Near-infrared contrast agents and their uses

Abstract

Near-infrared imaging agents comprise a targeting peptide that specifically binds to 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 the cancer cell microenvironment; an optional spacer directly linked to the targeting peptide; and a near-infrared fluorophore directly or indirectly linked to the targeting peptide or optional spacer by a natural or non-natural bond.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 376,377, filed September 20, 2022, the entire contents of which are incorporated herein by reference.

[0002] Government research support This invention was made with government support under Grant No. CA217956 awarded by the National Institutes of Health. The federal government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. This XML copy was created on September 20, 2023, is named CWR-031969st.26, and is 63,464 bytes in size. [Background technology]

[0004] Glioblastoma multiforme (GBM) is the most common adult malignant astrocytoma and has the lowest survival rate among malignant brain tumors. The prognosis for GBM is extremely poor, with a median survival of 12 to 15 months. Multiple biological characteristics contribute to the lethality of GBM tumors, including their uncontrolled growth within the limited cranial space, their angiogenic nature, and their widespread dispersal throughout the brain. Surgical resection remains the primary treatment. While near-complete resection of the gadolinium-enhancing portion of the tumor improves survival, it is widely recognized that microscopic infiltrating cells ("fingers" or "tentacles") may remain locally within 2 to 3 cm of the original tumor, resulting in a recurrence rate of up to 90% in patients. Currently, magnetic resonance imaging (MRI) is used to delineate tumor boundaries before resection. Unfortunately, it is an imperfect tool. Because the contrast agent gadolinium has variable enhancement, it is merely a marker of leaky blood vessels and does not visualize GBM cells migrating outward from the main tumor mass. Neurosurgeons use the naked eye and their own judgment to identify tumors. The development of specific molecular contrast agents would enable the detection of these tentacle-infiltrating cells during surgery and improve the extent of resection. Summary of the Invention

[0005] The embodiments described herein include: A near-infrared imaging agent for use in detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, dissemination, and / or invasion in a subject; A method of detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, dissemination, and / or invasion in a subject; Methods for determining and / or monitoring the effectiveness of cancer drugs and / or cancer therapies administered to a subject in need thereof; Methods for determining, monitoring, and / or imaging the effectiveness of surgical resection of cancer cells in a subject in need thereof, and / or methods for treating cancer in a subject in need thereof; Regarding.

[0006] In some embodiments, the near-infrared imaging agent is: a targeting peptide that specifically binds to and / or forms a complex with a 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; an optional spacer directly linked to the targeting peptide; and a near-infrared fluorophore linked directly or indirectly to a targeting peptide or an optional spacer by a natural or non-natural bond; may include:

[0007] In some embodiments, the near-infrared imaging agent administered to the subject has a signal-to-background ratio (SBR) during fluorescence imaging effective to distinguish cancer cells or other cells in the cancer cell microenvironment from surrounding tissue.

[0008] In some embodiments, the near-infrared fluorophore is hydrophobic or lipophilic.

[0009] In other embodiments, the natural or non-natural bond is not susceptible to proteolytic cleavage.

[0010] In some embodiments, the non-natural bond comprises an amide linking the targeting peptide or optional spacer to the near-infrared fluorophore.

[0011] In some embodiments, the near-infrared imaging agent is: [ka] or a pharmaceutically acceptable salt thereof; where R 1 is a near-infrared fluorophore; and R 2 comprises a targeting peptide and an optional spacer.

[0012] In some embodiments, the near-infrared fluorophore comprises at least one type of cyanine near-infrared fluorophore having fluorescence in a first near-infrared region or a second near-infrared region.

[0013] In some embodiments, the cyanine near-infrared fluorophore is a heptamethine cyanine near-infrared fluorophore, e.g., the near-infrared fluorophore comprises at least one of indocyanine green (ICG) or ICG-Osu.

[0014] In other embodiments, the near-infrared fluorophore is: [ka] or a pharmaceutically acceptable salt thereof; where R 2 comprises a targeting peptide and an optional spacer.

[0015] In some embodiments, the near-infrared fluorophore is directly linked to the targeting peptide by a non-natural bond.

[0016] In other embodiments, the near-infrared fluorophore is directly linked to the spacer by a non-natural bond.

[0017] In some embodiments, the spacer has a length and structure 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 near-infrared fluorophore.

[0018] In some embodiments, the spacer can include natural and / or unnatural amino acids. For example, the spacer can include at least three natural or unnatural amino acids.

[0019] In some embodiments, the spacer has a length of 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 unnatural amino acids.

[0020] 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.

[0021] In some embodiments, the spacer is a polyglycine or glycine / serine spacer. For example, the spacer can include at least one of the amino acid sequences (GS)a, (GGS)b, or (GGGS)c, or (GGGGS)d, where a, b, c, and d are each independently 2, 3, 4, 5, or 6.

[0022] In some embodiments, near-infrared imaging agents can be used in detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, dissemination, and / or invasion in a subject and / or to treat cancer.

[0023] In some embodiments, the near-infrared imaging agent can be configured for in vivo administration to a subject or ex vivo administration to a biological sample from a subject.

[0024] Other embodiments described herein relate to methods of detecting cancer cells and / or metastasis, migration, dissemination, and / or invasion of cancer cells in a subject in need thereof. The method may include administering to the subject an amount of a near-infrared imaging agent described herein. The agent that binds to and / or forms a complex with the cancer cells may be detected to determine the location and / or distribution of the cancer cells in the subject.

[0025] In some embodiments, the cancer cells include at least one of glioma, lung cancer, melanoma, breast cancer, or prostate cancer cells.

[0026] In other embodiments, the agent may be administered to the subject systemically, locally, or locally.

[0027] In some embodiments, the agent is administered to a subject to define tumor boundaries. can be detected.

[0028] Another embodiment relates to the use of near-infrared contrast agents in fluorescent image-guided surgery.

[0029] Yet another embodiment relates to the use of near-infrared contrast agents in the preparation of therapeutic agents for fluorescent image-guided surgery.

[0030] Another embodiment relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a near-infrared imaging agent described herein, wherein the agent bound to and / or complexed with the cancer cells can be irradiated at a wavelength effective to ablate the cancer cells.

[0031] In some embodiments, the cancer cells comprise at least one of glioma, lung cancer, melanoma, breast cancer, or prostate cancer cells.

[0032] In other embodiments, the agent may be administered to the subject systemically, locally, or locally.

[0033] Yet another embodiment relates to the use of near-infrared contrast agents in photodynamic or photothermal therapy.

[0034] Another embodiment is a compound of the formula: [ka] or a pharmaceutically acceptable salt thereof; where R2 comprises a targeting peptide that specifically binds to 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 the cancer cell microenvironment, and an optional spacer directly linked to the targeting peptide.

[0035] In some embodiments, R 2 consists of a targeting peptide.

[0036] In other embodiments, R 2 consists of a targeting peptide linked to a spacer, where the spacer separates the amide from the targeting peptide.

[0037] In some embodiments, the spacer has a length and structure effective to at least maintain or preserve the binding affinity of the linked targeting peptide for the proteolytically cleaved extracellular fragment.

[0038] In some embodiments, the spacer comprises natural and / or unnatural amino acids.

[0039] In other embodiments, the spacer may comprise at least three natural or unnatural amino acids.

[0040] In some embodiments, the spacer has a length of 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 unnatural amino acids.

[0041] 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.

[0042] In some embodiments, the spacer is a polyglycine or glycine / serine spacer. For example, the spacer can include at least one of the amino acid sequences (GS)a, (GGS)b, or (GGGS)c, or (GGGGS)d, where a, b, c, and d are each independently 2, 3, 4, 5, or 6. [Brief explanation of the drawings]

[0043] [Figure 1(AB)] Figure 1 shows in vivo tumor labeling of LN229 flank tumors with ICG-conjugated peptides over time. A: Flank tumor-bearing mice were injected with 300 nmol / kg of Scram-CLE-ICG, SBK2-CLE-ICG, Scram-ICG, and SBK2-ICG. Fluorescent images were acquired at the indicated times and are shown along with baseline (BL) images acquired before the start of the experiment for a representative set of animals. B: The average radiant efficiency (mean ± standard error) of the in vivo tumor signal in each cohort of animals is plotted over time. [Figure 2(AB)] Figure 1 shows in vivo tumor signals 24 hours after injection of ICG-conjugated peptides. A: LN229 flank tumor-bearing mice 24 hours after injection of Scram-CLE-ICG, SBK2-CLE-ICG, Scram-ICG, and SBK2-ICG at 300 nmol / kg. The top row shows photographs with the outlined tumor area, and the bottom row shows the corresponding fluorescent images. The mice are those shown in Figure 1, but with the scale optimized for the 24-hour time point. B: Average radiant efficiency (mean ± standard error) of in vivo tumor signals at 24 hours in mice administered the indicated agents at 300 nmol / kg or 400 nmol / kg. [Figure 3(AB)]Figure 1 shows the ex vivo fluorescent tumor signal detected 24 hours after administration of ICG-conjugated peptides at 300 nmol / kg and 400 nmol / kg. A: Fluorescence images of excised tumors in mice treated with each drug at 300 nmol / kg. Representative tumors are shown for the number of mice indicated. B: Average radiant efficiency (mean ± standard error) of ex vivo tumor signal at 24 hours in mice administered the indicated drug at 300 nmol / kg or 400 nmol / kg. Measurements were performed on tumors excised from the same mice used in Figure 2. [Figure 4(AB)] Figure 1 shows in vivo tumor labeling by IRDye800-conjugated peptides and TF8WS-conjugated peptides over time. A: Mice were injected with 400 nmol / kg of Scram-CCLE-IR800 or SBK2-CCLE-IR800, and images were acquired every 10 minutes for 60 minutes. The average radiant efficiency values ​​(mean ± standard error) of the tumor area for the two sets of mice are plotted. No significant differences were detected at any time point. B: Mice were injected with 400 nmol / kg of Scram-TF8WS or 400 nmol / kg of SBK2-TF8WS, and images were acquired at the indicated time points. No significant differences were observed between the two groups of mice at any time point. The inset plot shows data acquired for the TF8WS-conjugated peptide during the first 60 minutes, plotted alongside the data for the IR800-conjugated peptide shown in panel A. [Figure 5(AB)]In vivo tumor signal at 60 minutes for IRDye800-conjugated peptides and at 24 hours for TF8WS-conjugated peptides is shown. A: LN229 flank tumor-bearing mice either 60 minutes (IRDye800-conjugated peptides) or 24 hours (TF8WS-conjugated peptides) after injection of the indicated agent at 400 nmol / kg. The top row shows photographs with the tumor area outlined in each mouse. The bottom row shows fluorescent images overlaid with the photographic images. For each agent, a representative mouse from the cohort plotted in Figure 4 is shown. B: In vivo tumor fluorescence (mean ± standard error) at 60 minutes (IRDye800-conjugated peptides) or 24 hours (TF8WS-conjugated peptides) for the indicated agents. No significant differences were detected between Scram-CCLE-IR800 and SBK2-CCLE-IR800 or between Scram-TF8WS and SBK2-TF8WS. [Figure 6(AB)]

