PEPTIMMUNE CONFIDENTIAL IMMUNOMODULATORY THERAPY FOR PARKINSON’S DISEASE Induction of an anti-inflammatory immune response toward toxic species of alpha-synuclein 1 Eric Zanelli, PhD Bethesda, March 5, 2010

PEPTIMMUNE CONFIDENTIAL Parkinson’s Disease  Parkinson’s disease (PD) is a neurodegenerative disease with loss of dopamine-containing neurons in the substantia nigra  Suggested causes of PD include: –Mitochondrial dysfunction –Oxidative stress –Impaired protein degradation processes (misfolded  -synuclein)  Additionally, immune system involvement is established (either primary or secondary) –Innate immunity –Adaptive immunity  PD patients would benefit from an immunotherapy that –Clears toxic oligomers and protofibrils through  -syn-specific antibodies –Induces monocytes/microglia with anti-inflammatory phenotype 2

PEPTIMMUNE CONFIDENTIAL Immunomodulatory Approach for PD  Target Product Profile: –First-line disease modifying treatment –Weekly or bi-weekly subcutaneous administration in a pre-filled syringe/auto-injector –Exhibit excellent long-term safety and tolerability profile enjoyed by marketed copolymers such as Copaxone™ (Teva)  Dual Mechanisms of Action: –Induces antibody-mediated clearance of post-translationally modified, toxic alpha-synuclein oligomers and protofibrils found in PD patients –Induces an immunoregulatory, neuroprotective immune response capable of dampening inflammatory microenvironments found in PD patients 3

PEPTIMMUNE CONFIDENTIAL Amino Acid Copolymer Platform DEEP: Directed Expansion of Epitope Permutations 4 What is an amino acid copolymer? A single manufacturing peptide entity comprising multiple related antigenic determinants  Promiscuous MHC class II binding  Enhanced immunogenicity What is an amino acid copolymer? A single manufacturing peptide entity comprising multiple related antigenic determinants  Promiscuous MHC class II binding  Enhanced immunogenicity Broad Application  Therapeutic vaccines for various disorders  Prophylactic vaccines against highly mutating infectious agents  Ligands for antibody screening Immune Modulating Copolymer Specific Antigenic Determinant Epitope specific copolymer

PEPTIMMUNE CONFIDENTIAL Immune System Involvement in Parkinson’s Disease In Mouse  Th17 cells –promote neurodegeneration in MPTP model, Reynolds et al, J Immunol (2010) 184:2261  Vasoactive Intestinal peptide (VIP) –induces Treg which attenuate microglia-mediated inflammation, Reynolds et al, J Immunol (2010) 184:2261  FasL + CD4 + T cells –contribute to neurodegeneration in MPTP model, Brochard et al, J Clin Invest (2009) 119:182 In Man  CD4 + and CD8 + T cells – ten-fold increase in substantia nigra in PD patients as compared to age-matched controls, Brochard et al, J Clin Invest (2009) 119:182  Pro-inflammatory markers – Increased production of MCP-1, IL-8, IFN, TNF by PBMCs from PD patients, Reale et al, Brain Behav Immun (2009) 23:55 5

PEPTIMMUNE CONFIDENTIAL The Copaxone/PI-2301 Experience Copaxone™  Approved by FDA in 1996 for treatment of RR-MS  amino acid long peptides made of Y, E, A and K  Limited effect on monocytes  Induces regulatory T-cell response  Limited bioavailability  Suspected neuroprotective effect? PI-2301  Phase II in RR-MS initiated  52-amino acid-long peptides made of Y, F, A and K  Improved MHC class II binding  Induction of anti- inflammatory response in man demonstrated  Better preclinical efficacy  Better effect on monocytes  Improved bioavailability (N-terminal acetylation) 6

PEPTIMMUNE CONFIDENTIAL Copaxone in Animal Models of Neurodegeneration  Copaxone-specific T-cells protect mice from MPTP toxicity Benner et al, Proc Natl Acad Sci USA (2004) 101:9435 –Effect results in markedly decreased activation of microglia –Increased expression of Glial cell-Derived Neutrophic Factor (GDNF) might play a role  Copaxone vaccination reduces  amyloid accumulation in APP/PS1 transgenic mice Butovsky et al, Proc Natl Acad Sci USA (2006) 103:11784 –Induction of phenotype switch in microglia –Increased production of Insulin-like Growth Factor-1 (IGF-1) by microglia 7

PEPTIMMUNE CONFIDENTIAL Decreased Production of pro-inflammatory Cytokines by Macrophages cultured with PI-2301

PEPTIMMUNE CONFIDENTIAL Immune Response alone will not work  Concept of vaccine therapy for neurodegenerative diseases is currently tested in man –Anti-  amyloid (A  ) trials through either active or passive immunization in Alzheimer –6% of Alzheimer’s patients treated with AN1792 in Phase IIa (study 201) developed meningoencephalitis, Pride et al, Neurodegener Dis (2008) 5:194 –T-cell response to A  peptide was characterized as Th1 in contrast to Th2 response observed in study 102 Changes in formulation? –Antibody responses in both studies were similar –Use of adjuvant QS-21 probably promoted the Th1 response  Importance of maintaining the correct Th2 response as induced by Copaxone or PI

PEPTIMMUNE CONFIDENTIAL Proposed Design for  -syn Amino Acid Copolymer 10 DNEAYEMPSEEGYQDYE Tri-nitrationsTri-nitrations PhosphorylationPhosphorylation Species Alterations Target Region: -syn  Immune response targeted at a 17-amino acid region,  Specificity for toxic species guaranteed through use of phosphorylated Ser (S) and nitrated Tyr (Y),  Substitutions incorporated to account for interspecies variabilities,  Immunogenicity guaranteed by % Ala (A) incorporation at every position and compound length through tandem-repeats of the same region,  Tyr (Y) and Glu (A) also found in Copaxone provide anchoring residues to various MHC class II molecules and T-cell help,  Goal is to induce specific immune response to toxic species of -syn, only without need for strong adjuvant, while preserving anti-inflammatory properties found in Copaxone and PI  Immune response targeted at a 17-amino acid region,  Specificity for toxic species guaranteed through use of phosphorylated Ser (S) and nitrated Tyr (Y),  Substitutions incorporated to account for interspecies variabilities,  Immunogenicity guaranteed by % Ala (A) incorporation at every position and compound length through tandem-repeats of the same region,  Tyr (Y) and Glu (A) also found in Copaxone provide anchoring residues to various MHC class II molecules and T-cell help,  Goal is to induce specific immune response to toxic species of -syn, only without need for strong adjuvant, while preserving anti-inflammatory properties found in Copaxone and PI-2301.

PEPTIMMUNE CONFIDENTIAL A testable hypothesis  -synuclein amino acid copolymer induces:  In vitro –An expansion of anti-inflammatory monocytes and/or T-cells with regulatory properties, –Antibodies capable of clearing misfolded protein deposits.  ASO Mice –A reduction in alpha-synuclein burden, –Specific effects on motor and olfactory measurements in ASO mice, –Alterations in striata and ventral midbrain.  MPTP-induced Toxicity –Protection of nigrostriatal pathway. 11 From: SH Appel, J Clin Invest (2009) 119:13