Interleukin-15-armored GPC3-CAR T cells for patients with solid cancers

Abstract

Interleukin-15 (IL15) promotes the survival of T lymphocytes and enhances the antitumor properties of CAR T cells in preclinical models of solid neoplasms in which CAR T cells have limited efficacy^1-4. Glypican-3 (GPC3) is expressed in a group of solid cancers^5-10, and here we report the first evaluation in humans of the effects of IL15 co-expression on GPC3-CAR T cells. Cohort 1 patients (NCT02905188/NCT02932956) received GPC3-CAR T cells, which were safe but produced no objective antitumor responses and reached peak expansion at two weeks. Cohort 2 patients (NCT05103631/NCT04377932) received GPC3-CAR T cells that co-expressed IL15 (15.CAR), which mediated significantly increased cell expansion and induced a disease control rate of 66% and antitumor response rate of 33%. Infusion of 15.CAR T cells was associated with increased incidence of cytokine release syndrome, which was rapidly ameliorated by activation of the inducible caspase 9 safety switch. Compared to non-responders, tumor-infiltrating 15.CAR T cells from responders showed repression of SWI/SNF epigenetic regulators and upregulation of FOS and JUN family members as well as genes related to type I interferon signaling. Collectively, these results demonstrate that IL15 increases the expansion, intratumoral survival, and antitumor activity of GPC3-CAR T cells in patients.

Fig 1.. Safety characteristics of CAR and 15.CAR infusions:

A. Transgene maps of GPC3-CAR and iC9.NGFR.IL15 constructs used to co-transduce T cells to generate infusion products. B. Schematic representation of patient enrollment and treatment. C. Bubble plots representing frequency of the indicated adverse events for CAR (left) and 15.CAR (right) infused patients. Adverse events (AEs) were collected from Day −4 until Day +28 post-infusion and graded according to the Common Terminology Criteria of AEs v5. Color spectrum corresponds to AE grade 1–5, bubble size corresponds to frequencies. D. Comparison of frequency of AEs between CAR vs 15.CAR and E. Comparison of AEs between patients with and without CRS using two-tailed T test and two-way ANOVA with Sidac correction, respectively. Mean ± SD. F-H. Levels of GPC3-CAR and iC9.NGFR.IL15 expressing T cells quantified by qPCR (F) and flow cytometry (G) and changes in concentrations of indicated serum cytokines (H) in peripheral blood of patients treated with rimiducid, the chemical inducer of the iC9 safety switch.

Fig 2.. 15.GPC3-CAR T cells induce significant antitumor responses in patients.

Antitumor responses were determined by comparing pre- and post-infusion 3D imaging. A. Coronal CT chest (15.CAR 1), axial CT chest (15.CAR 4), PETCT (15.CAR 7), MRI abdomen (15.CAR 9) and axial CT abdomen (15.CAR 12) based images showing pre- and post-CAR T cell infusion. Red arrows and circles represent tumors. B. Waterfall plot representing changes in tumor volumes of patients treated with 3 × 10⁷/m² with CAR or 15.CAR T cells. Red line: 20% increase, Blue line: 20% decrease, Green line: 30% decrease. C. Serum alpha-feto protein (AFP) concentrations at indicated timepoints in responders with AFP secreting neoplasms. D. Pre- and post-infusion tumor biopsy assessed with hematoxylin-eosin staining showing near complete necrosis of patient 15.CAR 9’s liver tumor.

Fig 3.. Comparison of pre-infusion products and expansion in patients of CAR and 15.CAR T cells.

Products were first assessed with single cell RNA sequencing. A. Uniform Manifold Approximation and Projection (UMAP) identifying unique T cell clusters in integrated CAR and 15.CAR pre-infusion products. B-C. Differential representation of CAR vs 15.CAR T cells in clusters shown in UMAP projection (B) and proportion for each cluster (C). Center line: median, box limits: first and third quartiles, whiskers: 1.5x the interquartile range, dots: outliers. D. Differentially expressed genes in CAR vs 15.CAR products. E. Frequencies of CD4/8 and effector / memory subsets in CAR vs 15.CAR products by flow cytometry. Two-tailed, unpaired T test and two-way ANOVA with Šídák correction, respectively. F. Cytotoxicity of CAR vs 15.CAR products measured by ⁵¹Cr release assay. G. Polyfunctionality strength index comparing vs 15.CAR T cell product’s cytokine production by Isoplexis. Two-way ANOVA with Šídák correction. H. Differentially expressed cytokines in CD8 subsets of CAR and 15.CAR T cell products. I. Peripheral blood CAR T cell frequencies quantified by qPCR at indicated timepoints for each patient. J. Comparison of peak expansion on dose level 2 of CAR vs 15.CAR T cells post-infusion. Two-tailed, unpaired T test. Data represented as mean ± SD. K. Comparison of expansion of cells in responders vs non-responders and Mann-Whitney test. Data represented as mean ± SD.

Fig 4.. Comparing the single cell gene expression profile of tumor infiltrating 15.CAR T cells post-infusion in responders vs non-responders.

The transcriptomic profile of Infusion products and tumor infiltrating 15.CAR T cells were interrogated with single cell RNA sequencing. Differentially expressed genes (DEGs) for indicated groups were determined by comparing the product- with peripheral blood tumor-derived 15.CAR T cells. A. UMAP projection of tumor infiltrating 15.CAR T cells from responders and non-responders. B. Unsupervised clustering of cells corresponding of tumor infiltrating CAR T cells of responders and non-responders from the 15.CAR cohort. C. Differences in cluster proportions for indicated groups. D. DEG comparison (product vs tumor infiltrating 15.CAR T cells) in responders (x axis) vs non-responders(y axis) from the 15.CAR cohort. E. Gene sets enriched in tumor infiltrating 15.CAR T cells. F. Heatmap representing the genes with most differences in change from baseline in responders vs non-responders in 15.CAR T cells.