Immunological characterization of a rigid α-Tn mimetic on murine iNKT and human NK cells

Abstract

The ability of a rigid α-Tn mimetic (compound 1) to activate murine invariant natural killer T (iNKT) and human natural killer (NK) cells, two subsets of lymphocytes involved in cancer immunesurveillance, was investigated. For this purpose, the mimetic 1 was properly conjugated to a stearic acid containing glycerol-based phospholipid (compound 5) to be presented, in the context of the conserved non polymorphic major histocompatibility complex class I-like molecules (CD1d), to iNKT cells. On the contrary, the mimetic 1 was conjugated to a multivalent peptide-based scaffold (compound 6) to induce NK cell activation.

A Structurally Simple Vaccine Candidate Reduces Progression and Dissemination of Triple-Negative Breast Cancer

“…The outcome is the assembly of demanding constructs presenting immunodominant protein carriers, which often fail in inducing TACA-specific antibodies, eliciting undesired auto-immunity. In keeping these issues and capitalizing on the encouraging results obtained with TnThr mimetic 1 (Fallarini et al, 2017;Gracia et al, 2018;Manuelli et al, 2014;Richichi et al, 2014), we synthesized the differently activated derivatives 2 and 3, from 4 as starting material (Scheme 1), to decorate the clinically validated carrier-adjuvant protein CRM197 under mild conditions. The acetyl derivative 4, obtained as reported (Arda ´et al, 2015), was reacted with the mono Boc-protected 1,6-diaminohexane, in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate and N-methylmorpholine (NMM), in dimethylformamide (DMF), to afford derivative 5 (85%).…”

Recent advances on smart glycoconjugate vaccines in infections and cancer

“…Structures of: 14 a tripartite vaccine containing the unnatural amino acid a-methylserine glycosylated with GalNAc; [273] 15 GM3-lactone mimetic conjugated to KLH protein [275]. mimetic was also able to activate iNKT cells, when conjugated to a phospholipid carrier, which enabled the mimetic to be presented in the context of the conserved nonpolymorphic MHC class I-like molecules (CD1d) [279]. Modification at the N-acetamido position of the galactose residue of MUC antigens and in the sialic acid containing antigens (e.g., GM3, sTn, sTF) has likewise been widely investigated [280][281][282][283].…”

“…This vaccine prototype has been used in immunization studies in mice and elicited long‐lasting antibodies (e.g., IgG1, IgG2a, and IgG3) able to bind Tn expressing MCF‐7 human breast cancer cells [278]. The same mimetic was also able to activate iNKT cells, when conjugated to a phospholipid carrier, which enabled the mimetic to be presented in the context of the conserved nonpolymorphic MHC class I‐like molecules (CD1d) [279]. Modification at the N‐acetamido position of the galactose residue of MUC antigens and in the sialic acid containing antigens (e.g., GM3, sTn, sTF) has likewise been widely investigated [280–283].…”

“In vitro” studies on galectin-3 in human natural killer cells

“…Human NK cells were isolated from buffy coats of healthy donors (n ≥ 6), after their informed consensus, as previously described [26]. After cell separation through negative selection (Miltenyi Biotec, Calderara di Reno, Italy), the percentage of CD3 − CD56 + cells was routinely analyzed by FACS (S3 flow cytometer, Biorad, Segrate, Italy) using FITC anti-human CD56 (NCAM) Antibody [Clone: MEM-188 (BioLegend, San Diego, CA, USA)], PerCP anti-human CD3 [Clone: BW264/56 (Miltenyi Biotec)] and always resulted ≥97%.…”

“…Monensin (BD GolgiStop™ reagent, BD Bioscences, Milan, Italy) was added and cells incubated for 3 h at 37 °C. Cells were washed, stained using FITC anti-human CD56 (NCAM) Antibody [Clone: MEM-188 (BioLegend)], and CD107a expression on CD56 + cells evaluated by FACS [26]. To detect spontaneous NK cell degranulation, a negative control (NK cells without K562 target cells) was always included, while to measure the basal response, NK cells were stimulated only with IL-2 (100 U/ml) and IL-15 (20 ng/ ml) (positive controls).…”