Triple negative breast cancer (TNBC) is one of the lethal diseases affecting women and its lack of three marker receptors made it challenging for chemotherapy. In TNBC, CD24-Siglec10 (sialic-acid-binding Ig-like lectin 10) axis is identified as the innate immune checkpoint¹. CD24 is a glycoprotein over-expressing on breast cancer cell surface. Due to its intensive sialoside expression, Siglec-10 on tumor-associated macrophage (TAM) binds to CD24 with its IgV-like domain, activating “Don’t eat me signal” and assisting breast cancer cells away from the phagocytosis. In this study, to block CD24-Siglec10 interaction for recovering immune response, we developed macrocyclic peptides against Siglec-10. 

The Random nonstandard Peptides Integrated Discovery (RaPID) system was applied to select thioether-closed macrocyclic peptides binders to Siglec-10-Fc from the library with over 10¹² sequences in vitro². After 5th round of the selection, the hits were identified by next generation sequencing. However, the binding site of those hits is not necessarily to be the IgV-like domain which is the target to block CD24-Siglec10 interaction. To clarify the peptides’ binding preference, in vitro binding assay targeting the full and isolated IgV-like domain of Siglec-10 was performed. The strongest binding peptide, D2, shows binding affinities with K_ds of 24.2 and 188 nM to full and IgV domain of Siglec-10, respectively. Furthermore, the cell-based inhibition assay of CD24-Siglec10 was also performed. The breast cancer cell with CD24 overexpression was incubated with isolated Siglec-10-Fc in the presence of peptide D2. The fluorescent-labeled human IgG antibody was used as the probe to detect the binding amount of Siglec-10-Fc on the surface of breast cancer cell. The binding of Siglec-10-Fc was decreased in dose dependent manner with peptide D2. So far, combined with the surface plasmon resonance and cell-based assay, the identification of inhibitory macrocyclic peptides against CD24-Siglec10 was successfully achieved.