JP6111018B2 - Techniques and compositions for diagnosis and treatment of cancer (MUC1) - Google Patents

Description

Related applications

  This non-provisional application is filed in US Patent Act 119 (e) of US Provisional Application No. 60 / 498,260 (incorporated herein by reference), filed August 26, 2003, both of which are pending. Claim benefits.

  The present invention relates to a product for drug screening analysis, cancer diagnosis and cancer treatment evaluation, utilizing a part of the receptor remaining on the cell as a molecular target for cancer drug therapy, and against such a receptor cleavage product. It relates to binding peptides such as antibodies or antigen-binding fragments thereof, polypeptides comprising receptor cleavage products, and nucleic acid molecules encoding them.

Background of the Invention

  The molecular basis of programmed cell death, termed cell growth and apoptosis, is of great interest to pharmaceutical companies and cancer researchers in general. It appears that one or both of these processes have disappeared in cancer. Drug discovery for cancer is heavily focused on developing drug therapies that interfere with critical steps in the process of cell growth and programmed cell death. Of particular interest are substances that interfere with growth factor receptors. Growth factor receptors usually have an extracellular region that interacts in a highly specific manner with a cognate ligand that transmits a proliferation signal to the inside of the cell. Interactions and intracellular signaling pathways tend to be conserved and are not cell specific. Specificity is usually achieved via extracellular interactions. Substances that interfere with intracellular processes are undesirable in terms of drug therapy because they will have a wide range of effects on healthy cells as well as diseased cells. Conversely, drug therapies that target the extracellular portions of growth factor receptors are highly desirable because they specifically target cancer cells, especially if these portions are somewhat altered in the cancer cells.

  Thus, cell surface receptors associated with cancer constitute an important division of therapeutic targets. Many pharmaceutical companies are actively involved in screening drug libraries for compounds that bind to and sequester these cell surface receptors. For example, important drugs used to treat breast tumors include Herceptin (Pegram M, Lipton A, Hayes D, Webber B, Baselga J, Tripathy D, Baly D, Baughman S, Twaddell T, Glaspy. J, Slamon D: “Receptor-enhanced chemistry using recombinant humanized anti-p185 Her2 / neu monoclonal antibody and cisplatin in patients with Her-2 / neu-overexpressing metabolic breast tumors that are refractory to chemotherapy Phase II study of receptivity ", J. Clin. Oncol. 1998, 16 (8): 2659-2671). This drug is Her2 / neu (Ross J, Fletcher J: “Discussion, Her2 / neu Oncogene in Breast Tumors: Predictors, Predictors, and Targets for Treatment”, Stem Cells, 1998, 16 (6): 413. -428) and block. This is a cell surface receptor that is overexpressed in 30% of breast tumors.

  Other cell surface receptors are 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 nuclear protein is It is expressed on multiple myeloma cells and is induced by dexamethasone, "Blood, 1999, 93 (4): 1287-1298). The MUC1 receptor is a type I transmembrane glycoprotein obtained from the mucin family that is implicated in many human cancers. It is estimated that about 75% of solid tumors abnormally express the MUC1 receptor. The MUC1 + cancer group includes over 90% breast tumors, 47% prostate tumors and a high proportion of ovarian cancer, colorectal cancer, lung cancer and pancreatic cancer. MUC1 is normally expressed on glandular epithelial cells as well as the epithelium that supports the airways. Among the normal functions of the MUC1 receptor is evidence that it has a role in cell adhesion, fertility and immune response. The role of the MUC1 receptor in cancer has not yet been established in the literature. However, large differences in cell surface expression and receptor patterns in cancer are well documented. The most striking difference between MUC1 expression in healthy cells and expression in cancer cells is that receptors are clustered at the apical boundary in healthy cells, but receptors are evenly distributed on the entire surface of the cells in cancer cells. ing. Furthermore, there is evidence that the receptor is overexpressed on tumor cells in addition to abnormal patterning.

  The normal function of MUC1 as well as its association with cancer has not yet been clearly determined. What is known is that a portion of the extracellular region of MUC1 is fragmented or cleaved and can be detected in the serum of breast tumor patients. In breast tumor patients, the concentration of cleaved MUC1 in serum is sometimes measured to monitor the patient's response to treatment. The cytoplasmic tail of MUC1 is abundant in various signaling protein motifs. It has been reported in the literature that Grb2 and SOS, which are general signaling proteins, are associated with the cytoplasmic tail of MUC1. In cancer cells, the extracellular region is described in the scientific literature as not being fully glycosylated. Although the MUC1 receptor was cloned in 1990, its relevance to cancer remains unclear.

  The present invention describes a discovery that elucidates a critical aspect of the mechanism by which MUC1 triggers cell proliferation and tumorigenesis. These findings provide a novel molecular target for drug screening assays that we have used to identify compounds and binding peptides that inhibit MUC1-dependent tumorigenesis. These findings also allow for an early diagnostic analysis and an accurate way to follow the progression of cancer patients undergoing treatment.

Summary of invention

  We provide evidence herein that supports a mechanism by which a portion of the MUC1 receptor (proximal to the cell surface, ie, outside) functions as a growth factor receptor. The addition of a compound that binds to the PSMGFR portion of the MUC1 receptor is shown herein to inhibit cell growth, possibly by preventing dimerization of the MGFR portion of the receptor. The inventors also described that monovalent antibodies raised against the MGFR portion of the MUC1 receptor also inhibit cell growth by binding and blocking the association of the cognate ligand with the MGFR portion of the receptor. Prove it in the book.

  The invention may in some embodiments be a shorter form of the MUC1 receptor, a proteolytic fragment consisting essentially of the natural sequence of PSMGFRTC (ie, nat-PSMGFRTC—SEQ ID NO: 37 below), or another splice such as MUC1-Y. Any of the isoforms (SEQ ID NO: 40) is described as functioning as a growth factor receptor. As used herein, dimerization of the shorter form (ie truncated form) of the MUC1 receptor consisting essentially of nat-PSMGFRTC (SEQ ID NO: 37) transduces the signal inside the cell, and then Provides evidence to support the hypothesis of activating the cell growth signaling cascade. The present invention also inhibits receptor dimerization, and thus can be utilized as a cancer therapy for MUC1 + cancers, targeting a portion of the MUC1 receptor close to the cell surface (eg, MGFR) Monovalent fragments and monovalent single chain antibodies are described. A cell line that mimics MUC1 + cancer cells for use as a research tool for drug discovery is described. The present invention also provides experimental evidence that the major MUC1 species of breast tumors are cleavage products consisting essentially of nat-PSMGFRTC (SEQ ID NO: 37). Also provided are methods of using anti-PSMGFR antibodies, or antigen-binding fragments thereof, labeled for cancer diagnosis and imaging purposes. In such embodiments, such labeled antibodies that can be visualized by the surgeon during surgery to remove MUC1 + cancer are used during operations to selectively stain cancer tissue, so that the surgeon can It is more possible to see when such cancerous tissue is excised from the patient.

  The present invention relates to various kits, methods, compositions, peptide species, antibodies or fragments thereof that specifically bind to peptide species, nucleic acid molecules encoding such peptide species, and articles involved in cell proliferation, particularly cancer. provide. The present invention basically relates to techniques and components relating to the diagnosis and treatment of cancer.

In one aspect, the present invention provides a series of kits.
One kit contains the antibody or antigen-binding fragment thereof provided by the present invention.
One kit includes a first article having a surface and a peptide sequence immobilized on or applied to the surface. The peptide sequence includes a portion of a cell surface receptor that interacts with an active ligand, such as a growth factor or denaturing enzyme, that promotes cell proliferation. Also included in the kit are candidate agents that affect the ability of the peptide sequence to bind directly or indirectly to other same peptide sequences in the presence of the active ligand. Said portion sufficiently comprises a cell surface receptor that interacts with an active ligand.

Other kits of the invention can be immobilized on or immobilized on a species that can be immobilized to an interchain binding region of a disrupted cell surface receptor. Signal transduction material applied.
Another kit of the present invention provides a species capable of binding to a portion of the cell surface receptor remaining attached to the cell surface after the cleavage of the interchain binding region of the cell surface receptor and the species It includes a signaling agent that is immobilized or adapted to be immobilized.

Other kits of the invention are adapted to be or are immobilized to a species capable of binding to a portion of a cell surface receptor comprising an interchain binding region and to that species. Signal transduction substances.
Another kit of the invention is a fragment of a sequence corresponding to a part of a cell surface receptor that interacts with an article (which is a particle) and at least an active ligand such as a growth factor or denaturing enzyme that promotes cell proliferation. , Including fragments that are detected from any cell and fixed to or applied to the article.

In another aspect, the present invention provides a series of methods.
One method is a peptide comprising a portion of a cell surface receptor that interacts with an active ligand, such as a growth factor that promotes cell proliferation, sufficiently comprising the active ligand and a cell surface receptor that interacts with the portion And producing an antibody or antigen-binding fragment thereof that specifically binds to the peptide. Also described are antibodies or antigen-binding fragments thereof produced by the above methods.

  In another embodiment, a method of treating a patient suffering from a cancer characterized by abnormal expression of MUC1 comprising administering to the subject an amount of an antibody or antigen-binding fragment thereof effective to ameliorate the cancer. Is described.

  In still other embodiments, a portion of a cell surface receptor that interacts with an active ligand, such as a growth factor that promotes cell proliferation, comprising a portion that has sufficient cell surface receptors to interact with the active ligand. A method of treating a subject suffering from or at risk of developing cancer comprising administering to a subject an antibody or antigen-binding fragment thereof that specifically binds to a peptide is described.

  In other embodiments, a sample obtained from a subject suffering from or suspected of having cancer is treated with an antibody or antigen-binding fragment thereof that specifically binds to a peptide expressed on the cell surface, or the like Described is a method for determining the pathogenicity and / or metastatic potential of a cancer comprising contacting a recognizing substance and measuring the amount of an antibody or antigen-binding fragment or cognate ligand that specifically binds to a sample. Is done.

  In still other embodiments, a portion of a cell surface receptor comprising a portion sufficiently having a cell surface receptor that interacts with an active ligand such as a growth factor or denaturing enzyme and promotes cell proliferation, An expression vector that encodes an amino acid sequence containing a cell surface peptide that does not have the interchain binding region of the cell surface receptor to the extent necessary to prevent spontaneous binding between the parts, to transfect or transform host cells. And a method comprising promoting expression of the peptide by the cell so that the cell displays the peptide on its surface.

  In another embodiment, a portion of a cell surface receptor comprising a portion sufficiently having a cell surface receptor that interacts with an active ligand such as a growth factor or denaturing enzyme and promotes cell proliferation, Preparing a peptide that does not have an interchain binding region of a cell surface receptor to the extent necessary to prevent spontaneous binding, and expressing an expression vector containing a nucleic acid molecule encoding the peptide. Methods involving are described. Also described are expression vectors produced by the above methods.

  In still other embodiments, a portion of a cell surface receptor comprising a portion sufficiently having a cell surface receptor that interacts with an active ligand, such as a growth factor, to promote cell proliferation, between the portions A cell that expresses on its surface a peptide that does not have an interchain binding region of the cell surface receptor to the extent necessary to prevent spontaneous binding and affects the ability of the active ligand to interact with the peptide A method comprising: contacting a cell with a candidate agent and an active ligand that provides and determining whether an intracellular protein that is phosphorylated upon interaction of the activating ligand and the peptide is phosphorylated .

  In another embodiment, a cell is provided that expresses a peptide comprising MGFR on its surface, a candidate agent and an active ligand that affect the ability of the active ligand to interact with MGFR are contacted with the cell, and the intracellular A method is described that includes determining whether ERK-2 is phosphorylated.

  In yet other embodiments, the drug candidate presumed to have the ability to interfere with binding of the active ligand to the cell surface receptor interferes with binding of the active ligand to the cell surface receptor, and the drug candidate A method for simultaneously determining whether or not interacts with a cell surface receptor or ligand is described.

  In another embodiment, when the biological molecule is in the first denatured state to a different extent than the biological molecule is in close proximity to the biological molecule and in the second denatured state. A method for determining a denatured state of a biological molecule is described, comprising preparing colloidal particles arranged to be immobilized against and detecting the immobilization of the colloidal particles to the biological molecule. The

Another method of the invention relates to a method of treating a subject suffering from or at risk of developing cancer, the method comprising administering to the subject a substance that reduces cell surface receptor cleavage. Including doing.
Another method of the present invention for treating a subject suffering from or at risk of developing cancer is to administer a substance that reduces the cleavage of the interchain binding region of the cell surface receptor from the cell surface. including.

Another method of the invention involves measuring the amount of cleavage of the interchain binding region of the cell surface receptor from the cell surface and evaluating the signs of cancer or the likelihood of cancer based on the measurement process.
Another method of the invention involves measuring the cleavage site of a cell surface receptor in a sample obtained from a subject and assessing the signs of cancer or the likelihood of cancer based on the measurement process.

  Another method of the invention involves measuring the cleavage site on the cell surface. This method contacts a cell with a substance that specifically binds to the cleavage site of one potential cell surface receptor and another substance that specifically binds to the cleavage site of another potential cell surface receptor. Including that. The binding rate of the two substances to the cell surface is compared in this way.

  Another method of the invention involves measuring a first amount of cleavage of an interchain binding region of a cell surface receptor from the cell surface of a sample obtained from a subject. A second amount of cleavage of the cell surface receptor interchain binding region from the cell surface of the sample obtained from the subject is also measured, and the first amount is compared to the second amount.

Other compositions comprise an antibody or antigen-binding fragment thereof provided by the present invention.
Other compositions comprise an antibody or antigen-binding fragment thereof that specifically binds to MGFR.

  The present invention also provides peptide species. One peptide species of the present invention is a portion of a cell surface receptor that interacts with an active ligand such as a growth factor that promotes cell proliferation and has an affinity for a fragment of the sequence corresponding to the portion that is desorbed from any cell. Contains at least a tag.

In other embodiments, antibodies or antigen-binding fragments thereof that specifically bind to MGFR are described.
In yet another embodiment, an isolated protein or peptide comprising PSMGFR at the N-terminus, wherein the isolated protein does not comprise any of the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 6, or 7. Or a peptide is described.
In another embodiment, an isolated protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 7 at the N-terminus is described.

In another embodiment, an isolated protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 64 at the N-terminus is described.
In another embodiment, an isolated protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 2 is described.
In another embodiment, an isolated protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 60 is described.

In another embodiment, an isolated protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 7 is described.
In another embodiment, an isolated protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 64 is described.

In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to the amino acid sequence set forth in SEQ ID NO: 8 is described.
In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to the amino acid sequence set forth in SEQ ID NO: 65 is described.
In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to a unique region of the amino acid sequence set forth in SEQ ID NO: 39 is described.
In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to a region that bridges the N-terminus of the amino acid sequence set forth in SEQ ID NO: 39 and amino acid No. 104 is described.

  In another series of embodiments, the antibody or antigen-binding fragment thereof described herein is applied to the sample, the interaction between the sample and the antigen-binding fragment is observed, and the information observed during the observation act A method comprising diagnosing the presence of cancer or the pathogenicity of cancer is described based at least in part.

In another embodiment, an isolated protein or peptide comprising His-PSMGFR is described that does not include any of the amino acid sequences set forth in SEQ ID NOs: 1, 2, or 3.
In yet another embodiment, an isolated protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 7 is described.

  The present invention also provides a series of isolated nucleic acid molecules, an expression vector comprising the nucleic acid molecule, and a cell transformed with the expression vector or nucleic acid molecule. In one embodiment, isolated nucleic acid molecules and functional variants encoding PSMGFRTC and fragments thereof are described.

In other embodiments, isolated nucleic acid molecules and functional variants and fragments thereof that encode the amino acid sequence set forth in SEQ ID NO: 37 are described.
In still other embodiments, an expression vector is described comprising any of the above-described isolated nucleic acid molecules operably linked to a promoter.
In another embodiment, a host cell transfected or transformed with an expression vector comprising any of the isolated nucleic acid molecules described above is described.

In still other embodiments, an isolated nucleic acid molecule that hybridizes to the nucleic acid sequence set forth in SEQ ID NO: 37 under highly stringent conditions and its complement are described.
In another embodiment, an expression vector is described comprising an isolated nucleic acid molecule as described above or a complement thereof operably linked to a promoter.
In still other embodiments, host cells transfected or transformed with an expression vector comprising the above-described isolated nucleic acid molecule or complement thereof are described.

Detailed Description of the Invention

Definition:
The term “MUC1 growth factor receptor” (MGFR) is a functional definition that refers to a portion of the MUC1 receptor that interacts with an active ligand such as a growth factor or a denatured enzyme such as a cleavage enzyme that promotes cell proliferation. is there. The MGFR region of MUC1 is the extracellular portion that is closest to the cell surface and defined as most or all of PSMGFR, as defined below. MGFR includes both native peptides and peptides that have undergone enzymatic modifications such as phosphorylation, glycosylation, and the like. The effect of the present invention is consistent with the mechanism by which the part is involved in tumor formation that causes the release of all or part of the IBR from the cell and this part is brought close to the ligand upon MUC1 cleavage.

  The term “interchain binding region” (IBR) is strongly associated with the same region of other MUC1 molecules that give MUC1 the ability to aggregate (ie, self-aggregate) other MUC1 receptors via the IBR of each receptor. Functional definition meaning the portion of the MUC1 receptor that binds. This self-aggregation will contribute to the MUC1 receptor clustering observed in healthy cells.

  In a preferred embodiment, the IBR is a full-length human MUC1 receptor defined as comprising amino acids 507-549 of the extracellular sequence of the MUC1 receptor (SEQ ID NO: 10), particularly preferably amino acids 525-540 and 525-549. At least 12 to 18 amino acid sequences within the region or a fragment, functional variant or conservative substitution thereof defined in more detail below (numbers are Andrew Spicer et al. J. Biol. Chem. Vol. 266, No. 23. 1991, 15099-15109; these amino acid numbers correspond to the numbers 1067, 1109, 1085, 1100, 1085, 1109 of Genbank accession number P15941. PID G547937, SEQ ID NO: 10).

  The term “cleavage IBR” means IBR (or a portion thereof) released from a receptor molecule segment that remains attached to the cell surface. Release is by enzyme or by other cleavage of IBR. As used herein, when IBR is “on the surface of a cell”, this means that the IBR is attached to a portion of the cell surface receptor that has not been disrupted or cleaved. A cleaved IBR of interest is a “disease-related cleavage”, ie a cleavage type that results in cancer.

The term “constant region” (CR) is a non-repetitive sequence of MUC1 that is present in a 1: 1 ratio with IBR and forms part of the portion of MUC1 that is disrupted during cleavage in healthy and tumorigenic cells. It is.
The term “repeat” has its ordinary meaning in the art.

  The term “MUC1 growth factor receptor primary sequence” (PSMGFR) refers in some cases to peptide sequences defined as most or all of MGFR, and functional variants defined below and fragments of the peptide sequences. is there. PSMGFR is defined as SEQ ID NO: 36 below, and all functional variants and fragments thereof are integers up to 20 (ie, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid substitutions and / or addition or deletion of up to 20 integer amino acids at the N-terminus and / or C-terminus . In the above text, “functional variant or fragment” refers to a ligand that specifically binds to or otherwise specifically interacts with a peptide of SEQ ID NO: 36 or not. Variants that have the ability to interact specifically, while not binding strongly to the same region of these same other peptide molecules, resulting in the ability of the peptide molecule to aggregate (ie self-aggregate) with other same peptide molecules Or call a fragment. One example of PSMGFR, a functional variant of the PSMGFR peptide of SEQ ID NO: 36 (nat-PSMGFR, referred to as “natural”) is SEQ ID NO: 7 (var-PSMGFR, natural —SRY-- It differs from nat-PSMGFR by including a -SPY-sequence instead of (see). var-PSMGFR will enhance structural stability, which is important for certain applications such as antibody production, compared to the natural form. PSMGFR includes both native peptides and peptides that have undergone enzymatic modifications such as phosphorylation, glycosylation, and the like. Histidine-binding PSMGFR (see, eg, SEQ ID NO: 2) is abbreviated herein as His-PSMGFR. The His-binding peptide sequence is usually attached to the C-terminus. In certain embodiments, the invention is an isolated protein or peptide comprising, or consisting of, PSMGFR, eg, at the N-terminus of the protein or peptide, as set forth in SEQ ID NOs: 1, 2, 3, 6 or 7 below. An isolated protein or peptide that does not contain any of the amino acid sequences described above is provided. In one embodiment, the present invention is an isolated protein or peptide comprising or consisting of His-PSMGFR at the N-terminus of the protein or peptide, for example, as set forth in SEQ ID NO: 1, 2 or 3 below. An isolated protein or peptide that does not contain any of the amino acid sequences described above is provided.

  The term “MUC1 growth factor receptor extension sequence” (ESMGFR) is the peptide sequence defined below (see SEQ ID NO: 3) defined as all His-var-PSMGFR plus 9 amino acids at the proximal end of PSIBR. .

  The term “MUC1 growth factor receptor tumor-specific extension sequence” (TSEMGFR) is found in tumor cells that remain attached to the cell surface and interact with the active ligand in a manner similar to PSMGFR. Is a peptide sequence that defines a MUC1 cleavage product (see, eg, SEQ ID NO: 66 for an example).

PSIBR is the following peptide sequence (see SEQ ID NO: 8) that defines most or all of IBR.
“Cleaved interchain junction region” (TPSIBR) is a peptide sequence (see SEQ ID NO: 65) defined below as the smaller portion of IBR released from the cell surface after receptor cleavage of some tumor cells.

  PSMGFRTC can be N-terminal 30 or up to about 30 (ie 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids) and a truncated containing the full-length MUC1 receptor transmembrane and cytoplasmic sequences MUC1 receptor isoform. As used herein, the term “at the N-terminus” which refers to the position of the described sequence within a larger molecule, such as a polypeptide or receptor, is 30 from the N-terminal amino acid of the molecule. Such sequences are referred to as the following amino acids. Optionally, as discussed below, PSMGFRTC as well as other truncated MUC1 receptor isoforms are typically 20 to 30 amino acids long MUC1 N-terminal signaling sequence (SEQ ID NOs: 47, 58). Or 59), or a functional fragment or variant thereof. Such sequences are usually encoded by a nucleic acid construct encoding a truncated MUC1 receptor isoform, translated, and usually cleaved in the cell membrane of the cell prior to or upon insertion of the receptor. Such a PSMGFRTC, ie a sequence comprising the desired signaling sequence, would be a peptide or protein “having a PSMGFR sequence” “N-terminal” as defined above. An example is nat-PSMGFRTC (SEQ ID NO: 37, most N-terminal SEQ ID NO: 47) with nat-PSMGFR (SEQ ID NO: 36) at the N-terminal end (ie, most N-terminal or within its 30 amino acid range). With or without 58 or 59 signaling peptides).

  The term “separation” means physical separation from a cell, ie a situation in which a part of MUC1 immobilized on a cell is no longer immobilized on that cell. For example, in the case of cleavage of a portion of MUC1, if it migrates freely from the cell and is subsequently detected in body fluid, the cleaved portion is “separated” or separated from the cleaved cell. It is fixed at a position such as another cell or lymph node.

  The term “binding” refers to a mutual affinity or binding ability, usually a specific or non-specific binding or an interaction between corresponding pairs of molecules, including biological, physiological and / or pharmaceutical interactions. An interaction. Biological binding is defined as the type of interaction that occurs between pairs of molecules including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, and the like. Specific examples include antibody / antigen, antibody / hapten, enzyme / substrate, enzyme / inhibitor, enzyme / cofactor, binding protein / substrate, carrier protein / substrate, lectin / carbohydrate, receptor / hormone, receptor / Activator, complementary strand of nucleic acid, protein / nucleic acid repressor / inducer, ligand / cell surface receptor, virus / ligand and the like.

  The term “binding partner” refers to a molecule capable of binding to a specific molecule. An example is a biological binding partner. For example, protein A is a binding partner for the biological molecule IgG and vice versa.

  The term “aggregate” (noun) refers to a plurality of cell surface receptors or fragments thereof (eg, MUC1) that are immobilized to each other with or without auxiliary mediators to the host system. Means. Self-aggregation of healthy receptors on the cell surface; self-aggregation of cleaved receptors or fragments bound to each other; cleaved receptors or fragments bound to receptors or fragments attached to the cell surface; Receptors or fragments that are immobilized on each other via a mediator auxiliary to the host, whether or not cleaved. “Auxiliary mediators to the host system” includes synthetic species such as polymers, dendrimers, or naturally occurring species, such as IgM antibodies, which are not naturally occurring in the host system, but are external sources of the host system. To be added to the host system. This excludes aggregation that is the result of mediators naturally present in host systems such as growth factors that can cause disease-associated aggregation (inducible multimerization). “Aggregate” (verb) or “aggregation” means the process of forming an aggregate (noun).

  “Inducible multimerization” refers to aggregation in which formed aggregates can induce cells to grow or proliferate. Inducible multimerization usually involves dimerization or tetramerization of cell surface receptors, for example by growth factors or other active ligands, but the degree of multimerization forms natural receptor clusters in specific cell types It includes higher dimensional multimerization unless it is very large to mimic. This prevents the receptor from signaling to the cell to grow or proliferate.

  “Prophylactic clustering” refers to receptor multimerization to form aggregates involving a sufficient number of receptors that mimic the natural receptor cluster formation of a particular cell type. This prevents the receptor from signaling to the cell to grow or proliferate, for example by a mediator auxiliary to the host system.

  A “ligand” to a cell surface receptor refers to a substance that can interact with the receptor to change its structure and / or function either temporarily or permanently. Examples include, but are not limited to, receptor binding partners (eg, antibodies or antigen-binding fragments thereof) and substances (eg, denaturing enzymes) that can alter the chemical structure of the receptor. .

  An “active ligand” refers to a ligand that can interact with a receptor that transmits a signal to a cell. Active ligands include species that induce inductive multimerization of cell surface receptors, such as a single molecular species that has one or more active sites capable of binding to the receptor; dimerization, tetramerization, and more Examples include, but are not limited to, large multimers, bivalent antibodies or bivalent antigen-binding fragments thereof, or complexes comprising multiple molecular species. Active ligands can also include species that modify the receptor such that the receptor transmits a signal. The enzyme also activates the ligand when the active ligand modifies the receptor to create a new recognition site for other active ligands. For example, glycosylases activate a ligand when the addition of carbohydrate enhances the affinity of the ligand for the receptor. A cleavage enzyme is an active ligand when the cleavage product is the more active form of the receptor, for example by making the recognition site of the ligand more sensitive. Within the MUC1 tumor cells, the active ligand is a species that cleaves MUC1 and chemically modifies the receptor, or transmits a signal to cells that stimulate proliferation, with MGFR on the surface of the MUC1 tumor cell. Interacting species, such as species that undergo inductive multimerization.

  A “growth factor” is a species that falls in or does not fall within the previously identified class of growth factors, which acts as a growth factor that acts as an active ligand.

  “MUC1 display cell” refers to both non-cancerous and cancerous cells that express MUC1 and / or MGFR on their surface. “MUC1 tumor cell” or “MUC1 cancer cell” or “cancerous MUC1 cell” refers to a cancerous tumor cell that abnormally expresses MUC1 and / or MGFR on its surface.

  As used herein, “colloid” means nanoparticles, ie, very small self-suspendable or liquid-suspendable particles, such as inorganic or organic, polymers, ceramics, semiconductors, metals (eg, Gold), non-metallic, crystalline, amorphous, or combinations thereof. In general, the colloidal particles used in the present invention have a cross section of 250 nm or less in any dimension, more typically a cross section of 100 nm or less, most often about 2 to 30 nm. One class of colloids suitable for use in the present invention has a cross section of 10-30 nm, and others have a cross section of about 2-10 nm. As used herein, this term includes definitions commonly used in the field of biochemistry.

  As used herein, a component that is “immobilized to” another component binds to the other component, eg, by binding to a third component that is also bound to the other component. Or indirectly bound to other components or transitioned to and associated with other components. For example, if a signaling agent is bound to a binding species, bound to a colloidal particle to which the binding species are bound, or bound to a dendrimer or polymer to which the binding species are bound, the signaling agent is bound to the binding species. On the other hand, it is fixed. If the species bound to the surface of the first colloidal particle is attached to a substance, the species on the surface of the second colloidal particle is attached to the same substance, and this substance is a substance, a mixture of substances, cells, For example, colloidal particles are immobilized on other colloidal particles.

