Software Technology for “Molecular imaging of cancer cells: in vivo”

Animal Health – David L. Morse, Robert J. Gillies, Amanda Huynh, Josef Vagner, Arizona Board Of Regents On Behalf Of University Of /arizona, Arizona Board of Regents of University of Arizona, H Lee Moffitt Cancer Center and Research Institute Inc

Abstract for “Molecular imaging of cancer cells: in vivo”

Targeted molecular imaging can use targeted cell targets to detect cancer cells in vivo. These non-invasive methods can be used to detect cancer cells such as metastasized cells. Compositions and kits that can be used in the disclosed methods are also provided.

Background for “Molecular imaging of cancer cells: in vivo”

“Microscopic-sized cancer cells can spread or migrate in most small tumors. A surgeon will usually try to remove the tumor using normal tissue as a border. This is so that these small cells can be removed. This is known as cancer at the margin or positive margins. Cancer cells found near the edges of resected tissue are considered to be cancer. Negative resection margins can reduce the chance of cancer recurrence in patients with cancer. The current methods for assessing surgical margins include post-operative evaluations of pathology specimens taken during surgery. Additional surgery may be necessary if the pathology specimen is positive. A method to assess the intraoperative margin of the resection before actual resection can reduce the risk of a positive microscopical margin and the need to have additional surgery.

However, ILM techniques lack biomarkers to target these agents against cancer cells. These agents are distributed non-specifically across SLNs, providing only an anatomical and non-functional map (Galanzha E.I. et al. J Biophotonics 2:528-539 (2009); McElroy, M. et al. J Surg Res 151, 68-73 (2009). SLN biopsy is therefore required in order to detect potential cancer cells. SLN biopsy requires a multidisciplinary team that has access to specialized imaging and surgical equipment. Histopathology 55:107-113 (2009); Krag, D. et al. N Engl J Med 339:941-946 (1998); McMasters, K. M. et al. J Clin Oncol 18 :2560-2566 ((2000); Ung O. A. et al. Asian J Surg 27.284-290 (2004)). These complications include lymphedema and seroma formation, sensory neuro injury, and limited range of motion (Purushotham A. D. et. al. J Clin Oncol 23.4312-4321 (2006). A majority (74%) of breast cancer patients who have undergone SLN biopsy are negative for pathological reasons (Krag, D. N. et. al. Lancet Oncol 8:881-888 (2007)). Biopsies are not able to detect axillary disease in between 5-10% of patients (McMasters K. M. and al. J Clin Oncol 18 :2560-2566 ((2000); Ung O. A. et al. Asian J Surg 27.284-290 (2004). A non-invasive method of detecting cancer is needed with increased sensitivity and specificity, and to eliminate unnecessary surgeries.

“It is the object of the invention that compositions and methods are provided for non-invasive detection and treatment of cancer in a subject.”

“It is an object of the invention to provide compositions for in-vivo molecular imaging (in vivo) of cancer cells such as pancreatic and metastatic.

“It is another object of the invention that compositions and methods are provided for label-guided cancer surgery.”

“It is an object of the invention to provide compositions, methods and procedures for label-guided pancreatic cancer surgery.”

“It is another object of the invention that cell-surface markers can be used for the detection of cancer cells in vivo.

“It is an object of the invention to provide cell surface markers that can be used in vivo to detect pancreatic carcinoma cells.”

“A non-invasive method has been developed to detect cancer cells in in vivo subjects. This involves giving the subject one or more targeted imaging probs that bind to a cellular target. The probes can be chosen from the following groups: carbonic anhydrase 9, carbonic anhydrase 12, carbonic anhydrase12 (CAXII), mammaglobin A, CX-C motif chemokine 10, CXCL10, matrix metallopeptidase 9, and MMP-9. To detect the targeted imaging probes in the subject, a molecular imaging tool can be used to image the subject. This method can detect cancer cells in the organs or tissues of the subject by detecting the targeted imaging probe(s).

“Cancer cells may be metastasized or primary tumors. In some embodiments, targeted imaging probes are administered to patients with primary tumors in order to detect metastasized cancer cells. Other embodiments involve administering targeted imaging probes in order to detect primary and occult cancers. The disclosed methods can detect non-small-cell carcinoma cells as well as breast cancer cells.

“In preferred embodiments the cellular target may be CAIX or CAXII. Combining these cellular targets can identify 100% of lymph node metastasis in breast cancer patients. These embodiments can include administering to the subject a targeted imaging probe specifically binding CAIX and another targeted imaging probe specifically binding CAXII. The detection of either one or both of these probes in an organ is indicative of cancer cells.

Mammaglobin A has been shown to detect malignant breast cancer cells in the breast and lymph nodes. In some embodiments, mammaglobin A is the cellular target. These embodiments can include administering to the subject a targeted image probe that specifically binds mammaglobin A. The detection of mammaglobin A in the organ of a subject indicates that there are breast cancer cells within the organ.

“Devices that can be used in molecular imaging are well-known in the art. They include devices for optical imaging, magnetic resonance imaging (MRI), computed tomography(CT) and nuclear medicine imaging. These devices can be used with the disclosed methods. The molecular imaging system is an optical device that detects near-infrared light. It is preferred in certain embodiments.

Preferable embodiments of the antibody can be monoclonal antibodies or fragments thereof that bind cellular targets. Commercially available monoclonal antibodies that bind specifically CAIX, CAXII and mammaglobin A are known. Routine methods can also be used to produce additional antibodies that are suitable for in-vivo detection. Some embodiments allow for the production of antibodies or fragments thereof that can be chimeric, humanized and recombinant.

“The targeted imaging probe that specifically binds CAIX should contain an antibody with the idiotype monoclonal antibody-clone 315602 linked to a detectable tag. Targeted imaging probe that specifically binds CAXII should preferably contain an antibody with the idiotype monoclonal anti-clone 315602 linked via a detectable label. A targeted imaging probe that specifically binds mammaglobinA should contain an antibody with the idiotype monoclonal antibody Clone 304-1A5/clone 315602 linked to a detectable Label. In some embodiments, the targeted image probe includes monoclonal antibody, clone 315602 or clone 30411, clone 30411, or clone 31A5.

“The targeted imaging probe should be an antibody that is linked to a detectable marker. Based on the molecular imaging devices, it is possible to choose suitable detectable labels. Preferably, the detectable label for optical imaging is a near infrared (NIR), fluorophore. The preferred method involves, for instance, a first antibody with the idiotype monoclonal anti-body clone303123 linked to an NIR fluorophore, and a second antibody with the idiotype monoclonal anti-body clone315602 linked to another NIR fluorophore.

“Also included is a composition that contains a first antibody with the idiotype monoclonal anti-body clone303123 linked to an NIR fluorophore, and a second antibody with the idiotype monoclonal anti-body clone 315602 linked a second NIR fluorescent. A third monoclonal antibody can be added to the composition. It could contain either clone 31A5 or clone 306102. The composition can also contain monoclonal antibodies clone 305302, clone 315602 or clone 215600, or any combination thereof. For example, different targeted imaging probes can have different detectable labels. They can have different excitation frequencies and/or excitation frequencies. Different targeted imaging probes can be identified with different labels to allow them to be distinguished during imaging.

“A kit that includes two or more antibodies that bind to a specific cellular target from the group of CAIX and CAXII is also available. The kits contain at least one antibody linked to a first NIR fluorescent and at most one antibody linked to another NIR fluorescent. Preferably, light is not excited by the second NIR fluorescent dye when it passes through either the absorption spectrum or the emission spectrum of the first NIR fluorescent. Monoclonal antibody, clone 315602 or 303123 is preferred. Monoclonal antibody Clone 304-1A5 and clone 315602 can be added to the kit.

“I. Definitions”

“Targeted molecular imaging” is a term that refers to targeted molecular imaging. The in vivo detection and analysis of biological processes, such as biodistribution at the molecular and cellular levels, is called targeted molecular imaging. In vivo detection can be achieved using a targeted imaging probe, which binds to a molecular target or cell. An imaging device detects the probe in real time.

“The term “targeted imaging probe” is used. A molecule that binds specifically to a molecular target or cellular target in vivo and can be detected by in vivo imaging techniques. The binding molecule is linked to a detectable label. This allows for detection. The most preferred binding molecules are antibodies, peptides and peptidomimetics.

“Antibody” is a term that refers to a specific antibody. “Antibody” can be used to refer to any polyclonal or monoclonal, synthetic, recombinant or immunoglobulin molecule which specifically binds to a target antigen. This term covers intact immunoglobulin molecules as well as fragments or polymers from those immunoglobulins, chimeric antibodies that contain sequences from more then one species, class or subclass of immunoglobulin and humanized or humanized immunoglobulins molecules or fragments thereof, which at least have the idiotype of the immunoglobulin that binds to the target antigen.

