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.2011 May;60(5):629-38.
doi: 10.1007/s00262-011-0970-1. Epub 2011 Jan 26.

Enantiospecific adjuvant activity of cationic lipid DOTAP in cancer vaccine

Affiliations

Enantiospecific adjuvant activity of cationic lipid DOTAP in cancer vaccine

Elizabeth A Vasievich et al. Cancer Immunol Immunother.2011 May.

Abstract

Commercially available DOTAP is a racemic mixture of two enantiomers. The adjuvanticity of each isomer was examined using a peptide/lipid complex as a therapeutic vaccine in an established murine cervical cancer model. This simple vaccine consists of a cationic lipid (DOTAP) and a major histocompatibility complex (MHC) class I-restricted epitope of the Human Papillomavirus (HPV) 16 protein E7. Dose-dependent tumor regression experiments have been completed for racemic DOTAP/E7, (R)-DOTAP/E7 and (S)-DOTAP/E7. Tumor-bearing mice treated with (R)-DOTAP/E7 complexes have shown tumor regression in a dose-dependent manner comparable to those mice treated with a racemic DOTAP with E7 peptide. These data are supported by IFN-γ production by CD8(+) splenocytes, in vivo cytotoxic T-lymphocytes (CTL) response, CD8(+) tumor-infiltrating lymphocytes (TIL), and IFN-γ production by CD8(+) TIL in (R)-DOTAP/E7-vaccinated mice. When (S)-DOTAP/E7 is delivered, tumor progression is delayed. While IFN-γ production is absent from CD8(+) splenocytes in mice vaccinated with (S)-DOTAP/E7, IFN-γ production by CD8(+) TIL is present, supporting our hypothesis that (S)-DOTAP has limited activity. Activation of bone marrow-derived dendritic cells by the enantiomeric formulations has also been evaluated, as well as cytokine production and toxicity with no considerable differences between the groups. The results show the DOTAP enantiomers act differently as adjuvants in vivo, with (R)-DOTAP being more effective at stimulating a CD8(+) anti-tumor response.

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Conflict of interest statement

Grants from PDS Biotechnology Corporation and the NIH have funded this research. LH is a co-founder and on the scientific board of advisors of PDS Biotechnology Corporation, in addition to financial interests in the company. EAV, WC, and LH are named in a US pending patent on the technology described in this paper.

Figures

Fig. 1
Fig. 1
TC-1 tumor growth inhibition by vaccine delivery in vivo.a TC-1 tumor growth inhibition in mice by lipid/E7 complexes. Six-week-old female C57BL/6 mice were inoculated with 10,000 TC-1 cells s.c. in the abdomen on day zero. On day 6, treatments were s.c. injected into the opposite side of the abdomen. Lipid formulations delivered contained 100 nmol lipid and 20 μg of E7 peptide. Sample size and statistics are as follows:n = 5–6, *:P < 0.05, **:P < 0.01, ***:P < 0.0001.b Dose-dependent antitumor activity of racemic DOTAP/E7, (R)-DOTAP/E7, or (S)-DOTAP/E7 complexes. Six-week-old female C57BL/6 mice were injected with 10,000 TC-1 cells s.c. as in previous studies. On day 6, treatments were s.c. injected into the opposite side of the abdomen. The dose of lipid was varied while the dose of E7 peptide was kept constant at 20 nmol.n = 5–6 per dose
Fig. 2
Fig. 2
Chiral lipid interaction with BMDC. BMDC were treated with (R)-DOTAP or (S)-DOTAP liposomes (free of peptide) for 18 h anda dose-dependent activation of CD86 was evaluated (n = 3), as well asb dose-dependent CCL2 secretion after 24 h treatment (n = 3).c BMDCs were also evaluated for toxicity by PI staining of cells after 18 h treatment (one representative experiment shown, repeated in triplicate)
Fig. 3
Fig. 3
IFN-γ production from splenocytes of (R)-DOTAP/E7- or (S)-DOTAP/E7-treated mice.a Splenocytes isolated (on day 14) from tumor-bearing mice treated with (R) or (S)-DOTAP/E7 at 300 nmol/20 nmol (on day 6). Splenocytes were pulsed with E7 or an irrelevant peptide (NP) for 6 h, 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. One representative experiment is shown.b IFN-γ-secreting CD8+ cells per 104 CD8+ T cells after 6 h pulse with peptide.n = 6–8. **:P < 0.01c IFN-γ production after 12 h pulse with either NP or E7 peptide.d IFN-γ-secreting CD8+ cells per 104 CD8+ T cells after 12 h pulse with peptide.n = 6-8. *:P < 0.02
Fig. 4
Fig. 4
In vivo cytotoxic T-lymphocyte (CTL) assay. a Targets pulsed with E7 or an irrelevant peptide (NP) were stained with high (E7) or low (NP) concentrations of CFSE, injected into treated mice and in vivo killing was allowed for 16-20 h, then spleens removed and analyzed by flow cytometry.b Percent specific lysis,P < 0.05 between (R)-DOTAP and (S)-DOTAP groups. No statistical difference exists between the two (R)-DOTAP-treated groups, or the (S)-DOTAP tumor-bearing group and tumor-bearing-untreated group.n = 15–18
Fig. 5
Fig. 5
Tumor-infiltrating lymphocytes. Tumor-infiltrating lymphocytes assayed from tumors of mice treated with (R) or (S)-DOTAP/E7a CD4+ and CD8+ cellsb CD4+ or CD8+ cells per 10,000 cells in the solid tumor.n = 3–4. When comparing (R)-DOTAP to (S)-DOTAP or tumor only,P < 0.01 for CD8+ cells; however, (S)-DOTAP compared to tumor alone,P = 0.57. Looking at the groups with regard to CD4+ cells, all comparisons yieldP values greater than 0.3
Fig. 6
Fig. 6
IFN-γ production by tumor-infiltrating lymphocytes. TIL were isolated from tumors of mice vaccinated with (R)-DOTAP/E7 or (S)-DOTAP/E7 and pulsed for 6 h with E7 or an irrelevant peptide (NP).a Percentages are IFN-γ+CD8+ cells out of all CD8+ cells. One dot plot is shown for each group that is representative of three experiments.b IFN-γ+-producing cells per 10,000 CD8+ cells,P < 0.05.n = 6–7
Fig. 7
Fig. 7
Proposed tumor interaction with DOTAP enantiomers. In vitro (R)- and (S)-DOTAP show little difference in stimulating BMDC, shown by the size of the straight arrows. Due to an unknown interaction between the immune system and the tumor, the presence of a tumor in the in vivo system amplified the difference between (R)- and (S)-DOTAP reflected by the size of the ribbon arrows. The distinction between the two formulations is further amplified via talk back from the tumor to the immune system
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