Interleukin-7 expression by CAR-T cells improves CAR-T cell survival and efficacy in chordoma

Abstract

Chordoma is a rare bone tumor that frequently recurs after surgery, and the prognosis is poor with current treatments. This study aimed to identify potential novel immunotherapeutic targets for chordomas by identifying target proteins in clinical samples as well as tumor microenvironmental factors to enhance efficacy. Fourteen chordoma samples were analyzed by single-cell RNA sequencing, and B7-H3 and IL-7 were identified as potential targets and potentiators, respectively. B7-H3-targeted chimeric antigen receptor T (CAR-T) cells and B7-H3 CAR-T cells expressing IL-7 were synthesized and their anti-tumor activity evaluated in vitro, including in primary chordoma organoid models. The B7-H3 CAR-T/IL-7 therapy showed enhanced cytotoxicity and prolonged duration of action against tumor cells. Additionally, IL-7 modulated favorable subpopulations of cultured CAR-T cells, diminished immune checkpoint expression on T-cell surfaces, and enhanced T-cell functionality. The incorporation of IL-7 molecules into the B7-H3 CAR structure augmented CAR-T-cell function and improved CAR-T-cell efficacy, thus providing a novel dual therapeutic strategy for chordoma treatment.

Keywords: B7–H3; Chimeric antigen receptor T cells; Chordoma; Interleukin-7.

Fig. 1

Single-cell transcriptomic analysis of chordoma and control samples. A–C UMAP clustering, grouping, and cell type identification for chordoma and NP cells. D–E Expression of TBXT and CD276 was predominantly seen in tumor cells. F–G Expression of IL7 was seen in tumor cells, while expression of IL7R was seen in T cells; H Violin plot of TBXT and CD276 expression in chordoma and NP cells

Fig. 2

Single-cell transcriptomic analysis of chordoma and control samples. A Schematic of the construction of B7–H3-specific CARs. B Representative flow cytometry analysis showing CAR expression on T cells transduced with B7–H3 or B7–H3/IL-7 on day 7. C CAR expression on T cells transduced with B7–H3 or B7–H3/IL-7 on day 7. D Competitive binding of CAR-T-cell supernatants to IL-7. Data shown are mean ± SD (n = 3). **p < 0.01 and ***p < 0.001 (two-way ANOVA)

Fig. 3

B7–H3 CAR-T/IL-7 cells enhance anti-tumor cytotoxicity in vitro. A Expression of surface B7–H3 on two chordoma cell lines, JHC-7 and UCH2. B B7–H3 CAR-T and B7–H3 CAR-T/IL-7 cells were co-cultured with JHC-7, a chordoma cell line, at different E/T ratios (from 0.5:1 to 8:1). Cytotoxicity was measured by the LDH release assay after a 20-h incubation. C-F ELISA data showing the quantification of cytokines (IL-6, IFN-γ, IL-2, and TNF-α) in the supernatants after B7–H3 CAR-T and B7–H3 CAR-T/IL-7 cells were co-cultured with JHC-7 at an E:T ratio of 8:1 for 20 h. Data shown are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-way ANOVA). Not significant (ns)

Fig. 4

IL-7 improves the persistence of CAR-T cells during in vitro tumor re-challenges. A A schematic of the re-challenge experiment to assess CAR-T-cell persistence in vitro. B Sustained anti-tumor capacity of CAR-T cells against JHC-7 cells. C Sustained anti-tumor capacity of CAR-T cells against UCH2 cells. Data shown are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-way ANOVA). Not significant (ns)

Fig. 5

IL-7 regulates CAR-T-cell expression. A Expansion of T cells transduced with B7–H3 CAR and B7–H3 CAR/IL-7 cells. B Population assay of the CD8⁺ and CD4⁺ subpopulations in B7–H3 CAR-T or B7–H3 CAR-T/IL-7 cells after incubation for 12 days. C–F Subpopulations of memory T cells in the final B7–H3 CAR-T or B7H3 CAR-T/IL-7 cells were measured by flow cytometry on days 7–10. Subpopulations of different T cells were identified as follows: T_EM, naïve T, effector T, and T_CM. G PD-1 expression in B7–H3 CAR-T or B7–H3 CAR-T/IL-7 cells. H LAG-3 expression in B7–H3 CAR-T or B7–H3 CAR-T/IL-7 cells. Data shown are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-way ANOVA). Not significant (ns)

Fig. 6

The therapeutic efficacy of B7–H3 CAR-T/IL-7 cells is superior to B7–H3 CAR-T cells when targeting primary chordoma cells. A Flow cytometry analysis showing B7–H3 expression in chordoma organoids derived from patients. B B7–H3 CAR-T and B7–H3 CAR-T/IL-7 cells were co-cultured with chordoma organoids derived from patients at different E/T ratios (from 0.5:1 to 8:1). Cytotoxicity was measured by the LDH release assay after a 20-h incubation. C B7–H3 CAR-T and B7–H3 CAR-T/IL-7 cells were co-cultured with multiple rounds of chordoma organoids. Long-term cytotoxicity was measured by the LDH release assay after 1–11 days of incubation. Data shown are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-way ANOVA). Not significant (ns)