Conventional CARs versus modular CARs

Abstract

The clinical application of immune effector cells genetically modified to express chimeric antigen receptors (CARs) has shown impressive results including complete remissions of certain malignant hematological diseases. However, their application can also cause severe side effects such as cytokine release syndrome (CRS) or tumor lysis syndrome (TLS). One limitation of currently applied CAR T cells is their lack of regulation. Especially, an emergency shutdown of CAR T cells in case of life-threatening side effects is missing. Moreover, targeting of tumor-associated antigens (TAAs) that are not only expressed on tumor cells but also on vital tissues requires the possibility of a switch allowing to repeatedly turn the activity of CAR T cells on and off. Here we summarize the development of a modular CAR variant termed universal CAR (UniCAR) system that promises to overcome these limitations of conventional CARs.

Keywords: BiTE; Chimeric antigen receptor; Immunotherapy; T cells; TIMO XIV; UniCAR.

Fig. 1

Construction of conventional CARs. A conventional CAR consists of an Ab-based extracellular domain, a transmembrane domain and intracellular signaling domains. The extracellular domain is directed against the tumor-associated antigen (TAA) on the surface of the target cell. It can be constructed from the variable domains of the heavy and light chains of a mAb. After transduction of the T cell with the CAR gene, the resulting CAR T cell can recognize tumor cells via its extracellular Ab domain. Cross-linkage of the tumor cell with a CAR T cell leads to the formation of a synapse-like structure by clustering of the CAR receptors and thereby to an activation of the CAR T cell via its signaling domain(s) and finally to the destruction of the target cell. Both CD4- and CD8-positive T cells work equally well as killer cells though CD4-positive T cells need longer time for cytotoxic activity [29]

Fig. 2

Different CAR concepts. The activation domain in first-generation CARs is taken from the zeta chain of the CD3 complex. To improve survival and reactivity of CAR T cells, second- and third-generation CARs contain, in addition to the activation motif, one or two costimulatory motifs. CM costimulatory motif, TAA tumor-associated antigen, TMD transmembrane domain

Fig. 3

The UniCAR system, a modular switchable CAR platform. a Based on anti-La mAbs (either 5B9 or 7B6 directed towards the amino acid (aa) sequence (S)KPLPEVTDEY (UniCAR epitope, note the N-terminal serine (S) is not necessarily required for immune reactivity of the 5B9 mAb but is commonly present in E5B9-containing UniCAR TMs due to G₄S linkers between the scFv and the UniCAR epitope) or the aa EKEALKKIIEDQQESLNK we constructed the respective UniCAR domain. After adoptive transfer, UniCAR T cells remain in an “Off” mode in the absence of a TM until a TM is infused, which can form a cross-linkage between a UniCAR T cell and the target cell. Consequently, UniCAR T cells can be switched “On” in the presence of a TM and will automatically shut “Off” after elimination of the TM. Prerequisite for a rapid shut down is a short half-life of the TM. An example of selected experimental data for an anti-E5B9 UniCAR system is shown in b–d. b T cells transduced with a non-functional UniCAR (a UniCAR lacking intracellular signaling domains) do not kill target cells neither in the absence (gray bar) nor presence of a TM (black bar). c T cells transduced with a signaling UniCAR also do not attack target cells in the absence of a TM (gray bar). However, lysis occurs in the presence of a TM (black bar). b, c Untransduced T cells (white bars) also fail to destroy target cells. d Tumor cells transduced with luciferase can be detected in untreated and treated mice at day 0 by optical imaging. In the absence of a TM (untreated mice), the tumor cells can still be detected 4 days after co-injection of signaling UniCAR T cells. However, no more tumor cells can be detected in treated mice (tumor cells were co-injected with UniCAR T cells in the presence of a TM). Lysis assays and optical imaging were performed as described previously [44]. TM target molecule, UniCAR universal chimeric antigen receptor