BIOMARKER DISCOVERY IN COMPLEX BIOLOGICAL FLUID USING BEAD OR PARTICLE BASED LIBRARIES AND DIAGNOSTIC KITS AND THERAPEUTICS

• US 20120270741A1
• Filed: 03/23/2012
• Published: 10/25/2012
• Est. Priority Date: 03/24/2011
• Status: Active Grant

• Current Assignee: VACO Technology AG
• Original Assignee: VACO Technology AG
• Inventors: MOOLA, Muralidhar Reddy, SCHILKE, Jessica
• Granted Patent: US 9,804,168 B2
• Time in Patent Office: Days
• US Class Current: 506/6
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/20 Hypnotics; Sedatives
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C40B 40/04 : Libraries containing only organic compounds
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/12 : for screening libraries
• CPC Class Codes: G01N 2800/104 : Lupus erythematosus [SLE]
• CPC Class Codes: G01N 2800/2821 : Alzheimer
• CPC Class Codes: G01N 33/566 : using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/57438 : of liver, pancreas or kidney
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention is useful in screening for biomarkers associated with any other disease or condition. Such diseases and conditions range from the neurological diseases, autoimmune diseases and cancers identified above as well as any other disease or condition that has a biomarker such as an antibody or other characterizing protein or biomolecule associated with the disease or progression of the disease. The large ligand libraries of the invention can be used directly in biological fluid, under the appropriate experimental conditions and according to the processes recited herein, to screen for such markers and without the need to use fewer support members (e.g. about 100,000 or less) or without the need to transfer such peptoids or ligands to a microarray before screening the biological fluid. In addition, the ligand libraries may also be used to screen for cell based receptors that specifically relate to a particular cell surface marker.

Claims

1. A method of screening a complex biological fluid for biomarkers comprising,

(a) providing a random ligand library comprising a plurality of supports having at least one ligand bound to the support;

(b) exposing the random ligand library to a control complex biological fluid having ligand binding moieties;

(c) removing any supports having ligand binding moieties bound thereto to form a purified ligand library;

(d) screening the purified ligand library against a non-control complex biological fluid having biomarkers;

(e) exposing the solution formed in step (d) to a detection reagent or system to permit the detection of ligands having biomarkers bound thereto and, optionally,

(f) identifying the ligands bound to the biomarkers.

2. The method according to claim 1 wherein the random ligand library is pretreated before step (b).

3. The method according to claim 1 wherein the ligands are selected from peptides, carbohydrates, nucleic acid oligomers, peptoids, α

-substituted peptoids or small molecules.

4. The method according to claim 1 wherein the complex biological fluid is derived from a mammal.

5. The method according to claim 1 wherein the complex biological fluid comprises blood serum or plasma.

6. The method according to claim 5 wherein the blood serum or plasma is diluted.

7. The method according to claim 6 wherein the serum is diluted to a concentration range of between about 500 ug/ml to about 1 ug/mL.

8. The method according to claim 1 wherein the purified ligand library comprises about 0.2 to 2.0 grams per screen.

9. The method according to claim 1 wherein the disease associated biomarker is selected from a protein or cell surface receptor.

10. The method according to claim 1 wherein the disease associated biomarker is selected from an antibody.

11. The method according to claim 1 wherein the detection reagent is selected from a labeled secondary antibody.

12. The method according to claim 1 wherein the random ligand library comprises about 500,000 to about 150 MM supports and about 500,000 to about 150 MM ligands.

13. The method according to claim 1 wherein there is a PEG linker between the support and the random ligand.

14. The method according to claim 13 wherein the PEG linker is less than 10 monomeric units.

15. The method according to claim 2 wherein the support is a bead or resin.

16. The method according to claim 15 wherein the bead is a tentagel bead.

17. The method according to claim 16 wherein the tentagel bead is between about 5 to about 1,000 microns.

18. The method according to claim 17 wherein the tentagel bead having a ligand is pre-treated to a process step comprising exposing said bead to a swelling step.

19. The method according to claim 1 wherein the disease is selected from a cardiovascular disease, an autoimmune disease, cancer or a neurological disease.

20. The method according to claim 1 wherein the disease or condition is selected from an inflammatory disease, a metabolic disease or disorder or an infectious disease.

21. The method according to claim 1 wherein the biomarker is associated with a chemical toxin or environmental contaminant.