  “Signal transmitter” means a substance capable of showing its presence in a specific sample or at a specific position. The signal transducing substance of the present invention can be confirmed with the human naked eye, is not visible when released, but can be detected with the human naked eye if it is in a sufficient amount (for example, colloidal particles), Substances that absorb or emit electromagnetic radiation within a certain concentration or wavelength range, so that they can be easily detected visually (using a microscope, including the naked eye or an electron microscope) or by spectral analysis A material that is detected electrically or electrochemically, such as a redox-active molecule that exhibits a characteristic oxidation / reduction pattern when exposed to a suitable activation energy (“electron signaling substance”). Examples include dyes, pigments, electroactive materials such as redox active molecules, fluorescent molecules (including phosphorescent molecules in the definition), upstream control phosphorus, chemiluminescent materials, electrochemiluminescent materials, or horseradish peroxidase And enzyme linked signaling substances including alkaline phosphatase. “Signal transmitter precursors” do not themselves have the ability to transduce signals, but may interact with other species through chemical, electrochemical, electrical, magnetic, or physical interactions with other species. Become. Examples include chromogens that have the ability to emit radiation within a specific detectable wavelength range only upon chemical interaction with another molecule. Signaling agent precursors are distinguished from “signaling agents” as used herein, but are included in this definition.

  As used herein, “attached or applied to be bound” in another species or species relative to the surface of an article refers to covalent attachment, specific biology. It means that the species are chemically or biochemically bound, such as by attachment by chemical binding (eg, biotin / streptavidin), coordination bonds such as chelate / metal bonds, and the like. In this context, for example, “coupled” includes multiple chemical bonds, multiple chemical / biochemical bonds, etc., binding species such as peptides synthesized on polystyrene beads, proteins attached to the beads A binding species that is specifically biologically bound to an antibody bound to a protein such as A, a binding species that forms part of a molecule (by genetic engineering) such as GST or phage, and is covalently bound to the surface. However, it is not limited to those which are biologically bound one after another specifically (for example, glutathione in the case of GST). As another example, since thiol is covalently bonded to gold, molecules that are covalently bonded to thiol are applied to bind to the gold surface. Similarly, a species with a metal binding tag is applied to be bound to a surface with a molecule covalently bound to that surface (such as a thiol / gold bond). The molecule also exhibits a chelate that coordinates to gold. If the surface has a specific nucleotide sequence, the species is also applied to be bound to the surface, and the species includes a complementary nucleotide sequence.

  “Immobilized by covalent bonds” means immobilized by nothing but one or more covalent bonds. For example, a species that is covalently bound by EDC / NHS chemistry to an alkylthiol-indicating carbohydrate that is sequentially immobilized on the gold surface is covalently immobilized on the surface.

  “Specifically immobilized” or “applied to be specifically immobilized” as described above with respect to the definition of “immobilized or applied to be immobilized” In addition, it means that a species chemically or biochemically binds to another species or surface, but excludes all non-specific binding.

  Certain embodiments of the present invention utilize articles such as colloidal particles having a self-assembled monolayer (SAM) on the surface, such as the surface of the colloidal particles, and a SAM-coated surface. In one set of preferred embodiments, a SAM fully formed of synthetic molecules completely covers a surface or a region of the surface, eg, completely covers the surface of a colloidal particle. In the present text, “synthetic molecule” means a molecule that does not exist in nature, but rather is synthesized under human guidance or human creation or control involving humans. As used herein, “completely covering” means that there is no portion of the surface or area that is in direct contact with other species that prevent complete and direct coating with proteins, antibodies or SAM. That is, in a preferred embodiment, among other things, the surface or region comprises a SAM consisting entirely of non-natural molecules (eg, synthetic molecules). SAMs form SAM-forming species that form closely packed SAMs on the surface, or these species in combination with molecular wires, or other species that can facilitate electronic signaling in the SAM (in the SAM (Including defect-promoting species that can participate), or other species that can participate in SAM, and combinations thereof. Preferably, all of the species involved in the SAM include functionality that is optionally covalently attached to the surface, such as a thiol covalently attached to the gold surface. In the present invention, a self-assembled monolayer on a surface is a mixture of species that can essentially display (expose) any chemical or biochemical functionality (eg, a thiol species when gold is the surface). Consists of. To resist non-specific adsorption, for example, a triethylene glycol-terminated species (eg, triethylene glycol-terminated thiol), and other species (eg, thiol) that terminate with an affinity tag binding partner, eg, nickel Complexes with atoms include species terminated with a chelate capable of coordinating a metal such as nitrilotriacetic acid that captures a metal binding tag species such as a histidine tag binding species. The present invention provides a method for tightly controlling the concentration of chemical or biochemical species presented on a colloidal surface or other surface. Without tightly controlling the peptide density on each colloidal particle, the co-immobilized peptides aggregate easily with each other, even if there are no aggregate-forming species present in the sample. Forms a hydrophobic domain. This is an advantage of the invention over existing colloid agglutination analyses. In many embodiments of the invention, the self-assembled monolayer is formed on gold colloidal particles.

  The kits described herein include one or more containers that can contain compounds such as the species, signaling agents, biomolecules and / or particles described above. The kit may also include instructions for mixing, diluting and / or administering the compound. The kit may also include one or more solvents, surfactants, preservatives and / or diluents (eg, other containers containing normal saline (0.9% NaCl, or 5% dextrose), As well as the need for such treatment, it also includes a container for mixing, diluting the components into a sample, or for administering to a patient.

  The compounds in the kit may be provided as a liquid solution or a dry powder. Where the provided compound is a dry powder, the powder may be reconstituted by the addition of a suitable solvent. This is also provided. Liquid compounds are concentrated or used immediately. The solvent depends on the compound and the use or mode of administration. Suitable solvents are well known in pharmaceutical compounds and are available from the literature.

  As used herein, the term “cancer” includes biliary tract cancer; bladder cancer; brain cancer including glioblastoma and medulloblastoma; breast tumor; cervical cancer; choriocarcinoma; Endometrial cancer; Esophageal cancer; Gastric cancer; Hematological neoplasms including acute lymphocytic leukemia, myeloid leukemia; Multiple myeloma; AIDS-related leukemia and adult T-cell leukemia lymphoma; Epithelium including Bowen's disease and Paget's disease Liver cancer; lung cancer; lymphoma including Hodgkin's disease and lymphocyte lymphoma; neuroblastoma; oral cancer including squamous cell tumor; epithelial cells, stromal cells, germ cells and mesenchymal cells Pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell Skin cancer including cancer; Testicular cancer, including embryonic tumors such as thyroid, nonseminomas (teratomas, choriocarcinoma), stromal and germ cell tumors; thyroid cancer, including thyroid and medullary cancers; and adenocarcinoma and Wilms tumor Rectal cancer including, but not limited to. Preferred cancers include breast tumors, prostate cancer, lung cancer, ovarian cancer, colorectal cancer, and brain cancer.

  As used herein, the term “cancer treatment” includes, but is not limited to, chemotherapy, radiation therapy, adjuvant therapy, or a combination of these methods. Treatment embodiments that may vary include, but are not limited to, dosage, administration timing, or duration or therapy, and may or may not be combined with other treatments. They may also vary in dosage, timing, or duration. Another treatment for cancer is surgery, which can be used alone or in combination with any of the above treatment methods. Those skilled in the medical arts will determine the appropriate treatment.

  By “substance that inhibits cancer or tumor formation” is meant a substance that prevents any of the methods involved in cancer or tumor formation described herein. For example, a substance that interacts with (eg binds to) MGFR reduces or prevents the interaction of MGFR with a substance that promotes tumorigenesis by interacting with MGFR.

  As used herein, a “substance that reduces the cleavage of the interchain binding region of a cell surface receptor” refers to the cleavage of the MUC1 receptor between the N-terminus of MGFR and IBR that occurs in the absence of that substance. A composition that inhibits or reduces. Cleavage of the receptor between the MGFR and the N-terminus of IBR can occur by the activity of enzymes that are membrane related or soluble, such as matrix metalloptases (MMP and MT-MMP). Some of these enzymes react directly to cleavage. Other enzymes modify MUC1 with sugar groups or phosphates that mask recognition epitopes associated with cleavage, affecting the cleavage (eg, blocking cleavage at specific locations). Other enzymes can denature MUC1 with sugar groups or phosphates that create a recognition motif for cleavage at a specific position and promote cleavage at that position. Other enzymes can activate other cleavage enzymes to promote receptor cleavage. One method for selecting substances that reduce the cleavage of the cell surface receptor IBR is to first identify the enzymes that affect the cleavage as described above, and substances and analogs related to the ability to alter the activity of these enzymes Screening. Other methods test substances known to affect the activity of similar enzymes (eg, from the same family) with respect to their ability to alter the cleavage site of MUC1, and similarly test analogs of these substances That is. Alternatively, a substance is screened by a cell-free assay containing an enzyme and a MUC1 receptor, and the cleavage rate or position is measured by antibody probing, polymerase chain reaction (PCR), or the like. Alternatively, without first identifying an enzyme that affects MUC1, the substance is screened against cells displaying MUC1 for the ability of the substance to alter the cleavage site or rate of MUC1. For example, the substance can be screened with an assay that includes whole cells displaying MUC1, and is an indicator of the ability of the cell supernatant to aggregate, the amount of IBR remaining attached to the cleaved portion of MUC1, ie, between MGFR and IBR Measure the degree of cleavage. In other techniques, substances are screened with an assay that includes whole cells displaying MUC1. Cells remaining after removal of the supernatant are tested for accessibility of the MGFR moiety, eg, using a labeled antibody against MGFR. Substances can be identified from commercially available sources such as molecular libraries or rationally designed based on known substances with the same functional capabilities and tested for activity using screening assays.

  A “substance that reduces the cleavage of MUC1 receptor” is a composition that prevents or reduces the cleavage of MUC1 receptor at any position. Such substances can be used to treat a subject suffering from or at risk of developing cancer. If cleavage is blocked, then the contact of a functional receptor associated with cancer, MGFR, is reduced or blocked. Such substances are selected by exposing the cells to the candidate drug and measuring the amount of cleaved MUC1 receptor in the supernatant relative to the control.

  As used herein, a subject is any mammal (preferably a human) and preferably a mammal susceptible to tumorigenesis or cancer associated with abnormal expression of MUC1. . Examples include humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, or cats. In general, the present invention is directed to human use.

  The sample used in the present specification is a body tissue or body fluid sample obtained from a subject. Preferred are body fluids such as lymph, saliva, blood, urine, milk and breast secretions. Blood is most preferred. Tissue and / or cell samples used in the various methods described herein include tissue biopsies, including punch biopsies and cell scrapes, needle biopsies, and blood or other body fluids by aspiration or other methods. Obtained by standard methods including, but not limited to, collection.

  The following patent applications and publications describing compositions, articles and methods useful for practicing the present invention; US Patent Application Publication No. 2003/0036199, International Publication Number 02 / 056022A2, International Patent Application No. PCT / US00 / 01997 (filed Jan. 25, 2000, title of invention "rapid and sensitive detection of abnormal protein aggregation in neurodegenerative diseases", published as WO 00/43791), international patent application number PCT / US00 / 01504 (2000) Filed on Jan. 21st, entitled “Analysis involving colloidal and non-colloidal structures”, published on Jul. 27, 2000 as International Publication No. WO 00/34783, US Patent Application No. 09 / 631,818 (Filing Date 2000) Aug. 3, 1980, entitled "Rapid and Sensitive Detection of Protein Aggregation"), US Provisional Patent Application No. 6 by Bamdat et al. / 248,865 (filing date November 15, 2000, title of invention "Endostatin-like angiogenesis inhibition"), and US patent application (filing date November 15, 2001, filing of the same invention name) are: Introduced herein by reference. Each of the aforementioned patents, publications and applications is incorporated herein by reference.

  In one aspect, the present invention is involved in the treatment and diagnosis of novel molecular targets for drug screening and cancers characterized by abnormal expression of a class of cell surface receptors characterized by interchain binding regions. One set of cancers is characterized by abnormal expression of MUC1. Much of the description of the invention involves cells that abnormally express MUC1. In these examples, the description should be considered as an example, and the principles of the invention apply to other cell surface receptors that function by a similar mechanism. The description herein allows those skilled in the art to readily identify other cell surface receptors that function by this or similar mechanisms, and for these cancers characterized by abnormal expression of the receptors. The present invention can be applied. The present invention is based on a novel mechanism involving a cell surface receptor having a self-aggregating region represented by MUC1. This has been demonstrated by the inventors. MUC1 includes several regions described herein as follows. These regions in turn start from the region closest to the cell surface and progress away from the cell. US Patent Application Publication No. 2003/0036199, International Publication No. 02 / 056022A2, “Previous Application” filed by the same inventor, defined certain areas of MUC1 differently. It should be understood that the definition of the invention supersedes. In the earlier application mentioned above, the term “PSMGFR” relates to the exemplary peptide sequence of SEQ ID NO: 7 (currently referred to as “var-PSMGFR”). The expanded definition of PSMGFR described above is intended to apply to this application. The basic structure of the MUC1 receptor is shown in FIG. The receptors shown include 1) cytoplasmic tail, 2) membrane permeation cross section, 3) MGFR, 4) IBR, 5) intrinsic region, 6) repeat, and N-terminal region containing signal peptide.

  In healthy cells, the MUC1 receptor clusters in one part of the cell surface. Conversely, MUC1-positive tumor cells are characterized by a loss of this “healthy” cluster formation. The present invention contemplates use for detecting and treating abnormal expression of the MUC1 receptor in situations other than cancer. For example, the MUC1 receptor is an important factor for immune responses and fertilization. In the case of fertilization, it may be beneficial that a portion of the extracellular domain is cleaved to induce embryo transfer. The methods of the invention may be used in non-cancerous conditions that promote or inhibit receptor cleavage. Furthermore, the method of the present invention may be used to diagnose fertilization status. In tumor cells, the MUC1 receptor no longer clusters, but instead is a series of cells that are normally distributed on the whole cell surface or in some cancer types, and the receptor is expressed on a significant portion of the cell surface. Form a clustered island. Loss of MUC1 receptor clustering is associated with oncopathogenicity, metastatic potential and patient end result. The inventors herein referred to as MGFR and have shown that the cleavage product of the MUC1 receptor that remains attached to the cell surface functions as a growth factor receptor. When this part of the receptor is utilized for ligand activity, cell proliferation is stimulated. The MGFR portion of the receptor can be accessed to the active ligand in various ways. For example, cleavage of a receptor that releases some or all of IBR brings MGFR closer to the active ligand. Agents that reduce the cell surface expression of the complete MUC1 receptor greatly disengage the receptor from the cluster and thus increase the effectiveness of PSMGFR and ESMGFR as active ligands that signal and proliferate to the cell. Substances that essentially completely block the expression of the MUC1 receptor are good therapeutic candidates and are provided by one aspect of the present invention. Examples of such inhibitors include, but are not limited to, antisense oligos and RNA or inhibitory RNA.

  In some cases, the MUC1 receptor is cleaved to release IBR or TPSIBR from the cell surface. Alternatively, the cleavage can result in the release of a sufficient portion of IBR that causes the self-aggregation ability of the MUC1 receptor to be impaired. Loss of MUC1 aggregation will be a consequence of several events. Release of a sufficient portion of IBR from the IBR or the cell surface allows the receptor to ultimately distribute on the cell surface and freely bind the cytoplasmic tail to intracellular signaling proteins. External substances such as denatured enzymes and / or active ligands can then bind to the remaining extracellular portion of the receptor, undergoing a multimerization state, i.e. through changes in inducible multimerization, or inducible high Induces disease-related signals as subsequent structural changes. As will be appreciated by those skilled in the art, ligands such as growth factors or hormones often induce receptor dimerization that in turn causes an intracellular signaling cascade. Further support for this mechanism is that bivalent ligands such as MUC1 + tumor cell lines and transfected cells expressing a truncated isoform of the MUC1 receptor lacking IBR, a bivalent antibody against MGFR cause signaling, and ERK2 kinase phosphorylation. As shown by the detection of oxidation, it is shown in the data indicating that cell proliferation is due to the well-known MAP (mitogen-activated protein) kinase signaling cascade. Significantly, such phosphorylation and proliferation was not present or apparent in similar cells treated with a monovalent ligand to MGFR, such as a single chain antibody or a monovalent antigen-binding fragment of an antibody. .

  Cell proliferation will be the access of the MGFR moiety to an active ligand capable of interacting with the MGFR moiety. For example, the self-aggregating IBR of the MUC1 receptor forms a dense network that sterically blocks ligands such as growth factors from interacting with the MGFR portion of the receptor. In cancerous or tumor cells, this reticulum is lost, allowing the ligand to interact with MGFR.

  The model of the above mechanism is consistent with a mechanism in which a part of the MUC1 receptor remaining attached to the cell surface after the IBR region or TPSIBR is disrupted, that is, MGFR functions as a receptor for a ligand that causes cell proliferation. (A) Interaction between a portion of the MUC1 receptor (MGFR) that dimerizes the ligand and receptor causes cell proliferation, and (b) Interaction of this portion of the MUC1 receptor (MGFR) by the ligand Evidence is also provided herein that proves that blocking the cell block cell proliferation. When tumor cell lines in which the MUC1 receptor is uniformly expressed across the entire cell surface are treated with an IgG antibody of the invention against the MGFR portion of the MUC1 receptor (eg, PSMGFR), the rate of cell proliferation is greatly increased. Since the untreated IgG antibody is divalent, ie one antibody binds simultaneously to two adjacent MGFR moieties on the cell surface, these results indicate that the antibody acts as an active ligand and the MGFR moiety is dimerized. It demonstrates that it mimics the effects of growing growth factors and causes cell proliferation signaling consistent with signaling through the cytoplasmic tail of the receptor. This is further supported by the following experiments showing that dimerization of two adjacent MGFR moieties on the cell surface induces ERK-2 phosphorylation indicative of MAP kinase cell proliferation signaling (eg, (See FIG. 15). This discovery leads to two conclusions. First, an active ligand that binds to the MGFR portion of the MUC1 receptor results in inducible multimerization of the receptor. Second, an effective therapeutic strategy is therefore to block the MGFR portion of the receptor with a monomeric composition, preventing inductive multimerization and subsequent signaling cascades. For example, a single chain or monovalent antibody raised against the MGFR portion of the MUC1 receptor (eg raised against PSMGFR or against a peptide containing a PSMGFR sequence at the N-terminus), or untreated 2 Monovalent fragments of a titer antibody will function as an effective anti-cancer therapeutic (see discussion of antibodies and antigen-binding fragments thereof below). Data and examples supporting this discussion are given below. Another therapeutic strategy is to block the enzyme activity that denatures the receptor required for certain ligand binding.

  We show evidence that MUC1 receptor dimerization caused cell growth of T47D breast tumor cells. Dimerization is achieved by generating an IgG antibody to MGFR, which reminds us that the IgG antibody is divalent and can therefore dimerize a portion of the MUC1 receptor proximal to the cell surface. . A portion of the MUC1 receptor used was MGFR, and the peptide sequence used to generate the antibody was SEQ ID NO: 7 (var-PSMGFR). The present specification supports the premise that dimerization of MUC1 receptor causes cell proliferation in many MUC1 + tumor cell lines, but on cells that do not express or minimally express MUC1 receptor. Shows further experimental results that are virtually ineffective. MUC1 + breast tumor cells, T47D, 1500, 1504 and BT-474 were obtained from ATCC as described in Example 5 below. As controls, MUC1-breast tumor cells, MD-MB453, HEK (human embryonic kidney) cell line K293 and HeLa were also obtained from ATCC as described in Example 5 below. Western blot analysis as described in Example 5 below confirmed that all T47D, 1500 and 1504 cells expressed high levels of cleaved as well as uncleaved MUC1 and no control cells. Our analysis showed that cell line BT-474 did not express detectable concentrations of uncleaved MUC1, but expressed intermediate amounts of cleaved MUC1.

  As described in Example 8 below, a rabbit polyclonal antibody was produced against a synthetic peptide having a sequence derived from PSMGFR (var-PSMGFR-SEQ ID NO: 7) from a commercial antibody manufacturer (Zymed, CA). . The resulting antibody was purified by affinity chromatography on a column derivatized with the same peptide used to immunize rabbits. In order to confirm that the obtained antibody recognized MGFR of MUC1 receptor, the antibody was used as a cognate probe for Western blot. See Example 5. Here, samples of immune peptide and protein preparations obtained from MUC1 positive and MUC1 negative cell lines were run on a 15% polyacrylamide gel. A divalent antibody (which can be dimerized) was added along with a control cell line that was not expressed in a panel of breast tumor cell lines expressing the MUC1 receptor. The addition of antibody stimulated cell proliferation only in cells that expressed the MUC1 receptor. 1504 breast tumor cells treated with bivalent anti-PSMGFR antibody for 5 or 6 days caused a 400-600% enhancement of cell growth. See FIG. 6 and Example 11. Still referring to FIG. 6, control cells K293 and Hela were not affected by the same dose of the same antibody, anti-PSMGFR. The growth enhancement curve discusses that the antibody dimerizes the receptor. Since each receptor binds to a single antibody rather than one bivalent antibody bound to two receptors, the rate of cell growth is reduced at very high antibody concentrations. FIG. 7 shows that cells obtained from breast tumor cell line 1500 caused a 200% enhancement of cell proliferation after 3 days of treatment with bivalent anti-PSMGFR. When bivalent anti-PSMGFR treatment was extended for 4 days, the percent enhancement of cell proliferation increased to 300%. See FIG. Breast tumor cells obtained from the T47D cell line were tested for the ability of bivalent anti-PSMGFR antibodies to cause cell proliferation. FIG. 9 shows that, like the 1500 and 1504 cell lines, these cells also caused about 125% enhancement of cell growth. This reaction was dependent on antibody concentration. The breast tumor cell line BT-474 showed a similar stimulation of cell growth (150-200%) in response to divalent anti-PSMGFR. See FIG. The MUC1-cell line MDA-MB-453 was not affected by addition of anti-PSMGFR at any concentration (data not shown).

  The monovalent anti-PSMGFR fragment blocks cell growth in MUC1-positive tumor cells. As already described, MUC1 + tumor cells are induced to proliferate when the MUC1 receptor is dimerized. In particular, a proliferating signal occurs when a portion of the MUC1 receptor close to the cell surface is dimerized. As described above, one way in which the receptor dimerizes is through a bivalent antibody raised against the MUC1 receptor. In preferred embodiments, antibodies are raised against MGFR, and in yet preferred embodiments, raised against at least a portion of PSMGFR. As indicated above and below, substances that bind to the MGFR moiety of the monomeric MUC1 receptor rather than the dimer can block receptor dimerization, and thus cell proliferation. Inhibits. The following discussion describes several compounds that inhibit the growth of MUC1 + tumor cells by binding the MUC1 receptor to the MGFR moiety.

  As described above, another method of providing a substance that blocks dimerization of the MUC1 receptor is to generate a monovalent antibody produced against the MUC1 receptor or a monovalent antigen-binding fragment of an antibody. is there. Monovalent antibodies / fragments produced against the MUC1 receptor would be excellent therapeutic agents in MUC1-positive cancers. Monovalent antibodies / fragments that target the portion of the receptor close to the cell surface are preferred. Monovalent antibodies / fragments that bind to a portion of the MUC1 receptor that is the C-terminus of the beginning of the repeat are particularly preferred. Even more preferred are monovalent antibodies / fragments that target a portion of the receptor C-terminus to the unique region of the MUC1 receptor, and even more preferred are monovalent antibodies / fragments produced against the PSMGFR sequence.

  The peptide used for antibody production may or may not be glycosylated prior to immunizing the animal. The sequence of these peptides need not accurately reflect the sequence of the MUC1 receptor so that it exists in the general population. For example, the inventors have developed a PSMGFR peptide variant having a “-SPY-” motif, an antibody raised against var-PSMGFR (SEQ ID NO: 7), a real native sequence having a “-SRY-” motif. It was observed to have higher affinity and greater specificity for the MUC1 protein than the antibody produced against (ie, nat-PSMGFR, SEQ ID NO: 36). In some embodiments, mutations are introduced into the PSMGFR peptide sequence to produce a more robust peptide that enhances antibody production. For example, an R to P mutation in var-PFMGFR of SEQ ID NO: 7 actually gave a more robust peptide and was therefore immunogenic. Other methods of generating antibodies to regions of the peptide that are not particularly immunogenic, such as IBR or TPSIBR, attach an irrelevant sequence whose amino acids are D-forms to specific peptide sequences to stimulate the host animal's immune response. Is to work. Peptide sequences used to immunize animals in antibody production may also be glycosylated. The MUC1 peptide sequence that was used for drug screening and generated the cognate antibody was derived from the human species MUC1. Since there is considerable conservation across species in some parts of PSMGFR and IBR and UR, MUC1 peptides whose sequences are derived from other species can also be used for drug screening and to generate antibodies of these same purpose It is expected that it can be used. The present invention also relates in certain embodiments to the generation of bispecific antibodies and the bispecific antibodies formed thereby. Those of skill in the art are familiar with methods for generating antibodies that bind to adjacent sites of essentially the same antigen, although each recognition fragment of a divalent antibody is different.

  As described in more detail below, the MUC1 monovalent antibody / fragment used as a cancer therapeutic agent is a polyclonal or monoclonal antibody and is obtained by immunizing many different animal species, such as rabbits, goats and the like. It is done. In addition, techniques for generating hybridoma cells are known to those skilled in the art, which are then grown and collected to provide a supply of antibodies without having to repeatedly immunize the animal. Alternatively, humanized monovalent antibodies / fragments targeting these portions of the MUC1 receptor, as described in more detail below, may be used as effective anticancer agents that are less likely to utilize the patient's immune response. . The methods of the invention also include antibodies not involved in animal immunity and recombinant methods in Fab production.

  As described below, methods for generating monovalent antibodies and monovalent antigen-binding fragments of antibodies are known to those of skill in the art. The standard method is controlled proteolysis of divalent antibodies. The present inventors produced a monovalent PSMGFR-specific antibody fragment by proteolysis of the bivalent anti-PSMGFR of the present invention. Monovalent anti-PSMGFR competes with a divalent anti-PSMGFR antibody for the same binding site within the MGFR portion of the MUC1 receptor.

  The present invention details how in some embodiments a monovalent antibody / fragment to a portion of the MUC1 receptor close to the cell surface inhibits the growth of MUC1 + tumor cells. Monovalent antibodies / fragments targeting the PSMGFR portion of the MUC1 receptor were produced by proteolytically divalent anti-PSMGFR antibodies. This probably dimerized the MUC1 receptor and induced cell growth. Thus, the monovalent form of this antibody will bind to the MGFR moiety and block cell proliferation and prevent cognate ligand binding and / or dimerization.

  In this specification, we show experimental results demonstrating that monovalent antibody fragments targeting PSMGFR actually inhibit the growth of MUC1-positive tumor cells and have virtually no effect on control cell lines. provide. Referring to FIG. 6, note that the addition of divalent anti-PSMGFR induced a 600% enhancement of cell growth. In the method described in Example 11, FIG. 11 shows that the addition of the same anti-PSMGFR monovalent form to MUC1-positive breast tumor cells 1504 shows inhibited cell growth and induced cell death by about 150%. The opposite effect was shown. The addition of monovalent anti-PSMGFR showed a similar effect in breast tumor cell line 1500, which is also MUC1 +. See FIG.

  Monovalent anti-PSMGFR confirms in vitro drug screening. As described in detail below, this inhibits the color change of PSMGFR-immuno nanoparticles resulting from the addition of tumor cell lysate to the nanoparticles. It should be noted that monovalent antibodies / fragments can also inhibit PSMGFR peptide dimerization in vitro. Nanoparticulate drug screening assays that identify compounds that inhibit the dimerization of the MGFR portion of the MUC1 receptor are described in US patent publication 2003/0036199, hereby, and co-owned. 056022A2. In these analyses, histidine-added PSMGFR peptide (eg, SEQ ID NO: 2) was immobilized on NTA-Ni ++-SAM-coated gold nanoparticles. Lysates and supernatants obtained from MUC1-positive tumor cells, possibly containing the MUC1 receptor cognate ligand, were added to the Nao particles. Upon addition of the lysate / supernatant mixture, the color of the nanoparticle solution changes from a characteristic pink to blue, perhaps when the cognate ligand dimerizes the MUC1 receptor peptide on two different nanoparticles. Since the bivalent antibody also dimerizes the two PSMGFR peptides on different nao particles, adding the bivalent anti-PSMGFR antibody instead of the lysate / supernatant solution will change the nanoparticle solution from pink to blue To change. However, the addition of monovalent anti-PSMGFR to drug screening assays that also add lysate / supernatant probably inhibits the color change, competing with the natural cognate ligand that binds to the PSMGFR peptide. FIG. 13 shows that the characteristic nanoparticle color change that occurs upon addition of divalent anti-PSMGFR was inhibited upon addition of monovalent anti-PSMGFR.

