WO2014011327A1 - Sondes radiomarquées pour la détection et l'imagerie non invasives de la mort cellulaire - Google Patents

Sondes radiomarquées pour la détection et l'imagerie non invasives de la mort cellulaire Download PDF

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Publication number
WO2014011327A1
WO2014011327A1 PCT/US2013/043067 US2013043067W WO2014011327A1 WO 2014011327 A1 WO2014011327 A1 WO 2014011327A1 US 2013043067 W US2013043067 W US 2013043067W WO 2014011327 A1 WO2014011327 A1 WO 2014011327A1
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dehydrogenase
conjugate
ldh
apoptosis
functional fragment
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PCT/US2013/043067
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English (en)
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Andrew Kung
Timothy DEGRADO
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Dana-Farber Cancer Institute, Inc.
The Brigham And Women's Hospital, Inc.
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Publication of WO2014011327A1 publication Critical patent/WO2014011327A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification

Definitions

  • Cell death is a feature of many diseases as well as therapeutic methods, for example in the treatment of cancer. It is therefore desirable to be able to measure cell death, to assess either the status of a disease that involves cell death or the effect of a treatment that induces cell death.
  • apoptosis and necrosis two distinct mechanisms of cell death known as apoptosis and necrosis have been described. While distinct in certain aspects, these two forms of cell death can also be viewed as extremes along a continuum, such that late-stage apoptosis overlaps with necrosis.
  • Apoptosis which is also known as programmed cell death, is generally characterized as an energy-dependent, genetically controlled process by which cell death is activated through an internally regulated suicide program.
  • Features of apoptosis generally include phosphatidylserine externalization, loss of membrane integrity, cytoplasm shrinkage, chromatin and nucleus condensation, DNA degradation, and fragmentation of the cell into smaller apoptotic bodies by a budding process. Normally the resulting apoptotic bodies are phagocytosed by macrophages and neighboring cells without inducing an inflammatory response.
  • Methods frequently used to assess apoptosis include staining by fluorescently labeled annexin A5 (also known as Annexin V) and terminal uridine deoxynucleotidyl end-labeling (TUNEL) assay.
  • annexin A5 also known as Annexin V
  • TUNEL terminal uridine deoxynucleotidyl end-labeling
  • Necrosis which is also known as accidental cell death, is typically induced by any of a variety of sudden, severe, non-physiological insults, for example physical, chemical, and ischemic insults.
  • the process is generally characterized by progressive cell swelling, denaturation and coagulation of cytoplasmic proteins, disintegration of subcellular organelles and irreversible collapse of the plasma membrane integrity. This latter feature permits leakage of cytotoxic and other cellular components, inducing a local inflammatory response.
  • LDH lactate dehydrogenase
  • an isolated conjugate comprising dehydrogenase or functional fragment thereof linked to Zirconium-89 (Zr) that allows sensitive detection of cell death over a course of several days.
  • an isolated conjugate comprising dehydrogenase or functional fragment thereof linked to Zirconium-89
  • the isolated conjugate is linked to Zr, using a chelator.
  • the chelator is selected from the group consisting of desferoxamine, hydroxypyridinone, and catechol.
  • the dehydrogenase or functional fragment thereof is selected from the group of dehydrogenases shown in Table 1.
  • the dehydrogenase is a lactate dehydrogenase (LDH).
  • the LDH is a human LDH.
  • the dehydrogenase or functional fragment thereof is selected from the group consisting of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and malate dehydrogenase (MDH).
  • ADH alcohol dehydrogenase
  • ADH aldehyde dehydrogenase
  • MDH malate dehydrogenase
  • the dehydrogenase is a human dehydrogenase.
  • an isolated conjugate comprising
  • dehydrogenase or functional fragment thereof linked to a detectable label is provided.
  • the dehydrogenase or functional fragment thereof in the conjugate is in the monomeric form.
  • the dehydrogenase or functional fragment thereof is selected from the group of dehydrogenases shown in Table 1.
  • the dehydrogenase is a lactate dehydrogenase (LDH).
  • the LDH is a human LDH.
  • the dehydrogenase or functional fragment thereof is selected from the group consisting of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and malate dehydrogenase (MDH).
  • the dehydrogenase is a human dehydrogenase.
  • the detectable label is selected from the group consisting of a fluorescent label, a radioisotope, a nanoparticle, a chromophore, a dye, an enzyme, and a contrast agent.
  • the label is a fluorescent label.
  • the label is a nanoparticle.
  • the label is a radioisotope.
