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Laboratory for Thrombosis Research Interdisciplinary Research Center

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  1. Antibodies that interfere with the collagen-vWF-GPIb axis • anti-GPIb antibody 6B4 • anti-vWF A3 domain antibody 82D6A3 • epitope mapping • antithrombotic effect Laboratory for Thrombosis Research Interdisciplinary Research Center KU Leuven Campus Kortrijk, Belgium

  2. GPIb/IX/V vWF collagen Platelet adhesion

  3. von Willebrand Factor Propeptide Mature vWF B1 B2 D1 D2 D’ D3 A1 A2 A3 D4 C1 C2 N C GPIb Collagen Heparin Collagen GPIIb/IIIa Heparin FVIII

  4. vWF Platelet GPIb/V/IX complex GP Ib GP Ib GP V GP Ib GP Ib GP IX GP IX S-S S-S plasma membrane

  5. 6B4 A3 vWF A1 82D6A3 collagen

  6. Dynamic : Perfusion chamberShear-induced platelet adhesion control - PBS perfusion chamber imaging collagen coverslip anti-GPIb MoAb 6B4 pump + anti-GPIb MoAb 6B4 Blood water bath 37°C

  7. Low (650 s-1 ) IC50 :3.3 µg/ml Mid (1300 s-1 ) IC50 : 1.0 µg/ml High (2600 s-1) IC50 :0.5 µg/ml Shear dependent effect of anti-GPIb Fab 6B4 on platelet adhesion to collagen 100 80 60 maximal platelet adhesion (%) 40 20 0 0 1 2 3 4 5 20 µg/mL

  8. 30 20 10 0 40 30 20 10 0 650 1300 2600 82D6A3 inhibits platelet adhesion to collagen under flow 2600 s-1 % surface coverage 2.5 5 10 20 0 82D6A3 Fab (µg/ ml) 5 µg/ml % surface coverage shear s-1

  9. I. Inhibitory anti-GPIb antibodies

  10. I. Inhibitory anti-GPIb antibodies Epitope mapping of inhibitory antibodies against platelet GPIba reveals interaction between the leucine-rich repeat N-terminal and C-terminal domains of GPIba N. Cauwenberghs, K. Vanhoorelbeke, S. Vauterin, G. Romo, DF. Westra, E. Huizinga, J. Lopez, MC. Berndt, H.Deckmyn Blood 98:652-660, 2001

  11. vWF Platelet GPIb/V/IX complex GP Ib GP Ib GP V GP Ib GP Ib GP IX GP IX S-S S-S plasma membrane

  12. Structure of the N-terminal domain of GPIba Uff et al, J Biol Chem. 2002;277:35657 Huizinga et al, Science 2002; 297: 1176

  13. botrocetin ristocetin 26D1 0,4 26D1 0,5 27A10 0,4 0,3 12G1 vWF binding (OD 492nm) 12E4 vWF binding (OD 492 nm) 0,3 0,2 27A10 0,2 12G1 6B4 6B4 12E4 24G10 24G10 0,1 0,1 0,0 0,0 0 2 4 6 8 10 12 0 5 10 15 20 25 µg/mL µg/mL Static: Inhibition of vWF - GPIb interaction in ELISA by anti-GPIb MoAbs

  14. LJIb1 AK2 TM60 6B4 12G1 12E4 27A10 24G10 biotinylated - - - ++ - - 12G1 - - - ++ - - 12E4 - - - 27 A10 - - ++ - - - ++ 24G10 ++ ++ ++ ++ - - - ++ ++ ++ ++ ++ 6B4 - - - ++ ++ ++ ++ ++ TM60 ++ ++ - - - ++ ++ ++ ++ ++ LJIb1 ++ ++ - - - ++ ++ ++ ++ ++ AK2 ++ ++ Cross-blocking ELISA’s between MoAbs for binding to platelets Where ? bind to two regions on GPIb Two groups of intercompeting MoAbs

  15. Human - canine chimeras of GPIb(Shen et al., Blood 2000) N-flank C-flank aa 1 35 59 81 104 128 152 176 200 268 282 LRR 1 LRR 2 LRR 3 LRR 4 LRR 5 LRR 6 LRR 7 Dog Human C 35 C 59 C 81 ... Human Dog H 35 H 59 H 81 ... Expression on CHO-cells Binding of Abs: FACS analysis

  16. 6B4, TM60 AK2 24G10 LJIb1 27A10, 12E4, 12G1 Chimeras : results

  17. GPIba230-245 Vincente JBC 1990, 265, 274 GPIba 251-265 S D K F P V Y K YP GK G C P W K QG V DV K A MT S N N V A S Selected 6B4-binding sequences C7 and L15 library: a consensus sequence C N K P G E R T C C D T L K P G E C C A D Q K P G E C C Y K P G E W A C C K P G E V Q Q C C K P V E N R A C C K P G E V Q Q C K P G E M R K P G E M R G A A K P G D P S A L H V V R C W I C L15 library D G R R D V V V R S A T F Y L E G L Y S P W W P R S L P V L S S K T R S F G V H L V G P Y L P P G L H V F P L A S N R S

