SHAPE Society

1. Passive immunization using
recombinant IgG against apo B-
100 peptides
Jan Nilsson
Department of Clinical Sciences
Malmö, Lund University

2. Adaptive immune responses to oxidized LDL

3. Effect of oxidized LDL peptide
vaccines on atherosclerosis in mice
0
0,05
0,1
0,15
0,2
0,25
0,3
Control Peptide 45 Peptide 210 Peptide 240
%atherosclerosis
P<0.01
P<0.005
Nordin Fredrikson et al, Autoimmunity 2005

4. Phage display selection
Infection of E.Coli
Amplification of phage
MDA apo B peptides
- biotinylated B
Isolation
phage-antigen-biotin
complex with streptavidin
B
B SA
Additional rounds of selection or
Isolation of desired antibody
Phage antibody
The Antibody

5. Two mAbs used in in vivo
experiments
The Antibody
• Peptide 4 corresponds to MDA apo B-100 peptide 45 (amino
acids 661-680)
• Peptides 1-3, 5 and 6 are different MDA Apo B-100 peptides
• Affinity was estimated with Biacore on Apo B-100
0
20
40
60
80
100
120
2D03 IEI-E3
Normalizedratio
Peptide 1
Peptide 2
Peptide 3
Peptide 4
Peptide 5
Peptide 6
A
0
200
400
600
800
1000
IgG (ng/ml)
RLUx10
4
2D03 (KD = 3x10-9
)
IEI-E3 (KD = 2x10-8
)
Control antibody
200
2
400 600 8000
B

6. Immunohistochemistry (IHC)
The Antibody
FITC-8IEI-E32D03
•Specific binding to human and mouse
plaque
2D03 FITC-8IEI-E3
Human
Mouse

7. 125
I radiolabelled 2D03
localizes to mouse plaques
2D03
2D03 FITC-8

8. Passive immunization strategy
• Apo E knockout mice were given high cholesterol diet
from 6 weeks
• Immunization with 0.5 mg antibody at 20, 21 and 22
weeks
• Sacrificed at 25 weeks
• Effects assessed by ORO staining of descending aorta,
Trichrome Masson staining and macrophage
immunostaining of subvalvular plaques.

9. Effect of passive immunization with recombinant
human IgG against peptide 45 on early
atherosclerosis in mice

10. Passive immunization with recombinant human
IgG against peptide 45 induces regression of
existing atherosclerotic plaques
The ability of inducing reduction of pre-existing plaques was
investigated in LDLR-/- apobec-1-/- mice.
0
2
4
6
8
10
12
14
16
FITC-8 2D03 IEI-E3 Baseline
25w
OilRedstainedarea
*** **
*** **

11. Reduction of macrophages in plaque
after treatment with antibody
0
5
10
15
20
25
30
FITC-8 2D03 IEI-E3
Macrophagestainingarea(%)
*

12. Effect of the 2D03 antibody on carotid athero-
sclerosis in LDL receptor-/-
/human apo B 100+/+
mice
0
50
100
150
200
250
300
350
PlasmaoxidizedLDL
(Units)
FITC-8 2D03
P<0.05
Ström et al, unpublished results
100
1000
10000
100000
Lesionarea(um
2
)
p<0.01
FITC-8 2D03
C
FITC-8 2D03

13. 2D03 efficiently blocks MCP-1 secretion in
vitro from macrophages
Freshly isolated human monocytes were incubated with
different concentrations of anti-oxApo B-100 IgG clones IEI-
E3 or 2D03, or FITC-8 negative control IgG in the presence
of human serum for 48 hours.

14. Short time cultured macrophages lower
their production and release of MCP-1

15. Recombinant human MDA-apo
B-100 peptide specific IgG1
• Binds to oxidized but not native LDL in vitro
• Recognizes epitopes in atherosclerotic plaques
• Inhibits the development of atherosclerosis by up to
50%
• Induces regression of existing atherosclerotic
plaques by up to 50%
• Reduces inflammatory activity in plaques

16. Department of Clinical Sciences, UMAS, Lund University
Alexandru Schiopu Marie Lindholm
Ingrid Söderberg Jan Nilsson
Gunilla Nordin Fredrikson Irena Ljungcrantz
BioInvent International, Lund
Roland Carlsson Bo Jansson
Zufan Araya
Division of Cardiology, Cedars-Sinai Medical Center, UCLA
Prediman K Shah Kuang-Yuh Chyu