Optimized dithranol-imiquimod-based transcutaneous immunization enables tumor rejection

Abstract

Introduction: Transcutaneous immunization (TCI) is a non-invasive vaccination method promoting strong cellular immune responses, crucial for the immunological rejection of cancer. Previously, we reported on the combined application of the TLR7 agonist imiquimod (IMQ) together with the anti-psoriatic drug dithranol as novel TCI platform DIVA (dithranol/IMQ based vaccination). In extension of this work, we further optimized DIVA in terms of drug dose, application pattern and established a new IMQ formulation.

Methods: C57BL/6 mice were treated on the ear skin with dithranol and IMQ-containing ointments together with ovalbumin-derived peptides. T cell responses were determined by flow cytometry and IFN-ɤ ELISpot assay, local skin inflammation was characterized by ear swelling.

Results: Applying the adjuvants on separate skin sites, a reduced number of specific CD8⁺ T cells with effector function was detectable, indicating that the local concurrence of adjuvants and peptide antigens is required for optimal vaccination. Likewise, changing the order of dithranol and IMQ resulted in an increased skin inflammatory reaction, but lower frequencies of antigen-specific CD8⁺ T cells indicating that dithranol is essential for superior T cell priming upon DIVA. Dispersing nanocrystalline IMQ in a spreadable formulation (IMI-Sol+) facilitated storage and application rendering comparable immune responses. DIVA applied one or two weeks after the first immunization resulted in a massive increase in antigen-specific T cells and up to a ten-fold increased memory response. Finally, in a prophylactic tumor setting, double but no single DIVA treatment enabled complete control of tumor growth, resulting in full tumor protection.

Discussion: Taken together, the described optimized transcutaneous vaccination method leads to the generation of a strong cellular immune response enabling the effective control of tumor growth and has the potential for clinical development as a novel non-invasive vaccination method for peptide-based cancer vaccines in humans.

Keywords: cytotoxic T lymphocytes (CTL); dithranol (anthralin); imiquimod (IMQ); trancutaneous immunization; tumor rejection.

Figure 1

Optimization of DIVA in terms of application order in a single treatment. (A) Mice were immunized on both ears with dithranol petrolatum (25 mg [0.6 µg/mg) and IMQ (2.5 mg imiquimod/ear) together with SIINFEKL (OVA_257-264) and OVA_323-337 as indicated or left untreated. (B) Ear thickness was determined on day 8 using a micrometer (n= 5-10). (C) On day 7, the frequency of SIINFEKL-specific CD8⁺ T cells was determined in the blood of the mice using tetramer-staining (n=9, n=2 untreated) and (D) the effector function of the peptide-specific T cells was assessed after peptide restimulation for 20 h in an ELISpot assay (n=10, n=4 untreated). (E) Mice were immunized on one ear or on both ears as indicated with dithranol petrolatum (16 µg dithranol/ear) on day 0 and IMQ (2.5 mg imiquimod/ear) together with SIINFEKL (OVA_257-264) and OVA_323-337 on day 1 or left untreated and frequency of SIINFEKL-specific CD8⁺ T cells was assessed on day 7 in the blood of the mice (n=2-5). (F) Cytolytic activity of the specific T cells was determined 20h after adoptive transfer of SIINFEKL-labelled spleen cells (n=2.5). Shown are the median values (bars) with the individual measured values of the animals. *p < 0.05 by Kruskal-Wallis test and Dunn’s post-test.

