Characterising the molecular interactions in the CTLA-4 and PD-1 pathways as a basis for novel strategies in immune checkpoint blockade

免疫疗法使癌症的治疗发生了革命性的变化。2018年诺贝尔医学奖授予了Allison和Honjo，这是对我们癌症治疗方法的突破性转变的认可，即使用抗体来消除免疫系统的“刹车”，而不是针对癌症组织的细胞毒性药物。这种针对免疫系统而不是癌症的方法代表了一种思想上的根本改变，认识到癌症可以被免疫系统靶向，因此操纵免疫反应是非常有益的。迄今为止，靶向的两个主要检查点是CTLA-4和PD-1途径，它们分别被靶向，也越来越多地被联合靶向。尽管对这些途径进行了20多年的研究，但我们对这些途径的实际工作原理仍然知之甚少。此外，现在正在出现的是，这些途径之间在分子水平上存在明确的生物物理相互作用，但尚未得到适当的表征。Sansom实验室在这一领域取得了重大进展，发现CTLA-4具有非凡的捕获和破坏其配体（CD80和CD86）的能力，使用被称为Transendocytosis（TE）的细胞生物学过程，为我们的研究提供了一个平台。由于CTLA-4不寻常的细胞生物学，包括细胞内递送到受限的免疫突触，快速内化，回收和降解，因此CTLA-4抗体靶向的最佳策略尚未确定。最近的数据还显示，CTLA-4配体之一（CD80）也直接与PD-1途径的配体（PD-L1）相互作用。当考虑抗体调节时，这会产生许多额外的场景。例如，CTLA-4的表达水平可能通过控制CD80的可用性，直接调节PD-L1- PD-1通路。因此，拟议的项目将探索操纵这些检查点的不同策略，包括在CTLA-4和PD-1途径中单独或联合使用多种抗体配方(高亲和力与低亲和力，阻断与非阻断，不同Fc区域和不同大小（Fab2, Fab'， Fv等）的影响。该项目将评估分子水平（结合、解离、运输、降解效应）和细胞水平（免疫调节、T细胞活性、ADCC等）的影响，以便更好地理解、操纵和预测它们在治疗方案中的使用。总的来说，该项目将提供免疫检查点生物学方面的重点培训，以及与抗体工程有关的制药业的直接经验。因此，在这个项目中，我们的目标是：1)。对相关合作伙伴进行研究方面的培训，包括使用纯化蛋白质和基因操纵细胞系和原代人类细胞的功能影响测量其生物物理特性。2)。探索可以调节上述相互作用并确定其作用模式的新蛋白质（抗体变体，可溶性模拟物，配体突变体）的设计。通过CD80与PD-L1.4的相互作用，确定跨内吞作用过程对PD-1通路调控的影响。描述临床可用的抗ctla -4和抗pd - l1抗体对上述过程的影响，并将它们与本项目中产生的新变体进行比较。由于CTLA-4和PD-1通路都影响肿瘤生物学，是常见自身免疫性疾病的关键控制点，因此了解这些通路的分子细节以及如何在治疗上最好地操纵它们与疾病主题的基本机制高度相关。

Immunotherapy has revolutionised the treatment of cancer. The 2018 Nobel Prize in medicine to Allison and Honjo recognised this groundbreaking shift in our approach to cancer treatment whereby instead of cytotoxic drugs targeting cancer tissue, antibodies are used to remove the "brakes" on the immune system. This targeting of the immune system and not the cancer represents a fundamental change in thinking, recognising that cancer can be targeted by the immune system and therefore manipulating the immune response can be highly beneficial.The two main checkpoints targeted to date have been the CTLA-4 and PD-1 pathways, which have been targeted both separately and increasingly together. Despite over two decades of research into these pathways we still understand remarkably little about how these pathways actually work. In addition, it is now emerging that there are clear biophysical interactions between these pathways at the molecular level which have yet to be properly characterised. The Sansom lab has made significant progress in this area by identifying that CTLA-4 has the remarkable ability to capture and destroy its ligands (CD80 and CD86) using a cell biological process known as Transendocytosis(TE) providing a platform for our studies. Because of the unusual cell biology of CTLA-4 involving intracellular delivery to a confined immune synapse, rapid internalisation, recycling and degradation the best strategies for antibody targeting of CTLA-4 have yet to be determined.Recent data has also revealed that one of the CTLA-4 ligands (CD80) also directly interacts with a ligand from the PD-1 pathway (PD-L1). This produces numerous additional scenarios when considering antibody modulation. For example, it is likely that the level of CTLA-4 expression, by controlling the availability of CD80, directly regulates the PD-L1- PD-1 pathway. The proposed project will therefore explore different strategies for manipulating these checkpoints including the impact of multiple formulations of antibodies (high affinity vs low affinity, blocking vs non-blocking, different Fc regions and different sizes (Fab2, Fab', Fv, etc) used alone alone and in combination within the CTLA-4 and PD-1 pathways. The project will evaluate impacts at both the molecular level (binding, dissociation, trafficking, degradation effects) and cellular levels (immune modulation, T cell activity, ADCC etc.) in order to better understand, manipulate and predict their use in treatment scenarios. Overall the project will provide a focused training on the biology of immune checkpoints as well as direct experience of the pharmaceutical industry relating to antibody engineering.In this project we therefore aim to:1). Provide training in the study of the relevant partners involved, including measurement of their biophysical characteristics using purified proteins and functional impacts genetically manipulated cell lines and primary human cells. 2). Explore the design of new proteins (antibody variants, soluble mimics, ligand mutants) that can act to modulate the above interactions and determine their mode of action.3). Determine the influence of transendocytosis process on the regulation of the PD-1 pathway via CD80 interaction with PD-L1.4). Characterise the impact of clinically available anti-CTLA-4 and anti-PD-L1 antibodies for their ability to influence the above processes and compare them with novel variants produced in this programme.Since both the CTLA-4 and PD-1 pathways affect cancer biology and are key control points for common autoimmune diseases, the understanding of these pathways in molecular detail and how to best manipulate them therapeutically is highly relevant to the fundamental mechanisms of disease theme.