IL-24 armored CAR19-T cells show enhanced antitumor activity and persistence

Dear Editor, Nowadays, two autologous CAR19-T drugs, Tisagenlecleucel (KymriahTM) and axicabtagene ciloleucel (YescartaTM), have been approved for the treatment of B cell leukemia and lymphoma and achieved unprecedented successes. However, about 10–20% of BALL patients receiving CAR19-T drugs didn’t achieve complete remission (CR), while 30~50% of patients achieved CR would relapse mainly within 1 year. Moreover, the high CR rate of CAR19T therapy for B-ALL can’t be recaptured in other B-NHLs, such as Burkitt’s lymphoma (BL). Therefore, there is an urgent need to improve the therapeutic efficacy of CAR19-T cells. Cytokines play a fundamental role in modulating CAR-T cell functions. Based on our previous antitumor studies with Interleukin-24 (IL-24), we hypothesized that armoring CAR19-T cell with IL-24 might promote its functions. IL-24 is mainly expressed in the immune cells, acting as a potent and nearubiquitous cancer suppressor in various tumors, such as breast cancer, lung cancer, and lymphoma, etc. Moreover, IL-24 embodied anticancer activity and safety in patients with advanced cancer following direct injection with an adenovirus (Ad.mda-7; INGN-241) into tumors. However, either the effects of IL-24 on CAR19-T cells or the antitumor synergy of IL-24 and CAR19-T cells has not been tested. In this study, we confirmed that variable concentrations (0.78–200 ng/mL) of recombinant IL-24 protein (rIL-24) significantly inhibited the viability and proliferation of both B-ALL cell line Nalm6 and BL cell line Raji (Supplementary Fig. S1a~d). Moreover, Raji cells treated with rIL-24 showed slower growth in a long-term culture (Supplementary Fig. S1e), which might be due to cell cycle arrest and apoptosis (Supplementary Fig. S1f, g). Then we investigated the effects of IL-24 on human primary T cells, the most commonly used cell resource for CAR-T products. The vitality of T cells isolated from healthy donors by negative sorting was enhanced by approximately 20% at a wide range of rIL-24 treatment (0.78–200 ng/ml) (Supplementary Fig. S2a) without influencing the proliferation (Supplementary Fig. S2b, c). FMC63scFv-28ζ (CAR19), which is frequently used in CAR-T study, was selected as a comparison. Similarly, there was no adverse effects of rIL-24 on either the proliferation (Supplementary Fig. S2d, e) or the ratio and intensity of GFP (reflecting CAR19+) in CAR19-T cells (Supplementary Fig. S2f~h), suggesting the feasibility of armoring IL-24 to CAR19-T cells. Subsequently, a modified IL-24 cDNA was linked to CAR19 leading to a novel CAR19-IL-24 (Fig. 1a), which was packaged into a lentiviral vector as CAR19. CAR19-IL-24-T cells were prepared under the same process as CAR19-T cells (Supplementary Fig. S3a). Compared to CAR19-T cells, CAR19-IL-24-T cells showed similar expression of CAR19 (Supplementary Fig. S3b) and co-expression pattern of CAR19 and GFP (Fig. 1b), whereas expressed higher level of IL-24 (Supplementary Fig. S3b, c). Besides, the distribution of Th (CD4+) and Tc (CD8+) (Supplementary Fig. S3d, e), GFP+% in the Th and Tc (Supplementary Fig. S3f, g) and the expression of exhaustion-related markers (Supplementary Fig. S3h, i) in CAR19IL-24-T cells were similar to those in CAR19-T cells. Intriguingly, CAR19-IL-24-T cells possessed more Tn (50% > 30%) and less Teff (30% < 40%) than CAR19-T cells (Supplementary Fig. S3j, k). Collectively, CAR19-IL-24-T cells elevated the expression of IL-24 and tended to be more naive. To explore whether CAR19-IL-24-T cells enhanced antitumor activity, we tested their short-term cytotoxicity using LDH release experiments. As expected, CAR19-IL-24-T cells showed higher cytotoxicity to Nalm6, Raji, and K562-CD19 cells than CAR19-T cells in a dose-dependent