Development of a modular chemical linking strategy for bispecific antibodies to suppress neuroinflammation in Alzheimer's disease via microglial polar

博士项目战略主题：小胶质细胞是大脑的常驻巨噬细胞，由于大脑中缺乏抗体，它们在巨噬细胞中具有独特的反应性。小胶质细胞可以被极化成两种状态，促炎M1和抗炎M2状态，并且可以位于M1和M2状态之间的光谱上的任何位置。在阿尔茨海默病（AD）中，小胶质细胞极化为促炎性M1表型（其为吞噬细胞）是由致病性错误折叠蛋白介导的。补体系统能够通过多种途径（经典、凝集素或替代途径）进行吞噬作用。传统上认为，小胶质细胞补体系统仅用于清除病原体和细胞碎片。在AD中，β-淀粉样蛋白斑块通过小胶质细胞极化为炎性M1表型激活补体途径，后者既激活经典补体系统，又释放细胞因子激活反应性（A1）星形胶质细胞，后者介导替代补体系统。在组合中，小胶质细胞补体系统的异常激活导致AD患者中突触的调理作用和伴随的吞噬作用以及随后的神经变性。已经显示，PD-1缺陷在PD-1敲除小鼠中的脊髓损伤（SCI）后诱导促炎性M1小胶质细胞。而在野生型小鼠中，PD-1表达上调，并且存在抗炎性M2小胶质细胞，导致SCI更好地恢复。基于此，我们提出，上调AD脑中的PD-1信号传导，可以通过抑制小胶质细胞补体系统来实现神经炎症和神经变性的减少。从表达PD-L1以通过激活PD-1信号通路逃避T细胞免疫应答的癌细胞中获得灵感，据信小胶质细胞补体应答可以以类似的方式被抑制。通过设计具有淀粉样蛋白-β斑块靶向结构域和PD-L1结构域的双特异性抗体（bsAb），提出了将迁移至淀粉样蛋白-β斑块并极化为炎性M1表型的小胶质细胞可以在斑块周围的局部环境中交替地活化为抗炎M2状态。这将通过bsAb的PD-L1结构域与小胶质细胞的PD-1结构域的相互作用来实现，从而抑制神经炎症。目前在临床试验中的大多数bsAb是通过使用柔性肽连接结构域来重组表达bsAb来产生的，然而，这种方法并不适用于所有的bsAb构建体。因此，目的是设计一种稳健的化学连接策略，其不仅可以用于所述的模型bsAb，而且还可以应用于使用当前基于表达的连接策略不可访问的许多bsAb。这将通过模块化连接策略实现，位点特异性靶向将被工程化到单独的蛋白质结构域中或天然存在于单独的蛋白质结构域中的独特反应性残基。如果成功，这种方法可以扩展到其他疾病模型，这些疾病模型受益于用于靶向治疗的bsAb构建体的生产，例如其他神经退行性疾病或癌症。

PhD project strategic theme: Biosciences for an integrated understanding of healthMicroglia are the resident macrophages of the brain and due to the lack of antibodies in the brain, they are uniquely reactive amongst macrophages. Microglia can be polarised into two states, a pro-inflammatory, M1, and anti-inflammatory, M2 state, and can sit anywhere on the spectrum between the M1 and M2 states. In Alzheimer's disease (AD), microglial polarisation to a pro-inflammatory, M1 phenotype which is phagocytic, is mediated by pathogenic, misfolded proteins. The complement system enables phagocytosis through multiple pathways (classical, lectin or alternative). Traditionally it was believed that the microglial complement system was utilised solely to clear pathogens and cellular debris. However, it has been found to have a crucial role in the proper maturation of neuronal circuits in the developing brain through synaptic pruning via opsonisation of neurons.In AD, amyloid-beta plaques activate the complement pathway through polarisation of microglia to inflammatory M1 phenotypes which both activates the classical complement system and releases cytokines which activate reactive (A1) astrocytes, which mediate the alternative complement system. In combination, the aberrant activation of the microglial complement system leads to the opsonisation and concomitant phagocytosis of synapses and subsequent neurodegeneration in AD patients. It has been shown that PD-1 deficiency induces pro-inflammatory M1 microglia after spinal cord injury (SCI) in PD-1 knockout mice. Whilst in wildtype mice, PD-1 expression is upregulated and anti-inflammatory M2 microglia were present, leading to better recovery from SCI. Based on this, we propose that upregulating PD-1 signalling in the AD brain, a reduction in neuroinflammation and neurodegeneration could be achieved through suppression of the microglial complement system. Taking inspiration from cancer cells, which express PD-L1 to evade the immune response of T cells through activating the PD-1 signalling pathway, it is believed the microglial complement response could be suppressed in a similar way. By designing a bispecific antibody (bsAb) with an amyloid-beta plaque targeting domain and a PD-L1 domain, it is proposed that microglia which would migrate to amyloid-beta plaques and become polarised to the inflammatory M1 phenotype could be alternatively activated to the anti-inflammatory M2 state in the local environment surrounding the plaques. This would be achieved through the interaction of the PD-L1 domain of the bsAb with the PD-1 domain of the microglia, and thus suppress neuroinflammation.The majority bsAbs currently in clinical trials are produced through recombinant expression of bsAbs using a flexible peptide to link the domains, however, this approach is not suitable for all bsAb constructs. Therefore, the intention is to design a robust chemical linking strategy that can be used not only for the model bsAb described but can also be applied to a number of bsAbs which are inaccessible using current expression based linking strategies. This will be achieved via a modular linking strategy, site-specifically targeting uniquely reactive residues which will be engineered into, or naturally occuring, in the separate protein domains. If successful, this approach could be extended to other disease models which benefit from the production bsAb constructs for targeted therapies, such as other neurodegenerative conditions or cancers.