Tumor-Targeted Nonablative Radiation Promotes Solid Tumor CAR T-cell Therapy Efficacy

Abstract

Infiltration of tumor by T cells is a prerequisite for successful immunotherapy of solid tumors. In this study, we investigate the influence of tumor-targeted radiation on chimeric antigen receptor (CAR) T-cell therapy tumor infiltration, accumulation, and efficacy in clinically relevant models of pleural mesothelioma and non-small cell lung cancers. We use a nonablative dose of tumor-targeted radiation prior to systemic administration of mesothelin-targeted CAR T cells to assess infiltration, proliferation, antitumor efficacy, and functional persistence of CAR T cells at primary and distant sites of tumor. A tumor-targeted, nonablative dose of radiation promotes early and high infiltration, proliferation, and functional persistence of CAR T cells. Tumor-targeted radiation promotes tumor-chemokine expression and chemokine-receptor expression in infiltrating T cells and results in a subpopulation of higher-intensity CAR-expressing T cells with high coexpression of chemokine receptors that further infiltrate distant sites of disease, enhancing CAR T-cell antitumor efficacy. Enhanced CAR T-cell efficacy is evident in models of both high-mesothelin-expressing mesothelioma and mixed-mesothelin-expressing lung cancer-two thoracic cancers for which radiotherapy is part of the standard of care. Our results strongly suggest that the use of tumor-targeted radiation prior to systemic administration of CAR T cells may substantially improve CAR T-cell therapy efficacy for solid tumors. Building on our observations, we describe a translational strategy of "sandwich" cell therapy for solid tumors that combines sequential metastatic site-targeted radiation and CAR T cells-a regional solution to overcome barriers to systemic delivery of CAR T cells.

Fig. 1.. Tumor-targeted radiation prior to chimeric antigen receptor (CAR) T-cell administration results in superior antitumor efficacy.

(A) Mesothelin-targeted CAR construct with a CD28 costimulatory domain (M28z) with LNGFR tag or Myc tag for flow cytometric detection of CAR T cells. A prostate-specific membrane antigen–targeted CAR construct with CD28 costimulatory domain (P28z) served as a negative control. (B) M28z CAR T cells, but not P28z, lysed mesothelin-positive tumor cells, as measured by cytotoxicity assay (chromium-release assay). Tumor cell radiation (4 Gy) before CAR T-cell treatment did not enhance cytolysis. (C) NOD/SCID gamma mice with pleural mesothelioma monitored for tumor progression or regression by bioluminescence imaging. Mice received tumor-targeted thoracic radiation (4 Gy) followed by a single low dose of intravenous CAR T cells (5×10⁴). Tumors progressed without treatment (control) or with radiation alone. Tumors regressed in 2 of 6 mice after CAR T cells alone and in all 6 mice (up to 60 days) after preconditioning radiation and CAR T cells. (D) Radiation with CAR T cells yielded potent early tumor regression, compared with CAR T cells alone (50-fold difference at treatment day 16; P<0.001). (E-F) Kaplan-Meier survival analysis demonstrates superior antitumor efficacy of radiation with CAR T cells at multiple doses. (E) At a dose of 5×10⁴ CAR T cells, median survival in mice that received radiation with CAR T cells was 94 days, compared with 25 days in mice that received CAR T cells alone (P<0.05). (F) At a dose of 1×10⁵ CAR T cells, median survival was not reached in mice that received radiation with CAR T cells, compared with 57 days in mice treated with CAR T cells alone (P<0.05) and 27 days in control mice (n=6–8 mice/group). Student’s t tests were performed, and survival was analyzed using the log-rank test. *P<0.05; ***P<0.001. Error bars represent ± standard error of the mean. † indicates death.

Fig. 2.. Tumor-targeted radiation facilitates earlier and higher accumulation of chimeric antigen receptor (CAR) T cells.

(A) Quantification of CD3+CD45+ T cells by flow cytometry demonstrated enhanced accumulation of T cells in irradiated tumor, compared with nonirradiated tumor. Tumor-bearing mice were sacrificed at the first sign of antitumor efficacy on tumor bioluminescence imaging (day 7). (B-D) Serial T-cell bioluminescence imaging in tumor-bearing mice. T cells were doubly transduced with CAR followed by enhanced firefly luciferase (effLuc). Flow cytometry demonstrated 50% effLuc transduction of CAR T cells—a 3-fold higher transduction than for untransduced T cells. There is a linear correlation between T-cell quantity and effLuc-luciferase signal intensity. (D) Mice with pleural mesothelioma treated with CAR T cells with or without radiation were serially imaged. (E) Mice that received radiation before CAR T-cell treatment had early and enhanced accumulation of T cells (P<0.01; n=5 mice/group), predominantly CAR T cells (80%). (F) RT did not increase sequestration of T cells or engraftment of CAR T cells in lungs and other organs, as measured in harvested tissues on day 8 post-RT. *P<0.05; **P<0.01 by Student’s t test. Error bars represent ± standard error of the mean.

