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

364 17 Immunotoxins and Recombinant Immunotoxins in Cancer Therapy
fic binding sites. Such time-consuming procedures were rapidly replaced by the
third seminal development: the provision of a link between the phenotype and the
genotype using phages. In 1990, McCafferty et al. showed that antibody fragments
could be displayed on the surface of filamentous phage particles by fusion of the
antibody variable genes to one of the phage coat proteins [149]. Multiple rounds of affinity
selection could subsequently enrich antigen-specific phage antibodies, because
the phage particle carries the gene encoding the displayed antibody. This was originally
reported for scFv fragments [149], and later for Fab fragments [150±152] and
other antibody derivatives such as diabodies [153], as well as extended to various display
systems. With these advances in place, it became possible to make phage antibody
libraries by PCR cloning of large collections of variable region genes expressing
each of the binding sites on the surface of a different phage particle and harvesting
the antigen-specific binding sites by in vitro selection of the phage mixture on a chosen
antigen. In the early 1990s, Clackson et al. showed for the first time that phagedisplay
technology could be used to select antigen-specific antibodies from libraries
made from the spleen B cells of immunized mice [154], thereby bypassing the requirement
to immortalize the antigen-specific B cells, as in the hybridoma technology.
Similarly, libraries were made from human B cells taken from animals or individuals
immunized with antigen [155], exposed to infectious agents [156], with autoimmune
diseases [157]or with cancer [158]. Thus, phage-display technology in the early
1990s had already shown the potential to replace hybridoma technology by rescuing
V genes from immune B cells. Further advances were reported in the mid-1990s that
would bypass the use of immunization and animals altogether. First, it was shown
that antibodies against many different antigens could be selected from non-immune
libraries, made from the naive light chain and heavy chain IgM V gene pools of B
cells of a non-immunized, healthy individual [159]. Second, libraries of synthetic
antibody genes, with variable genes not harvested from immune sources but consisting
of germline segments artificially provided with diversity by oligonucleotide cloning
[150, 160], were shown to behave in a similar way to naive antibody libraries. It
thus became possible to use primary antibody libraries, with huge collections of
binding sites with different specificities, to select in vitro binding sites against most
antigens, including non-immunogenic molecules, toxic substances and targets conserved
between species [161].
Since these key discoveries, there have been numerous reports on applications of
phage antibody libraries [162, 163], ranging from basic research to drug development.
In addition, many novel, related molecular display methods for antibodies have been
described, including display systems on ribosomes [164], bacteria [165] and yeast cells
[166]. These technologies follow similar concepts for in vitro selection and improvement
of binding sites. Novel selection strategies of phage-display libraries and other
molecular display systems are being developed for the identification of novel antigenbinding
fragments. These include selection for binding using purified or non-purified
antigen, selection for function, selection based on display capability and phage infectivity,
subtractive selection procedures, and also using high-throughput selection and
screening [163, 167]. The use of phage-display systems will revolutionize the field of
targeted drug therapy in general and the recombinant immunotoxin field in particu-