**2. Materials and methods**

#### **2.1. Growth factors, recombinant human cytokines and medium**

Flt-3 ligand (Flt-3L), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-α) and stem cell factor (SCF) were purchased from CellGenix, Freiburg, Germany. Interferon-alpha (INF- α) was from Roche, Basel, Switzerland. Serum free medium CellGro/SCGM and CellGro/DC medium (CellGenix, Freiburg, Germany) were employed during the culture. To compare the serum free growth conditions with serum containing medium, CellGro/SCGM with 25% human albumin or Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FCS were used. Penicillin and Streptomycin was added to all mediums.

**Figure 1.** CD34+ cells (three different samples) cultured in serum-free CellGro SCGM/DC medium with added cytokines (as shown in materials and methods). The graphs indicate the fold expansion over time

#### **2.2. CD34+ cell isolation**

Leukapheresis-harvested samples were obtained from cancer patients undergoing peripheral blood (PB) stem cell mobilization after informed consent, using a CS 3000 Fenwall Cell Separator (Baxter, Deerfield, IL, USA). The isolation of CD34+cells was carried out using an Isolex 300i magnetic cell selector (Nexell, Irvine, CA, version 2.5CE/2.5CE+) as described earlier (16). The CD34+ samples were frozen in liquid nitrogen using PBS with 10% DMSO and 40% human serum albumin. The purity and viability of thawed CD34+ cells used for DC production was >98% and >95% respectively.

#### **2.3. Generation of CD34+-derived DCs**

CD34+ cells were rapidly thawed in a 37ºC water bath and washed once with culture medium. Then cells (0.5-1x105/ml) were transferred into VueLifeTM FEP Teflon bags (CellGenix, Freiburg, Germany) with serum containing DMEM/10% FCS medium, serumfree CellGro/SCGM medium or CellGro/SCGM/25% human albumin respectively. The following cytokine cocktail was added: GM-CSF 1000u/ml, IL-4 500u/ml, TNF-α 50ng/ml, Flt-3L 150ng/ml and SCF 50ng/ml. The bags were cultured for 14 days at 37ºC/5% CO2. To keep a cell concentration of 105cells per ml through the entire culture period, re-feeding of the cells with culture medium employing equal concentration of cytokines was performed at weekly intervals. The serum-free medium cultures were from day 7, supplemented with CellGro DC medium instead of CellGro SCGM medium.

78 Immunodeficiency

**2. Materials and methods** 

**2.2. CD34+ cell isolation** 

DC production was >98% and >95% respectively.

**2.3. Generation of CD34+-derived DCs** 

experience and have developed a similar serum-free culture system for CD34+ cell derived

Flt-3 ligand (Flt-3L), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-α) and stem cell factor (SCF) were purchased from CellGenix, Freiburg, Germany. Interferon-alpha (INF- α) was from Roche, Basel, Switzerland. Serum free medium CellGro/SCGM and CellGro/DC medium (CellGenix, Freiburg, Germany) were employed during the culture. To compare the serum free growth conditions with serum containing medium, CellGro/SCGM with 25% human albumin or Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FCS were used.

**Figure 1.** CD34+ cells (three different samples) cultured in serum-free CellGro SCGM/DC medium with added cytokines (as shown in materials and methods). The graphs indicate the fold expansion over time

Leukapheresis-harvested samples were obtained from cancer patients undergoing peripheral blood (PB) stem cell mobilization after informed consent, using a CS 3000 Fenwall Cell Separator (Baxter, Deerfield, IL, USA). The isolation of CD34+cells was carried out using an Isolex 300i magnetic cell selector (Nexell, Irvine, CA, version 2.5CE/2.5CE+) as described earlier (16). The CD34+ samples were frozen in liquid nitrogen using PBS with 10% DMSO and 40% human serum albumin. The purity and viability of thawed CD34+ cells used for

CD34+ cells were rapidly thawed in a 37ºC water bath and washed once with culture medium. Then cells (0.5-1x105/ml) were transferred into VueLifeTM FEP Teflon bags

DC and investigated the optimal immunological properties of these cells.

**2.1. Growth factors, recombinant human cytokines and medium** 

Penicillin and Streptomycin was added to all mediums.