Fig. 3.. Preconditioning radiation promotes migration, infiltration, proliferation, memory, and persistence of chimeric antigen receptor (CAR) T cells.

(A) Chemotaxis of CAR T cells was statistically significantly higher (with CD8 predominance, right panel) toward media from irradiated tumor cells than toward media from tumor cells alone, as assessed using a Boyden chamber assay. Unconditioned media served as controls. (B-C) Preconditioning radiation facilitated CD8 CAR T-cell infiltration (B) and proliferation in the tumor (C). Following preconditioning radiation (4 Gy), mice bearing orthotopic mesothelioma were administered carboxyfluorescein succinimidyl ester–labelled CAR T cells. Flow-assisted cell sorting analysis at day 7 demonstrated higher infiltration of CD8 CAR T cells (Generation 0) in irradiated tumor, compared with nonirradiated tumor. Higher CD8 CAR T cell proliferation (Generation ≥1) by site of radiation was noted in the tumor but not in the spleen. (D-E) Radiation was associated with a preserved central memory phenotype of CAR T cells. Phenotyping, as quantified by flow cytometry (D), demonstrated a higher fraction of central memory cells (P<0.01) and lower fraction of terminal effector cells (P<0.05) in irradiated tumor, compared with nonirradiated tumor, in vivo. (E) Memory-specific gene expression (CD62L, CD127, and CD45RO) was higher in CAR T cells harvested from irradiated tumor, compared with nonirradiated tumor (P<0.05, P<0.01, and P<0.05, respectively). (F) Radiation promoted long-term persistence of CAR T cells. In mice treated with or without radiation and 2×10⁶ CAR T cells, tumor was eradicated. (G) CAR T cells eradicated pleural tumor by day 18, with no relapse until sacrifice on day 56. Analysis of harvested spleen by flow cytometry showed robust CAR T-cell persistence in mice that received radiation before CAR T cells, compared with mice that received CAR T cells alone (P<0.05; n=4–8 mice/group). *P<0.05; **P<0.01, by Student’s t test with Bonferroni correction. Error bars represent ± standard error of the mean.

Fig. 4.. Radiation promotes tumor secretion of chemokines, and chimeric antigen receptor (CAR) T cells infiltrating irradiated tumor upregulate chemokine receptors.

(A-B) Tumor-secreted chemokines and cytokines, as quantified by Luminex assay, had dose-dependent (A, in vitro, baseline values normalized to 1) (B, in vivo, serial serum sampling after tumor-targeted radiation) chemokine secretion, with peak secretion 3 to 5 days after radiation. (C) Radiation-induced chemokine secretion was observed clinically. A patient with pleural mesothelioma received tumor-targeted palliative radiation. Serum chemokines, particularly corresponding ligands for CXCR3 (CXCL9 and CXCL11), increased after radiation. CAR T cells reappeared in peripheral blood following RT, as measured by vector copy number. (D-E) CXCR3 expression in CAR T cells infiltrating irradiated tumors. Tumor-bearing mice that received CAR T cells with or without radiation were sacrificed at the first sign of antitumor efficacy on tumor bioluminescence imaging (day 12; n=8 mice/group). Flow cytometry demonstrated a higher fraction of CXCR3-expressing CAR T cells (percent positive and quantity per gram of tumor; P<0.05 and P<0.01, respectively) in irradiated tumor, compared with nonirradiated tumor. The increase in CXCR3 expression was CAR T cell specific, as the untransduced T cells in the same tumors had no upregulation in CXCR3 expression. In irradiated tumor, the median fluorescent intensity of CXCR3 expression in CAR T cells was associated with an increase in CAR median fluorescence intensity; this was not observed in nonirradiated tumor (P<0.001). (E) Two representative flow cytometry plots demonstrate positive correlation between CXCR3 and CAR median fluorescence intensity. (F) By mRNA gene expression, CXCR3, CXCR6, CCR5, CCR2, and CX3CR1 were higher in CAR T cells in irradiated tumor, compared with nonirradiated tumor (≥2-fold; n=4 mice/group). (G) Furthermore, by mRNA gene expression, CAR T cells in irradiated tumor expressed higher markers of transendothelial migration (trend was noted, not statistically significant), compared with CAR T cells in nonirradiated tumor. *P<0.05; **P<0.01; ***P<0.001, by Student’s t test. Error bars represent ± standard error of the mean.

