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WO2016001671A1 - Agent d'imagerie de peptides ciblé sur la partie extracellulaire de cd115 - Google Patents

Agent d'imagerie de peptides ciblé sur la partie extracellulaire de cd115 Download PDF

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Publication number
WO2016001671A1
WO2016001671A1 PCT/GB2015/051931 GB2015051931W WO2016001671A1 WO 2016001671 A1 WO2016001671 A1 WO 2016001671A1 GB 2015051931 W GB2015051931 W GB 2015051931W WO 2016001671 A1 WO2016001671 A1 WO 2016001671A1
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WIPO (PCT)
Prior art keywords
seq
peptide
imaging agent
expression
agent according
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English (en)
Inventor
René Michael BOTNAR
Gabriella PASSACQUALE
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Kings College London
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Kings College London
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • 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
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the invention relates to an imaging agent which specifically targets Cluster of Differentiation 115 (CD115), also known as Colony Stimulating Factor 1 Receptor (CSFR1), and Macrophage Colony-Stimulating Factor Receptor (M-CSFR).
  • CD115 Cluster of Differentiation 115
  • CSFR1 Colony Stimulating Factor 1 Receptor
  • M-CSFR Macrophage Colony-Stimulating Factor Receptor
  • i8 FDG positron emission tomography-computed tomography
  • 18 F-fluorodeoxyglucose (i8 FDG)-PET which is currently used clinically, especially for cancer imaging, has been proposed as an alternative approach to detect arterial inflammation, based on pre-clinical evidence that macrophages take up 18 FDG more avidly than other cell types, thus providing intense signal within inflamed tissues.
  • i8 FDG uptake can be altered in patients with metabolic disorders (obesity and diabetes), since it depends on cellular glucose transport, which is deranged in such conditions; and this potentially constitutes an important limiting factor to its usefulness in patients at high cardiovascular risk.
  • image acquisition of the coronary arteries is compromised by background noise due to myocardial tracer uptake.
  • the inventors have developed a new imaging agent comprising a portion of the amino acid sequence which encodes for the human macrophage colony-stimulating factor (M-CSF), which is the autologous ligand of CD115 (Chitu et al, Curr Opin Immunol. 2006 Feb; 18(1): 39.48).
  • Chen X, et al. (Chen X, et al. Proc Natl Acad Sci USA 2008; 105:18267-18272) conducted an X-ray crystallograpbic analysis of the interface between murine M-CSF and its cognate ligand.
  • the human M-CSF protein in the form of a truncated isoform binding the human CD 115, has been characterised by Panditi J et al (Panditi J, et al. Science 1992; 258(5086): 1358-1362).
  • the imaging agent of the present invention is targeted to the extracellular portion of CD115, which is a universal marker of myeloid cell lineage, equally and highly expressed on both human and murine monocytes (Ingersoll MA, et al. Blood 2010; 115: el0-el9). Low levels of expression also are found on granulocytes.
  • CD115 on their extracellular membrane, including Kupffer cells, microglia, and osteoclasts.
  • abnormal expression of CD 115 has been described in breast and ovarian cancer and myeloid leukemic blast cells.
  • Increased expression of CD115 has also been reported in astrocytes and neurons following injury.
  • the imaging agent of the present invention specifically localises within, and enhances, inflamed atherosclerotic vessels without any evidence of accumulation within disease-free regions of the arterial wall.
  • the localisation of the imaging agent within atherosclerotic plaques is not attributable to non-specific accumulation of the peptide in the vascular wall, but rather to a targeted action. Indeed, this imaging agent has high specificity for myeloid cells given its ability to bind to the extracellular domain of CD115.
  • Targeted MRI-imaging agents have been developed and described in the literature by other groups, but they generally consist of iron-oxide/microcelle particles which, due to their molecular mass, show poor tissue permeability.
  • the present imaging agent In addition, they generally undergo cellular intemalisation by targeted cells so that accumulation within tissues persists for weeks before removal via the reticuloendothelial system.
  • the present imaging agent remains localised within the extracellular space and has a low molecular mass.
  • the present imaging agent is suitable for MR1 imaging which provides improved anatomical resolution.
  • Other targeted MRI imaging agents have been developed (US2006/0239913), but the specificity of the present imaging agent for CD115 makes it uniquely specific for the detection of myeloid cells.
  • an imaging agent comprising:
  • SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence that differs from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by 1, 2 or 3 amino acids, wherein the peptide binds to a human
  • the inventors have surprisingly found that this imaging agent is specifically targeted to the extracellular portion of CD115.
  • the specificity of the imaging agent for CD115 makes it uniquely specific for detection of monocytes/macrophages, as well as of other cell types belonging to the myeloid cell lineage and expressing CD115.
  • the imaging agent of the present invention localises within atherosclerotic plaques and gives plaque enhancement without any evidence of accumulation within healthy vessels.
  • the imaging agent of the present invention has a low molecular mass ( ⁇ 1 kDa) and does not bind to plasma proteins such as albumin, (molecules with molecular mass ⁇ 10 kDa are freely filtered by the glomeruli when unbound to plasma proteins) and this not only allows for excellent tissue penetration but also accounts for a favourable pharmacokinetic profile by undergoing rapid (within hours) renal clearance. Binding to CD115 by the imaging agent does not cause receptor activation. These are important safety considerations. Additionally, the imaging agent of the present invention is suitable for MRI imaging which provides optimal anatomical resolution in a non-invasive manner.
