Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor

Abstract

Infusions of antigen-specific T cells have yielded therapeutic responses in patients with pathogens and tumors. To broaden the clinical application of adoptive immunotherapy against malignancies, investigators have developed robust systems for the genetic modification and characterization of T cells expressing introduced chimeric antigen receptors (CARs) to redirect specificity. Human trials are under way in patients with aggressive malignancies to test the hypothesis that manipulating the recipient and reprogramming T cells before adoptive transfer may improve their therapeutic effect. These examples of personalized medicine infuse T cells designed to meet patients' needs by redirecting their specificity to target molecular determinants on the underlying malignancy. The generation of clinical grade CAR(+) T cells is an example of bench-to-bedside translational science that has been accomplished using investigator-initiated trials operating largely without industry support. The next-generation trials will deliver designer T cells with improved homing, CAR-mediated signaling, and replicative potential, as investigators move from the bedside to the bench and back again.

Figure 1

Modular structure of prototypical CAR. CAR shown dimerized on the cell surface demonstrating the key extracellular (A-B) and intracellular (C-E) domains. CARs may express 1, 2, or 3 signaling motifs within an endodomain to achieve a CAR-dependent fully-competent T-cell activation signal. The modular structure of the CAR's domains, for example, the scFv (V_L linked to V_H) region (A) and the flexible hinge and spacer, for example, from IgG4 hinge, C_H2, and C_H3 regions (B), allow investigators to change specificity through swapping of exodomains and achieve altered function by varying transmembrane and intracellular signaling moieties (C-E).

Figure 2

Timeline for in vitro gene transfer and propagation of CAR⁺ T cells. The electrotransfer of transposon/transposase systems has narrowed the gap between nonviral and viral-based gene transfer for the amount of time in tissue culture needed to generate a clinically sufficient number of genetically modified CAR⁺ T cells. Cells transduced with virus (blue text) are typically propagated for 3 weeks before infusion.^, T cells that undergo nonviral gene transfer with the SB system (red text) can be typically harvested within 4 weeks.

Figure 3

Schematic of vector systems to express CAR transgenes used in clinical trials. (A) Two SB DNA plasmids expressing a (CAR) transposon and a hyperactive transposase (eg, SB11). Transposition occurs at a TA dinucleotide sequence when the transposase enzymatically acts on the internal repeat flanking the transposon. (B) A recombinant retroviral vector showing the long terminal repeats (LTR) containing the promoter flanking the CAR. SD and SA are the splice donor and splice acceptor sites, respectively, and ψ is the viral packaging signal. (C) A self-inactivating recombinant lentiviral vector construct containing the LTR, ψ, SD and SA sites, HIV Rev response element (RRE), HIV central polypurine tract (cPPT), CAR under control of an internal promoter, and the wood-chuck hepatitis virus posttranscriptional regulatory element (WPRE).