“The term “idiotype” refers to the portion of an immunoglobulin molecule that confers its ability to bind an antigen. The immunoglobulin molecule whose ability to bind antigens is determined by its idiotype. The complementarity determining areas (CDRs), of the immunoglobulin variable domains (VL, and VH) determine the idiotype of an antigen.

“Peptide” is a term that refers to a natural or synthetic molecule. “Peptide” can refer to a natural or artificial molecule that contains two or more amino acid linked by the carboxyl groups of one amino acid to another. The length of a peptide does not matter; therefore,?peptide? Polypeptides can be combined with proteins.”

“The term ‘peptidomimetic? A mimetic of a protein that alters the normal peptide chemical. Peptidomimetics are often used to enhance the properties of the original peptide. These include increased stability, improved efficacy, enhanced delivery and increased half-life.

“The term ‘aptamer’ is a synonym for “oligonucleic acid molecules that specifically bind to a target molecule.” “Attamer” refers to oligonucleic acids molecules that bind specifically to a target molecule.

“Small molecule” is the term used herein. A compound with a molecular mass of less than 1000 Daltons and usually between 300 and 700 Daltons. This term can include monomers, primary metabolites, secondary metabolisms, biological amines, and synthetic or non-peptide biological molecules. Targeted imaging probes with small molecules can bind to the molecular target.

“Specifically Binds” is a term that refers to the binding of a molecule to a target molecule. The binding of a molecule or molecule to a target molecule (e.g., an antibody to its cognate Antigen) without significant binding to other molecules is called “specifically binds”. A molecule that?specifically binds is preferred. To a target molecule having an affinity constant (Ka?) greater than about 105?mol?1 (e.g. 106?mol?1,107?mol?1,108?mol?1,109?mol?1,109?mol?1,1010 mol.1, 1011?mol.1, and 1012?mol.1 or more) with that target molecule.

“The term “neoplasm” refers to a cell that is experiencing abnormal cell proliferation. A cell that is experiencing abnormal cell proliferation. Neoplastic cell growth is a form of abnormal cell proliferation that exceeds normal tissue growth. The excessive growth often persists even after the stimulation has been stopped and can lead to the formation of tumors. Neoplasms can be benign, malignant, or premalignant.

“Cancer” is a term that refers to cancer. or ?malignant neoplasm? Refers to cells that exhibit uncontrolled growth and invasion of adjacent tissues.

“Metastatic” is a term that refers to the phenomenon of metastatic. or ?metastasized? These are cancer cells that have spread from their original site (primary site), to another location (metastatic spot) within the body.

“The term “occult cancer” is used to describe a form of metastasized cancer cells. “Metastasized cancer cells from unknown origin” refers to them.

“The term’subject? or ?patient? Any individual who is the subject of administration. A subject could be a vertebrate or a mammal. The subject could also be a person. You can have the subject domesticated, farm, or keep them in a zoo or circus. Dogs, cats, rabbits and other domesticated animals include guinea-pigs, ferrets as well as ferrets, ferrets. For example, agricultural animals are horses, mules and donkeys, burros cattle, cows pigs, sheep, alligators, and pigs. For example, circus-maintained animals can include lions and tigers, bears as well as camels, giraffes and hippopotamuses. This term does not indicate a specific age or sex.

“Effective amount” is a term that refers to the quantity of targeted imaging probes sufficient for in vivo detection of the probes in an organ or tissue. “Effective amount” refers to the number of targeted imaging probes that are sufficient to detect probes in tissue or organs by in vivo imaging. The amount of targeted imaging probes required for in vivo detection of the probes in an organ or tissue will vary from one subject to the next. This depends on the individual subject’s age and general condition, as well as the type of administration and the specific probes being used. The appropriate amount is determined by a person of ordinary skill in the art using only routine experimentation. One of ordinary skill can determine the appropriate?effective amount by using only routine experimentation.”

“The term “detectable label” is used herein. “Detectable label” is a term that refers to any molecule which can be detected using in-vivo imaging techniques.

“The term “near-infrared” fluorophore (NIR) is used. A molecule with an emission and absorption wavelength in the NIR spectrum of 680 to 900 nm. The ‘biological window’ is a preferred wave range for in-vivo fluorescence imaging. These molecules are able to be detected further while minimizing fluorescence absorption by tissues.

“The terms “label-guided surgical” are used interchangeably. ?fluorescent-guided surgery,? Fluorescent-guided surgery, or the like, is surgery in which the location of relevant tissue/or cells has been marked with a label. This is intraoperative imaging. Label-guided surgery where the label is visualized via an image of tissue and/or cells can be referred to as image-guided-surgery.”

“II. Compositions”

“A. “A.

Targeted imaging probes are available to detect cancer cells. They can bind specific cellular targets on in vivo cancer cells. The cellular targets are proteins found on the surfaces of cancer cells. Imaging probes can access these targets and bind them in vivo. Targeted imaging probes disclosed preferably don’t bind normal tissue (non-cancerous). The targeted imaging probes may be used to detect metastasized cells of cancer or cells that are about to become metastasized from the primary tumor.

For detection of metastasized cancer cells, probes that specifically bind carbonic and anhydrase 9 or 12 (CAIX), mammaglobin A, C-XC motif chemokine 10, (CXCL10), carbonic anhydrase 9, (CAIX), and matrix metallopeptidase (9 (MMP-9) are available. Targeted imaging probes should preferably be able to bind the cell targets in areas that can be accessed from the circulation (e.g. blood or lymph) in vitro.

To detect cancer cells that express either one or both of these proteins, “targeted imaging probes” specifically binding CAIX/CAXII should be used in combination. These probes may also be combined with tissue-specific probes to increase specificity. To detect breast cancer cells that have metastasized, CAIX or CAXII probes should be used together with probes that specifically bind mammaglobin A.

“Probes that bind Toll like receptor 2 (TLR2) specifically in vivo have been disclosed to be used in the detection of cancer cells such as pancreatic, pancreatic, and metastasized pancreatic carcinoma cells.

“In some instances, the disclosed targeted imaging probes can be used with other targeting agents such as cancer-specific targeting probes. A targeting agent that binds tumor-associated glucoprotein-72 (TAG-72) can be used in conjunction with the disclosed targeted image probes. TAG-72, a glycoprotein that is found on many types of cancer cells including those in the breast, colon and pancreatic, is disclosed. TAG-72 is specifically bound to murine monoclonal antibody (CC49MAb, Minretumomab). It also reacts strongly with LS-174T breast cancer extract and a breast cancer extract.

Targeted imaging probes can generally contain a cellular targeting domain and a detectable tag. Routine methods can link the detectable label and cellular target binding area.

“In certain embodiments, the cellular target domain binding domain and detectable labels can be chemically crossedlinked using protein-crosslinking agents. Crosslinking agents, such as succinimidyl esters, are commonly found on commercially available labels such as fluorophores. These agents can be used to conjugate with proteins such as antibodies. Non-limiting examples of suitable protein crosslinkers include DSS (Disuccinimidylsuberate), DSP (Dithiobis(succinimidylpropionate)), DTSSP (3,3?-Dithiobis (sulfosuccinimidylpropionate)), SULFO BSOCOES (Bis[2-(sulfosuccinimdooxycarbonyloxy) ethyl]sulfone), BSOCOES (Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST (Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene glycolbis(sulfosuccinimidylsuccinate)), DPDPB (1,2-Di[3?-(2?-pyridyldithio) propionamido]butane), BSSS (Bis(sulfosuccinimdyl) suberate), SMPB (Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB (Sulfosuccinimdyl-4-(p-maleimidophenyl) butyrate), MBS (3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS (3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB (N-Succinimidyl(4-iodoacetyl) aminobenzoate), SULFO SIAB (N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC (Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), SULFO SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), NHS LC SPDP (Succinimidyl-6-[3-(2-pyridyldithio) propionamido) hexanoate), SULFO NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio) propionamido) hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio) propionate), NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate), NHS IODOACETATE (N-Hydroxysuccinimidyliodoacetate), MPBH (4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH (4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid hydrazide hydrochloride), MBH (m-Maleimidobenzoic acid hydrazidehydrochloride), SULFO EMCS (N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide), EMCS (N-(epsilon-Maleimidocaproyloxy) succinimide), PMPI (N-(p-Maleimidophenyl) isocyanate), KMUH (N-(kappa-Maleimidoundecanoic acid) hydrazide), LC SMCC (Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate)), SULFO GMBS (N-(gamma-Maleimidobutryloxy) sulfosuccinimide ester), SMPH (Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS (N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS (N-(gamma-Maleimidobutyrloxy) succinimide), DMP (Dimethylpimelimidate hydrochloride), DMS (Dimethylsuberimidate hydrochloride), MHBH(Wood’s Reagent) (Methyl-p-hydroxybenzimidate hydrochloride, 98%), and DMA (Dimethyladipimidate hydrochloride).”

