The cutting edge: apoptosis and therapeutic opportunity - Artificial Cells, Cell Engineering and Therapy - page 122

of free radicals as cells take up high amounts of oxygen at this stage. As a result,
current research involving quenching of free radicals has become an important
area of study in order to prevent the damaging side effects of strokes. One
pharmacological area of study involves the prevention of free radical production
through cyclo-oxygenase 2 (COX-2) inhibitors such as nimesulide and NS-398.
Candelario-Jalil et al. (2004) have reported on the neuroprotective potential of
these drugs in ischemic conditions of rat brains, showing both histological and
functional recovery even after administering the drugs 24 h after stroke onset.
These results are quite exciting since there are no current treatments which are
able to treat the later phase of this condition. It is suggested that COX-2
inhibition may delay the progression of the ischemic legion, limiting the size of
the penumbra region although the exact mechanism of COX-2 activation in
cases of ischemia in humans is still unclear (Candelario-Jalil et al., 2004).
Other antioxidant therapies involve searching out and quenching free radicals
released in ischemic areas. One such compound is the glutathione precursor N-
acetylcysteine (NAC) which, as reported by Khan et al. (2004), is able to limit
infarct size in rat stroke models and improve neurologic scores while limiting
apoptotic cell death and inflammation. Such optimistic results were observed
even 6 h after the onset of stroke and because of the previously demonstrated
safety and efficacy of NAC in other neurodegenerative diseases, this treatment
offers strong potential for preventing the effects of ischemic stroke.
Research in our laboratory focuses on preventing apoptosis in postmitotic
cells such as neurons by limiting ROS production, which results from mito-
chondrial permealization by the Bax protein. Because of the central role which
Bax plays in inducing apoptosis we are currently attempting to block the pro-
apoptotic action of this protein through the use of specific anti-Bax llama single
domain antibodies (sdAb).
Unlike conventional human antibodies which contain several components
(variable light (VL) and heavy (VH) chains and constant light and heavy chains)
llama and camelid antibodies do not contain any of the light chains, significantly
reducing their overall size. Typically, the smallest functional units of human
antibodies (the antigen binding site) are the fragments Fvs (VH and VL) or
scFvs (VH and VL connected by a peptide linker) (Fig. 5.4(a)). In contrast, the
antigen-binding sites of the llama (and camelid) species comprise only the VH
region (Fig. 5.4(b)). As a result these smaller fragments, termed single domain
antibodies (sdAbs), which contain the specificity of the parent antibody, are
significantly smaller and offer greater stability when working intra-cellularly
(Gueorguieva et al., 2006).
A sdAb to any target antigen can be isolated from a naive llama phage display
library, a process known as panning. Using Bax as an antigen of interest, in our
panning assay results we were able to identify of six unique anti-Bax sdAbs,
with which we have been able to successfully quench the activity of this pro-
apoptotic protein, both in vitro and in vivo. Specifically, we have been able to