Pseudomonas exotoxin

Like diphtheria toxin. Pseudomonas aeruginosa exotoxin A requires low pH to act (FitzGerald ef al., 1980). In spite of this, it has not been possible to induce translocation of Pseudomonas toxin across the surface membrane by exposure to low pH. It appears that the toxin must be transported beyond the endosomes, possibly to the trans-Golgi network or even to the endoplasmic reticulum to find conditions required for translocation (Chaudhary et al., 1990). In fact domain III ends with an amino acid sequence that (after removal of a terminal 

The toxins mentioned above have the disadvantage as targeting vehicles that they can only be used on toxin-resistant cells as they will kill normal cells. It is possible to make mutant toxins with highly reduced toxicity, but so far such mutants have proved to be less active in translocation than the wild-type toxins. In future work it may therefore be necessary to look for other toxins that do not kill the cells. 

Cholera toxin and E. call heat-labile toxin ADP-ribosylate the a-subunit of trimeric G-proteins (Gj) so that they are unable to hydrolyze GTP (Casey and Gilman, 1988 Neer and Claphan, 1988). As a result, the adenylate cyclase stays in a persistently active state. The toxin is taken up from the apical pole of the enterocytes, whereas the adenylate cyclase is located at the basolateral side. Since membrane glycolipids such as the toxin receptor (ganglioside GMi) tend not to cross tight junctions, it is unlikely that the toxin reaches the basolateral side by lateral diffusion. Most likely it enters the cytosol and diffuses to the target which is also located at the cytosolic side of the membrane. 

Pertussis toxin ADP-ribosylates a cysteine residue of a-subunits of G-proteins involved in the regulation of adenylate cyclase (Gi, Go) and of transducin, with the result that the G-protein is unable to interact with the receptors and the signal is therefore not transmitted. This toxin consists of 5 subunits that are linked by noncovalent bonds to the enzymatically active Si-subunit (Casey and Gilman, 1988 Neer and Claphan, 1988). The Spsubunit is activated by reduction of an internal disulfide bond. 

A number of toxins that act on intracellular targets have a somewhat different organization, being reconstituted at the cell surface from two different proteins (bipartite toxins). This type of organization was first discovered in the toxin from Bacillus anthracis (Leppla, 1982). This toxin has a three-component structure, consisting of two subunits 

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Diphtheria toxin, Pseudomonas exotoxin A Elongation factor 2 ADP-ribosylation Inhibition of protein synthesis (diphtheria, Pseudomonas infection)... 

Bjorn, M.J., Croetsema, G., and Scalapino, L. (1986) Antibody-Pseudomonas exotoxin A conjugates cytotoxic to human breast cancer cells in vitro. Cancer Res. 46, 3262. 

C. botulinum toxins belong to the AB group of toxins, which also includes diphtheria toxin, pseudomonas exotoxin A, anthrax toxin, Shiga(like) toxin, cholera toxin, pertussis toxin, and plant toxins, e.g., ricin. Moiety A has an enzymatic activity and usually modified cellular-target entering cytosol. Moiety B consists of one or more components and binds the toxin to surface receptors, and is responsible for translocation of the A component into cells. AB toxins are produced in a non-active form and are activated by a split between two cysteine residues within a region (Falnes and Sandvig, 2000). 

Pseudomonas exotoxin A Bacterium One ADP ribosylation of elongation factor-2 ... 

IL-4 PE38[KDEL] Interleukin-4 Binds to IL-4 receptor overexpressed on myeloma and glioma cells PE38[KDEL] is a binding-deficient variant of Pseudomonas exotoxin (PE) Cytotoxicity Under investigation as a potential intratnmor treatment for patients with glioma [104]... 

Puri, R.K., Development of a recombinant interleukin-4-Pseudomonas exotoxin for therapy of glioblastoma. Toxicol Pathol, 1999. 27(1) 53-7. 

Rozemuller, H., W.I Rombouts, IP. Touw, D.I FitzGerald, R.I Kreitman, I. Pastan, A. Hagenbeek, and A.C. Martens, Treatment of acute myelocytic leukemia with interleukin-6 Pseudomonas exotoxin fusion protein in a rat leukemia model. Leukemia, 1996.10(11) 1796-803. 

Pai, L.H., R. Wittes, A. Setser, M.C. Willingham, and I. Pastan. 1996.Treatment of advanced solid tumors with immunotoxin LMB-1 an antibody linked to Pseudomonas exotoxin. Nat. Med. 2 350-353. 

As previously discussed, CD25 is a component of the IL-2 receptor on activated T cells. A recombinant anti-CD25 immunotoxin, LMB-2, contains fused Fv and Pseudomonas exotoxin A sequences and is currently in clinical trials (186). 

This chapter will outline the development of immunotoxins and describe the preclinical development required for advancing VB4-845, an anti-EpCAM targeting scFv linked to a truncated form of Pseudomonas exotoxin A(252-608), into the clinic. 

FitzGerald D, Pastan I. Pseudomonas exotoxin recombinant conjugates as therapeutic agents. Biochem Soc Transact 1992 20 731-4. 

FitzGerald DJ, Waldmann TA, Willingham MC, Pastan I. Pseudomonas exotoxin-anti-TAC. cell-specific immunotoxin active against cells expressing the human T cell growth factor receptor. J Clin Invest 1984 74 966-71. 

Kondo K, FitzGerald DJ, Chaudhary V. Activity of immunotoxins constructed with modified pseudomonas exotoxin a lacking the cell recognition domain. / Biol Chem 1988 263 9470-5. 

Pastan I. Immunotoxins containing pseudomonas exotoxin A a short history. Cancer Immunol Immunother 2003 52 338-41. 

Pastan I, FitzGerald D. Pseudomonas exotoxin chimeric toxins. / Biol Chem 1989 264 15157-60. 

Siegall CB, Chaudhary V. Functional analysis of donaims II, lb and III of pseudomonas exotoxin. J Biol Chem 1989 264 14256-61. 

Siegall CB, Liggett D. Characterization of vascular leak syndrome induced by the toxin component of pseudomonas exotoxin-based immunotoxin and its protential inhibition with nonsteroidal anti-inflammatory drugs. Clin Cancer Res 1997 3 339-45. 

An example of an expanded bed process for recovery of a Pseudomonas exotoxin from an E. coli system has been recently reported in pilot plant scale.28 A single-step recovery of a secreted recombinant protein has been carried out in expanded-bed mode directly from the fermentation broth without prior cell removal. The fusion protein was designed to have relatively low isoelectric point to enable anionic exchange adsorption at pH 5.5 where most of the E. coli host proteins are not adsorbed. The gene product was secreted to the culture medium of E. coli in high yield and the recovery of the protein was 90% in one step.29... 

Fig. 3.3 shows the principle behind the design of immunotoxins. A number of protein toxins of bacterial and plant origin are useful for the production of immunotoxins. These include the diphtheria toxin and pseudomonas exotoxin from bacteria, and ricin, arbin, pokeweed antiviral proteins, saporin, and gelonin from plants (Pastan et al, 1986 Pastan and FitzGerald, 1991). All of these toxins kill cells by entering the cells, and enzymatically inactivating the translational machinery of the cells. Some, such as diphtheria toxin, arbin, and ricin, are composed of two protein chains, A and B. The B chains bind to the cell-surface... 

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