Source: http://www.google.com/patents/US7700715?dq=7222078
Timestamp: 2015-11-27 15:39:14
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Matched Legal Cases: ['application No. 60', 'application No. 60', 'application No. 60', 'application No. 60', 'application No. 60', 'application No. 60', 'application No. 60', 'application No. 60', 'application No. 60', 'application No. 60', 'application No. 60', 'application No. 60']

Patent US7700715 - Diagnostic tumor markers, drug screening for tumorigenesis inhibition, and ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe invention provides a series of compositions, methods, kits, articles and species associated primarily with the diagnosis and/or treatment of cell proliferation, specifically cancer. Cell proliferation associated with aberrant expression of MUC1 is particularly focused upon. Mechanisms associated...http://www.google.com/patents/US7700715?utm_source=gb-gplus-sharePatent US7700715 - Diagnostic tumor markers, drug screening for tumorigenesis inhibition, and compositions and methods for treatment of cancerAdvanced Patent SearchPublication numberUS7700715 B2Publication typeGrantApplication numberUS 09/996,069Publication dateApr 20, 2010Filing dateNov 27, 2001Priority dateNov 27, 2000Fee statusPaidAlso published asCA2430060A1, EP1354196A2, EP1354196B1, EP2241887A2, EP2241887A3, EP2241887B1, EP2322929A1, EP2386859A2, EP2386859A3, EP2386859B1, US8344113, US20030036199, US20100136017, US20130323755, WO2002056022A2, WO2002056022A3Publication number09996069, 996069, US 7700715 B2, US 7700715B2, US-B2-7700715, US7700715 B2, US7700715B2InventorsCynthia C. Bamdad, R. Shoshana BamdadOriginal AssigneeMinerva Biotechnologies CorporationExport CitationBiBTeX, EndNote, RefManPatent Citations (25), Non-Patent Citations (19), Classifications (45), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetDiagnostic tumor markers, drug screening for tumorigenesis inhibition, and compositions and methods for treatment of cancer
US 7700715 B2Abstract
The invention provides a series of compositions, methods, kits, articles and species associated primarily with the diagnosis and/or treatment of cell proliferation, specifically cancer. Cell proliferation associated with aberrant expression of MUC1 is particularly focused upon. Mechanisms associated with MUC1 cell proliferation are discussed.
a first article having a surface; and
a peptide sequence immobilized relative to or adapted to be immobilized relative to the surface, the peptide sequence comprising a portion of a cell surface receptor, in which an interchain binding region has been removed to the extent necessary to prevent self-aggregation of the peptide, wherein the peptide sequence interacts with an activating ligand to promote cell proliferation, wherein the primary sequence of said cell surface receptor consists of residues 1110-1154 of SEQ ID NO:10.
2. The kit as in claim 1, further comprising a second article having a surface and the peptide sequence immobilized relative to or adapted to be immobilized relative to the surface of the second article.
3. The kit according to claim 1, wherein the activating ligand is 14-3-3 protein, cathepsin D protein, or NM23 protein.
4. The kit according to claim 1, wherein the peptide sequence comprises at least 12 contiguous amino acids from the sequence residues 1110-1154 of SEQ ID NO:10.
5. The kit according to claim 1, wherein the peptide sequence comprises at least 12 contiguous amino acids from the sequence residues 1110-1142 of SEQ ID NO:10.
6. The kit according to claim 1, wherein the article is a colloid particle.
7. The kit according to claim 6, wherein the colloid is a gold colloid.
8. The kit according to claim 1, wherein the peptide is adapted to be fastened to the article by an affinity tag attached to the peptide.
9. The kit according to claim 8, wherein the affinity tag is polyamino acid tag, polyhistidine tag, GST tag, biotin, or thioredoxin.
