Cloning, isolation and characterization of human tumor in situ monoclonal antibodies

Abstract.

A bacterially expressed human antibody (Ab) library (diversity ≈10⁵) was generated from tumor-infiltrating B lymphocytes present in tissue isolated from a colon tumor. Immunoglobulin (IgG) heavy and light chain variable regions were amplified without isolating or enriching B cells, cloned into a phage-expression vector, and soluble antigen-binding fragment (Fabs) from >10⁵ members of the library were screened rapidly by two distinct and complementary methodologies. In the first approach, soluble Fabs were screened by enzyme-linked immunosorbent assay (ELISA) on tumor cell monolayers. Alternatively, tumor cell surface antigens were selectively biotinylated with a plasma membrane-impermeable reagent, solubilized with non-ionic detergent, and were used to screen >10⁵ members of the Ab library by capture lift. Reactive Fabs were partially characterized for tumor cell specificity and cross-reactivity, resulting in the identification of multiple Abs that bind cultured tumor cells but not normal human fibroblasts. The Fabs clustered into at least three distinct epitope specificity groups based on multiple criteria, including differential reactivity on two tumor cell lines and distinct antigen recognition patterns on western blot and immunoprecipitation. Moreover, DNA sequencing of the Ab variable regions demonstrated that the majority of the tumor-reactive Fabs were distinct and substantially different from the corresponding most homologous Ab germline gene. The relatively small size of the tumor-derived library allowed direct screening of soluble Fab of every member of the library, permitting the characterization of multiple human monoclonal antibodies (mAbs) that might not be discovered using alternative approaches, such as hybridoma technology or phage-display.