Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects

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.


Dear Editor,
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. IL-15 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 evaluate the anti-tumor effects of 19BB-CAR-T cells cultured using IL-2 in vivo, CD19 + Raji cells labeled with fluorescent luciferase fusion protein were engrafted to immunodeficient mice for lymphoma formation. Then human T cells transduced with 19BB-CAR or GFP vectors were infused into the mice (Fig. S1G). Mice receiving 19BB-CAR-T cells showed effective tumor regression, while mice infused with T cells harboring empty vector had progressive tumor growth ( Fig. S1G-H). Long-term monitoring showed that mice infused with 19BB-CAR-T cells had a significantly higher survival rate and longer survival period compared with mice receiving empty-vector T cells (Fig. S1I). These data provide evidence that 19BB-CAR-T cells can effectively remove tumor cells in vivo. However, lymphoma recurrence was observed in some mice treated with 19BB-CAR-T cells, and nearly half of the 19BB-CAR-T mice died within 60 days of infusion due to the tumor burden. These phenotypes are consistent with clinical data, which calls for optimization of CAR-T cells for more efficient tumor killing.
Proliferation and apoptosis are two major aspects to be considered for in vitro expansion of CAR-T cells. In the twoweek in vitro culture assays, we found that the 19BB-CAR-T cells were more efficiently expanded with IL-7/IL-15 than with IL-2 ( Fig. 1A). Satisfactorily, there were no differences in the CAR transduction efficiency of T cells cultured in IL-2 or IL-7/IL-15 (Fig. S2). 19BB-CAR-T cells cultured with IL-7/IL-15 showed higher proliferation and a lower apoptosis rate compared to cells cultured with IL-2 ( Fig. 1B and 1C). Consistent with this phenotype, the expression of the antiapoptosis protein BCL-2 is higher in 19BB-CAR-T cells cultured with IL-7/IL-15 than with IL-2 ( Fig. 1D). Together, these data suggest that IL-7/IL-15 provide a better environment than IL-2 for CAR-T cell expansion.
We next investigated the functional properties of CAR-T cells expanded in IL-7/IL-15 or IL-2. The results showed no significant difference in immune cytokine release (IL-2, IFNγ, TNF-α) or specific lysis ( Fig. 1E and 1F). We also investigated the cytokine secretion and cytotoxicity of 19BB-CAR-T cells after serial antigen stimulation to mimic tumor encounter in vivo, and found no significant differences between the two culture systems (Fig. S3A-C).
We then infused the IL-7/IL-15-or IL-2-expanded 19BB-CAR-T cells into mice with lymphoma for detection of tumor suppression effects. The antitumor effects were similar in the first 3 weeks. However, the 19BB-CAR-T cells expanded in IL-7/IL-15 showed superior anti-tumor activity, and the longterm survival of the tumor burden mice was significantly improved (Fig. 1G-I).
Chemokine receptor CCR7 is involved in lymph-node homing of T N and T CM cells, as well as lymph-node migration of dendritic cells. The expression levels of chemokine receptors CCR7 and CXCR4 are higher in 19BB-CAR-T cells expanded in IL-7/IL-15 than in IL-2 (Figs. 2D and S4). Accordingly, in a gradient of chemokine CCL21, 19BB-CAR-T cells cultured in IL-7/IL-15 showed enhanced migration ability compared to cells cultured in IL-2 (Fig. 2E).
Regulatory T (Treg) cells play an important role in immunosuppression. We found that IL-2 mediated a smaller increase of the CD4 + Foxp3 + 19BB-CAR-T cell population  (Fig. 2F).
In addition, we found decreased expression of PD-1 in 19BB-CAR-T cells cultured in IL-7/IL-15 compared to IL-2 upon serial antigen stimulation by Raji cells, which indicates that IL-7/IL-15 decrease CAR-T cell exhaustion (Fig. 2G). These data provide supporting evidence that IL-7/IL-15 induce a superior anti-tumor activity in 19BB-CAR-T cells compared to IL-2. We further evaluated the survival and memory T cell phenotype of 19BB-CAR-T cells cultured with different cytokines after infusion into tumor-bearing mice. 19BB-CAR-T cells cultured with IL-7/IL-15 showed enhanced engraftment in mice compared to cells cultured with IL-2 ( Fig. 2H-J). After infusion into mice, 19BB-CAR-T cells cultured in IL-7/IL-15 generated more CD8 + central memory T cells than CAR-T cells treated with IL-2, while cells cultured in IL-2 generated more CD8 + effector memory T cells than cells cultured in IL-7/IL-15 (Fig. 2K). This is consistent with the in vitro results. These data provide evidence that CAR-T cells cultured in IL-7/IL-15 have superior anti-tumor activity in vivo.
Generating optimized CAR-T cells in vitro is an important strategy to enhance the clinical efficacy of CAR-T cells in cancer immunotherapy. Recently, Xu et al. reported that IL-7/ IL-15 are better than IL-2 for preserving the CD8 + CD45RA + CCR7 + population in ex vivo-cultured CAR-T cells, and endow the CAR-T cells with superior proliferation and survival capability upon serial antigen stimulation (Xu et al., 2014). Another study showed that IL-7/IL-15 instruct the expansion of CD62L + CD45RA + memory T cells from naïve precursors (Cieri et al., 2013). In contrast, we found that IL-7/IL-15 promotes CAR-T cell proliferation directly without antigen stimulation in vitro, and the level of apoptosis is low. CAR-T cells cultured with IL-7/IL-15 expanded around 2-fold more within two weeks than cells cultured with IL-2, which will favor the generation of CAR-T cells for certain patients whose lymphocytes have limited expansion ability.
In conclusion, we systematically compared the effects of IL-7/IL-15 and IL-2 on CAR-T cell culture, and demonstrated that CAR-T cells expanded in the presence of IL-7/IL-15 showed enhanced proliferation and superior antitumor activity. IL-7/IL-15 selectively expanded naïve and central memory T cells, which help CAR-T cell engraftment in tumorbearing mice. Apart from IL-2, IL-7 and IL-15, many other cytokines are important for T cell development, differentiation and function. IL-12 is involved in the differentiation of naïve Th0 cells into Th1 cells, and augments the activity of cytotoxic T cells. IL-18 regulates the immune response by enhancing the secretion of IFN-γ and augmenting cytolytic