“In other embodiments, a targeted imaging probe could be a fusion protein or protein containing a cellular target binding region and a detectable tag. Fusion proteins are proteins that combine two or more genes, coding regions or proteins that were originally coded for peptides. This fusion gene or code region is translated into a single peptide, or polypeptide, with the functional properties of each original peptide. Recombinant DNA technology can create artificially recombinant fusion proteins or peptides. This involves removing the stop codon in a cDNA sequence that codes for the first protein or peptide, and then adding the cDNA sequence to the second protein or peptide in frame by ligation or overlap extensionPCR. Alternately, the coding regions may be synthesized and joined or even fusion coding. A cell can then express the fusion DNA sequence as a single protein or peptide. You can either include all of the original peptides and proteins in the protein, or just a fraction. Linker (or?spacer?) is often used if the entities are proteins. You can also add peptides to increase the likelihood that the proteins will behave and fold as expected. Internal ribosome entrance sites (IRES), elements can also be used to create multigene or polycistronic messages. The ribosome scanning model of 5 is bypassed by IRES elements. methylated Cap dependent translation can be started at internal sites. Multiple open reading frames can all be transcribed together. Each one is separated by an IRES. This creates polycistronic messages. Each open reading frame can be accessed by ribosomes to facilitate efficient translation thanks to the IRES element. There are many IRES sequences that are well-known in the art. These include those from encephalomycarditisvirus (EMCV). (Ghattas I. R. et. al., Mol. Cell. Biol. 11:5848-5849 (1991); biP protein (Macejak and Sarnow Nature, 353 :91 (1991); Antennapedia gene (exons d and e), [Oh et al. Genes & Development], 6:1643-1653 (1992); Antennapedia gene in drosophilia [Oh et., Genes & Development], 6:1643-1653 (1992); those in poliovirus [Pelletier Nature, 334:3203251988]; see also Mountford and Smith TIG, TIG, 11 184 (1985). There are many other recombinant or fusion methods that can be used to produce the peptides and proteins.

“Also disclosed were compositions comprising a first antibody consisting of the idiotype monoclonal anti-mouse antibody clone303123 linked with a first near infrared (NIR), fluorophore, and a 2nd antibody consisting of the idiotype monoclonal anti-mouse clone 315602 linked with a 2nd NIR fluorophore.”

“The composition may also include a third antibody that is either monoclonal antibody cloned 304-1A5 (or clone 31A5 linked with a third NIR fluorescent). Monoclonal antibody clone 315602 can be used as the first antibody. Monoclonal antibody 315602 can be the second. The third antibody may consist essentially of monoclonal anti-clone 315602 or 304-1A5.

“1. Cellular Target Binding Domain”

“a. Antibodies”

“Preferred embodiments use targeted imaging probes that specifically bind cellular targets. For use in the disclosed compositions or methods, antibodies that bind CAIX and CAXII, mammaglobin A, CEACAM6, CXCL10 and MMP-9 have been disclosed.

“The anti-CAIX antibody should preferably specifically bind to human CAIX protein (Accession Number. NP_001207). The anti-CAIX antibody may specifically bind the N-terminus or extracellular domain of human CAIX in some embodiments. Particularly preferred embodiments of the anti-CAIX antibodies specifically bind amino acids 59-414 from human CAIX protein. The anti-CAIX antibody may be either the monoclonal antibody mAb clone303123 (R&D Systems) or the idiotype. It is possible to identify anti-CAIX antibodies that bind the same epitope with this clone.

“The anti-CAXII antibody should preferably specifically bind to human CAXII (Accession Number. NP_001209). The anti-CAXII antibody binds to the N-terminus of human CAXII and the extracellular domain in some embodiments. Particularly preferred embodiments of the anti-CAXII antibodies specifically bind to amino acids 25-291 in human CAXII proteins. The anti-CAXII antibodies can either be monoclonal antibodies (mAbs) clone 315602 (R&D Systems) or have the idiotype. It is possible to identify anti-CAXII antibodies that bind to the same epitope than this clone.

“The anti-Mammaglobin A antibody should preferably specifically bind human Mammaglobin A (Accession Number. NP_002402.1). For example, anti-Mammaglobin A antibody could be monoclonal antibody mAb clone 31A5 or 304-1A5 (Zeta Corp. California, Sierra Madre), or it can have the idiotype one of these clones. It is also possible to identify anti-Mammaglobin A antibodies that bind the same epitope with this clone.

“Antibodies can be used in the disclosed compositions or methods include whole immunoglobulins (i.e. an intact antibody), fragments thereof and synthetic proteins that contain at least the antigen binding variable domain of an antibody. Variable domains are different in their sequence and used to determine the specificity and binding of each antibody to a particular antigen. The variability is not always evenly distributed in the variable domains. It is usually concentrated in three segments known as complementarity determining areas (CDRs), or hypervariable zones in both the light and heavy chain variable domains. The framework (FR) is the name given to the most conserved parts of the variable domains. Each of the variable domains for native heavy and lightweight chains has four FR regions. They generally adopt a beta-sheet layout and are connected by three CDRs that form loops connecting to, or in some cases, making up part of, this beta-sheet structure. Each chain’s CDRs are held in close proximity by the respective FR regions. They, along with the CDRs of the other chain, help to form the antigen binding site for antibodies.

“Antibodies that are used in the disclosed compositions or methods can be any type, including IgG and IgA. IgG antibodies can further be subdivided into IgG1, IgG2, IgG3, or IgG4 subtypes. IgA antibodies can further be subdivided into IgA1 or IgA2 subtypes.

“Fragments of antibodies that have bioactivity are also disclosed. These fragments can be attached to any sequence or not. They include insertions and deletions as well as substitutions and other selected modifications to specific regions or amino acid residues. However, the activity of the fragment must not be significantly impaired or altered from the nonmodified antibody. A fragment of an antigen-binding antibody that contains a monovalent antibody-binding fragment is called Fab. A Fab fragment is made by digesting whole antibodies with papain. This will yield an intact light and part of one heavy chain. Fab? The fragment of an antibody molecule is obtained by treating the whole antibody with pepsin and then reducing to give an intact light chain. There are two Fab? Two Fab? fragments can be obtained for each antibody molecule. Fab? Fab? (Fab? (Fab???? )2 is the fragment from an antibody that can obtained by treating whole antibody using the enzyme pepsin, but without further reduction. F(ab? F(ab?2)2 is a dimer from two Fab. Two disulfide bonds hold the fragments together. Fv is the minimum amount of antibody fragments that contain a binding site and an antigen recognition site. This dimer consists of a dimer consisting of one heavy and one lighter chain variable domains in a tight, noncovalent association (VH?VL dimer). This configuration is where the CDRs from each variable domain work together to create an antigen-binding spot on the VH-VL dimer’s surface. The six CDRs give antigen-binding speciality to the antibody. Even though a single variable domain, or half of an Fv containing only three CDRs that are specific for antigens, can recognize and bind the antigen, it has a lower affinity than the whole binding site.

“Techniques are also available to produce single-chain antibodies that target specific cellular targets. Single chain antibody (?SCA) Single chain antibody (?SCA) is a genetically engineered molecule that contains the variable region (VL) and the variable (VH) regions of the heavy chains (VH), which are linked by a suitable protein linker to form a single chain molecule. These single-chain antibodies are also known as “single chain Fv?” These single-chain antibodies are also known as?single-chain Fv? Antibody fragments. The Fv polypeptide also contains a linker polypeptide between the VH-VL domains, which allows the sFv structure to be formed for antigen binding. The methods for producing single-chain antibodies are well-known to those skilled in the art. One chain antibodies can be made by joining the variable domains of heavy and light chains with a short peptide linking agent. This allows for reconstituting the antigen binding site on one molecule. Single-chain antigen variable fragments (scFvs), in which the Cterminus of one variabledomain is linked to the N-terminus the other variabledomain via a 15-25 amino acid peptide, peptide linker, have been created without significantly disrupting the antigen binding or specificity. The linker allows the heavy and light chains to bind in their correct conformational orientation.

By linking two scFvs, it is possible to engineer divalent single-chain variable fractions (diScFvs). You can achieve tandem scFvs by creating a single peptide-chain with two VH regions and two VL areas. ScFvs can also have linker peptides designed that are too short to allow the two variable regions (about five amino acid) to fold together. This causes scFvs not to dimerize. This is called diabodies. The dissociation constants of diabodies have been found to be up to 40 times lower than the corresponding scFvs. This means that they are more closely related to their target. Even shorter linkers, one or two amino acids, can lead to the formation trimers (triabodies and tribodies). Also, tetrabodies can be made. They have a higher affinity for their targets than diabodies.

