Strategies to Address Chimeric Antigen Receptor Tonic Signaling Adam Ajina1,2 and John Maher1,2,3,4

Review Molecular Cancer Therapeutics Strategies to Address Chimeric Antigen Receptor Tonic Signaling Adam Ajina1,2 and John Maher1,2,3,4 Abstract Adoptive cell transfer using chimeric antigen receptors Here, we review the mechanisms underpinning CAR tonic (CAR) has emerged as one of the most promising new ther- signaling and highlight the wide variety of effects that can apeutic modalities for patients with relapsed or refractory B- emerge after making subtle structural changes or altering the cell malignancies. Thus far, results in patients with advanced methodology of CAR transduction. We highlight strategies to solid tumors have proven disappointing. Constitutive tonic prevent unconstrained tonic signaling and address its delete- signaling in the absence of ligand is an increasingly recognized rious consequences. We also frame this phenomenon in the complication when deploying these synthetic fusion receptors context of endogenous TCR tonic signaling, which has been and can be a cause of poor antitumor efficacy, impaired shown to regulate peripheral tolerance, facilitate the targeting survival, and reduced persistence in vivo. In parallel, ligand- of foreign antigens, and suggest opportunities to coopt ligand- dependent tonic signaling can mediate toxicity and promote T- dependent CAR tonic signaling to facilitate in vivo persistence cell anergy, exhaustion, and activation-induced cell death. and efficacy. Mol Cancer Ther; 17(9); 1795–815. Ó2018 AACR. Background subtle differences in CAR design can have amplified effects both in vitro and particularly in vivo and that the optimal selection of the Adoptive cell transfer (ACT), utilizing autologous T cells engi- CAR's extracellular targeting moiety, hinge, spacer, transmem- neered to express chimeric antigen receptors (CAR), has proven to brane domain (TMD), and intracellular costimulatory domain(s) be a highly efficacious strategy for the management of patients (ICD) is crucial. with relapsed or refractory B-cell malignancies (1–3). Indeed, It has become evident since the 1990s that nonactivated following the recent FDA approvals of the second-generation basal state T cells (and indeed B cells) exhibit low-level con- CD19-directed autologous CAR T-cell products: tisagenlecleucel stitutivetonicsignalingthatisabletoregulatetheirfunction (tradename KYMRIAH) for the management of pediatric and and survival in a homeostatic manner (12–14). More specifi- young adult patients with B-cell acute lymphoblastic leukemia cally, it is now understood that T-cell receptor (TCR)–mediated (ALL; refs. 4, 5) and axicabtagene ciloleucel (tradename YES- tonic signaling in nonengineered na€ve endogenous T cells, CARTA) for adult patients with relapsed or refractory large B-cell mediated by routine nonantigen-specific interactions with lymphoma following two or more lines of systemic therapy (6), mature antigen-presenting dendritic cells (DC), is able to CAR T-cell therapy is now a standard of care and can no longer be enhance their subsequent ability to react to foreign peptides regarded as a purely experimental therapeutic modality. However, (such as tumor neoantigens; refs. 12, 13). This is controlled, at the field remains in its infancy and these great strides are yet to be least in part, by interactions between the TCRs of na€ve T cells replicated in patients with advanced solid tumors (7–9). Much and self-peptide presented on MHC molecules expressed on the work remains to be undertaken to more fully appreciate how CAR surface of DCs and appears to be an important physiologic structure determines function and delineate the complexity of mechanism to ensure the homeostatic control of T-cell toler- CAR intracellular signaling as well the web of interactions ance in the periphery (15, 16). Despite considerable progress in between CAR T cells and other protagonist cells within the tumor understanding the molecular events involved in B-cell receptor microenvironment (TME) in vivo. Considerable effort continues (BCR)-mediated tonic signaling, which is a regulator of B-cell to be applied to the optimization of the CAR construct itself to maturation and survival (14, 17), our understanding of TCR- enhance antitumor potency, metabolism, proliferative capacity, mediated T-cell tonic signaling, which shares many of the and persistence (10, 11). It is becoming increasingly apparent that hallmarks of the former, remains poorly defined (14). CAR tonic signaling, however, may be defined as a noncoordi- 1CAR Mechanics Group, King's College London, London, United Kingdom. nated and sustained activation of the T cells in either a ligand- 2School of Cancer and Pharmaceutical Studies, Guy's Hospital, London, United independent or dependent fashion. In the absence of spatial and/ Kingdom. 