1. Plasmin Fibrin Fibrin degradation products Plasminogen Plasminogen activators (t-PA, u-PA) Plasminogen activator inhibitors (PAI) February 19, 2004 ProMMP MMP ECM Circulation

2. Plasminogen activator inhibitors Plasminogen activator inhibitor 1 (endothelial cell type) Plasminogen activator inhibitor 2 (placental type) Plasminogen activator inhibitor 3 (protein C inhibitor) February 19, 2004

3. Molecular properties of PAI-1 Member of the SERPIN superfamily P1’ P1 February 19, 2004

5. Molecular properties of PAI-1 Member of the SERPIN superfamily Labile (active latent) February 19, 2004

6. P1’ Active PAI-1 P1 Latent PAI-1 P1’ P1 February 19, 2004

7. Molecular properties of PAI-1 Member of the SERPIN superfamily Labile (active latent) Inhibits t-PA and u-PA Active PAI-1 binds to vitronectin February 19, 2004

8. Occurrence of PAI-1 Plasma Platelets Extracellular matrix [vitronectin !] Cells February 19, 2004

9. Plasma: mainly active PAI-1 Platelets: mainly latent PAI-1 Extracellular matrix: only active PAI-1 Antigen Activity 90 10 Plasma Platelets % of total PAI-1 in blood February 19, 2004

10. Pathophysiological role of PAI-1 increased levels are associated with thrombotic disorders role in development and progression of atherosclerosis causative role in cancer is still controversial (dual effects) role in tissue fibrosis circulating vs localized ! February 19, 2004

11. Inactivation of PAI-1 February 19, 2004

12. Conformational transitions I l I s + EEI s s ’ E +I c I a + EEI a a ’ EI c Active Substrate Latent February 19, 2004

13. Mechanism of inactivaction Direct interaction with reactive site Active latent Active substrate Active substrate latent P1’ P1 P1’ P1 February 19, 2004

14. Inactivation of PAI- 1 Polyclonal antibodies Monoclonal antibodies “Low molecular weight” inhibitors February 19, 2004

15. Monoclonal Antibodies Detailed characterization Epitope localizaton Cloning of smaller fragments ( scFv) February 19, 2004

16. PAI-1 neutralizing properties of monoclonal antibodies molar ratio (MA:PAI-1) % neutralization of PAI-1 activity 51015 0 50 100 MA-8H9D4 MA-33H1 MA-55F4 MA-33B8 MA-37H7 t-PA February 19, 2004

17. MA- 31C9 MA- 56A7C10 MA- 55F4 MA No MA t- PA Complex PAI-1 /t-PA non-reactive cleaved PAI-1 kD 14 20 30 43 67 94 PAI-1 neutralizing effect February 19, 2004

18. Monoclonal Antibodies Detailed characterization Epitope localizaton Cloning of smaller fragments ( scFv) February 19, 2004

19. Epitopes ConformationalLinear February 19, 2004

20. MA-33H1F7 and MA-55F4C12  neutralizing monoclonal antibodies  epitope between Glu 128 and Ala 156 February 19, 2004

22. Species cross-reactivity Human + Porcine- Rabbit- Rat+ Murine+ 154 KQEKKKQEKK 1379 Human PAI-1 128156 128-131 E V E R E M D R D V Q R E V E R February 19, 2004

23. K EVER A AAAA February 19, 2004

24. VariantsMutated residues (1) K A (1/M) (2) MA-55F4C12MA-33H1F7 PAI-1-wtnone2.7 ± 1.6 10 9 5.4 ± 1.7 10 9 (1) residues mutated into an alanine (2) mean  SD (n = 4 to 11) Affinity constants for PAI-1 mutants PAI-1-EVERE 128 V 129 E 130 R 131 9.9 ± 6.0 10 6 4.2 ± 4.4 10 8 PAI-1-KK 154 4.0 ± 3.1 10 7 3.0 ± 2.0 10 7 PAI-1-EVER/KE 128 V 129 E 130 R 131 / K 154 no bindingno binding February 19, 2004

