Methods and reagents for detecting and treating cancer

Abstract

An agent for detecting, monitoring and / or imaging cancer cells and / or cancer cell metastasis, migration, spread and / or invasion and / or for treating cancer in a subject, comprising a targeting peptide, at least one of a detectable moiety, a therapeutic agent or a diagnostic agent, and a peptide or peptidomimetic spacer directly or indirectly linking the targeting peptide to the at least one of a detectable moiety, a therapeutic agent or a diagnostic agent.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 062,053, filed August 6, 2020, the subject matter of which is incorporated herein by reference in its entirety. BACKGROUND

[0003] Cancer detection and treatment are hindered by the inability to distinguish cancer cells from normal cells. Early diagnosis of cancer requires better cancer detection tools or tumor imaging. Molecular recognition of tumor cells would help guide surgical resection. To improve surgical resection, targeted imaging tools must specifically label tumor cells, not only in the main tumor, but also in the tumor margin and small clusters of tumor cells dispersed throughout the body.

[0004] Targeted imaging tools designed to label molecules that accumulate in the tumor microenvironment can also be advantageous as therapeutic targeting agents, as they can identify the main tumor cell population and areas with infiltrating cells that lead to tumor recurrence. The ability to directly target tumor cells and / or their microenvironment would improve the specificity and sensitivity of current treatments, thus reducing the non-specific side effects of chemotherapy drugs that affect cells throughout the body. SUMMARY

[0005] Embodiments described herein relate to an agent and its use in detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, spread, and / or invasion and / or treating cancer in a subject. The agent can include a targeting peptide that specifically binds and / or complexes with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule expressed by a cancer cell or another cell in a cancer cell microenvironment, at least one of a detectable moiety, a therapeutic agent, or a theranostic agent, and a peptide or peptoid spacer that directly or indirectly links the targeting peptide to the at least one of a detectable moiety, a therapeutic agent, or a theranostic agent. The peptide or peptoid spacer has a length and structure that is effective to at least maintain or preserve the binding affinity of the linked targeting peptide to the proteolytically cleaved extracellular fragment and the activity of the linked at least one of a detectable moiety, a therapeutic agent, or a theranostic agent.

[0006] In some embodiments, the agent is configured for in vivo administration to a subject or ex vivo administration to a biological sample of a subject.

[0007] In some embodiments, the spacer includes natural amino acids and / or non-natural amino acids.

[0008] In other embodiments, the spacer comprises at least 3 natural amino acids or non-natural amino acids. For example, the spacer can be 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, or 30 natural amino acids or non-natural amino acids in length.

[0009] In some embodiments, the spacer comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% glycine residues and / or serine residues.

[0010] In other embodiments, the spacer comprises at least 50%, at least 60%, at least 70%, or at least 80% glycine residues.

[0011] In some embodiments, the spacer is a polyglycine spacer or a glycine / serine spacer.

[0012] In some embodiments, the spacer comprises the amino acid sequence of at least one of (GS)a, (GGS)b, or (GGGS)c or (GGGGS)d, and wherein a, b, c, and d are each independently 2, 3, 4, 5, or 6. For example, the spacer can have the following amino acid sequences: GGG (SEQ ID NO: 9), GGGG (SEQ ID NO: 10), GGGGG (SEQ ID NO: 11), GGGGGG (SEQ ID NO: 12), GGGGGGG (SEQ ID NO: 13), GGGGGGGG (SEQ ID NO: 14), GGGGGGGGG (SEQ ID NO: 15), GSGS (SEQ ID NO: 16), GSGSGS (SEQ ID NO: 17), GSGSGSGS (SEQ ID NO: 18), GSGSGSGSGS (SEQ ID NO: 19), GGSGGS (SEQ ID NO: 20), GGSGGSGGS (SEQ ID NO: 21), GGSGGSGGSGGS (SEQ ID NO: 22), GGGSGGGS (SEQ ID NO: 23), GGGSGGGSGGGS (SEQ ID NO: 24), GGGSGGGSGGGSGGGS (SEQ ID NO: 25), GGGGSGGGGS (SEQ ID NO: 26), or GGGGSGGGGSGGGGS (SEQ ID NO: 27).

[0013] In some embodiments, the agent further comprises at least one coupling agent that connects the spacer to the targeting peptide and / or at least one of: a detectable moiety, a therapeutic agent, or a diagnostic agent.

[0014] In some embodiments, the cell adhesion molecule can comprise a cell surface receptor protein tyrosine phosphatase (PTP) type IIb.

[0015] In some embodiments, the extracellular fragment can comprise the amino acid sequence of SEQ ID NO: 2, and the targeting peptide can comprise a polypeptide that specifically binds and / or complexes with SEQ ID NO: 2.

[0016] In some embodiments, the targeting peptide can comprise a polypeptide having an amino acid sequence with at least 80% sequence identity to about 10 to about 50 contiguous amino acids of SEQ ID NO: 3.

[0017] In other embodiments, the targeting peptide can comprise a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0018] In some embodiments, the detectable moiety can comprise a chelator, a contrast agent, an imaging agent, a radiolabel, a semiconductor particle, a nanoparticle, a nanobubble, or a nanochain. The detectable moiety can be detectable by at least one of magnetic resonance imaging (MRI), positron emission tomography (PET) imaging, computed tomography (CT) imaging, gamma imaging, near-infrared imaging, ultrasound, or fluorescence imaging.

[0019] In some embodiments, the diagnostic agent or the therapeutic agent comprises at least one of a photosensitizer, an ultrasound sensitizer, a heat sensitizer, a radiosensitizer, a radiotherapy agent, a chemotherapy agent, or an immunotherapy agent.

[0020] In some embodiments, the cancer detected or treated with the agent can be any type of cancer, including but not limited to bone cancer, bladder cancer, brain cancer, neuroblastoma, breast cancer, cancer of the urinary tract, carcinoma, cervical cancer, astrocytoma, brain stem glioma, glioblastoma, neuroendocrine tumor, NCS atypical teratoid / rhabdoid tumor, CNS embryonal tumor, CNS germ cell tumor, craniopharyngioma, ependymoma, kidney tumor, acute lymphoblastic leukemia, acute myeloid leukemia, and other types of leukemia; Hodgkin lymphoma, non-Hodgkin lymphoma, Ewing sarcoma, osteosarcoma and malignant fibrous histiocytoma of bone, rhabdomyosarcoma, soft tissue sarcoma, Wilms' tumor, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, hepatocellular cancer, liver cancer, lung cancer, lymphoma and leukemia, melanoma, ovarian cancer, endometrial cancer, pancreatic cancer, pituitary cancer, prostate cancer, rectal cancer, renal cancer, sarcoma, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer.

[0021] In some embodiments, the cancer cells can be, for example, metastatic, migratory, invasive, and / or aggressive cancer cells, such as metastatic, migratory, invasive, and / or aggressive brain cancer cells (e.g., glioma cells, particularly, glioblastoma multiforme (GBM) cells), lung cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, endometrial cancer cells, and / or melanoma.

[0022] Other embodiments described herein relate to a method of detecting cancer cells and / or cancer cell metastasis, migration, invasion, and / or aggression in a subject in need thereof. The method includes administering to the subject an amount of an agent described herein, wherein the agent comprises a diagnostic agent or a theranostic agent. The agent bound and / or complexed with the cancer cells can be detected to determine the location and / or distribution of the cancer cells in the subject.

[0023] In some embodiments, the cancer cells comprise at least one of glioma, lung cancer, melanoma, breast cancer, ovarian cancer cells, endometrial cancer cells, or prostate cancer cells.

[0024] In some embodiments, the agent can be administered systemically to the subject.

[0025] In some embodiments, the agent can be detected to determine the tumor margin of the subject.

[0026] Other embodiments described herein relate to a method of treating cancer in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of an agent described herein, which comprises a therapeutic agent or a theranostic agent.

[0027] In some embodiments, the therapeutic or diagnostic agent is a photosensitizer, radiosensitizer, or radiotherapeutic agent, and the method can further comprise irradiating the cancer cells with which the agent is bound or internalized, thereby inducing photosensitization or radiosensitization of the photosensitizer or radiotherapeutic agent and apoptosis and / or necrosis of the cancer cells. Photosensitizers can include, for example, porphyrin, biscarbocyanine, or pthalocyanine compounds.

[0028] In other embodiments, the therapeutic or diagnostic agent is a nanobubble, and the method can further comprise sonicating the nanobubbles bound to the cancer cells or internalized by the cancer cells with ultrasound energy effective to promote inertial cavitation and apoptosis and / or necrosis of the cancer cells and / or release of a chemotherapeutic drug to the cancer cells. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 A graph showing the in vivo average radiance efficiency of a first agent with no peptide spacer, a second agent with a peptide spacer, and a control agent administered to mice bearing ectopic xenograft flank U87 tumor implants is shown.

[0030] Figure 2 A graph showing the in vivo average radiance efficiency of a second agent with a peptide spacer and a third agent with a different peptide spacer administered to mice bearing ectopic xenograft U87 flank tumor implants or mice bearing ectopic xenograft flank U87 tumor implants overexpressing PTPmu is shown.

[0031] Figure 3 A graph showing the in vivo average radiance efficiency of a third agent with a peptide spacer and a fourth agent with a different peptide spacer administered to mice bearing ectopic xenograft U87 flank tumor implants or mice bearing ectopic xenograft flank U87 tumor implants overexpressing PTPmu is shown.

[0032] Figure 4 Ex vivo images and graphs showing the average radiance efficiency of a first agent and a control agent administered in vivo to mice bearing ectopic xenograft U87 flank tumor implants are shown.

[0033] Figure 5 Ex vivo images and graphs showing the average radiance efficiency of a second agent, a third agent, and a control agent administered in vivo to mice bearing ectopic xenograft U87 flank tumor implants are shown.

[0034] Figure 6 Ex vivo images and graphs showing the average radiance efficiency of a second agent, a third agent, and a control agent administered in vivo to mice bearing ectopic xenograft U87 flank tumor implants overexpressing PTPmu are shown.

[0035] Figure 7 Ex vivo images and graphs showing the mean radiant efficiency of the third agent, the fourth agent, and the control agent after being administered in vivo to mice bearing ectopic xenograft U87 flank tumor implants with overexpression of PTPmu.

[0036] Figure 8 Ex vivo images and graphs showing the mean radiant efficiency of the first agent after being administered in vivo to mice bearing orthotopic xenograft U87 intracranial tumors compared to the control agent

[0037] Figure 9 Ex vivo images and graphs showing the mean radiant efficiency of the third agent after being administered in vivo to mice bearing orthotopic xenograft U87 intracranial tumors compared to the control agent.

[0038] Figure 10 Ex vivo images of orthotopic xenograft U87 intracranial tumors or orthotopic xenograft LN229 intracranial tumors after the third agent, the fourth agent, and the control agent were administered in vivo to mice.

[0039] Figure 11 Ex vivo maestro images overlaid on black and white photographs of the brain after the third agent, the fourth agent, and the control agent were administered in vivo to mice.

[0040] Figure 12 Graphs showing the maximum signal intensity of the third agent, the fourth agent, and the control agent after being administered in vivo to mice bearing orthotopic xenograft U87 intracranial tumors. DETAILED DESCRIPTION

[0041] Methods involving routine molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detailed in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (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 this application belongs. Commonly understood definitions of molecular biology terms can be found, for example, in 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.

[0042] The articles "a" and "an" are used herein 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.

[0043] The terms "comprise," "comprising," "include," "including," and "have," "having" are used in the inclusive, open sense, meaning that other elements can be included. The term "such as," as used herein, is non-limiting and is merely used to illustrate a possible embodiment. "Include" and "including" are interchangeable with "comprise" and "comprising."

[0044] As used herein, the term "or" is understood to mean "and / or" unless the context clearly indicates otherwise.

[0045] The term "reagent" is used herein to mean a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material.

[0046] The term“cancer” or“tumor” refers to any neoplastic growth in a subject, including the original tumor and any metastases. The cancer can be a liquid or solid tumor type. Liquid tumors include tumors of blood origin, including, for example, myelomas (e.g., multiple myeloma), leukemias (e.g., Waldenstrom’s syndrome, chronic lymphocytic leukemia, other leukemias), and lymphomas (e.g., B-cell lymphoma, non-Hodgkin’s lymphoma). Solid tumors can originate in organs and include cancers of the lung, brain, breast, prostate, ovary, uterus, colon, kidney, and liver.

[0047] The term“cancer cell” or“tumor cell” can refer to a cell that is dividing at an abnormal (i.e., increased) rate. Cancer cells include, but are not limited to, epithelial cancers 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, hepatocellular carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, breast cancer, gastrointestinal cancer, colon cancer, bladder cancer, prostate cancer, and squamous cell carcinoma of the neck and head region; sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synovial sarcoma, and mesotheliosarcoma; hematological cancers such as myeloma, leukemia (e.g., acute myelogenous leukemia, chronic lymphocytic leukemia, granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), lymphoma (e.g., follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, malignant lymphoma, plasmacytoma, reticulum cell sarcoma, or Hodgkin’s disease), and nervous system tumors including glioma, glioblastoma multiforme, meningioma, medulloblastoma, schwannoma, and epidymoma.

[0048] The term“chimeric protein” or“fusion protein” is a fusion of a first amino acid sequence encoding a polypeptide with a second amino acid sequence defining a domain (e.g., polypeptide portion) foreign and substantially heterologous to any domain of the first polypeptide. The chimeric protein can exist in a foreign domain found in an organism that also expresses the first protein (albeit in a different protein), or it can be a fusion of protein structures expressed by different species of organisms, “interspecies,”“intergenic,” etc.

[0049] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0050] The term "gene" or "recombinant gene" refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including exon and (optionally) intron sequences.

[0051] The terms "homology" and "identity" are used synonymously herein and refer to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in the two sequences, which can be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. A degree of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0052] The term "mutant" refers to any change in the genetic material of an organism, particularly 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." While it is generally accepted that a change in genetic material results in a change in the function of a protein, the terms "mutant" and "variant" refer to a change in the sequence of a wild-type protein, regardless of whether the change alters the function of the protein (e.g., increases, decreases, imparts a new function), or whether the change has no effect on the function of the protein (e.g., the mutation or variation is silent).

[0053] The term "nucleic acid" refers to polynucleotides, such as deoxyribonucleic acid (DNA), and where appropriate ribonucleic acid (RNA). The term is also understood to include (as equivalents) analogs of either RNA or DNA made from nucleotide analogs and is applicable to single- stranded polynucleotides (sense or antisense) and double-stranded polynucleotides.

[0054] The phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than oral and topical administration, injection, and includes, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardiac, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0055] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" mean that the compound, agent, or other material is administered so that it enters the system of the animal and thus undergoes metabolism and other similar processes (e.g., subcutaneous administration), rather than being administered directly to a particular tissue, organ, or region (e.g., the brain) of the subject being treated.

[0056] The terms "patient," "subject," "mammalian host," and the like are used interchangeably herein and refer to a mammal, including human subjects and veterinary subjects.

[0057] The terms "peptide," "protein," and "polypeptide" are used interchangeably herein. As used herein, "polypeptide" refers to any peptide or protein (i.e., a peptide isomer) comprising two or more amino acids linked to one another by peptide or modified peptide bonds. "Polypeptide" refers to both short chains, generally referred to as peptides, oligopeptides, or oligomers, as well as longer chains generally referred to as proteins.

[0058] The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

[0059] As used herein, "recombinant" means that the protein is derived from a prokaryotic or eukaryotic expression system.

[0060] The terms "therapeutic agent," "drug," "medicament," and "biologically active substance" are art-recognized and include molecules and other agents that are substances having biological, physiological, or pharmacological activity that act locally or systemically in a patient or subject to treat a disease or condition. The term includes, but is not limited to, pharmaceutically acceptable salts and prodrugs thereof. Such agents can be acidic, basic, or salts; they can be neutral molecules, polar molecules, or molecular complexes capable of hydrogen bonding; they can be prodrugs in the form of ethers, esters, amides, and the like that are biologically active when administered to a patient or subject.

[0061] The phrase "therapeutically effective amount" or "pharmaceutically effective amount" is an art-recognized term. In certain embodiments, the term refers to the 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 the amount required to eliminate, reduce, or maintain a target of a particular therapeutic regimen, or an amount sufficient to eliminate, reduce, or maintain a target of a particular therapeutic regimen. The effective amount can vary depending on such factors as the disease or condition being treated, the particular targeted construct being administered, the size or disease or condition severity of the subject, and the like. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without undue experimentation. In certain embodiments, the therapeutically effective amount of a therapeutic agent for in vivo use can depend on a number of factors, including: the rate at which the agent is released from the polymeric matrix, which depends in part on the chemical and physical properties of the polymer; the properties of the agent; the mode and method of administration; and any other materials incorporated into the polymeric matrix in addition to the agent.

