Surgery, chemotherapy, and radiation remain the cornerstones of cancer treatment. However, many patients are not cured by these approaches and those that are cured may incur significant morbidities, demonstrating an urgent need for the development of novel therapeutic options. Cancer cells subvert normal metabolic pathways to favor their growth and evasion of the immune system. Altered metabolism of the tumor microenvironment (TME) plays a critical role in tumorigenesis by creating an immunosuppressive milieu (1–4). Several therapeutic approaches to modify and restore the immune system are currently being developed and applied. Genetically engineered T cells constitute a powerful new therapeutic approach in the treatment of cancer. Chimeric Antigen Receptors (CARs) are synthetic receptors that graft a defined specificity onto an immune effector cell, typically a T cell, and augment T cell function. Once infused into the patient they expand and kill tumor cells. They also prevent tumor recurrence by promoting immune surveillance in conjunction with tumor infiltrating lymphocytes or by their own persistence (5–7). While significant strides have been made in CAR T cell therapy for hematologic malignancies leading to FDA approval of multiple products, durable responses in solid tumors remain limited (8, 9). Strategies to improve CAR T cell function are actively being sought. Metabolic manipulation represents a potential approach for improving the former given that T cell function is closely tied to cellular metabolism. Energetic demands and consequences of T cell activation, cytokine production, proliferation, and survival are facilitated by metabolic rewiring (10, 11). Furthermore, each stage within the CAR T manufacture process can influence the eventual metabolic profile of the infusion product, while metabolic features of the latter are directly linked to in vivo efficacy and persistence (8). The goal of this manuscript is to review known aspects of T cell metabolism, in the context of CAR T therapy, and present potential metabolic interventions that can be undertaken at each step of the manufacture process, including CAR design, priming, and expansion to leverage metabolic fitness of CAR T cells to augment therapeutic outcomes. In general, CAR T products containing memory like cells with enhanced mitochondrial fitness and high reliance on oxidative phosphorylation (OXPHOS) along with fatty acid oxidation (FAO) have been shown to have superior in vivo anti-tumor efficacy and long-term persistence. While effector function is tied to glycolysis that is engaged upon antigen-driven activation, a high proportion of memory cells that can be maintained in diminishing levels of antigen are associated with sustained anti-tumor responses. Thus, strategies that favor mitochondrial biogenesis along the manufacture process of the CAR T cells and maintenance of this metabolic phenotype after infusion are discussed in this review.

T cells are specific effectors of the adaptive immune system, which continuously survey and eliminate pathogen infected cells as well as tumors. To elicit a robust immune response, T cells differentiate into diverse functional subsets, such as effector T cells (Teff) and memory T cells (Tmem), which can further differentiate into more diverse subsets based on cytokine milieu (12, 13). Such subsets have different functional and metabolic requirements. In the absence of antigen, naïve T cells are quiescent, rarely divide, have a low energetic demand, and continuously circulate through secondary lymphoid tissues (11, 14). Memory T cells are also mostly quiescent but display a greater mitochondrial mass that provides a bioenergetic advantage to support rapid recall responses upon antigen re-exposure. Both sets of T cells rely almost completely on the energy derived from mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) to maintain their basal energy level, cellular function, and viability (15–18). Following antigen exposure, T cell activation is orchestrated by TCR/peptide-MHC interaction providing the first signal forming an immune synapse. Further interaction at the synapse with costimulatory molecules provides the required second signal. Thus, a complete TCR-based activation of T cells requires two signals (15, 19, 20). Once activated, T cells predominantly engage in aerobic glycolysis, the pentose phosphate pathway (PPP), one-carbon metabolism, fatty acid oxidation (FAO), and glutaminolysis to facilitate proliferation and enable subsequent effector functions (17, 21–24), Figure 1A .

