Rapid in vitro assessment of the immunogenicity potential of engineered antibody therapeutics through detection of CD4+ T cell interleukin-2 secretion.

Therapeutic antibodies sometimes elicit anti-drug antibodies (ADAs) that can affect efficacy and safety. Engineered antibodies that contain artificial amino acid sequences are potentially highly immunogenic, but this is currently difficult to predict. Therefore, it is important to efficiently assess immunogenicity during the development of complex antibody-based formats. Here, we present an in vitro peripheral blood mononuclear cell-based assay that can be used to assess immunogenicity potential within 3 days. This method involves examining the frequency and function of interleukin (IL)-2-secreting CD4+ T cells induced by therapeutic antibodies. IL-2-secreting CD4+ T cells seem to be functionally relevant to the immunogenic potential due to their proliferative activity and the expression of several cytokines. The rates of the donors responding to low and high immunogenic proteins, mAb1, and keyhole limpet hemocyanin were 1.3% and 93.5%, respectively. Seven antibodies with known rates of immunogenicity (etanercept, emicizumab, abciximab, romosozumab, blosozumab, humanized anti-human A33 antibody, and bococizumab) induced responses in 1.9%, 3.8%, 6.4%, 10.0%, 29.2%, 43.8%, and 89.5% of donors, respectively. These data are comparable with ADA incidences in clinical settings. Our results show that this assay can contribute to the swift assessment and mechanistic understanding of the immunogenicity of therapeutic antibodies.

Collagen vitrigel promotes hepatocytic differentiation of induced pluripotent stem cells into functional hepatocyte-like cells.

Differentiation of stem cells to hepatocytes provides an unlimited supply of human hepatocytes and therefore has been vigorously studied. However, to date, the stem cell-derived hepatocytes were suggested to be of immature features. To obtain matured hepatocytes from stem cells, we tested the effect of culturing human-induced pluripotent stem (hiPS) cell-derived endoderm cells on collagen vitrigel membrane and compared with our previous reported nanofiber matrix. We cultured hiPS cell-derived endoderm cells on a collagen vitrigel membrane and examined the expression profiles, and tested the activity of metabolic enzymes. Gene expression profile analysis of hepatocytic differentiation markers revealed that upon culture on collagen vitrigel membrane, immature markers of AFP decreased, with a concomitant increase in the expression of mature hepatocyte transcription factors and mature hepatocyte markers such as ALB, ASGR1 Mature markers involved in liver functions, such as transporters, cytochrome P450 enzymes and phase II metabolic enzymes were also upregulated. We observed the upregulation of the liver markers for at least 2 weeks. Gene array profiling analysis revealed that hiPS cell-derived hepatocyte-like cells (hiPS-hep) resemble those of the primary hepatocytes. Functions of the CYP enzyme activities were tested in multi-institution and all revealed high CYP1A, CYP2C19, CYP2D6, CYP3A activity, which could be maintained for at least 2 weeks in culture. Taken together, the present approach identified that collagen vitrigel membrane provides a suitable environment for the generation of hepatocytes from hiPS cells that resemble many characteristics of primary human hepatocytes.

Stronger Uricosuric Effects of the Novel Selective URAT1 Inhibitor UR-1102 Lowered Plasma Urate in Tufted Capuchin Monkeys to a Greater Extent than Benzbromarone.