  The results of the present invention also suggest that the MUC1 receptor is involved in apoptosis. The addition of monovalent anti-PSMGFR not only inhibits cell growth but also induces cell death. This indicates that the MUC1 receptor also mediates signal transduction pathways involved in the process of programmed cell death known as apoptosis.

  Current cancer research literature presents a confusing situation as to whether the total amount of MUC1 receptor produced by cells is related to metastatic potential or tumor pathogenicity. The results described herein support the notion that an important mechanism of cell growth in MUC1-positive cancers is dependent on the amount of MUC1 cleavage that occurs rather than the total amount of expressed MUC1 receptor. . Low molecular weight species that migrate on acrylamide gels with apparent molecular weights (some are glycosylated) of about 20-30 kD are present in MUC1-positive tumor cells but should be detected in non-tumor MUC1 cells There are not enough. We identified two cleavage sites for the MUC1 receptor in tumor cells. The first cleavage site occurs in the middle of IBR, and the second cleavage site that our evidence indicates is more of a tumor form occurs at the C-terminus of IBR. The first cleavage site is located at the N-terminus of TPSIBR (SEQ ID NO: 8) and the second cleavage site is located at the N-terminus of nat-PSMGFR having SEQ ID NO: 60. When cleavage occurs at the first site, the portion of the receptor that remains attached to the cell surface is similar to TSESMGFR (see SEQ ID NO: 66, with native SRY sequence). When cleavage occurs at the second site, the remaining portion is PSMGFR as shown in SEQ ID NO: 63. This low molecular weight tumor-specific species consists essentially of the native PSMGFR sequence, and in some cases the TSESMGFR sequence, which is useful as a cognate ligand, ie self Does not aggregate. In support of this conclusion, it was determined in the present text that the sensitivity of proliferating tumor cells was a function of the amount of the shorter form of the MUC1 receptor.

  A comparison of the results of the present invention obtained by Western blot analysis, which quantifies the amount of low molecular weight MUC1 species produced by each cell type tested, and the cell proliferation data described above is relative to antibody-induced cell growth. It shows that the sensitivity of breast tumor cells is proportional to the amount of low molecular weight MUC1 species (25-30 Kd glycosylated, 19-20 Kd non-glycosylated) produced by the cells. Referring to FIG. 14, breast tumor cell lines 1500 and 1504 produce significantly more MUC1 cleavage products that run at 19-20 Kd than BT-474BT cells or control K293 and HeLa cells. Correspondingly, the anti-PSMGFR that induced an increase during growth of cell lines 1500 and 1504 was about 400% or less (FIG. 8) and about 600% or less (FIG. 6), respectively. On the other hand, in the control cells (FIG. 6), there was no increase detected in the rate of cell growth, and the growth of BT-474 cells increased only to about 200% or less. See FIG.

  To further support the conclusion that the cleavage product of the MUC1 receptor functions as a growth factor receptor in tumor cells, either behind PSMGFR (SEQ ID NO: 7) or the entire interchain binding region (PSIBR) (SEQ ID NO: MUC1 mutants ending after 38) were transfected into HEK cells. Cells transfected with a receptor containing PSIBR grew at a rate 4-6 fold slower than cells transfected with a MUC1 mutant ending after PSMGFR (eg, SEQ ID NO: 37). These results support the conclusion that the portion of the MUC1 receptor that acts as a growth factor receptor is a cleavage product from which many or all of the IBR is released from the cell surface. Furthermore, these results indicate that tumors in which a large percentage of the MUC1 receptor is cleaved to release TPSIBR (SEQ ID NO: 65) are particularly aggressive cancers, leaving PSMGFR (SEQ ID NO: 63) attached to the cell surface as a whole. Those cleaved to release IBR support the conclusion that they are even more aggressive. Thus, antibodies raised against the TPSIBR (SEQ ID NO: 65) portion of the MUC1 receptor can be used to assess the virulence of cancers that are MUC1-positive.

  Consistent with these findings, the amount of MGFR approaching on the cell (tissue) predicts tumor virulence and metastatic potential. Thus, antibodies that recognize the MGFR portion of the receptor diagnose cancer or propensity to develop cancer, predict cancer pathogenicity and metastatic potential, suggest treatment protocols, and track the progression of treatment protocols Can be used for.

  As a result, the virulence or metastatic potential of tumor cells can be assessed by measuring the amount of low molecular weight MUC1 species produced by the cells. This can be measured, for example, by SDS-PAGE analysis or Western blot analysis using antibodies raised against the MGFR portion of the receptor or antigen-binding fragments thereof. In certain embodiments, the colloidal analysis techniques of the invention described herein are used, wherein the antibody / fragment is attached to a carrier that is a nanoparticle or colloid. In a preferred embodiment, patient cells are probed using antibodies raised against MGFR or antigen-binding fragments thereof. Importantly, this method reveals the amount of MGFR-containing MUC1 that remains attached to the cell surface, regardless of access to the cognate ligand. In a still preferred embodiment, the patient's cells are probed using an antibody raised against PSMGFR or an antigen-binding fragment thereof. In fact, cells presenting a high degree of MUC1 receptor that reacts with an antibody or antigen-binding fragment thereof that recognizes PSMGFR or a portion thereof will undergo a large degree of cleavage of the MUC1 receptor present on these cells, This is an indication that the PSMGFR moiety is in close proximity to a cognate ligand that activates the cell growth pathway. Tumors composed of such characteristic cells have a higher metastatic potential and / or are more aggressive. Thus, using an antibody or antigen-binding fragment thereof that recognizes PSMGFR or a portion thereof can be used to diagnose the metastatic potential or pathogenicity of a patient tumor.

  In certain aspects, the present invention provides antibodies or antigen-binding fragments thereof. In one embodiment, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to MGFR. In certain embodiments, such an antibody or antigen-binding fragment thereof is bivalent, and in other embodiments it is monovalent. In certain embodiments, the above-described antibodies or antigen-binding fragments thereof specifically bind to PSMGFR. In such embodiments, the antibody or antigen-binding fragment thereof has the amino acid sequence set forth in SEQ ID NO: 36, or an addition or deletion of no more than 15 amino acids at the N-terminus, or substitution of no more than 20 amino acids. It specifically binds to a functional variant having or a fragment thereof. In other embodiments, it specifically binds to an amino acid sequence set forth in SEQ ID NO: 36 or a functional variant having a substitution of 10 amino acids or less, or a fragment thereof. In other embodiments, the antibody or antigen-binding fragment thereof specifically binds to an amino acid sequence set forth in SEQ ID NO: 36 or a functional variant having a substitution of 5 amino acids or less, or a fragment thereof. In still other embodiments, the antibody or antigen-binding fragment thereof specifically binds to the amino acid sequence set forth in SEQ ID NO: 36. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are human, humanized, xenogeneic or chimeric human-non-human antibodies or antigen-binding fragments thereof. In certain embodiments, an antibody of the invention or antigen-binding fragment thereof comprises an untreated antibody or an untreated single chain antibody. In certain embodiments, for antibodies or antigen-binding fragments thereof that are monovalent, they comprise a single chain Fv fragment, Fab ′ fragment, Fab fragment or Fd fragment. For an antibody of the invention or antigen-binding fragment thereof that is bivalent, one embodiment includes an antigen-binding fragment that is F (ab ′) 2.

  The invention also provides compositions comprising, in one embodiment, the antibody of the invention or an antigen-binding fragment thereof as a component. In certain embodiments, such compositions comprise a pharmaceutical composition and further comprise a pharmaceutically acceptable carrier. In such compositions, the antibody or antigen-binding fragment thereof is a polyclonal antibody, and in other embodiments is a monoclonal antibody.

  The present invention also provides various kits comprising, in one embodiment, any of the above-described antibodies of the invention or antigen-binding fragments thereof. In certain embodiments, such a kit also provides an article having a surface. In such embodiments, the antibody or antigen-binding fragment thereof is immobilized on the surface of the article or can be adapted to be immobilized. In certain embodiments, the article includes particles. In such embodiments, the kit further comprises a second particle and a peptide sequence comprising a portion of the cell surface receptor that remains attached to the cell surface after disruption of the interchain binding region of the cell surface receptor. This peptide sequence is desorbed from the cell and immobilized on the second particle or applied to be immobilized. In certain embodiments, the kit further comprises a candidate agent that affects the ability of the peptide sequence to bind to the same other peptide sequence and / or antibody or antigen-binding fragment thereof in the presence of the antibody or antigen-binding fragment thereof. The provided peptide sequences can include MGFR in certain embodiments. In some of the kits described above comprising particles, the kit further comprises a portion of the cell surface receptor that remains attached to the cell surface after disruption of the interchain binding region of the cell surface receptor, such peptides The sequence is desorbed from any cell and immobilized on the particle or applied to be immobilized. In some embodiments, the kit further comprises a second particle and is immobilized on the second particle or has the peptide sequence described above applied to be immobilized. Such kits are useful in the context of the present invention to perform various diagnostics, drug screening and other analyses. These are involved in colloid-colloid interactions and / or aggregation, as described in detail herein.

  The present invention also relates, in certain embodiments, to methods of producing or generating a peptide, eg, an antibody or antigen-binding fragment thereof that specifically binds to a peptide of the invention described herein. In one particular embodiment, an antibody or antigen-binding fragment thereof is generated against a peptide comprising a portion of a cell surface receptor that interacts with an active ligand such as a growth factor that promotes cell proliferation. Such moieties are sufficient to contain cell surface receptors that interact with the active ligand and do not include interchain binding regions to the extent necessary to prevent spontaneous binding between such moieties. In such methods, the cell surface receptor comprises MUC1, in other embodiments MGFR, and in still other embodiments the peptide comprises PSMGFR at its N-terminus, and in still other embodiments the peptide Includes an amino acid sequence set forth in SEQ ID NO: 36 at its N-terminus, or a functional derivative or fragment thereof containing an addition or deletion of 15 amino acids or less and substitution of 20 amino acids or less at the N-terminus. In certain embodiments of producing an antibody of the invention or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof is generated against PSMGFR. In such embodiments, such a peptide used to generate an antibody or antigen-binding fragment thereof has an amino acid sequence set forth in SEQ ID NO: 36 or 37 or an addition of no more than 15 amino acids at the N-terminus or It consists of a functional variant containing a deletion and a substitution of up to 20 amino acids or fragments thereof.

  In still other embodiments, the present invention provides methods of treating a subject exhibiting cancer or other symptoms in need of treatment with one or more antibodies of the present invention or antigen-binding fragments thereof. In one such embodiment, the present invention provides a method of treating a subject suffering from a cancer characterized by abnormal expression of MUC1. The method includes administering to the subject an amount of an antibody or antigen-binding fragment thereof effective to ameliorate the cancer. In such embodiments, an amount of antibody or antigen-binding fragment thereof effective to reduce tumor growth is administered. In certain embodiments, any of the above antibodies or antigen binding fragments thereof, particularly those that specifically bind to MGFR, PSMGFR, etc. may be used. In one preferred embodiment, after disruption of the interchain binding region of the MUC1 receptor, an amount of antibody effective to block the interaction of the portion of the MUC1 receptor that remains attached to the cell, eg, MGFR, and the natural ligand, or The antigen binding fragment is administered. In other embodiments, the method involves administering an antibody or antigen-binding fragment thereof that is effective to reduce disruption of the interchain binding region of the MUC1 receptor. In many such embodiments of this method, particularly those in which an antibody or antigen-binding fragment thereof specifically binds to MGFR, such treatment methods induce induction of cancer-associated growth factor receptors such as abnormally cleaved MUC1. Involved in administering to the subject an effective amount of an antibody or antigen-binding fragment thereof to prevent targeted dimerization.

  In still other methods according to the invention, the antibody or antigen-binding fragment thereof can be used in a method for determining cancer pathogenicity and / or metastatic potential. In one such method, an antibody of the invention or antigen thereof that specifically binds a sample obtained from a subject suffering from or suspected of having cancer to a cancer-related peptide expressed on the cell surface Contact with the binding fragment. This method involves measuring the amount of antibody or antigen-binding fragment thereof that specifically binds to a sample. Such an amount is an indication of cancer virulence and / or metastatic potential. In such embodiments, the sample used comprises the subject's cells and / or solubilized lysates thereof. In such embodiments, the cell surface expressed peptide can comprise a portion of a cell surface receptor that interacts with an active ligand such as a growth factor that promotes cell proliferation. Here, the portion sufficiently includes a cell surface receptor that interacts with the active ligand, and does not include any interchain binding region within a range necessary to prevent spontaneous binding between the portions. In such embodiments, the cell surface receptor is MUC1 and the peptide is MGFR or a peptide comprising PSMGFR at the N-terminus. In certain preferred embodiments of such methods, the antibody or antigen-binding fragment thereof is immobilized on or is immobilized to a signaling agent, such as any of the signaling agents described above. Can be applied. In such embodiments, the signaling agent can include one or more particles, such as colloidal particles.

  Accordingly, the present invention includes peptide binding substances that are antibodies or antibody fragments that have the ability to selectively bind to, for example, PSMGFR and / or MGFR. Antibodies include polyclonal and monoclonal antibodies prepared by conventional methods.

  Significantly, as is well known in the art, only a small portion of the antibody molecule, the paratope, is involved in binding of the antibody to its epitope (generally Clark, WR (1986) The experimental Foundation of Modern Immunology Wiley & Sons, Inc. New York; Roitt, I. (1991) Essential Immunology, 7th Ed. Blackwell Scientific Publications, Oxford). For example, pFc ′ and the Fc region are agonists of the complement cascade, but are not involved in antigen binding. Antibodies produced without the pFc ′ region cleaved by the enzyme or the pFc ′ region, termed F (ab ′) 2 fragment, retain both antigen-binding sites of the untreated antibody. Similarly, antibodies produced by the Fc region being cleaved by the enzyme or without the Fc region, termed Fab fragment, retain one of the antigen binding sites of the untreated antibody molecule, Contains one of the antibody fragments. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of an antibody heavy chain meaning Fd. The Fd fragment is a major determinant of antibody specificity (a single Fd fragment may not associate with antibody specificity and may associate with up to 10 different light chains) and the Fd fragment is separated However, it retains the epitope-binding ability. Thus, in one embodiment of the invention, the monovalent antibody fragment may be an Fd fragment.

  As is well known in the art, within the antigen-binding portion of an antibody are a complementarity determining region (CDR) that interacts directly with the epitope of the antigen and a framework region (FR) that maintains the three-dimensional structure of the paratope. (See generally Clark, 1986; Roitt, 1991). There are four framework regions (FR1 to FR4), each separated by three complementarity determining regions (CDR1 to CDR3) in both the heavy chain Fd fragment and the light chain of an IgG immunoglobulin. The CDRs, particularly the CDR3 region, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.

  As is now well known in the art, the non-CDR region of a mammalian antibody may be replaced with a similar region of a homologous or heterologous specific antibody while retaining the epitope specificity of the original antibody. This is most clearly shown in the development and use of “humanized” antibodies in which non-human CDRs are covalently linked to human FRs and / or Fc / pFc ′ regions to produce functional antibodies. See, for example, U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,762, and 5,859,205. Such antibodies or fragments thereof are within the scope of the present invention.

  In certain embodiments, fully human monoclonal antibodies can also be prepared by immunizing mice that have been genetically engineered with most of the heavy and light chain sites of human immunoglobulins. Following immunization of these mice (eg, XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared by standard hybridoma technology. These monoclonal antibodies have human immunoglobulin amino acid sequences and thus will not cause human anti-mouse antibodies (HAMA) when administered to humans.

  In certain embodiments, the invention provides a chimeric antibody, humanized antibody, single chain antibody, Fab fragment, F (ab ′) 2 fragment, bispecific antibody, fusion antibody, labeled antibody, or analogs of any of these A method for producing an antibody of the present invention or an antigen-binding fragment thereof, comprising any of the steps of: Corresponding methods are known to those skilled in the art and are described, for example, in Harlow and Lane "Antibodies, Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Production of chimeric antibodies is described, for example, in WO89 / 09622. Methods for producing humanized antibodies are described, for example, in EP-A 10239400 and WO 90/07861. A further source of antibodies to be used in the present invention is so-called cross-species antibodies. General principles for the production of cross-species antibodies such as human antibodies in mice are described, for example, in WO 91/10741, WO 96/34096 and WO 96/33735. As described below, the antibodies of the present invention exist in various forms (in addition to untreated antibodies, eg, antigen binding fragments such as Fv, Fab and F (ab ′) 2, and single chains (ie, Single chain antibody). See WO88 / 09344.

  That is, as will be apparent to those skilled in the art, the present invention also provides, in certain embodiments, F (ab ′) 2, Fab, Fv and Fd fragments; Fc and / or FR and / or CDR1 and / or CDR2 and / or Or a chimeric antibody in which the light chain CDR3 region has been replaced by homologous human or non-human sequences; FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences (Ab ′) 2 fragment antibodies; chimeric Fab fragment antibodies in which FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences; FR and / or CDR1 and / or CDR2 Textures in which the region is replaced by homologous human or non-human sequences To provide a Fd fragment antibodies. The present invention also includes so-called single chain antibodies.

  Furthermore, the present invention, in one embodiment, relates to a composition comprising the above-described antibody or antigen-binding fragment of the present invention or a chemical derivative thereof. The composition of the present invention may further contain a pharmaceutically acceptable carrier. The term “chemical derivative” refers to a molecule that contains additional chemical moieties that are not normally a part of the base molecule. Such a moiety improves the solubility, half-life, absorption, etc. of the base molecule, or this part attenuates undesirable side effects of the base molecule or reduces the toxicity of the base molecule. Examples of such portions are described in various texts such as Remington's Pharmaceuticals Science. Suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil / water emulsions, various wetting agents, sterile water and the like. Compositions containing such carriers can be prepared by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of suitable compositions is accomplished in a variety of ways, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or endothelial administration. Aerosols such as spray nasal sprays contain purified aqueous solutions or other solutions of the active agent along with preservatives and isotonic agents. Such formulations are adjusted, for example, to intranasal administration, preferably to a pH and isotonic state compatible with the nasal mucosa. Rectal or vaginal dosage forms are provided as suppositories with a suitable carrier.

  A therapeutically effective dose refers to that amount of an antibody and / or antigen-binding fragment of the invention that ameliorates a symptom or condition of cancer or other treated disease. The therapeutic effects and toxicity of such compositions are determined by standard pharmaceutical procedures in cell cultures or laboratory animals, such as ED50 (dose that has a therapeutic effect in 50% of the population) and LD (50% lethal dose of the population). ). The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50.

  The biological activity of the antibodies and / or antigen-binding fragments thereof of the present invention indicates that they have sufficient affinity to be candidates for drug localization to appropriate surface structures, eg, cells expressing MGFR. Show. Accordingly, agents wherein the targeting and binding of antibodies and / or antigen-binding fragments thereof of the present invention to cells are therapeutically or diagnostically active (targeted drugs, DNA sequences, RNA sequences, lipids, proteins and gene therapy / gene delivery Etc.) are useful. Accordingly, the antibodies and / or antigen-binding fragments thereof of the present invention are labeled (eg, fluorescence, radioactivity, enzymes, nuclear magnetism, colloids, other signaling agents, etc.) and include in vivo including “immunochemistry” -like analysis. Or used to detect specific targets in vitro. In vivo, they can be used in methods similar to nuclear drug imaging techniques to detect tissues, cells, or other substances that express MGFR. Other methods include delivering a therapeutically active agent to the patient. The method includes administering to the patient at least one antibody or antigen-binding fragment thereof and a therapeutically active agent. Preferably, the therapeutically active substance is selected from pharmaceuticals, DNA sequences, RNA sequences, proteins, lipids, and combinations thereof.

  In certain embodiments, the present invention also includes drug screening assays, therapeutic protocol screening, including determining whether intracellular proteins are chemically denatured in a manner that indicates their involvement in intracellular signaling pathways. Provides analytical methods, diagnostic analysis methods, etc. One such method involves providing a cell that expresses on its surface a peptide that can act as a growth factor receptor, such as MUC1. Analytical methods contact such cells with candidate agents that affect the ability of the active ligand of the cell surface peptide to interact with the peptide in the presence of the active ligand, and if the active ligand interacts with the cell surface peptide, Determining whether the intracellular protein to be phosphorylated is actually phosphorylated. Such a method is particularly useful in the present invention in cells that express a peptide containing PSMGFR at the N-terminus, such as MGFR or PSMGFRTC.

  As described below, in embodiments involving MUC1-expressing cells, intracellular cell proliferation signaling is responsible for the interaction of the MAP kinase pathway and its ligands with cell surface receptors and concomitant inductive multimerization. And is involved in phosphorylation of intracellular proteins including ERK-2. In such cases, the screening method of the present invention comprises a sample comprising a plurality of cells that may be solubilized or permeabilized in certain embodiments after being exposed to a candidate agent and an activating ligand. use. In certain embodiments, after exposure to a candidate agent and an active ligand, followed by cell solubilization or permeabilization, the method can be performed in the intracellular contents of cells on a gel using, for example, Western blot techniques. Separating the protein contained in the protein and visualizing or otherwise detecting the separated protein. Such detection can be performed as is well understood by those of skill in the art using antibodies or other molecules that specifically bind to the intracellular protein to be detected. In certain embodiments, such a molecule is preferably an antibody or antigen-binding fragment thereof comprising an auxiliary signaling agent that allows detection or visualization, such as one of those already described. In some embodiments, detecting phosphorylation of intracellular proteins after gel separation can be achieved by phosphorylating intracellular proteins (eg, phos-ERK-2, but if not phosphorylated, intracellular proteins Including, but not limited to, contacting the gel-separated protein with a biological molecule that specifically binds to the antibody. Western blot techniques useful for accomplishing the above methods are well known to those skilled in the art and are described in more detail in Example 5 below.

  In one embodiment, instead of using the gel-based method described above to determine phosphorylation of intracellular proteins within the context of the analytical method of the invention, it is described elsewhere and herein. A colloid-based agglutination assay similar to that described above can be used to detect the presence of phosphorylated forms of intracellular proteins. In such an embodiment, after the above-described step of solubilizing or permeabilizing the cells exposed to the activating ligand and the candidate agent, the intracellular contents are contacted with a plurality of colloidal particles. The plurality of colloidal particles preferably bind specifically to a phosphorylated form of an intracellular protein, but when not phosphorylated, a first biological molecule such as an antibody or antigen-binding fragment thereof that does not bind to an intracellular protein. A first subset that is immobilized to and other biological molecules that specifically bind to intracellular proteins at an epitope that is different from the epitope to which the first biological molecule specifically binds, eg, A second subset of colloidal particles immobilized to the antibody or antigen-binding fragment thereof. If the sample contains a phosphorylated form of intracellular protein, the colloid will aggregate and a color change will be observed. However, if the intracellular protein is not phosphorylated, no aggregation indicating the cross-linking of the colloidal particles to each other will be observed.

  Such a method as described above determines whether a drug candidate presumed to have the ability to interfere with binding of an active ligand to a cell surface receptor interferes with binding of the active ligand to the cell surface receptor, and These methods facilitate the simultaneous determination of whether a drug candidate acts by interacting with a cell surface receptor or ligand. That is, when the present invention is achieved under conditions of excess ligand concentration (relative to drug candidate concentration), the above method for drug screening based on detection of intracellular protein phosphorylation or other variants is most advantageous. In contrast to the candidate agent acting through a mode of action where it binds or otherwise interacts with the ligand, the candidate agent interacts directly with, for example, a cell surface receptor, eg, is immobilized. Only when acting in this way tends to show a positive test result. This one-step behavior will be best observed when a screening assay is achieved utilizing cell surface receptor ligands sufficiently excessively. As a result, any binding of the candidate agent to the ligand that prevents binding of the ligand to the receptor does not decrease the ligand used in receptor binding and inductive multimerization within the assay system.

  Furthermore, as described immediately above, such colloid-based analytical methods are not limited to analytical methods and analytical systems that detect intracellular signal transduction via phosphorylation of intracellular proteins. The colloid-based analysis methods described above are more generally applicable. For example, such analytical methods can include screening tests to determine the denaturation state of essentially any biological molecule. Such screening methods include, in one embodiment, analysis involving the detection of colloidal particle immobilization to a biological molecule. Here, the colloidal particles are configured to be immobilized relative to the biological molecule when the biological molecule is in a different degree of the first modified state than when the biological molecule is in a differently modified state.

  Denatured states determined using such methods include whether a particular biological molecule is phosphorylated, glycosylated, acetylated, or the like. Furthermore, in more preferred embodiments, such colloid-based assays involve colloid-colloid aggregation for detection, while in other embodiments, the colloid used is the biological being assessed. It is simply used as a signaling agent to detect whether the molecule is denatured, such as using Western blots or other gel-based assays. For example, in one such assay, a biological molecule whose denatured state is to be detected binds specifically to the biological molecule when in the first denatured state, but in the second denatured state. At some point, it can be contacted with a substance that does not bind, such as an antibody. In some embodiments, such materials are immobilized, for example, on colloidal particles that provide a detectable signaling agent in the gel, or the colloid is attached to a biological molecule whose denatured state is determined. It is immobilized to another binding substance such as a secondary antibody having specificity for the substance to be bound.

  In one embodiment of such a method using a colloidal agglutination detection assay for determining the denaturation state of a biological molecule, a sample containing the biological molecule is a plurality of colloidal particles of at least first and second types. Contact. A first subset (type) of colloidal particles specifically binds to a biological molecule when in a first state of denaturation, and a first substance that does not bind to a biological molecule when in a second state of denaturation. The second set (type) of colloidal particles is immobilized to a second substance that specifically binds to a biological molecule with an epitope different from the epitope to which the first substance specifically binds. . As described above, in such a test, if the biological molecule or a subset thereof is in a first state of denaturation, the plurality of colloidal particles described above is proportional to the concentration of the biological molecule in the first state of denaturation. Will tend to agglomerate. This causes a color change in the analytical sample. However, if the biological molecule is present only in the second state of activity, the first type of colloid will not be bound to it and no colloidal crosslinking or aggregation or color change derived therefrom will occur. In certain embodiments of such analytical methods described above, the first substance on the first subset of colloidal particles is an antibody or other binding that specifically binds to a biological molecule when in the first state of denaturation. And a second substance on the second subset of colloidal particles will be a substance that binds to a biological molecule in either the first state of denaturation or the second state of denaturation.

  As described immediately above, such colloidal aggregation assays are advantageous, for example, to determine which of the multiple intracellular signaling pathways are activated when an active ligand binds to a cell surface receptor. Can be used. In such analytical methods, a plurality of different types of colloidal particles can be used, including a plurality of different first and second colloid types having substances immobilized thereon. Thereby, in one embodiment of one analytical method, it is possible to determine the activity of the various cellular signaling pathways within the cell to be determined, and the multiple different intracellular signaling proteins It is possible to detect a denatured state.

  As described above, one aspect of the present invention involves the discovery that dimerization of the extracellular portion of the MUC1 receptor activates the intracellular MAP kinase pathway, a known signaling cascade that induces cell proliferation. The present invention also relates to various discoveries relating to the discovery by the inventors that in some embodiments, dimerization of the MUC receptor causes cell proliferation via the MAP (mitogen-activated protein) kinase cell proliferation signaling pathway. Includes diagnostic assays, drug and therapeutic screening protocols, and more. More specifically, dimerization of the MGFR portion of the receptor is necessary and sufficient to activate the MAP kinase pathway and induce cell proliferation. The MAP kinase signaling cascade is one of the well-understood intracellular signaling pathways. In summary, mitogens bind to the extracellular portion of the transmembrane receptor and then change its structure so that the signal is transmitted inside the cell. As described above, the downstream step in this cascade is ERK2 phosphorylation. It is well known in the art that cell growth proceeds once ERK2 is phosphorylated. We show that the addition of a bivalent antibody that recognizes MGFR dimerizes the MUC1 receptor and in some way generates or indicates a binding site for a signaling protein that binds to the cytoplasmic tail of the MUC1 receptor. Demonstrate. FIGS. 15-17 show that dimerization of MUC1 receptor results in phosphorylation of ERK2 via divalent anti-PSMGFR in breast tumor cells T47D, 1504 and 1500, respectively. See Example 12. This effect is dose dependent and time dependent. Furthermore, the synthetic compounds we have already shown bind to the MGFR portion of the MUC1 receptor and compete with bivalent anti-PSMGFR in the binding of this region to the MUC1 receptor. In competitive inhibition analyses, this compound also (in a dose-dependent manner) effectively blocks the binding of the divalent antibody to MGFR, resulting in decreased receptor dimerization and decreased ERK2 phosphorylation. See FIG. 18 and Example 12.

