WO2002047537A2 - Compositions et procedes de representation in situ et in vivo de cellules et de tissus - Google Patents

Compositions et procedes de representation in situ et in vivo de cellules et de tissus Download PDF

Info

Publication number
WO2002047537A2
WO2002047537A2 PCT/US2001/048157 US0148157W WO0247537A2 WO 2002047537 A2 WO2002047537 A2 WO 2002047537A2 US 0148157 W US0148157 W US 0148157W WO 0247537 A2 WO0247537 A2 WO 0247537A2
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
imaging
pharmaceutical formulation
image
chimeric molecule
Prior art date
Application number
PCT/US2001/048157
Other languages
English (en)
Other versions
WO2002047537A3 (fr
Inventor
Arul M. Chinnaiyan
Alnawaz Rehemtulla
Brian D. Ross
Original Assignee
Regents Of The University Of Michigan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regents Of The University Of Michigan filed Critical Regents Of The University Of Michigan
Priority to AU2002232568A priority Critical patent/AU2002232568A1/en
Priority to EP01992094A priority patent/EP1501476A2/fr
Publication of WO2002047537A2 publication Critical patent/WO2002047537A2/fr
Publication of WO2002047537A3 publication Critical patent/WO2002047537A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo

Definitions

  • compositions and methods for imaging cells, tissues and organs in vivo and in situ hi particular, compositions and methods are provided to enhance the imaging of cells and tissues by, e.g., computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or bioluminescence imaging (BLI).
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • BBI bioluminescence imaging
  • the invention provides compositions and methods for imaging normal and abnormal tissues, including, e.g., sites of primary and metastatic tumors and tumor neovasculature.
  • Neovascularization is essential for tumor growth and metastasis. It is now widely held that acquisition of an angiogenic phenotype is an early event in tumorigenesis, allowing tumors to grow beyond the size otherwise limited by the diffusion of oxygen and other nutrients through tissue. The identification of families of endogenous pro- and anti- angiogenic factors has fueled great excitement about therapeutic approaches aimed at interrupting tumor blood supply. Furthermore, proliferating endothelial cells forming tumor neovasculature express a number of proteins that are not found in mature vessels. These include endothelial cell surface alpha v integrins, which tightly bind peptides containing the Arg-Gly-Asp (RGD) motif.
  • RGD Arg-Gly-Asp
  • tumor neovasculature endothelium can express tumor-specific molecules (see, e.g., Pasqualini (1997) Nature Biotechnol. 15:542- 546). Phage displaying RGD peptides on their surface can selectively accumulate in tumor blood vessels and RGD peptides can inhibit metastasis in vivo (see, e.g., Fujii (1995) Biol. Pharm. Bull. 18:1681-1688; Saiki (1990) Jpn. J. Cancer Res. 81:1003-1011).
  • the invention provides compositions and methods for imaging cells and tissues in vivo and in situ; which can be particularly useful for imaging normal and abnormal tissue and organs, including sites of primary and metastatic tumors and tumor neovasculature.
  • the invention provides a chimeric molecule comprising a first domain comprising a fluorescent, bio luminescent or chemiluminescent polypeptide, or a heterologous (i.e., non-endogenous) kinase, and a second domain comprising a member selected from the group consisting of an RGD motif-comprising polypeptide; a selectin-binding polypeptide; a matrix metalloproteinase (MMP)-binding polypeptide, and a chondroitin sulfate proteoglycan-binding polypeptide.
  • the fluorescent or chemiluminescent polypeptide is luciferase, aequorin, obelin, mnemiopsin or berovin or equivalents or combinations thereof.
  • the heterologous (non-endogenous) kinase can be a non-mammalian kinase, e.g., a herpes simplex virus- 1 thymidine kinase (HSN-1 TK).
  • HSN-1 TK herpes simplex virus- 1 thymidine kinase
  • the RGD motif-comprising polypeptide (and thus the chimeric molecule) specifically binds to a cell-specific polypeptide, a tissue-specific polypeptide or an organ-specific polypeptide.
  • the cell-specific polypeptide can be a tumor- specific polypeptide.
  • the cell-specific polypeptide can be expressed on tumor neovasculature, including vascular endothelium.
  • the RGD motif-comprising polypeptide can comprise an integrin polypeptide or fragment thereof.
  • the RGD motif-comprising polypeptide can comprise an CDCRGDCFC (SEQ ID NO: 1) amino acid sequence.
  • the selectin can be an E-selectin.
  • the selectin binding polypeptide can comprise an IELLQAR (SEQ ID NO:2) amino acid sequence.
  • the matrix metalloproteinase is a gelatinase A or a gelatinase B.
  • the matrix metalloproteinase binding polypeptide can comprise an CTTHWGFTLC (SEQ ID NO:3) amino acid sequence.
  • the chondroitin sulfate proteoglycan comprises a high molecular weight (MW) human melanoma-associated antigen.
  • the chondroitin sulfate proteoglycan binding polypeptide can comprise a TAASGNRSMH (SEQ ID ⁇ O:4) or LTLRWVGLMS (SEQ ID NO:5) sequence.
  • the chimeric molecule is capable of specifically binding to a lumen-expressed vascular endothelial cell composition, such as a protein, carbohydrate, proteoglycan; the composition may be expressed exclusively during tumor neovascularization.
  • a lumen-expressed vascular endothelial cell composition such as a protein, carbohydrate, proteoglycan
  • the invention provides a pharmaceutical formulation comprising a chimeric molecule and a pharmaceutically acceptable excipient, wherein the chimeric molecule comprises a first domain comprising a fluorescent, bioluminescent or chemiluminescent polypeptide, or a kinase (e.g., a heterologous kinase), and a second domain comprising a member selected from the group consisting of an RGD motif-comprising polypeptide; a selectin-binding polypeptide; a matrix metalloproteinase (MMP)-binding polypeptide, and a chondroitin sulfate proteoglycan-binding polypeptide, wherein the formulation is suitable for administration as an imaging enhancing agent (e.g., for intravenous injection) and the chimeric molecule is present is an amount sufficient to enhance a computer assisted tomography (CAT) image, a magnetic resonance spectroscopy (MRS) image, a magnetic resonance imaging (MRI) image, a positron emission
  • the invention provides a pharmaceutical formulation comprising a composition comprising a first domain comprising an imaging enhancing agent and a second domain comprising a polypeptide that binds to a cell, a tissue or an organ in a cell-, tissue-, or organ-specific manner, and, a pharmaceutically acceptable excipient suitable for administration as an imaging enhancing agent, wherein composition is not an antibody and is present is an amount sufficient to enhance a computer assisted tomography (CAT) image, a magnetic resonance spectroscopy (MRS) image, a magnetic resonance imaging (MRI) image, a positron emission tomography (PET) image, or a single-photon emission computed tomography (SPECT) image, bioluminescence imaging (BLI) or equivalent, when the pharmaceutical formulation is administered to an individual, or applied to a tissue or organ in situ, in a sufficient amount.
