Construction and Characterization of MA-T94H3 Derivatives, a TAFI Neutralizing Antibody
Abstract number: P1365
Develter J, Gils A, Buelens K, Declerck PJ
Introduction: TAFIa exerts an antifibrinolytic effect by removing C-terminal lysines from partially degraded fibrin degradation products thereby abolishing their cofactor function in the activation of plasminogen by t-PA.
Objective: To construct and characterize derivatives of the TAFI inhibiting monoclonal antibody MA-T94H3.
Methods and results: A previous study identified 14 monoclonal antibodies, raised against human TAFI, that exert a TAFI inhibitory effect by impairment of the activation of TAFI into TAFIa by thrombin/thrombomodulin. Of these antibodies, MA-T94H3 could also hamper substantially the activation by plasmin (71% inhibition using an 8-fold molar excess of MA). Epitope analysis suggested that Val41 is a major residue for binding of MA-T94H3 to TAFI. To elucidate the working mechanism of this unique antibody, Fab and scFv fragments are generated. Papain digestion of MA-T94H3, followed by protein A purification, resulted in Fab-T94H3. Preliminary experiments revealed that Fab-T94H3 has virtually the same inhibitory effect as the MA-T94H3 (52% inhibition using an 8-fold molar excess of Fab). For the construction of scFv, cDNA was isolated from MA-T94H3 producing hybridomas, and VH and VL regions were amplified with the appropriate primers and assembled using a DNA linker segment (encoding (Gly4Ser)3). As expected sequencing of this scFv reveals the typical presence of CDR regions. The functional properties (i.e. affinity and inhibitory effect), will be evaluated and compared to the effects of the parental MA and Fab.
Conclusion: Characterization of Fab and scFv derived from the unique MA-T94H3 will provide valuable information regarding the working mechanism of TAFI inhibiting MA. The availability of the cloned scFv and its sequence also allows to further explore the paratope characteristics contributing to the functional effects of MA-T94H3.
To cite this abstract use the following format:
Journal of Thrombosis and Haemostasis 2005; Volume 3, Supplement 1: abstract number
|Session name:||XXIst ISTH Congress|
|Subject:||Poster Session Wednesday|
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