grompe Location: Portland, Oregon Products and Property • Primary Human Hepatocytes • FRG on C57Bl/6 • FRG on NOD • FAH Rat and Pig Company Aim: To create a method of culturing high quality human hepatocytes and other primary cells for use in cell therapy applications utilizing animal bioreactor technologies.

In this presentation: Part I: Introduction and Background • Tyrosine catabolic pathway • The Yecuris FRG/c57 Bl6 mouse • The Yecuris FRG/NOD mouse Part II: Liver repopulation with human hepatocytes • Principals of Humanization • Characterization of FRG KO Humanized Livers • Liver Model Comparison Part III: Application Areas • In Vitro Cells • Infectious Disease • Safety/Toxicology • Cancer • Stem Cells

Part I: Introduction and Background Tyrosine Catabolic Pathway Yecuris FRG KO/c57 Bl6 Model Yecuris FRG KO/NOD Model

Tyrosine Catabolic Pathway Part I: Introduction and Background Tyrosine TAT 4-Hydroxyphenylpyruvic Acid NTBC PPD Liver Disease & Renal Injury Homogentisic Acid HGD Maleylacetoacetate MAI Fumarylacetoacetate Fumarylacetoacetate Succinylacetoacetate Succinylacetone FAR FAH FAH HT1 Fumarate Succinate + + Acetoacetate Acetoacetate

Core Technology – The Yecuris FRG Mouse Part I: Introduction and Background • Yecuris FRG KO/c57 Bl6 mouse contains three mutations: • FAH knockout induces liver disease • Rag2 (recombinant activating gene 2) knockout induces T and B cell deficiency • Il2rg (Interleukin 2 subunit γ-chain) knockout induces NK cell deficiency

Core Technology – Next Generation Yecuris Models Part I: Introduction and Background • Yecuris FRG KO/NOD (Beta Testing in Process) • FAH-/- / Rag2-/- / Il2rg-/- • Ability to integrate humanization of liver and immune systems • Improved model health and robustness

Core Technology – Next Generation Yecuris Models Part I: Introduction and Background • New Species of Humanized Models • FRG KO Rat: • Large single animal cell isolation • Industry standard species for toxicology studies • Larger liver and body weight for DMPK studies • FAH KO in the Pig: • Hepatocytes for cell therapy and liver assist • Human liver transplant

Part II: Liver Repopulation with Human Hepatocytes Principles of Humanization Characterization of FRG KO Humanized Livers Liver Model Comparison

Human Hepatocyte Repopulation in FRG Mice Part II: Liver Repopulation with Human Hepatocytes Cell Engraftment .5-1M Cells 4 Weeks 1-5M Cells 8 Weeks 5-10M Cells 12+ Weeks 50-150M Cells 20-50 μg/mL HSA >> 1% Liver Repopulation 200-500 μg/mL HSA 1-5% Liver Repopulation 2000-5000+ μg/mL HSA 20-95% Liver Repopulation 14

log2 Scale of Human Albumin vs. Percent Repopulation Part II: Liver Repopulation with Human Hepatocytes

Histology of Various Liver Repopulation Levels Part II: Liver Repopulation with Human Hepatocytes < 2% repopulation 20-30% repopulation 70-80% repopulation 70-80% repopulation 10-20% repopulation 20-30% repopulation 30-50% repopulation 70-80% repopulation

Integration of Human Hepatocytes into Mouse Liver Part II: Liver Repopulation with Human Hepatocytes Human hepatocytes Tertiary structure Mouse hepatocytes • Human cell expansion occurs in an ordered fashion, and does not exhibit invasive growth characteristics

Integration of Human Hepatocytes into Mouse Liver Part II: Liver Repopulation with Human Hepatocytes Bile Cannaliculi Tight Junctions • Electron Microscopy imaging of both bile duct and tight junctions between human cells in a human repopulated FRG KO mouse.

Yecuris Models Span a Wide Range of Donors Part II: Liver Repopulation with Human Hepatocytes • Yecuris has accumulated a large repertoire of donors that range in age from 5M to 83 years of age.

Recapitulation of Genetic Disorders for Studies Part II: Liver Repopulation with Human Hepatocytes • Cryopreserved cells of rare genetic disorders have been archived and are being used to create new models for rare diseases. • Custom models are frequently created to meet specific research requirements.

