Oral rice-derived lactoferrin reduced the severity of Crohn's disease through anti-inflammatory regulation of T cells

描述(申请人提供)：这项拟议的研究的范围是证明重组人乳铁蛋白(RhLF)通过T细胞调节在不同的炎症性肠病(IBD)小鼠模型中作为免疫调节剂的有效性，该模型最好地概括为克罗恩样回肠炎和结肠炎。克罗恩病(CD)在美国影响着多达70万人，目前的治疗方法依赖于抗炎药物和免疫调节剂(充其量是缓解的)，或者昂贵的肿瘤坏死因子抗体疗法，每种疗法都有相关的毒性。因此，我们建议在临床前模型中研究Ventria的大米来源的rhLF的使用，以证明其作为治疗CD的一种经济有效的治疗方案的有效性。 乳铁蛋白(Lactoferrin，LF)是一种内源性抗炎分子，分泌于体内粘膜部位。虽然乳铁蛋白最初因其抗微生物特性而被确定为先天性免疫的关键组成部分，但越来越多的证据表明，乳铁蛋白具有额外调节适应性免疫系统的能力，使其成为CD等慢性粘膜炎症疾病的理想治疗靶点。我们的初步研究已经有效地证明了重组人乳铁蛋白在CD小鼠模型中的治疗潜力，从而否定了它与肠道微生物区系和毒素的直接相互作用。20wk龄肿瘤坏死因子Δ小鼠与CD患者相似，表现为自发性慢性跨壁性肠炎，其肠道固有层、肠系膜淋巴结和脾内幼稚T细胞的浸润和增殖明显减少。RhLF治疗的小鼠也表现出组织学指标的改善和肠道屏障功能的改善，这表明rhLF在CD临床前模型中减轻炎症是有效的。 鉴于CD涵盖的疾病表型范围很广，从轻度到重度，包括严格的回肠炎、结肠炎或两者兼而有之，在多个临床前模型中验证这些发现是谨慎的。Ventria Bioscience目前使用大米表达系统以低成本、高纯度的规模生产高纯度的重组人LF，并已在临床试验中证明了rhLF在人体试验中的安全性和有效性。基于已证实的重组人乳铁蛋白的安全记录和Ventria公司大规模生产这种重组蛋白的能力，这些第一阶段试验的成功将使我们能够迅速将这些发现转化为针对儿科患者的第二阶段临床试验。然而，我们必须首先证明，口服重组人乳铁蛋白仍然有效，更关键的是，在另一种临床前CD模型中验证重组人乳铁蛋白的保护作用。 具体地说，我们试图调查以下内容：在相关的慢性CD临床前模型中，研究口服重组人乳铁蛋白的有效性，并通过其增强亲调节环境的能力来论证重组人乳铁蛋白的作用机制。以前的研究已经使用严酷和非常急性的溃疡性结肠炎(UC)的化学模型来证明纯化的牛或人LF在口服时具有保护作用。这些研究没有试图证明低频的作用机制，模型本身也不能很好地概括人类的情况。 因此，我们将在一种最能显示人类疾病特征的慢性肿瘤坏死因子驱动的回肠炎(ΔARE)模型上测试口服重组人乳铁蛋白诱导缓解的效果。我们将研究重组人乳铁蛋白在特定T细胞表型调节中的作用，该表型以其加重或抑制炎症反应的能力为特征。通过进一步研究其在T细胞介导的结肠炎模型中的免疫调节作用，rhLF的活性并不局限于单一的CD临床前模型。为了证明rhLF的疗效不是模型特异性的，也为了评估rhLF对CD常见的克罗恩样结肠炎模型的疗效，我们选择了一种T细胞驱动的结肠炎模型(CD4+CD45RB高过继转移模型)。在这个模型中，我们将测试rhLF对T细胞群的调节能力，从最早的炎症阶段到普遍的炎症阶段，特别是调节性T细胞(Treg)的作用及其抑制致病免疫反应的能力。 综上所述，这些模型将在两种不同的CD慢性模型中定义rhLF的免疫调节作用，验证rhLF部分通过诱导肠道内的亲调节环境发挥保护作用的假设。

DESCRIPTION (provided by applicant): The scope of this proposed study is to demonstrate the efficacy of recombinant human lactoferrin (rhLF) to act as an immune modulator through T cell regulation in distinct murine models of inflammatory bowel disease (IBD) which best recapitulate Crohn's-like ileitis and colitis. Crohn's disease (CD) affects as many as 700,000 people in the United States, with current therapies relying on anti-inflammatories and immune modulators which are palliative at best, or expensive TNF antibody therapies, each of which have associated toxicities. Therefore, we propose to investigate the use of Ventria's rice-derived rhLF in preclinical models to demonstrate its efficacy as a cost effective therapeutic option in th treatment of CD. Lactoferrin (LF) is an endogenous anti-inflammatory molecule secreted at mucosal sites within the body. While originally identified as a key component of innate immunity because of its antimicrobial properties, increasing evidence points to the ability of lactoferrin to additionally modulate the adaptive immune system making it an ideal therapeutic target for chronic mucosal inflammatory conditions such CD. Our preliminary studies have effectively demonstrated the therapeutic potential of subcutaneous rhLF in a murine model of CD, thereby negating its direct interaction with the intestinal microbiota and toxins. Specifically, rhLF administration significanly decreased naïve T cell infiltration and proliferation in the intestinal lamina propria, mesenteric lymph nodes and spleen