SS1P Immunotoxin Induces Markers of Immunogenic Cell Death and Enhances the Effect of the CTLA-4 Blockade in AE17M Mouse Mesothelioma Tumors

Abstract

SS1P is an anti-mesothelin immunotoxin composed of a targeting antibody fragment genetically fused to a truncated fragment of Pseudomonas exotoxin A. Delayed responses reported in mesothelioma patients receiving SS1P suggest that anti-tumor immunity is induced. The goal of this study is to evaluate if SS1P therapy renders mesothelioma tumors more sensitive to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) immune checkpoint blockade. We evaluated the ability of SS1P to induce adenosine triphosphate (ATP) secretion and calreticulin expression on the surface of AE17M mouse mesothelioma cells. Both properties are associated with immunogenic cell death. Furthermore, we treated these tumors with intra-tumoral SS1P and systemic CTLA-4. We found that SS1P increased the release of ATP from AE17M cells in a dose and time-dependent manner. In addition, SS1P induced calreticulin expression on the surface of AE17M cells. These results suggest that SS1P promotes immunogenic cell death and could sensitize tumors to anti-CTLA-4 based therapy. In mouse studies, we found that the combination of anti-CTLA-4 with intra-tumoral SS1P induced complete regressions in most mice and provided a statistically significant survival benefit compared to monotherapy. The surviving mice were protected from tumor re-challenge, indicating the development of anti-tumor immunity. These findings support the use of intra-tumoral SS1P in combination with anti-CTLA-4.

Keywords: ATP; Calreticulin; SS1P; anti-CTLA-4; immunogenic cell death; immunotherapy; immunotoxins; mesothelin; mesothelioma.

Figure 1

The cytotoxic activity of SS1P and LMB-100 in AE17 M cells. WST-8 cytotoxicity assays in AE17M cells after 3-day incubation with either SS1P or LMB-100.

Figure 2

The anti-mesothelin immunotoxins promote the secretion of ATP. (A,B) Extracellular ATP in the media of AE17M cells after 24 h of incubation with either (A) SS1P or (B) LMB-100 at various concentrations. (C) Extracellular ATP in the media of AE17M cells incubated with 200 ng/mL LMB-100 at various time durations. (D) Extracellular ATP in the media in untreated AE17M cells or after 24 h of incubation with either 25, 250, or 2500 ng/mL of docetaxel or 13.4 µg/mL doxorubicin. * p < 0.05, ** p < 0.01. This experiment was repeated with similar results.

Figure 3

SS1P increases cell surface calreticulin. (A,B) Representative dot plots of (A) untreated AE17M cells or (B) cells exposed to 100 ng/mL SS1P for 24 h and stained with 7AAD and antibodies to calreticulin. (C) Percentage of calreticulin positive cells out of AE17M live cells (7AAD-) after exposure to 65 µM tunicamycin (24 h) or 5 µg/mL Oxaliplatin (24 h) or 100 ng/mL SS1P (21 or 24 h). (D) Percentage of 7AAD positive cells in the treatment conditions described in C. Pool data from two separate experiments is presented. CRT calreticulin, Tu Tunicamycin, Ox oxaliplatin, * p < 0.05, ** p < 0.01.

Figure 4

The in vivo anti-tumor effect of SS1P. (A) Average volume of AE17M tumors after receiving no treatment (control) or given intravenously SS1P (4 µg) every two days. (B) Average volume of AE17M tumors receiving direct intratumoral. SS1P (10 µg) or phosphate buffered saline (PBS) every four days. Treatment days are noted by the arrows. ** p < 0.01.

Figure 5

The SS1P and anti–CTLA-4 effect on AE17M tumor growth in mice. Individual tumor growth curves of AE17M tumors treated with (A) 8 µg SS1P i.t. alone, (B) PBS i.t. and 25 µg anti-CTLA-4 i.p., and (C) 8 µg SS1P i.t. and 25 µg anti-CTLA-4 i.p. (D) Long-term survival of mice described in (A–C). Survival of mice treated with SS1P and anti-CTLA-4 was significantly longer than that in the other groups (p < 0.05). (E) Mice that completely eradicate their tumor in (A–D) were re-challenged with additional AE17M cells implanted on the contralateral flank. The average tumor size is presented and compared to AE17M tumors growing on naïve mice (n = 5). The number of mice in complete regression and the total mice per group are shown in parentheses.