Institute of Biotherapy, School of Biotechnology, Southern Medical University, Guangzhou, China.
Acta Oncol. 2011 Oct;50(7):1111-8. doi: 10.3109/0284186X.2010.549838. Epub 2011 Jan 19.
Intravesical administration is an important treatment against superficial bladder cancer and CD40L is essential for the protective anti-tumor immunity. In situ gene therapy with CD40L was demonstrated to successfully inhibit tumor cell growth in the orthotopic mouse model of bladder cancer. In the present study, we prepared streptavidin (SA)-tagged sCD40L and developed a novel immunotherapy for superficial bladder cancer based on the strong interaction between streptavidin and biotin.
The SA-sCD40L fusion protein was expressed in E. coli and purified on the Ni-NTA column. After refolding with dialysis, the bi-function of the fusion protein was determined by flow cytometric analysis for streptaidin-mediated surface modification of MB49 bladder cancer cells and a mouse B cell CD40L-dependent proliferation assay. The mouse orthotopic model of MB49 superficial bladder cancer was used to evaluate the efficacy of SA-sCD40L immunotherapy.
The SA-sCD40L fusion protein exhibited both full biotin-binding property and CD40L bioactivity. After intravesical instillation, the SA-sCD40L bi-functional fusion protein was durably immobilized on the biotinylated mucosal surface of bladder wall for up to four days. The SA-sCD40L treatment significantly prolonged the survival of MB49 tumor-bearing mice and cured 50% of mice with MB49 superficial bladder cancer without significant adverse effects. In addition, more tumor-infiltrating CD4(+)or CD8(+) T cells were observed in SA-sCD40L-treated group.
Intravesical immobilization of SA-sCD40L elicited a strong and long-lasting immunity against the MB49 bladder cancer.
膀胱内给药是治疗浅表性膀胱癌的重要手段，而 CD40L 对于保护性抗肿瘤免疫至关重要。CD40L 的原位基因治疗已被证明可成功抑制膀胱癌的原位小鼠模型中的肿瘤细胞生长。在本研究中，我们制备了链霉亲和素（SA）标记的 sCD40L，并基于链霉亲和素与生物素之间的强相互作用，开发了一种治疗浅表性膀胱癌的新型免疫疗法。
在大肠杆菌中表达 SA-sCD40L 融合蛋白，并在 Ni-NTA 柱上进行纯化。通过透析进行复性后，通过流式细胞术分析来确定融合蛋白的双功能，用于链霉亲和素介导的 MB49 膀胱癌细胞表面修饰和小鼠 B 细胞 CD40L 依赖性增殖测定。使用 MB49 浅表性膀胱癌的小鼠原位模型来评估 SA-sCD40L 免疫疗法的疗效。
SA-sCD40L 融合蛋白具有完整的生物素结合特性和 CD40L 生物活性。经膀胱内灌注后，SA-sCD40L 双功能融合蛋白可在长达四天的时间内持久地固定在膀胱壁的生物素化黏膜表面上。SA-sCD40L 治疗可显著延长 MB49 荷瘤小鼠的存活时间，并治愈 50%的 MB49 浅表性膀胱癌小鼠，而无明显不良反应。此外，在 SA-sCD40L 治疗组中观察到更多的肿瘤浸润性 CD4(+)或 CD8(+)T 细胞。
SA-sCD40L 的膀胱内固定可引发针对 MB49 膀胱癌的强烈且持久的免疫反应。
