Switch receptor T3/28 improves long-term persistence and antitumor efficacy of CAR-T cells

Abstract

Background: Chimeric antigen receptor (CAR) T cells have been successfully used in tumor immunotherapy due to their strong antitumor responses, especially in hematological malignancies such as B cell acute lymphoid leukemia. However, on-target off-tumor toxicity and poor persistence severely limit the clinical application of CAR-T cell therapy.

Methods: T-cell immunoglobulin mucin domain molecule 3 (TIM-3) was used to develop a second-generation 41BB CD19 CAR linked with a T3/28 chimera, in which truncated extracellular TIM-3 was fused with the CD28 transmembrane and cytoplasmic domains. The efficacy of T3/28 CAR-T cells was evaluated in vitro and in vivo.

Results: We demonstrated that the switch receptor T3/28 preserved the T_CM phenotype, improved proliferative capacity, and reduced exhaustion of CAR-T cells, resulting in superior in vitro and in vivo antitumor activity in B lymphoma. Importantly, the switch receptor T3/28 substantially prolonged the persistence of CAR-T cells, and the interleukin-21/Stat3 axis probably contributed to the enhanced cytotoxicity of T3/28 CAR-T cells.

Conclusion: Overall, the T3/28 chimera significantly prolonged the persistence of CAR-T cells, and T3/28 CAR-T cells possessed potent antitumor activity in mice, shedding new light on potential improvements in adoptive T cell therapies.

Keywords: immunity; immunotherapy.

Figure 1

The cytotoxicity and cytokine secretion of T3/28 CAR-T cell are enhanced. (A) The cytotoxicity of transfected CAR-T or control cells to B lymphoma cell lines including Daudi, Raji, and Namalwa was evaluated. Effector cells and target cells (1×10⁵) were co-cultured for 18 hours at E:T ratios of 0.25:1, 0.5:1, 1:1 and 2:1. Then we analyzed the cytotoxicity of UNT, 19BBz and T3/28 CAR-T cells by LDH release assay. IFN-γ and granzyme B released by T cells co-cultured with Daudi (B), Raji (C) and Namalwa (D) were detected using ELISA kit. (E) The CD107a expression of UNT, 19BBz and T3/28 CAR-T cells co-cultured with Daudi cells was analyzed using flow cytometry. (F) The K562 cells and K562 cells overexpressing CD19 (K562-CD19) were co-cultured with UNT, 19BBz, or T3/28 CAR-T cells for 18 hours followed by LDH release assay. Data presented are the mean±SD of three separate experiments. ns means no significant difference, *p＜0.05, **p＜0.01, ***p＜0.001 compared with indicated group. CAR, chimeric antigen receptor; E:T, effector-to-target; IFN, interferon; LDH, lactate dehydrogenase; UNT, untreated T cells.

Figure 2

Switch receptor T3/28 enhances the cytotoxicity of 19BBz CAR-T cells depending on TIM-3 ligands. (A) The cytotoxicity of CAR-T cells against Raji-CEACAM1 and Namalwa-CEACAM1 cells was evaluated. (B) CAR-T cells and target cells (1×10⁵) were co-cultured for 18 hours at E:T ratios of 0.5:1 and 1:1. IFN-γ and granzyme B released by CAR-T cells co-cultured with Raji-CEACAM1 and Namalwa-CEACAM1 were detected using ELISA kit. (C) Flow cytometry was performed to detect PtdSer. The tumor cells were blocked by different amount of TIM-3 Fc chimera at 0, 1, and 5 μg mL⁻¹ for 1 hour at RT, and then co-incubated with Annexin V. (D) The cytotoxicity of 19BBz and T3/28 CAR-T cells against tumor cells. Target cells and CAR-T cells were co-incubated with different amounts of TIM-3 Fc chimera for 1 hour; subsequently, the tumor cells were co-cultured with CAR-T cells for 8 hours at E:T ratio of 1:1. Data presented are the mean±SD of three separate experiments. ns means no significant difference, *p＜0.05, **p＜0.01, ***p＜0.001 compared with indicated group. CAR, chimeric antigen receptor; E:T, effector-to-target; IFN, interferon; PtdSer, phosphatidylserine; RT, room temperature; TIM-3: T-cell immunoglobulin mucin domain molecule 3.

Figure 3

T3/28 CAR-T cells expanded ex vivo and maintain a superior phenotype. Flow cytometry was performed to detect expression of activation markers CD69 (A) and CD127 (B) and low-differentiation associated costimulatory molecules CD27 (C) and CD28 (D) on T cells stimulated with tumor cells. T3/28 CAR-T cells, 19BBz CAR-T cells, and UNT cells were expanded until day 18, and activation and differentiation markers were evaluated at indicated time points. (E) The T_CM compartment, with a CD45RO⁺CD62L⁺ phenotype, was assayed using flow cytometry. (F) CD4:CD8 ratio of CAR-T cells stimulated with tumor cells at day 9 post transduction was evaluated using flow cytometry. (G) Quantitative PCR was used to analyze memory stem-like-associated transcription factors TCF7 and LEF1 in CAR-T and control cells. Data presented are the mean±SD of three separate experiments. ns means no significant difference, *p＜0.05, **p＜0.01, ***p＜0.001 compared with indicated group. CAR, chimeric antigen receptor; mRNA, messenger RNA; T_CM, central memory T; T_E, effector T; T_EM, effector memory T; T_N, naive T; UNT, untreated T cells.

