Galactose oxidase treatment of dendritic cells to improve their immunogenicity

Abstract

The present invention relates to a method for producing dendritic cells with increased capability to activate T cells, to dendritic cells obtainable by such a method, and to a pharmaceutical composition comprising such dendritic cells.

Claims

1. A method for producing dendritic cells with increased capability to activate T cells, wherein said method comprises obtaining dendritic cells from bone marrow, or from monocytes or other hematopoietic dendritic cell precursor or progenitor cells by in vitro differentiation, and treating said dendritic cells with galactose oxidase in vitro, wherein said dendritic cells are human cells.

2. The method according to claim 1, wherein said dendritic cells to be treated with galactose oxidase are mature dendritic cells.

3. The method according to claim 1, wherein said dendritic cells are not treated with any maturation stimulus before, during or after said treatment with galactose oxidase.

4. The method according to claim 1, wherein said dendritic cells are not treated with neuraminidase before, during or after said treatment with galactose oxidase.

5. The method according to claim 1, wherein said dendritic cells with increased capability to activate T cells present at least one tumor antigen.

6. The method according to claim 1, wherein said galactose oxidase treatment is for 1-5 hours at a concentration of 0.1-20 U/ml.

7. The method according to claim 1, wherein said galactose oxidase treatment is for 1-2 hours at a concentration of 1-5 U/ml.

Description

(1) In the following, reference is made to the figures:

(2) All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.

(3) FIGS. 1A-1B show experimental data addressing the question whether GOX treatment alters the expression of MHC II, CD86, CD80, 4-1BBL or PD-L1.

(4) 1A. Murine BM-DCs (bone-marrow derived dendritic cells) were generated until day 8, treated with GOX for 90 min, washed and matured overnight with TNF, LPS or LPS+anti-CD40. Then FACS analysis was performed for the indicated markers. Representative dot plots (left) and statistical evaluation as bar graphs (right) are shown. As is evident from the data, murine BM-DCs treated with GOX do not show altered expression of MHC II or CD86.

(5) 1B. Also, GOX treatment of immature DCs for 90 min and culture overnight does not alter CD80, 4-1BBL or PD-L1 expression as shown by FACS analysis.

(6) All experiments shown are representative for three independent experiments with similar results.

(7) FIG. 2 shows experimental data addressing the question whether GOX treatment of murine BM-DCs leads to an increase in cytokine production of these cells.

(8) BM-DCs were treated at day 8 for 90 min with GOX, washed and subsequently matured with the indicated stimuli overnight before IL-6 and IL-12p40 analysis by ELISA. The data are representative for three independent experiments with similar results.

(9) As the data shows, GOX treatment of murine BM-DCs does not lead to an increase in cytokine release by these cells.

(10) FIGS. 3A-3B show experimental data addressing the question whether immature and mature murine BM-DCs show increased T cell priming capability in vitro and in vivo after GOX treatment.

(11) BM-DCs were generated until day 8, treated with GOX for 90 min and matured as indicated overnight.

(12) 3A. Then allogeneic lymph node cells were added as responder T cells for 3 days before proliferation was measured by [.sup.3H]-thymidine incorporation.

(13) 3B. For in vivo priming the DCs were additionally loaded with KLH (keyhole limpet hemocyanin) antigen together with the maturation stimuli for 16 h and before GOX treatment. Then the DCs were washed and injected s.c. into syngeneic mice. After 11 days lymph nodes and spleen were restimulated with KLH and pulsed with [.sup.3H]-thymidine to detect antigen-specific T cell priming. CPM=counts per minute. The data are representative for three independent experiments with similar results.

(14) Thus, immature and mature murine BM-DCs show increased T cell priming capability in vitro and in vivo after GOX treatment.

(15) FIGS. 4A-4C show experimental data addressing the question whether human DCs mature by GOX treatment alone.

(16) Human DCs were generated from monocytes and treated with different maturation stimuli or GOX or their combination overnight.

(17) 4A. Then cells were analyzed by FACS for surface marker expression as indicated from one representative donor.

(18) 4B. The results of FACS analyses of the CD83 and CD25 maturation markers of DCs from different donors are displayed (PIC=Poly I:C). Each data point represents the cytokine release of one donorGOX treatment.