10. The kit according to claim 8, wherein the affinity tag is fastened to the C-terminus of the portion of the peptide.
11. The kit according to claim 10, further comprising the activating ligand.
12. The kit according to claim 11, wherein the activating ligand is NM23.
This non-provisional application claims the benefit under Title 35, U.S.C. �119(e) of U.S. provisional application No. 60/253,361, filed Nov. 27, 2000, U.S. provisional application No. 60/255,370, filed Dec. 13, 2000, U.S. provisional application No. 60/256,027, filed Dec. 15, 2000, U.S. provisional application No. 60/258,157, filed Dec. 22, 2000, U.S. provisional application No. 60/259,615, filed Jan. 3, 2001, U.S. provisional application No. 60/260,186, filed Jan. 5, 2001, U.S. provisional application No. 60/266,169, filed Feb. 2, 2001, U.S. provisional application No. 60/289,444, filed May 7, 2001, U.S. provisional application No. 60/266,929, filed Feb. 6, 2001, U.S. provisional application No. 60/278,093, filed Mar. 23, 2001, U.S. provisional application No. 60/294,887, filed May 31, 2001, and U.S. provisional application No. 60/298,272, filed Jun. 14, 2001, each of which is incorporated by reference herein.
The invention relates to assays using shed cell surface receptor interchain binding regions and cleavage products for cancer diagnosis, and for the evaluation of cancer treatment and using the portion of the receptor that remains on the cell as a molecular target for cancer therapeutics.
Many of the biomolecular interactions that promote tumorigenesis involve cell surface proteins that mediate both intra- and intercellular signaling. “Tumor markers” are proteins on the surface of a cell that are exclusively expressed, over-expressed or show an altered expression pattern as a result of transformation to a neoplastic state. The surface concentration of certain tumor markers has been correlated to the progression of cancer. For example, the interaction between the cell surface receptor αVβ3 and the cell adhesion molecule vitronectin has been implicated in angiogenesis (Varner J, Cheresh D: Integrins and cancer. Curr Opin Cell Biol, 1996, 8(5): 724-730; Vailhe B, Ronot X, Tracqui P, Usson Y, Tracqui L: In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to αVβ3 integrin localization. In Vitro Cell Dev Biol Anim, 1997, 33(10): 763-773; Horton M: The aVb3 integrin “vitronectin receptor”. Int J Biochem Cell Biol, 1997, 29(5): 721-725) and the increased concentration of αVβ3 on melanoma cells has been correlated with poor prognosis (Hieken T, Farolan M, Ronan S, Shilkaitis A, Wild L, Das Gupta T: β3 integrin expression in melanoma predicts subsequent metastasis. J Surg Res, 1996, 63(1): 169-173).
Cell surface receptors, that have been linked to cancer, make up an important class of therapeutic targets. Many pharmaceutical companies are actively involved in screening drug libraries for compounds that bind to and block these cell surface receptors. For example, an important drug used to treat breast cancer is Herceptin (Pegram M, Lipton A, Hayes D, Webber B, Baselga J, Tripathy D, Baly D, Baughman S, Twaddell T, Glaspy J, Slamon D: Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185 Her2/neu monoclonal antibody plus cisplatin, in patients with Her2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment, J Clin Oncol, 1998, 16(8): 2659-2671). This drug binds to and blocks HER2/neu (Ross J, Fletcher J: review, The Her2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy. Stem Cells, 1998, 16(6): 413-428) which is a cell surface receptor that is over-expressed on 30% of breast tumors.
Another cell surface receptor, called MUC1 (Treon S, Mollick J, Urashima M, Teoh G, Chauhan D, Ogata A, Raje N, Hilgers J, Nadler L, Belch A, Pilarski L and Anderson K: MUC1 core protein is expressed on multiple myeloma cells and is induced by dexamethasone. Blood, 1999, 93(4): 1287-1298), is especially interesting since it is aberrantly expressed on many human tumors, including 80% of breast tumors, and on a significant percentage of prostate, lung, ovarian, colorectal and perhaps brain, cancers. On healthy secretory epithelium, MUC1 is clustered at the apical border and is not expressed over other portions of the cell. However, in tumor cells, the receptor is homogeneously over-expressed over the entire cell surface (Kufe D., Inghirami G., Abe M., Hayes D, Justi-Wheeler H, Schlom J: Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma, 1984, 3: 223-232), rather than just at the apical border. It is also known that women with breast cancer have elevated levels of shed MUC1 receptor in their blood stream. Extracellular portions of the MUC1 receptor are cleaved or “shed”, by at least one enzyme, and released into the blood stream. Levels of shed MUC1 receptor in serum are measured to track breast cancer patients for recurrence. However, the method is too variable and insensitive to be used as a general diagnostic.
