A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells

Abstract

Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure−activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.

Rationally Designed Highly Potent NKT Cell Agonists with Different Cytokine Selectivity through Hydrogen-Bond Interaction

“…As the prototype NKT cell-activating ligand, α-galactosylceramide (αGalCer) has been extensively studied for applications in immunotherapy , and vaccine as adjuvant, − among which conjugating αGalCer to antigens has shown effective enhancement in vaccine efficacy as other conjugate vaccines. − On the other hand, scientists are still making efforts to pursue new αGalCer analogues, as structural alterations to αGalCer have been found to profoundly shape the balance between Th1 and Th2 responses. , However, the fundamental principles governing the preferential generation of either Th1 or Th2 cytokines still remain uncovered. Nonetheless, an intensified interaction between glycolipids and CD1d has been reported to promote Th1-skewed polarization. , In line with this premise, several representative Th1-biasing ligands have been designed regarding the structure–activity relationship (SAR) of glycolipid binding to CD1d, such as αGalCer analogues with aromatic groups introduced in the fatty acyl chain, − sphingosine chain, , or hydroxyl groups on the pyranose ring. − In addition, altering the stability of the CD1d/glycolipid binary or the association between the binary and T cell receptor (TCR) was also reported to favor the production of IFN-γ. − A collection of representative αGalCer analogues with Th1-biased modifications is shown in Table S1, most of which are designed to regulate the interaction between ligands and CD1d.…”

Stereospecific Synthesis and Biological Evaluation of KRN7000 Analogues with Thio-modifications at the Acyl Moiety

“…The introduction of a relatively large sulfur atom compared with an oxygen atom influences these two key hydrogen bond interactions, thus adjusting the stability of the glycolipid-CDld complex. Although compound S34 displayed a more superior Th1-biased immune response in mice, in view of differences in the recognition of specific glycolipids by CD1d in wild type mice and humans, the activities of KRN7000 and its analogues still need to be further evaluated on human iNKT cells to provide comprehensive and more accurate information for drug discovery and development. Taken together, this work demonstrates a practical and facile approach for expeditious synthesis of various α-GalCer analogues and discovers that S34 could have the potential to develop antitumor agents and vaccine adjuvants.…”