Patent Application
20170119910
The present invention relates generally to the field of cancer diagnostics and therapeutics, including an enzymatically responsive product and its use for sorting cancerous and noncancerous cells, diagnosing cancerous conditions, and treating cancerous conditions.
Cell sorting, the isolation of certain types of cells from mixed cell population of organs or tissues, has become an increasingly important sampling method that has already contributed to many advances in biology and medicine (Claros et al., Eur. J. Biochem. 241:779 (1996); Panyam et al., Adv. Drug Deliver. Rev. 55:329 (2003); Cormack et al., Gene 173:33 (1996)). While the capture of bacteria is relatively easy by the magnetic nanoparticles decorated by a readily accessible ligand (e.g., vancomycin) (Xing et al., J. Am. Chem. Soc. 124:14846 (2002); Xing et al., Chem. Commun. 2224 (2003); Liu et al., Nat. Commun. 2 (2011)), the sorting of mammalian cells requires more complicated and expensive instruments and reagents. Fluorescent activated cell sorting (FACS) (Julius et al., Proc. Natl. Acad. Sci. U.S.A 69:1934 (1972); Kreth et al., Cell Immunol. 12:396 (1974)), the most widely-used cell sorting method, uses expensive hardware and requires labeling the cells of interest by fluorescent antibodies or cellular proteins (Cormack et al., Gene 173:33 (1996); Orlic et al., Nature 410:701 (2001)). The current magnetic cell sorting still requires cell specific antibodies to be conjugated to the magnetic beads, which is a less well-defined process due to the non-specific binding of proteins to the beads. Moreover, the high cost of FACS and cell specific antibodies is rather prohibitive for the applications of cell sorting for disease diagnosis in developing regions, thus, there is an unmet need to apply chemistry for the development of inexpensive cell sorting method that will contribute to low-cost diagnostics for the developing world (Martinez et al., Anal. Chem. 82:3 (2010); Martinez et al., Lab Chip 8:2146 (2008); Sia et al., Angew. Chem. Int. Edit. 43:498 (2004); Kuo et al., Lab Chip 11:2656 (2011); Kuo et al., Electrophoresis 25:3796 (2004)).
The present invention overcomes these and other deficiencies in the art.
A first aspect of the invention relates to an enzymatically responsive product that includes an amino acid residue conjugated to a magnetic particle, wherein the amino acid residue is phosphorylated or sulfated or comprises an ester-moiety linked via peptide bond.
A second aspect of the invention relates to a composition that includes an aqueous carrier and a product according to the first aspect of the invention.
A third aspect of the invention relates to a method of separating distinct types of cells that includes exposing a product according to the first aspect of the invention or a composition according to the second aspect of the invention to a mixed population of cells including a first cell type that expresses an ectoenzyme that hydrolyzes the phosphate group, the sulfate group, or the ester-moiety of the product, and a second cell type that lacks an ectoenzyme that hydrolyzes the phosphate group, the sulfate group, or the ester-moiety of the product, whereby the product labels the first cell type after said exposing; and separating the labeled first cell type from the second cell type. In certain embodiments, the cells are mammalian cells, particularly cancerous and non-cancerous cells.
A fourth aspect of the invention relates to a method for treating a cancerous condition that includes administering to a subject having a cancerous condition a therapeutically effective amount of the product according to the first aspect of the invention or a composition according to the second aspect of the invention, wherein said administering is effective to cause selective adherence of the dephosphorylated, desulfated, or de-esterified product to cancer cells, whereby said selective adherence affects growth or survival of cancer cells.
In one embodiment, the selective adherence of the dephosphorylated, desulfated, or de-esterified product to cancer cells is sufficient, without more, to affect growth or survival of those cancer cells.
In another embodiment, the magnetic particle is used to implement hyperthermic treatment of the cancer cells to which the dephosphorylated, desulfated, or de-esterified product is adhered. Because the dephosphorylated, desulfated, or de-esterified product selectively adheres to cancer cells that express or secrete an ectoenzyme that hydrolyzes the phosphate group, sulfate group, or ester moiety, exposing a tumor-containing region of the subject's body to an energy source (e.g., ultrasound, laser light, near infrared light, or alternating magnetic field) suitable to cause thermal heating of the magnetic particles adhered to the cancer cells and, thus, destruction of the cancer cells.
A fifth aspect of the present invention relates to a method for imaging cancer cells. This method includes administering to a subject having a cancerous condition a product according to the first aspect of the invention or a composition according to the second aspect of the invention, wherein the administering is effective to cause the product to contact cancer cells that express a cell surface-bound phosphatase or sulfatase, secrete a phosphatase or sulfatase, or both, and cause selective adherence of the dephosphorylated, desulfated, or de-esterified product to cancer cells; and generating an image of a part of said subject where cancer cells labeled by the dephosphorylated, desulfated, or de-esterified product reside, whereby enhanced contrast between normal tissue and cancerous tissue is provided by the dephosphorylated, desulfated, or de-esterified product.
The accompanying examples demonstrate that an underexplored generic difference between cancer and normal cells—verexpression of ectoenzymes having hydrolase activity, such as an ectophosphatase—triggers the D-tyrosine phosphate decorated magnetic nanoparticles (Fe3O4-p(D-Tyr)) to adhere selectively on cancer cells upon catalytic dephosphorylation. This enables magnetic separation of cancer cells from a mixed population of cells (e.g., co-cultured cancer cell and stromal cells), as well as other uses that involve selective labeling of cancer cells. Moreover, the Fe3O4-p(D-Tyr) nanoparticles also selectively inhibit cancer cells in the co-culture. As a general method to broadly target cancer cells without highly specific ligand-receptor interactions (e.g., antibodies), the use of the enzymatic reaction to spatiotemporally modulate the state of various nanostructures in cellular environment affords the theranostic applications of these nanomaterials. In addition to inhibiting survival of cancer cells decorated by the nanoparticles, these nanoparticles will also facilitate the hyperthermic killing of selectively labeled cancer cells in vivo by promoting localized heating of the tumor site. Finally, the enzymatically responsive nanoparticles afford a new type of contrast agent that can be used in imaging of tumors.
