Counteracting CAR T cell dysfunction

Abstract

In spite of high rates of complete remission following chimeric antigen receptor (CAR) T cell therapy, the efficacy of this approach is limited by generation of dysfunctional CAR T cells in vivo, conceivably induced by immunosuppressive tumor microenvironment (TME) and excessive antigen exposure. Exhaustion and senescence are two critical dysfunctional states that impose a pivotal hurdle for successful CAR T cell therapies. Recently, modified CAR T cells with an "exhaustion-resistant" phenotype have shown superior antitumor functions and prolonged lifespan. In addition, several studies have indicated the feasibility of senescence delay in CAR T cells. Here, we review the latest reports regarding blockade of CAR T cell exhaustion and senescence with a particular focus on the exhaustion-inducing pathways. Subsequently, we describe what potential these latest insights offer for boosting the potency of adoptive cell transfer (ACT) therapies involving CAR T cells. Furthermore, we discuss how induction of costimulation, cytokine exposure, and TME modulation can impact on CAR T cell efficacy and persistence, while potential safety issues associated with reinvigorated CAR T cells will also be addressed.

Fig. 1. Development of CAR generations.

CAR structure in CAR T cells compose of a single-chain variable fragment (scFv), a hinge and transmembrane (TM) region, costimulatory domain(s) (e.g., OX40, CD28, ICOS, 4-1BB), and a CD3ζ signaling domain. The 1st generation CARs contained only CD3ζ as intracellular domain, while the 2nd or 3rd generations have one or two costimulatory domains linked to CD3ζ, respectively. The 4th generation CARs are known as “TRUCK” CARs. These CARs are structurally similar to the 2nd generation CARs, but with an inducible cytokine expression (e.g., IL-12) through NFAT-responsive promoter.

Fig. 2. A scheme for induction of exhaustion and senescence.

The principal feature of exhaustion (left) is upregulation of inhibitory receptors such as PD-1. Engagement of PD-1 to its ligand (PD-L1) results in the inhibition of intracellular signaling pathways involved in the regulation of cell proliferation such as PI3K/AKT pathway. Inactivation of AKT (due to the lack of site-specific phosphorylation mediated by PI3K, mTORC (on Serine473), and PDK1 (on Threonine308)) in turn lifts the block on FOXO transcription factors and thereby activates p27, a repressor of cell cycle (G1-S transition). Senescence (right) can be induced by either AKT inhibitory receptors (e.g., KLRG-1) or DNA damage response mediators including ATM and ATR. KLRG-1 prevents AKT phosphorylation (on Serine473) removing its block on p27, and thereby results in cell cycle arrest. The DNA damage response components activate p53, p21, p38, and p16 which inhibit cell cycle progression by blocking the function of cyclins and cyclin-dependent kinases (CDKs). The figure adapted from [16].

Fig. 3. Intracellular network between exhaustion or senescence regulators.

Competition between exhaustion or senescence regulators and inhibitors determines the fate of CAR T cell in the TME. Inhibitory receptors transfer exhaustion/senescence signal to the inhibitory transcription factors (i.e., FOXO, Eomes, BATF, BLIMP1, TOX, NR4A, IRF4, NFAT, etc.) via downstream signaling pathways, whereas exhaustion/senescence delaying transcription factors (i.e., c-FOS, TCF1, T-bet, NFκB, etc.) counteract these inhibitory signals. The green plus and red minus signs refer to the enhanced/upregulated and blocked/downregulated genes in CAR T cells, respectively.

Fig. 4. Costimulation and coinhibition of CAR T cell.

Upregulation of costimulatory and coinhibitory receptors on the surface of CAR T cells is associated with effector polyfunctional and exhausted hypofunctional phenotypes, respectively. The final functional outcome depends on the number and diversity of relevant receptors. Although differentiation of functional CAR T cells to the exhausted/senescent phenotype is a common phenomenon in the tumor site, the possibility of reinvigorating exhausted/senescent CAR T cells is still a question.