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

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.

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

“…Finally, plasmids were transfected by electroporation in Pichia pastoris and the transfected colonies selected, as indicated in Materials and Methods. As indicated in our previous work [21], the yield of production of recombinant GRNLY was good, around 5 mg/L in the different batches, but the yield of production of SM3GRNLY was lower, around 2 mg/L. Taking into account the difference in molecular weight, this difference in weight yield meant a difference of 10-fold in molar yield.…”

“…The construct coding for the 9 kDa granulysin (GRNLY) gene alone was described in our previous works [19,21]. We designed constructs to encode for the two anti-Tn immunotoxins formed by granulysin bound through a flexible linker of 23 amino acids (formed by Ser and Gly residues) to the anti-Tn scFv SM3 or AR20.5, respectively.…”

“…For this reason, various steps of the production and purification process were modified in search of an optimized protocol. In addition, we used a non-thermic technology, yeast electroporation by pulsed electric fields (PEF), to obtain the intracellular protein and increase the yield of production [21].…”

Antibody-Based Immunotoxins for Colorectal Cancer Therapy

“…In these ITX, the foreign toxins are replaced by cytotoxic human proteins, thus avoiding the immune-mediated reaction against the toxin [ 24 , 25 , 26 ]. Several human proteins have been used to generate these fourth-generation ITX, including the microtubule-associated protein tau, RNases, granzyme B, death-associated protein kinase [ 26 ] and, more recently, granulysin [ 27 , 28 ]. Among those approaches, granzyme B-based ITX have been the most studied and further improved, demonstrating a therapeutic effect in several preclinical models in vivo [ 24 ].…”

“…In that study, an innovative procedure based on pulsed electric field technology was used to improve the yield of production of the ITX from yeast. It was demonstrated that SM3GRNLY retained its specific binding to the Tn antigen and increased the cytotoxicity against these Tn + cell lines compared with granulysin alone [ 27 ]. However, no in vivo studies with this ITX have been reported yet.…”