Allogeneic Dendritic Cells For Use In Cancer Treatment

Abstract

Disclosed is a pro-inflammatory dendritic cell with improved ability to activate allo-geneic T-cells through the direct pathway of allorecognition. The dendritic cell is infected with an adenovirus. Further, the pro-inflammatory dendritic cell has been obtained by: providing an immature dendritic cell; infecting the dendritic cell with an adenovirus; and maturating the immature dendritic cell into a pro-inflammatory dendritic cell by addition of the Toll-like receptor 3 (TLR3)-ligand poly-I:C, a TLR7/8-ligand, such as Resiquimod, and interferon gamma (IFN-) to induce maturation of the immature dendritic cell.

Claims

1. A pro-inflammatory dendritic cell with ability to activate allogeneic T-cells through the direct pathway of allorecognition, said dendritic cell being infected with an adenovirus, wherein said pro-inflammatory dendritic cell has been obtained by: providing an immature dendritic cell; infecting the dendritic cell with an adenovirus; and maturating the immature dendritic cell into a pro-inflammatory dendritic cell, wherein said maturation is performed by addition of the Toll-like receptor 3 (TLR3)-ligand poly-I:C, a TLR7/8-ligand, and interferon gamma (IFN-) to induce maturation of the immature dendritic cell.

2. The pro-inflammatory dendritic cell according to claim 1, wherein the adenovirus is an adenovirus serotype-5 (Ad5) with fiber shaft and/or knob from adenovirus serotype-35 (Ad35) in place of the Ad5 fiber shaft and/or knob; and/or wherein the hexon protein of the adenovirus is modified to comprise at least one protein transduction domain (PTD) of the transactivator of transcription (TAT) from human immunodeficiency virus (HIV); and/or wherein the adenovirus is an E1-deleted adenovirus.

3. (canceled)

4. (canceled)

5. The pro-inflammatory dendritic cell according to claim 1, wherein the adenovirus does not comprise any gene encoding for a tumor antigen, whereby the pro-inflammatory dendritic cell being infected but not transduced.

6. The pro-inflammatory dendritic cell according to claim 1, wherein the adenovirus does comprise at least one gene encoding for a tumor associated or tumor-specific antigen, whereby the infected dendritic cell being transduced to express the tumor associated or specific antigen.

7. The pro-inflammatory dendritic cell according to claim 1, wherein the maturation and the infection with the adenovirus, respectively, are performed simultaneously.

8. The pro-inflammatory dendritic cell according to claim 1, wherein the TLR7/8-ligand is Resiquimod.

9. The pro-inflammatory dendritic cell according to claim 1, wherein said step of maturating the immature dendritic cell into the pro-inflammatory dendritic cell further comprise the addition of at least one substance selected from the group consisting of TLR2-ligands, TLR4-ligands, TLR9-ligands, Interferon alpha (IFN-), interleukin 1 (IL-1), and tumor necrosis factor alpha (TNF-) to induce activation; and/or said step of maturating the immature dendritic cell into the pro-inflammatory dendritic cell does not comprise addition of prostaglandin E2 (PGE2).

10. The pro-inflammatory dendritic cell according to claim 1, wherein the immature dendritic cell is provided by differentiating monocytes into immature dendritic cells by use of interleukin 4 (IL-4) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), the monocytes having been isolated from peripheral blood leukocytes.

11. A pharmaceutical composition comprising the pro-inflammatory dendritic cell according to claim 1 and at least one pharmaceutical acceptable carrier.

12. A method of treating cancer, said method comprising administering the pro-inflammatory dendritic cell according to claim 1 to a subject suffering from cancer, wherein the pro-inflammatory dendritic cell is allogeneic to the subject.

13. The method according to claim 12, wherein the pro-inflammatory dendritic cell is administered intra-tumorally.

14. The method according to claim 12, wherein the method further includes administration of a drug decreasing the immunosuppresive tumor environment or activating the immune system to the subject, said drug being selected from the group consisting of sunitinib, nivolumab, pembrolizumab, pidilizumab, atezolizumab, avelumab, gemcitabine and 5-fluorouracil.

15. The method according to claims 12, wherein the adenovirus does comprise at least one gene encoding for a tumor associated or tumor-specific antigen, whereby the infected dendritic cell being transduced to expresses the tumor associated or specific antigen; and wherein the pro-inflammatory dendritic cell is administered intradermally, subcutaneously or intranodally to the subject.

16. A method of providing pro-inflammatory dendritic, said pro-inflammatory dendritic cells having ability to activate allogeneic T-cells through the direct pathway of allorecognition, and said dendritic cells being infected with an adenovirus, wherein said method comprises the steps of: providing immature dendritic cells; infecting the dendritic cells with an adenovirus; and maturating the immature dendritic cells into a pro-inflammatory dendritic cell, wherein said maturation is performed by addition of the Toll-like receptor 3 (TLR3)-ligand poly-I:C, a TLR7/8-ligand, and interferon gamma (IFN-) to induce maturation of the immature dendritic cell.

17. The method according to claim 16, wherein the adenovirus is an adenovirus serotype-5 (Ad5) with fiber shaft and/or knob from adenovirus serotype-35 (Ad35) in place of the Ad5 fiber shaft and/or knob; and/or wherein the hexon protein of the adenovirus is modified to comprise at least one protein transduction domain (PTD) of the transactivator of transcription (TAT) from human immunodeficiency virus (HIV); and/or wherein the adenovirus is an E1-deleted adenovirus.

18. (canceled)

19. (canceled)

20. The method according to claim 16, wherein the adenovirus does not comprise any gene encoding for a tumor antigen, whereby the pro-inflammatory dendritic cell being infected but not transduced.

