Targeted dianthin is a powerful toxin to treat pancreatic carcinoma when applied in combination with the glycosylated triterpene SO1861

Abstract

Targeted cancer therapy provides the basis for the arrest of tumor growth in aggressive pancreatic carcinoma; however, a number of protein-based targeted toxins lack efficacy due to insufficient endosomal escape after being endocytosed. Therefore, we tested a fusion protein of the ribosome-inactivating protein dianthin and human epidermal growth factor in combination with a glycosylated triterpene (SO1861) that serves as an endosomal escape enhancer. In vitro investigations with the pancreatic carcinoma cell lines BxPC-3 and MIA PaCa-2 revealed no significant differences to off-target cells in the half maximal inhibitory concentration (IC₅₀ ) for the fusion protein. In contrast, combination with SO1861 decreased the IC₅₀ for BxPC-3 cells from 100 to 0.17 nm, whereas control cells remained unaffected. Monotherapy of BxPC-3 xenografts in CD-1 nude mice led to a 51.7% average reduction in tumor size (40.8 mm³ ) when compared to placebo; however, combined treatment with SO1861 resulted in a more than 13-fold better efficacy (3.0 mm³ average tumor size) with complete regression in 80% of cases. Immunohistochemical analyses showed that tumor cells with lower target receptor expression are, in contrast to the combination therapy, able to escape from the monotherapy, which finally results in tumor growth. At the effective concentration, we did not observe liver toxicity and saw no other side effects with the exception of a reversible skin hardening at the SO1861 injection site, alongside an increase in platelet counts, plateletcrit, and platelet distribution width. In conclusion, combining a targeted toxin with SO1861 is proven to be a very promising approach for pancreatic cancer treatment.

Keywords: SO1861; endosomal escape; epidermal growth factor receptor; pancreatic carcinoma; targeted toxin; xenograft.

Figure 1

SDS/PAGE (12%) of the pellet obtained after ultrasonication and purified fractions of proteins obtained after (A) Ni‐NTA chromatography and (B) chitin column affinity chromatography. FT, flow‐through; W, washout; lane ‘1’: 125 mm imidazole eluate; lane ‘2’: 250 mm imidazole eluate. Arrows point to ^HisDianthin‐EGF.

Figure 2

SO1861‐mediated augmentation of ^HisDianthin‐EGF (DE). (A, D) BxPC‐3, (B, D) MIA PaCa‐2, and (C, D) NIH‐3T3 cells were seeded in 96‐well plates and grown for 24 h. Cells were then treated with (A–C) varying concentrations of ^HisDianthin‐EGF in DMEM (MIA PaCa‐2, NIH‐3T3) or RPMI 1640 (BxPC‐3) in the presence and absence of SO1861 (0.5 μg·mL⁻¹), or (D) at a fixed concentration of ^HisDianthin‐EGF corresponding to the IC ₅₀ shown in Table 1 and varying concentrations of SO1861. Cells were further incubated for 48 h. Viability was determined by an MTT assay. Each value represents the mean of three (two for panel (D)) independent experiments performed in quadruplicate. Single outliers defined as outside ± 3‐fold standard deviation were omitted in (D). A statistical significant effect between single and combination treatments was observed in MIA PaCa‐2 (Student's t‐test; P < 0.05) and BxPC‐3 (Student's t‐test; P < 0.01) cell lines. The vertical line in (D) indicates the SO1861 concentration used in (A–C) to determine the IC ₅₀ of ^HisDianthin‐EGF and is thus the expected IC ₅₀ for the variation in SO1861.

Figure 3

Real‐time cell analysis showing the dose‐dependent increase in cytotoxicity in monotherapy with ^HisDianthin‐EGF (DE) (A, C, E) and in combination therapy together with SO1861 (B, D, F) in EGFR‐overexpressing MIA PaCa‐2 cells (C, D) and BxPC‐3 cells (E, F) as compared to NIH‐3T3 cells (A, B), which is an off‐target cell line. The y‐axis shows the impedance‐based cell index that was normalized to 1.0 after 28 h in case of NIH‐3T3 and MIA PaCa‐2 cells and, due to faster cell proliferation, after 19 h in case of BxPC‐3 cells just before treatment started. The cell index can be assumed to be proportional to the number of living cells.

Figure 4

Body weight loss observed after single application of different doses of ^HisDianthin‐EGF. The values refer to the body weight just before treatment. Significance in body weight changes is indicated by * (P < 0.05) and ** (P < 0.01).

Figure 5

Xenograft tumor volume evaluated by the use of a digital vernier caliper for placebo (PBS only), single (^HisDianthin‐EGF; 0.35 μg per treatment), and combination (^HisDianthin‐EGF; 0.35 μg per treatment + SO1861; 30 μg per treatment) therapy. Arrows point to the days of treatment. A statistical significant decrease in tumor volume was observed in the combination treatment for the curves as a whole (P < 0.05 versus single therapy and versus placebo).

Figure 6

Images depicting tumor volumes and lesion‐free skin after six therapy cycles of (A) placebo, (B) ^HisDianthin‐EGF, and (C) ^HisDianthin‐EGF and SO1861. Arrows point to the injection site of tumor cells. The pictures show one representative animal of each group comprising five mice.

Figure 7

Histological and immunohistochemical evaluation of (A) liver and (B–G) tumor tissue samples. Hematoxylin and eosin (H&E) staining (A) showed single‐cell necrosis (white arrows) and necrosis of small groups of hepatocytes (white box); that is, moderate damage of liver tissues isolated from mice administered with the highest single dose of ^HisDianthin‐EGF (40 μg). (B) High EGFR expression level (arrow) was observed in untreated tumors, whereas (C) a weak expression of EGFR (arrow) was observed in the treatment group depicting curative effect of therapy as compared to untreated. High Ki67 proliferation index (D) was observed in all tumors except the one obtained after combination therapy [50%; (E)]. The right row of photographs (F, G) represents H&E‐stained samples corresponding to areas of the immunohistologically EGFR‐ and Ki‐67‐stained samples. Part (F) shows the tumor of an untreated mouse, whereas part (G) depicts the tumor of the sole mouse that retained a tumor after treatment with ^HisDianthin‐EGF + SO1861. The immunohistochemical samples are counterstained by hematoxylin. All scale bars represent a length of 100 μm.

Figure 8

Platelet‐derived parameters in different groups (healthy, placebo, ^HisDianthin‐EGF, and ^HisDianthin‐EGF + SO1861 treated). Significance is indicated as * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). PLT counts, platelet counts; PCT, plateletcrit; PDW, platelet distribution width; MPV, mean platelet volume.