Development of an immunotoxin incorporating the Australian jumper ant toxin, pilosulin 1
- Publication Type:
- Thesis
- Issue Date:
- 2007
Open Access
Copyright Clearance Process
- Recently Added
- In Progress
- Open Access
This item is open access.
Immunotoxins are therapeutic agents that directly target toxins to specific cells and are
generally comprised of an antibody or antibody fragment (Fab or scFv) linked to a toxic
moiety. The assessment of a number of immunotoxins in recent Phase I and 11 clinical
trials has been very promising, particularly for the treatment of haematological
malignancies. Since the majority of these incorporate large, potent bacterial or plant
toxins, their therapeutic potential is limited by dose-limiting non-specific toxicity,
immunogenicity and the need to be endocytosed. An alternative approach is to
incorporate cytolytic toxins such as melittin and pilosulin that are smaller, less toxic
molecules that act at the cell membrane. Both melittin- and pilosulin-based
immunotoxins (mel-IT and pil-IT) have been developed by our research group. These
cytolytic immunotoxins, which incorporate a scFv moiety specific for the human kappa
myeloma antigen (KMA) expressed on human kappa myeloma cells and the human
lymphoblastoid cell line, HMy2, display specific cytotoxic activity at micromolar
concentrations against the target cell line. In contrast, immunotoxins in clinical and late
stage pre-clinical studies are active at picomolar concentrations and thus it was deemed
necessary to enhance the specific activity of mel-IT and pil-IT to ensure they could be
effective at relevant clinical doses.
The pil-IT displayed greater cytotoxic potential as peptide studies indicated that
pilosulin was four times more potent than melittin against white blood cells (WBCs)
and additionally, the pil-IT was shown to be twice as toxic as the mel-IT on a molar
basis. In order to identify the regions of pilosulin essential for cytolytic activity and
thus develop a smaller imrnunotoxin, two recombinant constructs were generated that
encoded truncated toxin domains; P₁-₂₂F (incorporating the N-terminal helix of
pilosulin, amino acid residues 1 to 22) and P₂₃-₅₆F (incorporating the C-terminal helix,
residues 23 to 56). Unexpectedly, both recombinant constructs displayed reduced
cytolytic activity compared to the parent construct (pil-IT/ Pt₁-₅₆F), due to reduced
specific binding of the immunotoxins to the target cells, presumably as a result of
incorrect tertiary folding of the expressed proteins. In a further attempt to increase the
specific activity of the pilosulin-based immunotoxin, two additional constructs were
generated; P₁₂₁F which had a longer linker arm between the full-length pilosulin peptide
and the antigen-specific scFv moiety to enhance steric access of the toxin to the target
cell membrane, and P A₃₃K₄₁F which contained the C-terminal helical region of pilosulin
in an enhanced helical conformation to aid membrane interaction and penetration.
While preliminary studies indicated that both constructs had cytotoxicity comparable
the parent, neither exhibited enhanced cytolytic activity.
Contaminant proteins were observed to be co-purifying with the immunotoxins raising a
question as to whether these contaminants may have had the potential to affect the
cytolytic activity of pil-IT. The most significant contaminant was identified as apoA-1,
a 27-kDa hydrophobic serum protein that had previously been shown to inhibit the
activity of cytolytic peptides and also to stabilise damaged membranes. As the FBS
used to supplement the expression culture medium was identified as the source of apoA-
1, the pil-IT immunotoxin was expressed in serum-free medium. The recombinant
protein expressed under these conditions was extremely susceptible to proteolysis in the
cell culture medium and attempts to block this proteolysis by supplementing the
cultures with BSA or a2-macroglubulin were ineffective. However, supplementing the
serum-free expression cultures with E-64, a specific cysteine protease inhibitor, blocked
the majority of the proteolytic degradation of pil-IT and allowed affinity purification of
a very pure immunotoxin preparation.
Unexpectedly, pil-IT expressed in this manner displayed significant non-specific
toxicity compared to previous immunotoxin batches. It is possible that the presence of
E-64 in the culture supernatant affected the tertiary fold of the immunotoxin so that it
acted independently of the antigen binding specificity encoded by the scFv moiety, or
that pil-IT in a very pure form is very toxic and non-specific (i.e. a true result of an
'apoA-1 free'-immunotoxin preparation). Another issue requiring consideration was
that the insect cell line used for expression of the non-specific toxic batches of
immunotoxin in serum-free medium (High Five cells) was different to that used to
express batches of specifically cytotoxic immunotoxin in serum-containing medium
(Sf21 insect cells).
To address this question, the pil-IT was expressed in High Five insect cells in the
presence of FBS and then affinity purified with a Tween-20 wash step to dissociate the
apoA-1 from the immunotoxin. While this produced a pure preparation, non-specific
cytolytic activity was again observed, although to a lesser degree than that observed for
the batches expressed with E-64 supplementation. Tween-20 was found to contribute to
some of this non-specific activity but was not the sole factor, as pil-IT expressed in
High Five cells in the presence of FBS and purified without Tween also exhibited nonspecific
cytotoxicity. Thus it was likely to be a result of either (i) expressing the
immunotoxin in High Five insect cells which, in contrast to Sf21 cells, may generate a
tertiary fold in the immunotoxin that allows it to act non-specifically, or (ii) the absence
(or low levels) of apoA-1, which when present in the immunotoxin preparation, may
have inhibited its non-specific cytolytic activity and/or repaired toxin-induced cell
membrane damage, with the strength of this effect varying for different cell membranes.
Please use this identifier to cite or link to this item: