Chimeric antigen receptor generations. First generation CARs include a single-chain variable region from a monoclonal antibody paired with an intracellular signaling domain, the CD3 z chain from the CD3 TCR or FcR g . Second and third generation CARs include an additional one or two co-stimulating domains (e.g. CD28, 4-1BB, OX-40) that increase signal strength and persistence, with increased proliferation and cytokine production. TM: transmembrane domain; H: heavy chain; L: light chain. (A color version of this figure is available in the online journal.) 

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... safer alternative to other targets for adoptive TCR therapy, with a decreased risk of ‘‘on-target, off-tumor’’ effects. However, when TCR therapy against NY-ESO-1 was used in patients with synovial cell carcinoma and melanoma, the clinical results were mixed. One year after therapy, two of 11 patients with melanoma had complete tumor regression, and one patient with synovial cell carcinoma mounted a partial response. 44 Fortunately, there was no toxicity associated with the transferred cells. Because CTAs are highly specific to cancer cells, they were expected to be a safer target for TCR therapy. However, highly specific CTA expression on target cells only decreases the chances of an on-target, off-tumor event. If the affinity-enhanced TCR cross-reacts with a distinct, albeit similar, antigen expressed in normal tissue, then an ‘‘off-target, off-tumor event’’ is possible. Off-target, off- tumor effects are very concerning because they progress rapidly, with potentially lethal consequences, and are notoriously hard to predict. For instance, melanoma-asso- ciated antigen 3 (MAGE-A3) is a CTA that is specifically expressed in more than 30% of common epithelial malignancies, including melanoma, breast, lung, esophageal, and head and neck cancers. 45 In a TCR study targeting the MAGE-A3 antigen, two patients undergoing treatment experienced cardiogenic shock, resulting in death. Autopsy revealed severe myocardial damage and histo- pathological analysis revealed T-cell infiltration. Alarmingly, despite preclinical screening for cross- reactivity, MAGE-A3 was found to have a similar structure to a human cardiac protein, Titin, suggesting that the TCR-modified T-cell mistook a Titin-derived peptide on cardiomyocyte MHCs for MAGE-A3. In the follow-up, the TCR-modified T-cells reacted in vitro with beating human cardiomyocytes, thus confirming that Titin was the cross reactive protein. 46 In short, in the MAGE-A3 TCR trial, an off-target, off-tumor event led to the fatal destruction of cardiac tissue in the two patients. 47 Off-target, off-tumor toxicity is fast progressing and devastating. As evidenced by the MAGE-A3 trial, off- target, off-tumor effects are hard to completely rule out in preclinical studies due to variable protein sequence hom- ology between animal models and humans. As a result, it is far more difficult to identify potential off-target, off-tumor effects than it is to determine on-target off-tumor effects. This is as relevant to CAR applications as it was for TCR therapy for solid tumors. In order for CAR therapy targeting CTAs or other TAAs to succeed in the clinic, the risk of off-target off-tumor effects needs to be addressed using various safety strategies. For example, prior to a clinical trial, in-vitro studies could use human genome data to synthesize and test candidate cross reactive proteins for reactivity. Additionally, future CAR safety studies could potentially deploy high-throughput proteomics to screen for cross-reactivity with the selected tumor-specific antigens, to test off-target off-tumor anti- genic candidates, or even to select for the safest epitope. Another potentially advantageous safety strategy would be to include small molecule-inducible suicide switches in the modified T-cells. 48 Finally, perhaps the best conceptual approach in terms of therapeutic safety would be to use mRNA-modified T-cells in CAR applications. The CAR mRNA modifying the T-cell would be short-lived and would degrade a short time after infusion, allowing for enhanced dosage control and a safer therapy. As previously discussed, CARs consist of an antigen- derived binding motif, linked with a hinge to transmembrane and intracellular signaling domains, the latter derived from the z chain of CD3 or the FcR receptor g chain. In other words, CAR T-cells do not require MHC-mediated recogni- tion of the target antigen, allowing for a far greater range of potential cellular targets. Furthermore, modified CAR T- cells have increased power relative to TCR-based therapies, as they are immune to down-regulation of MHC expression in neoplastic cells. The correlate is that, by bypassing the MHC, CAR T-cells can only target cell surface markers. It is important to note that CAR affinity for the target epitope is a key determinant of CAR therapy efficacy. A murine antibody single chain variable fragment (Fv) is articulated to the transmembrane domain via a hinge region, which allows for greater flexibility of the antigen-receptor config- uration. Since CAR T-cells are not MHC-restricted, their interaction with antigen presenting cells (APCs) is limited, and they do not receive a co-stimulatory signal from an