and CD19-specific manner (Supplementary Fig. S4a and Fig. 1c). Furthermore, we evaluated their long-term cytotoxicity using a new Flow-Cytometry-based-Cytotoxicity-Assay (Supplementary Fig. S4b), in which tumor cells were respectively cocultured with NT cells, CAR19-T cells and CAR19-IL-24-T cells for 7 days. CAR19-IL-24-T cells induced apoptosis of Nalm6 cells and Raji cells similar to CAR19-T cells, while triggering a higher apoptosis in K562-CD19 cells (Supplementary Fig. S4c). Since the initial proportion of tumor cells among cocultured mixtures were almost the same, tumor cells cocultured with CAR19-IL-24-T cells showed minimal residue (Supplementary Fig. S4d and Fig. 1d), indicating that CAR19-IL-24-T cells showed stronger competence in eliminating tumor cells. Notably, only CAR19-IL-24-T cells could effectively lower the ratio of K562-CD19 cells (Fig. 1d). Contrasting to Nalm6 cells, Raji cells were relatively insensitive to CAR19-T cells (Fig. 1d), suggesting that BL was more challenging than B-ALL for CAR19-T therapy. The expansion and survivability of CAR-T cells affect the outcomes of CAR-T therapy. We noticed that CAR19-IL-24-T cells expanded faster than CAR19-T cells (Supplementary Fig. S4e). To determine the enhanced proliferation in responding to tumor, CAR19-IL-24-T cells were cocultured with Nalm6 cells, Raji cells, and K562-CD19 cells, respectively. The GFP+% in the CAR19-IL-24T group gradually surpassed that in CAR19-T group (Fig. 1e). Generally, the proliferation of CAR19-IL-24-T cells was more robust (Supplementary Fig. S4f). In short, CAR19-IL-24-T cells improved proliferation irrespective of tumor stimulus, implying a better persistence in vivo. We further investigated the apoptotic fate of CAR19-IL-24-T cells. Both NT cells and CAR19-T cells were high apoptotic after cocultured with tumor cells, while the apoptotic rate of CAR19-IL-24-T cells was significantly lower (Fig. 1f and Supplementary S4g, h), indicating that CAR19-IL-24-T cells were more anti-apoptotic, which would contribute to their survival and antitumor potential under the pressure of tumor cells. Clinical data showed that IL-24 was downregulated in tumor tissues and low IL-24 expression was identified as a predictor of poor prognosis in BL patients. In our study, we observed that CAR19-IL-24-T cells secreted IL-24 eight times higher than CAR19-T cells (Fig. 1g). The safety concern that CAR19-IL-24-T cells would elevate the level of IL-24 in the entire body and then evoke


Dear Editor,
Nowadays, two autologous CAR19-T drugs, Tisagenlecleucel (Kymriah™) and axicabtagene ciloleucel (Yescarta™), have been approved for the treatment of B cell leukemia and lymphoma and achieved unprecedented successes. However, about 10-20% of B-ALL patients receiving CAR19-T drugs didn't achieve complete remission (CR), while 30~50% of patients achieved CR would relapse mainly within 1 year. Moreover, the high CR rate of CAR19-T therapy for B-ALL can't be recaptured in other B-NHLs, 1 such as Burkitt's lymphoma (BL). 2 Therefore, there is an urgent need to improve the therapeutic efficacy of CAR19-T cells. 3 Cytokines play a fundamental role in modulating CAR-T cell functions. Based on our previous antitumor studies with Interleukin-24 (IL-24), we hypothesized that armoring CAR19-T cell with IL-24 might promote its functions. IL-24 is mainly expressed in the immune cells, acting as a potent and nearubiquitous cancer suppressor in various tumors, such as breast cancer, lung cancer, and lymphoma, etc. Moreover, IL-24 embodied anticancer activity and safety in patients with advanced cancer following direct injection with an adenovirus (Ad.mda-7; INGN-241) into tumors. However, either the effects of IL-24 on CAR19-T cells or the antitumor synergy of IL-24 and CAR19-T cells has not been tested.