One aspect of the present invention relates to an enzymatically responsive product that includes an amino acid residue conjugated to a magnetic particle, wherein the amino acid residue is phosphorylated or sulfated or comprises an ester-moiety linked via peptide bond. Upon enzymatic cleavage of the phosphate, sulfate, or ester group from the amino acid by, e.g., an ectoenzyme expressed by or secreted by certain cells, the so-modified product is rendered capable of adsorption to the cell that expresses/secretes the ectoenzyme.
As used herein, “magnetic particle” refers to a microparticle or nanoparticle that exhibits magnetic behavior under one or more conditions. The magnetic particle can be ferromagnetic, paramagnetic, or superparamagnetic. Paramagnetic and superparamagnetic particles can be magnetized when exposed to an external magnetic field, but in the absence of the external magnetic field these particles do not retain their magnetic properties. Paramagnetic materials have a small, positive susceptibility to magnetic fields. The magnetic susceptibility of superparamagnetic particles is much larger than that of paramagnetic particles.
Any of a variety of magnetic particles can be employed in the present invention. In certain embodiments, the magnetic particles can be single material such as iron oxide (e.g., maghemite, γFe2O3, or magnetite, Fe3O4) or a mixture of a polymer and iron oxide. In alternative embodiments, the magnetic particle is in the form of a core/shell particle. Exemplary core materials include, without limitation, cobalt, iron or iron oxide, a cobalt/iron alloy, a nonmagnetic polymer, or a mixture of such metals or alloys with a polymer. Exemplary shell materials include, without limitation, graphene, an oxide, gold, silver, platinum, a polymer, or a mixture of a polymer and iron oxide.
A number of magnetic particles are commercially available including, without limitation, iron core/graphene shell particles available under the tradename TurboBeads® (Turbobeads LLC, Zurich, Switzerland), iron oxide/polymer particles available under the tradename Dynabeads® (Life Technologies, Eugene, Oreg.), iron oxide/polymer latex particles available under the tradename Estapor® (Merck Millipore, Billerica, Mass.), and magnetite/polystyrene shell on polystyrene core particles available under the tradename SPHERO™ Magnetic Particles (Spherotech, Lake Forest, Ill.).
In one embodiment, the product includes a single amino acid that is conjugated directly to the magnetic particle, and the amino acid is phosphorylated, sulfated, or linked by peptide bond to an ester moiety.
In another embodiment, the product includes a polypeptide containing from 2 to about 35 amino acids, and at least one of the amino acids is phosphorylated, sulfated, or linked by peptide bond to an ester moiety. In certain embodiments, two or more of the amino acids are phosphorylated or sulfated, or one or more amino acids is phosphorylated or sulfated and another amino acid is linked by peptide bond to an ester moiety. The phosphorylated, sulfated, or ester-linked amino acid residue can be conjugated directly to the magnetic particle or it can be linked via other amino acid residues in the polypeptide, one of which is conjugated directly to the magnetic nanoparticle.
The polypeptide can contain up to about 35 amino acids, up to about 30 amino acids, up to about 25 amino acids, up to about 20 amino acids, up to about 15 amino acids, up to about 10 amino acids, or between 2 to 10 amino acids.
In certain embodiments, the polypeptide can include anti-angiogenesis peptides.
In certain embodiments, the polypeptide can contain an amino acid sequence that includes a metalloprotease (MMP) cleavage site. Exemplary polypeptide sequences with an MMP cleavage site are shown in the table below.
In certain embodiments, the polypeptide includes one or more aromatic amino acids. In alternative embodiments, the polypeptide contains only one aromatic amino acid. Exemplary aromatic amino acids include, without limitation, phenylalanine, phenylalanine derivatives, tyrosine, tyrosine derivatives, tryptophan, tryptophan derivatives, 1-naphthylalanine, and 2-naphthylalanine. In certain embodiments, not more than one aromatic amino acid is included or not more than two aromatic amino acids are included. In these embodiments, the polypeptide is generally not capable of acting as a hydrogelator, and self-assembling into nanostructures. Any known or hereinafter developed phenylalanine derivatives, naphthylalanine derivatives, tyrosine derivatives, or tryptophan derivatives can be used in the present invention. Exemplary derivatives of these amino acids include the addition of one or more ring substituents.
Other peptide variants include those described at Formula I in PCT Publication Nos. WO 2012/166705 and WO 2012/166706, both to Xu et al., which are hereby incorporated by reference in their entirety, where the nucleobase at the N-terminus of Formula I is instead replaced by the magnetic particle. Further peptide variants include those described at Formulae I and II in PCT Publication No. WO 2014/074789 to Xu et al., which is hereby incorporated by reference in its entirety, where the magnetic particle replaces the N-terminal group described therein or is instead conjugated to a Lys sidechain.
In certain embodiments, a phosphorylated amino acid residue is present alone (i.e., as a single amino acid conjugated to the magnetic particle) or present in the polypeptide. In larger polypeptides, more than one amino acid can be phosphorylated. The phosphorylated amino acid residue prevents selective adsorption of the magnetic particle to a cancer cell in the absence of dephosphorylation. Exemplary amino acids residues that are readily phosphorylated and catalytically dephosphorylated by an enzyme possessing hydrolase activity include, without limitation, serine, threonine, tyrosine, and histidine.
