CD26 is a type II glycoprotein known as dipeptidyl peptidase IV and has been identified as one of the cell surface markers associated with various types of cancers and a subset of cancer stem cells. Recent studies have suggested that CD26 expression is involved in tumor growth, tumor invasion, and metastasis. The CD26 is shown in an extensive intracellular distribution, ranging from the cell surface to the nucleus. We have previously showed that the humanized anti-CD26 monoclonal antibody (mAb), YS110, exhibits inhibitory effects on various cancers. However, functions of CD26 on cancer cells and molecular mechanisms of impaired tumor growth by YS110 treatment are not well understood. In this study, we demonstrated that the treatment with YS110 induced nuclear translocation of both cell-surface CD26 and YS110 in cancer cells and xenografted tumor. It was shown that the CD26 and YS110 were co-localized in nucleus by immunoelectron microscopic analysis. In response to YS110 treatment, CD26 was translocated into the nucleus via caveolin-dependent endocytosis. It was revealed that the nuclear CD26 interacted with a genomic flanking region of the gene for POLR2A, a subunit of RNA polymerase II, using a chromatin immunoprecipitation assay. This interaction with nuclear CD26 and POLR2A gene consequently led to transcriptional repression of the POLR2A gene, resulting in retarded cell proliferation of cancer cells. Furthermore, the impaired nuclear transport of CD26 by treatment with an endocytosis inhibitor or expressions of deletion mutants of CD26 reversed the POLR2A repression induced by YS110 treatment. These findings reveal that the nuclear CD26 functions in the regulation of gene expression and tumor growth, and provide a novel mechanism of mAb-therapy related to inducible translocation of cell-surface target molecule into the nucleus.
Bone remodeling is maintained by the delicate balance between osteoblasts (OBs) and osteoclasts (OCs). However, the role of CD26 in regulating bone remodeling has not yet been characterized. We herein show that CD26 is preferentially expressed on normal human OCs and is intensely expressed on activated human OCs in osteolytic bone alterations. Macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of NF-kB ligand (sRANKL) induced human OC differentiation, in association with CD26 expression on monocyte-macrophage lineage cells. CD26 expression was accompanied by increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which is crucial for early human OC differentiation. The humanized anti-CD26 monoclonal antibody, huCD26mAb, impaired the formation and function of tartrate-resistant acid phosphatase (TRAP)/CD26 positive multi-nucleated (nuclei > 3) OCs with maturation in the manner of dose-dependency. It was revealed that huCD26mAb inhibits early OC differentiation via the inactivation of MKK3/6, p38 MAPK and subsequent dephosphorylation of microphthalmia-associated transcription factor (mi/Mitf). These inhibitions occur immediately after RANKL binds to RANK on the human OC precursor cells and were demonstrated using the OC functional assays. huCD26mAb subsequently impaired OC maturation and bone resorption by suppressing the expression of TRAP and OC fusion proteins. In addition, p38 MAPK inhibitor also strongly inhibited OC formation and function. Our results suggest that the blockade of CD26 signaling impairs the development of human functional OCs by inhibiting p38 MAPK-mi/Mitf phosphorylation pathway and that targeting human OCs with huCD26mAb may have therapeutic potential for the treatment of osteolytic lesions following metastasis to alleviate bone destruction and reduce total skeletal-related events (SREs).
