Engineering multi-functional protein biologics through PAL-mediated hydrazide ligation

Attributed to the -effects, hydrazides are nucleophilic despite being non-basic. Herein, we report that the hydrazide nucleophiles are effective acyl acceptors in the ligation reactions catalyzed by peptidyl asparaginyl ligases (PALs). Because hydrazides are easily functionalizable, hydrazide ligation is highly versatile. Interestingly, the linkages formed with the hydrazide substrates have varying degrees of liability toward the PAL enzyme, with some being remarkably resistant and thus ensuring nearly irreversible ligation. Using the hydrazide ligation method, we labelled an EGFR-targeting affibody-Fc fusion protein with various functional moieties to generate selective and potent cancer-imaging and therapeutic agents. Irreversible hydrazide ligation also allowed a sequential ligation scheme to be conducted on a protein. Using this scheme, quadruple FITC labels were introduced onto the N- and C-termini of the affibody-Fc protein to yield a bi-specific engager for Car-NK cell therapy. Our work expands the substrate scope of PAL enzymes and further point to their promise as a precision manufacturing tool for multi-functional protein biologics.