In certain embodiments, a sulfated amino acid is present alone (i.e., as a single amino acid conjugated to the magnetic particle) or in the polypeptide. The sulfated amino acid residue prevents selective adsorption of the magnetic particle to a cancer cell in the absence of desulfation. Exemplary amino acids residues that are readily sulfated and catalytically desulfated by an enzyme possessing hydrolytic activity include, without limitation, serine, threonine, tyrosine, and hydroxyproline.
In certain embodiments, a single amino acid is present alone (i.e., as a single amino acid conjugated to the magnetic particle) with an ester-moiety linked via peptide bond, or an amino acid in the polypeptide possesses an ester-moiety linked via peptide bond. The amino acid residue containing the ester-linked moiety prevents selective adsorption of the magnetic particle to a cancer cell in the absence of de-esterification. The amino acid residue to which an ester-moiety is linked via peptide bond can be any amino acid, whether or not the amino acid contains an aromatic side chain. In these embodiments, the ester-moiety can be any ester-containing compound that also possesses a primary amino group that can react with the C-terminal carboxylic acid to form a peptide bond. For example, 4-(2-aminoethyl)-4-oxobutanoic acid; 5-aminovaleric acid; 4-[(8-aminooctyl)amino]-4-oxobutanoic acid; 4-[(5-amino-1-oxopentyl)amino]butanoic acid; and 4-[(5-aminopentyl)amino]-4-oxobutanoic acid are suitable ester-containing moieties.
In certain embodiments, the amino acid residue can be catalyzed by all the ectoenzymes (e.g., ectohydrolase, ectophosphatase, ectosulfatase, and ectocarboxylesterase).
Although numerous oligopeptides are known to form supermolcular hydrogels, those containing multiple aromatic groups facilitate aromatic-aromatic interactions that likely stabilize the intermolecular hydrogen bonding in water to afford the hydrogels (Du et al., Chem. Asian J. 9(6):1446-1472 (2014), which is hereby incorporated by reference in its entirety). Examples include, without limitation, the conjugation of aromatic moieties (e.g., phenyl, naphthyl, fluorenyl, pyrenyl, cinnamoyl) via simple amide bond to both aromatic amino acids (e.g., phenylalanine, naphthylalanine, tyrosine, tryptophan) and non-aromatic amino acids residues. Thus, in certain embodiments, the peptide includes one or more amino acids and conjugates, or two or more amino acids, which may promote hydrogelation of the peptides.
In certain embodiments of the present invention, the peptide includes two or more amino acids that are incapable of hydrogelation, and the peptide is incapable of hydrogelation.
The peptides can include all D-amino acids, all L-amino acids, or a mixture of L-amino acids and D-amino acids. In preferred embodiments, the peptide includes only D-amino acids or a mixture of D-amino acids and L-amino acids where the D-amino acid content is greater than 50%, 60%, 70%, 80%, 90%, or 95%. Similarly, where only one amino acid is used, the amino acid can be a D-amino acid or an L-amino acid.
As a consequence of utilizing entirely D-amino acids or a high proportion of D-amino acids, it is possible to render the peptide protease resistant, e.g., resistant to proteinase K digestion.
In certain embodiments, the peptide can include one or more amino acids whose side-chain is easily conjugated to the magnetic particle.
In certain embodiments, the peptide can include one or more amino acids whose side-chain is easily conjugated to, e.g., a fluorophore, a chemotherapeutic agent, an antiangiogenic agent, an immunomodulating agent, or an antigen. Numerous examples of each of these categories are well known in the art.
Exemplary amino acids that can be derivatized include lysine or arginine, whose terminal amino group of its side chain is reactive in conjugation procedures of the type described in the accompanying examples. Other conjugation protocols can be utilized with other amino acids, including aspartic and glutamic acid whose carboxylic acid groups are reactive in known conjugation procedures. Similarly, cysteine and cysteine derivatives can be used to form disulfide bonds during conjugation procedures. Allyl glycine can also be used in this regard.
In general, amino groups present in lysine side chains, as well as the N-terminal amino group, can be reacted with reagents possessing amine-reactive functional groups using known reaction schemes. Exemplary amine-reactive functional groups include, without limitation, activated esters, isothiocyanates, and carboxylic acids. Reagents to be conjugated include those listed above.
In general, guanidine groups present in arginine can be reacted with reagents possessing guanidine-reactive groups using known reaction schemes. Exemplary guanidine-reactive functional groups include, without limitation, NHS esters using gas phase synthesis (McGee et al., J. Am. Chem. Soc., 134 (28):11412-11414 (2012), which is hereby incorporated by reference in its entirety).
In general, thiol groups present in cysteine (or cysteine derivative) side chains can be reacted with reagents possessing thiol-reactive functional groups using known reaction schemes. Exemplary thiol-reactive functional groups include, without limitation, iodoacetamides, maleimides, and alkyl halides. Reagents to be conjugated include those listed above.
In general, carboxyl groups present in glutamic or aspartic acid side chains, or at the C-terminal amino acid residue, can be reacted with reagents possessing carboxyl-reactive functional groups using known reaction schemes. Exemplary carboxyl -reactive functional groups include, without limitation, amino groups, amines, bifunctional amino linkers. Reagents to be conjugated include those listed above.
In each of the types of modifications described above, it should be appreciated that the conjugate can be directly linked via the functional groups of the peptide and the reagent to be conjugated, or via a bifunctional linker that reacts with both the peptide functional groups and the functional groups on the reagent to be conjugated.
