Disconnecting CR2/CD21 from Its C3d Ligand to Ameliorate Lupus

描述(由申请人提供)：补体受体2型(CR2/CD21)及其主要的C3激活片段衍生配体C3d在针对外来抗原的高亲和力抗体的发展中发挥核心作用。小鼠对抗原结合的C3片段的主要B细胞受体是CR2和补体受体1型(CR1/CD35)。与人类不同，人类有独特的基因编码这些蛋白质，而老鼠的CR2和CR1是通过从一个名为Cr2的共同基因进行选择性剪接而产生的。较大的CR1蛋白是C3b形式的C3和抗原结合的C4b的主要受体，而较小的CR2蛋白仅以高亲和力结合C3d。高亲和力自身抗体和B淋巴细胞在系统性红斑狼疮(SLE)的免疫发病机制中发挥重要作用。原则上，根据我们目前对SLE发生的免疫基础的理解，通过颠覆正常的耐受检查点和发展自身反应，人们可能会预期，在SLE中，CR2与C3d结合的自身抗原的相互作用将起到与外来抗原类似的增强作用。然而，先前在SLE小鼠模型中使用缺乏CR2和CR1的基因靶向的Cr2-/-小鼠的研究并不支持这一假设，并表明除了CR1之外，CR2的表达对于维持对狼疮相关自身抗原的耐受是必要的。很容易理解CR1是如何作为C4b的高亲和力受体发挥这一作用的，C4b本身是维持小鼠和人类对自身抗原的耐受性所必需的。然而，CR2没有类似的实验支持的作用，因此如何需要这种特殊的受体的表达来保护SLE免受自身免疫性疾病的发展仍然是个谜。为了解决这个问题，我们已经克服了小鼠CR2/CR1受体系统中存在的技术挑战，开发了新的高度特异的小鼠抗小鼠单抗(MAbs)。第一种是非B细胞耗竭单抗，它只识别和阻断CR2/CD21功能，而不直接影响CR1与C4b或C3b的相互作用。第二个单抗在其受体结合部位识别C3d片段，并阻止其与CR2的相互作用，而不影响C3b或C4b与CR1的相互作用。有了这些新开发的工具，并追求以下具体目标，我们将第一次能够检验假设 仅破坏关键的C3d-CR2配体-受体结合步骤就会有所改善，而不是 在人类狼疮的MRL/LPR模型中，评估特异性单抗介导的阻断CR2/CD21功能对自身免疫进化和临床结果的影响；以及特定目的#2.使用一种新的C3d特异性单抗来阻断携带C3d的自身抗原复合体与CR2/CD21的相互作用，表征这一策略在MRL/LPR模型中对自身免疫进化和临床结果的改善作用。

DESCRIPTION (provided by applicant): Complement receptor type 2 (CR2/CD21) and its primary C3 activation fragment-derived ligand designated C3d play a central role in the development of high affinity antibodies to foreign antigens. The primary B cell receptors for antigen-bound C3 fragments in mice are CR2 and complement receptor type 1 (CR1/CD35). As opposed to humans, where unique genes encode these proteins, mouse CR2 and CR1 are derived through alternative splicing from a common gene designated Cr2. The larger CR1 protein is the primary receptor for the C3b form of C3 as well as antigen-bound C4b, while the smaller CR2 protein only binds C3d with a high affinity. High affinity autoantibodies and B lymphocytes play central roles in the immunopathogenesis of human systemic lupus erythematosus (SLE). In principle, given our current understanding of the immune basis for the development of SLE through subversion of normal tolerance checkpoints and development of autoreactive responses, one might expect a similar enhancing role as found with foreign antigens to be played by CR2 interactions with C3d-bound self-antigens in SLE. However, prior studies using gene-targeted Cr2-/- mice, which lack both CR2 and CR1, in murine models of SLE have not supported this presumption and have suggested that CR2, in addition to CR1, expression is necessary to maintain tolerance to lupus-related self- antigens. It is straightforward to understand how CR1 could play this role as a high affinity receptor for C4b, which is itself necessary to maintain tolerance to self-antigen in mice and humans. However, no similar experimentally supported role exists for CR2, and thus how expression of this particular receptor could be needed to protect from autoimmune disease development in SLE remains enigmatic. To address this question, we have overcome the technical challenges that exist in the murine CR2/CR1 receptor system by developing new highly specific mouse anti-mouse monoclonal antibodies (mAbs). The first is a non-B cell depleting mAb that recognizes and blocks only CR2/CD21 function without directly affecting CR1 interactions with C4b or C3b. The second mAb recognizes the C3d fragment at its receptor binding site and blocks its interaction with CR2 without affecting C3b or C4b interactions with CR1. With these newly developed tools and pursuing the following specific aims, we will for the first time be able to test the hypothesis that disruption of the critical C3d- CR2 ligand-receptor binding step alone will ameliorate, rather than enhance, autoimmunity and clinical disease in SLE: Specific Aim #1. Evaluate the effects of specific monoclonal antibody-mediated blockade of CR2/CD21 function on the evolution of autoimmunity and clinical outcomes in the MRL/lpr model of human lupus; and Specific Aim #2. Using a novel C3d-specific monoclonal antibody to interrupt the interaction by C3d-bearing autoantigen complexes with CR2/CD21, characterize the ameliorative effects of this strategy on the evolution of autoimmunity and clinical outcomes in the MRL/lpr model.