21. The method according to claim 16, wherein the adenovirus does comprise at least one gene encoding for a tumor associated or tumor-specific antigen, whereby the infected dendritic cell being transduced to express the tumor associated or specific antigen.

22. The method according to claim 16, wherein the maturation and the infection with the adenovirus, respectively, are performed simultaneously.

23. The method according to claim 16, wherein the TLR7/8-ligand is Resiquimod.

24. The method according to claim 16, wherein said step of maturating the immature dendritic cell into the pro-inflammatory dendritic cell further comprise the addition of at least one substance selected from the group consisting of TLR2-ligands, TLR4-ligands, TLR9-ligands, Interferon alpha (IFN-), interleukin 1 (IL-1), and tumor necrosis factor alpha (TNF-) to induce activation; and/or said step of maturating the immature dendritic cell into the pro-inflammatory dendritic cell does not comprise addition of prostaglandin E2 (PGE2).

25. The method according to claims 16, wherein the immature dendritic cells are provided by differentiating monocytes into immature dendritic cells by use of interleukin 4 (IL-4) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), the monocytes having been isolated from peripheral blood leukocytes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1. COMBIG/Ad5M-matured DCs express a mature phenotype and Th1-polarized cytokine secretion profile. (Data are shown as meanSEM (n.s. p0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001)).

[0067] (A) CD14.sup.+ monocytes were isolated from healthy donor PBMCs, differentiated into imDCs by GM-CSF/IL-4 for 5 days, matured under different conditions for 18h, washed and further cultured for 24 h and analyzed.

[0068] (B) DCs were characterized for HLA-DR, CD40, CD80, CD86 and CD83 expression by flow cytometry. Mean fluorescence intensity (MFI) for each marker on DCs (CD14.sup.CD1a.sup.+) produced from eight donors are shown.

[0069] (C) ELISA was used to measure IL-12p70, IL-6 and CXCL10 secretion for each PBMC donor.

[0070] FIG. 2. Allo-COMBIG/Ad5M-DCs activate innate and adaptive immune cells in vitro. (Data are shown as meanSEM (n.s. p0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001))

[0071] (A) After maturation for 18 h and washing (samples as described in FIG. 1A) the DCs, here called alloDCs, were co-cultured with allogeneic PBMCs (meaning from a different donor). After 24 h of co-culture, activation of T cells in the PBMC pool was characterized by the expression of CD69.

[0072] (B) Mean fluorescence intensities for CD69 on T-cells (CD3.sup.+CD56.sup.) from sixteen individual combinations of eight unrelated donors are shown.

[0073] (C) The MLR (mixed leukocyte reaction) supernatants (allo-SN) from the alloDC-PBMC co-cultures were used as maturation stimuli for bystander immature (im) DCs (DCs from the same donor as the PBMCs) to mimic a scenario for host bystander imDCs. Bystander DC maturation was assessed by the upregulation of HLA-DR, CD40, CD80, CD86 and CD83 as shown in MFI scatter plots for each marker on bystander-DCs (CD14.sup.CD1a.sup.+). Six individual combinations from three unrelated donors were evaluated.

[0074] FIG. 3. Activation and expansion of antigen-specific T-cells induced by cross-presentation of the antigen by autologous bystander-DCs matured by the MLR supernatant (allo-SN) from PMBC allo-COMBIG/Ad5M-DC co-cultures.

[0075] (A) Bystander im DCs from HLA-A2.sup.+ donors (bystander-DCs) were pulsed for 2 h with lysate from tumor cells expressing the CMV-pp65 antigen (A549(pp65)) and matured for 38 h by allo-SN from the various co-cultures (FIG. 2A). The cross-presenting bystander-DCs were then mixed for 18h with autologous T-cells, engineered with a TCR against the HLA-A2-restricted CMV-pp65.sub.495-503 peptide.

[0076] (B) ELISA was used to measure the concentration of IFN- released by the engineered antigen-specific T-cells into the supernatants. Six individual combinations from three unrelated donors were examined.

[0077] (C) Bystander-DCs from CMV-seropositive, HLA-A2.sup.+ donor were prepared as mentioned above and used to stimulate autologous (with respect to the bystander DCs), non-engineered, T cells for 12 days in the presence of low dose IL-2 (20IU/ml).

[0078] (D) CMV-pp65-specific expansion of non-engineered T cells was quantified by flow cytometry using PE-conjugated HLA-A*0201/pp65.sub.495-503 tetramer. FACS plots from one representative individual combination out of four using the allo-SN from PBMC allo-COMBIG/Ad5M-DC co-cultures as maturation stimuli are shown.

[0079] (E) The expanded non-engineered T-cells were then re-stimulated by exposure to T2 cells loaded either with CMV-pp65.sub.495-503 peptide (relevant target) or TARP.sub.4-13 peptide (irrelevant target). ELISA was used to measure the concentration of IFN- in supernatants harvested 18 h after re-stimulation. The dotted line indicates the background IFN- release from T-cells stimulated by immature bystander-DCs. Data are shown as meanSEM (*P<0.05; **P<0.01; ****P<0.0001).

[0080] FIG. 4. Prophylactic vaccination with COMBIG-DC transduced with the Ad5M vector coding for gp-100 indirectly prime gp-100 specific T cells

[0081] (A) The ability of COMBIG DCs (alloDCs) transduced with the Ad5M vector coding for the tumor antigen pg100 (Ad5M(gp100)-alloDCs) to indirectly stimulate gp100-specific T-cells in non-tumor-bearing mice was assessed by combining s.c. injection of Ad5M(gp100)-alloDCs (day 0) with intravenous (i.v.) adoptive cell transfer of gp100-specific Thy1.1.sup.+ pmel-1 splenocytes (day 2). T-cell read out was performed five days later by Thy1.1 staining (day 7).

