Clary B M, Coveney E C, Blazer D G, Philip R, Philip M, Morse M, Gilboa E, Lyerly H K
Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Immunother. 1997 Jan;20(1):26-37. doi: 10.1097/00002371-199701000-00003.
Ex vivo genetically engineered cytokine-secreting tumor cell vaccines have been shown to prevent metastatic disease in animal models of lung and breast cancer. Because of the inefficiency of existing modes of gene delivery in transducing primary human tumor cells, it has been difficult to clinically apply this strategy. In this study, liposome-mediated delivery of an adeno-associated virus (AAV)-based plasmid containing the sequence for murine gamma-interferon (gamma-IFN) (pMP6A-mIFN-gamma) was used to generate cytokine-secreting murine tumor cell vaccines. High levels of gamma-IFN and elevated class I major histocompatibility complex expression after transfer of pMP6A-mIFN-gamma into the murine lung cancer cell line, D122, was demonstrated. The efficiency of gene transfer was determined by two different methods and was estimated to be 10-15%. Irradiated gamma-IFN D122 cells generated by this novel gene delivery system (D122/pMP6A-mIFN-gamma) and also by standard retroviral methods (DIF2) were administered as weekly vaccinations by intraperitoneal injection to animals bearing 7-day-old intrafootpad D122 tumors. Hindlimb amputation was performed when footpad diameters reached 7 mm, and lungs were harvested 28 days later. Animals vaccinated with gamma-IFN-secreting D122 cells produced by AAV-based plasmids delivery demonstrated a significant delay in footpad tumor growth when compared with controls and DIF2 cells. Fifty-seven percent of animals vaccinated with D122/pMP6A-mIFN-gamma were free of pulmonary metastases 28 days after amputation, significantly improved from the 0, 7, and 15% observed in animals vaccinated with irradiated parental D122 cells, irradiated D122 cells lipofected with an empty-cassette vector (pMP6A), or DIF2 cells, respectively. These results and the ability to transfer genes with this delivery system to a broad range of tumor types support its use in the generation of cytokine-secreting tumor cell vaccinations for use in clinical trials.
体外基因工程改造的分泌细胞因子的肿瘤细胞疫苗已被证明可在肺癌和乳腺癌动物模型中预防转移性疾病。由于现有基因递送方式在转导原代人肿瘤细胞方面效率低下，因此难以将该策略应用于临床。在本研究中，使用脂质体介导递送基于腺相关病毒（AAV）的质粒，该质粒包含鼠γ干扰素（γ-IFN）序列（pMP6A-mIFN-γ），以生成分泌细胞因子的鼠肿瘤细胞疫苗。将pMP6A-mIFN-γ转入鼠肺癌细胞系D122后，证明有高水平的γ-IFN和I类主要组织相容性复合体表达升高。通过两种不同方法确定了基因转移效率，估计为10 - 15%。通过这种新型基因递送系统（D122/pMP6A-mIFN-γ）以及标准逆转录病毒方法（DIF2）产生的经辐照的γ-IFN D122细胞，通过腹腔注射作为每周一次的疫苗接种给予携带7日龄足垫内D122肿瘤的动物。当足垫直径达到7毫米时进行后肢截肢，并在28天后收获肺部。与对照和DIF2细胞相比，接种基于AAV质粒递送产生的分泌γ-IFN的D122细胞的动物足垫肿瘤生长明显延迟。接种D122/pMP6A-mIFN-γ的动物中有57%在截肢后28天无肺转移，与接种经辐照的亲本D122细胞、用空载体（pMP6A）脂质转染的辐照D122细胞或DIF2细胞的动物中分别观察到的0%、7%和15%相比有显著改善。这些结果以及使用该递送系统将基因转移到广泛肿瘤类型的能力支持其用于生成用于临床试验的分泌细胞因子的肿瘤细胞疫苗。