CARs are synthetic receptors that redirect T cells against a defined target in a major histocompatibility complex (MHC)-independent fashion. CAR T cell therapy aims to eliminate specific tumor cells in a sustained manner. The first step of the CAR T manufacturing process is the collection of PBMCs through leukapheresis, either patient’s own or from a donor (62, 63), followed by isolation of T cells. T cells are then activated with anti-CD3/CD28 magnetic beads to promote proliferation and differentiation. At this point, T cells transition from a naïve or quiescent state to an activated state, with a concurrent metabolic switch from FAO to glycolysis (20, 26, 29), whereby T cells differentiate into either high glucose requiring Teff cells and low glucose requiring Tmem cells (11, 36). The next step in the manufacturing process is viral transduction, where T cells are incubated with a lentiviral vector encoding the CAR construct. The final step in manufacture includes expansion within cytokine enriched media and represents yet another opportunity for T cells to differentiate into distinct functional phenotypes, i.e., Teff, Tem, Tscm, Tcm, based on culture conditions. CAR T products containing a high proportion Tcm or Tscm subsets have been shown to have an enhanced subsequent metabolic adaptability, mediated mainly through mitochondrial metabolism, and are able to maintain a long-term anti-tumor response in vivo (8, 10, 14, 64–67). Optimization of CAR T cell metabolism for the maintenance of early memory phenotypes, Tscm and Tcm, to improve CAR persistence and cytotoxic function can potentially be carried out at several stages, as described hereafter, Figures 1A, B .

CAR constructs: The first T cell activating receptors were CD3ζ chain fusions, which also elucidated the role of the ζ chain (68, 69). These early studies showed that T cell activation signaling, and initiation of cytotoxicity were possible by cross-linking the fusion receptors. Eshhar et al. then incorporated the immunoglobulin-derived single chain variable fragment (scFv) onto these receptors to direct and lyse hapten coated cells (5, 70), generating the first-generation CAR T. Since then, multiple generations of CAR T cells have been engineered. The current FDA approved CAR T cell receptor has four parts: 1) an extracellular target antigen-binding domain composed of a single chain variable fragment (scFv) of an antibody targeting the specific tumor antigen; 2) a hinge region; 3) a transmembrane domain; and 4) the intracellular domain, i.e., T cell receptor (TCR) signaling domain comprised of CD3ζ and costimulatory domains. The CD3ζ domain in the CAR structure serves as signal 1 and a costimulatory domain provides signal 2 (15). Second and third generation CARs have multiple costimulatory domains, such as immunoglobulin (Ig) superfamily members, CD28 (B7.1/B7.2-CD28) and inducible T cell costimulatory (ICOS, B7RP-1-ICOS) (71), and tumor necrosis factor receptor (TNFR) superfamily members 4-1BB, OX40 and CD27 (10, 15). Depending on the costimulatory domains incorporated into the CAR construct, different downstream signaling pathways are activated that impact in vivo persistence, susceptibility to exhaustion, generation of memory, and anti-tumor potency (10). CD28 and 4-1BB signaling domains are the most widely used and studied. Hence, their effects on CAR T cell metabolism are described below.

Metabolic phenotypes linked to CAR constructs: Kawalekar et al. found that 4-1BB based (BBζ) CAR T cells had higher proliferative capacity and persistence than CD28 based (28ζ) CAR T cells. Both sets of CARs started with a uniformly increased expression of CD69, an activation marker, on Day 1. Subsequently, the BBζ CAR T cells proliferated and persisted in culture for over 4 weeks, while the 28ζ CAR T cells had done so for 2 weeks (72). Even more striking persistence differences were observed in vivo, where CD28-based CAR T cells were detected for 30 days (73–75), while 4-1BB based CAR T cells persisted for years (76, 77). The increased persistence of BBζ CAR T cells was attributed to their differentiation into CD45RO⁺CCR7⁺ Tcm cells, and this phenotype was maintained through the culture process. In contrast, 28ζ T cell expansion resulted in a higher proportion of CD45RO⁺CCR7^- Tem cells following stimulation through the CAR (20, 72, 78).

To characterize cellular metabolism changes upon CAR signaling, Kawalekar et al. measured the oxygen consumption rate (OCR), a surrogate measure of OXPHOS, before activation, as well as at 7- and 21-days post antigen stimulation; including following serial addition of an inhibitor of ATP synthesis, an uncoupling ionophore, and blocking agents of the ETC. The OCR profiles were similar in both groups on Day 0. On days 7- and 21-post antigenic stimulation an ~10-fold increase in OCR was observed in both groups. Maximal respiratory capacity of BBζ CAR T cells showed a robust increase following decoupling of the mitochondrial membrane. The extracellular acidification rate (ECAR), a surrogate of lactic acid production during glycolysis, was elevated in 28ζ CAR T cells. 28ζ CARs were found to be rapidly consuming glucose and generating lactate, consistent with observed high ECAR levels. Additionally, Glut1, PDK1 (26), G6PD, and phosphoglycerate kinase (PGK), and have also been shown to be elevated in 28ζ cells. These findings support the conclusion that 28ζ CAR T cells rely on glycolysis, Figure 1B , for their energy needs (26, 72), a characteristic of Teff cells (72, 78).

