Preclinical Studies of Granulysin-Based Anti-MUC1-Tn Immunotoxins as a New Antitumoral Treatment

Abstract

Two granulysin (GRNLY) based immunotoxins were generated, one containing the scFv of the SM3 mAb (SM3GRNLY) and the other the scFv of the AR20.5 mAb (AR20.5GRNLY). These mAb recognize different amino acid sequences of aberrantly O-glycosylated MUC1, also known as the Tn antigen, expressed in a variety of tumor cell types. We first demonstrated the affinity of these immunotoxins for their antigen using surface plasmon resonance for the purified antigen and flow cytometry for the antigen expressed on the surface of living tumor cells. The induction of cell death of tumor cell lines of different origin positive for Tn antigen expression was stronger in the cases of the immunotoxins than that induced by GRNLY alone. The mechanism of cell death induced by the immunotoxins was studied, showing that the apoptotic component demonstrated previously for GRNLY was also present, but that cell death induced by the immunotoxins included also necroptotic and necrotic components. Finally, we demonstrated the in vivo tumor targeting by the immunotoxins after systemic injection using a xenograft model of the human pancreatic adenocarcinoma CAPAN-2 in athymic mice. While GRNLY alone did not have a therapeutic effect, SM3GRNLY and AR20.5GRNLY reduced tumor volume by 42 and 60%, respectively, compared with untreated tumor-bearing mice, although the results were not statistically significant in the case of AR20.5GRNLY. Histological studies of tumors obtained from treated mice demonstrated reduced cellularity, nuclear morphology compatible with apoptosis induction and active caspase-3 detection by immunohistochemistry. Overall, our results exemplify that these immunotoxins are potential drugs to treat Tn-expressing cancers.

Aberrant Glycosylation as Immune Therapeutic Targets for Solid Tumors

“…Another feature of AR20.5 is to form immune complexes with circulating MUC1 and/or MUC1-expressing tumor cells in vivo, implying that it may stimulate a dendric cell-mediated T cell response [247]. Recently, those SM3 and AR20.5-based immunotherapies in conjugation with immunotoxin (mAb-conjugated with Granulysis, GRNLY) have been tested in Tn + cancer cell lines including a pancreatic cell line, Capan-2, and a leukemic cell line, Jurkat, in both in vitro and in vivo mouse models, and resulted in targeted lysis of tumors with Tn + MUC1 [248]. Another novel mAb targeting MUC1 is PankoMab-GEX, which is a well-defined mAb from screening with a synthetic Tn + glycopeptides [249].…”

Conjugation of the 9-kDa Isoform of Granulysin with Liposomes Potentiates Its Cytotoxicity

“…Systemic administration of the immunotoxin demonstrated a decrease in tumor growth in a CEA+ tumor-bearing mouse model, whereas GRNLY did not exhibit a therapeutic effect [ 17 ]. Other GRNLY-based immunotoxins directed against the MUC1-Tn tumor antigen have been applied, demonstrating in vitro and in vivo efficacy in pre-clinical models [ 18 , 19 ]. These immunotoxins could have a broader application to different solid tumors and leukemia than the anti-CEA immunotoxin [ 20 ].…”