ErbB2 Targeted Epigenetic Modulation: Anti-tumor Efficacy of the ADC Trastuzumab-HDACi ST8176AA1

Abstract

Targeted therapy using monoclonal antibodies conjugated to toxins is gaining space in the treatment of cancer. Here, we report the anti-tumor effect of a new antibody drug conjugate (ADC) delivering a HDAC inhibitor to ErbB2+ solid tumors. Trastuzumab was partially reduced with tris [2-carboxyethyl] phosphine (TCEP) and conjugated to ST7464AA1, the active form of the prodrug HDAC inhibitor ST7612AA1, through a maleimide-thiol linker to obtain the Antibody Drug Conjugate (ADC) ST8176AA1. The average drug/antibody ratio (DAR) was 4.5 as measured by hydrophobic interaction chromatography (HIC). Binding of ST8176AA1 to ErbB2 receptor and internalization in tumor cells were investigated by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), cytofluorimetry, and High Content Screening (HCS) Imaging. The biological activity of the ADC was evaluated in vitro and in vivo by measuring cell proliferation/cell cycle, apoptosis/DNA damage, tubulin, and histone acetylation and modulation of Epithelial/Mesenchymal Transition (EMT) markers. Receptor binding and internalization of ST8176AA1 were confirmed to be similar to trastuzumab. Higher anti-tumor activity of ST8176AA1 compared to trastuzumab was observed in vitro in tumor cell lines. Such higher activity correlated with increased acetylation of histones and alfa-tubulin as a consequence of HDAC inhibitor-mediated epigenetic modulation that also induced increased expression of ErbB2 and estrogen receptor in triple negative breast cancer cells. Consistently with in vitro data, ST8176AA1 exhibited higher tumor growth inhibition than trastuzumab in xenograft models of ovary and colon carcinoma and in two patient-derived xenograft (PDX) models of pancreatic carcinoma. Immunohistochemistry analysis of tumor masses showed lower expression of the proliferation marker Ki67 and higher expression of cleaved caspase-3 in mice treated with the ADC compared to those treated with trastuzumab and results correlated with increased acetylation of both histones and tubulin. Collectively, present data indicate that ADC ST8176AA1 can target epigenetic modulation to ErbB2+ tumors. Interestingly, the amount of HDACi estimated to be delivered at the ST8176AA1 effective dose would correspond to ~1/1,000 of ST7612AA1 effective dose. Therefore, ST8176AA1 is an attractive new therapeutic candidate because it exhibits increased anti-tumor potency compared to trastuzumab by exerting epigenetic modulation at a much safer dose compared to standard HDACi-based therapeutic protocols.

Keywords: ADC (antibody drug conjugate); ErbB2; HDACi (Histone deacetylase inhibitor); solid tumors; trastuzumab.

Figure 1

Generation and preliminary characterization of trastuzumab-HDACi ADCs. (A) Schematic representation of ADC ST8176AA1 and ST8178AA1. (B) Size exclusion chromatography. (C) ErbB2 binding affinity by Surface Plasmonic Resonance (SPR). Analysis on BiaCore T200. Test items were flowed at 0.41–300 nM concentrations. Sensorgrams fitting 1:1 model provided K_D values that are reported in each graphic as mean of three independent determinations ± SD. One representative sensorgram of each test item is shown.

Figure 2

Receptor binding and cellular internalization of ST8176AA1. (A) Immunoreactivity of ST8176AA1 compared to trastuzumab by ErbB2-specific ELISA. Results are the mean of two independent experiments ± SD. (B) Binding of ST8176AA1 (red line) compared to trastuzumab (black line) by cytofluorimetry to human lung (NCI-H1975 and A549), breast (SKBR3), pancreas (Capan-1 and MiaPaCa2), ovary (SKOV3), colon (LS174T), and gastric (N87) carcinoma cell lines. Cell pellets were incubated with test items and then stained with FITC-conjugated mouse anti-human Ig and propidium iodide. Gray peaks refer to cells without primary antibody. (C) Internalization of ST8176AA1 and trastuzumab (both at 5 μg/mL) by SKBR3 breast cancer cells, as measured by HCS fluorescence imaging after 1 h incubation. After washing, the cells were fixed and stained by using FITC-conjugated mouse anti-human Ig (blue signal). Draq5 dye staining of nucleus (gray). Each image is representative of at least five fields of duplicate wells. Magnification 60X. Data are from one representative experiment out of two.

