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Nanoparticle-Based Immune Tolerance Therapies for Treatment of Multiple Sclerosis Miller Lab

Nanoparticle-Based Immune Tolerance Therapies for Treatment of Multiple Sclerosis Miller Lab Rosenblum MS Foundation Presentation Chicago, IL June 4, 2014 Stephen D. Miller, Ph.D. Department of Microbiology-Immunology and The Interdepartmental Immunobiology Center

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Nanoparticle-Based Immune Tolerance Therapies for Treatment of Multiple Sclerosis Miller Lab

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  1. Nanoparticle-Based Immune Tolerance Therapies for Treatment of Multiple Sclerosis Miller Lab Rosenblum MS Foundation Presentation Chicago, IL June 4, 2014 Stephen D. Miller, Ph.D. Department of Microbiology-Immunology and The Interdepartmental Immunobiology Center Northwestern University Medical School Chicago, IL

  2. Multiple Sclerosis HEALTHY NERVE nerve cell nerve axon scarred myelin myelin sheath DAMAGED NERVE Multiple sclerosis (MS) is a demyelinating disease characterized by inflammatory, autoimmune-mediated attacks resulting in variable relapsing or progressive central nervous system deficits. • MS is a heterogeneous disease which affects 400,000 in the United States and 2.5 million people around the world. • Cause of MS is unknown - but it is associated with an infectious etiology

  3. Protocol for Induction of Relapsing Experimental Autoimmune Encephalomyelitis (R-EAE) in SJL Mice Day 0: 20-100 g Myelin Protein or m Peptide/CFA s.c. 11-21 Days Clinical Signs: 1 limp tail 2 limp tail & hindlimb weakness 3 partial hindlimb paralysis 4 complete hindlimb paralysis 5 moribund

  4. MBP84-104 a a PLP178-191 PLP139-151 a a a PLP139-151 MBP84-104 PLP178-191 a a ? a MAG/ MOG a ? MAG/ MOG IFN-g IL-17 TNF-a LT IL-4, IL-10 CD4+CD25+ Tregs T cell apoptosis a o ? a PLP 3 epitope o a ? PLP 3 epitope Endogenous Ags Intra- and Intermolecular Epitope Spreading in R-EAE in SJL Mice: A Model of Relapsing-Remitting Multiple Sclerosis 1 RELAPSE 2 RELAPSE o o ACUTE PHASE 2 RELAPSE o ACUTE PHASE 1 RELAPSE o CLINICAL SEVERITY CLINICAL SEVERITY 10 20 30 40 50 60 70 10 20 30 40 50 60 70 DAYS POST-INDUCTION DAYS POST-INDUCTION PLP139-151 PRIMED PRIMED • T cells specific for spread epitopes can be isolated from the periphery and the CNS of mice with ongoing disease following recovery from acute disease

  5. Activated T Cells, Microglia and Macrophages Accumulate in EAE Lesions - CARS Microscopy 20µm Control tissue EAE tissue (score 2) • Red: myelin sheath • Green: IB4-FITC (microglia and macrophage). Ji-Xen Chen Purdue Univ.

  6. Tolerance Strategies for Treatment of Immune-Mediated Diseases • Immunologic Tolerance - antigen-specific inhibition of selective immune responses is the‘holy grail’ and the ‘future’ for the treatment of autoimmune diseases, allergy and tissue transplantation • Question -can ‘induced’ peripheral immune tolerance be used to treat Th1/17-mediated autoimmune disease, Th2-mediated allergic responses or prevent graft rejection in the absence of the application of immune subset depleting agents or prolonged use of immunosuppressive drugs/Abs/biologics • GOAL – ‘cure’ disease by specifically targeting ‘only’ the immunopathologic T cellsrather than continuously ‘suppressing’ the effector molecules and trafficking of autoreactive T cells with mAbs/biologics/small molecules Former Lab People Carol Vanderlugt Todd Eagar Cassandra Smith Emma Feeney Danielle Turley Aaron Martin Adam Kohm Current Lab People Daniel Getts Joe Podojil Suchitra Prasad Charles Smarr Derrick McCarthy Chris Harp Zoe Hunter Rachael Terry • FSM Collaborators • Xunrong Luo – Islet Transplant • Taba Kheramand • Sushen Wang • Paul Bryce – Allergy • Chia-Lin Hsu • Lonnie Shea – Nanoparticles • Woon Teck Yap • Kelan Hlavaty

