512 the cytokine release syndrome crucially contributes to the anti leukemic effects of cd44v6 car t cells

Background: Despite the remarkable clinical results of CD19 CAR-T cells in B-cell leukemias, their long-term efficacy is limited by the emergence of CD19-loss escape variants. Moreover, whether the cytokine release syndrome (CRS) is necessary for durable remissions is a matter of debate. Currently available xenograft models in NSG mice are not suited for studying the antitumor effects of CAR-T cells beyond 3-4 weeks, because of xenograft-versus-host disease (X-GVHD). Moreover, since NSG mice lack functional myeloid cells, the CRS does not develop. Aim: To verify whether the CRS contributes to the antileukemic effects of CAR in an innovative xenotolerant mouse model.Results: NSG mice triple transgenic for human IL-3, GM-CSF and SCF (NSG-3GS) were sub-lethally irradiated and injected intra-liver with human HSCs soon after birth, enabling an accelerated and better balanced lympho-hematopoietic reconstitution compared with NSG mice. Reconstituting human T cells were single CD4+/CD8+ T cells, representing all memory sub-populations. After ex vivo isolation and activation with CD3/CD28-beads and IL-7/IL-15, NSG-3GS T cells were transduced with a CD44v6 CAR, retaining an early-differentiated (stem-cell/central-memory) phenotype and full antitumor functionality against acute myeloid leukemia (AML). NSG-3GS-derived CD44v6 CAR T cells were subsequently infused in tumor-bearing secondary recipients previously humanized with autologous HSCs. CAR-T cells persisted in vivo for at least 6 months and mediated durable leukemia remissions (P<0.001 vs controls) in the absence of X-GVHD. Tumor clearance associated with an acute malaise syndrome, characterized by high fevers and a surge in human IL-6 levels, which was lethal in 30% of the mice. Differently from CD19 CAR-T cells, the CRS by CD44v6 CAR-T cells was significantly anticipated (3 vs 8 days), coinciding with human CD44v6+ monocyte depletion. In humanized mice, previous myeloid-cell depletion by clodronate administration completely prevented this syndrome, but associated with late leukemia relapses. Conversely, mice developing the CRS entered a state of durable and profound remission, as demonstrated by prolonged observation times and secondary transplantation. Conclusions: By using an innovative xenotolerant mouse model, we have demonstrated that the CRS is needed for sustained antileukemic effects by CD44v6 CAR-T cells

Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting