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. At first, we developed rat duodenal organoids according to a previous report, but they were not able to generate a confluent monolayer. Therefore, we modified organoid culture protocols and developed cyst-enriched organoids; these strongly promoted the formation of a confluent monolayer. Furthermore, adding valproic acid to the culture accelerated the differentiation of the monolayer, which possessed an effective barrier function and apicobasal cell polarity. Drug transporter P-gp function as well as CYP3A activity and nuclear receptor function were confirmed in the model. We expect our novel monolayer model to be a useful tool for elucidating drug absorption processes in detail, enabling the development of highly absorbable drugs.

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. Here, we established a three-dimensional (3D) culture model with a squamous epithelial structure using LUSC cell lines PLR327F-LD41 and MCC001F, established in-house. When PLR327F-LD41 cells were cultured in a mixture of Matrigel and collagen I, they generated 3D colonies (designated cancer organoids, or COs) with involucrin (IVL)-positive keratinizing cells in the center (IVLinner COs). COs with uniform size were generated by seeding PLR327F-LD41 cells in a form of small cell aggregates. Since Notch signaling induces the differentiation of squamous epithelium, we confirmed the effect of γ-secretase inhibitor in inhibiting Notch signaling in IVLinner COs. Surprisingly, γ-secretase inhibitor did not block induction of IVL-positive cells; however, cells residing between the CK5-positive basal-like layer and IVL-positive layer decreased significantly. Thus, our 3D culture model with uniform size and structure promises to be a useful tool for elucidating the biology of LUSC and for screening drug-candidates.

Continuous formation of small clusters with LGR5-positive cells contributes to tumor growth in a colorectal cancer xenograft model.

New cancer characteristics can be discovered by focusing on the process of tumor formation. Cancer stem cells (CSCs) are a key subpopulation, as they are theorized to be at the apex of the tumor hierarchy. We can better understand their function in the tumor hierarchy by using sectioned samples to observe the growth of tumors from their origins as CSCs. In this study, we evaluated the growth of moderate differentiated colorectal cancer from LGR5-positive cells, which is a CSC marker of colorectal cancer, using xenograft and three-dimensional culture models spatiotemporally. These cells express LGR5 at high levels and show CSC phenotypes. To detect them, we used a previously generated antibody that specifically targets LGR5, and were therefore able to observe LGR5-positive cells aggregating into small clusters (sCLs) over the course of tumor growth. Because these LGR5-expressing sCLs formed continuously during growth mainly in the invasive front, we concluded that the structure must contribute significantly to the expansion of CSCs and to tumor growth overall. We confirmed the formation of sCLs from gland structures using a three-dimensional culture model. In addition, sCLs exhibited upregulated genes related to stress response and partial/hybrid epithelial-mesenchymal transition (EMT), as well as genes reported to be prognosis factors. Finally, sCLs with high LGR5 expression were identified in clinical samples. Based on these results, we elucidate how sCLs are an important contributors to tumor growth and the expansion of CSCs.

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. It was expressed at all estrous cycles of the vagina with changes in distribution patterns along with the structural changes in each cycle, and expression was reduced in ovariectomized (OVX) mice. On the administration of the antibody, there was disarrangement of the vaginal mucosal epithelium with formation of miroabscess, increased granulocyte infiltration, and single cell necrosis. Despite similar expression levels of DSG3 in other tissues, histopathological changes were limited to the vagina. The severity of the changes was reduced by ovariectomy. From these findings, the lesions were thought to be related to the drastic change in the histological structure of the vaginal mucosa accompanying the estrous cycle. Thus, we have shown that the changing expression of target antigen distribution and its relationship with physiological changes in tissue structure are important features for estimating the toxic potential of cytotoxic antibody therapy.

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. The conditions were determined by comparing the phenotypes in the models with PLR123 mouse xenografts colonising lung and liver. StemCO resembled LGR5-positive undifferentiated tumours in the lung, and diffCO had lumen structures composed of polarised cells that were similar to the ductal structures found in differentiated tumours in the liver. In a case using the models for biomedical research, treatment with JAG-1 peptide or a γ-secretase inhibitor modified the Notch signaling and induced changes indicating that the signal participates in lumen formation in the models. Our results demonstrate that culture conditions affect the stemness of 3D culture models generated from CSCs and show that comparing models with different phenotypes is useful for studying how the tumour environment regulates cancer.