(19) 4C. The results from B were expressed as % increase of GOX-treated versus untreated cells. Thus, human DCs mature by GOX treatment alone.

(20) FIGS. 5A-5B show experimental data addressing the question whether GOX treatment causes increased IL-6 production of human DCs.

(21) Human DCs were generated from monocytes and treated with different maturation stimuli or GOX or their combination overnight.

(22) 5A. Then the culture supernatants were tested by ELISA for their cytokine content.

(23) 5B. The results from A were expressed as fold increase of GOX-treated versus untreated cells. Each data point represents the cytokine release of one donorGOX treatment.

(24) Thus, GOX treatment heavily increases IL-6 production of human DCs.

(25) FIGS. 6A-6B show experimental data comparing the allogeneic and syngeneic T cell priming capability of immature and mature human DCs in vitro upon GOX treatment.

(26) Human DCs were generated from monocytes and treated with different maturation stimuli overnight. Then the DCs were treated with GOX, washed and added to allogeneic (6A) or syngeneic (6B) T cell cultures. After 3 days the cultures were pulsed with [.sup.3H]-thymidine to measure T cell proliferation. CPM=counts per minute. The data are representative for four and three independent experiments with similar results, respectively.

(27) Thus, GOX treatment increases preferentially the allogeneic T cell priming capability of immature and mature human DCs in vitro.

(28) FIG. 7 shows experimental data addressing the question if GOX treatment of DCs alters surface lectin stainings.

(29) The indicated lectin stainings on immature or LPS- or cocktail-matured DCs with or without additional GOX treatment were performed together with CD83 staining. Histograms for immature DCs were gated as CD83.sup. and for mature DCs as CD83.sup.+ cells. Data are representative of three independent experiments with similar results.

(30) As can be seen from the data, GOX treatment of DCs does not alter surface lectin stainings.

(31) FIG. 8 shows experimental data addressing the question if GOX treatment of human DCs induces clustering with allogeneic T cells. Immature or mature DCs treated or not with GOX were co-cultured with allogeneic T cells for 1 h or 42 h in 96-well flat bottom plates before photographs were taken under phase contrast conditions. n=2

(32) As can be seen from the data, GOX treatment of human immature DCs induces strong and rapid clustering with allogeneic T cells unlike DCs without GOX. GOX treatment of mature DCs induces larger cluster formation as compared without GOX treatment.

(33) Thus, cluster formation with T cells can be used as a marker to distinguish GOX-treated from untreated immature DCs and increased clustering do detect GOX treatment of mature DCs.

(34) FIG. 9 shows experimental data to explore if the effects of GOX treatment of human DCs are stable. Human DCs were generated from monocytes and treated with different maturation stimuli overnight. Then the DCs were treated with GOX, washed and cultured for 0 h or 6 h at RT or 16 h at 37 C. before added to allogeneic T cells. After 3 days the cultures were pulsed with [.sup.3H]-thymidine to measure T cell proliferation. CPM=counts per minute. The graphs represent pooled cpm values from three independent experiments with each performed in triplicates.

(35) As can be seen from the data, GOX treatment of human DCs remain stable for 6 and 16 hours. Thus, the T cell stimulatory effects by GOX treatment as part of a clinical DC preparation procedure is retained, even when the cells may have to be transported for some hours from the cell culture laboratory to the patient until injection.

(36) FIG. 10 shows experimental data to investigate if GOX-treatment of mature DCs further improves the effects of therapeutic vaccination with such DCs in a mouse tumor model. As can be seen from the data, GOX treatment of mature peptide-loaded DCs further improves their potential as anti-tumor vaccines.

(37) In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.

EXAMPLES

Example 1

Generation and Maturation of Dendritic Cells

(38) Murine DC Generation and Maturation

(39) BM-DCs were generated as described in detail before (9). At d8 (day 8) cells were matured by addition of LPS (0.1-1 g/ml, SIGMA), TNF (500 U/ml, Peprotech) or anti-CD40 (5 g/ml, Biolegend) to the cultures for 16 h or as otherwise indicated.