22. A method of screening a complex biological fluid for disease associated biomarkers comprising,

(a) providing a large random ligand library comprising a plurality of supports having ligands bound to the support;

(b) exposing the random ligand library to a healthy control complex biological fluid having ligand binding moieties;

(c) removing any supports having ligand binding moieties bound thereto to form a purified ligand library;

(d) exposing the purified ligand library to a non-control complex biological fluid having disease associated biomarkers;

(e) detecting ligands having disease associated biomarkers bound thereto; and

(f) identifying the ligands having disease associated biomarkers bound thereto and, optionally,

(g) purifying and/or characterizing the disease associated biomarker.

23. A method of detecting high affinity ligands for disease associated biomarkers comprising,

(a) providing a large random ligand library comprising a plurality of supports having ligands bound to the support;

(b) exposing the random ligand library to a healthy control complex biological fluid having ligand binding moieties;

(c) removing any supports having ligand binding moieties bound thereto to form a purified ligand library;

(d) exposing the purified ligand library to a non-control complex biological fluid having disease associated biomarkers;

(e) detecting ligands having disease associated biomarkers bound thereto.

24. The method according to claim 23 wherein the large random library comprises between about 150,000 to about 150 MM supports having a ligand bound thereto.

25. The method according to claim 23 wherein the complex biological fluid is diluted to a concentration range of between about 1,000 ug/mL to about 1 ug/mL.

26. The method according to claim 23 wherein the number of distinct detected ligands bound to a biomarker ranges from about 5 to about 100.

27. A method of identifying high affinity ligands for disease associated biomarkers comprising,

(a) providing a large random ligand library comprising a plurality of beads having ligands bound to the bead;

(b) exposing the random ligand library to a healthy control complex biological fluid having ligand binding moieties;

(c) removing any beads having ligand binding moieties bound thereto to form a purified ligand library;

(d) exposing the purified ligand library to a non-control complex biological fluid having disease associated biomarkers;

(e) detecting ligands having disease associated biomarkers bound thereto; and

(f) identifying the ligands having disease associated biomarkers bound thereto.

28. A random ligand library for screening a complex biological fluid comprising a compound of formula I on a support,

29. A random ligand library for screening a complex biological fluid according to claim 28 comprising a compound of formula I on a support,

30. The ligand library according to claim 29 comprising about 200,000 to about 150 MM distinct ligands.

31. The ligand library according to claim 30 wherein the support is selected from a bead or resin.

32. The ligand library according to claim 31 wherein the bead or resin comprises a PEG linker of less than 10 monomeric units.

33. A compound of formula X

34. The compound according to claim 33 wherein R₄ is selected from -n-butylamine.

35. The compound according to claim 34 wherein R₁ is selected from —

CH₂CH₂SCH3 or —

CH₂SH.

38. The compound according to claim 35 wherein said compound of formula II is selected from the group having R₉-R₁₆ as

(a) R₉ is n-butylamine;

R₁₀ is 1-yl-n-butylamine;

R₁₁ is piperonyl;

R₁₂ is methylbenzyl;

R₁₃ is piperonyl;

R₁₄ is methylbenzyl;

R₁₅ is 1-yl-2-(4(benzenesulfonamide)ethyl and R₁₆ is 1-yl-2-methoxyethyl;

(b) R₉ is 1-yl-n-butylamine;

R₁₀ is 1-yl-2-(4(benzenesulfonamide)ethyl;

R₁₁ is 1-yl-n-butylamine;

R₁₂ is cyclohexyl;

R₁₃ is 1-yl-2-methoxyethyl;

R₁₄ is 1-yl-2,2-dimethylethyl (isobutyl);

R₁₅ is 1-yl-2-(4(benzenesulfonamide)ethyl and R₁₆ is methylbenzyl;

(c) R₉ is 1-yl-n-butylamine;

R₁₀ is 1-yl-n-butylamine;

R₁₁ is piperonyl;

R₁₂ is 1-yl-2-(4(benzenesulfonamide)ethyl;

R₁₃ is 1-yl-n-butylamine;

R₁₄ is methylbenzyl;

R₁₅ is methylbenzyl and R₁₆ is cyclohexyl;

(d) R₉ is piperonyl;

R₁₀ is 1-yl-n-butylamine;

R₁₁ is isobutyl;

R₁₂ is 1-yl-2-(4(benzenesulfonamide)ethyl;

R₁₃ is methylbenzyl;

R₁₄ is cyclohexyl;

R₁₅ is isobutyl and R₁₆ is 1-yl-n-butylamine;

(e) R₉ is 1-yl-n-butylamine;

R₁₀ is 1-yl-n-butylamine;

R₁₁ is methylbenzyl;

R₁₂ is 1-yl-2-methoxyethyl;

R₁₃ is cyclohexyl;

R₁₄ is cyclohexyl;

R₁₅ is methylbenzyl and R₁₆ is piperonyl;

(f) R₉ is piperonyl;

R₁₀ is 1-yl-n-butylamine;

R₁₁ is isopropyl;

R₁₂ is isopropyl;

R₁₃ is 1-yl-2-methoxyethyl;

R₁₄ is cyclohexyl;

R₁₅ is 1-yl-2-(4(benzenesulfonamide)ethyl and R₁₆ is 1-yl-2-(4(benzenesulfonamide)ethyl

and pharmaceutically acceptable salts thereof.

