Enhancing anti-tumour t cell function by controlled inhibition of checkpoint receptor signalling

在过去的十年中，免疫检查点阻断已经成为免疫治疗的一项重大治疗进展。然而，只有一小部分癌症患者对检查点封锁有反应，这表明对免疫检查点受体信号传递的基本机制缺乏基本了解。必须开发新的治疗药物。这个PHD项目旨在开发一种新的方法来增强T细胞功能，并了解检查点受体如何抑制T细胞功能。免疫检查点(如PD-1和CTLA-4)对T细胞信号的调节一直是最近癌症免疫治疗突破的核心。PD-1和CTLA-4的信号传递降低了T细胞的活性，导致“疲惫”的表型，严重影响了抗肿瘤反应。在PD-1的情况下，与PD-L1/2结合触发信号基序的酪氨酸磷酸化，并导致胞浆磷酸酶如SHP1/2的募集，从而减少TCR和CD28信号转导。值得注意的是，几种免疫受体的信号传递依赖于ITAM/ITIM/ITSM信号基序的Tyr磷酸化。我们假设紧张性受体的磷酸化和“配体经验”受体的持续信号影响T细胞功能，并且不能被细胞外拮抗剂抗体控制。为了解决这个问题，我们以前设计了一个双特异性分子，在PD-1附近招募CD45，一种丰富和混杂的受体酪氨酸磷酸酶。在这种方法中，CD45的磷酸酶结构域在顺式细胞内作用于PD-1 ITIM/ITSM基序的p-Tyr残基，从而抑制持续的信号转导。这表明，在体外存在和不存在PD-1配体结合的情况下，通过磷酸酶募集(RIPR)的受体抑制(RIPR)增强了T细胞的活性，超过了PD-1/PD-L1拮抗剂抗体，并减少了小细胞肺癌和结肠腺癌模型中的肿瘤生长(Fernandes等人，自然，2020)。在这个PHD项目中，我们建议扩展这一新的方法，通过关键的免疫和癌症特异性受体来关闭信号，目的是产生基于RIPR的新的抗肿瘤分子。我们建议开发新的RIPR蛋白，通过细胞毒性T细胞中表达的抑制性检查点受体来关闭信号，包括BTLA、CTLA-4、ILT2和ILT4。我们将使用各种抗CD45纳米抗体系统地测试新产生的RIPR蛋白的效力。针对免疫细胞中发现的CD45亚型和其他磷酸酶的纳米抗体也将被开发出来，并测试它们关闭抑制信号的能力。新开发的RIPR分子将在生物物理测试中进行表征。结合的开启速率、关闭速率和亲和力将由表面等离子体共振决定。下一步，将使用T细胞、NK细胞或巨噬细胞，通过体外激活分析来确定信号效力的早期读数。候选的RIPR分子还将通过与T细胞和靶癌细胞的共培养试验来测试增强T细胞细胞毒功能的能力。T细胞活化的标志物将使用流式细胞术、Western blotting、ELISA和RNA-Seq进行纵向定量。这一综合方法有望确定各种检查点受体的RIPR活性决定因素。这些信息将指导未来具有强大治疗应用潜力的检查点受体信号拮抗剂的设计。

In the past decade, immune checkpoint blockade has emerged as a major therapeutic advance in immunotherapy. However, only a small subset of cancer patients respond to checkpoint blockade, suggesting a fundamental understanding of the basic mechanisms of immune checkpoint receptor signalling is lacking. Novel therapeutic drugs must be developed. This PhD project aims to develop a novel approach to potentiate T cell function and to understand how checkpoint receptors dampen T cell function. Regulation of T-cell signalling by immune checkpoints such as PD-1 and CTLA-4 has been at the centre of recent breakthroughs in cancer immunotherapy. Signalling by PD-1 and CTLA-4 reduces T cell activity and contributes to an "exhausted" phenotype, severely compromising antitumor responses. In the case of PD-1, binding to PD-L1/2 triggers the tyrosine phosphorylation of signalling motifs and results in the recruitment of cytosolic phosphatases such as SHP1/2, which in turn reduces TCR and CD28 signalling. Strikingly, signalling by several immune receptors relies on the Tyr phosphorylation of ITAM/ITIM/ITSM signalling motifs. We hypothesise that tonic receptor phosphorylation and sustained signalling by 'ligand-experienced' receptors impact T cell function and fails to be controlled by extracellular antagonist antibodies. To address this issue, we previously engineered a bi-specific molecule to recruit CD45, an abundant and promiscuous receptor tyrosine phosphatase, within close proximity of PD-1. In this approach, the phosphatase domain of CD45 acts intracellularly, in cis, on the p-Tyr residues of the PD-1 ITIM/ITSM motif, thus inhibiting sustained signalling. This demonstrated that Receptor Inhibition by Phosphatase Recruitment (RIPR) potentiates T cell activity beyond that seen with PD-1/PD-L1 antagonist antibodies, both in the presence and absence of PD-1 ligand-binding in vitro, and reduces tumour growth in mouse models of small cell lung cancer and colon adenocarcinoma (Fernandes et al., Nature, 2020). In this PhD project, we propose to expand this novel approach to shut down signalling by key immune and cancer-specific receptors aimed at generating novel antitumor, RIPR-based, molecules.We propose to develop novel RIPR proteins to shut down signalling by inhibitory checkpoint receptors expressed in cytotoxic T cells, including BTLA, CTLA-4, ILT2 and ILT4. We will systematically test the potency of newly generated RIPR proteins using various anti-CD45 nanobodies. Nanobodies against CD45 isoforms and other phosphatases found in immune cells will also be developed and tested for their ability to shut down inhibitory signalling. Newly developed RIPR molecules will be characterized in biophysical assays. Binding on-rate, off-rate and affinity will be determined by surface plasmon resonance. Next, early readouts for signalling potency will be determined with in vitro activation assays, using T cells, NK cells or macrophages. Candidate RIPR molecules will also be tested for the ability to potentiate T cell cytotoxic functions using co-culture assays with T cells and target cancer cells. Markers of T cell activation will be quantified longitudinally using flow cytometry, western blotting, ELISAs and RNA-Seq. This comprehensive approach is expected to identify determinants of RIPR activity for various checkpoint receptors. This information will guide the design of future antagonists of checkpoint receptor signalling with strong potential for therapeutic applications.