[go: up one dir, main page]

WO2020081454A1 - Sondes d'imagerie activées par de multiples enzymes ainsi que compositions et méthodes associées - Google Patents

Sondes d'imagerie activées par de multiples enzymes ainsi que compositions et méthodes associées Download PDF

Info

Publication number
WO2020081454A1
WO2020081454A1 PCT/US2019/056126 US2019056126W WO2020081454A1 WO 2020081454 A1 WO2020081454 A1 WO 2020081454A1 US 2019056126 W US2019056126 W US 2019056126W WO 2020081454 A1 WO2020081454 A1 WO 2020081454A1
Authority
WO
WIPO (PCT)
Prior art keywords
enzyme
imaging probe
activated imaging
quencher
activated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2019/056126
Other languages
English (en)
Inventor
Matthew Bogyo
John Widen
Joshua YIM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leland Stanford Junior University
Original Assignee
Leland Stanford Junior University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leland Stanford Junior University filed Critical Leland Stanford Junior University
Publication of WO2020081454A1 publication Critical patent/WO2020081454A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids

Definitions

  • FGS fluorescence-guided surgery
  • removing excess healthy tissue can also have a major negative impact on patient outcomes that affect quality of life.
  • Patients with NSCLC that undergo surgery have a significant risk of mortality or decreased lung function after surgery. Removal of unnecessary lymphatic tissue during breast tumor resection often leads to lymphedema.
  • a primary challenge for improving surgical resection treatment is developing better ways to visualize tumors so that residual cancer cells are not left behind, resulting in relapse of disease.
  • One major drawback of all current targeted contrast agents is that they rely on affinity or activation by a single target, thus resulting in high background signal in non-tumor tissues as a result of the fact that no single protein is expressed exclusively in tumors.
  • the probes include a label, a first quencher or FRET dye operably coupled to the label via a first enzyme cleavable substrate, and a second quencher or FRET dye operably coupled to the label via a second enzyme cleavable substrate.
  • the first enzyme cleavable substrate and the second enzyme cleavable substrate are cleavable substrates for different enzymes.
  • pharmaceutical compositions including the multi-enzyme-activated imaging probes of the present disclosure, as well as methods of in vivo imaging of a tissue in an individual, which methods include administering to the individual a pharmaceutical composition of the present disclosure. Methods that include contacting a cell or tissue with a multi-enzyme-activated imaging probe of the present disclosure are also provided.
  • FIG. 1 Schematic illustration of a complete tumor resection (left) having a negative margin and an incomplete tumor resection (right) having a positive margin due to inadequate visualization of cancerous versus normal tissue during the resection procedure.
  • FIG. 2 Examples of cathepsins and their upregulated expression in particular cancer types.
  • FIG. 3 A non-limiting list of labels (including their excitation and emission wavelengths) and compatible quenchers which may be employed in the imaging probes of the present disclosure.
  • FIG. 4 Panel A: single parameter quenched-fluorescent substrate probe 6-QC.
  • Panel B Schematic illustration of probe 6-QC cleaved by Cats resulting in a fragment containing the fluorophore that produces a signal and becomes trapped in lysosomes of tumor cells and macrophages due to protonation of the free amino group on the fragment.
  • Panel C Non-specific activation of 6-QC in healthy tissues.
  • FIG. 5 Panel A: schematic illustration of a single-activated (or“single parameter”) probe.
  • Panel B schematic illustration of a dual-activated (or“two parameter”) probe according to one embodiment of the present disclosure.
  • FIG. 6 Panel A: Schematic illustrations of AND-Gate 1 and its respective negative controls.
  • Panel B Schematic illustrations of AND-Gate 2 and its respective negative controls.
  • FIG. 7 Fluorogenic assay progress curves for AND-Gate 1 and its respective negative controls (top row) and AND-Gate 2 and its respective negative controls (bottom row).
  • FIG. 8 Flow cytometric assay results for evaluation of AND-Gate probes in RAW macrophages.
  • FIG. 9 Fluorescence microscopy results of AND-Gate 1 and negative controls in RAW macrophages.
  • FIG. 10 Top: Results of in vivo imaging of mice 2 h post I.V. tail vein injection of 6- QC, AND-Gate 1 and AND-Gate 2 (images normalized).
  • Female Balb/c mice were orthotopically injected with 100 mI_ of 100,000 4T1 cells/m L into the 2 and 7 mammary fat pads and allowed to develop tumors for 10-12 days. Probes were injected (100 mI_, 20 nmol) I.V. tail vein, hair was removed from the mice to expose tumors, and imaged using a LiCor Pearl imager system under isoflurane anesthesia.
  • FIG. 11 Results of ex vivo imaging of mice 2 h post I.V. tail vein injection of 6-QC, AND-Gate 1 and AND-Gate 2 (images normalized).
  • Female Balb/c mice were orthotopically injected with 100 pL of 100,000 4T1 cells/mL into the 2 and 7 mammary fat pads and allowed to develop tumors for 10-12 days. Probes were injected (100 pL, 20 nmol) I.V. tail vein. Mice were then sacrificed, their tumors and organs excised, and imaged using a LiCor Pearl imager.
  • FIG. 12 Testing of AND-Gate 1 , AND-Gate 2, and their respective negative control probes, in a 4T1 orthotopic breast cancer mouse model.
  • FIG. 13 Examples of non-peptidic linkers which may be employed in the multi- enzyme-activated imaging probe of the present disclosure.
  • FIG. 14 Examples of cleavable substrate sequences which may be employed in the multi-enzyme-activated imaging probe of the present disclosure.
  • FIG. 15 Schematic illustration of a scheme for synthesizing a multi-enzyme- activated imaging probe of the present disclosure according to some embodiments.
  • FIG. 16 The chemical structure of the AND-Gate 1 probe.
  • FIG. 17 The chemical structure of the AND-Gate 1 P1 (D)-Asp Casp3 negative control.
  • FIG. 18 The chemical structure of the AND-Gate 1 P2 (D)-Phe Cats negative control.
  • FIG. 19 The chemical structure of the AND-Gate 2 probe.
  • FIG. 20 The chemical structure of the AND-Gate 2 P1 (D)-Asp Casp3 negative control.
  • FIG. 21 The chemical structure of the AND-Gate 2 P2 (D)-Phe Cats negative control.
  • FIG. 22 The chemical structures of the fluorophore (Cy5) and quencher (QSY2) employed in the AND-Gate 1 and AND-Gate 2 probes.
  • FIG. 23 Solid phase synthesis of the Cats- and Casp3-cleavable substrates.
  • FIG. 24 Schematic illustration of the scheme employed for synthesizing AND-Gate
  • FIG. 25 Schematic illustration of the scheme employed for synthesizing AND-Gate
  • FIG. 26 An example scheme for spermidine non-peptidic linker AND-Gate probe synthesis.
  • FIG. 27 Chemical structure of the IRDye® QC-1 quencher (NHS ester).
  • FIG. 28 Images from robotic fluorescence-guided surgery with the da Vinci ® Xi Surgical System equipped with Firefly ® detection and quantification of fluorescence in healthy organs
  • (a) Images of 4T1 breast tumors in mice injected with AND-Gate-FNIFt, 6- QC-ICG, or 6-QC-NIFt (20 nmol, I.V.). Tumors are outlined with white dotted line
  • FIG. 29 Detection of residual tumor margins using the AND-Gate-FNIR probe and Firefly® detection system
  • FIG. 30 Synthesis of AND-Gate-FNIR containing a (D)-Glu central linker with an FNIR-Tag fluorophore and QC-1 quencher system.
  • FIG. 31 Fluorogenic substrate assays with AND-Gate-FNIR. Probe was incubated with either protease first followed by addition of the second protease. The probe is fluorescently activated only after incubation with both proteases regardless of order of addition whereas addition of a single protease or buffer does not produce a signal. DETAILED DESCRIPTION
  • the probes include a label, a first quencher or FRET dye operably coupled to the label via a first enzyme cleavable substrate, and a second quencher or FRET dye operably coupled to the label via a second enzyme cleavable substrate.
  • the first enzyme cleavable substrate and the second enzyme cleavable substrate are cleavable substrates for different enzymes.
