A multimodal assay identifies optimized binders for a bicistronic CAR against GPC2 OR CD276 to overcome heterogeneity in Neuroblastoma

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and remains deadly in patients with high-risk disease. Single chimeric antigen receptor T-cell therapies (CAR-T) for solid tumor have shown poor efficacy in clinical trials due, in part, to T cell exhaustion and uneven expression of target antigens in tumors. Here, we attempted to target Glypican-2(GPC2) and CD276(B7-H3), both highly but heterogeneously expressed on NB cell surface, to overcome these challenges in single CAR T- therapies. First, to identify optimal binders for CAR T- cell targeting each antigen, we made individual CAR constructs using multiple binders against these antigens and utilized a multimodal approach including simultaneous protein and transcriptome measurement at single cell level to characterize each individual CAR T- cell in a pooled strategy. Combined with cell expansion and cytotoxicity assays, we identified the most effective CAR construct for each target. Overall design: To identify individual CAR T- cells and parallel characterize their functions in the CAR T- competition assay, we designed a multimodal single cell profiling approach(surface protein staining (Biolegend TotalSeq-C lyophilized panel), mRNA and CAR T- binders) using 10x Genomics Chromium system. This assay simultaneously identify CARs by sequencing the binding domain of CARs and assess T cell functions using simultaneous 28 protein markers (plus 3 isotype control anitibodies) and transcriptome measurement at the single cell level (CITE-seq).