“Preferably, if an antibody is to be administered in humans, it is either a human antibody, or a?humanized? antibody. an antibody that is derived from non-human animals. Humanizing non-human antibodies is a well-known art. They have been described in U.S. Pat. Nos. Nos.

“b. Peptides”

“In some cases, the targeted imaging probe may contain a peptide which binds to the cellular target CAIX or CAXII, mammaglobin A, CEACAM6, CXCL10 or MMP-9. Some embodiments contain the idiotype or antibody of an antibody. Another embodiment allows the identification of the peptide by screening a list of peptides that are compatible with the cellular target.

“c. Peptidomimetics”

“In certain embodiments, the targeted image probe may contain a peptidometic that binds CAIX or CAXII. A peptidometic is a small, protein-like chain that mimics a peptide. These systems are usually created by modification of an existing protein or by creating similar systems to mimic peptides such as peptoids or 0-peptides. The altered chemical structure can be used to improve molecular properties, such as stability and biological activity. This could be used to create drug-like compounds using existing peptides. Modifications are changes that occur in a peptide that cannot be found naturally, such as altering backbones or the incorporation non-natural amino acids.

U.S. Pat. 102/399 describes “Methods for making peptidomimetics that are based on a known sequence of polypeptides.” Nos. Nos. 5,631,280, 5,612,895 and 5,579.250. Non-amino acids can be found in peptididomimetics. They may have non-amide links at certain positions. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include ?-alanine, L-?-amino butyric acid, L-?-amino butyric acid, L-?-amino isobutyric acid, L-?-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N-?-Boc-N-?-CBZ-L-lysine, N-?-Boc-N-?-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-?-Boc-N-?CBZ-L-ornithine, N-?-Boc-N-?-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.”

“d. Aptamers”

“In certain embodiments, the targeted image probe may contain an aptamer that binds CAIX or CAXII. Aptamers can be single-stranded DNA or DNA oligonucleotides between 15 and 60 bases in length, which bind with high affinity for specific molecular targets. Most aptamers to proteins are compatible with monoclonal antibodies. They bind to Kds (equilibrium constant), which is in the 1 pM- 1 nM range. These nucleic acids ligands can be bound to proteins, nucleic acids, and small organic compounds. They can even bind to entire organisms.

You can select aptamers by incubating your target molecule in large pools of oligonucleotide (usually 40-60mers). Because of its large pool, the oligonucleotide is able to isolate the specific aptamer. Aptamers are able to distinguish between closely related, but not identical members of a protein families or between different functional and conformational states. The systematic evolution of ligands through exponential enrichment (SELEX), is a protocol that allows for modification and variation to select aptamers. This process can be used to create new aptamers within two weeks.

“e. Sulfonamide-Based Inhibitors”

“In certain embodiments, the targeted image probe may contain a carbonic acidase inhibitor that binds CAIX or CAXII. Carbonic anhydrase inhibits are pharmaceuticals that inhibit the activity of carbonic acidase by binding directly to its catalytic sites. A sulfonamide is a common component of suitable carbonic anhydrase inhibiters. Acetazolamide and Brinzolamide are all examples of carbonic-anhydrase inhibitors.

“f. Synthetic Analogues and Natural Ligands”

“In certain embodiments, the targeted image probe may contain a natural binding agent of the cellular targets for cancer cells or a fragment thereof.”

“Tell-like receptor 2 or TLR2 (for example) recognizes cell-wall components like peptidoglycan and lipoteichoic acids from gram-positive bacteria, lipoarabinomannan, and lipoproteins from mycobacteria and zymosan, which is found in the yeast cell wall. In some embodiments, the natural binding agent is a component of the cell wall of a microorganism such as yeast or bacteria.

“In other embodiments, the targeted image probe contains a synthetic analog of a natural drug ligand. Synthetic diacylated lipoproteins that correspond to the N-terminal partial structures bacterial lipoproteins have been also developed. They bind TLR2. In some embodiments, the targeted imaging probe contains the TLR2 ligand dipalmitoyl-S-glyceryl-L-Cys-Ser-(Lys)4 (Pam2CSK4), which has the following structure:”

“”

“In some embodiments, the targeted imaging probe contains the TLR2 ligand tripalmitoyl-S-glyceryl-L-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4), which has the following structure:”

“”

“In some embodiments, the targeted imaging probe contains the lipolanthionine peptide (2R,6R)-Pam2LanHda-Ser-(Lys)4-NH2 (lipolan).”

“In certain embodiments, the targeted image probe contains MALP-2 (a diacylated lipopeptide that was isolated from Mycoplasma fermentationans).

“In some embodiments, the targeted imaging probe contains a cellular target binding domain having the formula:\nX-Dhp(Pam2)-peptide MALP2,\nwhere ?X? There is an addition of: Palmitoyl and Fluorescein; Dhc=1,2-dihydroxypropylcysteine; Aha=epsilon-aminohexanoic acid; Aun=epsilon-aminoundecanoic acid; Ac-PEGO20 (Pam2)-peptide MALP2,nwhere?X? Ac=acetyl; Dhc=1,2-dihydroxypropylcysteine; Aha=epsilon-aminohexanoic acid; Aun=epsilon-aminoundecanoic acid; PEGO20=20 atoms long polyethelene glycol (4 Peg units); Pam2=dipalmitoyl-S-glyceryl; Pam3=tripalmitoyl-S-glyceryl.”

“In certain embodiments, the targeted image probe can have the structure.”

“”

“Where R1 is?CO.R6 or H, and R6 is C12-18 alkyl or alkenyl;

“Where R2 is??CH2?O??CO?R7 or?H?, wherein C12 to C18 alkyls, alkenyls, amines, alkyl-amines, alkylether, polyether and R7 are any C12 or C18 alkyls, alkenyls, amines, alkylamines, alkyls amines, alkyl ketol amines, alkylether, polyether or alkylamine;

“where R3 is R8-label, wherein R8 is acetyl-PEGO-(Ac-PEGO), palmitoyl-, fluorescein-, acetyl-6-aminohexanoyl-(Ac-Aha), adapalenoyl-, acetyl-11-aminoundecanoyl-(Ac-Aun), or tretinoyl-;”

“where R4 is -Gly-DSer-PEGO-NH2, -Gly-DSer-NH2, -Cys-Ser-(Lys)4-NH2, -Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys-NH2, -Ser-Arg-Phe-Asp-Glu-Asp-Asp-Leu-Glu-NH2, -Gly-Ser-Gln-Asn-Leu-Ala-Ser-Leu-Glu-Glu-NH2, or -serine methyl ester; and”

“where R5?S? “where R5 is?S??” or?Se?

“In some embodiments R1 can be H. R2 could then be?CH2O?CO?R7. R7 could be any C12-C18 alkyl or an alkenyl.

“In some embodiments, R1 can be ?CO?R6, wherein R6 is C12 to C18 alkyl; R2 can be ?CH2?O?CO?R7, wherein R7 is any C12 to C18 alkyl; R3 can be R8-label, wherein R8 is acetyl-PEGO-(Ac-PEGO), palmitoyl-, fluorescein-, acetyl-6-aminohexanoyl- (Ac-Aha), adapalenoyl-, acetyl-11-aminoundecanoyl- (Ac-Aun), or tretinoyl-; R4 can be -Gly-DSer-PEGO-NH2, -Gly-DSer-NH2, -Cys-Ser-(Lys)4-NH2, -Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys-NH2, -Ser-Arg-Phe-Asp-Glu-Asp-Asp-Leu-Glu-NH2, -Gly-Ser-Gln-Asn-Leu-Ala-Ser-Leu-Glu-Glu-NH2, or -serine methyl ester; and R5 can be ?S?.”

“2. Detectable Labels”

“The disclosed targeted imaging probes should be linked to a detectable marker. Fluorescent molecules, also known as fluorescent molecules, are suitable for labeling imaging agents. Fluorophores and fluororochromes, chemiluminescent agents (e.g. luminol), bioluminescent and chemiluminescent substances (e.g. luciferin, green fluorescent protein (GFP), and metals (e.g. gold nanoparticles) are all suitable for detectably labeling imaging agents. Based on the imaging method chosen, suitable detectable labels may be selected. Preferable embodiments include a near-infrared fluorescent dye, a Gadolinium chelate, SPECT or PET imaging radionuclides, and a gold nanoparticle or CT imaging.

“a. Fluorophores”

“b. Radioisotopes”

A radioisotope can either be integrated into or attached to a targeted imaging agent. Tritium, 11C and 13N are some examples of radioisotopes that can be useful. Some embodiments attach the radioisotope to the targeted imaging agent through halogenation. Some embodiments attach the radioisotope to the targeted image agent using a linking group or bound with a chelating agent. This is either attached directly to the agent or via a linker.

“3. Adjuvants”

“The TLR2 agonists described can be used as adjuvants in order to increase stimulation of an immune reaction, such as in a vaccine.”

Summary for “Molecular imaging of cancer cells: in vivo”

“Microscopic-sized cancer cells can spread or migrate in most small tumors. A surgeon will usually try to remove the tumor using normal tissue as a border. This is so that these small cells can be removed. This is known as cancer at the margin or positive margins. Cancer cells found near the edges of resected tissue are considered to be cancer. Negative resection margins can reduce the chance of cancer recurrence in patients with cancer. The current methods for assessing surgical margins include post-operative evaluations of pathology specimens taken during surgery. Additional surgery may be necessary if the pathology specimen is positive. A method to assess the intraoperative margin of the resection before actual resection can reduce the risk of a positive microscopical margin and the need to have additional surgery.

However, ILM techniques lack biomarkers to target these agents against cancer cells. These agents are distributed non-specifically across SLNs, providing only an anatomical and non-functional map (Galanzha E.I. et al. J Biophotonics 2:528-539 (2009); McElroy, M. et al. J Surg Res 151, 68-73 (2009). SLN biopsy is therefore required in order to detect potential cancer cells. SLN biopsy requires a multidisciplinary team that has access to specialized imaging and surgical equipment. Histopathology 55:107-113 (2009); Krag, D. et al. N Engl J Med 339:941-946 (1998); McMasters, K. M. et al. J Clin Oncol 18 :2560-2566 ((2000); Ung O. A. et al. Asian J Surg 27.284-290 (2004)). These complications include lymphedema and seroma formation, sensory neuro injury, and limited range of motion (Purushotham A. D. et. al. J Clin Oncol 23.4312-4321 (2006). A majority (74%) of breast cancer patients who have undergone SLN biopsy are negative for pathological reasons (Krag, D. N. et. al. Lancet Oncol 8:881-888 (2007)). Biopsies are not able to detect axillary disease in between 5-10% of patients (McMasters K. M. and al. J Clin Oncol 18 :2560-2566 ((2000); Ung O. A. et al. Asian J Surg 27.284-290 (2004). A non-invasive method of detecting cancer is needed with increased sensitivity and specificity, and to eliminate unnecessary surgeries.

“It is the object of the invention that compositions and methods are provided for non-invasive detection and treatment of cancer in a subject.”

“It is an object of the invention to provide compositions for in-vivo molecular imaging (in vivo) of cancer cells such as pancreatic and metastatic.

“It is another object of the invention that compositions and methods are provided for label-guided cancer surgery.”

“It is an object of the invention to provide compositions, methods and procedures for label-guided pancreatic cancer surgery.”

“It is another object of the invention that cell-surface markers can be used for the detection of cancer cells in vivo.

“It is an object of the invention to provide cell surface markers that can be used in vivo to detect pancreatic carcinoma cells.”

“A non-invasive method has been developed to detect cancer cells in in vivo subjects. This involves giving the subject one or more targeted imaging probs that bind to a cellular target. The probes can be chosen from the following groups: carbonic anhydrase 9, carbonic anhydrase 12, carbonic anhydrase12 (CAXII), mammaglobin A, CX-C motif chemokine 10, CXCL10, matrix metallopeptidase 9, and MMP-9. To detect the targeted imaging probes in the subject, a molecular imaging tool can be used to image the subject. This method can detect cancer cells in the organs or tissues of the subject by detecting the targeted imaging probe(s).

“Cancer cells may be metastasized or primary tumors. In some embodiments, targeted imaging probes are administered to patients with primary tumors in order to detect metastasized cancer cells. Other embodiments involve administering targeted imaging probes in order to detect primary and occult cancers. The disclosed methods can detect non-small-cell carcinoma cells as well as breast cancer cells.

“In preferred embodiments the cellular target may be CAIX or CAXII. Combining these cellular targets can identify 100% of lymph node metastasis in breast cancer patients. These embodiments can include administering to the subject a targeted imaging probe specifically binding CAIX and another targeted imaging probe specifically binding CAXII. The detection of either one or both of these probes in an organ is indicative of cancer cells.

Mammaglobin A has been shown to detect malignant breast cancer cells in the breast and lymph nodes. In some embodiments, mammaglobin A is the cellular target. These embodiments can include administering to the subject a targeted image probe that specifically binds mammaglobin A. The detection of mammaglobin A in the organ of a subject indicates that there are breast cancer cells within the organ.

“Devices that can be used in molecular imaging are well-known in the art. They include devices for optical imaging, magnetic resonance imaging (MRI), computed tomography(CT) and nuclear medicine imaging. These devices can be used with the disclosed methods. The molecular imaging system is an optical device that detects near-infrared light. It is preferred in certain embodiments.

Preferable embodiments of the antibody can be monoclonal antibodies or fragments thereof that bind cellular targets. Commercially available monoclonal antibodies that bind specifically CAIX, CAXII and mammaglobin A are known. Routine methods can also be used to produce additional antibodies that are suitable for in-vivo detection. Some embodiments allow for the production of antibodies or fragments thereof that can be chimeric, humanized and recombinant.

“The targeted imaging probe that specifically binds CAIX should contain an antibody with the idiotype monoclonal antibody-clone 315602 linked to a detectable tag. Targeted imaging probe that specifically binds CAXII should preferably contain an antibody with the idiotype monoclonal anti-clone 315602 linked via a detectable label. A targeted imaging probe that specifically binds mammaglobinA should contain an antibody with the idiotype monoclonal antibody Clone 304-1A5/clone 315602 linked to a detectable Label. In some embodiments, the targeted image probe includes monoclonal antibody, clone 315602 or clone 30411, clone 30411, or clone 31A5.

“The targeted imaging probe should be an antibody that is linked to a detectable marker. Based on the molecular imaging devices, it is possible to choose suitable detectable labels. Preferably, the detectable label for optical imaging is a near infrared (NIR), fluorophore. The preferred method involves, for instance, a first antibody with the idiotype monoclonal anti-body clone303123 linked to an NIR fluorophore, and a second antibody with the idiotype monoclonal anti-body clone315602 linked to another NIR fluorophore.

“Also included is a composition that contains a first antibody with the idiotype monoclonal anti-body clone303123 linked to an NIR fluorophore, and a second antibody with the idiotype monoclonal anti-body clone 315602 linked a second NIR fluorescent. A third monoclonal antibody can be added to the composition. It could contain either clone 31A5 or clone 306102. The composition can also contain monoclonal antibodies clone 305302, clone 315602 or clone 215600, or any combination thereof. For example, different targeted imaging probes can have different detectable labels. They can have different excitation frequencies and/or excitation frequencies. Different targeted imaging probes can be identified with different labels to allow them to be distinguished during imaging.

“A kit that includes two or more antibodies that bind to a specific cellular target from the group of CAIX and CAXII is also available. The kits contain at least one antibody linked to a first NIR fluorescent and at most one antibody linked to another NIR fluorescent. Preferably, light is not excited by the second NIR fluorescent dye when it passes through either the absorption spectrum or the emission spectrum of the first NIR fluorescent. Monoclonal antibody, clone 315602 or 303123 is preferred. Monoclonal antibody Clone 304-1A5 and clone 315602 can be added to the kit.

“I. Definitions”

“Targeted molecular imaging” is a term that refers to targeted molecular imaging. The in vivo detection and analysis of biological processes, such as biodistribution at the molecular and cellular levels, is called targeted molecular imaging. In vivo detection can be achieved using a targeted imaging probe, which binds to a molecular target or cell. An imaging device detects the probe in real time.

“The term “targeted imaging probe” is used. A molecule that binds specifically to a molecular target or cellular target in vivo and can be detected by in vivo imaging techniques. The binding molecule is linked to a detectable label. This allows for detection. The most preferred binding molecules are antibodies, peptides and peptidomimetics.

“Antibody” is a term that refers to a specific antibody. “Antibody” can be used to refer to any polyclonal or monoclonal, synthetic, recombinant or immunoglobulin molecule which specifically binds to a target antigen. This term covers intact immunoglobulin molecules as well as fragments or polymers from those immunoglobulins, chimeric antibodies that contain sequences from more then one species, class or subclass of immunoglobulin and humanized or humanized immunoglobulins molecules or fragments thereof, which at least have the idiotype of the immunoglobulin that binds to the target antigen.

“The term “idiotype” refers to the portion of an immunoglobulin molecule that confers its ability to bind an antigen. The immunoglobulin molecule whose ability to bind antigens is determined by its idiotype. The complementarity determining areas (CDRs), of the immunoglobulin variable domains (VL, and VH) determine the idiotype of an antigen.

