Peptide PET / SPECT Probe Specific for Tumor Proteins in the Tumor Extracellular Matrix

Abstract

The PET / SPECT probe comprises the following formula (I), where P is an EDB-FN targeting peptide, C is a PET / SPECT imaging agent, and L is an optional linker that covalently attaches the peptide to the imaging agent.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 793,789, filed on January 17, 2019, the subject matter of which is incorporated herein by reference in its entirety.

[0002] Government Funding This invention was made with government support under grant numbers CA211762 and CA194518 awarded by the National Institutes of Health (NIH). The U.S. government has certain rights in this invention.

Background Art

[0003] Cancer detection and treatment are hampered by the inability to distinguish between cancer cells and normal cells. Excellent detection tools for imaging cancer or tumors are needed for early cancer diagnosis. Molecular recognition of tumor cells would facilitate induced surgical resection. To improve surgical resection, targeted imaging tools must specifically label tumor cells not only in the main tumor but also along the margins of the tumor and in small tumor cell clusters dispersed throughout the body. Targeted imaging tools designed to label molecules accumulating in the tumor microenvironment can also be effective as therapeutic targeting agents because they can identify both the main tumor cell population and regions with infiltrating cells involved in tumor recurrence. The ability to directly target tumor cells and / or their microenvironment would increase both the specificity and sensitivity of current treatments and thus reduce the non - specific side effects of chemotherapeutic agents that affect cells throughout the body.

[0004] Positron emission tomography (PET) imaging is mainly applied to the clinical examination of prostate cancer based on the increased glucose metabolism of prostate cancer compared to normal tissue. However, 18 F]-FDG. However, 18F]-FDG PET has not been demonstrated to be able to distinguish between benign and aggressive prostate cancer. A PSMA-specific PET probe has recently been developed for prostate cancer. Clinical trials have demonstrated the ability of the PSMA probe in the effective detection of PSMA-positive prostate tumors. However, recent studies have warned that the PSMA probe may not be able to distinguish benign tissue from prostate cancer. PET probes are required to detect aggressive cancers and stratify risks and meet the clinical requirements of non-invasive diagnostic modalities for the accurate clinical management of cancer.

Summary of the Invention

[0005] Embodiments described herein relate to peptide positron emission tomography (PET) / single photon emission computed tomography (SPECT) probes for tumorigenic proteins in tumors and / or cancer extracellular matrix, and can be used to detect the location and / or distribution of cancer in a subject's tissue, the aggressiveness of cancer in a subject, and / or the effectiveness of cancer treatment and / or cancer therapy administered to a subject in need of cancer treatment and / or cancer therapy.

[0006] In some embodiments, the PET / SPECT probe can include the following formula,

Chemical formula

[0007] In some embodiments, the linker is a non-peptide linker. The non-peptide linker can be a non-peptide aliphatic, heteroaliphatic, cyclic, and / or heterocyclic linker. The non-peptide linker can include, for example, an alkylene, alkylene oxide, arylene, or alkylene arylene linker that covalently attaches a peptide to a contrast agent.

[0008] The PET / SPECT contrast agent can include at least one of a metal chelating agent or a metal fullerene and a positron or gamma ray emitting radionuclide. The metal chelating agent can include, for example, at least one of diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazadodecane tetraacetic acid (DOTA), 1,4,7,10-tetraazadodecane-1,4,7-triacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclotridecane tetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazadodecane tetramethylacetic acid (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetic acid (DO3MA), N,N’,N’’,N’’’-tetraphosphonatomethyl-1,4,7,10-tetraazacyclododecane (DOTP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene methylphosphonic acid) (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phenylphosphonic acid) (DOTPP), N,N’-ethylenedi-L-cysteine, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane (TACN), N,N’-bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N’-diacetic acid (HBED-CC), and derivatives thereof. The positron or gamma ray emitting radionuclide can include, for example, 67 Ga, 68 Ga, 64 Cu, 99m Tc, 111 In, 89Zr, 90 Y, 153 Sm, or 89 Sr can be included.

[0009] In some embodiments, the PET / SPECT probe can have the following formula, [Chemical formula] In the formula, P1 is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and their retro-inverso type amino acid sequences, R 1 is optional, and when present, -(CH2) n -, -(OCH2CH2) n , or can include an alkylene, alkylene oxide, arylene, or alkylene arylene linker such as arylene, and n is an integer from 1 to 18, M is 67 Ga, 68 Ga, 64 Cu, 99m Tc, 111 In, 89 Zr, 90 Y, 153 Sm, or 89 Sr, a metal selected from the group consisting of, or a salt thereof.

[0010] In yet other embodiments, the PET / SPECT probe can be systemically administered to a subject to detect the distribution and / or location of cancer in the subject, as well as the aggressiveness of the cancer. The cancer can include, for example, at least one of breast cancer, liver cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, prostate cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, brain cancer, head and neck cancer, or skin cancer. Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] Methods including conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodological treatises such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Commonly understood definitions of molecular biology terms can be found, for example, in Rieger et al., Glossary of Genetics, Classical and Molecular, 5th Edition, Springer-Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994.

[0013] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0014] The terms "comprise", "comprising", "include", "including", "have", and "having" are used in an inclusive and open sense and mean that additional elements may be included. The terms "such as" and "for example" used herein are non-limiting and are for illustrative purposes only. "Comprising" and "including but not limited to" are used interchangeably.

[0015] The term "or" as used herein is to be understood to mean "and / or" unless the context clearly indicates otherwise.

[0016] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract consisting of biological materials.

[0017] The term "cancer" or "tumor" refers to any neoplastic growth in a subject, including primary tumors and any metastases. Cancer can be of a liquid or solid tumor type. Liquid tumors include tumors of hematological origin, such as myeloma (e.g., multiple myeloma), leukemia (e.g., Waldenström's macroglobulinemia, chronic lymphocytic leukemia, other leukemias), and lymphoma (e.g., B-cell lymphoma, non-Hodgkin lymphoma). Solid tumors can occur in organs and include cancers of the lung, brain, breast, prostate, ovary, colon, kidney, and liver.

[0018] The term "cancer cell" or "tumor cell" can refer to cells that divide at an abnormal (i.e., increased) rate. Cancer cells include carcinomas such as squamous cell carcinoma, non-small cell carcinoma (e.g., non-small cell lung cancer), small cell carcinoma (e.g., small cell lung cancer), basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchiogenic carcinoma, melanoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, cholangiocellular carcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, breast cancer, gastrointestinal cancer, colon cancer, bladder cancer, pancreatic cancer, prostate cancer, and squamous cell carcinoma of the head and neck region; sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synovial sarcoma, and mesenchymal sarcoma; hematological cancers such as myeloma, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia, granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), lymphoma (e.g., follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, malignant lymphoma, plasmacytoma, reticulosarcoma, or Hodgkin's disease), and tumors of the nervous system including glioma, glioblastoma multiforme, meningioma, medulloblastoma, schwannoma, and epidermoid tumor, but are not limited thereto.

[0019] As used herein, the term "isolated" with respect to nucleic acids such as DNA or RNA, or amino acids, refers to a molecule separated from other DNA, or RNA, polypeptides, or proteins, respectively, that exist in the natural source of the polymer. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Further, "isolated nucleic acid" or "isolated peptide" means a nucleic acid fragment or peptide fragment that does not exist as a fragment in nature and is not found in its natural state.

[0020] The term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term also includes, as equivalents, RNA or DNA analogs made from nucleotide analogs, and should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides as applicable to the described embodiments.

[0021] The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

[0022] The phrases "parenteral administration" and "administered parenterally" are terms recognized in the art and include modes of administration other than enteral and topical administration such as injection, and include, but are not limited to, intravenous, intramuscular, intrapleural, intravascular, intracardiac, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subdural, intraspinal, and intrasternal injections and infusions.

[0023] The terms "patient", "subject", "mammalian host", etc. are used interchangeably herein and refer to mammals including human and veterinary subjects.

[0024] The term "polypeptide" refers to polymers consisting of naturally occurring structural variants, and amino acid residues related to their synthetic non-naturally occurring analogs linked via peptide bonds or modified peptide bonds (i.e., peptide isosteres), related naturally occurring structural variants, and their synthetic non-naturally occurring analogs, glycosylated polypeptides, and all "mimetic" and "peptide mimetic" polypeptide forms. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term can refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment, portion, or subunit of any of these. The term "protein" usually refers to large polypeptides. The term "peptide" usually refers to short polypeptides.

[0025] A "portion" of a polypeptide or protein means at least about three contiguous amino acid residues of the polypeptide. It is understood that a portion of a polypeptide can include all of the amino acid residues of the polypeptide.

[0026] "Mutants", "derivatives", and "variants" of a polypeptide (or the DNA encoding it) are polypeptides that can be modified or altered by one or more amino acids (or one or more nucleotides) such that the peptide (or nucleic acid) is not identical to the wild-type sequence but has homology to the wild-type polypeptide (or nucleic acid).