Generation of an anti-desmoglein 3 antibody without pathogenic activity of pemphigus vulgaris for therapeutic application to squamous cell carcinoma.

It is ideal for the target antigen of a cytotoxic therapeutic antibody against cancer to be cancer-specific, but such antigens are rare. Thus an alternative strategy for target selection is necessary. Desmoglein 3 (DSG3) is highly expressed in lung squamous cell carcinoma, while it is well-known that anti-DSG3 antibodies cause pemphigus vulgaris, an autoimmune disease. We evaluated DSG3 as a novel target by selecting an epitope that exerts efficacy against cancer with no pathogenic effects in normal tissues. Pathogenic anti-DSG3 antibodies induce skin blisters by inhibiting the cell-cell interaction in a Ca2+-dependent manner. We screened anti-DSG3 antibodies that bind DGS3 independent of Ca2+ and have high antibody-dependent cell cytotoxicity (ADCC) activity against DSG3-expressing cells. These selected antibodies did not inhibit cell-cell interaction and showed ADCC activity against squamous cell carcinoma cell lines. Furthermore, one of the DSG3 antibodies showed anti-tumour activity in tumour mouse models but did not induce adverse effects such as blister formation in the skin. Thus it was possible to generate an antibody against DSG3 by using an appropriate epitope that retained efficacy with no pathogenicity. This approach of epitope selection may expand the variety of druggable target molecules.

A simple method for histopathological evaluation of organoids.

In vitro-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. The morphology was well preserved, and the various morphological types and sizes of organoid structures were identified. This method was also applicable for special staining, immunohistochemistry, and immunofluorescence staining. This method makes it possible to utilize the advantages of conventional pathological methods when studying organoids.

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. Hybridomas were screened by flow cytometry using LGR6-transfected cells. Because the molecules of LGR4, LGR5, and LGR6 are 50% homologous at the amino acid level, specificity of the mAbs was confirmed by transfectants expressing LGR4, LGR5, or LGR6. Three LGR6-specific mAbs were generated. Two of the three mAbs (designated 43A6 and 43D10) recognized the large N-terminal extracellular domain of LGR6, and competitively blocked the binding of R-spondin 1, which is known to be the ligand for LGR6. The other mAb, 43A25, recognized the seven-pass transmembrane domain of LGR6, and was able to be used for immunoblot analysis. In addition, mAbs 43A6 and 43D10 detected endogenous expression of LGR6 in cancer cell lines. We expect that our mAbs will contribute to widening our understanding of LGR6-positive cells in humans.

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. In this study, we evaluated ADCC by YB-AHM and Len in combination against MM cells and their progenitors. YB-AHM was able to selectively kill via ADCC MM cells in bone marrow samples from patients with MM with low effector/target ratios, which was further enhanced by treatment with Len. Interestingly, Len also up-regulated HM1.24 expression on MM cells in an effector-dependent manner. HM1.24 was found to be highly expressed in a drug-resistant clonogenic "side population" in MM cells; and this combinatory treatment successfully reduced SP fractions in RPMI 8226 and KMS-11 cells in the presence of effector cells, and suppressed a clonogenic potential of MM cells in colony-forming assays. Collectively, the present study suggests that YB-AHM and Len in combination may become an effective therapeutic strategy in MM, warranting further study to target drug-resistant MM clonogenic 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. In this study, we produced defucosylated AHM and evaluated its potency for clinical application by performing autologous ADCC assays against primary MM cells from patients. Defucosylated AHM that was produced in rat myeloma YB2/0 cells expressing a low level of fucosyltransferase (FUT8) showed significant ADCC activity against three out of six primary MM cells in the presence of autologous PBMC, whereas conventional AHM did not. The results indicate that the potency of AHM to induce ADCC against primary MM cells was insufficient, but was significantly augmented by defucosylation. To generate more homogenous defucosylated monoclonal antibodies (mAb) for fermentation, we disrupted the GFT gene that encodes a GDP-fucose transporter in a CHO/DXB11 cell line by sequential homologous recombination. Analysis of the N-linked oligosaccharide in the defucosylated AHM produced by the established GFT(-/-)CHO cell line showed that a majority (93.4%) of the oligosaccharide was fucose free. The GFT(-/-) cells stably produced defucosylated mAb over passages. These results demonstrate that GTF(-/-)CHO-produced defucosylated AHM (GFTKO-AHM) will be a promising new therapeutic antibody against MM in the clinical setting.

