WO2008005942A2 - Sondes susceptibles d'être activées et procédés d'utilisation - Google Patents

Sondes susceptibles d'être activées et procédés d'utilisation Download PDF

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WO2008005942A2
WO2008005942A2 PCT/US2007/072680 US2007072680W WO2008005942A2 WO 2008005942 A2 WO2008005942 A2 WO 2008005942A2 US 2007072680 W US2007072680 W US 2007072680W WO 2008005942 A2 WO2008005942 A2 WO 2008005942A2
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tumor
fluorescence
gsa
gmsa
target
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PCT/US2007/072680
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WO2008005942A3 (fr
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Hisataka Kobayashi
Peter L. Choyke
Yasuteru Urano
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The Govt. Of The Usa As Represented By The Secretary Of The Dept. Of Health And Human Services.
The University Of Tokyo
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Publication of WO2008005942A2 publication Critical patent/WO2008005942A2/fr
Publication of WO2008005942A3 publication Critical patent/WO2008005942A3/fr

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • 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
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the disclosure provides target activatable fluorescent probes that are useful for detecting biologically active cells including tumor cells.
  • imaging techniques capable of detecting small clusters of living cells, such as tumor cells can be useful in screening high risk populations as defined by family history or proteomic screening. Moreover, such imaging can improve therapy by defining the extent of disease more accurately than has previously been possible.
  • the present disclosure provides improved probes for imaging cells and methods of using such probes.
  • TAFPs Target-specific activatable fluorescent probes
  • the change in fluorescent signal state can minimize the problems associated with background signal that result from the presence of unbound probe.
  • Methods of using TAFPs are also provided, for example by contacting the TAFP with a biologically active cell, such as a tumor cell.
  • the increase in signal state from the first extracellular state to the second intracellular state can be at least 200, 250, 1,000, or 10,000%, which allows for detection of small cell clusters or even individual cells.
  • the TAFPs described herein can include a targeting moiety that specifically binds to a cell and a labeling moiety.
  • the TAFP also includes a linker.
  • Methods are also provided for using the TAFPs to detect and/or remove tumors in subjects.
  • these methods can include administering the TAFP to a subject and removing the detected tumor.
  • Figs. IA and IB show graphs depicting the optical characteristics of GSA- BDP and GSA-detBDP.
  • Fig. IA shows emission spectra of GSA-BDP and GSA- detBDP in phosphate buffers with different pH values (2.3, 3.3, 5.2, 6.4 and 7.4). Both GSA-BDP and GSA-detBDP have the same emission peak at a wavelength of 570 nm regardless of pH changes when stepped in 10 nm increments.
  • Fig. IB shows a log plot of fluorescence intensities of GSA-BDP (Upper curves) and GSA- detBDP (Lower curves) at different pH values on the spectral unmixed image. The error bar indicates standard deviation.
  • Figs. 2A-2D shows serial flow cytometry of SHIN3 cancer cells labeled with (i.e. instilled) GSA-BDP or GSA-detBDP.
  • FIGs. 3A-3F show schematic illustrations of the concept of fluorescent activation of Av-3ROX.
  • Av-0.5ROX has 0.5 rhodamineX molecules per avidin whereas Av-3ROX has 3 rhodamineX molecules per avidin
  • Fig. 3A depicts how immediately after administration of Av-0.5ROX the background fluorescence from the unbound reagent is high.
  • Fig. 3B depicts how one hour later the AvO.5ROX is internalized and catabolized into monomers or smaller peptides.
  • FIG. 3C shows how after administration of Av-3ROX the fluorescence from both the cells and the background is weak due to self-quenching.
  • Fig. 3D depicts how after Av-3ROX is internalized and catabolized within the endoplasmic vesicles into degradation products such as monomers and peptides it is fluorescently activated by "de-quenching" and strong fluorescence signal is observed within the cells. Since the background fluorescence remains weak, a high signal-to- background ratio can be achieved.
  • Figs. 3C shows how after administration of Av-3ROX the fluorescence from both the cells and the background is weak due to self-quenching.
  • Fig. 3D depicts how after Av-3ROX is internalized and catabolized within the endoplasmic vesicles into degradation products such as monomers and peptides it is fluorescently activated by "de-quenching" and strong fluorescence signal is observed within the cells. Since the background fluorescence remains weak, a high
  • FIG. 3E and 3F depict how if the catabolism in the cell is blocked by crosslinking, the Av-3ROX is not activated either immediately (Fig. 3E) or 1 hour after administration (Fig. 3F).
  • Fig. 4 shows fluorescence emission spectra of Av-0.5ROX (1 nM) and Av-
  • Figs. 5A-5D show that crosslinking the three rhodamineX molecules of Av- 3ROX suppresses in vitro and in vivo intracellular activation of fluorescence signal.
  • Fig. 5B shows fluorescence emission spectra of crosslinked (CL+) and non-crosslinked (CL-) Av- 3ROX with or without SDS.
  • Crosslinking decreased the fluorescence intensities of Av-3R0X (775 ng/mL) when placed in 5% SDS and PBS at pH 7.4 at a wavelength of 603 nm.
  • the crosslinking did not affect the fluorescence intensity of Av-3R0X (775 ng/mL) when SDS was not used at a wavelength of 603 nm.
  • FIG. 5C shows a histogram of fluorescence intensity of an ROI drawn on each of the peritoneal membranes instilled with crosslinked (CL+) and non-crosslinked (CL-) Av-3R0X.
  • the dynamic range of the fluorescence intensity was split into equal- sized 256 bins (0-255). Then for each bin (horizontal axis) the number of pixels from the data set that fall into each bin (vertical axis) are counted.
  • Fig. 5D shows regression lines of crosslinked (CL+) and non-crosslinked (CL-) Av-3ROX. The regression lines were calculated from the data sets (fluorescence threshold values 40-240 total number of pixels within the threshold rage 1-10000 in common logarithm). The slopes of crosslinked and non-crosslinked Av-3ROX were 0.0375 and -0.0110, respectively.
  • foci positive forAv-3ROX were defined as those whose fluorescence intensities >1 (a.u.) on spectral unmixed Av-3ROX images, sensitivity and specificity of spectral unmixed Av3ROX images to detect the presence of cancer foci were 92% and 98%, respectively.
  • Fig. 7 shows the structures of two alkylB ODIPY molecules, Di- ethylaminophenyl BODIPY, and Di-methylaminophenyl BODIPY.
  • Figs. 8A-8D show the relative fluorescent intensities of various alkylBODIPY molecules.
  • Fig. 8A shows the scheme for aniline based alkylBODIPY probes.
  • Fig. 8B shows fluorescence images for pH profiles of
  • H 2 NBDP (a), DiMeNBDP (b), DiEtNBDP (c), and PhBDP (d). pH profiles range from pH 2 (left) to pH 9 (right) in 1 pH unit-steps.
  • 8D shows fluorescence enhancement of BODIPY-Herceptin conjugates at acidic pH, determined in a way that fluorescence intensity, at 533 nm, of BODIPY-labeled Herceptin at given pH is divided by that at pH 7.4. Excitation wavelength is 520 nm.
  • accession numbers are the accession numbers from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A. The accession numbers are as provided in the database on June 30, 2006.
  • administration refers to providing or giving a subject an agent, such as a composition that includes a target-specific activatable fluorescent probe (TAFP), alone or in combination with another agent, by any effective route.
  • an agent such as a composition that includes a target-specific activatable fluorescent probe (TAFP), alone or in combination with another agent, by any effective route.
  • TAFP target-specific activatable fluorescent probe
  • Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), intraperitoneal wash, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • injection such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous
  • intraperitoneal wash sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • an "asialoglycoprotein receptor” or “ASGPR” is a C-type animal lectin that mediates the removal of desialylated serum glycoproteins containing terminal galactose residues.
  • the ASGPR has also been termed the ⁇ -D-galactose receptor.
  • the ASGPR assembles as a hetero-oligomer consisting of two highly homologous subunits termed hepatic lectins Hl and H2. Both subunits contain an N-terminal cytoplasmic domain, a single transmembrane segment, a stalk domain, and a C- terminal carbohydrate recognition domain.
  • ASGPRl and ASGPR2 which encode the Hl and H2 subunits, respectively.
  • Exemplary nucleotide and amino acid sequences of human Hl and H2 subunits of ASGPR are publicly available from GENB ANK® (Accession Nos. M10058 and Ml 1025, respectively).
  • ASGPR nucleic acid and protein molecules can vary from those publicly available, such as ASGPR sequences having one or more substitutions, deletions, insertions, or combinations thereof, while still retaining the ability to mediate the removal of desialylated serum glycoproteins containing terminal galactose residues accordingly these sequences are considered ASGPR.
  • ASGPR sequences having at least 80%, at least 90%, or at least 95% sequence identity compared to those sequences provided under Accession Nos. M10058 and Ml 1025 are also considered ASGPR.
  • ASGPR molecules include fragments that retain the ability to bind to asialoglycoprotein receptor-binding ligand.
  • an "asialoglycoprotein receptor-binding moiety” or “ASGPR-binding moiety” is any targeting moiety that specifically binds to, or is bound by, an asialoglycoprotein receptor.
  • immunoglobulin A and fibronectin are natural targeting moieties for ASGPR.
  • ASGPR-binding moiety also includes artificial moieties, such as avidin, galactosyl human or bovine serum albumin (BSA), and other glycosylated (such as ligands possessing galactose, N- acetylgalactosamine and/or N-acetylglucosamine side chains) carrier proteins, such as serum proteins, including for example, glycosylated immunoglobulin proteins and micro- and macro-globulin proteins or fragments thereof.
  • BSA bovine serum albumin
  • glycosylated carrier proteins such as serum proteins, including for example, glycosylated immunoglobulin proteins and micro- and macro-globulin proteins or fragments thereof.
  • R 1 is an amino group which can be substituted by one or two alkyl groups (said alkyl group can be substituted by an amino group);
  • R , R 2 , R 3 , R 4 and R can be respectively and independently an alkyl group (said alkyl group can have a substitution group);
  • R 6 and R 7 respectively and independently indicate a monocarboxy alkyl group), a salt thereof or an ester thereof.
  • alkylBODIPY is a salt or ester thereof wherein R is an amino group which can be substituted by one or two C 1 ⁇ alkyl groups (said alkyl group can be substituted by a carboxyl group); R 2 , R 3 and R 4 and R 5 are respectively and independently a Ci_ 4 alkyl group; the aforementioned compound, a salt or an ester thereof in which R 6 and R 7 are respectively and independently a monocarboxy C M alkyl group; and R 1 is an amino group in which R 1 can be substituted by one or two Ci- 4 alkyl groups; R 1 , R 2 , R 3 , R 4 and R 5 are a methyl group; the compound, a salt or ester thereof in which R 6 and R 7 are respectively and independently a carboxy C 1- 4 alkyl group is provided.