The peptides of the present invention can be synthesized using standard peptide synthesis operations. These include both FMOC (9-Fluorenylmethyloxy-carbonyl) and tBoc (tert-Butyl oxy carbonyl) synthesis protocols that can be carried out on automated solid phase peptide synthesis instruments including, without limitation, the Applied Biosystems 431A, 433A synthesizers and Peptide Technologies Symphony or large scale Sonata or CEM Liberty automated solid phase peptide synthesizers. This can be followed with standard HPLC purification to achieve a purified peptide product.
Regardless of the embodiment, conjugation of the magnetic particles to the amino acid or polypeptide is carried out using carboxylic acid functionalized magnetic nanoparticles, which are activated using N-hydroxysuccinimide to form a peptide bond between the carboxylic acid on the magnetic particle and the amino group on the single amino acid residue or the N-terminal amino group of the polypeptide. This is illustrated schematically below, where the amino acid is shown in phosphorylated form (as opposed to sulfated or ester-linked):
Similarly, a polypeptide containing a protected N-terminal group and bearing a reactive lysine sidechain can be coupled to the carboxylic acid functionalized magnetic nanoparticles, which are activated using N-hydroxysuccinimide to form a peptide bond between the carboxylic acid on the magnetic particle and reactive amino group of the lysine sidechain. This is illustrated schematically below, where a dipeptide containing phospho-tyrosine at the N-terminus of the polypeptide is shown:
Deprotection of the illustrated dipeptide will afford a pTyr-Lys-MNP. This same reaction can be used, for example, with the dipeptide Lys-pTyr to afford MNP-Lys-pTyr with the MNP coupled to the sidechain rather than the N-terminus of the dipeptide. In addition, pTyr can be placed at any location of the peptides, which means that MNP-pTyr-Lys can be synthesized by using similar reactions. pTyr can also be replaced by pSer and pThr to generate MNP-Lys-pSer or MNP-Lys-pThr, respectively.
In certain embodiments, the magnetic particle includes an outer surface that is a polymer functionalized with one or more carboxylic acid groups, which are activated using N-hydroxysuccinimide to form a peptide bond between the carboxylic acid on the particle and the amino group on the single amino acid residue or the N-terminal amino group of the polypeptide. This is illustrated schematically below, where the amino acid is shown in phosphorylated form (as opposed to sulfated or ester-linked):
A further aspect of the present invention relates to pharmaceutical compositions that include a pharmaceutically acceptable carrier and an enzymatically responsive product of the present invention.
In certain embodiments, more than one peptide or amino acid can be provided per magnetic particle. For example, the magnetic particles can be labeled with from about 10 up to about 500 amino acids or peptides per particle, or about 50 up to about 400 amino acids or peptides per particle, or about 80 up to about 300 amino acids per particle. As demonstrated in the accompanying Examples, an average of about 124 phospho-D-Tyr were present on the magnetic particles used in those examples. In these embodiments, it is contemplated that multiple peptides or amino acids can be conjugated in a single step reaction. The different peptides can be similar in structure, but possess different conjugated agents as described above. In alternative embodiments, the peptides can be structurally distinct, including different primary amino acid sequences, and different conjugates.
In certain embodiments, the carrier is an aqueous medium that is well tolerated for administration to an individual, typically a sterile isotonic aqueous buffer. Exemplary aqueous media include, without limitation, normal saline (about 0.9% NaCl), phosphate buffered saline (PBS), sterile water/distilled autoclaved water (DAW), as well as cell growth medium (e.g., MEM, with or without serum), aqueous solutions of dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), and/or dextran (less than 6% per by weight.)
To improve patient tolerance to administration, the pharmaceutical composition preferably has a pH of about 6 to about 8, preferably about 6.5 to about 7.4. Typically, sodium hydroxide and hydrochloric acid are added as necessary to adjust the pH.
The pharmaceutical composition suitably includes a weak acid or salt as a buffering agent to maintain pH. Citric acid has the ability to chelate divalent cations and can thus also prevent oxidation, thereby serving two functions as both a buffering agent and an antioxidant stabilizing agent. Citric acid is typically used in the form of a sodium salt, typically 10-500 mM. Other weak acids or their salts can also be used.
The composition may also include solubilizing agents, preservatives, stabilizers, emulsifiers, and the like. A local anesthetic (e.g., lidocaine) may also be included in the compositions, particularly for injectable forms, to ease pain at the site of the injection.
Effective amounts of the enzymatically responsive product will depend on the nature of use, including whether it is used for in vitro sorting of cancerous and noncancerous cells, or the nature of the cancerous condition which is being treated or imaged, as well as tumor volume, stage, and location(s). By way of example only, suitable concentrations may range from about 1μM to about 500 mM, preferably about 10 μM to about 300 mM, about 50 μM to about 200 mM, or about 100 μM to about 100 mM. The volume of the composition administered, and thus, dosage of the peptide administered can be adjusted by one of skill in the art to achieve optimized results. This can be adjusted lower to identify the minimal effective dose, or tailored higher or lower according to the nature of the tumor to be treated or imaged.
According to one aspect the enzymatically responsive product can be used for separating distinct types of cells, i.e., those expressing or secreting an ectoenzyme from those that do not. This method includes exposing the enzymatically responsive product (or a composition containing the same) to a mixed population of cells including a first cell type that expresses an ectoenzyme that hydrolyzes the phosphate group, the sulfate group, or the ester-moiety of the enzymatically responsive product, and a second cell type that lacks an ectoenzyme that hydrolyzes the phosphate group, the sulfate group, or the ester-moiety of the enzymatically responsive product, whereby the product, after enzymatic cleavage of the phosphate, sulfate, or ester, selectively labels the first cell type after said exposing. Once selective labeling of the first cell type has occurred, it is then possible to separate the labeled first cell type from the second cell type. As noted above, in one embodiment the first cell type that expresses or secretes an ectoenzyme is a cancer cell and the second cell type is a non-cancerous cell.
