Applying metabolic control strategies to engineered T cell cancer therapies

Abstract

Chimeric antigen receptor (CAR) T cells are an engineered immunotherapy that express synthetic receptors to recognize and kill cancer cells. Despite their success in treating hematologic cancers, CAR T cells have limited efficacy against solid tumors, in part due to the altered immunometabolic profile within the tumor environment, which hinders T cell proliferation, infiltration, and anti-tumor activity. For instance, CAR T cells must compete for essential nutrients within tumors, while resisting the impacts of immunosuppressive metabolic byproducts. In this review, we will describe the altered metabolic features within solid tumors that contribute to immunosuppression of CAR T cells. We'll discuss how overexpression of key metabolic enzymes can enhance the ability of CAR T cells to resist corresponding tumoral metabolic changes or even revert the metabolic profile of a tumor to a less inhibitory state. In addition, metabolic remodeling is intrinsically linked to T cell activity, differentiation, and function, such that metabolic engineering strategies can also promote establishment of more or less efficacious CAR T cell phenotypes. Overall, we will show how applying metabolic engineering strategies holds significant promise in improving CAR T cells for the treatment of solid tumors.

Keywords: CAR T cell therapies; Cellular engineering; Immunotherapy; Metabolic engineering; Tumor microenvironment.

Figure 1.. Gene modulations in engineered T cells to aid in amino acid metabolism.

Engineered CAR T cells have been modified to overexpress amino acid transporters and intracellular enzymes to improve their function. Proteins in bolded blue represent an increase in expression, whereas proteins red indicate decreased expression. Circles are amino acids with three letter identifications. Curved arrows near the membrane indicate the transport of amino acids. Amino acid-related metabolites are represented in blue boxes, and enzymes are listed next to corresponding arrows. Up or down black arrows show the impact of the low amino acid concentration, and the squiggly arrows show the metabolic engineering approach to overcome the negative impacts of amino acid depletion. The SLC7A5 transporter mediates the transport of large neutral amino acids, including tryptophan and leucine, whereas the SLC7A11 transporter imports cystine. Overexpression of these transporters increased CAR T cell proliferation in low tryptophan or cystine environments. Overexpression of NGK7 (which is downregulated by low methionine levels) in CAR T cells improved their activity. Arginase 1/2 overexpression increased CAR T cell proliferation and tumor control. Similarly, knockin or overexpression of PRODH2, the enzyme responsible for the first step in the hydroxyproline catabolic pathway, in CAR T cells enhanced both in vitro and in vivo cytotoxicity and promoted memory T cell formation. Overexpression of ornithine transcarbamylase (OTC) and argininosuccinate synthase (ASS) in CAR T cells increased arginine production, and overexpression of cystathionase (CTH), an important enzyme of the transsulfuration pathway for producing cysteine, improved tumor control by CAR T cells. Finally, deletion of GCN2 kinase (which is activated in response to low amino acid levels) in T cells improved their function in low tryptophan conditions. Abbreviations: Arg, arginine; Arg1/Arg2, arginase 1/2; ASA, argininosuccinate; ASS, argininosuccinate synthase; ATF4, activating transcription factor 4; CBS, cystathionine-beta-synthase; Cit, citrulline; CTH, cystathionase; Cys, cysteine; Cystath, cystathionine; eIF2α, eukaryotic translation initiation factor 2 alpha; GCN2, general control nonderepressible 2; Gln, glutamine; Hcy, homocysteine; Hyp, hydroxyproline; Leu, leucine; Met, methionine; NKG7, natural killer cell granule protein 7; Orn, ornithine; OTC, ornithine transcarbamylase; P5C, pyrroline-hydroxy-5-carboxylate; PRODH2, proline dehydrogenase 2; SLC1A5, solute carrier family 1 member 5; SLC3A2, solute carrier family 3 member 2; SLC7A11, cystine/glutamate transporter; SLC7A5, large neutral amino acids transporter small subunit 1; Trp, tryptophan

Figure 2.. Metabolic gene manipulation of glycolytic transporters and enzymes within T cells to boost anti-tumor function.

The process of glycolysis starting with glucose import is shown. Transporters and enzymes in blue bolded font signify overexpression, whereas red bold text indicate knocked down genes. Compounds of glycolysis are represented in blue boxes, and enzymes are listed next to corresponding straight arrows. Curved arrows indicate transport of metabolites. GLUT1 overexpression in CAR T cells led to better tumor control. GLUT3 overexpression also improved CD8+ T cell fitness, by increasing glucose uptake and energy storage in the T cells, and increased tumor control and survival of mice with B16-OVA tumors. On the other hand, overexpression of miR-143, which inhibits GLUT1, was shown to improve the differentiation of central memory T cells and enhanced the HER2-CAR T cell-mediated killing of esophageal cancer. Hexokinase II genetic deletion was correlated to small increases in proliferation. Finally, overexpression of PEPCK in T cells increased their PEP production, and due to a boost in effector functions, improved the tumor control and survival of mice with melanoma tumors. Abbreviations: 1,3-BPG, 1,3-biphosphoglycerate; 2-PG, 2-phosphoglycerate; 3-PG, 3-phosphoglycerate; ALD, aldolase; DHAP, dihydroxyacetone phosphate; ENO, enolase; F-1,6-BP, fructose 1,6-biphosphate; F6P, fructose 6-phosphate; G6P, glucose 6-phosphate; GAPDH, glyceraldehyde phosphate dehydrogenase; Glu, glucose; GLUT1, glucose transporter 1; GLUT3, glucose transporter 3; G3P, glyceraldehyde 3-phosphate; HK, hexokinase; Lac, lactate; LDHA, lactate dehydrogenase A; miR-143, mir-143 microRNA; OAA, oxaloacetic acid; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PFK, phosphofructokinase; PGM, phosphoglycerate mutase; PGI, phosphoglucose isomerase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; Pyr, pyruvate; TPI, triose phosphate isomerase

