Anti-NME antibody and method for treating cancer or cancer metastasis

Abstract

The present application discloses anti-NME antibodies and their use in treating or preventing diseases. The present application relates to a method for treating or preventing cancer in a subject, comprising administering to the subject an antibody raised against an NME family member. The NME family may be the NME7 family.

Description

Background Art

[0001] 1. Field of the Invention The present invention relates to NME proteins, peptides derived from NME proteins, antibodies generated from these peptides, or antibodies or antibody fragments selected by their ability to bind to the above peptides. The present invention also relates to the treatment or prevention of diseases associated with the expression of NME in patients.

[0002] 2. Background Art and Current Technology NDPK (nucleoside diphosphate protein kinase) proteins are a family of proteins grouped together because they all contain an NDPK domain. The first discovered NME proteins, previously called NM23 proteins, were NM23-H1 and NM23-H2. For decades, it was not clear whether they induced or inhibited the differentiation of hematopoietic cells. The present inventors previously found that NM23-H1, when it is a dimer, binds to the MUC1 * growth factor receptor and inhibits differentiation, but at higher concentrations of NM23-H1, it forms hexamers, which do not bind to MUC1 * and induce differentiation. NM23 was called a metastasis suppressor when it was found to be underexpressed in certain very aggressive cancers. The present inventor previously showed that the NM23-H1 dimer binds to the extracellular domain of the MUC1 * growth factor receptor, which is overexpressed in most cancers, dimerizes it, and such binding promotes the growth of cancer cells. Conversely, at higher concentrations, NM23 forms tetramers and hexamers that do not bind to MUC1 * and do not promote tumor formation. To date, their functions have not been elucidated, but more recently, more NME family proteins (NME1-10) have been discovered. NME7 is a newly discovered NME family protein, but its NDPK domain, unlike other NME family members, does not have enzymatic activity. NME7 is not expressed at all or is expressed at very low levels in adult tissues.

SUMMARY OF THE INVENTION

[0003] The present invention relates to a method of treating or preventing cancer in a patient, comprising administering an antibody generated against an NME family member. The NME family can be the NME7 family. The antibody can bind to NME7. The antibody can bind to NME7 AB or an NME7 AB -like protein. The antibody can bind to NME7-X1. The antibody can inhibit the binding between NME7 and its cognate binding partner. The cognate binding partner can be MUC1 * or can be the PSMGFR portion of the extracellular domain of MUC1 * In one aspect, the antibody can be generated or selected by its ability to bind to a peptide selected from those listed in FIGS. 6-9 (SEQ ID NOs: 88-145). Preferably, the peptide can be selected from those listed in FIG. 9 (SEQ ID NOs: 141-145).

[0004] The peptide can be highly homologous to or have, at the N-terminus or C-terminus, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid residues added or deleted relative to the peptides listed in FIGS. 6-9 (SEQ ID NOs: 88-145). In one aspect, the antibody can be selected for its ability to bind to NME7 AB or NME7-X1 rather than to NME1. The antibody can be polyclonal, monoclonal, bivalent, monovalent, bispecific, an antibody fragment containing a variable domain, or an antibody mimetic. The antibody can be a human antibody or a humanized antibody. The antibody can be a single-chain scFv

[0005] In another aspect, the present invention relates to NME7 ABA method for treating or preventing cancer in a subject, comprising administering to the subject a peptide that is highly homologous or identical to a peptide in the region. The peptide can be at least 80% homologous to one or more of the peptides listed in FIG. 6. The peptide can be at least 80% homologous to one or more of the peptides listed in FIG. 7. The peptide can be at least 80% homologous to one or more of the peptides listed in FIG. 8. The peptide can be at least 80% homologous to one or more of the peptides listed in FIG. 9. The peptide can be selected from the peptides listed in FIGS. 6-9 (SEQ ID NOs: 88-145). The peptide can be selected from those listed in FIG. 9 (SEQ ID NOs: 141-145). Alternatively, the peptide is highly homologous to the peptides listed in FIGS. 6-9 (SEQ ID NOs: 88-145), or at the N-terminus or C-terminus thereof, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid residues can be added or deleted. The peptide can be connected to another peptide via a spacer or linker.

[0006] In another aspect, the present invention relates to a chimeric antigen receptor (CAR) for the treatment or prevention of cancer, wherein the target extracellular portion of the CAR comprises at least a peptide fragment of a member of the NME family. The NME family can be the NME7 family. A member of the NME7 family can be NME7. Alternatively, a member of the NME7 family is NME7 AB or NME7 ABIt may be a protein of the NME7 family. Members of the NME7 family may also be NME7-X1. The extracellular target portion of the CAR may include one peptide of a plurality of peptides listed in FIGS. 6-9 (SEQ ID NOs: 88-145). The peptide may be selected from those listed in FIG. 9 (SEQ ID NOs: 141-145). The peptide may include a peptide that is highly homologous to the peptides listed in FIGS. 6-9 (SEQ ID NOs: 88-145), or to which up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid residue is added or deleted at the N-terminus or C-terminus. The peptide may be connected to another peptide via a spacer or linker.

[0007] In yet another aspect, the invention relates to a method of treating or preventing cancer or cancer metastasis, comprising engineering a chimeric antigen receptor as recited in claim 3 into immune system cells and administering the cells to a subject in need thereof.

[0008] In another aspect, the invention relates to a chimeric antigen receptor (CAR) for the treatment or prevention of cancer, wherein the extracellular target portion of the chimeric antigen receptor comprises a part of an antibody that binds to an NME7 AB , NME7 AB -like protein or NME7-X1. The part of the antibody may be a single-chain scFv or a part of a human or humanized antibody.

[0009] In yet another aspect, the invention relates to a method of vaccinating a human against cancer or metastatic cancer, comprising immunizing the human with a peptide fragment of a member of the NME family. The NME family may be the NME7 family. Members of the NME7 family may be NME7 or NME7b. Members of the NME7 family may be NME7 AB or NME7 ABIt can be a protein of interest. The NME7 family can be NME7-X1. The immunizing peptide can be one peptide from the plurality of peptides listed in FIGS. 6-9 (SEQ ID NOs: 88-145). Preferably, the peptide can be selected from those listed in FIG. 9 (SEQ ID NOs: 141-145). The immunizing peptide can be highly homologous to the peptides listed in FIGS. 6-9 (SEQ ID NOs: 88-145), or can include a peptide with up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid residue added or deleted at the N-terminus or C-terminus thereof. The immunizing peptide can be connected to another peptide by a spacer or linker.

[0010] In yet another aspect, the present invention relates to a method for treating or preventing cancer in a subject, comprising administering to the subject a nucleic acid that suppresses the expression of NME7, NME7b, NME7 AB -like protein or NME7-X1. The nucleic acid can be an antisense nucleic acid that suppresses the expression of NME7, NME7 AB -like protein or NME7-X1. The nucleic acid can be a suppressive RNA, siRNA, RNAi, or shRNA that suppresses the expression of NME7, NME7 AB -like protein or NME7-X1.

[0011] In another aspect, the present invention relates to a method for treating or preventing cancer in a subject, comprising administering to the subject a nucleic acid that has been gene-edited to suppress the expression of NME7, NME7b, NME7 AB -like protein or NME7-X1. The nucleic acid that suppresses the expression of NME7, NME7b, NME7 AB -like protein, or NME7-X1 can be inserted into cells that can then be administered to a patient. The nucleic acid that suppresses the expression of NME7, NME7b, NME7 AB -like protein, or NME7-X1 can be inserted into cells using a viral vector. The viral vector can be a lentiviral system.

[0012] In another aspect, the present invention relates to a method of growing cancer cells comprising contacting the cells with NME7 AB , NME7b, NME7 AB -like proteins, or NME7-X1, 2i or 5i. The method comprises culturing the cells in a medium comprising NME7 AB , NME7b, NME7 AB -like proteins or NME7-X1, 2i or 5i, or growing the cells in an animal in which human NME7 AB , NME7b, NME7 AB -like proteins, or NME7-X1 is expressed, or in which NME7 AB , NME7b, NME7 AB -like proteins or NME7-X1 is administered. The cancer cells can be breast, prostate, ovarian, colorectal, pancreatic, liver, melanoma or brain cancer cells. Drug candidates can be tested in the cells. The effectiveness of the drug is evaluated by comparing cancer growth to a drug-free control, or by comparing the expression levels of metastasis markers or stem cell markers to a drug-free control, or by comparing the ability of the cells obtained from low cell copy numbers to form tumors in animals compared to a drug-free control, and determining the effectiveness of candidate drugs for the treatment of cancer or metastasis. The cells are obtained from a patient being evaluated for the treatment of cancer, and drugs that are likely to be effective for that patient are selected based on the results using the methods described above. The cells are not obtained from a patient being evaluated for the treatment of cancer, but drugs that are likely to be effective for that patient are selected based on the results using the methods described above.

[0013] In another aspect, the present invention relates to a method for generating an antibody or antibody-like molecule from a peptide or peptidomimetic having a sequence derived from the sequence of NME. The NME can be NME7. The peptide can be used as an immunogen to generate an antibody or antibody-like molecule. The peptide can be administered to an animal to generate an anti-NME7 antibody. The peptide can be administered to a human to generate an anti-NME7 antibody. The peptide can have the sequences listed in FIGS. 6-9 (SEQ ID NOs: 88-145). Preferably, the peptide can be selected from those listed in FIG. 9 (SEQ ID NOs: 141-145). The peptide can include a peptide that is highly homologous to the peptides listed in FIGS. 6-9 (SEQ ID NOs: 88-145), or to which up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid residue is added or deleted at the N-terminus or C-terminus.

[0014] In another aspect, the present invention relates to a method for detecting the presence or progression of cancer, comprising the following steps: 1) obtaining a sample from a patient having cancer or at risk of developing cancer; 2) subjecting the sample to an assay capable of detecting or measuring the level of a member of the NME7 family or the level of a nucleic acid encoding a member of the NME7 family; 3) comparing the measured level of a member of the NME7 family in the test sample, or the level of a nucleic acid encoding a member of the NME7 family, with the level in a control patient or control cell; 4) determining that the level of a member of the NME7 family, or the level of a nucleic acid encoding a member of the NME7 family, is elevated compared to the control; and 5) A step of concluding that cancer is progressing when the donor of the test sample is a donor who has cancer or a donor previously diagnosed with cancer against which the test substance is being compared. In this method, the detection of members of the NME7 family in the circulatory system or in tissues can be an indicator of cancer in a patient. Members of the NME7 family can be NME7, NME7b, NME7-X1, or NME7 AB like proteins.

[0015] In yet another aspect, the present invention relates to a method comprising the steps of detecting the presence of a member of the NME7 family or MUC1 * in a patient; and administering an anti-NME7 or anti-MUC1 * antibody or antibodies to a patient who exhibits expression of a member of the NME7 family or MUC1 * Members of the NME7 family can be NME7, NME7b, NME7-X1, or NME7 AB like proteins.

[0016] In yet another aspect, the present invention relates to a method for treating or preventing cancer, comprising: 1) Obtaining a sample from a patient who has cancer, is at risk of developing cancer, or is at risk of developing metastatic cancer; 2) Measuring the amount of a member of the NME7 family or a nucleic acid encoding a member of the NME7 family, wherein the measured level is significantly higher than that measured in a control sample; 3) Determining that the patient has or has progressed to a higher grade or metastatic cancer; 4) Administering to the patient an effective amount of a therapeutic agent that suppresses the expression of a member of the NME7 family, suppresses the cleavage of NME7, or suppresses the binding of NME7 to its target. The target of a member of the NME7 family can be MUC1 * The target of a member of the NME7 family can be MUC1 *It may be the PSMGFR portion of the extracellular domain. Members of the NME7 family may be NME7, NME7b, NME7-X1, or NME7-like proteins. AB It may be a protein.

[0017] In any of the above methods related to cancer, the cancer may include cancers of the breast, prostate, ovary, colorectal, pancreas, liver, melanoma, or brain tumor.

[0018] In one aspect, the present invention relates to an NME7-specific antibody or a fragment thereof that binds to the NME7 B3 peptide of SEQ ID NO: 145 or SEQ ID NO: 169. The antibody can be a monoclonal antibody or a bivalent, monovalent, Fab, or single-chain variable fragment. The antibody can be conjugated to an antibody-drug conjugate. The drug can be conjugated to a toxin or a protoxin.

[0019] The present invention also relates to an isolated nucleic acid encoding the antibody.

[0020] The present invention also relates to an isolated hybridoma that expresses the above monoclonal antibody. The antibody can specifically bind to NME7 or NME7-X1, but does not specifically bind to NME1. The antibody can disrupt the interaction between NME7 and the extracellular domain of MUC1, or the interaction between NME7-X1 and the extracellular domain of MUC1. Alternatively, the antibody can disrupt the binding between NME7 and PSMGFR, or the binding between NME7-X1 and PSMGFR. Furthermore, the antibody can disrupt the binding between NME7 and N-10, or the binding between NME7-X1 and N-10. AB It may not specifically bind to NME1, but can specifically bind to NME7 or NME7-X1. The antibody can disrupt the interaction between NME7 and the extracellular domain of MUC1, or the interaction between NME7-X1 and the extracellular domain of MUC1. Alternatively, the antibody can disrupt the binding between NME7 and PSMGFR, or the binding between NME7-X1 and PSMGFR. Furthermore, the antibody can disrupt the binding between NME7 and N-10, or the binding between NME7-X1 and N-10. AB and MUC1 * between the extracellular domains, or the interaction between NME7-X1 and MUC1 * between the extracellular domains. Alternatively, the antibody can disrupt the binding between NME7 AB and PSMGFR, or the binding between NME7-X1 and PSMGFR. Furthermore, the antibody can disrupt the binding between NME7 AB and N-10, or the binding between NME7-X1 and N-10.

[0021] In another aspect, the antibody may not disrupt the interaction between NME7 AB and MUC1 * between the extracellular domains, or the interaction between NME7-X1 and MUC1 * between the extracellular domains. NME7 ABAlternatively, NME7-X1 binds to the N-10 peptide (SEQ ID NO: 170), but does not bind to the C-10 peptide (SEQ ID NO: 171). In particular, the antibody can be 5A1, 4A3 or 5D4.

[0022] The antibody comprises an amino acid sequence in the heavy chain variable region comprising: YTFTNYGMN (SEQ ID NO: 439) in the CDR1 region; WINTYTGEPTYVDDFKG (SEQ ID NO: 440) in the CDR2 region; LRGIRPGPLAY (SEQ ID NO: 441) in the CDR3 region; and an amino acid sequence in the light chain variable region comprising: SASSSVSYMN (SEQ ID NO: 444) in the CDR1 region; GISNLAS (SEQ ID NO: 445) in the CDR2 region; QQRSSYPPT (SEQ ID NO: 446) in the CDR3 region and may include.

[0023] In another aspect, the antibody comprises an amino acid sequence in the heavy chain variable region comprising: NTFTEYTMH (SEQ ID NO: 429) in the CDR1 region; GFNPNNGVTNYNQKFKG (SEQ ID NO: 430) in the CDR2 region; RYYHSTYVFYFDS (SEQ ID NO: 431) in the CDR3 region; and an amino acid sequence in the light chain variable region comprising: SASQGISNYLN (SEQ ID NO: 434) in the CDR1 region; YTSSLHS (SEQ ID NO: 435) in the CDR2 region; QQYSKLPYT (SEQ ID NO: 436) in the CDR3 region and may include.

[0024] In another aspect, the antibody comprises an amino acid sequence in the heavy chain variable region comprising: NTFTEYTMH (SEQ ID NO: 388) in the CDR1 region; GFNPNNGVTNYNQKFKG (SEQ ID NO: 389) in the CDR2 region; RYYHSLYVFYFDY (SEQ ID NO: 390) in the CDR3 region; and an amino acid sequence in the light chain variable region comprising: ITSTDIDDDMN (SEQ ID NO: 393) in the CDR1 region; EGNTLRP (SEQ ID NO: 394) in the CDR2 region; LQSDNLPLT (SEQ ID NO: 395) in the CDR3 region may be included.

[0025] The antibody may be a human antibody, a humanized antibody, or an engineered antibody mimetic. The antibody may be a non-human antibody, e.g., a mouse or camelid antibody.

[0026] The present invention also relates to a method of administering to a patient for the prevention or treatment of cancer, the method comprising administering to the patient a composition comprising the above antibody.

[0027] The present invention also relates to a method for preventing or treating cancer metastasis in a patient, the method comprising administering to the patient a composition comprising the above antibody.

[0028] The present invention also relates to a method for diagnosing cancer or cancer metastasis, the method comprising contacting a patient sample and a normal sample with the above antibody, and comparing the results obtained from both samples, the presence of positive binding to the antibody in the patient sample indicating the presence of cancer or cancer metastasis in the patient. The antibody may be conjugated to an imaging agent. The patient sample may be in vitro, in vivo including during surgery, blood, body fluid, tissue, circulating cells.

[0029] The present invention also relates to AB cells engineered to express an anti-NME7 antibody or a fragment thereof. The cells may be immune cells such as T cells or NK cells, or stem cells or progenitor cells, preferably stem cells or progenitor cells that subsequently differentiate into T cells.

[0030] The cell may contain a chimeric antigen receptor (CAR) that recognizes a tumor-associated antigen. The expression of the anti-NME7 antibody may be inducible. The anti-NME7 AB nucleic acid encoding the antibody may be inserted into the Foxp3 enhancer or promoter. The anti-NME7 AB antibody may be present in the NFAT induction system. The NFATc1 response element may be inserted upstream of the antibody sequence inserted into the enhancer or promoter region.

[0031] The anti-NME7 AB antibody or its fragment may bind to the NME7 B3 peptide, or may disrupt the binding of NME7 AB or NME7-X1 to PSMGFR in the extracellular domain of MUC1 * The CAR may recognize a tumor-associated antigen and the anti-NME7 antibody. The tumor-associated antigen may be MUC1

[0032] The tumor-associated antigen may be MUC1. * It may be.

[0033] The present invention also relates to an anti-cancer vaccine comprising, as an immunogenicity-inducing portion, one or more peptides derived from NME7 listed in FIGS. 6 to 9, or peptides having at least 80%, 85%, 90%, 95%, 97% sequence identity thereto. The peptide may be the peptide of SEQ ID NOs: 141 to 145, or a peptide having at least 80%, 85%, 90%, 95%, 97% sequence identity thereto. The peptide may be the peptide of SEQ ID NO: 145, or a peptide having 80%, 85%, 90%, 95%, 97% sequence identity thereto. AB In another aspect, the present invention relates to a BiTE comprising the above antibody.

[0034] In yet another aspect, the present invention relates to a method for generating an anti-NME7

[0035] antibody, wherein the cysteine residue of the NME7 B3 peptide is mutated to avoid disulfide bonding. AB The cysteine residue of the NME7 B3 peptide is mutated to avoid disulfide bonding.

[0036] In yet another aspect, the present invention relates to a method of generating cells having enhanced metastatic ability, which comprises culturing cells with NME7 AB or NME7-X1.

[0037] The present invention also relates to cells engineered to express NME7 AB or NME7-X1, and transgenic animals expressing NME7 AB or NME7-X1, wherein NME7 AB or NME7-X1 can be human, and the expression of NME7 AB or NME7-X1 can be inducible.

[0038] This patent or application file contains at least one color drawing. Copies of this patent or patent application publication, which include a color drawing, will be provided by the Patent Office upon request and payment of the necessary fee. The present invention may be more fully understood from the detailed description given below and from the accompanying drawings which are given by way of illustration only and are not intended to limit the present invention.

Brief Description of the Drawings

[0039]

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DETAILED DESCRIPTION OF THE INVENTION

[0040] Definitions In this application, "a" and "an" are used to refer to both single and plural objects.

[0041] As used herein, "about" or "substantially" generally allows for some leeway from being limited to an exact number. For example, when used in the context of the length of a polypeptide sequence, "about" or "substantially" indicates that the polypeptide is not limited to the described number of amino acids. A small number of amino acids added or removed from the N-terminus or C-terminus may be included as long as functional activities such as its binding activity are present.

[0042] As used herein, "combined" administration with one or more additional therapeutic agents includes simultaneous (synergistic) administration and sequential administration in any order.

[0043] As used herein, "amino acid" and "amino acids" all refer to L-α-amino acids of natural origin. This definition is intended to include norleucine, ornithine, and homocysteine.

[0044] As used herein, generally, the term "amino acid sequence variant" refers to a molecule that has some differences in its amino acid sequence as compared to a reference (e.g., native sequence) polypeptide. The amino acid changes can be substitutions, insertions, deletions, or any desired combination of such changes in the native amino acid sequence.

[0045] A substitution variant is one in which at least one amino acid residue in the native sequence is removed and a different amino acid is inserted in its place at the same position. The substitution may be a single substitution in which only one amino acid in the molecule is replaced, or it may be a multiple substitution in which two or more amino acids in the same molecule are replaced.

[0046] Substitutions of amino acids within a sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present invention are proteins or fragments or derivatives thereof that exhibit the same or similar biological activity, and derivatives that are differently modified during or after translation, for example, by glycosylation, proteolytic cleavage, binding to antibody molecules or other cellular ligands.

[0047] An insertion variant is one that has one or more amino acids inserted immediately adjacent to an amino acid at a specific position in the native amino acid sequence. Immediately adjacent to an amino acid means connected to either the α-carboxy or α-amino functional group of that amino acid.

[0048] A deletion variant is one in which one or more amino acids in the natural amino acid sequence have been removed. Usually, deletion variants have one or two amino acid deletions in a specific region of the molecule.

[0049] As used herein, "fragment" or "functional derivative" refers to biologically active amino acid sequence variants and fragments of the polypeptides of the invention, as well as derivatives obtained by reaction with organic derivatizing agents, post-translational modifications, derivatives containing non-protein polymers, and covalent modifications including immunoadhesins.

[0050] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer, which are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations used. In many cases, the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum, albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG) and PLURONICS®.

[0051] As used herein, "pharmaceutically acceptable carrier and / or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is known in the art. These uses in therapeutic compositions are intended, except where any conventional media or agent is incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions.

[0052] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, a dosage unit form refers to physically discrete units suitable as unit dosages for the subject to be treated, each unit containing a predetermined quantity of the active substance calculated to produce the desired therapeutic effect incorporated in the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are defined by and directly depend on (a) the particular characteristics of the active substance and the specific therapeutic effect to be achieved, and (b) the unique limitations in the art of pharmacy, such as those for the treatment of diseases in a living body with a pathological condition where the health of the body is impaired, for the active substances for the treatment of such diseases.

[0053] The principal active ingredient, in conjunction with a suitable pharmaceutically acceptable carrier in dosage unit form, is formulated in an effective amount for convenient and effective administration. The dosage unit forms include, for example, an amount of the principal active ingredient in the range of from 0.5 μg to about 2000 mg. Expressed as a proportion, the active compound is usually present in the carrier at about 0.5 μg / ml or more. In the case of compositions containing supplementary active ingredients, the dosage is determined by reference to the usual dosage and method of administration of the ingredients.

[0054] As used herein, "vector", "polynucleotide vector", "construct", and "polynucleotide construct" are used interchangeably. The polynucleotide vectors of the invention may be in any of several forms, including but not limited to RNA, DNA, RNA encapsulated in a retroviral coat, DNA encapsulated in an adenoviral coat, DNA encapsulated in another virus or virus-like form (such as herpes simplex and adenoid structures such as polyamides).

[0055] As used herein, "host cell" includes individual cells or cell cultures that can be or have been recipients of the vectors of the invention. A host cell includes the progeny of a single host cell, and the progeny need not be identical, either morphologically or in total DNA complement, to the original parent cell, due to natural, accidental, or deliberate mutations and / or changes.

[0056] As used herein, "subject" is a vertebrate, preferably a mammal, more preferably a human.

[0057] As used herein, "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic animals, and zoo, sports, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, etc. Preferably, the mammal is a human.

[0058] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction of the degree of a disease, a stable (i.e., non-worsening) state of a disease, delay or slowing of disease progression, improvement of a disease state, or alleviation and remission (partial or total), as compared to survival that would be predicted in the absence of treatment. "Treatment" can also mean prolonging survival as compared to survival that would be predicted in the absence of treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventive treatment. Humans in need of treatment include humans already suffering from a disorder as well as humans in whom a disorder is to be prevented. "Alleviating" a disease means that the degree of the disease state and / or the undesirable clinical symptoms are reduced and / or the progression over time is slowed or extended as compared to a situation without treatment.

[0059] As used herein, the "A1" peptide, "A2" peptide, "B1" peptide, "B2" peptide and "B3" peptide refer to peptides that bind to human NME7 AB but do not (or only very weakly) bind to human NME1. The peptides used to generate these antibodies are common to both NME7 AB and NME7-X1 and are defined as follows. A1 is NME7A peptide 1 (A domain): MLSRKEALDFHVDHQS (SEQ ID NO: 141) A2 is NME7A peptide 2 (A domain): SGVARTDASES (SEQ ID NO: 142) B1 is NME7B peptide 1 (B domain): DAGFEISAMQMFNMDRVNVE (SEQ ID NO: 143) B2 is NME7B peptide 2 (B domain): EVYKGVVTEYHDMVTE (SEQ ID NO: 144) B3 is NME7B peptide 3 (B domain): AIFGKTKIQNAVHCTDLPEDGLLEVQYFF (SEQ ID NO: 145) Further, for clarity, NME7A (with a capital “A”) refers to the subunit A portion of NME7. NME7a (with a lower case “a”) refers to the full-length NME7 as otherwise described herein. And, NME7B (with a capital “B”) refers to the subunit B portion of NME7. NME7b (with a lower case “b”) refers to an NME7 species that is partially lacking in the DM10 region, which is otherwise described herein.

[0060] As used herein, the term “antibody-like” means a molecule that contains a portion of an antibody but has been engineered to contain a portion of an antibody that does not occur naturally in nature. Examples include, but are not limited to, CAR (chimeric antigen receptor) T cell technology and Ylanthia® technology. CAR technology uses an antibody epitope fused to a portion of a T cell so that the body's immune system can be directed to attack a specific target protein, or cell. Ylanthia® technology consists of a collection of synthetic human fabs, an “antibody-like” library, that are screened to bind to peptide epitopes derived from a target protein. The selected Fab regions can then be engineered into a scaffold or framework so that they resemble an antibody.

