The development of high-specificity and high-content targeting molecules with excellent performance is the basis for the realization of in vivo chemical measurements and is also the frontier of modern analytical chemistry. Compared with other types of recognition molecules, targeting polypeptides with endogenous activity have higher information content and better biocompatibility. Therefore, targeting peptides have unique advantages in precise molecular targeting in vivo and are powerful in vivo measurement and identification tools. However, there are currently limited methods for obtaining targeted peptides, and conventional molecular screening and molecular evolution methods are difficult to achieve breakthroughs in screening efficiency, screening accuracy, and peptide performance and conformation. Therefore, new strategies for targeted peptide screening are urgently needed.

Based on the above problems, Wang Weizhi’s research group from the Beijing Institute of Technology recently cooperated with Chen Xiaoyuan’s research group from the National University of Singapore to develop a new method for the multi-dimensional evolution of targeted peptide molecules based on microfluidic chips. This method can simultaneously screen target peptides with high affinity, high specificity, and high stable assembly performance against multiple tumor-related target molecules, which shows great advantages in tumor diagnosis and treatment. In addition, due to the molecular-directed evolution in the tumor tissue in situ environment, the obtained molecules are more stable in the complex living environment. The results were published in the top international journalAdvanced Materials(IF: 32.086) under the title of Totipotent 'All-in-one' Peptide Sequentially Blocks Immune Checkpoint and Reverses Immunosuppressive Tumor Microenvironment. Beijing Institute of Technology is the first correspondence unit, Zhang Limin, a doctoral student from the School of Chemistry and Chemical Engineering, is the first author of the paper, and researcher Wang Weizhi of Beijing Institute of Technology is the last corresponding author. Professor Chen Xiaoyuan from the National University of Singapore, Researcher Hu Zhiyuan from the National Center for Nanoscience, Researcher Zhao Minzhi, and Dr. Jiang Zhenqi from the Beijing Institute of Technology are the co-corresponding authors.

First, by analyzing the crystal structures of the target proteins PD-L1 and Rbm38, a peptide library with a capacity of 105 was designed and constructed, and TPE molecules with aggregation-induced emission (AIE) properties were introduced into the peptide library to indicate the stable assembly of the peptides performance. Then, the three-dimensional evolution strategy on the microarray chip was used to screen the peptide library in the three dimensions of binding ability, self-assembly performance, and specificity, and a targeted peptide TAP with high content performance was optimized.

Further, the biological properties of TAP were studied, and it was proved at the molecular and cellular levels that TAP can not only block the formation of Rbm38-eIF4E complex and upregulate p53 expression but also block PD-1/PD-L1 interaction. Notably, TAP has the ability to comprehensively downregulate intracellular and extracellular PD-L1 levels, which further enhances its in vivo therapeutic effect. At the same time, the rapid formation of stable nanofibers under the recruitment of PD-L1 due to the assembly properties of TAP further promotes its stable and continuous regulation of related pathways. In conclusion, TAP self-assembles on the cell membrane to block PD-L1 while downregulating PD-L1 expression throughout the cell, which, combined with its ability to activate p53, makes TAP a potentially powerful antitumor lead molecule.

Furthermore, the self-assembly behavior of TAP was studied by means of AFM, TEM, CD, XRD, and molecular dynamics simulation. It is demonstrated that it can be converted into an α-helix structure in the tumor microenvironment and rapidly assembled at extremely low concentrations through the specific recognition and recruitment of PD-L1.

At the level of normal mice and immunosuppressed mice, it was confirmed that TAP can specifically target tumor tissues, and showed an anti-tumor effect superior to that of antibodies.

To further understand the molecular mechanisms of TAP tumor suppressor, they used RNA sequencing (RNA-seq)-based transcriptomic and mass spectrometry-based proteomic approaches to map genetic and proteomic profiles.

Finally, the antitumor effect of TAP was validated in a patient-derived tumor xenograft (PDX) model.

In general, this study successfully screened novel targeted peptide molecules with multiple properties through the multi-dimensional evolution method of targeted peptides based on microfluidic chips. The anti-tumor effect and mechanism were studied in detail at the molecular, cellular, living, and PDX levels. This work provides a new method for the screening of high-content targeting peptides, and also provides a new idea for the precise diagnosis and treatment of cancer-based on molecular recognition.

Shanxi Provincial Hospital of Traditional Chinese Medicine, Ruijin Hospital Affiliated with Shanghai Jiaotong University, and Longhua Hospital Affiliated with Shanghai University of Traditional Chinese Medicine, etc. have given strong support in patient tissue samples and medical ethics evaluation.