Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
2000-03-08
2004-09-14
Ewoldt, G. R. (Department: 1644)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S007200, C435S030000, C435S325000, C435S332000, C435S346000, C435S372000, C436S501000, C436S548000, C530S350000, C530S388200, C530S388750
Reexamination Certificate
active
06790662
ABSTRACT:
Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
FIELD OF THE INVENTION
This invention relates to a positive selection method for isolating CD8
+
cells using certain CDB-specific antibodies. The isolated CD8
+
cells have importance as vehicles for combating viral infections and tumors.
BACKGROUND OF THE INVENTION
In humans, CD8
+
cells play a vital role in the immune system's ability to defend against potentially harmful foreign entities, such as bacteria and viruses [1]. CD8
+
cells circulate in the blood and possess on their surface the CD8 protein. When necessary, these cells are converted into cytotoxic cells (i.e. cell-killing cells) which proceed to destroy foreign cells, viruses, and other harmful pathogens present in the subject [2]. Because of CD8
+
cells' effective role in host defense, they hold great potential in isolated form as therapeutics for treating disorders such as viral infections and malignancies [3].
In the past, purification of human CD8
+
cells has been achieved by negative selection. Specifically, peripheral blood mononuclear cells (“PBMC's”) are incubated with a cocktail of monoclonal antibodies specific for non-CDS sub-populations. These sub-populations include, for example, B-cells, CD4
+
cells, NK cells, macrophages and neutrophils, and each contains specific, non-CD8 “markers”. The sub-populations are then removed from the resulting antibody cocktail using magnetic beads [4]. This technique has certain major disadvantages. The first is that several monoclonal antibodies are required for removing non-CD8
+
cells. The second is that the resulting CD8
+
population suffers from contamination from non-CD8
+
cells that possess relatively low levels of non-CD8 markers. Finally, when a magnetic separation procedure is used to remove all non-CD8
+
cells, a large number of magnetic beads are needed.
SUMMARY OF THE INVENTION
This invention provides a method of isolating CD8
+
cells which comprises the steps of
(a) contacting a sample of isolated peripheral mononuclear blood cells with a first antibody which specificall bind to the sequence AAEGLDTQRFSG, (SEQ ID NO:1) or portion thereof, on CD8 molecules present on the surface of CD8
+
cells but does not activate the CD8
+
cells once bound thereto, under conditions permitting the formation of a first complex between the CD8
+
cell and first antibody;
(b) separating from the sample any first antibody not present in the resulting first complex;
(c) contacting the sample with a second, immobilized antibody which specifically binds to the first antibody in the first complex, under conditions permitting the formation of an immobilized, second complex between the first complex and the second antibody, thereby immobilizing the CD8
+
cells present in the sample;
(d) separating from the resulting immobilized second complex the cells present in the sample which were not immobilized in step (c);
(e) contacting the immobilized second complex under suitable conditions with an agent which causes the dissociation of the second complex into CD8
+
cells and an immobilized third complex between the first antibody and second antibody; and
(f) separating the immobilized third complex from the CD8
S+
cells, thereby isolating the CD8
+
cells.
This invention also provides a hybridoma cell line which produces a monoclonal antibody which specifically binds to CD8 molecules present on the surface of CD8
+
cells but does not activate the CD8
+
cells. This invention further provides monoclonal antibodies produced by each of the instant hybridoma cell lines. Finally, this invention provides related polypeptides, isolated CD8
+
cells and kits.
REFERENCES:
patent: 5645837 (1997-07-01), Jameson et al.
patent: 6001962 (1999-12-01), Ramer et al.
patent: 0 699 907 (1996-03-01), None
patent: WO95/34817 (1995-12-01), None
patent: WO 99/54345 (1999-10-01), None
VanderVegt, FP et al. J. Exp. Med. 177:1587-1592.*
Whiteside, TL et al. Blood. 81(8):2085-2092.*
Nabholz M. and H.R. Macdonald (1983) Annual Review of Immunology 1:273-306.
Riddell S.R. and P.D. Greenberg (1994) Current Topics in Microbiology and Immunology 189:9-34.
Riddell, S. R. and P. D. Greenberg (1995) Annual Review of Immunology 13:545-586.
Horgan K. and S. Shaw (1994) Current Protocols in Immunology 2:7.4.1.
Lea, T. et al., (1988) Journal of Molecular Recognition 1(1) :9-18.
Kanof M.E. (1994) Current Protocols in Immunology 2:7.1.1.
Knaof M.E. (1994) Current Protocols in Immunology 2:7:3:1.
Eichmann et al., “Affinity Enhancement and Transmembrane Signaling Are Associated with Distinct Epitopes on the CD8 a&mgr; Heterodimer”, The Journal of Immunology, vol. 147, No. 7, pp 2075-2081 (Oct. 1991).
Littman et al., “The Isolation and Sequence of the Gene Encoding TB: A Molecule Defining Functional Classes of T Lymphocytes”, Cell, vol. 40, No. 2, pp 237-246 (1985), GenPept Accession P01732, Version P01732 GI: 116035.
Litessier et al., “enrichment in Tumor-Reactive CD8+ T-Lymphocytes by Positive Selection from the Blood and Lymph Nodes of Patients with Head and Neck Cancer”, Cancer Research, vol. 51, pp 3891-3899 (Aug. 1991).
Prince et al., “Preparation of CD8 bright and CD8 dim Lymphocyte Populations Using Two Positive Selection Methods in Tandem”, The Journal of Immunological Methods, vol. 165, pp 139-148 (1993).
Stanciu et al., “Production of IL-8 and IL-4 By Positively and Negatively Selected CD4+ and CD8+ Human T Cells Following a Four-Step Cell Separation Method Including Magnetic Cell Sorting (MACA)”, The Journal of Immunological Methods, vol. 189, pp 107-115 (1996).
Ewoldt G. R.
Ortho-McNeil Pharmaceutical , Inc.
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