Until now, the mechanistic link between the MUC1 receptor and tumorigenesis has not been understood. Attempts to correlate the number of repeat units, which varies from person to person, and susceptibility to cancer failed. Investigations of a possible connection, between glycosylation of the MUC1 receptor and cancer, produced conflicting results. Importantly, until now, a functional ligand(s) for the extracellular portion of the MUC1 receptor has not been identified.
Absent an understanding of the mechanism of the MUC1 receptor, and how it triggers tumorigenesis, it has not been possible to design or identify therapeutics that interfere with the disease-associated function of this receptor. Indeed, currently there is no drug in use or, to our knowledge, in clinical trials that is known to target the MUC1 receptor.
The present invention describes discoveries that elucidate critical aspects of the mechanism by which MUC1 triggers cell proliferation and tumorigenesis. These discoveries provide novel molecular targets for drug screening assays which the inventors have used to identify compounds that inhibit the MUC1-dependent tumorigenesis. These discoveries also enable an early diagnostic assay.
The present invention provides a variety of kits, methods, compositions, peptide species and articles associated with cell proliferation, specifically cancer. The invention involves primarily techniques and components for the diagnosis and treatment of cancer.
In one aspect, the invention provides a series of kits. One kit includes a first article having a surface, and a peptide sequence immobilized relative to or adapted to be immobilized relative to the surface. The peptide sequence includes a portion of a cell surface receptor that interacts with an activating ligand such as a growth factor to promote cell proliferation. Also included in the kit is a candidate drug for affecting the ability of the peptide sequence to bind to other identical peptide sequences in the presence of the activating ligand. The portion includes enough of the cell surface receptor to interact with the activating ligand and the portion is free of interchain binding region to the extent necessary to prevent spontaneous binding between the portions.
Another kit of the invention comprises a species able to become immobilized relative to a shed cell surface receptor interchain binding region, and a signaling entity immobilized relative to or adapted to be immobilized relative to the species.
Another kit of the invention comprises a species able to bind to a portion of a cell surface receptor that remains attached to the cell surface after shedding of a cell surface receptor interchain binding region, and a signaling entity immobilized relative to or adapted to be immobilized relative to the species.
Another kit of the invention comprises a species able to bind to a portion of a cell surface receptor that includes the interchain binding region, and a signaling entity immobilized relative to or adapted to be immobilized relative to the species.
Another kit of the invention comprises an article (which can be a particle), and at least a fragment of the sequence that corresponds to that portion of a cell surface receptor that interacts with an activating ligand such as a growth factor to promote cell proliferation, the fragment being detached from any cell, fastened to or adapted to be fastened to the article.
Another kit of the invention comprises an article having a surface, and a biomolecule that binds to a portion of a cell surface receptor that interacts with an activating ligand such as a growth factor to promote cell proliferation. The biomolecule is fastened to or adapted to be fastened to the surface of the article.
In another aspect, the invention provides a series of methods. One method comprises providing a peptide including a portion of a cell surface receptor that interacts with an activating ligand such as a growth factor to promote cell proliferation, exposing the peptide to a candidate drug for affecting the ability of the activating ligand to interact with the peptide, and to the activating ligand, and determining the ability of the candidate drug to prevent interaction of the activating ligand with the peptide. The portion includes enough of the cell surface receptor to interact with the activating ligand and the portion is free of interchain binding region to the extent necessary to prevent spontaneous binding between portions.
Another method of the invention involves treating a subject having cancer or being at risk for developing cancer, the method comprises administering to the subject an agent that reduces cleavage of a cell surface receptor.
Another method of the invention for treating a subject having cancer or at risk for developing cancer comprises administering to the subject an agent that reduces cleavage of a cell surface receptor interchain binding region from the cell surface.
Another method of the invention comprises determining an amount of cleavage of a cell surface receptor interchain binding region from a cell surface, and evaluating indication of cancer or potential for cancer based upon the determining step.
Another method of the invention comprises determining a site of cleavage of a cell surface receptor in a sample from a subject, and evaluating an indication of cancer or potential for cancer based upon the determining step.
