MOLECULAR ENGINEERING OF IL-2 FOR THE TREATMENT AND PREVENTION OF TYPE I DIABETES

描述（由申请人提供）：I型糖尿病（T1 D）是一种常见的破坏性疾病，其特征是胰腺产生胰岛素的β细胞的自身免疫性破坏。随后的病症使受影响的患者无法产生足够的胰岛素以进行适当的代谢和血糖控制，从而导致急性和慢性并发症，引起广泛的发病率和死亡率。然而，有相当大的希望，T1 D可以预防，甚至逆转，及时给予免疫抑制治疗。尚未批准此类疗法，并且存在开发新型T1 D疗法的相当大的需求。白细胞介素-2（IL-2）是一种免疫调节细胞因子，对T1 D的病因学至关重要：虽然IL-2对维持预防疾病的调节性T细胞（T细胞）至关重要，但它也刺激攻击胰腺并引起T1 D的自身反应性淋巴细胞。该提案描述了通过分离T1 D中发生的IL-2的对立功能来设计用于预防和治疗T1 D的新型基于IL-2的治疗剂。IL-2类似物将通过基本原理、定点诱变（Aim 1）和将酵母表面展示与磷酸流式细胞术结合的组合方法设计，以基于信号输出筛选数千种IL-2变体（Aim 2）。这些合成IL-2分子的功能表征将包括IL-2依赖性细胞功能的体外测定，如T细胞增殖和NK细胞细胞毒性。这些IL-2类似物的生物物理表征将采用表面等离子体共振来测量与IL-2受体亚基的亲和力和结合动力学，并采用X射线晶体学来确定IL-2/IL-2受体复合物的分子结构。最后，最重要的是，将在T1 D的非肥胖糖尿病（NOD）小鼠模型中评估通过上述方法开发的候选IL-2治疗剂在预防和治疗糖尿病中的功效。因此，通过这些多学科和转化研究，我们将推进新的候选T1 D疗法的临床开发。! 公共卫生相关性：I型糖尿病（T1 D）是一种常见的自身免疫性疾病，对发病率和死亡率有重大影响。然而，人们普遍认为，如果在胰腺发生不可逆损伤之前给予适当的治疗，这种疾病是可以预防或逆转的。该项目的目标是设计和表征新的免疫抑制疗法，靶向蛋白质白细胞介素-2（IL-2），适用于治疗和预防T1 D。

DESCRIPTION (provided by applicant): Type I diabetes (T1D) is a common and devastating disease characterized by autoimmune destruction of the insulin-producing beta cells of the pancreas. The ensuing condition leaves the affected patients unable to produce sufficient insulin for proper metabolic and glycemic control, resulting in acute and chronic complications that cause extensive morbidity and mortality. There is considerable hope, however, that T1D may be preventable, or even reversed, with the timely administration of immunosuppressive therapy. No such therapy has yet been approved, and considerable need exists for the development of novel T1D therapeutics. Interleukin-2 (IL-2) is an immunoregulatory cytokine with critical importance to the etiology of T1D: while IL-2 is essential to maintain the regulatory T cells (Tregs) that prevent the disease, it also stimulates autoreactive lymphocytes that attack the pancreas and cause T1D. This proposal describes the engineering of novel IL-2 based therapeutics tailored for the prevention and treatment of T1D by splitting the antithetical functions of IL-2 that occur in T1D. IL-2 analogues will be designed through rationale, site-directed mutagenesis (Aim 1) and through a combinatorial approach that couples yeast-surface display to phosphoflow cytometry to screen thousands of IL-2 variants on the basis of signaling output (Aim 2). Functional characterization of these synthetic IL-2 molecules will include in vitro asays of IL-2 dependent cellular functions, such as T cell proliferation and NK cell cytotoxicity. Biophysical characterization of these IL-2 analogues will employ surface plasmon resonance to measure affinities and binding kinetics to the IL-2 receptor subunits and x-ray crystallography to determine the molecular structures of the IL-2/IL-2 receptor complexes. Ultimately, and most importantly, candidate IL-2 therapeutics developed through the methods above will be assessed for efficacy in the prevention and treatment of diabetes in the non-obese diabetic (NOD) mouse model of T1D. Thus, through these multidisciplinary and translational studies, we will advance new candidate T1D therapies for clinical development. ! PUBLIC HEALTH RELEVANCE: Type I diabetes (T1D) is a common autoimmune disease with major impacts on morbidity and mortality. It is widely believed, however, that the disease could be prevented or reversed if an appropriate therapeutic were administered before irreversible damage to the pancreas occurred. The goal of this project is to engineer and characterize new immunosuppressive therapies targeting the protein Interleukin-2 (IL-2) that would be suitable for the treatment and prevention of T1D.