[0082] (B) Representative scatter plots of the specific proliferation of Thy1.1.sup.+ pmel-1 T-cells (Thy1.1.sup.+) in response to alloDCs or Ad5M(gp100)-alloDCs from two mice are shown.

[0083] FIG. 5. Therapeutic vaccination with COMBIG-DC transduced with the Ad5M vector coding for gp-100 decreases tumor growth and prolongs survival

[0084] (A) In a therapeutic setting, C57BL/6NRj mice were first injected s.c. into the hind flank with 110.sup.5 B16-F10 cells (day 0) and received 110.sup.7 pmel-1 splenocytes i.v. (day 8). The treatment was combined with i.t. injections of 110.sup.6 Ad5M(gp100)-alloDCs (day 8 and 14) or PBS as negative control. Tumor growth was monitored by caliper measurement.

[0085] (B) Average tumor growth is presented for each treatment (n=8 per group).

[0086] (C) Mice survival is shown by the Kaplan-Meier survival curve and compared by log-rank test (n=8 per group) (*P<0.05).

[0087] FIG. 6. COMBIG/Ad5-HPV16E6E7-matured DCs express a mature phenotype.

[0088] (A) CD14.sup.+ monocytes were isolated from healthy donor PBMCs, differentiated into imDCs by GM-CSF/IL-4 for 5 days, matured under different conditions for 18 h, washed and further cultured for 24 h and analyzed.

[0089] (B) DCs were characterized for CD40, CD80, CD86 and HLA-DR expression by flow cytometry. Mean fluorescence intensity (MFI) for each marker on DCs (CD14.sup. CD1a.sup.+) produced from eight donors are shown.

[0090] FIG. 7. Allo-COMBIG/Ad5-HPV16E6E7-DCs activate innate and adaptive immune cells in vitro.

[0091] (A) After maturation for 18h and washing (samples as described in FIG. 6A) the DCs, here called alloDCs, were co-cultured with allogeneic PBMCs (meaning from a different donor). After 24 h of co-culture, activation of T cells in the PBMC pool was characterized by the expression of CD69.

[0092] (B) Mean fluorescence intensities for CD69 on T-cells (CD3.sup.+CD56.sup.) from sixteen individual combinations of eight unrelated donors are shown.

[0093] (C) The MLR (mixed leukocyte reaction) supernatants (allo-SN) from the alloDC-PBMC co-cultures were used as maturation stimuli for bystander immature (im) DCs (DCs from the same donor as the PBMCs) to mimic a scenario for host bystander imDCs. Bystander DC maturation was assessed by the upregulation of CD40, CD80, CD86, and HLA-DR as shown in MFI scatter plots for each marker on bystander-DCs (CD14.sup.CD1a.sup.+). Six individual combinations from three unrelated donors were evaluated.

EXPERIMENTAL

[0094] The following examples are mere examples and should by no mean be interpreted to limit the scope of the invention. Rather, the invention is limited only by the accompanying claims.

[0095] EXAMPLE 1

COMBIG-DCs Transduced with an Adenovirus Vector without any Transgene

[0096] In example 1, the impact of infecting immature DCs with an empty adenoviral vector (Ad5M) on activation/maturation induced by the Combig-cocktail (Poly-I:C, R848 and IFN-gamma) alone was evaluated (e.g. phenotypic maturation and secretion of inflammatory factors, e.g. IL-6, IL-12 and CXCL10, were studied). In brief, it was seen that Ad5M infection does not impair the ability of the Combig-cocktail to induce maturation of pro-inflammatory DCs (cf. FIG. 1). These data are in line with the prevailing opinion. However, in contrast to prevailing opinion it was found that infection with Ad5M improves, and not impairs, as presumed (cf. e.g. Jonuleit et al in Gene Therapy (2000) 7, 249-254; Tan et al in BLOOD, (2005), 105(10), 3824- 3832; and Newton et al in Immunology, 125, 469-479), the ability of Combig-DCs to stimulate the direct pathway of allorecognition, as measured by the activation (CD69 expression) of allogeneic T cells during a mixed leukocyte reaction (MLR) (cf. FIG. 2B)

[0097] Moreover, the finding that activation/maturation of bystander-DCs with MLR-supernatants from co-cultures of allo COMBIG-Ad5M DCs and PBMCs was increased (FIG. 2C) and in turn provides improved capacity of bystander DCs to activate autologous antigen-specific T cells producing the Th1 associated cytokine IFN-gamma (cf. FIG. 3B and 3D) implies that COMBIG-DCs transduced with the Ad5M vector are useful as an improved cell-based allogeneic immune enhancer in which the mode of action is crucially dependent on the direct pathway of allorecognition.

[0098] Materials and Methods

[0099] Cell Lines

[0100] K562(Luc), A549(pp65) and T2 cells (ATCC) were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% PeSt and 1% HEPES. All components and culture media were from Thermo Fisher Scientific. K562(Luc) cells were engineered with a lentivirus vector to express firefly luciferase and A549(pp65) cells to express the cytomegalovirus (CMV)-pp65 antigen. All cells were cultured in a humidified incubator with a 5% CO.sub.2 atmosphere at 37 C.

[0101] Production of the Recombinant Virus

[0102] The Ad5M vector was constructed and produced as previously described (Yu, D., C. Jin, J. Leja, N. Majdalani, B. Nilsson, F. Eriksson, and M. Essand. 2011. Adenovirus with hexon Tat-protein transduction domain modification exhibits increased therapeutic effect in experimental neuroblastoma and neuroendocrine tumors. J Virol 85: 13114-13123). Ad5M is an E1-deleted human adenovirus serotype-5 (Ad5) vector with fiber shaft and knob from serotype-35 and a hexon modification to enhance transduction efficacy (Yu, D. et al 2013. Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures. PLoS One 8: e54952). Ad5M does not encode any transgene. Titers were determined by quantitative PCR as encapsidated virus genomes (evg) per ml (Yu, D. et al 2013. Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures. PLoS One 8: e54952).

[0103] Isolation of Human PBMCs and Generation of DCs

[0104] Buffy coats from healthy donors were obtained from the blood bank at the Uppsala University Hospital, Uppsala, Sweden. PBMCs were isolated by Ficoll-Paque Premium separation (GE Healthcare Life Science) and cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% PeSt, 0.5% L-glutamine, 1% HEPES and 20 mM -mercaptoethanol (DC medium).

[0105] Generation and Treatment of DCs.

[0106] Monocytes isolated from PBMCs by CD14.sup.+ positive magnetic selection (Miltenyi Biotec) were differentiated to immature DCs (imDCs) using 20 ng/mL human IL-4 and 100 ng/mL GM-CSF (Gentaur) for 5 days. Medium was replaced every 2 days. On day 5, cells were either left untreated (imDCs) or matured for 18 h with a cocktail of maturation stimuli (Napolitani, G. et al. 2005. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat Immunol 6: 769-776; Lovgren, T. et al 2017. Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-gamma and multiple toll-like receptor agonists. Cancer Immunol Immunother) consisting of 2.5 g/mL R848 (InvivoGen), 20 g/mL polyinosinic:polycytidylic acid (polyI:C) (Sigma-Aldrich) and 1000IU/mL IFN- (Shenandoah Biotechnology). This maturation cocktail was named Combined Toll-like receptor ligands with IFN- (COMBIG). DCs matured with COMBIG used as allogeneic stimuli are referred to as allo-COMBIG-DCs. Immature DCs treated with Ad5M (2000 evg/cell) for 18 h are referred to as allo-Ad5M-DCs while allo-COMBIG/Ad5M-DCs were treated with both COMBIG and Ad5M for 18 h. After washing, cells were further cultured in fresh DC medium.

[0107] Human Cytokine Array and Cytokine Release Assay

[0108] Supernatants from DC cultures (2.5-510.sup.5 cells/well in 48-well plates) were collected. ELISA kits were used to study the production of IL-12p70 (Mabtech), IL-6 (BioLegend) and CXCL10 (BioLegend).

[0109] Flow Cytometry

[0110] Human DC phenotyping. Anti-CD1a-BV510 (BD Biosciences), anti-CD14-APC/Cy7, anti-HLA-DR-perCP (MHC class II), anti-CD40-FITC, anti-CD80-PE, anti-CD83-APC and anti-CD86-BV421 were used to evaluate the DCs.

[0111] Activation of T-Cells in Allogeneic Mixed Leukocyte Reaction.

[0112] AlloDCs were co-cultured with PBMCs from unrelated donors (MLR) at ratio 1:5 (alloDCs:PBMCs). Activation was assessed 24 h later by flow cytometry with anti-CD3-FITC and anti-CD69-BV510. ELISA was used for the detection of IFN- (Mabtech) in the allogeneic co-culture supernatant (allo-SN).

[0113] Activation of Bystander Immature DCs.

[0114] AlloDCs were co-cultured with PBMCs at ratio 1:5 (alloDCs:PBMCs) for 24 h and the allo-SN was used as a maturation stimuli for immature bystander-DCs. Bystander-DC maturation was assessed 48 h later by flow cytometry as before.

[0115] All antibodies were purchased from BioLegend, unless specified elsewhere. Data acquisition was performed using a FACSCanto II (BD Biosciences) flow cytometer, and the analysis was performed using FlowJo software (version 7.6.5; Tree Star).

[0116] DC Antigen Cross Presentation and T-Cell Stimulation Assays

[0117] Cross-presentation of CMV-pp65 by DCs for specific stimulation of autologous CMV-pp65.sub.495-503 TCR-modified T-cells.

[0118] Immature DCs (HLA-A2.sup.+) were cultured for 2 h at 37 C. with freeze/thawed cell-lysate from A549(pp65) tumor cells, as a mean of providing CMV-pp65 protein to DCs exogenously. The DCs were subsequently matured in supernatant from alloDC/PBMCs (allo-SNs) for 38 h. Autologous T-cells engineered to express an HLA-A*0201-restricted T-cell receptor (TCR) for the CMV-pp65.sub.495-503 epitope (Hillerdal, V., et al 2016. Avidity characterization of genetically engineered T-cells with novel and established approaches. BMC Immunol 17: 23) were then mixed with CMV-pp65 cross-presenting DCs at ratio 5:1 (T-cells:DCs) and cultured in fresh medium for 18 h. TCR-specific T-cell activation was assessed by the secretion of IFN-.

[0119] Expansion and Re-Stimulation of T-Cells by Cross Presenting DCs.

[0120] T-cells (non-modified) from CMV-seropositive, HLA-A2.sup.+ donors were mixed with autologous CMV-pp65 cross-presenting DCs prepared as above at ratio of 20:1 (T-cells:DCs) and cultured in fresh medium for a total of 12 days. Medium with 30IU/ml IL-2 (Proleukin, Novartis) and 20 ng/ml IL-7 (Nordic Biosite) was replaced after 7 days. CMV-pp65-specific T-cell expansion was detected with an HLA-A*0201/pp65.sub.495-503 tetramer (Beckman Coulter).

[0121] T2 cells (HLA-A2.sup.+) were pulsed with 5 g/ml CMV-pp65.sub.495-503 peptide or the HLA-A2 binding irrelevant TARP(P5L).sub.4-13 peptide. The pulsed T2 cells were used to re-stimulate the expanded CMV-pp65-specific T-cells for 18 h. IFN- release was measured as an indicator of T-cell activation.

[0122] Statistics

[0123] The data are reported as meanSEM. Statistical analysis was performed by GraphPad prism software version 6.01 (La Jolla). Statistical analyses were performed using parametric One-way ANOVA with Holm-Sidak test for multiple comparison correction. Student t-test was used when only two groups were evaluated. Values with P<0.05 were considered to be statistically significant.

[0124] Results and Discussion

[0125] COMBIG-Matured and COMBIG/Ad5M-Matured DCs Exhibit a Phenotype and Cytokine Secretion Profile Associated with T-Helper-Cell (Th1) Polarization

[0126] The maturation status of DCs is very crucial for the induction of optimal effector responses in autologous DC cancer vaccination (Sabado, R. L., and N. Bhardwaj. 2010. Directing dendritic cell immunotherapy towards successful cancer treatment. Immunotherapy 2: 37-56.). Interestingly, it has also been found important for the activation of allogeneic T-cells (Jonuleit, H. et al 2000. Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med 192: 1213-1222.). In our experiments, immature DCs (imDCs) were left untreated or matured for 18 h by COMBIG, Ad5M or their combination. The use of the infection-enhanced adenoviral vector Ad5M can facilitate loading of DCs with tumor antigens, a standard ex vivo practice to modify patient-derived DCs (Tacken, P. J., and C. G. Figdor. 2011. Targeted antigen delivery and activation of dendritic cells in vivo: steps towards cost effective vaccines. Semin Immunol 23: 12-20.). For the sake of having a simple system to assess the maturation effect of Ad5M on DCs, in this study Ad5M was used without a transgene. Cells were washed and cultured for another 24 h in fresh DC medium without addition of any cytokines. DC phenotypic changes were assessed by flow cytometry and the supernatants (SN) were collected to map the secretion profiles (FIG. 1A).

[0127] COMBIG maturation, alone or combined with Ad5M, induced upregulation of HLA-DR, CD40, CD80, CD86 and CD83, implying a mature and activated phenotype (FIG. 1B). COMBIG-matured and COMBIG/Ad5M-matured DCs demonstrated also elevated secretion of pro-inflammatory cytokines and chemokines. Thus, confirming the transduction didn't impair the maturation of imDCs into pro-inflammatory DCs.

[0128] Taken together, our data indicate that Ad5M/COMBIG-maturation is well tolerated by human monocyte-derived DCs and resulted in the generation of a fully matured and pro-inflammatory DC phenotype.

[0129] DCs Matured with the COMBIG-Cocktail Combined with Ad5M Infection Increase their Capacity to Activate Allogeneic T-Cells

[0130] T-cell activation in the allogeneic PBMC pool by co-cultured Combig-DCs or COMBIG/Ad5M DCs was assessed by the upregulation of the early activation protein CD69. Interestingly, in marked contrast to prevailing opinion T-cell activation was higher in co-cultures with allo-COMBIG infected with the Ad5M virus vector compared to those of non-infected allo-COMBIC-DCs.

[0131] Supernatants from Co-Cultures of Allo-COMBIG/Ad5M-DCs and PBMCs Induce an Efficient Maturation Bystander-DCs

[0132] MLR supernatants (Allo-SN) from co-cultures of PBMCs with allo-COMBIG-DCs or allo-COMBIG/Ad5M-DCs was found to significantly induce upregulation of CD40 on immature bystander-DCs. In line with the increased capacity of allo COMBIG/Ad5 DCs to activate allogeneic T cells, MLR supernatants from PBMC allo-COMBIG/Ad5M-DC co-cultures, but not MLR supernatants from PBMC non-infected allo-COMBIG-DC co-cultures, additionally led to significant upregulation of the maturation markers CD80 and CD86 on the bystander-DCs (FIG. 2E).

[0133] Bystander-DCs, Matured by the MLR-Supernatant from PMBC Allo-COMBIG/Ad5M-DC Co-Cultures, Efficiently Cross Present Antigens for T-Cell Expansion

[0134] Beside DC maturation, antigen-uptake and antigen-presentation play important roles in inducing adaptive tumor immunity (Vandenberk, L. et al 2015. Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines. Front Immunol 6: 663).

[0135] In order to test antigen-uptake and antigen-presentation we used an in vitro model system where DCs from HLA-A2.sup.+ donors (bystander-DCs) were incubated with cell lysate from tumor cells (A549(pp65)) expressing the full length CMV-pp65 protein, mimicking the condition of lysed tumor cells (FIG. 3A, C). These antigen-loaded immature bystander-DCs were then matured with MLR supernatants from PBMC-alloDC co-cultures. The exogenous pp65 antigen needs to be processed by the bystander-DCs into the pp65.sub.495-503 peptide and cross-presented on HLA-A2 (MHC class I) in order to stimulate autologous T-cells, engineered with a TCR specific for CMV-pp65.sub.495-503. We found that antigen-loaded bystander-DCs matured in MLR supernatants could efficiently cross-present the antigen and stimulate engineered CMV-pp65.sub.495-503 TCR-specific T-cells (FIG. 3A) to secrete high amounts of IFN-, with MLR supernatants from PBMC allo-COMBIG/Ad5M-DC co-cultures yielding significantly better activation compared to the MLR supernatants from PBMCs co-cultured with untransduced allo-COMBIG-DC (FIG. 3B).

[0136] We next wanted to test whether endogenous CMV-pp65-specific T-cells (non-engineered) could be activated and expanded. In this case we started with bystander-DCs from HLA-A2.sup.+ CMV-seropositive donors, loaded with pp65-containing tumor cell lysate and matured with allo-SN from PBMC allo-COMBIG/Ad5M-DC co-cultures (FIG. 3C). These bystander-DCs yielded a 5-fold expansion of autologous CMV-pp65-specific T-cells from CMV-seropositive donors (FIG. 3D). The ability of those T-cells to recognize their target and exert effector functions was tested in a re-stimulation assay. The expanded T-cells were exposed to T2 cells pulsed with either the HLA-A2-restricted pp65.sub.495-503 peptide or an irrelevant the HLA-A2-restricted peptide and the IFN- release was measured. Upon re-stimulation with the pp65-positive target cells, high IFN- release could be detected from the T-cells expanded by pp65-presenting bystander-DCs matured with allo-SN from PBMC allo-COMBIG/Ad5M-DCs co-cultures (FIG. 3E). The expanded T-cells were also confirmed as antigen-specific since exposure to T2 cells pulsed with an irrelevant peptide led only to background amounts of IFN- release (FIG. 3E).

[0137] In summary, we postulate a rationale for using allo COMBIG DCs infected with the adenovirus Ad5M (allo COMBIG/Ad5M DCs) as an immune enhancer inducing bystander DC maturation and subsequent priming of antigen-specific T cells. When compared to non-infected COMBIG-DCs, COMBIG-DCs infected with the

[0138] Ad5M virus (COMBIG/Ad5M DCs) were surprisingly found to create a pro-inflammatory milieu with enhanced capacity to induce bystander-DC maturation when co-cultured with allogeneic PBMCs. When a model antigen was provided exogenously, mimicking tumor cell lysate, bystander DCs matured with MLR supernatants from co-cultures of allo COMBIG/Ad5M DCs and PBMCs could efficiently digest and cross-present the model antigen and provoke antigen-specific cytotoxic T-cell expansion and activation.

EXAMPLE 2

In Vivo Mouse Studies with Mouse COMBIG DC:s Infected and Transduced with Adenovirus Vector Coding for a Tumor Associated Antigen (Glycoprotein 100)

[0139] In example 2, alloDCs infected with the Ad5M virus with genes encoding for the tumor antigen gp100 were evaluated. It was found that subcutaneous administration of Ad5M(gp100)-alloDCs stimulated the proliferation of adoptively transferred pmel-1 (gp-100-directed) CD8+ T-cells in the prophylactic setting. Moreover intratumoral administration of Ad5M(gp100)-alloDCs in the therapeutic setting resulted in delayed tumor growth and prolonged survival.

[0140] Materials and Methods

[0141] Production of the Recombinant Virus

[0142] The Ad5M(MOCK) and Ad5M(gp100) vectors were constructed and produced as previously described (Yu D, et al. Adenovirus with hexon Tat-protein transduction domain modification exhibits increased therapeutic effect in experimental neuroblastoma and neuroendocrine tumors. J Virol 2011; 85(24):13114-23 doi 10.1128/JVI.05759-11). They are E1 deleted adenovirus serotype 5 (Ad5) vectors having the fiber shaft and knob replaced with the serotype-35 counterpart. They also contain a cell-penetrating peptide from HIV Tat in the hypervariable loop of their hexon proteins (major capsid protein). They are infection-enhanced vectors with improved ability to transduce most hematopoietic cells including DCs (Yu D, et al. Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures. PLoS One 2013; 8(l):e54952 doi 10.1371/journal.pone.0054952). Ad5M(MOCK) does not encode any transgene, while Ad5M(gp100) encodes the human melanoma antigen glycoprotein 100 (gp100, amino acids 25-33). Titers were determined by quantitative polymerase chain reaction as encapsidated virus genomes (evg) per ml (Yu D, et al. Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures. PLoS One 2013; 8(l):e54952 doi 10.1371/journal.pone.0054952).

[0143] Cell Lines

[0144] The 911 cell line (Crucell, Leiden, The Netherlands) used for adenovirus production was cultured in DMEM supplemented with 10% heat inactivated FBS, 1% penicillin/streptomycin (PeSt) and 1% sodium pyruvate. The mouse melanoma cell line B16-F10 (ATCC, Rockville, MD) were cultured in DMEM supplemented with 10% heat inactivated FBS, 1% PeSt and 1% sodium pyruvate. The mouse melanoma cell lines Hcme13 and Hcmel12 are derived from a primary HGF-CDK4.sup.(R24C) melanoma model (Bald T, et al. Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma. Nature 2014; 507(7490):109-13; Landsberg J, et al. Melanomas resist T-cell therapy through inflammation-induced reversible dedifferentiation. Nature 2012; 490(7420):412-6) and are a kind gift from Prof. Thomas Tting, University Hospital Magdeburg. Both Hcmel3 and Hcmel12 were cultured in RPMI-1640 supplemented with 10% heat inactivated FBS. All components and culture media were from Thermo Fisher Scientific (Carlsbad, Calif.). All cells were cultured in a humidified incubator with a 5% CO.sub.2 atmosphere at 37 C.

[0145] Isolation and Maturation of Mouse Bone Marrow-Derived Allogeneic DCs

[0146] Bone marrow-derived DCs were generated from the femur and tibia of female 7-8 week old wild-type (wt) BALB/c mice (H-2D.sup.d) (The Janvier Labs, France) by exposing the bone marrow and flushing out the cells with a sterile syringe. The harvested cells were cultured in IMDM supplemented with 10% heat inactivated FBS, 1% PeSt, 1% HEPES, 50 M -mercaptoethanol. Culture medium was supplemented with 20 ng/mL recombinant murine IL-4 and 20 ng/mL recombinant murine GM-CSF (Nordic BioSite, Stockholm, Sweden). Bone marrow cells were plated on non-treated Petri dishes (Sarstedt, Nmbrecht, Germany). Medium was replaced every 3 days. On day 7 the non-adherent imDCs were harvested and treated for 18 h with a cocktail of combined Toll-like receptor ligands with IFN- (COMBIG), consisting of 2.5 g/mL R848 (InvivoGen, Sam Diego, Calif.), 20 g/mL polyinosinic:polycytidylic acid (polyI:C) (Sigma-Aldrich, St. Louis, Mo.) and 1000 IU/mL IFN- (Nordic Biosite) (Napolitani G, Rinaldi A, Bertoni F, Sallusto F, Lanzavecchia A. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat Immunol 2005; 6(8):769-76 doi 10.1038/ni1223) in order to obtain mature alloDCs. Ad5M(gp100)-alloDCs were obtained by transducing pelleted imDCs for 2 h, at 37 C. with 2000 evg/cell Ad5M(gp100) and matured as above. Cells were cultured in a humidified incubator with a 5% CO.sub.2 atmosphere at 37 C.

[0147] Murine Immune Cell Phenotyping

[0148] Flow cytometry was used to phenotype murine T-cells using anti-CD3-PB, anti-CD8a-APC, anti-Thy1.1-PE, anti-V-13-FITC, H-2D.sup.b/hgp100.sub.25-33-PE tetramer (Beckman Coulter, San Diego, Calif.). All antibodies were purchased from BioLegend (San Diego, Calif.). Data acquisition was performed using a FACSCanto II (BD Biosciences) flow cytometer, and the analysis was performed using FlowJo software (version 7.6.5; Tree Star, Ashland, Oreg.).

[0149] Animal Experiments

[0150] The Uppsala Research Animal Ethics Committee (C215/12) and the Northern Stockholm Research Animal Ethics Committee (N170/13, N164/15) have approved the animal studies. All in vivo experiments were performed with female 6-7 weeks old C57BL/6NRj (H-2D.sup.b) mice receiving vaccinations with treated allogeneic DCs (vaccine cells) generated from the bone marrow of 7-8 weeks old female BALB/c (H-2D.sup.d) mice and treated as described above. All mice were obtained from Janvier Labs.

[0151] Adoptive Transfer of pmel-1 (gp100-Specific) Splenocytes.

[0152] Mice were injected s.c. with 110.sup.6 vaccine cells. Pmel-1 splenocytes (1010.sup.6 per mouse) were injected intravenously (i.v.) 48 hours later. The transgenic pmel-1 mice have a C57BL/6NRj (H-2D.sup.b) background and around 20% of their splenocytes are Thy1.1.sup.+ CD8.sup.+ T-cells that carry a gp100.sub.25-33-specific TCR. Pmel-1 mice were originally obtained from the Jackson Laboratory (Bar Harbor, Me., USA) and kept in breeding by our group. Draining (inguinal) lymph nodes were harvested 7 days post pmel-1 adoptive splenocyte transfer and digested into single-cell suspensions.

[0153] In Vivo Assessment of Therapeutic Efficacy in a B16 Melanoma Model after Intratumoral Injection with alloDCs

[0154] Mice were injected s.c. into the hind flank with 110.sup.5 B16-F10 tumor cells. For the pmel-1 T-cell transfer experiment, mice received i.v. injection of 110.sup.8 pmel-1 splenocytes on day 8 and i.t. injections of 10.sup.6 alloDCs on day 8 and 14 post B16-F10 tumor cell inoculation. Tumors were measured with caliper and the tumor size was calculated as the formula: Volume=lengthwidth.sup.2/6. Mice were sacrificed when the tumor volume exceeded 1 cm.sup.3 or if bleeding ulcers developed. Cells were obtained from spleen, blood and lymph node of the sacrificed mice on day 15 post B16-F10 inoculation and stained for expression of the Thy1.1 and V13 pmel-1 T-cell markers.

[0155] Statistics

[0156] The data are reported as mean and SEM. Statistical analysis was performed by GraphPad prism software version 6.01 (La Jolla, Calif., USA). Statistical analyses were performed using parametric One way ANOVA test (>2 experimental groups) with Holm-Sidak test for multiple comparison correction and Mann-Whitney U test (only 2 experimental groups). Association of gp100-TCR.sup.+ T-cells and tumor burden was evaluated by a linear regression model computing Spearman correlation. Statistical comparison of the Kaplan-Meier survival curve was performed using log-rank test. Values with P<0.05 were considered to be statistically significant.

[0157] Results

[0158] Ad5M(gp100)-alloDCs Vaccination in a Mouse Melanoma Model Augments the Proliferation and Percistence of Adoptively Transferred pmel-1 T-cells, Delays Tumor Growth and Prolongs Survival

[0159] We sought to examine if Ad5M(gp100)-alloDCs in combination with adoptive T-cell transfer from pmel-1 splenocytes has a positive survival effect in melanoma-bearing mice. Nave C57BL/6NRj mice were injected s.c on the right hind limb with 110.sup.6 alloDCs or Ad5M(gp100)-alloDCs, followed by intravenous (i.v) adoptive transfer of 1010.sup.6 pmel-1 splenocytes (FIG. 4A). In contrast to non-transfected COMBIG-DCs, COMBIG/Ad5M(gp100)-DCs were able to stimulate the proliferation of the adoptively transferred gp100-specific Thyl.1.sup.+ CD8.sup.+ T-cells and sustain their presence five days after transfer (FIG. 4B).

