Background Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a single-pass type I membrane glycoprotein that functions as an anti-tumor immune suppressor. Siglec-15 expression is low in normal tissue but upregulated across many cancer indications. Pre-clinical studies have demonstrated that Siglec-15 promotes tumor outgrowth by inhibiting T cell functionality,¹ suggesting that blockade of this molecule could serve as a novel cancer immunotherapy. Our data show that PYX-106, a fully human IgG1 monoclonal antibody, binds to human Siglec-15 at a unique epitope site with high specificity and affinity. Importantly, our data demonstrates that PYX-106 is a potent antagonistic antibody that reverses Siglec-15 mediated immune suppression and enhances T cell functionality.

Methods The binding and biophysical properties of PYX-106 and another anti-Siglec-15 antibody (Clone 5G12) were determined and compared by plate and cell-based assays. Structural modeling and site-directed mutagenesis were used to identify the key residue that PYX-106 interacts with Siglec-15. The functional activity of PYX-106 and 5G12 were also tested by in vitro assays involving stimulated peripheral blood mononuclear cells (PBMC) from healthy donors and cancer patients.

Results Biolayer interferometry analysis revealed that PYX-106 binds to human Siglec-15 with a dissociation constant of 13 pM, 10-fold stronger than 5G12, indicating extremely high affinity. Remarkably, site-directed mutagenesis data demonstrated that PYX-106 uniquely targets an ‘essential’ sialic acid recognition residue (R143) on human Siglec-15 for efficient sialoglycan ligand blocking. Compared to 5G12, PYX-106 also demonstrates superior specificity to human Siglec-15 with no detectable binding to other Siglec family members, as well as low multi-target reactivity in a poly-specificity reagent assay using HEK cell lysates. Using stimulated PBMC from multiple healthy donors, both PYX-106 and 5G12 reversed Siglec-15 mediated immunosuppression and enhanced T cell proliferation. However, PYX-106 induced significantly higher levels of IFNγ and TNFα in T cells compared to 5G12. Interestingly, similar results were also observed using stimulated PBMC from cancer patients, suggesting that PYX-106 could be an effective immunotherapy in certain indications. Finally, PYX-106 additionally induced potent cytotoxic T cell responses that effectively killed target cells in cell-based assays involving bi-specific T cell engager molecules.

Conclusions These findings support that PYX-106 could serve as a potent therapeutic antibody for a broad array of cancer indications, including those non-responsive to conventional immunotherapies. PYX-106 is a promising immunotherapy which is currently under investigation in a Phase I clinical trial (NCT05718557).

Reference

1. Wang J, Sun J, Liu LN, Flies DB, Nie X, Toki M, et al. Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy. Nat Med 2019;25:656–66. https://doi.org/10.1038/s41591-019-0374-x.