Drug Targeting Organ-Specific Strategies

212 8 Strategies for Specific Drug Targeting to Tumour Cells
8.5.1.3.3 Phage Display Library
As an alternative to immunization and hybridoma construction procedures, it is possible now
to construct large (synthetic) human antibody gene repertoires entirely in vitro (see also
Chapter 10). This procedure can generate a huge library of recombinant filamentous bacteriophages
that express hundreds of millions of different human scFvs on their tips fused to
the phage minor coat protein III [38,54]. The scFvs displayed by these phages can show antigen
binding activity and phages with the desired binding characteristics and specificity can be
selected by panning on the antigen.The selected phage (including the genetic information of
the displayed scFv inside) can be rescued and grown after each round of panning after which
the ‘enriched’ phage library is again subjected to selection so that even rare phages (< 1/10 8 )
can be isolated. Using this strategy human antibodies and/or their fragments have been isolated
with specificities against foreign and self antigens [55].
8.5.1.3.4 Transgenic ‘Human’ Animals
A further advance in antibody technology is the development of transgenic mouse ‘human’
strains. XenoMouse animals have been engineered in such a way that they now produce exclusively
human antibodies rather than murine antibodies when immunized. The use of
XenoMouse animals to produce MAbs avoids the need for any engineering of the antibody
genes, since the products are already 100% human protein. XenoMouse animals are fully
compatible with standard hybridoma technology and can be readily adopted by laboratories
experienced in monoclonal antibody production [56].
8.5.1.3.5 Considerations for Recombinant Antibody Production
When the biodistribution of scFv (Figure 8.1c), Fab′, (Fab) 2′ (Figure 8.1b), and IgG (Figure
8.1a) were compared, most of the intact IgG delivered to tumours was concentrated in
the region immediately adjacent to the blood vessels.The Fab′ and F(ab) 2′ fragments demonstrated
intermediate degrees of tumour penetration, while the scFv was distributed more
evenly throughout the tumour [57].
In mice with human breast carcinoma xenografts, a humanized IgG anti-HER-2 MAb
eradicated well-established tumours [58]. In addition, a humanized version of an IgG anti-
CD33 MAb (HuM 195) mediated ADCC in vitro [59] and had an 8.6-fold higher avidity than
the parent murine Mab. Recombinant antibody fragments may have valuable properties as
discussed above, but their biophysical behaviour, production yield and low thermostability
leaves much to be desired and thereby limits their usefulness for in-vivo applications so far
[60]. One possibility to improve these characteristics of scFv fragments with suboptimal stability
and/or folding yield, is the grafting of their CDRs onto the framework of a different,
more stable scFv [61,62].
Another valuable tool for the development of scFv-based therapeutics consists of a versatile
expression vector for the rapid construction and evaluation of scFv-based fusion proteins and
bispecific scFv [63]. The vector was used for grafting a number of biological effector princi-