  • AlkylBODIPY molecules display different fluorescent signals depending upon the pH of the surrounding environment.
  • AlkylBODIPY molecules show at least a 250% change in fluorescence intensity when they are changed from a relatively neutral pH medium, for example a pH of greater than 5, 6, 7, 8, or 9, to a relatively acidic pH medium having a pH of less than 4, less then 3, less than 2, or less than 1.
  • a relatively neutral pH medium for example a pH of greater than 5, 6, 7, 8, or 9, to a relatively acidic pH medium having a pH of less than 4, less then 3, less than 2, or less than 1.
  • the selection of the R groups and the substitution of the R groups can impact the change in fluorescent signal.
  • the alkylBODIPY molecule when used as a labeling moiety as described herein the alkylBODIPY molecule can increase in fluorescence intensity by at least 250% after internalization into a cell. Methods of determining the change in fluorescence intensity are known in the art and particular examples are provided in Example 2, below.
  • the alkyl part of the substitution group (for example, an alkyl carbonyl group) comprising an alkyl group or an alkyl part indicates an alkyl group which can be straight-chain, branching-chain, cyclic or a combination of these having 1 to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.
  • a lower alkyl group (alkyl group with 1 to 6 carbon atoms) is used.
  • the lower alkyl group can be a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a cyclopropylmethyl group, an n-pentyl group, an n-hexyl group and the like.
  • substitution groups alkoxy groups
  • R 1 indicates an amino group which has been substituted by a non-substitution amino group or by one or two alkyl groups.
  • the alkyl group substituted in the amino group can have a substitution group (however, this does not include an amino group as a substitution group). For example, it can have a carboxyl group and other substitution group.
  • a Ci_ 4 alkyl group is suitable as said alkyl group.
  • Examples of this are a non-substitution amino group, monomethyl amino group, dimethyl amino group, monoethyl amino group, ethyl methyl amino group, diethyl amino group, mono n-propyl amino group, n- propyl methyl amino group, carboxy substitution ethyl amino group and the like.
  • suitable alkyl groups are not necessarily restricted to these.
  • R which is substituted on the benzene ring, and R can also be a para position.
  • the alkyl group indicated by R 1 , R 2 , R 3 , R 4 and R 5 can be a Ci_ 4 alkyl group and particularly a methyl group.
  • the monocarboxy alkyl group indicated by R 6 and R 7 can be a single carboxyl group substituted on the end of the alkyl group, such as a monocarboxy Ci_ 4 alkyl group.
  • a monocarboxy ethyl group, monocarboxy propyl group and the like are particularly suitable.
  • AlkylBODIPY can be present as an acid added salt or a salt group added salt.
  • the salt of the amine can be formed using hydrochloric acid, sulfate, nitrate and other mineral acid salts or methane sulfonic acid, p-toluene sulfonate, oxalate, citrate, tartrate and other organic acid salts.
  • the salt of the carboxyl can be sodium salt, potassium salt, calcium salt, magnesium salt and other metal salts, ammonium salt or toluene ethyl amine salt and other organic amino salts. In addition to these, glycerol and other salts of amino acid can be formed.
  • AlkylBODIPY or a salt thereof is sometimes present as a solvate and these substances are included within the parameters of the present invention.
  • alkylBODIPY which is expressed by the aforementioned general formula (I) can be used as an ester.
  • an alkylBODIPY when used as a labeling moiety can be added to proteins and other molecules by using this as a succine imidyl ester.
  • An organism- related substance for example, proteins, antibodies and the like
  • the alkylBODIPY conjugate when internalized within a cell it can be activated by conversion of an alkyl ester to a methyl ester and converting an alkyl ether to a methoxy methyl ester. This type of ester undergoes hydrolysis through the action of the intracellular esterase after incorporation in the cell and the alkylBODIPY can be retained intracellularly thereby making it possible to measure the acid regions inside the cells.
  • AlkylBODIPY molecules can have one, two or more asymmetric carbon atoms depending on the type of substitution group. However, in addition to the optical activators based on one, two or more asymmetric carbon atoms and diastereo-isomers and other types of stereoisomers based on two or more asymmetric carbon atoms, any mixture of stereoisomers, racemic modification and the like can be included in the alkylBODIPY molecule.
  • Exemplary alkylBODIPY molecules include for example, NMeEtBODIPY, NMe 2 BODIPY, and NEt 2 BODIPY.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • a naturally occurring antibody e.g., IgG, IgM, IgD
  • IgG, IgM, IgD includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • H heavy
  • L light
  • binding fragments encompassed within the term antibody include (i) a Fab fragment consisting of the VL, VH, CL and CHl domains; (ii) an Fd fragment consisting of the VH and CHl domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al, Nature 341:544-546, 1989) which consists of a VH domain; (v) an isolated complimentarity determining region (CDR); and (vi) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • Suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al., Science 246: 1275-1281, 1989; and Ward et al., Nature 341: 544-546, 1989. "Specific" monoclonal and polyclonal antibodies and antisera (or antiserum) will usually bind to an appropriate antigen with a kD of at least about 0.1 ⁇ M, for example, at least about 0.01 ⁇ M, at least 0.001 ⁇ M, or better.
  • Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (e.g., see U.S. Patent No. 4,745,055; U.S. Patent No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125,023; Faoulkner et al, Nature 298:286, 1982; Morrison, J. Immunol. 123:793, 1979; Morrison et ah, Ann Rev. Immunol 2:239, 1984). Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S. Patent 5,482,856.
  • Bioly active cell(s) are cells that are actively metabolizing compounds. For example, a cell that is actively producing proteins (e.g. proteases) and converting carbon sources (such as glucose) to other molecules.
  • biologically active cells are capable of endocytosis such that a TAFP can be internalized and shift from a first extracellular state of fluorescence intensity to a second intracellular state of fluorescence intensity.
  • TAFP molecules can be used to assess the degree of biological activity displayed by a cell. For example, the speed at which a TAFP is internalized by a cell and shifts from a first state to a second state can indicate the cell's biological activity. Used in this way the TAFP can be used to determine if agents (such as chemotherapeutic agents) are impacting tumor cell biological activity.
  • binding refers to an association between two or more molecules, wherein the two or more molecules are in close physical proximity to each other, such as the formation of a complex.
  • An exemplary complex is a receptor-ligand pair or an antibody antigen pair.
  • Specific binding refers to a preferential binding between an agent and a specific target.
  • specific binding refers to when a TAFP that includes a labeling moiety specific for a tumor cell antigen binds to the tumor cell, but does not bind to other cells in close proximity to the tumor cell.
  • Other examples of specific binding include the binding between an ASGPR and an ASGPR targeting moiety.
  • binding can be a specific non-covalent molecular interaction between the ligand and the receptor.
  • binding is assessed by detecting the fluorescent signal emitted from a fluorophore conjugated to an ASGPR targeting moiety, after the ASGPR targeting moiety has been placed in contact with ASGPR.
  • cancer refers to a malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and is capable of metastasis.
  • Residual cancer is cancer that remains in a subject after any form of treatment given to the subject to reduce or eradicate a cancer.
  • Metastatic cancer is a cancer at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer is derived. In the case of a metastatic cancer originating from a solid tumor, one or more (for example, many) additional tumor masses can be present at sites near or distant to the site of the original tumor.
  • disseminated metastatic nodules or “disseminated metastatic tumors” refers to a plurality (typically many) metastatic tumors dispersed to one or more anatomical sites.
  • disseminated metastatic nodules within the peritoneum that is a disseminated intraperitoneal cancer
  • Such metastatic tumors can themselves be discretely localized to the surface of an organ, or can invade the underlying tissue.
  • contacting refers to the relatively close physical proximity of one object to another object.
  • contacting involves placing two or more objects in close physical proximity to each other to give the objects and opportunity to interact.
  • contacting a TAFP with a biologically active cell can be accomplished by placing the TAFP (which can be in a solution) in proximity to the cell, for example by injecting the TAFP into a subject having the tumor.
  • a TAFP can be contacted with a cell in vitro.
  • detect refers to determining if an agent is present or absent. In some examples this can further include quantification.
  • use of the disclosed probes permits detection of the presence of ASGPR. Any means of detection can be used, for example visual detection, flow cytometry, microscopy and spectrophotometry. In particular examples, detection is accomplished by exciting a fhiorophore with a laser and then detecting the resulting emission fluorescence.
  • detection is accomplished by exciting a fhiorophore with a laser and then detecting the resulting emission fluorescence.
  • the wavelength of excitation will depend on the fluorophore being detected.
  • a “detectable label” or “label” or “labeling moiety” is an agent capable of detection, for example by visual inspection, spectrophotometry or microscopy.
  • labels include fluorescent molecules that are self quenching or environmentally activatable.
  • labeling moieties can contain different self-quenching molecules placed in close proximity to each other via the use of various linkers or by conjugating multiple self-quenching molecules to a targeting moiety.
  • Environmentally activatable molecules include molecules that are pH sensitive, such as alkylBODIPYs, BODIPYfI and rhodamin-X, alkyl- rhodsminBs.
  • the labeling moiety that is included in the TAFP will function to increase in fluorescence intensity by at least 250% upon internalization into a cell.
  • the labeling moiety will have a first extracellular signal state and a second intracellular signal state that displays a fluorescence intensity for example, that is at least 250% greater than the first extracellular signal state.
  • labeling moieties described herein as environmentally activatable can also display self quenching characteristics when placed in close proximity to other labeling moieties, and that self-quenching labeling moieties can also display pH sensitivity.
  • the fluorescence intensity can be measured after the TAFP is initially (i.e. within 10 minutes of administration into a subject or within 10 minutes of placement into a reaction vessel) contacted with a cell that is thought to display the ligand to the targeting moiety included in the TAFP.
  • the fluorescence intensity can then be measured again at various time points.
  • the fluorescent signal from the TAFP will increase, eventually plateau and decrease.
  • the signal strength at the highest level of fluorescence can then be compared to the initial signal strength to determine the % increase.
  • Example 2 provides an exemplary method of determining the % increase in signal strength.
  • This example involves determining the serial fluorescence intensity of SHIN3 cancer cells using one-color flow cytometry.
  • the fluorescence intensity of a region of interest (ROI) within an interperitoneal cavity can be determined within 10 minutes of administration of a TAFP and then the ROI can be observed until the fluorescence intensity plateaus and decreases. The initial level of fluorescent intensity can then be compared to the maximum to determine the % increase in intensity.
  • ROI region of interest
  • the term "eliminate” with respect to a tumor or cancer refers to the substantial, and in some cases total, eradication of tumor cells. In many cases, where a single performance of a method diminishes or inhibits a tumor, repeated practice of the method can eliminate the tumor. Accordingly, the methods disclosed herein for the purpose of treating (that is, reducing, diminishing, inhibiting or eliminating) tumors can be performed one or more (multiple) times, at the discretion of the practitioner to achieve the desired reduction in tumor size and number.
  • a “fluorophore” is a luminescent chemical compound, which when excited by exposure to a particular stimulus, such as a defined wavelength of light, emits light (luminesces or fluoresces), for example at a different wavelength.
  • Activatable fluorophores are fluorophores that alone, or in combination, under one set of conditions emit a first signal intensity and under a second set of conditions emit a second signal intensity.
  • the shift from one signal intensity to another is not necessarily an all or none response, meaning that the fluorophore displays a continuum of intensities.
  • the conditions that cause the shift can be the structural, such as the physical proximity of fluorophores to other fluorophores or it can be environmental, such as the pH of the medium.
  • Examples of particular activatable fluorophores include those fluorophores that can change intensity by at least 250%.
  • Exemplary fluorophores include, rhodamine molecules, alkylBODIPY, and other BODIPY derivatives such as BODIPYfI, and combinations thereof.
  • intraperitoneal and “intraperitoneally” refer to the area and to objects within the area typically bounded by or associated with the peritoneum.
  • the peritoneum consists of two layers: the outer layer, called the parietal peritoneum, is attached to the wall of the abdominal cavity and the inner layer, the visceral peritoneum, is wrapped around the organs that are located inside the cavity.
  • the peritoneum both supports the abdominal organs and serves as a conduit for their blood and lymph vessels and nerves.
  • organs commonly categorized as intraperitoneal stomach, jejunum, ileum, superior horizontal part of duodenum, appendix, spleen, transverse colon, sigmoid colon, rectum, liver, uterus, fallopian tubes, ovaries
  • intra-retroperitoneal cecum, ascending colon, descending colon
  • retroperitoneal portions of the duodenum and rectum, kidneys, pancreas, suprarenal glands, ureters, renal and gonadal blood vessels
  • intraperitoneal portions of the rectum, urinary bladder
  • a “linker” is a molecule that is used to connect one or more agents to one or more other agents.
  • a linker can be used to connect one or more labels to one or more targeting moieties.
  • linkers include dendrimers, such as synthetic polymers, peptides, proteins and carbohydrates.
  • Linkers additionally can contain one or more protease cleavage sites or be sensitive to cleavage via oxidation and/or reduction.
  • Optically detectable labels include luminescent labels that generate a light signal (other than by heating).
  • Luminescent compounds include photoluminescent compounds (such as fluorescent and phosphorescent compounds), chemiluminescent compounds and electroluminescent compounds.
  • pharmaceutically acceptable carriers refers to pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic or diagnostic agents, such as one or more of the TAFP molecules provided herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • compositions to be administered can contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.
  • nontoxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.
  • a “targeting moiety” is any compound that binds to a ligand (such as a structure that binds preferentially to a TAFP). Typically, targeting molecules selectively bind to one type of cell displaying a ligand more effectively than they bind to other types of cells that do not display the ligand.
  • Targeting moieties can be chosen to selectively bind to tissues, such as organ tissues to assess the biological activity of those tissues. Targeting moieties can also be chosen to selectively bind to tumors.
  • Targeting moieties include asialoglycoprotein receptor-binding moieties, antibodies (including fragments of antibodies), immunoglobulin A, fibronectin, and artificial binding moieties, such as avidin, galactosyl serum albumin (GSA), and glycosylated carrier proteins such as immunoglobuline family proteins, macro- or micro-globulins.
  • GSA galactosyl serum albumin
  • TAFPs can be made to detect the tumors.
  • standard methods of making antibodies to the identified marker can be used to make targeting moieties specific for the tumor cell marker, thus, allowing for the detection of the tumor and potentially its removal from a subject.
  • a tumor is a neoplasm or an abnormal mass of tissue that is not inflammatory, which arises from cells of preexistent tissue.
  • a tumor can be either benign (noncancerous) or malignant (cancerous). Tumors can be solid or hematological.
  • hematological tumors include, but are not limited to: leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelogenous leukemia, and chronic lymphocytic leukemia), myelodysplastic syndrome, and myelodysplasia, polycythemia vera, lymphoma, (such as Hodgkin's disease, all forms of non-Hodgkin's lymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.
  • acute leukemias such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocy
  • solid tumors such as sarcomas and carcinomas
  • solid tumors include, but are not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, melanoma, and
  • the phrase "under conditions sufficient for” is used to describe any environment that permits the desired activity.
  • the phrase includes incubating tumor cells in the presence of a TAFP such that the TAFP binds to the cell.
  • the phrase includes contacting tumor cells with a TAFP such that the TAFP binds to the cell and is internalized. II. Making Target Activatable Fluorescence Probes
  • TAFPs Target activatable fluorescence probes
  • a ligand hereinafter a molecule that the targeting moiety binds to
  • TAFPs have at least one targeting moiety, which can be any molecule that preferentially binds to one or more ligands.
  • TAFPs can also include one or more linkers that function to bind one or more targeting moieties to one or more labeling moieties. Labeling moieties provide a detectable signal, such as a fluorescent signal.
  • the TAFP can be conjugated in vitro prior to addition to a sample, or the components of a TAFP can be added separately to a sample and the TAFP can be formed in the presence of the sample.
  • the targeting moiety can be added to a sample, or subject, and then the labeling moiety can be added such that the labeling moiety binds to the targeting moiety.
  • the sensitivity of a TAFP can be increased, for example, by having a single targeting moiety bound to one or more labeling moieties, either directly or through one or more linkers, hence providing a strong signal from the single targeting moiety being bound to a single ligand.
  • TAFPs include one or more targeting moieties that localize the TAFP to a cell.
  • a broad range of targeting moieties are suitable in the context of the TAFPs disclosed herein, including for example, targeting moieties that bind to organic molecules such as peptides, carbohydrates and combinations thereof, that are presented on the extracellular membrane.
  • a single variety of targeting moiety can be used to make a TAFP or more than one variety of targeting moiety can be used. Multiple targeting moieties are useful when the detection of cells displaying a combination of ligands is desired.
  • targeting moieties include for example, antibodies, fragments of antibodies, peptides, phages, affibodies, aptamers, glycosylated molecules, oligomers, nano-particles, and the like. More specifically, a targeting moiety can be a monoclonal antibody or a fragment thereof. In the case of an antibody targeting moiety, the TAFP binds to cells expressing the cognate antigen. For example, a TAFP using Herceptin ® (Genentech, San Francisco, California) as a targeting moiety specifically binds to HER2neu receptors on the surface of certain cancer cells, such as breast or ovarian cancer cells.
  • Herceptin ® Herceptin ®
  • targeting moieties include galactosyl serum albumin (GSA) which binds to an asialoglycoprotein receptor (ASGPR).
  • GSA galactosyl serum albumin
  • ASGPR asialoglycoprotein receptor
  • the TAFPs include an asialoglycoprotein receptor (ASGPR) targeting moiety attached to one or more activatable fluorophores.
  • ASGPR asialoglycoprotein receptor
  • the ASGPR-binding moiety binds to an asialoglycoprotein receptor (ASGPR) that is highly expressed on the surface of tumor cells. Because, the ASGPR is not expressed to a substantial level on the surface of most normal cells, the ASGPR-binding moiety and its attached label are localized to tumor cells, with minimal to negligible binding to normal tissues.
  • ASGPR-binding moieties are also useful for detecting liver cells which display ASGPR.
  • the TAFP can be internalized (e.g. via endocytosis) and concentrated into lysozymes.
  • Binding of a targeting moiety to the ASGPR is mediated via sugar (and sugar derivative) side chains attached to a carrier molecule (any molecule that can be bound to one or more sugar side chains).
  • the carrier molecule is a polypeptide, which is heavily glycosylated (that is, numerous sugar side chains are attached to the carrier molecule). Binding affinity of the targeting moiety to the ASGPR increases with the density of glycosylation, such that more highly glycosylated carrier polypeptides are bound more strongly to the ASGPR than are less highly glycosylated carrier polypeptides.
  • Avidin binds strongly to ASGPRs via glucosamine and mannose side chains. The ASGPR also binds to galactose and N-acetylgalactosamine with high affinity. Avidin is widely available commercially, and can be obtained from numerous sources conjugated to a variety of fluorophores (for examples, see, Molecular Probes, Eugene OR). Avidin is well suited as a targeting agent to localize activatable fluorophores that are subject to self-quenching when multiple fluorophores are located in close proximity. Avidin is a tetramer that is quickly dissociated following internalization into the lysozome/endosome.
  • fluorescence of the individual fluorophore moieties is quenched.
  • fluorophores unquench, resulting in increased fluorescence.
  • Glycosylated serum albumin binds strongly and specifically to ASGPRs but is not immunogenic in human subjects.
  • glycosylated (for example, galactosylated) serum albumins derived from the species in which the methods are to be practiced can be selected.
  • any immunologically neutral glycosylated ligand can be used as a targeting moiety in the context of the methods disclosed herein.
  • carrier proteins functionalized with galactose, N- acetylgalactosamine, glucosamine and mannose residues are specifically bound to ASGPRs with high affinity.
  • the ASGPR targeting moiety is assembled into a multimeric macromolecular complex using a linker, such as a dendrimer or other artificial particle.
  • a linker such as a dendrimer or other artificial particle.
  • Suitable dendrimer particles for use in the methods disclosed herein are known in the art.
  • Exemplary avidin dendrimers based on polyamidoamine (ethylenediamine) cores are described in Kobayashi et al., Bioconjug. Chem. 12:587-593, 2002 and Mamede et al., Clin. Can. Res. 9:3756- 3762, 2003, both of which are incorporated herein by reference.
  • Dendrimers are synthetic chemical polymers that can have any one of a number of different functional groups of their surface (Tomalia, Aldrichimica Acta, 26:91: 101, 1993; and U.S. Patent Nos 6,177,414 and 5,919,442 among many others). Methods for conjugating polypeptides to dendrimers and other artificial particles (including nano- and micro-particles or beads) are described, for example, in U.S. Patent Nos. 6,485,718 and 6,083,708. B. Labeling moieties
  • Labeling moieties function to facilitate the detection of the TAFP bound to or localized in a cell that presented the ligand.
  • Labeling moieties can include a single fluorophore or multiple fluorophores.
  • activatable fluorescent labels that change in signal intensity are particularly useful for imaging.
  • Activatable fluorescent labels are labels that fluoresce at a certain intensity (first state) when under one set of physical conditions and a different intensity (second state) under a second set of physical conditions.
  • Many activatable fluorescent labels display multiple intensity states, which can be described as a continuum of fluorescence intensity (e.g. signal strength).
  • Activatable fluorescent labels can be self-quenching, meaning multiple fluorophore molecules when placed in close proximity to each other display a relatively low level of fluorescence (i.e. they are quenched). Upon separation of the fluorophores from each other (for example by increasing the distance between the fluorophores) the signal from the fluorophore molecules increases (i.e. they become de-quenched).
