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

Baratelli F, Takedatsu H, Hazra S, Peebles K, Luo J, Kurimoto PS, Zeng G, Batra RK, Sharma S, Dubinett SM, Lee JM - J Transl Med (2008)

Bottom Line: In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity.Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein.Viable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.

View Article: PubMed Central - HTML - PubMed

Affiliation: UCLA Lung Cancer Research Program of the Jonsson Comprehensive Cancer Center, Division of Pulmonary and Critical Care Medicine, Department of Medicine, Los Angeles, CA 90095, USA. fbaratelli@mednet.ucla.edu

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.

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Related in: MedlinePlus

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.
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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.

Mentions: We have previously reported that transduction of DC with laboratory-grade AdCCL21 vector did not impair the secretion of IL-12 [29], a critical cytokine for induction of Th1-mediated anti-tumor immune responses. To further investigate the immunological properties of CCL21-DC, IL-12 production was analyzed from these cells upon stimulation with IFN-γ and LPS. CCL21-DC and CV-DC maintained the ability to become activated upon stimulation and secrete IL-12 in comparable levels to non-transduced, control DC (Figure 5A).


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

Baratelli F, Takedatsu H, Hazra S, Peebles K, Luo J, Kurimoto PS, Zeng G, Batra RK, Sharma S, Dubinett SM, Lee JM - J Transl Med (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2507704&req=5

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.
Mentions: We have previously reported that transduction of DC with laboratory-grade AdCCL21 vector did not impair the secretion of IL-12 [29], a critical cytokine for induction of Th1-mediated anti-tumor immune responses. To further investigate the immunological properties of CCL21-DC, IL-12 production was analyzed from these cells upon stimulation with IFN-γ and LPS. CCL21-DC and CV-DC maintained the ability to become activated upon stimulation and secrete IL-12 in comparable levels to non-transduced, control DC (Figure 5A).

Bottom Line: In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity.Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein.Viable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.

View Article: PubMed Central - HTML - PubMed

Affiliation: UCLA Lung Cancer Research Program of the Jonsson Comprehensive Cancer Center, Division of Pulmonary and Critical Care Medicine, Department of Medicine, Los Angeles, CA 90095, USA. fbaratelli@mednet.ucla.edu

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.

Show MeSH
Related in: MedlinePlus