Allogeneic chimeric antigen receptor invariant NKT cells for the treatment of canine and human solid tumors

Introduction Invariant Natural Killer T (iNKT) cells are CD1d-restricted lymphocytes that do not cause graft-versus-host disease, have natural tumor tropism and recognize CD1d on tumor-associated macrophages as well as malignant cells. Using a comparative approach in immunocompetent dogs, an unrivaled large-animal model for adoptive cell therapies (ACT), we investigated feasibility of allogeneic (allo-)iNKT ACT to improve clinical outcomes in solid tumor patients. Methods iNKTs from 10 canine and 10 human healthy donors were isolated, expanded and characterized by killing, proliferative and cytokine immunoassays alongside single-cell RNA-sequencing (scRNA-seq). To determine allo-iNKT safety, immune effects and persistence, unedited canine allo-iNKTs were infused into healthy, MHC-mismatched non-lymphodepleted recipients and monitored by clinical pathology, flow cytometry and RT-qPCR in blood and bone marrow. Canine and human iNKTs were engineered with CARs targeting the solid tumor antigens B7H3 and IL13Ra2 and characterized by scRNA-seq and killing assays against human and canine osteosarcoma and brain tumor lines. Results We revealed that canine iNKTs recapitulate unique genomic, immunophenotypic and transcriptomic features of human iNKTs. When comparing different donors, we identified donor-specific functional and transcriptome signatures of immunological fitness. Infusion of 4x108 unedited iNKTs into immunocompetent, non-lymphodepleted recipients was safe, and functional allo-iNKTs persisted for at least 118 days. Canine CAR-iNKTs exhibited greater killing and proliferative reactivity than unedited iNKTs against B7H3 and IL13Ra2-expressing lines, with variable anti-tumor activity associated with donor-specific molecular profiles. Conclusion We established a powerful canine iNKT model, proving the feasibility of allo-iNKTs therapy with long-lasting therapeutic potential. Canine allo-CAR-iNKTs recapitulate clinically relevant features of human CAR-iNKTs. Donor-specific signatures can inform infusion of optimal allo-CAR-iNKTs for maximal responses. Significance Our model will accelerate clinical development of iNKT-based therapies. Allo-CAR-iNKTs can be leveraged to realize a potent, non-toxic and globally accessible ACT to improve outcomes in canine and pediatric solid cancer patients.