The use of nonfactor products such as emicizumab, a bispecific antibody that mimics the cofactor function of activated factor VIII (FVIIIa) by bridging activated factor IX (FIXa) and factor X (FX), has significantly reduced bleeding episodes in people with hemophilia A. However, emicizumab high species-specificity in its FVIIIa-mimetic cofactor activity has limited in vivo functional evaluation to an acquired hemophilia A(HA) non-human primate model or congenital HA mouse model with infusion of human FIX (hFIX) and FX (hFX) proteins. To overcome the limitations of existing models, we generated hFIX/hFX double knock-in congenital HA model (Humanized HA; HuHA) mice. HuHA mice were established by the replacement of mouse FIX/FX genes with their human homologs and by the disruption of mouse FVIII. In HuHA mice, functional hFIX and hFX are constitutively expressed at levels equivalent to or higher than human plasma concentrations. Thrombin generation assays demonstrated dose-dependently improved peak thrombin generation by both recombinant human FVIII (rhFVIII) and emicizumab. These HuHA mice were found to express sufficient levels of hFIX and hFX to support functional evaluation of rhFVIII and emicizumab activities. Furthermore, the tail-clip assay suggested potential hemostatic effects of both therapeutic agents in vivo, although the findings should be interpreted with caution given the limitations of the model. This novel mouse model, in which functional hFIX and hFX are constitutively expressed on an FVIII-deficient background, may provide a new platform for the preclinical evaluation of human-specific therapeutics for HA.

Abstract Background In haemophilia, recurrent joint bleeding often results in joint destruction, which in turn leads to reduced physical activity (PA) and decreased quality of life (QOL). While maintaining high PA levels is important in the care of people with haemophilia (PwH), it remains unclear whether PwH without joint disease can maintain adequate activity levels. Aim This descriptive cross-sectional study sought to compare moderate-to-vigorous physical activity (MVPA) levels among 19 PwH with haemophilic arthropathy (HA), 12 PwH without HA, and 15 non-PwH. Methods A total of 46 males wore a triaxial accelerometer (wGT3X-BT, ActiGraph) during daily activities for seven consecutive days. A bout of MVPA was defined as at least 10 consecutive minutes of moderate-intensity or higher activity. MVPA was expressed as the percentage of the total wear time spent in MVPA, and the total number of MVPA bouts was also recorded. Results Both PwH groups demonstrated significantly lower MVPA percentages and fewer MVPA bouts than the controls (p < 0.01). No MVPA bouts were recorded among 52.6% of PwH with HA versus 25% of PwH without HA. No significant differences were observed in sedentary or light activity between the PwH groups. Conclusions PA levels were significantly reduced in PwH, regardless of joint status, suggesting that factors beyond joint damage, such as fear of bleeding or behavioural avoidance, may play a role. These findings highlight the need for individualized, evidence-based interventions to safely promote physical activity and improve long-term health outcomes in PwH.

BACKGROUND:Intra-cranial haemorrhage (ICH) in neonates with haemophilia A (HA) remains a serious perinatal complication. Prophylactic administration of emicizumab (Emi) to pregnant HA carriers has been discussed as a potential preventive strategy, although current clinical evidence is limited to one case report. This study evaluated whether ex vivo addition of Emi further augments coagulation potential in late pregnancy. METHODS:Plasma samples were obtained from 62 healthy pregnant women and five pregnant HA carriers during late gestation. Emi (50 µg/mL) was added ex vivo, and coagulation potential was assessed using the thrombin generation assay (TGA) and clot waveform analysis (CWA). TGA parameters included time to thrombin peak (ttPeak), peak thrombin (PeakTh) and endogenous thrombin potential (ETP). CWA parameters included clot time (CT) and adjusted coagulation velocity (Ad|min1|). RESULTS:FVIII activity was elevated in both groups. Emi induced modest shortening of ttPeak in healthy women (9.3-8.5 min) and in carriers (9.3-8.3 min). PeakTh was unchanged in healthy women before and after Emi addition and showed a small reduction in carriers. ETP did not change in healthy women, whereas a slight decrease was observed in carriers. CWA demonstrated a small but statistically significant shortening in CT and increases in Ad|min1| in both groups. Overall, Emi-induced changes of coagulation potential across assays were modest and generally within assay variability. CONCLUSION:These findings ex vivo indicated that Emi at clinically relevant concentrations does not meaningfully enhance the hypercoagulable state characteristic of late pregnancy. In vivo studies are required to confirm the safety of maternal prophylaxis.

