Circumventing resistance within the Ewing sarcoma microenvironment by combinatorial innate immunotherapy

Abstract

Background: Pediatric patients with recurrent/metastatic Ewing sarcoma (ES) have a dismal 5-year survival. Novel therapeutic approaches are desperately needed. Natural killer (NK) cell number and function are low in ES patient tumors, in large part due to the immunosuppressive tumor microenvironment (TME). Melanoma cell adhesion molecule (MCAM) is highly expressed on ES and associated with ES metastasis. NKTR-255 is a polymer-conjugated recombinant human interleukin-15 (IL-15) agonist improving NK cell activity and persistence. Magrolimab (MAG) is a CD47 blockade that reactivates the phagocytic activity of macrophages.

Methods: Transcriptome profiling coupled with CIBERSORT analyses in both ES mouse xenografts and human patient tumors were performed to identify mechanisms of NK resistance in ES TME. A chimeric antigen receptor (CAR) NK cell targeting MCAM was engineered by CAR mRNA electroporation into ex vivo expanded NK cells. In vitro cytotoxicity assays were performed to investigate the efficacy of anti-MCAM-CAR-NK cell alone or combined with NKTR-255 against ES cells. Interferon-γ and perforin levels were measured by ELISA. The effect of MAG on macrophage phagocytosis of ES cells was evaluated by in vitro phagocytosis assays. Cell-based and patient-derived xenograft (PDX)-based xenograft mouse models of ES were used to investigate the antitumor efficacy of CAR-NK alone and combined with NKTR-255 and MAG in vivo.

Results: We found that NK cell infiltration and activity were negatively regulated by tumor-associated macrophages (TAM) in ES TME. Expression of anti-MCAM CAR significantly and specifically enhanced NK cytotoxic activity against MCAM^high but not MCAM-knockout ES cells in vitro, and significantly reduced lung metastasis and extended animal survival in vivo. NKTR-255 and MAG significantly enhanced in vitro CAR-NK cytotoxicity and macrophage phagocytic activity against ES cells, respectively. By combining with NKTR-255 and MAG, the anti-MCAM-CAR-NK cell significantly decreased primary tumor growth and prolonged animal survival in both cell- and PDX-based ES xenograft mouse models.

Conclusions: Our preclinical studies demonstrate that immunotherapy via the innate immune system by combining tumor-targeting CAR-NK cells with an IL-15 agonist and a CD47 blockade is a promising novel therapeutic approach to targeting MCAM^high malignant metastatic ES.

Keywords: Chimeric antigen receptor - CAR; Macrophage; Natural killer - NK; Tumor microenvironment - TME.

Figure 1. Identification of mechanisms of resistance to NK therapy in ES xenograft tumors. (A, B) Top differentially expressed murine (A) and human (B) genes comparing NK cell treated peripheral and central tumor sections. ES xenograft tumors (N=5 per condition) were harvested 24 hours after the treatment with PBS or ex vivo expanded NK cells and dissected into peripheral and central tumor sections. Total RNA was extracted from the tumor sections and subjected to RNA-seq and subsequent data analyses for differentially expressed genes. Each cell represents the gene expression of each sample. (C, D) DAVID functional annotation analyses of differentially expressed murine (C) and human (D) genes comparing treated peripheral and central tumor samples. Top functional annotation terms were plotted with enrichment scores (−lg (p value)). (E, F) Ingenuity pathway analysis of differentially expressed murine (E) and human (F) genes comparing treated peripheral and central tumor samples. Red and blue colors indicate higher and lower expression in treated peripheral tumor samples, respectively. Octagon represents the regulated functions; other shapes indicate the types of molecules. Solid and dash lines represent direct and indirect interactions. DEGs, differentially expressed gene; ES, Ewing sarcoma; NK, natural killer; PBS, phophate buffered saline.