(40) Human DC Generation and Maturation

(41) DC generation from human PBMCs (peripheral blood mononuclear cells) was adapted from (10) and was principally performed under conditions that fulfill GMP (good manufacturing practice) requirements as used for injection of DCs in patients. Peripheral blood was obtained from Blood Bank of the University of Wrzburg and depleted of thrombocytes and plasma by using the Trima Accel apheresis system (CaridianBCT, #80300) from healthy blood donors with consent of the Ethical Committee of the University of Wrzburg. PBMCs were isolated by centrifugation in Lymphocyte Separation Medium LSM 1077 (PAA Laboratories GmbH, Clbe) and plated in sterile Tissue culture dishes 100 mm (Greiner No 664160) at a density of 5010.sup.6 cells per dish in 10 ml culture medium RPMI 1640 without L-Glutamine (PAA Laboratories GmbH, Clbe) with 1% heat-inactivated (30 min., 56 C.) human Serum, of the clot, Type AB (PAA Laboratories GmbH, Clbe), 2 mM L-Glutamin (PAA 200 mM), Penicillin/Streptomycin (PAA 100) and incubated at 37 C., 5% CO.sub.2 for 1 h. After 1 h the non-adherent fraction (NAF) was removed and deep-frozen at 80 C. for later use as T cell source for the allogeneic mixed lymphocyte reaction (MLR). The dishes were washed twice with PBS without Ca.sup.2+ or Mg.sup.2+. Afterwards 10 ml of fresh, warm culture medium was added on the dish with the adherent monocytes and incubated for 16 h at 37 C., 5% CO.sub.2. At day one the culture medium was taken off carefully so that loosely adherent cells were not removed and new culture medium containing 800 U/ml GM-CSF (Granulocyte macrophage colony-stimulating factor; Leukine Sargramostim Bayer) and 250 U/ml IL-4 (Strathmann Biotec AG Hamburg) was added. Cytokines were added again at day 3 in 3 ml fresh medium (containing GM-CSF 800 U/ml and IL-4 25-250 U/ml) per dish and cultured until day 5. Maturation of human DCs was performed at 510.sup.5 cells/well in 24 well plates with a maturation cocktail consisting of PGE.sub.2 (1 g/ml, SIGMA), TNF (1 ng/ml), IL-6 (1000 U/ml), and IL-1 (2 ng/ml, all Strathmann Biotec, Hamburg) as described (11). Alternatively, 0.1 g/ml LPS (E. coli, 0127:B8, SIGMA) or 50 g/ml Poly I:C (InvivoGen, Toulouse) were used.

GOX Treatment

(42) Murine and human DCs were treated with GOX (2 U/ml, SIGMA, from Dactylium dendroides) for 90 min at 37 C. and washed with PBS always before adding maturation stimuli. For in vitro and in vivo priming with DCs GOX treatment was performed for 90 min (2 U/ml) after maturation of the DCs just before the injection.

FACS Analyses

(43) For the staining 0.5-510.sup.5 cells were incubated with the antibodies and respective isotype controls with buffer (PBS with 0.1% BSA, 0.2% sodium azide, 2 mM EDTA at 4 C. for 30 min. Then cells were washed and analyzed with an LSR II flow cytometer (BD) and FlowJo Cytometry Analysis Software (Tree Star Inc. Olten).

(44) Antibodies to stain mouse cells were used at 1:300 dilution for MHC II (M5-114, PE, BD Pharmingen and Biolegend), and 1:100 dilution for CD80/FITC, CD86/FITC, 4-1BBL/PE and PD-L1/PE (Biolegend and eBioscience). Secondary staining was performed with streptavidin-PE conjugates (1:200, BD Pharmingen). Antibodies for human FACS analyses were HLA-DR/PE (1:50, BD Biosciences), CD83/APC (1:50, BD Biosciences), CD86/PE (1:25, BD Biosciences) and CD25/PE (1:50, Miltenyi Biotec).

(45) The lectins PNA (peanut agglutinin, from Arachis hypogaea, recognizing 1-3-linked galactose/N-acetylgalactosamine), SNA-1 (Sambucus nigra lectin type-1, recognizing 2-6-linked sialic acid/galactose) and MAA (Maackia amurensis lectin, recognizing 2-3-linked sialic acid/galactose) were used for detection of sugar linkages of the glycocalix on the DC surface as FITC-conjugates (EY laboratories).

