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

描述(申请人提供)：I型糖尿病(T1D)是一种常见的破坏性疾病，其特征是胰腺中产生胰岛素的β细胞受到自身免疫破坏。随之而来的情况使受影响的患者无法产生足够的胰岛素来适当控制代谢和血糖，导致急性和慢性并发症，导致广泛的发病率和死亡率。然而，通过及时给予免疫抑制治疗，T1D可能是可以预防的，甚至是可以逆转的，这是相当有希望的。目前还没有这种疗法被批准，开发新的T1D疗法有相当大的需求。白介素2(IL-2)是一种免疫调节细胞因子，对T1D的病因具有至关重要的作用：虽然IL-2对维持预防疾病的调节性T细胞(Treg)至关重要，但它也刺激攻击胰腺并导致T1D的自身反应性淋巴细胞。这项建议描述了基于IL-2的新型疗法的工程设计，该疗法是通过分离发生在T1D中的IL-2的相反功能来预防和治疗T1D的。IL-2类似物将通过原理、定点突变(AIM 1)和将酵母表面展示与磷酸盐结合的组合方法(AIM 1)设计，以基于信号输出筛选数千种IL-2变体(AIM 2)。这些人工合成的IL-2分子的功能特征将包括体外对IL-2依赖的细胞功能的测试，如T细胞增殖和NK细胞杀伤。这些IL-2类似物的生物物理表征将使用表面等离子体共振来测量与IL-2受体亚单位的亲和力和结合动力学，并用X射线结晶学来确定IL-2/IL-2受体复合体的分子结构。最终，也是最重要的是，通过上述方法开发的候选IL-2疗法将在T1D非肥胖糖尿病(NOD)小鼠模型中进行糖尿病预防和治疗的有效性评估。因此，通过这些多学科和转化性研究，我们将为临床开发提出新的候选T1D疗法。好了！

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. !