[0062] The term "wild-type" refers to a naturally occurring polynucleotide sequence encoding a protein or portion thereof, or a protein sequence or portion thereof, as is typically found in vivo.

[0063] Throughout this specification, 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, processes or process steps are described as having, including, or comprising specific process steps, it is contemplated that the processes also consist essentially of, or consist of, the recited process steps. Further, it shall be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods remain operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0064] Embodiments described herein relate to agents for detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, spread, and / or invasion in a subject, methods of detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, spread, and / or invasion in a subject, methods of determining and / or monitoring efficacy of a cancer treatment and / or cancer therapy administered to a subject in need thereof, and methods of using the agents to treat cancer in a subject in need thereof.

[0065] The agents described herein include a targeting peptide that specifically binds and / or complexes with an extracellular fragment proteolytically cleaved from an immunoglobulin (Ig) superfamily cell adhesion molecule expressed by a cancer cell or an endothelial cell in a cancer cell microenvironment, which supports survival of the cancer cell, at least one of a detectable moiety, a therapeutic agent, or a diagnostic and therapeutic agent, and a peptide or peptoid spacer that directly or indirectly links the targeting peptide to the at least one of a detectable moiety, a therapeutic agent, or a diagnostic and therapeutic agent.

[0066] It has been discovered that the peptide or peptidomimetic spacer that directly or indirectly links the targeting peptide to at least one of a detectable moiety, a therapeutic agent, or a theranostic agent can be selected to have a certain length and structure that is effective to at least maintain, preserve, or not interfere with the binding affinity of the linked targeting peptide to the proteolytically cleaved extracellular fragment and the activity of the linked at least one of a detectable moiety, a therapeutic agent, or a theranostic agent. The activity of the detectable moiety or the theranostic agent means, for example, the ability of the detectable moiety or the theranostic agent to be detected or imaged in vivo, ex vivo, or in vitro by magnetic resonance imaging (MRI), positron emission tomography (PET) imaging, computed tomography (CT) imaging, gamma imaging, near-infrared imaging, ultrasound imaging, fluorescence imaging, or other detection means. The activity of the therapeutic agent or the theranostic agent means, for example, the biological, physiological, or pharmacological activity of the therapeutic agent or the theranostic agent to treat a disease or condition (e.g., to treat cancer).

[0067] For example, when the agent includes a detectable moiety that is directly or indirectly linked to the targeting peptide by a peptide or peptidomimetic spacer, it was discovered that the agent can clearly distinguish tumor cells in tissue sections and tumor "margins" samples, which indicates that the agent can be used as a molecular imaging diagnostic tool for metastatic, disseminated, migratory, or invasive cancer or tumor margins. Systemic introduction of the agent as described herein results in rapid and specific labeling of flank and intracranial tumors within minutes. Labeling occurs primarily within the tumor, however, a gradient of the agent is also observed at the tumor margins. There is also a signal amplification effect as the extracellular fragment accumulates over time.

[0068] The agent can be systemically administered to a subject and readily target cancer cells, such as metastatic, migratory, disseminated, and / or invasive cancer cells, that are associated with proteolytically cleaved extracellular fragments of immunoglobulin (Ig) superfamily cell adhesion molecules. In some embodiments, the agent can cross the blood-brain barrier upon systemic administration to determine cancer cell location, distribution, metastasis, dissemination, migration, and / or invasion and tumor cell margins in a subject. In other embodiments, the agent can inhibit and / or reduce cancer cell survival, proliferation, and migration upon systemic administration.

[0069] Accordingly, the agents described herein can be used in methods of detecting cancer cells and / or cancer cell metastasis, migration, dissemination, and / or invasion, and in methods of treating cancer in a subject in need thereof. These methods can include administering to the subject an agent that includes a targeting peptide that binds and / or complexes with a proteolytically cleaved extracellular fragment of an Ig superfamily cell adhesion molecule in a cancer cell or tumor cell microenvironment, at least one detectable moiety, and a peptide or peptidomimetic spacer that directly or indirectly links the targeting peptide to the at least one detectable moiety. The agent that binds and / or complexes with the cancer cell can be detected to determine the location and / or distribution of the cancer cell in the subject.

[0070] In some embodiments, the Ig superfamily cell adhesion molecule can include an extracellular homophilic binding portion that can bind or participate in homophilic binding in the subject in a homophilic manner. In one example, the Ig superfamily cell adhesion molecule includes a RPTP type IIb cell adhesion molecule. In another example, the Ig superfamily cell adhesion molecule can include a RPTP of the PTP mu-like subfamily, such as PTP mu, PTP K PTP rho, and PCP-2 (also known as PTP lambda). PTP mu-like RPTPs include a MAM (Memrin / A5-protein / PTP mu) domain, an Ig domain, and a FNIII repeat. PTP mu can have an amino acid sequence of SEQ ID NO: 1, which is identified by Genbank accession number AAI51843.1. It should be appreciated that the PTP mu gene can produce splice variants, such that the amino acid sequence of PTP mu can differ from SEQ ID NO: 1. In some embodiments, PTP mu can have an amino acid sequence identified by Genbank accession number AAH51651.1 and Genbank accession number AAH40543.1.

[0071] Cancer cells and / or endothelial cells that support cancer cell survival express Ig superfamily cell adhesion molecules and can be proteolytically cleaved to produce detectable extracellular fragments, which can include, for example, other cells in the cancer cell and / or tumor microenvironment, such as stem cells, endothelial cells, stromal cells, and immune cells that promote their survival.

[0072] Cancers detected and / or treated with the reagents described herein can include the following: leukemias, such as, but not limited to, acute leukemias, acute lymphocytic leukemias, 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, and hairy cell leukemia; polycythemia vera; lymphomas, such as, but not limited to, Hodgkin's disease, non-Hodgkin's disease; multiple myeloma, 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 unknown 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 sarcoma, angiosarcoma, fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, such as, but not limited to, glioma, astrocytoma, glioblastoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pinealoma, primary brain lymphoma; breast cancer, including, but not limited to, ductal carcinoma, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast carcinoma, mucinous breast carcinoma, tubular breast carcinoma, papillary breast carcinoma, 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 cancer, such as, but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipidus; eye cancer, such as, but not limited to, ocular melanoma, such as iridal melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, 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 cancer, such as, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancer, such as, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancer, such as, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor;esophageal cancer, 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; gastric cancer, such as, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, such as, but not limited to, hepatocellular carcinoma and hepatoblastoma; gall bladder cancer, such as adenocarcinoma; bile duct cancer, such as, but not limited to, papillary, nodular, and diffuse; lung cancer, such as non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; testicular cancer, such as, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocyte tumor, non-seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma (yolk sac tumor), prostate cancer, such as, but not limited to, prostatic intraepithelial neoplasm, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer; oral cancer, such as, but not limited to, squamous cell carcinoma; basal cancer; salivary gland cancer, such as, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, such as, but not limited to, squamous cell carcinoma and verrucous carcinoma; skin cancer, such as, but not limited to, basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma; kidney cancer, such as, but not limited to, renal cell carcinoma, adenocarcinoma, nephroblastoma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter); Wilms tumor; bladder cancer, such as, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteosarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioendothelioma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (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).

[0073] These agents can also be used to detect and / or treat a variety of cancers or other abnormal proliferative disorders, including (but not limited to) the following: carcinomas of epithelial origin, including bladder, breast, prostate, rectal, colon, kidney, liver, lung, ovary, uterine, pancreatic, stomach, cervical, thyroid, and skin; squamous cell carcinomas; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphoblastic leukemia, acute lymphoblastic lymphoma, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, glioblastoma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer, and tetratocarcinoma. Cancers resulting from abnormalities in apoptosis are also contemplated to be treated by the methods and compositions of the application. Such cancers can include, but are not limited to, follicular lymphoma, carcinomas of epithelial origin, hormone-dependent tumors of the breast, prostate, and ovary, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndrome. In particular embodiments, malignant or dysproliferative changes (e.g., metaplasia and dysplasia) or hyperproliferative disorders in the skin, lung, colon, rectum, breast, prostate, bladder, kidney, pancreas, ovary, or uterus are detected, treated, or prevented. In other particular embodiments, sarcomas, melanomas, or leukemias are detected and / or treated.

[0074] In other embodiments, the cancer cells detected and / or treated can include glioma cells, lung cancer cells, breast cancer cells, prostate cancer cells, and melanoma cells, e.g., invasive, disseminated, active, 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 cells and / or endothelial cells that support the survival of cancer cells express Ig superfamily cell adhesion molecules and can be proteolytically cleaved to produce detectable extracellular fragments that can be identified or determined by, e.g., using an immunoassay that detects Ig superfamily cell adhesion molecules expressed by the cancer cells or endothelial cells.

[0075] In some embodiments, the targeting peptide (or targeting polypeptide) can include a polypeptide (or targeting polypeptide) that binds to and / or complexes with a proteolytically cleaved extracellular fragment of an 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 or identical to about 10 to about 50 contiguous amino acids of a cognate binding portion or domain of a proteolytically cleaved extracellular fragment of an Ig superfamily cell adhesion molecule. By substantially homologous is meant that the targeting polypeptide has an amino acid sequence that is at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a portion of the amino acid sequence of the binding portion of a proteolytically cleaved extracellular fragment of an Ig superfamily cell adhesion molecule.

[0076] In one example, the cognate binding portion of an Ig superfamily cell adhesion molecule can include, for example, an Ig domain of a cell adhesion molecule. In another example, where the Ig superfamily cell adhesion molecule is PTPμ, the cognate binding portion can include an Ig binding domain and a MAM domain.

[0077] In another aspect, the targeting peptide can have an amino acid sequence that is substantially homologous to about 10 to about 50 contiguous amino acids of an Ig binding domain and / or a MAM domain of PTPμ (e.g., SEQ ID NO: 1), and readily crosses the blood-brain barrier when administered systemically to a subject. Development of PTPμ targeting peptides can be based on a large amount of structural and functional data. Sites required for cognate adhesion mediated by PTPμ have been well characterized. In addition, the crystal structure of PTPμ can provide information about which regions of each functional domain are likely to be exposed to the external environment, and thus available for cognate binding, for detection by a peptide.

[0078] In some embodiments, the proteolytically cleaved PTPmu ectodomain fragment (e.g., SEQ ID NO: 1) can include the amino acid sequence of SEQ ID NO: 2, the Ig and MAM binding regions can comprise the amino acid sequence of SEQ ID NO: 3, and the polypeptide can have an amino acid sequence that is substantially homologous to about 10 to about 50 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 3. Examples of polypeptides that can specifically bind to 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 contiguous 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. Polypeptides comprising SEQ ID NO: 4, 5, 6, or 7 can recognize or bind to MAM, Ig domains, or FNIII repeat sequences. In specific embodiments, the targeting peptide is an SBK2 polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0079] In other embodiments, the polypeptide that binds and / or complexes with a proteolytically cleaved ectodomain fragment of an Ig superfamily CAM expressed by a cancer cell or another cell in the microenvironment of a cancer cell or its receptor can have the amino acid sequence of SEQ ID NO: 8. SEQ ID NO: 8 is substantially homologous to a portion of SEQ ID NO: 1 or SEQ ID NO: 2 and can specifically bind to SEQ ID NO: 2 or SEQ ID NO: 3.

[0080] The targeting peptide can be subjected to various changes, substitutions, insertions, and deletions that provide certain advantages in its use. In this regard, the targeting peptide that binds and / or complexes with a proteolytically cleaved ectodomain portion of an Ig superfamily cell adhesion molecule can be substantially homologous to the sequence of the polypeptide described, wherein one or more changes have occurred therein, rather than identical thereto, and it retains the ability to specifically bind and / or complex with a proteolytically cleaved ectodomain portion of an Ig superfamily cell adhesion molecule.

[0081] The targeting peptide can be any of a variety of forms of polypeptide derivatives, including amides, conjugates with proteins, cyclized polypeptides, polymeric polypeptides, retro-inverso peptides, analogs, fragments, chemically modified polypeptides, and like derivatives.

[0082] The term "analog" includes any polypeptide having a sequence of amino acid residues substantially identical to a sequence specifically shown herein, wherein one or more residues are conservatively substituted for functionally similar residues, and specifically binds 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 (e.g., isoleucine, valine, leucine or methionine) for another; one polar (hydrophilic) residue for another (e.g., in the case of arginine and lysine, in the case of glutamine and asparagine, in the case of glycine and serine); one basic residue (e.g., lysine, arginine or histidine) for another; or one acidic residue (e.g., aspartic acid or glutamic acid) for another.

[0083] The phrase "conservative substitutions" also includes the use of chemically derivatized residues in place of non-derivatized residues, provided that such peptides exhibit the requisite binding activity.

[0084] "Chemical derivative" refers to a polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, those in which a free amino group is derivatized to form an amine salt, a p-toluensulfonyl, a benzyloxy, a t-butoxy carbonyl, a chloroacetyl or a formyl group. A free carboxyl group can be derivatized to form a salt, a methyl and ethyl esters or other types of esters or a hydrazide. A free hydroxyl group can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those naturally occurring amino acid derivatives which contain one or more of the twenty standard amino acids. For example: 4-hydroxyproline can be substituted for proline; 5- hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine. The polypeptides described herein also include any polypeptide having one or more additions and / or deletions of residues relative to the sequence of the polypeptide whose sequence is shown herein, provided that the requisite activity is maintained.

[0085] Retro-inverso peptides are linear peptides whose amino acid sequence is reversed and whose a- center chirality of the amino acid subunits is also inverted. These types of peptides are designed by including D-amino acids in the reverse sequence to help maintain similar side chain topology as the original L-amino acid peptide and make them more resistant to proteolytic degradation. D-amino acids represent the conformational mirror image of the natural L-amino acids that exist in natural proteins in biological systems. Peptides containing D-amino acids have advantages over peptides containing only L-amino acids. Generally, these types of peptides are less susceptible to proteolytic degradation and have a longer effective time when used as drugs. In addition, the insertion of D-amino acids in selected sequence regions as sequence blocks containing only D-amino acids or containing D-amino acids between L-amino acids, allows the design of peptide-based drugs that have biological activity in addition to being resistant to proteolysis, with elevated bioavailability. In addition, if designed properly, retro-inverso peptides can have similar binding properties as L-peptides.

[0086] The term "fragment" refers to a polypeptide whose amino acid residue sequence is shorter than that of the subject polypeptide whose amino acid residue sequence is set forth herein.

[0087] Any polypeptide or compound can also be used in the form of a pharmaceutically acceptable salt. Acids capable of forming salts with polypeptides include inorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid (HC1), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, 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, p-aminobenzoic acid, and the like.

[0088] Bases capable of forming salts with 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).

[0089] Targeting peptides can be synthesized by any technique known to those skilled in the art of peptides, including recombinant DNA techniques. For reasons of purity, antigen specificity, absence of undesired by-products, ease of production, and the like, synthetic chemistry techniques can be used, such as solid phase Merrifield-type synthesis. A summary of many of the available techniques 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. Patent No. 4,244,946, which relates to solid phase peptide synthesis, and Schroder et al., "The Peptides", Vol. 1, Academic Press (New York), 1965, which relates to classical solution synthesis, each of which is incorporated herein by reference. Suitable protecting groups useful in such syntheses are described in the above and in J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973, which is incorporated herein by reference.

[0090] In general, the contemplated solid phase synthesis method involves the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Typically, the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. Different, selectively removable protecting groups are used for amino acids containing reactive side groups, such as lysine.

[0091] By way of example with solid phase synthesis, a protected or derivatized amino acid can be attached via its unprotected carboxyl or amino group to an inert solid support. The protecting group of the amino or carboxyl group can then be selectively removed, the next amino acid in the sequence with the complementary (amino or carboxyl) group appropriately protected is mixed, and reacted under conditions appropriate to form an amide bond 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 (appropriately protected) is added, and so on. After all of the desired amino acids have been linked in the correct order, any remaining terminal and side group protecting groups (and the solid support) can be removed sequentially or simultaneously to provide the final linear polypeptide.

[0092] It will be appreciated that in addition to PTPs, the targeting peptides can bind and / or complex with the homophilic binding domains of proteolytically cleaved extracellular fragments of other Ig superfamily cell adhesion molecules. For example, the analogous molecular detection strategies described herein can be used for any other Ig superfamily CAM whose cell surface protein is known to bind a ligand at a site. A 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 protein 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 that can be readily used by the skilled person to form therapeutic polypeptides useful in the treatment of cancer (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).

