Development of rat duodenal monolayer model with effective barrier function from rat organoids for ADME assay.

The in-depth analysis of the ADME profiles of drug candidates using in vitro models is essential for drug development since a drug's exposure in humans depends on its ADME properties. In contrast to efforts in developing human in vitro absorption models, only a limited number of studies have explored models using rats, the most frequently used species in in vivo DMPK studies. In this study, we developed a monolayer model with an effective barrier function for ADME assays using rat duodenal organoids as a cell source.

Generation of a lung squamous cell carcinoma three-dimensional culture model with keratinizing structures.

Tumor nests in lung squamous cell carcinoma (LUSC) have a hierarchical structure resembling squamous epithelium. The nests consist of basal-like cells on the periphery and layers of keratinocyte-like cells that differentiate towards the center of the nest, forming keratin pearls. Reproducing this spatial heterogeneity in in vitro models would be useful for understanding the biology of LUSC.

Tissue-specific effects of an anti-desmoglein-3 ADCC antibody due to expression of the target antigen and physiological characteristics of the mouse vagina.

Desmoglein-3 (DSG3) is a potential target of cytotoxic antibody therapy for squamous cell carcinomas but is also expressed in various normal squamous epithelia. We obtained information about DSG3 distribution in mouse tissues by immunohistochemistry and conducted an intravenous multiple-dose study in mouse to estimate the toxic potential of anti-DSG3 therapy. DSG3 was expressed in the squamous epithelium of several organs including the skin, esophagus, tongue, forestomach, eye, and vagina.

Three-dimensional culture models mimic colon cancer heterogeneity induced by different microenvironments.

Colorectal cancer demonstrates intra-tumour heterogeneity formed by a hierarchical structure comprised of cancer stem cells (CSCs) and their differentiated progenies. The mechanism by which CSCs are maintained and differentiated needs to be further elucidated, and there is evidence that the tumour microenvironment governs cancer stemness. Using PLR123, a colon cancer cell line with CSC properties, we determined the culture conditions necessary to establish a pair of three-dimensional (3D) culture models grown in Matrigel, designated stemCO and diffCO.

A simple method for histopathological evaluation of organoids.

-cultured 3D structures called organoids have become important tools for biological research, but there is little information concerning simple and efficient methods to evaluate organoid morphology. To address this issue, we attempted to establish a simple method by applying conventional histopathology that enables observation of multiple organoids on a single cross section, maintains good morphology, and is applicable to various histopathological stains. By centrifugation in unsolidified agarose solution, we were able to accumulate the organoids onto a single plane.

Generation and characterization of monoclonal antibodies against human LGR6.

Leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6) is a seven-pass transmembrane protein known to be a marker of stem cells in several organs. To deepen our understanding of the cell biology of LGR6-positive cells, including stem cells, we generated monoclonal antibodies (mAbs) against human LGR6. DNA immunization followed by whole-cell immunization with LGR6-expressing transfectants was performed to obtain mAbs that recognized the native form of LGR6.

Combination with a defucosylated anti-HM1.24 monoclonal antibody plus lenalidomide induces marked ADCC against myeloma cells and their progenitors.

The immunomodulatory drug lenalidomide (Len) has drawn attention to potentiate antibody-dependent cellular cytotoxicity (ADCC)-mediated immunotherapies. We developed the defucosylated version (YB-AHM) of humanized monoclonal antibody against HM1.24 (CD317) overexpressed in multiple myeloma (MM) cells.

A defucosylated anti-CD317 antibody exhibited enhanced antibody-dependent cellular cytotoxicity against primary myeloma cells in the presence of effectors from patients.

The humanized monoclonal antibody (mAb) against CD317 antigen (anti-HM1.24 antibody; AHM), which is highly expressed on multiple myeloma (MM), induces antibody-dependent cellular cytotoxicity (ADCC). However, the antitumor activity of AHM in the clinical setting has not been clearly demonstrated.

Interferon-alpha enhances CD317 expression and the antitumor activity of anti-CD317 monoclonal antibody in renal cell carcinoma xenograft models.