“Peptide” is a term that refers to a natural or synthetic molecule. “Peptide” can refer to a natural or artificial molecule that contains two or more amino acid linked by the carboxyl groups of one amino acid to another. The length of a peptide does not matter; therefore,?peptide? Polypeptides can be combined with proteins.”

“The term ‘peptidomimetic? A mimetic of a protein that alters the normal peptide chemical. Peptidomimetics are often used to enhance the properties of the original peptide. These include increased stability, improved efficacy, enhanced delivery and increased half-life.

“The term ‘aptamer’ is a synonym for “oligonucleic acid molecules that specifically bind to a target molecule.” “Attamer” refers to oligonucleic acids molecules that bind specifically to a target molecule.

“Small molecule” is the term used herein. A compound with a molecular mass of less than 1000 Daltons and usually between 300 and 700 Daltons. This term can include monomers, primary metabolites, secondary metabolisms, biological amines, and synthetic or non-peptide biological molecules. Targeted imaging probes with small molecules can bind to the molecular target.

“Specifically Binds” is a term that refers to the binding of a molecule to a target molecule. The binding of a molecule or molecule to a target molecule (e.g., an antibody to its cognate Antigen) without significant binding to other molecules is called “specifically binds”. A molecule that?specifically binds is preferred. To a target molecule having an affinity constant (Ka?) greater than about 105?mol?1 (e.g. 106?mol?1,107?mol?1,108?mol?1,109?mol?1,109?mol?1,1010 mol.1, 1011?mol.1, and 1012?mol.1 or more) with that target molecule.

“The term “neoplasm” refers to a cell that is experiencing abnormal cell proliferation. A cell that is experiencing abnormal cell proliferation. Neoplastic cell growth is a form of abnormal cell proliferation that exceeds normal tissue growth. The excessive growth often persists even after the stimulation has been stopped and can lead to the formation of tumors. Neoplasms can be benign, malignant, or premalignant.

“Cancer” is a term that refers to cancer. or ?malignant neoplasm? Refers to cells that exhibit uncontrolled growth and invasion of adjacent tissues.

“Metastatic” is a term that refers to the phenomenon of metastatic. or ?metastasized? These are cancer cells that have spread from their original site (primary site), to another location (metastatic spot) within the body.

“The term “occult cancer” is used to describe a form of metastasized cancer cells. “Metastasized cancer cells from unknown origin” refers to them.

“The term’subject? or ?patient? Any individual who is the subject of administration. A subject could be a vertebrate or a mammal. The subject could also be a person. You can have the subject domesticated, farm, or keep them in a zoo or circus. Dogs, cats, rabbits and other domesticated animals include guinea-pigs, ferrets as well as ferrets, ferrets. For example, agricultural animals are horses, mules and donkeys, burros cattle, cows pigs, sheep, alligators, and pigs. For example, circus-maintained animals can include lions and tigers, bears as well as camels, giraffes and hippopotamuses. This term does not indicate a specific age or sex.

“Effective amount” is a term that refers to the quantity of targeted imaging probes sufficient for in vivo detection of the probes in an organ or tissue. “Effective amount” refers to the number of targeted imaging probes that are sufficient to detect probes in tissue or organs by in vivo imaging. The amount of targeted imaging probes required for in vivo detection of the probes in an organ or tissue will vary from one subject to the next. This depends on the individual subject’s age and general condition, as well as the type of administration and the specific probes being used. The appropriate amount is determined by a person of ordinary skill in the art using only routine experimentation. One of ordinary skill can determine the appropriate?effective amount by using only routine experimentation.”

“The term “detectable label” is used herein. “Detectable label” is a term that refers to any molecule which can be detected using in-vivo imaging techniques.

“The term “near-infrared” fluorophore (NIR) is used. A molecule with an emission and absorption wavelength in the NIR spectrum of 680 to 900 nm. The ‘biological window’ is a preferred wave range for in-vivo fluorescence imaging. These molecules are able to be detected further while minimizing fluorescence absorption by tissues.

“The terms “label-guided surgical” are used interchangeably. ?fluorescent-guided surgery,? Fluorescent-guided surgery, or the like, is surgery in which the location of relevant tissue/or cells has been marked with a label. This is intraoperative imaging. Label-guided surgery where the label is visualized via an image of tissue and/or cells can be referred to as image-guided-surgery.”

“II. Compositions”

“A. “A.

Targeted imaging probes are available to detect cancer cells. They can bind specific cellular targets on in vivo cancer cells. The cellular targets are proteins found on the surfaces of cancer cells. Imaging probes can access these targets and bind them in vivo. Targeted imaging probes disclosed preferably don’t bind normal tissue (non-cancerous). The targeted imaging probes may be used to detect metastasized cells of cancer or cells that are about to become metastasized from the primary tumor.

For detection of metastasized cancer cells, probes that specifically bind carbonic and anhydrase 9 or 12 (CAIX), mammaglobin A, C-XC motif chemokine 10, (CXCL10), carbonic anhydrase 9, (CAIX), and matrix metallopeptidase (9 (MMP-9) are available. Targeted imaging probes should preferably be able to bind the cell targets in areas that can be accessed from the circulation (e.g. blood or lymph) in vitro.

To detect cancer cells that express either one or both of these proteins, “targeted imaging probes” specifically binding CAIX/CAXII should be used in combination. These probes may also be combined with tissue-specific probes to increase specificity. To detect breast cancer cells that have metastasized, CAIX or CAXII probes should be used together with probes that specifically bind mammaglobin A.

“Probes that bind Toll like receptor 2 (TLR2) specifically in vivo have been disclosed to be used in the detection of cancer cells such as pancreatic, pancreatic, and metastasized pancreatic carcinoma cells.

“In some instances, the disclosed targeted imaging probes can be used with other targeting agents such as cancer-specific targeting probes. A targeting agent that binds tumor-associated glucoprotein-72 (TAG-72) can be used in conjunction with the disclosed targeted image probes. TAG-72, a glycoprotein that is found on many types of cancer cells including those in the breast, colon and pancreatic, is disclosed. TAG-72 is specifically bound to murine monoclonal antibody (CC49MAb, Minretumomab). It also reacts strongly with LS-174T breast cancer extract and a breast cancer extract.

Targeted imaging probes can generally contain a cellular targeting domain and a detectable tag. Routine methods can link the detectable label and cellular target binding area.

“In certain embodiments, the cellular target domain binding domain and detectable labels can be chemically crossedlinked using protein-crosslinking agents. Crosslinking agents, such as succinimidyl esters, are commonly found on commercially available labels such as fluorophores. These agents can be used to conjugate with proteins such as antibodies. Non-limiting examples of suitable protein crosslinkers include DSS (Disuccinimidylsuberate), DSP (Dithiobis(succinimidylpropionate)), DTSSP (3,3?-Dithiobis (sulfosuccinimidylpropionate)), SULFO BSOCOES (Bis[2-(sulfosuccinimdooxycarbonyloxy) ethyl]sulfone), BSOCOES (Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST (Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene glycolbis(sulfosuccinimidylsuccinate)), DPDPB (1,2-Di[3?-(2?-pyridyldithio) propionamido]butane), BSSS (Bis(sulfosuccinimdyl) suberate), SMPB (Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB (Sulfosuccinimdyl-4-(p-maleimidophenyl) butyrate), MBS (3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS (3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB (N-Succinimidyl(4-iodoacetyl) aminobenzoate), SULFO SIAB (N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC (Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), SULFO SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), NHS LC SPDP (Succinimidyl-6-[3-(2-pyridyldithio) propionamido) hexanoate), SULFO NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio) propionamido) hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio) propionate), NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate), NHS IODOACETATE (N-Hydroxysuccinimidyliodoacetate), MPBH (4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH (4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid hydrazide hydrochloride), MBH (m-Maleimidobenzoic acid hydrazidehydrochloride), SULFO EMCS (N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide), EMCS (N-(epsilon-Maleimidocaproyloxy) succinimide), PMPI (N-(p-Maleimidophenyl) isocyanate), KMUH (N-(kappa-Maleimidoundecanoic acid) hydrazide), LC SMCC (Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate)), SULFO GMBS (N-(gamma-Maleimidobutryloxy) sulfosuccinimide ester), SMPH (Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS (N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS (N-(gamma-Maleimidobutyrloxy) succinimide), DMP (Dimethylpimelimidate hydrochloride), DMS (Dimethylsuberimidate hydrochloride), MHBH(Wood’s Reagent) (Methyl-p-hydroxybenzimidate hydrochloride, 98%), and DMA (Dimethyladipimidate hydrochloride).”

“In other embodiments, a targeted imaging probe could be a fusion protein or protein containing a cellular target binding region and a detectable tag. Fusion proteins are proteins that combine two or more genes, coding regions or proteins that were originally coded for peptides. This fusion gene or code region is translated into a single peptide, or polypeptide, with the functional properties of each original peptide. Recombinant DNA technology can create artificially recombinant fusion proteins or peptides. This involves removing the stop codon in a cDNA sequence that codes for the first protein or peptide, and then adding the cDNA sequence to the second protein or peptide in frame by ligation or overlap extensionPCR. Alternately, the coding regions may be synthesized and joined or even fusion coding. A cell can then express the fusion DNA sequence as a single protein or peptide. You can either include all of the original peptides and proteins in the protein, or just a fraction. Linker (or?spacer?) is often used if the entities are proteins. You can also add peptides to increase the likelihood that the proteins will behave and fold as expected. Internal ribosome entrance sites (IRES), elements can also be used to create multigene or polycistronic messages. The ribosome scanning model of 5 is bypassed by IRES elements. methylated Cap dependent translation can be started at internal sites. Multiple open reading frames can all be transcribed together. Each one is separated by an IRES. This creates polycistronic messages. Each open reading frame can be accessed by ribosomes to facilitate efficient translation thanks to the IRES element. There are many IRES sequences that are well-known in the art. These include those from encephalomycarditisvirus (EMCV). (Ghattas I. R. et. al., Mol. Cell. Biol. 11:5848-5849 (1991); biP protein (Macejak and Sarnow Nature, 353 :91 (1991); Antennapedia gene (exons d and e), [Oh et al. Genes & Development], 6:1643-1653 (1992); Antennapedia gene in drosophilia [Oh et., Genes & Development], 6:1643-1653 (1992); those in poliovirus [Pelletier Nature, 334:3203251988]; see also Mountford and Smith TIG, TIG, 11 184 (1985). There are many other recombinant or fusion methods that can be used to produce the peptides and proteins.

“Also disclosed were compositions comprising a first antibody consisting of the idiotype monoclonal anti-mouse antibody clone303123 linked with a first near infrared (NIR), fluorophore, and a 2nd antibody consisting of the idiotype monoclonal anti-mouse clone 315602 linked with a 2nd NIR fluorophore.”

“The composition may also include a third antibody that is either monoclonal antibody cloned 304-1A5 (or clone 31A5 linked with a third NIR fluorescent). Monoclonal antibody clone 315602 can be used as the first antibody. Monoclonal antibody 315602 can be the second. The third antibody may consist essentially of monoclonal anti-clone 315602 or 304-1A5.

“1. Cellular Target Binding Domain”

“a. Antibodies”

“Preferred embodiments use targeted imaging probes that specifically bind cellular targets. For use in the disclosed compositions or methods, antibodies that bind CAIX and CAXII, mammaglobin A, CEACAM6, CXCL10 and MMP-9 have been disclosed.

“The anti-CAIX antibody should preferably specifically bind to human CAIX protein (Accession Number. NP_001207). The anti-CAIX antibody may specifically bind the N-terminus or extracellular domain of human CAIX in some embodiments. Particularly preferred embodiments of the anti-CAIX antibodies specifically bind amino acids 59-414 from human CAIX protein. The anti-CAIX antibody may be either the monoclonal antibody mAb clone303123 (R&D Systems) or the idiotype. It is possible to identify anti-CAIX antibodies that bind the same epitope with this clone.

“The anti-CAXII antibody should preferably specifically bind to human CAXII (Accession Number. NP_001209). The anti-CAXII antibody binds to the N-terminus of human CAXII and the extracellular domain in some embodiments. Particularly preferred embodiments of the anti-CAXII antibodies specifically bind to amino acids 25-291 in human CAXII proteins. The anti-CAXII antibodies can either be monoclonal antibodies (mAbs) clone 315602 (R&D Systems) or have the idiotype. It is possible to identify anti-CAXII antibodies that bind to the same epitope than this clone.

“The anti-Mammaglobin A antibody should preferably specifically bind human Mammaglobin A (Accession Number. NP_002402.1). For example, anti-Mammaglobin A antibody could be monoclonal antibody mAb clone 31A5 or 304-1A5 (Zeta Corp. California, Sierra Madre), or it can have the idiotype one of these clones. It is also possible to identify anti-Mammaglobin A antibodies that bind the same epitope with this clone.

“Antibodies can be used in the disclosed compositions or methods include whole immunoglobulins (i.e. an intact antibody), fragments thereof and synthetic proteins that contain at least the antigen binding variable domain of an antibody. Variable domains are different in their sequence and used to determine the specificity and binding of each antibody to a particular antigen. The variability is not always evenly distributed in the variable domains. It is usually concentrated in three segments known as complementarity determining areas (CDRs), or hypervariable zones in both the light and heavy chain variable domains. The framework (FR) is the name given to the most conserved parts of the variable domains. Each of the variable domains for native heavy and lightweight chains has four FR regions. They generally adopt a beta-sheet layout and are connected by three CDRs that form loops connecting to, or in some cases, making up part of, this beta-sheet structure. Each chain’s CDRs are held in close proximity by the respective FR regions. They, along with the CDRs of the other chain, help to form the antigen binding site for antibodies.

“Antibodies that are used in the disclosed compositions or methods can be any type, including IgG and IgA. IgG antibodies can further be subdivided into IgG1, IgG2, IgG3, or IgG4 subtypes. IgA antibodies can further be subdivided into IgA1 or IgA2 subtypes.

“Fragments of antibodies that have bioactivity are also disclosed. These fragments can be attached to any sequence or not. They include insertions and deletions as well as substitutions and other selected modifications to specific regions or amino acid residues. However, the activity of the fragment must not be significantly impaired or altered from the nonmodified antibody. A fragment of an antigen-binding antibody that contains a monovalent antibody-binding fragment is called Fab. A Fab fragment is made by digesting whole antibodies with papain. This will yield an intact light and part of one heavy chain. Fab? The fragment of an antibody molecule is obtained by treating the whole antibody with pepsin and then reducing to give an intact light chain. There are two Fab? Two Fab? fragments can be obtained for each antibody molecule. Fab? Fab? (Fab? (Fab???? )2 is the fragment from an antibody that can obtained by treating whole antibody using the enzyme pepsin, but without further reduction. F(ab? F(ab?2)2 is a dimer from two Fab. Two disulfide bonds hold the fragments together. Fv is the minimum amount of antibody fragments that contain a binding site and an antigen recognition site. This dimer consists of a dimer consisting of one heavy and one lighter chain variable domains in a tight, noncovalent association (VH?VL dimer). This configuration is where the CDRs from each variable domain work together to create an antigen-binding spot on the VH-VL dimer’s surface. The six CDRs give antigen-binding speciality to the antibody. Even though a single variable domain, or half of an Fv containing only three CDRs that are specific for antigens, can recognize and bind the antigen, it has a lower affinity than the whole binding site.

“Techniques are also available to produce single-chain antibodies that target specific cellular targets. Single chain antibody (?SCA) Single chain antibody (?SCA) is a genetically engineered molecule that contains the variable region (VL) and the variable (VH) regions of the heavy chains (VH), which are linked by a suitable protein linker to form a single chain molecule. These single-chain antibodies are also known as “single chain Fv?” These single-chain antibodies are also known as?single-chain Fv? Antibody fragments. The Fv polypeptide also contains a linker polypeptide between the VH-VL domains, which allows the sFv structure to be formed for antigen binding. The methods for producing single-chain antibodies are well-known to those skilled in the art. One chain antibodies can be made by joining the variable domains of heavy and light chains with a short peptide linking agent. This allows for reconstituting the antigen binding site on one molecule. Single-chain antigen variable fragments (scFvs), in which the Cterminus of one variabledomain is linked to the N-terminus the other variabledomain via a 15-25 amino acid peptide, peptide linker, have been created without significantly disrupting the antigen binding or specificity. The linker allows the heavy and light chains to bind in their correct conformational orientation.

By linking two scFvs, it is possible to engineer divalent single-chain variable fractions (diScFvs). You can achieve tandem scFvs by creating a single peptide-chain with two VH regions and two VL areas. ScFvs can also have linker peptides designed that are too short to allow the two variable regions (about five amino acid) to fold together. This causes scFvs not to dimerize. This is called diabodies. The dissociation constants of diabodies have been found to be up to 40 times lower than the corresponding scFvs. This means that they are more closely related to their target. Even shorter linkers, one or two amino acids, can lead to the formation trimers (triabodies and tribodies). Also, tetrabodies can be made. They have a higher affinity for their targets than diabodies.

“Preferably, if an antibody is to be administered in humans, it is either a human antibody, or a?humanized? antibody. an antibody that is derived from non-human animals. Humanizing non-human antibodies is a well-known art. They have been described in U.S. Pat. Nos. Nos.