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single
  • the image enhancing agent comprises a kinase, e.g., a heterologous kinase.
  • the kinase can comprise a herpes simplex virus- 1 thymidine kinase (HSN-1 TK).
  • the imaging enhancing agent comprises a bioluminescent or chemiluminescent polypeptide and the composition is a chimeric recombinant protein.
  • the bioluminescent or chemiluminescent compound can comprise a luciferase, an aequorin, an obelin, a mnemiopsin, a berovin, a phenanthridinium ester, or an equivalent thereof or a combination thereof.
  • the imaging- enhancing agent of the pharmaceutical formulation can comprise a radioactive isotope, such as 131 1, 125 I, 123 1, 18 F, U C, 75 Br, 76 Br, 19 F, 13 C, 14 C or 3 H.
  • a radioactive isotope such as 131 1, 125 I, 123 1, 18 F, U C, 75 Br, 76 Br, 19 F, 13 C, 14 C or 3 H.
  • the imaging- enhancing agent can comprise a paramagnetic compound, such as a polypeptide chelated to a metal, e.g., a metalloporphyrin.
  • the paramagnetic compound can also comprise a monocrystalline nanoparticle, e.g., a nanoparticle comprising a lanthanide
  • the paramagnetic compound can comprise a neodymium iron oxide (NdFeO 3 ) or a dysprosium iron oxide (DyFeO 3 ).
  • the imaging- enhancing agent can comprise a synthetic compound, a peptidomimetic or a peptide (as used herein, the term polypeptide includes peptidomimetic and a peptide, as discussed below).
  • the compound of the second domain can comprise a member selected from the group consisting of an RGD motif- comprising polypeptide; a selectin-binding polypeptide; a matrix metalloproteinase (MMP)- binding polypeptide, and a chondroitin sulfate proteoglycan-binding polypeptide.
  • RGD motif- comprising polypeptide a selectin-binding polypeptide
  • MMP matrix metalloproteinase
  • the pharmaceutical formulation of the invention can further comprise a cytotoxic agent, such as an antitumor agent, an anti-angiogenic agent or a therapeutic radionuclide.
  • the pharmaceutical composition can further comprise a lipid, such as a liposome.
  • the pharmaceutically acceptable excipient of the pharmaceutical formulation can be a buffered saline, or equivalent.
  • the concentration of the composition in the pharmaceutical formulation is about 1 to about 1000 ⁇ g/ml, or, about 10 to about 100 ⁇ g/ml.
  • the pharmaceutical formulation of the invention can further comprise a substrate for the bioluminescent or chemiluminescent polypeptide (e.g., an enzyme, such as luciferase) or the heterologous kinase (in one aspect the substrate is altered by the enzyme or kinase; it is the altered molecule that is detected).
  • a substrate for the bioluminescent or chemiluminescent polypeptide e.g., an enzyme, such as luciferase
  • the heterologous kinase in one aspect the substrate is altered by the enzyme or kinase; it is the altered molecule that is detected.
  • the chemiluminescent polypeptide can be luciferase and the substrate is luciferin.
  • the invention also provides a nucleic acid encoding a chimeric polypeptide of the invention.
  • the nucleic acid comprises an open reading frame operably linked to a promoter, wherein the open reading frame encodes a chimeric polypeptide of the invention.
  • the invention also provides an expression vector comprising a nucleic acid encoding a chimeric polypeptide of the invention.
  • the invention provides a cell, e.g., a transformed or a viral vector-infected cell, comprising a nucleic acid encoding a chimeric polypeptide of the invention.
  • the cell can be a bacterial, a yeast, an insect, or a mammalian cell.
  • the invention provides a recombinant chimeric polypeptide produced by a cell of the invention.
  • the recombinant polypeptide can be isolated to varying degrees.
  • the invention also provides a method for in situ or in vivo imaging of a cell, a tissue, an organ or a full body comprising administration of a pharmaceutical formulation of the invention in an amount sufficient to enhance the image, wherein the image is generated by a computer assisted tomography (CAT) image, a magnetic resonance spectroscopy (MRS) image, a magnetic resonance imaging (MRI) image, a positron emission tomography (PET) image, a single-photon emission computed tomography (SPECT) image, a bioluminescence image (BLI) or equivalent.
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • BBI bioluminescence image
  • the invention also provides a method for in situ or in vivo imaging of a cell, a tissue, an organ or a full body comprising the following steps: (a) providing a pharmaceutical formulation of the invention (b) providing an imaging device, wherein the imaging device is computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), bioluminescence image (BLI) or equivalent; (c) administering the pharmaceutical formulation in an amount sufficient to generate the cell, tissue or body image; and, (d) imaging the distribution of the pharmaceutical formulation of step (a) with the imaging device, thereby imaging the cell, tissue or body.
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • BLI bioluminescence image
  • the pharmaceutical formulation is administered to a human, such as a cancer patient or a patient suspected of having or being screened for cancer.
  • the tissue can be a tumor tissue, such as a solid tumor, a metastasis or the neovasculature of a tumor.
  • the pharmaceutical formulation can be administered intravenously.
  • the image can be taken between about 2 minutes and about 24 hours after administering the pharmaceutical formulation.
  • the methods of the invention can further comprise providing a reagent (a substrate) for the bioluminescent or chemiluminescent polypeptide or the heterologous kinase, and administering the reagent with or after administration of the bioluminescent or chemiluminescent polypeptide or the heterologous kinase.
  • the chemiluminescent polypeptide can be luciferase and the reagent luciferin.
  • the invention provides a method for in vivo imaging tumor neovasculature in an individual comprising the following steps: (a) providing a pharmaceutical formulation of the invention; (b) providing an imaging device, wherein the imaging device is computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), bioluminescence image (BLI) or equivalent; (c) administering the pharmaceutical formulation in an amount sufficient to image the tumor neovasculature; and, (d) imaging the distribution of the pharmaceutical formulation of step (a) with the imaging device, thereby imaging the tumor neovasculature.
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • BLI bioluminescence image
  • the invention provides a method for in situ or in vivo screening for an anti- tumor agent by imaging a tumor neovasculature in an individual comprising the following steps: (a) providing a composition comprising a chimeric polypeptide as set forth in claim 1 or a pharmaceutical formulation as set forth in claim 18, and a test compound; (b) providing an imaging device, wherein the imaging device is computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), bioluminescence image (BLI) or equivalent; (c) administering the composition of step (a) in an amount sufficient to image the tumor neovasculature and imaging the distribution of the composition with the imaging device, thereby imaging the tumor neovasculature; (d) administering the test compound; and, (e) imaging the distribution of the composition with the imaging device, thereby imaging the tumor neovasculature, wherein