[0027] A "mutation" of a polypeptide (or the DNA encoding it) is a modification or alteration of one or more amino acids (or one or more nucleotides) such that the peptide (or nucleic acid) is not identical to the sequences listed herein but has homology to the wild-type polypeptide (or nucleic acid).

[0028] As used herein, "recombinant" means that a protein is derived from a prokaryotic or eukaryotic expression system.

[0029] As used herein, the terms "systemic administration", "administered systemically", "peripheral administration", and "administered peripherally" mean the administration of a compound, agent, or other substance that is not direct to a particular tissue, organ, or region (e.g., the brain) of the subject being treated, which enters the animal's system and is subject to metabolism and other similar processes, e.g., subcutaneous administration.

[0030] The term "wild-type" refers to a naturally occurring polynucleotide sequence that encodes a protein or a part thereof, or a protein sequence or a part thereof, respectively, and is normally present in vivo.

[0031] Throughout this specification, where a composition is described as having, including, or comprising a particular component, it is contemplated that the composition also consists essentially of, or consists of, the recited components. Similarly, where a method or process is described as having, including, or comprising a particular process step, the process also consists essentially of, or consists of, the recited process steps. Further, it should be understood that, insofar as the compositions and methods described herein are practicable, the order of the steps or the order for performing a particular operation is not critical. Further, two or more steps or operations can be carried out simultaneously.

[0032] The embodiments described herein can be used to detect, monitor, and / or image the distribution and / or location of cancer, and / or the metastasis, migration, and / or invasion of cancer cells in a subject, to detect and / or monitor the aggressiveness and / or malignancy of cancer cells in a subject, and / or to determine and / or monitor the effectiveness of a cancer treatment and / or cancer therapy administered to a subject in need thereof, with respect to a peptide positron emission tomography (PET) / single photon emission computed tomography (SPECT) probe for a tumorigenic protein in a tumor and / or extracellular matrix of cancer cells.

[0033] The PET / SPECT probes described herein include a targeting peptide having a peptide sequence that specifically binds to and / or forms a complex with a cancer fetal fibronectin (onfFN) isoform, extra domain-B fibronectin (EDB-FN), or extra domain-A fibronectin (EDA-FN). Cancer, particularly malignant cancer, has a unique tumor microenvironment that promotes the survival, proliferation, and metastasis of cancer cells. The presence of onfFN has been observed in various human cancer types, including prostate cancer, breast cancer, and pancreatic cancer. High expression of onfFN, EDB-FN, and / or EDA-FN has been correlated with cancer aggressiveness and inversely correlated with patient survival. It has been recognized that cancer cells in a subject's tissue can be detected, monitored, and / or imaged using a PET / SPECT probe that includes a targeting peptide that specifically binds to EDB-FN and / or EDB-FN, and similarly, the aggressiveness, malignancy, metastasis, migration, dissemination, and / or invasion of cancer cells can be determined.

[0034] The PET / SPECT probe containing the targeting peptide can be systemically administered to a subject, such as by intravenous or parenteral administration, easily targeting the extracellular matrix protein EDB-FN and / or EDA-FN, and can clarify the location, distribution, and / or aggressiveness of cancer cells in the subject, as well as the margin of the tumor.

[0035] In some embodiments, the PET / SPECT probe can include the following formula, [Chemical formula] wherein P is a targeting peptide, C is a PET / SPECT contrast agent, and L is an optional linker that covalently attaches the peptide to the contrast agent.

[0036] In some embodiments, the targeting peptide can specifically bind to EDB-FN. Targeting peptides that specifically bind to EDB-FN include linear peptides having the amino acid sequences of TVRTSAD (SEQ ID NO: 1), NWGDRIL (SEQ ID NO: 2), NWGKPIK (SEQ ID NO: 3), SGVKSAF (SEQ ID NO: 4), GVKSYNE (SEQ ID NO: 5), IGKTNTL (SEQ ID NO: 6), IGNSNTL (SEQ ID NO: 7), IGNTIPV (SEQ ID NO: 8), and LYANSPF (SEQ ID NO: 9), cyclic peptides having the amino acid sequences of CTVRTSADC (SEQ ID NO: 10), CNWGDRILC (SEQ ID NO: 11), CNWGKPIKC (SEQ ID NO: 12), CSGVKSAFC (SEQ ID NO: 13), CGVKSYNEC (SEQ ID NO: 14), CIGKTNTLC (SEQ ID NO: 15), CIGNSNTLC (SEQ ID NO: 16), CIGNTIPVC (SEQ ID NO: 17), or CLYANSPFC (SEQ ID NO: 18) , shi It can include linear peptides with a stay linker or retro-inverso peptides having the retro-inverso type amino acid sequences of those linear peptides.

[0037] In other embodiments, the targeting peptide can specifically bind to EDA-FN. Targeting peptides that specifically bind to EDA-FN include linear peptides having the amino acid sequence of WNYPFRL (SEQ ID NO: 19), SNTSYVN (SEQ ID NO: 20), SFSYTSG (SEQ ID NO: 21), WSPAPMS (SEQ ID NO: 22), TREHPAQ (SEQ ID NO: 23), or ARIIDNA (SEQ ID NO: 24), and cyclic peptides having the amino acid sequence of CWNYPFRLC (SEQ ID NO: 25), CSNTSYVNC (SEQ ID NO: 26), CSFSYTSGC (SEQ ID NO: 27), CWSPAPMSC (SEQ ID NO: 28), CTREHPAQC (SEQ ID NO: 29), or CARIIDNAC (SEQ ID NO: 30). , shi It can include a linear peptide with a stain linker or a retro-inverso peptide having the retro-inverso type amino acid sequence of these linear peptides.

[0038] The targeting peptide can be subject to various modifications, substitutions, insertions, and deletions, and such modifications provide specific advantages in its use. In this regard, the targeting peptide that binds to and / or forms a complex with EDB-FN and / or EDA-FN can be substantially homologous rather than identical to the sequence of the listed peptides, with one or more modifications made that maintain its ability to specifically bind to and / or form a complex with EDB-FN and / or EDA-FN.

[0039] The targeting peptide can be any of various forms of polypeptide derivatives, including amides, conjugates with proteins, cyclic polypeptides, polymerized polypeptides, retro-inverso peptides, analogs, fragments, chemically modified polypeptides, and similar derivatives.

[0040] Retro-inverso peptides are linear peptides in which the amino acid sequence is reversed and the chirality at the α-centre of the amino acid subunits is also reversed. These types of peptides maintain a side-chain topology similar to that of the original L-amino acid peptides and are designed by including D-amino acids in reverse sequence so as to render them more resistant to proteolysis. D-amino acids exhibit the conformational mirror image of the natural L-amino acids present in native proteins 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 the effects of proteolysis and have a longer duration of effectiveness when used. Furthermore, the insertion of D-amino acids in selected sequence regions as either blocks of only D-amino acids or containing L-amino acids in the middle enables the design of targeted peptides that are not only resistant to proteolysis but also bioactive and have increased bioavailability. Furthermore, when appropriately designed, retro-inverso peptides can have binding properties similar to those of L-peptides.

[0041] The term "analogue" includes any peptide having an amino acid residue sequence substantially identical to the sequences specifically shown herein, in which one or more residues have been conservatively substituted with functionally similar residues and which specifically binds to and / or forms a complex with the EDB-FN and / or EDA-FN described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue, such as isoleucine, valine, leucine, or methionine, for another, the substitution of one polar (hydrophilic) residue, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, etc., for another, the substitution of one basic residue, such as lysine, arginine, or histidine, for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid, for another.

[0042] The phrase "conservative substitution" also includes the use of chemically derivatized residues in place of non-derivatized residues, provided that such peptides exhibit the requisite binding activity.

[0043] "Chemically modified derivative" refers to a target peptide having one or more residues that are chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, molecules in which a free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group or formyl group. A free carboxyl group can be derivatized to form a salt, methyl and ethyl esters, or other types of esters or hydrazides. A free hydroxyl group can be derivatized to form an O-acyl or O-alkyl derivative. The imidazole nitrogen of histidine can be derivatized to form N-benzylhistidine. Polypeptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids are also included in chemically modified derivatives. For example, 4-hydroxyproline can be used in place of proline, 5-hydroxylysine can be used in place of lysine, 3-methylhistidine can be used in place of histidine, homoserine can be used in place of serine, and ornithine can be used in place of lysine. The peptides described herein also include any peptide having one or more additions and / or deletions or residues relative to the sequence of the peptides whose sequences are shown herein, as long as the necessary binding specificity or activity is maintained.

[0044] The term "fragment" refers to any target peptide having an amino acid residue sequence that is shorter than that of the polypeptide whose amino acid residue sequence is shown herein.

[0045] Any polypeptide or compound can also be used in the form of a pharmaceutically acceptable salt. Acids capable of forming salts with polypeptides include inorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric 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, sulfanilic acid, etc.

[0046] Bases capable of forming salts with the polypeptide include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, mono-, di-, and tri-alkyl and aryl-amines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, etc.) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, etc.) and other organic bases.

