Self-healing microcapsules synergetically modulate immunization microenvironments for potent cancer vaccination

Abstract

Therapeutic cancer vaccines that harness the immune system to reject cancer cells have shown great promise for cancer treatment. Although a wave of efforts have spurred to improve the therapeutic effect, unfavorable immunization microenvironment along with a complicated preparation process and frequent vaccinations substantially compromise the performance. Here, we report a novel microcapsule-based formulation for high-performance cancer vaccinations. The special self-healing feature provides a mild and efficient paradigm for antigen microencapsulation. After vaccination, these microcapsules create a favorable immunization microenvironment in situ, wherein antigen release kinetics, recruited cell behavior, and acid surrounding work in a synergetic manner. In this case, we can effectively increase the antigen utilization, improve the antigen presentation, and activate antigen presenting cells. As a result, effective T cell response, potent tumor inhibition, antimetastatic effects, and prevention of postsurgical recurrence are achieved with various types of antigens, while neoantigen was encapsuled and evaluated in different tumor models.

Fig. 1. Strategy of using self-healing microcapsules to modulate immunization microenvironments for cancer vaccination.

Through a diffusion manner and a healing process, the antigen could be efficiently loaded in the microcapsules. The corresponding characterizations of gigaporous microspheres and antigen-loaded microcapsules are displayed below: (A and A′) Scanning electron microscopy (SEM) images in a magnified view. Scale bars, 10 μm. (B and B′) SEM images in a local feature. Scale bars, 1 μm. (C and C′) Confocal laser scanning microscopy (CLSM) images in two-dimensional (2D) cut view. Scale bars, 5 μm. (D and D′) 3D reconstruction. Scale bars, 10 μm. Although without traditional molecular adjuvant, these microcapsules could still create in situ beneficial immunization microenvironments at the vaccination site, wherein sustained antigen release, constant APC recruitment, and favorable acidic surrounding collaborated effectively. As a result, a potent T cell response and tumor elimination could be achieved. S.C., subcutaneous.

Fig. 2. Spatiotemporal confluence of antigen release and APC recruitment maximized antigen utilization.

(A) Quantitative fluorescence intensity (left) and corresponding representative images (right) of antigen (blue)/microcapsules (red) at different time points. (B) Representative histological images of local tissues trapping microcapsules at different time points. PBS, phosphate-buffered saline. Scale bars, 50 μm. (C) The ratio of recruited cells versus local microcapsules. (D) Heat map representation of typical chemokine secretion at local injection site after 5 days of injection of different treatments. The color of the respective box in one row represents the value of the chemokine secretion in one sample compared with the normal expression level in untreated mice tissues. GRO-α, growth-regulated oncogene-alpha; IP-10, IFN-gamma-Inducible protein 10. (E) Comparison of number of OVA⁺ cells (left), intracellular OVA-Cy5 fluorescence intensity (middle; the number of mean fluorescence intensity was showed in corresponding colors), and utilization of OVA (right) after 5 days of injection of different formulations. The utilization of OVA at different formulations was calculated by formula: number of OVA⁺ cells × fluorescence intensity and normalized by utilization of free OVA. All bars represent means ± SD (n = 3).

Fig. 3. Local AM improved antigen uptake, cross-presentation, and APC activation.

(A) Two-photon fluorescent images of pH-sensitive dye loaded microcapsules in vivo (left) and pH value quantification during microcapsule degradation (right). (B) Activation of DCs induced by acidic environment in vitro. (C) Activation of DCs before/after acidification of NM microcapsules in vitro. (D) Comparison of recruited DCs and macrophages number at day 5 under different microenvironments [AM represents an acidic microenvironment with pH 6.5, and NM represents a neutral microenvironment with pH 7.2)]. (E) In vivo concentrations of the typical chemokines under different microenvironments. (F) Comparison of antigen uptake amounts in DCs and macrophages after 5 days of injection (mean fluorescence intensity was shown in corresponding colors). (G) Comparative evaluation of cross-presentation (MHC-1) in DCs and macrophages. (H) In vivo concentration of the indicated cytokines from explanted microcapsule tissues at AM and NM (left) and their one-on-one ratios (right, value > 1, represented up-regulated cytokines; value < 1, represented down-regulated cytokines). GM-CSF, granulocyte-macrophage colony-stimulating factor. All bars represent means ± SD (n = 3).

Fig. 4. Potent cellular immune response against OVA model antigen.

