Controlled Reversible N-Terminal Modification of Peptides and Proteins.

A reversible modification strategy enables a switchable cage/decage process of proteins with an array of applications for protein function research. However, general N-terminal selective reversible modification strategies which present site selectivity are specifically limited. Herein, we report a general reversible modification strategy compatible with 20 canonical amino acids at the N-terminal site by the palladium-catalyzed cinnamylation of native peptides and proteins under biologically relevant conditions. This approach broadens the substrate adaptability of N-terminal modification of proteins and shows a potential impact on the more challenging protein substrates such as antibodies. In the presence of 1,3-dimethylbarbituric acid, palladium-catalyzed deconjugation released native peptides and proteins efficiently. Harnessing the reversible nature of this protocol, practical applications were demonstrated by precise function modulation of antibodies and traceless enrichment of the protein-of-interest for proteomics analysis. This novel on/off strategy working on the N-terminus will provide new opportunities in chemical biology and medicinal research.

Design and synthesis of novel site-specific antibody-drug conjugates that target TROP2.

The approval of Trodelvy® validates TROP2 as a druggable but challenging target for antibody-drug conjugates (ADCs) to treat metastatic triple-negative breast cancer (mTNBC). Here, based on the TROP2-targeted antibody sacituzumab, we designed and developed several site-specific ADC candidates, which employ MMAE (monomethyl auristatin E) as the toxin, via IgG glycoengineering or affinity-directed traceless conjugation. Systematic evaluation of these site-specific ADCs in homogeneity, hydrophilicity, stability, and antitumor efficiency was conducted. The results indicate that the site-specific ADCs gsADC 3b made from one-step glycoengineering exhibit good aggregation stability and in vivo efficacy, providing a new format of ADCs that target TROP2.

Hiding Payload Inside the IgG Fc Cavity Significantly Enhances the Therapeutic Index of Antibody-Drug Conjugates.

The inadequate understanding of the structure-activity relationship (SAR) of glycosite-specific antibody-drug conjugates (ADCs) hinders its design and development. Herein, we revealed the systemic SAR and structure-toxicity relationship (STR) of gsADCs by constructing 50 gsADC structures bearing three glycan subtypes and diverse linker-drug combinations. According to the results, extra hydrophilic linkers are indispensable for the intact glycan-based gsADCs to achieve better in vivo efficacy. Meanwhile, the gsADCs that conjugate linker-drug complexes onto the terminal sialic acid are more stable and potent than the ones conjugated onto the terminal galactose in vivo. Notably, the LacNAc-based gsADCs, which shortened the spacer and located the linker-drug more inside the immunoglobulin class G (IgG) Fc cavity, showed excellent hydrophilicity, in vivo activity, pharmacokinetics, and safety. Conclusively, we found that hiding the linker-toxin into the Fc cavity can significantly enhance the therapeutic index of LacNAc-based gsADCs, which will benefit the further design of ADCs with optimal druggability.