CAR-T cell potency: from structural elements to vector backbone components

Abstract

Chimeric antigen receptor (CAR) T cell therapy, in which a patient’s own T lymphocytes are engineered to recognize and kill cancer cells, has achieved remarkable success in some hematological malignancies in preclinical and clinical trials, resulting in six FDA-approved CAR-T products currently available in the market. Once equipped with a CAR construct, T cells act as living drugs and recognize and eliminate the target tumor cells in an MHC-independent manner. In this review, we first described all structural modular of CAR in detail, focusing on more recent findings. We then pointed out behind-the-scene elements contributing to CAR expression and reviewed how CAR expression can be drastically affected by the elements embedded in the viral vector backbone.

New cell sources for CAR-based immunotherapy

“…All cells discussed in this review are predominantly used for generation CARs bearing scFv and not for VHH-based CARs, while CARs containing nanobodies have shown promising clinical outcomes; as evidence, the sixth CAR-T cell product (CARVYKTI, Janssen-Cilag International NV) recently approved by US FDA has utilized a VHH dimer as its antigen recognition domain [ 5 ]. The VHH domain, alternatively known as nanobody, is the smallest fragment with an antigen-binding capability similar to conventional antibodies in affinity and specificity [ 6 ]. Characteristics such as small size, high solubility and stability, low immunogenicity, high tissue penetration, and no need for additional folding and assembly steps or linker optimization due to the lack of variable light chain make nanobodies a promising alternative to scFvs in CARs.…”

“…Characteristics such as small size, high solubility and stability, low immunogenicity, high tissue penetration, and no need for additional folding and assembly steps or linker optimization due to the lack of variable light chain make nanobodies a promising alternative to scFvs in CARs. Furthermore, as VHHs can access epitopes that are hard or impossible to reach by scFvs, they are more favorable than scFv to be used as the antigen recognition domain of CARs, particularly for solid tumors [ 6 ]. The first report about the successful use of nanobodies in the CAR constructs emerged from our lab, where CAR-modified T cells used an anti-MUC1 VHH as the target-binding domain [ 255 ].…”

“…We have observed promising results using VHH-based CAR-T cells against solid tumor cell lines in our lab [ 256 , 257 ]. As VHHs own advantages over scFvs for solid tumors [ 6 ], transferring VHH-based CAR into cells other than T cells that can penetrate deep tumors may provide an ideal immunotherapy platform against solid tumors.…”

“…Chimeric antigen receptor (CAR) T cell therapy, in which a patient’s T lymphocytes are genetically modified to recognize and eradicate tumor cells independent of the major histocompatibility complex (MHC), has achieved remarkable success in some hematological malignancies, leading to the regulatory approval of six CAR-T products, including Tisagenlecleucel (Kymriah®), Axicabtagene ciloleucel (Yescarta®), Brexucabtagene autoleucel (Tecartus®), Lisocabtagene maraleucel (Breyanzi®), Idecabtagene vicleucel (ABECMA®), and Ciltacabtagene autoleucel (CARVYKTI®), that are currently available in the market [ 1 – 6 ]. CARs are synthetic immune receptors that benefit from both humoral and adaptive immunity through their unique structure at which an extracellular antigen recognition domain, typically a single-chain variable fragment (scFv) derived from a monoclonal antibody, is linked to an intracellular signaling domain of T cells.…”

Strategies to enhance CAR-T persistence

“…This structure has undergone constant remodeling, encompassing five generations of evolution and optimization since the origin of CAR-T cells [ 28 , 29 ]. These five generations have been comprehensively studied and applied and thus are not introduced in detail in this review [ 29 – 31 ]. The second-generation CAR is the most widely adopted structure, which includes one costimulatory domain within the intracellular domain that is most commonly derived from CD28 or 4-1BB and can enhance TCR signaling [ 3 , 32 – 36 ].…”