Figure 4

T3/28 CD19 CAR-T cells effectively eliminate clinically derived B lymphoma cells. (A) Clinically derived samples highly expressing CD19 antigen from several patients. (B) B lymphoma cells were mixed with effector cells followed by staining with anti-CD3 and anti-CD19 antibodies, and 12 hours later detection was performed by a flow cytometer. (C) B lymphoma cells (1×10⁵) were prepared in cell culture medium, then B lymphoma cells were co-incubated with CAR-T cells for 12 hours at E:T ratio of 1:1 in a total volume of 500 µL. The cytotoxicity of UNT, 19BBz, and T3/28 CAR-T cells was evaluated by LDH release assay. (D) IFN-γ and granzyme B released by T cells co-cultured with B lymphoma cells were assayed using ELISA kit. Data presented are the mean±SD of three separate experiments. **p＜0.01, ***p＜0.001 compared with indicated group at the same E:T ratio. CAR, chimeric antigen receptor; E:T, effector-to-target; IFN, interferon; LDH, lactate dehydrogenase; UNT, untreated T cells.

Figure 5

Switch receptor T3/28 mediates superior antitumor cytotoxicity in vivo. (A) The scheme of experimental design. (B) Body weights of the mice were measured two to three times per week. The values are presented as the mean±SE of the mean. (C) Survival was evaluated from the first day of tumor cell injection until death. Statistical analysis was performed using the log-rank (Mantel-Cox) text. **p＜0.01, ***p＜0.001 compared with indicated group. (D) Tumor burden was determined by weekly bioluminescent imaging (BLI) (n=5 mice per group). The T cells (E) and Daudi-luc cells (F) from visceral organs (BM (bone marrow), liver, spleen, and kidney were analyzed on a flow cytometer. (G) Serum levels of granzyme B and IFN-γ was determined using ELISA kit. Data presented are the mean±SD of three separate experiments. ns means no significant difference, *p＜0.05, **p＜0.01, ***p＜0.001 compared with indicated group. IFN, interferon; PBS, phosphatic buffer solution.

Figure 6

T3/28 CAR-T cells possess more potent persistence ability in vivo. (A) The scheme of experimental design. (B) Tumor burden was determined by bioluminescent imaging (n=5 mice per group). (C) Survival was evaluated from the first day of tumor injection until death. Statistical analysis was performed using the log-rank (Mantel-Cox) text. (D) The UNT and CAR-T cells from blood were monitored every 5 days. (E) The change of CD4: CD8 ratio was monitored every 5 days. Quantitative PCR analysis of exhaustion associated transcription factors (Blimp-1 and T-bet) (F) and memory stem-like-associated transcription factors (LEF1 and TCF-7) (G) on 19BBz and T3/28 CAR-T cells was performed. Data presented are the mean±SD of three separate experiments. ns means no significant difference, *p＜0.05, **p＜0.01, ***p＜0.001 compared with indicated group. CAR, chimeric antigen receptor; i.v., intravenously; IVIS, In Vivo Imaging System.

Figure 7

IL-21/Stat3 axis contributes to enhanced cytotoxicity of T3/28 CAR-T cells. (A) At 24 hours and 48 hours of co-culture at lower E:T ratios of 1:20 and 1:10, the cytotoxicity of CAR-T cells with or without IL-21 siRNA targeting B lymphoma cell lines (Raji and Namalwa) was analyzed using LDH assay kit. (B) After co-culture for 24 hours and 48 hours, the supernatants were harvested and an assay was performed to measure IFN-γ and granzyme B release by T cells with or without IL-21 siRNA co-cultured with Raji and Namalwa cells using ELISA kit. (C) After antigen stimulation, downstream signaling of IL-21 was altered as determined by measuring Stat3/5 phosphorylation using western blot. (D) After antigen stimulation, changes in T_CM subpopulations and CD25 expression on UNT, 19BBz and T3/28 CAR-T cells with or without IL-21 siRNA were analyzed. (E) The proliferation ability of T3/28 CAR-T cells was obviously affected with IL-21 knockdown. The statistical difference is presented at 78 hours only. (F) At 24 hours and 48 hours of co-culture at lower E:T ratios of 1:20 and 1:10, the cytotoxicity of CAR-T cells, with or without Stat3 inhibitor (static), targeting B lymphoma cell lines (Raji and Namalwa) was analyzed using LDH assay kit. Data presented are the mean±SD of three separate experiments. ns means no significant difference, *p＜0.05, **p＜0.01, ***p＜0.001 compared with indicated group. CAR, chimeric antigen receptor; E:T, effector-to-target; IFN, interferon; IL, interleukin; LDH, lactate dehydrogenase; OD, optical density; siRNA, small interferring RNA; T_CM, central memory T; UNT, untreated T cells.