Tethered IL15-IL15Rα augments antitumor activity of CD19 CAR-T cells but displays long-term toxicity in an immunocompetent lymphoma mouse model

Abstract

Background: Adoptive cell therapy using genetically modified T cells to express chimeric antigen receptors (CAR-T) has shown encouraging results, particularly in certain blood cancers. Nevertheless, over 40% of B cell malignancy patients experience a relapse after CAR-T therapy, likely due to inadequate persistence of the modified T cells in the body. IL15, known for its pro-survival and proliferative properties, has been suggested for incorporation into the fourth generation of CAR-T cells to enhance their persistence. However, the potential systemic toxicity associated with this cytokine warrants further evaluation.

Methods: We analyzed the persistence, antitumor efficacy and potential toxicity of anti-mouse CD19 CAR-T cells which express a membrane-bound IL15-IL15Rα chimeric protein (CD19/mbIL15q CAR-T), in BALB/c mice challenged with A20 tumor cells as well as in NSG mice.

Results: Conventional CD19 CAR-T cells showed low persistence and poor efficacy in BALB/c mice treated with mild lymphodepletion regimens (total body irradiation (TBI) of 1 Gy). CD19/mbIL15q CAR-T exhibits prolonged persistence and enhanced in vivo efficacy, effectively eliminating established A20 B cell lymphoma. However, this CD19/mbIL15q CAR-T displays important long-term toxicities, with marked splenomegaly, weight loss, transaminase elevations, and significant inflammatory findings in some tissues. Mice survival is highly compromised after CD19/mbIL15q CAR-T cell transfer, particularly if a high TBI regimen is applied before CAR-T cell transfer.

Conclusion: Tethered IL15-IL15Rα augments the antitumor activity of CD19 CAR-T cells but displays long-term toxicity in immunocompetent mice. Inducible systems to regulate IL15-IL15Rα expression could be considered to control this toxicity.

Keywords: Adoptive cell therapy - ACT; Chimeric antigen receptor - CAR; Cytokine; Hematologic Malignancies; Immune related adverse event - irAE.

Figure 1

In vitro and in vivo characterization of CD19 CAR-T cells. (A) Schematic representation of CAR constructs. (B) Example of flow cytometric analysis of CAR transduction percentage in murine CD8⁺ T cells (GFP⁺ as indicator of CAR⁺ T cells). Example of CAR detection in the membrane of transduced CD19 CAR CD8⁺ T cells (double-positive cells for GFP and Anti-Mouse IgG, F(ab')₂ staining were considered CAR⁺). (C–G) In vitro functional assays after stimulation of CD8⁺ CAR-T cells with recombinant mCD19-Fc protein-coated plates. (C) CAR-T proliferation assay measured by ³H-thymidine incorporation (cpm), (D–F) IFN-γ production of CAR-T cells in response to recombinant mCD19-Fc protein (D) or to A20 tumor cells (E) measured by ELISA (D–E) or by ELISPOT (F). (G) Relative percentage of A20 tumor cell lysis at different effector CAR-T: target ratios (after substraction of unspecific lysis with irrelevant CAR-T cells). (H–J) In vivo effect of CD19 CAR-T cells in the percentage of CD19⁺ cells (left y-axis) and percentage of CAR-T cells (right y-axis) in the blood of naïve mice preconditioned with high TBI (H), Low TBI (I) or no TBI (J) at different time points after CAR-T cell administration (n=7 mice per group). Graphs represent mean±SEM. The results are representative of 2–3 experiments performed independently. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. TBI, total body irradiation.

Figure 2

In vitro characterization of CD19/mbIL15q CAR-T cells. (A) Schematic representation of CD19/mbIL15q CAR construct. (B) Expression of mbIL15q in the CAR-T cells was measured by flow cytometry using anti-FLAG antibodies (C) pSTAT5 staining in CD4⁺ and CD8⁺ CAR-T cells. (D) Phenotypic characterization of CAR-T cells regarding CD44 and CD62L staining measured by flow cytometry. (E–G) In vitro characterization of CD19/mbIL15q CAR-T cells function. (E) IL2 production by CD4⁺ or CD8⁺ CAR-T cells in response to recombinant mCD19-Fc protein coated to the culture plate. (F) Number of IFN-γ producing CD4⁺ or CD8⁺ CAR-T cells and specific lytic activity (G), in response to A20 tumor cells. Graphs represent mean±SEM. The results are representative of 2–3 experiments performed independently. NL: non-labeled, Irrelevant protein: irrelevant protein (ovalbumin). One-way or two-way ANOVA with Bonferroni multiple test correction was employed for statistical analysis. ****p<0.0001; ***p<0.001; *p<0.05. ANOVA, analysis of variance.

