Enhanced Hemostatic Efficacy of a Membrane and Factor IXa Binding Antibody Fragment with Cellular Recyclability in Hemophilia Α
Authors: Kumar Shekhar , Krishnaswamy Sriram 
Published: December 2024

Factor VIIIa (FVIIIa) mimetic bispecific antibodies, emicizumab and mim8, mimic one of the key functions of FVIIIa in the intrinsic tenase complex by approximating the substrate factor X (FX) and protease factor IXa (FIXa). However, unlike FVIIIa, these mimetics do not bind to procoagulant membranes, which likely contributes to their significantly lower cofactor activity on a molar basis when compared to that of FVIIIa. Although single chain antibodies derived from the FIXa binding arm of emicizumab or mim8 retain some ability to enhance FIXa function, the rate of FX activation is greatly augmented in the bispecific format in which the antibodies engage both FIXa and FX. We have continued exploration of how the addition of membrane binding features to monospecific FIXa binding derivatives of emicizumab or mim8 could surpass the cofactor mimetic activities of the bispecific antibodies. Similar to one-arm antibodies derived from the FIXa binding arm of emicizumab that can only bind FIXa and impart a modest cofactor mimetic activity, our emicizumab FIXa arm derived single chain antibody fragment (scFv), EVH9VL, facilitated FIXa mediated FX activation at a rate several times lower than that of the bispecific emicizumab. The monospecific antibody fragment EVH9VL served as a foundation to introduce membrane binding properties by fusing it with the factor V C2-domain. A repeating Gly-Ser linker separated the antibody fragment from the C2-domain, allowing the variable regions to potentially orient correctly on the membrane surface for optimal engagement with FIXa. The resulting construct, EVH9VLC2 (GR8), demonstrated a substantial amplification in cofactor activity compared to EVH9VL. GR8 bound to synthetic phospholipid vesicles containing phosphatidylcholine (PC) and phosphatidylserine (PS) (PC:PS, 75:25 %) with an estimated affinity constant (Kd) of ~ 0.5 µM and strongly colocalized with FIXa at the membrane. In kinetic assays with purified proteins, GR8 showed approximately 22-fold faster FX activation as compared to EVH9VL. Remarkably, despite lacking FX binding ability, GR8 showed a cofactor mimetic activity about ten-fold higher than emicizumab in activated partial thromboplastin time (APTT) assays using FVIII-deficient hemophilia A (HA) plasma. This potent enhancement of FX activation by GR8 was also evident in thrombin generation assays (TGA). At 100 nM concentration, GR8 normalized thrombin generation in HA plasma supplemented with 4 μM PC:PS, activated by low tissue factor (0.1 pM) or factor XIa (0.1 nM). The C2 domain-mediated increase in cofactor activity of GR8 was effectively reversed by an antibody fragment (E9scFv) that impairs C2-domain binding to the membrane, providing a built-in reversal mechanism for GR8's cofactor activity if necessary. Further modifications, including changing the C-terminus 6-His tag to HPC4 or removing the tag entirely, increased cofactor mimetic activity by an additional 3-fold, suggesting that the histidine charge cluster at the C2-domain's C-terminus may hinder optimal membrane interaction. Substituting the FV C2-domain in GR8 with lactadherin's C2-domain maintained similar cofactor activity, indicating that membrane dependent enhancement of cofactor activity is not limited to FV C2-domain. Similarly, mim8 FIXa binding arm-derived scFv fused with the FV C2-domain also showed several-fold increased cofactor activity compared to its unmodified counterpart. These engineered FIXa binding variants were able to overcome FVIII inhibitors in HA plasma. To prolong half-life and introduce inherent latency until activation, human albumin was fused to GR8 at the N-terminus, linked with a FIX activation peptide. This fusion aimed to exploit cellular recycling via neonatal Fc receptor interaction, potentially maintaining a latent state until triggered by a procoagulant stimulus. The albumin-fused GR8 retained cofactor mimetic activity similar to GR8 and was cleaved by human FXIa in vitro. The engineered scFv with a factor V C2 domain fusion, demonstrates that simply adding a membrane binding feature to the FIXa arm of emicizumab surpasses the mimetic activity achieved by a bispecific antibody that exploits both bridging and allostery for effective cofactor function. This advancement underscores the potential of single-chain, monospecific, membrane-anchored FVIIIa mimetics to significantly enhance FIXa's catalytic activity.