Abstract
BackgroundAdapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy. In the UniCAR system, the safety switch is controlled via a target module (TM) which is characterized by a small-size and short half-life. The rapid clearance of these TMs from the blood allows a quick steering and self-limiting safety switch of UniCAR T-cells by TM dosing. This is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. For long-term UniCAR therapy, the continuous infusion of TMs may not be an optimal setting for the patients. Thus, in later stages of treatment, single infusions of TMs with an increased half-life might play an important role in long-term surveillance and eradication of residual tumor cells. Given this, we aimed to develop and characterize a novel TM with extended half-life targeting the tumor-associated carbohydrate sialyl-Tn (STn).MethodsThe extended half-life TM is composed of the STn-specific single-chain variable fragment (scFv) and the UniCAR epitope, fused to the hinge region and Fc domain of a human immunoglobulin 4 (IgG4) antibody. Specific binding and functionality of the αSTn-IgG4 TM as well as pharmacokinetic features were assessed using in vitro and in vivo assays and compared to the already established small-sized αSTn TM.ResultsThe novel αSTn-IgG4 TM efficiently activates and redirects UniCAR T-cells to STn-expressing tumors in a target-specific and TM-dependent manner, thereby promoting the secretion of proinflammatory cytokines and tumor cell lysis in vitro and in experimental mice. Moreover, PET-imaging results demonstrate the specific enrichment of the αSTn-IgG4 TM at the tumor site, while presenting a prolonged serum half-life compared to the short-lived αSTn TM.ConclusionIn a clinical setting, the combination of TMs with different formats and pharmacokinetics may represent a promising strategy for retargeting of UniCAR T-cells in a flexible, individualized and safe manner at particular stages of therapy. Furthermore, as these molecules can be used for in vivo imaging, they pose as attractive candidates for theranostic approaches.
Highlights
Adapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy
A novel Immunoglobulin 4 (IgG4)-based target module (TM) format targeting STn was constructed (Fig. 1). The structure of this construct is similar to the single-chain variable fragment (scFv)-based TM with the additional insertion of the hinge and Fc (CH2-CH3) regions derived from human IgG4 antibodies
These regions were introduced between the binding domain derived from the αSTn monoclonal antibody (mAb) L2A5 [28] at the Nterminus, and the E5B9 epitope used for Universal CAR (UniCAR) Tcell recognition (Fig. 1b)
Summary
Adapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy. The rapid clearance of these TMs from the blood allows a quick steering and self-limiting safety switch of UniCAR T-cells by TM dosing This is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. The cross-linkage between UniCAR effector cells and target cells is mediated via a target module (TM) composed of a binding moiety against a specific tumor antigen fused to the E5B9 epitope (Fig. 1a) [7, 8] This in turn leads to the specific activation of the otherwise inert UniCAR T-cells and allows a controlled on/off switch of the UniCAR T-cells in case severe side effects are observed. Multiple targeting strategies can be considered given that TMs can be constructed and interchanged using different formats and specificities [8,9,10,11,12]
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