Background The use of humanized or murine/human chimeric monoclonal antibodies (MoAbs) is applied to clinical use as anticancer therapy like HER-2-specific trastuzumab (Herceptin) against breast cancer, showing increased overall survival. These antibodies are characterized as humanized or murine/human chimeric MoAbs, which will cause adverse events with immune response recognizing these antibodies as foreign bodies in patients’ body. Fully human MoAb (HuMoAb) is ideal strategy to prevent such immune response, and at the present, HuMoAb is not yet reported clinically. In the present study, we show a method of successful production of HuMoAbs, reacting specifically to cancer cells, not to normal cells, utilizing human hybridoma technique with tumor infiltrating lymphocytes (TILs). Materials and Methods TILs were collected from clinical specimen of lung, gastric, pancreatic, or colonic carcinoma patients. Hybridomas were established using these TILs and mouse myeloma cell, P3U1. After selecting hybridoma clones, which binds to cancer cells, fluorescence microscope and immunohistochemical staining were used to examine antibodies’ ability to attach on cell surface of various kinds of specimen. To evaluate inhibitory effect of each antibody on cancer cell growth, human lung cancer cell line HLC-1, the human stomach cancer cell line MKN-45, and the human pancreas cancer cell line PANC-1 were examined using MTT method. Cells were incubated with 100, 50, 25, 12.5, and 6.25μg/ml of each antibodies or non-specific human IgM as a control vehicle for 6 days, followed by calculating inhibition rate compared with the control. Results Lympocytes were obtained from 28 patients and total 43 fusions were performed. Total growing clones were 9450, in which tumor-reactive immunoglobins were 36. The origins of these antibodies were 23 from lung, 8 from stomach, 4 from pancreas, and 1 from colon. 27 of these antibodies were IgM, and 9 were IgG. The 36 antibodies were evaluated according to reactivity to cancer and normal cell, resulting that 14 antibodies showed specific binding to cancer cells (Used cell lines: HLC-1, A-549, PC-3, MKN-45, HSC, MKN-74, SKS, PK-8, HPAC, PANC-1, LoVo, DLD-1, Colo, HCT-15) with high intensity, not to normal cells. To examine the inhibitory effect on cell growth by these antibodies, HLC-1, MKN-45, PANC-1 cells were incubated with each antibody, resulting in 11 antibodies inhibited cell growth of at least a cell line. Inhibitory effect determined by MTT assay was from 22% to 94% by means of the 11 antibodies. Conclusion HuMoAbs were successfully produced using a hybridoma technique with TILs, and we demonstrated specific reactivity to cancer cells, not to normal cells and tissue in vitro, with inhibitory effect on cell growth against malignant cells. These results will be encouraging to establish a novel anticancer HuMoAb, which is less toxic for host immune response in clinical use.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]