Anthrax toxin fusion proteins for intracellular delivery of macromolecules

Abstract

The dominant role played by the anthrax toxin in Bacillus anthracis pathogenesis shows that the toxin has evolved to be an efficient system for delivering its two catalytic protein components, oedema factor and lethal factor (LF), into the cytosol of host cells. This system involves binding of the protective antigen (PA) toxin component to a ubiquitous (and still unidentified) receptor, proteolytic activation at the cell surface, internalization by endocytosis and translocation through an early endosome membrane to the cytosol ( Leppla 1995). We and colleagues showed that the system can be exploited to deliver heterologous polypeptides to the cytosol ( Arora et al. 1992 ; Milne et al. 1995 ). This work used the catalytic domains of other toxins which are normally translocated across membranes ( Arora & Leppla 1994) . Immunity to intracellular pathogens depends on the cytosolic processing of antigens to produce peptides that are presented on the cell surface bound to MHC Class I molecules. The anthrax toxin delivery system provides a way to mimic this process. We made a fusion protein containing the (non‐catalytic) amino terminal domain of LF and the gp120 envelope glycoprotein of HIV‐1. Administration of this recombinant protein along with PA to antigen‐presenting cells sensitized them to cytolysis by cytotoxic T‐cells specific to gp120 peptides ( Goletz et al. 1997 ). Further exploitation of the anthrax toxin system as a cell‐targeting reagent would be facilitated by achieving cell type specificity. The recent determination of the PA structure ( Petosa et al. 1997 ) allows rational engineering to modify or replace the receptor‐binding domain with specific ligand structures. A model system was produced by fusing a c‐Myc peptide to the carboxyl terminus of PA so as to target hybridoma cells expressing cell surface antibodies to this peptide. Killing of the hybridoma cells was shown to be specific by competition with the peptide and with non‐toxic mutants of PA ( Varughese et al. 1998 ).

Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits

“…A possible explanation for why rPA preparations rich in more negatively charged isoforms are less biologically active in vitro may be the improper folding of one or more of the four functional domains. Elucidation of the extent of deamidation within each of the rPA domains, i.e., receptor-binding domain, or lethal/edema factor binding domain [30][31][32][33][34][35][36], may provide additional information, but is beyond the scope of this report. GMP grade rPA does not contain the more negatively charged isoforms present in the R&D grade material, which resulted in observable differences in biological activity.…”

Protection against Anthrax Toxemia by Hexa- d -Arginine In Vitro and In Vivo

“…Cleavage occurs at a surface loop and releases PA 20 , the N-terminal fragment of PA. Loss of this N-terminal fragment results in self-association of PA 63 , the C-terminal fragment of PA, forming membrane-inserting heptamers capable of binding LF and/or EF (20). These complexes are taken up into cells and enter the lumen of the acidic endosomal compartment, from which LF emerges to initiate enzymatic reactions that ultimately result in cell death (1,12,15).Through the use of combinatorial chemistry techniques, workers in our laboratory have developed the compound hexa-D-arginine (D6R) as a potential therapeutically useful furin inhibitor (4). Studies that used Pseudomonas aeruginosa exotoxin A (PEA), a bacterial toxin which also requires furin processing, have shown that D6R effectively blocks PEA-induced cell death in vitro and in vivo (21).…”

“…Cleavage occurs at a surface loop and releases PA 20 , the N-terminal fragment of PA. Loss of this N-terminal fragment results in self-association of PA 63 , the C-terminal fragment of PA, forming membrane-inserting heptamers capable of binding LF and/or EF (20). These complexes are taken up into cells and enter the lumen of the acidic endosomal compartment, from which LF emerges to initiate enzymatic reactions that ultimately result in cell death (1,12,15).…”

EphrinA1 inhibits malignant mesothelioma tumor growth via let-7 microRNA-mediated repression of the RAS oncogene

“…The prognosis of patients with advanced and metastatic MM is poor. [1][2][3] Approximately 3000 new cases are reported each year in the United States, and more than 10 000 are reported worldwide. 2,4 As mesothelioma symptoms do not arise until late stages, the majority of mesothelioma patients are diagnosed in an advanced stage of malignancy.…”

“…[1][2][3] Approximately 3000 new cases are reported each year in the United States, and more than 10 000 are reported worldwide. 2,4 As mesothelioma symptoms do not arise until late stages, the majority of mesothelioma patients are diagnosed in an advanced stage of malignancy. 5 MM is resistant to all currently available treatments; therefore, understanding the mechanisms involved in the growth of MM may provide insights for the development of new therapeutic strategies to treat patients with MM.…”