(P 241) MACROPHAGE INHIBITION ENHANCES PERIPHERAL CAR T CELL EXPANSION AND TUMOR CONTROL IN A PATIENT-DERIVED HUMANIZED MODEL OF AUTOLOGOUS CAR T CELL EFFICACY IN PEDIATRIC OSTEOSARCOMA (OS)

Objective: CAR T cell therapy holds promise for treatment of refractory pediatric solid tumors, but early phase clinical trials have not realized preclinical promise, in part due to the lack of preclinical models that recapitulate T cell – immune interactions impacting CAR T cell efficacy. We report the first humanized mouse model derived from OS patient hematopoietic CD34+ stem cells (Hu-pHSC) and compare performance of autologous CAR T cells in Hu-pHSC versus nonhumanized immunodeficient mice.

Methods: CD34+ HSCs and autologous CD3+ T cells were obtained from patient bone marrow historically discarded during primary tumor resection. CD34+ cells were assessed for colony formation and in vivo engraftment. B7-H3 CAR T cells were generated by lentiviral transduction and assessed for phenotype and in vitro function. NSG-SGM3 mice were humanized by injection of patient HSCs after busulfan ablation and autologous CAR T cell efficacy was assessed against orthotopic LM7 OS tumors. Macrophage inhibition with the small molecule BLZ945 was assessed in combination with B7-H3 CAR T cells in Hu-pHSC and nonhumanized mice.

Results: CD34+ HSCs engrafted into NSG-SGM3 mice with mean peripheral human engraftment of 10% after 8 weeks. Autologous B7-H3 CAR T cells were equally potent in in vitro cytokine production and killing assays as standard healthy peripheral donor-derived pBMCs. In an orthotopic OS model, B7-H3 CAR T cells had minimal antitumor activity in Hu-pHSC compared to nonhumanized mice. CAR T cell performance was rescued in Hu-pHSC mice by combination with the macrophage inhibitor BLZ945, which also enhanced peripheral CAR T cell expansion and cytokine expression including IFN-γ, IL4, IL6, and IL10. On histology, treated tumors in Hu-pHSC mice contained fibrosis, activated histiocytes, and immune infiltrates consistent with human patient treatment effects, while treated tumors in nonhumanized mice showed only bony remodeling.

Conclusion: Hu-pHSC mice developed from OS patient bone marrow products allow investigation of the effect of the patient’s inhibitory immune environment on CAR T cell efficacy. In this model, CAR T cells had limited antitumor activity, as in early phase clinical trials, unless macrophages were depleted. This model improves on standard xenograft models and has the potential to serve as a reliable avatar for modeling immunotherapeutics for OS.