Yui Suzuki

Differential renal glomerular changes induced by 5/6 nephrectomization between common marmoset monkeys (Callithrix jacchus) and rats.

We have been investigating the relevance and availability of 5/6 nephrectomized (Nx) common marmoset monkeys (Callithrix jacchus) as a chronic renal failure model. As a part of this investigation, renal glomerular changes in the Nx marmosets were histopathologically and immunohistochemically evaluated, and then compared with those in 5/6 Nx SD rats. In the Nx marmosets, the blood and urine parameters were elevated, excluding urine protein; histopathologically, enlargement of Bowmans capsule and atrophy of the glomeruli were observed in all animals, and other slight changes were also observed in 1 or 2 marmosets. There were no significant changes in the mesangial matrix injury score, vimentin and desmin positivity or the number of WT1 positive cells between the control and Nx marmoset groups. On the other hand, in the Nx rats, the blood and urine parameters were elevated; histopathologically, various changes were observed in the glomeruli, and the mesangial matrix injury score, vimentin and desmin positivity were increased, while the number of WT1 positive cells was decreased; these histopathological impacts on the renal glomerulus at 13 weeks after Nx in rats were more severe than that in the Nx marmosets. Because the glomerular basement membrane (GBM) was much thicker in the marmosets than in the rats in electron microscopy, the weaker pathological changes in the Nx marmosets might be due to the GBM thickness. This study showed for the first time glomerular lesions developed in the Nx marmosets, and the possible pathogenesis of the glomerular lesions was discussed.

Therapeutic activity of glycoengineered anti-GM2 antibodies against malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a rare and highly aggressive neoplasm that arises from the pleural, pericardial, or peritoneal lining. Although surgery, chemotherapy, radiotherapy, and combinations of these therapies are used to treat MPM, the median survival of such patients is dismal. Therefore, there is a compelling need to develop novel therapeutics with different modes of action. Ganglioside GM2 is a glycolipid that has been shown to be overexpressed in various types of cancer. However, there are no published reports regarding the use of GM2 as a potential therapeutic target in cases of MPM. In this study, we evaluated the efficacy of the anti‐GM2 antibody BIW‐8962 as an anti‐MPM therapeutic using in vitro and in vivo assays. Consequently, the GM2 expression in the MPM cell lines was confirmed using flow cytometry. In addition, eight of 11 cell lines were GM2‐positive (73%), although the GM2 expression was variable. BIW‐8962 showed a significant antibody‐dependent cellular cytotoxicity activity against the GM2‐expressing MPM cell line MSTO‐211H, the effect of which depended on the antibody concentration and effector/target ratio. In an in vivo orthotropic mouse model using MSTO‐211H cells, BIW‐8962 significantly decreased the incidence and size of tumors. Additionally, the GM2 expression was confirmed in the MPM clinical specimens. Fifty‐eight percent of the MPM tumors were positive for GM2, with individual variation in the intensity and frequency of staining. These data suggest that anti‐GM2 antibodies may become a therapeutic option for MPM patients.

Histopathological characterization of renal tubular and interstitial changes in 5/6 nephrectomized marmoset monkeys (Callithrix jacchus)

Common marmosets (Callithrix jacchus) have become a useful animal model, particularly for development of biopharmaceuticals. While various renal failure models have been established in rodents, there is currently no acceptable model in marmosets. We analyzed the damaged renal tubules and tubulointerstitial changes (inflammation and fibrosis) of 5/6 nephrectomized (Nx) common marmosets by histopathological/immunohistochemical methods, and compared these findings to those in 5/6 Nx SD rats. In Nx marmosets and rats sacrificed at 5 and 13 weeks after Nx, variously dilated and atrophied renal tubules were seen in the cortex in common; however, the epithelial proliferating activity was much less in Nx marmosets. Furthermore, the degrees of inflammation and fibrosis seen in the affected cortex were more severe and massive in Nx marmosets with time-dependent increase. Interestingly, inflammation in Nx marmosets, of which degree was less in Nx rats, consisted of a large number of CD3-positive T cells and CD20-positive B cells (occasionally forming follicles), and a few CD68-positive macrophages. Based on these findings, lymphocytes might contribute to the progressive renal lesions in Nx marmosets. Fibrotic areas in Nx marmosets comprised myofibroblasts expressing vimentin and α-smooth muscle actin (α-SMA), whereas along with vimentin and α-SMA expressions, desmin was expressed in myofibroblasts in Nx rats. This study shows that there are some differences in renal lesions induced by Nx between marmosets and rats, which would provide useful, base-line information for pharmacology and toxicology studies using Nx marmosets.

