A HUMAN MONOCLONAL ANTIMELANOMA SINGLE-CHAIN FV ANTIBODY DERIVED FROMTUMOR-INFILTRATING LYMPHOCYTES

With the development of recombinant DNA technology, it has become feas ible to clone, construct, and express fully human immunoglobulin molec ules. Here we report a novel methodology to make human antitumor singl e-chain Fv (scFv) antibodies from tumor-infiltrating B lymphocytes. We isolated and expanded tumor-infiltrating B lymphocytes from melanomas in the presence of Epstein-Barr virus. The transformed B cells secret ing tumor-specific antibodies were identified and cloned by Limiting d ilution. From one B cell clone with specific melanoma reactivity, we c aptured the immunoglobulin variable region genes V-H and V-k by PCR, s equenced the genes, and linked them together by PCR assembly,vith the use of a (Gly4Ser), linker. The scFv gene was then cloned into the pET 21d vector and expressed, The obtained scFv protein with a M(r) of 29, 000 was purified and biotinylated for further characterization. The sc Fv demonstrated specific tumor reactivity to 21 of 23 different melano ma cell lines and not to 14 nonmelanoma tumor cell lines, such as brea st, ovarian, and colon cancer cells lines; normal human melanocytes as wed as normal human leukocytes. These results were obtained in (a) a tumor cell ELISA, (b) fixed cell immunofluorescence, and (c) live cell flow cytometry. The immunoprecipitation results indicated that a prot ein antigen of M(r) 45,000 was recognized by the scFv. Since we report ed previously that about 70% of human tumors of different histological types contain tumor-infiltrating B lymphocytes producing specific ant itumor antibodies, this approach offers a rapid, effective method by c ombining in vitro B-cell expansion and PCR gene cloning to elucidate t he repertoire of the human antitumor immune response and to make human monoclonal antitumor antibody molecules.