[0033] Figure 1 shows ex vivo tumor fluorescence signals in mice treated with 400 nmol / kg IRDye800-conjugated peptide at 60 minutes or 400 nmol / kg TF8WS-conjugated peptide at 24 hours. A: Fluorescence images obtained in tumors excised from mice 60 minutes or 24 hours after injection of the indicated agent at 400 nmol / kg. The tumors shown are from representative mice. B: Average radiant efficiency (mean ± standard error) of ex vivo tumor signals at 24 hours in mice administered the indicated agent at 400 nmol / kg. No significant differences were observed between any of the agent pairs. [Figure 7]The molecular structures of the functionalized near-infrared fluorophores used to generate the PTPμ targeting agent and the control peptide-labeling agent are shown. (Top row) ICG-Osu contains an N-hydroxysuccinimide (NHS) ester and was conjugated to the primary amine of SBK2 or a scrambled peptide, while the peptide remained attached to the resin. The molecular weights (MWs) indicated by the manufacturer and the optimal excitation and emission wavelengths for fluorescence are shown. Analytical RP-HPLC chromatograms obtained at 220 nm for SBK2-CLE-ICG and SBK2-ICG are shown in the top right corner. These agents eluted with a high percentage of acetonitrile, with SBK2-CLE-ICG exhibiting a retention time (RT) approximately 1.3 minutes shorter than that of SBK2-ICG. The amide bond between the fluorophore and the peptide is shown in the inset in the chromatogram. (Middle) An additional cysteine ​​was added to the N-terminus of the SBK2-CLE and Scram-CLE peptides to allow conjugation of the hydrophilic IRDye 800CW maleimide in aqueous solution. The molecular weights and wavelengths associated with optimal fluorescence, as determined by the manufacturer, are shown. This chromatogram shows the expected SBK2-CCLE-IR800 molecule with an intact succinimide moiety eluting at 25.4 minutes. The asterisk (*) highlights the initial peak, which represents SBK2-CCLE-IR800 with an open succinimide ring, eluting at 24.4 minutes. The open and closed succinimide ring bonds between the fluorophore and the peptide are shown in the inset. (Bottom) The unique structure of the acidic form of the Tide Fluor 8WS fluorophore is shown, along with the molecular weight and wavelength of optimal fluorescence as indicated by the manufacturer. TF8WS acid was conjugated to the N-terminal amine of the peptide on the resin during the final step of the synthesis. Analytical RP-HPLC chromatogram shows the elution of the SBK2-TF8WS agent (with an amide bond between the fluorophore and the peptide) at 25.5 min (inset). [Figure 8(AB)]Ex vivo fluorescence in the kidney, spleen, and liver 24 hours after administration of Scram-CLE-ICG, SBK2-CLE-ICG, Scram-ICG, and SBK2-ICG at 300 nmol / kg, or ICG alone at 400 nmol / kg, is shown. A: Fluorescence detected at Ex 745 nm / Em 820 nm in organs removed from mice injected with the indicated ICG-conjugated peptides at 300 nmol / kg. Representative organs from the mice used in Figures 1 and 2 are shown. B: The average radiant efficiencies detected in different organs for each agent at 300 nmol / kg and for ICG alone at 400 nmol / kg were compared and plotted. For the kidneys, the signals from the right and left kidneys from each mouse were averaged and used as the kidney signal for that animal. Fluorescence signals were compared using unpaired t-tests with Welch's correction, and the resulting p-values ​​are summarized in Table 5. [Figure 9(AB)]Ex vivo fluorescence in the kidney, spleen, and liver after injection of 400 nmol / kg of IR800- or TF8WS-conjugated peptides is shown. A: Fluorescence detected at Ex 745 nm / Em 800 nm in kidneys, spleens, and livers removed from animals treated with Scram-CCLE-IR800 (upper left) and SBK2-CCLE-IR800 (upper right) for 60 minutes, and with Scram-TF8WS (lower left) and SBK2-TF8WS (lower right) for 24 hours. (a = right kidney, b = left kidney, c = spleen, d = liver). Representative organs are shown for each cohort. B: Plot of the average radiant efficiency measured in kidneys, spleens, and livers. For kidneys, the right and left kidney signals from each mouse were averaged and used as the kidney signal for that animal. Significantly stronger fluorescence was detected in the kidneys and spleens of animals administered Scram-CCLE-IR800 compared to SBK2-CCLE-IR800 after 60 minutes. p<0.005 for kidney; p<0.01 for spleen. No significant differences in liver fluorescence were detected in mice treated with Scram-CCLE-IR800 compared to SBK2-CCLE-IR800. No significant differences in mean radiant efficiency were observed in the kidneys, spleen, and liver in mice treated with Scram-TF8WS and SBK2-TF8WS for 24 hours. [Figure 10(AB)]Ventral images of mice treated with PTP μ-targeted drugs and control near-infrared imaging agents are shown at various time points to demonstrate drug biodistribution. A: Mice were imaged 60 minutes after injection of 300 nmol / kg ICG-conjugated peptide (top row, Ex 745 nm / Em 820 nm) or 400 nmol / kg IR800-conjugated and TF8WS-conjugated peptides (bottom row, Ex 745 nm / Em 800 nm) to demonstrate drug clearance patterns. B: Mice shown in A 24 hours after injection of 300 nmol / kg ICG-conjugated peptide (top row) or 400 nmol / kg TF8WS-conjugated peptide (bottom row). Mice injected with IR800-conjugated peptides were sacrificed after the 60-minute image acquisition and therefore did not receive 24-hour images. DETAILED DESCRIPTION OF THE INVENTION

[0044] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodological literature (such as "Current Protocols in Molecular Biology," edited by Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (regularly updated)). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. Commonly understood definitions of molecular biology terms can be found, for example, in Rieger et al., "Glossary of Genetics: Classical and Molecular," 5th ed., Springer-Verlag: New York, 1991; and Lewin, "Genes V," Oxford University Press: New York, 1994.

[0045] 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.

[0046] The term "log P" or "logP" refers to the log(base 10) of the n-octanol / water partition coefficient (i.e., log P ow ) refers to

[0047] The terms "comprise," "comprising," "include," "including," "have," and "having" are used in an inclusive, open-ended sense and mean that additional elements may be included. As used herein, the terms "such as," "for example," and "for example" are open-ended and for illustrative purposes only. "Including" and "including but not limited to" are used synonymously.

[0048] The term "or" as used herein should be understood to mean "and / or" unless the context clearly indicates otherwise.

[0049] As used herein, the term "agent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract obtained from biological materials.

[0050] The term "cancer" or "tumor" refers to any neoplastic growth in a subject, including primary tumors and metastases. Cancer can be liquid or solid tumor type. Liquid tumors include tumors of hematological origin, such as myeloma (e.g., multiple myeloma), leukemia (e.g., Waldenstrom syndrome, chronic lymphocytic leukemia, other leukemias), and lymphomas (e.g., B-cell lymphoma, non-Hodgkin's lymphoma). Solid tumors originate from organs and include cancers of the lung, brain, breast, prostate, ovary, colon, kidney, and liver.

[0051] The term "cancer cell" or "tumor cell" may refer to a cell that divides at an abnormal (i.e., increased) rate. Cancer cells include 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, liver cancer - hepatocellular carcinoma, bile duct carcinoma, and the like. Cancers such as 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 head and neck region; sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, synovial sarcoma, and mesothelial sarcoma; myeloma, leukemia (e.g., acute myeloid leukemia, chronic myeloid leukemia, Hematological cancers, such as 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 tumors of the nervous system, including glioma, glioblastoma multiforme, meningioma, medulloblastoma, schwannoma, and epidymoma.

[0052] The terms "homology" and "identity" are used synonymously throughout and refer to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which may be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous or identical at that position. The degree of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0053] The term "mutant" refers to any change in the genetic material of an organism, particularly a change (i.e., a deletion, substitution, addition, or modification) in the wild-type polynucleotide sequence or any change in the wild-type protein. The term "variant" is used synonymously with "mutant." Although changes in genetic material are often thought to result in a change in the function of the protein, the terms "mutant" and "variant" refer to a change in the sequence of a wild-type protein, regardless of whether the change alters protein function (e.g., increases, decreases, confers a new function) or whether the change does not affect protein function (e.g., the mutation or change is silent).

[0054] The term "nucleic acid" refers to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). This term also includes, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and is understood to be applicable to the described embodiments, single-stranded (sense or antisense) and double-stranded polynucleotides.

[0055] The expressions "parenteral administration" and "administered parenterally" are terms well known in the art and include modes of administration other than enteral administration and topical application, such as injection, including, but not limited to, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, and intrasternal injection and infusion.

[0056] As used herein, the expressions "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" refer to the administration of a compound, drug, or other substance other than by direct administration to a particular tissue, organ, or region of the subject being treated (e.g., the brain), whereby it enters the animal's system and is therefore subject to metabolism and other similar processes (e.g., subcutaneous administration).

[0057] The terms "patient," "subject," "mammalian host," and the like are used interchangeably herein and refer to mammals, including human and veterinary subjects.

[0058] The terms "peptide," "protein," and "polypeptide" are used synonymously herein. As used herein, "polypeptide" refers to any peptide or protein containing two or more amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isomers). "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides, or oligomers, and to longer chains, commonly referred to as proteins.

[0059] The terms "polynucleotide sequence" and "nucleotide sequence" are also used synonymously herein.

[0060] As used herein, "recombinant" means that a protein is derived from a prokaryotic or eukaryotic expression system.

[0061] The expressions "therapeutically effective amount" or "pharmaceutically effective amount" are terms well known in the art. In certain embodiments, the term refers to an amount of a therapeutic agent that produces a desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. In certain embodiments, the term refers to an amount necessary or sufficient to eliminate, reduce, or maintain the target of a particular therapeutic regimen. The effective amount may vary depending on factors such as the disease or condition being treated, the particular targeted construct being administered, the subject's weight, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without necessitating undue experimentation. In certain embodiments, the therapeutically effective amount of a therapeutic agent for in vivo use will likely depend on many factors, including the rate of drug release from the polymer matrix (which depends in part on the chemical and physical properties of the polymer); the identity of the drug; the mode and method of administration; and other substances incorporated into the polymer matrix along with the drug.

[0062] The term "wild-type" refers to a naturally occurring polynucleotide sequence encoding a protein, or a portion thereof, or a protein sequence, or a portion thereof, respectively, as it normally exists in vivo.

[0063] Throughout this specification, when a composition is described as having, including, or comprising certain components, it is intended that the composition also consists essentially of, or consists of, the recited elements. Similarly, when a method or process is described as having, including, or comprising certain process steps, the process also consists essentially of, or consists of, the recited process steps. Furthermore, it should be understood that the order of steps or the order for performing particular actions is immaterial so long as the compositions and methods described herein remain operable. Additionally, two or more steps or actions may be performed simultaneously.

[0064] The embodiments described herein include: Near-infrared imaging agents for use in detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, dissemination, and / or invasion in a subject; Methods of detecting, monitoring, and / or imaging cancer cells and / or cancer cell metastasis, migration, dissemination, and / or invasion in a subject; Methods for determining and / or monitoring the effectiveness of cancer drugs and / or cancer therapies administered to a subject in need thereof; Methods for determining, monitoring, and / or imaging the effectiveness of surgical resection of cancer cells in a subject in need thereof and / or methods for treating cancer in a subject in need thereof; Regarding.

[0065] Surgical resection of solid tumors is often the first step in cancer treatment. Much effort has been made recently in developing surgical tools that allow for better detection of tumor borders and identification of invasion and metastasis. Recent advances in this area include the development of molecularly targeted fluorescent contrast agents that assist surgeons in accurately distinguishing neoplastic from normal tissue in real time. Of particular importance is the use of near-infrared (NIR) fluorophores, which are detected at wavelengths that provide greater optical penetration and minimize autofluorescence or interference from hemoglobin and other endogenous components. Glioblastoma (GBM) is one of the most difficult tumor types to surgically remove due to its highly aggressive and infiltrative properties. In GBM, tumor-specific extracellular fragments of PTPμ are produced. PTPμ is a cognate cell adhesion molecule and a receptor protein tyrosine phosphatase that is normally localized at cell-cell boundaries. These extracellular fragments of PTPμ remain associated with both the main tumor mass and migrating tumor cells and serve as tumor biomarkers for GBM and other tumor types. Peptides that bind to this PTPμ biomarker have been effectively used as recognition and targeting moieties in preclinical tumor imaging by optical imaging, contrast-enhanced magnetic resonance imaging, and ultrasound imaging. We have discovered that selected NIR fluorophores conjugated to PTPμ-derived peptides by natural or non-natural linkages can be used as tumor-targeted fluorescent contrast agents for fluorescence-guided resection (FGR) with high specificity and favorable kinetic properties, and are promising for use in the operating room during FGR.

[0066] Furthermore, it has been found that near-infrared fluorophores linked directly or indirectly to targeting peptides can be selected such that the near-infrared imaging agent has a structure 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 near-infrared fluorophore. The activity of a near-infrared fluorophore refers to the ability of the near-infrared fluorophore to be detected or imaged in vivo, ex vivo, or in vitro by fluorescence imaging, for example, to provide a signal-to-background ratio (SBR) during fluorescence imaging effective to distinguish cancer cells or other cells in the cancer cell microenvironment from surrounding tissue.

[0067] For example, the near-infrared contrast agents described herein were found to clearly delineate tumor cell boundaries in tissue sections and tumor "periphery" samples, suggesting that near-infrared contrast agents can be used as molecular imaging diagnostic tools for metastatic, disseminated, migratory, or invasive cancers or tumor margins. Systemic introduction of near-infrared contrast agents as described herein resulted in rapid and specific labeling of flank and intracranial tumors within minutes. Labeling occurred primarily within the tumor, but a gradient of agent at the tumor border was also observed. There was also signal amplification as extracellular fragments accumulated over time.

[0068] Thus, in some embodiments, the near-infrared imaging agents described herein are: a targeting peptide that specifically binds to and / or complexes with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule expressed by the cancer cell or another cell in the cancer cell microenvironment; an optional spacer directly linked to the targeting peptide; and a near-infrared fluorophore linked directly or indirectly to a targeting peptide or optional spacer via a natural or non-natural bond; may include:

[0069] Near-infrared imaging agents can be administered locally (e.g., topically) or systemically (e.g., intravenously) to a subject and can readily target cancer cells (e.g., metastatic, migratory, dispersing, and / or invasive cancer cells) that are bound to proteolytically cleaved extracellular fragments of immunoglobulin (Ig) superfamily cell adhesion molecules. In some embodiments, the near-infrared imaging agent after systemic administration can cross the blood-brain barrier to reveal the location, distribution, metastasis, dispersal, migration, and / or invasion of cancer cells and tumor cell boundaries in a subject. In other embodiments, near-infrared imaging agents can be used in image-guided phototherapy to inhibit and / or reduce cancer cell survival, proliferation, and migration.

[0070] In some embodiments, the near-infrared imaging agents described herein can be used in methods for detecting cancer cells and / or cancer cell metastasis, migration, dissemination, and / or invasion in a subject in need thereof, as well as in methods for treating cancer. The methods can include administering to a subject a near-infrared imaging agent comprising a targeting peptide that binds to and / or complexes with a proteolytically cleaved extracellular fragment of an Ig superfamily cell adhesion molecule in the microenvironment of cancer cells or tumor cells, an optional spacer directly linked to the targeting peptide, and a near-infrared fluorophore directly or indirectly linked to the targeting peptide or optional spacer via a natural or non-natural bond. The near-infrared imaging agent bound to and / or complexed with cancer cells can be detected to determine the location and / or distribution of the cancer cells in the subject, and / or irradiated for phototherapy and ablation of the cancer cells.