(A) Flow cytometric analysis of OVA-specific CD 8⁺ T cell (from OT-1 mice) proliferation in lymph nodes after different treatments [G1, PBS control; G2, pure antigen group (200 μg); G3, antigen mixed with gigaporous microspheres; G4, antigen encapsulated in healed microcapsules; the total antigen dose in G2 equaled one single administration in G3/G4]. (B) Comparison of CD8⁺ granzyme B⁺ T cells in splenocyte population analyzed by the FC. (C) Proliferation of OVA-specific CD8⁺ T cells (from OT-1 mice) at different time points (the curve was analyzed using a nonlinear regression). (D) Comparison of OVA-specific CD8⁺ T cell amounts using pentamer at day 14 after vaccination. (E) Calculation of accumulated proliferation performance (left) and corresponding half cycles (right; the half-cycles for G1 was not applicable). (F) In vitro killing assay showing the percentage of specific lysis using EL4 cells (mock control) or E.G7 cells (specific target) at different time points and the comparison of corresponding accumulated lysis performance. All bars represent means ± SD (n = 3).

Fig. 5. Antitumor efficacy and safe evaluation in an E.G7-OVA tumor model.

(A) The scheme of E.G7 tumor inhibition. (B) E.G7-OVA tumor volume development after different vaccinations [G1, PBS control; G2, pure peptides group (60 μg); G3, free antigen mixed with gigaporous microspheres group (60 μg); G4, antigen encapsulated in healed microcapsules group (60 μg); G4⁺, antigen encapsulated in healed microcapsules with an increased dose group (200 μg)]. Each line represents one animal. Mice were euthanized as their tumor volumes had reached 3000 mm³. (C) The survival time of immunized mice in E.G7-bearing mouse model. (D) Measurement of cytokine storm–related cytokines, interleukin-6 (IL-6), interferon-γ (IFN-γ), and tumor necrosis factor–α (TNF-α). (E) Body temperature changes of mice in different groups. (F) Representative H&E images of tissue slices in G4⁺. All bars represent means ± SD (n = 6).

Fig. 6. Antimetastasis performance in malignant B16-MUC1 mouse models.

(A) Scheme of B16 antimetastasis model (several mice per group were dissected, photographed and sliced at day 18). G1, PBS control; G2, pure antigen group (200 μg); G4, antigen (200 μg) encapsulated in healed microcapsules; G5: antigen (200 μg) encapsulated in healed microcapsules with 3 μg of MPLA. (B) Representative lung photographs, H&E-stained lung slices, and corresponding quantification of lung metastasis nodules after different treatments. Scale bars, 500 μm. (C) Representative kidney photographs, H&E-stained kidney slices. Scale bars, 500 μm. Corresponding tumor volumes of metastasis nodules (right) after different treatments. (D) Body weight changes after different treatments. (E) The survival time of mice with different treatments. All bars represent means ± SD (n = 6). i.v., intravenous.

Fig. 7. Evaluation of vaccination performance with neoantigen peptides.

(A) Scheme of neoantigen production and vaccination in primary tumor model. G1, PBS control; G2, pure antigen group (200 μg); AS04, antigen (200 μg) mixed with AS04 adjuvant group. G4, antigen (200 μg) encapsulated in healed microcapsules; G5, antigen (200 μg) encapsulated in healed microcapsules with 3 μg of MPLA. IF, immunofluorescence. (B) Enumeration of the numbers of polyfunctional CD8⁺ T cells in splenocytes from mice immunized with various vaccine formulations. Polyfunctional: secreted two or more effector cytokines for killing tumor cells. (C) Representative immunofluorescence image of tumor slices, corresponding portion of CD8⁺ T cells, and phenotyping of infiltrated CD8⁺ T cells (by FCS analysis) in 4T1 tumor after different treatments. GrB⁺, granzyme B⁺; DAPI, 4′,6-diamidino-2-phenylindole. (D) Representative immunofluorescence image of tumor slices, corresponding portion of foxp3 T cells in 4T1 tumor after different treatments. (E) Individual tumor growth kinetics of primary tumors and tumor growth inhibition rates of different treatments. (F) The scheme vaccination in postsurgical recurrence model. (G) All bioluminescence images (BLI) of 4T1-luc tumors before/after surgical resection and quantitative statistics of BLI tumor burden at day 14. BLI values are represented as photons per second per square centimeter per steradian in regions of recurrent tumors (n = 6). (H) Individual tumor growth kinetics of postsurgical recurrence models and tumor growth inhibition (TGI) rates of different treatments (n = 6). All bars represent means ± SD.