Generation of Hinge-Modified Human CD19 Chimeric Antigen Receptor (CAR) T Cells for the Treatment of CD19-Low Leukemia

CAR T cell therapy targeting CD19 has emerged to show spectacular success in the treatment of B leukemias and lymphomas. This strategy is mainly achieved by potentiating T-cells to recognize and target tumor cells. However, CD19 CAR T cell therapy can result in neurotoxicity ¹ and cytokine-associated toxicity as well as relapse with antigen dim or negative disease ²; therefore, strategies to mitigate these side effects are needed.

Recent trials in adults with lymphoma using fully human CAR (Hu19-28) T cells resulted in the secretion of lower levels of cytokines and conferred lower neurologic toxicity. ³ To assess the potential responsiveness of this Hu19-28 CAR T cells against B-acute lymphocytic leukemia (B-ALL), T cells engineered with the Hu19-28 CAR were compared with murine FMC63-based CARs. Hu19-28 and FMC63-based CAR T cells exhibited a rapid increase in IFNg secretion in response to the CD19 ⁺ NALM6 ALL cell line (CD19 ^High). Interestingly, and consistent with the lower toxicity of Hu19-28 CAR T cells in patients with lymphoma, IFNg secretion by Hu19-28 CAR T cells in response to a CD19 ^Low NALM6 line was significantly reduced as compared to FMC63-28 CAR T cells. Hu19-28 CAR T cells also exhibited high in vitro cytotoxicity against CD19 ^High NALM6. Notably though, Hu19-28 CAR T cells demonstrated lower in vitro cytotoxicity against CD19 ^Low NALM6 as compared to FMC63-28 CAR T cells. These important differences were also detected in vivo; both Hu19-28 and FMC63-28 CAR T cells eradicated CD19 ^High leukemia in an NSG mouse model but only the latter efficiently controlled the in vivo growth of CD19 ^Med leukemia.

As we and others have shown that the hinge domain of the CAR plays a critical role in its function, especially against low antigen density tumors ^4,5,6, we evaluated the impact of the hinge on the function of the Hu19-28 CAR T cells. Compared with the initial construct with a CD8a hinge length of 54 amino acids (Hu19-8H ⁵⁴-28), Hu19 constructs with 45 (Hu19-H ⁴⁵-28), 25 (Hu19-H ²⁵-28), and 13 (Hu19-8H ¹³-28) amino acids were generated. All CAR constructs were highly expressed at the surface of transduced T lymphocytes and were efficient in the killing of CD19 ^High leukemia. However, only the Hu19-H ²⁵-28 and Hu19-8H ¹³-28 CAR constructs exhibited high cytotoxicity against CD19 ^Low leukemia ( Figure 1). Together these data support the preclinical evaluation of Hu19-28 CAR constructs with shorter CD8a hinge lengths for the treatment of pediatric patients with relapsed/refractory ALL.

References

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Chen X/ Mirazee JM et al (2022) Journal of Magnetic Resonance 340, 107234. https://doi.org/10.1016/j.jmr.2022.107234

Mirazee et al., in preparation