Fig. 5.. Infiltrating chimeric antigen receptor (CAR) T cells from irradiated tumor show superior abscopal antitumor efficacy and tumor immunity after repeated tumor rechallenge using the tumor treadmill test.

(A) Graphic summary of a potential mechanism for abscopal efficacy after tumor-targeted radiation. Radiation-induced tumor secretion of chemokines enhanced accumulation of CAR T cells in the tumor, and infiltrating CAR T cells showed upregulated chemokine receptor expression and markers of transendothelial migration, which in turn promoted abscopal infiltration and efficacy of T cells in nonirradiated tumor. (B) Pleural tumor-targeted radiation and antitumor immunity at a distal tumor. Mice established with pleural and right flank mesothelioma tumors received either thoracic tumor-targeted radiation followed by intrapleural CAR T cell administration or CAR T cells alone without preconditioning radiation. In flank tumors, accumulation of CD8 CAR T cells was higher in mice that received thoracic radiation than in mice that did not receive thoracic radiation. Infiltrating CAR T cells also had higher CXCR3 expression in irradiated flank tumor than in nonirradiated flank tumor. Mice that received thoracic radiation had superior antitumor efficacy at the flank, as measured by tumor volume and bioluminescence imaging, compared with mice that did not receive radiation (P<0.05; n=6 mice/group). (C-F) Tumor treadmill test. The above results were reproduced in a model with repeated tumor rechallenges at a distal site. Thoracic radiation showed distal tumor antitumor efficacy of the CAR T-cell constructs currently in use in our clinical trial ([C] M28z1XXPD1DNR; NCT04577326). The CAR co-expresses a PD-1 dominant negative receptor, a PD-1 receptor lacking an inhibitory signaling domain, and a CD3ζ domain containing a single functional immunoreceptor tyrosine-based activation motif, termed 1XX. (D) In vivo, a single low dose of 1XXPD1DNR CAR T cells (5×10⁴) eradicated pleural tumor with or without radiation. At 120 days after treatment, mice underwent a tumor treadmill test, where increasing doses of nonirradiated tumor were administered into the peritoneal cavity every 3 to 6 days. By tumor imaging, no difference in tumor regression was observed at initial lower tumor rechallenge doses (1–5 × 10⁶ tumor cells per dose). At higher doses (20–40 × 10⁶ tumor cells per dose), efficacy was observed in mice that received a single dose of chest radiation, compared with mice that did not receive radiation. (D, inset) Fold-difference in tumor bioluminescence imaging 3 days after each tumor challenge. After the sixth rechallenge (40×10⁶ tumor cells), mice that received thoracic radiation had an 11-fold lower tumor burden, compared with mice that did not receive radiation (P<0.01; n=8 mice/group). (E-F) Persistent CAR T cells demonstrated a high central memory phenotype and CXCR3 expression. After the tumor treadmill test, mice were sacrificed, and CAR T cells in the spleen and peritoneum were quantified by flow cytometry on day 160, 5 days after tumor rechallenge. (E) In the spleen of irradiated mice, CAR T cells had a 2-fold higher central memory fraction and higher CXCR3 expression, compared with nonirradiated mice (P<0.05). (F) In the peritoneum, terminal effector CD8 CAR T-cell expression of CXCR3 was higher in irradiated mice than in nonirradiated mice (P<0.01). *P<0.05; **P<0.01 by Student’s t test with Bonferroni correction. Error bars represent ± standard error of the mean.

Fig. 6.. Preconditioning radiation enhances the efficacy of chimeric antigen receptor (CAR) T cells in non-small cell lung cancer (NSCLC).

(A) Immunohistochemistry for mesothelin (MSLN) in human NSCLC showed heterogeneity in MSLN cell-surface expression. (B) Flow cytometry plots of high-, low-, and mixed-MSLN-expressing NSCLC cells. Human A549 cells were transduced with MSLN and/or green fluorescent protein–luciferase to generate high- and low-MSLN-expressing tumor cells. Mixed-MSLN-expressing NSCLC tumors were generated by mixing 50% high-MSLN-expressing and 50% low-MSLN-expressing tumor cells. Only low-MSLN-expressing cells in the heterogeneous tumor were transduced with green fluorescent protein–luciferase, so that tumor progression or regression of low-MSLN-expressing tumor alone could be monitored. (C) CAR T cells lysed tumor cells in an antigen expression density–dependent manner, as measured by cytotoxicity assay (chromium-release assay). CAR T cells lysed low-MSLN-expressing cells only at effector to target ratios >10:1. (D-E) Antitumor efficacy in high-MSLN-expressing NSCLC. Mice with established NSCLC after administration of tumor cells by tail vein received thoracic radiation followed by a single low dose of CAR T cells (5×10⁴) administered systemically. Tumor progressed in mice treated with untransduced T cells (control) or with preconditioning radiation and untransduced T cells. With CAR T-cell administration alone, tumors initially regressed but relapsed in all mice by 35 days after treatment. Radiation with CAR T cells resulted in potent tumor regression and long-term antitumor efficacy for >100 days in 6 of 8 mice (P<0.01). By average bioluminescence imaging, tumor burden at day 60 was 10-fold lower with radiation than without radiation. (F) Mice treated with preconditioning radiation followed by CAR T cells had superior antitumor efficacy (median survival, not reached at day 160 vs 107 days with CAR T-cell treatment alone vs 21 days in the control group vs 25 days in the radiation group) (P<0.001; n=8–16 mice/group). (G) Antitumor efficacy in mixed-MSLN-expressing NSCLC. Mice with established mixed-MSLN-expressing tumors received a low dose (5×10⁴) of CAR T cells systemically. Tumor bioluminescence imaging showed low-MSLN-expressing tumor burden only (only low-MSLN-expressing tumor cells were transduced with luciferase). Radiation with CAR T cells resulted in prolonged tumor regression and delayed tumor relapse, compared with CAR T cells alone (P<0.01). (H) Mice treated with preconditioning radiation and CAR T cells had prolonged survival (median survival, 63 vs 43 days in mice treated with CAR T cells alone; P<0.001; n=8 mice/group). (I-J) Treatment of mice with NSCLC with the clinical construct M28z1XXPD1DNR CAR T cells. Tumor regression measured by tumor bioluminescence imaging of mice bearing high-, low-, or mixed-MSLN-expressing tumor. Mice with established NSCLC received thoracic radiation followed by a low dose (5×10⁴) of 1XXPD1DNR CAR T cells. CAR T cells eradicated high-mesothelin-expressing tumor with or without radiation (I, left). In low- and mixed-MSLN-expressing tumor, in which only low-MSLN-expressing cells were imaged, CAR T cells had better efficacy with radiation than without radiation (I, middle and right; P<0.01 and P<0.001, respectively). (J) At this dose, mice with heterogeneous NSCLC treated with preconditioning radiation and CAR T cells had prolonged survival, compared with mice treated with CAR T cells alone (median survival, not reached at 100 days vs 72 days; P<0.05; n=6–8 mice/group). Student’s t tests were performed, and survival was analyzed using the log-rank test with Bonferroni correction. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Error bars represent ± standard error of the mean. † indicates death.

Fig. 7.. Tumor-targeted radiation promotes chemokine expression in tumor, and irradiated tumor-infiltrating T cells upregulate chemokine receptors in a syngeneic model of lung cancer.

Lung orthotopic tumors generated via injecting 1.5 × 105 HKP1 cells through tail vein into 7-week-old female C57BL6/J mice were monitored for tumor progression or regression by bioluminescence imaging (BLI). Mice received tumor-targeted thoracic radiation (4 Gy) or mock radiation. Tumors progressed without treatment (control) and with radiation alone. GSA analysis of mRNA expression (day 13, around 5000 cells per group) shows (A) increased tumor specific cheekiness CXCL9 and CXCL11 in CD45- tumor cells and (B) higher expression of CXCR3, CCR2, and CCR5 occurred in T cells within irradiated tumor relative to non-irradiated tumor. (C) The TME (including CD45+ immune cells and CD45- cells) harvested from the lungs of mice at day 13. RT promotes increased chemokine expression in the tumor microenvironment by GSA analysis of single cell gene expression for all CD45+ cells. (D) Macrophages express increased chemokines (CCL12 and CCL2) and markers of transendothelial migration. GSA: Gene-set analysis, TME: tumor microenvironment, t-SNE: t-distributed stochastic neighbor embedding. Data represents the means of triplicates ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. not significant (One-way ANOVA).