Another method of the invention involves determining a cleavage site of a cell surface. The method comprises contacting a cell with an agent that binds specifically to one potential cell surface receptor cleavage site and another agent that binds specifically to another potential cell surface receptor cleavage site. The ratio of binding of the two agents to the cell surface is compared in the method.
Another method of the invention comprises determining a first amount of cleavage of a cell surface receptor interchain binding region from a cell surface of a sample from a subject. A second amount of cleavage of cell surface receptor interchain binding region from a cell surface of a sample from the subject is also determined, and the first amount is compared to the second amount.
Another method of the invention involves treating a subject to reduce the risk of or progression of cancer. The method comprises administering to a subject, who is known to be at risk for cancer or is diagnosed with cancer, an agent for inhibiting interaction of an activating ligand with a portion of a cell surface receptor that interacts with the activating ligand to promote cell proliferation.
Another method of the invention involves treating a subject to reduce the risk of or progression of cancer. The method comprises administering to a subject, who is known to be at risk of cancer or is diagnosed with cancer, an agent for preventing clustering of portions of cell surface receptors that interact with an activating ligand such as a growth factor to promote cell proliferation.
Another method of the invention comprises exposing a ligand capable of binding with a portion of a cell surface receptor that remains attached to the cell after shedding of the cell surface receptor interchain binding region, and an agent capable of blocking this binding, to a candidate drug for disruption of interaction between the ligand and the agent. Disruption of the interaction by the candidate drug is determined.
Another method of the invention comprises exposing a portion of a cell surface receptor that remains attached to the cell surface after shedding of a cell surface receptor interchain binding region which is capable of binding with a ligand, and an agent capable of blocking this binding, to a candidate drug for disruption of interaction between the portion and the agent, and determining disruption of the interaction by the candidate drug.
Another method of the invention comprises exposing a synthetic drug, and a biological target of the synthetic drug, to a candidate drug which may interact with a biological target to a degree greater than the interaction between the synthetic drug and the target, and determining disruption of the interaction by the candidate drug.
Another method involves diagnosing a physiological state indicative of cancer or potential for cancer. The method comprises determining a specific cleavage site of MUC1 distinguishable from a different cleavage state of MUC1.
Another method of the invention involves treating a subject having a cancer characterized by the aberrant expression of MUC1, comprising administering to the subject etomoxir in an amount effective to reduce tumor growth.
Another method of the invention involves treating a subject having a cancer characterized by the aberrant expression of MUC1, comprising administering to the subject L-α-methyl-dopa in an amount effective to reduce tumor growth.
Another method of the invention for treating a subject having cancer characterized by the aberrant expression of MUC1, comprises administering to the subject calcimycin in an amount effective to reduce tumor growth.
Another method for treating a subject having a cancer characterized by the aberrant expression of MUC1, comprises administering to the subject butylindazole in an amount effective to reduce tumor growth.
In another aspect, the invention provides compositions. One composition of the invention comprises at least a portion of a shed cell surface receptor interchain binding region, and a signaling entity immobilized relative to or adapted to be immobilized relative to the portion.
The invention also provides peptide species. One peptide species of the invention comprises at least a fragment of a sequence that corresponds to that portion of a cell surface receptor that interacts with an activating ligand such as a growth factor to promote cell proliferation, the portion being detached from any cell, and an affinity tag.
