4 and and 5= 0

4 and and 5= 0.021) at 24 h and 7.9-fold (= 0.014) at 48 h in response to LMB-100 (Fig.?5= 0.28) and 48 h (4.27-fold = 0.014) (Fig. LMB-100, was designed. LMB-100 consists of a humanized Fab that targets human mesothelin (hMSLN) and a B-cell epitope silenced, 24 kD fragment of PE (PE24) (10). PE-based immunotoxins have a unique mechanism of cell killing. The toxin catalyzes the irreversible ADP-ribosylation of the diphthamide residue of EF2 and arrests protein synthesis. This mechanism of cell killing differs from that of currently approved chemotherapies for solid tumors (11). Though it is generally believed that this anti-tumor effects of immunotoxins are attributed to their direct tumor cell killing through arresting protein synthesis, there is growing evidence that immunotoxins may also induce anti-tumor immune responses. For example, delayed CR was observed in some patients treated with Moxetumomab pasudotox 6 mo after the end of treatment, suggesting that this immunotoxin does more than direct tumor cells killing (8, 12). A delayed response was also observed in some SS1P-treated mesothelioma patients (13). Additionally, a recent study reveals that treatment with LMB-100 results in systemic inflammatory response in some patients with mesothelioma (14). Preclinical animal studies in a 66C14-M murine breast cancer model (15) and AE17-M murine mesothelioma model (16), showed that mice achieved CRs and developed anti-tumor immunity after combination treatment of anti-MSLN immunotoxin injected into the tumor and anti-CTLA-4 given systemically. However, the subcutaneous tumor model in these studies does not resemble the microenvironment of human mesothelioma. To study the immune response induced by immunotoxin, a clinical-relevant animal model is needed. In the present study, we established an orthotopic mouse mesothelioma model and use it to study how immunotoxin-mediated cell death produces immune-related signals and reshapes the tumor immune microenvironment. We implanted mouse mesothelioma cells expressing hMSLN (AB1-L9) into the peritoneal cavity of Balb/c mice that are tolerant to hMSLN (TPO-MSLN mice) and treated the mice with EC-PTP LMB-100 given intraperitoneally. We showed here that immunotoxin-mediated cell death reshapes the tumor immune microenvironment, induces tertiary lymphoid structures (TLS) development, and contributes to the establishment of anti-tumor immunity. Results LMB-100 Completely Eradicates Orthotopic Mesothelioma and Produces an Anti-Tumor Immunity. To determine whether immunotoxins alone can induce anti-tumor immunity and to study the mechanism of this immunity, we generated an orthotopic mouse T56-LIMKi model in which the mouse mesothelioma cell line AB1 expressing hMSLN (AB1-L9) grows in the peritoneal cavity of mice. To ensure the tumor cells expressing hMSLN are not rejected, the mice, termed TPO-MSLN mice, express a human transgene (MSLN) in their thyroid gland (17). These mice have a normal immune system, do not recognize hMSLN as foreign, and can safely be used to study anti-tumor immune responses to LMB-100. The data in Fig. 1show that AB1-L9 cells are killed by LMB-100 with an IC50 of 19 ng/ml. AB1 cells not expressing hMSLN are resistant to LMB-100 with high viability up to 1 1,000 ng/ml. AB1-L9 cells are not killed by LMB-255, an inactive anti-hMSLN immunotoxin that has a deletion of the furin cleavage site and a mutation at residue 553 of the toxin. AB1-L9 cells are also not killed by LMB-34, an immunotoxin containing the same toxin moiety as LMB-100, but target human B-cell maturation antigen (hBCMA), which is not expressed in the tumors. These data indicate a specific killing effect of LMB-100. Open in a separate T56-LIMKi window Fig. 1. Test the therapeutic effect of LMB-100 on an orthotopic mesothelioma T56-LIMKi TPO-MSLN mouse model. < 0.005, ****< 0.001 compared with LMB-100 group. Plasma hMSLN concentration in mice treated by either LMB-100 (i.p. 3 mg/kg, day 5) or PBS (i.p. 200 l, day 5). Mean SD. ****< 0.001 compared with LMB-100 group. shows a representative IHC image of an AB1-L9 tumor stained for MSLN that is growing on the GS-ligament 5 d after implantation. On day 5, 90% of the cells on the GS-ligament are hMSLN positive (Fig. 1 and and and and and Table?1). Then, we explored the effect of delaying dosing (Fig. 2 and and Table 1). Survival was significantly decreased if the two-dose treatment was given on day 7 and day 11 (3/11 CRs; 27%) or on day 9 and day 13 (1/11 CRs; 9%). In addition, treatment efficacy was diminished if mice only received a single dose of LMB-100 on day 7 or day 9 (and < 0.001 compared with LMB-100 two doses group. < 0.05, ***< 0.005, ****< 0.001 compared with LMB-100 D5 + D9 group. < 0.005, ****< 0.001 compared with LMB-100 group. < 0.005 compared with LMB-100 group. and and and and and < 0.05.