Engineering Next-Generation CAR-T Cells for Better Toxicity Management

Abstract

Immunoadoptive therapy with genetically modified T lymphocytes expressing chimeric antigen receptors (CARs) has revolutionized the treatment of patients with hematologic cancers. Although clinical outcomes in B-cell malignancies are impressive, researchers are seeking to enhance the activity, persistence, and also safety of CAR-T cell therapy-notably with a view to mitigating potentially serious or even life-threatening adverse events like on-target/off-tumor toxicity and (in particular) cytokine release syndrome. A variety of safety strategies have been developed by replacing or adding various components (such as OFF- and ON-switch CARs) or by combining multi-antigen-targeting OR-, AND- and NOT-gate CAR-T cells. This research has laid the foundations for a whole new generation of therapeutic CAR-T cells. Here, we review the most promising CAR-T cell safety strategies and the corresponding preclinical and clinical studies.

Keywords: CAR-T cell; cancer; chimeric antigen receptor; cytokine release syndrome; engineering; immunotherapy; toxicity.

Figure 1

Structure of a chimeric antigen receptor (CAR)—a cell-surface receptor comprising an extracellular ligand binding domain (which recognizes a specific tumor-associated antigen (TAA)), a transmembrane (TM) domain, and an intracellular signaling domain. The latter consists of a T cell activation domain and one or more co-stimulatory domains (CD27, CD28, ICOS, 4-1BB, and/or OX40) (Adapted from Lee and Kim, 2019).

Figure 2

General coding sequence for a CAR.

Figure 3

The four generations of CAR-T cells. First-generation CARs were composed of antibody single-chain variable fragments (scFvs) fused through a TM domain to the cytoplasmic tail of the TCR signaling component CD3ζ. Second-generation CARs had improved CAR-T cell proliferation and cytotoxicity, thanks to the addition of co-stimulatory signaling domains (such as CD27, CD28, CD134, and CD137). Third-generation CAR-T cells were generated by adding a second co-stimulatory signaling domain (such as OX-40, also known as CD134, or 4-1BB, also known as CD137) to second-generation constructs. Fourth-generation CAR-T cells, also known as “T cells redirected for universal cytokine-mediated killing” (TRUCKs) were then engineered to secrete transgenic cytokines (like interleukin-12) within the targeted cancer and thus attract more immune cells (such as natural killer (NK) cells and macrophages). (Adapted from Smith et al., 2016).

Figure 4

Activation of iCasp9 induces the death of transduced cells. iCasp9 is synthetized in the transduced CAR-T cell as a homodimer. Administration of the chemical inducer of dimerization (CID) AP1903 causes iCasp9 dimerization and thus activation, which in turn triggers a signaling cascade leading to the apoptosis of the CAR-T cells (Adapted from Li and Zhao, 2017).

Figure 5

CAR T cells with an elimination marker. huEGFRt and CD20 have been tested as elimination marker systems co-delivered with CAR-T cells. The huEGFRt suicide molecule can be activated with an anti-EGFR cetuximab. In contrast, RQR8 (a 136-amino-acid marker with epitopes from CD34 and CD20) has a dual role, by acting simultaneously as a selection marker when CD34 is targeted with an anti-CD34 MAb (QBEnd/10) and as a suicide molecule when CD20 is targeted with rituximab (Adapted from Li and Zhao, 2017).

Figure 6

Schematic representation of the SWIFF-CAR principle (Adapted from Juillerat et al., 2019).

Figure 7

Schematic representation of the tyrosine kinase inhibitor (TKI)-based OFF-switch. The TKI dasatinib inhibits the phosphorylation of CD3ζ and ZAP70, which have a key role in the T-cell receptor (TCR) signaling pathway (Adapted from Wu et al., 2019).

Figure 8

Schematic representation of switchable adaptors. (1) Tag-specific adaptors CARs: (a) biotin-binding immune receptors (BBIRs), (b) split, universal, and programmable (SUPRA) CARs, (c) α-FITC CARs, (d) UniCARs, (e) α-peptide-neoepitope (α-PNE) CARs, (f) SpyCatcher, (g) fusion protein (FP) CARs, (2) bispecific antibody-binding (BsAb) adaptor CARs: (h) BsAb immune receptors (BsAb-IRs), (i) synthetic agonistic receptors (SARs), (3) Fc-binding CARs: (j) ADCC-mediating immune receptor (Adapted from Arndt et al., 2020).

Figure 9

Combinatorial antigen targeting for solid tumors. (a) AND-gate CAR SynNotch (b) AND-gate/CAR Split, (c) NOT-gate CAR, and (d) OR-gate/tandem CAR (Adapted from Schmidts et al., 2018).