[0047] The target peptide can be synthesized by any of the techniques known to those skilled in the art of polypeptides, including recombinant DNA technology. Synthetic chemical techniques such as solid-phase Merrifield-type synthesis can be used for reasons such as purity, antigen specificity, liberation from unwanted by-products, and ease of manufacture. A summary of many of the available techniques can be found for solid-phase peptide synthesis in Steward et al., "Solid Phase Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969, Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976, J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York), 1983, Merrifield, Adv. Enzymol., 32:221-96, 1969, Fields et al., int. J. Peptide Protein Res., 35:161-214, 1990, and U.S. Patent No. 4,244,946, and for conventional solution synthesis in Schroder et al., "The Peptides", Vol. 1, Academic Press (New York), 1965, each of which is incorporated herein by reference. Suitable protecting groups that can be used in such syntheses are described in the above text and in J.F.W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973, which is incorporated herein by reference.

[0048] Generally, the contemplated solid-phase synthesis method involves the sequential addition of one or more amino acid residues or appropriately protected amino acid residues to the growing peptide chain. Usually, either the amino group or the 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.

[0049] As an example, using solid-phase synthesis, a protected or derivatized amino acid can be attached to an inert solid support via its unprotected carboxyl or amino group. Next, the protecting group of the amino or carboxyl group is selectively removed, and the next amino acid in the sequence with an appropriately protected complementary (amino or carboxyl) group is mixed and reacted under conditions suitable for forming an amide bond with the residue already attached to the solid support. Next, the protecting group of the amino or carboxyl group can be removed from this newly added amino acid residue, and the next amino acid (appropriately protected) is added, and so on. After all the desired amino acids are linked in the appropriate sequence, the remaining terminal and side-chain protecting groups (and the solid support) can be removed sequentially or simultaneously to obtain the final linear polypeptide.

[0050] Furthermore, the targeted peptides described herein can be used as a starting point for developing higher affinity small molecules, peptides, antibodies, and / or antibody fragments that have similar ligand-binding capabilities. For example, the development and screening of small molecules from the pharmacophore of a peptide using computer-aided screening can be readily carried out, and the binding affinity of the molecules so identified can be readily screened against the targeted peptide using the assays described herein for selecting small molecule drugs.

[0051] Additional residues can also be added to either end of the peptide for the purpose of providing a "linker" that can conveniently link and / or immobilize the peptide to other polypeptides, proteins, detectable moieties, labels, solid matrices, or carriers.

[0052] The amino acid residue linker is usually at least 1 residue, can be 40 residues or more, and more often 1 to 10 residues. Typical amino acid residues used for ligation are glycine, tyrosine, cysteine, lysine, glutamic acid, aspartic acid, and the like. Further, the targeting peptide agent can differ in the sequence to be modified by terminal NH2 acylation, for example, acetylation, or by terminal carboxyl amidation, for example, terminal modification with thioglycolic acid amide, ammonia, methylamine, and the like. As is well known, terminal modification is useful for reducing susceptibility to protease digestion and thus serves to extend the half-life of the polypeptide in solution, especially in biological fluids where proteases may be present. In this regard, polypeptide cyclization is also a useful terminal modification and is particularly preferred from the perspective of the stable structure formed by cyclization and the biological activities observed for cyclic peptides as described herein.

[0053] When the linker is a peptide linker, the polypeptide linker can be produced as a single recombinant polypeptide using conventional molecular biological / recombinant DNA methods.

[0054] For example, the targeting peptide can contain lysine that can react with a carbonyl-containing group such as an anhydride or acid halide, or an alkyl group containing a good leaving group (e.g., halide). The targeting peptide can also contain cysteine that promotes chemical coupling via thiol-selective chemistry (e.g., maleimide-activated compounds). Further, the targeting peptide can contain tyrosine that can be modified using a diazonium coupling reaction. In an exemplary embodiment, the amino acid residue linker is a cysteine-glycine (CG) linker.

[0055] In other embodiments, chemical linker groups can be used. The linker group can serve to increase the chemical reactivity of the substituent, and thus the coupling efficiency, either on the targeting peptide or on a compound or molecule to which the targeting peptide is attached. Linker chemistries can include maleimidyl linkers that can be used to attach to thiol groups, isothiocyanates and succinimidyl (e.g., N-hydroxysuccinimidyl (NHS)) linkers that can attach to free amine groups, diazonium that can be used to attach to phenols, and amines that can be used to attach to free acids such as carboxylic acid groups using carbodiimide activation.

[0056] Useful functional groups are present on the targeting peptide based on the particular amino acids present, and additional groups can be designed. It will be apparent to those skilled in the art that a variety of both homo- and hetero-functional bifunctional or polyfunctional reagents (such as those described in the catalog of Pierce Chemical Co., Rockford, Ill.) can be used as linker groups. Coupling can be accomplished, for example, via amino, carboxyl, sulfhydryl, or oxidized carbohydrate residues.

[0057] Other types of conjugation chemistries can also be utilized. For example, methods for conjugating polysaccharides to peptides include using squaric acid diester (1,2-diethoxycyclobutene-3,4-dione) as a coupling reagent (Tietze et al. Bioconjug Chem. 2:148-153 (1991)), coupling to NaIO4-activated oligosaccharides via α or ε-amino groups (Bocher et al., J. Immunol. Methods 27, 191-202 (1997)), coupling via a peptide linker where the polysaccharide has a reducing end and does not contain a carboxyl group (U.S. Patent No. 5,342,770), and coupling by a synthetic peptide carrier derived from human heat shock protein hsp65 (U.S. Patent No. 5,736,146), but are not limited thereto. Further methods for conjugating polysaccharides, proteins, and lipids to peptides are described in U.S. Patent No. 7,666,624.

[0058] In some embodiments, the linker is a non-peptide linker. The non-peptide linker can be a non-peptide aliphatic, heteroaliphatic, cyclic, and / or heterocyclic linker. The non-peptide linker can include, for example, an alkylene, alkylene oxide, arylene, or alkylene arylene linker that covalently attaches a peptide to a contrast agent.

[0059] In other embodiments, the linker can be a PEG molecule linker. The PEG molecules can have various lengths and molecular weights and can include, for example, PEG200, PEG1000, PEG1500, PEG4600, PEG10,000, or combinations thereof.

[0060] The PET / SPECT contrast agent can be directly conjugated to the targeting peptide or linked to the targeting peptide via a linker. The role of the contrast agent is to facilitate the detection step of the detection or diagnostic method by enabling visualization of the complex formed by the binding of the PET / SPECT probe containing the targeting peptide to EDB-FN and / or EDA-FN. The contrast agent can be selected to produce a signal that can be measured and is related to (preferably proportional to) the amount of the PET / SPECT probe bound to the tissue whose intensity is analyzed.

[0061] In certain embodiments, the contrast agent comprises a chelating agent and a metal ion. The chelating agent generally has one or more groups capable of forming a covalent bond with a linker. Many different chelating agents known in the art can be used herein. In one aspect, the chelating agent comprises an acyclic or cyclic compound containing at least one heteroatom (e.g., oxygen, nitrogen, sulfur, phosphorus) having a lone pair of electrons capable of coordinating with an imaging agent. Metal chelating agents include, for example, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazadodecane tetraacetic acid (DOTA), 1,4,7,10-tetraazadodecane-1,4,7-triacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclotridecane tetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazadodecane tetramethylacetic acid (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetic acid (DO3MA), N,N’,N’’,N’’’-tetraphosphonatomethyl-1,4,7,10-tetraazacyclododecane (DOTP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenemethylphosphonic acid) (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephenylphosphonic acid) (DOTPP), N,N’-ethylenedi-L-cysteine, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane (TACN), N,N’-bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N’-diacetic acid (HBED-CC), and at least one of their derivatives. The term "derivative" is defined herein as the corresponding salts and esters of the chelating agent.

[0062] The choice of metal ion can vary depending on the detection technique (e.g., PET or SPECT). Metal ions useful for PET and SPECT imaging include 67 Ga, 68Ga, 64 Cu, 99m Tc, 111 In, 89 Zr, 90 Y, 153 Sm, or 89 Sr is included.

[0063] In some embodiments, the PET / SPECT probe can have the following formula,

Chemical formula

[0064] In other embodiments, the PET / SPECT probe has the following formula,

Chemical formula

[0065] In yet other embodiments, the PET / SPECT probe has the following formula, [Chemical formula] and 67 Ga, 68 Ga, 64 Cu, 99m Tc, 111 In, 89 Zr, 90 Y, 153 Sm, or 89 Sr, or can have a PET / SPECT radionuclide that is a salt thereof, selected from the group consisting of.

[0066] The PET / SPECT probes described herein can be administered to a subject by, for example, systemic, local, and / or parenteral administration methods. These methods include, for example, injection, infusion, deposition, implantation, or topical administration, or any other administration method where access to tissue by the molecular probe is desired. In one example, administration of the molecular probe can be by intravenous injection of the molecular probe in the subject. Single or multiple administrations of the probe can be performed. As used herein, "administered" means the provision or delivery of the molecular probe in an amount and for a period effective to label cancer cells in the subject.