In contrast, BBζ CAR T cells rely on mitochondrial oxidative phosphorylation (72). When glucose uptake and fatty acid utilization rates were evaluated by measuring residual glucose, lactate, and heavy-carbon-labeled long chain fatty acid, palmitic acid, in the media at different time points, BBζ cells showed high utilization of palmitic acid, measured by heavy-carbon-labeled acetyl CoA levels. As β-oxidation of fatty acids generates acetyl CoA, an increase in heavy-carbon-labeled acetyl-Co-A pool indicated that BBζ CAR T cells use FAO to fuel their bioenergetic needs. Also, carnitine palmitoyl transferase (CPT1A), which controls a rate limiting step in mitochondrial FAO and promotes mitochondrial biogenesis (20), was significantly elevated in BBζ cells. Additionally, fatty acid binding protein (FABP5) that is involved in fatty acid uptake, transport, and metabolism was also elevated in these cells. Together, these findings indicate that BBζ CAR T cells use fatty acids for their energy needs (23, 72). Finally, BBζ CAR T cells have a survival advantage due to their ability to generate increased mitochondrial mass (61, 72). BBζ CAR T cells consistently demonstrated high spare respiratory capacity (SRC), which is a characteristic of natural CD8⁺ T cell memory and supports T cell function in the hostile tumor environment (20, 72, 79–81).

Selection of CAR costimulation systems based on associated metabolic re-wiring: As discussed, the costimulatory signals in the CAR constructs that are necessary for T cell activation, expansion, cytokine secretion, cytotoxic function, memory formation, and survival are mediated through metabolic reprogramming. Depending on the costimulatory domain incorporated into the construct, different signaling pathways are triggered upon antigen activation (82–84). As already discussed, the CD28 domain in CARs leads to the activation of PI3K/Akt pathway, Figure 1A and aerobic glycolysis as the predominant metabolic program. In contrast, T cells with CAR constructs comprising of 4-1BB domain activate the NF-κB, MAPK, and ERK pathways. These CAR T cells exhibit enhanced OXPHOS and SRC derived from fatty acid oxidation. Mitochondrial biogenesis and oxidative metabolism associated with Tcm phenotype are preferred in CAR T therapy given enhanced in vivo persistence and function (72), Figure 1B .

Another costimulatory domain, ICOS, has been shown to lead to higher PI3K/Akt pathway activation compared to CD28, and increased secretion of IL-21, IL-17, and INF-gamma (85). OX40, a member of the TNFR family which is upregulated with T cell activation via the OX40L has a broad effect on T cell activation, proliferation, differentiation, and survival. It stimulates glycolysis and OXPHOS via PI3K/Akt, MAPK and NF-κB pathways, and induces antiapoptotic genes, Bcl-1and Bcl-xl, to promote T cell expansion and survival, respectively (86–88). To summarize, selection of co-stimulatory domains has a significant impact on persistence and antitumor function of CAR T therapies.

Improving CAR T cell efficacy using CAR systems encoding cytokines and/or chemokines: Cytokine- and chemokine- encoding genes can be added into the CAR construct, which may lead to distinct cellular metabolism features. While the metabolic consequences of such modifications have not been fully explored, they have been implemented to improve CAR T entry into and function within the TME. While first-generation CARs had a single CD3ζ signaling domain they were ultimately not very effective (89–91). Second and third generation CAR T cells have incorporated one or two costimulatory signals, respectively. Genetic modifications to CARs for co-expression of cytokine (92–98), chemokine (99–101), or both factors (102–104) have been successfully used to enhance therapeutic efficacy CAR T cells.

