Targeting Siglec-7: A Novel Immunotherapeutic Approach to Potentiate the Cytotoxic Functions of Natural Killer Cells Against Multiple Myeloma

Background: MultipleMyeloma (MM) is a malignant plasma cell disorder, accounting for approximately 10% of all haematological malignancies. The intrinsic ability of the malignant cells to evade the host immune system is one of the key hallmarks of the disease, and is associated with poor overall survival of MM patients. The immune-suppressive functions of sialylated glycans, which are abundantly expressed on the surface of myeloma cell, has not previously been addressed. We hypothesize that hypersialylation of MM cells is a key mechanism enabling evasion of cytotoxic Natural Killer (NK) cells within the MM bone-marrow (BM) niche. Furthermore, we hypothesize that disrupting the immune-suppressive interactions between sialylated glycans on MM cells, and their cognate sialic acid-binding lectin (Siglec) receptors on NK cells could pave the way for the development of novel immunotherapeutic strategies for treating MM patients.

Methods and Results: Using a recombinant Siglec-7 Fc chimera, we observed that Siglec-7 ligands (Siglec-7L) are highly expressed (>85%) across a panel of MM cell lines (RPMI 8226, H929 and MM1S). Furthermore, Siglec-7L expression was also observed on CD38⁺ CD138⁺ primary MM cells isolated from BM aspirate of MM patient. To determine if Siglec-7L expression on MM cells plays a role in immune-evasion, we next analysed the cognate Siglec-7L receptor (Siglec-7R) on primary BM NK cells from MM patients (n=5). Interestingly, immunophenotyping analysis revealed that CD3^- NKp46⁺ NK cells circulating within the BM of MM patients have significant expression of Siglec-7R on their cell surface (78 ±27%).

To investigate the immune-suppressive effects of Siglec-7L and Siglec-7R interaction in dampening the cytotoxic functions of NK cells, we treated MM cell lines RPMI-8226, H929 and MM1S with a sialyltransferase inhibitor (SIA) (3Fax-Peracetyl Neu5Ac; dose-200µM) for 5 days. As expected, this treatment resulted in a complete eradication of Siglec-7L from the surface of MM cells. Subsequently, SIA pre-treated cells were co-cultured with the human cytotoxic NK cell line KHYG-1 at E:T ratios of 1:1, 2.5:1 and 5:1 for 12 hours. Flow cytometry based analysis of cell viability using Annexin V and Sytox Green revealed that abolishing the expression of sialylated glycans from the cell surface of MM cells results in a 1.3-1.6 fold increase (p<0.05) in NK cell induced cell death in MM cell lines RPMI 8226 and H929 (n=3) (Figure 1). However, a significant increase in NK cell mediated killing was not observed for MM1S cells (n=3) pre-treated with SIA, suggesting a role of "classical" NK cell inhibitory signals in NK cell anti-tumor immunity in the case of MM1S.

Furthermore to validate our observations we knocked-down (KD) Siglec-7R in KHYG-1 cells using an siRNA approach. KHYG-1 has relatively lower expression of Siglec-7R (20-25%), as compared to peripheral blood NK cells (>90%), and other NK cell lines such as NK-92 (>60%). siRNA targeting Siglec-7R reduced the expression of Siglec-7R from 25% in the scrambled siRNA control to 15% in the Siglec-7R siRNA experimental condition. Upon co-culture of the scrambled siRNA or Siglec-7R siRNA KD KHYG-1 cells with RPMI-8226 at E:T ratio of 0.25:1 and 0.5:1, we observed a 7% increase in NK cell mediated cytotoxicity towards the myeloma cells (n=2) .

Conclusion: We observed that sialylated Siglec-7L is abundantly expressed on the surface of MM cells. The cognate receptors of the ligands are significantly expressed on primary NK cells circulating within the BM of MM patients. Functional assays revealed that Siglec-7L and Siglec-7R receptor interactions may have significant immune-suppressive effects on the cytotoxicity of NK cells. Thus, we can leverage the findings of this study to develop NK cell based cellular therapies to target Siglec-7 ligand- receptor interaction in MM. This could be achieved by knocking down Siglec-7R in the setting of an autologous or allogeneic blood derived NK cell therapy. A more efficient approach, both therapeutically and logistically, would be the use of Siglec-7R deficient NK cell lines, such as KHYG-1 or NK-92 for the treatment of patients with MM.

Conflict Of Interest: Michael O'Dwyer: Founder, Board of Directors, Equity in Onkimmune. Subhashis Sarkar is supported by a fellowship from Onkimmune.

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