MOLECULAR CLONING OF HLA CLASS II TUMOR ANTIGENS

我们的主要目标是开发一种新的技术，利用一种独特的丝状噬菌体克隆方法来鉴定人类白细胞抗原II类肿瘤抗原，这在过去还没有被应用于II类抗原的克隆。与现有的II类抗原克隆或生化多肽分离方法相比，该方法具有许多潜在的优势。这种独特的克隆方法将适用于来自不同组织类型肿瘤的各种肿瘤相关的人类白细胞抗原II类抗原，也适用于与感染性疾病有关的II类抗原。主动特异性免疫治疗在治疗黑色素瘤或结直肠癌(CRC)方面具有巨大的潜力。鉴于CD4+、HLAII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。到目前为止，几乎没有发现与肿瘤相关的TH抗原或具有疫苗潜力的多肽。这项拟议研究的长期目标是确定黑色素瘤和结直肠癌患者具有疫苗潜力的TH抗原。Th抗原可被现有的Th系和克隆识别。利用丝状噬菌体克隆哺乳动物HLAII类依赖Th抗原的新方法将被开发。肿瘤细胞的cDNA文库通过噬菌体表达，然后通过抗原提呈细胞将文库噬菌体呈递给TH细胞，并在细胞因子释放试验中鉴定相关的Th抗原。为了开发这一方法，我们提供了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。具体地说，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初步研究)；2)优化在初步研究下开发的丝状噬菌体HLAII类抗原克隆方法，以克隆黑色素瘤和结直肠癌相关Th抗原；随后将对这些抗原进行鉴定和鉴定(R33)。通过用表达不同抗原的噬菌体免疫小鼠，成功地诱导了抗原特异性的Th细胞依赖性抗体，从而强调了丝状噬菌体方法用于克隆人类白细胞抗原II类抗原的可行性。这项拟议的研究将开发一种新的技术，利用丝状噬菌体识别人类白细胞抗原II类依赖的Th抗原，从而导致使用这些抗原进行主动特异性癌症免疫治疗的新方法。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.