A human monoclonal anti-melanoma single chain Fv (scFv) antibody derived from tumor-infiltrating B lymphocytes.

Abstract

The development of recombinant DNA technology has made it feasible to clone, construct and express fully human immunoglobulin molecules. Here we report a novel methodology to make human antitumor scFv antibodies from tumor-infiltrating B lymphocytes (TIL-B). We isolated and expanded TIL-B from melanomas in the presence of EBV. The transformed B cells secreting tumor-specific antibodies were identified and cloned by limiting dilution. From one B cell clone with specific melanoma reactivity, we captured the immunoglobulin variable region genes, V(H) and V(k), by polymerase chain reaction (peR), sequenced the genes and linked them together by peR assembly using a (Gly₄Ser)₃ linker to form the scFv gene that was subsequently cloned into the pET21d vector and expressed. The scFv protein, obtained, with a molecular weight of 29 KD was purified and biotinylated for further characterization. The scFv demonstrated specific tumor reactivity to 21 of 24 different melanoma cell lines and not to 14 non-melanoma tumor cell lines, including breast, ovarian and colon cancer cell lines, normal human melanocytes as well as normal human leukocytes. These results were obtained using 1) a tumor cell ELISA, 2) fixed cell immunofluorescence and 3) live cell flow cytometry. The immunoprecipitation results indicated that a protein antigen of 45 KD was recognized by the scFv. Since we previously reported that about 70% of human tumors of different histologic types contain tumor-infiltrating B lymphocytes producing specific anti-tumor antibodies, this approach offers a rapid. effective method by combining in vitro B cell expansion and peR-gene cloning to elucidate the repertoire of the human anti-tumor immune responses and to make human monoclonal anti-tumor antibody molecules.