Engineered iPSC-derived NK cells targeting the SIGLEC7-glyco-immune checkpoint improves anti-tumor activity in solid cancers

Abstract Sialylated glycans are typically over-expressed on the surface of tumor cells compared to normal cells. These glycans engage inhibitory sialic acid-binding immunoglobulin-like lectins (SIGLECs) on immune cells to dampen anti-tumor immunity. SIGLEC7, a key glyco-immune checkpoint receptor on natural killer (NK) cells, suppresses NK cell-mediated cytotoxic function via recruitment of SHP1/SHP2 phosphatases following engagement with tumor-expressed sialo-glycans. This interaction contributes to immune evasion in the tumor microenvironment (TME), especially in solid tumors characterized by glycan-rich surfaces. To overcome SIGLEC7-mediated inhibition, we studied the impact of genetically deleting SIGLEC7 on human induced pluripotent stem cell (iPSC)-derived NK cells. Using a CRISPR-Cas9 gene editing approach, we engineered WT-iPSCs with three independent guide RNAs (gRNAs) targeting exon 2 of the SIGLEC7gene. SIGLEC7 deletion was confirmed by PCR, Sanger sequencing, and western blot analysis. After confirming SIGLEC7 deletion, we selected three SIGLEC7-KO clones (Clones 1, 4, and 5) derived by single clonal selection. Using standard methods from our group, hematopoietic differentiation was done by spin embryoid body (EBs) (also called hematopoietic organoid) formation using defined cytokines. EBs are analyzed by flow cytometry for CD34, CD31, CD43, and CD45 expression at 6 days of differentiation. These studies using KO and WT-iPSCs demonstrated that SIGLEC7 deletion did not impair early hematopoietic differentiation potential. Day 6 EBs were transferred into NK cell differentiation conditions. This leads to production of CD56+CD45+ NK cells. During NK cell expansion, the phenotypic characterization of WT and SIGLEC7 KO iPSC NK cells demonstrated similar expression patterns of key receptors, including CD94, CD16, NKp30, NKp44, NKp46, NKG2D, DNAM-1, FasL, and TRAIL, indicating successful differentiation and expansion of SIGLEC7-KO iPSC-derived NK cells. Deletion of SIGLEC7 was further validated by flow cytometry and western blotting on the SIGLEC7-KO iPSC NK cells compared to WT iPSC NK cells to demonstrate loss of SIGLEC7 expression. We hypothesized that deletion of SIGLEC7 in iPSC derived NK cells will improve anti-tumor activity and in vivo persistence. We have tested the killing activity of WT and SIGLEC7-KO iPSC-derived NK cells against various tumor cell lines including K562 (leukemia), NBLS and SKN-AS (neuroblastoma), A1847 and SKOV3 (ovarian cancer), HepG2 and SNU-449 (hepatocellular carcinoma), and CAL27 (head and neck squamous cell carcinoma). Functional activity of WT and SIGLEC7 KO iPSC NK cells was analyzed using an Incucyte-based long term killing assay, as well as flow cytometry-based analysis of CD107a, IFN-γ and TNF-α expression. SIGLEC7-KO NK cells exhibited significantly increased degranulation (CD107a expression) and increased production of IFN-γ and TNF-α when compared to WT iPSC NK cells against all tumor types. Interestingly, we compared the killing activity of SIGLEC7-KO and TGFBR2-KO iPSC NK cells (derived previously by our group) and found the SIGLEC7-KO iPSC NK cells demonstrated a similar degree of anti-tumor activity, even against tumor cells known to express high levels of TGF-β. Indeed, these results show anti-tumor activity of SIGLEC7-KO iPSC NK cells similar to TGFBR2-KO iPSC NK cells to suggest a link between SIGLEC7 and TGF-β signaling which is under investigation. Collectively, our data demonstrate that SIGLEC7-deletion is a viable and potent engineering strategy to reverse glyco-immune checkpoint-mediated NK cell inhibition. By genetically disrupting SIGLEC7, we were able to restore NK cell effector functions and improve anti-tumor activity against hematological and solid tumors. Studies to test the in vivo anti-tumor activity and persistence of these SIGLEC7 KO iPSC NK cells in a tumor xenograft model are now underway. This will provide critical translational insights into the therapeutic relevance of glyco-immune checkpoint inhibition in solid tumors. Our findings validate SIGLEC7 as a functional glyco-immune checkpoint that limits NK cell-mediated anti-tumor responses and establishes SIGLEC7 KO as a promising engineering strategy to improve the efficacy of engineered NK cell immunotherapies.