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. Therefore, we examined the effects of IFN on the expression of CD317 and on the antitumor activity of AHM and mAHM in RCC and melanoma. Flow cytometry and in vitro ADCC assays with human or mouse effector cells demonstrated that IFN-alpha markedly increased the amount of cell surface CD317 and augmented the ADCC activity of mAHM and AHM in RCC cells and to a lesser extent in melanoma cells. Administration of IFN-alpha to mice bearing RCC xenografts also increased the expression of CD317 in tumor cells. When coadministered with IFN-alpha, mAHM exhibited more profound antitumor activity in both IFN-alpha-sensitive and -insensitive RCC xenograft models. Thus, AHM in combination with IFN-alpha may be an effective therapy for the treatment of RCC.

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. Because hexameric IgM activates complement more effectively than pentameric IgM, we developed and evaluated a hexamer-dominant recombinant IgM for clinical applications. EXPERIMENTAL DESIGN: Chinese hamster ovary (CHO) cells were transfected with heavy- and light-chain genes of L612, with or without the joining-chain gene. Antitumor effects of the recombinant IgM secreted from CHO cells were evaluated in vitro and in vivo. RESULTS: Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was approximately 80% hexameric, whereas recombinant IgM from CHO cells transfected with heavy-, light-, and joining-chain genes (designated CJ45) was about 90% pentameric. Both CA19 and CJ45 recombinant IgMs caused complement-dependent cytotoxicity against human and mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller. I.v. injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing a GM3-positive mouse melanoma xenograft. CONCLUSIONS: A hexamer-dominant human IgM against GM3 may provide a more potent treatment option for patients with GM3-positive melanoma.

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. Here, we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells, lymphocytes, or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors, suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely, 2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs, melphalan, or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore, administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma.

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. Target genes up-regulated specifically in hepatocellular carcinoma were validated by immunohistochemical analysis and complement-dependent cytotoxicity assay using monoclonal antibodies generated against target molecules. RESULTS: The human homologue of the Drosophila Roundabout gene, axon guidance receptor homologue 1, ROBO1/DUTT1, a member of the immunoglobulin superfamily, was highly expressed in hepatocellular carcinoma, whereas it showed only a limited distribution in normal tissues. On immunohistochemical analysis using a newly generated anti-ROBO1 monoclonal antibody, positive signals were observed in 83 of 98 cases of hepatocellular carcinoma (84.7%). The mAb B2318C induced complement-dependent cytotoxicity in ROBO1-expressing cell lines and in the liver cancer cell line PLC/PRF/5. Strikingly, the ectodomain of ROBO1 was detected not only in the culture medium of liver cancer cell lines (PLC/PRF/5, HepG2, etc.) but also in sera from hepatocellular carcinoma patients (6 of 11). CONCLUSIONS: This is the first report that ROBO1 is overexpressed in hepatocellular carcinoma and shed into serum in humans. These observations suggest that ROBO1 is a potential new serologic marker for hepatocellular carcinoma and may represent a new therapeutic target.

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 HM1.24 using fluorescein-labeled AHM. More than 10(4) molecules/cell of HM1.24 were detected in 12 out of 14 patients' MM cells, and a linear correlation was found between ADCC by AHM and the amounts of HM1.24. Thus, AHM is likely to be more efficacious against MM cells with high levels of HM1.24. This conventional non-RI method to quantify HM1.24 will be useful to select patients most likely to respond to AHM.

