Vector-based Generation of Monoclonal Antibodies against the CB2 Receptor

描述（由申请人提供）：最近的研究表明，通过免疫细胞上的大麻素受体2型（CB 2）的信号传导可能在宿主免疫的产生和表达中发挥重要的调节作用。然而，对CB 2的分布和功能的研究受到缺乏在蛋白质水平鉴定该受体的试剂的限制。特别地，还没有开发出单克隆抗体（mAb）来检测活细胞中的CB 2蛋白。单克隆抗体为标记细胞表面受体、表征表达水平以及将受体表达与其他细胞特征和功能联系起来提供了一种可重复的灵活试剂。单克隆抗体还可以提供用于中和受体功能的高度选择性方法。该I期尖端基础研究应用（CEBRA）的主要目标是使用新的细胞和分子方法来产生针对CB 2细胞外序列的抗CB 2 mAb，并验证其识别和中和免疫细胞表达CB 2的能力。提出了以下两个具体目标：1）在CB 2受体敲除小鼠中采用基于载体的免疫方案以产生针对人CB 2的细胞表面表位的mAb，和2）验证所选mAb鉴定和/或中和靶免疫细胞上的CB 2表达的能力。将解决可能阻止产生所需抗CB 2 mAb的几个障碍。缺乏天然CB 2表达的CB 2受体敲除小鼠将被用作绕过自身耐受和改善免疫反应性的创新策略。我们还制备了表达人CB 2 cDNA的慢病毒载体，其将用于初免-加强免疫接种方案以诱导高抗CB 2 Ab升。此外，我们将使用已转导以表达高水平人CB 2的中国仓鼠卵巢（CHO）细胞作为靶细胞，用于鉴定针对细胞外结构域的CB 2抗体。有了这些目标和新方法，CEBRA第一阶段拨款将开发重要的新mAb试剂，作为CEBRA第二阶段拨款的一部分，这些试剂将促进未来对免疫细胞中CB 2受体的表达、调节和功能的研究。这些试剂还将促进对CB 2感兴趣的其他研究者的研究，并提供疫苗接种和mAb筛选方案，该方案可用于生成其他mAb，包括针对CB 1的mAb。这项工作有能力大大加快大麻素的研究。

DESCRIPTION (provided by applicant): Recent studies suggest that signaling through cannabinoid receptor type 2 (CB2) on immune cells may play an important regulatory role in the generation and expression of host immunity. However, research into the distribution and function of CB2 has been limited by a lack of reagents to identify this receptor at the protein level. In particular, no monoclonal antibodies (mAbs) have been developed to detect CB2 protein in ive cells. Monoclonal Abs provide a reproducible and flexible reagent for tagging cell surface receptors, characterizing expression levels, and linking receptor expression to other cell features and functions. Monoclonal Abs can also provide a highly selective approach for neutralizing receptor function. The primary goal of this Phase I Cutting-Edge Basic Research Application (CEBRA) is to use novel cellular and molecular approaches to generate anti-CB2 mAbs directed against the extracellular sequences of CB2 and to validate their capacity to identify and neutralize CB2 expression by immune cells. The following two specific aims are proposed: 1) to employ a vector-based immunization protocol in CB2 receptor knockout mice to generate mAbs directed against cell surface epitopes of human CB2, and 2) to validate the capacity for selected mAbs to identify and/or neutralize CB2 expression on target immune cells. Several obstacles that may prevent the generation of desirable anti-CB2 mAbs will be addressed. CB2 receptor knockout mice, which lack expression of native CB2, will be used as an innovative strategy for bypassing self-tolerance and improving immune responsiveness. We have also prepared a lentiviral vector that expresses human CB2 cDNA that will be used in a prime-boost vaccination protocol to induce high anti-CB2 Ab liters. In addition, we will use Chinese hamster ovary (CHO) cells that have been transduced to express high levels of human CB2 as a target cell for identifying CB2 antibodies directed against the extracellular domains. With these aims and novel approaches, this Phase I CEBRA grant will develop important new mAb reagents that will facilitate future studies into the expression, regulation and function of CB2 receptors in immune cells as part of a Phase II CEBRA grant. These reagents will also facilitate research by other investigators interested in CB2, and provide a vaccination and mAb screening protocol that can be adapted to generate other mAbs, including mAbs directed against CB1. This work has the capacity to significantly accelerate cannabinoid research.