Figure 2. Macrophages negatively regulate NK cells in the immune microenvironment of Ewing sarcoma (ES) tumors. (A) Abundance of M1 and M2 mouse macrophages in the peripheral and central sections of ES xenograft tumors. The abundance of mouse immune cells was deconvoluted using RNA-seq gene expression data and ImmuCC signature matrix by CIBERSORT analysis. The relative counts of M1 (orange) and M2 (blue) macrophages in central and peripheral tumor sections were plotted with each tumor sample. In x-axis sample names, T1C means treated central tumor 1; U1C, untreated central tumor 1; T1P, treated peripheral tumor 1; U1P, untreated peripheral tumor 1. (B) Percentage of resting and activated human NK cells in the peripheral and central tumor sections. RNA-seq gene expression data were used as input matrix and LM22 as signature matrix in CIBERSORT analysis to deconvolute the abundance of human immune cells in the xenograft tumors. The relative counts of resting (blue) and activated (orange) NK cells in central and peripheral tumor sections were plotted with each tumor sample. In x-axis sample names, T1C means treated central tumor 1; T1P, treated peripheral tumor 1. Aliquots of ex vivo expanded NK cells injected into the mice (NK1 and NK2) were used as control samples. (C), Cytokine array analysis for identification of cytokines/chemokines secreted by ES cells and PDX tumor. ES A673 and SKNMC cells and PDX tumor (NCH-EW-1) were cultured in DMEM media for 48 hours before the conditioned media were harvested and assayed by Human Cytokine Antibody Array (Membrane, 42 Targets, Abcam). Fresh DMEM media was used as a negative control. The duplicate black dots at specific positions show positive signals of certain cytokines. Asterisk: internal control; 1: MCP-1/CCL2; 2: IL-8; 3: GRO; and 4: GRO-α. Representative images are shown. The same trend was seen in two independent biological repeats. (D) Quantification of the dot intensity in (C) by ImageJ (https://imagej.nih.gov/ij). Columns represent the relative average density of the dots compared with the internal control. Error bars indicate the SD of duplicate samples in a representative experiment. The same trend was seen in two independent biological repeats. *p<0.05, **p<0.01 (two-tailed Student’s t-test). (E) Comparison of NK cell receptor expression levels on ex vivo expanded NK cells incubated with or without tumor-associated macrophages (TAMs) isolated from ES xenograft tumors. Macrophages were enriched from the single cell suspension of ES xenograft tumors using the anti-F4/80 MicroBeads (Miltenyi Biotec). Ex vivo expanded NK cells were incubated with (orange) or without (blue) isolated TAMs at a ratio of 1:1 for 4 hours followed by evaluation of NK cell receptor expression via flow cytometry. *p<0.05, **p<0.01 (two-tailed Student t-test). Representative results are shown. The same trend was seen in two independent biological repeats. (F) Correlations of immune cells in the TME of ES patient tumors. CIBERSORT analysis was used to deconvolute immune cell composition in ES patient tumors using publicly available ES microarray dataset (GSE17679). CIBERSORT counts were used to analyze the correlations of abundance between various immune cells by Pearson’s correlation method. Red and blue dots represent negative and positive correlations, respectively. The darker the color is, the stronger the correlation is. (G) The relative abundance of NK cells and macrophages in individual tumors are represented as open circles in scatterplots. Pearson correlation coefficient r=−0.41 (95% CI –0.55 to –0.24), p=6.49×10⁻⁶. N=98 in GSE17679. PDX, patient-derived xenograft; TME, tumor microenvironment.

Figure 3. Expression of anti-MCAM CAR significantly increased cytotoxic activity of NK cells against MCAM^high ES cells in vitro. (A) Expression of MCAM on ES cells. Cells were stained with isotype or MCAM antibody and subject to flow cytometry analyses. Histograms from a representative experiment were shown. Three biological repeat experiments were performed with similar results (n=3). (B) Schematic representation of the MCAM CAR construct. (C) Electroporation of CAR mRNA resulted in CAR expression on ex vivo expanded NK cells. Two micrograms of CAR mRNA per 10⁶ of ex vivo expanded NK cells were used for electroporation using Maxcyte GTx electroporator. CAR expression was detected at 1, 2, 4, 6 days postelectroporation by a biotinylated MCAM protein, followed by FITC-streptavidin staining and flow cytometry analyses. The numbers at the right corner of the dot plots are percent of CAR positive cells. Dot plots from a representative experiment were shown. Three biological repeat experiments (three donors) were performed with similar results. (D) In vitro cytotoxicity of unmodified expanded NK cells (Mock) and anti-MCAM-CAR-NK cells (CAR) against MCAM^high ES cells A673, TC32, and SKNMC. Mock or CAR-NK cells were incubated with luciferase-expressing ES cells at various effector to target (E:T) ratios (0.2:1, 0.5:1, 1:1) for 4 hours before cytotoxicity was analyzed by addition of D-luciferin to the cells and bioluminescence was measured with a luminometer. Asterisks indicate p<0.05, **p<0.01 (two-tailed Student’s t-test). Error bars indicate the SD of triplicate samples in a representative experiment. The same trend was seen in three independent biological repeats. (E, F), Secretion of cytokines IFN-γ (E) and perforin (F) from mock and anti-MCAM-CAR-NK cells when incubated with ES cells. Mock or CAR-NK cells were incubated with ES A673, TC32 or SKNMC cells at E:T ratio of 0.2:1 for 4 hours and cytokine secretion in the media was analyzed by ELISA. Tumor cells with no NK cell incubation were used as a negative control. *p<0.05, **p<0.01 (two-tailed Student’s t-test). Error bars indicate the SD of triplicate samples in a representative experiment. The same trend was seen in three independent biological repeats. (G) CRISPR/Cas9 knockout of MCAM in ES cells. MCAM expression in wildtype (WT) and knockout (KO1 and KO2) A673, TC32 and SKNMC cells were detected by flow cytometry using MCAM specific antibody. (H) In vitro cytotoxicity of mock and anti-MCAM-CAR-NK cells against MCAM WT and KO ES cells. Mock or CAR-NK cells were incubated with luciferase-expressing MCAM WT or KO (KO1 and KO2) ES cells (A673, TC32, SKNMC) at E:T ratio of 0.2:1 for 4 hours before cytotoxicity was analyzed by addition of D-luciferin to the cells and bioluminescence was measured with a luminometer. *p<0.05 (two-tailed Student’s t-test). Error bars indicate the SD of triplicate samples in a representative experiment. The same trend was seen in three independent biological repeats. ES, Ewing sarcoma; MCAM, melanoma cell adhesion molecule; NK, natural killer.

