Amide-Linked C4″-Saccharide Modification of KRN7000 Provides Potent Stimulation of Human Invariant NKT Cells and Anti-Tumor Immunity in a Humanized Mouse Model

Abstract

Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses. In silico analysis suggested that the tether length and degree of flexibility of the amide substituent affected the recognition by iNKT cell antigen receptors of the C4″-amide substituted glycolipids in complex with their antigen presenting molecule CD1d. Our findings establish the use of stable C4″-amide linked additions to the sugar moiety for further exploration of the immunological effects of structural modifications of iNKT cell activating glycolipids and highlight the critical need for more accurate animal models to assess these compounds for immunotherapeutic potential in humans.

A humanized mouse model for in vivo evaluation of invariant Natural Killer T cell responses

“…This pattern was repeated in a test of anti-tumor activity of these glycolipids in the three mouse strains ( Figure 8 ). Using the B16-F10 experimental lung metastasis model, which is well established to be responsive to treatment with KRN7000 or other forms of αGalCer ( 22 , 24 , 37 , 38 ), we observed marked reductions in lung tumor burdens based on calculated areas of melanization on the lung surface following treatment with either 7DW8-5 or AH03-1 in both WT and hCD1dKI mice. In striking contrast, only 7DW8-5 was effective in VαKI mice, whereas AH03-1 showed no detectable effect on tumor burden.…”

“…The B16-F10 melanoma cell line was obtained from ATCC (passage 3), and the experimental metastasis assay was performed as previously described ( 37 , 38 ). C57BL / 6 wild type mice, hCD1dKI, or VαKI mice were injected i.v.…”

Chemical Biology of αGalCer: A Chemist's Toolbox for the Stimulation of Invariant Natural Killer T (iNKT) Cells

“…Finding past screening efforts inadequate for identifying the activity of C-4'' modified analogs in human iNKT cells, recently the groups of Porcelli and Howell exploited human iNKT cell clones cultured with human CD1d transfected cell lines and hCD1d-KI mice to characterize the activity of C-4'' amide bearing analogs, in vitro and in vivo, respectively. [130] The compounds were surprisingly found to be active in stimulating human iNKT cells, as opposed to mouse iNKT cells. In particular, analog 38 displayed enhanced T H 1 cytokine bias, via sustained transactivation of NK cells, and was found to have anti-tumor properties in a mice tumor model.…”

“…While not explored by the authors, we speculate that a glucosyl analog of 39 may be employed for accessing 4''-amino-4''-deoxy d-Gal derivatives. Following a more classical strategy, Saavedra-Avila et al [130] introduced the azide substituent at C-4 of d-Glc precursor 40, via inversion of configuration, followed by glycosylation with the N-acylated phytosphingosine moiety 3 (method A of αGalCer synthesis), which proceeded in acceptable yield. Deprotection afforded the C-4'' functionalized precursor 43 for further 4''-N-derivatization (Scheme 4B).…”

“…found that α‐glucosyl (αGlc) variants of AGP , 7DW8‐5 , and C34 , namely their C‐4’’ epimers, were more potent in activating human iNKT cells and exhibited more marked T H 1 biasing properties, once again highlighting the species‐specific character of the iNKT‐ligand‐CD1d interaction, in this case with respect to the position of C‐4’’. Finding past screening efforts inadequate for identifying the activity of C‐4’’ modified analogs in human iNKT cells, recently the groups of Porcelli and Howell exploited human iNKT cell clones cultured with human CD1d transfected cell lines and hCD1d‐KI mice to characterize the activity of C‐4’’ amide bearing analogs, in vitro and in vivo , respectively [130] . The compounds were surprisingly found to be active in stimulating human iNKT cells, as opposed to mouse iNKT cells.…”