ELISA

(46) Murine and human cytokine detection were performed with commercial kits according to the manufacturer's instructions from the following sources: mouse IL-6 (BD Biosciences), mouse IL-12p40 (BD Biosciences), human IL-6 (R&D Systems), human IL-12p40 (BD Biosciences).

Allogeneic MLR (Mixed Lymphocyte Reaction)

(47) Mouse: DCs were cultured as described until d8, treated for 90 min with GOX, then transferred into a 24 well plate at 510.sup.5 cells/well and stimulated overnight for 16 h with maturation reagents. After washing titrated numbers of DCs were added to 210.sup.5 total lymph node cells as a source of T cells in a 96-well flat bottom plate (FALCON).

(48) Human: At day 5 of DC culture after PBMC (peripheral blood mononuclear cells) preparation NAF from other blood donors (allogeneic) were prepared for allogeneic MLR and cultured over night. At day 6 the stimulated DC were harvested and seeded into two 96-well flat-bottom plates as triplicates at titrated amounts of DCs as indicated. Then 210.sup.5 NAF from different donor were added.

(49) Both human and mouse: After 3 days cultures were pulsed with 1 Ci/well [.sup.3H]-Thymidin (Hartmann Analytic, Braunschweig) for 16 h and then harvested onto glass fiber filter mats (PerkinElmer, USA), scintillation wax added and counted in a 1450 Microbeta Counter (Tomtec) to measure cell proliferation.

In Vivo Priming of Mice with KLH

(50) BM-DCs from C57BL/6 mice were treated at d8 with KLH (50 g/ml, high purity, endotoxin-free, sterile, Calbiochem/Merck Millipore #374825) and with or without LPS overnight. Then cells were treated directly with GOX as mentioned above, washed and injected with 410.sup.5 cells/mouse s.c. into the footpads of syngeneic mice. After 11 days the popliteal and inguinal lymph nodes and spleens were removed. Spleens were treated for erythrocyte lysis with 1.66% NH.sub.4Cl in PBS for 3 min at 37 C. Single cell suspensions from both types of organs were restimulated with KLH at titrated doses in serum-free HL-1 medium (Bio Whittaker/Lonza) for three days before cultures were pulsed with 1 Ci/well [.sup.3H]-Thymidin (Hartmann Analytic, Braunschweig) for 16 h and then harvested onto glass fiber filter mats (PerkinElmer, USA), scintillation wax added and counted in a 1450 Microbeta Counter (Tomtec) to measure cell proliferation.

Therapeutic Vaccination with (GOX-Treated) DCs in Mice

(51) MO4-10 tumor cells (kindly provided by Natalio Garbi, Bonn), which represent a subclone of B16-ova originally generated by Kenneth L. Rock (15), were injected at 110.sup.6 cells/mouse s.c. into the flanks of C57BL/6-albino mice (each group n=5 mice). Seven days later 110.sup.7 OT-I and 110.sup.7 OT-II pooled lymph node and spleen cells were injected i.v. into each mouse.

(52) BM-DC were generated from C57BL/6 mice as described in detail before (9). DCs were harvested at days 6, 8 and 10, at each time point stimulated with LPS (0.1 g/ml SIGMA) plus anti-CD40 (5 g/ml, clone 1C10, Biolegend) plus OVA.sub.323-339 peptide (10 M) plus OVA.sub.257-264 peptide (10 M) for 4 h at 37 C. and 5% CO.sub.2 as described before (13, 14). Parallel DC cultures remained untreated or received the additionally treatment with GOX (2 U/ml) for the last 90 min of the above-mentioned stimulation. After washing the two types of DCs were counted and injected at 210.sup.6 DCs/200 l s.c. into the contra-lateral flank. Thus, each mouse received three DC injections at 1, 3 and 5 days after the T cell transfer. Tumor growth was followed by caliper measurement every second day after the first DC injection.

Example 2

Murine BM-DCs do not Show Changes in Maturation Markers or Cytokine Production after GOX Treatment

(53) All methods mentioned in this example were carried out as described in Example 1.

