Development and activity of canine B7-H3-CAR.CIK lymphocytes against sarcomas: preclinical evidence and perspectives for human clinical translation.

Abstract

Advanced-stage sarcomas pose a major challenge in oncology, as they are often resistant to conventional therapies and associated with poor prognosis. CAR-based cellular immunotherapy is emerging as a very promising therapeutic option; however, clinically relevant animal models are urgently needed to accelerate the clinical development of these approaches. B7-H3 molecule, due to its low expression in normal tissue, high prevalence in multiple human cancers, and association with cancer stemness and aggressiveness, represents one of the most attractive targets for CAR-immunotherapy. In this study, we established a preclinical cellular immunotherapy platform based on canine cytokine-induced killer cells (CIK) redirected by an antihuman B7-H3 CAR against canine sarcoma cells and 3D sarcoma spheroids. B7-H3 was consistently detected across all analyzed canine sarcoma subtypes, including osteosarcoma, soft tissue sarcoma, and hemangiosarcoma, although with variable levels of expression intensity. We successfully generated canine B7-H3-CAR.CIK, achieving a mean CAR expression of 39%&#x2009;&#xb1;&#x2009;2 with an immune phenotype unmodified (NTD) control (CD3&#x2009;=&#x2009;92%&#x2009;&#xb1;&#x2009;3, CD8&#x2009;=&#x2009;87%&#x2009;&#xb1;&#x2009;7; CD4&#x2009;=&#x2009;49%&#x2009;&#xb1;&#x2009;5; CD5&#x2009;=&#x2009;77%&#x2009;&#xb1;&#x2009;0.1; NKp46&#x2009;=&#x2009;83%&#x2009;&#xb1;&#x2009;5). Canine B7-H3-CAR.CIK efficiently killed canine sarcoma cell lines compared with NTD.CIK, even at low effector/target (E/T) ratios (B7-H3-CAR.CIK: 45% vs 8%; E:T 1:1; 48&#xa0;h; N&#x2009;=&#x2009;7, n&#x2009;=&#x2009;8; p&#x2009;<&#x2009;0.0001), and demonstrated significant cytotoxicity against 3D sarcoma spheroids (58% vs 13%; E:T 2:1; 48&#xa0;h; N&#x2009;=&#x2009;3, n =&#x2009;4; p&#x2009;<&#x2009;0.01). Our findings establish a clinically relevant and translationally valuable platform for evaluating B7-H3-CAR.CIK therapy in dogs with incurable sarcomas, providing a bridge toward the development of novel CAR-based immunotherapies for human incurable sarcomas.