ACTIVATABLE PROBES AND METHODS OF USE

FIELD OF THE DISCLOSURE

The disclosure provides target activatable fluorescent probes that are useful for detecting biologically active cells including tumor cells.

CROSS REFERENCE TO RELATEDAPPLICATIONS

This application claims priority from U.S. provisional applications 60/922,801 filed April 10, 2007, and 60/818,134, filed June 30, 2006, which are herein incorporated by reference in their entirety.

BACKGROUND

The detection of cells using optically detectable probes is helpful in diagnostic and therapeutic settings. Additionally, 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.

One difficulty in imaging optically detectable probes that are bound to cells is that the unbound probe creates a high background signal, which can mask the signal from the bound probe. Hence the sensitivity of the probe may be insufficient to provide significant therapeutic and diagnostic benefits. The present disclosure provides improved probes for imaging cells and methods of using such probes.

SUMMARY OF THE DISCLOSURE

Target- specific activatable fluorescent probes (TAFPs) are disclosed that change from an extracellular fluorescent signal state to an intracellular fluorescent signal state. 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. In some examples the TAFP also includes a linker.

Methods are also provided for using the TAFPs to detect and/or remove tumors in subjects. In some examples, these methods can include administering the TAFP to a subject and removing the detected tumor.

The foregoing and other features of the disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

DESCRIPTION OF THE FIGURES 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 (Figs 3A and 3B depict Av-0.5ROX and Figs. 3C and 3D depict Av-3ROX). Figs. 3E and 3F depict Av- 3ROX with crosslinking. 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. Some increase in fluorescence from dissociated Av- 0.5ROX can be expected, however, the low signal-to-background fluorescence ratio

is still problematic. 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. 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-

3ROX (1 nM) in PBS at pH 7.4. The fluorescence intensity of Av-0.5ROX is 2- fold higher than that of Av-3R0X at a wavelength of 603 nm due to self quenching of the latter. However, after incubation with PBS and 5% SDS the fluorescence intensities of Av-0.5ROX and Av-3ROX were increased 3- and 39-fold, respectively due to "de-quenching."

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. 5A is a graph showing the fluorescence intensities per molecule of monomer (17 kD), dimer (34 kD), trimer (51 kD) and tetramer (68 kD) were calculated by dividing the fluorescence intensity of each band by the corresponding total protein amount in arbitrary units (a.u.). The fluorescence intensity of crosslinked Av- 3ROX monomer was 8-fold higher than that of crosslinked Av-3R0X tetramer. Error bars indicate one standard deviation from the mean (n = 11). 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. Conversely, 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.

Fig. 6 shows two in vivo fluorescence intensity plots of the foci detected by unmixed Av-3ROX images, unmixed RFP images or both (n = 514) and non- tumorous areas (n =499). All foci with signal intensities >60 (a.u.) on spectral unmixed RFP images and diameters >0.8 mm were defined as cancer foci (n = 507). For comparison, 499 ROIs were drawn in the surrounding non-tumorous areas on the unmixed RFP images. When the 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. Fig. 8C shows pH Profiles of BODIPY-Herceptin conjugates around physiological pH range. Fitting curves to pH-sensitive BODIPY-labeled Herceptin are based on modified Henderson- Hasselbalch equation. DOL: PhBDP-IgG = 3.0, DiEtNBDP-IgG = 2.8, DiMeNBDP- IgG = 2.7. Fig. 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.

DETAILED DESCRIPTION I. Abbreviations and Terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

"Accession Numbers" provided herein 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.

The term "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.

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.

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 β-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. At least two human ASGPR genes have been described, designated 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). One skilled in the art will appreciate that 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. Similarly, 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. In addition, 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. For example, immunoglobulin A and fibronectin are natural targeting moieties for ASGPR. However, the term 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.

An "alkylBODIPY labeling moiety" is a compound represented by the following chemical formula (I) [Chemical Formula 1]

where 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.

In some examples 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. In particular examples, 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. One of ordinary skill in the art will appreciate that the selection of the R groups and the substitution of the R groups can

impact the change in fluorescent signal. In some examples, 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.

In some examples, 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. In some examples, 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. The same holds for the alkyl part of other substitution groups (alkoxy groups) having an alkyl part.

In the aforementioned general formula (I), R ¹ indicates an amino group which has been substituted by a non-substitution amino group or by one or two alkyl groups. When two alkyl groups are substituted, these can be the same or different. 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. However, suitable alkyl groups are not necessarily restricted to these. There are no particular restrictions on the position of 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.

In addition, alkylBODIPY which is expressed by the aforementioned general formula (I) can be used as an ester. For example, 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), which is conjugated with an alkylBODIPY, can be manufactured by using this ester. Alternatively, when the alkylBODIPY conjugate is 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.

The term "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) includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term "antibody." Specific, non-limiting examples of 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.

Methods of producing polyclonal and monoclonal antibodies are known to those of ordinary skill in the art, and many antibodies are available. See, e.g., Coligan, Current Protocols in Immunology Wiley/Greene, NY, 1991; and Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY,1989; Stites et al., (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY1986; and Kohler and Milstein, Nature 256: 495-497, 1975. Other 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 μM, for example, at least about 0.01 μM, at least 0.001 μM, 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. Additional details on humanization and other antibody production and engineering techniques can be found in Borrebaeck (ed), Antibody Engineering, 2nd Edition Freeman and Company, NY, 1995; McCafferty et al., Antibody Engineering, A Practical Approach, IRL at Oxford Press, Oxford, England, 1996, and Paul Antibody Engineering Protocols Humana Press, Towata, NJ, 1995.

"Biologically 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. In some examples, 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.

The term "binds" or "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. Generally, the stronger the binding of the molecules in a complex, the slower their rate of dissociation. Specific binding refers to a preferential binding between an agent and a specific target. For example, 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. Such binding can be a

specific non-covalent molecular interaction between the ligand and the receptor. In a particular example, 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.

The term "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. The phrase "disseminated metastatic nodules" or "disseminated metastatic tumors" refers to a plurality (typically many) metastatic tumors dispersed to one or more anatomical sites. For example, disseminated metastatic nodules within the peritoneum (that is a disseminated intraperitoneal cancer) can arise from a tumor of an organ residing within or outside the peritoneum, and can be localized to numerous sites within the peritoneum. Such metastatic tumors can themselves be discretely localized to the surface of an organ, or can invade the underlying tissue.

The term "contact" or "contacting" refers to the relatively close physical proximity of one object to another object. Generally, contacting involves placing two or more objects in close physical proximity to each other to give the objects and opportunity to interact. For example, contacting a TAFP with a biologically active cell, such as a tumor 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. Similarly, a TAFP can be contacted with a cell in vitro.

The term "detect" refers to determining if an agent is present or absent. In some examples this can further include quantification. For example, 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. One of ordinary skill in the art will appreciate that 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. Specific, non-limiting examples of labels include fluorescent molecules that are self quenching or environmentally activatable. Moreover, 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. These molecules are sometimes referred to as reversible because they will increase in fluorescence intensity under one set of conditions and can then be exposed to another set of conditions to lessen the fluorescence intensity. 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. For example, 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. One of ordinary skill in the art will appreciate that 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.

To determine the relative amount of fluorescence intensity increase from a first extracellular intensity state to a second intracellular intensity state, 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. In another example, 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.

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.

The terms "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. In the context of the current disclosure, 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); and intraperitoneal (portions of the rectum, urinary bladder) are encompassed within the term intraperitoneal.

A "linker" is a molecule that is used to connect one or more agents to one or more other agents. For example, a linker can be used to connect one or more labels to one or more targeting moieties. Particular non-limiting examples of 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.

The phrase "optically detectable" in reference to a labeling moiety indicates that the label can be directly or indirectly visualized under appropriate light conditions. 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.

The term "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. In addition to biologically-neutral carriers, pharmaceutical 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.

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. As additional research is performed new tumor specific markers will be identified. These additional markers can be used as ligands for binding to

targeting moieties and TAFPs can be made to detect the tumors. One of ordinary skill in the art will appreciate that once a marker is known, 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. Examples of 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.

Examples of solid tumors, such as sarcomas and carcinomas, 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 CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, meningioma, neuroblastoma and retinoblastoma).

A "subject" is a living multi-cellular vertebrate organisms, a category that includes human and non-human mammals (such as laboratory or veterinary subjects).

The phrase "under conditions sufficient for" is used to describe any environment that permits the desired activity. In one example, the phrase includes incubating tumor cells in the presence of a TAFP such that the TAFP binds to the cell. In another example, 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

Target activatable fluorescence probes (TAFPs) function to bind to a ligand (hereinafter a molecule that the targeting moiety binds to) and produce a signal, such that the ligand and/or the object that the ligand is bound to can be detected. Generally, 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. For example, 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.

A. Targeting moieties

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.

Specific exemplary 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.

Additional examples of targeting moieties include galactosyl serum albumin (GSA) which binds to an asialoglycoprotein receptor (ASGPR). Thus, in certain examples the TAFPs include an asialoglycoprotein receptor (ASGPR) targeting moiety attached to one or more activatable fluorophores. Following introduction into the subject, 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. Once bound to the receptor on the cell surface, 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). Typically, 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.

One exemplary targeting moiety is avidin. 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. When multiple flurophores, such as rhodamineX, are attached to avidin (Av-3ROX), fluorescence of the individual fluorophore moieties is quenched. Upon internalization into a cell the fluorophores unquench, resulting in increased fluorescence.

Glycosylated serum albumin (GSA) binds strongly and specifically to ASGPRs but is not immunogenic in human subjects. Similarly, for diagnostic and therapeutic applications in other species, glycosylated (for example, galactosylated) serum albumins derived from the species in which the methods are to be practiced can be selected. Essentially any immunologically neutral glycosylated ligand can be used as a targeting moiety in the context of the methods disclosed herein. In particular examples, carrier proteins functionalized with galactose, N- acetylgalactosamine, glucosamine and mannose residues are specifically bound to ASGPRs with high affinity. In some instances, the ASGPR targeting moiety is assembled into a multimeric macromolecular complex using 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. As described herein, 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. In some applications TAFPs are designed to display a significant intensity difference between a relatively quenched state and a relatively de-quenched state. For example, 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.

In another example, activatable fluorophores are reversibly activatable. For example, 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). 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). In specific examples, TAFP display a significant intensity difference between a first extracellular state and a second intracellular state. For example, 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. C. Linkers 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.

In some instances, 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. In some instances, 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). Next, a labeling moiety conjugated to a molecule that binds to the conjugated targeting moiety can be added. For example, 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. Thus, the TAFP is formed in the presence of the cells it is intended to detect. One of ordinary skill in the art will appreciate that linkers used in this way can be any binding pair of molecules. For example, linkers can be used that include avidin, biotin, antibodies (including functional equivalents of antibodies) and their congnate epitopes. In some examples, a bi-functional antibody can be used that binds to the ligand on the target cell, as well as to the labeling moiety. In other examples, 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. In yet other examples 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. Hence, after the targeting moiety binds to the cell of interest the secondary antibody conjugated to the label can be added and the secondary antibody will bind to the primary antibody, thus, allowing for detection.

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

Target Activatable Fluorescence Probes (TAFP) provided herein can be used for diagnostic purposes, such as for determining the extent of biological activity displayed by targeted cells or for detecting the targeted cells. TAFPs are also useful for detecting tumors or for determining the effectiveness of a treatment regime a subject has received. These and other uses are described herein. A. Compositions

This disclosure provides diagnostic compositions (for example, medicaments). In one particular example, the diagnostic composition includes a TAFP. In a specific example, 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. In some particular examples, a 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.,

63:3919-3922, 2003). In other examples, 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.

Diagnostic compositions of this disclosure can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for administration to mammals, such as humans. For example, the composition that includes a TAFP can be present in a pharmaceutically acceptable carrier. As mentioned above, the

term "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. In a particular example when the composition is administered intravenously, 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. A more complete explanation of 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. For example, the TAFP can be used to visualize the impact that a therapy is having upon a tumor. In particular examples, 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. For example, 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. Examples of anti-proliferative/anti-neoplastic agents are alkylating agents, antimetabolites, antimitotic agents, natural products, or hormones and their antagonists. Examples of 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). Examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine. Examples of 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, and ceratamine B) and microtubule-destabilizing agents (such as, vincristine, vinblastine, vinorelbine, and colchicine). 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). Examples of 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). Examples of 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). 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, Hectoral, DN-IOl, Rocaltrol® (Roche Laboratories), Calcijex® injectable calcitriol, investigational drugs from Leo Pharmaceutical including EB 1089 (24a,26a,27a- trihomo-22,24-diene-lαa,25-(OH) ₂ -D ₃ ), KH 1060 (20-epi-22-oxa-24a,26a,27a- trihomo-lα,25-(OH) ₂ -D ₃ ), MC 1288 and MC 903 (calcipotriol), Roche Pharmaceutical drugs including l,25-(OH) ₂ -16-ene-D ₃ , l,25-(OH) ₂ -16-ene-23-yne- D ₃ , and 25-(OH) ₂ -16-ene-23-yne-D ₃ , Chugai Pharmaceuticals' 22-oxacalcitriol (22- oxa-lα,25-(OH)2-D ₃ ; Ia-(OH)Ds from the University of Illinois; and drugs from the Institute of Medical Chemistry-Schering AG that include ZK 161422 and ZK 157202).

The methods of using 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. B. Methods of administration

Any method known in the art can be used to introduce the TAFP into the subject. Specific examples of methods of administering a TAFP to a mammalian subject include, but are not limited to, intraperitoneal (ip), intravenous (iv), subcutaneous, intradermal, intramuscular, epidural, intranasal, and oral routes. The 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. In addition, it can be desirable to introduce 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. In specific examples, 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. In other specific embodiments, 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.

In some cases, it 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. In one example, 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. In another example, 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).

In yet another example, the pharmaceutical composition is delivered in a controlled release system. In one example, 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). In another embodiment, 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. 25, 351, 1989; Howard et al, J. Neurosurg. 71, 105, 1989). Other controlled release systems, such as those discussed in the review by Langer (Science 249, 1527 1990), can also be used.

In particular examples, the disclosed methods include contacting a tumor with a TAFP For example, if the tumor is in a subject, 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

Optionally, additional diagnostic reagents are administered to the subject For example, in addition to a TAFP, one or more additional fluorescently labeled diagnostic probes can be administered to the subject In some examples, the second or other additional diagnostic probe is selected to aid in the diagnostic characterization of the tumors For example, antibodies useful for the identification or characterization (for example, with respect to cellular characteristics observed in malignant but not benign tumors, or other relevant properties), can be fluorescently labeled and administered in combination with the TAFP to further enhance diagnostic and prognostic capabilities Typically, 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)

Typically, after the TAFP is administered to the subject or to a cell culture sufficient time must pass to allow the TAFP to be internalized Upon internalization, the signal will increase and allow the cells to be detected One of ordinary skill in the art will appreciate that 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 the case of in vivo methods, 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 for Detecting and Removing Tumors

Comprehensive resection of disseminated metastases, particularly disseminated intraperitoneal metastases, is complicated by the inability of the surgeon to exhaustively visualize all the tumor foci dispersed throughout and attached to the intraperitoneal organs, the mesentery, and peritoneum. In many cases, following removal of a primary tumor mass, numerous tumors of various sizes, including nodules smaller than 1 mm in diameter, can be spread throughout the abdominal cavity, organs and membranes. In particular example, methods are disclosed for visualizing tumors within the peritoneum and elsewhere in the body, that are compatible with diagnostic and surgical visualization and imaging procedures, and can be employed in "real-time" during surgery, for example to facilitate resection by surgical personnel.

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. In particular examples, the subject is known or suspected of having a tumor.

In certain examples, the methods are performed prior to or at the time of surgery. Optionally, 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).

In particular examples, at effective concentrations of the TAFP, 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. In addition, once the tumor is detected, 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.

In examples where 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. In other examples where the targeting moiety is Herceptin (D (Genentech, San Fransisco, California) certain breast cancer cells can be detected.

In a particular example, 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.

In some examples, 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. For example, the area can be exposed to a wavelength of light that will excite the desired fluorophore, such as ambient light or a laser. For example, when 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. (Thus, 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. For example, 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. For example, if 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. If the selected fluorophore emits in the red light range (e.g., Texas-Red, Rhodamine-X, etc.), a filter is selected that permits passage of light in a wavelength range between about 620 nm and about 780 nm.

When the fluorescence emission is to be visualized by surgical personnel for the purpose of directing or monitoring surgical removal of the tumor, the emission filter can be located on an optical assistance device. Such a device can be worn by the individual performing the surgery in the form of eyeglasses (monocular or binocular), goggles, visors, or the like. Alternatively, the emission filter can be placed on an overhead or floor mounted lens. Typically, such a lens is variable in position, and can be interposed between the surgeon and the subject as convenience dictates. Optionally, the optical assistance device also includes a lens providing magnification of the operating field.

Surgical personnel can also visualize the fluorescence emission indirectly. For example, such 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.

Based on visualization (detection and localization) of the tumor(s), the surgeon can resect (that is, surgically remove) the tumor. Generally, a complete excision of all visible tumors is desirable. However, in certain circumstances, for example, if the tumor cannot be safely removed due to proximity to blood vessels or the like, the resection can be a partial removal of the tumor or tumors. In such cases, 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). In some applications, the methods are used for non-invasive (or minimally invasive) diagnostic procedures. In such cases, 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. In such cases, the diagnostic fluorophore is selected to have excitation and emission spectra compatible with commonly used diagnostic equipment.

