Elsevier

Cytotherapy

Volume 6, Issue 6, December 2004, Pages 563-570
Cytotherapy

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

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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 electroporated and transfected with mRNA isolated from melanoma tissue. DC were characterized by flow cytometry for the expression ofCD83, 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.

Introduction

As sentinels of corporeal integrity, DC are activated in inflammatory environments where they capture antigens. Consequently they mature and migrate to lymph nodes to stimulate antigen-specific T cells [1]. Clinical trials in immunotherapy of cancer have attempted to benefit from these DC features by ex vivo manufacturing and exposing large numbers of these cells to cancer-associated antigens and injecting them to patients [2]. However, DC products have been manufactured for different clinical trials from different precursors by different manufacturing methods, resulting in different phenotypes and functional characteristics. This has resulted in difficulties in interpreting the resulting clinical and laboratory data and inspired the need for standardization of DC vaccines 3., 4., 5..

There has been a growing consensus that only fully differentiated (mature) DC (MDC) have the capacity to migrate to lymph nodes [6] and are less likely to induce CD4+ CD25+ regulatory T cells that putatively attenuate immunity [7]. In addition, admixture of immature DC may also reduce the desired clinical and immune effects [8]. Thus, highly enriched and fully mature DC may engender more interpretable clinical and immune effects and facilitate progress in immunotherapy Recently, we have manufactured such highly enriched myeloid MDC 9., 10., 11. and administered them in phase I clinical trials of immunotherapy of CML (MR Litzow et al., manuscript in preparation) and stage IV malignant melanoma.We have previously standardized clinical-scale and clinical-grade DC manufacturing from normal CD14+ cells [11]. We used immunomagnetic adsorption of CD14+ cells to maximize the purity of the final DC product 9., 10., 11.. However, there is very little experience in the large-scale manufacturing of standardized MDC products required for more extensive clinical studies. Here we report an evaluation of our manufacturing record and an examination of manufacturing criteria, including sterility, purity, phenotype, viability and stability for 28 clinical-scale manufacturing procedures. Additionally, we optimized an electroporation procedure for transfection of transcripts isolated from melanoma tissue into autologous DC and report the effects of this procedure on the final MDC product.

Section snippets

Patients

All subjects gave informed consent to be admitted to the study as approved by the Mayo Foundation Institutional Review Board (Federal Wide Assurance Number 00005001; Rochester, MN). In addition, clinical trials were conducted under Investigational New Drug exemptions granted by the USA Food and Drug Administration (FDA).

Apheresis

PBMC were collected from nine CML patients and five melanoma patients employing a Fenwal CS3000 (Baxter, Round Lake, IL, USA) or a Cobe Spectra (Gambro BCT, Lakewood, CO, USA)

Clinical grade immunomagnetic selection of CD14+ precursors is highly efficacious

We measured the number and phenotype of cells before and after selection of CD14+ cells. Data in Table 1 show that the percentage of CD14+ cells among apheresis products obtained from normal subjects and patients suffering from CML and melanoma did not differ and was 14.4 ± 6.2 (mean ± SD) overall. Following immuno-magnetic isolation, purity of CD14+ cells was indistinguishable among groups and was 98.3 ± 3.6% overall. We calculated the efficiency of CD14+ cell selection by dividing the number of

Discussion

Numerous recent cancer immunotherapy clinical trials have employed DC of widely different phenotypes and purity, compounding the problems in interpretation of results [3,4]. Consequently, standardization of DC products has been proposed as a means to facilitate progress in the field [5]. To allow for an easier interpretation of the clinical and immunologic effects of DC vaccination, we sought to manufacture the cells at the highest feasible level of purity and maturity. For this reason we used

Acknowledgements

Supported by NIH Grant R01CA-84368 and Mayo Clinic Comprehensive Cancer Center Support Grant CA-15083. Stem Cell Laboratory has been supported by Mrs Adelyn L. Luther, Singer Island, FL; Commonwealth Cancer Foundation for Research, Richmond, VA; and Glen and Florence Voyles Foundation, Terre Haute, IN. We thank Dr Alan D. King and Mr Richard E. Walters, Cyto Pulse Sciences Inc., for electroporation equipment, supplies and helpful advice, and Drs Franklyn G. Prendergast and S. Breanndan Moore

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