A novel CAR T cell blend targeting PDPN and GD2 to overcome glioblastoma heterogeneity

Abstract

BackgroundWhile chimeric antigen receptor (CAR) T cells have achieved encouraging remission rates in hematological malignancies, they demonstrated limited success in treating Glioblastoma (GBM), particularly due to high intra- and intertumoral heterogeneity. In this study, we identified a relevant and preserved target antigen, Podoplanin (PDPN), and evaluated the potential of a PDPN- and GD2-CAR T cell blend to overcome GBM heterogeneity. MethodsTarget antigen screening included clinical samples, healthy tissues and cell lines, as well as publicly available RNA sequencing datasets. The anti-tumor function of CAR T cells were examined in co-culture experiments with GBM cell lines and patient-derived organoids (PDOs), and in vivo after locoregional delivery in orthotopic xenograft models. ResultsThe generated CAR T cells demonstrated strong anti-tumor activity against several cell lines and PDOs from multiple patients. PDPN and GD2 expression was detectable in all PDOs at varying densities and regardless of the antigenic profile, the CAR T cell blend induced significantly higher levels of apoptosis in organoids than single antigen targeting counterparts. In vivo, we observed efficient tumor regression after locoregional administration of monospecific CAR T cells. While heterogeneous orthotopic tumors eventually relapsed in these groups, blended therapy resulted in a significantly increased overall survival and even achieved cure in the majority of mice. ConclusionThis novel PDPN-/GD2-CAR T cell blend demonstrated strong efficacy in advanced preclinical models of glioblastoma. The results suggest that this approach can overcome GBM heterogeneity in clinical application and address previous limitations of single antigen CAR T cell therapies. KEYPOINTSO_LIPDPN is a relevant and consistent CAR T cell target antigen in primary and recurrent GBM C_LIO_LIPDPN- and GD2-CAR T cells display synergistic anti-tumor activity in patient-derived organoids C_LIO_LILocal delivery of our CAR T cell blend confers long-term survival and cure in GBM-bearing mice C_LI IMPORTANCE OF THE STUDYThe highly variable landscape of tumor antigens in GBM represents a serious obstacle to single antigen CAR T cell therapies. In this study, we identified Podoplanin (PDPN) and GD2 as the most preserved target antigens in a screening campaign, with an even increased density in recurrent GBM compared to primary GBM. We combined PDPN- and GD2-CAR T cells in a blended treatment approach to counter antigen heterogeneity, and achieved a substantial gain in efficacy against patient-derived organoids (PDOs). In an orthotopic xenograft model, locoregional administration of the PDPN- and GD2-CAR T cell blend conferred long-term complete remission and increased survival compared to single antigen targeting. This study provides a hierarchy of CAR target antigens for treating GBM and illustrates the therapeutic potential of combinatorial antigen targeting using a PDPN/GD2-CAR T cell blend. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/636223v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@109a9aaorg.highwire.dtl.DTLVardef@16215faorg.highwire.dtl.DTLVardef@910623org.highwire.dtl.DTLVardef@129f79c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACT:C_FLOATNO C_FIG