Immunotherapy for Infectious Diseases

82 Kunert and Katinger
HUMAN/MOUSE HYBRID MYELOMAS AS FUSION PARTNERS
Human/mouse heteromyeloma cell lines efficient for human B-cell immortalization
have been described by different authors (93). The CB-F7 cell line was derived from
xenogeneic somatic cell hybridization between normal human B-lymphocytes and the
murine hypoxanthine/aminopterine/thymidine (HAT)-sensitive P3X63Ag8/653 cell
line (94). It displays rapid cell growth, high cloning efficiency, and a hybridizing efficiency
of 2–6 clones/10 5 seeded lymphocytes. CB-F7 is ouabain-resistant and is therefore
suitable for fusion with EBV-transformed lymphoblastoid cell lines, which, as
human cells, are not resistant to ouabain. Several human monoclonal antibodies directed
against HIV-1 were expressd from CB-F7 hybrids (95).
Selection Screening and Stabilization for Antibody-Producing Hybridomas
After cell fusion, the primary hybrids contain a pool of primary transformants. These
are mostly generated by random fusion events. In these inhomogenous cell pools, only
a limited number of hybrids have the capacity to express antibodies of particular specificities.
Furthermore, during fusion of the two karyons, the chromosomes capable of
antibody expression may be lost. Therefore screening must be combined with an efficient
selection system that eliminates all lymphocyte/lymphocyte and myeloma/
myeloma primary hybrids. The HAT selection system is usually applied (96), in which
only hybrids fused between myelomas and lymphocytes can survive. Since only a
minor fraction of the lymphocytes are antibody-producing B-lymphocytes, efficient
screening procedures for antibody-secreting hybridomas are essential. Using, for example,
human peripheral blood as a source of lymphocytes, it is often not possible to get
more than 5 � 10 7 lymphocytes with approximately 10% B-cells. Calculating a fusion
frequency of 1 � 10 �4 , the fusion of 5 � 10 7 lymphocytes will yield approx. 5000
surviving hybrids containing only 500 B-cell hybridomas.
The first screening step is performed after 2 weeks when hybrids are starting to grow
exponentially in the selective HAT medium. The surviving cell pool is then further stabilized
by repeated subcloning with limited dilution techniques. Other immunologic methods
have been established to screen the hybridoma supernatant, either with immunomagnetic
catcher beads (97) or by dot immunobinding assay (98,99). Repeated subcloning after every
screening step is necessary to ensure that specific, high-producing subclones are selected.
To establish monoclonal cell lines capable of large-scale industrial manufacture, an extensive
stabilization procedure is essential. The stabilization procedure, by limiting dilution
plating, can be assumed to be finished if at least 90% of the subclones show comparable
IgG production as well as constant productivity over a period of 50–100 passages.
Recombinant Antibodies
In vivo application of murine MAbs shows reduced half-lives and induces human
anti-mouse antibodies (HAMAs). Biochemical or recombinant engineering techniques
are used to create more or less chimeric or humanized antibodies. Various methods can
be used to generate functional antibody fragments or to replace regions of the protein
backbone. To replace the murine Fc part of mouse-derived antibodies, the immunoglobulin
molecule can be cleaved proteolytically by applying enzymes such as papain or
pepsin. The smaller fragments can be separated chromatographically. According to the
specificity of enzymes applied, Fab and/or F(ab) 2 fragments are generated. The F(ab) 2