Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2001-01-31
2003-07-08
Witz, Jean C. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C424S530000, C424S534000
Reexamination Certificate
active
06589526
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for the enrichment of dendritic cells from the peripheral blood of a mammal, to purified dendritic cell populations, and to kits useful for enrichment of dendritic cells.
BACKGROUND OF THE INVENTION
Dendritic cells are antigen-presenting cells that are found in all tissues and organs, including the blood. Specifically, dendritic cells present antigens for T lymphocytes, i.e., they process and present antigens, and stimulate responses from naive and memory T cells. In addition to their role in antigen presentation, dendritic cells directly communicate with non-lymph tissue and survey non-lymph for an injury signal (e.g., ischemia, infection, or inflammation) or tumor growth. Once signaled, dendritic cells initiate the immune response by releasing IL-1 which triggers lymphocytes and monocytes.
Dendritic cells can play either a positive or negative role in various immunologically related situations. For example, dendritic cells are thought to be involved in transplant rejection and autoimmune diseases, and it may be desirable to block their function in such situations. On the other hand, various immunodeficiencies, e.g., towards tumors, may result from deficient dendritic cells. In addition, there is evidence that HIV can infect certain populations of dendritic cells.
It is desirable to obtain substantially pure populations of dendritic cells, e.g., in order to exploit the important role of dendritic cells in various immunotherapies. Dendritic cell isolation is particularly difficult, however, because it is believed that they are very low in frequency in blood and other tissues, and because there is no reported surface marker expressed by fresh and cultured dendritic cells which distinguishes it from monocytes. Conventional methods for isolation which enrich subpopulations of cell mixtures include, e.g., density gradient separation, fluorescence activated cell sorting, immunological cell separation techniques such as panning, complement lysis, rosetting, magnetic cell separation techniques, and nylon wool separation. Different patterns of expression of cell surface antigens have been used in some cases to identify different cell types. Certain disadvantages of many of these reported methods are that they can be time-consuming, labor-intensive, costly, require large amounts of reagent, can result in low specificity, low sensitivity, contaminated mixtures, poor and/or inaccurate separation, loss of desired cells, or can change the properties, functions, or viability of the desired cells. Thus, prior methods generally are inefficient, time-consuming, expensive and do not optimize for pure populations.
SUMMARY OF THE INVENTION
It is an object of the invention to provide an easy, effective, and inexpensive method for isolating dendritic cells from tissue, e.g., peripheral blood, of a mammal.
It is yet another object of the invention to provide a method for isolating dendritic cells which are viable and substantially unaltered in cell function.
It is yet another object of the invention to provide a method for isolating dendritic cells which are substantially pure.
It is yet another object of the invention to provide a method for isolating dendritic cells which can be used in various immunotherapeutic regimens.
It is yet another object of the invention to provide a method for isolating dendritic cells which express high levels of CD14.
It is yet another object of the invention to provide a method for isolating dendritic cells from monocytes.
It is yet another object of the invention to provide a method for isolating dendritic cells from tissue, e.g., peripheral blood, of a mammal which are unactivated.
Still another object of the invention is to provide a method for isolating monocytes from the tissue, e.g., peripheral blood, of a mammal which are unactivated.
According to the invention, a method for the enrichment of dendritic cells from the peripheral blood of a mammal is provided. Peripheral blood having mononuclear cells from a mammal is provided, and the mononuclear cells are separated from the peripheral blood. These mononuclear cells are separated into a first cell population having substantially lymphocytes, e.g., T cells, NK cells, B cells or mixtures thereof, and a second cell population having substantially myeloid cells. These myeloid cells are separated into a third cell population having substantially monocytes and a fourth cell population having substantially dendritic cells.
In certain embodiments, the separation of the mononuclear cells into a first cell population having substantially lymphocytes and a second cell population having substantially myeloid cells comprises contacting the mononuclear cells with antibodies against the lymphocytes so as to form an antibody-lymphocyte complex, and selectively separating the antibody-lymphocyte complex from the myeloid cells. The antibodies used, e.g., monoclonal antibodies, are directed against one or more antigens which are expressed by one or more of the lymphocytes. For example, T cell antibodies include anti-CD3 antibodies, anti-CD8 antibodies, and mixtures thereof; NK cell antibodies include, e.g., anti-CD16/56 antibodies; and B cell antibodies include, e.g., anti-CD19 or anti-CD20 antibodies.
In certain embodiments, the antibody-lymphocyte complex that is formed is selectively separated from the myeloid cells by contacting the antibody-lymphocyte complex and the myeloid cells with a matrix such that the antibody-lymphocyte complex is substantially retained by the matrix, e.g., greater than 20%, 40%, 60%, 80%, 90%, 95%, 98%, or 99% retained, and the myeloid cells are substantially not retained by the matrix, e.g., greater than 20%, 40%, 60%, 80%, 90%, 95%, 98%, or 99% not retained.
Preferably, the antibody-lymphocyte complex further comprises magnetic beads, e.g., superparamagnetic microparticles. The magnetic beads can be attached, e.g., to the antibody or to the lymphocyte or to both. In embodiments in which the antibody-lymphocyte complex has magnetic beads, separation of such a complex from the myeloid cells preferably comprises contacting the myeloid cells and the complex with a magnetic matrix, e.g., magnetized steel wool, such that the antibody-lymphocyte complex having the magnetic beads is substantially retained by the magnetic matrix and the myeloid cells are substantially not retained by the magnetic matrix.
A variation of this method is separating the mononuclear cells into a first cell population having substantially lymphocytes and a second cell population having substantially myeloid cells by centrifugation, e.g., density gradient centrifugation.
In certain embodiments, the separation of the mononuclear cells into a third cell population having substantially monocytes and a fourth cell population having substantially dendritic cells comprises contacting the myeloid cells with antibodies against the dendritic cells so as to form an antibody-dendritic cell complex, and selectively separating the antibody-dendritic cell complex from the monocytes. The antibodies used, e.g., monoclonal antibodies, are directed against one or more antigens which are expressed by the dendritic cells, e.g., anti-CD2 antibodies, anti-CD5 antibodies or mixtures thereof. In certain embodiments, the myeloid cells are cultured prior to contacting the myeloid cells with the antibodies, and anti-CD83 antibodies are used.
In certain embodiments, the antibody-dendritic cell complex that is formed is selectively separated from the monocytes by contacting the antibody-dendritic cell complex and the monocytes with a matrix such that the antibody-dendritic cell complex is substantially retained by the matrix and the monocytes are substantially not retained by the matrix. Preferably, the retained antibody-dendritic cell complex is then eluted from the matrix.
Preferably, the antibody-dendritic cell complex further comprises magnetic beads. In such embodiments, separation of the antibody-dendritic complex from the monocytes preferably comprises contacting the monocytes and the antibody-d
Alper Chester A.
Crawford Keith D.
Center for Blood Research
Ropes & Gray LLP
Witz Jean C.
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