[0067] The PET / SPECT probe comprising the targeting peptide described herein can be administered to a subject with a detectable amount of a pharmaceutical composition comprising the molecular probe or a pharmaceutically acceptable water-soluble salt thereof.

[0068] "Detectable amount" means that the amount of the molecular probe administered is sufficient to enable detection of the binding or complex formation of the probe to EDB-FN and / or EDA-FN expressed by cancer cells or other cells in the cancer cell microenvironment. "Imaging effective amount" means that the amount of the PET / SPECT probe administered is sufficient to enable imaging of the binding or complex formation of the molecular probe to EDB-FN and / or EDA-FN of cancer cells or other cells in the cancer cell microenvironment.

[0069] The formulation of the PET / SPECT probe administered will vary depending on the selected route of administration (e.g., solution, emulsion, capsule, etc.). Suitable pharmaceutically acceptable carriers may contain inert components that do not overly inhibit the biological activity of the compound. Pharmaceutically acceptable carriers should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic, and lacking other undesirable reactions upon administration to a subject. Standard formulation techniques can be used, such as those described in Remington’s Pharmaceutical Sciences, supra. 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, and the like.

[0070] The preparation of a pharmacological composition containing a dissolved or dispersed active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, although solid forms suitable for solution or suspension can also be prepared into a liquid prior to use. The formulation varies according to the selected route of administration (e.g., solution, emulsion, capsule).

[0071] Any polypeptide or compound can also be used in the form of a pharmaceutically acceptable salt. Acids capable of forming salts with polypeptides include inorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric 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, sulfanilic acid, etc.

[0072] Bases capable of forming salts with polypeptides include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc., mono-, di-, and tri-alkyl and aryl-amines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, etc.) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, etc.) and other organic bases.

[0073] The PET / SPECT probes described herein can be used in a method for detecting and / or determining the presence, location, and / or distribution of cancer cells expressing EDB-FN and / or EDA-FN in a target organ, tissue, or body region. The presence, location, and / or distribution of the probe in animal tissues, such as prostate tissue, can be visualized (e.g., using the in vivo imaging modalities described above). As used herein, "distribution" is a spatial property that is scattered over an area or volume. In this case, the "distribution of cancer cells" is the spatial property of cancer cells scattered over an area or volume contained in animal tissues, such as prostate tissue. And the distribution of the molecular probe can be correlated with the presence or absence of cancer cells in the tissue. The distribution can be a clue for the presence or absence of cancer cells, or can be combined with other factors and symptoms by those skilled in the art to clearly detect the presence or absence of migrating or dispersed cancer cells, cancer metastasis, or to clarify the tumor margin in the subject.

[0074] In one aspect, a PET / SPECT probe is administered to a subject to evaluate the distribution of malignant or metastatic cancer cells in the subject and correlate the distribution to specific locations. Surgeons routinely use localization techniques and intraoperative MRI (iMRI) in surgical resections. This enables them to specifically identify and sample tissue from different regions of the tumor, such as the tumor margin or the tumor center. Often, they also sample tissue regions at the tumor margin outside the tumor margin that is infiltrated by tumor cells that are dispersed upon histological examination but appear normal to the naked eye.

[0075] A PET / SPECT probe that specifically binds to and / or forms a complex with EDB-FN and / or EDA-FN associated with malignant or metastatic cells is used in intraoperative imaging techniques to guide surgical resection and eliminate the "knowledge and experience-based speculation" of the location of the tumor margin by the surgeon. Past studies have determined that more extensive surgical resection improves patient survival. Therefore, a probe that functions as a diagnostic molecular imaging agent has the potential to increase the survival rate of patients.

[0076] In some embodiments, intraoperative imaging (IOI) techniques can be combined with the systemically or locally administered PET / SPECT probes described herein to identify and facilitate the removal of cancer cells. The PET / SPECT probe upon administration to the subject can target, detect, and / or determine the presence, location, and / or distribution of cancer cells, i.e., cancer cells associated with EDB-FN and / or EDA-FN expression, in the organs or body regions of the patient. In one example, the probe can be combined with IOI to identify malignant cells that have infiltrated and / or are beginning to infiltrate the tumor margin. The method can be performed in real time during surgery. The method can include the topical or systemic application of a PET / SPECT probe that includes a detectable moiety such as a PET or SPECT contrast agent. Next, an imaging modality can be used to detect and subsequently collect the image data. The obtained image data can be used to at least partially determine surgical and / or radiological treatment. Alternatively, this image data can be used to at least partially control an automated surgical device (e.g., a laser, scalpel, micromachine) or assist in the manual guidance of the surgery. Further, the image data can be used to plan and / or control the delivery of a therapeutic agent (e.g., by microelectronics or micromachines).

[0077] Another embodiment described herein relates to a method for determining the aggressiveness or malignancy of cancer cells in a subject. The binding strength of a PET / SPECT probe to cancer has been found to correlate with the aggressiveness of the cancer. Enhanced binding correlates with more aggressive cancers, while decreased or reduced binding correlates with less aggressive or benign tumors. In one example, the binding of a probe to a prostate tumor section correlated with the Gleason score based on the aggressiveness of the tumor, and the enhanced binding strength of the molecular probe was distinguished from benign prostatic hyperplasia that showed lower binding of the probe, correlating with aggressive or malignant prostate cancer. Using the methods and molecular probes described herein, the aggressiveness of cancer in a subject before, during, or after administration of cancer treatment or cancer therapy can be monitored and / or compared.

[0078] Another embodiment described herein relates to a method for monitoring the effectiveness of cancer treatment or cancer therapy administered to a subject. Using the methods and PET / SPECT probes described herein, the aggressiveness, invasion, migration, dissemination, and metastasis of cancer in a subject before, during, or after administration of cancer treatment or cancer therapy can be monitored and / or compared.

[0079] As used herein, "cancer treatment" or "cancer therapy" can include any agent or treatment regimen that can have a negative impact on cancer in an animal, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to the tumor or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of an animal having cancer. Cancer treatment can include one or more therapies such as, but not limited to, chemotherapy, radiation therapy, hormonal therapy, and / or biological therapy / immunotherapy. For example, a decrease in the volume, growth, movement, and / or spread of cancer in a subject can indicate the effectiveness of a given therapy. This can provide a direct clinical efficacy endpoint measurement of cancer treatment. Thus, in another aspect, a method of monitoring the effectiveness of cancer treatment is provided. More specifically, embodiments of the present application provide a method of monitoring the effectiveness of cancer therapy.

[0080] A method of monitoring the effectiveness of cancer treatment can include the steps of administering a PET / SPECT probe in vivo to an animal as described herein, then visualizing the distribution of the probe in the animal (e.g., using an in vivo imaging modality as described herein), and then correlating the distribution of the probe with the effectiveness of cancer treatment. The administration step is intended to be able to determine the effectiveness of a selected treatment regimen by occurring before, during, and after the course of the treatment regimen. One way to evaluate the effectiveness of cancer treatment is to compare the distribution of the probe before and after cancer therapy.

[0081] In some embodiments, a PET / SPECT probe that is bound and / or complexed to EDB-FN and / or EDA-FN is detected in a subject and detects and / or provides information about the invasiveness, location, and / or distribution of cancer cells in the subject. Next, the invasiveness, location, and / or distribution of cancer cells in the subject can be compared to a control to determine the effectiveness of cancer treatment and / or cancer therapy. The control can be the location and / or distribution of cancer cells in the subject prior to administration of cancer treatment and / or cancer therapy. The location and / or distribution of cancer cells in the subject prior to administration of cancer treatment and / or cancer therapy can be determined by administering the probe to the subject and detecting the probe that has bound and / or complexed to cancer cells in the subject prior to administration of cancer treatment and / or cancer therapy.

[0082] In certain embodiments, the methods and PET / SPECT probes described herein can be used to measure the effectiveness of a therapeutic agent administered to a subject for treating metastatic or aggressive cancer. In this embodiment, the probe can be administered to the subject before, during, or after administration of a treatment regimen, and the distribution of cancer cells can be imaged to determine the effectiveness of the treatment regimen. In one example, the treatment regimen can include surgical resection of metastatic cancer, and the probe can be used to clarify the distribution of metastatic cancer before and after surgery to determine the effectiveness of the surgical resection. Optionally, the methods and probes can be used in intraoperative surgical procedures such as surgical tumor resection to more readily clarify and / or image the mass or volume of cancer cells during surgery.

[0083] In other embodiments, the targeting peptide can be conjugated to a therapeutic agent and administered to a subject for treating cancer such as metastatic cancer. In this embodiment, the targeting peptide conjugated to the therapeutic agent can be administered to the subject, and metastatic cells can be targeted with the therapeutic agent.

[0084] The therapeutic agent can include an anti-proliferative agent that exerts anti-tumor, chemotherapy, anti-viral, anti-mitotic, anti-tumorigenic, and / or immunotherapeutic effects. For example, through a cell growth inhibitory or cell destructive effect, it can directly act on tumor cells, for example, preventing the generation, maturation, or spread of neoplastic cells, without indirectly mediating mechanisms such as alteration of biological responses. There are numerous anti-proliferative agents available for commercial use, clinical evaluation, and pre-clinical development. For the sake of discussion, anti-proliferative agents are classified into the following classes, subtypes, and species. ACE inhibitors, alkylating agents, angiogenesis inhibitors, angiostatin, anthracycline / DNA intercalators, anti-cancer antibiotics or antibiotic-type drugs, antimetabolites, anti-metastatic compounds, asparaginase, bisphosphonates, cGMP phosphodiesterase inhibitors, calcium carbonate, cyclooxygenase-2 inhibitors, DHA derivatives, DNA topoisomerases, endostatin, epipodophyllotoxins, genistein, hormonal anti-cancer agents, hydrophilic bile acids (URSO), immunomodulators or immunological drugs, integrin antagonists, interferon antagonists or drugs, MMP inhibitors, various anti-neoplastic agents, monoclonal antibodies, nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors, pBATT, radiation / chemosensitizers / protectors, retinoids, selective inhibitors of endothelial cell growth and migration, selenium, stromelysin inhibitors, taxanes, vaccines, and vinca alkaloids.

[0085] The main categories into which some anti-proliferative agents are classified include the categories of antimetabolites, alkylating agents, antibiotic-type drugs, hormonal anti-cancer agents, immunological drugs, interferon-type drugs, and various anti-neoplastic agents. Some anti-proliferative agents function via multiple or unknown mechanisms and can therefore be classified into two or more categories.

[0086] In some embodiments, the targeting peptide can bind to the therapeutic agent using a linking molecule. The linking molecule can be a linker. Alternatively, the linking molecule can be a non-peptide linker.

Examples

[0087] Example 1 The inventors developed a Cu-DOTA conjugate as a PET probe for EDB-FN and evaluated its effectiveness for PET imaging in mice bearing aggressive PC3 and slow-growing LNCaP human prostate tumor xenografts. The inventors showed that EDB-FN was highly expressed in aggressive PC3 tumors and expressed at negligible levels in slow-growing, non-metastatic LNCaP tumors. MRI using the EDB-FN-targeted contrast agent ZD2-Gd(HP-DO3A) showed stronger contrast enhancement in PC3 tumors than in LNCaP tumors. 64 The use of Cu is particularly attractive because of its half-life of 12.74 hours, providing an extended imaging time frame for cancer detection in the prostate with minimal background subtraction from the bladder. The PET probe was synthesized by conjugating the ZD2 peptide to the macrocyclic ligand DOTA, followed by 64 complexation with CuCl2. The ability of the PET probe in cancer detection and tumor aggressiveness characterization was evaluated in mice bearing PC3 and LNCaP tumors. 64 Materials and Methods

[0088] Synthesis of ZD2-PEG-DOTA and Chelates Reagents used for chemical synthesis were purchased from Sigma-Aldrich (Saint Louis, MO, USA) unless otherwise specified. Fmoc-protected amino acids and 2-chlorotrityl chloride resin were obtained from Chem-Impex International, Inc. (Wood Dale, IL). The spacer Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-NH-(CH2CH2O)2-CH2COOH) was obtained from Chempep (Wellington, FL). 1,4,7,10-Tetraazacyclododecane-1,4,7-tris-tert-butyl acetate-10-acetic acid (DOTA-tris(t-Bu)) was purchased from TCI America (Portland, OR). ​

[0089] The precursor ZD2-DA-DOTA containing the ZD2 peptide (sequence: TVRTSAD), two repeats of NH2-(CH2CH2O)2-CH2COOH, and DOTA was synthesized by sequentially adding the corresponding protected amino acid, Fmoc-NH-(CH2CH2O)2-CH2COOH, and t-Bu-DOTA on a solid-phase resin using standard Fmoc-peptide chemistry. The product was then cleaved from the resin using trifluoroacetic acid / triisopropylsilane / H2O (96.5:1:2.5), stirred at room temperature for 3 hours, and precipitated in ether to obtain the crude product. The final product was purified using preparative HPLC on an Agilent 1100 HPLC system equipped with a semi-preparative C18 column (Agilent Technologies, Santa Clara, CA). ZD2-PEG-DOTA was characterized by MALDI-TOF mass spectrometry on a Voyager DE-STR spectrometer (PerkinElmer, Waltham, MA) in linear mode using R2,5-dihydroxybenzoic acid as the matrix (M+1: 1425.8, measured; 1425.7, calculated).

[0090] Cell culture and animal models Animal experiments were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University (CWRU), and all subjects signed an informed consent form. PC3 and LNCaP cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Roswell Park Memorial Institute medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin in a humidified incubator maintained at 37 °C and 5% CO2. Male athymic nude mice (4 - 6 weeks old) were obtained from the Case Comprehensive Cancer Center (Cleveland, OH, USA) and housed at the CWRU Animal Core Facility. Three million cells in high-concentration Matrigel (Corning, Tewksbury, MA) were used for tumor inoculation. LNCaP cells were subcutaneously inoculated into the left flank of the mice. Four weeks later, PC3 cells were inoculated into the right flank of the same mice for PET imaging.

[0091] Radioactive label Radioisotope 64 Cu(II) was obtained from the University of Wisconsin - Madison (Madison, WI). Chelation of ZD2 - PEG - DOTA with Cu(II) was first tested using non - radioactive CuCl2 in 0.1 N HCl aqueous solution under the same conditions as for radioactive labeling. Equimolar amounts of ZD2 - DA - DOTA and CuCl2 solutions in PBS buffer (pH 7.4) were mixed and stirred at 45 °C for 30 minutes. The formation of ZD2 - DA-(Cu - DOTA) was verified by MALDI - TOF mass spectrometry (M + 1: 1487.8, measured; 1486.04, calculated). For radioactive labeling, 10 mCi of 64 Cu(II) was dissolved in 200 μL of 0.1 N HCl. Twenty microliters of64 A Cu(II) solution (about 1 mCi) was mixed with 480 μL of ZD2-DA-DOTA (0.05 mg / mL, in large excess, PBS) in a 1.5 mL microcentrifuge tube. Next, the vessel was maintained at 45 °C with intermittent shaking for 30 minutes. The final pH of the solution was adjusted to neutral using NaOH solution prior to injection.

[0092] PET imaging All in vivo imaging studies were performed according to protocols and guidelines approved by the CWRU Animal Research Committee. Mice were anesthetized with 2% isoflurane in oxygen and injected with approximately 200 μCi [about 7.4 MBq] of ZD2-DA- 64 Cu(DOTA) via the tail vein. Mice were subjected to a 10-minute static PET scan after PET scans (Inveon microPET, Siemens Medical Solutions USA Inc.) at uptake periods of 4 hours and 22 hours. Images were reconstructed using 3D-OSEM with a 3D histogram and a zoom factor of 1.0 (2 iterations followed by 18 iterations of MAP). A CT scan (Siemens Medical Solutions USA Inc.) was performed after the PET procedure for anatomical co-registration. PET / CT images g were analyzed using AMIDE version 1.0.557 and AMIRA software. ROIs were drawn for PC3 and LNCaP tumors and the ratio of specific binding to non-specific (muscle) binding was calculated.

[0093] Biodistribution After the final microPET / CT imaging at 22 hours post-injection, three mice were euthanized and organs and blood were collected, weighed, and radioactivity was determined with a gamma counter. The percentage of the injected dose per gram of tissue was calculated using a standard containing 2% of the injected dose.

[0094] Histological analysis After image acquisition, the mice were euthanized. Tumors were harvested, embedded in optimal cutting temperature medium, frozen at -80 °C, cryosectioned at 5 μm, and permeabilized with cold acetone. Tissues were blocked with bovine serum albumin (1%) in PBS for 1 h at room temperature. Anti-EDB-FN BC1 antibody (Abcam, Cambridge, MA) was incubated with tissue sections of PC3 and LNCaP tumors. After extensive washing, secondary anti-mouse Alexa Fluor488 antibody was incubated for 1 h. Tissue sections were counterstained with Prolong Gold antifade mounting medium with 4’,6-diamidino-2-phenyl-indole (Thermo Fisher, Waltham, MA). Stained tissues were imaged with an Olympus FV1000 confocal laser scanning microscope.

[0095] Results ZD2 64 The Cu-DOTA conjugate was synthesized by conjugating the ZD2 peptide to the macrocyclic chelator DOTA using solid-phase peptide chemistry, followed by 64 complex formation with CuCl2 (Figure 1). A short spacer with two repeats of 8-amino-3,6-dioxaoctanoic acid was introduced between the peptide and the chelator. The targeted ligand ZD2-DA-DOTA was purified by preparative high-performance liquid chromatography (HPLC) and characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry [m / z = 1425.8 (M+1), measured; 1425.5, calculated]. Preparation of the targeted PET probe was demonstrated by equimolar complex formation of ZD2-DA-DOTA in phosphate-buffered saline (PBS) buffer (pH 7.4) and non-radioactive CuCl2 in dilute HCl (0.1 N) at 45 °C for 30 min, and the same conditions were used for radiolabeling. Formation of ZD2-DA-(Cu-DOTA) was verified by MALDI-TOF mass spectrometry [m / z = 1487.8 (M+1), measured; 1486.04, calculated].