[0061] As used herein, “effective amount of an agent for suppressing an NME family protein” refers to the effective amount of an agent in preventing the activation interaction between an NME family protein and its cognate receptor.

[0062] As used herein, “NME-derived fragment” refers to a fragment of NME or a peptide sequence that is highly homologous to a peptide sequence that is a fragment of NME.

[0063] As used herein, “MUC1” *」The extracellular domain is mainly defined by the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6)). The exact site of MUC1 cleavage depends on the enzyme that cleaves it, and since the cleavage enzyme appears to vary depending on cell type, tissue type, or time in the evolution of the cell, MUC1 * The exact sequence of the extracellular domain may vary at the N-terminus.

[0064] As used herein, the term "PSMGFR" is an acronym for the primary sequence of the MUC1 growth factor receptor, defined as follows; GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6). In this regard, "N-10 PSMGFR", or simply "N-10", "N-15 PSMGFR" or simply "N-15", or "N-20 PSMGFR" or simply "N-20", etc., "N-number" refers to the number of amino acid residues deleted at the N-terminus of PSMGFR. Similarly, "C-10 PSMGFR" or simply "C-10", "C-15 PSMGFR" or simply "C-15", or "C-20 PSMGFR" or simply "C-20", etc., "C-number" refers to the number of amino acid residues deleted at the C-terminus of PSMGFR. Mixtures of deletions and additions are also possible. For example, N+20 / C-27 refers to a peptide fragment of wild-type MUC1 * in which 20 amino acids are added at the N-terminus of PSMGFR and 27 amino acids are deleted from the C-terminus.

[0065] As used herein, "MUC1 * extracellular domain" refers to the extracellular portion of the MUC1 protein lacking the tandem repeat domain. In most cases, MUC1 * is a cleavage product, and that MUC1 * portion consists of a short extracellular domain lacking the tandem repeat, transmembrane domain, and cytoplasmic tail. The exact location of MUC1 cleavage is not known, probably because it appears to be cleavable by more than one enzyme. MUC1* The extracellular domain of contains most of the PSMGFR sequence, but may have an additional 10-20 N-terminal amino acids.

[0066] As used herein, "high homology" is considered to be at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity in a specified overlapping region between any two polypeptides.

[0067] As used herein, the "NME family proteins" numbered 1-10, or "NME family member proteins", are proteins grouped together because they all have at least one NDPK (nucleoside diphosphate kinase) domain. In some cases, the NDPK domain is not functional in terms of being able to catalyze the conversion of ATP to ADP. NME proteins were previously known as NM23 proteins numbered H1 and H2. Recently, ten NME families have been identified. Herein, the terms NM23 and NME are interchangeable. Herein, the terms NME1, NME2, NME5, NME6, NME7, NME8, and NME9 are used in the sense of native proteins as well as NME variants. In some cases, these variants are more soluble, express better in E. coli, or are more soluble than the native sequence protein. For example, NME7 as used herein has excellent commercial applicability because the variation allows for high-yield expression of a soluble, properly folded protein in E. coli. AB such as native proteins, or variants, meaning NME7 ABIt mainly consists of the NME7 A and B domains, but lacks most of the DM10 domain (SEQ ID NO: 39) at the N-terminus of the native protein. When referred to herein, "NME1" is interchangeable with "NM23-H1". It is also intended that the present invention is not limited by the exact sequence of the NME protein. The mutant NME1-S120G, also called NM23-S120G, is used throughout this application in the same sense. The S120G mutant and the P96S mutant are preferred due to their orientation towards dimer formation, and herein are referred to as NM23 dimers, NME1 dimers or dimeric NME1 or dimeric NM23.

[0068] When NME7 is referred to herein, it is intended to mean native NME7 having a molecular weight of about 42 kDa.

[0069] The "family of NME7" refers to full-length NME7, as well as truncated forms having a molecular weight of about 30 kDa, 33 kDa, or a truncated form having a molecular weight of about 25 kDa, of natural origin or artificially made; NME7b, NME7-X1, NME7 AB , or deletion or partial deletion variants of the DM10 leader sequence (SEQ ID NO: 162) of NME7 amino acids 1-91 of NME7 represented by SEQ ID NO: 82 or 147, such as recombinant NME7 protein; or variants whose sequences can be modified to allow efficient expression or variants that enhance yield, solubility, or other properties that make NME7 more effective or more commercially promising. The "family of NME7" may also include "NME7 AB -like" proteins, which are proteins in the range of 30-33 kDa expressed in cancer cells.

[0070] As used herein, the term "agent that maintains stem cells in a naive state or returns primed stem cells to a naive state" refers to a protein, small molecule, or nucleic acid that maintains stem cells, alone or in combination, in a naive state that resembles the cells of the inner cell mass of an embryo. Examples include, but are not limited to, human NME1 dimer, bacterial, fungal, yeast, viral or parasitic NME proteins that have high sequence identity to human NME proteins, particularly NME1, NME7, NME7-X1, NME7 AB , nucleic acids such as siRNAs that suppress the expression of 2i (Silva J et al, 2008; Hanna et al, 2010), 5i (Theunissen TW et al, 2014); MBD3, CHD4 (Rais Y1 et al, 2013), BRD4 or JMJD6 (Liu W et al, 2013).

[0071] As used herein, the term "NME7 AB ", "NME7AB" and "MNE-AB" are used interchangeably.

[0072] As used herein, the term "agent that promotes pluripotency" or "agent that returns somatic cells to a stem-like or cancer-like state" refers to a protein, small molecule, or nucleic acid that, alone or in combination, induces or suppresses the expression of specific genes such that the gene signature changes to one that is closely similar to that of stem cells or cancer cells. Examples include, but are not limited to, NME1 dimer, NME7, NME7-X1, NME7 AB , nucleic acids such as siRNAs that suppress the expression of 2i, 5i; MBD3 or CHD4 or BRD4 or JMJD6; microbial NME proteins that have high sequence identity to human NME1, NME2, NME5, NME6, NME7, NME8 or NME9, preferably having high sequence identity to the region containing the NDPK domain.

[0073] As used herein, with respect to an agent referred to as a "small molecule", it can be a synthetic chemical molecule or a chemistry-based molecule having a molecular weight between 50 Da and 2000 Da, more preferably between 150 Da and 1000 Da, and even more preferably between 200 Da and 750 Da.

[0074] As used herein, with respect to an agent referred to as a "natural product", as long as the molecule exists in nature, it can be a chemical molecule or a biological molecule.

[0075] As used herein, FGF, FGF-2 or bFGF refers to fibroblast growth factor (Xu RH et al, 2005; Xu C et al, 2005).

[0076] As used herein, a "Rho-associated kinase inhibitor" can be a small molecule, a peptide or a protein (Rath N, et al, 2012). Rho kinase inhibitors are abbreviated as ROCi or ROCKi, or Ri, here and elsewhere. The use of specific Rho kinase inhibitors is intended to be exemplary and can be replaced with any other Rho kinase inhibitor.

[0077] As used herein, the term "cancer stem cell" or "tumor-initiating cell" refers to a cancer cell that expresses the level of genes associated with a more metastatic or more invasive cancer. "Cancer stem cell" or "tumor-initiating cell" also refers to a cancer cell that can give rise to a tumor with a very small number of cells when transplanted into an animal. Cancer stem cells and tumor-initiating cells are often resistant to chemotherapeutic drugs.

[0078] As used herein, the terms “stem / cancer,” “cancer-like,” and “stem-like” refer to a state in which a cell has acquired the characteristics of a stem cell or cancer cell or shares important elements of the gene expression characteristics of a stem cell, cancer cell, or cancer stem cell. A stem-like cell can be a somatic cell that is undergoing induction to a less mature state, such as an increasing expression of pluripotency genes. A stem-like cell also refers to a cell that has undergone some degree of dedifferentiation or is in a metastable state where they can change to terminal differentiation. A cancer-like cell is a cancer cell that has not yet been fully characterized but exhibits the morphology and characteristics of a cancer cell and can grow anchorage-independently or cause tumors in animals.

[0079] As used herein, “spacers,” or “linkers” of different lengths can be incorporated anywhere in the peptide. Spacer linkages are usually through amide bonds, but other functional groups are possible.

[0080] The NME, NME7, and NME7 protein families The present inventors have found that NME7 and NME7-X1 are highly expressed in early human stem cells and furthermore in most cancer cells (FIGS. 17, 18, 19A-19F, 22, 23, 39, 40, 41, 47, 48). FIG. 17 shows a graph of RT-PCR measurement of the expression of NME7-X1 in a series of human stem cells and cancer cells. FIG. 18 shows a graph of RT-PCR measurement of the expression of NME7, NME7a, NME7b and NME7-X1 in a series of human stem cells and cancer cells. NME7a is full-length NME7, NME7b lacks only a part of the DM10 domain, and NME7-X1 lacks all of the DM10 domain and only a part of the N-terminus of the first NDPK A domain. The bar labeled NME7 means that primers detecting both NME7a and NME7b were used. FIGS. 19A-19F show Western blot photographs in which the expression of NME7 species in various cancer cell lines is detected using antibodies generated by immunization with NME7-derived short peptides. FIG. 19A shows a Western blot detected using the antibody #52 of the present invention that binds to the NME7-derived peptide A1. FIG. 19B shows a Western blot detected using the antibody #56 of the present invention that binds to the NME7-derived peptide B1. FIG. 19C shows a Western blot detected using the antibody #61 of the present invention that binds to the NME7-derived peptide B3. FIGS. 22A-22E show Western blot photographs of co-immunoprecipitation experiments. T47D breast cancer cell extracts were incubated with an antibody against the cytoplasmic side terminal of MUC1, Ab-5, or a control antibody, IgG, and co-immunoprecipitated. The gels were blotted with two different commercially available anti-NME7 antibodies B9 (FIG. 22A) and CF7 (FIG. 22B). Both gels show specific NME7 bands at about 33 kDa and about 30 kDa. The gels were stripped and re-detected with an antibody against the extracellular domain of MUC1, anti-PSMGFR ((FIG. 22C) and (FIG. 22D)). This indicates that NME7 species and MUC1 * interact. Recombinant NME7 * AB ​and recombinant NME7-X1 were mixed together and run on a gel, then detected with an anti-NME7 antibody. * Two unique NME7 species that interact with NME7 AB Figures 23A-23C show photographs of Western blots of co-immunoprecipitation experiments. Human induced pluripotent stem cell, iPS7 cell, or embryonic stem cell, HES3 cell extracts were incubated with an antibody against the cytoplasmic tail of MUC1, Ab-5, or a control antibody, IgG, and co-immunoprecipitated. Gels were blotted with the commercially available anti-NME7 antibody B9 (Figure 23A). Both cell types show distinctive NME7 bands at approximately 33 kDa and approximately 30 kDa. Gels were stripped and MUC1 was stained for NME7 bands at approximately 33 kDa and approximately 30 kDa. * This was redetected with anti-PSMGFR (Figure 23B), an antibody against the extracellular domain of NME7 and MUC1. * Recombinant NME7-AB and recombinant NME7-X1 were mixed together, run on a gel, and then detected with an anti-NME7 antibody. * Figures 39A-39C show human lung tissue specimens stained with anti-NME7 antibodies that bind to the B3 peptide. This figure shows the lack of NME7 expression in normal tissues, increasing expression of NME7 with tumor grade and increasing metastasis. Figures 40A-40C show human small intestine tissue specimens stained with anti-NME7 antibodies that bind to the B3 peptide. This figure shows the lack of NME7 expression in normal tissues, increasing expression of NME7 with tumor grade and increasing metastasis. Figures 41A-41D show human colon tissue specimens stained with anti-NME7 antibodies that bind to the B3 peptide. This figure shows the lack of NME7 expression in normal tissues, increasing expression of NME7 with tumor grade and increasing metastasis. Figures 47 and 48 show immunofluorescence photographs demonstrating that NME7 is secreted by and binds to extracellular receptors on a variety of cancer cell lines.

[0081] Furthermore, the inventors have demonstrated that, like NM23-H1, NME7 binds to and dimerizes growth factor receptors in both stem cells and cancer cells. Figure 5 shows the sequence alignment of the A and B domains of NME1 and NME7. * The inventors have recently found that NME7 is a primitive form of NME1 (NM23-H1) that is expressed in very early embryonic stem cells. NME7 is either not expressed at all or is expressed at very low levels in adult tissues. However, the inventors have found that NME7 is expressed at high levels in cancer cells and tissues, and at even higher levels in metastatic cancer cells and tissues. The cleaved form of NME7 can be secreted, which allows it to bind to and activate extracellular receptors. We detected full-length NME7, MW 42 kDa, as well as NME7 species of approximately 33 kDa and 30 kDa. The 33 kDa and 30 kDa species are secreted from cancer cells. Western blots detect full-length NME7 in cell lysates, but detect smaller 30-33 kDa NME7 species in their conditioned media. Western blots detected with either an antibody that recognizes NME7 or an antibody that recognizes only the DM10 domain indicate that the lower molecular weight NME7 species secreted into the conditioned media lack the DM10 domain. These data are consistent with the idea that naturally occurring NME7 species are comparable to the recombinant NME7 we produced, both having approximately the same molecular weight, both being secreted, and both lacking the 91 amino acids of the DM10 domain that can retain proteins intracellularly.

[0082] We have discovered a new NME7 isoform, NME7-X1, and further found that it is overexpressed in stem cells and cancer cells, particularly in prostate cancer (Figures 17, 18, 19, and 22). NME7-X1, with a molecular weight of approximately 30 kDa, contains NME7 amino acids 125-376, while the recombinant NME7 we produced AB contains amino acids 1-376.

[0083] contains amino acids 1-376. AB, with a molecular weight of approximately 33 kDa, spans amino acids 92 - 376 and thus contains 33 more N-terminal amino acids. NME7b spans amino acids 37 - 376, lacks only 37 amino acids of the DM10 domain, and is overexpressed in prostate cancer (Figure 18). We generated human recombinant NME7-X1, which is a secreted 30 kDa NME7 species in cancer cells with a molecular weight slightly smaller than the approximately 33 kDa NME7 species of natural origin that is thought to be a "NME7" AB -like protein of natural origin or a cleavage product or selected isoform.

[0084] We tested a series of cancer cell lines and found that they highly express cleavable low molecular weight species similar to NME7 AB -like proteins such as NME7 AB , or selected isoforms such as NME7-X1.

[0085] Unlike NM23-H1 (alias NME1) which must be a dimer, NME7 is a monomer with two binding regions for the MUC1 * extracellular domain. We generated recombinant human NME7 lacking the DM10 domain and named it NME7 AB . A sandwich ELISA binding assay shows that recombinant NME7 AB can simultaneously bind to two PSMGFR peptides whose extracellular domains contain most or all of the PSMGFR sequence (Figure 1). A nanoparticle binding assay also showed that NME7 can bind to the PSMGFR portion of the MUC1 * extracellular domain and can dimerize it. * Agents that inactivate NME7, block its interaction with its binding partners, or suppress its expression are potent anti-cancer therapeutics. Such agents can be antibodies, small molecules, or nucleic acids. They can act directly on NME7, on molecules that control NME7 expression, or on enzymes that cleave NME7 into a cancer-promoting form.

[0086]

[0087] We have found that recombinant NME7 monomers, such as the NM23-H1 dimer, can fully support the proliferation of pluripotent human stem cells in the absence of other growth factors, cytokines, or serum. Competitive inhibition of the interaction between NME7 and the extracellular domain of MUC1, which essentially contains the PSMGFR sequence, induces stem cell differentiation and indicates that it is the interaction between NME7 and MUC1 that promotes stem cell proliferation and inhibits differentiation. AB Next, we further showed that NME7 alone can fully support human cancer cell proliferation. When added to normal cancer cell growth medium, NME7 stimulates cancer cell proliferation, particularly that of MUC1-positive and MUC1 * -positive cancer cells. Inhibition of the interaction between MUC1 and NME7 suppressed cancer cell proliferation. Blocking of the MUC1 growth factor receptor by anti-MUC1 Fab strongly suppressed cancer cell proliferation. Similarly, antibodies that bind to NME7 suppressed cancer cell proliferation. In one example of cancer growth inhibition by an anti-NME7 antibody, a polyclonal antibody was generated by immunizing animals with a portion of NME7 in the range of amino acids 100 to 376 (Figures 12 and 13). However, we have also found that antibodies generated by immunization with shorter peptides from NME7 or from NME7-X1 also suppress cancer growth. In particular, they suppress the growth of MUC1 and MUC1 * -positive cancers. The anti-NME7 antibodies of the present invention suppressed the formation of non-adherent "floating" cells that can migrate from primary tumors and form metastatic tumor spheres (Figures 14, 16, 29). The anti-NME7 antibodies of the present invention suppressed the upregulation of metastasis and stem cell markers, which are currently also considered characteristic of metastasis (Figures 15, 30, 31, 32).

[0088] Next, we AB showed that NME7 alone can fully support human cancer cell proliferation. NME7 stimulates cancer cell proliferation when added to normal cancer cell growth medium, particularly that of MUC1-positive and MUC1 AB -positive cancer cells. Inhibition of the interaction between MUC1 and NME7 suppressed cancer cell proliferation. Blocking of the MUC1 growth factor receptor by anti-MUC1 Fab strongly suppressed cancer cell proliferation. Similarly, antibodies that bind to NME7 suppressed cancer cell proliferation. In one example of cancer growth inhibition by an anti-NME7 antibody, a polyclonal antibody was generated by immunizing animals with a portion of NME7 in the range of amino acids 100 to 376 (Figures 12 and 13). However, we have also found that antibodies generated by immunization with shorter peptides from NME7 or from NME7-X1 also suppress cancer growth. In particular, they suppress the growth of MUC1 and MUC1 * -positive cancers. The anti-NME7 antibodies of the present invention suppressed the formation of non-adherent "floating" cells that can migrate from primary tumors and form metastatic tumor spheres (Figures 14, 16, 29). The anti-NME7 antibodies of the present invention suppressed the upregulation of metastasis and stem cell markers, which are currently also considered characteristic of metastasis (Figures 15, 30, 31, 32). * and NME7 suppressed cancer cell proliferation. Blocking of the MUC1 growth factor receptor by anti-MUC1 Fab strongly suppressed cancer cell proliferation. Similarly, antibodies that bind to NME7 suppressed cancer cell proliferation. In one example of cancer growth inhibition by an anti-NME7 antibody, a polyclonal antibody was generated by immunizing animals with a portion of NME7 in the range of amino acids 100 to 376 (Figures 12 and 13). However, we have also found that antibodies generated by immunization with shorter peptides from NME7 or from NME7-X1 also suppress cancer growth. In particular, they suppress the growth of MUC1 and MUC1 * Fab suppressed cancer cell proliferation strongly. Similarly, antibodies that bind to NME7 suppressed cancer cell proliferation. In one example of cancer growth inhibition by an anti-NME7 antibody, a polyclonal antibody was generated by immunizing animals with a portion of NME7 in the range of amino acids 100 to 376 (Figures 12 and 13). However, we have also found that antibodies generated by immunization with shorter peptides from NME7 or from NME7-X1 also suppress cancer growth. In particular, they suppress the growth of MUC1 and MUC1 * -positive cancers. The anti-NME7 antibodies of the present invention suppressed the formation of non-adherent "floating" cells that can migrate from primary tumors and form metastatic tumor spheres (Figures 14, 16, 29). The anti-NME7 antibodies of the present invention suppressed the upregulation of metastasis and stem cell markers, which are currently also considered characteristic of metastasis (Figures 15, 30, 31, 32). AB from, or antibodies generated by immunization with shorter peptides from NME7-X1, also suppress cancer growth. In particular, they suppress the growth of MUC1 and MUC1 * -positive cancers. The anti-NME7 antibodies of the present invention suppressed the formation of non-adherent "floating" cells that can migrate from primary tumors and form metastatic tumor spheres (Figures 14, 16, 29). The anti-NME7 antibodies of the present invention suppressed the upregulation of metastasis and stem cell markers, which are currently also considered characteristic of metastasis (Figures 15, 30, 31, 32).

[0089] NME7 causes cancer metastasis The present inventors have further found that culturing cancer cells in a minimal medium containing NME7 AB induces various cancer cells to transform into a more metastatic state. Evidence of this induced metastatic state includes a change from adherent cell growth to non-adherent cell, alias "floating" cell growth, and in particular, upregulation of specific metastasis markers that are upregulated in floating cells. These metastasis markers that are upregulated after culturing in NME7 AB include, but are not limited to, CXCR4, CHD1: alias E-cadherin, MUC1, ALDH1, CD44, and pluripotent stem cell markers such as OCT4, SOX2, NANOG, KLF2 / 4, FOXa2, TBX3, ZEB2, and c-Myc (Figure 2, Figure 3, Figure 20, Figure 49, Figure 51). NME7 AB Cancer cells cultured in NME7 have a very high engraftment rate of over 90% when xenografted into test animals. Furthermore, a very small number of transplanted cancer cells form tumors in test animals, which is evidence that NME7 AB transformed them into cancer stem cells, which are known as metastatic cancer cells. NME7 AB Cancer cells cultured in NME7 and injected into NOD / SCID / GAMMA mice containing estrogen-releasing pellets metastasized from a small number of cells in the animals compared to the parental cells grown in normal medium (Figures 33 - 38). Since the cancer cells produce NME7 cleavage products or alternative isoforms that are essentially equivalent to NME7 AB , the methods described herein are not limited to the use of NME7 AB and other NME7 species may function similarly. For example, we have found that another NME7 isoform, NME7-X1, is expressed by cancer cells. It is identical to our recombinant NME7 AB except that the X1 isoform lacks 33 amino acids from the N-terminus. NME7-X1 is predicted to function like NME7 AB . An "NME7 AB -like" protein was also detected in cancer cells as a ~33 Da species.

[0090] In our previous work as inventors, NME7 AB alone has been shown to be able to return human stem cells to their previous naive state. The inventors have found that culturing cancer cells in the presence of other reagents that return stem cells to a more naive state transforms the cancer cells into a more metastatic state. We have shown that culturing cancer cells in NME7 AB (Figure 2), or NME1 dimer (Figure 3), or "2i" inhibitor (Figure 4) can each transform normal cancer cells into metastatic cancer cells called cancer stem cells "CSC" or tumor initiating cells "TIC". However, NME7 AB induced cancer cells to enter a higher metastatic state than NME1, also known as NM23-H1, which was better than 2i.

[0091] 2i is the name given to two biochemical inhibitors that researchers have found can return human stem cells to a more naive state. 2i are the MEK and GSK3 beta inhibitors, PD0325901 and CHIR99021, which are added to the medium at final concentrations of approximately 1 mM and 3 mM, respectively. NME7 AB and NME7-X1 function well at both lower and higher concentrations, in the range of approximately 1 nM to 16 nM, but are used at a final concentration of approximately 4 nM when added to separate batches of minimal medium to transform cancer cells into metastatic cells. Human or bacterial NME1 dimers are used at final concentrations of 4 nM to 32 nM and are typically used at 16 nM in these experiments, with the human NME having the S120G mutation. Lower concentrations are required when using the wild type. These exact concentrations are not intended to be critical. Certain mutations allow for the presence of the dimer at higher concentrations, but it is important that the NME1 protein is a dimer and the range of concentrations at which this occurs is in the low nanomolar range. Similarly, the concentration of the NME7 protein can vary. NME7 AB and NME7-X1 are monomers, and the concentrations used to transform cancer cells into metastatic cells need to allow the protein to remain as a monomer.

[0092] NME7, NME7 AB , NME7-X1, and in addition to 2i inhibitors, MEKi and GSK3i, other reagents and inhibitors have been shown by other researchers to return stem cells to a more naive state. These inhibitors "i" include JNKi, p38i, PKCi, ROCKi, BMPi, BRAFi, SRCi, as well as growth factors activin and LIF (Gafni et al 2013, Chan et al 2013, Valamehr et al 2014, Ware et al 2014, Theunissen et al 2014). These reagents can also be used to progress cancer cells to a more metastatic state. Cells induced to transform into a more metastatic state by using inhibitors or growth factors that return stem cells to a more naive state, either alone or in combination, can then be used as a discovery tool to identify or test drugs for treating or preventing cancer metastasis.

[0093] Various molecular markers have been proposed as indicators of metastatic cancer cells. Different cancer types may have different molecules that are upregulated. For example, the receptor, CXCR4, is upregulated in metastatic breast cancer, while E-cadherin, also known as CHD1, is more upregulated in metastatic prostate cancer. In addition to these specific metastasis markers, typical markers of pluripotency such as OCT4, SOX2, NANOG, and KLF4 are upregulated when cancer becomes metastatic. Starting cancer cells and subsequent metastatic cancer cells are assayed by PCR to measure the expression levels of these genes. We have demonstrated that these cancer cells cultured in agents such as NME7 AB that transform them into a more metastatic state, as evidenced by increased expression of metastasis markers and pluripotent stem cell markers, function as metastatic cancer cells.

[0094] A functional test of whether a cancer cell population is metastatic is to inject a very small number of cells, for example 200 cells, into immunodeficient mice and determine whether they progress to tumors. Typically, 5 to 6 million cancer cells are required to form tumors in immunodeficient mice. We have shown that as few as about 50 NME-induced metastatic cancer cells formed tumors in mice. Furthermore, human NME7 AB , NME1 or NME7-X1-injected mice during the test period developed distant metastases.

[0095] In one particular experiment, T47D human breast cancer cells were cultured in standard RPMI medium for 14 days, with the medium changed every 48 hours, and were subjected to trypsin treatment at an approximate confluence density of 75%. The cells were then seeded into 6-well plates and cultured in minimal stem cell medium (see Example 1) supplemented with 4 nM NME7 AB . The medium was changed every 48 hours. By about day 4, some cells detached from the surface and became floating. Since these are the cells with the highest metastatic potential as revealed by RT-PCR measurement of metastasis markers, the medium was carefully changed so as to retain the "floaters". On day 7 or 8, the floaters were collected and counted. Samples were retained for RT-PCR measurement. The important marker measured was CXCR4, which is upregulated 40 - 200-fold after short-term culture in NME7 AB .

