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

352 17 Immunotoxins and Recombinant Immunotoxins in Cancer Therapy
(the binding subunit), however, immunotoxins made with poorly internalized antibodies
are not cytotoxic.
Immunotoxins containing the ricin A chain are rapidly cleared from the circulation
by the liver by the binding of mannose and fucose residues of the A chain to receptors
present on the reticuloendothelial system and hepatocytes. To circumvent this problem,
these carbohydrate residues were chemically modified [35], resulting in a deglycosylated
A chain (dgA) molecule. In preclinical studies, dgA-containing immunotoxins
were found to have longer half-lives in the circulation and better antitumor efficacy
in vivo [36]. Also, recombinant A chain produced in E. coli can be used in place
of dgA because it is devoid of carbohydrate and is not rapidly cleared by the liver.
Another strategy to decrease the non-specific toxicity of native ricin is to block the galactose-binding
sites of the B chain by cross-linking with glycopeptide [37]or to use
short cross-linkers to connect the antibody to the toxin so that the galactose-binding
site is sterically blocked by the antibody [38]. Blocked ricin retains a low affinity for
galactose-binding sites, which enhances internalization and cytotoxicity of an antibody
that binds to a poorly internalized antigen.
Other plant toxins commonly used for clinical immunotoxin construction are saporin
and pokeweed antiviral protein, which are single polypeptide chains that inactivate
ribosomes in a similar fashion to ricin. Because these toxins lack the binding
chain (B chain), they are relatively non-toxic to cells and are used for immunotoxin
production [39±42].
17.3.1.2 Bacterial toxins: DTand DT derivatives
DT is a 58-kDa protein, secreted by pathogenic Corynebacterium diphtheria, which
contains a lysogenic b phage [43]. DT ADP-ribosylates eukaryotic elongation factor
(EF)-2 at a ªdiphthamideº residue located at His415, using NAD + as a cofactor [44].
This modification arrests protein synthesis and subsequently leads to cell death [45].
Only a few, and perhaps only one, DT molecules need to reach the cytosol in order to
kill a cell. When DT is isolated from the culture medium of C. diphtheria it is composed
of an N-terminal 21-kDa A subunit and a C-terminal 37-kDa B subunit held
together by a disulfide bond. DT is the expression product of a single gene [43],
which when secreted into the medium is processed into two fragments by extracellular
proteases. When DT is produced as a recombinant single-chain protein in E. coli,
it is not cleaved by the bacteria, but is instead cleaved by a protease in the target cells
[46]. The A domain of DT contains its enzymatic activity. The N-terminus of the
B subunit of DT (or the region between A and B in single-chain DT) mediates translocation
of the A subunit into the cytoplasm. The B domain, especially its C-terminus,
is responsible for the binding of DT to target cells. Deletions or mutations in
this part of the molecule abolish or greatly diminish the binding and toxicity of DT
[47±49]. DT enters cells via coated pits and is proteolytically cleaved within the endocytic
compartment, if it is not already in the two-chain form, and reduced. It also undergoes
a conformational change at the acidic pH present in endosomes, which
probably assists translocation of the A chain into the cytosol, perhaps via a pore-like
structure mediated by the B chain [50±52]. Derivatives of DT that are used to make
immunotoxins have the C-terminus altered by mutations or partially deleted