THERAPEUTIC CANCER VACCINES DERIVED FROM A NOVEL DENDRITIC CELL LINE

Abstract

The invention is in the field of medical sciences. It provides means and methods for the treatment of cancer. More in particular, it provides cells and cell lines that can be developed into fully functional dendritic cells. These cells endogenously express cancer-specific antigens, which makes them particularly suited for the treatment of different kinds of cancer. More in particular, the invention relates to a precursor cell line for dendritic cells called DC-One as deposited at the DSMZ under accession number DSMZ ACC3189 on Nov. 15, 2012.

Claims

1-5. (canceled)

6. A method for treating cancer comprising administering to a subject in need thereof modified cells that are CD34-positive, CD1a-positive, CD83-positive, and CD14-negative, thereby treating cancer in the subject.

7. The method according to claim 6 wherein the cancer is acute myeloid leukemia (AML).

8. The method according to claim 6, wherein the modified cells are administered as a therapeutic cancer vaccine.

9. (canceled)

10. The method according to claim 6, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, head and neck cancer, chronic myeloid leukemia, ovarian cancer, colon cancer, multiple myeloma, prostate cancer, skin cancer, myelodysplastic syndrome, brain cancer and bladder cancer.

11. The method according to claim 6, wherein the modified cells are combined with a pharmaceutically acceptable carrier to form an immunogenic composition.

12. The method of claim 6, wherein the modified cell is differentiated from a precursor cell line of leukemic origin.

13. The method according to claim 11, wherein the precursor cell line is as deposited at the DSMZ under accession number DSMZ ACC3189 on Nov. 15, 2012.

14. The method according to claim 6, wherein the modified cells are obtained by a method comprising the steps of incubating the precursor cell line under conditions that allow differentiation of the progenitor cells into immature cells and incubating said immature cells under conditions that allow maturation of the immature cells into modified cells.

15. The method according to claim 14, wherein the method for obtaining the modified cells additionally comprises a step of altering the phenotype of the modified cells by introducing genetic material into the cells and/or by knocking out endogenous genes of the cells.

16. The method of claim 15, wherein the genetic material is a gene encoding an immunotherapeutic agent.

17. The method of claim 16, wherein the immunotherapeutic agent is selected from the group consisting of a tumor antigen, a viral antigen, and an antigen from a parasite, bacteria, or other microorganism.

18. The method of claim 14, wherein conditions that allow differentiation of the precursor cells comprise contacting the precursor cells with a composition comprising at least one molecule selected from the group consisting of IL-4, IL-6, PGE-2, TNF-α, TGF-β, and GM-CSF.

19. The method of claim 14, wherein differentiation of the precursor cells comprises contacting the precursor cells with a composition comprising GM-CSF, IL-4, and TNF-α.

20. The method of claim 14, wherein conditions that allow maturation of the immature cells comprise contacting the immature cells with a composition comprising at least one molecule selected from the group consisting of IL-6, PGE-2, TNFα and IL1β.

21. The method of claim 14, wherein maturation of the immature cells comprises contacting the immature cells with a composition comprising TNFα, PGE2, and IL1fβ.

22. The method of claim 14, further comprising combining the modified cell with a pharmaceutically acceptable carrier to form an immunogenic composition.

23. The method of claim 14, further comprising loading the cell with at least one further antigen.

Description

LEGEND TO THE FIGURES

[0056] FIG. 1: Graphic representation of the expression profile of dendritic cell differentiation markers and maturation markers on DCOne progenitor cells, immature DCOne cells and mature DCOne dendritic cells.

[0057] FIG. 2: Expression of WT-1 by DCOne derived cells. Photographic representation of a Western blot analysis demonstrating protein expression of WT-1 in DCOne progenitors (lane 1), immature DCOne (lane 2) and mature DCOne dendritic cells (lane 3). An anti-actin antibody is used as the control, showing equivalent protein loading in each lane.

[0058] FIG. 3: Schematic representation of the process of proliferation, differentiation and maturation of DCOne cells leading to fully functional DCOne Dendritic Cells.

[0059] FIG. 4: Graph representing the percentage expression of various cell markers found on mature DCOne dendritic cells (mDCOne) versus peripheral blood monocyte derived dendritic cells (moDC).