Recent advancements in haemophilia treatment have dramatically reduced the frequency of symptomatic bleeding. However, minor, asymptomatic bleeding may contribute to the progression of joint damage and decrease quality of life. Detecting small amounts of blood with minimal flow is difficult using existing testing methods, necessitating the development of new diagnostic approaches. To determine whether tiny amounts of blood injected subcutaneously, intramuscularly, and intra-articularly can be detected in a pig to assess the feasibility of detecting microbleeds in people with haemophilia. Pig whole blood was injected subcutaneously, intramuscularly, and intra-articularly into the isolated hind limbs of pigs. Photoacoustic imaging (PAI) was performed using an Acoustic X device (CYBERDYNE INC, Tsukuba, Japan) to detect blood signals. Following the injection of 200 µL of blood, conventional ultrasound imaging revealed no discernible changes, such as echo-free spaces. Conversely, on PAI, a signal indicating the presence of blood was detected. We have demonstrated that even very small amounts of nonflowing blood injected into the subcutaneous tissue, muscle, and joint of the hind limbs of pigs can be detected using PAI. We believe that the results of this study show the potential of this technique for detecting microbleeds in people with haemophilia. Not applicable.

Neovascularization is a major cause of blindness in various retinal diseases, and inflammation aggravates the pathological and clinical conditions of these diseases. VEGFA or ANG-2 neutralizing antibodies have been used to block pathological neovascularization. In this work, the effects of intraocular administration of neutralizing antibodies against VEGFA, ANG-2, or bispecific to these two factors on pathological findings were examined in the oxygen-induced retinopathy (OIR) mouse model. At both postnatal day (P)17 and P19, anti-VEGFA and -ANG-2 administration suppressed neovascularization, and the bispecific antibody attenuated neovascularization more efficiently. However, oxygen-induced vaso-obliteration was not modified by these antibodies. Numbers of photoreceptor, amacrine, and bipolar cells were reduced in the OIR retina, and the antibodies reversed these changes. Microglia-specific gene expression increased in the OIR retina, and administration of the antibodies reduced the IBA1-positive area in the OIR retina, although these antibodies did not affect Iba1 gene expression. Labeled VEGFA and ANG-2 were found to be co-localized with microglia, suggesting that VEGFA and ANG-2 affect microglia activation directly. Taken together, neutralizing antibody to VEGFA or ANG-2 attenuated oxygen-induced neovascularization and inflammation, and the bispecific antibody more efficiently suppressed some features than the single antibody to VEGFA or ANG-2.