  18. 6B4 epitope : Phage display peptide libraries phage peptides ‘gain-of-function’ Platelet-type vWD aa G233V, M239V 6B4

  19. 6B4 24G10 RU RU 300 Mutants Wild-type 250 G233V 250 200 M239V 200 150 Wild-type Mutants 150 100 G233V 100 50 M239V 0 50 350 450 550 650 750 850 450 550 650 750 850 Time (sec) Time (sec) 6B4: reduced binding to PT-vWD mutants 24G10: enhanced binding to PT-vWD mutants Biacore binding of 6B4 and 24G10 to wild-type and mutant rec GPIb (1-280)

  20. Mutants: results ‘gain-of-function’ Platelet-type vWD aa G233V, M239V 6B4 24G10

  21. Two regions on GPIb important for binding of vWF : close proximity ‘platelet-type von Willebrand’s disease mutations Conclusions • Two groups of anti-GPIb MoAbs with distinct inhibitory characteristics I. Leucine-rich repeat N-terminal flanking region and first adjacent LRRs (aa 1-81) II. Leucine-rich repeat C-terminal flanking region (aa 200-268) Regulates binding properties of aa 1-81 of GPIb

  22. Structure of the N-terminal domain of GPIbaand the vWF A1-domain- GPIba complex Uff et al, J Biol Chem. 2002 Jun 26 Huizinga et al, Science 2002; 297: 1176-1179

  23. I. Inhibitory anti-GPIb antibodies Antithrombotic effect Cauwenberghs N, Meiring M, Lamprecht S, Roodt JP, Vauterin S, Deckmyn H, Kotzé HF Antithrombotic effect of platelet glycoprotein Ib blocking monoclonal antibody Fab-fragments in a baboon model. Arterioscl. Thromb. Vasc Biol, 20, 1347-1353, 2000 D. Wu, M. Meiring, HF Kotzé, H Deckmyn, N. Cauwenberghs Inhibition of platelet GPIb, GPIIb/IIIa or both by monoclonal antibodies, prevents arterial thrombus formation in baboons. Arterioscl. Thromb. Vasc Biol,22, 323-328, 2002

  24. Baboon Extracorporeal Shunt Model Teflon tubing Collagenic surface Silicone rubber tubing 3 mm 111-In labeled platelets Detector baboon Computer

  25. 3 3 IgG IgG ) ) 4 4 i.v. anti-GPIb antibody 6B4 IgG profound thrombocytopenia (Fab’)2 thrombocytopenia Fab minor drop in platelet counts 2 2 (Fab’) (Fab’) 2 2 Fab Fab Molecules/platelet (x 10 Molecules/platelet (x 10 1 1 0 0 1 10 100 1 10 100 nmol/L nmol/L Baboon Models

  26. 80 µg/kg 160 µg/kg 320 µg/kg 640 µg/kg Baboon Extracorporeal Shunt Model Inhibition of Platelet Deposition by anti-GPIb Fab 6B4 4 control 3 2 Platelets deposited (x 109) 1 0 10 20 30 Time (minutes)

  27. Baboon femoral artery Folts’ Model • High shear model • 2. Comparison anti-GPIb (6B4) • anti-GPIIb/IIIa (16NC72) • anti-GPIb + anti-GPIIb/IIIa

  28. 0.6 mg/kg 6B4 Baboon femoral artery Folts’ Model Inhibition of CFR by anti-GPIb Fab 6B4 2.0 mg/kg 6B4

  29. *** * 140 *** ** 120 * 100 * * 80 CFR (% of pre-control) 60 0.1 40 20 Anti-Ib 0 0.6 2.0 0.3 0.0 0.0 Anti-Ib ( 0.6)+ Anti-IIb/IIIa( 0.1) Anti-IIb/IIIa (mg/kg) Baboon femoral artery Folts’ Model ***

  30. 30 60 150 300 Plasma levels, receptor occupancy and RiPA 2.0 mg/kg 6B4 100 50 80 40 % of GPIb receptor occupancy % inhibition of ristocetin-induced 60 platelet aggregation Plasma concentration of 6B4 Fab (µg/ml) 30 40 20 20 10 0 0 0 24 h Time (min)

  31. 100 80 60 40 20 0 GPIb-occupancy needed for Ripa and CFR 100 80 60 32% 20% % CFR ( ) % ristocetin-induced agglutination ( ) 40 20 0 60 0 20 40 80 % GPIb Receptor occupancy

  32. Template bleeding time in baboons 25 20 * * • Anti-IIb/IIIa 0.3 mg/kg 15 Bleeding time (min) 10 • Anti-IIb/IIIa 0.1 mg/kg • Anti-Ib 0.6 mg/kg • Anti-Ib 2.0 mg/kg • 0.6 Anti-Ib + 0.1 Anti-IIb/IIIa 5 0 0 60 30 Time after iv (min)

  33. Conclusions 1. Inhibition of GPIb is antithrombotic in two baboon models • 2. Administration of anti-GPIb Fab-fragments does not provoke thrombocytopenia 3. Inhibition of GPIb has minor effects on the bleeding time • Combination of low dose anti-GPIb and anti-GPIIb/IIIa • antibodies is antithrombotic with moderate effects on • the bleeding time

