PB1059 - Single Amino Acid Substitutions of an Antibody Specific for a Non-Active Site of Activated Protein C (APC) Yielded an Antibody Library Capable of Modulating and Uncoupling APC Pleiotropic Functions

Background: Activated protein C (APC) is a coagulation protease with pleiotropic effects on anticoagulant, anti-inflammatory, and cytoprotective activities. Its in vitro half-life in plasma is controlled by serine protease inhibitors (SERPINs). Antibodies that can selectively modulate these APC activities lend themselves to therapeutic application.

Aims: We constructed an IgG library based on an APC non-active-site reference antibody, whose epitope was determined to include the autolysis loop, to elucidate the effect of antigen binding site amino acid substitutions on the antibody ability to modulate APC’s pleiotropic activities.

Methods: Two rounds of optimization were performed on the reference antibody (TPP-24727). The antibody most potent for inhibiting APC anticoagulant activity (TPP-26870) in Round 1 underwent a Round 2 of single amino acid substitutions in its antigen binding sites for further KD improvements.

Results: Each of the 21 IgGs from Round 2 differed from the Round 1 Final IgG TPP-26870 by only 1 amino acid. Twelve had higher APC anticoagulant activity inhibition than TPP-26870, while 9 had lower inhibition despite having improved KDs (Table 1). Interestingly, while all antibodies were directed to the same epitope, affinity optimization generated antibodies that exerted different combinations of activity modulation of APC. For example, a gain in inhibition of APC anticoagulant activity relative to TPP-26870 led to an increase in APC H3 cleavage activity (50%) and plasma half-life (70%) for TPP-29850 but led to a decrease in H3 cleavage (23%) and half-life (18%) for TPP-29858.

Conclusion(s): APC functions are very sensitive to the epitope-paratope interaction with a non-active site binding IgG. Even single amino acid substitutions within the antigen binding site are sufficient to generate diverse antibodies capable of modulating and uncoupling APC pleiotropic functions for potential therapeutic effects such as inhibition of APC’s anticoagulant activity, enhancement of histone proteolysis, or reduced interaction with SERPINs.