  • the radioisotope is selected from the group consisting of Zirconium-89 ( 89 Zr), Copper-64 ( 64 Cu), Niobium-90 ( 90 Nb), Titanium-45 ( 45 Ti),and Gallium-68 ( 68 Ga).
  • compositions comprising the conjugates described herein are provided.
  • a method for detecting cells undergoing cell death in a subject comprises administering to a subject in need thereof
  • the dehydrogenase or functional fragment thereof is selected from the group of dehydrogenases shown in Table 1.
  • the dehydrogenase or functional fragment thereof is a lactate dehydrogenase (LDH).
  • the LDH is a human LDH.
  • the dehydrogenase or functional fragment thereof is selected from the group consisting of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and malate dehydrogenase (MDH).
  • ADH alcohol dehydrogenase
  • ADH aldehyde dehydrogenase
  • MDH malate dehydrogenase
  • the dehydrogenase is a human dehydrogenase.
  • the cell death is apoptosis. In some embodiments, the cell death is necrosis.
  • the subject has or is suspected of having a condition associated with apoptosis and/or necrosis. In some embodiments, the condition associated with
  • apoptosis/necrosis is selected from the group consisting of cancer; chemotherapy-, radiation-, or hormone-induced apoptosis in solid and hematological tumors; tumor resistance to therapy; acute cardiac allograft rejection; acute myocardial infarction; anthracycline-induced
  • cardiotoxicity arrhythmogenic right ventricle dysplasia; skeletal muscle apoptosis; congestive heart failure; coronary artery disease; atherosclerosis; infectious endocarditis; myocarditis;
  • myocardial dysfunction myocardial ischemia-reperfusion injury; non-cardiac allograft rejection; bacterial infection; viral infection; multiple organ dysfunction syndrome; septic shock; cerebral ischemia-reperfusion injury; macular degeneration; neurodegenerative disease; central nervous system trauma; autoimmune diabetes mellitus; rheumatoid arthritis; systemic lupus
  • erythematosus erythematosus
  • inflammatory bowel disease multiple sclerosis; other autoimmune diseases; annexinopathies; osteoarthritis; renal failure; chronic renal atrophy and renal fibrosis; glomerular injury; and polycystic renal disease.
  • FIG. 1 is a SDS-PAGE gel of Zr-LDH showing high radiochemical purity of the tracer.
  • FIG. 2 shows the uptake of Zr-LDH by control and staurosporine (STS) treated 293T cells.
  • FIG. 3 shows images of IgG control and anti-FAS treated mice. Anti-FAS treatment resulted in
  • Dehydrogenases such as LDH, alcohol dehydrogenase, aldehyde
  • dehydrogenase malate dehydrogenase, and various isoforms of human LDH (LDH-A, LDH-B, and LDH-C), are taken up and retained by cells undergoing apoptosis and necrosis.
  • This phenomenon is specific for dehydrogenases because glutathione S-transferase (GST), a non- dehydrogenase enzyme of nearly identical molecular weight to LDH, is not taken up and retained by dying cells, whereas GST-LDH conjugates are taken up and retained by dying cells (WO 2010/014222).
  • GST glutathione S-transferase
  • This invention is based, at least in part, on the discovery of a conjugate comprising
  • Zr is a radioisotope of zirconium with a half-
  • positron emission tomography imaging for example, with Zr labeled antibodies (immuno-PET) (Van Dongen GA, Vosjan MJ. "Immuno-positron emission tomography: shedding light on clinical antibody therapy”. Cancer Biother Radiopharm. 2010 Aug;25(4):375-85).
  • the Zr is attached to the dehydrogenase using a chelator.
  • chelator can both chelate the Zr and be conjugated to the dehydrogenase.
  • the chelator is selected from the group consisting of desferoxamine (DFO), hydroxypyridinone (HOPO), and catechol.
  • DFO desferoxamine
  • HOPO hydroxypyridinone
  • catechol catechol
  • DFO also known as desferal
  • DFO is a bacterial siderophore (iron chelating compound) produced by the actinobacteria Streptomyces pilosus. It has three hydroxamate groups that are
  • Dehydrogenases are ubiquitous, highly conserved enzymes that catalyze the transfer of a proton and a pair of electrons from a donor molecule to an acceptor molecule, thereby oxidizing the donor and reducing the acceptor. These enzymes typically use NAD/NADP or a flavin coenzyme such as FAD or FMN as the receptor.
  • Dehydrogenases specifically include, but are not limited to, lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, and malate dehydrogenase. Additional representative dehydrogenases are listed in Table 1.