  • the peptide has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that differs from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by 1, 2 or 3 amino acids.
  • This means that the peptide is selected from the amino acid sequence of SEQ ID NO: 1 with 1, 2 or 3 amino acid changes, the amino acid sequence of SEQ ID NO: 2 with 1, 2 or 3 amino acid changes, the amino acid sequence of SEQ ID NO: 3 with 1, 2 or 3 amino acid changes, or the amino acid sequence of SEQ ID NO: 4 with 1, 2 or 3 amino acid changes.
  • the peptide is selected from a peptide having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that differs from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by 1 or 2 amino acids.
  • the peptide is selected from a peptide having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that differs from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by one amino acid.
  • the peptide has the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In a particularly preferred embodiment, the peptide has the sequence of SEQ ID NO: 1 or SEQ ID NO: 4.
  • the imaging agent may comprise more than one peptide as defined above.
  • the imaging agent may comprise a single signal entity which is bound to a plurality of peptides, preferably two peptides.
  • the plurality of peptides may, or may not be, identical.
  • peptide refers to any peptide comprising amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • the term "peptide” as used herein is generally between 5 and 15 amino acids in length.
  • the peptide generally will contain naturally occurring amino acids but may include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts. Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini.
  • a given peptide may contain many types of modifications.
  • the peptide may comprise amino acid isomers. Such isomers are well known to one skilled in the ait and may include, for example, norleucine and norvaline.
  • a "deletion" is defined as a change in an amino acid sequence in which one or more amino acid residues, respectively, are absent.
  • an "insertion” or “addition” is a change in an amino acid sequence which has resulted in the addition of one or more amino acid residues, respectively, as compared to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • substitution is defined as the replacement of one or more amino acids by different amino acids, respectively.
  • the peptide may have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent peptide, i.e., a peptide that binds to human CDl 15.
  • amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicit , and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the peptide has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that differs from SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by 1, 2 or 3 amino acids.
  • the amino acid differences include any substitution of, variation of, modification of, replacement of, deletion of or addition of an amino acid.
  • a number of different changes can be made, for example, one amino acid could be substituted and another amino acid added, provided the peptide only differs by 3 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • One skilled in the art can determine which amino acids to change. For example, one skilled in the art would be able to determine which amino acids can be changed without affecting the peptide's tertiary structure.
  • the peptide binds to a human CDl 15 receptor.
  • Methods for determining that the peptide binds to the human CDl 15 receptor are known in the art, and are described in detail elsewhere in this document.
  • the signal entity is any signal entity that enables imaging.
  • the signal entity is suitable for medical imaging.
  • Medical imaging includes magnetic resonance imaging (MRI), nuclear medicine, x-ray, ultra-sound, optical imaging and fluoroscopy.
  • the medical imaging is MRI.
  • suitable signal entities include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles, and the like.
  • the signal entity is a metal chelate.
  • the chelate may be a linear or macrocyclic chelate.
  • Chelates for magnetic resonance imaging are selected to form stable complexes with paramagnetic metal ions, such as Gd (III), Dy (III), Fe (III), Mn (III) and Mn (II), and include the residue of a polyaminopolycarboxylic acid, either linear or cyclic, in racemic or optically active form, such as ethylenediaminotetracetic acid (EDTA), diethylenetriammopentaacetic acid (DTP A), N-[2- [bis(carboxymethyl)amino]-3 -(4-ethoxyphenyI)propyl] -N- [2-
  • paramagnetic metal ions such as Gd (III), Dy (III), Fe (III), Mn (III) and Mn (II)
  • EDTA ethylenediaminotetracetic acid
  • DTP A diethylenetriammopentaacetic acid
  • DTPA-GLU N,N-bis[2- [bis(carboxymethyl)amino]ethyl]-L-lysine
  • DTPA-LYS N,N-bis[2- [bis(carboxymethyl)amino]ethyl]-L-lysine
  • DTPA-BMA 4-carboxy-5,8,l l-tris(carboxymethyl)-l-phenyl-2-oxa-5,8,l 1- triazatridecan-13-oic acid (BOPTA), 1,4,7, 0-tetraazacyclododecan- 1,4,7,10-tetraacetic acid (DOTA), 1 ,4,7, 10-tetraazacyclododecan- 1,4,7- triacetic acid (D03A), 10-(2-hydroxypropyl)- l,4,
  • Usable chelates may also be DOTA gadofluorins, D03A, HPD03A, TETA, TRITA, HETA, DOTA-NHS, M4DOTA, M4D03A, PCTA and their derivatives, 2-benzyl-DOTA, alpha- (2-phenethyl) l,4,7,10,tetraazacyclododecane-l-acetic-4,7,10-tris (methylacetic) acid, 2benzyl- cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6methyl-DTPA, and 6,6"-bis [N, N, N " , N” teti'a(carboxymethyl)aminomethyI)-4'-(3 -amino-4-methoxyphenyl)-2,2' : 6',2 " - terpyridine, N,N'-bis-(pyridoxal-5-phosphate) ethylenediamine-N,N'-
  • Appropriate chelates are not limited to this list; other chelates with good efficiency in imaging diagnostics may be used.