Figure 4. Anti-MCAM-CAR-NK cells significantly decreased lung metastasis and prolonged animal survival in an ES orthotopic xenograft mouse model. (A) Schematic representation of the animal work schedule. Luciferase-expressing A673 cells (A673-luc) were injected into the tibia of NSG mice on day 0. 24 hours later, PBS or mock or CAR-NK cells were administered through tail vein (10⁷ cells/animal, once a week for 6 weeks). Tumor growth was monitored by IVIS imaging once a week. (B) Effects of NK/CAR-NK treatment on lung metastasis. Lungs were harvested at the endpoint. Graphs show the percentage of mice with pulmonary lesions in each indicated condition. **Fisher’s exact test p<0.01. (C) Representative images of lungs from two of the animals treated with PBS, mock NK, or CAR-NK cells. (D) Kaplan-Meier survival curves for comparison of survival among all groups. Animal survival was followed after therapy initiation using animal death or sacrifice as the terminal event using the Prism program V.8.0 (GraphPad Software). *p<0.05 (log-rank test). N=7 for each group. ES, Ewing sarcoma; IVIS, In Vivo Imaging System; MCAM, Melanoma cell adhesion molecule; NK, natural killer; PBS, phosphate buffered saline.

Figure 5. Effects of NKTR-255 on NK/CAR-NK cells and magrolimab (MAG) on macrophage phagocytosis of Ewing sarcoma (ES) cells in vitro. (A) Effects of NKTR-255 on the expression of NK cell receptors. Ex vivo expanded natural killer (NK) cells were incubated with NKTR-255 (40 ng/mL) for various periods of time (0–6 days) in the absence of IL-2 with replenishment of media and NKTR-255 at day 3. Flow cytometry was performed to detect the expression of NK cell receptors (CD94, NKp30, NKG2D, NKG2A, KIR and NKp44). Representative results from 4 days of incubation were shown. The same trend was seen in three independent biological repeats. (B) Effects of NKTR-255 on NK cell survival and expansion in the absence of IL-2 in vitro. NK cells (2.5×10⁶) at 14 days of expansion were incubated with NKTR-255 (40 ng/mL) for various periods of time (0–6 days) in the absence of IL-2 with replenishment of media and NKTR-255 at day 3. Viable cells were counted by the method of trypan blue staining. Error bars indicate the SD of triplicate samples in a representative experiment. The same trend was seen in three independent biological repeats. *p<0.05, **p<0.01 (two-tailed Student’s t-test). (C) In vitro cytotoxic activity of anti-MCAM-CAR-NK cells in combination with NKTR-255 against ES cells. CAR-NK cells were incubated with or without NKTR-255 (40 ng/mL) in the absence of IL-2 for 3 days before incubating with luciferase-expressing ES cells (A673, TC32, SKNMC) at E:T ratio of 0.2:1 for 4 hours. Cytotoxicity was analyzed by addition of D-luciferin and bioluminescent was measured with a luminometer. *p<0.05, **p<0.01 (two-tailed Student’s t-test). Error bars indicate the SD of triplicate samples in a representative experiment. The same trend was seen in three independent biological repeats. (D) CD47 expression on ES cells. CD47 expression levels were detected by flow cytometry on ES A673, TC32, SKNMC cells. (E) CD47 expression on ES PDX tumors. Immunofluorescent staining by CD47 antibody was performed to detect CD47 (green) expression on MSK-3 and NCH-EW-1 ES PDX tumors. The nuclei were counterstained with DAPI (blue). (F) MAG significantly enhanced macrophage phagocytosis of ES cells. Macrophages were derived from healthy donor peripheral blood mononuclear cells by recombinant human M-CSF (100 ng/mL). ES A673, TC32, SKNMC cells were labeled with CMFDA dye (recognized by FITC channel) and co-cultured with macrophages at a 2:1 ratio together with IgG or MAG (1 µg/mL) for 4 hours. Cells were harvested and stained with α-CD11b followed by flow cytometry analysis. The gated population (CD11b+FITC+) in the dot plots (left) show macrophages phagocytosed ES cells. Right, significantly enhanced macrophage phagocytosis of ES cells (A673, TC32, EWS502) by MAG (blue) compared with IgG control (orange). Quantification of the results in three biological repeats is shown. *p<0.05, **p<0.01 (two-tailed Student’s t-test). E:T, effector-to-target; PDX, patient-derived xenograft.