BackgroundMalignant Mesothelioma (MM) is a highly aggressive tumor with poor prognosis. Multimodal treatments and novel molecular targeted therapies against MM are in high demand in order treat this disease effectively. We have developed a humanized monoclonal antibody YS110 against CD26 expressed in 85 % of MM cases. CD26 is thought to be involved in tumor growth and invasion by interacting with collagen and fibronectin, or affecting signal transduction processes.MethodsWe evaluated the direct anti-tumor effect of YS110 against MM cell lines, NCI-H2452 and JMN, and investigated its effects on cell cycle and on the cell cycle regulator molecules. In addition, we investigated synergistic effects of YS110 and anti-tumor agent pemetrexed (PMX) against MM cell line both in vitro and in vivo.ResultsYS110 suppressed the proliferation of NCI-H2452 cells by approximately 20 % in 48 h. Based on cell cycle analysis, percentage of cells in G2/M phase increased 8.0 % on the average after YS110 treatment; in addition, cell cycle regulator p21 cip/waf1 was increased and cyclin B1 was decreased after YS110 treatment. Inhibitory phosphorylation of both cdc2 (Tyr15) and cdc25C (Ser216) were elevated. Furthermore, activating phosphorylation of p38 MAPK (Thr180/Tyr182) and ERK1/2 (Thr202/Tyr204) were augmented at 24 h after YS110 treatment. PMX rapidly induced CD26 expression on cell surface and the treatment with both YS110 and PMX inhibited in vivo tumor growth accompanied by a synergistic reduction in the MIB-1 index.ConclusionThis is a first report of a novel anti-proliferative mechanism of the humanized anti-CD26 monoclonal antibody YS110, which resulted in G2/M cell cycle delay through regulation of quantity and activity of various cell cycle regulating molecules.
Here, we report a novel antibody drug conjugate (ADC) with the humanized anti-CD26 monoclonal antibody YS110 and triptolide (TR-1). YS110 has an inhibitory activity against the CD26-positive tumor growth via the immunological and direct pathway, such as intra-nuclear transportation of CD26 and YS110, and suppressed transcription of RNA polymerase II (Pol II) subunit POLR2A. The ADC conjugated with YS110 and an antitumor compound triptolide (TR-1), which is an inhibitor for TFIIH, one of the general transcription factors for Pol II was developed. YS110 and triptolide were crosslinked by the heterobifunctional linker succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and designated Y-TR1. Antitumor efficacy of Y-TR1 against malignant mesothelioma and leukemia cell lines were assessed by the in vitro cell viability assay and in vivo assay using xenografted mouse models. Y-TR1 showed significant cytotoxicity against CD26-positive cell lines but not CD26-negative counterparts in a dose-dependent manner via suppression of mRNA synthesis by impairment of the Pol II activity. The tumors in xenografted mice administered Y-TR1 was smaller than that of the unconjugated YS110 treated mice without severe toxicity. In conclusion, the novel compound Y-TR1 showed antitumor properties against CD26-positive cancer cell lines both in vitro and in vivo without toxicity. The Y-TR1 is a unique antitumor ADC and functions against Pol II.
A 110-kDa type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 has a multitude of biological functions and plays an important role in the regulation of inflammatory responses and tumor biology. Our work has focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and we have recently developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which has promising safety profile and clinical activity in patients with malignant pleural mesothelioma. The development of an anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in formalin-fixed paraffin-embedded (FFPE) tissues in the clinical setting is therefore of the utmost importance. To develop novel anti-CD26 mAbs capable of binding to denatured CD26, we immunized mice with urea-treated CD26 protein. Hybridoma supernatants were screened for specific reactivity with human CD26 by immunostaining through the use of a set of FFPE human CD26-positive or negative tumor cell lines. This screening method enables us to develop novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in FFPE non-tumor and tumor tissue sections with reliable clarity and intensity. Specifically, these mAbs display strong binding affinity to denatured human CD26 rather than undenatured human CD26, and are capable of detecting denatured human CD26 in decalcified specimens. These novel anti-CD26 mAbs are potentially useful for the analysis of CD26 expression in cancer patients with bony metastasis, and may help decide the appropriateness of YS110 therapy for future cancer patients.
