Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

17.3
The Development of Recombinant DNA-based Immunotoxins: Design of Recombinant
Immunotoxins
17.3.1
The Toxin Moiety
17.3 The Development of Recombinant DNA-based Immunotoxins
The toxins that are most commonly used to make immunotoxins are ricin, DT and
PE. These toxins belong to a group of polypeptide enzymes that catalytically inactivate
protein synthesis leading to cell death. Some of these toxins have been shown to
induce apoptosis [24, 25].
The genes for these toxins have been cloned and expressed in Escherichia coli, and
the crystal structures of all three proteins have been solved [50, 55]. This information,
in combination with mutational studies, has elucidated which toxin subunits
are involved in their biological activity and, most importantly, the different steps of
the cytocidal process. DT, PE, ricin and their derivatives have all been successfully
used to prepare immunotoxin conjugates [3, 26], but only PE- and DT-containing fusion
proteins generate active recombinant immunotoxins [1, 27]. This is because the
toxic moiety must be separated from the binding moiety after internalization [28,
29]. PE and DT fusion proteins generate their free toxic moieties by proteolytic processing.
Ricin does not possess such a proteolytic processing site and therefore cannot
be attached to the targeting moiety with a peptide bond without losing cytotoxic
activity. Recently, proteolytic processing sites were introduced into ricin by recombinant
DNA techniques to try to overcome this problem [30].
17.3.1.1 Plant toxins
Ricin, or the ricin A chain fragment, has been a commonly used toxin for conjugation
to antibodies. Ricin is synthesized as single polypeptide chains and processed posttranslationally
into two subunits. A and B linked though a disulfide bond. Ricin is a 65kDa
glycoprotein purified from the seeds of the castor bean (Ricinus communis). It is
composed of an A subunit which kills cells by catalytically inactivating ribosomes. The
A subunit is linked by a disulfide bond to a B subunit which is responsible for cell binding.
The B chain is a galactose-specific lectin that binds to galactose residues present on
cell surface glycoproteins and glycolipids [31]. Once the B subunit of ricin binds to the
cell membrane, the protein enters the cell through coated pits and endocytic vesicles.
The A and B subunits of ricin are separated by a process involving disulfide bond reduction.
The A subunit of ricin translocates across an intracellular membrane to the cell cytosol,
probably with the assistance of the B subunit. In the cytosol, it arrests protein
synthesis by enzymatically inactivating the 28S subunit of eukaryotic ribosomes [32,
33]. Because native ricin is highly toxic and lacks specificity, several modified forms of ricin
have been developed to prepare immunotoxins that are better tolerated by patients.
To decrease the non-specific binding of whole ricin, the A chain alone has been
coupled to antibodies. The A chain is obtained by reducing the disulfide bond that
links it to the B chain. Immunotoxins composed of the ricin A chain coupled to wellinternalized
antibodies can be highly cytotoxic [34]. In the absence of the B chain
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