[0093] In some embodiments, the targeting peptides described herein can include additional residues that can be added to either end of the polypeptide to provide a "linker" through which the polypeptide can be conveniently linked and / or immobilized to a peptide or peptidomimetic spacer. Typical amino acid residues for linking are glycine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, among others. In addition, the polypeptide can be differentially sequence modified by acylation (e.g., acetylation, or thioglycolic acid amidation) of the terminal -NH2, amidation of the terminal -carboxyl (e.g., end modification with ammonia, methylamine, and the like). It is well known that end modification can be used to reduce susceptibility to protease digestion, and thus can be used to prolong the half-life of the polypeptide in solution, particularly in biological fluids where proteases can be present. In this regard, cyclization of the polypeptide is also a useful end modification, and is particularly preferred because cyclization forms a stable structure, and in view of the biological activity observed on such cyclic polypeptides, as described herein.

[0094] The peptide or peptidomimetic spacer linking the targeting peptide directly or indirectly to at least one of a detectable moiety, a therapeutic agent, or a diagnostic agent can include additional natural and / or non-natural amino acid residues (or a linker peptide with the target peptide) added at either end of the targeting peptide. The peptide or peptidomimetic spacer can include at least three natural or non-natural amino acids and have a structure effective to at least maintain or preserve the binding affinity of the extracellular fragment of the linked targeting peptide to the protein and the activity of the linked at least one of a detectable moiety, a therapeutic agent, or a diagnostic agent. Typical amino acid residues for the spacer are glycine, serine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, among others.

[0095] In some embodiments, the selection of the peptide or peptidomimetic spacer is based in part on its ability to alter hydrophobicity (e.g., to make the agent more hydrophilic or hydrophobic), depending on the desired use.

[0096] In some embodiments, the spacer can be a flexible peptide or peptidomimetic spacer that links the targeting peptide directly or indirectly to other polypeptides, proteins, and / or molecules, such as a detectable moiety, a label, a therapeutic agent, a diagnostic agent, a solid matrix, or a carrier. The flexible peptide or peptidomimetic spacer can be, for example, at least about 3 to about 30 or fewer natural or non-natural amino acids in length. For example, the spacer can be 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, or 30 natural or non-natural amino acids in length. Where the spacer is a peptide spacer, the peptide spacer can be produced as a single recombinant polypeptide using conventional molecular biology / recombinant DNA methods.

[0097] In some embodiments, the spacer comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% glycine and / or serine residues.

[0098] In other embodiments, the spacer comprises at least 50%, at least 60%, at least 70%, or at least 80% glycine residues. In some embodiments, the remainder of the spacer comprises serine residues.

[0099] In some embodiments, the spacer is a polyglycine spacer or a glycine / serine spacer consisting of pure glycine residues or of glycine and serine residues. The small size of glycine residues provides flexibility and allows mobility of the attachment of the targeting peptide to at least one of the detectable moiety, the therapeutic agent, or the diagnostic agent. Incorporation of serine can maintain stability of the spacer in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the spacer and the targeting peptide.

[0100] In some embodiments, the spacer comprises an amino acid sequence of at least one of (GS)a, (GGS)b, or (GGGS)c or (GGGGS)d, and wherein a, b, c, and d are each individually 2, 3, 4, 5, or 6. For example, the spacer can have an amino acid sequence of GGG (SEQ ID NO: 9), GGGG (SEQ ID NO: 10), GGGGG (SEQ ID NO: 11), GGGGGG (SEQ ID NO: 12), GGGGGGG (SEQ ID NO: 13), GGGGGGGG (SEQ ID NO: 14), GGGGGGGGG (SEQ ID NO: 15), GSGS (SEQ ID NO: 16), GSGSGS (SEQ ID NO: 17), GSGSGSGS (SEQ ID NO: 18), GSGSGSGSGS (SEQ ID NO: 19), GGSGGS (SEQ ID NO: 20), GGSGGSGGS (SEQ ID NO: 21), GGSGGSGGSGGS (SEQ ID NO: 22), GGGSGGGS (SEQ ID NO: 23), GGGSGGGSGGGS (SEQ ID NO: 24), GGGSGGGSGGGSGGGS (SEQ ID NO: 25), GGGGSGGGGS (SEQ ID NO: 26), or GGGGSGGGGSGGGGS (SEQ ID NO: 27).

[0101] In some embodiments, the spacer can be a contiguous portion of the targeting peptide that is directly coupled to the N-terminal or C-terminal residue of the targeting peptide, with or without the inclusion of a linker peptide.

[0102] For example, a polyglycine or glycine / serine spacer coupled to an SBK2 targeting peptide having SEQ ID NO:5 can have the following amino acid sequences: GGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:28), GGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 29), GGGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 30), GGGGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 31), GGGGGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 32), GGGGGGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:33), GGGGGGGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:3 ), GSGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 35), GSGSGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 36), GSGSGSGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:), GSGSGSGSGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:38), GGSGGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:39), GGSGGSGGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:40), GGSGGSGGSGGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:41), GGGSGGGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:42), GGGSGGGSGGGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:43), GGGSGGGSGGGSGGGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:44), GGGGSGGGGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:45), or GGGGSGGGGSGGGGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:46).

[0103] It should be noted that there seems to be a small error in the original text where "SEQ ID NO:3 " is likely a typo and should probably be "SEQ ID NO:34". This has been maintained as is in the translation for the purpose of following the instructions.It is understood that other peptide or peptidomimetic spacers can be attached to SBK2 or other targeting peptides described herein at the N-terminal or C-terminal portion of the targeting peptide.

[0104] In some embodiments, a targeting peptide having a contiguous spacer can be produced as a recombinant polypeptide. For production of recombinant polypeptides, a variety of host organisms can be used. Examples of hosts include, but are not limited to: bacteria, e.g., E. coli, yeast cells, insect cells, plant cells, and mammalian cells. The skilled artisan will understand how to consider certain criteria in selecting a suitable host for production of a recombinant polypeptide. Factors influencing host selection include, for example, patterns of post-translational modification, e.g., phosphorylation and glycosylation, as well as technical factors, e.g., general expected yield and ease of purification. Host-specific post-translational modifications of a targeting peptide or spacer peptide to be used in vivo should be carefully considered, as certain post-translational modifications are known to be highly immunogenic.

[0105] In other embodiments, a spacer can be a non-contiguous portion of a targeting peptide that is indirectly coupled or conjugated to the targeting peptide by a coupling or conjugating agent. By "non-contiguous portion" it is meant that the targeting peptide and the spacer are linked by an additional element that is not part of the targeting peptide or the spacer and / or a peptide residue that is not contiguous in nature and functions as a linker.

[0106] Coupling and / or conjugating agents can include, for example, maleimide-based binders that can be used to bind to thiol groups, isothiocyanate and succinimidyl (e.g., N-hydroxysuccinimidyl (NHS)) binders that can bind to free amine groups, diazonium salts that can be used to bind to phenols, and amines that can be used to bind to free acids (e.g., carboxylate groups) through carbodiimide activation. Useful functional groups can be present on a peptide or peptidomimetic spacer based on the particular amino acids present, and additional groups can be designed. It will be apparent to those skilled in the art that a variety of bifunctional or multifunctional reagents, homobifunctional and heterobifunctional reagents (such as those described in the catalog of Pierce Chemical Co., Rockford, Ill.), can be employed as coupling agents. Coupling can be achieved, for example, through amino, carboxyl, sulfhydryl, or oxidized carbohydrate residues.

[0107] Examples of coupling and / or conjugating agents are described in Means and Feeney, CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp. 39-43. These agents include, for example, N-hydroxysuccinimide 3-(2-pyridyldithio)propionate (SPDP) or N,N'-(l,3-phenylene)dimaleimide (both of which have high specificity for sulfhydryl groups and form irreversible bonds); N,N'-ethylene-bis-(iodoacetamide) or other reagents with six to eleven carbon methylene bridges (which have relative specificity for sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversible bonds with amino and tyrosine groups). Other coupling or conjugating agents include: p,p'-difluoro-m,m'-dinitrobenzene sulfone (which forms irreversible bonds with amino and phenolic groups); dimethyl adipimidate (which has specificity for amino groups); phenol-1,4-disulfonyl chloride (which reacts primarily with amino groups); hexamethylene diisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which react primarily with amino groups); glutaraldehyde (which reacts with several different side chains); and diazobenzenes (which react primarily with tyrosine and histidine).

[0108] The coupling or conjugating agent can be homobifunctional, i.e., have two functional groups that react the same way. An example of a homobifunctional crosslinking agent is bis-maleimido hexane ("BMH"). BMH contains two maleimide functional groups that react specifically with sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). The two maleimide groups are linked by a hydrocarbon chain. Thus, BMH can be used to irreversibly link polypeptides containing cysteine residues.

[0109] The coupling or conjugating agent can also be heterobifunctional. Heterobifunctional coupling or conjugating agents have two different functional groups, such as an amine-reactive group and a thiol-reactive group, which will crosslink two proteins with free amines and thiols, respectively. Examples of heterobifunctional crosslinking agents are succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate ("SMCC"), m-maleimidobenzoyl-N-hydroxysuccinimide ester ("MBS"), and succinimidyl 4-(p-maleimidophenyl) butyrate ("SMPB", an extended-chain analogue of MBS). The succinimidyl group of these crosslinking agents reacts with primary amines, and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine residue.

[0110] Many of the coupling or conjugation agents produce conjugates that are essentially non-cleavable under cellular conditions. However, some agents contain covalent bonds that are cleavable under cellular conditions, such as disulfide bonds. For example, the Traut's reagent, dithiobis(succinimidyl propionate) ("DSP") and N-succinimidyl 3-(2-pyridyldithio)propionate ("SPDP") are well known cleavable cross-linking agents. The use of cleavable coupling or conjugation agents allows for separation of the targeting peptide, spacer and / or detectable moiety, therapeutic and / or diagnostic agent after delivery to the target cell. Direct disulfide bonds can also be useful.

[0111] Many of the coupling agents, including those discussed above, are commercially available. Detailed instructions for their use can be readily obtained 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).

[0112] In some embodiments, the peptide or peptidomimetic spacer can be coupled, directly or indirectly, to a detectable moiety, therapeutic agent and / or diagnostic agent using, for example, a coupling or conjugation agent described herein.

[0113] In some embodiments, the detectable moiety can include any contrast agent or detectable label to facilitate the detection step of the diagnostic or therapeutic method by visualization of the complex formed by binding of the extracellular fragment of the proteolytic cleavage of the Ig superfamily cell adhesion molecule to the agent comprising the targeting peptide, spacer and detectable moiety and / or diagnostic agent. The detectable moiety can be selected so that it produces a signal that can be measured and the intensity of which is related (preferably proportional) to the amount of agent bound to the tissue being analyzed. Methods for labeling biological molecules (e.g., polypeptides) are well known in the art.

[0114] Any of a number of detectable moieties can be linked to the targeting peptide by the peptide or peptidomimetic spacer described herein. Examples of detectable moieties include, but are not limited to: various ligands, radionuclides, fluorescent agents and dyes, infrared and near infrared agents, chemiluminescent agents, microparticles or nanoparticles (e.g., quantum dots, nanocrystals, semiconductor particles, nanoparticles, nanobubbles or nanochains, etc.), enzymes (e.g., those used in ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, chelators, biotin, dioxanes, or other haptens for which antisera or monoclonal antibodies are available, and proteins.

[0115] In some embodiments, the agents comprising the detectable moieties described herein can be used in conjunction with non-invasive imaging (e.g., neuroimaging) techniques for in vivo imaging of the agents, such as magnetic resonance spectroscopy (MRS) or 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 that allows detection of the labeled agent, as described above. For gamma imaging, the radiation emitted from the organ or region being examined is measured and expressed as total binding or as a ratio normalized (e.g., divided by) total binding in one tissue relative to total binding in another tissue in the same subject during the same in vivo imaging procedure. In vivo total binding is defined as the total signal detected in a tissue by the in vivo imaging technique without correction by a second injection of the same amount of agent and a large amount of unlabeled but otherwise chemically identical compound.

[0116] For the purposes of in vivo imaging, the type of detection instrument available is a major factor in the selection of a given detectable moiety. For example, the type of instrument used will guide the selection of stable isotopes. The half-life should be long enough to be detectable at the time of maximum uptake of the target, but short enough so that the host is not detrimentally affected.

[0117] In one example, the detectable moiety can comprise a radiolabel that is attached (e.g., attached or complexed) directly or indirectly to the peptide or peptidomimetic spacer using general organic chemistry techniques. The radiolabel can be, for example, 68 Ga, 123 I, 131 I, 125 I, 18 F, 11 C, 75 Br, 76 Br, 124 I, 13 N, 64 Cu, 32 P, 35 S. Such radiolabels can be detected by PET techniques as described in Fowler, J. and Wolf, A. in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota, J., and Schelbert, H. eds.) 391-450 (Raven Press, NY 1986), the contents of which are incorporated herein by reference. The detectable moiety can also comprise a radiolabel for SPECT 123 I. 123I can be coupled to the peptide spacer by any of several techniques known in the art. See, e.g., Kulkarni, Int. J. Rad. Appl. & Inst. (Part B) 18:647 (1991), the contents of which are incorporated herein by reference. In addition, the detectable moiety can include any of the radioiodine isotopes, such as, but not limited to 131 I, 125 I or 123 I. The radioiodine isotopes can be coupled to the peptide spacer by direct iodination of diazotized amino derivatives via diazotized iodides, see Greenbaum, F. Am. J. Pharm. 108:17 (1936), or by conversion of unstable diazotized amines to stable triazenes, or by conversion of non-radioactive halogenated precursors to stable trialkyl tin derivatives, which can then be converted to iodinated compounds by a variety of methods well known in the art.

[0118] The detectable moiety can further include known metal radio labels, such as Technetium-99m (Tc), 99m Gadolinium (Gd), 153 Indium (In), 111 Gallium (Ga), 67 Thallium (Tl), 201 Rubidium (Rb), 82 Copper (Cu), 64 Yttrium (Y), 90 Rhodium (Rh), Tritium (T), 188 Samarium (Sm), 153 Strontium (Sr), and 89 211 ​At ordinary skill in the art of radiolabeling would be able to modify the targeting peptide to incorporate a ligand that binds such metal ions without undue experimentation. Metal radiolabeled reagents can then be used to detect cancer, e.g., GBM in a subject. The preparation of radiolabeled derivatives of Tc99m is well known in the art. See, e.g., Zhuang et al., "Neutral and stereospecific Tc-99m complexes: [99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT)" Nuclear Medicine & Biology 26(2):217-24, (1999); Oya et al., "Small and neutral Tc(v)O BAT, bisaminoethanethiol (N2S2) complexes for developing new brain imaging agents" Nuclear Medicine & Biology 25(2): 135-40, (1998); and Hom et al., "Technetium-99m-labeled receptor-specific small-molecule radiopharmaceuticals: recent developments and encouraging results" Nuclear Medicine & Biology 24(6):485-98, (1997).

[0119] In some embodiments, the detectable moiety can include a chelator (with or without a chelated radiolabeled metal group). Examples of chelators can include those disclosed in U.S. Patent No. 7,351,401, which is incorporated by reference herein in its entirety. In some embodiments, the chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0120] Fluorescently labeled reagents or infrared reagents include those known in the art, many of which are commonly commercially available, e.g., fluorophores such as ALEXA 350, PACIFIC BLUE, MARINA BLUE, ACRIDIN, EDANS, COUMARI, BODIPY 493 / 503, CY2, BODIPY FL-X, DANSYL, ALEXA 488, FAM, OREGON GREEN, RHODAMINE GREEN-X, TET, ALEXA 430, CAL GOLD.TM., BODIPY R6G-X, JOE, ALEXA 532, VIC, HEX, CALORANGE.TM., ALEXA 555, BODIPY 564 / 570, BODIPY TMR-X, QUASAR.TM. 570, ALEXA 546, TAMRA, RHODAMINE RED-X, BODIPY 581 / 591, CY3.5, ROX, ALEXA 568, CAL RED, BODIPY TR-X, ALEXA 594, BODIPY 630 / 650-X, PULSAR 650, BODIPY 630 / 665-X, ALEXA 647, IR700, IR800, INDOCYANINE GREEN (ICG), TEXAS RED, or QUASAR 670.

[0121] Fluorescently labeled reagents can also include other known fluorophores or proteins known in the art, e.g., green fluorescent protein. The disclosed targeting peptides and peptide or peptoid spacers can be directly or indirectly coupled to a fluorescently labeled reagent, administered to a subject or sample, and the subject / sample examined by fluorescence spectroscopy or imaging to detect the labeled compound.

[0122] In some embodiments, the detectable moiety comprises a fluorescent dye. Exemplary fluorescent dyes include fluorescein isothiocyanate, a cyanine such as Cy5, Cy5.5, and analogs thereof (e.g., sulfo-cyanine 5 NHS ester and Cy5.5 maleimide). See also Handbook of Fluorescent Probes and Research Chemicals, 6th Ed., Molecular Probes, Inc., Eugene Oreg., which is incorporated herein by reference.

[0123] The detectable moiety can further comprise a near infrared imaging group. Near infrared imaging groups are disclosed, for example, in Tetrahedron Letters 49 (2008) 3395-3399; Angew. Chem. Int. Ed. 2007, 46, 8998-9001; Anal. Chem. 2000, 72, 5907; Nature Biotechnology vol 23, 577-583; Eur Radiol (2003) 13: 195-208; and Cancer 67: 1991 2529-2537, which are incorporated by reference in their entirety. Applications can include use of a NIRF (near infrared) imaging scanner. In one example, the NIRF scanner can be handheld. In another example, the NIRF scanner can be miniaturized and embedded in a device (e.g., a micro-machine, a scalpel, a neurosurgical cell removal device).

[0124] Quantum dots (e.g., semiconductor particles) can also be used as detectable moieties, as described in Gao, et al "In vivo cancer targeting and imaging with semiconductor quantum dots", Nature Biotechnology, 22, (8), 2004, 969-976, the entire teachings of which are incorporated by reference. The disclosed targeting peptides and peptide or peptoid spacers can be coupled to quantum dots, administered to a subject or sample, and the subject / sample examined by fluorescence spectroscopy or imaging to detect the labeled compound.

[0125] In certain embodiments, the detectable moiety comprises an MRI contrast agent. MRI relies on the variation of magnetic dipoles to perform detailed anatomical imaging and functional studies. MRI can employ dynamic quantitative Tl mapping as an imaging method to measure the longitudinal relaxation time of protons in a magnetic field after excitation by a radiofrequency pulse, i.e., the Tl relaxation time. The Tl relaxation time, in turn, can be used to calculate the concentration of the agent in the region of interest, thereby allowing quantification of the retention or clearance of the agent. In this case, retention measures the binding of the molecular contrast agent.

[0126] Many magnetic resonance imaging (MRI) contrast agents are known in the art, for example, positive contrast agents and negative contrast agents. The disclosed targeting peptides and peptide or peptoid spacers can be coupled to an MRI agent, administered to a subject or sample, and the subject / sample examined by MRI or imaging to detect the labeled compound. Positive contrast agents (typically exhibiting a predominantly bright color on MRI) can generally include small molecular weight organic compounds that chelate or contain active elements with unpaired outer shell electron spins, such as gadolinium, manganese, iron oxide, and the like. Typical contrast agents include gadolinium (III) chelates of macrocyclic structures, such as gadopentetic acid meglumine (gadopentetic acid), gadopentate dimeglumine, gadoteridol, mangafodipir trisodium, gadodiamide, and others known in the art. In certain embodiments, the detectable moiety includes gadopentetic acid meglumine. Negative contrast agents (typically exhibiting a predominantly dark color on MRI) can include small particulate aggregates composed of superparamagnetic materials, such as superparamagnetic iron oxide (SPIO) particles. Negative contrast agents can also include compounds that lack hydrogen atoms associated with signal in MRI imaging, such as perfluorocarbons (perfluorides).

[0127] In some embodiments, the targeting peptide and peptide or peptoid spacer can be coupled or linked to a chelator (e.g., macrocyclic chelator DOTA) and a single metal radiolabel.

[0128] In other embodiments, the targeting peptide and peptide or peptoid spacer or multiple targeting peptides and peptide or peptoid spacers can be coupled or linked to a nanobubble for diagnostic and / or therapeutic applications. Nanobubbles can include a lipid membrane defining an internal void comprising at least one gas. Examples of nanobubbles that can be coupled to a targeting peptide and peptide or peptoid spacer are described, for example, in U.S. Patent Nos. 10,375,575, 10,434,194, and 10,973,935, and U.S. Patent Application Publication Nos. 2029 / 0061220 and 2021 / 0106699, all of which are incorporated by reference in their entirety.

[0129] The agents comprising the detectable moieties described herein can be administered to a subject by, for example, systemic, topical, and / or parenteral methods of administration. These methods include, for example, injection, infusion, deposition, implantation, or topical application, or any other method of administration that achieves entry of the agent into the tissue. In one example, administration of the agent can be performed by intravenous injection of the agent in the subject. Single or multiple administrations of the probe can be performed. As used herein, “administration” means providing or delivering the agent in an amount and for a period of time effective to label cancer cells of the subject.

[0130] The agents comprising the detectable moieties described herein can be administered to a subject in a pharmaceutical composition containing a detectable amount of the agent or a pharmaceutically acceptable water-soluble salt thereof for a patient.

[0131] The formulation of the agent to be administered will vary depending on the chosen route of administration (e.g., solution, emulsion, capsule, etc.). Suitable pharmaceutically acceptable carriers can contain inert ingredients that do not unduly inhibit the biological activity of the compound. The pharmaceutically acceptable carrier should be biologically compatible, e.g., non-toxic, non-inflammatory, non-immunogenic, and free of other undesirable reactions when administered to a subject. Standard pharmaceutical formulation techniques can be employed, e.g., those described in Remington's Pharmaceutical Sciences, as previously noted. Pharmaceutical carriers suitable for parenteral administration include, e.g., 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.

[0132] The preparation of a pharmaceutical composition that contains active ingredients dissolved or dispersed therein is well understood. Generally, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution or suspension in liquid prior to use can also be prepared. The formulation will depend on the chosen route of administration (e.g., solution, emulsion, capsule).

[0133] "Detectable amount" means an amount of detectable compound administered sufficient to enable detection of the binding of the compound to cancer cells. "Imaging effective amount" means an amount of detectable compound administered sufficient to enable imaging of the binding of the agent to cancer cells.

[0134] An agent comprising a detectable moiety administered to a subject can be used in a method of detecting and / or determining the presence, location, and / or distribution of cancer cells (i.e., cancer cells associated with extracellular fragments of proteolytic cleavage of Ig superfamily cell adhesion molecules) in an organ or body region of a patient (e.g., at least one region of interest (ROI) of the subject). The ROI can include a particular region or portion of the subject, in some cases, two or more regions or portions of the subject including the subject as a whole. The ROI can include a region to be imaged for diagnostic and therapeutic purposes. The ROI is typically internal; however, it should be understood that the ROI can additionally or alternatively be external.

[0135] The presence, location, and / or distribution of the agent in the animal tissue (e.g., brain tissue) can be visualized (e.g., using the in vivo imaging modalities described above). As used herein, “distribution” is a spatial property of something spread over an area or volume. In this case, the “distribution of cancer cells” is a spatial property of cancer cells spread over an area or volume included in the animal tissue (e.g., brain tissue). The distribution of the agent can then be correlated with the presence or absence of cancer cells in the tissue. The distribution can determine the presence or absence of cancer cells, or it can be combined by one of skill in the art with other factors and symptoms to positively detect or define the tumor margin for the presence or absence of cancer cell migration or spread, cancer metastasis in the subject. It will be appreciated that the imaging modalities can be used to generate baseline images prior to administration of the composition. In this case, the baseline and post-administration images can be compared to determine the presence, absence, and / or extent of a particular disease or condition.

[0136] In one aspect, an agent including a detectable moiety can be administered to a subject to assess the distribution of cancer cells in the subject and correlate the distribution with a particular location. Surgeons often use stereotactic techniques and intraoperative MRI (iMRI) in surgical resection. This allows them to specifically identify and sample tissue from different regions of the tumor (e.g., tumor margin or tumor center). Often, they also sample regions of the brain outside the tumor margin that appear quite normal but are infiltrated with disseminated tumor cells upon histological examination. For example, in glioma (brain tumor) surgery, drugs can be given intravenously about 24 hours prior to preoperative stereotactic localization MRI. These agents can be imaged on gradient echo MRI sequences as contrast agents to localize gliomas.

[0137] The agents described herein, which comprise a detectable moiety and specifically bind to and / or complex with a proteolytically cleaved cell-associated Ig superfamily cell adhesion molecule (PTPμ), can be used in intraoperative imaging (IOI) techniques to guide surgical resection and eliminate the surgeon's "educated guess" of the location of the tumor margin. Previous studies have determined that more extensive surgical resection can improve patient survival Stummer W, Novotny A, Stepp H, Goetz C, Bise K, Reulen HJ (2000) Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. J Neurosurg 93: 1003-1013. Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. Stummer W, Novotny A, Stepp H, Goetz C, Bise K, Reulen HJ (2000) Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. J Neurosurg 93: 1003-1013. Thus, agents used as diagnostic molecular imaging agents have the potential to improve patient survival.

[0138] In some embodiments, to identify and facilitate removal of cancer cells, imaging in microscopy (IOI) techniques can be combined with the systemically or locally administered reagents described herein. After administration to a subject, the reagents can target and detect and / or determine the presence, location, and / or distribution of cancer cells (i.e., cancer cells associated with proteolytic cleavage of extracellular fragments of Ig superfamily cell adhesion molecules) in an organ or region of the body of a patient. In one example, the reagents can be combined with IOI to identify malignant cells that have infiltrated and / or are beginning to infiltrate the tumor brain margin. This method can be performed in real time during brain or other surgical procedures. The method can include local or systemic application of a target reagent described herein, which includes a detectable moiety, such as a fluorescent or MRI contrast moiety. Detection can then be performed using an imaging modality and subsequent image data acquisition. The imaging modality can include one or a combination of known imaging techniques capable of visualizing the reagents. The resulting image data can be used to determine, at least in part, surgical treatment and / or radiation therapy. Alternatively, the image data can be used to at least partially control automated surgical instruments (e.g., lasers, scalpels, micro-machines) or assist surgical personnel with manual guidance. In addition, the image data can be used to plan and / or control delivery of therapeutic agents (e.g., by microelectronic or micro-machines).

[0139] In one example, a reagent including a targeting peptide and a peptide or peptidomimetic spacer linked to a fluorescently detectable moiety can be applied locally during a surgical procedure as needed to interactively guide the surgeon and / or surgical instruments to remaining abnormal cells. The reagent can be applied locally at low concentrations such that it is unlikely to reach pharmacologically relevant concentrations. In one example, excess material can be removed (e.g., washed away) after a period of time (e.g., incubation period).

[0140] Another embodiment described herein relates to methods of monitoring the efficacy of a cancer treatment or cancer therapy administered to a subject. The methods and reagents described herein can be used to monitor and / or compare the invasion, migration, spread, and metastasis of a cancer in a subject before administration of a cancer treatment or cancer therapy, during administration, or after a treatment regimen.

[0141] As used herein, a "cancer treatment" or "cancer therapy" can include any agent or treatment regimen capable of having a negative effect on a cancer in an animal, for example, by killing cancer cells, inducing cancer cell apoptosis, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to a tumor or cancer cell, promoting an immune response against a cancer cell or tumor, preventing or inhibiting progression of a cancer, or prolonging the life of an animal with a cancer. A cancer treatment can include one or more therapies, such as, but not limited to, chemotherapy, radiation therapy, hormone therapy, and / or biological therapy / immunotherapy. For example, a reduction in cancer volume, growth, migration, and / or spread in a subject can be indicative of the efficacy of a given therapy. This can provide a direct clinical efficacy endpoint measurement for a cancer treatment. Thus, in another aspect, methods of monitoring the efficacy of a cancer treatment are provided. More specifically, embodiments of the present application provide methods of monitoring the efficacy of a cancer treatment.

[0142] The cancer treatment agent can be in the form of a biologically active ligand, a small molecule, a peptide, a polypeptide, a protein, a DNA fragment, a DNA plasmid, an interfering RNA molecule, such as an siRNA, an oligonucleotide, and a DNA encoding a shRNA.

[0143] The method of monitoring the efficacy of a cancer treatment can include the steps of administering an agent described herein to an animal in vivo, then visualizing the distribution of the agent in the animal (e.g., using an in vivo imaging modality described herein), then correlating the distribution of the agent with the efficacy of a cancer treatment. It is contemplated that the administration step can occur before, during, and after a treatment regimen to determine the efficacy of a selected treatment regimen. One method of assessing the efficacy of a cancer treatment is to compare the distribution of the agent before and after a cancer therapy.

[0144] In some embodiments, an agent that binds and / or complexes with an extracellular fragment proteolytically cleaved from an Ig superfamily cell adhesion molecule is detected in a subject to detect and / or provide the location and / or distribution of cancer cells in the subject. The location and / or distribution of cancer cells in the subject can then be compared to a control to determine the efficacy of a cancer treatment and / or cancer therapy. The control can be the location and / or distribution of cancer cells in the subject prior to administration of the cancer treatment and / or cancer therapy. The location and / or distribution of cancer cells in the subject prior to administration of the cancer treatment agent and / or cancer therapy can be determined by administering the agent to the subject and detecting the agent bound and / or complexed with cancer cells in the subject prior to administration of the cancer treatment and / or cancer therapy.

[0145] In certain embodiments, the methods and reagents described herein can be used to measure the efficacy of a therapeutic agent administered to a subject to treat a metastatic, invasive, or disseminated cancer. In this embodiment, the reagent can be administered to the subject prior to, during, or after administration of a therapeutic regimen, and the distribution of cancer cells can be imaged to determine the efficacy of the therapeutic regimen. In one example, the therapeutic regimen can comprise surgical resection of metastatic cancer, and the reagent can be used to determine the distribution of metastatic cancer pre- and post-surgery to determine the efficacy of the surgical resection. Optionally, these methods and reagents can be used in intraoperative surgical procedures, such as surgical tumor resection, to more easily delineate and / or image cancer cell mass or volume during surgery as described above.

[0146] In other embodiments, the targeting peptide and the peptide or peptidomimetic spacer can be directly or indirectly linked to a therapeutic or diagnostic agent. In one example, the diagnostic or therapeutic agent linked to the targeting peptide and the peptide or peptidomimetic spacer can be used in a method of treating a cancer or tumor (e.g., a brain cancer or tumor). In one embodiment, the therapeutic or diagnostic agent can comprise a photosensitizer, and the reagent comprising the targeting peptide, the spacer, and the photosensitizer can be used in photodynamic therapy.

[0147] Photodynamic therapy (PDT) is a site-specific treatment modality that requires the presence of a photosensitizer, light, and sufficient amounts of molecular oxygen to destroy the target tumor (Grossweiner, Li, The science of phototherapy. Springer: The Netherlands, 2005). Upon light exposure, the photoactivated sensitizer transfers energy to molecular oxygen, which leads to the production of singlet oxygen (O2) and other reactive oxygen species (ROS), triggering cancer cell apoptosis and oxidative damage. Only cells that are simultaneously exposed to the diagnostic PDT drug (non-toxic in the dark) and light are destroyed, while surrounding healthy, non-targeted, and non-irradiated cells are spared from photodamage. In addition, the fluorescence of the photosensitizer molecule enables simultaneous diagnostic optical imaging, which can be used to guide PDT cancer treatment.

[0148] Methods for performing photodynamic therapy are known in the art. See, e.g., Thierry Patrice. Photodynamic Therapy; Royal Society of Chemistry, 2004. Pharmaceutical compositions comprising a reagent comprising a targeting peptide, a spacer, and a diagnostic or therapeutic agent directly or indirectly linked to the spacer can be applied to an organ or tissue as a step in PDT. In certain embodiments, the composition is administered to an epithelial, mesothelial, synovial, fascial, or serosal surface, including but not limited to the epithelial surface of the eye, esophagus, mucosa, bladder, joint, tendon, ligament, bursa, gastrointestinal tract, genitourinary system, pleura, pericardium, lung, or urinary tract.

[0149] The diagnostic or therapeutic agent for PDT directly or indirectly linked to the spacer and the targeting peptide can be administered to a cancer subject by systemic administration, e.g., intravenous administration. Upon administration, the targeting agent can localize and / or accumulate at the site of the target tumor or cancer. In some embodiments, specific binding and / or complexing with the proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule expressed by a cancer cell or another cell in the cancer cell microenvironment allows the agent comprising the targeting peptide, the spacer, and the PDT agent to bind, complex, and / or be taken up by the target cell, e.g., by endocytosis. This binding and / or uptake is specific for the target cell, which allows the targeting agent to selectively target cancer cells and / or cells in the cancer cell microenvironment in the subject.

[0150] After administration and localization of the agent comprising the targeting peptide, the spacer, and the diagnostic or therapeutic agent to the target cancer cell, the target cancer cell can be exposed to a therapeutic amount of light, which causes cancer cell damage and / or inhibition of cancer cell growth. Light capable of activating the PDT agent can be delivered to the target cancer cell using, e.g., a semiconductor laser, a dye laser, an optical parametric oscillator, and the like. It will be appreciated that any light source can be used so long as the light is capable of exciting the hydrophobic PDT agent.

[0151] For example, the agent comprising the targeting peptide, the spacer, and the PDT agent can provide image guidance for a glioma tumor resection and allow for subsequent PDT to eliminate cancer cells that could not be resected or that remain. In certain embodiments, the targeting moiety can comprise a peptide having SEQ ID NO: 5.