Use of cytokines in expansion media to promote CAR T cell metabolic rewiring: Cytokine composition in culture media impacts efficacy of the CAR T cell product. Cytokines that have been broadly investigated to date are IL-2, IL-7, IL-15, and IL-21 (105). IL-2 is a T cell growth factor that promotes effector differentiation and glycolysis in CD8 T cells (22, 106, 107). When CAR T cells are expanded using IL-2, they differentiate to effector CD8+ cells via Akt-m TOR pathway. However, aryl hydrocarbon receptor activation was also observed under such expansion conditions, suggesting that IL2 signaling contributes to CAR T cell exhaustion (105, 108). IL-7 and IL-15 can cause differentiation to memory T cells (20, 109, 110). IL-7 can induce glycerol channel expression and triglyceride (TAG) synthesis that results in a product with a high CD8 memory proportion. It also promotes Glut-1 cell surface expression thereby increasing glucose uptake and promoting cell survival (111). The combination of IL-2 and IL-7 in the ex vivo culture media during CAR T cell expansion enhances glycolysis and differentiation of T cells towards the effector phenotype necessary for cytotoxicity (112). Another cytokine used for CAR T expansion is IL-15. It downregulates mTORC1 activity and expression of several glycolysis enzymes, thereby improving mitochondrial fitness and maintenance of the Tscm phenotype (105, 113). Consequently, recent trials have used CAR T cells expanded in IL-7 and IL-15 and have demonstrated superior anti-tumor efficacy. Notably, CAR T cells expanded in IL-2 can show phenotypic features similar to those expanded in IL-15, when mTORC1 inhibition is provided by concurrent exposure to rapamycin (24).

Optimizing nutrients within expansion media: Optimization of expansion media, aside from cytokine milieu, can also impact on T cell differentiation and subsequent function. For example, L-arginine is consumed rapidly in activated T cells; hence, when exogenous L-arginine was supplemented, a shift from glycolysis to OXPHOS was observed in cultured cells (34, 114). A potential explanation for this switch is that with increased L-arginine leads to an upregulation of the serine biosynthesis pathway, fueling the TCA cycle and enhancing OXPHOS (114, 115). The decrease in glycolysis combined with increased L-arginine levels potentiates the generation of the Tcm subset, promoting anti-tumor activity in vivo (29, 36, 114). Another feature of T cells cultured in excess L-arginine is prolongation of survival (114). Other nutrients in the media that could be altered are fatty acids, especially short chain fatty acids (SCFA), such as butyrate, propionate, and acetate (116), which diffuse passively into the mitochondria. Depending on the concentration, the SCFAs have been shown to favor a memory-like T cell phenotype (116–120).

Following infusion, CAR T have to traffic to tumor sites, penetrate the TME, and persist in the patient to generate sustained anti-tumor activity. Strategies aimed at improving trafficking and persistence of CAR T, including nanoparticle RNA vaccines as well as oncolytic viruses have been described, albeit so far in pre-clinical studies, with clinical testing currently ongoing for the latter. While these approaches have the potential to enhance CAR T in vivo performance, they are not metabolic in nature and are reviewed elsewhere (121).

Cancer involves abnormal cell growth, diminished apoptosis, and evasion of the normal host defenses that facilitate local invasion and potential distant metastasis (122). The high replication rate of tumor cells requires a continuous source of energy necessitating modification of normal metabolism. Cancer cells engage aerobic glycolysis to meet their metabolic requirements and ATP production (1, 123). Increased aerobic glycolysis by the tumor cells creates a glucose deprived TME, impairing effector T cell function within the endogenous anti-tumor immunity and adaptive cell therapy (1, 123). Tumor derived lactate accumulation via aerobic glycolysis leads to an acidified TME. This in turn impedes monocarboxylate transporter-1 mediated, gradient-dependent export of lactate from CD8+ T cells (1, 124, 125). Malignant cells within a solid tumor rapidly proliferate and organize, which can lead to poor vascularization that exacerbates TME hypoxia. In addition to the decreased oxygen delivery, TME hypoxia can be further enhanced by high tumor oxygen consumption. Hypoxia inhibits differentiation, proliferation, and cytokine production of cytotoxic T lymphocytes (CTL) and T helper (Th1) cells (10, 125, 126). Under hypoxic conditions, tumor cells respond with rapid induction of transcription factor hypoxia-inducible factor 1 alpha (HIF1α) and NFκB (1, 127). Both factors are involved in the regulation of genes implicated in inflammation and adaptation to hypoxia. These pathways control mitochondrial dynamics and mitophagy, promote TME acidification, and regulate the expression of cytokines and angiogenic factors (1, 10, 128–131). These transcription factors regulate the expression of a plethora of cytokines and angiogenic factors, including, IL-6, IL-10, and vascular endothelial growth factor (VEGF) (10, 127). These molecules then recruit cells with immunosuppressive function, including myeloid-derived suppressor cells (MDSCs), T regulatory (Treg) cells, innate lymphoid cells (ILCs), tumor associated macrophages (TAMs), and cancer associated fibroblasts (CAFs). This establishes an immunosuppressive TME and suppresses antigen processing and presentation (10, 132), leading to immune evasion. Furthermore, HIFα controls the expression of checkpoint molecule programmed death-ligand 1 (PD-L1) on the surface of cancer cells and PD-1 expression on T cells, which negatively impacts T cell survival and effector functions via the PD1/PD-L1 axis (10, 125). Additionally, hypoxia dampens activity of adenosine kinase and adenosine deaminase leading to adenosine accumulation in the TME that favors immunosuppressive cells and inhibits anti-tumor T cell function (125). As described, the TME plays an important role in augmenting endogenous T cell and CAR T cell function. Several excellent reviews on TME metabolism as a barrier to immunotherapy and metabolic strategies to manipulate the former have been recently published (3, 4, 133). The focus of this review is the manipulation of CAR T metabolism to improve in vivo function and persistence, which is described hereafter.

