Immunotherapy for Infectious Diseases

84 Kunert and Katinger
are expressed as soluble or fusion proteins, thus allowing linkage of genotypic and phenotypic
properties of one antibody on each single phage particle. Phages are easy to
handle and can be propagated in E. coli. A single phage library consists of at least 10 8
clones, each expressing one antibody attached to solid surfaces. Phages presenting antibody
fragments of particular specificity can be selected from the library by a panning
technique on surfaces on which the antigen of interest is immobilized. Afterward, lowaffinity
antibodies from such libraries can be randomly mutated by PCR under imperfect
conditions yielding higher affinity variants. This procedure is called in vitro
affinity maturation (105). Several high-affinity antibodies, some of potential clinical
interest, have been developed from such libraries (106).
Host Cell Lines for Monoclonal Antibody Production
The choice of the proper expression system for antibody production depends very
much on the intended use of the antibody. E. coli is an option for most of the nonglycosylated
forms of antibody fragments. Mainly single-chain Fv fragments or their
fusion proteins are expressed in E. coli, intracellularly or in the periplasmic space.
High-level expression in E. coli tends to generate inclusion bodies in which the antibody
fragments are accumulated at rather high purity but in a denatured form. Refolding
to activate the protein is required. Different factors influence the protein folding,
stability, and export of the antibodies (107). E. coli can be grown in very large volumes
and at high cell densities in simple defined media and with tightly controlled transcriptional
regulation.
Yeasts such as Saccharomyces cerevisiae and Pichia pastoris have been tested as
hosts, but little real progress has been described as yet (108,109).
Attempts have been made to develop alternative production systems. The use of
insect cells as a production vehicle is based on infection with recombinant baculoviruses;
expression titers of around 30 mg/L are given (110). The glycosylation pattern
of such cells differs from that of mammalian cells. They process the so-called
high-mannose type of glycosylation (111–113).
The milk of transgenic animals has been reported to yield as much as 4 g IgG/L. The
cloning of transgenic animals will probably open a new era of recombinant protein production.
However, aspects of product quality assessment are still a serious concern.
Another novel approach for the production of antibodies is the use of transgenic
plants as a production system (114). Transgenic tobacco plants (Nicotiana tabacum)
were first used to show stable accumulation of recombinant antibody in the seed (115).
Antibody production in a transgenic crop bears a potential of nearly unlimited mass
production at low cost (116). The expression and accumulation of up to 280 mg of
secretory IgA antibodies per corn cob have been reported. Furthermore, corn is provided
with the repertoire of housekeeping genes necessary to properly process complicated
protein structures such as soluble IgA (sIgA) into their functional form.
Up to now antibodies for therapeutic application have been produced in mammalian
cell culture. Generally, these are considered to confer proper posttranslational processing
in order to achieve optimal induction of antibody effector functions (117), pharmacokinetics,
and biodistribution in patients. A variety of different mammalian cell
lines are used, most commonly hybridomas. Hybridomas are easily grown in suspen-