α-Galactosylceramides (α-GalCer) are potent immunomodulatory glycolipids that specifically activate iNKT cells (invariant natural killer T), a subclass of T-lymphocytes. Their activation requires the formation of a trimolecular complex involving the iNKT cell T-cell receptor (TCR), α-GalCer, and the CD1d molecule expressed on antigen-presenting cells (APCs). Formation of this ternary complex triggers rapid iNKT cell activation, expansion, and the secretion of a broad range of cytokines that orchestrate both innate and adaptive immune responses.

The functional outcome of iNKT cell activation, including T_H1 or T_H2 immune polarization, is strongly influenced by the stability of the TCR/α-GalCer/CD1d ternary complex. Extensive structure–activity relationship (SAR) studies, together with molecular modeling and crystallographic analyses, have demonstrated a direct correlation between ternary complex stability and the orientation of the immune response. This relationship provides a key framework for the rational design of novel α-GalCer analogues with optimized immunomodulatory properties for a range of therapeutic applications, with a particular focus on cancer immunotherapy in our group.

SAR Study: Our work in this field focuses on the synthesis and evaluation of novel α-GalCer analogues bearing modifications on the sphingosine and/or N-acyl chains, including polyfluorinated derivatives, to investigate how electronic effects influence the stability of the ternary complex. We developed an efficient synthetic strategy involving 10–15 steps. Biological evaluation, together with molecular modeling studies, enabled the identification of highly potent analogue displaying activity up to 100 times greater than that of the parent compound, KRN7000.

Vectorization approach: Our research subsequently evolved toward the vectorization of this derivative. We hypothesized that coupling a therapeutic antibody with an immunostimulatory payload would enable selective tumor targeting and enhance therapeutic efficacy through synergistic immunostimulation.

Anti-EGFR monoclonal antibodies (mAbs), including cetuximab and panitumumab, are part of the standard treatment for several solid tumors, particularly RAS wild-type colorectal cancer. Beyond blocking EGFR signaling, these antibodies also trigger immune effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, these immune functions alone are not sufficient to achieve durable tumor control, especially in patients presenting RAS mutations.

Our project proposed to explore an alternative strategy, initially inspired by the Antibody-Directed Enzyme Prodrug Therapy (ADEPT) concept, by covalently coupling therapeutic anti-EGFR antibodies to our optimised GalCer immunostimulants. The antibody serves as a selective tumor-targeting moiety, delivering the immunostimulatory payload directly to the tumor microenvironment to enhance local immune activation while preserving the therapeutic properties of the antibody. The project focused on the design, synthesis and preclinical evaluation of glycoconjugated anti-EGFR antibodies based on the clinically approved mAbs cetuximab. The objective was to combine targeted EGFR pathway inhibition with localized immune stimulation, thereby providing a complementary mechanism of action to improve anti-tumor efficacy, reduce off-target toxicity through selective tumor delivery, and potentially overcome resistance associated with RAS mutations.

Our preclinical biological results are highly encouraging and are largely driven by the remarkable activity of the optimized GalCer derivative previously identified through our initial SAR study. These findings are currently awaiting publication and further development of the project depends now on obtaining additional funding. Given its broad applicability, the overall strategy could readily be extended to other cancer types.