Declerck, P. J.

Develter, J.

Gils, A.

KU ScholarWorks

Laboratory for Pharmaceutical BiologyBiochemical characterization of derivatives of MA-T12D11, a TAFI neutralizing antibodyDevelter J, Declerck PJ, Gils ACoagulation FibrinolysisFibrinogen Fibrin Fibrin degrad-ation products Thrombin PlasminProthrombin           PlasminogenThrombinThrombomodulinAct. protein CProtein CTAFIaTAFI( - )( - )Results  TAFI/TAFIa is a risk factor for thrombosis and related diseases.2  TAFI inhibitors can be a powerful tool in thrombosis models.  Already known inhibitors of TAFI(a) are not selective orpowerful enough:−Chelating agents (e.g. EDTA)−Reduction of disulfide bridges (e.g. DTT)−Arginine and lysine analogs (e.g. GEMSA)−Naturally occurring inhibitors (e.g. Carboxypeptidaseinhibitor or CPI)  Monoclonal Antibodies (MA) towards human TAFI wereraised in our lab.  MA-T12D11 inhibits the activation of TAFI by T/TM.  Figure 1 Scheme of blood coagulation and fibrinolysisIntroductionObjectiveMaterials & MethodsConclusions1 Bajzar, Thrombin activatable fibrinolysis inhibitor and an antifibrinolytic pathway. Arterioscler Thromb Vasc Biol. 2000; 20: 2511-8. Review2 Leurs and Hendriks, Carboxypeptidase U (TAFIa): a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thromboticdisease. Thromb Haemost. 2005; 94: 471-87. ReviewLaboratory for Pharmaceutical Biology, KULeuven, BelgiumTo characterize the biochemical properties of antibody fragments derived from MA-T12D11.  Fab-T12D11 was generated by papain digestion of MA-T12D11, followed by protein A purification.   The scFv-T12D11 fragment was constructed by isolation of the cDNA of MA-T12D11 producing hybridomas, followed by amplification and assembling of VH and VL with a (Gly4Ser)3linker.  Affinity between TAFI and the antibody (fragments) was measured with surface plasmon resonance technique.   The inhibitory effect of MA (derivatives) was tested in a chromogenic assay by activating TAFI with T/TM.  ResudualTAFIa activity was determined using a chromogenic assay in which hippuryl-arginine is used as a substrate.  The profibrinolytic effect of MA (derivatives) was investigated with an in vitro clot lysis assay in which CaCl2was used to start clot formation and tPA to initiate clot lysis.  VH:QVQLQQSGAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDVKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCHHGNWAAWFAYWGQGTTVTVSS  VL:DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDDYLNWYQQRPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCHQSNEDPFTFGSGTKLEIKRFigure 2: VH and VL chain of MA-T12D11 (CDR regions of VH and VLin red and blue, respectively)    1.7 ± 0.75 x1092.8 ± 1.0 x1095.4 ± 1.5 x109KA(1/M)3.2 ± 0.46 x10-44.3 ± 0.33 x10-42.5 ± 0.28 x10-4kd(1/s)5.4 ± 2.8 x1051.2 ± 0.38 x1061.3 ± 0.32 x106ka(1/Ms)scFvFabMAT12D110 1 2 3 4 5 6 7 8 90102030405060708090100110MA T12D11Fab T12D11scFvT12D11Molar excess of MA/Fab/scFv% inhibition of TAFIaactivityTable 1: Binding parameters (ka, kd and KA) of MA-T12D11 and its derivatives for binding to TAFIThe results represent mean ± SD (n 3)Figure 4: % inhibition of TAFIa activity by MA/Fab/scFvTAFI was activated with T/TM in presence of MA, Fab or scFv. The results represent mean ± SD (n 3).−Fab-T12D11 and scFv-T12D11 show a similar maximum inhibitory effect as MA-T12D11 i.e. 99.0 ±0.72 % and 91.4 ± 3.0 % (Fab and scFv, respectively) vs. 96.7 ± 1.3 % for MA. −The inhibitory effect of Fab-T12D11 and  especially that of scFv-T12D11 shows a shift towardshigher concentrations, in accordance to a slightly decreased affinity towards TAFI (table 1).Figure 5: Profibrinolytic effect of MA, Fab and CPI during in vitro clot lysisClot formation and dissolution is shown in presence of 8-fold molar excess of MA-T12D11 (•), 8-fold molar excess of Fab-T12D11 (•), 45-fold molar excess of CPI (•) and 0,9% NaCl(negative control, • ). All concentrations are finalconcentrations.(n = 3). Note: concentration of scFv-T12D11 was too low to test it in a clot lysis assay.0 50 100 150 200 250 3000.850.951.051.151.251.351.45 MA-T12D11Fab-T12D11CPI0.9% NaClTime (min)OD (405 nm)−MA, Fab and CPI shorten clot lysis time significantly (i.e. 28.3 ± 2.9 min, 29.2 ± 10 min and 34.3 ±1.2 min, respectively) vs. 137 ± 25.2 min for the negative control, demonstrating the powerful effect of both the MA and Fab fragment.  scFv-T12D11 as well as Fab-T12D11 have similar affinity constants as the parental MA.  Fab-T12D11 has similar functional effects in the chromogenic assay and in the clot lysis.  scFv-T12D11 has a similar functional effect in the chromogenic assay.   Thrombin Activatable Fibrinolysis Inhibitor (TAFI) provides a link between the coagulation and the fibrinolytic cascade.1  TAFI can be activated to TAFIa by the thrombin-thrombomodulin complex (T/TM).  TAFIa has carboxypeptidase activity and exerts anantifibrinolytic effect.Figure 3: Model of scFv-T12D11, obtained bymodeling with AbM software (WAM server)  The amino acid sequence of the VH and VL region of MA-T12D11 is shown in Figure 2.  Based on this sequence, a model of scFv-T12D11 was obtained using the AbM software (Figure 3).  The rate constants for association (ka) and dissociation (kd) and the affinity constant (KA) as well as the inhibitory effect of the antibody fragments on TAFI activity was compared to the parental MA-T12D11.  Effect of MA-T12D11 and Fab-T12D11 in clot lysis.  This effect is compared to that of CPI (a non-specific TAFIa inhibitor).

Biochemical characterization of derivatives of MA-T12D11, a TAFI neutralizing antibody

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Biochemical characterization of derivatives of MA-T12D11, a TAFI neutralizing antibody

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