Constitutive IL-7 signaling promotes CAR-NK cell survival in the solid tumor microenvironment but impairs tumor control

Abstract

Background: Adoptive transfer of chimeric antigen receptor (CAR)-expressing natural killer (NK) cells has demonstrated success against hematological malignancies. Efficacy against solid tumors has been limited by poor NK cell survival and function in the suppressive tumor microenvironment (TME). To enhance efficacy against solid tumors, stimulatory cytokines have been incorporated into CAR-NK cell therapeutic approaches. However, current cytokine strategies have limitations, including systemic toxicities, exogenous dependencies, and unwanted TME bystander effects. Here, we aimed to overcome these limitations by modifying CAR-NK cells to express a constitutively active interleukin (IL)-7 receptor, termed C7R, capable of providing intrinsic CAR-NK cell activation that does not rely on or produce exogenous signals nor activate bystander cells.

Methods: We examined persistence, antitumor function, and transcriptional profiles of CAR-NK cells coexpressing C7R in a novel tumor immune microenvironment (TiME) co-culture system and against hematologic and solid tumor xenografts in vivo.

Results: Peripheral blood NK cells expressing a CAR directed against the solid tumor antigen GD2 and modified with C7R demonstrated enhanced tumor killing and persistence in vitro compared with CAR-NK cells without cytokine support and similar functions to CAR-NK cells supplemented with recombinant IL-15. C7R.CAR-NK cells exhibited enhanced survival and proliferation within neuroblastoma TiME xenografts in vivo but produced poor long-term tumor control compared with CAR-NK cells supplemented with IL-15. Similar results were seen using C7R-expressing CD19.CAR-NK cells against CD19+leukemia xenografts. Gene expression analysis revealed that chronic signaling via C7R induced a transcriptional signature consistent with intratumor stressed NK cells with blunted effector function. We identified gene candidates associated with chronic cytokine-stressed NK cells that could be targeted to reduce CAR-NK cell stress within the solid TME.

Conclusion: C7R promoted CAR-NK cell survival in hostile TMEs independent of exogenous signals but resulted in poor antitumor function in vivo. Our data reveals the detrimental role of continuous IL-7 signaling in CAR-NK cells and provides insights into proper application of cytokine signals when attempting to enhance CAR-NK cell antitumor activity.

Keywords: Adoptive cell therapy - ACT; Chimeric antigen receptor - CAR; Cytokine; Natural killer - NK; Tumor microenvironment - TME.

Figure 1. C7R transduction increases CAR-NK persistence and short-term function. (A) Representative flow plots demonstrating transduction efficiencies in untransduced (UTD), CAR, and C7R.CAR-NK cells. (B) Representative flow plot of pSTAT5 after overnight cytokine starvation. For exIL-15, cytokine-starved CAR-NK cells were stimulated for 30 min with 10 ng/mL IL-15. (C) Pooled data from (B), n=4 independent donors. Data presented as mean±SD; p*<0.05; p**<0.01. (D) Representative flow plots of intracellular IFN-γ and CD107a in 4-hour flow co-culture assays with LAN1 tumor cells at a 2:1 effector-to-target ratio ratios. (E) Pooled data from (D), n=4 independent donors. presented as mean±SD; p*<0.05; p**<0.01; p***<0.001. (F) CAR-NK fold expansion in the absence (black) or presence of IL-7 (10 ng/mL; orange), IL-15 (10 ng/mL; green), or transduced with C7R. CAR, chimeric antigen receptor; exIL, exogenous interleukin; IFN, interferon; NK, natural killer; pSTAT, phosphorylated signal transducer and activator of transcription.

Figure 2. C7R augments CAR-NK function in in vitro TME mimics. (A) Specific cytotoxicity of indicated NK cells after 4-day co-culture with CHLA255 and autologous CD14+monocytes. Briefly, 2.5×10⁵ CHLA255 and 2.5×10⁵ autologous CD14+ monocytes were cultured in a 24-well plate for 3 days prior to the addition of NK cells after which 1×10⁵ UTD or CAR-NK cells were added directly into the cell culture. For exIL-15, IL-15 was supplemented at a concentration of 10 ng/mL and was replenished each day. (B) CAR+ NK fold-expansion after 4 days of TME co-culture. CAR+ NK fold expansion was quantitated via absolute CAR+ NK counts calculated from counting beads added to flow cytometry samples prior to acquisition. Each unique shape represents a different donor. (C) Cell culture supernatants were analyzed for IFN-γ, TNF-α, and GM-CSF after 4 days of TME co-culture in indicated CAR-NK conditions. Data presented as mean±SD of independent donors (n=6, A; n=4, B, C). *p<0.05, **p<0.01, ****p<0.001 (one-way paired analysis of variance, A, B, C). CAR, chimeric antigen receptor; exIL, exogenous interleukin; GM-CSF, granulocyte-macrophage colony stimulating factor; IFN, interferon; NK, natural killer; TME, tumor microenvironment; TNF, tumor necrosis factor; UTD, untransduced.