[0060] FIG. 5: Graph showing the population doubling time (PDT) of MUTZ-3 progenitor cells (squares) versus DCOne progenitors (circles). Horizontal axis represents passage numbers.

[0061] FIG. 6: Graph showing the expression of different cell markers in mature DCOne (mDCOne) and MUTZ-3 cells.

[0062] FIG. 7: Graph showing the stimulation of WT-1 specific Cytotoxic T-lymphocytes by mDCOne as demonstrated by IFN release.

[0063] FIG. 8: Graph showing percentage lysis of target cells by WT-1 specific cytotoxic T-lymphocytes (CTL) that have been stimulated by mDCOne. Such stimulated CTL specific for WT1 can kill cells of the myeloid leukemia cell line K562 when K562 is transfected with HLA-A2, since HLA-A2 is required for WT-1 antigen presentation. As a negative control, the untransfected K562 cell line that lacks HLA-A2 was used. Also cells from another AML cell line, AML progenitors, could be killed.

[0064] FIG. 9: Graph showing the percent migration of immature DCOne, mature DCOne and monocyte derived dendritic cells under the influence of two lymph node homing cytokines (6Ckine (CCL21) and MIP3beta (CCL19)). Culture medium is used as the control.

[0065] FIG. 10: Graph and photograph showing the relation between mean induration and pre- and post vaccination potential of mDCOne cells to induce a systemic immune response in patients, as documented by the delayed type hypersensitivity (DTH) response (Upper Panel). Picture showing a typical delayed type hypersensitivity response as documented by erythema and induration (Lower Panel). As a negative control, the formulation fluid used to cryopreserve mDCOne cells is used.

[0066] FIG. 11: Pre- and post-vaccination PBMC were in-vitro stimulated with PRAME peptide mix and cultured for 10 days. The cells were restimulated with PRAME peptides and PRAME reactive IFN-γ-producing T-cells were analysed in IFN-γ ELISpot assay. Medium control served as negative control.

EXAMPLES

Example 1: Proliferation, Differentiation and Maturation of DCOne Cells

[0067] DCOne progenitor cells may be differentiated into cells with all characteristics of immature dendritic cells, and subsequently matured into cells with all characteristics of mature dendritic cells.

[0068] DCOne progenitor cells were cultured (expanded) in routine maintenance medium, consisting of MEM-α (Minimum essential medium, Lonza, Verviers, Belgium) containing 10% fetal calf serum (FCS) (Hyclone, Perbio Science, Etten-Leur, The Netherlands), 100 IU/ml sodium-penicillin (pen), 100 μg/ml streptomycin (strep), 2 mM L-glutamine (glut), 50 μM β-mercaptoethanol (2ME) and GM-CSF (5 ng/ml). This is the first step, resulting in expansion of the progenitor cells.

[0069] Progenitor cells were allowed to differentiate into immature DCOne for 6 days by adding 1000 IU/ml GM-CSF, 20 ng/ml IL-4 and 120 IU/ml TNF-α. Fresh cytokines were added on day 3.

[0070] Next, maturation was induced by adding mimic mix (24001 U/ml TNF-α, 100 ng/ml IL-6, 1 ug/ml PGE2 and 25 ng/ml IL1-β) for 2 days.

Example 2: Phenotyping

[0071] Cells were immunophenotyped using the following FITC- and/or PE-conjugated Mabs reactive against: CD1a (1:25), CD80 (1:25), CD86 (1:25), CD40 (1:10) (PharMingen, San Diego, Calif.), CD14 (1:25), DC-SIGN (1:10) (BD Biosciences, San Jose, Calif.), CD83 (1:10), CD34 (1:10), Langerin (1:10) (Immunotech, Marseille, France). 2.5 to 5.Math.10.sup.4 cells were washed in PBS supplemented with 0.1% BSA and 0.02% NaN.sub.3 and incubated with specific or corresponding control Mabs for 30 minutes at 4° C. Cells were washed and analyzed on a FACS-Calibur flow cytometer (Becton and Dickinson, San Jose, Calif.) equipped with CellQuest analysis software. Results were expressed as the percentage of positive cells. Mature DCOne cells exhibit the prototypical expression of the mature DC marker CD83, and strongly increased expression of CD1a and CD40, as can be observed in FIG. 1.