Figure 3.. Metabolic engineering strategies within the Krebs cycle to improve CAR T cell anti-tumor efficacy.

Pyruvate dehydrogenase activity is controlled by pyruvate dehydrogenase kinase (PDK), which can phosphorylate pyruvate dehydrogenase to deactivate it. Overexpression of PDK1 can divert the flux towards lactate production during T cell activation. PDK1-overexpressing CAR T cells demonstrated enhanced effector T cell function and improved CAR T cell cytotoxicity over short periods of time but led to the development of a terminally exhausted T cell population. In addition, isocitrate dehydrogenase 2 (IDH2) limits glucose use through the pentose phosphate pathway and reduces cytosolic acetyl-coA, which is necessary in promoting memory cell formation. The PPP is also important because it alleviates oxidative stress towards CAR T cells by producing antioxidants. Enzymes in bold blue text indicate overexpression, whereas red bolded text refers to knockdown. Compounds of the TCA cycle are represented in blue boxes, and enzymes are listed next to corresponding arrows. Abbreviation: PPP, pentose phosphate pathway

Figure 4.. Metabolic engineering strategies to reduce the effects of immunosuppressive metabolites that accumulate in the TME on CAR T cells.

These approaches typically involve overexpressing immunosuppressive metabolite-degrading enzymes or knocking down proteins responsible for their production or inhibitory effects. Enzymatic reactions are indicated by standard arrows, and dashed arrows represent the activation or association of one molecule with another. Enzymes are listed next to corresponding standard arrows. Overexpressed enzymes or proteins are in blue bolded text, whereas knockdown or deletion are in red bolded text. Immunosuppressive metabolites are identified with bold text. Lactate is an immunosuppressive metabolite formed during fermentation to regenerate NAD+. Transient inhibition of LDH during culturing promoted oxidative phosphorylation and the generation of memory T cells, whereas complete LDHA genetic deletion prevents the formation of potent cytotoxic T cells. Overexpression of LDHB in CD4 T cells helped these T cells to produce cytokines despite lactic acid buildup. IDO1 helps converts tryptophan into kynurenine, and deletion of IDO1 in CAR T cells improves their control of murine pancreatic cancers. Also, reducing kynurenine concentrations by overexpressing kynureninase has improved the anti-tumor traits of CAR T cells. Levels of adenosine, another potent immunosuppressive metabolite, can be reduced by overexpression of ADA1 and ADA2. Overexpressing ADA1 and CD26, which upon activation increases ADA1 secretion, provided CAR T cells with inosine, while reducing adenosine levels. In addition, deletion of the A_2AR adenosine receptor improved CAR T cell activity. Adenosine and PGE2 both activate protein kinase A, which can then travel to the immune synapse after binding to the membrane protein ezrin to have inhibitory effects. Overexpression of RIAD blocked this interaction and enabled better signaling and cancer cell killing by CAR T cells. Knockout of the DGK gene also allowed for increased TCR signaling. In addition, overexpression of D2HGDH, which can convert D2HG, an immunosuppressive metabolite, into 2-oxoglutarate, produced CAR T cells with greater cytotoxicity. Finally, expression of thioredoxin-1, glutaredoxin-1, and catalase can help T cells overcome the impact of reactive oxygen. Abbreviations: 2OG, 2-oxoglutarate; A2AR, adenosine A2A receptor; AA, anthranilic acid; ADA1/ADA2, adenosine deaminase 1/2; ADO, adenosine; CAT, catalase; D2HG, D-2-hydroxyglutarate; D2HGDH, D-2-hydroxyglutarate dehydrogenase; DAG, diacylglycerol; DGK, diacylglycerol kinase; GRX1, glutaredoxin-1; IDO1, indoleamine 2,3-dioxygenase-1; INO, inosine; Kyn, kynurenine; KYNU, kynureninase; Lac, lactate; LDHA/LDHB, lactate dehydrogenase A/B; PA, phosphatidic acid; PGE2, prostaglandin E2; PKA, protein kinase A; Pyr, pyruvate; RIAD, regulatory subunit I anchoring disruptor; ROS, reactive oxygen species; scFv, single-chain variable fragment; TRX1, thioredoxin-1; Trp, tryptophan