[0096] The newly recovered floating metastatic cells were xenografted into the flanks of female nu / nu athymic mice implanted with a 90-day slow-release estrogen pellet, with the floating cells xenografted with 10,000, 1,000, 100, or 50 cells each. Half of the mice in each group of 6 were also injected daily with 32 nM NME7 AB near the original implantation site. NME7 ABParental T47D cells cultured in RPMI medium without were also transplanted into mice at 6 million, 10,000, or 100 cells as controls. Mice transplanted with NME7-induced floating cells developed tumors even when only 50 cells were transplanted. Floating cells were transplanted and NME7 AB Mice that received daily injections of also developed distant tumors or distant metastases in various organs. NME7 AB After transplantation of cultured cancer cells, human NME7 AB Of the 12 mice injected with, 11, or 92%, developed tumors at the injection site. Only 7 of the 12 mice, or 58%, that were not injected with human NME7 AB after transplantation developed tumors. Nine of the 11 mice (82%) that showed tumors and were injected with human NME7 AB developed multiple tumors distantly from the injection site. None of the mice not injected with NME7 AB developed multiple visible tumors.

[0097] After sacrifice, RT-PCR and Western blot showed that the distant nodules in mice injected with NME7 AB were indeed human breast tumors. Similar analysis of those organs showed that in addition to the distant nodules, the mice had randomly metastasized to the liver and lungs with the human breast cancer characteristics of the transplanted human breast cancer cells. As expected, only the mice injected with 6 million cells had tumor growth.

[0098] We showed that the size and sequence of human recombinant NME7 AB were equivalent to NME7-X1 and the NME7 cleavage products of 30 - 33 kDa. We, NME7 ABhas been shown to promote cancerous growth and accelerate cancer cells into a highly metastatic cancer stem cell (CSC) state, also known as tumor-initiating cells (TIC). Therefore, we concluded that NME7-X1, and NME7 cleavage products lacking the DM10 domain, also promote cancerous growth and accelerate cancer cells into a highly metastatic cancer stem cell (CSC) state, also known as tumor-initiating cells (TIC). In one example, NME7 AB was added to cancer cells in serum-free medium and in the absence of any other growth factors or cytokines. Within 7 - 10 days, the cancer cells reverted to highly metastatic CSC / TIC, as evidenced by more than a 100-fold increase in the expression of molecular markers such as CXCR4, which are indicators of metastatic cancer cells. In one example, T47D breast cancer cells were cultured in standard RPMI medium or in minimal stem cell medium (Example 1) supplemented with recombinant NME7 AB at a final concentration of 16 nM. After 10 days, the cells were harvested and analyzed by RT-PCR for the expression of CSC molecular markers that were increased 10 - 200-fold (Figure 2). This is a specific and detailed example of how we transformed one type of cancer cell into a more metastatic state. There is a series of cancer cells transformed in this way, a series of agents that revert stem cells to a more naive state and further progress cancer cells to a more metastatic state, and a series of concentrations of the added agent that transform cancer cells, so the present invention is not intended to be limited by these details. Other types of cancer cells required a longer culture period in NME7 AB due to a dramatic increase in the expression of metastasis markers and the ability to form tumors from a very small number of transplanted cancer cells. For example, prostate cancer cells cultured in NME7 AB , 2i, human NME1, or bacterial NME1, which has high homology to human NME1 or human NME7, showed a dramatic increase in metastasis markers after 2 - 3 passages.

[0099] The metastasis marker CXCR4 was particularly elevated in metastatic breast cancer cells, while CHD1 was particularly elevated in metastatic prostate cancer. Here, we show that pluripotent stem cell markers such as OCT4, SOX2, NANOG, KLF2 / 4, and TBX3 are also upregulated when cancer cells transform into more metastatic cells.

[0100] DU145 prostate cancer cells were similarly cultured and NME7 AB These cells cultured in also showed a dramatic increase in CSC marker expression (Figure 3). In prostate cancer cells, CHD1 (also known as E-cadherin) and CXCR4 were upregulated along with other pluripotent stem cell markers compared to control cancer cells that did not proliferate in NME7 AB Figure 20A - 20C show that all of the ovarian cancer cell lines SK-OV3, OV-90, and the breast cancer cell line MDA-MB migrated from adherent to non-adherent floating cells and increased the expression of metastasis markers after 72 or 144 hours of culture with NME7 AB Ovarian cancer cells, prostate cancer cells, pancreatic cancer cells, and melanoma cells also cultured in NME7 AB were transformed into a more metastatic state after only 3 days of culture. Figure 21 shows that breast cancer, ovarian cancer, prostate cancer, pancreatic cancer cells, and melanoma cells express MUC1 and MUC1 * Here, we have shown that NME7

[0101] Here, we show that NME7 AB transforms a wide range of cancer cells into a more metastatic state. We also show that the cancer cells have a native origin species with a molecular weight almost the same as recombinant NME7 33 kDa (Figures 17, 18, 19, and 22) and NME7 AB like that lacking the DM10 domain, and except for lacking 33 amino acids from the N-terminus NME7 AB AB ​It was also shown that the alternative isoform NME7-X1 30 kDa, which has the same array, is expressed. Co-immunoprecipitation experiments were performed in T47D breast cancer cells. Cell extracts were incubated with an antibody against the cytoplasmic side terminal of MUC1, Ab-5 or a control antibody, IgG, for co-immunoprecipitation. The immunoprecipitated species were separated by gel electrophoresis. The gels were blotted with two different commercially available anti-NME7 antibodies. Both gels showed specific NME7 bands at approximately 33 kDa and approximately 30 kDa (Figure 22A and Figure 22B). The gels were stripped and redetected with an antibody against the extracellular domain of MUC1 * , anti-PSMGFR (Figure 22C and Figure 22D). This indicates that the NME7 species and MUC1 * interact. The recombinant NME7 AB and recombinant NME7-X1 that we produced were mixed together, developed on a gel, and then detected with an anti-NME7 antibody. Two specific NME7 species that occur naturally in breast cancer cells and interact with MUC1 * were shown to be the NME7 AB -like species and NME7-X1 (Figure 22E). Similar experiments were performed on human stem cells. Figures 23A-23C show photographs of Western blots of co-immunoprecipitation experiments. Human induced pluripotent stem cells, iPS7 cells, or embryonic stem cells, HES3 cell extracts were incubated with an antibody against the cytoplasmic side terminal of MUC1, Ab-5, or a control antibody, IgG, for co-immunoprecipitation. The gels were blotted with a commercially available anti-NME7 antibody B9 (Figure 23A). Both cell types showed specific NME7 bands at approximately 33 kDa and approximately 30 kDa. The gels were stripped and redetected with an antibody against the extracellular domain of MUC1 * , anti-PSMGFR (Figure 23B). This indicates that the NME7 species and MUC1 * interact. The recombinant NME7 AB and recombinant NME7-X1 that we produced were mixed together, developed on a gel, and then detected with an anti-NME7 antibody. Two specific NME7 species that occur naturally in breast cancer cells and interact with MUC1 * were shown to be the NME7 AB -like species and NME7-X1 (Figure 23C). NME7AB Since it is a recombinant protein, it is not clear whether naturally occurring species contain 1 to 15 additional amino acids or lack 1 to 15 additional amino acids compared to recombinant NME7 AB However, it migrates with the same apparent molecular weight. "NME7 AB -like" is intended to be an NME7 species that migrates with an apparent molecular weight of approximately 33 kDa and can function as recombinant NME7 does in that it can stimulate cancer cell proliferation, induce the migration of cancer cells to a more metastatic state, and fully support the development of pluripotency in human stem cells. AB We conclude that cancer cell lines and cancer cell populations expressing NME7 and lower molecular weight NME7 species contain some cancer cells that are CSCs or metastatic cancer cells. These cancers can become more metastatic or increase the population of metastatic cells by culturing the cells in NME7

[0102] , NME7-X1, or lower molecular weight NME7 species. Figure 19 shows Western blots of a series of cancer cells expressing all NME7 as well as NME7 AB -like at 33 kDa and NME7-X1 at 30 kDa, which are lower molecular weight species. Figure 21 shows that all of the T47D breast cancer cell line, PC3 and DU145 prostate cancer cell lines, BT-474 breast cancer cell line, CHL-1 and A2058 melanoma cell lines, and CAPAN-2 and PANC-1 pancreatic cancer cell lines express MUC1, MUC1 AB In Figure 21A, the BT474 cells appear not to express MUC1 or MUC1 * ; however, we have previously shown that these HER2-positive breast cancer cells become metastatic when they become resistant to chemotherapeutic drugs, and that they become metastatic by increasing the expression of MUC1 * (Fessler et al 2009) (Figure 21D). By anti-MUC1 * Fab, MUC1 * by *Receptor blockade reversed their resistance to Herceptin (Figure 21E), taxol (Figure 21F), and other chemotherapeutic agents. These cancer types and other cancer types that express NME7 and lower molecular weight NME7 species such as 33 kDa and 30 kDa are more metastatic or can increase cell populations that are metastatic by culturing cells in NME7 AB , NME7-X1, or lower molecular weight NME7 species.

[0103] Conversely, the metastatic potential of these and other cancer types that express NME7 and lower molecular weight NME7 species such as 33 kDa or 30 kDa can be reversed by treating the cells with anti-NME7 antibodies. An anti-NME7 antibody or an antibody that binds to NME7 AB or NME7-X1 is administered to a patient for the treatment or prevention of cancer including breast, prostate, ovarian, pancreatic, and liver cancers. We have shown that NME7 AB binds to the MUC1 * growth factor receptor and activates it, thereby exerting its tumorigenic effect. Therefore, anti-NME7 antibodies are thought to be effective against any MUC1 * positive cancer including, but not limited to, breast, lung, liver, pancreas, stomach, colorectal, prostate, brain, melanoma, kidney, etc. An anti-NME7, anti-NME7 AB , or anti-NME7-X1 antibody is administered to a patient for the treatment or prevention of cancer that is NME7 AB , NME7 AB -like, or NME7-X1 positive, or MUC1 * positive.

[0104] Testing of patient cancer cells for effective treatment NME7 AB, NME7-X1, as well as 2i and other reagents that revert stem cells to a more naive state, also induce cancer cells to transform into a more metastatic state. After treatment with any one or combination of these reagents, cancer cells have a higher engraftment rate and require up to 100,000-fold fewer cells to produce tumors in test animals. Thus, the methods described in this disclosure can be used to enable xenotransplantation of a patient's primary tumor cells into test animals.

[0105] Candidate therapeutic agents can then be tested in recipient animals. The effective therapeutic agents thus identified can be used to treat donor patients or other patients having a similar cancer. In one embodiment, a method for identifying an effective therapeutic agent for a particular patient or a particular type of cancer includes the following steps: 1) obtaining cancer cells from a cell line, a patient, or a patient to whom a test therapeutic agent is to be administered; 2) culturing the cancer cells in NME7 AB , NME7-X1, human NME1, bacterial NME1 having high homology to human NME1 or NME7, 2i, or other reagents shown to revert stem cells to a more naive state; 3) transplanting the obtained cancer cells into a test animal to which human NME7 AB , NME7-X1, human NME1, bacterial NME1 having high homology to human NME1 or NME7, 2i, or other reagents shown to revert stem cells to a more naive state can likewise be administered, or the animal is transduced with human NME7 AB or NME7-X1; 4) administering a candidate anti-cancer therapeutic agent to the animal; 5) evaluating the effectiveness of the therapeutic agent; and 6) administering the effective therapeutic agent to a donor patient or another patient having a similar cancer.

[0106] anti-NME7 antibody Anti-NME7 antibodies are potent anti-cancer agents. NME7 is a growth factor that promotes cancer cell proliferation and further promotes their progression to a more metastatic or more highly malignant state. NME7, and a cleaved form of NME7 that is approximately 33 kDa or 30 kDa, have been shown to fully support cancer growth even in serum-free media lacking any other growth factor or cytokine. In pull-down assays, ELISA, and nanoparticle binding experiments, we have shown that the growth factor receptor MUC1 * is a binding partner of NME7 and NME7 AB . Promotion of this interaction by the exclusion of all other growth factors or cytokines increased the expression of cancer stem cell markers. Blockade of the interaction using polyclonal antibodies that specifically bind to NME7 killed cancer cells actively even in the presence of serum. Therefore, anti-NME7 or anti-NME7 AB antibodies are potent anti-cancer agents that can be administered to patients for the treatment or prevention of cancer. More than 75% of all cancers are MUC1 * positive. MUC1 * is a transmembrane cleavage product of MUC1, with most of the extracellular domain deleted, leaving a part of the extracellular domain that contains most of the PSMGFR sequence and may contain an additional 9 - 20 amino acid N-terminus at the boundary of the PSMGFR sequence.

[0107] One aspect of the invention is a method of treating or preventing cancer in a subject, comprising administering to the subject an effective amount of an anti-NME7 antibody. In one example, the anti-NME7 antibody can bind to NME7 AB . In another example, the anti-NME7 antibody can bind to NME7-X1. In yet another example, the anti-NME7 antibody administered to a patient inhibits or prevents binding to its target in the promotion of cancer. In one example, the target is the extracellular domain of cleaved MUC1. More specifically, the NME7 target that promotes cancer is the PSMGFR region of the MUC1 * extracellular domain. In one aspect, an effective therapeutic agent consists mainly of the NME7 species consisting of the PSMGFR portion or PSMGFR peptide of MUC1 * , and MUC1 *It is an agent that disrupts or prevents the interaction with the extracellular domain. An agent for the treatment or prevention of cancer is an agent that directly or indirectly suppresses the expression or function of NME7, NME7 AB -like cleavage products, or alternative isoforms including NME7-X1. In one example, an effective anti-cancer therapeutic agent binds to NME7 species or inactivates its tumor activity. An effective therapeutic agent for the treatment or prevention of cancer is an agent that binds to or inactivates NME7, NME7 AB -like cleavage products, or alternative isoforms, or NME7-X1. In one aspect, the therapeutic agent that binds to NME7 species is an antibody. The antibody can be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv or an antibody mimetic of animal origin, a human-animal chimera, humanized, or a human antibody. The antibody can be generated by inoculating or immunizing with NME7 species or a fragment thereof, or can be selected from an antibody library or pool based on their binding ability to NME7, NME7 AB -like cleavage products or alternative isoforms including NME7-X1.

[0108] Generation of anti-NME7 antibodies Anti-NME7 antibodies can be generated outside the patient, such as in a host animal, or within the patient. The antibody can be a natural, synthetic, full-length antibody or antibody fragment, generated by immunization with NME7 or an NME7 fragment, or selected from an antibody library or pool based on their binding ability to a desired NME7 species such as NME7 AB or NME7-X1. In one aspect, the antibody is generated by immunization with a peptide selected from those listed in FIGS. 6-9, or selected for its binding ability to the peptide. In another aspect, the antibody is generated from a peptide whose sequence is not identical to that of human NME1, or the antibody is selected for its binding ability to NME7 species and its inability to bind to human NME1.

[0109] One method used to identify NME7 or NME7-X1-derived peptides that give rise to antibodies that suppress cancer growth and inhibit metastasis, or peptides that are inhibitory per se, is as follows: 1) The protein sequences of human NME1, human NME7, human NME7-X1, and several bacterial or fungal NME proteins having high sequence homology to human NME1 or human NME7 are aligned; 2) Regions of high sequence homology in all NMEs are identified; 3) Peptide sequences that are specific to NME7 or NME7-X1 but adjacent to the regions of high sequence homology are identified. The peptides are then synthesized and used to generate antibodies in humans or host animals. The resulting antibodies are selected for therapeutic use if: 1) they bind to NME7 AB or NME7-X1 but not to NME1; 2) they have the ability to suppress cancer growth; 3) they have the ability to inhibit the migration of cancer cells to a more metastatic state; or 4) they suppress metastasis in vivo. In some cases, antibodies for therapeutic use are selected for their ability to disrupt binding to the extracellular domain of MUC1 AB of NME7 * or NME7-X1, binding to the PSMGFR peptide, or binding to the N-10 peptide.

[0110] Use of anti-NME7 antibodies for the treatment of cancer Antibodies that suppress cancer growth or transition to a more metastatic state are selected for use as anti-cancer therapeutics and can be administered to a patient for the treatment or prevention of cancer. The selected antibodies can be further optimized, for example, by designing or producing human chimeric antibodies or fully human antibodies. To demonstrate the effectiveness of this approach, we selected NME7 peptides from regions of NME7 that are thought to be essential for its cancerous function. We then used these peptides to generate antibodies and subsequently tested both the resulting antibodies, as well as the immunizing peptides, for their ability to a) suppress cancerous growth; and b) inhibit the induction of migration from cancer cells to metastatic cancer cells. The NME7 peptides were selected as immunizing agents for antibody production and as inhibitors themselves (Figure 9 and Example 7). Peptides A1 (SEQ ID NO: 141), A2 (SEQ ID NO: 142), B1 (SEQ ID NO: 143), B2 (SEQ ID NO: 144) and B3 (SEQ ID NO: 145), where A means the domain from which the peptide is derived, i.e., the NDPK A domain, and B means that the peptide is derived from the NDPK B domain (Figure 5). Each peptide was used as an immunogen and injected into each of two rabbits for the production of polyclonal antibodies. Antibodies collected from the immunized rabbit blood were purified on columns derivatized with the immunizing peptides. The purified antibodies were then tested for their ability to bind to human NME7. All of the resulting antibodies bound to human NME7 as desired, but did not bind to human NME1 (Figures 10A - 10B, Example 8). These results show that by selecting peptides whose sequences are found in NME7 but not exactly identical to NME1, antibodies that specifically bind to NME7 but not to NME1 are generated. Since NME1 has healthy functions, it is most often desirable to generate antibodies that do not interfere with NME1. The antibodies were also tested for their ability to suppress the binding of NME7 to the MUC1 * extracellular domain peptide. The ELISA experiments shown in Figure 11 show that the antibodies bind much more of the MUC1 * extracellular domain peptide to NME7 ABindicates inhibition of the binding. Recall that each of the NME7 A and B domains can bind to the PSMGFR peptide. Thus, NME7 AB complete suppression of binding to the PSMGFR peptide cannot be achieved with a single antibody or a peptide from just one domain. These antibodies and their respective immunizing peptides also inhibited cancer cell growth (Figs. 12 - 13). These antibodies also inhibited the formation of non - adherent "floating" cells arising from cancer cells growing in NME7 AB (Fig. 14). As can be seen from the figure, the polyclonal antibody purified by immunization with the B3 peptide reduced the number of metastatic floating cells by 95%, indicating that the anti - NME7 antibody that binds to the B3 peptide is the most effective in suppressing cancer metastasis. Similarly, the antibody inhibited the expression of the metastasis marker CXCR4 (Fig. 15A). Again, the B3 antibody was the most efficient in suppressing the expression of CXCR4; the bar labeled NME7 FL (NME7 floating cells) shows a 70 - fold increase in CXCR4, which was reduced 20 - fold (bar labeled NME7+61FL) by the B3 antibody 61. In addition, the immunizing peptide itself inhibited the up - regulation of CXCR4 and other metastasis markers when T47D cancer cells were grown in NME7 AB or 2i.

[0111] This is only an example of a selected peptide that generates an antibody that suppresses the cancerous functions of NME7 and NME7 species. Sequence alignment between human NME1, human NME7, human NME7-X1, and bacterial NME proteins having high sequence homology to human NME1 or NME7 identified five homologous domains. The fact that peptides A1, A2, B1, B2, and B3 all generated antibodies that inhibited their transition to a cancer-proliferative or metastatic state means that the five regions from which these peptides are derived are regions of NME7 that are important for its function in promoting cancer. Other peptides from these regions will also suppress cancer growth and metastasis and thus give rise to anti-NME7 antibodies that are potent anti-cancer therapeutics. Antibodies generated from peptides A1, A2, B1, B2, and B3 were shown to suppress cancer growth and inhibit transition to a more metastatic state. Monoclonal antibodies generated by immunization with the same or similar peptides and subsequent testing of the monoclonal antibodies will identify antibodies that can be administered to patients for the treatment or prevention of cancer after humanization or other manipulations known to those skilled in the art.

[0112] In certain experiments, antibodies generated by immunization with peptides A1, A2, B1, B2, and B3, as well as the immunizing peptides themselves, were added to cancer cells in culture to determine whether the addition of the antibody or immunizing peptide inhibited cancer cell growth. At low concentrations and added separately, the antibody as well as the immunizing peptide inhibited cancer cell growth (Figure 12 for one example). However, when added at higher concentrations or in combination, the antibody as well as the immunizing peptide strongly inhibited cancer cell growth (Figure 13). The corresponding human NME7 amino acid numbers for immunizing peptides A1, A2, B1, B2, and B3 are 127-142, 181-191, 263-282, 287-301, 343-371, respectively, from human full-length NME7 having SEQ ID NO: 82 or 147.

[0113] For clarity, when residue numbers of NME7 are discussed, they refer to the residue numbers of NME7 shown by SEQ ID NO: 82 or 147.

[0114] One of the antibodies used in the cancer growth inhibition experiment and the antibody shown in FIG. 12 was generated by immunizing with an NME7 peptide corresponding to amino acids 100-376 (SEQ ID NO: 82 or 147) of NME7. To generate anti-NME7 antibodies with higher affinity and specificity, the following steps were continued: animals were immunized with a peptide containing human NME7 amino acids 100-376, and then: 1) antibodies that bind to human NME1 were removed; 2) antibodies that suppress NME7, 2i, or other NMEs that induce the migration of cancer cells to a more metastatic state were selected; 3) antibodies that suppress the growth of cancer cells were selected; 4) antibodies that suppress the growth of MUC1-positive cancer cells were selected; 5) antibodies that suppress the binding of NME7 or NME7-X1 to the extracellular domain and substantially suppress the binding to the PSMGFR peptide were selected; and / or 6) antibodies that bind to one or more of the peptides A1, A2, B1, B2, or B3 listed in FIG. 9 were selected. AB 、2i or other NMEs that induce the migration of cancer cells to a more metastatic state were selected; 3) antibodies that suppress the growth of cancer cells were selected; 4) * antibodies that suppress the growth of MUC1-positive cancer cells were selected; 5) * to the extracellular domain of NME7 AB or NME7-X1 and substantially suppress the binding to the PSMGFR peptide were selected; and / or 6) antibodies that bind to one or more of the peptides A1, A2, B1, B2, or B3 listed in FIG. 9 were selected.

[0115] Higher affinity monoclonal antibodies, or monoclonal antibodies generated from longer peptides, may be more effective antibody therapeutics. Alternatively, combinations of anti-NME7, anti-NME7, AB or anti-NME7-X1 antibodies may be administered to patients to enhance efficacy.

[0116] Anti-NME7 antibodies suppress the migration of cancer cells to metastatic cancer cells Anti-NME7 antibodies suppress the migration of cancer cells to metastatic cancer cells or tumor-initiating cells (TICs) called cancer stem cells (CSCs). We would like to recall that culturing various cancer cells in the presence of NME7 has been demonstrated to cause those cancer cells to transition from normal cancer cells to metastatic CSCs or TICs. Therefore, antibodies that bind to NME7, NME7 AB or NME7-X1 will likely suppress the progression of cancer cells to a more metastatic state. AB or NME7-X1 will likely suppress the progression of cancer cells to a more metastatic state.

[0117] When cancer cells are cultured in the presence of an agent that returns stem cells to a more naive state, they transform into a more metastatic state. We have shown that culturing cancer cells in NME7 AB , human NME1 dimer, bacterial NME1 dimer, or MEK and GSK3 beta inhibitors (referred to as "2i") causes the cells to become more metastatic. As the cells transition to a more metastatic state, they become non-adherent or poorly adherent and detach from the culture dish to float. These floating cells, the "floaters", were collected separately from the adherent ones and the following was shown: a) they express much higher levels of metastatic genes; and b) they formed tumors when xenografted into mice at very low copy numbers. RT-PCR measurements of specific metastasis markers such as CXCR4 for breast cancer, CHD1 for prostate cancer, and other pluripotent stem cell markers such as OCT4, SOX2, NANOG, KLF4 were dramatically overexpressed in cancer cells cultured in NME7 AB and were most overexpressed in the non-adherent cells, herein and in the figures referred to as "floaters".

[0118] In one example, NME7 AB specific antibodies generated by immunization with NME7-derived peptides A1, A2, B1, B2, and B3, as well as the immunizing peptides themselves, were added to the medium to determine whether they inhibit the transformation of normal cancer cells into metastatic cancer stem cells. NME7 AB or 2i was added to the medium either alone. The antibodies and peptides were added separately with an agent that causes metastatic transformation, in this case NME7 AB or the 2i inhibitors PD0325901 and CHIR99021. NME7 AB and 2i were used separately to induce cancer cells to transform into a more highly malignant metastatic state. 2i was used so that it could not be claimed that the antibody added to the medium simply blocked all of the NME7 AB and thus the causative agent was effectively absent (Example 10).

[0119] Visual observations were independently recorded by two scientists as the experiment progressed (Figure 14). The most prominent observation was that the antibody and peptide dramatically reduced the number of floating cells, which was the first indication that the antibody and peptide suppressed the transformation of metastatic cancer cells. In particular, cells generated from immunization with the B3 peptide against which the antibody was generated produced few floating cells. mRNA was extracted from both floating cells, adherent cells, and control cancer cells. The amount of mRNA indicating cell survival and proliferation was measured. Cells treated with the antibody had far less mRNA and showed fewer viable dividing cells (Figure 16), which is anti-NME7 AB confirming that the antibody suppresses cancer cell proliferation and their transition to a more metastatic state. Using RT-PCR, the expression levels of metastasis markers including CXCR4 were measured. Treatment with the anti-NME7 antibody greatly reduced the amount of metastasis markers such as CXCR4, indicating that the anti-NME7 antibody or peptide suppressed the transition to metastatic cancer (Figures 15A - 15C). These results indicate that an antibody that binds to NME7 AB can be administered to a patient for the treatment or prevention of metastatic cancer.