**Figure 2.** Expansion fold on Day 14. Different initial concentrations of CD34+ cells incubated in different culture medium. Cell concentration 105/ml are expanded better than 104/ml especially in serum free medium

#### **2.4. Isolation of mRNA from cell line of human prostate cancer origin**

Prostate cancer cell line DU 145 obtained from American Type Culture Collection (ATCC), was cultured in RPMI 1640 supplemented with 10% FCS. The method employed for isolation of mRNA from the tumor cell line has been described earlier (15). Briefly, 5x107 cells were washed with cold PBS and transferred to a 1.5 ml microtube. Five hundred microliter ice-cold 2% IGEPAL (polyoxyethylene 9 nonylphenyl ether) (Sigma-Aldrich) was added to lyse the cells. The supernatant containing the cytosol fraction was obtained after centrifugation (10000xG for 1 minute at 4°C) and transferred to a 1.5 ml tube placed on a cooling block (4°C). To the supernatant 80 μl 10% LiDS (Lithium Lauryl Sulfate) (Sigma-Aldrich), 80 ul 5M LiCl (Lithium Chloride) (Sigma-Aldrich) and 0.5 ml Lysis & Binding Solution (Geno Vision) was added. Samples were frozen and stored at –80ºC until use. Isolation and purification of mRNA from the frozen or fresh samples were prepared in a GenoMTM-48 Robotic Workstation (Genovision AS, Oslo, Norway) following the procedure as described by the manufacturer (GenoMTM-48, Automated mRNA Isolation Handbook, http:// www.qiagen.com /genovision/technical.htm). Denaturing agarose/formaldehyde gel electrophoresis was used to evaluate the quality of mRNA. The prepared mRNA was either used fresh or stored at -80° C until use.

Is Anticancer Vaccine Possible: Experimental Application of Produced mRNA Transfected Dendritic Cells Derived from Enriched CD34+ Blood Progenitor Cells 81

C. The prepared samples were thereafter transferred to liquid nitrogen

C with compensation for heat of fusion,

C/min to -40º

and stored until use. The quality control of the frozen DCs consisted of sterility tests, phenotyping and viability testing by trypan blue staining before freezing and after thawing.

Immature and mature DCs were phenotyped using the following panel of monoclonal antibodies: fluorescence isothiocyanate (FITC)- or phycoerythrin (PE)- conjugated antihuman CD1a, CD14, CD40, CD33, CD34, HLA DR, CD80 (Becton Dickinson), and CD83, CD86 (Immunotech). Negative controls were isotype-matched irrelevant antibodies (Dakocytomation). Cells were analyzed by flow cytometry using a FACSsort (Becton

Autologous-T cells were stimulated four times with weekly intervals in vitro by transfected DCs as described before (15). Briefly, CD34- PBMC from the same patient were thawed and plated in 6-well plates to get rid of adherent cells. Non-adherent cells containing high numbers of T lymphocytes were collected and used as responder cells. Thawed mRNAtransfected DCs used as stimulator cells were washed and irradiated with 3000 cGy. They were co-incubated in 24-well plates at a ratio 10:1 in serum-free CellGroDC-medium with 20 ng/ml of IL-7 and 100 pg/ml of IL-12. After 7 days incubation at 37C in 5% CO2, 1 ml of the suspension from each well was replaced with 1 ml of fresh DC medium containing 20 ng/ml of IL-7. On day 12, 19 and 26 the responder cells were restimulated by new batches of thawed and irradiated transfected DCs as on day 0. On day 14, 21 and 28 the cell cultured were given 1 ml of DC medium containing 20 IU/ml of IL-2. Finally on day 33 the cells were

The conditions for the ELISPOT assay have been described previously (15). A 96 well plate (Millipore-MAIP N45) were coated with 75 μl antibodies against human IFN-γ (Mabtech 1- D1K, 1 mg/ml diluted with PBS to a final concentration of 2 μg/ml) and incubated overnight at 4C. The plate was left at room temperature (RT) for 1 hour and washed six times with PBS, 200 μl/well. Then RPMI-1640 + 1% human albumin was added 100 μl/well and incubated for 1- 2 hours at 37C to block unspecific binding of the antibody. The responder cells and stimulator cells were transferred to the precoated wells in different cell concentrations. As control mocktransfected DC, responder cells alone or medium alone was used. After incubation over night at 37C, the plates were washed six times with PBS/0.05% Tween. To each well 75 μl of a stock solution of 0.75 μg/ml Biotinylated antibody against human IFN- γ (Mabtech, 7-B6-1-biotin, 1 mg/ml) was added and incubated for 2 hours at RT. Following six repeated washings the plate was incubated for one hour with 75μl per well of Streptavidin-ALP (Mabtech, 3310-8) from a stock solution (diluted 1:1000 in PBS+1%BSA). The plate was again washed 5 times with

**2.9. Generation of T-cell responses in vitro by transfected DCs** 

harvested and tested using ELISPOT assay as described below.

rate freezer giving a rate of cooling of 1º

**2.8. Immunophenotyping of the cells** 

C/min to -90º

Dickinson, San Jose, Ca, USA).