(54) BM-DCs were generated and treated with various maturation stimuli alone or in combination with GOX. GOX treatment was performed on untreated DCs or after the overnight maturation of DCs. Analysis of surface MHC II and CD86 expression showed activity of the maturation stimuli as expected, but no further up-regulation by GOX (FIG. 1A). Additional analyses on other co-stimulatory or co-inhibitory markers such as CD80, 4-1BBL or PD-L1 on immature DCs revealed no effects (FIG. 1B).

(55) Since secretion of pro-inflammatory cytokines by DCs is another critical component not only for DC maturation but also T helper cell polarization, the levels of IL-6 and IL-12 secreted by BM-DCs after various stimulations alone or in combination with GOX was tested. Also here, no effects of GOX on the DCs could be observed (FIG. 2). Together, mouse BM-DCs seem unresponsive to GOX treatment with respect to various maturation criteria.

Example 3

GOX Treatment of Murine DCs Increases T Cell Priming Capability In Vitro and In Vivo

(56) All methods mentioned in this example were carried out as described in Example 1.

(57) To test whether GOX-treated DCs are functionally modified by treatment with GOX, in vitro and in vivo T cell priming assays were carried out.

(58) Immature and mature BM-DCs were treated or not with GOX and then titrated into cultures containing allogeneic T cells. After 3 days T cell proliferation was measured. Interestingly, both immature and mature DCs improved their T cell stimulatory potential in vitro (FIG. 3A). For therapeutical application of DCs, their in vivo priming potential for exogenously pulsed antigens and their successful migration into the T cell areas of lymphoid organs are more relevant. Thus, BM-DCs were loaded with KLH antigen that is also used in clinical studies as a bystander antigen supporting tumor peptide vaccinations with DCs (12). The DCs were matured with LPS or not for the period of the 4 h simultaneously during KLH pulse before the 90 min GOX treatment and subcutaneous injection into syngeneic mice. After 11 days the draining lymph nodes and the spleens were removed and single cell suspensions restimulated with KLH. Immature DC were largely unable to prime the mice, while LPS-DCs showed a clear response in the lymph nodes but little in the spleen (FIG. 3B). In contrast, both immature and mature DCs that were additionally treated with GOX showed an increased priming potential, also by immature DCs and in the spleen, indicating that GOX-aided DC vaccination provokes not only a stronger but also more systemic T cell response against an antigen.

Example 4

Human Monocyte-Derived DCs Mature Upon GOX Treatment

(59) All methods mentioned in this example were carried out as described in Example 1.

(60) To test also human DCs for their response to GOX, the murine experiments (Examples 2 and 3) were repeated with human immature and mature DCs. Surprisingly, while murine DC surface markers remained unaffected after GOX treatment, human immature DCs responded on GOX by upregulating HLA-DR, CD83, CD86 and CD25 (FIGS. 4A, 4B). Combined GOX treatment with different typical maturation stimuli such as by cytokine cocktail, Poly I:C or LPS could only weakly further improve the maturation state that was reached with GOX alone, when the cells from the same donor were compared (FIG. 4C).

(61) When testing the supernatants of these cultures, it was found that GOX alone could not induce IL-12p40, but stimulated IL-6 release (FIG. 5A). Combination of GOX with the other maturation stimuli dramatically further augmented IL-6 release, but did only marginally improve IL-12p40 production when cells from the same donor were compared (FIG. 5B).

(62) Together, GOX treatment of human DCs induces strong maturation as detected by surface markers and IL-6 release, but does not substantially provoke IL-12p40 production. Combination with typical maturation stimuli had mild effects on further surface marker and IL-12p40 up-regulation, but enhanced IL-6 release between 163- and 2763-fold.

Example 5

Human GOX-Treated DCs Show Increased T Cell Priming Potential In Vitro

(63) To test how the GOX-mediated maturation translates into T cell priming, the differentially treated DCs were cultured with allogeneic or syngeneic naive T cells and their proliferation after 3 days in culture was measured. GOX treatment of immature DCs clearly enabled T cell priming but combination of GOX with any maturation stimulus enhanced T cell proliferation (FIG. 6A, B). In fact 1000 DCs after GOX plus maturation stimulus treatment were as or even more effective than 10000 DCs treated with maturation stimuli alone, indicating an at least 10-fold improved T cell priming capability. Also the proliferation of syngeneic T cells could be induced (FIG. 6B), but did not exceed the levels of allogeneic T cell proliferation stimulated by immature DCs (FIG. 6A). Thus, a preferential expansion of allogeneic over syngeneic T cells could be achieved by GOX treatment of DCs.

