Farnesylthiosalicylic acid-derivatized PEI-based nanocomplex for improved tumor vaccination

Abstract

Cancer vaccines that make use of tumor antigens represent a promising therapeutic strategy by stimulating immune responses against tumors to generate long-term anti-tumor immunity. However, vaccines have shown limited clinical efficacy due to inefficient delivery. In this study, we focus on vaccine delivery assisted by nanocomplexes for cancer immunotherapy. Nanocomplex-mediated vaccination can efficiently deliver nucleic acids encoding neoantigens to lymphoid tissues and antigen-presenting cells. Polyethylenimine (PEI) was conjugated with farnesylthiosalicylic acid (FTS) to form micelles. Subsequent interaction with nucleic acids led to formation of polymer/nucleic acid nanocomplexes of well-controlled structure. Tumor transfection via FTS-PEI was much more effective than that by PEI, other PEI derivatives, or naked DNA. Significant numbers of transfected cells were also observed in draining lymph nodes (LNs). In vivo delivery of ovalbumin (OVA; a model antigen) expression plasmid (pOVA) by FTS-PEI led to a significant growth inhibition of the OVA-expressing B16 tumor through presentation of OVA epitopes as well as other epitopes via epitope spreading. Moreover, in vivo delivery of an endogenous melanoma neoantigen tyrosinase-related protein 2 (Trp2) also led to substantial tumor growth inhibition. FTS-PEI represents a promising transfection agent for effective gene delivery to tumors and LNs to mediate effective neoantigen vaccination.

Keywords: farnesylthiosalicylic acid; immunotherapy; nanocomplex; neoantigen; polyethylenimine; vaccination.

Graphical abstract

Figure 1

Synthesis scheme and chemical characterization of FTS-PEI polymers (A) FTS-PEI was synthesized via a condensation reaction from linear PEI. PEI was reacted with FTS at different ratios in DMF at room temperature with DCC as a condensing reagent and DMAP as a catalyst. (B) ¹H nuclear magnetic resonance (NMR) spectra of the FTS-PEI (5% FTS, PEI MW = 2.5 kDa). (C) The matrix-assisted laser desorption/ionization (MALDI) spectra of free PEI (PEI MW = 2.5 kDa, black line) and FTS-PEI (5% FTS, PEI MW = 2.5 kDa, blue line).

Figure 2

In vitro and in vivo characterizations of DNA/FTS-PEI nanocomplexes (A) The hydrodynamic sizes and zeta potentials of FTS-PEI micelles (5% FTS, PEI MW = 2.5 kDa) and expression plasmid (p)GFP/FTS-PEI nanocomplexes formed at various N/P ratios. (B) Gel retardation assay of pGFP/FTS-PEI nanocomplexes at different N/P ratios. Samples were incubated for 20 min at room temperature prior to electrophoresis on a 1.5% (w/v) agarose gel (120 V, 20 min). (C) Gel electrophoresis assay of DNA displacement from pGFP/FTS-PEI nanocomplexes (N/P = 1) by dextran sulfate at various S/P ratios. (D) Linear PEI (MW = 2.5 kDa or 25 kDa) conjugated with different percentages of FTS (1% or 5%) were complexed with pGFP at N/P ratios of 0.5/1, 1/1, and 2.5/1, respectively. A total of 12 nanocomplexes were obtained that vary in the FTS/PEI (m/m) as well as N/P ratios. Nanocomplexes were individually administered via intratumor injection into tumor-bearing mice. Tumor tissues were collected after 48 h, and transfection efficiency was evaluated by fluorescence microscopic examination of GFP expression.

Figure 3

In vivo transfection of FTS-PEI (A) GFP expression after 48 h in tumor tissues and LNs. (B) Immunofluorescence staining of GFP in tumors and LNs. (C) Luciferase expression after 48 h in tumors and LNs. Data are presented as mean ± SEM, n = 3. p values were generated by two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (D) Fluorescence-activated cell sorting (FACS) analyses of GFP expression in fluorescence-labeled tumor cells (tdTomato⁺) in tumor tissues and DCs (CD11b⁺, MHC class II⁺) in LNs. (E) Comparison of pGFP transfection efficiency in tumors and LNs by PEI, OA-PEI, LA-PEI, and FTS-PEI, respectively.

Figure 4

FTS-PEI nanocomplex-mediated anti-tumor immunity, antigen presentation, and epitope spreading (A) PBS, pcDNA/FTS-PEI, free p-ovalbumin (pOVA), and pOVA/FTS-PEI were injected locally (intratumor) to B16F10-OVA (local tumors)-bearing mice once every 6 days for three times. Tumor growth was monitored once every 2 days. (B) Re-challenged tumors (distant tumors) were established via inoculation of tumor cells at the contralateral side after the 3^rd vaccination of the primary tumors, and tumor growth in both groups were monitored. Values reported are the mean ± SEM, n = 5. p values were generated by one-way ANOVA using the Tukey test for multiple comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) SIINFEKL-H2K(b) presentation by B16F10-OVA tumors treated with pOVA/FTS-PEI or PBS. Cells were stimulated with recombinant mouse IFN-γ (10 ng/mL) overnight to induce OVA surface expression. MFI, mean fluorescence intensity. Data are presented as the mean ± SEM, n = 5. p values were generated by two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (D) CD8⁺ T cells were isolated from B16F10-OVA tumors treated with PBS, pcDNA/FTS-PEI, free pOVA, or pOVA/FTS-PEI and co-cultured with B16F10 or MC38 for 6 h. Tumor cells were collected afterward for flow cytometry analysis for CFSE intensity. Data are presented as the mean ± SEM, n = 3. p values were generated by two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (E) CD8⁺ T cells were isolated from B16F10-OVA tumors treated with PBS, pcDNA/FTS-PEI, free pOVA, or pOVA/FTS-PEI and co-cultured with B16F10 or MC38 for 6 h. Culture medium was collected, and the level of IFN-γ in the supernatant was measured by ELISA.

Figure 5

FTS-PEI nanocomplex-mediated neoantigen vaccination induces tumor inhibition (A) PBS, pcDNA/FTS-PEI, free pTrp2, and pTrp2/FTS-PEI were injected locally (intratumor) to B16F10 (local tumors)-bearing mice, respectively, once every 6 days for three times. (B) Re-challenged tumors (distant tumors) were established via inoculation of B16F10 cells at the contralateral side, 2 days after the final vaccination, and tumor growth in all groups was monitored. Values reported are the mean ± SEM, n = 5. p values were generated by one-way ANOVA using the Tukey test for multiple comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

Effect of FTS treatment on PEI-mediated in vitro transfection (A) GFP expression of cultured B16F10 tumor cells without transfection (negative control [NC]), cells transfected with pGFP/PEI without FTS pre-treatment, and cells transfected with pGFP/PEI and FTS pre-treatment (0.625 μM, 1.25 μM, 2.5 μM, and 5 μM). (B) Flow cytometry analysis of GFP⁺ cells transfected with pGFP/PEI, with or without pre-treatment of FTS (0.625−5 μM), doxorubicin (0.3−2.4 μM), and paclitaxel (0.5−4 μM). Values reported are the mean ± SEM, n = 5. p values were generated by one-way ANOVA using the Tukey test for multiple comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.