**2.10. ELISPOT assay** 

then 1-2º

#### **2.5. Transfection of tumor mRNA into immature DCs**

Teflon bags containing immature DCs (day14) were concentrated by centrifugation (600xG, 5 min., 4°C), the supernatant was removed using a plasma extractor (FENWAL Laboratories, USA) and the cell pellet was transferred by a syringe to a 50 ml tube. After one additional wash by centrifugation, the DCs were resuspended in cold culture medium to give a final volume of 0.6-0.8 ml and placed in a 4°C cooling block until use. mRNA transfection was performed as described earlier (17) using a BTX ECM 830 square-wave electroporator (Genetronics Inc.,San Diego,CA). Electroporation settings were adjusted to single pulse, 500 volt and 2 ms. The BTX-4mm electroporation cuvette (Genetronics Inc.) was washed twice with sterile DC culture medium. Then mRNA extracted from 5x107 cells (40 μl) was added to the prepared immature DCs and transferred to the electroporation cuvette . After eletroporation, DCs were transferred back into the tube and stored on the cooling block for 1 min. before further incubation and maturation. All mock-mRNA transfected DCs used as control underwent electroporation following the same procedure as described above. The cell processing and electroporation procedure took place in a sterile laminar hood inside the GMP (good manufacturing practice) laboratory facility. In order to assess the transfection efficacy, immature DCs were also electroporated with enhanced green fluorescence protein (EGFP) mRNA as a reporter gene instead of mRNA from tumor. The experimental conditions used and the flow cytometry measurement has been described previously (17).

#### **2.6. Maturation of DCs in sterile VueLifeTM FEP Teflon bags**

The two cells, mRNA-transfected and mock-transfected DCs, were removed from the tube by a syringe and injected through a sterile sampling site coupler into VueLifeTM FEP Teflon bags. In order to mature the DCs, serum-free or serum-containing medium was supplemented with a mixture of the cytokines: 50 ng/ml TNF-α (CELLGenix, Freiburg) and 1000u/ml INF-α (Sigma-Aldrich). The final cell concentration during the incubation at 37º C with 5% CO2 for 72 hours was 5x105cells per ml.

#### **2.7. Cryopreservation of mature DCs**

The bag containing matured DC was centrifuged at 600xG for 10 minutes at room temperature. By the use of plasma extractor the supernatant was removed and the remaining DCs were transferred to a 50 ml tube. Following cell enumeration and sterility testing, cells were transferred into Nunc vials. The cryoprotectant solution was CellGro DC medium with 50% human albumin and 10% DMSO. The final cell concentration was 1x107 cells/ml. Total volume in each Nunc vial was 500 μl. Freezing was performed in a control rate freezer giving a rate of cooling of 1º C/min to -40º C with compensation for heat of fusion, then 1-2º C/min to -90º C. The prepared samples were thereafter transferred to liquid nitrogen and stored until use. The quality control of the frozen DCs consisted of sterility tests, phenotyping and viability testing by trypan blue staining before freezing and after thawing.

#### **2.8. Immunophenotyping of the cells**

80 Immunodeficiency

used fresh or stored at -80°

as described by the manufacturer (GenoMTM-48, Automated mRNA Isolation Handbook, http:// www.qiagen.com /genovision/technical.htm). Denaturing agarose/formaldehyde gel electrophoresis was used to evaluate the quality of mRNA. The prepared mRNA was either

Teflon bags containing immature DCs (day14) were concentrated by centrifugation (600xG, 5 min., 4°C), the supernatant was removed using a plasma extractor (FENWAL Laboratories, USA) and the cell pellet was transferred by a syringe to a 50 ml tube. After one additional wash by centrifugation, the DCs were resuspended in cold culture medium to give a final volume of 0.6-0.8 ml and placed in a 4°C cooling block until use. mRNA transfection was performed as described earlier (17) using a BTX ECM 830 square-wave electroporator (Genetronics Inc.,San Diego,CA). Electroporation settings were adjusted to single pulse, 500 volt and 2 ms. The BTX-4mm electroporation cuvette (Genetronics Inc.) was washed twice with sterile DC culture medium. Then mRNA extracted from 5x107 cells (40 μl) was added to the prepared immature DCs and transferred to the electroporation cuvette . After eletroporation, DCs were transferred back into the tube and stored on the cooling block for 1 min. before further incubation and maturation. All mock-mRNA transfected DCs used as control underwent electroporation following the same procedure as described above. The cell processing and electroporation procedure took place in a sterile laminar hood inside the GMP (good manufacturing practice) laboratory facility. In order to assess the transfection efficacy, immature DCs were also electroporated with enhanced green fluorescence protein (EGFP) mRNA as a reporter gene instead of mRNA from tumor. The experimental conditions used

C until use.

and the flow cytometry measurement has been described previously (17).