Often, the mixed population of cells is obtained in the form of a biopsy of tissue suspected to contain cancerous tissue. The recovery and preparation of biopsy samples is well known in the art. Once the biopsy sample is obtained, the mixed cell population can be suspended in a suitable medium (e.g., buffer or growth medium) for subsequent exposure to the enzymatically responsive product or composition of the present invention.
The separation step involves introducing the exposed, mixed cell population to a magnetic field, wherein the labeled first cell type is retained within the magnetic field and the second cell type is not retained within the magnetic field, thereby separating the first and second cell types. This can be accomplished using a flow-through column, whereby the magnetic field retains the first cell type in the column and allows the second cell type to pass through the column for collection. Alternatively, a container containing aqueous medium can be exposed to the magnetic field and the first cell type collected in a region of the container, e.g., the bottom of the container, by manipulating the container within the magnetic field.
Thereafter, it is possible to remove from the container a supernatant containing the second cell type and then recover the second cell type from the supernatant. In certain embodiments, the supernatant is substantially free of or excludes the first cell type. If it is not, then this process can be repeated more than once.
Recovery of the second cell type may include, e.g., centrifuging the supernatant and obtaining a cell pellet that includes the second cell type. In certain embodiments, the cell pellet is substantially free of or excludes the first cell type. If it is not, then this process can be repeated more than once. Once the second cell type of the supernatant is recovered, it can be introduced into a growth medium and the second cell type grown for a period of time.
The retentate in the container is then collected and optionally re-processed to ensure that the second cell type is not present in the population of cells. In certain embodiments, the retentate is substantially free of or excludes the second cell type. Once the first cell type of the retentate is recovered, it can be introduced into a growth medium and the first cell type grown for a period of time.
Assessment of the cancerous state of the first cell type can be carried out using otherwise conventional procedures, including genotyping and cell surface marker analyses.
Various types of cancer cells express or secrete ectoenzymes of the types described above, which are suitable to activate the products of the present invention.
Other cell types that express or secrete ectoenzymes of the types described above are mammalian progenitor cells, virus-infected cells, bacterial pathogen, protozoa, and fungi. Some of the bacterial pathogens expressing an ectoenzyme are described in PCT Publication No. WO 02/10442 to Zyskind, which is hereby incorporated by reference in its entirety. Ectophosphatase activities have been reported in several microorganisms (Freitas-Mesquita et al., Int. J. Mol. Sci. 15:2289-2304 (2014), which is hereby incorporated by reference in its entirety), including protozoa such as Leishmania (Remaley et al., Exp. Parasitol. 60:331-341 (1985); De Almeida-Amaral et al., Exp. Parasitol. 114:334-340 (2006), which are hereby incorporated by reference in their entirety), Trypanosoma (Fernandes et al., Z. Naturforschung 52C:351-358 (1997); Meyer-Fernandes et al., Z. Naturforschung 54:977-984 (1999); Dos-Santos et al., Int. J. Parasitol. 42:819-827 (2012), which are hereby incorporated by reference in their entirety), and bacteria, such as Mycobacterium bovis (Braibant et al., FEMS Microbiol. Lett. 195:121-126 (2001), which is hereby incorporated by reference in its entirety). In fungi, ectophosphatases have been described in a large number of species (Freitas-Mesquita et al., Int. J. Mol. Sci. 15:2289-2304 (2014), which is hereby incorporated by reference in its entirety), including Aspergillus fumigatus (Bernard et al., Microbiology 148:2819-2829 (2002), which is hereby incorporated by reference in its entirety), and Candida albicans (Portela et al., Oral Dis. 16:431-437 (2010), which is hereby incorporated by reference in its entirety).
A further aspect of the invention relates to a method of treating cancer. This method includes administering to a subject having a cancerous condition a therapeutically effective amount of the enzymatically responsive product of the present invention or a composition containing the same, wherein the administration thereof is effective to cause selective adherence of the dephosphorylated, desulfated, or de-esterified product to cancer cells. As demonstrated in the accompanying examples, the selective adherence of the enzymatically modified product to cancer cells affects the growth or survival of cancer cells.
Exemplary subjects include any mammal that is susceptible to cancerous conditions including, without limitation, rodents, rabbits, canines, felines, ruminants, and primates such as monkeys, apes, and humans.
Administration of the enzymatically selective product or pharmaceutical composition can be carried out using any suitable approach. By way of example, administration can be carried out parenterally, subcutaneously, intravenously, intradermally, intramuscularly, intraperitoneally, by implantation, by intracavitary or intravesical instillation, intraarterially, intralesionally, intradermally, peritumorally, intratumorally, or by introduction into one or more lymph nodes. In certain embodiments, administration is carried out intralesionally, intratumorally, intradermally, or peritumorally. This administration can be repeated periodically, e.g., one or more times per month, one or more times per week, once daily, or twice daily. Optimal administration protocols can be established using routine skill.
In these several aspects of the invention, the cancer cells express a cell surface-bound phosphatase, secrete a phosphatase, or both; express a cell surface-bound sulfatase, secrete a sulfatase, or both; express a cell surface-bound esterase, secrete an esterase, or both; or any combination thereof. In these embodiments, the enzyme produced by the cancer cells is an ectoenzyme having hydrolytic activity, i.e., the enzyme hydrolyzes a phosphate group, a sulfate group, or a (carboxyl)ester group.
