Malignant 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. We 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. YS110 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. This 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.

CD26 is a type II, cell surface glycoprotein known as dipeptidyl peptidase (DPP) IV. Previous studies have revealed CD26 expression in T cell leukemia/lymphoma and malignant mesothelioma, and an inhibitory effect of anti-CD26 monoclonal antibody (mAb) against the growth of CD26+ cancer cells in vitro and in vivo. The function of CD26 in tumor development is unknown and the machinery with which the CD26 mAb induces its anti-tumor effect remains uncharacterized. The localization of CD26 in the nucleus of T cell leukemia/lymphoma cells and mesothelioma cells was shown by biochemical and immuno-electron microscopic analysis. The DPPIV enzyme activity was revealed in the nuclear fraction of T cell leukemia/lymphoma cells. These expressions of intra-nuclear CD26 were augmented by treatment with the CD26 mAb, 1F7, with anti-tumor effect against the CD26+ T cell leukemia/lymphoma cells. In contrast, the CD26 mAb, 5F8, without anti-tumor effect, did not augment CD26 expressions in the nucleus. Biotin-labeled, cell surface CD26 translocated into the nucleus constantly, and this translocation was enhanced with 1F7 treatment but not with 5F8. These results indicate that the intra-nuclear CD26 which moves from plasma membrane may play certain roles in cell growth of human cancer cells.