Toxin-targeted Constructs

As discussed in Section 11.8.1, many toxins of bacterial or plant origin are built up of different moieties or subunits which mediate binding, membrane translocation and catalytic or 

Anthrax toxin is a bacterial toxin from Bacillus anthracis consisting of three parts protective antigen (PA), lethal factor (LF) and edema factor (EF). Both LF and EF compete for binding sites on the PA protein. The PA protein binds with high affinity to an as yet unknown receptor on macrophages and related cell types. When PA is internalized by the target cells, it functions as a shuttle protein for either EF or LF. Intracellularly, in the acidic environment of the endosome, EF and LF are capable of entering the cytosol by pH-dependent pore formation [139]. 

A second example of a toxin that has been used as targeting device is tetanus toxin. Tetanus toxin is a potent neurotoxin, which can undergo uptake in the nerve endings of motor neurones and subsequent retrograde transport into the central nervous system. The nontoxic C-fragment of tetanus toxin (TTC,451 amino acids), has been used to increase the neuronal uptake of the therapeutic protein SOD [57]. Following intravenous infusion, the recombinant hybrid protein reduced the occurrence of ischaemia-induced cerebral infarction in rats [58]. 

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Fig. 1. Outline of the strategy to construct GST-fused expression plasmids by the in vitro recombination-assisted method. Am, Gm, and Cm are abbreviations for ampicillin-, gentamicin-, and chloramphenicol-resistance, respectively. The figure also indicates the ccdB gene encoding a toxin targeting the co//essential DNA gyrase and the phage X recombination sites (attB, attP, attL, and attR).

Olsnes, S. and Pihl, A. (1986) Construction and properties of chimeric toxins target specific cytotoxic agents, in Domer, F. and Drews, J. (Eds.) Pharmacology of Bacterial Toxins, 1st ed, Oxford, England, New York, NY, Pergamon Press. 

A variant of the original immunotoxin approach is the so-called immunocytokines. In these constructs the antibody targeting moiety is maintained, but the toxin as the effector molecule is replaced by a cytokine. In contrast to toxins, cytokines are often proteins endogenously produced in man. If both the antibody and cytokine are of human origin, then no foreign proteins are introduced which could provoke an antibody response from the host immune system when the drug targeting preparation is clinically applied. 

Phage display is the best system for random peptide library construction used for affinity ligand selection. Several examples are reported in Table 16.1. Libraries of 6- to 15-mers are usually used, and peptides are isolated to a variety of targets including receptors, enzymes, antibodies, DNA, toxins, lectines, and even organs. 

Fully human antibodies can be constructed from phage libraries. In addition to the diversity of engineered antibodies, other molecules can be attached to the antibody, such as enzymes, toxins, viruses, radionuclides, and biosensors for targeting, imaging, or diagnosing. These are commonly referred to as conjugated" monoclonal antibodies. 

Monoclonal antibodies can vary tremendously in terms of isotype, construction (animal derived, chimeric, humanized, bound to toxin), ability to activate complement, binding avidity, target specificity, and whether it binds and blocks or binds and activates the receptor. Monoclonal antibodies may be directed toward soluble or membrane bound receptors or receptor ligands, tumor antigens, growth factor or their receptors. Therefore toxicity and side effects are equally variable [68]. 

Compound 117 is also a key intermediate in the synthesis of diplodiatoxin, a toxin produced by Diplodia maydis on maize and responsible for cattle disease [99]. The two chiral centers present in 117 are transferred in the target compound and induce all other chiralities of this bicyclic strucmre constructed via an intramolecular Diels-Alder reaction (Scheme 11.29). 

There is now a substantial literature on the synthesis and testing of chimeric molecules. These substances are each composed of at least two functional domains, such as a tissue-targeting domain and a pharmacologically active domain. One common strategy in constructing chimeric toxins is to isolate the binding domain of one toxin (e.g., diphtheria toxin) and attach this to the poisoning domain of another (e.g., ricin). This chimeric molecule attaches only to cells that have the diphtheria toxin receptor, and it expresses only the intracellular effects of ricin. 

Fusion proteins have been constructed from peptide epitopes from influenza A antigens and the binding and translocation domains of Pseudomonas exotoxin A (Donelly ef al., 1993). When target cells were incubated with these fusion proteins, and subsequently exposed to cytotoxic T lymphocytes (CTLs) specific for the relevant epitopes, a CTL mediate lysis of the target cells was observed. These experiments suggest that the translocation machinery supplied by protein toxins may be useful tools for bringing peptides into cells for presentation via the major histocompatibility class I (MHC I) system. It should be noted that no direct evidence was provided that the translocation occurred by the toxin pathway, and it cannot be excluded that the toxin was only instrumental in accumulating the peptide on the surface of the cells and in the endocytic pathway. 

Viruses may be constructed that express the HIV-receptor CD4 and coreceptor on the viral surface, either as part of the virus itself, or as the result of viral pseudotyping (55,56). These molecules then define the target-cell specificity of the virions, and they bind to and infect target cells expressing the HIV envelope on the cell surface (i.e., HIV-infected cells). If the virus itself is cytopathic, then it may kill the target cell (55). Alternatively, these viruses may be used to package nucleic acids for gene therapy, toxins, or other anti-HIV materials. 

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