For example, 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. Optionally, an excitation filter can be employed that narrows the range of wavelengths used to excite the fluorophore. For example, 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. Alternatively, the diagnostic instrument (for example, a laparoscope or an endoscope) 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. D. Detection of biological activity

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. In the case of pH activatable TAFPs, 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. For example, upon internalization into a lysosome or other intracellular organelle, a TAFP can change from a first to a second state.

Because 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. When a TAFP is introduced along with an additional non-activatable probe, 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.

EXAMPLES Example 1: Making alkylBODIPY

The following schematic provides numbered steps in the synthesis of alkylBODIPY. The description of each step is provided below using the same numbering scheme. [Chemical Formula 2]

TFA

(1) methyl 2,4-dimethyl-3-pyrrole-propionate (1) methyl 5-(benzoyloxy-carbonyl)-2,4-dimethyl-3-pyrrole-propionate

Methyl 5-(benzoyloxy-carbonyl)-2,4-dimethyl-3-pyrrole propionate (1.55 g. 4.9 mmol) was dissolved 150 mL of acetone containing 10 % palladium / carbon and stirred at room temperature for 12 hours in an oxygen atmosphere. The reaction solution was filtered and depressurized and the solvent was removed. The reaction was immediately dissolved in 10 mL of trifluoro acetate (TFA) and stirred for 10 minutes in an argon atmosphere at room temperature. 30 mL of dichloromethane was added to the reaction solution. The reaction was successively washed with water and then with a 1 mol/L NaHCO ₃ aqueous solution. The reaction was then dried using anhydrous Na ₂ SO ₄ , depressurizing and removing to obtained a pale brown oily substance 1 (0.835 g, 94 % yield).

¹ H NMR (300 HHz, CDCl ₃ ) δ 2.02 (s, 3H, NHCHCCH ₃ ), 2.16 (s, 3H, NHCCH ₃ ), 2.42-2.48 (m, 2H, COCH ₂ ), 2.69 - 2.74 (m, 2H, COCH ₂ H ₂ ), 3.66 (s, 3H, OCH ₃ ), 6.36 (s, IH, NHCH), 7.64 (br s, IH, NH). ¹³ C NMR (75 MHz, CDCl ₃ ) δ 10.2, 11.1, 19.9, 35.3, 51.4, 113.0, 116.5, 117.7, 124.1, 173.9

LRMS (ESL ⁺ ) m/z 182 (M+H) ⁺ .

(2) l,3,5,7-tetramethyl-2,6-bis-(2-methoxy carbonyl ethyl)-8-(4-aminophenyl)- 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene (2a) Compound 1 (0.542 g, 2.99 mmol), 4-aminobenzaldehyde (0.153 g, 1.49 mmol) was dissolved in 300 mL of dichloromethane containing a catalytic amount of TFA and stirred in an argon atmosphere at room temperature overnight. 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). 3 mL of BF3 • OEt ₂ were slowly dropped in while stirring at room temperature. Stirring was then continued for 10 minutes. The reaction solution was washed with water, dried with anhydrous Na ₂ SO ₄ , and depressurized. The compound obtained was refined using silica gel column chromatography using dichloromethane/methanol (95:5) as an effluent solvent and a red solid was obtained (compound 2a, 40.1 mg, 5.2 % yield). ¹ H NMR (300 MHz, CDCl ₃ ) δ 1.41 (s, 6H, NCCCH ₃ ), 2.33 -2.28 (m, 4H, COCH ₂ ), 2.53 (s, 6H, NCCH ₃ ), 2.61-2.67 (m, 4H, COCH ₂ CH ₂ ), 3.65 (s, 6H, OCH ₃ ), 3.93 (br s, 2H, NH ₂ ), 6.76-6.78 (m, 2H, NH ₂ CCHCH), 6.96-6.99 (m, 2H, NH ₂ CCH).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 12.0, 12.4, 19.2, 34.2, 51.5, 115.3, 124.7, 128.7, 128.9, 131.4, 139.5, 141.8, 147.1, 153.4, 173.0 HRMS (EST ⁺ ) calculated for [M+Na] ^4" m/z 534.23516, found 534.23846 (δ 3.30 mmu).

(3) 1,3,5,7-tetra methyl-2,6-bis-(2-carboxyl ethyl)-8-(4-aminophenyl)-4,4- difluoro-4-bora-3a, 4a-diaza-s-indacene (3a)

Compound 2a (40.1 mg, 78.4 μmol) was dissolved in 1 mL of dichloromethane, 20 mL of methanol and 5 mL of a 1 mol/L NaoH aqueous solution were successively added and stirred at room temperature overnight. 30 mL of water was added to the reaction solution and the reaction was washed with dichloromethane. 1 mol/L HCL aqueous solution (approximately 5 mL) was added until the solution emitted a green-colored fluorescence under UV irradiation (365 nm). The reaction was extracted with dichloromethane, dried using anhydrous Na ₂ SO ₄ , and depressurized. The compound obtained was refined twice under the following conditions using a semi-preparative HPLC; A/B = 50/50 (o min) to 0/100 (20 min), then A/B = 70/30 (0 min) to 0.100 (30 min) (solvent A: H ₂ O, 0.1 % TFA; solvent B: acetonitrile /H ₂ O = 80/20, 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).

¹ H NMR (300 MHz, CD ₃ OD) δ 1.37 (s, 6H, NCCCH ₃ ), 2.24 (t, 4H, J = 7.4, 8.0 Hz, COCH ₂ ), 2.38 (s, 6H, NCCH ₃ ), 2.54 (t, 4H, J = 7.4, 8.0 Hz, COCH ₂ CH ₂ ), 6.74-6.75 (m, 2H, NCCHCH), 6.83-6.86 (m, 2H, NCCH).

¹³ C NMR (75 MHz, CD ₃ OD) δ 12.5, 12.7, 20.3, 35.3, 116.6, 125.2, 130.1, 130.4, 132.7, 140.8, 144.0, 150.2, 154.6, 176.5.

HRMS (ESI ⁺ ) calculated for [M+Na] ⁺ m/z 506.20386, found 506.20749 (δ 3.43 mmu).

(4) 1,3,5,7 -tetramethyl-2,6-bis-(2-methoxy carbonyl ethyl)-8-[4-(N,N-dimethyl amino) phenyl]-4,4-difluoro-4-bora-3a, 4a-diaza-s-indacence (2b)

Compound 1 (0.574.g, 3.17 mmol), 4-(N,N-dimethyl amino) benzaldehyde (0.236 g, 1.58 mmol) was dissolved in 300 mL of dichloromethane containing a catalytic amount of TFA and stirred at room temperature in an argon atmosphere for one day. p-chloranil (0.384 g, 1.56 mmol) was added and stirred for another 10 minutes. The reaction solution was washed with water, dried with anhydrous

Na ₂ SO ₄ , and depressurized and removed. The resulting compound was refined by repeatedly using alumina column chromatography using dichloromethane/methanol (9:1) containing 1 % TEA as an effluent solvent and a green solid was obtained. The resulting compound was dissolved in 100 mL of toluene containing 5 mL of DIEA. 5 mL of BF ₃ • OEt ₂ were dropped in while stirring at room temperature and stirring then continued for 10 minutes. The reaction solution was washed in water, dried in anhydrous Na ₂ SO _4, and depressurized. The compound was refined three times using silica gel column chromatography using dichloromethane/methanol (#1 97:3; #2 99:1; #3 100.0) and a brown solid (compound 3a, 228 mg, 27 % yield) was obtained.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.40 (s, 6H, NCCCH ₃ ), 2.33-2.38 (m, 4H, COCH ₂ ), 2.53 (s, 6H, NCCH ₃ ), 2.61-2.67 (m, 4H, COCH ₂ CH ₂ ), 3.02 (s, 6H, NCH ₃ ), 3.65 (s, 6H, OCH ₃ ), 6.75-6.80 (m, 2H, NCCHCH), 7.01-7.06 (m, 2H, NCCH). ¹³ C NMR (75 MHz, CDCl ₃ ) δ 12.1, 12.5, 19.4, 34.3, 40.3, 51.6, 112.5, 122.5, 128.7, 128.8, 131.6, 139.6, 142.4, 150.7, 153.3, 173.1

HRMS (EST ⁺ ) calculated for [M+N] ⁺ m/z 562.26656, found 562.26315 (δ -3.32 mmu).