  • dehydrogenases including their nucleotide and amino acid sequences, have been extensively studied and are well known in the art.
  • preparations of these and other dehydrogenases including various individual isoenzymes (isozymes), are available from a number of commercial sources, including, for example, Sigma- Aldrich, St. Louis, Missouri.
  • dehydrogenases which include full-length counterparts, as well as functional fragments thereof, are embraced by the invention, to the extent that they retain the ability to be taken up by dying cells (e.g., apoptotic and necrotic cells) as described herein.
  • conjugates and fusion proteins comprising full-length
  • dehydrogenases or functional fragments of dehydrogenases to the extent that they retain the ability to be taken up by dying cells (e.g., apoptotic and necrotic cells) as described herein.
  • the dehydrogenase used in the invention is a human dehydrogenase. In humans there are at least as many as 205 dehydrogenase genes.
  • the dehydrogenase used in the invention is a lactate dehydrogenase (LDH).
  • LDH lactate dehydrogenase
  • the dehydrogenase used in the invention is a mammalian LDH.
  • lactate dehydrogenase or, equivalently, “LDH” as used herein encompasses the A, B and C forms of LDH as well as cytoplasmic, mitochondrial, or otherwise compartmentalized forms of the enzyme (e.g., in the endoplasmic reticulum or membrane -bound within the cytoplasm).
  • lactate dehydrogenase or, equivalently, “LDH” in one embodiment also includes modified (e.g., mutated, truncated) forms of LDH that retain the ability to be taken up by dying cells (e.g., apoptotic and necrotic cells) in accordance with the invention.
  • lactate dehydrogenase or, equivalently, “LDH” refers to a single subunit (e.g., LDH-A, LDH-B, or LDH-C) or a multimer of subunits (e.g., the mature tetrameric enzyme), or both, depending upon the context.
  • Mammalian LDH is a tetrameric enzyme composed of A, B, and/or C subunits. There are five isozymes of LDH enzyme resulting from the assembly of homotetramers (AAAA (A 4 ) or BBBB (B 4 )) or heterotetramers (ABBB, AABB, AAAB) of the A and B isoforms.
  • the B isoform predominates in heart muscle and facilitates the aerobic oxidation of pyruvate.
  • the A subunit predominates in skeletal muscle and liver and is primarily implicated with anaerobic metabolism and pyruvate reduction to lactate. To date, only the A form has been identified in islet ⁇ -cells.
  • Another LDH isoform, LDH-C is a homotetramer of C (C 4 ) and has been identified in spermatozoa.
  • LDH shares structural similarities with other NAD-binding enzymes.
  • the polypeptide chain of each subunit folds into two clearly separated domains.
  • the two domains have different functions and appear to each comprise a separate module.
  • One of the domains (domain 1) binds to the coenzyme, NAD/NADH, and the second (domain 2) binds the substrate (e.g., pyruvate or lactate) and also provides the amino acid residues that are involved in catalysis.
  • the coenzyme- binding domain is in the amino-terminal portion of the polypeptide.
  • the active site of the enzyme is the cleft or "vacuole" that is formed between the two domains.
  • the coenzyme- binding site on the one domain and the substrate-binding site on the other are oriented so that the C4 position of the nicotinamide ring is in close proximity to the hydrogen atom to be transferred between the substrate and coenzyme.
  • the dehydrogenase used in the invention is a human LDH.
  • lactate dehydrogenase A (muscle), B (heart), and C (testis) polypeptide chains are encoded by individual genes.
  • Human LDH-A and LDH-C genes are on chromosome 11 and human LDH-B gene is on chromosome 12. The expression of these three LDH genes is developmentally regulated and tissue-specific. Markert CL et al.
  • a cDNA sequence for human LDH-A is available as GenBank Accession No. NM_005566; the corresponding amino acid sequence is available as GenBank Accession No. NP_005557.
  • a cDNA sequence for human LDH-B is available as GenBank Accession No. NM_002300; the corresponding amino acid sequence is available as GenBank Accession No. NP_002291.
  • a cDNA sequence for human LDH-C is available as GenBank Accession No. NM_002301; the corresponding amino acid sequence is available as GenBank Accession No. NP_002292.
  • the dehydrogenase used in the invention is an alcohol dehydrogenase (ADH).
  • ADH alcohol dehydrogenase
  • the dehydrogenase used in the invention is a mammalian ADH.
  • the dehydrogenase used in the invention is a human ADH.