[0096] Next, ZD2-DA-( 64The efficacy of (Cu-DOTA) was investigated in male nude mice bearing both PC3 and LNCaP human prostate cancer xenografts. The inventors have previously shown that EDB-FN is highly expressed in aggressive PC3 tumors and expressed at negligible levels in slow-growing, non-metastatic LNCaP tumors. Using tumor models representative of high- and low-risk prostate tumors, the ability of the probe to detect and stratify aggressive prostate cancer was tested. The radiolabeling was performed by mixing 20 μL of 64 a Cu(II) solution (0.1 N HCl, approximately 1 mCi or 37 MBq) with 480 μL of ZD2-DA-DOTA (0.05 mg / mL, large excess, PBS, pH = 7.4) in a 1.5 mL microcentrifuge tube and maintaining at 45 °C for 30 min with intermittent shaking. The reaction mixture was then diluted 1:2 with PBS and tested with pH test paper to ensure a neutral pH for intravenous injection. The radiotracer was injected intravenously at a dose of 7.4 MBq (200 μCi) per mouse. PET images of the mice were acquired at 4 h and 22 h post-injection in groups of 4 mice bearing both PC3 and LNCaP tumor xenografts.

[0097] Figure 2 shows representative three-dimensional (3D) volume renderings and axial PET / computed tomography (CT) images of two tumor-bearing mice at 4 h and 22 h post-injection of ZD2-DA-( 64 Cu-DOTA). In aggressive PC3 tumors, a stronger signal was visually apparent compared to slow-growing LNCaP tumors. The location and size of the PC3 tumors were clearly delineated in the PET images. The tracer uptake or signal intensity was quantitatively analyzed in the region of interest (ROI) at 4 h and 22 h. As shown in Figure 3, ZD2-DA-( 64(Cu-DOTA) resulted in higher probe uptake in PC3 tumors than in LNCaP tumors. At 22 h, PET revealed more than two-fold higher accumulation of the PET tracer in the more aggressive PC3 tumors (7711 ± 1994 Bq / mL) compared to the less aggressive LNCaP tumors (3213 ± 1511 Bq / mL) (N = 4, P < 0.05, two-sided Student's t-test). Other organs that showed substantial radioactive tracer uptake were the liver, stomach, and kidneys, which showed elimination of the radioactive tracer via the liver and kidney pathways.

[0098] The biodistribution of the radioactive tracer was measured at 24 h post-injection after anesthetizing the mice (Figure 4). The biodistribution pattern was consistent with the results of the PET imaging and had strong uptake in the tumors, liver, and kidneys. Other organs such as the brain and muscle showed low uptake of the radioactive tracer, which is a desirable property of the radioactive tracer. Comparison of the uptake of the radioactive tracer in PC3 and LNCaP tumors showed that the accumulation of the radioactive tracer in PC3 (1. 64 ID% / g) was higher than that in LNCaP tumors (0.86 ID% / g) (N = 3, P = 0.32, two-sided Student's t-test), confirming that the probe preferentially accumulates in the more aggressive PC3 tumors than in the non-metastatic LNCaP tumors.

[0099] The expression of EDB-FN in prostate tumors was determined by immunofluorescent staining of tissue sections of PC3 and LNCaP tumors with anti-EDB-FN monoclonal antibody BC1 after PET imaging. The BC1 antibody and EDB-FN were stained using Alexa Fluor488-conjugated anti-mouse antibody. Figure 5 shows the fluorescence images of tumor sections obtained with an Olympus FV1000 confocal laser scanning microscope. Intense fluorescent staining was visible by eye in the PC3 tumor sections, while little staining was observed in the LNCaP tumors. Consistently, the inventors have already shown that the EDB mRNA level in LNCaP cells was lower than that in PC3 cells. The EDB-FN expression levels in two different prostate tumors were in good correlation with the observations by PET molecular imaging. The results suggest that ZD2-DA-( 64 Cu-DOTA) is effective for the sensitive and quantitative visualization of EDB-FN expression in prostate cancer.

[0100] In this example, the possibility of PET imaging of the ECM tumor protein EDB-FN with the peptide probe ZD2-DA-( 64 Cu-DOTA) for the detection and characterization of prostate cancer was shown. The inventors have already shown that EDB-FN is highly expressed in rapidly proliferating PC3 tumors and lowly expressed in slowly proliferating LNCaP tumors. The ZD2 peptide-targeted MRI contrast agent was able to produce a stronger signal in PC3 tumors than in LNCaP tumors. The results of PET molecular imaging of EDB-FN with ZD2-DA-( 64 Cu-DOTA) were consistent with MR molecular imaging using the ZD2 peptide-targeted MRI contrast agent, especially at 22 hours after injection. Comparing the probe uptake in tumors, a strong PET signal was detected in rapidly proliferating PC3 tumors with higher EDB-FN expression than in slowly proliferating LNCaP tumors. However, a significant signal intensity was still observed in the LNCaP tumors in the PET images. This may be due to 64 the relatively low chelation stability of the Cu-DOTA monoamide. The free 64Cu(II) has been shown to accumulate in prostate tumors in animal models. The relatively high signal intensity in LNCaP tumors may be due to the accumulation of free 64 Cu(II) released from the probe. Nevertheless, the targeting effect of the ZD2 peptide of the probe still resulted in a significantly higher signal intensity in PC3 tumors than in LNCaP tumors. Compared with MR molecular imaging, PET imaging results in a sensitive and quantitative visualization and measurement of the EDB-FN expression level in prostate cancer, providing a more accurate risk stratification of aggressive prostate cancer.

[0101] In general, PET imaging with probes of relatively short half-lives is plagued by significant signal extrapolation from the bladder when imaging primary tumors in the prostate due to the limited imaging window. Cu with a relatively long half-life 64 enables emptying of the bladder and sufficient time to minimize potential signal interference from the bladder, which is important for the early detection of primary tumors in the prostate. A substantial signal was still visually apparent in the tumor at 22 hours post-injection with little signal in the bladder. A significant signal intensity was observed in the liver by ZD2-DA-( 64 Cu-DOTA), which may also be due to the relatively low stability of the Cu-DOTA monoamide. Release of free 64 Cu-(II) from the chelate can lead to non-specific accumulation of the radioisotope in the liver.

[0102] Antibodies and antibody fragments have been developed to target EDB-FN for the detection of cancers including prostate cancer. This study showed that small peptide-targeted PET probes specific for EDB-FN also have the potential for prostate cancer imaging. Compared with antibody-based probes, small peptide PET probes have several advantages, including cost-effective production, excellent tumor penetration by diffusion and perfusion, and rapid excretion of unbound probes from the circulation.

[0103] Example 2 The inventors have shown that EDB-FN is highly expressed in PaCa tissues from human pancreatic cancer (PaCa) specimens and mouse PaCa models, and is not expressed in normal pancreatic tissues in either case. The presence of EDB-FN in the PaCa tumor ECM enables rapid and specific binding of a targeting tracer for highly sensitive molecular imaging and PaCa diagnosis. The peptide sequence of the EDB fragment is conserved in all mammalian species.

[0104] A peptide ZD2 (Thr-Val-Arg-Thr-Ser-Ala-Asp) that specifically binds to EDB-FN was identified. The ZD2 peptide showed strong binding affinity for high-grade prostate tumors, weak binding affinity for low-grade tumors, and non-binding in normal tissues. In this example, it is shown that the ZD2 peptide can be used to develop a PET probe for highly sensitive and quantitative molecular imaging of EDB-FN for accurate detection and risk stratification of pancreatic cancer. A ZD2 peptide-targeted Ga(III) PET probe was designed and synthesized by conjugating NOTA to the ZD2 peptide using the linker 6-aminohexanoic acid. The efficacy of the targeted Ga(III) tracer was evaluated for PET imaging in male nude mice bearing aggressive and rapidly growing PC3 and slow-growing LNCaP prostate cancer xenografts.

[0105] Experiment Materials The protected amino acids for peptide synthesis were purchased from Novabiochem (Burlington, MA, USA). N,N-Diisopropylethylamine (DIPEA) was purchased from MP Biomedical LLC (Santa Ana, CA, USA). O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluorophosphate (HBTU) was purchased from Anaspec Inc (Fremont, CA, USA). Fmoc-6-aminohexanoic acid was purchased from Chem-IMPEX International (WD, IL, USA). t-Butyl bromoacetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemical reagents were purchased from Thermo Fisher. 1 1H-NMR spectra were acquired on a 500 MHz Varian Inova NMR spectrometer (supplier and address) using TMS as the internal standard. Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectra were acquired in linear mode on a Voyager DE-STR spectrometer (PerSeptive BioSystems) using 2,5-dihydroxybenzoic acid as the matrix. Agilent 1100 equipped with a ZORBAX 300 SB-C18 column semi-preparative HPLC was used for the purification of the ligand under the following conditions: eluent A, H2O / TFA (0.1%); B, MeCN / TFA (0.1%); 0% B for 15 min, 0 - 50% B for 30 min, 50% B for 5 min, 50% - 100% B for 2 min, 100% B for 5 min, flow rate 2 mL / min, UV detection at 210 nm. Ga was eluted with 0.1 M HCl 68 Ge / 68 Ga generator (ITG isotope technologies Garching GmbH, Germany).