(5) 1,3,5,7-tetra methyl-2,6-bis-(2-carboxy ethyl)-8-[4-(N,N-dimethyl amino) phenyl]-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (3b)

Compound 2b (50.8 mg. 94.4 μ mol) was dissolved in 1 niL of dichloromethane. 20 mL of methanol and 5 mL of a 1 mol/L NaOH aqueous solution were successively added and stirred at room temperature for 2 hours. 30 mL of water was added to the reaction solution and the reaction solution was washed in dichloromethane. 1 mol/L HCl aqueous solution (approximately 5 mL) was added until the solution emitted a green fluorescence at UV irradiation (365 nm). The reaction solution was extracted with dichloromethane, dried with anhydrous Na ₂ SO ₄₁ and depressurized. The resulting compound was refined under the following conditions using a semi-preparative HPLC; A/B = 60/40 (9 min) to 0/100 (30 min) (solvent A: H ₂ O, 0.1 % TFA; solvent B: acetonitrile/H ₂ 0 = 80/40, 0.1 % TFA). 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. ¹ H NMR (300 MHz, CD ₃ OD + DMF-d ₇ ) δ 1.34 (s, 6H, NCCCH ₃ ), 2.21-2.26 (m, 4H, COCH ₂ ), 2.39 (s, 6H, NCCH ₃ ), 2.51-2.56 (m, 4H, COCH ₂ CH ₂ ), 2.91 (s, 6H, NCH ₃ ), 6.77-6.80 (m, 2H, NCCHCH), 6.94-6.97 (m, 2H, NCCH). ¹³ C NMR (75 MHz, CD ₃ + DMF-d ₇ ) δ 12.6, 12.8, 20.3, 35.3, 40.5, 113.6, 123.5, 130.1, 130.6, 132.7, 140.8, 143.9, 152.5, 154.6, 175.9 HRMS (EST ^" ) calculated for [M-H] ^" m/z 510.23757, found 510.23776 (δ 0.19 mmu).

(6) l,3,5,7-tetramethyl-2-(2-carboxy ethyl)-6-(2-succine imidyl oxy carbonyl ethyl)-8-[4-(N,N-dimethyl amino)-phenyl]-4,4-difluoro-4-bora-3a, 4a-diaza-s- indacene (4b) Compound 3b ( 11.1 mg, 21.7 μ mol) was dissolved in 2 mL of N,N-dimethyl formamide (DMF). 100 mM of a N-hydroxy succine imide (NHS) DMF solution and 100 mM of a water-soluble carboxyimide (WSCD) DMF solution (respectively

32.6 μ mol) was successively added while being cooled. The reaction solution was gradually returned to room temperature and stirred for 24 hours. The reaction solution was depressurized, the solvent was removed and the reaction solution was refined under the following conditions using semi-preparative HPLC: A/B = 50/50 (0 min) to 0/100 (20 min) (solvent A: H ₂ O, 0.1 % TFA; solvent B : acetonitrile / H ₂ O = 80/20, 0.1 % TFA). The fraction containing the desired compound was extracted. The reaction solution was dried with anhydrous Na ₂ SO ₄ , and depressurized. A red solid (compound 4b, 2.6 mg, 20 % yield) was obtained. The original material yield was 41 %. HRMS (ESI ⁺ ) calculated for [M+Na] ⁺ m/z 631.25154, found 631.25518 (δ 3.64 mmu).

(7) 1,3,5,7 -tetra methyl-2,6-bis-(2-methoxy carbonyl ethyl)-8-[4-(N,N-diethyl amino) phenyl)]-4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene (2c). Compound 1 (0.542 g, 2.99 mmol), 4-(N,N-diethyl amino) benzaldehyde

(0.265 g., 1.49 mmol) was dissolved in 300 mL of dichloromethane containing a catalytic amount of TFA. The resulting reaction solution was stirred in an argon atmosphere at room temperature overnight, p-chloranil (0.370 g,. 1.51. mmol) was added and stirring continues for another 10 minutes. The reaction solution was washed in water and dried using anhydrous Na ₂ SO ₄ The resulting compound was refined three times using alumina column chromatography with dichloromethane/methanol (# 95:5; # 2 98.2; #3 100:00) containing 1 % TEA as an effluent solvent and a green solid was obtained. The compound obtained was dissolved in 100 mL of toluene containing 5 mL of DIEA. 5 mL of BF ₃ • OEt ₂ was dropped in while stirring at room temperature and stirring continued. The reaction solution was washed in water, dried in anhydrous Na ₂ SO ₄ , and depressurized. The resulting compound was refined three times using silica gel column chromatography using dichloromethane/methanol (#1 98.2; #2 100:0; #3 95:5) as an effluent solvent and an orange solid (compound 2c, 136 mg, 16% yield) was obtained. ¹ H NMR (200 MHz, CDCl ₃ ) δ 1.22 (t, 6H, J = 7.0 Hz, NCH ₂ CH ₃ ), 1.44 (s, 6H,

NCCH ₃ ), 1.44 (s, 6H, NCCCH ₃ ), 2.36 (t, 4H, J = 7.3, 8.4 Hz, COCH ₂ ), 2.53 (s, 6H, NCCH ₃ ), 2.65 (t, 4H, J = 7.3, 8.4 Hz, COCH ₂ CH ₂ ), 3.41 (q, 4H, J = 7.0 Hz, NCH ₂ ),

3.65 (s, 6H, OCH ₃ ), 6.74 (d, 2H, J = 8.6 Hz, NCCHCH), 6.99 (d, 2H, J = 8.6 Hz, NCCH).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 12.1, 12.3, 12.5, 19.3, 34.3, 44.3, 51.6, 112.0, 121.6. 128.6, 129.0, 131.7, 139.6, 142.6, 148.2, 153.1, 173.1 HRMS (EST ⁺ ) calculated for [M+H] ⁺ m/z 568.31582, found 568.31626 (δ 0.44 mmu)

(8) l,3,5,7-tetramethyl-2,6-bis-(2-carboxy ethyl)-8-[4-(N,N-diethyl amino) phenyl]-4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene (3c) Compound 2c (136 mg, 239 μ mol) was dissolved in 3 mL of dichloromethane, 20 mL of methanol and 1 mol/L of an NaOH aqueous solution was added successively and stirred for 4 hours at room temperature. 30 mL of water was added to the reaction solution and the reaction solution was washed in dichloromethane. 1 mol/L HCl aqueous solution (approximately 5 mL) was added until the solution emitted a green fluorescence under UV irradiation (365 nM). The reaction solution was extracted using dichloromethane, dried in anhydrous Na ₂ SO ₄ , and depressurized. The resulting compound was refined using a preparative TLC using dichloromethane/acetone (1: 1) as an effluent solvent and an orange solid (compound 3c, 118 mg, 91% yield) was obtained. ¹ H NMR (300 MHz, CD ₃ OD) δ 1.10 (t, 6H, J = 7.0 Hz, NCH ₂ CH ₃ ), 1.39 (t, 6H,

NCCCH ₃ ), 2.25 (t, 4H, J = 7.5, 7.9 Hz, COCH ₂ ), 2.39 (s, 6H, NCCH ₃ ), 2.56 (t, 4H, J = 7.5, 7.9 Hz, COCH ₂ CH ₂ ), 3.33 (q, 4H, J = 7.0 Hz, NCH ₂ ), 6.75 (d, 2H, J = 8.8 Hz, NCCHCH), 6.93 (d, 2H, J = 8.8 Hz, NCCH). ¹³ C NMR (75 MHz, CD ₃ OD) δ 12.5, 12.7, 20.4, 35.3, 45.4, 113.3, 122.8, 130.3 (representing two different carbons), 132.8, 140.8, 144.2, 149.7, 154.4, 176.5. HRMS (EST ^" ) calculated for [M-H] ^" m/z 538.26887, found 538.26446 (δ -4.40 mmu).

(9) l,3,5,7-tetramethyl-2-(2-carboxy ethyl)-6-(2-succine imidyl oxycarbonyl ethyl)-8-[4-(N,N-diethyl amino)-phenyl]-4,4-difluoro-4-bora-3a, 4a-diaza-s- indacene (4c)

Compound 3c (25.7 mg, 47.6 μ mol) was dissolved in 2 mL of DMF. 100 mM of a NHS DMF solution and 100 mM of a WSCD DMF solution (47.6 μm respectively) was added successively while being cooled. The reaction was stirred for 14 hours while gradually returning it to room temperature. The solvent was depressurized and the reaction was refined using a TLC preparative using dichloromethane/acetone (1: 1) as a developing solvent and a red solid (compound 4c, 13.4 mg, 44 % yield) was obtained.

HRMS (EST ⁺ ) calculated for [M+H] ⁺ m/z 637.30090, found 637.30278 (δ 1.89 mmu).

