Vector-based Generation of Monoclonal Antibodies against the CB2 Receptor

描述(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(CB2)发出的信号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，由于缺乏在蛋白质水平上识别CB2受体的试剂，对CB2的分布和功能的研究一直受到限制。特别是，还没有开发出用于检测Ive细胞中CB2蛋白的单抗。单抗为标记细胞表面受体、确定表达水平以及将受体表达与其他细胞特性和功能联系起来提供了一种可重复性和灵活性的试剂。单抗也可以为中和受体功能提供一种高度选择性的方法。这一第一阶段前沿基础研究应用(CEBRA)的主要目标是使用新的细胞和分子方法来产生针对CB2胞外序列的抗CB2单抗，并验证它们识别和中和免疫细胞表达CB2的能力。我们提出了以下两个具体目标：1)在CB2受体基因敲除小鼠中使用载体免疫方法，以产生针对人CB2细胞表面表位的单抗；2)验证所选单抗识别和/或中和靶免疫细胞上CB2表达的能力。将解决可能阻碍产生理想的抗CB2单抗的几个障碍。CB2受体基因敲除小鼠缺乏天然CB2的表达，将被用作绕过自我耐受和提高免疫反应性的创新策略。我们还制备了表达人CB2基因的慢病毒载体，该载体将用于初始-增强免疫方案，以诱导高水平的抗CB2抗体滴度。此外，我们将使用已转导高水平表达人CB2的中国仓鼠卵巢(CHO)细胞作为靶细胞，以识别针对胞外区的CB2抗体。有了这些目标和新的方法，这项第一阶段的CEBRA赠款将开发重要的新单抗试剂，作为第二阶段CEBRA赠款的一部分，这些试剂将促进未来对免疫细胞中CB2受体的表达、调节和功能的研究。这些试剂还将促进对CB2感兴趣的其他研究人员的研究，并提供一种疫苗接种和单抗筛选方案，该方案可用于产生其他单抗，包括针对CB1的单抗。这项工作有能力显著加快大麻类药物的研究。

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.