  34. II. Inhibitory anti-vWF A3 domain antibody

  35. II. Inhibitory anti-vWF A3 domain antibody Epitope mapping Vanhoorelbeke K, Depraetere H,  Romijn RAP, Huizinga E,  De Maeyer M, Deckmyn H J. Biol. Chem. 278, 37815–37821, 2003

  36. 82D6A3 binding phages 1.2 1.0 0.8 OD (490-630) Phage binding to 82D6A3 0.6 0.4 0.2 0 0 5 10 Phages (1010/ml) 100 Inhibition of phage binding to 82D6A3 by vWF 80 60 % inhibition 40 20 0 0 10 20 30 vWF (mg/ml)

  37. 82D6A3 binding phages 1.2 1.0 0.8 OD (490-630) Phage binding to 82D6A3 0.6 0.4 0.2 0 0 5 10 Phages (1010/ml) 100 Inhibition of phage binding to 82D6A3 by vWF 80 60 % inhibition 40 20 0 0 10 20 30 vWF (mg/ml)

  38. Peptides displayed on 82D6A3 binding phages C6-libraryCMTSPWRC 8/13 clones CRTSPWRC 4/13 CYRSPWRC 1/13 L15-library GDCFFGFLNSPWRVC RSSYWVYSPWRFISR vWF(974-989) SITTIDVPWNVVPEK

  39. Localisation of PW on vWF-A3-domain Pro 981 Trp 982

  40. Pro981Ala 82D6A3 binding to 28 vWF point mutants 125 100 75 Percentage 82D6A3 antibody bound 50 25 0 A3 wt D mock Ile975Ala Pro981His His990Ala Arg963Ala Val985Ala Pro962His Gln966Ala Glu987Ala Val980Ala Val984Ala Val997Ala Ser974Ala Thr977Ala Ser993Ala Gln999Ala Asn983Ala Asp979Ala His1023Ala Arg1016Ala Glu1021Ala Glu1001Ala Gln1006Ala Ser1020Ala Pro1027His Asp1009Ala Met1022Ala

  41. Epitope of 82D6A3 on vWF-A3 domain Asp 1009 Arg 1016 Pro 981 Trp 982 His 1023 Met 1022 Ser 1020

  42. Epitope of 82D6A3 on vWF-A3 domain

  43. modeled cyclic CMTSPWRC mapped onVWF-A3 domain “buried form” of Trp982 (crystal) “exposed form” of Trp982

  44. modeled cyclic CMTSPWRC mapped on VWF-A3 domain • Fluorescence decay measurements • yielded two lifetime components • of 2,08 ns and of 6,26 ns • Energy minimalisation calculation • Shows two rotamer clusters • M Hellings, Y Engelborghs, H Deckmyn, • ME Schiphorst, JW Akkerman, M De Maeyer • submitted

  45. 0 10 20 30 40 50 60 70 80 90 100 Correlation between mutant binding to collagen and 82D6A3 S974A I975A T977A V997A E1001A 100 80 60 82D6A3 binding R1016A S1020A 40 D1009A 20 P981H S968T H1023A 0 Coll III binding

  46. Conclusions • 82D6A3 prevents vWF binding to collagen in vitro • 82D6A3 binds to the vWF-A3 domain • by phage display aSer-Pro-Trp-Arg-mimotopesequence is identified • by analysis of mutant-vWF the binding area is identified at the ‘front face’ • of A3, with Pro981, Asp1009, Arg1016, Ser1020, Met1022, His1023 • as dominant residues • His1023,Ser1020 and lessArg1016,(Pro981)are necessary • for binding to both 82D6A3 and to collagen confirming • the localisation of the collagen binding site within vWF-A3 • and explaining the inhibitory activity of 82D6A3

  47. II. Inhibitory anti-vWF A3 domain antibodyAntithrombotic effect D. Wu, K. Vanhoorelbeke, N. Cauwenberghs, G. Vandecasteele, S. Vauterin, M. Meiring, S. Lambrecht, JP Rood, H. Depraetere, H. Kotzé, H. DeckmynBlood 99, 3623-3628, 2002 Laboratory for Thrombosis Research, KU Leuven Campus Kortrijk, Kortrijk, Belgium Department of Hematology and Cell Biology, U Orange Free State, Bloemfontein, South-Africa

  48. Inhibition of baboon vWF binding to collagen by 82D6A3 Removal of collagen-bound baboon vWF by 82D6A3 ( ) • Further in vitro characterization of 82D6A3 120 0.7 0.6 100 0.5 80 0.4 60 % vWF binding 0.3 OD 490 nm 40 0.2 0.1 20 0.0 0 0.1 1 10 100 1000 0 2 4 6 8 10 ng/ml 82D6A3 µg/ml 82D6A3

  49. 0.1 mg/kg 82D6A3 In vivo inhibition of arterial thrombus formation : Folts’ model • e.g. administration of 100 µg/kg 82D6A3