The cancer cells to be treated in accordance with these aspects can be present in a solid tumor, present as a metastatic cell, or present in a heterogenous population of cells that includes both cancerous and noncancerous cells. Exemplary cancer conditions include, without limitation, cancers or neoplastic disorders of the brain and CNS (glioma, malignant glioma, glioblastoma, astrocytoma, multiforme astrocytic gliomas, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma), pituitary gland, breast (Infiltrating, Pre-invasive, inflammatory cancers, Paget's Disease, Metastatic and Recurrent Breast Cancer), blood (Hodgkin's Disease, Leukemia, Multiple Myeloma, Lymphoma), lymph node cancer, lung (Adenocarcinoma, Oat Cell, Non-small Cell, Small Cell, Squamous Cell, Mesothelioma), skin (melanoma, basal cell, squamous cell, Kapsosi's Sarcoma), bone cancer (Ewing's Sarcoma, Osteosarcoma, Chondrosarcoma), head and neck (laryngeal, pharyngeal, and esophageal cancers), oral (jaw, salivary gland, throat, thyroid, tongue, and tonsil cancers), eye, gynecological (Cervical, Endometrial, Fallopian, Ovarian, Uterine, Vaginal, and Vulvar), genitourinary (Adrenal, bladder, kidney, penile, prostate, testicular, and urinary cancers), and gastrointestinal (appendix, bile duct (extrahepatic bile duct), colon, gallbladder, gastric, intestinal, liver, pancreatic, rectal, and stomach cancers).
Use of the disclosed products and compositions can be coordinated with previously known therapies. For instance, chemotherapeutic agents, immunotherapeutic agents, or radiotherapeutic agents, as well as surgical intervention can be used in a coordinated manner with the enzymatically selective product or pharmaceutical compositions of the present invention. Thus, a chemotherapeutic agent, an immunotherapeutic agent, or a radiotherapeutic agent can be administered to a patient before or after treatment with the peptides or pharmaceutical compositions of the present invention. Alternatively, surgical resection of a tumor can be carried out before or after treatment with the peptides or pharmaceutical compositions of the present invention.
Alternatively, hyperthermic treatment of tumors can be performed using the enzymatically selective product. After administration of the enzymatically selective product to an individual, a tumor-containing region of the subject's body can be exposed to an energy source suitable to cause thermal heating of the magnetic particles associated with the cancer cells and destruction of cancer cells. Exemplary energy sources include ultrasound, laser light, near infrared light, or alternating magnetic field (Chichel et al., Rep. Pract. Oncol. Radiother. 12(5):267 (2007); Day et al., J. Biomech. Eng. 131(7):074001 (2009); Kaddi et al., Nanomedicine 8(8):1323 (2013); Elsherbini et al., Internat'l J. Nanomed. 6:2155 (2011), which are hereby incorporated by reference in their entirety).
Magnetic hyperthermia provides increased specificity by selectively accumulating nanoparticles in target tissues. The present invention presents an entirely novel approach for accumulation of the magnetic particles on tumor cell surfaces.
Magnetic particles induce local hyperthermia due to hysteresis heating in the presence of an alternating magnetic field (Alexiou et al., J. Nanosci. Nanotech. 6:2762 (2006), which is hereby incorporated by reference in its entirety). The resulting induced flux reversal dissipates energy as heat only in the region of magnetic material localization allowing a highly localized increase in temperatures thus avoiding the normal tissues. The extent of temperature rise depends upon magnetic properties of the particles, magnetic field strength, frequency of oscillation, and cooling capacity of the blood flow in the tumor site (Gupta and Gupta, Biomaterials 26:3995 (2005), which is hereby incorporated by reference in its entirety). Cellular inactivation is achieved when temperatures exceed 42° C. for >30 minutes; temperatures>46° C. cause extensive cellular necrosis (thermoablation) (Pankhurst et al., J. Physics D: Applied Physics 36:R167 (2003), which is hereby incorporated by reference in its entirety).
Yet another aspect of the invention relates to a method for imaging cancer cells. This method includes administering to a subject having a cancerous condition an enzymatically selective product of the invention or a composition containing the same, wherein said administering is effective to cause the product to contact cancer cells that express a cell surface-bound phosphatase or sulfatase, secrete a phosphatase or sulfatase, or both, and cause selective adherence of the dephosphorylated, desulfated, or de-esterified product to cancer cells. Thereafter, it is possible to use the magnetic particles, now selectively adsorbed to the cancer cells, as a contrast agent during magnetic resonance imaging. Generating a magnetic resonance image of a part of the subject where labeled cancer cells reside allows for enhanced contrast between normal tissue and cancerous tissue.
Superparamagnetic contrast agents have greater magnetic susceptibility than traditional MRI contrast agents (e.g., gadolinium) and some are commercially available, such as suspensions of polymer-coated ferromagnetic nanoparticles in water (Alexiou et al., J. Nanosci. Nanotechnol. 6:2762-2768 (2006), which is hereby incorporated by reference in its entirety). Their presence significantly weakens the MRI signal and creates a negative enhancement effect on images. MRI-enhancing contrast agents include superparamagnetic iron oxide (SPIO, >50 nm) and ultrasmall superparamagnetic iron oxide (USPIO, <50 nm) particles (Couvreur and Vauthier, Pharmaceutical Research 23:1417 (2006), which is hereby incorporated by reference in its entirety). Sequestration of SPIO nanoparticles by the reticulo-endothelial system provides high contrast imaging of splenic/hepatic tumors and metastases. USPIO nanoparticles have longer circulation times in the blood and broader tissue distribution because they avoid reticulo-endothelial system sequestration; they are ideal for detecting metastases in lymph nodes. The present invention presents an entirely novel approach for accumulation of the magnetic particles on tumor cell surfaces.