  • Preferred chelators are DTP A, DOTA, DTPA BMA, BOPTA, D03A, HPD03A, TETA, TRITA, HETA, M4DOTA, DOTMA, MCTA, PCTA and the derivatives thereof.
  • the chelate is preferably DOTA.
  • the signal entity is a chelate
  • the signal entity comprises a chelate complexed with a metal ion.
  • Preferred metal ions are paramagnetic metal ions including ions of transition and lanthanide metals (i.e. metals having atomic number of 21 to 29, 42 to 44, or 58 to 70).
  • ions of Mn, Fe, Co, Ni, Eu, Gd, Dy, Tm, and Yb are preferred, with those of Mn, Fe, Eu, Gd, and Dy being more preferred and Gd being the most preferred.
  • Gd is preferred for use in clinical MRI,
  • the metal chelate is selected to form stable complexes with the metal ion chosen for the particular application.
  • Chelators or bonding moieties for diagnostic radiopharmaceuticals are selected to form stable complexes with the radioisotopes that have imageable gamma ray or positron emissions such as 99 Tc, 1 !7 Sn, H1 In, 97 Ru, 7 Ga, 68 Ga, 8 3 ⁇ 4r, 177 Lu, 47 Sc, 105 Rh; 188 Re, 60 Cu, 62 Cu, 64 Cu, 67 Cu, 90 Y, 159 Gd, l49 Pr, 166 Ho.
  • Chelators for technetium are selected preferably from diaminedithiols, monoamine- monoamidedithiols, triamide- monothiols, monoamine-diamide-monothiols, diaminedioximes, and hydrazines.
  • the chelators are generally tetradentate with donor atoms selected from nitrogen, oxygen and sulfur.
  • Preferred reagents are comprised of chelators having amine nitrogen and thiol sulfur- donor atoms and hydrazine bonding units.
  • the thiol sulfur atoms and the hydrazines may bear a protecting group which can be displaced either prior to using the reagent to synthesize a radiopharmaceutical or preferably in situ during the synthesis of the radiopharmaceutical.
  • Chelators for 11 ' ⁇ , ⁇ , copper and gallium isotopes are typically selected from cyclic and acyclic polyaminocarboxylates such as DTP A, DOTA, D03A, 2benzyl-DOTA, alpha- (2- phenethyl) 1,4,7,10-tetraazazcyclododecanel -acetic-4,7,10-tris(methylacetic)acid, 2- benzylcyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl DTP A, and 6,6"-bis [N, N, N", N"-tetra(carboxymethyl)aminomethyl4'-(3-amino-4-meth
  • the signal entity is a fluorescent agent for time-resolved fluorescence assays.
  • the unique fluorescent properties of lanthanide metals which are long- lived luminescence molecules, make them attractive for application in cellular imaging due to the lack of overlapping spectra with autofiuorescent molecules.
  • Lanthanide chelates have been successfully applied to in vivo cellular imaging (microscopy and spectroscopy) as well as in vitro bioassays for protein detection.
  • the fluorescent signal emitted by lanthanides upon excitation is generally detected by time-resolved fluorescence or used as a source of excitation light for near infrared fluorescent (NIRF) molecules. The excitation light is absorbed by the NIRF molecule that then emits light (fluorescence) spectrally distinguishable (longer wavelength) from the excitation light.
  • NIRF near infrared fluorescent
  • Fluorescent agents are suited for studying biological phenomena, as has been done extensively in fluorescence microscopy. If fluorescent probes are to be used in living systems, the choice is generally limited to the near infrared spectrum (600-1000 nm) to maximize tissue penetration by minimizing absorption by physiologically abundant absorbers such as hemoglobin ( ⁇ 550 nm) or water (>1200 nm). Typically the fluorochromes are designed to emit at 800+/-50 nm.
  • NIRF molecules A variety of NIRF molecules have been described and/or are commercially available, including: Cy5.5 (Amersham, Arlington Heights, III.); NIR-1 (Dojindo, umamoto, Japan); 1RD382 (LI-COR, Lincoln, Nebr.); La Jolla Blue (Diatron, Miami, Fla.); ICG (Akorn, Lincolnshire, III.); and ICG derivatives (Serb Labs, Paris, France). Quantum dots derivatives (inorganic fluorophores comprising nanocristal) may also be used.
  • Lanthanide chelates have also been used as energy donors to excite NIRF molecules, such a technique being applied to both in vivo imaging and in vitro bioassays.
  • the peptide is directly bound to the signal entity.
  • the peptide is bound to the signal entity by a linker.
  • the peptide is directly bound to the signal entity.
  • the signal entity may be bound to the peptide at any suitable position provided the peptide remains capable of binding to the human CD115 receptor.
  • the signal entity may be bound to the N-terminus or the C-terminus of the peptide.
  • the signal entity may be bound at some point between the N-terminus and the C-teiminus of the peptide.
  • the peptide is bound to the N-terminus.
  • the signal entity is bound to both the N-terminus and the C-terminus.
  • the signal entity may be bound to the peptide via a linker.
  • Any suitable linker may be used including peptide linkers and chemical linkers. Suitable linkers are well known to those skilled in the ait.