  • pharmaceutical compositions including the multi-enzyme-activated imaging probes of the present disclosure, as well as methods of in vivo imaging of a tissue in an individual, which methods include administering to the individual a pharmaceutical composition of the present disclosure. Methods that include contacting a cell or tissue with a multi-enzyme-activated imaging probe of the present disclosure are also provided.
  • the present disclosure provides multi-enzyme-activated imaging probes.
  • the probes include a label, a first quencher or FRET dye operably coupled to the label via a first enzyme cleavable substrate, and a second quencher or FRET dye operably coupled to the label via a second enzyme cleavable substrate.
  • the first enzyme cleavable substrate and the second enzyme cleavable substrate are cleavable substrates for different enzymes.
  • the probes find use in a variety of applications in which detecting/imaging a cell or tissue is desirable.
  • the probe produces a signal that indicates when both the first and second substrates have been cleaved by their respective enzymes.
  • a probe of the present invention acts as an 'AND Gate' that reports on the presence of multiple enzyme activities. For example, if the first and second substrates are processed by enzymes (e.g., proteases) that are only active together within the context of the tumor microenvironment, then the probe will provide a highly selective readout of the location of tumor cells. As such, in addition to diagnostic applications, the probes enable direct visualization, e.g., of the tumor margin during surgery, which will in turn enable more complete resection of tumors without taking healthy normal tissue (that is - reduces under- and over-sampling), thereby resulting in improved surgical outcomes.
  • the probes of the present disclosure constitute an advancement over existing single parameter substrate reporters as they combine the activity of multiple enzymes (2, 3, or more enzymes, e.g., proteases) to activate the signal, e.g., fluorescent signal.
  • the probes may be designed to be more specific for a target tissue or disease indication of interest (e.g., tumor tissue or particular type thereof) based on a multiple enzyme“signature” of the target tissue or disease indication. Further details regarding the probes of the present disclosure will now be described.
  • FIG. 5 Schematic illustrations of a single-activated (or“single parameter”) probe and a dual- activated (or“two parameter”) probe according to one embodiment of the present disclosure are provided in FIG. 5, panels A and B, respectively.
  • the dual-enzyme activated probe acts as an AND Gate, in that activation of the probe requires the presence of both enzymes (e.g., indicative of tumor tissue), whereas the presence of only one enzyme is inadequate to activate the label (indicative of healthy tissue).
  • the multi-enzyme-activated imaging probes of the present disclosure may include a third quencher or FRET dye operably coupled to the label via a third enzyme cleavable substrate. That is, a multi-enzyme- activated imaging probe of the present disclosure includes at least two quencher- or FRET dye-cleavable substrate pairs, and may include 3, 4, or more of such pairs. The pairs may be designed to interrogate a cell or tissue for a multi-enzyme signature of interest.
  • the imaging probes of the present disclosure include a label, a first quencher or FRET dye, and a second quencher or FRET dye.
  • the label and quenchers or FRET dyes are selected such that they are compatible with one another.
  • the label and the one or more quenchers are selected such that the one or more quenchers are capable of absorbing energy from the label (e.g., a fluorophore, such as a fluorescent dye) and re-emitting much of that energy as either non- radiative energy (in the case of a dark quencher) or visible light (in the case of a fluorescent quencher).
  • an imaging probe of the present disclosure employs a label-quencher pair among those provided in FIG. 3. Shown in FIG. 3 is a non-limiting list of labels (including their excitation and emission wavelengths) and compatible quenchers which may be employed in the imaging probes of the present disclosure.
  • the label is a fluorescent label, such as Cy5, indocyanine green (ICG), a heptamethine cyanine fluorophore FNIR-Tag (FNIR-Tag), or the like.
  • the label is a fluorescent label and the first quencher or FRET dye, the second quencher or FRET dye, or both, is a QSY® quencher.
  • QSY® 7, QSY® 9 and QSY® 21 dyes are essentially non-fluorescent diarylrhodamine chromophores with strong absorption in the visible wavelength region, and they have proven to be extremely effective fluorescence quenchers.
  • QSY® 7, QSY® 9 and QSY® 21 dyes complement the QSY® 35 dye, a non-fluorescent quencher based on the NBD fluorophore that absorbs maximally near 475 nm, and the dabcyl quencher.
  • the label is a fluorescent label and one or both of the quenchers is a IRDye ® QC-1 quencher.
  • the label is indocyanine green (ICG) and one or both of the quenchers is a IRDye ® QC-1 quencher.
  • the label is an FNIR-Tag and one or both of the quenchers is a IRDye ® QC-1 quencher.
  • the IRDye ® QC-1 quencher is a nonfluorescent (dark) quencher compatible with a wide range of visible and near-infrared fluorophores (-500-800 nm) available from Li-Cor®.
  • the IRDye ® QC-1 quencher exhibits efficient nonfluorescent quenching of near-infrared (NIR) dyes.
  • NIR near-infrared
  • the NHS ester of this quencher has the chemical formula: C 53 H 62 CIN4Na 3 0i 6 S4 and a molecular weight of 1243.7589. Further details regarding label-quencher pairs which may be employed in the imaging probes of the present disclosure are described, e.g., in Marras S.A.E. (2006) Methods in Molecular Biology, vol. 335.
  • FRET fluorescence resonance energy transfer
  • FRET is a distance-dependent interaction between the electronic excited states of two molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.
  • the efficiency of FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable to the dimensions of biological macromolecules.
  • the donor and acceptor molecules must be in close proximity (typically 10-100 A), the absorption spectrum of the acceptor must overlap the emission spectrum of the donor, and the donor and acceptor transition dipole orientations must be approximately parallel.
  • the distance at which energy transfer is 50% efficient is defined by the Forster radius (R o ).
  • R o The magnitude of R o is dependent on the known spectral properties of the donor and acceptor dyes.
  • the first quencher or FRET dye is a FRET dye
  • the label and the FRET dye are a FRET pair.
  • in the second quencher or FRET dye is a second FRET dye
  • the label and the second FRET dye are a FRET pair.
  • the one or more FRET dyes when one or more FRET dyes are employed, alter the label’s emission such that the label may be detected as a first color, and upon cleavage of the first and second enzyme substrates, the one or more FRET dyes are no longer in sufficient proximity to the label to alter its emission, such that the label may be detected as a second color different from the first color.
  • the first enzyme cleavable substrate and the second enzyme cleavable substrate operably couple the first quencher or FRET dye and the second quencher or FRET dye to the label.
  • “operably couple” is meant each quencher or FRET dye is connected to the label via the cleavable substrate (and any optional linkers employed in the imaging probe) such that each quencher or FRET dye is within sufficient proximity of the label to quench emission from the label (in the case of a quencher) or participate in FRET with the label (in the case of a FRET dye).
  • the cleavable substrate positions its corresponding quencher or FRET dye such that the quencher or FRET dye alters the emission from the label.
  • “operably coupled” may be used interchangeably herein with“optically coupled”.
  • the length of the cleavable substrate is selected such that the cleavable substrate operably couples its corresponding quencher or FRET dye to the label.
  • a suitable distance between the label and quencher or FRET dye may vary depending upon the particular label- quencher pair or label-FRET dye pair employed.
  • the quenchers or FRET dyes are no longer within sufficient proximity of the label to quench emission from the label or participate in FRET with the label, respectively, such that emission from the label is no longer altered.