  • the term "alcohol dehydrogenase” or, equivalently, “ADH” in one embodiment also includes modified (e.g., mutated, truncated) forms of ADH that retain the ability to be taken up by dying cells (e.g., apoptotic and necrotic cells) in accordance with the invention.
  • Human ADH is a homodimer or heterodimer composed of ⁇ , ⁇ , and/or ⁇ polypeptides encoded by separate gene loci, ADH 1; ADH 2 , and AD3 ⁇ 4.
  • a cDNA sequence for human ADH a is available as GenBank Accession No. NM_000667; the corresponding amino acid sequence is available as GenBank Accession No. NP_000658.
  • a cDNA sequence for human ADH ⁇ is available as GenBank Accession No. NM_000668; the corresponding amino acid sequence is available as GenBank Accession No. NP_000659.
  • a cDNA sequence for human ADH y is available as GenBank Accession No. NM_000669; the corresponding amino acid sequence is available as GenBank Accession No. NP_000660.
  • dehydrogenase used in the invention is a mammalian ALDH. In one embodiment the dehydrogenase used in the invention is a human ALDH.
  • the term "aldehyde dehydrogenase” or, equivalently, “ALDH” in one embodiment also includes modified (e.g., mutated, truncated) forms of ALDH that retain the ability to be taken up by dying cells (e.g., apoptotic and necrotic cells) in accordance with the invention.
  • Aldehyde dehydrogenases are a group of enzymes that catalyze the oxidation (dehydrogenation) of aldehydes.
  • Mitochondrial Aldehyde Dehydrogenase is a polymorphic enzyme responsible for the oxidation of aldehydes to carboxylic acids, which leave the liver and are metabolized by the body's muscle and heart.
  • class 1 low K m , cytosolic
  • class 2 low K m , mitochondrial
  • class 3 high K m , such as those expressed in tumors, stomach and cornea.
  • ALDH1 and ALDH2 are the most important enzymes for aldehyde oxidation, and both are tetrameric enzymes composed of -54 kDa subunits.
  • the dehydrogenase used in the invention is a malate dehydrogenase (MDH).
  • MDH malate dehydrogenase
  • the dehydrogenase used in the invention is a mammalian MDH.
  • the dehydrogenase used in the invention is a human MDH.
  • the term "maltate dehydrogenase” or, equivalently, “MDH” in one embodiment also includes modified (e.g., mutated, truncated) forms of MDH that retain the ability to be taken up by dying cells (e.g., apoptotic and necrotic cells) in accordance with the invention.
  • Malate dehydrogenase (EC 1.1.1.37) is an enzyme in the citric acid cycle that catalyzes the conversion of malate into oxaloacetate (using NAD + ) and vice versa. Malate dehydrogenase is also involved in
  • gluconeogenesis the synthesis of glucose from smaller molecules. Pyruvate in the mitochondria is acted upon by pyruvate carboxylase to form oxaloacetate, a citric acid cycle intermediate.
  • mitochondrial malate dehydrogenase reduces it to malate, which then traverses the inner mitochondrial membrane. Once in the cytosol, the malate is oxidized back to oxaloacetate by cytosolic malate dehydrogenase.
  • phosphoenol-pyruvate carboxy kinase converts oxaloacetate to phosphoenol pyruvate. Banaszak LJ et al.
  • any of the dehydrogenases (e.g., LDH, ADH, ALDH and MDH) suitable for use according to the invention may be a full-length dehydrogenase or a truncated counterpart (e.g., functional fragments as described herein) thereof.
  • the structure-activity relationship of dehydrogenase probes comprising varied fragments or length and their ability to detect apoptotic cells can be established by techniques known in the art. Understanding the structural requirements for binding of dehydrogenase probes to dead cells is important for subsequent optimization of the particular probes for in vivo use.
  • all hLDH subunits appropriately bind to apoptotic cells, even when fused to GST.
  • GST-hLDHA can be systematically mutated to identify the regions that are necessary for binding to apoptotic cells. Then, the domain(s) identified to as necessary can be fused to ectopic proteins, followed by systematic truncation, to determine which domains are sufficient to confer binding to apoptotic cells.
  • protein size limits may be determined for appropriate detection of apoptotic cells, since this may be a critical determinant of in vivo pharmacokinetics.
  • the protein domain structure of a dehydrogenase of interest may be deduced from the amino acid sequence of the protein using appropriate programs that are known in the art, such as the InterPro database, which may provide guidance for designing a truncated dehydrogenase (e.g., a fragment) for use according to the invention.