(10) l,3,5,7-tetramethyl-2,6-bis-(2-methoxy carbonyl ethyl)-8-phenyl-4,4- difluoro-4-bora-3a, 4a-diaza-s-indacene (2d)

Compound 1 (0.634g, 3.50 mmol) and benzaldehyde (0.185 g, 1.74 mmol) was dissolved in 300 mL of CH ₂ Cl ₂ . The resulting compound was stirred at room temperature in an argon atmosphere overnight, p-chloranil (0.428 g, 1.74 mmol) was added and stirring continued for another 10 minutes. The reaction solution was washed in water, dried with anhydrous Na ₂ SO ₄ , and depressurized. The resulting compound was refined repeatedly using alumina column chromatography with dichloromethane containing 1 % TEA as an effluent solvent and a green solid 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. ¹ H NMR (300 MHz, CDCl ₃ ) δ 1.29 (s, 6H, NCCCH ₃ ), 2.32-2.38 (m, 4H, COCH ₂ ), 2.54 (s, 6H, NCCH ₃ ), 2.61-2.66 (m, 4H, COCH ₂ CH ₂ ), 3.65 (s, 6H, OCH ₃ ), 7.25- 7.28 (m, 2H, benzene), 7.46-7.49 (m, 3H, benzene).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 11.8, 12.6, 19.3, 34.2, 51.6, 128.0, 128.9, 129.1,

130.9, 135.4, 139.4, 140.9, 154.0, 173.0.

HRMS (ESI ⁺ ) calculated for [M+N] ⁺ m/z 519.22426, found 519.22433 (δ 0.07 mmu).

(11) l,3 _» 5,7-tetramethyl-2,6-bis-(2-carboxy ethyl)-8-phenyl-4,4-difluoro-4-bora-

3a, 4a-diaza-s-indacene (3d)

Compound 2d (40.1 mg, 78.4 // m) was dissolved in 1 mL of dichloromethane. 20 mL of methanol and 5 mL of a 1 mol/L NaOH aqueous solution was successively added and stirred at room temperature overnight. 30 mL of water was added to the reaction solution and the reaction solution was washed using dichloromethane. 1 mol/L of an HCl aqueous solution (approximately 5 mL) was added until the solution emitted a green fluorescence under UV irradiation (365 nm). The reaction was extracted using dichloromethane, dried using anhydrous Na ₂ SO _4, depressurized and removed. The resulting compound was refined under the following conditions using a semi-preparative HPCL: A/B = 50/50 (0 min) to 0/100

(20 min), then A/B = 70/30 (0 min) to 0/100 (30 min) (solvent A: H ₂ O, 0.1 % TFA; solvent B: acetonitrile /H ₂ O = 80/20, 0.1 % (TFA). The aqueous solution of the fraction containing the desired compound was extracted. The reaction solution was dried using anhydrous Na ₂ SO ₄ and depressurized. A red solid (compound 3d, 32.0 mg, 84 % yield) was obtained.

¹ H NMR (300 MHz, CD ₃ OD) δ 1.19 (s, 6H, NCCCH ₃ ), 2.23 (t, 4H, J = 8.1 Hz,

COCH ₂ ), 2.40 (s, 6H, NCCH ₃ ), 2.55 (t, 4H, J = 8.1 Hz, COCH ₂ CH ₂ ), 7.21-7.46 (m,

5H, benzene). ¹³ C NMR (75 MHz, CD ₃ OD/NaOD) δ 12.2 (representing two different carbons),

22.0, 39.3, 129.5, 130.2, 130.4, 132.0, 132.2, 136.9, 140.4, 142.1, 155.2, 181.8.

HRMS (ESI ⁺ ) calculated for [M+Na] ⁺ m/z 491.19296, found, 491.18910 (δ -3.87 mmu)

(12) l,3,5,7-tetramethyl-2-(2-carboxy ethyl)-6-(2-succine imidyl oxy carbonyl ethyl)-8-phenyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (4d)

Compound 3d (12.4 mg, 26.5 μmol) was dissolved in 2 mL of DMF. 100 mM NHS DMF solution and a 100 mL WSCD DMF solution (39.7 μmol respectively) was added successively while being cooled. The reaction was stirred for 24 hours while gradually returning the reaction to room temperature. The reaction was depressurized and solvent was removed. The reaction was refined under the following conditions using semi-preparative HPLC; A/B = 50/50 (0 min) to 0/100 (20 min) (solvent A: H ₂ O, 0.1 % TFA; solvent B: acetonitrile / H ₂ O, = 80/20, 0.1 % (TFA). The fraction containing the desired compound was extracted. The extracted reaction was dried using anhydrous Na ₂ Sθ ₄ and depressurized. A red solid (compound 4d, 4.0 mg, 27 %) was obtained. The raw material yield was 24 %. HRMS (ESI ^" ) calculated for [M-H] ^" m/z 564.21175, found 564.21392 (δ 2.18 mmu).

Example 2: Using AlkylB ODIPY to detect biological activity

This example describes the use of a target- specific activatable fluorescent probe (TAFP) that contains diethyl-BODIPY as a labeling moiety to detect biological activity in vitro and in vivo. One of ordinary skill in the art will appreciate that other alkylBODIPY labeling moieties can be used to obtain similar results. A summary of the results from the in vitro and in vivo examples is provided followed by a detailed description of the materials and methods used.

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 μL). Both 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.

On the spectral unmixed image, 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 fluorescence intensity of GSA-BDP changed little among different pH values (slope = -3.7 a.u./pH: where a.u. is arbitrary unit). Whereas, the fluorescence intensity of GSA-detBDP increased >21-fold when the pH changed from 7.4 to 2.3 (slope = -30.4 a.u./pH). That is, when the pH changed from 7.4 to 2.3, the fluorescence intensity of GSA-BDP increased by 10%, whereas the fluorescence intensity of GSA-detBDP increased as much as 2113%. For comparison of pH dependent fluorescence intensity changes between pH 2.3 and 7.4, 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 correlation coefficients and the slopes of GSA-BDP were -0.83 and -3.7, and those of GSA-detBDP were -0.99 and -30.4, respectively. These results indicate that GSA-detBDP is substantially activated under acidic conditions and the degree of activation is highly correlated with pH, while GSA-BDP is stable and shows little changes in fluorescing capability under acidic conditions. Intracellular fluorescence production and distribution, was analyzed using serial observation of SHIN3 ovarian cancer cells (Mellman et ah, Ann. Rev. Biochem. 55, 663-700, 1986) incubated with 200 nmol/L GSA-BDP or GSA- detBDP. 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. Unlike the large temporal changes observed in GSA-detBDP, 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.

To compare the serial fluorescence intensity of SHIN3 cancer cells, one- color flow cytometry was performed at 30 minutes, 1 hour, and 3 hours after incubation with 200 nmol/L GSA-BDP or GSA-detBDP. Both GSA-BDP and GSA-detBDP showed a significant rightward shift (>one order shift) as compared with SHIN3 control cells as early as 30 minutes after incubation (Figs. 2A and 2B). The mean fluorescence index (MFI) for both GSA-BDP and GSA-detBDP consistently increased during the incubation period of 3 hours (Fig. 2C). The relative MFI, calculated from the MFI of GSA-detBDP divided by the MFI of GSA- BDP at the specific time point, demonstrated linear increase in relative MFI (r = 0.98, slope of regression line = 0.11 a.u./hour) (Fig. 2D). These results indicate that both GSA-BDP and GSA-detBDP are internalized into SHIN3 cancer cells and then accumulate into intracellular endosomes or lysosomes, where GSA-BDP and GSA- detBDP are exposed to acidic conditions (Weissleder et al., Nat Med. 9:123-8, 2003). However, GSA-detBDP is progressively activated by the acidic conditions and becomes highly fluorescent over the incubation times.

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 Maestro™ 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. However, the background signal in GSA-BDP was very high due to incomplete clearance of the GSA-BDP in the peritoneal fluid. In contrast, 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. These results indicate that GSA-detBDP is activated within the cancer cells and became highly fluorescent, while unbound GSA-detBDP remains minimally fluorescent in the peritoneal fluid where it is under neutral conditions (pH -7.4).

The observed high signal-to-background imaging was further investigated using dying cancer cells after instillation with GSA-detBDP and GSA-BDP. 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. For objective comparison, a region of interest (ROI) as large as the peritoneal membrane was drawn inside the bowel, and the serial fluorescence intensity of each ROI was measured. The signal intensity of GSA-detBDP consistently decreased during the observation period and became almost undetectable at 60 minutes after sacrifice. GSA-BDP showed minimal fluorescence intensity changes during the observation period. These results indicate that GSA- detBDP is highly fluorescent exclusively in the living cells, but after cell death, it becomes less fluorescent due to the decreased endosomal or lysosomal pH gradient. These results demonstrate that GSA-detBDP can be used as a marker for testing biological activity.