[0071] In some embodiments, the Ig superfamily cell adhesion molecule can include an extracellular homotypic binding moiety, which can bind homotypically or participate in homotypic binding in a subject. In one example, the Ig superfamily cell adhesion molecule includes a type IIb RPTP cell adhesion molecule. In another example, the Ig superfamily cell adhesion molecule can include RPTPs of the PTPμ-like subfamily, such as PTPμ, PTPκ, PTPρ, and PCP-2 (also known as PTPλ). PTPμ-like RPTPs include a MAM (meprin / A5 protein / PTPμ) domain, an Ig domain, and FNIII repeats. PTPμ can have the amino acid sequence of SEQ ID NO: 1, identified by GenBank accession number AAI51843.1. It should be understood that the PTPμ gene can produce splice variants, whereby the amino acid sequence of PTPμ can differ from SEQ ID NO: 1. In some embodiments, PTP μ can have the amino acid sequence identified by Genbank Accession No. AAH51651.1 and Genbank Accession No. AAH40543.1.

[0072] Cancer cells and / or endothelial cells that support cancer cell survival that express Ig superfamily cell adhesion molecules and can be proteolytically cleaved to produce detectable extracellular fragments may include, for example, cancer cells and / or other cells in the tumor microenvironment (such as stem cells, endothelial cells, stromal cells, and immune cells that promote their survival).

[0073] Cancers detected and / or treated by the near-infrared imaging agents described herein may include: leukemias (such as, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia), myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia, leukemia, etc., and myelodysplastic syndromes; Chronic leukemia (including, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia); Polycythemia vera; lymphoma (including but not limited to Hodgkin's disease and non-Hodgkin's disease); multiple myeloma (including, but not limited to, smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma); Waldenström macroglobulinemia; Monoclonal gammopathy of unknown significance; Benign monoclonal gammopathy; heavy chain disease; Sarcomas of bone and connective tissue (including, but not limited to, osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, and synovial sarcoma); Brain tumors (including, but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, non-glioma, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary cerebral lymphoma); breast cancer (including, but not limited to, ductal carcinoma, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary carcinoma, mucinous carcinoma, ductal carcinoma, papillary carcinoma, Paget's disease, and inflammatory breast cancer); adrenal cancer (including, but not limited to, pheochromocytoma and adrenocortical carcinoma); thyroid cancer (including, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer); pancreatic cancer (including, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or insulinoma); pituitary cancers (including, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus); eye cancers (such as, but not limited to, intraocular melanoma (iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma)); vaginal cancer (including squamous cell carcinoma, adenocarcinoma, and melanoma); vulvar cancer (including squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease); cervical cancer (including, but not limited to, squamous cell carcinoma and adenocarcinoma); uterine cancer (including, but not limited to, endometrial cancer and uterine sarcoma); ovarian cancer (including, but not limited to, epithelial ovarian cancer, borderline tumors, germ cell tumors, and stromal tumors); Esophageal cancer (including, but not limited to, squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, myeloma, verrucous carcinoma, and oat cell (small cell) carcinoma); Gastric cancer (including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma); colon cancer; rectal cancer; liver cancer (including, but not limited to, hepatocellular carcinoma and hepatoblastoma); gallbladder cancer (e.g., adenocarcinoma); Cholangiocarcinoma (including, but not limited to, papillary, nodular, and expanding); Lung cancer (including non-small cell lung cancer, squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer); Testicular cancer (including, but not limited to, germ cell tumor, seminoma, undifferentiated, classic (typical), spermatocyte, non-seminoma, embryonal carcinoma, teratocarcinoma, and choriocarcinoma (yolk sac tumor)); prostate cancer (including, but not limited to, prostatic intraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma); Kidney cancer; oral cancer (including, but not limited to, squamous cell carcinoma); basal carcinoma; salivary gland cancers (including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma); pharyngeal cancer (including, but not limited to, squamous cell carcinoma and verrucous); skin cancer (including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentiginous melanoma); kidney cancer (including, but not limited to, renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter)); Wilms tumor; Bladder cancer (including, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma).

[0074] Additionally, cancers include: Myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangioendothelial sarcoma, mesothelioma, synovial tumor, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (for reviews of such disorders, see Fishman et al., 1985, Medicine, 2nd 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 USA, Inc., USA).

[0075] Near-infrared imaging agents may also be used to detect and / or treat various cancers or other abnormal proliferative diseases; such cancers and diseases include, but are not limited to: Cancer (including cancer of the bladder, breast, prostate, rectum, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, and skin); (including squamous cell carcinoma); Hematopoietic neoplasms of lymphoid lineage (including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and Burkitt lymphoma); Hematopoietic neoplasms of myeloid lineage (including acute and chronic myeloid leukemia and promyelocytic leukemia); tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma); Other tumors (including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma); tumors of the central and peripheral nervous system (including astrocytoma, neuroblastoma, glioma, and schwannoma); tumors of mesenchymal origin (including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma); and other tumors (including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma). Cancers resulting from abnormalities in apoptosis are also contemplated to be treated by the methods and compositions of the present invention. Such cancers include, but are not limited to, follicular lymphoma; carcinoma; hormone-dependent tumors of the breast, prostate, and ovary; and precancerous lesions (such as familial adenomatous polyposis and myelodysplastic syndrome). In certain embodiments, malignant tumors or abnormal proliferative changes (such as metaplasia and dysplasia), or hyperproliferative diseases, are detected, treated, or prevented in the skin, lung, colon, rectum, breast, prostate, bladder, kidney, pancreas, ovary, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia are detected and / or treated.

[0076] In yet other embodiments, the cancer cells to be detected and / or treated may include glioma cells, lung cancer cells, breast cancer cells, prostate cancer cells, and melanoma cells (such as invasive, dispersed, motile, or metastatic cancer cells), and may include glioma cells, lung cancer cells, breast cancer cells, prostate cancer cells, and melanoma cells. It should be understood that other cancer cells and / or endothelial cells that support the survival of cancer cells, which express Ig superfamily cell adhesion molecules and can be proteolytically cleaved to produce detectable extracellular fragments, can be identified or determined, for example, by using immunoassays that detect Ig superfamily cell adhesion molecules expressed by cancer cells or endothelial cells.

[0077] In some embodiments, the targeting peptide (or targeting polypeptide) can comprise a polypeptide (or targeting polypeptide) that binds to and / or forms a complex with a proteolytically cleaved extracellular fragment of an Ig superfamily cell adhesion molecule. The targeting peptide can comprise about 10 to about 50 The target polypeptide may comprise, consist essentially of, or consist of about 10 to about 50 amino acids, and may have an amino acid sequence substantially homologous to about 10 to about 50 contiguous amino acids of the cognate binding portion or domain of a proteolytically cleaved extracellular fragment of an Ig superfamily cell adhesion molecule. Substantially homologous means that the target polypeptide has at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity with a portion of the amino acid sequence of the binding portion of a proteolytically cleaved extracellular fragment of an Ig superfamily cell adhesion molecule.

[0078] In one example, the cognate binding portion of an Ig superfamily cell adhesion molecule can include, for example, the Ig domain of the cell adhesion molecule. In another example, if the Ig superfamily cell adhesion molecule is PTPμ, the cognate binding portion can include the Ig binding domain and the MAM domain.

[0079] In another aspect, the targeting peptide can have an amino acid sequence substantially homologous to about 10 to about 50 contiguous amino acids of the Ig-binding domain and / or MAM domain of PTPμ (e.g., SEQ ID NO:1) and can readily cross the blood-brain barrier when administered systemically to a subject. The development of PTPμ targeting peptides can be based on extensive structural and functional data. The sites required for PTPμ-mediated homotypic binding have been extensively characterized. Furthermore, the crystal structure of PTPμ can provide information regarding which regions of each functional domain are likely to be exposed to the external environment and therefore accessible for homotypic binding and detection by the peptide.

[0080] In some embodiments, a proteolytically cleaved extracellular fragment of PTPμ (e.g., SEQ ID NO:1) can comprise the amino acid sequence of SEQ ID NO:2, the Ig and MAM binding region can comprise the amino acid sequence of SEQ ID NO:3, and the polypeptide can have an amino acid sequence 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 SEQ ID NO:2 or SEQ ID NO:3 and have an amino acid sequence substantially homologous to about 10 to about 50 contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:3 are polypeptides comprising 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 a MAM, an Ig domain, or an FNIII repeat. In certain embodiments, the targeting peptide is an SBK2 polypeptide comprising the amino acid sequence of SEQ ID NO:5.

[0081] In other embodiments, a polypeptide or its receptor that binds to and / or complexes with a proteolytically cleaved extracellular fragment of an Ig superfamily CAM expressed by a cancer cell or another cell in the cancer cell microenvironment 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.

[0082] The targeting peptide may be subjected to various modifications, substitutions, insertions, and deletions, and such modifications may provide specific advantages in its use. In this regard, a targeting peptide that binds to and / or complexes with the proteolytically cleaved extracellular portion of an Ig superfamily cell adhesion molecule may be substantially homologous to, rather than identical to, the sequence of a recited polypeptide, with one or more modifications made thereto, so that it retains the ability to function when specifically binding to and / or complexing with, the proteolytically cleaved extracellular portion of an Ig superfamily cell adhesion molecule.

[0083] The targeting peptide can be any of a variety of forms of polypeptide derivatives, including amides, conjugated to proteins, cyclized polypeptides, polymerized polypeptides, retro-inverso peptides, analogs, fragments, chemically modified polypeptides, and other derivatives.

[0084] The term "analog" includes any polypeptide having substantially identical amino acid residue sequences to those specifically set forth herein, in which one or more residues have been conservatively substituted with functionally similar residues, which specifically bind to and / or complex with the proteolytically cleaved extracellular portion of an Ig superfamily CAM as described herein. Examples of conservative substitutions are: substitution of one non-polar (hydrophobic) residue, such as isoleucine, valine, leucine, or methionine, for another; substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine; Substitution of one basic residue, such as lysine, arginine, or histidine, for another; or substitution of one acidic residue for another, such as aspartic acid or glutamic acid; Includes.

[0085] The language "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue, provided that such peptide exhibits the requisite binding activity.

[0086] "Chemical derivative" refers to a subject polypeptide having one or more chemically derivatized residues by reaction of a functional side group. Such derivatized molecules include those molecules in which free amino groups are derivatized to form, for example, amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups, or formyl groups. Free hydroxyl groups may be derivatized to form salts, methyl and ethyl esters, or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N im The polypeptides may be derivatized to form -benzylhistidine. Chemical derivatives also include those polypeptides containing derivatives of one or more naturally occurring amino acids of the 20 standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. The polypeptides described herein also include any polypeptides whose sequences have one or more additions and / or deletions of residues relative to the sequences of the polypeptides shown herein, so long as the essential activity is maintained.

[0087] Retro-inverso peptides are linear peptides whose amino acid sequence is reversed and whose α-center chirality of the amino acid subunits is also inverted. These types of peptides are designed by incorporating D-amino acids into the reverse sequence to help maintain a side-chain topology similar to that of the original L-amino acid peptide and to make them resistant to proteolysis. D-amino acids mirror the conformations of natural L-amino acids in natural proteins present in biological systems. Peptides containing D-amino acids have advantages over peptides containing only L-amino acids. In general, these types of peptides are less susceptible to proteolysis and have a longer shelf life when used as pharmaceuticals. Furthermore, the insertion of D-amino acids in selected sequence regions, such as D-amino acid-only blocks, or between L-amino acids allows for the design of peptide drugs that are biologically active and have enhanced bioavailability in addition to being resistant to proteolysis. Furthermore, if properly designed, retro-inverso peptides can have binding properties similar to those of L-peptides.

[0088] The term "fragment" refers to any subject polypeptide whose amino acid residue sequence is shorter than that of the polypeptides shown herein.

[0089] Any polypeptide and compound may also be used in the form of a pharmaceutically acceptable salt. Acids that can form salts with the polypeptide include inorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid (HCl), 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, naphthalenesulfonic acid, and sulfanilic acid.

[0090] Bases that can form salts with polypeptides include sodium hydroxide, ammonium hydroxide, potassium hydroxide, and the like; and inorganic bases such as organic bases (such as mono-, di-, and trialkyl) and arylamines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, and the like).

[0091] Targeting peptides can be synthesized by any of the techniques known to those skilled in the art of peptide technology, including recombinant DNA techniques. Synthetic chemistry techniques, such as solid-phase Merrifield synthesis, can be used for reasons of purity, antigen specificity, freedom from unwanted by-products, ease of preparation, etc. A summary of the many techniques available can be found in the following references: Steward et al., "Solid Phase Peptide Synthesis," W.H. Freeman Co., San Francisco, 1969; Bodanszky et al., "Peptide Synthesis," John Wiley & Sons, 2nd ed., 1976; J. Meienhofer, "Hormonal Proteins and Peptides," Vol. 2, p. 46, Academic Press (New York), 1983; Merrifield, Adv. Enzymol., 32:221-96, 1969; Fields et al., Int. J. Peptide Protein Res., 35:161-214, 1990; and U.S. Pat. No. 4,244,946 regarding solid-phase peptide synthesis; and Schroder et al., "The Peptides," Vol. 1, Academic Press (New York), 1965 regarding classical solution synthesis; each of these references is incorporated herein by reference. Suitable protecting groups that can be used in such syntheses are described above as well as in JFW MacOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, 1973, which reference is incorporated herein by reference.

