Kharkwal et al. showed that covalent conjugation of the CD1d agonist, α-galactosylceramide (α-GalCer), to a soluble CD1d/Β2m fusion protein linked to mouse or human HER2-specific ScFvs enabled durable iNKT cell activation. In vitro and in wild-type, CD1d-/-, or human CD1d knock-in mice, α-GalCer/HER2 BiTEs directly stimulated iNKT cells to induce DC maturation, NK and T cell activation, and epitope spreading for tumor-specific CD8+ T cell responses. BiTEs regressed grafted human Her2+ metastatic melanoma and colorectal tumor lines, synergized with anti-CTLA-4, and induced tumor-specific recall responses. Repeated BiTE injection did not induce anergy.
Contributed by Paula Hochman
ABSTRACT: CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong anti-tumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogs of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T cell responses. The anti-tumor effects of iNKT cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT anergy and exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens.