Trp2 peptide vaccine adjuvanted with (R)-DOTAP inhibits tumor growth in an advanced melanoma model

Abstract

Previously we have shown cationic lipid (R)-DOTAP as the immunologically active enantiomer of the DOTAP racemic mixture, initiating complete tumor regression in an exogenous antigen model (murine cervical cancer model). Here, we investigate the use of (R)-DOTAP as an efficacious adjuvant delivering an endogenous antigen in an aggressive murine solid tumor melanoma model. (R)-DOTAP/Trp2 peptide complexes showed decreasing size and charge with increasing peptide concentration, taking a rod shape at highest concentrations. The particles were stable for 2 weeks at 4 °C. A dose of 75 nmol of Trp2 (formulated in (R)-DOTAP) was able to show statistically significant tumor growth delay compared to lower doses of 5 and 25 nmol, which were no different than untreated tumors. (R)-DOTAP/Trp2 (75 nmol) treated mice also showed increased T cell IFN-γ secretion after restimulation with Trp2, as well as CTL activity in vivo. This vaccination group also showed the highest population of functionally active tumor-infiltrating lymphocytes, indicated by IFN-γ secretion after restimulation with Trp2. Thus, (R)-DOTAP has shown the ability to break tolerance as an adjuvant. Its activity to enhance immunogenicity of other tumor associated antigens should be studied further.

Figure 1. R-DOTAP/Trp2 characterization and stability

(A) Size, charge and (B) entrapment of Trp2 was evaluated on (R)-DOTAP/Trp2 complexes with varying concentrations of Trp2, n=3. (R)-DOTAP/Trp2 nanoparticles were stored at 4°C and (C) size measured over a period of two weeks, n=3. Experiment was repeated twice. In (C), the size measured from 0nmol Trp2 was the same as that with 5nmol Trp2, overlaying completely at every time point. (D) TEM image of (R)-DOTAP/Trp2 (75nmol) complexes. A total of 210 particles from 3 different fields were measured. Particle dimensions were calculated by measuring the longest dimension and the perpendicular diameter.

Figure 2. B16F10-luc tumor growth inhibition by (R)-DOTAP/Trp2 complexes in vivo

Six week old female C57BL/6 mice were inoculated with 3x10⁵ B16F10-luc cells s.c. in the abdomen on day 0. On day 6, treatments were s.c. injected into the opposite side of the abdomen. Lipid formulations delivered contained 300nmol (R)-DOTAP and varying doses of Trp2 peptide. Tumors were measured with calipers with area calculated as length times width. The inset represents body weight tracked over time. Seven mice were used per group. Experiment was repeated twice. *: P<0.005.

Figure 3. Multi-organ IFN-γ production from (R)-DOTAP/Trp2 vaccinated mice

(A) Splenocytes, (B) vaccine draining lymph nodes, or (C) tumor draining lymph nodes were isolated (on day 13) from tumor-bearing mice treated with (R)-DOTAP/Trp2 (300nmol (R)-DOTAP/ varying Trp2 doses) (on day 6). Resulting cells were pulsed with Trp2 or an irrelevant peptide (Ova) for 6h, washed, and stained for CD8 and IFN-γ. The numbers on the contour plots indicate percentage of IFN-γ⁺CD8⁺ T cells out of all CD8⁺ T cells. Data from one representative mouse is shown, with five to seven total animals per group. Experiment was repeated twice.

Figure 4. In vivo CTL shows specific lysis of Trp2-pulsed targets elicited by (R)-DOTAP/Trp2 (75nmol) vaccination

Targets pulsed with Trp2 or an irrelevant peptide (Ova) were stained with high (Trp2) or low (Ova) concentrations of CFSE, injected into (R)-DOTAP/Trp2 vaccinated mice and in vivo killing was allowed for 22h, then spleens removed and analyzed by flow cytometry. Percent specific lysis, P<0.05 between (R)-DOTAP/Trp2 (75nmol) and other groups. Three to five mice were used per group. Experiment was repeated twice.

Figure 5. CD3⁺ Tumor Infiltrating lymphocytes are higher numbers with higher vaccination doses

CD4⁺ and CD8⁺ tumor infiltrating T cells assayed from tumors of mice treated with varying doses of (R)-DOTAP/Trp2. Cells were gated on CD3⁺, and percentages represent CD3⁺CD4⁺ or CD3⁺CD8⁺ out of total numbers of cells in the tumors. Data from one representative animal is shown, three to four mice were used per group. Experiment was repeated twice.

Figure 6. Tumor infiltrating lymphocytes respond to Trp2 after a high vaccination dose

TIL were isolated from tumors of mice vaccinated with varying doses of (R)-DOTAP/Trp2, pulsed for 6h with Trp2 or an irrelevant peptide (Ova) and analyzed for intracellular IFN-γ in CD8⁺ cells. Percentages are IFN-γ⁺CD8⁺ cells out of all CD8⁺ cells., *: P<0.005. Three mice were used per group. Experiment was repeated twice.