Introduction: Humanized mouse models have been developed to elucidate human-specific responses in various research applications. The TK-NOG mouse model enables human hepatocyte engraftment following ganciclovir-induced hepatic injury, allowing replacement of mouse hepatocytes with human hepatocytes. Since many coagulation and anticoagulation factors are produced in the liver, we expected that the coagulation cascade in humanized TK-NOG mice would more closely resemble that of humans than that of mice. We therefore hypothesized that developing a hemophilia A model using these mice would provide a unique experimental model with functional human coagulation-related factors. In this study, we generated a novel hemophilia A mouse model (TK-NOG F8KO mice) and subsequently humanized these mice by transplanting human hepatocytes. We then characterized the phenotypic and functional properties of these humanized mice.Methods: Animal experiments were conducted with institutional approval. We first replaced the genetic background of B6;129S-F8tm1Kaz/J (F8KO) mice with NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic (NOG) to generate NOG F8KO mice, then crossbred TK-NOG mice with NOG F8KO mice to create TK-NOG F8KO mice. These mice received 0.1 mg/mL valganciclovir hydrochloride in drinking water for 48 hours. Seven days after initiation of water intake, each mouse received 0.76-1.10×10^6 viable normal human hepatocytes via splenic portal injection following recombinant human (rh) FVIII administration for hemostasis. Plasma and liver samples were collected 8 weeks post-transplantation and analyzed for human hepatocyte engraftment, chimerism rate, gene expression of coagulation factors, concentrations of human factor IX (hFIX) and human factor X (hFX), and coagulation activity using histological assessment, enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), electrochemiluminescence (ECL), and global coagulation assays.Results: Human hepatocyte engraftment was confirmed by histological assessment using human mitochondria staining, with chimerism rates ranging from 12.6% to over 95% in 25 humanized TK-NOG F8KO mice as determined by plasma human albumin ELISA. qPCR analysis of liver tissue showed no human gene expression in TK-NOG F8KO control mice, whereas humanized TK-NOG F8KO mice expressed human coagulation and anticoagulation factor genes (F2, F7, F8, F9, F10, F11, protein C (PC), antithrombin (AT)). Both human and mouse FVIII genes were detected, consistent with previous reports of low FVIII gene expression in human hepatocytes (Tatsumi, Cell Transplant., 2012) and residual expression in F8KO mice (Bi, Blood, 1996). Global coagulation assay revealed that 11 humanized TK-NOG F8KO mice showed FVIII activity below the limit of quantification, while one mouse demonstrated 5.3% FVIII activity. Additionally, 20 mice, including controls, showed less than 4% FVIII activity. PC levels were below the detection limit in 3 humanized TK-NOG F8KO mice, but all other factors showed measurable activity. Although the assay cannot differentiate between human and mouse activities, some factors such as FX, factor XI (FXI), AT, and PC showed tendencies toward 100% activity as chimerism increased, suggesting that human factors were becoming dominant. This phenomenon was supported by ECL measurements, which confirmed hFIX and hFX expression with positive correlation to chimerism rate, indicating that humanized TK-NOG F8KO mice with higher chimerism rates express greater quantities of human coagulation factors. Finally, activated partial thromboplastin time (APTT) and clot waveform analysis were performed in mice with sufficient plasma volume to evaluate the effects of rhFVIII and emicizumab, that has no cross reactivity to mouse FIX and FX. Both treatments shortened APTT and increased adjusted maximum coagulation velocity. Notably, with emicizumab treatment, APTT tended to shorten as chimerism rate increased, demonstrating the functionality of human coagulation factors in this model.Conclusions: We successfully developed a novel humanized mouse model expressing human coagulation factors derived from transplanted human hepatocytes. This model demonstrates that human-derived coagulation factors increase proportionally with chimerism rate, enabling effective evaluation of human factor-specific therapeutics. With stable production of high-chimerism mice, applications to various disease research areas can be anticipated.

Diabetic retinopathy (DR) is known as a microvascular complication, in which various inflammatory symptoms, including activation of microglia, are observed. A model of hyperglycemia resembling type 1 diabetes mellitus (DM) induced in mice by intraperitoneal injection of streptozotocin (STZ) has been widely used. We examined the effects of anti- vascular endothelial growth factor A (VEGFA) and anti-angiopoietin-2 (ANGPT2) antibodies in addition to a bispecific antibody against VEGFA and ANGPT2 by intravitreously administrated to the eyes on early signs, especially the activation of microglia in STZ-treated mice eyes. After 14 weeks of STZ administration, alterations in activity by ERG and CD31 staining patterns were not observed. Although a difference in the number of microglia in the retina between normal and STZ-model retinas was not observed, the morphology of microglia had changed from ramified in control to amoeboid shape in STZ model at 4 days after the antibodies injection. Detailed morphological examination showed decreases in area, ramification index, and tree length in the STZ-model retinas compared with normal retinas. Recovery from these decreases was demonstrated mainly by the administration of the bispecific antibody. These results suggest that anti-VEGFA/ANGPT2 therapy may suppress the activation of microglia in the early stages of DR.