  • the intensity of fluorescence can be controlled by the number of fluorophores placed in close proximity to each other. Moreover, the number of fluorophores that produce a minimum amount of fluorescence (the most quenched state) depends of the fluorophore, or combination of fluorophores used to make the TAFP.
  • TAFPs are designed to display a significant intensity difference between a relatively quenched state and a relatively de-quenched state.
  • TAFPs that can display at least a 225, 250, 275, 300, 325, 350, 400, 450, 500, 1000, 2000, 3000, 4000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, or at least a 12,000 percent increase in fluorescence when changed from a first extracellular state to a second intracellular state are desirable for some applications.
  • activatable fluorophores are reversibly activatable.
  • a reversibly activatable probe under a first pH will display a certain fluorescence intensity (first state) and when at a different pH will have a different intensity (second state).
  • first state fluorescence intensity
  • second state different intensity
  • These fluorophores are reversible, meaning they can be changed from one intensity to another by changing the pH (such as changing pH 6g at least 0.5, at least 1, at least 2, at least 3, or at least 4 pH units).
  • Many reverse activatable fluorescent labels will display multiple intensity states, which can be described as a continuum of fluorescence intensity (signal strength).
  • TAFP display a significant intensity difference between a first extracellular state and a second intracellular state.
  • TAFPs can display at least a 200, 225, 250, 275, 300, 325, 350, 400, 450, 500, 1000, 2000, 3000, 4000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, and 75,000 percent increase in fluorescence when changed from a first state to a second state.
  • Linkers are molecules that function to join one or more targeting moieties and to one or more labeling moieties (such as multiple fluorophores). In some examples linkers can be selected to include sites that are sensitive to protease degradation so that upon degradation the fluorophores attached thereto are released and become unquenched. In other instances, linkers are used to connect more than one individual labeling moiety to a targeting moiety, thus increasing the signal strength from the TAFP.
  • the TAFP is assembled into a multimeric macromolecular complex, such as a dendrimer or other artificial particle.
  • Suitable dendrimer particles for use in the methods disclosed herein are known in the art.
  • Exemplary avidin dendrimers based on polyamidoamine (ethylenediamine) cores are described in Kobayashi et al., Bioconjug. Chem. 12:587-593, 2002 and Mamede et al., Clin. Can. Res. 9:3756-3762, 2003, both of which are incorporated herein by reference.
  • Dendrimers are synthetic chemical polymers that can have any one of a number of different functional groups of their surface (Tomalia, Aldrichimica Acta, 26:91: 101, 1993; and U.S.
  • Patent Nos 6,177,414 and 5,919,442 among many others Methods for conjugating polypeptides to dendrimers and other artificial particles (including nano- and micro-particles or beads) are described, for example, in U.S. Patent Nos. 6,485,718 and 6,083,708.
  • Linkers can be made using a labeling moiety conjugated to a first binding molecule and targeting moiety conjugated to a second binding molecule, wherein the first and second binding molecules bind to each other and form a binding pair ⁇ see, Hama et al., Cancer Res. 67:3809-3817, 2007, which is herein incorporated by reference).
  • Targeting molecule is added to a sample, or subject, and then the labeling moiety is added after the targeting moiety has had an opportunity to bind to its target cell.
  • the targeting moiety then binds to the labeling moiety through their respective binding molecules and the TAFP is formed in the presence of the cell it is intended to detect.
  • the targeting moiety can include a linker, such as biotin.
  • the targeting moiety can then be added to a sample, or a subject, under conditions sufficient to allow the targeting moiety to bind to its ligand on a cell (i.e. pre-target the cell).
  • a labeling moiety conjugated to a molecule that binds to the conjugated targeting moiety can be added.
  • a labeling moiety conjugated to avidin, or its deglycosylated form neutravidin can be added to the sample, or subject, under conditions sufficient to allow the avidin to bind to the biotinylated targeting moiety.
  • the TAFP is formed in the presence of the cells it is intended to detect.
  • linkers used in this way can be any binding pair of molecules.
  • linkers can be used that include avidin, biotin, antibodies (including functional equivalents of antibodies) and their congnate epitopes.
  • a bi-functional antibody can be used that binds to the ligand on the target cell, as well as to the labeling moiety.
  • either the labeling moiety or the targeting moiety can be conjugated to an artificial epitope such as biotin, small chelates such as DTPA or DOTA and the other TAFP moiety can be bound to an antibody that recognizes the artificial epitope.
  • antibodies conjugated to the targeting moiety are used and referred to as a primary antibodies, and the labeling moiety is conjugated to a secondary antibody that is specific for the primary antibody.
  • the targeting moiety can be conjugated to a labeling moiety using any method known in the art.
  • One of ordinary skill in the art will appreciate that the method used to conjugate the targeting moiety to the labeling moiety will vary depending on the specific moieties that are being conjugated. Conjugation methods will also vary if a linker used. Exemplary methods of conjugating targeting moieties to labeling moieties are provided in the Examples below. III. Using Target Activatable Fluorescence Probes
  • TAFP Target Activatable Fluorescence Probes
  • the diagnostic composition includes a TAFP.
  • a composition includes an optically detectable TAFP and additional compounds, such as pharmaceutically acceptable carriers or excipients.
  • the amount of the TAFP that is needed to detect a tumor cell can vary depending on the physical location of the tumor within the body, the means of detecting the TAFP that will be used, as well as the method used to deliver the
  • TAFP The effective amount of labeling moieties conjugated to targeting moieties can be determined through in vitro testing of the fluorescence intensity of the particular flurophore.
  • the effective amount of the TAFP needed to detect a particular tumor can also be determined by testing the sensitivity of the particular TAFP to an excised sample of the tumor.
  • Exemplary amounts TAFP that can be used include a dosage range of 0.001 to 200 mg/kg body weight in single or divided doses. Another example of a dosage range is 0.01 to 100 mg/kg body weight in single or divided doses.
  • composition containing a TAFP is administered to a subject, such as a human, by intraperitoneal injection at a dosage of 0.01 to 0.150 mg/kg/day (see, for example, Filleur et ah, Cancer Res.,
  • the TAFP is administered to a subject at a dosage of at least 0.01 mg/kg/day, 0.02 mg/kg/day, 0.03 mg/kg/day, 0.04 mg/kg/day, 0.05 mg/kg/day, or at least 1.0 mg/kg/day.
  • compositions of this disclosure can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for administration to mammals, such as humans.
  • the composition that includes a TAFP can be present in a pharmaceutically acceptable carrier.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the TAFP is administered.
  • Such pharmaceutical carriers include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • water is a carrier.
  • Saline solutions, blood plasma medium, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, for example for injectable solutions.
  • the composition can also contain conventional pharmaceutical adjunct materials such as, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.
  • Examples of pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the pharmaceutical composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the pharmaceutical composition can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • the pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • parenteral pharmaceutical carriers can be found in Remington: The Science and Practice of Pharmacy (19th Edition, 1995) in chapter 95.
  • Other exemplary compositions are prepared with conventional pharmaceutically acceptable counterions, as would be known to those of skill in the art.
  • the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to reduce pain at the site of the injection.
  • the disclosed TAFP compositions can be used as a diagnostic or to assess the effectiveness of a treatment, such as an anti-neoplastic treatment.
  • the TAFP can be used to visualize the impact that a therapy is having upon a tumor.
  • TAFPs are used in combination with an anti-tumor therapy, such as a therapy that includes anti-proliferative agents, anti-neoplastic agents, radiological agents, surgery, or combinations thereof.
  • an anti-tumor therapy such as a therapy that includes anti-proliferative agents, anti-neoplastic agents, radiological agents, surgery, or combinations thereof.
  • the subject can receive one or more anti-proliferative agents, anti-neoplastic agents, radiological agents, or combinations thereof, and also receive (for example at a subsequent time) a TAFP to determine the effect of the one or more agents on the tumor to be treated.
  • anti-proliferative/anti-neoplastic agents are alkylating agents, antimetabolites, antimitotic agents, natural products, or hormones and their antagonists.
  • alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).
  • antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.
  • antimitotic agents include microtubule- stabilizing agents (such as, paclitaxel and its analogues, docetaxel, abraxane, epothilones (such as epothilone A, B, D, and others), discodermolide, patupilone (EPO906), eleutherobins, laulimalide and its analogues (such as, C(16)-C(17)-des-epoxy laulimalide and C(20)-methoxy laulimalide), WS9885B, C-7 substituted eleutheside analogues (e.g., Castoldi et ah, Tetrahedron, 61(8):2123-2139, 2005), ceratamine A
  • Examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase).
  • vinca alkaloids such as vinblastine, vincristine, or vindesine
  • epipodophyllotoxins such as etoposide or teniposide
  • antibiotics such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C
  • enzymes such as L-asparaginase
  • miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).
  • platinum coordination complexes such as cis-diamine-dichloroplatinum II also known as cisplatin
  • substituted ureas such as hydroxyurea
  • methyl hydrazine derivatives such as procarbazine
  • adrenocrotical suppressants such as mitotane and aminoglutethimide
  • hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acdtate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fhioxymesterone).
  • adrenocorticosteroids such as prednisone
  • progestins such as hydroxyprogesterone caproate, medroxyprogesterone acdtate, and magestrol acetate
  • estrogens such as diethylstilbestrol and ethinyl estradiol
  • antiestrogens such as tamoxifen
  • androgens such as testerone proprionate and fhioxymesterone
  • Examples of the most commonly used chemotherapy drugs that could be used in combination with the disclosed TAFP agents include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-I l), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and Vitamin D drugs (such as, calcitriol
  • TAFPs in conjunction with various treatment regimes are not limited to the lists provided in these examples, but include any composition for the treatment of diseases or conditions for which the TAFP is targeted.
  • compositions containing TAFP can be administered by any convenient route, including, for example, infusion or bolus injection, topical, absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, and the like) ophthalmic, nasal, and transdermal, and can be administered together with other biologically active agents. Administration can be systemic or local.
  • a pharmaceutical composition by intraventricular or intrathecal injection; intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir.
  • Pulmonary administration can also be employed (for example, by an inhaler or nebulizer), for instance using a formulation containing an aerosolizing agent.
  • a fluorescently labeled TAFP is injected or infused directly into the vasculature of a subject, such as a human; for example, a TAFP can be injected intravenously.
  • Methods for intravascular injection of pharmaceutical compositions are well known.
  • the TAFP is topically applied (for example, through a wash) or infused intraperitoneally. Methods for intraperitoneal injection and infusion are well known in the art.
  • a pharmaceutical composition can be desirable to administer a pharmaceutical composition locally to the area in need of treatment.
  • This can be achieved by, for example, by local or regional infusion or perfusion during surgery, topical application (for example, as part of a wound dressing), injection, catheter, suppository, or implant (for example, implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like.
  • administration is by direct injection at the site (or former site) of a tissue that is to be treated, such as the site from which a tumor is surgically resected.
