Clinical Trials with IL12

虽然延长的IL-12治疗已显示增加表达记忆表型标志物的外周血CD 8 + T细胞的数量，但尚未检查这些细胞的抗原特异性。本申请的目的之一是进行系列ELISPOT和四聚体分析，以确定全身性IL-12治疗是否影响能够在体外识别确定的黑素瘤肽抗原的循环CD 8 + T细胞的数量或表型。我们已经表明，IFN γ的诱导，IL-12诱导的肿瘤消退的关键决定因素，随着连续的IL-12注射而逐渐下降，并且这种下调的程度与临床结果呈负相关。尽管这种对IL-12的明显脱敏很可能是设计用于限制TH 1免疫应答的持续时间和严重性的先天生理机制，但它也可能限制IL-12作为抗肿瘤剂的治疗效用。因此，阐明未能维持IL-12诱导的IFN γ产生的分子机制是本申请的优先事项和目标之一。初步数据表明，这种失败不能归因于表达IL-12受体的细胞的消失或PBL中STAT的整体损失。本申请中提出的研究将评估受体接合下游的事件，例如IL-12和其他细胞因子在IL-12治疗过程中的不同时间点获得的PBL中体外诱导STAT磷酸化的能力。研究还将评估IL- 12治疗对细胞因子信号传导抑制剂（例如细胞因子信号传导抑制剂或SOCS家族成员）表达和Janus激酶（JAK）家族可调控成员的影响。最后，由于TH 2和病毒感染的T细胞的IFN γ的有限产生已被证明是继发于IFN γ启动子的超甲基化，我们建议评估在IL-12治疗的整个过程中连续获得的PBL中该启动子的甲基化状态。总的来说，这些研究可以提供对IL-12反应调节的见解，并提供如何增强这种重要细胞因子的抗肿瘤活性的线索。

Although prolonged IL-12 treatment has been shown to increase the number of peripheral blood CD8' T cells expressing memory phenotypic markers, the antigenic specificity of these cells has not been examined. One of the objectives of this application is to perform serial ELISPOT and tetramer analyses to determine if systemic IL-12 treatment affects the number or phenotype of circulating CD8+ T cells capable of recognizing defined melanoma peptide antigens in vitro. We have shown that the induction of IFNgamma, a critical determinant of IL-12-induced tumor regression, progressively declines with successive IL-12 injections and the extent of this down modulation correlates negatively with clinical outcome. Although this apparent desensitization to IL-12 is most likely an innate physiologic mechanism designed to restrict the duration and severity of TH 1 immune responses, it may also limit the therapeutic utility of IL-12 as an antitumor agent. The elucidation of the molecular mechanism underlying the failure to sustain IL-12-induced IFNgamma production is therefore a priority and one of the objectives of this application. Preliminary data suggest that this failure cannot be attributed to a disappearance of cells expressing IL-12 receptors or a global loss of STATs in PBL. The studies proposed in this application will evaluate events downstream of receptor engagement such as the ability of IL-12 and other cytokines to induce STAT phosphorylation in vitro in PBL obtained at various time points throughout the course of IL-12 treatment. The studies will also assess the effects of IL- 12 treatment on the expression of inhibitors of cytokine signaling (e.g. the Suppressor of Cytokine Signaling or SOCS family members) and on the regulable members of Janus Kinase (JAK) family. Finally, since the limited production of IFNgamma by TH2 and virus-infected T cells has been shown to be secondary to hypermethylation of the IFNgamma promoter, we propose to evaluate the state of methylation of this promoter in PBL obtained serially throughout the course of IL-12 treatment. Collectively, these studies may provide insights into the regulation of IL-12 responses and offer clues as to how the antitumor activity of this important cytokine might be enhanced.