BACKGROUND:Haemophilia, caused by deficiencies in factor VIII or IX, leads to deep tissue bleeding and haemophilic arthropathy. Bleeding, arthropathy, and the perioperative period of surgery for arthropathy are associated with severe pain. To avoid this pain, patients may compensate by overloading other parts of the body, potentially resulting in pain or secondary damage in areas distant from the primary site. This study investigates the use of infrared thermography (IRT) to monitor whole-body surface temperatures in people with haemophilia and to explore the potential of infrared thermography for early detection of compensatory overload. METHODS:A cross-sectional study involved 24 people with haemophilia aged 6-76 years old, experiencing pain after bleeding, post-orthopaedic surgery, or haemophilic arthropathy. Thermal images were captured with IRT and analyzed using software that utilises deep learning for whole-body temperature mapping. RESULTS:Increases in temperature indicative of overload were observed in areas distant from the affected site, either on the same side (vertical pattern) or on the opposite side (diagonal pattern) relative to the site of pain. These patterns were observed in 13 of 14 participants with haemophilic arthropathy, all post-orthopaedic surgery participants, and 5 of 6 after bleeding. CONCLUSIONS:Temperature increases occurred in areas beyond the painful area, suggesting strain even in asymptomatic regions. People with haemophilia experiencing pain may require careful monitoring and treatment of surrounding areas. Our findings could aid in diagnosing haemorrhage and local inflammation while informing treatment decisions. CLINICAL TRIAL REGISTRATION:Not applicable.

Corneal neovascularization (CNV) is frequently observed after various corneal injuries and corneal transplantation, leading to impairment of corneal transparency. Lymphangiogenesis and the inflammatory response often accompany CNV. Chemical injury is one of the most common causes of CNV, and alkali injury has been widely used as an animal model of this pathological state. We examined the effects of subconjunctival injection of an anti-VEGFA antibody (anti-VEGFA_ab), anti-ANGPT2 antibody (anti-ANGPT2_ab), and bispecific antibody against VEGFA and ANGPT2 (BsAb) in CNV using the alkali injury mouse model. The pathological indexes were examined using anterior segment optical coherence tomography (OCT) and whole-mount immunostaining, and gene expression patterns were examined using RT-qPCR. Treatment with anti-VEGFA_ab, anti-ANGPT2_ab, or BsAb did not affect the swelled thickness of the cornea; however, angiogenesis, but not lymphangiogenesis, was hampered by the treatment of either one of the antibodies. We observed an increase in the mRNA levels of Vegfa, Angpt2, Il1b, and Cx3cr1 following alkali injury. The administration of BsAb suppressed the induction of Vegfa and Cx3cr1. Additionally, BsAb treatment enhanced the mRNA levels of Angpt1 in this model. This study demonstrates the potential of dual VEGFA and ANGPT2 inhibition as a therapeutic strategy for CNV.