Figure 6. Combinatorial antitumor effects of anti-MCAM-CAR-NK cells with magrolimab (MAG) and NKTR-255 in ES xenograft mouse models. (A) Schematic representation of the animal work schedule in the cell-based xenograft mouse model. A673-luc cells were injected into the tibia of NSG mice on day 0. 24 hours later, vehicle control or CAR-NK (10⁷ cells/animal, i.v., once a week for 6 weeks) or NKTR-255 (0.3 mg/kg, i.v., once every 2 weeks for three times) or MAG (100 µg/animal, i.p., once every day for 12 days) alone or in combination were administered. Tumor growth was monitored by IVIS imaging once a week. (B) Anti-MCAM-CAR-NK cells in combination with NKTR-255 and MAG significantly reduced ES primary xenograft tumor growth in the ES cell-based orthotopic mouse model. Growth rates between groups were analyzed using mixed-effect model. N=10 for each group. Mice were followed until death or sacrificed if any tumor size reached 1.5 cm in any dimension. **p<0.01, ***p<0.001, ****p<0.0001 (ANOVA). (C) Anti-MCAM-CAR-NK cells in combination with NKTR-255 and MAG significantly reduced lung metastasis. Lungs were harvested at the endpoint. Graphs show the percentage of mice with pulmonary lesions in each indicated condition. *Fisher’s exact test p<0.05, ***p<0.001, ****p<0.0001. (D) Anti-MCAM-CAR-NK cells in combination with NKTR-255 and MAG significantly prolonged animal survival of ES xenografted NSG mice. Kaplan-Meier method was used for comparison of survival among all groups. Animal survival was followed after therapy initiation using animal death or sacrifice as the terminal event using the Prism program V.8.0 (GraphPad Software). *p<0.05 (log-rank test), ***p<0.001, ****p<0.0001. N=10 for each group. (E) Schematic representation of the animal work schedule in the PDX mouse model. PDX tumors (NCH-EW-1) were implanted into the flanks of NSG mice. When PDX tumors reach 5–7 mm in diameter (day 14), vehicle control or CAR-NK (10⁷ cells/animal, i.v., once a week for 3 weeks) or NKTR-255 (0.3 mg/kg, i.v., once every 2 weeks for twice) or MAG (100 ug/animal, i.p., once every day for 12 days) alone or in combination were administered. Tumor size was measured by caliper every day. (F) Combination of anti-MCAM-CAR-NK cells with NKTR-255 and MAG significantly reduced ES PDX tumor growth. Growth rates between groups were analyzed using mixed-effect model. n=9 for each group. Mice were followed until death or sacrificed if any tumor size reached 1.5 cm in any dimension. ****p<0.0001 (ANOVA). (G) MCAM-CAR-NK cells in combination with NKTR-255 and MAG significantly prolonged animal survival of ES PDX xenografted NSG mice. Kaplan-Meier method was used for comparison of survival among all groups. Animal survival was followed after therapy initiation using animal death or sacrifice as the terminal event using the Prism program V.8.0 (GraphPad Software). **p<0.01 (log-rank test), ****p<0.0001. n=9 for each group. ANOVA, analysis of variance; ES, Ewing sarcoma; i.v., intravenous; MCAM, Melanoma cell adhesion molecule; PDX, patient-derived xenograft.