Experimental chemotherapy of anti-human melanoma antibody conjugated antitumor agent.

将纯化的抗人牙周黑色素瘤细胞株(HMG)的单抗(IA6-C2)及其Fab‘组分偶联到顺铂(CDDP)脂质体上。研究了脂质体(Lip-CDDP、抗-Lip-CDDP、Fab‘-Lip-CDDP)对HMG细胞的杀伤作用。用蛋白A琼脂糖亲和层析法纯化了单抗，并对其进行了药代动力学研究。用N-hydroxysuccinimidyl-3-(pridylditio)-propionate将单抗与脂质体偶联，用羟基自由基将单抗与脂质体偶联。以磷脂酰胆碱、胆固醇和3-(2-吡啶二硫)丙酰二棕榈酰磷脂酰乙醇胺(摩尔比，26/10/0.4)混合制成脂质体。用原子吸收光谱仪测定铂浓度时，CDDP的包封率为3%。在37ﾟC培养液(RPMI1640含20%胎牛血清)和4ﾟC PBS培养液中检测Lip-CDDP的释放量。在PBS中24 h和48 h的释放率分别为56%和72%，在PBS中24 h和48 h的释放率分别为12%和14%。Lip-CDDP在4ﾟC PBS中稳定48 h。单抗-Lip-CDDP体外抗HMG细胞活性为Lip-CDDP的2.1倍和CDDP的20倍。裸鼠体内铂浓度在裸鼠体内分别为Lip-CDDP和Lip-CDDP的1.4倍和2倍，肾组织中则分别为Lip-CDDP和CDDP的0.7倍和0.5倍。

A purified monoclonal antibody (IA6-C2) to a human melanoma cell line from gingiva (HMG) and its Fab' fraction were coupled on Cisplatinum (CDDP) entrapped liposome. The effect of liposomes (Lip-CDDP, Ab-Lip-CDDP, Fab'-Lip-CDDP) against HMG cell was investigated. The Pharmocokinetics of these liposomes in HMG tumor xenografted nude mice were also examined.A MoAb was purified with protein A agarose affinity chromatography. N-hydroxysuccinimidyl-3-(pridylditio)-propionate (SPDP) was used for conjugation of MoAb to the liposome, and SH radical of MoAb was used for conjugation of Fab' to the liposome. The liposome was produced, mixing of phosphatidylcholine, cholesterol and 3-(2-pyridyldithio) propionyldipalmitoyl-phosphatidylethanolamine (molar ratio, 26/10/0.4). The encapsulated ratio of CDDP was 3% when platinum concentration was measured with atomic absorption spectrophotometer. The releasing CDDP from Lip-CDDP was tested in 37ﾟC culture medium (RPMI 1640 contained 20% FBS) and in 4ﾟC PBS for stability. The releasing ratio was 56% after 24 hrs and 72% after 48 hrs in culture medium, and 12% after 24 hrs and 14% after 48 hrs in PBS.The Lip-CDDP showed to stabilize in 4ﾟC PBS for 48 hrs.No effect of PBS encapsulated liposome against HMG cell and normal fibloblast indicates components of liposome to be harmless for cell growth. The anti-HMG cell effects of Ab-Lip-CDDP significantly increased to 2.1-fold of Lip-CDDP and 20-fold of CDDP in vitro. The Fab'-Lip-CDDP effect was, however, almost similar with Lip-CDDP.The tissue platinum concentration of nude mice was increased to 1.4-fold of Lip-CDDP and 2-fold of CDDP in HMG tumor, and decreased to 0.7-fold of Lip-CDDP and 0.5-fold of CDDP in kidney.