  • the resulting lack of alteration of the label’s stand-alone emission indicates the presence of the multi-enzyme signature which the imaging probe is designed to detect.
  • the multi-enzyme-activated imaging probes include a label which is a near-infrared (NIR) dye.
  • NIR near-infrared
  • NIR dyes offer important advantages over traditional visible light dyes. Because cellular or tissue components produce minimal autofluorescence in the near-IR region, near-IR dyes have the potential to offer highly specific and sensitive detection in complex biological systems. Light with wavelength in the near-IR region has strong tissue penetration, allowing the use of near-IR dyes for in vivo fluorescence imaging.
  • the NIR dye is a cyanine NIR dye, a phthalocyanine NIR dye, a porphyrin NIR dye, a squaraine NIR dye, a BODIPY NIR dye, a benzo[c]heterocyclic NIR dye. Further details regarding NIR dyes that may be included in the imaging probes of the present disclosure are described, e.g., in Escobedo et al. (2010) Curr Opin Chem Biol. 14(1 ):64.
  • a multi-enzyme-activated imaging probe of the present disclosure includes a label-quencher or label-FRET dye pair as described in U.S. Patent Application Publication No. US 2018/0002375, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • the imaging probes of the present disclosure may include one or more linkers.
  • One or more linkers may be incorporated into the image probes to connect one or more of: the label to first enzyme cleavable substrate, the label to the second enzyme cleavable substrate, the label to the first and second enzyme cleavable substrates, the first quencher or FRET dye to the first enzyme cleavable substrate, the second quencher or FRET dye to the second enzyme cleavable substrate, and any combinations thereof.
  • the label is attached to a central linker, and the first enzyme cleavable substrate and the second enzyme cleavable substrate are attached to the central linker.
  • the one or more linkers may include one or more linkers which are not themselves enzyme substrates, e.g., enzyme cleavable substrates.
  • Types of linkers which may be employed in the imaging probes include, but are not limited to, peptidic linkers and non-peptidic linkers.
  • A“peptidic” linker is a linker that includes or consists of one or more amino acids.
  • a peptidic linker may consist of only one or more natural amino acids, or a non-natural peptidic linker which includes or consists of one or more non-natural amino acids may be employed.
  • Non natural amino acids include, but are not limited to, those which reduce or preclude cleavage of a linker by proteases and/or other enzymes.
  • non-natural amino acids include D-amino acids.
  • Amino acids except glycine
  • L- and D- forms analogous to left- handed and right-handed configurations.
  • Only L-amino acids are manufactured in cells and incorporated into proteins.
  • one or more linkers which include (or consist of) one or more D-amino acids to render the linker incompatible as a substrate for proteolytic cleavage are employed.
  • Non-limiting examples of multi-enzyme-activated imaging probes which include such linkers are described in the Experimental section below and schematically illustrated in FIG. 6 (panel B). As demonstrated in the Experimental section below, it was surprisingly found that a non-natural peptidic linker substantially increased the stability of the multi-enzyme imaging probe.
  • a multi-enzyme activated imaging substrate of the present disclosure includes one or more non-peptidic linkers.
  • non-peptidic linkers which may be employed include a trifunctional linker including three functional group derivatives independently selected from the group consisting of: an alkyne, an azide, a hydroxyl, a thiol, a thia-Michael acceptor, an amine, a carboxylic acid, 1 ,2,4,5-tetrazine, cyclopropene, cyclooctyne, cyclooctene, an aldehyde, and a ketone.
  • non-peptidic linkers which may be employed include a spermidine linker, a 4- oxoheptanedioic acid linker, or a 5-aminoisophthalic acid linker.
  • spermidine, 4- oxoheptanedioic acid, and 5-aminoisophthalic acid, as well as example probes including a linker based on the same, are schematically illustrated in FIG. 13.
  • An example scheme for spermidine non-peptidic linker AND-Gate probe synthesis is shown in FIG. 26. As shown, the primary amines of spermidine are protected with two orthogonal protecting groups and then the secondary amine is reacted with para-nitrophenyl chloroformate.
  • the para- nitrophenol is then displaced by a single protected diamine linker.
  • One protecting group is removed and coupled to a protected peptide substrate via a urea linkage using para- nitrophenyl chloroformate.
  • the protecting group for the other primary amine is removed and coupled to the second protected peptide substrate in the same manner.
  • the middle linker protecting group is then removed and coupled to the fluorophore.
  • the protecting groups on both peptide sequences re then removed and each are coupled to a quencher molecule.
  • the first and second enzyme cleavable substrates are selected to detect a cell or tissue having a particular multi-enzyme signature of interest.
  • a multi-enzyme- activated imaging probe of the present disclosure may include a first enzyme cleavable substrate and a second enzyme cleavable substrate selected to detect the presence of first and second enzymes which in combination are characteristic of cell type or tissue of interest, e.g., a cancer cell and/or tumor tissue.
  • the first enzyme cleavable substrate is a substrate for a first hydrolase and the second enzyme cleavable substrate is a substrate for a second hydrolase different from the first hydrolase.
  • hydrolase is meant an enzyme that catalyzes the hydrolysis of a particular substrate.
  • the first and second hydrolases are independently selected from the group consisting of: a protease, an esterase, a lipase, a nuclease, and a glycosidase.
  • the first hydrolase is a protease
  • the second hydrolase is independently selected from the group consisting of: a protease, an esterase, a lipase, a nuclease, and a glycosidase.
  • each of the first and second hydrolases are proteases.
  • the first and second proteases are independently selected from the group consisting of: a cathepsin, a caspase, a matrix metalloproteinase (MMP), fibroblast activating protein (FAP), prostate specific membrane antigen (PSMA), type II transmembrane serine protease (TTSP), urokinase-type plasminogen activator (uPA), a deubiquitinase, an ADAM protease, a tissue factor, a granzyme, and a kallikrein.
  • MMP matrix metalloproteinase
  • FAP fibroblast activating protein
  • PSMA prostate specific membrane antigen
  • TTSP type II transmembrane serine protease
  • uPA urokinase-type plasminogen activator
  • ADAM protease an ADAM protease
  • tissue factor a tissue factor
  • the first hydrolase is a cathepsin.
  • Cathepsins are members of the family of papain-like cysteine proteases. In the lysosomal system, protein degradation is a result of the combined random and limited action of various proteases (also termed peptidases or proteolytic enzymes).
  • cysteine cathepsins In addition to the aspartic cathepsin D, cysteine cathepsins have a key role among the lysosomal proteases.
  • cathepsin expression and activity has been implicated in the development of various pathological conditions, such as neurological disorders, cardiovascular diseases, obesity, inflammatory diseases, such as rheumatoid arthritis, and cancer, among others.
  • pathological conditions such as neurological disorders, cardiovascular diseases, obesity, inflammatory diseases, such as rheumatoid arthritis, and cancer, among others.
  • the roles of cathepsins in these pathological conditions are reviewed in Turk et al. (2012) Biochimica et Biophysics Acta (BBA) - Proteins and Proteomics 1824(1 ):68-88. It is now clear that the cathepsins have an important role in both tumor progression and invasion, which is supported by the numerous clinical reports and results from experimental mouse-cancer models. Elevated cathepsin expression and/or activity has been shown to be associated with cancer progression in a number of different types of tumors.
  • cathepsin expression positively correlated with a poor prognosis for cancer patients and was suggested to be a potential prognostic marker.
  • serum cathepsin levels have been positively correlated with metastasis.