[0152] The PDT agent photosensitizer compound for use in the agents described herein can include a compound that is excited by an appropriate light source to produce free radicals and / or reactive oxygen species. Generally, when a sufficient amount of the photosensitizer is present in a diseased tissue (e.g., tumor tissue), the photosensitizer can be activated by exposure to light for a particular period of time. The light dose provides enough energy to stimulate the photosensitizer, but not enough to damage adjacent healthy tissue. The free radicals or reactive oxygen produced upon excitation of the photosensitizer kill target cells (e.g., cancer cells). Light treatment of tissue that has accumulated the PDT drug can also induce an immune response. In some embodiments, the target tissue can be irradiated locally. For example, light can be delivered to the photosensitizer via an argon or copper-pumped dye laser coupled to a fiber, a dual laser consisting of a KTP (potassium titanyl phosphate) / YAG (yttrium aluminum garnet) medium, an LED (light-emitting diode), or a solid-state laser.

[0153] PDT sensitizing agents used as diagnostic or therapeutic agents can include first generation photosensitizers (e.g., hematoporphyrin derivatives (HpD), such as Photofrin (porfimer sodium), Photogem, Photosan-3, etc.). In some embodiments, the PDT sensitizing agents can include second and third generation photosensitizers, such as porphyrin-like derivatives and precursors. Porphyrin-like derivatives and precursors can include porphyrins and metalloporphyrins (e.g., meso-tetra(hydroxyphenyl)porphyrin (m-THPP), 5,10,15,20-tetra(4-sulfonatophenyl)-21H,23H-porphyrin (TPPS4), and precursors of endogenous protoporphyrin IX (PpIX): 5-aminolevulinic acid (5-ALA, which has been used for photodynamic therapy (PDT) of gliomas with some success (Stummer, W. et al. J Neurooncol. 2008, 87(1): 103-9.), methyl aminolevulinate (MAL), hexyl aminolevulinate (HAL)), chlorins (e.g., benzoporphyrin derivative monacid ring A (BPD-MA), meso-tetra(hydroxyphenyl)chlorin (m-THPC), N-aspartyl chlorin e6 (NPe6), and tin ethyl etioporphyrin (SnET2)), de- methoxychlorins (e.g., 2-(1-hexyloxyethyl)-2-devymethyl pyrode-methoxychlorin (HPPH)), bacterial de-methoxychlorins (e.g., bacteriochlorophyll a, WST09, and WST11), Texaphyrin (e.g., motexafin lutetium (Lu-Tex)), and phthalocyanines (PC) (e.g., aluminum phthalocyanine tetrasulfonate (AlPcS4) and silicon phthalocyanine (Pc4)). In some embodiments, the PDT sensitizing agents can include cationic zinc ethynylphenyl porphyrin. Although porphyrin-like structures comprise the majority of photosensitizers, some non-porphyrin chromophores exhibit photodynamic activity. These compounds include anthracenes, phenothiazines, xanthenes, fluorins, and curcuminoids. Alternatively, the photosensitizers can include indocyanine green (ICG).

[0154] In some embodiments, the diagnostic or therapeutic agents described herein can include a phthalocyanine compound. Phthalocyanines (hereinafter also abbreviated as “Pc”) are a group of photosensitizer compounds with a phthalocyanine ring system. Phthalocyanines are azaporphyrins consisting of four benzindole groups connected by nitrogen bridges to form a 16-membered ring with carbon and nitrogen atoms alternating (i.e., C32H16N8), which forms stable chelates with metal and metalloid cations. In these compounds, the center of the ring is occupied by a metal ion (diamagnetic or paramagnetic ion), which can carry one or two ligands depending on the ion. In addition, the periphery of the ring can be unsubstituted or substituted. Phthalocyanine compounds strongly absorb clinically useful red or near-IR radiation with absorption peaks falling between about 600 nm and 810 nm, which can allow light to penetrate deep into tissues. The synthesis and use of various phthalocyanines in photodynamic therapy are described in International Publication No. WO 2005 / 099689.

[0155] In some embodiments, the phthalocyanine compound is Pc4. Pc4 is relatively light resistant and almost non-toxic. In some embodiments, the phthalocyanine compound is an analog of the PDT photosensitizing drug Pc4, which has been found to be effective in cancer-targeted bioimaging and targeted PDT in a subject, see, e.g., U.S. Patent No: 9,889,199, the contents of which are incorporated herein by reference. In some embodiments, the Pc4 analog can include Pc413.

[0156] In other embodiments, the therapeutic or diagnostic agent is a nanobubble directly or indirectly linked to a targeting peptide via a peptide or peptoid spacer. The nanobubble can have a membrane defining at least one internal void comprising at least one gas and, optionally, at least one therapeutic agent contained within or conjugated to the membrane of each nanobubble. The therapeutic agent can include, for example, at least one chemotherapeutic agent, anti-proliferative agent, biocide, biostatic agent, or antimicrobial agent.

[0157] The agent comprising the targeting peptide, the peptide or peptoid spacer, and the nanobubble can be administered to a cancer subject. The targeting peptide can bind to a target cancer cell, and the nanobubble can have a size, diameter, and / or composition to facilitate the cancer cell-targeted nanobubble being internalized by the target cancer cell upon binding of the targeting peptide to the cancer cell. Upon administration of the target nanobubble to the subject, the cell-targeted nanobubble internalized into the target cell can be sonicated with ultrasound energy to effectively promote inertial cavitation of the internalized nanobubble and apoptosis and / or necrosis of the target cancer cell and / or release of the therapeutic agent (e.g., chemotherapeutic agent) from the nanobubble to the cancer cell.

[0158] In other embodiments, the therapeutic agent linked to the peptide or peptidomimetic spacer and targeting peptide can include an anti-cancer or anti-proliferative agent that exerts an anti-tumor, chemotherapeutic, anti-viral, anti-mitotic, anti-neoplastic, and / or immunotherapeutic effect, e.g., directly on tumor cells to prevent the development, maturation, or spread of tumor cells, e.g., by cytostatic or cytotoxic effects, rather than indirectly through mechanisms such as biological response modification. There are a large number of anti-proliferative agents in commercial use, clinical evaluation, and pre-clinical development. For ease of discussion, anti-proliferative agents are divided into the following categories, subtypes, and classes: ACE inhibitors, alkylating agents, angiogenesis inhibitors, angiostatin, anthracyclines / DNA intercalators, anticancer antibiotics or antibiotic-like drugs, antimetabolites, anti-metastatic compounds, asparaginase, bisphosphonates, cGMP phosphodiesterase inhibitors, calcium carbonate, cyclooxygenase-2 inhibitors, DHA derivatives, DNA topoisomerase, endostatin, epipodophyllotoxins, genistein, hormonal anticancer agents, hydrophilic bile acids (URSO), immunomodulatory or immunological agents, integrin antagonists, interferon antagonists or agents, MMP inhibitors, miscellaneous antineoplastic agents, monoclonal antibodies, nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors, pBATT, radiation / chemosensitizers / protectors, retinoids, selective inhibitors of endothelial cell proliferation and migration, selenium, solubility matrix inhibitors, taxanes, vaccines, and vinca alkaloids.

[0159] Some of the major classes of anti-proliferative agents include antimetabolites, alkylating agents, antibiotic-like drugs, hormonal anticancer agents, immunological agents, interferon-like drugs, and a miscellaneous class of antineoplastic agents. Some anti-proliferative agents act through multiple or unknown mechanisms, and thus can be classified in more than one class.

[0160] Examples of anti-cancer therapeutic agents that can be directly or indirectly linked to a targeting peptide in an agent described herein include taxol, adriamycin, dactinomycin, bleomycin, vinblastine, cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anagrelide; anastrozole; anthramycin; asparaginase; aspergillin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; efomithine hydrochloride; elsamitrucin; epirubicin hydrochloride; epithilone; estrogen; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; floxuridine; fludarabine; fluorouracil; fluvoxamine maleate; flutamide; fostriecin sodium salt; gandotinim; gemcitabine; gemtuzumab ozogamicin; gimatecan; gliozyme; hepsul-famide; herceptin; hexamethylmelamine; hydroxyurea; ibandronic acid; idarubicin; ifosfamide; ilmofosine; imiquimod; interferon alfa2a; interferon alfa2b; interferon alfa-nl; interferon alfa-n3; interferon alfaconl; interferon alpha-nl; interferon alpha-n3; interferon gamma-lb; interferon gamma-lb; itracenanth; ixabepilone; lentinan; letrozole; leucovorin; leuprolide acetate; liarozole; linear plutonium plutonyl complex; lobaplatin; lomustine; losoxantrone; lovastatin; loxorubicin; lurtotecan hydrochloride; lutetium texaphyrin; lysofyllate; macharidine A;Enloplatin; Enpromate; Epipropidine; Epirubicin hydrochloride; Erbulozole; Esorubicin hydrochloride; Estramustine; Estramustine phosphate sodium; Etanidazole; Etoposide; Etoposide phosphate; Etotrine; Fadrozole hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine phosphate; Fluorouracil; Fluorocitabine; Fosquidone; Fostriecin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Ilmofosine; Interleukin II (including recombinant interleukin II or rIL2), Interferon alpha-2a; Interferon alpha-2b; Interferon alpha-n1; Interferon alpha-n3; Interferon beta-I a; Interferon gamma-Ib; Iproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprolide acetate; Liarozole hydrochloride; Lometrexol sodium; Lomustine; Losoxantrone hydrochloride; Masoprocol; Maytansine; Mechlorethamine hydrochloride; Megestrol acetate; Melengestrol acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole;Nogalamycin; Ormaplatin; Oxisuran; Pegaspargase; Peliomycin; Pentamustine; Peplomycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; plicamycin; Plomestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium sodium); tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin;Tubulozole hydrochloride; Uracil mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vinglycinate sulfate; Vinleurosine sulfate; Vinorelbine tartrate; Vinrosidine sulfate; Vinzolidine sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin hydrochloride.

[0161] Other anticancer agents include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; arubicin; acylfulvene; adenocyclopentanol; adozelesin; aldesleukin; ALL-TK antagonists; hexamethylmelamine; ambamustine; amidox; amifostine; aminopentanoic acid; amrubicin; acridine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1. Protein-1); anti-androgen, prostate cancer; anti-estrogen; anti-tumor ketone; antisense oligonucleotide; afenidimycin glycine; apoptosis gene regulator; cell apoptosis regulator; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonist; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid acid); bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; butthionine sulfoximine; calcipotriol; calcium phosphoprotein C; camptothecin derivatives; canarypox IL-2; capecitabine; formamide-amino-triazole; carboxyamine-triazole; CaRest M3; CARN 700; chondroitin inhibitor; carzelesin; casein kinase inhibitors (ICOS);Iminosugar castanospermine; cecropin B; cetrorelix; chlorins; chloroquine sulfonalmines; cicaprost; cis-porphyrin; clabrivine; clomifene analogs; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogs; conagenin; cram bescidin 816; crisnatol; macrolide 8; macrolide A derivative; curacin A; cyclopentane quinone; cycloplatam; cypemycin; cytarabine ocfosfate; cytolysin; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemen; emitefur; epirubicin; epitiostanol; estramustine analogs; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;Galocitabine; Galunserin; Gelatinase inhibitor; Gemcitabine; Glutathione inhibitor; Hepsulfam; Heregulin; Hexamethylene bisacetamide; Hypericin; Ibandronate; Idarubicin; Idoxifene; Idramantone; Ilmofosine; Ilomastat; Imidazoacridones; Imiquimod; Immunostimulant peptide; Insulin-like growth factor-1 receptor inhibitor; Interferon agonists; Interferons; Interleukins; Iobenguane; Iododoxorubicin; Ipomeanol, 4-; Iroplact; Isoarrazine; Isobengazole; Isohomohalicondrin B; Itasetron; Jasplakinolide; Kahalalide F; Lamellarin-N triacetate; Lanreotide; Leinamycin; Lenograstim; Lentinan; Leptolstatin; Letrozole; Leukemia inhibitory factor; Leukocyte alpha interferon; Leuprolide + estrogen + progestin; Leuprolide; Levamisole; Liarozole; Linear polyamine analogues; Lipophilic bisnaphthalenylmaleamide; Lipophilic platinum compounds; Lissoclinamide 7; Lobaplatin; Lombricine; Lometrexol; Lonidamine; Losoxantrone; Lovastatin; Loxoribine; Lurtotecan; Lutetium texaphyrin; Lysofylline; Lytic peptide; Maitansine; Mannostatin A hydrochloride; Marimastat; Masoprocol; Mastsatin; Matrix metalloproteinase inhibitor; Menogaril; Merbarone; Meterelin; Methioninase; Metoclopramide; MIF inhibitor; Mifepristone; Miltefosine; Mirimostim; Misfolded double- chain RNA; Mitoguazone; Mitolactol; Mitomycin analogues; Mitonafide;Mitomycin Fibroblast Growth Factor-Saporin; Mitoxantrone; Mofarotene; Molgramostim; Monoclonal Antibody, Human Chorionic Gonadotropin; Monophosphoryl Lipid A + Mycobacterial Cell Wall Sk; Mopidamol; Multiple Drug Resistance Gene Inhibitor; Multitargeted Therapy Based on Tumor Suppressor 1; Mustard Anti-Cancer Agent; Mycaperoxide B; Mycobacterial Cell Wall Extract; Myriaporone; N-acetyldinaline; N-Substituted Benzamides; Nafarelin; Nagrestip; Naloxone + Pentazocine; Napavin; Naphterpin; Nartograstim; Nedaplatin; Nemorubicin; Neridronic Acid; Neutral Endopeptidase; Nilutamide; Nisamycin; Nitric Oxide Modulators; Nitric Oxide Antioxidants; Nitrullyn; 06-Benzylguanine; Octreotide; Okicenone; Oligonucleotide; Onapristone; Ondansetron; Ondansetron; Oracin; Oral Cytokine Inducers; Ormaplatin; Osaterone; Oxaliplatin; Oxaunomycin; Palauamine; Palmitoylrhizoxin; Pamidronic Acid; Panaxydol; Panomifene; Parabactin; Pazelliptine; Pegaspargase; Peldesine; Pentosan Polysulfate Sodium; Pentostatin; Pentrozole; Perflubron; Perfosfamide; Perillyl Alcohol; Phenazinomycin; Phenylacetates; Phosphoguanidin; Picibanil; Pilocarpine Hydrochloride; Pirarubicin; Piritrexim; Placetin A; Placetin B; Plasminogen Activator Inhibitor; Platinum Complexes; Platinum Compounds; Platinum-Triamine Complexes; Porfimer Sodium; Porfiromycin; Prednisone; Propyl Bis-Acridone; Prostaglandin J2; Proteasome Inhibitors; Protein A-Based Immuno-Modulators; Protein Kinase C Inhibitor; Protein Kinase C Inhibitor; Protox; Protein Tyrosine Phosphatase Inhibitors; Purine Nucleoside Phosphorylase Inhibitors; Purpurins; Pyrazoloacridine; Pyridoxylated Hemoglobin Polyoxyethylene Conjugate;raf antagonists; raltitrexed; ramucirumab; ras farnesyl-protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamides; rohitukine; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 analogues; semustine; senolytic 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single-chain antigen-binding proteins; silicon phthalocyanine (PC4) sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; spicamycin D; spiroplatin; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitors; stipiamide; stromelysin inhibitors; sulfonylureas; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodectin; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimics; thymalfasin; thymopoietin receptor agonists; thymotrinan; thyrotropin; tin ethyl etiopurpurin; tirapazamine; titanium dichloride; toptsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin;Tolcapone; Tolperisone; Turosteride; Tyrosine kinase inhibitors; Tyrosine protease; UBC inhibitors; Uracil mustard; Urodynamic growth inhibitory factor; Urokinase receptor antagonists; Vapreotide; Variolin B; Vector systems, red blood cell gene therapy; Velaresol; Veramine; Verdins; Verteporfin; Vinorelbine; Vinxaltine; Vitaxin; Vorozole; Zanoterone; Zeniplatin; Zilascorb; and Zinostatin stimalamer.