[0120] NME7 AB , or an NME7-X1-derived peptide, competitively inhibits the binding of intact NME7 AB and NME7-X1 and is an anti-cancer agent. In another aspect of the invention, a therapeutic agent for the treatment or prevention of cancer is a peptide derived from the NME7 sequence, which is administered to a patient for the treatment or prevention of cancer. In one aspect, an NME7-derived peptide is administered to a patient, whereby a peptide that is shorter than full-length NME7 and should not be able to confer the carcinogenic activity of NME7 binds to the target of NME7 and competitively inhibits the cancer-promoting interaction between its target and intact NME7. Since NME7 AB can fully confer carcinogenic activity, NME7 ABThe array is preferably a shorter peptide source, in which case it is necessary to confirm that the peptide itself cannot promote cancerous growth or other tumor or carcinogenic activities. In a preferred embodiment, NME7 AB One or more peptides having a partial sequence of, preferably about 12 to 56 amino acids in length, are administered to a patient. To increase the half-life, the peptide can be a peptidomimetic, such as a peptide having a non-natural backbone or D-amino acids instead of L-type amino acids. In yet another case, the anti-cancer therapeutic agent is a peptide or a peptidomimetic, and the peptide is NME7, NME7 AB , or NME7-X1, or the PSMGFR peptide, which is also referred to as "FLR" in some cases herein and is its target, includes MUC1 * and has a sequence highly homologous to at least a part of the extracellular domain of.

[0121] Figures 6-9 provide a list of preferred amino acid sequences that are expected to inhibit the binding of NME7 to its cognate target. In an even more preferred embodiment, the peptides selected for administration to a patient suffering from cancer or at risk of developing cancer are selected because they bind to the NME7 binding partner and the peptide itself does not confer tumor activity. In an even more preferred embodiment, the NME7 binding partner is the extracellular domain of MUC1 * . In an even more preferred embodiment, the NME7 binding partner is the PSMGFR peptide.

[0122] The term "confer tumor or carcinogenic activity" means that the peptide itself cannot assist or promote the growth of cancer. Another way to test whether a peptide or peptides derived from NME7 can promote tumorigenesis is to test whether the peptide can assist the pluripotent growth of human stem cells. NME proteins and peptides that assist pluripotent human stem cell growth also assist cancer growth. In yet another way, peptides are deselected if they can return somatic cells to a lower state of maturity.

[0123] NME7 AB The fragments of AB suppress cancer cell proliferation and the migration of cancer cells to a more metastatic state. As an example, NME7 peptides A1, A2, B1, B2, and B3 added separately (Figure 12) or in combination (Figure 13) suppress the proliferation of cancer cells. Furthermore, NME7 peptides A1, A2, B1, B2, and B3 suppressed the migration of cancer cells to a more metastatic state (Figure 15).

[0124] Therefore, antibodies generated by immunization with peptides specific to NME7 and to NME7 AB or NME7-X1 will block the cancerous effects of the NME7 species and would be potent anti-cancer agents. Similarly, these results indicate that peptides specific to NME7 and to NME7 AB or NME7-X1 block the cancerous effects of the NME7 species. In one aspect of the invention, the peptide is selected from the list shown in Figure 6. In one aspect of the invention, the peptide is selected from the list shown in Figure 7. In one aspect of the invention, the peptide is selected from the list shown in Figure 8. Further in another aspect of the invention, the peptide is selected from the list shown in Figure 9. These antibodies can be generated by immunization or can be generated or selected by other means and are then selected for their ability to bind to NME7, NME7 AB , NME7-X1 or NME7-derived peptides including, but not limited to, NME7-derived peptides A1 (SEQ ID NO: 141), A2 (SEQ ID NO: 142), B1 (SEQ ID NO: 143), B2 (SEQ ID NO: 144) or B3 (SEQ ID NO: 145). Such antibodies can be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, human, or humanized antibodies or can be antibody mimetics such as protein scaffolds that provide a recognition region that binds to a specific target.

[0125] Anti-NME7 antibodies for use in the treatment or prevention of cancer can be prepared by standard methods known to those of skill in the art, and these methods include NME7, NME7 ABor NME7 lacking an additional 10 to 25 amino acids from the N-terminus AB It is used to generate antibodies or antibody-like molecules that recognize shorter forms of AB . Such antibodies can be human antibodies or humanized antibodies. Such antibodies can be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, human, or humanized antibodies, or can be antibody mimetics such as protein scaffolds that provide recognition regions that bind to specific targets.

[0126] Anti-NME7 antibodies generated by immunization with NME7-derived peptides A1 (SEQ ID NO: 141), A2 (SEQ ID NO: 142), B1 (SEQ ID NO: 143), B2 (SEQ ID NO: 144), or B3 (SEQ ID NO: 145), or antibodies that bind to the A1, A2, B1, B2, or B3 peptides, bind to NME7 AB and NME7-X1, but resist binding to NME1, which may be required for the function of some healthy cells. Such antibodies bind to NME7 AB or their target receptor for NME7-X1, MUC1 * to suppress binding. Antibodies that bind to the A1, A2, B1, B2, or B3 peptides can be administered to patients diagnosed with cancer or metastasis, or at risk of developing cancer or metastasis. Such antibodies can be human antibodies or humanized antibodies. Such antibodies can be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, or can be antibody mimetics such as protein scaffolds that provide recognition regions that bind to specific targets.

[0127] Anti-NME7 antibodies generated by immunization with the B3 peptide, or antibodies that bind to the B3 peptide, are particularly specific with respect to the recognition of NME7 AB and NME7-X1. Such antibodies bind to NME7 AB or their target receptor for NME7-X1, MUC1 *It is also extremely efficient in suppressing binding thereto. Antibodies that bind to the B3 peptide are also extremely efficient in terms of preventing, suppressing, and recovering from cancer or cancer metastasis. Such antibodies can be human antibodies or humanized antibodies. Such antibodies can be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, or can be antibody mimetics such as protein scaffolds that provide a recognition region that binds to a specific target.

[0128] Note that polyclonal antibody #61 generated by immunization with the B3 peptide in rabbits suppressed the transformation of cancer cells into cancer stem cells, as was also evident from antibody #61 that blocked the increased expression of the metastasis marker CXCR4 (Figure 15).

[0129] The B3 peptide derived from NME7 (SEQ ID NO: 145) has a cysteine at position 14, which complicates the production of anti-NME7 antibodies. We mutated cysteine 14 to serine to generate AIFGKTKIQNAVHSTDLPEDGLLEVQYFF (SEQ ID NO: 169), immunized animals, and generated anti-NME7 monoclonal antibodies. The antibodies obtained bind to the native B3 sequence as well as the B3Cys14Ser peptide. Seven high-affinity and specific monoclonal antibodies: 8F9A5A1, 8F9A4A3, 5F3A5D4, 5D9E2B11, 5D9E10E4, 5D9G2C4, and 8H5H5G4 were generated. However, sequence alignment showed that only three unique sequence antibodies: 8F9A5A1, 8F9A4A3, and 5F3A5D4 were present, as shown below.

[0130] Heavy chain alignment

Chemical formula

[0131] Light chain alignment

Chemical formula

[0132] The monoclonal antibodies 5D9E2B11, 5D9E10E4, 5D9G2C4, and 8H5H5G4 all have the same sequence as 5F3A5D4, also known as 5D4. Here, when we refer to the antibody 5F3A5D4, also known as 5D4, it is understood to apply also to 5D9E2B11, 5D9E10E4, 5D9G2C4, and 8H5H5G4. As can be seen from FIGS. 24 and 25, the anti-NME7 antibodies 8F9A5A1, 8F9A4A3, and 5F3A5D4 all bind to NME7 AB but do not bind to NME1. This is important because the A domain of NME7 has high homology to NME1, which is necessary for normal cell function. In the case of an anti-cancer therapeutic or an anti-metastasis therapeutic, it will be essential to inhibit NME7 AB and not inhibit NME1.

[0133] FIGS. 26, 27, and 28 show that these anti-NME7 antibodies can also disrupt the binding of NME7 AB to the MUC1 * PSMGFR peptide and the N-10 PSMGFR peptide. As can be seen from the figure, there is no overall replacement of NME7 from the MUC1 * peptide. However, it should be recalled that NME7 AB consists of an A domain and a B domain, each of which can bind to MUC1 AB . These antibodies are designed to disrupt the binding of the B domain to MUC1 * ; the A domain of NME7 will still be able to bind to the MUC1 * peptide on the plate surface. For a useful therapeutic, it is sufficient for the antibody to only disrupt the binding to one domain of MUC1 AB , and by doing so, ligand-induced dimerization and MUC1 * * * ​​Activation of growth factor receptors can be blocked. An antibody or antibody mimetic that binds to the NME7 B3 peptide or the B3Cys14Ser peptide (SEQ ID NO: 169) is an antibody that can be administered to a patient diagnosed with cancer or metastasis, or a patient at risk of developing cancer or metastasis.

[0134] It is well known in the art that it is difficult to generate cancer cells that metastasize in animal models. In human tumors, only 1 in 100,000 cancer cells, or 1 in 1,000,000 cancer cells, are estimated to separate from the tumor, implant somewhere, and initiate metastasis [Al-Hajj et al., 2003]. Some researchers have reported that T47D breast cancer cells injected into mice with reduced immune function metastasize after about 12 weeks [Harrell et al 2006]. Other researchers have reported that AsPC-1 pancreatic cancer cells metastasize after about 4 weeks [Suzuki et al, 2013].

[0135] Here, we show that T47D breast cancer cells grew for 10 days in serum-free medium containing recombinant NME7 AB as the sole growth factor. When grown in NME7 AB about 25% of the cancer cells began to float and stopped dividing, but were still viable. PCR measurements showed that these "floating" cells highly upregulated the expression of the breast cancer metastatic factor CXCR4.

[0136] In some of the figures provided in this specification, these floating cells are called cancer stem cells (CSCs). Estrogen-releasing pellets were implanted into female nu / nu mice with reduced immune function for 90 days. 500,000 T47D-wt cells or 10,000 T47D-CSCs (cancer stem cells) were injected into the subcutaneous (s.c.) tail vein (i.vi) or into the intraperitoneal space (i.p.) of nu / nu mice. These cancer cells were engineered to express luciferase. To visualize the tumors or cancer cells, luciferin was injected into the animals and visualized 10 minutes later with an IVIS imager. As can be seen from the IVIS measurements in FIGS. 33A - 33B, by day 6, 500,000 T47D-wt cells injected into the tail vein showed no signs of viable cancer cells or cell engraftment.

[0137] In stark contrast, 10,000 T47D-CSCs injected into the tail vein metastasized. Prior to the IVIS measurement on day 6, 32 nM of recombinant NME7 AB was injected into the T47D-CSC mice. The next day, one of two CSC mice was injected with a cocktail of anti-NME7 monoclonal antibodies 8F9A5A1, 8F9A4A3, and 5F3A5D4 at a concentration equivalent to 15 mg / kg in a volume of 200 μL. The almost simultaneous injection of NME7 AB and the anti-NME7 antibody is likely to nullify the effect of the antibody. FIG. 34 shows that by day 10, the treated mice are almost completely metastatic. As can be seen from the figure, the mice selected for treatment are more metastatic than equivalent T47D-CSC mice.

[0138] The animals were reinjected with the anti-NME7 antibody on the 10th day. IVIS measurements on the 12th day (Figure 35) indicate that the antibody-treated mice began to remove metastases. By the 14th day (Figure 36), the untreated mice had died due to severe metastases, while the treated mice had eliminated the metastases. Figure 37 shows the time-course of IVIS measurements for mice injected with 500,000 T47D-wt cells and T47D-CSCs that received anti-NME7 treatment until the 17th day when antibody treatment was discontinued. As can be seen from the figure, by the 17th day, small clusters of cancer cells remained, which grew larger by the 19th day. By the 21st day, the metastases had spread, and antibody treatment was restarted. As shown in the figure, after the resumption of anti-NME7 antibody treatment, the animals eliminated all metastases and showed no signs of ill health.

[0139] Figure 38 shows the time-course of IVIS for animals injected subcutaneously or intraperitoneally. Antibody injection for animals injected subcutaneously or intraperitoneally with CSCs was also performed with anti-NME7 antibody by s.c. or i.p. In these animals, antibody injection was stopped on the 17th day and not restarted. Figures 39 and 40 show that polyclonal anti-NME7 antibodies generated by immunization with the B3 peptide strain promoted the progression of advanced cancer and metastatic cancer but did not promote normal tissue or low-grade cancer, indicating that 1 out of 100,000 cancer cells or 1 out of 1,000,000 cancer cells would be metastatic cancer cells. Collectively, these data suggest that anti-NME7 antibodies 8F9A5A1, 8F9A4A3, and 5F3A5D4 or 8F9A5A1, or 8F9A4A3, or 5F3A5D4 administered to patients diagnosed with cancer or patients at risk of developing cancer will prevent, suppress, or reverse the formation of cancer metastases.

[0140] In addition to treatment with metastatic animal anti-NME7AB antibody cocktails, we have also shown that monoclonal anti-NME7AB antibodies can be administered individually to prevent and reverse cancer metastasis. In one demonstration experiment, estrogen-releasing pellets were implanted into female nu / nu mice, each approximately 20 g and 8 - 10 weeks old, for 90 days. Cancer cells were made metastatic by culturing them in serum-free medium supplemented with the growth factor NME7 AB for 10 - 15 days. Both adherent and floating cells showed upregulation of metastasis markers and were able to metastasize in animals within 4 - 7 days. In this case, floating cells were harvested on day 11 of in vitro culture and injected into the tail vein of test animals. To test the prevention model, anti-NME7 AB antibody 8F9A4A3 was injected into the tail vein of one group of animals at 15 mg / kg, 24 hours before injection of metastatic cancer cells, and then the same dose was injected approximately every 48 hours. Figures 42A - 42F show photographs of female nu / nu mice injected with 10,000 luciferase-positive T47D metastatic breast cancer stem cells into the tail vein and treated with anti-NME7 AB antibody 4A3 (also known as 8F9A4A3). To image the cancer cells, the luciferase substrate, luciferin, was injected intraperitoneally 10 minutes before imaging with an IVIS imager. Figures 42A - 42C show IVIS photographs of the animals facing downwards. Figures 42D - 42F show IVIS photographs of the animals facing upwards. Figures 42A and 42D show control animals injected with phosphate-buffered saline. Figures 42B and 42E show the prevention model where animals were injected with anti-NME7 AB antibody 4A3 24 hours before injection of metastatic cancer cells and then received a total of 12 antibody injections over 22 days, approximately once a day. Figures 42C and 42F show the reversal model where animals were injected with anti-NME7 AB antibody 4A3 24 hours after injection of metastatic cancer cells and then received a total of 11 antibody injections over 20 days, approximately once a day. As can be seen from the figures, anti-NME7 AB antibody 8F9A4A3 can prevent and reverse established metastases.

[0141] Anti-NME7 antibodies 5A1 and 5D4 were also tested in a metastasis prevention model and were shown to significantly suppress cancer metastasis. Figures 43A - 43F show 10,000 luciferase-positive T47D metastatic breast cancer stem cells injected into the tail vein of female nu / nu mice weighing approximately 20 g each, and treated with anti-NME7 AB antibodies 5A1 (also known as 8F9A5A1), and 5D4 (also known as 5F3A5D4). To image the cancer cells, the luciferase substrate luciferin was injected intraperitoneally 10 minutes prior to imaging with an IVIS imager. Figures 43A - 43C show IVIS images of the animals facing downwards. Figures 43D - 43F show IVIS images of the animals facing upwards. Figures 43A and 43D show control animals injected with phosphate-buffered saline. Figures 43B, 43E, 43C, and 43F show a prevention model where the animals were injected with 15 mg / kg of anti-NME7 AB antibody 24 hours prior to injection of metastatic cancer cells, and then injected with the antibody a total of 12 times over 22 days, approximately once every other day. The images were taken on day 24 or 27. Specifically, mouse #1 in the group treated with antibody 5A1 was imaged on day 27, while mice #2 and #3 were imaged on day 24. The reason is that the animal died on day 26.

[0142] Anti-NME7 AB antibodies 5A1 and 5D4 were also tested in a metastasis reversal model and were shown to significantly suppress established cancer metastasis. In this experiment, the animals were injected with 10,000 T47D metastatic cancer cells mixed with NME7 AB at a final concentration of 32 nM into the tail vein on day 0. Additionally, the animals were injected with more NME7 AB on days 3 and 4, twice, which has been shown in our experiments to form metastases that are more difficult to reverse. The first antibody injection was on day 7. Since the degree of metastasis varied somewhat among the test animals, we needed to confirm that the apparent elimination of metastatic cancer cells was due to anti-NME7 AB treatment. Therefore, we treated the animals using high and low doses alternately. As can be easily seen from Figure 44, high-dose anti-NME7 ABresulted in the removal of metastases and, when complete removal was not achievable, went back and increased further with a lower dosage. This experiment was conducted on all three anti-NME7 AB antibodies, 5A1, 4A3, and 5D4, which were shown to be able to bind to NME7-B3 and suppress cancer metastasis in a concentration-dependent manner. Figures 44A - 44D are photographs of female nu / nu mice injected with 10,000 luciferase-positive T47D metastatic breast cancer stem cells mixed with NME7 AB at a final concentration of 32 nM via the tail vein. The animals were then treated with individual anti-NME7 AB antibodies after being injected with 32 nM of NME7 AB via the tail vein. Figure 44A shows control animals injected with phosphate-buffered saline. Figure 44B shows animals treated with the anti-NME7 AB monoclonal antibody 8F9A5A1. Figure 44C shows animals treated with the anti-NME7 AB monoclonal antibody 8F9A4A3. Figure 44D shows animals treated with the anti-NME7 AB monoclonal antibody 5F3A5D4. Green arrows indicate the antibody dosage (5 - 7 mg / kg) over the indicated period, and red arrows indicate the high dosage (15 mg / kg). As can be seen from the figure, when the antibody is administered at 15 mg / kg, the metastases disappear significantly.

[0143] In addition to demonstrating that the anti-NME7 AB antibodies of the present invention can suppress metastases, we tested their effects on metastases from primary tumors. This would more closely mimic the physiology of cancer metastasis. We used T47D metastatic breast cancer cells, also known as cancer stem cells (CSCs), and mixed them with NME7 ABGenerated by culturing cancer cells in serum-free minimal medium for 10 - 15 days. These T47D CSCs were then transplanted subcutaneously into the right flank of NSG mice implanted with estrogen-releasing pellets for 90 days. Since the transplanted cancer cells are luciferase-positive, after injection of the luciferase substrate, luciferin, the cancer cells emit photons and can be photographed with an IVIS imager to measure and locate the transplanted cancer cells. Figures 45A and 45B show, on day 0, NME7 AB mixed to a final concentration of 32 nM and then mixed 1:1 vol:vol with Matrigel, and 10,000 luciferase-positive T47D metastatic breast cancer stem cells were subcutaneously injected into the right flank of female nu / nu mice. Tumor engraftment was allowed to proceed from day 0 to day 6. The animals were then treated i.v. by tail vein injection of anti-NME7 AB antibody. Control animals were injected with PBS. Figure 45A shows the IVIS photograph of the control animals. Figure 45B shows the IVIS photograph of the animals injected with a cocktail of anti-NME7 AB antibodies 5A1, 4A3, and 5D4 at a total concentration of 15 mg / kg via the tail vein. The antibody or PBS was administered 4 times between day 7 and day 18. As can be seen from the figure, the animals treated with anti-NME7 AB antibody show fewer metastases (blue dots throughout the body) than the control group. Among the treated group, 2 out of 5 animals have primary tumors larger than those in the control group. This is probably because the anti-NME7 AB antibody inhibits the spread of cancer cells and thus remains concentrated in the primary tumor. In this experiment, PCR analysis performed before injection of the cancer cells showed that after 11 days in cultures containing NME7 AB the T47D breast cancer cells upregulated CXCR4 by 109-fold, OCT4 by 2-fold, NANOG by 3.5-fold, and MUC1 by 2.7-fold.

[0144] In another experiment, we tested the effect of the anti-NME7 AB antibody of the present invention on metastasis from the primary tumor to the organs to which breast cancer usually metastasizes. Breast cancer usually metastasizes to the liver, lungs, bones, and brain, in that order. We used T47D metastatic breast cancer cells, NME7 ABGenerated by culturing cancer cells in serum-free minimal medium for 11 days. These T47D CSCs were then transplanted subcutaneously into the right flank of NSG mice implanted with estrogen-releasing pellets for 90 days. Figures 46A - 46P show that on day 0, after being mixed with NME7 at a final concentration of 32 nM, 10,000 luciferase-positive T47D metastatic breast cancer stem cells, mixed with Matrigel at a 1:1 vol:vol ratio, were subcutaneously injected into the right flank of female nu / nu mice. Tumor engraftment was allowed to proceed from day 0 to day 6. The animals were then treated i.v. by tail vein injection of anti-NME7 AB antibody. Control animals were injected with PBS. On day 38, the animals were sacrificed, the livers were harvested, and analyzed by IVIS to detect cancer cells that had metastasized to the liver. Figures 46A and 46B show whole-body IVIS images of control animals injected with PBS only. Figures 46C and 46D show whole-body IVIS images of control animals injected with anti-NME7 AB antibody 5A1. Figures 46E and 46F show whole-body IVIS images of control animals injected with anti-NME7 AB antibody 4A3. Figures 46G and 46H show whole-body IVIS images of control animals injected with anti-NME7 AB antibody 5D4. Figures 46A, 46C, 46E, and 46G are all IVIS images taken 7 days before all treatments. Figures 46B, 46D, 46F, and 46H are IVIS images taken 31 days after anti-NME7 AB antibody treatment or mock treatment. As can be seen from the figures, animals in the PBS control group showed metastases in the whole-body IVIS images (blue dots), while animals treated with anti-NME7 AB antibody did not show metastases. Figures 46I - 46P show photographs and IVIS images of the livers and lungs harvested from the sacrificed animals. Figures 46I, 46K, 46M, and 46O are normal photographs. Figures 46J, 46L, 46N, and 46P are IVIS images that reveal cancer cells that have metastasized there. As can be seen from the figures, anti-NME7 AB antibody... ABThe antibody significantly suppressed metastasis to the liver, which is a primary site for breast cancer metastasis. Figure 46Q is a bar graph of the measured photons emitted and counted by an IVIS imager for the livers collected from control animals and treated animals. As can be seen from the inset graph of the IVIS measurement, the suppression of metastasis to the liver, when the cells were injected into the tail vein, followed the order of metastasis, which is NME7 AB -MUC1 * and also matched the order of the efficacy to disrupt the interaction.

[0145] We performed immunofluorescence imaging of many cancer cell lines to determine whether the cultured cancer cell lines expressed NME7 AB As clearly shown in Figures 47A - 47F and 48A - 48I, each MUC1 - positive cancer cell line we tested was positive for NME7AB, and the binding was membranous, consistent with NME7AB secreted from cancer cells. As a result, it binds to the extracellular domain of MUC1 * . Figures 47A - 47F show photographs of immunofluorescence experiments in which various cancer cell lines were stained for the presence of NME7AB. Figure 47A shows T47D breast cancer cells stained with anti - NME7AB antibody 5D4 at various concentrations. Figure 47B shows ZR - 75 - 1 breast cancer cells (also known as 1500s) stained with anti - NME7AB antibody 5D4 at various concentrations. Figure 47C shows H1975 non - small cell lung cancer cells stained with anti - NME7AB antibody 5D4 at various concentrations. Figure 47D shows H292 non - small cell lung cancer cells stained with anti - NME7 AB antibody 5D4 at various concentrations. Figure 47E shows HPAFII pancreatic cancer cells stained with anti - NME7 AB antibody 5D4 at various concentrations. Figure 47F shows DU145 prostate cancer cells stained with anti - NME7AB antibody 5D4 at various concentrations. As can be seen from the figures, all the cancer cell lines we tested showed strong membranous staining for NME7AB. The monoclonal antibody used in these experiments was 5D4. In parallel, the same cell lines were stained with NME7AB antibodies 5A1 and 4A3, and the same results were obtained.

[0146] Figures 48A to 48I show photographs of immunofluorescence experiments in which various human lung cancer cell lines were stained for the presence of NME7 AB . Figures 48A to 48C show H1975 non-small cell lung cancer cells, which are adenocarcinomas, stained with anti-NME7 AB antibody 5D4 at various concentrations. Figure 48A is an overlay of DAPI and anti-NME7 AB staining. Figure 48B shows anti-NME7 AB staining alone. Figure 48C is an enlarged view of the overlay of DAPI and anti-NME7 AB staining. Figures 48D to 48F show H292 non-small cell lung cancer cells, which are pulmonary mucinous epidermoid carcinomas, stained with anti-NME7 AB antibody 5D4 at various concentrations. Figure 48D is an overlay of DAPI and anti-NME7 AB staining. Figure 48E shows anti-NME7 AB staining alone. Figure 48F is an enlarged view of the overlay of DAPI and anti-NME7 AB staining. Figures 48G to 48I show H358 non-small cell lung cancer cells, which are metastatic bronchioloalveolar carcinomas, stained with anti-NME7 AB antibody 5D4 at various concentrations. Figure 48G is an overlay of DAPI and anti-NME7 AB staining. Figure 48H shows anti-NME7 AB staining alone. Figure 48I is an enlarged view of the overlay of DAPI and anti-NME7 AB staining.