  • Figure 1 shows the chimeric RGD-luciferase protein attached to the surface of cultured cells as detected using BLI, as described in detail in Example 1, below.
  • Figure 2 shows a nude mouse with an orthotopic mammary tumor imaged with an in vivo bioluminescent imaging system after injection of RDG-luciferase, as described in detail in Example 1, below. The presence of the tumor was detected by the emission of luciferase-produced photons from the tumor site (see arrow).
  • the invention provides compositions and methods for imaging cells, tissues and organs in vivo and in situ.
  • the compositions and methods of the invention are used to identify sites of primary and metastatic tumors and tumor neovasculature.
  • the invention also provides a non-invasive means to evaluate the effectiveness of a therapy for a disorder of cell growth, such as cancer.
  • the compositions and methods enhance the imaging of cells, tissues, organs, entire bodies and angiogenesis by, e.g., computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or bioluminescence imaging (BLI).
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • BBI bioluminescence imaging
  • antibody or “Ab” includes both intact antibodies having at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds and antigen binding fragments thereof, or equivalents thereof, either isolated from natural sources, recombinantly generated or partially or entirely synthetic.
  • antigen binding fragments include, e.g., Fab fragments, F(ab')2 fragments, Fd fragments, dAb fragments, isolated complementarity determining regions (CDR), single chain antibodies, chimeric antibodies, humanized antibodies, human antibodies made in non-human animals (e.g., transgenic mice) or any form of antigen binding fragment.
  • non-endogenous kinase or “heterologous kinase” means a kinase not normally associated with a cell or tissue, as described in further detail, below.
  • an exemplary kinase used in the compositions and methods of the invention includes herpes simplex virus-1 thymidine kinase (HSN-1 TK).
  • HSN-1 TK herpes simplex virus-1 thymidine kinase
  • a chimeric polypeptide comprising a domain comprising a non-mammalian kinase is administered to a mammal, e.g., a human.
  • bioluminescence imaging includes all bioluminescence, fluorescence or chemiluminescence or other photon detection systems and devices capable of detecting bioluminescence, fluorescence or chemiluminescence or other photon detection systems. Since light can be transmitted through mammalian tissues at a low level, bioluminescent and fluorescent proteins can be detected externally using sensitive photon detection systems; see, e.g., Contag (2000) ⁇ eoplasia 2:41-52; Zhang (1994) Clin. Exp. Metastasis 12: 87-92. The methods of the invention can be practiced using any such photon detection device, or variation or equivalent thereof, or in conjunction with any known photon detection methodology, including visual imaging.
  • An exemplary photodetector device is an intensified charge-coupled device (ICCD) camera coupled to an image processor. See, e.g., U.S. Patent No. 5,650,135. Photon detection devices are manufactured by, e.g., Xenogen, Hamamatsue.
  • ICCD intensified charge-coupled device
  • a "computer assisted tomography (CAT)” or a “computerized axial tomography (CAT)” incorporates all computer-assisted tomography imaging systems or equivalents and devices capable of computer assisted tomography imaging.
  • the methods of the invention can be practiced using any such device, or variation of a CAT device or equivalent, or in conjunction with any known CAT methodology. See, e.g., U.S. Patent Nos. 6,151,377; 5,946,371; 5,446,799; 5,406,479; 5,208,581; 5,109,397.
  • Animal imaging modalities are also included, such as MicroCATTM (ImTek, Inc.).
  • PET positron emission tomography imaging
  • the methods of the invention can be practiced using any such device, or variation of a PET device or equivalent, or in conjunction with any known PET methodology. See, e.g., U.S. Patent Nos. 6,151,377; 6,072,177; 5,900,636; 5,608,221; 5,532,489; 5,272,343; 5,103,098.
  • Animal imaging modalities are included, e.g. micro-PETs (Corcorde Microsystems, Inc.).
  • single-photon emission computed tomography (SPECT) device incorporates all single-photon emission computed tomography imaging systems or equivalents and all devices capable of single-photon emission computed tomography imaging.
  • the methods of the invention can be practiced using any such device, or variation of a SPECT device or equivalent, or in conjunction with any known SPECT methodology. See, e.g., U.S. Patent Nos. 6,115,446; 6,072,177; 5,608,221; 5,600,145; 5,210,421; 5,103,098.
  • Animal imaging modalities are also included, such as micro-SPECTs.
  • magnetic resonance imaging (MRI) device incorporates all magnetic resonance imaging systems or equivalents and all devices capable of magnetic resonance imaging.
  • the methods of the invention can be practiced using any such device, or variation of an MRI device or equivalent, or in conjunction with any known MRI methodology.
  • a static magnetic field is applied to a tissue or a body under investigation in order to define an equilibrium axis of magnetic alignment in a region of interest.
  • a radio frequency field is then applied to that region in a direction orthogonal to the static magnetic field direction in order to excite magnetic resonance in the region.
  • the resulting radio frequency signals are detected and processed.
  • the exciting radio frequency field is applied.
  • the resulting signals are detected by radio- frequency coils placed adjacent the tissue or area of the body of interest.
  • MRI and supporting devices are manufactured by, e.g., Bruker Medical GMBH; Caprius; Esaote Biomedica; Fonar; GE Medical Systems (GEMS); Hitachi Medical Systems America; Intermagnetics General Corporation; Lunar Corp.; MagneNu; Marconi Medicals; Philips Medical Systems; Shimadzu; Siemens; Toshiba America Medical Systems; including imaging systems, by, e.g., Silicon Graphics. Animal imaging modalities are also included, such as micro-MRIs.
  • a computer/ processor can be a conventional general-purpose digital computer, e.g., a personal "workstation” computer, including conventional elements such as microprocessor and data transfer bus.
  • the computer / processor can further include any form of memory elements, such as dynamic random access memory, flash memory or the like, or mass storage such as magnetic disc optional storage.
  • chondroitin sulfate proteoglycan includes those proteoglycan molecules expressed by immature cells and tumor cells, including the human melanoma proteoglycan also known as the high molecular weight melanoma-associated antigen, see, e.g., Desai (1998) Cancer Res. 58:2417-2425; Chattopadhyay (1991) Cancer Res. 51 :3183-3192. Chondroitin sulfate proteoglycan binding polypeptides are discussed below.
