Chimeric antigen receptor T (CAR-T) cells have produced unprecedented results in blood cancers, but clinical responses in solid tumors are rare due to the detrimental effects of the hostile, immunosuppressive tumor microenvironment (TME) and the use of patient derived, dysfunctional T cells adversely affected by advanced disease state and previous chemotherapy. Invariant Natural Killer T cells (iNKTs) are a distinct lineage of CD1d-restricted T lymphocytes with natural tissue (and tumor) tropism, direct cytolytic effect on CD1d+ cancer cells and tumor associated macrophages and adjuvant effects on endogenous anti-tumor immunity. Unlike conventional T cells, allogeneic iNKTs mediate a robust graft-versus tumor effect without inducing graft-versus-host disease, thus eliminating the need for gene editing to maintain tolerance. Moreover, preclinical studies in mouse models suggest that CAR-enhanced iNKTs can eradicate solid and solid-like hematological tumors where CAR-T cells fail. We hypothesize that allogeneic CAR-engineered iNKTs will overcome the barriers to successful CAR-therapy and provide a powerful off-the-shelf universal platform with curative potential for solid tumors. Yet, unsolved questions related to allo CAR-iNKT safety, optimal preconditioning regimens to promote persistence, cell dose and therapeutic efficacy in solid tumors remain. Furthermore, while healthy human donors for allogeneic iNKT clinical trials are randomly selected, cells generated from different donors have different immunomodulatory capacities that may affect their engraftment and survival and it is currently unknown which products are “best” for adoptive cell therapy (ACT). These questions cannot be adequately addressed in mice due to the dissimilarity between murine and human iNKT cells. In contrast, our preliminary studies demonstrate that canine and human iNKT cells share remarkable phenotypic and functional similarities and can be CAR engineered and expanded to clinical scale for trial use. Here we will use immunocompetent pet dogs with spontaneous osteosarcoma (OSA), which is remarkably similar to pediatric OSA, to advance allogeneic IL-13Rα2-targeting CAR-iNKT cells into the human clinic. We will first characterize CAR-iNKT cells from different donor dogs and generate a master cell bank of canine alloCAR-iNKT products with different immunomodulatory capacities. Next, we will address the safety and effects of 3 strategically designed pre-conditioning regimens, including a combination of cytotoxic chemotherapies, low dose total body irradiation and a clinical grade iNKT glycolipid agonist, RGI-2001, on allo-CAR-iNKT engraftment and persistence in dogs with metastatic OSA. Finally, we will determine the maximum tolerated dose of alloCAR-iNKT cells using an accelerated dose escalation trial design and evaluate their effects on the TME, systemic immunome and disease free interval in dogs with appendicular OSA. This work addresses pivotal questions for the advancem...