MOLECULAR CLONING OF HLA CLASS II TUMOR ANTIGENS

我们的主要目标是开发一种新的技术来识别HLA II类肿瘤抗原，使用一种独特的克隆方法在丝状真菌，这在过去还没有应用于II类抗原的克隆。 这种方法与现有的II类抗原克隆或生化肽分离方法相比具有许多潜在的优点。这里提出的独特的克隆方法将适用于各种肿瘤相关的HLA II类抗原来自不同的组织学类型的肿瘤，也涉及感染性疾病的II类抗原。主动特异性免疫治疗在黑色素瘤或结直肠癌（CRC）患者的治疗中具有巨大的潜力。 鉴于CD 4+、HLA II类依赖性辅助性T（TH）细胞在控制肿瘤生长中发挥的重要作用，需要开发使用TH细胞定义的抗原的主动免疫疗法的方法。 到目前为止，已经鉴定出非常少的具有疫苗潜力的肿瘤相关TH抗原或肽。 拟议研究的长期目标是鉴定具有黑色素瘤和CRC患者疫苗潜力的TH抗原。 Th抗原被可用的Th系和克隆识别。 一种新的方法克隆哺乳动物HLA II类依赖性Th抗原将开发使用丝状真菌。肿瘤细胞cDNA文库在噬菌体中表达，然后通过抗原提呈细胞将文库提呈给Th细胞，并在细胞因子释放试验中鉴定相应的Th抗原。 为了开发这种方法，我们有一个独特的模型系统，包括Th细胞对破伤风类毒素和编码破伤风类毒素相关的Th表位的cDNA片段。 具体而言，我们将：1）使用丝状噬菌体和破伤风类毒素作为模型系统开发Th抗原克隆技术（R21中试研究）;和2）优化在中试研究下开发的丝状噬菌体HLA II类抗原克隆方法，以克隆黑素瘤和CRC相关Th抗原;随后将鉴定和表征这些抗原（R33）。丝状噬菌体方法克隆HLA II类抗原的可行性，强调了成功诱导抗原特异性，Th细胞依赖性抗体在小鼠中通过用表达各种抗原的抗体免疫小鼠的示范。拟议的研究将开发一种新的技术，以确定HLA II类依赖性Th抗原使用丝状菌导致新的方法，主动特异性癌症免疫治疗使用这些抗原。 这些抗原也可能具有诊断价值。

Our major goal is to develop a novel technology to identify HLA class II tumor antigens using a unique cloning approach in filamentous phages which has not been applied to the cloning of class II antigens in the past. This approach has numerous potential advantage over existing approaches to class II antigen cloning or biochemical peptide isolation. The unique cloning approach proposed here will be applicable to a variety of tumor-associated HLA class II antigens derived from tumors of different histologic types, and also to class II antigens involved in infectious diseases. Active specific immunotherapy has great potential for the treatment of patients with melanoma or colorectal carcinoma (CRC). In light of the important role that CD4+, HLA class II-dependent helper T (TH) cells play in the control of tumor growth, approaches to active immunotherapy with TH cell-defined antigens need to be developed. Very few tumor- associated TH antigens or peptides with vaccine potential have been identified thus far. The long-term goal of the proposed studies is to identify TH antigens with vaccine potential for melanoma and CRC patients. The Th antigens are recognized by available Th lines and clones. A novel approach for the cloning of mammalian HLA class II-dependent Th antigens will be developed using filamentous phages. Tumor cells cDNA libraries will be expressed by the phages, followd by library phage presentation to TH cell by antigen- presenting cells and identification of the relevant Th antigen in cytokine release assay. To develop this approach, we have available a unique model system including Th cells against tetanus toxoid and a cDNA fragment encoding the tetanus toxoid-associated Th epitope. Specifically we will: 1) develop Th antigen cloning technology using filamentous phages and tetanus toxoid as a model system (R21 pilot study); and 2) optimize the filamentous phage HLA class II antigen cloning approach developed under the pilot study to clone melanoma and CRC-associated Th antigens; this will be followed by the identification and characrterization of these antigens (R33). The feasibility of the filamentous phage approach for the cloning of HLA class II antigens is emphasized by the demonstration of successful induction of antigen-specific, Th cell-dependent antibodies in mice by immunizing the mice with phages expressing various antigens. The proposed studies will develop a novel technology to identify HLA class II-dependent Th antigens using filamentous phages leading to novel approaches to active specific cancer immunotherapy using these antigens. The antigens also may be of diagnostic value.