  7. Clone 1 2 3 4 5 Clone 1 2 3 4 5 . . . 10-100x106 . . . 10-100x106 Anti- Influenza Virus Anti- Salmonella Anti- Hepatitis Virus Anti- Lung Cancer Protein Anti- Myelin Anti- Influenza Virus Anti- Salmonella Anti- Hepatitis Virus Anti- Lung Cancer Protein Anti- Myelin MS Composition of the Immune Repertoire in a Healthy Individual vs. a MS Patient Healthy Individual MS Patient

  8. Clone Clone 1 1 2 2 3 3 4 4 5 5 . . . 10-100x106 . . . 10-100x106 X X X X X Anti- Influenza Virus Anti- Influenza Virus Anti- Salmonella Anti- Salmonella Anti- Hepatitis Virus Anti- Hepatitis Virus Anti- Lung Cancer Protein Anti- Lung Cancer Protein Anti- Myelin Anti- Myelin MS MS Comparison of Immunosuppressive vs. Tolerance Therapies for Treatment of MS Immunosuppressive Therapy Tolerance Therapy X

  9. Protocol for Inducing T Cell-Mediated Immunity and Antigen-Specific Tolerance Using ECDI-Fixed Splenocytes (Ag-SP) RBCs lysed Single Cell Suspension Spleens removed from naïve syngeneic mice Peptide 1 hour, 4o C ECDI • Covalent attachment of peptide • Apoptosis s.c. DTH i.v. Antigen-coupled Splenocytes (Ag-SP) Tolerance Miller, et. al. J. Exp. Med. 149:758-773, 1979

  10. Ag-SP Tolerance Therapy in Regulation Induction and Progression of Relapsing EAE in SJL Mice MBP84-104 a a PLP178-191 PLP139-151 a a a PLP139-151 MBP84-104 PLP178-191 a a ? a MAG/ MOG a ? MAG/ MOG IFN-g IL-17 TNF-a LT IL-4, IL-10 CD4+CD25+ Tregs T cell apoptosis a o ? a PLP 3 epitope o a ? PLP 3 epitope PLP178-SP Tolerance PLP139-SP Tolerance PLP139-SP Tolerance PLP139 Tolerance 1 RELAPSE 2 RELAPSE o o ACUTE PHASE 2 RELAPSE o ACUTE PHASE 1 RELAPSE o CLINICAL SEVERITY CLINICAL SEVERITY 10 20 30 40 50 60 70 10 20 30 40 50 60 70 DAYS POST-INDUCTION PLP178-191 DAYS POST-INDUCTION PLP139-151 PRIMED PRIMED • Autoimmune diseases are moving targets due to epitope spreading – also true for pathogenesis of spontaneous T1D in NOD mice • Tolerance-based immunotherapy can be used to treat ongoing autoimmune disease • Caveat - Inhibition of relapses by peptide-specific tolerance administered during disease remission is only induced by the spread epitope

  11. Potential Clinical Applications of Antigen-Coupled APC Tolerance • Tolerance to alloantigens or xenoantigens- to promote long-term acceptance of organ and tissue grafts and bone marrow transplants without the need for immunosuppressive therapy • CD4 Th2-mediated diseases - allergic asthma, topical allergies and food allergies • Tolerance induction to recombinant enzymes, proteins or humanized antibody therapeutics- preceding initiation of enzyme replacement or virus-delivered gene therapy in genetic enzyme deficiency disorders [e.g.Pompe disease ( glucosidase); Fabry disease ( galactosidase); hemophilia (Factor VIII or IX); AAV-delivered dystrophin, e.g. in MD]; antibody therapy [e.g.Remicaid, etc.]; or cytokine therapy [e.g. IFN-β] • CD4 Th1/Th17-mediated autoimmune diseases: • MS - myelin protein/peptide coupled PBLs • NMO – aquaporin 4 (AQP4)-coupled PBLs • Type 1 Diabetes - insulin-coupled PBLs • Atherosclerosis - Ox-LDL/Hsp60-coupled PBLs • Celiac Disease - gluten-coupled PBLs

  12. EstablishTolerance In MS (ETIMS) - MRI-Controlled Phase I*/IIA Trial for Treatment of Relapsing MS Using Peptide-Coupled PBL Tolerance Stephen Miller, Ph.D. – Northwestern Univ. Medical School Roland Martin, M.D. & Andreas Lutterotti, M.D. – Univ. of Hamburg/Zürich Early R-R MS Patient Leukocyta-phaeresis PBLs (Pre-existing T cell response To 1-2 myelin epitopes) MBP13-32 MBP83-99 MBP111-129 MBP146-170 MOG1-20 MOG35-55 PLP139-154 Cocktail of Immunodominant Myelin Peptides ECDI i.v. re-infusion of autologous Ag-PBLs (3x109) Antigen-coupled PBLs (Ag-PBL) Funding: German Government Cumming Foundation Myelin Repair Foundation *Phase I successfully completed 11/2011, Phase IIa will start 9/2012

  13. ETIMS Phase I Design and Overall Results • Administration of antigen-coupled PBMCs had a favorable safety profile and was well tolerated in MS patients. • Compared to the pre-treatment observation period there was no increase in clinical and MRI parameters of disease activity and in most patients no new MRI lesions were observed in the 6 month follow-up. • Patients receiving the higher doses (>109) of peptide-coupled PBMCs had a decrease in antigen-specific T cell responses following cell therapy. Lutterotti, et al. 2013. Sci. Transl. Med. In press.