APC. Indeed, whereas first generation CAR design consists of the CAR fused to the intracellular portion of the TCR domain, second generation CAR design includes one co-stimulatory domain, e.g. CD28 or 4-1BB, and third generation CARs include two or more co-stimulatory signaling domains, e.g. CD28 and 4-1BB (Figure 3). The first successful CAR therapies were directed against hematologic cancers, notably B-cell malignancies. B-cell ablation is considered safe for patients in the context of the prompt recovery of other blood counts. For that reason, the CD19 marker, highly specific to B-cells including those transformed to lymphoma and leukemia, was chosen as target for CAR T-cell therapy. The first CAR targeting CD19 was dramatically effective in a patient with B-cell lymphoma, with eradication of B-lineage cells for a prolonged period of time, with concomitant marked lymphoma regression. 49 In order to further strengthen CAR T-cell activation, a co- stimulatory domain from CD137 (4-1BB) was added in a subsequent trial for advanced B-cell chronic lymphocytic leukemia (CLL), with two out of three patients undergoing complete remission. CAR-modified T-cell lines successfully expanded ex vivo more than 1000-fold, engrafted in the bone marrow and could establish CAR memory for at least six months. 50 The combination of a CAR with a co-stimulatory signal led to a robust response: each CAR-expressing T-cell was estimated to lyze at least 1000 CLL cells. 50 Consequently, tumor lysis syndrome was reported as common serious adverse event in both CLL and B-cell acute lymphoid leukemia (AML) trials. 51,52 The lesson was that, during CAR therapy, patients need to be closely monitored for signs of tumor lysis syndrome since CAR T-cells ablate large amounts of target cells at levels vastly superior to those of past immunotherapies. CAR T-cell therapy has succeeded where conventional therapies have failed, and CAR T-cell therapy has led to remission in patients with refractory disease. In a landmark clinical trial, autologous T-cells transduced with a CD19- directed CAR led to a 90% remission rate (27/30 cases) for patients with relapsed or refractory acute lymphoblastic leukemia (ALL), including 15 patients for whom stem cell transplantation had previously failed. 2 The six-month event-free survival rate was 67%; overall survival rate was 78%. All patients experienced cytokine-release syndrome, with severe cytokine-release syndrome observed in 27% of patients. Severe cases of cytokine-release syndrome were associated with a higher tumor burden and were treated effectively with tocilizumab, the anti-IL-6 receptor antibody. The results of this trial were without precedent and demonstrated that CAR T-cells can overcome mechanisms of B-cell neoplasm resistance in subsets of patients with abysmally low prognosis. Indeed, 90% remission was a quasi-reversal of the odds for these patients, and firmly made the case that CAR therapy, if done correctly, can completely upend longstanding cancer treatment paradigms, providing hope for millions of patients with dire prognosis. Although CAR T-cell therapy was first attempted against a solid tumor, CAR therapy for solid tumors has seen limited progress due to safety concerns. In a 2008 neuroblastoma trial, Epstein-Barr virus (EBV)-specific T-cells were engineered to also co-express a CAR targeted at diasialoganglio- side GD2, an antigen expressed by neuroblastoma cells. 53 Half the patients (4/8) had evidence of tumor regression. Intriguingly, the virus-specific CAR T-cells survived longer than T-cells with CAR alone. Additionally, on-target, off-tumor toxicity is an especially critical safety concern for CAR therapy, even more so than for TCR therapy because CAR T-cell destruction of cells is not MHC- mediated, leading to a stronger and faster response during activation. For instance, a CAR therapy derived from a mAb against carboxyanhydrase-IX (CAIX), a marker of clear cell renal cell carcinoma, also destroyed normal bile duct epithelial cells, which happened to also express CAIX. 53 It was a striking example of an on-target, off-tumor effect in a CAR therapy, and a potent reminder that future CAR strategies will need to further guarantee targeting specificity. 53 The power of CAR therapy resides in the ability to choose any surface marker as a target, and to hit that target with high efficacy. Nonetheless, not all patients respond completely to CAR therapy and the mechanisms of failure will need to be defined in non-responding patients. For instance, the chimeric murine mAb Fv presents the risk of immunogenicity, potentially resulting in CAR neutraliza- tion. Tumor antigen loss and the development of antigen variants could lead to tumor resistance to CAR therapy. Another challenge is the development of treatment-related toxicity, cytokine-release syndrome, which has been linked to massive macrophage and IL-6 activation. Although IL-6 blockade has been successful in managing the most severe manifestations of cytokine-release syndrome, regulating the level or dose of CAR expression would allow for greater control over toxicity as well. CAR T-cell therapy targeting CD19 risks selecting for neoplastic clones that express low or no CD19. 52 Beyond absence of CD19, dynamic or cyclical CD19 ...