[0106] Synthesis Synthesis of 1,4-bis(tert-butoxycarbonylmethyl)-1,4,7-triazanonane 1,4,7-Triazacyclononane (1.5 g, 11.62 mmol) was dissolved in anhydrous CHCl3 (15 mL) in an ice bath, and tert-butyl bromoacetate (4.98 g, 25.56 mmol) in CHCl3 (30 mL) was slowly added over 1.5 h. The mixture was stirred at room temperature for 24 h, and the solvent was removed. The residue was treated with DI water (15 mL), adjusted to pH 3 with 1 M HCl, and extracted with ether (50 mL × 2). The organic phase was removed, the aqueous phase was adjusted to pH 8 - 9 with 1 M NaOH, and extracted again with CH2Cl2 (25 mL × 3). Finally, the organic phase was evaporated to obtain the product. Yield: 36%, 1 H NMR (500 MHz, CDCl3): δ = 1.48 (s, 18H), 2.79 (s, 4H), 3.03 - 3.07 (m, 4H), 3.24 (s, 4H), 3.37 (s, 4H), 9.46 (s, H).

[0107] Synthesis of NOTA-bis(t-Bu ester) 1,4-Bis(tert-butoxycarbonylmethyl)-1,4,7-triazanonane (0.3 g, 0.84 mmol) and bromoacetic acid (0.415 g, 3 mmol) were dissolved in methanol (3 mL), and K2CO3 (0.53 g, 3.84 mmol) in water (3 mL) was added. The mixture was stirred at room temperature overnight and concentrated. Next, the residue was dissolved in water and adjusted to pH 4 with 1 M HCl. Water was removed by rotary evaporation, and the product was purified by flash chromatography (methanol:ethyl acetate 6.5:3.5). Yield: 64 %, 1 H NMR (500 MHz, D2O): δ = 1.48 (s, 18H), 2.84 (s, 4H), 3.08 (m, 4H), 3.35 (s, 4H), 3.47 (s, 4H).

[0108] Synthesis of ZD2-HA-NOTA ZD2-HA was synthesized using solid-phase chemistry. A mixture of 6-aminohexanoic acid (1.5 equiv), HBTU (1.5 equiv), and DIPEA (1.5 equiv) in 10 mL of anhydrous DMF was added to the resin at the end of peptide synthesis (0.5 mmol peptide), and the mixture was shaken until the ninhydrin no longer changed color (Kaiser test). Next, the resin was washed using DMF (10 mL × 3) and DCM (10 mL × 3). Subsequently, ZD2-HA was cleaved from the resin using a cocktail of TFA:H2O:TIBS (96.5:2.5:1) for 3 h. ZD2-HA was precipitated in cold ethyl ether and centrifuged and lyophilized. The product was characterized by MALDI-TOF mass spectrometry: calculated m / z for [M], C 47 H 83 N 15 O 18 , 1146.25; found (M+H + ), 1147.56.

[0109] Synthesis of non-radioactive Ga-ZD2-HA-NOTA To a solution of ZD2-HA-NOTA (0.11 g, 0.1 mmol) dissolved in 10 mL of NaAc-Ac buffer (0.1 M, pH 5.5), Ga(NO3)3 (0.076 g, 0.3 mmol) was added. The solution was stirred overnight at room temperature, and finally, the product was purified using preparative HPLC and lyophilized to obtain a fluffy white powder. Yield: 43%. The product was characterized by MALDI-TOF mass spectrometry: calculated m / z for [M], C 47 H 81 GaN 15 O 18 , 1212.51; found (M+H + ), 1213.54.

[0110] Results and discussion Chemistry and radiochemistry The synthesis of ZD2-HA-NOTA was shown in Figure 6. NOTA-bis(t-Bu ester) was prepared from TACN as the starting material by two rounds of displacement. Then, the precursor ZD2-HA-NOTA was successfully synthesized by conjugating with NOTA-bis(t-Bu ester) and ZD2-HA using solid-phase peptide chromatography and purified by RP-HPLC. The purified ZD2-HA-NOTA was characterized by MALDI-TOF (m / z=1147.56) and HPLC (purity: about 98%). Nat Ga-ZD2-HA-NOTA was also prepared and characterized by MALDI-TOF (m / z = 1213.54) and RP-HPLC (purity: approximately 96%).

[0111] Non-radioactive ZD2-(Ga-NOTA) was first synthesized according to the procedure shown in Figure 6. The macrocyclic ligand NOTA was used under relatively mild conditions. 68 This is because it can easily form a stable chelate with Ga(III), which is important for preserving the binding properties of the peptide. The ZD2 peptide was synthesized using standard solid-phase peptide synthesis, and then 6-aminohexanoic acid (HA) was conjugated to the N-terminus of the peptide as a spacer. NOTA-bis(t-Bu ester) was finally conjugated to the amino group on the resin, and the targeting ligand ZD2-NOTA was obtained by treating the resin with a cocktail of TFA:H2O:TIBS (96.5:2.5:1). The final product was purified by preparative HPLC. The purified ZD2-NOTA was characterized by MALDI-TOF (m / z = 1147.56 [M+1], observed; 1146.25, calculated) and had a purity of about 98% (HPLC), Figure 7A,B. ZD2-( Nat Ga-NOTA) was prepared by reacting the ligand with excess GaCl3 in acetate buffer (0.1 M, pH 5.5) at room temperature. Nat The peptide and ZD2-(Ga-NOTA) were purified using preparative HPLC and characterized by MALDI-TOF (m / z=1213.54 [M+1], observed; 1212.51, calculated) with a purity of about 96% (HPLC), FIG.Nat Ga-NOTA has high water solubility, which is an advantageous property for minimizing non-specific tissue binding.

[0112] Radioactive tracer ZD2-( 68 Ga-NOTA) was radiosynthesized in the radiopharmaceutical laboratory of University Hospitals, Cleveland (UH) in collaboration with Dr. Avril by reacting ZD2-NOTA with GaCl3 in sodium acetate buffer solution (0.1 M, pH 5.5) at 90 °C for 15 minutes. The pH of the reaction solution was finally adjusted with NaOH. The radiochemical yield was approximately 77% when determined by HPLC (gradient of water + 0.1% TFA / acetonitrile + 0.1% TFA, UV at 220 nm) equipped with a radioactive detector and a Zorbax Eclipse C18 column. The radiolabeled tracer was purified using reverse-phase HPLC equipped with a C-18 column before imaging. ZD2-( 68 The HPLC chromatogram of Ga-NOTA) is shown in Figure 8, and the product parameters are summarized in Table 3. The small peaks around the main peak may be 68 due to the complex formation of Ga(III) and the peptide, which is commonly observed in radiolabeled peptide products. The yield of the radiolabel is comparable to that of clinical tracers. The purity of the product is also equivalent to that of clinical-grade products.

Table 1

[0113] Expression of EDB-FN in human pancreatic cancer cells and tumor xenografts The expression of EDB-FN was first demonstrated by Western blotting in four different human pancreatic cancer cell lines, including BXPC3, Capan-1, Panc10.05, and Panc-1 cells. These human PaCa cell lines are commonly used to develop mouse PaCa cancer models in preclinical trials. All cancer cell lines tested had high expression of EDB-FN, Figure 9A. Tumor models were developed by subcutaneous transplantation of cancer cells in the flanks of female nude mice according to the ATCC description. The expression of EDB-FN was demonstrated in tumor xenografts of human PaCa cells using immunofluorescent staining with the BC-1 anti-EDB-FN monoclonal antibody. As shown in Figure 9B, substantial expression of EDB-FN was observed in all four PaCa subtypes, and no expression was observed in normal pancreas and muscle, consistent with reported results. High expression of EDB-FN was observed in the ECM of PaCa tumors. The results indicate that EDB-FN is highly expressed by PaCa cells and tumors and is a promising tumor protein target for molecular imaging and detection of PaCa.

[0114] ZD2 peptide that binds to EDB-FN in PaCa tumors The ZD2 peptide (Thr-Val-Arg-The-Ser-Ala-Asp)-targeted fluorescent tracer ZD2-Cy5.5 was synthesized according to the reported method to evaluate the peptide binding with EDB-FN in PaCa tumors. The binding specificity of the ZD2 peptide with EDB-FN in pancreatic cancer was tested by incubation of ZD2-Cy5.5 with the tumor slides of the above-mentioned tumor xenografts. As shown in Figure 10, strong binding of ZD2-Cy5.5 (red) was observed in all four tumor tissues tested, similar to the immunofluorescence staining in Figure 9B. No significant binding of ZD2-Cy5.5 with normal pancreas and muscle was observed. The strong binding of ZD2-Cy5.5 with EDB-FN in PaCa was blocked by the BC-1 anti-EDB-FN monoclonal antibody (BC-1 / ZD2). In PaCa specimens pre-incubated with the BC-1 antibody followed by ZD2-Cy5.5 (BC-1 / ZD2), almost no red fluorescence staining was observed. The results suggest that both ZD2-Cy5.5 and BC-1 specifically bind to the same EDB-FN protein target in tumor tissues. The ZD2 peptide is a promising targeting agent for specific binding of EDB-FN in PaCa tumors.

