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

在过去的十年中，免疫检查点阻断已成为免疫治疗的主要进展。然而，只有一小部分癌症患者对检查点阻断有反应，这表明对免疫检查点受体信号传导的基本机制缺乏基本的理解。必须开发新的治疗药物。该博士项目旨在开发一种增强T细胞功能的新方法，并了解检查点受体如何抑制T细胞功能。通过免疫检查点（如PD-1和CTLA-4）调节t细胞信号传导一直是癌症免疫治疗最近突破的核心。PD-1和CTLA-4的信号传导降低了T细胞的活性，导致“耗尽”表型，严重影响抗肿瘤反应。在PD-1的情况下，结合PD-L1/2触发信号基序的酪氨酸磷酸化，并导致细胞质磷酸酶（如SHP1/2）的募集，从而减少TCR和CD28信号传导。引人注目的是，几种免疫受体的信号传导依赖于ITAM/ITIM/ITSM信号基序的Tyr磷酸化。我们假设强化剂受体磷酸化和“配体经验”受体的持续信号传导影响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，我们之前设计了一个双特异性分子来招募CD45，这是一种丰富且混杂的酪氨酸磷酸酶受体，靠近PD-1。在这种方法中，CD45的磷酸酶结构域在细胞内顺式作用于PD-1 ITIM/ITSM基序的p-Tyr残基上，从而抑制持续的信号传导。这表明，在体外存在或不存在PD-1配体结合的情况下，通过磷酸酶募集（RIPR）抑制受体可以增强T细胞活性，而不是PD-1/PD-L1拮抗剂抗体，并降低小细胞肺癌和结肠腺癌小鼠模型中的肿瘤生长（Fernandes等人，Nature, 2020）。在这个博士项目中，我们建议扩展这种新方法，以关闭关键免疫和癌症特异性受体的信号，旨在产生新的抗肿瘤，基于ripr的分子。我们建议开发新的RIPR蛋白来关闭细胞毒性T细胞中表达的抑制性检查点受体的信号传导，包括BTLA， CTLA-4， ILT2和ILT4。我们将使用各种抗cd45纳米体系统地测试新生成的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体也将被开发出来，并测试它们关闭抑制性信号传导的能力。新开发的RIPR分子将在生物物理分析中进行表征。表面等离子体共振决定了结合的接通率、关闭率和亲和力。接下来，信号效力的早期读数将通过体外激活试验确定，使用T细胞，NK细胞或巨噬细胞。候选RIPR分子还将通过与T细胞和靶癌细胞共培养试验来测试增强T细胞细胞毒性功能的能力。T细胞活化的标记物将使用流式细胞术、western blotting、elisa和RNA-Seq进行纵向定量。这种综合的方法有望确定各种检查点受体的RIPR活性的决定因素。这一信息将指导未来检查点受体信号拮抗剂的设计，具有很强的治疗应用潜力。

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.