Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-04-28
2002-04-09
Bansal, Geetha P. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388100, C530S388200, C530S387100, C530S350000, C435S007100
Reexamination Certificate
active
06369204
ABSTRACT:
FIELD OF THE INVENTION
This application relates to hybrid cell lines (lymphocyte hybridomas) for the production of monoclonal antibodies to &agr;v&bgr;3 integrin, to such homogeneous antibodies, and to the use of such antibodies for diagnostic and therapeutic purposes.
BACKGROUND OF THE INVENTION
&agr;v&bgr;3 is a member of the integrin supergene family of cell-surface glycoprotein receptors that promote cellular adhesion. Each cell has a specific repertoire of receptors that define its adhesive capabilities. The integrins are expressed as heterodimers of noncovalently associated &agr; and &bgr; subunits. According to the nomenclature proposed by Hynes, R. O. [
Cell
48,875-886 (1987)], the integrins can be divided into families each with a common &bgr;-subunit and a set of variable &agr;-subunits known to associate with the common &bgr;-subunit. The different a chains are denoted by the original cell type, by a subscript used by the original discoverer, or, as in the case of the &agr;v&bgr;3 receptor, by the nature of the ligand (i.e. &agr;
v
stands for a vitronectin receptor &agr;-chain). Many, but not all, integrin receptors have been shown to interact with proteins via a tripeptide sequence, Arg-Gly-Asp (or RGD using the single letter amino acid code), originally defined from studies of the cell binding domains of fibronectin [Ruoslahti, E. and Pierschbachter, M. D.,
Cell
44, 5170518 (1986); Ruoslahti, E. and Pierschbachter, M. D.,
Science
238, 491-497 (1987)].
&agr;v&bgr;3 (also referred to as vitronectin receptor or VNR) is a member of the &bgr;3 integrin subfamily and is expressed on a variety of cells, including endothelial, melanoma, smooth muscle cells and, along with another integrin &agr;2&bgr;1 (VLA-2) (the receptor for Type I collagen and laminin), on the surface of osteoclasts [Horton, M. A. and Davies, J.,
J. Bone Min. Res.
4, 803-808 (1989); Davies, J. et al.,
J. Cell. Biol.
109, 1817-1826(1989); Horton, M.,
Int. J. Exp. Pathol.
71 741-759 (1990)]. &agr;v&bgr;3 mediates cell adhesion to vitronectin, fibrinogen, fibronectin, thrombospondin, osteopontin, bone sialo protein II and von Willebrand factor.
Osteoclasts are the main type of bone cells involved in the resorption of bone tissues. The resorption process involves the proliferation and chemotaxis of developing osteoclasts to the skeleton from hematopoietic sites migration of mature cells to sites of subsequent resorption, attachment of osteoclasts to bone substrate and the eventual formation of the polarized, functional mature end cells which are directly involved in bone resorption. The &agr;v&bgr;3 integrin mediates adhesion of osteoclasts to RGD sequence-containing bone matrix proteins.
Antibodies to &agr;v&bgr;3 are expected to be valuable diagnostic and therapeutic tools in studying the biological role and the structural/functional relationships of this integrin with its various ligands. In particular, monoclonal antibodies (Mabs) detecting unique epitopes on osteoclasts would be of great value in understanding of the development of osteoclasts. Even more importantly, neutralizing Mabs specific for &agr;v&bgr;3 that inhibit the osteoclast binding to the bone matrix proteins have great potential as therapeutic agents useful in the treatment of conditions associated with excessive bone resorption.
There are several monoclonal antibodies known in the art that bind to various epitopes on &agr;v&bgr;63. Immunizing with osteoclasts from osteoclastomas (giant cell tumors of bone), Horton, M. A. et al. [
Cancer Res.
45, 5663-5669 (1985)] produced eleven mouse hybridomas secreting monoclonal antibodies which bind to osteoclasts in normal human fetal bone and a variety of neoplastic and non-neoplastic bone lesions. One of these, designated 23C6, was subsequently shown to bind the &agr;v&bgr;3 complex, and was demonstrated to be able to disrupt osteoclast function [Horton, M. A. et al.,
Exp. Cell. Res.
