Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-04-18
2004-08-24
Moran, Marjorie (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S300000, C530S350000, C530S388100, C702S019000, C702S020000
Reexamination Certificate
active
06780598
ABSTRACT:
TECHNICAL FIELD
The present invention relates to locating protein epitopes and more particularly to novel methods for identifying, determining the location, and the optimal length of immunobiologically active amino acid sequences.
BACKGROUND OF INVENTION
Epitopes or antigenic determinants of a protein antigen represent the sites that are recognized as binding sites by certain immune components such as antibodies or immunocompetent cells. While epitopes are defined only in a functional sense i.e. by their ability to bind to antibodies or immunocompetent cells, it is usually accepted that there is a structural basis for their immunological reactivity.
Epitopes are classified as either being continuous and discontinuous (Atassi and Smith, 1978
, Immunochemisty
, vol 15 p. 609). Discontinuous epitopes are composed of sequences of amino acids throughout an antigen and rely on the tertiary structure or folding of the protein to bring the sequences together and form the epitope. In contrast, continuous epitopes are linear peptide fragments of the antigen that are able to bind to antibodies raised against the intact antigen.
Many antigens have been studied as possible serum markers for different types of cancer because the serum concentration of the specific antigen may be an indication of the cancer stage in an untreated person. As such, it would be very advantageous to develop immunological reagents that react with the antigen, and more specifically, with the epitopes of the protein antigen.
To date, methods using physical-chemical scales have attempted to determine the location of probable peptide epitopes which includes looking at the primary structure, that being the amino acid sequence, secondary structure such as turns, helices, and even the folding of the protein in the tertiary structure. Continuous epitopes are structurally less complicated and therefore may be easier to locate, however, the ability to predict the location, length and potency of the site is limited.
Various methods have been used to identify and predict the location of continuous epitopes in proteins by analyzing certain features of their primary structure. For example, parameters such as hydrophilicity, accessibility, and mobility of short segments of polypeptide chains have been correlated with the location of epitopes (see Pellequer et al. 1991
, Method in Enzyology
, vol 203, p. 176-201).
Hydrophilicity, has been used as the basis for determining protein epitopes by analyzing an amino acid sequence in order to find the point of greatest local hydrophilicity as disclosed in U.S. Pat. No. 4,554, 101. Hopp and Woods (See
Proc. Natl. Acad. Sci. USA
, vol. 78, No. 6, pp. 3824-3828, Jun. 1981) have shown that by assigning each amino acid a relative hydrophilicity numerical value and then averaging local hydrophilicity so that the location of the highest local average hydrophilicity values represent the locations of the continuous epitopes. However, this method does not provide any information as to the optimal length of the continuous epitope.
Likewise, the amino acid sequence of a protein as measured by the Kyte-Doolittle (Kyte and Doolittle, 1982
, J. Mol. Biol
. vol. 72, p. 105) scale, is commonly used to evaluate the hydrophilic and hydrophobic tendencies of polypeptide chains by using a hydropathy scale. Each amino acid in the polypeptide chain is assigned a value reflecting its relative hydrophilicity and hydrophobicity which are averaged across a moving section of the sequence. This method offers a graphic visualization of the hydropathic character of the amino acid chain. It is theorized that by using the hydropathic character of the sequence, interior sequence regions which are usually composed of hydrophobic amino acids can be distinguished from hydrophilic exterior sequence regions. This information offers the ability to evaluate the possible secondary structure. However this model, does not predict the optimal length of the epitope or indicate if the effective size of epitopes is unique for each protein molecule.
Accordingly, what is needed is a simple method to identify immunobiologically-active peptide epitopes, determine their optimal length, and locations of these epitopes within a polypeptide.
SUMMARY OF THE INVENTION
In accordance with this invention there is provided methods for identifying immunobiologically-active linear peptide epitopes of a protein antigen and determining the optimal length of amino acid residues of the epitope.
TERMS
For purposes of this invention, the terms and expressions below, appearing in the specification and claims, are intended to have the following meanings:
“Window” as used herein means the number of amino acid residues in a curve segment.
“Lagging” as used herein means to move across the entire amino acid residues sequence increasing by one (1) in each step.
“Period number” as used herein means the number of amino acids assigned as the period between −180° to +180° in the negative cosine function plot.
“Fit-Correlation Value” as used herein means a numerical value which is indicative of the fit between the hydropathy plot curve and a negative cosine function wherein the value may be positive or negative depending on the fit. The better the fit the more positive the value.
“Epitope” as used herein means the portion of an antigen that binds specifically with the binding site of an antibody or a receptor on a lymphocyte.
“Potential Ho-Hi-Ho epitope” as used herein means an epitope wherein the curve segment of the hydrophilicity plot correlates with the negative cosine function giving a fit-correlation value.
“Potential Ho-Hi-Ho epitope set” as used herein means a set of epitopes having a positive fit-correlation value for a specific period assigned to the negative cosine curve.
“Ho-Hi-Ho theoretical epitopes” as used herein means the epitopes in the potential epitope set that have ranking values that exhibit the most oscillating behavior about an equilibrium position and either converge towards or diverge away from this equilibrium position and are deemed the most immunobiologically-active linear peptides.
“Number Range” as used herein means the numerated amino acid sequence number region of the amino acid sequence having a length equal to a period number, i.e. if the period is 10, then the sequence number ranges could be 1-10, 2-11, 3-12 and so on until (n−(m−1)) where n is equal to the number of amino acid residues in the entire polypeptide and m is the period number.
Immune responses arise as a result of exposure to foreign stimuli. The compound that evokes the response is referred to as antigen or as immunogen. An immunogen is any agent capable of inducing an immune response. In contrast, an antigen is any agent capable of binding specifically to components of the immune response, such as lymphocytes and antibodies. The smallest unit of an antigen that is capable of binding with various immune components, either cells, such as T and B lymphocytes, or antibodies, is called an epitope. Compounds may have one or more epitopes capable of reacting with immune components. The methods of the present inventions provide an in silica methodology for determining the antigen-binding site of an antibody or a receptor on a lymphocyte that has a unique structure that allows a complementary “fit” to some structural aspect of the specific antigen.
Thus understood, a primary object of the present invention is to provide a method for determining immunobiologically-active linear peptide epitopes and their optimal length.
Another object of the present invention is to identify immunobiologically-active linear peptide epitopes without the need for time consuming and expensive testing regimes to determine immunogenic activity, such as in vivo animal testing and/or in vitro assay testing.
A further object of this invention is to determine the immunopotency of an epitope and provide a ranking system delineating between dominant and subdominant epitopes.
A still further object of the invention is to provide monoclonal and polyclonal antibodies highly specific
Clow Lori A.
Moran Marjorie
The Bilicki Law Firm P.C.
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