Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in non-small cell lung cancer

Abstract

Background: Our previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability. In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity. These results have provided the rationale to generate a clinical grade adenoviral vector encoding CCL-21 for ex vivo transduction of human DC in order to assess intratumoral administration in late stage human lung cancer.

Methods: In the current study, human monocyte-derived DC were differentiated by exposure to GM-CSF and IL-4 from cryopreserved mononuclear cells obtained from healthy volunteers. Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein.

Results: CCL21 protein production from transduced DC was detected in supernatants (24-72 hours, 3.5-6.7 ng/4-5 x 10(6) cells). DC transduced with the clinical grade adenoviral vector were > 88% viable (n = 16), conserved their phenotype and maintained integral biological activities including dextran uptake, production of immunostimulatory cytokines/chemokines and antigen presentation. Furthermore, supernatant from CCL21-DC induced the chemotaxis of T2 cells in vitro.

Conclusion: Viable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.

Figure 1

Susceptibility of human DC to adenoviral transduction. DC on day 6 of culture were transduced with an adenovirus encoding the marker gene GFP under the same conditions utilized to transduce CCL21-DC. After transduction, DC were cultured up to 48 h. GFP expression was analyzed by flow cytometry at 24 h (A, B) and 48 h culture (C, D), as described in Materials and Methods. A population of GFP⁺ CD86⁺ DC at 24 h (B) and 48 h (D) is shown. Staining with isotype control is indicated in A and C. The percentage of GFP⁺ CD86⁺ DC is indicated in the upper right corner, and MFI is also reported for D and C. The density plots for one representative experiment of four are shown.

Figure 2

DC phenotype is conserved after transduction with AdCCL21. DC (A), and CCL21-DC (B), were analyzed for expression of the following surface markers: CD40, CD54, CD80, CD83, CD86, HLA-DR and CCR7, as indicated. Histograms show the expression of each surface molecule individually. White lines contour the isotype control. The percentage of positive cells and MFI are indicated. A representative experiment of at least three is shown.

Figure 3

Dextran uptake by CCL21-DC. DC (A and B) and CCL21-DC (C and D) were incubated with DX-FITC at 4°C and 37°C for 15 minutes, as described in Materials and Methods. DX expression was analyzed in CD86⁺ DC. A population of DX⁺ CD86⁺ obtained at 4°C and at 37°C is shown. Values indicate the percentage and MFI of DX⁺ CD86⁺. Density plots of a representative experiment of three are shown.

Figure 4

CCL21-DC are efficient APC in the absence of KLH. (A) Allogeneic MLR: Briefly, culture medium mock-transduced DC (DC) and CCL21-DC (AdDC) were analyzed for their ability to induce allogeneic T cell proliferation. The effect of pulsing 100% (DC KLH 100 and AdDC KLH 100) or 30% of cells (AdDC KLH 30) with 10 μg/ml KLH prior to initiating the MLR assay was assessed. After pre-treatment with mitomycin C and KLH, where indicated, DC and CCL21-DC were mixed with allogeneic T cells at several ratios, as shown. Cell proliferation was assessed by BrdU incorporation following 5 days incubation at 37°C. One representative experiment of three is shown. (B-G) Autologous T cell proliferation assay: T cells were cultured for 6 days with IL-2 (15 U/ml) prior to the TT presentation assay. After mitomycin-C treatment and KLH pulsing where indicated, DC (B, E), CCL21-DC (C, F), or CV-DC (D, G) were mixed at 1:10 ratio to autologous T cells in the presence of TT (2 μg/ml) or BSA (2 μg/ml). In (B-D), IFN-γ production was measured in the cell supernatant collected after 48 hours of cell proliferation. The measured values of IFN-γ detected by ELISA were within the linear portion of the IFN-γ concentration curve. The concentration of IFN-γ is expressed in pg/ml with basal IFN-γ production subtracted. In (E-G), T cell proliferation in response to TT and BSA was measured by BrdU incorporation following 5 days co-culture at 37°C and is expressed as a percentage of proliferating cells. One representative experiment of at least three is shown. P value ≤ 0.05 was considered significant.

Figure 5

CCL21-DC secrete immunostimulatory cytokines. Human monocyte-derived DC were transduced with culture medium, AdCCL21 (1167 VP/cell), or AdCV by the centrifugation method. 2.0 × 10⁵ DC, CCL21-DC, or CV-DC were seeded into a 48 well plate in 1 ml of culture medium in the presence or absence of the indicated stimuli. For IL-12 assays, cells were stimulated with IFN-γ + LPS, and for IP-10 and MIG assays cells were stimulated with LPS only. The supernatants from stimulated and unstimulated DC were harvested after 48 h culture. IL-12p₇₀ (A), IP-10 (B), and MIG (C) were measured by ELISA. Values refer to cytokine concentration expressed in ng/million cells. One representative experiment of four is shown.

Figure 6

Supernatants from CCL21-DC stimulate chemotaxis of T2 cells. (A) The supernatants from DC, CCL21-DC and CV-DC from different individuals (n = 12) were utilized in a standard chemotaxis assay to assess their ability to induce chemotaxis of T2 cells. Briefly 2.0 × 10⁵ T2 cells were loaded in the upper chamber of a 24-well transwell apparatus and allowed to migrate through a 3 μm insert for 2 and 1/2 h at 37°C. In A, the lower chambers of the transwell had been loaded with the supernatant from DC or CCL21-DC (supernatant #1–11) from the same individual, or DC or CV-DC (#12) from the same individual, as indicated. Recombinant CCL21 (600 ng/ml) and 10% AB medium were added to the wells as positive and negative controls, respectively. A summary of chemotaxis from 12 different samples is shown. The CCL21 protein concentration in ng/ml is indicated over each column. Migrated cells were analyzed by flow cytometry by counting the number of events per minute (expressed as migrated cell number) within a pre-determined T cell gate. One-way ANOVA p < 0.0001. In blocking experiments (B), the supernatants from CCL21-DC or recombinant CCL21 were pre-treated with a neutralizing concentration of anti-CCL21 antibody (1.5 μg/50 ng CCL21) or isotype immunoglobulin, prior loading in the lower chamber of the transwell apparatus. The supernatant from CV-DC and medium containing 10% AB were also assessed. To determine total T2 cell migration, T2 cells were loaded without inserts. A representative experiment of a total of seven is shown. The number of migrated cells was analyzed by flow cytometry as above, and is expressed as percentage of total T2 cell migration. Asterisks denote statistical significance (p < 0.05). The surface expression of CCR7 is analyzed by flow cytometry in T2 cells (C) and in immature DC (D) cultured for 6 days in GM-CSF and IL-4. Histograms are shown: dotted lines indicate isotype control, bold lines indicate marker expression. The percentage of positive cells and MFI are indicated. A representative experiment of two is shown.