Naoko A. Wada

Inorganic phosphate homeostasis in sodium-dependent phosphate cotransporter Npt2b⁺/⁻ mice.

An inorganic phosphate (P(i))-restricted diet is important for patients with chronic kidney disease and patients on hemodialysis. Phosphate binders are essential for preventing hyperphosphatemia and ectopic calcification. The sodium-dependent P(i) (Na/P(i)) transport system is involved in intestinal P(i) absorption and is regulated by several factors. The type II sodium-dependent P(i) transporter Npt2b is expressed in the brush-border membrane in intestinal epithelial cells and transports P(i). In the present study, we analyzed the phenotype of Npt2b(-/-) and hetero(+/-) mice. Npt2b(-/-) mice died in utero soon after implantation, indicating that Npt2b is essential for early embryonic development. At 4 wk of age, Npt2b(+/-) mice showed hypophosphatemia and low urinary P(i) excretion. Plasma fibroblast growth factor 23 levels were significantly decreased and 1,25(OH)(2)D(3) levels were significantly increased in Npt2b(+/-) mice compared with Npt2b(+/+) mice. Npt2b mRNA levels were reduced to 50% that in Npt2b(+/+) mice. In contrast, renal Npt2a and Npt2c transporter protein levels were significantly increased in Npt2b(+/-) mice. At 20 wk of age, Npt2b(+/-) mice showed hypophosphaturia and reduced Na/P(i) cotransport activity in the distal intestine. Npt2b(+/+) mice with adenine-induced renal failure had hyperphosphatemia and high plasma creatinine levels. Npt2b(+/-) mice treated with adenine had significantly reduced plasma P(i) levels compared with Npt2b(+/+) mice. Intestinal Npt2b protein and Na(+)/P(i) transport activity levels were significantly lower in Npt2b(+/-) mice than in the Npt2b(+/+) mice. The findings of the present studies suggest that Npt2b is an important target for the prevention of hyperphosphatemia.

Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression—not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.

Novel genetically-humanized mouse model established to evaluate efficacy of therapeutic agents to human interleukin-6 receptor

For clinical trials of therapeutic monoclonal antibodies (mAbs) to be successful, their efficacy needs to be adequately evaluated in preclinical experiments. However, in many cases it is difficult to evaluate the candidate mAbs using animal disease models because of lower cross-reactivity to the orthologous target molecules. In this study we have established a novel humanized Castlemans disease mouse model, in which the endogenous interleukin-6 receptor gene is successfully replaced by human IL6R, and human IL6 is overexpressed. We have also demonstrated the therapeutic effects of an antibody that neutralizes human IL6R, tocilizumab, on the symptoms in this mouse model. Plasma levels of human soluble IL6R and human IL6 were elevated after 4-week treatment of tocilizumab in this mouse model similarly to the result previously reported in patients treated with tocilizumab. Our mouse model provides us with a novel means of evaluating the in vivo efficacy of human IL6R-specific therapeutic agents.

In vivo evidence that DNA polymerase kappa is responsible for error-free bypass across DNA cross-links induced by mitomycin C

Translesion DNA synthesis (TLS) is an important pathway that avoids genotoxicity induced by endogenous and exogenous agents. DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in TLS but its protective roles against DNA damage in vivo are still unclear. To better understand these roles, we have established knock-in mice that express catalytically-inactive Polk and crossbred them with gpt delta mice, which possess reporter genes for mutations. The resulting mice (inactivated Polk KI mice) were exposed to mitomycin C (MMC), and the frequency of point mutations, micronucleus formation in peripheral erythrocytes, and γH2AX induction in the bone marrow was determined. The inactivated Polk KI mice exhibited significantly higher frequency of mutations at CpG and GpG sites, micronucleated cells, and γH2AX foci-positive cells than did the Polk wild-type (Polk(+)) mice. Recovery from MMC-induced DNA damage, which was evaluated by γH2AX induction, was retarded in embryonic fibroblasts from the knock-in mice when compared to those from the Polk(+) mice. These results suggest that Polk mediates TLS, which suppresses point mutations and DNA double-strand breaks caused by intra- and interstrand cross-links induced by MMC treatment. The established knock-in mice are extremely useful to elucidate the in vivo roles of the catalytic activity of Polk in suppressing DNA damage that was induced by a variety of genotoxic stresses.

