Fluorescent nuclide-containing agents for detecting and treating cancer

Abstract

An agent includes a targeting peptide that specifically binds to and / or complexes with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule that is expressed by a cancer cell or another cell in the cancer cell microenvironment; an optional spacer directly linked to the targeting peptide; a fluorophore that is directly or indirectly linked to the targeting peptide or optional spacer; and an optional chelating agent directly or indirectly linked to the targeting peptide, optional spacer, or fluorophore; wherein at least one of the targeting peptide, optional spacer, or fluorophore is directly labeled with a nuclide and / or the optional chelating agent includes a chelated nuclide.

Description

FLUORESCENT NUCLIDE-CONTAINING AGENTS FOR DETECTING AND TREATING CANCER RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application No.63 / 652,206, filed May 28, 2024, the subject matter of which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 28, 2025, is named CWR-033400WO ORD.st.26 and is 10,930 bytes in size. BACKGROUND

[0003] The lifetime risk of developing cancer for men and women in the United States is approximately 40%. An estimated 2 million people will be diagnosed with cancer in 2023, and approximately 600,000 people will die of cancer. Glioblastoma (GBM) is the most common primary brain tumor with a median survival of one year from diagnosis due to its invasive nature. The treatments are often non-specific and ineffective. Furthermore, there are limited imaging modalities to recognize or “see” a tumor or assess response to therapy. Detection of solid tumors is often done by anatomical imaging techniques followed by biopsy. Anatomical imaging, such as magnetic resonance imaging (MRI), visualizes the main tumor mass but detection of invasion is difficult. In addition, once the tumor is resected and treated with radiation and chemotherapy MRI cannot distinguish inflammation or general tissue damage from effective treatment and tumor shrinkage. Repeat biopsies are often performed to detect tumor recurrence. SUMMARY

[0004] Embodiments described herein relate to targeted fluorescent nuclide-containing agents and their use in detecting and / or treating cancer cells and / or cancer cell metastasis, migration, dispersal, and / or invasion in a subject, and particularly their use in methods of fluorescent image-guided surgery, imaging, targeted radionuclide diagnosis or therapy, and / or photothermal therapy (PTT) and / or photodynamic therapy (PDT).

[0005] We developed PTPµ-targeted nuclide-containing agents that can be detected by fluorescent imaging as well as positron emission tomography / single-photon emission computed tomography (PET / SPECT) or magnetic resonance imaging (MRI) to specifically detect intractable tumors, such as PTPμ-expressing human brain tumors, and then utilized the same agent with a matched therapeutic radionuclide to treat the intractable tumors. Our precise targeting can deliver lethal radiation to neoplastic cells while sparing tissue that is otherwise healthy.

[0006] In some embodiments, the fluorescent nuclide-containing agent can include a targeting peptide that specifically binds to and / or complexes with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule that is expressed by a cancer cell or another cell in the cancer cell microenvironment, an optional spacer directly linked to the targeting peptide, a fluorophore that is directly or indirectly linked to the targeting peptide or optional spacer; and an optional chelating agent directly or indirectly linked to the targeting peptide, optional spacer, or fluorophore. At least one of the targeting peptide, optional spacer, or fluorophore is directly labeled with a nuclide and / or the optional chelating agent includes a chelated nuclide.

[0007] In some embodiments, the fluorescent nuclide-containing agent can include an amide linkage that links the targeting peptide or optional spacer to the fluorophore.

[0008] In some embodiments, the agent can have the formula (I): a pharmaceutically acceptable salt thereof;targeting peptide; R2is absent or the optional spacer, which is optionally directly labeled with the nuclide; R3is the fluorophore; R4or R5are each independently absent or an optional chelating agent with an optional linker; wherein one of R4or R5is the optional chelating agent with the optional linker if R2is not directly labeled with the nuclide; alternatively both of R4and R5are absent and R2is present and is directly labeled with a nuclide; andwherein NH is an amino of the spacer or the targeting peptide and C=O is a carboxyl group of the fluorophore.

[0009] In other embodiments, the agent can have the formula (II): (II) or a pharmaceutically acceptable salt thereof; wherein R1includes the targeting peptide; R3is the fluorophore; R4is a chelating agent with an optional linker; wherein NH is an amino group of the targeting peptide and C=O is a carboxyl group of the fluorophore.

[0010] In yet other embodiments, the agent can have the formula (III): a pharmaceutically acceptable salt thereof; the targeting peptide;R2is the spacer; R3is the fluorophore; R5is the chelating agent with an optional linker; and wherein NH is an amino group of the spacer or the targeting peptide and C=O is a carboxyl group of the fluorophore.

[0011] In still other embodiments, the agent can have the formula (IV):(IV) or a pharmaceutically acceptable salt thereof; wherein R1includes the targeting peptide; R2is the spacer, which is directly labeled with the radionuclide; R3is the fluorophore; and wherein NH is an amino group of the spacer or the targeting peptide and C=O is a carboxyl group of the fluorophore.

[0012] In some embodiments, the fluorophore of the agents described herein can be hydrophobic or lipophilic.

[0013] In some embodiments, the of the agents described herein can include at least one of a cyanine near-infrared fluorophore having a fluorescence in the first near infrared region or second near infrared region. For example, the cyanine near-infrared fluorophore can be a heptamethine cyanine near-infrared fluorophore.

[0014] In some embodiments, the fluorophore can include at least one of indocyanine green (ICG) or ICG-Osu.

[0015] In some embodiments, the fluorophore of the agents described herein can be directly labeled with a nuclide.

[0016] In some embodiments, the optional spacer can include an optional peptide spacer optionally directly labeled with the nuclide. For example, the optional spacer can include an amino acid residue, such as tyrosine, directly labeled with the nuclide.

[0017] In some embodiments, the targeting peptide can have the amino acid sequence of GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 5) or GIDVRDAPLKEIKVTSSR (SEQ ID NO: 7).

[0018] In some embodiments, the chelating agent can be configured to chelate a diagnostic or therapeutic nuclide.

[0019] In some embodiments, the diagnostic or therapeutic nuclide includes at least one of Ga, I, In, Y, Lu, Bi, Ac, Re, Th, Tc, Tl, Tb, Zr, Cu, Rb, At, Pb, Gd, Sm, or Sr.

[0020] In some embodiments, the diagnostic nuclide can be a diagnostic radionuclide selected from123I,124I,125I,64Ga,18F,11C,13N,76Br,149Tb,161Tb,99mTc,153Gd,111In,67Ga,68Ga,201Tl,82Rb,64Cu,67Cu,89Zr,90Y, T(tritium),149Tb,161Tb,153Sm, or89Sr.

[0021] In some embodiments, the therapeutic nuclide can be a therapeutic radionuclide selected from131I,225Ac,226Ac,227Th,211Bi,212Bi,213Bi,203Pb,212Pb, or177Lu.

[0022] In some embodiments, the chelating agent is selected from 1,4,7- triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazadodecanetetraacetate (DOTA), 2,2’,2”-(10-(pyridin-2-ylmethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-1Py), 2,2’-(7,10-(pyridin-2-ylmethyl)-1,4,7,10-tetraazacyclododecane- 1,4-diyl) diacetic acid (DOTA-2Py), 2-(4,7,10-tris (pyridin-2-ylmethyl)-1,4,7,10- tetraazacyclododecane-1-yl) acetic acid (DOTA-3Py), 1,4,7,10-tetraazadodecane-1,4,7- triacetate (DO3A), ethylenediaminetetraacetate (EDTA), 1,4,7,10- tetraazacyclotridecanetetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11- tetraacetic acid (TETA), 1,4,7,10-tetraazadodecanetetramethylacetate (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetate , N,N',N'',N'''-tetraphosphonatomethyl- 1,4,7,10-tetraazacyclododecane (DOTP), 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetrakis(methylene methylphosphonic acid) (DOTMP), 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetrakis(methylene phenylphosphonic acid) (DOTPP), N,N'-ethylenedi-L-cysteine, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacylododecane tetracetic acid (p-SCN-Bn- DOTA), 2-(4-isothiocyanatobenzyl-1,4,7,10-tetraaza-1,4,7,10,tetra-(2-carbamonylmethyl)- cyclododecane (p-SCN-Bn-TCMC), MeO-DOTA-NCS, [(R)−2-Amino-3-(4- isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (CHX- A’’-DTPA-NCS), 2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N'''' - pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N''''-hexaacetic acid (HEHA), desferrioxamine B (DFO), macropa, macropa-NCS, macropid, bispa2, EuK- 106, 7-[2-(bis-carboxymethyl-amino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza- cyclododec-1-yl-acetic acid (DEPA), 3p-C-DEPA, or derivatives thereof.

[0023] In some embodiments, the fluorescent nuclide-containing agent can include a compound selected from: ;(VII); wherein R1is the targeting peptide; R6and R7are each absent or a nuclide; and at least one of R6or R7is a nuclide.

[0024] In some embodiments, R6and / or R7is selected from123I,124I,125I, or131I.

[0025] In some embodiments, compounds (VI) and (VII) or pharmaceutically acceptable salts thereof can further include a chelated diagnostic or therapeutic nuclide.

[0026] In some embodiments, the diagnostic or therapeutic nuclide can include at least one of Ga, I, In, Y, Lu, Bi, Ac, Re, Th, Tc, Tl, Tb, Zr, Cu, Rb, At, Pb, Gd, Sm, or Sr.

[0027] In some embodiments, the nuclide is a diagnostic radionuclide selected from123I,124I,125I,64Ga,18F,11C,13N,76Br,149Tb,161Tb,99mTc,153Gd,111In,67Ga,68Ga,201Tl,82Rb,64Cu,67Cu,89Zr,90Y, T(tritium),149Tb,161Tb,153Sm, or89Sr.

[0028] In some embodiments, selected from131I,225Ac,226Ac,

[0029] In some embodiments, R1has the amino acid sequence of GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 5) or GIDVRDAPLKEIKVTSSR (SEQ ID NO: 7).

[0030] In some embodiments, the fluorescent nuclide-containing agent administered to a subject has a signal-to-background ratio (SBR) upon imaging effective to delineate the cancer cell or another cell in the cancer cell microenvironment from surrounding tissue.

[0031] Other embodiments described herein relate to a fluorescent nuclide-containing agent as described herein for use in detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, dispersal, and / or invasion, and / or for treating cancer in a subject.

[0032] Other embodiments described herein relate to a complex mixture comprising a plurality of the fluorescent nuclide-containing agents described herein, and optionally other fluorescent agents structurally similar to the fluorescent nuclide-containing agents described herein but devoid of a diagnostic and / or therapeutic nuclide and / or chelated nuclide.

[0033] In some embodiments, the complex mixture can further include a pharmaceutically acceptable carrier.

[0034] Other embodiments described herein relate to a method of detecting cancer cells and / or cancer cell metastasis, migration, dispersal, and / or invasion in a subject in need thereof. The method can include administering to the subject the complex mixture as described herein and detecting the fluorescent nuclide-containing agents and optionally the other fluorescent agents devoid of a diagnostic and / or therapeutic nuclide and / or chelated nuclide bound to and / or complexed with the cancer cells to determine the location and / or distribution of the cancer cells in the subject.

[0035] In some embodiments, the cancer cells can include at least one of a glioma, lung cancer, melanoma, breast cancer, ovarian cancer, endometrial cancer, or prostate cancer cell.

[0036] In some embodiments, the complex mixture can be administered systemically, locally, or topically to the subject.

[0037] In some embodiments, the optionally the other agents can be detected to define a tumor in a subject.

[0038] Other embodiments relate to a method of treating cancer cells and / or cancer cell metastasis, migration, dispersal, and / or invasion in a subject in need thereof. The method can include administering to the subject an amount of the complex mixture described herein effective to ablate the cancer. Optionally, the fluorescent nuclide-containing agents and the other fluorescent agents devoid of a diagnostic and / or therapeutic nuclide and / or chelated nuclide bound to and / or complexed with the cancer cells can be detected to determine the location and / or distribution of the cancer cells in the subject.

[0039] In other embodiments, the cancer cells can include at least one of a glioma, lung cancer, melanoma, breast cancer, ovarian cancer, endometrial cancer, or prostate cancer cell.

[0040] In some embodiments, the complex mixture can be administered systemically, locally, or topically to the subject.

[0041] In some embodiments, the agents described herein and optionally the other agents can be detected to define a tumor in a subject.

[0042] In some embodiments, the method further includes surgically resecting the cancer cells.

[0043] In other embodiments, the method further includes ablating remaining or residual cancer cells after surgical resection.

[0044] Other embodiments relate to the use of the fluorescent nuclide-containing agents described herein or a complex mixture described herein in a fluorescent image-guided surgery, imaging and / or targeted radionuclide diagnosis or therapy.

[0045] In some embodiments, the fluorescent nuclide-containing agents described herein or the complex mixture described herein can be used in the preparation of a medicament for fluorescent image-guided surgery, imaging and / or targeted radionuclide diagnosis or therapy. BRIEF DESCRIPTION OF DRAWINGS

[0046] Fig.1 illustrates a schematic showing imaging agents targeting PTPμ for diagnosis and treatment of Tumors.

[0047] Fig.2 illustrates PET images of the brain.

[0048] Figs.3(A-H) illustrate 3D views of dispersed cells from U-87 MG, Gli36Δ5, LN- 229 and CNS-1 intracranial tumors with distinct levels of invasion. Mouse brains containing orthotopic xenografts were cryo-imaged and reconstructed in 3D to show the main tumormass (green), dispersed tumor cells , vasculature (red) and illustrate that the dispersing cells often migrate on blood vessels (C,D). The dispersing cell population is extensively co-labeled with the PTPµ agent (F,H pink). PTPµ co-labeled cells are detected far away from the main tumor mass (F,H).

[0049] Fig.4 illustrates PTPµ biomarker binding agent (PTPµ-ICG / SBK2) targets intracranial tumors (top row), flank tumors (bottom row). Scales for A and B are shown.

