News Resource: School of Chemistry and Chemical Engineering

Editor: News Agency of BIT

Translator: Li Wenlin, News Agency of BIT

Under the precise regulation of signal pathway, the state of proteins in cells has constantly been in an orderly dynamic change. However, the occurrence of some major diseases is accompanied by the cellular localization and content expression of protein disorder. For instance, in cancer treatment research, though PD-L1 immune checkpoint blockade cancer therapy is a cancer treatment strategy that has attracted much attention in recent years, but in clinical research it has been proved that there are few target-audience and high recurrence rate. Research has confirmed that PD-L1’s receptor PD-1 has other ligands, and PD-L1 itself has the behavior of ‘repeated compensation’ in cells, disturbing its location and content at the subcellular level. Therefore, it’s crucial to identify and regulate the location and content of PD-1 pathway protein thoroughly, accurately and orderly. Presently, enhanced tumor combined treatment platform can be constructed through multifunctional protein recognition, drug delivery, etc. However, hardly any intelligent recognition systems can spatially regulate the location of immune checkpoint proteins.

Based on the above issues, Professor Wang Weizhi's team of BIT has recently developed a click chemistry-driven polypeptide functional recognition and regulation "cell space station" system to realize spatiotemporal manipulation of immune checkpoints. The function of this "Cell Space Station (CSS)" is similar to that of a space station. It can complete tasks such as "landing at a designated location", "releasing goods or astronauts", and "docking with boosters" at the cellular or subcellular level, so as to achieve a comprehensive blockade of PD-1 at the cellular level and release drugs, directionally transporting PD-L1 on the cell membrane to the lysosome at the subcellular level, decreasing its abundance on the cell surface. The whole process is controlled by click chemistry, targeted, controllable and orderly. Relevant achievements were published on ACS Nano（DOI: 10.1021/acsnano.2c05483）, an authoritative international chemical journal, under the title of "A" Cell Space Station "for Spatial Molecular Manipulation of Immune Checkpoint". Li Lingyun, a postgraduate of the School of Chemistry and Chemical Engineering of BIT, is the first author of this paper, researcher Wang Weizhi is the last corresponding author, and Professor Zhang Jiatao and Associate Professor Ma Bing of BIT are the co corresponding authors.

Fig 1. "Cell Space Station" is used for spatiotemporal manipulation of immune checkpoints

The main contents of the study are as follows: The peptide libraries are constructed with PD-L1 and PD-L2 as targets, respectively. The targeted peptides KC (KHTDYEYNC) and HC (HVSKTEGRHRNC) with high affinity (KD ≈ 10-7 M) and specificity are screened out by high throughput microfluidic chips, while functional liposomes are prepared to construct CSS. Through CSS, PD-L1/PD-L2 are simultaneously identified and landed on the cell surface, enhancing cell uptake of loaded drugs. The overall blocking of PD-1 is achieved through the collaborative blocking of PD-L1/PD-L2-PD-1.

Fig 2. Comprehensively blocking of PD-1 through the CSS system.

In order to specifically transport PD-L1 from cell membrane to lysosome, a lysosomal chaperone autophagy motif KQ (KFERQKILDQRFFEC) is introduced. Sequential molecular recognition and regulation in time and space are realized through click chemistry in organisms. From the spatial dimension, PD-L1 on the cell membrane is transported to the lysosome for rapid and continuous degradation to break its "repeated compensation" behavior and reduce its content on the cell surface. From the time dimension, on the one hand, all links of this complex regulation process, including the blocking of immune checkpoint, drug release, PD-L1 transport, etc., occur orderly and controllable. On the other hand, it makes protein regulation occur within the "residence time" of ligand receptor interaction of targeted peptide KC/PD-L1 to improve the efficiency of subsequent protein transport and degradation. Specifically, azide is introduced into the KC side chain to prepare CSS that can generate click chemistry, and alkynyl is introduced into the KQ side chain as another component of click chemistry "Lysosome Booster (LB).” By identifying PD-L1/PD-L2, CSS stay on the cell surface can be controlled to have in situ click chemical reaction with LB at the specified time point to activate the spatiotemporal manipulation of PD-L1, degrading it continuously from lysosome. The protein sorting and regulation strategy based on click chemistry is named "click chemistry driven protein sorting (PROCLISORT)”. Both in vitro and in vivo, this strategy has been proved to be more efficient than CSS/LB in acting on proteins at the same time, and has enhanced immune activation ability, showing anti-tumor potential.

Fig 3. "Cell Space Station" realizes space-time control by clicking chemistry

In this paper, a docking process of "cell space station CSS" and "lysosomal booster LB" is developed (CSS@LB), realizing the spatiotemporal manipulation of the entire PD-1 pathway. In terms of treatment, CSS@LB could enhance the effect of tumor immunotherapy by comprehensively blocking PD-1 and regulating the subcellular location and content of PD-L1. In terms of strategy development, our proposed PROCLISORT strategy provides an effective reference for the spatiotemporal manipulation of proteins in living cells, and is expected to be applied to other protein pathways that need to be regulated.