  • fluorescent include all known polypeptides known to be fluorescent, bioluminescent or chemiluminescent, or, acting as enzymes on a specific substrate (reagent), can generate a fluorescent, bioluminescent or chemiluminescent molecule. They include, e.g., isolated and recombinant luciferases, aequorin, obelin, mnemiopsin, berovin and variations thereof and combinations thereof, as discussed in detail, below.
  • the pharmaceutical formulation of the invention can further comprise a substrate for the bioluminescent or chemiluminescent polypeptide.
  • the chemiluminescent polypeptide can be luciferase and the reagent luciferin.
  • the substrate can be administered before, at the same time (e.g., in the same formulation), or after administration of the chimeric polypeptide (including the enzyme).
  • lanthanide metal ions include, e.g., lanthanum [La], cerium [Ce], praseodyme [Pr], gadolinium [Gd], dysprosium [Dy], ytterbium [Yb], and lutetium [Lu].
  • Non-lanthanides that can be imaged are also used in the methods of the invention and include lead [Pb] and bismuth [Bi]. See, e.g. Krause (1996) Invest. Radiol. 31:502-511.
  • matrix metalloproteinase includes interstitial collagenases, stromelysins, gelatinases and membrane-type metalloproteinases and MMPs secreted by cancer cells. These later MMPs degrade extracellular matrices and allow for tumor infiltration of tissues and metastasis; see, e.g., Yip (1999) Invest. New Drugs 17:387- 399. MMPs and MMP -binding polypeptides are discussed in detail, below.
  • selectin includes L-, E-, and P-selectins, which are membrane-anchored, C-type lectins that initiate tethering and rolling of flowing leukocytes on endothelial cells, platelets, or other leukocytes during inflammation.
  • Selectins bind to sialylated, fucosylated, sulfated glycans on glycoproteins, glycolipids, or proteoglycans.
  • E- selectin which are expressed on vascular endothelial cells, can bind to carbohydrate determinants, e.g., sialyl Lewis A and sialyl Lewis X, which are frequently expressed on human cancer cells. See, e.g., McEver (1997) Glycoconj. J. 14:585-591; Kannagi (1997) Glycoconj. J. 14:577-584. Selectin-binding polypeptides are discussed in detail, below.
  • composition refers to a composition suitable for pharmaceutical use in a subject (including human or veterinary).
  • the pharmaceutical compositions of this invention are formulations that comprise a pharmacologically effective amount of a composition comprising, e.g., a chimeric composition, a recombinant polypeptide, a nucleic acid encoding a chimeric polypeptide of the invention, a vector comprising a nucleic acid of the invention, or a cell of the invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical formulation of the invention can further comprise a substrate for the bioluminescent or chemiluminescent polypeptide.
  • the chemiluminescent polypeptide can be luciferase and the reagent luciferin.
  • the substrate reagent can be co-administered or administered before or after the chimeric polypeptide (enzyme) formulation.
  • promoter includes all sequences capable of driving transcription of a coding sequence in an expression system.
  • promoters used in the constructs of the invention include cts-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a nucleic acid of the invention.
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • recombinant refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide.
  • nucleic acid or “nucleic acid sequence” refers to a deoxy- ribonucleotide or ribonucleotide oligonucleotide, including single- or double-stranded, or coding or non-coding (e.g., "antisense") forms.
  • the term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides.
  • the term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Mull (1996) Antisense Nucleic Acid Drug Dev 6: 153-156.
  • polypeptide As used herein the terms “polypeptide,” “protein,” and “peptide” are used interchangeably and include compositions of the invention that also include “analogs,” or “conservative variants” and “mimetics” (e.g., “peptidomimetics”) with structures and activity that substantially correspond to the polypeptides of the invention, including the peptide sequences as set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
  • the terms “conservative variant” or “analog” or “mimetic” also refer to a polypeptide or peptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the polypeptide's (the conservative variant's) structure and/or activity (e.g., binding specificity), as defined herein.
  • conservatively modified variations of an amino acid sequence i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity.
  • one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): ala/gly or ser; arg/ lys; asn/ gin or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gin; ile/leu or val; leu/ile or val; lys/arg or gin or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu.
  • An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T)
  • mimetic and “peptidomimetic” refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the polypeptides of the invention (e.g., ability to bind to integrins, selectins, MMPs, chondroitin sulfate proteoglycans, etc.).
  • the mimetic can be either entirely composed of synthetic, non- natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetics' structure and/or activity.
  • Polypeptide mimetic compositions can contain any combination of non- natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a secondary structural mimicry i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
  • Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, ⁇ - hydroxysuccinimide esters, bimnctional maleimides, ⁇ , ⁇ '-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC).
  • a polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature.
  • Fluorescent, bioluminescent or chemiluminescent polypeptides The invention provides a chimeric molecule, e.g., a recombinant polypeptide, and a pharmaceutical composition, comprising a fluorescent, bioluminescent or chemiluminescent polypeptide.
  • these polypeptides include enzymes that act on a specific reagent to generate a molecule that can be imaged (e.g., luciferase reacting with luciferin in situ).
  • these polypeptides include, e.g., luciferase, aequorin, halistaurin, phialidin, obelin, mnemiopsin or berovin, or, equivalent photoproteins, and combinations thereof.
  • the compositions and methods of the invention also include recombinant forms of these polypeptides as recombinant chimeric or "fusion" proteins, including chimeric nucleic acids and constructs encoding them. Methods of making recombinant forms of these polypeptides are well known in the art, e.g., luciferase reporter plasmids are described, e.g., by Everett (1999) J. Steroid Biochem. Mol.
  • the monomeric Ca2+-binding protein aequorin is described by, e.g., Kurose (1989) Proc. Natl. Acad. Sci. USA 86:80-84; Shimomura (1995) Biochem. Biophys. Res. Commun. 211:359-363.
  • the aequorin-type photoproteins halistaurin and phialidin are described by, e.g., Shimomura (1985) Biochem J. 228:745-749. Ward (1975) Proc. Natl. Acad. Sci USA 72:2530-2534, describes the purification of mnemiopsin, aequorin and berovin.