[0050] Fig.5 illustrates a schematic showing an imaging protocol of heterotopic model of GBM and orthotopic brain tumor model.

[0051] Fig.6 illustrates graphs and plots showing Mean tumor normalized T1values and slope analysis following intravenous administration of scrambled-Gd (gray), or PTPµ-Gd (SBK2-black) contrast agents in mice with glioma flank tumors (left panel). Note the sustained decrease in normalized T1for PTPµ-Gd as well as the significant difference in slope due to agent retention. The slope of PTPµ-Gd in orthotopic brain tumors (right panel) shows significant binding and retention.

[0052] Fig.7 illustrates the chemical structures of fluorescent nuclide-containing agents in accordance with an embodiment described herein.

[0053] Fig.8 illustrates images showing in vivo tumor labeling of LN229 flank tumors with ICG-DOTA conjugated peptides over time.

[0054] Fig.9 illustrates images showing in vivo tumor labeling of LN229 flank tumors with ICG-DOTA conjugated peptides over time. DETAILED DESCRIPTION

[0055] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. Commonly understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Edition, Springer-Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994.

[0056] The articles "a" and "an" are to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0057] The terms "comprise," "comprising," "include," "including," "have," and "having" are used in the inclusive, open sense, meaning that additional elements may be included. The terms "such as", "e.g.,", as used herein are non-limiting and are for illustrative purposes only. "Including" and "including but not limited to" are used interchangeably.

[0058] The term "or" as used herein should be understood to mean "and / or”, unless the context clearly indicates otherwise.

[0059] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0060] The term “sample” can refer to a specimen or culture obtained from any source, as well as clinical, research, biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass blood, serum, urine, saliva, stool, biopsy, cells, fluids, solids, tissues, and organs, and whole organisms.

[0061] The terms “cancer” or “tumor” refer to any neoplastic growth in a subject, including an initial tumor and any metastases. The cancer can be of the liquid or solid tumor type. Liquid tumors include tumors of hematological origin, including, e.g., myelomas (e.g., multiple myeloma), leukemias (e.g., Waldenstrom's syndrome, chronic lymphocytic leukemia, other leukemias), and lymphomas (e.g., B-cell lymphomas, non-Hodgkin’s lymphoma). Solid tumors can originate in organs and include cancers of the lungs, brain, breasts, prostate, ovaries, colon, kidneys and liver.

[0062] The terms “cancer cell” or “tumor cell” can refer to cells that divide at an abnormal (i.e., increased) rate. Cancer cells include, but are not limited to, carcinomas, such as squamous cell carcinoma, non-small cell carcinoma (e.g., non-small cell lung carcinoma), small cell carcinoma (e.g., small cell lung carcinoma), basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinalcarcinoma, colonic carcinomas, bladder prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; hematologic cancers, such as myelomas, leukemias (e.g., acute myelogenous leukemia, chronic lymphocytic leukemia, granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), lymphomas (e.g., follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkin’s disease), and tumors of the nervous system including glioma, glioblastoma multiform, meningoma, medulloblastoma, schwannoma and ependymoma.

[0063] The term “chelating agent” refers to a molecule containing two or more electron donor atoms that can form coordinate bonds to a single central metal ion, e.g., to a radionuclide. Typically, chelating agents coordinate metal ions through oxygen or nitrogen donor atoms, or both. After the first coordinate bond is formed, each successive donor atom that binds creates a ring or cage containing the metal ion. A chelating agent may be bidentate, tridentate, tetradentate, etc., depending on whether it contains 2, 3, 4, or more donor atoms capable of binding to the metal ion. However, the chelating mechanism is not fully understood and depends on the chelating agent and / or radionuclide. For example, it is believed that DOTA can coordinate a radionuclide via carboxylate and amino groups (donor groups) thus forming complexes having high stability (Dai et al. Nature Corn.2018, 9, 857). The expression “chelating agent” is to be understood as including the chelating agent as well as salts thereof. Chelating agents having carboxylic acid groups, e.g., DOTA, TRITA, HETA, HEXA, EDTA, DTPA etc., may, for example, be derivatized to convert one or more carboxylic acid groups to amide groups for attachment to the compound, i.e., to the reactive moiety or the linker, alternatively, for example, said compounds may be derivatized to enable attachment to the compound via one of the CH2 groups in the chelate ring.

[0064] The term "homology" and "identity" are used synonymously throughout and refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous or identical at thatposition. A degree of homology or sequences is a function of the number of matching or homologous positions shared by the sequences.

[0065] The term "mutant" refers to any change in the genetic material of an organism, in particular a change (i.e., deletion, substitution, addition, or alteration) in a wild type polynucleotide sequence or any change in a wild type protein. The term "variant" is used interchangeably with "mutant". Although it is often assumed that a change in the genetic material results in a change of the function of the protein, the terms "mutant" and "variant" refer to a change in the sequence of a wild type protein regardless of whether that change alters the function of the protein (e.g., increases, decreases, imparts a new function), or whether that change has no effect on the function of the protein (e.g., the mutation or variation is silent).

[0066] The term "nucleic acid" refers to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double- stranded polynucleotides.

[0067] The phrases "parenteral administration" and "administered parenterally" are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0068] The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, agent or other material other than directly into a specific tissue, organ, or region of the subject being treated (e.g., brain), such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

[0069] The terms "patient", “subject”, "mammalian host," and the like are used interchangeably herein, and refer to mammals, including human and veterinary subjects.

[0070] The terms "peptide(s)", "protein(s)" and "polypeptide(s)" are used interchangeably herein. As used herein, “polypeptide” refers to any peptide or proteincomprising two or more amino acids other by peptide bonds or modified peptide bonds (i.e., peptide isomers). “Polypeptide(s)” refers to both short chains, commonly referred as peptides, oligopeptides or oligomers, and to longer chains generally referred to as proteins.

[0071] A "portion" of a polypeptide or protein means at least about three sequential amino acid residues of the polypeptide. It is understood that a portion of a polypeptide may include every amino acid residue of the polypeptide.

[0072] The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

[0073] The term “radionuclide” as used herein refers to an atom with an unstable nucleus, which is a nucleus characterized by excess energy available to be imparted either to a newly created radiation particle within the nucleus or to an atomic electron. Radionuclides occur naturally or can be artificially produced.

[0074] "Recombinant," as used herein, means that a protein is derived from a prokaryotic or eukaryotic expression system.

[0075] The terms “treating” or “treatment” of a disease can refer to executing a treatment protocol to eradicate at least one diseased cell. Thus, “treating” or “treatment” does not require complete eradication of diseased cells.

[0076] An “effective amount” can refer to that amount of a therapeutic agent that results in amelioration of symptoms or a prolongation of survival in the subject and relieves, to some extent, one or more symptoms of the disease or returns to normal (either partially or completely) one or more physiological or biochemical parameters associated with or causative of the disease.

[0077] Therapeutic agents can include any agent (e.g., molecule, drug, pharmaceutical composition, etc.) capable of preventing, inhibiting, or arresting the symptoms and / or progression of a disease.

[0078] The phrase "therapeutically effective amount" or “pharmaceutically effective amount” is an art-recognized term. In certain embodiments, the term refers to an amount of a therapeutic agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain a target of a particular therapeutic regimen. The effective amount may vary depending on such factors as the disease orcondition being treated, the particular being administered, the size of the subject or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In certain embodiments, a therapeutically effective amount of a therapeutic agent for in vivo use will likely depend on a number of factors, including: the rate of release of an agent from a polymer matrix, which will depend in part on the chemical and physical characteristics of the polymer; the identity of the agent; the mode and method of administration; and any other materials incorporated in the polymer matrix in addition to the agent.

[0079] The term "wild type" refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo.

[0080] Throughout the description, where compositions are described as having, including, or comprising, specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remain operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0081] Embodiments described herein relate to targeted fluorescent nuclide-containing agents and their use in detecting and / or treating cancer cells and / or cancer cell metastasis, migration, dispersal, and / or invasion in a subject, and particularly their use in methods of fluorescent image-guided surgery, imaging, targeted radionuclide diagnosis or therapy, and / or photothermal therapy (PTT) and / or photodynamic therapy (PDT).

[0082] We developed PTPµ-targeted fluorescent nuclide-containing agents that can be detected by fluorescent imaging as well as positron emission tomography / single-photon emission computed tomography (PET / SPECT) or magnetic resonance imaging (MRI) to specifically detect intractable tumors, such as PTPμ-expressing human brain tumors, and then utilized the same agent with a matched therapeutic radionuclide to treat the intractable tumors. Our precise targeting can deliver lethal radiation to neoplastic cells while sparing tissue that is otherwise healthy.

[0083] In some embodiments, the nuclide-containing agent can include a targeting peptide that specifically binds to and / or complexes with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule that is expressed by a cancer cell or another cell in the cancer cell microenvironment, an optional spacer directly linked to the targeting peptide, a fluorophore that is directly or indirectly linked to the targeting peptide or optional spacer; and an optional chelating agent directly or indirectly linked to the targeting peptide, optional spacer, or fluorophore. At least one of the targeting peptide, optional spacer, or fluorophore is directly labeled with a nuclide, and / or the optional chelating agent includes a chelated nuclide.

[0084] Advantageously, the fluorescent nuclide-containing agent can be administered locally (e.g., topically) or systemically (e.g., intravenously) to a subject and readily target cancer cells associated with proteolytically cleaved extracellular fragments of the immunoglobulin (Ig) superfamily cell adhesion molecule, such as metastatic, migrating, dispersed, and / or invasive cancer cells.

[0085] In some embodiments, the fluorescent nuclide-containing agent after systemic administration can cross the blood-brain barrier to define cancer cell location, distribution, metastases, dispersions, migrations, and / or invasion as well as tumor cell margins in the subject.

[0086] In other embodiments, the fluorescent nuclide-containing agent can be used in fluorescent image-guided surgery, imaging, targeted radionuclide diagnosis or therapy, and / or targeted photodynamic therapy (PDT) and / or photothermal therapy (PTT) to inhibit and / or reduce cancer cell survival, proliferation, and migration.

[0087] In some embodiments, the fluorescent nuclide-containing agents described herein can be used in a method of detecting cancer cells and / or cancer cell metastasis, migration, dispersal, and / or invasion as well as in a method of treating cancer in a subject in need thereof. The method can include administering to a subject the fluorescent nuclide- containing agent and detecting the fluorescent nuclide-containing agent bound to and / or complexed with the cancer cells to determine the location and / or distribution of the cancer cells in the subject. In some embodiments, the administered fluorescent nuclide-containing agent can provide targeted radionuclide therapy, and / or targeted PDT and PTT and ablation of the cancer cells. In other embodiments, additional or other fluorescent nuclide-containingagents including a therapeutic be administered to the subject to provide targeted radionuclide therapy, and / or targeted PDT and PTT and ablation of the cancer cells

[0088] In some embodiments, the fluorescent nuclide-containing agent can have the formula (I): a pharmaceutically acceptable salt thereof; targeting peptide;or spacer, which is optionally directly labeled with the nuclide; R3is the fluorophore; R4or R5are each independently absent or an optional chelating agent with an optional linker; wherein one of R4or R5is the optional chelating agent with the optional linker if R2is not directly labeled with the nuclide; alternatively both of R4and R5are absent and R2is present and is directly labeled with a nuclide; and wherein NH is an amino group of the spacer or the targeting peptide and C=O is a carboxyl group of the fluorophore.

[0089] In other embodiments, the fluorescent nuclide-containing agent can have the formula (II): (II) or a pharmaceutically acceptable salt thereof; wherein R1includes the targeting peptide; R3is the fluorophore; R4is a chelating agent with an optional linker; wherein NH is an amino group of the targeting peptide and C=O is a carboxyl group of the fluorophore.

[0090] In yet other embodiments, the fluorescent nuclide-containing agent can have the formula (III):acceptable salt thereof; peptide; R3is the fluorophore; R5is the chelating agent with an optional linker; and wherein NH is an amino group of the spacer or the targeting peptide and C=O is a carboxyl group of the fluorophore.

[0091] In still other embodiments, the fluorescent nuclide-containing agent can have the formula (IV): (IV) or a pharmaceutically acceptable salt thereof; wherein R1includes the targeting peptide; R2is the spacer, which is directly labeled with the radionuclide; R3is the fluorophore; and wherein NH is an amino group of the spacer or the targeting peptide and C=O is a carboxyl group of the fluorophore.

[0092] In some embodiments, the Ig superfamily cell adhesion molecule to which the targeting peptide binds can include an extracellular homophilic binding portion, which can bind in homophilic fashion or engage in homophilic binding in a subject. In one example, the Ig superfamily cell adhesion molecule includes RPTP type IIb cell adhesion molecules. In another example, Ig superfamily cell adhesion molecules can include RPTPs of the PTPµ-like subfamily, such as PTPµ, PTPĸ, PTPρ, and PCP-2 (also called PTPλ). PTPµ-like RPTPs include a MAM (Meprin / A5-protein / PTPµ) domain, an Ig domain, and FNIII repeats. PTPµ can have the amino acid sequence of SEQ ID NO: 1, which is identified by Genbank Accession No. AAI51843.1. It will be appreciated that the PTPµ gene can generate splice variants such that the amino acid sequence of PTPµ can differ from SEQ ID NO: 1. In some embodiments, PTPµ can have an amino acid sequence identified by Genbank Accession No. AAH51651.1 and Genbank Accession No. AAH40543.1.

[0093] Cancer cells and / or endothelial which support cancer cell survival, that express an Ig superfamily cell adhesion molecule and that can be proteolytically cleaved to produce a detectable extracellular fragment can include, for example, cancer cells and / or other cells in the tumor microenvironment, such as stem cells, endothelial cells, stromal cells and immune cells that promote their survival.

[0094] The cancers detected and / or treated by the fluorescent nuclide-containing agents described herein can include the following: leukemias, such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias, such as, myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia leukemias and myelodysplastic syndrome; chronic leukemias, such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including but not limited to ductal carcinoma, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactin-secreting tumor,acromegaly, and diabetes insipius; eye as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancers such as non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, prostatic intraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers such as but not limited to renal cell carcinoma, adenocarcinoma, hypemephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or uterer); Wilms' tumor; bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).