U.S. Pat. No. 5,869,023 to Ericcson, which is hereby incorporated by reference in its entirety, describes a method for magnetic resonance imaging using positive and negative contrasting agents. The same approach can be used with the products of the present invention as contrast agents.
The following examples are intended to illustrate the present invention, but are not intended to limit the scope of the appended claims.
Transmission electron microscope (TEM) images were taken on Morgagni 268 transmission electron microscope. Confocal images were obtained on a Leica TCS SP2 Spectral Confocal Microscope. Magnetic studies were carried out using a Lakeshore 7404 high sensitivity vibrating sample magnetometer (VSM). Samples were dried with Labconco Freezone 4.5 Plus vacuum lyophilizer. The cells were counted by Bio-Rad TC 20™ Automated cell counter.
The phosphate assay kit (colorimetric) (ab65622, abcam) was utilized to quantify the amount of phosphate on MNP_pY. First, the phosphate standard curve was obtained. 10 μl of the 10 mM phosphate standard was diluted with 990 μl dH2O and mixed well to generate 100 μM working phosphate standard. After adding 0, 10, 20, 30, 40, 50 μl of the 100 μM working phosphate standard to one 96 well plate, the volume was adjusted to 200 μl with dH2O to generate 0, 1, 2, 3, 4, 5 nmol of phosphate standard. Prior to reading the absorbance at 620 nm using a plate reader, 30 μl phosphate reagent was added into all standard wells and the mixture was incubated at room temperature for 30 minutes. When the amount of phosphate on MNP_pY was quantified by following similar protocol. First, 40 μg MNP_pY was incubated with 100 μl dH2O or 100 μl dH2O containing 30 U alkaline phosphatase (“ALP”) for 24 hours. After centrifugation, 20 μl of the treated solution was taken out and the volume was adjusted to 200 μl with dH2O. After the treatment with 30 μl phosphate reagent for 30 minutes at room temperature, the absorbance was read by using a plate reader.
The result indicated that the absorbance of 20 μl solution treated with ALP is 1.36 and that of 20 μl solution just with dH2O is 0.2843. According to the phosphate standard curve shown in
All cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Va., USA). The HeLa-GFP and HS-5 cells were propagated in Dulbecco's Modified Eagle Medium (DMEM, high glucose, Invitrogen Life Technologies 10829-018) supplemented with 10% fetal bovine serum (FBS, Invitrogen Life Technologies 10082-147), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (Invitrogen Life Technologies 15070-063) in a fully humidified incubator containing 5% CO2 at 37° C.
1.0×106 cells in exponential growth phase were seeded in 6 cm cell culture dish. The cells were allowed for attachment for 12 hours at 37 ° C., 5% CO2. The culture medium was removed, and new culture medium containing MNP_pY or MNP at 40 μg/mL was added. After 4 hours of incubation, cells were washed with growth medium 3 times and detached with 0.25% (w/v) Trypsin-0.53 mM EDTA solution. After harvesting the cells (
Similar to the method for confocal microscopy, 1.0×106 cells were treated with growth medium containing 200 μg MNP_pY or MNP for 4 hours, and then washed with growth medium 3 times and detached with 0.25% (w/v) trypsin-0.53 mM EDTA solution. The collected cell pellets after centrifugation were dried with the vacuum lyophilizer. Magnetic studies were carried out using a Lakeshore 7404 high sensitivity vibrating sample magnetometer (VSM) with fields up to 1.5 tesla at room temperature.
Cells in exponential growth phase were seeded in a 96 well plate at a concentration of 1.0×104 cell/well. The cells were allowed to attach to the wells for 12 hours at 37° C., 5% CO2. The culture medium was removed and 100 μL of the culture medium containing compounds (immediately diluted from fresh prepared stock solution of 10 mM) at gradient concentrations (0 μM as the control) was placed into each well. After culturing at 37 ° C., 5% CO2 for 24, 48, 72 hours, the growth medium from each well was removed and 50 μL 0.25% (w/v) trypsin-0.53 mM EDTA solution was added. 4 minutes later, the cells were counted by using automated cell counter. Data represents the mean±standard deviation of three independent experiments.
Encouraged by the seminal work on the DNA linked gold nanoparticles to report DNA hybridization (Mirkin et al., Nature 382:607 (1996); Cao et al., Science, 297:1536 (2002); Rosi et al., Chem. Rev. 105:1547 (2005); Taton et al., Science 289:1757 (2000), which are hereby incorporated by reference in their entirety) and the recent work on the dispersion of peptide coated gold nanoparticles to detect a specific enzyme (Jia et al., Anal. Chem. 74:2217 (2002); Liu et al., J. Am. Chem. Soc. 125:6642 (2003); Laromaine et al., J. Am. Chem. Soc. 129:4156 (2007), which are hereby incorporated by reference in their entirety), and based on unexpected observation of selective formation of pericellular nanonets on cancer cells upon dephosphorylation of D-peptides catalyzed by ectophosphatases (Kuang et al., Angew. Chem., Int. Ed. 53(31):8104 (2014), which is hereby incorporated by reference in its entirety), enzymatic transformation (“ET”) was used to trigger the adhesion of iron oxide nanoparticles on cells for sorting cancer cells. Enzymatic reactions were selected over antibodies to distinguish cancer and normal cells for three reasons: (i) the overexpression of ectophosphatases on the surface of cancer cells represents a generic difference between many cancer and normal cells (Fishman et al., Nature 219:697 (1968); Pospisil et al., BMC Bioinformatics 7:11 (2006), which are hereby incorporated by reference in their entirety); (ii) the omission of antibodies reduces the cost and increases the stability of agents; (iii) being highly efficient and specific, enzymatic reactions offer a simple, fast yet fundamentally new way to modulate the surface chemistry of magnetic nanoparticles (Sun et al., Nano Lett. 3:955 (2003); Sun et al., Anal. Chem. 74:5297 (2002); Yang et al., . Am. Chem. Soc. 136:8153 (2014), which are hereby incorporated by reference in their entirety) for spatiotemporally defining the magnetic nanoparticles in cellular environment, which is less explored.
