Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology.

Patricia Guerrero-Ochoa,Ramón Hurtado-Guerrero,Javier Macías-León,Javier Raso,Raquel Ibáñez-Pérez,Diederich Aguilar-Machado,Alberto Anel

doi:10.3390/ijms21176165

Abstract

Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of the intratumoral injection of recombinant granulysin and of the systemic injection of an immunotoxin between granulysin and the anti-carcinoembryonic antigen single-chain Fv antibody fragment MFE23, which were produced in the yeast Pichia pastoris. In the present work, we developed a second immunotoxin combining granulysin and the anti-Tn antigen single-chain Fv antibody fragment SM3, that could have a broader application in tumor treatment than our previous immunotoxin. In addition, we optimized a method based on electroporation by pulsed electric field (PEF) to extract the remaining intracellular protein from yeast, augmenting the production and purificiation yield. The immunotoxin specifically recognized the Tn antigen on the cell surface. We also compared the thermal stability and the cytotoxic potential of the extracellular and intracellular immunotoxins on Tn-expressing human cell lines, showing that they were similar. Moreover, the bioactivity of both immunotoxins against several Tn+ cell lines was higher than that of granulysin alone.

Highlights

Granulysin is a protein contained in the granules of cytotoxic T lymphocytes (CTLs) and human natural killer (NK) cells that possesses cytolytic potential on microbes and on tumor cells [1,2,3]
We generated a new granulysin-based immunotoxin directed against the Tn antigen
We demonstrated the enhanced cytotoxicity of the immunotoxin compared with granulysin alone on the pancreatic carcinoma Capan-2 cells, showing that 10 μM of SM3GRNLY induced 70% of cell death, while GRNLY did not induce more than 45% (Figure 6B, left upper panel)

Summary

Introduction

Granulysin is a protein contained in the granules of cytotoxic T lymphocytes (CTLs) and human natural killer (NK) cells that possesses cytolytic potential on microbes and on tumor cells [1,2,3]. We characterized this mechanism in more detail, demonstrating that it was initiated by an increase in cytoplasmic Ca2+ concentration, followed by the generation of mitochondrial reactive oxygen species that initiated, in turn, the mitochondrial apoptotic pathway, with caspase 3 activation and nuclear AIF translocation [4,6,7]. In these studies, we demonstrated that granulysin was able to kill cells from B-cell lymphocytic leukemia patients while being nontoxic against lymphocytes from healthy donors [4]. We demonstrated that the antitumor action of granulysin was accompanied by a massive infiltration of NK cells in the tumor, providing evidence of immunogenicity, as well as the absence of side effects [8]

Methods

Results

Conclusion