SHAPE Scientific Symposium, Dallas, TX 2013 Concept of a Vaccine for Vascular Disease

1. SHAPE Scientific Symposium, Dallas, TX 2013 Concept of a Vaccine for Vascular Disease Modulating Autoimmunity in Atherosclerosis Using a Vaccine Kuang-Yuh Chyu, MD, PhD Associate Director, CICU Cedars Sinai Heart Institute Cedars-Sinai Medical Center Disclosures: Co-Inventor of ApoB-100 Peptide Vaccines for Atherosclerosis. Patent rights are assigned to Cedars-Sinai Medical Center.

2. Atherosclerosis is an Auto-Immune Mediated Inflammatory Disease Pro-atherogenic Adaptive Immunity APC Inflammatory Cytokines IFN- γ and TNF-α Th1 Auto antigen NKT MHC/CD1 Atherosclerosis Naïve T cell Inflammatory Cytokine IL 17 Th 17 Activated CD4+ T-cell

3. Athero-protective Adaptive Immune Response Treg Antigen Suppression of Autoimmune Response APC Regulatory T cell CD25+, Foxp3+ Secrete anti-inflammatory cytokines IL-10/TGF-β Naive T cell Th1 Tolerogenic Dendritic Cell Athero-protective Effect

4. Oxidized LDL in Human Coronary Plaques Colocalizing with Macrophages Auto-antigens in Atherosclerosis LDL/Ox-LDL Hsp-60 2GP1 OxLDL Macrophages

5. Would Immunization with LDL/oxLDL Worsen or Reduce Atherosclerosis?

6. Immunization of Cholesterol-fed Rabbits with Homologous LDL Reduces Aortic Atherosclerosis Despite Hypercholesterolemia Extent of Plaque (mm2) P<0.05 Control N=7 Immunized N=9 Ameli, Shah, Nillson et al: ATVB 1996 Nilsson, Ameli, Shah et al: JACC 1997 Cholesterol 1259 mg/dl 1181 mg/dl

7. Effect of Immunization with Native and Oxidized Homologous LDL On Experimental Atherosclerosis in Hypercholesterolemic Mice Immunization of LDL-R null mice with homologous native or MDA-LDL reduces atherosclerosis (Freigang et al ATVB 1998) Immunization of apo E null mice with homologous MDA-LDL reduces atherosclerosis (George et al : Atherosclerosis 1998) Timing affects the athero-protective effects of LDL-immunization (Chyu, Shah et al. Atherosclerosis 2004)

8. Apo B-100 550kD 4536 AA • Peptides, 20 amino acids long with 5 amino acid overlap simulating the entire amino acid sequence of human Apo B 100 were synthesized. • Using an ELISA with peptides sequences as antigens, antibodies to 101 of these peptide sequences were identified in pooled human sera • Several peptide sequences were then used to create vaccines for Immunization in apo E null mice fed a high cholesterol diet Amino acid 3136-3155 KTTKQ SFDLS VKAQY KKNKH p210 Johs, A. et al. JBC 2006;281:19732-19739

9. Apo B100 Related Peptide (p210) Vaccine Reduces Atherosclerosis in Hyperlipidemic Mice 61% Chyu KY, Shah PK et al: PLoS One 2012 P210: apo B-100 amino acids 3136-3155

10. Apo B100 Related Peptide (p210) Vaccine Reduces Plaque Macrophages in ApoE-/- Mice * p<0.05 PBS Adjuvant p210 Control Control Vaccine Chyu KY, Shah PK et al: PLoS One 2012

11. Apo B100 Related Peptide (p210) Vaccine Reduces Plaque Dendritic Cells in ApoE-/- Mice p < 0.05 PBS Adjuvant p210 Control Control Vaccine Chyu KY, Shah PK et al: PLoS One 2012

12. Adoptive Transfer of CD8 T-cells from p210 Immunized Mice Conveys Athero-protection to Unimmunized Mice p < 0.05 Chyu KY et al : PLoS One 2012

13. Adoptive Transfer of p210 loaded Dendritic Cells reduces Atherosclerosis in Apo E-/- Mice Nasal delivery of p210-CTB Fusion Construct reduces Atherosclerosis in Apo E-/- Mice Prolonged subcutaneous delivery of p210 reduces Atherosclerosis in Apo E-/- Mice

14. Abdominal aortic aneurysm formation after AngII infusion in apoE-/- mice Daugherty et al Br J Pharmacol. 2001 October; 134(4): 865–870.

15. % Total Aneurysmal Segment number of aneurysmal segment/number of total segment (%) 1 2 (Aneurysmal segment = diameter>1.35mm) 3 4 p<0.05 5 8 6 7 Number of aneurysmal segment (severity of disease) was significantly reduced in p210 immunized mice. Honjo T: Circulation (abst) 2011

16. p210 Vaccine Reduces Mortality in AngiotensinII Induced Aneurysm Model in ApoE-/- Mice Survival Curve Mortality P210 = 6.2% PBS = 32.1% cBSA = 29.7% p<0.05 vs. PBS p<0.05 vs. cBSA by Logrank test Honjo T: Circulation (abst) 2011

17. p210 Vaccine Reduces Pro-inflammatory IL-17 Positive CD4 T cells * * CD4+IL-17A+ p < 0.05 N: PBS=8, cBSA=6, p210=6 Honjo T et al : unpublished data 2013

18. P<0.05 AngII P<0.05 AngII Honjo T: Circulation (abst) 2011

19. P210 Vaccine Reduces AT1R Expression in Aorta IB; AT1R IB; GAPDH N=6, P<0.05 PBS cBSA p210 p210 vaccine reduced the expression of AT1R in whole aorta. Honjo T: Circulation (abst) 2011

20. Conclusions • Immunization with selected ApoB-100 peptides reduces atherosclerosis and favorably modifies plaque phenotype in hypercholesterolemic mice. • Immunization with selected ApoB-100 peptides also reduced AngII induced hypertension and aneurysm formation and mortality due to rupture. • These experimental observations provide proof of concept regarding the feasibility and efficacy of apoB-100 peptide based vaccine for vascular diseases.

21. Lund University, Sweden Oppenheimer Atherosclerosis Research Center Jan Nilsson, MD, PhD GN Fredriksson,PhD Cardiovax P.K.Shah, MD Kuang-Yuh Chyu, MD, PhD Paul Dimayuga,PhD Tomoyuki Honjo, MD,PhD Xiaoning Zhao,PhD Jianchang Zhou, PhD Nicole Lio Bojan Cercek, MD, PhD Cheryl Chan Juliana Yano Funding NIH-NHLBI Eisner Foundation Cardiovax The HeartFoundation Spielberg Foundation Corday Foundation

22. Potential Mechanisms of Action of p210 Vaccine Increased Lysis of APC p210 Suppression of Antigen Specific adverse Autoimmune Response APC CD8+ Regulatory T cell CD25+, IL-10+ CD8+T cell

23. IL-17 Deletion Protects Mice from AngiotensinII Induced Aortic Aneurysm Formation Survival Incidence 0% 46.7% Honjo T et al : unpublished data 2013

24. Abdominal Aortic Aneurysm Pathophysiology Inflammation Immune activation Oxidative stress Matrix proteolysis SMC death