Figure 3

RNA-seq transcriptomic analysis in CD4⁺ CAR-T cells after co-culture with A20 cells. RNA-seq analysis was performed in in vitro samples of PSMA/mbIL15q, CD19 and CD19/mbIL15q CAR-T cells after co-culture with A20 cells at a 1:1 (CAR-cell:tumor cell) ratio. After 48 hours of co-culture, CD4⁺ and CD8⁺ CAR-T cells were purified separately for RNA-seq analysis. (A) Venn’s diagram of genes with differential expression (DE), p<0.05. (B) Principal component analysis (PCA) of the RNA seq. (C) Heat map of gene expression across the analyzed samples. (D) Pathways enrichment analysis for the different CAR-T.

Figure 4

In vivo characterization of CD19/mbIL15q CAR-T cells. (A) Schematic representation of the experimental setting. (B) In vivo effect of CAR-T cells in percentage of CD19⁺ cells in the blood of naïve mice preconditioned with Low TBI (1 Gy) at different time points after CAR-T cell administration (n=7 mice per group). (C, D) % of CD19⁺ cells in the bone marrow (C) and the spleen (D) at day 40 after treatment. A representative example of flow cytometry plots for CD19⁺ staining for each experimental group is shown. (E) CAR-T cell numbers were measured in the spleen of mice at day 58 after CAR-T cell administration. Graphs represent mean±SEM. The results are representative of two experiments performed independently. One-way or two-way ANOVA with Bonferroni multiple test correction was employed for statistical analysis.****p<0.0001. ANOVA, analysis of variance; TBI, total body irradiation.

Figure 5

Therapeutic efficacy of CD19/mbIL15q CAR-T cells in the A20 lymphoma murine model. (A) Schematic representation of the experimental setting. Mice were challenged intravenously with 10⁶ A20 tumor cells, and, 7 days later, they were treated with low TBI and CAR-T cells (CD19 CAR-T or CD19/mbIL15q CAR-T cells). (B) Percentage of CD19⁺ cells in the blood of mice preconditioned with Low TBI at different time points after CAR-T cell administration (n=7 mice per group). (C) Mice survival after CAR-T cell therapy. (D) H&E staining of the spleens and macroscopic aspect of the spleen in mice were sacrificed when the symptoms of the disease were appearing (from days 25 to 50). (E) Schematic representation of the experimental setting for (F–I). (F) Number of IFN-γ-producing cells in the splenocytes of mice in response to stimulation with A20 tumor cells (ratio 10:1, lymphocytes: tumor). (G–I) Percentage of IFN-γ⁺/GrzB⁺ cells (G) PD1⁺ cells (H) or CD137⁺ cells (I) in the tumor-draining lymph node cells of mice treated with the different CAR-T. Graphs represent mean±SEM. One-way or two-way ANOVA with Bonferroni multiple test correction was employed for statistical analysis. ***p<0.001; *p<0.05. ANOVA, analysis of variance; TBI, total body irradiation.

Figure 6

Toxicity caused by CD19/mbIL15q CAR-T cells in immunocompetent mice preconditioned with high or low TBI. (A, B) Schematic representation of the experimental setting and evolution of mice weight, survival and histological findings in mice treated with CD19 CAR-T cells or CD19/mbIL15q CAR-T cells after preconditioning with high TBI (A) or low TBI (B). (C) TCR Clonal diversity in mice treated with CD19/mbIL15q CAR-T cells. TCR Analysis was done in endogenous T cells (upper pie charts) or in the CD19/mbIL15q CAR-T cells (lower pie charts) isolated from the spleen of mice at days between 50 and 70 after CAR-T administration. One naïve mouse was also included as a representative control for TCR diversity in endogenous T cells. TBI, total body irradiation.

Figure 7

Dose-dependent CD19/mbIL15q CAR-T cell toxicity. Follow-up of BALB/c mice after preconditioning with high TBI and the injection of 6, 2 or 0.2×10⁶ CAR-T cells. (A) Schematic representation of the experimental setting. (B) Evolution of mice weight and survival. (C) Follow-up of the percentage of CD19⁺ cells and total numbers of CD4 and CD8 T cells in the blood of mice after CD19/mbIL15q CAR-T cell treatment. (D) Histological analyses (anti-CD3 immunostaining) of tissues from mice treated with different doses of CD19/mbIL15q CAR-T cells at days 25 (in the TBI and the group treated 6×10⁶ CAR-T cells), 52 (in the group receiving 2×10⁶ CAR-T cells) and 70 (in the group treated with 0.2×10⁶ CAR-T cells).