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CAR Signaling Networks Interactive Pathway

Access the full library of downloadable pathway diagrams, along with recommended products for each signaling pathway.

Activation Domain Costimulatory Domain 2 Costimulatory Domain 1 TM CD28 4-1BB CD3ζ VL VH Peripheral T-Cell Actin Polymerization scFv (Antigen Recognition) Hinge/ Spacer Linker CD8αCD28 OX-40 CD27 ICOS DAP10 ITAM1 (Y72, Y83) ITAM2 (Y111, Y123) ITAM3 (Y142, Y153) CAR Signaling Networks Cytoplasm Nucleus CaM NCK CaMKIV Calcineurin Zap70 Itk PLCγ1 SLP-76 MEKK1 MKK4/7 JNK1 TAK1 MKK7 JNK2 CREB JUN JUN STAT3 FOS FOS Ras Raf IKKα IKKγ IKKβ IκB IκB NF-κB MEK1/2 ERK1/2 PI3K GRB2 LAT PDK1 PKCθ Carma1 Bcl10 MALT1 AKT Rac/cdc42 GADS WASP ADAP Lck VAV SOS GRB2 SOS mTORC1 Cell Proliferation Survival IKKα IKKγ IKKβ IκB NF-κB • Effector Memory Phenotype• Decreased Persistance • Central Memory Phenotype• Enhanced Proliferaion Capacity• Enhanced Persistance Gene Expression Linked to T-Cell Effector Functions (T-Cell Activation, Proliferation, Chemokines, Cytokines, Cytotoxicity, Metabolic Alterations) NFAT NFAT ATF2 NF-κB NF-κB Intracellular Ca2+ Store IP3R PIP 2 IP 3 DAG RasGRP DAG Ca2+ Proteasomal Degradation MEKK1 NIK MKKs JNK p38 MAPK TRAF2 ASK1 PLCγ1 PIP 2 IP 3 DAG IκB Proteasomal Degradation CRACChannel Ca2+ Key CostimulatoryDomain 1 CostimulatoryDomain 2 ActivationDomain 191 scFv Targets: CD19 Diffuse Large B-Cell Lymphoma Acute Lymphoblastic LeukemiaBCMA Multiple MyelomaMSLN Ovarian CancerCD38 Multiple MyelomaEGFRvIII GlioblastomaSLAMF7 Multiple MyelomaIL13Rα2 GlioblastomaCD30 Hodgkins/Non-Hodgkins LymphomaCLEC12A Acute Myelogenous Leukemia rev. 12/05/19

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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.

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Created November 2019.

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