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Summary for Patent: 5,366,862
|Title:||Method for generating and screening useful peptides|
|Abstract:||The invention allows the generation and screening of a large population of peptides for the presence of peptides which bind a particular macromolecule or macromolecular complex with high affinity, and further allows the favored net synthesis of analyzable quantities of such peptides, by using as the \"trap\" a macromolecule or macromolecular complex for which binding of the peptide is desired. The starting mixture is preferably spiked with a peptide having some affinity for the target macromolecule so that mutation of the spike or \"lead\" peptide is favored. The development of improved binding peptides through scrambling may be dynamically monitored by initially binding the target with an insolubilized ligand, and then looking for an increase in the concentration of the target in the soluble phase as a result of the displacement of the reference ligand by scrambled peptides.|
|Inventor(s):||Venton; Duane L. (Lombard, IL), Hopfinger; Anton J. (Lake Forest, IL), Le Breton; Guy (Oak Park, IL)|
|Assignee:||Receptor Laboratories, Inc. (Chicago, IL)|
|Patent Claims:||1. A method of identifying peptides which bind specifically to a predetermined target, comprising the steps of:
(a) subjecting a mixture initially comprising a starting protein and/or a plurality of starting peptides to scrambling conditions, said scrambling conditions comprising exposure to proteolytic enzymes in a scrambling zone, under which the starting proteins and/or peptides and derivatives thereof in said zone undergo both random degradation into smaller peptide and free amino acid derivatives, and random recombination of the starting proteins and/or peptides, and/or their peptide and amino acid derivatives, into new scrambled peptide derivatives, the component amino acids of the starting mixture being scrambled as a result of the activity of said proteolytic enzymes to generate a diverse population of scrambled peptides of different sequences, said scrambled peptides not being characterized by a predetermined amino acid sequence;
(b) allowing the scrambled peptides to contact a specific predetermined target and to compete with each other to bind therewith to form a specifically bound peptide-target complex;
(c) protecting only those peptides bound to the target from further scrambling by physically removing or withholding the bound peptides from said scrambling zone so that they are no longer exposed to said scrambling conditions, while allowing peptides which were contacted with the target but did not become bound ("rejected peptides") to return to the scrambling zone and be scrambled therein, steps (b) and (c) then being repeated with respect to said scrambled rejected peptides; wherein repeated step (b) includes contact with specific predetermined target and, if already formed, specifically bound peptide-target complex;
(d) recovering the specifically bound peptide from the peptide-target complex; and
(e) sequencing the specifically bound peptide.
2. The method of claim 1 wherein the proteolytic enzymes are selected from the group consisting of papain, pepsin, bromelain, thermolysin, trypsin, pronase, chymotrypsin, subtilisin and dipeptidyl peptidase IV.
3. A method according to claim 1 wherein the protection from further scrambling is provided by interposing a semipermeable membrane between said scrambling reagents and said target, said membrane being permeable to said peptides and impermeable to said target and said scrambling reagents.
4. The method of claim 3 wherein the semipermeable membrane has a permeability cutoff of about 1-3.5 kDa.
5. The method of claim 1 wherein protection from further scrambling is provided by immobilizing the scrambling reagents in a first zone and the targets in a second, spatially separated zone, the unbound peptides generated by the scrambling reaction being allowed to circulate between the first and second zones.
6. The method of claim 1, further comprising monitoring the peptide size distribution from time to time while the scrambling reaction is in progress.
7. The method of claim 6, further comprising adjusting the peptide size distribution during the course of the scrambling reaction by adding more amino acids, peptides or protein, or by diluting the reaction mixture.
8. The method of claim 1 wherein the average length of the peptides of the starting mixture is in the range of 7 to 10 amino acids.
9. The method of claim 1 wherein substantially all of the twenty genetically encoded amino acids are represented in the peptides of the starting mixture.
10. The method of claim 1, wherein the starting mixture is biased in favor of or against certain predetermined amino acids.
11. The method of claim 1, wherein the starting mixture is spiked with a peptide of known sequence and having an affinity of at least about 10.sup.-4 for the target.
12. A method according to claim 1 wherein the target is a receptor involved in a physiological process.
13. The method of claim 1, wherein the scrambled peptides are simultaneously screened for affinity for each of a plurality of different targets.
14. The method of claim 1 wherein the target is a macromolecule or a macromolecular complex.
15. A method according to claim 14 wherein the specific binding macromolecule or macromolecular complex is fibrinogen, sickle cell hemoglobin, collagenase IV, renin, GpII.sub.b III.sub.a or phospholipase A.sub.2.
16. The method of claim 3 wherein said semipermeable membrane defines a scrambling zone and a binding zone, the target is provided in soluble form but cannot pass the membrane, and the binding of the scrambled peptides to the predetermined target in the binding zone is monitored by:
(i) incubating an insolubilized ligand having a known affinity for the target with said target, prior to commencing the scrambling of step (a), and
(ii) whenever it is desired to monitor the binding of the scrambled peptides to the target, sampling the soluble fraction from binding zone and assaying the sample for the presence of the soluble target.
17. The method of claim 1 wherein at least one of the target-binding peptides which is recovered is a peptide which was not a starting peptide or a peptide obtainable solely by fragmentation of a starting peptide or protein.
|Applicant||Tradename||Biologic Ingredient||Dosage Form||BLA||Number||Approval Date||Patent No.||Assignee||Estimated Patent Expiration||Status||Orphan||Source|
|Advance Biofactures||SANTYL||collagenase||VIAL||101995||001||1965-06-04||Try a Free Trial||Receptor Laboratories, Inc. (Chicago, IL)||2011-11-22||RX||search|
|>Applicant||>Tradename||>Biologic Ingredient||>Dosage Form||>BLA||>Number||>Approval Date||>Patent No.||>Assignee||>Estimated Patent Expiration||>Status||>Orphan||>Source|
|Country||Patent Number||Publication Date|
|World Intellectual Property Organization (WIPO)||9112331||Aug 22, 1991|
|World Intellectual Property Organization (WIPO)||9304079||Mar 04, 1993|
|World Intellectual Property Organization (WIPO)||9404558||Mar 03, 1994|
|United States of America||5814460||Sep 29, 1998|
|Japan||H05506774||Oct 07, 1993|
|>Country||>Patent Number||>Publication Date|
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