Protein & Cell | 2019
Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects
Abstract
Genetic engineering of T cells to express chimeric antigen receptors (CARs) is an efficient approach for clinical therapy of hematological malignancies (Kuwana et al., 1987; Eshhar et al., 1993; Barrett et al., 2014). The CARs endow T cells with the ability to recognize specific antigens and bind them in an MHC-independent manner, thereby overcoming some of the mechanisms that mediate tumor immune escape. In addition, by providing co-stimulatory signals, CARs endow T cells with enhanced cytotoxicity and persistence compared with primary T cells. A typical CAR comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb) for antigen recognition and signaling domains for coactivation (Eshhar et al., 1993; Sadelain et al., 2013). To date, CAR-T cell therapy has been most effective in immunotherapy of CD19 B cell acute lymphoblastic leukemia, with a complete response in more than 75% of cases (Sadelain et al., 2013). However, there are still some challenges for CAR-T-mediated treatments. Side effects like offtargeting, cytokine release syndrome (CRS) and neuronal toxicities have been reported, and these may induce lethal responses (Morgan et al., 2010; Park et al., 2011). In addition, no response, incomplete tumor regression, and tumor recurrence were also observed after CAR-T treatment. For example, 10%–20% of patients were non-responsive to CD19 CAR-T clinical therapy (Lee et al., 2015; Park et al., 2018). Even in cases with a complete response, about 50% of them suffered tumor recurrence in one year, and one third of them had a CD19 relapse (Maude et al., 2018; Orlando et al., 2018). These disappointing results are associated with early CAR-T cell disappearance or poor cell function, which leads to incomplete tumor regression or loss of long-term antitumor effects. Cytokines are important factors for T cell development and homeostasis. In addition to the TCR and costimulatory receptors, cytokines provide stimulatory signals for full T cell activation, and have pleiotropic effects on T cell proliferation, differentiation and function. Currently, IL-2 is the main cytokine used to culture cells for adoptive cell therapy, as it plays an important role in the proliferation and functional effect of T cells. However, T cells cultured with IL-2 are phenotypically heterogeneous, being predominantly composed of effector memory cells which have sufficient functional effect but are sensitive to death. IL-7 has a critical role in the development and maturation of T cells. It promotes the generation of naïve and central memory T cell subsets and regulates their homeostasis. IL15 mediates the formation and homeostasis of CD8 memory T cells. It has been reported that IL-7 and IL-15 are able to instruct T cells toward memory stem-like phenotypes, which are less differentiated and have a superior capacity for expansion and survival (Cieri et al., 2013). Here, we systematically compared the effects of IL-7/IL-15 and IL-2 on the expansion, apoptosis and anti-tumor responses of CAR-T cells. We first constructed the anti-CD19 CAR (19BB-CAR) using an anti-CD19 mAb (clone FMC63)-derived scFv linked to the CD8α hinge and transmembrane regions, followed by a 4-1BB intracellular signaling domain and the CD3ζ signaling moiety. The 19BB-CAR and enhanced green fluorescent protein (eGFP) sequences were ligated and subcloned into the lentiviral vector FUW with a substitutive EF1α promoter (Fig. S1A). The cultured primary T cells were stimulated with anti-CD3/anti-CD28 Dynabeads and cytokine IL-2 before transduction with 19BB-CAR lentiviral particles. Using Protein L binding to the variable immunoglobulin light chains of the CAR, we found that CAR expression is directly correlated to eGFP expression (Fig. S1B). The CAR was highly expressed in IL-2-cultured T cells three days after infection (Fig. S1C). The CAR-T cells were expanded 100-fold in 2 weeks under IL-2 stimulation (Fig. S1D). To test the specificity of 19BB-CAR-T cells, we co-incubated them with two human leukemia cell lines, Raji (CD19) and K562 (CD19). The secretion of IL-2, IFN-γ and TNF-α by 19BB-CAR-T cells was significantly increased upon coincubation with CD19 Raji but not CD19 K562 cells (Fig. S1E). Accordingly, cytotoxicity assays showed that 19BB-CAR-T cells specifically lysed CD19 Raji but not CD19 K562 cells (Fig. S1F). These data suggest that 19BBCAR-T cells specifically recognize the CD19 molecule.