Example 6

GOX Treatment of DCs does not Modify Glycocalix Structure

(64) All methods mentioned in this example were carried out as described in Example 1.

(65) In this experiment, it was tested whether GOX treatment of DCs would modify galactose moieties of the glycocalix in such a way that the binding of FITC-conjugated plant lectins to the DC surface would be modified. Lectins were selected that can detect distal disaccharide linkage structures of galactose with other sugars. The results indicate that neither binding of PNA (1-3-linked galactose/N-acetylgalactosamine), SNA-1 (2-6-linked sialic acid/galactose) nor MAA (2-3-linked sialic acid/galactose) was altered by GOX treatment (FIG. 7). These data suggest that the three-dimensional structure of the glycocalix is not modified by GOX.

Example 7

Rapid and Strong Clustering with Allogeneic T Cells Marks GOX-Treated DCs

(66) All methods mentioned in this example were carried out as described in Example 1.

(67) Increased T cell stimulation may result from more intense interactions between DCs and T cells that can be visualized as cluster formations. Cultures prepared as for the mixed lymphocyte reactions were photographed after 1 h or 42 h. The data show the absence of clusters when T cells were cultured alone or with immature DC that were not treated with GOX (FIG. 8). However, immature DC treated with GOX rapidly clustered with T cells already after 1 h, thereby marking them for a preceding GOX treatment. As expected cluster formation appeared with mature DCs and GOX treatment further increased with GOX treatment (FIG. 8). Thus, GOX treatment of immature and mature DCs promotes more intense interactions during antigen presentation as indicated by cluster formation.

Example 8

The Increased T Cell Stimulatory Effect of DCs by GOX-Treatment Remains Stable

(68) All methods mentioned in this example were carried out as described in Example 1.

(69) The enzymatic modification of the DC surface by GOX may be transient and reversible after removal of GOX by the turnover of cell surface molecules and metabolic activity. Therefore it was tested whether the increased T cell stimulation of GOX-treated mature DCs remained stable after maturation and GOX treatment for 6 h at RT or 16 h at 37 C. before adding to allogeneic T cells (FIG. 9). The data reveal that no loss of T cell stimulatory capacity occurs under these conditions, allowing the GOX treatment of DCs also under clinical conditions during a transfer of GOX-DCs from the cell culture facility to the patient in the clinic.

Example 9

GOX-Treatment of LPS-Plus Anti-CD40-Matured DCs Improves their Anti-Tumor Vaccination Capacity in Mice

(70) All methods mentioned in this example were carried out as described in Example 1.

(71) In this experiment, it was tested whether GOX-treatment of mature DCs would further improve the therapeutic vaccination success of DCs in a pre-clinical tumor model of therapeutic vaccination with mice that carried an established tumor. To show this, OVA.sup.+B16 melanoma cells (subclone MO4-10) were subcutaneously injected into the flanks of mice. One week later OVA-specific CD4.sup.+ (OT-II) and CD8.sup.+ (OT-I) T cells were adoptively transferred i.v. and one day later the therapeutic DC vaccinations started. DCs were loaded with the MHC I- and MHC II-restricted OVA peptides plus LPS and anti-CD40 antibodies for 4 h before s.c. injection into the contralateral flank. One experimental group of DCs received additional GOX treatment. The combination of maturation reagents LPS plus anti-CD40 was chosen due to its rather high immunogenicity (13, 14). Here it was tested whether GOX-treatment of these mature DCs would further improve their therapeutic potential. Injection of LPS/CD40-matured DC delayed tumor growth between days 4 and 8, while the same DCs additionally treated with GOX fully controlled further tumor growth over a period of 2 weeks. These results indicate that GOX treatment of mature peptide-loaded DCs further improve their potential as anti-tumor vaccines.

(72) The results of all 9 time points were analyzed together by a paired, two-tailed Students's t test. The result was significant as p=0.0105.

(73) The features of the present invention disclosed in the specification, the claims, and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.

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