The Structural and Functional Basis of HLA-associated Drug Hypersensitivity

描述（由申请人提供）：特异质药物不良反应不可预测，与剂量无关，可能危及生命;这使其成为药物开发成本和不确定性的主要因素。临床数据表明，许多此类反应涉及免疫机制，遗传相关性研究已确定对几种药物的药物超敏反应与特定HLA等位基因之间存在强烈联系。抗病毒药物阿巴卡韦（abacavir）发现了这种最强的遗传关联之一，阿巴卡韦仅在表达HLA分子变体B*57：01的患者中引起严重的不良反应。阿巴卡韦诱导这种病理性T细胞反应的机制尚不清楚。我们有强有力的初步数据表明，系统性药物不良反应可能是由改变的肽库形成引起的，该肽库在HLA相关药物超敏反应中触发T细胞免疫。使用体外结合测定、MHC配体洗脱和X射线晶体学，我们已经表明阿巴卡韦结合在HLA B*57：01的F口袋内，从而改变其特异性。这支持了一种新的假设，即全身性药物不良反应是由于形成了一种改变的肽库，在HLA相关的药物超敏反应中触发T细胞免疫。 目的是利用这种方法来定义三种药物在改变与HLA结合的肽中的作用，并指导开发具有降低引起HLA相关药物超敏反应可能性的药物变体。我们的具体目标是：1）鉴定以药物依赖性方式与HLA-B分子结合的肽。我们将在存在或不存在药物的情况下对组合肽文库进行与相关HLA-B分子的亲和力测量，并使用活细胞的MHC配体洗脱实验确认在MHC结合测定中检测到的药物作用。2)确定与超敏反应相关的HLA-B分子复合的药物结构。我们将通过X射线晶体学确定药物/肽/HLA复合物的结构。如可用，我们将利用目标1中鉴定的肽进行结晶，我们发现其在阿巴卡韦的情况下促进药物/肽/HLA复合物的形成。3)a.药物诱导的改变的肽库的功能后果的表征。B.药物变异对HLA相关药物识别的功能影响的表征。我们将利用Aim 2的结构信息来设计不结合HLA分子的药物变体，因此预计不会引起T细胞免疫反应。我们将使用来自过敏患者的样本测试药物变体的T识别。在分子结合试验中测试特定药物HLA组合对自身库的影响的能力可以显著提高检测HLA连锁药物超敏反应的能力。 此外，药物变异方法可能对全球医疗保健工作具有吸引力，在这些工作中，对已证实的疗法进行潜在的微小改变将简化治疗。

DESCRIPTION (provided by applicant): Idiosyncratic adverse drug reactions are unpredictable, dose independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggests that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkage between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations has been found for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. The mechanism by which abacavir induces this pathologic T cell response remains unclear. We have strong preliminary data that systemic adverse drug reactions can be caused by the formation of an altered peptide repertoire that triggers T cell immunity in HLA associated drug hypersensitivity. Using in vitro binding assays, MHC ligand elution and X- ray crystallography, we have shown that abacavir binds inside the F-pocket of HLA B*57:01, thereby altering its specificity. This supports a novel hypothesis that systemic adverse drug reactions are due to the formation of an altered peptide repertoire that triggers T cell immunity in HLA associated drug hypersensitivity. The objective is to utilize this approach to define the roles of three drugs in altering peptide binding to HLA and to guide development of drug variants that have a reduced likelihood of causing HLA linked drug hypersensitivity. Our specific aims are: 1) Identify peptides that bind to HLA-B molecules in a drug dependent manner. We will perform affinity measurements of a combinatorial peptide library in the presence or absence of the drug to the relevant HLA-B molecule and confirm drug effects detected in the MHC binding assays using MHC ligand elution experiments with live cells. 2) Determine structures of drugs complexed to HLA-B molecules associated with hypersensitivity. We will determine the structure of drug/peptide/HLA complexes by X-ray crystallography. As available, we will utilize the peptides identified in Aim 1 for crystallization, which we have foun to facilitate drug/peptide/HLA complex formation in the case of abacavir. 3) a. Characterization of functional consequences of the drug-induced altered peptide repertoire. b. Characterization of the functional effects of drug variation on HLA associated drug recognition. We will utilize the structural information from Aim 2 to design drug variants that do not bind HLA molecules and are therefore predicted to not cause T cell immune responses. We will test the drug variants for T recognition using samples from hypersensitive patients. The ability to test specific drug HLA combinations in molecular binding assays for effects on the self repertoire could drastically improve the ability to detect HLA linked drug hypersensitivities. Moreover, the drug variation approach may be attractive to global healthcare endeavors where a potentially small change in a proven therapy would simplify treatment.