Developing bispecific antibodies is laborious because they require proper assembly of two Fab domains, maintaining stability, and achieving the desired binding affinity and specificity against both targets. Our group has developed Lasso-grafting technology, which could facilitate the development of bispecific antibodies by introducing cyclic peptide pharmacophores into the Fc domain of IgG1 to maintain both parental peptide's and the antibody's function [1]. In this study, we embedded the pharmacophore of an anti-SIRPα macrocyclic peptide identified by the Random nonstandard Peptides Integrated Discovery (RaPID) system into the anti-CD20 antibody to develop bifunctional antibody for cancer treatment.
The SIRPα expressed on macrophage suppressed its activity through the CD47-SIRPα immune checkpoint pathway. Our previous study confirmed that inhibiting this interaction raises the antibody-dependent cellular phagocytosis (ADCP) by anti-CD20 antibody against CD47-expressing tumor cells [2, 3]. Therefore, we lasso-grafted the pharmacophore of the anti-SIRPα cyclic peptide into an anti-CD20 antibody to enhance ADCP. The engineered antibody's specificity against CD20 and FcγR was confirmed using Surface Plasmon Resonance. Cellular CD47 binding assay showed that the engineered antibody inhibited CD47-SIRPα interaction.
However, we found that the lasso-grafting process dramatically decreased the binding affinity of the pharmacophore in engineered antibody compared to that of the original macrocycles. This would be mainly caused by the collapse of target-binding conformation of the pharmacophore in Fc domain because our previous study proved that affinity loss could be recovered by optimizing the spacer sequence between the pharmacophore and the protein scaffold [4]. We are currently working on the in vitro selection of proper spacer sequence in the Fc domain by mRNA display.
In conclusion, integrating the RaPID system, lasso-grafting technology, and spacer optimization has great potential to design bispecific antibodies. This new construction facilitates the development of cancer immunotherapy and paves the way for developing future therapeutic proteins with multiple functions.