Fenton R G, Keller C J, Hanna N, Taub D D
Division of Clinical Sciences, National Cancer Institute, National Cancer Institute-Frederick Cancer Research and Development Center (NCI-FCRDC), MD 21702, USA.
J Natl Cancer Inst. 1995 Dec 20;87(24):1853-61. doi: 10.1093/jnci/87.24.1853.
Point mutations in the ras proto-oncogene that activate its oncogenic potential occur in approximately 30% of human cancers. Previous studies have demonstrated that T-cell immunity against some forms of mutant Ras proteins could be elicited, and some effectiveness against tumors expressing activated Ras has been reported.
The goal of this study was to determine if immunization of mice with two forms of mutant Ras protein can induce high levels of Ras mutation-specific T-cell immunity in vitro and tumor regression in vivo.
Mice (BALB/c or C3H/HeJ) were immunized subcutaneously at 2-week intervals with purified Ras oncoproteins mixed with the immunologic adjuvants Antigen Formulation or QS-21, both of which have been shown to enhance the induction of T-cell-mediated immunity when included as components of soluble protein vaccines. In some experiments, mice were immunized directly with heat-killed Escherichia coli that had been induced to express one of the mutant Ras proteins. Spleen cells plus lymph node cells from Ras-immunized mice were tested in vitro for lysis of syngeneic Ras-expressing tumor cells and proliferation in response to mutant Ras peptides. For some of the cytolytic activity experiments, the spleen cells were grown under TH1 conditions (growth in presence of interleukin 2, interferon gamma, and an antibody directed against interleukin 4 to stimulate a cell-mediated immune response) or TH2 conditions (growth in presence of interleukins 2 and 4 to stimulate a humoral immune response). The specificity of immunity was examined in vivo by challenge of Ras-immunized mice with syngeneic tumor cells expressing mutant Ras oncoproteins (HaBalb, i.e., BALB/c mouse cells expressing Ras with arginine substituted at amino acid position 12 [Arg 12 Ras]; C3HL61, i.e., C3H/HeJ mouse cells expressing Ras with leucine substituted at position 61 [Leu 61 Ras]). Ten mice per group were used in each experiment.
Proliferative and cytolytic T-cell responses directed against the Arg 12 Ras protein were generated in BALB/c mice, resulting in protection against challenge with cells expressing Arg 12 Ras and therapeutic benefit in mice bearing established tumors expressing this protein. In C3H/HeJ mice, high levels of cytolytic and proliferative responses were induced against Leu 61 Ras. Immunization with heat-killed E. coli genetically engineered to express Leu 61 Ras also led to the induction of anti-Ras T-cell immunity. T cells grown under TH1 conditions were cytolytic against Ras-transformed tumor cells, whereas those grown under TH2 conditions were not.
Immunization as described here leads to Ras mutation-specific antitumor immunity in vitro and in vivo, with therapeutic efficacy in an established tumor model.
ras原癌基因中的点突变激活其致癌潜能，约30%的人类癌症中会出现这种情况。先前的研究表明，可以引发针对某些形式突变Ras蛋白的T细胞免疫，并且已报道对表达活化Ras的肿瘤有一定疗效。
本研究的目的是确定用两种形式的突变Ras蛋白免疫小鼠是否能在体外诱导高水平的Ras突变特异性T细胞免疫，并在体内诱导肿瘤消退。
以2周的间隔对小鼠（BALB/c或C3H/HeJ）进行皮下免疫，免疫原是与免疫佐剂抗原制剂或QS-21混合的纯化Ras癌蛋白，这两种佐剂作为可溶性蛋白疫苗的成分时，均已显示能增强T细胞介导免疫的诱导。在一些实验中，直接用经诱导表达其中一种突变Ras蛋白的热灭活大肠杆菌免疫小鼠。对来自经Ras免疫的小鼠的脾细胞和淋巴结细胞进行体外测试，检测其对同基因表达Ras的肿瘤细胞的裂解作用以及对突变Ras肽的增殖反应。对于一些细胞溶解活性实验，脾细胞在TH1条件下培养（在白细胞介素2、干扰素γ和抗白细胞介素4抗体存在的情况下培养以刺激细胞介导的免疫反应）或在TH2条件下培养（在白细胞介素2和4存在的情况下培养以刺激体液免疫反应）。通过用表达突变Ras癌蛋白的同基因肿瘤细胞（HaBalb，即表达在氨基酸位置12处精氨酸被取代的Ras的BALB/c小鼠细胞；C3HL61，即表达在位置61处亮氨酸被取代的Ras的C3H/HeJ小鼠细胞）攻击经Ras免疫的小鼠，在体内检测免疫的特异性。每个实验每组使用10只小鼠。
BALB/c小鼠产生了针对精氨酸12 Ras蛋白的增殖性和细胞溶解性T细胞反应，从而对表达精氨酸12 Ras的细胞攻击产生保护作用，并对携带表达该蛋白的已建立肿瘤的小鼠有治疗益处。在C3H/HeJ小鼠中，诱导了针对亮氨酸61 Ras的高水平细胞溶解和增殖反应。用经基因工程改造以表达亮氨酸61 Ras的热灭活大肠杆菌免疫也导致了抗Ras T细胞免疫的诱导。在TH1条件下培养的T细胞对Ras转化的肿瘤细胞具有细胞溶解性，而在TH2条件下培养的T细胞则没有。
此处所述的免疫在体外和体内均可导致Ras突变特异性抗肿瘤免疫，并在已建立的肿瘤模型中具有治疗效果。