[0095] The fluorescent nuclide-containing agents can also be used to detect and / or treat a variety of cancers or other abnormal proliferative diseases, including (but not limited to) the following: carcinoma, including that of the bladder, breast, prostate, rectal, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoictic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyclocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and teratocarcinoma. It is also contemplated that cancers caused by aberrations in apoptosis would also be treated by the methods and compositions of the invention. Such cancers may include but not be limited to follicular lymphomas, carcinomas, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are detected, treated ,or prevented in the skin, lung, colon, rectum, breast, prostate, bladder, kidney, pancreas, ovary, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is detected and / or treated.

[0096] In still other embodiments, the cancer cells that are detected and / or treated can include glioma cells, lung cancer cells, breast cancer cells, prostate cancer cells, and melanoma cells, such as invasive, dispersive, motile or metastatic cancer cells can include glioma cells, lung cancer cells, breast cancer cells, prostate cancer cells, and melanoma cells.It will be appreciated that other cancer endothelial cells, which support cancer cell survival, that express an Ig superfamily cell adhesion molecule and that can be proteolytically cleaved to produce a detectable extracellular fragment can identified or determined by, for example, using immunoassays that detect the Ig superfamily cell adhesion molecule expressed by the cancer cells or endothelial cells.

[0097] In some embodiments, the targeting peptide (or targeting polypeptide) can include a polypeptide (or targeting polypeptide) that binds to and / or complexes with the proteolytically cleaved extracellular fragment of the Ig superfamily cell adhesion molecule. The targeting peptide can include, consist essentially of, or consist of about 10 to about 50 amino acids and have an amino acid sequence that is substantially homologous to about 10 to about 50 consecutive amino acids of a homophilic binding portion or domain of the proteolytically cleaved extracellular fragment of the Ig superfamily cell adhesion molecule. By substantially homologous, it is meant the targeting polypeptide has at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity with a portion of the amino acid sequence of the binding portion of the proteolytically cleaved extracellular fragment of the Ig superfamily cell adhesion molecule.

[0098] In one example, the homophilic binding portion of the Ig superfamily cell adhesion molecule can include, for example, the Ig domain of the cell adhesion molecule. In another example, where the Ig superfamily cell adhesion molecule is PTPµ, the homophilic binding portion can include the Ig binding domain and the MAM domain.

[0099] In another aspect, the targeting peptide can have an amino acid sequence that is substantially homologous to about 10 to about 50 consecutive amino acids of the Ig binding domain and / or MAM domain of PTPµ (e.g., SEQ ID NO: 1) and readily cross the blood brain barrier when systemically administered to a subject. The development of the PTPµ targeting peptides can be based on a large body of structural and functional data. The sites required for PTPµ-mediated homophilic adhesion have been well characterized. In addition, the crystal structure of PTPµ can provide information regarding which regions of each functional domain are likely to be exposed to the outside environment and therefore available for homophilic binding and thus detection by a peptide.

[0100] In some embodiments, the proteolytically cleaved extracellular fragment of PTPµ (e.g., SEQ ID NO: 1) can include an amino acid sequence of SEQ ID NO: 2, the Ig and MAM binding region can comprise the amino acid sequence of SEQ ID NO: 3, and thepolypeptide can have an amino acid is substantially homologous to about 10 to about 50 consecutive amino acids of SEQ ID NO: 2 or SEQ ID NO: 3. Examples of polypeptides that can specifically bind SEQ ID NO: 2 or SEQ ID NO: 3 and have an amino acid sequence that is substantially homologous to about 10 to about 50 consecutive amino acids of SEQ ID NO: 2 or SEQ ID NO: 3 are polypeptides that include an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 (SBK2), SEQ ID NO: 6, and SEQ ID NO: 7 (SBK4). Polypeptides comprising SEQ ID NO: 4, 5, 6, or 7 can recognize or bind to the MAM, Ig domain, or the FNIII repeats. In particular embodiments, the targeting peptide is a polypeptide comprising an amino acid sequence of SEQ ID NO: 5 (SBK2) or a polypeptide comprising an amino acid sequence of SEQ ID NO: 7 (SBK4).

[0101] The targeting peptides can be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use. In this regard, targeting peptides that bind to and / or complex with a proteolytically cleaved extracellular portion of an Ig superfamily cell adhesion molecule can be substantially homologous with, rather than be identical to, the sequence of a recited polypeptide where one or more changes are made and it retains the ability to function as specifically binding to and / or complexing with the proteolytically cleaved extracellular portion of an Ig superfamily cell adhesion molecule.

[0102] The targeting peptides can be in any of a variety of forms of polypeptide derivatives, which include amides, conjugates with proteins, cyclized polypeptides, polymerized polypeptides, retro-inverso peptides, analogs, fragments, chemically modified polypeptides, and the like derivatives.

[0103] The term "analog" includes any polypeptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and that specifically binds to and / or complexes with the proteolytically cleaved extracellular portion of an Ig superfamily CAM as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue, such as isoleucine, valine, leucine, or methionine for another, the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one such as aspartic acid or glutamic acid for another.

[0104] The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite binding activity.

[0105] "Chemical derivative" refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those polypeptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 4- hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. Polypeptides described herein also include any polypeptide having one or more additions and / or deletions or residues relative to the sequence of a polypeptide whose sequence is shown herein, so long as the requisite activity is maintained.

[0106] Retro-inverso peptides are linear peptides whose amino acid sequence is reversed and the α-center chirality of the amino acid subunits is inverted as well. These types of peptides are designed by including D-amino acids in the reverse sequence to help maintain side chain topology similar to that of the original L-amino acid peptide and make them more resistant to proteolytic degradation. D-amino acids represent conformational mirror images of natural L-amino acids occurring in natural proteins present in biological systems. Peptides that contain D-amino acids have advantages over peptides that just contain L-amino acids. In general, these types of peptides are less susceptible to proteolytic degradation and have a longer effective time when used as pharmaceuticals. Furthermore, the insertion of D-amino acids in selected sequence regions as sequence blocks containing only D-amino acids or in- between L-amino acids allows the design of peptide based drugs that are bioactive andpossess increased bioavailability in being resistant to proteolysis. Furthermore, if properly designed, retro-inverso peptides can have binding characteristics similar to L- peptides.

[0107] The term "fragment" refers to any subject polypeptide having an amino acid residue sequence shorter than that of a polypeptide whose amino acid residue sequence is shown herein.

[0108] Any polypeptide or compound may also be used in the form of a pharmaceutically acceptable salt. Acids, which are capable of forming salts with the polypeptides, include inorganic acids such as trifluoroacetic acid (TFA) hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or the like.

[0109] Bases capable of forming salts with the polypeptides include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl-amines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine and the like).

[0110] The targeting peptides can be synthesized by any of the techniques that are known to those skilled in the peptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a solid-phase Merrifield-type synthesis, can be used for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production and the like. A summary of the many techniques available can be found in Steward et al., "Solid Phase Peptide Synthesis", W. H. Freeman Co., San Francisco, 1969; Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976; J. Meienhofer, "Hormonal Proteins and Peptides", Vol.2, p.46, Academic Press (New York), 1983; Merrifield, Adv. Enzymol., 32:221-96, 1969; Fields et al., int. J. Peptide Protein Res., 35:161-214, 1990; and U.S. Pat. No.4,244,946 for solid phase peptide synthesis, and Schroder et al., "The Peptides", Vol.1, Academic Press (New York), 1965 for classical solution synthesis, each of which is incorporated herein by reference. Appropriate protective groups usable in such synthesis are described in the above texts and in J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973, which is incorporated herein by reference.

[0111] In general, the solid-phase methods contemplated comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.

[0112] Using a solid phase synthesis as an example, the protected or derivatized amino acid can be attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group can then be selectively removed and the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino or carboxyl group can then be removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) can be removed sequentially or concurrently, to afford the final linear polypeptide.

[0113] It will be appreciated that the targeting peptide can bind to and / or complex with homophilic binding domains of proteolytically cleaved extracellular fragments of other Ig superfamily cell adhesion molecules, besides PTPs. For example, a similar molecular detection strategy described herein can be used with any other Ig superfamily CAM having a homophilic binding cell surface protein whose ligand binding site is known. A large variety of cell surface proteins, including other phosphatases, are cleaved at the cell surface (Streuli M, Saito H (1992) Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region. EMBO J 11:897- 907; Anders L, Ullrich A (2006) Furin-, ADAM 10-, and gamma-secretase-mediated cleavage of a receptor tyrosine phosphatase and regulation of beta-catenin's transcriptional activity. Mol Cell Biol 26:3917-3934; Haapasalo A, Kovacs DM (2007) Presenilin / gamma- secretase-mediated cleavage regulates association of leukocyte-common antigen-related (LAR) receptor tyrosine phosphatase with beta-catenin. J Biol Chem 282:9063-9072; Chow JP, Noda M (2008) Plasmin-mediated processing of protein tyrosine phosphatase receptor type Z in the mouse brain. Neurosci Lett 442:208-212; Craig SE, Brady-Kalnay SM. Tumor-derived extracellular fragments of receptor tyrosine phosphatases (RPTPs) as cancer molecular diagnostic tools. Anticancer Agents Med Chem.2011 Jan;11(1):133-40. Review. PubMed PMID: 21235433; PubMed Central PMCID: PMC3337336; Craig SE, Brady- Kalnay SM. Cancer cells cut homophilic cell adhesion molecules and run. Cancer Res.2011 Jan 15;71(2):303-9. Epub 2010 Nov 17. PubMed PMID: 21084269; PubMed Central PMCID: PMC3343737; Phillips-Mason PJ, Craig SE, Brady-Kalnay SM. Should I stay or should I go? Shedding of RPTPs in cancer cells switches signals from stabilizing cell-cell adhesion to driving cell migration. Cell Adh Migr.2011 Jul 1;5(4):298-305. Epub 2011 Jul 1. PubMed PMID: 21785275; PubMed Central PMCID: PMC3210297). These proteins represent additional targets for that can be readily used by the skilled artisan for forming therapeutic polypeptides that can be used to treat cancers (Barr AJ, Ugochukwu E, Lee WH, King ON, Filippakopoulos P, Alfano I, Savitsky P, Burgess-Brown NA, Muller S, Knapp S (2009) Large-scale structural analysis of the classical human protein tyrosine phosphatome. Cell 136:352-363).

[0114] In some embodiments, the targeting peptides described herein can include additional residues that may be added at either terminus of a polypeptide for the purpose of providing a "linker" by which the polypeptides can be conveniently linked and / or affixed to the optional spacer or the fluorophore. Typical amino acid residues used for linking are glycine, tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. In addition, a subject polypeptide can differ by the sequence being modified by terminal-NH2acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications. Terminal modifications are useful, as is well known, to reduce susceptibility by proteinase digestion, and therefore serve to prolong half-life of the polypeptides in solutions, particularly biological fluids where proteases may be present. In this regard, polypeptide cyclization is also a useful terminal modification, and is particularly preferred because of the stable structures formed by cyclization and in view of the biological activities observed for such cyclic peptides as described herein.

[0115] The optional spacer directly linked to the targeting peptide can include additional natural and / or non-natural amino acid residues added at either terminus of a targeting peptide (or target peptide with linker peptide). The spacer can include at least one natural and / or non-natural amino acid and can have a structure effective to at least maintain or preservebinding affinity of the linked targeting the proteolytically cleaved extracellular fragment and activity of the fluorophore or optional chelating agent. Typical amino acid residues used for use in the spacer are glycine, serine tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.

[0116] In some embodiments, the optional spacer is selected in part based on its ability to alter the phobicity (e.g., to cause the agent to become more hydrophilic or hydrophobic) depending on its desired use.

[0117] In other embodiments, the optional spacer can be selected such that it can be directly labeled with a nuclide, such as a radionuclide. For example, the optional spacer can include an amino acid residue, such as tyrosine, which is directly labeled with a nuclide, preferably, a halide, such as I,23I,124I,125I, or131I. Radioactive iodine isotopes can be coupled to the optional spacer by iodination of a diazotized amino derivative directly via a diazonium iodide, see Greenbaum, F. Am. J. Pharm.108: 17 (1936), or by conversion of the unstable diazotized amine to the stable triazene, or by conversion of a non-radioactive halogenated precursor to a stable tri-alkyl tin derivative which then can be converted to the iodo compound by several methods well-known to the art.

[0118] In other embodiments, the optional spacer can be a flexible peptide that directly or indirectly links the targeting peptide to the fluorophore and / or optional chelating agent. A flexible peptide or peptidomimetic spacer can be, for example, at least about 1 to about 10 or fewer natural and / or non-natural amino acids in length. For example, the spacer can have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 natural and / or non-natural amino acids. Where the spacer is a peptide spacer, the peptide spacer may be produced as a single recombinant polypeptide using a conventional molecular biological / recombinant DNA method.

[0119] The fluorophore is directly or indirectly linked to the targeting peptide or optional spacer via, for example, a natural or non-natural linkage. In some embodiments, the natural or non-natural linkage is not susceptible to proteolytic cleavage. For example, compounds (I, II, III, and IV) include an amide linkage formed by an amino group of the targeting peptide or optional spacer and a carboxy group of the fluorophore that is used to link the fluorophore to the targeting peptide or optional spacer and that is not susceptible to proteolytic cleavage.

[0120] In some embodiments, the fluorophore can include an organic small molecule fluorophore that fluoresces upon irradiation in the first near-infrared region (NIR-I, 650-1000 nm) or the second near-infrared region (NIR-II, 1000-1700 nm). The fluorophore can besufficiently or effectively hydrophobic or such that when directly or indirectly conjugated to the targeting peptide or optional peptide spacer with the natural or non-natural linkage, the fluorescent nuclide-containing agent has a signal-to-background ratio (SBR) (or signal to noise ratio (SNR)) upon fluorescent imaging effective to delineate the cancer cell or another cell in the cancer cell microenvironment from surrounding tissue.