of 20wk old TNFΔARE mice which, similar to CD patients, develop a spontaneous chronic transmural intestinal inflammation. rhLF-treated mice also exhibited improved histological indices and had improved intestinal barrier function, indicating efficacy for rhLF to decrease inflammation in a preclinical model of CD. Given the broad spectrum of disease phenotypes that CD encompasses, from mild to severe disease involving strict ileitis, colitis or both, it is prudent to validate these findings in multple preclinical models. Ventria Bioscience currently uses a rice expression system to produce recombinant human LF at high levels of purity at a cost-effective scale, and has demonstrated the safety and efficacy of rhLF in clinical trials when administered to human subjects. Based on the proven safety record of rhLF and the capacity within Ventria for large scale production of this recombinant protein, success in these phase I trials will allow us to rapidly translate these findings into a phase II clinical trial in pediatric patients. Nonetheless we must first demonstrat that rhLF remains effective when administered orally and more crucially validate the protective effect of rhLF in an additional preclinical model of CD. Specifically, we seek to investigate the following: Investigate the efficacy of oral rhLF in a relevant pre-clinical model of chronic CD, and demonstrate the mechanism of action of rhLF through its ability to enhance a pro-regulatory environment. Previous studies have used harsh and very acute chemical models of ulcerative colitis (UC) to demonstrate a protective effect of purified bovine or human LF, when orally administered. These studies did not attempt to demonstrate a clear mechanism of action of LF, nor do the models themselves recapitulate the human condition very well. Therefore, we will test the efficacy of oral rhLF to induce remission in a chronic model of TNF- driven ileitis (TNFΔARE), which best displays the hallmarks of human disease. We will investigate the role of rhLF in the regulation of specific T cell phenotypes characterized by their ability to exacerbate or suppress the inflammatory response. The activity of rhLF is not limited to a single pre-clinical model of CD, by further investigating its immunoregulatory role in a T cell mediated model of colitis. In an effort both to demonstrate that the effect of rhLF is not model-specific, ad to evaluate rhLF's efficacy a model of Crohn's-like colitis, a common feature of CD, we have chosen a T cell driven model of colitis (CD4+CD45RBhigh adoptive transfer model). In this model, we will test the ability for rhLF to regulate T cell populations from the earliest to pervasive staes of inflammation, with particular regard to the role of regulatory T cells (Tregs) and their abilityto suppress the pathogenic immune response. Together, these models will define an immunomodulatory role of rhLF in two distinct chronic models of CD, testing the hypothesis that rhLF exerts its protective effect in part by induction of a pro-regulatory environment within the intestine.