The adoptive transfer of T cells that are engineered to express chimeric antigen receptors (CARs) has shown remarkable success in treating B-cell malignancies but only limited efficacy against other cancer types, especially solid tumours. Compared with haematological diseases, solid tumours present a unique set of challenges, including a lack of robustly expressed, tumour exclusive antigen targets as well as highly immunosuppressive and metabolically challenging tumour microenvironments, that limit treatment safety and efficacy. Here, we review protein-and cell-engineering strategies that seek to overcome these obstacles and produce next-generation T cells with enhanced tumour-specificity and sustained effector function for the treatment of solid malignancies.T-cell targeting of other TAAs has similarly led to undesired but clinically tractable adverse events. Melanoma antigen recognized by T cells 1 (MART-1) and glycoprotein 100 (gp100) are TAAs expressed not only in melanomas but also in healthy melanocytes in the skin, eyes, and ears 12 .Patients with metastatic melanoma who received T cells engineered to express TCRs specific for MART-1 or gp100 experienced transient melanocyte toxicity, resulting in damages to skin, eyes, or ears that were treatable with steroid applications 12 . Notably, a subset of patients experienced melanocyte toxic effects without appreciable tumour clearance 12 , indicating that on-target, offtumour toxicities can happen even in the absence of robust anti-tumour response. Patients with metastatic colorectal cancer who received T cells expressing carcinoembryonic antigen (CEA)targeted TCRs experienced severe transient colitis due to CEA expression on healthy epithelial cells in the gastrointestinal tract, with limited anti-tumour responses 13 . Similarly, treatment with carboxy-anhydrase-IX (CAIX) CAR-T cells in patients with metastatic renal carcinoma resulted in dose-limiting toxicity to the liver and bile-duct epithelial cells despite being a first-generation CAR, which is expected to provide limited tumour-killing efficacy 14 . These cases highlight the delicate balance between eliciting potent anti-tumour activities with preventing severe off-target toxic effects.In certain clinical studies, unanticipated off-target toxic effects have resulted in life-threatening complications. Melanoma associated antigens (MAGE) are cancer-testes antigens (CTAs) that are absent from healthy adult tissue but overexpressed in a variety of cancers 15 . However, three out of nine patients treated with MAGE-A3-targeted TCR-T cells experienced severe neurotoxicity, resulting in two fatalities 15 . This was attributed to cross-reactivity of the MAGE-A3 TCR to unanticipated MAGE-A12 expression in the brain 15 . In a separate MAGE-A3 TCR study, two patients experienced lethal cardiac toxicity due to myocardial damage induced by TCR crossreactivity with the protein titin, which is found in myocardium 16,17 . It should be noted that the tested MAGE-A3 TCRs were avidity-and affinity-enhanced with the intenti...

Chimeric antigen receptors (CARs) are fusion proteins whose functional domains are often connected in a plug-and-play manner to generate multiple CAR variants. However, CARs with highly similar sequences can exhibit dramatic differences in function. Thus, approaches to rationally optimize CAR proteins are critical to the development of effective CAR T-cell therapies. Here, we report that as few as two amino-acid changes in non-signaling domains of a CAR were able to significantly enhance in vivo antitumor efficacy. We demonstrate juxtamembrane alanine insertion and single-chain variable fragment (scFv) sequence hybridization as two strategies that could be combined to maximize CAR functionality, and describe a CD20 CAR that outperformed the CD19 CAR in antitumor efficacy in preclinical in vitro and in vivo assays. Precise changes in the CAR sequence drove dramatically different transcriptomic profiles upon antigen stimulation, with the most efficacious CAR inducing an enrichment in highly functional memory T cells upon antigen stimulation. These findings underscore the importance of sequence-level optimization to CAR T-cell function, and the protein-engineering strategy described here may be applied to the development of additional CARs against diverse antigens.

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SUMMARYChimeric antigen receptors (CARs) are modular proteins capable of redirecting immune cells toward a wide variety of disease-associated antigens. Here, we explore the effects of CAR protein sequence and structure on CAR-T cell function. Based on the empirical observation that CD20 CARs with similar sequences exhibit divergent tonic-signaling and anti-tumor activities, we devised engineering strategies that aimed to improve CAR-T cell function by tuning the intensity of tonic signaling. We found that CARs designed to exhibit low but non-zero levels of tonic signaling show robust effector function upon antigen stimulation while avoiding premature functional exhaustion by CAR-T cells. Through alterations of the CAR’s ligand-binding domain and overall protein conformation, we generated CD20 CAR variants that outperform the CD19 CAR in mouse models of human lymphoma. We further demonstrate that rational modification of protein confirmation can be generalized to improve GD2 CAR-T cell efficacy against neuroblastoma. These findings point to tonic signaling and basal T-cell activation as informative parameters to guide the rational design of next-generation CARs for cancer therapy.

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