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Encyclopedia of Physical Science and Technology EN007K-319 July 2, 2001 17:53

438 Hybridomas, Genetic Engineering of

the genes of a multimeric protein structure in a single antibody against Streptococcus mutans to prevent binding
genetically stable hybrid plant. In one example of the of the bacteria to the surface of teeth and thus reducing
use of this technique, the assembly of secretory IgA was tooth decay.
achieved. This is a multimeric immunoglobulin which Plant cells are eukaryotes and therefore capable of post-
consists of two Ig units dimerized by a small polypeptide translational modification of proteins including N-linked
chain (J). Four transgenic plants were produced initially glycosylation. However, although the plant glycan struc-
for the expression of a light chain (kappa), a heavy chain, tures have not been analyzed in detail it is likely that these
a J chain, and a secretory component. A series of sex- structures are significantly different from those in mam-
ual crosses between these plants enabled the generation malian systems. For example, the commonly found mam-
of a hybrid in which all four components were expressed malian terminal sialic acid (N-acetyl neuraminic acid)
simultaneously. In this hybrid the four recombinant pro- residueisastructurenotfoundinplants.Alsothealpha-1,3
teins were assembled into the fully functional secretory core fucose structure appears to be unique to plants and has
immunoglobulin. The antibody expressed in these plants been implicated in human allergies to pollen. The poten-
is secreted and accumulates in the apoplasm which is a tial presence of such unusual glycan structures in plant-
large aqueous space external to the cells. This is a sta- derived antibodies might not have an effect on antigen
ble environment unlikely to cause any proteolysis of the binding but for a therapeutic antibody they are likely to
accumulated protein. increase the chance of an adverse immunogenic reaction
An alternative method to the production of transgenic during human treatment. This could limit the use of plant-
plants is the infection of a wild-type plant with a suit- derived antibodies in certain applications particularly if
able recombinant virus vector. Using this method a mono- systemic long-term administration is required.
clonal antibody can be expressed in the leaves of a tobacco
plant (Nicotiana benthamiana) by infection with two vi-
ral vector constructs of tobacco mosaic virus (TMV). In XVIII. HUMANIZED ANTIBODIES
one example, these two constructs contained the genes FROM TRANSGENIC MICE
for the heavy and light chains of an antibody (CO17-1A)
against a colorectal cancer associated antigen. A func- Transgenic mice strains have been produced capable of
tional full-length antibody was detected in extracts of synthesizing human monoclonal antibodies. Xenomouse
the leaves of the plants infected with these recombinant strains have large portions of human variable region genes
viruses. The use of plant virus vectors may have several incorporated into the germ line via a yeast artificial chro-
advantages over the development of transgenic plants. The mosome (YAC). The megabase-sized YAC allows the
long generation time associated with plant transformation genes for human heavy and light chain immunoglobu-
is avoided. The process also avoids the time-consuming lin to be incorporated as transgenes into a mouse strain
process of crossing transgenic plants to produce hybrids deficient in the production of murine Ig. The large hu-
for the expression of proteins with multiple subunits. Dif- man variable region repertoire incorporated as transgenes
ferenthostplantscanbeinfectedbythesamevirusvectors, allows the mice to generate a diverse immune response
thus allowing screening for the maximum efficiency of comparable to that in humans. These human genes are
expression. also compatible with the mouse enzymes that allow class
One major advantage of monoclonal antibodies from switching from IgM to IgG. The immunoglobulin genera-
plants is the potential low cost of large-scale production. tion will also undergo somatic hypermutation and affinity
There are commercial companies (such as EPIcyte Phar- maturation, a natural process that enhances the affinity of
maceutical Inc) who are planning clinical trials for plant- the antibody for the antigen. Thus an antigen introduced
produced secretory antibodies for human therapy. These intoaXenomouseproducesahumanmonoclonalantibody
so-called “plantibodies” can be produced at an estimated with high specificity for its corresponding antigen. Such
cost of $0.01 to $0.1/mg as opposed to $1 to $5/mg for antibodies have already proved their potential in clinical
production from cell culture processing of animal-derived trials.
hybridomas. The cost of microbial fermentation is lower
than that of mammalian cell culture but bacteria lack the
abilityforefficientmultimericproteinassemblyandofany XIX. THE IMPORTANCE OF
post-translational modification. A further potential advan- GLYCOSYLATION TO
tageoftheplantibodiesisdeliverybyconsumptionofplant THERAPEUTIC ANTIBODIES
tissue and thus avoiding any need of purification. These
possibilities are particularly applicable in certain cases The glycosylation pattern of the immunoglobulin struc-
such as the previously shown ability of a plant-produced ture has particular relevance to the production and use of