In vitro characterization of CT-001-a short-acting factor VIIa with enhanced prohemostatic activity

Abstract

Background: Traumatic injury and the associated acute bleeding are leading causes of death in people aged 1 to 44 years. Acute bleeding in pathological and surgical settings also represents a significant burden to the society. Yet there are no approved hemostatic drugs currently available. While clinically proven as an effective pro-coagulant, activated factor VII (FVIIa) use in acute bleeding has been hampered by unwanted thromboembolic events. Enhancing the ability of FVIIa to quickly stop a bleed and clear rapidly from circulation may yield an ideal molecule suitable for use in patients with acute bleeding.

Objectives: To address this need and the current liability of FVIIa, we produced a novel FVIIa molecule (CT-001) with enhanced potency and shortened plasma residence time by cell line engineering and FVIIa protein engineering for superior efficacy for acute bleeding and safety.

Methods: To address safety, CT-001, a FVIIa protein with 4 desialylated N-glycans was generated to promote active recognition and clearance via the asialoglycoprotein receptor. To enhance potency, the gamma-carboxylated domain was modified with P10Q and K32E, which enhanced membrane binding.

Results: Together, these changes significantly enhanced potency and clearance while retaining the ability to interact with the key hemostatic checkpoint proteins antithrombin and tissue factor pathway inhibitor.

Conclusions: These results demonstrate that a FVIIa molecule engineered to combine supra-physiological activity and shorter duration of action has the potential to overcome the current limitations of recombinant FVIIa to be a safe and effective approach to the treatment of acute bleeding.

Keywords: blood coagulation; factor VIIa; hemorrhage; hemostasis; protein engineering.

FIGURE 1

Structures of FVIIa variants. Wild‐type FVIIa (WT FVIIa) is composed of a Gla domain, 2 EGF domains, and a protease domain (PD). It is glycosylated with 2 N‐glycosylation sites (N145, N322). The N145 and N322 sites are composed of di‐ and triantennary carbohydrate chains with terminal GalNac capped with sialic acid. Highlighted in red are the amino acid substitutions relative to WT FVIIa. CT‐001(Sial) is engineered with P10Q and K32E in the Gla domain for enhanced affinity to negatively charged phospholipids and two additional N‐glycan structures T106 N and V253 N capped with sialic acid for providing a prolonged circulating half‐life to the molecule. CT‐001 is a desialylated variant of CT‐001(Sial) with additional terminal Gal and GalNAc moieties in comparison to dWT FVIIa to further accelerate plasma clearance and enhanced activity versus wild‐type sequences. dWT, desialylated wild‐type; EGF, epidermal growth factor; FVIIa, activated factor VII

FIGURE 2

CT‐001 and CT‐001(Sial) demonstrate enhanced binding to human platelets activated with human thrombin and convulxin. Activated platelets from three healthy independent donors were washed and exposed to FVIIa variants, and the binding to cell surfaces was detected by Alexa488‐anti‐FVII (clone AA3) using flow cytometry. The mean FL1 of platelet population was measured and expressed as a percent of maximum FL1 achieved by each study (n=3 independent healthy donors). CT‐001 showed significantly increased binding to activated platelets in comparison to dWT FVIIa and WT FVIIa. ANOVA, analysis of variance; dWT, desialylated wild‐type; FVIIa, activated factor VII

FIGURE 3

Inactivation of FVIIa variants by AT and TFPI. (A) There is no significant difference among CT‐001, CT‐001(Sial), dWT FVIIa, and WT FVIIa in response to inhibition by the presence of 125 nM of AT and heparin. Data are displayed as an average of three independent experiments. (B) CT‐001, CT‐001(Sial), dWT FVIIa, and WT FVIIa exhibited similar sensitivity to TFPI inhibition. Data are displayed as an average of three independent experiments. AT, antithrombin; dWT, desialylated wild‐type; FVIIa, activated factor VII; TFPI, tissue factor pathway inhibitor