In this study, we confirmed that variable concentrations (0.78-200 ng/mL) of recombinant IL-24 protein (rIL-24) significantly inhibited the viability and proliferation of both B-ALL cell line Nalm6 and BL cell line Raji ( Supplementary Fig. S1a~d). Moreover, Raji cells treated with rIL-24 showed slower growth in a long-term culture ( Supplementary Fig. S1e), which might be due to cell cycle arrest and apoptosis ( Supplementary Fig. S1f, g). Then we investigated the effects of IL-24 on human primary T cells, the most commonly used cell resource for CAR-T products. The vitality of T cells isolated from healthy donors by negative sorting was enhanced by approximately 20% at a wide range of rIL-24 treatment (0.78-200 ng/ml) ( Supplementary Fig. S2a) without influencing the proliferation ( Supplementary Fig. S2b, c). FMC63scFv-28ζ (CAR19), which is frequently used in CAR-T study, was selected as a comparison. Similarly, there was no adverse effects of rIL-24 on either the proliferation ( Supplementary Fig.  S2d, e) or the ratio and intensity of GFP (reflecting CAR19+) in CAR19-T cells ( Supplementary Fig. S2f~h), suggesting the feasibility of armoring IL-24 to CAR19-T cells.
To explore whether CAR19-IL-24-T cells enhanced antitumor activity, we tested their short-term cytotoxicity using LDH release experiments. As expected, CAR19-IL-24-T cells showed higher cytotoxicity to Nalm6, Raji, and K562-CD19 cells than CAR19-T cells in a dose-dependent and CD19-specific manner (Supplementary Fig. S4a and Fig. 1c). Furthermore, we evaluated their long-term cytotoxicity using a new Flow-Cytometry-based-Cytotoxicity-Assay ( Supplementary Fig. S4b Fig. S4c). Since the initial proportion of tumor cells among cocultured mixtures were almost the same, tumor cells cocultured with CAR19-IL-24-T cells showed minimal residue (Supplementary Fig. S4d and Fig. 1d), indicating that CAR19-IL-24-T cells showed stronger competence in eliminating tumor cells. Notably, only CAR19-IL-24-T cells could effectively lower the ratio of K562-CD19 cells (Fig. 1d). Contrasting to Nalm6 cells, Raji cells were relatively insensitive to CAR19-T cells (Fig. 1d), suggesting that BL was more challenging than B-ALL for CAR19-T therapy.

), in which tumor cells were respectively cocultured with NT cells, CAR19-T cells and CAR19-IL-24-T cells for 7 days. CAR19-IL-24-T cells induced apoptosis of Nalm6 cells and Raji cells similar to CAR19-T cells, while triggering a higher apoptosis in K562-CD19 cells (Supplementary
The expansion and survivability of CAR-T cells affect the outcomes of CAR-T therapy. We noticed that CAR19-IL-24-T cells expanded faster than CAR19-T cells ( Supplementary Fig. S4e). To determine the enhanced proliferation in responding to tumor, CAR19-IL-24-T cells were cocultured with Nalm6 cells, Raji cells, and K562-CD19 cells, respectively. The GFP+% in the CAR19-IL-24-T group gradually surpassed that in CAR19-T group (Fig. 1e). Generally, the proliferation of CAR19-IL-24-T cells was more robust (Supplementary Fig. S4f). In short, CAR19-IL-24-T cells improved proliferation irrespective of tumor stimulus, implying a better persistence in vivo. We further investigated the apoptotic fate of CAR19-IL-24-T cells. Both NT cells and CAR19-T cells were high apoptotic after cocultured with tumor cells, while the apoptotic rate of CAR19-IL-24-T cells was significantly lower (Fig. 1f and Supplementary S4g, h), indicating that CAR19-IL-24-T cells were more anti-apoptotic, which would contribute to their survival and antitumor potential under the pressure of tumor cells.