[0147] In addition, culturing these cell lines in serum-free medium containing NME7 AB further increased the expression of their stem cell and metastasis markers. In particular, here, the non-adherent cells, called floating cells, have a higher expression of stem cell and metastasis markers than their adherent counterparts. Figures 49A to 49I show NME7 ABPCR graphs of cancer cell lines: breast T47D, lung H1975, lung H358, and pancreatic HPAFII, before and after culture are shown. Figure 49A is the measured breast cancer metastasis marker CXCR4. Figure 49B is the measured stem cell marker OCT4. Figure 49C is the measured metastasis marker ALDH1. Figure 49D is the measured stem cell marker SOX2. Figure 49E is the measured stem cell marker NANOG. Figure 49F is the measured metastasis marker CDH1 (also known as E-cadherin). Figure 49G is the measured metastasis marker CD133. Figure 49H is the measured stem cell marker ZEB2. Figure 49I is the measured stem, cancer, and metastasis marker MUC1. Suspended cells (also known as tumor spheres) can grow independently on a substrate and show increased metastasis markers compared to adherent cells. Animals injected with cancer stem cells are NME7 AB Animals injected with proliferating suspended cells. As can be seen from the figure, metastasis markers, stem cell markers, or markers of epithelial-mesenchymal transition (EMT) are NME7 AB have increased after culture in, indicating a conversion to a higher metastatic state. Figure 50 is NME7 AB IVIS images on day 6 of NSG mice injected with 10,000 H358 lung cancer parental cells or H358 cells into the tail vein after 10 - 12 days in culture containing NME7 AB As can be seen from the figure, NCI-H358 lung cancer cells grown in NME7 AB showed a significantly enhanced metastatic ability compared to the parental cells which are reported to be metastatic cells themselves. The functional increase in metastasis at day 6 from 10,000 NCI-H358 NME7 AB metastatic cancer stem cells is consistent with Figure 49 and indicates that H358 significantly increases the expression of metastasis markers after culture in NME7

[0148] Figure 51 is the dimer NM23-H1 (also known as NME1), or NME7 ABThe PCR graphs of MUC1-negative prostate cancer cell line PC3 before and after 2 or 3 passages in culture are shown. The graphs show the fold differences of stem cell, cancer cell markers as well as metastasis markers. As can be seen from the figure, NME1 or NME7 AB Repeated culturing in induces upregulation of stem, cancer and metastasis markers, but also results in 5- to 8-fold upregulation of MUC1 expression.

[0149] Collectively, these data demonstrate that NME7 lacking the DM10 domain is selected by cancer cells, binds to the extracellular domain of MUC1 lacking the tandem repeat domain, and as a result NME7 * dimerizes the extracellular domain, which leads to increased cancer cell proliferation and increased cancer cell metastatic ability. NME7 AB and MUC1 * It stands to reason that an antibody that disrupts the interaction between the extracellular domains of NME7 AB and MUC1 * can inhibit cancer cell proliferation and cancer metastasis. Here, we show that an anti-NME7 AB antibody that inhibits the interaction between the extracellular domains of NME7 AB and MUC1 actually inhibits cancer cell proliferation and cancer metastasis. Therefore, the anti-NME7

[0150] antibody can be administered to patients diagnosed with cancer or metastasis, or patients at risk of developing cancer or metastasis, for the treatment or prevention of cancer. AB NME1 is expressed in the cytoplasm of all cells and is lethal when knocked out. Importantly, the NME1 A domain has high sequence homology to the NME7 A domain. Therefore, the anti-NME7 AB antibody for therapeutic use is important in that it binds to NME7 AB or NME7-X1 but not to NME1. In one aspect of the invention, antibodies that are thought to be optimal for therapeutic use are selected for their ability to bind to peptides that are specific for NME7 AB or NME7-X1 and are not present in the NME1 sequence. Figures 6-9 list peptides specific for NME7

[0151] In a preferred embodiment, for the treatment or prevention of cancer or cancer metastasis, antibodies suitable for administration to a patient are selected from the group of antibodies that bind to the NME7 B3 peptide. In a more preferred embodiment, for the treatment or prevention of cancer or cancer metastasis, antibodies suitable for administration to a patient are selected from the group of antibodies that bind to the NME7 B3 peptide and NME7 AB but do not bind to NME1. Examples of antibodies suitable for therapeutic use for the treatment or prevention of cancer or cancer metastasis that have demonstrated such anti-cancer and anti-metastatic activity in vitro and in vivo include anti-NME7 antibodies 5A1, 4A3, and 5D4. These are merely examples, and antibodies generated as described herein and antibodies selected as described herein will have the same anti-cancer and anti-metastatic activity. Such antibodies can be full antibodies or fragments thereof, including scFv or antibody mimetics, in which the variable domain of the antibody is incorporated into a protein scaffold that mimics the antibody. The antibody can be of human or non-human origin, including mouse, camelids, llama, human or humanized, and can be monoclonal, polyclonal, scFv or fragments thereof.

[0152] Anti-NME7 antibodies for the treatment or prevention of cancer or metastasis can be used in many different therapeutic formats. For example, any of the antibodies described herein, or fragments thereof, can be administered to a patient as an independent antibody or antibody fragment, or conjugated to a toxin such as an antibody-drug conjugate (ADC), or incorporated into a bispecific antibody or a BiTE (bispecific T cell engager), or engineered to be incorporated into a chimeric antigen receptor (CAR) or expressed by cells that also express the CAR. The cells can be immune cells, T cells, NK cells or stem cells or progenitor cells, which can then differentiate into T cells or NK cells.

[0153] Any of the antibodies described herein, or fragments thereof, are NME7, which is thought to be an indicator of sensitivity to cancer or cancerAB Alternatively, it can be used as a diagnostic agent for detecting body fluids, cells, tissues or body specimens for the presence of NME7-X1. Diagnostic antibodies can be conjugated to imaging agents, nucleic acid tags, and can be of any species including camels, and can be used for whole body applications or for body fluids such as blood, cells, or tissues, or in vitro, in vivo, or during surgery.

[0154] The selection criteria for therapeutically or diagnostically useful anti-NME7 antibodies depend on the format or modality of the therapy or diagnosis in which the antibody is incorporated. When an antibody or antibody fragment is administered to a patient as a stand-alone agent for the treatment or prevention of cancer or cancer metastasis, the antibody should: i) bind to NME7 AB or NME7-X1 but not to NME1; ii) bind to the PSMGFR peptide; iii) bind to the N-10 peptide; and iv) AB be selected for its ability to disrupt the interaction between NME7 * or NME7-X1 and the extracellular domain of MUC1, AB or the interaction between NME7 AB or NME7-X1 and N-10. The antibody can also be selected for its ability to bind to the NME7 B3 peptide. This therapeutic format also includes cells engineered to express a CAR and a selected anti-NME7 antibody.

[0155] Other formats require other selection criteria for anti-NME7 antibodies. When an anti-NME7 antibody is incorporated into an ADC, the ADC must be internalized by the target cells in order to induce target cell death. Recall that NME7 AB or NME7-X1 binds to the extracellular domain of MUC1. * If the antibody disrupts the binding of NME to the extracellular domain of MUC1, * the toxin-conjugated antibody will not be internalized and the cells will not be killed. Similarly, when an anti-NME7 antibody is incorporated into a CAR or BiTE, NME7 ABOr the interaction between NME7-X1 cannot be disrupted, or immune cells will no longer be able to direct cytotoxic agents towards cancer cells. When the anti-NME7 antibody is used as a diagnostic agent, NME7 AB Or the interaction between NME7-X1 cannot be disrupted, or the antibody and associated labels will be washed away. Thus, for ADC, CAR T, or CAR-NK, BiTE or diagnostic applications, the anti-NME7 antibody is such that i) it binds to NME7 AB Or binds to NME7-X1 but not to NME1; ii) binds to the PSMGFR peptide; iii) binds to the N-10 peptide; and iv) binds to the extracellular domain of MUC1 * Or the interaction between NME7 AB Or between NME7-X1 and the N-10 peptide without disrupting the interaction, and is selected for its ability to bind to NME7 AB Or NME7-X1. The antibody may also be selected for its ability to bind to the NME7 B3 peptide.

[0156] In one aspect of the invention, the cell is engineered to express the anti-NME7 AB Antibody or a fragment thereof of the invention. The cell may be an immune cell such as a T cell, NK cell, or may be a stem cell or progenitor cell, which can differentiate into more mature immune cells such as T cells or NK cells. In a preferred embodiment, the cell engineered to express the anti-NME7 AB Antibody is also engineered to express a chimeric antigen receptor (CAR). In a preferred embodiment, the CAR recognizes a tumor-associated antigen. In a preferred embodiment, the CAR targets MUC1 * . In a more preferred embodiment, the CAR is directed to the tumor by the anti-MUC1 * Antibody MNC2. In another aspect of the invention, the cell engineered to express the CAR is also engineered to express the anti-NME7 antibody in an inducible manner. In one example, the nucleic acid encoding the anti-NME7 AB Antibody is inserted into the Foxp3 enhancer or promoter. In another example, the anti-NME7 ABThe antibody is present in the NFAT induction system. In one aspect, the NFAT induction system incorporates an NFATc1 response element inserted upstream of the anti-NME7 AB antibody sequence. They can be inserted into the IL2 promoter, the Foxp3 enhancer or promoter, or other suitable promoters or enhancers.

[0157] In another aspect of the invention, NME7 AB or a peptide specific to NME7-X1 is incorporated into a substance used to immunize or vaccinate humans against cancer or cancer metastasis. In a preferred embodiment, the peptide comprises all or part of the NME7 B3 peptide, which may be an NME7 B3 peptide having a Cys-14-Ser mutation.

[0158] Another aspect of the invention includes a method of generating an anti-NME7 AB antibody in a host animal, wherein the animal is immunized with the NME7 B3 peptide. In a preferred embodiment, the NME7 B3 peptide has cysteine 14 mutated to serine (SEQ ID NO: 169) to avoid the formation of disulfide bonds that suppress the generation of NME7-specific antibodies.

[0159] Another aspect of the invention includes a method of generating cells with enhanced metastatic ability, which includes culturing cells with NME7 AB or NME7-X1. These cells can then be used in many aspects of drug discovery.

[0160] Another aspect of the invention includes cells that are also engineered to express anti-NME7 AB or NME7-X1. NME7 AB or NME7-X1 is a human sequence. Their expression can be inducible. In one aspect, the cells are eggs, which can grow into animals that are transgenic animals capable of expressing human NME7 AB or NME7-X1.

[0161] NME7 is MUC1 *It binds to the extracellular domain of growth factor receptors and dimerizes them. Tissue studies have shown that MUC1 * Here, we show that NME7 expression increases with increasing tumor grade and metastasis (Fig. 39-Fig. 41). Here, we show that NME7 and MUC1 expression increase with increasing tumor grade and metastasis (Fig. 39-Fig. 41). * Antibodies that inhibit this interaction have been shown to inhibit tumor growth and metastasis.

[0162] Another NME family member is MUC1 * They can bind to and dimerize the extracellular domain of growth factor receptors. For example, we have shown that NME1, NME2, and NME6 exist as dimers and that they bind to MUC1. * We showed that it binds to the extracellular domains and dimerizes them. AB and NME7-X1, MUC1 * They have two domains that can bind to the extracellular domain, so that they form MUC1 as monomers. * NME7 dimerizes and activates growth factor receptors. We have now shown that anti-NME7 antibodies inhibit cancer and cancer metastasis. Similarly, antibodies or antibody mimetics that bind to these other NME proteins can be anti-cancer or anti-metastasis therapeutics that can be administered to patients diagnosed with cancer or metastasis or at risk of developing cancer or metastasis. In one embodiment of the invention, the antibodies that can be used therapeutically for the treatment of cancer or metastasis are antibodies that bind to NME1, NME2, NME3, NME4, NME5, NME6, NME7, NME8, NME9 or NME10. In one embodiment of the invention, the therapeutic antibody or antibody mimetics inhibit the binding of the NME protein to its cognate growth factor receptor. In one embodiment of the invention, the therapeutic antibody or antibody mimetics inhibit the binding of the NME protein to MUC1 * In another embodiment of the invention, the therapeutic antibody or antibody mimetic binds to a peptide derived from NME1, NME2, NME3, NME4, NME5, NME6, NME7, NME8, NME9 or NME10, wherein the peptide is homologous to the NME7 A1, A2, B1, B2 or B3 peptide.

[0163] The following is a sequence alignment showing the homology and identity alignment between NME7 and other NME family members. The underlined or underlined + bold sequences correspond to NME7 peptides A1 (SEQ ID NO: 141), A2 (SEQ ID NO: 142), B1 (SEQ ID NO: 143), B2 (SEQ ID NO: 144) or B3 (SEQ ID NO: 145).

[0164] Nucleoside diphosphate kinase 7 isoform a [Homo sapiens] (Hu_7)

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[0165] As an example, an antibody or antibody mimetic that binds to a peptide homologous to NME7 (the "homologous peptide"), in particular a peptide homologous to the A1, A2, B1, B2 or B3 peptide, can be administered to a patient diagnosed with cancer or cancer metastasis, or a patient at risk of developing cancer or cancer metastasis.

[0166] A peptide homologous to the A1, A2, B1, B2 or B3 peptide A peptide homologous to the A1, A2, B1, B2 or B3 peptide may include, but is not limited to: NME2A1 (amino acids) RASEEHLKQHYIDLKD (SEQ ID NO: 247)

[0167] NME2A2 (amino acids) PADSKPGT (SEQ ID NO: 248)

[0168] NME2B1 (amino acids) QKGFRLVAMKFLRASEEHLK (SEQ ID NO: 249)

[0169] NME2B2 (amino acids) IDLKDRPFPGLVKY (SEQ ID NO: 250)

[0170] NME2B3 (amino acids) GDFCIQVGRNIIHGSDSVKSAEKEISLWF (SEQ ID NO: 251)

[0171] NME3A1 (Amino acid) QASEELLREHYVELRE (SEQ ID NO: 252)

[0172] NME3A1 (Amino acid) PGDATPGT (SEQ ID NO: 253)

[0173] NME3B1 (Amino acid) RKGFKLVALKLVQASEELLR (SEQ ID NO: 254)

[0174] NME3B2 (Amino acid) VELRERPFYSRLVKY (SEQ ID NO: 255)

[0175] NME3B3 (Amino acid) GDFCVEVGKNVIHGSDSVESAQREIALWF (SEQ ID NO: 256)

[0176] NME4A1 (Amino acid) QAPESVLAEHYQDLRR (SEQ ID NO: 257)

[0177] NME4A2 (Amino acid) SAEAAPGT (SEQ ID NO: 258)

[0178] NME4B1 (Amino acid) RRGFTLVGMKMLQAPESVLA (SEQ ID NO: 259)

[0179] NME4B2 (Amino acid) QDLRRKPFYPALIRY (SEQ ID NO: 260)

[0180] NME4B3 (Amino acid) GDFSVHISRNVIHASDSVEGAQREIQLWF (SEQ ID NO: 261)

[0181] NME5A1 (Amino acid) RLSPEQCSNFYVEKYG (SEQ ID NO: 262)

[0182] NME5A2 (Amino acid) SLVAKETHPDS (SEQ ID NO: 263)

[0183] NME5B1 (Amino acid) RSGFTIVQRRKLRLSPEQCS (SEQ ID NO: 264)

[0184] NME5B2 (Amino acid) VEKYGKMFFPNLTAY (SEQ ID NO: 265)

[0185] NME5B3 (Amino acid) AIYGTDDLRNALHGSNDFAAAEREIRFMF (SEQ ID NO: 266)

[0186] NME6A1 (Amino acid) LWRKEDCQRFYREHEG (SEQ ID NO: 267)

[0187] NME6A2 (Amino acid) VFRARHVAPDS (SEQ ID NO: 268)

[0188] NME6B1 (Amino acid) SNKFLIVRMRELLWRKEDCQ (SEQ ID NO: 269)

[0189] NME6B2 (Amino acid) REHEGRFFYQRLVEF (SEQ ID NO: 270)

[0190] NME6B3 (Amino acid) GSFGLTDTRNTTHGSDSVVSASREIAAFF (SEQ ID NO: 271)

[0191] NME8A1 (Amino acid) VLSEKEAQALCKEYEN (SEQ ID NO: 272)

[0192] NME8A2 (Amino acid) VEEAIEYFPES (SEQ ID NO: 273)

[0193] NME8A3 (Amino acid) FLTPEQIEKIYPKVTG (SEQ ID NO: 274)

[0194] NME8A4 (Amino acid) PEEAKLLSPDS (SEQ ID NO: 275)

[0195] NME8A5 (Amino acid) VLTEEQVVNFYSRIAD (SEQ ID NO: 276)

[0196] NME8B1 (Amino acid) EAGFDLTQVKKMFLTPEQIE (SEQ ID NO: 277)

[0197] NME8B2 (Amino acid) PKVTGKDFYKDLLEM (SEQ ID NO: 278)

[0198] NME8B3 (Amino acid) AQFGISKLKNIVH (SEQ ID NO: 279)

[0199] NME8B4 (Amino acid) DEDFKILEQRQVVLSEKEAQ (SEQ ID NO: 280)

[0200] NME8B5 Amino acid KEYENEDYFNKLIEN (SEQ ID NO: 281)

[0201] NME8B6 Amino acid AQFAMDSLPVNQLYGSDSLETAEREIQHFF (SEQ ID NO: 282)

[0202] NME8B7 Amino acid KAGFIIEAEHKTVLTEEQVV (SEQ ID NO: 283)

[0203] NME8B8 Amino acid SRIADQCDFEEFVSF (SEQ ID NO: 284)

[0204] NME9A1 Amino acid TMTEAEVRLFY (SEQ ID NO: 285)

[0205] NME9B1 Amino acid EAGFEILTNEERTMTEAEVR (SEQ ID NO: 286)

[0206] NME10A1 Amino acid SMKAEDAQRVFREK (SEQ ID NO: 287)

[0207] NME10A2 Amino acid GQRQKSSDES (SEQ ID NO: 288)

[0208] NME10A3 Amino acid IQDCENCNIYIFDHSA (SEQ ID NO: 289)

[0209] NME10B1 ELAFQFKDAGLSIFNNTWSNIH (SEQ ID NO: 290)

[0210] In some cases, peptides from other NME proteins can be made homologous to the NME7 A1, A2, B1, B2, or B3 peptides by shifting the frame or by extending the NME7 peptide such that the extended peptide gives rise to an antibody that suppresses cancer or cancer metastasis. As another example, an antibody or antibody mimetic that binds to an extended peptide (an "extended peptide") homologous to NME7 can be administered to a patient diagnosed with cancer or cancer metastasis or a patient at risk of developing cancer or cancer metastasis.

[0211] An extended peptide homologous to the A1, A2, B1, B2, or B3 peptides

[0212] Peptides homologous to the A1, A2, B1, B2, or B3 peptides that are extended peptides may include, but are not limited to:

[0213] NME2A1 (amino acids) RASEEHLKQHYIDLKDRPFFPGL (SEQ ID NO: 291)

[0214] NME2A2 (amino acids) LGETNPADSKPGTIRGDF (SEQ ID NO: 292)

[0215] NME2B1 (amino acids) GLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHY (SEQ ID NO: 293)

[0216] NME2B2 (amino acids) YIDLKDRPFFPGLVKYMNSGPVVAM (SEQ ID NO: 294)

[0217] NME2B3 (amino acids) PGTIRGDFCIQVGRNIIHGSDSVKSAEKEISLWF (SEQ ID NO: 295)

[0218] NME3A1 (Amino acid) LKLVQASEELLREHYVELRERPFYSRL (SEQ ID NO: 296)

[0219] NME3A1 (Amino acid) LIGATDPGDATPGTIRGDF (SEQ ID NO: 297)

[0220] NME3B1 (Amino acid) LVGEIVRRFERKGFKLVALKLVQASEELLRE (SEQ ID NO: 298)

[0221] NME3B2 (Amino acid) EHY-VELRERPFYSRLVKYMGSGPVVAM (SEQ ID NO: 299)

[0222] NME3B3 (Amino acid) PGTIRGDFCVEVGKNVIHGSDSVESAQREIALWF (SEQ ID NO: 300)

[0223] NME4A1 (Amino acid) GFTLVGMKMLQAPESVLAEHYQDLRRKPF (SEQ ID NO: 301)

[0224] NME4A2 (Amino acid) GHTDSAEAAPGTIRGDF (SEQ ID NO: 302)

[0225] NME4B1 (Amino acid) LVGDVIQRFERRGFTLVGMKMLQAPESVLAEHY (SEQ ID NO: 303)

[0226] NME4B2 (Amino acid) EHYQDLRRKPFYPALIRYMSSGPVVAM (SEQ ID NO: 304)

[0227] NME4B3 (Amino acid) PGTIRGDFSVHISRNVIHASDSVEGAQREIQLWF (SEQ ID NO: 305)

[0228] NME5A1 (Amino acid) GFTIVQRRKLRLSPEQCSNFYVEKYGKMFF (SEQ ID NO: 306)

[0229] NME5A2 (Amino acid) LLGPNNSLVAKETHPDSLRAIYGTD (SEQ ID NO: 307)

[0230] NME5B1 (Amino acid) IQDIILRSGFTIVQRRKLRLSPEQCSNFY (SEQ ID NO: 308)

[0231] NME5B2 (Amino acid) FYVEKYGKMFFPNLTAYMSSGPLVAM (SEQ ID NO: 309)

[0232] NME5B3 (Amino acid) PDSLRAIYGTDDLRNALHGSNDFAAAEREIRFMF (SEQ ID NO: 310)

[0233] NME6A1 (Amino acid) FLIVRMRELLWRKEDCQRFYREHEGRFFYQRL (SEQ ID NO: 311)

[0234] NME6A2 (Amino acid) LMGPTRVFRARHVAPDSIRGSFG (SEQ ID NO: 312)

[0235] NME6B1 (Amino acid) ILSNKFLIVRMRELLWRKEDCQRFY (SEQ ID NO: 313)

[0236] NME6B2 (Amino acid) FYREHEGRFFYQRLVEFMASGPIRA (SEQ ID NO: 314)

[0237] NME6B3 (Amino acid) ARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFF (SEQ ID NO: 315)

[0238] NME8A1 (Amino acid) FKILEQRQVVLSEKEAQALCKEYENEDYFNKLI (SEQ ID NO: 316)

[0239] NME8A2 (Amino acid) WKQLLGPRTVEEAIEYFPESLCAQFAMD (SEQ ID NO: 317)

[0240] NME8A3 (Amino acid) AGFDLTQVKKMFLTPEQIEKIYPKVTGKDFYKDL (SEQ ID NO: 318)

[0241] NME8A4 (Amino acid) EWRRLMGPTDPEEAKLLSPDSIRAQFG (SEQ ID NO: 319)

[0242] NME8A5 (Amino acid) KAGFIIEAEHKTVLTEEQVVNFYSRIADQCDFEE (SEQ ID NO: 320)

[0243] NME8B1 (Amino acid) ILKIVKEAGFDLTQVKKMFLTPEQIEKIY (SEQ ID NO: 321)

[0244] NME8B2 (Amino acid) YPKVTGKDFYKDLLEMLSVGP (SEQ ID NO: 322)

[0245] NME8B3 (Amino acid) DPEEAKLLSPDSIRAQFGISKLKNIVH (SEQ ID NO: 323)

[0246] NME8B4 (Amino acid) LRIIKDEDFKILEQRQVVLSEKEAQ (SEQ ID NO: 324)

[0247] NME8B5 (Amino acid) KEYENEDYFNKLIENMTSGPSLA (SEQ ID NO: 325)

[0248] NME8B6 (Amino acid) PESLCAQFAMDSLPVNQLYGSDSLETAEREIQHFF (SEQ ID NO: 326)

[0249] NME8B7 (Amino acid) IKRKITKAGFIIEAEHKTVLTEEQVVNFY (SEQ ID NO: 327)

[0250] NME8B8 (Amino acid) FYSRIADQCDFEEFVSFMTSG (SEQ ID NO: 328)

[0251] NME9A1 (Amino acid) AGFEILTNEERTMTEAEVRLFY (SEQ ID NO: 329)

[0252] NME9B1 (Amino acid) IIMKIQEAGFEILTNEERTMTEAEVRLFY (SEQ ID NO: 330)

[0253] NME10A1 (amino acid) GFFLVQTKEVSMKAEDAQRVFREKAP (SEQ ID NO: 331)

[0254] NME10A2 (amino acid) EANRSIVPISRGQRQKSSDESCLVVLFAGD (SEQ ID NO: 332)

[0255] NME10A3 (amino acid) IQDCENCNIYIFDHSA (SEQ ID NO: 333)

[0256] NME10B1 ELAFQFKDAGLSIFNNTWSNIHDFTPVDCT (SEQ ID NO: 334)

[0257] Some NME proteins perform functions required for normal cell growth or development. For example, NME1 is thought to be required for normal cell function. Other NME proteins have catalytic domains whose functions are required in normal cells or tissues. In these cases, therapeutic antibodies can be selected based on their ability to bind to the targeted cancer-associated NME but not to the non-targeted NME. For example, the anti-NME7 antibodies, 8F9A5A1, 8F9A4A3, and 5F3A5D4 presented herein were selected for their ability to bind to NME7 AB but not to NME1; they were further selected based on their ability to suppress cancer and cancer metastasis.

[0258] In another aspect of the invention, anti-NME7 antibodies, antibody fragments, e.g., scFv, or fragments of antibody mimetics are incorporated into chimeric antigen receptors (CARs) engineered to be expressed in immune cells. The immune cells are anti-NME7 CAR, anti-MUC1 *CAR, or both, can be engineered to be expressed. One of the CARs can be expressed from an inducible promoter. Alternatively, the immune cells can be engineered to express a CAR such as * and an inducible anti-NME7 antibody or antibody fragment. In some cases, the inducible promoter can contain an NFAT response element. In one aspect, these engineered species are expressed in T cells, NK cells or dendritic cells. The immune cells can be obtained from a patient or a donor. In some cases, immune molecules such as MHC, checkpoint inhibitors or receptors for checkpoint inhibitors are mutated or removed using, for example, CrisPR or CrisPR-like technologies. In another aspect, ITAM molecules, Fos, or Jun are mutated or genetically excised in immune cells derived from a patient or a donor using Talen, Sleeping Beauty, CrisPR or CrisPR-like technologies. * CARs such as * and inducible anti-NME7 antibodies or antibody fragments can be engineered to be expressed. In some cases, the inducible promoter can contain an NFAT response element. In one aspect, these engineered species are expressed in T cells, NK cells or dendritic cells. The immune cells can be obtained from a patient or a donor. In some cases, immune molecules such as MHC, checkpoint inhibitors or receptors for checkpoint inhibitors are mutated or removed using, for example, CrisPR or CrisPR-like technologies. In another aspect, ITAM molecules, Fos, or Jun are mutated or genetically excised in immune cells derived from a patient or a donor using Talen, Sleeping Beauty, CrisPR or CrisPR-like technologies.

