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 receptor (CAR) T cells use re-engineered cell surface receptors to specifically bind to and lyse oncogenic cells. Two clinically approved CAR-T–cell therapies have significant clinical efficacy in treating CD19-positive B cell cancers. With widespread interest to deploy this immunotherapy to other cancers, there has been great research activity to design new CAR structures to increase the range of targeted cancers and anti-tumor efficacy. However, several obstacles must be addressed before CAR-T–cell therapies can be more widely deployed. These include limiting the frequency of lethal cytokine storms, enhancing T-cell persistence and signaling, and improving target antigen specificity. We provide a comprehensive review of recent research on CAR design and systematically evaluate design aspects of the four major modules of CAR structure: the ligand-binding, spacer, transmembrane, and cytoplasmic domains, elucidating design strategies and principles to guide future immunotherapeutic discovery.

A chimeric antigen receptor (CAR) that responds to transforming growth factor beta (TGF‐β) enables the engineering of T cells that convert this immunosuppressive cytokine into a potent T‐cell stimulant. However, clinical translation of TGF‐β CAR‐T cells for cancer therapy requires the ability to productively combine TGF‐β responsiveness with tumor‐targeting specificity. Furthermore, the potential concern that contaminating, TGF‐β?producing regulatory T (Treg) cells may preferentially expand during TGF‐β CAR‐T cell manufacturing and suppress effector T (Teff) cells demands careful evaluation. Here, we demonstrate that TGF‐β CAR‐T cells significantly improve the anti‐tumor efficacy of neighboring cytotoxic T cells. Furthermore, the introduction of TGF‐β CARs into mixed T‐cell populations does not result in the preferential expansion of Treg cells, nor do TGF‐β CAR‐Treg cells cause CAR‐mediated suppression of Teff cells. These results support the utility of incorporating TGF‐β CARs in the development of adoptive T‐cell therapy for cancer.

Objective. Tumor necrosis factor ␣ (TNF␣) blockade provides substantive reduction of the symptoms of rheumatoid arthritis (RA). While the biologic actions of TNF␣ have been well characterized in immune and synovial cells, which are known to be major contributors to the progression of cartilage destruction in RA, the current studies were designed to assess the direct effects of TNF␣ on chondrocytes.Methods. We examined the expression of several groupings of messenger RNA (mRNA) that define key biologic pathways that have previously been associated with either the general actions of TNF␣ or cartilage destruction, in murine articular chondrocytes isolated from wild-type mice and TNF␣ receptor-null (p55/ p75 ؊/؊ ) mice. Results. TNF␣ induced the expression of multiple mRNA that facilitate apoptosis and lead to apoptosisinduced cell death. The induction of apoptosis was accompanied by the increased expression of several factors involved in the regulation of skeletal tissue proteolysis and resorption. Quantitative increases from 2-fold to >10-fold were seen for inducible nitric oxide synthase, matrix metalloproteinase 3, macrophage colony-stimulating factor, and osteoprotegerin mRNA expression. The dependence of the induction of these mRNA on TNF␣ was confirmed by comparison with the effects of TNF␣ on chondrocytes isolated from receptornull mice.Conclusion. These findings demonstrate that TNF␣ alters the expression of a complex array of genes within murine chondrocytes that contribute to the destruction of joint surfaces, independent of its actions on synovial and immune cells. Further studies are needed to clarify the biologic actions of TNF␣ in human cartilage cells.

The expression of synthetic receptors in primary T cells enables the programming of user-defined responses when designing T-cell therapies. Chimeric antigen receptors (CARs) are synthetic receptors that have demonstrated efficacy in cancer therapy by targeting immobilized antigens on the surface of malignant cells. We have recently shown they can also rewire T-cell responses to soluble ligands. In contrast to other synthetic receptors, CARs are not only readily engineered by rational design, but also clinically translatable with robust function in primary human T cells. This protocol discusses design principles for CARs responsive to soluble ligands and delineates steps for producing T cells expressing synthetic receptors. Functional assays for quantifying the ability of CART cells to sense and respond to soluble ligands are also presented. This protocol provides a framework for proficient immune-cell researchers to test novel T-cell therapies targeting soluble ligands in under two weeks. KEYWORDS Chimeric antigen receptor, primary human T cells, soluble factors Related manuscripts: 1. Chang, Z. L. et al. Rewiring T-cell responses to soluble factors with chimeric antigen receptors.