  • the InterPro database may provide guidance for designing a truncated dehydrogenase (e.g., a fragment) for use according to the invention.
  • hLDHA is analyzed as an example.
  • the InterPro database identifies the following conserved domains in the hLDH amino acid sequence, which may be deleted by site-directed mutagenesis:
  • any other dehydrogenase probes comprising truncated (e.g., fragment) dehydrogenase proteins. Accordingly, to identify the domains of a dehydrogenase probe necessary for detecting dying cells, an expression plasmid encoding the dehydrogenase or fragment thereof (e.g., hLDHA, B, and C) is used, and a truncated form of a probe may be designed based initially on evolutionarily-conserved domains.
  • the expression plasmid is a bacterial expression vector.
  • suitable expression systems are available and known in the art, including a GST-based system.
  • Certain aspect of the invention provide methods for detecting cells undergoing cell death in a subject.
  • the method comprises administering to a subject in need thereof a conjugate of
  • the conjugate in the subject is detected 6, 9, 12, 24, 36, 48, 72, 96, 120 or 144 hours after conjugate administration. In some embodiments, the conjugate in the subject is detected 24 hours or more after conjugate administration.
  • the cells to be detected by the method are cells undergoing apoptosis, which can include early-stage, late-stage, or a combination of early- and late-stage apoptosis.
  • the cells to be detected by the method are cells undergoing necrosis.
  • the cells to be detected by the method are undergoing both apoptosis and necrosis.
  • the Positron Emission Tomography (PET) imaging technology may be employed for detecting the conjugate in the subject.
  • PET/CT imaging technology is known in the art, and the skilled artisan can readily adapt a suitable system, such as a Siemens Inveon preclinical multimodality system. This scanner has been previously shown to provide high resolution and high sensitivity small animal molecular imaging (Visser, E.P. et al., J Nucl Med, 2009. 50: 139- 47).
  • the Inveon CT component provides high resolution anatomical images with intrinsic image co-registration that can be used for definition of regions-of-interest (ROI), and PET attenuation and scatter correction.
  • ROI regions-of-interest
  • a subject in need of detecting cells undergoing cell death is a subject having or suspected of having a condition associate with apoptosis and/or necrosis.
  • a "subject having a condition associated with apoptosis and/or necrosis” is a subject diagnosed with such a condition or having at least one objective feature characteristic of a condition associated with apoptosis and/or necrosis.
  • apoptosis/necrosis is a subject not yet diagnosed with such condition or suspected of having at least one objective feature characteristic of a condition associated with apoptosis and/or necrosis.
  • the subject can be any vertebrate. In some embodiments the subject is a mammal. In some embodiments the subject is a human.
  • the conjugate can be administered to the subject by oral or parenteral routes.
  • a "condition associated with apoptosis and/or necrosis” is a condition such as a disease or disorder which has apoptosis and/or necrosis as a feature characteristic of that condition.
  • An important condition associated with apoptosis is cancer.
  • Additional conditions associated with apoptosis and/or necrosis include, without limitation, chemotherapy-, radiation-, or hormone-induced apoptosis in solid and hematological tumors; tumor resistance to therapy; acute cardiac allograft rejection; acute myocardial infarction; anthracycline-induced cardiotoxicity; arrhythmogenic right ventricle dysplasia; skeletal muscle apoptosis; congestive heart failure; coronary artery disease; atherosclerosis; infectious endocarditis; myocarditis;
  • myocardial dysfunction myocardial ischemia-reperfusion injury; non-cardiac allograft rejection; graft-versus-host disease; bacterial infection; viral infection; multiple organ dysfunction syndrome; septic shock; cerebral ischemia-reperfusion injury; macular degeneration;
  • rheumatoid arthritis systemic lupus erythematosus; inflammatory bowel disease; multiple sclerosis; other autoimmune diseases; annexinopathies; osteoarthritis; renal failure; chronic renal atrophy and renal fibrosis; glomerular injury; and polycystic renal disease.
  • Cancer specifically includes solid malignant tumors and hematological malignancies as well as localized and metastatic cancer. Cancers include, but are not limited to,
  • adenocarcinoma basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and other central nervous system cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small cell and non-small cell lung cancer); lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cancer; cancer of the respiratory system;
  • Transplant rejection specifically includes but is not limited to acute and chronic immune- mediated rejection of kidney, heart, lung, liver, pancreas, small bowel, and skin allografts.
  • Neurodegenerative diseases specifically include but are not limited to Parkinson's disease, Alzheimer's disease, and Huntington's disease.
