Direct activation of iNKT cells by CD1d bound glycolipid antigens leads to a different kinetics of iNKT activation and expansion

Abstract

Abstract Invariant Natural Killer T cells (iNKT) are innate like T lymphocytes that recognize glycolipid antigens bound to CD1d molecule expressed by antigen presenting cells (APCs). iNKT cells modulate many aspects of immune responses and hence are targeted in many immunotherapeutic strategies. However, the prototypical iNKT agonist α-galactosylceramide (α-GC) and its synthetic analogues have limitations that include but are not limited to toxicity and iNKT anergy. Here, we report the development of an iNKT activating tumor targeted tertiary complex composed of a CD1d-antitumor ScFv antibody fusion protein covalently coupled to novel benzophenone linked α-GC analogs (BPGCs). Covalent complexes have dramatically improved stability and higher avidity to iNKT TCR and hence up to ten times lower effective dosage of the complex can stimulate strong immune response. Notably, these complexes directly activate iNKT cells bypassing the need for APCs. Hallmarks of covalent complex mediated iNKT activation include strong and sustained IFN-y secretion with no extensive iNKT proliferation or upregulation of co-inhibitory markers such as PD-1, even after multiple treatments as opposed to free α-GC and non-covalent complexes. In vitro, anti Her2 ScFv.CD1d.BPGC complexes lead to iNKT mediated targeted killing of MC38 cells overexpressing human Her2. Preliminary antitumor immunotherapy experiments in mice have shown that one third of the covalent complex treated mice show substantial reduction in tumor growth, including apparent eradication of tumors in some cases. Altogether, our approach enables a strong iNKT-based immune activation without the need for APCs and provides long lived and localized NKT activation in the tumor microenvironment.