Five-sixth Nephrectomy in Female Common Marmosets (Callithrix jacchus) as a Chronic Renal Failure Model:—A Longitudinal Course of Serum Biochemical, Hematological and Histopathological Changes—

To assess the relevance and availability of subtotal nephrectomized common marmoset monkeys as a chronic renal failure (CRF) model, we observed for 26 weeks the pathophysiological condition of female marmosets subjected to five-sixth surgical nephrectomy (5/6Nx) by a two-step surgical method. The 5/6Nx marmosets showed a significant increase in serum levels of urea nitrogen, creatinine and cystatin-C immediately after 5/6Nx surgery. These renal disorder parameters subsequently tended to decrease with the passage of time but remained higher than the control levels by the end of the study. Hyperplastic parathyroid glands, a high turnover state of osteodystrophy in the femoral bone with higher serum ALP activity and anemia with hypocellularity of bone marrow were evident. The 5/6Nx marmosets showed a stable CRF condition for a long time and some characteristic disorders similar to those observed in CRF patients. These diagnostic aspects might be a species-specific anatomical and physiological signature, reflecting the nutritional condition. The CRF model using 5/6Nx marmosets might become a useful method of evaluating the unique mechanism of CRF development.

A Simple Protocol for the Myocardial Differentiation of Human iPS Cells

We have developed a simple protocol for inducing the myocardial differentiation of human induced pluripotent stem (iPS) cells. Human iPS cell-derived embryonic bodies (EBs) were treated with a combination of activin-A, bone morphogenetic protein-4 and wnt-3a for one day in serum-free suspension culture, and were subsequently treated with noggin for three days. Thereafter, the EBs were subjected to adherent culture in media with 5% serum. All EBs were differentiated into spontaneously beating EBs, which were identified by the presence of striated muscles in transmission electron microscopy and the expression of the specific cardiomyocyte markers, NKX2-5 and TNNT2. The beating rate of the beating EBs was decreased by treatment with a rapidly activating delayed rectifier potassium current (Ikr) channel blocker, E-4031, an Ikr trafficking inhibitor, pentamidin, and a slowly activating delayed rectifier potassium current (Iks) channel blocker, chromanol 293B, and was increased by treatment with a beta-receptor agonist, isoproterenol. At a low concentration, verapamil, a calcium channel blocker, increased the beating rate of the beating EBs, while a high concentration decreased this rate. These findings suggest that the spontaneously beating EBs were myocardial cell clusters. This simple protocol for myocardial differentiation would be useful in providing a sufficient number of the beating myocardial cell clusters for studies requiring human myocardium.

Potent Therapeutic Activity Against Peritoneal Dissemination and Malignant Ascites by the Novel Anti-Folate Receptor Alpha Antibody KHK2805

Many ovarian cancer patients often show peritoneal metastasis with malignant ascites. However, unmet medical needs remain regarding controlling these symptoms after tumors become resistant to chemotherapies. We developed KHK2805, a novel anti-folate receptor α (FOLR1) humanized antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The primary aim of the present study was to evaluate whether the anti-tumor activity of KHK2805 was sufficient for therapeutic application against peritoneal dissemination and malignant ascites of platinum-resistant ovarian cancer in preclinical models. Here, both the ADCC and CDC of KHK2805 were evaluated in ovarian cancer cell lines and patient-derived samples. The anti-tumor activity of KHK2805 was evaluated in a SCID mouse model of platinum-resistant peritoneal dissemination. As results, KHK2805 showed specific binding to FOLR1 with high affinity at a novel epitope. KHK2805 exerted potent ADCC and CDC against ovarian cancer cell lines. Furthermore, primary platinum-resistant malignant ascites cells were susceptible to autologous ADCC with KHK2805. Patient-derived sera and malignant ascites induced CDC of KHK2805. KHK2805 significantly reduced the total tumor burden and amount of ascites in SCID mice with peritoneal dissemination and significantly prolonged their survival. In addition, the parental rat antibody strongly stained serous and clear cell-type ovarian tumors by immunohistochemistry. Overall, KHK2805 showed cytotoxicity against both ovarian cancer cell lines and patient-derived cells. These translational study findings suggest that KHK2805 may be promising as a novel therapeutic agent for platinum-resistant ovarian cancer with peritoneal dissemination and malignant ascites.

