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CAR Signaling Networks Interactive Pathway

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Pathway Description:

Chimeric antigen receptor T-cell (CAR-T) therapy is a promising new immunotherapy that uses genetically modified cells to treat cancer. Through the ex vivo introduction and expression of a chimeric antigen receptor (CAR), a patient’s own T cells are engineered to target a specific surface antigen that serves as a molecular beacon on cancer cells. Upon infusion, CAR-T cells act as a “living drug” that identify and kill cells expressing this marker by harnessing the cytotoxic abilities of T cells. A key area of research in the ongoing development of CARs is to fully understand how they activate downstream signaling pathways to maximize clinical efficacy while reducing toxicity.

CARs are synthetic proteins with a modular design intended to engage the endogenous cell signaling cascades that elicit effector T-cell functions—including enhanced proliferation, cytokine release, and cytotoxicity. CARs bind to a target of interest via an extracellular antigen recognition domain (ARD), which consists of a single-chain variable fragment (scFv) that links the variable light and variable heavy regions of a monoclonal antibody. The scFv is tethered to the transmembrane portion of the receptor by a spacer domain, the length of which directly influences the binding affinity of the ARD. A transmembrane domain, typically derived from CD8 or CD28, anchors the CAR to the T-cell membrane and connects the ARD to the intracellular signaling portions of the receptor. The optimal composition of the intracellular region of CARs is an active area of research, as variations in the number and length of these domains can dramatically alter CAR-T antitumor effects. The current generation of receptors consists of an activation domain and one or multiple costimulatory domains that serve to communicate ligand binding events to alter T-cell transcriptional programs via the engagement of an array of downstream signaling networks. The activation domain derived from the T-cell receptor CD3ζ chain is a common feature of the intracellular portion of CARs, capable of initiating signaling to drive T-cell cytotoxic functions. The addition of costimulatory domains—from either the CD28 receptor family or the tumor necrosis factor receptor family (4-1BB, OX40, or CD27)—is thought to enhance CAR-T efficacy by enhancing cytokine secretion as well as CAR-T proliferation and persistence.

The inclusion of distinct functional domains on the intracellular portion enables CARs to recapitulate the integrated events of T-cell receptor signaling with a single receptor chain. A key post-translational modification that is created upon ligand binding is the phosphorylation of CD3ζ, which in turn recruits zeta-chain associated protein kinase 70 (Zap-70) to promote the assembly of downstream adaptor and scaffold proteins. In parallel, costimulatory modules initiate signaling via the PI3K/AKT, TNF receptor-associated factor 2 (TRAF2)/p38MAPK, and JNK pathways. Collectively, these signaling events converge on critical transcriptional modulators—including NF-κB, NFAT, STAT3, JUN, and FOS—to drive changes in gene expression related to T-cell activation and effector function.

While previous theories hypothesized that distinct costimulatory domains signaled through divergent mechanisms, a recent phosphoproteomic anaylsis of CAR signaling suggests that they instead alter the activation kinetics and intensity of many of the same signaling molecules (Salter et al., 2018). However, the effects observed in this study may be context dependent, as an independent evaluation of the CAR interactome and signalsome identified significant differences in association with signaling molecules and pathway activation between CARs containing variable intracellular regions (Ramello et al., 2019). These findings underscore the need to fully evaluate the relationship between CAR design and the intracellular signaling events they control in order to optimize CAR-T cell therapy efficacy.

Selected Reviews:

Created November 2019.

Acetylase
Metabolic Enzyme
Adaptor
Methyltransferase or G-protein
Apoptosis/Autophagy Regulator
Phosphatase
Cell Cycle Regulator
Protein Complex
Deacetylase or Cytoskeletal Protein
Receptor
Growth Factor/Cytokine/Development Protein
Transcription Factor or Translation Factor
GTPase/GAP/GEF
Ubiquitin/SUMO Ligase or Deubiquitinase
Kinase
Other
 
Direct Stimulatory Modification
Direct Stimulatory Modification
Transcriptional Stimulatory Modification
Transcriptional Stimulation
Direct Inhibitory Modification
Direct Inhibitory Modification
Transcriptional Inhibition
Transcriptional Inhibition
Multistep Stimulatory Modification
Multistep Stimulatory Modification
Joining of Subunits
Joining of Subunits
Multistep Inhibitory Modification
Multistep Inhibitory Modification
Translocation
Translocation
Tentative Stimulatory Modification
Tentative Stimulatory Modification
Separation of Subunits or Cleavage Products
Separation of Subunits or Cleavage Products
Tentative Inhibitory Modification
Tentative Inhibitory Modification