[0259] In one aspect of the invention, the anti-NME7 antibody or antibody mimetic for use in a CAR T format is selected from the group of antibodies or antibody mimetics that are specific for NME7 but do not disrupt the binding of NME7 to the extracellular domain of MUC1 * Thus, the anti-NME7 antibody or antibody mimetic that targets CAR T to the tumor not only simply deprives the receptor of the ligand, but in so doing, the T cells will not be able to inject granzyme B into the target cancer cells. Such antibodies or antibody mimetics are generated by immunizing animals with NME7 peptides such as NME7 peptide A1, A2, B1, B2 or B3, or selected by their ability to bind to NME7 peptides A1, A2, B1, B2 or B3. Antibodies or antibody mimetics can be screened for their ability to specifically bind to NME7 but not to NME1 or NME2, and for their inability to disrupt the binding between NME7 and the extracellular domain of MUC1 * For example, in an ELISA device, the PSMGFR peptide is immobilized on the surface. Labeled NME7 AB is added to surface-immobilized MUC1 *Bound to the extracellular domain, NME7 AB Detection of the label is measured in the presence or absence of the test antibody or antibody mimetic. In one aspect of the invention, NME7 AB and surface-immobilized MUC1 * An antibody that does not reduce the binding between the extracellular domain peptide is selected as the antibody to be incorporated into the CAR, engineered to be expressed in immune cells, and then administered to a patient for the treatment or prevention of cancer or cancer metastasis.

[0260] In one aspect of the invention, the anti-NME7 antibody or fragment thereof is administered to a patient diagnosed with cancer or cancer metastasis, or a patient at risk of developing cancer or cancer metastasis. In one aspect, the anti-NME7 antibody or antibody fragment binds to the NME peptides discussed above, particularly in the sections "Peptides homologous to the A1, A2, B1, B2 or B3 peptides" and "Extended peptides homologous to the A1, A2, B1, B2 or B3 peptides".

[0261] In another aspect, the antibody, antibody fragment or antibody mimetic binds to an NME7-derived peptide selected from A1, A2, B1, B2 or B3 (SEQ ID NOs: 141-145). In yet another aspect, the antibody, antibody fragment or antibody mimetic binds to an NME7 peptide that includes most or all of the B3 peptide. In one aspect of the invention, the anti-NME7 antibody, antibody fragment or antibody mimetic comprises a sequence derived from the variable domain of the following anti-NME7 antibodies 8F9A4A3 (SEQ ID NOs: 1001-1015), 8F9A5A1 (SEQ ID NOs: 1016-1030), or 8H5H5G4 (SEQ ID NOs: 1031-1045).

[0262] Anti-NME7 B3 peptide monoclonal antibody 8F9A4A3 heavy chain variable region sequence mouse (DNA) Gtccagctgcaacagtctggacctgaactggtgaagcctggggcttcagtgaagatatcctgcaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggcaccactctcacagtctcctca(SEQ ID NO: 335) (Amino acid) VQLQQSGPELVKPGASVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSLYVFYFDYWGQGTTLTVSS(SEQ ID NO: 1001)

[0263] IGHV1-24 * 01 V-region sequence human (closest match hu antibody sequence) (DNA) Caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaaca(SEQ ID NO: 336) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT (SEQ ID NO: 1002)

[0264] Human (closest match hu antibody sequence) IGHJ4*01 J region sequence (DNA) tactttgactactggggccaaggaaccctggtcaccgtctcctca (SEQ ID NO: 337) (Amino acid) YFDYWGQGTLVTVSS (SEQ ID NO: 1003)

[0265] Humanized heavy chain variable sequence (SEQ ID NO: 1001 + SEQ ID NO: 1002 + SEQ ID NO: 1003) Humanized 8F9A4A3 heavy chain variable region sequence (DNA) caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggaaacacattcactgaatacaccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggaaccctggtcaccgtctcctca (SEQ ID NO: 338) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1004)

[0266] Humanized heavy chain variable array (codon-optimized version of 1004) Humanized 8F9A4A3 heavy chain variable region array (codon-optimized) (DNA) caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggacttgaatggatgggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggccgcgtgaccatgaccgaggacacaagcacagacaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttct (SEQ ID NO: 339) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1005)

[0267] Humanized heavy chain variable array (the "modified" SEQ ID NO: 1005 sequence, the modification means that certain amino acids considered important for binding or structure have been reverted to the mouse sequence) Modified humanized 8F9A4A3 heavy chain variable region sequence (DNA) caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggaaacacattcactgaatacaccatgcactgggtgcgacaggctcctggaaaagggcttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaagtcaccctgaccgtggacacatctagcagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggaaccctggtcaccgtctcctca(SEQ ID NO: 340) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS(SEQ ID NO: 1006)

[0268] Humanized heavy chain variable sequence (codon optimization of SEQ ID NO: 1006) Modified humanized 8F9A4A3 heavy chain variable region sequence (codon optimization) (DNA) caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttct(SEQ ID NO: 341) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS(SEQ ID NO: 1007)

[0269] 8F9A4A3 Mouse light chain variable region sequence (DNA) gaaacaactgtgacccagtctccagcatccctgtccatggctataggagaaaaagtcaccatcagatgcataaccagcactgatattgatgatgatatgaactggtaccagcagaagccaggggaacctcctaagctccttatttcagaaggcaatactcttcgtcctggagtcccatcccgattctccagcagtggctatggtacagattttgtttttacaattgaaaacatgctctcagaagatgttgcagattactactgtttgcaaagtgataacttgcctctcacgttcggctcggggacaaagttggaaataaaacgg(SEQ ID NO: 342) (Amino acid) ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGSGTKLEIKR (SEQ ID NO: 1008)

[0270] Human (closest match hu antibody sequence) IGKV5-2 * 01 V region sequence (DNA) gaaacgacactcacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaacatctcctgcaaagccagccaagacattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattttcattattcaagaagctactactctcgttcctggaatcccacctcgattcagtggcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgt (SEQ ID NO: 343) (Amino acid) ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQKPGEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFC (SEQ ID NO: 1009)

[0271] Human (closest match hu antibody sequence) IGKJ4 * 02 J region sequence (DNA) ctcacgttcggcggagggaccaaggtggagatcaaa (SEQ ID NO: 344) (Amino acid) LTFGGGTKVEIK (SEQ ID NO: 1010)

[0272] Humanized light chain variable sequence (SEQ ID NO: 1008 + SEQ ID NO: 1009 + SEQ ID NO: 1) Humanized 8F9A4A3 light chain variable region sequence (DNA) gaaacgacactcacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaacatctcctgcataaccagcactgatattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattttcattattcaagaaggcaatactcttcgtcctggaatcccacctcgattcagtggcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtttgcaaagtgataacttgcctctcacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 345) (Amino acid) ETTLTQSPAFMSATPGDKVNISCITSTDIDDDMNWYQQKPGEAAIFIIQEGNTLRPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (SEQ ID NO: 1011)

[0273] Humanized light chain variable sequence (codon-optimized version of SEQ ID NO: 1011) Modified humanized 8F9A4A3 light chain variable region sequence (codon-optimized) (DNA) Gagacaaccctgacacagagccctgccttcatgtctgccacacctggcgacaaagtgaacatcagctgcatcaccagcaccgacatcgacgacgacatgaactggtatcagcagaagcctggcgaggccgccatcttcatcatccaagagggcaacacactgcggcctggcatccctcctagattttctggcagcggctacggcaccgacttcaccctgaccatcaacaacatcgagagcgaggacgccgcctactacttctgcctgcaaagcgacaacctgcctctgacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 346) (Amino acid) ETTLTQSPAFMSATPGDKVNISCITSTDIDDDMNWYQQKPGEAAIFIIQEGNTLRPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (SEQ ID NO: 1012)

[0274] Humanized light chain variable sequence (the "modified" SEQ ID NO: 1012 sequence, the modification means that certain amino acids considered important for binding or structure have been reverted to the mouse sequence) Modified humanized 8F9A4A3 light chain variable region sequence (DNA) gaaacgacagtgacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaccatctcctgcataaccagcactgatattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattctgctgattagcgaaggcaatactcttcgtcctggaatcccacctcgattcagtagcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtttgcaaagtgataacttgcctctcacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 347) (Amino acid) ETTVTQSPAFMSATPGDKVTISCITSTDIDDDMNWYQQKPGEAAILLISEGNTLRPGIPPRFSSSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (SEQ ID NO: 1013)

[0275] Humanized light chain variable sequence (codon optimization of SEQ ID NO: 1013) Modified humanized 8F9A4A3 light chain variable region sequence (codon optimization) (DNA) gagacaaccgtgacacagagccctgccttcatgtctgccacacctggcgacaaagtgaccatcagctgcatcaccagcaccgacatcgacgacgacatgaactggtatcagcagaagcctggcgaggccgccatcctgcttatctctgagggaaacacactgcggcctggcatccctcctagattttccagcagcggctacggcaccgacttcaccctgaccatcaacaacatcgagagcgaggacgccgcctactacttctgcctgcaaagcgacaacctgcctctgacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 348) (Amino acid) ETTVTQSPAFMSATPGDKVTISCITSTDIDDDMNWYQQKPGEAAILLISEGNTLRPGIPPRFSSSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR(SEQ ID NO: 1014)

[0276] Humanized heavy and light chains linked via a flexible linker. Modified humanized 8F9A4A3 sequence (codon-optimized) (DNA) Caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttctggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgaaacgacagtgacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaccatctcctgcataaccagcactgatattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattctgctgattagcgaaggcaatactcttcgtcctggaatcccacctcgattcagtagcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtttgcaaagtgataacttgcctctcacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 349) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSETTVTQSPAFMSATPGDKVTISCITSTDIDDDMNWYQQKPGEAAILLISEGNTLRPGIPPRFSSSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (SEQ ID NO: 1015)

[0277] 8F9A5A1 Heavy Chain Variable Region Sequence (DNA) atccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgttgatgacttcaagggacggtttgccttctctttggaaacctctgccaccactgcctatttgcagatcaacaacctcaaaaatgaggacacgtctacatatttctgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 350) (Amino Acid) IQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYVDDFKGRFAFSLETSATTAYLQINNLKNEDTSTYFCARLRGIRPGPLAYWGQGTLVTVSA (SEQ ID NO: 1016)

[0278] IGHV7-81 * 01 V Region Sequence (DNA) caggtgcagctggtgcagtctggccatgaggtgaagcagcctggggcctcagtgaaggtctcctgcaaggcttctggttacagtttcaccacctatggtatgaattgggtgccacaggcccctggacaagggcttgagtggatgggatggttcaacacctacactgggaacccaacatatgcccagggcttcacaggacggtttgtcttctccatggacacctctgccagcacagcatacctgcagatcagcagcctaaaggctgaggacatggccatgtattactgtgcgaga(SEQ ID NO: 351) (Amino acid) QVQLVQSGHEVKQPGASVKVSCKASGYSFTTYGMNWVPQAPGQGLEWMGWFNTYTGNPTYAQGFTGRFVFSMDTSASTAYLQISSLKAEDMAMYYCAR(SEQ ID NO: 1017)

[0279] IGHJ4 * 03 J region sequence (DNA) tactttgactactggggccaagggaccctggtcaccgtctcctca(SEQ ID NO: 352) (Amino acid) YFDYWGQGTLVTVSS(SEQ ID NO: 1018) SEQ ID NO: 1019

[0280] Humanized 8F9A5A1 heavy chain variable region sequence (DNA) Caggtgcagctggtgcagtctggccatgaggtgaagcagcctggggcctcagtgaaggtctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgccacaggcccctggacaagggcttgagtggatgggatggataaacacctacactggagagccaacatatgttgatgacttcaagggacggtttgtcttctccatggacacctctgccagcacagcatacctgcagatcagcagcctaaaggctgaggacatggccatgtattactgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggaccctggtcaccgtctcctca(SEQ ID NO: 353) (Amino acid) QVQLVQSGHEVKQPGASVKVSCKASGYTFTNYGMNWVPQAPGQGLEWMGWINTYTGEPTYVDDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARLRGIRPGPLAYWGQGTLVTVSS(SEQ ID NO: 1019)

[0281] Humanized 8F9A5A1 heavy chain variable region sequence (codon-optimized) (DNA) caggttcagctggtgcagtctggccacgaagtgaaacagcctggcgcctctgtgaaggtgtcctgtaaagccagcggctacacctttaccaactacggcatgaactgggtgccccaggctcctggacaaggcttggaatggatgggctggatcaacacctacaccggcgagcctacctacgtggacgacttcaagggcagattcgtgttcagcatggacaccagcgccagcacagcctacctgcagatcagctctctgaaggccgaggatatggccatgtactactgcgccagactgagaggcatcagacctggacctctggcctattggggacagggcacactggtcacagtgtcctct(SEQ ID NO: 354) (Amino acid) QVQLVQSGHEVKQPGASVKVSCKASGYTFTNYGMNWVPQAPGQGLEWMGWINTYTGEPTYVDDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARLRGIRPGPLAYWGQGTLVTVSS (SEQ ID NO: 1020)

[0282] Modified humanized 8F9A5A1 heavy chain variable region sequence (DNA) cagatccagctggtgcagtctggccccgaggtgaagcagcctggggcctcagtgaaggtctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggcccctggacaagggcttgagtggatgggatggataaacacctacactggagagccaacatatgttgatgacttcaagggacggtttgccttctccatggacacctctgccagcacagcatacctgcagatcagcagcctaaaggctgaggacaccgccacctattactgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 355) (Amino acid) QIQLVQSGPEVKQPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYVDDFKGRFAFSMDTSASTAYLQISSLKAEDTATYYCARLRGIRPGPLAYWGQGTLVTVSS (SEQ ID NO: 1021)

[0283] Modified humanized 8F9A5A1 heavy chain variable region sequence (codon optimized) (DNA) cagattcagctggtgcagtctggccccgaagtgaaacaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccaactacggcatgaactgggtcaagcaggcccctggacaaggcctggaatggatgggctggatcaacacctacaccggcgagcctacctacgtggacgacttcaagggcagattcgccttcagcatggacaccagcgccagcacagcctacctgcagatcagctctctgaaggccgaggacaccgccacctactactgtgccagactgagaggcatcagacccggacctctggcctattggggacagggaacactggtcaccgtgtcctct(SEQ ID NO: 356) (Amino acid) QIQLVQSGPEVKQPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYVDDFKGRFAFSMDTSASTAYLQISSLKAEDTATYYCARLRGIRPGPLAYWGQGTLVTVSS(SEQ ID NO: 1022)

[0284] Variable region sequence of the light chain of 8F9A5A1 (DNA) gaaattttgctcacccagtctccagcaatcatagctgcatctcctggggagaaggtcaccatcacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaaccaggatcctcccccaaaatatggatttatggtatatccaacctggcttctggagttcctgctcgcttcagtggcagtgggtctgggacatctttctctttcacaatcaacagcatggaggctgaagatgttgccacttattactgtcagcaaaggagtagttacccacccacgttcggaggggggaccaagctggaaataaaacgg(SEQ ID NO: 357) (Amino acid) EILLTQSPAIIAASPGEKVTITCSASSSVSYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFSFTINSMEAEDVATYYCQQRSSYPPTFGGGTKLEIKR (SEQ ID NO: 1023)

[0285] IGKV3D-15 * 02 V region sequence (DNA) gaaatagtgatgatgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcaacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccaccagggccactggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtataataac (SEQ ID NO: 358) (Amino acid) EIVMMQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNN (SEQ ID NO: 1024)

[0286] IGKJ4 * 02 J region sequence (DNA) ctcacgttcggcggagggaccaaggtggagatcaaa (SEQ ID NO: 359) (Amino acid) LTFGGGTKVEIK (SEQ ID NO: 1025)

[0287] Humanized 8F9A5A1 light chain variable region sequence (DNA) gaaatagtgatgatgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaacctggccaggctcccaggctcctcatctatggtatatccaacctggcttctggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcaaaggagtagttacccacccacgttcggcggagggaccaaggtggagatcaaacgg(SEQ ID NO: 360) (Amino acid) EIVMMQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLLIYGISNLASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR(SEQ ID NO: 1026)

[0288] Humanized 8F9A5A1 light chain variable region sequence (codon-optimized) (DNA) gagatcgtgatgatgcagagccccgccacactgagtgtgtctccaggcgaaagagccacactgtcctgtagcgccagcagcagcgtgtcctacatgaactggtatcagcagaagcccggacaggcccctagactgctgatctacggcatcagcaatctggccagcggcatccctgccagattttctggctctggctccggcaccgagttcaccctgacaatctctagcctgcagagcgaggacttcgccgtgtactactgccagcagagaagcagctaccctcctacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 361) (Amino acid) EIVMMQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLLIYGISNLASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR (SEQ ID NO: 1027)

[0289] Modified humanized 8F9A5A1 light chain variable region sequence (DNA) gaaatagtgctgacccagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaacctggccaggctcccaggctctggatctatggtatatccaacctggcttctggcatcccagccaggttcagtggcagtgggtctgggacaagcttcagcctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcaaaggagtagttacccacccacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 362) (Amino acid) EIVLTQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLWIYGISNLASGIPARFSGSGSGTSFSLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR (SEQ ID NO: 1028)

[0290] Modified humanized 8F9A5A1 light chain variable region sequence (codon optimized) (DNA) gagatcgtgctgacacagtctcccgccacactgagtgtgtctccaggcgaaagagccacactgtcctgtagcgccagcagcagcgtgtcctacatgaactggtatcagcagaagcccggacaggcccctagactgtggatctacggcatcagcaatctggccagcggcatccctgccagattttctggctctggctccggcaccagcttcagcctgacaatcagcagcctgcagagcgaggacttcgccgtgtactactgccagcagagaagcagctaccctcctacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 363) (Amino acid) EIVLTQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLWIYGISNLASGIPARFSGSGSGTSFSLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR(SEQ ID NO: 1029)

[0291] Modified humanized 8F9A5A1 scFv sequence (codon-optimized) (DNA) Cagattcagctggtgcagtctggccccgaagtgaaacaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccaactacggcatgaactgggtcaagcaggcccctggacaaggcctggaatggatgggctggatcaacacctacaccggcgagcctacctacgtggacgacttcaagggcagattcgccttcagcatggacaccagcgccagcacagcctacctgcagatcagctctctgaaggccgaggacaccgccacctactactgtgccagactgagaggcatcagacccggacctctggcctattggggacagggaacactggtcaccgtgtcctctggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgagatcgtgctgacacagtctcccgccacactgagtgtgtctccaggcgaaagagccacactgtcctgtagcgccagcagcagcgtgtcctacatgaactggtatcagcagaagcccggacaggcccctagactgtggatctacggcatcagcaatctggccagcggcatccctgccagattttctggctctggctccggcaccagcttcagcctgacaatcagcagcctgcagagcgaggacttcgccgtgtactactgccagcagagaagcagctaccctcctacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 364) (Amino acid) QIQLVQSGPEVKQPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYVDDFKGRFAFSMDTSASTAYLQISSLKAEDTATYYCARLRGIRPGPLAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLWIYGISNLASGIPARFSGSGSGTSFSLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR(SEQ ID NO: 1030)

[0292] 8H5H5G4 heavy chain variable region sequence (DNA) gtccagctgcaacagtctggacctgatctggtgaagcctgggacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctcctca(SEQ ID NO: 365) (Amino acid) VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVSS(SEQ ID NO: 1031)

[0293] IGHV1-24 * 01 V region sequence (DNA) caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaaca(SEQ ID NO: 366) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT(SEQ ID NO: 1032)

[0294] IGHJ4 * 03 J region sequence (DNA) tactttgactactggggccaagggaccctggtcaccgtctcctca(SEQ ID NO: 367) (Amino acid) YFDYWGQGTLVTVSS(SEQ ID NO: 1033)

[0295] Humanized 8H5H5G4 heavy chain variable region sequence (DNA) caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggaaacacattcactgaatacaccatgcacTgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcAgagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtGcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaagggaccctggtcaccgtctcctca(SEQ ID NO: 368) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS(SEQ ID NO: 1034)

[0296] Humanized 8H5H5G4 heavy chain variable region sequence (codon-optimized) (DNA) caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggacttgaatggatgggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggccgcgtgaccatgaccgaggacacaagcacagacaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtcacagtttcttct(SEQ ID NO: 369) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS(SEQ ID NO: 1035)

[0297] Modified humanized 8H5H5G4 heavy chain variable region sequence (DNA) caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggaaacacattcactgaatacaccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatcggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggtcaccctgaccgtggacacatctagcagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaagggaccctggtcaccgtctcctca(SEQ ID NO: 370) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS(SEQ ID NO: 1036)

[0298] Modified humanized 8H5H5G4 heavy chain variable region sequence (codon optimized) (DNA) caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtcacagtttcttct(SEQ ID NO: 371) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS(SEQ ID NO: 1037)

[0299] 8H5H5G4 light chain variable region sequence (DNA) gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtttacattcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaacgg(SEQ ID NO: 372) (Amino acid) DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR (Sequence No. 1038)

[0300] IGKV1-27 * 01 V region sequence (DNA) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcagggcattagcaattatttagcctggtatcagcagaaaccagggaaagttcctaagctcctgatctatgctgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaagtataacagtgcccct (Sequence No. 373) (Amino acid) DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAP (Sequence No. 1039)

[0301] IGKJ4 * 02 J region sequence (DNA) ctcacgttcggcggagggaccaaggtggagatcaaa (Sequence No. 374) (Amino acid) LTFGGGTKVEIK (Sequence No. 1040)

[0302] Humanized 8H5H5G4 light chain variable region sequence (DNA) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcagtgcaagtcagggcattagcaattatttaaacTggtatcagcagaaaccagggaaagttcctaagctcctgatctattacacatcaagtttacattcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcagcagtatagtaagcttccttacacgttcggcggagggaccaaggtggagatcaaacgg(SEQ ID NO: 375) (Amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(SEQ ID NO: 1041)

[0303] Modified humanized 8H5H5G4 light chain variable region sequence (codon optimized) (DNA) gacatccagatgacacagagccctagcagcctgtctgccagcgtgggagacagagtgaccatcacatgtagcgccagccagggcatcagcaactacctgaactggtatcagcagaaacccggcaaggtgcccaagctgctgatctactacaccagcagcctgcacagcggcgtgccaagcagattttctggcagcggctctggcaccgacttcaccctgaccatatctagcctgcagcctgaggacgtggccacctactactgtcagcagtacagcaagctgccctacacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 376) (Amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR (SEQ ID NO: 1042)

[0304] Modified humanized 8H5H5G4 light chain variable region sequence (DNA) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcagtgcaagtcagggcattagcaattatttaaactggtatcagcagaaaccagggaaagttcctaagctcctgatctattacacatcaagtttacattcaggggtcccatctcggttcagtggcagtggatctgggacagattacactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcagcagtatagtaagcttccttacacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 377) (Amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR (SEQ ID NO: 1043)

[0305] Modified humanized 8H5H5G4 light chain variable region sequence (codon-optimized) (DNA) gacatccagatgacacagagccctagcagcctgtctgccagcgtgggagacagagtgaccatcacatgtagcgccagccagggcatcagcaactacctgaactggtatcagcagaaacccggcaaggtgcccaagctgctgatctactacaccagcagcctgcacagcggcgtgccaagcagattttctggcagcggctctggcaccgactacaccctgaccatatctagcctgcagcctgaggacgtggccacctactactgtcagcagtacagcaagctgccctacacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 378) (Amino acid) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(SEQ ID NO: 1044)

[0306] Modified humanized 8H5H5G4 scFv sequence (codon-optimized) (DNA) Caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtcacagtttcttctggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgacatccagatgacacagagccctagcagcctgtctgccagcgtgggagacagagtgaccatcacatgtagcgccagccagggcatcagcaactacctgaactggtatcagcagaaacccggcaaggtgcccaagctgctgatctactacaccagcagcctgcacagcggcgtgccaagcagattttctggcagcggctctggcaccgactacaccctgaccatatctagcctgcagcctgaggacgtggccacctactactgtcagcagtacagcaagctgccctacacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 379) (Amino acid) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(SEQ ID NO: 1045)

[0307] Human IgG1 heavy chain constant region sequence: (for the production of full antibodies - paired with a kappa or lambda constant region; 2 plasmids, expressed together) (DNA) (Amino acid) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1046)

[0308] Human IgG2 heavy chain constant region sequence: (for the production of full antibodies - paired with a kappa or lambda constant region; 2 plasmids, expressed together) (DNA) gcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatag(SEQ ID NO: 381) (Amino acid) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 1047)

[0309] Human kappa light chain constant region sequence: (DNA) aggacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 382) (Amino acid) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 1048)

[0310] Human lambda light chain constant region sequence: (DNA) ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatag(SEQ ID NO: 383) (Amino acid) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO: 1049)

[0311] Human IgG1 Fc region sequence: (Fused to scFv for homodimerization) (DNA) gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga(SEQ ID NO: 384) (Amino acid) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO: 1050)

[0312] Human IgG2 Fc region sequence: (DNA) gagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatag (SEQ ID NO: 385) (Amino acid) ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO: 1051)

[0313] In another aspect of the invention, immune cells engineered to express a CAR are administered to a patient diagnosed with cancer or cancer metastasis, or a patient at risk of developing cancer or cancer metastasis, and the immune cells are also engineered to express an anti-NME7 antibody or antibody fragment, which can be expressed from an inducible promoter. In one aspect, the CAR is MUC1 * induced by an antibody fragment. In one example, the CAR is huMNC2-CAR44. In one aspect, the anti-NME7 antibody or antibody fragment binds to an NME peptide listed above in the section "Peptides homologous to A1, A2, B1, B2 or B3 peptides" and "Extended peptides homologous to A1, A2, B1, B2 or B3 peptides". In another aspect, the antibody or antibody fragment binds to an NME7-derived peptide selected from A1, A2, B1, B2 or B3 (SEQ ID NOs: 141-145). In yet another aspect, the antibody, antibody fragment or antibody mimetic binds to an NME7 peptide containing the B3 peptide. In one aspect of the invention, the anti-NME7 antibody, antibody fragment or antibody mimetic comprises a sequence derived from the variable domain of anti-NME7 antibody 8F9A4A3 (SEQ ID NOs: 1001-1015), 8F9A5A1 (SEQ ID NOs: 1016-1030), or 8H5H5G4 (SEQ ID NOs: 1031-1045).