[0092] Generally, 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 nascent peptide chain. Usually, either 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 utilized for amino acids containing reactive side groups, such as lysine.

[0093] As an example, using solid-phase synthesis, a protected or derivatized amino acid can be linked to an inert solid support via its unprotected carboxyl or amino group. The amino or carboxyl protecting group can then be selectively removed, and the adjacent amino acid in the sequence, bearing a suitably protected complementary (amino or carboxyl) group, is mixed and reacted under suitable conditions to form an amide bond with the residue already attached to the solid support. The amino or carboxyl protecting group can then be removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so on. After all the desired amino acids have been linked in the appropriate sequence, any remaining terminal and side-group protecting groups (and the solid support) can be removed sequentially or simultaneously, thereby yielding the final linear polypeptide.

[0094] It is understood that targeting peptides, in addition to PTPs, can bind to and / or complex with the homophilic binding domains of proteolytically cleaved extracellular fragments of other Ig superfamily cell adhesion molecules. For example, similar molecular detection strategies described herein can be used with any other Ig superfamily CAMs that have homophilic binding cell surface proteins whose ligand binding sites are known. A variety of cell surface proteins, including other phosphatases, are cleaved at cell surface sites (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 PMC Number: 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 PMC Number: 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 PMC Number: PMC3210297. These proteins are further targets that can be readily used by those skilled in the art to prepare therapeutic polypeptides that can be used in cancer treatment (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);

[0095] In some embodiments, the targeting peptides described herein may contain additional residues that may be added at either end of the polypeptide to provide a "linker" by which the polypeptide can be conveniently linked and / or attached to a spacer or near-infrared fluorophore. Typical amino acid residues used for linking include glycine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid. Furthermore, the subject polypeptides may differ in sequence, which may be modified by terminal NH2 acylation (e.g., acetylation) or thioglycolic acid amidation, by terminal carboxylamidation (e.g., with ammonia, methylamine), and similar terminal modifications. Terminal modifications are well known to be useful for reducing susceptibility to proteolytic enzyme digestion and thus for extending the half-life of polypeptides in solution, particularly biological fluids where proteases may be present. In this regard, polypeptide cyclization is also a useful terminal modification and is particularly preferred due to the stable structures formed by cyclization and the observed biological activity of cyclic peptides as described herein.

[0096] The optional spacer may contain additional natural and / or unnatural amino acid residues added at either end of the targeting peptide (or the targeting peptide with the linker peptide). The spacer may contain at least three natural or unnatural amino acids and has a structure 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 near-infrared fluorophore. Typical amino acid residues for use in spacers include glycine, serine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid.

[0097] In some embodiments, a spacer is selected in part based on its ability to modify affinity (eg, make the agent more hydrophilic or hydrophobic) depending on its desired application.

[0098] In some embodiments, the spacer can be a flexible peptide that directly or indirectly links the targeting peptide to the near-infrared fluorophore. The flexible peptide or peptidomimetic spacer can be, for example, at least about 3 to about 30 or fewer natural or unnatural amino acids in length. For example, the spacer can have a length of 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 unnatural amino acids. When the spacer is a peptide spacer, the peptide spacer can be produced as a single recombinant polypeptide using conventional molecular biology / recombinant DNA methods.

[0099] In some embodiments, the peptide 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.

[0100] In other embodiments, the peptide spacer comprises at least 50%, at least 60%, at least 70%, or at least 80% glycine residues, hi some embodiments, the remainder of the peptide spacer comprises serine residues.

[0101] In some embodiments, the peptide spacer is a polyglycine or glycine / serine spacer, consisting purely of glycine residues or consisting of glycine and serine residues. The small size of the glycine residues provides flexibility and allows mobility of at least one of the targeting peptide and the detectable moiety, therapeutic agent, or theranostic agent to which it is attached. The incorporation of serine can maintain the stability of the spacer in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing unfavorable interactions between the spacer and the targeting peptide.

[0102] In some embodiments, the peptide spacer comprises at least one amino acid sequence of (GS)a, (GGS)b, or (GGGS)c, or (GGGGS)d, where a, b, c, and d are each independently 2, 3, 4, 5, or 6. For example, the spacer may have the 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). obtain.

[0103] In some embodiments, the peptide spacer can be a contiguous portion of the targeting peptide that is directly linked to the N-terminal or C-terminal residue of the targeting peptide with or without a linker peptide.

[0104] For example, a polyglycine or glycine / serine spacer linked to an SBK2 targeting peptide having SEQ ID NO:5 may be GGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 28), GGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 29), GGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 30), GGGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 31), GGGGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 32), GGGGGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 33), GGGGGGGGG.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 34), GSGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 35), GSGSGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 36), GSGSGSGS.GEGDDFNWEQVNTLTKPTSD (SEQ ID NO: 37), 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) The amino acid sequence may be:

[0105] It is understood that other spacers can be linked to SBK2 or other targeting peptides described herein at the N-terminal or C-terminal portion of the targeting peptide.

[0106] In some embodiments, a targeting peptide having a continuous peptide spacer can be produced as a recombinant polypeptide. A variety of host organisms can be used to produce recombinant polypeptides. Examples of hosts include, but are not limited to, bacteria such as E. coli, yeast cells, insect cells, plant cells, and mammalian cells. Those skilled in the art will understand how to consider specific criteria when selecting a suitable host for producing a recombinant polypeptide. Factors that influence the selection of a host include, for example, post-translational modifications (such as phosphorylation and glycosylation patterns) and technical factors (such as general expected yield and ease of purification). Host-specific post-translational modifications of targeting peptides or spacer peptides used in vivo should be carefully considered, as certain post-translational modifications are known to be highly immunogenic.

[0107] Alternatively, the spacer may be a non-amino or non-peptide linker. For example, the non-peptide linker may be a biocompatible polymer comprising two or more repeating units linked together. Examples of non-peptide polymers include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol (PPG), copoly(ethylene / propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacrylamide, polyacrylic acid, polycyanoacrylates, lipid polymers, chitin, hyaluronic acid, and heparin. Typically, such linkers will have a molecular weight ranging from about 1 kDa to 50 kDa, depending on the particular linker. For example, a typical PEG has a molecular weight of about 1 to 5 kDa, while a polyethylene glycol has a molecular weight of about 5 kDa to 50 kDa, and more preferably about 10 kDa to 40 kDa.

[0108] Near-infrared fluorophores directly or indirectly linked to a targeting peptide or optional spacer via natural or non-natural linkages can include small organic fluorophores that fluoresce in the first near-infrared region (NIR-I, 650-1000 nm) or the second near-infrared region (NIR-II, 1000-1700 nm) upon irradiation. The near-infrared fluorophores, when directly or indirectly linked to a targeting peptide or optional peptide spacer via natural or non-natural linkages, can be sufficiently or effectively hydrophobic or lipophilic so that the near-infrared imaging agent has a signal-to-background ratio (SBR) (or signal-to-noise ratio (SNR)) during fluorescence imaging that is effective for distinguishing cancer cells or other cells in the cancer cell microenvironment from surrounding tissue.

[0109] It has been surprisingly discovered that the hydrophobicity or lipophilicity of small organic near-infrared fluorophores can substantially affect the SBR of near-infrared contrast agents. More hydrophobic and lipophilic near-infrared fluorophores, when linked directly or indirectly to targeting peptides that specifically bind to and / or complex with proteolytically cleaved extracellular fragments of immunoglobulin (Ig) superfamily cell adhesion molecules, or to an optional spacer via natural or non-natural linkages, can provide or form near-infrared contrast agents that can readily distinguish cancer cells from surrounding tissue, in contrast to more hydrophilic small organic near-infrared fluorophores that, when linked directly or indirectly to targeting peptides or optional spacers via natural or non-natural linkages, cannot distinguish cancer cells from surrounding tissue.

[0110] In some embodiments, more hydrophobic, lipophilic near-infrared fluorophores that can provide or form near-infrared imaging agents with targeting peptides that can readily distinguish cancer cells from surrounding tissue can include small organic molecule near-infrared fluorophores that have a logP of at least 0, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6 or more, e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or any range therebetween.

[0111] In some embodiments, the near-infrared fluorophore can include a cyanine near-infrared fluorophore that fluoresces in a first near-infrared region or a second near-infrared region. Cyanine near-infrared fluorophores are broadly defined as two heterocyclic nitrogen atoms connected by an electron-deficient polymethine bridge. Monomethine cyanines exhibit one methine unit, defined in this case as (=C-), between the heterocyclic structures; this class of compounds exhibits absorption in the ultraviolet and visible regions with low fluorescence quantum yields. Extending the length of the central chromophore with a set of two methylene groups results in tri-, penta-, and heptamethine cyanines. The wavelength of trimethine cyanines is too low to be useful for NIR imaging in biological systems; on the other hand, penta- and heptamethine cyanines possess near-infrared absorption and fluorescence properties that are a function of their heterocyclic structures and moieties within the polymethine chain, and can be tuned to provide high quantum yields and molecular brightness.

[0112] In some embodiments, cyanine near-infrared fluorophores having sufficient hydrophobicity, lipophilicity, and / or logP to provide or form near-infrared contrast agents with targeting peptides that can readily distinguish cancer cells from surrounding tissue can include indocyanine green (ICG) or ICG-Osu.

[0113] In some embodiments, the natural or non-natural linker used to directly or indirectly link the near-infrared fluorophore to the targeting peptide may include any natural or chemical linker that is insensitive to proteolytic cleavage and that is a non-contiguous portion of the targeting peptide or optional spacer. By "non-contiguous portion" is meant that the targeting peptide and spacer are linked via a portion of the targeting peptide or spacer that is continuous in nature and function as a linker and / or an additional element that is not a peptide residue.

[0114] In some embodiments, non-natural linkers can be created using coupling agents attached to or containing a moiety of the near-infrared fluorescent imaging agent. Coupling and / or linking agents can include, for example, maleimidyl linking agents, which can be used to couple to thiol groups; isothiocyanate and succinimidyl (e.g., N-hydroxysuccinimidyl (NHS)) linking agents, which can couple to free amine groups; diazonium linking agents, which can be used to couple to phenols; and amines, which can be used to couple to free acids, such as carboxylate groups, using carbodiimide activation. Useful functional groups can be present on the targeting peptide or optional spacer based on the specific 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 (both homo- and heterofunctional; such as those listed in the catalog of Pierce Chemical Co., Rockford, 111) can be used as coupling agents. Coupling can be achieved, for example, via amino groups, carboxyl groups, sulfhydryl groups, or oxidized carbohydrate residues.

[0115] Examples of coupling and / or conjugation agents are described in "CHEMICAL MODIFICATION OF PROTEINS" by Means and Feeney (Holden-Day, 1974, pp. 39-43). These reagents include, inter alia, J-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N,N'-(1,3-phenylene)bismaleimide (both of which are highly specific for sulfhydryl groups and form irreversible bonds); N,N'-ethylene-bis-(iodoacetamide) or other such reagents with a 6- to 11-carbon methylene bridge (which are relatively specific for sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversible bonds with amino and tyrosine groups). Other coupling or complexing agents may include p,p'-difluoro-m,m'-dinitrodiphenyl sulfone (which forms irreversible bonds with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4-disulfonyl chloride (which reacts primarily with amino groups); hexamethylene diisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts primarily with amino groups); glutaraldehyde (which reacts with several different side chains) and disdiazobenzidine (which reacts primarily with tyrosine and histidine).

[0116] Coupling agents can be homobifunctional (i.e., have two functional groups that undergo the same reaction). One example of a homobifunctional cross-linking reagent is bismaleimidohexane ("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. Therefore, BMH is useful for irreversible linkage of polypeptides containing cysteine ​​residues.

[0117] Coupling agents can also be heterobifunctional. Heterobifunctional coupling or linking agents have two different functional groups (e.g., an amine-reactive group and a thiol-reactive group; they crosslink two proteins with free amines and thiols, respectively). Examples of heterobifunctional crosslinkers are succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC"), m-maleimidobenzoyl-N-hydroxysuccinimide ester ("MBS"), and succinimidyl 4-(p-maleimidophenyl)butyrate ("SMPB") (an extended-chain analog of MBS). The succinimidyl group of these crosslinkers reacts with primary amines, and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine ​​residue.

[0118] The coupling agent can produce a conjugate of the targeting peptide or optional spacer and the near-infrared fluorophore that is essentially non-cleavable under cellular conditions. Many coupling agents, including those listed above, are commercially available. Detailed instructions for their use are readily available from commercial suppliers. A general reference for protein crosslinking and preparation of conjugates is Wong, "CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING" (CRC Press, 1991).

[0119] In some embodiments, the near-infrared imaging agent has the formula: [ka] or a pharmaceutically acceptable salt thereof; where R 1 is a near-infrared fluorophore described herein; and R 2comprises a targeting peptide that specifically binds to and / or forms a complex with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule expressed by the cancer cell or another cell in the cancer cell microenvironment, and an optional spacer directly linked to the targeting peptide.

[0120] In other embodiments, the near-infrared imaging agent has the formula: [ka] or a pharmaceutically acceptable salt thereof; where R 2 comprises a targeting peptide that specifically binds to and / or forms a complex with a 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, and an optional spacer directly linked to the targeting peptide.

[0121] In some embodiments, R 2 consists of a targeting peptide.

[0122] In other embodiments, R 2 consists of a targeting peptide linked to a spacer, where the spacer separates the amide from the targeting peptide.

