Department of Urology, Experimental Urology, University Hospital Freiburg, Freiburg, Germany.
Prostate. 2011 May;71(6):588-96. doi: 10.1002/pros.21274. Epub 2010 Oct 13.
For redirecting T-lymphocytes to induce prostate cancer cell lysis, we constructed a novel bispecific single-chain (bsc) diabody directed to the prostate specific membrane antigen (PSMA) and the T-cell receptor (TCR)-associated CD3 molecule on T-cells.
The PSMA × CD3 bsc diabody was generated from an anti-CD3 single chain Fv fragment (scFv) and the anti-PSMA scFv D7. It was expressed in E. coli and purified from the periplasmic extract and culture supernatant by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST-1) was used and activation of T-cells was determined by measuring the surface marker expression of CD25 and CD69. For in vivo evaluation, the diabody was administered in combination with human peripheral blood lymphocytes (Ly) in a C4-2 xenograft-SCID mouse model.
Specific binding of the PSMA × CD3 bsc diabody both to CD3-positive Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the PSMA × CD3 bsc diabody proved to be a potent agent for retargeting CD4+ and CD8+ human lymphocytes to lyse C4-2 prostate cancer cells. Treatment of SCID mice bearing C4-2 tumor xenografts with the diabody and human lymphocytes efficiently inhibited tumor growth.
The PSMA × CD3 bsc diabody bears a high potential for the immunotherapy of prostate cancer.
为了使 T 淋巴细胞转向以诱导前列腺癌细胞裂解，我们构建了一种新型双特异性单链（bsc）二联体，该二联体针对前列腺特异性膜抗原（PSMA）和 T 细胞上的 T 细胞受体（TCR）相关 CD3 分子。
PSMA-CD3 bsc 二联体由抗-CD3 单链 Fv 片段（scFv）和抗-PSMA scFv D7 构建而成。它在大肠杆菌中表达，并通过固定金属亲和层析（IMAC）从周质提取物和培养上清液中纯化。通过流式细胞术在表达 PSMA 的前列腺癌细胞和 PSMA 阴性细胞系以及 Jurkat 细胞上测试结合特性。为了进行体外功能分析，使用细胞活力测试（WST-1），并通过测量 CD25 和 CD69 的表面标记表达来确定 T 细胞的激活。为了进行体内评估，将二联体与人类外周血淋巴细胞（Ly）联合在 C4-2 异种移植-SCID 小鼠模型中给药。
通过流式细胞术显示 PSMA-CD3 bsc 二联体特异性结合 CD3 阳性 Jurkat 细胞和表达 PSMA 的 C4-2 细胞。体外，PSMA-CD3 bsc 二联体被证明是一种有效的试剂，可将 CD4+和 CD8+人类淋巴细胞重定向以裂解 C4-2 前列腺癌细胞。用二联体和人类淋巴细胞治疗携带 C4-2 肿瘤异种移植物的 SCID 小鼠可有效抑制肿瘤生长。
PSMA-CD3 bsc 二联体具有很高的潜力可用于前列腺癌的免疫治疗。