[0314] Such antibodies can be human antibodies or humanized antibodies. Such antibodies can be polyclonal, monoclonal, bispecific, bivalent, monovalent, single-chain, scFv, or can be antibody mimetics such as protein scaffolds that provide a recognition region that binds to a specific target. As will be understood by those skilled in the art, antibodies can be of non-human origin, human or humanized antibodies. Methods of humanizing an antibody include fusing all or a portion of the murine variable region to the V and J regions of the closest matching human antibody sequences, such as those represented by SEQ ID NOs: 1001-1045. Full antibodies can also be made, rather than single-chain constructs. For example, the murine heavy chain variable sequence is fused to human V and J regions and then fused to IgG1, IgG2, or IgG3 of the human heavy chain constant region. Similarly, the murine light chain variable sequence is fused to human V and J regions and then fused to IgG1, IgG2, or IgG3 of the human kappa or lambda constant region. The plasmids are co-expressed and bind to form a full antibody (SEQ ID NOs: 1047-1051).

[0315] In another aspect of the invention, the small molecule is selected for its ability to inhibit the tumorigenic effects of NME7, NME7 AB or NME7-X1. For example, high-throughput screening identifies small molecules that treat cancer. In a multi-well plate, the small molecules are added separately to wells in which cancer cells are cultured in a medium containing NME7 AB . If a small molecule reduces the amount of cells that become floating and / or reduces the expression of a metastasis marker or pluripotent stem cell marker such as CXCR4, CHD1, etc., then that small molecule is a cancer drug candidate. Another method of identifying a small molecule that is a cancer drug is to select a small molecule that binds to NME7, NME7 AB or NME7-X1 or inhibits the expression of NME7 species. Yet another high-throughput screening is to select small molecules that inhibit the binding of NME7 * to the PSMGFR peptide of the extracellular domain of MUC1 AB , and these small molecules are cancer drugs.

[0316] NME7 AB and the sequence of NME7-X1 is AB The only difference is that it lacks some of the N-terminal sequence found in NME7. AB We have shown that there are seven naturally occurring NMEs that are nearly identical to NMEs. AB Antibodies that bind to NME7-X1 are called NME7 variants unless there is a conformational difference that the antibody can distinguish. AB It can also bind to naturally occurring species that mimic NME7. AB If it is desired to inhibit NME7-X1 but not other species, or vice versa, siRNA, antisense nucleic acid, or gene editing techniques can be used to inhibit expression in one species but not the other.

[0317] In one example, the anti-cancer therapeutic is NME7, NME7-X1 or NME7. AB The nucleic acid directly or indirectly suppresses the specific expression of various species. Such nucleic acid can be siRNA, RNAi, antisense nucleic acid, etc. that directly suppresses NME7 species. In another aspect of the present invention, the nucleic acid can indirectly suppress NME7 species, for example, by changing the expression of a molecule that regulates it. For example, the super enhancer BRD4 suppresses the expression of NME7. Therefore, an effective therapeutic agent for treating or preventing cancer is an agent that increases the expression of BRD4. An effective therapeutic agent can be an agent that increases the expression of JMJD6, a cofactor of BRD4.

[0318] NME7 AB Alternatively, NME7-X1 derived peptides or the entire protein can be used to generate anti-NME7 and anti-NME7-X1 antibodies in animals, which we have demonstrated to inhibit cancer growth and inhibit the migration of cancer cells to metastatic cancer cells. Similarly, NME7 derived peptides can be administered to humans to generate antibodies to treat or prevent cancer or inhibit the migration of cancer cells to metastatic cancer cells. The NME7 peptide or protein can be administered to humans to generate anti-NME7, anti-NME7, anti-NME7 antibodies in the recipient. ABor is administered to a human as a vaccine so as to stimulate the production of an anti-NME7-X1 antibody. The results shown in FIGS. 12 and 13 are from NME7, particularly NME7-X1 or NME7 AB Immunizing a human with a group of peptides in the sequence may be a more effective vaccine than immunization with a single peptide, indicating that it can be. The peptide or protein may be further conjugated to a carrier protein or other adjuvant known to those skilled in the art to assist in stimulating an immune response.

[0319] NME7 peptides located outside the DM10 domain are preferred for generating antibodies for cancer treatment or prevention. Peptides that can be administered to a patient for cancer or metastasis prevention include the peptide sequences listed in FIGS. 6-9. A1, A2, B1, B2, and B3 are NME7 AB and NME7-X1, and are examples of peptides that bind to and generate antibodies that are administered to a patient for cancer treatment or prevention. The present invention is not limited to peptides of exact sequences such as the naturally occurring sequences in NME7 or NME7-X1. As known to those skilled in the art, some amino acid substitutions in peptide sequences can still generate antibodies that specifically recognize the native protein sequence. It is not intended that the present invention be limited to the peptides demonstrated herein that inhibit cancer growth or inhibit the migration of normal cancer cells to metastatic cancer cells. The methods used herein to identify peptides A1, A2, B1, B2, and B3 can also be used to identify other peptide sequences that may be equal to or more effective than the peptides demonstrated herein.

[0320] Human NME7 AB or a part of NME7-X1, or an antibody fragment that binds to NME7 AB or NME7-X1, the chimeric antigen receptor molecule is an anti-cancer therapeutic agent and is administered to a patient for the treatment or prevention of cancer or cancer metastasis.

[0321] In one example, the recognition unit or variable region of the anti-NME7 antibody is fused to a molecule of a T cell using a technique known as CAR (chimeric antigen receptor) technology or CAR T technology. A prominent feature of an antibody or a fragment thereof that can be used therapeutically to treat or prevent cancer is the identification of an antibody-like variable region that recognizes NME7 and prevents its interaction with a target that promotes cancer. In one example, the target is the PSMGFR region of MUC1 * is.

[0322] An antibody, antibody fragment, or single-chain antibody can be engineered into a chimeric molecule that includes a chimeric antigen receptor, also known as a CAR, which is then transfected or transduced into immune system cells such as T cells and administered to a patient. A humanized antibody or antibody fragment, typically an scFv, comprises much of the extracellular domain of the CAR. The antibody fragment is biochemically fused to an immune system signaling molecule such as a cytoplasmic signaling motif such as CD8 as a transmembrane domain, a T cell receptor signaling molecule also called an activation domain, or a costimulatory domain including, but not limited to, CD3 zeta, CD28, 41bb, OX40. The CAR can be transfected into a T cell or other cell, preferably an immune system cell, and administered to a patient. Here we describe a CAR whose extracellular portion comprises an anti-NME7, anti-NME7 AB or anti-NME7-X1 antibody, antibody fragment or single-chain, scFv antibody fragment. In a preferred embodiment, the antibody or antibody fragment is a human or humanized antibody.

[0323] An effective anti-NME7 or anti-NME7-X1 antibody, or fragment, has the ability to bind to native NME7, NME7 AB or NME7-X1. Indeed, the parent antibody from which the extracellular domain of the CAR is engineered binds NME7, NME7 ABOr it is generated by immunizing an animal with a peptide derived from NME7-X1. In one aspect of the present invention, the immunizing peptide is composed of NME7 amino acids 1 to 376. In one aspect of the present invention, the immunizing peptide is composed of NME7 amino acids 92 to 376. In another aspect of the present invention, the immunizing peptide is composed of NME7 amino acids 125 to 376. In yet another aspect of the present invention, the immunizing peptide is composed of the sequences listed in FIGS. 6-8. In another aspect of the present invention, the immunizing peptide is composed of the sequence listed in FIG. 9. Alternatively, the parental antibody or antibody fragment is selected from a natural, synthetic or any fragment-containing antibody library or pool, which are NME7, NME7 AB or NME7-X1, and are selected for their ability to bind to the peptides listed in FIGS. 6-8 or the peptide listed in FIG. 9.

[0324] The targeting moiety of the CAR need not be an antibody or antibody fragment. Here we describe a CAR in which the extracellular domain contains an NME7 fragment. NME7-derived peptides are engineered into different types of CARs where the targeting moiety of the extracellular domain is not an antibody or antibody fragment, but a protein fragment or peptide. The peptide CAR is genetically introduced or transduced into immune cells, typically T cells. NME7 fragments or NME7-derived peptides bind to their cognate binding partners, but are selected for their ability not to function like intact NME7, NME7 AB , or NME7-X1, and not to confer oncogenic activity. NME7 fragments or NME7-derived peptides are biochemically fused to immune signaling molecules such as CD8 as a transmembrane domain, cytoplasmic signaling motifs such as T cell receptor signaling molecules also called activation domains, or co-stimulatory domains including, but not limited to, CD3 zeta, CD28, 41bb, OX40.

[0325] In one aspect of the present invention, the NME7 fragment is most or all of the NME7 NDPK B domain. In another aspect of the present invention, the NME7 fragment is an NME7 peptide comprising one or more of the peptide sequences listed in FIGS. 6-9. Experimental results show that NME7 or NME7, NME7 AB or a fragment of NME7-X1 as a strategy for using, NME7 AB or a fairly large fragment of NME7-X1 may be more effective than shorter peptides, e.g., peptides less than 15 amino acids in length. Alternatively, a group of CARs each having a different NME7 AB derived peptide can be collectively transduced or transfected into immune system cells and administered to a patient for the treatment or prevention of cancer. The experiments shown in FIGS. 12 and 13 support the validity of this approach.

[0326] CARs containing an NME7 fragment in the extracellular domain are transduced or transfected into immune system cells, typically T cells, and administered to a patient for the treatment or prevention of cancer. In one aspect, the cancer is MUC1 * positive cancer. In another aspect, the cancer is metastatic cancer.

[0327] Agents that inhibit enzymes that cleave NME7 can be used to treat or prevent cancer. Some forms of NME7 are sequestered intracellularly and thus not secreted from the cell, where they can act as growth factors to promote cancer. Full-length NME7 is 42 kDa. However, we have found that an NME7 species of approximately 33 kDa that lacks the DM10 domain and appears substantially identical to the recombinant NME7 AB we generated is secreted from cancer cells and stem cells. This approximately 33 kDa NME7 species and another approximately 25 kDa NME7 species may be cleavage products removed by the use of agents that suppress the cleavage of NME7.

[0328] The detection of high levels of NME7, an approximately 33 kDa NME7 species (which we refer to as the NME7 AB -like species), or NME7-X1 in patient samples is an indicator of the presence of cancer or its progression to a higher malignancy or metastatic state. The inventors have found that at both early stages, naive stem cells and cancer cells, particularly MUC1 * -positive cancer cells, express high levels of an approximately 33 kDa NME7 and NME7-X1 that lack the DM10 domain.

[0329] NME7-X1 was recently listed in the protein database as a theoretical alternative isoform of NME7. However, it has not been detected in tissues or cells. We designed primers to distinguish NME7-X1 from NME7 by PCR. The expression levels of human NME7, NME7a, NME7b, and NME7-X1 were measured by PCR in a series of cells including fibroblasts, human embryonic stem cells, human iPS cells, T47D human breast cancer cells, DU145 human prostate cancer cells, PC3 human prostate cancer cells, HEK295 human fetal liver cells, and other human stem cell lines. NME7 is expressed at higher levels in cancer cells than in stem cells. In particular, NME7-X1 is expressed 10-fold higher in prostate cancer cells and 3-fold higher in breast cancer cells than in fibroblasts or stem cells. NME7-X1 is expressed approximately 5-fold higher in HEK293 fetal liver cells than in fibroblasts or stem cells, and thus, NME7-X1 is expected to be elevated in liver cancer. NME7b is expressed 17 - 25-fold higher in prostate cancer cells than in stem cells.

[0330] The detection of high levels of NME7 in patient samples is an indicator that the patient has or is at risk of developing cancer. The levels of NME7 can be measured or evaluated by PCR, hybridization schemes, cycling probe technology, FISH, immunocytochemistry, IHC, Western blot, immunoprecipitation, sandwich analysis, ELISA assays, etc. Patient samples can be liquid samples, blood samples, milk, urine, cells, liquid biopsies, biopsies, etc. In patients diagnosed with cancer, high levels of NME7 are indicators of increased metastatic potential. High levels of NME7-X1 are indicators of prostate cancer. The antibodies of the present invention are used to detect and distinguish NME7 species and are used as diagnostic tools.

[0331] Since adult human cells and tissues do not express or secrete NME7 at significant levels, an effective way to diagnose cancer or a more aggressive or metastatic type or a shift to a more aggressive type is to compare the NME7 levels in patient samples from cell or tissue collections or cultured cells from patients to the NME7 levels in healthy samples and / or to the levels of NME7 known to be present in healthy adult cells or tissues. Increased levels of NME7 indicate the presence of cancer, metastatic cancer, or the onset of metastasis. Also, increased levels of NME7 are associated with MUC1 *Indicates positive cancer. The sample assayed for the presence of NME7 can be a collection of cells that can be cells from a patient, a cultured cell line, a body fluid, a blood sample, a tissue specimen, or a biopsy material. Thus, a diagnostic assay for detecting the presence or progression of cancer includes the following steps: 1) obtaining a sample from a cancer patient or a patient at risk of developing cancer; 2) subjecting the sample to an assay capable of detecting or measuring the level of NME7 or the level of nucleic acid encoding NME7; 3) comparing the level of measured NME7 protein or NME7-encoding nucleic acid in the test sample to the level of a control patient or control cells; 4) identifying that the level of NME7 or the level of nucleic acid encoding NME7 is elevated compared to the control; and 5) concluding that cancer is progressing if the donor of the test sample has cancer or the control to which the test substance is compared is from a donor previously diagnosed with cancer.

[0332] In this assay, the control sample to which the test sample is compared can be a non-cancerous cell, a cultured cell, a sample from a healthy donor, a non-cancerous sample from the donor, or a sample from the donor of the test sample that is a control sample taken from the donor at a previous time point. Such sample sources can be any specimen taken from a patient being tested for the presence or progression of cancer, including body fluids, cerebrospinal fluid, bone marrow samples, blood, tissue, cells, biopsy tissue or cells, cultured cells derived from patient cells, etc. The sample source to which the test sample is compared can be body fluids, cerebrospinal fluid, bone marrow samples, blood, tissue, cells, biopsy tissue or cells, or cultured cells derived from a healthy donor or the test patient from whom the sample was obtained at a previous time point. The measured levels to which the test sample is compared can be from previously recorded data and are accumulated in a list for comparison to the test sample.

[0333] Cerenostics Patients diagnosed with high levels of NME7 protein or nucleic acid encoding NME7 are then treated with a therapeutic agent that suppresses NME7 expression, suppresses NME7 cleavage, or suppresses NME7 binding to its target (such interactions promote cancer). An important target of NME7 or a cleavage product of NME7 is MUC1 * . NME7 binds to the extracellular domain of MUC1 * and dimerizes it. Thus, patients diagnosed with high levels of NME7 benefit from treatment with a therapeutic agent that suppresses NME7 and / or a therapeutic agent that suppresses the dimerization of a cleaved form of MUC1 whose extracellular domain consists of some or all of the PSMGFR sequence. Thus, evaluating the suitability of cancer treatment and administering an effective amount of a therapeutic agent for the treatment or prevention of cancer consists of the following steps: 1) obtaining a sample from a patient suspected of having cancer, or at risk of developing cancer, or at risk of developing metastatic cancer; 2) measuring the amount of NME7, or its cleavage product, or NME7-encoding nucleic acid, wherein the measured level is significantly higher than the amount measured in a control sample; 3) identifying that the patient has cancer or has progressed to a higher-grade or metastatic cancer; 4) administering to the patient an effective amount of a therapeutic agent that suppresses NME7 expression, suppresses NME7 cleavage, or suppresses NME7 binding to its target, and / or an effective amount of a therapeutic agent that suppresses MUC1 expression, suppresses MUC1 * cleavage to MUC1, or suppresses MUC1 * binding to its target. In a preferred embodiment, the therapeutic agent that suppresses NME7 binding to its target suppresses its interaction with MUC1 * . In a more preferred embodiment, it suppresses its interaction with the extracellular domain of MUC1 * substantially composed of the PSMGFR sequence. In a preferred embodiment, the therapeutic agent that suppresses MUC1 * binding to its target suppresses the interaction between MUC1 * and NME7. In a more preferred embodiment, MUC1 *Therapeutic agents that inhibit the interaction between NME7 and NME7 AB inhibit the binding of MUC1 to the portion of NME7 that is substantially composed of the sequence of NME7 * .

[0334] Chemically modified peptides Polypeptides or antibody therapeutics have problems with short circulating half-lives and proteolysis and low solubility. To improve the pharmacokinetic and pharmacodynamic properties of the biopharmaceuticals of the present invention, methods such as manipulation of the amino acid sequence can reduce or increase immunogenicity and reduce proteolytic cleavage; fusion or conjugation of peptides to immunoglobulins such as albumin and serum proteins can be done; incorporation of biopharmaceuticals such as the peptides and antibodies of the present invention into drug delivery carriers for protection and sustained release can also be done; and conjugation to natural or synthetic polymers can also be considered. In particular, for synthetic polymer conjugation, acylation such as pegylation or N-acylation, S-acylation, etc. can also be considered.

[0335] Nucleic acid constructs Expression vectors containing the nucleic acid molecules of the present invention as described herein are also provided, wherein the nucleic acid molecules are operably linked to expression control sequences. Also provided is a host-vector system for the production of polypeptides comprising the expression vectors of the present invention introduced into host cells suitable for the expression of the polypeptides. Suitable host cells can be bacterial cells such as Escherichia coli, yeast cells such as Pichia pastoris, insect cells such as Spodoptera frugiperda, or mammalian cells such as COS, HEK or CHO cells.

[0336] The present invention also provides a method for producing the polypeptides of the present invention by growing the cells of the host-vector system described herein under conditions that permit the production of the polypeptides, and a method for recovering the polypeptides so produced. The polypeptides useful for practicing the present invention can be prepared by expression in prokaryotic or eukaryotic expression systems.

[0337] The recombinant gene is expressed and the polypeptide is purified using a number of methods. The gene can be subcloned into a bacterial expression vector such as, but not limited to, pZErO.

[0338] The polypeptide can be purified by any technique that allows for the formation of a stable biologically active protein. For example, but not limited to, the factor can be recovered from the cells either as a soluble protein or as an inclusion body, from which it is quantitatively extracted with 8 M guanidine hydrochloride and dialyzed. To further purify the factor, numerous purification methods are used, including, but not limited to, conventional ion exchange chromatography, affinity chromatography, differential sugar chromatography, hydrophobic interaction chromatography, reverse phase chromatography, or gel filtration.

[0339] As used herein, polypeptide includes functionally equivalent molecules in which amino acid residues are substituted within the sequence to result in silent or conservative changes. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of similar polarity that acts as a functional equivalent, resulting in a silent or conservative change. Substitutions of amino acids within the sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Potentially glycosylated amino acids include serine, threonine, and asparagine. Also included within the scope of the present invention are proteins or fragments or derivatives thereof that exhibit the same or similar biological activity, and derivatives that are modified differently, for example, by glycosylation, proteolytic cleavage, or linkage to antibody molecules or other cell ligands, during or after translation.

[0340] Any method known to those of ordinary skill in the art for inserting a DNA fragment into a vector can be used to construct an expression vector encoding the polypeptide of the present invention using appropriate transcriptional / translational control signals and protein coding sequences. These methods can include in vitro recombinant DNA and synthetic techniques, and in vivo recombination (genetic recombination). Expression of the nucleic acid sequence encoding the polypeptide of the present invention can be regulated by a second nucleic acid sequence, whereby the polypeptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of the polypeptides described herein can be controlled by any promoter / enhancer sequence known in the art. Promoters that can be used to control polypeptide expression include, but are not limited to: long terminal repeats described by Squinto et al. (1991, Cell 65:1-20); the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the CMV promoter, the M-MuLV 5' terminal repeat, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:144-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A.(80:21-25), see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; promoter sequences from yeast or other fungi, such as the gal4 promoter, ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerate kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions that show tissue specificity and have been used in transgenic animals: the elastase I gene control region that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region that is active in pancreatic β cells (Hanahan, 1985, Nature 315:115-122), the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), the mouse mammary tumor virus control region that is active in the testis, breast, lymphoid, and mast cells (Leder et al., 1986, Cell 45:485-495), the Sendai virus, lentivirus, albumin gene control region that is active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), the alpha-fetoprotein gene control region that is active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); the alpha1-antitrypsin gene control region that is active in the liver (Kelsey et al., 1987, Genes and Devel.The β-globin gene regulatory region that is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene regulatory region that is active in oligodendrocyte cells of the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene regulatory region that is active in skeletal muscle (Shani, 1985, Nature 314:283-286), and the gonadotropin-releasing hormone gene regulatory region that is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0341] Accordingly, by the present invention, an expression vector containing a nucleic acid encoding a polypeptide described herein that can replicate in a bacterial or eukaryotic host is used for gene introduction into the host, thereby inducing the expression of such nucleic acid to produce a polypeptide that can be recovered in a biologically active form. As used herein, biologically active form includes forms that can bind to a relevant receptor, give rise to a differentiation function, and / or affect the phenotype of cells expressing the receptor.

[0342] Expression vectors containing nucleic acid inserts can be identified by, but not limited to, at least three general methods: (a) DNA-DNA hybridization, (b) the presence or absence of "marker" gene function, and (c) expression of the inserted sequence. In the first method, the presence of the heterologous nucleic acid inserted into the expression vector can be detected by DNA-DNA hybridization using a probe containing a sequence homologous to the inserted nucleic acid sequence. In the second method, recombinant vector / host systems can be identified and selected based on the presence or absence of a specific "marker" gene function (e.g., thymidine kinase activity, antibiotic resistance, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a heterologous nucleic acid sequence into the vector. For example, if an efl nucleic acid sequence is inserted within the marker gene sequence of the vector, recombinants containing the insert can be identified by the absence of the marker gene function. In the third method, recombinant expression vectors can be identified by assay of the heterologous nucleic acid product expressed by the recombinant construct. Such assays can be based on, for example, the binding of a ligand to a receptor or a portion thereof that can be tagged with a detectable antibody or a portion thereof, or the binding of an antibody produced against the protein or a portion thereof of interest, or on the physical or functional properties of the nucleic acid product of interest.

[0343] Polypeptides, particularly the modified polypeptides of the present invention, can be expressed transiently, constitutively or permanently in host cells.

[0344] An effective amount useful for treating the diseases or disorders shown in the present invention can be determined by methods known to those skilled in the art (for example, Fingl, et al., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds. Macmillan Publishing Co, New York, pp. 1 - 46 (1975)). The pharmaceutical composition for use according to the present invention comprises the polypeptide described above in a pharmaceutically acceptable liquid, solid, or semi - solid carrier, is linked to a carrier or a targeting molecule (for example, an antibody, a hormone, a growth factor, etc.), and / or is incorporated into liposomes, microcapsules, and sustained - release formulations before in vivo administration. For example, the pharmaceutical composition can contain the polypeptide in an aqueous solution such as sterile water, physiological saline, phosphate buffer, or dextrose solution. Alternatively, the active agent can be contained in a solid (for example, wax) or semi - solid (for example, gelatin) formulation that can be implanted into a patient in need of such treatment. The route of administration can be any method of administration known in the art and includes, but is not limited to, intravenous, intrathecal, subcutaneous, intrauterine, injection into the diseased tissue, intra - arterial, intranasal, oral, or via an implant device.

[0345] Administration can result in the distribution of the active agent of the present invention over a systemic or local area. For example, in some conditions including distant regions of the nervous system, intravenous or intrathecal administration of the agent may be desirable. In some cases, an implant tablet containing the active agent can be placed in or near the damaged area. Suitable implant tablets include, but are not limited to, Gelfoam, wax, spray, or microparticle - based implant tablets.

[0346] The present invention also provides a pharmaceutical composition comprising the polypeptide described herein in a pharmaceutically acceptable carrier. The composition can be administered systemically or locally. Any method of administration known in the art can be used, and the methods of administration include, but are not limited to, intravenous, intrathecal, intra - arterial, intranasal, oral, subcutaneous, intraperitoneal, or by local injection or surgical implant. Sustained - release formulations are also provided.

[0347] Gene therapy Gene therapy refers to a therapy that is carried out by administration of an expressed or expressible nucleic acid to a subject. In this embodiment of the present invention, the nucleic acids produce their encoded proteins that mediate a therapeutic effect.

[0348] Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

[0349] For an overview of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods generally known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0350] Delivery of the nucleic acid to the patient can be direct or indirect. In the case of direct delivery, the patient is directly exposed to the nucleic acid or nucleic acid-bearing vector. In the case of indirect delivery, cells are first transformed in vitro with the nucleic acid and then transplanted into the patient. These two approaches are known as in vivo or ex vivo gene therapy, respectively.

[0351] In certain embodiments, the nucleic acid sequences are administered directly in vivo, where they are expressed to produce the encoded product. This can be accomplished by any of a number of methods known in the art, for example, by constructing them as part of an appropriate nucleic acid expression vector and administering them such that they enter cells; for example, by infection with a defective or attenuated retrovirus or other viral vector, or by direct injection of naked DNA, or by coating with lipids or cell surface receptors or gene delivery agents, encapsulation in liposomes, microparticles or microcapsules, or by conjugating them to peptides known to enter the nucleus and administering them, or by conjugating them to ligands that are exposed to receptor-mediated endocytosis (see, for example, Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used, in particular, to target cell types that express the receptor), etc. In another embodiment, a nucleic acid-ligand complex is formed, where the ligand comprises a fusogenic viral peptide that disrupts endosomes, and the nucleic acid is able to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be in vivo targeted for cell-specific uptake and expression by targeting a particular receptor. Alternatively, the nucleic acid can be introduced into the cell interior and integrated into the host cell DNA for expression by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

[0352] In certain embodiments, a viral vector comprising a nucleic acid sequence encoding a polypeptide is used. The nucleic acid sequence encoding the polypeptide used in gene therapy is cloned into one or more vectors, which facilitates delivery of the gene into the patient. Lentiviral vectors such as retroviral vectors, and other vectors such as adenoviral vectors and adeno-associated viruses are examples of viral vectors that can be used. Retroviral vectors contain the components necessary for the accurate packaging of the viral genome and integration into the host cell DNA.