  • a second aspect of the invention relates to the use of the imaging agent of the present invention for identifying the level of CD115 expression in an individual.
  • an altered level of CD115 expression is identified.
  • an altered level of CD 115 expression it is meant an altered level compared to the level observed in a normal, healthy individual.
  • an increase in the level of CD115 expression is identified.
  • an increased level of CD115 expression indicates the presence of tissue inflammation as indicated by the presence of CD115 -expressing cells, predominately myeloid cells such as monocytes and macrophages.
  • Increased CD115 expression, or the presence of myeloid cells can be identified in an individual or in a sample (e.g., tissue sample) obtained from an individual, in particular, a human.
  • blast cells expressing CD115 characterise acute myeloid leukemia; myeloid cell infiltration of solid tumours contributes to neoangiogenesis and cancer vascularisation and growth, thus influencing the individual's prognosis; ovarian and breast cancers can display increased level of CD1 15 on tumorigenic cells and this is considered a negative prognostic factor; and liver cancer/metastasis replaces normal hepatic tissue with absence of Kupffer cells, thus leading to a loss of hepatic CD 115 expression. Finally, increased colonisation of transplanted organs by myeloid cells predict the risk of allograft rejection.
  • the peptide and the signal entity are as defined above.
  • the invention provides a method of using the imaging agent of the present invention for identifying the level of CD115 expression in an individual comprising administering to an individual the imaging agent of the present invention and acquiring an image of a site of concentration of said imaging agent in the individual by a diagnostic imaging technique.
  • an altered level of CD115 expression is identified.
  • an altered level of CD115 expression it is meant an altered level compared to the level observed in a normal, healthy individual.
  • an increase in the level of CD 1 15 expression is identified.
  • an increased level of CD115 expression indicates the presence of tissue inflammation as indicated by the presence of CD115 -expressing cells, predominately myeloid cells such as monocytes and macrophages.
  • the diagnostic imaging technique may include magnetic resonance imaging (MRI), nuclear medicine, x-ray, ultra-sound, optical imaging and fluoroscopy.
  • the digital imaging technique is MRI.
  • the invention provides a method of acquiring an image of a site of concentration of the imaging agent of the present invention, wherein the imaging agent has been previously administered to an individual.
  • the imaging is for non-diagnostic purposes.
  • the image is acquired without any physical contact with the individual's body, e.g., by scanning the body.
  • the image is obtained on a tissue sample.
  • the image which is acquired will not lead to an immediate diagnosis but will contribute to a diagnosis.
  • a third aspect of the invention relates to the imaging agent of the present invention for use in diagnosing a disease or disease risk in which levels of CD115 expression are altered.
  • the levels of CD115 expression are increased.
  • Increase in the level of CD115 within organs indicates the presence of tissue inflammation as determined by the presence of CD115-expressing cells, predominately myeloid cells such as monocytes and macrophages.
  • tissue inflammation as determined by the presence of CD115-expressing cells, predominately myeloid cells such as monocytes and macrophages.
  • Particular cardiovascular diseases, neurological diseases, autoimmune disorders, cancers and organ transplantation where tissue infiltration by myeloid cells is clinically relevant are listed above.
  • the peptide and the signal entity are as defined above.
  • a fourth aspect of the invention relates to the imaging agent of the present invention in the manufacture of an agent for diagnosing a disease or disease risk in which levels of CD115 expression are altered.
  • the levels of CD115 expression are increased.
  • Increase in the level of CD115 within organs his indicates the presence of tissue inflammation as determined by the presence of CD115-expressing cells, predominately myeloid cells such as monocytes and macrophages.
  • tissue inflammation as determined by the presence of CD115-expressing cells, predominately myeloid cells such as monocytes and macrophages.
  • Particular cardiovascular diseases, neurological diseases, autoimmune disorders, cancers and organ transplantation where tissue infiltration by myeloid cells is clinically relevant are listed above, The peptide and the signal entity are as defined above.
  • a fifth aspect of the invention relates to use of an imaging agent for diagnosing a disease or disease risk in which levels of CD 115 expression are altered.
  • the levels of CD 115 expression are increased.
  • Increase in the level of CD115 within organs indicates the presence of tissue inflammation as indicated by the presence of CD115-expressing cells, predominately myeloid cells such as monocytes and macrophages.
  • tissue inflammation as indicated by the presence of CD115-expressing cells, predominately myeloid cells such as monocytes and macrophages.
  • Particular cardiovascular diseases, neurological diseases, autoimmune disorders, cancers and organ transplantation where tissue infiltration by myeloid cells is clinically relevant are listed above.
  • the peptide and the signal entity are as defined above.
  • diagnosis a disease or disease risk refers to determining whether an individual has a certain disease or has an increased risk of developing the disease compared to the general population.
  • high level of vascular tissue infiltration by myeloid cells is an indicator of certain cardiovascular diseases, especially atherosclerosis, as discussed above.
  • abnormal tissue infiltration by myeloid cells is indicative of a inflammatory disorder.
  • a further aspect of the invention relates to a method of identifying a patient at high risk of an acute ischemic attack by determining the degree of inflammation within atherosclerotic plaques in the patient comprising administering to the patient the imaging agent of the present invention and acquiring an image of a site of concentration of said imaging agent in the patient by MRI.
