Pharmaceutical compositions, vaccines and their uses in the prevention or treatment of a persistent infection or of cancer

Abstract

Pharmaceutical compositions and pharmaceutical combination preparations. The pharmaceutical compositions and the pharmaceutical combination preparations comprise at least one oxidative stressor selected from the group consisting of dithranol (anthralin, cignolin), or other anthrones or hydroxyanthracenes, at least one Toll-like receptor 7 (TLR7) ligand, and at least one peptide antigen. The pharmaceutical compositions or combination preparations may be used in the prevention or treatment of a persistent viral, bacterial or fungal infection or of cancer. The pharmaceutical compositions or combination preparations may find use for topical application on the skin of a human or animal body.

Claims

1. A pharmaceutical composition, comprising: (a) at least one oxidative stressor selected from the group consisting of dithranol (anthralin, cignolin), or other anthrones or hydroxyanthracenes, (b) at least one Toll-like receptor 7 (TLR7) ligand, (c) at least one peptide antigen.

2. The pharmaceutical composition according to claim 1, wherein the at least one TLR7 ligand is imiquimod (R837), loxoribine and/or resiquimod (R848).

3. The pharmaceutical composition according to claim 1, wherein the at least one TLR7 ligand is Pam3Cys, poly-(I:C) or CpG-DNA

4. The pharmaceutical composition according to claim 1, wherein the at least one peptide antigen is selected from the group consisting of (i) major histocompatibility complex class I (MHCI) ligands, (ii) major histocompatibility complex class II (MHCII) ligands, or (iii) peptides that comprise one or more of said MHCI and/or MHCII ligands.

5. The pharmaceutical composition according to claim 1, wherein the at least one peptide antigen is a synthetic peptide or isolated naturally occurring peptide comprising the amino acid sequence of SEQ ID NO: 1 (PLDGEYFTL), SEQ ID NO: 2 (YMNGTMSQV),SEQ ID NO: 3 (AAGIGILTV), SEQ ID NO: 4 (VLRENTSPK), SEQ ID NO: 5 (SPSSNRIRNT), SEQ ID NO: 6 (RLVTLKDIV), SEQ ID NO: 7 (AVFDRKSDAK), SEQ ID NO: 8 (GLSPTVWLSV), SEQ ID NO: 9 (ILKEPVHGV), SEQ ID NO: 10 (KIRLRPGGK), SEQ ID NO: 11 (TPGPGVRYPL), SEQ ID NO: 12 (ILGFVFTLTV), SEQ ID NO: 13 (VLTDGNPPEV), SEQ ID NO: 14 (TQHFVQENYLEY), SEQ ID NO: 15 (WRRAPAPGAKAMAPG), SEQ ID NO: 16 (FRKQNPDIVIQYMDDLYVG), SEQ ID NO: 17 (RIHIGPGRAFYTTKNIIGTI), SEQ ID NO: 18 (PGPLRESIVCYFMVFLQTHI), SEQ ID NO: 19 (PYYTGEHAKAIGN),), SEQ ID NO: 20, (IAFNSGMEPGVVAEKV), SEQ ID NO: 21 (KQEELERDLRKTKKKI), SEQ ID NO: 22 (GRDIKVQFQSGGNNSPAV), SEQ ID NO: 23 (SIINFEKL), SEQ ID NO: 24 (SIIQFEHL), SEQ ID NO: 25 (SGPSNTPPEI) and modified forms thereof in which one or more amino acids in said amino acid sequences are linked to a chemical group.

6. The pharmaceutical composition according to claim 1, wherein the composition comprises 0.3125% to 4% by weight of the at least one oxidative stressor, 0.1% to 10% by weight of the at least one Toll-like receptor 7 (TLR7) ligand and 0.01% to 30% by weight of the at least one peptide antigen.

7. The pharmaceutical composition according to claim 1, wherein the three components of the composition are present in a form which is adapted for topical administration on intact or lesional skin regions.

8. The pharmaceutical composition according to claim 1, wherein the composition further comprises a pharmaceutically acceptable vehicle, diluent, adjuvant or excipient.

9. The pharmaceutical composition according to claim 1, wherein the composition is provided in a form of a vaccine.

10. A pharmaceutical composition according to claim 1 for use in the prevention or treatment of a persistent viral, bacterial or fungal infection.

