Plant toxins in targeted cancer therapy : Construction, characterization and intracellular trafficking of an abrin-a A chain immunotoxin

Abstract

Despite the development of several new drugs for cancer treatment, challenges such as drug resistance and toxic side effects necessitate newer therapeutic approaches. One promising strategy is immunotherapy, where antibodies or ligands targeting cell surface molecules deliver toxins specifically to cancer cells, thereby increasing treatment efficacy. Such conjugates are termed immunotoxins.

Immunotoxins can be generated either as:

Chemical conjugates using cross-linkers to attach antibodies to toxins.

Fusion proteins, where antibody fragments (Fab or Fv) are genetically fused with toxin genes and expressed recombinantly.

Several toxins, particularly ribosome-inactivating proteins (RIPs) such as ricin, saporin, diphtheria toxin, and Pseudomonas exotoxin, have been explored. Among them, abrin, a type II RIP from Abrus precatorius, is one of the most potent, inhibiting translation and inducing apoptosis at picomolar concentrations.

Aim of the Study This study focuses on constructing immunotoxins using recombinant abrin A chain (rABRa-A) and antibodies targeting GnRH receptors (GnRH-R) and EpCAM, both of which are overexpressed in certain carcinomas. Recombinant expression avoids contamination risks associated with native toxin purification and improves specificity.

Chapter Summaries Chapter 1 - Introduction

Overview of cancer and current treatment modalities.

Introduction to immunotherapy and immunotoxins.

Review of toxins used in immunotoxin construction.

Chapter 2 - Construction of Immunotoxins

Recombinant abrin A chain (rABRa-A) was expressed and purified, showing activity comparable to wild-type protein.

Antibodies targeting GnRH-R and EpCAM were selected for conjugation.

Chapter 3 - Characterization of Immunotoxins

Three immunoconjugates were constructed using rABRa-A.

mAb F1G4-rABRa-A (GnRH-R specific) inhibited translation and induced apoptosis in GnRH-R-bearing MCF-7 (human breast carcinoma) cells, but not in KB (nasopharyngeal carcinoma) or HFF (normal fibroblast) cells.

Mutant rABRa-A (R167L), with reduced catalytic activity, confirmed that inhibition was due to the recombinant A chain.

EpCAM-targeting conjugates failed to inhibit translation, indicating lack of receptor internalization.

The immunoconjugate showed higher efficacy in MCF-7 cells compared to normal breast cells (MCF-10A) and also inhibited translation in HepG2 (human hepatocarcinoma) cells, though with delayed kinetics.

Chapter 4 - Intracellular Trafficking

Comparative studies of trafficking between native abrin and immunotoxin in HepG2 cells revealed distinct pathways.

Native abrin followed the classical type II RIP pathway: binding via B chain, endocytosis, ER transport, disulfide cleavage, and cytosolic release.

Novel findings: nuclear localization of abrin A chain in HepG2 and KB cells, but not in OVCAR-3 cells, suggesting cell-specific trafficking.

Immunotoxin trafficking differed: rABRa-A was localized predominantly in the cytosol, bypassing ER localization.

Release of A chain from the immunoconjugate occurred via the thioredoxin system, confirming a distinct cellular pathway.