Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-21
2003-06-17
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007370, C435S069300, C435S252300, C435S325000, C435S455000, C435S462000
Reexamination Certificate
active
06579676
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of molecular biology and immunology. It relates to a novel method of selecting for and analyzing mutants. The present invention also relates to the use of this method to identify antigenic domains, epitopes or binding domains of cell surface or intracellular proteins or polypeptides.
BACKGROUND
Resting human T cells bind sheep erythrocytes via a T cell specific 50 kD cell surface protein called CD2 (Bach, J. F., et al., Transplantation 8:265-280 (1969); Howard, F. D., et al., J. Immunol. 126:2117-2122 (1981)). This phenomenon has long had practical utility, but, until recently, little known physiological significance. However, parallel studies of the practical utility, but, until recently, little known physiological significance. However, parallel studies of the interaction between T cells and sheep erythrocytes (Hunig, T., J. Exp. Med. 162:890-901 (1985); Hunig, T. R. , J. Immunol. 136:2103-2108 (1986)), and T cells and their physiological targets (Shaw, S., et al., Nature 323:262-264 (1986)), have led to the identification of a specific molecular ligand for CD2 which is a widely distributed surface protein called, in the human case, LFA-3. CD2/LFA-3 interactions mediate cytolytic target conjugation (Shaw, S., et al., Nature 323:262-264 (1986)), thymocyte-epithelial adhesion (Vollger, et al., (1987)), and the mixed lymphocyte reaction (Martin, P. J., et al., J. Immunol. 131:180-185 (1983)). In addition, a broader role for the CD2 antigen has been suggested by the discovery that certain combinations of anti-CD2-monoclonal antibodies can directly activate mature T cells via an antigen-independent pathway.
An understanding of the molecular interaction between CD2 and LFA-3 or anti-CD2 antibodies would be useful in correlating physiological function with structure. This type of information is useful in designing compounds that can mediate killer T cell or other immune response mechanisms. At present, the most common method for mapping protein epitopes requires the synthesis of an array of short synthetic peptides spanning the protein sequence, and the use of these peptides in multiple binding assays (Geysen, H. M., et al., Science 235:1184-1190 1987)). In order to identify specific residues important for antibody binding, variants of the peptide are synthesized with substitutions at each position. The synthetic peptide strategy has several limitations. If the antibody derives its affinity from interaction with disparate portions of the polypeptide backbone or with a novel conformation of the backbone, the peptide will be unable to mimic the entire protein in binding to the antibody. In order to identify individual residues contacted by the antibody, an extremely large number of peptide variants must be synthesized. The most exhaustive study to date involved the assay of over 1500 individual peptides (Getzoff, E. D., et al., Science 235:1191-1196 (1987)).
Monoclonal antibodies have been used to select against viral envelope determinants (Yewdell, J. W., and Gerhard, W., Ann. Rev. Microbiol. 35:185-206 (1981)). Such selections are both less convenient and less sensitive than desired for the determination of epitope loss mutants because the mutational alterations must be extracted from the viral genome, and mutations leading to viral inviability cannot be detected.
Thus, an efficient method for mapping epitopes or binding domains by identification of epitope or binding domain loss mutants is needed. Such method should utilize an expression host whose viability is not impaired by the introduced mutations. Additionally, the method should permit simple extraction of mutant sequences from the expression host. Further, the method should provide for rapid production, expression and screening of a large number of mutant sequences.
SUMMARY OF THE INVENTION
The present invention relates to a rapid and simple method for mapping protein binding domains including epitopes by identification of substitution mutations that result in the loss of binding capacity. In one embodiment, the rapid mutational analysis technique of the present invention involves the selection of antigen cDNA mutations which lead to a loss of antigen-antibody reactivity. In other embodiments, the rapid mutational analysis technique involves the selection of cDNA encoding protein mutations which lead to a loss of enzyme-substrate or ligand-receptor binding. The method employs cDNA binding domain loss mutants and allows the sampling of a very large number of amino acid substitutions in the native molecule. The mutation frequency is high enough, with the oligonucleotide directed random mutagenesis method used, that rare variants can be efficiently isolated. The technique of the present invention is rapid and simple enough to allow a very large number of mutants to be isolated and can be applied to any surface or intracellular protein for which a cDNA and at least two ligands (e.g., substrates or monoclonal antibodies) are available.
The method of isolating mutant cDNAs encoding protein binding domain loss mutants comprises expressing a vector containing the mutant cDNAs in host cells; exposing the host cell expression products to a negative selection agent (e.g., a first ligand or antibody); discarding those cells containing expression product that binds to the negative selection agent; exposing the host cell expression product to a positive selection agent (e.g., a second ligand or antibody); recovering the host cells whose product binds to the positive selection agent; and recovering vector and cDNA from the recovered host cells. The first ligand or antibody binds to a first binding domain of the native or naturally occurring protein. The second ligand or antibody binds to a second binding domain of the native protein. The selection of host cells which express product that binds only to the positive selection ligand can be accomplished by methods known in the art, e.g., fluorescence activated cell sorting, or by the novel panning method described herein.
The rapid mutation method of the present invention is an oligonucleotide directed random mutagenesis method, in which preselected regions of the cDNA are synthesized with mixtures of wild type and contaminant nucleotide precursors. Preferably, the concentration of said contaminants is selected to produce about one amino acid mutation on average per preselected region and protein. The randomly mutant oligonucleotide is then annealed to a cDNA template, and a duplex cDNA is synthesized. The duplex is amplified under conditions selecting against amplification of the template sequence, and favoring the amplification of the mutant sequence containing the randomly mutant oligonucleotide sequence.
The ability to easily obtain a large number of binding domain loss mutants allows detailed mapping of the accessible surfaces of proteins. The mapping process comprises identifying the nucleotide substitutions by sequencing the cDNAs recovered from host cells expressing product that binds only to the positive selection ligand. The collection of nucleotide substitutions which result in protein binding domain loss for the negative selection ligand define the binding domain for the negative selection ligand.
Using the methods of the present invention, negative selection ligands can be used to identify residues important for ligand or substrate/protein interaction which may occur in pockets inaccessible to antibodies. The identification of ligand binding sites will facilitate structural studies leading to the design of new drugs and ligands that are less antigenic but have greater biological effect.
The method of the present invention has been used to define the regions through which the CD2 antigen binds to anti-CD2 monoclonal antibodies and to define the binding sites on the CD4 antigen for the human immunodeficiency virus (HIV).
CD2 cDNA mutations were selected which lead to loss of CD2-antibody reactivity. The pattern of amino acid substitutions in the mutants defines three distinct regions of the CD2 molecule: one epitopic region rec
Peterson Andrew
Seed Brian
Greenlee Winner and Sullivan PC
Scheiner Laurie
The General Hospital Corporation
LandOfFree
Rapid mutational analysis method and compositions does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Rapid mutational analysis method and compositions, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Rapid mutational analysis method and compositions will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3088528