Light-emitting fusion proteins and diagnostic and therapeutic methods therefor

• US 7,919,274 B2
• Filed: 03/19/2002
• Issued: 04/05/2011
• Est. Priority Date: 03/20/2001
• Status: Expired due to Term

• Current Assignee: Dana-Farber Cancer Institute, Howard Hughes Medical Institute
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Kim, Tae-You, Kaelin, William G. Jr., Livingston, David M.
• Primary Examiner(s): GABEL, GAILENE
• Application Number:
  US10/101,662
• Publication Number:
  US 20030022198A1
• Time in Patent Office: 3,304 Days
• Field of Search: 435/7.72, 435/7.8, 435/7.9, 435/8, 435/29, 435/68.1, 435/69.7, 435/69.8, 435/183, 435/115, 435/212, 435/219, 435/968, 435/91.52, 435/69.9, 436/172, 436/800, 424/9.34, 424/9.6, 424/9.61, 424/94.5, 424 9463- 9467, 530/356, 530/387.3, 530/387.7
• US Class Current: 435/69.7
• CPC Class Codes: A61K 49/0045 the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
• CPC Class Codes: A61K 49/0047 Green fluorescent protein [GFP]
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 2333/90245 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Light-generating fusion proteins having a ligand binding site and a light-generating polypeptide moiety and their use as diagnostics, in drug screening and discovery, and as therapeutics, are disclosed. The light-generating fusion protein has a feature where the bioluminescence of the polypeptide moiety changes upon binding of a ligand at the ligand binding site. The ligand may be, for example, an enzyme present in an environment only under certain conditions, e.g., ubiquitin ligase in a hypoxic state, such that the light-generating fusion protein is “turned on” only under such conditions.

Claims

1. A light-generating fusion protein comprising a ligand binding site and a light-generating polypeptide moiety, wherein said ligand binding site is a mammalian HIF1α

polypeptide moiety comprising the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α

, wherein a) residue 402 is hydroxylated proline, b) residue 564 is hydroxylated proline, or c) both residue 402 and residue 564 are hydroxylated proline; and

wherein said light-generating polypeptide moiety is luciferase, and wherein said ligand is pVHL, whereupon ligand binding to said ligand binding site alters the luminescence of said light-generating polypeptide moiety.

2. The light-generating fusion protein of claim 1, further comprising a targeting moiety.

3. A light-generating fusion protein comprising a ligand binding site and a light-generating polypeptide moiety, wherein said ligand binding site is a mammalian HIF1α

polypeptide moiety,

wherein said light-generating polypeptide moiety is luciferase, and wherein said ligand is pVHL, wherein said ligand binding site comprises the amino acid sequence Y-X₁-Leu-X₂-Pro_h-X₃-X₄-X₅-X₆-Y′

(SEQ ID NO;

     12), wherein

Pro_h is hydroxylated proline;

X₁, X₂, X₃, X₄, X₆, and X₆ are amino acids selected so as to not modify or alter VHL binding properties, wherein X₁, X₂, X₄, X₆, and X₆ are independently Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, or Trp, and wherein X₃ is Ser, Thr, or Tyr; and

Y and Y′

are independently present or absent and, if present, independently comprise a peptide having from 1 to 600 amino acids; and

wherein a) the amino acid at position 402 when numbered in accordance with wild type HIF1α

is hydroxylated proline, b) the amino acid at position 564 when numbered in accordance with wild type HIF1α

is hydroxylated proline, or c) both the amino acid at position 402 and the amino acid at position 564 when numbered in accordance with wild type HIF1α

are hydroxylated proline; and

whereupon ligand binding to said ligand binding site alters the luminescence of said light-generating polypeptide moiety.