Immunomodulatory therapy for parkinson’s disease

1. Eric Zanelli, PhD Bethesda, March 5, 2010 Induction of an anti-inflammatory immune response toward toxic species of alpha-synuclein Immunomodulatory therapy for parkinson’s disease

2. Parkinson’s Disease • Parkinson’s disease (PD) is a neurodegenerative disease with loss of dopamine-containing neurons in the substantianigra • Suggested causes of PD include: • Mitochondrial dysfunction • Oxidative stress • Impaired protein degradation processes (misfoldeda-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 a-syn-specific antibodies • Induces monocytes/microglia with anti-inflammatory phenotype

3. 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

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

5. Immune System Involvement in Parkinson’s Disease In Mouse 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, IFNg, TNFa by PBMCs from PD patients, Reale et al, Brain Behav Immun (2009) 23:55 • 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

6. The Copaxone/PI-2301 Experience Copaxone™ 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) • Approved by FDA in 1996 for treatment of RR-MS • 20-200 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?

7. 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 b 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

8. Decreased Production of pro-inflammatory Cytokines by Macrophages cultured with PI-2301

9. Immune Response alone will not work • Concept of vaccine therapy for neurodegenerative diseases is currently tested in man • Anti-bamyloid (Ab) 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, NeurodegenerDis (2008) 5:194 • T-cell response to Ab 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-2301

10. Proposed Design for a-syn Amino Acid Copolymer Tri-nitrations DNEAYEMPSEEGYQDYE Target Region: a-syn 121-137 Species Alterations Phosphorylation • 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 a-syn, only • without need for strong adjuvant, • while preserving anti-inflammatory properties found in Copaxone and PI-2301.

11. A testable hypothesis a-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. From: SH Appel, J Clin Invest (2009) 119:13