Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer

<p>Abstract</p> <p>Background</p> <p>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 <it>ex vivo </it>transduction of human DC in order to assess intratumoral administration in late stage human lung cancer.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>CCL21 protein production from transduced DC was detected in supernatants (24–72 hours, 3.5–6.7 ng/4–5 × 10⁶cells). 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 <it>in vitro</it>.</p> <p>Conclusion</p> <p>Viable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.</p

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

BackgroundOur 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.MethodsIn 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.ResultsCCL21 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 &gt; 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.ConclusionViable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC

Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer-2

DC. A population of DXCD86obtained at 4°C and at 37°C is shown. Values indicate the percentage and MFI of DXCD86. Density plots of a representative experiment of three are shown.<p><b>Copyright information:</b></p><p>Taken from "Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer"</p><p>http://www.translational-medicine.com/content/6/1/38</p><p>Journal of Translational Medicine 2008;6():38-38.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2507704.</p><p></p

Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer-0

After transduction, DC were cultured up to 48 h. GFP expression was analyzed by flow cytometry at 24 h () and 48 h culture (), as described in . A population of GFPCD86DC at 24 h () and 48 h () is shown. Staining with isotype control is indicated in and . The percentage of GFPCD86DC 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.<p><b>Copyright information:</b></p><p>Taken from "Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer"</p><p>http://www.translational-medicine.com/content/6/1/38</p><p>Journal of Translational Medicine 2008;6():38-38.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2507704.</p><p></p

Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer-4

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-12p), IP-10 , and MIG were measured by ELISA. Values refer to cytokine concentration expressed in ng/million cells. One representative experiment of four is shown.<p><b>Copyright information:</b></p><p>Taken from "Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer"</p><p>http://www.translational-medicine.com/content/6/1/38</p><p>Journal of Translational Medicine 2008;6():38-38.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2507704.</p><p></p

Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer-3

Ell 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. 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 (), CCL21-DC (), or CV-DC () were mixed at 1:10 ratio to autologous T cells in the presence of TT (2 μg/ml) or BSA (2 μg/ml). In (), 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 (), 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.<p><b>Copyright information:</b></p><p>Taken from "Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer"</p><p>http://www.translational-medicine.com/content/6/1/38</p><p>Journal of Translational Medicine 2008;6():38-38.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2507704.</p><p></p