Abstract

Background

Anti-tumor immunity is critically regulated by immune checkpoints such as TIM-3, PD-1 or TIGIT. Notwithstanding the remarkable success of the antibody-mediated immune checkpoint blockade in the clinics, many key immune receptors remain orphan or poorly characterized, hindering the development of novel therapeutics. This is partially due to technical challenges associated with the study of membrane-expressed proteins and identification of receptor binding partners. Here, we present a new technology for receptor-ligand discovery, which we apply to investigate interactions established by key inhibitory immune receptors.

Methods

A new platform was developed for high sensitivity receptor-ligand discovery in high throughput, the Conditioned Media Alpha Screen. Using this method, prominent immune receptors were screened for binding to a library consisting of ≈1,200 single transmembrane proteins in the human genome. The new interactions were confirmed using biophysical and cell-based methods for functional characterization of select receptor-ligand pairs.

Results

Using this technology Interleukin-20 receptor subunit alpha (IL20RA) was identified as the first counter-receptor for B7-H3. Furthermore, the natural killer cell receptor KIR2DL5A is identified as a new binding partner for PVR, an interaction that is fully validated using orthogonal methods and cellular assays. Here we show that KIR2DL5A engagement by PVR expressed on tumor cells results in reduced natural killer cell cytotoxicity. PVR deletion or treatment with an anti-KIR2DL5A antibody restored NK-mediated cytolysis of tumor cells. Additionally, competition assays show that PVR interacts with both TIGIT and KIR2DL5A on the cell surface, suggesting a previously unrecognized mechanism of action.

Conclusion

Here we developed a new platform for elucidation of secreted or membrane protein interactomes. The power of this technology is demonstrated by identification of new interactors for emerging therapeutic targets such as PVR or B7-H3. These results provide insights into immune receptor biology and highlight potential new cellular targets, ultimately serving as a unique tool to inform therapeutic development.

Legal entity responsible for the study

The authors.

Funding

Genentech Inc.

Disclosure

All authors have declared no conflicts of interest.

中文翻译：

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99P细胞外相互作用组发现的平台为免疫受体B7-H3 / CD276和PVR / CD155鉴定了新型功能性结合伴侣

抽象的

背景

抗肿瘤免疫力由TIM-3，PD-1或TIGIT等免疫检查点严格调节。尽管抗体介导的免疫检查点封锁在临床上取得了显著成功，但许多关键的免疫受体仍然是孤儿或特征欠佳，阻碍了新型疗法的发展。这部分是由于与膜表达蛋白的研究和受体结合伴侣的鉴定相关的技术挑战。在这里，我们提出了一种新的受体配体发现技术，我们将其应用于研究关键抑制性免疫受体建立的相互作用。

方法

开发了用于高通量高灵敏度受体-配体发现的新平台，即条件培养基阿尔法筛选。使用这种方法，筛选出突出的免疫受体与人基因组中约1200个单跨膜蛋白组成的文库结合。使用生物物理和基于细胞的方法对所选受体-配体对进行功能表征，证实了新的相互作用。

结果

使用该技术，白介素20受体亚基α（IL20RA）被确定为B7-H3的第一个抗受体。此外，天然杀伤细胞受体KIR2DL5A被鉴定为PVR的新结合伴侣，该相互作用已使用正交方法和细胞测定法充分验证。在这里我们显示，PVR在肿瘤细胞上表达的KIR2DL5A参与导致减少自然杀伤细胞的细胞毒性。PVR缺失或用抗KIR2DL5A抗体治疗可恢复NK介导的肿瘤细胞的细胞溶解。此外，竞争分析表明PVR与细胞表面的TIGIT和KIR2DL5A都相互作用，提示了以前无法识别的作用机制。

结论

在这里，我们开发了一个新的平台，用于阐明分泌或膜蛋白相互作用组。通过为新兴的治疗靶标（例如PVR或B7-H3）鉴定新的相互作用子，证明了这项技术的力量。这些结果提供了对免疫受体生物学的见解，并突出了潜在的新细胞靶标，最终成为了指导治疗发展的独特工具。

负责研究的法人实体

作者。

资金

基因泰克公司

揭露

所有作者均声明没有利益冲突。