Abstract C123: A novel anti-FOLR1 antibody developed with AccretaMab® technology, KHK2805, exhibits markedly high ADCC/CDC activity and a tolerable safety profile in preclinical models

Introduction: Folate receptor alpha (FOLR1) is a member of the folate transporter family expressed on normal tissues and overexpressed in multiple types of tumors, such as ovarian cancer, uterine cancer, non-small cell lung cancer, gastric cancer, breast cancer and kidney cancer. Currently, several clinical trials of FOLR1-targeting drugs [conventional IgG1 antibodies, which exhibit antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity (ADCC/CDC) activities, folic acid or antibody-drug conjugates and vaccines] have been conducted for ovarian and lung cancer. Therefore, FOLR1 is a remarkable target for cancer therapy under ongoing investigation. AccretaMab® technology involves combining both the POTELLIGENT®, a clinically validated ADCC-enhanced technology, and COMPLEGENT®, a new CDC-enhanced technology, systems to result in a superior technology for enhancing the killing activity of antibodies. KHK2805 is a novel humanized and CDR-altered anti-FOLR1 antibody developed with AccretaMab® technology. In this study, we evaluated the anti-cancer activity of KHK2805 in preclinical ovarian cancer models, both in vitro and in vivo, and confirmed the safety profile of KHK2805 in cynomolgus monkeys, since KHK2805 cross-reacts to cynomolgus monkey FOLR1. Materials and Methods: The binding kinetics of KHK2805 against recombinant FOLR1 (rFOLR1) were measured using the Biacore system. The epitope was determined with an ELISA against rFOLR1s. The in vitro ADCC and CDC activities against FOLR1-positive ovarian cancer cells were evaluated using PBMCs and serum from healthy volunteers. The in vivo anti-tumor activity of KHK2805 was examined using a SCID mouse model. The safety profile of KHK2805 was evaluated in cynomolgus monkeys. Results: KHK2805 induced potent ADCC and CDC activities against FOLR1-positive ovarian cancer cells. The ADCC activity of KHK2805 was significantly higher than that of the conventional anti-FOLR1 antibody. Furthermore, KHK2805 showed a potent ADCC activity against ovarian cancer cells with a low FOLR1 expression or low folic acid-uptake activity, which may be difficult to target with current FOLR1-targeting drugs. The results also showed that the markedly higher ADCC activity of KHK2805 was caused by its super-high affinity, unique epitope and use of AccretaMab® technology. In addition, the CDC activity of KHK2805 was also clearly higher than that of the conventional anti-FOLR1 antibody. This indicates that the higher CDC activity of KHK2805 is due to the application of protein engineering of CDR alterations and AccretaMab® technology. Moreover, the potent anti-tumor activity of KHK2805 was observed in a peritoneal dissemination model in SCID mice. Finally, we completed preliminary safety experiments with KHK2805. A repeated-dose toxicity study of KHK2805 (weekly 100 mg/kg for 4 weeks, intravenously) showed an acceptable tolerability profile in cynomolgus monkeys. Conclusions: KHK2805 may be a promising novel anti-FOLR1 therapeutic agent with a potent anti-tumor activity and tolerable safety profile for patients with the FOLR1 expression. Citation Format: Munetoshi Ando, Keiko Nagata, Hiroshi Ando, Mariko Nakano, Naoya Kameyama, Tsuguo Kubota, Maiko Adachi, Yui Suzuki, Kazuyasu Nakamura, Toshihiko Ishii, Ryuichiro Nakai, Takeshi Takahashi. A novel anti-FOLR1 antibody developed with AccretaMab® technology, KHK2805, exhibits markedly high ADCC/CDC activity and a tolerable safety profile in preclinical models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C123.

Abstract C124: A novel anti-FOLR1 antibody developed with AccretaMab® technology, KHK2805, exhibits potent anti-cancer activity against ovarian cancer samples with the FOLR1 expression