The Protein C (PC) system maintains proper control of fibrin formation in hemostasis by downregulating the coagulation propagation phase, making its inhibition a candidate for “rebalancing” approaches in hemophilia treatment. It is also known that decreased PC-function in non-hemophilia individuals causes purpura fulminans, a severe thrombotic condition. In people with hemophilia, coagulation potential sometimes reaches non-hemophilic levels during on-demand therapeutic intervention for breakthrough bleeds. With the expectation of achieving both prophylaxis of bleeding and mitigation of the purpura fulminans risks, we developed a partial neutralizing antibody to human PC (termed PPC85N), and evaluated it in nonhuman primates. In in vitro enzymatic assays, PPC85N demonstrated incomplete inhibition of up to approximately 75% against human activated PC (APC) activity in the presence of Protein S, and up to approximately 60% against human PC activation by thrombin on endothelial cell sheets. Consistent with these results, PPC85N partially restored thrombin generation capacity in PC-activated human and primate plasma in vitro. For in vivo evaluation in a hemophilia A state, we used our established sub-acute bleeding model of primates with acquired hemophilia A, in which specific bleeding symptoms progressed over 3 days following artificial injury procedures. Previously, it was prospectively demonstrated that in vivo results of emicizumab against such bleeding symptoms roughly predicted its clinical efficacy. Results of a small-scale primate study showed that preventive dosing of PPC85N (3 mg/kg IV on Days 0 and 2, n = 2) tended to suppress the sub-acute bleeding symptoms, hemorrhagic anemia (indicated by % hemoglobin (Hb) levels relative to pre-values) and bruised area on body surface (cm²): 82.8% [mean of 2 animals] vs 42.5 ± 7.3% [mean ± SE] in the control group (n = 8), and 62.0 cm² vs 78.4 ± 20.6 cm², respectively. The degree of suppression was comparable to twice-daily dosing of recombinant porcine factor (F)VIII (rpoFVIII) at 1 or 3 U/kg IV (n = 3 for each) that would maintain its plasma levels equivalent to moderate or mild-to-moderate disease: 80.1 ± 6.3% or 92.9 ± 2.8%, and 65.6 ± 8.1 cm² or 28.5 ± 22.7 cm², respectively. Additionally, we induced joint bleeds in their right knees by intra-articular needling and evaluated joint swelling. While peak increase of knee joint diameter (% increase from pre-values) was 10.2 ± 3.5% in the control group, it was 2.6%, 3.0 ± 1.4% or 1.4 ± 0.7% in the PPC85N group, the rpoFVIII 1 U/kg group, or the 3 U/kg group, respectively, suggesting that PPC85N tended to suppress joint bleeds. To more rigorously examine these suppressive effects against sub-acute progressive bleeding symptoms observed in primates, we reproduced such bleeding symptoms in FVIII-deficient mice. The mice were crossbred with a nude strain to visualize bruising without damage from hair removal procedure. In this model, preventive dosing of anti-mouse PC antibody (3 or 10 mg/kg IV, n = 9 for each) significantly improved both Hb level and bruised area: 12.1 ± 0.7 g/dL* or 11.6 ± 1.2 g/dL vs 8.0 ± 1.6 g/dL in the vehicle group (n = 8), and 1.11 ± 0.48 cm²* or 0.73 ± 0.46 cm²* vs 5.44 ± 1.21 cm², respectively [*P < 0.05 by Dunnett's test]. These mouse results supported the effects of PPC85N observed in primates, although this anti-mouse PC antibody had a potential to fully neutralize APC activity, unlike PPC85N. Finally, we examined whether PPC85N would induce purpura fulminans-like symptoms in non-hemophilic primates. To minimize immune reactions caused by PPC85N itself, we prepared and used a chimeric antibody with human IgG1 in which reactivity to Fcγ receptors and C1q was silenced (termed chPPC85N). As a result, chPPC85N (1, 3, or 10 mg/kg IV, n = 3 for each) induced purpura fulminans-like symptoms (skin redness, irritation, wounds etc.) in more than half of the animals at each dosage, and also thrombotic pathological changes in several animals. In conclusion, we demonstrated the bleeding preventive effects of a partial neutralizing antibody to PC, PPC85N, in non-human primates with acquired hemophilia A. However, we also found in the non-hemophilic primate study that the purpura fulminans risk was not avoided even with this partial inhibition approach. Our in vivo research may highlight difficulties in balancing efficacy and safety for clinical application of anti-PC intervention therapy in hemophilia.