Figure 3. C7R.CAR-NK fail to control tumor long-term in in vivo xenograft. (A) In vivo schematic. Briefly, 1×10⁶ CHLA255 and 0.5×10⁶ autologous CD14+peripheral blood mononuclear cells were implanted subcutaneously in the right flank of NSG mice. 2 weeks later, 5×10⁶ CAR-NK cells were administered intravenously followed by a second dose 3 days later. PBS (CAR, C7R.CAR) or IL-15 (CAR exIL-15) was administered i.p. three times per week. (B) Spider plots of tumor volume in each indicated NK condition overlaid with control PBS (gray) condition. Tumor volumes were determined by caliper measurement. (C) Tumor volumes from (B) plotted at respective timepoints in indicated CAR-NK condition. (D). Sequential bioluminescent imaging of NK cells administered intravenously. (E) Quantitated bioluminescent signal of NK cells over time from (D). Four mice were used for each NK group and three for control in B, C. Three mice were used for each NK group in D, E. Data presented as averages between mice±SD (n=4, C; n=3, E). *p<0.05, **p<0.01 (one-way analysis of variance, C, E). CAR, chimeric antigen receptor; exIL, exogenous interleukin; i.p., intraperitoneal; NK, natural killer; PBS, phosphate-buffered saline.

Figure 4. C7R.CAR-NK cells fail to control tumor despite localization and survival in the tumor microenvironment. (A) In vivo schematic. Briefly, 1×10⁶ CHLA255 and 0.5×10⁶ autologous CD14+ peripheral blood mononuclear cells were implanted subcutaneously in the right flank of NSG mice. 2 weeks later, 5×10⁶ CAR-NK cells were administered intratumorally (i.t.) followed by a second dose 3 days later. PBS (CAR, C7R.CAR) or IL-15 (CAR exIL-15) was administered i.p. three times per week. (B) Spider plots of tumor volume in each indicated CAR-NK condition overlaid with control PBS (gray) condition. Tumor volumes were determined by caliper measurement. (C) Gating strategy to determine GFP+ NK phenotype in digested tumors, red boxes indicate gated population for downstream analysis in (D), (E), (F), (G), (H), (I). Cell surface receptors and intracellular transcription factors expression from cell populations gated in red from (C). Three mice were used for each group in B, C. Data presented as averages between mice±SD (n=3, B, C, D, E, F, G, H, I). *p<0.05, **p<0.01 (two-tailed t-test, D, E, F, G, H, I). CAR, chimeric antigen receptor; exIL, exogenous interleukin; GFP, green fluorescent protein; i.p., intraperitoneal; NK, natural killer; PBS, phosphate-buffered saline.

Figure 5. C7R-CD19.CAR-NK fail to control tumor long-term in B-cell leukemia. (A) In vivo schematic. Briefly, on day −1, 5×10⁴ Nalm6 were administered intravenously in NSG mice. On day 0, 4×10⁶ CAR-NK cells were administered intravenously followed by a second dose 7 days later. IL-15 (1 µg/dose) was administered i.p. only in the CAR exIL-15 treated mice two to three times per week. (B) Representative imaging of Nalm6 growth over time. (C) Quantitated bioluminescent signal of Nalm6 over time from (C). (D) Quantitated Nalm6 bioluminescence on day 35. Four mice were used for each group in B, C. Data presented as averages between mice±SD (n=4, D; two-way t-test). CAR, chimeric antigen receptor; exIL, exogenous interleukin; i.p., intraperitoneal; NK, natural killer; PBS, phosphate-buffered saline.

Figure 6. Long-term C7R signaling induces phenotype associated with NK dysfunction. (A) Experimental schematic. Briefly, C7R.CAR or exIL-15 CAR-NK cells were cultured for 14 days followed by total RNA isolation. RNA was also isolated from day 0 samples. Gene expression analysis was subsequently performed using the Human Immune Exhaustion and nCounter Analysis System (NanoString). (B) On day 14, CAR exIL-15 or C7R.CAR-NK cells were stimulated with the 1A7 anti-idiotype antibody for 16 hours to stimulate CAR-mediated production of granzyme B measured via ELISA. (C) Day 14 CAR-NK cells were cultured at a 1:1 ratio to assess CHLA255 tumor growth analyzed via live cell imaging using IncuCyte. (D) Area under the curve (AUC) from (C). (E) Soluble cytokine concentrations in different CAR-NK conditions after 48 hours anti-idiotype 1A7 stimulation. (F) Principal component analysis in day 14 CAR.exIL-15 versus day 14 C7R.CAR. (G) Volcano plot demonstrating differentially expressed genes in day 0 versus day 14 C7R.CAR NK cells. Horizontal dashed line indicates p value<0.05; vertical dashed lines indicate genes that are <log2 fold change (−1) or >log2 fold change. (H) Directed global significance scores of indicated pathways at day 14 RNA. Data presented as mean±SD of different donors. (n=3, B–H). p*<0.05, p**<0.01, p****<0.001 (paired two-sided t-test, B, E; one-way analysis of variance, D). CAR, chimeric antigen receptor; CCL, C-C motif ligand; exIL, exogenous interleukin; GM-CSF, granulocyte-macrophage colony stimulating factor; IFN, interferon; NK, natural killer; TNF, tumor necrosis factor.