3Department of Clinical Immunology and Allergy, King's College or temporal control of CAR cell surface expression, this consti- Hospital NHS Foundation Trust, London, United Kingdom. 4Department of tutive or chronic cell signaling may have a substantial deleterious Immunology, Eastbourne Hospital, East Sussex, United Kingdom. impact on CAR T-cell effector function and survival, and may lead Corresponding Author: Adam Ajina, CAR Mechanics Group, King's College to a significant disparity between in vitro cytolytic capacity and in London, 3rd Floor Bermondsey Wing, Guy's Hospital Campus, Great Maze Pond, vivo antitumor efficacy (18–21). This review highlights the current London SE1 9RT, United Kingdom. Phone: 207-188-1473; Fax: 207-188-0919; research being undertaken to identify and address CAR tonic E-mail: [email protected] signaling in all its forms, drawing attention to data that are at doi: 10.1158/1535-7163.MCT-17-1097 times conflicting and hypothesis-generating. At least four major Ó2018 American Association for Cancer Research. overlapping patterns of ligand-independent CAR tonic signaling www.aacrjournals.org 1795 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Ajina and Maher Figure 1. Iterative design of first-, second-, third-, and fourth-generation CARs. CARs are modular fusion receptor dimers that comprise (from N-terminus to C-terminus) an extracellular targeting moiety (typically an scFv) fused to a spacer (such as an IgG1 hinge and CH2-CH3 domains), a transmembrane domain (such as CD8a or CD28), and a signaling endodomain. First-generation CARs fused the scFv to a CD3z, CD3e,orFcgR activation domain. Second-generation CARs contain an additional intracellular costimulatory domain (such as CD28, 4-1BB, OX40, or ICOS) to recapitulate signal 2 for T-cell activation. Third-generation CARs combine two or more costimulatory domains in cis. Fourth-generation CARs are engineered with an activation inducible element such as an NFAT-responsive expression cassette to facilitate secretion of a transgenic cytokine such as IL12. CSD, costimulatory domain; ICD, intracellular domain; NFAT, nuclear factor of the activated T-cell; scFV, single-chain variable fragment; TMD, transmembrane domain. are presented and a variety of strategies designed to ameliorate the transducing subunit of either the immunoglobulin receptor negative consequences of these are expounded. Finally, through (FcgR) or the TCR CD3e or CD3z chains (28). First-generation the prism of endogenous T-cell tonic signaling and its important CAR T cells tended to elicit only weak antitumor activity and were regulatory role in immune tolerance and cell-mediated adaptive highly prone to anergy (29). The fusion of costimulatory ICDs immunity, we posit a number of hypothetical strategies designed with the cytoplasmic tail of CD3z-containing first-generation to harness the potential benefits of CAR tonic signaling to improve constructs has led to the emergence of second-generation CAR T-cell antitumor efficacy and in vivo persistence. [comprising a single costimulatory ICD such as CD28 (30), 4- 1BB (CD137; ref.31), inducible T-cell costimulator (ICOS); CAR structure ref. 32, OX40 (33), CD27 (34), or DNAX-activating protein 10 Conventionally designed CARs exploit the specificity of an (DAP10); ref. 35] and third-generation CARs [comprising multi- antibody-derived extracellular binding domain while harnessing ple costimulatory ICDs, aligned in cis (36, 37)]. Incorporation of the effector and memory capacity of T cells to target tumors (22). costimulatory ICDs can recapitulate signal 2 required for T-cell CAR T cells may thus deliver the promise of "living drugs," capable activation, leading to enhanced effector function, proliferation, of targeting tumor-associated or tumor-specific antigens (TAAs or survival, and ultimately enhanced tumor killing (38). Fourth- TSAs) over a prolonged period of time (23). Given that CARs generation CAR T cells (termed "TRUCKs") containing CAR- function in the absence of HLA/TCR interactions, they have inducible transgenes and "armored CARs" capable of constitu- considerable applicability across patient groups and are ideally tively producing cytokines (such as IL12, IL15, and IL18) in placed to address the growing problem of acquired resistance to secreted or membrane-tethered form have been engineered to immune checkpoint inhibition due to disrupted antigen proces- recapitulate signal 3 in an autocrine and paracrine manner (39– sing and/or presentation (24). Furthermore, with the advent of 42). These designs are illustrated in Fig. 1. Further modifications allogeneic HLA and TCR-edited CAR T cells, the potential exists for have been explored with respect to the CAR TMD (43) and hinge/ scalable

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