Non–clinical efficacy,safety and stable clinical cell processing of induced pluripotent stem cell‐derived anti–glypican‐3 chimeric antigen receptor‐expressing natural killer/innate lymphoid cells

The use of allogeneic, pluripotent stem‐cell‐derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator‐initiated clinical trials will soon begin for ovarian cancer treatment using human leukocyte antigen (HLA)‐homozygous‐induced pluripotent stem cell (iPSC)‐derived anti–glypican‐3 (GPC3) chimeric antigen receptor (CAR)‐expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder‐free production of CAR‐expressing NK/ILC cells from CAR‐transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8‐3.6 × 10⁶ iPSC within 7 weeks was 1.8‐4.0 × 10⁹. These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN‐γ) production against GPC3‐expressing tumor cells. When the CAR‐NK/ILC cells were injected into a GPC3‐positive, ovarian‐tumor‐bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non–clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR‐NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell‐based immune cell cancer therapies.     

Diagnostic yield of endoscopic retrograde cholangiography and of EUS-guided fine needle aspiration sampling in gallbladder carcinomas

Background

Obtaining histological evidence of gallbladder carcinoma (GBC) is difficult due to its extraductal nature, and pathological confirmation remains challenging. We compared the diagnostic value and safety of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) with endoscopic retrograde cholangiography (ERC) in patients with suspected GBC.

Patients

Eighty-three patients with GBC were evaluated. Prior to definitive management, pathological evidence of GBC was obtained through either ERC cytopathologic sampling (n?=?33), EUS-FNA (n?=?24) or both (n?=?26).

Results

Among the 83 patients, 59 (71.0%) with biliary obstruction were sampled using ERC with 47.4% (28/59) sensitivity. In 19 of the remaining 31 cases, EUS-FNA sampling had 100% diagnostic sensitivity. Likewise, 50 (60.2%) of the 83 patients with suspected GBC underwent EUS-FNA of regional lymph nodes or the gallbladder (GB) mass itself with 94.8% sensitivity. The overall diagnostic sensitivity rates of ERC and EUS-FNA were 47.4 and 96%, respectively (P?<?0.001). Post-procedural complications were seen in 6.7% of the ERC group (4/59, all were mild pancreatitis), and in none of the EUS-FNA group (P?=?0.10).

Conclusions

Gallbladder carcinoma sampling using ERC and EUS-FNA should be incorporated into the diagnostic workup of GB lesions as complementary tools, and EUS-FNA should be applied in the setting of failed or not indicated ERC.     

Contribution of ubiquitous calpains to cataractogenesis in the spontaneous diabetic WBN/Kob rat

To determine the involvement of calpains in human cataractogenesis, studies in aged animal models are needed. Aged, male WBN/Kob rats spontaneously develop cataract along with severe, persistent diabetes with hyperglycemia and nephropathy. The purpose of present experiments was to provide a biochemical mechanism for the involvement of ubiquitous calpains in cataractogenesis in WBN/Kob rats. Serum and urinary glucose were measured to confirm diabetes, and cataracts were observed by slit lamp biomicroscopy. Calcium determinations were performed on lens samples from several ages of WBN/Kob and Wistar rats. Casein zymography, immunoblot analysis for alpha-spectrin, calpain 2, and calpain 10 were performed to detect activation of calpain in lens samples. Serum glucose levels increased and cortical cataract developed in male WBN/Kob rats within 1 year, indicating diabetic cataract. Cataract was accompanied by several presumptive biochemical indicators of calpain activation, including increased calcium, proteolysis of alpha-spectrin, and decreased caseinolytic activity for calpains suggesting calpain activation followed by autolytic degradation. Activation of ubiquitous calpains may contribute to biochemical mechanism of cataractogenesis in spontaneously diabetic WBN/Kob rats. The WBN/Kob model may be useful for elucidating the roles of calpain 2 and calpain 10 in human cataractogenesis.     

Role of hypomagnesemia in chronic cyclosporine nephropathy

BACKGROUND: Hypomagnesemia is a common finding of cyclosporine (CsA)-treated patients and has been proposed as both a cause and a consequence of CsA-induced nephrotoxicity. This experiment was conducted to elucidate the role of hypomagnesemia in the pathogenesis of chronic CsA nephropathy. METHODS: CsA (15 mg/kg/day subcutaneously) was administered to rats maintained on a low-sodium diet for 1, 2, and 4 weeks, and the effects of magnesium (Mg) supplementation on renal function, renal histology, and renal gene expression profile of fibrogenic molecules and vasoconstrictors was examined. RESULTS: CsA elicited hypomagnesemia and induced a progressive decline in glomerular filtration. At 28 day, renal tubular atrophy and cortical striped interstitial fibrosis were evident with CsA treatment. Dietary supplementation of Mg ameliorated CsA-induced hypomagnesemia and almost completely abolished CsA-induced chronic fibrotic lesions. Neither CsA nor Mg supplementation affected blood pressure. Renal cortical mRNA of transforming growth factor beta, plasminogen activator inhibitor (PAI)-1, and extracellular matrix started to increase at 14 days and elevated further at 28 days. In contrast, the increase in mRNA of tissue inhibitor of matrix metalloproteinase-1 and renin was evident early at 7 days and reached peak at 14 days. These mRNA increases, except that of renin, were almost abolished when hypomagnesemia was corrected. Magnesium supplementation also improved glomerular dysfunction, at least in part, through inhibition of up-regulated mRNA of endothelin-1. CONCLUSION: CsA-induced hypomagnesemia contributes to chronic renal fibrotic lesions seen during CsA treatment through up-regulation of fibrogenic molecules, most notably early activation of tissue inhibitor of matrix metalloproteinase-1 expression.     

