How CAR T Cells Breathe

Abstract

The manufacture of efficacious CAR T cells represents a major challenge in cellular therapy. An important aspect of their quality concerns energy production and consumption, known as metabolism. T cells tend to adopt diverse metabolic profiles depending on their differentiation state and their stimulation level. It is therefore expected that the introduction of a synthetic molecule such as CAR, activating endogenous signaling pathways, will affect metabolism. In addition, upon patient treatment, the tumor microenvironment might influence the CAR T cell metabolism by compromising the energy resources. The access to novel technology with higher throughput and reduced cost has led to an increased interest in studying metabolism. Indeed, methods to quantify glycolysis and mitochondrial respiration have been available for decades but were rarely applied in the context of CAR T cell therapy before the release of the Seahorse XF apparatus. The present review will focus on the use of this instrument in the context of studies describing the impact of CAR on T cell metabolism and the strategies to render of CAR T cells more metabolically fit.

Determination of CAR T cell metabolism in an optimized protocol

“…Still, to our knowledge, no optimization studies had been properly conducted on primary human CAR T cells and the increasing use of Seahorse urges for a harmonization of the Here we described the optimized conditions to evaluate the metabolism of CAR T cells using a Sea-horse XF96 analyzer. This report complements our previous review discussing Seahorse use in the context of CAR T cell studies (Forcados et al, 2022), where we noticed that although successful results were often reported (van der Windt et al, 2016;Milam et al, 2020;Mercier-Letondal et al, 2021;Kong et al, 2022), the conditions between labs dramatically varied. To our knowledge, no optimization studies had been properly conducted on primary human CAR T cells and the increasing use of Seahorse urges for a harmonization of the protocols.…”

Targeting of chimeric antigen receptor T cell metabolism to improve therapeutic outcomes

“…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 .…”

“…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 ).…”

“…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 ).…”