[0162] Other anticancer drugs can include the following marketed and developing drugs: Erbulozole (also known as R-55104), Dolastatin 10 (also known as DLS-10 and NSC-376128), Mivobulin isethionate (also known as CI-980), Vincristine, NSC-639829, Discodermolide (also known as NVP-XX-A-296), ABT-751 (Abbott, also known as E-7010), Altorhyrtins (e.g., Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g., Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (also known as LU-103793 and NSC-D-669356), Epothilones (e.g., Epothilone A, Epothilone B, Epothilone C (also known as desoxyepothilone A or dEpoA), Epothilone D (also known as KOS-862, dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide, 16-Aza-epothilone B, 21-aminoepothilone B (also known as BMS-310705), 21-hydroxyepothilone D (also known as desoxyepothilone F and dEpoF), 26-fluoroepothilone, Auristatin PE (also known as NSC-654663), Soblidotin (also known as TZT-1027), LS-4559-P (Pharmacia, also known as LS-4577), LS-4578 (Pharmacia, also known as LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, also known as WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF,also known as ILX-651 and LU-223651), SAH-49960 (Lilly / Novartis), SDZ-268970 (Lilly / Novartis), AM-97 (Armad / Kyowa Hakko), AM-132 (Armad), AM-138 (Armad / Kyowa Hakko), IDN-5005 (Indena), Macrolide 52 (also known as LY-355703), AC-7739 (Ajinomoto, also known as AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCl and RPR-258062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (also known as NSC-106969), T-138067 (Tularik, also known as T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), Anticancerin Al (also known as BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, also known as SPIKET-P, 3-IAABU (Cytoskeleton / Mt. Sinai School of Medicine, also known as MF-569), Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton / Mt. Sinai School of Medicine,also known as MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, Inanocine (also known as NSC-698666), 3-IAABE (Cytoskeleton / Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tularik, also known as T-900607), RPR-115781 (Aventis), Eleutherobin (e.g., Desmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (-)-Phenylahistin (also known as NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris, also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (also known as SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCl), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi).

[0163] Other anti-cancer therapeutic agents include alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, chlorambucil, melphalan, etc.), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozotocin, etc.), or triazenes (decabenzene, etc.), antimetabolites, such as folic acid analogs (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxuridine, cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin), vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide, teniposide), platinum coordination complexes (e.g., cisplatin, carboplatin), anthracenediones (e.g., mitoxantrone), substituted ureas (e.g., hydroxyurea), methyl hydrazine derivatives (e.g., procarbazine), adrenocortical suppressors (e.g., mitotane, amino glutethimide).

[0164] In some embodiments, cytotoxic compounds are included in the agents described herein. Cytotoxic compounds include small molecule drugs, such as doxorubicin, mitoxantrone, methotrexate, and pyrimidine and purine analogs, referred to herein as antineoplastic agents.

[0165] The agents described herein, including targeting peptides, spacers, and therapeutic agents, can be administered to a subject by any of the conventional methods of pharmacological administration, e.g., oral 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 meals), nasally (e.g., solutions, suspensions), transdermally, intradermally, topically (e.g., creams, ointments), transmucosally (e.g., intrabronchially, intranasally, orally, or intranasal drops), or rectally. Delivery can also be by injection into the brain or body cavity of the patient or by use of a timed release or sustained release matrix delivery system, or by use of micelles, gels, and liposomes for in situ delivery. Nebulizing devices, powder inhalers, and nebulized solutions can also be used to administer such formulations to the respiratory tract. Delivery can be in vivo or ex vivo. Administration can be local or systemic, as indicated. If desired, more than one route can be used simultaneously. The preferred mode of administration can vary depending on the particular disclosed compound chosen. In particular embodiments, oral, parenteral, or systemic administration is the preferred mode of treatment.

[0166] The agent comprising a targeting peptide, peptide, or peptidomimetic spacer and a therapeutic agent described herein can be administered alone as a monotherapy or in conjunction or in combination with one or more additional therapeutic agents. For example, an agent comprising a targeting peptide described herein linked to a therapeutic agent can be administered to a subject prior to, during, or after administration of an additional therapeutic agent, and can target the distribution of metastatic cells with the therapeutic agent. The agent can be administered to an animal as part of a pharmaceutical composition comprising the agent and a pharmaceutically acceptable carrier or excipient and, optionally, one or more additional therapeutic agents. The agent comprising a targeting peptide, peptide, or peptidomimetic spacer and a therapeutic agent described herein and the additional therapeutic agent can be components of separate pharmaceutical compositions, they can be mixed together prior to being administered or administered separately. The agent comprising a targeting peptide, peptide, or peptidomimetic spacer and a therapeutic agent described herein, for example, is administered in a composition containing the additional therapeutic agent, so as to be administered concurrently with the agent. Alternatively, the agent comprising a targeting peptide, peptide, or peptidomimetic spacer and a therapeutic agent described herein can be administered concurrently without mixing (e.g., by delivering the agent on an intravenous line also used to administer the therapeutic agent, or vice versa). In another embodiment, the agent comprising a targeting peptide, peptide, or peptidomimetic spacer and a therapeutic agent described herein can be administered separately (e.g., not mixed), but within a short time frame (e.g., within 24 hours) of administration of the therapeutic agent.

[0167] The methods described herein contemplate single administrations as well as multiple administrations, either concurrently or over an extended period of time. The agent comprising a targeting peptide, peptide, or peptidomimetic spacer and a therapeutic agent described herein (or a composition containing the agent) can be administered continuously at regular intervals depending on the nature and extent of the inflammatory condition effect. As used herein, administration at regular intervals means that a therapeutically effective amount is administered periodically (as opposed to a one-time dose). In one embodiment, the agent and / or additional therapeutic agent is administered periodically, e.g., at regular intervals (e.g., once every two months, once a month, once every two weeks, once a week, twice a week, once a day, twice a day, or three or more times a day).

[0168] The intervals of administration for individual subjects can be fixed or can vary over time, depending on the needs of the subject. For example, the dosing interval can be shortened when physical illness or stress occurs, or if the symptoms of the disease worsen. Depending on the half-life of the detectable moiety, therapeutic agent, or diagnostic agent in the subject, the agent can be administered, for example, once a day or once a week.

[0169] For example, administration of the agent and / or additional therapeutic agent can be performed at least once on days 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, optionally, at least once on weeks 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 every 60 hours, 48 hours, 36 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, or 2 hours. Administration can be performed at any time of day (e.g., in the morning, afternoon, or evening). For example, administration can be performed in the morning (e.g., between 6:00 AM and 12:00 PM); in the afternoon (e.g., after noon and before 6:00 PM); or in the evening (e.g., between 6:01 PM and midnight).

[0170] Agents and / or additional therapeutic agents comprising a targeting peptide, peptide, or peptidomimetic spacer and a therapeutic agent described herein can be administered at a dose of, for example, 0.1 to 100 mg / kg per day, e.g., 0.5, 0.9, 1.0, 1.1, 1.5, 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, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg. Dosage forms (compositions) adapted for oral administration typically contain from about 0.1 mg to about 500 mg of active ingredient per unit. In these pharmaceutical compositions, the active ingredient will ordinarily be present in an amount of from about 0.5 to 95% weight based on the total weight of the composition.

[0171] The amount of the disclosed agents and / or additional therapeutic agents comprising a targeting peptide, peptide, or peptidomimetic spacer and a therapeutic agent described herein administered to a 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 extent, severity, and type of rejection. The skilled artisan will be able to determine appropriate dosages using standard clinical techniques. Generally, the dose employed will fall within the range of from about 0.1 to 100 mg / kg, e.g., 0.5, 0.9, 1.0, 1.1, 1.5, 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, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg.

[0172] In addition, in vitro or in vivo assays can be employed to determine the appropriate dosage range. The dosage employed can also depend on the route of administration, the severity of the disease, and the condition of the subject. Effective doses can be extrapolated from dose- response curves derived from in vitro or animal model test systems. The amount of agent, including the targeting peptide, peptide or peptidomimetic spacer and therapeutic agent described herein, to be administered can also depend on the disease state or condition being treated as well as clinical factors and the route of administration of the compound.

[0173] The disclosed agents and / or additional therapeutic agents described herein can be administered to a subject with an acceptable pharmaceutical carrier or diluent as part of a pharmaceutical composition for treatment. The formulation of the compound to be administered will vary depending on the route of administration chosen (e.g., solution, emulsion, capsule, etc.). Suitable pharmaceutically acceptable carriers can contain inert ingredients that do not unduly inhibit the biological activity of the compound. The pharmaceutically acceptable carrier should be biologically compatible, e.g., non-toxic, non-inflammatory, non-immunogenic, and free of other undesirable reactions when administered to a subject. Standard pharmaceutical formulation techniques can be employed, e.g., those described in Remington's Pharmaceutical Sciences, as previously noted. Pharmaceutical carriers suitable for parenteral administration include, e.g., sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate buffered saline, Hank's solution, Ringer's lactated solution, etc. Methods for encapsulating compositions, e.g., in coatings of hard gelatin or cyclodextrin, are known in the art (Baker, et al., "Controlled Release of Biological Active Agents", John Wiley and Sons, 1986).

[0174] The preparation of a pharmaceutical composition that contains active ingredients dissolved or dispersed therein is well understood. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution or suspension in liquid prior to use can also be prepared. The formulation will depend on the route of administration chosen (e.g., solution, emulsion, capsule).

[0175] Pharmaceutically acceptable carriers for pharmaceutical compositions can also include delivery systems known in the art for entrapping or encapsulating drugs (e.g., anticancer drugs). In some embodiments, the disclosed compounds can be used with such delivery systems, including, for example, liposomes, nanoparticles, nanospheres, nanodisks, dendrimers, and the like. See, e.g., 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 of the references cited in this paragraph are incorporated herein by reference.

[0176] The following examples are included to demonstrate preferred embodiments.

[0177] Examples

[0178] Methods

[0179] Peptide synthesis and conjugation

[0180] SBK-targeting peptides (e.g., GEGDDFNWEQVNTLTKPTSD (SEQ ID NO:5)) and disordered peptides (GTQDETGNFDWPVSEDLNKT (SEQ ID NO:47)) were synthesized on a synthesizer at Case Western Reserve University or purchased from PolyPeptide Group (San Diego, CA). During synthesis, N-terminal glycine or glycine / serine spacers were added to the peptides. After synthesis, the N-terminal glycine residue of each peptide spacer was specifically coupled to Texas Red (TR)-X (a single isomer), which has a five-carbon spacer between the succinimide group coupled to the N-terminal amine and the fluorophore. Optionally, the peptide was coupled to indocyanine green (ICG). Amino acid spacers of various lengths can also be added during peptide synthesis. N-terminal cysteine ​​residues can also be used for conjugation.

[0181] Cell culture and orthotopic xenograft flank tumor implantation

[0182] Human U87-MG and LN-229 glioma cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured. NIH athymic nude mice (5-8 weeks old and weighing 20-25g; NCI-NIH) were housed at the Athymic Animal Core Facility at Case Western Reserve University in accordance with institutional policy. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC). Cells were diluted in a 1:1 mixture of PBS and BDMATRIGEL matrix (BD Biosciences, Franklin Lakes, NJ, USA) and injected into the right flank of athymic nude mice (NCr-nu / +, NCr-nu / nu, 20-25g each). The Matrigel-cell mixture was loaded into a 1ml syringe fitted with a 26-gauge needle and kept on ice. The mixture was subcutaneously injected into the right flank region of mice. The flank tumor grew for 2 to 3 weeks. 1.4–2 × 10⁸ mcg was implanted into each flank. 6 To correlate tumor location with GFP fluorescence, mice were imaged using the Perkin-Elmer MAESTRO FLEX in vivo imaging system. For in vivo analysis, mice were anesthetized with inhaled isoflurane / oxygen and then imaged. For ex vivo analysis, mice were euthanized by decapitation. The flank tumor was then removed and imaged.

[0183] In vivo imaging of flank tumors

[0184] Nude mice bearing ectopic (flank) tumors are typically imaged 3 to 6 weeks after tumor induction. Nude mice bearing orthotopic (intracranial) tumors are typically imaged 7 to 14 days after tumor cell implantation. Fluorophore-conjugated PTPm peptides are diluted to 100-200 mM and injected via lateral tail vein to administer the desired dose of reagent, typically between 100-400 nmol / kg. In animals using tumor cells expressing green fluorescent protein (GFP) to facilitate monitoring of tumor growth and / or migration, a fluorophore with limited spectral overlap with GFP is used. In vivo and ex vivo images of specific tissues are obtained using an IVIS Spectrum In Vivo Imaging System (Perkin Elmer, Waltham, MA, USA) using the manufacturer’s recommended excitation and emission filters for the given fluorophore and the built-in auto-exposure function. The following are some examples of filter pairs used for different fluorophores: 465 / 520 for GFP; 465 / 520 for GFP; Texas Red, 570 / 620; Cy5, 640 / 680; IR800CW, 745 / 800; Indocyanine Green (ICG), 745 / 820. A background image is acquired prior to injection of any fluorescent peptide for baseline measurements. Animals are imaged ten minutes after peptide injection and every ten minutes as needed up to 2 h. For some fluorescent peptides, additional in vivo images are acquired between 8 h and 24 h. After the last in vivo imaging, mice are euthanized. Flank tumors or the entire brain with intracranial tumors and other organs of interest are excised and imaged ex vivo. Data are imported into Living Image software (Perkin Elmer) for image analysis and binning is set to 1. Region of interest (ROI) analysis is used to examine the fluorescent signal obtained in a specific location (using a ROI of defined size) or in the whole mouse or organ (using a ROI encompassing the body or organ). The mean radiance units per ROI are calculated by Living Image software. Each fluorescent PTPm peptide is tested on at least three tumor-bearing animals. Statistical analysis is performed using Microsoft Excel and unpaired Student’s t test.Spectrum or MAESTRO FLEX In vivo Imaging System (Cambridge Research & Instrumentation (CRi), Woburn, MA) as previously described. The excised whole brains were imaged using the filters described for GFP (tumor cells) and the various fluorophores.

[0185] Ex vivo imaging of tumors

[0186] Ex vivo imaging was performed after injection of SBK peptide reagents in live tumor-bearing mice to allow for clearance of unbound reagents. After different time intervals for clearance of unbound reagents, animals were sacrificed and brains excised for ex vivo optical imaging and histology. Imaging was performed using Spectrum or MAESTRO FLEX In vivo Imaging System (Cambridge Research & Instrumentation (CRi), Woburn, MA) as previously described. Excised whole brains were imaged using the filters described for GFP (tumor cells) and the various fluorophores.

[0187] Orthotopic xenograft intracranial tumors

[0188] NIH-bred athymic female nude mice (NCr-nu / +, NCr-nu / nu) were bred at the Athymic Animal Core Facility and housed at the Case Center for Imaging Research at Case Western Reserve University according to an animal protocol approved by the Institutional Animal Care and Use Committee. Human U-87MG glioma cells were obtained from the American Type Culture Collection. CNS-1 rodent glioma cells were obtained from Mariano S. Viapiano. SJ-GBM2 cells were derived from post-mortem 5-year-old female GBM patients and were obtained from pediatric tumor group cell lines and xenograft banks. Cells were infected with lentiviruses to express green fluorescent protein (GFP) or m-Cherry, as directed, and intracranial implantation of tumor cells was performed as described. Briefly, 6- to 7-week-old mice were anesthetized and placed in stereotactic rodent frames (David Kopf Instruments, Tujunga, California). Small holes were drilled 0.7 mm anterior to and 2 mm lateral to the anterior fontanelle. Cells were collected for intracranial implantation and placed into the right striatum at a depth of -3 mm from the dura mater using a 10 μL syringe. A total of 2 × 10⁶ cells were injected. 5 4.5 × 10 U-87MG cells 4 One CNS-1 cell or 3 × 10 5 SJ-GBM2 cells were collected. The needle was slowly withdrawn, and the incision was sutured. Mice were imaged as described below, and then sacrificed 8–21 days after tumor implantation. Brain tissue was collected for imaging and histological processing.

[0189] In vivo labeling of intracranial tumors

[0190] Nude mice bearing intracranial tumors were imaged 9 to 12 days after GBM cell implantation. Fluorophore-conjugated PTPm peptides were injected via the tail vein. After a 25-minute incubation to clear unbound PTPm peptides, animals were sacrificed, brains were removed and imaged whole or cut into coronal sections at 1 mm intervals. Individual brain sections containing tumors were placed on black slides and examined using the Spectrum or Maestro FLEX in vivo imaging system as described above. Untreated brains containing intracranial tumors were used to provide a spectrum of autofluorescence. ROIs were selected in the tumor region of each brain section. Pixel values for peptide signal were determined within these ROIs to measure photons counted from the section. Multispectral fluorescence images were background subtracted and analyzed using Maestro software as previously described. Statistical analysis was performed using Microsoft Excel and unpaired Student's t test.

[0191] Results

[0192] Figures 1 to 7 The binding and in vivo average radiant efficiencies of various imaging agents administered to mice bearing ectopic xenograft flank U87 tumor implants were compared. The imaging agents included SBK targeting peptides linked to a fluorophore via a polyglycine or glycine / serine spacer, or control scrambled peptides linked directly to a fluorophore or via a polyglycine or glycine / serine spacer.

[0193] Figure 1 A graph showing the in vivo average radiant efficiencies of a first agent (Peptide 1, which includes SBK linked to a fluorophore without a peptide spacer), a second agent (Peptide 2, which includes SBK linked to a fluorophore via a polyglycine peptide spacer), and a control agent (Scrambled, which includes a scrambled peptide linked to a fluorophore via a polyglycine peptide spacer) administered to mice bearing ectopic xenograft flank U87 tumor implants is shown. In vivo imaging of the mice's flank tumors showed that the second agent (including a polyglycine peptide spacer) had a higher average radiant efficiency compared to the first agent (without a spacer) and the control agent (including a polyglycine peptide spacer).