[0353] Adenoviruses are particularly attractive carriers for delivering genes to the airway epithelium because they naturally infect the airway epithelium, causing mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Additionally, adeno-associated virus (AAV) has also been proposed for use in gene therapy.

[0354] Another approach to gene therapy involves introducing the gene into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, the method of introduction involves the introduction of a selectable marker into the cells. The cells are then subjected to a selection process to isolate the cells that have taken up and are expressing the transgene. Those cells are then delivered to the patient.

[0355] In this embodiment, the nucleic acid is introduced into the cells prior to in vivo administration of the resulting recombinant cells. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral vector or bacteriophage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques for introducing foreign genes into cells are known in the art and can be used according to the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide stable transfer of the nucleic acid into the cells, such that the nucleic acid is expressed by the cells and preferably is heritable and can be expressed by the progeny of those cells.

[0356] Cells into which nucleic acids can be introduced for the purposes of gene therapy include any desired available cell type, including but not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; and various stem or progenitor cells, particularly hematopoietic stem or progenitor cells, obtained from, for example, bone marrow, cord blood, peripheral blood, fetal liver, etc.

[0357] In a preferred embodiment, the cells used for gene therapy are of patient autologous origin.

[0358] In embodiments where recombinant cells are used for gene therapy, the nucleic acid sequences encoding the polypeptides are introduced into the cells such that they can be expressed by those cells or their progeny, and the recombinant cells are then administered in vivo for a therapeutic effect. In certain embodiments, stem cells or progenitor cells are used. Any stem cells and / or progenitor cells that can be isolated and maintained in vitro can likely be used according to this embodiment of the present invention.

[0359] In certain embodiments, the nucleic acid introduced for the purposes of gene therapy comprises an inducible promoter operably linked to the coding region, whereby the expression of the nucleic acid is controllable by modulating the presence or absence of a suitable transcriptional inducer.

[0360] Therapeutic composition Formulations of therapeutic compounds are generally known in the art and reference can conveniently be made to Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, from about 0.05 ng to about 20 mg per kg body weight per day can be administered. The dosing regimen can be adjusted to obtain the optimal therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compounds can be administered by convenient routes such as oral, intravenous (when water-soluble), intramuscular, subcutaneous, intranasal, intraocular, intradermal or suppository routes or by implantation (e.g., using sustained release molecules by the intraperitoneal route or using cells such as monocytes or dendritic cells sensitized in vitro and adoptively transferred to the recipient). Depending on the route of administration, the peptide may need to be coated with a material that will protect it from the action of enzymes, acids and other natural conditions that may inactivate the components.

[0361] For example, the low lipophilicity of peptides allows them to be destroyed in the digestive tract by enzymes capable of cleaving peptide bonds or in the stomach by acid hydrolysis. In order to administer peptides by methods other than parenteral administration, the peptides are coated with a substance that prevents their inactivation or are administered with such a substance. For example, the peptides can be administered in an adjuvant, by co-administration with an enzyme inhibitor, or in liposomes. Adjuvants contemplated herein include resorcinol; nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEP), and trasylol. Liposomes include water-in-oil-in-water type CGF emulsions, as well as conventional liposomes.

[0362] The active compounds can also be administered parenterally or intraperitoneally. The dispersants can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0363] Dosage forms suitable for injectable use include sterile aqueous solutions (when soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersing agents. In all cases, the form must be sterile and fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof and vegetable oils. Appropriate fluidity can be maintained by the use of coatings such as lecithin, and by the use of surfactants, in the case of dispersions, to maintain the required particle size. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents (such as chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferred to contain isotonic agents (such as sucrose or sodium chloride). Sustained absorption of the injectable composition can be brought about by the use of absorption delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0364] Sterile injectable solutions are prepared by incorporating the required amount of the active compound in a suitable solvent, with the various other ingredients enumerated above, and, as required, subsequently subjecting the mixture to sterile filtration. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile carrier containing a basic dispersion medium and the necessary other ingredients selected from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a mixed powder of the active ingredient derived from its previously sterile-filtered solution and any additional desired ingredients.

[0365] When the peptide is properly protected as described above, the active compound is administered, for example, with an inert diluent or assimilable edible carrier, or it is enclosed in hard or soft shell gelatin capsules, or it is compressed into tablets, or it is directly incorporated into food for diet and administered orally. In the case of oral therapeutic administration, the active compound is incorporated with excipients and can be used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions and formulations should contain at least 1% by weight of the active compound. The percentages of the compositions and formulations can, of course, vary and can preferably be between about 5% and about 80% by weight units. The amount of the active compound in such therapeutically useful compositions is an amount such that an appropriate dosage is obtained. Preferred compositions or formulations according to the present invention are prepared such that the oral dosage unit form contains an active ingredient between about 0.1 μg and 2000 mg.

[0366] Tablets, pills, capsules, etc. may also include the following: binders such as tragacanth gum, gum arabic, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose or saccharin, or flavoring agents such as peppermint, wintergreen oil or cherry flavor. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the substances of the above types. Various other substances may be present as coating agents or for other reasons to change the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sucrose, or both. Syrups or elixirs may contain the active compound, sucrose as a sweetener, methyl and propyl parabens as preservatives, and flavoring agents such as cherry or orange flavor. Of course, any substance used in the preparation of any dosage unit form must be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active compound can be incorporated into sustained release formulations and combinations.

[0367] Delivery system A variety of delivery systems are known, for example, encapsulation into liposomes, microparticles, microcapsules, recombinant cells capable of expressing a compound, receptor-dependent endocytosis, nucleic acid constructs as part of a retrovirus or other vector, etc., can be used to administer the compounds of the present invention. The methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, and oral routes. The compound or composition can be administered by any convenient route, for example, by injection or bolus injection, by absorption through epithelial or skin mucosal surfaces (such as oral mucosa, rectal and intestinal mucosa, etc.), and can also be administered together with other bioactive substances. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compound or composition of the present invention into the central nervous system by any suitable route including intracerebroventricular injection and intrathecal injection. Intracerebroventricular injection can be facilitated, for example, by an intracerebroventricular catheter attached to a reservoir such as an Ommaya reservoir. For example, pulmonary administration can also be employed by the use of an inhaler or nebulizer and a formulation having an aerosolizing agent.

[0368] In certain embodiments, it may be desirable to locally administer the pharmaceutical compounds or compositions of the invention to the area in need of treatment; this can be achieved, for example, but not limited to, by local injection during surgery, topical application, for example, in combination with a postoperative wound dressing, by injection, by catheter, by suppository, or by implant. The implant can be a porous, non-porous, gelatinous material containing a membrane such as a silicone membrane or fibers. Preferably, when administering a protein comprising an antibody or peptide of the invention, care must be taken to use a substance that the protein will not absorb. In another embodiment, the compound or composition can be delivered in vesicles, particularly liposomes. In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed in the vicinity of the therapeutic target, thereby requiring only a very small amount of the systemic dose.

[0369] Sequence Listing Free Text For the use of nucleotide abbreviations other than a, g, c, and t, they shall conform to the rules defined in WIPO Standard ST.25, Supplementary Annex 2, Table 1, where k represents t or g; n represents a, c, t, or g; m represents a or c; r represents a or g; s represents c or g; w represents a or t, and y represents c or t.

[0370] MTPGTQSPFF LLLLLTVLTV VTGSGHASST PGGEKETSAT QRSSVPSSTE KNAVSMTSSV LSSHSPGSGS STTQGQDVTL APATEPASGS AATWGQDVTS VPVTRPALGS TTPPAHDVTS APDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDNRPALGS TAPPVHNVTS ASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHS SVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQI YKQGGFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN ​​YGQLDIFPAR DTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL (SEQ ID NO: 1) represents the full length MUC1 receptor (mucin 1 precursor, GenBank Accession Number 15941).

[0371] MTPGTQSPFFLLLLLTVLT (SEQ ID NO: 2) MTPGTQSPFFLLLLLTVLT VVTA (SEQ ID NO: 3) MTPGTQSPFFLLLLLTVLT VVTG (SEQ ID NO: 4) SEQ ID NOs:2, 3 and 4 are N-terminal MUC-1 signaling sequences for targeting the MUC1 receptor and truncated isoforms to the cell membrane surface. Up to three amino acid residues can be deleted at the C-terminus, as shown by the variants in SEQ ID NOs:2, 3 and 4.

[0372] GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQ ID NO:5) represents a truncated MUC1 receptor isoform with the native PSMGFR at its N-terminus and contains the transmembrane and cytoplasmic sequences of the full-length MUC1 receptor.

[0373] GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6) represents the extracellular domain of the native primary sequence of the MUC1 receptor (nat-PSMGFR - an example of "PSMGFR").

[0374] TINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 7) represents the extracellular domain of the native primary sequence of the MUC1 growth factor receptor (nat-PSMGFR - an example of "PSMGFR") having a single amino acid deletion at the N-terminus of SEQ ID NO: 6.

[0375] GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 8) represents the extracellular domain of the "SPY" functional variant of the native primary sequence of the MUC1 growth factor receptor (var-PSMGFR - an example of "PSMGFR") having enhanced stability.

[0376] TINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 9) represents the extracellular domain of the "SPY" functional variant of the MUC1 growth factor receptor having enhanced stability and having a single amino acid deletion at the C-terminus of SEQ ID NO: 8 (var-PSMGFR - an example of "PSMGFR").

[0377] tgtcagtgccgccgaaagaactacgggcagctggacatctttccagcccgggatacctaccatcctatgagcgagtaccccacctaccacacccatgggcgctatgtgccccctagcagtaccgatcgtagcccctatgagaaggtttctgcaggtaacggtggcagcagcctctcttacacaaacccagcagtggcagccgcttctgccaacttg (SEQ ID NO: 10) represents the MUC1 cytoplasmic domain nucleotide sequence.

[0378] CQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQ ID NO: 11) represents the MUC1 cytoplasmic domain amino acid sequence.

[0379] gagatcctgagacaatgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggtatgttgaatacactatattcagtacattttgttaataggagagcaatgtttattttcttgatgtactttatgtatagaaaataa (SEQ ID NO: 12) represents the NME7 nucleotide sequence (NME7: GenBank accession number AB209049).

[0380] DPETMNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGMLNTLYSVHFVNRRAMFIFLMYFMYRK (SEQ ID NO: 13) represents the NME7 amino acid sequence (NME7: GenBank accession number AB209049).

[0381] atggtgctactgtctactttagggatcgtctttcaaggcgaggggcctcctatctcaagctgtgatacaggaaccatggccaactgtgagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatcaagcgttttgagcagaaaggattccgccttgttggtctgaaattcatgcaagcttccgaagatcttctcaaggaacactacgttgacctgaaggaccgtccattctttgccggcctggtgaaatacatgcactcagggccggtagttgccatggtctgggaggggctgaatgtggtgaagacgggccgagtcatgctcggggagaccaaccctgcagactccaagcctgggaccatccgtggagacttctgcatacaagttggcaggaacattatacatggcagtgattctgtggagagtgcagagaaggagatcggcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactggatctatgaatga (SEQ ID NO: 14) represents the NM23-H1 nucleotide sequence (NM23-H1: GenBank accession number AF487339).

[0382] MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVESAEKEIGLWFHPEELVDYTSCAQNWIYE(SEQ ID NO:15) NM23-H1 represents the amino acid sequence (NM23-H1: GenBank accession number AF487339).

[0383] atggtgctactgtctactttagggatcgtctttcaaggcgaggggcctcctatctcaagctgtgatacaggaaccatggccaactgtgagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatcaagcgttttgagcagaaaggattccgccttgttggtctgaaattcatgcaagcttccgaagatcttctcaaggaacactacgttgacctgaaggaccgtccattctttgccggcctggtgaaatacatgcactcagggccggtagttgccatggtctgggaggggctgaatgtggtgaagacgggccgagtcatgctcggggagaccaaccctgcagactccaagcctgggaccatccgtggagacttctgcatacaagttggcaggaacattatacatggcggtgattctgtggagagtgcagagaaggagatcggcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactggatctatgaatga(SEQ ID NO:16) represents the nucleotide sequence of the NM23-H1 S120G mutant (NM23-H1: GenBank accession number AF487339).

[0384] MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGGDSVESAEKEIGLWFHPEELVDYTSCAQNWIYE (SEQ ID NO: 17) represents the NM23-H1 S120G mutant amino acid sequence (NM23-H1: GenBank accession number AF487339).

[0385] atggccaacctggagcgcaccttcatcgccatcaagccggacggcgtgcagcgcggcctggtgggcgagatcatcaagcgcttcgagcagaagggattccgcctcgtggccatgaagttcctccgggcctctgaagaacacctgaagcagcactacattgacctgaaagaccgaccattcttccctgggctggtgaagtacatgaactcagggccggttgtggccatggtctgggaggggctgaacgtggtgaagacaggccgagtgatgcttggggagaccaatccagcagattcaaagccaggcaccattcgtggggacttctgcattcaggttggcaggaacatcattcatggcagtgattcagtaaaaagtgctgaaaaagaaatcagcctatggtttaagcctgaagaactggttgactacaagtcttgtgctcatgactgggtctatgaataa (SEQ ID NO: 18) represents the NM23-H2 nucleotide sequence (NM23-H2: GenBank accession number AK313448).

[0386] It represents the amino acid sequence of MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE (SEQ ID NO: 19), which is the NM23-H2 amino acid sequence (NM23-H2: GenBank accession number AK313448).

[0387] Human NM23-H7-2 sequence optimized for expression in E. coli: (DNA) (Amino acid) MHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN(SEQ ID NO: 21)

[0388] Human NME7-A: (DNA) atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttttga(SEQ ID NO: 22) (Amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF (SEQ ID NO: 23)

[0389] Human NME7-A1: (DNA) atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttacttga (SEQ ID NO: 24) (Amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT (SEQ ID NO: 25)

[0390] Human NME7-A2: (DNA) atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttttga(SEQ ID NO: 26) (Amino acid) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF(SEQ ID NO: 27)

[0391] Human NME7-A3: (DNA) atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttacttga(SEQ ID NO: 28) (Amino acid) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT(SEQ ID NO: 29)

[0392] Human NME7-B: (DNA) atgaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga(SEQ ID NO: 30) (Amino acid) MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF(SEQ ID NO: 31)

[0393] Human NME7-B1: (DNA) atgaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga(SEQ ID NO: 32) (Amino acid) MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN(SEQ ID NO: 33)

[0394] Human NME7-B2: (DNA) atgccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga(SEQ ID NO: 34) (Amino acid) MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF(SEQ ID NO: 35)

[0395] Human NME7-B3: (DNA) atgccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga(SEQ ID NO: 36) (Amino acid) MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN(SEQ ID NO: 37)

[0396] Human NME7, also known as NME7-AB AB : (DNA) atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga(SEQ ID NO: 38) (Amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN--(SEQ ID NO: 39)

[0397] Human NME7-AB1: (DNA) atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga(SEQ ID NO: 40) (Amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF(SEQ ID NO: 41)

[0398] Human NME7-A sequence optimized for E. coli expression: (DNA) atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttctga(SEQ ID NO: 42) (Amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF(SEQ ID NO: 43)

[0399] Human NME7-A1 sequence optimized for E. coli expression: (DNA) atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttacctga(SEQ ID NO: 44) (Amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT(SEQ ID NO: 45)

[0400] Human NME7-A2 sequence optimized for E. coli expression: (DNA) atgaatcactccgaacgctttgtttttatcgccgaatggtatgacccgaatgcttccctgctgcgccgctacgaactgctgttttatccgggcgatggtagcgtggaaatgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttctga(SEQ ID NO: 46) (Amino acid) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF(SEQ ID NO: 47)

[0401] NME7-A3 sequence optimized for E. coli expression: (DNA) atgaatcactccgaacgctttgtttttatcgccgaatggtatgacccgaatgcttccctgctgcgccgctacgaactgctgttttatccgggcgatggtagcgtggaaatgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttacctga(SEQ ID NO: 48) (Amino acid) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT(SEQ ID NO:49)

[0402] Human NME7-B sequence optimized for E. coli expression: (DNA) atgaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga(SEQ ID NO:50) (Amino acid) MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF(SEQ ID NO:51)

[0403] Human NME7-B1 sequence optimized for E. coli expression: (DNA) atgaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga(SEQ ID NO: 52) (Amino acid) MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN(SEQ ID NO: 53)

[0404] Human NME7 - B2 sequence optimized for E. coli expression: (DNA) atgccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga(SEQ ID NO: 54) (Amino acid) MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF(SEQ ID NO: 55)

[0405] Human NME7 - B3 sequence optimized for E. coli expression: (DNA) atgccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga(SEQ ID NO: 56) (Amino acid) MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN(SEQ ID NO: 57)

[0406] NME7 optimized for E. coli expression AB The human NME7-AB sequence, also known as: (DNA) atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga(SEQ ID NO: 58) (Amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN (SEQ ID NO: 59)

[0407] NME7 optimized for E. coli expression AB The human NME7-AB1 sequence also known as 1: (DNA) Atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga(SEQ ID NO: 60) (Amino acid) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF(SEQ ID NO: 61)

[0408] Mouse NME6 (DNA) Atgacctccatcttgcgaagtccccaagctcttcagctcacactagccctgatcaagcctgatgcagttgcccacccactgatcctggaggctgttcatcagcagattctgagcaacaagttcctcattgtacgaacgagggaactgcagtggaagctggaggactgccggaggttttaccgagagcatgaagggcgttttttctatcagcggctggtggagttcatgacaagtgggccaatccgagcctatatccttgcccacaaagatgccatccaactttggaggacactgatgggacccaccagagtatttcgagcacgctatatagccccagattcaattcgtggaagtttgggcctcactgacacccgaaatactacccatggctcagactccgtggtttccgccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaggaaccccagctgcggtgtggtcctgtgcactacagtccagaggaaggtatccactgtgcagctgaaacaggaggccacaaacaacctaacaaaacctag(SEQ ID NO: 62) (Amino acid) MTSILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRTRELQWKLEDCRRFYREHEGRFFYQRLVEFMTSGPIRAYILAHKDAIQLWRTLMGPTRVFRARYIAPDSIRGSLGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVHYSPEEGIHCAAETGGHKQPNKT (SEQ ID NO: 63)

[0409] Human NME6: (DNA) Atgacccagaatctggggagtgagatggcctcaatcttgcgaagccctcaggctctccagctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtgctatagcccagagggaggtgtccactatgtagctggaacaggaggcctaggaccagcctga (SEQ ID NO: 64) (Amino acid) MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGVHYVAGTGGLGPA (SEQ ID NO: 65)

[0410] Human NME6 1: (DNA) Atgacccagaatctggggagtgagatggcctcaatcttgcgaagccctcaggctctccagctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtga (SEQ ID NO: 66) (Amino acid) MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV (SEQ ID NO: 67)

[0411] Human NME6 2: (DNA) Atgctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtga (SEQ ID NO: 68) (Amino acid) MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV (SEQ ID NO: 69)

[0412] Human NME6 3: (DNA) Atgctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtgctatagcccagagggaggtgtccactatgtagctggaacaggaggcctaggaccagcctga(SEQ ID NO: 70) (Amino acid) MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGVHYVAGTGGLGPA(SEQ ID NO: 71)

[0413] Human NME6 sequence optimized for E. coli expression: (DNA) Atgacgcaaaatctgggctcggaaatggcaagtatcctgcgctccccgcaagcactgcaactgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctgttattctccggaaggtggtgtccattatgtggcgggcacgggtggtctgggtccggcatga(SEQ ID NO: 72) (Amino acid) MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGVHYVAGTGGLGPA(SEQ ID NO: 73)

[0414] Human NME6 1 sequence optimized for E. coli expression: (DNA) Atgacgcaaaatctgggctcggaaatggcaagtatcctgcgctccccgcaagcactgcaactgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctga(SEQ ID NO: 74) (Amino acid) MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV(SEQ ID NO: 75)

[0415] Human NME6 2 sequence optimized for E. coli expression: (DNA) Atgctgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctga(SEQ ID NO: 76) (Amino acid) MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV(SEQ ID NO: 77)

[0416] Human NME6 3 sequence optimized for E. coli expression: (DNA) Atgctgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctgttattctccggaaggtggtgtccattatgtggcgggcacgggtggtctgggtccggcatga(SEQ ID NO: 78) (Amino acid) MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGVHYVAGTGGLGPA(SEQ ID NO: 79)

[0417] OriGene-NME7-1 full length (DNA) (Amino acid) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDNTRTRRLEQKLISEEDLAANDILDYKDDDDKV(SEQ ID NO: 81)

[0418] Abnova NME7-1 full length (Amino acid) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN(SEQ ID NO: 82)

[0419] Abnova partial NME7-B (Amino acid) DRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKT...

Claims

1. An NME7-specific monoclonal antibody or an NME7-binding fragment thereof that binds to the NME7 B3 peptide of SEQ ID NO:

169.

2. The antibody or NME7-binding fragment thereof according to claim 1, which is a bivalent antibody.

3. The antibody or NME7-binding fragment thereof according to claim 1, which is a monovalent, Fab, or single-chain variable fragment antibody (scFv).

4. NME7 ＡＢ Or an antibody or NME7-binding fragment thereof according to any one of claims 1-3 that specifically binds to NME7 or NME7-X1 but does not specifically bind to NME1.

5. NME7 ＡＢ And MUC1 ＊ An antibody or NME7-binding fragment thereof according to any one of claims 1-4 that disrupts the interaction between the extracellular domains of NME7 and MUC1 or between NME7-X1 and MUC1. ＊

6. An antibody or NME7-binding fragment thereof according to any one of claims 1-5 that disrupts the binding between NME7 and the PSMGF peptide or between NME7-X1 and the PSMGF peptide. NME7 ＡＢ

7. An antibody or NME7-binding fragment thereof according to claim 6 that disrupts the binding between NME7 and the N-10 peptide of PSMGF or between NME7-X1 and the N-10 peptide. NME7 ＡＢ

8. An amino acid sequence in the heavy chain variable region consisting of: A CDR1 region consisting of YTFTNYGMN (SEQ ID NO: 439), A CDR2 region consisting of WINTYTGEPTYVDDFKG (SEQ ID NO: 440), and A CDR3 region consisting of LRGIRPGPLAY (SEQ ID NO: 441), and ​ An amino acid sequence in a light chain variable region consisting of the following, A CDR1 region consisting of SASSSVSYMN (SEQ ID NO: 444), A CDR2 region consisting of GISNLAS (SEQ ID NO: 445), and A CDR3 region consisting of QQRSSYPPT (SEQ ID NO: 446) The antibody according to any one of claims 1 - 7 or an NME7-binding fragment thereof, comprising the same.

9. An amino acid sequence in a heavy chain variable region consisting of the following, A CDR1 region consisting of NTFTEYTMH (SEQ ID NO: 429), A CDR2 region consisting of GFNPNNGVTNYNQKFKKG (SEQ ID NO: 430), and A CDR3 region consisting of RYYHSTYVFYFDS (SEQ ID NO: 431), and An amino acid sequence in a light chain variable region consisting of the following, A CDR1 region consisting of SASQGISNYLN (SEQ ID NO: 434), A CDR2 region consisting of YTSSLHS (SEQ ID NO: 435), and A CDR3 region consisting of QQYSKLPYT (SEQ ID NO: 436) The antibody according to claim 1 or an NME7-binding fragment thereof, comprising the same.

10. The antibody according to any one of claims 1 - 7 or an NME7-binding fragment thereof, which is a humanized antibody.

11. The antibody according to any one of claims 1 - 10 or an NME7-binding fragment thereof, for use in the diagnosis of cancer or cancer metastasis.

12. The antibody according to claim 11 or an NME7-binding fragment thereof, wherein the diagnosis of the cancer is in a tissue.

13. The antibody according to any one of claims 1 - 12 or an NME7-binding fragment thereof, wherein the antibody is conjugated to an imaging agent.

14. A composition comprising the antibody according to any one of claims 1 - 10 or its NME7-binding fragment for use in the prevention or treatment of cancer.

15. A composition comprising the antibody according to any one of claims 1 - 10 or its NME7-binding fragment for use in the prevention or treatment of cancer metastasis.

16. An isolated nucleic acid encoding the monoclonal antibody according to any one of claims 1 - 10 or its NME7-binding fragment.

17. NME7 ＡＢ A cell engineered to express a nucleic acid comprising a sequence encoding a NME7-specific monoclonal antibody or its NME7-binding fragment that binds to the NME7 B3 peptide of SEQ ID NO:

169.

18. Said NME7 ＡＢ wherein the specific monoclonal antibody or its NME7-binding fragment disrupts the binding of NME7 to the PSMGF peptide of the extracellular domain of MUC1 ＊ or the binding of NME7-X1, the cell according to claim 17. ＡＢ

19. The cell according to claim 17 or 18, wherein the cell is an immune cell or its progenitor cell.

20. The immune cell according to claim 19, comprising a CAR that recognizes a tumor-associated antigen.

21. The tumor-associated antigen is MUC1 ＊ The immune cell according to claim 20.

22. Said NME7 ＡＢ The cell according to any one of claims 17 - 21, wherein the expression of the specific monoclonal antibody is inducible.

23. A method for generating an NME7-specific monoclonal antibody that binds to an NME7 B3 peptide, comprising the step of injecting a peptide consisting of a continuous sequence having 90% sequence identity to SEQ ID NO: 39 into a non-human animal, a. the N-terminus of the peptide is within 7 amino acids from the N-terminus of SEQ ID NO: 145, b. the C-terminus of the peptide is within 7 amino acids from the C-terminus of SEQ ID NO: 145, c. the peptide contains a mutation at cysteine 14 of SEQ ID NO: 145, Method.

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