  • the recombinant fluorescent, bioluminescent or chemiluminescent chimeric polypeptides of the invention can be made by any method, see, e.g., U.S. Patent No. 6,087,476, that describes making recombinant, chimeric luminescent proteins.
  • U.S. Patent Nos. 6,143,50; 6,074,859; 6,074,859, 5,229,285, describe making recombinant luminescent proteins.
  • the fluorescent or chemiluminescent activity of the chimeric recombinant polypeptides of the invention can be assayed, e.g., using assays described in, e.g., U.S. Patent Nos.
  • the invention provides a chimeric molecule, e.g., a recombinant polypeptide, and a pharmaceutical composition, comprising a fluorescent or chemiluminescent polypeptide and an RGD motif-comprising polypeptide, such as the peptide comprising an CDCRGDCFC (SEQ LD NO:l) amino acid sequence.
  • a chimeric molecule e.g., a recombinant polypeptide
  • a pharmaceutical composition comprising a fluorescent or chemiluminescent polypeptide and an RGD motif-comprising polypeptide, such as the peptide comprising an CDCRGDCFC (SEQ LD NO:l) amino acid sequence.
  • the tripeptidic sequence Arg-Gly- Asp, or "RGD” is often the primary site of recognition by integrins that are expressed on tumor cells and are responsible for tumor invasion and metastasis.
  • Tumor cell-expressing polypeptides that recognize RGD-comprising polypeptides mediat
  • RGD-comprising polypeptides mediate the invasion and migration of tumor cells.
  • Pasqualini (1997) Nat. Biotechnol. 15:542-546 showed that alpha v integrins present in tumor blood vessels can bind circulating RGD-expressing peptide ligands selective for these integrins.
  • imaging of cells that can specifically bind to RGD-expressing peptide and polypeptide ligands in vivo can identify tumor cells and tumor blood vessels.
  • Selectin binding compositions The invention provides a chimeric molecule, e.g., a recombinant polypeptide, and a pharmaceutical composition, comprising a fluorescent or chemiluminescent polypeptide and a selectin- (e.g., E-selectin-) binding polypeptide, such as the peptide comprising an IELLQAR (SEQ ID NO:2) amino acid sequence.
  • selectin-binding ligands include carbohydrates, such as sialyl Lewis X, that bind in a calcium-dependent manner. Significantly, some selectin carbohydrate ligands are expressed on tumor cells.
  • Imaging of ligands that can specifically bind to selectin in vivo can identify cells, e.g., immune cells, such as NK cells, that can target tumor cells.
  • immune cells such as NK cells
  • tumor cells adhere to vascular endothelia.
  • E-selectin is an adhesive protein expressed by cytokine-activated endothelium that can support adhesion of some cancer cells (e.g., colon cancer cells) through the recognition of specific carbohydrate ligands that can selectively bind to a selectin polypeptide can be done by routine screening using in vitro and in vivo techniques, including the use of high throughput screening of various peptide and other combinatorial libraries. For example, phage display libraries expressing libraries of peptides can be routinely screened for selectin-binding members.
  • screening of selectin-binding compounds can also be done indirectly using "anti-idiotype" strategies; for example, antibodies that bind to carbohydrate ligands that are selectin ligands can be screened by, e.g., combinatorial or phage display libraries. See, e.g., Fukuda (2000) Cancer Res. 60:450-456, who used to method to identify the peptide IELLQAR (SEQ ID NO:2) as a selectin-binding ligand.
  • Matrix metalloproteinase binding compositions The invention provides a chimeric molecule, e.g., a recombinant polypeptide, and a pharmaceutical composition, comprising a fluorescent or chemiluminescent polypeptide and a matrix metalloproteinase- (MMP-) binding polypeptide, such as the peptide comprising an CTTHWGFTLC (SEQ ID NO:3) amino acid sequence.
  • MMPs matrix metalloproteinases
  • MMP-9 matrix metalloproteinase 9
  • imaging of cells that can specifically bind to MMP polypeptides in vivo can identify tumor cells and site of metastasis.
  • Identification of compounds that can selectively bind to MMP polypeptides can be done by routine screening using in vitro and in vivo techniques, including the use of high throughput screening of various peptide and other combinatorial libraries. For example, phage display libraries expressing libraries of peptides can be routinely screened for MMP- binding members.
  • the invention provides a chimeric molecule, e.g., a recombinant polypeptide, and a pharmaceutical composition, comprising a fluorescent or chemiluminescent polypeptide and a chondroitin sulfate proteoglycan-binding polypeptide, such as a peptide comprising the sequence TAASGNRSMH (SEQ ID ⁇ O:4) or LTLRWVGLMS (SEQ ID NO:5).
  • TAASGNRSMH SEQ ID ⁇ O:4
  • LTLRWVGLMS SEQ ID NO:5
  • Chondroitin sulfate proteoglycans can be expressed by immature cells and tumor cells.
  • a human melanoma proteoglycan also known as the high molecular weight melanoma-associated antigen
  • Another melanoma proteoglycan is NG2, a rat homologue of this human melanoma proteoglycan.
  • NG2 is a 500 kDa integral membrane chondroitin sulfate proteoglycan. It is found on the surfaces of several different types of immature cells and is most prominent in immature blood vessels during development.
  • NG2 is associated with multipotential glial precursor cells (O2A progenitor cells), chondroblasts of the developing cartilage, brain capillary endothelial cells, aortic smooth muscle cells, skeletal myoblasts and human melanoma cells (see, e.g., Levine (1996) Perspect. Dev. Neurobiol. 3:245-259).
  • O2A progenitor cells multipotential glial precursor cells
  • chondroblasts of the developing cartilage e.g., brain capillary endothelial cells
  • aortic smooth muscle cells e.g., aortic smooth muscle cells
  • skeletal myoblasts e.g., Levine (1996) Perspect. Dev. Neurobiol. 3:245-259).
  • NG2 is expression can again be detected within newly forming blood vessels. It is not clear if expression of NG2 within the neovasculature can be attributed to endothelial cells or mesenchy
  • Identification of compounds that can selectively bind to tumor-expressing chondroitin sulfate proteoglycans can be done by routine screening using in vitro and in vivo techniques, including the use of high throughput screening of various peptide and other combinatorial libraries.
  • phage display libraries expressing libraries of peptides can be routinely screened for proteoglycan-binding members.
  • An exemplary technique is described by Burg (1999) Cancer Res.
  • NG2-binding peptides by screening a phage-displayed random peptide library on purified NG2, including the decapeptides TAASGNRSMH (SEQ ID ⁇ O:4) and LTLRWVGLMS (SEQ ID NO:5).
  • the invention provides a chimeric polypeptide having a first domain comprising a non-endogenous kinase, such as a herpes simplex virus- 1 thymidine kinase (HSN-1 TK), and a second domain comprising an RGD motif-comprising polypeptide; a selectin-binding polypeptide; a matrix metalloproteinase (MMP)-binding polypeptide or a chondroitin sulfate proteoglycan-binding polypeptide.
  • the chimeric polypeptide can be used as a reporter for administration in quantitative assays to non-invasively image expression of the polypeptide living animals using MRI, PET, SPECT, BRI and the like.