  • the roles of cathepsins in the individual tumor- biological processes were also confirmed ex vivo using cell-based systems. As such, the cathepsins B, L and some others have been shown to promote the migration and invasion of tumor cells. Cathepsins have a well-known function associated with the ECM degradation and remodeling in the tumor microenvironment. Examples of cathepsins and their upregulated expression in particular cancer types are shown in FIG. 2.
  • the enzyme cleavable substrate for the cathepsin is a substrate for one or more of cathepsin S (CatS), cathepsin B (CatB), and cathepsin L (CatL).
  • CatS cathepsin S
  • CatB cathepsin B
  • CatL cathepsin L
  • FIG. 14 A non-limiting example of a cathepsin cleavable substrate is provided in FIG. 14. Further details regarding cathepsins and cathepsin cleavable substrates which may be included in the multi-enzyme-activated imaging probes of the present disclosure are described, e.g., in Turk et al. (supra).
  • the first hydrolase is a caspase.
  • Caspases are proteases at the heart of networks that govern apoptosis and inflammation.
  • the name“caspase” derives from cysteine-dependent aspartate-specific protease.
  • Catalysis is governed by a critical conserved Cys side chain of the enzyme and by a highly stringent selectivity for cleaving peptides and proteins on the carboxy-terminal side of Asp residues.
  • the primary specificity pockets of caspases are almost identical, being formed by the side chains of the strictly conserved residues Arg-179, Arg-341 , and Gln-283 (caspase-1 numbering convention).
  • the enzyme cleavable substrate for the caspase is a substrate for caspase 3 (Casp3).
  • the first cleavable substrate is a substrate for a caspase (e.g., Casp3) and the second cleavable substrate is a substrate for a cathepsin, e.g., a cleavable substrate for one or more of Cats, CatB and/or CatL.
  • a Casp3 cleavable substrate is provided in FIG. 14. Further details regarding caspases and caspase cleavable substrates which may be included in the multi-enzyme-activated imaging probes of the present disclosure are described, e.g., in Poreba et al. (2013) Cold Spring Harb Perspect Biol. 5(8):a008680; Song et al. (2010) Bioinformatics 26(6):752-760; and Talanian et al. (1997) The Journal of Biological Chemistry 272:9677-9682.
  • the first hydrolase is a matrix metalloproteinase (MMP).
  • MMPs consist of a multigene family of zinc-dependent extracellular matrix (ECM) remodeling endopeptidases implicated in pathological processes, such as carcinogenesis.
  • ECM extracellular matrix
  • their activity plays a pivotal role in tumor growth and the multistep processes of invasion and metastasis, including proteolytic degradation of ECM, alteration of the cell-cell and cell-ECM interactions, migration and angiogenesis.
  • 21 human MMPs are known, and they can be divided into subgroups based on their structure and substrate specificity.
  • MMP-2 (gelatinase A) and MMP-9 (gelatinase B) have three tandem repeats of 58 amino acid residue long fibronectin type ll-like modules in the catalytic domain.
  • gelatinases degrade components of basement membranes, they are believed to play a crucial role in processes requiring basement membrane disruption, such as tumor invasion and tissue infiltration of T lymphocytes.
  • MMP-2 is also thought to be important in malignancies, as its activation correlates with tumor spread and poor prognosis.
  • MMP-2- deficient mice show reduced angiogenesis and tumor progression, and MMP-9-deficient mice show impaired metastasis formation and tumor growth.
  • the enzyme cleavable substrate for the MMP is a substrate for MMP-2 or MMP-9.
  • the first cleavable substrate is a substrate for MMP- 2 and the second cleavable substrate is a substrate for MMP-9.
  • MMP-2 and MMP-9 cleavable substrates are provided in FIG. 14. Further details regarding MMPs and MMP cleavable substrates which may be included in the multi-enzyme-activated imaging probes of the present disclosure are described, e.g., in Reunanen & Kahari, Madame Curie Bioscience Database Austin (TX) Austin (TX) Austin (TX) Austin (TX) Austin (TX) Austin (TX) Austin (TX) Austin (40):E4148-E4155; and Turk et al. (2001 ) Nature Biotechnology 19:661 -667.
  • the cell type or tissue of interest is a cancer cell and/or tumor tissue.
  • cancer cell is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth and/or development in an immunocompromised non-human animal model, and/or any appropriate indicator of cellular transformation.
  • cancer cell may be used interchangeably herein with “tumor cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of tumor tissue.
  • Tumor tissue includes tissue of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, and the like.
  • tumor tissue not only includes a tissue made up exclusively of cancer cells, but also a tissue that includes cancer cells and one or more additional cell types, including but not limited to, immune cells (e.g., tumor associated macrophages (TAMs)) associated with (e.g., infiltrated within) the tissue.
  • TAMs tumor associated macrophages
  • the first and second enzyme cleavable substrates are selected to detect tumor tissue selected from the group consisting of: a carcinoma, a sarcoma, a melanoma, a lymphoma, a leukemia, any combinations thereof, and any sub-types thereof.
  • the first and second enzyme cleavable substrates are selected to detect tumor tissue of a cancer selected from the group consisting of: breast cancer, melanoma, lung cancer, colorectal cancer, prostate cancer, glioma, bladder cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, non-Hodgkin lymphoma, pancreatic cancer, thyroid cancer, any combinations thereof, and any sub-types thereof.
  • the imaging probes may vary depending upon the particular dye and quencher- or FRET dye-cleavable substrate pairs to be included in the imaging probe.
  • the imaging probe includes a central linker (e.g., a peptidic (e.g., non-natural peptidic linker) or a non-peptidic linker as described above), and the imaging probe is made by attaching the first enzyme cleavable substrate, the second enzyme cleavable substrate, and the label to the central linker, in any desired order.
  • a central linker e.g., a peptidic (e.g., non-natural peptidic linker) or a non-peptidic linker as described above
  • the cleavable substrates may already be attached to their respective quencher or FRET dye when the cleavable substrates are attached to the central linker, or the quencher or FRET dye may be attached to its respective cleavable substrate subsequent to the cleavable substrate being attached to the central linker.
  • FIG. 15 A general synthetic scheme for making a multi-enzyme-activated imaging probe of the present disclosure which includes a central linker is schematically illustrated in FIG. 15.
  • the circle, square, and triangle represent orthogonal functional groups.
  • functional groups include alkyne, maleamide, and tetrazine functional groups.
  • Protecting groups (“PGs”) are used to block reactivity on the enzyme cleavable substrates (“Seq 1” and“Seq 2”).
  • the circle including an“F” represents the label (here, a fluorophore) and the circle including a “Q” represents the quencher or FRET dye (here, a quencher).
  • the central linker includes three orthogonal functional groups each of which are reacted with one of the first enzyme cleavable substrate, the second enzyme cleavable substrate, and the label, in any convenient order.
  • the two enzyme cleavable substrates (peptide sequences) are deprotected to become competent for attachment of a corresponding quencher or FRET dye.
  • the enzyme cleavable substrate portions of the probes may be made, e.g., using solid phase peptide synthesis. Examples of suitable approaches for solid phase peptide synthesis of enzyme cleavable substrates are provided in the Experimental section below and illustrated in FIG. 23.
  • the present disclosure also provides compositions that include any of the multi-enzyme-activated imaging probes of the present disclosure.
  • the compositions include a multi-enzyme-activated imaging probe of the present disclosure present in a liquid medium.
  • the liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like.
  • One or more additives such as a salt (e.g., NaCI, MgCh, KOI, MgS0 4 ), a buffering agent (a Tris buffer, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-
