Improved Immunotoxins with Novel Functional Elements

Abstract

Immunotoxins (ITs) are protein-based drugs combining a target-specific binding domain (usually derived from an antibody) and a cytotoxic domain to kill target cells. They are among the most promising new therapeutic tools to fight cancer, and several clinical trials have been completed with encouraging results. Although the targeted elimination of malignant cells is an elegant concept, there are numerous practical challenges that limit the clinical use of ITs, including inefficient cellular uptake, low cytotoxicity and off-target effects. Here we present some of the strategies that have been developed to improve the efficacy of ITs, particularly those involving the incorporation of functional peptide sequences into recombinant ITs to improve target binding, modify plasma half life and distribution, boost tumor penetration, enhance cellular uptake and increase cytotoxic efficiency.

Targeting antibodies to the cytoplasm

“…176,177 Photochemical internalization enhances endosomal release by means of "photosensitizers", which are agents that translocate through intracellular membranes, e.g., some ribonucleases or toxins. This principle has been extensively studied for immunotoxins or targeted RNases [195][196][197] that both require cell binding and uptake into target cells by a ligand or antibody recognizing internalizing surface receptors. After endocytosis, the toxin or RNase moiety must be transferred into the cytosol in order to engage with their cellular substrates and ultimately cause cell death.…”

Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein

“…However, marked advances have been achieved in minimizing these drawbacks and enhancing immunotoxin potential. 85,86 Efforts to increase the circulation time of these agents include the design of fusion proteins in which the immunotoxins are linked to Fabs 87 or to full-length immunoglobulins. 88 The immunogenicity problem is being addressed by identifying and eliminating B cell epitopes using site-directed mutagenesis 89 or by choosing human-derived proteins as the cytotoxic moieties.…”

Diving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody-Targeted Anti-Tumor Toxins

“…The former approach can be achieved by the insertion or addition of drug-releasing elements directly into the targeted toxin [36]. These additional elements can comprise peptides with new functions or chemical characteristics [10]. The latter approach can be specific for the internalized molecule or represent a more general leakage that tolerates the transfer of other molecules from the lumen of the vesicles.…”

“…An increase in efficacy can also be achieved when only the furin-site is exerted without a CPP, which is probably caused by the resulting drug release [56,57], however, this must be tested in individual cases since the addition of either one of two furin cleavage sites to a targeted gelonin was less efficient in a xenograft tumor model compared to a flexible non-cleavable linker [58]. Further strategies to increase the endosomal escape of targeted toxins include the use of the translocation motif of diphtheria toxin or Pseudomonas exotoxin A instead of a CPP demonstrated for ricin and granzyme B, and cellular protein retention signals such as REDLK and KDEL as shown for ricin and ribonucleases [10].…”

“…Where the toxin is a protein, the drug can also be expressed as a fusion protein generated by recombinant DNA technology [5]. Insertion of an acid-sensitive bond or a cleavage site for endosomal proteases between the toxin and the antibody can result in the release of the toxin in acidic endosomes or lysosomes, thus allowing the passage of the toxin across the endosomal/lysosomal membrane where this is permissible [2,10]. Mutation of targeted toxins to avoid lysosomal degradation is another option to increase cytotoxicity, but seems to be limited to lymphocytes [11].…”