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Hybridomas, Genetic Engineering of 435

C. Affinity Binding TABLE II Human or Human Hybrid Fusion Partners
Certain bacterial cell wall proteins (called Protein A and Immunoglobulin
Cell type Cell line expression
Protein G) bind with high affinity to mammalian im-
munoglobulins. Protein A is derived from Staphylococcus Human myeloma SK007 Yes
aureus and has a strong affinity for antibodies. This al- Human lymphoblastoid RH-L4 Yes but nonsecretor
lows antibodies to be isolated by chromatography columns Human lymphoblastoid GM1500 Yes
which contain inert beads conjugated to Protein A or G. Human lymphoblastoid KR4 Yes
If a sample (such as Mab-containing culture medium) is Human lymphoblastoid LICR-LON Yes
run through the column at neutral pH only antibodies will
Human/human hybridoma KR12 Yes
bind, allowing all other components to be washed out.
Human/mouse hybridoma SHM-D33 Yes but nonsecretor
Pure antibody can then be eluted from the column by a
low pH buffer.
Suitable affinity columns of this type have been de- with humans and the source of human lymphocytes is lim-
signed for use with HPLC and this offers an extremely ited to samples of peripheral blood. These can be taken
rapid method of analyzing or purifying antibodies. How- from patients who have acquired an immunity against a
ever, the method will detect any mammalian immunoglob- particular compound or disease. Alternatively, methods of
ulin which means that the immunoglobulin content of in vitro immunization of human lymphocytes are possible.
the serum used in the growth medium may interfere with This approach requires the optimization of conditions for
analysis. human B-lymphocyte activation by use of the appropriate
HPLC affinity columns (such as ProAnaMabs from cytokines and growth factors.
Hyclone) offer a rapid assay for measuring antibodies in
serum-free culture medium and they could be used instead
B. Immortalization and Chromosome
of ELISA. Affinity chromatography can also be used for
Instability
large-scale antibody extraction, although the preparative
Protein A or G columns are expensive. There must be a suitable human fusion partner to immor-
talize the B-lymphocytes. Human myeloma cell lines are
difficult to grow in culture. Human lymphoblastoid cell
XIII. HUMAN MONOCLONAL ANTIBODIES lines have been used as fusion partners but the frequency
of cell fusion and genetic stability of the resulting hybrido-
Although murine-derived monoclonal antibodies are mas is low compared with equivalent fusions with mouse
widely used as laboratory reagents, in affinity purifica- cells. An alternative approach is to immortalize the acti-
tion and clinical diagnostic tests, they have had limited vated human lymphocytes by transfection with oncogenic
success in human therapy. Immunoglobulins synthesized DNA or by transformation by a virus.
from mice and humans have different constant regions
and so any antibody of mouse origin injected into a hu-
C. Antibody Secretion of Human
man could elicit an undesirable immune reactions. First,
although the antigen-binding site might be appropriate for Parental Fusion Partners
the target the antibody will not produce appropriate hu- Mouse myelomas commonly used in fusion are nonan-
man effector responses such as those of complement and tibody secretors. The value of this is that the resulting
Fc receptor binding. Second, the human immune system hybridomas only secrete the antibody associated with the
will produce antibodies against the murine immunoglob- fused B-lymphocyte. Therefore, the culture product will
ulin. This is referred to as the human anti-murine antibody be a single selected antibody type. However, most of the
immune response (HAMA). human myeloma or lymphoblastoid cells commonly used
This presents an obstacle in developing therapeutic an- for hybridization are immunoglobulin secretors (Table II).
tibodies from a murine source. However, there are at least This means that the resulting selected human hybridoma
three major difficulties in producing human hybridoma will secrete at least two antibodies, which are those asso-
cells capable of secreting human monoclonal antibodies. ciated with each of the parental cells.

A. The Source of Antibody-Secreting XIV. RECOMBINANT ANTIBODIES
Lymphocytes
In generating murine hybridomas, the spleen of an immu- A further possibility is the humanization of monoclonal
nized mouse is used as a source of the mixed lymphocyte antibodies originally produced from mice. This pro-
population for cell selection. Clearly this is not possible cess involves antibody engineering which relies on the