Figure 2

Optimization of DIVA in terms of application dosis in a single treatment. (A) Mice were immunized with 16 µg dithranol dispersed in petrolatum [25mg, 0.6 µg/mg w/w] and different amounts of imiquimod ointment (2.5 - 0.5 mg imiquimod/ear) together with SIINFEKL (OVA_257-264) and OVA_323-337 or left untreated. On day 7, the frequency of SIINFEKL-specific CD8⁺ T cells was determined in the blood of the mice using tetramer-staining (n=4-13, n=3 untreated). (B) the cytolytic activity was determined 20 h after adoptive transfer of peptide-labelled target cells (n=4-13, n=3 untreated) (C) Mice were immunized as indicated with different doses of dithranol dispersed in petrolatum [16-2 µg dithranol/ear] on day 0 and IMQ (2.5mg imiquimod/ear) together with SIINFEKL (OVA_257-264) and OVA_323-337 on day 1 or left untreated and frequency of SIINFEKL-specific CD8⁺ T cells was assessed on day 7 in the blood of the mice (n=2-5). (D) Mice were immunized with dithranol petrolatum [16 µg dithranol/ear] and imiquimod ointment (2.5 mg imiquimod/ear) together with different amounts of ovalbumin peptides (SIINFEKL (OVA_257-264) and OVA_323-337, 1-100 µg each) or left untreated. On day 7, 14, and 35, the frequency of SIINFEKL-specific CD8⁺ T cells was determined via tetramer staining of the blood (n=5-16) and on day 35 the effector function of the CD8 T cells was assessed in an ELISpot assay after restimulation of splenocytes with (E) OVA_257-264 or (F) OVA_323-337 peptide for 20 h (n=2-6). Shown are the median values (bars) with the individual measured values of the animals. *p < 0.05 by Kruskal-Walli’s test and Dunn’s post-test.

Figure 3

IMI-Sol+ reveals equal efficiency in DIVA approach compared to IMI-Sol. (A) Imiquimod nano-emulsion IMI-Sol and (B) IMI-Sol+. (C) Mice were immunized with dithranol petrolatum [16 µg dithranol/ear] and the two different imiquimod ointments IMI-Sol or IMI-Sol+ (2.5 mg imiquimod/ear) together with SIINFEKL (OVA_257-264) and OVA_323-337 or left untreated. The frequencies of SIINFEKL-specific T cells were determined via tetramer staining of the blood on day 7 (n=6-12) and (D) their cytolytic function was assessed 20 h after adoptively transfer of peptide-labelled target cells (n=8-13). (E) In the memory phase on day 35 the frequencies of SIINFEKL-specific CD8⁺ T cells were determined and (F) the effector function was revealed after restimulation of splenocytes for 20 h in an ELISpot assay to determine specific IFN-ɤ production (n=6-9). (G) Ear thickness was measured after treatment with dithranol and or imiquimod formulations on day 8 (n=4-20). Dithranol/IMI-Sol values from Figure 1A . Bars represent median value with single values depicted as dots. *p < 0.05 by Kruskal-Walli’s test and Dunn’s post-test.

Figure 4

Second immunization one week after the initial treatment induces a ten-fold increased memory response. Mice were immunized with dithranol petrolatum [8 µg dithranol/ear] (day 0) and IMI-cream (2.5 mg Imiquimod/ear) together with SIINFEKL (OVA_257-264) and OVA_323-337 (100 µg per ear) (day 1) (A) in one single application (DIVA) or (B) boosted on day 7/8 or (C) on day 14/15. One week after the first treatment, tetramer staining of blood cells was performed to assess the frequency of SIINFEKL-specific CD8⁺ T cells once a week until day 49 (n=5 for each group). Bars represent median value with single values depicted as dots. (D) Analysis of the effector function of specific CD8⁺ T cells in the memory phase (CD8⁺, H2-Kb-SIINFEKL⁺, CD44^high/CD62L^low cells). Gating strategy is depicted in Supplemental Figure 2A . (E) Restimulation of splenocytes with SIINFEKL (OVA_257-264) and OVA_323-337 (1µM each) for 20 h to determine specific IFN-ɤ production of memory T cells (n=2-5). Bars represent median value with single values depicted as dots. Statistical analysis by Kruskal-Walli’s test and Dunn’s post-test (*p < 0.05).

Figure 5

Twofold immunization completely controls tumor growth in a prophylactic tumor model. (A) Schematic overview of the application pattern for DIVA² in a prophylactic tumor setting. On day 0 5x10⁴ MC38mOVA tumor cells were inoculated subcutaneously. (B) Tetramer staining of pre-immunized mice at the day of tumor cell inoculation (day 0, n=12). (C) Tumor volumes were assessed three times per week. Every curve represents the tumor volume of one individual mouse (n=12). (D) Kaplan-Meier survival curve. Statistical analysis of tetramer staining using Kruskal-Wallis with Dunn`s post-test. Comparisons of survival curves were performed by Log-rank (Mantel-Cox test) (*p < 0.05).