[0121] It was found that the hydrophobicity or lipophilicity of the organic small molecule fluorophore can substantially affect the SBR of the fluorescent nuclide-containing agent. More hydrophobic and lipophilic fluorophores when directly or indirectly linked to the targeting peptide, which specifically binds to and / or complexes with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule, or optional spacer via a natural or non-natural linkage can provide or form a fluorescent nuclide- containing agent that can readily delineate cancer cells from surrounding tissue in contrast to more hydrophilic organic small molecule fluorophores that when directly or indirectly linked to the targeting peptide or optional spacer via a natural or non-natural linkage could not delineate cancer cells from surrounding tissue.

[0122] In some embodiment, the more hydrophobic and lipophilic fluorophores, which can provide or form a fluorescent nuclide-containing agent with the targeting peptide and can readily delineate cancer cells from surrounding tissue, can include organic small molecule fluorophores that have a logP of at least 0, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6 or more, for example, a logP of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or any range therebetween.

[0123] In some embodiments, the fluorophore can include a cyanine near-infrared fluorophore having a fluorescence in the first near infrared region or second near infrared region. Cyanine near-infrared fluorophores are broadly defined as two heterocyclic nitrogen atoms that are connected via an electron deficient polymethine bridge. Monomethine cyanines display one methine unit, in this case defined as (=C-), between the heterocyclic structures; this class of compounds displays absorbance within the ultraviolet and visible regions with low fluorescence quantum yield. Elongating the central chromophore length by sets of 2 methylene groups yields tri-, penta-, and heptamethine cyanines. The wavelengths of trimethine cyanines are too low to be effective in NIR imaging in biological systems; however, penta- and heptamethine cyanines have near-infrared absorbance and fluorescencecharacteristics that are a function of their structure and moieties within the polymethine chain, which can be tuned to offer high quantum yield and molecular brightness.

[0124] In some embodiments, a cyanine near-infrared fluorophore, which has sufficient hydrophobicity, lipophilicity, and / or logP to provide or form a fluorescent nuclide-containing agent with the targeting peptide and can readily delineate cancer cells from surrounding tissue, can include indocyanine green (ICG) or ICG-Osu.

[0125] In some embodiments, the optional chelating directly or indirectly linked to the targeting peptide, optional spacer, or fluorophore is selected from 1,4,7-triazacyclononane- 1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazadodecanetetraacetate (DOTA), 2,2’,2”-(10- (pyridin-2-ylmethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-1Py), 2,2’-(7,10-(pyridin-2-ylmethyl)-1,4,7,10-tetraazacyclododecane-1,4-diyl) diacetic acid (DOTA-2Py), 2-(4,7,10-tris (pyridin-2-ylmethyl)-1,4,7,10-tetraazacyclododecane-1-yl) acetic acid (DOTA-3Py), 1,4,7,10-tetraazadodecane-1,4,7-triacetate (DO3A), ethylenediaminetetraacetate (EDTA), 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10- tetraazadodecanetetramethylacetate (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7- trimethylacetate (DO3MA), N,N',N'',N'''-tetraphosphonatomethyl-1,4,7,10- tetraazacyclododecane (DOTP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene methylphosphonic acid) (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetrakis(methylene phenylphosphonic acid) (DOTPP), N,N'-ethylenedi-L-cysteine, S-2-(4- isothiocyanatobenzyl)-1,4,7,10-tetraazacylododecane tetracetic acid (p-SCN-Bn-DOTA), 2- (4-isothiocyanatobenzyl-1,4,7,10-tetraaza-1,4,7,10,tetra-(2-carbamonylmethyl)- cyclododecane (p-SCN-Bn-TCMC), MeO-DOTA-NCS, [(R)−2-Amino-3-(4- isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (CHX- A’’-DTPA-NCS), 2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N'''' - pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N''''-hexaacetic acid (HEHA), desferrioxamine B (DFO), macropa, macropa-NCS, macropid, bispa2, EuK- 106, 7-[2-(bis-carboxymethyl-amino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza- cyclododec-1-yl-acetic acid (DEPA), 3p-C-DEPA, or derivatives thereof.

[0126] In some embodiments, the optional metal chelating agent is directly or indirectly linked to an amino acid of the targeting peptide or optional spacer or the fluorophore via, for example, a natural or non-natural linker. The natural or non-natural linker used to directly orindirectly link the optional chelating agent targeting peptide, optional spacer, or fluorophore can include any natural or chemical linker that is not susceptible to proteolytic cleavage. The non-natural linker can be formed using a coupling agent that is attached to or comprises a portion of the optional chelating agent, fluorophore, optional spacer, or targeting peptide. The coupling agent and / or conjugating agent can include, for example, maleimidyl binders, which can be used to bind to thiol groups, isothiocyanate and succinimidyl (e.g., N- hydroxysuccinimidyl (NHS)) binders, which can bind to free amine groups, diazonium which can be used to bind to phenol, and amines, which can be used to bind with free acids such as carboxylate groups using carbodiimide activation. Useful functional groups can be present on the targeting peptide or optional spacer based on the particular amino acids present, and additional groups can be designed. It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, Ill.), can be employed as a coupling agent. Coupling can be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues.

[0127] Examples of coupling agents and / or conjugating agents are described in Means and Feeney, CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp.39-43. Among these reagents are, for example, J-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or N,N'-(1,3-phenylene) bismaleimide (both of which are highly specific for sulfhydryl groups and form irreversible linkages); N,N'-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11 carbon methylene bridges (which relatively specific for sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversible linkages with amino and tyrosine groups). Other coupling agents or conjugating can include: p,p'- difluoro-m,m'-dinitrodiphenylsulfone (which forms irreversible linkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4- disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts principally with amino groups); glutaraldehyde (which reacts with several different side chains) and disdiazobenzidine (which reacts primarily with tyrosine and histidine).

[0128] The coupling agent may be homobifunctional, i.e., having two functional groups that undergo the same reaction. An example of a homobifunctional cross-linking reagent is bismaleimidohexane ("BMH"). BMH contains two maleimide functional groups, which reactspecifically with sulfhydryl-containing under mild conditions (pH 6.5-7.7). The two maleimide groups are connected by a hydrocarbon chain. Therefore, BMH is useful for irreversible linking of polypeptides that contain cysteine residues.

[0129] Coupling agents may also be heterobifunctional. Heterobifunctional coupling or conjugating agents have two different functional groups, for example an amine-reactive group and a thiol-reactive group, that will cross-link two proteins having free amines and thiols, respectively. Examples of heterobifunctional cross-linking agents are succinimidyl 4- (N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC"), m-maleimidobenzoyl-N- hydroxysuccinimide ester ("MBS"), and succinimide 4-(p-maleimidophenyl) butyrate ("SMPB"), an extended chain analog of MBS. The succinimidyl group of these cross-linkers reacts with a primary amine, and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine residue.

[0130] The coupling agents can yield a conjugate of the targeting peptide or optional spacer and the fluorophore that is essentially non-cleavable under cellular conditions. Numerous coupling agents, including the ones discussed above, are commercially available. Detailed instructions for their use are readily available from the commercial suppliers. A general reference on protein cross-linking and conjugate preparation is: Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING, CRC Press (1991).

[0131] In some embodiments, the optional chelating agent directly or indirectly linked to the targeting peptide, optional spacer, or fluorophore can be configured to chelate a metal nuclide, such as a diagnostic or therapeutic nuclide. The diagnostic or therapeutic nuclide can include at least one of Ga, I, In, Y, Lu, Bi, Ac, Re, Th, Tc, Tl, Tb, Zr, Cu, Rb, At, Pb, Gd, Sm, or Sr.

[0132] In some embodiments, the diagnostic nuclide can be a diagnostic radionuclide selected from123I,124I,125I,64Ga,18F,11C,13N,76Br,149Tb,161Tb,99mTc,153Gd,111In,67Ga,68Ga,201Tl,82Rb,64Cu,67Cu,89Zr,90Y, T(tritium),149Tb,161Tb,153Sm, or89Sr.

[0133] In some embodiments, the therapeutic nuclide can be a therapeutic radionuclide selected from131I,225Ac,226Ac,227Th,211Bi,212Bi,213Bi,203Pb,212Pb, or177Lu.

[0134] In still other embodiments, a chelated radionuclide can be converted from an imaging or diagnostic radionuclide to a therapeutic radionuclide. For example, a fluorescent nuclide-containing agent that includes a diagnostic radionuclide can be injected into the subject’s body and targeted to the cancer in the subject by the targeting peptide. Alpha orgamma irradiation treatment can be used to the radionuclide from an imaging or diagnostic isotope of lower energy to a therapeutic isotope of higher energy. Switching the isotope converts the agent from an imaging agent to an alpha or gamma emitting therapeutic agent

[0135] In some embodiments, the metal nuclide can be bound to a chelating agent under mild temperature conditions, e.g., less than about 65°C, 60°C, 55°C, 50°C, 45°C, 40°C, 35°C or 30° C as well as elevated temperatures, e.g., greater than about 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C. In some embodiments, the mild temperature conditions are between about 10°C and 65°C, including any value or subrange therebetween, for example, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C. In some embodiments, the metal nuclide can be conjugated to the chelating agent at room temperature, i.e., in the range of about 15°C to about 25°C, including any temperature value therebetween.

[0136] In some embodiments, the metal nuclide can be combined with the metal chelating agent to form a metal chelate under mild pH conditions, e.g., between about 6.0 and about 8.0, including any value or subrange therebetween, e.g., 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, or 7.8. In some embodiments the metal nuclide is a radionuclide conjugated to a chelating agent at approximately neutral pH, i.e., a pH of approximately 7.0, e.g., between about 6.8 and 7.2 including any value therebetween, e.g., 6.9, 7.0 or 7.1. In some embodiments, the radionuclide is conjugated to the chelating agent at approximately physiological pH, i.e., at approximately pH 7.4, e.g., between about 7.2 and 7.6 including any value therebetween, e.g., 7.3, 7.4 or 7.5.

[0137] In some embodiments, the nuclide is combined with the metal chelating agent for an incubation period to allow a chelated metal complex to form. In some embodiments, the incubation period is between about 5 minutes and about 6 hours, including any period therebetween, e.g., 10, 15, 20, 25, 30, 45, 60 or 90 minutes, or 2, 3, 4 or 5 hours.

[0138] In other embodiments, the fluorescent nuclide-containing agent can include compounds selected from:; ;(VII); or wherein R1is the targeting peptide; R6and R7are each absent or a nuclide; and at least one of R6or R7is a nuclide.

[0139] In some embodiments, R6and / or R7is selected from123I,124I,125I, or131I.

[0140] In some embodiments, compounds (VI) and (VII) or pharmaceutically acceptable salts thereof can further include a chelated diagnostic or therapeutic nuclide.

[0141] In some embodiments, the diagnostic or therapeutic nuclide can include at least one of Ga, I, In, Y, Lu, Bi, Ac, Re, Th, Tc, Tl, Tb, Zr, Cu, Rb, At, Pb, Gd, Sm, or Sr.

[0142] In some embodiments, the diagnostic nuclide is a diagnostic radionuclide selected from123I,124I,125I,64Ga,18F,11C,13N,76Br,149Tb,161Tb,99mTc,153Gd,111In,67Ga,68Ga,201Tl,82Rb,64Cu,67Cu,89Zr,90Y, T(tritium),149Tb,161Tb,153Sm, or89Sr.

[0143] In some embodiments, the therapeutic nuclide is a therapeutic radionuclide selected from131I,225Ac,226Ac,227Th,211Bi,212Bi,213Bi,203Pb,212Pb, or177Lu.

[0144] In some embodiments, R1has the amino acid sequence of GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 5) or GIDVRDAPLKEIKVTSSR (SEQ ID NO: 7).

[0145] The fluorescent nuclide-containing agents described herein can be administered to the subject by, for example, systemic, topical, and / or parenteral methods of administration. These methods include, e.g., injection, infusion, deposition, implantation, or topical administration, or any other method of administration where access to the tissue by the fluorescent nuclide-containing agent is desired. In one example, administration of thefluorescent nuclide-containing agent can intravenous injection of the fluorescent nuclide-containing agent in the subject. Single or multiple administrations of the agent can be given. “Administered”, as used herein, means provision or delivery of the fluorescent nuclide-containing agent in an amount(s) and for a period of time(s) effective to label cancer cells in the subject.

[0146] The fluorescent nuclide-containing agents described herein can be administered to a subject in a detectable quantity of a pharmaceutical composition containing the fluorescent nuclide-containing agent or a pharmaceutically acceptable water-soluble salt thereof, to a patient.

[0147] Formulation of the fluorescent nuclide-containing agent to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule, and the like). Suitable pharmaceutically acceptable carriers may contain inert ingredients which do not unduly inhibit the biological activity of the fluorescent nuclide-containing agents. The pharmaceutically acceptable carriers should be biocompatible, e.g., non-toxic, non- inflammatory, non-immunogenic and devoid of other undesired reactions upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, ibid. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like.

[0148] The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically, such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. Formulation will vary according to the route of administration selected (e.g., solution, emulsion, capsule).

[0149] A "detectable quantity" means that the amount of the fluorescent nuclide- containing agent that is administered is sufficient to enable detection of binding of the fluorescent nuclide-containing agent to the cancer cells. An "imaging effective quantity" means that the amount of the fluorescent nuclide-containing agent that is administered is sufficient to enable fluorescent, PET / SPECT, and / or MRI imaging of binding of the fluorescent nuclide-containing agent to the cancer cells.

[0150] The fluorescent nuclide- agent administered to a subject can be used in a method to detect and / or determine the presence, location, and / or distribution of cancer cells, i.e., cancer cells associated with proteolytically cleaved extracellular fragments of Ig superfamily cell adhesion molecules, in an organ or body area of a patient, e.g., at least one region of interest (ROI) of the subject. The ROI can include a particular area or portion of the subject and, in some instances, two or more areas or portions throughout the entire subject. The ROI can include regions to be imaged for both diagnostic and therapeutic purposes. The ROI is typically internal; however, it will be appreciated that the ROI may additionally or alternatively be external.

