Novel Antibody-Drug Conjugate with Anti-CD26 Humanized Monoclonal Antibody and Transcription Factor IIH (TFIIH) Inhibitor, Triptolide, Inhibits Tumor Growth via Impairing mRNA Synthesis

Abstract

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.

Keywords: CD26; RNA polymerase II; antibody drug conjugate; malignant mesothelioma; targeted therapy; triptolide.

Figure 1

Structural formula of triptolide and conjugation protocol of Y-TR1 (SMCC). Triptolide was modified by a sulfhydryl (SH) group and was provided as an S-S dimer for chemical stability. The SH group-induced triptolide was designated TR1. The S-S bond was reduced to the SH monomer by the tris(2-carboxyethyl) phosphine (TCEP) reducing gel just before reaction. The humanized anti-CD26 monoclonal antibody YS110 was modified by the heterobifunctional linker SMCC. SMCC-modified YS110 and TR1 monomers were mixed and allowed to react overnight and purified. See Methods for details.

Figure 2

In vitro cytotoxic effect of Triptolide and TR1against MM cells and leukemia cells. Horizontal axis shows the concentration of the compounds in nM. Vertical axis shows the percent of control of the absorbance value in the WST-1 assay. The representative results of at least three independent experiments are shown. (A) Triptolide against MSTO wt; (B) triptolide against MSTO clone12; (C) triptolide against JMN; (D) triptolide against Jurkat (−); (E) triptolide against Jurkat CD26(+); (F) TR-1 against MSTO wt; (G) TR-1 against MSTO clone12.

Figure 3

Biochemical analysis of Y-TR1. (A,B): The intact mass of unconjugated YS110 and Y-TR1 (SMCC) measured by the MALDI-TOF mass analysis. (A) Unconjugated YS110: 147,012.7; (B) Y-TR1 (SMCC): 151,815.6; (C) binding of Y-TR1 to the multiple myeloma (MM) cell line MSTO clone12 (CD26 positive). First antibody: Y-TR1 second antibody: Anti-human rabbit IgG FITC conjugate. Y-TR1 over 10 µg/mL showed intact binding to CD26-positive cells.

Figure 4

In vitro cytotoxicity of Y-TR1 against MM and leukemia cell lines. The representative results of at least three independent experiments are shown. (A) Comparison of cytotoxicity of Y-TR1 using various linkers against the CD26-positive MM cell line MSTO clone12. Y-TR1 using SMCC showed the highest cytotoxicity. The horizontal axis shows the concentration of the compounds in μg/mL. The vertical axis shows the percent of control of the absorbance value in the WST-1 assay; (B) comparison of IC50 of Y-TR1 using three linkers, SPDP, GMBS, and SMCC against CD26-positive MM cell lines MSTO clone12 and JMN; (C) in vitro cytotoxicity of TR-1 against the CD26-positive MM cell line JMN compared to unconjugated YS110. The horizontal and vertical axes show the same as indicated in (A); (D and E) higher in vitro cytotoxicity of Y-TR1 against the CD26-positive MM cell line MSTO clone12 compared to the CD26-negative counterpart MSTO wt (D) and CD26-positive leukemia cell line Jurkat CD26 (+) compared to the CD26-negative counterpart Jurkat (−) (E) at Y-TR1 concentration of 20 μg/mL. The vertical axis of the graph shows the percent of control in the WST-1 assay; (F) in vitro cytotoxicity of Y-TR1 against CD26-positive non-cancer adult dermal human microvascular endothelial cells (dHMVECs). Horizontal and vertical axes show the same as indicated in (A); (G) IC50 of unconjugated TR1 and Y-TR1 compared in molar concentration.

Figure 5

Nuclear translocation of Y-TR1. (A) Western blot analysis using anti-human IgG antibody detected Y-TR1 in the cytoplasm and the nuclear fraction of Y-TR1 treated CD26 positive JMN cells after 30 min and 60 min. Lamin B1 and Na-K ATPase were used as loading controls of the nuclear and cytoplasm fraction; (B) immunofluorescence staining observed under confocal laser microscopy of fixed JMN cells following 1 h of Y-TR1 treatment with the Alexa Fluor 488 labeled anti-human IgG antibody. Nuclear staining was done with Hoechst 33342. Localization of Y-TR1 (green) was observed in the nucleus (blue) as indicated by the white arrows. Scale bar: 10μm.

Figure 6

Investigation into the cytotoxic effect of TR-1. (A) Caspase 3/7 activity (represented in fluorescence) after 48 h of treatment with YS110, triptolide, TR1, and Y-TR1 in the CD26-positive MM cell line MSTO clone12. The activity is elevated in triptolide-, TR1-, and Y-TR1-treated cells. The vertical axis shows the intensity value measured by the fluorometer; (B, C) effects of Y-TR1 on the mRNA levels of HSP70 after heat induction. Relative mRNA levels of HSP70 after heat induction (45 °C, 2 h) were significantly lower in Y-TR1-treated cells compared to unconjugated YS110-treated cells in CD26-positive MM cell lines. A t-test at the p = 0.05 level was carried out as statistical analysis (n = 10). The error bar indicates one standard deviation; (B) MSTO clone12; (C) JMN.

Figure 7

In vivo anti-tumor effect of Y-TR1 in the NOD/SCID mouse xenograft model using the CD26 positive MM cell line JMN. (A) Y-TR1 was administered intraperitoneally 4 mg/kg/dose, three times per week, for a total of nine doses from day zero of subcutaneous inoculation of 1 × 107 JMN cells. The average estimated tumor volume on day 55 was compared among three groups (control, YS110, Y-TR1, n = 10) with Fisher’s protected least protected difference multiple comparison test. The mean tumor volume of the Y-TR1 group was significantly lower (* p < 0.05) than that of the control or YS110 group. The mean tumor volume of the YS110 group was not significantly altered compared with the control. An experiment out of two with similar results is shown; (B) Y-TR1 was administered intraperitoneally 8 mg/kg/dose, three times per week, for a total of nine doses. The average estimated tumor weight on day 42 was compared among three groups (control, 14D10, YS110, Y-TR1, n = 10) with Fisher’s protected least protected difference multiple comparison test. The mean tumor weight of the YS110 or Y-TR1 groups was significantly lower (* p < 0.05 or ** p < 0.025, respectively) than that of the control group. The mean tumor weight of the Y-TR1 group was significantly lower (* p < 0.05) than that of the YS110 group. An experiment out of two with similar results is shown; (C) histological analysis of xenograft tumors of JMN cells. JMN-derived tumors show histopathology of sarcomatoid mesothelioma. (×20). a: Hematoxylin and eosin staining. b: Immunohistochemical staining with anti-human CD26 antibody revealed CD26 expression in tumor cells. c–e: MIB-1 (Ki67) staining showed a decreased number of MIB-1-positive cells in Y-TR1-treated tumors compared to IgG1- or YS110-treated tumors. Scale bar: 10 μm.

Figure 8

Y-TR1 has multiple anti-tumor effects as follows; (1) introduction of cell death via immunological cytotoxicity such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), (2) retarded cell cycling of both G1/S and G2/M, (3) suppression of POLR2A transcription by increased amount of intranuclear CD26, (4) inhibition of TFIIH by TR1 carried into the nucleus using conjugation of TR1 to YS110.