CARs are composed of four major domains: antigen binding, hinge, transmembrane and intracellular signaling. Each domain plays a role in recognizing the CAR target and relaying this signal to activate the T cell. Determining the optimal components to drive CAR-T cell function is a major area of CAR-T cell research. First generation CARs contained an extracellular antigen binding domain composed of the single- chain variable fragment (scFv) of an antibody sequence attached to a trans membrane domain and CD3ζ intracellular signaling domain from the endogenous T cell receptor (Fig. 1a, b). These CARs were designed so that when the scFv region binds its target antigen, CD3ζ transduces an activating signal for the T cell to expand, kill the target cell, and persist for long-term tumor control. However, clinical trials using first generation CARs showed little efficacy in patients due to limited expansion and persistence of the CAR-T cells.
Fig1. Schematic of CAR design by generation. (a) Canonical chimeric antigen receptor (CAR) constructs contain an extracellular binding domain derived from an antibody single-chain variable fragment (scFv) sequence, including variable light (VL) and variable heavy (VH) regions of the antigen binding fragment (Fab) of an antibody. Intracellular signaling domains are derived from sequences in the endogenous T cell receptor (TCR) and vary by generation (b). First generation CAR-T cells contain only CD3ζ sequence, however, resulting T cell expansion and persistence is inadequate for clinical use. Second generation constructs harbor an additional co-stimulatory sequence—typically either CD28 or 4-1BB—and have shown improved efficacy and persistence in humans. Third generation CAR-T cells feature multiple co-stimulatory domains. (c) The improved efficacy of second generation CARs led to the FDA approval of four CD19- targeted and one BCMA targeted engineered T cell therapies in 2017, including CD28-containing Axicabtagene ciloleucel and 4-1BB-containing Tisagenleleucel. Brexucabtagene autoleucel, approved for use in mantle cell lymphoma in 2020, uses the same CAR construct as axi-cel, but differs in the method of T cell isolation. Lisocabtagene maraleucel uses a 4-1BB costimulatory domain with a CD28 transmembrane domain and contains a 1:1 mixture of transduced CD4 and CD8 T cells. Idecabtagene vicleucel, approved this year for use in multiple myeloma, is another second generation CAR with an anti-BCMA scFv and a 4-1BB costimulatory domain
To improve their function, second generation CARs were developed with additional intracellular signaling domains aimed at boosting the activation signal. Second-generation CARs typically contain either a CD28 or 4-1BB co-stimulatory domain placed between the transmembrane domain and CD3ζ Fig. 1b. Either combination implemented in CD19-targeted CAR-T cells demonstrated anti-tumor efficacy in patients, with profound and durable responses achieved for relapsed or refractory B cell leukemia. These results led to the FDA approval of two second-generation CAR-T cells in 2017 (Fig. 1c). Axicabtagene ciloleucel (axi-cel, brand name Yescarta, manufactured by Kite Pharma Inc.) is composed of a CD19- specific scFv, CD28 hinge and transmembrane domain, CD28 costimulatory domain, and CD3ζ activation domain. Axi-cel is approved for relapsed or refractory large B-cell lymphoma, and more recently for relapsed or refractory follicular lymphoma. Tisagenleleucel (tisa-cel, brand name Kymriah, manufactured by Novartis) harbors the same scFv and activation domain but contains a CD8 hinge and transmembrane domain with a 4-1BB costimulatory domain. Tisa-cel is approved for pediatric and young adult relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) and adult relapsed or refractory large B-cell lymphoma.
Since then, three more second generation CAR-T cells have been approved by the FDA (Fig. 1c). In 2020 and 2021, two additional second-generation CD19- targeted CAR-T cells were approved by the FDA. Brexucabtagene autoleucel (brexu-cel, brand name Tecartus, manufactured by Kite Pharma Inc), approved for use in mantle cell lymphoma, is identical to the axi-cel CAR construct, but varies in the manufacturing process. During manufacturing, the T cells are isolated to ensure that no leukemia or lymphoma cells are included in the product. Lisocabtagene maraleucel (liso-cel, brand name Breyanzi, manufactured by Juno Therapeutics Inc./Bristol-Myers Squibb) harbors a similar 4-1BB costimulatory domain as tisa- cel, but also has a CD28 hinge and transmembrane domain. During the manufacturing process, genetically modified lymphocytes are allocated into a 1:1 mixture of CD4:CD8 T cells prior to infusion into the patient. Finally, in March of 2021, idecabtagene vicleucel (ide-cel, brand name Abecma, manufactured by Bristol- Myers Squibb) was approved by the FDA for treatment of relapsed or refractory multiple myeloma. Ide-cel is a second generation CAR with an anti-BCMA scFv, a 4-1BB costimulatory domain, and a CD8 ∝ hinge and transmembrane domain.
Third generation CARs contain two co-stimulatory domains adjacent to CD3ζ to further enhance the strength of the intracellular signal. Other costimulatory domains that have been tested in CAR-T cells include OX40, CD27, and inducible T cell co-stimulator (ICOS) (Fig. 2). The type of costimulatory domain included in the CAR renders different CAR-T cells with varying functional properties, which will be discussed later in this section.
Fig2. CAR domain components. Chimeric antigen receptor (CAR) proteins consist of an extra cellular antigen-binding domain, a hinge, a transmembrane domain, a co-stimulatory domain, and an activation domain. The extracellular portion of the CAR is most often composed of a single- chain variable fragment (scFv) molecule, though camelid nanobodies and humanized natural ligands or cytokines have been used. The intracellular regions, which function in activation upon antigen binding, typically harbor a T cell activation domain derived from the CD3ζ chain of the T cell receptor. Co-stimulatory domains often include CD28 or 4-1BB and can influence CAR-T cell memory, phenotype, and metabolism. Other co-stimulatory domains have been tested including OX40, CD27, and inducible T cell co-stimulator (ICOS). The activation domain, composed of CD3ζ, DAP12, or other sequences, contains immunoreceptor tyrosine-based activation motif (ITAM) regions that can be mutated to attenuate downstream activity
