Development of Conformer-Specific Anti-HLA Class II mAbs

摘要： 小鼠MHC II类分子以两种不同的构象状态存在，这些状态是由 第二类跨膜(TM)结构域GxxxG二聚基序(即，M1和M2配对的第二类)，以及这些 两种构象与不同的免疫功能有关。此外，我们最近报道：1) 人类白细胞抗原II类-DR、DP和DQ分子在拥有三个TM结构域GxxxG中反映了II类小鼠 Motif，2)在电子分子模拟中发现，人类白细胞抗原-DR和DQ TM结构域可以被大力采用 良好的M1和M2配对状态，以及3)具有HLA-DR或DQ TM结构域的混合II类分子可以 同时采用M1和M2构象。在这项提案中，我们展示了一种商业上可用的抗人类白细胞抗原 II类单抗(H0002)对野生型与HLADR M1 GxxxG&gt；VxxxV突变体的反应性不同 分子(它将具有改变的构象状态)。这些发现有力地表明，像老鼠一样 第二类，人类第二类分子也以两种不同的构象状态存在(M1和M2配对的第二类)。 除了已发表的关于正常B类小鼠M1和M2配对II类信号的差异信号的工作外 细胞，我们的初步研究表明，M1和M2 II类与不同的信号通路有关 肿瘤B细胞。在这里，我们表明，虽然M1 II类的选择性结合抑制了B细胞淋巴瘤 M1和M2 II类分子的增殖、共连接可阻断这一作用。这一点很重要，因为三种抗人类白细胞抗原-DR 单抗(IMMU-114、1D09C3和Hu1D10)已经进行了临床试验，用于治疗B细胞 白血病/淋巴瘤，结果喜忧参半。由于这三种抗DR单抗的构象特异性是 尚不清楚的是，这增加了抗DR单抗治疗效果与单抗构象反应有关的可能性。 这些发现导致了一种假设，即与小鼠II类分子一样，人类HLAII类分子存在于两个 基于跨膜结构域GxxxG二聚交替配对的不同构象状态 并且这两个不同的人类白细胞抗原II类构象与不同的临床相关的B细胞有关 信号通路。为了检验这一核心假设，我们将：1)定义大型 可用的抗人类白细胞抗原II类单抗的小组，包括那些已经进行了临床试验的单抗 B细胞白血病/淋巴瘤，以及2)开发一组新的单抗，识别 M1和M2的构象差异使人类的HLA-DR和HLA-DQ II类分子配对。 这些目标的完成将有助于将M1/M2 II类构象范例引入人类白细胞抗原领域，提供 深入了解临床相关抗DR单抗的构象特异性之间是否存在相关性 并展示了治疗效果，并为该领域提供了一套新的工具(即，构象特异性抗- 以进一步研究M1和M2在人类健康中的作用 和疾病。

Summary: Murine MHC class II molecules exist in two distinct conformational states formed by differential pairing of class II transmembrane (TM) domain GxxxG dimerization motifs (i.e., M1 and M2 paired class II), and these two conformers are linked to different immunological functions. Moreover, we have recently reported that: 1) HLA class II HLA-DR, DP and DQ molecules mirror mouse class II in possessing three TM domain GxxxG motifs, 2) in silico molecular modeling reveals that HLA-DR and DQ TM domains can adopt energetically favorable M1 and M2 paired states, and 3) hybrid class II molecules with an HLA-DR or DQ TM domain can adopt both an M1 and M2 conformation. In this proposal, we show that a commercially-available anti-HLA class II mAb (H0002) exhibits differential reactivity to wild type vs. an HLA-DR M1 GxxxG>VxxxV mutant molecule (which would have an altered conformer state). These findings strongly suggesting that like mouse class II, human class II molecules also exist in two distinct conformational states (M1 and M2 paired class II). In addition to published work on the differential signaling of M1 vs. M2 paired class II in normal B murine cells, our preliminary studies show that M1 and M2 class II are linked to distinct signaling pathways in neoplastic B cells. Herein, we show that while selective engagement of M1 class II inhibits B cell lymphoma proliferation, co-ligation of M1 and M2 class II blocks this effect. This is important because three anti-HLA-DR mAbs (IMMU-114, 1D09C3 and Hu1D10) have undergone clinical trials as treatment for B cell leukemia/lymphoma with mixed results. Since the conformer specificity of these three anti-DR mAbs is unclear, it raises the possibility that anti-DR mAb therapeutic efficacy is linked to mAb conformer reactivity. These findings lead to the hypothesis that like mouse class II, human HLA class II molecules exist in two distinct conformational states based on alternative pairing of transmembrane domain GxxxG dimerization motifs and that these two distinct HLA class II conformers are linked to different clinically-relevant B cell signaling pathways. To test this core hypothesis, we will; 1) Define the HLA-DR conformer specificity of a large panel of available anti-HLA class II mAbs, including those that have undergone clinical trials as therapeutics for B cell leukemia/lymphoma, and 2) Develop a panel of new monoclonal antibodies that recognize the conformational differences between M1 and M2 paired human HLA-DR and HLA-DQ class II molecules. Completion of these aims will help bring the M1/M2 class II conformer paradigm to the HLA field, provide insight into whether there is a correlation between the conformer-specificity of clinically-relevant anti-DR mAbs and demonstrated therapeutic effect, and provide the field with a new set of tools (i.e., conformer-specific anti- HLA-DR and anti-HLA-DQ mAbs) to further investigate the role of M1 and M2 HLA class II in human health and disease.