  Furthermore, the monovalent anti-PSMGFR competed with the bivalent antibody and effectively inhibited ERK2 phosphorylation. When an excess amount of monovalent anti-PSMGFR was added to breast tumor cells with an amount of bivalent anti-PSMGFR shown to be sufficient to stimulate ERK2 phosphorylation, the monovalent antibody was probably the MUC1 receptor Since its dimerization was blocked, its phosphorylation was blocked. FIG. 19 shows that monovalent anti-PSMGFR competes with the bivalent antibody to block phosphorylation of ERK2 in cell line 1500.

  Thus, in one embodiment of the invention. The phosphorylation status of ERK2 can be monitored as a way to identify therapeutics for MUC1-positive cancers. It was mentioned above that monovalent anti-PSMGFR bound to a monovalent compound and MGFR competed with the bivalent antibody for its binding site, inhibited activation of the MAP kinase signaling pathway and did not result in ERK2 phosphorylation. Yes. This suggests drug screening to identify substances that affect signal transduction by the MUC1 receptor. In this drug screening, bivalent anti-PSMGFR is added to MUC1-positive tumor cells. Drug candidates are also added and the phosphorylation status of ERK2 is measured as already described. Cells in which ERK2 phosphorylation is inhibited indicate that the drug candidate successfully competed with the bivalent antibody for binding to MGFR and inhibited its dimerization and subsequent activation of the MAP kinase signaling pathway. . This drug screening method can also identify compounds that act on intracellular proteins that affect the ERK2 arm of the MAP kinase signaling pathway.

  Monitoring the extent of ERK2 phosphorylation induced by the addition of bivalent anti-PSMGFR also shows that any compound identified as being able to inhibit MUC1 cell proliferation is less than related factors such as ligands Rather, it provides a method for determining whether to inhibit such by directly binding to MGFR.

  Furthermore, a more effective MUC1 + cancer treatment protocol is to treat patients simultaneously with MUC1 receptors, drugs that target signaling elements in the MAP kinase / ERK2 pathway, and / or drugs that target ligands to MGFR. Can be born from.

  Another aspect of the present invention provides agents that bind together the MGFR portion of MUC1 following disease-related cleavage that carries out prophylactic clustering of receptors. This material may be any species that contains multiple sites, each capable of binding to the MGFR moiety, and may be immobilized with respect to each other. For example, polymers or dendrimers or other contiguous substances can contain multiple sites that can bind to MGFR moieties, respectively, and other structural limitations that inhibit association with these moieties or factors that promote cell proliferation. Cluster formation occurs. Alternatively, IgM-type monoclonal antibodies or polyclonal antibodies raised against MGFR or PSMGFR can be used. Each anti-MGFR IgM antibody was able to aggregate 10 MGFR on the cell surface to form a prophylactic cluster.

  Furthermore, the above-described antibody or antigen-binding fragment thereof that specifically binds to the MGFR portion of the MUC1 receptor may be a cytoplasmic agent or other substance (eg, radioactive) that can selectively kill cells to which the ligand is immobilized. By attaching a substance, the antibody or antigen-binding fragment thereof can be modified to act as a target delivery substance. In this way, such therapeutic agents can be directed to tumor cells. For example, a substance that binds to the MGFR portion of the MUC1 receptor can be denatured with a radioactive substance to destroy tumor cells that abnormally express the MUC1 receptor. Other toxic substances, such as ricin, as well as other therapeutic agents can be attached to substances that bind to MGFR. Alternatively, the antibody or antigenic determining fragment thereof that binds to MGFR can be denatured to present an imaging agent for use in diagnostic images of MUC1 + tumors and metastases. Such antibodies or antigen-binding fragments thereof can also be denatured to act as agents that are useful for the prevention and / or treatment of cancer. In one embodiment, an antibody or antigen-binding fragment thereof that binds multiple MGFR that produces inducible multimerization in a native form is denatured to remove or inactivate all but one of the active binding sites of MGFR. It becomes. As a result, each denatured antibody or antigen-binding fragment can bind to only one receptor. A particular example of this would be the production of monovalent fragments of anti-MGFR IgG, for example via enzymatic or other cleavage methods. In other embodiments, individual antibodies or antigen-binding fragments are immobilized to another ligand molecule / peptide that is capable of binding to MGFR, eg, via covalent bonding, non-covalent bonding, co-immobilization to a substrate, etc. To be denatured. As a result, the modified multi-unit ligand can perform prophylactic clustering of the receptor to which it binds.

  Identification of ligands for the portion of MUC1 that remains bound to cells after cleavage allows the development of powerful assays to screen for agents that disrupt this interaction. The interaction of potential binding partners with the extracellular part of MUC1 remaining after cleavage is determined by conventional techniques (Western blotting, ELISA, MALDI, etc.) and by our colloid-colloid color change assay or colloid bead color assay. Can be used to study both. The remaining extracellular portion of the peptide sequence of MUC1 can be attached to the beads or colloid via histidine addition. Potential binding partners can be histidine added, attached to a second set of colloids, and analyzed for binding to the colloid-immobilized portion of MUC1. Alternatively, potential binding partners can be attached to beads or colloids by EDC / NHS binding, or nonspecifically adsorbed to beads for analysis. The interaction of the MUC1 peptide and the potential binding partner can be detected either by solution color change (in colloid-colloid analysis) or by colloidal clot formation on the bead that makes the bead red. The entire cDNA library can be screened using this technique in a short time to identify the remaining extracellular MUC1 natural ligand. (PCT / US00 / 01997, WO00 / 34783, and 09/63818, filed Nov. 15, 2000, "Detection of binding species having colloidal and non-colloidal structures", inserted above)

  In certain embodiments of the invention, biopsy species are studied in common operations or tissues are studied (eg, tissue at the surgical site is covered) to determine tumor formation or the likelihood of tumor formation. Can be studied without removing tissue from the examiner). In either of these studies, the primary indicator of tumor formation or tumor formation potential is the amount of cell surface MGFR that is susceptible to interaction with external substances such as growth factors. This determination can be made, for example, either by using standard antibody binding research techniques or by exposing the sample to a colloid immobilized with an antibody specific for the MGFR region, and the above referenced international patent publication. Using the techniques described in the numbers WO 00/34788 and WO 00/43791, it can be done by measuring the binding of the colloid to the sample and measuring the amount of antibody to the MGFR region that binds to the sample. In other techniques (possibly more suitable for excised samples), antibodies to the MGFR region and IBR are exposed to the sample and the respective binding ratio to the sample is measured. Healthy samples will show little or no antibody binding to the MGFR region. Samples showing tumor formation or potential tumor formation will show that the ratio of MGFR antibody binding to IBR binding is not zero.

  One aspect of the invention is the identification of an antibody or antigen-binding fragment thereof that binds directly to the MGFR portion of the MUC1 receptor. Thus, a sensitive method for early tumor diagnosis is to administer to a patient an antibody or antigen-binding fragment thereof that binds to PSMGFR that has been induced with contrast or imaging agents. These antibodies or antigen-binding fragments thereof will aggregate on the tumor to which this part of the MUC1 receptor is approached. The antibodies described herein or antigen-binding fragments thereof that bind to the MGFR region, as well as other compounds that can be identified using the methods of the present invention, can be readily modified to carry imaging agents. Such imaging agents include, but are not limited to, technetium, 123I and other control agents or radioactive materials commonly used in imaging technology. Imaging techniques include, but are not limited to, single photon computed tomography (SPECT), MRI, and microscope. In some applications, the accessory colloid can act as an imaging agent. Since the imaging agent carrier can also be a therapeutic agent, this technique can combine early diagnosis with direct treatment.

  According to another aspect of the invention, a series of isolated proteins or peptides are provided. The peptides of the present invention include those defined above as PSMGFR and PSMGFRTC, and SEQ ID NOs: 1, 2, 3, 4, 5, 6, 36, 7, 8, 9, 37, 38, 39, 40, 41, 47, 60-66, and 14-35, but are not limited to these. Furthermore, the present invention encompasses all proteins or peptides not specifically described above, encoded by any of the isolated nucleic acid molecules of the present invention described below. The invention also encompasses unique fragments of the protein or peptide.

  Proteins can be isolated from biological samples including tissue or cell homogenates and expressed by recombinant techniques in various prokaryotic and eukaryotic expression systems. In the recombinant technique, an expression vector suitable for the expression system is constructed, the expression vector is introduced into the expression system, and the protein expressed by the recombinant technique is isolated. Short polypeptides, including antigenic peptides (such as those displayed by MHC molecules on the surface of cells in immune recognition) can also be chemically synthesized using well-defined peptide synthesis methods.

  That is, as used herein with respect to proteins, “isolated” means separated from the natural environment and present in an amount sufficient to permit its identification or use. When referring to a protein or polypeptide, isolation means, for example, (i) selectively produced by expression of a recombinant nucleic acid, or (ii) purified by chromatography or electrophoresis. . An isolated protein or polypeptide is substantially pure, but need not be. The term “substantially pure” means that the protein or polypeptide is free of other substances. Together with other substances, they will be found in nature and to the extent practical and appropriate for their intended use in nature or in vivo systems. A substantially pure protein will be produced by techniques well known in the art. Since the isolated protein is mixed in a pharmaceutical preparation with a pharmaceutically acceptable pharmaceutical preparation carrier, the protein will contain very little preparation. However, the protein has been isolated from other proteins, for example, so that it is separated from substances that associate in biological systems.

  The invention also encompasses unique fragments of the protein or peptide of the invention. A fragment of any one of the proteins or peptides of the invention has fragment aspects and features, including, for example, the unique fragments described herein, generally in association with a nucleic acid molecule. As those skilled in the art will appreciate, the size of a fragment that is unique will depend on factors such as whether the fragment constitutes a portion of a conserved protein region. That is, certain regions of the proteins or peptides of the present invention require longer segments that are unique, while others typically have 5-12 amino acids (eg, 5, 6, 7, 8, 9 Only a short segment that is 10, 11, and 12 amino acids long) will be required.

Intrinsic fragments of proteins are preferably those fragments that retain the distinct functional capabilities of the protein. Functional capabilities that can be retained in a fragment of a protein include interaction with an antibody, interaction with other proteins or fragments thereof, selective binding of nucleic acid molecules, and enzymatic activity. One important activity is the ability to act as a signature to identify the polypeptide.
Those skilled in the art are generally well versed in methods for selecting unique amino acid sequences based on the ability of fragments to selectively distinguish sequences of interest from non-family members. Usually, it is necessary to compare the sequence of fragments with those of known databases.

  The invention encompasses variants of the proteins or peptides described herein. As used herein, a “variant” of a protein includes one or more modifications of the primary amino acid sequence of such protein. Modifications that create protein variants allow such proteins to 1) produce, increase, decrease or eliminate protein activity; 2) protein stability such as protein stability or protein-protein binding stability in the expression system. By providing a novel activity or property to the protein, such as adding an antigenic epitope or adding a detectable moiety; and / or 4) providing equal or better binding to a ligand molecule is there. Modifications to the protein can be made through modifications to the nucleic acid molecule encoding the protein. And can include deletions, point mutations, truncations, amino acid substitutions, and addition of amino acid or non-amino acid moieties. Alternatively, modifications can be made directly to the protein by cleavage, substitution of one or more amino acids during chemical synthesis, addition of linker molecules, addition of detectable moieties such as biotin, addition of fatty acids, and the like. Denaturation also encompasses fusion proteins comprising all or part of the amino acid sequences of the present invention. Those skilled in the art will be familiar with methods for predicting the effects of amino acid sequence changes on protein structure. Thus, variant polypeptides can be “designed” by known methods. One example of such a method is the method described in Dahiyat and Mayo, Science 278: 82-87, 1997, where proteins can be newly designed. This method can be applied to known proteins to mutate only a portion of the protein sequence. Using Dahiyat and Mayo calculation methods, specific variants of the DOS protein can be proposed and tested to determine whether the variant retains the desired structure.

  In certain embodiments, the variant comprises a protein that has been specifically denatured to alter the aspect of the protein independent of the desired physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide bonds. Similarly, certain amino acids can be altered to remove proteolysis by proteases in the expression system to enhance protein expression (eg, dibasic amino acid residues in yeast expression systems where KEX2 protease activity is present) .

  Modification of the nucleic acid molecule encoding the protein or peptide of the present invention preferably preserves the amino acid reading frame of the coding sequence and preferably is a secondary structure such as a hairpin or loop that is detrimental to the expression of various proteins. Does not create a region in the nucleic acid that appears to hybridize to form.

  Denaturation can be done by selecting amino acid substitutions or by random mutagenesis of selected sites in the nucleic acid encoding the protein. The various proteins are then expressed and tested for one or more activities to determine which denaturation results in a variant protein having the desired properties. Furthermore, denaturation can be made to asymptomatic mutants (or non-mutant proteins) with respect to the amino acid sequence of the protein. However, this provides preferred codons for translation in specific hosts, well known to those skilled in the art. Still other modifications can be made to non-coding sequences of genes or cDNA clones that express proteins that enhance protein expression. Mutational activity of a specific protein is determined by cloning the gene encoding the mutant protein into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the mutant protein, and the function of the protein. Can be tested by testing for their ability to bind, interact with or interact with a specific ligand, or to act as a ligand for a specific biomolecule, such as a receptor. The preparation of other mutant proteins aids other activity tests, as is well known to those skilled in the art.

  Those skilled in the art also provide “functional variants” of certain protein or peptides in which certain amino acid substitutions, such as conservative amino acid substitutions, ie, variants that retain the functional capabilities of the corresponding protein or peptide of the invention. Therefore, it will be clearly understood that this is done in the protein or peptide of the present invention. As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Conservative substitutions of amino acids include the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e ) S, T; (f) Q, N; and (g) substitutions made between amino acids in E, D.

For example, in one embodiment, amino acid substitutions can be made, eg, conservative amino acid substitutions on the amino acid sequence of the protein or peptide of the invention. The substituted peptides can then be tested for one or more of the desired functions of the unsubstituted peptide in vivo and / or in vitro. These variants can be tested, for example, for improved stability or other desired properties. These make them especially more useful, for example, in pharmaceutical compositions.

  Functional variants of the protein or peptide of the present invention, ie, protein or peptide variants that retain the functionality of the original protein or peptide are described, for example, in the literature collecting such methods, Molecular Cloning: A Laboratory Manual, J Sambrook, et al., Eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbar, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., Eds., John Wiley & Sons, It can be prepared according to methods of altering polypeptide sequences known to those skilled in the art, such as those found in Inc., New York. Conservative amino acid substitutions are usually made by changes in the nucleic acid molecule that encodes the protein or peptide. Such substitutions can be made by various methods known to those skilled in the art. For example, amino acid substitution may be performed by PCR-specific mutation or site-directed mutagenesis according to Kunkel's method (Kunkel, Pro. Nat. Acad. Sci. USA 82: 488-492, 1985) or by a gene encoding a protein. The chemical synthesis of When amino acid substitutions are made on small unique fragments of the protein or peptide of the invention, the substitution can be made directly by synthesizing the peptide. The activity of a functional variant or fragment of the protein or peptide of the present invention is altered by cloning a gene encoding the altered protein into a bacterial or mammalian expression vector, introducing the vector into a suitable host cell, and Can be tested by expressing and testing for the functional ability of the proteins described herein.

  The method comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions. In order to obtain a functional variant having, for example, it is achieved by sequence repeats. Similarly, the above or other functional variants have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, at their C- and / or N-terminus. It can be prepared by adding or deleting 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Protein or peptide variants prepared by the above methods can be sequenced to determine the amino acid sequence, if desired, and thereby to deduce the nucleotide sequence encoding such a variant.

  In another aspect, the invention is directed to nucleic acid sequences encoding various truncated MUC1 receptor proteins, or functional variants or fragments thereof, and other nucleic acid sequences that hybridize to the nucleic acid sequences under highly stringent conditions. I will provide a. The sequences of certain nucleic acid molecules of the present invention are shown as the following SEQ ID NOs: 42-46. The predicted amino acid sequences of the protein products of these genes, including the truncated MUC1 receptor protein isoforms, are shown below. Accordingly, in one aspect, the present invention provides peptide sequences that show truncated isoforms of MUC1 receptor, genes and functional modifications encoding these peptide sequences and the variants, useful fragments and therapeutic and diagnostic products. And related methods. Peptides referred to herein as truncated MUC1 receptor proteins include fragments of the full length MUC1 receptor but do not include the full length MUC1 receptor protein (ie, SEQ ID NO: 10). Similarly, nucleic acid molecules encoding the various truncated isoforms of the MUC1 receptor described herein include fragments of the MUC1 gene coding region but do not include the full length MUC1 coding region.

In one embodiment of the invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the following group.
(A) a nucleic acid molecule encoding the MUC1 truncated receptor isoform set forth in SEQ ID NOs: 37, 38, 39, 40 and 41, or for example the nucleotide sequence: SEQ ID NOs: 42, 43, 44, 45 and 46, respectively A functional variant thereof or a fragment thereof, and (b) a nucleic acid molecule that hybridizes under high stringency conditions to the nucleic acid molecule of (a),
(C) a deletion, addition or substitution of the nucleic acid molecule of (a) or (b),
(D) a nucleic acid molecule different from the nucleic acid molecule of (a), (b) or (c) in the codon sequence due to the degeneracy of the genetic code, and (e) (a), (b), (c) or Complement of (d).

  Certain isolated nucleic acids of the invention may be a truncated isoform of the MUC1 receptor, or a functional fragment or variant thereof, or a functional equivalent thereof (eg, one of the nucleic acid sequences, eg, the sequence described below Nucleic acid molecule encoding the same protein encoded by number 42). However, functional fragments and equivalents encode proteins that exhibit the functional activity of the truncated isoform of the MUC1 receptor encoded by such sequences. As used herein, the functional activity of a truncated isoform of MUC1 receptor specifically interacts with a ligand of MGFR, and cell growth or cell proliferation as a response to such interaction. Refers to the ability of a truncated isoform of the MUC1 receptor peptide sequence to regulate. In certain embodiments, the isolated nucleic acid molecule is SEQ ID NO: 42.

  The present invention provides a nucleic acid molecule that hybridizes under high stringency conditions to a nucleic acid molecule consisting of the nucleotide sequences set forth in SEQ ID NOs: 42 to 46. Such nucleic acids may be DNA, RNA consisting of mixed deoxyribonucleotides and ribonucleotides, or may incorporate synthetic unnatural nucleotides. Various methods for measuring nucleic acid expression and / or polypeptides in normal or tumor cells are known to those skilled in the art.

  As used herein, the term “high stringency conditions” or “high stringency conditions” refers to parameters familiar to those skilled in the art. Nucleic acid hybridization parameters can be found in literature summarizing such methods, for example, Molecular Cloning; A Laboratory Manual, edited by J. Sambrook et al., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols. in Molecular Biology, FM Ausubel et al., found in John Wiley & Sons, Inc. New York. More specifically, stringent conditions used herein include, for example, hybridization buffer (3.5 × SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Hybridization at 65 ° C. in bovine serum albumin, 2.5 mM NaH 2 PO 4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7; SDS is sodium dodecyl sulfate and EDTA is ethylenediaminetetraacetic acid. After hybridization, the membrane to which the DNA has been transferred is washed with 2 × SSC at room temperature and then with 0.1 × SSC / 0.1 × SDS at temperatures up to 68 ° C.

  The set of hybridization conditions is only one example of highly stringent hybridization conditions known to those skilled in the art. There are other conditions and reagents that can be used that produce highly stringent hybridization. Skilled professionals will be familiar with such conditions and are not listed here. However, it will be appreciated that the skilled practitioner will be able to manipulate the conditions in a manner that allows unambiguous identification of homologues and alleles of the nucleic acid molecules of the present invention. Skilled professionals are also familiar with the methodology for screening cells and libraries for expression of such molecules isolated on a regular basis, and isolating and sequencing related nucleic acid molecules.

  In general, homologues and alleles of a particular SEQ ID NO listed herein are usually at least 40% nucleotide identity and / or at least 50% of such nucleotide or amino acid sequence, respectively. Will share amino acid identity. In some examples share at least 50% nucleotide identity and / or at least 65% amino acid identity, and in other examples share at least 60% nucleotide identity and / or at least 75% amino acid identity Will do. Preferred homologues and alleles are SEQ ID NO: 42 and SEQ ID NO: 37; respectively, SEQ ID NO: 43 and SEQ ID NO: 38; or SEQ ID NO: 44 and SEQ ID NO: 39 respectively; or SEQ ID NO: 45 and SEQ ID NO: 40 respectively; Polynucleotides sharing the nucleotide and amino acid identity of SEQ ID NO: 46 and SEQ ID NO: 41, and having an identity of 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more. Encodes the peptide. The percent identity can be calculated using various publicly available software tools. This tool was developed by NCBI (Bethesda, Maryland) and is available from the Internet (ftp: /ncbi.nlm.nih.gov/pub/). An example of a tool is the BLAST system available from http://www.ncbi.nlm.nih.gov using an algorithm developed by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). Is mentioned. Pairwise and Clustal W alignment (BLOSUM30 matrix settings) and Kyte-Doolittle hydropathy analysis can be obtained using Mac Vector sequence analysis software (Oxford Molecular Group). The Watson-Crick complement of the nucleic acid molecule is also encompassed by the present invention.

  The invention also includes degenerate nucleic acid molecules that contain separate codons for those present in the natural material. For example, serine residues are encoded by codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purpose of encoding a serine residue. Thus, it will be apparent to one skilled in the art that any serine-encoding nucleotide triplet will be used in a protein synthesizer in vitro or in vivo to introduce a serine residue into the extending peptide sequence of the present invention. I will. Similarly, nucleotide sequence triplets encoding other amino acid residues include CCA, CCC, CCG and CCT (proline codon); CGA, CGC, CGG, CGT, AGA and AGG (arginine codon); ACA, ACC, ACG And ACT (threonine codon); AAC and AAT (asparagine codon); and ATA, ATC and ATT (isoleucine codon). Other amino acid residues are similarly encoded by multiple nucleotide sequences. Accordingly, the present invention encompasses degenerate nucleic acids that differ from nucleic acids biologically isolated in codon sequences due to the degeneracy of the genetic code.

  The invention also provides isolated unique fragments of SEQ ID NOs: 42-46 and / or complements of SEQ ID NOs: 42-46. A unique fragment is one that is a “signature” of a larger nucleic acid. For example, if it is sufficiently long, its exact sequence cannot be found exactly outside the molecule of the nucleic acid molecule of the invention defined above. Those skilled in the art will only use routine methods to determine whether a fragment is unique within a human or mouse chromosome.

  The unique fragment can be used as a probe in Southern blot analysis to identify such nucleic acid molecules, or can be used in amplification analysis methods such as analysis methods using PCR. Unique fragments can also be used to produce fusion proteins to generate antibodies, to determine the binding of polypeptide fragments, or to generate immunoassay components. Similarly, unique fragments can be used to produce unfused fragments of certain polypeptides of the invention that are useful, for example, in the preparation of antibodies in immunoassays. The unique fragment can further be used as an antisense oligonucleotide to inhibit nucleic acid expression and polypeptides, particularly for therapeutic purposes. The present invention also includes antisense oligonucleotides of the above-described nucleic acid molecules of the present invention.

  In general, as used herein, the term “antisense oligonucleotide” or “antisense” hybridizes to DNA containing a particular gene, or mRNA transcript of that gene, under physiological conditions. Oligonucleotides that are oligonucleotides, oligodeoxyribonucleotides, modified oligonucleotides, or modified oligodeoxyribonucleotides that soy, thereby inhibiting transcription of the gene and / or translation of the mRNA. One skilled in the art will depend on the exact length of the antisense oligonucleotide and the degree of complementarity with its target, depending on the target label and the specific label chosen, including the specific base containing the sequence. Antisense oligonucleotides are constructed and arranged to selectively bind to the target under physiological conditions, i.e., to substantially hybridize with the target sequence over other sequences in the target cell under physiological conditions. It is preferable that One skilled in the art can readily select and synthesize any of a number of suitable antisense molecules for use in the present invention. In order to be sufficiently selective and potent in inhibition, although in some cases short modified oligonucleotides of 7 bases in length are successfully used as antisense oligonucleotides (Wagner et al., Nature Biotechnology 14: 840-844). , 1996), such antisense oligonucleotides should contain at least 10, more preferably at least 15 conserved bases that are complementary to the target. In certain embodiments, antisense oligonucleotides of the invention may include “modified” oligonucleotides. That is, oligonucleotides do not prevent them from hybridizing to their target, but are modified in a number of ways known in the art to enhance their stability or targeting, or enhance their therapeutic efficacy. May be. Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such pharmaceutical compositions may comprise an antisense oligonucleotide with any standard physiological and / or pharmaceutically acceptable carrier known in the art. The composition should be sterile and should contain a therapeutically effective amount of the antisense oligonucleotide, which is a unit of weight or volume suitable for administration to a patient.

  As those skilled in the art will appreciate, the size of the unique fragment will depend on the conservation of the genetic code. Thus, some regions of SEQ ID NOs: 42-46 and their complements will require longer segments that are unique, while others are typically 12-32 nucleotides or longer in length (eg, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 9 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, or more) And would require a short segment less than the full length of the sequence described. Many segments of a polynucleotide coding region or its complement that are 18 or more nucleotides in length will be unique. Those skilled in the art are generally well versed in methods for selecting such sequences based on the ability of the unique sequence to selectively distinguish the sequence of interest from other unrelated nucleic acid molecules. Comparing the sequence of a fragment with that of a known database is usually necessary even if in vitro confirmatory hybridization and sequencing analysis has been achieved.

  A unique fragment is a functional fragment. Functional fragments of the nucleic acid molecules of the present invention encode several functional properties of larger nucleic acid molecules, such as encoding functional polypeptides, binding proteins, and controlling transcription of operably linked nucleic acid molecules. Is a fragment that holds Those skilled in the art are familiar with the analytical methods described herein and in the art to determine whether a fragment is a functional fragment of a nucleic acid molecule without using anything other than routine experimentation. Can be easily determined using various methods.

  The term “isolated” as used herein for a nucleic acid molecule is (i) amplified in vitro, eg, by polymerase chain reaction (PCR); (ii) produced by recombinant technology by cloning. (Iii) purified, such as by cleavage and gel separation; or (iv) means synthesized, for example, by chemical synthesis. Isolated nucleic acid molecules are those that can be readily manipulated by recombinant DNA techniques well known in the art. Thus, it is believed that the nucleotide sequence contained in the vector in which the 5 'and 3' restriction enzyme sites are known or the polymerase chain reaction (PCR) primer sequences are described has been isolated. Nucleic acid sequences that are naturally present in the host have not been isolated. An isolated nucleic acid molecule is substantially pure but need not be purified. For example, a nucleic acid molecule isolated in a cloning or expression vector is not pure because it contains only a small percentage of the material in the cells where it remains. Such nucleic acid molecules can be isolated because the term is used in the specification and is readily manipulable by standard techniques known to those skilled in the art. An isolated nucleic acid molecule as used herein is not a naturally occurring chromosome.

  In other aspects of the invention, the invention relates to the use of sequences such as those just described encoding the peptides of the invention or fragments or variants thereof in expression vectors, as well as prokaryotic cells (eg, E. coli) or true. It includes host cells and their use to transfect cell lines that are nuclear cells (eg, CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression systems in insect cells). In particular, mammalian cells such as humans, mice, hamsters, pigs, goats, and primates are useful. They have a wide range of tissue types and they may be primary cells or cell lines. An expression vector includes a nucleic acid of the present invention that encodes a related sequence, ie, the peptide of the present invention operably linked to a promoter.

  In certain embodiments, an expression vector comprising any of the isolated nucleic acid molecules of the invention, preferably operably linked to a promoter, is provided. In related embodiments, host cells transformed or transfected with such expression vectors are also provided. Expression vectors containing all the elements necessary for expression are commercially available and known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are engineered by genetic engineering by introducing foreign DNA (RNA), fragments or variants thereof encoding the protein of the present invention into the cells. Heterologous DNA (RNA) is placed under the feasible control of transcription elements that allow the expression of the foreign DNA in the host cell.