[0151] In some embodiments, the presence, location, and / or distribution of the fluorescent nuclide-containing agents in the animal’s tissue, e.g., brain tissue, can be visualized with a near-infrared fluorescence (NIRF) scanner. In one example, the NIRF scanner may be handheld. In another example, the NIRF scanner may be miniaturized and embedded in an apparatus (e.g., micro-machines, scalpel, neurosurgical cell removal device).

[0152] In other embodiments, where the fluorescent nuclide-containing agents include a diagnostic nuclide or diagnostic radionuclide, the fluorescent nuclide-containing agents can be detected by other in vivo imaging modalities, such as magnetic resonance imaging (MRI), or gamma imaging, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT). The term "in vivo imaging" refers to any method, which permits the detection of the fluorescent nuclide-containing agents, as described above. For gamma imaging, the radiation emitted from the organ or area being examined is measured and expressed either as total binding or as a ratio in which total binding in one tissue is normalized to (for example, divided by) the total binding in another tissue of the same subject during the same in vivo imaging procedure. Total binding in vivo is defined as the entire signal detected in a tissue by an in vivo imaging technique without the need for correction by a second injection of an identical quantity of molecular probe along with a large excess of unlabeled, but otherwise chemically identical compound.

[0153] For purposes of in vivo imaging, the type of detection instrument available is a major factor in selecting a given detectable moiety. For instance, the type of instrument used will guide the selection of the stable isotope. The half-life should be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that the host does not sustain deleterious effects.

[0154] “Distribution” as used herein spatial property of being scattered about over an area or volume. In this case, “the distribution of cancer cells” is the spatial property of cancer cells being scattered about over an area or volume included in the animal’s tissue, e.g., brain tissue. The distribution of the fluorescent nuclide-containing agent may then be correlated with the presence or absence of cancer cells in the tissue. A distribution may be dispositive for the presence or absence of a cancer cells or may be combined with other factors and symptoms by one skilled in the art to positively detect the presence or absence of migrating or dispersing cancer cells, cancer metastases or define a tumor margin in the subject. It will be appreciated that the imaging modality may be used to generate a baseline image prior to administration of the fluorescent nuclide-containing agent. In this case, the baseline and post-administration images can be compared to ascertain the presence, absence, and / or extent of a particular disease or condition.

[0155] In one aspect, the fluorescent nuclide-containing agent may be administered to a subject to assess the distribution of cancer cells in a subject and correlate the distribution to a specific location. Surgeons routinely use intra-operative in vivo imaging in surgical resections. This allows them to specifically identify and sample tissue from distinct regions of the tumor such as the tumor edge or tumor center. Frequently, they also sample regions of brain on the tumor margin that are outside the tumor edge that appear to be grossly normal but are infiltrated by dispersing tumor cells upon histological examination. For example, in glioma (brain tumor) surgery, the fluorescent nuclide-containing agent can be given intravenously prior to pre-surgical localization imaging. The agents can be imaged using near-infrared fluorescent, MRI, and / or PET / SPECT imaging that localizes with the glioma.

[0156] The fluorescent nuclide-containing agents described herein that specifically bind to and / or complex with proteolytically cleaved Ig superfamily cell adhesion molecules (e.g., PTPµ) associated with cells can be used in intra-operative imaging (IOI) techniques to guide surgical resection and eliminate the “educated guess” of the location of the tumor margin by the surgeon. Previous studies have determined that more extensive surgical resection improves patient survival. Thus, fluorescent nuclide-containing agents that function as diagnostic molecular imaging agents have the potential to increase patient survival rates.

[0157] In some embodiments, the fluorescent nuclide-containing agent upon administration to the subject can target and detect and / or determine the presence, location,and / or distribution of cancer cells, i.e., associated with proteolytically cleaved extracellular fragments of Ig superfamily cell adhesion molecules, in an organ or body area of a patient. In one example, the fluorescent nuclide-containing agent can be combined with intraoperative imaging (IOI) to identify malignant cells that have infiltrated and / or are beginning to infiltrate at a tumor brain margin. The method can be performed in real-time during brain or other surgery. The method can include local or systemic application of the fluorescent nuclide-containing agent described herein. A fluorescent, MRI, PET, and / or SPECT imaging modality can then be used to detect and subsequently gather image data. The resultant image data may be used to determine, at least in part, a surgical and / or radiological treatment. Alternatively, this image data may be used to control, at least in part, an automated surgical device (e.g., laser, scalpel, micromachine) or to aid in manual guidance of surgery. Further, the image data may be used to plan and / or control the delivery of a therapeutic agent (e.g., by a micro-electronic machine or micro-machine).

[0158] In one example, the fluorescent nuclide-containing agent can be topically applied as needed during surgery to interactively guide a surgeon and / or surgical instrument to remaining abnormal cells. The fluorescent nuclide-containing agent may be applied locally in low concentration, making it unlikely that pharmacologically relevant concentrations are reached. In one example, excess material may be removed (e.g., washed off) after a period of time (e.g., incubation period).

[0159] Another embodiment described herein relates to a method of monitoring the efficacy of a cancer therapeutic or cancer therapy administered to a subject. The methods and agents described herein can be used to monitor and / or compare the invasion, migration, dispersal, and metastases of a cancer in a subject prior to administration of a cancer therapeutic or cancer therapy, during administration, or post therapeutic regimen.

[0160] A "cancer therapeutic” or “cancer therapy”, as used herein, can include any agent or treatment regimen that is capable of negatively affecting cancer in an animal, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of an animal with cancer. Cancer therapeutics can include one or more therapies such as, but not limited to, chemotherapies, radiation therapies, hormonaltherapies, and / or biological A reduction, for example, in cancer volume, growth, migration, and / or dispersal in a subject may be indicative of the efficacy of a given therapy. This can provide a direct clinical efficacy endpoint measure of a cancer therapeutic. Therefore, in another aspect, a method of monitoring the efficacy of a cancer therapeutic is provided. More specifically, embodiments of the application provide for a method of monitoring the efficacy of a cancer therapy.

[0161] The method of monitoring the efficacy of a cancer therapeutic can include the steps of administering in vivo to the animal the fluorescent nuclide-containing agent as described herein, then visualizing a distribution of the fluorescent nuclide-containing agent in the animal (e.g., with an in vivo imaging modality as described herein), and then correlating the distribution of the fluorescent nuclide-containing agent with the efficacy of the cancer therapeutic. It is contemplated that the administering step can occur before, during, and after the course of a therapeutic regimen in order to determine the efficacy of a chosen therapeutic regimen. One way to assess the efficacy of the cancer therapeutic is to compare the distribution of the fluorescent nuclide-containing agent pre and post cancer therapy.

[0162] In some embodiments, the fluorescent nuclide-containing agent bound to and / or complexed with the proteolytically cleaved extracellular fragment of the Ig superfamily cell adhesion molecule is detected in the subject to detect and / or provide the location and / or distribution of the cancer cells in the subject. The location and / or distribution of the cancer cells in the subject can then be compared to a control to determine the efficacy of the cancer therapeutic and / or cancer therapy. The control can be the location and / or distribution of the cancer cells in the subject prior to the administration of the cancer therapeutic and / or cancer therapy. The location and / or distribution of the cancer cells in the subject prior to the administration of the cancer therapeutic and / or cancer therapy can be determined by administering the fluorescent nuclide-containing agent to the subject and detecting the agent bound to and / or complexed with cancer cells in the subject prior to administration of the cancer therapeutic and / or cancer therapy.

[0163] In certain embodiments, the methods and agents described herein can be used to measure the efficacy of a therapeutic administered to a subject for treating a metastatic, invasive, or dispersed cancer. In this embodiment, the fluorescent nuclide-containing agent can be administered to the subject prior to, during, or post administration of the therapeutic regimen and the distribution of cancer cells can be imaged to determine the efficacy of thetherapeutic regimen. In one example, the regimen can include a surgical resection of the metastatic cancer and the fluorescent nuclide-containing agent can be used to define the distribution of the metastatic cancer pre-operative and post-operative to determine the efficacy of the surgical resection. Optionally, the methods and the fluorescent nuclide- containing agents can be used in an intra-operative surgical procedure as described above, such as a surgical tumor resection, to more readily define and / or image the cancer cell mass or volume during the surgery.

[0164] In other embodiments, the fluorescent nuclide-containing agent can be used in a method of treating cancer or tumors (e.g., brain cancer or tumors). For example, where the fluorescent nuclide-containing agents include therapeutic nuclide or radionuclide as described herein, the fluorescent nuclide-containing agents can be used to carry out targeted radionuclide therapy. For example, the fluorescent nuclide-containing agents may be administered to a subject in any suitable manner, and the targeting effect imparted by the targeting peptide can be used to deliver a radionuclide or chelated radionuclide to a proteolytically cleaved Ig superfamily cell adhesion molecules (e.g., PTPµ) associated with cancer cells within the subject's body. In some embodiments, radiation from radionuclide can be used to kill cancer cells associated with the proteolytically cleaved Ig superfamily cell adhesion molecules (e.g., PTPµ) at the desired location. In some embodiments, cancer cells that are killed at the desired location are PTPμ-expressing human brain tumor cells. In some embodiments, fluorescent nuclide-containing agents can be used to perform targeted radionuclide therapy, such as targeted alpha therapy, targeted beta therapy, targeted gamma therapy, or targeted auger therapy.

[0165] The fluorescent nuclide-containing agents can be administered alone as a monotherapy, or in conjunction with or in combination with one or more additional therapeutic agents. In some embodiments, the fluorescent nuclide-containing agents that include a therapeutic nuclide or radionuclide described herein can be administered to the subject in combination with an additional anti-cancer agent.

[0166] The term "in conjunction with" or “in combination with” indicates that the fluorescent nuclide-containing agents are administered at about the same time as the additional agent. The fluorescent nuclide-containing agents can be administered to the subject in need thereof as part of a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier or excipient and, optionally, one or more additionaltherapeutic agents. The compound and therapeutic agent can be components of separate pharmaceutical compositions, which can be mixed together prior to administration or administered separately. The fluorescent nuclide-containing agents can, for example, be administered in a composition containing the additional therapeutic agent, and thereby, administered contemporaneously with the agent. Alternatively, the fluorescent nuclide- containing agents can be administered contemporaneously, without mixing (e.g., by delivery of the compound on the intravenous line by which the compound is also administered, or vice versa). In another embodiment, the fluorescent nuclide-containing agents can be administered separately (e.g., not admixed), but within a short timeframe (e.g., within 24 hours) of administration of the compound.

[0167] In other embodiments, the fluorescent nuclide-containing agents can be used in imaging-mediated phototherapy to ablate cancer cells or another cell in the cancer cell microenvironment.

[0168] Image-mediated phototherapy can include imaging-guided photothermal therapy (PTT) and imaging-guided photodynamic therapy (PDT). In PTT, the fluorophore of fluorescent nuclide-containing agents bound to the cancer cell or another cell in the cancer cell microenvironment can be irradiated with a wavelength of light effective to convert light energy into heat and ablate the cancer. Advantageously, the as-produced heat can potentially cause thermal expansion of the cancer tissue to generate a photoacoustic imaging (PAI) signal. Alternatively, the fluorophore of the fluorescent nuclide-containing agents bound to the cancer cell or another cell in the cancer cell microenvironment can be irradiated with a wavelength of light effective to produce singlet oxygen (O2) or other reactive oxygen species (ROS) under laser irradiation to induce apoptosis or necrosis of cancer cells, which can be applied for imaging-guided photodynamic therapy (PDT) or further to achieve synergistic PDT / PTT. Only the cells that are exposed simultaneously to the fluorescent nuclide- containing agent and light are destroyed while surrounding healthy, non-targeted and nonirradiated cells are spared from photodamage. Furthermore, the fluorescence of the fluorescent nuclide-containing agent enables simultaneous diagnostic optical imaging that can be used to guide cancer treatment.

[0169] Methods for conducting PTT and / or PDT are known in the art. See for example Thierry Patrice. Photodynamic Therapy; Royal Society of Chemistry, 2004. Apharmaceutical composition including nuclide-containing agents described herein can be applied to an organ or tissue as a step in PTT and / or PDT.

[0170] A fluorescent nuclide-containing agent for PTT and / or PDT can be administered to a subject with cancer by systemic administration, such as intravenous administration. Upon administration, the fluorescent nuclide-containing agent described herein can localize to and / or accumulate at the site of the targeted tumor or cancer. In some embodiments, specific binding and / or complexing with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule that is expressed by a cancer cell or another cell in the cancer cell microenvironment allows the agent to be bound to, complexed with and / or taken up by the targeted cells. This binding and / or uptake is specific to the targeted cells, which allows selective targeting of the cancer cells and / or cells in the cancer cell microenvironment in the subject by the targeted agents.

[0171] Following administration and localization of the fluorescent nuclide-containing agents to the targeted cancer cells, the targeted cancer cells can be exposed to a therapeutic amount of light that causes cancer cell ablation, damage and / or suppression of cancer cell growth. The light, which is capable of activating the fluorescent nuclide-containing agents for PTT and / or PDT agent can delivered to the targeted cancer cells using, for example, semiconductor laser, dye laser, optical parametric oscillator or the like. It will be appreciated that any source light can be used as long as the light excites the fluorescent nuclide- containing agent.