Figure 3

ST8176AA1 inhibits ErbB2 signaling and upregulates acetylation of histones and alpha-tubulin in tumor cells. (A) HCS imaging showing phosphorylation of ErbB2 and downstream molecules in heregulin (20 ng/mL)-activated LS174T human colon carcinoma cells. ST8176AA1 and trastuzumab were used at 30 μg/mL (24-h incubation). (B) HCS imaging showing acetylation of alpha-tubulin (red) and histone H3 (fuchsia) in Capan-1 (pancreas carcinoma) cells. ST8176AA1 and trastuzumab were used at 5 μg/mL (3 h incubation). All panels, insets show specific fluorescence signals within the cells. Draq5 dye staining of nucleus (gray). Each image is representative of at least five fields of duplicate wells. Magnification 60X. Data are from one representative experiment out of two. (C) Effect of trastuzumab (lanes 2–3), ST8178AA1 or ST8176AA1 (lanes 4–5 and 6–7, respectively) at 10 or 40 μg/mL on acetylation of α-tubulin and histone H3 in LS174T cells after 3 h incubation. β-actin was used for normalization. One representative blot is shown.

Figure 4

ST8176AA1 inhibits tumor cell proliferation and induces apoptosis by perturbing the cell cycle. (A) LS174T cells were counted by nucleocounter at indicated time points after exposure to ST8176AA1 or trastuzumab (both at 500, 250, and 125 nM). Data are the average ± SD (n = 3) of counted cells. (B) Cellular doubling time (hours) calculated in the experimental conditions as in (A). (C) Assessment of cell cycle phases after 6 days cultivation as in (A) and staining with PI. Bar graph of the average ± SD (n = 3) of the number of cells/ml in G0/G1 (blue), S (red), and G2/M (green) phases gated on total events. (D) Flow cytometry of cells stained with PI after 6-day cultivation as in (A). The Sub-G1 fraction of total cell population in orange. Representative results of one out of three independent samples. Student's t-test: ^***P ≤ 0.001; ^**P ≤ 0.01, and ^*P ≤ 0.05 vs. vehicle.

Figure 5

ST8176AA1 induces cell damage and reduces apoptosis resistance in tumor cells. (A) HCS imaging analysis showing that LS174T cells treated with ST8176AA1 exhibit increased expression of damage markers HSP70, phospho-P21 (pP21) and phospho-P53 (pP53), compared to trastuzumab. ST8176AA1, and trastuzumab were used at 5 μg/mL (3 h incubation). (B) HCS imaging analysis showing that SKOV3 cells treated with ST8176AA1 exhibit reduced expression of the apoptosis resistance biomarkers Bcl2 and BclXL and increased pro-apoptotic cleaved-caspase3 and cleaved-PARP proteins, compared to trastuzumab. ST8176AA1 and trastuzumab were used at 40 μg/mL (3 days incubation). In all panels, insets show specific fluorescence signals within the cells. Draq5 dye staining of nucleus (gray). Each image is representative of at least five fields of duplicate wells. Magnification 60X. Data are from one representative experiment out of two.

Figure 6

ST8176AA1 induces reversal of epithelial/mesenchymal transition in tumor cells. HCS imaging analysis showing that SKOV3 cells treated with ST8176AA1 exhibit increased expression of the claudin2 and E-cadherin (epithelial) proteins and reduction of vimentin and fibronectin (mesenchymal) proteins. ST8176AA1 and trastuzumab were used at 15 μg/mL (3 days incubation). In all panels, insets show specific fluorescence signals within the cells. Draq5 dye staining of nucleus (gray). Each image is representative of at least five fields of duplicate wells. Magnification 60X. Data are from one representative experiment out of two.

Figure 7

ST8176AA1 induces the expression of ERα and ErbB2 in ErbB2+ and triple negative breast cancer cells. (A) HCS fluorescence imaging analysis show that ErbB2-positive (SKBR3) and triple negative (BT549) breast cancer cells treated with ST8176AA1 exhibit increased expression of ERα (purple) and ErbB2 (green) proteins, compared to trastuzumab. ST8176AA1 and trastuzumab were used at 5 μg/mL (3 days incubation). Basal ErbB2 expression scored by cytofluorimetry in brackets. In all panels, insets show specific fluorescence signals within the cells. Draq5 dye staining of nucleus (gray). Each image is representative of at least five fields of duplicate wells. Magnification 60X. Data are from one representative experiment out of two. (B) SAG-induced Hh target gene expression measured by qPCR in LS174T cells after 24 h-treatment with ST7612AA1 (100 nM), ST8176AA1 or trastuzumab (250 nM) and with the reference Hh inhibitor GANT58 (10 μM). Graph displays the relative expression of the indicated target genes (after normalization to the housekeeping gene GUSB) as compared to not stimulated cells. Data are mean of triplicates ± SD. Student's t-test: ^***P ≤ 0.001; ^**P ≤ 0.01, and ^*P ≤ 0.05 vs. SAG.