Clinical data showed that IL-24 was downregulated in tumor tissues and low IL-24 expression was identified as a predictor of poor prognosis in BL patients. 4 In our study, we observed that CAR19-IL-24-T cells secreted IL-24 eight times higher than CAR19-T cells (Fig. 1g). The safety concern that CAR19-IL-24-T cells would elevate the level of IL-24 in the entire body and then evoke unexpected side effects could be addressed by using adjustable expression strategies. Knowing that IL-24 is a multidimensional anti-cancer therapeutic, such as transforming the TME by promoting T cells infiltration, IL-24 secreted by CAR19-IL-24-T cells would affect both tumor cells and T cells, which in turn enhance the antitumor ability of CAR19-IL-24-T cells. Therefore, targeted delivery of IL-24 by CAR19-IL-24-T cells would be more precise and effective compared to systemic or local administration of rIL-24. Moreover, CAR19-IL-24-T cells produced higher levels of both IL-2 and TNF-α and lower level of IFN-γ compared to CAR19-T cells (Fig. 1h~j). A lower level of IFN-γ might be safer according to previous reports. 5  -K562-CD19  CAR19-T-K562-CD19  CAR19-IL-24-T-K562-CD19  NT-K562  CAR19-T-K562  CAR19-IL-24-T We preliminarily evaluated the short-term efficacy and safety of CAR19-IL-24-T cells in vivo (Supplementary Fig. S5a). CAR19-IL-24-T cells reduced BL tumor volumes in M-NSG mice as effectively as CAR19-T cells (Fig. 1k). Besides, no obviously unfavorable effects on the body weight were observed after a large dose of CAR19-IL-24-T cells treatment (Supplementary Fig. S5b). We analyzed the embedded T cells in the blood of xenograft mice ( Supplementary  Fig. S5c), finding a higher T cell population and more GFP+T cells in the CAR19-IL-24-T treatment group compared to CAR19-T treatment group although there was no statistical significance ( Supplementary  Fig. S5d~f). We assumed that IL-24 incurred profound impacts on CAR19-IL-24-T cells by providing with a unique expression profile of key regulators that involved in autophagy and RNA epigenetic modification ( Supplementary Fig. S5g). Next, we will employ the xenograft model to explore the antitumor mechanisms of CAR19-IL-24-T cells and evaluate the potential side effects. It is also needed to understand the limitations and meet the challenges for extending this approach to other tumors.
In summary, we provided a successful proof of concept for IL-24 armored CAR19-T cells to enhance antitumor efficiency, while improving proliferation and persistence with anti-apoptotic properties. Moreover, CAR19-IL-24-T cells secreted excess IL-24 as a powerful weapon to antitumor synergistically. In brief, CAR19-IL-24-T cells exhibit potential in clinical application, suggesting a new path forward to the improvement of CAR-T therapy. Fig. 1 Enhanced antitumor activity and persistence of CAR19-IL-24-T cells. a Schematic diagram of FMC63scFv-28ζ (CAR19) and CAR19-IL-24. SP, signal peptide; 2A, 2A "self-cleaving" peptide; EGFP, enhanced green fluorescent protein. b Representative flow cytometry data showed coexpression of CAR19 and EGFP in CAR19-T cells and CAR19-IL-24-T cells at days 10 post-transduction. CAR-T cells were confirmed by GFP and labeling with Alexa Fluor 647-AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG (H+L) that recognizes scFv portion of the CAR19. c The cytotoxicity of T cells to target cells was measured with CytoTox 96® Non-Radioactive Cytotoxicity Assay. T cells were cocultured with K562 cells (CD19−) or K562-CD19 cells (CD19+) for 18 h at the indicated ratios. d Quantification and statistical analysis of residue Nalm6 cells, Raji cells, and K562-CD19 cells ratio in living cells subset during coculture. e Changes in GFP+ ratio of CAR19-T cells and CAR19-IL-24-T cells after cocultured with tumor Nalm6 cells, Raji cells and K562-CD19 cells at ratio = 1:1, respectively. f Quantification and statistical analysis of the apoptosis of GFP+ T cells in CAR19-T group and CAR19-IL-24-T group after challenged with Nalm6 cells, Raji cells, and K562-CD19 cells, respectively. g-j T cells were cocultured with tumor cells at ratio 1:1 for 24 h, without exogenous rIL-2. The supernatant was collected for ELISA assay of IL-24 (g), IL-2 (h), TNF-α (i) and IFN-γ (j) according to the manufacturer's suggestions. k Analysis of Burkitt lymphoma tumor volumes. NSG mice were inoculated with Raji tumor cells (10 6 cells/mouse) at day 0, then treated with NT cells, CAR19-T cells and CAR19-IL-24-T cells (normalized to 5% CAR19+, 3 × 10 7 cells/mouse, n = 3/group) at day 8, respectively. All data with error bars were analyzed with GraphPad Prism8 and presented as mean ± SEM. e, f Unpaired and non-parametric Mann-Whitney test with two-tailed, (c, d, g-k) One-way analysis of variance with Bonferroni correction. ns not significant; p > 0.05; *p < 0.05, **p < 0.01, ***p < 0.001