25. Molecular explanation neutralizing properties mobility of the hinge is restricted t-PA not locked in covalent complex Epitope hinge region between  -helix F and main part of PAI-1 February 19, 2004

27.  neutralizing monoclonal antibody  specific binding to the active conformation  no reactivity with rat PAI-1 MA-56A7C10 February 19, 2004

28. + Human PAI-1 - Rat PAI-1 Rat26hum-PAI-1 26 - Human/rat PAI-1 chimeras February 19, 2004

29. 26 187 1379 81 277 327 187 327 Human PAI-1 Rat PAI-1 Rat26hum-PAI-1 Rat81hum-PAI-1 Rat187hum-PAI-1 Rat327hum-PAI-1 Hum81rat-PAI-1 Hum187rat-PAI-1 Hum277rat-PAI-1 Hum327rat-PAI-1 Human/rat PAI-1 chimeras February 19, 2004

30. Mean  SD (n = 2 to 6) PAI-1 variant MA-33H1F7 MA-56A7C10 Human PAI-1 4.9  0.06 Rat PAI-1 2.7  0.05 Affinity constants K A (10 9 1/M) Rat 26 Hum 2.9  0.05 Rat 81 Hum 8.5  0.02 Rat 187 Hum 6.2  0.84 Rat 327 Hum 2.0  0.03 Hum 81 Rat 2.9  0.06 Hum 187 Rat 5.2  0.70 Hum 277 Rat 4.6  0.12 Hum 327 Rat 6.7  0.44 1.2  0.05 no binding 0.61  0.02 1.0  0.07 1.3  0.16 0.61  0.004 no binding February 19, 2004

31. MA-56A7C10 1.2  0.05 no binding 3791 Affinity of MA-56A7C10 (10 9 1/M) 26 0.61  0.02 1.0  0.07 1.3  0.16 0.61  0.004 187 81 327 81 277 187 327 no binding February 19, 2004

32. Species cross-reactivity 1 379 Human PAI-1 327 Human + Porcine + Rat - Murine - EETTEETT 350 February 19, 2004

33. Alanine-scanning E350 February 19, 2004

34. Alanine-scanning E350 EKE242-4 RR185-7 HK190-1 D193 DR355-6 February 19, 2004

35. Variants Mutated residues (1) K A (1/M) MA-56A7C10 (2) PAI-1-wtnone5.4 ± 1.7 10 9 (1) residues mutated into an alanine (2) mean  SD (n = 2 to 6) EKE242-4AE 242 K 243 E 244 0.11 ± 0.01 10 9 R356AR 356 0.067 ± 0.003 10 9 E350AE 350 0.18 ± 0.01 10 9 Affinity constants for PAI-1 mutants February 19, 2004

36. Molecular explanation neutralizing properties MA-56A7C10 Epitope in the vicinity and comprising the distal hinge of the reactive site loop higher affinity for active PAI-1 February 19, 2004

37. Active PAI-1 Latent PAI-1 Higher affinity for PAI-1 in the active form February 19, 2004

38. Monoclonal Antibodies Detailed characterization Epitope localizaton Cloning of smaller fragments ( scFv) February 19, 2004

39. VHVH VLVL

40. Neutralization of PAI-1 by MA-8H9D4 and scFv-8H9D4 molar excess % neutralization of PAI-1 activity 012345 0 50 100 MA-8H9D4 scFv-8H9D4 February 19, 2004

41. Paratope of scFv-8H9D4 February 19, 2004

42. General conclusions PAI-1 is a putative therapeutic target PAI-1 can be targeted at various positions February 19, 2004

44. General conclusions PAI-1 is a putative therapeutic target PAI-1 can be targeted at various positions Molecular and structural information on epitopes and paratopes allows further rational design of PAI-1 neutralizing compounds February 19, 2004