  As used herein, a “vector” is a number of sequences into which a desired sequence is inserted by restriction degradation or ligation for delivery between different genetic environments or expression in a host cell. Any of the nucleic acid molecules. RNA vectors are also available, but vectors usually consist of DNA. Vectors include, but are not limited to, plasmids, phagemids and viral genomes. A cloning vector is one that can replicate in a host cell, and further features one or more endonuclease restriction enzyme sites in which the vector is cleaved in a defined manner, in which the desired DNA sequence is contained. Ligated so that the new recombinant vector retains the ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence occurs many times because the plasmid either increases copy number within the host bacterium or increases only once per host before the host is reproduced by mitosis. In the case of phage, replication occurs either actively in the lytic phase or passively in the lysogenic phase.

  An “expression vector” is one in which the desired DNA sequence is inserted by restriction or ligation, so that it is operably linked to regulatory sequences and expressed as an RNA transcript. The vector further includes one or more marker sequences suitable for use in identifying cells that have been transformed, transfected, or not with the vector. Markers can be detected by, for example, a gene encoding a protein that increases or decreases either resistance or sensitivity to antibiotics or other compounds, the standard activity of which the activity is known in the art Genes that encode enzymes (eg, β-galactosidase or alkaline phosphatase) and genes that visually affect the phenotype of transformed, transfected cells, hosts, colonies or plaques (eg, green fluorescent protein) Can be mentioned. Preferred vectors are those that allow the expression of structural gene products present in DNA segments that are operably linked to autonomous replication.

  As used herein, a coding sequence and a control sequence are “implemented” when they are covalently linked in such a way as to subject the expression or transcription of the coding sequence to the influence or control of the control sequence. It is said to be “possibly connected”. If it is desired that the coding sequence be translated into a functional protein, the two DNA sequences can be transcribed if the induction of the promoter in the 5 'regulatory sequence results in the coding sequence and between the two DNA sequences. The binding properties of (1) do not introduce frameshift mutations, (2) do not interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) of the corresponding RNA transcript to be translated into protein If it doesn't interfere with capability, it is said to be operably coupled. Thus, a promoter region is operably linked to a coding sequence if the promoter region can affect transcription of the DNA sequence so that the resulting transcript is translated into the desired protein or polypeptide. Will.

  The exact nature of the regulatory sequences required for gene expression varies between species or cell types, but is generally involved in the initiation of each transcription and translation, such as a TATA box, cap-forming sequence, CAAT sequence, etc. 'Non-transcribed and 5'-translated sequences should be included. In particular, such 5′-non-transcribed control sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably linked gene. Control sequences also optionally include enhancer sequences or upstream activator sequences. The vectors of the present invention optionally include a 5 ′ leader or signal sequence. Selection and design of appropriate vectors are within the ability and discretion of those skilled in the art.

  Control elements that allow expression in eukaryotic host cells include, for example, the PL, lac, trp, or tac promoter in E. coli, and examples of control elements that allow expression in prokaryotic host cells include AOX1 in yeast. Or GAL1 promoter or CMV-promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or globin intron in mammalian or other animal cells.

  In addition to elements that are important in the initiation of transcription, such regulatory elements also contain transcription termination signals, such as SV40-poly-A or tk-poly-A sites, downstream of the polynucleotide. In addition, depending on the expression system used, a leader sequence capable of directing the polypeptide to a cell compartment, eg, the cell plasma membrane, and secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention. This is well known in the art. The leader sequence is assembled in a suitable phase with translation, initiation and termination sequences, and in certain embodiments can direct the polypeptide to the cellular compartment, or the translated protein or a portion of the periplasmic space or extracellular medium A leader sequence that directs secretion into the medium. In this context, suitable expression vectors are known in the art such as the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (GIBCO BRL).

  The invention further relates to host cells transformed with the polynucleotides or vectors of the invention. Such host cells may be prokaryotic cells or eukaryotic cells. A polynucleotide or vector of the invention present in a host cell may be integrated into the genome of the host cell or may be retained extrachromosomally. Host cells are prokaryotic or eukaryotic cells such as bacteria, insects, fungi, plants, animals or human cells. Some fungal cells are, for example, those of the genus Saccharomyces, in particular S. cerevisiae. It is of the cerevisiae species. The term “prokaryotes” is meant to include all bacteria that can be transformed or transfected with DNA or RNA molecules for expression of the peptide sequences of the invention. Prokaryotic hosts are e.g. Includes gram-negative and gram-positive bacteria such as typhimurium, Serratia marcescens and Bacillus subtilis. The term “eukaryote” is meant to include yeast, higher plant, insect and preferably mammalian cells such as NSO and CHO cells. Depending on the host used for recombinant production techniques, the peptide sequences encoded by the polynucleotides of the invention may or may not be glycosylated. The polynucleotides of the invention can be used to transform or transfect a host using any technique commonly known to those of skill in the art. In addition, methods for preparing fused and operably linked genes and expressing them in, for example, mammalian cells and bacteria are well known in the art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring). Harbor Laboratory, Cold Spring Harbor, NY, 1989). The genetic constructs and methods described therein, or candid modifications thereof, can be used to express the polypeptides of the invention in eukaryotic or prokaryotic hosts. In certain embodiments, expression vectors containing promoter sequences that facilitate efficient transcription of the inserted polynucleotide are used in connection with the host. Expression vectors usually contain an origin of replication, a promoter and terminator, and specific genes that can result in selection of the phenotype of the transformed cell. DNA sequences for polypeptide expression and suitable source cells of the host cell are the American Type Culture Collection ("Catalogs of Cell Lines and Hybridomas", Fifth edition (1985) Rockville, Maryland, USA, which is incorporated herein by reference. And can be obtained from many sources.

  In one embodiment, a viral vector for delivering a nucleic acid molecule encoding a peptide sequence of the invention is a poxvirus, including adenovirus, adeno-associated virus, vaccinia virus and attenuated poxvirus, Semliki Forest virus. Selected from the group consisting of Venezuelan equine encephalitis virus, retrovirus, Sindbis virus and Ty virus-like particles. Examples of viruses and virus-like particles that have been used to deliver exogenous nucleic acids include replication defective adenoviruses (eg, Xiang et al., Virology 219: 220-227, 1997; Eloit et al., J. Virol. 7: 5375-5381, 1997; Chengalvara et al., Vaccine 15: 335-339, 1997), modified retrovirus (Townsend et al., J. Virol. 71: 3365-3374, 1997), non-replicating retrovirus (Irwin et al., J. Virol 68: 5036-5044, 1994), replication-defective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA 92: 3009-3013, 1995), canary pox virus and highly attenuated vaccinia virus derivatives (Paoletti Natl. Acad. Sci. USA 93: 11349-11353, 1996), non-replicating vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA 93: 11341-11348, 1996), replicating vaccinia virus (Moss, Dev. Biol. Stand. 82: 55-63, 1994), Venezuela equine encephalitis virus (Davis et al., J. Virol. 70: 3781-3787, 1996), De bis virus (Pugachev et al., Virology 212: 587-594, 1995), and Ty virus-like particle (Allsopp et al., Eur J. Immunol 26:.. 1951-1959, 1996) can be mentioned. In certain embodiments, the viral vector is an adenovirus.

  Other viruses that are potentially used in certain applications are adeno-associated viruses, double-stranded DNA viruses. Adeno-associated viruses can infect a wide range of cell types and species and can be engineered to be replication deficient. In addition, it has advantages such as heat and lipid solvent stability, high transduction frequency in various lineages of cells including hematopoietic cells and lack of superinfection suppression that allows multiple sequential transductions. Adeno-associated viruses are incorporated into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability in inserted gene expression. Furthermore, wild-type adeno-associated virus infection implies that in the absence of selective pressure, tissue culture continues for more than 100 passages and adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also function in extrachromosomal manner.

  Other viral vectors are based on non-cytopathic eukaryotic viruses in which a non-essential gene is replaced with a gene of interest. Non-cytopathic viruses include retroviruses whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral incorporation into host cell DNA. Adenoviruses and retroviruses are approved for human gene therapy. In general, retroviruses are replication deficient (ie, can direct the synthesis of the desired protein but cannot produce infectious particles). Such genetically altered retroviral expression vectors can have general utility in high efficiency transduction of genes in vivo. Standard protocols for producing replication-defective retroviruses (incorporation of foreign genetic material into plasmids, transfection of packaging cell lines with plasmids, production of recombinant retroviruses with packaging cell lines, viruses from tissue culture media) Particle collection and infection of target cells with viral particles) is described in Kriegler, M. Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990) and Murry, EJ, Ed. in Molecular Biology ", vol. 7, Humana Press, Inc., Cliffton, New Jersey (1991).

  In one embodiment, the nucleic acid delivery vector is (1) transcribed and translated in a mammal, localized and oriented in the cytoplasmic membrane of a cell, resulting in a peptide sequence (eg, PSMGFR) An exogenous genetic material capable of producing the peptide sequence of the present invention wherein an extracellular receptor moiety is expressed on the outer surface of the cytoplasmic membrane of the cell, and (2) the surface of a target cell such as a mammalian cell A ligand that selectively binds the receptor thereon is included on the surface and thereby enters the target cell.

  Various techniques may be used to introduce a nucleic acid molecule of the invention into a cell, depending on whether the nucleic acid molecule is introduced into a host in vitro or in vivo. Such techniques include transfection of a nucleic acid molecule-calcium phosphate precipitate, transfection of an EDTA-associated nucleic acid molecule, transfection or infection with the virus containing the nucleic acid molecule of interest, liposome-mediated transfection, and the like.

  For some applications, it is preferred to target nucleic acid molecules to specific cells. In such examples, the medium used to deliver the nucleic acid molecule of the invention into a cell (eg, a retrovirus or other virus; liposome) can have a target molecule attached thereto. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound or introduced into the nucleic acid molecule delivery vehicle. In particular, monoclonal antibodies are preferred. When liposomes are used to deliver the nucleic acid molecules of the present invention, proteins that bind to surface membrane proteins involved in endocytosis may be introduced into a liposome preparation for targeting and / or uptake. Such proteins include capsid proteins or fragments thereof for specific cell types, antibodies to proteins that undergo internalization during cycling, and proteins that target intracellular localization and increase intracellular half-life. Can be mentioned. Polymeric delivery systems have also been used to deliver nucleic acid molecules into cells, as is known to those skilled in the art. Such a system allows oral delivery of nucleic acid molecules.

  In addition to delivery by use of vectors, the nucleic acids of the invention may be delivered to cells without vectors using methods known to those skilled in the art, for example, as “naked” nucleic acid delivery.

  In another aspect of the present invention, a genetically modified non-human animal comprising the expression vector of the present invention is provided. As used herein, a “genetically modified non-human animal” includes non-human animals that have one or more exogenous nucleic acid molecules introduced into germline and / or somatic cells. . Therefore, genetically modified animals include animals having an expression vector introduced into an episome or chromosome. In general, such expression vectors may employ various promoters that provide the desired gene expression pattern (eg, temporal or spatial). A conditional promoter is also operably linked to the nucleic acid molecule of the invention to increase or decrease the expression of the polypeptide molecule encoded in a controlled or conditional manner. Trans-positive or negative regulators of polypeptide activity or expression can also be operably linked to the conditional promoters described above. Such trans-acting regulators include antisense nucleic acid molecules, nucleic acid molecules encoding dominant negative molecules, transcription factors, ribozyme molecules specific for nucleic acid molecules, and the like. Genetically modified non-human animals are useful, for example, in experiments directed to testing the biological or physiological effects of diagnosis or treatment for cancers characterized by abnormal expression of MUC1. Other uses will be apparent to those skilled in the art.

  The present invention also includes so-called expression kits that allow an expert to prepare the desired expression vector or vector. Such an expression kit includes at least one of the nucleotide sequences of the present invention described above encoding the peptide sequence of the present invention. Other components may be added as desired as long as the above sequence is included.

  The effect shown herein is that, within the context of the present invention, a MUC1 transfectant (ie a cell transfected with a nucleic acid molecule encoding the MUC1 receptor isoform on the cell surface) is a native MUC1 + breast. It proves that it behaves like a tumor cell.

  The results presented in the context of the present invention also suggest that PSMGFR is a functionally necessary and sufficient part of the MUC1 receptor that mediates cell growth. The evidence presented here suggests that the MUC1 receptor, which is abnormally expressed in about 75% of all human solid tumors, is a major receptor that mediates tumor cell growth. The results shown here and described above and in the examples show that MUC1 cells in which the portion of the MUC1 receptor that remains attached to the cell surface after cleavage is sufficient and necessary for MUC1-dependent cell growth. Provide evidence that is part of the outer domain. As previously shown and discussed, dimerization of the MGFR portion of the MUC1 receptor induced cell proliferation. Although the exact site of receptor cleavage has not yet been determined, the present invention requires that all necessary and sufficient portions of the MUC1 receptor that need to stimulate cell proliferation be native PSMGFR (nat-PSMGFR, SEQ ID NO: 36). Experimental results suggesting that it is part of a receptor that essentially contains.

  MUC1 + breast tumor cells, T47D, 1500, 1504, and BT-474 were obtained from ATCC (see Example 5). As controls, MUC1-breast tumor cells, MDA-MB-453, HEK (human H embryonic kidney) cell line K293 and HeLa cells were also obtained from ATCC (see Example 5). Western blot analysis was performed to determine the expression level of MUC1 in each cell type (see Example 5). High (15%) as well as low (6%) polyacrylamide gels were used to visualize uncleaved as well as cleaved MUC1 receptors. A low-concentration gel capable of visualizing a protein having a molecular weight range of 50 kDa to 350 kDa is obtained with an antibody VU4H5 (Santa Cruz Biotechnology, Santa Cruz, CA) that recognizes an ADDTR sequence present in the terminal repeat portion of the extracellular domain. Explored. The high concentration gel used to visualize the 6.5-50 kDa protein was probed with the rabbit polyclonal antibody targeting PSMGFR described above. FIG. 20 of the process described in Example 5 is a Western blot showing that cell line 1504 expressed a high concentration of uncleaved MUC1, followed by T47D, 1500 cells, BT-474, and K293 And undetectable amounts of uncleaved MUC1 are shown in HeLa cells. FIG. 14 of the process described in Example 5 shows other Western tumors showing that all three breast tumor cell lines 1504, 1500 and T47D expressed similar amounts of proteolytic MUC1 with an apparent molecular weight of 20-30 kDa. It is a blot. Wreschner et al. Published data showing that this MUC1 cleavage product is not expressed in normal healthy breast cells. BT-474 expressed a fairly low concentration of cleaved MUC1. Furthermore, the protein band of BT-474 shows a low intensity at about 15 kDa and is concentrated at 20 kDa (see FIG. 14). K293 cells did not show MUC1 expression as expected, while HeLa cells showed 20-30 kDa minimal MUC1 as already reported in the literature. MDA-MB-453 also did not express detectable concentrations of cleaved or uncleaved MUC1 (data not shown).

  Cellular proteins often undergo post-translational modifications such as cleavage, glycosylation and phosphorylation. In particular, in some situations, the extracellular domain of the MUC1 receptor is cleaved (at an unknown location) and allowed to flow into the bloodstream. The extracellular portion of the receptor can also be glycosylated in tumor cells, although it has often been reported that it is not glycosylated. The fact that the MUC1 receptor experiences these unknown denaturations makes it difficult to characterize the portion of the receptor that remains on the cell surface after cleavage in length. It should be noted that the extent of glycosylation changes the molecular weight of the receptor, for example when analyzed by Western blot. Therefore, it is advantageous to deglycosylate protein samples prior to Western gel analysis in order to compare the expression levels of MUC1 in different cell types and obtain a better measure of its true molecular weight. In FIG. 21, the process described in Example 5, after deglycosylation, the MUC1 protein bands gather to form a protruding band with an apparent molecular weight of approximately 20 kDa. These results suggest that all of the breast tumor cell lines tested produced MUC1 cleavage products with approximately the same length but with different glycosylation. Note that the non-deglycosylated 1504, 1500 and T47D samples showed three distinct MUC1 protein bands. The PSMGFR sequence contains three arginine residues that are glycosylated. Further analysis suggests that the MUC1 proteolyte was in fact N-glycosylated but not O-glycosylated. See FIG. 21 (lanes 3 to 4).

  To determine which amino acids were glycosylated, 1500 cell lines were deglycosylated using an enzyme that specifically removed O- or N-linked glycol units. FIG. 21 shows that changes in molecular weight occur only after treatment with N-specific deglycosylase. This suggests that the PSMGFR region of the MUC1 receptor is uniquely N-glycosylated.

  In another set of embodiments, MUC1 isoform constructs of different lengths are transfected into HEK cells and analyzed by Western blot using anti-PSMGFR to determine the cleavage pattern of various MUC1 constructs. Were determined. To investigate whether any part of the extracellular domain of the MUC1 receptor is necessary and sufficient for the growth factor-like functions described herein, or after cleavage in tumor cells In order to investigate the sequence of the receptor protein remaining on the cell surface, a HEK (human embryonic kidney) cell is transformed with a plasmid designed to generate MUC1 receptor variants with different lengths. Introduced. FIG. 22 shows the generated MUC1 construct. See Examples 6-7. In short, the construct consists of the complete MUC1 receptor (SEQ ID NO: 10), a MUC1 receptor variant with only 1.3 kilobases of the repeat section (“Rep isoform”, SEQ ID NO: 41), IBR (interchain binding domain). MUC1 receptor variant that terminates later (“CM isoform”, SEQ ID NO: 38), MUC1 receptor variant that terminates after PSMGFR (“nat-PSMGFRTC isoform”, SEQ ID NO: 37) and the complete MUC1-Y alternative splice variant To produce the body (“Y isoform”, SEQ ID NO: 40). FIG. 23 shows that all of the MUC1 variants are clearly cleaved, except for the nat-PSMGFRTC isoform and the CM isoform construct. There is evidence in the literature that a C-terminal sequence to the IBR terminus is required for the enzymatic cleavage of the receptor.

  Cells transfected with the nat-PSMGFRTC isoform grew faster than the parental cell line and much faster (about 2-fold) than the full length or repeat (Rep isoform) construct. Furthermore, the present inventors have observed that HEK cells transfected with the nat-PSMGFRTC isoform are capable of anchorage-dependent cell growth. Anchorage-dependent cell growth is a phenomenon that is not yet understood, but is a prominent feature of true tumor cells. Attempts to temporarily transfect cells with full-length or repetitive (Rep isoform) constructs were difficult and often failed. In striking contrast, the nat-PSMGFRTC isoform construct was easily transfected into each and every time it was attempted. These results support the assumption that this is the PSMGFR portion of the receptor that acts as a growth factor receptor.

  To determine if the MUC1 truncated receptor behaves in HEK cells as it does in tumor cells, a series of cell proliferation stimuli and inhibitions were performed. In particular, bivalent antibodies and monovalent antigen binding fragments were added to these cells and the effects on cell growth and phosphorylation status of ERK2 were measured. FIG. 24 shows that the addition of bivalent anti-PSMGFR (grey bars) to cells transfected with the nat-PSMGFRTC isoform construct caused an 80% enhancement of cell growth, whereas the addition of monovalent anti-PSMGFR was untransformed. Shows up to 50% inhibition of transfected cell growth. This figure also shows that monovalent antibodies compete with divalent antibodies to reduce the degree of enhancement of cell growth. FIG. 25 shows that the addition of a bivalent antibody induces ERK2 phosphorylation, while FIG. 26 shows that the monovalent antibody competes with the bivalent and blocks ERK2 phosphorylation. Therefore, the nat-PSMGFRTC isoform-transfected cell line constitutes an excellent research tool for drug discovery for MUC1 + cancer. Because they behave similarly to tumor cells, this type of receptor appears to be essentially active.

  In summary, the extracellular domains presented in the transfected cells produced in the present invention include: 1) PSMGFR only (nat-PSMGFRTC isoform-SEQ ID NO: 37); 2) PSMGFR and PSIBR (CM isoform) -SEQ ID NO: 38); 3) PSMGFR, PSIBR and unique sequence terminated immediately before the repeat region (UR isoform-SEQ ID NO: 39); 4) PSMGFR, PSIBR, unique sequence and 1 kb repeat (Rep isoform-sequence) No. 41); 5) Complete MUC1 extracellular domain (full length MUC1 receptor—SEQ ID NO: 10); and 6) Complete extracellular domain of Y isoform (SEQ ID NO: 40). Cells were grown and processed according to standard protocols for molecular weight analysis of specific proteins by SDS-PAGE followed by Western blot (see Example 5). The antibodies of the invention used in the Western blot stage of the analysis specifically recognize the PSMGFR sequence of the MUC1 receptor. Various transfectants were then analyzed by Western blot.

  As previously mentioned, constructs that are essentially expressed in the MUC1 + cancer cell line are believed to have MUC1 receptors terminated at or near the N-terminus of PSMGFR within that cell line, but 20 at the time of glycosylation. A series of protein bands were generated between 30 and 30 Kd (see FIG. 21). After deglycosylation, the band changed to a molecular weight of about 20 Kd (FIG. 21). Significantly, the calculated molecular weight of the nat-PSMGFRTC isoform transfection construct is 19 Kd.

  The MUC1 construct, referred to as the “Y isoform,” produced a series of protein bands that reacted with anti-PSMGFR in Western blots that migrated through SDS-PAGE gels with apparent molecular weights ranging from 35 to 45 Kd (FIG. 23). After deglycosylation, protein motility changed to apparent molecular weights ranging from 29-41 Kd. The calculated molecular weight of the transfected MUC1-Y isoform is 29 Kd. A faint protein band was seen at 20 Kd, consistent with the idea that some minimal cleavage of the Y isoform occurred, resulting in a proteolytic fragment whose molecular weight matched that of the nat-PSMGFRTC isoform. Comparison of the glycosylated protein lanes in FIG. 23 shows a clear difference between the MUC1 protein from a breast cancer patient (1500 cell line) and the Y isoform transfected into HK cells (“Y” lane). Still referring to FIG. 23, the lane loaded with the breast tumor cell sample does not show a protein band between 35 and 45 Kd; however, the protein band of the glycosylated Y isoform is clearly between 35 and 45 Kd. It is visible and dark. These results do not exclude the possibility that patient-derived breast tumor cells produce another spliced isoform, such as the Y isoform, in addition to MUC1. Because it is a low concentration, it may not be visible by Western blot analysis. However, these results clearly show that the dominant MUC1 species produced by these breast tumor cell lines are not Y isoforms.

  The CM isoform construct produced a doublet that flowed at approximately 28 Kd. After deglycosylation, the band changed to a low molecular weight of about 25 Kd. The comparison shows that this construct is resistant to cleavage as the low 20 Kd band found in the nat-PSMGFRTC isoform construct is absent.

  Similar to the CM isoform construct, the UR isoform construct produced a MUC1 protein band flowing at 29-30 Kd. After deglycosylation, the band changed to a molecular weight of approximately 24 Kd. A faint band of 20 Kd is visible, indicating that some cleavage of the construct occurs.

  The “full length” and Rep isoform constructs appear to behave similarly and produce a PsMGFR-specific band at 25-30 Kd that changes to about 23 Kd after deglycosylation. It is impossible to distinguish these species from those produced by breast tumor cells from patients by Western blot.

  The (calculated) molecular weight of the portion of the MUC1 receptor that includes the cytoplasmic tail, transmembrane domain and PSMGFR (non-glycosylated) is approximately 19 Kd (ie, nat-PSMGFRTC isoform—see SEQ ID NO: 37). In addition, as discovered in the present invention, normal breast cells do not express, and breast tumor cells express a short form of MUC1 receptor that flows on an SDS-PAGE gel at about 20 Kd (deglycosylated). , Suggesting that this tumor-specific type of extracellular domain consists essentially of PSMGFR.

  To ascertain whether breast tumor cells from actual breast cancer patients produce a MUC1 cleavage product similar to the transfection construct described above, we used the same Western blot method described above and in Example 5. MUC1 protein from several breast tumor cell lines was analyzed. Breast tumor cell lines 1500, 1504, T47D, BT-474 and MDA-MB-453 were tested. The molecular weight of the MUC1 receptor fragment was then determined by performing standard Western blot analysis again using antibodies raised against PSMGFR as described above. Western blots showed that the breast tumor cell line produced several MUC1 protein bands that flow between 25 and 30 Kd. As with the nat-PSMGFRTC isoform construct, deglycosylation of the breast tumor-derived sample resulted in a series of MUC1 protein bands (25-30 Kd) and changed to a band with an approximate molecular weight of 20 Kd, see FIG. These Western blots show that the low molecular weight MUC1 species produced by breast tumor cells are similar to the MUC1 construct described above in which the receptor is cleaved behind PSMGFR. These results indicate that the part of the MUC1 receptor that remains attached to the cell surface after cleavage temporarily consists of the PSMGFR sequence. It should be noted that 1500 and T47D cells appear to produce what flows as a doublet on a gel with two cleavage products, a band of about 19-20 Kd and another band at about 22 Kda. The 22 Kda band appears to correspond to the band produced by the CM isoform, which contains PSIBR at its N-terminus. Significantly, (1) the absence of the 19-20 Kda band is evident for the CM isoform construct, and (2) the 22 Kd band is also a “healthy” type (ie, full-length receptor and Rep isoforms). -See FIG. 28) in the MUC1 cleavage product produced by transfected cells expressing. These results support the argument that the 22Kd band product represents a normal and non-abnormal cleavage product and that abnormal cleavage (ie, the generation of 19-25 Kd band) may be blocked by the presence of IBR. To do. The ability to discriminate molecular weight by SDS-PAGE analysis is only accurate to about 1 Kd, which is about 9 amino acids, and the actual portion of the MUC1 receptor remaining on the surface of tumor cells is the end of PSMGFR Up to +/- 9 to 15 amino acids N-terminal, possibly +/- 20 amino acids as well. However, the gel shows that the protein flows with approximately the same molecular weight. These bands are considered cleavage products because the same gel is also probed by antibodies that recognize the repetitive regions of the receptor. A protein band that stains positive for the repeats flows at a molecular weight between 250 and 300 Kd, indicating that the long receptor was cleaved after expression. The portion of the receptor that stains positive for PSMGFR flows with a molecular weight consistent with the cytoplasmic tail, the transmembrane region, and the calculated molecular weight of PSMGFR. Furthermore, the data we have presented here suggests that the unique protein band produced by the Y isoform construct is not identical to the MUC1 fragment produced by patient-derived or naturally occurring breast tumor cells. .

  As mentioned above, one aspect of the present invention is a method for treating a subject diagnosed with or at risk of developing a cancer or tumor characterized by abnormal expression of MUC1. Is targeted. The treatment of the present invention includes the use of agents or “substances” as described herein. That is, one aspect includes a series of compositions useful for the treatment of cancer or tumors characterized by abnormal expression of MUC1, and a kit comprising instructions for use of the composition for the treatment of such conditions. Including these compositions. That is, the kit includes a description of the use of the composition involved in the biological or chemical mechanism disclosed herein associated with cancer or tumor. The kit also includes instructions for use in combination of two or more compositions of some embodiments of the invention. Instructions can be provided for administering the drug orally, intravenously, or other known routes of drug delivery. These and other embodiments of the invention can also include promoting the treatment of cancer or tumors by any of the techniques and compositions and composition combinations described herein.

  In one set of embodiments, the indication of treatment with one of the compositions of the present invention in a condition that is not related to cell proliferation, or a condition that is different from cancer or tumor, including symptoms that may be associated with cell proliferation, cancer or tumor. Even if the patient shows, the patient can be treated with the composition of the present invention. That is, if the composition of the present invention is known for the treatment of different conditions, some embodiments of the present invention also provide the composition for treatment with symptomatic cell proliferation, cancer or tumor disease Including the use of These and other embodiments of the present invention may vary depending on dose, delivery technique, or vehicle, lack of combination with other pharmaceutical compositions or combination with other pharmaceutical compositions, rate of administration, timing of administration, or other factors. May include treatment different from the use of the composition for the treatment of conditions different from cell proliferation, cancer, or tumor. In another set of embodiments, treatment of cell proliferation, cancer, or tumor with the composition of the invention is similar to or overlaps with the use of the composition of the invention for the treatment of different conditions. May occur under symptoms. However, the compositions of the invention are propelled for treatment involving cell proliferation, cancer, or tumor, or include instructions for treatment involving cell proliferation, cancer, or tumor as described above. As used herein, “promoted” refers to educational methods, hospitals and other clinical methods involving compositions of the invention related to treatment involving cell proliferation, cancer, or tumors. Includes instructions and all methods of conducting transactions involving pharmaceutical industry activities, including pharmaceutical sales, including any form of advertising, including all forms of document, verbal, electronic communications or other promotional activities. “Instructions” can be defined and often defined as a component of a promotion, and typically include instructions for documents on or associated with the packaging of the composition of the present invention. The instructions can also include any oral or electronic instructions provided in any manner. A “kit” is typically and preferably defined as a package containing both one or a combination of the compositions of the invention and instructions, but the composition of the invention and instructions thereof. Any form of instructions provided in connection with this composition can be included in such a way that clinical experts can clearly recognize that should be involved in a particular composition.