  • Central nervous system trauma specifically includes but is not limited to spinal cord injury and brain injury.
  • the present invention provides an isolated conjugate comprising dehydrogenase or functional fragment thereof linked to a detectable label, wherein the dehydrogenase in the conjugate is in the monomeric form.
  • conjugates are taken up and retained by dying cells, and are useful for detecting and monitoring cells undergoing cell death, both in vivo as well as in vitro.
  • a test population of cells is contacted with the conjugate, under conditions suitable for uptake of the conjugate by the test population of cells; and the label is detected in the test population of cells, wherein presence of the label in the test population of cells indicates that the cells are undergoing cell death.
  • conjugate is a molecule or complex formed by linking a molecule to at least one other molecule or other moiety.
  • the conjugate is stable under physiological conditions, i.e., the linked components remain linked together under physiological conditions.
  • the linkage can be any suitable chemical or physicochemical linkage, including a covalent bond and a noncovalent bond.
  • the conjugate is a recombinant fusion protein.
  • the linkage between any two components of the conjugate can be direct, i.e., without a linker moiety, or it can be indirect, i.e., it can include a linker moiety connecting the two components.
  • the linkage involves a biotin- strep tavidin interaction.
  • the dehydrogenase is linked to a detectable label via an enzymatically cleavable linkage.
  • the dehydrogenase in the conjugate is in a monomeric form.
  • the term "monomeric" is intended to mean that the dehydrogenase is present in the conjugate is a non- polymeric form.
  • monomeric forms of dehydrogenase include, but are not limited to, a monomeric LDH subunit such as the A form monomer, the B form monomer, or the C form monomer.
  • the conjugate, wherein the dehydrogenase in the conjugate is in the monomeric form is prepared under reducing conditions so that the dehydrogenase subunits do not cross-link.
  • the form of the dehydrogenase in the conjugate can be ascertained by determining the molecular weight of the dehydrogenase in the conjugate using, for example, SDS-polyacrylamide gel electrophoresis under reducing conditions.
  • the dehydrogenase is linked to a detectable label.
  • a detectable label is a molecular or atomic tag or marker that generates or can be induced to generate an optical or other signal or product that can be detected visually or by using a suitable detector.
  • Detectable labels are well known in the art and include, without limitation, fluorescent labels, radioisotopes, nanoparticles, chromophores, dyes, enzymes, and contrast agents.
  • Fluorescent labels commonly used include Alexa, cyanine such as Cy5TM and Cy5.5TM, and indocyanine, and fluorescein isothiocyanate (FITC), but they are not so limited. In general such fluorescent labels and their derivatives can react with carboxyl, amino, or sulfhydryl functional groups of the dehydrogenase. Fluorescent labels useful in the practice of the invention can include, also without limitation, 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichloro fluorescein; 5-Carboxyfluorescein (5-FAM); 5-
  • Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5- Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7- Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin;
  • ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange + DNA; Acridine Orange + RNA; Acridine Orange, both DNA & RNA; Acridine Red; Acridine Yellow; Acriflavin;
  • Acriflavin Feulgen SITS A Aequorin (Photoprotein); Alexa Fluor 350TM; Alexa Fluor 430TM; Alexa Fluor 488TM; Alexa Fluor 532TM; Alexa Fluor 546TM; Alexa Fluor 568TM; Alexa Fluor 594TM; Alexa Fluor 633TM; Alexa Fluor 647TM; Alexa Fluor 660TM; Alexa Fluor 680TM; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA
  • AMCA Aminomethylcoumarin
  • Anilin Blue Anthrocyl stearate
  • APC Allophycocyanin
  • APC-Cy7 APC-Cy7
  • APTRA-BTC Ratio Dye, Zn 2+ ; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAGTM CBQCA;
  • Coelenterazine ; Coelenterazine cp (Ca 2+ Dye); Coelenterazine f; Coelenterazine fcp;
  • Coelenterazine h Coelenterazine hep
  • Coelenterazine ip Coelenterazine n
  • Coelenterazine O Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2TM; Cy3.1 8; Cy3.5TM; Cy3TM; Cy5.1 8; Cy5.5TM; Cy5TM; Cy7TM; Cyan GFP; cyclic AMP
  • Fluorosensor (FiCRhR); CyQuant Cell Proliferation Assay; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR
  • Dihydorhodamine 123 Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-DM6-ASP);
  • DCFH Dichlorodihydrofluorescein Diacetate
  • DiD DiD - Lipophilic Tracer
  • DiD DiD (DiIC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; Red fluorescent protein; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin;
  • LysoSensor Green LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo- 1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF;
  • Noradrenaline Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon GreenTM; Oregon GreenTM 488; Oregon GreenTM 500; Oregon GreenTM 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP- Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL;
  • Rhodamine 5 GLD Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L;
  • Sulphorhodamine B can C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYT; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44;
  • the fluorescent label is a fluorescent protein.
