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（T1D）是一种常见的破坏性疾病，其特征是胰腺产生胰岛素的β细胞的自身免疫破坏。随后的情况使受影响的患者无法产生足够的胰岛素来进行适当的代谢和血糖控制，从而导致急性和慢性并发症，导致广泛的发病率和死亡率。然而，有相当大的希望，T1D可以预防，甚至逆转，及时给予免疫抑制治疗。目前还没有这样的治疗方法被批准，并且对新型T1D治疗方法的开发有相当大的需求。白细胞介素-2 （IL-2）是一种对T1D病因至关重要的免疫调节性细胞因子：虽然IL-2对于维持预防疾病的调节性T细胞（Tregs）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。该提案描述了通过分裂IL-2在T1D中发生的对立功能，为预防和治疗T1D量身定制的新型基于IL-2的治疗方法的工程。IL-2类似物将通过基本原理、定点诱变（Aim 1）和结合酵母表面显示和磷酸流式细胞术的组合方法来设计，以信号输出为基础筛选数千种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实验，如T细胞增殖和NK细胞的细胞毒性。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受体亚基的亲和和结合动力学，并使用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疗法将在非肥胖糖尿病（NOD） T1D小鼠模型中评估其预防和治疗糖尿病的功效。因此，通过这些多学科和转化研究，我们将推进新的候选T1D治疗方法的临床开发。！

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. !