  A subject who is not intended for a treatment method of the present invention (with a specific composition for cell proliferation, cancer, or tumor) is diagnosed by a condition that already requires treatment with this specific composition. People. Accordingly, one aspect of the present invention includes the treatment of cell proliferation, cancer, or tumor with the specific compositions disclosed herein for that purpose, for use in the treatment of cell proliferation, cancer, or the tumor itself. Includes treatments that are not in combination with other substances already taught. Other embodiments include treatment of cell proliferation, cancer, or tumors that are only this particular composition and not in combination with any other active agent. Other embodiments are according to this particular composition, where the use of the composition in therapy is specifically described for the treatment of cell proliferation, cancer, or tumor (eg, according to written instructions that may accompany the composition) Involved in the treatment of cell proliferation, cancer, or tumors. In a preferred embodiment of this aspect, the present invention provides specific compositions that are specifically illustrated that the use of the composition in therapy affects the mechanisms involved in cell proliferation, cancer, or tumor disclosed herein. Including cell growth by cancer, treatment of cancer or tumor. In yet another set of embodiments, the agents and substances of the invention can be used for disease prevention purposes. In this context, the present invention is particularly directed to patient populations that have not previously been treated with agents useful in certain methods of the invention, which patients are not suffering from cell proliferation, cancer or tumors. And patients with or without suspected cell proliferation, cancer or tumor. In other words, prophylactic treatment is preferably directed to a patient population that does not have disease symptoms that require active treatment with any of the agents described herein as useful according to the present invention.

  In one embodiment, the invention relates to a specific antibody and antigen-binding fragment thereof, particularly a monovalent antibody or a monovalent antigen-binding fragment of an antibody (eg, nat-PSMGFR (SEQ ID NO: 36) or var-PSMGFR (as generated against PSMGFR such as SEQ ID NO: 7)) can block the interaction of MGFR and its ligand that promotes tumorigenesis by binding to MGFR. In this aspect, the invention includes treatment of a subject associated with a tumor or cancer involved in abnormal expression of MUC1 with these substances or combinations.

  The method comprises any of the above antibody-derived or antibody-type agents (eg, a monovalent anti-MGFR antibody or a monovalent MGFR-binding portion of an anti-MGFR antibody) in an amount effective to provide a medically desirable result. Administration to a subject. In one embodiment, the method treats any one of the aforementioned antibody-derived or antibody-type agents (eg, a monovalent anti-MGFR antibody or a monovalent MGFR binding portion of an anti-MGFR antibody) with MUC1 abnormalities. Administration to a subject in an amount effective to reduce / prevent these / reduce / inhibit the risk of a tumor or cancer involved in expression.

  Effective amounts include the specific condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the combination therapy (if any), the specific route of administration, It depends on factors such as knowledge and competence of the practitioner. For example, in connection with a tumor or cancer involved in abnormal expression of MUC1, an effective amount is an amount that blocks the interaction between MGFR and a ligand that promotes cell proliferation (a substance that acts according to its mechanism, (For example, a substance containing a monovalent anti-MGFR antibody or a monovalent MGFR-binding portion of an anti-MGFR antibody) derived from an antibody.

  According to another mechanism of drug activity, an effective amount is that which maintains the self-aggregation of the MUC1 receptor (for substances such as polymers or dendrimers that act according to that mechanism). Alternatively, an effective amount is one that reduces the concentration of cleaved MUC1 IBR or maintains a low concentration of cleaved MUC1 IBR (for substances that act according to the mechanism). Similarly, an effective amount of treatment is sufficient to reduce or completely inhibit the concentration of cleaved MUC1 IBR to slow or stop the onset or progression of a tumor or cancer involved in abnormal expression of MUC1. Quantity (for substances that act according to their mechanism). Similarly, an effective amount of treatment is sufficient to reduce or completely inhibit the concentration of cleaved MUC1 IBR in order to slow or stop the development or progression of a tumor or cancer involved in abnormal expression of MUC1. (A substance that acts according to its mechanism). It is generally preferred that the maximum dose, ie the maximum safe dose according to reliable medical judgment, be used.

  When used therapeutically, the substances of the invention are administered in a therapeutically effective amount. In general, a therapeutically effective amount means that amount necessary to delay the onset of, inhibit the progression of, or totally halt the particular condition being treated. In general, a therapeutically effective amount will vary depending on the subject's age, symptoms, sex and the nature and extent of the disease in the subject, all of which can be determined by one skilled in the art. The dose is adjusted by the individual physician or veterinarian, especially in the case of complications. A therapeutically effective amount typically varies from 0.01 mg / kg to about 1000 mg / kg. It is expected that doses in the range of 1-500 mg / kg and preferably doses in the range of 1-50 mg / kg will be suitable. In other embodiments, the substance is at a dose in the range of 1 μg / kg / day to 10 mg / kg / day, more preferably 1-200 μg / kg / day, 1-100 μg / kg / day, 1-50 μg / kg / day. The dose is administered daily or in the range of 1-25 μg / kg / day. In other embodiments, the dose may range from about 0.1 mg / kg to about 200 mg / kg, and most preferably from about 0.2 mg / kg to about 20 mg / kg. For these administrations, one or more doses may be applied daily, every day or more.

  The substance of the invention should be administered for a period of time sufficient to provide the subject with either a therapeutic or prophylactic benefit, or both. In general, the substance is administered for at least one day. In certain instances, the substance may be administered for the remainder of the subject's life. The rate at which the substance is administered varies depending on the needs of the subject and the mode of administration. For example, in certain instances, if administration of a substance to a higher dose and higher frequency of a subject still achieves a medically desirable result, for example, during an event associated with a tumor or cancer, or Immediately thereafter, it may be necessary. On the other hand, lower dose administration may be desired to maintain the medically desirable results once achieved. In still other embodiments, the same dose of substance may be administered during a treatment period that spans the lifetime of the subject as described herein. The frequency of administration varies depending on the characteristics of the subject. Substances are described daily, every 2 days, every 3 days, every 4 days, every 5 days, every week, every 10 days, every 2 weeks, every month, or more, or explicitly stated herein During any such period.

  In one embodiment, the daily dose of active compound is about 0.01 mg / kg / day to 1000 mg / kg / day. It is expected that oral administration in the range of 50 to 500 mg / kg will produce the desired result in one or several doses per day. The dosage may be adjusted appropriately to achieve the desired drug concentration locally or systemically, depending on the mode of administration. In cases where such a dose is inadequate for the subject, higher doses (or higher doses that are more effective depending on the different and more locally delivered routes) are used to the extent that patient tolerance allows. Multiple doses per day are scheduled to achieve the appropriate systemic concentration of the compound.

  Preferably, such substances are used in doses, formulations and dosing schedules that favor the activity of the substance and, if any, do not significantly impact normal cell function.

  In one embodiment, the degree of activity of the drug is at least 10%. In other embodiments, the degree of activity of the drug is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. .

  When administered to a subject for therapeutic purposes, the formulations of the present invention are applied in a pharmaceutically acceptable amount and in a pharmaceutically acceptable composition. Such pharmaceutical compositions can contain the substances of the invention in combination with any standard physiologically and / or pharmaceutically acceptable carrier known in the art. The composition should be sterile and contain a therapeutically effective amount of the substance in weight or volume units suitable for administration to a patient. As used herein, the term “pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid excipients, diluents, or encapsulating materials, Suitable for administration to humans or other animals. The term “carrier” denotes an organic or inorganic component, a natural or synthetic component, whereby the active ingredients are combined to facilitate application. The components of the pharmaceutical composition can also be mixed with each other and with the molecules of the invention in such a manner that there is no interaction that substantially impairs the desired pharmaceutical effect. Pharmaceutically acceptable means a non-toxic material that is further compatible with biological systems such as cells, cell cultures, tissues, or organs. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, excipients, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

  Such formulations may usually contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic ingredients. For use in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used in the preparation of the pharmaceutically acceptable salt and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, odorous acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, Salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts such as sodium, potassium, calcium salts of carboxylic acid groups.

  Suitable buffering agents include: acetic acid and salt (1-2% W / V); citric acid and salt (1-3% W / V); boric acid and salt (0.5-2. 5% W / V); and phosphoric acid and salts (0.8-2% W / V).

  Suitable preservatives include benzalkonium chloride (0.003-0.03% W / V); chlorobutanol (0.3-0.9% W / V); paraben (0.01-0.25). % W / V) and thimerosal (0.004-0.02% W / V).

  A variety of administration routes are available. The particular form selected will, of course, depend on the particular combination of drugs selected, the severity of the cancer symptoms being treated, the condition of the patient, and the dosage required for the therapeutic effect. The methods of the present invention can be performed using any form of administration that is generally medically acceptable and generally produces an effective concentration of the active compound that does not cause clinically unacceptable side effects. Means any form of Such modes of administration include oral, rectal, topical, nasal, other mucosal types, direct injection, transdermal, sublingual or other routes. “Parenteral” routes include subcutaneous, intravenous, intramuscular, or infusion. Direct injection is preferred for local arrival at the site of cancer. Since oral administration is convenient for patients and administration schedules, it is preferable for preventive treatment in subjects at risk of developing cancer, for example.

  Chemical / physical vectors can also be used to deliver a substance of the invention to a target (eg, a cell), thereby facilitating absorption. “Chemical / physical vectors” as used herein are natural or synthetic molecules, other than those derived from bacterial or viral sources, that are capable of delivering a substance of the invention to a target (eg, a cell). Show what you can do.

  A preferred chemical / physical vector of the present invention is a colloidal dispersion system. Colloidal dispersion systems include oil-in-water emulsifiers, micelles, mixed micelles and lipid type systems including liposomes. The preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vessels that are useful as delivery vectors in vivo or in vitro. Large single film vessels (LUV) range in size from 0.2 to 4.0 mμ, which has been shown to be able to encapsulate large polymers. RNA, DNA, and intact virus can be encapsulated within an aqueous interior and delivered to cells in a biologically active form (Fraley et al., Trends Biochem. Sci., V. 6, p. 77 (1981)). . In order for liposomes to be an effective gene delivery vector, one or more of the following properties must be present: (1) Encapsulate the gene of interest with high efficacy while retaining biological activity; (2) binds favorably and substantially to target cells compared to non-target cells; (3) delivers aqueous content of vesicles to the cytoplasm of target cells with high efficacy; and (4) genetic information. Accurate and effective expression.

  Liposomes can be targeted to specific (eg, tissue) (eg, vascular cell walls) by binding the liposome to specific ligands such as monoclonal antibodies, sugars, glycolipids or proteins.

  Liposomes are commercially available from Gibco BRL, for example as LIPOFECTINTM and LIPOFECTACETM, which are N- [1- (2,3dioleyloxy) -propyl] -N, N, N-trimethylammonium chloride (DOTMA) and It is formed from a cationic lipid such as dimethyl dioctadecyl ammonium bromide (DDAB). Methods for producing liposomes are well known in the art and are described in numerous references. Liposomes are also reviewed in Gregoriadis, G. Trends in Biotechnology, V. 3, p. 235-241 (1985).

  In one particular embodiment, the preferred vehicle is a biocompatible microparticle or a graft suitable for implantation into a mammalian recipient. A specific biodegradable graft useful by this method is described in PCT International Application No. PCT / US / 03307 (Publication No. WO 95/24929, name “Polymerizable Gene Delivery System”, US Patent Application No. 213,668, 1994. March 15 priority claim). PCT / US / 03307 describes a biocompatible, preferably biodegradable polymeric matrix for containing exogenous genes under the control of a suitable promoter. The polymeric matrix is used to achieve sustained release of exogenous genes in the patient. In accordance with the present invention, the materials of the present invention are encapsulated or dispersed within a biocompatible, preferably biodegradable polymeric matrix as disclosed in PCT / US / 03307. The polymer matrix is preferably in the form of microparticles, such as microspheres (the material is dispersed in a solid polymer matrix) or microcapsules (the material is stored in the core of the polymer shell). Other forms of polymeric matrices containing the materials of the present invention include films, coatings, gels, grafts and stents. The size and composition of the polymeric matrix device is selected to produce favorable release kinetics in the tissue in which the matrix device is implanted. The size of the polymeric matrix device is further selected by the delivery method used, typically by injection into the tissue or administration of an aerosol suspension to the nasal and / or pulmonary region. The polymeric matrix composition can be selected to have both desirable degradation rates and formed from a bioadhesive material to further increase transport efficiency when the device is administered to the vascular surface. The matrix composition can also be selected so that it does not degrade, but rather is released by diffusion over time.

  Both non-biodegradable and biodegradable polymeric matrices can be used for delivery of the substances of the invention to a subject. A biodegradable matrix is preferred. Such a polymer may be a natural or synthetic polymer. Synthetic polymers are preferred. The macromolecule is selected based on the desired duration of release and is generally on the order of a few hours to one year or more. Typically, release over a period ranging between 2, 3 hours and 3-12 months is most desirable. The polymer is optionally in the form of a hydrogel that can absorb up to about 90% weight in water, and is optionally crosslinked with multivalent ions or other polymers.

  In general, the substances of the invention are delivered using biodegradable implants by diffusion or more preferably by degradation of the polymeric matrix. Specific synthetic polymers used to form the biodegradable delivery system include polyamide, polycarbonate, polyalkylene, polyalkylene glycol, polyalkylene oxide, polyalkylene terephthalate, polyvinyl alcohol, polyvinyl ether, polyvinyl ester, polyvinyl Halide, polyvinylpyrrolidone, polyglycolide, polysiloxane, polyurethane and copolymers thereof, alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, acrylic acid and methacrylic acid ester polymer, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose , Hydroxy-propylmethylcellulose, hydroxybutylmethylcellulose , Cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyl ethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl Methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly ( Octadecyl acrylate), polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene glycol) Sid), poly (ethylene terephthalate), poly (vinyl alcohol), polyvinyl acetate, polyvinyl chloride, polystyrene and polyvinylpyrrolidone and the like.

  Examples of non-biodegradable polymers include ethylene vinyl acetate, poly (meth) acrylic acid, polyamides, copolymers and mixtures thereof.

  Examples of biodegradable polymers include polymers of lactic acid and glycolic acid, polyanhydrides, poly (ortho) esters, polyurethanes, poly (butyric acid), poly (valeric acid), and poly (lactide-cocaprolactone) Synthetic polymers of, and other polysaccharides including alginate and dextran and cellulose, collagen, chemical derivatives thereof (for example, substitution, addition, hydroxylation, oxidation of chemical groups such as alkyl, alkylene, etc.) Other modifications), natural polymers such as albumin and other hydrophilic proteins, zein and other prolamins and hydrophobic proteins, copolymers, and mixtures thereof. Generally, these materials degrade by surface or bulk erosion due to enzymatic hydrolysis or exposure to water in vivo.

  Bioadhesive polymers of particular interest include biodegradable hydrogels described in HS Sawhney, CP Pathak and JA Hubell, Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference. Polyhyaluronic acid, casein, gelatin, glutin, polyanhydride, polyacrylic acid, alginate, chitosan, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (Hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), and poly (octadecyl acrylate). It is done. Thus, the present invention provides a composition of the above-mentioned substances for pharmaceutical use, a method for preparing a medicament, and a method for sustained release of a medicament in vivo.

  The compositions are conveniently provided in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of bringing the therapeutic agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately associating the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and, if necessary, shaping the product.

  Compositions suitable for parenteral administration conveniently comprise a sterile aqueous formulation of the therapeutic agent, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation is also a sterile injectable solution or suspension, such as a nontoxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Suitable carrier preparations for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

  Compositions suitable for oral administration may be presented as discrete units, such as capsules, encapsulants, tablets or troches, each containing a predetermined amount of the therapeutic substance. Other compositions include water-soluble liquids or suspensions in water-insoluble liquids such as syrups, elixirs, or emulsions.

  Other delivery systems can include sustained release, delayed release, sustained release delivery systems. Such a system prevents repeated administration of the therapeutic substance of the present invention and improves convenience for the subject and the physician. Many types of release delivery systems are available and are known to those skilled in the art. They include macromolecular systems such as polylactic and polyglycolic acids, poly (lactide-glycolides), copolyoxalates, polyanhydrides, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polycaprolactone. The aforementioned polymeric microcapsules containing a drug are described, for example, in US Pat. No. 5,075,109. Lipids containing sterols such as cholesterol, cholesterol esters, and fatty acids, or neutral fats such as mono-, di- and tri-glycerides; liposomes; phospholipids; hydrogel release systems; silastic systems; peptide-type systems; Non-polymeric systems, which are compressed tablets using conventional binders and excipients, are partially fused to an implant or the like. Specific examples include: (a) an erosion system in which the polysaccharide is included in the form within the matrix found in US Pat. Nos. 4,452,775, 4,675,189, and 5,736,152, and (B) a diffusion system in which the active ingredient penetrates at a controlled rate from the polymer as described in US Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. For example, but not limited to. In addition, pump-type hardware delivery systems are used, some of which are adapted for implantation.

  The use of long-term sustained-release implants is particularly suitable for the treatment of subjects at risk of established cancer symptoms as well as cancer progression. As used herein, “long term” release means that the implant is constructed and adjusted to deliver therapeutic levels of the active ingredient for at least 7 days, preferably 30-60 days. The graft can be placed at the site of the tumor. Long-term sustained release implants are well known to those skilled in the art and include several of the release systems described above.

  The therapeutic agent can be administered alone or in combination with an anti-cancer agent. When the therapeutic agents are administered in combination, the compounds can be administered in the same manner, for example intravenously, orally, or the therapeutic agent is administered orally and the anticancer agent is administered intravenously. Depending on the form, they can be administered separately. In one embodiment of the invention, the therapeutic agent and the anticancer agent are administered together intravenously. In other embodiments, the therapeutic agent and the anticancer agent are administered separately.

  Anti-cancer agents that can be administered with the compounds of the present invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronin; adriamycin; adzelesin; aldesleukin; Ambomycin; amethanetron acetate; aminoglutetimide; amsacrine; anastrozole; anthromycin; asparaginase; asperginase; azacitidine; azetepa; azotomycin; Biseresin; Bleomycin sulfate; Brequinal sodium; Bropyrimine; Busulfan; Kactinomycin; Carsterone; Caracemide; -Carboplatin; Carmustine; Carubicin hydrochloride; Calzesin; Sedefingol; Chlorambucil; Silolemycin; Cisplatin; Cladribine; Crisatol mesylate; Dezaguanine; desaguanine mesylate; diaziquan; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; Enpromate; epipropidine; epirubicin hydrochloride; Esolubicin hydrochloride; Estramustine; Estramustine phosphate sodium; Etanidazole; Etoposide; Etoposide phosphate; Etoprine; Fadrozol hydrochloride; Synsodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosin; interferon alfa-2b; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta-Ia; interferon gamma-Ib Iproplatin; Lanotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; riarosol hydrochloride; lometrexol sodium; lomustine; rosoxantrone hydrochloride; masoprocol; Melengestrol acetate; melphalan; menogalyl; mercaptopurine; methotrexate; methotrexate sodium; methoprin; Nogaramicin; Ormaplatin; Oxythran; Paclitaxel; Pega Pergomycin; Peromycin; Pentamustine; Pepromycin Sulfate; Perphosphamide; Pipobroman; Piposulfan; Pyrazofurin; Ribopurine; Logretimide; Saphingol; Saphingol Hydrochloride; Semustine; Simtrazene; Taxol; Tecogalan sodium; Tegaflu; Temoxorphine hydrochloride; temoporfin; teniposide; teloxilone; test lactone; thiampurine; thioguanine; thiotepa; thiafazomine; Nitrate; Triptorelin; Tubrosol hydrochloride; Uracil Mustard; Uledepa; Vapretide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vinrosidine sulfate; vinzolidine sulfide Over preparative; vorozole; Zenipurachin; zinostatin; sol bicine hydrochloride. Additional anti-neoplastic substances include Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner) Chapter 52 and Goodman and Gilman's “Pharmacological Basis of Therapeutics”, Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division), 1202-1263, for example.

  Peptide sequence (written from N-terminal to C-terminal):

Histidine-added truncated receptor (His-TR) (having the “SPY” sequence of var-PSMGFR):
GTINVHDVETQFNQYKTEAASPYNLTISDVSVSHHHHHH (SEQ ID NO: 1)

Example of histidine-added primary sequence of the MUC1 growth factor receptor (His-var-PSMGFR) (having the “SPY” sequence of var-PSMGFR):
GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGAHHHHHH (SEQ ID NO: 2)

Example of histidine tag primary sequence of the MUC1 growth factor receptor with a single amino acid deletion at the C-terminus of SEQ ID NO: 2 (His-var-PSMGFR) (having the “SPY” sequence of var-PSMGFR):
TINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGAHHHHHH (SEQ ID NO: 60)

The histidine addition extension sequence (ESMGFR) of the MUC1 growth factor receptor (having the “SPY” sequence of var-PSMGFR):
VQLTLAFREGTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFHHHHHH (SEQ ID NO: 3)

The histidine-tagged tumor-specific extension sequence (TSESMGFR) of the MUC1 growth factor receptor (having the “SPY” sequence of var-PSMGFR):
SVVVQLTLAFREGTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGAHHHHHH (SEQ ID NO: 61)

Histidine tag primary sequence (His-PSIBR) in the interchain binding region:
HHHHHHGFLGLSNIKFRPGSVVVQLTLAFRE (SEQ ID NO: 4)

Histidine tag cleavage type interchain binding region (His-TPSIBR):
HHHHHHSVVVQLTLAFREG (SEQ ID NO: 62)

Histidine tag repeat motif 2 (His-RM2):
PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAHHHHH (SEQ ID NO: 5)

Truncated PSMGFR receptor (TR) (having the “SPY” sequence of var-PSMGFR):
GTINVHDVETQFNQYKTEAASPYNLTISDVSVS (SEQ ID NO: 6)

Wild type primary sequence of MUC1 growth factor receptor (example of nat-PSMGFR- “PSMGFR”):
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 36)

Wild-type primary sequence of the MUC1 growth factor receptor having a single amino acid deletion at the C-terminus of SEQ ID NO: 36 (example of nat-PSMGFR— “PSMGFR”):
TINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (sequence number 63)

A "SPY" functional variant of the wild-type primary sequence of the MUC1 growth factor receptor with enhanced stability (example of var-PSMGFR- "PSMGFR"):
GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (sequence number 7)

A "SPY" functional variant of the wild-type primary sequence of the MUC1 growth factor receptor with enhanced stability having a single amino acid deletion at the C-terminus of SEQ ID NO: 7 (example of var-PSMGFR- "PSMGFR") :
TINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 64)

Primary sequence of interchain binding region (PSIBR):
GFLGLSNIKFRPGSVVVQLTLAFRE (SEQ ID NO: 8)

Cleaved interchain binding region (TPSIBR):
SVVVQLTLAFREG (SEQ ID NO: 65)

Repeat motif 2 (RM2):
PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 9)

Tumor-specific extension sequence of the MUC1 growth factor receptor (TSESMGFR) (having the “SPY” sequence of var-PSMGFR):
SVVVQLTLAFREGTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 66)

Full length MUC1 receptor mucin 1 precursor, Genbank accession number: P15941 (Table 1 below)

A truncated MUC1 receptor isoform having a nat-PSMGFR at the N-terminus and including the transmembrane and cytoplasmic sequence of the full-length MUC1 receptor (“nat-PSMGFRTC isoform” —an example of “PSMGFRTC” —any N- Shown excluding the terminal signal sequence-after translation, before cleavage of the receptor on the cell surface (SEQ ID NO: 47, 58 or 59):
G TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIALAVCQCRRKN YGQLDIFPARDTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL
(SEQ ID NO: 37)

A truncated MUC1 receptor isoform (“CM isoform” —with any nat-PSMGFR and PSIBR at the N-terminus and containing the full-length MUC1 receptor transmembrane and cytoplasmic sequences—the signal sequence of any N-terminus Shown excluded-SEQ ID NO: 47, 58, or 59) which is cleaved after translation and before expression of the receptor on the cell surface
GFLGLSNIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPARDTYHPMSEYP TYHTHGRYVP PSSTDRSPYEKVSAGNGGSSNLSYPA
(SEQ ID NO: 38)

A truncated MUC1 receptor isoform having a nat-PSMGFR + PSIBR + native region at the N-terminus and including the transmembrane and cytoplasmic sequence of the full-length MUC1 receptor (“UR isoform” —SEQ ID NO: 47, 58, or 59) (Except for any N-terminal signal sequence):
ATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSTVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYP TYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL
(SEQ ID NO: 39)

A truncated MUC1 receptor isoform containing transmembrane and cytoplasmic sequences of the full-length MUSC1 receptor (“Y isoform” —shown except for any N-terminal signal sequence—post-translational receptor on the cell surface SEQ ID NO: 47, 58, or 59) that is cleaved before expression in the body:
GSGHASSTPGGEKETSATQRSSVPSSTEKNAFNSSLEDPSTDYYQELQRDISEMFLQI YKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDMETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCLVALAIVYHTGPS
(SEQ ID NO: 40)

A truncated MUC1 receptor isoform (“Rep isoform” —which has a nat-PSMGFR + PSIBR + specific region + repeat at the N-terminus and contains the transmembrane and cytoplasmic sequence of the full-length MUC1 receptor—any N-terminal Shown excluding the signal sequence-SEQ ID NO: 47, 58, or 59) which is cleaved after translation and before expression of the receptor on the cell surface:
LDPRVRTSAPDTRPAPGSTAPQAHGVTS (APDTRPAPGSTAPPAHGVTS) 25APDTRP APGSTAPPAHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARAT TTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYH THGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL
(SEQ ID NO: 41)

N-terminal MUC-1 signal sequence for directing MUC1 receptor and truncated isoform to the cell membrane surface (optionally present in whole or in part-eg, N-terminal of MUC1 truncated receptor isoform listed above) Up to three amino acids at the C-terminus, as shown by the variants of SEQ ID NOs: 47, 58 and 59):
MTPGTQSPFFLLLLLTVLT (SEQ ID NO: 47)
MTPGTQSPFFLLLLLTVLT VVTA (SEQ ID NO: 58)
MTPGTQSPFFLLLLLTVLT VVTG (SEQ ID NO: 59)

Proopiomelanocortin (adrenocorticotropin / beta-lipotropin / alpha-melanocyte stimulating hormone / beta melanocyte stimulating hormone / beta endorphin) [Homo sapiens]
Deposit number: XP_002485

RGD
HHHHHHSSSSGSSSSGSSSSGGRGDSGRGDS (SEQ ID NO: 12)

  Nucleic acid sequence encoding a truncated isoform of MUC1 receptor (listed from 5′-end to 3′-end):

Examples of nucleic acid molecules encoding nat-PSMGFRTC of SEQ ID NO: 37:
acgggcacgg ccggtaccat caatgtccac gacgtggaga cacagttcaa tcagtataaa acggaagcag cctctcgata taacctgacg atctcagacg tcagcgtgag tgatgtgcca tttcctttct ctgcccagtc tggggctggg gtgccaggct ggggcatcgc gctgctggtg ctggtctgtg ttctggttgc gctggccatt gtctatctca ttgccttggc tgtctgtcag tgccgccgaa agaactacgg gcagctggac atctttccag cccgggatac ctaccatcct atgagcgagt accccaccta ccacacccat gggcgctatg tgccccctag cagtaccgat cgtagcccct atgagaaggt ttctgcaggt aacggtggca gcagcctctc ttacacaaac ccagcagtgg cagccgcttc tgccaacttg tagggcacgt cgccgctgag ctgagtggcc agccagtgcc attccactcc actcaggttc ttcaggccag agcccctgca ccctgtttgg gctggtgagc tgggagttca ggtgggctgc tcacagcctc cttcagaggc cccaccaatt tctcggacac ttctcagtgt gtggaagctc atgtgggccc ctgaggctca tgcctgggaa gtgttgtggg ggctcccagg aggactggcc cagagagccc tgagatagcg gggatcctga actggactga ataaaacgtg gtctcccact g
(SEQ ID NO: 42)

