Inflammation-Triggered Cancer Immunotherapy by Programmed Delivery of CpG and Anti-PD1 Antibody

Abstract

Inflammation-triggered combination delivery of anti-PD-1 antibody and CpG oligodeoxynucleotides (CpG ODNs) has been demonstrated to prevent cancer relapse utilizing postsurgical inflammatory response. The controlled release of anti-PD1 and CpG ODN by CpG DNA-based "nano-cocoons" can induce considerable immune response, which in turn significantly prolongs the survival time of mice.

Keywords: CpG; anti-PD1; cancer immunotherapy; drug delivery; nanobiotechnology.

Figure 1

Schematic illustration of delivery of CpG and anti-PD1 antibody (aPD1) by DNA nano-cocoon under an inflammation condition. (A) CpG-sequence containing DNA nano-cocoon (DNC) loaded with anti-PD1 antibody and caged restriction enzyme for inflammation-trigged fragmentation of DNC for releasing both CpG DNA and aPD1. (B) A schematic representation of in vivo tumor immunotherapy after primary tumor resection, local injection and treatment of DNC-based delivery system. (C) Released CpG DNA activates dendritic cells (DCs) to drive T cell response with aPD1 for PD 1 blockade.

Figure 2

Characterization of CpG DNCs loaded with aPD1 and caged enzyme and enzyme-responsive drug release. (A) TEM imaging of HhaI-TGMS-DNCs-aPD1 nanocomposites (Scale bar: 500 nm). Inset: zoom-in image (Scale bar: 200 nm). (B) Dynamic light scattering characterization of HhaI-TGMS-DNCs-aPD1 nanocomposites. (C) Schematic of LPS activation of RAW264.7 macrophages for mimicking inflammatory conditions. (D) Gel electrophoresis of HhaI-TGMS-DNCs-aPD1 nanocomposites incubated with cell culture supernatant from the activated and non-activated macrophages at the difference time points (Lane 0, 0min; Lane 1, 30min; Lane 2, 1h; Lane 3, 2h; Lane 4, 4h; Lane 5, 6h; Lane M, DNA ladder). (E-F) Percentage of DNA and aPD1 released from TGMS-DNC nanocomposites when incubated with cell culture supernatant from activated and non-activated macrophages at different time points. (G-H) AFM images and hydrodynamic size of HhaI-TGMS-DNCs-aPD1 nanocomposites when incubated with cell culture supernatant from non-activated macrophages. (I-J) AFM images and hydrodynamic size of HhaI-TGMS-DNCs-aPD1 nanocomposites when incubated with cell culture supernatant from activated macrophages (Scale bar: 500 nm). The error bars are based on the standard deviations (SD) of triplicated samples.

Figure 3

In vivo tumor therapy to reduce post-surgical tumor relapse via CpG DNC delivery system. (A) In vivo bioluminescence imaging of the B16F10 tumors of the different groups after removal of primary tumor. (Group 1, PBS control; G2, HhaI-TGMS-DNCs; G3, HhaI-TGMS-cDNCs-aPD1; G4, free aPD1/free CpG nucleotides; G5, HhaI-TGMS-DNCs-aPD1) (B) Quantified tumor signals and (C) mean tumor growth of different groups of mice after various treatments indicated. Pie chart shows percent CR rate (orange) (n=10). The black arrow indicates the surgery time. (D) Representative plots of T cells in relapsed tumors analyzed by the flow cytometry. (Gated on CD3+ T cells). (E) Representative plots of activated CD8 T cells (CD44+CD62L-) in relapsed tumors analyzed by the flow cytometry (gated on CD8+ T cells). (F) Immunofluorescence of relapsed tumors showed CD4+ T cells and CD8+ T cells infiltration (Scale bar: 100 μm). (G) Absolute number of the activated CD8 cells present in tumors for the study shown in C&D. (H) Ratios of the tumor-infiltrating CD8+ T cells and effective CD4+ T cells over regulatory T cells in the relapsed tumors upon various treatments. Statistical significance was calculated by 2-way ANOVA using the Tukey post-test. P value: *, P<0.05; **, P<0.01; ***P<0.005.

Figure 4

Systemic antitumor efficacy could be obtained by the local injection of DNC delivery system at the surgical site. (A) In vivo bioluminescence imaging of the B16F10 metastasis of different groups after removing of primary tumors at different time points. (G1, PBS control; G2, HhaI-TGMS-DNCs; G3, HhaI-TGMS-cDNCs-aPD1; G4, free aPD1 / free CpG nucleotides; G5, HhaI-TGMS-DNCs-aPD1). (B) Quantified tumor signals according to A. Every line represents one animal and each dot shows the whole animal photon count (n=3). (C) Kaplan Meier survival curves for treated and control mice. Shown are ten mice per treatment group. (D) IFN-γ CD8 CTLs T-cell in splenocytes of mice with various treatments indicated in A. (E) Quantified IFN-γ CD8 CTL T-cell in splenocytes from three independent experiments. (F) CTL-mediated immune responses measured by incubating restimulated splenocytes with B16 tumor cells. The error bars are based on the standard error of the mean (SEM) of three mice.

Figure 5

Antitumor efficacy by the local injection of DNC delivery system in spontaneous metastasis model. (A) In vivo bioluminescence imaging at different time points of the B16F10 metastasis of different groups after removal of primary tumors. (G1: PBS control, G2: aPD1 i.v. Inj., G3: aPD1+CpG local Inj., G4: HhaI-TGMS-DNCs-aPD1) (B) Representative lung photographs and H&E-stained lung slices collected from mice post different treatments indicated (Scale bar: 150 μm). (C) Average weights of relapsed tumors collected from mice in the end of various treatments indicated. (D) Quantification of lung metastasis nodules after different treatments. (E) Quantified tumor signals according to A. (F) Kaplan Meier survival curves of mice in after various treatments indicated. Shown are ten mice per treatment group. P value: *, P<0.05.