Immune-modulative nano-gel-nano system for patient-favorable cancer therapy

Abstract

Current cancer immunotherapies exhibit low response rates attributed to suppressive tumor immune microenvironments (TIMEs). To address these unfavorable TIMEs, supplementation with tumor-associated antigens and stimulation of immune cells at target sites are indispensable for eliciting anti-tumoral immune responses. Previous research has explored the induction of immunotherapy through multiple injections and implants; however, these approaches lack consideration for patient convenience and the implementation of finely tunable immune response control systems to mitigate the side effects of over-inflammatory responses, such as cytokine storms. In this context, we describe a patient-centric nano-gel-nano system capable of sustained generation of tumor-associated antigens and release of adjuvants. This is achieved through the specific delivery of drugs to cancer cells and antigens/adjuvants to immune cells over the long term, maintaining proper concentrations within the tumor site with a single injection. This system demonstrates local immunity against tumors with a single injection, enhances the therapeutic efficacy of immune checkpoint blockades, and induces systemic and memory T cell responses, thus minimizing systemic side effects.

Keywords: In situ cancer vaccine; Injectable hydrogel; Multi-targetable; Nanocomplexes; Patient-favorable.

Graphical abstract

Fig. 1

Schematic illustration of a nano-gel-nano (NGN) system for local and long-term modulation of a suppressive TIME and enhancement of ICB therapeutic efficacy with a single injection for patient-favorable immunotherapy, including the mechanism underlying its action.

Fig. 2

Characterizations of the RCP hydrogel-based NGN system A) Illustration of concept of nano-gel-nano (NGN) system. B) Gel retardation assay of the RCP/CpG complexes as a function of w/w ratios. C) The photo images of PTX solubility in RCP solution in wide range of concentration. D) Sizes and morphologies of the nanocomplexes (RCP, RCP-P, RCP-C, and RCP-PC; n = 3). Scale bar is 50 nm ∗∗∗ indicates P$<$ 0.001 by one-way ANOVA followed by Tukey's post hoc analysis. E) Temperature-dependent sol-gel transition of 10 wt% aqueous RCP solutions. F) The storage modulus (G′) and loss modulus (G″) of the RCP at 4 and 37 °C. G) In vitro release profile of dissociated nanocomplexes from the RCP-P and RCP-C hydrogel using PTX and FITC-tagged CpG, respectively. H) IVIS images showing CpG retention within the RCP-C hydrogel of 10 % and CpG alone using FITC-tagged CpG in a B16F10 tumor-bearing mouse. I) Transmission electron microscope (TEM) image of dissociated nanocomplexes from the PCP-PC hydrogel after 7 days of incubation at 37 °C. The scale bar is 200 nm ∗∗∗ indicate P$<$ 0.001 by one-way ANOVA followed by Tukey's post hoc analysis.

Fig. 3

In vitro selective PTX delivery-mediated cancer cell death for TAA release A) Schematic illustration of active intracellular uptake of the nanocomplexes into cancer cells through RGD moiety and release of DAMPS by immunogenic cell death. B) Representative images of RGD moiety-dependent cellular uptake of the RCP nanocomplexes with Nile Red (NR) in B16F10 cancer cells. The scale bar represents 50 μm. C) Quantification of NR fluorescence intensity (n = 5). D) Percentages of apoptosis in B16F10 cells (Annexin-V positive cells: early and late apoptosis stage) after treatment of the nanocomplexes (RCP, RCP-P, RCP-C, RCP-PC; PTX 5 μg/mL, CpG 5 μg/mL). E) Flow cytometry for the quantification of intracellular ROS level using DCFH-DA 24 h following treatment. F) Quantification of HMGB1 in supernatant of B16F10 cells by ELISA 24 h following treatment. G) Western blot assay of the HSP70 expression in B16F10 cells after 24 h of treatment. The graph under the western bands shows the relative protein expression of HSP70 by quantifying the greyscale values with ImageJ. H) Quantification of calreticulin expression on the cell surface by flow cytometry analysis 24 h following treatment. I) Immunofluorescence images and dot plots of CRT expression on the B16F10 cells after 24 h with different treatments. J) Cell viability for checking the reducing cancer cell growth in the B16F10 cell line by PTX that was released from 10 % RCP-PC hydrogel after 7 days at 37 °C. ∗ ∗∗, ∗∗∗, and ns indicate P$<$ 0.05, P$<$ 0.01, P$<$ 0.001 and not significant, respectively by one-way ANOVA followed by Tukey's post hoc analysis.