The two cells, mRNA-transfected and mock-transfected DCs, were removed from the tube by a syringe and injected through a sterile sampling site coupler into VueLifeTM FEP Teflon bags. In order to mature the DCs, serum-free or serum-containing medium was supplemented with a mixture of the cytokines: 50 ng/ml TNF-α (CELLGenix, Freiburg) and 1000u/ml INF-α (Sigma-Aldrich). The final cell concentration during the incubation at 37º

The bag containing matured DC was centrifuged at 600xG for 10 minutes at room temperature. By the use of plasma extractor the supernatant was removed and the remaining DCs were transferred to a 50 ml tube. Following cell enumeration and sterility testing, cells were transferred into Nunc vials. The cryoprotectant solution was CellGro DC medium with 50% human albumin and 10% DMSO. The final cell concentration was 1x107 cells/ml. Total volume in each Nunc vial was 500 μl. Freezing was performed in a control

**2.6. Maturation of DCs in sterile VueLifeTM FEP Teflon bags** 

with 5% CO2 for 72 hours was 5x105cells per ml.

**2.7. Cryopreservation of mature DCs** 

**2.5. Transfection of tumor mRNA into immature DCs** 

Immature and mature DCs were phenotyped using the following panel of monoclonal antibodies: fluorescence isothiocyanate (FITC)- or phycoerythrin (PE)- conjugated antihuman CD1a, CD14, CD40, CD33, CD34, HLA DR, CD80 (Becton Dickinson), and CD83, CD86 (Immunotech). Negative controls were isotype-matched irrelevant antibodies (Dakocytomation). Cells were analyzed by flow cytometry using a FACSsort (Becton Dickinson, San Jose, Ca, USA).

#### **2.9. Generation of T-cell responses in vitro by transfected DCs**

Autologous-T cells were stimulated four times with weekly intervals in vitro by transfected DCs as described before (15). Briefly, CD34- PBMC from the same patient were thawed and plated in 6-well plates to get rid of adherent cells. Non-adherent cells containing high numbers of T lymphocytes were collected and used as responder cells. Thawed mRNAtransfected DCs used as stimulator cells were washed and irradiated with 3000 cGy. They were co-incubated in 24-well plates at a ratio 10:1 in serum-free CellGroDC-medium with 20 ng/ml of IL-7 and 100 pg/ml of IL-12. After 7 days incubation at 37C in 5% CO2, 1 ml of the suspension from each well was replaced with 1 ml of fresh DC medium containing 20 ng/ml of IL-7. On day 12, 19 and 26 the responder cells were restimulated by new batches of thawed and irradiated transfected DCs as on day 0. On day 14, 21 and 28 the cell cultured were given 1 ml of DC medium containing 20 IU/ml of IL-2. Finally on day 33 the cells were harvested and tested using ELISPOT assay as described below.

#### **2.10. ELISPOT assay**

C

The conditions for the ELISPOT assay have been described previously (15). A 96 well plate (Millipore-MAIP N45) were coated with 75 μl antibodies against human IFN-γ (Mabtech 1- D1K, 1 mg/ml diluted with PBS to a final concentration of 2 μg/ml) and incubated overnight at 4C. The plate was left at room temperature (RT) for 1 hour and washed six times with PBS, 200 μl/well. Then RPMI-1640 + 1% human albumin was added 100 μl/well and incubated for 1- 2 hours at 37C to block unspecific binding of the antibody. The responder cells and stimulator cells were transferred to the precoated wells in different cell concentrations. As control mocktransfected DC, responder cells alone or medium alone was used. After incubation over night at 37C, the plates were washed six times with PBS/0.05% Tween. To each well 75 μl of a stock solution of 0.75 μg/ml Biotinylated antibody against human IFN- γ (Mabtech, 7-B6-1-biotin, 1 mg/ml) was added and incubated for 2 hours at RT. Following six repeated washings the plate was incubated for one hour with 75μl per well of Streptavidin-ALP (Mabtech, 3310-8) from a stock solution (diluted 1:1000 in PBS+1%BSA). The plate was again washed 5 times with

PBS/0.05% Tween and one additional time with PBS alone. Then, after adding 75 μl of substrate BCIP/NBT (Sigma B911) to each well the plate was incubated for 4-5 minutes. When spots appeared, water was added to stop the reaction. The number of spots per well was counted under a stereomicroscope and the frequency of reactive T cells was calculated.

Is Anticancer Vaccine Possible: Experimental Application of Produced mRNA Transfected Dendritic Cells Derived from Enriched CD34+ Blood Progenitor Cells 83

**Figure 3.** Immunophenotyping profile of: A) Immature DC; B) Mature DC generated from enriched monocytes. Overlay histograms show the expression of relevant antigens of immature (blue) and mature DC (green) versus isotype-matched control (red). The percentage of positive cells and mean

fluorescence intensity value is shown too.
