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

靶向上皮 GM1 神经节苷脂的口服治疗先导蛋白治疗溃疡性结肠炎的临床前验证

• 批准号: 10596495
• 负责人: Nobuyuki Matoba
• 金额: $ 44.57万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-20 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10596495
• 关键词: Acceleration; Acute; Affect; Affinity; Anti-Inflammatory Agents; Antibodies; Azoxymethane; Binding; Biochemical; Biological; Biological Assay; Biological Response Modifier Therapy; Biophysics; Biopsy; C-terminal; C57BL/6 Mouse; Caco-2 Cells; Cells; Cholera Toxin Protomer B; Cholera Vaccine; Chronic; Clinical Trials; Colectomy; Colitis; Colon; Crohn' s disease; Data; Detection; Development; Disease; Disease remission; Dose; Dryness; Dysplasia; E-Cadherin; Endoplasmic Reticulum; Endoscopy; Enteral; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Epithelium; Excision; Exposure to; Fibrosis; Formulation; Frequencies; Ganglioside GM1; Gene Expression; Genes; Goals; High Pressure Liquid Chromatography; Histopathology; Human; Immune; Immune System Diseases; Immunohistochemistry; Immunologic Surveillance; Immunosuppressive Agents; Impairment; In Vitro; Individual; Inflammation; Inflammatory Bowel Diseases; Interleukin-10; Intestines; Knockout Mice; Lamina Propria; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Modeling; Modification; Molecular; Monitor; Mononuclear; Mucous Membrane; Mus; Natural regeneration; Nicotiana; Oral; Oral Administration; Organoids; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Phase I Clinical Trials; Piroxicam; Plants; Postoperative Complications; Process; Proliferating; Proteins; Recombinants; Recording of previous events; Recovery; Rectum; Refractory; Relapse; Research; Safety; Scanning; Series; Sodium Dextran Sulfate; Specimen; System; TNF gene; Testing; Therapeutic; Therapeutic Agents; Therapeutic Effect; Tissues; Treatment Efficacy; Ulcerative Colitis; Vaccine Antigen; Validation; Variant; analog; cell type; clinical care; colitis associated cancer; comparative efficacy; curative treatments; dextran sulfate sodium induced colitis; efficacy study; efficacy validation; epithelial injury; epithelial repair; first-in-human; gut inflammation; healing; holotoxins; immunoregulation; improved; indexing; infection risk; inflammatory marker; mouse model; murine colitis; mutant; novel; novel therapeutic intervention; overexpression; pre-clinical; prototype; rectal; response; retrograde transport; safety study; screening; tissue culture; translational goal; tumorigenesis; wound healing

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.

项目摘要 目前，溃疡性结肠炎（UC）是一种炎症性肠病， 影响直肠和结肠最内层粘膜的疾病。虽然粘膜愈合是主要的 治疗目标，许多患者未能实现粘膜愈合与可用的UC药物，如抗炎， 免疫调节剂和抗TNF剂。由于上皮修复是粘膜愈合的关键过程， 促进这一过程的药物将提供一种与现有临床护理不同的新的治疗策略， 为UC。我们的治疗先导是口服霍乱疫苗抗原霍乱毒素B亚单位（CT B）的变体， 用包括内质网滞留基序（CTBSEKDEL）的C-末端延伸修饰。我们 最近表明口服CTBSEKDEL而不是天然CTB促进结肠上皮修复 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，双周 CTBSEKDEL的口服给药显著降低了结肠炎的氧化偶氮甲烷/DSS模型中的肿瘤发生。 相关癌症基于这些发现，我们假设CTBSEKDEL提供了一种原型口腔生物制剂， 促进UC的粘膜愈合。本翻译R 01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 在急性结肠炎模型中，我们将立即在慢性结肠炎模型中进行进一步验证。同时，在 目的1，我们将创建在C-末端序列中具有修饰的CTBSEKDEL变体（CTB（X）H/KDEL），以改善CTBSEKDEL的功能。 肠溶包衣制剂喷雾干燥后的分子稳定性。我们将使用一种 本氏烟草植物瞬时过表达系统的建立及一系列生化指标的筛选 和生物物理测定，以及小鼠急性DSS结肠炎模型。在目标2中，我们将验证疗效， 基于重复DSS暴露的两种慢性结肠炎模型中CTBSEKDEL和选定CTB（X）H/KDEL的安全性 在C57 bl/6小鼠和吡罗昔康暴露的IL-10敲除小鼠中，与抗TNF α抗体相比。 疗效和安全性将通过疾病活动指数、组织病理学、 炎症、隐窝再生、上皮屏障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，我们将采用人结肠外植体模型来进一步验证粘膜 CTBSEKDEL和CTB（X）H/KDEL的愈合潜力。结肠活检和结肠切除术组织将从 不同疾病史和生物学背景的患者。将根据伤口评价疗效 愈合相关基因表达和上皮增殖/再生标记物的免疫组织化学。细胞 将在结肠固有层单核细胞和结肠类器官中研究类型特异性应答。 总的来说，该项目将产生关键的临床前数据，支持开发一流的口服 诱导结肠上皮修复的生物候选物，用于UC治疗的I期临床试验。

项目成果

Pharmacokinetics and Safety Studies in Rodent Models Support Development of EPICERTIN as a Novel Topical Wound-Healing Biologic for Ulcerative Colitis.

啮齿动物模型中的药代动力学和安全性研究支持将 EPICERTIN 开发为治疗溃疡性结肠炎的新型外用伤口愈合生物制剂。

• DOI: 10.1124/jpet.121.000904
• 发表时间: 2022
• 期刊: The Journal of pharmacology and experimental therapeutics
• 作者: Tusé,Daniel;Reeves,Micaela;Royal,Joshua;Hamorsky,KrystalT;Ng,Hanna;Arolfo,Maria;Green,Carol;Trigunaite,Abhishek;Parman,Toufan;Lee,Goo;Matoba,Nobuyuki
• 通讯作者: Matoba,Nobuyuki

The Unfolded Protein Response and Its Implications for Novel Therapeutic Strategies in Inflammatory Bowel Disease.

• DOI: 10.3390/biomedicines11072066
• 发表时间: 2023-07-23
• 期刊: Biomedicines
• 影响因子: 4.7

Spray-Dried Formulation of Epicertin, a Recombinant Cholera Toxin B Subunit Variant That Induces Mucosal Healing.

• DOI: 10.3390/pharmaceutics13040576
• 发表时间: 2021-04-18
• 期刊: Pharmaceutics
• 影响因子: 5.4
• 作者: Reeves MA;Royal JM;Morris DA;Jurkiewicz JM;Matoba N;Hamorsky KT
• 通讯作者: Hamorsky KT

Characterization and utility of two monoclonal antibodies to cholera toxin B subunit.

• DOI: 10.1038/s41598-023-30834-2
• 发表时间: 2023-03-15
• 期刊: SCIENTIFIC REPORTS
• 影响因子: 4.6
• 作者: Verjan Garcia, Noel;Santisteban Celis, Ian Carlosalberto;Dent, Matthew;Matoba, Nobuyuki
• 通讯作者: Matoba, Nobuyuki

Isolation and detection of a KDEL-tagged recombinant cholera toxin B subunit from Nicotiana benthamiana.

• DOI: 10.1016/j.procbio.2020.10.018
• 发表时间: 2021-03
• 期刊: Process biochemistry (Barking, London, England)
• 作者: Morris DA;Reeves MA;Royal JM;Hamorsky KT;Matoba N
• 通讯作者: Matoba N