  • the pharmaceutical composition is delivered in a vesicle, such as liposomes (see, e.g., Langer, Science 249, 1527, 1990; Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365, 1989).
  • a vesicle such as liposomes
  • the pharmaceutical composition is delivered in a controlled release system.
  • a pump is used (see, e.g., Langer Science 249, 1527, 1990; Sefton Crit. Rev. Biomed. Eng. 14, 201, 1987; Buchwald et al, Surgery 88, 507, 1980; Saudek et al, N. Engl. J. Med. 321, 574, 1989).
  • polymeric materials can be used (see, e.g., Ranger et al, Macromol. ScL Rev. Macromol. Chem. 23, 61, 1983; Levy et al, Science 228, 190, 1985; During et al., Ann. Neurol.
  • the disclosed methods include contacting a tumor with a TAFP
  • the method can include administering the TAFP-contaimng composition to the subject, for example mtraperitoneally Following introduction into the subject, the TAFP binds a specific ligand (the ligand will vary depending upon the type of cancer) that is expressed on the surface of the tumor cells Because, the specific ligand is not significantly expressed on the surface of most normal (non-tumor) cells, the TAFP is localized to tumor cells, with minimal to negligible binding to normal (non-tumor) tissues Upon entry into the cell the TAFP emission fluorescent signal intensifies permitting identification of tumor cells in the background of unbound TAFP
  • additional diagnostic reagents are administered to the subject
  • one or more additional fluorescently labeled diagnostic probes can be administered to the subject
  • the second or other additional diagnostic probe is selected to aid in the diagnostic characterization of the tumors
  • antibodies useful for the identification or characterization can be fluorescently labeled and administered in combination with the TAFP to further enhance diagnostic and prognostic capabilities
  • the one or more additional diagnostic reagents is labeled with a fluorophore other than that with which the TAFP is labeled (such as a fluorophore having an emission spectra that is distinguishable from the emission spectra of the label moiety in the TAFP)
  • the amount of time necessary for internalization will vary depending upon the type and health of the cells Exemplary times include at least 30 seconds, at least 1 minute, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes or at least one hour
  • in vitro testing to establish the optimum time for detection can be used Standard times can then be established for various types of cell detection C.
  • Methods of detecting the tumors can include contacting a TAFP with a cell expressing a ligand specific for the TAFP and detecting the label.
  • the TAFP binds to the ligand on the tumor cell, thereby permitting detection of the labeled- ligand bound to the tumor cell.
  • the subject is known or suspected of having a tumor.
  • the methods are performed prior to or at the time of surgery.
  • the methods further include surgically removing, e.g., excising or otherwise ablating (for example by the direct application of laser energy) the detected tumor(s).
  • the methods are non-toxic, highly sensitive, and can be used both in minimally invasive diagnostic procedures (such as endoscopy and laparoscopy) and during surgery to identify and localize tumors, such as tumors having a diameter of less than 1 mm.
  • the methods can permit real time visualization of tumors and metastatic foci during surgery, for example under ambient light conditions.
  • the method can further include treating the tumor (for example metastatic cancer, including dispersed or single cells and very small foci or clusters of cells) using photodynamic therapy. In some examples, this allows a surgeon to remove all metastatic foci.
  • the targeting moiety binds to ASGPR
  • cancer cells such as ovarian, gastric, colon, bladder, synovial, pleural and pancreatic cancers, and disseminated metastatic nodules can be detected.
  • the targeting moiety is Herceptin (D (Genentech, San Fransisco, California) certain breast cancer cells can be detected.
  • the disclosed methods of detecting cells permit detection of relatively small tumors, hi some examples, the tumor detected is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or less than 0.05 mm in diameter.
  • TAFP is contacted with the tumor cells under conditions sufficient to permit binding of the TAFP to the ligand, the relevant anatomical area is exposed to a light source that emits a wavelength capable of exciting the fluorophore associated with the TAFP.
  • the area can be exposed to a wavelength of light that will excite the desired fluorophore, such as ambient light or a laser.
  • the method is practiced in conjunction with the surgical removal of tumors (for example, intraperitoneal tumors)
  • the subject's exterior abdominal tissues and peritoneum are surgically incised to expose the interior of the peritoneum and mesentery. Most typically abdominal surgery is performed using a high intensity halogen light source that emits light across the visible spectrum.
  • exemplary fluorophores that can be conjugated to a TAFP include those with excitation spectra in the visible range, e.g., between about 400 and about 700 nm).
  • Light emitted from the ambient surgical light source illuminates the abdominal region, and excites the fluorophore attached to the TAFP.
  • the resulting fluorescence emission can then be detected, precisely localizing the tumor. Due to the high ambient light level, background light in the visible spectrum can overwhelm the fluorescence emission making tumor localization difficult. Accordingly, visualization of the localized fluorophore can be performed using an emission filter that permits transmission of light corresponding to the emission spectra of the fluorophore, and that reduces light scatter and blocks the high background that would otherwise make detection of the fluorescent signal difficult.
  • the emission filter can be a band pass filter that permits transmission of light in a selected wavelength range, while blocking light outside that selected range or band.
  • the emission filter and the fluorophore are selected to be compatible, such that the fluorescent emission of the fluorophore is within the range "passed" by the emission filter.
  • the fluorophore emits in the green light range (e.g. , Rhodamine- green, or BODIPY® FL, etc.)
  • the emission filter is selected to pass wavelengths between about 490 nm and about 575 nm.
  • a filter is selected that permits passage of light in a wavelength range between about 620 nm and about 780 nm.
  • the emission filter can be located on an optical assistance device.
  • a device can be worn by the individual performing the surgery in the form of eyeglasses (monocular or binocular), goggles, visors, or the like.
  • the emission filter can be placed on an overhead or floor mounted lens.
  • the optical assistance device also includes a lens providing magnification of the operating field.
  • Surgical personnel can also visualize the fluorescence emission indirectly.
  • indirect visualization can be performed by means of a camera, such as a charge-coupled device (CCD) camera interfaced with a monitor for displaying visual images captured by the CCD camera.
  • CCD charge-coupled device
  • the surgeon can resect (that is, surgically remove) the tumor.
  • a complete excision of all visible tumors is desirable.
  • the resection can be a partial removal of the tumor or tumors.
  • surgery can be succeeded by photodynamic therapy, for example, using the photodynamic therapeutic methods described below (or by other treatment modalities, including radiation therapy or chemotherapy).
  • the methods are used for non-invasive (or minimally invasive) diagnostic procedures.
  • the fluorescence emission is visualized using a diagnostic apparatus, such as an endoscope or a laparoscope, depending on the tissues or organs to be visualized.
  • a diagnostic apparatus such as an endoscope or a laparoscope, depending on the tissues or organs to be visualized.
  • the diagnostic fluorophore is selected to have excitation and emission spectra compatible with commonly used diagnostic equipment.
  • the methods can be performed using a commercially available endoscope, such as the D-light equipped endoscope from Karl Storz (Tuttlingen), which uses a high intensity xenon lamp as a light source.
  • an excitation filter can be employed that narrows the range of wavelengths used to excite the fluorophore.
  • the aforementioned endoscope is equipped to elicit autofluorescence and uses an excitation filter to restrict the excitation wavelength to between about 375 and 450 nm.
  • a fluorophore is selected that has an excitation spectrum maximum within the range transmitted by the filter.
  • the endoscope can be equipped with a suitable emission filter that permits detection of fluorescent emissions generated by excitation of the fluorophore, but blocks scattered light and autofluorescence of other wavelengths.
  • the diagnostic instrument for example, a laparoscope or an endoscope
  • the diagnostic instrument is equipped with customized filters that are selected to optimize excitation and detection of emission of the selected diagnostic fluorophore.
  • the selection of suitable filters based on the excitation and emission spectra of fluorophores can be made by one of ordinary skill in the art, without undue experimentation.
  • TAFPs can be designed so that they are at a first state when they are extracellular and not bound to a ligand and then change to a second state when they bind to a ligand or change to a second state upon internalization into a cell.
  • TAFPs can be activated via enzymatic activity (activation mechanism). Enzymatic activity can be found in or displayed on the cell membrane or can be found in an intercellular organelle (such as a lysosome or endosome). Hence, how a TAFP changes from a first state to a second state will depend upon its construction the proximity of its ligand to the activation mechanism.
  • the change from a first state to a second state can occur upon movement of the TAFP from the extracellular space to the intracellular space.
  • a TAFP upon internalization into a lysosome or other intracellular organelle, a TAFP can change from a first to a second state.
  • TAFPs can be activated upon internalization into cells (for example via endocytosis), TAFPs can be used to detect cells that are capable of performing internalization. This allows TAFPs to detect biological activity.
  • the intensity of fluorescence attributable to the TAFP can be compared to the intensity of fluorescence attributable to the non-activatable probe, and the relative health of the cell population can be ascertained. For example, if the TAFP signal is weak compared to that of the non-activatable probe, this indicates that the targeted cells are not biologically active.
  • Tetrachloro-l,4-benzoquinone (p-chloranil) (0.361 s, 1.47 mmol) was added and stirring continued for another 10 minutes.
  • the reaction solution was washed in water, dried using anhydrous Na 2 SO 4 , and depressurized.
  • the resulting compound was refined repeatedly using silica gel column chromatography using dichloromethane/methanol (9: 1) containing 1 % triethyl amine (TEA) as an effluent solvent and a reddish green solid was obtained.
  • the resulting compound was dissolved in 100 mL of toluene containing 3 mL of N,N-diisopropyl ethyl amine (DIEA).
  • DIEA N,N-diisopropyl ethyl amine
  • solvent A H 2 O, 0.1 % TFA
  • the fraction containing the desired compound was extracted with dichloromethane.
  • the compound was dried using anhydrous Na 2 SO 4 , and depressurized and an orange solid was obtained (compound 3a, 32.0 mg, 84 % yield).
  • the fraction that contained the desired compound was extracted using dichloromethane.
  • the desired compound was dried with anhydrous Na 2 SO 4 , depressurized, and a red solid (compound 3b, 40.3 mg, 84 % yield) was obtained.
  • the compound obtained was dissolved in 100 mL of toluene containing 5 mL of DIEA. BF 3 • OEt 2 was dropped in slowly while stirring at room temperature. The resulting solution was stirred for 10 minutes. The reaction solution was washed with water, dried using anhydrous Na 2 SO 4 and depressurized. The resulting compound was refined using silica gel column chromatography using dichloromethane as an effluent solvent and an orange solid (compound 2d, 273 mg, 32 % yield) was obtained.
  • Example 2 Using AlkylB ODIPY to detect biological activity
  • TAFP target-specific activatable fluorescent probe
  • a pH activatable TAFP was synthesized (galactosyl serum albumin conjugated with diethyl-BODIPY: GSA-detBDP), as well as a non- activatable fluorescent probe (galactosyl serum albumin conjugated with BODIPY-R6G®: GSA-BDP).