FIG. 1 is a schematic illustration of the MUC1 receptor;
FIG. 2 (color) is a colloid-based, color change binding experiment that shows which portions of soluble MUC1 bind to each other or self-aggregate;
FIG. 3 (color) is a colloid-based color change experiment in which the ability of peptides to self-aggregate was used to help determine a boundary between a portion of the MUC1 receptor that self-aggregates the cell-proximal portion that does not; results imply a disease-related cleavage site on the MUC1 receptor;
FIG. 4 is a graph of percent cell proliferation that shows that an antibody against an epitope of the MUC1 receptor which is proximal to the cell surface, and that dimerizes the receptor, enhances cell proliferation in a manner typical of a growth factor/receptor-antibody interaction;
FIG. 5 is a graph of percent cell proliferation that shows that an antibody against an epitope of the MUC1 receptor which is proximal to the cell surface, and that dimerizes the receptor, dramatically enhances cell proliferation;
FIG. 6 (color) is an image of a section of a 96-well plate illustrating a color change assay in which a ligand(s) present in the lysates of cells that express MUC1, binds to and dimerize the His-PSMGFR peptide, derived from MUC1, which is immobilized on gold colloids, while lysates from cells that do not express MUC1 do not;
FIG. 7 (color) is an image of 96-well plate illustrating a colloid-based color-change binding assay between a MUC1-derived peptide and a ligand(s) present in a crude cell lysate; addition of imidazole, which releases the probe peptide from the colloid, causes a reversal of the color change, which argues that the color change is the result of a specific interaction rather than random colloid aggregation;
FIGS. 8A-D (color) shows a colloid-based color change assay in 96-well plates in which a ligand present in a cell lysate caused dimerization of a MUC1-derived peptide and that the degree of color change, which indicates an amount of ligand present, was a function of which cell line supplied the lysate;
FIG. 9 (color) is a silver-stained gel showing ligands that were fished out of cell lysates using the PSMGFR peptide, in the presence of the protease inhibitor PMSF;
FIG. 10 (color) is a silver-stained gel showing ligands that were fished out of cell lysates using the PSMGFR peptide, in the absence of the protease inhibitor PMSF;
FIG. 11 (color) is an image of 96-well plate illustrating a color-change binding assay between a MUC1-derived peptide and a ligand(s) present in a crude cell lysate from cells that overexpress MUC1;
FIG. 12 (color) is an image of a 96-well plate illustrating a color-change drug-screening assay used to detect inhibitors of the MUC1-Ligand interaction;
FIG. 13 shows a histogram illustrating the selective inhibition of proliferation of tumor cells that aberrantly express the MUC1 receptor, in response to treatment with compounds of the invention, and lack of an effect on cells that do not express MUC1;
FIG. 14 (color) is an image of a 96-well plate illustrating a color-change drug-screening assay identifying several compounds that intefere with the interaction of the MGFR portion of the MUC1 receptor and a multimerizing ligand(s);
FIG. 15 shows a histogram illustrating the selective inhibition of proliferation of tumor cells that aberrantly express the MUC1 receptor, in response to treatment with drugs that specifically inhibit MUC1 positive cells;
FIG. 16 shows a histogram illustrating the nonselective inhibition of proliferation of cells in response to treatment with drugs that non-specifically inhibit cell proliferation;
FIG. 17 shows a histogram illustrating that drugs that selectivly inhibit proliferation of tumor cells that aberrantly express the MUC1 receptor bind to the PSMGFR, while drugs that non-selectively inhibit cell proliferation do not;
FIG. 18 is a graph showing that the inhibition of MUC1-dependent cell proliferation induced by an anti-tumor drug identified in accordance with the invention, is modulated when a synthetic peptide, corresponding to the portion of MUC1 that remains at the cell following cleavage, competitively inhibits the drug-cell surface receptor interaction;
FIG. 19 (color) is a comassie blue-stained gel showing that the PSMGFR peptide runs at an apparently higher molecular weight after incubation with cell;
FIG. 20 (color) is an image of a 96-well plate illustrating a color-change ligand binding assay illustrating that inhibiting enzymatic modification of PSMGFR prevents it binding to ligands.
The term “MUC1 Growth Factor Receptor” (MGFR) is a functional definition meaning that portion of the MUC1 receptor that interacts with an activating ligand, such as a growth factor, to promote cell proliferation. The MGFR region of MUC1 is that portion that is closest to the cell surface and is defined by most or all of the PSMGFR. The MGFR is inclusive of both unmodified peptides and peptides that have undergone enzyme modifications, such as, for example, phosphorylation, glycosylation, etc. Results of the invention are consistent with a mechanism in which this portion is made accessible to the ligand upon MUC1 cleavage at a site associated with tumorigenesis that causes release of the IBR from the cell.
The term “Interchain Binding Region” (IBR) is a functional definition meaning that portion of the MUC1 receptor that binds strongly to identical regions of other MUC1 molecules giving MUC1 the ability to aggregate (i.e. self-aggregate) with other MUC1 receptors via the IBRs of the respective receptors. This self-aggregation may contribute to MUC1 receptor clustering, observed in healthy cells.