1348 Bone disease is a hallmark of malignancy with osteolytic bone metastasis, including multiple myeloma (MM) and targeting osteoclasts (OCs) to alleviate bone destruction is a component of the standard care for MM. CD26 is a 110-kDa multifunctional membrane-bound glycoprotein, with dipeptidyl peptidase IV (DPPIV) enzyme activity in its extracellular domain and is critical in T-cell activation and several tumor developments, including malignant lymphoma. However, little is known about the role of CD26 in regulating bone remodeling. In this study, we show that CD26 is expressed on normal human osteoclasts and moreover, intensely expressed on activated human osteoclasts with osteolytic bone metastasis, including MM. We explore the function of CD26 in osteoclastgenesis (OCG) and investigate the effects of humanized anti-CD26 monoclonal antibody (huCD26mAb), which has shown promising clinical activity in T-cell lymphoma, on human OC differentiation, maturation and function. We further define the molecular targets of CD26 signaling cascade in OCG and explore the therapeutic potential of huCD26mAb for treating osteolytic bone metastasis. Human bone marrow mononuclear cells (BMMs) were cultured with human M-CSF (25ng/ml) plus sRANKL (50ng/ml) in the absence or presence of huCD26mAb for the indicated times. Then, M-CSF and sRANKL stimulate CD26 expressions during OCG, in a dose-dependent manner. The expression of CD26 up-regulates mitogen-activated protein kinase14 (p38MAPK) phosphorylation. P38MAPK phosphorylation also occurs downstream of RANK signaling in OCs and stimulates its downstream activation of microphthalmia-associated transcription factor (mi/Mitf), which plays an important role in OC function. Importantly, huCD26mAb decreased the number of multinucleated OCs (>3 nuclei) by tartrate-resistant phosphates (TRAP)/CD26 staining and the secretion of TRAP-5b and type 1 collagen; specific mature OC markers. It decreased the size of OCs and the number of nuclei per OC, with significantly defective bone resorption activity, as evidenced by diminished pit formation on fluoresceinated calcium phosphate-coated plates. In contrast, huCD26mAb added after 4- or 7- days' BMM cultures with M-CSF plus sRANKL did not have significant effects on mature osteoclast formation and function. Given these dual roles of CD26 in OCG, we next examined the effects of huCD26mAb on the phosphorylation of p38MAPK in OC precursor cells and mature OCs. At first, in the absence of huCD26mAb, similar amounts of p38MAPK and MKK3/6 (a molecule that is upstream of p38MAPK) were present in OC precursor cells and OCs. In response to RANKL, MKK3/6-p38MAPK was phosphorylated within 15 minutes in OC precursor cells and reached a maximal level within 30 minutes, and was maintained up to 60 minutes. Moreover, mi/Mitf was subsequently rapidly activated and persisted for 24hours. In the presence of huCD26mAb, when huCD26mAb bound to CD26 on OC precursor cells, only the MKK3/6-p38MAPK pathway was specifically rapidly inactivated, as shown by the persistent decrease in the phosphorylation of p38MAPK, together with MKK3/6, starting within 15 minutes of RANKL stimulation. Subsequent mi/Mitf phosphorylation was also persistently inhibited. In contrast, MKK3/6-p38MAPK was not phosphorylated at all in mature OCs after RANKL stimulation, regardless of the absence or presence of huCD26mAb. These results suggest that huCD26mAb suppressed RANKL induced p38MAPK phosphorylation in OC precursor cells, but not in OCs. The activation of other MAPKs including ERK and SAPK/JNK, or NFκB were rapidly induced in response to RANKL both in OC precursor cells and OCs, regardless of the absence or presence of huCD26mAb. Moreover, p38MAPK inhibitor also strongly inhibited OC formation and function through the suppression of p38 MAPK phosphorylation and subsequent mi/Mitf activation in OC precursor cells, but not in OCs. In conclusion, these data demonstrate that targeting CD26 on OC precursor cells with huCD26mAb suppressed human osteoclast differentiation, via the inhibition of MKK3/6-p38MAPK-mi/Mitf phosphorylation pathway and impaired subsequent mature osteoclast formation and function. Our results strongly suggest that targeting OCs with huCD26mAb has a promising alternative therapeutic potential for the treatment of osteolytic bone metastasis, including MM, to reduce the occurrence of total skeletal-related events. Disclosures: No relevant conflicts of interest to declare.
