Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells

Abstract

MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)–A*01–restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.

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“…Lentiviral vector containing high affinity MAGE-A3 a3a TCR 24 and corresponding packaging and envelope plasmids (VSVG, REV and gag/pol) was generously provided under MTA.…”

Recent advances in T‐cell immunotherapy for haematological malignancies

“…In vivo replication of the interaction between MAGE-A3 TRCR-transduced T-cells and cardiomyocytes replicated these findings. (Cameron et al 2013) This unexpected off-target toxicity highlights the importance of confirming the specificity of engineered TCRs to the best of our capabilities prior to clinical investigation.…”

The Flipside of the Power of Engineered T Cells: Observed and Potential Toxicities of Genetically Modified T Cells as Therapy

“…Further investigation revealed that the affinity enhancement of the TCR resulted in recognition of a similar peptide expressed in cardiac muscle, leading to acute cardiac rejection. 33 Following these unexpected deaths, an extensive investigation was undertaken, with initial experiments demonstrating no cross-reactivity with standard myocyte cell culture models. Only the use of highly purified human cardiomyocytes derived from induced pluripotent stem cells revealed the reactivity with the titin peptide expressed in beating cardiac myocytes.…”