  • a kinase substrate (a "reporter probe") is administered, e.g., the positron-emitting 8-[18F] fluoroganciclovir (FGCN).
  • FGCN positron-emitting 8-[18F] fluoroganciclovir
  • HSN1-TK herpes simplex virus 1 thymidine kinase enzyme
  • Adenovirus-directed hepatic expression of the HSV-1 TK gene in living mice has been shown to be detectable by PET. See, e.g., Tjuvajev (1995) Cancer Res. 55:6126-6132; Gambhir (1999) Proc. ⁇ atl. Acad. Sci.
  • the invention provides compositions and methods to enhance the imaging of cells and tissues by, e.g., bioluminescence imaging (BLI).
  • BBI Bioluminescence imaging
  • This modality consists of the detection of a photoprotein (i.e., an optical reporter), such as luciferase from the firefly, using a sensitive photon detection system.
  • the number of photons emitted from cells expressing the photoprotein can be quantitatively detected and overlay ed (projected) onto a visual picture of the animal (including humans).
  • This imaging approach provides a two-dimensional image data set and thus provides some spatial information as to the origin of the signal within the animal.
  • An exciting aspect of BLI is its excellent sensitivity along with its ability to report on "molecular events" using specifically designed luciferase reporter constructs.
  • the invention provides pharmaceutical formulations comprising the chimeric molecules of the invention that can further comprise imaging contrast agents (see, e.g., U.S. Patent No. 4,731,239).
  • the pharmaceutical formulations and/or the contrast agents can be administered by nanoencapsulation, e.g., by hydrogel nanoparticles (and liposomes, which are discussed below). Nanoencapsulation can be used to manipulate the environment surrounding the pharmaceutical formulation and/or the contrast agent.
  • nanoencapsulation can be used to manipulate the environment surrounding the pharmaceutical formulation and/or the contrast agent.
  • MRI magnetic resonance imaging
  • Contrast agents for magnetic resonance imaging typically affect the protons on adjacent water molecules shortening either the T_ or T 2 signals generated in the magnetic field.
  • the most important factor in enhancement of relaxation is the difference between Ti and T 2 .
  • Enhancement of T 2 effects requires clustering of the contrast agent and proximity to each other. This clustering of magnetic contrast agent exerts a greater influence ovei a much larger localized field. Thus incorporation into liposomes increases the proximity of T 2 contrast agents and enhances their effectiveness. Incorporation of contrast agents into the body o] hydrogel nanoparticles has with it the potential advantages of both immobilizing and clustering the contrast agent and providing a material through which water can freely diffuse.
  • the pharmaceutical compositions of the invention can further comprise monocrystalline iron oxide nanoparticles (MION), which have been successfully used in a variety of biological and clinical applications.
  • MION has an average diameter of approximately 18 to 24 nm and thus are able to penetrate endothelial fenestrations throughout the body and are cleared through the reticuloendothelial system and are disposed of by hepatic metabolism of iron.
  • MION has excellent contrast characteristics in vivo and out-performs the most effective dendrimer- conjugated contrast agents. Although conjugation of MION to antibodies has been achieved, this strategy leaves considerable room for improvement with regard to contrast enhancement and specificity/uniformity of targeting.
  • This invention provides improved strategies for efficient biotargeting of multifunctional imaging and therapeutic nanodevices.
  • the chimeric polypeptides of the invention can also be associated with gold or other equivalent metal particles (such as nanoparticles), either attached to the nanoparticles or as ; further composition in the pharmaceutical formulations of the invention.
  • Any metal particle system can be used, e.g., the gold nanoparticle NanogoldTM (Nanoprobes, Yaphank, NY), which can be covalently attached to peptides via, e.g., a sulfo-succinimidyl- 4-N-maleimido- cyclohexane- 1-carboxylate (sulfo-SMCC) linkage (see, e.g., Du (1998) Neuroscience 84:37-48).
  • Polypeptides and Peptides e.g., Polypeptides and Peptides
  • the invention provides a chimeric polypeptide comprising a fluorescent, bioluminescent or chemiluminescent domain and an RGD motif-comprising polypeptide; a selectin-binding polypeptide; a matrix metalloproteinase (MMP)-binding polypeptide or a chondroitin sulfate proteoglycan-binding polypeptide.
  • MMP matrix metalloproteinase
  • chondroitin sulfate proteoglycan-binding polypeptide comprise sequences as set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 (as noted above, the term polypeptide includes peptides and peptidomimetics, etc.).
  • Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides of the invention can be made and isolated using any method known in the art.
  • Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA.
  • peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol.
  • Peptides and peptide mimetics of the invention can also be synthesized using combinatorial methodologies.
  • Various techniques for generation of peptide and peptidomimetic libraries are well known, and include, e.g., multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymol.
  • Modified peptides of the invention can be further produced by chemical modification methods, see, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896.
  • Peptides and polypeptides of the invention can also be synthesized and expressed as chimeric or "fusion" proteins with one or more additional domains linked thereto for, e.g., to more readily isolate a recombinantly synthesized peptide, and the like.
  • Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
  • metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp, Seattle WA.
  • the inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego CA) between the purification domain and GCA-associated peptide or polypeptide can be useful to facilitate purification.
  • an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34: 1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-14).
  • the histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein.
  • This invention provides nucleic acids encoding the chimeric polypeptides of the invention.
  • the genes and vectors of the invention can be made and expressed in vitro or in vivo, the invention provides for a variety of means of making and expressing these genes and vectors.
  • desired phenotypes associated with altered gene activity can be obtained by modulating the expression or activity of the genes and nucleic acids (e.g., promoters) within the vectors of the invention. Any of the known methods described for increasing or decreasing expression or activity can be used for this invention.
  • the invention can be practiced in conjunction with any method or protocol known in the art, which are well described in the scientific and patent literature.
  • nucleic acid sequences of the invention and other nucleic acids used to practice this invention may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to bacterial cells, e.g., mammalian, yeast, insect or plant cell expression systems.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med.
