Immunogenic Chemotherapy Enhances Recruitment of CAR-T Cells to Lung Tumors and Improves Antitumor Efficacy when Combined with Checkpoint Blockade

Abstract

Adoptive therapy using chimeric antigen receptor-modified T cells (CAR-T cells) is effective in hematologic but not epithelial malignancies, which cause the greatest mortality. In breast and lung cancer patients, CAR-T cells targeting the tumor-associated antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) infiltrate tumors poorly and become dysfunctional. To test strategies for enhancing efficacy, we adapted the Kras^LSL-G12D/+;p53^f/f autochthonous model of lung adenocarcinoma to express the CAR target ROR1. Murine ROR1 CAR-T cells transferred after lymphodepletion with cyclophosphamide (Cy) transiently control tumor growth but infiltrate tumors poorly and lose function, similar to what is seen in patients. Adding oxaliplatin (Ox) to the lymphodepletion regimen activates tumor macrophages to express T-cell-recruiting chemokines, resulting in improved CAR-T cell infiltration, remodeling of the tumor microenvironment, and increased tumor sensitivity to anti-PD-L1. Combination therapy with Ox/Cy and anti-PD-L1 synergistically improves CAR-T cell-mediated tumor control and survival, providing a strategy to improve CAR-T cell efficacy in the clinic.

Trial registration: ClinicalTrials.gov NCT02706392.

Keywords: CAR-T cells; CXCR3; CXCR6; KP; ROR1; alveolar macrophage; immunogenic cell death; lung adenocarcinoma; oxaliplatin.

Figure 1.. ROR1 CAR-T cells infiltrate tumors poorly and become dysfunctional in patients with ROR1⁺ TNBC and NSCLC.

A) Treatment scheme. Cy=cyclophosphamide. Flu=fludarabine. B) Frequency of tEGFR⁺ ROR1 CAR-T among total CD8⁺ or CD4⁺ T cells in blood of ROR1⁺ TNBC or NSCLC patients. C) Left: flow analysis of tEGFR and inhibitory receptor expression on CD8⁺ and CD4⁺ T cells in blood of patient X552. Right: summary of inhibitory receptor expression on CD8⁺tEGFR⁺ and CD4⁺tEGFR⁺ CAR-T cells in blood of patients in (B) day 14 post-transfer relative to infusion product (IP). N=3 per group. Paired Student’s two-way t-test. D) Luminex analysis of cytokine secretion by CD8⁺tEGFR⁺ and CD4⁺tEGFR⁺ ROR1 CAR-T cells from IP or blood of patients 14 days post-transfer after re-stimulation ex vivo with anti-CD3/CD28. N=2–3 per group. E) Flow analysis of live cells from PBMC and tumor 21 days post-infusion from patient X475. See also Table S1.

Figure 2.. ROR1 CAR-T cells modestly control tumor growth in KP^ROR1 mice.

A) Treatment scheme. I.T.=intratracheally. Ffluc=firefly luciferase. B) Percent change in total tumor (left) or individual tumor volume (right) quantified by MRI in KP^ROR1 mice. N=3–4 mice per group. Two-way ANOVA with Tukey’s post-test. C) Survival of KP^ROR1 mice. N=3–4 mice per group. Log-rank Mantel-Cox test. D) Frequency of CD45.1⁺CD8⁺GFP⁺ control (red) or CAR-T cells (blue) in blood. N=6–8 mice per group. E) Left: flow analysis of GFP and Fc-ROR1 (CAR) expression on PE⁻CD8⁺ non-vascular T cells (top) and PD-1 and LAG-3 expression on PE⁻CD8⁺GFP⁺ non-vascular control or CAR-T cells (bottom) from lungs of KP^ROR1 mice 10 days post-transfer. Right: Summary of PE⁻ control and CAR-T cell frequency and inhibitory receptor expression in WT or KP^ROR1 lungs 10 days post-transfer. N=6–8 mice per group. Unpaired Student’s two-way t-test. F) Intracellular cytokine analysis in CD45.1⁺CD8⁺GFP⁺ control (red) or CAR-T cells (blue) from lungs of WT or KP^ROR1 mice 10 days post-transfer and re-stimulated with PMA/ionomycin. N=5 mice per group. Student’s unpaired two-way t-test. G) Flow analysis of hROR1 expression on primary CD45⁻EpCAM⁺ lung epithelial cells from control or CAR-T cell-treated KP^ROR1 mice 10 weeks post-transfer or on KP cell lines overexpressing hROR1. N=5 mice per group. H) ROR1 IHC staining on control or CAR-T cell-treated KP^ROR1 lungs 10 weeks post-transfer. Data are representative of 4 independent experiments. See also Figures S1–S3.

Figure 3.. ROR1 CAR-T cells do not efficiently infiltrate all KP^ROR1 tumors.

A) Representative MRI scans of KP^ROR1 mice treated as indicated. B) Left: CD3 IHC staining showing T cell localization patterns. Right: percentage of individual tumors per mouse with indicated T cell localization pattern in control- and CAR-T cell-treated KP^ROR1 mice. N=4 mice per group. C) Flow analysis of chemokine receptor expression on CD45.1⁺CD8⁺GFP⁺ CAR-T cells or CD45.2⁺CD8⁺ endogenous T cells in blood of KP^ROR1 mice 3 days post-transfer. N=4 mice per group. Paired Student’s two-way t-test. D) qPCR of chemokines relative to housekeeping genes in untreated KP^ROR1 tumors 13 weeks post-infection. N=4 mice per group. Data are representative of 2 independent experiments.

Figure 4.. Ox/Cy enhances chemokine expression and CAR-T cell accumulation in KP^ROR1 tumors.