  • a salt e.g., NaCI, MgCh, KOI, MgS0 4
  • a buffering agent a Tris buffer, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES)
  • Morpholino)ethanesulfonic acid MES
  • 2-(N-Morpholino)ethanesulfonic acid sodium salt MES
  • 3-(N-Morpholino)propanesulfonic acid MOPS
  • N-tris[Hydroxymethyl]methyl-3- aminopropanesulfonic acid TAPS
  • a solubilizing agent e.g., a non-ionic detergent such as Tween-20, etc.
  • a ribonuclease inhibitor glycerol
  • a chelating agent chelating agent
  • the pharmaceutical compositions include any of the multi-enzyme-activated imaging probes of the present disclosure, and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions generally include an effective amount of the multi-enzyme-activated imaging probe.
  • An“effective amount” is meant an amount sufficient to produce a desired result, e.g., imaging of a tissue of interest (e.g., tumor tissue), if present, in an individual.
  • An effective amount can be administered in one or more administrations.
  • a multi-enzyme-activated imaging probe of the present disclosure can be incorporated into a variety of formulations for administration to an individual. More particularly, the multi-enzyme-activated imaging probe can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.
  • Formulations of the multi-enzyme-activated imaging probes of the present disclosure suitable for administration to an individual are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.
  • the multi-enzyme-activated imaging probe can be administered alone or in appropriate association, as well as in combination, with a pharmaceutically active compound, e.g., an anti-cancer agent (including but not limited to small molecule anti-cancer agents), an immune checkpoint inhibitor, and any combination thereof.
  • a pharmaceutically active compound e.g., an anti-cancer agent (including but not limited to small molecule anti-cancer agents), an immune checkpoint inhibitor, and any combination thereof.
  • an anti-cancer agent including but not limited to small molecule anti-cancer agents
  • an immune checkpoint inhibitor an immune checkpoint inhibitor
  • the multi-enzyme-activated imaging probe can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium
  • the multi-enzyme-activated imaging probe can be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration.
  • parenteral e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.
  • the multi-enzyme-activated imaging probe is formulated for injection by dissolving, suspending or emulsifying the multi-enzyme-activated imaging probe in an aqueous or non- aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous or non- aqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
  • solubilizers isotonic agents
  • suspending agents emulsifying agents
  • stabilizers and preservatives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • compositions that include the multi-enzyme-activated imaging probe may be prepared by mixing the multi-enzyme-activated imaging probe having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents.
  • Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as ge
  • the pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration.
  • the standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration.
  • An aqueous formulation of the multi-enzyme-activated imaging probe may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5.
  • buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers.
  • the buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
  • a tonicity agent may be included in the formulation to modulate the tonicity of the formulation.
  • Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
  • isotonic denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
  • Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.
  • a surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption.
  • Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).
  • suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20TM) and polysorbate 80 (sold under the trademark Tween 80TM).
  • Suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188TM.
  • suitable Polyoxyethylene alkyl ethers are those sold under the trademark BrijTM.
  • Example concentrations of surfactant may range from about 0.001 % to about 1 % w/v.
  • a lyoprotectant may also be added in order to protect the multi-enzyme-activated imaging probe against destabilizing conditions during a lyophilization process.
  • known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 nM.
  • the pharmaceutical composition includes a multi-enzyme- activated imaging probe of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m- cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof.
  • a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
  • the present disclosure also provides methods of using the multi-enzyme-activated imaging probes of the present disclosure.
  • methods of in vivo imaging of a tissue in an individual comprising administering to the individual a pharmaceutical composition of the present disclosure.
  • the first enzyme cleavable substrate is a substrate for a first enzyme expressed in the tissue
  • the second enzyme cleavable substrate is a substrate for a second enzyme expressed in the tissue.
  • Such methods may further include imaging the tissue upon cleavage of the first and second enzyme cleavable substrates by the first and second enzymes, respectively.
  • the tissue may be tumor tissue.
  • the methods further include diagnosing the individual as having a medical condition based on the imaging of the tissue.
  • the methods further include performing a resection procedure on the tissue based on the imaging of the tissue.
  • at least a portion of the resection procedure is performed during the imaging of the tissue.
  • An image- guided surgical device may be employed to perform the resection procedure.
  • a non-limiting example of an image-guided surgical device which may be employed to perform the resection procedure is a da Vinci ® surgical system available from Intuitive Surgical, such as a da Vinci ® Si TM surgical system, a da Vinci ® Xi TM surgical system, a da Vinci ® X TM surgical system, or a da Vinci ® SP TM surgical system.
  • This system has integrated fluorescence imaging capability providing real-time, image-guided identification of key anatomical landmarks using near-infrared technology.
  • This system can be used to perform minimally-invasive laparoscopic surgical procedures.
  • the system is equipped with a NIR camera that can be used in addition to a white light imaging system to visualize contrast agents carrying NIR signals. Resection studies in multiple models of cancer using this system are described, e.g., in U.S. Patent Application Publication No. 20180002375, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • kits include one or more (e.g., 1 , 2, 3, 4, or more) containers including an amount of a pharmaceutical composition of the present disclosure.
  • a kit may further include instructions for in vivo imaging of a tissue in an individual using the multi-enzyme-activated imaging probe present in the pharmaceutical composition.
  • kits that include any of the multi-enzyme- activated imaging probes of the present disclosure, and instructions for contacting a cell or tissue using the multi-enzyme-activated imaging probe.
  • the instructions may further include instructions for detecting/imaging the cell or tissue using the multi-enzyme-activated imaging probe.
  • kits may be present in separate containers, or multiple components may be present in a single container.
  • Suitable containers include individual tubes (e.g., vials), one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), or the like.
  • the instructions included in the kits may be recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, DVD, CD-ROM, diskette, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded.
  • the means for obtaining the instructions is recorded on a suitable substrate.
  • a multi-enzyme-activated imaging probe comprising:
  • first enzyme cleavable substrate wherein the first enzyme cleavable substrate and the second enzyme cleavable substrate are cleavable substrates for different enzymes.
  • non-peptidic linker is a trifunctional linker comprising three functional group derivatives independently selected from the group consisting of: an alkyne, an azide, a hydroxyl, a thiol, a thia-Michael acceptor, an amine, a carboxylic acid, 1 ,2,4,5-tetrazine,