[0123] The near-infrared contrast agent described herein can be administered to a subject by, for example, systemic, local, and / or parenteral administration. These methods include, for example, injection, infusion, deposition, implantation, or external administration, or any other administration method that is desired for the near-infrared contrast agent to access tissue. In one example, the administration of the near-infrared contrast agent can be performed by intravenous injection of the near-infrared contrast agent in the subject. A single dose or multiple doses of the probe can be administered. As used herein, "administered" refers to the provision or delivery of the near-infrared contrast agent in an amount and for a time period effective to label cancer cells in the subject.

[0124] The near-infrared contrast agents described herein can be administered to a patient in a detectable amount of a pharmaceutical composition comprising the near-infrared contrast agent or a pharmaceutically acceptable water-soluble salt thereof.

[0125] The formulation of the administered near-infrared contrast agent will vary depending on the route of administration selected (e.g., solution, emulsion, capsule, etc.). Suitable pharmaceutically acceptable carriers may contain inactive ingredients that do not unduly inhibit the biological activity of the near-infrared contrast agent. Pharmaceutically acceptable carriers should be biocompatible (e.g., non-toxic, non-inflammatory, non-immunogenic, and free of other undesirable reactions upon administration to a subject). Standard pharmaceutical formulation techniques can be used (e.g., those described in Remington's Pharmaceutical Sciences, ibid.). Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate-buffered saline, Hank's solution, lactated Ringer's solution, etc.

[0126] The preparation of pharmacological composition that contains dissolved or dispersed active ingredient is well understood in the art.Typically, such composition is prepared as an injectable, either as a solution or suspension, but also can be prepared in solid form suitable for solution or suspension in liquid before use.Preparation varies according to the route of administration selected (for example, solution, emulsion, capsule).

[0127] By "detectable amount" is meant that the amount of near-infrared contrast agent administered is sufficient to allow detection of binding of the near-infrared contrast agent to cancer cells. By "imaging effective amount" is meant that the amount of near-infrared contrast agent administered is sufficient to allow fluorescence imaging of binding of the near-infrared contrast agent to cancer cells.

[0128] Near-infrared imaging agents administered to a subject can be used in methods to detect and / or determine the presence, location, and / or distribution of cancer cells, i.e., cancer cells associated with proteolytically cleaved extracellular fragments of Ig superfamily cell adhesion molecules, in at least one region of interest (ROI) in a patient's organ or body. The ROI can include a specific region or portion of the subject, and in some instances, two or more regions or portions throughout the subject. The ROI can include regions imaged for both diagnostic and therapeutic purposes. The ROI is typically internal; however, it should be understood that the ROI may additionally or alternatively be external.

[0129] The presence, location, and / or distribution of near-infrared contrast agents in animal tissue, such as brain tissue, can be visualized by a near-infrared fluorescence (NIRF) scanner. In one example, the NIRF scanner can be handheld. In another example, the NIRF scanner can be miniaturized and embedded in an instrument (e.g., a micromachine, a scalpel, a neurosurgical cell removal device).

[0130] As used herein, "distribution" refers to the spatial property of being scattered over an area or volume. In this case, "distribution of cancer cells" refers to the spatial property of cancer cells scattered over an area or volume contained in an animal tissue, such as brain tissue. The distribution of the near-infrared contrast agent can then be correlated with the presence or absence of cancer cells in the tissue. The distribution can be a clue to the presence or absence of cancer cells, or can be combined with other factors and symptoms by those skilled in the art to reliably detect migrating or dispersing cancer cells, the presence or absence of cancer metastasis, or to determine tumor boundaries in a subject. It should be understood that an imaging modality can be used to generate a baseline image before administration of the near-infrared contrast agent. In this case, the baseline and post-administration images can be compared to confirm the presence, absence, and / or severity of a particular disease or condition.

[0131] In one aspect, near-infrared contrast agents can be administered to a subject to assess the distribution of cancer cells in the subject and correlate the distribution to specific locations.Surgeons routinely use intraoperative fluorescence imaging during surgical resection.This allows them to specifically identify and examine tissues from different areas of a tumor, such as the tumor margin or tumor center.Frequently, surgeons also examine brain regions adjacent to the tumor border, outside the tumor margin, which appear normal to the naked eye but are infiltrated by dispersed tumor cells in histological examination.For example, in glioma (brain tumor) surgery, near-infrared contrast agents can be administered intravenously before performing pre-surgical localization imaging.The agent can be imaged using near-infrared fluorescence imaging to localize with glioma.

[0132] The agents described herein, which contain near-infrared contrast agents and specifically bind to and / or complex with cell-associated proteolytically cleaved Ig superfamily cell adhesion molecules (e.g., PTPμ), can be used in intraoperative imaging (IOI) techniques to guide surgical resection, eliminating surgeons' "educated guesses" regarding the location of tumor borders. Previous studies have demonstrated that more extensive surgical resection improves 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. Therefore, near-infrared contrast agents that function as diagnostic molecular contrast agents have the potential to increase patient survival.

[0133] In some embodiments, upon administration to a subject, the near-infrared imaging agent can target cancer cells, i.e., cancer cells associated with proteolytically cleaved extracellular fragments of Ig superfamily cell adhesion molecules, in a patient's organ or body region, allowing the presence, location, and / or distribution of the cancer cells to be detected and / or determined. In one example, the near-infrared imaging agent can be combined with intraoperative imaging (IOI) to identify malignant cells that have already infiltrated and / or are beginning to infiltrate the tumor-brain border. The method can be performed in real time during brain surgery or other surgical procedures. The method can include local or systemic administration of the near-infrared imaging agent described herein. A fluorescence imaging modality can then be used to detect and subsequently collect image data. The resulting image data can be used, at least in part, to determine surgical and / or radiation treatment. Alternatively, the image data can be used, at least in part, to control automated surgical devices (e.g., lasers, scalpels, micromachines) or to assist in manual guidance of surgical procedures. Additionally, the image data may be used to plan and / or control the delivery of therapeutic agents (eg, by microelectronic devices or micromachines).

[0134] In one example, a near-infrared contrast agent can be applied locally as needed during the surgical procedure to interactively guide the surgeon and / or surgical instruments to any remaining abnormal cells. The near-infrared contrast agent can be applied locally at a low concentration so as not to reach pharmacologically relevant concentrations. In one example, excess material can be removed (e.g., washed away) after a period of time (e.g., an incubation period).

[0135] Another embodiment described herein relates to a method for monitoring the efficacy of a cancer drug or therapy used in a subject. The methods and agents described herein can be used to monitor and / or compare cancer invasion, migration, dissemination, and metastasis in a subject before, during, or after the administration of a cancer drug or therapy.

[0136] As used herein, a "cancer therapeutic agent" or "cancer therapy" can include any agent or treatment regimen that can negatively affect cancer in an animal, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the rate of cancer cell proliferation, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting cancer progression, or prolonging the survival of an animal with cancer. A cancer therapeutic agent can include one or more therapies, such as, but not limited to, chemotherapy, radiation therapy, hormonal therapy, and / or biological therapy / immunotherapy. A reduction in, for example, cancer volume, growth, migration, and / or dissemination in a subject can indicate the effectiveness of a given therapy. This can provide a direct clinical efficacy endpoint measure for a cancer therapeutic agent. Accordingly, in another aspect, a method for monitoring the effectiveness of a cancer therapeutic agent is provided. More specifically, embodiments of the present application provide a method for monitoring the effectiveness of a cancer therapy.

[0137] A method for monitoring the effectiveness of a cancer therapeutic agent can include administering a near-infrared contrast agent as described herein to an animal in vivo, then visualizing the distribution of the near-infrared contrast agent in the animal (for example, using an in vivo imaging modality as described herein), and then correlating the distribution of the near-infrared contrast agent with the effectiveness of the cancer therapeutic agent.The administration step can occur before, during, and after a selected treatment regimen to determine the effectiveness of the treatment regimen.One method for evaluating the effectiveness of a cancer therapeutic agent is to compare the distribution of the near-infrared contrast agent before and after cancer therapy.

[0138] In some embodiments, a near-infrared imaging agent bound to and / or complexed with a proteolytically cleaved extracellular fragment of 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 effectiveness of the cancer therapeutic agent 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 therapeutic agent and / or cancer therapy. The location and / or distribution of cancer cells in the subject prior to administration of the cancer therapeutic agent and / or cancer therapy can be determined by administering a near-infrared imaging agent to the subject and detecting the agent bound to and / or complexed with cancer cells in the subject prior to administration of the cancer therapeutic agent and / or cancer therapy.

[0139] In certain embodiments, the methods and agents described herein can be used to evaluate the effectiveness of a therapeutic agent administered to a subject to treat metastatic, invasive, or disseminated cancer. In this embodiment, the agent can be administered to the subject before, during, or after a therapeutic regimen, and the distribution of cancer cells can be imaged to determine the effectiveness of the therapeutic regimen. In one example, the therapeutic regimen can include surgical resection of metastatic cancer, and a near-infrared imaging agent can be used to reveal the distribution of metastatic cancer before and after surgery to determine the effectiveness of the surgical resection. Optionally, the method and near-infrared imaging agent can be used in intraoperative surgical procedures such as surgical tumor resection, as described above, to more easily reveal and / or image the amount or volume of cancer cells during surgery.

[0140] In other embodiments, the near-infrared imaging agents can be used in methods of treating cancer or tumors (e.g., brain cancers or tumors). In one embodiment, the near-infrared imaging agents can be used in imaging-mediated phototherapy to ablate cancer cells or other cells in the cancer cell microenvironment.

[0141] Image-mediated phototherapy can include imaging-guided photothermal therapy (PTT) and imaging-guided photodynamic therapy (PDT). In PTT, near-infrared fluorophores of a near-infrared imaging agent bound to cancer cells or other cells in the cancer cell microenvironment can be irradiated with a wavelength of light effective to convert light energy into heat and ablate the cancer. Advantageously, the generated heat can potentially cause thermal expansion of the cancer tissue, generating a photoacoustic imaging (PAI) signal. Alternatively, near-infrared fluorophores of a near-infrared imaging agent bound to cancer cells or other cells in the cancer cell microenvironment can be irradiated with a wavelength of light effective to generate singlet acid (O) or other reactive oxygen species (ROS) under irradiation, inducing apoptosis or necrosis of the cancer cells, which can be applied to imaging-guided photodynamic therapy (PDT) and further to achieve synergistic PDT / PTT. Only cells simultaneously exposed to the near-infrared contrast agent and light are destroyed, while surrounding healthy non-target and non-irradiated cells are spared photodamage. Furthermore, the fluorescence of the near-infrared contrast agent allows for simultaneous diagnostic optical imaging that can be used to guide cancer treatment.

[0142] Methods for performing PTT and / or PDT are known in the art. See, for example, Thierry Patrice, "Photodynamic Therapy," Royal Society of Chemistry, 2004. The pharmaceutical composition comprising the near-infrared agent described herein can be applied to organs or tissues as a step in PTT and / or PDT. In certain embodiments, the composition is applied to epithelial, mesothelial, synovial, fascial, or serous surfaces (including, but not limited to, the surfaces of the eye, esophagus, mucous membrane, bladder, joint, tendon, ligament, bursa, gastrointestinal tract, urogenital tract, pleura, pericardium, lung, or urethral epithelium).

[0143] Near-infrared imaging agents for PTT and / or PDT can also be administered to a subject with cancer by systemic administration (e.g., intravenous administration). Upon administration, the near-infrared imaging agents described herein can localize and / or accumulate at the site of the targeted tumor or target cancer. In some embodiments, specific binding and / or complexation with a 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 to bind to, complex with, and / or be internalized by the target cell. This binding and / or internalization is specific to the target cell, allowing the targeted agent to selectively target cancer cells in the subject or cells in the cancer cell microenvironment.

[0144] Following administration and localization of the near-infrared contrast agent to the targeted cancer cells, the targeted cancer cells can be exposed to a therapeutic amount of light that causes cancer cell ablation, damage, and / or inhibition of cancer cell growth. Light capable of activating the near-infrared contrast agent as a PTT agent and / or a PDT agent can be delivered to the targeted cancer cells using, for example, a semiconductor laser, a dye laser, an optical parametric oscillator, etc. It should be understood that any light source can be used as long as the light excites the near-infrared contrast agent.

[0145] The near-infrared contrast agents described herein can be administered to a subject by any conventional method of drug administration, for example, orally in capsules, suspensions, or tablets, or by parenteral administration. Parenteral administration can include, for example, intramuscular, intravenous, intraventricular, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration. The disclosed compounds can also be administered orally (e.g., via capsules, suspensions, tablets, or meals), intranasally (e.g., solutions, suspensions), transdermally, intradermally, topically (e.g., creams, ointments), by inhalation (e.g., intrabronchial, intranasal, oral inhalation, or nasal drops), transmucosally, or rectally. Delivery can also be by injection into the patient's brain or body cavity, by using a slow-release or sustained-release matrix delivery system, or by on-site delivery using micelles, gels, and liposomes. Nebulizers, powder inhalers, and aerosolized solutions can also be used to administer such preparations to the respiratory tract. Delivery can be in vivo or ex vivo. Administration can be local or systemic as described above. Two or more routes can be used simultaneously, if necessary. The preferred mode of administration varies depending on the specific disclosed compound selected. In certain embodiments, oral, parenteral, or systemic administration is the preferred mode of administration for treatment.