A. Synthesis of GSA-conjugated BDP or detBDP

Galactosylated bovine serum albumin (GSA), which contained 23 galactoses on a single albumin molecule, was purchased from Sigma Chemical (St. Louis, MO), and amido-reactive 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3- propionic acid, succinimidyl ester (BODIPY-R6G®, BDP) was purchased from Molecular Probes Inc. (Eugene, OR, USA). pH sensitive amido-reactive diethyl BODIPY (detBDP) was prepared as described in Example 1. At room temperature, 400 μg (5.9 nmol) of GSA in 196 μL of Na ₂ HPO ₄ was incubated with 24 nmol (4 μL/ 6mM) of BDP ester or detBDP-succinoimidyl, respectively, in DMSO for 15 minutes. The mixture was purified with Sephadex G50 (PD-IO; GE Healthcare, Milwaukee, WI, USA). GSA-conjugated BDP and detBDP samples (GSA-BDP and GSA-detBDP, respectively) were kept at 4°C in the refrigerator as stock solutions. 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 μg/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.

B. Measurement of fluorescence intensity and emission spectra of fluorophore conjugates

Prior to administering the optical probes in vitro or in vivo, fluorescence intensity and emission spectra of GSA-BDP and GSA-detBDP were measured by the Maestro™ In- Vivo Imaging System (CRI Inc., Woburn, MA, USA) in arbitrary units (a.u.). GSA-BDP and GSA-detBDP (50 pmol) with 400 μL phosphate buffers with different pH values (2.3, 3.3, 5.2, 6.4 and 7.4) 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. The common logarithm (Log) values of mean fluorescence intensity as well as SD were calculated and plotted as a function of pH. The regression line was calculated from the data sets, pH and common logarithm value of mean fluorescence intensity, using Microsoft Excel 2003. To evaluate the pH dependent fluorescence intensity changes, the correlation coefficients and the slopes of regression lines were compared between GSA-BDP and GSA-detBDP. C. Fluorescence microscopy

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.

D. Flow cytometry

One-color flow cytometry was performed for the assessment of fluorescing capability of GSA-BDP and GSA-detBDP. SHIN3 cells (5 x 10 ⁴ ) were plated on a 12-chamber 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 once with PBS, trypsinized and flow cytometry was performed. The argon ion 488 nm laser was employed for excitation. Signals from cells were collected using a 530/30 nm band-pass filter. Cells were analyzed in a FACScan cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and all data were analyzed using CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). The fluorescing capability of each fluorophore was referred as Mean Fluorescence Index (MFI). The relative MFI was calculated from the MFI value of GSA-detBDP divided by the MFI value of GSA-BDP at the specific time point. The correlation coefficient (r) and the regression line of relative MFI was calculated as a function of incubation times. If the absolute value of r is >0.9, the relative MFI is considered strongly correlated with the incubation times.

E. Tumor model

The intraperitoneal tumor implants were established by intraperitoneal injection of 2 x 10 cells suspended in 200 μL 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.

F. In vivo spectral fluorescence imaging

700 pmol each of GSA-BDP and GSA-detBDP were diluted in 500 μL BSA 5% solution and injected into the peritoneal cavities of mice with peritoneally disseminated cancer implants. Three hours after injection of each fluorophore, each mouse was sacrificed with carbon dioxide. Immediately after sacrifice, the

abdominal cavity was exposed and spectral fluorescence images were obtained using the Maestro™ In- Vivo Imaging System (CRi Inc., Woburn, MA, USA).

To compare the dynamic spectral imaging between GSA-BDP and GSA- detBDP, 2 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 Maestro™ 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. Using the unmixed fluorescence images of the peritoneal membranes, serial fluorescence intensity of the cancer implants was compared between GSA-BDP and GSA-detBDP. An ROI as large as the peritoneal membrane was drawn inside the bowel, and the fluorescence intensity of each ROI was measured by using ImageJ software. The regression lines as well as the correlation coefficient (r) were calculated from the data sets, the time after sacrifice and the fluorescence intensity, using the Microsoft Excel 2003. If the absolute value of r was >0.9, then the time after sacrifice was considered highly correlated with the signal intensity of the tumor.

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. One of ordinary skill in the art will appreciate that other nRO (as described above nRO refers to more than one rhodamine molecule) labeling moieties can be used to obtain similar results. A summary of the results from the in vitro and in vivo examples is provided followed by a detailed description of the materials and methods used.

Av-0.5ROX has 0.5 rhodamineX molecules per avidin whereas Av-3ROX has 3 rhodamineX molecules per avidin. As a consequence 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). After incubation with PBS with or without 5% sodium dodecyl sulphate (SDS) for 30 minutes at room temperature, fluorescence intensities of Av-0.5ROX and Av-3ROX increased 3-fold and 39-fold, respectively (Fig. 4). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) under reducing conditions showed that the avidin monomers (17kD) of Av-3ROX emitted 5-fold greater fluorescence than that of Av-0.5ROX.

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).

To demonstrate that Av-3ROX could be activated within the target tumor and to achieve target- specific cancer imaging with high signal-to-background ratio, in vivo spectral fluorescence imaging with Av-3ROX and Av-0.5ROX was performed in a mouse cancer model of intraperitoneal metastases using SHIN3 ovarian cancer cells. Immediately after intraperitoneal injection of 30 μg Av- 0.5ROX or 30 μg Av-3ROX in 300 μL PBS in tumor bearing mice with peritoneal implants the fluorescence intensity of Av-0.5ROX was higher than that of Av- 3ROX, but tumor nodules were difficult to detect due to the high background signals produced by Av-0.5ROX. Av-3ROX produced minimal background and tumor fluorescence, resulting in poor tumor detection. However, one hour after intraperitoneal injection, the fluorescence intensity arising from tumor nodules was higher with Av-3ROX compared to Av-0.5ROX and the background signal was lower. Three hours after injection 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.

Since the fluorescence intensity of the two probes at the same concentration of avidin is different for Av-0.5R0X and Av-3R0X, a comparison was performed in which the two optical agents had the same initial fluorescence. This was achieved by using 7 μg Av-O.5ROX and 22 μg Av-3R0X. The fluorescence intensity of Av-0.5ROX from the tumor nodules was slightly higher than that of Av-3R0X 1 hour after injection. By 3 hours however, the fluorescence signal arising from Av-3R0X was much higher than that of A V-O.5ROX. These results indicate that a higher signal-to-background ratio can be obtained with Av-3R0X compared with Av-0.5R0X in the settings of the same avidin dose or the same fluorescence intensity.

Av-3R0X was stabilized by crosslinking with disuccinimidyl suberate (DSS) which covalently bound the avidin tetramer using amide residues on lysine molecules (Fig. 5A). 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 (ImageJ™ 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). To verify that crosslinking decreases the fluorescence intensity of Av-3R0X in vivo, 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.

To make an objective comparison of fluorescence intensity of the peritoneal implants between crosslinked Av-3R0X and non-crosslinked Av-3R0X 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. These results indicate that the activation of Av-3ROX could be hampered by stabilizing the Av- 3ROX tetramer with crosslinking.

To investigate the effectiveness of Av-3ROX, the sensitivity and specificity of spectral unmixed Av-3ROX imaging to detect peritoneal cancer foci were assessed using RFP-transfected SHIN3 cancer cells. 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. Additionally, 499 ROIs were created in the non-tumorous areas (i. e. where no tumors were visible on the RFP images). RFP-positive foci (positive standard) were defined as those whose fluorescence intensities were >60 (a.u.) on unmixed RFP images, and 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). 497 foci showed Av-3ROX fluorescence intensities < 1 (a.u.) amongst the 506 RFP-negative foci (i.e. fluorescence intensities < 60 (a.u.) on unmixed RFP images). Thus, the spectral unmixed Av-3ROX imaging had a sensitivity of 92% (465/507) and a specificity of 98% (497/506). Without injection of Av-3ROX, 0 focus showed Av-3ROX fluorescence intensities > 1 (a.u.) amongst the 312 RFP-positive foci.

A. Synthesis of avidin-rhodaminX conjugate

Avidin was purchased from Pierce Biochemical Inc. (Milwaukee, WI, USA), rhodamineX succinoimidyl ester was purchased from Molecular Probes Inc. (Eugene, OR, USA). Av-0.5ROX has 0.5 rhodamineX molecules per avidin whereas Av-3ROX has 3 rhodamineX molecules per avidin. At room temperature, 400 μg (5.9 nmol) of avidin in 80 μL of Na ₂ HPO ₄ was incubated with 6 μg (9 nmol) (for Av-0.5ROX) or 65 μg (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.

B. Crosslinking study Disuccinimidyl suberate (DSS) (Pierce, Rockford, IL) was used to crosslink the avidin tetramer. At room temperature 300 μg of Av-0.5ROX or Av-3ROX in 1 mL PBS was incubated with 100 μg/20 μL 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.

