A Cross-Reactive Small Protein Binding Domain Provides a Model to Study Off-Tumor CAR-T Cell Toxicity

Abstract

Tumor-targeted chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR-T cells) have demonstrated striking clinical success, but their use has been associated with a constellation of toxicities. A better understanding of the pathogenesis of these toxicities is required to improve the safety profile of CAR-T cells. Herein, we describe a xenograft model of off-tumor CAR-T cell-associated toxicity. Human CAR-T cells targeted against HER2 using a small-protein binding domain induced acute, dose-dependent toxicities in mice. The inclusion of a CD28 or 4-1BB co-stimulatory domain in the CAR was required to produce toxicity; however, co-stimulation through CD28 was most toxic on a per-cell basis. CAR-T cell activation in the lungs and heart was associated with a systemic cytokine storm. The severity of observed toxicities was dependent upon the peripheral blood mononuclear cell (PBMC) donor used as a T cell source and paralleled the CD4 + -to-CD8 + T cell ratio in the adoptive transfer product. CD4 + CAR-T cells were determined to be the primary contributors to CAR-T cell-associated toxicity. However, donor-specific differences persisted after infusion of a purified CD4 + CAR-T cell product, indicating a role for additional variables. This work highlights the contributions of CAR-T cell-intrinsic variables to the pathogenesis of off-tumor toxicity.

Expanded human NK cells armed with CAR uncouple potent anti-tumor activity from off-tumor toxicity against solid tumors

“…In vivo studies show that these HER2 CAR-T cells have the capacity to infiltrate into the lungs and heart, leading to cross-reactivity and activation causing lethal toxicities in tumor xenograft mice (Hammill et al, 2020). Since MHC class I molecules are constitutively expressed on normal cells while also often downregulated on tumors, our findings suggest that CAR-NK cells will overcome the current on-target, off-tumor toxicity concerns with CAR-T cells redirected against solid tumors without compromising anti-tumor function.…”

p16INK4a Regulates Cellular Senescence in PD-1-Expressing Human T Cells

“…We next used a clinically relevant approach to investigate cell fate in context of initial CD3/CD28 stimulation followed by serial antigenic encounters. We generated B-cell maturation antigen (BCMA)-specific CAR T cells ( 40 ) that were activated and expanded for three weeks with anti-CD3/CD28 beads and subsequently transduced with p16 INK4a - targeting or non-targeting shRNAs ( Figures 7E, F ). While exposure to repetitive stimulations with BCMA-expressing human KMS-11 multiple myeloma cells ( 64 ) led to the expression of inhibitory receptors such as PD-1 and KLRG1 ( Supplementary Figure 5 ), p16 INK4a knockdown increased the fraction of Ki67 + cells and restricted the development of SA-β-Gal-expressing CAR T cells ( Figures 7G, H ).…”

“…Second generation B-cell maturation antigen (BCMA)-specific CAR construct [on the CD28ζ-CAR backbone and expressing the NGFR marker as described in ( 39 , 40 )] is a kind gift from Jonathan Bramson (McMaster University, Hamilton, Canada). Lentiviral transduction of T cells was performed 24 hours after stimulation with CD3/CD28-coated beads.…”

ERBB2-CAR-Engineered Cytokine-Induced Killer Cells Exhibit Both CAR-Mediated and Innate Immunity Against High-Risk Rhabdomyosarcoma

“…However, the T cells among CIK cells are non-classical terminally differentiated T lymphocytes with an NK cell phenotype representing diverse T and NK cell receptor specificities, which are not comparable to young T cells (31,48). Furthermore, CIK cells include classical NK cells as well as CD4 + and CD8 + T lymphocytes with naïve T, effector memory, and central memory T phenotypes, and this may represent an advantage with respect to the unselected CAR-transduced T cell populations used so far, which contain significant amounts of CD4 + T cells that may contribute to the dramatic cytokine storm sometimes observed in vivo (49). In contrast to the high specificity and memory of CAR T cells, CIK cells are capable of eliminating tumor cells by recognizing pathogen patterns through a variety of receptors (DNAM-1, NKG2D, NKp30, and TCR/CD3) (24), suggesting that ERBB2-CAR CIK cells may provide NK cell-like activities, mainly NKG2D-mediated functions, and specific anti-ERBB2mediated cytotoxicity in combination, as indicated by NK and T-NK cells which were present at potential tumor sites in all ERBB2-CAR CIK cell-treated mice.…”