“b. Peptides”

“In some cases, the targeted imaging probe may contain a peptide which binds to the cellular target CAIX or CAXII, mammaglobin A, CEACAM6, CXCL10 or MMP-9. Some embodiments contain the idiotype or antibody of an antibody. Another embodiment allows the identification of the peptide by screening a list of peptides that are compatible with the cellular target.

“c. Peptidomimetics”

“In certain embodiments, the targeted image probe may contain a peptidometic that binds CAIX or CAXII. A peptidometic is a small, protein-like chain that mimics a peptide. These systems are usually created by modification of an existing protein or by creating similar systems to mimic peptides such as peptoids or 0-peptides. The altered chemical structure can be used to improve molecular properties, such as stability and biological activity. This could be used to create drug-like compounds using existing peptides. Modifications are changes that occur in a peptide that cannot be found naturally, such as altering backbones or the incorporation non-natural amino acids.

U.S. Pat. 102/399 describes “Methods for making peptidomimetics that are based on a known sequence of polypeptides.” Nos. Nos. 5,631,280, 5,612,895 and 5,579.250. Non-amino acids can be found in peptididomimetics. They may have non-amide links at certain positions. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include ?-alanine, L-?-amino butyric acid, L-?-amino butyric acid, L-?-amino isobutyric acid, L-?-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N-?-Boc-N-?-CBZ-L-lysine, N-?-Boc-N-?-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-?-Boc-N-?CBZ-L-ornithine, N-?-Boc-N-?-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.”

“d. Aptamers”

“In certain embodiments, the targeted image probe may contain an aptamer that binds CAIX or CAXII. Aptamers can be single-stranded DNA or DNA oligonucleotides between 15 and 60 bases in length, which bind with high affinity for specific molecular targets. Most aptamers to proteins are compatible with monoclonal antibodies. They bind to Kds (equilibrium constant), which is in the 1 pM- 1 nM range. These nucleic acids ligands can be bound to proteins, nucleic acids, and small organic compounds. They can even bind to entire organisms.

You can select aptamers by incubating your target molecule in large pools of oligonucleotide (usually 40-60mers). Because of its large pool, the oligonucleotide is able to isolate the specific aptamer. Aptamers are able to distinguish between closely related, but not identical members of a protein families or between different functional and conformational states. The systematic evolution of ligands through exponential enrichment (SELEX), is a protocol that allows for modification and variation to select aptamers. This process can be used to create new aptamers within two weeks.

“e. Sulfonamide-Based Inhibitors”

“In certain embodiments, the targeted image probe may contain a carbonic acidase inhibitor that binds CAIX or CAXII. Carbonic anhydrase inhibits are pharmaceuticals that inhibit the activity of carbonic acidase by binding directly to its catalytic sites. A sulfonamide is a common component of suitable carbonic anhydrase inhibiters. Acetazolamide and Brinzolamide are all examples of carbonic-anhydrase inhibitors.

“f. Synthetic Analogues and Natural Ligands”

“In certain embodiments, the targeted image probe may contain a natural binding agent of the cellular targets for cancer cells or a fragment thereof.”

“Tell-like receptor 2 or TLR2 (for example) recognizes cell-wall components like peptidoglycan and lipoteichoic acids from gram-positive bacteria, lipoarabinomannan, and lipoproteins from mycobacteria and zymosan, which is found in the yeast cell wall. In some embodiments, the natural binding agent is a component of the cell wall of a microorganism such as yeast or bacteria.

“In other embodiments, the targeted image probe contains a synthetic analog of a natural drug ligand. Synthetic diacylated lipoproteins that correspond to the N-terminal partial structures bacterial lipoproteins have been also developed. They bind TLR2. In some embodiments, the targeted imaging probe contains the TLR2 ligand dipalmitoyl-S-glyceryl-L-Cys-Ser-(Lys)4 (Pam2CSK4), which has the following structure:”

“”

“In some embodiments, the targeted imaging probe contains the TLR2 ligand tripalmitoyl-S-glyceryl-L-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4), which has the following structure:”

“”

“In some embodiments, the targeted imaging probe contains the lipolanthionine peptide (2R,6R)-Pam2LanHda-Ser-(Lys)4-NH2 (lipolan).”

“In certain embodiments, the targeted image probe contains MALP-2 (a diacylated lipopeptide that was isolated from Mycoplasma fermentationans).

“In some embodiments, the targeted imaging probe contains a cellular target binding domain having the formula:\nX-Dhp(Pam2)-peptide MALP2,\nwhere ?X? There is an addition of: Palmitoyl and Fluorescein; Dhc=1,2-dihydroxypropylcysteine; Aha=epsilon-aminohexanoic acid; Aun=epsilon-aminoundecanoic acid; Ac-PEGO20 (Pam2)-peptide MALP2,nwhere?X? Ac=acetyl; Dhc=1,2-dihydroxypropylcysteine; Aha=epsilon-aminohexanoic acid; Aun=epsilon-aminoundecanoic acid; PEGO20=20 atoms long polyethelene glycol (4 Peg units); Pam2=dipalmitoyl-S-glyceryl; Pam3=tripalmitoyl-S-glyceryl.”

“In certain embodiments, the targeted image probe can have the structure.”

“”

“Where R1 is?CO.R6 or H, and R6 is C12-18 alkyl or alkenyl;

“Where R2 is??CH2?O??CO?R7 or?H?, wherein C12 to C18 alkyls, alkenyls, amines, alkyl-amines, alkylether, polyether and R7 are any C12 or C18 alkyls, alkenyls, amines, alkylamines, alkyls amines, alkyl ketol amines, alkylether, polyether or alkylamine;

“where R3 is R8-label, wherein R8 is acetyl-PEGO-(Ac-PEGO), palmitoyl-, fluorescein-, acetyl-6-aminohexanoyl-(Ac-Aha), adapalenoyl-, acetyl-11-aminoundecanoyl-(Ac-Aun), or tretinoyl-;”

“where R4 is -Gly-DSer-PEGO-NH2, -Gly-DSer-NH2, -Cys-Ser-(Lys)4-NH2, -Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys-NH2, -Ser-Arg-Phe-Asp-Glu-Asp-Asp-Leu-Glu-NH2, -Gly-Ser-Gln-Asn-Leu-Ala-Ser-Leu-Glu-Glu-NH2, or -serine methyl ester; and”

“where R5?S? “where R5 is?S??” or?Se?

“In some embodiments R1 can be H. R2 could then be?CH2O?CO?R7. R7 could be any C12-C18 alkyl or an alkenyl.

“In some embodiments, R1 can be ?CO?R6, wherein R6 is C12 to C18 alkyl; R2 can be ?CH2?O?CO?R7, wherein R7 is any C12 to C18 alkyl; R3 can be R8-label, wherein R8 is acetyl-PEGO-(Ac-PEGO), palmitoyl-, fluorescein-, acetyl-6-aminohexanoyl- (Ac-Aha), adapalenoyl-, acetyl-11-aminoundecanoyl- (Ac-Aun), or tretinoyl-; R4 can be -Gly-DSer-PEGO-NH2, -Gly-DSer-NH2, -Cys-Ser-(Lys)4-NH2, -Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys-NH2, -Ser-Arg-Phe-Asp-Glu-Asp-Asp-Leu-Glu-NH2, -Gly-Ser-Gln-Asn-Leu-Ala-Ser-Leu-Glu-Glu-NH2, or -serine methyl ester; and R5 can be ?S?.”

“2. Detectable Labels”

“The disclosed targeted imaging probes should be linked to a detectable marker. Fluorescent molecules, also known as fluorescent molecules, are suitable for labeling imaging agents. Fluorophores and fluororochromes, chemiluminescent agents (e.g. luminol), bioluminescent and chemiluminescent substances (e.g. luciferin, green fluorescent protein (GFP), and metals (e.g. gold nanoparticles) are all suitable for detectably labeling imaging agents. Based on the imaging method chosen, suitable detectable labels may be selected. Preferable embodiments include a near-infrared fluorescent dye, a Gadolinium chelate, SPECT or PET imaging radionuclides, and a gold nanoparticle or CT imaging.

“a. Fluorophores”

“b. Radioisotopes”

A radioisotope can either be integrated into or attached to a targeted imaging agent. Tritium, 11C and 13N are some examples of radioisotopes that can be useful. Some embodiments attach the radioisotope to the targeted imaging agent through halogenation. Some embodiments attach the radioisotope to the targeted image agent using a linking group or bound with a chelating agent. This is either attached directly to the agent or via a linker.

“3. Adjuvants”

“The TLR2 agonists described can be used as adjuvants in order to increase stimulation of an immune reaction, such as in a vaccine.”

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