Lactate oxidase nanocapsules boost T cell immunity and efficacy of cancer immunotherapy

Abstract

Cancer immunotherapy has reshaped the landscape of cancer treatment. However, its efficacy is still limited by tumor immunosuppression associated with the excessive production of lactate by cancer cells. Although extensive efforts have been made to reduce lactate concentrations through inhibition of lactate dehydrogenase, such inhibitors disrupt the metabolism of healthy cells, causing severe nonspecific toxicity. We report herein a nanocapsule enzyme therapeutic based on lactate oxidase, which reduces lactate concentrations and releases immunostimulatory hydrogen peroxide, averting tumor immunosuppression and improving the efficacy of immune checkpoint blockade treatment. As demonstrated in a murine melanoma model and a humanized mouse model of triple-negative breast cancer, this enzyme therapeutic affords an effective tool toward more effective cancer immunotherapy.

Fig. 1.. n(LOx) retains the enzyme activity with enhanced stability.

(A) Representative transmission electron micrograph (TEM) of n(LOx). Scale bar, 50 nm. (B) Gel electrophoresis of native LOx and n(LOx). (C) Enzyme kinetics of native LOx and n(LOx) in generating H₂O₂. Km, Michaelis constant. K_cat, turn over number. (D) Lactate concentration in the culture media of MDA-MB-231 cells after incubating with native LOx or n(LOx) (n=3). (E) H₂O₂ generation in the culture media of MDA-MB-231, 4T1, or B16F10 cells after incubating with native LOx or n(LOx) (0.5 μg/mL) for 30 minutes (n=5). a.u., arbitrary units. H2DCFDA is used as an indicator of H₂O₂, which can be oxidized by H2O2 to produce highly fluorescent 2’,7’-dichlorofluorescein (DCF). The fluorescence intensity of oxidized H₂DCFDA (DCF) was used to evaluate the generation of H₂O₂. (F) Fluorescence imaging of intracellular H₂O₂ in MDA-MB-231 tumor spheroids after incubating with native LOx or n(LOx). H₂O₂ and nuclei were stained by 6-chloromethyl (CM)-H₂DCFDA (green) and DAPI (blue), respectively. Scale bar, 100 μm. (G) Biodistribution of native LOx and n(LOx) after intratumoral injection. 4T1-Luc cells were used for luminescent imaging. Native LOx and n(LOx) were labeled with DyLight 755 for fluorescence imaging. (H and I) Concentrations of lactate (H) and H₂O₂ (I) in tumors from mice treated as indicated (n=6 mice per group). H₂O₂ was measured with Amplex Red reagent. All data are presented as mean ± s.d. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.. n(LOx) suppresses T_reg cell proliferation and promotes cytokine production by T cells.

(A and B) Relative cell proliferation (A) and percentages (B) of FOXP3⁺CD25^hi Treg cells in CD4⁺ T cells after incubating with n(LOx) in the presence or absence of catalase (CAT) (n=6). (C and D) Percentages of IFN-γ-producing CD4⁺ (C) and CD8⁺(D) T cells after incubating with MDA-MB-231 cells treated with n(LOx) in the presence or absence of CAT (n=6). (E and F) Percentages of IL-2-producing CD4⁺ (E) and CD8⁺ (F) T cells after incubating with MDA-MB-231 cells treated with n(LOx) in the presence or absence of CAT (n=6). (G) Immunohistochemistry (IHC) staining of CD4, CD8, and IFN-γ in triple-negative breast cancer (4T1) tissues from the indicated treatment groups. Mice were intratumorally injected with PBS or n(LOx) at day 7, 10, 13, and 16 after tumor engraftment. Tumors were isolated for IHC 25 days after tumor engraftment. Scale bar, 50 μm. All data are presented as mean ± s.d. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison test. ***P < 0.001, ****P < 0.0001; ns, not significant.

Fig. 3.. n(LOx) synergizes with aPD-L1 therapy to suppress melanoma.

