OC 76.1 - Directed Evolution of An Antibody Specific for a Non-Active Site of Activated Protein C (APC) - Identification of Positions Within Each CDR for Selective Modulation of APC Pleiotropic Activities

Wednesday, June 26, 2024

10:15 – 10:30 ICT

Room: 210 A-D

Presenting Author(s)

Derek S. Sim, PhD

Chief Scientific Officer
Coagulant Therapeutics
Berkeley, California, United States

Co-Author(s)

MS

Meenal Shukla

Scientist II
BioLegend/PerkinElmer
San Diego, California, United States
CM

Cornell R. Mallari

Scientist
Coagulant Therapeutics
Berkeley, California, United States
JF

Jose A. Fernández

Staff Scientist
The Scripps Research Institute
La Jolla, California, United States
XX

Xiao Xu

Scientist
The Scripps Research Institute
La Jolla, California, United States
DS

Doug Schneider

Consultant
Coagulant Therapeutics
Berkeley, California, United States
MB

Maxine Bauzon

Consultant
Coagulant Therapeutics
Berkeley, California, United States
Laurent O. Mosnier

Professor
Scripps Research
La Jolla, California, United States
TH

Terry W. Hermiston

Chief Executive Officer
Coagulant Therapeutics
Berkeley, California, United States

Background: Activated protein C (APC) is a pleiotropic protease with anticoagulant, anti-inflammatory, and cytoprotective activities. The selectively uncoupling of these APC activities by antibodies may have therapeutic application in indications such as traumatic bleeding.

Aims: We aim to characterize antibodies from the directed evolution of a parental antibody, TPP-26870, a non-active site binder of APC, to understand the role of its complementarity-determining regions (CDRs) in the modulation of APC’s pleiotropic activities.

Methods: TPP-26870 underwent CDR-directed evolution to identify amino acid substitutions with improved K¬D for subsequent APC functional characterization.

Results: Directed evolution resulted in 21 antibodies with improved KD’s and diverse effects on the modulation and uncoupling APC pleiotropic activities. Six of the mutations within the CDR1 and CDR2 of the heavy chain as well as the CDR2 and CDR3 of the light chain exhibited improved APC anticoagulant activity inhibition and enhanced PAR1 cleavage in comparison to TPP-26870, a combination of properties desirable for ameliorating bleeding without perturbing anti-inflammatory signaling and cytoprotection (Figure 1). Of these 6 mutations, light chain CDR2 substitutions (N57R and N57W) and light chain CDR3 substitutions (Y100F and Y100W) improved APC plasma half-life through the increase in APC resistance to SERPINs (Figure 2). Among them, N57W and Y100F also enhanced histone H3 cleavage in comparison to TPP-26870.

Conclusion(s): The pleiotropic functions of APC are very sensitive to epitope-CDR interactions. Single amino acid mutations within the CDRs of TPP-26870 were sufficient to elicit diverse functional effects on APC. N57W and Y100F, in the light chain CDR2 and CDR3 respectively, contributed unique properties to TPP-26870 with therapeutic potential for bleeding indications by enhancing the inhibition of anticoagulant activity, increasing histone H3 cleavage, and prolonging the half-life of APC by conferring the resistance to SERPINs with minimal perturbation of PAR1 cleavage activity.