Clinical-grade manufacturing of DC from CD14+ precursors: Experience from phase I clinical trials in CML and malignant melanoma

Allan B Dietz, D. J. Padley, G. W. Butler, M. L. Maas, C. W. Greiner, D. A. Gastineau, Stanimir Vuk-Pavlović

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Background. We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14+ precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials. Methods. We isolated CD14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15™ medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-α, IL-1β, IL-6 and prostaglandin E2 for 3 days. Some cells were electraporated and translected with mRN4 isolated from melanoma tissue. DC were characterized by flow cytometry for the expression of CD83, CD86 and CD14. Results. CD14+ cells constituted 14.4±6.2% (mean±SD) of nucleated cells in apheresis products and 98.3±3.6% of isolated cells. Normal DC and CML DC were 77.4±7.3% CD83+ and 93.5±7.0% CD86+. Corresponding values for electroporated DC from melanoma patients were 66.1±7.2% and 94.1±7.8%. The yield of CD83+ DC from isolated CD14+ cells was 18.1+7.2% for normal and CML patients and 9.8±3.7% for melanoma patients. DC viability was 92.7±5.8%; after cryopreservation and thawing it was 77± 13.5%. Discussion. Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product.

Original languageEnglish (US)
Pages (from-to)563-570
Number of pages8
JournalCytotherapy
Volume6
Issue number6
DOIs
StatePublished - 2004

Fingerprint

Clinical Trials, Phase I
Melanoma
Blood Component Removal
Cryopreservation
Vaccines
Immunomagnetic Separation
Interleukin-7
Mycoplasma
Granulocyte-Macrophage Colony-Stimulating Factor
Clinical Protocols
Interleukin-1
Dinoprostone
Interleukin-4
Interleukin-6
Flow Cytometry
Tumor Necrosis Factor-alpha
Tissue Donors
Bacteria
Phenotype
Serum

Keywords

  • CD14 cells
  • Clinical-grade manufacturing
  • CML
  • DC
  • Electroporation
  • Immunomagnetic separation
  • Immunotherapy
  • Malignant melanoma

ASJC Scopus subject areas

  • Immunology

Cite this

Clinical-grade manufacturing of DC from CD14+ precursors : Experience from phase I clinical trials in CML and malignant melanoma. / Dietz, Allan B; Padley, D. J.; Butler, G. W.; Maas, M. L.; Greiner, C. W.; Gastineau, D. A.; Vuk-Pavlović, Stanimir.

In: Cytotherapy, Vol. 6, No. 6, 2004, p. 563-570.

Research output: Contribution to journalArticle

Dietz, Allan B ; Padley, D. J. ; Butler, G. W. ; Maas, M. L. ; Greiner, C. W. ; Gastineau, D. A. ; Vuk-Pavlović, Stanimir. / Clinical-grade manufacturing of DC from CD14+ precursors : Experience from phase I clinical trials in CML and malignant melanoma. In: Cytotherapy. 2004 ; Vol. 6, No. 6. pp. 563-570.
@article{9b760ccc9edd428aae1e93fcfbf39171,
title = "Clinical-grade manufacturing of DC from CD14+ precursors: Experience from phase I clinical trials in CML and malignant melanoma",
abstract = "Background. We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14+ precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials. Methods. We isolated CD14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15™ medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-α, IL-1β, IL-6 and prostaglandin E2 for 3 days. Some cells were electraporated and translected with mRN4 isolated from melanoma tissue. DC were characterized by flow cytometry for the expression of CD83, CD86 and CD14. Results. CD14+ cells constituted 14.4±6.2{\%} (mean±SD) of nucleated cells in apheresis products and 98.3±3.6{\%} of isolated cells. Normal DC and CML DC were 77.4±7.3{\%} CD83+ and 93.5±7.0{\%} CD86+. Corresponding values for electroporated DC from melanoma patients were 66.1±7.2{\%} and 94.1±7.8{\%}. The yield of CD83+ DC from isolated CD14+ cells was 18.1+7.2{\%} for normal and CML patients and 9.8±3.7{\%} for melanoma patients. DC viability was 92.7±5.8{\%}; after cryopreservation and thawing it was 77± 13.5{\%}. Discussion. Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product.",
keywords = "CD14 cells, Clinical-grade manufacturing, CML, DC, Electroporation, Immunomagnetic separation, Immunotherapy, Malignant melanoma",
author = "Dietz, {Allan B} and Padley, {D. J.} and Butler, {G. W.} and Maas, {M. L.} and Greiner, {C. W.} and Gastineau, {D. A.} and Stanimir Vuk-Pavlović",
year = "2004",
doi = "10.1080/14653240410005357",
language = "English (US)",
volume = "6",
pages = "563--570",
journal = "Cytotherapy",
issn = "1465-3249",
publisher = "Informa Healthcare",
number = "6",

}

TY - JOUR

T1 - Clinical-grade manufacturing of DC from CD14+ precursors

T2 - Experience from phase I clinical trials in CML and malignant melanoma

AU - Dietz, Allan B

AU - Padley, D. J.

AU - Butler, G. W.

AU - Maas, M. L.

AU - Greiner, C. W.

AU - Gastineau, D. A.

AU - Vuk-Pavlović, Stanimir

PY - 2004

Y1 - 2004

N2 - Background. We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14+ precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials. Methods. We isolated CD14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15™ medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-α, IL-1β, IL-6 and prostaglandin E2 for 3 days. Some cells were electraporated and translected with mRN4 isolated from melanoma tissue. DC were characterized by flow cytometry for the expression of CD83, CD86 and CD14. Results. CD14+ cells constituted 14.4±6.2% (mean±SD) of nucleated cells in apheresis products and 98.3±3.6% of isolated cells. Normal DC and CML DC were 77.4±7.3% CD83+ and 93.5±7.0% CD86+. Corresponding values for electroporated DC from melanoma patients were 66.1±7.2% and 94.1±7.8%. The yield of CD83+ DC from isolated CD14+ cells was 18.1+7.2% for normal and CML patients and 9.8±3.7% for melanoma patients. DC viability was 92.7±5.8%; after cryopreservation and thawing it was 77± 13.5%. Discussion. Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product.

AB - Background. We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14+ precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials. Methods. We isolated CD14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15™ medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-α, IL-1β, IL-6 and prostaglandin E2 for 3 days. Some cells were electraporated and translected with mRN4 isolated from melanoma tissue. DC were characterized by flow cytometry for the expression of CD83, CD86 and CD14. Results. CD14+ cells constituted 14.4±6.2% (mean±SD) of nucleated cells in apheresis products and 98.3±3.6% of isolated cells. Normal DC and CML DC were 77.4±7.3% CD83+ and 93.5±7.0% CD86+. Corresponding values for electroporated DC from melanoma patients were 66.1±7.2% and 94.1±7.8%. The yield of CD83+ DC from isolated CD14+ cells was 18.1+7.2% for normal and CML patients and 9.8±3.7% for melanoma patients. DC viability was 92.7±5.8%; after cryopreservation and thawing it was 77± 13.5%. Discussion. Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product.

KW - CD14 cells

KW - Clinical-grade manufacturing

KW - CML

KW - DC

KW - Electroporation

KW - Immunomagnetic separation

KW - Immunotherapy

KW - Malignant melanoma

UR - http://www.scopus.com/inward/record.url?scp=11144260243&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=11144260243&partnerID=8YFLogxK

U2 - 10.1080/14653240410005357

DO - 10.1080/14653240410005357

M3 - Article

C2 - 15773024

AN - SCOPUS:11144260243

VL - 6

SP - 563

EP - 570

JO - Cytotherapy

JF - Cytotherapy

SN - 1465-3249

IS - 6

ER -