[0115] Expression of EDB-FN in human PaCa tumors The expression of EDB-FN in human pancreatic cancer is shown by staining human pancreatic cancer specimens with ZD2-Cy5.5. As shown in Figure 11, strong red fluorescence was observed in human PaCa specimens, almost no fluorescence was observed in normal pancreas, but a certain degree of fluorescence intensity was shown in pre-cancerous pancreatic intraepithelial neoplasms. The fluorescence intensity suggests high EDB-FN expression in PaCa, low expression in pre-cancerous tissues, and no expression in normal pancreas.

[0116] ZD2-( 68 Ga-NOTA)-mediated PET imaging of PaCa Sensitivity of molecular imaging of EDB-FN and efficacy of ZD2-( 68 Ga-NOTA) in detection of PaCa was evaluated by microPET / CT in a mouse model bearing Capan1 and BXPC3 human PaCa xenografts. The tumor model was developed in female nude mice as in C.1. The tracer synthesized using the above method was intravenously injected at a dose of 300 μCi per mouse. Figure 12 shows representative 2D coronal PET / CT images showing tumors at 1 hour and 2 hours after injection of the tracer. Strong uptake of the tracer was observed at both time points in the tumors and the bladder. In normal tissues and organs, especially the brain, liver, and lungs, little uptake was observed at 1 hour after injection. Background noise increased slightly at 2 hours after injection, probably due to the decay of radioactivity and the extension of the scan time. The signal intensity of both tumors was approximately 5 times that of muscle at 1 hour and 2 hours after injection. The 3D PET images also showed strong uptake into the tumors with little nonspecific uptake into the surrounding normal tissues and organs other than the kidneys and bladder, Figure 13. The high signal intensity in the kidneys and bladder indicates that the tracer is mainly excreted via renal filtration. These results demonstrate the efficacy and high specificity of ZD2-( 68 Ga-NOTA) in molecular imaging of EDB-FN and early detection of PaCa, and further verify the specific expression of EDB-FN in PaCa. The ZD2 peptide-targeted 68 Ga chelate is promising for highly sensitive early detection of pancreatic cancer in clinical practice.

[0117] Example 3 Synthesis of ZD2-HBED-CC The ZD2 peptide was synthesized using standard solid-phase chemistry. Next, HBED-CC-tris(tBu) ester was conjugated to the N-terminus of the ZD2 peptide on the resin. Then, the peptide was cleaved from the resin using a cocktail of TFA / water / TIBS (96.5 / 2.5 / 1). The product was precipitated in ethyl ether, purified by preparative HPLC, lyophilized, and characterized by MALDI-TOF mass spectrometry. (M+1) m / z, found 1264.02; calculated for C55H82N12O22 1264.32.

[0118] Synthesis of ZD2-AH-HBED-CC The ZD2 peptide was synthesized using standard solid-phase chemistry. Next, Fmoc-6-aminohexanoic acid was conjugated to the N-terminus of the ZD2 peptide on the resin. Subsequently, HBED-CC-tris(tBu) ester was reacted with the peptide, and cleavage from the resin was continued using a cocktail of TFA / water / TIBS (96.5 / 2.5 / 1). The product ZD2-AH-HBED-CC was precipitated in ethyl ether, purified by preparative HPLC, lyophilized, and characterized by MALDI-TOF mass spectrometry. (M+1) m / z, found 1377.1; calculated for C61H93N13O23 is 1377.48.

[0119] Synthesis of ZD2-(Ga-HBED-CC) The ligand, linker-free ZD2-HBED-CC, and gallium nitrate were mixed in PBS at 90 °C for 2 minutes. Next, the product ZD2-(Ga-HBED-CC) was purified by preparative HPLC and characterized by MALDI-TOF mass spectrometry. (M+1) m / z, found 1329.8; calculated for C55H79GaN12O22 is 1329.47.

[0120] Synthesis of ZD2-AH-(Ga-HBED-CC) The ligand, linker-containing ZD2-HBED-CC, and gallium nitrate were mixed in PBS at 90 °C for 2 minutes. Next, the product ZD2-AH-(Ga-HBED-CC) was purified by preparative HPLC and characterized by MALDI-TOF mass spectrometry. (M+1) m / z, found 1442.9; calculated for C61H90GaN13O23 is 1442.55.

[0121] PET Imaging of Mice with Tumors All in vivo imaging studies were performed in accordance with protocols and guidelines approved by the CWRU Animal Research Committee. Mice bearing BxPC3 or Capan-1 human pancreatic xenografts were anesthetized with 2% isoflurane in oxygen. The tracer ZD2-(68 Ga-HBED-CC) or ZD2-AH- 68 Ga-HBED-CC) was injected via the tail vein at a dose of 100 - 300 μCi [5.3 - 13.0 MBq]. Next, the mice were subjected to static PET scans (Inveon microPET, Siemens Medical Solutions USA Inc.) for 10 or 20 minutes after a 30- or 60-minute uptake period. After all PET procedures, CT scans were continued for anatomical co-registration. PET / CT images were analyzed using Inveon Research Workplace version 3.0 and Horos software. Regions of interest (ROIs) were delineated for tumors, major organs, and muscles to calculate the ratios of specific and non-specific tissue uptake. Images were processed with 3D-OSEM iterated 2 times followed by MAP iterated 18 times for 3D reconstruction with a zoom factor of 1.0.

[0122] High-sensitivity molecular imaging of EDB-FN and detection of pancreatic cancer by ZD2- 68 The efficacy of Ga-HBED-CC) was evaluated by microPET / CT in a mouse model bearing Capan-1 and BxPC3 human pancreatic cancer xenografts. The figures show representative 2D and 3D whole-body PET / CT images of mice bearing tumors at 30 or 60 minutes after injection. Strong uptake of the tracer was observed in tumors, kidneys, and bladder at 30 or 60 minutes after injection, as shown in the whole-body PET images. Tracer uptake in both tumors was significantly higher than that in normal organs and tissues including the brain, heart, liver, and muscle. High signal intensity in the kidneys and bladder indicates that the tracer is mainly excreted via renal filtration.

[0123] Quantitative analysis revealed that uptake in both BxPC3 and Capan-1 tumors was significantly higher than that in normal tissues including the brain, heart, liver, and muscle at 30 or 60 minutes after injection. ZD2-AH- 68In Ga-HBED-CC, tumor uptake was approximately 18.3-fold and 13-fold that of muscle at 60 minutes post-injection in BxPC3 and Capan-1 tumors, respectively (p < 0.01). Without a linker, ZD2-( 68 In Ga-HBED-CC, tumor uptake was approximately 10.2-fold and 7.3-fold that of muscle at 60 minutes post-injection in BxPC3 and Capan-1 tumors, respectively (p < 0.01). Tumor uptake remained significantly higher than that in normal tissues in both tumor models (p < 0.05). These results demonstrate that both ZD2-( 68 Ga-HBED-CC) and ZD2-AH-( 68 Ga-HBED-CC) are highly specific for pancreatic cancer tumors with minimal uptake in normal tissues including the liver.

[0124] From the foregoing description of the invention, those skilled in the art will recognize improvements, changes, and modifications. Such improvements, changes, and modifications within the art are intended to be covered by the appended claims. All references, publications, and patents cited in this application are hereby incorporated by reference in their entirety. As described in connection with the present invention, the following is claimed.

Claims

**Claim 1** A PET / SPECT probe comprising the following formula, 【Chemical 1】 wherein P is a peptide comprising the amino acid sequence of SEQ ID NO: 1, C is a PET / SPECT contrast agent, L is any 6-aminohexanoic acid linker that covalently attaches the peptide to the PET / SPECT contrast agent, the contrast agent comprises at least one of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), N,N'-bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N'-diacetic acid (HBED-CC), and derivatives thereof, a metal chelating agent, and a chelated metal, the PET / SPECT probe is for use in detecting, monitoring, and / or imaging cancer cells including pancreatic cancer cells. A PET / SPECT probe.

2. The chelate metal is ６７ Ga, ６８ Ga, ６４ Cu, ９９ｍ Tc, １１１ In, ８９ Zr, ９０ Y, １５３ Sm, or ８９ Sr, and the probe according to claim 1 is selected from the group consisting of. **Claim 3** The probe according to claim 1, for use in detecting, monitoring, and / or imaging cancer cells and / or cancer cell invasiveness. **Claim 4** The probe according to claim 3, for use in determining cancer invasiveness.

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