Immunotargeting-therapy for bile duct carcinoma using human type bispecific antibody

我们产生人单克隆抗体（HMAb）的策略包括亲本B细胞系之间的细胞融合（Kudo等人，1994）和来自胆管癌患者或健康志愿者的转移淋巴细胞的EBV转化的B细胞系进入SCID小鼠，以允许体内刺激人淋巴细胞对抗同种异体胆管癌细胞系TFK-1（西条等人，手稿正在准备中）。目前，我们已经获得了几种HMAs，但它们与TFK-1表面的反应很弱。这些HMAb反应性较弱，不适合作为特异性靶向治疗。因此，我们无法开始研究针对TFK-1的双特异性抗体（BsAb）的作用。我们需要更多的时间来开发与TFK-1具有强反应性的HMAbs。本实验以抗肝癌单克隆抗体（L-7-6）为实验材料，研究了抗肝癌单克隆抗体（BsAb）在体内外的作用。用化学偶联法制备了L-7-6 x抗CD 3和L-7-6 x抗CD 16两种BsAb， ...更多信息 离子。如下获得作为效应细胞的LAK细胞。简而言之，通过密度梯度离心从胆管癌患者或健康志愿者的肝素化血液中分离的外周血单核细胞在培养瓶中在补充有10%FBS和100 U/ml重组人IL-2的RPMI-1640中以1 × 10^6/ml的细胞密度培养48小时，结果表明：在体外实验中，仅用0.1 μ g/ml的BsAb，即使在低E/T比（0.3-4）下，对三种肝癌细胞系的杀伤率也在20- 80%之间。另一方面，在没有BsAb的情况下，在相同的E/T比下，细胞毒性百分比仅为5-20%。使用BsAb和不使用BsAb之间的最大差异达到约60%。本研究进一步证实了BsAb具有较强的体外细胞毒作用。在Winn的体内试验中，我们得到的印象是，在SCID小鼠上，有LAK细胞和BsAb的人肝癌细胞系的生长比没有BsAb的人肝癌细胞系的生长慢。下一步，我们将通过静脉注射含BsAb的LAK细胞建立小鼠皮下移植人肝癌细胞系，验证含BsAb的LAK细胞是否具有杀伤肿瘤细胞的作用。少

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 … More 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