Iron oxide nanoparticles (
The synthesis of MNP_pY is fast and straightforward. Using the well-established iron oxide nanoparticles (Sun et al., J. Am. Chem. Soc. 126:273 (2004); Hyeon et al., J. Am. Chem. Soc. 123:12798 (2001), which are hereby incorporated by reference in their entirety) that are surface-functionalized with carboxylic acid groups (and are commercially available), the nanoparticles were directly coupled to D-tyrosine phosphate using N-hydroxysuccinimide (NHS) to activate the carboxylic acid groups. Following three times rinsing by methanol and water, respectively, centrifugation separated the final MNP_pY dispersed in water for use. Transmission electron microscopy confirmed that there was little morphological change of the iron oxide nanoparticles before and after functionalization by D-tyrosine phosphates (
A process for the sorting of cancer cells from the cell mixture is shown in Stage I of
To further confirm the selectivity of MNP_pY towards cancer cells, HeLa-GFP and HS-5 cells were used separately as the control cells and the procedure shown in
To quantify the efficiency of cell capture of MNP_pY, the cell numbers were counted in the extraction or the supernatant fractions. As shown in
Magnetic properties of the iron oxide nanoparticles were studied by using a vibrating sample magnetometer (“VSM”) for quantifying the amount of MNP_Y remained on the cells. As shown in
Besides selectively capturing cancer cells in co-culture, MNP_pY selectively inhibited the proliferation of cancer cells. As shown in
To further demonstrate that enzymatic transformation of MNP_pY is the key point for selective sorting of cancer cells, MNP_Y was utilized in a separate experiment. MNP_Y resulted from the treatment of MNP_pY with ALP prior to incubating with cells, and the procedure shown in
These studies show that an underexplored generic difference—overexpression of ectophosphatases—between cancer and normal cells enabled the magnetic nanoparticles to adhere selectively on cancer cells upon catalytic dephosphorylation for inhibiting cancer cells via extrinsic cell death pathways (e.g., Fas, DR3, or DR5). See
In conclusion, this work, for the first time, demonstrates the use of enzymatic transformation of magnetic nanoparticles for selectively sorting and inhibition of cancer cells from stromal cells without involving specific ligand-receptor interactions or the use of antibodies. The high capture efficiency of cancer cells from the co-culture demonstrated the expression level of enzymes as a new paradigm for exploring strategies that target cancer cells. This strategy relied on an enzymatic reaction (e.g., catalytic dephosphorylation), but not specific enzyme inhibition, to target cancer cells selectively. The same principle should be useful for developing a relatively inexpensive, simple, and selective method for sampling other biological specimens. By mimicking the essence of biological signaling processes (e.g., kinase/phosphatase enzymatic switch (Lodish et al., Molecular Cell Biology; Seventh Edition edition ed.; Freeman, W. H. (2012), which is hereby incorporated by reference in its entirety)), the use of enzymatic transformation to control the formation or state of nanostructures (Zhou et al., J. Am. Chem. Soc. 136:2970 (2014); Toledano et al., J. Am. Chem. Soc. 128:1070 (2006); Yang et al., J. Am. Chem. Soc. 129:266 (2007); Yang et al., Chem. Commun. 2424 (2004); Lovell et al., Angew. Chem. Int. Edit. 51:2429 (2012); Kim et al., Angew. Chem. Int. Edit. 47:8438 (2008); Kim et al., J. Am. Chem. Soc. 128:688 (2006); Lee et al., Nat. Med. 13:95 (2007); Zhao et al., J. Am. Chem. Soc. 135:12940 (2013); Xia et al., Adv. Mater. 15:353 (2003); Jin et al., Nat. Nanotechnol. 4:571 (2009); Yang et al., J. Am. Chem. Soc. 130:5286 (2008); Chien et al., J. Am. Chem. Soc. 135:18710 (2013); Ku et al., J. Am. Chem. Soc. 133:8392 (2011), which are hereby incorporated by reference in their entirety) ultimately may lead to new approaches for detecting and treating other diseases.
Having thus described the basic concept of the invention, it will be rather apparent to those skilled in the art that the foregoing detailed disclosure is intended to be presented by way of example only, and is not limiting. Various alterations, improvements, and modifications will occur and are intended to those skilled in the art, though not expressly stated herein. These alterations, improvements, and modifications are intended to be suggested hereby, and are within the spirit and scope of the invention. Additionally, the recited order of processing elements or sequences, or the use of numbers, letters, or other designations therefore, is not intended to limit the claimed processes to any order except as may be specified in the claims. Accordingly, the invention is limited only by the following claims and equivalents thereto.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/977,484 filed Apr. 9, 2014 and U.S. Provisional Patent Application Ser. No. 62/063,773, filed Oct. 14, 2014, each of which is hereby incorporated by reference in its entirety.
The present invention was made with support from the National Institutes of Health under grant R01CA142746 and from the National Science Foundation under grant DMR-0820492. The U.S. government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/25137 | 4/9/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61977484 | Apr 2014 | US | |
| 62063773 | Oct 2014 | US |