11. A pharmaceutical composition according to claim 1 for use in the prevention or treatment of cancer.

12. A pharmaceutical combination preparation, comprising at least the following components: (a) at least one oxidative stressor selected from the group consisting of dithranol (anthralin, cignolin), or other anthrones or hydroxyanthracenes, (b) at least one Toll-like receptor 7 (TLR7) ligand, (c) at least one peptide antigen, wherein component (a) and components (b) and (c) are present in at least two separate formulations.

13. (canceled)

14. The pharmaceutical combination preparation according to claim 12, wherein the formulation of component (a) is adapted to be applied to a human or animal body simultaneously or successively with the formulations of components (b) and (c).

15. A pharmaceutical combination preparation according to claim 12 for use in the prevention or treatment of a persistent viral, bacterial or fungal infection.

16. A pharmaceutical combination preparation according to claim 12 for use in the prevention or treatment of cancer.

17. The pharmaceutical composition according to claim 5, wherein the chemical group is NH.sub.2 or CH.sub.3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1

[0039] Dithranol (Anthralin) enhances T cell responses after imiquimod based transcutaneous immunization (primary T cell response).

[0040] C57BU6 mice (n=4-12 per group) were immunized by transcutaneous immunization, TCI, performed as described by Lopez A. et al. (2017) using 50 mg IMI-Sol together with 100 μg of the synthetic peptides OVA.sub.257-264 (SIINFEKL; SEQ ID NO: 23) and OVA.sub.223-237 (SIIQFEHL; SEQ ID NO: 24; obtained from peptides & elephants, Potsdam, Germany) dissolved in DMSO and mixed with cremor basalis officinalis (Pamela Aranda Lopez et al., “Transcutaneous Immunization with a Novel Imiquimod Nanoemulsion Induces Superior T Cell Responses and Virus Protection.,” Journal of Dermatological Science 87, no. 3 (September 2017): 252-59, doi:10.1016/j.jdermsci.2017.06.012). TCI treatments were applied via the ear skin on day 1.

[0041] Where indicated, dithranol (=anthralin at 0.0625% in Vaseline) was applied in the ear skin 24 h before. (A) The frequency of peptide-specific CD8.sup.+ T cells was assessed on day 7 via flow cytometry. (B) The in vivo cytolytic activity was analyzed on day 7, 20 h after transfer of peptide-loaded splenocytes. (C) IFN-γ production of splenocytes on day 7 post the indicated treatments. Splenocytes were re-stimulated with the indicated peptides for 24 h before harvest and analysis by ELISpot assay. (D) C57BU6 mice (n=5 per group) were immunized as in (A) and infected with OVA transgenic vaccinia virus (VV-OVA, 2×10.sup.6 pfu i.p. 7 days later. 5 days after infection, mice were sacrificed and ovarial viral loads were determined by BSC-40 plaque assay.

[0042] *Significance by One Way ANOVA with Bonferroni Posttest analysis; p<0,05 (*).

[0043] Conclusion:

[0044] The results demonstrate that dithranol treatment of the skin in combination with TCI has a synergistic effect on T cell activation on CD8 positive as well as CD4 positive T cells. Only the combined anthralin/TCI leads to a protective immune response against VV-OVA.

[0045] FIG. 2

[0046] Combined dithranol (anthralin) and imiquimod based transcutaneous immunization induces a memory immune response (secondary T cell response).

[0047] C57BU6 mice (n=3-8 per group) were immunized by TCI, performed as described in FIG. 1 (A) The frequency of peptide-specific CD8.sup.+ T cells was assessed on Day 35 via flow cytometry. (B) The in vivo cytolytic activity was analyzed on Day 35, 20 h after transfer of peptide-loaded splenocytes. (C) IFN-γ production of splenocytes on Day 35 post the indicated treatments. Splenocytes were re-stimulated with the indicated peptides for 24 h before harvest and analysis by ELISpot assay. *Significance by One Way ANOVA with Bonferroni Posttest analysis; p<0.05 (*).

[0048] Conclusion:

[0049] The results demonstrate that dithranol treatment of the skin in combination with TCl has a synergistic effect on T cell activation on CD8 positive as well as CD4 positive T cells including memory T cell formation.

[0050] FIG. 3

[0051] Dithranol (anthralin), but not danthron enhances T cell responses after imiquimod based transcutaneous immunization (primary T cell response).