Example 3: Comparison with Dendritic Cells Prepared from CD34+ Precursor Cells

[0072] The phenotypical characteristics of mature DCOne cells are comparable to those of mature dendritic cells prepared from CD34+ precursor cells. CD34.sup.+ haematopoietic precursor r cells were isolated from blood of healthy donors and expanded for 2-5 weeks with 25 ng/ml fms-like tyrosine kinase-3 ligand (Flt3-L) and 10 ng/ml stem cell factor (SCF). Thereafter, they were differentiated and matured as described above. This process is illustrated in FIG. 3. DCOne progenitor cells were expanded, differentiated and matured as described above using medium supplemented with specific cytokines. Cells were subsequently tested using flow cytometry for expression of the markers CD1a, CD80, CD86, CD40 and CD83, using antibodies as described under Example 2. The phenotypic characteristics of the monocyte-derived mature DC population and mature DCOne cells are comparable, as can be seen in FIG. 4. Mature DCOne showed a higher expression of CD1a in comparison to moDC. CD1a is a characteristic molecular marker on DCs that are differentiated and is involved in antigen-presenting functions. MoDCs and DCOne were comparable in their expression of CD80, CD86, CD40 and CD83. CD83 is a co-stimulatory molecule and is the most specific marker of mature DCs, since it is absent from immature DC (see also FIG. 1).

Example 3: DCOne as Well as iDCOne and mDCOne Express WT-1

[0073] Western blotting was performed with a commercial SDS-PAGE Electrophoresis System. Cells (10E6) were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X100 and proteinase inhibitor PMSF)

[0074] Ten microgram protein samples were resuspended in a reduced SDS PAGE sample buffer, and then electrophoresed on 4 to 10% Tris gel with Tris running buffer and blotted to PVDF membrane. The blots were blocked for 1 hour at room temperature in 10% non-fat dry milk in Tris buffered Saline containing 0.1% Tween-20 (TBST). Blots were probed with a polyclonal anti WT-1 antibody diluted in TBST containing 0.5% non-fat dry milk in TBST. A horseradish peroxidase-conjugated goat anti-rabbit antibody was then added, and secondary antibodies were detected through autoradiography using enhanced chemiluminescence (ECL Plus, General Electric Healthcare, Milwaukee, Wis.). Beta actin was used as a protein loading control and detected by an anti-beta actin antibody.

Example 4: Reproducibility of Proliferation of DCOne in Comparison with MUTZ-3

[0075] Progenitor cells obtained from the DCOne and MUTZ-3 cell line (obtained from DSMZ) progenitors were cultured in T175 culture flasks at 0.2×10E6 cells/ml in MEM-alpha 20% FCS plus penicillin/streptomycin supplemented with 20% conditioned medium of 5637 supernatant (Kurtzberg et al., 1989 and Welte et al., 1985). Cells are incubated in a CO2 incubator at 37 degrees Celsius+5% CO2 and passed every 3 to 4 days. Population Doubling Times (PDT) were assessed by cell counting.

[0076] To obtain conditioned medium from 5637 cells, 5637 cells of a growing and confluent culture were seeded at 0.9×10E6 cells/25 ml/T175 culture flask and incubated in a CO2 incubator at 37 degrees Celsius and 5% CO2. After 72 hours, all conditioned medium was collected by centrifugation for 10 minutes at 3000 rpm at 4 degrees Celsius.

Example 5: Activation of WT-1 Specific T Cells

[0077] It appeared that mature DCOne cells were able to directly activate WT-1 specific T-cells, without any deliberate loading with WT-1 antigen of exogenous origin, and such WT-1 specific T cells stimulated by the DCOne product can kill WT-1 positive leukemic tumour cells in vitro.

[0078] Due to its WT-1 expression, the mature DCOne product can stimulate WT1-specific T cells as demonstrated by their ability to induce IFN release by T-cells specific for WT-1, as well as induce killing of leukemia cells by such DCOne-stimulated WT-1 specific T cells. A sample of the data is shown in FIGS. 7 and 8.

