Methods and compositions for obtaining mature dendritic cells

Details Methods and compositions for obtaining mature dendritic cells Methods and compositions for obtaining mature dendritic cells

1998-10-27

2001-08-14

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S325000, C435S366000, C435S375000

Reexamination Certificate

active

06274378

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
This invention relates to methods and compositions useful for activating cells of the immune system. The methods and compositions provided by this invention are useful for causing the maturation of non-proliferating immature dendritic cells to mature dendritic cells capable of processing and presenting antigen. This invention also relates to culture mediums which promote maturation of immature dendritic cells to mature dendritic cells. In addition, this invention relates to assays useful for detecting the presence in a test substance of a dendritic cell maturation factor.
BACKGROUND OF THE INVENTION
Dendritic cells are specialized antigen presenting cells, critical for eliciting T cell mediated immune responses (Steinman, 1991; Caux et al. 1995a; Hart and McKenzie, 1990; Austyn, 1987). These specialized antigen presenting cells elicit both CD4+ helper cells (Inaba et al. 1990a; Inaba et al. 1990b; Crowley et al. 1990) and CD8+ killer cells (Porgador and Gilboa, 1995; Mayordomo et al. 1995; Zitvogel et al. 1995) in vivo.
Because of the potent activity of dendritic cells to activate T cells, the art, e.g. Flanand et al.,
Env. J. Immunol.,
24:605-610, 1994, has accepted the characterization of dendritic cells as “nature's adjuvant”. It is therefore desirable to be able to use dendritic cells to process and present protein antigens as a means of modulating an individual's immune response, and in particular to activate an individual's T cells in connection with the treatment or prevention of disease.
The use of primed dendritic cells to activate cytotoxic T cells has been reported. For example, Paglia et al., recently reported that murine dendritic cells cultured from bone marrow precursors and exposed to antigen in vitro provide effective resistance to challenge with live tumor cells. Paglia et al., “Murine Dendritic Cells Loaded In Vitro With Soluble Protein Prime Cytotoxic T Lymphocytes Against Tumor Antigen In Vivop”,
J. Exp. Med.,
183:317-322 (1996).
The in vitro observations and the murine results have recently been extended to the treatment of humans with B cell lymphoma using mature dendritic cells which were primed in vitro with tumor antigens. Hsu et al., “Vaccination of Patients With B-cell Lymphoma Using Autologous Antigen-Pulsed Dendritic Cells”,
Nature Med.,
2:52-58 (1996). According to this report, the antigen used to prime the dendritic cells was idiotypic antibody obtained from hybridomas made from the fusion of lymph node tumor cells with a mouse cell line. All of the four patients involved in this study exhibited significant antitumor idiotype PBMC proliferative responses. Positive clinical responses were also observed including one patient who experienced complete tumor regression. Hsu et al., however, express uncertainty whether dendritic cells obtained from expansion of dendritic cell cultures in the presence of GM-CSF, TNF-&agr; or IL-4 would function equivalently in their ability to process and present antigen or to stimulate cellular immune responses as the freshly isolated cells.
Prior studies have identified proliferating dendritic cell progenitors within the small CD34+ subfraction of cells in human blood (Inaba et al. 1992; Caux et al. 1994; Caux et al. 1992; Caux et al. 1995). Methods have been developed for expanding these proliferating cells in culture to obtain sufficient numbers of cells to be useful for priming with antigen and administering to an individual to activate an immune response. Steinman et al. International patent application WO 93/20185. These dendritic cells can be stimulated with cytokines, particularly GM-CSF and, optionally TNF&agr; or other cytokines, to develop into potent dendritic cells over 1-2 weeks in culture (Caux et al. 1992; Inaba et al. (1992)). Although useful dendritic cells can be produced from the proliferating precursors, it is desirable to obtain alternative methods of obtaining suitable numbers of mature dendritic cells for therapeutic purposes where proliferating progenitors are infrequent.
The removal of monocytes and lymphocytes from human blood has uncovered a small population of nonproliferating progenitors that require cytokines to develop into typical dendritic cells. These progenitor cells exist at a concentration of at most about 10
6
cells per 450-500 ml of blood (O'Doherty et al. 1994; O'Doherty et al. 1993; Thomas et al. 1993). More recently, the combination of GM-CSF and IL-4 has been shown to facilitate the generation of significantly larger numbers of dendritic cells from adherent blood mononuclear fractions, about 3-8×10
6
per 40 ml of blood (Romani et al. 1994; Sallusto and Lanzavecchia, 1994 both of which are incorporated herein by reference). However, we have now determined that when the cytokines are removed, the cells revert to an adherent and less stimulatory state, that is, they do not have the properties of mature, stable dendritic cells. If the latter reversion were to take place in vivo during adoptive immunotherapy, the cells could be ineffective as adjuvants. In addition, it is desirable to develop a culture system independent of fetal calf serum.
Engleman et al. International Patent Application WO 95/34638 refers to a method of activating an immune response in a human patient by administering to the patient dendritic cells primed with antigen. Engleman et al. however refer to using dendritic cells isolated from the individual which exist in small numbers. Accordingly, it would be desirable to be able to obtain dendritic cells from a large population of cells present in an easily accessible tissue such as blood.
Two antigens, have recently been reported which, in addition to other antigens or phenotypic characteristics, distinguish mature dendritic cells from other types of white blood cells. Zhou and Tedder (Zhou and Tedder, 1995) reported that CD83, a member of the immunoglobulin superfamily that was cloned from an EBV induced B cell library, is expressed on dendritic cells in blood cells that were cultured for 2 days. This culture period is sufficient to allow a small subset of immature dendritic cells to mature (O'Doherty et al. 1993). CD83 has also been detected on some presumptive dendritic cells in the T cell areas of lymphoid organs, and on some B cells in germinal centers (Zhou et al. 1992). Langhoff and coworkers found that p55, an actin bundling protein, also marks the dendritic cells that are found in 2 day cultures of human blood (Mosialos et al. 1995). p55 is an intracellular protein that was discovered as an EBV induced host cell product. It is expressed by interdigitating cells and at high levels in the brain (Mosialos et al. 1995).
SUMMARY OF THE INVENTION
This invention provides methods of preparing large numbers of stable, mature dendritic cells. The stable mature dendritic cells provided by this invention retain characteristics of a mature phenotype, including expression of dendritic cell markers p55 and CD83 and high levels of accessory molecules like CD86 and CD40, even when removed from contact with cytokines which promote the maturation of pluripotential peripheral blood mononuclear cells (PBMCs) to non-stable immature dendritic cells. As used herein, “stable” refers to retention of a mature dendritic cell phenotype for three days when mature dendritic cells are cultured in the absence of cytokines. Dendritic cells are considered “mature” according to this invention when they have an increased expression of one or more phenotypic markers, such as for example non-adherence to plastic and CD86 or other antigen markers, associated with the accessory function of dendritic cells following contact with a dendritic cell maturation factor.
In one embodiment of the invention, mature dendritic cells are produced in vivo or in vitro from immature dendritic cells derived from PBMC pluripotential cells having the potential of expressing either macrophage or dendritic cell characteristics. The method, according to the invention, comprises contacting the immature dendritic cells with a

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