cells compared to cells cultured with IL-2. Bars show the distribution of CD4 + and CD8 + T cells in 19BB-CAR-T cells cultured
with IL-7/IL-15 or IL-2 at day 3 (D3) and day 11 (D11). Data are presented as mean ± SEM from 4 independent experiments. (B) 19BB-CAR-T cells cultured with IL-7/IL-15 generate a higher percentage of CD8 + naïve cells (T N ) compared to cells cultured with IL-2. Expression of CD45RA and CD62L was assessed by flow cytometry analysis of 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 at day 5 (D5). The percentages of T N (CD45RA + CD62L + ), T CM (CD45RA − CD62L + ), T EM (CD45RA − CD62L − ), and T RAEM (CD45RA + CD62L − ) in CD8 + lymphocytes (left) and CD4 + lymphocytes (right) are shown. Results are presented as mean ± SEM from 4 independent experiments, *P < 0.05. (C) 19BB-CAR-T cells expanded with IL-7/IL-15 generate a larger population of central memory T cells during culture. The percentages of T N (CD45RA + CD62L + ), T CM (CD45RA − CD62L + ), T EM (CD45RA − CD62L − ), and T RAEM (CD45RA + CD62L − ) in CD8 + lymphocytes (left) and CD4 + lymphocytes (right) in 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 at day 11 are shown. Data are presented as mean ± SEM from 4 independent experiments, *P < 0.05. (D) IL-7/IL-15 enhance CCR7 expression compared to IL-2. The expression of CCR7 on 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 at day 11 was detected by flow cytometry. Results are presented as mean ± SEM from 4 independent experiments, *P < 0.05. (E) 19BB-CAR-T cells cultured with IL-7/IL-15 show higher migration ability compared to cells cultured with IL-2. Results are presented as mean ± SEM from 3 independent experiments, **P < 0.01. (F) IL-7/IL-15 decreases the Foxp3 + CD4 + T cell population. The expression of Foxp3 in 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 at day 11 was detected by flow cytometry. Results are shown as mean ± SEM from 4 independent experiments, *P < 0.05. (G) The percentage of 19BB-CAR-T cells expressing the inhibitory receptor PD-1 is lower after culture with IL-7/IL-15 than with IL-2. The expression of PD-1, LAG-3 and TIM-3 on 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 was determined by flow cytometry. Results are presented as mean ± SEM from 3 independent experiments, *P < 0.05. (H) IL-7/IL-15 increase the survival rate of 19BB-CAR-T cells in peripheral blood of tumor-bearing mice. CD3 + GFP + cells were detected in peripheral blood by flow cytometry in lymphoma-bearing mice at days 14 and 21 after infusion. Results are presented as mean ± SEM from 5 independent experiments, *P < 0.05. (I) IL-7/IL-15 enhance survival of 19BB-CAR-T cells in spleen of tumor-bearing mice after infusion. CD3 + GFP + cells were detected by flow cytometry in the spleen of lymphoma-bearing mice at day 21 after infusion. Results are shown as mean ± SEM from 5 independent experiments, *P < 0.05. (J) Copy numbers of 19BB-CAR vector per microgram genomic DNA in the peripheral blood of mice receiving 19BB-CAR-T cells at day 60 after infusion. Results are shown as mean ± SEM from 4 independent experiments, *P < 0.05. (K) IL-7/IL-15 maintain the CD8 + T CM (CD45RA -CD62L + ) population in 19BB-CAR-T cells from peripheral blood in tumor-bearing mice. Flow cytometry results are presented as mean ± SEM from 4 independent experiments, *P < 0.05. b activity. These cytokines could be potentially investigated for optimization of CAR-T expansion.