While the conventional understanding of haemostatic plug formation is that coagulation proceeds efficiently on the surface of activated platelets at the vascular injury site to form a robust haemostatic plug, this understanding does not explain the clinical reality that platelet dysfunction results in a mild bleeding phenotype, whereas coagulation disorders exhibit severe bleeding phenotypes, particularly in deep tissues. Here, we introduce an in vivo imaging method to observe internal bleeding and subsequent haemostatic plug formation in mice and report that haemostatic plug formation after internal bleeding, coagulation occurs primarily outside the blood vessel rather than on platelets. Experiments in mice with impaired platelet surface coagulation, depleted platelets, haemophilia A or reduced tissue factor expression suggest that this extravascular coagulation triggers and regulates haemostatic plug formation. Our discovery of the important role of extravascular coagulation in haemostasis may contribute to refining the treatment of haemostatic abnormalities and advancing antithrombotic therapy.

Arthropathy is a common complication in haemophilia and decreases quality of life. It has been known that concentrations of β-hydroxybutyrate (BHB) in blood are increased by a ketogenic diet, and elevated levels of circulating BHB restricts the progression of inflammation-mediated joint pathological changes. We hypothesized that elevation of blood BHB concentrations could be effective for reducing the progression of bleeding-induced arthropathy by moderating the inflammatory responses of macrophages. In this study, we investigated whether BHB alleviates the arthropathy caused by repeated intra-articular blood injection in rats. To increase blood BHB levels, rats were fed with ketogenic diet. Repeated intra-articular blood injection induced significant joint swelling, whereas ketogenic diet intake significantly increased blood BHB concentrations and ameliorated the joint swelling. The periarticular tissue-fibrosis observed in the control diet intake group appeared to be significantly alleviated in the ketogenic diet intake group. In addition, the IL-1β, which is involved in the progression of arthropathy, levels in the supernatants of blood-exposed macrophages derived from THP-1 cell line were significantly suppressed by BHB supplementation. In summary, BHB moderated the pathological joint changes caused by intra-articular blood exposure.

Background: Haemophilia is a bleeding disorder caused by deficiencies in factor VIII or IX, leading to bleeding in deep tissues and haemophilic arthropathy (HA). Surgical treatments like synovectomy or joint replacement can alleviate symptoms but often result in severe pain. Secondary injuries may occur in non-affected body parts due to compensatory overloading. This study explores using infrared thermography (IRT) to monitor whole-body surface temperatures in haemophilia patients to detect early signs of overload and prevent further injury by visualizing compensatory load distribution. Methods: A descriptive cross-sectional study was conducted involving 24 haemophilia participants aged 6-76 years, experiencing pain after active bleeding, after orthopaedic surgery, or with HA. Thermal images were captured using IRT and analysed with deep learning for whole-body mapping and temperature comparison between symmetrical body regions. Results: Temperature increases indicating overload were observed in body areas ipsilateral (vertical pattern) or contralateral (diagonal pattern) to the painful site. These patterns were detected in 13 of 14 participants with HA, all participants after orthopaedic surgery, and 5 of 6 participants with active bleeding. Conclusion: The temperature increases, which may reflect the physical load, occurred in areas other than the painful area. When PwH present with pain, the surrounding area, especially in the diagonal and/or vertical position, may be under strain, even if it is asymptomatic, and requires careful observation and treatment. Our findings may be useful not only for supporting the diagnosis of haemorrhage and local inflammation but also for supporting such treatment decisions.

