Chimeric Antigen Receptor-modified T Cells Against Ewing’s Sarcoma Related Nervous System Tumors

Ewing’s sarcoma/peripheral primitive neuroectodermal tumor(ES/PNET),member of the Ewing’s sarcoma family of tumors(ESFTs),is a malignant soft tissue tumor with characteristics of small undifferentiated neuroectodermal cells.Genetically,ES is most commonly characterized by a reciprocal chromosomal translocation between chromosomes 11 and 22,t(11;22)(q24;q12)as present in about 85% of these tumors.ES is the second most common sarcomas in the pediatric-young adult age range following osteosarcoma,and accounts for approximately 3% of all malignancies in pediatric patients,with a male predominance of 3:2 and a median age of 15 years.All bones can be affected.With the application of modern multimodal therapeutic regimens including induction chemotherapy,surgery and radiotherapy,or a combination of the above,approximately70% cure rate can be achieved in ES patients with localized disease.However,patients with aggressive neoplasms have a low survival rate despite these combination treatments.Targeted therapeutics of ES has potentials to improve treatment outcomes.Chimeric antigen receptor-modified T cell(CAR-T)therapy holds a great promise as a novel cancer therapy approach.CAR-T cells meet this need by utilizing the immune system’s surveillance capacity and potent cytotoxic mechanisms against tumor cells with exquisite specificity.In this study,we developed advanced lentiviral vectors(LVs)carrying a universal promoter(EF1α)driving the expression of green fluorescent protein(GFP)or luciferase.We chose the SK-N-MC cell line as a representative cell line for all subsequent experiments which were analyzed for surface expression of CD276,Lewis Y,Her2,GD2 and PD-L1 by flow cytometry;the expression of the above antigens was detected at 78%,100%,97%,25% and 0,respectively.ES patients who have primary or local recurrence or distant metastasis were enrolled in the study.ES patients’ tumor sections were immunohistochemically stained with different antibodies including CD276,Lewis Y,Her2,GD2,PSMA,PD-L1 and CD70.An advanced CAR-T therapy regimen,CAR2.0,has been developed,which involves a primary and a booster CAR-T cell infusions targeting multiple antigens identified on each patient’s tumor.T cells were collected from healthy donor and transduced with an apoptosis-inducible,safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/4-1-BB or CD27/CD3ζ-i Casp9(4SCAR).SKNMC-GFP was established and co-cultured with CAR-Ts to investigate the efficacy of CAR-T killing in vitro.The co-culture experiments revealed the effective killing of SKNMC-GFP cells by CD276/Lewis Y-CAR T cells,but not by control CD19-CAR T cells.To potentiate CAR T cell killing activity against ES,we investigated chemotherapy drugs and histone deacetylase(HDAC)/proteasome inhibitor based on MTT assay.To evaluate the efficacy of CAR-T against ES in vivo,SK-N-MC cells transduced with lentivirus vectors expressing luciferase(LV-EF1α-luciferase)were injected subcutaneously into the right back of male NOD-SCID mice(ES-CDX mouse model)to enable longitudinal monitoring of tumor burden via caliper measurement or in vivo imaging system(IVIS).The ES-CDX mouse model has been established while CAR-T killing assay in vivo and the expanded study is ongoing.