Transcript Determinant-specific Amino Acid Copolymers Induce Innate

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
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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 a-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
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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
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Amino Acid Copolymer Platform
DEEP: 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
Broad Application
Immune
Modulating
Copolymer
Epitope
specific
copolymer
Specific
Antigenic
Determinant
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 Therapeutic vaccines
for various disorders
 Prophylactic vaccines
against highly mutating
infectious agents
 Ligands for antibody
screening
Immune System Involvement in Parkinson’s
Disease
In Mouse
In Man
 Th17 cells
 CD4+ and CD8+ T 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
– 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,
 FasL+ CD4+ T cells
– contribute to neurodegeneration
in MPTP model,
Brochard et al, J Clin Invest (2009) 119:182
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Reale et al, Brain Behav Immun (2009) 23:55
The Copaxone/PI-2301 Experience
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Copaxone™
PI-2301
 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?
 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 antiinflammatory 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 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
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Decreased Production of pro-inflammatory
Cytokines by Macrophages cultured with PI-2301
IL-6 concentration in culture supernatant
of bone marrow-derived macrophages
- Study day 9 -
TNFa concentration in culture supernatant
of bone marrow-derived macrophages
- Study day 9 2750
15,000
2500
[TNFa ] culture supernatant (pg/mL)
[IL-6] culture supernatant (pg/mL)
12,500
10,000
7,500
5,000
2250
2000
1750
1500
1250
2,500
Copaxone
PI-2301
1000
PLP139-151
0
0
3
6
9
12
0
[Compound] (M)
3
6
9
[Compound] (M)
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Immune Response alone will not work
 Concept of vaccine therapy for neurodegenerative
diseases is currently tested in man
– Anti-b amyloid (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, Neurodegener Dis (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
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Proposed Design for a-syn Amino Acid Copolymer
Tri-nitrations
Target Region: a-syn 121-137
DNEAYEMPSEEGYQDYE
Species Alterations






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
•
•
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Phosphorylation
without need for strong adjuvant,
while preserving anti-inflammatory properties found in Copaxone and PI-2301.
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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
–
From: SH Appel, J Clin Invest (2009) 119:13
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Protection of nigrostriatal pathway.