[0146] The near-infrared imaging agents described herein can be administered alone as monotherapy, or together or in combination with one or more additional therapeutic agents. For example, the near-infrared imaging agents described herein can be administered to a subject before, during, or after administration of an additional therapeutic agent, so that the distribution of metastatic cells can be targeted by the therapeutic agent. The near-infrared imaging agents can be administered to an animal as part of a pharmaceutical composition containing the near-infrared imaging agent and a pharmaceutically acceptable carrier or excipient, and optionally one or more additional therapeutic agents. The near-infrared imaging agents described herein and the additional therapeutic agent can be components of different pharmaceutical compositions that can be mixed together before administration or administered separately. The near-infrared imaging agents described herein can be administered, for example, as a composition containing the additional therapeutic agent, thereby being administered simultaneously with the agent. Alternatively, the near-infrared imaging agents described herein and the therapeutic agent can be administered simultaneously without mixing (e.g., by delivery of the agent through an intravenous line, through which the therapeutic agent is also administered, or vice versa). In another embodiment, the near-infrared imaging agents described herein can be administered separately (e.g., unmixed), but within a short time frame (e.g., within 24 hours) after administration of the therapeutic agent.

[0147] The methods described herein contemplate single administration as well as multiple administrations (either given simultaneously or over an extended period of time). The near-infrared imaging agent (or a composition comprising the agent) described herein can be administered at regular intervals, depending on the nature and extent of the impact of the inflammatory disorder and the current situation. As used herein, administration at "regular intervals" refers to the periodic administration of a therapeutically effective amount (as distinguished from a single administration). In one embodiment, the near-infrared imaging agent is administered periodically, for example, at regular intervals (e.g., every two months, every month, every two weeks, weekly, twice weekly, daily, twice or three times a day, or more frequently).

[0148] The dosing interval for a single individual may be fixed or may vary over time according to the needs of that individual, for example, the dosing interval may be shortened in times of physical ill-health or stress, or if disease symptoms worsen.

[0149] For example, administration of the near-infrared imaging agent and / or additional therapeutic agent may be performed on at least the following days: day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, day 42, day 43, day 44, day 45, day 46, day 47, day 48, day 49, day 50, day 51, day 52, day 53, day 54, day 55, day 56, day 57, day 58, day 59, day 60, day 61, day 62, day 63, day 64, day 65, day 66, day 67, day 68, day 69, day 70, day 71, day 72, day 73, day 74, day 75, day 76, day 77, day 78, day 79, day 80, day 81, day 82, day 83, day 84, day 85, day 86, day 87, day 88, day 89, day 90, day 91, day 92, day 93, day 94, day 95, day 96, day 97, day 98, day or once on day 40, or once on at least week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, or in divided doses every 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, or 2 hours, or any combination thereof. Administration can be at any time of day, for example, in the morning, afternoon, or evening. For example, administration can be performed in the morning, eg, between 6:00 AM and 12:00 PM (noon); in the afternoon, eg, after noon and before 6:00 PM; or in the evening, eg, between 6:00 PM and midnight.

[0150] The near-infrared imaging agents and therapeutic and / or additional therapeutic agents described herein can be administered at a dose of, for example, 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 per day). Dosage forms (compositions) suitable for internal administration generally contain about 0.1 milligrams to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions, the active ingredient is typically present in an amount of about 0.5 to 95% by weight based on the total weight of the composition.

[0151] The amount of near-infrared imaging agent described herein and therapeutic agent and / or additional therapeutic agent described herein administered to a subject may depend on the characteristics of the subject (such as general health, age, sex, weight, and drug tolerance, as well as the degree, severity, and type of rejection). One of ordinary skill in the art would be able to determine appropriate dosages depending on these and other factors using standard clinical techniques.

[0152] Furthermore, in vitro or in vivo assays can be used to determine the desired dosage range. The dosage used can also depend on the route of administration, the severity of the disease, and the condition of the subject. The effective dosage can be estimated from a dose-response curve using an in vitro or animal model test system. The amount of the near-infrared contrast agent described herein can also depend on clinical factors and the route of administration of the near-infrared contrast agent, as well as the disease state or condition being treated.

[0153] The near-infrared contrast agents disclosed and 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 administered near-infrared contrast agent will vary depending on the selected route of administration (e.g., solution, emulsion, capsule, etc.). Suitable pharmaceutically acceptable carriers may contain inactive ingredients that do not unduly inhibit the biological activity of the compound. Pharmaceutically acceptable carriers must be biocompatible (e.g., non-toxic, non-inflammatory, non-immunogenic, and free of other undesirable reactions) upon administration to a subject. Standard pharmaceutical formulation techniques (such as those described in Remington's Pharmaceutical Sciences, ibid.) can be used. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, saline, bacteriostatic saline (saline containing approximately 0.9% mg / ml benzyl alcohol), phosphate-buffered saline, Hank's solution, lactated Ringer's solution, and the like. Methods for encapsulating compositions (such as with a coating of hard gelatin or cyclodextran) are known in the art (Baker et al., "Controlled Release of Biological Active Agents," John Wiley and Sons, 1986).

[0154] The preparation of pharmacological compositions containing an active ingredient dissolved or dispersed therein is well known in the art. Typically, such compositions are prepared as either injectable solutions or suspensions, although solid forms suitable for solution or suspension in liquid prior to use can also be prepared. Formulations vary depending on the selected route of administration (e.g., solution, emulsion, capsule).

[0155] Pharmaceutically acceptable carriers for pharmaceutical compositions can also include delivery systems known in the art for incorporating or encapsulating agents, such as anticancer agents, in some embodiments, the disclosed compounds can be used with delivery systems including, for example, liposomes, nanoparticles, nanospheres, nanodiscs, 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 tumors." 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. Each reference cited in this paragraph is incorporated herein by reference in its entirety.

[0156] The following examples are for illustrative purposes only and are not intended to limit the scope of the claims appended hereto.

[0157] Example Recent research has focused on molecular imaging agents incorporating cancer-specific targeting moieties, which allow for more precise localization of fluorescent labels to tumors in vivo. Ideally, these agents would more clearly distinguish tumor borders and aid in the identification of invading or metastatic cells. The PTPμ biomarker, derived from the full-length receptor protein tyrosine phosphatase μ (PTPμ), is found in GBM both at the tumor margin and associated with dispersed cells. In normal cells, PTPμ is a homotypic cell adhesion molecule that functions in contact inhibition. In GBM and other cancers, PTPμ-mediated cell-cell adhesion is altered by aberrant protease activity within the tumor microenvironment, which cleaves the extracellular domain of PTPμ into fragments. Peptides targeting the homotypic binding region within the PTPμ biomarker successfully bind GBM both in vitro and in vivo using multiple imaging modalities. These "SBK" peptides demonstrated labeling of the main tumor mass as well as migrating and infiltrating glioma cells using a unique cryo-imaging technique after conjugation to Cy5 fluorophores.

[0158] Four PTP μ-derived peptides were utilized as tumor imaging agents. One of these peptides, SBK2, was selected as the targeting moiety for the agent described herein based on previous studies. Accurately predicting the in vivo behavior of molecular imaging agents is challenging. In addition to the choice of targeting moiety, both the fluorophore and the spacer or linker significantly influence biodistribution. We tested the insertion of a six-amino acid linker, GGSGGS (SEQ ID NO: 20), between the targeting peptide and the fluorophore. This linker was designed to allow flexibility between the peptide and the fluorophore. Three NIR fluorophores were selected to examine the effect of fluorophore hydrophobicity / hydrophilicity on in vivo behavior: ICG, IRDye® 800CW, and Tide Fluor® 8WS. ICG is a more hydrophobic molecule and has been used safely in medical facilities for decades. In contrast, both IRDye® 800CW and Tide Fluor® 8WS are hydrophilic dyes according to their manufacturers. As controls, scrambled peptide versions of all agents were synthesized and tested in mice to confirm the specificity of SBK2-containing agents for the PTPμ biomarker in vivo.

[0159] Materials and Methods material Fmoc-protected amino acids were purchased from either Aapptec (Louisville, KY, USA) or Chem-Impex (Wood Dale, IL, USA). Other reagents obtained from Chem-Impex included 2-chlorotrityl chloride resin and (O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HCTU). Solvents N,N-dimethylformamide (DMF), dichloromethane (DCM), diethyl ether, HPLC-grade water, and HPLC-grade acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA, USA). Anhydrous N,N-diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), 2,2′-(ethylenedioxy)diethanethiol (DODT), triisopropylsilane (TIS), 4-methylpiperidine, dimethyl sulfoxide (DMSO), and ammonium bicarbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS), pH 7.4, or ICU from Life Technologies (Grand Island, NY, USA) were purchased from ICU. 0.9% sodium chloride (saline) from A&M Medical (Lake Forest, IL, USA) was used to prepare and administer the contrast agent. The fluorophore IRDye® 800CW maleimide was purchased from LI-COR Biosciences (Lincoln, NE, USA). The fluorophores Tide Fluor® 8WS, indocyanine green-OSu (ICG-OSu), and ICG acid were purchased from AAT Bioquest (Pleasanton, CA, USA).

[0160] Synthesis of near-infrared contrast agents The PTPμ targeting peptide SBK2 and a control scrambled peptide ("Scram") have been previously described. For some agents, an additional six amino acids (GGSGGS) were incorporated at the N-terminus. For brevity, these additional amino acids are referred to as "CLE" in the agent name. Peptides were conjugated to ICG-OSu on resin. A detailed description of the in-house method used is provided in this example. Several ICG-labeled peptides were produced by PolyPeptide Group (San Diego, CA, USA) using the same method.

[0161] The stock concentration was calculated based on the A780nm of multiple dilutions made in DMSO and the 230,000 M concentration indicated on the manufacturer's website. -1 cm -1 The extinction coefficient (ε) was determined using a multiple dilution method to ensure measurements were made in the linear range.

[0162] The hydrophilic nature and pH sensitivity of IRDye® 800CW precluded on-resin coupling in the organic solvents and TFA-based cleavage cocktails used for ICG-labeled molecules. IRDye® 800CW maleimide was conjugated to a cysteine ​​residue at the N-terminus of the peptide in aqueous solution according to the manufacturer's recommended protocol and was purchased at greater than 95% purity from PolyPeptide Group. Further LC-MS / MS analysis of these agents was performed by the Lerner Research Institute Proteomics Core at Cleveland Clinic using an Orbitrap Elite LC-MS instrument purchased through NIH Common Fund funding (number 1S10RR031537-01).

[0163] The Tide Fluor® 8WS-conjugated peptides "SBK2-TF8WS" and "Scram-TF8WS" were generated by Bachem Americas (Torrance, CA, USA) by attaching the acidic form of the Tide Fluor® 8WS fluorophore to the N-terminus of SBK2 and the scrambled peptide as the final step of synthesis after activation with a coupling agent in solid-phase peptide synthesis. The drugs were obtained with a purity of >95%.

[0164] Detailed methods for the synthesis of ICG-labeled SBK2 / SBK2-CLE peptides and scrambled / scrambled CLE peptides For ICG labeling of peptides, dry peptidyl resin lacking an N-terminal Fmoc group was weighed to obtain the desired amount of crude peptide, swollen in DMF for at least 2 hours, and then incubated in 100 mM triethylamine in anhydrous DMF for 10 minutes. Excess solvent was removed from the swollen resin, and a 1.5-2x molar excess of ICG-OSu (20 mM in DMSO) relative to the peptide was added along with a 10x molar excess of triethylamine. The resin / dye mixture was mounted on a rotator, protected from light, and allowed to react overnight at room temperature. The peptidyl resin / dye mixture was transferred to a fritted syringe (Torviq; Tucson, AZ, USA), washed extensively with DMF to remove unreacted fluorophores, equilibrated in DCM, and incubated for 1 hour at room temperature in a cleavage cocktail of 92.5% TFA / 2.5% TIS / 2.5% DODT / 2.5% water. After 1 hour, the cleaved peptide was precipitated in ice-cold diethyl ether, thoroughly washed with additional diethyl ether, and then dried. The ICG-conjugated peptide was evaluated by reverse-phase HPLC (water / 0.1% TFA (solvent A) and acetonitrile / 0.1% (solvent B)) using an analytical C18 column (Eclipse XDB-C18, 5 μm, 4.6 x 150 mm; Agilent; Santa Clara, CA, USA). HPLC runs were monitored at both 220 nm and 780 nm using an SPD-M20A diode array detector (Shimadzu Scientific Instruments; Columbia, MD, USA). The ICG-conjugated peptide was purified to >95% purity using a preparative C-18 column (ZORBAX 300SB-C18 PrepHT, 21.2 x 250 mm, 7 μm column; Agilent; Santa Clara, CA, USA) and the same solvents.

[0165] Mouse and experimental tumor models All applicable institutional and / or NIH guidelines for the care and use of animals were followed. The Case Western Reserve University Institutional Animal Care and Use Committee reviewed and approved the animal protocol. Outbred homozygous female nude mice (Fox1 nu / Fox1 nu) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Details of the human glioblastoma LN-229 cell line have been described above. LN-229 cells express the PTPμ biomarker and have been used in previous studies to evaluate PTPμ-targeting agents using different imaging modalities. Flank tumors were grown at 2x10 6 Tumors were induced in the right flank of approximately 7-week-old mice by injecting a mixture of cells and Matrigel® matrix (Corning®, Corning, Inc., Corning, NY, USA). Experiments were performed on mice 4 to 8 weeks after tumor implantation.