Comparison of Humanized Mouse Models Part II: Liver Repopulation with Human Hepatocytes • Stable transgenic knockout yielding no reversion of mouse cells • Robust expansion of hepatocytes of all ages • Ability to easily serially transplant and expand hepatocytes • Ability to ship and use models at client sites or third party CRO’s • Large healthy animals offer improved survivability, increased serial sampling, and lower cost per data point

Part III: Application Areas In Vitro In Vivo Infectious Disease Safety & Toxicology Stem Cells and Lines

Yecuris Model Allows for Serial Expansion Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines Human Donor 800K Mice 4K Mice 20 Mice 4 Months 4 Months 4 Months 1 Vial 5M Cells 200 Vials 2B Cells 40000 Vials 400B Cells 8M Vials 80T Cells

Humanized Mouse Model Yields High Quality Cells Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines 12 Days 7 Days .25 Day • Cryopreserved human hepatocytes from FRGTM KO mice, plated on collagen, no Percol, no Matrigel • 100% of isolations yield plateable cryopreserved cells

Cryopreserved Cells Exhibit Normal Phase II Activity Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines (UGT1A1, UGT1A6, UGT1A9, UGT2B15) (SULT1A1, SULT1A3/4, SULT1E1) • Cryopreserved human hepatocytes from FRGTM KO mice demonstrate normal transporter and robust Phase II activity

Hu-FRG KO Mice Express Human-Like Lipid Profiles – In Vivo Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines • Serum analysis conducted on control mice versus human cell repopulated FRG mice

Human Apo-E is detected in the serum of Hu-FRG KO Mice Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines human ApoE mouse ApoE

Hu-FRG KO Mice Express Human-Like Bile Acid Profiles – In Vivo Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines T-CA = Taurine conjugated cholic acid; G-CA = Glycine conjugated cholic acid; CA = total cholic acid • LC-MS/MS analysis of conjugated cholic acid in the gallbladder bile of human repopulated and control FRG KO mice. • Humanized FRG KO mice show glycine conjugation of cholic acid which is human specific.

Case Study 2 – Diclofenac Clearance Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines Diclofenac • Diclofenac is more rapidly cleared in humans than in rodents. Chimeric FRG mice recapitulate this clearance with high reproducibility

Case Study 1 – Lamotrigine Metabolism Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines WT C57Bl/6 Major Human Metabolite M3 (N2 Glucuronide) Lamotrigine Routes of Formation: UGT, GST, 2A6 (epoxide) Rag2-/- / Il2rg-/- Fah-/- / Rag2-/- / Il2rg-/- M8 M3 M2/M7 P M1/M5 M6

Case Study 1 – Lamotrigine Metabolism Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines Fah-/- / Rag2-/- / Il2rg-/- Major Human Metabolite M3 (N2 Glucuronide) Lamotrigine hFRG (50%) • At low levels of chimerism, the major human metabolite is observed in modest quantities • At levels of chimerism in excess of 80%, high levels of human metabolite are observed hFRG (90%) M3 P

Case Study 3 – Propafenone Metabolism Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines 4OH Derivatives 5OH Derivatives Propafenone Rodent Human • Propafenone metabolism is 2D6 mediated, and is highly conserved between species. It is cleared at least an order of magnitude more rapidly by mouse hepatocytes than human.

Case Study 3 – Propafenone Metabolism Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines Propafenone Drug Metab. Pharmacokinet. 25 (3): 223-235 (2010) 16th North American ISSX Meeting, 2009 • Despite efforts by Astellas, Merck, and Phoenix Bio, the 5-hydroxy human metabolite has never been observed in chimeric models.

Case Study 3 – Propafenone Metabolism Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines • 5-hydroxy metabolite is observed in highly chimerized animals, with no observed metabolite generation in control animals.

FRG as a Model for Hepatitis B and C – In Vivo Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines HBV • • Chimeric FRG KO mice were susceptible to both HBV and HCV infections. • HBV infections could be detected in mice that had only 10% human liver repopulation. • • HCV infections persisted for more than 34 weeks: • - Treatment with known HCV drugs reduced viral RNA. • Serial infection from mouse to mouse demonstrated. HCV FAH: Green; Hepatitis: Red

FRG as a Model for Malaria – In Vivo Part III: Application Areas In Vitro In Vivo Infectious Disease Safety / Toxicity Stem Cells and Lines • • P. falciparum sporozoites were injected by tail vein into a Chimeric FRG KO mouse and imaged at days three and seven. • Current efforts involve recapitulation of full cycle liver to blood stage infections in FRG-NOD KO mice.

Summary Part III: Application Areas • Large-scale single donor primary hepatocyte culture utilizing chimeric animals as bioreactors produces consistent high quality cells in large quantities • Use of same donor pools for cells and in vivo models provides a new level of data parity and integration of preclinical workflows • Chimeric systems can be used for clearance, metabolism, and toxicology, but: • - Quasi-humanized systems have shown compensatory mechanisms that may confound analysis • - Not all parameters and correction factors are known and much more study is necessary before • - Humanized systems are not (yet) fully human, so care must be taken to develop experimental parameters that isolate effects • Next generation systems are bringing disease state, metabolism, and toxicology into juxtaposition, which will allow for the development of novel platforms for biologics and vaccine development

Acknowledgements and Accolades Collaborators Milton Finegold, Department of Pathology, Texas Children’s Hospital Stephen Strom, University of Pittsburgh Cedo Bagi and Jiri Aubrecht at Pfizer Ashley Vaughan and Stefan Kappe at Seattle Biomed Xenotech, BioPredic, and Xenoblis for Analytical Studies Industrial Images Certain images and content courtesy of Pfizer and Seattle Biomed

Confidential. All Rights Reserved. 2012