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

17.7 Isolation of New and Improved Antibody Fragments as Targeting Moieties
17.7
Isolation of New and Improved Antibody Fragments as Targeting Moieties:
Display Technologies for the Improvement of Immunotoxin Activity
The generation of mAbs made by immunizing animals and allowing in vivo processes,
such as immune tolerance and somatic hypermutation, to shape the antigencombining
site is a key issue for the generation of specific antibodies. The unique
features required from these molecules were already described in the Introduction
to this chapter. These antibodies created in vivo can be used for many research and
diagnostic applications. Mouse mAbs might be made less immunogenic and more
effective for human therapy by reformatting the binding site into chimeric or complementary-determining
region (CDR)-grafted antibodies [73]. The advances in recombinant
DNA technology and antibody engineering have also lead to the ability to
manipulate the size of the antigen-binding domain as described in this chapter. The
two variable domains of the binding site can be cloned and arranged into a large array
of possible molecular formats and sizes, and expressed in a variety of hosts, ranging
from bacteria, lower eukaryotes such as yeast and fungi, to the higher eukaryotes,
including mammalian cells, transgenic animals and plants [140, 141]. Extraordinary
progress in engineering and selecting small antibody fragments for immunotherapeutic
approaches has been made over the past decade, when molecular display
technologies have been developed that allow us to create very large repertoires
of mouse or fully human antibodies that are displayed on filamentous phage or
other molecular display systems. These technologies are now revolutionizing the
way in which we can build high-affinity binding sites from scratch, from any species
(including humans) and use them for clinical applications such as the targeting of a
drug or toxin to cancer cells as in recombinant Fv±immunotoxins.
The concept of molecular display technology relays on the physical linkage between
the genotype (the antibody variable region genes) and the phenotype (antigen-binding
capability) to allow simultaneous selection of the genes that encode a protein
with the desired binding function. This concept can be viewed as an in vitro mimicking
system for the natural antibody response function of the immune system. This
concept was first applied by George Smith in 1985 to small peptides [142]. The display
of functional antibody repertoires on phages required several additional discoveries.
First, a procedure for accessing large collections of antibody variable domains
was needed; this was first described in 1989, when partially degenerate oligonucleotides
priming to the 5' and 3' end of variable region genes and the polymerase chain
reaction (PCR) were used to amplify hybridoma [143, 144]or large collections of variable
genes [145, 146]. Second, as whole antibodies cannot yet be functionally expressed
in bacteria, a crucial discovery was that antibody fragments (Fab or scFv)
were functionally expressed in E. coli when they were secreted into the periplasm of
the bacteria, which simulated the naturally oxidizing environment of the endoplasmic
reticulum [147, 148]. By providing restriction sites in the oligonucleotides used
for PCR amplification, antibody libraries could thus be cloned for expression in E.
coli. Initially, such antibody libraries were expressed from phage l vectors [146]; a
plaque-screening assay with labeled antigen was then used to identify antigen-speci-
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