Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Catalytic antibody
Reexamination Certificate
1995-06-07
2003-02-18
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Catalytic antibody
C530S388900, C530S389800
Reexamination Certificate
active
06521432
ABSTRACT:
FIELD OF THE INVENTION
This application is a continuation-in-part of commonly assigned applications U.S. Ser. No. 190,271, filed May 4, 1988, PCT/US89/01950, filed May 4, 1989, and PCT/US89/01951, filed May 4, 1989. The subject matter of said applications is incorporated by reference.
This invention relates generally to methods for enhancing the rate of cleavage or formation of peptide bonds. More particularly, this invention relates to methods for enhancing the rate of cleavage or formation of specific metastable peptide bonds within protein or peptide molecules by contacting such molecules with a rate-enhancing antibody.
Several publications are referenced in this application by Arabic numerals within parenthesis. Full citation for these references are found at the end of the specification immediately preceding the claims. The references more fully describe the state of the art to which this invention pertains as well as certain aspects of the invention itself.
BACKGROUND OF THE INVENTION
It is known that certain peptide sequences in proteins are metastable. These sequences, also referred to as “sensitopes,” are susceptible to spontaneous chemical reactions such as deamidation, isomerization, racemization, and in some cases peptide bond cleavage. As such, they may be target sites for antibodies that enhance the rate of chemical reactions that occur at such sensitive sites.
It is known that certain synthetic peptide sequences are particularly susceptible to spontaneous reactions (1). Asparagine, aspartic acid, glutamine and glutamic acid are amino acid residues that are frequently associated with susceptible sequences, and it has been proposed that the amino acid residues flanking these particular side chains can determine the particular susceptibility of these sites within peptides (2). It has also been observed that the structural features around these sites in intact proteins can also influence the stability of these sites to spontaneous chemical modification (3).
More specifically, it is known that polypeptides containing the dipeptide sequences, ASN-PRO, ASN-GLY, ASP-PRO, ASP-GLY GLN-X or GLU-X, wherein X is any amino acid, undergo hydrolysis at a much higher rate than other dipeptides. This instability is due to the formation of a cyclic structure resulting from intramolecular attack of the side chain amide or acid on the peptide bond between the two amino acids. However, these metastable bonds are reported to be more stable in native (not denatured) proteins (2).
Antibodies have previously been generated that catalyze acyl transfer reactions (4), sigmatropic rearrangements (5), intramolecular cyclization (6), and peptide bond hydrolysis (7). It has been speculated that such antibodies may be particularly suited to performing substrate assisted catalysis—i.e., catalyzing the reaction of a substrate containing a reactive nucleophile or catalyst within the molecule undergoing transformation.
It is known that antibodies raised against peptides are able to bind to the same sequence when the latter are located within an intact protein. For example, antibodies elicited against a peptide comprising amino acids 1-15 of tumor necrosis factor (TNF) are able to bind to native tumor necrosis factor and in doing so, inhibit its interaction with a cell surface receptor (8). Similarly, antibodies against a peptide comprising amino acids of the gp 120 coat protein from HIV cross-react with the intact virus and inhibit the interaction of the virus with its cellular receptor, CD4 (9). In another example, monoclonal antibodies raised against a peptide comprising amino acids 67-83 of hen egg lysozyme were able to cross-react with the intact protein and are able to recognize other avian species of lysozyme whose sequences within the epitope are substantially similar (10).
While methods for preparing catalytic antibodies have been described, and while methods for binding noncatalytic and catalytic antibodies to antigens or substrates of interest have been described, the art has heretofore not provided methods of enhancing the rate of cleavage or formation of certain metastable peptide bonds known to undergo spontaneous hydrolysis.
OBJECTS OF THE INVENTION
It is a primary object of this invention to provide methods for enhancing the rate of cleavage or formation of metastable peptide bonds within protein or peptide molecules.
It is a further object of the invention to provide methods for enhancing the rate of cleavage or formation of metastable peptide bonds, e.g., ASN-PRO, ASN-GLY, ASP-PRO, ASP-GLY, GLN-X or GLU-X, wherein X is any amino acid, by contacting the peptide or protein molecule containing the metastable peptide bond with a rate-enhancing antibody which is prepared by a rational design method according to the invention.
It is still a further and related object of the invention to provide methods for enhancing the rate of hydrolysis of specific peptide bonds in protein or peptide molecules by contacting such molecules with a rate enhancing antibody which promotes the natural tendency of these bonds to form a cyclic intermediate structure by intramolecular attack of the amide or acid group of the aspartic or glutamic acid or asparagine or glutamine side chains on the peptide bond.
SUMMARY OF THE INVENTION
These and other objects of the invention are achieved in an antigen for elicitation of a rate-enhancing antibody, said antigen containing a hapten having a metastable bond.
One embodiment of the invention is an antigen for elicitation of an antibody capable of enhancing the rate of reaction of a substrate of interest at the site of a metastable bond, said antigen containing a hapten which mimics said substrate of interest at or near the said site of said metastable bond.
A further embodiment of the invention is an antibody which enhances the rate of modification of a metastable bond in a substrate of interest, said antibody having been prepared by a process comprising the steps of: selecting the specific metastable bond to be modified; selecting an antigen comprising a hapten which mimics said substrate at or near the said site of said metastable bond; exposing cells capable of producing antibodies to said antigen and thereby generating antibody producing cells; hybridizing said antibody producing cells with myeloma cells and thereby generating a plurality of hybridoma cells each producing monoclonal antibodies; and screening said plurality of monoclonal antibodies to identify a monoclonal antibody which binds to an epitope at or near the metastable bond to be modified and enhances the rate of modification of said metastable bond.
A further embodiment of the invention is a method for preparing antibodies which enhance the rate of cleavage or formation of a metastable bond of interest comprising the steps of: selecting the specific metastable bond to be cleaved or formed in a protein or peptide molecule substrate of interest; selecting an antigen comprising a hapten which mimics said substrate at or near the said site of said metastable bond; exposing cells capable of producing antibodies to said antigen and thereby generating antibody producing cells; hybridizing said antibody producing cells with myeloma cells and thereby generating a plurality of hybridoma cells each producing monoclonal antibodies; and screening said plurality of monoclonal antibodies to identify a monoclonal antibody which binds to an epitope at or near the metastable bond to be modified so as to enhance the rate of modification of said metastable bond.
A yet further embodiment of the invention is a method for enhancing the rate of modification of a specific metastable bond within a protein or peptide molecule substrate of interest which comprises contacting said substrate with an antibody under conditions sufficient for said antibody to bind to said substrate at an epitope at or near said specific metastable bond and to enhance the rate of reaction.
A still further embodiment of the invention is a method for enhancing the rate of modification of a specific metastable bond within a protein or peptide molecule s
Booth Paul M.
Hong Wonpyo
Massey Richard J.
Powell Michael J.
Rees Anthony R.
Evans, Esq. Barry
IGEN International Inc.
Kramer Levin Naftalis & Frankel LLP
Patterson Jr. Charles L.
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