[0052] C57BU6 mice (n=5 per group, untreated n=2) were immunized by TCl, performed as described in FIG. 1. TCl treatments were applied via the ear skin on day 1. Where indicated Danthron (0.0625% in Vaseline) or dithranol (=anthralin at 0.0625% in Vaseline) applied in the ear skin 24 h before. (A) The frequency of peptide-specific CD8.sup.+ T cells was assessed on day 7 via flow cytometry. (B) The in vivo cytolytic activity was analyzed on day 7, 20 h after transfer of peptide-loaded splenocytes. *Significance by One Way ANOVA with Bonferroni Posttest analysis; p<0.05 (*).

[0053] Conclusion:

[0054] The results demonstrate that dithranol treatment of the skin in combination with TCl has a synergistic effect on T cell activation. The oxidized analogue danthron has no impact on T cell activation suggesting that anthralin acts by oxidative mechanisms.

[0055] FIG. 4

[0056] α-Tocopherol antagonizes the enhanced cytolytic activity induced by dithranol (anthralin) TCl without affecting CTL frequency.

[0057] C57BU6 mice (n=4-10 per group) were immunized by TCl, performed as described in FIG. 1. TCl treatments were applied via the ear skin on day 1 either on both ears as indicated. Where indicated α-Tocopherol (600 U/ kg) as antioxidant was injected i.p. 1 h before the application of anthralin and/or TCl. (A) The frequency of peptide-specific CD8+ T cells was assessed on day 7 via flow cytometry. (B) The in vivo cytolytic activity was analyzed on day 7, 20 h after transfer of peptide-loaded splenocytes. (C) IFN-y production of splenocytes on Day 7 post the indicated treatments.

[0058] *Significance by One Way ANOVA with Bonferroni Posttest analysis; p<0.05 (*).

[0059] Conclusion:

[0060] The adjuvant effect of anthralin on TCl is dependent on oxidative mechanisms.

[0061] FIG. 5

[0062] Anthralin (dithranol) inhibits the TLR7 mediated activation of splenic dendritic cells in vitro.

[0063] CD11c positive Dendritic cells (DCs) were purified from spleens from C57BU6 mice) as described in Weber M et al. (2014) and activated as indicated with the TLR7/8 agonist R-848 (10 μg/ml), anthralin (dithranol), N-acetyl cysteine (NAC 5 mM) or left untreated in Iscoves's medium+5% FCS (Michael Weber et al., “Donor and Host B Cell-Derived IL-10 Contributes to Suppression of Graft-Versus-Host Disease.,” European Journal of Immunology 44, no. 6 (June 2014): 1857-65, doi:10.1002/eji.201344081).

[0064] After 24 h incubation, the cells were harvested, labeled with specific mAbs and analyzed by flow cytometry for the activation markers CD40, CD80 and CD86 gating on live (propidium iodide negative, CD11c/MHC class II positive DCs).

[0065] Conclusion:

[0066] Anthralin suppresses the TLR7 mediated activation of DCs in vitro. The activation phenotype is restored by adding NAC as an antioxidant. Therefore, the synergistic effect of anthralin on T cell activation in vivo is surprising and unexpected. The restoration of DC activation in the presence of NAC indicates that anthralin acts by oxidative mechanisms

[0067] FIG. 6

[0068] Dithranol (anthralin) and TCl are required at the same location to enhance T cell responses (primary T cell response).

[0069] C57BU6 mice (n=2-9 per group) were immunized by TCl, performed as described in FIG. 1. TCl treatments were applied via the ear skin on day 1 either on both ears or only one ear as indicated. (A) The frequency of peptide-specific CD8.sup.+ T cells was assessed on day 7 via flow cytometry. (B) The in vivo cytolytic activity was analyzed on day 7, 20 h after transfer of peptide-loaded splenocytes.

[0070] *Significance by One Way ANOVA with Bonferroni Posttest analysis; p<0.05 (*).

[0071] Conclusion:

[0072] Anthralin and TCl are required at the same site (ear) to mediate synergistic T cell activation. The magnitude of the induced T cell response upon Anthralin/TCl correlates with the area treated (dose-response relationship). Therefore, the synergistic effect of anthralin on T cell activation in vivo is initiated by the local activation of immune cells.

[0073] FIG. 7

[0074] Bone-marrow derived macrophages (BMM) upregulate CD80/CD86 expression following Anthralin/R848 or Anthralin/Imiquimod stimulation.

