Preclinical validation of oral therapeutic lead proteins targeting epithelial GM1 ganglioside for ulcerative colitis therapy

项目总结 目前，还没有治愈溃疡性结肠炎(UC)的药物，UC是一种炎症性肠炎 影响直肠和结肠最内层粘膜的疾病。尽管粘膜愈合是一项重要的 治疗目标，许多患者用现有的UC药物如抗炎、 免疫调节剂和抗肿瘤坏死因子药。由于上皮修复是粘膜愈合的关键过程， 促进这一过程的试剂将提供一种有别于现有临床护理的新治疗策略 适用于UC。我们的治疗线索是口服霍乱疫苗抗原霍乱毒素B亚单位(CTB)的变体，它 是用包括内质网保留基序(CTBSEKDEL)的C末端延伸修饰的。我们 最近发现口服CTBSEKDEL而非天然CTB可促进结肠上皮修复 在葡聚糖硫酸钠(DSS)诱导的急性结肠炎小鼠模型中的粘膜愈合。此外，每两周一次 口服CTBSEKDEL可显著降低偶氮甲烷/DSS结肠炎模型的肿瘤形成。 相关癌症。基于这些发现，我们假设CTBSEKDEL提供了一种口腔生物学原型 促进UC的粘膜愈合。此翻译版R01项目的目标是优化和验证 CTBSEKDEL在临床前UC模型中的治疗潜力。由于CTBSEKDEL已经在一个 对于急性结肠炎模型，我们将立即在慢性结肠炎模型中进行进一步的验证。同时，在 目标1，我们将创建在C-末端序列(CTB(X)H/KDEL)中进行修改的CTBSEKDEL变体以改进 肠溶制剂喷雾干燥时的分子稳定性。我们将使用一种 烟草植物瞬时过表达系统及其基于一系列生化指标的筛选 和生物物理检测，以及小鼠急性DSS结肠炎模型。在目标2中，我们将验证疗效和 CTBSEKDEL和选择的CTB(X)H/KDEL在两种基于重复DSS暴露的慢性结肠炎模型中的安全性 在C57BL/6小鼠和吡罗昔康暴露的IL-10基因敲除小鼠中，与抗肿瘤坏死因子α抗体进行比较。 治疗效果和安全性将由疾病活动性指数、组织病理学、 炎症、隐窝再生、上皮屏障的免疫组织化学和分子生物学分析 恢复和纤维化。在目标3中，我们将使用人类结肠外植体模型来进一步验证粘膜 CTBSEKDEL和CTB(X)H/KDEL的愈合潜能。结肠活检和结肠切除组织将从 有不同病史和生物学背景的患者。疗效将根据伤口情况进行评估 愈合相关基因的表达和上皮细胞增殖/再生标志物的免疫组织化学检测。细胞 类型特异性反应将在结肠固有层、单核细胞和结肠类器官中进行研究。 总的来说，该项目将产生关键的临床前数据，支持开发一流的口腔 诱导结肠上皮修复的生物学候选药物用于UC治疗的I期临床试验。

PROJECT SUMMARY Currently, there is no curative medication available for ulcerative colitis (UC), a type of inflammatory bowel disease affecting the innermost mucosal layer of the rectum and the colon. Although mucosal healing is a major treatment goal, many patients fail to achieve mucosal healing with available UC drugs such as anti-inflammatory, immuno-modulatory and anti-TNF agents. Since epithelial repair is a critical process towards mucosal healing, an agent facilitating this process will provide a novel therapeutic strategy differentiating from existing clinical care for UC. Our therapeutic lead is a variant of the oral cholera vaccine antigen cholera toxin B subunit (CTB), which was modified with a C-terminal extension including an endoplasmic reticulum retention motif (CTBSEKDEL). We have recently shown that oral administration of CTBSEKDEL, but not native CTB, facilitates colon epithelial repair and mucosal healing in a dextran sodium sulfate (DSS)-induced acute colitis mouse model. Moreover, biweekly oral administration of CTBSEKDEL significantly reduced tumorigenesis in the azoxymethane/DSS model of colitis- associated cancer. Based on these findings, we hypothesize that CTBSEKDEL provides a prototype oral biologic facilitating mucosal healing in UC. The goal of this translational R01 project is to optimize and validate the therapeutic potential of CTBSEKDEL in preclinical UC models. Since CTBSEKDEL has already shown feasibility in an acute colitis model, we will immediately proceed with further validation in chronic colitis models. In parallel, in Aim 1, we will create CTBSEKDEL variants with modifications in the C-terminal sequence (CTB(X)H/KDEL) to improve molecular stability upon spray dry for enteric-coated formulations. We will produce these proteins using a Nicotiana benthamiana plant transient overexpression system and screen them based on a series of biochemical and biophysical assays, as well as a mouse acute DSS colitis model. In Aim 2, we will validate the efficacy and safety of CTBSEKDEL and a selected CTB(X)H/KDEL in two chronic colitis models based on repeated DSS exposure in C57bl/6 mice and piroxicam-exposed IL-10 knockout mice, in comparison to an anti-TNFα antibody. Therapeutic efficacy and safety will be determined by disease activity index, histopathology, immunohistochemistry and molecular biological analysis of inflammation, crypt regeneration, epithelial barrier recovery and fibrosis. In Aim 3, we will employ a human colon explant model to further validate the mucosal healing potential of CTBSEKDEL and CTB(X)H/KDEL. Colon biopsy and colectomy tissues will be obtained from patients with different disease history and biological backgrounds. Efficacy will be evaluated based on wound healing-related gene expression and immunohistochemistry for epithelial proliferation/regeneration markers. Cell type-specific responses will be investigated in colon lamina propria mononuclear cells and colonic organoids. Collectively, the project will generate pivotal preclinical data supporting the development of a first-in-class oral biologic candidate inducing colon epithelial repair for UC treatment towards a Phase I clinical trial.