Impaired infiltration of tumor-specific cytolytic T cells in the absence of interferon-gamma despite their normal maturation in lymphoid organs during CD137 monoclonal antibody therapy

Engagement of CD137 receptor by agonistic monoclonal antibodies (mAb) stimulates IFN-gamma production and eradicates established tumors in syngeneic mouse models. Using IFN-gamma-deficient mice or neutralizing mAb, we demonstrate that IFN-gamma is an absolute requirement for the antitumor effect of CD137 mAb. Despite progressive tumor growth in IFN-gamma-depleted mice, a fully competent CD8(+) cytolytic T cell (CTL) response developed in the lymph nodes. In addition, tumor cell sensitivity to IFN-gamma was not required because expression of a dominant-negative IFN-gamma receptor on the tumor did not affect the therapeutic effect of CD137 mAb. However, in the absence of IFN-gamma, the number of tumor-infiltrating CD8(+) CTLs was drastically decreased. Our results demonstrate that IFN-gamma is a critical factor regulating the infiltration of antigen-specific CTL into the tumor.     

Strategies for antigen loading of dendritic cells to enhance the antitumor immune response

Dendritic cells (DCs) primed with tumor antigens can effectively mediate the regression of a variety of established solid malignancies in both murine and human models. Despite such clinical efficacy, the optimal means of DC priming is unknown. The goal of this study was to compare three methods of tumor preparation: irradiation, boiling, or freeze thaw lysis for DC priming. Mouse bone marrow-derived DCs were loaded with defined ratios of E.G7 tumor cells expressing a model tumor antigen, OVA. Sensitized DCs were used for stimulation of OVA-specific CTLs derived from OT-1 T-cell receptor transgenic mice. IFN-gamma release, determined by ELISA at 24 and 48 h, was used to assess the expression of antigens by DCs. DCs loaded with irradiated tumors were effective stimulators for OT-1 CTLs, whereas DCs stimulated with freeze-thawed or boiled tumors did not stimulate IFN-gamma production. Freeze-thaw lysis appeared to inhibit CTL activity in vitro and in two of three cases, this effect was not overcome by the addition of OVA. The ability to load irradiated tumor cells was reproduced in two analogous human melanoma models using melanoma cell lines expressing gp100 and CTL clones specific for a gp100 melanoma antigen. Consistent with the in vitro data, only DC/irradiated tumor vaccines were effective in preventing or delaying outgrowth of E.G7 and a poorly immunogenic murine squamous cell carcinoma (SCCVII), on local tumor challenge. These data demonstrate that the method of tumor cell preparation clearly influences the ability of DCs to present antigen to T cells. Correlation of in vitro data with the generation of protective immunity in vivo suggests the utility of irradiated tumor-primed DCs as a means to generate protective immunity in patients with solid malignancies.     

Glucose monitoring by reverse iontophoresis

Glucose can be extracted through intact skin by electro-osmotic flow (a process called 'reverse iontophoresis') upon the application of a low-level electrical current. Recently we have combined iontophoretic extraction with an in situ glucose sensor in a device called the GlucoWatch biographer. Clinical results with this device show close tracking of blood glucose over a range of 2.2 to 22.2 mmol/l for up to 12 h using a single blood glucose value as calibration. The biographer readings lag behind blood glucose values by an average of 18 min. An analysis of data from 92 diabetic subjects in a controlled clinical setting shows a linear relationship (r=0.88) between GlucoWatch biographer readings and blood glucose. The mean absolute relative difference between the two measurements was 15.6% and more than 96% of the data fell in the (A+B) regions of the Clarke error grid. Similar results have been obtained from subjects using the GlucoWatch biographer in an uncontrolled home environment. The automatic, frequent, and non-invasive measurements obtained with the GlucoWatch biographer provide substantially more information about glucose levels than do the current fingerstick methods. This information can be used for improved decisions about all aspects of diabetes management.     

Multiple bile duct biopsies using a sheath with a side port: usefulness of intraductal sonography

OBJECTIVE: We clarified the number of biopsies required to determine malignancy of the biliary tract on the basis of the type of bile duct tumor. SUBJECTS AND METHODS: Patients with a biliary tract malignancy (n = 33) and a benign biliary stenosis (n = 3) underwent biopsy via the percutaneous transhepatic route. We performed intraductal sonography using a 20-MHz probe with a 2.0-mm diameter. The sonographic findings were prospectively classified as polypoid, circular, or semicircular. The tip of a long 9-French sheath with a side port was wedged into the stenosis, and six specimens were obtained with a 1.8-mm-diameter forceps with serrated cups. RESULTS: When cholangiography or intraductal sonography showed a polypoid lesion, the sensitivity of two biopsies was 100% (6/6). When cholangiography showed a stenotic lesion, the sensitivity of nine biopsies (96%, 26/27) was superior to that of two biopsies (74%, 20/27; p < 0.05). When intraductal sonography showed a circular lesion, the sensitivity of three biopsies (100%, 14/14) was superior to that of a single biopsy (64%, 9/14; p < 0.05). When it showed a semicircular lesion, the sensitivity of nine biopsies (92%, 12/13) was superior to that of two biopsies (54%, 7/13; p < 0.05). CONCLUSION: Bile duct biopsy using a sheath with a side port has a high sensitivity. However, the number of biopsies required depends on the cholangioscopic and intraductal sonographic appearance of the tumor.