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. 1

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

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

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

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

Immunization of Cholesterol-fed Rabbits with Homologous LDL Reduces Aortic Atherosclerosis Despite Hypercholesterolemia Ameli, Shah, Nillson et al: ATVB 1996 Nilsson, Ameli, Shah et al: JACC 1997 Immunized N=9 Extent of Plaque (mm 2 ) Cholesterol1259 mg/dl 1181 mg/dl Control N=7 P<0.05 6

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) Effect of Immunization with Native and Oxidized Homologous LDL On Experimental Atherosclerosis in Hypercholesterolemic Mice 7

Apo B kD 4536 AA Johs, A. et al. JBC 2006;281: 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 KTTKQ SFDLS VKAQY KKNKH Amino acid p210

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

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

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

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

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

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

% Total Aneurysmal Segment number of aneurysmal segment/number of total segment (%) Number of aneurysmal segment (severity of disease) was significantly reduced in p210 immunized mice. (Aneurysmal segment = diameter>1.35mm) Honjo T: Circulation (abst) p<0.05

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

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

P<0.05 AngII Honjo T: Circulation (abst)

N=6, P<0.05 IB; AT1R IB; GAPDH PBScBSAp210 p210 vaccine reduced the expression of AT1R in whole aorta. P210 Vaccine Reduces AT1R Expression in Aorta Honjo T: Circulation (abst)

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. 20

Lund University, Sweden Oppenheimer Atherosclerosis Research Center 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 Jan Nilsson, MD, PhD GN Fredriksson,PhD Cardiovax Funding NIH-NHLBI Eisner Foundation Cardiovax The HeartFoundation Spielberg Foundation Corday Foundation 21

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

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

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