Increased persistence and survival are characteristics of an effective CAR T anti-tumor response (134). Presence of specific T cell subsets, such as those with minimal differentiation and increased self-renewal capacity, can enhance CAR T cell anti-tumor efficacy (135). As already described, metabolism plays a major role in this process, with effector T cells depending more on glycolysis and memory T cells relying on mitochondrial oxidation and FAO for their bioenergetic needs (136). Pre-clinical studies have shown that an infusion of a high proportion of naïve T cells (Tn), Tscm, or Tcm leads to superior anti-tumor efficacy compared to products with higher Tem or Teff content (137, 138). However, during the ex vivo expansion process, a large proportion of Tem and Teff are generated due to a high rate of glycolysis induced during activation and expansion that drives T cells towards terminally differentiated phenotypes (36). The cytokine milieu during ex vivo expansion, antigen activation, and costimulation, contribute to this process, as previously described. Given that the functional phenotype, i.e., Tscm, and favorable metabolic features are linked, maintenance of this phenotype represents a promising strategy for generating effective adoptive T cell therapies, Figure 1B .

Inhibition of glycolysis: 2-deoxyglucose (2-DG) is a prototypical inhibitor of the glycolytic pathway that blocks hexokinase (139), Figure 1A . Fraietta et al. showed inhibition of glycolysis with 2-DG decreased T cell effector and promoted memory T cell generation (140). Additionally, the same group demonstrated that a glycolytic gene signature characterized CAR T cells obtained from patients with partial or no response to therapy. The former also displayed a higher uptake of a glucose analog than CAR T cells isolated from patients with complete responses (140). Thus, employing strategies to interfere with glycolysis is a potential solution to improve downstream in vivo CAR T cell efficacy. At high concentrations, however, 2-DG may interfere with T cell proliferation and cytolytic function. Cham et al. showed that 2-DG at 10-50 mM concentrations in culture interfered with T cell proliferation and almost completely abolished cytolytic ability of CD8⁺ T cells (123). Shi et al. showed that inhibition of glycolysis by 2-DG at a concentration of 1mM had minimal inhibitory effects on cell proliferation (139). Similarly, Sukumar et al. used 2 mM concentrations of 2-DG, which sufficiently inhibited glycolysis without interfering with cell proliferation and successfully induced OXPHOS. They also demonstrated that T cells primed in the presence of 2-DG accumulated at higher numbers in tumors (36). In conclusion, inducing a memory T cell phenotype by inhibiting glycolysis with relatively low levels of 2-DG during CAR T cell manufacture is a potential strategy to improve CAR T cell efficacy, Figure 1B .

Improving mitochondrial function: As already discussed, mitochondria play a key role in the regulation of T cell metabolism, biosynthesis, migration, cell fate and programmed cell death. Regulating mitochondrial OXPHOS is one approach to improve CD8⁺ T cell function. Lactate dehydrogenase (LDH) is an enzyme just distal to glycolysis that converts pyruvate into lactate. Hermans et al. demonstrated that a small molecule LDH inhibitor, at a concentration of 1 uM led to metabolic rewiring, blocking generation of lactate and promoting pyruvate entry into the TCA cycle, and ultimately enhancing OXPHOS (51), Figure 1A . The latter can inhibit terminal effector differentiation and exhaustion. The same group also demonstrated that LDH inhibition in combination with IL-21 exposure increased the formation of Tscm cells leading to an improved anti-tumor response and persistence. Interleukin-21 (IL-21) is a cytokine that uses the common cytokine receptor γ chain (γ_c) as a receptor component (141). It primarily activates STAT3 (142), along with IL-7 and IL-15 expands CD8+ T cells (143). Thus, transient inhibition of LDH in combination with IL-21 supplementation during expansion phase generated a more effective cell therapy product (51). Another strategy for improving mitochondrial function is through the peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α that belongs to a family of transcription coactivators (144). PGC-1α overexpression in CD8 T cells has been found to boost mitochondrial biogenesis and memory phenotype, enhancing anti-tumor immunity (121, 145, 146), Figure 1A . Additionally, overexpression of PGC-1α in exhausted T cells improved their mitochondrial function, restoring functionality (121, 147). CD8⁺ T cells with PGC-1α overexpression secondary to exposure to bezafibrate, a PGC-1α agonist, in the presence of PD-1 blockade, upregulated mitochondrial OXPHOS and increased FAO, which enhanced their survival (148). Bezafibrate is a drug already in clinical use for hypercholesterolemia and could potentially be deployed in the setting of adoptive immunotherapy, following CAR T infusion.