SGLT5 Reabsorbs Fructose in the Kidney but Its Deficiency Paradoxically Exacerbates Hepatic Steatosis Induced by Fructose

Although excessive fructose intake is epidemiologically linked with dyslipidemia, obesity, and diabetes, the mechanisms regulating plasma fructose are not well known. Cells transfected with sodium/glucose cotransporter 5 (SGLT5), which is expressed exclusively in the kidney, transport fructose in vitro; however, the physiological role of this transporter in fructose metabolism remains unclear. To determine whether SGLT5 functions as a fructose transporter in vivo, we established a line of mice lacking the gene encoding SGLT5. Sodium-dependent fructose uptake disappeared in renal brush border membrane vesicles from SGLT5-deficient mice, and the increased urinary fructose in SGLT5-deficient mice indicated that SGLT5 was the major fructose reabsorption transporter in the kidney. From this, we hypothesized that urinary fructose excretion induced by SGLT5 deficiency would ameliorate fructose-induced hepatic steatosis. To test this hypothesis we compared SGLT5-deficient mice with wild-type mice under conditions of long-term fructose consumption. Paradoxically, however, fructose-induced hepatic steatosis was exacerbated in the SGLT5-deficient mice, and the massive urinary fructose excretion was accompanied by reduced levels of plasma triglycerides and epididymal fat but fasting hyperinsulinemia compared with fructose-fed wild-type mice. There was no difference in food consumption, water intake, or plasma fructose between the two types of mice. No compensatory effect by other transporters reportedly involved in fructose uptake in the liver and kidney were indicated at the mRNA level. These surprising findings indicated a previously unrecognized link through SGLT5 between renal fructose reabsorption and hepatic lipid metabolism.

Inhibitory FcγRIIb-Mediated Soluble Antigen Clearance from Plasma by a pH-Dependent Antigen-Binding Antibody and Its Enhancement by Fc Engineering

Fc engineering can modulate the Fc–FcγR interaction and thus enhance the potency of Abs that target membrane-bound Ags, but it has not been applied to Abs that target soluble Ags. In this study, we revealed a previously unknown function of inhibitory FcγRII in vivo and, using an Ab that binds to Ag pH dependently, demonstrated that the function can be exploited to target soluble Ag. Because pH-dependent Ab dissociates Ag in acidic endosome, its Ag clearance from circulation reflects the cellular uptake rate of Ag/Ab complexes. In vivo studies showed that FcγR but not neonatal FcR contributes to Ag clearance by the pH-dependent Ab, and when Fc binding to mouse FcγRII and III was increased, Ag clearance was markedly accelerated in wild-type mice and FcR γ-chain knockout mice, but the effect was diminished in FcγRII knockout mice. This demonstrates that mouse FcγRII efficiently promotes Ab uptake into the cell and its subsequent recycling back to the cell surface. Furthermore, when a human IgG1 Fc variant with selectively increased binding to human FcγRIIb was tested in human FcγRIIb transgenic mice, Ag clearance was accelerated without compromising the Ab half-life. Taken together, inhibitory FcγRIIb was found to play a prominent role in the cellular uptake of monomeric Ag/Ab immune complexes in vivo, and when the Fc of a pH-dependent Ab was engineered to selectively enhance human FcγRIIb binding, the Ab could accelerate soluble Ag clearance from circulation. We assume such a function would enhance the therapeutic potency of Abs that target soluble Ags.

Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3–mediated therapeutics

T cell–mediated immunotherapy is an attractive strategy for treatment in various disease areas. In this therapeutic approach, the CD3 complex is one of the key molecules to modulate T cell functions; however, in many cases, we cannot evaluate the drug candidates in animal experiments because the therapeutics, usually monoclonal antibodies specific to human CD3, cannot react to mouse endogenous Cd3. Although immunodeficient mice transfused with human hematopoietic stem or precursor cells, known as humanized mice, are available for these studies, mice humanized in this manner are not completely immune competent. In this study we have succeeded in establishing a novel mouse strain in which all the three components of the Cd3 complex — Cd3ε, Cd3δ, and Cd3γ — are replaced by their human counterparts, CD3E, CD3D, and CD3G. Basic immunological assessments have confirmed that this strain of human CD3 EDG–replaced mice are entirely immune competent, and we have also demonstrated that a bispecific antibody that simultaneously binds to human CD3 and a tumor-associated antigen (e.g. ERBB2 or GPC3) can be evaluated in human CD3 EDG–replaced mice engrafted with tumors. Our mouse model provides a novel means to evaluate the in vivo efficacy of human CD3–mediated therapy.

Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock‐in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP‐induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven‐week‐old inactivated Polk KI and wild‐type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP‐induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644–653, 2017.

Corrigendum: Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3–mediated therapeutics

This corrects the article DOI: 10.1038/srep45839.

Abstract 1482: Anti-GPC3 TRAB, a first-in-class T cell-redirecting bispecific antibody targeting glypican-3 with potent in vitro and in vivo antitumor efficacy against solid tumors

We present efficacy data for the T cell-redirecting antibody (TRAB) with highly potent anti-tumor efficacy. Anti-Glypican-3 (GPC3) TRAB is a humanized IgG4 bispecific antibody that simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface. Anti-GPC3 TRAB utilizes T cells as effectors to induce strong TRAB dependent cellular cytotoxicity (TDCC) in the presence of GPC3-expressing cells. Treatment with anti-GPC3 TRAB first activates T cells by increasing the expression of CD25 and CD69 and also upregulating cytokines IL-2, IL-4, IL-6, IL 10, IFNγ, and TNF, and then it enhances the proliferation of T cells. Anti-GPC3 TRAB showed antitumor activity against xenograft tumors derived from various cancer types — MKN-74 (human gastric adenocarcinoma), PC-10 (human lung squamous cell carcinoma), TOV-21G (human ovarian clear cell carcinoma), and KYSE70 (human esophageal squamous cell carcinoma) — in a NOD-SCID mouse model injected with human T cells. Although recent immunotherapy, as represented by immune check point inhibitors PD-1, PD-L1, and CTLA-4 antibodies, showed promising efficacy in human, not every patient can benefit from this immunotherapy, because the significant efficacy shown in patients by a blockade of immune checkpoints is closely related to the tumor microenvironment. The immune check point inhibitors show high efficacy against inflamed tumors, because these have been sufficiently infiltrated by cytotoxic T cells that recognize cancer-specific antigens. However, they do not have efficacy against non inflamed tumors. In an immunocompetent mouse model using human CD3 transgenic mice, neither the inhibitors that block immune checkpoints (such as PD-1, PD-L1 and CTLA-4) nor a conventional ADCC antibody recognizing GPC3 could show significant efficacy against a poorly immunogenic LLC1/hGPC3 tumor. However, anti-GPC3 TRAB showed efficacy against this poorly immunogenic tumor by utilizing any kind of T cell as effectors irrespective of TCR specificity, including not only CD8-positive but also CD4-positive T cells. The studies we present show that anti-GPC3 TRAB is a promising drug with high efficacy utilizing all kinds of T cells as effectors. The compound is expected to have efficacy even in patients with poorly immunogenic tumors, in which an immune checkpoint blockade fails to show efficacy. Citation Format: Yasuko Kinoshita, Takahiro Ishiguro, Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Natsuki Ono, Yoko Kayukawa, Otoya Ueda, Naoko A. Wada, Hiroshi Hino, Koichi Jishage, Hirotake Shiraiwa, Mika Kamata-Sakurai, Junichi Nezu, Mika Endo. Anti-GPC3 TRAB, a first-in-class T cell-redirecting bispecific antibody targeting glypican-3 with potent in vitro and in vivo antitumor efficacy against solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1482.