MOLECULAR CLONING OF HLA CLASS II TUMOR ANTIGENS

我们的主要目标是开发一种新的技术，利用独特的丝状噬菌体克隆方法来鉴定HLA II类肿瘤抗原，这在过去还没有应用于II类抗原的克隆。与现有的II类抗原克隆或生化肽分离方法相比，这种方法具有许多潜在的优势。本文提出的独特克隆方法将适用于来自不同组织学类型肿瘤的多种肿瘤相关HLAⅱ类抗原，也适用于涉及感染性疾病的ⅱ类抗原。主动特异性免疫疗法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CD4+、HLA ii类依赖的辅助性T （TH）细胞在控制肿瘤生长中的重要作用，需要开发利用TH细胞定义的抗原进行主动免疫治疗的方法。迄今为止，很少有肿瘤相关的TH抗原或具有疫苗潜力的肽被确定。拟议研究的长期目标是鉴定具有黑色素瘤和结直肠癌患者疫苗潜力的TH抗原。可用的Th系和克隆都能识别Th抗原。一种利用丝状噬菌体克隆哺乳动物HLA ii类依赖Th抗原的新方法将被开发出来。噬菌体首先表达肿瘤细胞cDNA文库，然后由抗原呈递细胞将文库噬菌体呈递至TH细胞，并通过细胞因子释放试验鉴定相关TH抗原。为了开发这种方法，我们有一个独特的模型系统，包括抗破伤风类毒素的Th细胞和编码破伤风类毒素相关Th表位的cDNA片段。具体而言，我们将：1)开发以丝状噬菌体和破伤风类毒素为模型系统的Th抗原克隆技术（R21中试研究）；2)优化前期研究开发的丝状噬菌体HLAⅱ类抗原克隆方法，克隆黑色素瘤和crc相关Th抗原；随后将对这些抗原（R33）进行鉴定和鉴定。通过用表达多种抗原的噬菌体免疫小鼠成功诱导抗原特异性、Th细胞依赖性抗体，强调了丝状噬菌体方法克隆HLA II类抗原的可行性。拟议的研究将开发一种利用丝状噬菌体鉴定HLA ii类依赖的Th抗原的新技术，从而为利用这些抗原进行活性特异性癌症免疫治疗提供新方法。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.