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Background Chimeric antigen receptor (CAR) T cell therapy is an attractive novel immunotherapy for pediatric OS, but homing of CAR T cells to target tumors remains a challenge for clinical translation. In previous work, we identified the chemokine CXCL16 in human OS patient samples and demonstrated enhanced survival of mice with OS xenografts treated with CXCR6-modified B7-H3.CAR T cells. 1 Here, we evaluated the efficacy of CXCR6.B7-H3.CAR T cells in a spontaneously metastatic immune competent model of OS.Methods Two GEMM-derived OS cell lines, F420 and 1332, were screened for mCXCL16 production and mB7-H3 expression, and mB7-H3 overexpressing derivative lines were generated. 2 Murine CD3+ T cells were isolated from healthy donor spleens and transduced with retroviral vectors to generate mCXCR6.mB7-H3.CAR and mB7-H3.CAR T cells active against murine B7-H3.3 We evaluated CAR T cell function including transduction, phenotype, effector cytokine production, cytotoxicity, and homing capability to mCXCL16 and OS cell lines. For in vivo modeling, we performed serial passaging of cell line 1332 through murine strains of increasing immunologic complexity (NSG, Rag1KO, and C57Bl/6) to generate a spontaneously metastatic line (figure 1) and used this to evaluate mCXCR6.B7-H3.CAR antitumor activity in tail vein pulmonary colonization and primary tumor orthotopic implantation 4 models.Results F420 and 1332 were successfully labeled with mCherry and ffluc and mB7-H3-overexpressing derivatives of each line were developed. mB7-H3 overexpression enhanced cytokine production in both cell lines. mCXCL16 was poorly expressed by all cell lines in 2D culture, but robust expression was seen in whole tumor samples, indicating that tumor stroma is required for mCXCL16 production ( figure 2A). mCXCR6 expression enhanced migration of CAR T cells towards mCXCL16 (figure 2B), but had minimal impact on phenotype, cytokine production, or in vitro cytotoxicity. Mice treated with mCXCR6.mB7-H3.CAR T cells demonstrated enhanced survival in a tail vein model of pulmonary colonization as well as reduced pulmonary metastasis as measured by bioluminescence in an orthotopic primary tibial implantation model.Conclusions In an immune competent model of OS, mCXCL16 is produced in tumors only in association with tumor stroma. Transgenic mCXCR6 expression enhanced mCXCL16 dependent migration of CAR T cells with minimal impact on antigen-specific cytokine production and cytotoxicity. mCXCR6.mB7-H3.CAR T cells demonstrated enhanced control of pulmonary metastasis in both tail vein and primary orthotopic models compared to unmodified mB7-H3.CAR T cells. We conclude that CXCR6-mediated homing is a viable strategy to enhance CAR T cell efficacy in pediatric OS.Acknowledgements Dr. Deanna Langfitt, Mrs. Amanda GeorgeReferences Talbot LJ, et al. Redirecting B7-H3.CAR T cells to chemokines expressed in osteosarcoma enhances homing and antitumor activity in preclinical models. Clin Cancer Res 2024;30(19):4434-4449.Zhao S, et al. NKD2, a negative regulator of Wnt signaling, suppresses tumor growth and metastasis in osteosarcoma. Oncogene 2015;34(39):5069-79.Haydar D, et al. Cell-surface antigen profiling of pediatric brain tumors: B7-H3 is consistently expressed and can be targeted via local or systemic CAR T-cell delivery. Neuro Oncol 2021;23(6):999-1011.Talbot LJ, et al. A novel orthotopic implantation technique for osteosarcoma produces spontaneous metastases and illustrates dose-dependent efficacy of B7-H3-CAR T cells. Front Immunol 2021;12:691741.Ethics Approval This study was approved by St. Jude Children’s Research Hospital’s IACUC Committee, protocol number 3095.Abstract 279 Figure 1Derivation of spontaneously metastatic OS cell line on C57Bl/6 backbone. A) Derivation strategy. B) Bioluminescent images demonstrating 1332.m2R and 1332.m3C metastatic activity post primary tumor amputation. C) Quantification of pulmonary bioluminescence of 1332.m2R and 1332.m3C cell lines post primary tumor amputationAbstract 279 Figure 2CXCL16-mediated migration of mCXCR6.mB7-H3.CAR murine T cells. A) Whole tumor supernatant versus 2D cell line supernatant quantification of mCXCL16 by ELISA. B) Migration of mCXCR6.mB7-H3.CAR versus standard mB7-H3.CAR and NT cells in semihalo migration assay