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. AHM bound to HM1.24 antigen with a dissociation constant of 0.35 nM, and its epitope resided between Leu116 and Leu127 of the HM1.24 antigen. Single intravenous injection of AHM significantly inhibited tumor growth in both orthotopic and ectopic human MM xenograft models. AHM reduced serum M protein levels and prolonged survival of mice intravenously inoculated with KPMM2 and ARH-77 cells. The number of KPMM2 cells in bone marrow or tumor volume of subcutaneously inoculated RPMI 8226 cells was also inhibited by AHM. The antitumor activity of AHM against tumor cells in bone marrow was diminished when the mice were pretreated with anti-Fcgamma receptor III/II antibody, demonstrating that antitumor activity by AHM requires effector cell functions in vivo. Experiments involving in vitro ADCC assays indicated that NK cells and monocytes/macrophages serve as effector cells for AHM-induced ADCC in mouse and human. Thus, AHM will provide an additional treatment option for MM.

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 2.54+/-0.12 nmol/L by Scatchard analysis. In addition, tocilizumab had the ability to dissociate IL-6 and sIL-6R from their preformed complex. The immune complex of tocilizumab and sIL-6R did not transmit signaling. Moreover, tocilizumab suppressed the IL-6/sIL-6R complex-induced proliferation of human gp130-transfected cell, BAF-h130. In addition, tocilizumab had the ability to bind to human IL-6R expressing COS-7 cells and to suppress the growth of the IL-6-dependent myeloma cell line, KPMM2. Finally, to analyze the specificity of this antibody, the effects on signal transduction of IL-6 family cytokines such as interleukin-11 (IL-11), oncostatin M (OSM), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF) were examined using murine transfectant cell lines (BaF/IL-6R, BaF/IL-11R, BaF/OSMR, BaF/LIFR and BaF/CNTFR) that proliferate depending on IL-6, IL-11, OSM, LIF and human CNTF, respectively. Tocilizumab inhibited the proliferation of BaF/IL-6R induced by IL-6, but did not inhibit the proliferation of BaF/IL-11R, BaF/OSMR, BaF/LIFR and BaF/CNTFR cells induced by their corresponding cytokines. These lines of evidence indicate that tocilizumab is able to bind to both sIL-6R and mIL-6R and to inhibit IL-6 binding to its receptors, leading to the blockade of the IL-6 signaling through both sIL-6R and mIL-6R, but not block the signaling of other IL-6 family cytokines.

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. RESULTS: HM1.24-primed T cells responded selectively to HM1.24-loaded autologous peripheral blood mononuclear cells (PBMC) in an IFN-gamma ELISPOT assay (median, 342; range, 198-495 IFN-gamma-producing cells/10(5) cf. unloaded PBMC median, 98; range, 7-137; P < 0.05, n = 5) and also to autologous malignant plasma cells (MPC; median, 227; range, 153-335; P < 0.05 when compared with the response to allogeneic MPC median, 57; range, 22-158; n = 5). HM1.24-primed T cells lysed autologous MPC (at 20:1 E/T ratio: median, 48% specific killing; range, 23-88%; at 10:1 E/T ratio: median, 43%; range, 15-80%; n = 12) but not allogeneic MPC. Lysis of autologous MPC was inhibited by anti-MHC class I but not anti-MHC class II antibodies and was blocked by Concanamycin A. Lysis of autologous MPC was blocked by competition with autologous HM1.24-transfected dendritic cells (10:1 ratio with autologous MPC). Unmanipulated, or control plasmid-transfected dendritic cells had no effect on lysis of autologous MPC. CONCLUSION: Our results indicate that HM1.24 is a promising target for immunotherapy of multiple myeloma.

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. As a notable characteristic feature, the lethal effect of 2D7 diabody was quite rapid, mediated by a caspase-independent death pathway. Furthermore, 2D7 diabody also showed cytotoxicity against several leukemia and lymphoma cell lines, and mitogen-activated peripheral blood mononuclear cells (PBMC), but not for normal resting PBMC and adherent cell lines such as HUVEC. These results suggest that 2D7 diabody could be expected as a novel therapeutic antibody for hematological malignancies as well as inflammatory diseases.