[0194] Figure 2A graph showing in vivo average radiance efficiency of a second agent (peptide 2, which includes SBK linked to a fluorophore by a polyglycine spacer) and a third agent (peptide 3, which includes SBK linked to a fluorophore by a glycine / serine spacer) displayed by mice administered with either an ectopic xenograft U87 flank tumor implant or a mouse with an ectopic xenograft U87 flank tumor implant overexpressing PTPmu. In vivo imaging of U87 flank tumors and U87 flank tumors overexpressing PTPmu showed that the third agent (including a glycine / serine peptide spacer) had a higher average radiance efficiency in U87 flank tumors and U87 flank tumors overexpressing PTPmu compared to the second agent (with a polyglycine spacer).

[0195] Figure 3 A graph showing in vivo average radiance efficiency of a third agent (peptide 3, which includes SBK linked to a fluorophore by a glycine / serine peptide spacer) and a fourth agent (peptide 4, which includes SBK linked to a fluorophore by a second glycine / serine spacer) displayed by mice administered with either an ectopic xenograft U87 flank tumor implant or a mouse with an ectopic xenograft U87 flank tumor implant overexpressing PTPmu.

[0196] Figure 4 Ex vivo images and graphs showing average radiance efficiency of a first agent (peptide 1, which includes SBK linked to a fluorophore without a peptide spacer) and a control agent (scramble 1, which includes a scrambled peptide linked to a fluorophore by a polyglycine peptide spacer of peptide 1) after being administered in vivo to mice with an ectopic xenograft U87 flank tumor implant.

[0197] Figure 5 Ex vivo images and graphs showing average radiance efficiency of a second agent (peptide 2, which includes SBK linked to a fluorophore by a polyglycine peptide spacer), a third agent (peptide 3, which includes SBK linked to a fluorophore by a glycine / serine peptide spacer), and control agents (scramble 2, which includes a scrambled peptide linked to a fluorophore by a polyglycine spacer of peptide 2, and scramble 3, which includes a scrambled peptide linked to a fluorophore by a glycine / serine spacer of peptide 3) after being administered in vivo to mice with an ectopic xenograft U87 flank tumor implant.

[0198] Figure 6Ex vivo images and graphs showing the mean radiance efficiency after in vivo administration of a second agent (peptide 2, which includes SBK connected to a fluorophore by a polyglycine peptide spacer), a third agent (peptide 3, which includes SBK connected to a fluorophore by a glycine / serine peptide spacer), and control agents (scramble 2, which includes a scrambled peptide connected to a fluorophore by the polyglycine spacer of peptide 2, and scramble 3, which includes a scrambled peptide connected to a fluorophore by the glycine / serine spacer of peptide 3) to mice bearing ectopic xenograft U87 flank tumor implants overexpressing PTPmu.

[0199] Figure 7 Ex vivo images and graphs showing the mean radiance efficiency after in vivo administration of a third agent (peptide 3, which includes SBK connected to a fluorophore by a glycine / serine peptide spacer), a fourth agent (peptide 4, which includes SBK connected to a fluorophore by a second glycine / serine spacer), and control agents (scramble 3, which includes a scrambled peptide connected to a fluorophore by the glycine / serine spacer of peptide 3, and scramble 4, which includes a scrambled peptide connected to a fluorophore by the glycine / serine spacer of peptide 4) to mice bearing ectopic xenograft U87 flank tumor implants overexpressing PTPmu.

[0200] Figures 8 to 12 The binding and in vivo mean radiance efficiency of various imaging agents administered to mice bearing orthotopic xenograft U87 intracranial tumors is compared. The imaging agents include SBK targeting peptides connected to a fluorophore by a polyglycine or glycine / serine spacer, or control scrambled peptides connected directly to a fluorophore or by a polyglycine or glycine / serine spacer.

[0201] Figure 8 Ex vivo images and graphs showing the mean radiance efficiency after in vivo administration of a first agent (peptide 1, which includes SBK connected to a fluorophore without a peptide spacer) compared to a control agent (scramble, which includes a scrambled peptide connected to a fluorophore by a polyglycine peptide spacer) to mice bearing orthotopic xenograft U87 intracranial tumors.

[0202] Figure 9 Ex vivo images and graphs showing the mean radiance efficiency after in vivo administration of a third agent (peptide 3, which includes SBK connected to a fluorophore by a glycine / serine peptide spacer) compared to a control agent (scramble 3, which includes a scrambled peptide connected to a fluorophore by the glycine / serine spacer of peptide 3) to mice bearing orthotopic xenograft U87 intracranial tumors.

[0203] Figure 10Ex vivo maestro images overlaid on brain black and white photos showing the third agent (peptide 3, which includes SBK attached to a fluorophore via a glycine / serine peptide spacer), the fourth agent (peptide 4, which includes SBK attached to a fluorophore via a second glycine / serine spacer), and control agents (scramble 3, which includes a scrambled peptide attached to a fluorophore via the glycine / serine spacer of peptide 3, and scramble 4, which includes a scrambled peptide attached to a fluorophore via the glycine / serine spacer of peptide 4) after in vivo administration to mice bearing orthotopic U87 intracranial tumors.

[0204] Figure 11 Ex vivo maestro images overlaid on brain black and white photos showing the third agent (peptide 3, which includes SBK attached to a fluorophore via a glycine / serine peptide spacer), the fourth agent (peptide 4, which includes SBK attached to a fluorophore via a second glycine / serine spacer), and control agents (scramble 3, which includes a scrambled peptide attached to a fluorophore via the glycine / serine spacer of peptide 3, and scramble 4, which includes a scrambled peptide attached to a fluorophore via the glycine / serine spacer of peptide 4) after in vivo administration to mice bearing orthotopic U87 intracranial tumors.

[0205] Figure 12 Graphs showing the maximum signal intensity of the third agent (peptide 3, which includes SBK attached to a fluorophore via a glycine / serine peptide spacer), the fourth agent (peptide 4, which includes SBK attached to a fluorophore via a second glycine / serine spacer), and control agents (scramble 3, which includes a scrambled peptide attached to a fluorophore via the glycine / serine spacer of peptide 3, and scramble 4, which includes a scrambled peptide attached to a fluorophore via the glycine / serine spacer of peptide 4) after in vivo administration to mice bearing orthotopic U87 intracranial tumors.