Examples of nucleic acid molecules encoding the CM isoform of SEQ ID NO: 38:
acggccggtt ttctgggcct ctccaatatt aagttcaggc caggatctgt ggtggtacaa ttgactctgg ccttccgaga aggtaccatc aatgtccacg acgtggagac acagttcaat cagtataaaa cggaagcagc ctctcgatat aacctgacga tctcagacgt cagcgtgagt gatgtgccat ttcctttctc tgcccagtct ggggctgggg tgccaggctg gggcatcgcg ctgctggtgc tggtctgtgt tctggttgcg ctggccattg tctatctcat tgccttggct gtctgtcagt gccgccgaaa gaactacggg cagctggaca tctttccagc ccgggatacc taccatccta tgagcgagta ccccacctac cacacccatg ggcgctatgt gccccctagc agtaccgatc gtagccccta tgagaaggtt tctgcaggta acggtggcag cagcctctct tacacaaacc cagcagtggc agccgcttct gccaacttgt agggcacgtc gccgctgagc tgagtggcca gccagtgcca ttccactcca ctcaggttct tcaggccaga gcccctgcac cctgtttggg ctggtgagct gggagttcag gtgggctgct cacagcctcc ttcagaggcc ccaccaattt ctcggacact tctcagtgtg tggaagctca tgtgggcccc tgaggctcat gcctgggaag tgttgtgggg gctcccagga ggactggccc agagagccct gagatagcgg ggatcctgaa ctggactgaa taaaacgtgg tctcccactg
(SEQ ID NO: 43)

Examples of nucleic acid molecules encoding the UR isoform of SEQ ID NO: 39:
acggccgcta ccacaacccc agccagcaag agcactccat tctcaattcc cagccaccac tctgatactc ctaccaccct tgccagccat agcaccaaga ctgatgccag tagcactcac catagctcgg tacctcctct cacctcctcc aatcacagca cttctcccca gttgtctact ggggtctctt tctttttcct gtcttttcac atttcaaacc tccagtttaa ttcctctctg gaagatccca gcaccgacta ctaccaagag ctgcagagag acatttctga aatgtttttg cagatttata aacaaggggg ttttctgggc ctctccaata ttaagttcag gccaggatct gtggtggtac aattgactct ggccttccga gaaggtacca tcaatgtcca cgacgtggag acacagttca atcagtataa aacggaagca gcctctcgat ataacctgac gatctcagac gtcagcgtga gtgatgtgcc atttcctttc tctgcccagt ctggggctgg ggtgccaggc tggggcatcg cgctgctggt gctggtctgt gttctggttg cgctggccat tgtctatctc attgccttgg ctgtctgtca gtgccgccga aagaactacg ggcagctgga catctttcca gcccgggata cctaccatcc tatgagcgag taccccacct accacaccca tgggcgctat gtgcccccta gcagtaccga tcgtagcccc tatgagaagg tttctgcagg taacggtggc agcagcctct cttacacaaa cccagcagtg gcagccgctt ctgccaactt gtagggcacg tcgccgctga gctgagtggc cagccagtgc cattccactc cactcaggtt cttcaggcca gagcccctgc accctgtttg ggctggtgag ctgggagtg gctct ggctgga ctct
(SEQ ID NO: 44)

Examples of nucleic acid molecules encoding the Y isoform of SEQ ID NO: 40:
acaggttctg gtcatgcaag ctctacccca ggtggagaaa aggagacttc ggctacccag agaagttcag tgcccagctc tactgagaag aatgctttta attcctctct ggaagatccc agcaccgact actaccaaga gctgcagaga gacatttctg aaatgttttt gcagatttat aaacaagggg gttttctggg cctctccaat attaagttca ggccaggatc tgtggtggta caattgactc tggccttccg agaaggtacc atcaatgtcc acgacgtgga gacacagttc aatcagtata aaacggaagc agcctctcga tataacctga cgatctcaga cgtcagcgtg agtgatgtgc catttccttt ctctgcccag tctggggctg gggtgccagg ctggggcatc gcgctgctgg tgctggtctg tgttctggtt gcgctggcca ttgtctatct cattgccttg gctgtctgtc agtgccgccg aaagaactac gggcagctgg acatctttcc agcccgggat acctaccatc ctatgagcga gtaccccacc taccacaccc atgggcgcta tgtgccccct agcagtaccg atcgtagccc ctatgagaag gtttctgcaggcgactactcccactccc
(SEQ ID NO: 45)

Example of a nucleic acid molecule encoding the Rep isoform of SEQ ID NO: 41:
ctcgacccac gcgtccgctc gacccacgcg tccgcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac accaggccgg ccccgggctc caccgccccc ccagcccatg gtgtcacctc ggccccggac aacaggcccg ccttgggctc caccgcccct ccagtccaca atgtcacctc ggcctcaggc tctgcatcag gctcagcttc tactctggtg cacaacggca cctctgccag ggctaccaca accccagcca gcaagagcac tccattctca attcccagcc accactctga tactcctacc acccttgcca gccatagcac caagactgat gccagtagca ctcaccatag ctcggtacct cctctcacct cctccaatca cagcacttct ccccagttgt ctactggggt ctctttcttt ttcctgtctt ttcacatttc aaacctccag tttaattcct ctctggaaga tcccagcacc gactactacc aagagctgca gagagacatt tctgaaatgt ttttgcagat ttataaacaa gggggttttc tgggcctctc caatattaag ttcaggccag gatctgtggt ggtacaattg actctggcct tccgagaagg taccatcaat gtccacgacg tggagacaca gttcaatcag tataaaacgg aagcagcctc tcgatataa c ctgacgatct cagacgtcag cgtgagtgat gtgccatttc ctttctctgc ccagtctggg gctggggtgc caggctgggg catcgcgctg ctggtgctgg tctgtgttct ggttgcgctg gccattgtct atctcattgc cttggctgtc tgtcagtgcc gccgaaagaa ctacgggcag ctggacatct ttccagcccg ggatacctac catcctatga gcgagtaccc cacctaccac acccatgggc gctatgtgcc ccctagcagt accgatcgta gcccctatga gaaggtttct gcaggtaacg gtggcagcag cctctcttac acaaacccag cagtggcagc cgcttctgcc aacttgtagg gcacgtcgcc gctgagctga gtggccagcc agtgccattc cactccactc aggttcttca ggccagagcc cctgcaccct gtttgggctg gtgagctggg agttcaggtg ggctgctcac agcctccttc agaggcccca ccaatttctc ggacacttct cagtgtgtgg aagctcatgt gggcccctga ggctcatgcc tgggaagtgt tgtgggggct cccaggagga ctggcccaga gagccctgag atagcgggga tcctgaactg gactgaataa cactgg
(SEQ ID NO: 46)

Examples of nucleic acid molecules encoding the full length MUC1 receptor of SEQ ID NO: 10:
acaggttctg gtcatgcaag ctctacccca ggtggagaaa aggagacttc ggctacccag agaagttcag tgcccagctc tactgagaag aatgctgtga gtatgaccag cagcgtactc tccagccaca gccccggttc aggctcctcc accactcagg gacaggatgt cactctggcc ccggccacgg aaccagcttc aggttcagct gccacctggg gacaggatgt cacctcggtc ccagtcacca ggccagccct gggctccacc accccgccag cccacgatgt cacctcagcc ccggacaaca agccagcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggccc c gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctccacc gcccccccag cccacggtgt cacctcggcc ccggacacca ggccggcccc gggctcca cc gcccccccag cccatggtgt cacctcggcc ccggacaaca ggcccgcctt gggctccacc gcccctccag tccacaatgt cacctcggcc tcaggctctg catcaggctc agcttctact ctggtgcaca acggcacctc tgccagggct accacaaccc cagccagcaa gagcactcca ttctcaattc ccagccacca ctctgatact cctaccaccc ttgccagcca tagcaccaag actgatgcca gtagcactca ccatagctcg gtacctcctc tcacctcctc caatcacagc acttctcccc agttgtctac tggggtctct ttctttttcc tgtcttttca catttcaaac ctccagttta attcctctct ggaagatccc agcaccgact actaccaaga gctgcagaga gacatttctg aaatgttttt gcagatttat aaacaagggg gttttctggg cctctccaat attaagttca ggccaggatc tgtggtggta caattgactc tggccttccg agaaggtacc atcaatgtcc acgacgtgga gacacagttc aatcagtata aaacggaagc agcctctcga tataacctga cgatctcaga cgtcagcgtg agtgatgtgc catttccttt ctctgcccag tctggggctg gggtgccagg ctggggcatc gcgctgctgg tgctggtctg tgttctggtt gcgctggcca ttgtctatct cattgccttg gctgtctgtc agtgccgccg aaagaactac gggcagctgg acatctttcc agcccgggat acctaccatc ctatgagcga gtaccccacc taccacaccc atgggcgcta tgtgccccct agcagtaccg atcgtagccc ctatgagaag gtttctg cag gtaacggtgg cagcagcctc tcttacacaa acccagcagt ggcagccgct tctgccaact tgtagggcac gtcgccgctg agctgagtgg ccagccagtg ccattccact ccactcaggt tcttcaggcc agagcccctg caccctgttt gggctggtga gctgggagtt caggtgggct gctcacagcc tccttcagag gccccaccaa tttctcggac acttctcagt gtgtggaagc tcatgtgggc ccctgaggct catgcctggg aagtgttgtg ggggctccca ggaggactgg cccagagagc cctgagatag cggggatcct gaactggact gaataaaacg tggtctccca ctg
(SEQ ID NO: 48)

  The following examples are intended to illustrate the advantages of the present invention, but do not embody the full scope of the invention.

Examples Colloid Preparation / Drug Screening Methods Used in the Examples In certain examples and embodiments of the present invention, self-assembled monolayers (SAMs) are made and used on the surface of colloidal particles. The colloid has been derivatized with SAM and published in International Publication No. WO 00/43791, July 27, 2000, titled “Rapid and hypersensitive detection of abnormal protein aggregation in neurodegenerative diseases” (incorporated herein by reference). Prepared for drug screening in a manner similar to that described in (incorporated).

  In a typical example, 1.5 ml of commercially available gold colloid (Auro Dye, Amercham) was pelleted by centrifugation in a microcentrifuge tube at high speed for 10 minutes. The pellet was resuspended in 100 μL of storage buffer (sodium citrate and tween-20), 100 μL dimethylformamide solution (DMF) containing thiol. After 3 hours incubation in thiol solution, the colloid was pelleted and the supernatant was discarded. They were then subjected to a thermal cycle with 100 μL of 400 μM tri-ethylene glycol terminated thiol in DMF at 55 ° C. for 2 minutes, 37 ° C. for 2 minutes, 55 ° C. for 1 minute, 37 ° C. for 2 minutes, and room temperature for 10 minutes. It was. Thermal cycling results in the elimination of all species not at the lowest energy check, resulting in a stable, tightly enclosed self-assembled monolayer. Thermal cycling can be performed by any of the species that form a broad self-assembled monolayer. The colloid was then pelleted and 100 μL of 100 mM NaCl phosphate buffer was added. The colloid was then diluted 1: 1 with 180 μM NiSO4 in colloid storage buffer.

  The thiols used in the coating colloid are typically about 40 μM nitrilotriacetic acid (NTA) -thiol and methyl-terminated thiols (HS— (CH 2) 15 CH 3), 40% tri-ethylene glycol terminated thiol, HS (CH 2) 11 ( Derived from a solution containing CH2CH2) 3OH, (Chemical Formula) and other thiols such as 50% poly (ethynylphenyl) thiol (Cl6H10S). Various thiols have been used to selectively inhibit optimal non-specific binding.

  Colloidal aggregation was detected sensitively by monitoring the color change of the colloidal particles initially dispersed in the suspension. Aggregation results in a color change to blue. Ancillary signaling substances are not necessary. In drug screening, aggregation (or deletion thereof) is observed in the presence of the candidate drug.

Example 1: Dimerization of the MGFR portion of the MUC1 receptor induces enhanced cell proliferation consistent with the mechanism presented to MUC1 tumor cells.
This example demonstrates the effect of dimerization on the MUC1 receptor. In this example, exposure of cells to the bivalent antibody of the invention generated against the MGFR region of the MUC1 receptor resulted in enhanced cell proliferation consistent with the mechanism presented to MUC1 tumor cells at various concentrations. (Or its deletion). Bivalent antibodies were raised against either var-PSMGFR or nat-PSMGFR shown in the sequence listing (ie, one antibody with the ability to bind to two MGFRs at the same time was produced). MUC1 tumor cells (T47D) were exposed to this antibody and cell proliferation was studied as a function of antibody concentration. A typical growth / response curve of growth factor / receptor-antibody response was observed. In particular, cell growth was low at concentrations sufficiently low that only a fraction of the cells were exposed to the antibody. At a sufficiently high antibody concentration that one antibody can bind to the adjacent MGFR, cell proliferation was maximal. In high excess of antibody, each antibody bound to only one MGFR rather than dimerizing adjacent MGFR, and proliferation was reduced.

  A human breast cancer cell line overexpressing T47D (HTB-133) cells, MUC1, was cultured to 30% confluence. An antibody of the invention raised against the PSMGFR portion of the MUC1 receptor, ie an antibody to MFGR (from nat-PSMGFR or var-PSMGFR supplied by the inventor by Zymed, San Francisco, California, USA) (Produced under contract with the inventors) were added to the cells in multi-well cell culture plates at various concentrations. As a negative control, a second set of T47D cells was treated with an irrelevant antibody (anti-streptavidin). Cells were counted (time zero) before adding antibody. All experiments were performed in triplicate. Cells were grown in a CO2 incubator under normal conditions. Cells were counted using a hemocytometer at 24 hours and again at 48 hours (3 times per well). The results show in a concentration dependent manner that the addition of the antibody caused an increase in cell proliferation compared to the growth of the same cells treated with the control antibody. See FIG. FIG. 2 is a graph plotting cell growth measured at 24 and 48 hours as a function of anti-MGFR concentration. At an optimal antibody concentration, cell growth is maximal when a putative one antibody binds bivalently to the two MGFR portions of the MUC1 receptor, ie when the receptor is dimerized.

  In a similar experiment, a concentration of anti-MGFR antibody specified to maximize cell proliferation was added to a first group of T47D tumor cells grown as described above. The same amount of anti-MGFR antibody was added to one set of control cells, K293 cells. FIG. 3 shows that the addition of anti-MGFR antibody to MUC1 tumor cells (T47D) enhanced proliferation by 180% in 24 hours but had no effect on control cells. The growth of T47D cells peaked at saturation, and was 48 hours for antibody-added cells. Control cells did not reach saturation within the experimental time frame and were 70% confluent at 48 hours.

Example 2: Identification of a ligand that binds to the MGFR portion of the MUC1 receptor In an attempt to identify a ligand for the MUC1 receptor, a synthetic His-var-PSMGFR peptide, GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGAHHHHHH (SEQ ID NO: 2), part of the MUC1 receptor It is representative and remains on the cell surface after cleavage of the interchain junction region, but this is loaded onto NTA-Ni beads (Catalog No. 100630; commercially available from Qiagen GmbH, Germany) to produce a protease inhibitor Incubation with cell lysate in the presence (FIG. 4) or absence (FIG. 5) of PMSF (phenylmethylsulfonyl fluoride). A lysate obtained from T47D cells was used because it is known that this breast tumor cell line overexpresses MUC1 and MUC1 ligand. T47D cells were cultured and then sonicated for 1 minute to solubilize the cells. The lysate was mixed with PSMGFR peptide-presenting beads and incubated for 1 hour on ice with intermittent mixing. As a negative control, an irrelevant peptide HHHHHHRGEFTGTYITAVT (SEQ ID NO: 13) was attached to NTA-Ni beads and treated in the same manner. Both sets of beads were washed twice with pH 7.4 phosphate buffer. The bound protein species was eluted by adding 100 μL phosphate buffer containing 250 mM imidazole three times. For both peptides, a portion of the original eluate was removed and stored to run as a separate sample, while the remainder was combined with two additional eluates and concentrated by TCA (trichloroacetic acid) -titration. (Chen, L. et al., Anal. Biochem. Vol 269; pgs 179-188; 1999). The eluate was run on a 12% SDS gel, see FIG. The gel was then silver stained (Schevchenko, A et al. Anal. Chem., Vol. 68; pg 850-858; 1996). The lanes were loaded as follows (from left to right): (1) Benchmark pre-stained protein ladder (Gibco); (2) First eluate from MUC1 peptide; (3) 1/10 of TCA enriched sample; (4) Blank; (5) 9/10 of the TCA-enriched sample; (6) Negative control peptide of the first eluate; (7) 1/10 of the TCA-enriched sample obtained from the negative control peptide; 5 pmol BSA (as standard); (9) 9/10 of TCA enriched sample obtained from negative control peptide; (10) Silver stained SDS page standard (BioRad catalog number 1610314). Referring to FIG. 4, when comparing lanes 2 and 6 (control), it can be seen that the PSMGFR peptide of MUC1 was separately bound to the three peptides: the first unique peptide flowing at an apparent molecular weight of 17 kD; A second peptide (darker band) that flows with an apparent molecular weight of 23 kD. It should be noted that in lane 5 where the sample is most concentrated, a third characteristic band is seen at about 35 kD.

  FIG. 5 shows the results of an experiment similar to that shown in FIG. 4 with the exception that the protease inhibitor PMSF was not added. PMSF binds to several enzymes such as proteases and blocks their action. This experiment was performed to determine if the enzyme in the lysate was a MUC1 receptor ligand in the absence of PMSF. Referring to FIG. 5, comparing lanes 3 (control) and 7, it can be seen that the MUC1 and PSMGFR peptides bound to one peptide having an apparent molecular weight of 35 kD. It should be noted that this band can be seen in FIG. 4 (with PMSF) but was very faint and eluted only from the most concentrated sample. These results are consistent with the idea that the PFMGFR portion of the MUC1 receptor is a substrate for an apparent molecular weight ligand of approximately 35 kD, which is an enzyme. As noted elsewhere in this specification, drug screening based on inhibition of binding of PSMGFR to this ligand or to a ligand in crude cell lysate can identify compounds that inhibit the action of this enzyme. .

  Cell lines were purchased from ATCC (American Type Culture Collection, Manasses, VA) and are all breast adenoma cell lines. Several strains are known for the expression or overexpression of the tumor marker receptors MUC1, Her2 / neu or the carcinogenic enzyme cathepsin K.

Example 3: Demonstration that ligands that interact with MUC1 cancer cells are multimers In this example, it was demonstrated that the ligands produced by MUC1 cancer cells that cause cell proliferation in MUC1 cancer cells are multimers.

  Protein bands at 17 kD, 23 kD and 35 kD were excised from the gel described above in Example 2 and subjected to peptide analysis. These gel bands intentionally included ligands to the MGFR region of the MUC1 receptor. Recall that the 17 kD and 23 kD species bound to the MGFR peptide in the presence of the protease inhibitor PMSF, while the 35 kD species bound when PMSF was not added to the cell lysate mixture.

  The following peptide analysis was performed. The sample from the gel piece was degraded by proteolysis. The fragments were then separated by microcapillary HPLC, which was directly coupled to the nanoelectrospray ionization source of the ion trap mass spectrometer. MS / MS spectra were obtained online. The SEQUEST® algorithm was then used in combination with other algorithms to associate these fragmented spectra with known sequences. The results were then manually reviewed to confirm consensus with known protein sequences.

  The peptide sequence contained in both the 17 kD and 23 kD bands (PMSF added to the lysate) corresponds to a protein known as the metastasis inhibitor NM23, which is involved in both the promotion and inhibition of human cancer metastasis. Yes. Whether the role of NM23 is a tumor suppressor or a promoter depends on the type of cancer. NM23 overexpression has been linked to a malignant phenotype in ovarian, colon, and neuroblastoma tumors (Schneider J, Romero H, Ruiz R, Centeno MM, Rodriguez-Escudero FJ, “advanced and borderline ovarian cancer NM23 expression ", Anticancer Res, 1996; 16 (3A): 1197-202). However, breast cancer studies have shown that decreased expression of NM23 is associated with poor prognosis (Mao H, Liu H, Fu X, Fang Z, Abrams J, Worsham MJ, Expect metastasis and low survival rates ", Int. J. Oncol. 2001 Mar; 18 (3): 587-91).

  The sequences derived from a number of cancer-related proteins identified from the protein gel bands shown in FIGS. 4 and 5 and called metastasis inhibitor NM23 are shown in Table 5 below. NM23 exists as a hexamer and can recognize the native form of the MGFR portion of the MUC1 receptor.

  Peptide sequences identified from the 35 kD gel band (no PMSF added to the lysate) are 14-3-3, a signal transduction protein associated with many cancers, and proteases, associated with tumor progression Corresponded to one or more protein species containing cathepsin D. 14-3-3 exists as a dimer and can bind to two identical phospho-serine peptides at the same time. This dimerizes the MGFR portion of the MUC1 receptor that causes cell proliferation, which is consistent with the mechanism presented herein. Cathepsin D is a protease and may be required for cleavage of the MUC1 receptor.

  The identity of these ligands is consistent with the MUC1-dependent cell proliferation mechanism disclosed herein, ie, ligands that dimerize the MGFR portion of the MUC1 receptor induce cell proliferation and MUC1 Cleavage of only a portion of the extracellular domain exposes the functional portion of the receptor identified by most or all of the nat-PSMGFR sequence (SEQ ID NO: 36) set forth in the sequence listing.

  Consistent with the methods of the present invention, therapeutic strategies identify compounds that block the interaction between the MGFR portion of the MUC1 receptor and one ligand, or identify compounds that bind to a ligand and block the action. It is.

Prophetic Example 4: MUC1 IBR release associated with screening for agents that affect MUC1 cleavage status may be associated with cancer progression. The following is a complete description of cell analysis to identify drug candidates that affect the cleavage status of these receptors. Screening also includes, but is not limited to, enzymatic cleavage, receptor production, expression, stability, transport or secretion, resulting in a decrease in the self-aggregated portion of the receptor that is disrupted and released from the cell. Identify drug candidates that directly or indirectly modulate the process.

  Tumor-derived cells that express cell surface receptors of the type described above are cultured and treated with drug candidates. After some incubation period, peptide aggregation analysis is performed in the pericellular solution. For example, colloids with binding peptides such as antibodies to the constant region of the receptor are distant from the enzyme cleavage site (amino acids 425-479 of MUC1; numbered Andrew Spicer et al., J. Biol. Chem Vol 266 No. 23, 1991). pgs. 15099-15109; the numbers of these amino acids correspond to Genbank deposit number P15941; PID G547937 numbers 985-1039) and are added to the solution. If the fragmented portion of the receptor contains a self-aggregating moiety, the receptor in solution aggregates and causes aggregation of attached colloids, visibly detectable changes in the solution, eg: color Causes changes or visible aggregate formation. Inhibition of this visual change represents a substance that is effective in treating the disease state.

  The sequence listing is representative of sequence fragments that are found within the entire sequence of the full-length peptide in certain instances or are fragments or variants thereof. For example, any set of at least 10 contiguous amino acids from any of the sequence fragments in the sequence listing may be sufficient to identify a cognate binding motif. The sequence listing is meant to encompass one sequence of each, and when referring to fragment size, the range is intended to encompass the entire sequence from the smallest fragment (less than one amino acid is a fragment) It is intended to be able to specifically list the length of any and all fragments. Thus, if the fragment length is between 10 and 15, this clearly means that it is 10, 11, 12, 13, 14 or 15 in length.

  With respect to the sequence listing, the receptor is cleaved at a number of different sites to produce peptide fragments with alternative initiation and termination sites. For these fragments of the sequence listing, any stretch of, for example, 8 to 10 contiguous amino acids, either upstream or downstream, is sufficient to identify the specific fragment that is a conjugate referred to herein. It is.

Example 5 FIG. Western blot analysis protocol Cell culture:
All cell lines were obtained from ATCC (American Type Culture Collection) and cultured according to the ATCC recommended method attached to the cell lines. The cells used include the following [name of applicant (ATCC number)]: T-47D (HTB-133), 1500 (CRL-1500), 1504 (CRL-1504), HeLa (CCL-2), HEK-293 (CRL-1705), BT-474 (HTB-20), MDA-MB-453 (HTB-131).

Solubilization preparation:
In preparing the cell lysate, healthy cells were seeded onto 100 mm culture dishes and incubated until the cells were approximately 80-90% confluent. The medium was then removed and the monolayer was washed twice with 5 mL cold PBS. All remaining PBS was completely removed. 1 ml of cold high salt RIPA solubilization buffer [400 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 × protease inhibitor cocktail (Roche Applied Sciences Indianapolis, IN)] was added to the monolayer and the cells were solubilized by incubating on ice for 5 minutes with occasional agitation. The cells were scraped off and the lysate was collected in a 1.5 mL Eppendorf tube. The lysate was centrifuged at 10,000 rpm, the clear supernatant was removed, placed in a new tube and stored at −20 ° C. until use.

Protein concentration:
Protein concentrates were obtained using Pierce BCA Protein Assay (Rockford, L). The manufacturer's protocol was followed. Absorbance was read using a U-2010 UV / Vis-absorbance meter from Hitachi (Randolph, MA). The data was plotted and the curve was fitted by Microsoft Excel.

Deglycosylation:
Samples were deglycosylated using the Enzymatic Protein Deglycosylation Kit from Sigma (St. Louis, MO). The kit includes O-glycosidase, PNGase-F and α-neuraminidase enzymes along with reaction and denaturing buffers. Each deglycosylation was performed using 100 μg of total protein from the solubilized solution, and the enzyme and buffer were added according to the manufacturer's protocol attached to the kit.

SDS-PAGE:
SDS-PAGE electrophoresis was used to separate lysate proteins. The cell lysate was thawed and diluted to an appropriate SDS loading buffer with a total protein amount of 50 μg to prepare a sample with a final volume of 60 μl. A 15% polyacrylamide gel was used to separate the low molecular weight band of the cleaved MUC1 moiety. The gel was electrophoresed on BioRad Mini-Protean 3 (Hercules, CA).

PVDF transcription:
After electrophoresis was completed, gels were prepared for semi-dry transfer using Immobilon-P PVDF membrane from Millipore (Bedford, Mass.) And Trans-Blot SD Semi-Dry Transfer Cell from BioRad (Hercules, Calif.). . Gels, PVDF membranes, and blotting paper (BioRad; Hercules, CA) were equilibrated in Tris / glycine transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 15 minutes. Sandwiches were prepared on the apparatus as described by the manufacturer. Electrophoretic transfer was performed at 15V for 45 minutes.

Western blot:
The membrane was removed and immediately placed in a 25 ml blot (PBS, 0.05% Tween-20, 5% milk) and incubated for 2 hours with gentle agitation. Blot was removed and 25 ml primary antibody solution [1: 1000 dilution of α-PSMGFR antibody of the invention or 1: 200 dilution of VU-4H5 antibody (Santa Cruz Biotechnologies; Santa Cruz, CA) in the blot] And incubated overnight at 4 ° C. Thereafter, the solution was discarded, and the membrane was washed with PBS-T (PBS, 0.05% Tween-20) five times for 10 minutes each. The membrane was then washed with a secondary antibody solution [in blot, HRP (horseradish peroxidase) -conjugated goat-alpha-rabbit IgG antibody or HRP-conjugated rabbit-alpha-mouse IgG antibody (Jackson Immunoresearch; West Grove, PA). : 20000 dilution] for 1 hour at room temperature. The solution was discarded and the membrane was washed 5 times in PBS-T for 10 minutes each. The membrane was then placed in a 1: 1 mixture of Immun-Star HRP Luminol / Enhancer and peroxidase buffer from BioRad Laboratories (Hercules, CA) for 5 minutes. The substrate was removed and the membrane was placed on Saran wrap, exposed to film, and developed with Kodak X-OMAT.

