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Laboratory for Thrombosis Research Interdisciplinary Research Center KU Leuven Campus Kortrijk, Belgium Antibodies that interfere with the collagen-vWF-GPIb.

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Presentation on theme: "Laboratory for Thrombosis Research Interdisciplinary Research Center KU Leuven Campus Kortrijk, Belgium Antibodies that interfere with the collagen-vWF-GPIb."— Presentation transcript:

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

2 collagen GPIb/IX/V vWF Platelet adhesion Platelet adhesion

3 von Willebrand Factor NC D1D2D’D3A1A2A3D4 B1 B2 B2 C1C2 Propeptide Mature vWF CollagenGPIbCollagenHeparin HeparinFVIII GPIIb/IIIa

4

5 Platelet GPIb/V/IX complex GP Ib  GP V GP IX GP Ib  GP Ib  plasma membrane vWFS-S S-S

6 collagen A1A3vWF 6B4 6B4 82D6A3

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

8 maximal platelet adhesion (%) maximal platelet adhesion (%) µg/mL Mid (1300 s- 1 ) IC 50 : 1.0 µg/ml Mid (1300 s- 1 ) IC 50 : 1.0 µg/ml Low (650 s- 1 ) IC 50 : 3.3 µg/ml Low (650 s- 1 ) IC 50 : 3.3 µg/ml High (2600 s -1 ) IC 50 : 0.5 µg/ml High (2600 s -1 ) IC 50 : 0.5 µg/ml Shear dependent effect of anti-GPIb Fab 6B4 on platelet adhesion to collagen

9 % surface coverage shear s % surface coverage D6A3 Fab (µg/ ml) 82D6A3 inhibits platelet adhesion to collagen under flow 2600 s -1 5 µg/ml

10 I. Inhibitory anti-GPIb antibodies I. Inhibitory anti-GPIb antibodies

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

12 Platelet GPIb/V/IX complex GP Ib  GP V GP IX GP Ib  GP Ib  plasma membrane vWFS-S S-S

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

14 Static: Inhibition of vWF - GPIb interaction in ELISA by anti-GPIb MoAbs

15 Cross-blocking ELISA’s between MoAbs for binding to platelets TM60 LJIb1 AK TM60LJIb1AK2 bind to two regions on GPIb Two groups of intercompeting MoAbs Where ? G1 12E4 27 A10 24G10 6B4 24G106B412G112E427A10 biotinylated

16 Human - canine chimeras of GPIb  (Shen et al., Blood 2000) LRR 1 LRR 2 LRR 3 LRR 4 LRR 5 LRR 6 LRR aa N-flank Human Dog C 35 C 59 C C-flank DogHuman H 35 H 59 H Expression on CHO-cells Binding of Abs: FACS analysis

17 6B4, TM60 AK224G10 LJIb1 27A10, 12E4, 12G1 Chimeras : results

18 Selected 6B4-binding sequences C7 and L15 library: a consensus sequence C N K P G E R T C 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 K P G E V Q Q C C Y K P G E W A 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 K P G E M R G A A 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 K P G D P S A L H V V R C W I C 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 S D K F P V Y K Y P G K G C P GPIb  W K Q G V D V K A M T S N N V A S GPIb  Vincente JBC 1990, 265, 274 L15 library

19 6B4 phage peptides ‘gain-of-function’ Platelet-type vWD aa G 233 V, M 239 V 6B4 epitope : Phage display peptide libraries

20 Biacore binding of 6B4 and 24G10 to wild-type and mutant rec GPIb  (1-280) 6B RU Time (sec) Wild-type M239V G233VMutants 6B4: reduced binding to PT-vWD mutants RU Time (sec) 24G10Wild-type M239V G233V Mutants 24G10: enhanced binding to PT-vWD mutants

21 6B4 ‘gain-of-function’ Platelet-type vWD aa G 233 V, M 239 V 24G10 Mutants: results

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

23 Uff et al, J Biol Chem Jun 26 Structure of the N-terminal domain of GPIb  and the vWF A1-domain- GPIb  complex Huizinga et al, Science 2002; 297:

24 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, , 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, , 2002

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

26 Baboon Models i.v. anti-GPIb antibody 6B4 IgG profound thrombocytopenia (Fab’) 2 thrombocytopenia Fab minor drop in platelet counts Molecules/platelet (x ) ) nmol/L IgG (Fab’) 2 2 Fab

27 Time (minutes) Time (minutes) control 160 µg/kg µg/kg 320 µg/kg Platelets deposited (x 10 9 ) Baboon Extracorporeal Shunt Model Inhibition of Platelet Deposition by anti-GPIb Fab 6B4 640 µg/kg 640 µg/kg 80 µg/kg 80 µg/kg

28 Baboon femoral artery Folts’ Model 1.High shear model 2. Comparison anti-GPIb (6B4) anti-GPIIb/IIIa (16NC72) anti-GPIb + anti-GPIIb/IIIa

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

30 Anti-Ib ( 0.6) + Anti-IIb/IIIa ( 0.1) **** * *** CFR (% of pre-control) Anti-Ib (mg/kg) * *** Baboon femoral artery Folts’ Model ** Anti-IIb/IIIa 0.0 *