[0166] Fluorescence imaging experiments An IVIS Spectrum in vivo imaging system and bioimage analysis software (Perkin Elmer; Hopkinton, MA, USA) were used to acquire and analyze mouse images. Before the experiment, mice were weighed, and baseline images were acquired using appropriate filter and autoexposure settings. Three excitation / emission filter sets were used: 745 nm / 800 nm, 745 nm / 820 nm, and 745 nm / 840 nm. The filter set showing the highest fluorescence intensity of a fluorophore was used throughout for that dye. For ICG, 745 nm / 820 nm was used; for IRDye® 800CW and Tide Fluor® 8WS, 745 nm / 800 nm was used. Drug stocks were diluted to approximately 100 μM in saline or PBS, and the volume required for the desired dose (300 nmol / kg or 400 nmol / kg) was calculated. Each dose was placed in polyurethane tubing and connected to a 1 mL syringe as described above. Mice were anesthetized with 2% isoflurane in oxygen in an induction chamber and transferred to a 37°C heated platform equipped with a nose cone. A 26-gauge veterinary catheter was placed in each tail, and the pre-loaded tubing was connected. Drugs were infused over 30 seconds, the catheters were removed, and the animals were placed in the prepared Spectrum imaging chamber. Images were acquired beginning 10 minutes after drug injection. For SBK2-CCLE-IR800 and Scram-CCLE-IR800, images were acquired every 10 minutes for 60 minutes. For ICG- and TF8WS-labeled peptides, images were acquired at 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours. At the end of the imaging period, animals were euthanized by CO2 asphyxiation, and tumors and other organs were removed and imaged on the Spectrum instrument.

[0167] Data analysis In vivo image analysis software was used to analyze regions of interest (ROIs) in the optical data. Images acquired for each time point consisted of a fluorescent image overlaid on a photographic image. Binning was set to 1, and in vivo tumor ROIs were manually drawn based on the visible contours in the monochrome photographs for each time point. After delineating the tumor ROI, fluorescence was measured using the built-in spectral function. For excised tumors, kidneys, spleens, and livers, ROIs were drawn along the periphery of the tumor or organ.

[0168] LivingImage software measures fluorescence intensity in units of radiant efficiency (photons / sec / cm). 2 / steradian) / (μW / cm 2 Measurements of mean radiative efficiency at 1000 nm were used for further comparisons to account for differences in tumor and organ size. GraphPad Prism 9 was used for plotting and statistical analysis. Where indicated, mean values ​​for pairs of drugs were compared using a two-tailed t-test with Welch's correction, with p<0.05 considered statistically significant. Welch's correction was used to remove assumptions about variance between the groups being compared.

[0169] result A series of near-infrared imaging agents targeting the PTPμ biomarker, as well as appropriate control agents using scrambled peptides, were generated by conjugating the NIR fluorophores ICG, IRDye® 800CW, or Tide Fluor® 8WS to the peptide N-terminus. These agents are listed in Table 1. Additional structural information and properties are provided in Supplementary Table 1. ICG and Tide Fluor® 8WS were conjugated to the N-terminal amine of resin-bound peptides, resulting in the formation of an amide bond between the fluorophore and the peptide (Figure 7). Even when conjugated in a similar manner, the difference in hydrophobicity of the fluorophores significantly impacts their reversed-phase (RP) HPLC retention time (as shown by the ICG- and TF8WS-labeled peptides in Figure 7). Both SBK2-ICG and SBK2-CLE-ICG required >45% acetonitrile / 0.1% TFA to elute from the column, exhibiting RTs of 44.9 and 43.6 min, respectively (Figure 7).

[0170] [Table 1]

[0171] Interestingly, the insertion of a six-amino acid "CLE" between ICG and SBK2 resulted in a shorter retention time of approximately 1.3 min compared to SBK2-ICG (Figure 1). Tide Fluor® 8WS is described by the manufacturer as an alternative to IRDye® 800CW, which has similar hydrophilic properties but a broader pH compatibility. The more hydrophilic nature of SBK2-TF8WS compared to the CG-labeled peptide is evident by its much shorter retention time (RT) of 25.5 min.

[0172] A different strategy was used to generate drugs bearing the hydrophilic, low-pH-sensitive IRDye® 800CW. Because the SBK2 and scrambled peptides contain lysines, an additional cysteine ​​residue was added to the N-terminus of each peptide, and the dye was attached in aqueous solution via a thiol-maleimide reaction with IRDye® 800CW maleimide (Table 1, Figure 7). A closed cyclic succinimide ring was formed linking the dye to the peptide.

[0173] Similar to Tide Fluor® 8WS, the hydrophilic nature of IRDye® 800CW resulted in the elution of SBK2-CCLE-IR800 at approximately 25 minutes (Figure 7). However, evaluation of the IRDye® 800CW-labeled drugs by RP-HPLC revealed that a second peak appeared upon warming of stocks of SBK2-CCLE-IR800 and scrambled-CCLE-IR800 to room temperature. LC-MS / MS analysis confirmed the characteristics of the main peak as belonging to the drug with a closed succinimide ring, while a second peak, representing approximately 30% of this material and eluting slightly earlier than the main peak, was identified as containing an open succinimide ring (Figure 1). Conversion of a closed succinimide ring to a more stable open ring has also been reported to occur in other conjugates generated using thiol-maleimide reactions.

[0174] The ICG-labeled peptides were evaluated in vivo by injecting each of the four drugs into nude mice bearing LN-229 glioma flank tumors and acquiring images at various time points over a 24-hour period. As shown in Figure 1A, overall fluorescence rapidly increased after injection of the ICG drug at 300 nmol / kg and continued to increase for approximately 2 hours. While the fluorescence induced by the control scrambled drug remained diffuse throughout the imaging period, the signal became much more pronounced specifically in the tumor region of SBK2-CLE-ICG and SBK2-ICG-treated animals. At 30 minutes, fluorescent tumors were evident in SBK2-CLE-ICG-treated animals and remained highly fluorescent at 4 hours (Figure 1). At 2 and 4 hours, some fluorescence was observed in the kidneys of SBK2-CLE-ICG-treated mice as the drug disappeared (Figure 1A). For mice treated with SBK2-ICG, similar in vivo tumor labeling was observed with slightly different kinetics. Tumor-specific fluorescence was observed at 30 min, became more pronounced by 1 h after SBK2-ICG administration, and persisted for up to 24 h (Figures 1A and 2A). Figure 1B shows the mean radiant efficiency (mean ± standard error) of in vivo tumor signal over time in mice administered 300 nmol / kg of each drug. p values ​​obtained from unpaired t-tests between drugs at each time point are summarized in Tables 2 and 3. SBK2-ICG produced significantly higher in vivo tumor fluorescence compared with either Scram-ICG or Scram-CLE-ICG from 10 min to 24 h (Figure 1B). SBK2-ICG-treated animals also had significantly higher in vivo tumor fluorescence compared with SBK2-CLE-ICG-treated animals at 8 and 24 h (Figure 1B). SBK2-CLE-ICG administration produced significantly higher in vivo tumor signal compared to Scram-CLE-ICG from 10 min to 8 h and compared to Scram-ICG from 10 min to 4 h (Figure 1B). Injection of ICG dye at 400 nmol / kg (approximately 0.34 mg / mL) produced little in vivo tumor fluorescence, which disappeared much more rapidly than either ICG-conjugated peptide (Figure 1B).Peak ICG in vivo tumor signal occurred by 10 min. In vivo tumor fluorescence was significantly higher in animals treated with ICG-labeled peptides compared to the base ICG dye, except for Scram-CLE-ICG at 24 h (Figure 1B, Table 3).

[0175] Figure 2 shows images and measurements obtained 24 hours after administration of the ICG peptide. Figure 2A shows the 24-hour time point shown in Figure 1A at a different scale, along with the corresponding photographs. Much stronger tumor-specific fluorescence is detected in animals treated with 300 nmol / kg of SBK2-ICG compared to animals injected with other drugs (Figure 2A). Figure 2B shows the in vivo tumor signal (mean ± standard error) at 24 hours from mice treated with 300 nmol / kg or 400 nmol / kg of each drug or with 400 nmol / kg of ICG. Mean values ​​were compared as above, and p values ​​are summarized in Tables 2–4. SBK2-ICG at 300 nmol / kg and 400 nmol / kg produced significantly more tumor fluorescence at 24 hours compared to all other ICG-conjugated peptides at the same dose (Figure 2B, Tables 2 and 4). At 400 nmol / kg, SBK2-CLE-ICG produced significantly more in vivo tumor fluorescence than Scram-CLE-ICG (Figure 2B). Scram-ICG, when used at 400 nmol / kg (N = 6), appeared to disappear more slowly than either CLE-containing agent, and significantly higher in vivo tumor signals were detected in animals treated with this agent compared with either SBK2-CLE-ICG or Scram-CLE-ICG (Figure 2B, Table 4). At 400 nmol / kg, all four ICG peptides produced significantly higher in vivo tumor signals at 24 hours compared with ICG dye alone (Figure 2B, Table 4).

[0176] [Table 2]

[0177] [Table 3]

[0178] [Table 4]

[0179] After 24 hours of imaging, mice were euthanized, and tumors, kidneys, and livers were removed, along with the spleen, which served as a control and non-cleared organ. A representative example of ex vivo tumors obtained at 24 hours from animals injected with 300 nmol / kg is shown in Figure 3A, along with a plot of the average radiant efficiency (mean ± standard error) of ex vivo tumors using the drug at either 300 nmol / kg or 400 nmol / kg. Figure 3A shows the substantially stronger ex vivo tumor fluorescence present in mice treated with 300 nmol / kg SBK2-ICG. The ex vivo tumor fluorescence in mice treated with 300 nmol / kg SBK2-ICG was more than 3.5-fold stronger than that in the other groups (Figure 3B). At the higher dose of 400 nmol / kg, the fluorescence intensity ratio was lower in mice administered SBK2-ICG compared to the other drugs. At 400 nmol / kg, the SBK2-ICG signal was The SBK2-ICG signal was approximately 1.8-fold higher than the Scam-ICG signal, suggesting that stronger specificity was achieved at lower doses. Compared to ICG alone, tumor fluorescence from mice treated with SBK2-ICG varied from 52-fold higher (300 nmol / kg) to 70-fold higher (400 nmol / kg), demonstrating the importance of including an ICG-labeled scrambled peptide as a control agent (Figure 3). Statistical comparisons are summarized in Table 5.

[0180] [Table 5]

[0181] Tumors from SBK2-ICG-treated mice treated with 300 nmol / kg and 400 nmol / kg were significantly more fluorescent than tumors excised from mice treated with other drugs at the same doses or with 400 nmol / kg ICG (Figure 3). At 300 nmol / kg, no significant differences were detected between the other drugs. At 400 nmol / kg, tumors excised from mice treated with both SBK2-CLE-ICG and Scram-ICG were significantly more fluorescent than tumors excised from mice treated with Scram-CLE-ICG.

[0182] ICG dye is eliminated from the body primarily via the liver. As shown in Figure 8, some fluorescence was detected in the kidneys and livers excised from mice treated with ICG-labeled peptides, with little residual fluorescence detected in the spleen. Significantly stronger fluorescence was detected in the kidneys, liver, and spleen of mice treated with SBK2-ICG compared to other mice, likely related to the overall slower clearance rate observed in these animals (Figure 8, Table 5).

[0183] In addition to ICG-labeled peptides, peptides conjugated to the more hydrophilic dyes IRDye® 800CW and Tide Fluor® 8WS were tested for their ability to specifically label LN-229 flank tumors in nude mice. Figure 5 shows the time course of in vivo tumor fluorescence (mean ± standard error) in mice administered 400 nmol / kg of SBK2-CCLE-IR800 or Scram-CCLE-IR800 (Figure 4A) and SBK2-TF8WS or Scram-TF8WS (Figure 4B). In contrast to the ICG-conjugated peptides, specific tumor labeling was not observed with either SBK2-CCLE-IR800 or SBK2-TF8WS. Highly nonspecific whole-body fluorescence with the IR800 agent was observed (Figure 4A). The inset panel in Figure 4B shows data obtained from mice administered the TF8WS-conjugated peptide, plotted on the same scale as in Figure 4A. All four agents were injected at 400 nmol / kg, and similar levels of peak in vivo tumor fluorescence were observed 10 minutes after injection. Although both fluorophores are characterized as hydrophilic and similar RTs were obtained by RP-HPLC, different clearance characteristics were evident between the two classes of agents. The IRDye® 800CW-labeled peptide disappeared rapidly, and 60 minutes was determined to be the appropriate imaging endpoint. On the other hand, the TF8WS-conjugated agent peaked rapidly but disappeared more slowly than the IR800 agent. As seen in the inset plots, in vivo tumor fluorescence for both SBK2-TF8WS and Scram-TF8WS was relatively stable for the first 60 minutes and then began to gradually disappear.

[0184] Representative mice receiving 400 nmol / kg Scram-CCLE-IR800 and SBK2-CCLE-IR800 are shown 60 min post-injection in Figure 5A (left), along with animals 24 h after receiving 400 nmol / kg Scram-TF8WS or SBK2-TF8WS (Figure 5A, right). In contrast to the IR800-conjugated peptide, both TF8WS-labeled peptides produced strong signals localized to the tumor, suggesting a strong EPR effect. Plots of in vivo tumor fluorescence (mean ± standard error) at 60 min (IR800-drug) and 24 h (TF8WS-drug) are shown in Figure 5B. No significant differences were detected between the IR800- or TF8WS-labeled drugs.

