Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-12-05
2004-09-21
Celsa, Bennett (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S161000, C436S518000, C436S528000, C530S413000
Reexamination Certificate
active
06794148
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the integrated, multi-dimensional, rapid analysis of solutions of a large number of mixed molecular species, commonly called “libraries”. More specifically, the invention is directed toward methods for the discovery of molecular entities useful in a variety of biological contexts using hi-flux screening of natural and synthetic libraries to select ligands having a desired affinity for a target molecule of interest.
BACKGROUND
Multi-dimensional systems, i.e., systems involving the application of multiple distinct physico-chemical separation steps, are (n)own to be useful for many applications. The purification of proteins, for example, frequently is accomplished using multiple passes through different chromatographic columns exploiting differential partitioning such as adsorption and size exclusion. Inherent in any multi-dimensional process is the necessity to identify the desired component, from the output of the first partition, to collect it, and to introduce it into the next dimension of the system. Disadvantages of such systems include slow analysis, solvent incompatibility between successive partitioning phases, the necessity of labor intensive handling, and consequent contamination or loss of sample.
The recent prior art discloses various new methods for implementing the search for novel agents such as, for example, pharmacological or therapeutic agents (i.e., drug discovery) agents useful in animal care or management, agriculturally useful chemicals, selective biocides for insects, weeds, or other pests, and catalytic and other entities useful in industrial processes. Collections of molecules or “libraries” are prepared and screened for molecules having a specified bioactivity, as indicated initially by detection of binding between one or more species or “ligands” in the library and a “target” molecule with which it reacts to influence some biological process. More specifically, libraries consist of a complex assortment of molecules containing one or more ligands which may bind to a target of interest. The identification of ligands which bind may provide a lead for identifying compounds with a desired biological activity, e.g., as a potential drug candidate. As methods have become available to screen these complex mixtures more effectively, interest in exploiting this new “rational design” or “directed molecular evolution” approach has increased.
Libraries of biopolymers may be prepared by the sequential synthesis based on randomized addition of amino acid, nucleotide, or sugar residues, or combinations thereof, to form peptides, RNAs, polysaccharides, glycosaminoglycans or the like, thereby to prepare a random mixture of oligomers. Techniques suitable for preparing protein or peptide libraries at the nucleic acid level by phage display and similar technologies also are known. Likewise, these general synthesis approaches could be adapted to prepare, peptide nucleic acid (PNA) libraries, or libraries of PNA/DNA, or PNA/RNA chimeras, and indeed other complex mixtures of synthetic molecules.
Screening of soluble peptide libraries frequently is performed either by immunoassay or by laboriously assaying for a particular biological function (e.g. blocking of viral replication). These methods are not necessarily target based and in most cases, involve tedious set up. See Scott and Craig, Curr. Opin. Biotech. 5, 40-48 (1994); Dooley et. al.
Proc. Natl. Acad. Sci
., 90:10811-10815 (1993); Dooley et. al.,
Life Sciences
, 92:1509-1517 (1990); Houghton et. al.,
Biorg. Med. Chem. Lett
., 3:405-412 (1993). For example, inhibitors of HIV protease have been identified by screening sets of equimolar peptide mixtures, together containing more than 240,000 soluble tetrapeptides. See Owens et. al.
Biochem. Biophys. Res. Comm
., 181:402-408 (1991). It has also been suggested to use a phosphopeptide library to determine the sequence specificity of the peptide-binding sites of SH2 domains by employing the GST-SH2 fusion protein immobilized onto a column. See Songyang et al. Cell, 72, 767-778 (1993).
The screening methods described immediately above are based upon identifying which ligand in a mixture binds to a target of interest. Binding typically is assayed with either the ligands of the library or the target immobilized on some form of solid support. Various solution parameters may be adjusted to emulate different binding conditions and to obtain different ligands. Often, peptides which are obtained through procedures involving their immobilization to a support have disappointing affinity, i.e., have a binding constant too low to be useful. Traditionally, antibodies are used for the affinity purification of proteins and other biomolecules. However, the cost of generating antibodies, the potential for antibody leaching, and the need for relatively harsh eluting conditions pose problems for the routine use of antibodies in affinity purification.
Screening methods known in the art thus are not entirely satisfactory. Prior methods for detecting or identifying ligands which bind to a target of interest often fail to provide ligands of sufficiently high affinity to be useful, and additionally suffer from the loss of sample, the need for large amounts of ligands, and the need to vary loading, binding, or elution conditions to obtain useful results. Additionally, existing systems are unable selectively to screen a library while simultaneously determining the affinity of selected ligand(s) for the target under relevant conditions.
A major hurdle in the exploitation of current screening techniques of the type described above is effective chemical characterization of ligands identified in these processes. Chemical characterization, e.g., determining the sequence of an identified biopolymer, is at best time-consuming and complex. A major focus of prior art screening techniques is to enable the collection of enough of or enough information about a ligand of interest so as to permit determination of its structure and to enable synthesis of larger amounts for testing and further empirical structural refinement.
Accordingly, there is a need for integrated, multi-dimensional screening, selection and analysis systems and methods which permit automated, direct transfer of samples without dilution or loss between various dimensions, and efficiently screen for, and subsequently permit characterization and recovery of ligands to a target of interest, even when present at low concentration.
SUMMARY OF THE INVENTION
Accordingly, the present invention is directed to rapid, efficient and automated, multi-dimensional systems, methods and apparatus for screening libraries to select, recover and characterize a candidate ligand with a desired or preselected affinity K for a preselected target molecule. Additionally, the present invention is directed to certain combination of individual dimensions of such a system, which can be used to obtain a desired result, and, specifically to a method of detecting a ligand to a target of interest which overcomes the disadvantages of the methods known in the art.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description and drawing, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the process particularly pointed out in the written description, drawing, and appended claims.
To achieve these and other advantages, and in accordance with the invention as embodied and broadly described, the invention provides novel methods for screening a sample to select a ligand to a target of interest and for obtaining information about the ligand and its binding characteristics. Specifically, the claimed multi-dimensional methods involve combining a solution of heterogeneous ligands with the target of interest to screen the ligands on the basis of one or more binding characteristics. Ligands having the first binding characteristic will bind to the target of interest th
Afeyan Noubar
Evans David
Jindal Satish
Paliwal Sandeep
Pingali Aruna
Celsa Bennett
PerSeptive Biosystems Inc.
Testa Hurwitz & Thibeault LLP
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