In a preferred embodiment, the IBR may be approximately defined as a stretch of at least 12 to 18 amino acid sequence within the region of the human MUC1 receptor defined as comprising amino acids 507 to 549 of the extracellular sequence of the MUC1 receptor, with amino acids 525 through 540 and 525 through 549 especially preferred (numbers refer to Andrew Spicer et al., J. Biol. Chem Vol 266 No. 23, 1991 pgs. 15099-15109; these amino acid numbers correspond to numbers 1067, 1109, 1085, 1100, 1085, 1109 of Genbank accession number P15941; PID G547937, SEQ ID NO: 10) or fragments, functional variants or conservative substitutions thereof.
The term “cleaved IBR” means the IBR (or a portion thereof) that has been released from the receptor molecule segment which remains attached to the cell surface. The release may be due to enzymatic or other cleavage of the IBR. As used herein, when the IBR is “at the surface of a cell”, it means the IBR is attached to the portion of the cell surface receptor that has not been shed, or cleaved. The cleaved IBR of interest is a “disease-associated cleavage”, i.e. that type of cleavage that can result in cancer.
The term “Constant Region” (CR) is any non-repeating sequence of MUC1 that exists in a 1:1 ratio with the IBR and forms part of the portion of MUC1 that is shed upon cleavage in healthy and tumorigenesic cells.
The term “Repeats” is given its normal meaning in the art.
The term “Primary Sequence of the MUC1 Growth Factor Receptor” (PSMGFR) is a peptide squence, defined below (See Table 1—SEQ ID NO: 7), that defines most or all of the MGFR. The PSMGFR is inclusive of both unmodified peptides and peptides that have undergone enzyme modifications, such as, for example, phosphorylation, glycosylation, etc. The histidine-tagged PSMGFR (See Table 1—SEQ ID NO: 2) is abbreviated herein as His-PSMGFR.
The term “Extended Sequence of the MUC1 Growth Factor Receptor” (ESMGFR) is a peptide squence, defined below (See Table 1—SEQ ID NO: 3), that defines all of His-PSMGFR plus 9 amino acids of the proximal end of PSIBR.
PSIBR is a peptide squence, defined below (See Table 1—SEQ ID NO: 8), that defines most or all of the IBR.
The term “separation” means physical separation from a cell, i.e. a situation in which a portion of MUC 1 that was immobilized with respect to a cell is no longer immobilized with respect to that cell. E.g. in the case of cleavage of a portion of MUC 1, the portion that is cleaved is “separated” if it is free to migrate away from the cell and thereafter may be detected in a bodily fluid, or immobilized at a location remote from the cell from which it was cleaved such as another cell, a lymph node, etc.
The term “aggregate” (noun) means a plurality of cell surface receptors or fragments thereof (e.g. MUC 1) immobilized with respect to each other with or without an intermediate auxialliary to the host system. This includes self-aggregation of healthy receptors at a cell surface; self-aggregation of cleaved receptors or fragments bound to each other; cleaved receptors or fragments bound to receptors or fragments attached to a cell surface; receptors or fragments, whether attached to a cell or cleaved, immobilized with respect to each other via an intermediate auxialliary to the host. “Intermediate auxialliary to the host system” includes a synthetic species such as a polymer, dendrimer, etc., or a naturally-occurring species, for example an IgM antibody, which is not simply naturally present in the host system but is added to the host system from a source external to the host system. This excludes aggregation that is the result of an intermediate naturally present in the host system such as a growth factor that can cause disease-associated aggregation (“Inductive multimerization”). “Aggregate” (verb) or “aggregation” means the process of forming an aggregate (noun).
“Inductive multimerization” refers to aggregation wherein the aggregate formed can act to induce the cells to grow or proliferate. Inductive multimerization typically involves dimerization or tetramerization of cell surface receptors, for example by a growth factor or other activating ligand, but can also involve higher order multimerization, so long as the degree of multimerization is not so great as to mimic natural receptor clustering, in a particular cell type, which prevents receptors from signalling the cell to grow or proliferate.
“Preventative clustering” refers to multimerization of receptors to form an aggregate involving a sufficient number of receptors to mimic natural receptor clustering, in a particular cell type, which prevents receptors from signalling the cell to grow or proliferate, for example with an intermediate auxialliary to the host system.
