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Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate
S Debrock1, L Sironi, P J Declerck
1Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit, Leuven, Belgium.
Abstract:
Increased levels of plasminogen activator inhibitor-1 (PAI-1) are a well-known risk for cardiovascular diseases. A significant number of investigations are aimed at lowering plasma levels of PAI-1 to enhance endogenous fibrinolysis. We have recently generated monoclonal antibodies that neutralize PAI-1 activity by switching the inhibitory conformation to a substrate conformation. However, intact murine antibodies have quite some disadvantages for therapeutic use in man. In the current study, we describe the construction of a smaller antibody fragment derived from a monoclonal antibody (MA-8H9D4) with PAI-1 neutralizing properties. The cDNAs encoding the variable domains of the heavy and light chain were amplified, linked and cloned into a phagemid vector. Resulting clones were expressed as a single-chain variable fragment (scFv, VH-(Gly4Ser)3-VL) on the surface of a phage and selected for binding to PAI-1. Subsequently, a positive phage was used for the production of soluble scFv-8H9D4. Following purification, the characteristics of the scFv-8H9D4 were compared to those of the original MA-8H9D4. The scFv inhibited PAI-1 activity to a similar extent as MA-8H9D4 and by a similar mechanism, i.e., induction of a conformational switch. Thus, this smaller antibody fragment, exhibiting the same properties as the parent molecule may constitute a useful starting point for the design of PAI-1 neutralizing therapeutics.
Insights
Researchers developed a smaller antibody fragment that effectively neutralizes plasminogen activator inhibitor-1 (PAI-1), a key risk factor for cardiovascular disease. This engineered antibody fragment offers a promising therapeutic strategy for enhancing fibrinolysis and managing heart conditions.
Area of Science:
- Biochemistry
- Immunology
- Cardiovascular Research
Background:
- Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are a significant risk factor for cardiovascular diseases.
- Current therapeutic strategies focus on reducing plasma PAI-1 levels to improve endogenous fibrinolysis.
- Monoclonal antibodies neutralizing PAI-1 activity have been developed but face limitations for human therapeutic use.
Purpose of the Study:
- To construct and characterize a smaller, potentially more suitable antibody fragment with PAI-1 neutralizing properties.
- To evaluate the efficacy and mechanism of action of the engineered antibody fragment compared to the parent monoclonal antibody.
Main Methods:
- Generation of a single-chain variable fragment (scFv) by linking heavy and light chain variable domains from a PAI-1 neutralizing monoclonal antibody (MA-8H9D4).
- Cloning the scFv into a phagemid vector for surface display on phage.
- Selection of PAI-1 binding phage and subsequent production and purification of soluble scFv-8H9D4.
- Comparison of scFv-8H9D4 characteristics with MA-8H9D4.
Main Results:
- A soluble single-chain variable fragment (scFv-8H9D4) was successfully produced.
- The scFv-8H9D4 demonstrated comparable inhibition of PAI-1 activity to the parent monoclonal antibody MA-8H9D4.
- The mechanism of PAI-1 inhibition by the scFv involved inducing a conformational switch, similar to the parent antibody.
Conclusions:
- The engineered scFv-8H9D4 retains the PAI-1 neutralizing properties and mechanism of action of the original monoclonal antibody.
- This smaller antibody fragment represents a viable starting point for developing novel PAI-1 neutralizing therapeutics for cardiovascular diseases.