Immunotherapy for Infectious Diseases

86 Kunert and Katinger
missing metabolite or a related agent be added to the growth medium or that genes
capable of supplementing the metabolic deficit be introduced into the host. For example,
many mammalian cell lines are glutamine auxotrophs, i.e., they require glutamine
as an essential supplement in the growth medium.
The glutamine auxotrophic phenotype is also compensated for by transfection of the
glutamine synthetase (GS) gene together with the antibody genes. Growing the cells in
glutamine-free media thus ensures both the survival of the transfected host cell and stable
antibody expression. The copy number of the gene encoding GS synchronously
with the antibody genes can be further amplified by a gradually increasing addition of
analogs inhibiting the activity of GS. Those cells that acquire increasing copies of the
GS gene will survive and also coamplify the antibody genes. Thus high-expression cell
clones are selected. This amplification system was shown to work very efficiently with
the NSO cell line (124).
Another widely used marker system is the dihydrofolate reductase (DHFR) system.
DHFR converts folate to tetrahydrofolate, which is a critical metabolite in amino acid
and purine synthesis. CHO cells lacking endogenous DHFR have been generated (125)
by means of mutation and selection. CHO DHFR � cells require supplemention with
adenosine, desoxyadenosine, and thymidine in the growth medium. Transfection of an
exogenous DHFR gene together with the antibody genes and omitting said supplements
form the basis for selecting recombinant cells expressing DHFR and antibodies. A further
amplification of the respective gene copy numbers can be achieved by a selection
of surviving cells in the presence of methotrexate (MTX), which is an inhibitor of
DHFR. High-producing recombinant cell clones can be selected by a stepwise increase
of the MTX concentration. The DHFR marker/MTX selection system can also generally
be used as a dominant marker. In this case an exogenous mutant DHFR with lower
MTX affinity and sensitivity is used for transfection into DHFR-positive host cells.
Thus the action of endogenous DHFR becomes negligible, and the transfectant cells
survive in the presence of higher concentrations of MTX (126,127).
Subsequent to the selection procedure, single high-expression cell clones are isolated.
Various techniques have been established that are more or less labor-intensive.
Fluorescence-activated cell sorting can be helpful (128). The limiting dilution method
in microtiter plates is very useful in obtaining cell clones of monoclonal origin. The
assumption that monoclonality can be reached after only one round of subcloning is
rather theoretical (129,130). Repeated subcloning and intensive screening is necessary
to establish stable, high-producing cell lines for industrial manufacture.
INDUSTRIAL PRODUCTION OF MONOCLONAL ANTIBODIES
Monoclonal antibodies for the prevention or therapy of infectious diseases are
administered in doses of up to several milligrams per kilogram of body weight. Other
biopharmaceuticals such as tissue plasminogen activator and erythropoietin, which are
also manufactured by comparable recombinant technologies, are applied in only
nanogram or microgram ranges per kilogram body weight. In other words, a beneficial
antibody treatment dose requires an amount of at least a 1000-fold more of the recombinant
protein. This simple comparison clearly shows the challenge and the necessity
for the development of cost-effective manufacturing technologies. In fact, marketing
and manufacturing costs as well as quality assurance have been the driving forces