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: 10055139
• 负责人: Nobuyuki Matoba
• 金额: $ 46.13万
• 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 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10055139
• 关键词: 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; Dysplasia; E-Cadherin; Endoplasmic Reticulum; Endoscopy; Enteral; Enzyme-Linked Immunosorbent Assay; Epithelial; 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; Impairment; In Vitro; Individual; Inflammation; Inflammatory Bowel Diseases; Interleukin-10; Intestines; Knock-out; 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; 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; base; cell type; clinical care; colitis associated cancer; comparative efficacy; curative treatments; efficacy study; epithelial injury; first-in-human; healing; holotoxins; immunoregulation; improved; indexing; infection risk; inflammatory disease of the intestine; inflammatory marker; mouse model; mutant; novel; novel therapeutics; overexpression; pre-clinical; prototype; repaired; response; retrograde transport; safety study; screening; tissue culture; 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.

项目总结