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 Kds 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.