A murine (mAHM) and a humanized (AHM) monoclonal antibody against CD317 (also called tetherin, BST2, or HM1.24 antigen), expressed preferentially in neoplastic B cells such as multiple myeloma, exhibited antitumor effects as a result of antibody-dependent cellular cytotoxicity (ADCC). The putative interferon (IFN) response elements IRF-1/2 and ISGF3 are present in the promoter of the CD317 gene, and IFN has been used for the treatment of not only myeloproliferative diseases but also solid tumors such as renal cell carcinoma (RCC) and melanoma.

Recombinant human hexamer-dominant IgM monoclonal antibody to ganglioside GM3 for treatment of melanoma.

Purpose: L612, a human IgM monoclonal antibody produced by an EBV-transformed human B-cell line, binds to ganglioside GM3 and kills GM3-positive human melanoma cells in the presence of complement. It has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (about 20%), pentameric IgM (about 74%), and other minor IgM molecules.

A single-chain Fv diabody against human leukocyte antigen-A molecules specifically induces myeloma cell death in the bone marrow environment.

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However, clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody, we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A.

Identification of ROBO1 as a novel hepatocellular carcinoma antigen and a potential therapeutic and diagnostic target.

Purpose: Hepatocellular carcinoma is the most common primary malignancy of the liver and accounts for as many as one million deaths annually worldwide. The present study was done to identify new transmembrane molecules for antibody therapy in hepatocellular carcinoma.

Experimental Design: Gene expression profiles of pooled total RNA from three tissues each of moderately differentiated and poorly differentiated hepatocellular carcinoma were compared with those of normal liver, noncancerous liver tissue in hepatocellular carcinoma patients, 30 normal tissue samples, and five fetal tissue samples.

Construction of a conventional non-radioisotope method to quantify HM1.24 antigens: correlation of HM1.24 levels and ADCC activity of the humanized antibody against HM1.24.

A humanized monoclonal antibody (mAb) against HM1.24 (AHM) caused antibody-dependent cellular cytotoxicity (ADCC) against multiple myeloma (MM) cells. Here, we constructed a conventional non-radioisotope method that quantifies the amount of HM1.

Antitumor activity of humanized monoclonal antibody against HM1.24 antigen in human myeloma xenograft models.

A humanized monoclonal antibody against HM1.24 antigen (AHM), which is highly expressed on multiple myeloma (MM) cells, induced antibody-dependent cellular cytotoxicity (ADCC) in vitro. In this study, we further characterized AHM and evaluated its potency for clinical application.

Tocilizumab inhibits signal transduction mediated by both mIL-6R and sIL-6R, but not by the receptors of other members of IL-6 cytokine family.

To characterize the biological activity of tocilizumab, a humanized anti-human interleukin-6 receptor (IL-6R) monoclonal antibody, we examined its binding activity to both soluble IL-6R (sIL-6R) and membrane bound IL-6R (mIL-6R) and its neutralizing activity to other IL-6 family cytokines. ELISA assay demonstrated that tocilizumab bound to sIL-6R and inhibited IL-6 binding to sIL-6R in a dose-dependent manner. The dissociation constant (Kd value) for IL-6R was determined to be 2.

Generation of potent antitumor CTL from patients with multiple myeloma directed against HM1.24.

Purpose: The purpose of this work was to test the suitability of the HM1.24 antigen as a CTL target for immunotherapy of patients with multiple myeloma.

Experimental Design: Antigen-specific T cells were generated from patients with multiple myeloma using stimulation with protein-pulsed dendritic cells and tested in ELISPOT and CTL assays.

2D7 diabody bound to the alpha2 domain of HLA class I efficiently induces caspase-independent cell death against malignant and activated lymphoid cells.

A mouse monoclonal antibody (2D7 mAb), which specifically bound to the alpha2 domain of HLA class I, rapidly induces cell aggregation accompanied by weak cytotoxicity against ARH-77 cells, suggesting that 2D7 mAb had a potential for agonist antibody. In order to enhance this cytotoxicity, 2D7 mAb was engineered to be a small bivalent antibody fragment, 2D7 diabody. The resultant 2D7 diabody showed a strong cytotoxicity against ARH-77 cells.