  • the peptide and the signal entity are as defined above.
  • the invention also provides a pharmaceutically acceptable composition comprising the imaging agent of the present invention and one or more excipients.
  • the composition may comprise a plurality of signal entities, each signal entity having one or more peptides bound to it.
  • the peptides bound to each signal entity may, or may not be, identical.
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise one or more suitable excipients. Acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as, or in addition to, the excipient any suitable binder, lubricant, suspending agent, coating agent or solubilising agent. Preservatives, stabilizers and dyes may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid. Antioxidants and suspending agents may be also used
  • the pharmaceutical composition of the present invention may be formulated to be delivered parenterally in which the composition is formulated in an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the disease, age, weight and response of the particular patient.
  • the appropriate dosage can be determined by one skilled in the art.
  • compositions of the present invention may be administered by parental or mucosal routes.
  • the composition may be formulated for parenteral (i.e. intramuscular, intravenous or subcutaneous) and mucosal administration.
  • parenteral i.e. intramuscular, intravenous or subcutaneous
  • mucosal administration i.e. intramuscular, intravenous or subcutaneous
  • the invention provides a method of preparing an imaging agent comprising coupling the peptide to the signal entity.
  • the peptide may be coupled to the signal entity directly or via a linker as described above. Techniques for doing so are well known to one skilled in the art.
  • the peptide and the signal entity are as defined above.
  • a further aspect of the invention relates to a peptide having the amino acid sequence of SEQ ID NO: I, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that differs fiom SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by 1, 2 or 3 amino acids, wherein the peptide binds to a human CD 1 15 receptor.
  • the peptide has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that differs SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO; 4 by 1 or 2 amino acids, wherein the peptide binds to a human CD115 receptor.
  • the peptide has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that differs fiom SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by one amino acid, wherein the peptide binds to a human CD115 receptor.
  • the peptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the peptide is SEQ ID NO: 1 or SEQ ID NO: 4.
  • the invention also provides for a kit comprising a peptide as defined above, a signal entity as defined above, and components for coupling the peptide to the signal entity.
  • the components are generally reagents for enabling the coupling of the peptide to the signal entity, and may comprise a linker.
  • Figure 1 shows the secondary structure of human and murine recombinant M-CSF (truncated isoforms). Areas of homology between the two species are highlighted in grey.
  • Figure 2 shows the results of a fluorescence antibody competition binding assay.
  • the left panel shows a dose-dependent decrease in the percentage of CD14 + CD115 + monocytes as detected by whole blood flow cytometry following pre-treatment of human samples with Gd- MoCA at the different concentrations as indicated. (*p ⁇ 0.05 vs 0 mM (control).)
  • Statistical analysis was by repeated measures ANOVA with Dunnett's post-test correction.
  • the right panel shows a time-course performed with Gd-MoCA (1.5 mM), M-CSF1 (100 ng/ml) and Magnevist (1.5 mM) as indicated.
  • the dotted line indicates level of expression of CDl 15 in untreated blood at 37°C for 60 minutes (control), which was further reduced by M-CSF1 and Gd-MoCA, but not Magnevist treatment.
  • Figure 3 illustrates CyTOF-based characterisation of cellular distribution of Gd-MoCA in human blood.
  • Post-acquisition analysis was gated for all cell types as identified by DNA-Ir staining (i,ii,iii; upper box).
  • Staining of whole blood with Gd-MoCA identified two distinct cell populations: highly-expressing cells were mainly constituted of CD14 + cells (monocytes) whilst low-expressing cells mainly included CD15 + cells (neutrophils).
  • Figure 4 shows a DELFIA-based binding assay.
  • HSA human serum albumin
  • CDl 15 human recombinant CDl 15
  • No binding was found for HSA.
  • Figure 6 shows CDl 15 expression in total cell protein lysates from human PBMCs.
  • Figure 7 shows in vivo Magnevist-based DE-MRI in wild-type mice.
  • Panel A shows an abdominal scan of a wild-type mouse injected with Magnevist (0.2 mmol/kg of body weight). Acquisition was pre-injection (baseline) and at 30 and 60 minutes post-injection.
  • Panel B reports SNR (signal-to-noise ratio) in the different organs and at the different time points as indicated. (* p ⁇ 0.05, ** p ⁇ 0.01, repeated measures ANOVA with Bonferroni's post test correction.)
  • Figure 8 shows in vivo Gd-MoCA-based DE-MRI in wild-type mice.
  • Panel A shows an abdominal scan of wild-type mice injected with Gd-MoCA (0.2 mmol/kg of body weight). Acquisition was pre-injection (baseline) and at 30 and 60 minutes post-injection.
  • Panel B reports SNR (signal-to-noise ratio) in the different organs and at the different time points as indicated. (* p ⁇ 0.05, ** p ⁇ 0.01, repeated measures ANOVA with Bonferroni's post test correction.)
  • Figure 9 shows in vivo Gd-MoCA-based DE-MRI in ApoE _ " mice.
  • Panel A shows an abdominal scan of ApoE "/_ mice injected with Gd-MoCA (0.2 mmol/kg of body weight). Acquisition was pre-injection (baseline) and at 30 and 60 minutes post-injection.
  • Panel B reports SNR (signal-to-noise ratio) in the different organs and at the different time points as indicated.
