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

小胶质细胞是大脑中的巨噬细胞，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。小胶质细胞可以分化成两种状态，促炎状态M1和抗炎状态M2，并且可以处于M1和M2状态之间的任何位置。在阿尔茨海默病（AD）中，小胶质细胞极化为促炎M1表型，是吞噬性的，是由致病性错误折叠蛋白介导的。补体系统通过多种途径（经典途径、凝集素途径或其他途径）实现吞噬作用。传统上，人们认为小胶质补体系统仅用于清除病原体和细胞碎片。然而，已经发现它在发育中的大脑中通过神经元的调理突触修剪神经元回路的适当成熟中起着至关重要的作用。在AD中，淀粉样斑块通过小胶质细胞向炎性M1表型的极化激活补体途径，这既激活经典补体系统，又释放细胞因子，激活反应性（A1）星形细胞，介导替代补体系统。综上所述，小胶质补体系统的异常激活导致AD患者突触的调谐和伴随的吞噬以及随后的神经退行性变。研究表明，PD-1基因敲除小鼠脊髓损伤（SCI）后，PD-1缺乏可诱导促炎性M1小胶质细胞。而在野生型小鼠中，PD-1表达上调，存在抗炎M2小胶质细胞，导致脊髓损伤后更好的恢复。基于此，我们提出通过抑制小胶质补体系统上调AD脑中的PD-1信号，减少神经炎症和神经退行性变可以实现。癌细胞通过激活PD-1信号通路来表达PD-L1以逃避T细胞的免疫反应，受此启发，我们认为小胶质补体反应可能以类似的方式被抑制。通过设计一种具有淀粉样β斑块靶向结构域和PD-L1结构域的双特异性抗体（bsAb），研究人员提出，在斑块周围的局部环境中，小胶质细胞可能会迁移到淀粉样β斑块并极化为炎症M1表型，从而被激活为抗炎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。目前临床试验中的大多数bsAb是通过重组表达bsAb，使用柔性肽连接结构域来产生的，然而，这种方法并不适用于所有的bsAb结构。因此，我们的目的是设计一种强大的化学连接策略，不仅可以用于描述的bsAb模型，还可以应用于使用当前基于表达的连接策略无法访问的许多bsAb。这将通过模块化连接策略来实现，位点特异性靶向独特的活性残基，这些残基将被设计到单独的蛋白质结构域中，或自然发生。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bsAb结构的生产，用于靶向治疗，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.