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Nilsson

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Nilsson

  1. 1. Passive immunization using recombinant IgG against apo B- 100 peptides Jan Nilsson Department of Clinical Sciences Malmö, Lund University
  2. 2. Adaptive immune responses to oxidized LDL
  3. 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. 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. 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. 6. Immunohistochemistry (IHC) The Antibody FITC-8IEI-E32D03 •Specific binding to human and mouse plaque 2D03 FITC-8IEI-E3 Human Mouse
  7. 7. 125 I radiolabelled 2D03 localizes to mouse plaques 2D03 2D03 FITC-8
  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. 9. Effect of passive immunization with recombinant human IgG against peptide 45 on early atherosclerosis in mice
  10. 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. 11. Reduction of macrophages in plaque after treatment with antibody 0 5 10 15 20 25 30 FITC-8 2D03 IEI-E3 Macrophagestainingarea(%) *
  12. 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. 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. 14. Short time cultured macrophages lower their production and release of MCP-1
  15. 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. 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

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