Figure 8

Anti-tumor efficacy of ST8176AA1 in tumor xenografts. (A) Subcutaneous tumors were induced in nude mice (10/group) by injecting human SKOV3 cells ovary carcinoma. When tumor masses reached an average size of 50 mm³ mice were treated i.p. (4 doses of 15 or 30 mg/kg once every 4 days, starting 10 days after tumor cell transplantation) with ST8176AA1 or trastuzumab. One group received vehicle (PBS) with the same schedule. Tumor growth was monitored using a Vernier caliper. (B) Body weight. Data are the mean ± SE. Statistical analysis by Mann-Whitney's test *P < 0.05 vs. vehicle and °P < 0.05 vs. trastuzumab; (C–F) Tumor masses at the end of study in (A) were analyzed by immunohistochemistry. Cells positive for Ki67 (C), cleaved-caspase 3 (D), acetyl-H3 (E), acetyl-α-tubulin (F) were counted by two independent observers in five randomly selected fields. Data in the graph are expressed as the mean of positive cells × 100/total cells ± SE or as score with negative staining, score 0; 1-20% positive cells, score 1+; 21-50% positive cells, score 2+ >50% positive cells, score 3+. Statistical analysis by Mann-Whitney's test. **P < 0.01 and ****P < 0.0001 vs. vehicle-treated group; °°P < 0.01, °°°P < 0.001, and °°°°P < 0.0001 vs. trastuzumab. (G) Orthotopic tumor models of ovarian and colon (H) cancer by tumor cell injection in the peritoneum of mice (10/group). Mice were treated ip with ST8176AA1 or trastuzumab (4 doses of 15 mg/kg once every 4 days, starting 3 days after tumor cell transplantation). One group received vehicle (PBS) with the same schedule. Median survival time and Kaplan Myers were evaluated. Data are expressed as mean ± SE, *P < 0.05 and **P < 0.01 vs. vehicle and °P < 0.05 vs. trastuzumab.

Figure 9

Immunohistochemistry of tumor masses from SKOV3 xenografts. SKOV3 xenografts collected 24 h after the last drug administration and analyzed by immunohistochemistry for the expression of acetylated-α-tubulin and acetylated-histone H3 (A), or Ki67 and cleaved-caspase-3 (B). Images captured using 40 × magnification. Scale bar: 500 μm. One representative section, out of 6, for each group is shown.

Figure 10

Anti-tumor efficacy of ST8176AA1 in PDX pancreatic carcinoma model of patient PA5363. (A) NODSCID mice (10/group) bearing patient-derived pancreatic carcinomas PA5363 were treated i.p. with ST8176AA1 or trastuzumab (5 doses of 15 mg/kg once every 4 days, starting 28 days after tumor transplantation as indicated by arrows in the graph). One group received vehicle (PBS) with the same schedule. Tumor growth was monitored using a Vernier caliper. Body weight in panel (B). Data are expressed as mean ± SE. Statistical analysis by Mann-Whitney's test. *P < 0.05 vs. vehicle and °P < 0.05 vs. trastuzumab. (C,D) Tumor masses at the end of the study were analyzed by immunohistochemistry. Cells positive for acetyl-histone H3 (C) and for acetyl-α-tubulin (D) from PA5363 were counted by two independent observers in five randomly selected fields. Data in the graph are expressed as the mean of positive cells × 100/total cells ± SE or as score with negative staining, score 0; 1-20% positive cells, score 1+; 21-50% positive cells, score 2+ >50% positive cells, score 3+. Statistical analysis by Mann-Whitney's test. *P < 0.05 vs. vehicle-treated group; °°P < 0.01 vs. trastuzumab. (E) Representative immunohistochemistry pictures for acetyl-α-tubulin and acetyl-histone H3 are shown. Images captured using 40 × magnification. Scale bar: 500 μm.

Figure 11

Anti-tumor efficacy of ST8176AA1 in PDX pancreatic carcinoma model of PA5366. (A) NODSCID mice (10/group) bearing patient-derived pancreatic carcinomas PA5366 were treated i.p. with ST8176AA1 or trastuzumab (5 doses of 15 mg/kg once every 4 days, starting 36 days after tumor transplantation as indicated by arrows in the graph). One group received vehicle (PBS) with the same schedule. Tumor growth was monitored using a Vernier caliper. Data are expressed as mean ± SE. Statistical analysis by Mann-Whitney's test. *P < 0.05 vs. vehicle and °P < 0.05 vs. trastuzumab. (B) Tumor masses at the end of the study were analyzed by immunohistochemistry. Cells positive for acetyl-histone H3 from PA5366 were counted by two independent observers in five randomly selected fields. Data in the graph are expressed as the mean of positive cells × 100/total cells ± SE or as score with negative staining, score 0; 1-20% positive cells, score 1+; 21-50% positive cells, score 2+ >50% positive cells, score 3+. Statistical analysis by Mann-Whitney's test. *P < 0.05 vs. vehicle-treated group; °°P < 0.01 vs. trastuzumab. (C) Representative immunohistochemistry pictures for acetyl-histone H3 are shown. Images captured using 40 × magnification. Scale bar: 500 μm.