195 368-375 (1991)]. Another monoclonal antibody, LM609 (produced in hybridoma LM609 ATCC HB 9537) disclosed in PCT Application Publication No. WO 89/05155 (published Jun. 15, 1989) and Cheresh et al.
J. Biol. Chem.
262:17703-17711 (1987) was also found to bind the &agr;v&bgr;3 complex and, due to its ability to inhibit the binding of ECr molecules present on the surface of tumor cells and blood vessel forming endothelial cells to vitronectin, fibrinogen and von Willebrand factor, was proposed for therapeutic use as tumor growth inhibitor. Monoclonal antibody 13C2 (Horton, M. A. et al.,
Cancer Res.
1985, Supra) was shown to bind the &agr;v portion of the &agr;v&bgr;3 molecule, whereas several other monoclonal antibodies were reported to recognize the &bgr;3 portion [Nesbitt, S. et al., Epitope Analysis of the Vitronectin Receptor (CD51), In “Leukocyte Typing IV” White Cell Differentiation Antigens, Knapp, W. et al. (eds.) 1991, p.1037]. The specific monoclonal antibodies variously reported in the art were shown to also bind to endothelial cells and various melanoma cell lines.
There is a need for high affinity monoclonal antibodies to the &agr;v&bgr;3 integrin that are capable of effective inhibition of the binding of &agr;v&bgr;3 expressing cells to &agr;v&bgr;3 ligands, such as vitronectin and fibronectin.
It would be further desirable to provide monoclonal antibodies to &agr;v&bgr;3 that bind osteoclasts and optionally other cells known to express &agr;v&bgr;3.
It would be particularly desirable to provide monoclonal antibodies that are effective inhibitors of &agr;v&bgr;3 binding to its ligands and which specifically bind osteoclasts without binding to other cells known to express &agr;v&bgr;3, i.e., which are more specific for the target integrin on osteoclasts.
SUMMARY OF THE INVENTION
The present invention is based on successful research involving the production and extensive characterization of monoclonal antibodies to &agr;v&bgr;3 integrin. Accordingly, the present invention is directed to monoclonal antibodies, and derivatives thereof, which are capable of recognizing unique epitopes on &agr;v&bgr;3 and/or which exhibit high affinity for &agr;v&bgr;3. The invention is specifically directed to monoclonal antibodies recognizing unique epitopes on the &agr;v&bgr;3 complex or the &bgr;3 portion thereof. The invention is further directed to monoclonal antibodies effectively inhibiting the binding to vitronectin and fibrinogen of &agr;v&bgr;3 expressing cells. In a particularly important aspect, the invention is directed to monoclonal antibodies specifically binding &agr;v&bgr;3 on osteoclasts but not other &agr;v&bgr;3 on other cells (e.g. melanoma cells C32R, M-21, HA-A, HA-L and HT-144 and human umbilical vein endothelial cells).
In one aspect, the invention concerns an anti-&agr;v&bgr;3 monoclonal antibody that is capable of: (1) inhibiting the binding of &agr;v&bgr;3 expressing cells to fibrinogen, (2) binding osteoclasts, and (3) binding to substantially the same epitope recognized by any one of a monoclonal antibody selected from the group consisting of 10C4.1.3, 9G2.1.3 and 9D4.9.1 or which has an affinity for &agr;v&bgr;3 which is about equal to or greater than that of the foregoing three antibodies.
In another aspect, the invention concerns isolated nucleic acid encoding such antibodies, and hybridoma or recombinant cells producing such antibodies.
In a further aspect, the invention concerns the therapeutic or diagnostic use of such antibodies. The monoclonal antibodies of the invention are useful as therapeutic agents, either by themselves or in conjunction with (chemo)therapeutic agents, to treat diseases or conditions that are characterized by excessive bone resorption and/or to inhibit tumor growth. The monoclonal antibodies of the invention also are useful in diagnostic and analytical assays for determining the presence of &agr;v&bgr;3 on cells, cell typing and in histochemical tissue staining.
These and further aspects will be apparent from the following detailed description.
REFERENCES:
patent: 4707352 (1987-11-01
Bodary Sarah C.
Chuntharapai Anan
Horton Michael A.
Kim Kyung Jin
Bansal Geetha P.
Carpenter David A.
Genentech Inc.
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