Hirschowitz E A, Leonard S, Song W, Ferris B, Leopold P L, Lewis J J, Bowne W B, Wang S, Houghton A N, Crystal R G
Division of Pulmonary and Critical Care Medicine, New York Hospital-Cornell Medical Center, NY 10021, USA.
Gene Ther. 1998 Jul;5(7):975-83. doi: 10.1038/sj.gt.3300668.
Melanocyte differentiation antigens, such as the brown locus protein gp75, are potential biological targets for immunotherapy. We investigated whether expression of the murine gp75 cDNA mediated by an adenovirus (Ad) vector could induce melanoma rejection using this model self antigen that usually induces tolerance, and whether Ad vector-directed production of interleukin-2 (IL2) might augment this response. To evaluate this approach, Ad vectors were constructed containing the murine gp75 cDNA (Ad.gp75) and the human IL2 cDNA (Ad.IL2). Efficacy was evaluated in C57BI/6 mice challenged i.v. with 10(5) B16 cells, using the number of lung metastases as the efficacy parameter. Naive control mice developed 175 +/- 12 metastases by day 14. Controls receiving intranasal Ad.IL2 1 day after B16 cell injection, intraperitoneal (i.p.) mitomycin-C-treated B16 cells +/- i.p. Ad.IL2 before B16 cell challenge and Ad.beta gal-treated mice had similar numbers of metastases as controls (P > 0.1). In marked contrast, preimmunization with intradermal Ad.gp75 provided dramatic reduction in the number of lung metastases (52 +/- 7, 29% of control). Addition of regional (intranasal delivery to the lung) Ad.IL2 to intradermal Ad.gp75 preimmunization 1 day following tumor challenge provided further protection (18 +/- 6, 10% of control). Depletion of CD4+ and CD8+ T-cell subsets effectively blocked the protective effect seen following immunization. Adoptive transfer of macrophage-depleted splenocytes from Ad.gp75-immunized mice similarly afforded significant protection against B16 tumor cell challenge. Further, serum obtained 21 days following Ad.gp75 immunization showed no detectable anti-gp75 antibody by immunoprecipitation. These results suggest that immunization with Ad.gp75 induces cellular immune responses that are capable of rejecting B16 melanoma in a host that is usually tolerant to gp75 antigen.
黑素细胞分化抗原，如棕色基因座蛋白gp75，是免疫治疗的潜在生物学靶点。我们利用这种通常诱导耐受的模型自身抗原，研究了腺病毒（Ad）载体介导的鼠gp75 cDNA表达是否能诱导黑色素瘤排斥反应，以及Ad载体定向产生的白细胞介素-2（IL-2）是否会增强这种反应。为评估该方法，构建了包含鼠gp75 cDNA（Ad.gp75）和人IL-2 cDNA（Ad.IL2）的Ad载体。在经静脉注射10⁵个B16细胞攻击的C57BI/6小鼠中评估疗效，以肺转移灶数量作为疗效参数。未处理的对照小鼠在第14天出现175±12个转移灶。在B16细胞注射后1天接受鼻内Ad.IL2、在B16细胞攻击前接受腹腔（i.p.）丝裂霉素-C处理的B16细胞±腹腔内Ad.IL2以及接受Ad.β半乳糖苷酶处理的小鼠的转移灶数量与对照相似（P>0.1）。与之形成显著对比的是，皮内注射Ad.gp75进行预免疫可使肺转移灶数量显著减少（52±7，为对照的29%）。在肿瘤攻击后1天，将局部（经鼻递送至肺部）Ad.IL2添加到皮内Ad.gp75预免疫中可提供进一步的保护作用（18±6，为对照的10%）。CD4⁺和CD8⁺T细胞亚群的耗竭有效地阻断了免疫后所见的保护作用。从Ad.gp75免疫小鼠中分离出巨噬细胞耗竭的脾细胞进行过继转移，同样能提供显著的保护作用以抵抗B16肿瘤细胞攻击。此外，Ad.gp75免疫后21天获得的血清经免疫沉淀未检测到可检测的抗gp75抗体。这些结果表明，用Ad.gp75免疫可诱导细胞免疫反应，该反应能够在通常对gp75抗原耐受的宿主体内排斥B16黑色素瘤。