Example 6 Transfectants: Construction of 6 Mammalian Expression Plasmids Encoding 6 Different Lengths of Muc1 Receptor pMucl-Full Encoding Full Length MUC1 Receptor
The pMucl-Full construct contains the complete DNA of MUC1 and encodes the full length Mucl protein (Figure 22 and SEQ ID NO: 10). The pMucl-Full plasmid was ligated from two separate plasmids containing different parts of the MUC1 cDNA. The amino-terminus of MUC1 was obtained from EST0039670 obtained from the Genome Research Center and the Center for Functional Analysis of Human Genome (GRC / CFAHG) of Korea Life Science and Biological Science Research Institute (Ota, Korea). EST0039670 contained up to about 800 base pairs of cDNA from the amino-terminus of the MUC1 open reading frame to the tandem repeat segment of MUC1. The carboxy-terminus of the MUC1 cDNA was obtained from clone number 2428103 of Integrated Molecular Analysis of Genomes and their Expression (IMAGE) obtained from American Type Culture Collection (ATCC). This plasmid contains the remaining 1300 base pairs of tandem repeats through the carboxy-terminus of MUC1. The full length Mucl construct (Mucl-Full) was generated by restriction enzyme digestion of the plasmid containing IMAGE2428103 with SalI and subcloning into EST0039670 digested with XhoI. The resulting subclone, pESTMucI, was confirmed by restriction enzyme digestion and agarose gel electrophoresis, and was found to have a band of the expected size for a correctly constructed plasmid. The pESTMucl plasmid was used for subcloning into the mammalian expression vector pIRES2-GFP.

  The expression vector used for all of the MUC1 constructs was pIRES2-EGFP obtained from Becton Dickinson Clontech (Palo Alto, Calif.). This vector expresses MUC1 from the cytomegalovirus (CMV) early promoter. This vector also contains an internal ribosome entry site (IRES) in the expression of green fluorescent protein (GFP) from the same message as MUC1.

  Full-length Mucl containing plasmid pESTMucl was digested with EcoRI and XhoI, and a DNA fragment containing MUC1 was gel purified. The expression vector, pIRES2-GFP, was digested with EcoRI and SalI and gel purified. The two portions of DNA were ligated to generate pMucl-Full containing the full length MUC1 protein encoded by the mammalian expression vector pIRES2-EGFP. A subclone of mammalian expression vector with the correct insertion was selected for sequencing. The appropriate construct of the desired plasmid was confirmed by sequencing.

  All sequencing was performed by GeneWiz (North Brunswick, NJ). Sequencing was performed by PCR sequencing. All results were confirmed by sequence comparison with the National Center for Biotechnology (NCBI) database.

PMucl-Rep encoding the Rep isoform
The pMucl-Rep (SEQ ID NO: 46) construct encodes a Mucl protein with the amino terminus deleted (FIG. 1; SEQ ID NO: 41). The Rep isoform is missing the amino terminus and contains only about half of the terminal repeat of MUC1. The first pSP-Rep was generated by subcloning the PCR amplified signal peptide of MUC1 into the EST plasmid (IMAGE2428103). The signal peptide of MUC1 was subcloned at the amino terminus to ensure protein expression in the plasma membrane. PCR amplification of the normal MUC1 signal peptide obtained from IMAGE clone number 4695020 was performed using the primers in Table 6. The PCR product was digested with EcoRI and XhoI and subcloned into EcoRI and SalI digested EST plasmids (IMAGE2428103). The appropriate construct of the resulting subclone, pSP-Rep, was confirmed by degradation and agarose gel electrophoresis. The pSP-Rep plasmid and pIRES2-GFP were then digested with EcoRI and BamHI and ligated together. The appropriate construct of pMucl-Rep was confirmed by restriction enzyme digestion followed by gel electrophoresis. Sequencing also showed that the pMucl-Rep plasmid was created correctly.

PMucl-UR encoding the UR isoform: pMucl-CM encoding the CM isoform and pMucl-PSMGFRTC encoding the nat-PSMGFRTC isoform:
These three constructs encode the various amino terminal deleted isoforms of MUC1, the carboxy terminus up to the tandem repeat (see Figure 1. pMucl-UR (SEQ ID NO: 44) encodes SEQ ID NO: 39; pMucl- CM (SEQ ID NO: 43) encodes SEQ ID NO: 38; and pMucl-PMMGFRTC (SEQ ID NO: 42) encodes SEQ ID NO: 37). pMucl-UR encodes an isoform comprising amino acids 981 to 1255 of MUC1. pMucl-CM encodes an isoform that includes amino acids 1085 to 1255 of MUC1 and encodes a peptide of 168 amino acids. pMucl-PSMGFRTC encodes a peptide from amino acid 1110 to amino acid 1255 of MUC1 and is 143 amino acids in length. All three of these plasmids were constructed by the same procedure below. First, from IMAGE clone number 4695020, the signal peptide of MUC1 was amplified by PCR using primers each containing a restriction enzyme site of either EcoRI or EagI. Subsequently, the carboxy-terminal portion of the construct was amplified by PCR with a primer containing an EagI or BamHI site. The primers used for PCR are listed in Table 6. The PCR product was digested with EagI and then ligated. Ligation was again amplified by PCR using the primers containing EcoRI and BamHI used previously. This amplified the ligated PCR product. The resulting DNA fragment was digested with EcoRI and BamHI and subcloned into similarly degraded pIRES2-EGFP. The desired PCR product and plasmid size were confirmed by agarose gel electrophoresis. Sequencing confirmed that the correct plasmid was produced.

PMucl-Y encoding the Y isoform:
The pMucl-Y plasmid encodes an alternatively spliced form of MUC1 (see Figure 1. pMucl-UR (SEQ ID NO: 45) encodes SEQ ID NO: 40). Mucl-Y cDNA was obtained from IMAGE clone no. 4695020. This clone was sequenced and found to contain an extra 9 amino acids. These were deleted by PCR. The front part of the Mucl-Y cDNA was amplified with a primer containing the restriction enzyme site EcoRI and a primer containing the AlwNI restriction enzyme site. The end portion of Mucl-Y was amplified with a primer containing the AlwNI restriction enzyme site and a primer containing the restriction enzyme site BamHI. After digesting both PCR products with AlwNI, the two fragments were ligated. Ligation was again amplified by PCR using the primers containing EcoRI and BamHI used previously. This amplified the ligated PCR product. The resulting DNA fragment was digested with EcoRI and BamHI and subcloned into similarly degraded pIRES2-EGFP. The desired PCR product and plasmid size were confirmed by agarose gel electrophoresis. Sequencing confirmed that the correct plasmid was created with 9 amino acids deleted.

PCR primers

Method-DNA manipulation:
Polymerase chain reaction (PCR) was performed using MiniCylcer from MJ Research (Watertown, MA). The following steps were used for PCR. First step: 94 ° C., 2 minutes, second modification step: 94 ° C., 30 seconds, third annealing step: 55 ° C., 30 seconds, fourth extension step: 68 ° C., 1 minute, fifth step: second step To 4th step 34 times, 6th step: 68 ° C, 5 minutes, and maintained at 4 ° C. Platinum Pfx DNA Polymerase from Invitrogen (Carlsbad, Calif.) Was used for amplification. The PCR reaction contained 2 ng plasmid DNA, 1 × Pfx amplification buffer, 1 mM MgSO 4, 0.3 μl of each primer, 1.25 units of Pfx polymerase, and 0.3 μM of each dNTP in a total 50 μl reaction volume. It was. The PCR product was purified using Qiagen's Qiaquick PCR removal kit, removing the primer and buffer. The PCR product was processed in this way prior to restriction enzyme digestion.

  All DNA restriction enzymes were purchased from New England BioLabs (Beverly, MA). Restriction enzyme digestion was performed in the supplied reaction buffer at 37 ° C. as recommended by the manufacturer. In order to purify the DNA fragment, the band on the agarose gel was visualized with ethidium bromide, and the gel was cut out. The fragments were then purified using Qiagen (Valencia, CA) Qiaquick gel purification columns according to the manufacturer's recommended protocol.

  A small amount of plasmid DNA was prepared by alkaline solubilization as outlined in Ausubel (ref). Large amounts of DNA were prepared using Qiagen Maxi Prep columns. The protocol was performed as recommended by the company except that an additional step of phenol chloroform extraction was added after the DNA was isolated. The DNA concentration was determined by measuring the absorbance of the diluted sample at 260 nm with a Hitachi 3000 absorbance meter. The DNA fragments were ligated using a Rapid DNA Ligation Kit provided by Roche (Indianapolis, IN). The ligated DNA was transformed into chemically competent E. coli strain JM101 by heat shock. DNA sequencing was performed by PCR sequencing and performed at GeneWiz (North Brunswick, NJ).

Example 7: Transfection of HEK293 cells capable of expressing MUC1 isoform on the cell surface:
The goal is to transfect HEK293 cells with various isoforms of MUC1 protein, having proteins expressed on the cell surface. HEK293 cells were transfected with MUC1 isoform and the expression of MUC1 isoform was shown on the surface of the transfected cells.

Transfection procedure:
Human embryonic kidney (HEK) 293 cells were seeded to produce 90% confluency. Cells were transfected with Lipofectamine 2000 from Invitrogen (Carlsbad, CA). The manufacturer's protocol was followed. Both DNA and lipofectamine were diluted in medium without serum and antibiotics. After 5 minutes, DNA and lipofectamine were mixed and incubated at room temperature for 30 minutes. Cells were washed once with sterile 1 × PBS, after which the transfection mixture was added to the cells. The medium was incubated for 4-6 hours before changing the cells with DNA: lipofectamine complex to medium containing 10% serum. These cells were analyzed for MUC1 isoform expression after 24 or 48 hours. Stable cell lines were selected with Invitrogen's 600 μg / ml Geneticin (G418) for 48 days 48 hours after transfection.

Example 8: Polyclonal anti-PSMGFR antibody production The peptide sequence used for immunization was GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (var-PSMGFR SEQ ID NO: 7). Two rabbits were immunized using the Polyquick ™ method, which is a manufacturer's own commercial technique. After 4 weeks, blood was evaluated for PSMGFR specific antibodies. Rabbits received an antigen boost two weeks prior to antibody recovery. The antibody was affinity purified using a peptide bound to a column support.

Example 9 Production of Monovalent Antibody Fragment Antibody fragmentation of the polyclonal antibody produced in Example 8 was performed using papain degradation by Maine Biotechnology Servises Inc. (Portand, ME). Fragmented antibody was purified from uncleaved antibody and Fc fragment. The cleavage reaction and purification were evaluated by SDS PAGE.

Example 10: Evaluation of transfectants in MUC1 isoform expression:
To verify that the MUC1 isoform transfectants expressed the protein on the surface of the cells, flow cytometry was performed on the transfected cells. The indirect cell staining protocol continues briefly as follows. 100 million cells were stained with 1 mg primary anti-PSMGFR antibody. Thereafter, the cells were washed with 1 × PBS. Cells were then stained with Jackson Laboratories' phycoerythrin (PE) -labeled-Fab-fragment, anti-rabbit secondary antibody. Cells were washed and stained with PI. Flow cytometry was performed on a FACS Calibur facility at Beckton Dickinson (Palo Alto, Calif.). Cell populations were explored for live cells by forward and side scatter and propidium iodine (PI) negative staining. Cells were evaluated for levels of GFP showing transfectants and PE showing MUC1 staining. Transfectants stained PSMGFR above the level of vector control (data not shown). It was observed that the MUC1-PSMGFRTC and MUC1-Y constructs were stained to a higher level compared to other transfectants. Most other transfectants have low GFP, which may indicate low transfection levels.

  We observed the localization of the MUC1 isoform on the surface of the transfected cells using a fluorescence microscope. The procedure for cell staining is as follows. Cells were grown on sterile cover pieces. Cells were washed with 1 × PBS. Cells were fixed with 4% paraformaldehyde in 1 × PBS. Thereafter, the cover piece was washed with 1 × PBS. Anti-PSMGFR antibody was added to the cover strip at 2 μg / ml. The cover pieces were washed again with 1 × PBS. Zymed's anti-rabbit tetramethylrhodamine isothiocyanate (TRITC) labeled secondary antibody was added to the cover strip and then washed with 1 × PBS. The nuclei were stained with 4 ', 6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) from Sigma (St. Louis, MO). The cover piece was placed on the slide using mounting media. The cover piece was sealed with nail polish. The slides were viewed using an Olympus IX70 microscope 60 × objective equipped with an Applied Precision (Issaquah, WA) DeltaVision® microscope system. Pictures were taken using Applied Precision's SoftwareRx® software. The staining pattern seen with the anti-PSMGFR antibody was unique to the proteins localized on the cell surface.

Example 11-Stimulatory / inhibitory effect of cell proliferation by bivalent versus monovalent anti-PSMGFR antibody Reagent:
* Serial dilutions of ANTI-PSMGFR antibodies (1 ×, 1 × 1/10, 1/50, 1/250, 1/1000) produced as described in Examples 8 and 9 in serum-free RPMI medium. )
* 60-70% confluent flask with desired cells * 10% and 0.1% cell specific media * Trypsin * 96 well plate

Method:
1. Place 100 μl of medium in the peripheral wells of a 96 well plate. 6000 cells per well are placed in the inner well (in a 100 μl volume of growth medium containing 10% serum).
2. The next day, the medium is changed to 0.1% serum growth medium and BT474 cells changed to medium containing 2.5% serum are removed and incubated overnight (12-24 hours).
3. The next day, add antibody (1 μl per well) to the desired well, mix gently, and return to the incubator. Antibody is added every 24 hours. For tumor cell lines 1500, 1504, and BT474, the antibody was added 5 times and the cells were counted 24 hours after the addition of the last antibody. For tumor cell line T47D, antibodies were added two or three times. For the K293 cell stable transfectants produced as described above in Example 7, the antibody was added twice. In a competition experiment between a monovalent antibody fragment and a divalent antibody, the monovalent antibody fragment was added for 10-15 minutes prior to the addition of the bivalent antibody.
4). The desired analysis (BrdU, individual cell counting) was performed to count the cells.
5. The percentage growth was calculated using the following formula:
Growth% = 100 {final cell number-starting cell number} / starting cell number Normalized growth was calculated as follows:
Normalized growth% = 100 {final cell number regardless of presence or absence of antibody} / final cell number without antibody

  Human breast adenoma CRL-1500 cells were obtained from the American Type Culture Collection (ATCC). Cells were seeded in T75 vented flasks containing RPMI 1604 containing 10% fetal calf serum, Pen / Sterp, 1 mM sodium pyruvate, 0.5% glucose, and 0.15% sodium bicarbonate. In addition, cells were passaged 10 times before the experiment. When ready, cells are seeded in 96-well plates containing RPMI 1640 containing 10% fetal bovine serum, Pen / Sterp, 1 mM sodium pyruvate, 0.5% glucose and 0.15% sodium bicarbonate (100 μl medium). 6000 cells per well), incubated for 24 hours at 5% CO 2 and 37 ° C. The next day, the growth medium was added to RPMI medium containing 0.1% fetal bovine serum, Pen / Sterp, 1 mM sodium pyruvate, 0.5% glucose and 0.15% sodium bicarbonate overnight at 5% CO2 and 37 ° C. Replaced. To test the effect of MUC1 receptor dimerization on 1500 cell growth, various concentrations of affinity purified bivalent anti-PSMFGR polyclonal antibody, various concentrations of affinity purified monovalent anti-PSMFGR polyclonal Cells were incubated with either antibody fragment or both. Antibody / fragment was added to the cells 5 times every 24 hours. To demonstrate the specificity of the anti-PSMFGR antibody, a control experiment was performed with an irrelevant antibody (anti-streptavidin polyclonal antibody). Cells were harvested 24 hours after the last antibody addition with 50 μl trypsin and the number of cells per well was determined using a hemocytometer (3 wells counted per condition).

  The same protocol described above for CRL-1500 cells was used for CRL-1504 cells. For BT474 (HTB-20) cells, the protocol used was the same as that of CRL-1500 cells with two exceptions: (1) ATCC Hybridare supplemented with 10% fetal bovine serum and penicillin / streptomycin (catalog number) 46-X) the cells were cultured in medium, and (2) during the experiment, the cells were cultured in the presence of medium containing 2.5% or 5.0% serum as specified in the individual experiment. What you did.

  For T47-D (HTB-133) cells, the media used were 10% fetal bovine serum, 1 mM sodium pyruvate, penicillin / streptomycin, 1.25 g glucose / 500 ml medium, 0.2 units / ml bovine insulin and 1. RPMI 1640 medium supplemented with 5 g / l sodium bicarbonate. For antibody stimulation / inhibition experiments, no insulin was added and the experiments were performed in medium containing 0.1% fetal bovine serum.

  HeLa (CCL-2) cells were cultured in MEM Earle's BSS medium (ATCC No. 30-2003) supplemented with penicillin / streptavidin and 10% fetal calf serum. Antibody experiments were performed as described for CRL-1500 cells.

  HEK293 (CRL-1705) cells were cultured in DMEM (BioWhittaker # BW12-640F) medium supplemented with penicillin / streptomycin and 10% fetal calf serum. Antibody experiments were performed as described for CRL-1500 cells.

  HEK293 (CRL-1705) cell transfectants were cultured in DMEM (BioWhittaker number BW12-640F) medium supplemented with penicillin / streptavidin, 10% fetal bovine serum and 600 μg / ml geneticin (GibcoBRL # 11101-011). did. The selective drug geneticin was not added to the medium during the experiment. Antibody stimulation / inhibition experiments were performed in the presence of 0.1% serum-containing medium, and one antibody / multiple antibodies were added twice at 24 hour intervals.

The percentage of cell growth was calculated using the following formula:
Growth% = 100 × {number of final cells−number of starting cells} / number of starting cells Normalized growth percentage was calculated as follows:
Normalized growth% = 100 × {number of final cells regardless of presence / absence of antibody} / number of final cells without antibody

Example 12 Measurement of ERK2 Phosphorylation Status in Breast Tumor Cells as a Determinant of MGFR Dimerization Human breast adenoma CRL-1500 cells were obtained from American Type Culture Collection (ATCC). Cells were seeded in T75 vented flasks containing RPMI 1604 containing 10% fetal bovine serum, Pen / Sterp, 1 mM sodium pyruvate, 0.5% glucose and 0.15% sodium bicarbonate. In addition, cells were passaged 10 times before the experiment. When ready, cells per 60 mm plate in RPMI 1640 containing 10% fetal bovine serum, Pen / Sterp, 1 mM sodium pyruvate, 0.5% glucose and 0.15% sodium bicarbonate and insulin (10 μg / ml) 1 × 10 6 seeds were seeded. The next day, the cells were carefully washed (once) in serum-free RPMI medium without disturbing the 70% confluent cells and incubated overnight in 2 ml serum-free RPMI medium at 5% CO 2 and 37 ° C. The next day, cells were stimulated with 5 μl of affinity purified bivalent anti-PSMGFR polyclonal antibody, either monovalent anti-PSMFGR alone (10 μl), or both together. Untreated cells were used as a control. After activation, the cells were immediately washed twice with ice-cold PBS and buffered (1% Triton X-100, 10% glycerol, 20 mM HEPES, pH 7.2, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 μg). / Ml aprotinin, 10 μg / ml leupeptin and 1 mM Na3VO4). The cell lysate was incubated on a rotating plate for 15 minutes and then centrifuged for 10 minutes (10 g) to remove detergent insoluble material. An equal amount of protein (100 μg per lane) was mixed with SDS-PAGE sample buffer, boiled for 5 minutes, separated on a 10% SDS-polyacrylamide gel, and then transferred to a polyvinylidene difluoride membrane. The membrane was then blocked in phosphate buffered saline (PBS) containing 5% non-fat dry milk for 1 hour at room temperature. Membranes are incubated overnight with anti-ERK2 or anti-ppERK1 / 2 antibodies (Cell Signaling Technology Inc., Beverly, MA, USA) (diluted 1: 1000 in PBS containing 0.5% milk) at 4 ° C. did. The immunoblot was washed once for 20 minutes, twice for 5 minutes with PBS, incubated with a secondary antibody conjugated with horseradish peroxidase (diluted 1: 20000) for 1 hour, once for 20 minutes, 2 for 5 minutes. Washed once with PBS and visualized by chemiluminescence using enhanced chemiluminescent reagent (BioRad).

Example 13 Colloidal analysis to test for competition of mono-bivalent antibodies:
Colloids were prepared as described above with 25 μM NTA thiol. Next, histidine-tagged PSMGFR peptide (eg, His-var-PSMGFR SEQ ID NO: 2) or control RGD peptide was bound to the colloid.
55 μl of phosphate buffer was added to the wells of the microtiter plate, followed by 5 μl of 100 μM BSA (fetal bovine serum albumin).
One antibody / multiple antibodies (minimum 10 μl of undiluted stock, approximately 1 μg / μl) were then added to the wells and the contents mixed by pipetting.
30 μl of either control RGD colloid or His-PSMGFR colloid was added followed by subsequent mixing. The antibody-peptide interaction was allowed to proceed for 3-4 hours.
Wells were scored by measuring absorbance at 650 nm.

  In addition to the diagnosis and screening analysis of the present invention, the present invention involves therapeutic methods and related products for the treatment and prevention of cancer. For example, in one aspect, the present invention relates to a subject suffering from cancer, or the risk of developing cancer, by administering to a subject a substance that reduces IBR cleavage of a cell surface receptor from a cell surface receptor. Relates to a method of treatment of a subject having

  Those skilled in the art will simply mean that all parameters listed herein are typical, and that the actual parameters will depend on the specific application in which the method and apparatus of the present invention is used. Can be predicted. Therefore, it will be understood that the foregoing embodiments are presented by way of example only, and are within the scope of the appended claims and their equivalents, and the invention may be practiced otherwise than as specifically described. In particular, one of ordinary skill in the art can recognize, or ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

  Several methods for administering a compound to a subject for the prevention or treatment of a particular condition are disclosed herein. In each such embodiment of the invention, the invention also includes a compound for use in the treatment or prevention of a particular condition, as well as the use of the compound in the manufacture of a medicament for the treatment or prevention of a particular condition. It will be understood.

  In the claims, all transitional phrases such as “include”, “include”, “have”, “include”, “involve”, etc., mean openly, that is, including but not limited to Then it will be understood. Only the transition phrases “consisting of” and “consisting essentially of” are closed or semi-closed transition phrases, respectively.

1 is a schematic diagram of MUC1 receptor (top) and various truncated MUC1 receptor isoforms produced by the present invention. FIG. The antibodies of the present invention against the epitope of the MUC1 receptor that dimerizes the receptor in close proximity to the cell surface, ie outside the cell, enhance cell proliferation in a typical method of growth factor / receptor-antibody interaction It is a graph of the cell growth rate which shows doing. It is a graph of the cell proliferation rate which shows that the antibody of this invention with respect to the epitope of the MUC1 receptor which dimerizes this receptor close | similar to a cell surface dramatically enhances cell proliferation. FIG. 5 is a silver stained gel showing ligand removed from cell lysates using specific PSMGFR peptides in the presence of protease inhibitor PMSF. FIG. 5 is a silver stained gel showing ligand removed from cell lysate using the PSMGFR peptide of FIG. 4 in the presence of protease inhibitor PMSF. 2 is a graph showing that bivalent anti-PSMGFR antibody stimulates cell growth of MUC1 + breast tumor cell line 1504. 2 is a graph showing that bivalent anti-PSMGFR antibody stimulates cell growth of MUC1 + breast tumor cell line 1500. 4 is another graph showing that bivalent anti-PSMGFR antibody stimulates cell growth of MUC1 + breast tumor cell line 1500. FIG. 2 is a graph showing that bivalent anti-PSMGFR antibody stimulates cell growth of MUC1 + breast tumor cell line T47D. FIG. 2 is a graph showing that bivalent anti-PSMGFR antibody stimulates cell growth of MUC1 + breast tumor cell line BT-474. FIG. 6 is a graph showing that monovalent anti-PSMGFR inhibits cell growth of MUC1 + breast tumor cell line 1504. 1 is a graph showing that monovalent anti-PSMGFR inhibits cell growth of MUC1 + breast tumor cell line 1500. FIG. 5 is a bar graph showing that monovalent anti-PSMGFR competes with divalent anti-PSMGFR and prevents color changes in nanoparticle analysis. It is a Western blot showing that breast tumor cells produce a MUC1 cleavage product with an apparent molecular weight of 20-30 kDa. FIG. 2 is a western blot showing that bivalent anti-PSMGFR dimerizes MUC1 in T47D cells and activates the intracellular MAP kinase cell proliferation pathway. FIG. 2 is a Western blot showing that bivalent anti-PSMGFR activates the intracellular MAP kinase cell proliferation pathway of 1504 breast tumor cells. Bivalent anti-PSMGFR is a Western blot showing activation of the intracellular MAP kinase cell proliferation pathway of 1500 breast tumor cells. 2 is a Western blot showing that drug compounds compete with bivalent anti-PSMGFR and block activation of the intracellular MAP kinase cell proliferation pathway. FIG. 5 is a western blot showing that monovalent anti-PSMGFR competes with divalent anti-PSMGFR and blocks activation of the intracellular MAP kinase cell proliferation pathway. Western blot showing breast tumor cells displaying full length as well as cleaved MUC1. It is a Western blot showing that the MUC1 cleavage product is N-glycosylated. It is a schematic diagram of the MUC1 receptor mutant transfected into HKE cells. Western blot showing MUC1 tumor-specific cleavage product flowing as an approximately 20 kDa band. FIG. 6 is a bar graph showing that monovalent anti-PSMGFR inhibits cell growth of nat-PSMGFRTC transfectants. FIG. 5 is a Western blot showing that bivalent anti-PSMGFR antibody induces ERK2 phosphorylation in HEK cells transfected with nat-PSMGFRTC isoform. It is a Western blot which shows that a bivalent anti-PSMGFR antibody induces ERK2 phosphorylation and a monovalent anti-PSMGFR antibody inhibits ERK2 phosphorylation in a nat-PSMGFRTC transfectant. FIG. 5 is a western blot showing receptor cleavage products of MUC1 + tumor cells and transfectants. FIG. 3 is a wetan blot showing that breast tumor cells produce two MUC1 cleavage products.

Claims (13)

Comprising the following, how the transmembrane peptide presented on the cell surface:
Amino acids according to a cell surface receptor interchain binding region chromatic without and SEQ ID NO: 37 in order to prevent spontaneous binding, substitution of amino acids up to 15 of the amino acid, or N- terminus or C- terminus Transfecting or transforming a host cell in vitro with an expression vector encoding a functional variant or fragment having up to 15 amino acid additions or deletions; and
A method comprising promoting expression of a peptide in the cell, and allowing the cell to display a transmembrane peptide on its surface.
The method of claim 1, wherein the host is a cell line.
The method of claim 1, wherein the host is a primary cell.
Determining whether the molecules administered to the transfected or transformed cells obtained by the method according to any one of claims 1 to 3 are reduced in enhanced cell growth involving MUC1 + cells. including, method.
The method according to claim 4, wherein the MUC1 + cell is a cancer cell or a mimicking cancer cell.
Comprising the following, how the transmembrane peptide presented on the cell surface:
Amino acids set forth in SEQ ID NO: 37, expressing encodes a functional variant or fragment having addition or deletion of amino acids up to 15 substituted or N- terminus or C- terminus of the amino acid of up to 15 of the amino acid Injecting the vector into a non-human animal host, and
A method comprising promoting expression of a peptide in the cell, and allowing the cell to display a transmembrane peptide on its surface.
A method for testing biochemical or physiological effects of diagnostic or therapeutic molecules on cells characterized by abnormal expression of MUC1, comprising a genetically modified non-human animal obtained by the method of claim 6 To administer the diagnostic or therapeutic molecule to the animal and measure the biochemical or physiological effect of the diagnostic or therapeutic molecule on the animal Method.
The method according to any one of claims 1 to 5, wherein the amino acid further comprises a signal sequence.
The method according to any one of claims 1 to 5 and 8, wherein the nucleic acid sequence of the peptide to be expressed is the nucleic acid sequence of SEQ ID NO: 42.
The method according to any one of claims 1 to 5, 8, and 9, wherein the nucleic acid is operably linked to a promoter.
The expression vector does not have an interchain binding region of a cell surface receptor to prevent spontaneous binding, and the amino acid set forth in SEQ ID NO: 37, addition of up to 10 amino acids at the N-terminus of the amino acid, or The method according to any one of claims 1 to 5, and 8 to 10, wherein the method encodes a functional mutant or fragment having a deletion.
8. The method of claim 7, wherein the diagnosis or treatment is a method characterized by abnormal expression of MUC1.
  9. The method of claim 8, wherein the signal sequence is a MUC1 + N-terminal signal sequence.

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