FIGURE 4

CT‐001 binding affinities to sEPCR and flTF are not altered but are lowered toward sTF versus WT FVIIa. sEPCR, flTF, and sTF sEPCR were immobilized on sensor chips, and FVIIa variants of varying concentrations were injected over the surface of flow cells. The substitution of amino acids (P10Q, K32E, T106 N, V253 N) in CT‐001(Sial) led to lower K_Ds toward sEPCR, flTF, and sTF relative to WT FVIIa. The desialylation of CT‐001(Sial) to become CT‐001appears to restore some of the lowered affinity of the molecule towards sEPCR, flTF, and sTF. This increase in affinity by desialyation was also observed in dWT FVIIa in comparison to WT FVIIa. dWT, desialylated wild‐type; flTF, full‐length tissue factor; FVIIa, activated factor VII; sEPCR, soluble endothelial protein C receptor; sTF, soluble tissue factor; WT, wild‐type

FIGURE 5

In vitro and ex vivo activity characterization of CT‐001. FXa generation was measured in both (A) TF independent, and (B) TF dependent reactions to determine the ability of the FVIIa variants to generate FXa. FXa generation was determined using chromogenic substrate S‐2765 and a purified FXa standard curve. Independent of TF, CT‐001 had two times higher catalytic rate constant (k_cat), maximum reaction rate (V_max), and specificity constant (k_cat/K_M) in comparison to WT FVIIa. dWT FVIIa and WT FVIIa have similar k_cat, Michaelis constant (K_M), and V_max under this experimental condition. The data are displayed as the average of three experiments (ANOVA; *P < 0.01; **P < 0.0005). Under TF‐dependent conditions, similar k_cat, K_M, and V_max were observed among CT‐001, CT‐001(Sial), dWT FVIIa, and WT FVIIa. The data are displayed as the average of three to six experiments. (C) CT‐001 has enhanced thrombin generation activity relative to other FVIIa variants. Thrombin generation was measured in normal human plasma with 0.024 – 1000 nM of FVIIa in the presence 4 μM phospholipid. Thrombin generation was initiated with CaCl₂, and in the presence of thrombin fluorogenic substrate. The endogenous thrombin potential (ETP) was measured. CT‐001 had two times higher potency than CT‐001(Sial), dWT FVIIa, and WT FVIIa. The data are represented by the average of three independent experiments using a normal human plasma pool. (D‐G) CT‐001 has superior clotting activity to WT FVIIa across species. aPTT (STA‐PTT A) assays were performed in normal plasma pool from C57Bl/6 mice (D), cynomolgus monkeys (E), and humans (F). For all species, CT‐001 and CT‐001(Sial) were similar and demonstrated superior clotting time relative to dWT FVIIa or WT FVIIa. (G) In PT assays (STA‐Neoplastine CI Plus 5, rabbit brain thromboplastin), human plasma has an average PT clotting time of 12.8 seconds. CT‐001, CT‐001(Sial), dWT FVIIa, and WT FVIIa had similar potency, requiring 0.5, 0.6, 0.8, and 0.7 nM respectively to shorten PT clotting time to 10 seconds. (H) CT‐001 and CT‐001(Sial) showed enhanced clotting activity in rotational thromboelastometry assay in comparison to dWT FVIIa and WT FVIIa. INTEM assays were performed with ellagic acid for activating the intrinsic pathway. While all FVIIa variants shortened the average clotting time, CT‐001 and CT‐001(Sial) were superior to either dWT FVIIa or WT FVIIa (n=4 independent normal donors). ANOVA, analysis of variance; aPTT, activated partial thromboplastin time; dWT, desialylated wild‐type; FVIIa, activated factor VII; FXa, activated factor X; INTEM, intrinsic pathway thromboelastometry; PT, prothrombin time; TF, tissue factor; WT, wild‐type

FIGURE 6

Desialylated FVIIa variants have increased clearance by hepatocytes in vitro. CT‐001, which has four desialylated N‐glycans, was cleared by hepatocytes from supernatant significantly faster than CT‐001(Sial) and WT FVIIa. dWT FVII, which has two desialylated N‐glycans, was removed at a slower rate than CT‐001. The majority of CT‐001(Sial) and WT FVIIa was recovered in the supernatant after 60 minutes of incubation with hepatocytes. Three independent studies were performed. dWT, desialylated wild‐type; FVII, factor VII; FVIIa, activated factor VII; WT, wild‐type