  • the first enzyme cleavable substrate is a substrate for a first hydrolase and the second enzyme cleavable substrate is a substrate for a second hydrolase different from the first hydrolase.
  • first and second hydrolases are independently selected from the group consisting of: a protease, an esterase, a lipase, a nuclease, and a glycosidase.
  • the multi-enzyme-activated imaging probe of embodiment 1 1 wherein the first hydrolase is a protease, and wherein the second hydrolase is independently selected from the group consisting of: a protease, an esterase, a lipase, a nuclease, and a glycosidase.
  • first and second proteases are independently selected from the group consisting of: a cathepsin, a caspase, a matrix metalloproteinase (MMP), fibroblast activating protein (FAP), prostate specific membrane antigen (PSMA), type II transmembrane serine protease (TTSP), urokinase-type plasminogen activator (uPA), a deubiquitinase, an ADAM protease, a tissue factor, a granzyme, and a kallikrein.
  • MMP matrix metalloproteinase
  • FAP fibroblast activating protein
  • PSMA prostate specific membrane antigen
  • TTSP type II transmembrane serine protease
  • uPA urokinase-type plasminogen activator
  • deubiquitinase an ADAM protease
  • tissue factor a tissue factor
  • a granzyme a kallikrein
  • CatS cathepsin S
  • CatB cathepsin B
  • CatL cathepsin L
  • the label is a heptamethine cyanine fluorophore FNIR-Tag and the first quencher or FRET dye, the second quencher or FRET dye, or both, is an IRDye® QC-1 quencher.
  • NIR near-infrared
  • a pharmaceutical composition comprising:
  • the multi-enzyme-activated imaging probe of any one of embodiments 1 to 34 ; and a pharmaceutically acceptable carrier.
  • the first enzyme cleavable substrate is a substrate for a first enzyme expressed in the tissue
  • the second enzyme cleavable substrate is a substrate for a second enzyme
  • a method comprising contacting a cell or tissue with the multi-enzyme-activated imaging probe of any one of embodiments 1 to 34.
  • a kit comprising:
  • a kit comprising:
  • the multi-enzyme-activated imaging probe of any one of embodiments 1 to 34 and instructions for contacting a cell or tissue using the multi-enzyme-activated imaging probe.
  • multi- quenched (in the examples herein, dual-quenched) orthogonal protease substrates referred to herein as“AND-Gate” probes.
  • These probes require input from two independent proteases that are elevated in tumors to produce a fluorescent signal.
  • the inventors have demonstrated in situ that two orthogonal proteases are required for activation of the AND- Gate probes.
  • studies with the probes in a mouse model of breast cancer confirms that the AND-Gate strategy results in higher overall fluorescence intensity at the tumor site while dramatically reducing non-specific activation in healthy tissues compared to a single-protease-targeted probe designated 6-QC.
  • Probe 6-QC is processed by cysteine cathepsin proteases (Cats), which are overexpressed and secreted predominantly by immune cells that enter into the tumor microenvironment and play important roles in initiation and progression.
  • Probe 6-QC is schematically illustrated in FIG. 4, panel A.
  • panel B probe 6-QC is cleaved by Cats resulting in a fragment containing the fluorophore that produces a signal and becomes trapped in lysosomes of tumor cells and macrophages due to protonation of the free amino group on the fragment.
  • the quenched substrate probe 6-QC can quickly produce fluorescent signals (within 30-120 min) at the tumor site after intravenous injection or topical administration to tumor tissues.
  • quenched protease substrates reduce background signal compared to affinity-based approaches, they still suffer from non-specific activation due to Cat activity in healthy tissues. This becomes problematic when the tumor of interest is located in areas of high background such as the lungs, liver, and kidneys, as shown in FIG. 4, panel C.
  • the multi-enzyme-activated imaging probes of the present disclosure overcome this challenge by identifying a unique proteolytic“fingerprint” that results from multiple targets that are simultaneously active only in the context of an environment of interest, such as a tumor microenvironment.
  • the aforementioned drawbacks of single parameter quenched substrate probes are overcome by the probes of the present disclosure as demonstrated in the following examples.
  • Single parameter contrast agents have an inherent limit of selectivity for tumor tissue compared to normal tissue because generally every protein found in tumors is also found in healthy tissue, thus contrast is driven only by differences in expression or activity.
  • the AND-Gate strategy takes advantage of two active proteases that are only found together with elevated expression/activity in the tumor microenvironment.
  • “AND-Gate” probes that require activity of both cathepsins (Cats) and Casp3 were designed and synthesized, as Cats are highly active in the liver and kidneys, but Casp3 is only active in tissues with increased programmed cell death, such as the stomach and intestinal lining. Yet, both proteases are activated within a tumor.
  • FIG. 6 A first generation AND-Gate probe requiring activation by both Cats and Casp3 is schematically illustrated in FIG. 6, panel A (top molecule). This probe (designated“AND-
  • Gate 1 includes a Casp3 cleavable substrate (left) attached to a quencher and a Pan-Cats cleavable substrate (right) attached to a quencher.
  • the cleavable substrates are attached to a central linker.
  • the label (“F”) is also attached to the central linker.
  • the intact cleavable substrates (in combination with the central linker) operably couple the quenchers to the label.
  • the linker employed was a (L)-Glu linker.
  • panel A are negative controls used in experimental testing of the AND- Gate 1 probe.
  • the negative controls include a Casp3 negative control containing a P1 (D)- Asp to block Casp3 cleavage, and a Cat negative control containing a P2 (D)-Phe to block Cat cleavage.
  • AND-Gate 1 contains a peptide sequence that is efficiently cleaved by Cats and a second peptide sequence cleaved by Casp3. These two proteases were chosen because they have highly orthogonal substrate specificities and both are active in tumor tissues. Cats are active in TAMs and Casp3 is active in tumor tissues due to significant populations of cells undergoing cell death due to ischemia. (35)
  • the probe design includes two QSY21 quenchers that are attached to each peptide through a lysine sidechain.
  • AND-Gate 1 contains a (L)-Glu central linker attached to a Cy5 fluorophore.
  • the full chemical structures of the AND-Gate 1 probe, the P1 (D)-Asp Casp3 negative control, and the P2 (D)-Phe Cats negative control are provided in FIGs. 16, 17 and 18, respectively.
  • the scheme for synthesizing the AND-Gate 1 probes is shown in FIG. 24.
  • the synthesis of AND-Gate 1 began by coupling the protected Casp3 sequence (1 equiv.) with Fmoc-Glu(OH)-OAII (3 equiv.) using HCTU (3 equiv) and 2,4,6-collidene (5 equiv.) in DMF for 16 h at room temperature.
  • the reaction mixture was diluted in 1 :1 H20:MeCN (0.1 % TFA) and purified using reverse phase semi-preparative HPLC. The desired fractions were collected and lyophilized overnight to obtain a white powder.
  • the resulting product (1 equiv.) was then deprotected using palladium acetate (1 equiv.), triphenyl phosphine (2 equiv.), and phenylsilane (5 equiv.) in THF over 16 h at RT.
  • the reaction solvent was reduced in vacuo, suspended in 1 :1 H20:MeCN (0.1 % TFA), filtered through a 0.2 urn filter, and purified using reverse phase chromatography. The fractions collected were lyophilized over night to obtain a white powder.
  • the product (1 equiv.) from reaction 3 was reacted to sulfo-Cy5-OSu (1 .5 equiv.) in the presence of DIPEA in DMF for 24 h at RT.
  • the subsequent reaction solution was diluted in 1 :1 H20:MeCN (0.1 % TFA), purified using reverse phase chromatography, and the resulting fractions of product were concentrated and then dissolved in 85:15 TFA:DCM for 2 h and protected from light.
  • the solution was then concentrated in vacuo, dissolved in 1 :1 H20:MeCN (0.1 % TFA) and lyophilized to obtain a blue powder.
  • reaction 5 The product (1 equiv.) of reaction 5 was reacted with sulfo-QSY21 (4 equiv.) in the presence of DIPEA (10 equiv.) in DMSO for 48 h at RT.
