GPR160 antibody development for cancer treatment

用于癌症治疗的 GPR160 抗体开发

基本信息

批准号：
10711206
负责人：
James D Brien
金额：
$ 21.25万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-16 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10711206
关键词：
Affinity Affinity Chromatography Alloys Amino Acid Sequence Antibodies Antibody Therapy Avidity Binding Biological Assay Biological Products Breast Breast Cancer Cell Breast Cancer cell line Cancer cell line Cause of Death Cell Culture Techniques Cell Line Cell Survival Cells Chemosensitization Cisplatin Clinical Colon Colon Carcinoma Combined Modality Therapy Cytotoxin Data Development Dose Dose Limiting Elements Esophagus Flow Cytometry G-Protein-Coupled Receptors Genes Goals Human Human Cell Line Hybridomas Immunize Immunodeficient Mouse In Vitro Keyhole Limpet Hemocyanin Ligands Light Lung MDA MB 231 Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of pancreas Malignant neoplasm of prostate Measures Mediating Modeling Monoclonal Antibodies Monoclonal Antibody Therapy Mus Oryctolagus cuniculus Paclitaxel Patients Prostate Publications Role SW480 Stomach Testing Therapeutic Therapeutic Monoclonal Antibodies Toxic effect Transgenic Mice Translating Trastuzumab Treatment-Related Cancer Treatment-related toxicity Uncertainty Work Xenograft Model Xenograft procedure anti-cancer anticancer treatment cancer cell cancer therapy clinical effect clinical efficacy clinical predictors colon cancer cell line cytotoxic experimental study extracellular human monoclonal antibodies improved in vitro Model in vivo malignant breast neoplasm mouse model neoplastic cell overexpression oxaliplatin pharmacologic polyclonal antibody programs receptor response scale up side effect small molecule success therapy development triple-negative invasive breast carcinoma tumor

项目摘要

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.

我们发现针对人 G 蛋白第二个胞外环产生的兔多克隆抗体偶联受体160（hGPR160/ECL2）有两个显着的作用：（1）对人体有直接的抗癌作用结肠癌和三阴性乳腺癌 (TNBC) 细胞系表达受体而不改变正常结肠癌细胞系的活力，以及（2）小分子细胞毒性作用的极大增强化疗药物奥沙利铂本身没有显着活性。与hGPR160/ECL2抗体联合可能会显着改善临床效果与目前使用的细胞毒素剂量相比，产生的副作用更少。将观察结果转化为实际疗法，我们建议开发一种人单克隆 hGPR160/ECL2 抗体进一步表征其抗癌作用，并作为开发具有治疗作用的人类的一步目前的提案是为了响应 PAR-22-216，旨在开发单克隆 hGPR160/ECL2 抗体。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。不知道我们最近发表的文章1 和 GPR160 的任何市售小分子配体。这里提供的初步数据表明 hGPR160/ECL2 单克隆抗体是一种可行的然而，我们的初步研究使用了兔多克隆抗体，它具有不可还原性。因此，人单克隆 hGPR160/ECL2 的结合位点存在不确定性。抗体将解决这个问题，并允许对人类抗癌作用进行明确的体外实验癌细胞系和免疫缺陷小鼠体内人细胞系来源的异种移植物（CDX）的体内实验。这里提出的三个目标工作的成功将导致进一步的工作，以开发一种治疗方法在目标1中，我们将通过免疫来开发针对hGPR160/ECL2的人单克隆抗体。表达具有 hGPR160 的 ECL2 氨基酸序列的人类重基因和轻基因的转基因小鼠（或其子序列）与 KLH 缀合并通过 PEG 融合开发杂交瘤。通过流式细胞术检测抗体，并使用结合阻断测定对约 10 组抗体进行向下选择 hGPR160/ECL2 mAb 克隆用于进一步的体外和体内研究，然后我们将测试抗癌细胞活性。在目标 2 中，排名前 2-3 的候选抗体（对配体的最佳结合亲和力）。和体外细胞活力测定中的 LC50）将被推进（1）体内原位细胞系衍生的异种移植 (CDX) 结肠癌和 TNBC 小鼠模型在目标 3 中，最佳候选药物将在体外进行奥沙利铂测试。 hGPR160/ECL2 mAb 可能被证明是对紫杉醇的显着改善。目前的抗癌疗法对于大部分患者来说是不够的，但是小分子疗法。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其细胞毒性作用最大化，同时使用副作用少得多的剂量。