[0075] BMM precursor cells were isolated from the bone-marrow and differentiated in culture medium supplemented with 10% FCS and 10% L929 fibroblast culture supernatants (containing M-CSF). On day 6 macrophages were harvested with a cell scraper and 2.5×10.sup.5 BMMs were seeded in 96-U-wells. BMMs were then stimulated with either Anthralin alone (ranging from 0.25 to 1 μM), the TLR7 ligands Resiquimod (R848, 10 μg/ml) and Imiquimod (10 μg/ml) or the combination of both Anthralin and TLR7 ligation. After 24h of stimulation BMMs were harvested and activation state of living MHCII.sup.+F4/80.sup.+CD11b.sup.+ BMMs was assesed by FACS analysis of the co-stimulatory surface markers CD80 and CD86. Furthermore, supernatants of stimulated BMMs were collected after 24 h and IL-6 ELISA was performed subsequently.

[0076] Conclusion:

[0077] The experiments summarize the results of the inventive combination using imiquimod (R837) or resiquimod (R848) as TLR7 ligand together with dithranol (anthralin) plus peptide antigen. As shown, the application of dithranol (anthralin), a TLR7 ligand and a peptide antigen results in a cooperative activation of BMM cells, which is significantly more effective as compared to the application of anthralin and/or peptide antigen alone.

[0078] Material & Methods

[0079] Mice

[0080] 6 to 8 weeks old C57BL/6 (wildtype) mice were used for vaccination experiments and purchased from the animal facility of the University of Mainz (TARO) or the Harlan Laboratories. All animal procedures were conducted according to the institutional guidelines and were reviewed and confirmed by an institutional review board headed by the local animal welfare officer (Prof. Dr. O. Kempski) of the University Medical Center (Mainz, Germany). All assays were approved by the responsible authority (National Investigation Office Rheinland-Pfalz, Koblenz, Germany). The Approval ID assigned by this authority: AZ 23 177-07/ G13-1-012.

[0081] Transcutaneous Immunization

[0082] Transcutaneous immunizations were performed as described previously (Lopez et al. 2017) using 50 mg IMI-Sol (own production) together with 100 μg of the synthetic peptides OVA.sub.257-264 (SIINFEKL; SEQ ID NO: 23) and OVA.sub.223-237 (SIIQFEHL; SEQ ID NO: 24; obtained from peptides & elephants Potsdam, Germany) dissolved in DMSO and mixed with cremor basalis officinalis. TCl treatments were applied via the ear skin on day 1.

[0083] Foregoing to TCl, ears were pretreated with 25 mg of an anthralin or danthron containing (1/16%) vaseline creme on day 0 when indicated. α-Tocopherol was administered in a weight adjusted dosage (600 U/ kg body weight, i.p.) 1 h preceding the application of anthralin and/or TCl. Mice were anesthetized previous to all procedures using a dilution of Ketamin/Rompun in a weight adjusted dosage.

Flow Cytometric Analyses and In Vivo Cytotoxicity Assay

[0084] Flow cytometric analyses and evaluation of in vivo cytotoxicity were performed as described previously (Lopez et al. 2017). Blood samples were collected via tail vein incision, subjected to hypotonic lysis and incubated with specific mAbs as indicated. Antibodies used for FACS analysis were PB-conjugated anti-CD8 (clone 53-6.7; eBioscience, San Diego, USA), FITC-conjugated anti-CD62L (clone 17A2; eBioscience, San Diego, USA) and APC-conjugated anti-CD44 (clone GK1.5; BioLegend, San Diego, USA). To measure the frequency of peptide-specific CD8.sup.+ T cells, blood samples were stained with PE-conjugated OVA.sub.257-264-H2-K.sup.b (tetrameric labeling, own production).

[0085] For detecting in vivo cytolytic activity, splenocytes of syngenic wildtype mice were labeled with different amounts of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Carlsbad, USA) resulting in a CFSE.sup.low and a CFSE.sup.high population. The cells labeled with CFSE.sup.low were additionally labeled with 1 μM of OVA.sub.257-264. Adoptive transfer of 3×10.sup.7 cells in a 1:1 ratio (CFSE.sup.low:CFSE.sup.high) into immunized and untreated mice was performed via intravenous injection into the tail vein. For assessment of cytolytic activity during the primary response, blood was drawn and subjected to flow cytometry on day 7 post immunization, 20 hours after transfer of target cells. Specific lysis was evaluated as following: Specific lysis (%)=100×(1−(CFSE.sup.low of immunized mouse/CFSE.sup.low of untreated control). All analyses were performed with a LSRII Flow Cytometer and FACSDiva Software (Becton Dickinson, Franklin Lakes, USA).