A) Volcano plot of genes significantly upregulated (red) or downregulated (green) in tumors excised and pooled from Ox/Cy-treated or untreated KP^ROR1 mice, 6 hours post-Ox/Cy injection. N=4–5 mice per group. B) Network plot of top KEGG pathways enriched among genes upregulated by Ox/Cy. Node size is proportional to number of genes within each gene set; thickness of grey line between nodes indicates proportion of shared genes between genesets. C) Heatmap of leading-edge genes in “Cytokine-Cytokine Receptor Interaction” pathway. Genes encoding chemokines are highlighted, with those involved in T cell recruitment indicated in bold. D) Treatment scheme. E) Frequency of CD45.1⁺CD8⁺GFP⁺ control and CAR-T cells in spleens and tumors excised and pooled from KP^ROR1 mice 10 days post-transfer. N=4 mice per group. One-way ANOVA with Tukey’s post-test. F) Lymphocyte counts in peripheral blood of KP^ROR1 mice treated with Cy or Ox/Cy. N=4 mice per group. Data are representative of 2 independent experiments.

Figure 5.. Ox/Cy enhances accumulation of tumor-infiltrating CAR-T cells with an activated phenotype.

A) Unsupervised clustering of cells derived from tumors excised and pooled from untreated (day 0), Ox/Cy-treated (day 0, 6 hours post-Ox/Cy), Cy + CAR-T cell-treated (day 10), and Ox/Cy + CAR-T cell-treated (day 10) KP^ROR1 mice and analyzed by scRNA-seq. B) Clusters in (A) colored by tumor sample type. C) Percent of indicated clusters comprised of indicated tumor sample. D) Expression of indicated genes in CAR-T cell infusion product and tumor-infiltrating T cell clusters. See also Figures S4 and S5.

Figure 6.. Ox/Cy activates expression of T cell-recruiting chemokines by tumor macrophages.

A) Unsupervised clustering of mononuclear phagocyte (MNP) cluster from Fig. 5A. B) MNP clusters in (A) colored by tumor sample type. C) Unsupervised clustering of macrophage cluster in (A). D) Fraction of macrophage cluster comprised of each tumor sample type. E) Violin plots of select genes in macrophage sub-clusters. F) Geneset scores of enriched pathways in macrophage sub-clusters. G) Violin plots of geneset score of indicated KEGG pathways in macrophage sub-clusters. H) Frequency of iNOS⁺ cells among F4/80⁺ macrophages in tumors excised and pooled from KP^ROR1 mice 10 days after treatment. N=6–10 mice per group. One-way ANOVA with Tukey’s post-test. I) Frequency of iNOS⁺ cells among F4/80⁺ macrophages (left) and CAR-T cells among CD8⁺ T cells (right) in spleens and tumors excised and pooled from KP^ROR1 mice 10 days after treatment. N=5 mice per group. One-way ANOVA with Tukey’s post-test. J) Violin plots of chemokine expression in macrophage sub-clusters. K) Frequency of WT (red) and chemokine receptor knockout (blue) CAR-T cells in tumors excised and pooled from Ox/Cy- or vehicle-treated KP^ROR1 mice 2 days (top) or 10 days (bottom) after transfer. N=3–4 mice per group. Paired Student’s two-way t-test. Data in 6H, 6I, and 6K are representative of 2–3 independent experiments. See also Figure S6.

Figure 7.. Ox/Cy-enhanced CAR-T cell infiltration sensitizes tumors to anti-PD-L1.

A, B) Expression of Cd274 among scRNA-seq clusters from Fig. 5A (A) and macrophage subclusters from Fig. 6A (B). C) Median fluorescence intensity of PD-L1 on F4/80⁺ macrophages in lungs from KP^ROR1 mice 10 days after treatment. N=4 mice per group. One-way ANOVA with Tukey’s post-test. D) Frequency and PD-1 expression on CD45.1⁺CD8⁺GFP⁺PE⁻ transferred control or CAR-T cells in lungs of KP^ROR1 mice treated as indicated 10 days after the second infusion of T cells. N=4 mice per group. One-way ANOVA with Tukey’s post-test. E) Percent change in tumor volume in KP^ROR1 mice treated as indicated. N=4 mice per group. Two-way ANOVA with Tukey’s post-test. Data are representative of 2 independent experiments.

Figure 8.. Ox/Cy and anti-PD-L1 improve CAR-T cell-mediated tumor control and survival.

A) Percent change in total (left) or individual tumor volume (right) in KP^ROR1 mice treated as indicated. N=4 mice per group. Two-way ANOVA with Tukey’s post-test. B) Representative MRI scans of KP^ROR1 mice. C) CD3 IHC staining on lungs from KP^ROR1 mice 10 days after the second infusion of T cells. D) Quantification of CD3⁺ T cells in individual KP^ROR1 tumor nodules and fraction of tumors in each mouse showing intratumoral localization. N=4 mice per group. One-way ANOVA with Tukey’s post-test. E) Flow analysis of CD45.1⁺CD8⁺GFP⁺PE⁻ control and CAR-T cells in lungs of KP^ROR1 mice 10 days after the second infusion of T cells. N=7–8 mice per group. One-way ANOVA with Tukey’s post-test. F) Intracellular cytokine analysis of CD45.1⁺CD8⁺GFP⁺PE⁻ control and CAR-T cells isolated from lungs of KP^ROR1 mice 10 days after the second infusion of T cells and re-stimulated with PMA/ionomycin. N=7–8 mice per group. One-way ANOVA with Tukey’s post-test. G) Survival of KP^ROR1 mice. N=6–8 mice per group. Log-rank Mantel-Cox test. A-F: Data are representative of two independent experiments. G: Data are combined from two independent experiments. See also Figures S7 and S8.