36. A compound having a formula Ia

37. A compound of the formula:

39. A screening kit for screening for the presence of disease associated biomarkers in a patient complex biological fluid having or suspected of having Alzheimer'"'"'s disease comprising at least one peptoid according to claim 37 and a detection platform.

40. The screening kit according to claim 39 wherein the kit comprises a bead based platform or a microarray platform or an ELISA platform.

41. The screening kit according to claim 40 wherein the bead based platform is selected from a TentaGel platform or a Luminex platform.

42. The screening kit according to claim 40 wherein the concentration range of the peptoid in a test solution ranges from 0.1 mM to about 20 mM.

43. The screening kit according to claim 40 wherein the complex biological fluid is diluted in a range of between about 1:

100 and about 1;

50,000 fluid/diluent.

44. The screening kit according to claim 41 wherein the complex biological fluid is diluted to a concentration range of between about 10 to about 200 ug/mL in a diluent.

45. The screening kit according to claim 44 wherein the TentaGel beads have a micron diameter range of between about 10 um and 50 um.

46. The screening kit according to claim 45 wherein the TentaGel beads have a PEG linker linked to the peptoid.

47. A method of screening for the presence or absence of biomarkers associated with Alzheimer'"'"'s disease in a complex biological fluid comprising contacting the complex biological fluid with at least one peptoid according to claim 37 and detecting the presence or absence of binding of said biomarker in the complex biological fluid.

52. A screening kit for screening for the presence of disease associated biomarkers in a patient complex biological fluid having or suspected of having pancreatic cancer comprising at least one peptoid according to claim 37 and a detection platform.

53. The screening kit according to claim 52 wherein the kit comprises a bead based platform or a microarray platform or an ELISA platform.

54. The screening kit according to claim 53 wherein the bead based platform is selected from a TentaGel platform or a Luminex platform.

55. The screening kit according to claim 53 wherein the concentration range of the peptoid in a test solution ranges from 0.1 mM to about 20 mM.

56. The screening kit according to claim 53 wherein the complex biological fluid is diluted in a range of between about 1:

100 and about 1;

50,000 fluid/diluent.

57. The screening kit according to claim 54 wherein the complex biological fluid is diluted to a concentration range of between about 10 to about 200 ug/mL in a diluent.

58. The screening kit according to claim 54 wherein the TentaGel beads have a micron diameter range of between about 10 um and 50 um.

59. The screening kit according to claim 54 wherein the TentaGel beads have a PEG linker linked to the peptoid.

63. A screening kit for screening for the presence of disease associated biomarkers in a patient complex biological fluid having or suspected of having SLE (Lupus) comprising at least one peptoid according to claim 37 and a detection platform.

64. The screening kit according to claim 63 wherein the kit comprises a bead based platform or a microarray platform or an ELISA platform.

65. The screening kit according to claim 64 wherein the bead based platform is selected from a TentaGel platform or a Luminex platform.

66. The screening kit according to claim 64 wherein the concentration range of the peptoid in a test solution ranges from 0.1 mM to about 20 mM.

67. The screening kit according to claim 64 wherein the complex biological fluid is diluted in a range of between about 1:

100 and about 1;

50,000 fluid/diluent.

68. The screening kit according to claim 65 wherein the complex biological fluid is diluted to a concentration range of between about 10 to about 200 ug/mL in a diluent.

69. The screening kit according to claim 68 wherein the TentaGel beads have a micron diameter range of between about 10 um and 50 um.

70. The screening kit according to claim 68 wherein the TentaGel beads have a PEG linker linked to the peptoid.

71. A method of screening for the presence or absence of biomarkers associated with SLE (Lupus) in a complex biological fluid comprising contacting the complex biological fluid with at least one peptoid according to claim 37 and detecting the presence or absence of binding of said biomarker in the complex biological fluid.

48. A compound of the formula:

49. A compound of the formula:

60. A method of screening for the presence or absence of biomarkers associated with pancreatic cancer in a complex biological fluid comprising contacting the complex biological fluid with at least one peptoid according to claim 49 or 51 and detecting the presence or absence of binding of said biomarker in the complex biological fluid.

50. A compound of the formula:

51. A compound of the formula:

61. A compound of the formula:

62. A compound of the formula: