Transl Stroke Res. (44%) topics following active treatment and 3 of 5 (60%) subjects following placebo. AB023 did not increase bleeding or prothrombin times. Anticoagulation was verified by a saturable ~2-fold prolongation of the partial thromboplastin time for over one month after the highest dose. Conclusions: AB023, which inhibits contact activation-initiated blood coagulation and experimental thrombus formation in primates, produced a dose-dependent duration of limited anticoagulation without drug-related MDRTB-IN-1 adverse effects in a phase 1 trial. When put in context with earlier observations suggesting that FXI contributes to venous thromboembolism and cardiovascular disease, while contributing minimally to hemostasis, our data further justify clinical evaluation of AB023 in conditions where contact-initiated FXI activation is suspected to have a pathogenic role. models including device-associated thrombosis, arterial thrombosis, and polymicrobial or listeria sepsis.10C14 This work culminated in the development of AB023 (also referred to elsewhere as xisomab 3G3, or 3G3), a recombinant, humanized anti-FXI A2 domain antibody with ~70% homology to the 14E11 antibody, which we evaluated in preclinical safety and efficacy studies as well as the herein described phase 1 clinical MDRTB-IN-1 trial. The aims of this first\in\human, dose\escalation study were to describe the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and immunogenicity of a single ascending, intravenous bolus dose of AB023 in healthy volunteers as compared to placebo. Methods Because of the sensitive nature of the data collected for this study, requests to access the dataset from qualified researchers trained in human subject confidentiality protocols may be sent to the corresponding author at Aronora, Inc. The AB023 phase 1 clinical study was preregistered prior to conducting research under the unique identifier, “type”:”clinical-trial”,”attrs”:”text”:”NCT03097341″,”term_id”:”NCT03097341″NCT03097341. Please see the Major Resources Table in the Supplemental Material. AB023 humanization and manufacturing cell line development The antibody AB023 was generated by CDR-grafting technology at Antitope, Ltd (Cambridge, UK). The variable region genes from MDRTB-IN-1 the monoclonal mouse anti-human FXI antibody 14E11 were sequenced to design a series of humanized antibody variants. MDRTB-IN-1 Humanized variable region genes were designed based on human germline sequences with the closest homology to the murine sequences and constructed by gene synthesis. These were then cloned into separate vectors containing human IgG4 genes. The humanized antibody was stably expressed and tested for binding to human FXI by Rabbit polyclonal to PFKFB3 competition ELISA against biotinylated 14E11. The lead candidate antibody was chosen based on the competition binding ELISA and activity in the activated partial thromboplastin (aPTT) assay, and a stable manufacturing cell line was generated at Antitope, Ltd (Cambridge, UK) using Composite CHO? Technology. AB023 manufacturing The antibody AB023 was manufactured at Bayer Healthcare LLC, Berkeley, CA. Briefly, the cells used to inoculate production MDRTB-IN-1 bioreactor originated from the AB023 master cell bank. Cells were cultured using a fed-batch process and harvested at 14 days. Purification consisted of three chromatography column steps, two specific viral clearance steps, followed by concentration and diafiltration. After final formulation in a buffer containing histidine, arginine-HCl, methionine, sucrose, and polysorbate 80, the AB023 was filtered into the drug substance containers and stored frozen until fill/finishing as a lyophilized drug product (15 mg/mL after reconstitution). Binding affinity for FXI and FXIa The antibody AB023 was biotinylated using the EZ-Link? Sulfo-NHS-Biotinylation Kit (Thermo Scientific) as per instructions. FXI or FXIa (2 g/mL, 100 L/well) in 50 mM Na2CO3 pH 9.6 was incubated overnight at 4C in Immulon 2HB microtiter plates (Thermo Scientific). Wells were blocked with 150 L phosphate buffered saline (PBS) with 2% BSA for one hour (h) at RT. 100 L biotinylated AB023 (0.7 pM to 6.7 M) in 90 mM HEPES pH 7.2, 100 mM NaCl, 0.1% BSA, 0.1% Tween-20 (HBS) was added, and incubated for 90 min at room temperature (RT). After washing with PBS-0.1% Tween-20 (PBS-T), 100 L streptavidin-HRP (Thermo Scientific, 1:8000 dilution in HBS) was added, with incubation at RT for 90 min. After washing, 100 L substrate solution (12 mL 30 mM citric acid, 100 mM Na2HPO4 pH 5.0, 1 o-phenylenediamine dihydrochloride (OPD) tablet, 12 L 30% H2O2) was added. Reactions were stopped after 10 min with 50 L 2.5.