(A) Schematic illustration of the treatment schedule in the mouse model of B16F10 melanoma. (B) Tumor volume changes of mice treated with PBS, aPD-L1, n(LOx), or the combination therapy of n(LOx) and aPD-L1 (n(LOx)/aPD-L1). The error bars indicate s.e.m. (n=6). (C) Kaplan-Meier survival curve of mice in different groups (n=6 to 8 mice per group; Mantel-Cox test. **P < 0.01, ****P < 0.0001). (D to G) Percentages of CD45⁺ cells (D), CD11c⁺ dendritic cells (E), F4/80⁺CD11b⁺ macrophages (F), and CD3⁺ T cells (G) in tumors from different groups. (H and I) Percentages of CD69⁺ cells in CD4⁺ (H) and CD8⁺ (I) T cells in tumors from different groups. (J and K) Percentages of IFN-γ ⁺ cells in CD4⁺ (J) and CD8⁺ (K) T cells in tumors from different groups. In (D to K), the error bars indicate s.d.; n=6 mice per group. (L) Schematic illustration of the engraftment of secondary tumor after n(LOx) treatment. (M) Tumor volume changes of the secondary tumors from mice treated with PBS or n(LOx). The error bars indicate s.e.m. (n=6). (N) Fold change of the population of different immune cells in the secondary tumors from mice treated with n(LOx). M1 macrophages were identified as CD80^hiCD206^low macrophages, and M2 macrophages were identified as CD80^lowCD206^hi macrophages. Statistical significance in (D to K) was calculated by one-way ANOVA with Tukey’s multiple comparison test. Statistical significance in (M) was calculated by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.. n(LOx) treatment promotes T cell activation and anti-tumor responses in melanoma.

(A) scRNA-Seq gene ontologies (GO) for genes significantly impacted after n(LOx) treatment versus control (PBS) in B16F10 cancer cells. (B) Pathway analysis of upregulated genes relative to T cell activation in cancers after n(LOx) treatment. (C) scRNA-Seq GO terms for genes significantly impacted after n(LOx) treatment versus control (PBS) in T cells. (D) Heatmap of gene expression values in cancer cells and T cells relative to cytokine production after n(LOx) treatment. (E) Heatmap of gene expression values related to Th1 type immune responses after n(LOx) treatment. (F) Scaled expression of chemokine genes (Cxcl1, Cxcl3, and Cxcl10) in different cell types after n(LOx) treatment.

Fig. 5.. n(LOx) inhibits TNBC in a humanized mouse model.

(A) Schematic illustration of the treatment schedule in the humanized mouse model of MDA-MB-231 TNBC. (B) Luminescence imaging of H₂O₂ in the tumor from different groups. L-012 was used as an in vivo H2O2 probe. White circles indicate the tumors. (C) Tumor volume changes of mice treated with PBS, aPD-L1, n(LOx) and CAT (n(LOx)/CAT), the combination therapy of n(LOx) and CAT with aPD-L1 (n(LOx)/CAT/aPD-L1), n(LOx), or the combination therapy of n(LOx) and aPD-L1 (n(LOx)/aPD-L1). The error bars indicate s.e.m. (n=6). (D) Percentages of CD4⁺ T cells and CD8⁺ T cells in the tumors from mice receiving PBS, n(LOx), or n(LOx)/CAT. The error bars indicate s.d. (n=6). (E) Percentages of CD4⁺ T cells and CD8⁺ T cells in the tumors from mice receiving PBS, aPD-L1, n(LOx)/aPD-L1, or n(LOx)/CAT/aPD-L1. The error bars indicate s.d. (n=6). (F) Percentages of FOXP3⁺ T_reg cells in CD4⁺ T cells in the tumor from mice treated with n(LOx). The error bars indicate s.d. (n=6). (G) Percentages of PD-1⁺ cells in CD8⁺ T cells in the tumors from different groups. The error bars indicate s.d. (n=6). (H) Immunofluorescence staining of CD3 (cyan) and IFN-γ (magenta) in tumor tissues. Scale bar, 20 μm. (I) Percentages of CD4⁺ and CD8⁺ T cells expressing TNF-α. The error bars indicate s.d. (n=6). (J) Percentages of IL-4⁺ cells in CD4⁺ T cells in the tumor from mice treated with n(LOx). The error bars indicate s.d. (n=6). (K) Percentages of CD4⁺ and CD8⁺ T cells with effector memory phenotypes. The error bars indicate s.d. (n=6). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.