Background: CD26, a 110-kDa transmembrane glycoprotein with DPPIV activity, has been implicated in tumorigenesis and shown to be expressed in several tumor cells including malignant lymphoma, whereas its role has not been characterized in plasma cell malignancies, yet. We have recently shown that CD26 is intensely expressed in activated osteoclasts (OCs) in osteolytic bone tumors including multiple myeloma (MM). CD26 is not expressed in hematopoietic stem cells, but M-CSF and sRANKL induced human OC differentiation with the upregulation of CD26 expression in monocyte-macrophage lineage cells, OC precursor cells and OCs. Blockade of CD26 signaling with humanized anti-CD26 monoclonal antibody (YS110) impaired OC differentiation via inhibiting RANKL-induced MKK3/6-p38MAPK-mi/Mitf phosphorylation in OC precursor cells (Nishida et al., 2014). In the present study, we characterize the biological function of CD26 in MM cells in the bone marrow (BM) and investigate the therapeutic potential of YS110 to treat MM cell growth and reduce MM-induced osteolytic lesions. Methods and Results: Although CD26 expression was not detected any of 11 MM cell lines in mono-culture, CD26 expression levels were upregulated in all MM cell lines when co-cultured with OCs for 72 hours by flow cytometry and immunohistochemistry analysis. CD26 protein levels in MM cell lines were also enhanced in co-culture with OCs by immunoblotting. To determine the factors responsible for CD26 upregulation in MM cells, we examined CD26 expression in MM cell lines under transwell co-culture conditions with OCs or under stimulation with anti-apoptotic cytokines produced by OCs. CD26 expression in MM cell lines was reduced under transwell co-culture conditions with OCs. TNFα, BAFF, APRIL and SDF-1 slightly upregulated CD26 expression in MM cell lines. To further explore CD26 expression in BM of MM patients, we performed immunohistochemical stainig on decalcified bone sections of biopsy specimens of MM cases. CD26/CD138 positive plasma cells were detected around CD26 positive OCs and certain endothelial vascular cells in several cases. Next, to clarify the role of CD26 in MM cell survival, we examined the effects of YS110 on MM cell growth and related osteolytic bone lesions in vitro and in vivo. YS110 had no significant effects on viability of MM cell lines in mono-culture, but dose-dependently inhibited growth of MM cell lines in co-culture with OCs. YS110 immediately inhibited p38 activation and its downstream Hsp27 phosphorylation in MM cells. Contitnued treatment of MM cells with YS110 also led to the reduction of total Hsp27 protein at 12 hours and 16 hours after the treatment of YS110, with commensurate decrease of phospho-Hsp27 and to increased MM apoptosis correlated with the induction of p53, increased activation of caspases and PARP, as well as the reduction of Mcl-1 and c-Myc. In parallel, we constructed a xenograft of murine model of human MM. A total of 5x106 CD26 positive MM cell lines by co-culture with OCs were inoculated to mice (NOD/SCID-s.c. mice) or directly injected into the implanted human bone chips in mice (NOD/SCID-hu mice). Mice treated with YS110 (500μg/dose) showed a significant effect in inhibiting MM tumor weight compared with control mice 4 weeks after tumor cell inoculation. To further investigate whether YS110 could reduce MM-induced osteolysis as well as suppress MM cell growth, we used NOD/SCID-hu mice model. Mice were treated with YS110 after the first detection of tumor growth. Continuous YS110 treatment significantly suppressed MM cell growth after 4 weeks. Immunohistochemistry for CD138 and TRAP staining showed decreased numbers of MM cells and TRAP positive OCs, with decreased bone resorption activity in human bones from YS110-treated versus control mice. Lastly, we examined the effects of targeting CD26 with YS110 in CD26 positive myeloma stem-like cells (SP cells). CD26 expression was demonstrated in SP cells of several MM cell lines in co-culture with OCs. YS110 dose-dependently inhibited cell viability of CD26 positive SP cells co-cultured with OCs, indicating targeting CD26 might reduce chemoresistance in MM. Conclusions: Theses results suggest that CD26 may be a promising novel target in MM, strongly supporting YS110 to inhibit MM cell growth in the BM as well as its related osteolytic bone lesions. Our results provide the framework for clinical development of YS110 as a novel therapeutic option in MM. Disclosures Morimoto: Y's Therapeutics: Consultancy, Research Funding. Yamada:Y's Therapeutics: Consultancy, Research Funding.
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