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

通过控制检查点受体信号传导的抑制来增强抗肿瘤 T 细胞功能

• 批准号: 2743035
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2743035
• 关键词: Enhancing; anti; tumour; cell; function

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.

在过去的十年中，免疫检查点阻断已成为免疫疗法的一个主要治疗进展。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了解。必须开发新的治疗药物。这个博士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。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中心。PD-1和CTLA-4的信号传导降低了T细胞活性，并导致“衰竭”表型，严重损害了抗肿瘤反应。在PD-1的情况下，与PD-L1/2的结合触发信号传导基序的酪氨酸磷酸化，并导致细胞溶质磷酸酶如SHP 1/2的募集，这反过来又减少TCR和CD 28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信号传导基序的Tyr磷酸化。我们假设，紧张性受体磷酸化和持续的信号传导的“配体经历”受体的影响T细胞的功能，并未能被控制的细胞外拮抗剂抗体。为了解决这个问题，我们先前设计了一种双特异性分子，以在PD-1附近招募CD 45，这是一种丰富且混杂的受体酪氨酸磷酸酶。在该方法中，CD 45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，从而抑制持续的信号传导。这表明，在存在和不存在PD-1配体结合的情况下，通过磷酸酶募集的受体抑制（RIPR）增强T细胞活性超过用PD-1/PD-L1拮抗剂抗体所见的T细胞活性，并且减少小细胞肺癌和结肠腺癌的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的方法来关闭关键免疫和癌症特异性受体的信号传导，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，ILT 2和ILT 4。我们将使用各种抗CD 45纳米抗体系统地测试新生成的RIPR蛋白的效力。还将开发针对免疫细胞中发现的CD 45亚型和其他磷酸酶的纳米抗体，并测试其关闭抑制性信号传导的能力。新开发的RIPR分子将在生物物理测定中表征。结合结合速率、解离速率和亲和力将通过表面等离子体共振测定。接下来，将使用T细胞、NK细胞或巨噬细胞通过体外活化测定来确定信号传导效力的早期读数。还将使用与T细胞和靶癌细胞的共培养测定来测试候选RIPR分子增强T细胞细胞毒性功能的能力。将使用流式细胞术、蛋白质印迹、ELISA和RNA-Seq纵向定量T细胞活化标志物。这种全面的方法，预计将确定各种检查点受体的RIPR活性的决定因素。这些信息将指导未来检查点受体信号传导拮抗剂的设计，具有强大的治疗应用潜力。