C. Fluorescence intensity analysis of Av-ROX with SDS-PAGE The fluorescence intensities of avidin monomer and tetramer were determined using SDS PAGE under reducing conditions (10% 2- mercoptoethanol/95°C/2 minutes) with a 4%-to-20% gradient polyacrylamide gel (Invitrogen, Novex, San Diego, CA). Immediately after separating the protein in the dark room the fluorescence intensity of wet gels were analyzed with a high

resolution fluorescence scanner system (FLA-5100, Fuμfϊlm 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 μm 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 Imagel™ software After obtaining the fluorescence intensity in each band, the fluorescence intensity per avidm was calculated by dividing the fluorescence intensity of each band by the corresponding protein concentration

D. Determination of fluorescence after "de- quenching" of Av-ROX with 5% SDS

To determine the fluorescence of each sample after "dequenchmg," 780 ng/mL Av-O 5ROX and 780 ng/mL Av-3ROX samples were incubated in PBS with or without 5% SDS for 30 minutes at room temperature The denaturation of avidin causes the rhodamineX molecules to ' de-quench ' resulting in fluorescence which was measured with Perkm-Elmer LS55 fluorescence spectrometer (PerkinElmer, Shelton, CT)

E. Cell culture

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 μg/mL Streptomycin in 5% CO ₂

F. Expression of red fluorescence protein (DsRed2) in SHIN3 cells RFP (DsRed2) expressing plasmid was purchased from Clontech

Laboratories Inc , (Mountain View, CA) 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 II™ (Bio- Rad Laboratories, Hercules, CA). Briefly, 3 μg of DsRed2-express plasmid was mixed with 2 million SHIN3 cells in 400 μL 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 μg/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. H. In vivo spectral fluorescence imaging study

30 μg Av-0.5ROX or 30 μg Av-3ROX (equivalent total avidin dose), or 7 μg Av-0.5ROX or 22 μg Av-3ROX (equivalent fluorescence intensity), or 50 μg crosslinked Av-3ROX or 50 μg non-crosslinked Av-3ROX (in the cross linking study) were diluted in 300 μL PBS and injected into the peritoneal cavities of mice with peritoneally disseminated cancer implants. Mice were sacrificed with carbon dioxide immediately, 1 hour, or 3 hours after injection (n = 3 in each group). The abdominal cavity was exposed and spectral fluorescence images were obtained using the Maestro™ 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 spectral fluorescence images consisting of autofluorescence spectra and rhodamineX spectra were obtained and then unmixed, based on their spectral patterns using commercial software (Maestro software, CRi Inc., Woburn, MA).

I. Semi-quantitative comparison of fluorescence intensity between crosslinked and non-crosslinked A V-3ROX in vivo

To compare the fluorescence intensities of tumor foci semi-quantitatively between the crosslinked Av-3ROX and non-crosslinked Av-3ROX, close-up peritoneal membrane images were obtained 3 hours after intraperitoneal injection of 50 μg crosslinked Av-3ROX or 50 μg non-crosslinked Av-3ROX. Using the unmixed fluorescence image of the 2 peritoneal membranes, region of interest (ROI) as large as the peritoneal membrane was drawn inside the bowel, and histograms (number of pixels at specific fluorescence intensity) were made using ImageJ software. A threshold was set in the fluorescence intensity above which a pixel is counted. The total number of pixels (N) within the threshold range was calculated at a threshold value of t, where i is the fluorescence intensity in arbitrary units, n is the number of pixels at the fluorescence intensity of i, t is the threshold value, and N is the total umber of pixels within the threshold range (i >= t) [Eq. I].

OO

∑ ⁿ ω i=t [Eq.l]

The common logarithm (Log) values of N were calculated and plotted as a function of t. The regression line and the correlation coefficient (r) were calculated from these data sets (t and Lo gN) by the Microsoft Excel 2003. For comparison of fluorescence intensity or "brightness", the slope of regression line was compared between the two agents. If the absolute value of r was >0.9, a comparison study using the slope values was performed.

J. Assessment of the sensitivity and specificity of Av-3ROX for the detection of peritoneal cancer foci 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 μL of PBS, in female nude mice (National Cancer Institute Animal Production Facility, Frederick, MD). Three hours after i.p. injection of 50 μg Av-3R0X diluted in 300 μL PBS, spectral fluorescence images of the peritoneal membranes were obtained by Maestro™ 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. For each mouse, 2-3 different parts of the peritoneal membranes were randomly selected and spread-out on a nonfluorescent plate, before close-up spectral fluorescence imaging was performed. 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). The minimum possible diameter for ROI drawing was 0.8 mm, thus all visible nodules with short- axis diameters > 0.8 mm on either image were included for analysis. 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.). The numbers of foci positive for both Av-3ROX and RFP, and the number positive only for Av-3ROX or RFP, were counted. 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. The resulting labeling moeites were almost non-fluorescent (φ _f i<0.002) in the neutral (non-protonated) form due to the photoinduced electron transfer (PeT) from the aniline moiety to the fluorophore, but turned to be highly fluorescent (φ _f i=0.55-0.60) in the protonated form, and their fluorescence activation ratio (φ _fl ratio between the non-protonated and the protonated form) exceeded over 300 (Fig. 8A). Fig. 8B shows their pH-dependent changes in emission intensity, and their pKa values differed according to the alkyl group on the nitrogen and were calculated as 3.8, 4.3, 6.0 for aniline, dimethylaniline, and diethylaniline conjugated probes, respectively.

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 resulting fluorophore-tagged proteins had varying φ _fl values according to the number of fluorophores per protein (DOL; Degree of labeling). Conjugates with DOL=2.7-3.0 were selected for further study, and examined for pH- dependent changes of fluorescence emission intensity. 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.

As shown in Fig. 8D, the TAFP activation ratio in fluorescence intensity (i.e. fluorescence intensity in acidic environment/intensity at pH 7.4) 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 results clearly showed that the control probe always strongly fluoresced, regardless of being internalized or not. In contrast 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. In order to compare the "always on" control and the TAFPs, side-by-side postmortem in situ imaging was performed 1 day after injection. The TAFPs labeled the HER2-positive tumors. In contrast, 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. In addition, green signal was also seen in HER2-negative tumors shown up in yellow, which was a mixed signal between red and green. In order to evaluate the signal-to background ratio, ex vivo spectrally resolved fluorescence imaging of fresh resected lungs was performed in side-by- side fashion 1 day after injection of either the TAFPs or the "always on" control. The tumor-to-heart ratio was calculated using tumors having the same size in both lungs. The control showed signal from the heart, as well as non-tumor bearing lung tissue. In contrast, the TAFP produced signal only from the HER2-positive tumors.

Example 5: Use of GmSA-rhodamine20 to detect biological activity

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.

To compare the serial fluorescence intensity of SHIN3 cancer cells, one- color flow cytometry was performed at 30 minutes, 1, 3 and 6 hours after incubation with 1 μg/mL galactosamine-conjugated bovine serum albumin (GmSA) GmSA- IROX or GmSA-20ROX. GmSA-IROX showed a small rightward shift during the incubation period of 6 hours, whereas, GmSA-20ROX showed a significant rightward shift (>one order shift) as compared with SHIN3 control cells at 6 hours after incubation. The mean fluorescence index (MFI) for both GmSA-IROX and GmSA-20ROX consistently increased during the incubation periods of 6 hours. 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. Whereas, 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. At 3 hours or later after incubation, the fluorescence intensity of SHIN3 cancer cells instilled with GmSA-20ROX became 5 times higher than that of GmSA-IROX. Slopes of regression lines, calculated from the MFI and incubation time, were 0.485 a.u./hour (r= 0.997) for GmSA-IROX and 5.574 a.uThour (r=0.986) for GmSA-20ROX. The results indicate that GmSA-20ROX became more fluorescent (~5 times) than GmSA-IROX after internalization into SHIN3 cancer cells.

To investigate the temporal sequence of intracellular fluorescence activation, serial images of SHIN3 ovarian cancer cells incubated with 1 μg/mL GmSA-IROX or GmSA-20ROX were obtained with a fluorescence microscopy and DIC imaging. Fluorescence microscopy initially demonstrated a number of fine fluorescent dots on the cell surface with minimal cytoplasmic fluorescence as early as 10 minutes after incubation with GmSA-IROX, however, fluorescent dots were barely seen with GmSA-20ROX. At 30 minutes after incubation, the number of fluorescent dots increased slightly with GmSA-IROX and GmSA-20ROX. Three hours later, the number of fluorescent dots produced by GmSA-IROX increased, however, unlike

the gradual increase in light intensity observed with GmSA-IROX, the size and the intensity of the fluorescent dots produced by GmSA-20ROX dramatically increased. These results indicate that once GmSA-20ROX is bound to the cell surface and internalized it becomes highly fluorescent due to lysosomal degradation of the complex and resulting de -quenching.