  • Figure 10 shows in vivo Tl mapping.
  • Images on the left-hand side and in the middle show the Tl and l maps respectively on the MRI abdominal scanning of ApoE " _ mice injected with either Magnevist or Gd-MoCA (both at a dose of 0.2 mmol/ kg of body weight) as indicated, in comparison with the pre-injection scans.
  • Corresponding DE-MRI on Tl-weigheted images are shown on the right-hand side.
  • the graph display the relaxivity (Rl) in the different organs. (*p ⁇ 0.05 vs pre-injection, repeated measures ANOVA with Bonferroni's post test correction.)
  • Figure 11 shows in vivo Gd-MoCA-based MRI of atherosclerotic plaques in ApoE ⁇ A mice.
  • A 3D reconstruction and axial view time of flight (TOF) MR images of the aortic arch and associated vessels, showing the level of the axial scanning (green line).
  • LSC left subclavian artery
  • LCC left common carotid artery
  • BC brachiocephalic artery
  • Ao aorta.
  • Delayed enhancement (DE) MRI image and composite DE and TOF images overlayed at 30 (top) and 60 minutes (bottom) post-Gd-MoCA injection. Arrow indicates the area of delayed enhancement corresponding to the posterior brachiocephalic wall.
  • Regions of interest were selected in the wall of the brachiocephalic, left common carotid and left subclavian arteries and an area outside of the scan defined as "noise", and the signal to noise ratios (SNRs) were calculated in three different slices of DE-MR images from each animal.
  • SNRs signal to noise ratios
  • C Modification of Gd-MoCA (1-DOTA) with a Gd-DOTA moiety also at the C-terminus via lysine addition to the amino acid sequence (2-DOTA) compromised the ability of the contrast agent to enhance atherosclerotic plaques.
  • Figure 12 shows a DELFIA-based binding assay, in particular, peptide binding to human recombinant CD 115. Best fitting curve was One site - Specific Binding with Hill slope for both peptides.
  • Figure 13 shows a DELFIA-based binding assay (panel A), in particular, peptide SEQ ID NO: 4 binding to human recombinant CD115. Best fitting curve was One site - Specific Binding with Hill slope.
  • Panel B shows in vivo MRI of atherosclerotic plaques in ApoE " " mice injected with SEQ ID NO: 4. 3D reconstruction and axial view time of flight (TOF) MR images of the aortic arch and associated vessels, showing the level of the axial scanning (middle line).
  • Figure 14 shows in vivo Gd-MoCA-based MRI of atherosclerotic plaques in ApoE _ " mice at different time points of disease progression as indicated.
  • 3D reconstruction on the top
  • TOF axial view time of flight
  • LS left subclavian artery
  • LCC left common carotid artery
  • BCA brachiocephalic artery
  • DE Delayed enhancement
  • Arrows indicates the area of delayed enhancement corresponding to the posterior brachiocephalic wall.
  • Gd-MoCA Monocyte Imaging agent
  • CD115 also laiown as c-fms or CSF-Rl
  • the peptide consists of amino acid residues 55-66 (SEQ ID NO: 1) of the human macrophage colony-stimulating factor (M-CSF, GeneBank: AAA59573.1), which is the autologous ligand of CD115.
  • the 12 mer-peptide was selected among regions of homology between the murine and human species (Fig.l).
  • the compound was synthesised in the form of a DOTA-peptide at the N-terminus to allow for Gd 3+ complexation, therefore providing paramagnetic properties to the molecule, thus making it suitable for an MR! application.
  • MoCA was synthesised by Peptide Synthetics (Peptide Protein Research Limited, UK) in the form of a DOTA-peptide.
  • the DOTA-peptide was complexed to the lanthanide metal gadolinium (III) (Gd3 + ) according to a previously published protocol incorporated herein by reference (Kotek J et al. Synthesis and Characterization of Ligands and their Gadolinium(III) Complexes, hi: The Chemistry of Imaging agents in Medical Magnetic Resonance Imaging, 2nd Edition, edited by Merbach A, Helm L and Toth E. London: John Wiley & Sons Ldt, 2013, p. 83-156.).
  • the DOTA- peptide was complexed to the lanthanide metal Europium (Eu 3+ ) and tested by DELFIA time- resolved fluorescence assay. Briefly, 1 mg of MoCA was reconstituted in 1 ml ddH 2 0 (5.74 x 10 "4 M), from which a stock solution of 2 x 10 "5 M was prepared. An equimolar concentration of EuCl 3 .6H20 (Sigma- Aldrich, UK) previously resuspended in ddFTiO was added to the stock solution of MoCA. In both cases, Ianthanide complexation of MoCA occurred under conditions of constant stirring, at room temperature and at pH 7.0. The reaction was terminated when no free lanthanides could be detected in the solution as determined by the xylenol orange test (Barge A, et ah Contrast Media & Molecular Imaging 1 : 184-188, 2006).
  • Fluorescence antibody competition binding assays and time of flight mass cytometry were used to determine the specificity of Gd-MoCA for the myeloid cell lineage and its ability to specifically target CD115.
  • Experiments were conducted in vitro on human whole blood obtained from healthy volunteers and collected in EDTA vacutainer tubes (Becton & Dickinson, BD, UK).
