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

358 17 Immunotoxins and Recombinant Immunotoxins in Cancer Therapy
fused to the C-terminus of DT [91, 92]. This corresponds to the inverse arrangement
of the functional modules of PE and DT (see Fig. 17.3). DT immunotoxins are active
only when the enzymatically active N-terminal domain is free to translocate into the
cytosol.
The expression vectors used for DT immunotoxins are very similar to those used
with PE with the exception that the DNA fragments encoding the binding moiety
are ligated to the 3'-end of the DT coding region. The cloning of the antibody variable
regions is performed using cloning techniques that are now well established
(Fig. 17.3) [73]. The plasmid vector for the expression of scFv±immunotoxins or the
components of dsFv±immunotoxins is a high-copy-number plasmid derived from
vectors made and described by Studier and Moffatt [93]. These contain the T7 promoter,
translation initiation signals and a transcription terminator, as well as an F+
phage replication origin to generate single-stranded DNA to be used for site-directed
mutagenesis.
When these plasmids are transformed into E. coli BL21/DE3 (which contain the T7
RNA polymerase gene under the control of the lacUV5 promoter) they generate large
amounts of recombinant protein upon IPTG induction. The recombinant scFv±immunotoxin
or the components of the dsFv±immunotoxin accumulate in insoluble
intracellular inclusion bodies. [dsFv±immunotoxins require two cultures, one expressing
the VH and one expressing the VL; the toxin moiety (PE38) can be fused to
either the VH or the VL.]The inclusion bodies are then isolated, purified, solubilized,
reduced and subsequently used in a refolding reaction that is controlled for oxidation
(redox shuffling). In the case of dsFv±immunotoxins, solubilized inclusion bodies of
VH and VL (with the toxin fused to either) are mixed in a 1:1 molar ratio into the refolding
solution. The formation of the interchain disulfide bond between the VH and
VL domains is promoted by inducing oxidation using excess oxidized glutathione or
by refolding at high pH. The immunotoxins are then purified from the refolding
mixtures by ion-exchange and size-exclusion chromatography. Approximately 20 mg
of clinical-grade active immunotoxin can be obtained from 1 l of a fermentor culture
induced with IPTG.
17.5
Preclinical Development of Recombinant Immunotoxins
A wide variety of recombinant immunotoxins have been made and tested against
cancer target cells. If found to be active and are considered to be tested in clinical
trials, they undergo several years of preclinical development to determine their efficacy
and toxicity in several in vitro and in vivo experimental models (Tab. 17.2).
The initial phase is the characterization of the biological activity of the immunotoxin
on cultured tumor cells. These assays include measurement of cell-free enzymatic
activity, i. e. ADP-ribosylation activity in the case of bacterial toxins, and the binding
affinity of the immunotoxin to the target antigen, which can be determined on purified
antigen, on cells by binding displacement assays or by surface plasmon resonance
assays. Cytotoxicity assays are performed on antigen-bearing cells and mea-