[0172] The fluorescent nuclide-containing agents described herein can be administered to a subject by any conventional method of drug administration, for example, orally in capsules, suspensions, or tablets or by parenteral administration. Parenteral administration can include, for example, intramuscular, intravenous, intraventricular, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration. The disclosed compounds can also be administered orally (e.g., in capsules, suspensions, tablets, or dietary), nasally (e.g., solution, suspension), transdermally, intradermally, topically (e.g., cream, ointment), inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops) transmucosally or rectally. Delivery can also be by injection into the brain or body cavity of a patient or by use of a timed release or sustained release matrix delivery systems, or by onsite delivery using micelles, gels, and liposomes. Nebulizing devices, powder inhalers, and aerosolized solutions may also be used to administer such preparations to the respiratory tract. Deliverycan be in vivo, or ex vivo. Administration local or systemic as indicated. More than one route can be used concurrently if desired. The preferred mode of administration can vary depending upon the particular disclosed compound chosen. In specific embodiments, oral, parenteral, or systemic administration are preferred modes of administration for treatment.

[0173] In some embodiments, the fluorescent nuclide-containing agents described herein can be administered alone as a monotherapy, or in conjunction with or in combination in a complex mixture with other agents structurally similar to the agents described herein but devoid of a diagnostic and / or therapeutic nuclide and / or chelated nuclide.

[0174] The methods described herein contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time. The fluorescent nuclide-containing agents described herein (or composition containing the agent) can be administered at regular intervals, depending on the nature and extent of the cancer, and on an ongoing basis. Administration at a "regular interval," as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). In one embodiment, the fluorescent nuclide-containing agent is administered periodically, e.g., at a regular interval (e.g., bimonthly, monthly, biweekly, weekly, twice weekly, daily, twice a day or three times or more often a day).

[0175] The administration interval for a single individual can be fixed, or can be varied over time, depending on the needs of the individual. For example, in times of physical illness or stress, or if disease symptoms worsen, the interval between doses can be decreased.

[0176] For example, the administration of the fluorescent nuclide-containing agents and / or the other agent can take place at least once on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least once on week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any combination thereof, using single or divided doses of every 60, 48, 36, 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. Administration can take place at any time of day, for example, in the morning, the afternoon, or evening. For instance, the administration can take place in the morning, e.g., between 6:00 a.m. and 12:00 noon; in the afternoon, e.g., after noon and before 6:00 p.m.; or in the evening, e.g., between 6:01 p.m. and midnight.

[0177] The fluorescent nuclide-containing agents and other agents described herein and / or additional therapeutic agent can be administered in a dosage of, for example, 0.1 to100 mg / kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day. Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions, the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.

[0178] In some embodiments, where the fluorescent nuclide-containing agent includes a therapeutic radionuclide, the fluorescent nuclide-containing agent may be administered to the subject at an amount effective to deliver a radiation dose, for example, at or below 1 mCi / kg (i.e., where the amount of the fluorescent nuclide-containing agent administered to the subject delivers a radiation dose of below 1000 μCi per kilogram of subject's body weight). According to certain aspects, the effective amount is at or below 900 μCi / kg, 800 μCi / kg, 700 μCi / kg, 600 μCi / kg, 500 μCi / kg, 400 μCi / kg, 300 μCi / kg, 200 μCi / kg, 150 μCi / kg, 100 μCi / kg, 80 μCi / kg, 60 μCi / kg, 50 μCi / kg, 40 μCi / kg, 30 μCi / kg, 20 μCi / kg, 10 μCi / kg, 5 μCi / kg, or 1 μCi / kg. According to certain aspects, the effective amount of the radiation dose from the fluorescent nuclide-containing agent is at least 1 μCi / kg, 2.5 μCi / kg, 5 μCi / kg, 10 μCi / kg, 20 μCi / kg, 30 μCi / kg, 40 μCi / kg, 50 μCi / kg, 60 μCi / kg, 70 μCi / kg, 80 μCi / kg, 90 μCi / kg, 100 μCi / kg, 150 μCi / kg, 200 μCi / kg, 250 μCi / kg, 300 μCi / kg, 350 μCi / kg, 400 μCi / kg or 450 μCi / kg. According to certain aspects, the fluorescent nuclide-containing agent may be administered at an amount effective to deliver a radiation dose that includes any combination of upper and lower limits as described herein, such as from at least 5 mCi / kg to at or below 50 μCi / kg, or from at least 50 mCi / kg to at or below 500 μCi / kg.

[0179] In other embodiments, the fluorescent nuclide-containing agent containing a therapeutic radionuclide may be administered to the subject at an amount effective to deliver a radiation dose and the effective amount of radiation dose delivered may be at or below 2 mCi (i.e., wherein the fluorescent nuclide-containing agent is administered to the subject in a non-weight-based dosage). According to certain aspects, the effective dose of the radiation delivered by the PSMA targeted compound may be at or below 1 mCi, such as 0.9 mCi, 0.8 mCi, 0.7 mCi, 0.6 mCi, 0.5 mCi, 0.4 mCi, 0.3 mCi, 0.2 mCi, 0.1 mCi, 90 μCi, 80 μCi, 70 μCi, 60 μCi, 50 μCi, 40 μCi, 30 μCi, 20 μCi, 10 μCi, or 5 μCi. The effective amount of PSMA targeted compound may be at least 2 μCi, such as at least 5 Ci, 10 μCi, 20 μCi, 30 μCi, 40 μCi, 50 μCi, 60 μCi, 70 μCi, 80 μCi, 90 μCi, 100 μCi, 200 μCi, 300 μCi, 400 μCi,500 μCi, 600 μCi, 700 μCi, 800 μCi, 900 mCi, 1.1 mCi, 1.2 mCi, 1.3 mCi, 1.4 mCi, or 1.5 mCi. According to certain aspects, the fluorescent nuclide-containing agent may be administered at an amount effective to deliver a radiation dose that includes any combination of upper and lower limits as described herein, such as from at least 2 μCi to at or below 1 mCi, or from at least 2 μCi to at or below 250 μCi, or from 75 μCi to at or below 400 μCi.

[0180] In other embodiments, the fluorescent nuclide-containing agent can be administered in a single dose that delivers less than 12Gy, or less than 8 Gy, or less than 6 Gy, or less than 4 Gy, or less than 2 Gy, such as doses of 2 Gy to 8 Gy, to the subject, such as predominantly to the targeted PTPμ expressing cancer.

[0181] The amount of the disclosed fluorescent nuclide-containing agent, other agent described herein, and / or additional therapeutic agent administered to the subject can depend on the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to drugs as well as the degree, severity and type of rejection. The skilled artisan will be able to determine appropriate dosages depending on these and other factors using standard clinical techniques.

[0182] In addition, in vitro or in vivo assays can be employed to identify desired dosage ranges. The dose to be employed can also depend on the route of administration, the seriousness of the disease, and the subject's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The amount of the fluorescent nuclide-containing agent described herein can also depend on the disease state or condition being treated along with the clinical factors and the route of administration of the fluorescent nuclide-containing agent.

[0183] The disclosed fluorescent nuclide-containing agent described herein can be administered to the subject in conjunction with an acceptable pharmaceutical carrier or diluent as part of a pharmaceutical composition for therapy. Formulation of the fluorescent nuclide-containing agent to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule, and the like). Suitable pharmaceutically acceptable carriers may contain inert ingredients which do not unduly inhibit the biological activity of the compounds. The pharmaceutically acceptable carriers should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic and devoid of other undesired reactions upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington'sPharmaceutical Sciences, ibid. Suitable carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like. Methods for encapsulating compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al., "Controlled Release of Biological Active Agents", John Wiley and Sons, 1986).

[0184] The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically, such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. Formulation will vary according to the route of administration selected (e.g., solution, emulsion, capsule).

[0185] A pharmaceutically acceptable carrier for a pharmaceutical composition can also include delivery systems known to the art for entraining or encapsulating drugs, such as anticancer drugs. In some embodiments, the disclosed compounds can be employed with such delivery systems including, for example, liposomes, nanoparticles, nanospheres, nanodiscs, dendrimers, and the like. See, for example Farokhzad, O. C., Jon, S., Khademhosseini, A., Tran, T. N., Lavan, D. A., and Langer, R. (2004). "Nanoparticle- aptamer bioconjugates: a new approach for targeting prostate cancer cells." Cancer Res., 64, 7668-72; Dass, C. R. (2002). "Vehicles for oligonucleotide delivery to tumours." J. Pharm. Pharmacol., 54, 3-27; Lysik, M. A., and Wu-Pong, S. (2003). "Innovations in oligonucleotide drug delivery." J. Pharm. Sci., 92, 1559-73; Shoji, Y., and Nakashima, H. (2004). "Current status of delivery systems to improve target efficacy of oligonucleotides." Curr. Pharm. Des., 10, 785-96; Allen, T. M., and Cullis, P. R. (2004). "Drug delivery systems: entering the mainstream." Science, 303, 1818-22. The entire teachings of each reference cited in this paragraph are incorporated herein by reference.

[0186] The following examples are for the purpose of illustration only and are not intended to limit the scope of the claims, which are appended hereto. Example 1

[0187] In this example, we disclose a new theranostic that enables radiodiagnostic selection of patients who will benefit from a novel radiotherapeutic treatment. Specific tumor detection and treatment is a critically important goal in oncology. However, the challenge inimaging tumors is that conventional in methods suffer from a lack of specificity and limited tumor contrast. Magnetic Resonance imaging (MRI) is the primary imaging modality for brain tumors yet is incapable of specifically recognizing tumors or distinguishing between pseudoprogression, radiation necrosis, and actual tumor recurrence. Our strategy is to develop a positron emission tomography (PET) imaging agent to provide signal sufficient to specifically detect tumors and then utilize the same agent (with a matched therapeutic radionuclide) to treat these intractable tumors. Our precise targeting can deliver lethal radiation to neoplastic cells while sparing brain tissue that is otherwise healthy.

[0188] We discovered a novel molecular targeting strategy using extracellular fragments of protein tyrosine phosphatase type mu (PTPµ) abundantly expressed in the tumor microenvironment. We showed proof-of-concept by creating mouse models of human brain tumors; developed cryo-imaging methods to visualize and quantify tumor size, cell dispersal, invasion on white matter tracts and blood vessels density in an entire brain in 3D reconstructions. We discovered, using optical and MR imaging, that PTPµ-targeted agents: 1) label invading tumor cells far from the main tumor; 2) exhibit sustained tumoral retention compared to conventional untargeted agents; and 3) achieve specific recognition of the full extent of the tumor. Thus, PTPµ provides a targetable biomarker that specifically and comprehensively detects and could potentially treat brain tumors.

[0189] Our previous work generated an agent for fluorescent-guided resection of brain tumors. We successfully developed an agent with a near-infrared fluorophore (ICG) with fast binding kinetics (minutes) and a stable peak from 2-8 hours. The agent is still found in the tumor beyond 24 hours. Extensive studies identified the optimal NIR fluorophore and chemical composition required to achieve this biodistribution, which is ideal for a theranostic. That agent (PTPµ-ICG) was tested in a two species GLP toxicology study and found to be safe at 25X the dose that we will use for imaging. Recent studies using a gadolinium-DOTA conjugated PTPµ agent indicate that we can also detect tumors using MRI and that agent can be complexed with radioactive metals to generate a radiotheranostic. We have three radiopharmaceuticals that will be tested to detect and treat cancer: 1) PTPµ-Lys-DOTA complexed with64Cu; 2) PTPµ-ICG directly iodinated (124I / 131I); and 3) a combined PTPµ- DOTA-ICG complexed with64Cu for PET imaging. Many radiometals are complexed by DOTA including161Tb,225Ac and177Lu for therapy.

[0190] Using mouse models of brain tumors, it was discovered that PTPµ-targeted agents: 1) label the main tumor mass within minutes; 2) label 99% of dispersing tumor cells regardless of the distance from the main tumor mass in orthotopic rodent xenograft models; 3) exhibit sustained tumoral binding in comparison to conventional untargeted agents; and 4) achieve specific recognition of the full extent of the tumor including invading tumor cells far away from the main tumor mass that lead to recurrence.

[0191] We determined that the ideal biodistribution of the PTPµ agent occurred when it was conjugated to the fluorophore ICG made by AAT Bioquest. A 20-amino acid peptide (SBK2), recognizing the PTPµ-derived tumor-specific biomarker, was synthesized by PolyPeptides with or without a linker. The peptides were then conjugated to three different near-infrared fluorophores: indocyanine green (ICG), IRDye® 800CW, and Tide Fluor™ 8WS. The in vivo specificity, time course, and biodistribution were evaluated using mice with human glioma tumors to identify component combinations with optimal properties for fluorescent-guided surgery. SBK2 conjugated to ICG demonstrated excellent specificity for gliomas and showed significantly higher in vivo tumor labeling from 10 min to 24 h. Inserting a six-amino acid hydrophilic linker between the targeting peptide and ICG increased the clearance rate and resulted in significantly lower in vivo tumor signal. Agents made with the more hydrophilic IRDye® 800CW and Tide Fluor™ 8WS showed no specific tumor labeling relative to the controls (31). The optimal PTPµ-ICG agent now serves as one strategy for a radiotheranostic in this application due to its ideal imaging and biodistribution.

[0192] A molecular peptide-based MRI contrast agent (PTPµ-Lys-Gd-DOTA) was developed targeting the PTPµ biomarker. Contrast enhanced MRI involves injecting an agent to accentuate the visibility of soft tissue. Nonspecific contrast agents show enhanced flow through tumors due to their increased vascular support, which is called the enhanced permeability and retention (EPR) effect. However, when used with molecular imaging agents, MRI can identify molecular and physiological properties of tumors through sustained or extended retention in the target tissue via molecular interactions. PTPµ-DOTA is easy to synthesize using standard chemistry and allows efficient, selectable conversion to MRI (gadolinium) or SPECT / PET imaging (gallium, copper, actinium, lutetium, terbium) modalities by complexing with distinct metals. At NeoIndicate, we hypothesized that PTPµ- DOTA can be chelated to copper (64Cu) then combined with PET to visualize tumors with great sensitivity and determine on-target binding, off target binding and biodistribution.64Cuis in widespread use as an imaging agent this case serves as a surrogate for radiotherapeutic nuclides to achieve an effective dose and appropriate timing.

