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Disclosures

Co-Inventor of Apo B Peptide Vaccines for Atherosclerosis,

Patent rights assigned to Cedars Sinai Medical Center

Y

YYYYYYY

Antigen

Antigen Presenting Cell

Y

Y

T cell

T cell proliferation

Inflammatory cytokine secretion (IFN gamma and TNF alpha)

Increased macrophage lipid uptake

Perpetuation of atherosclerosis Koltsova EK et al : JCI 2012

Dynamic T-cell and APC Interaction Perpetuates Vascular Inflammation and Atherosclerosis

Y

MHC TCR

Co-Stim

Co-Stim

Atherosclerosis is an Auto-Immune Mediated Inflammatory Disease

Pro-atherogenic Adaptive Immunity: The Yin

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

Adapted from Shah PK : Nature CPC 2005

Antigen

APC

Naive

T cell

Regulatory T cell

CD25+, Foxp3+

Secrete anti-inflammatory

cytokines

IL-10+/TGF β +

!"##$%&&'()*

(+*

,"-('..")%*/%&#()&%*

Athero-protective Adaptive Immune Response: The Yang

Treg

Tolerogenic

Dendritic Cell

,-0%$(1#$(-%2-'3%*

45%2-**

%$(1#$(-%2-'

Th1

Oxidized LDL in Human Coronary Plaques

Colocalizing with Macrophages

OxLDL

Macrophages

Auto-antigens

in

Atherosclerosis

LDL/Ox-LDL

Hsp-60

β2GP1 0

1

2

3

4

5

oxLDL - - + + + +

MC + - - + + +

MAb - - - - anti-DR Ctrl ab

T cells of Human Plaques

Recognize oxLDL as Antigen

Stemme et al

PNAS 1995

T-c

ell

pro

life

rati

ve i

ndex

Immunized

N=9

Immunization of Cholesterol-fed Rabbits with Homologous LDL Substantially

Reduces Aortic Atherosclerosis Despite Hypercholesterolemia

Exte

nt

of

Pla

que

(mm

2)

Ameli, Shah, Nillson et al :ATVB 1996

Nilsson , Ameli, Shah et al: JACC 1997

Cholesterol 1259 mg/dl 1181 mg/dl

Control

N=7

-Antigen: 280 mcg ox LDL or LDL

-Adjuvant: 700 mcg

-Primary SC Vaccination followed by 1 booster

-Animals euthanzied 16 weeks after vaccination

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

P<0.05

Palinski , Witztum et al : PNAS 1995

0

10

20

30

40

50

60

70

Control MDA-LDL

Rabbits Immunized Rabbits

(N=11) (N=14)

Immunization of LDL-Receptor Deficient (Watanabe Rabbits) with

Homologous Malondialdehyde (MDA) Modified LDL Reduces Atherogenesis

% of Aortic Surface with Plaque

P<0.005

CSMC--PKS

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

LDL Apo B-100

Apo B-100 550kD

4536 AA

Johs, A. et al. JBC 2006;281:19732-19739

302  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

20 Amino Acid Peptides

Identification of Antigenic Epitopes in Human Apo B-100

IgG and IgM antibodies were identified against 102 such peptide epitopes in pooled human plasma

Fredrikson et al. ATVB 2003;23:872-878

•  Human apo B-100 contains 4536 amino acids

•  Generate a library of 302 peptides to cover the complete

sequence of apo B-100

•  Each peptide is 20 amino acids long with 5 amino acids overlap

•  P2: ATRFK HLRKY TYNYE AESSS apo B-100 amino acids 16-35

•  P45: IEIGL EGKGF EPTLE ALFGK apo B-100 amino acids 661-680

•  P102 : SLTST SDLQS GIIKN TASLK apo B-100 amino acids 1516-1535

•  P210: KTTKQ SFDLS VKAQY KKNKH apo B-100 amino acids 3136-3155

Human Apo B-100 Related Peptides

Immunization of Apo E Null Mice with Apo B-100 related

Peptide Sequence Reduces Atherosclerosis

EEEMLENVSLVCPKDATRFK

ATRFKHLRKYTYNYQAQSSS

Peptide 1

Peptide 2

Mouse Apo B 100

Homology

75%

85%

Alum used as adjuvant

6-7 wks 8-9 wks 25 wks

Ist vaccination Booster Sacrifice

Chyu KY et al: BBRC 2005

(1-20)

(16-35)

Immunization of Apo E Null Mice with Apo B-100 related Peptide Sequence :