  14. Ag-bearing biodegradable PLGnanoparticlesserve as surrogates for apoptotic membranes for efficient induction of tolerance Antigen-coupled apoptotic cells 1979 Antigen-coupled carboxylated PLG Nanoparticle 2008 Tolerogenic Immune Modifying Particle (TIMP) 2012 - - - - Next Generation Next Generation - - - - - - - - - - - - - - - - - - - - - - - Miller Lab – Northwestern University Daniel Getts, Ph.D. Aaron Martin, Ph.D. Chris Harp, Ph.D. Derrick McCarthy, B.S. Zoe Hunter, Ph.D. Rachael Terry Lonnie Shea – Northwestern University Woon Teck Yap Kelan Hlavaty

  15. Antigen-Coupled Biodegradable PLG Nanoparticles Effectively Prevent, but more Importantly, Therapeutically Treat Established Autoimmune Disease in the EAE Model of MS d-7 Rx Preventative Tolerance: d+10 Rx Therapeutic Tolerance:

  16. Results of Double-Blind, Two-Arm Validation of Ag-PLG Nanoparticles Tolerance Performed by CBI Ability of PLP139-151-coupled PLG Nanoparticles to prevent and treat Active EAE is validated

  17. Proposed Mechanisms of Ag-Nanoparticle Tolerance (Recapitulates how tolerance is maintained in the hematopoietic compartment) BLOOD BLOOD SPLEEN/LIVER TIMPs bind to monocytes – divert to spleen Antigen Presenting Cells (APC) take up & process TIMP Control of Autoreactive T cells Peripheral T cell regulation 1 Proprietary surface modification: ζ = < 50 mV Disease specific autoreactive antigen SPLEEN LIVER 1 - - - Deletion 2 - 500 nm 2 Auto-reactive T cell Anergy MHC TCR MARCO 3 Naïve T Cell Naïve T cell APC PD1 Monocyte PD-L1 CD28 TFG-b IL-10 Naïve T Cell Naïve T Cell Naïve T Cell Naïve T Cell TIMP Naïve T Cell iTreg 3 iTreg iTreg iTreg • Apoptosis • Apoptosis of TIMP-monocytes in the spleen/liver • TIMP and apoptotic debris picked up by antigen presenting cells (APCs) in spleen/liver • Antigen Presentation • Autoreactive antigen is: • processed by APCs • presented to T cells • T cells get: • negative co-stimulation – PD-L1 • anti-inflammatory milieu (IL-10 / TGF-β) • Immune Tolerance • T cell deletion or anergyfrom: • negative co-stimulation • lack of positive stimulation • IL-10 and TGF-β induce Tregs • maintain tolerance Naïve T Cell iTreg Naïve T Cell

  18. Advantages of Ag-PLG NPs for Treatment of (Auto)Immune Diseases • Rapidity and simplicity of tolerogen preparation and induction using a GMP manufacturable off-the-shelf ‘universal’ tolerogenic carrier - theoretically useful for any immune-mediated disease by simply switching the Ag • No need to isolate and expand immature DCs (or Tregs) ex vivo • No need to be concerned with immature DCs being activated upon ex vivo manipulation and becoming ‘stimulatory’ rather than tolerogenic; or Tregs converting to Th1/17 after transfer • Directs Ag to ‘tolerogenic’ DCs in splenic MZ and liver via the MARCO scavenger receptor - ‘host’tolerogenic APCs process and represent the antigen in a tolerogenic manner,i.e. host APCs can select the relevant immunodominant self epitopes from PLG NPs encapsulating intact auto-Ags or tissue extracts(e.g.OVA encapsulated PLG NPs prevent OVA/Alum-induced AAD) • The protocol appears to be exquisitely Ag-specific (no apparent bystander suppression), non-toxic, safe, highly efficient and can induce unresponsiveness in both effector T cells (Th1, Th2, Th17 and CD8) and naïve T cells involved with epitope spreading • PLG Nanoparticles carry inherent anti-inflammatory properties - enhances their ability to induce tolerance

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