  • the reaction solution was then diluted in 1 :1 H20:MeCN (0.1% TFA) and purified using reverse phase chromatography. The fractions of product were lyophilized to obtain a blue solid.
  • Negative controls were synthesized in the same manner except the Casp3 sequence contains a (D)-Asp in the P1 position or for the Cat negative control a (D)-Phe in the P2 position.
  • the progress curves show the fluorescence increase over time for the positive control AND-Gate probes only after the addition of the second orthogonal protease.
  • the two negative controls, (D)-Phe 2 or (D)-Asp 2 were not activated under the same conditions.
  • AND-Gate 1 and the respective negative control probes were tested in a 4T1 orthotopic breast cancer mouse model. After injecting 100 pL of 100,000 4T1 cells/mL in 1 X PBS solution into the 2 and 7 mammary fat pads of Balb/c female mice tumors developed after 10-12 days. Probes were then injected via I.V. tail vein (20 nmol). Overall signal in tumors for each probe decreased at the 4 h time point. Fluorescent signal was measured in excised organs 2 h post injection and it was found that AND-Gate 1 and the (D)-Phe negative control showed high background signals in lungs and livers, but had reduced background in kidneys.
  • FIGs. 19, 20 and 21 The full chemical structures of the AND-Gate 2 probe, the P1 (D)-Asp Casp3 negative control, and the P2 (D)-Phe Cats negative control are provided in FIGs. 19, 20 and 21 , respectively.
  • the structures of the label (Ft 1 ) and quencher (R 1 ) employed in both the AND- Gate 1 and AND-Gate 2 probes are shown in FIG. 22.
  • the product (1 equiv.) was then subjected to palladium acetate (1 equiv.), triphenylphosphine (2 equiv.), and phenylsilane (5 equiv.) in THF at RT for 16 h.
  • the solvent was removed in vacuo and the resulting mixture was suspended in 1 :1 H20:MeCN (0.1 % TFA) and purified using reverse phase chromatography. The fractions containing product were collected and lyophilized to obtain a white powder.
  • reaction two The product (1 equiv.) of reaction two was then coupled to the Casp3 sequence (1 equiv.) using HCTU (1 .2 equiv.) and 2,4,6-collidene (3 equiv.) in DMF at RT for 16 h. Piperidine was then added to obtain a 20% v/v in DMF and allowed to react for 2 h at RT.
  • the resulting reaction solution was diluted with 1 :1 H20:MeCN (0.1 % TFA) and purified using reverse phase chromatography. The fractions containing product were collected and lyophilized to obtain a white solid.
  • reaction three The product (1 equiv.) of reaction three was reacted with sulfo-Cy5-Osu (2 equiv.) in the presence of DIPEA (5 equiv.) in DMF for 24 h at RT.
  • the reaction solution was diluted with 1 :1 H20:MeCN (0.1 % TFA) and purified using reverse phase chromatography.
  • the product fractions were concentrated in vacuo and dissolved in 80:20 TFA:DCM and stirred for 2 h at RT protected from light.
  • the resulting solution was concentration in vacuo and dissolved in 1 :1 H20:MeCN (0.1% TFA) for lyophilization to obtain a blue powder.
  • reaction four The product (1 equiv.) of reaction four was reacted with suflo-QSY21 -OSu (4 equiv.) in the presence of DIPEA (10 equiv.) in DMSO for 48 h at RT.
  • the reaction solution was diluted in 1 :1 H20:MeCN (0.1 % TFA) and purified using reverse phase chromatography.
  • the product was collected and lyophilized to obtain a blue solid.
  • the two negative controls containing (D)-Asp in the P1 position of the Casp3 sequence and (D)-Phe in the P2 position of the Cat sequence where synthesized in the same manner.
  • Results from testing of the AND-Gate 2 probe with recombinant enzymes confirmed that the probe functions as desired with activation only after addition of both proteases (FIG. 7, bottom row).
  • the AND-Gate 2 probe and negative controls were tested for stability in tumor lysate derived from orthotopic 4T1 murine breast tumors (FIG. 12, two right-most panels).
  • these second generation probes produced a robust fluorescent signal while both of the negative controls (D)-Phe 2 and (D)-Asp 2 remained fully quenched.
  • AND-Gate 2 produced a strong fluorescent signal in tumors and importantly, both respective negative controls showed only low background activation. This increased tumor signal for the AND-Gate 2 probe was accompanied by a marked reduction in fluorescent signal within healthy organs including liver and kidneys (FIG. 1 1 ). Comparison of signal in the liver, lungs, and kidneys to tumors also showed that AND-Gate 2 significantly reduces signal in all three organs compared to 6-QC.
  • an AND-Gate probe containing a fluorophore that has excitation and emission wavelengths compatible with the FDA approved Firefly ® detection system on the da Vinci ® Xi Surgical System was synthesized.
  • the Firefly ® fluorescence detection system is specifically tuned to the excitation/emission properties of indocyanine green (ICG, ex/em: 780/820 nm).
  • ICG indocyanine green
  • a heptamethine cyanine fluorophore FNIFt- Tag that was designed by the Schnermann group to resist aggregation and have better water solubility compared to ICG 56 was used.
  • the FNIR dye was successfully conjugated to the a-amine on the central (D)-Glu linker and subsequently two QC-1 quenchers were attached to the lysine side chains to produce AND-Gate-FNIFt.
  • the purified fractions were collected and General Procedure C was followed to obtain a dark green powder (2.6 mg, 65% yield). AND-Gate-FNIR.
  • the AND-Gate-FNIR had comparable fluorescent signal intensity to the 6-QC-ICG probe and much improved signal in comparison to 6-QC-NIR, which also has lower excitation/emission wavelengths than ICG (788/799 nm).
  • tumors and organs from mice injected with each probe were imaged ex vivo using the LiCor Pearl imaging system to quantify the fluorescent signal.
  • Representative images of excised tumors and organs confirmed a reduced signal in the liver and kidneys compared to the signal found in the tumors for AND-Gate-FNIR compared to both of the 6-QC probes (FIG. 28b).
  • Quantification of the signal in the organs normalized to the tumor signal confirmed a significant reduction in background in the liver for mice injected with AND-Gate-FNIR compared to both 6-QC probes and a significant reduction in background for all healthy organs compared to 6-QC-NIR (FIG. 28c).
  • Nguyen QT Fluorescence-guided surgery with live molecular navigation-a new cutting edge. Nature reviews Cancer. 2013;13(9):653-62. doi: 10.1038/nrc3566. PubMed PMID: 23924645; PubMed Central PMCID: PMC4427343.
  • PubMed PMID 18712739; PubMed Central PMCID: PMC2743602.
  • Jaattela M Multiple cell death pathways as regulators of tumour initiation and progression.
  • PubMed PMID 15077138. 47. Cederqvist K, Sorsa T, Tervahartiala T, Maisi P, Reunanen K, Lassus P, Andersson S. Matrix metalloproteinases-2, -8, and -9 and TIMP-2 in tracheal aspirates from preterm infants with respiratory distress. Pediatrics. 2001 ;108(3):686-92. PubMed PMID: 1 1533337.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des sondes d'imagerie activées par de multiples enzymes. Selon certains aspects, les sondes comprennent une étiquette, un premier extincteur ou colorant de FRET fonctionnellement couplé à l'étiquette par l'intermédiaire d'un premier substrat clivable par une enzyme, et un second extincteur ou colorant de FRET fonctionnellement couplé à l'étiquette par l'intermédiaire d'un second substrat clivable par une enzyme. Le premier substrat clivable par une enzyme et le second substrat clivable par une enzyme sont des substrats clivables pour différentes enzymes. L'invention concerne également des compositions pharmaceutiques comprenant les sondes d'imagerie activées par de multiples enzymes selon la présente invention, ainsi que des méthodes d'imagerie in vivo d'un tissu chez un individu, lesdites méthodes consistant à administrer à l'individu une composition pharmaceutique selon la présente invention. L'invention concerne également des méthodes qui consistent à mettre en contact une cellule ou un tissu avec une sonde d'imagerie activée par de multiples enzymes selon la présente invention.
PCT/US2019/056126 2018-10-15 2019-10-14 Sondes d'imagerie activées par de multiples enzymes ainsi que compositions et méthodes associées Ceased WO2020081454A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862745809P 2018-10-15 2018-10-15
US62/745,809 2018-10-15