  • Eu-MoCA was tested by DELFIA time-resolved fluorescence assay against a human recombinant His-tagged CD115 protein (Life Technologies, UK) to assess its binding affinity to the target.
  • Gd-MoCA 0.05 x 10 "6 , 5 x 10 "6 , 2 x 10 "5 , 5 x 10 "5 , 1 x lO 4 , 5 x 10 4 1 x 10 '3 , 1.5 xlO "3 M
  • a time course (0, 5, 15, 30 and 60 minute incubation) was also performed with Gd-MoCA at a final concentration of 1.5 x 10 ⁇ 3 M in blood.
  • Samples were washed as above in filtered 0.2% BSA/0.1% S A/PBS and then incubated for 30 minutes at room temperature with a cocktail of primary metal- conjugated antibodies (Er 170 -CD3, Pr l43 -CD1 , Dy 164 -CD1 , Sm 147 -CD20, and a secondary Gd-conjugated anti-PE antibody for detection of anti-human PE-CD115 antibody) (all from DVS Sciences Ltd., UK). After washing in filtered 0.2% BSA/0.1% SA PBS, samples were then fixed in 500 ⁇ 1% PFA overnight.
  • a cocktail of primary metal- conjugated antibodies Er 170 -CD3, Pr l43 -CD1 , Dy 164 -CD1 , Sm 147 -CD20, and a secondary Gd-conjugated anti-PE antibody for detection of anti-human PE-CD115 antibody
  • the cell pellet was subsequently resuspended in 500 ⁇ 0.2% BSA/0.1% SA/PBS containing 10% saponin for permeabilisation, and incubated with 1 ⁇ of an Ir-DNA intercalating dye for 20 minutes at room temperature. Samples were washed as above and fixed in 500 ⁇ 1.6% PFA for at least 24 hours at +4°C before data acquisition.
  • Multiwell plates were acquired at a spectiOfluorometer (TECA , Infinite 200 PRO) with the following instrument settings : excitation 340 urn; emission 617 nm; delay 400 msec; window 400 msec; gain 100; Z-position 20000 mm.
  • the intensity of fluorescence of the different peptide concentrations were subtracted by the non-specific binding. Values were plotted against concentrations and analysis was by GraphPad Prism using non linear regression analysis (Specific Binding with or without Hill slope). Similar experiments were performed to assess the binding of MoCA to human serum albumin (HSA).
  • HSA human serum albumin
  • the Eu-DOTA peptide was tested against immobilised HSA (Sigma, UK; 96-multiwell plate coating with HSA was overnight at 4 °C using a 4.3% solution made in PBS).
  • PBMCs peripheral blood mononuclear cells
  • PBMC pellet was either processed immediately for cell protein extraction (as "fresh blood") or incubated with either 50 ⁇ 0.9% w/v saline solution, Magnevist® (1.5 mM), M-CSF (100 ng ml) or Gd-MoCA (1.5 x 10 "3 M) for 60 minutes at +37°C.
  • Magnevist® 1.5 mM
  • M-CSF 100 ng ml
  • Gd-MoCA 1.5 x 10 "3 M
  • Protein concentration of the samples was determined by bicinchoninic acid (BCA) assay. Forty micrograms of protein were then loaded and separated in a 10% SDS-PAGE gel (30% acryiamide, 1.5 M Tris, 10% SDS, 10% APS & 0.004% TEMED) and transferred to a polyvinylflouride (PVDF) membrane (Millipore, UK) by semi-dry transfer at 30 mA for 2 hours.
  • BCA bicinchoninic acid
  • Membranes were subsequently incubated with rabbit anti-human CD 115 antibody targeting the infracellular portion of the protein (Cell Signalling, UK) (1 :250 in 5% milk-TBST, 2 hours at room temperature), followed by incubation with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1 :5000 in 5% milk-TBST). The signal was revealed by ECL chemiluminescence substrate (Thermo Fisher Scientific, UK). Protein normalisation was with rabbit anti-human ⁇ -actin (Cell Signalling Technologies, UK) (1 :5000 in 5% milk- TBST).
  • HRP horseradish peroxidase
  • the high-resolution DE-MRI scan was preceded by a two-dimensional Look-Locker (LL) sequence to determine the optimal inversion recovery time for blood signal nulling during the DE-MRI scan.
  • LL Look-Locker
  • mice Following the baseline (pre-injection) scanning, animals (two wild-type and one ApoE “7" mice) were injected with a dose of 0.2 mmol/kg of either Magnevist ® (preparation diluted 1 :20 in PBS) or Gd-MoCA. The above imaging protocol was then repeated at 30 and 60 minutes post imaging agent injection.
  • Magnevist ® preparation diluted 1 :20 in PBS
  • Gd-MoCA Gd-MoCA
  • ROIs were selected in at least three consecutive slices within each animal.
  • Tl and relaxivity values of Gd-MoCA in vitro and in water were analysed with the same programme following acquisition at the 3 Tesla scanner of increasing concentration of the imaging agent (0-1.5 raM) using Tl mapping sequences. 4.2. Imagine of atherosclerotic plaques
  • Gd-MOCA was modified by the addition of a lysine residue at the C-terminus to allow binding to a second DOTA moiety at the C-terminus, so that the contrast agent could carry a double dose of Gd3 + per molecule [Gd-DOTA-IDSQMETSCQIT (K)-DOTA-Gd].