  • the invention provides pharmaceutical formulations comprising the chimeric molecules of the invention and a pharmaceutically acceptable excipient suitable for administration an imaging enhancing agents, and methods for making and using these compositions.
  • These pharmaceuticals can be administered by any means in any appropriate formulation. Routine means to determine drug regimens and formulations to practice the methods of the invention are well described in the patent and scientific literature. For example, details on techniques for formulation, dosages, administration and the like are described in, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA.
  • the formulations of the invention can include pharmaceutically acceptable carriers that can contain a physiologically acceptable compound that acts, e.g., to stabilize the composition or to increase or decrease the absorption of the agent and/or pharmaceutical composition.
  • Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of any co-administered agents, or excipients or other stabilizers and or buffers. Detergents can also used to stabilize the composition or to increase or decrease the absorption of the pharmaceutical composition.
  • Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known, e.g., ascorbic acid.
  • compositions for administration comprises a chimeric polypeptide of the invention in a pharmaceutically acceptable carrier, e.g., an aqueous carrier.
  • a pharmaceutically acceptable carrier e.g., an aqueous carrier.
  • carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well-known sterilization techniques.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration and imaging modality selected.
  • the pharmaceutical formulations of the invention can be administered in a variety of unit dosage forms, depending upon the particular cell or tissue or cancer to be imaged, the general medical condition of each patient, the method of administration, and the like. Details on dosages are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences. The exact amount and concentration of chimeric molecule or pharmaceutical of the invention and the amount of formulation in a given dose, or the "effective dose” can be routinely determined by, e.g., the clinician.
  • the "dosing regimen,” will depend upon a variety of factors, e.g., whether the cell or tissue or tumor to be imaged is disseminated or local, the general state of the patient's health, age and the like.
  • contrast-enhanced CT studies of the head and neck are performed frequently using contrast material volumes of about 30 g iodine and a scan delay of 30 to 45 seconds, see, e.g., Groell (1999) AJNR Am. J. Neuroradiol. 20:1732-1736. Desser (1999) Acad. Radiol. 6:176-183, described use of three different doses of liposomal iodixanol to image livers by CAT. Hamm (1994) J. Magn. Reson.
  • the concentration of the composition in the pharmaceutical formulation is about 1 to about 1000 ⁇ g/ml, or, 10 to about 100 ⁇ g/ml. However, higher or lower concentrations are used when appropriate and the invention is not limited by any particular dosage range.
  • compositions of the invention can be delivered by any means known in the art systemically (e.g., intravenously), regionally, or locally (e.g., intra- or peri-tumoral or intracystic injection, e.g., to image bladder cancer) by, e.g., intraarterial, intratumoral, intravenous (IV), parenteral, intra-pleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa), intra-tumoral (e.g., transdermal application or local injection).
  • intraarterial, intratumoral, intravenous (IV), parenteral, intra-pleural cavity, topical, oral, or local administration as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vagina
  • intra-arterial injections can be used to have a "regional effect," e.g., to focus on a specific organ (e.g., brain, liver, spleen, lungs).
  • a specific organ e.g., brain, liver, spleen, lungs.
  • intra-hepatic artery injection or intra-carotid artery injection If it is desired to deliver the preparation to the brain, it can be injected into a carotid artery or an artery of the carotid system of arteries (e.g., occipital artery, auriculiar artery, temporal artery, cerebral artery, maxillary artery, etc.).
  • the pharmaceutical formulations of the invention can be presented in unit- dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • sterile liquid excipient for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • compositions can also be administered in a lipid formulation, e.g., complexed with liposomes or in lipid/nucleic acid complexes or encapsulated in liposomes, as in immunoliposomes directed to specific cells.
  • lipid formulations can be administered topically, systemically, or delivered via aerosol. See, e.g., U.S. Patent Nos. 6,149,937; 6,146,659; 6,143,716; 6,133,243; 6,110,490; 6,083,530; 6,063,400; 6,013,278; 5,958,378; 5,552,157.
  • kits comprising the compositions, e.g., the pharmaceutical compositions, nucleic acids, cells, of the invention.
  • the kits also can contain instructional material teaching methodologies, e.g., how and when to administer the pharmaceutical compositions, how to apply the compositions and methods of the invention to imaging systems, e.g., computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single- photon emission computed tomography (SPECT) or bioluminescence imaging (BLI).
  • Kits containing pharmaceutical preparations e.g., chimeric polypeptides, vectors, nucleic acids
  • Example 1 In Vivo Targeting to Angiogenic Vasculature
  • An exemplary chimeric recombinant polypeptide of the invention an RGD-containing-luciferase fusion protein was produced in a bacterial expression system.
  • the RGD-luciferase protein and, as a negative control, the reverse sequenced DGR fusion protein (DGR-luciferase) were expressed.
  • the RGD-luciferase protein had a strong tendency to multimerize (cyclize) due to reactive SH-groups on the terminal end of the peptide. This required production of the protein under dilute conditions.
  • the recombinant proteins were purified and added to MDA-435 human breast carcinoma cells (see, e.g., Rahman (1989) J. Natl. Cancer Inst. 81:1794-1800) in culture wells.
  • the MDA-435 cells which express the RGD receptor on their cell surface, were useful for in vitro testing of this targeting approach for bioluminescent imaging (BLI).
  • BLI bioluminescent imaging
  • the chimeric RGD-luciferase protein attached to the surface of the cells and could be detected using BLI. DGR-luciferase binding, serving as a control for possible nonspecific binding, could not be detected.
  • the RDG-luciferase was also injected into a nude mouse with an orthotopic mammary tumor. Luciferin was administered. The animal was imaged in an in vivo bioluminescent imaging system. As shown in Figure 2, the presence of the tumor was detected by the emission of luciferase-produced photons from the tumor site (see arrow).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pathology (AREA)
  • Radiology & Medical Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Magnetic Resonance Imaging Apparatus (AREA)

