Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-07-28
2001-02-27
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S395000
Reexamination Certificate
active
06194549
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel cell surface structures that are selectively expressed on a subpopulation of natural killer cells and to antibodies that bind to unique epitopes on these structures.
BACKGROUND OF THE INVENTION
Natural killer cells (hereinafter sometimes referred to as “NK cells”) are large granular lymphocytes (“LGLs”) comprising 2-15% of peripheral blood mononuclear cells in healthy individuals. Although NK cells do not rearrange or express either of the known T cell receptor complexes, they can recognize and kill certain virus-infected and transformed cells in a non-MHC-restricted fashion, without prior sensitization. With the exception of CD
16
, an Fc receptor for immunoglobulin that recognizes antibody-coated target cells, the NK cell surface receptors responsible for target cell recognition have not been identified. The lack of a defining surface receptor requires NK cells to be identified by a combination of phenotypic and functional characteristics.
Although most NE cells are CD
3
:TCR−, CD
16
+, CD
56
+ LGLs, there is considerable phenotypic and functional heterogeneity within this population (Trinchieri,
Adv. Immunol.,
47:187 (1989)). For example, the surface density of CD
56
has been shown to define functionally distinct NK cell populations. CD
56
bright
NK cells are largely CD
16
+
, agranular lymphocytes deficient in cytolytic effector function that proliferate vigorously in response to exogenous IL-
2
. CD
56
dim
NK cells are CD
16
+
LGLs possessing potent cytolytic effector function that do not proliferate in response to IL-
2
. Because some T cells express both CD
16
and CD
56
, these molecules, by themselves, cannot define the NK cell population. (Trinchieri, 1989). Furthermore, because the expression of CD
56
on the functionally differentiated population of NK cells is low, monoclonal antibodies reactive with CD
56
cannot be used to reliably distinguish this subpopulation of NK cells from other cells in a sample.
It is an object of the present invention to identify a novel cell surface structure that is preferentially expressed on functionally differentiated natural killer cells, which can be used to reliably identify this subpopulation of peripheral blood mononuclear cells.
It is another object of the invention to provide antibodies that will bind to unique epitopes present on a cell surface structure selectively expressed on functionally differentiated NK cells.
SUMMARY OF THE INVENTION
These as well as other objects and advantages are achieved in accordance with the present invention, which provides a partially purified preparation of a novel natural killer cell-specific molecule, to monoclonal antibodies and immunoreactive fragments and derivatives thereof that bind to unique epitopes present on this NK cell-specific molecule, and to hybridomas that produce the monoclonal antibodies. Methods of using the antibodies and fragments and derivatives are also provided.
The novel NK cell-specific molecule of the invention consists essentially of a pair of polydispersed glycoproteins, designated herein as PEN
5
&agr; ( and PEN
5
&bgr;, having apparent molecular weights of 120-150 and 210-245 kdal, respectively, as determined by SDS polyacrylamide gel electrophoresis on a 6% polyacrylamide gel under non-reducing conditions. The unique epitopes of the PEN
5
&agr;/PEN
5
&bgr; glycoprotein pair are preferentially expressed on the subpopulation of peripheral blood NK cells which are of the phenotype CD
16
+
CD
56
dim
relative to their expression on peripheral blood NK cells having the phenotype CD
16
+
CD
56
bright
and are not present on CD
3
+
T lymphocytes or CD
20
+
B lymphocytes. In preferred embodiments of the invention, the antibody is unreactive with peripheral blood T cells, activated T cells, thymocytes, peripheral blood B cells, splenic B cells, activated B cells, monocytes, granulocytes, platelets, and red blood cells. The antibodies of the invention are preferably monoclonal antibodies and in particularly preferred embodiments, are of mouse or human origin, or they are chimeric antibodies having at least the constant region thereof of human origin.
All monoclonal antibodies having the above specificity and characteristics are encompassed by the present invention. The monoclonal antibodies are produced by hybrid cell lines using conventional hybridization and screening techniques, such as those described in Anderson et al,
J. Immunol.,
143:1899 (1989), which is hereby incorporated by reference. As is well known in the monoclonal antibody art, independently produced hybrid cell lines that produce monoclonal antibodies specific for a given antigenic determinant are typically distinct from one another, as is each of the monoclonal antibodies so produced. Thus, while repetition of the procedure described herein can result in the production of a hybrid cell line that produces a useful monoclonal antibody in accordance with the invention, it is unlikely that it will produce a hybrid cell line that produces a monoclonal antibody that is chemically an exact copy of the monoclonal antibody described below.
In another embodiment of the invention, the epitope recognized by the antibodies of the invention is a sulfated polylactosamine carbohydrate related to keratan sulfate glycosaminoglycan.
In yet another embodiment, the antibodies have the characteristics of the monoclonal antibody, alternatively referred to herein as either anti-PEN
5
or mAb
5
H
10
, secreted by a hybridoma identified by ATCC Accession No. HB11441.
The antibodies and/or immunoreactive fragments or derivatives of the invention can be labeled, e.g. with a radioactive, enzymatic, or fluorescent label and used to detect, enumerate, and/or purify functionally differentiated NK cells in a mixed population of cells and to distinguish these cells from non-NK cells and NK cells that are not functionally differentiated. Identification of the functionally differentiated subpopulation of NK cells involves (a) contacting a suitable sample that contains a mixed population of cells, which can be, for example, peripheral blood, bone marrow aspirate, or lymphoid tissue, with an antibody of the invention or an immimoreactive fragment or derivative thereof, and (b) detecting immune complex formation. Immune complex formation can be detected by any of the techniques that are conventional and well known in the art.
The antibodies of the invention can also be used to selectively eliminate functionally differentiated NK cells that are of the phenotype CD
16
+
CD
56
dim
in a sample comprising a mixed population of cells. Thus, in another aspect of the invention, methods are provided for selectively eliminating or removing functionally differentiated natural killer cells from a suitable sample, preferably a biological sample, which involve (a) contacting the sample with an antibody of the invention or an immunoreactive fragment or derivative thereof, which is optionally linked to a radionucleotide or a toxin, and (b) removing from the sample the cells that bind to the antibody, fragment or derivative. In preferred embodiment of the invention, the biological sample is bone marrow aspirate.
These as well as other features and advantages of the present invention will be apparent to persons skilled in the art from the following detailed description and the claims.
REFERENCES:
patent: 4772552 (1988-09-01), Hercend et al.
patent: 4797475 (1989-01-01), Terasaki et al.
patent: 4831122 (1989-05-01), Buchsbaum et al.
patent: 5068223 (1991-11-01), Lipsky et al.
patent: 5169939 (1992-12-01), Gefter et al.
patent: 5215927 (1993-06-01), Berenson et al.
patent: 5786160 (1998-07-01), Anderson
Baume et al., “Differential Responses to Interleukin 2 Define Functionally Distinct Subsets of Human Natural Killer Cells,”Eur. J. Immunol., 22:1-6 (1992).
Caterson et al., “Identification of a Monoclonal Antibody That Specifically Recognizes Corneal and Skeletal Keratan Sulfate,”J. Biol. Chem., 258(14):8848-8854 (1983).
Chou et al., “Stru
Anderson Paul
Vivier Eric
Dana-Farber Cancer Institute
Onofrio, Esq. Dara L.
Saunders David
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