  • the fluorescent protein can be linked to a dehydrogenase by conventional chemical methods, or it can be expressed together with the dehydrogenase as a fusion protein.
  • a wide variety of fluorescent proteins are available in a broad range of the light spectrum, including red, orange, yellow-green, green, cyan and UV-excitable green.
  • Non-limiting examples of fluorescent proteins that can be used as labels include mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, EYFP, Emerald, EGFP, CyPet, mCFPm, Cerulean, T-Sapphire, Citrine, mYFP, ECFP, mCFP, EBFP, AceGFP (available from Evrogen), AcGFPl (available from Clontech), AmCyanl (available from Clontech), AQ143 (available from Lukyanov), AsRed2 (available from Clontech), Azami-Green/mAG (available from MBL Intl.), cOFP (available from Stratagene), CopGFP (available from Evrogen), dimer2, tdimer2(12) (developed by Tsien), DsRed/DsRed2/Ds
  • the fluorescent label is a far-red fluorescent protein suitable for use in whole-body imaging, such as Katushka and derivatives thereof.
  • chromophore is a chemical group that produces color in a compound.
  • a dye is a colored compound, such as a stain, that includes a chromophore.
  • vitamin dyes There are many effective biological stains available in the art. Different stains react or concentrate in different parts of a cell or tissue, and these properties are used to advantage to reveal specific parts or areas. Generally, these dyes may be used with fixed cells and tissues, and some are particularly suitable for use with living organisms ("vital dyes").
  • Non-limiting examples of biological stains that are commonly used include: Bismarck brown, Carmine, Coomassie blue, Crystal violet, DAPI, Eosin, Ethidium bromide, Fuchsin, Haematoxylin, Hoechst stains, Iodine, Malachite green, Methyl green, Methylene blue, Neutral red, Nile blue, Nile red, Osmium tetroxide, Rhodamine, and Safranin.
  • the detectable label is a radioisotope.
  • Radioisotopes are well known in the art and can include Zirconium-89 ( 89 Zr), Copper-64 ( 64 Cu), Niobium-90 ( 90 Nb), Titanium-45 ( 45 Ti), and Gallium-68 ( 68 Ga).
  • the radioisotopes include 3 H, n C, 18 F, 35 S, 67 Ga, 99m Tc, m In, 123 I, 124 I, 125 I, and 131 I.
  • Attachment of any gamma emitting radioactive materials e.g., 99m Tc and U1 ln, which can react with carboxyl, amino, or sulfhydryl groups of dehydrogenase, is suitable for use in detection methods using gamma scintigraphy.
  • Attachment of radioactive compounds which can react with carboxyl, amino, or sulfhydryl groups of dehydrogenase are suitable for use in detection methods using PET/SPECT imaging.
  • the radioisotopes are attached to the dehydrogenase using chelators.
  • the chelator is selected from the group consisting of desferoxamine, hydroxypyridinone, and catechol.
  • Nanoparticle is a particle about 1 nm (10 ⁇ 9 m) to 250 nm in diameter that is capable of being taken up by cells.
  • Nanoparticles specifically include but are not limited to quantum dots, gold nanoparticles, and superparamagnetic particles. Quantum dots, which are also known as fluorescent semiconductor crystals or qdots, have recently been described as highly versatile agents for use in in vivo imaging, tumor targeting, and diagnostics. See, for example, Michalet X et al. (2005) Science 307:538-544. Nanoparticles, including quantum dots and their derivatives, either alone or in combination with other agents that can react with carboxyl, amino, or sulfhydryl groups of dehydrogenase, are suitable for use in detection methods using optical imaging.
  • Tl and T2 contrast agents useful in magnetic resonance imaging can include chelates of gadolinium which can react with carboxyl, amino, or sulfhydryl groups of dehydrogenase, as well as any superparamagnetic nanoparticles such as iron oxide and cobalt oxide which can react with carboxyl, amino, or sulfhydryl groups of dehydrogenase.
  • a detectable label in one embodiment is an enzyme.
  • the enzyme can act on an appropriate substrate to result in production of a detectable dye.
