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

_________________________________________________________________________________________________________________________ 项目-4：开发单克隆抗体以抑制胰腺癌中的p53活化 肿瘤（主要研究者：Estelle Leclerc博士） 项目摘要 胰腺癌（PC）是最具破坏性的疾病之一，生存率非常低。PC的特点是 由于存在非常致密的促结缔组织增生间质，这阻碍了药物向肿瘤核心的输送。 最近的证据表明，晚期糖基化终末产物受体（Receptor for Advanced Glycation End Products，CRN） 显著促进PC的进展和结缔组织增生的形成。β-淀粉样蛋白是一种细胞表面受体， 被细胞外配体激活。在PC中也发现了大量的高水平的配体 肿瘤间质我们已经鉴定出抗-β-CD抗体，其显著降低PC的体外增殖， 细胞鉴于此，本提案的目标是证明抗CD 4抗体可以减少PC肿瘤 通过减少PC细胞增殖和肿瘤结缔组织增生来促进生长。 我们将通过四个具体目标来验证我们的假设。在目标1中，我们将测量新的化合物的结合亲和力。 用于抗ESTA的抗ESTA抗体，并鉴定这些抗ESTA抗体的结合表位。这些 抗体将由杂交瘤产生。将通过ELISA和表面活性剂在体外进行测量。 等离子共振抗体的表位将通过使用寡核苷酸的肽阵列来确定。在这 目的，我们还将通过流式细胞术测量抗体与细胞表面结合， 荧光显微镜在目标2中，我们将测试抗-β-CD抗体是否可以抑制增殖， PC细胞的迁移或侵袭。我们还将通过以下方法确定抑制细胞凋亡的分子机制： 抗-β-内酰胺抗体。将确定在PC肿瘤中激活的关键信号分子的水平 通过Western印迹分析并在用抗体处理后进行比较。在目标3中，我们将测试 抗-β-内酰胺酶抗体以抑制3D-球状体中的成纤维细胞的活化。这些球体将由 PC细胞与成纤维细胞的共培养物。将通过以下方法研究抗体的抑制机制： 研究细胞外基质蛋白和趋化因子水平的变化， 成纤维细胞在目的4中，我们将测试我们的抗体在PC的KPC小鼠模型中的功效。KPC小鼠 会自发产生类似人类的胰腺肿瘤我们将比较PC的增长 用抗肿瘤抗体治疗和未治疗的动物中的肿瘤。我们还将调查是否治疗 具有抗CD 14抗体的荷瘤小鼠导致结缔组织增生减少和结缔组织增生增加， 对吉西他滨的敏感性 我们提出的研究将提供一个更好的理解的分子机制的PC发展 以及在PC中的角色。我们的项目也可以转化为一种新的治疗策略来治疗PC 患者 _________________________________________________________________________________________________________________________

_________________________________________________________________________________________________________________________ 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. _________________________________________________________________________________________________________________________