Efficiency of Immunotoxin Cytotoxicity Is Modulated by the Intracellular Itinerary

Lori L Tortorella,Frederick R Maxfield,David Fitzgerald,Sushmita Mukherjee,Nina H Pipalia,Ira Pastan,Wanjin Hong

doi:10.1371/journal.pone.0047320

Lori L Tortorella, Frederick R Maxfield + Show 5 more

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https://doi.org/10.1371/journal.pone.0047320

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Journal: PloS one	Publication Date: Oct 9, 2012
Citations: 13	License type: CC0 1.0

Affiliation: Cornell University, National Cancer Institute

Abstract

Pseudomonas exotoxin-based immunotoxins, including LMB-2 (antiTac(Fv)-PE38), are proposed to traffic to the trans-Golgi network (TGN) and move by a retrograde pathway to the endoplasmic reticulum, where they undergo translocation to the cytoplasm, a step that is essential for cytotoxicity. The retrograde transport pathways used by LMB-2 are not completely understood, so it is unclear if transit through specific organelles is critical for maximal cytotoxic activity. In this study, we used Chinese hamster ovary (CHO) cell lines that express chimeric constructs of CD25, the Tac antigen, attached to the cytoplasmic domain of the TGN-targeted transmembrane proteins, TGN38 and furin. These chimeras are both targeted to the TGN, but the itineraries they follow are quite different. LMB-2 was incubated with the two cell lines, and the efficiency of cell killing was determined using cell viability and cytotoxicity assays. LMB-2 that is targeted through the endocytic recycling compartment to the TGN via Tac-TGN38 kills the cells more efficiently than immunotoxins delivered through the late endosomes by Tac-furin. Although the processing to the 37 kDa active fragment was more efficient in Tac-furin cells than in Tac-TGN38 cells, this was not associated with enhanced cytotoxicity – presumably because the toxin was also degraded more rapidly in these cells. These data indicate that trafficking through specific organelles is an important factor modulating toxicity by LMB-2.

Highlights

Protein toxins have been developed as components of anticancer therapies due to their potent cell killing ability
LMB-2 is an immunotoxin comprised of a truncated form of Pseudomonas exotoxin A (PE) fused to the variable region of an antibody that binds the Interleukin 2 Receptor (IL2R) a-chain, which acts as the binding domain (Figure 1C) [1,2]
On day 2, they were treated with LMB-2 or negative control toxin Erb38, an immunotoxin that binds to Erb2/HER2, which is not expressed in Chinese hamster ovary (CHO) cells

Summary

Introduction

Protein toxins have been developed as components of anticancer therapies due to their potent cell killing ability. Immunotoxins contain a cell-binding moiety based on an antibody that has specificity for tumor cell antigens attached to a portion of a plant or bacterial toxin. LMB-2 is an immunotoxin comprised of a truncated form of Pseudomonas exotoxin A (PE) fused to the variable region of an antibody that binds the Interleukin 2 Receptor (IL2R) a-chain ( known as anti-Tac antibody), which acts as the binding domain (Figure 1C) [1,2]. LMB-2 inhibited protein synthesis in IL2R+ transfected epidermoid carcinoma cells and caused complete tumor regression in tumor-bearing nude mice [4]. In clinical trials this immunotoxin was shown to be effective against some IL2R+ hematologic malignancies, including refractory hairy cell leukemia [5]

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