  • the fluorescence intensities as well as the emission spectra of GSA- BDP and GSA-detBDP were measured at 5 different pH values (2.3, 3.3, 5.2, 6.4 and 7.4) using the same amount (50 pmol) of GSA-BDP or GSA-detBDP in the same volume of buffer solutions (400 ⁇ L).
  • GSA-BDP and GSA-detBDP have an emission peak at a wavelength of 570 nm when stepped in 10 nm increments (Fig. IA).
  • the peak fluorescence intensity of GSA-BDP changed little at different pH values, but the peak fluorescence intensity of GSA-detBDP increased from 150 to 2014 (>13-fold increase) in arbitrary unit (a.u.) when pH changed from 7.4 to 2.3.
  • the mean fluorescence intensities ( ⁇ SD) of GSA- BDP were 225.7 ⁇ 16.6, 223.3 ⁇ 18.9, 206.6 ⁇ 9.3, 216.7 ⁇ 6.6, 204.4 ⁇ 3.3 at pH 2.3, 3.3, 5.2, 6.4 and 7.4, respectively, while the mean fluorescence intensities of GSA-detBDP were 169.1 ⁇ 21.4, 99.0 + 9.5, 34.8 ⁇ 3.3, 20.1 + 2.2 and 7.6 + 1.4 at pH 2.3, 3.3, 5.2, 6.4 and 7.4, respectively (Fig. IB).
  • the regression lines were calculated in GSA-BDP and GSA-detBDP from the data sets of pH values and common logarithm values of mean fluorescence intensity (Fig. IB).
  • the probes were optically detected using fluorescence microscopy and transmitted light differential interference contrast (DIC) imaging.
  • Fluorescence microscopy demonstrated a large number of fluorescent dots within the cytoplasm as early as 30 minutes after incubation with both GSA-BDP and GSA-detBDP.
  • the fluorescent dots produced by GSA-detBDP were initially very small and minimally fluorescent, but became marked and bright later especially >1 hour after incubation.
  • the size and the intensity of fluorescent dots produced by GSA-BDP were consistent, although a slight increase in fluorescence was observed at 3 hours after incubation.
  • a mouse peritoneal cancer model was used to demonstrate the probes' in vivo activity.
  • the model was established 21 days after intraperitoneal injection of SHIN3 ovarian cancer cells into the mouse.
  • GSA-BDP or GSA-detBDP (700 pmol each) was injected into the peritoneal cavity of a tumor-bearing mouse, and spectral fluorescence imaging was performed using the MaestroTM In- Vivo Imaging System (CRi Inc., Woburn, MA, USA) 3 hours after injection. Sufficient fluorescence arising from tumor foci of the peritoneal cavities, as well as on the peritoneal membranes, was observed with both GSA-BDP and GSA-detBDP.
  • GSA-detBDP demonstrated high fluorescence from the tumor foci with minimal background signals, thereby permitting visualization of small peritoneal implants as well as the aggregated tumor foci.
  • the observed high signal-to-background imaging was further investigated using dying cancer cells after instillation with GSA-detBDP and GSA-BDP.
  • GSA-detBDP At 3 hours after intraperitoneal injection of 700 pmol GSA-BDP or GSA-detBDP, 2 mice intraperitoneally injected with GSA-BDP or GSA-detBDP were sacrificed with carbon dioxide.
  • the peritoneal membranes were washed once with PBS and placed side-by- side on a nonfluorescent plate, and serial spectral fluorescence imaging was performed every 5 minutes after sacrifice for 60 minutes. Immediately (0 min) after sacrifice, both GSA-BDP and GSA-detBDP depicted small cancer foci on the peritoneal membranes.
  • GSA Galactosylated bovine serum albumin
  • BODIPY-R6G® BDP
  • BDP pH sensitive amido-reactive diethyl BODIPY
  • the protein concentration of GSA-BDP and GSA-detBDP samples was determined with Coomassie Plus protein assay kit (Pierce Chemical Co., Rockford, IL, USA) by measuring the absorption at 595 nm with a UV- Vis system (8453 Value UV-Bis system, Agilent Technologies, Palo Alto, CA, USA) using GSA standard solutions of known concentrations (100, 200 and 400 ⁇ g/mL). BDP and detBDP concentrations were measured by absorption at 503 nm respectively with a UV- Vis system (8453 Value UV-Bis system, Agilent Technologies, Palo Alto, CA, USA) to confirm the number of fluorophore molecules conjugated with each GSA molecule. The numbers of BDP and detBDP molecules per GSA were 5.2 and 6.3, respectively.
  • fluorescence intensity and emission spectra of GSA-BDP and GSA-detBDP were measured by the MaestroTM In- Vivo Imaging System (CRI Inc., Woburn, MA, USA) in arbitrary units (a.u.).
  • GSA-BDP and GSA-detBDP 50 pmol
  • 400 ⁇ L phosphate buffers with different pH values were placed in a nonfluorescent 96-well plate (Costar, Corning Incorporated, NY, USA) and spectral fluorescence imaging was performed.
  • a band pass filter from 490 to 510 nm and a long pass filter over 530 nm were used for emission and excitation light respectively.
  • the tunable filter was automatically stepped in 10 nm increments from 500 to 800 nm while the camera captured images at each wavelength interval with constant exposure.
  • Spectral unmixing algorithms were applied to create the unmixed image.
  • a region of interest (ROI) as large as each well was drawn to determine the emission spectra of 2 probes using commercial software (Maestro software, CRi Inc. Woburn, MA USA).
  • the mean fluorescence intensity (a.u.) as well as the standard deviation (SD) of each probe at different pH values was measured using ImageJ software.
  • SHIN3 cells Imai et ah, Oncology 47, 177-184, 1990 (1 x 10 4 ) were plated on a cover glass bottom culture well and incubated for 16 hours.
  • GSA-BDP or GSA-detBDP was added to the medium (200 nmol/L) and the cells were incubated for 30 minutes, 1 hour, and 3 hours.
  • Cells were washed one time with PBS and fluorescent microscopy as well as transmitted light differential interference contrast (DIC) imaging was performed using an Olympus BX51 microscope (Olympus America Inc., Melville, NY, USA) equipped with the following filters: excitation wavelength 470-490 nm, emission wavelength 515 nm long pass.
  • DIC transmitted light differential interference contrast
  • MFI Mean Fluorescence Index
  • the intraperitoneal tumor implants were established by intraperitoneal injection of 2 x 10 cells suspended in 200 ⁇ L of PBS in female nude mice (National Cancer Institute Animal Production Facility, Frederick, MD, USA). Experiments with tumor-bearing mice were performed at 21 days after injection of the cells.
  • mice were sacrificed at a time with carbon dioxide 3 hours after injection with GSA-BDP or GSA-detBDP.
  • the peritoneal membranes were washed one time with PBS and placed side -by- side on a nonfluorescent plate, and serial spectral fluorescence imaging was performed using the MaestroTM In- Vivo Imaging System every 5 minutes for 60 minutes.
  • the image acquisition parameters such as excitation/emission filters, distance between the peritoneal membranes and the camera, spectrum used for unmixing and exposure time were the same during the dynamic study.
  • Example 3 Using rhodamineX to detect biological activity
  • This example describes the use of a target-specific activatable fluorescent probe (TAFP) that contains 3 rhodamineX molecules (3ROX) as a labeling moiety to detect biological activity in vitro and in vivo.
  • TAFP target-specific activatable fluorescent probe
  • 3ROX 3 rhodamineX molecules
  • Av-0.5ROX has 0.5 rhodamineX molecules per avidin whereas Av-3ROX has 3 rhodamineX molecules per avidin.
  • Av-0.5ROX is fluorescent while Av-3ROX is self-quenched. Therefore, at the same concentration (780 ng/mL), Av-0.5ROX was brighter than Av-3ROX in PBS even though it has fewer rhodamineX molecules (Fig. 4).
  • SDS sodium dodecyl sulphate
  • Av-3ROX showed more fluorescence than Av-0.5ROX once internalized by SHIN3 cells in vitro.
  • Serial observation of SHIN3 cells incubated with Av-0.5ROX or Av-3ROX was performed using fluorescence microscopy to compare the temporal changes of intracellular fluorescence production and distribution.
  • Fluorescence microscopy demonstrated fluorescent dots within the cytoplasm as early as 30 minutes after incubation with both Av-0.5ROX and Av-3ROX. However, the fluorescence intensity of Av-0.5ROX was initially higher than that of Av-3ROX. The number of fluorescent dots within the cytoplasm increased 6 hours after incubation in both groups. However, fluorescent dots produced by Av-3ROX became larger and brighter than those of Av-0.5ROX. However, the self-quenched Av-3ROX was activated once it was internalized and increased markedly in fluorescence, likely as a consequence of proteolysis in the lysosome (Fig. 3D).
  • Av-3ROX produced minimal background and tumor fluorescence, resulting in poor tumor detection.
  • the fluorescence intensity arising from tumor nodules was higher with Av-3ROX compared to Av-0.5ROX and the background signal was lower.
  • the fluorescence intensity of the tumor nodules was much higher with Av-3ROX than with Av-0.5ROX.
  • the background signal was comparable between the two groups.
  • Av-3R0X was stabilized by crosslinking with disuccinimidyl suberate (DSS) which covalently bound the avidin tetramer using amide residues on lysine molecules (Fig. 5A).
  • DSS disuccinimidyl suberate
  • Crosslinked or non-crosslinked Av-3R0X was separated into monomer, dimmer, trimer and tetramer with reducing conditions of SDS- PAGE.
  • the fluorescence intensity of Av-3R0X monomer was 8-fold stronger than that of crosslinked Av-3R0X tetramer at the same protein concentration (ImageJTM software).
  • imageJTM software To assess the influence of crosslinking on the fluorescence intensity, the fluorescence intensity of crosslinked Av-3ROX was compared with non- crosslinked Av-3R0X at the same concentration with or without 5% SDS.
  • Crosslinking decreased the fluorescence intensity of Av-3R0X with 5% SDS, although crosslinking did not affect the fluorescence intensity without SDS (Fig. 5B).
  • the fluorescence intensity of crosslinked Av-3R0X was compared with non-crosslinked Av-3R0X.
  • Non-crosslinked Av-3R0X clearly visualized submillimeter cancer implants with minimal background, whereas, the fluorescence signal of the crosslinked Av-3ROX arising from the tumor nodules was considerably lower.
  • a region of interest (ROI) as large as the peritoneal membrane was drawn inside the intestine using the unmixed Av-3ROX image and a histogram depicting the distribution of pixel intensities was created (Fig. 5C).
  • the dynamic range of signal intensity in the unmixed fluorescence image was set from 0 to 255 in arbitrary units (a.u.) and the threshold value (t) was changed from 40 to 240 in increments of 10, because the background signals such as the normal peritoneal membrane excluding tumors and the nonfluorescent plate were mostly less than 40 (a.u.).