[0160] As pmel-1 T-cells persist after vaccination with COMBIG/Ad5M(gp100)-DCs, we next evaluated the combinatorial effect of adoptive pmel-1 splenocyte transfer with i.t. injection of COMBIG/Ad5M(gp100)-DCs (FIG. 5A) in a therapeutic setting. The combined treatment delayed tumor growth (FIG. 5B) and significantly prolonged survival of tumor-bearing mice (FIG. 5C).

[0161] Discussion

[0162] Taken together the present example demonstrates that the use of COMBIG DCs infected with Ad5M coding for the tumor antigen gp100 can potentiate systemic gp100-specific T-cell responses, suppress tumor growth and prolong survival in a mouse melanoma model. Effective priming of T-cell responses by an immune primer/vaccine requires endogenous DCs to take up antigens, mature and migrate to dLNs where they can productively interact with nave T-cells (Dieu-Nosjean MC, et al Tertiary lymphoid structures in cancer and beyond. Trends Immunol 2014; 35(11):571-80; Kleindienst P, et al Endogenous dendritic cells are required for amplification of T cell responses induced by dendritic cell vaccines in vivo. J Immunol 2003; 170(6):2817-23), thus indicating that locally injected COMBIG-DC that have been infected with an Ad5M virus coding for a tumor antigens will similar to COBMIG DCs infected with and non-coding Ad5M virus (see Example 1) induce a pro-inflammatory response that stimulate maturation of endogenous bystander DCs and a subsequent efficient priming of tumor-specific T cells.

EXAMPLE 3

COMBIG-DCs Transduced with an Adenovirus Vector with Transgenes Coding for HPV-16 E6 and E7 Proteins

[0163] In example 3, the impact of infecting immature DCs with an adenoviral vector (Ad5) with transgenes coding for HPV-16 E6 and E7 proteins (Ad5-HPV16E6E7) on phenotypic activation/maturation induced by the COMBIG-cocktail (Poly-LC, R848 and IFN-gamma) alone was evaluated. In brief, it was seen that Ad5-HPV16E6E7 infection, similar to infection with the empty vector Ad5M (cf. example 1), does not impair the ability of the COMBIG-cocktail to induce phenotypic DC-maturation (cf. FIG. 6A, B). Moreover, the ability of COMBIG-DCs to stimulate the direct pathway of allorecognition, as measured by the activation (CD69 expression) of allogeneic T cells during a mixed leukocyte reaction (MLR) was increased (cf. FIG. 7B). A similar effect was seen in example 1 with the empty vector Ad5M (cf. FIG. 2B).

[0164] Moreover, the finding that activation/maturation of bystander-DCs with MLR-supernatants from co-cultures of allo COMBIG-Ad5M DCs and PBMCs was increased (FIG. 7C) implies that COMBIG-DCs transduced with the Ad5-HPV16-E6E7 vector are useful as an improved cell-based allogeneic immune enhancer in which the mode of action is crucially dependent on the direct pathway of allorecognition.

[0165] Materials and Methods

[0166] Production of Ad5-HPV16E6E7

[0167] Ad5 is an E1-deleted human adenovirus serotype-5 (Ad5) vector encoding the E6 and E7 transgenes. Titers were determined by quantitative PCR as encapsidated virus genomes (evg) per ml (Yu, D. et al 2013. Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures. PLoS One 8: e54952).

[0168] Isolation of Human PBMCs and Generation of DCs , Generation and Treatment of DCs,n Flow Cytometry, Human DC Phenotyping, Activation of T-cells in Allogeneic Mixed Leukocyte Reaction, Activation of Bystander Immature DCs and Statistics.

[0169] See Material and methods in Example 1

[0170] Results and Discussion

[0171] COMBIG/Ad5-HPV16E6E7-Stimulated DCs Exhibit a Mature Phenotype

[0172] To assess the maturation effect of Ad5-HPV-16-E6 and E7 on DCs, phenotypic changes were studied by flow cytometry (FIG. 6A, B). As shown in FIG. 6B, stimulation with COMBIG alone or combined with Ad5-HPV-16E6E7, induced upregulation of CD40, CD80, CD86, and HLA-DR, implying a mature and activated phenotype (FIG. 6B). Taken together, these data indicate that Ad5-HPV-16-E6E7/COMBIG-maturation is well tolerated by human monocyte-derived DCs and resulted in the generation of a fully matured DC phenotype.

[0173] DCs Matured with the COMBIG-Cocktail Combined with Ad5-HPV16E6E7 Infection Increase their Capacity to Activate Allogeneic T-cells

[0174] T-cell activation in the allogeneic PBMC pool by co-cultured COMBIG-DCs or COMBIG/Ad5-HPV16E6E7 DCs was assessed by the upregulation of the early activation protein CD69. Interestingly, in marked contrast to prevailing opinion, T-cell activation was higher in co-cultures with allo-COMBIG infected with the Ad5-HPV16E6E7 virus vector compared to those of non-infected allo-COMBIG-DCs (FIG. 7B).

[0175] Supernatants from Co-Cultures of allo-COMBIG/Ad5-HPV16E6E7 DCs and PBMCs Induce an Efficient Maturation Bystander-DCs

[0176] MLR supernatants (Allo-SN) from co-cultures of PBMCs with allo-COMBIG-DCs and allo-COMBIG/Ad5-HPV16E6E7 DCs, respectively, were found to induce upregulation of maturation markers (CD40, CD80 and CD86) on immature bystander-DCs. In line with the increased capacity of allo COMBIG/Ad5-HPV-16E6E7 DCs to activate allogeneic T cells (cf. FIG. 7B), MLR supernatants from PBMC allo-COMBIG/Ad5-HPV16-E6E7-DC co-cultures, provided increased upregulation of the maturation markers (CD40, CD80 and CD86) compared to MLR supernatants from PBMC non-infected allo-COMBIG-DC co-cultures (FIG. 7C).