Fenton R G, Taub D D, Kwak L W, Smith M R, Longo D L
Clinical Research Branch, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Md 21702-1201.
J Natl Cancer Inst. 1993 Aug 18;85(16):1294-302. doi: 10.1093/jnci/85.16.1294.
Activated forms of the ras proto-oncogene have been found in approximately 30% of human malignancies, including pancreatic, colon, and lung adenocarcinomas. Ras oncoproteins arise by somatic mutation and contain amino acid changes at residues 12, 13, or 61, thus generating unique tumor-specific proteins that are attractive targets for cancer therapy.
The goal of this study was to determine whether vaccination with mutant Ras protein could lead to the generation of cytotoxic T lymphocytes (CTLs) specific for the mutant epitope and to protection against challenge with tumor cells expressing the mutant oncoprotein.
To determine a methodology for generating CTL responses following immunization with soluble protein, ovalbumin was used as a model tumor antigen. C57BL/6 mice were immunized with soluble ovalbumin administered intraperitoneally at 2-week intervals or with intravenous injection of ovalbumin or osmotically loaded splenocytes. Immunized mice were challenged with E.G7 cells (which express a transfected ovalbumin gene), and tumor growth was monitored. Generation of ovalbumin-specific CTLs was determined by 51Cr release assays. Purified wild-type or mutant H-Ras proteins (containing single amino acid substitutions at position 12 converting Gly to Arg or Val) were used to immunize BALB/c mice intraperitoneally. Ras-immunized mice were challenged with tumor cells containing Arg 12 or Val 12 mutations or not harboring mutant forms of Ras. Cytolytic and proliferative responses to mutant forms of Ras were studied, and the effects of in vivo depletion of CD4+ or CD8+ T lymphocytes were determined.
In vivo challenge with E.G7 showed that intraperitoneal immunization with soluble ovalbumin was as effective as osmotic loading, resulting in long-term disease-free survival of some mice and the development of ovalbumin-specific CTLs. Immunization with Arg 12 Ras led to disease-free survival in nine of 10 animals challenged with tumor cells containing an Arg 12 mutation, while no protection was afforded against tumors expressing other forms of Ras or other oncogenes. Splenocytes from BALB/c mice immunized with Arg 12 Ras demonstrated cytolytic activity specific against tumor cells expressing Arg 12 Ras, with most of this activity residing in the CD8+ subset. Mutation-specific proliferation to Arg 12 Ras peptides was also observed. Immunization with Val 12 Ras did not elicit detectable Val 12-specific immunity.
Antigen-specific CTLs can be induced following intraperitoneal immunization of mice with purified, soluble proteins. For both ovalbumin and Arg 12 Ras, specific in vivo protection against tumor cell challenge was observed.
在大约30%的人类恶性肿瘤中发现了原癌基因ras的激活形式，包括胰腺癌、结肠癌和肺腺癌。Ras癌蛋白通过体细胞突变产生，在第12、13或61位氨基酸处发生变化，从而产生独特的肿瘤特异性蛋白，这些蛋白是癌症治疗的有吸引力的靶点。
本研究的目的是确定用突变型Ras蛋白进行疫苗接种是否能导致产生针对突变表位的细胞毒性T淋巴细胞（CTL），并预防用表达突变癌蛋白的肿瘤细胞进行攻击。
为了确定用可溶性蛋白免疫后产生CTL反应的方法，将卵清蛋白用作模型肿瘤抗原。C57BL/6小鼠每隔2周腹腔注射可溶性卵清蛋白，或静脉注射卵清蛋白或经渗透加载的脾细胞进行免疫。用E.G7细胞（表达转染的卵清蛋白基因）攻击免疫的小鼠，并监测肿瘤生长。通过51Cr释放试验确定卵清蛋白特异性CTL的产生。纯化的野生型或突变型H-Ras蛋白（在第12位含有单个氨基酸取代，将甘氨酸转换为精氨酸或缬氨酸）用于腹腔免疫BALB/c小鼠。用含有精氨酸12或缬氨酸12突变的肿瘤细胞或不含有Ras突变形式的肿瘤细胞攻击经Ras免疫的小鼠。研究了对Ras突变形式的细胞溶解和增殖反应，并确定了体内去除CD4+或CD8+T淋巴细胞的效果。
用E.G7进行体内攻击表明，腹腔注射可溶性卵清蛋白与渗透加载一样有效，导致一些小鼠长期无病生存，并产生卵清蛋白特异性CTL。用精氨酸12 Ras免疫导致10只接受含有精氨酸12突变的肿瘤细胞攻击的动物中有9只无病生存，而对表达其他形式Ras或其他癌基因的肿瘤没有提供保护。用精氨酸12 Ras免疫的BALB/c小鼠的脾细胞表现出对表达精氨酸12 Ras的肿瘤细胞的特异性细胞溶解活性，这种活性大部分存在于CD8+亚群中。还观察到对精氨酸12 Ras肽的突变特异性增殖。用缬氨酸12 Ras免疫未引发可检测到的缬氨酸12特异性免疫。
用纯化的可溶性蛋白腹腔免疫小鼠后可诱导抗原特异性CTL。对于卵清蛋白和精氨酸12 Ras，均观察到对肿瘤细胞攻击的特异性体内保护作用。