  • the total number of pixels (N) within the threshold range was calculated as a function of threshold (t) and regression line was calculated in each ROI (Fig. 5D).
  • the correlation coefficients of crosslinked Av-3R0X and non-crosslinked Av-3ROX were -0.9927 and 0.9997, respectively.
  • the slopes of crosslinked Av-3ROX and non-crosslinked Av3ROX were -0.0375 and -0.0110, respectively.
  • Av-3ROX was injected into RFP-transfected SHIN3 tumor-bearing mice and spectral fluorescence imaging was performed. Spectral resolved RFP images, Av-3ROX images, and composite images were made. Unmixed Av-3ROX images demonstrated ring-like accumulation around the foci which were depicted by the unmixed RFP images. A total of 514 peritoneal tumor foci in 4 mice were identified by the unmixed Av- 3ROX images, the unmixed RFP images, or both.
  • ROIs were created in the non-tumorous areas (i. e. where no tumors were visible on the RFP images).
  • RFP-positive foci positive standard
  • Av-3ROX-positive foci were defined as those whose fluorescence intensities were > 1 (a.u.) on unmixed Av-3ROX images.
  • 465 foci showed Av-3ROX fluorescence intensities > 1 (a.u.) amongst the 507 RFP-positive foci (Fig. 6).
  • Av-0.5ROX has 0.5 rhodamineX molecules per avidin whereas Av-3ROX has 3 rhodamineX molecules per avidin.
  • Av-0.5ROX has 0.5 rhodamineX molecules per avidin
  • Av-3ROX has 3 rhodamineX molecules per avidin.
  • 400 ⁇ g (5.9 nmol) of avidin in 80 ⁇ L of Na 2 HPO 4 was incubated with 6 ⁇ g (9 nmol) (for Av-0.5ROX) or 65 ⁇ g (100 nmol) (for Av-3ROX) of rhodamineX succinoimidyl ester in DMSO for 15 minutes.
  • the mixture was purified with Sephadex G50 (PD-IO; GE Healthcare, Milwaukee, WI, USA).
  • Av-3ROX samples were kept in the refrigerator for 3 days and the precipitated fraction was separated by centrifugation; the supernatant was used for further study.
  • the protein concentration of each sample was determined with Coomassie Plus protein assay kit (Pierce Chemical Co., Rockford, IL) by measuring the absorption at 595 nm with a UV-Vis system (8453 Value UV-Bis system, Agilent Technologies, Palo Alto, CA).
  • the rhodamineX concentration was measured by the absorption at 587 nm with a UV-Vis system (8453 Value UV-Bis system, Agilent Technologies, Palo Alto, CA) to confirm the number of rhodamineX molecules conjugated with each avidin molecule.
  • DSS Disuccinimidyl suberate
  • Av-0.5ROX or Av-3ROX in 1 mL PBS was incubated with 100 ⁇ g/20 ⁇ L DMSO of DSS in 0.1 M Na 2 HPO 4 at pH 8.5 for 1 hour.
  • the non-crosslinked control was prepared by exactly the same procedure without DSS.
  • Crosslinked and non-crosslinked samples were used for in vitro SDS- PAGE analysis and fluorescence spectrometry as well as in vivo imaging studies.
  • the fluorescence intensity of wet gels were analyzed with a high resolution fluorescence scanner system (FLA-5100, Fu ⁇ f ⁇ lm Medical Systems, Stanford, CT) An internal laser of 532 nm was used for excitation and a long pass filter over 575 nm was employed for light emission The lateral and longitudinal spatial resolution (pixel size) was 100 ⁇ m
  • the fluorescence intensity of each band was analyzed with commercial software (Multigage, Fujifilm Medical Systems USA, Inc , Stanford, CA) and the ratio of fluorescence intensities was determined
  • the gels were stained with Coomassie blue using a Colloidal Coomassie gel staining kit (In vitro gen, No vex, San Diego, CA), then dried, and digitally scanned (Epson 6300, Epson America Inc , Long Beach, CA), and the protein concentration in each band was determined with ImagelTM software After obtaining the fluorescence intensity in each band, the fluorescence intensity per avidm was calculated by dividing the fluorescence intensity
  • An established ovarian cancer cell line SHIN3 (Imai et al , Oncology 47 177-84, 1990) was used for generating intraperitoneal disseminated cancer rmcrofoci
  • the cell lines were grown in RPMI 1640 medium (Gibco, Gaithersberg, MD) containing 10% fetal bovine serum (FBS) (Gibco, Gaithersberg, MD), 0 03% L glutamme at 37°C, 100 Umts/mL Penicillin and 100 ⁇ g/mL Streptomycin in 5% CO 2
  • the plasmid was transfected into SHIN3 cells to validate the results with targeted fluorophores
  • the transfection of RFP was performed with an electroporation method using Gene Plus IITM (Bio- Rad Laboratories, Hercules, CA). Briefly, 3 ⁇ g of DsRed2-express plasmid was mixed with 2 million SHIN3 cells in 400 ⁇ L of the cell culture media (RPMI-1640 with 10% FCS), The cell suspension was then placed in a pulse cuvette (Bio-Rad Laboratories) and 250V pulses were delivered after 950 cycles, G. Fluorescence microscopy
  • SHIN3 cells (5 x 10 ) were plated on a coverglass bottom culture well and incubated for 16 hours, Av-0.5ROX or Av-3ROX was added to the medium (20 ⁇ g/mL) and the cells were incubated for either 30 minutes or 6 hours.
  • Cells were washed one time with PBS and fluorescence microscopy was performed using an Olympus BX51 microscope (Olympus America Inc., Melville, NY) equipped with the following filters: excitation wavelength 530-490 nm, emission wavelength 590 nm long pass. Transmitted light differential interference contrast (DIC) images were also acquired.
  • Olympus BX51 microscope Olympus America Inc., Melville, NY
  • DIC Transmitted light differential interference contrast
  • the abdominal cavity was exposed and spectral fluorescence images were obtained using the MaestroTM In- Vivo Imaging System (CRi Inc., Woburn, MA, USA). Whole abdominal images as well as close-up peritoneal membrane images were obtained.
  • a band pass filter from 503 to 555 nm and a long pass filter over 580 nm were used for emission and excitation light, respectively.
  • the tunable filter was automatically stepped in 10 nm increments from 550 to 800 nm while the camera captured images at each wavelength interval with constant exposure.
  • the sensitivity and specificity of spectral Av-3ROX imaging for the detection of peritoneal disseminated cancer foci was studied using four tumor- bearing mice.
  • the intraperitoneal (i.p.) tumor xenografts were established 14 days after i.p. injection of 2 x 10 RFP-transfected SH1N3 cancer cells suspended in 200 ⁇ L of PBS, in female nude mice (National Cancer Institute Animal Production Facility, Frederick, MD). Three hours after i.p.
  • spectral fluorescence images of the peritoneal membranes were obtained by MaestroTM In- Vivo Imaging System (CRI Inc., Woburn, MA).
  • a band pass filter from 445 to 490 nm and a long pass filter over 515 nm were used for emission and excitation light, respectively.
  • the tunable filter was automatically stepped-up in 5 nm increments from 500 to 800 nm while the camera captured images at each wavelength interval with a constant exposure.
  • Spectral unmixing algorithms were applied to create the unmixed images of fluorescein and autofluorescence.
  • ROIs were drawn within the nodules depicted by unmixed RFP images, unmixed Av-3ROX images, or both. Additional ROIs were drawn in the surrounding non-tumorous areas on the unmixed RFP images (standard reference for non-cancerous foci). The average fluorescence intensity of each ROI was calculated both on the RFP and the Av-3ROX spectral unmixed images using commercial software (Maestro software version 2, CRI Inc., Woburn, MA).
  • Av-3ROX-positive nodules were defined as having an average fluorescence intensity > 1 (a.u.) on the unmixed Av-3ROX images, whilst Av-ROX-negative nodules were defined as average fluorescence intensity ⁇ 1 (a.u.).
  • Sensitivity of Av-3ROX for the detection of peritoneal cancer foci was defined as the number of peritoneal foci positive for both Av-3ROX and RFP divided by the number of peritoneal foci positive for RFP.
  • Specificity of Av-3ROX was defined as the number of peritoneal foci negative for both RFP and Av-3ROX divided by the number of peritoneal foci negative for RFP.
  • Example 4 Using aniline reactive moieties in alkylBODIPY to detect biological activity
  • This example describes the results from using TAFPs that include NMe 2 BODIPY and NEt 2 BODIPY (Fig. 7) to detect biological activity.
  • Anilines were selected for inclusion in the labeling moiety because of their reactivity towards protons and 2,6-dicarboxyethyl-l,3,5,7-tetramethylBODIPY was included as a fluorophore.
  • Fig. 8A fluorescence activation ratio
  • the aniline probes were conjugated to herceptin and GSA. Briefly, one of the carboxy groups of the 2,6-substituents of the BODIPY was converted to the NHS (N-hydroxysuccinimidyl) ester, and tagged covalently to the antibody and the ligand by forming an amide bond with Lys residues according to methods well known in the art.
  • the resultant probe-antibody conjugates also worked as acidic environment sensitive fluorescence probes (Fig. 8C).
  • the pKa values were 4.3 and 5.9 for NMe 2 BODIPY, NEt 2 BODIPY tagged HERCEPTIN, respectively, which were almost identical to those of the unconjugated labeling moiety.
  • the TAFP activation ratio in fluorescence intensity is dependent on the pH value inside endosome or lysosome.
  • the TAFPs with the HERCEPTIN targeting moiety were then tested using NIH/3T3 HER2 cells that overexpress the HER2 receptor.
  • the TAVPs were added to the NIH/3T3 HER2 cells in a culture dish at a final concentration of 5 nM. Fluorescence images were then taken with a confocal microscope. A control that included a fluorophore that was always fluorescent regardless of the pH was conjugated to HERCEPTIN and imaged. The results showed confined bright fluorescence only on the plasma membrane in the image taken immediately after the addition of the probes. After 2 hours, small bright spots could be observed inside the cells. After 4 hours and 1 day, almost the same and slightly brighter images were obtained.
  • the control probe always strongly fluoresced, regardless of being internalized or not.
  • the TAFP showed almost no fluorescence in the images taken immediately after addition to the cells, because the media was at a relatively neutral pH. After 2 hours, some portions of the TAFPs attached and were internalized.
  • the NEt 2 BOD IPY-tagged TAFP sample showed fluorescent spots inside the cells, but not on the plasma membrane.
  • the NMe 2 BOD IPY-tagged TAFP showed few fluorescent spots inside the cells. After 4 hours, both activatable probes gave strong fluorescent spots only inside the cells, and this activation lasted at least 1 day.
  • the TAFPs were then used to detect HER2 overexpressing lung metastatic tumors in vivo.
  • Two HER2-positive and HER2-negative/RFP-positive tumors were injected intravenously to produce lung metastatic tumors.