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. When the regression lines were calculated as a function of pH values, the slopes of GmSA-IROX and GmSA- 20ROX were 0.023 and 0.105, respectively. These results indicate that the fluorescence intensity of GmSA-IROX is higher than that of GmSA-20ROX under the same dose and the same acidic conditions, but the fluorescence intensity of GmSA-20ROX is more strongly quenched by acidic conditions.

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. For GmSA-20ROX, 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. With respect to the current incubation protocols, only MMP-2 activated the fluorescence of GmSA-IROX whereas trypsin, cathepsin D and MMP-2 activated the fluorescence of GmS A-20ROX, with cathepsin D producing the strongest gain in fluorescence.

Since enzymatic activation of GmSA-IROX or GmSA-20ROX was slow and limited compared with the observed activation of GmSA-20ROX in SHIN3

cells, activation was induced by adding a detergent, SDS. 5% SDS activated GmSA-20ROX signal from 21 (a.u.) to 624 (a.u.), while GmSA-IROX was activated from 29 (a.u.) to 52 (a.u.). The activation happened immediately after mixing SDS with the samples and was stable for at least 30 minutes. This quick and intense activation of GmSA-20ROX with addition of SDS can be captured by video, and is most similar to the activation observed after the intracellular activation of GmSA-20ROX.

Immediately after intraperitoneal injection with GmSA-IROX or GmSA- 20ROX the fluorescence intensity of GmSA-20ROX was comparable to that of GmSA-IROX, but tumor nodules were hard to detect due to free complex in the peritoneal cavity. By 1 hour after intraperitoneal injection, the fluorescence intensity arising from tumor nodules using GmSA-20ROX was higher than GmSA- IROX and the background signal was comparable. By 3 hours after injection, the fluorescence intensity of the tumor nodules was visually higher with GmSA-20ROX than with GmSA-IROX. Although a small amount of fluorescence was noted from biliary excretion after intraperitoneal injection with GmSA- 20ROX due to transperitoneal systemic absorption, the peritumoral background signal was comparable between the two groups. Sub-millimeter cancer foci were clearly visualized by close-up imaging of the peritoneal membranes. To make an objective comparison of fluorescence intensity generated by

GmSA-IROX and GmSA-20ROX, 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.). Then, 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. These results indicate that the fluorescence of GmSA-IROX decreased over time, whereas that of GmSA-20ROX was maintained or even activated during the incubation time of 3 hours. Sensitivity and specificity of spectrally unmixed imaging to detect peritoneal cancer foci were compared between GmSA-IROX and GmSA-20ROX using RFP- transfected SHIN3 cancer cells. Both unmixed GmSA-IROX and GmSA-20ROX images demonstrated ring-like accumulation around the cancer foci which were depicted by the unmixed RFP images. 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. fluorescence intensities < 30 (a.u.) on unmixed RFP images). Thus, the spectral unmixed GmSA-IROX image had a sensitivity of 24% (49/207) and a specificity of 100% (181/181). In contrast, 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. Thus, the spectrally unmixed GmSA- 20ROX imaging had a sensitivity of 99% (189/190) and a specificity of 99% (144/146).

A. Making Galactosamine-conjugated bovine serum albumin (GmSA)

Galactosamine-conjugated bovine serum albumin (GmSA), which contained 23 galactosamine molecules on a single albumin molecule was purchased from Sigma Chemical (St. Louis, MO). Amido-reactive rhodamineX (ROX) was purchased from Molecular Probes Inc. (Eugene, OR, USA). At room temperature, 500 μg (7.0 nmol) of GmSA in 396 μL of Na ₂ HPO ₄ was incubated for 15 min with either 360 nmol (18 μL/20 niM) of ROX-succinoimidyl ester in DMSO for GmSA- 20ROX or either 360 nmol (18 μL/20 mM) for GmSA-20ROX or either 14 nmol

(0.7 μL/20 mM) for GmSA-IROX. The mixture was purified with Sephadex G50 (PD-IO; GE Healthcare, Milwaukee, WI, USA). GmSA-conjugated ROX samples (GmSA-ROX) was used within 6 hours after conjugate.

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. By changing the concentration of the GmSA solution, the number of fiuorophore molecules per GmSA 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 μg/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.

B. Cell culture, Trasfection of Red Fluorescence Protein (DsRed2) SHIN3 cells were cultured as described above in Example 3E and the red fluorescence protein (RFP DsRed2) was expressed as described in Example 3F above.

C. Flow cytometry

Flow cytometry was performed as described above in Example 2D, except signals from cells were collected using a 585/42 nm band-pass filter.

D. Fluorescence microscopy Fluorescence microscopy was performed as described above in Example 2C, except the filter excitation wavelength of 530-570 nm, and emission wavelength 590 nm long pass were used.

E. In vitro chemical analysis of activation

To compare the fluorescing capability of GmSA-IROX and GmSA-20ROX under the different acidic conditions in vitro, fluorescence intensity and emission spectra of GmSA-IROX and GmSA-20ROX were measured by the Maestro™ In- Vivo Imaging System (CRI Inc., Woburn, MA, USA) in arbitrary units (a.u.). 5 μg

GmSA-IROX or 5 μg GmSA-20ROX in 390 μL 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

(Maestro software, CRi Inc. Woburn, MA, USA). A regression line was calculated from the data sets of pH value and fluorescence intensity on unmixed images, and then plotted as a function of pH value using Microsoft Excel 2003 (Microsoft, Redmond, WA, USA). The fluorescence intensity value at each pH and the slope of the regression line were compared between GmSA-IROX and GmSA-20ROX. F. Enzyme activation of fluorescence signal Enzymatic activation of GmSA-IROX and GmSA-20ROX by trypsin (Invitrogen, Grand Island, NY, USA), cathepsin C (Sigma-Aldrich Inc., St. Louis, MO, USA), cathepsin D (Sigma-Aldrich), and matrix metalloproteinase-2 (MMP-2) (Sigma-Aldrich) were studied. Trypsin, cathepsin C, cathepsin D, or MMP-2 dissolved in 390 μL phosphate buffers (pH 7.4 for trypsin and MMP-2, pH 6.4 for cathepsin C, pH 4.5 for cathepsin D) was mixed with 2.5 μg GmSA-IROX or 2.5 μg GmSA-20ROX. Concentrations of trypsin, cathepsin C, cathepsin D and or MMP-2 were 0.05%, 1 U/mL, 1 U/mL and 1 U/mL, respectively. The mixture was then incubated at 37 ⁰ C for 0, 1, 3 and 6 hours. Spectral fluorescence images were obtained using the Maestro™ In- Vivo Imaging System (CRi Inc., Woburn, MA, USA) and the fluorescence signal was measured as the same method as described above. To compare the fluorescence activation of GmSA-IROX and GmSA- 20ROX, the fluorescence intensity immediately after incubation with each enzyme was adjusted to be 1 in arbitrary unit (a.u.) (relative signal intensity [SI]). 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.

G. Detergent activation of fluorescence signal In order to evaluate the dequenching ability of GmSA-20ROX, 90 μL of

20% SDS, a detergent, was added to 270 μL/5 μg of either GmSA-20ROX or GmSA-IROX in PBS at pH7.4 at the room temperature. As a control, 90 μL of PBS was added to another set of samples. Immediately after mixing the SDS, spectral fluorescence images were obtained using the Maestro™ In- Vivo Imaging System (CRi Inc., Woburn, MA, USA) and the fluorescence signal was measured as the same method as described above. The same experiment was repeated 5 times.

Additionally, 600 μL/75μg of either GmSA-20ROX or GmSA-IROX was placed in the plastic cuvette and excited from the bottom by a 380 nm UV lamp. The 200 μL of 20% SDS was added to each cuvette and mixed well immediately. The entire process of activation was recorded by a digital video camera. H. In vivo spectral fluorescence imaging.

Animal model peritoneal metastases was established as described in Example 3J, above.

50 μg GmSA-IROX or 50 μg GmSA-20ROX was diluted in 300 μL PBS and injected into the peritoneal cavities of mice with peritoneally disseminated cancer implants and images were collected as described in Example 2F.

I. Assessment of the sensitivity and specificity of GmSA-IROX and GmSA-20ROX for the detection of peritoneal cancer foci.

The sensitivity and specificity of GmSA-IROX and GmSA-20ROX imaging was assessed using the methods described in Examples 31 and 31, above.

In view of the many possible embodiments to which the principles of this disclosure can be applied, it should be recognized that the examples herein are only examples of the disclosure and should not be taken as a limitation on the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.