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430 Hybridomas, Genetic Engineering of

oratory technique but now can be performed on a large
commercial scale in bioreactors. The techniques described
can be performed in any cell culture laboratory with ac-
cess to animal facilities. Alternatively, a wide range of
antibody-secreting hybridomas are available from culture
collections.
In the 1970s the techniques of immunization, cell
hybridization, genetic selection, and cell cloning were
utilized to produce cells that were hybrids of B-
lymphocytes and myelomas. The B-lymphocytes are
antibody-secreting cells, whereas the myeloma cells are
transformed lymphocytes capable of growing indefinitely.
The resulting hybridomas were capable of continuous syn-
FIGURE 5 Glycoforms of IgG.
thesis of preselected antibodies. Kohler and Milstein ob-
tained the Nobel Prize in 1984 for their work on the devel-
be important for antigen binding with specific examples opment of Mab-secreting hybridomas. The original work
showing that the degree of binding may either increase or described the creation of a mouse–mouse hybridoma that
decrease. secreted antibody with affinity to sheep red blood cells
Although the level of glycosylation of IgG is small as antigens. The antibody could be easily detected by a
(2–3% by weight) compared to other proteins, the glycan hemolytic plaque assay that showed the capability of the
structures on immunoglobulins are known to have a sig- antibody to bind to and lyse sheep erythrocytes. Since
nificant effect on immune responses. Figure 5 shows the the 1970s, these methods have found wide application
common glycoforms of IgG with 0, 1, or 2 galactose termi- and have resulted in the large-scale production of kilo-
nal residues (G0, G1, and G2) and the possibility of a sialic gram quantities of some monoclonal antibodies. The term
acid terminal group on G2. The distribution of these gly- “monoclonal” indicates that the antibody is of a single
coforms changes under certain pathological conditions. type. This will bind to just one antigen.
For example, it is well established that a high propor- The method developed by Kohler and Milstein involves
tion of agalactosylated glycan structures in immunoglob- four stages which result in the production of a hybrid
ulin (G0) is associated with specific human disorders, no- lymphocyte with an infinite growth capacity and capable
tably rheumatoid arthritis. Here the predominant form of of continuous synthesis of a single antibody. The stages
the glycan attached to Asn-297 is a biantennary complex of this process are summarized in Fig. 6. The four stages
structure that terminates in N-acetylglucosamine residues involve
and lacks the usual galactose terminus. This altered gly-
Immunization
can structure results in a change in the interaction of the
Cell fusion
immunoglobulin with specific monocyte receptors. Also,
Genetic selection
there are changes to the structure of the immunoglobulin
Cell cloning
caused by the altered glycan that may explain changes in
immune response related to the disease condition.
The glycosylation of IgM is more complex with five
identifiable N-glycan sites in the heavy chain. Three of
these are complex biantennary, whereas the other two
are of a high mannose type glycan. Variations of these
glycan structures may also produce undesirable immune
responses if they are utilized as therapeutic products of
hybridomas.

IV. PRODUCTION OF
MONOCLONAL ANTIBODIES

This section describes the background to the methods used
to produce hybridomas that secrete monoclonal antibod-
ies (Mabs). This is a technology that developed as a lab- FIGURE 6 Production of Mab-secreting hybridomas.