双特异性单链抗体介导的T细胞对前列腺癌细胞的杀伤作用

Killing effects of T cells on prostate cancer cells mediated by two kinds of bispecific single-chain antibodies

目的研究并比较两种抗人γ-精浆蛋白/抗CD3双特异性单链抗体介导T细胞杀伤前列腺癌细胞的作用。方法利用流式细胞仪分析双特异性单链抗体(BsAb)及多价双特异性单链抗体(mBsAb)与LNCaP细胞和Jurkat细胞的亲和力;将LNCaP细胞作为靶细胞,分为抗体浓度固定组和效应细胞/靶细胞比例固定组,利用51Cr释放试验评价两种双特异性单链抗体在体外介导T细胞杀伤靶细胞的能力;将Jurkat细胞种植裸鼠后,建立裸鼠前列腺癌模型,并分为非治疗组、对照组、BsAb组和mBsAb组,进一步评价两种双特异性单链抗体在体内介导T细胞杀伤靶细胞的能力。结果流式细胞仪结果显示:BsAb和mBsAb均可特异性结合LNCaP细胞和Jurkat细胞,阳性结合率分别为56.3%、55.4%和74%、83%。51Cr释放试验结果显示:在体外,BsAb和mBsAb均可介导T细胞对前列腺癌细胞的杀伤,并且T细胞的杀伤效率与抗体浓度和效应细胞/靶细胞比例呈正相关。与非治疗组和对照组比较,接种前列腺癌细胞的裸鼠在体内注射激活的细胞毒T细胞的同时分别接受两种抗体的治疗后,肿瘤生长均明显受到抑制(P<0.05)。另外,体外介导杀伤作用和体内抑制肿瘤生长等方面,多价双特异性抗体的效果明显优于双特异性抗体。结论同时识别人γ-精浆蛋白和CD3分子的双特异性单链抗体可有效地介导T细胞对前列腺癌细胞的杀伤作用,并且双特异性单链抗体四聚体的形成可明显改善抗体介导杀伤作用的效率。

Objective To study the killing effects of T cells on prostate cancer cells mediated by two kinds of bispecific single-hain antibodies （BsAb and m BsAb） specific for human γ-seminoprotein and CD3 molecule. Methods Flow cytometry （FCM） was used to detect the binding activity of two antibodies to CD3^- positive cell line Jurkat and prostate carcinoma cell line LNCaP. LNCaP cells as target cells were divided into two groups： group with fixed antibody concentration and group with fixed effector/target cells ratio. In the two groups, the efficacy of the two antibodies in mediating the killing activity of T cells against target cells in v/tin was determined by the chromium^54 -release test. Mouse models of prostate cancer were contructed by inoculation of LNCaP cells, and then divided into four groups： untreated group, control group, BsAb group, and mBsAb. Tumor growth in the mouse models was measured weekly. Results The binding rates of BsAb to the Jurkat and LN- CaP cells were 54.1% and 53.7% respectively, while binding rates of mBsAb to the Jurkat and LNCaP cells were 70.4% and 81% respectively. In vitro, with activated CTLs as effector cells, a specific lysis of LNCaP cells mediated by both antibodies was confirmed by chromium51- release assay. In vivo, both antibodies could reduce tumor growth of mouse model significantly as compared to the group treated only with CRLs and the untreated control group （ P 〈 0.05）. The experiment results showed that the mBsAb has a better activity than BsAb in binding antigens, mediating lysis of LNCaP cells, and reducing tumor growth （ P 〈 0.05）. Conclusion Both kinds of bispecific single-chain antibodies specific for human γ-seminoprotein and CD3 molecule could mediate killing activity of T cells in vitro andin vivo, and formation tetrametization of single-chain antibody could improve the efficacy.