Urate-lowering therapy is indispensable for the treatment of gout, but available drugs do not control serum urate levels tightly enough. Although the uricosurics benzbromarone and probenecid inhibit a urate reabsorption transporter known as renal urate transporter 1 (URAT1) and thus lower serum urate levels, they also inhibit other transporters responsible for secretion of urate into urine, which suggests that inhibiting URAT1 selectively would lower serum urate more effectively. We identified a novel potent and selective URAT1 inhibitor, UR-1102, and compared its efficacy with benzbromarone in vitro and in vivo. In human embryonic kidney (HEK)293 cells overexpressing URAT1, organic anion transporter 1 (OAT1), and OAT3, benzbromarone inhibited all transporters similarly, whereas UR-1102 inhibited URAT1 comparably to benzbromarone but inhibited OAT1 and OAT3 quite modestly. UR-1102 at 3-30 mg/kg or benzbromarone at 3-100 mg/kg was administered orally once a day for 3 consecutive days to tufted capuchin monkeys, whose low uricase activity causes a high plasma urate level. When compared with the same dosage of benzbromarone, UR-1102 showed a better pharmacokinetic profile, increased the fractional excretion of urinary uric acid, and reduced plasma uric acid more effectively. Moreover, the maximum efficacy of UR-1102 was twice that of benzbromarone, suggesting that selective inhibition of URAT1 is effective. Additionally UR-1102 showed lower in vitro potential for mechanisms causing the hepatotoxicity induced by benzbromarone. These results indicate that UR-1102 achieves strong uricosuric effects by selectively inhibiting URAT1 over OAT1 and OAT3 in monkeys, and could be a novel therapeutic option for patients with gout or hyperuricemia.

Electrophysiological characterization of cardiomyocytes derived from human induced pluripotent stem cells.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) hold great promise for development of in vitro research tools to assess cardiotoxicity, including QT prolongation. In the present study, we aimed to clarify the electrophysiological/pharmacological characteristics of hiPS-CMs using the patch-clamp technique. The hiPS cells were differentiated into beating cardiomyocytes by the embryoid body method. The expression of genes related to cardiac ion channels and differentiation markers in cardiomyocytes were detected by RT-PCR. Whole-cell patch-clamp recordings were performed using single hiPS-CMs dispersed from beating colonies. We confirmed voltage-dependence of major cardiac ion currents (I(Na), I(Ca), I(Kr), and I(Ks)) and pharmacological responses to ion-channel blockers. Action potential duration (APD) was prolonged by both I(Kr)/hERG and I(Ks) blockers, whereas it was shortened by an I(Ca) blocker, indicating that these ion current components contribute to action potential generation in hiPS-CMs. As for multiple ion channel blockers, terfenadine prolonged APD, but verapamil did not, results which were identical to clinically relevant pharmacological responses. These data suggest that patch-clamp assay using hiPS-CMs could be an accurate method of predicting the human cardiac responses to drug candidates. This study would be helpful in establishing an electrophysiological assay to assess the risk of drug-induced arrhythmia using hiPS-CMs.

Modulation of retinal capillary endothelial cells by Müller glial cell-derived factors.

PURPOSE: The inner blood-retinal barrier (BRB) is a gliovascular unit in which macroglial cells surround capillary endothelial cells and regulate retinal capillaries by paracrine interactions. The purpose of the present study was to identify genes of retinal capillary endothelial cells whose expression is modulated by Müller glial cell-derived factors. METHODS: Conditionally immortalized rat retinal capillary endothelial (TR-iBRB2) and Müller (TR-MUL5) cell lines were chosen as an in vitro model. TR-iBRB2 cells were incubated with conditioned medium of TR-MUL5 (MUL-CM) for 24 h and subjected to microarray and quantitative real-time PCR analysis. RESULTS: TR-MUL5 cell-derived factors increased alkaline phosphatase activity in TR-iBRB2 cells, indicating that paracrine interactions occurred between TR-iBRB2 and TR-MUL5 cells. Microarray analysis demonstrated that MUL-CM treatment leads to a modulation of several genes including an induction of plasminogen activator inhibitor 1 (PAI-1) and a suppression of an inhibitor of DNA binding 2 (Id2) in TR-iBRB2 cells. Treatment with TGF-beta1, which is incorporated in MUL-CM, also resulted in an induction of PAI-1 and a suppression of Id2 in TR-iBRB2 cells. CONCLUSIONS: In vitro inner BRB model study revealed that Müller glial cell-derived factors modulate endothelial cell functions including the induction of anti-angiogenic PAI-1 and the suppression of pro-angiogenic Id2. Therefore, Müller cells appear to be one of the modulators of retinal angiogenesis.