Woska Joseph R, Last-Barney Kathleen, Rothlein Robert, Kroe Rachel R, Reilly Patricia L, Jeanfavre Deborah D, Mainolfi Elizabeth A, Kelly Terence A, Caviness Gary O, Fogal Steven E, Panzenbeck Maret J, Kishimoto Takashi K, Giblin Patricia A
Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., P.O. Box 368, Ridgefield, CT 06877, USA.
J Immunol Methods. 2003 Jun 1;277(1-2):101-15. doi: 10.1016/s0022-1759(03)00176-5.
The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.
β2整合素淋巴细胞功能相关抗原-1（CD11a/CD18）是一种白细胞特异性黏附分子，它通过与配体ICAM-1、ICAM-2和ICAM-3相互作用，介导白细胞外渗、抗原呈递以及T细胞介导的细胞溶解。我们最近报道了一种LFA-1的小分子拮抗剂（BIRT 377），它能抑制LFA-1/ICAM-1分子间相互作用、LFA-1依赖性黏附试验、抗原诱导的T细胞增殖以及小鼠体内超抗原诱导的IL-2产生。我们最近还报道了一种独特的单克隆抗体R3.1，它与BIRT 377及其类似物竞争结合纯化的全长LFA-1和纯化的重组I结构域模块。在本论文中，我们将这些研究扩展到基于细胞的系统，并利用这种独特的试剂开发一种受体占有率测定法。基于这些观察结果，我们设计并验证了一种测定法，可用于在体外测定猴和人外周血白细胞上的受体占有率，以及在给予小分子LFA-1拮抗剂的猴的全血中进行离体测定。对这些试剂的进一步优化导致了一种基于Fab的测定法的开发，该方法能够对全血样本进行快速且可重复的分析。这些优化后的试剂能够定量细胞表面表达的受体数量，并更准确地定量受体占有率。