[0193] Due to extraordinary sensitivity (down to the picomolar level), deep tissue penetration, and their quantitative nature, radionuclide-based imaging provides high clinical relevance. Radionuclide imaging includes dynamic positron emission tomography (PET) for detection and monitoring of therapeutic efficacy in tumors using radiolabeled agents. PET / CT or PET / MRI are available at most major medical centers. Clinical translation and FDA approval of radiotracers has fewer hurdles since they qualify for FDA microdosing guidance. Based our mutual goal of moving this quickly to therapy64Cu was selected as our radionuclide of choice for its PET imaging capability. We will also be using124I since we can directly label the SBK2-ICG agent without the need for a chelator as chelators can sometimes effect biodistribution.

[0194] The most widely used PET tracer is18F-labeled 2-fluoro-2-deoxy-D-glucose (18F- FDG), a radiolabeled glucose analog, which identifies tumors by their increased glucose metabolism. However, some tumors utilize substrates other than glucose and well differentiated or slow-growing tumors often have low glucose metabolism. Clinicians need to be able to specifically detect primary, invasive and metastatic tumors independent of their metabolic rates. A problem for brain tumors with FDG-PET imaging is that normal brain has a very high glucose metabolic rate making tumor visualization challenging due to poor contrast and low specificity. Molecular PET imaging agents that specifically recognize tumor cells are necessary for improved specificity and sensitivity of detection and subsequent therapeutic efficacy studies. The most important advantage of our agents is they are theranostic, i.e., you can both detect and treat with either the same or different radionuclide (Fig.1). For example, the PTPµ binding peptide (SBK2) is synthesized once with a chelation moiety (DOTA) attached using cGMP manufacturing. The molecule can then be “loaded” at the local hospital with radionuclides like64Cu to image tumors (Fig.1). Treatment often utilizes a distinct radionuclide such as alpha or beta emitting radionuclide (161Tb,225Ac or177Lu). Our124 / 131I strategy is elementally matched. We aim to translate the PTPµ biomarker recognition agents for PET imaging that will detect and ultimately treat GBM as well as additional invasive and metastatic tumors inside and outside the brain

[0195] We have used the PTPµ agents in 20 distinct preclinical multi-modality imaging studies that highlight its specificity for tumor detection and target engagement using multiplefluorophores, nanoparticles and contrast Using a fluorescent PTPµ agent, we determined that the PTPµ fragment is common to GBM, labels human invasive tumor “edges”, demarcates tumor cells in tissue sections and is prognostic. We also tested the ability of the PTPµ agent to image tumors in vivo in glioma xenografts. Systemic injection of the agent results in rapid, specific labeling of tumors within minutes and it recognizes the main tumor mass and invasive tumor cells throughout the brain (Fig.3). GBM is characterized by a main tumor mass with robust neovascularization surrounded by pseudo- palisades of migrating cells far away from the primary tumor. Migrating glioma cells follow characteristic pathways along perivascular, perifascicular, perineural and neuronophagic, subpial or “surface”, and intrafascicular growth. Cryo-imaging allows a quantitative and comprehensive 3D view based upon sectioning of an organ or organism while imaging of the block face of the tissue with bright-field as well as fluorescent imaging. Algorithms reconstruct the images in 3D that are co-registered with data from other imaging modalities. We characterized xenograft glioma models that highlight different aspects of tumor cell biology and unique tumor microenvironments similar to human GBM patients (Fig.3). This series of xenograft glioma models are more “standardized” than human tumors in that the cells are genetically identical, GFP-labeled and implanted with the same number and location in each animal resulting in rigor and reproducibility when developing new methods. These models create a recognizable pattern with similar extents of invasion and timing of tumor formation. We utilized cryo-imaging to demonstrate that over 99% of tumor cells in the brain are labeled with an optical PTPµ agent including invading tumor cells far away from the main tumor mass that lead to recurrence (Fig.3).

[0196] We developed molecular PTPµ imaging agents that are easy to synthesize using standard peptide chemistry and allow efficient, selectable conversion to MRI or PET imaging modalities. Targeted radionuclide therapy has been translated to the clinic with commercially available agents223RaCl2 (Xofigo®), [131I]tositumomab (Bexxar®), [90Y]ibritumomab tiuxetan (Zevalin®), and, most recently, [177Lu]-PSMA-617 (Pluvicto®). The novel PTPµ- PET imaging agents would identify tumors with high sensitivity and specificity. Unlike other nonradioactive imaging agents, PET imaging agents can be administered at a microdose level, allowing for FDA approval with limited toxicology data. Notably, there is not yet a commercially-approved targeted molecular imaging agent for brain cancer. Oncologists, radiologists and radiation oncologists will use this technology to detect and monitor tumors inresponse to various therapies including where it is currently difficult to distinguish response to therapy from tumor recurrence. In addition, nuclear medicine physicians would use this agent to treat patients with the specific targeting and concentrating alpha- or beta-emitting radionuclides in and near the tumor. The PTPµ therapeutic will be competitive due to high levels of the product’s molecular target in tumors and selectivity for cancer over normal tissue. PTPµ is also simple to commercially synthesize and conjugate to fluorophores or DOTA with our CRO (PolyPeptides), which is expected to yield a low cost of goods.

[0197] The PTPµ agents specifically visualize tumors as well as invasive and metastatic lesions. The most important advantage of PTPµ agents is that you can achieve specific molecular recognition of cancer to target therapy directly to tumors to avoid off target side- effects.

[0198] We recently performed toxicology studies to determine the safety of the PTPµ- ICG agent. During our two pre-IND meetings, the FDA agreed that testing of a PTPµ peptide, at 25X the optimal dose for imaging in mice, is satisfactory for doing a limited 24- hour and 14-day rat toxicology study. Only this single dose was required. They also indicated toxicology testing on 5 animals of a second species will satisfy their additional toxicology requirements. Selection of Radionuclide Table131Direct conjugation to agent (no Iodine ( I) √ Beta chelator)and therapeutic radionuclides. We already produced the GMP PTPµ-ICG that will be used in the iodination reaction. We have achieved the desired biodistribution with this agent. However, the DOTA chelator also has many positive features including the fact that many metals are able to be used by simple on-site chelation. Our selection for diagnostic imaging is64Cu. For therapeutic intervention,161Tb,177Lu and225Ac are clinically viable strategies.

[0200] While225Ac and177Lu are in clinical use as radiotherapeutics for multiple cancer types and serve as obvious choices of radionuclides for DOTA chelation, terbium (161Tb) has recently received attention as a radiotheranostic (17-25).161Tb is a radiolanthanide with a half-life of 6.89 days, which emits β-particles (Eβ-av = 154 keV) for a therapeutic effect and γ- radiation (Eγ = 49 keV, I = 17.0%; Eγ = 75 keV, I = 10.2%) that allows PET imaging.161Tb is similar to177Lu but emits lower energy γ-radiation. However,161Tb emits a substantial number of low-energy conversion and Auger electrons, making this an ideal therapeutic for invasive and metastatic cancers. A 3.5-fold increase was observed in Monte Carlo simulations when using161Tb compared to177Lu resulting in a 1.3-fold higher absorbed electron energy fraction per decay for161Tb (197 keV / decay) compared to177Lu (147 keV / decay). The elementally matched149Tb is visible with PET imaging and DOTA is a good chelator for terbium facilitating translation of our previous MRI results with the PTPµ-Lys-DOTA (SBK2) agent into a radionuclide theranostic.

[0201] Translation of theranostic agents requires characterization and optimization then testing the efficacy of therapy in our brain tumor models. PTPµ-ICG will be synthesized, characterized, and optimized for targeting imaging and treatment of tumors in vivo. In this Aim, we will synthesize the PTPµ peptides and conjugate the fluorophore. Once produced,a) the stability of the complex in serum tested; b) determine the dose and timeframe to maximize the accumulation in glioma tumors in vivo; c) assess the biodistribution; and d) results will be compared to a scrambled control. We generated an engineering batch and demonstrated that PTPµ-ICG (SBK2-ICG) labels heterotopic and orthotopic brain tumors, and tumor labeling is still observed ex vivo (Fig.4).

[0202] The same agent that we originally complexed with gadolinium (Gd) for MRI (Fig.6), PTPµ-Lys-DOTA, and a new combined PTPµ-ICG-DOTA will be used for complexation with radioactive metals to make a PET imaging agent. We will examine the agents in both heterotopic and orthotopic brain tumors. For orthotopic tumors, we will image with PET / MRI and register to cryo-images to determine both sensitivity and specificity in the brain as well as transit of the compromised blood brain barrier. Chemistry of agents with DOTA

[0203] We generated PTPµ-Lys-DOTA with a single Gd ion for use in MRI that recognizes tumors at the clinical dose of 0.1mmol / kg (Fig.5). The addition of a single Lys- DOTA to the N-terminus allows easy synthesis using conventional peptide chemistry opening up the possibility of swapping of the Gd ion for the radioactive (e.g.,64Cu) ion, to convert the PTPµ agent to a PET agent. Polypeptides synthesizes engineering or cGMP batches of the peptides conjugated to the ICG fluorophore and / or DOTA. The ICG or ICG-DOTA is supplied by AAT Bioquest. 3D Imaging will also do direct iodination of PTPµ-ICG via an oxidation reaction. Conditions for chemical complexation will depend upon the radionuclide, while buffer conditions, pH and temperature are often varied to achieve the highest and most stable radiolabeling.

[0204] The imaging / treatment window will depend upon the half-life of the radionuclide. Ultimately, patient (mouse) treatment must have a reasonable treatment window to exert a therapeutic effect. The radionuclide is simply complexed with the DOTA chelator at room temperature in optimized buffer conditions that we have established recently. Over the past year, NeoIndicate and RAD completed some pilot experiments testing a PTPµ-NHS-DOTA agent. We performed preliminary radiolabeling of 3 distinct PTPµ-NHS-DOTA peptides and with greater than 95% labeling. Like the experiments described for the fluorophore project, the first agent we tested, PTPµ-NHS-DOTA, was too hydrophilic and cleared quickly. We are planning to use the PTPµ-Lys-DOTA now, which is less hydrophilic, that we used in previous MRI studies that had sufficient residence time (8). PTPµ-DOTA final products arecharacterized by mass spectrometry. purity is examined by thin-layer chromatography (radio-TLC) and radio-HPLC. Serum stability will be evaluated. We will confirm that the radiolabeling efficiency is >95% then no further purification is required. Chemistry of Direct Iodination

[0205] We are synthesizing a cGMP batch of PTPµ-ICG now. Ideally, we could use that material for direct iodination as it is already clinical grade. PTPµ-ICG has not previously been radiolabeled with124I. The usual site for radiolabeling of peptides with124I is on the activated aromatic ring of tyrosine. While SBK2-ICG does not have tyrosine, it does contain tryptophan which is considered an alternate labeling site, as well as the dye moiety which has activated rings that share some resemblance to histidine, another typical labeling site. Therefore, there is a good chance that direct radiolabeling with124I using methods commonly employed by 3D Imaging to label peptides and proteins will be successful. It can be determined with a short pilot experiment whether the radiolabeling will proceed using an oxidative electrophilic aromatic substitution using124I iodide and a suitable oxidant such as chloramine-T, iodobeads, or iodogen. The reaction is performed at room temperature at neutral pH. An alternative method is the iodine can be attached using the Bolton-Hunter reagent (essentially tyrosine succinimide without the amino group) which can react with a free amine as would any activated amino acid. Another alternative is to add a tyrosine to the N-terminus of SBK2 during synthesis for addition of the124I. We have already made an engineering batch of the Tyr-SBK2. Imaging Studies

[0206] Two different tumor lines will be used to assess the imaging of the PTPµ agents on heterotopic flank tumor models of human glioma to ensure that the observed results are not only seen in one model system. These glioma models have been credentialed and validated using STR DNA Profiling as outlined in Authentication. We will utilize heterotopic flank tumors due to their size, long window for imaging studies due to the stability of the tumors over time, ease of monitoring and visualization as well as testing (re- testing) of multiple agents once the agent has decayed. Furthermore, it is easy to measure therapeutic effects such as changes in tumor size in heterotopic tumors with calipers. We will implant the glioma cell lines U87-MG or LN-229 into flanks.

[0207] PET scans will be obtained for mouse after 15-21 days. Mice bearing flank tumors will be injected into the tail vein with 150-200µCi dose of PTPµ agents or a scrambled peptide control agent. A cohort of male and female mice will be evaluated with whole-body micro-PET / MR imaging [n=12 / group (6M / 6F) x 2 human glioma models x 2 groups (scrambled, PTPµ)] per agent. Dynamic PET scans will determine the binding rate and the biodistribution over ~1-2 hours after intravenous injection of the PTPµ-targeted and untargeted PET agents to compare intra-tumoral binding and retention. Dosimetry will be performed in consultation with Dr. Muzic. Static scans at 5 minutes, 15 minutes, 30 minutes, 1 hour, 4 hours and 20 hours post-injection will allow the radionuclide clearance from the non-targeted tissues for increased tumor-to-background uptake ratio. Biodistribution will be observed from the PET images and by euthanizing a subset of animals for each condition followed by ex vivo analysis and scintigraphy.

