Immunotherapy for Infectious Diseases

Production of Igs and MAbs Targeting Infectious Diseases 81
duction, the B-lymphocytes are fused. In vitro immunization procedures for B-cells
producing high-affinity IgG have also been described (81). However, none of these
complex techniques have achieved widespread industrial application.
Amplification of human B-lymphocytes
Especially for the immortalization of human B-lymphocytes, very small amounts
of cells are often available. The enrichment of the desired population of human
B-lymphocytes prior to immortalization can be achieved by two methods. Peripheral
B-lymphocytes expressing the receptor for EBV (82) can be immortalized by
EBV transformation. Only a subset of 20% of the total B-cell population is actually
immortalized (83,84), whereas a large fraction of activated antibody-producing plasma
cells is resistant to transformation (85). These findings and the fact that fully differentiated
plasma cells lack the receptor for EBV indicate an increasing resistance of highspecificity,
antibody-producing B-cells to EBV transformation as these mature into
plasma cells following antigen or mitogen stimulation.
The second method for enrichment of primed B-lymphocytes uses growth factors
(IL-4, IL-6, IL-14), mitogens such as plant mitogens (pokeweed mitogen), bacterial
products (SAC), and chemical compounds such as phorbol myristate acetate or different
antigens (tetanus toxoid, Candida albicans). This method needs no viral agents, and the
cells are subsequently immortalized by fusion. Alternatively, primed B-lymphocytes can
be enriched in the cell population by catching the cells with surface-bound, antigenspecific
antibody (86).
Fusion Partners for the Generation of Hybridomas
Several laboratories are still engaged in developing human myelomas or lymphoblastoid
cells capable of establishing human hybridomas that stably produce human
MAbs with a high yield (87,88). However, only a few myeloma cell lines have been
established that are capable of generating human-human hybridomas. Generally, these
cell lines show low growth rates with doubling times of 40–60 hours. They also often
tend to become senescent, possibly because of their nearly normal diploid chromosomal
content (89,90). The fusion of human lymphocytes with non-human fusion partners
generates xenohybridomas preferentially segregating human chromosomes. Since
the chromosomal constitution of intraspecific human hybrids is much more stable,
human fusion partners for hybrid generation are preferable.
HUMAN FUSION PARTNERS
Plasmacytomas are differentiated human lymphoid neoplasias characterized in vivo by
myeloma protein expression and in vitro by immunoglobuline synthesis (91). These cell
lines are very difficult to establish in continuous cell culture. Lymphoblastoid cell lines
(LCLs) as fusion partners were taken into consideration because of the paucity of human
myeloma cell lines (92). LCLs are established from malignant or normal hematopoietic tissue
and are easily maintained in tissue culture. Most of the cell lines described for human
fusion are of lymphoblastoid origin. A comparison of different properties like hybridization
frequency, yield of antigen-specific hybridomas, cloning efficiencies, growth rate, and clone
stability shows no significant advantages of LCLs or plasmacytomas as fusion partners. The
limitation of LCLs seems to be the sustained level of human MAb production in these
hybrids. Cell morphology obviously correlates with the amount of antibody being produced;
abundant rough endoplasmatic reticulum correlates with higher specific expression.