  • This compound was injected at a dose of 0.2 mmol/kg in an ApoE _ " mouse. MRI was performed as described above.
  • Table 1 List of peptides including truncated and overlapping sequences of MoCA.
  • Gd-MoCA specifically targets CD115 expressed on the extracellular surface of human myeloid cells
  • the fluorescence antibody competition binding assays showed that Gd-MoCA is capable of displacing a specific anti -human CD115 antibody in a dose and time-dependent manner, thus demonstrating its specificity to the target when tested in human blood.
  • pre- treatment of the human blood with Gd-MoCA for 1 hour and at a dose of 1.5 mM prevented a PE-conjugated anti-human CD1 15 from binding to its target, leading to a statistically significant reduction of the percentage of CD115 + monocytes as detected by flow cytometry.
  • Reduction in the percentage of CD115 + monocytes was solely due to a competitive binding of Gd-MoCA to the CD 115 site engaged by the anti-CD115 specific antibody.
  • Gd-MoCA Specificity of Gd-MoCA in targeting CD115-expressing cells in human blood was confirmed by CyTOF analysis. The distribution of Gd-MoCA on the extracellular surface of the different leucocytes in human blood was consistent with the CD115 expression on the different cell types as detected by the specific PE-conjugated anti-human CD115 ( Figure 3).
  • Gd-MoCA specifically targets the human CD115 extracellular receptor
  • Gd-MoCA displayed similar accumulation within the kidney and bladder at 30 and 60 minutes post-injection compared to Magnevist, consistent with renal excretion of Gd-based imaging agents.
  • Gd-MoCA is able to enhance atherosclerotic, but not disease-free vessels, in ApoE ⁇ ' mice DE-MRI of the brachiocephlic artery in ⁇ " ⁇ mice on HFD showed selective and discrete accumulation of Gd-MoCA in atherosclerotic lesions, but not in healthy vessels, at 30 minutes post-injection (0.2 mmol kg of body weight) (Figure 11). Enhancement persisted at 60 minutes post-injection. Accumulation of the imaging agent within inflamed atherosclerotic lesions was not attributable to passive diffusion of the contrast agent from the bloodstream to the arterial wall, but it was rather due to a targeted localisation.
  • the peptide library screening identified 2 truncated sequences of Gd-MoCA that display specific binding to the CD115, namely DOTA-METSCQIT and DOTA-IDSQMETSCQI. These two peptides correspond to SEQ ID NO: 2 and SEQ ID NO: 3. Without wishing to be bound by theory, the analysis of the truncated sequences suggest that the primary binding site of Gd-MoCA to CD115 lays within the -METSCQI- sequence.
  • the lack of a consistent pattern in terms of ability to bind the target among the different C-terminus and/or N-terminus truncated peptides that also contain this sequence may point to a relevant role played by the other amino acid residues of Gd-MOCA (namely IDSQ) in determining a particular folded structure upon which it is able to bind to its target.
  • Modified MoCA peptide (SEQ ID NO: 4) retains specificity of binding to human CD115 and ability to image atherosclerotic plaques in vivo
  • a modified isoform of MoCA was generated with replacement of the Methionine residue with its synthetic analogue Norleucine (IDSQ(Nle)ETSCQIT).
  • This peptide corresponds to SEQ ID NO: 4.
  • the compound was synthesised by Peptide Synthetics with conjugation of DOTA at the N-terminus. It was tested by DELFIA assay to assess its binding affinity to human CD115 in comparison with the original sequence of MoCA (IDSQMETSCQIT) and by in vivo cardiovascular MRI in the ApoE _ " animal model of atherosclerosis, accordingly with the protocols described above.
  • the modified sequence of Gd-MoCA namely DOTA- IDSQ(Nle)ETSCQIT, retained binding affinity to its target human CD115.
  • Gd-MoCA is able to detect atherosclerotic disease progression by cardiovascular MRI
  • Gd-MoCA was tested in the ApoE 7" murine model of atherosclerosis at different stages of disease progression.

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Abstract

L'invention concerne des agents d'imagerie et des peptides qui se lient à un récepteur CD115 humain, ainsi que l'utilisation de ces agents d'imagerie pour identifier l'expression de CD115 et des patients présentant un risque élevé d'accident vasculaire aigu.
PCT/GB2015/051931 2014-07-03 2015-07-01 Agent d'imagerie de peptides ciblé sur la partie extracellulaire de cd115 Ceased WO2016001671A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
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US20060239913A1 (en) 2003-06-25 2006-10-26 Marc Port Peptide conjugate for magnetic resonance imaging
WO2012107725A1 (fr) * 2011-02-08 2012-08-16 King's College London Matériaux et méthodes associés à l'imagerie cardiovasculaire

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US20060239913A1 (en) 2003-06-25 2006-10-26 Marc Port Peptide conjugate for magnetic resonance imaging
WO2006049599A1 (fr) * 2004-10-28 2006-05-11 The General Hospital Corporation Methodes permettant de detecter et de traiter les plaques d'atherome par immunomodulation
WO2012107725A1 (fr) * 2011-02-08 2012-08-16 King's College London Matériaux et méthodes associés à l'imagerie cardiovasculaire

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