Fig. 4

In vitro selective adjuvant delivery-mediated immune modulation of BMDCs. A) Schematic illustration of activation of APCs by the nanocomplexes. B) Confocal images of in vitro cellular uptake of the RCP nanocomplexes with FITC tagged CpG in BMDM and BMDC after 2 h. Scale bar represents 20 μm. C) Western blot assay of the NF-κB (total p65 and phosphate p65) expressions in BMDCs. Cytokine levels of D) IL-6, E) IL-12p70, F) IFNγ, and G) TNFα in supernatant of BMDCs measured by ELISA (n = 3 from different culture wells). Flow cytometry for the surface markers of H) CD40 and I) MHCII expression in BMDCs (MFI, mean fluorescence intensity) J) CD40 expression in BMDCs by the RCP-PC nanocompelxes that was released from 10 % RCP-PC hydrogel after 7 days at 37 °C. All results were obtained from three different culture wells (n = 3). ∗ ∗∗, ∗∗∗, and ns indicate P$<$ 0.05, P$<$ 0.01, P$<$ 0.001 and not significant, respectively by one-way ANOVA followed by Tukey's post hoc analysis.

Fig. 5

Antitumor effects with TIME modulation. A) Schematic illustration of schedule for in vivo study. B) Survival rates as determined by Kaplan–Meier analysis (n = 6–8). C) Tumor volumes on day 10. D) Tumor volume changes until day 20. E) Individual tumor volumes until day 20. Numbers on the top side represent the number of tumor-free mice and median survival (m.s.). F) Percentages of CD40⁺ DCs in tumor on day 3 (n = 5). G) Percentages of CD80⁺ and H) MHCII ⁺ macrophages in tumor on day 3 (n = 5). I) Percentages of CD8⁺ T cells in tumor day 7 (n = 5). J) Representative dot plots for IFNγ⁺ on CD8⁺ T cells; K) Percentages of IFNγ⁺ on CD8⁺ T cells in tumor (n = 5). ∗ ∗∗, ∗∗∗, and ns indicate P$<$ 0.05, P$<$ 0.01, P$<$ 0.001, and not significant, respectively by one-way ANOVA followed by Tukey's post hoc analysis.

Fig. 6

Increased anti-tumor effects and inhibited over-inflammatory side effects of Ag/Adjuvant releases. A) Schematic illustration of schedule for anti-tumor therapeutic effect of the RCP-P hydrogel (10 %, 20 μL) and solution (2 %, 100 μL) in a B16F10 melanoma tumor model (RCP polymer 2 mg, PTX 20 μg per mouse, PBS as control). B) Tumor volume changes until day 14. n = 5 biologically independent animals per group. C) The individual tumor volumes until day 16, and numbers on the top side represent the number of survived mice. D) Schematic illustration of schedule for the administration of the RCP-PC (s.c and i.v.) and RCP (i.v.), mice peripheral blood collecting and analysis. E) The mice plasma level of inflammatory cytokines including IL-1β, IFNγ, IL-12p70, and IL-2 from day 0 (before administration) to day 14 (n = 3).

Fig. 7

Synergistic effect withαPD-1 by immune modulation and systemic immune activation-based inhibition of recurrence. A) Schematic illustration of schedule for the combined therapeutic procedure of the RCP hydrogel system with αPD-1 on B16F10. B) Survival rates represent Kaplan–Meier plots for all groups (n = 8). C) Tumor volume changes until day 20. D) The photographs of reinoculated B16F10 tumor in cured mice by RCP-PC with αPD-1 at day 75 following initiation of treatment. E) Treatment schedule for systemic anti-tumor immune response by RCP-PC with αPD-1. F) Primary tumor volume changes until day 20; n = 8 biologically independent animals per group. G) Representative images of mice at 10 days after intratumoral injection; numbers below the image represent the number of total mice/the number of dead mice/the number of mice that developed secondary tumors. H) Percentages of CD8⁺ T cells in TDLNs (n = 4), I) memory T cells in TDLNs (n = 4), and J) memory T cells in spleens (n = 4) on day 12 after the injection of RCP-PC. ∗ ∗∗, and ∗∗∗ indicate P$<$ 0.05, P$<$ 0.01, and P$<$ 0.001, respectively, by one-way ANOVA followed by Tukey's post hoc analysis.