Active Immunotherapy with Pox Vector Modified DC

这项修订的项目将测试编码肿瘤抗原癌胚抗原(CEA)的POX载体修饰的树突状细胞(DC)是否会在结肠癌患者中诱导肿瘤抗原特异性T细胞的临床有效频率。抗原特异性T细胞的激活和增殖对于临床有效的免疫反应至关重要。有希望的激活抗原特异性T细胞的方法包括使用抗原负载的DC。在资助的最初阶段，我们将携带编码CEA的mrna的DC应用于最小的 结肠癌肝转移切除后残留病变。虽然DC耐受性良好，但我们观察到CEA特异性免疫反应几乎没有增强，无复发生存率也没有改善。部分原因是DC内抗原表达和提呈不足，未成熟DC上低水平的共刺激分子(如CD80)导致T细胞活化不良，以及DC免疫原性有限 天然肿瘤抗原。为了克服这些问题，我们用表达三组共刺激分子(CD80、CD54和CD58，命名为Tricom)的重组复制缺陷痘载体(包括牛痘(Vvinia，RV)和鸡痘(Fowlpox，RF))以及修饰的肿瘤抗原CEA(6D)修饰DC，并通过氨基酸取代创建更有效的表位。在一项对CEA表达的恶性肿瘤患者进行的I期研究中，我们检测到0.1-0.5%的外周血单核细胞(PBMC)的抗原特异性T细胞应答。临床益处与最高水平的免疫反应有关。最近，人们观察到，异种Prime-Boost免疫对POX载体编码的抗原产生更高水平的免疫反应。建议先接种编码CEA的牛痘疫苗，然后再接种编码CEA的鸡痘疫苗，以提高晚期癌症患者的存活率。然而，诱导的CEA特异性免疫应答是PBMC的0.01%或更少。我们假设，通过提高CEA特异性T细胞活性水平，POX载体修饰的DC的初始增强策略将获得更大的临床益处。因此，我们提出了一项II期研究，在两种免疫策略中进行选择，即先感染RV-CEA(6D)-Tricom的DC，再感染RV-CEA(6D)-Tricom或RV-CEA(6D)-Tricom，然后是RF-CEA(6D)-Tricom，这与肝转移瘤切除和辅助化疗后2年的无瘤生存率有关。我们还将测量Elispot的CEA特异性免疫反应的速度和幅度。IL-2可增加CEA特异性T细胞对POX载体免疫的反应。因此，我们随后将探讨IL-2在最佳免疫策略下增强CEA特异性免疫的作用。

This revised project will test whether dendritic cells (DC) modified with pox vectors encoding the tumor antigen carcinoembryonic antigen (CEA) will induce a clinically-effective frequency of tumor antigen-specific T cells in patients with colon cancer. Antigen-specific T cell activation and proliferation are essential for clinically effective immune responses. Promising methods for activating antigen-specific T cells include the use of antigen loaded DC. During the initial period of funding, we administered DC loaded with mRNA encoding CEA to patients with minimal residual disease following resection of hepatic metastases of colon cancer. Although the DC were well-tolerated, we observed little augmentation of the CEA-specific immune response and no improvement in recurrence free survival. This can be explained partly by inadequate antigen expression and presentation within the DC, by poor T cell activation due to low levels of costimulatory molecules such as CD80 on the immature DC, and by limited immunogenicity of the native tumor antigen. To overcome these problems, we have modified DC with recombinant, replication-defective pox vectors (including vaccinia (rV) and fowlpox (rF)) that express a TRiad of COstimulatory Molecules (CD80, CD54, and CD58, designated TRICOM) as well as a modified tumor antigen CEA(6D), with an amino acid substitution that creates a more potent epitope. In a phase I study of patients with CEA-expressing malignancies who received ex vivo generated DC modified with rF-CEA(6D)-TRICOM, we detected antigen-specific T cell responses in the range of 0.1-0.5% of the peripheral blood mononuclear cells (PBMC). Clinical benefit was associated with the highest levels of immune response. Recently, it has been observed that greater levels of immune response to pox vector-encoded antigens occur with heterologous prime-boost immunizations. Priming with vaccinia encoding CEA followed by boosts with fowlpox encoding CEA has been suggested to improve survival in patients with advanced cancer. Nonetheless, the induced CEA-specific immune responses were 0.01% or less of the PBMC. We hypothesize that prime-boost strategies with pox vector-modified DC will achieve greater clinical benefit by increasing the level of CEA-specific T cell activity. Therefore, we propose a phase II study to choose between two immunization strategies, DC infected with rV-CEA(6D)-TRICOM followed by DC infected with rF-CEA(6D)-TRICOM or rV-CEA(6D)-TRICOM followed by rF-CEA(6D)-TRICOM, in terms of which is associated with a better rate of disease-free survival at 2 years following hepatic metastasis resection and adjuvant chemotherapy. We will also measure the rate and magnitude of the CEA-specific immune response by ELISPOT. IL-2 increases the magnitude of the CEA specific T cell responses to pox-vector immunizations. Therefore, we will subsequently explore the role of IL-2 in augmenting CEA-specific immunity when given with the optimal immunization strategy.