Immunotherapy for Infectious Diseases

80 Kunert and Katinger
Different procedures have been established to immortalize lymphocytes as vehicles
for the production of unlimited amounts of monoclonal antibodies. The first report
describing the immortalization of human antibody-expressing B-lymphocytes with a
distinct specificity with EBV was published by Steinitz et al. (76).
Although the immortalization of B-lymphocytes is a rather easy technique to perform,
an intrinsic problem is retaining stable antibody production in culture for prolonged
periods. The transformation of peripheral B-lymphocytes from an
antigen-primed donor with EBV generates expanded lymphocytes or even immortalized
lymphocytes. Generally 2–3 weeks after virus infection transformants producing
specific antibodies can be detected in the supernatant. However, with continued growth
of the culture, specific antibody levels invariably fall and become undetectable after
3–4 months—probably owing to the overgrowth of the culture with nonproducing cells.
Therefore EBV transformation is usually applied as a tool to enrich human antibodyproducing
B-lymphocytes for functional screening and as a means of amplifying the
genes of interest. Somatic cell hybridization for the creation of antibodies with predetermined
specificity was first described in 1975 (77). This technology—the hybridoma
technology—revolutionized immunology by allowing production of monoclonal antibodies
of virtually any specificity. The hybridoma technology—first established with
rodent species—was initially not used for the production of human MAbs. The application
of hybridoma technology to create human antibodies suffered from the variable
and often low fusion frequency of hybrids. Furthermore, the isolation and amplification
of antibody-producing B-cells prior to fusion was one of the most critical points.
In the following sections the main issues of immortalization of high-producing
hybridoma cells will be addressed.
Source of Lymphocytes Capable of Cell Fusion and MAb Production
After immortalization of lymphoblastoid B-cells, the yield of antibody-secreting
hybridomas very much depends on the status of the immunologic differentiation of the
B-lymphocytes prior to cell fusion. In the rodent system, an optimized scheme of
immunization can be applied, leading to the enrichment of antigen-stimulated
B-lymphoblasts in the spleen, which are activated to enter mitosis concurrently with the
fusion partner used for immortalization. By contrast, it is almost impossible to obtain
human spleen B-lymphocytes from antigen-primed donors. Usually only PBLs of vaccinated
or reconvalescent donors are available. Because of the lack of accessibility to
surgically removed tonsils or spleen cells, alternative techniques have been developed
to stimulate naive lymphocytes with the desired antigen outside human body.
In Vitro Antigen Priming
Techniques of in vitro antigen priming of B-cells are commonly refered to as in vitro
immunization. Many protocols for in vitro immunization have been established (78,79).
Those include the purification of the lymphocyte population by inactivation or irradiation
of T-suppressor cells and retaining T-helper cells and macrophages. After the antigen
priming step (80), the B-cells are amplified by incubation with B-cell mitogens
such as Staphylococcus aureus Cowan I (SAC) and stimulated with lymphokines to
secrete immunoglobulins. Often phytohemagglutinin is added to activate T-helper cells
secreting the B-cell lymphokines. After screening of the supernatants for antibody pro-