  • enzymes useful in the invention include, without limitation, alkaline phosphatase and horseradish peroxidase.
  • the enzyme can be, for example, luciferase.
  • the enzyme can be linked to a dehydrogenase by conventional chemical methods, or it can be expressed together with the dehydrogenase as a fusion protein.
  • detectable labels useful in the invention include other contrast agents, particularly radiocontrast agents such as barium or other metals, provided in a form which can react with carboxyl, amino, or sulfhydryl groups of dehydrogenase.
  • detectable labels also include any combination of the above-described detectable labels.
  • the detectable label excludes an immunoglobulin or fragment thereof which is capable of binding to the dehydrogenase. In a particular embodiment, the detectable label excludes an immunoglobulin or fragment thereof which is capable of binding to lactate dehydrogenase.
  • Conjugates of the invention can be isolated.
  • isolated means removed from the environment in which, and/or removed from other substances with which, the conjugate may be found in nature.
  • Conjugates of the invention can be purified.
  • the term "purified” means substantially free of other substances.
  • the term “purified” means that a preparation of conjugate is, by weight, at least 80 percent conjugate. In one embodiment such a preparation is, by weight, at least 90 percent conjugate. In one embodiment such a preparation is, by weight, at least 95 percent conjugate. In one embodiment such a preparation is, by weight, at least 99 percent conjugate.
  • Conjugates of the invention can be used in the preparation of compositions that include such conjugates.
  • Such compositions can be prepared by placing at least one conjugate of the invention in contact with at least one other moiety, such as a pharmaceutically acceptable carrier or solvent.
  • the invention accordingly embraces both compositions which include at least one conjugate of the invention, as well as methods for making same.
  • the composition is a pharmaceutical composition suitable for administration to a living subject.
  • the chelator DFO was conjugated to recombinant rabbit LDH.
  • the labeling procedure is as follows: Rabbit lactate dehydrogenase (LDH-B) was incubated for 30 min in a solution of DFO in DMSO at 37°C. Purification by size exclusion chromatography (SEC) and subsequent analysis of the eluent fractions by SDS-PAGE identified the fractions
  • Zr labeled LDH is taken up by cells undergoing apoptosis (FIG. 2).
  • Hepatocyte apoptosis is rapidly induced in mice treated with an anti-Fas antibody (Chang, B. et al. Arch Biochem Biophys, 2003. 411: 63-72; Feng, G. and N. Kaplowitz, J Clin Invest, 2000. 105: 329-39; Nishimura, Y. et al., Int Immunol, 1997. 9: 307-16; Ogasawara, J. et al., Nature, 1993. 364: 806-9).
  • NCr nude mice are injected with either the anti-Fas antibody (Jo2, Pharmingen) or an isotype-matched control antibody.
  • the synchronous induction of apoptosis in hepatocytes in this model system is well established (Chang, B. et al., Arch

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Abstract

L'invention concerne des composés et leurs procédés d'utilisation dans la détection de l'apoptose et de la nécrose. Les composés et procédés reposent sur des conjugués formés avec une déshydrogénase, telle que la lactate déshydrogénase, l'alcool déshydrogénase, l'aldéhyde déshydrogénase et la malate déshydrogénase. Les composés et procédés sont utiles dans le diagnostic d'états caractérisés par l'apoptose, comprenant le cancer, une maladie cardiaque, une maladie neurologique y compris l'accident vasculaire cérébral, et l'auto-immunité.
PCT/US2013/043067 2012-07-12 2013-05-29 Sondes radiomarquées pour la détection et l'imagerie non invasives de la mort cellulaire WO2014011327A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
US8999661B2 (en) 2008-07-30 2015-04-07 Dana-Farber Cancer Institute, Inc. Compositions for detecting cell death and methods of use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100034800A1 (en) * 2008-07-30 2010-02-11 Dana-Farber Cancer Institute, Inc. Compositions for detecting cell death and methods of use thereof
US20100111856A1 (en) * 2004-09-23 2010-05-06 Herman Gill Zirconium-radiolabeled, cysteine engineered antibody conjugates

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100111856A1 (en) * 2004-09-23 2010-05-06 Herman Gill Zirconium-radiolabeled, cysteine engineered antibody conjugates
US20100034800A1 (en) * 2008-07-30 2010-02-11 Dana-Farber Cancer Institute, Inc. Compositions for detecting cell death and methods of use thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8999661B2 (en) 2008-07-30 2015-04-07 Dana-Farber Cancer Institute, Inc. Compositions for detecting cell death and methods of use thereof

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