Generation of functional canine TIL products for solid tumors

Immunotherapy based on the adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) has proven effective in treating human metastatic melanoma patients, but success in tumors with lower mutational burdens remains a challenge. Preclinical evaluation of cellular therapies commonly relies on murine models, which often require implantation of tumors into immunocompromised mice and thus do not accurately reflect the complex tumor-immune interactions seen in patients. Alternatively, spontaneous tumors in client-owned dogs serve as an underutilized and valuable parallel patient population for investigating the effectiveness of adoptive cell therapy in an immunocompetent host. However, TILs have been largely unexplored in dogs. Leveraging canine cancer patients with naturally occurring low tumor mutational burden (TMB) cancer types to study TIL therapy aims to enhance preclinical translatability. To evaluate the feasibility of TIL therapy in the veterinary sector, we developed protocols to reliably expand TILs from canine oral melanoma and appendicular osteosarcoma, despite low T cell frequencies in tumor digests. A subset of these TIL products showed reactivity to autologous tumor cells from fresh tumor digests as well as early passage cell lines. Lack of TIL reactivity in a beta-2-microglobulin (B2M)-ablated canine melanoma sample confirmed that recognition was major histocompatibility complex (MHC) class I-dependent. Together, these data establish the feasibility of generating functional canine TIL products and pave the way for comparative trials to evaluate TIL efficacy and novel strategies to enhance responses in low-TMB malignancies.