Development of Monoclonal Antibodies to Inhibit RAGE Activation in Pancreatic Cancer tumors

_________________________________________________________________________________________________________________________ 项目4：研制抑制胰腺癌RAGE激活的单抗 肿瘤(PI：埃斯特尔·莱克勒克博士) 项目摘要 胰腺癌(PC)是最具破坏性的疾病之一，存活率很低。PC的特点是 由于存在非常密集的促结缔组织间质，这阻碍了药物向肿瘤核心的输送。 最近的证据表明，晚期糖基化终产物受体(RAGE)在 对PC的进展和结缔组织增生的形成有显著的作用。RAGE是一种细胞表面受体， 是由细胞外配体激活的。在PC中也发现了大量的RAGE配体 肿瘤间质。我们已经鉴定出显著降低PC体外增殖的抗RAGE抗体 细胞。鉴于此，本提案的目的是证明抗RAGE抗体可以减少PC肿瘤 通过抑制PC细胞的增殖和肿瘤的促结缔组织生长。 我们将通过四个具体目标来检验我们的假设。在目标1中，我们将测量新的结合亲和力 针对RAGE的抗RAGE抗体，并鉴定这些抗RAGE抗体的结合表位。这些 杂交瘤将产生抗体。测量将在体外、通过酶联免疫吸附试验和表面试验进行。 等离子激元共振。抗体的表位将通过使用RAGE的多肽阵列来确定。在这 目的：我们还将通过流式细胞仪检测抗体与细胞表面RAGE的结合 荧光显微镜。在目标2中，我们将测试抗RAGE抗体是否能抑制增殖， PC细胞的迁移或入侵。我们还将通过以下方式确定RAGE抑制的分子机制 抗愤怒抗体。PC肿瘤中被激活的关键信号分子的水平将被确定 经免疫印迹分析，并与治疗后的抗体进行比较。在目标3中，我们将测试以下能力 抗RAGE抗体抑制3D球体中成纤维细胞的激活。这些椭球体将从 PC细胞与成纤维细胞的共培养。抗体的抑制机制将通过以下方式进行研究 活化细胞分泌细胞外基质蛋白和趋化因子水平的变化 成纤维细胞。在目标4中，我们将在PC的KPC小鼠模型中测试我们的抗体的有效性。KPC小鼠 自发性地发展出与人类相似的胰腺肿瘤。我们将比较PC的增长 用抗RAGE抗体治疗和未治疗的动物肿瘤。我们还将调查是否治疗 携带抗RAGE抗体的荷瘤小鼠结缔组织增生减少，结缔组织增生增加 对吉西他滨的敏感性。 我们提出的研究将为更好地理解PC发展的分子机制提供帮助 以及RAGE在PC中的作用。我们的项目也可以转化为治疗PC的新治疗策略 病人。 _________________________________________________________________________________________________________________________

_________________________________________________________________________________________________________________________ Project-4: Development of Monoclonal Antibodies to inhibit RAGE Activation in Pancreatic Cancer Tumors (PI: Dr. Estelle Leclerc) Project Summary Pancreatic cancer (PC) is one of the most devastating disease with very low survival rates. PC is characterized by the presence of a very dense desmoplastic stroma, which hinders the delivery of drugs to the tumor's core. Recent evidence suggests that the Receptor for Advanced Glycation End products (RAGE) contributes significantly to the progression of PC and to the formation of desmoplasia. RAGE is a cell-surface receptor that is activated by extracellular ligands. High levels of RAGE ligands have also been found in abundance in PC tumor stroma. We have identified anti-RAGE antibodies that reduced significantly the in vitro proliferation of PC cells. Given this, the goal of this proposal is to demonstrate that anti-RAGE antibodies can reduce PC tumor growth by both decreasing PC cell proliferation and tumor desmoplasia. We will test our hypothesis through four Specific Aims. In Aim 1, we will measure the binding affinity of new anti-RAGE antibodies for RAGE, and identify the binding epitopes of these anti-RAGE antibodies. These antibodies will be generated from hybridomas. Measurements will be performed in vitro, by ELISA and surface plasmon resonance. The epitopes of the antibodies will be determined by using peptide arrays of RAGE. In this aim, we will also measure the binding of the antibodies to cell surface RAGE by flow cytometry and fluorescence microscopy. In Aim 2, we will test whether the anti-RAGE antibodies can inhibit the proliferation, migration, or invasion of PC cells. We will also determine the molecular mechanisms of RAGE inhibition by anti-RAGE antibodies. The levels of key signaling molecules that are activated in PC tumors will be determined by Western blot analysis and compared after treatment with the antibodies. In Aim 3, we will test the abilities of anti-RAGE antibodies to inhibit fibroblasts' activation in 3D-spheroids. These spheroids will be generated from co-cultures of PC cells with fibroblasts. The mechanisms of inhibition by the antibodies will be studied by investigating changes in the levels of extracellular matrix proteins and chemokines secreted by activated fibroblasts. In Aim 4, we will test the efficacy of our antibodies in the KPC mouse model of PC. KPC mice develop spontaneously pancreatic tumors that resemble human ones. We will compare the growth of PC tumors in animal treated and non-treated with anti-RAGE antibodies. We will also investigate whether treating the tumor-bearing mice with anti-RAGE antibodies results in decreased desmoplasia and in increased sensitivity toward gemcitabine. Our proposed research will provide a better understanding of the molecular mechanisms of PC development and of the role of RAGE in PC. Our project could also be translated into a new therapeutic strategy to treat PC patients. _________________________________________________________________________________________________________________________