| Summary: | The generation of human monoclonal antibodies that may be clinically useful is
hampered by the lack of an efficient, applicable technology. In this thesis I report the
development of the Selected Lymphocyte Antibody Method (SLAM) as an alternative
approach to generate monoclonal antibodies and to facilitate the analysis of the human
humoral immune response. SLAM technology is based upon the molecular cloning and
expression of immunoglobulin heavy and light chain variable region cDNAs from single B
lymphocytes that have been identified in a modified hemolytic plaque assay as secreting
immunoglobulin which is specific for a target antigen. SLAM technology was first
developed in a murine model. Immunoglobulin variable region cDNAs were RT-PCR
amplified from single murine B lymphocytes that secreted immunoglobulin which is
specific for an epitope found in gpll6 of the Human Cytomegalovirus (HCMV) or in
gpl20 of HIV-1. These immunoglobulin variable regions were then cloned and expressed
in the context of human immunoglobulin constant regions. The resulting chimeric
immunoglobulins were shown to reproduce the same specificity as the "parental"
immunoglobulin.
To generate human monoclonal antibodies that were specific for gpl 16, peripheral
blood B lymphocytes from a single HCMV immune donor were cultured at limiting dilution
in the "EL4.B5" B-cell culture system. 107 single B lymphocytes, corrresponding to 34
clones that secreted gpll6-specific immunoglobulin, were identified by hemolytic plaque
assay and then isolated by micromanipulation. Immunoglobulin variable region cDNAs
were successfully RT-PCR amplified from 29 of the 34 clones. Sequence analysis of the V
region cDNAs showed a remarkable restriction in the use of heavy chain variable and
joining gene segments as well as restriction in the use of kappa light chain variable and
joining gene segments in the associated light chains. Two independent B cell clonotypes
were identified based on the utilization of different diversity gene segments in the heavy
chain variable regions. Two recombinant monoclonal antibodies (mAbs) that represent each
clonotype were expressed and were shown to neutralize HCMV in vitro. Thus, SLAM
technology facilitated the generation of two potentially clinically useful antibodies and may
be used in the future to generate mAbs against a wide variety of pathogens.
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