|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1994 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1993 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1992 : ¥900,000 (Direct Cost : ¥900,000)
Our strategy to generate human monoclonal antibodies (HMAbs) consist of cell fusion between a parental B cell line (Kudo et al., 1994) and EBV-transformed B cell line from transferred lymphocytes of bile duct cancer patient or healthy volunteers into SCID mouse to allow in vivo stimulation of human lymphocytes against allogenic bile duct cancer cell line TFK-1 (Saijo et al., manuscript in preparation). At present, we obtain several HMAbs, but they weakly reacted with the surface of TFK-1. These HMAbs were not suitable for specific targeting therapy because of there weak reactivity. So we could not start to investigate the effect of a bispecific antibody (BsAb) against TFK-1. We need more time to develop strongly reactive HMAbs with TFK-1. We have investigated the effect of BsAb in vitro and in vivo using monoclonal antibody (L-7-6) against hepatocellular carcinomas (HCC) as a preliminary experiment. Two type of BsAb, L-7-6 x anti-CD3 and L-7-6 x anti-CD16, was made by chemical conjugat
ion. LAK cells, as effector cells, were obtained as follows. In brief, Peripheral blood mononuclear cells, isolated from heparinized blood of a bile duct cancer patient or a healthy volunteer by density gradient centrifugation, were cultured for 48 h in RPMI-1640 supplemented with 10% FBS and 100U/ml recombinant human IL-2 at a cell density of 1 x 10^6/ml, in a culture flask, where bottom was precoated with OKT-3 MoAb (10mg/ml) for induction of LAK cells.
Result as follows ; In vitro experiment, percent cytotoxicity for three HCC cell lines ranged from 20-80%, even at a low E/T ratio (0.3-4) with only 0.1 mu g/ml of BsAb. On the other hand, without BsAb, percent cytotoxicity was only 5-20% at the same E/T ratio. The maximal discrepancy between using BsAb an without BsAb was reached at about 60%. This indicted that LAK cells with BsAb greatly enhanced cytotoxicity.
In this study, we clearly demonstrate that BsAbs have a potent effect of cytotoxicity in vitro. At Winn's assay in vivo, we have an impression that the growth of human HCC cell lines with LAK cells and BsAb in more slower than without BsAb on SCID mouse. Next, we are going to verify whether LAK cells with BsAb can kill the tumor cells after establishing xenografted human HCC cell line into mouse subcutaneous tissue by intravenous injection of LAK cells with BsAb. Less