[0185] Representative excised tumors from mice treated with various agents, along with corresponding plots of mean radiant efficiency values ​​(mean ± standard error), are shown in Figure 6. No statistical differences were observed between the signals of ex vivo tumors obtained from mice administered either type of IR800-conjugated agent or between the signals of mice injected with Tide Fluor® 8WS-labeled agents.

[0186] In addition to tumors, kidneys, spleens, and livers were removed from animals administered IR800-labeled and TF8WS-conjugated drugs. Representative examples of these organs are shown in Figure 9A, and Figure 9B shows plots of the average radiant efficiency (mean ± standard error) for various organs. The highest fluorescent signal was observed in the 100% IR800-labeled and TF8WS-conjugated animals. Fluorescence was detected in the kidneys. Kidneys from animals treated with SBK2-CCLE-IR800 were significantly less fluorescent than those from animals treated with Scram-CCLE-IR800 (Figure 9B). No significant differences in liver fluorescence were observed, but spleen fluorescence in mice treated with Scram-CCLE-IR800, while low, was significantly higher than in mice administered SBK2-CCLE-IR800 (Figure 9B). For animals treated with SBK2-TF8WS and Scram-TF8WS, the highest fluorescent signal was detected in the kidneys, with lower values ​​measured in the spleen and liver. No significant differences between SBK2-TF8WS and Scram-TF8WS were observed in any of these three organs.

[0187] Ventral images show the biodistribution of the drug as it clears through the bladder. Ventral images were acquired at 1 hour for the entire set of animals and at 24 hours for animals treated with ICG- and TF8WS-labeled drugs ( FIG. 10 ). More diffuse fluorescence throughout the abdomen was observed for mice treated with ICG-conjugated peptides. In contrast, mice treated with the more hydrophilic IRDye® 800CW- and Tide Fluor® 8WS-conjugated peptides showed intense fluorescence localized to the bladder, suggesting a primary role for renal clearance of these drugs.

[0188] In recent years, fluorescence imaging-guided surgery (FIGS) using non-targeted agents has benefited many patients, prolonging their lives by enabling the removal of large portions of the tumor. However, tumor recurrence remains a problem, particularly at the margins of resection. Recent efforts have focused on creating molecularly targeted agents that enhance surgeons' ability to distinguish tumor boundaries from surrounding normal tissue. For small molecule contrast agents, identifying the ideal properties of the targeting moiety, linker, and fluorophore combination is an ongoing process, as it remains unpredictable how these components will work together to create a novel single agent that functions in vivo. Our study contributes to this active area of ​​research in two key areas. First, we describe two molecularly targeted NIR contrast agents, SBK2-ICG and SBK2-CLE-ICG, that exhibit specific in vivo tumor labeling compared with controls in a mouse model of human glioblastoma. The tumor labeling achieved by SBK2-ICG immediately after injection and for at least 24 hours is particularly attractive for FIGS. Second, by using three different fluorophores and linkers, these studies contribute important insights to the growing effort to understand how fluorophores and linkers contribute to the in vivo behavior of drugs.

[0189] ICG was first approved for clinical use in 1959 and continues to play an important role in many imaging applications. New insights into the utility of this fluorophore continue to emerge. Recent studies have revealed that, in addition to fluorescence in the NIR-I region, ICG fluorescence can also be detected in the NIR-II region (1000–1700 nm), suggesting opportunities for deeper imaging. Furthermore, surgeons have recently explored the administration of high doses of ICG (5 mg / kg) via FIGS at 24 hours in a technique called "second optical window ICG (SWIG)" to exploit the EPR effect. By comparison, the 300 nmol / kg (0.89 mg / kg) dose of SBK2-ICG used in these studies corresponds to a human equivalent dose of approximately 0.07 mg / kg (or 70 μg / kg). By combining an SBK2-targeting peptide with ICG, specific tumor labeling occurred rapidly and persisted for up to 24 hours, even at doses smaller than those used for SWIG.

[0190] These studies also tested agents utilizing the hydrophilic IRDye® 800CW and Tide Fluor® 8WS. IRDye® 800CW combined with different antibody-based targeting moieties has shown great promise, so the lack of any detectable specificity with SBK2-CCLE-IR800 was unexpected. Furthermore, clearance of both SBK2-CCLE-ICG and Scram-CCLE-ICG via the renal system occurred more rapidly than expected. Injection of IRDye® 800CW dye alone resulted in more overall fluorescence throughout the body than achieved with the IRDye® 800CW-labeled peptide at the same dose, and the resulting systemic fluorescence dissipated more slowly than with peptide-containing agents (data not shown). While unexpected, these results highlight the notion that the properties of the fluorophore have a significant impact on the biodistribution of small molecule contrast agents. It is not immediately clear why the IRDye® 800CW-labeled peptide showed an increased clearance rate compared to its own fluorophore, in contrast to the ICG-labeled peptide, which showed much slower clearance than ICG alone. The hydrophilic SBK2-TF8WS peptide also showed no specificity for tumors expressing the PTPμ biomarker, suggesting that hydrophilic dyes may not be the best option for use with these targeting peptides. Although nonspecific for PTPμ, administration of both SBK2-TF8WS and Scram-TF8WS resulted in high tumor-specific signals at 24 hours.

[0191] These data demonstrate specific in vivo labeling of human gliomas over a suitable time frame using a PTP μ-targeting peptide conjugated to the NIR fluorophore ICG. SBK2-ICG achieved significantly higher tumor labeling than the control drug within 10 minutes of administration, and the tumor-specific signal persisted for at least 24 hours. Similarly, SBK2-CLE-ICG, which contains an additional six-amino acid linker, demonstrated specific in vivo tumor labeling but disappeared more rapidly. In contrast, more hydrophilic NIR agents utilizing the IRDye® 800CW fluorophore and the Tide Fluor® 8WS fluorophore did not demonstrate specificity. Injection of the peptide conjugated to IRDye® 800CW resulted in rapid, diffuse fluorescence that was widely dissipated throughout the kidney by 60 minutes. Both the Tide Fluor® 8WS-labeled SBK2 and Scram peptides generated very strong, nonspecific fluorescent tumor signals that persisted for at least 24 hours. The properties of SBK2-ICG make it an ideal agent for the treatment of glioblastoma, and we are currently working on clinical trials utilizing this agent.

[0192] Those skilled in the art will perceive improvements, variations, and modifications from the above description of the invention. Such improvements, variations, and modifications within the scope of the art are also intended to be encompassed by the appended claims. All references, publications, and patents cited in this application are hereby incorporated by reference in their entirety.

Claims

1. a targeting peptide that specifically binds to and / or complexes with a proteolytically cleaved extracellular fragment of an immunoglobulin (Ig) superfamily cell adhesion molecule expressed by the cancer cell or another cell in the cancer cell microenvironment; an optional spacer directly linked to the targeting peptide; and a near-infrared fluorophore linked directly or indirectly to a targeting peptide or optional spacer by a natural or non-natural linkage; A near-infrared contrast agent comprising:

2. 2. The near-infrared imaging agent of claim 1, wherein the agent administered to the subject has a signal-to-background ratio (SBR) during fluorescence imaging effective to distinguish cancer cells or other cells in the cancer cell microenvironment from surrounding tissue.

3. 3. The near-infrared contrast agent of claim 1, wherein the near-infrared fluorescent dye molecule is hydrophobic or lipophilic.

4. The near-infrared imaging agent of any of claims 1 to 3, wherein the non-native linkage is not susceptible to proteolytic cleavage.

5. 5. The near-infrared imaging agent of any of claims 1 to 4, wherein the non-native linkage comprises an amide linking the targeting peptide or optional spacer to the near-infrared fluorophore.

6. Chemical formula: 【Chemical 1】 or a pharmaceutically acceptable salt thereof; R １ is a near-infrared fluorophore; and R ２ comprises a targeting peptide and an optional spacer; The near-infrared contrast agent according to any one of claims 1 to 5.

7. The near-infrared contrast agent according to any one of claims 1 to 6, wherein the near-infrared fluorescent dye molecule comprises at least one kind of cyanine near-infrared fluorescent dye molecule having fluorescence in the first near-infrared region or the second near-infrared region.

8. 8. The near-infrared imaging agent of claim 7, wherein the cyanine near-infrared fluorophore is a heptamethine cyanine near-infrared fluorophore.

9. The near-infrared contrast agent according to any one of claims 1 to 8, wherein the near-infrared fluorescent dye molecule comprises at least one of indocyanine green (ICG) and ICG-Osu.

10. Chemical formula: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof; R ２ The near-infrared imaging agent of any of claims 1 to 9, wherein said compound comprises a targeting peptide and an optional spacer.

11. 11. The near-infrared imaging agent of any of claims 1 to 10, wherein the near-infrared fluorophore is directly linked to the targeting peptide via a non-natural linkage.

12. 11. The near-infrared imaging agent of any of claims 1 to 10, wherein the near-infrared fluorophore is directly linked to the spacer via a non-natural linkage.

13. 13. The near-infrared imaging agent of claim 12, wherein the spacer has a length and structure 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 near-infrared fluorescent dye molecule.

14. 14. The near-infrared imaging agent of claim 12 or 13, wherein the spacer comprises a natural and / or unnatural amino acid.

15. 15. The near-infrared imaging agent of any of claims 12-14, wherein the spacer comprises at least three natural or unnatural amino acids.

16. 16. The near-infrared imaging agent of any of claims 12-15, wherein the spacer has a length of 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 unnatural amino acids.

17. 17. The near-infrared imaging agent of any of claims 12-16, wherein 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.

18. 18. The near-infrared contrast agent of any one of claims 12 to 17, wherein the spacer is a polyglycine or glycine / serine spacer.

19. 19. The near-infrared imaging agent of any of claims 12-18, wherein the spacer comprises at least one amino acid sequence of (GS)a, (GGS)b, or (GGGS)c, or (GGGGS)d, and a, b, c, and d are each independently 2, 3, 4, 5, or 6.

20. 20. The near-infrared imaging agent of any of claims 1 to 19 for use in detecting, observing and / or imaging cancer cells and / or metastasis, migration, dissemination and / or invasion of cancer cells and / or for treating cancer in a subject.

21. 21. The near-infrared imaging agent of any of claims 1 to 20, configured for in vivo administration to a subject or ex vivo administration to a biological sample from a subject.

22. 1. A method for detecting cancer cells and / or metastasis, migration, dissemination, and / or invasion of cancer cells in a subject in need thereof; administering to a subject an amount of the near-infrared contrast agent of any one of claims 1 to 21; and detecting agents bound to and / or complexed with cancer cells to determine the location and / or distribution of cancer cells in the subject; A method comprising:

23. 23. The method of claim 22, wherein the cancer cells comprise at least one of glioma, lung cancer, melanoma, breast cancer, or prostate cancer cells.

24. 24. The method of claim 22 or 23, wherein the agent is administered to the subject systemically, locally, or topically.

25. The method of any of claims 22 to 24, wherein the agent is detected to define tumor boundaries in the subject.

26. Use of the near-infrared contrast agent of any one of claims 1 to 21 in fluorescence imaging-guided surgery.

27. 22. The near-infrared contrast agent of any one of claims 1 to 21 for use in the preparation of a therapeutic agent for fluorescence image-guided surgery.

28. 1. A method of treating cancer in a subject in need thereof, comprising: administering to a subject an amount of the near-infrared contrast agent of any one of claims 1 to 21; and irradiating the agent bound to and / or complexed with the cancer cells at a wavelength effective to ablate the cancer cells; A method comprising:

29. 30. The method of claim 28, wherein the cancer cells comprise at least one of glioma, lung cancer, melanoma, breast cancer, or prostate cancer cells.

30. 30. The method of claim 28 or 29, wherein the agent is administered to the subject systemically, locally, or topically.

31. Use of a near-infrared contrast agent according to any one of claims 1 to 21 in photodynamic therapy or photothermal therapy.

32. Chemical formula: 【Chemistry 3】 or a pharmaceutically acceptable salt thereof; R ２ a targeting peptide that specifically binds to 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 the cancer cell microenvironment, and an optional spacer directly linked to the targeting peptide, or a pharmaceutically acceptable salt thereof.

33. R ２ 33. The compound of claim 32, wherein comprises a targeting peptide.

34. R ２ 34. The compound of claim 32 or 33, wherein comprises a targeting peptide linked to a spacer, and the spacer separates the amide from the targeting peptide.

35. 35. The compound of claim 34, wherein the spacer has a length and structure effective to at least maintain or retain the binding affinity of the linked targeting peptide to the proteolytically cleaved extracellular fragment.

36. 36. The compound of claim 34 or 35, wherein the spacer comprises a natural and / or unnatural amino acid.

37. The compound of any of claims 34 to 36, wherein the spacer comprises at least three natural or unnatural amino acids.

38. 38. The compound of any of claims 34-37, wherein the spacer has a length of 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 unnatural amino acids.

39. 39. The compound of any of claims 34-38, wherein 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.

40. 40. The compound of any one of claims 34 to 39, wherein the spacer is a polyglycine or glycine / serine spacer.

41. 41. The compound of any one of claims 34 to 40, wherein the spacer comprises at least one amino acid sequence of (GS)a, (GGS)b, or (GGGS)c, or (GGGGS)d, and a, b, c, and d are each independently 2, 3, 4, 5, or 6.

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