Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
1999-11-30
2002-11-05
Lankford, Jr., Leon B. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C435S372000
Reexamination Certificate
active
06475483
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to a method of culturing cells of the immune system. In particular a method is provided for culturing proliferating dendritic cell precursors and for their maturation in vitro to mature dendritic cells. This invention also relates to dendritic cell modified antigens which are T cell dependent, the method of making them, and their use as immunogens. Vaccines, methods of immunizing animals and humans using the mature dendritic cells of the invention, and the modified antigens are also described.
BACKGROUND OF THE INVENTION
The immune system contains a system of dendritic cells that is specialized to present antigens and initiate several T-dependent immune responses. Dendritic cells are distributed widely throughout the body in various tissues. The distribution of dendritic cells has been reviewed in (1). Dendritic cells are found in nonlymphoid organs either close to body surfaces, as in the skin and airways, or in interstitial regions of organs like heart and liver. Dendritic cells, possibly under the control of the cytokine granulocyte macrophage colony-stimulating factor, (hereinafter GM-CSF), can undergo a maturation process that does not entail cell proliferation (2,3). Initially, the dendritic cells process and present antigens most likely on abundant, newly synthesized MHC class II molecules, and then strong accessory and cell-cell adhesion functions are acquired (4-7). Dendritic cells can migrate via the blood and lymph to lymphoid organs (8-10). There, presumably as the “interdigitating” cells of the T-area (8,11-13), antigens can be presented to T cells in the recirculating pool (14). However, little is known about the progenitors of dendritic cells in the different compartments outlined above.
The efficacy of dendritic cells in delivering antigens in such a way that a strong immune response ensues i.e., “immunogenicity”, is widely acknowledged, but the use of these cells is hampered by the fact that there are very few in any given organ. In human blood, for example, about 0.1% of the white cells are dendritic cells (25) and these have not been induced to grow until this time. Similarly, previous studies (20, 21) have not reported the development, in culture, of large numbers of dendritic cells from bone marrow. A more recent report described the development of dendritic cells in GM-CSF supplemented marrow cultures, however no documentation as to the origin of the dendritic cells or use of proliferating aggregates as an enriched source of dendritic cells was observed. (Scheicher et al. (1992))
J. Immunol. Method
. 154:253-264. While dendritic cells can process foreign antigens into peptides that immunologically active T cells must recognize (4,6,7,14) i.e., dendritic cells accomplish the phenomenon of “antigen presentation”, the low numbers of dendritic cells prohibits their use in identifying immunogenic peptides.
Dendritic cells in spleen (15) and afferent lymph (16,17) are not in the cell cycle but arise from a proliferating precursor. Ultimately, dendritic cells emanate from the bone marrow (15,16,18,19), yet it has been difficult to generate these cells in culture except for two reports describing their formation in small numbers (20,21). Although a bone marrow precursor cell has been reported, conditions have not been reported that direct its proliferation in culture (Steinman, R. (1991)) “The Dendritic Cell System and Its Role In Immunogenicity”,
Ann. Rev. Immunol
., 9:271-96. Identification of proliferating dendritic cells in bone marrow, in contrast to blood, is difficult because there are large numbers of granulocytes that develop in, response, to GM-CSF and these crowd the immature dendritic cell cultures, preventing maturation of the dendritic precursors. The use of cell surface markers to enrich bone marrow dendritic cell precursors has been reported to result in only modest increases because the markers are also expressed by numerous non-dendritic bone marrow cells (Bowers, W. E. and Goodell (1989)), “Dendritic Cell Ontogeny”
Res. Immunol
. 140:880-883.
Relatively small numbers of dendritic cells have also been isolated from blood (Vakkila J. et al. (1990) “Human Peripheral blood-derived dendritic cells do not produce interleukin 1&agr;, interleukin 1&bgr;, or interleukin 6
” Scand. J. Immunol
. 31:345-352; Van Voorhis W. C. et al., (1982) “Human Dendritic Cells”,
J. Exp. Med
., 1172-1187.) However, the presence in blood of dendritic cell precursors has not been reported and as recently as 1989 the relationship between blood dendritic cells and mature dendritic cells in other tissues was uncertain. Furthermore, it was recognized that dendritic cells are “rare and difficult to isolate and have not as yet been shown to give rise to DC [dendritic cells] in peripheral tissues.” (MacPherson G. G. (1989) “Lymphoid Dendritic cells: Their life history and roles in immune responses”,
Res. Immunol
. 140:877-926).
Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a factor which modulates the maturation and function of dendritic cells. (Witmer-Pack et al, (1987) “Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells”.
J.Exp.Med
. 166:1484-1498; Heufler C. et al., (1988) “Granulocyte/macrophage colony-stimulating factor and interleukin 1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells”,
J. Exp. Med
. 167:700-705). GM-CSF stimulated maturation of dendritic cells in vitro suggests that the presence of GM-CSF in a culture of dendritic cell precursors would mediate maturation into immunologically active cells, but the important goal of achieving extensive dendritic cell growth has yet to be solved.
T-dependent immune responses are characterized by the activation of T-helper cells in the production of antibody by B cells. An advantage of T-dependent over T-independent immune responses is that the T-dependent responses have memory, i.e. cells remain primed to respond to antigen with rapid production of antibody even in the absence of antigen and the immune response is therefore “boostable”. T-independent immune responses are, in contrast, relatively poor in children and lack a booster response when a T-independent antigen is repeatedly administered. The immunologic memory of T cells likely reflects two consequences of the first, “primary” or “sensitizing” limb of the immune response: (a) an expanded number of antigen-specific T cells that grow in response to antigen-bearing dendritic cells, and (b) the enhanced functional properties of individual T cells that occurs after dendritic cell priming (Inaba et al., (1984) Resting and sensitized T lymphocytes exhibit distinct stimulatory (antigen presenting cell) requirements for growth and lymphokine release;
J.Exp.Med
. 160:868-876; Inaba and Steinman, (1985) “Protein-specific helper T lymphocyte formation initiated by dendritic cells”,
Science
229: 475-479; Inaba et al., (1985) “Properties of memory T lymphocytes isolated from the mixed leukocyte reaction”,
Proc.Natl.Acad.Sci
. 82:7686-7690).
Certain types of antigens characteristically elicit T-cell dependent antibody responses whereas others elicit a T-cell independent response. For example, polysaccharides generally elicit a T-cell independent immune response. There is no memory response and therefore no protection to subsequent infection with the polysaccharide antigen. Proteins, however, do elicit a T-cell dependent response in infants. The development of conjugate vaccince containing a polysaccharide covalently coupled to a protein converts the polysaccharide T-independent response to a T-dependent response. Unfortunately, little is known concerning the sites on proteins which confer their T-cell dependent character, therefore hampering the design of more specific immunogens.
As stated above, dendritic cells play a crucial role in the initiation of T-cell dependent responses. Dendritic cells bind and modify antigens in a manner such that the mo
Inaba Kayo
Schuler Gerold
Steinman Ralph M.
Hale and Dorr LLP
Lankford , Jr. Leon B.
Merix Bioscience, Inc.
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