Publications (1)

Publication Number Publication Date
WO2020081454A1 true WO2020081454A1 (fr) 2020-04-23

Family

ID=70284085

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/056126 Ceased WO2020081454A1 (fr) 2018-10-15 2019-10-14 Sondes d'imagerie activées par de multiples enzymes ainsi que compositions et méthodes associées

Country Status (1)

Country Link
WO (1) WO2020081454A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113444146A (zh) * 2021-06-01 2021-09-28 南方医科大学南方医院 靶向成纤维细胞活化蛋白探针、制备方法及其在制备pet显像剂中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020042394A1 (en) * 2000-05-31 2002-04-11 Hogenkamp Henricus P.C. Cobalamin compounds useful as antibiotic agents and as imaging agents
WO2016151297A1 (fr) * 2015-03-20 2016-09-29 The University Court Of The University Of Edinburgh Sonde optique pour thrombine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020042394A1 (en) * 2000-05-31 2002-04-11 Hogenkamp Henricus P.C. Cobalamin compounds useful as antibiotic agents and as imaging agents
WO2016151297A1 (fr) * 2015-03-20 2016-09-29 The University Court Of The University Of Edinburgh Sonde optique pour thrombine

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113444146A (zh) * 2021-06-01 2021-09-28 南方医科大学南方医院 靶向成纤维细胞活化蛋白探针、制备方法及其在制备pet显像剂中的应用

Similar Documents

Publication Publication Date Title
Ofori et al. Design of protease activated optical contrast agents that exploit a latent lysosomotropic effect for use in fluorescence-guided surgery
Zhang et al. Activatable molecular probes for fluorescence-guided surgery, endoscopy and tissue biopsy
Mai et al. Targeting legumain as a novel therapeutic strategy in cancers
Dragulescu-Andrasi et al. In vivo bioluminescence imaging of furin activity in breast cancer cells using bioluminogenic substrates
US12320801B2 (en) Methods to spatially profile protease activity in tissue and sections
US9695251B2 (en) Activatable cell penetrating peptides with quenched fluorophores
US8968700B2 (en) Imaging of protease activity in live cells using activity based probes
Chowdhury et al. Prodrug-inspired probes selective to cathepsin B over other cysteine cathepsins
Edgington et al. Functional imaging of legumain in cancer using a new quenched activity-based probe
Li et al. Activatable near-infrared fluorescent probe for in vivo imaging of fibroblast activation protein-alpha
KR101103548B1 (ko) 단백질분해효소 활성 측정용 나노입자 센서 및 그 제조방법
ES2828985T3 (es) Detección basada en la afinidad de biomarcadores sintéticos codificados por ligando
US20240280576A1 (en) Sensors for detecting and imaging of cancer metastasis
US9180209B2 (en) Non-peptidic quenched fluorescent imaging probes
EP3270977B1 (fr) Sondes optiques pour métalloprotéinases matricielles
Poreba Recent advances in the development of legumain-selective chemical probes and peptide prodrugs
EA030795B1 (ru) Молекулы для селективной доставки пары визуализирующих агентов в раковую ткань
WO2013036743A1 (fr) Agents d'imagerie pour imagerie d'activité protéase et leurs utilisations
Kasperkiewicz Peptidyl activity-based probes for imaging serine proteases
Luo et al. Fibroblast activation protein α activatable theranostic pro-photosensitizer for accurate tumor imaging and highly-specific photodynamic therapy
WO2020081454A1 (fr) Sondes d'imagerie activées par de multiples enzymes ainsi que compositions et méthodes associées
Gorzeń et al. Engineering unnatural amino acids in peptide linkers enables cathepsin-selective antibody-drug conjugates for HER2-positive breast cancer
JP2018511556A (ja) ｉｎ ｖｉｖｏ画像化のためのプロテアーゼ活性化コントラスト剤
WO2016151297A1 (fr) Sonde optique pour thrombine
Wang et al. Design and synthesis of fluorogenic substrate-based probes for detecting Cathepsin B activity

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19874197

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19874197

Country of ref document: EP

Kind code of ref document: A1