[0079] In these experiments, WT-1-specific T cell clones were analysed for cytotoxicity by flow cytometry using K562, K562-A2 and DCOne progenitors as targets labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE; ImmunoChemistry technologies, LLC, MA, USA). In short, 1-2×10E6 target cells were washed and labelled with 1:10 diluted CFSE stock solution for 15 minutes at room temperature (RT). Next, the target cells were washed with 1 ml Iscove's Modified Dulbecco's Media (IMDM, Gibco) supplemented with 100 U/mL penicillin/100 microliter/mL streptomycin, 2 mM L-glutamine (Life technologies, Grand Island, N.Y., USA; further referred as CTL medium). Cells were resuspended in 2 ml CTL medium and incubated in water bath for 30 minutes at 37 degrees Celsius. CFSE-labelled targets cells were then washed and co-cultured with WT-1 specific T cell clones at desired effector:target (E:T) ratios for 4 hours at 37 degrees Celsius in a fully humidified 5% CO2 atmosphere. After 4 hours co-cultured cells were stained with 7AAD for 15 minutes on ice in dark and then analysed on FACSCalibur (BDbiosciences) and the data were analysed using CellQuest software.

Example 6: Migration Assay

[0080] Mature DCOne were shown to have the capacity to migrate in response to chemokines that determine their homing to the para-cortical lymph node areas. Mature DCOne cells are equivalent or superior to conventional monocyte-derived DC, as can be seen in FIG. 9. Being able to migrate is an essential feature of DC. For in vitro trans-well migration assays, 10E5 mDCOne cells or MoDC were seeded in the upper compartment of Costar 24-well trans-wells with a pore-size of 6 μm. The lower compartment contained 600 μl serum free MEM-α supplemented with pen/strep, and 250 ng/ml CCL19 (Mip3beta; Peprotech, Huissen, The Netherlands) or CCL21 (6CKine; Invitrogen, Carlsbad, Calif.). Cells were allowed to migrate for 4 hours at 37° C. After migration, 500 μl medium was harvested from the lower compartment and migrated cells were quantified with flow-count fluorospheres (Beckman Coulter, Fullerton, Calif.) by flow cytometry.

Example 7: Clinical T Cell Responses in Patients Vaccinated with mDCOne as Measured by Induction of a DTH Response

[0081] The skin test injections are made on the volar side of the right arm of a human volunteer, at least 5 cm apart from each other, and delayed type hypersensitivity (DTH) reactions. (Induration and erythema areas) were recorded after 48 h, and calculated as circular areas based on the average of vertical and horizontal diameters. The results are shown in FIG. 10.

Example 8: Mature DCOne Cells Express Langerin at a Higher Level than MUTZ-3 Derived Dendritic Cells

[0082] Cells were immunophenotyped using the following FITC- and/or PE-conjugated Mabs reactive against: CD1a (1:25), CD80 (1:25), CD86 (1:25), CD40 (1:10) (PharMingen, San Diego, Calif.), CD14 (1:25), DC-SIGN (1:10) (BD Biosciences, San Jose, Calif.), CD83 (1:10), CD34 (1:10), Langerin (1:10) (Immunotech, Marseille, France). 2.5 to 5×104 cells were washed in PBS supplemented with 0.1% BSA and 0.02% NaN3 and incubated with specific or corresponding control Mabs for 30 minutes at 4 C. Cells were washed and analyzed on a FACS-Calibur flow cytometer (Becton and Dickinson, San Jose, Calif.) equipped with CellQuest analysis software. Results were expressed as the percentage of positive cells.

Example 9: DCOne Cells Carry a Deletion in Comparison with MUTZ-3

[0083] We have performed an array comparative genomic hybridization on a 180K array produced by Agilent to test chromosomal commonalities and differences in copy number between DCOne cells and MUTZ-3 cells. The array used is a custom array with probes spaces 17 kb as described by Tack et al 2010.

[0084] Cell line DCOne was found to be distinctly different from MUTZ-3 since DCOne has a large aberration from 11p15.5 to 11p12 encompassing approximately 16 Mb of genomic regions (20.7 Mb-36.6 Mb). The heterozygous loss contains close to 60 known and unknown genes. This is considered a large loss and makes the DCOne cell line different from MUTZ-3. We found that the copy number variable regions (CNVs) indicates that the DCOne cell line is clonal, this means that the deletions on 7q and 12p are the same in all cells obtained from the cell line.

REFERENCES

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