A “ligand” to a cell surface receptor, refers to any substance that can interact with the receptor to temporarily or permanantly alter its structure and/or function. Examples include, but are not limited to binding partners of the receptor and agents able to alter the chemical structure of the receptor (e.g. modifying enzymes).
An “activating ligand” refers to a ligand able to effect inductive multimerization of cell surface receptors. Activating ligands can include, but are not limited to, a single molecular species with greater than one active site able to bind to a receptor; a dimer, a tetramer, a higher multimer, or a complex comprising a plurality of molecular species. In the context of MUC1 tumor cells, an activating ligand can be a species produced by the cells that interacts with the MGFRs on the surface of the MUC1 tumor cells in a manner that effects inductive multimerization.
A “growth factor” refers to a species that may or may not fall into a class of previously-identified growth factors, but which acts as a growth factor in that it acts as an activating ligand.
A “MUC1 presenting cell” refers to both non-cancerous and cancerous cells expressing MUC1 and/or MGFRs on the surface. A “MUC1 tumor cell” or “MUC1 cancer cell” or “cancerous MUC1 cell” refers to a cancerous tumor cell that aberrantly expresses MUC1 and/or MGFR on its surface.
“Colloids”, as used herein, means nanoparticles, i.e. very small, self-suspendable or fluid-suspendable particles including those made of material that is, e.g., inorganic or organic, polymeric, ceramic, semiconductor, metallic (e.g. gold), non-metallic, crystalline, amorphous, or a combination. Typically, colloid particles used in accordance with the invention are of less than 250 nm cross section in any dimension, more typically less than 100 nm cross section in any dimension, and in most cases are of about 2-30 nm cross section. One class of colloids suitable for use in the invention is 10-30 nm in cross section, and another about 2-10 nm in cross section. As used herein this term includes the definition commonly used in the field of biochemistry.
As used herein, a component that is “immobilized relative to” another component either is fastened to the other component or is indirectly fastened to the other component, e.g., by being fastened to a third component to which the other component also is fastened, or otherwise is transitionally associated with the other component. For example, a signaling entity is immobilized with respect to a binding species if the signaling entity is fastened to the binding species, is fastened to a colloid particle to which the binding species is fastened, is fastened to a dendrimer or polymer to which the binding species is fastened, etc. A colloid particle is immobilized relative to another colloid particle if a species fastened to the surface of the first colloid particle attaches to an entity, and a species on the surface of the second colloid particle attaches to the same entity, where the entity can be a single entity, a complex entity of multiple species, a cell, another particle, etc.
“Signaling entity” means an entity that is capable of indicating its existence in a particular sample or at a particular location. Signaling entities of the invention can be those that are identifiable by the unaided human eye, those that may be invisible in isolation but may be detectable by the unaided human eye if in sufficient quantity (e.g., colloid particles), entities that absorb or emit electromagnetic radiation at a level or within a wavelength range such that they can be readily detected visibly (unaided or with a microscope including an electron microscope or the like), or spectroscopically, entities that can be detected electronically or electrochemically, such as redox-active molecules exhibiting a characteristic oxidation/reduction pattern upon exposure to appropriate activation energy (“electronic signaling entities”), or the like. Examples include dyes, pigments, electroactive molecules such as redox-active molecules, fluorescent moieties (including, by definition, phosphorescent moieties), up-regulating phosphors, chemiluminescent entities, electrochemiluminescent entities, or enzyme-linked signaling moieties including horseradish peroxidase and alkaline phosphatase. “Precursors of signaling entities” are entities that by themselves may not have signaling capability but, upon chemical, electrochemical, electrical, magnetic, or physical interaction with another species, become signaling entities. An example includes a chromophore having the ability to emit radiation within a particular, detectable wavelength only upon chemical interaction with another molecule. Precursors of signaling entities are distinguishable from, but are included within the definition of, “signaling entities” as used herein.
“Covalently fastened” means fastened via nothing other than one or more covalent bonds. E.g. a species that is covalently coupled, via EDC/NHS chemistry, to a carboxylate-presenting alkyl thiol which is in turn fastened to a gold surface, is covalently fastened to that surface.
“Specifically fastened” or “adapted to be specifically fastened” means a species is chemically or biochemically linked to another specimen or to a surface as described above with respect to the definition