Background: The hemostatic effect of recombinant (r) factor (F)VIIa after repetitive intermittent administration may be attenuated in patients with hemophilia A (PwHA) with inhibitors (PwHAwI) creating a clinically unresponsive status, although mechanism(s) remain to be clarified. In patients receiving prophylaxis treatment with emicizumab, concomitant rFVIIa is sometimes utilized in multiple doses for surgical procedures or breakthrough bleeding. Aim and methods: We identified 'unresponsiveness' to rFVIIa, based on global coagulation function monitored using rotational thromboelastometry (ROTEM) in 11 PwHAwI and 5 patients with acquired HA, and investigated possible mechanisms focusing on the association between plasma FX levels and rFVIIa-mediated interactions. Results: Our data demonstrated that FX antigen levels were lower in the rFVIIa-unresponsive group than in the rFVIIa-responsive group (0.46 +/- 0.14 IU/mL vs. 0.87 +/- 0.15 IU/mL, p < 0.01). This relationship was further examined by thrombin generation assays using a FX-deficient PwHAwI plasma model. The addition of FX with rFVIIa was associated with increased peak thrombin (PeakTh) generation. At low levels of FX (<0.5 IU/mL), rFVIIa failed to increase PeakTh to the normal range, consistent with clinical rFVIIa-unresponsiveness. In the presence of emicizumab (50 mu g/mL), PeakTh was increased maximally to 80 % of normal, even at low levels of FX (0.28 IU/mL). Conclusions: Unresponsiveness to rFVIIa was associated with reduced levels of FX in PwHAwI. Emicizumab exhibited in vitro coagulation potential in the presence of FX at concentrations that appeared to limit the clinical response to rFVIIa therapy.

(233/250) Retinal vein occlusion (RVO) causes macular edema and retinal ischemia resulting in visual field and vision loss. A bispecific antibody that blocks VEGF-A and angiopoietin-2 (Ang-2) has been recently launched and applied clinically to treat macular edema, but the role of Ang-2 in the pathogenesis of RVO is still unclear. In this study, we investigated the effects of the anti-VEGF-A/anti-Ang-2 bispecific antibody (BsAb) in a murine RVO model. By using RVO model mice, the expression of Ang-2 gene and protein was examined in the retina through real-time qPCR and Western blotting, respectively. A significant increase in Ang-2 was detected 1 day after occlusion. Immediately after occlusion, control IgG 400 μg/mL, anti-VEGF-A antibody 200 μg/mL, anti-Ang-2 antibody 200 μg/mL, and BsAb 400 μg/mL were intravitreally administered at 2 μL. Visual function was examined using electroretinograms, and apoptosis was examined using TUNEL staining. Interestingly, BsAb partially suppressed the decrease in amplitude of a and b waves compared to control IgG. Anti-Ang-2 antibody and BsAb reduced apoptosis-positive cells 1 day after occlusion. Comprehensive gene expression profiles were also examined using RNA sequencing analysis. RNA sequencing analysis of the retinal tissues showed that BsAb suppressed expression of gene groups associated with inflammatory response and vascular development compared to anti-VEGF-A antibody. Taken together, higher expression of Ang-2 contributes to the pathophysiology of RVO, providing a possible mechanism for the efficacy of BsAb in suppressing retinal dysfunction in RVO.

Emicizumab, a factor (F) VIII function-mimetic bispecific antibody, is used for the treatment with patients with hemophilia A (PwHA). Although the immunogenicity of emicizumab is low, potential of immunogenicity is still remained. Despite some cases of anti-drug antibodies (ADAs) reported, the characteristics of ADAs have not been fully elucidated. In this research, we evaluated the characteristics of ADAs by using repository samples collected in phase 1, phase 1/2 and bioavailability studies conducted in Japan. Ten plasma/serum samples from 6 healthy volunteers (HVs) and 4 PwHA who tested positive for ADAs in the clinical studies were used for the assessment of neutralizing activity, epitope analysis and pharmacokinetics (PK). Neutralizing activity of ADAs was observed in 3 HVs and 1 PwHA. Among these, 3 HVs developed ADAs which bound to the complement-determining region (CDR)1, 3 of the common light chain (cLC) of emicizumab and associated with shorter half-life. Epitopes of ADAs in 1 PwHA were on the Fab-regions of emicizumab, and the ADAs were not associated with decreased exposure in this PwHA. Neutralizing activity was undetectable in 3 HVs and 3 PwHA. Among these, ADAs in 2 HVs and 2 PwHA recognized the Fab-regions or the CDR 1, 3 of the cLC, and 1 of these 2 HVs showed shorter half-life of emicizumab. In conclusion, our analysis of ADAs demonstrated the various characteristics of ADAs, such as ADAs with either neutralizing activity or affected pharmacokinetics, or both properties.