Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
1998-02-24
2001-12-25
Chan, Christina Y. (Department: 1644)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C514S004300, C514S016700, C514S013800, C514S012200, C514S002600, C530S303000, C530S328000, C530S324000, C530S325000, C530S350000, C435S325000
Reexamination Certificate
active
06333031
ABSTRACT:
INTRODUCTION
1. Technical Field
The field of this invention is the modulation of activity of cell surface receptors, including both modulation of internalization of the receptor and modulation of activation of the receptors in the presence or absence of ligand.
2. Background
The complex regulatory balance between hormones, receptors and responding cells is critical to the correct functioning of multicellular organisms. Subtle environmental and genetic factors can disrupt this balance, sometimes resulting in disease. The advent of molecular biology has meant that medically important hormones can be made available in therapeutically useful amounts. Among them are human growth hormone, EPO, TPO, insulin-like growth factor, insulin, epidermal growth factor, and numerous others.
A condition of great economic and medical significance is insulin resistance, which is an essential feature of a great variety of clinical disorders, such as diabetes mellitus, obesity and certain types of hypertension. Individuals with non-insulin dependent diabetes present with insulin resistance in peripheral tissues. They have a subnormal glucose utilization in skeletal muscle, where glucose transport across the cell membrane of skeletal muscle is the rate limiting step in glucose metabolism. It is possible that a defect exists in insulin-dependent glucose transport in skeletal muscle in diabetic states, where decreased levels of the glucose transporter 4 protein (GLUT4) have been observed. In adipose and muscle cells, insulin stimulates a rapid and dramatic increase in glucose uptake, primarily by promoting the redistribution of the GLUT4 glucose transporter from its intracellular storage site to the plasma membrane.
Insulin resistance may also be attributed to a defect in insulin action at the cellular level. The insulin receptor is activated by binding of insulin to the alpha-subunit of the receptor, which causes autophosphorylation of the intracellular beta-subunit region. The activated insulin receptor couples to cytosolic receptor substrates that can affect signaling cascades, resulting in the pleiotropic hormone response. Most proteins involved in the signal transduction pathway are not known yet, but each of them might play a role in the various forms of insulin resistance. The heterogeneous nature of insulin resistance makes treatments that can act “upstream” of the signal transduction pathways very attractive, because a number of different pathologies could be treated with a single drug.
Specific peptides have been previously shown to enhance the cellular response to certain hormones. This effect has been attributed to inhibition of the internalization of the corresponding hormone receptors. Insulin-stimulated glucose uptake is increased by adding the peptides to responding cells, offering the possibility of improved therapy for insulin dependent and insulin resistant diabetes. The enhanced response may also be exploited in therapies involving other hormones. Improvements in the specificity of agents that enhance the activity of insulin and other hormones are of considerable interest for their therapeutic benefits. The site of action for such peptides on receptors molecules is of interest for drug evaluation and design.
In addition, there is great interest in finding ligand replacements, or mimetics, that could be used in place of the naturally-occuring ligand. This is of particular interest for ligand hormones that may have a number of biological functions of which only a subset are to be regulated.
Relevant Literature
Several groups have examined the glucose transporter and insulin receptor for residues that are involved in internalization. Rajagopalan et al. (1995)
Biochem. Biophys. Res. Commun.
211:714-8 found that residues GPYL950-953 served as the predominant endocytosis signal and the sequence NPEY957-960 as a secondary signal. Levy-Toledano et al. (1993)
Biochem. Biophys. Acta.
1220:1-14 suggest that the structural domain located 43-113 amino acids from the C-terminus is required in intact cells for insulin-stimulated autophosphorylation and signal transmission. Verhey et al. (1995)
J. Cell Biol.
130:1071-9 identified sequences involved in the differential subcellular localization and hormone-responsiveness of glucose transporter isoforms. The COOH-terminal 30 amino acids of GLUT4 are sufficient for its correct localization to an intracellular storage pool that translocates to the cell surface in response to insulin. In addition, there is a report of important leucine residues in insulin receptor endocytosis. See Hamer et al., J. Biol. Chem. 272:21685 (1997).
U.S. Pat. No. 5,385,888, issued Jan. 31, 1995, describes Class I MHC peptide modulation of surface receptor activity. Data presented in International patent application PCT/US94/09189 suggest that these peptides must be in an ordered conformation to be biologically active. The composition and uses of such peptides are further described in International application PCT/US93/01758. The peptides are further disclosed in International application PCT/US89/00876.
Regulation of receptor internalization by the major histocompatibility complex class I molecule is shown by Olsson et al. (1994)
Proc. Natl. Acad. Sci.
91:9086-90, and by a peptide derived from the insulin receptor in Naranda et al.,
Proc. Natl. Acad. Sci.
94:11692 (1997). Peptides derived from the alpha 1 domain of the major histocompatibility complex class I protein (MHC-I) inhibit internalization of some receptors, thereby increasing the steady-state number of active receptors on the cell surface. It is suggested that MHC-I participates in the regulation of cell surface receptor activity. Stagsted et al. (1993)
J. Biol. Chem.
268:22809-13 demonstrate that such peptides inhibit the internalization of glucose transporters (GLUT4) and insulin-like growth factor II (IGF-II) receptors in insulin-stimulated cells.
SUMMARY OF THE INVENTION
It is an object of the invention to provide compositions and methods useful in the modulation of receptor activity. Accordingly, the present invention provides oligopeptides comprising at least about 8 amino acids and less than about 40 amino acids which has an amino acid sequence corresponding to the activation sequence of the extracellular domain of a cell surface receptor. Also provided are oligopeptides at least about 90% homologous to an activation sequence of a cell-surface receptor.
In an additional aspect, the invention provides methods of modulating the internalization of a cell-surface receptor containing an activation sequence comprising binding an exogeneous compound to said activation sequence.
In a further aspect, the invention provides mammalian cells comprising a modified cell surface receptor, wherein the modification comprises an amino acid sequence substitution, insertion or deletion in an activation sequence of the region of the extracellular domain.
In an additional aspect, the invention provides methods of determining an activation sequence of a cell surface receptor comprising searching for a region of sequence similarity between the cell surface receptor and the sequence of an &agr;1-domain of an MHC Class I antigen.
In a further aspect, the invention provides methods for screening for a bioactive agent capable of binding to the activation sequence of a cell surface receptor, comprising combining a cell surface receptor and a candidate bioactive agent, and determining the binding of the candidate agent to the activation sequence of the cell surface receptor.
In an additional aspect, the invention provides methods for screening for an bioactive agent capable of modulating the internalization of a type-1 cell-surface receptor, the method comprising the steps of adding a ligand bound by the cell surface receptor and a candidate bioactive agent to a cell comprising the cell surface receptor. The effect of the candidate bioactive agent on the internalization of the receptor is then determined.
In a further aspect, the invention provides methods for screening for an bioactive agent capable of modulating the interna
Naranda Tatjana
Olsson Lennart
Chan Christina Y.
DiBrino Marianne
Reception, Inc.
Rowland Bertram I.
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