  • side-by-side postmortem in situ imaging was performed 1 day after injection.
  • the TAFPs labeled the HER2-positive tumors.
  • the "always on” control showed green fluorescence signal not only from the HER2-positive lung matsatasis but also from everywhere in the body including muscle, great vessels.
  • green signal was also seen in HER2-negative tumors shown up in yellow, which was a mixed signal between red and green.
  • This example provides another description of a nRO labeling moiety and its use to identify cancer cells.
  • a summary of the results from in vitro and in vivo examples is provided followed by a detailed description of the materials and methods used.
  • GmSA-IROX galactosamine-conjugated bovine serum albumin
  • GmSA-20ROX showed a significant rightward shift (>one order shift) as compared with SHIN3 control cells at 6 hours after incubation.
  • MFI mean fluorescence index
  • MFI values immediately, 30 minutes, 1 , 3 and 6 hours after incubation with GmSA- IROX were 4.0, 4.0, 4.4, 5.4 and 6.8, respectively.
  • MFI values immediately, 30 minutes, 1, 3 and 6 hours after incubation with GmSA-20ROX were 4.0, 4.9, 8.0, 24.3 and 35.4, respectively.
  • the fluorescence intensity of SHIN3 cancer cells instilled with GmSA-20ROX became 5 times higher than that of GmSA-IROX.
  • Spectra of GmSA-IROX at pH 3.3, 5.2, 6.4 and 7.4 contained an emission peak at a wavelength of 610 nm, while at pH 2.3, the spectrum contained an emission peak at a wavelength of 620 nm when stepped in 10 nm increments.
  • Spectra of GmSA-20ROX at pH 5.2, 6.4 and 7.4 contained an emission peak at a wavelength of 610 nm, whereas at pH 2.3 and 3.3, the spectra contained an emission peak at a wavelength of 620 nm.
  • the fluorescence signal intensity of GmSA-IROX changed little while the intensity of GmSA-20ROX decreased under acidic conditions.
  • the fluorescence intensities of GmSA-IROX at pH 2.3, 3.3, 5.2, 6.4 and 7.4 were 151, 225, 231, 227 and 213, and those of GmSA-20ROX were 41, 75, 93, 152 and 146 in arbitrary units, respectively.
  • the slopes of GmSA-IROX and GmSA- 20ROX were 0.023 and 0.105, respectively.
  • Enzymatic activation of GmSA-IROX or GmSA-20ROX was studied using trypsin, cathepsin C, cathepsin D and MMP-2.
  • the slopes of regression lines calculated from the data sets of serial relative fluorescent SI of GmSA-IROX and the incubation time were -0.060, -0.080, -0.074 and 0.151 (a.u./hour) for trypsin, cathepsin C, cathepsin D and MMP-2, respectively.
  • the slopes of trypsin, cathepsin C, cathepsin D and MMP-2 were 0.079, -0.050, 0.305 and 0.012 (a.u./hour), respectively.
  • region of interest (ROI) measurements were drawn on peritoneal surfaces using the unmixed fluorescence image and a histogram depicting the distribution of pixel intensities was created.
  • the dynamic range of signal intensity in the unmixed fluorescence image was set from 1 to 256 in arbitrary units and the threshold value (t) was changed from 31 to 241 in increments of 10, because the background signals, such as the normal peritoneal membrane excluding tumors and the black plate, were mostly less than 31 (a.u.).
  • the total number of pixels (N) within the threshold range was calculated as a function of threshold (t) and a regression line was calculated in each ROI.
  • the correlation coefficients of GmSA-IROX and GmSA-20ROX were -0.990 and -0.978 for immediately, -0.968 and -0.950 for 1 hour after, -0.946 and -0.988 for 3 hours after injection, respectively.
  • the slopes of GmSA-IROX and GmSA-20ROX were -0.009 and - 0.012 immediately after injection, -0.017 and -0.005 at 1 hour, and -0.074 and - 0.007 at 3 hours after injection, respectively.
  • RFP-positive foci (positive standard) were defined as those whose fluorescence intensities were > 30 (a.u.) on unmixed RFP images, and GmSA-ROX-positive foci were defined as those whose fluorescence intensities were > 10 (a.u.) on unmixed GmSA-IROX images or GmSA-20ROX images.
  • 49 foci showed GmSA-IROX fluorescence intensities > 10 (a.u.) amongst the 207 RFP-positive foci >0.8mm in diameter.
  • 181 foci showed GmSA-IROX fluorescence intensities ⁇ 10 (a.u.) amongst the 181 RFP-negative foci (i.e.
  • the spectral unmixed GmSA-IROX image had a sensitivity of 24% (49/207) and a specificity of 100% (181/181).
  • unmixed GmSA-20ROX images demonstrated that 189 foci showed GmSA-20ROX fluorescence intensities > 10 (a.u.) amongst the 190 RFP-positive foci.
  • 144 foci showed GmSA-20ROX fluorescence intensities ⁇ 10 (a.u.) amongst the 146 RFP-negative foci.
  • the spectrally unmixed GmSA- 20ROX imaging had a sensitivity of 99% (189/190) and a specificity of 99% (144/146).
  • GmSA Galactosamine-conjugated bovine serum albumin
  • ROX Amido-reactive rhodamineX
  • the ROX concentrations were measured by the absorption at 595 nm with a UV- Vis system (8453 Value UV-Bis system, Agilent Technologies, Palo Alto, CA, USA) to confirm the number of ROX molecules conjugated with each GmSA molecule.
  • a UV- Vis system 853 Value UV-Bis system, Agilent Technologies, Palo Alto, CA, USA
  • the concentration of the GmSA solution was adjusted to be either 1 (GmSA-IROX) or 20 (GmSA-20ROX).
  • the protein concentration of GmSA-ROX samples was determined by measuring the absorption at 280 nm with a UV- Vis system (8453 Value UV-Bis system, Agilent Technologies, Palo Alto, CA, USA) using GmSA standard solutions of known concentrations (100, 200 and 400 ⁇ g/mL). Then, the protein concentration was calculated using the absorbance value corrected by the absorbance of ROX molecule at 280 nm known from the ROX concentration as provided above.
  • a UV- Vis system 853 Value UV-Bis system, Agilent Technologies, Palo Alto, CA, USA
  • DsRed2 Red Fluorescence Protein
  • RFP DsRed2 red fluorescence protein
  • Example 2D Flow cytometry was performed as described above in Example 2D, except signals from cells were collected using a 585/42 nm band-pass filter.
  • fluorescence intensity and emission spectra of GmSA-IROX and GmSA-20ROX were measured by the MaestroTM In- Vivo Imaging System (CRI Inc., Woburn, MA, USA) in arbitrary units (a.u.). 5 ⁇ g GmSA-IROX or 5 ⁇ g GmSA-20ROX in 390 ⁇ L phosphate buffers with pH 2.3, 3.3, 5.2, 6.4 and 7.4 was put in a non-fluorescent 96-well plate (Costar, Corning Incorporated, NY, USA) and spectral fluorescence imaging was performed.
  • a band pass filter from 503 to 555 nm and a long pass filter over 580 nm were used for emission and excitation light, respectively.
  • the tunable filter was automatically stepped in 10 nm increments from 500 to 800 nm while the camera captured images at each wavelength interval with constant exposure.
  • Spectral unmixing algorithms were applied to create the unmixed image of the GmSA-IROX and GmSA-20ROX.
  • a region of interest (ROI) as large as each well was drawn to determine the emission spectra and the fluorescence intensity of 2 probes using commercial software
  • cathepsin D Sigma-Aldrich
  • matrix metalloproteinase-2 MMP-2
  • Trypsin, cathepsin C, cathepsin D, or MMP-2 dissolved in 390 ⁇ L phosphate buffers pH 7.4 for trypsin and MMP-2, pH 6.4 for cathepsin C, pH 4.5 for cathepsin D
  • phosphate buffers pH 7.4 for trypsin and MMP-2, pH 6.4 for cathepsin C, pH 4.5 for cathepsin D
  • a regression line was calculated from the data sets of relative SI and incubation time, and then plotted as a function of incubation time using Microsoft Excel 2003 (Microsoft, Redmond, WA, USA). The slope of the regression line was compared between GmSA-IROX and GmSA-20ROX. All experiments were performed in triplicate.
  • 50 ⁇ g GmSA-IROX or 50 ⁇ g GmSA-20ROX was diluted in 300 ⁇ L PBS and injected into the peritoneal cavities of mice with peritoneally disseminated cancer implants and images were collected as described in Example 2F.

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Abstract

La présente invention concerne des sondes fluorescentes, susceptibles d'activation et spécifiques d'une cible, (SFSASC), qui comportent des groupements de marquage et des groupements de ciblage. L'invention concerne également des procédés d'utilisation des SFSASC permettant de détecter des cellules biologiquement, ainsi que des exemples d'utilisation desdits procédés pour détecter des tumeurs in vitro et in vivo.
PCT/US2007/072680 2006-06-30 2007-07-02 Sondes susceptibles d'être activées et procédés d'utilisation WO2008005942A2 (fr)

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EP2151249A1 (fr) * 2008-07-28 2010-02-10 Canon Kabushiki Kaisha Indicateur de pH comprenant un polymère et un fluorophore
US8258171B2 (en) 2006-11-15 2012-09-04 The University Of Tokyo pH-sensitive fluorescent probe
WO2013162502A1 (fr) * 2012-04-23 2013-10-31 Empire Technology Development Llc Visualisation de tissu pour une résection
US10064943B2 (en) 2014-06-02 2018-09-04 Li-Cor, Inc. Therapeutic and diagnostic probes
US11364297B2 (en) 2010-07-09 2022-06-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Photosensitizing antibody-fluorophore conjugates
US11781955B2 (en) 2014-08-08 2023-10-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Photo-controlled removal of targets in vitro and in vivo

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8258171B2 (en) 2006-11-15 2012-09-04 The University Of Tokyo pH-sensitive fluorescent probe
EP2151249A1 (fr) * 2008-07-28 2010-02-10 Canon Kabushiki Kaisha Indicateur de pH comprenant un polymère et un fluorophore
US11364297B2 (en) 2010-07-09 2022-06-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Photosensitizing antibody-fluorophore conjugates
US11364298B2 (en) 2010-07-09 2022-06-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Photosensitizing antibody-fluorophore conjugates
WO2013162502A1 (fr) * 2012-04-23 2013-10-31 Empire Technology Development Llc Visualisation de tissu pour une résection
US10064943B2 (en) 2014-06-02 2018-09-04 Li-Cor, Inc. Therapeutic and diagnostic probes
US10588972B2 (en) 2014-06-02 2020-03-17 Li-Cor, Inc. Phthalocyanine probes and uses thereof
US11781955B2 (en) 2014-08-08 2023-10-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Photo-controlled removal of targets in vitro and in vivo

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