Effect on Cholesterol Levels and Aortic Atherosclerosis

0

0.5

1

1.5

2

2.5

3

3.5

Control)

1100 mg/dl

Peptide 1

700 mg/dl

Peptide 2

1400/mg/dl

% of Aortic Surface Covered by Plaque

P<0.01

N=9 N=10 N=10

Chyu : BBRC 2005

Cholesterol:

CSMC--PKS

Immunization of Apo E Null Mice with Apo B-100 related

Peptide Sequence Reduces Plaque Inflammation and Increases Collagen Content

0

2

4

6

8

10

12

14

Alum

(Control)

Peptide 1 Peptide 2

% Macrophage immunoreactivity

Immunization Group

p<0.05

N=9 N=10 N=10 0

5

10

15

20

25

30

35

40

45

Alum

(Control) Peptide 1 Peptide 2

% Collagen content (Trichrome)

Immunization Group

p<0.05

N=9 N=10 N=10

Chyu KY, et al : BBRC 2005 CSMC--PKS

0

0.5

1

1.5

2

2.5

3

3.5

Mice receiving

Splenocytes

From Alum

Immunized mice

% of Aortic Surface Covered by Plaque

P<0.01

N=9 N=9

Mice receiving

Splenocytes

From Peptide 2

Immunized mice

Adoptive Transfer of Splenocytes from Peptide 2 Immunized Mice Reduces

Atherosclerosis in Recipient Unimmunized Apo E Null Mice

Mice receiving

Splenocytes

From Peptide 1

Immunized mice

N=9

Chyu KY et al : BBRC 2005

Apo B-100 550kD

4536 AA

3136-3155

KTTKQ SFDLS VKAQY KKNKH

Amino acid #

p-210

Pentamer 5: QSFDLSVK sequence found in plaque derived microparticles.

•  p210: KTTKQSFDLSVKAQY KKNKH apo B-100 amino acids 3136-3155

CSMC--PKS

PBS

cBSA/A

lum

p210

-cBSA-A

lum

0

5

10

15P<0.05

% A

ort

ic l

esio

n

Apo B100 Related Peptide (p210) Vaccine Reduces Atherosclerosis in Hyperlipidemic Mice

61%

P210: apo B-100 amino acids 3136-3155 Chyu KY,Shah PK et al: PLoS1 2012

Additional Effects of P210 Vaccine

-  Reduced inflammation

-  Reduced plaque dendritic cells

-  No change in blood lipids

Adoptive Transfer of CD8 T-cells from p210 Immunized Mice Conveys Athero-protection to Unimmunized Mice

Chyu KY et al : PLos1 2012

P< ,0.05

p210 Vaccination Generates CD8+ T cells that Induce Lysis of Dendritic Cells loaded with P210

Chyu KY et al : PLos1 2012 Chyu KY et al : PLoS1 2012

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

Antigen

APC

Naive

T cell

Regulatory T cell

CD25+, Foxp3+

Secrete anti-inflammatory

cytokines

IL-10+/TGF β +

Suppression

of

Autoimmune Response

Potential Mechanisms of Athero-protective Effects of p210 Vaccine

Treg

Tolerogenic

Dendritic Cell

p210

Primed CD8 T cells

Athero-protective

Effect

ero-protecti

Th1

p210

Death of Dendritic Cells

Ath

tory

Th1

Autoim

JACC 2014

p210

Aortic Aneurysm Pathophysiology

Inflammation

Immune activation

Oxidative stress

Matrix proteolysis

SMC death

Daugherty et al Br J Pharmacol. 2001 October; 134(4): 865–870.

Abdominal aortic aneurysm formation after AngII

infusion in apoE-/- mice

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(798';"<<-,*'E*G2<*%'O216*)'.='>,*2)0%1"@'/*L1*,&%'Q'

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Honjo T: JACC 2015 Honjo T: JACC 2015

Honjo T: JACC 2015 Honjo T: JACC 2015

Conclusions

Immunization with selected Apo B-100 related peptide sequences

reduces atherosclerosis and favorably modifies plaque phenotype

in hypercholesterolemic apo E null mice

Apo B100 related peptide immunization also reduces AngII induced

aneuryms formation and rupture related mortality ,

These experimental observations provide proof of concept regarding

the feasibility and efficacy of peptide based vaccine for vascular disease

Cautiously

Optimistic

Lund University , Sweden Oppenheimer Atherosclerosis Research Center

P.K.Shah, MD

KY 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 Funding g NIH-NHLBI

Eisner Foundation

Cardiovax

The HeartFoundation

Spielberg Foundation

Corday Foundation