[0086] IFNγ Elispot Assay

[0087] 96 well plates (MultiScreen.sub.HTS IP, 0.45mm, Merck Millipore, Darmstadt, Germany) were coated over night at 4° C. with murine IFNy AN18 antibody (10 mg/ml, Mabtech, Nacka Strand, Sweden). After a blocking step 5×10.sup.5 cells were added. Therefore splenocytes were lysed and loaded either with 1mM OVA.sub.257-264, 1 mM OVA.sub.323-337 or left in medium. After over night incubation at 37° C. plates were washed and stained with the biotinylated IFNγ R4-6A2 antibody (2 mg/ml, Mabtech, 2 h, 37° C.). Afterwards Vectastain ABC Kit (Vector Laboratories, Burlingame, USA)/AEC (Sigma-Aldrich, Taufkirchen, Germany) complex was added as described in the manufacturer's instruction. After spots were visible plates were washed with water and dried overnight. Analysis was performed by an ImmunoSpot Analyzer (C.T.L. Europe, Bonn, Germany).

Dendritic Cell Isolation, Cultivation and Assessment of Activation Markers

[0088] Spleens of C57BL6/J wildtype mice were harvested, picked and digested for 45 min with type 2 collagenase (50 U/ml from Worcester, Pappenheim, Germany) and stopped by adding 2 mM EDTA (from Sigma-Aldrich, Taufkirchen, Germany). After a hypotonic lysis step DCs were isolated using CD11 c Micro Beads UltraPure (Miltenyi Biotec, Germany) following the manufacturer's instruction. 2×10.sup.5CD11c.sup.+ DCs/ well were plated in 96 well plates and cultivated for either 24 or 48 h in the presence of TLR7 ligand R-848 (10 ug/ml) and/or different concentrations of anthralin (solved in DMSO). Afterwards cells were washed and stained with following antibodies for FACS analysis: CD19-PerCP, CD90.2-PerCP, CD11c-APC or PE-Cy7, MHCII-BV241, CD86-PE, CD80-FITC or BV605, CD40-APC. Additionally, supernatants of DC cultivation after 24 or 48 h were collected and employed for cytokine bead array (CBA) analyses. Following cytokines were quantified using CBA technology: IL-1b; IL-6; IL-10; IL-12; IL-23; TNF-α.

[0089] Assessment of Virus Protection Via Plaques Assay

[0090] Prior to the infection female C57BL6/J mice were immunized as described above. On day 7 effective immunization was verified via tetrameric staining. Afterwards 2×10.sup.6 pfu Vaccinia OVA virus (VV OVA) were re-suspended in 200 pl medium and applied via intraperitoneal injection. 5 days after the infection mice were sacrificed and ovaries were removed and minced. Different concentrations of these virus containing samples were added to a layer of BSC-40 cells. Subsequent the plates were incubated for 24 h at 37° C. Following this incubation an examination under the light microscope revealed the appearance of plaques within the cell layer. Consecutively, the remaining BSC-40 cells were stained with a crystal violet solution and plaques were counted on a lightbox. Counted plaques were multiplicated with the dilution factor of the appropriate well to estimate the pfu/ovary.

[0091] Tumor Rejection Assay

[0092] MC38 colon adenocarcinoma cells were provided by H.C. Probst (Institute for Immunology, University Medical Center Mainz). Before inoculation, MC38 cells were cultured for one week in DMEM (Thermo Fisher Scientific), supplemented with 1% penicillin-streptomycin, 10% FCS, 2mM glutamine and 1mM sodium pyruvate. Tumor cells were subcutaneously injected into the lower left flank of anesthetized animals. Treatment was initiated at tumor size of approximately 25 mm.sup.2 (5-7 days after inoculation). Tumor growth was traced by measuring the tumor diameter in 2 dimensions three times a week using a caliper. Mice were sacrificed when tumor size exceeded a diameter of 400 mm.sup.2 or when bleeding ulceration occurred. The point of death was recorded as the day after sacrifice.