[0208] A MR scan will be performed using the PET / MR system for overlay display of PET / MR images. These image frames will be reconstructed using a vendor-supplied algorithm and will be overlaid on the aligned MR scans for region of interest (ROI) definition. Uptake in tumor ROI will be calculated as standardized uptake value (SUV) along the time frames, which is the radioactivity concentration within tissue normalized to injected dose and body weight. In addition, an ROI in the muscle will be defined for background uptake, and SUVtumor / SUVbackground ratio will be calculated for all time points. We will also validate the radionuclide activity concentrations in tumors by counting after euthanization at the end of a final scan. Blood, urine and organs will be removed and compared to control animals. Tissue samples will be collected to measure radioactivity concentrations by gamma counting. Tumor samples will also be preserved, fixed in formalin and embedded in paraffin for histopathology correlation with in vivo imaging data. We expect the PTPµ-targeted agents to more sensitively and specifically label and exhibit sustained retention times compared to the scrambled agent due to our extensive published results. If unexpected problems occur, additional PTPµ agents such as other SBK peptides or recombinant antibodies available in our collection of PTPµ reagents will be tested. Quantitative Metrics

[0209] A major quantitative metric for the aims is dosimetry including determining if the absorbed dose (measured in Gy) in the tumor meets therapeutic levels while the dose to Organs at Risk (OARs) is lower than the constraints. Pharmacokinetic parameters such astemporal resolution, absorbed dose, in the tumor, concentration in OARs, concentration over time with imaging as well as “cut and count” methods will be additional quantitative metrics. On-target labeling, off-target labeling (OARs), specificity, sensitivity, kinetics of binding / time course analysis, binding at key imaging time points, biodistribution, clearance and residence time for imaging will be additional quantitative milestones. There will likely be a trade-off between tumor labeling and organs at risk to preserve their normal function. The optimal effects depend upon time to wash out or a long residence time in tumor are key quantitative parameters. The measurements of concentration versus time allow integration of the area under curve (AUC), which permit calculation of absorbed dose. The tumor signal has to be minimally 2X background for imaging and often 20X for a therapeutic. We will utilize OLINDA / EXM 2 software, which has mouse dosimetry. For quantitation, we draw regions of interest in the image, measure the activity concentration vs. time and calculate the AUC, residence time, and the absorbed dose. We then extrapolate dose from mice to humans as described. Example 2

[0210] Fluorescence-guided resection (FGR) using non-specific agents has improved patient outcomes, particularly in the case of glioblastoma. Molecularly-targeted agents that recognize specific tumor biomarkers may further improve FGR. We synthesized a 20-amino acid peptide (SBK2) recognizing the receptor protein-tyrosine phosphatase mu (PTPmu)- derived tumor-specific biomarker. We added a near-infrared fluorophore called indocyanine green (ICG). A further modification was made to add the metal chelator DOTA. Two different versions were synthesized as shown in Fig.7. A scrambled peptide (Scram-ICG- DOTA) was used as a control. The in vivo tumor specificity, time course and biodistribution were evaluated for each agent using mice with heterotopic, PTPmu biomarker-expressing human glioma tumors.

[0211] SBK2 conjugated to either form of ICG-DOTA demonstrated excellent specificity for gliomas in heterotopic tumors. SBK2-ICG-DOTA showed significantly higher in vivo tumor labeling compared to control from 10min to 24h following injection. The second version of SBK2 (SBK2-Lys(DOTA)-ICG) also showed good tumor specificity but the labeling was not quite as strong as SBK2-ICG-DOTA and seemed to clear a little more quickly.

[0212] Fig.8 illustrates images vivo tumor labeling of LN229 flank tumors with ICG-DOTA conjugated peptides over time. Flank tumor-bearing mice were injected with 300nmol / kg of scrambled control peptide (Scram-ICG-DOTA) or SBK2-ICG-DOTA. Fluorescent images were acquired at the indicated times and are shown alongside baseline (BL) images acquired prior to the start of the experiment for a representative set of animals. The average radiant efficiency scale is shown on the right. Red corresponds to the highest intensity signal.

[0213] Fig.9 illustrates images showing in vivo tumor labeling of LN229 flank tumors with ICG-DOTA conjugated peptides over time. Flank tumor-bearing mice were injected with 300nmol / kg of SBK2-Lys(DOTA)-ICG or SBK2-ICG-DOTA. Fluorescent images were acquired at the indicated times and are shown alongside baseline (BL) images acquired prior to the start of the experiment for a representative set of animals. The average radiant efficiency scale is shown on the right. Red corresponds to the highest intensity signal.

[0214] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.

Claims

Having described the invention, the claimed:

1. An agent comprising: a targeting peptide that specifically binds to and / or complexes with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule that is expressed by a cancer cell or another cell in the cancer cell microenvironment; an optional spacer directly linked to the targeting peptide; a fluorophore that is directly or indirectly linked to the targeting peptide or optional spacer; and an optional chelating agent directly or indirectly linked to the targeting peptide, optional spacer, or fluorophore; wherein at least one of the targeting peptide, optional spacer, or fluorophore is directly labeled with a nuclide and / or the optional chelating agent includes a chelated nuclide.

2. The agent of claim 1, including an amide linkage that links the targeting peptide or optional spacer to the fluorophore.

3. The agent of claim 1 or 2 having the formula (I): a pharmaceutically acceptable salt thereof;targeting peptide; R2is absent or the optional spacer, which is optionally directly labeled with the nuclide; R3is the fluorophore; R4or R5are each independently absent or an optional chelating agent with an optional linker; wherein one of R4or R5is the optional chelating agent with the optional linker if R2is not directly labeled with the nuclide; alternatively both of R4and R5are absent and R2is present and is directly labeled with a nuclide; andwherein NH is an amino the spacer or the targeting peptide and C=O is a carboxyl group of the fluorophore.

4. The agent of claim 3 having the formula (II): (II) or a pharmaceutically acceptable salt thereof; wherein R1includes the targeting peptide; R3is the fluorophore; R4is a chelating agent with an optional linker; wherein NH is an amino group of the targeting peptide and C=O is a carboxyl group of the fluorophore.

5. The agent of claim 3 having the formula (III): a pharmaceutically acceptable salt thereof;the targeting peptide; R2is the spacer; R3is the fluorophore; R5is the chelating agent with an optional linker; and wherein NH is an amino group of the spacer or the targeting peptide and C=O is a carboxyl group of the fluorophore.

6. The agent of claim 1 or 2 having the formula (IV): (IV) or a pharmaceutically acceptable salt thereof;includes the targeting peptide; R2is the spacer, which is directly labeled with the radionuclide; R3is the fluorophore; andwherein NH is an amino the spacer or the targeting peptide and C=O is a carboxyl group of the fluorophore.

7. The agent of any of claims 1 to 6, wherein the fluorophore is hydrophobic or lipophilic.

8. The agent of any of claims 1 to 7, wherein the fluorophore includes at least one of a cyanine near-infrared fluorophore having a fluorescence in the first near-infrared region or second near-infrared region.

9. The agent of claim 8, wherein the cyanine near-infrared fluorophore is a heptamethine cyanine near-infrared fluorophore.

10. The agent of any of claims 1 to 9, wherein the fluorophore includes at least one of indocyanine green (ICG) or ICG-Osu.

11. The agent of any of claims 1 to 10, wherein the fluorophore is directly labeled with a nuclide.

12. The agent of any of claims 1 to 11, wherein the optional spacer is an optional peptide spacer optionally directly labeled with the nuclide.

13. The agent of any of claims 1 to 12, wherein the optional spacer includes an amino acid residue, such as tyrosine, directly labeled with the nuclide.

14. The agent of any of claims 1 to 13, where the targeting peptide has the amino acid sequence of GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 5) or GIDVRDAPLKEIKVTSSR (SEQ ID NO: 7).

15. The agent of any of claims 1 to 14, wherein the chelating agent is configured to chelate a diagnostic or therapeutic nuclide.

16. The agent of any of claims wherein the diagnostic or therapeutic nuclide includes at least one of Ga, I, In, Y, Lu, Bi, Ac, Re, In, Th, Tc, Tl, Tb, Zr, Cu, Rb, At, Pb, Gd, Sm, or Sr.

17. The agent of claim 16, wherein the diagnostic nuclide is a diagnostic radionuclide selected from123I,124I,125I,64Ga,18F,11C,13N,76Br,149Tb,161Tb,99mTc,153Gd,111In,67Ga,68Ga,201Tl,82Rb,64Cu,67Cu,89Zr,90Y, T(tritium),149Tb,161Tb,153Sm, or89Sr.

18. The agent of claim 16, wherein the therapeutic nuclide is a therapeutic radionuclide selected from131I,225Ac,226Ac,227Th,211Bi,212Bi,213Bi,203Pb,212Pb, or177Lu.

19. The agent of any of claims 1 to 18, wherein the chelating agent is selected from 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10- tetraazadodecanetetraacetate (DOTA), 2,2’,2”-(10-(pyridin-2-ylmethyl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-1Py), 2,2’-(7,10-(pyridin-2- ylmethyl)-1,4,7,10-tetraazacyclododecane-1,4-diyl) diacetic acid (DOTA-2Py), 2-(4,7,10-tris (pyridin-2-ylmethyl)-1,4,7,10-tetraazacyclododecane-1-yl) acetic acid (DOTA-3Py), 1,4,7,10-tetraazadodecane-1,4,7-triacetate (DO3A), ethylenediaminetetraacetate (EDTA), 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane- 1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazadodecanetetramethylacetate (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetate (DO3MA), N,N',N'',N'''- tetraphosphonatomethyl-1,4,7,10-tetraazacyclododecane (DOTP), 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetrakis(methylene methylphosphonic acid) (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phenylphosphonic acid) (DOTPP), N,N'-ethylenedi-L-cysteine, S-2-(4-isothiocyanatobenzyl)-1,4,7,10- tetraazacylododecane tetracetic acid (p-SCN-Bn-DOTA), 2-(4-isothiocyanatobenzyl- 1,4,7,10-tetraaza-1,4,7,10,tetra-(2-carbamonylmethyl)-cyclododecane (p-SCN-Bn-TCMC), MeO-DOTA-NCS, [(R)−2-Amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)- cyclohexane-1,2-diamine-pentaacetic acid (CHX-A’’-DTPA-NCS), 2-[4-nitrobenzyl]- 1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N'''' -pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N''''-hexaacetic acid (HEHA), desferrioxamine B (DFO), macropa, macropa-NCS, macropid, bispa2, EuK-106, 7-[2-(bis-carboxymethyl-amino)-ethyl]-4,10-bis- 1,4,7,10-tetraaza-cyclododec-1-yl- acetic acid (DEPA), 3p-C-DEPA, or derivatives thereof.

20. The agent of claim 1, including compounds selected from: ;(VII); wherein R1is the targeting peptide; R6and R7are each absent or a nuclide; and at least one of R6or R7is a nuclide.

21. The agent of claim 20, wherein R6and / or R7is selected from123I,124I,125I, or131I.

22. The agent of claim 20, wherein compounds (VI) and (VII) or pharmaceutically acceptable salts thereof further including a chelated diagnostic or therapeutic nuclide.

23. The agent of claim 20, wherein the diagnostic or therapeutic nuclide includes at least one of Ga, I, In, Y, Lu, Bi, Ac, Re, In, Th, Tc, Tl, Tb, Zr, Cu, Rb, At, Pb, Gd, Sm, or Sr.

24. The agent of claim 23, wherein the diagnostic nuclide is a diagnostic radionuclide selected from123I,124I,125I,64Ga,18F,11C,13N,76Br,149Tb,161Tb,99mTc,153Gd,111In,67Ga,68Ga,201Tl,82Rb,64Cu,67Cu,89Zr,90Y, T(tritium),149Tb,161Tb,153Sm, or89Sr.

25. The agent of claim 23, wherein the therapeutic nuclide is a therapeutic radionuclide selected from131I,225Ac,226Ac,227Th,211Bi,212Bi,213Bi,203Pb,212Pb, or177Lu.

26. The agent of any of claims 25, wherein R1has the amino acid sequence of GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 5) or GIDVRDAPLKEIKVTSSR (SEQ ID NO: 7).

27. The agent of any of claims 1 to 26, wherein the agent administered to a subject has a signal to background ratio (SBR) upon imaging effective to delineate the cancer cell or another cell in the cancer cell microenvironment from surrounding tissue.

28. The agent of any of claims 1 to 27, for use in detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, dispersal, and / or invasion, and / or for treating cancer in a subject.

29. A complex mixture comprising a plurality of the agents of any of claims 1 to 27, and optionally other agents structurally similar to the agents of any of claims 1 to 27 but devoid of a diagnostic and / or therapeutic nuclide and / or chelated nuclide.

30. The complex mixture of claim 29, further comprising a pharmaceutically acceptable carrier.

31. A method of detecting cancer cells and / or cancer cell metastasis, migration, dispersal, and / or invasion in a subject in need thereof, the method comprising: administering to the subject the complex mixture of claim 29 or 30; and detecting the agents and optionally the other agents devoid of a diagnostic and / or therapeutic nuclide and / or chelated nuclide bound to and / or complexed with the cancer cells to determine the location and / or distribution of the cancer cells in the subject.

32. The method of claim 31, the cancer cells comprising at least one of a glioma, lung cancer, melanoma, breast cancer, ovarian cancer, endometrial cancer, or prostate cancer cell.

33. The method of claim 31 or 32, the complex mixture being administered systemically, locally, or topically to the subject.

34. The method of any of to 33, the agents and optionally the other agents being detected to define a tumor in a subject.

35. A method of treating cancer cells and / or cancer cell metastasis, migration, dispersal, and / or invasion in a subject in need thereof, the method comprising: administering to the subject an amount of the complex mixture of claim 29 or 30 effective to ablate the cancer; and optionally detecting the agents and optionally the other agents devoid of a diagnostic and / or therapeutic nuclide and / or chelated nuclide bound to and / or complexed with the cancer cells to determine the location and / or distribution of the cancer cells in the subject.

36. The method of claim 35, the cancer cells comprising at least one of a glioma, lung cancer, melanoma, breast cancer, ovarian cancer, endometrial cancer, or prostate cancer cell.

37. The method of claim 35 or 36, the complex mixture being administered systemically, locally, or topically to the subject.

38. The method of any of claims 35 to 37, the agents and optionally the other agents being detected to define a tumor in a subject.

39. The method of any of claims 35 to 38, further comprising surgically resecting the cancer cells.

40. The method of claim 39, further comprising ablating remaining or residual cancer cells after surgical resection.

41. Use of the agents of any of claims 1 to 27 or a complex mixture of claim 29 or 30 in a fluorescent image-guided surgery, imaging and / or targeted radionuclide diagnosis or therapy.

42. The agent of any of claims or the complex mixture of claim 29 or 30 for use in the preparation of a medicament for fluorescent image-guided surgery, imaging and / or targeted radionuclide diagnosis or therapy.

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