GPR160 antibody development for cancer treatment

我们发现，一种针对人G蛋白第二胞外环的兔多克隆抗体- 偶联受体160(hGPR160/ECL2)有两个显著的作用：(1)直接抗癌作用 结肠癌和三重阴性乳腺癌(TNBC)细胞株表达受体而不改变 正常结肠癌细胞系的活性，以及(2)小分子对细胞毒作用的极大增强 化疗药物奥沙利铂，剂量本身没有明显的活性。这种增强表明 联合应用hGPR160/ECL2抗体可显著改善临床疗效。 与目前使用的相比，细胞毒素剂量更低，副作用更少。要翻译这些内容 观察到一种实用的治疗方法，我们建议研制一种人源性单抗hGPR160/ECL2用于治疗 进一步表征其抗癌作用，并作为开发治疗有用的人类的一步 HGPR160/ECL2单抗。目前的提案是对PAR-22-216的回应，旨在开发 并测试一种既能治疗癌症又能减轻癌症治疗相关毒性的新生物制剂。我们是 不知道GPR160有任何商业上可用的小分子配体。我们最近的出版物1和 初步数据表明hGPR160/ECL2单抗是一种可行的 化疗药物。然而，我们的初步研究使用了一种兔多克隆抗体，它具有不可还原的 关于结合地点的不确定因素。因此，人的单抗hGPR160/ECL2 抗体将解决这一问题，并允许毫不含糊地进行人体抗癌作用的体外实验 肿瘤细胞系和免疫缺陷小鼠人细胞系来源的异种移植(CDX)的体内实验。 在这里提出的三个目标的工作的成功将导致进一步的工作，开发一种治疗 单抗。在目标1中，我们将通过免疫的方法制备人源性抗hGPR160/ECL2的单抗 表达人类重基因和轻基因的转基因小鼠，带有hGPR160‘S ECL2氨基酸序列(或 其后续序列)连接到K1H并通过聚乙二醇化融合形成杂交瘤。我们将对克隆人进行筛选 通过流式细胞术检测抗体，并通过阻断结合试验对~10个抗体进行下选择 HGPR160/ECL2单抗克隆为进一步的体内外研究奠定了基础。然后我们将测试抗癌细胞的活性 我们的抗体的体外研究。在目标2中，前2-3个候选者(与配体的最佳结合亲和力 和体外细胞存活率测定中的半数致死浓度)将被用于(1)体内原位细胞系来源的异种移植 (CDX)结肠癌和TNBC小鼠模型。在目标3中，最佳候选方案将在体外进行奥沙利铂试验 和紫杉醇增强作用的研究。HGPR160/ECL2单抗可能证明是对 目前的抗癌治疗对很大比例的患者来说是不够的。然而，小分子 细胞毒素将继续发挥重要作用，并与hGPR160/ECL2抗体联合治疗 可以使它们的细胞毒性作用最大化，同时使用副作用少得多的剂量。

We discovered that a rabbit polyclonal antibody raised against the second extracellular loop of human G-protein- coupled receptor 160 (hGPR160/ECL2) has two remarkable actions: (1) direct anti-cancer effects on human colon cancer and triple negative breast cancer (TNBC) cell lines that express the receptor without altering the viability of a normal colon cancer cells line, and (2) great potentiation of the cytotoxic effects of the small molecule chemotherapeutic, oxaliplatin, at doses that by themselves have no significant activity. This potentiation indicates that combination with the hGPR160/ECL2 antibody might yield a considerable improvement in the clinical effect with cytotoxin doses that are lower and produce fewer side-effects than those in current use. To translate these observations into a practical therapy, we propose to develop a human monoclonal hGPR160/ECL2 antibody for further characterization of its anticancer effects and as a step towards developing a therapeutically useful human monoclonal hGPR160/ECL2 antibody. The current proposal is in response to PAR-22-216 with aims to develop and test a new biologic agent that both treats cancer and mitigates cancer treatment-related toxicities. We are unaware of any commercially available small molecule ligands for GPR160. Our recent publication1 and preliminary data presented here indicate that an hGPR160/ECL2 monoclonal antibody is a viable chemotherapeutic. However, our preliminary studies used a rabbit polyclonal antibody, which has an irreducible element of uncertainty concerning the locus of binding. Accordingly, a human monoclonal hGPR160/ECL2 antibody will address this issue and permit unambiguous in vitro experiments on anticancer effects in human cancer cell lines and in vivo experiments on human cell line-derived xenografts (CDX) in immunodeficient mice. Success with the work proposed here in three Aims would lead to further work to develop a therapeutic monoclonal antibody. In Aim 1, we will develop human monoclonal antibodies to hGPR160/ECL2 by immunizing transgenic mice expressing human heavy and light genes with the hGPR160’s ECL2 amino acid sequence (or subsequence thereof) conjugated to KLH and developing hybridomas via PEG-fusion. We will screen clonal antibodies by flow cytometry and down-select using a blocking-of-binding assay to a panel of ~10 hGPR160/ECL2 mAbs clones for further in vitro and in vivo studies. We will then test the anti-cancer cell activity of our antibodies with in vitro studies. In Aim 2, the top 2-3 candidates (best binding affinity against the ligand and LC50 in the in vitro cell viability assay) will be advanced for (1) in vivo orthotopic cell line-derived xenograft (CDX) mouse models of colon cancer and TNBC. In Aim 3, the top candidate will be tested in in vitro oxaliplatin and paclitaxel potentiation studies. An hGPR160/ECL2 mAb may prove to be a significant improvement to current anti-cancer treatments that are inadequate for a large percentage of patients. However, small molecule cytotoxins will continue to have an important role and combination therapy with an hGPR160/ECL2 antibody may allow their cytotoxic effects to be maximized while using doses with far fewer side effects.