Abstract

Cette invention concerne des compositions et des procédés permettant de représenter des cellules et des tissus in vivo et in situ. L'administration de ces compositions et l'utilisation de ces procédés permettent d'améliorer la qualité des images de cellules et de tissus obtenues, par exemple, par tomographie par ordinateur, spectroscopie à résonance magnétique, imagerie par résonance magnétique (IRM), tomographie par émission de positons (TEP), tomographie monophotonique d'émission ou imagerie par bioluminescence. Cette invention porte donc sur des compositions et des procédés permettant d'identifier des sites de tumeurs primaires et de métastases ainsi que des néovaisseaux tumoraux.
PCT/US2001/048157 2000-12-11 2001-12-11 Compositions et procedes de representation in situ et in vivo de cellules et de tissus WO2002047537A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002232568A AU2002232568A1 (en) 2000-12-11 2001-12-11 Compositions and methods for in situ and in vivo imaging of cells and tissues
EP01992094A EP1501476A2 (fr) 2000-12-11 2001-12-11 Compositions et procedes de representation in situ et in vivo de cellules et de tissus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US73462800A 2000-12-11 2000-12-11
US09/734,628 2000-12-11

Publications (2)

Publication Number Publication Date
WO2002047537A2 true WO2002047537A2 (fr) 2002-06-20
WO2002047537A3 WO2002047537A3 (fr) 2004-11-25

Family

ID=24952460

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/048157 WO2002047537A2 (fr) 2000-12-11 2001-12-11 Compositions et procedes de representation in situ et in vivo de cellules et de tissus

Country Status (3)

Country Link
EP (1) EP1501476A2 (fr)
AU (1) AU2002232568A1 (fr)
WO (1) WO2002047537A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007030012A2 (fr) * 2005-09-05 2007-03-15 Stichting Voor De Technische Wetenscahappen Mise en image d'une matrice extracellulaire
US7795386B2 (en) 2004-12-23 2010-09-14 Molmed Spa Peptides comprising an isoDGR motif
WO2016061142A1 (fr) 2014-10-14 2016-04-21 Novartis Ag Molécules d'anticorps de pd-l1 et leurs utilisations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6180084B1 (en) * 1998-08-25 2001-01-30 The Burnham Institute NGR receptor and methods of identifying tumor homing molecules that home to angiogenic vasculature using same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6180084B1 (en) * 1998-08-25 2001-01-30 The Burnham Institute NGR receptor and methods of identifying tumor homing molecules that home to angiogenic vasculature using same

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7795386B2 (en) 2004-12-23 2010-09-14 Molmed Spa Peptides comprising an isoDGR motif
WO2007030012A2 (fr) * 2005-09-05 2007-03-15 Stichting Voor De Technische Wetenscahappen Mise en image d'une matrice extracellulaire
WO2007030012A3 (fr) * 2005-09-05 2007-10-04 Stichting Tech Wetenschapp Mise en image d'une matrice extracellulaire
WO2016061142A1 (fr) 2014-10-14 2016-04-21 Novartis Ag Molécules d'anticorps de pd-l1 et leurs utilisations
EP4245376A2 (fr) 2014-10-14 2023-09-20 Novartis AG Molécules d'anticorps de pd-l1 et leurs utilisations

Also Published As

Publication number Publication date
AU2002232568A1 (en) 2002-06-24
WO2002047537A3 (fr) 2004-11-25
EP1501476A2 (fr) 2005-02-02

Similar Documents

Publication Publication Date Title
AU2019232847B2 (en) Bi-terminal pegylated integrin-binding peptides and methods of use thereof
Jaffer et al. Seeing within: molecular imaging of the cardiovascular system
Hsu et al. In vivo near-infrared fluorescence imaging of integrin α v β 3 in an orthotopic glioblastoma model
Neri et al. Tumour vascular targeting
Bogdanov et al. The development of in vivo imaging systems to study gene expression
US20020122806A1 (en) Compositions and methods for in situ and in vivo imaging of cells and tissues
Park et al. A new atherosclerotic lesion probe based on hydrophobically modified chitosan nanoparticles functionalized by the atherosclerotic plaque targeted peptides
ES2436424T3 (es) Agente de diagnóstico
AU2001292456B2 (en) Peptide-based compounds
Haubner et al. Radiotracer-based strategies to image angiogenesis
US9284381B2 (en) Methods and reagents for preparing multifunctional probes
US8188051B2 (en) Metadherin polypeptides, encoding nucleic acids and methods of use
US20030118585A1 (en) Use of protein biomolecular targets in the treatment and visualization of brain tumors
KR20210009421A (ko) 다중 특이적 항체 구조체
Ailuno et al. Peptide-based nanosystems for vascular cell adhesion molecule-1 targeting: A real opportunity for therapeutic and diagnostic agents in inflammation associated disorders
Jaffer et al. Molecular imaging of myocardial infarction
US8828355B2 (en) Imaging reporters of transgene expression
US6958140B2 (en) Methods of imaging and targeting vasculature
US20020073441A1 (en) Compositions and methods for detecting proteolytic activity
Capuana et al. Imaging of Dysfunctional Elastogenesis in Atherosclerosis Using an Improved Gadolinium-Based Tetrameric MRI Probe Targeted to Tropoelastin
WO2002047537A2 (fr) Compositions et procedes de representation in situ et in vivo de cellules et de tissus
US20050089470A1 (en) Labeled peptides for lectin-like oxidized low-density lipoprotein receptor (LOX-1)
US20090263320A1 (en) Peptide-Based Compounds
US7575738B2 (en) Heat shock protein as a targeting agent for endothelium-specific in vivo transduction
JP2024536369A (ja) 腫瘍または線維症の診断および療法における使用のためのフィブロネクチン結合ペプチド

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2001992094

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 2001992094

Country of ref document: EP

NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2001992094

Country of ref document: EP

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)