The PNGase F-mediated (Supplementary figure s14). killing in vivo we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual-functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that nature killer cells altered with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells Dye 937 and improved trafficking to sites where the malignancy cells reside, respectively. while leaving healthy B cells untouched. in a xenograft lymphoma model, however, the functionalized NK-92MI cells only show weak activities. In this model, E-selectin that is constitutively expressed in the bone marrow binds to lymphoma cells, facilitates their bone-marrow deposition, and shed them from attacking by NK-92MI cells. We discovered that NK-92MI cells that Dye 937 are functionalized with CD22 ligands can be further designed via fucosyltransferase-mediated installation of E-selectin ligands to enhance their bone marrow migration. In this way, significant suppression of B-lymphoma proliferation is usually achieved. Results Engineering CD22 Dye 937 ligands on live cells via chemoenzymatic glycan editing. To engineer killer cells for targeting CD22 positive tumors, we sought to install CD22 ligands onto their cell surfaces directly. Because the sialylated epitope Neu5Ac2-6Gal1-4Glc2,6ST (Pd2,6ST[34,35]), were evaluated for this endeavor using biotinylated CMP-Neu5Ac[36] as the donor. Approximately 2faged more biotin-Neu5Ac was incorporated onto Lec2 cells by ST6Gal1 compared to that added by Pd2,6ST (Supplementary physique s1). Therefore, ST6Gal1 was chosen for the follow-up cell-surface glycan engineering experiment (Physique 1a). Open in a separate window Physique 1. Chemo-enzymatic generation of sialyl ligands on live cells to increase CD22 binding. (a) ST6Gal1- and ST3Gal4-assisted incorporation of Neu5Ac onto type 2 Lacsialylation, even Dye 937 with proliferation- or turnover-associated dilution of cell-surface ligands the altered NK-92MI and CIK cells still showed approximately 10 and 15 fold better CD22-Fc binding than the untreated cells, respectively (Supplementary physique s7f and s8b). Desialylation by neuraminidase treatment followed by enzymatic sialylation of NK-92MI and CIK cells did not increase CD22 binding (Physique 2f, Supplementary physique s7b, s8h and s8i), but rather impaired CD22 binding especially when the natural donor substrate CMP-Neu5Ac was utilized for sialylation. This observation suggests that in the natural scenario, Neu5Ac2-6Gal1-4Glcscenario. RBC present nature ligands of CD22 on their cell surface as revealed by CD22-Fc staining (Supplementary physique s12).Interestingly, CD22-ligand-mediated targeting of NK-92MI and CIK cells to B-lymphoma cells was 2faged improved after docking 100 occasions of RBC cells into the co-culture systems (Figure 3i, ?,3j3j and Supplementary physique s8d). As expected, human RBCs functionalized with high-affinity CD22 ligands did not induce the killing of Raji and Daudi cells (Supplementary physique s13). Importantly, while the altered NK-92MI cells bound to normal B cells from healthy donors, there was no apparent specific lysis detected (Physique 3a and Supplementary physique s14). Together, these results suggest that killer cells armed with CD22-ligands may be a biocompatible tool for selective eradication of B-lymphoma. Exploiting glycan engineering for efficient NK-92MI-immunotherapy in a mouse model. The antitumor efficacies of NK-92MI armed with high-affinity CD22 ligands were next evaluated in vivo using a xenograft model. Raji cells expressing firefly luciferase (Raji-Luc) were intravenously (suggest that the altered NK cells may not be sufficiently trafficking to bone marrow to attack B lymphoma where they reside injection, the injected Raji-Luc cells primarily migrated to the bone marrow and the circulating Raji-Luc cells was only detectable in the bloodstream until late stages MMP10 (i.e. 14-16 days after Raji inoculation). By contrast, the majority of the injected NK-92MI cells were found in the lung, spleen and blood steam.[19,27] Based on this observation, we hypothesized that increasing bone marrow infiltration would increase NK-induced tumor cell targeting and killing. The rolling and tethering of leukocytes on inflamed vessel endothelium cells rely on the binding of cell-surface sialyl Lewis X (sLeX,.