PEPTIMMUNE CONFIDENTIAL

IMMUNOMODULATORY IMMUNOMODULATORY THERAPY FOR PARKINSON’S THERAPY FOR PARKINSON’S DISEASEDISEASE

Induction of an anti-inflammatory immune response toward toxic species of alpha-synuclein

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Eric Zanelli, PhDEric Zanelli, PhDBethesda, March 5, 2010Bethesda, March 5, 2010

PEPTIMMUNE CONFIDENTIAL

Parkinson’s Disease 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 thatPD patients would benefit from an immunotherapy that

– Clears toxic oligomers and protofibrils through Clears toxic oligomers and protofibrils through -syn-specific antibodies-syn-specific antibodies

– Induces monocytes/microglia with anti-inflammatory phenotypeInduces monocytes/microglia with anti-inflammatory phenotype

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Immunomodulatory Approach for PDImmunomodulatory 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

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Amino Acid Copolymer PlatformAmino Acid Copolymer PlatformDEEP: Directed Expansion of Epitope PermutationsDEEP: Directed Expansion of Epitope Permutations

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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

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Immune System Involvement in Parkinson’s Immune System Involvement in Parkinson’s Disease 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

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The Copaxone/PI-2301 ExperienceThe Copaxone/PI-2301 Experience

Copaxone™ 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 Suspected

neuroprotective effect?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)

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Copaxone in Animal Models of NeurodegenerationCopaxone 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

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IL-6 concentration in culture supernatantof bone marrow-derived macrophages

- Study day 9 -

0 3 6 9 12

0

2,500

5,000

7,500

10,000

12,500

15,000

[Compound] (M)

[IL

-6] c

ultu

re s

uper

nata

nt (

pg/m

L)

TNF concentration in culture supernatantof bone marrow-derived macrophages

- Study day 9 -

0 3 6 9 12

1000

1250

1500

1750

2000

2250

2500

2750

[Compound] (M)

[TN

F] c

ultu

re s

uper

nata

nt (

pg/m

L)

PI-2301

PLP139-151

Copaxone

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

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Immune Response alone will not workImmune 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 Importance of maintaining the correct Th2 response as induced by Copaxone or PI-2301as induced by Copaxone or PI-2301

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Proposed Design for Proposed Design for -syn Amino Acid Copolymer-syn Amino Acid Copolymer

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DNEAYEMPSEEGYQDYEDNEAYEMPSEEGYQDYE

Tri-nitrationsTri-nitrations

PhosphorylationPhosphorylationSpecies Species AlterationsAlterations

Target Region: Target Region: -syn 121-137-syn 121-137Target Region: Target Region: -syn 121-137-syn 121-137

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.

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A testable hypothesisA 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.

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From: SH Appel, J Clin Invest (2009) 119:13