Introduction: Folate receptor alpha (FOLR1) is a folate transporter expressed in many cancers, including ovarian cancer. Currently, several clinical trials of FOLR1-targeting drugs [conventional IgG1 antibodies, which exhibit antibody-dependent cellular cytotoxicity/complement dependent cytotoxicity (ADCC/CDC) activities, folic acid or antibody-drug conjugates and vaccines] have been conducted for ovarian and lung cancer. Therefore, FOLR1 is a remarkable target for cancer therapy under ongoing investigation. We established KHK2805, a novel anti-FOLR1 monoclonal antibody, using AccretaMab® technology to enhance both ADCC and CDC activities. Translational research (TR) using clinical samples is essential for determining whether a novel drug shows potent efficacy in clinical studies. In this study, we evaluated the anti-cancer activity of KHK2805 using malignant ascites and serum samples from patients with ovarian cancer. In addition, the FOLR1 expression was evaluated immunohistochemically using ovarian cancer tissues. Materials and Methods: An autologous ADCC assay was conducted using cells from the malignant ascites of ovarian cancer patients, in which both malignant cells (target cells) and immune cells (effector cells) were present. Similarly, the CDC activity was evaluated using supernatant of the malignant ascites obtained from the patients. Furthermore, a CDC assay using the serum of ovarian cancer patients was conducted. An immunohistochemical protocol was established using KM4193, the parental rat antibody of KHK2805, and formalin-fixed, paraffin-embedded ovarian cancer samples were immunohistochemically stained with KM4193. Results: KHK2805 showed potent ADCC activity against FOLR1-positive ovarian cancer cells in the autologous setting using the malignant ascites samples of the ovarian cancer patients, showing a clearly higher activity than that of the conventional anti-FOLR1 antibody. In addition, the CDC activity of KHK2805 was higher than that of the conventional anti-FOLR1 antibody under conditions using the supernatant of malignant ascites or serum from the ovarian cancer patients. Therefore, KHK2805 is thought to have markedly higher killing activity against tumor cells in patients with ovarian cancer. An immunohistochemical examination of the FOLR1 expression showed that the ovarian cancer tissues were positively stained with KM4193. Conclusions: TR using clinical samples from patients with ovarian cancer demonstrated that KHK2805 may be a promising novel anti-FOLR1 ovarian therapeutic agent with a potent antitumor activity. Citation Format: Kaito Nihira, Munetoshi Ando, Keiko Nagata, Maiko Adachi, Yui Suzuki, Yutaka Kanda, Takeshi Oshima, Ken-ichiro Nan-ya, Masanori Hiura, Toshihiko Ishii, Ryuichiro Nakai, Takeshi Takahashi. A novel anti-FOLR1 antibody developed with AccretaMab® technology, KHK2805, exhibits potent anti-cancer activity against ovarian cancer samples with the FOLR1 expression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C124.

Spontaneous Malignant T Cell Lymphoma in a Young Male Common Marmoset (Callithrix jacchus)

We histopathologically and immunohistochemically investigated a case of malignant lymphoma that spontaneously developed in a male common marmoset at two years of age. Beginning at two years four months of age, the animal had an enlargement of the submandibular and inguinal lymph nodes, small subcutaneous nodules near the right breast and an approximately fivefold increase in peripheral lymphocyte count compared with the previous examination value. The postmortem findings at two years eight months of age showed lymphadenopathy with enlargement of the thymus and spleen. Small- to intermediate-sized neoplastic lymphocytes had diffusely proliferated in the enlarged nodes. The neoplastic cells were pleomorphic and had irregularly shaped nuclei. The nuclear chromatin staining revealed hyperchromatism in the small-sized cells, and the intermediate-sized cells exhibited vesicular staining. An immunohistochemical examination indicated that the neoplastic lymphocytes were positive for CD3 and negative for CD20, thus suggesting that they had originated from T cells. In addition, the proliferation of high endothelial venules and reactive epithelioid histiocytes was observed. Scattered tingible body-laden macrophages were infrequently detected. Neoplastic lymphocytes were also observed in the thymus, spleen, heart, lungs, liver, kidneys, adrenal glands and femoral and sternal bone marrow. This malignant lymphoma in a young male common marmoset was considered to fit the category of “peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS)” according to the new WHO system of classification.

Spontaneous Adenosquamous Carcinoma with Rapid Growth and EMT-like Changes in the Mammary Gland of a Young Adult Female BALB/c Mouse

This study histopathologically and immunohistochemically investigated a spontaneously occurring single mass subcutaneously located in the left lower abdomen of a female BALB/cAJcl−nu/+ mouse at 10 weeks of age. The mass was about 20 × 15 × 10 mm in size after formalin fixation; nevertheless, it was not detected by clinical observations at 9 weeks of age. H&E staining revealed the tumor origin was epithelial and probably arose from the mammary gland, and the tumor cells demonstrated a squamous, acinar or polyhedral/basal pattern. A cell kinetics analysis revealed that many of the tumor cells of the squamous, acinar or polyhedral/basal component were positive for PCNA and cyclin D1, although there were a few of TUNEL-positive tumor cells in all of the components. An epithelial/mesenchymal analysis demonstrated that most of the tumor cells of the squamous and acinar components contained keratin and E-cadherin; however, most of the tumor cells of the polyhedral/basal component were less or very weakly positive for these markers. The tumor cells of the squamous component were negative for vimentin and SMA; however, many of the tumor cells of the polyhedral/basal component exhibited vimentin. In addition, expression of SMA was confirmed in some tumor cells of the acinar and basal components. Based on the microscopic and immunohistochemical characterizations, the tumor was diagnosed to be adenosquamous carcinoma that originated from the mammary gland with rapid growth, and the tumor cells demonstrated epithelial-mesenchymal transition-like changes.