Claims for Patent: 6,824,986

Summary for Patent: 6,824,986

Title:	Methods for measuring in vivo cytokine production
Abstract:	The present invention involves techniques for evaluating in vivo cytokine production through the in vivo capture of secreting cytokines by labeled cytokine-binding reagents, followed by in vitro measurement of serum levels of captured cytokine. The methods of the present invention make use of the ability of a neutralizing antibody to a cytokine, when injected into a person or expierimental animal, to bind that cytokine and prevent its catabolism, excretion, or binding to a cytokine receptor. This causes the cytokine, which may normally have a very short in vivo half life, to accumulate in vivo as a cytokine/anti-cytokine antibody complex. If the anti-cytokine antibody is either labeled with a molecule that can be bound by another molecule (e.g.; biotin, which is bound by avidin or streptavidin), or is itself capable of being bound by another molecule (e.g.; a rat anti-cytokine antibody could be bound by an anti-rat immunoglobulin antibody), and the cytokine can also be bound by an antibody that recognizes a site distinct on the cytokine molecule from the site bound by the injected, neutralizing antibody, than the concentration of the cytokine/anti-cytokine complex in serum or other biological fluid can easily be assayed by a modified ELISA. This assay may be used with target analytes other than cytokines, which may include hormones, drugs or other analytes in a human or aninial. The target analyte is preferably a macromolecule, more preferably a protein, and most preferably a cytokine.
Inventor(s):	Finkelman; Fred D. (Cincinnati, OH), Morris; Suzanne C. (Mason, OH)
Assignee:	University of Cincinnati (Cincinnati, OH)
Application Number:	09/167,088
Patent Claims:	1. A method of measuring the production of a secreted target analyte of interest in a human or animal, comprising the steps of: a. injecting the human or animal with an amount of labeled neutralizing targeting moiety, wherein the targeting moiety binds specifically to the target analyte, and wherein the targeting moiety is injected in sufficient quantity that a measurable fraction of target analyte is bound by the labeled neutralizing targeting moiety; b. allowing the targeting moiety to circulate through the injected human or animal for a defined period of time sufficient to bind to the target analyte of interest and form a targeting moiety:target analyte conjugate wherein the formation of the targeting moiety:target analyte conjugate decreases the clearing rate of the target analyte; c. obtaining a sample of blood from the human or animal after the defined period of time; d. combining the sample of blood with a capture moiety wherein the capture moiety binds specifically to the targeting moiety:target analyte conjugate in order to form an assay mixture; e. incubating the assay mixture of step d to allow the capture moiety to bind to the targeting moiety:target analyte conjugate and form targeting moiety:target analyte:capture moiety complexes in the assay mixture; f. removing any unbound and unconjugated targeting moiety and target analyte from the assay mixture; g. detecting the amount of labeled targeting moiety:target analyte:capture moiety complexes; wherein the amount of labeled targeting moiety:target analyte:capture moiety complexes detected provides a measure of the production of secreted target analyte in the sample during the defined period of time; and wherein the secreted target analyte is a secreted cytokine, secreted peptide or secreted protein hormone. 2. The method of claim 1, wherein the defined period of time is from about 1 hour to about 72 hours. 3. The method of claim 2, wherein the target analyte is a cytokine. 4. The method of claim 3, wherein the cytokine is selected from the group consisting of interleukins, interferons chemokines, growth factors, colony stimulating factors, lymphokines, lymphotoxins, and tumor necrosis factors. 5. The method of claim 3, wherein the cytokine is selected from the group consisting of interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interferon-alpha, interferon-beta, interferon-gamma, lymphotoxin, tumor necrosis factor-alpha, transforming growth factor (TGF)-beta, granulocyte macrophage-colony stimulating factor (GM-CSF), nerve growth factor (NGF), and epidermal growth factor (EGF). 6. The method of claim 1, wherein the blood is selected from the group consisting of whole blood, serum and plasma. 7. The method of claim 1, wherein the targeting moiety is selected from the group consisting of antibodies, soluble receptors, and recombinant molecules with binding sites for the target analyte. 8. The method of claim 7, wherein the targeting moiety is a monoclonal antibody. 9. The method of claim 8, wherein the targeting moiety is detectably labeled, wherein the label is selected from the group consisting of radioisotopes, affinity labels, enzymatic labels, and fluorescent labels. 10. The method of claim 9, wherein the targeting moiety is labeled by linking to a fluorescent labeling compound. 11. The method of claim 10, wherein the fluorescent labeling compound is selected from the group consisting of fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. 12. The method of claim 9, wherein the labeled targeting moiety comprises first and second members of a complimentary ligand/anti-ligand pair, wherein the first member of the complimentary ligand/anti-ligand pair is injected as the targeting moiety in step (a); wherein the second member of the complimentary ligand/anti-ligand pair is a detectable binding partner to the first member; and wherein the method fuither comprises the steps of (I) contacting the assay mixture after step (e) and before step (f) with the second member of the complimentary ligand/anti-ligand pair to allow binding of the first and second members; (II) removing any unbound second member; (III) detecting the amount of bound second member; and (IV) correlating the detected amount to the amount of targeting moiety:target analyte:capture moiety complexes in the assay mixture; wherein the amount of targeting moiety:target analyte:capture moiety complexes detected provides a measure of the production of secreted target analyte during the defined period of time. 13. The method of claim 12, wherein the first member of the complimentary ligand/anti-ligand pair is a monoclonal antibody. 14. The method of claim 13, wherein the capture moiety is an antibody. 15. The method of claim 14, wherein the capture moiety is a polyclonal antibody. 16. The method of claim 12, wherein the targeting moiety:target analyte:capture moiety complexes are detected by radioimmunoassay. 17. The method of claim 12, wherein the second member of the complimentary ligand/anti-ligand pair is detectably labeled by an enzymatic label. 18. The method of claim 17, wherein the label is a small molecule hapten. 19. The method of claim 18, wherein the hapten is biotin. 20. The method of claim 17, wherein the second member of the complimentary ligand/anti-ligand pair is selected from the group consisting of streptavidin, anti-biotin antibody, anti-hapten antibody, and anti-immunoglobulin antibody. 21. The method of claim 17, wherein the enzyme is selected from the group consisting of alkaline phosphatase, glucose oxidase, beta-galactosidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, asparaginase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. 22. The method of claim 12, wherein the second member of the complimentary ligand/anti-ligand pair is labeled with a fluorescent label. 23. The method of claim 22, wherein the fluorescent labeling compound is selected from the group consisting of fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. 24. The method of claim 1, wherein the capture moiety is an antibody. 25. The method of claim 24, wherein the antibody is a polyclonal antibody which recognizes many epitopes on the target analyte. 26. The method of claim 1, wherein the targeting moiety is labeled with a small molecule hapten and wherein the method fuirther comprises the step of binding the small molecule hapten to a binding partner which is conjugated to an enzyme. 27. The method of claim 26, wherein the hapten is biotin. 28. The method of claim 26, wherein the enzyme-conjugated binding partner is selected from the group consisting of streptavidin, anti-biotin antibody, anti-hapten antibody, and anti-immunoglobulin antibody. 29. The method of claim 26, wherein the enzyme is selected from the group consisting of alkaline phosphatase, glucose oxidase, beta-galactosidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, asparaginase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. 30. The method of claim 1 or 12, wherein the capture moiety is immobilized on a solid phase support.

Details for Patent 6,824,986

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Recordati Rare Diseases, Inc.	ELSPAR	asparaginase	For Injection	101063	01/10/1978		2017-10-06