31 h Time (min) % inhibition of ristocetin-induced platelet aggregation 2.0 mg/kg 6B4 Plasma concentration of 6B4 Fab (µg/ml) % of GPIb receptor occupancy Plasma levels, receptor occupancy and RiPA

32 GPIb-occupancy needed for Ripa and CFR % ristocetin-induced agglutination ( ) % GPIb Receptor occupancy % CFR ( ) 20% 20% 32% 32%

33 Bleeding time (min) Time after iv (min) * * Anti-IIb/IIIa 0.3 mg/kgAnti-IIb/IIIa 0.3 mg/kg Anti-IIb/IIIa 0.1 mg/kgAnti-IIb/IIIa 0.1 mg/kg Anti-Ib0.6 mg/kgAnti-Ib0.6 mg/kg Anti-Ib 2.0 mg/kgAnti-Ib 2.0 mg/kg 0.6 Anti-Ib Anti-IIb/IIIa0.6 Anti-Ib Anti-IIb/IIIa Template bleeding time in baboons

34 ConclusionsConclusions 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 4.Combination of low dose anti-GPIb and anti-GPIIb/IIIa antibodies is antithrombotic with moderate effects on antibodies is antithrombotic with moderate effects on the bleeding time the bleeding time

35 II. Inhibitory anti-vWF A3 domain antibody II. Inhibitory anti-vWF A3 domain antibody

36 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

37 % inhibition vWF (  g/ml) Phages (10 10 /ml) OD ( ) 82D6A3 binding phages Phage binding to 82D6A3 Inhibition of phage binding to 82D6A3 by vWF

38 % inhibition vWF (  g/ml) Phages (10 10 /ml) OD ( ) 82D6A3 binding phages Phage binding to 82D6A3 Inhibition of phage binding to 82D6A3 by vWF

39 C6-library CMTSPWRC 8/13 clones CRTSPWRC 4/13 CRTSPWRC 4/13 CYRSPWRC 1/13 CYRSPWRC 1/13 L15-library GDCFFGFLNSPWRVC RSSYWVYSPWRFISR RSSYWVYSPWRFISR Peptides displayed on 82D6A3 binding phages vWF( ) SITTIDVPWNVVPEK

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

41 wt  A3 mock Percentage 82D6A3 antibody bound 82D6A3 binding to 28 vWF point mutants Pro981His Pro981Ala Arg963Ala Pro962His Gln966Ala Thr977Ala Ile975Ala Ser974Ala Asp979Ala Val980Ala Asn983Ala Val984Ala Val985Ala Glu987Ala His990Ala Ser993Ala Val997Ala Gln999Ala Glu1001Ala Gln1006Ala Asp1009Ala Arg1016Ala Ser1020Ala Glu1021Ala Met1022Ala His1023Ala Pro1027His

42 Pro 981 Trp 982 Met 1022 His 1023 Ser 1020 Asp 1009 Arg 1016 Epitope of 82D6A3 on vWF-A 3 domain

43

44 modeled cyclic CMTSPWRC mapped on VWF-A3 domain “exposed form” of Trp982 “buried form” of Trp982 (crystal)

45 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

46 Coll III binding 82D6A3 binding S968T D1009A S974A I975A T977A V997A E1001A Correlation between mutant binding to collagen and 82D6A3 H1023A H1023A S1020A P981H R1016A

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

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

49 Further in vitro characterization of 82D6A OD 490 nm µg/ml 82D6A % vWF binding ng/ml 82D6A3 Inhibition of baboon vWF binding to collagen by 82D6A3 Removal of collagen-bound baboon vWF by 82D6A3 ( )

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

51 Antithrombotic effect of 82D6A µg/kg 82D6A3 µg/kg 82D6A3 CFRs % reduction in CFRs P<0.05 P<0.01 In vivo inhibition of CFRs by 82D6A3 Relation between in vivo reduction in CFRs and ex vivo vWF-collagen binding % reduction in CFR's % ex vivo collagen binding

52 82D6A3 in vivo Coagulation tests, platelet count, vWF-Ag levels:Coagulation tests, platelet count, vWF-Ag levels: no significant changes Bleeding time: no significant prolongation Bleeding time: no significant prolongation even not with ‘overdose’ (600 µg/kg) even not with ‘overdose’ (600 µg/kg)

53 RESULTS ’ 30’ 60’ 150’ 300’ 24h 48h Ex vivo analysis after administration of 300 µg/kg 82D6A3 % vWF-collagen binding ( ) % vWF-occ ( ) µg/ml 82D6A3 ( )

54 Conclusions – – Blocking VWF-A3 domain is anti-thrombotic – – Without major prolongation of the bleeding time reason why so far few patients with defect in vWF-collagen interaction have been identified? – –The importance of the vWF-collagen interaction in primary hemostasis under high shear was indicated by in vitro experiments This study provided the first in vivo proof

55 collagen General conclusions The collagen-vWF-GPIb axis is an interesting target for the develoment of new antithrombotic agents The collagen-vWF-GPIb axis is an interesting target for the develoment of new antithrombotic agents with little effect on bleeding time A1A3vWF VCL, 6B4 AJvW2 AJvW2 saratin 82D6A3


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