An exhausted T cell (Tex) phenotype has been described extensively in the setting of chronic infection but has also been recognized in tumor-resident endogenous T cells and in the setting of adoptive T cell therapy (105, 149). Tex vulnerability is at least in part attributable to tonic TCR stimulation, which leads to metabolic rewiring and epigenetic changes that can enforce terminal exhaustion (105). CAR T cells are thought to be especially susceptible to this process given continuous antigenic stimulation that occurs during their ex vivo expansion, ahead of exposure to tumor antigen in the TME. Enhancing mitochondrial fitness and the linked Tscm phenotype during priming and expansion appear to counteract this susceptibility (105). Tex display inhibitory receptors, diminished effector ability, and decreased proliferative capacity. Metabolically, these cells are characterized to mitochondrial dysfunction and decreased glycolysis utilization (105, 149). Interestingly, Tex functionality can be restored with metabolic manipulation, as described above, through PGC-1α overexpression (121, 147), further supporting the notion that ability to maintain OXPHOS and FAO metabolism are necessary to avoid Tex phenotype. Additionally, PD-1 blockade in T cells has been shown to drive increased FAO, enhancing their survival and memory phenotype (121, 150). However, terminally exhausted T cells may only partially respond to PD-1 blockade, presumably due to fixed epigenic modifications (149). Acetate supplementation, which can restore acetyl-CoA required for histone acetylation, can improve chromatic accessibility and restore functionality in CD8 T cells (149, 151). Other epigenetic modifiers may lower susceptibility or restore functionality in Tex and are explored in greater depth in other reviews (105, 149).

Sustained activation of the PI3K-Akt-mTOR pathway by activation beads, IL-2, or tonic signaling drives T cells towards terminal differentiation and inefficient tumor killing (152–154). mTOR is a main regulator of CD8 T cell differentiation. Inhibition of mTORC1 by rapamycin or metformin (AMPK activator) has been shown to enhance OXPHOS by increasing FAO, and ultimately promoting the CD8+ T memory phenotype (154, 155). In the setting of CAR T, use of rapamycin during priming and expansion promoted the memory phenotype and increased FAO metabolism (121, 152, 156, 157). The addition of PI3K inhibitors, including, MK2206, LY294002, IC87114, idelalisib, and TGR-1202, during expansion has been shown to maintain CAR T cells in a less differentiated state leading to increased downstream anti-tumor efficacy and persistence of CAR T cells in vivo (105, 121, 158).

To summarize, induction of mitochondrial biogenesis and promotion of T cell differentiation towards a memory phenotype is a promising strategy for improving anti-tumor efficacy and persistence of CAR T cells.

Aside from identifying tumor specific antigens and engineering appropriate CARs, several strategies to enhance in vivo CAR T function are currently being pursued. As greater understanding of T cell metabolism and immunosuppressive features of the TME is gained, this knowledge can be potentially leveraged to enhance anti-tumor responses. As described, manipulation of the T cell activation machinery that is linked to cellular metabolism reprogramming can enhance in vivo CAR T cell performance and persistence. Improved trafficking and function of CAR T cells in the TME have been observed with CAR T products containing distinct functional phenotypes that are linked to corresponding metabolic rewiring. These phenotypes can be generated via manipulations of the CAR construct itself, i.e., through selection of costimulatory elements, or during the expansion phase using cytokines and nutrients to skew CAR T products towards more favorable metabolic characteristics. Additionally, metabolic regulators can be used either during expansion or after infusion to generate and maintain metabolic phenotypes within CAR T cells, respectively. Metabolic rewiring of cellular therapies represents a promising clinically relevant approach to improve immunotherapy responses.

All authors contributed to the writing of this review after NB received the invitation to submit.