[0206] While this application has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the application encompassed by the appended claims. All patents, publications, and references cited in the above specification are herein incorporated by reference in their entirety. SEQUENCE LISTING <110> CASS Western Reserve University Susann, BRADY-KALNAY <120> Methods and agents for detecting and treating cancer <130> CWR-029758WO ORD <150> 63 / 062,053 <151> 2020-08-06 <160> 47 <170> PatentIn Version 3.5 <210> 1 <211> 1465 <212> PRT <213> Homo sapiens <400> 1 Met Arg Gly Leu Gly Thr Cys Leu Ala Thr Leu Ala Gly Leu Leu Leu 1 5 10 15 Thr Ala Ala Gly Glu Thr Phe Ser Gly Gly Cys Leu Phe Asp Glu Pro 20 25 30 Tyr Ser Thr Cys Gly Tyr Ser Gln Ser Glu Gly Asp Asp Phe Asn Trp 35 40 45 Glu Gln Val Asn Thr Leu Thr Lys Pro Thr Ser Asp Pro Trp Met Pro 50 55 60 Ser Gly Ser Phe Met Leu Val Asn Ala Ser Gly Arg Pro Glu Gly Gln 65 70 75 80 Arg Ala His Leu Leu Leu Pro Gln Leu Lys Glu Asn Asp Thr His Cys 85 90 95 Ile Asp Phe His Tyr Phe Val Ser Ser Lys Ser Asn Ser Pro Pro Gly 100 105 - 110 Leu Leu Asn Val Tyr Val Lys Val Asn Asn Gly Pro Leu Gly Asn Pro 115 120 125 Ile Trp Asn Ile Ser Gly Asp Pro Thr Arg Thr Trp Asn Arg Ala Glu 130 135 140 Leu Ala lie Ser Thr Phe Trp Pro Asn Phe Tyr Gin Val lie Phe Glu 145 150 155 160 Val lie Thr Ser Gly His Gin Gly Tyr Leu Ala lie Asp Glu Val Lys 165 170 175 Val Leu Gly His Pro Cys Thr Arg Thr Pro His Phe Leu Arg lie Gin 180 185 190 Asn Val Glu Val Asn Ala Gly Gin Phe Ala Thr Phe Gin Cys Ser Ala 195 200 205 lie Gly Arg Thr Val Ala Gly Asp Arg Leu Trp Leu Gin Gly lie Asp 210 215 220 Val Arg Asp Ala Pro Leu Lys Glu lie Lys Val Thr Ser Ser Arg Arg 225 230 235 240 Phe lie Ala Ser Phe Asn Val Val Asn Thr Thr Lys Arg Asp Ala Gly 245 250 255 Lys Tyr Arg Cys Met lie Arg Thr Glu Gly Gly Val Gly lie Ser Asn 260 265 270 Tyr Ala Glu Leu Val Val Lys Glu Pro Pro Val Pro lie Ala Pro Pro 275 280 285 Gln Leu Ala Ser Val Gly Ala Thr Tyr Leu Trp Ile Gin Leu Asn Ala 290 295 300 Asn Ser lie Asn Gly Asp Gly Pro lie Val Ala Arg Glu Val Glu Tyr 305 310 315 320 Cys Thr Ala Ser Gly Ser Trp Asn Asp Arg Gin Pro Val Asp Ser Thr 325 330 335 Ser Tyr Lys lie Gly His Leu Asp Pro Asp Thr Glu Tyr Glu lie Ser 340 345 350 Val Leu Leu Thr Arg Pro Gly Glu Gly Gly Thr Gly Ser Pro Gly Pro 355 360 365 Ala Leu Arg Thr Arg Thr Lys Cys Ala Asp Pro Met Arg Gly Pro Arg 370 375 380 Lys Leu Glu Val Val Glu Val Lys Ser Arg Gin lie Thr lie Arg Trp 385 390 395 400 Glu Pro Phe Gly Tyr Asn Val Thr Arg Cys His Ser Tyr Asn Leu Thr 405 410 415 Val His Tyr Cys Tyr Gin Val Gly Gly Gin Glu Gin Val Arg Glu Glu 420 425 430 Val Ser Trp Asp Thr Glu Asn Ser His Pro Gin His Thr lie Thr Asn 435 440 445 Leu Ser Pro Tyr Thr Asn Val Ser Val Lys Leu Ile Leu Met Asn Pro 450 455 460 Glu Gly Arg Lys Glu Ser Gln Glu Leu Ile Val Gln Thr Asp Glu Asp 465 470 475 480 Leu Pro Gly Ala Val Pro Thr Glu Ser Ile Gln Gly Ser Thr Phe Glu 485 490 495 Glu Lys Ile Phe Leu Gln Trp Arg Glu Pro Thr Gln Thr Tyr Gly Val 500 505 510 Ile Thr Leu Tyr Glu Ile Thr Tyr Lys Ala Val Ser Ser Phe Asp Pro 515 520 525 Glu Ile Asp Leu Ser Asn Gln Ser Gly Arg Val Ser Lys Leu Gly Asn 530 535 540 Glu Thr His Phe Leu Phe Phe Gly Leu Tyr Pro Gly Thr Thr Tyr Ser 545 550 555 560 Phe Thr Ile Arg Ala Ser Thr Ala Lys Gly Phe Gly Pro Pro Ala Thr 565 570 575 Asn Gln Phe Thr Thr Lys Ile Ser Ala Pro Ser Met Pro Ala Tyr Glu 580 585 590 Leu Glu Thr Pro Leu Asn Gln Thr Asp Asn Thr Val Thr Val Met Leu 595 600 605 Lys Pro Ala His Ser Arg Gly Ala Pro Val Ser Val Tyr Gin He Val 610 615 620 Val Glu Glu Glu Arg Pro Arg Arg Thr Lys Lys Thr Thr Glu He Leu 625 630 635 640 Lys Cys Tyr Pro Val Pro He His Phe Gin Asn Ala Ser Leu Leu Asn 645 650 655 Ser Gin Tyr Tyr Phe Ala Ala Glu Phe Pro Ala Asp Ser Leu Gin Ala 660 665 670 Ala Gin Pro Phe Thr He Gly Asp Asn Lys Thr Tyr Asn Gly Tyr Trp 675 680 685 Asn Thr Pro Leu Leu Pro Tyr Lys Ser Tyr Arg He Tyr Phe Gin Ala 690 695 700 Ala Ser Arg Ala Asn Gly Glu Thr Lys He Asp Cys Val Gin Val Ala 705 710 715 720 Thr Lys Gly Ala Ala Thr Pro Lys Pro Val Pro Glu Pro Glu Lys Gin 725 730 735 Thr Asp His Thr Val Lys He Ala Gly Val He Ala Gly He Leu Leu 740 745 750 Phe Val He He Phe Leu Gly Val Val Leu Val Met Lys Lys Arg Lys 755 760 765 Leu Ala Lys Lys Arg Lys Glu Thr Met Ser Ser Thr Arg Gin Glu Met 770 775 780 Thr Val Met Val Asn Ser Met Asp Lys Ser Tyr Ala Glu Gin Gly Thr 785 790 795 800 Asn Cys Asp Glu Ala Phe Ser Phe Met Asp Thr His Asn Leu Asn Gly 805 810 815 Arg Ser Val Ser Ser Pro Ser Ser Phe Thr Met Lys Thr Asn Thr Leu 820 825 830 Ser Thr Ser Val Pro Asn Ser Tyr Tyr Pro Asp Pro Phe Val Pro Thr 835 840 845 Ala Ile Leu Val Pro Ile Asn Asp Glu Thr His Thr Met Ala Ser Asp 850 855 860 Thr Ser Ser Leu Val Gin Ser His Thr Tyr Lys Lys Arg Glu Pro Ala 865 870 875 880 Asp Val Pro Tyr Gin Thr Gly Gin Leu His Pro Ala Ile Arg Val Ala 885 890 895 Asp Leu Leu Gin His Ile Thr Gin Met Lys Cys Ala Glu Gly Tyr Gly 900 905 910 Phe Lys Glu Glu Tyr Glu Ser Phe Phe Glu Gly Gin Ser Ala Pro Trp 915 920 925 Asp Ser Ala Lys Lys Asp Glu Asn Arg Met Lys Asn Arg Tyr Gly Asn 930 935 940 Ile Ile Ala Tyr Asp His Ser Arg Val Arg Leu Gin Thr Ile Glu Gly 945 950 955 960 Asp Thr Asn Ser Asp Tyr Ile Asn Gly Asn Tyr Ile Asp Gly Tyr His 965 970 975 Arg Pro Asn His Tyr Ile Ala Thr Gin Gly Pro Met Gin Glu Thr Ile 980 985 990 Tyr Asp Phe Trp Arg Met Val Trp His Glu Asn Thr Ala Ser Ile Ile 995 1000 1005 Met Val Thr Asn Leu Val Glu Val Gly Arg Val Lys Cys Cys Lys 1010 1015 1020 Tyr Trp Pro Asp Asp Thr Glu Ile Tyr Lys Asp Ile Lys Val Thr 1025 1030 1035 Leu Ile Glu Thr Glu Leu Leu Ala Glu Tyr Val Ile Arg Thr Phe 1040 1045 1050 Ala Val Glu Lys Arg Gly Val His Glu Ile Arg Glu Ile Arg Gin 1055 1060 1065 Phe His Phe Thr Gly Trp Pro Asp His Gly Val Pro Tyr His Ala 1070 1075 1080 Thr Gly Leu Leu Gly Phe Val Arg Gin Val Lys Ser Lys Ser Pro 1085 1090 1095 Pro Ser Ala Gly Pro Leu Val Val His Cys Ser Ala Gly Ala Gly 1100 1105 1110 Arg Thr Gly Cys Phe Ile Val Ile Asp Ile Met Leu Asp Met Ala 1115 1120 1125 Glu Arg Glu Gly Val Val Asp Ile Tyr Asn Cys Val Arg Glu Leu 1130 1135 1140 Arg Ser Arg Arg Val Asn Met Val Gin Thr Glu Glu Gin Tyr Val 1145 1150 1155 Phe Ile His Asp Ala Ile Leu Glu Ala Cys Leu Cys Gly Asp Thr 1160 1165 1170 Ser Val Pro Ala Ser Gin Val Arg Ser Leu Tyr Tyr Asp Met Asn 1175 1180 1185 Lys Leu Asp Pro Gin Thr Asn Ser Ser Gin Ile Lys Glu Glu Phe 1190 1195 1200 Arg Thr Leu Asn Met Val Thr Pro Thr Leu Arg Val Glu Asp Cys 1205 1210 1215 Ser lie Ala Leu Leu Pro Arg Asn His Glu Lys Asn Arg Cys Met 1220 1225 1230 Asp lie Leu Pro Pro Asp Arg Cys Leu Pro Phe Leu lie Thr lie 1235 1240 1245 Asp Gly Glu Ser Ser Asn Tyr lie Asn Ala Ala Leu Met Asp Ser 1250 1255 1260 Tyr Lys Gln Pro Ser Ala Phe lie Val Thr Gin His Pro Leu Pro 1265 1270 1275 Asn Thr Val Lys Asp Phe Trp Arg Leu Val Leu Asp Tyr His Cys 1280 1285 1290 Thr Ser Val Val Met Leu Asn Asp Val Asp Pro Ala Gin Leu Cys 1295 1300 1305 Pro Gin Tyr Trp Leu Glu Asn Gly Val His Arg His Gly Pro lie 1310 1315 1320 Gln Val Glu Phe Val Ser Ala Asp Leu Glu Glu Asp lie lie Ser 1325 1330 1335 Arg lie Phe Arg lie Tyr Asn Ala Ala Arg Pro Gin Asp Gly Tyr 1340 1345 1350 Arg Met Val Gin Gin Phe Gin Phe Leu Gly Trp Pro Met Tyr Arg 1355 1360 1365 Asp Thr Pro Val Ser Lys Arg Ser Phe Leu Lys Leu Ile Arg Gin 1370 1375 1380 Val Asp Lys Trp Gin Glu Glu Tyr Asn Gly Gly Glu Gly Arg Thr 1385 1390 1395 Val Val His Cys Leu Asn Gly Gly Gly Arg Ser Gly Thr Phe Cys 1400 1405 1410 Ala Ile Ser Ile Val Cys Glu Met Leu Arg His Gin Arg Thr Val 1415 1420 1425 Asp Val Phe His Ala Val Lys Thr Leu Arg Asn Asn Lys Pro Asn 1430 1435 1440 Met Val Asp Leu Leu Asp Gin Tyr Lys Phe Cys Tyr Glu Val Ala 1445 1450 1455 Leu Glu Tyr Leu Asn Ser Gly 1460 1465 <210> 2 <211> 477 <212> PRT <213> Homo sapiens <400> 2 Met Arg Gly Leu Gly Thr Cys Leu Ala Thr Leu Ala Gly Leu Leu Leu 1 5 10 15 Thr Ala Ala Gly Glu Thr Phe Ser Gly Gly Cys Leu Phe Asp Glu Pro 20 25 30 Tyr Ser Thr Cys Gly Tyr Ser Gin Ser Glu Gly Asp Asp Phe Asn Trp 35 40 45 Glu Gin Val Asn Thr Leu Thr Lys Pro Thr Ser Asp Pro Trp Met Pro 50 55 60 Ser Gly Ser Phe Met Leu Val Asn Ala Ser Gly Arg Pro Gin Gly Gin 65 70 75 80 Arg Ala His Leu Leu Leu Pro Gin Leu Lys Gin Asn Asp Thr His Cys 85 90 95 He Asp Phe His Tyr Phe Val Ser Ser Lys Ser Asn Ser Pro Pro Gly 100 105 110 Leu Leu Asn Val Tyr Val Lys Val Asn Asn Gly Pro Leu Gly Asn Pro 115 120 125 He Trp Asn He Ser Gly Asp Pro Thr Arg Thr Trp Asn Arg Ala Gin 130 135 140 Leu Ala He Ser Thr Phe Trp Pro Asn Phe Tyr Gin Val He Phe Gin 145 150 155 160 Val He Thr Ser Gly His Gin Gly Tyr Leu Ala He Asp Gin Val Gin 165 170 175 Val Leu Gly His Pro Cys Thr Arg Thr Pro His Phe Leu Arg He Gin 180 185 190 Asn Val Glu Val Asn Ala Gly Gin Phe Ala Thr Phe Gin Cys Ser Ala 195 200 205 Ile Gly Arg Thr Val Ala Gly Asp Arg Leu Trp Leu Gin Gly Ile Asp 210 215 220 Val Arg Asp Ala Pro Leu Lys Glu Ile Lys Val Thr Ser Ser Arg Arg 225 230 235 240 Phe Ile Ala Ser Phe Asn Val Val Asn Thr Thr Lys Arg Asp Ala Gly 245 250 255 Lys Tyr Arg Cys Met Ile Arg Thr Glu Gly Gly Val Gly Ile Ser Asn 260 265 270 Tyr Ala Glu Leu Val Val Lys Glu Pro Pro Val Pro Ile Ala Pro Pro 275 280 285 Gln Leu Ala Ser Val Gly Ala Thr Tyr Leu Trp Ile Gin Leu Asn Ala 290 295 300 Asn Ser Ile Asn Gly Asp Gly Pro Ile Val Ala Arg Glu Val Glu Tyr 305 310 315 320 Cys Thr Ala Ser Gly Ser Trp Asn Asp Arg Gin Pro Val Asp Ser Thr 325 330 335 Ser Tyr Lys Ile Gly His Leu Asp Pro Asp Thr Glu Tyr Glu Ile Ser 340 345 350 Val Leu Leu Thr Arg Pro Gly Glu Gly Gly Thr Gly Ser Pro Gly Pro 355 360 365 Ala Leu Arg Thr Arg Thr Lys Cys Ala Asp Pro Met Arg Gly Pro Arg 370 375 380 Lys Leu Glu Val Val Glu Val Lys Ser Arg Gln Ile Thr Ile Arg Trp 385 390 395 400 Glu Pro Phe Gly Tyr Asn Val Thr Arg Cys His Ser Tyr Asn Leu Thr 405 410 415 Val His Tyr Cys Tyr Gln Val Gly Gly Gln Glu Gln Val Arg Glu Glu 420 425 430 Val Ser Trp Asp Thr Glu Asn Ser His Pro Gln His Thr Ile Thr Asn 435 440 445 Leu Ser Pro Tyr Thr Asn Val Ser Val Lys Leu Ile Leu Met Asn Pro 450 455 460 Glu Gly Arg Lys Glu Ser Gln Glu Leu Ile Val Gln Thr 465 470 475 <210> 3 <211> 280 <212> PRT <213> Homo sapiens <400> 3 Met Arg Gly Leu Gly Thr Cys Leu Ala Thr Leu Ala Gly Leu Leu Leu 1 5 10 15 Thr Ala Ala Gly Glu Thr Phe Ser Gly Gly Cys Leu Phe Asp Glu Pro 20 25 30 Tyr Ser Thr Cys Gly Tyr Ser Gln Ser Glu Gly Asp Asp Phe Asn Trp 35 40 45 Glu Gln Val Asn Thr Leu Thr Lys Pro Thr Ser Asp Pro Trp Met Pro 50 55 60 Ser Gly Ser Phe Met Leu Val Asn Ala Ser Gly Arg Pro Glu Gly Gln 65 70 75 80 Arg Ala His Leu Leu Leu Pro Gln Leu Lys Glu Asn Asp Thr His Cys 85 90 95 Ile Asp Phe His Tyr Phe Val Ser Ser Lys Ser Asn Ser Pro Pro Gly 100 105 110 Leu Leu Asn Val Tyr Val Lys Val Asn Asn Gly Pro Leu Gly Asn Pro 115 120 125 Ile Trp Asn Ile Ser Gly Asp Pro Thr Arg Thr Trp Asn Arg Ala Glu 130 135 140 Leu Ala Ile Ser Thr Phe Trp Pro Asn Phe Tyr Gln Val Ile Phe Glu 145 150 155 160 Val Ile Thr Ser Gly His Gln Gly Tyr Leu Ala Ile Asp Glu Val Lys 165 170 175 Val Leu Gly His Pro Cys Thr Arg Thr Pro His Phe Leu Arg lie Gin 180 185 190 Asn Val Glu Val Asn Ala Gly Gin Phe Ala Thr Phe Gin Cys Ser Ala 195 200 205 Ile Gly Arg Thr Val Ala Gly Asp Arg Leu Trp Leu Gin Gly lie Asp 210 215 220 Val Arg Asp Ala Pro Leu Lys Glu lie Lys Val Thr Ser Ser Arg Arg 225 230 235 240 Phe lie Ala Ser Phe Asn Val Val Asn Thr Thr Lys Arg Asp Ala Gly 245 250 255 Lys Tyr Arg Cys Met lie Arg Thr Glu Gly Gly Val Gly lie Ser Asn 260 265 270 Tyr Ala Glu Leu Val Val Lys Glu 275 280 <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 4 Glu Thr Phe Ser Gly Gly Cys Leu Phe Asp Glu Pro Tyr Ser Thr Cys 1 5 10 15 Gly Tyr Ser Gin 20 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 5 Gly Glu Gly Asp Asp Phe Asn Trp Glu Gin Val Asn Thr Leu Thr Lys 1 5 10 15 Pro Thr Ser Asp 20 <210> 6 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 6 Thr Pro His Phe Leu Arg lie Gin Asn Val Glu Val Asn Ala Gly Gin 1 5 10 15 Phe Ala Thr <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 7 Gly lie Asp Val Arg Asp Ala Pro Leu Lys Glu lie Lys Val Thr Ser 1 5 10 15 Ser Arg <210> 8 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 8 Cys Gly Glu Gly Asp Asp Phe Asn Trp Glu Gin Val Asn Thr Leu Thr 1 5 10 15 Lys Pro Thr Ser Asp 20 <210> 9 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Gly Gly Gly 1 <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 10 Gly Gly Gly Gly 1 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 11 Gly Gly Gly Gly Gly 1 5 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 Gly Gly Gly Gly Gly Gly 1 5 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 13 Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 14 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 16 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 16 Gly Ser Gly Ser 1 <210> 17 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 17 Gly Ser Gly Ser Gly Ser 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 18 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 19 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 Gly Gly Ser Gly Gly Ser 1 5 <210> 21 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 21 Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 22 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 22 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 10 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 23 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 24 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 25 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 26 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 27 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 27 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 28 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 28 Gly Gly Gly Gly Glu Gly Asp Asp Phe Asn Trp Glu Gin Val Asn Thr 1 5 10 15 Leu Thr Lys Pro Thr Ser Asp 20 <210> 29 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 29 Gly Gly Gly Gly Gly Glu Gly Asp Asp Phe Asn Trp Glu Gin Val Asn 1 5 10 15 Thr Leu Thr Lys Pro Thr Ser Asp 20 <210> 30 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 30 Gly Gly Gly Gly Gly Gly Glu Gly Asp Asp Phe Asn Trp Glu Gin Val 1 5 10 15 Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 31 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 31 Gly Gly Gly Gly Gly Gly Gly Glu Gly Asp Asp Phe Asn Trp Glu Gln 1 5 10 15 Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 32 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 32 Gly Gly Gly Gly Gly Gly Gly Gly Glu Gly Asp Asp Phe Asn Trp Glu 1 5 10 15 Gln Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 33 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 33 Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Gly Asp Asp Phe Asn Trp 1 5 10 15 Glu Gln Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 34 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 34 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Gly Asp Asp Phe Asn 1 5 10 15 Trp Glu Gin Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 35 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 35 Gly Ser Gly Ser Gly Glu Gly Asp Asp Phe Asn Trp Glu Gin Val Asn 1 5 10 15 Thr Leu Thr Lys Pro Thr Ser Asp 20 <210> 36 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 36 Gly Ser Gly Ser Gly Ser Gly Glu Gly Asp Asp Phe Asn Trp Glu Gin 1 5 10 15 Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 37 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 37 Gly Ser Gly Ser Gly Ser Gly Ser Gly Glu Gly Asp Asp Phe Asn Trp 1 5 10 15 Glu Gln Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 38 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 38 Gly Ser Gly Ser Gly Ser Gly Ser Gly Glu Gly Asp Asp Phe 1 5 10 15 Asn Trp Glu Gln Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 30 <210> 39 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 39 Gly Gly Ser Gly Gly Ser Gly Glu Gly Asp Asp Phe Asn Trp Glu Gln 1 5 10 15 Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 40 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 40 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Glu Gly Asp Asp Phe Asn 1 5 10 15 Trp Glu Gin Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 41 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 41 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Glu Gly Asp 1 5 10 15 Asp Phe Asn Trp Glu Gin Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 30 <210> 42 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 42 Gly Gly Gly Ser Gly Gly Gly Ser Gly Glu Gly Asp Asp Phe Asn Trp 1 5 10 15 Glu Gin Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 <210> 43 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 43 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Glu Gly Asp 1 5 10 15 Asp Phe Asn Trp Glu Gin Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 30 <210> 44 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 44 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Glu Gly Asp Asp Phe Asn Trp Glu Gin Val Asn Thr Leu Thr Lys 20 25 30 Pro Thr Ser Asp 35 <210> 45 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Gly Asp Asp Phe 1 5 10 15 Asn Trp Glu Gin Val Asn Thr Leu Thr Lys Pro Thr Ser Asp 20 25 30 <210> 46 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 46 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Glu Gly Asp Asp Phe Asn Trp Glu Gin Val Asn Thr Leu Thr Lys Pro 20 25 30 Thr Ser Asp 35 <210> 47 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 47 Gly Thr Gin Asp Glu Thr Gly Asn Phe Asp Trp Pro Val Ser Glu Asp 1 5 10 15 Leu Asn Lys Thr 20

Claims

1. A reagent comprising: A targeting peptide that specifically binds to and / or complexes with extracellular fragments of proteolytically cleaved immunoglobulin (Ig) superfamily cell adhesion molecules expressed by cancer cells or another cell in a cancer cell microenvironment, wherein the targeting peptide comprises a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; It can detect at least one of the following: a component, a therapeutic agent, or a diagnostic agent; and A peptide or peptide-like spacer that directly or indirectly links the target peptide to at least one of the detectable moiety, therapeutic agent, or diagnostic agent, wherein the spacer is a polyglycine spacer or a glycine / serine spacer, the glycine / serine spacer comprising an amino acid sequence of at least one of (GS)a, (GGS)b, or (GGGS)c or (GGGGS)d and wherein a, b, c, and d are each independently 2, 3, 4, 5, or 6, and the spacer at least maintains or preserves the binding affinity of the linked target peptide to the extracellular fragment of the proteolytically cleaved protein and the activity of at least one of the linked detectable moiety, therapeutic agent, or diagnostic agent.

2. The reagent according to claim 1, used for detecting, monitoring and / or imaging cancer cells and / or cancer cell metastasis, migration, spread and / or invasion, and / or for treating cancer in a subject.

3. The reagent according to claim 1, which is configured as a biological sample for in vivo administration to a subject or ex vivo administration to the subject.

4. The reagent according to claim 1, wherein the spacer has the following amino acid sequence: GGG (SEQ ID NO: 9), GGGG (SEQ ID NO: 10), GGGGG (SEQ ID NO: 11), GGGGGG (SEQ ID NO: 12), GGGGGGG (SEQ ID NO: 13), GGGGGGGG (SEQ ID NO: 14), GGGGGGGGG (SEQ ID NO: 15), GSGS (SEQ ID NO: 16), GSGSGS (SEQ ID NO: 17), GSGSGSGS (SEQ ID NO: 18), GSGSGSGSGS (SEQ ID NO: 19), GGSGGS (SEQ ID NO: 20), GGSGGSGGS (SEQ ID NO: 21), GGSGGSGGSGGS (SEQ ID NO: 22), GGGSGGGS (SEQ ID NO: 23), GGGSGGGSGGGS (SEQ ID NO: 24), GGGSGGGSGGGSGGGS (SEQ ID NO: 25). ID NO:25), GGGGSGGGGS (SEQ ID NO:26) or GGGGSGGGGGSGGGGS (SEQ ID NO:27).

5. The reagent according to any one of claims 1 to 4, further comprising at least one coupling agent that links the spacer to the target peptide and / or at least one of the following: the detectable moiety, the therapeutic agent, or the diagnostic agent.

6. The reagent according to any one of claims 1 to 4, wherein the detectable portion comprises a chelating agent, a contrast agent, an imaging agent, a radiolabel, a semiconductor particle, a nanoparticle, a nanobubble, or a nanochain.

7. The reagent according to claim 6, wherein the detectable portion can be detected by at least one of magnetic resonance imaging (MRI), positron emission tomography (PET) imaging, computed tomography (CT) imaging, gamma imaging, near-infrared imaging, or fluorescence imaging.

8. The reagent according to any one of claims 1 to 4, wherein the diagnostic or therapeutic agent comprises at least one of photosensitizers, ultrasound sensitizers, thermosensitizers, radiosensitizers, radiotherapy agents, chemotherapeutic agents, or immunotherapy agents.

9. The reagent according to any one of claims 1 to 4, wherein the spacer has the following amino acid sequence: GGG (SEQ ID NO:9), GSGSGS (SEQ ID NO:17) or GGSGGS (SEQ ID NO:20).

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