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Background Emicizumab (HEMLIBRA®) is a factor (F) VIII function-mimetic therapeutic bispecific antibody (BsAb) to FIXa and FX able to prevent bleeding in persons with hemophilia A (PwHA) when injected subcutaneously once every 1, 2 or 4 weeks. To develop a next generation version, we sought an agent able to keep hemostatic potential in non-hemophilic range with more convenient dosing regimen (dosing frequency/volume). We successfully created the emicizumab-based engineered four-chain BsAbs, NXT series. Among these, we selected NXT007 as a clinical candidate. Objectives The aim of this study is to clarify the in vitro and in vivo properties of NXT007 and predict its therapeutic potency non-clinically. Methods We evaluated the pharmacological activities of NXT007 in vitro using a thrombin generation assay (TGA) with FVIII-deficient patient plasma, and in vivo by inducing bleeding in FVIII-neutralizing antibody-treated acquired hemophilia A cynomolgus monkey (cyno) model. To clarify the FVIII-cofactor activity of NXT007, we performed an enzymatic kinetics analysis of FIXa-catalyzed FX activation with and without NXT007, as well as surface plasmon resonance analysis to determine the dissociation constant (KD) of NXT007 to FIX, FIXa, FX and FXa. We obtained its pharmacokinetic (PK) profile in non-human primates in a single dose SC/IV study. Results In vitro addition of NXT007 at 30 μg/mL increased the peak height of TGA in FVIII-deficient plasma to the same levels achieved by recombinant human FVIII at 40-100 IU/dL (FXIa-triggering) or 100-150 IU/dL (tissue factor-triggering). A single bolus intravenous injection of NXT007 (0.075 mg/kg) ameliorated bleeding symptoms in the cyno model to similar as a twice daily intravenous injection of recombinant porcine FVIII (20 U/kg). The in vitro and in vivo results were roughly concordant. NXT007 increased the turnover rate (kcat) of FIXa-catalyzed FX activation by approximately 4,000-folds compared to the condition without cofactor. The impact of NXT007 on the kcat was similar to that of emicizumab. As for binding affinities, the KD values of NXT007 to FIX, FIXa, FX and FXa were 1.08, 0.728, 0.0538 and 0.0231 μM, respectively in buffer solution. Compared to emicizumab, NXT007 bound more strongly to FX/FXa and with similar affinity to FIX/FIXa. This means that NXT007 would have an ability to form more FIX-BsAb-FX ternary complex than emicizumab. Calculated using the above KD values, at 30 μg/mL of BsAb the estimated concentration of FIX-NXT007-FX ternary complex in plasma is approximately 10-fold higher than that of the FIX-emicizumab-FX ternary complex which is roughly concordant with the difference in their FVIII equivalent thrombin generation activity. Prothrombin time (PT) was not clearly prolonged suggesting minimal impact on FX function by in vitro addition of NXT007 at up to 30 μg/mL, which was enough to induce sufficient thrombin burst in FVIII-deficient plasma as described above. A half-life of NXT007 was 19.6 to 24.4 days (0.02-2 mg/kg, SC) and SC bioavailability was 84.4% (2 mg/kg) in the in vivo cyno PK study, in which no obvious change in plasma FIX or FX levels was observed after 0.02-2 mg/kg single SC administration. Conclusions Based on the nonclinical results, NXT007, delivered in every-4-week SC injections, will keep a non-hemophilic range of equivalent FVIII thrombin generation in PwHA, Compared with emicizumab, NXT007's improved cofactor activity may be attributed to its more efficient ternary complex formation while keeping turnover rate with minimal impact on FX function suggested by PT value and antigen accumulation. A phase 1/2 clinical study of NXT007 is now on-going (NXTAGE; JapicCTI-194919). Disclosures Yamaguchi: Chugai Pharmaceutical Co., Ltd: Current Employment. Soeda:Chugai Pharmaceutical Co., Ltd.: Current Employment. Sato:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shibahara:Chugai Pharmaceutical Co., Ltd.: Current Employment. Koga:Chugai Pharmaceutical Co., Ltd.: Current Employment. Ichiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Joyashiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Teranishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Nishimura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shiraiwa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitamura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Igawa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Konishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Current Employment.