T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice

Abstract

Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution.

NK cells and cancer: you can teach innate cells new tricks

“…However, a very serious concern with enhanced NKG2D CAR T cells is that NKG2DLs can be expressed on non-tumour tissues, which might cause autoimmune reactions. Indeed, a recent study reported that NKG2D CARs transduced into mouse T cells induced rapid lethal toxicity when adoptively transferred into an autologous mouse model 171 . As yet, there is limited experience in vivo with CAR-engineered NK cells to know the efficacy versus toxicity outcomes of this strategy.…”

Tumor Regression and Delayed Onset Toxicity Following B7-H4 CAR T Cell Therapy

“…However, this does not rule out initiation of lesions at earlier time points. Previous studies that observed toxicity in murine models of CAR therapy targeting fibroblast activating protein 49 and NKG2D ligands 50 report treatment-mediated pathology within days post T-cell transfer. The differences in kinetics seen in our study may have occurred for several reasons including differences in CAR T-cell dose, preconditioning regimes, distribution of target antigen in normal tissue, and murine strain variability.…”

“…Other models of CAR-mediated toxicity in mice tend to cause acute toxicity. 49,50 Our B7-H4 CAR model of toxicity could provide a unique preclinical model to evaluate safety approaches designed to limit long-term CAR-mediated toxicity following antitumor activity in vivo. Additionally, these studies reveal the endogenous expression pattern of B7-H4, advancing our understanding of the molecule and fostering intelligent design of novel B7-H4-specific therapeutics for the clinic.…”

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

“…With the development of the "humanized mouse" model 18,19 , the TCR retrogenic could further be expanded utilizing cloned human TCRs. Additionally, the 2A linked constructs can also be used to study other multi-proteins including the CD3 chains, interleukins and chimeric antigen receptors [20][21][22][23][24] .…”