Disruption of the sialic acid/Siglec-9 axis improves antibody-mediated neutrophil cytotoxicity towards tumor cells

Abstract

Upregulation of surface expressed sialoglycans on tumor cells is one of the mechanisms which promote tumor growth and progression. Specifically, the interactions of sialic acids with sialic acid-binding immunoglobulin-like lectins (Siglecs) on lymphoid or myeloid cells transmit inhibitory signals and lead to suppression of anti-tumor responses. Here, we show that neutrophils express among others Siglec-9, and that EGFR and HER2 positive breast tumor cells express ligands for Siglec-9. Treatment of tumor cells with neuraminidases or a sialyl transferase inhibitor significantly reduced binding of a soluble recombinant Siglec-9-Fc fusion protein, while EGFR and HER2 expression remained unchanged. Importantly, the cytotoxic activity of neutrophils driven by therapeutic EGFR or HER2 antibodies in vitro was increased by blocking the sialic acid/Siglec interaction, either by reducing tumor cell sialylation or by a Siglec-9 blocking antibody containing an effector silenced Fc domain. In vivo a short-term xenograft mouse model confirmed the improved therapeutic efficacy of EGFR antibodies against sialic acid depleted, by a sialyltransferase inhibitor, tumor cells compared to untreated cells. Our studies demonstrate that sialic acid/Siglec interactions between tumor cells and myeloid cells can impair antibody dependent tumor cell killing, and that Siglec-9 on polymorphonuclear cells (PMN) is critically involved. Considering that PMN are often a highly abundant cell population in the tumor microenvironment, Siglec-9 constitutes a promising target for myeloid checkpoint blockade to improve antibody-based tumor immunotherapy.

Dissecting the Ability of Siglecs To Antagonize Fcγ Receptors

“…It has been proposed that Siglecs may limit the effectiveness of tumor-targeting antibodies. , In recent years, there has been a surge of studies examining the role of Siglec-E (murine Siglec-9) in antitumor immune responses. Notably, the expression of Siglec-E on phagocytic cells has been linked to reduced survival in patients with brain tumors. ,, Further to this, the removal or blockade of Siglec-E has been seen to have a synergistic effect when used in combination with known immune checkpoint inhibitors such as anti-PD-1/PD-L1 therapy. , This previous evidence of Siglecs behaving as immune-checkpoint molecules, in combination with our findings that Siglec-9 can fully inhibit FcγR-mediated immune cell activation toward cells expressing Siglec-9 ligands, demonstrates the potential for targeting Siglecs in antitumor treatments.…”

“…It was recently proposed that Siglecs expressed on myeloid cells bind to sialic acid-containing ligands on tumor cells and limit the effectiveness of tumor-targeting antibodies by antagonizing FcγRs . Moreover, antibody-mediated neutrophil cytotoxicity against tumor cells can be improved by either removing sialic acid on the surface of tumor cells or using a Siglec-9 blocking antibody . Therefore, previous evidence of Siglecs limiting the efficacy of immune checkpoints inhibitors may be the result of Siglec-mediated FcγR inhibition, leading us to question whether Siglecs can directly inhibit FcγR-mediated activation of cells.…”

Sialic Acids on Tumor Cells Modulate IgA Therapy by Neutrophils via Inhibitory Receptors Siglec-7 and Siglec-9

“…CD47-blocking, SIRPα fusion protein was produced and purified in house as described in Chernyavska et al (2022) [28]. IgG2 FcKO Siglec-9 blocking antibody (mAbA) was produced and purified in house [23].…”

“…Furthermore, recognition is heavily dependent on the linkage orientation to the core glycan and subterminal carbohydrate moieties. Recent studies have identified specific sialoglycan moieties found on malignant cells to be ligands for Siglec-7 and -9 [ 19 , 20 , 21 , 22 , 23 ]. This, however, needs to be explored further.…”

“…This, however, needs to be explored further. Although studies on NK cells, T cells, and monocytes/macrophages have demonstrated that Siglecs can contribute to tumor growth, only a limited number of studies have addressed the role of Siglecs in neutrophils [ 20 , 23 , 24 , 25 , 26 ]. A recent study conducted by Lustig et al provided evidence supporting the suppressive role of Siglec interaction in neutrophils in the context of cancer therapy [ 23 ].…”